nBazni BLOOD CHEMISTRY COLORIMETRIC METHODS BLOOD CHEMISTRY COLORIMETRIC METHODS For the General Practitioner WITH CLINICAL COMMENTS AND DIETARY SUGGESTIONS By WILLARD J. STONE, M.D. Pasadena, California Attending Physician, Los Angeles General Hospital / Introduction by George Dock, M.D. Pasadena, California NEW YORK PAUL B. HOEBER, Inc. 1923 Copyright, 1923 By PAUL B. HOEBER, Inc. Published October, 1923 Printed in the United States of America PREFACE It is hoped that those interested in the chemistry of the more common blood constituents affected by impaired func- tion or disease will find this compilation useful because of the arrangement of the methods. The methods, which have been slightly modified to meet the demands of the clinical laboratory using relatively small quantities of blood, are dependent entirely upon the exhaustive researches of Folin and Wu, Denis, Lewis, Benedict, Van Slyke, Bloor, Myers, Bailey, and others. Certain clinical comments have been added. The steps to be followed in the determination of total nitrogen in the urine have been modified from the original method of Folin in order to conform to the same strength nitrogen standard used in the determination of non-protein nitrogen in the blood. It has been added for the convenience of those who may be interested in the comparison of blood nitrogen retention, of output nitrogen in the urine and the intake nitrogen from weighed diets. Folin’s method for the determination of urinary titratable acidity has been added. For the purpose of ready reference an outline of the essential facts to be determined in the study of impaired kidney function has also been included, together with dietary suggestions covering the treatment of certain disturbed metabolic states. Willard J. Stone. Pasadena, California August, 1923. INTRODUCTION Discoveries in biological chemistry have stimulated many valuable investigations, which are not utilized as much as they should be in clinical medicine and especially in private practice. This neglect has not been peculiar to chemical methods. Many other diagnostic procedures have been less used than they should be, for similar reasons, the chief of which has been igno- rance of the methods. All physicians, however, should be famil- iar with new methods, while those of recent laboratory training should be able to repeat them and become expert in their use. A frequent excuse for neglect has been the fear that the methods reported were imperfect and would be improved upon or modi- fied. This has been true also of many other means of diagnosis, but should not deter the physician with the investigative turn of mind from such work, since by means of it the results can be compared, the sources of error and the fallacies recognized, and improvements adopted. The time required for the methods, rather than the cost of apparatus or reagents, has been an important factor for the busy physician. A well-equipped small laboratory will, however, be able to do quite as satisfactory work as the large scale performance of tests in public laboratories, but with the added advantage that the physician may modify and improve his own methods and check his own work. An important reason for the wider application of chemical methods in diagnosis depends upon the importance of the findings as a guide to the condition of the body with normal or disturbed function. Many who have had such tests applied have been discouraged because diagnoses, especially names of diseases, have not been furnished. But this ignores the fact that in practice the name of the disease is not so important as accurate knowledge of the physiology of the patient. Also that the changes which may occur from day to day are of more importance than the results of a VIII INTRODUCTION single test. So in the case of many chemical methods the course of the various changes is essential just as a temperature curve is more useful than an isolated observation of the body heat. While some of the present methods may in the light of experi- ence be abandoned, it is certain that many others will become as necessary as the simpler clinical examinations. Dr. Stone has given the essential details of the most valu- able clinical methods of biochemistry, methods that have been extensively used by himself and others. Those already famil- iar with such work will find the book useful for reference, while those who have been discouraged by the mass of detail given in more exhaustive textbooks will find it a clear and accurate guide. The use of such methods of clinical study will add not only interest but greater accuracy to the work of the physician and enable him with satisfaction to take part in the general advance of clinical knowledge. The large field of clinical chem- istry is open to those who are interested in giving to their patients advice founded upon facts rather than fads or fancies. It should also be recalled]that many discoveries in the field of medicine have come from small laboratories, a reason which should give further stimulation to the more general adoption of such investigative methods of work. *• George Dock. Pasadena, California August, 1923. PAGE Introduction vii Preface ix I. Suggestions for Blood Chemistry Work i II. Preparation of Protein-Free Blood Filtrate used in Determination of Non-Protein Nitrogen, Urea, Uric Acid, Preformed Creatinin, Total Creatinin, Sugar and Chlorides 6 III. Uric Acid 16 IV. Preformed Creatinin 25 V. Blood Sugar 29 VI. Blood Chlorides 35 VII. Blood Cholesterol 39 VIII. Total Nitrogen Determination in the Urine ... 45 Titratable Acidity of Urine 46 Phenolsulphonephthalein Determination 47 IX. Clinical Comments on the Diagnosis of Impaired Kidney Function 48 X. TheDietary Control of Disturbances of Metabolism 53 XI. The Test and Maintenance Diets of Joslin in the Treatment of Diabetes Mellitus 61 Index 73 CONTENTS CHAPTER I Suggestions for Blood Chemistry Work 1. For blood chemistry work the fasting state is desirable, since practically all of the established normal values have been obtained from blood specimens taken twelve to fourteen hours after the ingestion of food. If the blood specimen is taken during the period of greatest absorption from the gastro- intestinal tract, within a period of three or four hours after a meal, the figures for non-protein nitrogen, urea, and sugar may be considerably increased, for which due allowance should be made in interpreting the results. As to the method of taking the blood specimen the following plan has been found satis- factory. About 6 c.c. of blood are drawn from one of the veins at the bend of the elbow by means of a dry glass syringe. One and one-half c.c. are expelled into a small tube for the routine Wassermann test while the balance is expelled into another tube containing 2 or 3 drops of a 20 per cent potassium oxalate solution to prevent clotting. This tube is inverted several times to mix thoroughly the oxalate with the blood. 2. It has been found most satisfactory to make the tests for non-protein nitrogen, urea, creatinin, uric acid, and sugar upon the blood filtrate as described in the methods of Folin and Wu. 3. The picric acid method of Lewis and Benedict (or its modification by Myers and Bailey) for blood sugar may give higher readings in many specimens of blood, primarily because of the absorption of creatinin by the picric acid. While not criticizing the usefulness of this test, it is believed that more uniform results are obtained by following a procedure such as that offered by the method of Folin and Wu. For verification 2 BLOOD CHEMISTRY METHODS of blood sugar in any given case the Lewis and Benedict picric acid method is recommended. 4. Chemically pure sodium tungstate and picric acid are essential. (See notes in the text.) Some preparations of sodium tungstate are not satisfactory because of an excess of carbonate. Sodium tungstate, Primrose Brand, or Merck’s c.p. sodium tungstate are satisfactory. The Nessler’s solution should be carefully prepared. Fig. i. Myers colorimeter. Fig. 2. Duboscq colorimeter. 5. For standard sugar solutions, chemically pure dextro- gucose (Pfanstiehl) may be recommended. The standard nitro- gen, uric acid, and creatinin solutions can be secured from any dealer in scientific supplies by those who do not have facilities for making their own solutions. SUGGESTIONS FOR BLOOD CHEMISTRY WORK 3 6. Discrepancies in the calibration of glassware, especially pipettes, burettes, graduates, and volumetric flasks, should be noted when equipping the laboratory. It is best to have on hand for comparison a few utensils the accuracy of which has been confirmed by the Bureau of Standards, Washington. Showing perfectly opaque black colorimetric cup with optically clear bottom Showing nephelomet- ric cup with optically clear sides and perfectly opaque black bottom Fig. 3. Kober colorimeter. 7. It is wise to calibrate frequently the colorimeter scale in order to determine that approximately identical readings are secured, using for comparison the standard solutions used in the tests. If the Kober colorimeter is used, the mirrors should be carefully adjusted to reflect the same amount of light into 4 BLOOD CHEMISTRY METHODS the cups, which should be filled about two-thirds full of the solutions. If discrepancies occur, the scale should be adjusted so that the readings coincide. 8. The blood pipettes and blood-sugar tubes of Folin, with bulb and constriction, as well as Pyrex ignition test-tubes, can be obtained from any dealer in scientific supplies. 9. The choice of the colorimeter will depend upon the individual. The Duboscq is now made in this country, as well as the Kober and the Bock-Benedict models. Fig. 4. Bock-Benedict colorimeter. Formula for Calculation in Colorimeter Work The following formula serves as the basis for computations in which different quantities of unknown and varying strengths of standard solutions are used: Standard Strength of . Reading Standard in Mg. fP. Amount Unknown Volume of Standard as ~ t°t • °?j Reading Compared with Unknown SUGGESTIONS FOR BLOOD CHEMISTRY WORK The Normal Blood Constituents of Clinical Importance Per ioo c.c. of Blood Urea 12.0 - 15.0 mg. Uric acid 1-5 - 3.0 mg. Preformed creatinin i-5 - 2.0 mg. Total creatinin (creatin plus creatinin) 4.0 - 6.0 mg. Amino-acid nitrogen 6.o - 8.0 mg. Ammonia nitrogen 0. I - 0.2 mg. Total non-protein nitrogen (theincoagu- Iable nitrogen) 25.0 - 35.0 mg. Sugar 80.0 -110.0 mg. Cholesterol 160.0 -200.0 mg. Chlorides (as NaCI) 600.0 -650.0 mg. CHAPTER II Preparation of Protein-Free Blood Filtrate Used in Determination of Non-Protein Nitrogen, Urea, Uric Acid, Preformed Creatinin, Total Creatinin, Sugar and Chlorides (Method of Folin and Wu1) I. Solutions Used in the Preparation of Protein-Free Blood Filtrate 1. Sodium tungstate, c. p 20.0 gm. Distilled water to 200.0 c.c. 2. % Normal Sulphuric Acid Sulphuric acid 35.0 gm. by weight Distilled water to 1000.0 c.c. This solution, while approximately correct, should be checked up by titration, since in the quantities used it is intended to be equivalent to the sodium content of the tungstate. II. Transfer exactly 4 c.c. of the oxalated blood to 100 c.c. flask. Avoid an excess of oxalate because of interference with uric acid precipitation. Add 28 c.c. of distilled water and shake to lake the blood. Add 4 c.c. of 10 per cent sodium tungstate solution and mix. Add from graduated burette, slowly and with constant shaking, 4 c.c. of normal sulphuric acid. Close flask with rubber stopper and shake. The shaking should produce but little foaming. Let stand for five minutes. The color should change from red to brown. If color does not change the coagula- tion is incomplete, usually because of too much oxalate. In such an event add 10 per cent sulphuric acid, one drop at a time, and shake after each drop until there is no foaming and the brown coloration has occurred. It is important to avoid IJ. Biol. Cbem., 1919, xxxviii, 81. THE BLOOD NON-PROTEIN NITROGEN 7 adding any excess of sulphuric acid beyond the amount required to secure thorough protein precipitation since an excess may also precipitate the uric acid and interfere with its subsequent determination. Pour the mixture slowly on filter paper and cover with watch glass. The filtrate should be clear.1 If filtrate is to be kept for a day or two add a few drops of xylol or toluol. Ten c.c. oj the blood filtrate equals 1 c.c. of blood. Non-Protein Nitrogen I. Solutions Used in the Determination of Non-Protein Nitrogen in the Blood 1. Acid Phosphoric-Sulphuric Digestion Mixture Copper sulphate (5 per cent solution) 50.0 c.c. Acid phosphoric (85 per cent) 300. o c.c. Acid sulphuric, c.p. (ammonia-free) 100.0 c.c. Distilled water 450.0 c.c. Keep well stoppered to prevent absorption of ammonia from the air. 2. Stock Nessler’s Solution KI 15.0 gm. Iodine n.ogm. in 100 c.c. flask; add Water 10.0 c.c. Metallic mercury 14.5 gm. Shake flask vigorously, using rubber cork, for seven to ten minutes until dissolved iodine has nearly all disappeared. The solution becomes quite hot. When the red iodine solution has begun to pale in color, cool in running water and continue shaking until red color of iodine has been replaced by greenish color of the double iodide (about ten minutes). Separate solution from surplus mercury by decant- ing and washing with distilled water. Dilute solution with washings to 200 c.c. 1 The blood filtrate, if the correct strength of acid has been used, should be only slightly acid to Congo-red paper. 8 BLOOD CHEMISTRY METHODS 3. Preparation oj Nessler’s Reagent Jrom Stock Nesslers Solution Put into 500 c.c. bottle: 10 per cent solution of NaOH 350.0 c.c. Stock Nessler’s solution 75 -0 c.c. Distilled water 75.0 c.c. (In the preparation of the 10 per cent solution of NaOH used in Nessler’s reagent it has been found much more satisfactory to make the solution accurately by weight instead of by volume.) 