I REVIEW OF INDUSTRIAL APPLlcATioNs
I I
I Clinical Chemistry I I G. R. KINGSLEY I Department of Physiological Chemistry, School of Medicine, University of California at 10s Angeles, and I Clinical Biochemistry laboratory, Veterans Administration Center, l o s Angeles 25, Calif.
T
review is an attempt to cover some of the significant developments in analytical clinical chemistry which have occurred since the last review in this journal by Archihald ( I d ) . Special :ittention is given to new or improved methods that have been used recently in :inintensified study of certain conditions wch as heart and mental diseases. Methods and techniques which appear to have practical applications in clinical chemistry are included. Attention was drawn t o the growing importance of clinical chemistry by the International Congress of Clinical Chemistry held in S e w Tork, September 9 to 14, 1956 (98). HIS
REVIEWS PERTAINING TO CLINICAL CHEMISTRY
The review, “Clinical Applications of Biochemistry” by Wootton, JIilne, and King ($44) covered precision in the clinical laboratory, plasma sodium and potassium, iodine-131 in the diagnosis of thyroid dysfunction, and aminoaciduria. Bodansky (29) continued this review. Thich included precision in the laboratory, improvements in analytical metliods, new principles in diagnostic procedures. and methods for newly dis(mered compounds in body fluids. Soljotka and Carr (Z05) reviewed “Laboratory Aids to Diagnosis and Therapy.” IF hich covered inorganic serum constituents, acetone and acetoacetic acid, pentosuria, serum bilirubin, ketosteroids, serum proteins, choice and economy of methods, and scope and function of the clinical chemistry laboratory. This review was continued the next year by Reinhold ( I 7 I ) , who discussed standardization of a uniform procedure for estimation of hemoglobin, problems and techniques of separating abnormal hemoglobins, amino acids and amines. calcium and phosphate, electrophoresis and proteins, lipoproteins and lipides. the problem of blood ammonia measurement and other tests for liver disease, enzymes in body fluids, and sugars. A review of the fundamental developments of biochemical analysis by Duggan (62) is of interest to the clinical chemist. ,‘Clinical Chemical Significance of Ionogmphy” by McDonald, Bermes, and
Spitzer (133))and “Paper Chromatography and Paper Electrophoresis” by Fisher (70) are informative for those who wish t o review filter paper chromatography in the measurement of electromigration, and the evaluation of paper chromatography and paper electrophoresis as diagnostic tools. NEW JOURNALS AND BOOKS
The increasing international interest in clinical chemistry has culminated in the formation in January 1956, of a new journal, Clinica Chimica Acta (Elsevier, New York, J. Awapara, editor), which made an auspicious start, as judged by the number of excellent articles appearing in its first five issues. Another new journal, as of May 1956, Journal of Neurochemistry (Bergman Press, New York, A. Engstrom, editor). is of interest to the clinical chemist because of the recent intensified investigation of mental disease. The second volume of “Standard Methods of Clinical Chemistry” (1%) should appear early in 1957. This volume covers about 24 clinical procedures, which will include fatty acids in stool and serum, hydroxy corticoids, serum iron, 17-ketosteroids, porphyrins, protein bound iodine, enzymatic uric acid, and urobilinogen. “Practical Clinical Biochemistry,” by Varley (2SS), included most of the modern methods, several functional tests, and a large number of procedures usually not found in routine manuals. “Clinical Chemistry, Principles and Procedures” by Annino ( I S ) is divided into two parts: basic techniques and fundamental information, and methods. The first part, which includes use of basic laboratory equipment, standardization of photometer, review oi quantitative analysis, and collection and preservation of samples, is excellent for the beginning technician. The second part, on methods, is limited t o about 25 of the usual clinical chemistry methods and is outlined as: principle, reagents, procedure, calculations, discussion, and physiological significance. Several of the methods included would not be the choice of the reviewer.
Glick (81) gave several excellent r(1views of methodology. Methods of particular interest to the clinical chemist in this series of reviews are: volume 1, chemical determination of ascorbic, dehydroascorbic, and diketogulonic acids, zone electrophoresis, the in vitro determination of hyaluronidase, ultracentrifugal analysis of serum lipoproteins, the assay of urinary neutral 17-ketosteroids; volume 2, chemical determination of adrenaline and noradrenaline in bod! fluids and tissues, lipide analysis, assay of proteolytic enzymes, determination of glutathione, determination of serum glycoproteins, new color reactions for the determination of sugars in pols saccharides; volume 3, phosphate analysis of organic compounds, determination of histamine, enzymatic microdetermination of uric acid and relatetl compounds, determination of zinc, and flame photometry and spectrometry. Hoffman’s (93) “Biochemistry of Clinical Xedicine” is one of the clearest, most eomprehensive books on this subject and is excellent for the interpretation of the data obtained on chemical analysis of human specimens. The eighth edition of “Official Methods of Analysis of the Association of Official Agricultural Chemists,” edited by Horwitz (95), was revised to October 1954, and includes several new methods, a section on hormone drugs, and an expanded microchemical chapter. Korey and Surnberger (121) have edited a series of contributions in “Xeurochemistry” which is the first volume of a new series of symposia entitled “Progress in Neurobiology.” NEW APPARATUS AND EQUIPMENT
An autoanalyzer (Figure 1) was devised by Skeggs (201) in which blood, serum, or urine specimens are introduced automatically, together with proper diluent and reagents by a continuous metered flow pump through a continuous dialyzer. The ultrafiltrate is transferred and mixed with suitable reagents in desired sequence to produce a specific color, which is measured photometrically in a flow cell and the density change measured and graphically recorded. VOL. 29, NO. 4, APRIL 1957
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The machine is constructed for rapid washing, and can be quickly changed for a number of sequences of reagents and physical conditions. One cycle for analysis requires about 6 minutes and about 40 analyses can be carried out per hour. Glucose, urea, and calcium methods have been adapted to the machine. A clinical study of the serum proteins of a variety of patients wits made by Mackay (134) by using the automatic scanner, Analytrol, Spinco Model R (Spinco Division, Beckman Instruments, Inc., Belmont, Calif.), Sor quantitative measurement of proteins on dyestrained paper strips on which serum proteins have been electrophoretically separated. The scanner was found to he accurate, and time saving in plotting electrophoretic patterns, when compared to a nonautomatic photometer Eliphor (Bender and Holbein, Munich, Germany). Bowman, Caulfield, and Udenfriend (91) made fluorometric measurements of serotonin with a spectrophotofluorometer for measurement of submicrogram quantities of substances which was devised by Duggan and Vasta (61). The spectrophotofluorometer consists of a high pressure xenon arc source emitting a continuum from 200 40 800 mg, two monochromators, one for the selection of monochromatic