Review of
APPLIED ANALYSIS
CLlNlCAL CHEMISTRY G. R. Kingsley Veterans Administration Center, Los Angeles 25, Calif.
T
a continuation of the author’s last one which appeared in this journal (347) on the significant developments in analytical clinical chemistry. HIS review is
REVIEWS, NEW BOOKS, A N D JOURNALS
The general subjects-blood electrolytes, porphyrins, standardization in clinical chemistry, enzymes, and proteins-presented in the Symposia of the International Congress of Clinical Chemistry (New York, September 1956) (618) give an indication of some of the principal interests of clinical chemists throughout the world. Specific papers of these symposia will be referred to under the appropriate sections of this review. Clinical applications of biochemistry have been reviewed by Reinhold (508) in which emphasis was placed upon chemicaI alterations obseIved in disease, especially those relating to chemical mechanisms. I n comments on the trend of clinical chemistry, Stewart (611) indicates that there is a “tendency of clinical chemistry to penetrate into new fields of inquiry” which “must continue as research extends the range of possible analyses and relates the results more closely to clinical conditions.” Examples are: increasing use of enzyme reactions; increasing specificity and speed in hormone assays; more critical appraisal of the real value of such procedures as electrophoresis; and greater use of chemical methods in controlling the dosage of synthetic therapeutic agents. Natelson’s “Microtechniques of Clinical Chemistry” (448) is an outgrowth of the author’s booklet, ‘Correlation of Clinical and Chemical Observations in the Immature Infant.” With an excellent introduction on techniques and equipment for microsampling of specimens, it is probably the best book on microtechniques in clinical chemistry to appear in recent years. A wide coverage is presented of most routine methods as well as procedures for toxicology (arsenic, barbiturates, carbon monoxide, cyanide, lead, mercury) and special determinations (protein-bound iodine, l’i.-ketosteroids, lactic acid, cholinesterase, blood ammonia, glucuronic acid, pyruvic acid, transaminase). Excellent microgasometric methods for oxygen, carbon monoxide, carbon dioxide, etc., are presented. The methods
656
ANALYTICAL CHEMISTRY
are outlined as procedures, procedure notes, reagents, and references. King’s third edition of “MicroAnalysis in Medical Biochemistry” (846) was expanded to include new chapters on control of laboratory accuracy, electrophoresis of plasma proteins, flame photometry of sodium, potassium, and calcium, and radioactive isotope tests. Several new procedures were also added and the chapter on colorimetric and spectrophotometric analyses was rewritten. The microaspects of analysis were still emphasized. “Clinical Biochemical Methods” by Tarnoky (635)presented methods which are typically in demand in a large hospital. “The methods selected yield results in a reasonable time and with an accuracy sufficient for clinical requirements. Procedures which need expensive and highly specialized apparatus have been avoided. Accurate stepwise directions are given for most of the routine clinical chemistry methods and also a few toxicological tests.” “Colorimetric Analysis” by Allport and Keyser (IS), which encompasses determinations of clinical and biological significance, contains most of the photometric methods which are of interest to the clinical chemist, including several not found in the usual laboratory manual-Le., adenine, adrenaline, adreno-cortical steroids, cytosine, ergothioneine, methionine, nicotinic acid, thiouracil, and vitamin E. Sobotka and Stewart edited the first volume of a new and much-needed series, “Advances in Clinical Chemistry” (697),to present “objectively critical discussions of methods, and of their rational, critical, and comparative evaluation of techniques, automation in clinical chemistry, and microanalytical procedures.” The authors “take cognizance of the fact that clinical chemistry plays an essential part in the progress of medical science in general by assisting in elucidating the fundamental biochemical abnormalities which underlie disease.” Two new journals appeared since the last review: Microchemical J o u r n a l , 1957 (.416), and J o u r n a l of Chromatogr a p h y , 1958 (879). NEW APPARATUS AND EQUIPMENT
Smith and Mencken (696)constructed a machine to reduce time required for adding quantities such as 0.02, 0.04
ml., etc., of fluid to sets of 20 to 30 tubes in serial dilutions of serums. Levenbook (586) described a quantitative method for automatic application of microliter quantities onto paper chromatograms. An automatic spotting apparatus which eliminates the concentration of specimens spotted to chromatographic paper was developed by Logothetis and Sherman (391). An automatic pipetting device was attached by Seligson and Marino (671) to the Van Slyke manometric apparatus for the determination of carbon dioxide. It is accurate, requires a small sample of serum, is self-cleaning, and is rapid. An apparatus for continuous electrophoretic separation of charged particles on a filter paper curtain was described by Selden and Westphal (568). Dixon described an adaptor to be used with the Coleman Jr. spectrophotometer for the direct photoelectric scanning of electrophoretograms of serum proteins (173). Aksnes and Rahn measured the total gas pressure of individual samples of arterial and mixed venous blood together with the partial gas pressure for oxygen, carbon dioxide, and nitrogen as they exist simultaneously in the alveolar gas ( 6 ) . Popp and Walcher (488) described an ion microscope for the investigation of biological tissues. The histological distribution of alkali atoms in biological tissue was made visible by incinerating a tissue section of 10-mp thickness which, in vacuo, emits alkali atoms a t a certain temperature. The ions are focused by an intense electrical field and made visible either on a fluorescent screen or on a photographic plate. Hadd and Perloff (667) improved an all-glass automatic extractor based on Cohen’s principle, which can be used for a wider variety of extraction problems. Kohn (358) developed a small-scale and micromembrane filter electrophoretic method, in which samples ranging from 0,0001 ml. up t o 0.01 ml. with a protein content from about 5 to 1000 y can be separated, giving a neat and distinct pattern, Carroll et al. (115) described a set of adaptors, each employing two Polaroid plates to convert the photoelectric colorimeter t o a sensitive polarimeter. Brada and KoEent (96) described an apparatus for the microfractionation of blood plasma by Cohn’s Method 10 which permits a complete fractionation of four samples simultaneously within a day. Skeggs
(687) developed an automatic method for colorimetric analysis in which samples are introduced in close succession by means of a pump and propelled through a small dialyzer, together with a constant proportion of a suitable diluent. The substance to be determined is transferred in the dialyzer to a flowing stream of reagent which is continuously processed to produce a specific change in color which is measured by passing the flowing stream through a colorimeter equipped for recording. CONTROL AND PRECISION OF CHEMISTRY METHODS
CLINICAL
Guillot (263), in discussing methods for standardization in clinical chemistry, stated that these methods should require very small samples and short performance time. He said attention should be given to: the need for national or international standardization of methods; the limitation of the number of methods used in routine clinical work; a need for classification of methods into elaborate specific analytic techniques, semispecific techniques, analytic techniques concerning a group of organic compounds similar in chemical behavior, and a group of biologic tests. Rice (616) made an international survey of clinical chemistry procedures used in 95 teaching hospitals of medical schools and found that the most popular in current use appeared to be the “old time-honored” procedures. Humphrey et al. (306) reviewed biological standards in biochemical analysis. Wootton (708) made a short review of the problems involved in the standardization of methods in clinical chemistry, such as the establishment of normal values, attempts to establish agreement by distribution of specimens for analysis, use of internal checking by the individual laboratory, and use of certified samples for controls. Seligson (669), in discussing the problem of improving quality of performance of clinical chemistry, maintained that a high quality of accuracy could be attained by running concurrent primary and secondary serum standards with specimens analyzed as well as improving the methods used. A lyophilized serum chemistry reference standard for this purpose (commercially available) was studied and found satisfactory by Klein and Weissman (366). The trend toward micromethods was repeatedly emphasized in numerous papers, recent books, and journals. Brown (99) described semimicromethods in pediatric biochemistry stating that these methods require no more time, show no loss of accuracy, and require 0.1 ml. or less of sample. Ultramicroprocedures were described for chloride, protein, sodium, potassium, hematocrit, and p H by Prather (492). Sanz (649) described new equipment which permits
easy and accurate work on the ultramicro scale using 5 to 50 pl. of sample for such determinations as chloride, proteinbound iodine, calcium, potassium, sodium, nonprotein nitrogen, pH, carbon dioxide, proteins, phosphorus, glucose, etc. Saifer et al. (641) developed a rapid, semiautomatic system of microchemical analysis for the clinical chemistry laboratory which had five basic elements : use of siliconated-heparinized plasma; use of the calibrated pipet-tip buret technique for measuring small samples; use of the decantation principle as a precision step in making quantitative transfer; use of automatic syringe pipets for adding constant volumes of reagents; and use of specific enzymic methods, whenever applicable. Copeland (148) outlined a practical means for the measurement and control of the precision of clinical laboratory determinations as follows: use of proper primary standards to control accuracy as related to absolute level of concentration, and use of a standard deviation formula to measure and control accuracy as it relates to precision. A detailed analysis of the measurement errors of the Coleman Jr. spectrophotometer was made by Kanner and Moss (334). An analytic study of work-load factors in clinical chemistry involving many elements was made by Finch et al. (a@), who found that clinical chemistry tests per man hour averaged 3.1 in a hospital servicing patients with chronic diseases and 4.7 in a general hospital. Amino Acids. Gale (233) reviewed the methods for determination of amino acids by use of bacterial amino acid decarboxylases. Buchanan (108) used piperidine in dilute solution as a desalting agent, as it efficiently displaces all common amino acids on cation exchange resins; and Verghese and Ramakrishnan (679) described a simple method for desalting biological fluids for paper chromatography of amino acids. Filteau and Martel (203) determined the normal distribution of 17 free amino acids of blood serum by twodimensional paper chromatography, and Parry (473) determined the positions of 56 amino compounds of urine by using phenol and butanol-acetic acid solvents with two-dimensional paper chromatography. Roberts and Kolor (622) assayed the accuracy and precision of the quantitative paper chromatographic procedure of McFarren and Mills and found that 11 amino acids gave average values within 2% of the theoretical value, 13 less than 3%, and 17 less than 5%. Only lysine, norleucine, and tyrosine had values in excess of 5%- Whitehead (701) described the separation of amino acids and their N-acetyl derivatives by paper chromatography and paper ionophoresis. Tuckerman (660) used a newly available sulfonic acid-type cat-
ion exchange resin paper which required smaller samples and shorter time for separation. Elimination of fraction collection analysis of arginine, histidine, and lysine from casein hydrolysates could be made by direct staining. Jirgl (316) described a method for chromatographic separation on paper of free amino acids in human serum without previous desalting. Meymiel et al. (424) used a resin, Dowex 1x2, 200 to 400 mesh in a 15 X 1.5 cm. column, for the qualitative and quantitative separation of iodine-containing amino acids. Hamilton (270), using particles of sulfonated polystyrene cationic resins of different micron-diameter classes, studied the effect of resin particle size on column performance in ion exchange chromatography of amino acids. Busson and Guth (117) chromatographically separated free amino acids of serum from proteins, carbohydrates, and lipides using Amberlite 1R-120. Inouye (310) used dinitrofluorobenzene and two-dimensional chromatography for the quantitative analysis of the amino acid composition of lysozyme and bacterial amylase. Ermakova (196) claimed the establishment of optimal conditions for the ninhydrin reaction, and Tsukamoto et al. (668) described a procedure to achieve highest possible color and stability with a new modified ninhydrin reagent. Two reagents were described by Barrollier et al. (47) employing isatin, zinc acetate, formic acid, and pyridine; and sodium lJ2-naphthoquinone-4-sulfonate, zinc acetate, and quinoline. When sprayed on paper chromatograms with different solvents and heated, these reagents were capable of identifying peptides, glucosamine, and amino acids a t a concentration of 3 x 10-8 to mole per sq. cm. iln isatin and silicate reagent was used by Boyarkin (94) to develop color with amino acids on paper chromatograms. The problems of colorimetric determination of amino nitrogen in colored solutions were studied by Weill and Bedekian (693). The diacetyl monoxide urea method of Wheatley was used by Rendi (610) for the quantitative determination of arginine in nonhydrolyzed proteins. Kuratomi et al. (369) used a simplified method of hydrazinolysis for the microdetermination of the total quantity of cysteine plus cystine. Tabor and Wyngarden (630) used a sensitive spectrophotometric method for the assay of N-formimidoylglutamic acid in urine. Sanahuja and Scoane (647) determined lysine colorimetrically with alkaline diazotized p-nitroaniline. Serum phenylalanine was determined by paper chromatographic methods by Berry (67) and Zahn and Marstaller (720) and by adsorption onto Dowex 50 by Henry et al. (686) before using the VOL. 31, NO. 4, APRIL 1959
c
657
Kapeller-Adler reaction. Petrovacki (480) used a colorimetric analytical method for the determination of phenylpyruvate and phenylalanine in phenylketonuric urine, serum, and cerebrospinal fluid. The et al. (640) determined phenylpyruvic acid in urine with a simple, rapid ferric chloride reagent. L-Tyrosine in urine was determined by Tashian (636) by conversion to tyramine with decarboxylase and assay to tyramine by the nitrosonaphthol method. A new spectrophotometric method for the determination of tyrosine and tryptophan in proteins based upon the measurement of absorbance in the range between 278 and 293 mp was described by Bencze and Schmid (54). TYaalkes and Udenfriend (683) utilized the fluorescence of 1-nitroso-2-naphthol derivative of tyrosine for a specific and sensitive measurement of microgram quantities of tyrosine in plasma and tissues. Ottaway (466) used a colorimetric method based on the method of Gerngross, employing 1-nitroso-2-naphtho1 in acetone for determination of 10 to 250 y of tyrosine. A method for determination of peptides in the presence of free amino acids was described by Markovitz and Steinberg (413). Balikov et al. (37) used a biuret reagent for determination of urinary peptides. Three methods were compared by Sobel et al. (595): a modification of the copper technique of Beauchene, the 8-naphthoquinone-4-sulfonate method of Cagan, and the gasometric ninhydrin procedure of Van Slyke. Cations and Anions. The Cation Workshop of the American Society of Clinical Pathologists published standardized methods for the determination of sodium, potassium, calcium, and total base by colorimetry and flame photometry (622). Sackner and Sunderman (539) employed a rapid and reliable conductometric method for the estimation of total base in serum. Several colorimetric micromethods were published recently for the direct determination of serum calcium. Baar (33) used nuclear Fast Red; Kingsley and Robnett (350, 351) employed disodium-1-hydroxy-4-chloro2,2 - diazobenzene 1,8 - hydroxynaphthalene-3,6-disulfonic acid; and Chilcote and Wasson (135) found buffered murexide satisfactory as a direct serum calcium reagent. Indirect serum calcium methods were described by Stern and Lewis (608) by reacting the precipitated calcium oxalate with o-cresolphthalein complexone; and Ferro and Ham (201, $02) precipitated calcium with sodium chloranilate and then formed a highly colored complex with (ethylenedinitri1o)tetratetrasodium acetic acid. Ferro and Ham’s method was modified by Chiamori and Henry (133) for the determination of calcium in urine and feces.
