IN MEMORIAM
DR. M.H. SIKMOHS
PHYSIOLOGY AND BIOCHEMISTRY IN MODERN MEDICINE
BY
J. J. E. MACLEOD, M.B.
PROFESSOR OF PHYSIOLOGY IN THE UNIVERSITY OF TORONTO, TORONTO, CANADA; FORMERLY
PROFESSOR OF PHYSIOLOGY IN THE WESTERN RESERVE UNIVERSITY,
CLEVELAND, OHIO
ASSISTED BY ROY G. PEARCE, A. C. REDFIELD, AND N. B. TAYLOR
AND BY OTHERS
THIRD EDITION
WITH 243 ILLUSTRATIONS, INCLUDING 9 PLATES IN COLORS
ST. LOUIS
0. V. MOSBY COMPANY
1920
COPYRIGHT, 1918, 1919, 1920, BY C. V. MOSBY COMPANY
(All Rights Strictly Reserved)
Press of
C. V. Mosby Company St. Louis, U.S.A.
TO M. W. M.
•w,.J
PREFACE TO THIRD EDITION
Many changes have been made in the present (third) edition of the book. The section on the nervous system has been entirely recast and re- written by my colleague, A. C. Redfield, who, besides bringing this part of the subject up to date, has incorporated with it an account of the funda- mental principles of neuromuscular physiology. Although no applica- tion of this subject may at present be apparent in the investigation of disease it is certain that such exists; but it can be made only after the clinical researcher has become familiar with the brilliant work which has been done in the field in recent years by Keith Lucas, Adrian, and others. It is the function of a volume of this nature to describe not merely what already has been achieved in the clinical applications of physiology, but also to anticipate where this application is likely soon to be made and to prepare the way by describing the physiological principles that may be involved.
Another section in which complete changes have been called for, is that relating to the chemistry of respiration. This has been rewritten and rearranged so as to incorporate the recent work on the effects of deficiency of oxygen on the respiratory center, as well as the interesting and important clinical applications of the subject. Several new chapters have been added dealing with such practical problems as the measurement of the functional capacity of the heart, the principles of ventilation and the therapeutic value of oxygen, and the chapters on vitamines, on the capillary circula- tion, on surgical shock, and on the interpretation of polysphygmograms have been rewritten.
In practically every other section of the book many additions have been made, particularly in that which deals with the endocrine organs, and several new figures and tables have been added. To make room for these changes some of the more technical details, appearing in the previous editions, have been put in small print and some of the figures removed. This has been done in order to keep the volume as near to its original bulk as possible.
I wish to take this opportunity to thank my colleagues here and else- where for many valuable suggestions and for their encouraging comments on the book. I am also greatly indebted to Dr. N. B. Taylor for his as- sistance in the preparation of this edition and for reading the proofs.
J. J. R. MACLEOD.
Toronto. Canada. 1920.
VI PREFACE
PREFACE TO SECOND EDITION
The opportunity has been taken in this second edition to eliminate typographical errors and to alter the wording in certain chapters where there was ambiguity of statement in the first edition. The most en- couraging reception afforded the volume has fully confirmed the author's conviction that acquaintance with modern physiology is fundamental to sound medical and surgical practice.
J. J. R. MACLEOD.
Toronto, Canada. 1919.
PREFACE TO FIRST EDITION
The necessity of allotting the various subjects of the medical curric- ulum to different periods, so that the more strictly scientific subjects are completed in the earlier years, has the great disadvantage that the student, being no longer in touch with laboratory work, fails to employ the scientific knowledge with full advantage in the solution of his clin- ical problems. He is apt to regard the first two or three years in the laboratory departments as inconsequential in comparison with the sup- posedly more practical instruction offered during the subsequent clinical years. He is taught by his laboratory instructors to observe accurately, and to correlate the observed facts, so that he may be enabled to draw conclusions as to the manner of working of the various functions of the animal body in health, and before proceeding to his clinical studies, he is required to show a proficiency in scientific knowledge, because it is recognized that this must serve as the basis upon which his knowledge of disease is to be built. When the clinic is reached, however, the meth- ods of the scientist are. not infrequently cast aside and an understanding of disease is sought for largely by the empirical method; namely, by the endeavor to see and examine innumerable patients, to diagnose the case according to the grouping of the signs and symptoms, and to treat it by the prescribed methods of experience. So much has to be learned and so much has to be seen during the clinical years, that the student gives little thought to the nature of the functional disturbance which' is responsible for the symptoms; he fails to realize that after all, there is no essen- tial difference between the condition brought about in his patient by some pathological lesion, and that which may be produced in the labora- tory by experimental procedures, by drugs or by toxins. It must of course be recognized that just as the science of medicine originated by the grouping of symptoms into more or less characteristic diseases for
PREFACE Vil
which the most favorable method of treatment had to be discovered by experience, so must a certain part of the medical training be more or less empirical but it should at the same time be realized that such a method is only a means to an end, and that the real understanding of disease can be acquired only when every abnormal condition is inter- preted, as a primary or secondary consequence of some perverted bodily function, and when the training in observation and the inductive method is carried from the laboratory into the clinic.
It is a constant experience of clinical instructors who would employ scientific methods of instruction, that they find the students not only indifferent to an analysis of their cases from the functional standpoint, but also that they are too inadequately prepared in fundamental phys- iological knowledge, to make the analysis possible. The student may have a superficial acquaintance with the main facts of physiological science but have failed to acquire the enquiring habit of mind which will en- able him, through reflection, comparison, and personal research, to ap- ply the knowledge in practical medicine and surgery.
For this lack of correlation between the laboratory and clinical stud- ies, the clinical instructors are not alone responsible. The laboratory courses are frequently given without any attempt being made to show the student the bearing of the subject in the interpretation of disease, or to train him so that in his later years he may be able to adapt the methods of investigation which he learned in the laboratory, to the study of morbid conditions. It is self-evident that (without any knowledge of disease) the extent to which the student in the earlier years of the course could be expected to appreciate the clinical significance of what he learns in the laboratory is limited, but this should not deter the in- structor from indicating whenever he can, the general application of scientific knowledge in the interpretation of diseased conditions. But the chief remedy of the evil undoubtedly lies partly in the continuance of certain of the laboratory courses into the clinical years, and partly in the study of medical literature in which the application of physiology and biochemistry in the practice of medicine is emphasized.
Notwithstanding the sufficient number of excellent textbooks in phys- iology available to the medical student, there is none in which partic- ular emphasis is laid upon the application of the subject in the routine practice of medicine. In the present volume the attempt is made to meet such a want, by reviewing those portions of physiology and bio- chemistry which experience has shown to be of especial value to the clinical investigator. The work is not intended to be a substitute, either for the regular textbooks in physiology, or for those in functional
Vlll PREFACE
pathology. It is supplementary to such volumes. It does not start like the modern test in functional pathology, with a consideration of the diseased condition, and then proceed to analyze the possible causes and consequences of the disturbances of function which this exhibits; but it deals with the present-day knowledge of human physiology in so far as this can be used in a general way to advance the understanding of disease. In a sense it is therefore an advanced text in physiology for those about to enter upon their clinical instruction, and at the same time, a review for those of a maturer clinical experience who may desire to seek the physiological interpretation of diseased conditions.
In attempting to fulfil these requirements, it has been deemed essen- tial to go back to the fundamentals of the subject, and to explain as simply as possible the physical and physicochemical principles upon which so large a part of physiological knowledge depends. Physiology may be considered as an application of the known laws and facts of physics and chemistry to explain the functions of living matter, and it is only after the extent to which this application can be made has been appreciated, that the knowledge may be used to serve as the foundation upon which a superstructure of clinical knowledge can be built.
In order that the volume might be maintained of reasonable size, it has been necessary to select certain parts of the subject for particular emphasis, the basis of selection being the degree to which our knowledge clearly shows the value of the application of physiological methods both of observation and of thought in the study of diseased conditions. This has not been done to the extent of omitting the apparently less essential parts, for these have been treated in sufficient detail to link the others together so as to preserve a logical continuity, and show the bearing of one field of knowledge on another. There are however certain parts of the science, particularly the physiology of nerve and muscle, of the special senses, and of reproduction, for which application in the general fields of medicine and surgery is limited, and these parts have been omitted entirely. It has been judged that this perhaps somewhat arbi- trary selection is justified on the ground that the ordinary text in physiology covers these subjects sufficiently, except for the specialist, for whom on the other hand, no adequate review would have been pos- sible within the limits of such a volume as this. With reference to bio- chemistry, no attempt is made to review the properties or describe the characteristic tests of the various chemical ingredients of the body tis- sues and fluids. This is already sufficiently done in the textbooks on biochemistry, and in the numerous manuals on clinical methods. Bio- chemical knowledge is treated rather from the physiologist's stand- point, as an integral part of his subject, particular attention, neverthe-
PREFACE IX
less, being paid to the far-reaching applications of this latest depart- ment of medical science, in the elucidation of many obscure problems of clinical medicine, such as those of diabetes, nephritis, acidosis, goiter and myxedema. To make the volume of value to those who may not have had time or opportunity to familiarize themselves with the techni- cal methods of the physiologist and biochemist as used in the modern clinic, a certain amount of space is devoted to a brief description of the methods that appear at present to be receiving most attention, and to be of greatest value.
Finally, it should be mentioned that the principles of serum diagnosis and therapy are omitted, since these belong to a highly specialized science requiring an intensive training of its own.
In the hope that the volume may be instrumental in arousing sufficient interest to stimulate a more intensive study of the various subjects which it introduces, a brief bibliography is given at the end of each section. The references selected are to papers that are more partic- ularly known to the author; they are not necessarily the most impor- tant publications on the subject, but are often chosen because of the useful reviews of previous work contained in them, rather than because of their own originality. Some of the papers, however, are referred to as authority for statements of fact which may arouse in the reader a desire to ponder for himself the evidence upon which these are based. The references are usually divided into two groups, "monographs" and "original papers," and it is only occasionally that specific reference is made to the former in the context. The original papers, on the other hand, are referred to by numbers. With the general field of the subject so well covered by such excellent textbooks as Bayliss' "Principles of General Physiology," Stewart's, HowelPs, Starling's, and Halliburton's "Human Physiologies," and Leonard Hill's "Recent and Further Ad- vances in Physiology," the author has felt free to pick and choose from the monographs and original papers, topics that are ordinarily passed over cursorily in the textbook, and when this has been done, the refer- ences are somewhat more extensive. Such is the case for example . in the chapters relating to the chemistry of respiration, to the metabolism of carbohydrates and fats, to the problems of dietetics and growth, to the physicochemical basis of neutrality regulation in the animal body, and to the action of enzymes.
Acknowledgment is gratefully made for the assistance and advice in the preparation of the book, particularly to Doctor R. G. Pearce, for the contribution of several chapters, to which his name is attached, and for which he is entirely responsible; and to Doctor E. P. Carter, whose criticisms, after patient perusal of the unfinished manuscript, were of
X PREFACE
inestimable value in its final revision. Acknowledgment is also made to Doctor R. W. Scott and Professor F. E. Lloyd, for valuable criticism and advice, and to the former for a chapter on the "Clinical Applica- tion of Electrocardiographs. " To Miss Achsa Parker, M.A., the author owes a great debt of gratitude for the thorough and painstaking way in which she prepared the manuscript for the press, and for her never- tiring endeavors to have the spelling and punctuation in conformity with Webster's Dictionary. For assistance in the preparation of the index thanks are due to Miss Marion Armour and Mrs. MacFarlane, and for permission to use certain of the figures and illustrations, to the various authors and publishers who granted it. For the excellent man- agement and careful execution of the presswork, the author wishes to thank the publishers, whose courteous and friendly dealings have always made the work easier.
J. J. R. MACLEOD.
University of Toronto, Toronto, Canada.
CONTENTS
PART I
THE PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL
PROCESSES
CHAPTER I PAGE
GENERAL CONSIDERATIONS 1
The Laws of Solution, 3; Gas Laws, 3; Osmotic Pressure, 4; Biological Meth- ods for Measuring Osmotic Pressure, 6; Hemolysis, 7; Plasmolysis, 8.
CHAPTER II
OSMOTIC PRESSURE (CONT'D.) 10
Measurement by Depression of Freezing Point, 10; The Role of Osmosis, Dif- fusion, and Allied Processes in Physiological Mechanisms, 11.
CHAPTER III
ELECTRICAL CONDUCTIVITY, DISSOCIATION, AND IONIZATION . . 10
Biological Applications, 19.
CHAPTER IV
THE PRINCIPLES INVOLVED IN THE DETERMINATION OF THE HYDROGEN-ION CONCEN- TRATION 22
Titrable Acidity and Alkalinity, 22; Actual Degree of Acidity or Alkalinity, 23; Mass Action, 23; Application to the Measurement of H-ion Concentration, 26; Application in Determining the Real Strength of Acids or Alkalies, 28.
CHAPTER V
THE PRINCIPLES INVOLVED IN THE MEASUREMENT OF HYDROGEN-ION CONCENTRA- TION (CONT'D) 29
The Electrical Method, 29; The Indicator Method, 32.
CHAPTER VI
REGULATION OF NEUTRALITY IN THE ANIMAL BODY AND ACIDOSIS 3G
Buffer Substances, 36 ; Theory of Acidosis, 38 ; Measurement of the Reserve Alkalinity, 41; Tit-ration Methods, 41; CO,-Combining Power, 42; Indirect Methods, 46.
CHAPTER VII . COLLOIDS 51
Characteristic Properties, 51 ; Characteristics of True Colloidal Solutions, 5L> ; Tyndall Phenomenon, 52; Relative Indiffusibility, 52; Electrical Properties, 56; Browuian Movement, 58; Osmotic Pressure, 58.
xi
Xll CONTENTS
CHAPTER VIII PAGE
COLLOIDS (CONT'D) Gl
Suspensoids and Emulsoids, 61; Gelatin ization, 62; Imbibition, 63; Action of Electrolytes on Colloids, 63; Proteins as Colloids, 64; Surface Tension, 65; Adsorption, 66; Reactions which Depend on Adsorption, 67; Conditions that Influence or are Influenced by Adsorption, 68; Biological Processes Depend- ing on Adsorption, 70.
CHAPTER IX
FERMENTS, OR ENZYMES 71
The Nature of Enzyme Action, 72 ; Properties of Enzymes, 73 ; Reversibility of Enzyme Action, 77 ; Specificity of Enzyme Action, 79 ; Peculiarities of Enzymes, 80 ; Types of Enzyme, 81 ; Enzyme Preparations, 82 ; Conditions for Enzymic Activity, 82.
PART II THE CIRCULATING FLUIDS
CHAPTER X
BLOOD: ITS GENERAL PROPERTIES (BY R. G. PEARCE) 85
Quantity of Blood in the Body, 85; Water Content, 87; Proteins,. 88; Fer- ments and Antifcrments, 90.
CHAPTER XI
THE BLOOD CELLS (BY R. G. PEARCE) 92
Red Blood Corpuscles, or Erythrocytes, 92 ; Origin, 93 ; Rates of Regeneration, 94; Hemolysis, 96; Leucocytes, 97; Blood Platelets, 98.
CHAPTER XII
BLOOD CLOTTING '. 99
Visible Changes in the Blood During Clotting, 99; Methods of Retarding Clotting of Drawn Blood, 100; Nature of the Clotting Process, 102; Influence of Calcium Salts, 104; Influence of Tissues, 105.
CHAPTER XIII
BLOOD CLOTTING (€ONT'D) 107
Theories of Blood Clotting, 107; Intravascular Clotting, 108; Measurement of the Clotting Time, 109; Blood Clotting in Various Physiological Conditions, 111; Blood Clotting in Disease, 111; Hemorrhagic Diseases, 113; Thrombus Formation, 113.
CHAPTER XIV
LYMPH FORMATION AND CIRCULATION — CEREBROSPINAL FLUID 115
General Considerations, 115; Experimental Investigations, 118; Edema, 120; Cerebrcspinal Fluid, 121.
CONTENTS Xlll
PART III CIRCULATION OF THE BLOOD
CHAPTER XV PAGE
BLOOD PRESSURE 124
The Mean Arterial Blood Pressure, 125; Mercury Manometer Tracings, 125; Spring Manometer Tracings, 128; Clinical Measurements, 129.
CHAPTEE XVI
THE FACTORS CONCERNED IN MAINTAINING THE BLOOD PRESSURE 135
Pumping Action of the Heart, 135 ; Peripheral Resistance, 135 ; Amount of Blood in the Body, 138; Effects of Hemorrhage and Transfusion, 140; Vis- cosity of the Blood, 141; Elasticity of Vessel Walls, 143.
CHAPTER XVII
THE ACTION OP THE HEART . . 145
The Pumping Action of the Heart, 145; Intracardiac Pressure Curves, 146; Comparison of the Curves, 148.
CHAPTER XVIII
THE PUMPING ACTION OF THE HEART (CONT'D) '. 151
Contour of the Intracardiac Curves, 151; Ventricular Curve, 151; Auricular Curve, 153; The Mechanism of Opening and Closing of the Valves, 154; The Heart Sounds, 156; Causes of Sounds. 157; Record of Heart Sounds (Elec- trophonograms), 158.
CHAPTER XIX
THE NUTRITION OF THE HEART 161
Blood Supply, 161; Ferfusion of the Heart Outside the. Body, 161; Heart- Lung Preparation, 163; Resuscitation of the Heart in Situ, 165; Relationship of the Chemical Composition of the perfusion Fluid in Cold-blooded and Warm-blooded Hearts, 166.
CHAPTER XX
PHYSIOLOGY OF THE HEARTBEAT 170
Origin and Propagation of the Beat, 170; Physiological Characteristics of Cardiac Muscle, 170; Myogenic Hypothesis, 171; Neurogenie Hypothesis, 172; The Pacemaker of the Heart and Heart-block, 174; Physiological Char- acteristics of Cardiac Muscle, 176.
CHAPTER XXI
PHYSIOLOGY OF THE HEARTBEAT (CONT'D) 182
Origin and Propagation of the Beat in the Mammalian Heart, 182; Conduct- ing Tissue in Mammalian Heart, 182; Site of Origin of Beat, 187. .
CHAPTER XXII
PHYSIOLOGY OF THE HEARTBEAT (CONT'D) 191
Mode of Propagation in the Auricles and from the Auricles to the Ventricles, 191 ; Spread of Beat in the Ventricle, 193 ; Fibrillation of the Ventricles and Auricles, 195.
XIV CONTENTS
CHAPTER XXIII PAGE
THE BLOODFLOW IN THE ARTERIES IDS
The Pulses, 198; General Characteristics, 198; Eate of Transmission of Pulse Waves, 198; Contour of the Pulse Curves, 200; Velocity Pulse, 200; Palpable Pulse, 202; Analysis of the Curve, 202; The Dicrotic Wave, 203; Causes of Disappearnce of the Pulse in the Veins, 205.
CHAPTER XXIV
RATE OF MOVEMENT OF THE B-LOOD IN THE BLOOD VESSELS 200
Velocity of Flow in a Vessel, 206; Mass Movement of the Blood in a Vas- cular Area, 208; The Visceral Bloodflow in Man, 212; Circulation, 212; Work of the Heart, 213; Circulation Time, 214; Movement of Blood in the Veins, 214.
CHAPTER XXV
THE OUTPUT OF THE HEART IN RELATION TO THE VENOUS INFLOW, CHANGE OF
RATE, ETC 21G
Output of the Heart per Beat, 216; Reserve Power, 218; Effect of Alteration in Rate of Heart Beat on Output of Blood, 218 ; Tone pi the Heart, 220.
CHAPTER XXVI
THE CONTROL OF THE CIRCULATION , 221
Nerve Control, 222; Vagus Control in the Cold-blooded and the Mammalian Heart, 217; Tonic Vagus Action, 226; Afferent Vagus Impluses, 227; Mech- anism of Action of Vagus on the Heart, 229; Termination of the Vagus Fi- bers in the Heart, 230 ; Sympathetic Control, 232.
CHAPTER XXVII
THE CONTROL OF THE CIRCULATION (CONT'D) 234
Nerve Control of the Peripheral Resistance, 234; Detection of Vasomotor Fi- bers in Nerves, 236'; Origin of Vasomotor Nerve Fibers, 237; Vasomotor Nerve Centers, 240; Independent Tonicity of B-lood Vessels, 241.
CHAPTER XXVIII
THE CONTROL OF THE CIRCULATION (CONT'D) 242
Control of the Vasomotor Center, 242 ; Hormone Control, 242 ; Nerve Control, 243; Pressor and Depressor Impulses, 243; Reciprocal Inncrvation of Vas- cular Areas, 247; Influence of Gravity on the Circulation, 248; Capillary Circulation, 251.
CHAPTER XXIX
PECULIARITIES OF BLOOD SUPPLY IN CERTAIN VISCERA 254
Circulation of the Brain, 254; Anatomical Peculiarities, 254; Physical Condi- tions of the Intracranial Circulation, 256; Physiological Conditions of the In- tracranial Circulation, 258; Vasomotcr Nerves, 262; Intracranial Pressure, 263; Circulation through the Lungs, 264; Circulation Through the Liver, 265; The Coronary Circulation, 267.
CHAPTER XXX
CLINICAL APPLICATIONS OF CERTAIN PHYSIOLOGICAL METHODS 270
Electrocardiograms, 270; The Ventricular Complex, 273; Interpretation of Electrocardiograms by the Triangle Method, 276.
CONTENTS XV
CHAPTER XXXI PAGE
CLINICAL APPLICATIONS OF CERTAIN PHYSIOLOGICAL METHODS (CONT'D) . . . 278 Electrocardiograms of the More Usual Forms of Cardiac Irregularities, 278; Sinus Arrhythmia, 278; Sinus Bratiycardia, 278; The Extrasystoles, 278; Paroxysmal Tachycardia, 281 ; Auricular Fibrillation, 281 ; Auricular Flutter, 281; Heart-block, 282.
CHAPTER XXXII
CLINICAL APPLICATIONS OF CERTAIN PHYSIOLOGICAL METHODS (CONT'D) . . . 285 Polysphygmograms, 285; Venous Pulse Tracings, 285; Abnormal Pulses, 291.
CHAPTER XXXIII
CLINICAL APPLICATIONS OF CERTAIN PHYSIOLOGICAL METHODS (CONT'D) . . . 296 Measurement of the Mass Movement of the Blood, 296; The Normal Flow, 297; Clinical Conditions Which Affect the Bloodflow, 298.
CHAPTER XXXIV
SHOCK 301
Varieties of Shock, 301; Experimental Investigations of Shock, 304; Hista- mine Shock, 307; Traumatic Toxemia Factor in Shock, 309; Cause of Second- ary Symptoms, 310; Treatment and Prognosis, 311.
PART IV RESPIRATION
CHAPTER XXXV
RESPIRATION 316
Mechanics of Respiration, 316; Pressure of the Air in the Lungs, 316; Respir- atory Tracings, 320; The Intrapleural Pressure, 321; Influence on Blood Pressure, 323.
CHAPTER XXXVI
THE MECHANICS OF RESPIRATION (CONT'D) (BY R. G. PEARCE) 327
Variations in Dead Space, Residual Air and Mid and Vital Capacities in Various Physiological and Pathological Conditions, 327.
CHAPTER XXXVII
THE MECHANICS OF RESPIRATION (CONT'D) .< ' . . . . 332
Mechanism of the Changes in Capacity of the Thorax and Lungs, 332; The Movements of the Ribs, 332; The Action of the Musculature of the Ribs, 336; The Action of the Diaphragm, 337; The Effects of the Respiratory Movements on the Lungs, 342.
