Clinical chemistry - ACS Publications

Clinical chemistry - ACS and Metcalfe (95(7) described an a...

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(524) W‘ada, O., etal., Brit. J . Ind. Med., 26,240 (1969). (525) Wales, P. J., et al., Analyst, 93, 691 (1968). (526) Walisch, W., Mikrochim. Acta, 1968,748. (527) Wallcave, L. Environ. Sci. Technol., 3,948 (1969): (528) Walker. A. 0.. U.S. Patent 3.453,081 (July 1969). ‘ (529) Walther, J. E., Amberg, H. R., Tappz, 51, 126A (1968). (530) Wells, B. J., Health Phys., 13, 1001 (1967). (531) Westfaelische Berg., French Patent 1,493,859 (1 Sept. 1967) 11 pp. (532) West, P. W., Jungreis, E., Anal. Chim. Acta, 45, 188 (1969). (533) West, P. W., Lyles, G. R., Miller, J. L., Environ. Sci. l’echnol. 4, 487 (1970). (534) West, P. W., Sachdev, S. L., J . Chem. Educ., 46, 96 (1969). (535) Whitby, K. T., NcFarland, A. R., J . Air Pollut. Contr. Ass., 18, 760 (1968). ,



(536) Whitnack, G. C., Brophy, R. G., Anal. Chim. Acta, 48, 123 (1969). (537) Wilbv, F. V., J . Air Pollut. Contr. Ass., 19,- 96 (1969). (538) Willis. C. P.. Olson. D. G.. Sill.‘ C. W:, ANAL:CHBM:,42, 124 (1970). (539) Wilson, L. G., Cavanagh, P., Atmos. Environ., 3, 47 (1969). (540) Winkel, A., Staub, 28, 1 (1968). (541) Wronski, M., CHEM.ANAL. (Warsaw), 15, 371 (1970). (542) Yamaguchi, R., Machida, T., Ueki, M., Yakugaku Zasshi, 89, 1534 (1969). (543) Yamada, V. M., Charlson, It. J., Envzron. Sci. Technol., 3,483 (1969). (544) Yamamoto, 1%.K., Cook, W. A., Amer. Ind. Hug. Ass. J., 29, 238 (1968). (545) Yamamoto, Y., et al., Anal. Lett., 1, 955 (1968). (546) Yamate, N., Matsumara, T., Tonomura, M., Eisei Shikenjo Hokoku, 86, 58 \ -

f 1968). \----

(547) Y’inagisawa, s., fi’titsuzawa, s., Mori, hl., Bunseki Kagaku, 17, 580 (1968).

(548) Yavorovskaya, S. F., N&. Obl. Prom.-Sanit. Khim., 166 (1969). (549) Young, R. A., SRI Project No, PAU-3895, 34 pp (1967). (550) Yuhi, K., Japan J . Hyg., 21, 407 (1967 - - ). (551) Zakrocki, Z., Gaz. Woda Tech. Sanit., 38,162 (1967). (552) Zawadski, S., Pr. Cent. Inst. Ochr. Pr., 18, 283 (1968). (553) Zawadzki, S., Sojecki, W., ibid., 19.79 (1969). (554j Zebel, G., Staub, 28, 1 (1968). (555) Zeman, A., Stary, J., Kratzer, K., Radiochem. Radioanal. Lett., 4, 1 (1970). (556) Zverev, Y. G., Derevyanko, D. G., Gasova, T. V., ,Vov. Obl. Prom-Sanit. Khim., 1969, 71. (557) Zverev, Y. G., Derevyanko, D. G., Gasova, T. V., Vses. iVauch.-Issled. Inst. Okhr. Tr., 1967, 264. \ - -

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of this review was supported

in part by U.S.E.P.A., APCO, Contract CPA 70-24, Mr. Tom Stanley, Project


Clinical Chemistry G. R. Kingsley, Clinical Chemistry Consultant, 62 1 Bonhill Road, Los Angeles Calif. 90049, and Lecturer, Department o f Pathology, School o f Medicine, University o f California, los Angeles, Calif. 90024


reviewed (7A) significant developments in clinical chemistry for the period December 1966 to December 1968, and continues this review to cover the period December 1968 to Sovember 1970. HE AUTHOR