activation and the second at right angles to the first to analyze the resulting fluorescence, and a nine-stage photomultiplier to detect the emitted light. An apparatus of two dual-analysis cells connected in series with one cell placed in the water bath of an electrophoresis instrument, Aminco Portable, (American Instrument Co., Inc., Silver Spring, Md.) and the other in an auxiliary water bath has been designed by Oreskes and Corey (152) to permit the performance of four experiments. Techniques for the Kopp-Natelson gasometric apparatus was reported by Natelson and Menning (148) for the microdetermination of oxygen and carbon monoxide in 0.03-ml. samples of blood. Experience has indicated that this gasometric apparatus permits more rapid determinations with accuracy equal to that obtained with the standard Van Slyke gasometric apparatus. The Kopp-Natelson microgasometer has been modified for automatic shaking and temperature control by Holaday and Verosky (94) who also described techniques for carbon dioxide, and combined carbon dioxide and oxygen determination in whole blood. An apparatus similar to the Kopp-Natelson microgasometer for the determination of carbon dioxide in 0.05 ml. of plasma was described hy Rappaport, Eichhorn, and Nutman (166). Lura (f29)described a simple microapparatus for the electrochemical measurement of dissolved oxygen in 3 to 4 616
ANALYTICAL CHEMISTRY
ml. of biological fluids and Clark, Wolf, Granger, and Taylor (46) found that a platinum cathode covered with a layer of cellophane was suitable for the direct measurement of oxygen tension in whole blood by polarographic procedures. A single automatic buret for ultramicro-, micro-, and macromeasurement of volumes of 0.1 to 10 ml. within an accuracy of 1% or less was designed by Natelson (147). The buret is constructed of a precision glass plunger, which moves through a Teflon gasket of special design into a chamber containing the liquid to he delivered. The movement of the plunger is measured by means of a dial indicator attached to a metal rod which moves with the plunger. When titration is complete, the dial is returned t o zero which sets the instrument for the next titration. The design of a new blood-pH cell assembly was described by Murray (144) for rapid routine determination of pH on large numbers of samples. The use of anion exchangers attached to pipets and syringes was suggested by Slade ($03)as a simple means to remove excessive iodides prior to protein bound iodine analysis. A filter-beaker was designed by Abrahamson (2) to permit precipitation and filtration in one vessel for the determination of hlood urea by the colorimetric xauthydrol method. An apparatus, the Drunkometer, was developed hy Harger, Forney, and Baker (86) for the determination of alcohol in rehreathed air from which blood-alcohol concentration may be estimated. CONTROL A N D PRECISION OF CLINICAL CHEMISTRY METHODS
Wootton (243) conducted an international survey of 133 laboratories in eight countries by submitting to them two freeze-dried horse serum specimens of different concentration for assay of sodium, potassium, calcium, phosphorus, nonprotein nitrogen, total protein, urea nitrogen, glucose, chloride, and bilirubin. The results of the survey indicated that
the laboratories could compare two solutions more accurately than they could determine the absolute value of a constituent. The preparation, storage, methods of standardizing, and estimation of precision of control serums have been presented by Benenson, Thompson, and Klugerman (19). A survey by Tonks and Allen (2%) of 19 clinical laboratories indicated that many laboratories were in need of better control of their Folin-MTu method for glucose determination. A review was made hy Kingsley (116) of principles and recent techniques in maintaining control and precision in clinical chemistry methods. Statistical evaluation was made of the relative precision (coefficient of variation) obtained in the use of different procedures by technicians and student technicians using pooled frozen serum standards. Allowable error was also determined from data obtained in an interlaboratory survey of standard laboratory procedures. A survey of 13 to 17 hospital laboratories by Saifer and Deutscher (181) of the use of protein- and sterol-free hlood serum nltrafiltrate as a standard for sugar, urea nitrogen, creatinine, uric acid, chloride, carbon dioxide, phosphorus, calcium, potassium, and sodium showed excellent agreement between the laboratories. A standard of this kind is stable, can be prepared in large lots, and is uniform. The sugar, phosphorus, calcium, and potassium of this standard were outside normal human range. Standardization of microtechniques for the clinical chemistry laboratory was described by Kaplan and Carmen (107). Spivek (807) describes the utilization of microtechniques in the determination of 11 blood constituents, with standard deviations ranging from 0.37 to 12.5, on samples from 5-day-old infants, with as little as 1.3 ml. of blood. Ultramicrotechniques employing 10 to 100 PI. of serum were worked out by Caraway and Fanger (38)for 17 routine clinical chemistry procedures. Standard deviations ranging from 0.11 to 6.8 for all deter-
niinations were obtained except for cholesterol (13.2) and amylase (10.7). Amino Acids. The photometric ninhydrin method for a-amino nitrogen of Yemm and Cocking (248) was successfully applied to tungstic acid filtrates of plasma by Kalant (106). Schwartz, Robertson, and Holmes (191) have adapted the niicrodiffusion method of Conway for the determination of glycine. Gilboe and Killiams (79)have established the best conditions for the colorimetric estimation of arginine by the Sakaguchi reaction. An improved colorimetric method for the determination of hydroxyproline in gelatin and collagen was developed by lliyada and Tappel ( I @ ) , by a study of the factors associated with the color reagent pdimethylaniinobenzaldehyde in 1propanol. Cifonelli and Smith (45) found that certain periodate sprays, with or without subsequent benzidine spray, are useful for the detection of amino acids on paper chromatograms. Bito (26) norked out a method dependent on spot area and color intensity for high precision quantitative determination of lysine, alanine, valine, phenylalanine, tyrosine, cystine, and proline by paper chromatography. SEthyl maleimide mas used to prevent oxidation by Smith and Tuller (205) in the paper chromatographic determination of sulfhydryl containing free amino acids and small peptides in whole blood and serum. A simple and practical technique, suitable for routine analysis of amino acids in urine by one-dimensional paper chromatography, was described by hwapara and Sato (17). A preliminary treatment of the urine with charcoal and chloroform followed by a desalting technique with ion exchange resin was used. Cations and Anions. Xatelson and Penniall (149) developed a rapid colorimetric ultramicromethod, which requires a n extraction, for the determination of calcium in 0.02 ml. of serum by complexing calcium with alizarin in noctyl alcohol in the presence of triethanolamine. A simple direct colorimetric determination of calcium and magnesium in 0.1 ml. of serum was carried out by YanagisaiTa (247) with the dye l-hydroxy-4-chloro-2,2-diazobenzene-1, 8-dihydroxynaphthalene-3,6disulfonic acid. Ionized calcium and magnesium may also be determined with this dye. Optimum conditions for the use of the dye for rapid direct serum and urine calcium determinations have been described by Kingsley and Robnett (119).