CHAPTER XXXVIII
THE CONTROL OF THE RESPIRATION 344
The Respiratory Centers, 344; Reflex Control of the Respiratory Center, 348.
XVI CONTENTS
CHAPTER XXXIX PAGE
THE CONTROL OF RESPIRATION (CONT'D) 352
Hormone Control of the Respiratory Center, 352; Tension of CO2 .and O., in Arterial Blood, 354; Tension of CO2 and O, in Alveolar Air, 356; Tension of CO2 in Venous Blood, 359.
CHAPTER XL
THE CONTROL OF RESPIRATION (CONT'D) 301
Estimation of the Alveolar Gases, 361; Method of Normal Subjects, 362; Clinical Method, 364.
CHAPTER XLI
THE CONTROL OF RESPIRATION (CONT'D) 366
The Nature of the Respiratory Hormone, 366 ; Relationship between CO, of Inspired Air and Pulmonary Ventilation, 367; Possibility that CO, Specifically Stimulates the Center, 368; Relationship between Alveolar CO, and Respira- tory Activity, 371.
CHAPTER XLII
THE CONTROL OF RESPIRATION (CONT'D) 373
Alveolar CO, Tension in Conditions of Anoxemia, 373 ; Constancy of the Al- veolar CO2 Tension under Normal Conditions, 373; The Nature of Changes Produced in the Body in Anoxemia, 378.
CHAPTER XLIII
THE CONTROL OF RESPIRATION (CONT'D) 382
Apnea, 382; Periodic Breathing, 385; Types of Periodic Breathing-, 385; Causes of Periodic Breathing, 386.
CHAPTER XLIV
INSPIRATION BEYOND THE LUNGS 391
Transportation of Gases by the Blood, 392; Transportation of Oxygen, 392; Dissociation Curve of CO2, 396; Difference between Curves of Blood and Hemoglobin Solutions, 396; Rate of Dissociation, 399; Dissociation Constant, 401.
CHAPTER XLV
RESPIRATION BEYOND THE LUNGS (CONT'D) 403
Means by Which the Blood Carries the Gases, 403 ; Oxygen Requirement of the Tissues, 408; Mechanism by which the Demands of the Tissues for Oxy- gen are met, 412.
CHAPTER XLVI
THE PHYSIOLOGY OF BREATHING IN RAREFIED AND COMPRESSED AIR 415
Mountain Sickness, 415; Compressed Air Sickness (Caisson Disease), 420; Application of Foregoing Laws in Practice, 424.
CHAPTER XLVII ADAPTATIONS OF THE CIRCULATORY AND RESPIRATORY SYSTEMS DURING MUSCULAR
EXERCISE 427
Circulatory Changes Accompanying Muscular Exercise, 427; .Mechanical Fac- tors, 428: Nervous Factor, 430; Hormone Factor, 431.
CONTENTS XV11
. CHAPTER XLVIII PAGE
ADAPTATIONS OF THE CIRCULATORY AND RESPIRATORY MECHANISMS DURING MUS- CULAR EXERCISE (CONT'D) 4-35
The Effect of Muscular Exercise on the Composition of the Alveolar Air, 435 ; Second-Wind, 438; Influence of Oxygen, 439; After Effects, 440; Effort Syn- drome, 441.
CHAPTER XLIX
OXYGEN UNSATURATION OF THE BLOOD CYANOSIS. THE THERAPEUTIC VALUE OF
OXYGEN 443
Oxygen ITnsaturation of the Blood, 443; Therapeutic Value of Oxygen, 445.
PART V DIGESTION
CHAPTER L
GENERAL PHYSIOLOGY OF THE DIGESTIVE GLANDS 453
Microscopic Changes During Activity, 453 ; Mechanism of Secretion, 455 ; Other Changes During Activity, 456 ; Control of Glandular Activity, 457 ; Nervous Control, 458.
CHAPTER LI
PHYSIOLOGY OF THE DIGESTIVE GLANDS (CONT'D) 460
Hormone Control, 460; Nervous Control of Pancreas, 462.
CHAPTER LII
PHYSIOLOGY OF THE DIGESTIVE GLANDS (CONT'D) 465
Normal Conditions of Secretion, 465; Normal Secretion of Saliva, 466; Se- cretion of Gastric Juice, 467; The Intestinal Secretions, 476.
CHAPTER LIII
THE MECHANISMS OF DIGESTION 478
Mastication, 478; Deglutition or Swallowing, 479; The Cardiac Sphincter, 482; Vomiting, 483.
CHAPTER LIV
THE MECHANISMS OF DIGESTION (CONT'D) 485
Movements of the Stomach, 485; Character of the Movements, 485; Effect of the Stomach Movement on the Food, 488; Emptying of the Stomach, 490; Control of the Pyloric Sphincter, 490; Rate of Emptying the Stomach, 492; Influence of Pathological Conditions on the Emptying, 494; Gastroenteros- tomy, 494.
CHAPTER LV
THE MECHANISMS OF DIGESTION (CONT'D) 497
Movements of the Intestines, 497; Movements of the Small Intestine, 497; Movements of the Large Intestine, 503; Effect of Clinical Conditions on the Movements, 504.
XV111 CONTENTS
CHAPTER LVI PAGE
HUNGER, APPETITE AND THIRST 506
Hunger, 506; Remote Effects of Hunger Contractions, 509; Hunger During Starvation, 510; Control of Hunger Mechanism, 511; Thirst, 514; Sensation of Thirst, 514.
CHAPTER LVII
BIOCHEMICAL PROCESSES OF DIGESTION 515
Digestion in the Stomach, 515; Functions of Hydrochloric Acid, 516; Amount of Acid, 516; Source of Acid, 517; Action of Pepsin, 519; Clotting of Milk in the Stomach, 521.
CHAPTER LVIII
BIOCHEMICAL PROCESSES OF DIGESTION (CONT'D) 52.°>
Digestion in the Intestines, 523; Pancreatic Digestion, 523; The Bile, 5-6; Chemistry of Bile, 528.
CHAPTER LIX
BACTERIAL DIGESTION IN THE INTESTINE ., 533
Bacterial Digestion of Protein, 535; Botulism, 537.
PART VI THE EXCRETION OF URINE
CHAPTER LX
THE EXCRETION OF URINE (BY R. G. PEARCE) 541
Structure of Kidney, 541; Mechanism of the Excretion of Urine, 544; Theories of Renal Function, 545; Diuretics, 552; Albuminuria, 552; Influence of the Nervous System on the Excretion of Urine, 553.
CHAPTER LXI
THE AMOUNT AND COMPOSITION OF THE, URINE TN HEALTH AND DISEASE (By R. G.
PEARCE) 555
Amount, 556; Specific Gravity, 556; Depression of Freezing Point, 557; Reaction, 558 ; Solid Constituents, 560 ; Quantitative Changes in the Blood and Urine in Disease, 567.
PART VII METABOLISM
CHAPTER LXII METABOLISM 570
Energy Balance, 571; Methods for Measuring Energy, 572; Normal Values, 573; Influence of Age and Sex, 577; Influence of Diseases, 578; Material Balance of the Body, 579; Methods for Measuring Outputs, 579.
CONTENTS XIX
CHAPTER LXIII PAGE
THE CARBON BALANCE 582
Respiratory Quotient, 582; Influence of Diet, 582; Influence of Metabolism, 584; Magnitude of the Respiratory Exchange, 585; Influence of Body Tem- perature, 586.
CHAPTER LXIV A CLINICAL MKTHOD FOR DETERMINING THE RESPIRATORY EXCHANGE IN MAN
(BY R. G. PEARCE) 589
The Valves, 590; Tissot Spirometers, 591; Douglas Bag, 592; Haldane Gas Apparatus, 593; Calculations, 596.
CHAPTER LXV
STARVATION 600
Excretion of Nitrogen, 600; Energy Output, 602; Nitrogenous Metabolites, 602; Excretion of Furines, 603; Excretion of Sulphur, 603; Normal Metab- olism, 604; Nitrogenous Equilibrium, 605; Protein Sparers, 605.
CHAPTER LXVI
NUTRITION AND GROWTH * . . . . 608
The Food Factor of Growth, 608; Relationship of Proteins to Growth and Maintenance of Life, 609.
CHAPTER LXVII
NUTRITION AND GROWTH (CONT'D) 617
Relationship of Carbohydrates and Fats to Growth, 617; Accessory Food Fac- tors, or Vitamines, 618.
CHAPTER LXVIII
DIETETICS 625
Calorie Requirement, 625; The Protein Requirement, 627; Accessory Food Factors, 630; Digestibility and Palatability, 630.
CHAPTER- LXIX THE METABOLISM OF PROTEIN 632
Introductory, 632; Chemistry of Protein and of the Amino Acids, 633.
CHAPTER LXX
THE METABOLISM OF PROTEIN (CONT'D) 641
Amino Acids in the Blood and Tissues, 641; Fate of the Amino Acids, 645.
CHAPTER LXXI
THE METABOLISM OF PROTEIN (CONT'D) 647
End Products of Protein Metabolism, 647; Urea and Ammonia, 649; Urea Ratio, 650; Influence of Liver on Ammonia-Urea Ratio, 651; Perf vision of Or- gans, 652; Clinical Observations, 654.
CHAPTER LXXII
THE METABOLISM OF PROTEIN (CONT'D) 656
Creatine and Creatinine, 656; Essential Chemical Facts, 656; Metabolism, 657; Influence of Food, Age, and Sex, 657; Origin of Creatine and Creatinine, 659.
XX CONTENTS
CHAPTER LXXIII PAGE
THE METABOLISM OF PROTEIN (CONT'D) 662
Undetermined Nitrogen and Dctoxication Compounds, (562; Ethereal Sul- phates and Glycuronates, 660.
CHAPTER LXXIV
URIC ACID AND THE PURINE BODIES 667
Chemical Nature of the P'urines, 667 ; Chemical Nature of the Substances Con- taining Purine and Pyrimidine Bases, 669 ; History of Nucleic Acid in the Animal Body, 671; Balance Between Intake and Output of Purine Substances under Various Physiological and' Pathological Conditions, 674.
CHAPTER LXXV
URIC ACID AND THE PURINE BODIES (CONT'D) 676
Source of Endogenous Purines, 676; Influence of Various Physiological Con- ditions, of Drugs, and of Disease on the Endogenous Uric-acid Excretion, 680; Uric Acid of Blood, 681.
CHAPTER LXXVI
THE METABOLISM OF THE CARBOHYDRATES 685
Capacity of the Body to Assimilate Carbohydrates, 685; Assimilation Limits, 685 ; Tolerance of the Body for Glucose, 688 ; Digestion and Absorption, 689 ; Sugar Level in the Blood, 690; Value of Blood Examination in Diagnosis of Diabetes, 691; Relationship Between Sugar Concentration of the Blood and the Occurrence of Glycosuria, 692.
CHAPTER LXXVII
THE METABOLISM OF THE CARBOHYDRATES (CONT'D) 694
Fate of Absorbed Glucose. Glueoneogenesis, 694; Shortage of Sugar, 694; Sources of Glycogen, 694; Glueoneogenesis in Normal Animals, 699.
CHAPTER LXXVIII
THE METABOLISM OF THE CARBOHYDRATES (CONT'D) 701
Fate 'of Glycogen, 701; Regulation of the Blood Sugar Level, 703; Nerve Con- trol and Nervous Experimental Diabetes, 704; Nervous Diabetes in Man, 706; Hormone Control and Permanent Diabetes, 707 ; Utilization of Glucose in Tis- sues, 708; Diabetes and the Ductless Glands, 710; Relationship of Pancreas to Sugar Metabolism, 710; Pathogenesis of Pancreatic Diabetes, 712; Dia- betic Acidosis or Ketosis, 715; Starvation Treatment, 716.
CHAPTER LXXIX
FAT METABOLISM 718
Chemistry of Fatty Substances, 718; Digestion of Fats, 721; Absorption of Fats, 722.
CHAPTER LXXX
FAT METABOLISM (CONT'D) . 726
Fat of Blood, 726; Methods of Determination, 726; Variations in Blood Fat, 727; Depot Fat, 730; Fat in the Liver, 731.
CONTENTS XXI
CHAPTER LXXXI PAGE
FAT METABOLISM (CONT'D) 736
Production of Fatty Acid out of Carbohydrate, 736; Method by which the Fatty Acid is Broken Down, 737.
CHAPTEE LXXXII
CONTROL OF BODY TEMPERATURE AND FEVER 742
Variations in Body Temperature, 742 ; Factors in Maintaining the Body Tem- perature, 743; Control of Temperature, 747; Fever, 748; Causes of Fever, 748; Changes in the Body During Fever, 750; Heat-regulating Center, 752; Significance of Fever in the Organism, 753.
CHAPTER LXXXIII
THE PHYSIOLOGICAL PRINCIPLES OF VENTILATION 754
Relationship Between Chemical Composition of the Air and the Well-being of the Body, 754; Relationship Between the Physical Conditions of the Air and the Well-being of the Body, 757; Relationship between the Conditions of Ventilation and Susceptibility to Infections, 759; Methods for Determining the Healthfulness of Air, 763.
PART VIII THE ENDOCRINE ORGANS, OR DUCTLESS GLANDS
CHAPTER LXXXIV
GENERAL CONSIDERATIONS, THE ADRENAL GLANDS 766
Methods of Investigation, 767; Adrenal Gland, 768; Cortex, 768; Medulla, 770 ; Adrenalectomy, 771 ; Adrenal Disease in Man, 772 ; Suprarenal Extracts, 773; Physiological Action, 774.
CHAPTER LXXXV
Tin-: ADRENAL GLANDS, (CONT'D) 779
Variations in Physiological Activity, 779; Assaying the Epmephriue Content of the Gland, 779; Epinephrine Content of the Blood, 780; Autoinjection Method, 784; Association of the Adrenal with Other Endocrine Organs, 788.
CHAPTER LXXXVI
THE THYROID AND PARATHYROID GLANDS 791
Structural Relationships, 791; Thyroid Gland, 792; Condition of Gland, 792; Experimental Thyroidectomy, 794; Disease of the Thyroid, 795; Relationship with Other Endocrine Organs, 800; Parathyroids, 800; Experimental Para- thyroidectomy, 800; Injury or Disease of the Parathyroids in Man, 801.
CHAPTER LXXXVII THE PITUITARY BODY . 806
Structural Relationships, 806; Functions, 807; Clinical Manifestations of Deranged Pituitary Function, 816; Relationship with Other Endocrine Or- gans, 818.
XX11 CONTENTS
CHAPTER LXXXVIII
PAGE
THE PINEAL GLAND, THE GONADS, AND THE THYMUS 820
Pineal Gland, 820; Goiiads or the Generative Organs, 821; Generative Glands of the Male, 821 ; Generative Organs of the Female,. 822 ; Thymus, 824.
PART IX
THE CENTRAL NERVOUS SYSTEM AND THE CONTROL OF MUSCULAR ACTIVITY
(Rewritten by A. C. Redfield)
CHAPTER LXXXIX
THE EVOLUTION OF THE NEUROMUSCULAR MECHANISM 827
Primitive Neuromuseular Mechanisms, 827 ; The Nerve Net, 830 ; The Cen- tral Nervous System, 830.
CHAPTER XC
THE CONDITION OF THE NERVOUS IMPULSE 836
Conduction in the Nerve Fiber, 837; The All or None Law, 837; Refractory Period, 839 ; Conduction between Neurons, 841 ; Resistance Due to Synapse, 842 ; Summation, 842 ; Inhibition, 843 ; Canalization, 844 ; Myoneurarl Junc- tion, 845.
CHAPTER XCI
THE NUTRITION OF NERVOUS TISSUE 846
Function of the Nerve Cell Body, 846; Degeneration and Regeneration of Nerve Fibers, 846; Metabolism of the Nerve Fiber, 850; Metabolism of the Central Nervous System, 851.
CHAPTER XCII THE RECEPTORS 854
The Evolution of Specialized Receptors, 854; Quality of Sensation and Its Local Sign, 855; Referred Pain, 858; Cutaneous and Deep Sensibility, 859; Touch, 860; Heat and Cold, 861; Pain, 862; Distribution of Sensitivity in the Body, 863.
CHAPTER XCIII
THE AFFERENT PATHS OF SENSORY IMPULSES 866
Segmental Distribution of Afferent Nerves, 866 ; Ascending Pathways in the Spinal Cord, 868; Afferent Paths in the Brain Stem, 871; Afferent Impulses Which Fail to Produce Sensation, 872.
CHAPTER XCIV
THE SENSORY CENTERS OF THE BRAIN 876
The Sensory Center of the Optic Thalamus, 876; The Sensory Centers of the Cerebral Cortex, 878; The Visual Areas, 880; Sensory Hallucinations, 883.
CONTENTS XX111
CHAPTEE XCV
PAGE
THE MOTOR AREAS OF THE CEREBRUM AND THE EFFERENT PATHWAY TO SKELETAL
MUSCLE 884
The Motor Area of the Cerebral Cortex, 885; The Visuo-Motor Areas, 886; The Efferent Pathway in the Brain and Cord, 888; Distribution of Efferent Nerves, 890; Spinal Ecflexes, 891.
CHAPTEE XCVI
THE AUTONOMIC NERVOUS SYSTEM, OR THE EFFERENT PATHWAY TO SMOOTH MUS- CLES AND GLANDS 893
The Organization of Efferent Nerves to the Viscera, 893; The Double Inner- vation of the Visceral Organs, 896; The Function of the Autonomic Nervous System, 897; The Axon Eeflex, 898; Function of the Bulbo-sacral Divisions, 899 ; The Mechanism for Emptying the Bladder, 900 ; Function of the Thorac- ico-Lumbar Division, 901 ; Effects of Impulses from the Viscera Upon Central Nervous Activity, 902.
CHAPTEE XCVII
MUSCULAR CONTRACTION 904
The Tonic Contraction of Skeletal Muscle, 905; Tetanic Contraction of Skeletal Muscle, 906; The All or None Law, 908; Chemistry of Tetanic Con- traction, 910; Smooth Muscle, 912.
CHAPTEE XCV11I POSTURAL COORDINATION 914
Eeflex Adjustment of Tone, 914; The Posture of the Body as a Whole, 917; Compensatory Movements of the Eyes, 918; Clinical Tests of Labyrinthine Mechanism, 920; The Tendon Jerks, 921.
CHAPTEE XCIX
THE CENTRAL CONTROL OF POSTURAL EEACTIONS; THE CEREBELLUM 921
The Influence of the Brain on the Local Tonic Eeflex, 924; Function of tl^ Cerebellum, 926; Localization of Function in the Cerebellum, 929; Compensa- tion for Cerebellar Injuries, 931.
CHAPTEE 0
THE INTEGRATION OF ACTION WITHIN THE EEFLEX ARC t);;;;
The Eeceptors, 933; Summation, 934; Refractory Period, 934; Reciprocal Inhibition, 937; Action of Strychnine and Tetanus Toxin on Beeiprocal In- hibition, 941; The Eeflex Figure, 941; Eules for the Spread of Spinal Be- flexes, 944.
CHAPTEB CI
THE INTEGRATION OF SIMULTANEOUS AND SUCCESSIVE BEFLEXES 945
Principle of the Final Common Path, 945; Integration of Allied Eeflexes, 946; Integration of Antagonistic Eeflexes, 947.
CHAPTEE CII
THE INTEGRATIVE ACTION OF THE CEREBRUM . . . 951
Eolation of the Cerebrum to the Distance Eeceptors, 952; Conditioned Be- flexes, 954.
XXIV CONTENTS
CHAPTEE GUI
PAGE
THE HIGHER FUNCTIONS OF THE CEREBRUM IN MAN; APHASIA 958
Psychopathological Applications, 960.
CHAPTER CIV
SUMMARY OF THE ORGANIZATION OF THE MAMMALIAN NERVOUS SYSTEM; SPINAL
SHOCK 9G3
Spinal Shock and the Recovery of Reflexes in Animals, 965; Spinal Shock and the Recovery of Reflexes in Man, 967; The Cause of Spinal Shock, 969.