Purdy and Melville (9.4) recently pointed out the manpower crisis t h a t will exist in the field of clinical chemist r y when the provisions of Title 20 of the Medicare Bill (Federal Health Insurance for the Aged) takes effect (July 1, 1971). Unfortunately, there are not enough well trained individuals in clinical chemistry t o properly staff existing independent laboratories, if these laboratories are to meet the specific requirements of Title 20 of the Medical Bill to qualify its services for reimbursement under the supplemental medical insurance of the Health Insurance for the Aged program. These authors suggested that analytical chemists may be oriented toward clinical chemistry to ease this crisis. I n the book, “Fundamentals of Clinical Chemistry,” Tietz together with 16 expert contributors (I6A) covered a wide range of laboratory techniques and current instrumentation of clinical chemistry and showed how to perform, interpret, and analyze chemical laboratory tests including those for bodily functions. Varley (17il) in the 4th edition of “Practical Clinical Biochemistry” presented a large amount of information on clinical chemistry methodology and its

interpretation. Searcy ( I d A ) in the book “Diagnostic Biochemistry” presented significant facts concerning physiological functions to unify biochemistry with the clinical practice of medicine to provide better diagnoses. Evaluations were made of current methods employed for assaying substances in the body. Henry et al. ( 5 A ) presented a format for description OP methods in clinical pathology which included : introduction, principle, sample for analysis, procedure, reagents, equipment, calculations, significant figures, notes, normal values, quality control, comparison of results with a reference method, discussion, and bibliography. Franke and Thiele ( 4 A ) published the book, “Physicochemical Methods in Clinical Laboratories,” Vols. I and I1 (Guide for ClinicalLaboratory Methods). Weygand (18A) reviewed the developments of analytical methods in biochemistry during the past 30 years. Reynolds (IOA) presented a book, “Clinical Chemistry for the Small Hospital Laboratory’’ to assist the laboratory in making available to the physician all clinical chemistries necessary to the welfare of the patient. Thalmann (15A) reviewed the application of methods and automation in the microliter range as applied to chemical analysis. The use of gas-liquid chromatography in clinical chemistry methodology was reviewed by Street ( I S A ) . Scott ( I I A ) oriented the book “Clinical Analysis by Thin-Layer Chromatography Techniques” t o the clinical chemistry labora-

tory, and devoted sections to: specific separations, carbohydrates, amino acids, steroid hormones, bile acids, lipids, organic acids, etc. Juvet and Cram ( 6 A ) reviewed gas chromatography covering: packed columns, liquid phase, solid supports, adsorption columns, carrier gases, trapping detectors, quantitative analysis, instrumentation interfacing, and specialized operations. Strickland ( I @ ) reviewed electrophoresis under the titles of interest t o the clinical chemist: fundamental developments, apparatus, stabilizing media, buffers and solvents, biological application, human serum interaction with plasma proteins, hemoglobin, lipoprotein, glycoproteins, enzymes and forensic, toxicological, and pharmaceutical applications. Emission spectrometry was reviewed by ;\largoshes and Scribner (8A)under the subjects: books and reviews, spectral descriptions and classifications, instrumentation, standards, sample, calibration and calculation, excitation source, trace analysis and other applications. Winefordner and Vickers (2OA) reviewed flame spectrometry under the subjects: reviews, books and bibliographies, fundamental studies, atomic and molecular emission spectrometry, atomic absorption spectrometry, and atomic fluorescence spectrometry. Infrared spectroscopy was reviewed by Fahr and Rohlfing (SA) who covered enzymes, proteins, fats, lipids, nucleic acids and their components, carbohydrates, steroids, and pharmaceutical compounds. Boltz and Mellon ( I A ) reviewed light absorption



spectrometry, covering: chemistry, metals, nonmetals, organic constituents, different types of instrumentation, and special applications instruments. White and Weissler ( I 9 A ) reviewed fluorometric analysis under the subjects: apparatus, inorganic, nonmetals, chemiluminescence, p H indicators, organic and biological, immunofluorescence, steroids and hormones, and pharmaceuticals. Ultraviolet spectrometry was reviewed by Crummett and Hummel ( I A ) under the titles: books and reviews, apparatus, spectral studies' of classes of compounds, adsorbed molecules and crystals, solvent, temperature and p H effect, solution equilibria, elucidation of structure, complexs, inorganic analysis, organic analysis, and biological and pharmaceutical analysis. APPARATUS AND EQUIPMENT