-
658
ANALYTICAL CHEMISTRY
Titration methods for the determination of calcium are still popular as judged by the number of publications on this subject. Golby et al. (243) and Yarbro and Golby (715) used a direct Versene titration with Calcon as indicator for serum calcium, and for urinary calcium used an excess of standard ethylene glycol bis(aminoethy1 ether)-N,N’-tetraacetic acid and backtitrated with calcium using Calcon as indicator. Andersch (16), Ashby and Roberts (24),and Baron and Bell (45) used (ethylenedinitri1o)tetraacetic acid (EDTA) with calcein indicator for serum calcium determination, the latter using calcein thymolphthalein indicator. Bachra et al. (34) used Gal-Red, and Wilkinson (703) used murexide indicators with EDTA for serum and urine calcium estimations. Simple dialysis methods for the determination of ionized, ultrafilterable, or diffusible plasma and serum calcium were described by Poulos (491), Toribara et al. (65R), Pasad and Flink (474, and Rose (669). Direct argentometric, electrometric chloride titration methods mere employed by Cotlove et al. (150), Hallaway (269), Seligson et al. (671), and Stern and Shmchman (610) for chloride determination in various biological fluids. hlercurimetric direct titration methods for chloride estimation were described by Rice (516) and Juliard (362). hfercuric chloranilate was used by Barney and Bertolacini (44) to determine chloride in aqueous solution within limits of detection of 0.2 p.p.ni. hfenis et al. (423) used an indirect flame photometric method for the deterniination of chlorides and other halides. A clinical review of magnesium] together with some experiences in its determination, was made by Hasselman and Van Kampen ($77). Masson (419) described a rapid colorimetric method for serum magnesium in which a red lake is formed with titanium yellow and magnesium oxide. Microgram quantities of magnesium were determined flame spectrophotometrically by use of 80% acetone by Manna et al. (411). Teloh (639) developed a self-standardizing method for flame photometric determination of serum magnesium by measuring intensity of spectral emission a t 372 mp and background emission a t 360 mp. Glick et al. (240) modified the Titan yellow method for the determination of magnesium in microgram samples of tissue. Teeri and Sesin (638) and Sunderman and Sunderman (623) proposed a turbidimetric method for serum potassium determination based on the reaction of potassium with sodium tetraphenylborate to form an insoluble potassium salt which is stabilized in turbid suspension with a buffer. A simplified colorimetric method was described by Marune (418) for the determination of
serum potassium by precipitation as potassium cobalt hexanitrite, decomposing the complex with sulfuric acid, and measuring the resulting cobalt thiocyanate acetone complex colorimetrically. Potassium in aqueous solutions was precipitated with an excess of sodium tetraphenylborate by Schall (669) and this excess was titrated with a quaternary ammonium salt. Flaschka et al. ($09) carried out an improved complexometric determination of serum potassium with a copper (ethylenedinitrilo) tetraacetate-a-pyridylazo-p- naphthol system which gave a sharper end point. Pavlova and Iordanov (475) made a polarographic determination of potassium in serum by precipitating potassium as potassium sodium-cobaltinitrite and measuring the polarographic reduction of the complex [Co(NH&]. Enzymes. Enzyme system methods were reviewed by Bruns (107). Bodman (84) discussed the measurement, physiological importance] and control of proteolytic enzyme inhibitors. Overbeek (467) noted the need for better methodology in a review on fatsplitting enzymes in blood. +A simple, direct spectrophotometric method was developed by Kramer and Ganison (562) for the determination of acetylcholinesterase activity using a new chromogenic substrate, indophenyl acetate. Harris (276) described a routine procedure for the microdetermination of serum aldolase, and Friedman and Lapan (262) standardized a colorimetric serum aldolase method based on the hydrolysis of fructose 1,6-diphosphate and color development of the resulting trioses with dihydroxyacetone. Plasma amine oxidase activity was determined by Humoller et al. (306) with a modification of Akerfeldt’s method and by Cotzias and Greenough (161) by use of the transfer rate of ammonia. Ultramicrodetermination of serum amylase activity in 0.01 ml. of serum mas achieved by Close and Street (141) by adaptation of their micromethod. Gomori (851) modified the serum amylase method of Huggins and Russell for 0.1 ml. of serum and 30-second incubation. hfcGeachin et al. (406) found that anticoagulants, oxalate, or citrate will lower amylase values 20% below that of sera collected mith heparin. Serum amylase was determined by Paget and Delaisse (469)by hydrolyzing starch, clearing the reaction mixture with zinc ferrocyanide, and determining the reducing sugar formed. Smith and Roe (591) made a micromodification of their amylase method which gave values 21% higher than the original method. Vargas et al. (672) compared the turbidimetric Peralta and Reinhold and the Somogyi-Nelson serum amylase methods and found a range and standard deviation of 34 to 260 =!z 4.25 in the former and 30 to 195 =k 2.87 in the latter.
Caspe and Pitcoff (127) determined blood codehydrogenase niiciobiologically by means of H . parainjluenzae. A simple, semiquantitative, bedside method was developed by Goiffon et al. (242) for the determination of fecal catalase activity within 15 seconds. A colorimetric quantitative method for plasma catalase activity, based on the reaction of undecomposed hydrogen peroxide with ferrocyanide and ferric gum ghatti t o give a Prussian blue color, was described by Dobkin and Glantz (174). Bodman (85) measured serum inhibitor to chymotrypsin by determining the inhibition of the release of biuretpositive, trichloroacetic acid-soluble material from casein by the action of chymotrypsin. Assay methods for cholinesterases v-ere revien-ed by Augustinsson (28). Cellular and plasma cholinesterases TTere measured by Biggs et al. (71)using a procedure based on the determination of the amount of acetic acid released from acetylcholine, according to the change in absorbance of bromothymol blue. The liberation of acetic acid from acetylcholine bj- serum cholinesterase was analyzed photometrically by measurement of the decrease in absorbance of phenol red indicator by Caraway (122). Thiocholine chloride ester substrates ivere developed by Gal and Roth (232) for spectrophotometric measurement of cholinesterase activity in 0.01- to 0.001-ml. samples of blood serum or tissue extracts. The colorimetric method was based on the reduction of indophenol by the thiocholine liberated during the enzymatic hydrolysis. Goshev (255) measured cholinesterase actiyity potentiometrically in 0.1 gram of tissue or fluid by measuring the changes in p H of a borate buffer containing specimen and acetylcholine with an antimony electrode. Inhibition curves for a large group of inhibitors of cholinesterase were run by Holmstedt (298). Cholinesterase in blood serum and m-hole blood was determined by Pokrovskii (436) using a method based on color change in cresol red when titrated with acetic acid formed by the enzymatic hydrolysis of acetylcholine. Jlethods for the determination of succinic dehydrogenase have been reviewed by Singer and Kearney (586). Bajusz and Kovary (36) determined the dehydrogenase activity of serum protein fractions by treating the paper strips after electrophoretic fractionation n ith buffered 2.3,5-triphenyltetrazolium chloride. Eckstein et al. (181) modified the dehydrogenase method of Kun-Abood for application to tissues using buffered neotetrazolium chloride. Tsukamura (659) used picric acid with success in place of tetrazolium salts in the determination of dehydrogenase activity.
Lazaroni et al. (378) found serum lactic dehydrogenase stable for 7 days a t 37" C. in 80% of cases studied. Cabaud and Wr6blewski (119) developed a practical spectrophotometric method for the colorimetric measurement of lactic dehydrogenase activity of body fluids by use of dihydrodiphosphopyridine nucleotide, buffered pyruvic acid substrate, and dinitrophenylhydrazine for color development. Robert and Samuel (521) described a spectrophotometric method for the determination of serum elastase inhibitor. Ramsey (502) used fatty acid esters of 2-naphthol as substrates for measurement of esterase activity in biologic tissues and fluids by photometric measurement of a diazonium salt coupled to the 2-naphthol liberated by the hydrolysis. Goldbarg et al. (245) used a substrate of 6-bromo-2-naphthyla-D-glucopyranoside for the colorimetric determination of a-D-glucosidase in serum and tissue. Tauber (637) prepared a stable olive oil emulsion for serum lipase determinations with the aid of Carbopol 934, a new hydrophilic colloid. Henry et al. (285) demonstrated the specificity of the lipolytic activity of serum of patients with acute pancreatitis for olive oil substrate which mas shown not to be true of tributyrin substrate. Nachlas and Blackburn (444) presented a urinary lipase method which employed 0-naphthyl caprylate as a substrate and colorimetric determination of a n azo dye of @-naphthol. Goldschniidt (248) measured N,Ndimethyl-p-phenylenediamine oxidase in heparinized plasma by oxidation of N , N dimethyl - p - phenglenediamine-hydrochloride. Courtois and Villiers-Huiban (153) assayed pepsin in gastric juice by determining the amount of unhydrolyzed edestin which can be precipitated by phytic acid. Bergesi (56) presented a new method for determination of the proteolytic activity of pepsin, using hemoglobin as a substrate and measuring the products of hydrolysis spectrophotometrically at 280 mp. Klotz and Duvall (357),by use of radioactive albumin and bovine albumin as carriers, determined pepsin in gastric juice. Bunte and Demling (112) determined peroxidase activity in serum by measuring photometrically the purpurogallin formed from pyrogallol in the substrate. Lyr (400) showed that peroxidase activity may be determined by measuring the time required for a color change to occur in a specimen to which benzidine and ascorbic acid were added. Fishman and Davidson (208) revien-ed the determination of acid phosphatases. 8tolbach et al. (613) simplified the technique of Fishman and Lerner for measurement of serum acid phosphatase of prostatic origin by eliminating deproteinization with a
diazotized coupling agent to measure the liberated phenol. A rapid stable color for phosphorus and phosphatase determination was obtained by Dryer et al. (178) with N-phenyl-p-phenylenediamine. Gupta and Chatterjee (264) developed techniques for the quantitative histochemical determination of alkaline phosphatase activity. Aspen and Meister (25) surveyed methods for the determination of transaminase. The clinical significance of alterations of transaminase activities of serum and other body fluids was reviewed by Kr6blewski (711). Humoller et al. (304) modified the method of Cabaud et a l . and eliminated the interference of unreacted dinitrophenylhydrazine by extracting the pyruvate dinitrophenylhydrazone with aqueous bicarbonate. Kaltenhach et al. (328) simplified the methods for measurement of glutamic oxalacetic transaminase and lactic dehydrogenase in plasma or seruni by use of a single blank of reduced cozymase (DPN-H). Several simplified colorimetric procedures for measurement of glutamic oxalacetic and glutamic pyruvic transaminase of sera were published by Mohun and Cook ( 4 3 4 , Reitman and Frankel (609), Sal1 et al. (542), and Wr6blewski and Cabaud (712). Schneider and Willis (564) studied several conditions which cause variation in the spectrophotometric assay of serum glutamic oxalacetic transaminase. Reichard (507) avoided the use of isotopic substrate by measuring the formation of ammonia from citrulline in the determination of ornithine carbamyl transferase by a microdiffusion technique. Reichard (506) also measured ornithine carbamyl transferase in human serum by measurement of C1402 formation from a citrulline~arbamy1-C'~substrate. Pantlitschko and Grundig (472) followed the extent of proteolysis by trypsin, papain, and pepsin on casein and serum by a modified biuret reaction. Houck (300) determined ribonuclease by measuring turbidity M hich developed after mixing ribonucleic acid with serum albumin and gelatin. Trypsin was assayed by spectrophotometric measurement of acid production by Rhodes et al. (513), by ammonia formation by Nardi (447), by modification of the Kunitz casein method by Bundy and Nehl ( I l l ) , and by measurement of the absorbance a t 280 mp of the hydrolysis products remaining in solution after precipitation of a casein digest by Christensen (137). A method for the estimation of plasminogen activator and trypsin inhibitor in animal and human tissues was described by Astrup and Albrechtsen (26). London et al. (392) assayed tripeptidase by measuring the difference in absorbance between the copper complex of VOL. 31, NO. 4, APRIL 1959
659
diglycyl-glycine and the copper complex of its enzymatic breakdown products. Christensen (136) estimated uropepsin by absorption measurement of the separated digestion products of an edestin substrate a t 280 mp. The methodology and clinical significance of the uropepsin test was critically discussed by Elsner (191). Dodgson and Spencer (176) reviewed methods for the assay of sulfatases. Hemoglobin. Huisman (301) wrote an excellent review on abnormal hemoglobins. -4 clinical and biochemical review of human hemoglobins was made by Vella (673). Goldberg (246) discussed the practical physical and chemical procedures available for the identification of hemoglobins. Bruckner (106) determined the oxyhemoglobin-hemoglobin ratio in blood by differential absorption in the infrared. The second and final report of Cannan (121) on a proposal for a certified standard for use in hemoglobinometry, as directed by the National Research Council, was reported in several scientific journals. Williams and Zak (705) standardized hemoglobin by wet-ashing a small sample of blood in a mixture of 100 parts of nitric acid, 50 of perchloric acid, and 5 of sulfuric acid, and using 2,2’-bipyridyl as a color reagent for the iron liberated. Gladishevska et al. (239) made a spectrophotometric determination of blood hemoglobin using a Soret spectrum with a 417-mp wave length a t which oxyhemoglobin solutions have a high absorbance. Zijlstra and Muller (725) devised a spectrophotometric method for the simultaneous determination of carboxyhemoglobin and methemoglobin in blood. Small differences in the transmittance spectra of derivatives or compounds of normal hemoglobin and hemoglobin S were observed by McCord and Gadsden (404). Clearer separations of hemoglobins A, S, C, E, and F were obtained with starch gel than by paper electrophoresis by De Grouchy et al. (166). The differences of the mobilities of abnormal human hemoglobin on Amberlite IRC-50 column and Whatman 3-mm. paper were reported by Huisman and Prins (302). A comparative study of different methods for hemolysis of human erythrocytes was made by Huisman and Schreuder (303).