ILLUSTRATIONS
FIG. PAGE
1. Diagram of osmometer 5
2. Hematocrite 7
3. Plasmolysis in cells from Tradeseantia discolor 9
4. Apparatus for measurement of the depression of freezing point of solution . 11
5. Diagram of conductivity cells 18
6. Wheatstone Bridge for the measurement of electric resistance 18
7. Diagram to show type of electrodes used in studying electromotive force . . 30
8. Diagram of apparatus for the measurement of the H-ion concentration . . 31
9. Chart of tints as used in colorimetric measurement of H-ion concentration
(Color Plate) 34
10. Diagram of apparatus for saturating blood and plasma with expired air . . 43
11. Van Slyke's apparatus for measuring the CO2-combining power of blood in
blood plasma 44
12. Ultramicroscope (slit type) for the examination of colloidal solutions ... 53
13. To show diffusion into gelatin of a crystalloid stain, and the nondiffusion of a
colloid stain 54
14. Diagram from W. Ostwald showing the relative size of various particles and
colloidal dispersoids compared with a red blood corpuscle and an anthrax
bacillus 55
15. Capillary analysis of colloids 57
16. Diagram to show structure of gels 62
17. Diagram to illustrate surface tension 65
18. Traube's stalagmometer 66
19. Diagram of the graphic coagulometer 110
20. Coagulometer 110
21. Mercury manometer and signal magnet, arranged for recording the mean ar-
terial blood pressure in a laboratory experiment 126
22. The arterial blood pressure recorded with a mercury manometer (lower trac-
ing) along with a tracing of the respiratory movement of the thorax . 127
23. Hiirthle's spring manometer 128
24. Normal curve of arterial blood pressure obtained with spring manometer . . 128
25. Diagram based on experiments on dogs to show the systolic, diastolic and
mean blood pressures at different parts of the circulatory system . . . 129
26. Apparatus for measuring the arterial blood pressure in man 131
27. Effect of cutting the vagus nerve on the arterial blood pressure 136
28. Effect of stimulating the peripheral end of the right vagus on the arterial
blood pressure 136
29. Effect of stimulation of the left splanchnic nerve on the arterial blood pressure 137
30. Composite curves to show effects of hemorrhage and transfusion of various
solutions on blood pressure 140
31. Diagram of experiment to show that the diastolic pressure depends on the
elasticity of the vessel wall 144
32. Diagram of Wiggers' optical manometer 147
XXVI ILLUSTRATIONS
TIG. PAGE
33. Optical records of intraventricular ^pressure 148
34. Superimposed pressure curves after being graduated 150
35. Von Frank's maximal and minimal valve, which is placed in the course of
the tube between heart and mercury manometer 152
36. Diagram to show the positions of the cardiac valves 156
37. Electrophonograms along with intraventricular pressure curves from three
different experiments 159
38. Arrangement of apparatus for heart-lung preparation . . . 164
39. Volume curve of ventricles of cat (lower curve) in a heart-lung perfusion
preparation 169
40. Heart and cardiac nerves of Limulus polyphemus . . . 173
41. Heart-block produced by applying clamp 175
42. Tracing of contraction of ventricle, showing the effect -of the local appli-
cation of heat to the auricle 175
43. Frog heart showing the position of the first and second ligatures of Stannius 176
44. Effects of stimuli of increasing strength on skeletal and cardiac muscle to
illustrate the "all or nothing" principle in the latter 177
45. The effects of successive stimuli on skeletal and cardiac muscle to show the
prominence of the staircase phenomenon, or treppe, in the latter . . . 178
46. The effects of successive stimuli and of tetanizing stimuli on skeletal muscle
and cardiac muscle 179
47. Myograms of frog's ventricle, showing effect of excitation by break induc-
tion shocks at various moments of the cardiac cycle 180
48. Heart of tortoise as suspended 183
49. Dissection of heart to show auriculoventricular bundle 184
50. Photograph of model of the auriculoventricular bundle and its ramifications,
constructed from dissections of the heart 184
51. Diagram of an auricle showing the arrangement of the muscle bands; the
concentration point; and the outline of the node 186
52. Diagram to show the general ramifications of the conducting tissue in the
heart of the mammal 186
53. Diagram to illustrate the development and spread of the wave of negativity
in a strip of muscle (curarized sartorius) when stimulated at the end . 188
54. Simultaneous electrocardiograms to show the cause for extrinsic deflections 190
55. Diagram of experiment by Lewis showing the times at which the excitation
wave appeared on the front of the heart 194
56. Diagram of Chauveau's dromograph 200
57. Diagram to show principle of Pitot's tubes for measuring velocity pulse . . 201
58. Cybulski's photohematotachometer 201
59. Dudgeon's sphygmograph 201
60. Pulse tracing (sphygmogram) taken by sphygmograph 202
61. Forms of apparatus for measurement of blood velocities 207
62. Plethysmograph for recording volume changes in the hand and forearm . . 210 03. Effect of venous supply on volume of heart . 217
64. Simultaneous tracings from auricle and ventricle of turtle's heart .... 223
65. Effect of vagus stimulation on heart of turtle 223
66. Tracing to show that vagus stimulation may diminish transmission from
auricles to ventricles . . 224
ILLUSTRATIONS XXV11
FIG. PAGE
67. Tracing to show that vagus stimulation may facilitate transmission from
auricles to ventricles 225
68. Diagram to show the innervation of the heart in the frog or turtle. (Color
Plate.) 228
69. Frog heart tracing showing the action of nicotine 231
70. Schematic representation of the innervation of the heart of the mammal.
(Color Plate.) .' 232
71. Tracings showing the effects on the heartbeat of the frog resulting from
stimulation of the sympathetic nerves prior to their union with the vagus
nerve 233
72. Boy's kidney oncometer 235
73. Pall of blood pressure from excitation of the depressor nerve 244
74. The effect of strong stimulation (heat) of the skin of the foot on the ar-
terial blood pressure and respiratory movements 245
75. Diagram showing the probable arrangements of the vasomotor reflexes . . 246
76. Aortic blood pressure, showing the effect of posture 249
77. Tracing to show the effect of gravity on the arterial blood pressure . . . 250
78. The effect of gravity on the aortic pressure after division of the spinal cord
in the upper dorsal region 250
79. Capillaries from abdominal wall of guinea pigs after injection of india ink . 252
80. Tracing showing simultaneous records of the arterial blood pressure, the
venous pressure, the intracranial pressure, the pressure in the venous
sinuses 260
81. Electrocardiographic apparatus as made by the Cambridge Scientific Ma-
terials Co. . 271
82. Normal electrocardiogram 272
83. Electrocardiogram (dog) taken simultaneously with curves from auricle and
ventricle 273
84. Records of electrocardiogram and movement of ventricle of frog showing that
when the apex is warmed a typical T-wave appears in place of a wave
in the opposite direction appearing when the apex is cooled ...... 275
85. Sinus bradycardia 279
86. Auricular extrasystole '. 279
87. Ventricular extra systoles arising in the right ventricle 279
88. Ventricular extrasystole arising in the left ventricle 279
89. Paroxysmal tachycardia 280
90. Auricular fibrillation . . 280
91. Auricular flutter 282
92. Delayed conduction 282
93. Partial dissociation 283
94. Complete dissociation 283
95. Tracings of the jugular pulse, apex beat, carotid and radial pulses .... 286
96. Polysphygmograph 288
97. Normal jugular tracing 288
97A. Superimposed pressure curves from aorta, ventricle and auricle, along Avith
electrocardiogram and phonocardiogram 289
98. Polysphygmograms including jugular, apex and radial tracings 290
99. Delayed conduction 291
100. Dropped beats . ,, . , 292
XXV111 ILLUSTRATIONS
FIG. PAGE
101. Premature beats (extrasy stoles) ventricular in origin 292
102. Paroxysmal tachycardia 293
103. Auricular flutter 294
104. Auricular flutter 294
105. Auricular fibrillation .295
106. Showing the appearance of the blood vessels in the ears of a rabbit in a
state of deep shock. (Color Plate.) . 304
107. Diagram showing amounts of air contained by the lungs in various phases
of ordinary and of forced respiration 318
108. Diagram of structure of air sacs, atria, alveolar ducts, etc 318
109* Pneumograph 321
110. Effect of abdominal and chest breathing on the pulse and blood pressure
of man 325
111. First dorsal vertebra, sixth dorsal vertebra and rib. Axis of rotation shown
in each case 333
112. Lower half of the thorax from the 6th dorsal to the 4th vertebra, seen from
the front ...... 335
113. Intercostal muscles of 5th and 6th spaces 336
114. Hamberger's schema to demonstrate the functional antagonism of internal
and external intercostals 336
115. Schema to demonstrate that the function of the internal intercartilaginous
intercostals is identical with that of the external interosseous intercostals 337
116. Diagram to show the effect of high and low positions of the diaphragm on
the costal angle 339
117. Diagram to show the effect of clinical displacements of the diaphragm on
the costal angle 340
118. Diagram to show cuts required for isolation of the phrenic center .... 345
119. Diagram to show certain positions in the medulla and upper cervical cord,
where sections may be made without seriously disturbing the respirations 346
120. Diagram to show where cuts are made to isolate the chief respiratory center
from afferent impulses 347
121. Diagram showing principle for measurement of the tension of CO2 in blood 355
122. The gas analysis pipette for the microtonometer shown in Fig. 123 . . . 356
123. Microtonometer, to be inserted into a blood vessel 356
124. Apparatus for collection of a sample of alveolar air by Haldane's method . 357
125. Fridericia's apparatus for measuring the CO2 in alveolar air 358
126. Curves to show the relationship between the O2 and CO2 tensions in alveolar
air and arterial blood 358
127. Same as Fig. 126, except that in this case the tension of CO, in the alveolar
air was experimentally altered 359
128. Arrangement of meters and connections of Pearce's method for measure-
ment of CO2 of alveolar air in normal subjects 363
129. Curve showing the respiratory response to CO2 in the decerebrate cat . . 368
130. The behavior of the respiratory volume, the blood pressure and the pulse
during progressive anoxemia 376
131. Curves showing variations in alveolar gas tensions after forced breathing for
two minutes 383
132. Various types of periodic breathing 385
ILLUSTRATIONS
FIG. PAGE
133. Quantitative record of breathing air through a tube 260 cm. long and 2 cm.
in diameter 388
134. Barcroft's tonometer for determining the curve of absorption of oxygen by
hemoglobin or blood 394
135. Barcroft's differential blood gas manometer 394
136. Barcroft blood gas manometer 395
137. Typical dissociation curve. (Color Plate.) 396
138. Average dissociation curves 397
139. Dissociation curves of hemoglobin 398
140. Dissociation curves of human blood 399
141. Curves showing relative rates of oxidation and reduction of blood as in-
fluenced by temperature and tension of CO2 400
142. Curve of CO2 tension in blood 405
142 A. CO2 tension at various altitudes 417
143. Cells of parotid gland showing zymogen granules 454
144. Parotid gland of rabbit in varying states of activity examined in fresh state 454
145. Diagrammatic representation of the innervation of the salivary glands in the
dog. (Color Plate.) 458
146. Pancreatic acini stained with hematoxylin 462
147. Three preparations of pancreatic acini stained by eosin orange toluidin blue 463
148. Diagram of stomach showing miniature stomach separated from the main
stomach by a double layer of mucous membrane 468
149. Typical curve of secretion of gastric juice collected in 5-minute intervals
on mastication of palatable food for 20 minutes 471
150. Cubic centimeters of gastric juice secreted after diets of meat, bread, and milk 475
151. Digestive power of the juice, as measured by the length of the protein column
digested in Mett's tubes, with diets of flesh, bread, and milk .... 475
152. Loop of intestine after tying off the portions, cutting the nerves running to
the middle portion and returning the loop to the abdomen for some time 477
153. The changes which take place in the position of the root of the tongue, the
soft palate, the epiglottis and the larynx during the second stage of swal- lowing 480
154. Schematic outline of the stomach 486
155. Diagrams of outline and position of stomach as indicated by skiagrams taken
on man in the erect position at intervals after swallowing food impreg- nated with subnitrate 4.86
156. Outlines of the shadows cast by the stomach at intervals of an hour each
after feeding a cat with food impregnated with bismuth subnitrate . . 487
157. Section of the frozen stomach (rat) some time after feeding with food given
in three differently colored portions 489
158. Outlines of shadows in abdomen obtained by exposure to x-rays 2 hours after
feeding with food containing bismuth subnitrate 492
159. Curves to show the average aggregate length of the food masses in the small
intestine at the designated intervals after feeding 493
160. Apparatus for recording contractions of the intestine 498
161. Diagrammatic representation of the process of segmentation in the intestine 499
162. Intestinal contractions after excision of the abdominal ganglia and section of
both vagi 500
XXX ILLUSTRATIONS
FIG. PAGE
163. The effect of excitation of both splanchnic nerves on the intestinal contrac-
tions . . 502
164. The effect of stimulation of light vagus nerve on the intestinal contractions 502
165. Diagram of time it takes for a capsule containing bismuth to reach the various
parts of the large intestine 504
166. Diagram of method for recording stomach movements 507
167. Tracing of the touus rhythm of the stomach three hours after a meal . . 508
168. Tracings from the stomach during the culmination of a period of vigorous
gastric hunger contractions 508
169. Showing augmentation of the knee-jerk during the marked hunger contrac-
tions 509
170. Diagram of the uriniferous tubules, the arteries, and the veins of the kidney 542
171. Cross section of convoluted tubules from kidney of rat 543
172. Diagram of blood supply of Malpighian corpuscle and of convoluted tubules
in amphibian kidney 549
173. Nerve supply of the kidney 553
174. Respiration calorimeter of the Russell Sage Institute of Pathology, Bellevue
Hospital, New York / 572
175. Chart of determining surface area of man in square meters from weight in
kilograms and height in centimeters according to formula 576
176. Diagram of Atwater-Benedict respiration calorimeter 580
177. Nose clip, face mask, and mouth piece 590
178. Diagram of respiratory valves 590
179. The Tissot spirometer 591
180. The Douglas bag method for determining the respiratory exchange . . . 592
181. Haldane gas apparatus and Pearce sampling tube 593
182. Curve constructed from data obtained from a man who fasted for- thirty-
one days 601
183. Curves of growth of rats on basal rations plus the various proteins indicated 610
184. Curves of growth of rats on basal rations plus the proteins indicated . . . 611
185. Photographs of rats of same brood on various diets 613
186. Curves of growth of rats as influenced by the accessory food factors . . . 620
187. Vividiffusion 'apparatus of J. J. Abel 642
188. Curves showing the amount of amino nitrogen taken up by different tissues
after the cutaneous injection of amino acids 643
189. Curves showing the concentrations of amino-acid nitrogen in the blood dur-
ing fasting and protein digestion 644
190. Curves showing the percentage of glucose in blood after a constant injection
of an 18 per cent solution into a mesenteric vein 691
191. Child aged 4^ years suffering from hypernephroma 769
192. Arrangement of apparatus for recording contractions of a uterine strip, intes-
tinal strip, or ring, etc 781
193. Tracing showing the effect of epinephrine on the intestinal contractions and
on the arterial blood pressure 782
194. Arrangement of apparatus for perfusion of the vessels of a brainless frog . 783
195. Microphotographs of thyroid gland of a dog 793
196. Cretin, nineteen years old 796
197. Case of myxedma before and after treatment : 797
19S. Drawing from a photograph of mesial sagittal section through the pituitary
gland of a human fetus 807
ILLUSTRATIONS XXXI
FIG. PAGE
199. Tracing showing the action of pituitrin on the uterine contractions and
blood pressure in a dog 812
200. Tracing showing the constructing action of pituitrin on the bronchioles and
its effect on blood pressure in a spinal dog 813
201. Showing the appearance before and after the onset of acromegalic symptoms 815
202. Hand of a person affected with acrornegaly 817
203. The evolution of the nervous system 829
204. Normal cell from the anterior horn, stained to show Nissl's granules . . . 831 20.1. Arborization of collaterals from the posterior root fibers around the cells of
the posterior horn 832
200. Part of an anterior cormial cell from the calf's spinal cord stained to show
neurofibrils 833
207. Schema of simple reflex arc 833
208. Diagram of nervous system of segmented invertebrate 834
209. Diagram illustrating the effect of areas of narcosis on the strength of the
nerve impulse 839
210. The recovery of excitability in the nerve fiber after the passage of a nerve
impulse 840
21.1. Degeneration and regeneration of a sectioned nerve fiber . , 847
212. Evolution of the sense organs 855
213. Cold spots and heat spots of an area of skin of the right hand 860
214. Diagram showing the segmented arrangement of the sensory nerves . . . 867
215. Diagram of the afferent paths followed by sensory impulses within the spinal
cord and brain 869
216. Afferent paths connecting the retina with the visual area of the cerebral
cortex 881
217. Outer aspect of the brain of the chimpanzee showing the position of the motor
centers 885
218. Diagram to illustrate the different arrangements of the internuncial neurons
of the voluntary and autonomic nervous systems 894
219. Diagram of the autonomic nervous system. (Color Plate.) 894
220. Diagram showing the main parts of the autonomic nervous system. (Color
Plate.) 896
221. Schematic representation of the autonomic nervous system. (Color Plate.) 898
222. Diagram of an axon reflex in a sensory nerve fiber of the skin 899
223. Electromyogram of the voluntary contraction of the flexor muscles . . . 907
224. The contraction of a single fiber of the sartorius muscle of the frog . . . 907
225. The all or none nature of the contraction of a single fiber of skeletal muscle 909
226. Eeciprocal inhibition 916
227. Kecords of the contraction of the isolated extensor muscle of the knee of
the cat 917
228. Compensatory movements of the eyes and fins of the dogfish 919
229. The semicircular canals of the ear 919
230. A tracing of the knee-jerks of a normal man and of a man with a cerebellar
injury 922
231. Schema of the parts of the mammalian cerebellum 929
232. Diagrams to represent a ventral view of the left half and a dorsal view of
the right half of the cerebellum 930
XXX11 ILLUSTRATIONS
FIG. . PAGE
233 and 234. The inferolateral and the posterior aspect of the human cerebellum
indicating certain cerebellar localizations according to Barany . . . 931
235. Footprints after destruction of the cerebellum in a dog 932
236. Tracing from the hind limb of a spinal dog during the scratching 'movements 935
237. Diagram showing the reflex arcs involved in the scratch reflex 936
238. The region of body of dog from which the scratch reflex can be elicited . . 936
239. Record from myograph connected with the extensor muscle of the knee . . 939
240. Sherrington's diagram illustrating the mechanism of reciprocal inhibition 940
241. Reflex figures 942
242. Successive induction illustrated by the crossed-extension reflex 949
243. Postures assumed by the robber fly when the eyes are unequally illuminated 953
PHYSIOLOGY AND BIOCHEMISTRY IN MODERN MEDICINE
PART I
THE PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL
PROCESSES
CHAPTER I GENERAL CONSIDERATIONS
The work of the physiologist consists, in large part, in ascertaining to what extent the known laws of physics and chemistry find application in explaining the phenomena of life. He gathers from the vast store- house of physical and chemical knowledge whatever is of value in the interpretation of the various mechanisms that work together to com- pose the living machine, and having added to this knowledge he passes it on for use by those who are concerned in the study and treatment of disease.
Many of the most important steps in the advance of physiological knowledge in recent years have depended upon the discovery of some hitherto unknown physical or chemical law, or upon the elaboration of some accurate method for the measurement of the phenomena upon which these or previously known laws depend. The discoveries of van't Hoff, Arrhenius, and OstwTald of the so-called laws of solution were soon followed by important observations on their relationship to the movement of fluids and dissolved substances through cell mem- branes; the discoveries of Hardy, Willard Gibbs, etc., of the behavior of colloids and of the phenomena of surface tension found application in explaining many hitherto inexplicable, peculiarities in the activities of ferments; the discovery by Nernst, etc., of methods for the measurement of the electro-motive force of dissolved substances was applied to de- termine the actual reaction or hydrogen-ion concentration of animal
2 PHYSICOCHKMICAL BASTS OP PHYSIOLOGICAL PROCESSES
fluids, and to explain the generation of the electric currents which ac- company muscular, nervous, and glandular activity.
It would be out of place here to devote much space to a detailed ac- count of such matters. They belong more properly in the domain of general than in that of human physiology. General physiology is con- cerned with the study of the essential nature of the vital processes; whereas human physiology is merely a branch of the subject in which special attention is devoted to the application of the truths of general physiology to the working of the human machine. For the physician and surgeon a knowledge of human physiology is as essential as is a knowledge of the construction of a piece of machinery for the engineer who attempts its repair, but obviously to acquire this knowledge the fundamental principles of general physiology must first of all be under- stood. For these reasons the introductory chapters are devoted to a brief review of the most important of the physkochemical principles upon which the working of the cell depends.
From the viewpoint of the physical chemist the cell consists of an envelope of more or less permeable material inclosing a solution of various crystalloids and colloids, in which these are in a state of equilibrium with one another. This equilibrium is readily altered by various influences that may act on the cell, and the resulting changes manifest themselves outwardly -by alterations in the shape and volume of the cell — growth and motion; by the extrusion of some of its contents — secretion; or by the propagation to other parts of the cell, or its processes, of the state of disturbed equilibrium — nervous impulse. Besides the activities that are dependent upon physicochem- ical changes, purely chemical processes go on in the cell. Many of these consist in the breakdown and oxidation of complex unstable organic molecules, a process identical with that occurring in combustion outside the cell. Others involve the building up, stage by stage, of complex substances out of the elements or out of simpler molecules. Chemical transformations occur in the cell which, in the chemical laboratory, re- quire the most powerful reagents and physicochemical forces, either the strongest of acids, alkalies, oxidizing agents, etc., or extreme degrees of heat, electrical energy, etc. But this is not all, for in the cell these chemical transformations are capable of being guided to a very remark- able degree of nicety so as to produce intermediate products that are used for some special purpose either by the cell that produced them or, after transportation by the blood, etc., by cells in other parts of the organism.
It is customary to speak of the cell as a chemical laboratory, but it
LAWS OF SOLUTION 3
is more than this ; it is a laboratory furnished not only with the equip- ment of the chemist but directed in the harmonious operation of its many activities by a guiding hand which far surpasses anything else known to man. Chemical transformations that require for their accomplishment the greatest skill proceed without apparent difficulty in the cell. To what are these changes due? What is the nature of the chemical rea- gents and forces, and what is the directive influence that guides them in their varied activities? To these, which are among the great ques- tions of general physiology, the reply may be given that the reagents are the ferments or enzymes, and that the directive influence operates through the susceptibility of enzymic activities to changes in the envi- ronment in which the enzymes are acting. In many cases these changes can be explained on a physicochemical basis as dependent upon the known laws of mass action or surface tension; in other cases they de- pend on purely chemical changes in the cell contents, such as changes in reaction or the accumulation of chemical substances that act like poisons on the enzyme. But there are still others that appear to depend on influences which as yet are quite unknown to the physical chemist, such as the changes in cell activity that can be brought about by the nerve impulse.
These preliminary remarks will serve to indicate the problems with which we must first occupy our attention. They concern the physico- chemical nature of saline solutions and of colloids, and the general na- ture of enzyme action. The knowledge which we acquire will be found to be of value, not only because it will help us to understand the nature of the workings of the normal healthy cell, but because, here and there, it will indicate possible causes for derangement in cellular function and suggest rational means by which we may attempt to rectify the fault.
THE PHYSICOCHEMICAL LAWS OF SOLUTION
The Gas Laws
Three fundamental principles of general chemistry serve as the basis for an understanding of the nature of solutions. The first is that if we take a quantity of any gas equal to its molecular weight in grams (called a gram-molecule or for sake of brevity a mol), it will occupy ex- actly 22.4 liters at a temperature of 0° C. and a pressure of 760 mm. Hg. ; the second is that, as we compress a gas, its pressure will increase in exactly the same proportion as the volume diminishes (the volume of a gas is in- versely proportional to its pressure) ; the third is that all gases expand by
4 PHYSICOCHTvMICAL BASTS OF PHYSIOLOGICAL PROCESSES
1/273 part of their volume at 0° C. for every degree C. that their tempera- ture is raised.*
The pressure of a gas is measured by connecting a pressure gauge or manometer with the vessel which contains the gas. Now, it is plain that if the 22.4 liters, which is the volume occupied by a gram-molecular quantity, were compressed so as to occupy a volume of 1 liter, its pressure would be 22.4 times that of 1 atmosphere, or 22.4 x 760 mm. Hg — the temperature remaining constant. Under these conditions we must im- agine that the molecules of gas are crowded together by the compression, and if we further conceive of these molecules as being in constant mo- tion, then we can understand why the pressure should increase just in proportion as we confine the space in which they can move.
One other property of gases must be borne in mind — namely, their tendency to diffuse from places where the pressure is high to places where it is low until the pressure is the same throughout.
OSMOTIC PRESSURE
These fundamental facts regarding the behavior of gases suggested to van't Hoff the hypothesis that molecules of dissolved substances must behave in a similar manner to those of gases. To put this hypothesis to the test, it is necessary that we have some method for measuring the pressure of dissolved molecules. We can not, as in the case of a gas, use an ordinary manometer, for this would measure only the pressure of the solvent on the walls of its container and would tell us nothing of the pressure of the dissolved molecules. We must use some filter or membrane that will allow the molecules of the solvent but not those of the dissolved substance to pass through it. It is evident that if such a filter is placed, for example, between a solution of sugar in water and water alone, the molecules of the latter will diffuse into the solution until this has become so diluted that the pressure of the dissolved mol- ecules is equal on both sides of the membrane. Such a membrane is called semipermeable; the diffusion of molecules through it is called osmosis, and the pressure which is generated, the osmotic pressure. If we prevent the water molecules from actually diffusing by opposing a pressure which is equal to that with which they tend to diffuse through the membrane, we can tell the magnitude of the osmotic pressure (Fig. 1).
In applying these facts to test the hypothesis that molecules in solution
*This implies that at -273° C. the gas would occupy no volume. Before this temperature is reached, however, the liquefaction of the gas sets in. The temperature -273° C. is known as absolute zero. An observed temperature plus 273° is called the absolute temperature. Another way of stat- ing the above law is therefore that the volume is directly proportional to the absolute temperature. At 273° C. the volume of a gas at 0° C. would be doubled, or if expansion were prevented the pressure would be doubled.