The application of tetraphenylboron as a reagent for the turbidimetric determination of potassium in urine and serum was studied by Power and Ryan (162). The sensitivity of the tetraphenylborate method for potassium was extended by a study by Pflaum and
Howick (159) of the absorption maxima a t 266 and 274 mp of the tetraphenylborate ion. I I a n n and Yoe (137) discovered a new, extremely sensitive reagent, sodium l-azo-2-hydroxy-3-(2,4dimethylcarboxanilido) - naphthalene1-(2-hydrovybenzene-5-suIfonate) for rapid photometric determination of magnesium. This reagent is about 10 times as sensitive as Titan yellow for measurement of magnesium. Unfortunately, interference of calcium must be corrected for and details for the use of the reagent in the analysis of biological fluids were not reported. The addition of calcium to magnesium standard solutions and the use of gum ghatti in place of hydroxylamine hydrochloride as a color stabilizer was suggested by Xeill and Neely (151) in the estimation of magnesium in serum using Titan yelloil-, A review of serum chloride methods was presented by Teloh (220). Davis and Simpson (54) improved the flame photometric method for the determination of serum bicarbonate. Kleeman, Taborsky, and Epstein (120) measured small amounts of inorganic sulfate (10 or more y) in serum or urine by utilizing powdered glass to prevent loss during precipitation with benzidine and washing of the precipitate with ethyl alcohol and ether prior to development of color 11-ith sodium 1,2-naphthoquinone-4-sulfonate. Stoa (210) made the pyridine n-benzidine reaction more specific for the determination of thiocyanate in serum by precipitating the thiocyanate as its silver salt and removing excess silver ions with sodium chloride. Enzymes. Karmen. Wroblewski, and La Due (109) first demonstrated transaminase activity in human serum by incubating aspartate or alanine with a-ketoglutarate. The rate of production of glutamate was taken as a measure of transaminase activity which was measured by separating the amino acids by chromatography, eluting, and measuring colorimetrically with ninhydrin. A more practical method for the measurement of transaminase activity in serum was devised by Karmen (108) by carrying out the transamination reaction nith aspartate. coupled to the reduction of oxalacetate to malate by reduced diphosphopyridine nucleotide in the presence of a n excess of purified malic dehydrogenase. The transaminase reaction was followed by measuring, a t room temperature, the decrease in light absorption a t 340 mp a t which the reduced pyridine nucleotides have an absorption peak. As the rate of reaction is markedly affected by temperature, Steinberg, Baldwin, and Ostrow (208) suggested a standard temperature of 37” C. Tonhazy, TVhite, and Umbreit (221) devised a method for estimation of transaminase activity in tissues based on the activity of glutamic oxalacetic transaminase to mediate the
transfer of the a-amino nitrogen of Laspartic acid to o-ketoglutaric acid which results in the synthesis of oxalacetic acid and L-glutamic acid. The oxalacetate formed was converted to pyruvate by aniline citrate. A dinitrophenylhydraaone of pyruvate is formed and then treated in toluene extract with alkali to produce a colored compound n hich is measured photometrieally a t 450 to 520 mp. Henley and Pollard (89) modified Tonhazy’s method by converting ovalacetic acid to pyruvic acid with heat. Thr pyruvic acid was estimated by the o d a t i o n of reduced coenzyme I ( D P S H ) nith lactic acid dehydrogenase as shon n by decreased absorbance a t 340 nip. Cabaud, Leeper, and TT7roblen.ski (34) made practical application of the niethod of Tonhazy in the study of patients \\ ith cardiac and hepatic disease. Further modification of the Tonhazy niethod wa5 made by Fales (65) by enhancing the rate of change of the oxalacetate concentration by using an initial glutamate concentration 20 times that of oxalacetate. T’ersene was also included in the reaction mixture to prevent the catalysis of oxalacetate breakdon n hy calcium and niagne.iuni. LTnibieit and associates (230) niodifird Tonhazy’s original colorimetric method for routine transaminase determinations and made a clinical comparison n ith the units of transaminase activity obtained n ith the spectrophotometric method of Karnien (108). Unfortunately, there is no agreement as to the definition of a unit of transaminase became of the different substrates, temperatures, tinie of reaction, and methods used in this determination. Siekert and Fleiqher (196) applied the deterniination of transaminase in spinal fluid and nerye tissue to the study of neurologic diseases. Peralta and Reinhold (1.56) used a stable starch substrate for the rapid (10- to 15-minute) determination of amylase by a turbidimetric method which agrees favorably v ith Somogyi’s saccharogenic method. A detailed method was described by Street and Close (212) for the preparation of amylose for w e as a sulxtrate in a 15minute photometric iodonietric determination of amylase. Optimum conditions were determined by Friedman and Becker (7’5) for hemolysis of red cells to determinr blood arginase. Best conditions for the estimation of arginase activity by the colorimetric method was investigated by Gilboe and Williams (80). Caraway ( 3 7 ) simplified and modified the photometric determination of serum cholinesterase to eliminate dye binding error. A method for the determination of cocarboxylase in blood was described by Kay and Murfitt (111 ) A satisfactory 4-hour serum lipase technique was worked out by Bunch and Emerson (33) by buffering olive oil and VOL. 29, NO. 4, APRIL 1957
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gum acacia with 0.066-I! phosphate buffer a t pH 7.5 which increased enzyme activity sufficiently to measure normal serum lipase in adults within a normal range of 0.31 =t0.17 (S.D.)ml. of 0.055 sodium hydroxide for 4-hour incubation a t 37 O C. A sensitive micromethod for the determination of a-keto acids using 3-quinolylhydrazine was utilized by Robins and coworkers (176) to determine malic, lactic, and glutamic dehydrogenases in 0.05 to 0.5 y dry weight of brain. Fishman, Bonner, and Homburger (71) proposed the use of L(+)tartrate inhibition for the detection of prostatic acid phosphatase and reported good correlation of increase of this enzyme with proved cancer of the prostate. This method was further simplified by Davis and Kood (53) by omitting protein precipitation and conducting the test with all reagents in a single tube. Bensley, Drysdale, and Osiek (60)and Hill (96) failed to confirm increased specificity of the tartrate inactivation test over the standard acid phosphatase test for diagnosis of prostatic carcinoma. Kind and King (113) determined phosphatase by a modification of the Grifols-Lucas method in which the amount of phenol liberated by enzymatic hydrolysis is determined colorimetrically by reaction with 4aminoantipyrine to produce a redcolored quinone. Proteins do not have to be removed t o carry out this reaction. -4method was designed by Cooper and Brown (50) in which a Bacto-Bgartyrosine substrate was prepared in a Petri dish, holes were cut, test solution was added, and melanin color formation \vas followed visually, and a semiquantitative determination of tyrosinase activity was made within 30 minutes. Hemoglobin. Standardization of methods for the determination of hemoglobin were reviewed by Reinhold (171) and a review, “The Human Hemoglobins in Health and Disease,” was prepared by Chernoff (42). A proposal for the distribution of a certified standard for standardization of the cyanmethemoglobin method as recommended by the National Academy of Sciences, National Research Council, was outlined by Cannon (55). Sunderman, and associates (214) have also recommended a standard detailed procedure for hemoglobin determination. Spectrophotometric determination of hemoglobin in 10 ml. of plasma was simply carried out by McCall (130) by converting hemoglobin to methemoglobin with ferricyanide, measuring its absorption at 540 mp, and then converting to cyanmethemoglobin with a drop of sodium cyanide solution and measuring absorption a t 540 mp, the change in absorbance observed being directly proportional to the total hemoglobin. Crosby and Furth (51) have 618