1 c.c. contains approximately: Hg metallic 0.1 gm. KI 0.01 gm. Iodine 0.001 gm. NaOH 0.07 gm. 4. Standard Nitrogen Solution Ammonium sulphate (highest purity). 0.9432 gm. Distilled water 1000.0 c.c. 1 c.c. = 0.2 mg. nitrogen II. Preparation of Unknown Solution To 2.5 c.c. of filtrate, in large Pyrex ignition test-tube, add 0.5 c.c. of the acid phosphoric-sulphuric digestion mixture. Boil gently over micro-burner until water has been nearly evaporated. Cover tube with watch glass and continue boiling gently for about two minutes. Dense fumes from the acid will rise in the tube. The solution will turn dark brown and upon heating slowly will soon turn nearly colorless. Allow tube to cool. Wash contents of tube carefully with 5 to 7 c.c. of distilled water into a 25 c.c. volumetric flask. This completes the preparation of the unknown solution with the exception of the addition of Nessler’s reagent. (See below.) III. Preparation of Standard Solution The standard usually required is about 0.2 mg. of nitrogen per 100 c.c. Place with measuring pipette 1 c.c. of standard THE BLOOD NON-PROTEIN NITROGEN 9 nitrogen solution (which contains 0.2 mg. per c.c.) in 50 c.c. volumetric flask, add 1 c.c. of the phosphoric-sulphuric acid digestion mixture to balance acid in the unknown, and then add about 15 c.c. of distilled water. IV. Final Step Add 8 to 10 c.c. of Nessler’s reagent slowly to flask contain- ing the unknown, and when full development of color has oc- curred, fill to 25 c.c. mark with distilled water. Insert clean rubber stopper in flask and mix. If solution is turbid, centrifuge small portion before comparing with standard. Add 12 to 15 c.c. of Nessler’s reagent slowly to standard solution in volumetric flask and when full development of color has occurred add distilled water to 50 c.c. mark. Insert clean rubber stopper and mix. (The Nessler’s reagent should be added as nearly simultaneously as possible to unknown and standard solutions.) Compare unknown in colorimeter with standard set at 20 mm. V. Calculation The equivalent of 0.25 c.c. of blood was used; the standard solution contained 0.2 mg. nitrogen; the volume of standard was twice the volume of the unknown solution. The calculation will be as follows, R indicating the reading of the unknown: 20 w 0.2 . . ,tt X —- mg. = mg. non-protein nitrogen in 0.25 c.c. blood xv 2 or 800 . . T r i = mg. non-protein nitrogen in 100 c.c. blood. The average of many analyses of normal blood specimens has been between 25 and 35 mg. of non-protein nitrogen per 100 c.c. of blood. 10 BLOOD CHEMISTRY METHODS Determination of Blood Urea (Method of Folin) I. Solutions Used i . Buffer Mixture to Activate the Urease Solution Sodium pyrophosphate (U. S. P.) 140.0 gm. Glacial phosphoric acid 20.0 gm. 2. Urease Solution Wash about 3 gm. of permutit in a flask with 2 per cent acetic acid, decant and repeat the process twice with water; add 5 gm. of Jack bean meal to 16 c.c. of 95 per cent alcohol and 84 c.c. of water. Shake for ten minutes, filter and collect the filtrate in 3 or 4 different small bottles. This solution will keep for one week at ordinary room temperature, but may be preserved for four to six weeks in an ice box. Direct sunlight exposure of the solution should be avoided. N 3. — Hydrochloric Acid Concentrated HCI 1. o c.c. Distilled water to 200. o c.c. (This solution is approximately correct.) 4. Saturated Borax Solution 5. Paraffin Oil 6. Standard Nitrogen Solution Ammonium sulphate (highest purity). 0.9432 gm. Distilled water ; 1000.0 c.c. 1 c.c. = 0.2 mg. nitrogen. II. Preparation of Unknown Solution To 2.5 c.c. of blood filtrate in a large Pyrex test tube (which must previously have been rinsed with nitric acid and then with water if it has contained Nessler’s solution), add one drop of buffer mixture and 0.5 c.c. of urease solution. Immerse THE BLOOD UREA 11 the tube in warm water (40°-55°C.) for five minutes or let stand at room temperature for fifteen minutes. To collect the ammonia formed from the urea without using a condenser, a test-tube with perforated rubber stopper con- taining a curved glass tube is used for distillation as in the accompanying illustration. The distillate is collected in a graduated 25 c.c. perforated rubber-stoppered receiving tube N containing 1 c.c. of — hydrochloric acid solution. Fig. 5. A at beginning, B toward end of distillation. From Folin’s Manual of Biological Chemistry. Courtesy of D. Appleton and Company. Add to the blood filtrate a dry pebble, a drop or two of par- affin oil and i c.c. of the borax solution. Boil, using a small flame at a uniform rate, for about four minutes. The boiling 12 BLOOD CHEMISTRY METHODS should not be so brisk that the emission of steam occurs from the receiving tube before three minutes. At the end of four minutes disconnect the receiving tube from the rubber stopper, let it rest in a slanting position and continue the distillation for a minute longer. Rinse out the delivery tube with a little water, and after cooling the distillate with running water add the washings to it and bring the volume to 10 c.c. by adding suffi- cient distilled water. III. Preparation of Standard Solution Transfer 1 c.c. of standard nitrogen solution to a 50 c.c. volumetric flask, dilute to about 40 c.c., add about 7.5 c.c. of Nessler’s solution, rotate flask until Nesslerization is com- plete and add water to the 50 c.c. mark. IV. Final Step Add 1.5 c.c. of Nessler’s solution to the unknown and after Nesslerization is complete dilute to the 12.5 c.c. mark with water. V. Calculation The equivalent of 0.25 c.c. blood was used; the standard solution contained 0.2 mg. nitrogen; the volume of standard was 50 c.c., while the volume of unknown was 12.5 c.c. Compare unknown in colorimeter with standard set at 20 mm. The calculation will be as follows, R indicating the reading of the unknown: 20 0.2 . T T T -p X —- = mg. in 0.25 c.c. blood, rv 4 or = mg. in ioo c.c. blood. VI. Clinical Comments on Non-Protein Nitrogen and Urea in the Blood Marked increase of non-protein nitrogen (urea constituting 70-80 per cent) in the blood may be expected in partial or com- BLOOD NITROGEN RETENTION 13 plete suppression of kidney function, whether acute or chronic. If complete, the condition is that usually described as uremia. It is also increased in such conditions as prostatic hypertrophy producing urinary retention and bilateral ureter compression or obstruction. The increase is especially marked in the type of nephritis brought about by poisoning with the heavy metals such as lead, arsenic and mercury. In many patients with arterial hypertension due to contracted arterioles (high dias- tolic blood-pressure) with its associated cardiorenal symp- toms, there may be little evidence of abnormal nitrogen reten- tion as long as the kidneys are permeable, fluids freely excreted and a low protein diet compatible with their excretory capabili- ties is being followed. When, however, as a result of infection, overindulgence, or poisoning, the capacity of the kidneys for the elimination of waste nitrogen is overtaxed, an acute exacerbation of a preexisting nephritis, giving perhaps few symptoms, may occur, with rapid increase in the quantities of retained nitrogen in the blood. In early kidney damage or impaired function the non-pro- tein nitrogen of the blood is usually moderately increased, i.e., to 40-50 mg. This increase is largely made up of urea, nitrogen and uric acid. During the process of digestion urea is formed in the liver from ammonia resulting from the breaking down of the protein food constituents into amino-acids. Urea is therefore of exogenous origin. The uric acid results from the action of enzymes or other glandular constituents upon amino and oxypurins. It is usually considered to be partly of endogenous and partly exogenous origin. In the condition de- scribed as acute nephritis the retention of non-protein nitrogen is higher, often reaching 150 mg. per 100 c.c. of blood. In the chronic types of diffuse or interstitial nephritis the retention may be less marked, especially if the patient is living within the functional capacity of his kidneys. In such chronic types the non-protein nitrogen retention will usually be found to vary from 50 to 100 mg. per 100 c.c. of blood. The types of chronic nephritis associated with severe anemia are partic- ularly subject to blood nitrogen retention. This type of anemia, 14 BLOOD CHEMISTRY METHODS which seems to bear no constant relation to edema, is often characterized by a high hemoglobin index, approaching 1 or 1 +, while the changes in the red cells usually expected in severe anemias, such as stippling and irregular shapes and sizes, are lacking. As has been suggested by Berg,1 this hyperchromat- ic type of anemia probably results from profound changes in the activity of the blood-forming organs. It is not improbable that prolonged nitrogen retention and associated acidosis may bear some etiologic relationship to it. In the condition described as parenchymatous nephritis or “nephrosis,” nitrogen retention is, except in the terminal stages of the disease, not markedly increased. In the terminal stage abnormal nitrogen retention usually occurs. In eclampsia there is usually a moderate increase in the non-protein nitrogen of the blood. This increase results from a marked retention of uric acid rather than retention of urea. The evidence points to a certain degree of impaired kidney function, not only because of the uric acid retention, but also because the threshold of sugar elimination is increased, which results in a moderate hyperglycemia. Eclampsia should not be described as a condi- tion due to “uremia” according to knowledge of the subject now available. In many chronic conditions, such as arteriosclerosis or malignancy, abnormal blood nitrogen retention may develop as evidence of the impaired function and associated changes involving the kidneys. Such retention, if persistent and if unin- fluenced by diet, undoubtedly influences the prognosis. In some severe acute infections, such as diphtheria and pneu- monia, blood nitrogen retention may occur. This seems to be especially marked if dehydration and acidosis is an associated condition. In acute intestinal obstruction the retention of non- protein nitrogen or urea may reach three or four times the normal figures. In prostatic obstruction, blood chemistry studies are im- portant in that knowledge of the associated kidney changes gives indication as to the most suitable time for any contem- 1 Berg. Am. J. M. Sc., 1922, clxiv, 88. BLOOD NITROGEN RETENTION plated operation for relief. If the non-protein nitrogen figure does not exceed 35 mg. per 100 c.c., the uric acid 3 mg. and creatinin 2 mg. per 100 c.c. of blood, the patient may be considered a reasonably good operative risk. If these figures are appreciably exceeded the evidence of kidney damage should indicate caution. Institution of measures designed to relieve gradually the hydrostatic back-pressure upon the kidneys, such as preliminary bladder drainage, are of great value in restoring impaired kidney function to these patients. The reten- tion of non-protein nitrogen, uric acid and creatinin in the blood may rapidly diminish under such treatment, the extent of the reduction depending, of course, upon the extent of the previous damage. CHAPTER III Uric Acid (New method of Folin1) L Solutions Used in the Determination of Blood Uric Acid 1. Silver Lactate Solution (io per cent) Dissolve 50 gm. of silver lactate in 350 c.c. of warm water and add a mixture consisting of 50 c.c. of 85 per cent lactic acid and 50 c.c. of 10 per cent sodium hydroxide. Add water to 500 c.c. The sediment present should be allowed to settle and only the clear super- natant solution used in the test. 2. Acidified Sodium Chloride Solution Concentrated HCI i.oc.c. 10 per cent sodium chloride sol 100.0 c.c. 3. Lithium Sulphate Solution (20 per cent) Dissolve 20 gm. of powdered lithium sulphate (Baker & Adamson’s) in 80 c.c. of cold water. Dilute to volume of 100 c.c. and filter. 4. 15 Per Cent (approx.) Solution of Sodium Cyanide Prepare enough to last three months, since this solution is believed to improve with age. Use white solid sodium cyanide not discolored or decomposed by exposure to air. Weigh out from 100 to 200 gm. of cyanide, transfer to beaker, add 6.7 c.c. of 0.1 normal sodium hydroxide solution for each gram of cyanide taken and stir until all has dissolved. The solution is opalescent. Transfer to bottle and keep at least two weeks before using. The 0.1 normal sodium hydroxide 1J. Biol. Cbem., October, 1922, Iiv, 153. URIC ACID 17 was added to prevent decomposition with discolora- tion. Ammoniacal decomposition of the cyanide sooner or later destroys its efficiency, since maximum color development is retarded and turbidity is produced. This can be prevented by covering the stock bottle with a beaker instead of using a cork stopper, or by boiling off the ammonia and then diluting to the orig- inal volume. Because of its toxicity it should, in per- forming the tests, be measured from a burette. To test for the blank due to impurity of the cyanide, transfer 5 c.c. of water, 2 drops of lithium sulphate solution and 2 c.c. of the 15 per cent cyanide solution to a test-tube. Add 1 c.c. of the uric acid reagent (given below) and let stand two minutes. The solution should remain colorless. Heat in boiling water for one and a half minutes. Some color will develop. To determine whether this color will materially affect the uric acid values in a test, repeat the above with two graduated test- tubes and with standard uric acid solution, 5 c.c. in one and 3 c.c. plus 2 c.c. of water in the other. Dilute to volume after heating and compare the colors. If the cyanide is suitable the weaker solution will give the theoretical reading, 33.5 mm., when the stronger solution is placed in colorimeter at 20 mm. 5. Stock Uric Acid-Formaldehyde Solution Transfer exactly 100 mg. uric acid1 to a funnel on a 100 c.c. volumetric flask. Dissolve 45 to 50 mg. of lithium carbonate in 15 c.c. of water by heating to about 6o°C. until all the carbonate has been dissolved. With the hot carbonate solution rinse the uric acid on the funnel into its flask and shake. The uric acid will promptly dissolve. Cool flask under running water by shaking and add 40 to 50 c.c. of water. Then add 2.5 c.c. of 40 per cent formaldehyde and, after shaking to insure thorough mixing, acidify by the addition of 0.3 c.c. of glacial acetic acid. Shake to remove most of the 1 Uric acid (Kahlbaum) is a satisfactory preparation. 