LAWS OF SOLUTION
obey the same laws as .those in gaseous form, we must employ a semi- permeable membrane which is rigid enough to withstand the pressure and which forms part of the walls of a closed vessel connected with a manometer. If we place in such an osmometer a solution containing the molecular weight in grams of some substance dissolved in one liter of solvent, a so-called gram-molecular solution, it is obvious that, if the gas laws are to apply, the osmotic pressure should equal that of 22.4 liters of a gas compressed to the volume of one liter; in other words, it should equal 22.4 x 760 = 17.024 mm. Hg. Although there are very considerable technical difficulties in making a semipermeable membrane that is strong enough to withstand such a pressure, yet this has been accom-
w
Fig. 1. — Diagram of osmometer. The cylindrical vessel (O), with a bottom of unglazed clay, the pores of which are filled with a precipitate of copper ferrocyanide to form a semi- permeable membrane, is suspended in an outer vessel, and is closed above by a tightly fitting stopper pierced by a tube leading to a manometer (M). O contains a strong solution of cane sugar, and W contains water. The water molecules tend to pass through the semipermeable membrane into the cane sugar solution, and since the cane sugar molecules can not pass in the opposite direction, the pressure in O rises and is recorded in M. This equals the osmotic pressure.
plished, and the fundamental principle has therefore been firmly estab- lished that substances in solution obey the same laws as gases.
Further proof that the gas laws apply to solutions has been secured by showing that the osmotic pressure (of a dilute solution) is directly pro- portional to the concentration of the dissolved substance (the solute) and to the absolute temperature. It also obeys the law of partial pres- sures, which states that the total pressure exerted by a mixture (of gases or dissolved molecules) is the sum of the pressures which each constit- uent of the mixture would exert were it alone present in the space occupied by the mixture.
PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES
Since the osmotic pressure is analogous to the pressure of a gas and is therefore proportional to the molecular concentration (i. e., number of molecules in unit space), it follows that a semipermeable membrane can be used to determine the relative concentration of two solutions of the same substance. When a watery solution of some substance is placed in an osmometer that is surrounded by a similar but more dilute solution, water molecules will diffuse into the osmometer until the pres- sure is equal on the two sides of the semipermeable membrane; that is, the water will pass from the solution having a lower osmotic pressure into the solution having the higher pressure. When two solutions have the same osmotic pressure, they are said to be isotonic; when that of one is greater than that of the other, it is hypertonic; and when less, hypotonic.
Biological Methods for Measuring Osmotic Pressure
A practical biological application of these principles can very readily be made if, instead of a rigid semipermeable membrane such as that figured in the diagram, we employ one that is extensible and takes the form of a closed sac ; then as diffusion of water occurs the sac will either distend when it contains a Stronger solution than that outside, or shrivel or crenate when the reverse conditions obtain. Many animal and veg- etable protoplasmic membranes are semipermeable, including the en- velope of red blood corpuscles. Thus, if we examine blood corpuscles under the microscope and add to them a saline solution of higher os- motic pressure than blood serum, they will visibly diminish in size and become irregular in shape; whereas if the solution is of lower osmotic pressure, they will distend. If no change occurs, the osmotic pressure of the cell contents must equal that of the saline solution in which the cells are immersed, from which it is clear that we can readily determine the magnitude of the osmotic pressure if we know the strength of the saline solution.
Instead of measuring the individual cells under the microscope, we can measure the space they occupy in the fluid in which they are suspended. For this purpose a portion of the suspension is placed in a graduated tube of narrow bore, which is rotated in a horizontal position by a cen- trifuge after being closed at one end. The graduation at which the upper edge of the column of cells stands after centrifuging is a measure of the relative amounts of cells and of fluid in the suspension. Having found this value for cells suspended in an isotonic solution, as for exam- ple, blood corpuscles in blood serum, we may then proceed to ascertain it for the same cells suspended in an unknown solution ; if we find that the now occupy a greater volume, the saline solution must have an os-
LAWS OF SOLUTION 7
motic pressure that is lower than that of serum in approximate proportion to the readings on the tube in the two cases, and vice versa.
The above apparatus, called a hematocrite (Fig. 2) has been very ex- tensively used in the collection of data concerning the relative osmotic pressures of different physiological fluids.
Hemolysis
Another way for determining the relative osmotic pressure of dif- ferent solutions consists in placing equal amounts (a few drops) of blood in a series of test tubes containing solutions of different strengths, and after allowing the tubes to stand for some time, noting in which of them laking of the blood corpuscles occurs. In solutions which are isotonic or hypertonic with the contents of the corpuscles, the latter will settle to the bottom of the tube and the supernatant fluid will be untinted with hemoglobin, but in solutions which are distinctly hypotonic, the sediment will be less distinct and the supernatant fluid red.
Fig. 2. — Hematocrite. The graduated glass tubes are filled with the two specimens of blood, or corpuscular suspension, and then rotated rapidly by a centrifuge. The relative heights at which the corpuscular sediment stands in the two tubes is proportional to the osmotic pressures of the fluid in which the corpuscles are suspended.
By noting (1) the lowest concentration (percentage composition) of the solutions in which the corpuscles sink to the bottom and leave the supernatant fluid colorless, and (2) the highest concentration in which the corpuscles when they settle leave the supernatant fluid tinted red, we can determine the limiting concentrations for solutions of different sub- stances. Thus, with bullock's blood the following results were obtained (Hamburger) :
SUBSTANCE PERCENTAGE STRENGTH OF SOLUTION IN WHICH
I II
SUPERNATANT FLUID SUPERNATANT FLUID
WAS COLORLESS WAS RED
|
KN03 |
1.04 |
0.96 |
|
NaCl |
0.60 |
0.56 |
|
K2S04 |
1.16 |
. 1.06 |
|
C^H^O,, (Cane sugar) |
6.29 |
5.63 |
|
CH3C~OOH (Pot. acetate) |
1.07 |
1.00 |
|
MgSO4.7H2O |
3.52 |
3.26 |
|
CaCU |
0.85 |
0.79 |
8 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES
The mean of these limiting concentrations is the critical concentration and indicates the strength of each solution that can be added to blood without causing any damage to the corpuscles. This critical concen- tration is not, as might at first sight be imagined, the same as that which is isotonic with the contents of the corpuscles, but distinctly below it. The reason for this becomes apparent if we observe the be- havior of corpuscles suspended in an isotonic solution which is then gradually diluted. As dilution proceeds, the corpuscles distend, until at last their envelopes burst and the hemoglobin is discharged. The lim- iting concentrations of a given salt vary for different corpuscles; thus, the concentration of sodium chloride solution that just causes laking of frog's blood corpuscles is 0.21 per cent, that of human blood 0.47 per cent, and that of horse blood 0.68 per cent. It is the strength of the corpuscular envelope rather than variations in the osmotic pressure of the contents that is responsible for these differences.
The above described method of hemolysis, as it is called, can not be used for comparisons of osmotic pressure in cases in which the solution contains substances which alter the permeability of the corpuscular envelop; for example, it can not be used when urea, or ammonium salts, or certain toxic bodies are present. We may therefore ascertain whether a given substance has a damaging influence on the corpuscular envelope by finding whether hemolysis occurs when we suspend the cor- puscles in a solution that is known by physical methods to be isotonic with the corpuscular contents. We can further determine the approximate degree of this toxic influence by estimating by color comparisons (colorimetry) the amount of hemoglobin that has diffused out of the corpuscles.
Plasmolysis
An analogous method for determining osmotic pressure is that of plasmolysis, in which the behavior of certain plant cells is observed microscopically while they are in contact with solutions of different strengths. When the surrounding solution is isotonic with the cell contents, the latter fill the cell and extend up to the more or less rigid cell wall (A in Fig. 3) ; but when the solution is hypotonic, the cell contents become detached from the cell wall at one or more places — plasmolysis (B and C). The semipermeable membrane in this case is therefore not the cell wall but the layer of protoplasm on the surface of the cell contents. The method can be used only for detecting solu- tions that are hypertonic, for wdth those that are hypotonic the cells merely become turgid and exert more pressure on the more or less rigid cell wall. Many of the conclusions that have been drawn from
LAWS OF SOLUTION
results obtained by the plasmolytic method have recently been called in question, because no regard has been taken of the power of the colloids of the cell to absorb (imbibe) water (see page 63).
The methods of hemolysis and plasmolysis have been used for the inves- tigation of many problems in medicine besides those pertaining strictly to osmotic pressure. In the case of certain toxic fluids, such as snake venom, tetanus toxin, etc., determination of the hemolytic power has proved of value in roughly assaying the damaging influence on other cells than blood corpuscles. Studies in hemolysis have also been especially valuable in working out the mechanism by which cellular toxins in general develop their action, and the conditions under which this action may be counter-
Fig. 3. — To show plasmolysis in cells from Tradescantia discolor. A, normal cell; B, plasmolysis in 0.22 M. cane sugar; C, pronounced plasmolysis in 1.0 M. KNO3; h, the cell wall; p, the protoplasm. (After De Vries.)
acted, as by the development of antibodies. Furthermore, any solution that is to be injected into the animal body, either intravenously or subcu- taneously, should first of all be tested by the above methods in order to find out whether it is isotonic with the body fluids. If a hypertonic so- lution is injected, it will result in the abstraction of water from the tissue cells, whereas a hypotonic solution will cause the water content of these to increase. Advantage has recently been taken of this water-abstracting effect of hypertonic solutions in the treatment of wounds. By constantly bathing them with strong saline solutions, an outflow of water is set up from the tissue cells that border on the wound, and this tends to bring to the focus of infection the defensive substances that are present in animal fluids.
CHAPTER II OSMOTIC PRESSURE (Cont'd)
MEASUREMENT BY DEPRESSION OF FREEZING POINT
The limitations in the use of the plasmolytic and hemolytic methods in the precise measurement of the osmotic pressure of the body fluids have rendered it necessary to find some physical method that will be generally applicable. Because of technical difficulties, it is impracticable to measure the pressure directly by employing an osmometer, so that some indirect method, depending on a readily measurable physical prop- erty which varies in proportion to the osmotic pressure of the dissolved substances, must be used. Fortunately, one such exists in the property which dissolved substances have in lowering the temperature at which the pure solvent solidifies; the freezing point of pure water, for example, is lowered when substances are dissolved in it, and the extent of this lowering, with certain reservations which will be explained later (page 16), is proportional to the molecular concentration of the solution and independent of the chemical nature of the substance dissolved. This lowering of temperature is designated by the Greek letter A, and to measure it a thermometer is used which is not only extremely sensitive but in which the level of the mercury column can be adjusted so that it stands at a convenient level on the scale corresponding to the freezing point of whatever solvent was used in making the solution under investi- gation (Beckmairs thermometer) (Fig. 4). Having ascertained the exact position on the scale of this thermometer at which the pure solvent freezes, the observation is repeated with the solution, the osmotic pressure of which is to be determined.
A gram-molecular solution in water (having therefore an osmotic pres- sure of 17.024 mm. Hg) has a freezing point that is 1.86° C. lower than that of pure water. This is known as the "freezing point constant," and it varies for different solvents, being 3.9 for acetic acid and 4.0 for benzene. If an unknown watery solution is found to have a freez- ing point that is A° C. loAver than that of water, its osmotic pressure
, Ax 17.024
will equal — ^-^ mm. Hg.
l.ob
The depression of the freezing points produced by the various body
10
OSMOTIC PRESSURE
11
fluids has been compared, the objects in view being to see whether osmotic pressure is a property which changes under different physiological and pathological conditions, and to find out by comparison of the osmotic pressures of the fluids in contact with a membrane, whether physical forces alone can be held responsible for the transference of substances through it from one fluid to the other.
THE ROLE OF OSMOSIS, DIFFUSION, AND ALLIED PROCESSES IN PHYSIOLOGICAL MECHANISMS
The Transference of Substances Through Cell Membranes. — An ac- count of some of the investigations in which the foregoing meth-
Fig. 4. — Apparatus for measurement of the depression of freezing point of solutions. The solution is placed in the large test tube with the side arm, and in it is suspended the bulb of a Beckmann thermometer with a platinum loop to serve for stirring. The upper end^of the mercury column of the thermometer is shown magnified at the upper left corner. The amount of mercury in the thermometer tube can be regulated by tapping the upper end with the thermometer in various positions. The test tube is protected by an outer tube, which is then placed in a vessel containing a freezing mixture.
ods have been used will illustrate their value in revealing the mech- anism involved in the transference of water and dissolved substances through cell membranes, as occurs in absorption of food in the intestine, in the formation of lymph and urine, and so forth. In em- ploying physical methods in the elucidation of such problems, it is always most necessary to proceed with great care, since the physical
12 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES
chemist works with pure solutions, while the physiologist has to use fluids that are always complicated and frequently very variable in com- position. We must simplify the problem as far as possible by having clearly before us the' exact nature of the biological problem which a com- parison of physicochemical values, such as osmotic pressure, may ena- ble us to elucidate, and we must consider the other physical forces which may assist or modify the particular one we are investigating.
In the physical experiments described above, the semipermeable mem- brane may be conceived of as composed of pores of such a size that they permit only the smallest of molecules — those of water — to pass through them. Semipermeable membranes with larger pores may, how- ever, exist — that is, membranes which permit water molecules and mole- cules of simple chemical substances to pass, but hold back those com- posed of large complex molecules. Such a semipermeable membrane would allow the saline constituents but not the proteins of blood serum to pass. It is, however, no longer semipermeable towards all of the dis- solved substances, and the process of diffusion through it is more gener- ally designated as one of dialysis than of osmosis.
Since the passage of dissolved molecules through membranes de- pends upon the principle of diffusion, its rate will be proportional to the osmotic pressures of the solutions on the two surfaces of the mem- brane and to the size of the molecules, small molecules diffusing more quickly than large ones. Suppose a membrane permeable to sodium chloride and water is placed between two fluids containing sodium chloride in solution, but in greater concentration in one of them than in the other: although the sodium chloride will diffuse from the stronger to the weaker solution, the water will tend to diffuse still more quickly (be- cause its molecules are smaller) in the opposite direction, until the number of sodium-chloride molecules in a given volume of solution is equal on both sides of the membrane. For a time, therefore, the volume of the stronger solution will increase. The differences which exist in the dif- fusibility of dissolved molecules are analogous to those which have long been known to exist in the diffusibility of gases, but the relation between rate of diffusibility and molecular weight is not so simple as the ratio between these tivo quantities in gases. These relationships, however, indicate several further possibilities in the explanation of the mechanism of exchange of substances through membranes, and must not be overlooked, as they often are, in the interpretation of physiological phenomena. An excellent review of the possible conditions is given by Starling in his "Human Physiology."4 For example, let us suppose the substances dissolved in the fluid on the two sides of a semipermeable membrane, such as the peritoneum, to be different in diffusibility, as cane
OSMOTIC PRESSURE 13
sugar, which does not readily diffuse, and sodium chloride, which diffuses quickly; the osmotic flow will take place from the sodium-chloride solu- tion to that of cane sugar even though the sodium-chloride solution is stronger than the sugar.
Furthermore, the simple laws of osmosis may be upset by an attrac- tive influence of the membrane toward certain substances [due to their becoming dissolved or adsorbed in it (see page 66)] but not toward others. Many membranes of this nature are known to the chemist (e. g., rubber membranes in contact with gases, pyridine solutions, etc.), and it is probable that such a property of selective solubility may play a not unimportant role in the transference of substances across animal membranes (Kahlenberg5).
These few conditions which may modify the direction of the osmotic flow, are indicated here to show how involved such problems are, and how careful we must be not to assume that, because a substance is trans- ferred through a living membrane contrary to the simpler laws of os- mosia and diffusion, it must involve the expenditure of forces different from those operating in dead membranes.
Another force comes into operation in causing transference of sub- stances through membranes — namely, that of filtration. This is a purely mechanical process, in which molecules are forced through the pores of a filter (i. e., membrane) by differences in pressure on its two sides.
We are now in a position to consider in how far the above phj^sical forces explain certain physiological problems.
The Physical Factors Involved in Absorption, Excretion and Lymph Formation. — 1. 7s the absorption, into the blood and lymph circulating in the intestinal walls, of substances in solution in the intestinal contents, entirely dependent upon the processes of filtration, diffusion and osmosis? The absorption of weak solutions of highly diffusible substances is probably very largely a matter of osmosis and diffusion, and water passes quickly into the blood because of osmotic attraction, but that other forces ordi- narily come into play is very clearly established by the following ob- servations. If a piece of intestine is isolated from the rest by placing two ligatures on it, and the isolated loop filled either with a solution con- taining the same saline constituents in similar proportions as in blood serum, or better still, with some of the same animal's blood serum, it will be found after some time that all of the solution becomes absorbed into the blood; the contents of the loop are therefore absorbed into the blood, even though the osmotic pressures of the dissolved substances are the same on both sides of the membrane (Weymouth Eeid6).
The intestinal membrane seems to possess towards readily diffusible
14 PIIYSICOCHEMTCAL BASTS OF PHYSIOLOGICAL PROCESSES
substances a permeability which varies, not at all with the physical diffusibility of the substance, but with its value from a physiological standpoint. Thus, sodium sulphate and sodium chloride diffuse through ordinary membranes with about equal facility, and yet if a solution con- taining these two salts is placed in the intestine, the chloride will be absorbed into the blood much more quickly than the sulphate. Sodium sulphate in watery solution diffuses through a membrane fifteen times more quickly than cane sugar, but from the intestinal lumen, cane sugar is absorbed ten times more quickly than sodium sulphate. If. however, the vitality of the epithelium is destroyed, as by first of all bathing it with a solution of sodium fluoride, then the sulphate and chloride will be absorbed at an equal rate.
Although diffusion and osmosis can not therefore play any significant role in the normal process of absorption from the intestine, we must not entirely discount them; under certain circumstances, these physical forces may assert their influence as, for example, when concentrated saline solutions are present. Such solutions will attract water from the blood, and, other things being equal, more will be attracted the less permeable the epithelium happens to be towards the saline employed. Sulphates and phosphates will attract more water than chlorides or acetates. This property of the saline solutions to attract water coun- teracts the natural tendency for the water to be absorbed, and the large volume of fluid stimulates peristalsis.
2. Do the physical processes of filtration, diffusion and osmosis suf- fice to account for the production of urine by the kidney sf Under normal conditions the molecular concentration of the urine, as determined by the depression of freezing point, is considerably greater than that of the blood. This indicates that excretion must have occurred contrary to the laws of diffusion and osmosis ; in other words, that the renal cells must have compelled dissolved molecules to be transferred from the blood to the urine, although the difference in concentration would cause them to pass in the opposite direction. This force, sometimes called for want of a better name " vital activity," must depend on the operation of processes that are quite distinct from those of diffusion, etc.; but that they are necessarily of a nonphysical nature (e. g., vital) is less probable than that they depend on some physical process the nature of which our present knowledge does not permit us to understand.
By comparing the osmotic pressures of urine and blood, attempts have been made to measure the work done by the kidney in the produc- tion of urine. Thus, it has been found that A for normal urine (human) is about 1.8, and for blood about 0.6, from which it may be calculated that in the production of 1 kilogram of urine 150 kilogrammeters of
OSMOTIC PRESSURE 15
work are expended.* But that such comparisons of the osmotic pres- sure of blood and urine are fallacious as an indication of the work of the kidney is evidenced, not alone by the results of the above calcula- tions, but also by the fact that under certain circumstances (as after copious diuresis) the osmotic pressure of the urine may be considerably loirrr than that of the blood.
For some time after the application of osmotic pressure measurements to the study of biological problems, it was thought that determination of A in urine might be of clinical value as a criterion of renal efficiency, especially in one kidney as compared with the other. For this purpose A was determined in samples of urine removed from each ureter by catheterization. The tests of renal efficiency based on the rate of excre- tion and on the specific gravity of the urine, following ingestion of a fixed amount of water, have been found of much greater value.
3. Is the formation of lymph purely a physical process? The osmotic pressure of normal lymph is nearly always somewhat below that of blood serum, although occasionally it has been found to be a trifle higher. Physical processes, such as filtration, might therefore suffice to account for its formation under most conditions. But when we con- sider the excessive production of lymph that occurs as a result of cel- lular activity or following the injection of certain substances, called "lymphagogues," it is not so easy to explain the production in such terms, although some interesting attempts have been made to do so by those that are wedded to the mechanistic view. For example, the very marked increase in lymph flow which occurs as a result of muscular exercise or glandular activity has been attributed to the fact that dur- ing such processes large molecules become broken down into small ones in the cell protoplasm, so that the osmotic pressure is raised and water is attracted into the the cell until the latter becomes distended and a process of filtration into the neighboring lymph spaces occurs (see page 119).
There are several other physiological processes of secretion and excre- tion which might be considered in the present relationship, but the above instances will suffice to illustrate the general principle upon which all of them have to be considered.
*Osmotic pressure corresponding to A = _0.6° C. enuals 5,662 mm. TTg (75 m. of TT..O), and that corresponding to A = ~1.8° C. equals 16,986 mm. Kg (225 m. H2O). The difference "is there- fore equal to a column of water 150 m. high. According to these calculations it would appear that the kidney in producing the average daily output of 1500 c.c. urine performs 225 kilogrammeters of work in comparison with the 14,000 kilogrammeters which the heart is computed to perform in the same time (page 213).
CHAPTER III
ELECTRICAL CONDUCTIVITY, DISSOCIATION, AND IONIZATION
The osmotic pressure is not infrequently found to be considerably greater than that expected from the strength of the solution. Although A of a gram-molecular watery solution of cane sugar (342 gm. to the liter) is 1.86 (see page 10), that of sodium chloride (58.5 gm. to the liter) is considerably greater. If the hypothesis regarding the relationship of molecular concentration to osmotic pressure is to hold good, it becomes necessary to explain this apparent inconsistency; xme must account for a greater number of dissolved units than is represented by the actual number of dissolved molecules (i.e., weight of dissolved substances).
It was observed that the power to conduct the electric current — electrical conductivity — in the case of solutions (e. g., of sugar) which have an osmotic pressure that corresponds to the weight of dissolved substances is practically nil, whereas the conductivity of those solutions which give higher osmotic pressure is quite pronounced. Arrhenius made the hy- pothesis that the conductivity depends on the splitting of molecules into two or more portions or ions, each of which carries either a positive or a negative electric charge, and that it is only when such dissociation occurs that the electric current can be conducted through the solution, the ions serving as it were as floats carrying the electric current. When sodium chloride is dissolved in water, it splits into Na carrying a positive charge and Cl carrying a negative charge, or Na+Cl", as it is written; on the other hand, when sugar is dissolved, the molecules remain unbroken and no electric charges are set free.
Substances which thus dissociate are called electrolytes, and those which do not, nonelectrolytes. When the electric current is passed through a solution of electrolytes, the ions which carry a positive charge move to the electrode or pole by which the current leaves the solution — that is, in the same directions as the current; and since this electrode is called the cathode, these are called cations. Hydrogen and the metals belong to this group. The ions carrying a negative charge go in the opposite direc- tion, against the current — that is, towards the electrode by which the cur- rent enters, or the anode; they are therefore called anions. They include oxygen, the halogens and the acid groups, such as S04, C03, etc.
It must be understood that this dissociation into ions is already present
16
ELECTRICAL CONDUCTIVITY, DISSOCIATION, IONIZATION 17
in the solution before any electric current passes through it, the ions being however uniformly distributed throughout — that is, arranged so that the negative charges of the anions precisely neutralize the positive charges of the cations. The electric current causes the electrodes to be- come charged, the one positively, the other negatively, so that an attrac- tive force is exerted on the ions of opposite sign. This causes the nega- tively charged ions to migrate towards the positive electrode, and the positively charged, towards the negative electrode. It is this migration of the ions that endows the solution with conducting qualities.