Iodine, Protein-Bound, and Gonadotropins. Silver (584) reviewed blood plasma levels of radioactive iodine-131
in the diagnosis of hyperthyroidism. Chaney (130) surveyed the techniques and problems concerned with the determination of protein-bound iodine (PBI) by the distillation method. Rosenkrantz (631) reviewed infrared analysis of hormones. Barker and Man (42) mod8ed Malkin’s screening test, and Grossman and Grossman (260) proposed
660
ANALYTICAL CHEMISTRY
a rapid test for iodine contaminants to prevent cross contamination during alkaline incineration in the serum PBI determinations. Several factors affecting the accuracy of the alkaline ashing method for the determination of serum protein-bound iodine were carefully investigated by Acland (1). Strickland and Maloney (620) proposed a 3-hour protein-bound iodine method in which the cerate is stabilized with mercuric mercury after incubation. Some factors affecting the determination of plasma protein-bound iodine using the alkaline fusion-ceric sulfate method, such as time and temperature of storage of plasma, standard error, and coefficient of variation, were studied by Lennon and Mixner (384). A new apparatus was described for the determination of protein-bound iodine by Rosenmund (632), in which the dried protein piecipitate was oxidized in a stream of oxygen and the gas evolved absorbed by sodium hydroxide. An ultramicromodification of the Barker protein-bound iodine method which required only 0.05 ml. of sample was achieved by Sanz et al. (550). Sensitivity was obtained by spectral measurement a t 317 mp, Thyroxine in 1 ml. of serum was determined chromatographically by Stolc (614) by spraying the chromatogram with ceric ammonium sulfate and arsenite, drying, treating with fluorescein, and observing under ultraviolet light. The purification of ceric salts for PBI determination was described by Todd (646). Thyroxine-binding capacity and measurement of circulating serum iodoproteins was accomplished by Block et al. (79) and Hofmann-Credner (291) with iodine-131. Bode and WaldSchmidt (83)used iodine-131 to measure the accuracy and per cent recovery of the Chaney distillation method for serum PBI. Fletcher (210) also used iodine-131 for fractionation of urinary iodine. Blanquet et al. (78) used Dowex 1 or 2 to separate inorganic iodide, thyroxine, monoiodotyrosine, and diidotyrosine from each other. Methods employing butyl alcohol for extraction of serum PBI were described by Maclagen et al. (Cor), Kumaoka and Toba (368),Kontaxis and Pickering (360), and Barna et al. (43). Methods for the extraction, filtration, purification, and measurement of urinary gonadotropins were described by Wagner ( 6 8 4 , Loraine (394), Kameya (329), Johnsen (317),Zacco et al. (719), and Albert et al. ( 7 ) . Antoniades et al. (18) described a procedure for the preparation and concentration of the pituitary gonadrotropins from human plasma. Piotti (482) described a chemical-biological method for the determination of thyrotropic hormone in human plasma. Lipides. Svennerholm and
Svennerholm (627) presented a quantitative method for estimation of true cerebrosides in plasma using orcinol reagent. Biesold and Strack (70)determined glycerol in blood or serum by cleaving glycerol with sodium periodate t o form formaldehyde and formic acid, The formaldehyde formed is measured photometrically with chromotropic acid. Methods for glycolipide determination were reviewed by %din (498). The measurement of polyunsaturated fatty acids was surveyed by Holman (296). The conversion of fatty acids and their esters to hydroxamic acid was used for the determination of fatty acids in blood, body fluids, and feces by Tompsett and Tennant (650), Tompsett (648), Gey and Schon (235), and Eggstein (184). Weigel (691) used a rapid colorimetric formaldehyde reaction method t o determine serum fatty acids. Unesterified plasma fatty acids were determined by Svanborg and Svennerholm (626) by a method based on total lipide extraction with methanol-chloroform, removal of nonlipide contaminants by phase partition, chromatographic separation of the phospholipides from the extract on silicic acid, and titrimetric determination of unesterified fatty acids. Improved separation and identification of higher fatty acids by paper chromatography were obtained by Alimova and Bolgova (10) by a preliminary treatment of the paper with hydrochloric acid, water, alcohol, and ether. An empirical isomerization method for the determination of polyunsaturated acids in plasma and tissue was proposed by Holman and Hayes (297). Serum lipides were analyzed by chromatography and infrared spectrophotometry by Freeman et al. (221). Tourtellotte et al. (655) determined total lipides in cerebrospinal Auid by an ultramicromethod based on the oxidation of an extract by a dichromatesulfuric acid reagent. Methods for serum and tissue lipide fractionation were described by Delsal (166),Folch-Pi et al. (211), Roboz et al. (525), and Schauenstein et al. (560). Scanu et al. (555) used heparin and phenol to form a precipitate in serum in which the ratio of cholesterol to phospholipide was similar to that of p-lipoproteins separated by ultracentrifugation or chemical fractionation. This provided a simple method for measuring 8-lipoprotein. Burstein and Praverman (116) used heparin in presence of nickel, cobalt, or manganese chlorides to precipitate totally serum p-lipoproteins. Dextran sulfate was used to separate p-lipoproteins from serum by Oncley et al. (464),Burstein (114), Burstein and Samaille (116), and Antoniades et al. (19). Bernfeld et al. (66) determined serum p-lipoprotein by a method based on the nephelometric measurement of a finely-dhpersed precipitate of p - l i p
protein with sulfated amylopectin a t p H 8.6. Burstein (113) used polyvinylpyrrolidone to precipitate 8-lipoproteins. Fischer (207) used Oil Red 0 for staining the lipoprotein fractions of serum protein electrophorograms. Moinat et d. (455) stained lipoproteins on paper with Sudan Black B using methyl and isopropyl alcohols as solvents in the determination of lipoproteins by electrophoresis. Kanabrocki et al. (332) prestained serum with Sudan Black B dye before paper electrophoretic separation of lipoproteins. Dileo et al. (172) also used the latter procedure. Solinas et al. (598) prestained serum lipoproteins with diacetin. Five serum lipoprotein fractions were recognized by Straus and Wurm (619) by staining with F a t Red 7B and using sodium hypochlorite decolorization. Talluto et nl. (632) used a 50% aqueous solution of diacetin saturated with Oil Red 0 as a dyeing method for quantitative determination of lipides and lipoproteins directly on filter paper. Starch block electrophoresis a t p H 8.6 was employed by Bernsohn (66) to separate serum CYand 8-lipoproteins. Pezold (481) found that lipoproteins fractionated by ultracentrifugation did not migrate identically on paper as compared to agar gel electrophoresis. Nury and Smith (462) modified the procedure of Langan, Durrum, and Jencks for rapid measurement of total serum cholesterol LYand @-lipoproteins. Weigel (692) separated the serum lipides from other lipide constituents with a n acetone-calcium chloride solution. Liver Function. The diazochlorides of certain amines were used successfully for the detection of bilirubin in bile, serum, and urine by Barakat et al. (41). A high-frequency technique !vas used by Johansson et al. (316) for continuous recording in the chromatographic analysis of bile acids. Colorimetric determination of bile acids was made by Usui et al. (668) by use of hydroxamic acids of bile acids to develop a purple color upon addition of ferric perchlorate reagent. Billing (79)found that direct-reacting bile pigment is a mixture of two components: Pigment I, which occurs in serum and urine, is the monoglucuronide of bilirubin; and pigment 11, the major one found in human bile, is the diglucuronide ester of bilirubin. Separate determination of directreacting urinary bilirubin was made by Gries and Gries (259) by preliminary adsorption and elution of bilirubin on calcium phosphate and measurement of the direct-reacting pigment extracted by selective coupling a t p H 3 to 4.2 with diazosulfanilic acid. Lathe and Ruthven (376) studied the effect of factors such as pH, solvent, and protein on the rate of coupling of bilirubin and conjugated bilirubin in the Van den Berg reaction
for their estimation in a small amount of serum. Pittera and Cassia (484) found it impossible to separate direct and indirect bilirubin chromatographically. Stoner and Weisberg (616) described an ultramicro diazo blue reaction method for serum bilirubin. A microcapillary method for measurement of bilirubin in 0.02 to 0.05 ml. of serum was used by White et al. (700). Bunch (110) has shown that incubation of the cephalin-cholesterol reaction mixture for 3 hours a t 37.5" C. results in approximately the same degree of flocculation as that observed in 24 hours a t room temperature. Yonan and Reinhold (717) observed decreased thymol and zinc sulfate turbidity values and increased phenol turbidity after serum was refrigerated overnight. Kuroda (370) removed indican from urine to improve the method for measurement of urobilinogen. Sat0 et al. (552) used formalin to prepare blanks to use in Watson's method for urinary urobilinogen determination. A new micromethod for determination of hippuric acid in urine was developed by Elliott (189),by which ultraviolet spectrophotometric measurement is made of hippuric acid partially separated from other urinary components by ion exchange chromatography. Metals. Contamination in trace element analysis and its control was reviewed by Thiers (641). Techniques were developed for the spectrochemical determination of magnesium, chromium, nickel, copper, and zinc in human plasma by Monacelli et al. (437). A number of organic colorimetric reagents of interest to the chemist for measurement of microgram quantities of most of the metals was described by Yoe (716). A technique for determination of trace amounts of cobalt, nickel, copper, and zinc by a two-solvent chromatographic separation, elution of ions from sections of a chromatogram, and their spectrophotometric determination was developed by Frierson and Rearick (223). Different complexing reagents for the microdetermination of copper in body fluids were used: [Cyclo-CeHlo:NNHC(: O ) ] *by Bohuon (88),diphenylcarbazone by Lapin (372), and Zincon by McCall and Davis (402). The latter reagent was also used for zinc determination. Saltzman and Keenan (545) reviewed methods for the microdetermination of cobalt in biological materials. Ramsay (499) reviewed the techniques of plasma iron determination. Ramsay (600) made further improvements in his 2,2'dipyridyl serum ion method. Umemot0 and Yamamoto (665) improved Torrii's serum iron method in which o-nitrosoresorcinolmonomethyl ether was used. Underwood (667) used a new reagent, ethylenediamine di(o-hydroxyphenylacetic acid), for spectrophotometric iron determination. Baily (35)
stabilized ferric thiocyanate color in aqueous solution by the addition of methyl ethyl ketone-acetone. Rosenthal et al. (537) suggested the inclusion of control serum samples for the estimation of serum iron by the KingsleyGetchell bathophenanthroline and the Ramsay dipyridyl methods. Palmieri and Giacca (471) made a critical review of serum iron methods. Roche et al. (526) described an isotopic tracer for measurement of iron lost into and reabsorbed from gastrointestinal bleeding lesions. A method for direct determination of the latent iron-binding capacity of blood was described by Klein (356). Ramsay (501) determined total ironbinding capacity of serum by a sensitive method based on the addition of excess ferric ion t o serum, followed by removal of unbound iron by adsorption on magnesium carbonate, and determination of bound iron. Bartley et al. (48) modified Sideris' manganese method in which manganese reacts with formaldoxime for a simple colorimetric method for estimation of manganese in tissue extract. Ventura and Candura (677) made a critical review of serum zinc methods. Stewart and Bartlet (612)proposed the utilization of 4-chlororesorcinol for the colorimetric determination of zinc in ashed biological material. Nitrogen Compounds. A simple method for microdetermination of Kjeldahl nitrogen in biological materials was described by Lang (371). Leuschner and Estler (585) developed a method for the determination of adenosinetriphosphate and adenosinediphosphate based on the enzymic synthesis of acetylcholine. Pirwitz et al. (483) described a method for the colorimetric determination of adenosine in human blood. Crane (154) detected the presence of basic organic nitrogen compounds and their salts by precipitation of the amine cations with sodium tetraphenylborate. Stone (615) used phenosafranin as a new colorimetric reagent for the microdetermination of ammonia following Conway microdiffusion. Seligson and Hirahara (670) developed a microdiffusion method for measurement of ammonia in blood for rapid multiple determinations. n'athan and Rodkey (460) used this method to determine ammonia in trichloroacetic acid filtrates of blood, making a final colorimetric determination with ninhydrin. Brown et al. (102) described a colorimetric micromethod for the determination of plasma ammonia in the presence of labile amides. Van Pilsum and Bovis (671) investigated the effect of various types of protein precipitation procedures upon the recovery of creatinine added to blood. Anderson et al. (17) determined creatine in biological fluids by first separating VOL. 31, NO. 4, APRIL 1959
661
with Amberlite IRC-50, IRA-401 , and IR-120, after which creatine was reacted with diacetyl in the presence of 1-naphthol for colorimetric measurement. The Raaflaub and Abelin diacetyl creatine method was modified by Kanig (533). Sullivan and Irreverre (621) showed that 1,4naphthoquinone2-potassium sulfonate has a greater specificity for creatinine in whole blood than other reagents. The principle of a method used by Avi-Dor and Lipkin (90) for the spectrophotometric determination of reduced glutathione was based upon the addition of borate t o the product of the interaction of fluoropyruvic and thiol compounds which have no free amino groups in the alpha or beta position, to produce a shift in the absorption peak from 275 to 290 mp. Methods for the assay of heparin were described by Serafin (574) and Freeman et al. (219). Methods for the determination of hexosamines were reviewed by Gardell (234). Clinical methods for the determination of histamine were described by Fukarek (226) and Adam et al. (2). A sensitized filmmethod for the detection of homogenetisic acid in urine was reported by Sommerfelt and Wynstroot (699). Gadebusch and Gabriel (629) modified Kovacs’ reagent for the detection of indol by substituting isoamyl alcohol for n-amyl alcohol to obtain better color and greater stability. A simple, more specific method for fluorometric determination of kynurenic and xanthurenic acids in human urine was developed by Satoh and Price (555). Miller (499) reviewed the microbiological assay of nucleic acids and their derivatives. Webb and Levy (688) reviewed new developments in the chemical determination of nucleic acids. Webb (687) investigated the estimation of total nucleic acids in trichloroacetic acid hydrolysates of biological material by ultraviolet-absorption methods. Specific fluorometric methods for the measurement of pyridine nucleotides were described by Lowry et al. (396)and Jacobson and Astrachan (314). Crook and Rabin (157) described a colorimetric procedure for the determination of dipeptides which depends upon the formation of oxygenated cobalt complexes of the dipeptide in the presence of alkaline cobaltous phosphate suspension. Markol-itz and Steinberg (413) described a method which permits the detection of small amounts of peptides or other bound amino acids in the presence of large excesses of free amino acids. Bergmann and Dikstein (57) reviewed new methods for purification and separation of purines. A technique has been described by Semenza (573) for the separation of purines and pyrimidines by paper electrophoresis. Loo and Michael (399) developed a colorimetric method for the determination of 6-mercaptopurine and 4aminopyrazolo [3,4d]pyrim-
662
ANALYTICAL CHEMISTRY
idine in blood and urine. A simple method was presented by Ling (390) for urinary taurine estimation as its dinitrophenyl derivative. Pentz et al. (477) used a dinitrofluorobenzene derivative for determination of taurine in urine. Improved diacetyl monoxine methods for the determination of blood urea were proposed by Sardou (551), Marsh et al. (416), and LeMar and Bootzin (380). A new method for the gasometric determination of urea in urine was proposed by Barakat et al. (40) in v hich N-bromosuccinimide reacts with urea in an alkaline medium to give nitrogen, carbon dioxide, succinimide, and bromide. Boutwell (93) used an iodinated Nessler’s reagent and a two-step nesslerization procedure to eliminate turbidity in the determination of blood and urine urea. A turbidimetric method for the estimation of blood urea was proposed by Lawrie ($77) who used a xanthydrol reagent. -4colorimetric determination of urea in pure solution was made by Rashkovan (504) by use of hypochlorite and phenol reagents. Watson and Pratt (686) used filter paper spot test technique with Feigl’s reagent to determine urea in 5 to 20 pl. of biological fluid. Archibald (20) made a more convenient modification of the Kern and Stransky uric acid method which was confirmed by klper and Seitchik (14). Bergmann et al. (58) prepared plasma filtrates with mercuric chloride for quantitative determination of xanthines and uric acids. Dikstein et al. (171) determined xanthines and uric acids in urine by forming mercury complexes of purines and adsorbing on an ion exchanger column loaded with mercuric ion. Procedures were proposed by Valori and Antonini (670), Hausman et al. (979), Henry et al. (187)’ and Steel (607) for the prevention of turbidity and improved color development in serum uric acid determinations. Shapiro et al. (576) isolated uric acid in biologic fluids by an ion exchange separation and determined the uric acid by an established procedure. Electromigration of uric acid from serum and synovial fluid was studied by Salteri et al. (544) and Salteri and Cirla (543), who found that uric acid present in serum and synovial fluid showed the same electrophoretic behavior as uric acid in free solution. Kanabrocki et al. (331) compared five different plasma uric acid methods and found that the method of Kern and Stransky gave the closest agreement with the uricase method.