Automated. Blackburn et al. (5B) patented an automatic chemical analyzer which consisted of a disposable testing container with a n area for reagents and an optical reaction chamber where programmed reagents were mixed with the test sample. Johnson et at. (23%) patented an automated device consisting of a number of premeasured amounts of reagents and an empty disposable test pouch contained between 2 thermosealed sheets of plastic material such as polyethylene and provided with a rigid tube and windows for colorimetric analysis. ilnderson (2B) described an automated multiple cuvet rotor (GeMSAEC) for the simultaneous analysis of a series of discrete samples. Jolley, Pitt, and Scott (24B) developed a method for metering liquid reagents without variations in flow rate for continuous colorimetric detection systems by use of hydraulic head or gas pressure. Hardy (19B) designed a limited function automation system for rapid, more accurate and reproducible clinical chemistry determinations. Rait (39B) described an apparatus which automatically prepares standards and sample solutions for colorimetric determinations. Vaills (45B) patented an automatic laboratory analyzer system which consisted of syringes with pileumatically controlled pistons vertically located above test tubes containing samples into which test solutions were delivered. Unicam Instruments (44B) designed an autoanalyzer for the continuous analysis of biological liquids in which a peristaltic pump delivered predetermined amounts of specific reagents. Xatelson (S5B) developed an automated system utilizing 1- to 1OO-pl samples which divided the sample into several aliquots and analyzed each one indepeiideiitly and simultaneously by a n independent phototube system. small automated high resolution analyzer 16R

for ultraviolet absorbing constituents of body fluids was developed by Pitt et al. (S7B). Stuart (/OB) described a 15-lb biosatellite urinalysis instrument which performed analyses for calcium, creatinine, and creatin, four times daily. Loebl (SIB) patented a n apparatus for sampling biological fluids successively or simultaneously from a series of several samples with multiple automatic pipets having a tip wiping action. Auphan and Perilhou (4B) patented an automatic analyzing device consisting of a number of flexible tubes which were attached to a moving belt and programmed so that the fluid in the tubes could be admitted, discharged, or intermixed with other reagents. Csizmas and Pate1 (1IB) invented a device for placing at' least 1 reagent-containing transferable article into a receiver to carry out chemical tests. Hughes, Tressel, and Flavell (22B) described an automated colorimeter for the analysis of biological materials which had four different' photodetecting circuits. Miscellaneous. Levkoff, Westphal, and Finklea ( S I B ) evaluated a direct reading spectrophotometer for neonatal bilirubiiiometry and found excellent correlation with the MalloyEvelyn method. Boehringer (6B) used polymer films on carriers to hold detection reagents for estimation of urobilinogen in the urine, and urea and glucose in urine and blood. Thacker et al. ( 4 I B ) developed miniature photometers for use on stream detectors in systems for liquid chromatography. Vestergaard (46%) developed a multicolumn system for high capacity liquid chromatography in the routine assay of urinary steroids and other applications. Kuz'min et al. (29B) developed a simple apparatus for the chromatographic analysis of mixtures of substances containing 1-5 x lo-* pg of the components using a double-beam micro densitometer. Lefar and Lewis (SOB) reviewed the precise and sensitive instrumentation available for the quantitative evaluation of thin-layer chromatograms. A method was developed by Kaffczyk et al. (26B) for thin-layer chromatography of urinary constituents without pretreatment of the urine. Kaffczyk, Helger, and Lang (25B) described the two dimensional thin-layer chromatography of amino acids on two-layer plates. Ertinghausen, Adler, and Reichler (15B) developed an automated high speed ion-exchange chromatographic system to separate amino acids present in protein hydrolysates. Burtis, Goldstein, and Scott (9B) developed high resolution anion-exchange gel chromatography for urine for resolving more than 150 constituents t h a t absorb ultraviolet light. Abadi ( I B ) described a n apparatus for discontinuous electrophoresis on a slab of polyacrylamide gel which resolves serum protein as distinct as