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improved, considerably, the usefulness and accuracy of the benzidine method of microchemoglobinometry . Indoles. Udenfriend, Titus, and Weissbach (267) have developed a fairly specific method for the determination of urinary 5-hydroxyindoleacetic acid by first removing interfering substances and developing a color with nitrosonaphthol reagent. Sjoerdsma, Weissbach, and Udenfriend (200) have used this method for the detection of malignant carcinoid of the small intestine (argentaffinoma). A simple paper chromatographic method was described by Jepson (102) for the identification and assay of urinary indoles and applied to the study of metastasizing carcinoid and phenylketouria. Iodine, Protein-Bound. A study by Blackburn and Power (27) of the diagnostic accuracies of protein-bound iodine (PBI), basal metabolic rate, and uptake of radio-iodine determinations of serum, indicated that protein-bound iodine (PBI) was the most valuable single test in the clinical evaluation of thyroid function. Grossman and Grossman (85) and several others have suggested the addition of brucine for stabilizing the cerate color a t a selected time interval, and mercuric acetate was proposed for the same purpose by Meyer and coworkers (139). The best stabilizing technique was discovered by Fischl (69) who employed brucine acetate to inhibit the ceric-arsenious system and greatly enhanced and stabilized the color by heating on the water bath for 5 minutes The color was enhanced sufficiently to permit protein-bound iodine determinations on 0.1 ml. of serum. Escobar. Morreale, and Kassenaar (63) have investigated the efficiency of butanol extractions of serum organic iodine compounds and showed that 98% extraction of organic bound iodine was obtained if 2 ml. of serum a t pH 2 to 3 was extracted 4 times with 5 ml. of 1-butanol. A simple screening test was proposed by hlalkin (136) to eliminate highly contaminated serum samples, before incineration, in order to prevent cross contamination. Lipides. Sperry and Brand (206) described a method for the extraction of the unmodified total lipides of serum or plasma by direct chloroformmethanol (2 t o 1) extraction, purification of extracts, and gravimetric determination. Brown (32) determined lipide phosphorus in 10 pl. of serum by a n ultramicromethod. Sachs and Danielson (180) separated serum phospholipides by paper electrophoresis and used a staining technique for measurement. Fractionation of total lipides and phospholipide in 8- and a-lipoproteins of normal serum proteins was made by Chapin (40) by paper electrophoresis. Techniques for the paper electrophoretic
analysis of serum lipoproteins, combined with chemical analysis of serum lipide partitions for the preparation of electropherograms, in which protein and lipide patterns of the same serum were plotted on the same graph for comparison, have been described by Bdlersberg and coworkers ( 3 ) . Techniques for preparing and staining paper strips prepared by electrophoresis for identification of proteins, fat, and cholesterol were described by Dangerfield and Smith (52). Dye Oil Red 0 was used by Jencks, Durrum, and Jetton (100) for semiquantitative estimation of serum lipoproteins by means of paper electrophoresis. Albrink and coworkers ( 7 ) in a study of the displacement of serum water by lipides worked out a new method for the rapid determination of serum rater. Liver Function. One of the most comprehensive reviews and critical evaluations of the chemical tests used for liver function was made by Reinhold (17 2 ) . A stable standard prepared from colloidal glass suspensions with similar optical absorption properties to those occurring in the thymol turbidity test was prepared by Reinhold (170). The reproducibility and stability of the thymol turbidity test were irnproved by Reinhold and Yonan (173) by buffering the thymol-barbital reagmt a t pH 7.80. The simple microcapillary tube method of Graham (84) for the separation of 0.1 ml. of plasma permits the determination of bilirubin on finger or ear blood. Evidence was presented by Childs and Home (43) that direct bilirubin exists in serum as a bilirubinmetal-albumin complex. Verschure (234) applied paper electrophoresis to the analysis of liver and gall bladder bile for detection of protein, bilirubin, lipides, bile acids, urobilin, cholesterol, and alkaline phosphatase. Metals. Middleton and Stuckey (140) improved the 1-nitroso-2-naphthol complexing method for the separation of cobalt in tissues by using alumina column to separate interfering metals and iron after reduction with alkaline hydroxylamine. Saltzman (184) complexed cobalt with 1-nitroso-2-naphthol, extracted with chloroform, washed, ashed, and developed a color with nitroso-R salt for photometric micromeasurement in biological materials. Keenan and Kopp (112) extended the sensitivity of the latter method. Peterson and Bollier (158) found biscyclohexanoneoxalyldihydrazone to be the most sensitive of all reagents so far investigated for the spectrophotometric determination of serum copper. Trinder (224) used sulfonated 4,7-diphenyl-1,lO-phenanthroline in the presence of thioglycollic acid for the determination of serum iron. Kingsley and Getchell (117) in the determination of serumiron, extracted the iron, complexed with 4,7-diphenyl-l,lO-phenanthroline,