18 BLOOD CHEMISTRY METHODS carbonic acid, dilute to ioo c.c. and mix. This stock solution should be kept tightly corked in a dark bottle. i c.c. = i mg. uric acid. 6. Standard Uric Acid Solution Place i c.c. of the stock solution, with a graduated pipette, in a 250 c.c. volumetric flask, add 125 c.c. of water and 10 c.c. of the % normal sulphuric acid used in blood protein precipitation, then add 1 c.c. of 40 per cent formaldehyde, dilute to 250 c.c. and shake to mix. 1 c.c. = 0.004 mg. uric acid. 7. Uric Acid Reagent oj Folin and Denis In flask place Water 75.0 c.c. Sodium tungstate 10.0 gm. Phosphoric acid (85 per cent) 8.0 c.c. Partly close flask with funnel and small watch glass and boil gently for two hours, then dilute with water to 100 c.c. II. The Test (Short Method)1 Have ready for use a wide-mouthed beaker containing boil- ing water. Place 5 c.c. of the blood filtrate and 2 c.c. of water in a test-tube graduated at 25 c.c. Place in a similar tube 5 c.c. of the standard uric acid solution and 2 c.c. of water. Add 2 or 3 drops of 20 per cent lithium sulphate solution to each. The lithium sulphate solution is added to prevent precipi- tate formation in the presence of potassium oxalate used for anticlotting purposes. It may be necessary at times to add 4 drops of the lithium sulphate solution to prevent precipitation. 1 The quantity of lithium sulphate necessary to prevent precipitation, together with the precipitation which may result after boiling for eighty seconds, has been found to be such a source of trouble in some specimens of blood that the more complete “silver lactate method” given on page 19 is preferred. URIC ACID 19 From the burette add 2 c.c. of 15 per cent sodium cyanide solution to each tube. With a graduated pipette add 1 c.c. of the uric acid reagent to each tube, mix and let stand two minutes. At the end of two minutes transfer both tubes to the boiling water for eighty seconds. Longer heating may cause precipitation. Cool the tubes, add water to the 25 c.c. mark and mix by inverting the tubes. III. Calculation Read the standard against itself set at 20 mm. If the two scales do not coincide, adjust to secure correct reading. Equal volumes of unknown and standard were used. The equivalent of 0.5 c.c. of blood was used. The standard contained 0.02 mg. uric acid. With R indicating the reading of unknown the calculation will be: X 0.02 mg. = mg. uric acid in 0.5 c.c. blood, or = mg. uric acid in ioo c.c. blood. This short method may give values from o.i to 0.2 mg. higher than those obtained by the following method, which employs more certain recovery by means of silver lactate precipitation. IV. The Test (Silver Lactate Method) Have ready for use a beaker containing boiling water. Place 5 c.c. of the blood filtrate in a centrifuge tube, add 7 c.c. of the io per cent silver lactate solution. Mix and centri- fuge. All the uric acid is contained in the precipitate. Decant the supernatant fluid as completely as possible. 20 BLOOD CHEMISTRY METHODS Add 1 c.c. of the acidified sodium chloride solution to the precipitate, stir thoroughly with a glass rod, add 4 c.c. of water and, after stirring, centrifuge. Pour the supernatant solution into a test-tube graduated at 25 c.c. Place 5 c.c. of standard uric acid solution (containing 0.004 mg. per c.c.) in a test-tube graduated at 25 c.c. To the contents of the unknown and to the standard tube, add 0.2 c.c. of 20 per cent solution of lithium sulphate, 2 c.c. of the 15 per cent sodium cyanide solution from burette, and 1 c.c. of the uric acid reagent. Shake each tube and let stand for two minutes. Heat the two tubes in boiling water for eighty seconds, cool, add water in each tube to the 25 c.c. mark and compare in colorimeter as in the short method. V. Calculation Read the standard against itself set at 20 mm. If the two scales do not coincide, adjust to secure correct reading. Equal volumes of unknown and standard were used. The equivalent of 0.5 c.c. of blood was used. The standard contained 0.02 mg. uric acid. With R indicating the reading of unknown the calculation will be: 20 p- X 0.02 mg. = mg. uric acid in 0.5 c.c. blood, or = mg. uric acid in ioo c.c. blood. Uric acid in the blood normally varies from 1.5 to 3.0 mg. per 100 c.c. The latter figure may be considered a high normal. URIC ACID 21 Uric Acid (Benedict’s Modification, using the blood filtrate method of Folin and Wu1) I. Solutions Used in the Determination of Blood Uric Acid 1. Arsenic-Phosphotungstic Acid Reagent2 Add ioo grams sodium tungstate, c.p., to 600 c.c. distilled water, and after dissolved add 50 gm. pure arsenic pentoxide, 25 c.c. of phosphoric acid (85 per cent) and 20 c.c. of concentrated hydrochloric acid. Boil the mixture for twenty minutes, cool and dilute to 1000 c.c. (This reagent keeps indefinitely and yields nearly seven times as much color as does the “uric acid reagent” of Folin and Denis. It is scarcely affected by polyphenols in the presence of uric acid.) 2. 5 Per Cent Sodium Cyanide Solution Sodium cyanide 12.5 gm. Concentrated ammonia 0.5 c.c. Distilled water to 250.0 c.c. This solution should be freshly prepared once in two months. It should be measured from a burette in performing the test because of its toxicity. 3. Stock Uric Acid Solution Dissolve 2.25 gm. of pure crystals of hydrogen di- sodium phosphate and 0.25 gm. of dihydrogen sodium phosphate in 75 c.c. of hot distilled water. Filter and make up to 125 c.c. with hot water. Pour this warm clear solution on 50 mg. pure dried uric acid (Kahl- baum) suspended in a few c.c. of water in a 250 c.c. volumetric flask. Agitate until completely dissolved and add at once exactly 0.35 c.c. of glacial acetic acid, 1 Benedict. J. Biol. Cbem., March, 1922, Ii, 187. 2 The arsenic pentoxide used in the “arsenic-phosphotungstic acid reagent” is marketed under the name “acid arsenic c.p. powdered.” 22 BLOOD CHEMISTRY METHODS then add distilled water nearly to the 250 c.c. mark, add 1.5 c.c. of chloroform and finally add distilled water to the 250 c.c. mark. The solution should be freshly prepared every two months. Before weighing it will be best to dry the uric acid at about ioo°C. in an oven for an hour or two. 1 c.c. of the stock solution = 0.2 mg. uric acid. 4. Standard Uric Acid Solution Measure 10 c.c. of the stock uric acid solution (containing 2 mg. uric acid) into a 500 c.c. volumetric flask and fill the flask about half full of distilled water, add 25 c.c. of dilute hydrochloric acid (one volume of concentrated acid diluted to ten volumes with distilled water) and dilute the solution to 500 c.c. This standard should be freshly prepared once in two weeks. 1 c.c. = 0.004 mg. uric acid. II. Preparation of Unknown Solution Transfer 5 c.c. of the blood filtrate to a graduated test-tube and add 5 c.c. of distilled water. Add 4 c.c. of the 5 per cent sodium cyanide solution. III. Preparation of Standard Solution Transfer 5 c.c. of the standard solution to a graduated test- tube and add 5 c.c. of distilled water. Add 4 c.c. of the 5 per cent sodium cyanide solution. IV. Final Step Add i c.c. of the arsenic-phosphotungstic acid reagent to the tube containing the unknown and to the tube containing the standard solution. Mix by inverting tubes and place them immediately in boiling water for three minutes. Then remove tubes and place in beaker of cool water for three minutes. Compare in colorimeter within five minutes because of tendency to turbidity. URIC ACID 23 V. Calculation Equal volumes of unknown and of standard solutions were used. The standard contained 0.02 mg. An equivalent of 0.5 c.c. of blood was used. Therefore if S equals the height of standard solu- S tion in mm. and R equals the reading of the unknown, X S x A 0.02 X 200 or —— = mg. of uric acid per 100 c.c. of blood. Note on Benedict’s Uric Acid Method. Benedict’s method has been found by Brown and Raiziss1 to give higher readings, due to interfering substances, than the original method of Folin and Wu. In the following table their comparative results are given when the same protein-free filtrate was used for the two methods: Uric Acid (Folin and Wu) mg. per ioo c.c. Uric Acid (Benedict) mg. per ioo c.c. I 2.2 3-2 2 1.9 2.8 3 i.8 4-7 4 2.6 3-6 5 5.2 6-3 6 3-o 3 9 7 2.0 3-i 8 3-2 4-5 9 4.0 5.8 10 6.2 6.6 VI. Clinical Comments on Uric Acid Retention Uric acid is probably the first non-protein nitrogen constitu- ent to be retained in abnormal amounts by the blood in early, 1 Brown, H., and Raiziss, G. W. The estimation of uric acid in blood. J. Lab. & Clin. M., November, 1922, viii, 129. 24 BLOOD CHEMISTRY METHODS temporary or permanent damage to the kidneys. A distinctly higher than normal value, above 3 mg. per 100 c.c., if persis- tent and not remedied by appropriate dietary restrictions and other possible contributing causes, should attract attention to the possibility of kidney impairment. Uric acid is almost invariably high in gout, while the total non-protein nitrogen value may be within normal limits. In acute gout the blood uric acid not uncommonly reaches 8 to 10 mg. if the patient is not on a low purin diet. In chronic gout, with which condition there is so many times evidence of associated or concomitant kidney impairment, the amount of uric acid commonly retained by the blood reaches 6 to 8 mg. per 100 c.c. This can be decreased in some instances by a low protein-low purin diet, although for many patients with chronic gout the influence of a Iow-purin diet on the blood uric acid is not marked. The administration of uric acid eliminants, such as cinchophen and tolysin or the salicylates, is many times of service. In suspected gouty arthritis without tophi, but in which an increased blood uric acid figure is obtained, if the non-protein nitrogen and creatinin are also increased, gout may be excluded and the increase in uric acid be more reasonably ascribed to impaired kidney function. In non-gouty arthritis without kidney impairment the blood uric acid is usually within normal limits. Since uric acid has its origin in the body partly from endogenous and partly from exogenous sources, disturbances of metabolism resulting in retention not uncommonly lead, in those whose elimination is impaired, to muscle pains, stiffness, and headaches. If confirmation of the suspected disturbance is obtained by finding an abnormal retention of uric acid in the blood, the condition, which is usually temporary, may in many instances be relieved by a Iow-purin diet and the administra- tion of cinchophen or tolysin. CHAPTER IV Preformed Creatinin (Method of Folin and Wu) I. Solutions Used in the Determination of Preformed Creatinin in the Blood 1. Alkaline-Pier ate Solution Saturated (1.2 per cent) picric acid solution 8.5 c.c. 10 per cent sodium hydroxide solution.. 1.5 c.c. 2. Stock Creatinin Solution Creatinin-zinc chloride 1.61 gm. Tenth-normal HCI solution.. 1000.0 c.c. 1 c.c. = 1 mg. creatinin. 3. Standard Creatinin Solution Stock creatinin solution 5.0 c.c. Tenth normal HCI solution 10.0 c.c. Distilled water to 100.0 c.c. Add two or three drops of xylol as a preservative. 1 c.c. = 0.05 mg. creatinin. II. Preparation of Unknown Solution To 5 c.c. of blood filtrate in graduated tube add 2.5 c.c. of alkaline-picrate solution. Mix and allow to stand six to eight minutes to develop color. III. Preparation of Standard Solution Measure with pipette 0.3 c.c. of standard creatinin solution (containing 0.05 mg. per c.c.) into graduated tube and add water, using same pipette, to 10 c.c. mark. Add with accurate pipette 5 c.c. of alkaline-picrate solution. Mix and let stand 26 BLOOD CHEMISTRY METHODS six to eight minutes to develop color. (The standard solution thus prepared contains 0.015 mg. creatinin.) IV. Calculation Note that both fields are equal when both cups of colorim- eter contain the standard set at 20 mm. The color comparison between standard and unknown should be made within fifteen minutes from the time the alkaline-pier ate was added. With standard set at 20 mm., R indicating the reading of the unknown solution, the computation will be as follows, since the equivalent of 0.5 c.c. of blood was used: 20 0.015 . r I r T p- X —-— = mg. in 0.5 c.c. 01 blood. or = mg. in ioo c.c. blood. The average of many analyses has shown the normal amount of preformed creatinin to be about 1.5 mg. per 100 c.c. of blood. Total Creatinin (Creatin plus Creatinin) I. Solutions Used Same as for preformed creatinin determination. II. Preparation of Unknown Solution Place 2 c.c. of blood filtrate in io c.c. volumetric flask, add 0.5 c.c. of normal hydrochloric acid. Cover mouth of flask Picric Acid Purity. To test purity of picric acid as used in creatinin and blood sugar determinations, Folin and Doisy (J. Biol. Chem., 1916-17, xxviii, 349) have suggested the following procedure: Add 1 c.c. of 10 per cent NaOH solution to 20 c.c. of a saturated (1.2 per cent) solution of picric acid in water. The color of the alkaline-picrate solution so prepared must not be more than twice as deep a color as that of the saturated picric acid solution. With unusually pure picric acid, the color of the alkaline-picrate solution will not be more than one and one- half times as deep as that of the picric acid solution, i.e., with picric acid solution set at 20 mm. in the colorimeter the alkaline-picrate solution will give a reading of 13 to 14 mm. CREATININ 27 with tinfoil and heat in cup of boiling water for twenty minutes. Cool. Add 2 c.c. of freshly prepared alkaline-picrate solution. Allow to stand five minutes and dilute with water to 10 c.c. mark. III. Preparation of Standard Solution Place 0.5 c.c. of the creatinin standard solution (contain- ing 0.05 mg. per c.c.) in a 20 c.c. volumetric flask. Add 1 c.c. of normal hydrochloric acid,1 and 4 c.c. of freshly prepared alkaline-picrate solution. Allow to stand five minutes, then add water to 20 c.c. mark. The standard so prepared contains 0.025 mg. creatinin. IV. Calculation Fill both colorimeter cups half full of standard solution as prepared above, and determine whether both fields are equal with tube length set at 20 mm. If both are not alike adjust vernier scale of right-hand tube by thumb-screw (Kober instru- ment) so that tube length corresponds to left-hand scale. Adjust mirrors so that reflected light is equal in each field. Empty the right-hand tube and wash. Also wipe solution from plunger. Fill this tube half full of unknown solution and make comparison. R indicates reading of the unknown. 