that with electrophoresis on a gel column. A versatile system was described by Brownstone (8B) for preparative electrophoresis on acrylamide gel with an intermittent collection system. Oliva and Schultz (S6B) designed an electrophoresis apparatus for clinical use in expendable single use kit form. Pressman (S8B) reviewed and discussed practical applications of ion specific electrodes and the specificity of the electrodes to H+, Na+, and K+. Arthur, Carlsen, and Stevenson (SB) patented a n apparatus for the electrochemical analysis of small liquid samples such as blood. Derr, Neff, and Sambucetti (12B) described an invention for the direct electrometric measurement of a component' of a liquid by forming its reaction product and measuring the resultant electrical potential by means of a pair of glass electrodes. Williams, Doig, and Korosi (49B), designed a n electrode t'o measure directly whole blood glucose by monitoring the electrooxidation of hydroquinone in the presence of glucose oxidase. Edwards (14B) measured cations of biological fluids by a patented reagent consisting of paper impregnated with a weakly basic or acidic ion exchange resin and a suitable p H indicator. Durst ( I S B ) edited a report on the proceedings of a symposium on ion selective electrodes which was a thorough review and application of this subject. A microelectrode was described by Kahn and Spracklen ( I 7 B ) for making intjracellular measurements of living plant and animal cells. Makin and Warren (34B) adapted a simple polarographic oxygen electrode for the specific estimation of blood glucose using glucose oxidase. Crouch (10B) described a n all-electronic versatile reciprocal time computer for both fast and slow reactions. Toren et al. (4SB) reported that interface instrumentation between computer and spectrophotometer for reaction rate measurement is readily constructed from commercially available components. Bowers and Haschemeyer (7B) described a versatile small volume ultra filtration cell. A microcalorimeter was described by Wadso (47B) for measurement of relatively fast reactions and enzyme kinetics. White and Offerman (48B)discussed the basic principles of liquid scintillation techniques and the applications of radioactivity counting in biological and clinical research. Harvey @OB) described a simple stopped flow photometer which employed a rapid mixing device for small sample volumes (20 pl) and featured a mixing time of four milliseconds. Haljamae and Larsson (18B) designed a dual channel integrating ultramicro flame photomet'er for analysis of nanoliter quantities of K and N a of single cells and biological fluids. Farese and Mager (16B)prepared serum ultrafiltrates by centrifuging through

size selective membranes. Glick (17B) described cytochemical analysis of one cell by laser microprobe-emission spectroscopy. Luckey (3%)patented a n apparatus for determination of blood alcohol content by analysis of breath, without prolonged detention of subject. Timmins and DeFilippi (42B) described a patented device for in vivo quantitative measurement of gases in the blood stream. Hughes (21B) patented a kit for performing certain types of chemical tests on blood. Kiess (28B) patented a reagent kit for colorimetric determinations which consisted of a group of equal size sealed containers, each of selected material so t h a t all containers had the same light transmittancy characteristics and contained a measured amount of reagents. Winkelman (50B) developed a novel device for the separation of plasma from a conventional syringe which eliminates the need of centrifugation. AUTOMATION

Equipment. Kelson (91C) reviewed work-load, space, staff, equipment, and the benefits of automation in the clinical chemistry laboratory. Broughton et al. (16C) recommended a scheme for the evaluation of instruments for automatic analyses and Alpert (SC) reviewed factors which may be useful to the clinical chemist in evaluating established and new automated systems. Ahuja and Basis ( I C ) evaluated the “Autochemist” with 28 analytical channels and 135 samples per hour capacity and obtained results within medically accepted limits. Ahuja and Basis (2C) compared the performance of this instrument with single channel “AutoAnalyzers” and did not obtain agreement with all analytical methods. Hamilton (56C) described a disposable reaction container with sealed in reagents which is suitable for the automatic chemical analysis of body fluids. Dahms (22C) patented an electrochemical apparatus which included a computing system and a series of electrodes for the automatic analysis of C1-, H+, Na+, K + , 02, and COz. Anderson (5C) reviewed the basic research necessary for the development of advanced methods for clinical chemistry and described a new analytical system (GeMSAEC) using centrifugal force t o transfer and mix fluids in a multiple cuvet rotor to produce data signals at intervals of 3.3 milliseconds. Anderson (6C) described the basic principles of the GeMSAEC fast analyzer, and also described (Hatcher and Anderson) (59C) the application of the biuret serum protein method t o this new analytical tool. Daly, Fabiny, and Ertingshausen (S4C) described the physical characteristics of CentrifiChem, a high speed