in the presence of hydrazine sulfate, with isoamyl alcohol to separate from other chromogens present in serum before photometric measurement. Schade and others (185) employed terpyridine and 1,7-diphenyl-l, 10-phenanthroline for the colorimetric determination of siderophilin-bound and free iron in serum. A simple technique Kas suggested by Peters and coworkers (157) for the determination of the serum iron-binding capacity by adding excess iron as ferric ammonium citrate and then removing the excess iron by the addition of a small amount of an anion exchange resin. Taylor and Paige (219) evaluated the optimum conditions for the determination of strontium with the Beckman DU flame photometer. Nitrogen Compounds. McDermott, Adams, and Riddell (132) used the microdiffusion technique of Conway for the determination of ammonia in blood and spinal fluid, and Seligson (194) improved this technique using potassium carbonate-bicarbonate buffer for alkalinization of the microdiffusion system which diminished hydrolysis and permitted better recoveries of added amnionia in blood or plasma. Taussky (218) improved the specificity of the Jaff6 reaction for the determination of creatinine and creatine in urine and blood plasma by ether extraction of urine and ether evtraction preceded by iodine treatment of serum to remove interfering substances. However, because of these additional techniques. the method does not lend itself well to routine determination. Jacobsson and Paulsen (99) found that creatinine in ethyl alcohol and water (4 to 1) gave a distinct niavimum ultraviolet absorption a t 235 to 236 mp which followed Lambert-Beer’s law. Bhattacharya, Robson, and Sten-art (24) adapted the glyoxalase method for the determination of glutathione content of blood. A paper chromatographic method for the determination of glutamine in spinal fluid was described by Whitehead and Whittaker (840). Van Pilsum and others (232)developed a nerr analytical method which employed a modified Sakaguchi color reaction for the determination of guanidiniuin compounds: creatinine, creatine, arginine, guanidinacetic acid, guanidine, and methyl guanidine, in hiological fluids. Connerty, Briggs, and Eaton (49)stabilized the color in the blood-urea-nitrogen method by adding 1 drop of a 2% aqueous solution of iodine t o the blood filtrate or to the Xessler’s reagent before use. Rosenthal (179)improved the method for the determination of urea in blood and urine by the condensation of urea with acid diacetyl monoxime by establishing the optimum concentrations of hydrochloric and arsenic acids to produce maximum linear color. Caraway(S6) substituted sodium carbonate for sodium cyanide to simplify and
make more reproducible the commonly used uric acid method of Brown. Bergmann and Dikstein (22)precipitated uric acid from urine or plasma with mercuric acetate and measured the absorbancy of uric acid in saline a t 290 mp. Uric acid was determined routinely in serum by Dubbs, Davis, and Adanis (60) and Feichtmeir and Wrenn (66) by spectrophotometric measurement of absorbance a t 293 mp before and after incubation u-ith uricase. A simple spectrophotometric determination of hypoxanthine and xanthine in plasma and urine was made by Jorgensen and Poulsen (104) by using xanthine oxidase and uricase. Organic Acids. Di Ferrante and Rich (57) determined urinary aminopolysaccharide by its glucuronic acid content after precipitation vith cetyl trimethyl ammonium bromide. Armstrong, Shaw, and Robinson (15) described a method for the estimation of o-hydroxyphenylacetic acid which is increased in the urine of patients with phenylketouria. Bonting (SO) modified the Barker and Summerson method for the microdetermination of 0.025 to 0.26 y of lactic acid in a volume of 25 pl. Bergerman and Elliot (21) determined oxalic acid photometrically in kidney stones by heating with 0.1% indole in concentrated sulfuric acid in a water bath to develop color. Henley, Wiggins, and Pollard (90) and Segal, Blair, and Wyngaarden (195’) used a niethod for plasma pyruvic acid based on the conversion of pyruvate to lactate in the presence of lactic acid dehydrogenase and diphosphopyridine nucleotide. Density change a t 340 mp indicated that the reduced coenzyme I ( D P S H ) oxidized was equiralent to the amount of pyruvate present. A geneIal description Tvas given by Osteuv and Laturaze (155) of a method of separation and identification of nonvolatile organic acids in human urine by paper chromatography. Forty-three different phenolic acids \\-ere observed by ilrnistrong. Shavi., and \Tall (16) in the urine of 400 individuals by paper chromatography. A study vias made by Biserte and Dassonville (25) of methods for the identification and estimation of keto acids in blood and urine by paper chromatography of their 2,4-dinitrophenylhydrazones Porphyrins. Forniijne and Poulie (73) studied the factors influencing the reaction between porphobilinogen and p-dimethylaminobenzaldehyde in water and urine. Investigation of the separation by paper electrophoresis of porphobilinogen by Heikel (88) and microseparation of urinary porphyrins was made by With (248). Horizontal paper chromatography was used by Rappoport and associates (167), with a modified solvent system for a rapid method for the complete separation of methyl esters of uroporphyrin I, coproporphyrins I and
111, and protoporphyrin I X for fluorometric analysis. hlauzerall and Granick (1%) quantitatively determined porphobilinogen using ion exchange resins Dresel, Rimington, and Tooth (59) directly extracted urinary uroporphyrins I and I11 with cyclohexanone a t pH 1.5 after prior removal of coproporphyrin with ether-acetic acid. A detailed study of the effect of solvent and pH on the spectrophotometric absorption of coproporphyrin I11 was made by Zondag and Kampen (250). Proteins. Von Frijtag Drabbe and Reinhold (2%) evaluated the precision and accuracy of the suspended horizontal paper strip electrophoretic separation of seruni proteins and found that i t compared favorably with the moving boundary method. Schultz and Holdcraft (189) critically evaluated the relative errors of the paper electrophoresis of serum proteins. Great uniformity in albumin mobilities was found by Gordon (85) in a study of 26 sera run in pairs with the hniinco Model B elcctrophoresis apparatus (American Instrument Co., Inc., Silver Spring, Md.) Rees and Laurence (169) have studied the technical factors affecting the absorbance relationship to surface protein concentration in fractionation of proteins by paper electrophoresis, and Walsh, Humoller, and Dunn (238) made comparative studies of different dyes in quantitative filter paper electrophoresis of serum proteins. Wurm and Epstein (246) evaluated electrophoretic patterns of normal human serum by densitometry of protein bands on paper stained with bromophenol blue or Amidoschn arz 10 B compared them ivith the patterns obtained by movingboundary electrophoresis. and found that the logarithm of the protein concentration was proportional to the absorbance, and that Beer’s l a x did not apply. .Jencks, Jetton, and Durruni (101) described a staining procedure Fhich gave a linear relationship betmen serum protein and dye concentration. Puls and Fiedler (164) made quantitatix-e estimations of protein carbohydrates in qeruni protein fractions following paper electrophoresis by carbohydrate-staining techniques. Ressler and Jacobson (174) used viscous film and Monty and associates (143) impregnated paper with cationic detergent to minimize “trailing” in the electrophoresis of serum proteins. The addition of small aniounts of calcium ion to buffer was found by Laurell, Laurell, and Skoog (1%) t o improve the separation of serum protein p- fraction by paper chromatography. Kingsley and Getchell (116, 11s) made an investigation of the use of the dye, tetrabromophenolphthalein ethyl ester and its potassium salt in the determination of protein in submicrogram amounts in serum and spinal fluid. The differentiation and detection of serum in VOL. 29, NO.