Organic Acids and
Compounds.
Nordmann et al. (459)determined organic acids in human plasma by use of Dowex 2, X-10, and two-dimensional paper chromatography. Zelnicek (723) separated the hydrazones of &-keto acid hydrazones of blood and urine electrophoretically in 0.05M bicarbonate and
measured the yellow color a t 380 mp. Gaitonde and Gordon (231) determined shikimic acid in all tissues except liver and muscle by elution from paper chromatograms. Bruce and Howard (104) determined biphenyl colorimetrically in biological materials by nitrating to form 4nitrobipheng1, then reducing and coupling the resulting amine with N-(1naphthy1)ethylenediamine to give a purple dye. lliettinen et al. (427) and Alrashi (6) determined glucuronic acid in plasma, blood, and serum with modifications of the naphthoresorcinol colorimetric method. Brown (103) determined urinary hydroxykynurenine by a method based on the increase in color caused by the addition of nitrite to a sample of urine previously purified by ion exchange chromatography. Roberts and Kolor (5%) improved the determination of hydroxyproline by paper chromatography, using ninhydrin-isatin-triethylamine instead of isatin. Grunbaum and Glick (261) simplified the procedure for the hydrolytic liberation of hydroxyproline from tissue for colorimetric determination. llatsch and Graft (421) determined urinary p-hydroxypropiophenone colorimetrically by a reverse Molisch reaction in which p-hydroxypropiophenone replaces naphthol and develops a blue-violet color with added glucose in Valthe presence of sulfuric acid. guarnera (669) suggested modifications of Parri’s reaction for a new method for detecting indoxyl in urine. Keish (469) reviened methods for determination of 3-keto acids. Save1 and Cariou (664) determined acetoacetic and @-hydroxybutyric acids in blood and serum by microdiffusion and colorimetric measurement with salicylaldehyde-ethyl alcohol-sodium hydroxide reagent. A similar method was used by Bloom (81). Levine and Taterka (387) formed a color complex of acetone and diacetic acid distilled from blood and urine with vanillin in an alkaline medium. Colorimetric methods for the determination of acetone and acetoacetic acid in blood and urine were proposed by Adler (4),Bohm and Zimmermann (86)’ Juhasz (321), and Schon and Lippach (565). Bevenue and Williams (68) detected 5-keto-aldonic acids on paper chromatograms by spraying with 0.5N hydrochloric acid and heating. Rabinowitz and Pricer (497) determined formic acid by a method based on the enzymic conversion of formic acid to 10-formyltetrahydrofolic acid and the spectrophotometric determination of 5,lOmethylidynetetrahydrofolic acid, which is formed by the action of acid on the enzymic products, ITieland (702) proposed a colorimetric routine lactic acid method employing the reduction of potasqium ferricyanide and a diphosphopyridine nucleotide-independent lactic acid dehydrogenase from yeast. Tompsett
(649, 6.47, 649) described colorimetric methods for tleterniinatioii of urinary phenols aiid phenolic acids. Kath a i d Mukherjee i$@) estiniated pyruvic acid in blood by the modified colorimetric niethod of Lu, Bueding, and Kortiq. Zelnicek (723) and Zeliiicek and Cernocli (?2.$)sepnr:ited the hydrazones of pyruTic, a-ketoglutaric, aiid osalacetic acids by paper electroplioresis. Dickeiis aiid Killianiwn ( 16s)describeda colorimetric method for the drterniination of hydros?-pyru\-ate a n d glj-coaldehyde by a method h n w l on a condensation reaction with nnplithoresorcinol in 2 3 5 sulfuric. ncitl. Porphyrins. Cliu and Cliu (135, 1%) separated tlie methyl esters of uroporyli!-riii. I ant1 111 isolated from urine on n Hyflo Super-Gel column by using cliloroforriiheiiz~.nr.. as the dereloping agent. Hcikcl (280) used an aiiioii eschange rc4n for the clet~erniiiiation of porphobiliiiogrn, copro-, and uroporphyrin in uriiir aiid tissue. Holecek (233) con.itlcwt1 the best n-ay to deterniiiie small amounts of coproporphyrin I11 in the pre-ence of large excess of coproporpli>-riii I n-ns to develop paper c hroiiia togra ins 17-it11lut idine-water in a n atniospherr containing ammonia vapors antl if thr ratio Tvere reversed a small amount of trichloroacetic was added. Fluoropliotoiiietric. chromatographic methods ere used by Kajimire (325) and T’c~g1ie.r (678)for the determination of porpli:\-rins in blood and urine. iiiig test for estiniatioii of ins in the clinical laboratory was prripmed by Schlenker and Iiiteliell (562). K i t h (707) could not tlmionstrate protein-binding of any porphyrins 11y mtwis of paper electrophoresis of normal serum to whirh porphyrins n-erc ad t lr rl . Proteins. O w n (468) reviewed papry Plecti,oplioresis of proteins and ilxtanccs in clinical undernian et al. (624) s e p a r a t d nornial serum proteins by coiitiiiuous flon- electrophoresis to determine their nitrogen content and found their average iiitrogeii per 100 grams of protein to Iic 13.3 -L 0.2. d factor of 6.54 instead of 6% \vas proposed to calculate total prott4ii from nitrogen content. Serum nlbiu-~iin n-as determined by Rlondheini (SO) I]>- use of broniophenol blue 11-1iic.hiq nlnioot entirely bound to albumin. Luliraii and Lloss (397) dcxscrilxd n precise isotope dilution method for the tletc.r~iiiiiatioiiof serum albumin in n-hicli io~liiie-131-labeled albuniin w i s adrlml t o R saniplc of seruni and the globulins w r e wparated n-it,hsodium siilfitv m i l citlier. Biuret nieasurenieiit of alhiiniin aiitl radioactivity count was made t o d c u l a t e albumin. liaiialxoc*liiet al. (330) obtained better agrecwcnt brtn.ecn albumin values obtained 1 ) ~ -clectrophoresis aiid saltingmit scymition n-hen excessire shaking in
the latter method n-as avoided. Boettcher et al. (87) separated a B1metal-combining globulin fraction of plasma by use of rivanol as a precipitating agent for albumin, remora1 of riJ anol n ith charcoal, and precipitation of globulin with 40y0 ethyl alcohol a t pH 5.8. Frattini (216) used mercuric chloride as n reagent for a turbidimetric test for y-globulineniia. A rapid method )!-as developed by Hans1 (272) for the preparative fractionation of human 7-globulins in a rontiiiuously developing p H gradient. The coupling of protein m ith stabilized tliazotized p-nitroaniline vas u v d as a method for the separation of globulin from albumin in cerebroapjnal fluid by Hall (268). Sethiia aiitl Tqao (575) clvaluated several n.cll-kiio~vii methods for deteriniiiatioii of proteinr in ccrchrospinal fluid i i w g thc micro-I(jPltiali1 method as a standard for coni11ari~oii. Several methods have been proposed for electrophoresiq of proteins in cerebrobpinal fluid by Llunimthaler and lIarli1 (.$@), Leninien et al. (381-3), and Riechert and Veber (518). Tourtclllotte et al. (654) described aii ultraniicroKjeldahl nitrogen procrdure for the determination of total protein in l ml. of cerebrospinal fluid. Bendiwn (55)conipared the methods of Kaddell, Kjeldahl. and Lowry et al., and found Waddell’s spectrophotometric niethod most suitable for determination of proteins in cerebrospinal fluid. X tracer method for the deterniination of microgram quantities of protein n a s described by Shefner et al. (577) in which protein tms precipitated by tungsten-185 (K2KO1) and the radioactivity present in filter membrane measured the protein tungsten-185 complex. An ultramicromethod. sensitire to 0.1 y of protein, using tetrabroniophenolpthalein ethyl ester and it. potassiuni salt mas described by Kingsley and Getchell (3.49) for the estimation of total protein in spinal fluid and serum. Laurent (376) reviewed studies on protein-bound carbohydrates in human serum and urine in proteinuria. Shetlar et al. (573) compared continuous and strip paper electrophoretic techniques for separation of serum glycoproteins and found both methods in good agreement. Xeuliauq and Letzring (453) separated glycoproteins of nornial human serum by electrophoresiq on starch and found 27.1% herosaniines in cyl-, 36.6% in CY?-, 21.1% iii PI-, and 19.2% in 7-globulin. Hollinger and Lansing (295) studied the reliability of the determination of serum protein in a series of 55 deterniinations by the biuret Kcichsclhaum method and found the day-to-day varia) ~ v larger a ~ than that en replicate deterniiiiations made on a single d q (reproducibility). Thr Gorn:zll biurt4, spec-
trophotometry a t 280 nipu,nep1ielonietr;iwith sulfosalicylic acid, and Lon-r;\-’s photometry methods were conipared I)>Cartier and Picard (126) n-ho found tlii. biuret and sulfosalicylic acid methods more specific. The biuret protcliii method was studied, modified, and ncnapplicat,ions were made by Krey et al. (365),De Biasi et al. (161)>Henry et n l . (284): Kibrick (3@), Rowiitlial sit1 Cundiff (534),aiid Roseiitlial and Kan.:ikami (535). Dcbro et al. (162) propos:rd a ii(w incthod for the detcrniinatioii of serum albuniin aiid globulin i~liichdepcnJs on the solubility of albuniin in alcoholic, trichloroacetic acid in n-hich glohuliii~ nre rendered insoluble by denaturatioii v i t h tlie reagent. Conimercial peptones, ivhich arc stable for 6 months. n-ere proposetl by Rappaport and LOCK (503) as standards for tlie biuret S P ~ L I I : ~ protein method. Hirsch nncl Carra1ic.r) (290) made n coniparati~-eanalj modern quantitative methods of plasn1a fibrinogen determinations, n-hich iiieluded the biuret, tyrosine, Sesslw, ant1 ninhydrin react,ions, nephelometric antl electrophoretic methods. The biiiwt, ninhydrin, and nephelomctric niethotli shoived the smallest deviation. Thrombin reagent for plasma fibrimgen determination n+as used by Clauss (140) in a fast coagulation method, aiid by Bang (3.9) for clot format,ion n-hich is dried and weighed. Coles and Roniaii (144) modified Pargentjev’s ammonium sulfate reagent for rapid fibrinogen determination. Graf and Kinibel (256) described methods for the determination of fibrinogen of plasma in which the fibrin clot obtained after recalcification is dissolved in 2 S sodium hydroxide and the protein is titrated n-ith a cationic detergent. Fibrinogen was determined by paper electrophoresis by Berke5 et n l . (61) and Reece and Jepson (505). Uehleke (663) used fluoresceinsulfonyl chloride and Scardi and Bonavita (558) used nigrosine for marking serum proteins for estiniatioii by electrophoretic separation. Electrophoretic separation methods for biologically iniportant substances were reviewed by Morris (440). Studies of agar gel electrophoresis for analysis of serum proteins \vert’ made by Giri et al. (237; R38), Zak antl Sun (722), Port,ocala and Boeru (@9). and Ban and Sobel (89). X fluid filni niethod for protein electrophoresis h>layering a thin fluid film over an electrophoretic medium (transparent vinyl sheet) w3s proposed by Ressler ef 01. (512). Poulik and Smithies (490) conipared t’he results obtained by electrophoresis of proteins of normal and abnormal serums by starch-gc.1 antl filterpaper methods. t-siii sional electrophoretic niension, filter paper; second, starch gel), more than 20 seruni protein coniponeiits \\-ere dmionstrable. Fine anti VOL. 31, NO. 4, APRIL 1959
663
Raszczenko (205) described the preparation of transparent starch gels after electrophoresis. A new electrophoretic buffer containing 60.5 grams of tris(hydroxymethy1)aminomethane (tris), 6 grams of (ethylenedinitri1o)tetraacetic acid, and 4.6 grams of boric acid per liter for improved separation of serum proteins was described by Aronsson and Gronwall (22). A quantitative method for the estimation of proteins separated by paper electrophoresis by polarographic determination was studied by Balle-Helaers (38). Some of the variables and errors involved in the fractionation of serum proteins by paper electrophoresis were studied and reported by Berkek et al. (62), Kon et al. (359), and Henry et al. (283). Kaplan et al. (356)made a coniparative study of protein fractionation by the precipitation method of Kolfson and Cohn and paper electrophoresis (Spinco Model R), and thry found the results of the two methods were not interchangeable. Schmid et al. (663) described a method for the chroniatographic fractionation of acidic proteins on carboxylated ion exchange resins. TT’allenius et al. (685) made ultracentrifugal studies of major nonlipide electrophoretic components of normal human serum and found, upon analysis of fractions obtained by this procedure, a t least ten major groups of proteins. Wurm and Straus ( 7 1 4 improved the resolution oi serum proteins by means of paper electrophoresis by reducing the period of electrophoresis to 9 hours and increasing the current to 10 ma. a t 470 volts. Eight serum protein fractions were identified. Various dyeing and staining techniques for quantitation of seruni proteins in electrophoresis were studied. Wurm and Epstein (713) used bromophenol blue or Amidoschwartz 10B; PuEar et al. (495),a triple coloration procedure: Formusa et al. (213),bromophenol blue; Gorringe @&), Lissamine Green; and Kessel and Sylvester (3$0), Light Green SF. Frattini (215) replaced ethyl ether with chloroform in the sodium sulfate salt fractionation of serum proteins. Goodman (252) demonstrated the feasibility of routine production in chickens of specific antihuman albumin and y-globulin serums for use in turbidimetric methods for hunian serum protein fractionation which were in close agreement with electrophoretic methods. Aull and XcCord (29) and Davis et al. (160) reported the use of a phosphate turbidity method for the rapid estimation of serum proteins. Larin (374)showed experimentally, as judgcd by paper electrophoresis, that serum protein is considerably denatured by ultraviolet irradiation. Grant (257) described the proteins of normal urine obtained by concentrating by ultrafiltration under negative pressure and 664