analyzer, and found kinetic and endpoint analytical methods adaptable t o the instrument. A modification of the Beckman discrete sample analyzer was made b y Gallwas and Gray (38C) t o provide multichannel readout for both end-point and kinetic determinations at 340 mp. Wagner-hfelka (135C) patented a n apparatus for automated routine analysis of biological fluids in which dosed amounts of sample and reagents were transported through a network of capillary tubing by means of air pressure. Husbands (64C) described the “Unicam AC 600” automatic analytical chemistry system. Buckle and Riley (17C) described a n apparatus for automatic sampling, dilution, addition of reagents, heating and photometry, etc. a t each of several stations for the analysis of 12-15 components of body fluids. Loebl (’75C) patented a system for automated colorimetry of biological samples. An apparatus was patented by Rait (99C) which automatically carried out analysis on biological fluids. Scott et al. (1132) developed a n automated high resolution chromatographic system capable of analyzing 150 UV-absorbing molecular constituents in body fluids. An automatic discrete sample-handling analytical instrument was developed by Westlake et al. (l42C) t o interface with a computer. Seifter, Kambosos, and Rettura (1l4C) diluted small serum samples with a protein precipitating agent to eliminate dialysis in automated systems. Bennet et al. (12C) studied the calibration, drift, and specimen interaction in the AutoAnalyzer systems. Finley et al. (35C) evaluated the technical performamce of the ShIA 12/60 AutoAnalyzer and found its precision and accuracy a s good as manual methods and single channel systems. Hoffmeister and Junge (SSC) investigated the reliability of the AutoAnalyzer SMA 12/60 under different conditions. Neill, Doggart, and Mitchell (9OC) studied the precision and accuracy and economic considerations of the Technicon SMA 12/60 hospital model to determine its usefulness for routine work in a large laboratory. Gardanier and Spooner (4OC) reported the effects of air bubbles, colorimetric flow rate, and circuit components on longitudinal mixing during continuous-flow analyses. Habig et al. (5SC) described a bubble-gating flow cell for improvement of continuous flow analysis. Jansen, Peters, and Zelders (66C) modified Skegg’s continuous flow system for colorimetric analysis by replacement of the sampling system based on time by a system based on volume using a 12-channel pump giving low pulsation without visible back flow. Walker, Pennock, and McGowan (136C) studied the practical considerations in kinetics of continuous flow analysis of

the AutoAnalyzer, especially contamination, shoulder peak, carry over, standard profile, sample to sample interaction, and first-order kinetics. Thiers, Meyn, and Wildermann (125C) developed a graphic analog computer program to correct errors resulting from interaction of continuous flow analysis of the AutoAnalyzer system. Whitby (14SC) discussed computer processing of clinical chemistry data from the AutoAnalyzer. Roberts and Zydlewski (1037) applied multiple wavelength spectrophotometry to chromatogram scanning. Gambino et al. (S9C) developed a n automatic instrument for the determination of hemoglobin in whole blood. Automated Methods. Amino Acids. Van D y k e a n d Szustkiewicz (133C) developed a n automated amino acid method based upon oxidative deamination of the amino acid coupled with oxidation of 0-dianisidine by H202. Palmer and Peters (94C) described a simple automated method for the determination of total free amino acids in blood based on the formation of colored complexes with 2,4,6-trinitrobenzene sulfonate. An automated method for the simultaneous quantitative analysis of urinary peptides and free amino acids was described by Ellis, Prescott, and Welch (SSC). Ambrose (4C) developed a n ultra micro automated fluorimetric procedure (AutoAnalyzer) for screening of phenylalanine in blood. Paterson and Metcalfe (96C) described an automated procedure for proline which employed a modified Chinard ninhydrin reagent. Carbohydrates (See 1 9 K ) . Scott et al. (112C) described two high resolution automated analytical systems for the analysis of UV-absorbing constituents and carbohydrates in body fluids. Fingerhut (SSC) automated the ferricyanide-phosphomolybdate glucose method (SMA 12/60). Baillod and Boyle (8C) assessed the effect of the instrumental lag on the recorded data to interpolate a continuous glucose concentration plotted by the AutoAnalyzer. Giiidler (44C) described an automated serum glucose method in which a reductive formation of lavender Cu(1)-2,2’bicinchoninate chelate occurred. An automated procedure based on the hexokinase method for the determination of glucose was described by Harding and Heinzel (58C). Gutteridge and Wright (51C) described an automated blood glucose method which employed a guaiacum reagent. A modified enzymatic serum glucose method was adapted to the AutoAnalyzer by Van Der Slik et al. (131C). Rosevear et al. (105C) developed a continuous automated monitoring method for blood glucose employing glucose oxidase-peroxidase-0dianisidine-HCl-H2SO4. Asrow (7c) carried out enzymic semi-automated