4, APRIL 1957
619
spinal fluid were made by use of this dye and its salt. Fuhr and Hinz (77) surveyed micromethods for the determination of protein in biological fluids. Benedict’s quantitative sugar reagent mas suggested by Goa (82) as a biuret reagent for the determination of serum proteins. Simple ultraviolet spectrophotometric determination of serum and spinal fluid protein was made by Waddell (236) by subtraction of the absorbance at 225 mp from that a t 215 mp of serum diluted 1 to 1000 and spinal fluid 1 to 10. Albumin may be determined by diluting the sodium sulfate filtrate from globulin precipitate 1 to 50. Schaffner, Schervel, and Lyttle (187) observed lower a-globulin in early viral hepatitis. Saifer and Zymaris (182) made use of the relative specific interaction of the cationic detergent, Octab (Rhodes chem.) a t p H 6.65 in collidinesodium chloride (0.0S.M)buffer on CYglobulins for their microdetermination in serum and spinal fluid. Blondheim (28) found that the binding of phenolsulfonephthalein by serum correlated closely with its albumin concentration, except when jaundice \vas present. Feichtmeir and Wrenn (67) measured albumin directly in 0.1 ml. of serum by its interaction with the anionic dye, 2-(4’hydroxybenzeneazo) benzoic acid. Wycoff (246) microassayed fibrinogen in 0.1 ml. of plasma by adding 0.2 mg. of thrombin (2 m1.-volume) and separated the fibrin formed n i t h a glass needle for assay with Sessler’s reagent. Using paper chromatography Rohoz and others (177) separated glycoproteins from spinal fluid and identified the carbohydrates: mannose, galactose, fucose, and glucosamine. Anderson and Maclagen (9) estimated urinary mucoids by absorption on benzoic acid and heating with acid diphenylamine reagent for 30 minutes a t 100’ C. to produce a color. Puls and Albauni (163) isolated and fractionated mucoproteins from serum by paper electrophoresis. A simple technique apparently more satisfactory and specific than older methods for urinary peptide determination was developed by Balikor and Castello (18) in which tungstate filtrates of urines \\-ere treated n ith biuret reagent. Sterols. Aldosterone n as identified chemically in urine bv Llaurado, Keher, and Wettstein (128) by conversion to its characteristic crystalline ylactone and by paper chromatography by Keher and Wettstein (150) with the blue tetrazolium reaction. il new method for the determination of cerebrosides was developed by Radin, Lavin, and Brown (165)by passing brain lipides through a Florisil column and a mixture of ion exchange resins followed by anthrone color development Zak and Ressler (249) made a critical review of cholesterol methods developed in recent years. Albers and L o m y (4) 620
ANALYTICAL CHEMISTRY
designed a centrifugal evaporator for processing small animal tissues for fluorometric measurement of 0.1 to 10 y of cholesterol. Leppanen (1%) found p-toluenesulfonic acid satisfactory as a colorimetric reagent for the determination of serum total cholesterol. Carr and Drekter (39) modified the Liebermann-Burchard reagent to obtain equal maximum color intensities with equimolar concentrations of free and esterified cholesterol in order to avoid saponification. The technique required careful addition of a special dehydrating reagent (sulfuric acid-acetic acid 1 to 3) to a glacial acetic-acetic anhydride extract of serum, before addition of sulfuric acidacetic acid (1 to I) for color development. A method for the estimation in 0.1 ml. of serum of cy- and P-lipoprotein cholesterols by paper electrophoresis and by cold ethyl alcohol precipitation was described by Anderson and Keys (10). Anker (12) reported a n improvement of the Allen modification of the Kober colorimetric assay method for estrogens and found that specimens could be stored for 2 years a t room temperature under a layer of 1-butanol. Anker (11) also described a technique for continuous extraction and hydrolysis of urinary estrogens with purified 1-butanol without destruction of free steroids. Strickler, Grauer, and Caughey (215) described techniques for the purification of urinary extracts by Engel’s method, countercurrent distribution, chromatography. and redistribution. B detailed review of chemical and biological techniques of human urinary gonadotropin assays ( 6 ) , and a brief technical outline of chemical and biological methods (6) were prepared by Albert. Diczfalusy and others (56) studied the factors influencing the estimation of urinary 17-ketosteroids. RIigeon and Plager (141) have fractioned dehydroepiandrosterone and androsterone in plasma by ethyl alcohol and ether extraction, chromatography on Florisil columns, paper chromatography, and micro-Zinimerman colorimetric estimation. Schedl and coworkers (188) made corrections for color differences between standards, urine extracts, and ketonic fractions in the Callow-Zimmerman reaction by comparing the absorption peak of the unknown with that of the standard a t three n ave lengths, 425, 510, and 595 mp. Johnsen (103) carried out separation of urinary IT-ketosteroids by repeated use of the same column of aluminum oxide for chromatographic separation. Paper chromatographic studies of adrenal cortical hormones were made by Dohan and associates (68). Quantitative analysis of small amounts of urinary corticosteroids was made by Talbot and others (217) by making absorption measurements at 302 mp of ethyl alcohol extracts of dried