20 0.025 . pr X ——— = mg. in 0.2 c.c. blood, or 12 5 -p- = mg. in ioo c.c. blood. The normal value for total creatinin by this method is about 6 mg. per ioo c.c. of blood. 1 Normal HCl. Concentrated HCI (molecular weight 36.46) is approximately ten times a normal solution, therefore 25 c.c. cone. HCI plus distilled water to 250 c.c. constitutes a normal solution; and 25 c.c. of this latter solution plus distilled water to 250 c.c. will make a tenth-normal solution sufficiently accurate for this test. 28 BLOOD CHEMISTRY METHODS V. Clinical Comments on Blood Creatinin Creatinin is considered to be the most easily eliminated non-protein nitrogen constituent of the blood under conditions of normal kidney function. For this reason considerable impair- ment of kidney function may exist without retention of creat- inin beyond normal limits. When extensive impairment of kidney function has occurred, retention of creatinin results. An increase of preformed creatinin in the blood to 4 or 5 mg. per 100 c.c. has therefore great diagnostic and prognostic importance. Its persistence at a high level indicates severe nephritis except in prostatic or bilateral ureteral obstruction or compression. With persistent retention of 5 mg. or more per 100 c.c. of blood, few patients live longer than a few months. In acute retention due to prostatic obstruction the preformed creatinin may reach 10 mg. with recovery when the obstruction is relieved. Behre and Benedict (J. Biol. Chem., May, 1922) have re- cently cast doubt upon the presence of creatinin in the blood. They believe that the blood does contain creatin, the source of which is muscle tissue and which represents in the blood a waste product for elimination by the kidneys. During the process of elimination it is converted into creatinin. They believe that it is creatin rather than creatinin which is retained in the blood when the renal function is disturbed. As a matter of correct phraseology involving physiological facts it is important that their work be confirmed. Such confirmation would not particularly change the clinical fact that creatin bodies are retained above the normal limits in the blood, particularly in the chronic forms of nephritis. CHAPTER V Blood Sugar (New Method of Folin and Wu1) I. Solutions Used in the Determination of Blood Sugar 1. Stock Sugar Solution, io mg. per c.c. Dextrose, c.p i .0 gm. Distilled water to ioo.o c.c. Add a few drops of xylol to preserve. 2. Standard Sugar Solution, o.i mg. per c.c. Stock sugar solution 5.0 c.c. Distilled water to 500.0 c.c. Add a few drops of xylol to preserve. 3. Molybdate-Phosphate Solution Molybdic acid (85 per cent) c.p 17.5 gm. Sod. tungstate, c.p 2.5 gm. 10 per cent sod. hydrate sol 100.0 c.c. Distilled water 100.0 c.c. Boil vigorously for twenty to thirty minutes to remove ammonia, cool, dilute to about 175 c.c., add 62.5 c.c. of concentrated (85 per cent) phosphoric acid, then dilute to 250 c.c. 4. Alkaline Copper Tartrate Solution Anhydrous sod. car- bonate, c.p 20.0 gm. Distilled water 200.0 c.c. in flask; Add Tartaric acid 3.75 gm., and when dissolved Add Crystallized copper sulphate 2.25 gm. 1 Folin and Wu. J. Biol. Cbem., 1920, xli, 367. 30 BLOOD CHEMISTRY METHODS Mix and make vol- ume to 500.0 c.c. (This is the same solution as that mentioned in Folin’s earlier method.) II. Preparation of Unknown Solution Place 2 c.c. of the blood filtrate in a Folin blood-sugar tube graduated at 25 c.c. Add 2 c.c. of alkaline copper tartrate solution. The surface of the mixture should reach the constricted part of the tube. If the bulb is too large for the volume (4 c.c.), not more than 0.5 c.c. of a diluted 1 to 1 alkaline copper tar- trate solution may be added. If this does not suffice to bring the contents to the narrow part, the tube should be discarded; likewise, if the bulb is so small that 4 c.c. fill it above the neck, the tube should be discarded. III. Preparation of Standard Solution Place in another similar tube 2 c.c. of standard sugar solu- tion equal to 0.2 mg. of dextrose and add 2 c.c. of the alkaline copper tartrate solution. IV. Final Step Place the two tubes in a cup of boiling water for six minutes. Then place tubes in a cup of tap water to cool for two or three minutes. Add to each tube 2 c.c. of the molybdate-phosphate solu- tion which dissolves the cuprous oxide usually within two min- utes. When dissolved, dilute the resulting blue solutions in the tubes to the 25 c.c. mark, insert a rubber stopper and invert the tubes to mix. This should be done carefully, since the greater part of the blue color has been formed in the bulb of the tube. It is important that the unknown and the standard tubes be heated the same length of time, and also that they be approxi- mately the same temperature when the molybdate-phosphate solution is added. In this method reoxidations of the cuprous compounds are excluded, the blank due to blue alkaline copper BLOOD SUGAR tartrate is eliminated, and the error due to so-called phenols in the blood filtrate is removed. V. Calculation The standard should be set at 10 mm. Equal volumes of unknown and standard solu- tions were used. The standard contained 0.2 mg. of dextrose. The equivalent of 0.2 c.c. of blood was used. The calculation will therefore be as follows, R indicating the reading of the unknown: 10 • ITT X 0.2 mg. = mg. in 0.2 c.c. blood, or IOOO . ITT = mg. in ioo c.c. blood. The average of many analyses of normal blood specimens has been from 80 to no mg. of sugar per 100 c.c. Blood Sugar (Picric Acid Method) (Modified from the Lewis-Benedict and Myers- Bailey Methods) Fig. 6. Folin Blood- Sugar Tube. I. Solutions Used in the Determination of Blood Sugar by the Picric Acid Method 1. Hydrochloric Acid, 2.5 per cent Solution Acid hydrochloric, cone 2.5 c.c. Distilled water to 100.0 c.c. 2. Picrate-Picric Acid Solution Dry powdered picric acid, c.p 36.0 gm. Sodium hydroxide, 1 per cent solution 500.0 c.c. Hot water 400. o c.c. 32 BLOOD CHEMISTRY METHODS Shake until dissolved and when cool add distilled water to 1000 c.c. 3. Myers-Bailey Picric Acid Sugar Standard Dextro-glucose, anhydrous, c.p 0.01 gm. Picric acid, c.p., saturated sol. in water.. 100.o c.c. 1 c.c. = 0.1 mg. sugar. (This solution keeps indefinitely.) 4. 20 Per Cent Sodium Carbonate Solution Sodium carbonate, anhydrous, c.p 20.0 gm. Distilled water to 100.0 c.c. II. Preparation of Unknown Solution Place i c.c. of oxalated blood in 15 c.c. tube; place 2 c.c. of 2.5 per cent hydrochloric acid solution in a small graduate and rinse pipette by drawing the solution up in it two or three times to remove blood adhering to wall; add the washings to the blood and shake to lake it.1 Then add exactly 7 c.c. of picrate-picric acid solution. Insert cork and shake to mix thoroughly. Filter through paper. Place 3 c.c. of the filtrate (equal to 0.3 c.c. blood) in graduated tube and add 1 c.c. of 20 per cent sodium carbonate solution. III. Preparation of Standard Solution (Myers and Bailey) Place in graduated tube 3 c.c. of the Myers-Bailey picric acid sugar standard; add 1 c.c. of 20 per cent sodium car- bonate solution. This standard contains 0.3 mg. glucose. IV. Final Step Place in cup of boiling water for ten minutes both the unknown and standard tubes. Cool the tubes to room tempera- xTwo c.c. of 2.5 per cent solution HCI, when added to i c.c. of blood when final mixture equals io c.c. will bring the acid dilution to 0.05 normal, which amount Benedict has shown (J. Biol. Cbem., 1919, xxxvii, 503) is necessary, for proper precipitation of the proteins, in addition to the picrate-picric acid solution used for that purpose. Three c.c. of the 2.5 per cent HCI will be necessary if 2 c.c. of blood are used and the final dilution equals 15 c.c. BLOOD SUGAR 33 ture and then add distilled water to each to the 10 c.c. mark. Allow ten minutes for the development of color and then com- pare in the colorimeter with standard set at 15 mm. V. Calculation R indicates the reading of the unknown. I c X 0.3 mg. = mg. in 0.3 c.c. blood, or 1500 • M T = mg. in ioo c.c. blood VI. Clinical Comments on Blood Sugar In diabetes occurring in patients with impaired kidney function the non-protein nitrogen constituents of the blood may also be increased in addition to the increase in blood sugar. The threshold of sugar excretion in the urine may also be higher. The renal threshold for sugar excretion, however, varies within quite wide limits. It has been believed that with so-called normal kidneys sugar will appear in the urine when the blood sugar reaches 170 to 175 mg. per 100 c.c. In diabetics of long standing, sugar may be absent in the urine when the blood sugar has reached 250 to 350 mg. per 100 c.c., due to the associated nephritis. In early diabetes the blood will be found to contain from 160 to 200 mg. of sugar per 100 c.c. The threshold of sugar excretion in the urine may be unimpaired when the blood sugar is only moderately increased to 160 to 170 mg. and no sugar may be found in the urine. It is important, however, to recog- nize in such patients the probability that an early diabetic state exists, regardless of the absence of sugar in the urine, since so much may be accomplished for their benefit by appro- priate dietary restrictions. For individuals with lowered ability to metabolize carbohydrates it is important to determine their capacity by performing the so-called glucose tolerance test. This consists in determining the sugar content of the blood in the fasting state (before breakfast). A urinary specimen is to be taken at this time. 34 BLOOD CHEMISTRY METHODS The patient is then given ioo gm. of anhydrous glucose dissolved in 250-300 c.c. of iced water to which the juice of a lemon has been added. Specimens of blood are taken for the determination of sugar at the end of one hour, two hours, and three hours. At the end of each of these periods the patient empties the bladder. Each specimen of urine is examined for sugar, and if found to be present the percentage is estimated in the polari- scope or by means of Benedict’s quantitative method. The patient should drink about 200 c.c. of water each hour while the test is in progress. The interpretation of the findings may be summarized as follows: For the normal individual the blood sugar is increased during the first hour after taking the glucose, reaching its maximum at that interval and falling again to normal at the end of two hours. In the pre-diabetic state the rise in blood sugar is most marked at the end of the first hour, but approaches the normal level more slowly at about the end of the three hour interval or longer. In the diabetic state the rise of the blood- sugar level is more slowly reached, the maximum amount being found in the blood at the end of two or three hours. The normal level (equal to the amount of sugar in the blood prior to the test) is reached more slowly, usually at the end of five to eight hours. In so-called “renal diabetes,” a rare condition, the blood sugar may be normal while the urine contains glucose which persists and is unaffected by carbohydrate restrictions. The kidneys are more permeable to sugar excretion than normal, that is, the “threshold” for sugar excretion is below the level of the normal sugar in the blood. In the condition known as “alimentary glycosuria” sugar may be found in the urine during the period of alimentary absorption after a meal rich in carbohydrates. The sugar will not be found in the urine during the fasting state ten to twelve hours after a meal. CHAPTER VI Blood Chlorides (Method of Whitehorn,1 using the protein-free filtrate of Folin and Wu) I. Solutions Used in the Determination of Blood Chlorides 1. Silver Nitrate Solution Dissolve 4.791 gm. of c.p. silver nitrate in distilled water and add up to the 1000 c.c. mark in volumetric flask. Preserve in dark bottles. 1 c.c. = 1 mg. Cl. 2. Sulphocyanate Solution This should be prepared volumetrically. Add about 3 gm. of potassium sulphocyanate or 2.5 gm. of am- monium sulphocyanate to 1000 c.c. of distilled water. By titration and dilution the solution should be standardized so that 5 c.c. are equivalent to 5 c.c. of the silver nitrate solution. 3. Nitric acid, concentrated, of a specific gravity of 1.42. 4. Sodium tungstate, used in the preparation of the protein- free blood filtrate. This should be free from chlorides. To test, mix one volume of sodium tungstate solution with two volumes of concentrated chloride-free nitric acid and filter into a test- tube containing silver nitrate solution. Turbidity indicates contamination with halogen. II. Method Accuracy is especially important in measuring the solutions, since slight variations in the amount of chlorides may be of significance. Volumetric flasks are recommended for the i-io dilution. With a pipette place io c.c. protein-free filtrate (equivalent to 1 c.c. blood) in a porcelain dish. Add 5 c.c. of the standard 1 Whitehorn, J. C. J. Biol. Chem., February, 1921, xlv, 449. 