ANALYTICAL CHEMISTRY
analyzing by the imniuno-electrophoretic technique of Grabar and Williams. Pagliardi et al. (470) proved the occurrence of processes of interaction between various protein fractions of sera by salting out sera to n hich known protein fractions had been added. Free et al. (218) used the “protein error of indicators” of bromophenol blue and tetrabromophenol blue for a colorimetric test for proteinuria. Effersge and Tidstrgm (183) detected myeloma protein in urine by a simple method based on the difference in solubility in certain qalt solutions between myeloma protein and protein occurring in the urine of patients 1%-ithnonmyelomatous diseases. Methods for the deproteinization of blood and other body fluids were studied by Hunter (307), Caraway (123),Isricescu et al. (311), and Henry and Berkman (282). Sterols. Kuchel et al. (367) reviewed t h e biological methods for estimating mineralocorticoid activity in urine. Baulieu and de Vigan (50) reported a physico-chemical method for the estimation of aldosterone in urine by a two-step descending paper chromatographic procedure. Aldosterone and cortisone were isolated on n’hatnian KO. 1 paper with formamide-methanol (1 : 1). The isolated zone was excised and eluted with methanol-methylene chloride ( I : 1) and spotted on Whatman KO.2 strips using toluene-ethyl acetate-methanolwater (9:1:5:5) as the dweloping solvent. The spots were fluorrsccd in ultraviolet light after spraj ing with trtrazolium blue. A. colorimetric method using the same general principles was described by hIoolenaar (459) for urinarv aldosterone. A. new method for the purification of crude neutral extracts of urine for the isolation and determination of aldosterone was reported by Xowaczinski et al. (461). Sowaczinski and Koiw (460)applied a new paper chromatographic system for the separation of polar corticosteroids in the analysis of urinary aldosterone, wing ethylene glycol-toluene. Dyrenfurth and Venning (179) found mild acid hydrolysis more effective than p-glucuronidase for the hydrolysis of aldosterone conjugates. Kellie (338) reviewed the dcterniination of 17-ketosteroids and 17-ketogenic steroids, Silber and Porter (583) surveyed the methods for the determination of 17,21-dihydrosy-20-ketosteroids in urine and plasma. Neher (451) reviewed the determination of individual adrenocortical steroids. -4 study of solvents, problems of extraction, hydrolysis, and purification of extracts for determination of neutral steroids in blood was reported by Tanini et al. (633). Different modified methods for the determination of blood 17-hvdroxycorticosteroids were published by Yudaev and Pankov (718), Tamm et al. (634), Silber et al. (582), Silber and Busch (581))Riondel et al. (520),Peter-
son (478), Peterson et al. (47‘3),Elk-Xes (185), and Brayer (96). Fujita (226) used a modified oxycellulose method for the determination of corticotropin in blood and urine. De Venuto et al. (167) described a procedure by which C-, steroids of tissues and blood of varying polarities were extracted through a dialysis inenibrane in a continuous process. Gold (244) reviewed the measurement and significance of blood corticoids. The Sorymberqki and sodium bismuthate methods were studied and modified by Sobel et al. (593) EdTrards and Kellie ( I R d ) , and Diczfalu-y et al. (169). The tetrazolium blue and the Porter :tnd Silher reactions for the determination of urinary steroidq n ere studied by Marks et al. (414), Izzo et al. (312), and D e Filippis (164). Chromatographic urinary corticosteroid methods werr studied, modified, and reported b? Weinmann and Jayle (696),Wotiz et al. (709), Wilson et 01. (706), Vermeulcn (680), Linford (3S9), Kellie and Wade (339), and Broobs (9s). Enzymatic methods for the estimation of urinary steroids by the quantitative reaction of ctrroid alcohols with dipho~phopyridinenucleotides in the presence of CY- or p - h ~drolysteroid hydrogenases m-ere described by Hurlock and Talalay (308, 309). 111kulaj and Stole (428) used p-glucuronidase from the snail for urinarl- 17-hyIsotopic droxycorticoid h! drolysis. methods for the determination of steroids in plasma and urine were studied by Avres et al. ( 3 2 ) . Hollander and T’inecour (294), and Berliner et al. (63, 6$). Fluorometric methods for microanalyses of corticosteroids in plasma and other fluids were investigated by X c I a i g h l i n et al. (.$OS), Kalant (326, 327), Goldzieher and Beech (2$3), Epstein et al. (194), El>- et al. (192), Bondy et at. (go), and Ayre- et al. (32). The Reddy and Sorymberqki urinary 17-hydrosycorticosteroid methods n-ere compared by Butt et al. (11s) and Golub et al. (%(I), who found the Reddy method less reliable, especially in urines n-ith small amounts of 17-hj droxycorticosteroids. Schopman et al. (566) modified the Retldy method to increase its accuracy a i d specificity. ,Johnson et al. (318) presented a method for the chromatographic fractionation of individual adrenocortical steroids of urine extracts by an automatic gradient elution technique. The tight binding of conjugated steroids to serum albumin was used for the efficient extraction of such steroids from urine by Slaunv hite and Sandberg (590). The isonicotinic acid hydrazide reaction was applied to extracts of human plasma by ST-eichselbaum and JIargraf (690) for the determination of A4-3-keto corticosteroids. Nazarella (422) proposed the use of 4,i-diphenyl-1.10-phenanthroline for colorimetric microde~
tcrmiiiation of cortirostc.roids. Caniber (120) investigated sa1ic;vloylhydrazido as n reagcnt for the characterization antl estimation of simple and steroidal aldchj-dcs and ketones. Bischoff and T u r n u (73) dctcrniined the spectral data ill the 320- to 7OO-nip range of the reaction products of 18 steroids subjected to tlie Zak color reaction for l m s i b k use in identifiration. lletliods of isolatioii and eetiination I J f clioll,stcrol n-err rc,vien-ed by Cook and Iinttlay (I@), C‘ostello and Curr:in (1$1) iiiotlifiid tlic turbidimetric niithotl of I’ollak and Kadlrr for the tli,trriiiiii:itioii of rliol!~stcrol in serum. \‘c~lu antl Tolu (si:. 6T6) added serum tiirwtl?. t o :i sodiinn alc~~il~olate reagent f ( J T thix cli,tc,rniiiiatioii of total cholestcwl :inti c ~ i i i ) a r dt h i r method (676) to tlic. I:!icw)l tot:il t~liol,~sterol method of Eiunk(L1. The ferric chloride colorimiJtric niethod I T ~ F investigated and modified by Hcnly ( % S I ) . Torres (655), Rosenthal et a/.(636). Rice and 1,ukasiewicz (617), Hcrrniann (E%),Hansen :ind I l a m (2?1), Cranford (155), and Zak (721). A. rapid fluorometric total seruni cliolcstt~rolmethod employing a mixed reagent of trichlorocthane, acetic nnhytlride, and sulfuric acid n-as described b!- l\IcDougal :ind Farmer (40;). Lapin (3%‘) introdiicd a new colorimetric blood cholesterol niethod eniployiiig furfural, acetic anhydride, and mlfuric ac.id. A direct serum cholwtcrol method n-:is described by Irc>frrenc.cto a calibration c w w .
METHODS USED IN STUDY OF HYPERTENSION, P H A E O C H RO M O C Y TO M A , A N D MENTAL DISEASE
Th(, fluortimetric niethock of I.untl, Yon E ~ l c r and , Flotliiig for the estinintion of :idrmaline and iioradrenalinr in ldootl and urine n-ere inr-estigatecl and modified by J-on Euler ant1 Flodiiig 16cy2).Yon Euler (6s1).Pricc and Price (.@?)) Pobcl and Henry (5.94))doncs aiitl Blakc (320), and Thonin~(642). Furthpr modifications of the fluorometric estimation of catrcliolaniinc~s iii blood xiid urine by thr method of Keil-3Ialherbe and Bone nxirc ninile I>>- TT'eilS I a l h d x and Bonc, (8.9.;. 6.9;)!Gray and Young (258), Pourke-. mid Drujnn (603).and KRgi et cr/ Goldenbwg (I.$$) study of the fluorcwPiicr spectra of ndrcnolutine and noratlrenolutiiie in order to determine them simultaneously in mixtures. .[-riiiary adrrn:ilinc, noradrcnalinp. and liytlrox>-tyramirie iwre adsorbcd on >.niberlitv IRC-SO by Kirshner and Gooddl (35.$):tnd CraiI-ford and Law (1.18). Blood and urine catechol amines n-ere adsorhccl on alumimini hydrosidc by Bri1niayc.r et a / . (8?) and .Johnson (31.9). Nethods for the fluoronicJtric tleterniinat,ion of adrcwaline nntl noraclrenal-
quantitat,ivc method for th(. detection of urinary D-lysergic acid clicthylamide hased on t h r inhibition of ieriini choliiiwtcrase. Dalgliesh (f 5,s) rc~1-icn-edhydroxyindoles. lfohler ( ~ $ 3 cwluated 3 a urinc t w t for serotonin iiwtabolites based on blie der-elopnieiit of :i purple
l a t d metabolites, enz!-inr-. mid tlmgs. Keissbach et al. (697) devrlopcd :I siniod for the determination of in tissue extract> in n-liich tisSUP protpin n-as preci1)itatc.d with zinc hydroxide, followd 1)- i1iwc.t fluorometric determination of serotonin. Sollcr arid Stelgens i$%) ~ c p a r n t c t lserotonin completely from sniitliurcnic acid, .j-li~-drosyrindole, jncloIqiyr~~-iracid: and indolmcetic acid by higli-tcnsion clcctropliorcsis.
cc,ntratid sulfuric, wid. and ethyl alcolid, and nie:i~~u.ing thc. resulting color. II:ili~ret nl. (@9) clvrrriliccl :L quantiI'robl(~n1sof arid suggestions fur d(>:ilt:itii-c ultraviolet rlJ('c'trop1iotonictrie ing with toxicologic problenis in n gcniiic~tliodfor t,hr tletc~rniinationof isonie r d hospital ~ ~ e preseiitecl r c by Tol)el :izitl and p-aminosalicylic arid in body ( 4 5 G ) . Fcldsteiii and h'lendshoj (200) flui(ls. Poolc ant1 1Io>-i~i. (.&C?) tleterclividid the volatile poi iiiiiictl isonicotinoyl li>-ilmzitle in s m i n i groups, liasrd on tlicir clj 13:. prepuring alco1iol:itc filtratc, hvnting tcristics, antl dcscrihed tlie detection :inrl estimation of tlicni in 1iologic:il f i l h tc c~orr(qmiii1iiigto 1 nil. of ~ c r u n i ~ i t h0.3 nil. of 0.4% l-fliioro-2.~-diiiirii:iterialh. T-ltraviolet, traiisniittancc. i:.olicwzc~nctin alrohol and 0.1 grani of cur\-es of nicothc, barbit:il, eplicilrinc 11fJr:lX. m d rr:itling color tlcvc~loprd a t quinine sulfate, dilantin, nntl 300 nip. Tc.dcy-Hadzija and ;ih:iffy ne n-cre stutliccl hy JIiwlling c t ). JIohrschulz (433) differircd isoiiicotinic acid photocntintcd soporifics drugs in iuinc, 11y y a nicthoti bascd on the for1j:iiirr ~~lir~~iiiatograpliy. Alha et c r l . (9) inntion of a y(>llo\\color, protluccd b>-the t l ( k t c d disulfirsm in lilootl hy coiiwrtLiitkJli of p-tlimrtli~l~iiiinobcnzaldeiiig t o c8:ipric tiit~th~-lrlitliioc~nl.il:rmatr, rtli antl Eiinavitn (557) and purifyiiig by paper c ~ h r o i i i a t o g r ~ ~ ~ i l i ~ ~ ~ 6') tlrtrrniiiird isoniazid i n :itit1 dissolving in ci:irlion t~ctrachloritl(~ logiral fluids ti>-a niethotl bascd upon for tho ~iic:isur~mcntof :ibsorption tlic r ~ ~ ~ ( . t iIicti~-cen iiii isoniazid and soIj('aIi5 :it 2T2, 291. and 436 nip(. tliuni I)i,iituc!