In water, or in a solution of a nonelectrolyte, molecules of H20 or non- electrolyte exist thus:
H20 H20 H20
H20 H20 H20 H20 H20 H20
In a solution of an electrolyte, many of the molecules split into ions thus :
Na+ Cl- Na* Cl- Na+ Cl-
Na* Cl- Na+ Cl- Na+ Cl- Na+ Cl- Na+ Cl- Na+ Cl-
When an electric current passes through a solution of an electrolyte, the ions tend to arrange themselves thus:
• Cathode- Anode*
Na+ Na+ Na* Cl- Cl- Cl-
Na* Na+ Na+ Cl- Cl- Cl-
Na+ Na+ Na+ Cl- Cl- Cl~
It follows from the above considerations that the conductivity of a sub- stance in solution will depend on the degree to which it undergoes dissocia- tion. Furthermore, if we assume that in so far as osmotic pressure phenomena are concerned, each ion behaves in the same way as a mole- cule, then it follows th.at the electrical conductivity must be proportional to the extent to which the osmotic pressure is greater than we should ex- pect it to be from the amount of substance actually dissolved.
In the Determination of the Conductivity it is obviously necessary to use standard conditions of depth and width of the fluid through which the current is passed, and to have some standard of comparison. The value is then known as the specific conductivity, the standard for comparison being the conductivity of a hypothetical liquid which, if enclosed in a centimeter cube, would offer a resistance of 1 ohm between two opposite sides of the cube acting as electrodes. The actual determination is usu-
18
PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES
ally made in a cylindrical vessel of hard glass (from soft glass enough alkali might be dissolved to affect the results), the electrodes being circu- lar plates of platinum firmly cemented at a known distance from each other (Fig. 5).* This conductivity cell, as it is called, is connected with a suitable apparatus for measuring the resistance offered by the
Fig. 5. — Diagram of conductivity cells. The platinum discs are represented by the thick black lines. They are held in position by thick-walled glass tubes, through which they are connected with the terminals by platinum wires. (From Spencer.)
solution to the passage of an electric current (Wheatstone Bridge) (see Fig. 6). The resistance is of course inversely proportional to the con- ductivity.
As a saline solution is progressively diluted, its specific conductivity naturally decreases (since there are now fewer molecules between the
Fig. 6. — Wheatstone Bridge for the measurement of electric resistance: a-b, bridge wire; c,
the movable contact.
two opposite faces of the centimeter cube, and the space between ions or molecules is increased). This result will not, however, tell us whether the salt itself is undergoing any alteration in conducting power as a con- sequence, for example, of greater dissociation. To ascertain this we must
*This distance is determined not by direct measurement but by calculation from results obtained by testing the actual resistance of a solution whose specific resistance is accurately known.
ELECTRICAL CONDUCTIVITY, DISSOCIATION, IONIZATION 19
obtain figures relating to the same quantity of salt at each dilution. If we multiply the specific conductivity by the volume of solution in c.c. which contains 1 gram-equivalent (see page 22), a value will be secured which represents the conducting power of a gram-equivalent. This is known as the equivalent or molecular conductivity * and is represented by the sign A. When it is determined for progressively diluted solutions, A gradually increases, indicating that the efficiency of the electrolyte itself as a conductor increases with dilution, because it dissociates more. The extent of this increase is found to become less and less as dilution proceeds. By plotting the values of the molecular conductivity of suc- cessive dilutions as a curve, the value at infinite dilution can be ascertained by extrapolation. This value is represented by A °c .
Now, let us see how these facts bear out the theory of electrolytic dissocia- tion. According to this hypothesis the conductivity depends on the num- ber of ions (see page 17), and since it is at a maximum at infinite dilu- tion, the value AOC must represent the total number of ions that can le pro- duced "by the dissociation of 1 gram-equivalent, and A thai at some other dilution. If, therefore, we divide A by AOC we obtain a value (called a) which must represent the degree to which the electrolyte is ionized at the various dilutions at which A is measured. From what has been said re- garding the osmotic pressure of similar solutions, it is evident that the value a could also be calculated by finding the extent to which the de- pression of freezing point A is greater than would be expected from the number of dissolved molecules. As a matter of fact, it has been found that the two methods yield practically identical values for many substances, thus furnishing almost incontrovertible proof in support of the dissociation hypothesis. In the cases of weak acids and bases, it is possible to secure a value, called the dissociation constant (K), which represents the rela- tive values of a at all dilutions. Since the activity of acids and bases is dependent upon the number of H- and OH-ions, respectively, set free by dissociation, it follows that it must be proportional to K. It will be necessary, however, to postpone a further consideration of the application of this constant until we have studied mass action (page 23).
Biological Applications. — The practical value of a knowledge of the laws of electrical conductivity rests, not so much on any direct application that can be made of it in explaining physiological processes, as on the es- sentially important bearing which it has in enabling us to understand the nature and operation of other physicochemical laws. Without a clear com- prehension of the elemental laws of dissociation, it is impossible to con- sider such problems as those which concern the activities of enzymes (mass
*In other words, the molecular conductivity is the specific conductivity divided by the number of gram-equivalents contained in 1 c.c.
20 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES
action, etc.), the occurrence of electrical currents during the physiological activity of muscles, glands, and nerves, and the all-important question of the reaction or H-ion concentration of the body fluids.
There are, however, several instances in which measurements of electrical conductivity and of dissociation have direct physiological value. The circu- lation time of the bloodflow through an organ can be determined by first finding the electrical resistance of a short piece of the vein of the organ, and then observing the change in resistance which is produced when the conductivity of the blood in the vein is altered by the arrival in it of saline injected into the artery. The interval elapsing between the injec- tion into the artery and the changes in resistance in the vein equals the circulation time (G. N. Stewart).
The same investigator has used measurements by electrical conductiv- ity to study the passage of electrolytes out of the red blood corpuscles into the serum. Under normal conditions the blood serum has a certain elec- trical conductivity equal to that of a 0.9 per cent sodium-chloride solution. The conductivity of the defibrinated blood is only about one-half that of serum, because it contains corpuscles which are nonconductors and there- fore obstruct the free passage of the ions, just as a suspension of quartz powder in a sodium-chloride solution lowers the conductivity of the lat- ter. If anything occurs therefore to occasion a passage of the saline con- tents of the corpuscles through their walls into the serum, an increase in the electrical conductivity will be produced. The value of this method in the investigation of changes in permeability of the red corpuscles is de- pendent on the fact that such migration of electrolytes out of the cor- puscles may occur before any of the less diffusible hemoglobin itself has escaped. The rise in conductivity precedes the hemolysis (see page 7).
Although determinations of the specific conductivity of blood and urine under various pathological conditions have also been made, the results have not been found to possess any diagnostic value or clinical signifi- cance. Measurements of the electrical conductivity of blood have, how- ever, been used by Wilson7 and by Priestley and Haldane8 to detect the degree of dilution when large quantities of water are ingested.
Another application of conductivity measurements in biochemistry has been made in studying the digestive action of proteolytic enzymes (Bay- liss). The general action of the enzymes is to break the large undisso- ciated molecules of the higher proteins (albumin, casein, etc.), into smaller molecules (ammo acids, etc.), which are partly ionized. As diges- tion proceeds, therefore, the conductivity of the digestion mixture pro- gressively increases, and is a measure of the rate of digestion.
Applications of the dissociation hypothesis in physiology concern the explanation of such phenomena as the production of electric currents
ELECTRICAL CONDUCTIVITY, DISSOCIATION, IONIZATION 21
during muscular, glandular, and nervous activity. The exact details of the application are not as yet sufficiently understood to warrant our at- tempting to do more than indicate the general lines along which the problems are being investigated. To do so we must delve a little further into physicochemical research, when we shall find that there are two further facts concerning ionized molecules that must be of importance in connec- tion with our problem. The first is that the contribution which each ion makes to the equivalent (or molecular) conductivity of a solution is inde- pendent of the other ion with which it is associated; and the second, that ions differ considerably in their conducting power. Since the univalent ions, K., Na., CL', N03', carry charges of equal magnitude,* and yet all do not conduct to the same degree, they must move at different velocities through the solution. We are driven, therefore, to the conclusion that, exposed to the same electrical force, different ions have different mobili- ties ; that is to say, when an electric current passes through a solution of an electrolyte, the positively charged ions move towards the cathode at a different rate from that at which, the negatively charged ions move towards the anode. Confirmation of this conclusion is obtained by exam- ination of the concentration changes around the two electrodes of an electrolytic cell. The actual velocity of each ion can be determined by experimental means. The inequality in concentration of ions in different regions of a tissue is no doubt the fundamental cause for the electrical currents that are set up by injury and activity.
*Thus Faraday showed that the amounts of the various ions liberated by electrolysis are in the same ratio as their chemical equivalents.
CHAPTER IV
THE PRINCIPLES INVOLVED IN THE DETERMINATION OF THE HYDROGEN-ION CONCENTRATION
TITRABLE ACIDITY AND ALKALINITY
All acids have one property in common — namely, that they contain hydrogen^and when the acid becomes neutralized, it is this element which becomes replaced by some other cation. Evidently, then, the strength of an acid is proportional to the number of displaceable hydro- gen atoms which it contains. It may contain other hydrogen atoms which are so bound up in the molecule that they do not become displaced when an alkali is mixed with the acid. For example, in organic acids like acetic, CH3COOH, it is only the H atom attached to the COOH group, but not those attached to the CH3 group, that is replaceable. It must therefore be possible to prepare for every acid a solution having exactly the same neutralizing power as that of any other acid; that is, the same volume of solution must be required in each case to neutralize a given quantity of alkali, the point of neutralization being judged by the change in color of indicators. As a standard a gram-molecular solu- tion of an acid with one displaceable H ion, such as hydrochloric, is chosen. This we call a "normal acid" (N). To prepare a normal solu- tion of acids having two displaceable H atoms, such as H2S04, we can not however use a gram-molecular quantity, but must take one-half of it; and similarly in the case of those with three H atoms, such as H3PO4, a one-third gram-molecular solution will be a normal acid solution. For practical purposes, use is very generally made of solutions that are some fraction of the normal, e. g., tenth or decinormal (written N/10), or hun- dredth or centinormal (N/100).
In a similar way, alkaline solutions can be prepared, a normal alkali being one which exactly corresponds in strength with a normal acid (i.e., can exactly neutralize it). Now, the characteristic of alkalies is that they produce in solution "OH" or hydroxyl ions; so that the process of neutralization must consist in the union of the H ions of the acid with the OH ions of the alkali to form water: KOH + HC1 = KC1 +H20. We can, therefore, prepare normal solutions of alkalies by dissolving in 1 liter of water such quantities of alkali (in grams) as will yield the OH required to react with the available hydrogen in normal acid solutions.
22
HYDROGEN-ION CONCENTRATION 23
Actual Degree of Acidity or Alkalinity. — According to the foregoing method of titration a normal solution of a powerful mineral acid, such as hydrochloric, is no stronger than a normal solution of a weak acid, such as acetic or lactic. It requires no fewer c.c. of N alkali to neutralize it. But the normal solution of the powerful acid is more acid to the taste, is more toxic, dissolves metals more readily, and in all its other chemical and physiological properties acts much more quickly than the weak acid, so that the titrable acidity or alkalinity can not express the real strength of the acid or alkali, or the actual degree of acidity or alkalinity. It is in this connection that the dissociation hypothesis aids us, for it suggests that the degree to which the acid becomes dissociated into H- and the remainder of the molecule will determine its real strength (see page 16). The question is, how are we to measure the latter ? One action of H ions which we may measure is that known as catalytic — that is, the power to accelerate reactions, such as the splitting of cane sugar (C^H^On) into glucose and levulose, which otherwise would proceed very slowly (see page 75). If then the real strength of an acid depends on the degree of dissociation which it undergoes, figures representing the catalytic power should correspond with those representing the relative conductivi- ties of the acids in equivalent concentration (see page 19). That this is actually the case is shown in the following table, in which the above values of various acids are given compared with HC1, which is taken as 100.
ACID CATALYTIC POWER RELATIVE CONDUCTIVITY
HC1 100 100
Dichloracetic 27 25
Monochloracetic 4.8 4.9
Formic 1.5 1.7
Acetic 0.40 0.42
It will be evident that, if we could measure the concentration of free H ions in a solution — that is, of H ions that are not matched by OH ions — we should have a faithful index of its real acidity. This measurement has been rendered possible by the application of two other physico- chemical principles — namely, those of mass action and electromotive force. Since the object of this volume is to present the scientific basis for the various methods that are used in modern medicine, it will be nec- essary for us to review the main principles of these two actions. We shall see that they apply, not only in the measurement of H-ion concentration, but in many other physiological processes.
Mass Action
When materials take part in a reaction, some molecules are decom- posing while others are being formed. After some time, however, a
24 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES
condition is reached in which the changes in one direction are exactly offset by those in the other. An equilibrium is said to have become estab- lished between the reacting substances. Bearing in mind that the ions and molecules entering into these reactions are constantly moving about and coming in contact with one another, it is easy to see that if we were to add an additional quantity of one kind of molecule or ion, there would be a change all along the line until a new equilibrium was established. If, on the other hand, we were to remove one kind of molecule or ion as fast as it is formed, the equilibrium could never be established, and the reaction would proceed until all of this material had disappeared. The natural rate at which any chemical reaction proceeds is dependent upon a number of conditions, such as chemical affinity, temperature, catalysis, and concentration. Of these conditions that of concentration is most readily measured. If we maintain all of the conditions other than that of concentration unchanged, and designate this combined in- fluence as K (constant), we shall find that the speed of the reaction is proportional to the molecular concentration of the reacting substances (i. e., the number of gram-molecular weights per liter). In other words, the speed with which two substances, a and b, unite to form other sub- stances, c and d, will be expressed by the equation,
k (a)x(b) *± k' (c)x(d);*
which means that, when the reaction is complete, the composition of the mixture will be dependent upon the ratio between k and k'. Since however these are both constants, their quotient is also constant (K), and
we have the equation, -y-r — ~f = K, indicating that no matter how
\ / ^ \ ^*- /
the concentrations a, b, c, and d are varied, reaction will take place in one direction or the other until the concentrations have become adjusted so that K remains unchanged.
As an example of the application of these laws, let us take the reaction which occurs between alcohols and organic acids to form the substances called esters — a reaction which is analogous to that between mineral alkalies and acids to form neutral salts, and which is of special interest to us because it is the reaction involved in the splitting of animal fats. The equation for the reaction is:
C2H5OH + CHSCOOII ?± C2HBOOCCH8 + H2O. (ethyl (acetic (ethyl acetate,
alcohol) acid) an ester)
Or expressed according to the law of mass action:
[C2H5OH] x [CH3COOH] [C2H6OOCCH3] x
*The brackets indicate that gram molecular quantities are used.
HYDROGEN-ION CONCENTRATION 25
Now it is clear that if we increase, say, H20 in the above equation, then in order that K may remain unchanged C2H5OOCCH3 must diminish or the substances which form the numerator of the equation must increase, or both these changes must occur. As a matter of fact, in such a case as the above, both of these adjustments take place, for, as the ester breaks down, it must thereby increase the concentration of acid and alcohol. Since in aqueous solutions the reaction occurs in the presence of an excess of water, it is evident that the tendency for an ester is to break down into alcohol and acid, and this must occur in all reactions in the body fluids in which water enters into the equation.
Physiological Applications. — The application of the law of mass action in the explanation of biochemical processes is very extensive. Most of the reactions which enzymes or ferments are capable of influencing are of the same general nature as that represented above, and the products of their activities are usually the substances on the side of the equation in which no water molecules appear — i. e., they are hydrolytic reactions. Enzymes merely accelerate the reaction (page 72), so that if we start with a mixture of the substances on either side of the equation, all they do is to accelerate the production of a sufficient concentration of those on the other side, until the equilibrium point is reached. For example, an enzyme present in pancreatic juice, called lipase, accelerates the breakdown of such esters as neutral fat, which consists of the triatomic alcohol, glycerol, combined with the fatty acids palmitic (C15H31COOH), stearic (C17H35COOH) and oleic (C7H33COOH):
C3H5 (O 00 C17H35)3 + 3H2O^3CnH35COOH + C3H5 (OH)8.
(the neutral fat, (the fatty acid, (glycerol)
tristearin) * stearic)
Under ordinary conditions the reaction proceeds until nearly all the neutral fat has become decomposed because of the preponderance of water, but if we start with a mixture of fatty acid and glycerol with just enough water to permit the enzyme to act, the reaction will pro- ceed in the opposite direction — i. e., so that some neutral fat will be synthesized. This is called the reversible action of enzymes (page 77).
Because of the universal presence of water, it is plain that such re- versible reactions could not alone be held responsible for the synthe- sis of neutral fat or of similar substances in the animal body. The only way by which synthesis could occur under these conditions would be if the substance produced along with the water were removed from the site of the reaction as soon as it was formed. This might occur by the precipitation of the substance or by its becoming surrounded by an en- velope of some inert material. In the synthesis of neutral fat which
26 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES
occurs in the epithelium of the intestine out of the fatty acid and glycerol absorbed from the intestinal contents, it is possible that the last men- tioned process occurs. In other cases the substance may be carried away by the blood or lymph or urine as fast as it is formed.
The Law of Mass Action as Applied to the Measurement of H-ion Concentration. — Let us now return to the reaction or H-ion concentration of substances in solution. As the standard of neutrality, pure water is chosen. Let us consider, then, how the laws of mass action can be applied in order to enable us to determine the H-ion concentration of pure water. It has been stated above that chemically pure water is in- capable of conducting the electric current. This however is not strictly the case, for it conducts to a very slight degree. According to the dis- sociation hypothesis, it must therefore be represented as containing molecules of H20 and ions of H • and OH', and according to that of mass action there must be a balanced reaction between the molecules and ions represented thus:
=.
Since the concentration of H • and OH' is extremely small, there must always be such an overwhelming preponderance of H20 molecules that no changes in the concentration of H • and OH' will be capable of appre- ciably affecting the concentration of H20 ; in other words, one may omit the denominator of the equation and write it [H •] x [OH'] = K. If then we know the value of K, it will only be necessary to measure the concentration of either H • or OH' in order to express in numerical terms the reaction of the solution. It has been found that the value of K is about -1 x 10'14,* and since the concentrations of H • and OH' are nec- essarily equal in pure wrater, it follows that [H] = [OH] = \flxlO~14, i. e., each ion has a concentration of 1 x 10 7. This means that water con- tains approximately 1 gram mol. each of H- and OH' ions, or 1 gram H- and 17 grams OH' ions, in 10+7 or 10,000,000 liters. A consequence of the above law is that no matter how much the concentration of one ion is increased by adding another substance, the solution must still contain some of the other ion. Thus, in acid solutions the concentration of H • must increase and the concentration of OH' must decrease in such pro- portion that the two multiplied together equals about 1 x 10~14. The H-ion concentration can ~be used therefore to express the reaction of neutral, acid and- alkaline solutions.
In place of water, let us substitute decinormal hydrochloric acid
*The sign 10-14 is simply a convenient way of expressing the degree of dilution. It gives the number of times the value standing in front of it must be divided by 10 in order to find the concentration.
HYDROGEN-ION CONCENTRATION 27
(0.1 N HC1) — that is, a hydrochloric acid solution containing one tenth of the molecular weight of hydrochloric acid in grams dissolved in a liter of water. At this dilution HC1 is 91 per cent dissociated; therefore the H-ion concentration (or CH as it is written for short) is 0.091 Nt or, in mathematical notation, 9.1 x 10 2.
Method of Expressing CH. — To avoid the necessity of having to use several figures to express CH, as has been done above, Sorenson has intro- duced a scheme by which only one figure is required. This figure, des- ignated by PH, is found by subtracting from the power of ten (i. e., the figure standing behind 10) the common logarithm of the figure ex- pressing the normality of the acid. In a decinormal HC1 solution, therefore, we must subtract from the power 2, the common log. of 9.1, which is .96 (ascertained from logarithm tables), leaving 1.04. . Take another example: decinormal acetic acid is dissociated only to the ex- tent of 1.3 per cent ; CH is therefore 0.0013 normal, or 1.3 x 10'3. Since the logarithm of 1.3 is .11, PH equals 3-.11, or -2.89.*
The only objection to the use of the exponent PH as an expression of the H-ion concentration is that it increases in magnitude as the acidity becomes less; this is because the negative sign of the power is disre- garded. As stated above, it is usual to express the strength of alkalies as well as acids in terms of CH, or PH, because it is easier to measure the concentration of H ions than of OH ions. A 0.1 NaOH solution is 84 per cent dissociated; therefore the "OH" ion is 0.084 N (i. e., 0.084 gram equivalents OH per liter), and since the product of the II • and OH' concentrations must always equal 10-14-14 (at 20° C.), it is clear that as the H ion increases in concentration, the OH ion must reciprocally de- crease. Expressed according to the above scheme, the 0.084 N NaOH solution gives PH 13.06; thus, 0.084 = 8.4 x 10'2; the log. of 8.4 is .924, and this subtracted from the power -2 = 1.08 as POH, or 14.14 - 1.08 = 13.06 as PH.**
Similarly, PH of 0.1 N NH4HO solution is 11.286. Its dissociation is 1.4 per cent; therefore the solution contains only 0.0014 gram equivalents HO— i. e., 1.4 x 10-3 POH = 3 - 0.146 = 2.854 . • . PH 14.14 - 2.854 = 11.286.f
*If we wish to express the value of Pit in ordinary notation, we must find the antilogarithm of the difference between the value of PH and the next higher whole numher; e. g., if PH = 7.45, the antilogarithm of 0.55 being 3.55, the CH is 3.55 x 10'8, or 0.000,000,0355 N, or 3.55 gm. mol. H ion in 100,000,000 liters.
**It must be remembered that the power of a number indicates the number of times by which that number must be multiplied by ten; thus, Pn-6 does not mean that the H ion is six times less than PH°, but 1 x 10 x 10 x 10 x 10 x 10 x 10, or 1,000,000 times less. Similarly, Pn'3 is 1000 times as great as PH-«, not twice as great.
A solution containing almost exactly 1 gram molecule of dissociated hydrogen in 10,000,000 liters constitutes a neutral solution (Pn — 7).
tThe expressions PH and CH may be used indiscriminately, but when the numerical value is given, it is most convenient to use the former.
28 PHYSICOCH^MICAL BASIS OF PHYSIOLOGICAL PROCESSES
Application of the Law of Mass Action in Determining the Real Strength of Acids or Alkalies. — We have seen that it is the degree of dissociation upon which the real strength of an acid depends and that this varies with dilution (page 19). The equilibrium between the un- dissociated and dissociated molecules may therefore be shifted in either direction by changing the concentration; in other words, the process of dissociation is a reversible reaction, and may be represented as AB ±5 A' + B •. The law of mass action must apply in such a case, and as a matter of fact it has, been found that a constant can be calculated, which is known as the dissociation constant.* It is an expression of the inherent ability of the acid to dissociate into ions, and is therefore the best measure of the strength of the acid. This is strictly the case for all of the weaker acids, but strong mineral acids (and bases) do not give a satisfactory constant, so that the comparison must not be made between them and weaker ones. That the dissociation constant expresses the rela- tive strength of organic acids can be shown by comparing its value with that of the rate at which cane sugar is inverted (see page 23), this being proportional to the concentration of the H ions present. K for some or- ganic acids is: Acetic, 0.000018; Formic, 0.000214; Benzoic, 0.00006; Sal- icylic, 0.00102.
a2 *The equation is -j- — r-rr- := K, where it is supposed that in volume V of the solution there is
1 gram-equivalent of electrolyte, and that the degree of dissociation is a; the quantity of undis- sociated electrolyte stated in a fraction of a gram-equivalent will be 1-a, and the quantity of each ion a. To illustrate, let us take acetic acid in various dilutions:
V a KxlO5
0.994 0.004 1.62
2.02 0.00614 1.88
15.9 0.0166 1.76
18.1 0.0178 1.78
CHAPTER V
THE PRINCIPLES INVOLVED IN THE MEASUREMENT OF THE HYDROGEN-ION CONCENTRATION (Cont'd)
THE METHODS OF MEASUREMENT
The Electrical Method
In order to understand the principle of the standard method used for measuring the H-ion concentration, it is necessary that a few words be said concerning the factors governing the development of electric cur- rents in chemical batteries. There may be a further application of this knowledge in connection with the generation of the electric currents which occurs during physiological activity, as in active glands and muscles.