36 BLOOD CHEMISTRY METHODS silver nitrate solution and stir thoroughly. Then add 5 c.c. of concentrated nitric acid, mix by stirring and let stand five minutes. Then add with a spatula an abundant amount (about 0.3 gm.) of powdered ferric ammonium sulphate as indicator. Titrate the excess of silver nitrate with the standard sulpho- cyanate solution until the definite salmon red (not yellow) color of the ferric sulphocyanate persists when stirred for a few seconds. III. Calculation Each c.c. of the sulphocyanate solution used in titration is equivalent to i c.c. of the silver nitrate solution. The difference between the number of c.c. of silver nitrate solution taken and the excess by titration, i.e., 5 minus the number of c.c. of sulpho- cyanate solution titrated, will represent the volume which reacted with chloride in the ratio of 1 c.c. to 1 mg. of Cl. Since 1 c.c. of blood was used, the calculation will be 5 minus the number of c.c. of standard sulphocyanate solution used = mg. of Cl per c.c. of blood. To convert Cl figures into NaCI divide by 0.606. Multiply by 100 to obtain the result per 100 c.c. of blood. Example: It required by titration 1 c.c. of standard sulphocyanate solution to produce the end reaction. The result would be 5 minus 1=4 mg. of Cl per c.c. of blood, or 6.6 mg. sodium chloride per c.c. or 660 mg. per 100 c.c. of blood. Blood Chlorides (Method of Rieger,1 using the protein-free filtrate of Folin and Wu and based upon the principle of Rappleye) I. Solutions Used in the Determination of Blood Chlorides i. A sodium tungstate solution prepared as follows to re- move chlorides: Prepare a 10 per cent solution of sodium tung- state and after acidifying with an equal volume of concentrated nitric acid filter off the lemon-yellow precipitate. To the fil- 1 Rieger, J. B. J. Lab. & Clin. M., October, 1920, vi, No. 1. BLOOD CHLORIDES 37 trate, if clear after the addition of a few more drops of nitric acid, a few drops of silver nitrate test solution are added, which should not be turbid when viewed by transmitted light if free from chlorides. To purify the tungstate solution it is poured into a cylinder containing an equal volume of 50 per cent sul- phuric acid. The precipitate is allowed to settle and the super- natant fluid is then siphoned or poured off. The precipitated acid is then washed by decantation until the test for chlorides is no longer given. The precipitate is then dissolved in the requisite amount of 40 per cent sodium hydroxide, using 7 c.c. for each 10 gm. of sodium tungstate taken. The reaction of the resulting solution should be adjusted with dilute sulphuric acid until neutral to litmus. Enough water is then added to make a solution with sp. gr. of 1.15. This is filtered and is then ready for use as a 10 per cent neutral chloride-free solution of sodium tungstate. 2. Standard Silver Solution Silver nitrate crystals 7.2653 gm. Nitric acid, sp. gr. 1.42 250.0 c.c. Sat. sol. iron ammonium alum 50.0 c.c. Distilled water to 1000.0 c.c. 3. Ammonium Sulphocyanate Solution Ammonium sulphocyanate 0.75 gm* Distilled water 1000.0 c.c. This should be adjusted by titration so that 25 c.c. equals 5 c.c. of the silver solution. 4. Sulphuric Acid % Normal Solution Sulphuric acid 35.0 gm. by weight Distilled water to 1000. o c.c. This solution is approximately correct. II. Method Place 5 c.c. of the sodium tungstate in a 50 c.c. volumet- ric flask, add 5 c.c. rof oxalated blood and 5 c.c. of % normal sulphuric acid. The flask is well agitated and allowed to stand for one hour. Distilled water is then added to the 50 c.c. mark, 38 BLOOD CHEMISTRY METHODS the flask agitated and the contents filtered. The filtrate should be water white and give no precipitate with an equal volume of nitric acid (absence of tungstate). The presence of tungstate greatly obscures the end point in the succeeding titration. Twenty c.c. of the filtrate, which represent 2 c.c. of blood, are placed in a 50 c.c. volumetric flask. To this are added 10 c.c. of distilled water and 10 c.c. of the standard silver solution. Distilled water is then added to the 50 c.c. mark. The flask is shaken vigorously to coagulate the silver chloride. The suspen- sion is then filtered. Twenty-five c.c. of the filtrate are then titrated with the ammonium sulphocyanate solution to the appearance of the first brown tinge. The reaction is quite sharp. III. Calculation The number of cubic centimeters of sulphocyanate solution used to secure the reaction is subtracted from 25. The difference is then multiplied by 50 to obtain the number of milligrams of sodium chloride per 100 c.c. of whole blood. Example. It required 12.2 c.c. of sulphocyanate solution by titration to secure the reaction of a brown tinge to the silver chloride filtrate. Subtracting this from 25 would equal 12.8, which, multiplied by 50, would equal 640 or the number of mg. of sodium chloride per 100 c.c. of blood. IV. Clinical Comments on Blood Chlorides Under normal conditions the blood contains about 650 mg. chlorides (as sodium chloride) per 100 c.c. In chronic nephritis the content may vary between 450 and 750 mg., depending upon the ability of the kidneys to excrete. In diabetes mellitus and insipidus the chloride content of the blood is decreased because of the diuresis. In edema associated with cardiac and renal disease the chloride concentrations are usually increased be- cause of the absence of diuresis. This is especially true in that form of kidney disease designated as chronic parenchymatous nephritis or “nephrosis.” In pneumonia the blood chlorides are relatively low and correspond with the lowered urinary chloride excretion in this disease. CHAPTER VII Blood Cholesterol (Modified Method of Bloor1) I. Solutions Used in the Determination of Blood Cholesterol 1. Cholesterol Stock Solution Cholesterol (Kahlbaum) 0.2 gm. Chloroform (pure) 200.0 c.c. 1 c.c. = 1 mg. cholesterol. 2. Cholesterol Standard Solution Cholesterol stock solution 10.0 c.c. Chloroform (pure) 90.0 c.c. 1 c.c. = 0.1 mg. cholesterol. 3. Alcohol (redistilled) 4. Ether 5. Chloroform (dry) 6. Acetic anhydride 7. Sulphuric acid (concentrated) II. Preparation of Unknown Solution Place 3 c.c. of whole blood slowly (with constant shaking of the flask) in a mixture of 60 c.c. of redistilled alcohol and 20 c.c. of ether in a 100 c.c. graduated flask. Shake thoroughly. The flask is placed in a water bath on an electric hot plate and the contents carefully raised to boiling. Care should be taken not to overheat, by frequently shaking the flask. After boiling point has been reached, cool flask to room temperature, fill to the 100 c.c. mark with the alcohol-ether mixture, thor- oughly mix and filter. (The filtered liquid will keep in a tightly stoppered dark bottle until the next day if necessary before completing the final determination.) 1 Bloor. J. Biol. Cbem., 1916, xxiv, 227; 1917, xxix, 437. 40 BLOOD CHEMISTRY METHODS Place io c.c. of the alcohol-ether filtrate in a small beaker and evaporate just to dryness on a water bath or electric plate. Care should be used not to heat beyond the point of dryness, as a brownish color is produced, which renders the determination difficult. The dry residue in the beakers is then extracted with succes- sive small amounts (2 to 3 c.c.) of dry chloroform.1 The residue and chloroform suspension are brought to a boil on a water bath. Decant each time after boiling to half volume into a 10 c.c. glass-stoppered graduated cylinder or graduated test-tubes. After cooling add chloroform up to 5 c.c. The solution should be colorless. Slight turbidity does not interfere. Add 2 c.c. of acetic anhydride and 0.2 c.c. of concentrated sulphuric acid.1 Mix by inverting cylinder several times. The unknown contains the equivalent of 0.3 c.c. blood. III. Preparation of Standard Solution Place 5 c.c. of the standard cholesterol solution in a 10 c.c. glass-stoppered graduated cylinder. Add 2 c.c. of acetic anhy- dride and 0.2 c.c. of concentrated sulphuric acid. Mix by invert- ing cylinder several times. The standard solution which contains 0.5 mg. cholesterol begins to fade in about twenty minutes, so that comparison should be made within an interval of fifteen minutes. IV. Final Step Set tubes containing unknown and standard solutions aside at room temperature for about five minutes, after which place in colorimeter cups for comparison with standard set at 15 mm. An average of three or four readings should be taken. 1 Georgine Luden in her work on Cholesterol (J. Lab. & Clin. M., September, 1919, iv, 719) found that any trace of water in the chloroform interfered with the subsequent color reaction. If the chloroform is kept in a wide glass bottle, into which has been placed a quantity of calcium chloride, any trace of water will be taken up by the latter substance. The chloroform should be filtered before use. Luden has found that adding 0.2 c.c. of concentrated sulphuric acid instead of o. 1 c.c., as generally recommended, produces a tone of green that can be matched more readily without interfering with the cholesterol values. BLOOD CHOLESTEROL 41 V. Calculation X 0.5 mg. = mg. in 0.3 c.c. blood, or = mg. in ioo c.c. blood VI. Clinical Comments on Blood Cholesterol The origin of blood cholesterol is not definitely known. Some of it arises from exogenous and some from endogenous sources. It is subject to quite wide variations under conditions of disturbed metabolism. The test should not be regarded as a diagnostic test, but rather as a clinical test similar to the deter- mination of hemoglobin or the test for albumin in the urine. The normal amount of cholesterol present in whole blood varies between 160 and 200 mg. per 100 c.c. In the plasma the figure is higher, averaging about 230 mg., while the corpuscle content averages about 200 mg. per 100 c.c. In mild diabetes the cholesterol content of whole blood is usually increased to 240 to 250 mg. per 100 c.c., while in severe types of the disease the content is increased to 350 to 410 mg. per 100 c.c. The increase of the cholesterol content in diabetes is relatively proportionate to the increase of fat and total fatty acids of the blood in this disease. In cholelithiasis due to cholesterin concretions, the blood cholesterol is sometimes increased to 280 mg. or more, 950 mg. being mentioned by Hawk. This latter figure may be considered unusual, for many instances of proven cholelithiasis have not shown an increase in blood cholesterol above the range of normal values. The problems of disturbed cholesterol metabolism are concerned especially with the activity and quantity of bile and pancreatic juice available to convert cholesterol into esters. The adrenals and other glands are also concerned in the process. De Zani’s experiments, mentioned by Georgine Luden in her work on cholesterol, have shown the importance of this lipoid 42 BLOOD CHEMISTRY METHODS to the cellular integrity of the organism. He fed mice on a cholesterin-free diet. During this time the animals drew upon their reserve deposits of cholesterol present in the body fat, brain, adrenal cortex and the liver, for cholesterol was present in the feces. The mice increased in size and weight, but they all died at the end of seventeen days. The recent work of Epstein and Lande1 has called attention to the importance of blood cholesterol studies in connection with protein deficiency and decreased basal metabolism, especially in parenchymatous nephritis (nephrosis). In their observations on 6 such patients the basal metabolism was sub- normal and was associated with high blood cholesterol figures. In these patients with large amounts of albumin in the urine and decreased output associated with extensive edema, and who did not improve upon salt-free, Karrell, and carbohydrate diets, an improvement in basal metabolism and decreased edema was noted under a high protein diet together with the administration of thyroid extract. In myxedema, which may be associated with nephrosis, the blood cholesterol may be increased. Under thyroid therapy with improvement in the basal metabolic rate in such cases the blood cholesterol has decreased. In exophthalmic goiter with marked increase in the basal metabolic rate, as well as in toxic adenomas of the thyroid, the blood cholesterol values are usually lower than normal. The following table arranged from the publication of Epstein and Lande recapitulates their findings: 1 Epstein, A. A., and Lande, H. The relation of cholesterol and protein defi- ciency to basal metabolism. Arcbiv. Int. Med., November, 1922. BLOOD CHOLESTEROL 43 Blood Cholesterol Content in Relation to Metabolic Rate Condition Number Observa- tions Basal Metabolic Rate Average Cholesterol in Mg. Per ioo c.c. Blood Method Parenchymatous nephritis (nephrosis). 6 subnormal 510 Bloor modification of Funk-Auten- Chronic diffuse nephritis. 4 subnormal 300 reith method. Myxedema i -19% 1350 (decreased to 206 under thy- roid therapy) Bloor modification of Funk-Auten- reith method. Myxedema i -14% 313 Non-toxic adenoma of thyroid. 6 normal 176 Bloor modification of Funk-Auten- Menopause IO normal 234 reith method. Exophthalmic goiter. 