-aiioa~iiiii(,fi,rroatc and Kirk et 02. used n combination iw:isurrnicnt of the yc.llon- chromogen. of :ic~ationantl distillation to iniprow Lutln-ig ant1 Hoffiiiaii i a c.licmiical and enzymat,ic procodurc for iiiined mcprolinmatc. in biological fluids the, tlctcrniinntion of blood al(~o1iol. hy a color rcwtion n-hirh takcs placc IIather (@O) proposed a simple steani n-lien mcprobaniatr is treattd with dirtillation arid colorimc+ie c1etermin:iiiirfur:il in tlir presence of antimony tion of 1)lootl alcohol. JIonnicr et d. (~lilorirl(~.Hurris antl R d (275) h>-(@,(') modificd a Coni~-a\-cc.11 for tlw tlrolyzctl urinary meprobamatc in basic solution and mc~asnrcdthv ammonia reisot1icrm:il ecparatioii of blood alcoliol Icaqed after acitlificxtion bj- ncxsslrriza:it 50" C., using an oxidizing solution of nitric acid :iiid potassium tlic~liroiiiatc~. tion. Ellman (190) clctcrmined mercaptan in blood by a colorimetric nictliod R(~sicIua1clichroniate, aft,c,r isotlicrnial n-liich tlcpcnrls on t distillation of'blood alcoliol into it, was l)i.;(p-iiitro~)lic~nyl)tl trcntetl with brucine to yicld a stable wptan anions :it pH sensitive c~hromophoreas suggcstctl by photomc+ic tletrrmiiiation of nictheKilliams et crl. (704). Turkel ant1 Gifmoglobin vas inr-cstigated and further ford (661) slion-ed that blood sainplcs i ~ f ~ n eby d Miillrr and Zijlstra (442). t n k m a t autopsy from tlic pericwtlial , Herka (5.9) 60) iiivestigatcTt1 t,liree SRI', but not those from thP fenior:il vein, clinical methods for thc drtcrmination of shou- false alcohol lrvels. Skraug cnrhoii nionoxide in l~loocl aiid found ) de\-eloped a rapid and stwsitiw that thr. method based on the rolor tcst for methanol in blood by mirrodischange of palladium eiliconiolybdate is tillation and oxidation t o form:rldehy&, fnstcr than thaw hascd on the. srparation which is measured by s color reactioli of nietallic palladiuni from solutions of with 2,i-naphtliole1icdiol. pnlladiuni c>hloridcor on tlic, blackening JIethods for the separation slid identiof indicator paljcr iniprcgnated n-ith the ficsation of barbiturates in biological niachloride. Bruckner and Desniond (106) tcrial bj- paper chromatography 11-ere mc.asurcd extinction values of a 1 to dwcribed by -4.llgen ( I I ) and 1Ioller20 blood dilution at five wave lengths to c&iiate carbon monoxide henioglobin bwg ( 4 3 3 , the latter using chloroforniin blood. Giacoino et al. (2%) nieaeammonia and amyl acetate-animonia u r d carbon nionosiclr in onr drop of systems for the srparatioa of 18 barbiblood by projecting and rcy&cring the turic acid duivativcs. Siineliiiic slid absorption spectra on a photogrziphic Hackett (62.5) correlated tlie blood and film. Harbor (27.4) showrd that extent iirinary barbital lewis clinically slid also of exposure to carbon nionosidc could iiinde blood and urinary cleararicv stlidbr tletcrmincd spcctropliotomctrically, ics. Freciiiari (220) nic~asuretl :iiiieewii aftcr 2- to 4-day storage of specitllropterin in plasma by fluorescent inen. Garnsler et al. (5'30) developed a mcasurenitmt before and aftcr oxidat,ioii. 11cw method for drtcrniinat,ion of carThompson (64.4)used the Bratton arid bon monoxide in blood by diluting the JIarshall method for the estiniatioii of extracted gas in a tonometcr and analyzthe oral Iiypoglyccniic agent, carbutaing tlic carbon monosick content n-ith an mide, in blood. Cyanoacrtic acid hyinfrared meter. drazide n-as determined in blood by Schilt (561) determined cyanitlc by JIaresch and Philippi (412) by treating reacting with tris(l,lO-phenantliroline)a blood filtrate n-ith a reagent coniposed iron(I1) to form a dicyano complex of p-dinietliylaniiiioher?zald~~liyi~~~, colin-liich ii; cxtractcd n-ith chlorofoi~ni.and TOXICOLOGY
the color i> i i ~ ~ ~ : i sat~ 5Oi i r ~ nip. ~I -intinioiiy n - a ~tlt~tcrniincdin biological material bj- Fiiznilov ( Green, 1)). dhcn et 01. (578) ivith rhotlaniinc. B :ind mnj-1 ncetatc,, and tq- T w i et a/. (6.76)with mn1:tchitc grcrii. Hill et crl. ( 2 8 9 ) improvod tlic nicthyl Ijor:ltc. distillatioii inc~thotl for the rlc+wiiiiintion of liorato in blood and tis+uc witli final cwiiiino tcst. >.d:m- cf ril. (9 inodificd thv ~.;niitli-C;:iitlii(~r-~~-illi:iIii~ dt-nc+l thorium fluoritlc iitraiioii, elimiiiatiiig tlic, necessity for tlir rilvtir chloritlc scparntion etcp of iiriiiary fluoriilt.. S i c tc~rniinctl tmcc amounts ues (1 to 10 y) by employing an ion haiigc tcv~liiiiqur for ro1iccntr:iting the fluoritlc and freezing tho sohition from inti~rforing inns. IIicrotl(tcmiination of fluorinc in bone+, twtli, ant1 bloorl K R P niadr 17y Sanix(,Ii>oii et rrl. (546)11)- microtlistillatioii :11id u s c of n nioclificil dizarin rcxgcmt for cdorimetric niwwrmieiit. -) tl(tc.ctotl .Y-forni11 win(, by :i procc,clurc ir-liic-h consists in the ( ~ i z ~ x i a t i r rcduc.tion of folic acid t'o tctrahytlrofolic acitl (THFJ.), enzymatic formylation of THF;\. by .~~-foriiii~iiitiogliita~iii~~ acid, the noneiizymatic convcwion of tlio prcdiict S1o-formyl THFJ. t o A175-fortiiyl THF,\. (c.itror-orui the iiiic~roljiologit.al et ol. (2.97). aftc,r a further purificaation, and find solution in citratc of tlw blood lcad prcwiit, ni:ide a po1arogr:ipIiic iiiicrocl(,ti,ri~iiiiatioiiiof the h a d . I'luni (@j) c4m:itrtl the approximate amount of lithium in *('rum hy thc formnt,ion of a colorotl (,oniplex, LiK(FeIO6), \ T i t h thc rc,:igc,nt cmnplcx tlissolwd in csw.s pot hydroxidc. Tlioniason (6.i.3) tlcterniinetl micrigrani anioiirits oi lithium spcctropliot~onietrically b>- using 0-12hydroxy - 3.6 - disulfo - 1 - naii1itliyl:rzo)bcnzencarsonic acid as t h e (,liroiiirigcmic :gent, in 11 potassium hytlroxitlc~rolution of water and acetonc. \Tet digestion arid tlithizont~ t d i iiiques \wrc u s c d for thc tlctermitiation of mercury in urine by I,upa~it-A.ndre (SQQ),JIiller antl Swaiibrrg (.'+31), and 3-ohrl ant1 S o b r l (457). IIcRryd? and IYilliams (401) dctmiiiiied 1 to 10 y of mercury in urine antl n-atw using a Gcncral Electric instantancotis mercury vapor nicrcurimrter. =\. volorinictric dithizoiic nictliod for microcstimation of int,act phenylmrrcury compounds in aninial tissue was described h>- JIillcr et 01. (@0). Cluctt and Yoe (I@) tlcterniinc~lsubmicrogram anlolints of iiirkrl in hunian blood spectropliotc~iiic~tric.ally by a procedure based on t h cmnples formed n-it h diet hy ltli t hioc~ir bania t e which is extracted into isoani>.l alcoliol for nieasurcmcnt at 325 nip. 3.rzaniastwv (23) 1istc.d 1J:isic reactions for tlir colorimetric tlvtcwiiination VOL. 31, NO. 4, APRIL 1959
667
of morphine, codeine, atropine, hyoscyamine, scopolomine, strychnine, caffeine, theobromine, and theophylline. E l Darany and Tompsett (188) determined alkaloids in biological material by compound formation with indicators. The method was based on the extraction of the indicator salt of the alkaloid into benzene to ebtablish the stoichiometry, followed by extraction of indicator into 0.1S sodium hydroxide for measurement of the indicator. Szcrb et d. (629) deterniined morphine in blood and tissues colorimetrically after removal of impurities by benzene precipitation and adsorption on ion exchange resin by use of the Fohn-Ciocalteu phenol reagent. Xadenu and Sobolemki ( 4 U j determined ver v small amounts of morphine by a method nhich depends on the fluorescence produced by reaction with concentrated sulfuric acid, followed by treatment n ith ammonia. Krynskq a (366) determined bound and free procninc>in urine by diazotization of the free S H 2 group of procaine and coupling with thymol to produce a red color for photonietric measurement. Chiang and Freeman (134) developed a inicroniethod for the determination of salicylic ncid b) direct separation of salic! lic acid from plasma by ascending paper chromtography n-ith ethyl alcohol, followd by tolorimetric estimation of the salicvlic acid after extraction with water and ferric nitrate reagent. Khon (3/t27j drterniincd sulfonamides b r a method bascd 011 the property of phosphotungstic ncid to form insoluble complex ( onipounds n-ith sulfonaniidca. Forist et ul. (212) determined plasma tolbutamide (orinase) by evtraction of weakly a ~ ~ t l i f i ~plasma tl with chloroform, concentration of the estract to d r y n w , solution of the residue in a mcasurcd volume of 9570 eth? 1 alcohol. treatment of the resulting solution n i t h Darco G-60, and nieasurenicnt of the abqorbancc of the filtrate a t 228 nip. Bladh and Sorden (76) used a modification of this method. Spinqlcr (604) developed a method for the determination of l-buty1-3-(p-tolylsulfonyl) urra in serum by formation of butylamine from butylurea a t a high temperature in an inert medium, followed by subscquent colorimetric estimation as the yellov dinitroaniline derivative. Hanson (279) determined microgram quantities of tellurium in urine by separation of tellurium from all but a few elements and quantitative recovery by extraction in n-amyl alcohol from solutions made approsimately 1A’ in hydrogen ion and 0.62TT in iodide, The extract was evaporated, and the residue, pure tellurium ealt, n.as diesolved in hydrochloric acid, then precipitated as the element in finely divided form for photometric determination. Hasselmann (278) formed a colored combination of quinine and quinidine
668
ANALYTICAL CHEMISTRY
with Rose Bengal for their microdetermination in serum.
(33) Baar, S., Clin. Chim. A c t a 2 , 567 f19571. ( 3 i ) Bachra, B. N., Dauer, Sobel, A. E., Clin. Chem. 4, 107 (1958). (35) Baily, P., A N ~ L . CHEY. 29, 1534 , ~ - - - ~ A \ . ,
ACKNOWLEDGMENT
Thc author wishes to thank Gloria Gdchell and Roscoe Schaffert for their assistance in the preparation of the nianuxcript. LITERATURE CITED
( 1 ) Acland, J. D., Biochem. J . 66, 177 (195Tl - , ( 2 ) -Adam, H. AI., Hardnick, D. C., \ -
Spencer. K. E. V., Brit. J . Pharmacol.
12, 397 (1957). ( 3 ) Adams, 11. F., Koppe,
R. K.,
)\lay-
hew. I>. J.. A u . 4 ~ . CHEJI. 29. 1108 (19*I,, >Ian, 1:. B., J . Lab. C l m . .Wed. 52, 659 (1958). (43) Barna, S., Szabo, G., Fener, G.,
Balogh, I. R., Acta X e d . d c n d . Sei. H m g . 10, 339 (1957). (44) Barney, J. E., 11, Bertolarini, R. J., . ~ s & L . CIIEX.29, 1187 (1957) 145) Baron. D. Y . .Bell. J. L.. C l m . C‘hirn. ’ Acta 2, i27 (1957). ’ (46) Barrollier, J . , Heilmann, J., 2. Phusiol. Chem., Hoppe-Seder‘s 309, 221
sen, E., Aird, R. B., Winston, S., Arch. Biochem. Biophps. 74, 100 (1958). (9) Alha, .I\. R., Hjelt, E., Tammimen, V.. Acta Pharrnacol. Toxicol. 13, 277 (1957).
(10) Alimova, E. IC., Bolgova, G. D., Biokhiiniva 22. 568 (1957). ( 1 1 ) Allgen,’ L. ’G.> dca72d.’ J . Clin. RLab. Incest. 9, 71 (1957). (12) .I\llington, RI. J., J . Clin. Pathol. 1 1 , ~
62 (1858). (13) Allport, ?;. I., Keysrr, J. W.,eds. “Colorimetric Analysis,” 2nd ed,, vol. 1 , Chapman R- Hall, London, 1957. (14) Alper, C., Seitchik, J.! Clin. Chem. 3, 95 (1957). (15) Alvarez, L. F., et al.: d r c h . nzCd. Cliba 8 , 319 (1957). (16) Andersrh. 11. A . J . Lab. Ciin. M e d . 49, 486 (1957). 7 ) .indemon, D. 13.. Williams, C. Krise, G. XI.> Don-den, R. \I.>Biochem. J . 67, 258 (1957). 8) hntoniades, H. S . , Pennell, R . B., hlchrthur, J. W.j Inperwdl, F. \I,,
1-lfedler. H.. OnclcT-. J. L.. J . B i d .
?hem. 228, 8ii3 (195ij. (19) .intoni:rdes, H . S . , Tiillis, J. L., Sargent, L. H., I’mnell, R. R., Onelel-, J. L., J . Lab. (‘/in.3 r d . 51, 630 (1058). (20) hrchihald, R . \I., Clin. C h e ~ n .3, 102 (1957). (21) Akovitch, J., Growowicz, X., Ibid., 4 , 22 (1958). (22) ,\ronsson, T., Gronrvnll, .\., Scand J . C‘lzn. R- Lab. Inzest. 9, 338 (1957). (23) hrzamastsev, .4.P., Apptechnoe Delo 6 , 7 3 (1957). (24) .4shh), R . O., Roberts, >I.,J . Lab. Clzn. M e d . 49, 958 (1957) (25) Aspen, .I\ J., XIeister, .I\, Methods of’ Bzochein. -4 nal. 6 , 131 (1958). (26) Astrup, T , Albrechtsen, 0. K., Scand. J . C1.n. & Lab. Inzest 9, 233 (1957). (27) Athanail, G , Cahand, P. C., J Lab C’lzn M e d . 51, 321 (1058). (28) iiugustinsson, K. B., Jfethods of Bzochein. Anal. 5 , 1 (195i). (29) hull, J. C., McCord, \T. hI., Clin. Chenz. 2 , 379 (1956). (30) Avi-Dor, I-,Lipkin, R., J . Bzol Chem. 233, G9 (1958). (31) Ayres, P. J., Garrod, O., Simpson, S. A,, Tait, J F., Bzochern. J . 65, 639 (1957). (32) hyres, P. J., Simpson, S. -\., Tait, J F Ibzd., 65, 647 (1057). ~
’ (1957). (56) Bergebi, G., Rnss elin. tetap. e sei. a f l n i 56, 26 (1957). (57) Bergmann, F., Dikstein, S.,X e t h o d s of’ Biochent. i l n a l . 6, 79 (1958). (58) ,Bergmann, F., Dikstein, S., Chnimovitz, >I,, J . Biol. Chem. 230, 193 (1958). 150) Berka, I., Acta X e d . Scand. 157, 129 (1957). (60) Berka, I., Pacocnt I C k a M i 9, 301 (1957). (61) Berkes!. I., BerkeS-Toninscvic*, P., Kaloakdzilan. 11.9Schu;e,i. med. Tl‘och-
-.. . _ .
(62) BerkeS,- I>:, Straus, B., Fiscr, >I., Acta dfed. 17iigoslav 9, 206 (1955). (63) Berliner, D. L., Proc. Soc. Erptl. Biol. J f e d . 94, 126 (1957). (64) Berliner, D. L., Dominguee, 0. \-., \Testenslto\v, G., ASAL. CHEJI. 29, 1797 (1957). (65) Bernfeld, P., Berkom-itz, W. E., Donahut, 1’. >I., J . Clin. Inzest. 36, 1363 (1957). (66) Bernsohn, J., J . Lab. Clzn. 3 f e d . ’ 49, 478 (1957). (6i’i RPrrv. H. K.. Proc. SOC. Exptl.