When a metal is immersed in a solution of one of its salts, it has a tendency to give off ions into the solution. Similar ions are, however, already present in this solution, and these, by their osmotic pressure, tend to oppose the passage of the ions coming from the metal. The force with w^hich the metal sends out its ions into the solution is called the electrolytic solution pressure. If this is equal to the osmotic pres- sure of the metallic ions in the solution, there will be no electric current generated, but if it is greater or less than the osmotic pressure of the metallic ion, an electric current will be set up. "When the solution pres- sure is the greater, the metal will become negatively charged, because its ions carry positive charges into the solution (cations) ; on the contrary, when the osmotic pressure is greater than the solution pressure, the metal will have a positive charge, owing to the receipt of the positive cations from the solution.
Because of a force called electrostatic attraction, the ions given off from the metal can not travel any measurable distance from the oppositely charged mass of metal, so that from one of the electrodes alone it is impossible for us to lead off any electric current. For this purpose we must form a circuit. This is done in the manner shown in Fig. 7 by connecting side tubes coming from the electrode vessels with an inter- mediate vessel containing a solution of high conductivity and by con- necting the metals by wires. If the circuit is formed between the same metals in solutions of the same concentration, no electric cur- rent will be generated, because the two electrode potentials will be
29
30
PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES
equal and in opposite directions to each other. On the other hand, should the concentration of the metallic ion in the solutions be unequal, the electromotive force will flow from the one electrode to the other, and the pressure with which it flows will be equal to the difference in con- centration of the two solutions. This is the principle of a concentration cell, and if we know the concentration of one of the solutions composing it, and then proceed to measure the. electromotive force, we can obtain the concentrations of the other solution by difference. To do this we must employ a formula which takes into consideration the relation be- tween the potential and the concentration of the solution.
The potential of an unknown electrode composed of a metal in con- tact with a solution of one of its salts may also be determined by making it one pole of a battery of which the other pole is composed of a stand- ard electrode of unchanging known potential. An electrode of the latter
Fig. 7. — Diagram to show type of electrodes used in studying electromotive force. The metal in each electrode is connected (through a glass tube) with a platinum wire, to which the apparatus for measurement of the voltage is connected. The metal dips into a solution contained in the electrode vessel and filling the side tube. The latter dips into an inter- mediate vessel containing saturated KC1 solution. The currents flow through the circuit under the following conditions: (1) dissimilar metals dipping into the same fluid; (2) similar metals dipping into different fluids; (3) dissimilar metals dipping into different fluids.
type can most readily be made by bringing pure mercury in contact with a -saturated solution of calomel (Hg2Cl2) in normal potassium chlo- ride solution (Fig. 8). Under suitable conditions (i. e., when the circuit is completed), a potential of .0560 v. is developed in this so-called calomel electrode* — that is, positive ions of mercury are deposited on the mercury from the calomel solution at this pressure. Suppose that we connect a calomel electrode, through the intermediation of some solution which
*The calomel electrode consists of a suitably shaped glass vessel containing pure mercury, con- nected by means of a platinum wire with a conductor, and filled with a saturated solution of pure mercurous chloride in normal KC1 solution up to such a level that it also fills a side tube connected with a vessel containing a saturated solution of potassium chloride. Into this vessel also runs a similar side tube from the unknown electrode. By having an excess of midissolved calomel in the solution in the calomel electrode its saturated condition is maintained during the chemical changes which accompany the production of the electric current.
HYDROGEN-ION CONCENTRATION
31
will serve as a good conductor, with another electrode, the two elec- trodes being also connected by wires with electrical apparatus for measuring the total potential of the battery; then by adding +0.560 v. to or subtracting this value from the total potential (depending on the sign of the unknown electrode) we can tell the potential of the unknown electrode.
We have discussed these principles of electrochemistry because they form the basis upon which depends the standard method for the deter- mination of the H-ion concentration of fluids. Suppose, for example, that in place of using a metal in the construction of one electrode, we use an electrode consisting of a layer of pure hydrogen gas in contact with a solution in which are free H ions; then the rate at which H ions
CapVVf&vy .. „_.— I
5C«v3e - —
, --(-Jh)-- /
AMMwlqkpr
Fig. 8. — Diagram of apparatus for the measurement of the H-ion concentration. The cur- rent generated in the battery (composed of calomel electrode, connecting vessel with KC1 solu- tion, and the H electrode) or that from the normal element is transmitted through a reversing key to the bridge wire, where the voltage is compared with a steady current flowing through the bridge wire from an accumulator. The capillary electrometer is used to detect the flow of current at various positions of the movable contact on the bridge wire. (Modified from Sorensen.)
become added to the solution from the H layer, or taken from it, will de- pend on the concentration of H ions in solution. In order to secure a hydrogen electrode fulfilling the above requirements, it is necessary to employ some means by which a layer of hydrogen may be furnished, and fortunately this can be done by taking advantage of the property which spongy platinum possesses of absorbing large quantities of this gas. It is also necessary to keep an atmosphere of pure H in contact with the fluid.
As is the case of the simpler cells described above, there are two types which we might use for measuring the electromotive force gen- erated in the unknown electrode: a concentration cell composed of two
32 PHYSICOCIIEMICAL BASIS OF PHYSIOLOGICAL PROCESSES
hydrogen electrodes, of which, one contains a solution of "known H-ion concentration, and the other the solution in which this is unknown; and a cell of which one electrode is a standard calomel electrode and the other, a hydrogen electrode containing the unknown solution.
The exact arrangement of the apparatus in which the calomel elec- trode is used will be seen in the accompanying sketch. The hydrogen electrode, it will be noticed, is a very small V-shaped tube, in which is suspended a platinum wire coated with spongy platinum and dipping into a solution which nearly fills the tube. The space above the solution is filled with pure hydrogen. This and the calomel electrode are con- nected with suitable electric measuring instruments, and the circuit is completed by connecting the two electrodes by means of an intermediate vessel containing a saturated solution of potassium chloride. This con- necting solution is used because it has been found that the electric cur- rents set up, at the contact between different solutions are so small that they can be disregarded.*
As outlined above, the hydrogen electrode is that which is used to determine the H-ion concentration of blood, the particular point about it, in comparison with the apparatus used for simpler solutions, being that the hydrogen is not changed in the course of the experiment. This precaution is to prevent the carbon dioxide of the blood from being "washed out" of it by a frequently changing atmosphere of hydrogen. Many inaccuracies in the earlier results obtained by this method were due to the removal of carbon dioxide, which, as we shall see later, is one of the chief acids contributing to the H-ion concentration of blood.
The Indicator Method
As pointed out in a previous chapter (page 22), the method of titra- tion for acidity or alkalinity in which a standard solution of alkali or acid is added until a certain change in the color of a suitable indicator is detected, does not afford any information regarding the H-ion con- centration actually present in the solution. It tells us the total con- centration of available acid or base, both dissociated and undissociated. By modification of the method of procedure, however, we may also use indicators for determining the H-ion concentration. The principle of this method depends on the fact that there are certain dyes which change quite distinctly in tint with very slight changes in the H-ion concentration, so that if we use dyes which possess this property at a point near that of neutrality (i. e., between PH6.5 and Pn8), we can es-
*A description of the technic for measuring the electric potential developed by the cell would be out of place here. Suffice to say that the strength of the current is compared with that of a current of known strength furnished by a normal cell, the comparison being made by a bridge wire F, a capillary electrometer II being employed to detect the direction and degree of current.
HYDROGEN-ION CONCENTRATION
33
timate the H-ion concentration of the body fluids with very remarkable accuracy, provided certain precautions are taken to circumvent the disturbing influence which the protein and salts in these fluids may have on the color change.
To understand this use of indicators, it is important to bear in mind that one solution reacting neutral to one indicator may have a H-ion concentration which differs very markedly from that of another solu- tion reacting neutral to another indicator. This is because indicators react to different H-ion concentrations. A solution that is neutral to phenolphthalein has a PH of about 9, whereas one neutral to methyl or- ange has a PH of about 4. This can be very clearly shown by titrating a solution of phosphoric acid with decinormal alkali. After a certain amount of alkali has been added it will be noticed that methyl orange changes from red to yellow, but after it has changed and is therefore alkaline as judged by this indicator, it still remains distinctly acid to- wards phenolphthalein (shows no red tint) even though considerably more alkali is added. The methyl orange is, therefore, itself unrespon- sive to weak acids such as remain after the greater part of the phos- phoric acid has been neutralized by the alkali.
The series of indicators which has been employed for this purpose is given in the accompanying table, along with the PH limits through which they change in color.
LIST OF INDICATORS
|
CHEMICAL NAME |
COMMON NAME |
CONCEN- TRATION |
COLOR CHANGE |
RANGE PH |
|
Thymol sulfon phthalein |
per cent |
|||
|
(acid range) |
Thymol blue |
0.04 |
Red-yellow |
1.2-2.8 |
|
Tetra bromo phenol sul- |
||||
|
fon phthalein |
Brom phenol |
|||
|
blue |
0.04 |
Yellow-blue |
3.0-4.6 |
|
|
Ortho carboxy benzene |
||||
|
azo di methyl |
||||
|
aniline |
Methyl red |
0.02 |
Red-yellow |
4.4-6.0 |
|
Ortho carboxy benzene |
||||
|
azo di propyl |
||||
|
aniline |
Propyl red |
0.02 |
Red-yellow |
4.8-6.4 |
|
Di bromo ortho cresol |
||||
|
sulfon phthalein |
Brom cresol |
|||
|
purple |
0.04 |
Yellow- |
||
|
purple |
5.2-6.8 |
|||
|
Di bromo thymol sulfon |
Brom thymol |
|||
|
phthalein |
blue |
0.04 |
Yellow-blue |
6.0-7.6 |
|
Phenol sulfon phthalein |
Phenol red |
0.02 |
Yellow-red |
6.8-8.4 |
|
Ortho cresol sulfon |
||||
|
phthalein |
Cresol red |
0.02 |
Yellow-red |
7.2-8.8 |
|
Thymol sulfon phthalein |
Thymol blue |
0.04 |
Yellow-red |
8.0-9.6 |
|
(see above) |
||||
|
Ortho cresol phthalein |
Cresol |
|||
|
phthalein |
0.02 |
Colorless-red |
8.2-9.8 |
These dyes may now be obtained in this country.
(W. M. Clark and H. A. Lul»s.)»
34 PHYSICOOIIKMICAL BASIS OF PHYSIOLOGICAL PROCESSES
Briefly stated the method for measuring the H-ion concentration con- sists in preparing a series of solutions containing known concentrations of H-ion — that is to say, of known PH — and adding to each solution an equal amount of an indicator which exhibits easily distinguishable changes in tint at H-ion concentrations approximating those believed to be present in the unknown solution. The same indicator is added to the unknown solution, which is then placed side by side with the stand- ards to find with which of them it most closely matches. The series of solutions of known H-ion concentration is prepared by mixing fif- teenth normal solutions of Na2IIP04 and KTT2P04 in varying propor- tions as given in the following table:
PREPARATION of STANDARD SOLUTIONS The solutions are mixed in the proportions indicated be-low to obtain the desired PH:*
PH 6.4 6.6 6.8 7.0 7.1 7.2 7.3 7.4 7.5 .7.6 7.7 7.8 8.0 8.2 8.4
I'rimary Potas. 73 63 51 37 32 27 23 19 15.8 13.2 11 8.8 5.6 3.2 2.0
Phos., c.c. Secondary Sodium 27 37 49 63 68 73 77 81 84.2 86.8 89 91.2 94.4 96.8 98.0
Phos., c.c.
(From Levy, Rowntree and Marriott.)
*Standard phosphate mixtures are prepared according to Sorensen's directions as follows: 1/15 mol. acid or primary potassium phosphate. — 9.078 grams of the pure recrystallized salt (KH2PO4) are dissolved in freshly distilled water and made up to 1 liter.
1/15 mol. alkaline or secondary sodium phosphate. — The pure recrystallized salt (NaslIPO,!. 12H2O) is exposed to the air for from ten days to two weeks, protected from dust. Ten molecules of water of crystallization are given off and a salt of the formulai Na2HPO4.2H2O is obtained; 11.876 grains of this are dissolved in freshly distilled water and made up to 1 liter. The solution should give a deep rose red color with phenolphthalein. If only a faint pink color is obtained, the salt is not sufficiently pure.
The indicator method is extremely accurate when used with pure solutions of acids, but, as mentioned above, it is apt to be inaccurate, at least with most indicators, when protein or inorganic salts are pres- ent in the solution, and of course it is quite unusable with colored fluids such as blood. In order to overcome these difficulties, the dialysis method has recently been evolved. It consists in placing the fluid — blood, for example — in a dialyser sac composed of celloidin and about as large as a small test tube. The sac is placed in a wider test tube of hard glass containing an isotonic solution of sodium chloride that has been carefully tested to ascertain that it is strictly neutral. The amount of blood or serum required for this method is only 2 or 3 c.c., and the amount of salt solution placed outside the sac should be about the same. It takes only from five to ten minutes for dialysis to occur. The celloidin sac is then removed, a few drops of the indicator are thoroughly mixed with the dialysate, and the tube compared with the series of standards until the corresponding tint is matched. This indicates the H-ion concentration in the dialysate. The tints produced by using sulphonephenolphthalein are reproduced as nearly as possible
PH7-o PH7-/
10
PH7-5 PH7-6
PH6-o
Fig. 9. — Chart showing approximately the tints produced by adding sulphophenolphthalein to a series of phosphate solutions of the H-ion concentrations indicated in each case by PH.
HYDROGEN-ION CONCENTRATION
35
in the accompanying chart. The H-ion concentration of the unknown solution is that of the tint with which it matches in the series.
It might be thought that this method would be inaccurate because of the loss of carbon dioxide from the blood. By actual experiment, how- ever, it has been found that, if the blood is collected with certain pre- cautions, the error is negligible. The method is, therefore, a most useful one clinically.
The following table gives the hydrogen-ion concentration or true reaction of the body fluids.
FLUID
PH
FLUID
PH
Blood 7.4
Urine (5.0
Saliva 6.9
Gastric juice (adult) 0.9-1.6
Gastric juice (infant) 5.0
Pancreatic juice (dog) 8.3
Small intestinal contents 8.3 Small intestinal contents (infant) 3.1
Bile from liver 7.8
Bile from gall bladder 5.3-7.4
Perspiration 7.1
Perspiration 4.5
Tears 7.2
Muscle juice (fresh) 6.8
Muscle juice (autolyzed) Variable
Pancreas extract 5.6
Peritoneal fluid 7.4
Pericardial fluid 7.4
Aqueous humor 7.1
Vitreous humor 7.0
Cerebrospinal fluid (fresh) 7.4 Cerebrospinal fluid (after standing) 8.3
Amniotic fluid 7.1
Amniotic fluid 8.1
Milk (human) 7*0-7.2
Milk (cow) 6.6-6.8
Milk (goat) 6.6
Milk (ass) 7.6
(W. M. Clark and H. A. Lubs.)
CHAPTER VI
THE REGULATION OF NEUTRALITY IN THE ANIMAL BODY
AND ACIDOSIS
Nothing is more constant in the animal economy than the H-ion con- centration (CH) of the fluids which bathe the tissues. This regulation is fundamentally of a physicochemical nature, depending on the inter- action of alkalies with acids, of which carbonic and phosphoric acids are the most important.* When different amounts of acids or al- kalies are added to water, the range of variation in H ion is very extensive, whereas in blood the range is very limited indeed, not extending beyond PH7 and PH7.52 (i. e., CH never goes above that of a 0.000,000,1 N solution or below that of a 0.000,000,03 N solution). In • other words blood can withstand considerable additions of acid or al- kali without much change.
Buffer Substances. — The chemical reactions upon which this remark- able constancy in reaction depends have been explained by Lawrence J. Henderson.10 The fundamental equations are as follows:
M ,HPO4 + HA = MII2PO4 -f MA, and MHCCX + HA z= H2C03 + MA,
when M — a basic radicle, and A, an acid radicle.
Now it has been discovered that weak acids, like carbonic and phos- phoric, possess the remarkable property of maintaining the reaction constant when they are present in a solution which also contains an excess of their salts. Under these circumstances the concentration of ionized hydrogen is almost exactly equal to the product of the dissocia- tion constant! of the acid (see page 1.9) multiplied by the ratio be- tween free acid and salt; in other words,
If carbonic acid is present in a solution of bicarbonates so that there
*Under certain circumstances, proteins may also act either as acids or as alkalies. They are therefore called amphoteric. The neutralizing properties of proteins are, however, of little conse- quence in the neutrality regulation in the animal body (Bayliss20).
tThe dissociation constant has already been referred to as a figure which expresses the tendency of a weak acid or base to dissociate in an aqueous solution. "It expresses the proportion in which the nondissociated part is capable of existing in the presence of its ions," and therefore is a gauge of the strength. The dissociation constant amounts to about 0.000,000,5 for carbonic acid ; that is, the dissociation of HoCOs into H' + ITCO?/ at room temperature will be such that the concentra- tion of H-ion equals a 0.000,000,5 N solution.
36
ACIDOSIS 37
are equivalent quantities of free H2C03 and bicarbonate — i. e., : A1 =—
L-BAJ 1
—the H-ion concentration will be exactly the same as the dissociation constant of carbonic acid; therefore 0.000,000,5 N (PH = 6.31), or about five times the value of neutrality, 0.000,000,1 N (PH = 7.31). If ten times as much free carbonic acid as bicarbonate is present, then the H-ion
concentration will be fifty times that of neutrality, i. e., rp . • =^r-
L-bAj . i
x 0.000,000,5 = 0.000,005 (PH = 5.31) ; if there is ten times less carbonic acid than bicarbonate, the H-ion concentration will be one-half that of
neutrality, i. e., -lT"- TX 0.000,000,5 = 0.000,000,05) (PH = 7.31) • or
if twenty times less, one fourth (PH = 7.6). Since a large amount of bicarbonate is actually present in blood (enough to yield from 50 to 65 c.c. C02 per 100 c.c. of blood), and the free carbonic acid undergoes fluctua- tions which are only trivial when compared with those which have been chosen in the above examples, it is clear that there must be very little change in the H-ion concentration of the blood in comparison with the variations which would occur were no bicarbonate present.
Another weak acid which acts like carbonic in maintaining neutral- ity is acid phosphate (MH2P04), and for the same reason — namely, that its dissociation constant is of similar magnitude to the H-ion concen- tration. Although the blood plasma itself contains much less phosphate than bicarbonate, the tissues contain a considerable amount, which en- ables them to maintain their neutrality. This action of bicarbonates and * phosphates is styled the buffer action, meaning that it serves to damp \ down the effect on the H-ion concentration which additions of acids or alkalies would otherwise have. As pointed out by Bayliss, however, a better word to use would be "tampon action," since the substances actually soak up much of the added H- or OH' ions. It is not confined to the fluids of the higher animals, but is very widely distributed throughout nature ; for example, in the ocean and in the fluids of marine organisms and animalcules (see L. J. Henderson).11
Although the actual reaction by which neutrality is maintained is purely of a physicochemical nature, some provision must obviously be made so that the acid and basic substances that take part in it may be supplied and those produced by the reactions removed as occasion re- quires. The source of supply is partly exogenous and partly endogenous. The exogenous source is the basic and acid substances present in the food; and although we do not ordinarily attempt to control the amounts of these substances ingested, we may do so, as, for example, by the persistent administration of soda in cases of pathological acidosis. The endogenous source depends on the constant production in metabolism
38 PHYSICOCHEMICAL BASIS OP PHYSIOLOGICAL PROCESSES
of acids such as carbonic, phosphoric, lactic, and sulphuric, and of alkalies such as ammonia and fixed alkali, a considerable reserve of which is undoubtedly available in the animal organism.
The removal is affected by three pathways: (1) through the lungs gaseous carbonic acid is eliminated; (2) through the kidneys, the fixed acids; and (3) through the intestines, some of the phosphoric acid.
Carbonic acid is produced in large amounts in the normal process of metabolism, and is excreted in a gaseous condition by the lungs. Varia- tion in its excretion is the most important mechanism for controlling temporary changes in CH. In order to make this clear, it may be well to revert for a moment to the physicochemical equation by which carbonic acid is enabled to maintain neutrality. This may be written: CH =
TT QQ
molecular ratio - * T^e rat*° may ^e "lcrease(^ either by adding
free carbonic acid to the blood (as by causing an animal to respire some of the gas), or by the addition of some other acid (e. g., oxybutyric, as in diabetes) which will decompose some of the NaHC03 and produce H2C03. The increase which these changes would cause in CH of the blood is prevented by the remarkable sensitivity of the respiratory cen- ter to changes in CH. An increase which is much less than can be measured by physicochemical means stimulates the center, causing in- creased pulmonary ventilation, so that the carbonic acid is immediately eliminated through the lungs. This elimination does not stop when the
old level of carbonic-acid concentration is reached, but proceeds until TT c*r\
the original ratio TjptA is again attained in the blood, and CH is JNaJtluUo
restored exactly to its original value. If it stopped at the old C02 con- centration, the ratio would be too high because there is less NaHC03.
THE THEORY OF ACIDOSIS
Although these considerations indicate that variations may occur in the bicarbonate content of the blood without any significant change in CH, they also show that the bicarbonate content must be a criterion of the acid-base balance of the blood, and probably of the body fluids in general. As pointed out by Van Slyke,12 bicarbonate represents the ex- cess of base which is left over after all the fixed acids have been neu- tralized. It represents the base that is available for the neutralization of any excess of such acids that may appear — a measure of the reserve of <( buffer substance" or, more specifically, the alkaline reserve of the body. Under normal conditions the amount of NaHCO3 in blood plasma is very constant (amounting to 50-65 vols. per cent C02), and when it is reduced, it indicates that an excess of fixed acid must be present? This is taken
ACIDOSIS 39
by Van Slyke and others to constitute the real definition of acidosis — namely, "a condition in which the concentration of bicarbonate in the blood is reduced below the normal level." If the respiratory center for any reason should not respond promptly enough to an increase in
TT p/~)
the molecular ratio—,. Vrnrr > an(^ ^H consequently become greater, the condition is called uncompensated acidosis, but if the center does respond so that CH is held constant (although NaHC03 is decreased), the condition is one of compensated acidosis.