residues obtained by iso-octane and 2butanol partition, performed according to Craig countercurrent distribution of chloroform extracts. Silber and Porter (198) modified and improved their method for the determination of l i hydroxycorticosteroids in urine and plasma. The specificity of the phenylhydrazine reaction with 17,21-dihydroxy20-ketosteroids has been studied by Silber and Busch (197). A detailed evaluation of the precision of the SelsonSamuels method was made by Harwood and Mason (87) to define the limits for interpretation of results and to determine the suitability of the method for the measurement of low hormone levels. After methylene chloride extraction and Chromatographic separation, Chen, Voegtli, and Freeman (42) measured the total free reducing steroids of plasma by means of the reduction of blue tetrazolium to the corresponding formazan in alkaline ethanolic solution. Touchstone and Hsu (223) measured a-ketolic steroids of urine after resolution on filter paper strips by spraying with blue tetrazolium and eluting with ethyl acetate-methanol for density determination. Recknagel and Litteria (168) also used blue tetrazolium reagent for measurement of steroids. A new color reagent, 50 grams of phosphorus pentoxide in 100 grams of concentrated sulfuric acid was proposed for spectrophotometric measurement of li-hydroxycorticoids by Steyermark and Kowaczynski (209). 17-Hydroxy- and 17ketosteroids were rendered more chrornogenic for microdetection by Wilson and Fairbanks (941) by chromic acid oxidation before application of the Zimmerman reaction. Clayson (47) made a detailed investigation of the Redd>method for 17-hydroxycorticoids and recommended certain practical modifications. Abelson and Bondy ( I ) have measured within 0.01 y, testosterone, progesterone, and adrenocortical steroids by a method based on a reaction with potassium lert-butoxide in lert-butyl alcohol. Sugars. Tygstrup and associates (226) found that a specific glucose oxidase, Xotatin, is superior to yeast in removing glucose in the galactose determination. King and Hainline (114) made a clinical trial of commercial glucose oxidase preparations and found they have a greater sensitivity than Benedict’s reagent in detection of glucose in urine. Free and others (74) used a paper strip containing glucose oxidase and o-tolidine-peroxidase as a specific qualitative test paper for urinary glucose and Froesch and Renold (76) extended the use of this enzyme to blood glucose determination. Cohen and Kantor (48)described a simplified paper chromatographic method for the separation and identification of glucose and fructose in urine
Fructose, 0.5 mg. or more, in proteinfree filtrates was measured by Karvonen and Malm (110) by treating with indole in the presence of concentrated hydrochloric acid. Patmalnieks and Gardell (154) employed thymol-hydrochloric acid reagent for specific colorimetric measurement of 25 to 400 y of fructose in blood and urine. The naphthoresorcinol method has been modified by Fishman and Green (72) for measurement of small amounts of glucuronides in blood by removing interfering substances by selective oxidation. Heyrovsk9 (91) employed a new method for the rapid determination of inulin in plasma and urine in which fructose and p-indolylacetic acid in concentrated hydrochloric acid form a purple-riolet color. Vitamins. A modification of t h e Roe-Kuether method, by incubating in a water bath to obtain rapid completion of color development in 5 minutes, was made by Schaffert and Kingsley (166). A mixture of 85% phosphoric acid and concentrated hydrochloric ( 2 to 3) was used by Schwartz and Williams (190) instead of 85% sulfuric acid as described by Roe and Kuether to increase color development. A new sensitive, specific method for the estimation of blood vitamin E in serum was carried out by Nair and Magar (146) by treating a petroleum etherextract residue from saponified serum n it11 phosphomolybdic acid reagent to produce color a t 725 mp. Miscellaneous. Pyrazinamide was determined in serum and plasma by a simple polarographic method by Svoboda (216). Chloroform was substituted by Lewis and Foord (127) for ether in the extraction of quinine from urine to obtain better recoveries in the “tubeless method” of gastric analysis. GASOMETRIC ANALYSIS
Singer, Shohl, and Bluemle (199)modified and improved the technique of Shock and Hastings in order to determine pH, carbon dioxide content, and packed cell volume in 0.1 ml. of blood. The technique was useful in studying acid-base balance in infants. The carbon dioxide content of 1 ml. of serum was calculated by Kahn (105) from a nomogram from data obtained by measuring the p H before and after equilibration with a gas mixture of 5 to 6% of carbon dioxide and 94 to 95% of nitrogen. The meter used must be capable of measuring under anaerobic conditions with an accuracy of rtO.01 pH unit. Segal (192) described a rapid electrotitrimetric method for the determination of carbon dioxide in serum and plasma which compares favorably with the gasometric method of Van Slyke and Cullen. A rapid spectrophotometric deter-
mination of oxygen saturation in 5 ml. of blood based on the absorption maximum of oxyhemoglobin and the isobestic point of oxyhemoglobin and hemoglobin was carried out by Tsao and others (225) by hemolyzing blood with saponin and measuring the absorption ratios a t 576 and 505 mp from TThich the oxygen saturation can be calculated. Agreement \\-ith the Roughton-Scholander method was obtained. Dempsey (65) modified the Roughton-Scholander method for the rapid microdetermination of oxygen content, capacity, and per cent saturation in 0.04 ml. of blood. Complete hemolysis by freezing and thawing for oxygen and ovyhenioglobin determination was suggested by Huckabee (97),who observedamolarextinction coefficient difference of uncontaminated oxy- and reduced hemoglobin in blood a t a higher TTave length, 660 mp, than that reported in the presence of saponin. METHODS USED IN STUDY OF HYPERTENSION, PHAEOCHROMOCYTOMA, AND MENTAL DISEASE
A study by West and Taylor (639) indicates the continued interest in the determination of urinary catecholamines, adrenaline, and noradrenaline in the study of hypertension and diagnosis of phaeochromocytoma. Pekkarinen and Pitkanen (155) determined adrenaline and noradrenaline in urine by absorption on aluminum oxide a t pH 8.5, elution by oxalic acid, and fluorescence measurement after manganese dioxide oxidation. The adrenalines ivere differentiated by treating with sodium hydroxide without the addition of manganese dioxide which produces maximum fluorescence of adrenaline while that of noradrenaline remains weak. Euler and Floding (64) determined adrenaline and noradrenaline after separation with aluminum oxide from urine by a method based on the differential oxidation of adrenaline and noradrenaline with potassium ferricyanide a t p H 3.5 and 6.0. Richardson, Richardson, and Brodie (175) made further study of and improved the Weil-Malherbe method for determination of adrenaline and noradrenaline in serum. Valk and Price (231) made a critical evaluation of the ethylenediamine condensation method (Weil-Malherbe) and compared it to the trihydroxyindole method (Euler and Floding) for the determination of adrenaline and noradrenaline in plasma, and found the latter method to be more specific for estimation of noradrenaline. Biological measurement of serotonin was made by Zucker and Borrelli in platelets (253) and in serum (261) of patients with thrombocytopathia, pseudohemophilia, and thrombocytosis. An extraction procedure for 5-hydroxytryptamine in biological material was described by Udenfriend, Weissbach,