(69) Bickoff, E. l f . > %ethoc A n a l . 4 , 1 (1956). (TO) Biesold, D., Strack, E., 2. Physiol. Chem.. Hoppe-Seyler‘s 311, 115 (1958). (71) Biggs, H-. G.,. Carey, S., Morrison, D. B., J . Clin. Pathol. 30, 181 (1958). (72) Billing, B. H., Biochem. J . 65, 774 (1957).
(73) Bischoff, F., Turner, J. G., Jr., Clin. Chem. 4, 300 (1958). (74) Bishop, J. G., Texas Repts. Biol. and M e d . 15, 833 (1957). (75) Bitman, J., Rosselet, J . P., Reddy, A. D., Lieberman, S., J . Bzol. Chem. 225, 39 (1957). (76) Bladh, E., ’Norden, A, A c t a pharmacol. et tozicol. 14, 188 (1958). (77) Blarikenhorn, D. H.. J . Biol. C h e m 227, 963 (1957). (78) Blanquet, P., Meyniel, G., llounier, J., Tobias, C. A,, Bull. soc. chini. biol. 39, 419 (1957). (79) Block, R. J., ITerner, S. C., hlandl, R. H.,Arch. Biochem. Biophys. 73, 9 (19581. ( 8 0 ) Blondheim, S. H., Bull. R e s e a d Council Israel 6E. 223 (1957). (81) Bloom, W. L:, J . L a b . Clin. X e d . 51, 824 (1958). (82) Blumenkrantz. S..Clin. Chem. 3. 696 (1957). (83) Bode, E., Waldschmidt, X, Z. Physiol. Chem., Hoppe-Seyler’s 308, 204 (1957 i.
(86) Bohm, M., Zimmermann, H., Z. Physiol. Cheni., Hoppe-Seyler’s 308,
132 (1957). ( 8 7 ) Boettcher, E:. IT., Kistler, P., Sitschmann, H., S u t u r e 181, 490 (1958). ( 8 8 ) B_ohuon, C., A n n . biol. clin. ( P a r i s ) 16. r3 119583. (89)‘Bon,‘\T*. F., Sobel, P. C., Rec. traz’. chim. 77, 47 (1958). (90) Bondy, P. K., hbelson, D ClarkeLen.is, A,! J . bid. Chem. 224, ;1‘7 (1957). (91 ) Bongiovnnni, A. AI., Eberlein, IT. R., L h - a ~CHEX . 30, 388 (1958). (92) Bongiovanni! A. M., Eherlein, IT. R., Thomas, P. Z.,J . Clin. Endocrinol. and Jfettrbolism 17, 331 (1957). (93) Boiitn-ell, J. H., Clin. Chenz. 3 , 205 119571. (94) Bo\ arkin, .I, S., Fzzzol. Xastenz‘l, Ahad. .Yazik. S . S . S . R . 5, 86 (1958). (95) Blada, Zbynek, &rent, A, Chena. lzaty 51, 2376 (1957). (96) Brajer, F. T., U. S. Atomic Energy Conim. UR-513. 13 (1957). (97) Brilmayer, C. H., Cdek, H. H., Littig, 11.) A f e d . Klzn. (Jlirnich) 52, 1184 (1957). ( 0 8 ) Brooks, R. V., Bzocheni. J 68, 50 1 19583. ($19) Bro\rn, E. L., Can. J . IIIed. Il’echriol. 19, 126 (1957). (100) Broirn, J. B., Bulbrook, R. D., (heennood, F. C., J . Endocrznol. 16, 11 (1%). (101) Ibztl.., Iu. 49. (102)Browi, It. H., I)udn, G. D., Korkes, S., Handler, P., .-lrch. Biochem. Biophys. 66, 301 (195i). (103) Brown, R. R., J . B i d . C h e m . 227, 649 (1057). (101) Bruce. R . B., Hon.ard, J. IJ-., .\?;AL. C H E I I . 28. 1973 11956). (105) Briirkner, J., Schweiz. med. Wochschr. 86, 1057 (1956). (106) Bruckner, J., Deqmond, F. B., C l ~ n Chini. . A c t a 3. 173 11958). (107) Bruns, F. H., Ibzd., 2, 257 (1957). (108) Buchanan, I). L., Ahar.. CHEX 29,1877 (1957). (109) Bulbrook, R. D., Greenwood, F. C., Williams, P. C., J . Endocrznol 15, 206 (1957). (110) Bunch, L. D., A m . J . Clan. Pathol. 28, 111 (1957). (111j Bundy, H. F., blehl, J . tT., J . Clzn. Incest. 37. 947 11958). (112) Bunt;, H., Demling, L., 2. Physiol. Chem., Hoppe-Seyler’s 310, 107 (1958).
(113) Burstein, AI., Compt. rend. 244, 3189 (1957). (114) Ibid.. 245. 586 11957).
(154) Crane, F. E., Jr., ANAL. CHEH. 30, 1426 (1958). (155) Cran-ford, N., Clin. Chini. Acta 3, 357 (1958). (156) Cralyford, T. B. B., Law, IT., J . P h a r m . and Pharmacol. 10. 179 Chim. Acta 3,320 (1958: (1958). (117) Busson, F., Guth, P., .1fCd. trop. (157) Crook, E. M., Rahin, B. R., B i o 16. 833 119561. chem. J . 68, 177 (1958). (118) Butt,’ IT. ’R., Kornel, L., Morris, (158) Dalgliesh C. E., in “Advances in Clinical khemistry,” Sobotka, H., R., Acta Endocrznol. 26, 65 (1957). (119) Cabaud, P. G., Wr6blewski, F., Stewart, C. P., eds., vol. 1 , p . 194, Am. J . Clin. Pathol. 30. 234 (1958). Academic Press, Seir- York, 1908. 1159) Date, J. IT.) Scand. J . Clin. 6 (120) Camber, B., Clin. ‘ C h i m : Acta 2, Lab. Invest. 10. 119 11958) 188 (1957). (160) Davis, D.’ R., Teague, F. A., (121) Cannan, R. K.! Clzn. Phem. 4, Dietz, B. J.. Budd, R. E.. J . Lab. Clin. 246 (1958): U.S. Armed Forces M e d . M e d . 51. 616 1 1958). J . 9; 683’(1958); Science 127, 1376 (161) De Biasi,‘S., Cora, D., Zagarese, (1958); Blood 13, 1101 (1958). R., Proyr. med. 13, 756 (1957). (122) Caraway, W. T., d m . J . Clin. Pathol. 26. 945 (1956). (162) Debro, J. R., Tarver. H., Iiorner, A,. J . Lab. Clin. X e d . 50. 728 (19571. (123) CaraIGay, 1iTi-. T.; Chemist .-lnalyst 47, 44 (1958). (l63j De Carvalho, C. X., Pogeli, B. hl., (124) Carpenter, IC. J., Gotsis, A,, Bzochim. et Biophys. Acta 26,206 (1957). Hegsted, D. X j Clin. Chem. 3 , 233 (164) De Filiuuis. V.,Proor. med. 13. 722 (1957). (1957). (125) Carroll, B., Tillem, H. B., Freeman, (165) De Grouchy, J., Schapira, C; , E. S., ASAL. CAEX 30, 1099 (1958). Dreyfus, J. C., Rev. f r a n c . Btiides clzn. et biol. 3, 377 (1958). (126) Cartier, P., Picard, J., Ann. biol. din. ( P a r i s ) 15, 459 (1957). (166) Delsal, J. L., Compt. rend. 244, 2252 (1957). (127) Caspe, S., Pitcoff, I). I)., Clin. 1167) De Venuto. F.. Mule. S. J.. WestChem. 4 , 37-1 (1958). . phal., U., A r c h . Biochem: Biophys. 73, (128) Catch, J. It.? .isa~..CHEM. 29, 1726 (1957). 451 (1958). (129) Cavina, G., Tentori, L., Clin. C h i m . (168) Dickens, D. F., Killiamaon, I).H., Acta 3 , 160 (1958). Biochem. J . 68, 84 (1958). (130) Chanev, .i. L., in “Advances in (169) Diczfalusy, E., Plantin, L. O., Clinical Chemistry,” Sohotka, H., StewBirke, G., Ingall, S. C., Norymberski, J. K., rlcta Endocrinol. 27, 275 (1958). art’, C. 1’~)eds., vol. 1, p. 82, Av:tdernic (170) Dieckert, J. IT-.> Morris, S. J., Press, S e w York, 1958. (131) Charonnat, R., Miocqiic,, >I., .4nn. - 4 S A L . CHEM. 29, 31 (1967). (171) Dikstein, S., Bergman, F., Chaimpharm. franc. 15, 593 (1957). ovitz. 11.. J . Biol. Chenz. 230. 203 (132) Cheronis, N. D., Zymaris! lr. C., 1 195d). Mikrochim. Acta 6, 769 (1957). (133) Chiamori, S . , Henry, R. J., Proc. (lf2) -I%leo, 1:. F. P., Betti, R., Solinas, Soc. E x p t l . Rid. J l e d . 97, 817 (1958). P., J l i n e r c n M e d . 2 , 3714 (1957). (173) Ilixon, A. C.; J r . , *-lnz. J . Clin. (134) Chiang, S., Freeman, S., J . Lab. Clin. M e d . 49, 481 (1957). Pathol. 28, 323 (1957). (135) Chilcote, 11. E., Wasson, 11, D., (174) Dobkin, G. B., Glantz, 11. D., Clin. Chem. 4, 200 (1958). Clin. Chem. 4, 316 (1958). (136) Christensen, L. IC, S c a d . J . (175) Dobrick, I,. A,, Harpcr, H. A . , Clin.h Lab. Inisest. 9, 377 (1957). J . B i d . Chein. 231. 408 119583. (137) Ibid., 10. 177 11958). (176) Dodgson, K. S., Spencer, B., (138) Chuj’ T.’ C., ‘Chu,‘ E. J., .ASAL. Methods qf Biochem. A n a l . 4 , 211 (1956). CHEU.30, 1678 (1958). (177) Dreskin, 0. H.! J . Lah. Piin. X e d . (139) Chu, T. C., Chu, E. J., J . Biol. 51, 312 (1958). Chem. 227, 505 (1957). (178) Dryer, R . L., Tammes, A . R., (140) Clams, .I.,Acta Haematol. 17, Routh, J. I., J . Biol. Cheni. 225, 177 237 (1957). 11957). (141) Close, J. R., Street! H. F-., Clin. ( l f 9 ) Ijyrenfiirth, I., 17enning, E. H., C h i m . Acta 3 476 119581. Endocrinology 60, 136 (1957). (112) Cluett, 11.L.: J. H., 1\-41.. (180) Ehwleiri, \V. I t . , Bongiovanni, .\. CHEX 29, 1265 (1957). AI., Plin. Chenz. 4, 350 (1958). 1143) Cohcn. G.. (hldPnhPrP, 11,, J . (181) E:rkstein, B., I < a h n ~D., Borut, A,, Bull. Research Corincii Israel 6E, -- , (144) Coles, l l . , Iionian, K., J ( ‘ / l t ~ , 189 (1957). Pathol. 10, 282 119573. (182) Edn-ards, 11 Ti-. H., Iirllie, -L E., (145) Constam, G. R.; Iliubcies 7 , 3G d c t a Endocrznol. 27, 262 (ll158). (1958). (183) Cffersde. P,, Tidstrdm. B., J . Lab. Clin. X e d . 50. 134 11937) (1.46) Cook, R . P., RattrgJ., J . 13. ll,, “Cholesterol; Chemist;!-, Biorhcmistry (184) Fggstein, ai.: ’ Fiocieni. ’Probiem. and Pathology,” p 118, . i ~ ~ : ~ ~ ! r i n i c Lipzden 3, 150 (19,:6). Press, Sen. York, I % ? , (1853 Eik-Ses. K. J . C(in. Endocrinol. (147) Cooper, J. .I. I ) , ! $.!;> 1 a / d Jletabolisni 17, 502 (1957). Borden, C., J . I,( b. ( ’ i,: . I / r . i . 2; il8G) E k > J., Hultman, E , .Yature 181, 129 119583. 780 (1958). 1187) Ek. J.. Hultman. r.. Scand. J . Clin.R. Lab. Zniesl. 9, ? ! 5 (1957). (149) Cortcllo, Ti. T,.,’ Curran, (;. L., (188) El Darsv,v, Z. I., Tompsett, S I,., Zbid., 27, 108 (195,’). Analyst 81, 601 (1056). (150) Cotlove, E., Trantham, H. B., (189) Elliott, H. C.. Jr.. h s a ~ .CHEJI. Bowman, R. L., J . Lab. Clin. .Ired. 29. 1712 119571. 51, 461 (1958). (190j Ellni$n, G. L., d r c h . Biochena. (151) Cotzias, G. C., Greenough, J. J., Biophys. 74, 443 (1958). Arch. Biocheni. Biophys. 75, 15 (1958). (191) Elsner. P., 2. Vitamin-, Hornzon(152) Coulthard, A. J., Waugh, I). A , , u. Fermentforsch. 8 , 98 (1956). Clin. Chim. Acta 2, 318 (1957). (192) Fly, R. S., Hughes, E. R., Kelley, (153) Conrtois, J. E., 17illiers-Hriihnn, V. C., J . Clin. Endocrinol. and MetuboH., Zbid., 2, 332 (1957). lisni 18, 190 (1958). \
I
-
*
\
I
YG>
I
\ -
-’
VOL. 31, NO. 4, APRIL 1959
.
669
(193) Emtree, S. I)., Amee! S. R., Lehman, R . IT.>Harris, P. L., Methods of Biochen!. A n a l . 4 , 43 (1956). (194) Eprtein, E., Jladdock, \I7. O., Boyle, .I. J., .$SAL. CHEL 29, 1548 (1957). (105) Erhe, S.,z.anal. ( ' h e m , 159, 327 11958). ( l h ) Erninkova, E. A., B i o k h i n i i p 22, 917 (1957). (197) Fabrc, It., Truhaut, 11.: Boudene, C., Chin?. anal. 39, 285 (1957). 1 198) Feldheim. W..l , i t e l , n . 2. T'itaniin
' foisch. 27, 8 (1956). ( I b 9 ) Feldnian, I., a$sa~..CHEX 28, 1859 (1956). (200) Feldctein, Klendshoj, S. C., J . Forensic Sei. 2. 39 (1957). (201) Ferro, P. V.,Ham,'A. B.. A m J . C k n . Patho/. 28, 208 (1957). (202) Zbid.. u. 689. (203j Filtek;, G., Martel, F., Rei. Can. Biol. 15, 212 (1956). (204) Finch, E. P., Onens, S. E., Bj-erg. JT. E., L'lzn. Chenz. 2 , 427 (1956). 1205'3 Fine. J. 11.. Waezczenko. Z. E..