21 +44% 144 Bloor modification Toxic adenoma of thyroid. IO +29% 182 of Funk-Auten- reith method. Since a large reserve of cholesterol is present in the cells of the body and since practically all staple foods contain it in varying amounts, it is not likely that a marked deficit occurs in the human body except under conditions of starvation, in the presence of wasting diseases such as carcinoma and Addison’s disease, or the fatal cases of exophthalmic goiter. The diet most suitable for patients with excessive amounts of blood cholesterol but with normal metabolic rates, should consist of fruit, vegetables, and milk, eliminating largely those foods which are known to contain high cholesterol values, such as eggs, meats, and fish, since with normal metabolic rates there probably will be no deficiency in protein utilization. An excess of carbohydrates in the diet calling for excess work on the part of the pancreas may under certain conditions prevent proper esterification of the blood cholesterol and its elimination, 44 BLOOD CHEMISTRY METHODS with resulting concentration of cholesterol in the blood. This was observed by Luden in her experimental oatmeal diet. For patients, however, who manifest disturbances associated with subnormal metabolic rates and increased blood cholesterol, the combination of high protein diet and thyroid therapy should be tried on the basis that the possible benefit from the adminis- tration of thyroid stimulates the rate of oxidation and promotes the assimilation of protein, the utilization of which is impaired. As mentioned by Epstein and Lande, the effect of thyroid in promoting utilization of protein may explain its influence on edema, especially in parenchymatous nephritis. CHAPTER VIII Total Nitrogen Determination in the Urine (Modified from the Method of Folin) 1. With a measuring pipette place 0.2 c.c. filtered urine and 0.5 c.c. of distilled water in a Pyrex ignition test tube. With an ordinary pipette add 0.5 c.c. of the phosphoric- sulphuric-acid digestion mixture, made as follows: Acid Phosphoric-Sulphuric Digestion Mixture Copper sulphate solution (5 per cent).... 50.0 c.c. Acid phosphoric (85 per cent) 300.0 c.c. Acid sulphuric, c. p. (free from ammonia).. 100.0 c.c. Distilled water 450.0 c.c. This should be kept well stoppered to prevent absorp- tion of ammonia from the air. (Same as Solution 1 for blood non-protein nitrogen determination.) 2. Heat slowly over a micro-burner until the water is driven off and the color changes to dark brown, then cover tube with watch glass and continue heating gently until dense fumes fill the tube. Continue boiling at such a rate that the tube con- tains fumes but almost no fumes escape. The color should become clear or bluish-green after partially cooling. A drop of hydrogen peroxide may be added to clear the solution while the tube is warm. Remove flame and let cool for about two min- utes. Rinse the tube thoroughly (the contents may be turbid from silica) with a little distilled water into a 25 c.c. volumetric flask. Shake to mix. 3. Transfer with a measuring pipette 1 c.c. of standard nitrogen solution (used in the determination of non-protein nitrogen in the blood, and which contains 0.2 mg. N per c.c.) into a 50 c.c. volumetric flask, add 1 c.c. of the phosphoric- sulphuric acid mixture to balance the acid in the unknown, 46 BLOOD CHEMISTRY METHODS add about 10 c.c. of distilled water and shake to mix. The standard so prepared contains 0.2 mg. nitrogen. 4. When ready give each flask a whirl and add about 10 c.c. of Nessler’s reagent to the unknown, and about 20 c.c. to the standard. When full development of color has been secured add sufficient distilled water to bring volume in the unknown to the 25 c.c. mark and in the standard to the 50 c.c. mark. 5. If the unknown Nesslerized mixture is turbid from silica, centrifuge a portion before the color comparison is made. The white sediment in the tube is silica. If the sediment is deeply colored the Nesslerization was not successful and should be discarded. Wait five minutes for color to develop before com- paring in the colorimeter. 6. With standard set at 20 mm., with R indicating the read- ing of the unknown solution, the calculation will be as follows 20 w 0.2 mg. XT . X -—-— = mg. N in 0.2 c.c. urine, or iooo ,T . — = mg. N in ioo c.c. urine. 7. Example: If the twenty-four hour quantity of urine equaled 1560 c.c. and the reading of the unknown was 15, the result would be *-*■ X 15.6 = 1040 mg. or 1.04 gm. l5 Titratable Acidity of Urine, in Terms of N/io NaOH (Method of Folin) 1. Place io c.c. of urine in a flask, add about 6 gm. of finely pulverized neutral potassium oxalate,1 and i to 2 drops of i per cent phenolphthalein solution as indicator. 2. Shake solution vigorously one to two minutes and titrate with N/io NaOH until the solution turns a faint pink which remains permanent when the solution is shaken. 3. If 1200 c.c. represents the twenty-four hour volume of urine, and 6 represents the number of cubic centimeters of N/10 NaOH used, the total acidity will be calculated as follows: 1 Potassium oxalate is added to precipitate the calcium which would interfere with the titration end-point when the urine is neutralized. PHTHALEIN ELIMINATION 47 10:6:: I200:x or, iox = 7200 or, x = 720, the acidity of the twenty-four hour urine in terms of cubic centimeters of N/10 NaOH. The acidity may also be represented in terms of percentage. In the example above the acidity per 100 c.c. in terms of N/10 NaOH would be 60. Phenolsulphonephthalein Determination of Kidney Function 1. Have patient empty bladder and then drink 300-400 c.c. of water. 2. Twenty minutes later inject 1 c.c. (from ampule con- taining 6 mg.) of phenolsulphonephthalein into gluteal or lumbar muscles. 3. One hour and ten minutes later have patient empty bladder (ten minutes of this time interval is to allow the dye to reach the kidneys). Add 200 c.c. water to urine and 1 c.c. of 10 per cent sodium hydroxide solution to bring out the deep purple color, then dilute to 1000 c.c. Label “first-hour” specimen. 4. One hour later have patient empty bladder. Add 200 c.c. of water to the urine and 1 c.c. of 10 per cent sodium hydroxide solution, then dilute to 1000 c.c. Label “second-hour” specimen. 5. The standard solution required contains 3 mg. phenol- sulphonephthalein and 1 c.c. of 10 per cent sodium hydroxide solution and volume made to 1000 c.c. 6. Set standard in colorimeter at 10 and make comparison with first- and second-hour specimens. R equals reading of unknown. The calculation will be X 50 or = per cent of dye excreted. 7. The first-hour specimen should equal 40-60 per cent; the second-hour specimen 20-25 per cent more, or a total in the two-hour period of 60-85 per cent. An average of many- so-called normal estimations has equaled 48 per cent for the first hour and 17 per cent for the second hour, or a total of 65 per cent. CHAPTER IX Clinical Comments on the Diagnosis of Impaired Kidney Function Degenerative processes affecting the cardiovascular renal systems, produced by many processes, including acute and chronic infections as well as the wear and tear incidental to life, are such important factors leading to death before the expira- tion of the normal life expectancy, and constitute such an impor- tant part of the work of the physician in his endeavor to prevent the development of such lesions, that an understanding of this phase of clinical medicine is essential in the care of prac- tically every patient who has reached the fifth decade of life. The endeavor to determine what factors are concerned in producing evidence of early damage to these organs involves in practically every case knowledge of the blood chemistry as well as an investigation of kidney function in a more compre- hensive manner than such tests are usually performed. A little time and a comparatively small outlay spent in equipping a laboratory, as well as training a young woman in the essentials of the work, will make available for every physician the information desired. The reward in added satisfaction that the advice given is founded upon fact, rather than upon the basis of a casual examination of a single urinary specimen, will be many times worth the time consumed and the effort used. Among the essential facts to be determined in an examination of kidney function are the following: The Urine Examination i. In the absence of edema the quantity of urine voided varies normally with the intake of fluid. Variations in humidity, the amount of physical exercise, and the occurrence of diarrhea may be mentioned as factors disturbing the normal ratio of CLINICAL COMMENTS 49 intake to output. Normally the quantity of urine voided during the twelve-hour night period should not exceed 50 per cent of the quantity voided during the twelve-hour day period. 2. The specific gravity should vary between 1.010 and 1.030, depending upon the amount of fluid taken, the humidity and other factors, such as exercise and nervous or emotional stress. Normally there should be evident no tendency toward so-called fixation of specific gravity toward either the higher or the lower points in a series of specimens passed at different periods during the twenty-four hours. 3. All specimens should normally be free from abnormal ingredients such as albumin, sugar, diacetic acid, casts, blood- cells and pus. A few leucocytes in specimens from patients who have suffered a previous inflammatory or traumatic lesion in any portion of the genitourinary tract may be found for long periods subsequently. Specimens from women who have borne children and who have relaxed bladder walls normally contain a considerable number of leucocytes. The finding of an occa- sional hyaline cast in a centrifugated specimen, especially from an individual near the middle point of life, may be ignored. 4. The urea content of any specimen should be 2 per cent or higher. The hypobromite method for urea is sufficiently accu- rate for clinical purposes. If the patient has been under super- vision with weighed diets so that the total intake of protein in grams is known (from which the nitrogen intake can be com- puted by dividing by 6.25) it aids in an understanding of the patient’s condition if the “nitrogen balance” is determined by a computation of the total nitrogen eliminated in the twenty- four hour specimen of urine. 5. The output of sodium chloride in any specimen varies according to intake from 1 to 2.5 per cent or higher. The Volhardt method is satisfactory. A modification of this method may be performed as follows: Dilute 10 c.c. of urine with 90 c.c. of water to which should be added 1 or 2 drops of 25 per cent nitric acid. The mixture should then be made alkaline with 10 per cent solution of sodium carbonate. A few drops of 50 BLOOD CHEMISTRY METHODS a io per cent solution of potassium chromate solution are added as an indicator. Titration is then performed with N/io silver chloride solution. Each cubic centimeter of the silver solution used equals 0.00583 gm. of sodium chloride. 6. The output of phthalein as a measure of the excretory function of the kidneys should under normal conditions approxi- mate 40 to 60 per cent during the first hour, and 20 to 25 per cent during the second hour, or a total of 60 to 85 per cent during the first two hours. 7. The usual tests for acetone bodies in the urine are, as Folin has shown, tests for diacetic acid. The sodium nitro- prusside test is more delicate than the ferric chloride reaction. These tests, when persistently positive, indicate disturbed metabolism dependent usually upon dehydration and so-called “acid poisoning.” The administration of salicylates and coal- tar products such as phenacetine may produce a diacetic reac- tion in the urine. The Blood Examination i. The test diet of Mosenthal designed to measure the excretory capacity of the kidneys has served a useful purpose in calling attention to phases of the subject not generally appre- ciated. For its proper interpretation most careful attention to detail is necessary, conditions being obtained, as a rule, only with the cooperation of a trained dietician during the period of obser- vation. Under this plan blood chemistry studies and deductions incidental to the test diet and measured fluid intake are correlated with examinations of the measured urinary speci- mens, all of which has for its purpose the determination of kidney function under more or less artificial conditions. There are many reasons for believing, as Mosenthal has recently stated,1 that if the various phases of renal function are studied while the patient is following his usual daily routine, a more valuable estimate of how he should adjust his habits to the condi- tions found to exist may be obtained than by carrying out arti- 1 Mosenthal, Herman O. Value of tests for renal function in clinical medicine. Ohio M. J.. Columbus, May, 1922. CLINICAL COMMENTS ficial dietetic tests that impose standards which may not be applicable or approximate the normal daily routine of the individual. It is obvious that in carrying out any investiga- tion the ordeal must be simplified as much as possible for the patient. For ambulatory patients the following plan is recom- mended: For the twenty-four hours preceding the time set for taking the sample of blood before breakfast, the patient collects the urine specimens as follows: Upon arising the bladder is emptied. This urine is discarded. All specimens passed from 8 a.m. to and including 8 p.m. are measured and a portion of each placed in bottles which are to be brought to the laboratory. The urine passed after 8 p.m. to 8 a.m., including the amount voided upon arising, is collected in one container, measured, and a portion labeled “ 8 p.m. to 8 a.m.” is brought to the laboratory. The patient is instructed to drink no fluid after the evening meal. If possible a record should be kept of the amounts of fluid taken during the day. For many patients it is only necessary to collect and measure the urine passed during the day and night twelve-hour periods, a portion of each being brought for examination. The data accumulated in such an examination may conveniently be summarized in the form of a chart which includes the blood chemistry findings. 52 BLOOD CHEMISTRY METHODS Day quantity After 8 a.m.-8 p.m. Night quantity After 8 p.m.