and Clark (228) in preparation for measurement with the photofluorometer described by Bowman, Caulfield, and Udenfriend (SI) and Duggan and Vasta (61). Serotonin was activated a t 295 mp and emitted fluorescent light a t 330 mp. After acetylation, serotonin was determined by Gaedtke and Schreier (78) in urine by paper chromatography with xanthydrol. Udenfriend, Weissbach, and Sjoudsma (229) have found serotonin elevated 0.6 to 3.0 y per ml. in the blood of carcinoid patients as compared to 0.1 to 0.3 y per ml. in normal individuals. Sakal and Xerrill (183) assayed reserpine in biological fluids by paper ionophoresis follor\*ed by ultraviolet spectrophotometric measurement, Colorimetric estimation of 3-chloropromazine in blood and urine was made by Leach and Crimniin (194). These investigators hydrolyzed the specimens with sodium hydroxide extracted with ether followed by 0.1-Y sulfuric acid extraction of the ether and final color development with ferric nitrate-sulfuric acid reagent. TOXICOLOGY
Kadeau (145) identified the alkaloids, papaverine, cocaine, atropine, dionine, and morphine, by paper chromatography. Huang (96) detected free and bound morphine in urine with Froehde’s reagent, and Feldstein and Klendshoj (68) rapidly isolated morphine from urine by butanol-benzene extraction and spectrophotometric measurement of its alkaline nitroso salt. Additional clinical data mere presented by Sunshine and Hockett (215) for the interpretation of blood and urine barbiturate levels. Maher and Puckett (135) mapped the ultraviolet absorption characteristics of 19 commercial barbiturates for their identification. Phenobarbital and diphenylhydantoin in blood were separated by Plaa and Hine (160) with cyclohexanebutanol and identified by ultraviolet absorption. Rapid accurate determination of trace quantities of boric acid (2 to 15 y) in 2 grams of blood, urine, or other biological material were carried out by Smith, Goudie, and Sivertson (204) by fusion with lithium carbonate, solution of carbonate in hydrochloric acid, sulfuric acid, and carminic acid added to develop color. Simple colorimetric methods were described by Chinn, Pawel, and Redmond (44),and Waggoner and Pernell (2%‘) for estimating carbon monoxide in small amounts of blood. An adaptation of the palladium chloride method was made by Allen and Root (8) for the quantitative photometric determination of carbon monoxide in blood, An accurate, but simplified, method for the determination of carbon monoxide in 5 ml. of blood was proposed by Lawther and Apthorp (123) in which blood gases are extracted under vacuum VOL. 29, NO. 4, APRIL 1957
621
after reduction of the hemoglobin by acid ferricyanide and measurement of carbon monoxide by infrared absorption McCord and Zemp (131) eliminated precipitation and ashing in urinary lead determination by extracting lead as iodide from acid solution with methyl isopropyl ketone, from which lead was removed by aqueous sodium hydroxide, and determined colorimetrically by dithizone Bessman and Lagne (23) used organic chelating compounds in the determination of lead in blood and urine. The rapid microdetermination of mercury in biological material was further simplified by Polley and Miller (161) by using the reaction between mercuric mercury and dithizone. A simple screening technique =as described by Leach, Evans, and Crimmin (124 for the estimation of mercury in urine using di-P-naphthylthiocarbazone. Stratton (211) improved the chromotropic-acid method for the determination of methanol in body fluids. Rapid screening of urine for vanadium was carried out by Rockhold and Talvitie (118) by a method which is based on the catalytic effect of vanadium on the oxidation of N , N-diethyl-p-phenylenediamine by potassium chlorate by which a deep magenta color is developed for photometric measurement.
Bensley, E. H., Drysdale, A,, Osiek, R.. Ibid.. 26. 247 (1996).
4Y7 I1 Q.56I \----
Chem. 2 , 242 (1956).
I)Uggan, E.L., -&SAL.
1
”
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Abraharnson, E. M., Am. J . Clin. Pathol. 26, 103 (1956).
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(4) Albers, R.
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CHEW27, 1829 (1955). (5) Albert, A., Proc. Sta$ Meetings Mayo Clinic 30, 552 (1955). (6) Albert, A., Recent Progr. in’Hormone Research 12, 227 (1956).
(7) Albrink, M. J., Hald, P. M., Man, E. B.. Peters. J. P., J . Clin. Invest.‘J4. 1483 (1955): (8) Allen, T. H:, Root‘, W.’S., J . Biol. Chem. 216, 319 (1955). (9) Anderson, A. J., Maclagan, hT.F., Biochem. J . 59, 638 (1955). (10) Anderson, J. T., Keys. A..‘ Clin. Chem. 2, 145 (1956): ‘ Anker, R. hl., Ibid., 2 , 184 (1956). Anker, R. hl., J . Clin. Endocrinol. and Metabolism 15, 210 (1955). Annino, J. S., “Clinical Chemistry Principles and Procedures,” 1 s t ed., Little, Brown, Boston, 1956. Archibald, R. hl., ANAL.CHERI.27, 677 (1955).
Armstrong, M. D., Shaw, K. N. F., Robinson, K. S., J . Biol. Chem. 213, 797 (1955).
’
Armstrong, M. D., Shaw, Il. X. F., Wall. P. E..Ibid.. 218.293 (19561. Awapaia, J.,’Sato,’ Y., ’CLin.‘Chih. Ada 1, 75 (1956).
Balikov, B., Castello, R. A,, Clin. Chem. 2, 83 (1956).
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BiopYhys. 56,’307 (1955). Bowman, R. I,., Caulfield, P. A , , Udenfriend, S., Science 122, 32 (1955). Broyn, W. D., Australian J . Ezptl. Biol. Med. Sci. 32,677 (1954). Bunch, L. D., Emerson, R. L., Clin. Chem. 2 . 75 119561. Cabaud, P., Lekper,‘ R., ’Wroblewski, F., A m . J . Clin. Pathol. 26, 1102 (1956).
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Dubbs, C. .4., Davis, F. RT., Adams, FV. S.,J . Baol. Chem. 218,
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