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microanalysis of blood glucose on filtrates. Moorehead and Sasse (87C) described an automated micro serum glucose method which employed a n improved, more sensitive 0-toluidine reagent. Wenk e t al. (14OC) adapted Dubowski's 0-toluidine procedure to the automated micro measurement of glucose. Sudduth, Widish, and Moore (121C) automated a direct method for serum glucose employing 0-toluidine reagent in concentrated CHaCOOH and stabilized with thiourea. An automated method utilizing a new O-toluidine reagent for the microdetermination of glucose was described by Sasse and Moorehead (109C). Frings, Ratliff, and D u m (37C) described a n automated 0-toluidine glucose method in which 0.04 ml of serum was used without dialysis. Sawyer (11OC) determined reducing sugars by replacing the colorimetric end point with a redox electrode system utilizing a Pt electrode for automated potential measurement. A new rapid instrumental method for glucose determination based upon a rate sensing oxygen measurement was developed by Sternberg (118C). Cations and Anions. H a k e (66C) determined serum calcium with the AutoAnalyzer by a method based on a color shift from orange t o blue when Ca2+is added to a n alkaline solution of 2-carboxy-2-hydroxy 5'-sulfoformazylbenzene (Zincon) containing small amounts of the Zn complex of EGTA. Frings, Cohen, and Foster (S6C) determined serum calcium by an automated method by measuring the red chromogenic complex formed b y calcium and alizarin. Burr (1%) improved the automated serum calcium method employing glyoxal-bis-(2-hydroxyanil) reagent by increasing the dye concentration and using E D T A in the buffer. Fingerhut, Poock, and Miller (342) developed an automated fluorimetric procedure to measure calcium over a wide range. Ruzicka and Tjell (IO7C) described the use of a Ca2+ selective electrode sensor in procedures for the automated determination of free calcium. Gochman and Givelber (47C) described a procedure for the simultaneous automatic determination of serum calcium and magnesium by atomic absorption. The rapid direct determination of serum chloride by automation using ferric thiocyanate reagent was described by Giraudet, Pre, and Cornillot (4SC). Neff et al. (89C) constructed a n automated experimental automatic computer assisted electrode system to determine blood pH, p02, pCO2, Na, and K . Van Belle (1SOC) developed a n automated method for the determination of serum inorganic phosphate by employing a new reaction between phosphomolybdate and the dye, Methyl Green 00. An automated procedure for the determination of total 18 R

and inorganic sulfate in serum and urine was described by Haff (54C). Lipids. An automated serum total lipid assay method based on the color produced when serum treated with concentrated Hi304 reacts with a phosphoric acid-vanillin solution was described by Ratliff, Culp, and Gevedon (1OOC). Tytko, Willis, and King (128C) described a semi-automated lipid profile analysis adapted to the AutoAnalyzer. A sensitive automated fluorimetric method for estimation of both serum and serum lipoprotein cholesterol was presented by Robertson and Cramp (104C). Kashket (67C) described an automation device by which on-line extraction of serum fatty acids into standard organic solvents was made for their determination. Lorch (77C) described a new device for extraction of free fatty acids from serum in a continuous flow system. Dalton and hlal1011 (SSC) assayed serum glycerol by a n automated glycerol kinase-coupled ADP-detecting system with subsequent measurement as loss of native fluoresAn automated cence of NADH. method for the determination of free glycerol in whole blood by the enzymic method of Willand was described by Harding and Heinzel (57C). KO and Royer (71C) developed two automated fluorimetric procedures for the determination of glycerol in plasma by the AutoAnalyzer. Block and Jarrett (147) and Noble and Campbell (92C) improved the automated procedure of Kessler and Ederer for the photofluorimetric determination of serum triglycerides. Royer and KO (106C) and Motegi, Shoji, and Toyoda (88C) described automated fluorimetric methods for serum triglycerides. Enzymes. Ihadley and Tappel (15C) designed, developed, and applied two automated multiple enzyme analysis systems. Trayser and Seligson (127C) proposed a new kinetic method for enzyme analysis suitable for automation. An automated determination of A T P based upon the measurement of the light emission produced during the oxidation of luciferin by molecular oxygen in the presence of A T P and M g ions was reported by Van Dyke et al. ( I S S C ) . Massod, Werner, and RfcGuire (8SC) presented a n automated multipoint spectrophotometric serum alkaline phosphatase procedure based on the liberation of p-nitrophenol from p-nitrophenylphosphate for its kinetic determination. Cornish, Neale, and Posen (SOC) described an automated micro assay for serum alkaline phosphatase which utilizes 10-aJf 4-methylumbelliferylphosphate as substrate. Small (115C) described an automated method for differentiating serum alkaline phosphatases. Lott and Mercier (78C) used dextrin substrate and 50 O C incubation and measured the liberated