-8 a.m. Urine Specimens Quantity c.c. Specific gravity of different specimens. Greatest variation in specific gravity = Total nitrogen output 24-hour quantity = Total sodium chloride 24-hour quantity = Abnormal ingredients, Albumin = Sugar = Diacetic acid = Chlorides = Microscopic Phthalein output, first hour = Fasting blood chemistry Non-protein nitrogen = Urea = Preformed creatinin = Uric acid = Sugar = Chlorides = Cholesterol = per cent; second hour = mg. per 100 c.c. blood. per cent CHAPTER X The Dietary Control of Disturbances of Metabolism I. Neutral, Alkali and Acid-Producing Foods The following lists of foods useful in the treatment of certain disturbed metabolic states are appended. They are based upon the ash analyses of Sherman and Gettler and have been tested on man by Blatherwick.The first three have been published by Sansum of the Potter Metabolic Clinic. i. Neutral Foods Butter Cream Lard Cornstarch Sugar Tapioca 2. Alkali-Producing Foods In the following list the excess of base or alkali over acid is expressed in terms of cubic centimeters of a normal solution. FRUITS Per 100 Gm. Raisins • 23.63 Muskmelons • 7-47 Pears, dried • 7-07 Oranges . 6.61 Currants • 5- 97 Bananas . 5.56 Lemons • 5-45 Peaches . 5.04 Apples • 376 VEGETABLES Beans, lima, dried . 41.6s Beans, dried • 23.87 Beets Carrots . 10.82 Celery • 7.78 Lettuce • 7-37 54 BLOOD CHEMISTRY METHODS VEGETABLES (Cont.) Per ioo Gm. Potatoes 7.19 Cauliflower 5-33 Cabbage 4-34 Radishes 2.87 Turnips 2.68 Asparagus 0.81 NUTS Almonds 12.38 Chestnuts 7.42 MISCELLANEOUS Cow’s milk 2.37 3. Acid-Producing Foods In the following list the total excess of acidity over base is expressed in terms of cubic centimeters of a normal solution. The ash of these foods is alkaline in reaction, but since they contain benzoic acid, which is changed to hippuric acid before elimina- tion, the body acidity is increased when these foods preponder- ate in the diet. MEATS AND FISH Per ioo Gtn. Oysters . 30.0 Chicken . 17.0 Haddock . l6.0 Rabbit . I4.8 Beef, lean • 13-9 Veal • 13-5 Pike . 11.8 Pork, lean . 11.8 Frog . 10.3 EGGS Yolk . 26.6 Eggs, whole . 11.1 CEREALS Oatmeal . 12.9 Rice . 8.1 Crackers . 7.8 Corn, sweet, dried.... • 5-9 Bread, whole wheat... . 3.0 Bread, white . 2.7 DIETARY CONTROL 55 NUTS Per. 100 Gm Peanuts 3-9 FRUITS Cranberries Prunes Plums For reference the following table has also been found useful. In it the excess of base or alkali over acid in ioo calories of each food is given: Spinach 113.0 Cucumbers 45-5 Celery 41.1 Chard 41.1 Lettuce 38.6 Rhubarb 37. o Figs 32.3 Tomatoes (canned) 24.5 Carrots 24.0 Beets (fresh) 23.6 Molasses 20.0 Muskmelons 19.0 Olives 18.8 Parsnips 18.2 Cabbage. 18.0 Cauliflower 17.4 Pineapple 15.7 Orange-juice 14.4 Beans (string) 13.0 Raspberry juice 13.0 Peaches (fresh) 12.2 Lemons 12.0 Oranges n.o Lemon-juice n.o Apricots n.o Peaches (canned) 10.o Radishes 9.8 Mushrooms 8.9 Watermelon 8.8 Potatoes (white) 8.6 Prunes 8.0 Cherries 7.8 Plums 7.3 Turnips 7.0 Raisins 6.8 Buttermilk 6.1 Squash 6.1 Apples (fresh) 6.0 Pumpkin 5.7 Bananas 5.6 Pears (fresh) 5.6 Potatoes (sweet) 5.4 Milk (skimmed) 5.0 Beans (baked) 5.0 Grape-juice 4.0 Potatoes (chips) 3.9 Cranberries 3.7 Asparagus 3.6 Chestnuts 3.2 Dates 3.2 Onions 3.1 Citron 3.0 Koumiss 2.9 Grapes 2.8 Milk (condens. unsw.) 2.7 Milk (whole) 2.6 Beans (kidney) 2.5 Pears (canned) 2.3 Almonds 1.8 Currants 1.8 Peas (canned) 1.5 Peas (dried) 1.5 Milk (condens. sweet) 1.4 Cocoanuts 1.2 Peas (green) 1.2 Cream (18% fat) 0.3 Cream (40% fat) 0.1 Marmalade o. 1 Cocoa 0.1 Koumiss 56 BLOOD CHEMISTRY METHODS II. Minimum Salt and Basic Alkali Diet For general purposes the following list of foods has been useful in the treatment of edema in chronic nephritis of the types associated with hyperarterial tension. In this list the content of salt is small, the protein restricted, and an excess of alkali-producing foods is present in the amounts ordinarily consumed. The amount of fluid intake should be restricted to 700-1000 c.c. daily. The following foods are allowed: Bread or toast (made without salt), sugar, farina, custard, fresh butter, rice, tea, milk (250 c.c. daily), baked apples or apple sauce, orange-juice, melons, grape-fruit, lettuce, jello, bananas, prunes. All other green vegetables may be allowed but should be boiled in two waters to remove the salt. III. Clinical Comments The accumulated experience of many physicians has shown that patients who have disturbed kidney function, definite nephritis, hyperarterial tension, rheumatic symptoms (when due to improper diet), headaches due to overfeeding, lack of exercise and improper elimination, obscure neuralgic and neuri- tis-Iike pains (not due to foci of chronic infection), and other similar conditions, are generally benefited by restriction of the meats and eggs. This benefit aside from the restricted albumin intake may with much reason be ascribed, especially in most typical types of chronic nephritis and the associated acidosis, to the influence of diminished acid formation, the deleterious effect of which upon the kidneys has been thoroughly investi- gated by Martin Fischer. The only apparent exceptions to such a regime as regards the protein intake are to be found in the treatment of certain types of chronic nephritis in which sub- normal basal metabolism may be present, and in which it may be believed that impaired utilization of protein exists. This has been discussed under blood cholesterol. It will be noted from the above list that certain cereals are acid-producing foods. From a practical standpoint, if the diet is largely made up of the base or alkali-producing foods given DIETARY CONTROL 57 above, the moderate consumption of breadstuffs will not greatly influence the result desired. In general, with the exception of cranberries, prunes and plums, all fruits, vegetables, and nuts (with the exception of peanuts) are basic (alkaline) in nature. The following foods, among those mentioned in the list above, have been found useful clinically in reducing the body acidity as measured by the reaction of the urine: Melons, apples, oranges, bananas, lemons, carrots, beets, lettuce, celery and potatoes. The fruit acids are largely converted into alkali carbonates in the intestine, hence they may be considered alkaline in nature. The neutral foods do not increase the body acidity and may be used in ordinary amounts unless special reason for restriction, such as acidosis, exists, in which case the intake of fats, such as cream, butter, and lard, should be limited. For ordinary clinical control I have found it useful to give to the patient or nurse a supply of methyl-red papers, after a suggestion by Martin Fis- cher, by means of which the reaction of the urine may be tested once or twice daily. IV. The Purin Constituents of Foods Since it appears highly desirable to limit the intake of purin- containing foods in the treatment of conditions due to abnormal retention of uric acid in the blood, the following table of Walker’s, quoted in the Presbyterian Hospital (New York) Diet Lists, is added: I Meats Purin bodies, gm. per kilo. Beef I.3-2-0 Pork 1.2 Chicken Ham Veal Salmon i. I Halibut Mutton Cod 0.5 Meat soups 58 BLOOD CHEMISTRY METHODS Vegetables Pur in bodies, gm. per kilo. Beans, kidney Oatmeal o-53 Peas 0.39 Lentils 0.38 Asparagus Drinks Per 500 c.c. Coffee 1.7 Tea 1.2 Cocoa 1.0 Chocolate 0.7 The foods listed in the above table should be limited in conditions associated with uric acid retention. In addition to those mentioned, the glandular meats, such as sweetbreads, liver, kidney, and brain; whole-wheat products, such as graham or whole-wheat bread or shredded wheat; and malt prepara- tions, such as beer, ale or porter, should be restricted. The following foods are purin-free or contain a negligible amount:1 Cereals Rice Hominy Farina Cream of Wheat Potatoes (Irish or sweet) Cauliflower Onions Cabbage Lettuce Vegetables Eggplant Spinach Brussels sprouts Corn Breadstuffs Flour (white) Bread (white) Corn meal Spaghetti Macaroni Biscuits (white) Crackers (white) Desserts Nuts Cheese (American, Swiss, and Cream) Ice cream and ices Cake (except coffee or chocolate) Puddings (bread, tapioca, or cornstarch) Pie (apple, custard, or cocoanut) Dairy Products Butter Milk Buttermilk Cream Miscellaneous Jam and marmalade Sugar and syrup Fresh and cooked fruits Bacon Soups (cream or vegetable) Eggs 1 From Von Noorden & the Vanderbilt Clinic Diet Lists. DIETARY CONTROL 59 Beverages Carbonated water Vichy Grape-juice Loganberry-juice Cider Malted milk The following “moderate protein-low purin” diet, consisting largely of neutral and alkali-producing foods has been found useful in the treatment of nephritis. It contains approximately 2000 calories, an amount sufficient to maintain efficiency for the ambulatory patient who is obliged to do a moderate amount of work. For obese patients a diet may be appropriately fol- lowed which consists largely of fruits and vegetables. Moderate Protein-Low Purin Nephritic Diet Wt. in gm. or c.c. Pro- tein Fat Carbo- hydrate Cal- ories Remarks Breakfast I egg 6.2 5-6 76.0 Farina (cooked) 100 1.7 0.2 11.5 56.0 If 100 gm. hominy are used prot. = 2.2; fat = 0.2; carbo. 17.8; calories 84. If 100 gm. boiled rice, prot. 2.8; fat 0.1; carbo. 24.4; calories 112 is ISO 60.0 Cream (20 per cent) 90 3.0 18.0 30 180.0 Usual market cream Toast (white bread 2 slices).. 60 5-5 0.8 32 .0 160.0 Butter is 0. IS 12.7 119.0 If edema is present use fresh butter Orange or peach marmalade (1 hp. tablespoonful) 30 0.18 25 -3 105.0 Luncheon Cream vegetable soup 250 6.4 18.0 14.6 242.0 Made without meat stock from com, potatoes, asparagus, celery, cauli- flower, thickened with cornstarch Butter is 0.15 12.7 119 0 Bread (white 2 slices) 60 5-5 0.8 32.0 160.0 If edema is present use bread made without salt 60 BLOOD CHEMISTRY METHODS Moderate Protein-Low Purin Nephritic Diet Dinner 6o I .0 2.0 12.0 Vegetables with 3 per cent available carbohydrate used, which include the following: Asparagus, cauliflower, Brussels sprouts, beet greens, cab- bage, rhubarb, spinach, string beans, eggplant. If vegetables with 6 per cent available carbohy- drate are used, such as the following: Onions, squash, turnips, carrots, mushrooms, or beets, add 1.0 gm. protein and 2.0 gm. carbohydrate to in- take and 12 calories Potato (baked) med. size.... 130 37 0.2 32.0 14s.0 180 6.0 7.2 9.0 130.0 so 0.6 0.15 1.5 10.0 French dressing (i tbsp.) 22 16.0 150.0 Made in proportion 4 tbsp. olive or other vegetable oil, 1 tbsp. lemon juice, teasp. salt 30 2.75 0.4 16.0 80.0 IS 0.15 12.7 119.0 120 0.6 0.6 30.0 128.0 3 heap. tbsp. apple sauce contains prot. 0.2s; fat 1.0; carbo. 46, calories 194 Totals 43-5 106.0 224.0 2051.0 CHAPTER XI The Test and Maintenance Diets of Joslin in the Treatment of Diabetes Mellitus So much improvement in the management of diabetic patients has followed the teachings of Joslin that a brief resume of his plan of treatment is appended. The following test diets were designed by him for use by the patient to reduce gradually the amount of sugar present in the urine until the urine is sugar- free, and to decrease the amount of sugar present in the blood. When this has occurred the Maintenance Diets are begun, to establish tolerance and a sugar-free state. As Joslin has stated, this plan is an arbitrary one and the majority of cases will require some modification of it. Much more can be accom- plished, however, by following such an established guiding principle than by attempting to secure the result desired by less laborious short-cut methods, which in the end react little to the benefit of the patient or the credit of the physician. The Test Diets are to be used for a period of five days during which the patient gradually becomes sugar-free. On successive days advances are to be made from Test Diet No. i to Test Diet No. 5. If on the fifth day the urine still contains sugar, fasting can be employed for one or more days. During the fast days clear broths, water, clear coffee, tea, and whiskey or brandy may be allowed. The Maintenance Diets are to be begun as soon as the patient’s urine does not contain sugar. If this should occur on the fifth day while the patient is taking Test Diet No. 5 he may begin the next day on Maintenance Diet No. 1. The plan will then carry him through on successive days the use of Main- tenance Diets 2, 3, 4, etc. until sugar appears in the urine. Certain patients can progress steadily day by day until Main- tenance Diet No. 10 or No. 12 is taken without showing sugar. If sugar should appear in the urine, for example, while the patient is taking Maintenance Diet No. 6, one should drop back 62 BLOOD CHEMISTRY METHODS in the amount of carbohydrate allowed to that contained in the diet two days earlier, i.e., to Maintenance Diet No. 4, while still continuing the same amounts of protein and fat allowed in Diet No. 6. Such a diet should perhaps be continued for two or three days or until the urine is again sugar-free. The patient’s approximate carbohydrate tolerance may then be assumed to have been established. The tolerance may not always be maintained at this point; for a variety of conditions, such as an acute infection, may upset it. The next step should be to increase the amount of protein and fat allowed as follows: If the patient can take the amount of carbohydrate allowed in Maintenance Diet No. 4 and the amount of protein and fat allowed in No. 6, the attempt should be made to increase the calories of protein and fat until he can take the amounts allowed in Diet No. 7 or Diet No. 8, the aim being to furnish a total of 52 to 30 calories for each kilogram (2.2 lbs.) of patient’s body weight, which amount is sufficient for maintenance “at rest.” If in carrying out the Test Diets it should be found that the patient becomes sugar-free on Diet No. 3, it will not be necessary to begin with Maintenance Diet No. 1, but instead one may begin with Maintenance Diet No. 6, which contains an amount of carbohydrate about equal to that contained in the Test Diet upon which the patient became sugar-free. Experience with the recently discovered substance insulin, which has been found of such great value in making it possible for the diabetic patient to utilize more carbohydrate, will modify these dietary suggestions according to the need of the patient and the judgment of the physician. test and maintenance diets 63 ** c. P. F. 5 per cent vegetables 10.0 5.0 0.0 Orange 26.0 1.8 0.0 Skimmed milk 25.7 17.2 i-5 Fish (halibut, mackerel) 0.0 24.0 4.8 Potato 48.0 8.0 0.0 Meat (cooked, lean) 0.0 24.0 15.0 Bread 54.0 9.0 0.0 Oatmeal (dry weight) 20.0 50 2.0 Cream “average” 2.1 2.25 12.0 Bacon 0.0 5.0 15.0 Butter 0.0 0.0 25.0 Cottage cheese 2.0 12.0 0.0 Egg (one) 0.0 6.0 6.0 Shredded wheat biscuit 22.6 30 0.4 Uneeda crackers 8.7 1.2 1.1 The total sugar-forming constituents of a diet should be computed as 100 per cent of the carbohydrate, 58 per cent of the protein and 10 per cent of the fat intake. The fat used should not exceed twice the carbohydrate plus one-half the protein (Woodyatt formula). * The computations are figured from the publications of Joslin and from Locke’s “Food Values.” Approximate Weight Equivalents* (Used in following tables) i gm. carbohydrate = 4 calories. 