For practical reasons, therefore, the study of pathological acidosis de- pends on an estimation of the bicarbonate content of the blood or, since it is simpler to carry out and is of equal value, of the plasma. When plasma is obtained by removing blood from a vein of the arm and cen- trifuged immediately out of contact with air (so that C02 may not be lost from it) it contains approximately 60 vols. per cent of C02. Since we know that the partial pressure of C02 in blood is equal to 42 mm. Hg (ascertained from determinations of the alveolar C02) (see page 361), we can calculate how much of the 60 vols. per cent must be in simple solution by application of the law of solution of gas in a liquid (page 353'). One cubic centimeter of plasma at body temperature and at 760 mm. Hg (atmospheric pressure) dissolves 0.54 c.c. C02, so that at
42
42 mm. it will dissolve T^TTX 100 x 0.54 = 3 vols. per cent. Transcribing
[H2C03] 3 1
the figures to our equation we get -- = — , or — .*
[NaHC03] 60 20 This definition of acidosis leaves out of regard all conditions that may
TT r^ri raise the ratio -A- V tne addition of H2C03 without decomposing
any of the NaHC03, such, for example, as occurs when an excess of free carbonic acid is present in the blood plasma. Since increases in free C02 are not infrequent in both health and disease — e. g., asphyxial con- ditions — the above definition is not sufficiently comprehensive. When we come to study the control of the respiratory center, we shall see that
FT C*O
an increase in the ratio -A- °^ sufficient magnitude to cause an
actual increase in CH can be brought about by causing an animal to respire air containing an excess of CO2 — a true acidosis, but one for which no place is found in the above definition.
*This agrees sufficiently with the result as calculated from the known values of the equation
'XT 2u/~A~ = — ^~- • Thus, if we take CH as 0.35 x 10-7, \ as 0.605 for blood conditions, and NaHCUj K
r- 1n 7 ,,,. , .. HoCO3 0.605 x 0.35 x 10-7 1
K as 4.4 xlO-7 (Michaehs and Rona), we get = 4.4 x IQ-T -- = JT
40 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES
Nevertheless, Van Slyke's definition has a real value, because it em- phasizes the importance of a determination of the bicarbonate as a cri- terion of the degree of the forms of acidosis usually met with in disease. The bicarbonate under such conditions may become reduced either be- cause of the appearance of improperly oxidized fatty acids, like /?-oxy- butyric and acetoacetic, when carbohydrate metabolism is upset as in diabetes or starvation, or because the acids produced by a normal metabolism are inadequately eliminated by the kidneys, as in nephritis.
Accordingly, if the respiratory mechanism and increased mass move- ment of the blood (for an increase in CH accelerates this also) should
TT f^Ci
fail to eliminate C02 quickly enough so as to keep the -n-pr? ratio at one twentieth, then CH will rise. This is not likely to happen until a large part of the NaHC03 has been used up, so that an estimation of that actually present must be a reliable index of the proximity to this condition.
A sustained increase in CH is incompatible with life. The NaHC03 is the buffer, the factor of safety which prevents its occurrence. Although it is only in arterial blood (i. e., after elimination of excess of C02 by
TT r^r\ the lungs has been accomplished) that constancy in the ratio 2 *
.vv'o
can be expected, it is fortunate, for practical reasons, that venous blood
collected during muscular rest and without stasis should be only slightly different.
When acids are added to the blood, they will first of all be neutralized by the "buffers" of the plasma — namely, NaHC03 (and protein), as we have seen. But this is only the first line of defense against acidosis, for buffer substances present in the corpuscles may also be used. This intra- corpuscular reserve of base is supplied partly by transference of K and Na from corpuscle to plasma, but mainly by that of HC1 from the plasma into the corpuscle, so releasing base in the former to combine with the added acid (e. g., H2C03), according to the equation: H2C03 + NaCl *± NaHC03 i- HC1. The HC1 on entering the corpuscle reacts with phosphates according to the equation: HCl + Na2HP04^ NaH2P04 + NaCl. This is a particularly important, detail of the buffer action of the blood, not only because it shows us how the phosphates of the corpuscles (of the blood of species in which much phosphoric acid is present) are rendered available for neutralizing acids added to the plasma, where there are practically no phosphates, but also because the transference of acid must go on with the other cells of the body, so that the plasma, itself rather poor in buffer substances, has all those of the body at its disposal.
ACIDOSIS 41
THE MEASUREMENT OF THE RESERVE ALKALINITY OF THE
BODY FLUIDS
Titration Methods
There are several methods by which the reserve alkalinity of the blood may be measured. The simplest in theory consists in seeing how much standard acid must be added to a measured quantity of blood plasma in order to reach the neutral point as judged by change in tint of some indicator. The indicators employed (e. g., methyl orange) are such as change their tints at H-ion concentrations that are well to the acid side of neutrality (i. e., at a high CH or low PH). To bring the plasma to this point of neutrality the added acid will need to neutralize, not only the bicarbonate of the plasma, but other acid-binding substances as well. This will give us a false impression of the acid-binding powers Of the plasma, since, at the normal CH of the blood, proteins do not absorb acids to anything like the extent they do at higher degrees of CH. Another objection to the method is that the proteins interfere with the sensitive- ness of the indicators.
The objections can be removed by determining the end point electro- metrically or by indicators that change tint at about PH7. The most practical way is to determine the change in CH produced by adding a known volume of standard acid to blood plasma. The resulting change in CH will then be greater the less the alkaline reserve. In the electro- metric method irregularities that might be caused by variable amounts of carbonic acid in the blood to start with are best controlled by removing the C02 from the plasma after adding the standard acid. The procedure therefore consists in mixing 1 c.c. plasma with 2 c.c. N/50 HC1 in a small separating funnel, wrhich is then evacuated so as to remove the C02, after which the fluid is transferred to a hydrogen electrode and CH measured (see page 29). In normal blood this should be 10 5-6 (PH5.6). In acidosis, where there is a depleted alkaline reserve, the 2 c.c. of acid will cause a much greater change in CH — in diabetic blood to below 5 or lower.
The technic involved in the above method is, however, too exacting for routine clinical work. For such purposes the colorimetric method of Levy and Kowntree may be employed.
The Method of Levy and Rowntree.is — A test tube made of hard ("nonsoT') glass of about 20 c.c. capacity, containing about a gram of powdered neutral potassium oxa- late, is filled with newly drawn blood, immediately stoppered and placed on ice. Quan- tities of 2 c.c. each of the blood are then placed in a series of seven small (nonsol) test tubes and allowed to stand for five to six minutes in order to permit a narrow layer of plasma to separate on the surface (this prevents laking of the blood during the sub-
42 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES
sequent addition of acid or alkali). The blood in the first tube is used for the de- termination of the normal H-ion. In each of the next three tubes are added respec- tively 0.1, 0.2 and 0.3 c.c. N/50 HC1, and to the last three, similar quantities of N/50 NaOH. After inverting the tubes so as to mix the contents, the blood in each is trans- ferred to celloidin sacs and the CH determined according to the method described else- where (page 32).
The tubes are noted in which a change in tint from that of the normal blood is evident, and the results are expressed as the c.c. of N/50 HC1 or NaOH which must be added to blood to change its OH. Thus, the alkali buffer is the c.c, of N/50 NaOH which can be added to 2 c.c. of blood without change of CR of the dialysate, and the acid buffer the c.c. of N/50 HC1.
The method suffers from the following drawbacks:
1. Very small quantities of acid and alkali are employed.
2. It is often difficult to tell just exactly when a slight difference in tint has been produced.
3. Even with the precautions described above, it is impossible to be sure that the amount of OX) in the different samples of blood is the same, which means, of course, that some bloods will, on this account alone, be able to bind more alkali than others.
The Method of Van Slyke. — A method based on somewhat the same principle, but which is more accurate because it meets the above objections, is that suggested by Van Slyke, Stillman and Cullen.14 Plasma is freed of CO2 by placing it in a vacuum, and is then mixed with an equal volume of N/50 HC1 (or NaOH) and the CH deter- mined by the electric method (see page 29). In the case of normal blood, after such an addition of acid, a practically normal CH will be found, whereas in the blood of cases of acidosis it will be very distinctly increased (i.e., PH lower).
C02-combining Power
The above objections to the titration of blood plasma or dialysate with standard solutions of acids are removed if we measure the com- bining power of the blood alkali towards carbonic acid itself at normal blood reaction. This may be done either in blood immediately after its removal from the animal or in blood that has been first of all saturated outside the body with- carbonic acid at a partial pressure equal to that existing in the body. Since for practical reasons venous blood must be used — in the clinic at least — the former of these methods suffers from the fault that varying amounts of carbonic acid^will be added to the blood during its passage through the tissues, and the error thereby incurred will become greatly aggravated if venous stasis has been pro- duced in drawing the specimen for analysis. But the chief reason why this method has not been extensively employed, as pointed out by Van Slyke, is the technical difficulty of making the necessary analysis.
It is most satisfactory to collect venous blood after a period (one hour at least) of muscular rest (so that there is no excess of CO ) and without venous stasis, and to centrifuge without permitting any considerable loss of carbonic acid. The latter precaution is necessary because there is a migration of acid radicles, e. g., HC1, from plasma into corpuscles when the CO,, of the former is increased, and in the reverse
ACIDOSIS
43
direction when the CO is decreased. If the COo in the blood were not the same dur- ing centrifuging as it is in the body, the separate plasma would not contain the same amount of alkali — i. e., its reserve alkalinity would be altered. Although theoretically, therefore, centrifuging should be performed in an atmosphere containing the same partial pressure of COs as exists in the body (i. e., the alveolar air) (see page 361), this has been found impracticable for general use, and is unnecessary if loss of CO, from the specimen of blood is prevented by allowing it to flow into the syringe very slowly (without any suction). It is mixed in the syringe with powdered (neutral) potassium oxalate (enough to make a 1 per cent solution with the blood), and imme- diately delivered into a centrifuge tube under paraffin oil, which by floating on its surface serves. to diminish free diffusion of CO2 to> the 'outside air (even though such oils dissolve more CO than water). To mix the blood with the oxalate, the syringe should be moved backward and forward several times, but it must not be shaken.
After centrifuging, ab'out 3 c.c. of plasma are removed and saturated with CO at the same tension as in alveolar air (i. e., 5.5 per cent). This is done by placing the plasma in a separating funnel of 300 c.c. capacity, laying the funnel on its side and displacing the air in it by alveolar air secured by quickly making as deep an inspira-
Fig. 10. — Diagram of apparatus for saturating blood or plasma with expired air. The glass beads in the bottle condense excess of moisture. The separating funnel, as soon as it has been filled with expired air, should be closed by a stopper and the stopcock turned off. It is then rotated so that the blood forms a film on its walls.
tion as possible through the tube and bottle containing glass beads (Big. 10). The glass beads remove excess of water vapor from the air. The funnel must be restop- percd before the end of the expiration, so that no outside air enters. It is then ro- tated, for about two minutes, in such a way that the plasma forms a film on its walls. If it is necessary to postpone the saturating of the plasma, this should me pipetted off from the corpuscles and preserved in hard glass test tubes coated with paraffin. From ordinary glass enough alkali is soon dissolved out to vitiate the results. After satura- tion of the plasma with C02, the funnel is placed in the upright position and the plasma allowed to collect in the narrow portion, after which 1 c.c. is removed with an accurate pipette and analyzed for CO,.
The analysis may be done by using either the Van Slyke or the ITaldanc-Barcroft apparatus. The Van Slylcc method is as follows:
The apparatus is filled to the top of the graduated tube with mercury (Fig. 11) by raising the mercury reservoir F, care being taken that D and E are also filled. One c.c. of the CO -saturated plasma is then delivered into A (which has been rinsed out with CO -free ammonia water), and the stopcock I turned so that by cautiously lowering the level of the reservoir F, the plasma runs into E (but no trace of air).
44
PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES
The same procedure is repeated with 1 c.c. water, so as to wash in all of the plasma, and finally 0.5 c.c. of 5 per cent H SO is sucked in, after which stopcock I is turned off. The reservoir F is then lowered sufficiently to allow all of the mercury, but none of the blood, to run out of B and C. A vacuum is thus produced in B and C.
As the level of the mercury falls in B and C, the plasma effervesces violently,* be- cause it is exposed to a vacuum. To be certain that all traces of CO have been dislodged from the solution, the apparatus is inverted several times. To ascertain how much CO has been liberated, stopcock II is now turned so as to bring C and E into communication, and by cautiously lowering the reservoir the fluid in C is allowed to rim into the bulb E. Stopcock II is thereafter turned so as to connect C and D, and the reservoir raised so that the mercury runs into C as far as the CO2 that has col-
Fig. 11. — Van Slyke's apparatus for measuring the CO2-combining power of blood in blood plasma. For description, see context.
lected in the burette will permit it to go. After bringing the level of the mercury in F to correspond to that in the burette, the graduation at which this stands is read. It gives the c.c. of CO liberated from the plasma. Under the above conditions normal plasma binds about 75 per cent of its volume of COo; therefore, since the total capacity of the pipette is 50 c.c., the mercury should stand at 0.375 e.c. on the burette. For accurate measurement it is necessary to allow for the CO9 that remains dissolved in the water, etc., as well as for barometric pressure and temperature. This is best done by the use of a table based on the known solubility of CO2 under the various condi- tions obtaining, which is given in Van Slyke 's paper.12
The Haldane-Barcroft apparatus that is most suitable for the above analysis is
'This may be prevented by adding a small drop of caprylic alcohol.
ACIDOSIS 45
shown in Fig. 136, page 395." One c.c. of CCX-free ammonia water is placed in the bottle and the 1 c.c. of plasma delivered beneath it. The bottle is then connected with the manometer with the precautions described elsewhere in this volume. When temperature conditions have been allowed for, saturated tartaric acid is mixed with the plasma solution and the gas evolved measured by the displacement of the fluid in the manometer. The apparatus may also be used with blood in place of plasma. In this case, however, it is necessary that the oxygen be removed before adding the tartaric acid. This precaution is necessary, since acid can dislodge some of the H, from hemoglobin. The blood is therefore first of all laked with ammonia containing some saponin, then shaken with 0.25 c.c. saturated potassium ferricyanide solution, and finally with the saturated acid solution. If blood is used, the estimations must be made on strictly fresh blood, since on standing the CO2 combining power greatly de- teriorates.
From what has been said in the introductory part of this chapter it is clear that the plasma furnishes only the first line of defense of the body against excess of acid; the corpuscles form the second line of defense, so that a truer estimate of the * ' reserve alkalinity ' ' is afforded when the C02- combining power of whole blood rather than that of plasma is used. The reason why Van Slyke recommends the latter is because the estimations are much easier, there being no hemoglobin to complicate the process, and there can be no doubt that his method has been of immense value in the elucidation of the acidosis problem, and that for the majority of diagnos- tic purposes it is perfectly satisfactory. For further advancement of knowledge it is advisable, however, to use the whole blood as has been done by Christiansen, Haldane and Douglas,22 and by Morawitz and Walker,23 and more recently by Haggard and Henderson21.
Another question remains to be considered, namely, whether arterial or venous blood should l>e employed. For various reasons arterial blood is preferable. In the first place the percentage of C02 actually present in it is proportionate to the alkaline reserve, because the respiratory center is so sensitive to the slightest excess of this acid (see page 353) that it stimu- lates respiration so as to remove the excess and thus maintain the C02 of the arterial blood at exactly the point which corresponds to its alkaline reserve. It is, therefore, unnecessary to expose the blood to an atmosphere
*This form of Haldane-Barcroft apparatus is not quite the same as the differential manometer that is used for measurement of the Oa-combining power of hemoglobin (page 395). In the form used for the present purpose, a side tube at the bend of the U-tube is connected with a small rub- ber bag, which can be compressed by a screw. When the gas is evolved in the bottle, it presses down the fluid in the proximal limb of the manometer correspondingly and raises that in the distal limb. Since the calculation of the amount of gas evolved depends on finding the pressure produced without any change in volume, it is necessary after the gas has been evolved to compress the rubber bag until the meniscus of fluid in the proximal limb of the manometer is brought back to its original level. The height at which the fluid stands in the distal limb then obviously corresponds to the pressure created by the evolved gas.
The equation for determining the amount of gas evolved depends on the gas law, which states that the pressure of a gas is inversely proportional to its volume (page 353). Suppose that the volume of gas evolved was equal to the volume of the bottle, then, since the volume has been kept constant, the pressure would be doubled — that is, the fluid in the distal limb would equal that of 1 atmosphere, or 10,400 mm. of water or 10,000 of clove oil, which is the fluid actually used to fill the manometer. Any other observed pressure would therefore correspond to the volume of evolved gas according to the equation,
... vol. of bottle (and tubing to meniscus)
V = nnn :- — ^ — X mm. Pressure m Manometer.
10,000 (when clove oil is used)
In using the apparatus in the above manner, only one of the bottles is employed, and the tartaric acid is added from a pocket in the stopper by a simple manipulation.
46 PHYSICOCHIvMICAL BASTS OF PHYSIOLOGICAL PROCESSES
containing C02 before measuring the C02 content. In the second place, the arterial blood represents the mixed blood of the body, and not that of one locality only, as is the case with blood removed from a peripheral vein. If venous blood is collected with the precaution that the muscles in the corresponding area have been at rest for some time it appears that there is practically no difference between the alkaline reserve of arterial and ve- nous blood; but if there has been any muscular contraction, the venous blood will have a lower reserve than the arterial, because of the lactic acid thrown into it by the active muscles. But even when we take the precau- tion of avoiding muscular action it is probable that there is not a strict parallelism between the buffer action of arterial and venous blood, as in cases in which the demands on the alkaline reserves are such that those of the tissues are being called on as well as those of the blood itself.
Even when the whole Wood is used, however, we do not necessarily measure the total reserve of the body, a final reserve being afforded by the alkalies and possibly .certain of the proteins of the tissue cells. Now it is clear that there can be no test tube method by which measurement of the magnitude of all of these defensive agencies is possible ; and we are there- fore compelled to supplement them by certain indirect methods.
Indirect Methods
The chief criticism against the use of the C02 carrying power of blood or blood plasma, is therefore, that it tells little if anything concerning the acid-absorbing powers of the tissues. Is there not, therefore, some test of the acid buffer which can be applied to the intact animal 1 One such is the percentage of C02 in alveolar air (see page 361).
1. Determination of the Tension of C02 in Alveolar Air. — Since this method is employed more particularly in investigating the hormone con- trol of the respiratory center, we shall defer a description of it until later (page 361). For the present, however, it should be remarked that the alve- olar C02 can be a precise gauge of the acid-base equilibrium only provided that the respiratory center is perfectly normal, and that there is no in- terference with the diffusion of C02 from the blood into the alveolar air. In order to place an estimate on the "relative value of this method com- parisons have been made between the C02 tension of the alveolar air and the C02 absorbing power of the blood. This has been done both in nor- mal and pathological subjects. In normal subjects the comparisons have been made under conditions, such as the taking of food and during mus- cular exercise, in which slight alterations in the acid-base equilibrium are known to occur. Van Slyke, Stillman and Cullen9b found that the ratio
— plasma C°2 varies from 1.27 to 1.80 in different resting individu-
mm. alveolar C02
als, there being apparently a characteristic ratio for each individual, and
ACIDOSIS 47
that the taking of food invariably raises the alveolar C02-combining power. This would seem to indicate that it must be the excitability of the respi- ratory center rather than the acid-base equilibrium that becomes altered so as to cause variations in alveolar C02.
Technical difficulties have also to be overcome in the collecting of the alveolar air, for it is now well established that the original method of Hal- dane and Priestley is approximately accurate only when it is carried out under strictly controlled conditions — so strict that they can not be prac- tised in the clinic — and even then, as R. G. Pearce, Carter, Krogh, Sie- beck and others have shown, we can not be certain of the results. At best, therefore, the alveolar C02 can serve as an accurate index of the acid-base equilibrium of the blood only under strictly controlled conditions.
2. The Measurement of the Acid Excretion by the Kidney. — As might be expected, the acid-base equilibrium of the body may also be gauged by measurement of the acid excretion of the urine, in which the acids are contained partly in combination with ammonia or a fixed base, and partly in a free state. We shall first of all consider the methods of acid excretion and then examine the evidence showing that the total acid excretion is proportional to the alkaline reserve as measured by the above described methods.
EXCRETION OF ACID IN COMBINATION WITH AMMONIA. — The production of ammonia is essentially an endogenous process, and when excessive quantities of acid make their appearance in the organism, the fixed alkali may not be sufficient to neutralize it all, so that ammonia, derived from the breakdown of amino acids (page 650), instead of being converted into urea is employed to neutralize the excess of acid. Most workers have in this way explained the very large ammonia excretion that has long been known to occur in such conditions as diabetic acidosis. Some recent workers are, however, inclined to question the significance of ammonia in this connection, believing that the increased ammonia ex- cretion is, like the acetone bodies themselves, a product of perverted metabolism. Be this as it may, it is no doubt true that ammonia is used for neutralizing acid in disease, although it may not be an important factor in the maintenance of neutrality under normal conditions. It is a factor of safety, in that it helps to care for an increase in acid when the normal mechanism of the body is overtaxed.
EXCRETION OF PHOSPHATES. — The more permanent control of neutrality depends on the excretion of phosphates by the kidney. The principle governing this process is exactly the same as that already discussed in connection with carbonic acid. In the one case it is the volatile acid C02, and in the other, the fixed phosphoric acid that is concerned in the reaction. The ratio between the acid salts of phosphoric acid, MH2P04,
48 PHYSICOCHIvMICAL BASIS OF PHYSIOLOGICAL PROCESSES
and the alkaline salts, M2HP04, in blood is approximately 1 to 5, but in the urine this ratio varies according to the amount of H ion that must be eliminated from the blood. In other words, a definite amount of phos- phoric acid is enabled to carry variable amounts of H ion out of the body by causing the amount of alkali excreted in combination with it to be- come altered. For example, in the form of MH2P04 a given amount of P04 obviously carries out more H ion than wrhen it is excreted as M2HP04. The adjustment between these two salts is a function of the kidney. We may accordingly measure the amount of alkali retained by the organism by finding how much standardized alkali must be added to a given quantity of urine until the reaction of the blood is obtained. Since the latter value is constant, the titration can be done simply by titrating the urine with an indicator such as sulphonephenolphthalein, which changes tint at about PH of blood.
A more serviceable indicator to use, however, is phenolphtkalein, be- cause its end point is such that when human urine just reacts neutral to it — that is, when the titrable acid approaches zero — the C02-absorb- ing power of the plasma is at its maximum of 80 vols. per cent and the ammonia excretion by the urine is zero (Van Slyke). It is advantageous, therefore, to use this indicator, because it happens to have its turning point situated for a reaction which is well to the alkaline side of neu- trality, and which is reached in urine when the blood is at its maximal acid-combining power and no ammonia is being used for neutralization purposes. As the C02-combining power of the blood decreases, there should, therefore, be a proportionate increase in ammonia and in the titrable acidity of the urine.
3. Determination of Alkali Retention. — Another valuable criterion of the alkaline reserve is the amount of alkali required to change the re- action of the urine. In health the CH of the urine varies from 0.000,016 N (PH = 4.8) to about 0.000,000,035 N (PH = 7.46) with a mean of about 0.000,001 N (PH = 6). These extremes are rarely overstepped in disease, but frequently the average is considerably different. In car- dio-renal disease, for example, the mean acidity may be approximately 0.000,005 N (PH = 5.3), or five times the normal value. A certain de- gree of acidosis is therefore common enough in this condition — a fact which has indicated the advisability of administering sodium bicarbon- ate. It has been found that 5 grams or less of soda, given by mouth to a normal person, causes a distinct diminution in the CH of the urine, whereas in pathologic cases it may be necessary to give more than 100 grams before a similar effect is observed (L. J. Henderson and Palmer15 and Sellards16).