sugars with alkaline potassium ferricyanide by a semi-automated method to determine serum and urine amylase activity. O'Neal and Gochman (9SC) adapted the Somogyi serum amylase method to automation by destroying glucose with glucose oxidase and catalase. Hathaway, Berrett, and Hunter (6OC) adapted the serum amylase method to automation by using amylase azure. A new simple, sensit,ive automated method for the determination of serum alpha-amylase in which a fluorescent starch substrate was used was developed by Rinderknecht and Marbach (102C). Miyahara and Usui (86C) presented a n automated micro method for the determination of plasma cholinesterase based on colorimetry of 5-thio-2-nit,ro-benzoic acid formed by reaction between 5,5'-dithiobis (2-nitrobenzoic acid) and thiocholine. An automated procedure for the determination of serum creatine phosphokinase (CPK) in which enzyme activity is measured by the creatine phosphate formed from the CPK-catalyzed reaction of creatine with adenosine triphosphate was described by Wright and Alexander (145C). Daly and Levine (26C) suggested an improvement of the automated C P K method of Siege1 and Cohen by substituting a more stable orcinol reagent for alpha-naphthol which resulted in a sixfold increase in sensitivity. Kruse-Jarres and Klingmueller (72C) described the discontinuous and continuous enzymic analysis of galactose by means of an AutoAnalyzer. Hochella and Hill (SSC) described a n automated fluorimetric screening procedure to detect persons lacking blood enzyme galactose transferase. Tan and Whitehead (I%%) developed an automated fluorometric microtechnique for the quantitative determination of glucose-6phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD). hlassod el al. (8%') automated the determination of serum lactic dehydrogenase (LDH) by spectrophotometric measurement of the conversion of NAD to NADH. Dube, Hunter, and Knight ($8'2) adapted the serum spectrophotometric analysis of L D H and hydroxybutyric dehydrogenase to the AutoAnalyzer. Schwartz and Burns (111C) described a sensitive reproducible automated method for the determination of serum malate dehydrogenase by measuring the disappearance of N A D H by fluorometry. BelfieId and Goldberg ( 1 f C ) described a procedure for the continuous spect'rophotometric assay of serum 5'-nucleotidase in serum. A semiautomated fluorimetric method for serum pepsinogen was described by Wenger and Munro (139C) in which precipitation and filtration were eliminated. Metals. Clarke and Nicklas (19c) adapted the colorimetric 2,4,6-t'ripy-

ridyl-S-triazine serum iron method to the AutoAnalyzer. Garry and Owen (41C) used sulfonated bathophenanthroline color reagent for the microdetermination of serum iron and iron binding capacity with the AutoAnalyzer. An automated procedure for serum iron and iron binding capacity utilizing a new iron-chelating ligand, a 5-pyridyl benzodiazepin-2-one derivative was developed by Klein, Kleinman, aiid Searcy (7OC). Bide (1%’) described a n automated method for the estimation of low levels of iron in biological fluids. Eckman e t al. (SOC) automated the quantitative immunochemical determination of human serum transferrin. Nitrogen Compounds. Van Belle (129C) described a new and fully automated colorimetric method for the determination of adenosine a t the micro level. Imler et al. (65C) studied automated methods for the determination of blood ammonia using dialysis for the separation of ammonium ion from the blood and Berthelot method for colorimetry. Dobbs, Castleberry, and Shaw (27C) applied the Berthelot reaction to the automated analysis of rg-quantities of N H 4 + in urine. Looyi! (76C) described a n automated method for the determination of p-acetylaminohippurate in serum simultaneously with inulin. A sensitive automatic method for the determination of creatin in body fluids based on the diacetyl-naphthol reaction was described by Gundlach, HoppeSeyler, and Johann (49C). Mather and Roland (84C) improved the sensitivity and linearity of the diacetyl monoxime reaction for urea by the addition of thiosemicarbazide. Hormones. Heistand ( S I C ) described a n automated method for the determination of catechols in urine based on the formation of a tungstatecatechol complex in acid and the formation of a red color with NaXOn in a n excess of KaOH. A partially automated fluorometric method for separate and direct measurements of epinephrine and norepinephrine was developed by Mabry and Warth (79C). Gutteridge (50C) described a partially automated method for the quantitative detection of 4-hydroxy-3-methoxymandelic acid in urine. An automated fluorometric method for the determination of dopamine (3-hydroxytyramine) was described by Craig et al. (21C). Van Stekelenburg, DeBree, and Van der Stam (IS4C) made a micromodification of the flow diagram for the determination of P B I with the AutoAnalyzer. Keller, Weidler, and Ammon (68C) improved the serum thyroxine (T4) method of Passen and von Salesky by eliminating column blank values, aiid increased the sensitivity of the automated method ten times by changing reaction conditions. Passen and von Salesky (95C) described a semiauto-