(206) Finkelgtein, '1I.,-Cox, R . I., Proc. Soc. Erpll. Bzol. M e d . 95, 297 (1957). (207) Fischer, F. IT,, Klzn. TT*ochschr. 34, 849 (1056). 1208) Fishman. W.H.. Davidson. H. 11.. JIethorls of Bzocheni Anal. 4 , 257 (1956). (209) Fla-clika, H., Holasek, A, Rosenthal, 11, Z . Physzol. Chenz., H o p p e Seyle?'s 308, 183 (1957). (210) Flrtrher, I;., Biocheni. J . 67, 136 (1957). (211) Folcah-Pi, J., Lees, 11, Stanley, C i . H., .I. Bzol. Chenz. 226, 497 (1957). (212) Forlit, A. A., Rfiller, \I7. L , Jr., IiraAe, J., Struck, JT-. -$., Proc. Soc. Esptl. Bzol. .lied. 96, 180 (1957). 1213'1 Forniusa. K. ;\I , Benerito. R. R.. Sii'igleton, IT'. s.,\Vhite, J. L.', ANAL: CHELI.29, 1816 (1957). (214) Fragner, J., Davidelc, J., Prdrnysl poiraiin 7, 76 (1956). (215) Frat,tini. J. F.. Reu. assoc. bioauini. . urg. u r j . 22, 201 (1957). (195i). 1216) Zbid.. p. 13. 211. (216) Zbid., (217j Frederick, Fredkr'ick, G. L., Anz. J . V e t . Re(217) search 18, 481 (1957). (218) Free, -$. H., Rupe, C. O., Rletzler, I., Clin. Chem. 3, 716 (1957). L., Posthuma. Posthuma, R., R.,Gor1219) (219) Freeman. Freeman, L.. don. L L... l l a r x . W..Arch.' A r c h . Biocheni. Biophys.' 70, 169 (1967). (220) Freeman, 11. V., J . Pharinacol. Ezptl. Therap. 120, 1 (1957). (221) Freeman, S. K., Lindgren, F. T., Sg..T. C.. Sichols. A. IT.. J . Biol. ('t&n. 227.'449 I'195f). (222) Friedman, AI. RI Lapan, B., J . Lab. Clzn. X e d . 51, 7 i k (1958). (223) Frierson, IT. J., Rearick, D. A,, AXAL.CHEV.30, 468 (1958). (224) Froesch, E. R., Reardon, J. B., Renoltl. A. E.. J . L a b . Clin. X e d . 50, 918 (1957). ' (225) Fujita, T., J . Cl, ~
(234) (iardell. S.. Ibid.. 6. 280 110t581 (235) (:e\, IC. F , Schon,", Z. Phiisid. Cheni
, Hoppe-Seyle?'~305,
1-19 (1956).
(236) Giacomo, P., Guillot, AI., Jacyuinot P., Compt. rend. 243, 985 (1956j. (237) Giri, K. V.,J . I n d i a n I n s f . Sci. 38A, 190 (1956). (238) Giri, K. I-., S a t h , 11.; Sriknntinh, H., Ibid., 39A, 224 (1957). (239) Gladishevska, T. 31.) 1Cvyak:itkivska, T. S., Sobchuk, B. h.,L-krafn. Biokhini. Zhitr. 29, 371 (1957). (240) Glick, I),, Freir, E. F., Ochs, 11.J.. J . B i d . Cheni. 226, 77 (1957). (241) ( h e c k , H. I., J . Lab. J . Hdlszde H o s p . 5 , 246 (1956). 1 2 W Goldschmidt, L.. C'izn. ('he))!.3, 738 (1957).
1249) Goldzielier. J. K..Besch.' P. IC.. ' hsaL. CHEX 30, 962 (1958). (250) Golub, 0 . J., Sobel, C., Henry. R. J., J . Clin. Endoc,.inol. u n d Jletaholima 18, 522 (1958). (251) Gomori, G.. A m . J . Clin. Pathoi. 27. 714 119571. (252j Goodman,' )I., J . L a b . Clin. X e d . 49, 151 (1957). (253) Gordy, E., Drahkin, 1). L., IIarqh. J. B., J . Biol. Cheni. 227, 285 (1957). (254) Gorrinee. .J, A . I,.. Clzn. Chzni. =Icta ' 2.'353 119971'1. (256) ~Goshev, I.: Vopros!j M e d . Khz'ni. 4, 149 (1958). (256) Graf, Rr.>Kimbel, K. H., 2. phusiol. Chem., Hoppe-Serjler's 304, 273 (1956). ( 2 5 7 ) Grant. G. H.. J . Clin. Pathol. 10.
(258) Gray,'I., Toung, J. G., Cltn. C'hem. 3 , 239 (1957). 1259'1 Gries. F. A, Gries..G.. Klzn. Wooch' schr. 35, SI (1957). (260) Grossman, 8.,Grossman, G. F., Clin. Chein. 4 , 296 (1958). (261) Grunbaum, B. IT7., (;lick, D., .-lrch. Biochem. Biophys. 65, 260 (1956). (262) Guillot,, >I.>A n n . biol. din. ( P a r i s ) 16. 105 11958). (263j Guillot, M. H., Clin. Chem. 3, 420 (1957). (264) Gupta, P. S.,Chatterjee, J., A n n . Biochenz. Emtl. X e d . ( C a l c u t f a ) 17. 129 (1957). (265) ,Guyton, A. C., Sichols, R. J., Jr., Farish, C., J . A p p l . Physiol. 10, 158 1195;). (266) Hacknev, J . L).> Sears, C. H., Collier, C. R., Ibid., 12, 428 (1958). (267) Hadd. H. E., Perloff, IT. H., J . Clzn. Endocrinol. and Metab. 17, 673 (1957). (268) Hall, R . J., J . J f e d . Lab. Technd. 14. 215 (1957). . , (269j Hallsway, B. E., . t n ~ J . J l e d . Technol. 22, 65 (1956). (270) Hamilton, P. B., ASAL (=HE\[. 1230) E. h.: Cadinan.' J. \ , Gaensler. 30: 914 (1958). B., J r . , Ellicott, RI. F:, Jones, R: H., ( 2 i l j Hanskn, P. K.,Dam, H., . I d a Marks. A,, J . Lab. Clin. J l e d . 49, Cheni. Scand. 11, 1658 (1957). &aitoAde, 31. K., Gordon, AI. W., (272) Hans1 Y. R., J . 97n. Cheni. Soc. 79, 1511 (ii57). 1273) Hanson. C. IC., r i s a ~ .CHEX 29. ' 1204 (msj. (274) Harboe, M.,Scand. J . Cliri. X. Lab. Inzest. 9 , 317 (195i). ,
~
670
ANALYTICAL CHEMISTRY
I
(275) Harris. E. S.. Reik, J J . Clin. Chenl. 4 , 241 (1958). (276) Harris, )I. L., A m . J . J f e d . Technol. 24, 99 (1958).
(277) Hasselmnn. J. J. F., Van Kampen, E. J., Clin. Chzni. Acta 3, 305 (1958). 12781 Hasselmann. 11.. C'onmt. rend. 244. 2860 (195i). (279) Hausman, E. R.. Lewis, 0. T., 31cA\nally. J. S.,Clin. C'heni. 3, 657 11957). (280) Heikel, T., Scund. J . Clin. &. Lab. Inrest. 10, 193 (1958). (281) Henly, h.,B n a l y s t 8 2 , 2 8 6 (1957). (282) Henry, R. J., Berkman, S., Clin. Cheni. 3, i l l (1957). (283) Henry, R. J., Goluh, 0.J., Sobel, A$.
C.. Ibid.. 3. 49 (1957).
(284.j Hen;,y,'R. J.: Sohel, C.) Berltnian, S.!A s ~ L .CHEX 29, 1491 (1957). (285) Henry, R. J., Sobel, C., Berkman, S . , C/in. rhein. 3, 77 (1957). (286) Henry, It. J., Sobel, C., Chianiori, S.,d.Jl..i. J . Diseases Children 94, GO4 (1957). (287) Henry> Ii. J., Sobel, C., Kim, J., A n i . ,I. C'li?~ Pathol. 28, 152 (1957). (288) Herrmann, R. G., Proc. Soc. Ezptl. B i d . J l e d . 94, 503 (1957). (289) Hill, IT-. H., JIerrill, J. M.,Nontiegel, E. C., Palm, B. J., Schmitt, J., Shiiltz, AI., -4.Jr.d. Arch. I n d . Health 17, 210 (1058). (290) Hirsch, A,, Carraneo, C., 2. Physiol. Chein., Hoppe-Seyler's 304, 53 (1956). (291) Hofmann-Credner, D., K l i n . It'ochscher. 35, 121 (1957). (292) Hofstra, S. Y., Van Sloten, E. -I., .\ed. Tijdschr. Geneesk. 102, 754 (1958). (298) Holecek, V., Cheiu. iisty 51, 315 (1957). (294) Hollander, V. P., Vinecour, J., XSAL. CHEX 30, 1429 (19%). (295) Hollinger, S . F., Lansing, R. K., A n i . J . J l e d . Technol. 22, 365 (1956). (296) Holman, R. T., Methods oJ Biochenr. d n a i . 4 , 99 (1956). (297) Holman, R. T., Hayes, H., .$SAL. CHEJI.30, 1422 (1958). (298) Holmstedt, B., -4cta Physiol. Scand. 40, 322 (1957). (299) Holthaus, J. ll., Humoller, F. I,., Kalsh, J . R., J . Lab. Clin. Mal. 51, 654 (1958). (300) Houck, J. C., -4rch. Biochern. Biop h y s . 73, 384 (1958). 13011 Huisman. T . H. J.. Clin. Chini. Beta 3,201 (1958): (302) Huisman, T. H. J., Prins, H. IC., Zbid., 2 , 307 (1957). (303) Huisman, T. H. J., Schreuder, J., Ibid.. 2. 210 11957). (304) Humollei, F. L., Holthaus, J. Rl., Walsh, J. R., McCann, E., Clin. Chenz. 3 , 703 (1957). (305) Humoller, F. L., Majka, F. A., Barak. A. J., Stevens, J. D., Holthaus, J . lI., Afatsumoto, Y., Ibid., 4 , 1 (1958). (306) Humphrey, J. H., Long, D. A, Perry, IT. L. Rl.. Methods of Bzocheni. Ami. 5 , 54 (1957). (307) Hunter, G., J . Clzn. Pathol. 10,
161 (1957). (308) Hiirlock, B., Talalay, P., Endoc/znology 62, 201 (1958). (309) Hurlock, B , Talalay, P., Proc. Soc. Esptl. Biol. M e d . 93, 560 (1956). (310) Inouve, K., Shionogz Kenkylisho .Yenip6 6 , 44 (1966). 1'311) Isgrescu. D. A.. Ionescu, P., blachvei, LI., Conaztn. acnd. rep. populare Ronikne 7, 845 (1957). (312) Izzo, .\. J., Keutmann, E. H., Bnrton, R. B., J . Clan. Endocrinol. and Jfetabolisnz 17, 889 (1957). (313) Jarobeen, C. F., Leonis, J., Lintierstr@ni-Lang, K., Ottesen, 11.) J f a t h o d s of Baocheni. Anal. 4 , 171 (1956).
(314) Jacobson, K. B., .1strachan, L., Arch. Biochem. Biophys. 71, 69 (1957). (315) Jirgl, V., Clin. Chem. 3, 154 (1957). (316) Johansson, G., Sorman, X.,Karrman, K. J., ;\N.4L. CHEX 30, 1397 (1958). (317) Johnsen, S. G., Acta Endocrinol. 28, 69 (1958). (318) Johnson, 11. F., Heftmnnn, E., Hayden, A . L., Ibid., 23, 341 (1956). (319) Johnson, R. B., Jr., J . Lab. C'lin. M e d . 51, 956 (1958). (320) Jones, R. T., Blake, K. l),, J . .-lpp/. Physiol. 12, 448 (1958). (321) Juhas7 B., KisPdetes O r m s t i i d o m n i g 8, 215 (19kG). (322) Juliard, A. I,., -1s.1~. CHEII. 30, 44 (1958). (323) Kabara, J. J., J . Lab. Clin. 3 1 e d . 50, 146 (1957). (324) Iiagi, J., Burger, >I., (iiger, Ii., A r c h . exptl. Pathol. Pharmakol., S a i ~ nyn-Schmiedebey's 230, 470 (1957).
(358) Kohn, J., Clzn. Chitti. +-letu 3 , 450 (1958). (359) Kon, S., Urata, T., Uchiyama, S., Iiimura, T., Ahe, T., Muto, .L,Haga, I' K',, Ohno, IC:, .Ikabori, S., J . Biocheni. ( T o k y o ) 44, 183 (1957). (370) Iiuroda, Y., Fukitoka, Iqaku-Zasshi
( 3 2 5 ) Kajin-are, T., Fukzioka-Igaku-Zasshi
R. L., Wilkoff, L., J . Lab. C'lin. X e d . 52, 661 (1958). (331) Kanabrocki, E. I,., Greco, J., ]Tilkoff, L., r7each, R., Clin. C'hetti. 3, I56 (1957). ( 3 3 2 ) Kanaii'rocki,
E. L., Kaplan, E., Kinnory, I).S., Imperato, .1.*I.,Berry, J. E., Baker. L. Ibid., 4 , 382 (1958). (:133) Kanig, IC., Z. Physiol. C h e w , HoppeSeyler's 306, 247 (1957). 1334) Kanner, O., Moss, C., -4n~.,I. C l i n . Path. 29. 310 11958). ( 3 3 5 ) Iiantor, S., Xlkerson, H. L. C., Diahetes 6 , 1'76 (1957). (336) Iiaplan, C. M.,Weisherg, H. F., L)OTV,I,.> Clin. C h e m 4 , 181 (1958). I
\
~~~
~
-
-
I
(837) Iinziro, Y., J . Biochenz. ( T o k y o ) 44, 827 (1957). (338) Kellie, -1. E., Analyst 82, 722 (1957). (339) Kellie, A. E., Wade, A. P., Biochein. J . 66, 196 (1957). (330) ICessel, M.,Sylvester, R., d r i t l . Wochschr. 13, 238 (1958). (341) Kesselman. P.. Rev. assoc. bioortii?i. arg. 22, 107 (1557). (342) Khon, En, Aptechnoe Delo 6 , 29 11057)
C'psa(iensis 82, 36 (1957). (3345) Kimura, I