This test has been found of particular value in the diagnosis of acidosis
ACIDOSIS 49
accompanying certain forms of renal disease (chronic interstitial nephri- tis), which raises the question as to whether the retention may not be due to faulty elimination of the bicarbonate rather than to its retention in order that a deficient alkaline reserve may be corrected. It has not been a very simple matter to entirely disprove this possible explanation, and ex- periments of a variety of types have had to be devised in connection with the problem. One of them consists in determining the effect of a second dose of bicarbonate administered to an acidosis patient to whom a suffi- cient amount had previously been given to render the urine just alkaline. It has been found that a few grams now suffice, indicating, apparently, that the alkaline reserve must have been restored to its normal level. Even to this experiment the objection can be raised, however, that the large doses 'were retained because the threshold of the kidney for the ex- cretion of bicarbonate was a very high one, and that the second, smaller administration just sufficed to overstep this threshold.
Sellards' careful work with this method seems quite clearly to establish its value, however, and for practical purposes it is probably the most prac- ticable test of acidosis at present available in routine clinical work. It has the important advantage, furthermore, of being simple and of requir- ing no elaborate apparatus.
It may be advantageous in this place to classify the possible causes which might lead to a want of stability in the CH of the blood; that is, to threatened acidosis or alkalosis, not of acidosis in the narrow sense im- plied in Van Slyke's definition, but in the broader sense of any disturb- ance in the acid-base equilibrium.
In general, a tendency to acidosis might be due to an increase in the numerator or decrease in the denominator of the molecular equation TT .r<(\ -i
= — , or to a proportionate decrease in both. In the latter
XT T-T
J\aH(JO3 20
case, there would be no actual change in CH, but the alkaline buffer would be depleted so that the change would very readily set in when foreign acids were added. Furthermore, it should be understood that NaHC03 only stands as a symbol for all substances that might serve as alkaline re- serves, for although this salt is no doubt the most important of these, the alkaline phosphates, of the corpuscles, and the protein of the blood and tissues must also be considered. A tendency to alkalosis — which is no doubt extremely rare as a pathologic condition — would be due to changes of a reverse character. A theoretic classification of the conditions which might cause these changes is given:
50
PHYSICOCHEMTCAL BASIS OP PHYSIOLOGICAL PROCESSES
Addition or accumula- tion of acid
Decrease of base
Addition or accumula- tion of base
Removal of acids
Increase of CH.
Accumulation of COo (asphyxial conditions).
Incomplete oxidation of carbohydrate (lactic and in mus- cular exercise).
Defective oxidation of fat (ketosis).
Eenal insufficiency (nephritis).
Decomposition of protein (as in acidosis of fever).
Intestinal fermentation.
Administration of acid (experimental).
Diarrhea and hemorrhage, respectively (may explain acido- sis in cholera and in certain forms of shock).
Decrease in €„.
xi
Ammonia (faulty metabolism of urea).
Intestinal putrefaction (infantile conditions).
Administration of alkalies (experimental).
Excretion of CO (excessive pulmonary ventilation, as in
faulty ether administration). Excretion of acid urine. '
CHAPTER VII
COLLOIDS
Substances which can be obtained in the crystalline state and which, when in solution, are capable of readily diffusing through membranes, are designated as crystalloids, and are to be distinguished from another, larger group of substances not having these characteristics or having them only in very minor degree — the colloids. In every field of chem- istry the properties of colloids have been studied extensively during recent years, but in no field more than in that which covers the chem- istry of biological fluids and tissues, into whose composition colloids enter much more extensively than crystalloids. The subject of colloidal chemistry has indeed become so extensive that an attempt to do more than indicate some of the most important characteristics of colloids would take us far beyond the limitations of this book. The far-reaching applications of the subject in physiology and medicine are only begin- ning to be realized.
The term "colloid," or "colloidal," does not refer to a class of chemical substances, but rather to a state of matter which is quite independent of the chemical composition of the substance. We are familiar with more colloids in the organic than in the inorganic world, yet they are plentiful in both, and the same substance may at one time be colloidal and at another noncolloidal. Indeed, under appropriate conditions prob- ably all substances may assume the colloidal state — not solids and liq- uids alone, but gases as well. It is mainly with liquids, however, that we are concerned in biochemistry.
CHARACTERISTIC PROPERTIES
The distinction between molecular* and colloidal solutions is a rela- tive one. Suppose, for example, that we take a piece of gold in water and divide it up into smaller and smaller parts. At a certain stage, the particles will be so fine that they will remain in suspension and be in- visible by ordinary means. They are then said to be in the colloidal state. If we divide them further until they become molecules of gold, a molecular solution will be obtained. In the colloidal state, there are
*Molecular solutions include those of nonelectrolytes, such as sugar, and electrolytes, such as inorganic salts.
51
52 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES
two distinct phases in the solution, one solid and the other liquid, and between the two, because of the great subdivision of the original par- ticle, is an enormous surface of contact. The solution is heterogeneous, and at the interface between the two " phases" the physical forces which depend on surface — e. g., surface tension (see page 65) — are enormously developed, and are responsible for the peculiar properties of colloidal solutions as compared with those of molecular solutions, which may, therefore, be styled homogeneous. The solutions of crystalline substances which we have hitherto been concerned with, are homogeneous.
Between these two groups of solutions is an intermediate one — namely, suspensions (as suspensions of quartz or carbon, or oil emulsions). Be- sides being turbid in transmitted light, the solutions may be seen by means of the ultramicroscope to contain particles. These can be sepa- rated by filtration from the fluid they are suspended in, except in the case of many emulsions in which the particles can squeeze their way through the filter pores by changing their shape.' On standing or being centrifuged suspensions may also separate into their constituents, al- though this can be greatly hindered by the addition of a suspending' substance such as gelatin or certain bodies having a so-called protec- tive action (as peptone, proteose, etc.).
True Colloidal Solutions
1. The Solution Is More or Less Turbid. — Frequently this can be recog- nized by holding the solution in a thin-walled glass vessel against a dark background, but the turbidity may be so slight that it requires for its detection the use of the Tyndall phenomenon. This is familiar to all in the effect of a beam of sunlight let in through a small aperture into an otherwise darkened room. In the course of the beam suspended dust particles, which are invisible in an equally illuminated room, be- come visible, and thus render very 'distinct the pathway of the beam. If a colloidal solution contained in a glass vessel, preferably with paral- lel sides, is held in the course of such a beam, the Tyndall phenomenon will be seen in the liquid, which is not the case with molecular solutions. Focused artificial light may be employed for intensifying the effect. The light that is sent out at right angles to the beam is plane-polarized, which means that the particles reflecting the light must be smaller than the mean wave length of the light forming the beam. It should be under- stood that the individual particles themselves may not be rendered visible to the naked eye by the beam, although in such cases they can often be seen by using intense illumination and a dark-field (ultramicro- scope) combined with suitable magnification (Fig. 12).
2. Colloids Do Not Readily Diffuse. — To demonstrate this, test tubes
COLLOIDS 53
are half filled with a 5 per cent solution of pure gelatin or a 1 per cent solution of pure agar, and, after the jelly is set, the solution under examination is poured on the surface; or, when it is of high spe- cific gravity, the tube of gelatin, etc., is placed mouth downwards in the solution. In the case of colloidal solutions very little if any diffu- sion into the gelatin or agar will occur, even after several days; whereas true molecular solutions will diffuse for a considerable distance. "When colored solutions are used, the diffusion can readily be recognized by inspection (see Pig. 13), but when they are colorless, the presence or absence of diffusion must be determined by removing the column of gelatin or agar and dividing it into slices of equal size, which are then examined chemically for the substance in question.
A further test is afforded by the failure of colloids to diffuse through membranes (dialysis). This was the method originally used by Thomas Graham to distinguish between molecular and colloidal solutions. The solution under examination is placed in a dialyzer, which is then im- mersed in a wide vessel containing the pure solvent. The older forms
Fig. 12. — Ultramicroscope (slit type) for the examination of colloidal solutions. The arrange- ment of diaphragms, etc., in this form removes the absorptive effects of the surfaces of the glass vessel or slide used to contain the colloidal solutions.
of dialyzer consisted in general of a bell-shaped glass vessel closed be- low with parchment paper, but more recently so-called diffusion sacs have been adopted. These consist of pig or fish bladders or of col- lodion sacs. The latter are made by placing some collodion dissolved in ether in a test tube, which is then tilted so that the collodion runs out except for a thin layer which remains adherent to the walls. When the collodion has set, the sac can be removed after loosening it by allow- ing a little water to flow between the sac and the walls of the test tube. The sac containing the colloidal solution is then suspended in water or some of the solvent used in preparing the colloidal solution, care being taken that the menisci of the fluids inside and outside of the sac stand at the same level. Sometimes, especially when collodion sacs are used, some colloid may at first diffuse through, but if the outer fluid (the dialysate) is renewed and the dialysis allowed to proceed, this ceases.
54
PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES
When a fluid solution exhibits both of the above properties (i. e., the Tyndall phenomenon and indiffusibility), there can be no doubt as to its being in a true colloidal state, but there are substances, such as congo red, or protein solutions of certain strengths, which may exhibit a very slight divisibility in a dialyzer but not show the Tyndall phenomenon. Substances of this group constitute transitional types between molecular and colloidal solutions, and to determine their true nature it is neces-
Fig. 13. — To show diffusion into gelatin of a crystalloid stain in b and the nondiffusion of a colloid stain in o. (From W. Ostwald.)
sary to employ refined methods sucht as those of ultramicroscopy, ultra- filtration, etc., which can not be described here.
3. The Size of Colloidal Particles. — It will be apparent that the essential property upon which the above-mentioned phenomena depend is the size of the particle. Particles which can still be seen under the microscope are called microns. They have been computed to have a dimension of 0.1 p (0.001 mm.) or more, and they form suspensions. Particles which are invisible microscopically under the ordinary conditions of illumina-
COLLOIDS
55
tion, but are still visible when the ultramicroscopic illumination is used, are called submicrons. They have a dimension between 0.1 /A and 1 w (0.000,001 mm.),* and they constitute the colloids. Particles smaller than 1 fj.fji are called amicrons, this term being used to include the mol- ecules and ions present in molecular solutions. (The amicron of hydro- gen is, for example, computed to be 0.067 to 0.159 /X/A, and that of water vapor, 0.113 /*/*.) This classification of dissolved substances according to the size of the particles and molecules shows the relationship of one
Fig. 14. — Diagram from W. Ostwald showing the relative size of various particles and colloidal dispersoids compared with a red blood corpuscle and an anthrax bacillus.
class of substances to others. An idea of the relative sizes of colloidal particles and molecules in comparison with such familiar objects as a blood corpuscle and an anthrax bacillus is given in Fig. 14. The fluid in which the "particle" is suspended is called the dispersion medium, or external phase, and the particle itself the dispersoid, or internal phase. It is the enormous development of surface which determines the dif-
*/t rz 0.001 mm., and fifi = 0.000,001 mm.
56 PHYSICOCHEMICAL .BASIS OF PHYSIOLOGICAL PROCESSES
ference in the properties of a colloidal solution from those of a suspen- sion of the same substance. Thus, the difference between a colloidal solution of platinum (prepared by allowing an electric arc to form be- tween platinum electrodes in water) and pieces of platinum in water depends on the fact that the surface of the platinum in the former case has been increased many million times. When the subdivision becomes still greater and the particles gain the size of molecules, the phenomena due to surface development become suppressed and those due to con- centration in unit volume become accentuated. The properties depend- ent on osmotic pressure, diffusibility, etc., are exhibited by all dispersoids, whether ions, molecules or particles, but some of these properties are much more pronounced when the dispersoids are of large dimensions, and others when they are small. In other words, the phenomena due to surface, such as those of surface tension (see page 65), become apparent only when the dispersoids have the properties of matter in mass; when the dispersoids become molecular in size, they manifest the properties characteristic of true solutions.
4. Electrical Properties of Colloids. — Most colloids carry a charge, which may be either positive or negative tOAvard the dispersion medium. Both crystalloids and colloids therefore carry electric charges; in the former case, however, the charge does not reveal itself until the molecules in solution have become dissociated, when each ion carries a charge of opposite sign (see page 16), whereas in the case of colloids, each col- loid particle usually carries a charge which is always of one sign, either positive or negative. Colloids may therefore be grouped into positive and negative, according to the charges which they carry, and there is a third group in which the charge may be either positive or negative ac- cording to the nature of the dispersion medium.
A colloid not carrying a charge to begin with can be caused to assume one by the action of electrolytes, for the electrical properties of colloids, as well as those of inert powders suspended in water, are readily in- fluenced by the charges present in the ions of the dispersion medium. The H- and OH' ions are especially liable to exert this influence. The particles of inert powders in suspensions (kaolin, sulphur, etc.) carry a positive charge when the water in which they are suspended is acidi- fied, and a negative charge when it is made alkaline. In general, it may be said that suspensions of most powders and of insoluble organic acids in water (e. g., charcoal, cellulose, kaolin, caseinogen, mastic, free acid of congo red, etc.) are electro-negative. Of true colloids ferric hydrox- ide (ferrum dialysatum) and serum globulin are positive in acid solu- tions; arsenious sulphide and serum globulin are negative in alkaline solution, and serum globulin in neutral solutions has no charge.
COLLOIDS
57
To ascertain the nature of the charge various methods may be em- ployed, of which the following are important:
1. The method of electrophoresis. The colloid solution is placed in a U-tube, each side of which carries a platinum electrode dipping into the solution. After a strong continuous electric current has been allowed to pass for some time through the solution, it will be found that the colloid collects at the anode (where the current enters) when it is a negative colloid (since unlike electric charges attract each other), and at the cathode when it is positive. In the case of colored solutions, the migration can be readily seen, but otherwise it may be necessary to ana- lyze the solution at the two poles.
Fig. 15. — Capillary analysis of colloids. Strips of filter paper, after being suspended with the lower ends dipping into colloidal solutions. Those on the right hand were positive colloids, which did not rise in the strips, but formed a sharp line of demarcation at the lower end on account of precipitation. Those on the left hand were negative colloids. (From W. Ostwald.)
2. The method of capillary analysis. For this purpose a long strip of filter paper is arranged vertically over the solution, with its lower end dipping into it. In the case of negative colloids the colloid, as well as the dispersion medium, rises uniformly on the strip of paper (it may be to a height of 20 cm.) ; whereas with positive colloids the dispersion medium alone rises, the colloid itself doing so only to a very slight ex- tent, but becoming so highly concentrated at the interface between the solution and the paper that it coagulates on the end of the strip of paper, where it forms a sharp line of demarcation (Fig. 15).
3. The method of mutual precipitation of colloids. When a positive
58 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES
and a negative colloid are mixed in such proportions that the electric charges are neutralized, precipitation usually occurs. When it does so, we can tell the nature of the electric charge of an unknown colloid by its behavior when a colloid of known electric sign is added to it. For example, if ferric hydroxide (positive) causes a precipitate to form when it is added to an unknown colloidal solution, the electric charge of the latter must be negative; if it does not precipitate with ferric hydroxide, but does so with arsenious sulphide (negative), it must be positive.
5. Brownian Movement, — Like the particles in fine mechanical suspen- sions, those of colloidal solutions, especially when examined ultra- microscopically, exhibit the so-called Brownian movements, which have been described as "dancing, hopping and skipping." These movements occur in straight lines, which are suddenly changed in direction and are quite independent of external sources of energy, such as change in temperature (although they become quicker as the temperature of the solution is raised), earth vibrations, chemical changes, or the electric charge of the colloid. The movements become more rapid the smaller the particles, and they become sluggish as the viscosity of the solution in- creases. Addition of electrolytes decreases the movement by causing the particles to clump together. The density and viscosity of the disper- sion medium, the electric charge of the dispersoid and the presence of Brownian movements, are the forces which operate together to prevent sedimentation of the particles in a colloidal solution.
6. Osmotic Pressure. — As one of the distinguishing properties of col- loids we have seen that their diffusibility, as into gelatin or agar jel- lies, is extremely slow when compared with that of a molecular solution. This does not mean, however, that colloids are possessed of no power of diffusibility if left long enough. Indeed the existence of the Brownian movement indicates that such diffusion must occur, and therefore it should be possible, by the application of the same principles as those which govern molecular solutions (e. g., by using a semipermeable mem- brane), to measure the osmotic pressure.
Many studies of the osmotic properties of colloidal solutions have been undertaken, especially by those who are interested in the possibility that the colloids of blood serum (serum albumin and globulin) may cre- ate an osmotic pressure. If this should prove to be the case, it would be necessary for the osmotic pressure to be overcome by mechanical pressure such as that supplied by the heart (i. e., the blood pressure) in the various physiological processes of filtration and diffusion taking place through cell membranes (as in the formation of urine in the kidney).
For measuring the osmotic pressure of colloids, osmometers similar
COLLOIDS 59
to those already described (page 4) can be employed. Most of the recent work has been done either with collodion sacs, or with unglazed clay cups impregnated with some gel, such as silica or gelatin. When such an osmometer, filled with some colloidal solution (like a solution of pure albumin) and provided with a vertical glass tube, is placed in an outer vessel containing water, the fluid will be seen to rise in the ver- tical tube, the height to which it rises being proportional to the osmotic pressure.
But the observed pressure does not necessarily give us the osmotic pressure of the pure colloid, for to this, even when highly purified, there is almost certain to be attached a considerable amount of inorganic salt, which may be responsible for the osmosis. It has indeed been maintained by some observers that electrolytes form an integral part of certain colloids, being bound to them perhaps by adsorption (sec page 66), and that they are essential to the maintenance of the colloidal state. In any case, since electrolytes are always present, the osmotic pressure of the pure colloid can be measured only when means are taken to discount their influence. Sev- eral devices have been used, of which the following may be mentioned:
1. Addition to the fluid outside the osmometer of a percentage of salt equal to that found by chemical analysis to be present in the colloid. (This method is untrust- worthy.)
2. The use of a limited quantity of fluid on the outside of the osmometer so that equality of saline content soon becomes established, by diffusion, in the fluids on the two sides of the membrane.
3. The use of a membrane which is permeable to electrolytes but not to colloids. Even when the greatest care is taken in its measurement, the osmotic pressure of
a given colloid has been found to vary considerably not only according to the method used in its preparation, but also according to the amount of mechanical agitation (shaking, stirring, etc.) to which the colloid solution has been subjected. Regarding the influence of the method of preparation, it was found in one series of experiments that albumin that had been repeatedly washed (but still contained considerable ash) gave no osmotic pressure, whereas another preparation that had been purified by crystal- lization twice (and contained much less ash) had a pressure of 3.38 mm. Hg. Ac- cording to these results the ash content of the colloid is not fundamentally responsible for its osmotic pressure. As to the influence of mechanical agitation, the osmotic pres- sure of a gelatin solution is increased by shaking, while that of a solution of egg albu- min is decreased.
The property upon which the osmotic pressure depends is undoubtedly the state of dispersion of the colloid particles, and until we know all of the factors which may in- fluence this, measurements of osmotic pressures of colloids can scarcely be of very much value. Nevertheless, that this property has some physiologic bearing is clear from the effect which colloids have in restoring the blood pressure after hemorrhage (page 141).
Further evidence that the osmotic pressure of colloids has not the significance that it has in the case of molecular solutions is furnished by the fact that the osmotic pres- sure is only approximately proportional to the concentration of the solution ; it may either increase or decrease relatively to the strength of the solution. Temperature also has quite a different influence on the osmotic pressure of colloids from that which it has on the osmotic pressure of molecular solutions, and it frequently has an influence which persists after the solution is brought back to its original level.
60 PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES
The influence of added substances on the osmotic pressure of colloidal solutions is of considerable interest to the biologist, for, whereas in the case of molecular solu- tions this is purely additive, in the case of colloids the added substance may at one time cause the osmotic pressure to increase, at another, to decrease. It has been found that the osmotic pressure of gelatin solutions at first decreases, then rapidly increases as the H-ion concentration is raised. The addition of alkali increases the osmotic pressure until a maximum is reached, beyond which it begins to fall. Both acids and alkalies lessen the osmotic pressure of egg albumin. Electrolytes always decrease the osmotic pressure of gelatin and albumin solutions, and the degree to which they exert this influence depends on the nature of the cation and anion composing the electrolyte. In the order of their depressing influence the cations arrange themselves:
Heavy metals > alkaline earths > alkalies; and the anions:
S04 > 01 > NOo > Br > I > CNS.
The influence of a given electrolyte varies extraordinarily with the reaction of the colloid, a fact which must be carefully regarded in all work in this field.
CHAPTER VIII COLLOIDS (Cont'd)
SUSPENSOIDS AND EMULSOIDS
According to whether colloids form solutions that are more or less viscid than the suspension medium, they are divided into emulsoids and suspensoids. Examples of the former class are silicates and gelatin, and of the latter, dialyzed iron and arsenious sulphide. The following char- acteristics are used to distinguish between suspensoids and emulsoids:
1. Measurement of the time it takes, at a standard temperature, for a given volume of the fluid to flow out of a standard pipette (10 c.c.) shows the viscosity to be, roughly, inversely proportional to the time of outflow. In the case of suspensoids the viscosity is no different from that of the dispersion medium alone, and does not vary much when the solution is cooled. The viscosity of emulsoids even in very dilute solutions is, on the other hand, considerably greater than that of the dispersion medium itself, and it becomes greatly increased by cooling.
2. Suspensoids are much more readily coagulated by the addition of electrolytes than emulsoids. This is particularly true when water is the dispersion medium (so-called hydrosols), and when electrolytes hav- ing a polyvalent ion (such as Al or Mg.) are employed. Thus, practically all suspensoids are coagulated in the presence of 1 per cent of alum, which has no influence on emulsoids. We shall return to this phase of our subject later on.
The division of colloids into emulsoids and suspensoids is more or less arbitrary, since one class may be changed into the other, the determining factor being the water content of the dispersoid. The water content of suspensoids is low (lyophobe), while that of emulsoids is high. By changing the relative amounts of water and solid of which a colloidal solution is composed, the nature of the dispersoid may be changed. If the water is diminished, the dispersoid behaves as a suspensoid and be- comes readily precipitated. The practical importance of this fact is that it explains the salting out of proteins — a process extensively used in their separation. Ordinarily these behave as emulsoids, but the addi- tion of salt raises the osmotic pressure of the dispersion medium, and thus attracts water from the dispersoids, with the result that they come
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PHYSICOCHEMICAL BASIS OF PHYSIOLOGICAL PROCESSES
to behave as suspensoids, and are accordingly precipitated by the elec- trolytes.
Another property of emulsoids of biological importance is the pro- tection which they can afford against the precipitating influence of electrolytes on suspensoids. If a colloidal solution of gold is mixed with a trace of gelatin, the subsequent addition of salts will be found to produce no precipitation. The explanation of this is that the emulsoid becomes distributed as a film on the suspensoid particles, thus practically converting them into emulsoids.
Gelatinization
One of the best known properties of emulsoids is that of gelatiniza- tion, which has an interesting bearing on many problems of biology. After the gel has set, an enormous pressure is required to squeeze out
any water from it, indicating that the water no longer forms the con- tinuous phase but must be enclosed in vesicles formed of more solid material.
By observing solutions of pure soaps under the ultramicroscope it has been