1 gm. protein = 4 calories. 1 gm. fat = 9 calories. 64 BLOOD CHEMISTRY METHODS Test diets Weight in grams Vege- tables, 5 per cent Orange Oat- meal* Shred- ded wheat Uneeda Potato Bread Egg Cream, 20 per cent Bacon But- ter Meat Fish Skimmed milk No. i Carbo 189 Protein 89 Fat is Calories 1247 300 300 I 240 90 90 120 480 No. 2 Carbo 102 Protein 58 Fat 0 Calories 640 300 300 I 120 180 300 No. 3 Carbo 64 Protein 33 Fat 0 Calories .... 388 300 300 60 90 240 No. 4 Carbo 36 Protein 27 Fat 0 Calories 252 300 200 90 120 No. s Carbo 15 Protein 5 Fat 0 Calories 80 300 50 Test Diets * Oatmeal always measured by dry weight. TEST AND MAINTENANCE DIETS Mainte- nance diets Weight in grams Carbohydrate Protein and Fat Vege- tables, S per cent Orange Oat- meal* Shred- ded wheat Uneeda Potato Bread Egg Cream, 20 per cent Bacon But- ter Meat Fish Skimmed milk No. i Carbo io Protein n Fat 6 Calories 138 300 1 No. 2 Carbo 22 Protein 13 Fat 18 Calories 302 300 100 I 60 No. 3 Carbo 32 Protein 24 Fat 24 Calories 440 600 100 2 60 No. 4 Carbo 42 Protein 29 Fat 39 Calories 63 s 600 200 2 60 30 No. 5 Carbo 52 Protein 32 Fat S3 Calories 813 600 200 IS 2 60 30 IS Maintenance Diets * Oatmeal always measured by dry weight. 66 BLOOD CHEMISTRY METHODS Mainte- nance diets Weight in grams Carbohydrate Protein and Fat Vege- tables, S per cent Orange Oat- meal* Shred- ded wheat Uneeda Potato Bread Egg Cream, 20 per cent Bacon But- ter Meat Fish Skim med milk No. 6 Carbo 63 Protein 43 Fat 6s Calories 1009 600 200 30 2 90 30 IS 30 No. 7 Carbo 73 Protein Si Fat 70 Calories 1126 600 300 30 2 90 30 IS 60 No. 8 Carbo 83 Protein 60 Fat 88 Calories 1364 600 300 30 2 2 90 30 30 90 No. 9 Carbo 96 Protein 63 Fat 94 Calories 1482 600 300 30 X 2 2 120 30 30 90 No. io Carbo 107 Protein 64 Fat 94 Calories 1530 600 300 30 I 2 2 120 30 30 90 Maintenance Diets (Continued) * Oatmeal al ways measured by dry weight. TEST AND MAINTENANCE DIETS 67 Mainte- nance diets Weight in grams Carbohydrate Protein and Fat Vege- tables, 5 per cent Orange Oat- meal* Shred- ded wheat Uneeda Potato, Bread Egg Cream, 20 per cent Bacon But- ter Meat Fish Skimmed milk No. ii Carbo 131 Protein 76 Fat 99 Calories 1719 600 300 30 1 2 120 2 120 30 30 120 No. 12 Carbo 155 Protein 80 Fat 99 Calories 1831 600 300 30 I 2 240 2 120 30 30 120 Maintenance Diets (Continued) * Oatmeal always measured by dry weight. 68 BLOOD CHEMISTRY METHODS Lettuce Cucumbers Spinach Asparagus Rhubarb Endive Marrow Sorrel Sauerkraut Beet greens Dandelion greens Swiss chard Celery Mushrooms Tomatoes List of 5 Per Cent Vegetables* and Fruits Brussels sprouts Water cress Sea kale Okra Cauliflower Egg plant Cabbage Radishes Leeks String beans, canned Broccoli Artichokes, canned Ripe olives Grape fruit List of 10 Per Cent Vegetables* and Fruits String Beans Pumpkin Turnip Kohl-rabi Squash Beets Carrots Onions Green peas, canned Watermelon Strawberries Lemons Cranberries Peaches Pineapple Blackberries Gooseberries Oranges List of i 5 Per Cent Vegetables* and Fruits Green peas Artichokes Parsnips Lima beans, canned Raspberries Currants Apricots Pears Apples Huckleberries Blueberries Cherries List of 20 Per Cent Vegetables* and Fruits Potatoes Shell beans Baked beans Green corn Boiled rice Boiled macaroni Plums Bananas Prunes * Fresh or canned. DIABETIC MANAGEMENT 69 Clinical Comments on Diabetic Management The discovery of insulin has made it no longer necessary to maintain diabetic patients in a state of undernutrition. In be- ginning treatment it has not been found wise to depend too much upon household or approximate measurements. It will be found much more satisfactory to use a food scale.1 In the treatment of patients with severe diabetes, especially those who manifest evidences of more or less disturbed kidney permeability, many difficulties may arise. In such patients it may be difficult to decrease the blood sugar to near the normal amount, although sugar may not be constantly present in the urine because of the higher renal threshold for sugar elimina- tion. If the endeavor is made to restrict the carbohydrates for many such patients to a minimum, serious symptoms of acido- sis may arise. The most important of such symptoms are: The presence of the so-called acetone odor to the breath, the presence of diacetic acid in the urine, the presence of hyperpnea or of nausea or vomiting, and an early tendency to sluggish speech or sleepiness. A high protein, high fat and low carbo- hydrate diet is a combination upon which many such patients do not do well. Patients with marked arteriosclerosis with or without hypertension will usually do better and be in safer condition if the protein is more or less restricted and the carbo- hydrates moderately increased, especially if the blood sugar is not high and sugar is present in the urine in only small amounts. For many patients of this type it has been found necessary in order to render them sugar-free to proceed very slowly in the restriction of carbohydrates. If the intake is very gradually restricted until the urine is sugar-free and then the tolerance is slowly built up it may be possible to keep the urine free from sugar for long periods without fasting. Should symptoms of acidosis arise, the fats should be restricted and the patient saturated with water. The attempt should be made to give one liter of water every four hours by mouth or by proctoclysis. 1A 500 gm. accurate food scale is recommended. 70 BLOOD CHEMISTRY METHODS The injection of Ringer’s solution into the vein is also a useful method. This should be given slowly. It is most important that diabetic patients, when ill, even from trivial causes, should go to bed and take a glass of hot water, tea, clear coffee, broth, orange-juice, or oatmeal water gruel every hour. The bowels should be emptied by an enema. The possibility of an impending coma should always be borne in mind. Should coma develop, insulin, if available, should be used at the earliest possible moment. The dosage will depend upon the clinical condition of the patient, which includes, if time allows, the blood-sugar percentage. In general, io units may be given every hour for two or three doses, then, if necessary, every two or three hours for two or three doses more. Experi- ence has indicated that not more than 60 to ioo units should be given during the first twelve hours. In addition a 5 per cent glucose and 2 per cent soda bicarbonate solution should be given by the drip method per rectum, while glu- cose and orange-juice or coffee may be given by mouth or by gavage. In order to prevent dehydration, fluids should be forced either by gavage or by hypodermoclysis of normal salt solution. When insulin becomes more available for general use, the matter of proper dosage of this valuable substance will be better understood. Experience has so far indicated that it is much safer to begin treatment with the small dosage of one unit, increasing the dose daily as rapidly as possible. One unit of insulin will enable the patient to utilize approximately from 2.5 to 4.0 gm. additional carbohydrate. Should too large a dose of insulin be given with resulting rapid reduction in blood sugar, symptoms of nervousness, weak- ness, hunger, increased pulse rate, and sweating may occur. These symptoms are relieved by giving sugar in some form, by the hypodermic injection of ten to fifteen minims of 1-1000 solution of adrenalin chloride, or, as Banting has recently reported, by the intravenous injection of calcium chloride. Banting has also reported that calcium lactate in 10-gr. doses DIABETIC MANAGEMENT may be given to children three times daily to prevent the development of shock during insulin treatment. In case of contemplated operation upon a diabetic patient, nitrous oxide gas-oxygen should be the anesthetic of choice. As a general rule ether anesthesia is unsafe. It may be consid- ered a safe rule before any operation upon a diabetic patient is performed, whether of major or minor degree, to restrict the intake of fats for a few days regardless of his apparent toler- ance and freedom from symptoms of acidosis. Nearly every physician has seen serious sequelae follow minor operations, such as may have been performed by chiropodists, leading to gangrene because of failure to consider the possibility of an impending acidosis. After operation upon a diabetic patient it usually is not wise, at least for the first few days, to give anything more than plenty of water by mouth or proctoclysis, or Ringer’s solution by vein, albumin water, orange-juice, fresh pineapple-juice, and oatmeal gruel. The 5 per cent vegetables are not as a rule well tolerated during this trying period because of the stomach dis- turbance which is apt to occur. INDEX Acetone, 50 Acidosis, 14, 50, 56, 69 Addison’s disease, 43 Adrenalin chloride, 70 Anemia, 13 hyperchromatic, 14 Arsenic pentoxide, 21 Arteriosclerosis, 14, 69 Banting, 70 Basal metabolism, 42, 43, 44 Behre and Benedict, 28 Berg, 14 Blatherwick, 53 Blood, chlorides, 5, 35, 36, 38 cholesterol, 5, 39 creatin, 5, 26 creatinin, 5, 25, 26, 28 examination, 50 filtrate, protein-free, 6 acidity of, 7 nitrogen, non-protein, 5, 7 sugar, 1, 5, 29, 31, 33, 69 urea, 5, 10, 12 uric acid, 5, 16, 21, 23, 24 Bloor, 39 Brown and Raiziss, 23 Calcium lactate, 70 Calories, 63 Carbohydrate tolerance, 62, 69 Carcinoma, 43 Chart, urine and blood chemistry, 52 Chlorides, 5, 35, 36, 38, 49 Cholelithiasis, 41 Cholesterol, 5, 39, 41 Cinchophen, 24 Colorimeters, Bock-Benedict, 4 Duboscq, 2 Kober, 3 Myers, 2 Coma, diabetic, 70 Creatin, 5, 26 Creatinin, blood, 5, 25, 26, 28 preformed, 5, 25 total, 5, 26 i Dehydration, 14 De Zani, 41 Diabetes mellitus, 33, 41, 61, 69 renal, 34 Diabetic management, 69 Diacetic acid, 50, 69 Diet lists, Presbyterian Hospital, 57 Vanderbilt Clinic, 58 Diets, carbohydrate, 42, 61 Karrell, 42 maintenance, 61, 65 minimum salt and basic alkali, 56 moderate protein-low purin nephrit- ic, 59 salt-free, 42, 56 test, 61, 64 Eclampsia, 14 Edema, 38, 42 Epstein and Lande, 42, 44 Fat, 62 Fischer, Martin, 56, 57 Folin, 1, 6, 10, 16, 21, 25, 29, 45, 46, 50 and Doisy, 26 blood-sugar tube, 30, 31 Foods, acid-producing, 54 alkali-producing, 53 neutral, 53 purin constituents of, 57 purin-free, 58 Formula for colorimeter work, 4 Glucose tolerance test, 33 Glycosuria, alimentary, 34 Goiter, exophthalmic, 42, 43 Gout, 24 74 INDEX Indicator, ferric ammonium sulphate, 36 phenolphthalein, 46 potassium chromate, 50 Insulin, 62, 70 Intestinal obstruction, 14 Joslin, 61, 63 Kidney function, 47, 48 Lande and Epstein, 42, 44 Locke, 63 Luden, 40, 41 Menopause, 43 Metabolism, 42, 43, 44, 53 Methods, Benedict, 1, 21, 23, 31, 34 Bloor, 39 Folin, 10, 16, 45, 46 and Wu, 1, 6, 21, 25, 29 hypobromite, 49 Lewis and Benedict, 1, 31 Myers and Bailey, 1, 31, 32 picric acid, 31 Rieger, 36 silver lactate, 19 Volhardt, 49 Whitehorn, 35 Mosenthal, 50 Myxedema, 42, 43 Nephritis, 13, 14, 56 Nephrosis, 14, 38, 42 Nitrogen, amino-acid, 5 ammonia, 5 balance, 49 incoagulable, 5 intake, 49 non-protein, 1, 5, 7, 12 total in urine, 45 Phenolsulphonephthalein, 47 Picric acid, 1, 26 Pneumonia, 38 Polyphenols, 21 Potassium oxalate, 1 Prostatic obstruction, 14 Protein, 62 deficiency, 42 Rappleye, 36 Reaction, ferric chloride, 50 Reagents, arsenic-phosphotungstic acid, 21 Nessler’s, 8, 46 uric acid of Folin and Denis, 18 Rieger, 36 Sansum, 53 Sherman and Gettler, 53 Solutions, acetic anhydride, 39 acid phosphoric-sulphuric digestion mixture, 7, 45 acidified sodium chloride, 16 alcohol, 39 alkaline copper tartrate, 29 alkaline-picrate, 25 ammonium sulphocyanate, 37 arsenic-phosphotungstic acid rea- gent, 21 buffer mixture, 10 chloroform, 39 cholesterol standard, 39 stock, 39 ether, 39 hydrochloric acid, 10, 31 lithium sulphate, 16 molybdate-phosphate, 29 Myers-Bailey picric acid sugar stand- ard, 32 Nessler’s, 7 nitric acid, 35 paraffin oil, 10 picrate-picric acid, 31 Ringer’s, 70 saturated borax, 10 silver lactate, 16 nitrate, 35 sodium carbonate, 32 cyanide, 16, 21 tungstate, 2, 35 standard creatinin, 25 nitrogen, 8, 10, 45 silver, 37 sugar, 29 uric acid, 18, 22 stock creatinin, 25 sugar, 29 INDEX 75 Solutions, stock uric acid, 21 uric acid-formaldehyde, 17 sulphocyanate, 35 sulphuric acid, 6, 37, 39 urease, 10 uric acid reagent of Folin and Denis, 18 Starvation, 43 Sugar, 1, 5, 61, 62, 69 renal threshold for, 33 Test, sodium nitro-prusside, 50 Test-tube distillation, 11 Thyroid adenoma, 43 therapy, 44 Tolysin, 24 Urea, 1, 5, 12 Uremia, 13 Uric acid, 1, 5, 16, 21 eliminants, 24 retention, 23 Urine, 45, 46, 48, 61, 69 examination, 48 Von Noorden, 58 Walker, 57 Weight equivalents, 63 Woodyatt, 63 Paul B. Hoeber, Inc., 67-69 East 59th St., New York.