mated method for the determination of T4 which does not require digestion for release of iodine from thyroxine. Kessler and Pileggi (69C) described a modified column chromatographic procedure for isolating Td and Ta and their subsequent determination by an automated technique using the ceric arsenite reaction with bromine. An improved automatic procedure for IZ5I-T3 was described by Webber, Johnstone, and Garnett (138C). Mardell (81C) reported a source of error in the automated serum T3 uptake test which was caused by a large and variable proportion of radioiodide in the dialysate from a mixture of radioactive triiodothyronine and serum. Organic Acids and Compounds. Salway (108C) determined acetoacetate in capillary blood by a n automated method in which acetoacetate was coupled with 2,5-dichlorobenzenediazonium chloride to produce formazan dyes. Pellet, Seigner, aiid Cohen (97C) converted citric acid to citraconic anhydride with acetic anhydride and condensed this product with CsH5N and measured fluorimetrically by automation. Mann and Shute (8OC) described a semiautomated method for the determination of L-lactic acid in whole blood after deproteinization by the Somogyi procedure. Hadjiioannou, Siskos, and Valkana (5%) determined lactic acid in blood by a n automatic reaction rate method in which L-lactic acid is selectively oxidized in the presence of L D H and diphosphopyridine nucleotide. Gindler (45C) described a new method for the automated determination of serum uric acid by the reductive formation of Lavender Cu(I)-B,b’-bicinchoninate chelate. An automated enzymatic spectrophotometric method for the determination of uric acid in biologic fluids was devised by Steele and Ahnsdorfer (117C). Proteins. Read (101C) described a n automated method for determining serum globulin by the turbidity produced by its precipitation in a sodium sulfate solution. A fully automated biuret method for the determination of total serum protein mas described by Ward and Hirst (137C). Lane and Maurides (7SC) presented a rapid automated biuret method for the determination of protein in concentrations of 20100 mg/ml by use of double strength biuret reagent. Gibbs and Bright (437) measured protein concentrations down to 0.2 pg/ml b y a n automated method based on the Folin and Ciocalteu phenol reagent. Steroids. Thysen et al. (I26C) developed a n automated method for the determination of urinary 17,21-dihydroxy-20-ketosteroids. Godse, Lyall, and Stanler (48C) modified the method of Lyall e t al. for continuous measurement of plasma cholesterol and ex-

tended i t for in vivo monitoring. Levine, Morgenstern, and Vlastelica (74C) applied the direct LiebermannBuchard cholesterol method to the AutoAnalyzer, using a stable one-picee reagent. Eberhagen (29C) recommended Zak’s serum cholesterol method for 2-step automation. Strickler and Stanchak (12OC) applied automated fluorometry to the assay of individual estrogens and their 3-methyl ether derivatives in purified urinary extracts. An improved automated fluorimetric method for the determination of urinary oestriol was developed by Barnard and Logan (E). Toxicology. Syed (122C) and Ellis and Hill ( S I C ) described automated fluorimetric procedures for the enzymatic determination of ethynol in blood, Terfloth and Wuermeling (lZ4C) described a method for continuous breath analysis for blood alcohol. Powell (98C) automated the K 2 C r 2 0 r H 2 S 0 4alcohol method. Stowe and Pelletier (119C) adapted the carbon monoxide method of Levaggi and Feldstein to the AutoAnalyzer for determination of blood CO. Vitamins. Garry and Owen (42C) described an automated screening technique for vitamin C assay in small quantities of blood. An automated colorimetric method for detemination of ascorbic acid which utilizes diazotized 4-methoxy-2-nitroanaline to produce a color directly in biological fluids was developed by Rilson and Guillan (144C). Millbank e t al. (85C) determined folate in serum by an automated method iii which deproteinization was not required. Davis, Nicol, and Kelly (26C) described a new approach to the automated estimation of folate activity in serum and whole blood by use of a chloramphenicol-resistant strain of L. casei as the test organism. Miscellaneous. Bartels aiid l3oehmer (1OC) determined serum bilirubin by a n automated ultramicro method in which an acid solution of diazotized 2-chloro-4-nitroaniline and Fehling solution I1 was used. R e n t and Whitehead (141C) investigated the automat’ed serum COz content determination as a screening test for acid-base state disorders. Spiegel and Tonchen (116C) described an automated method for serum H b which was based on the development of a green chromageii when H b react,s with benzidine arid H202. CONTROL A N D PRECISION OF CLINICAL CHEMISTRY METHODS

Kaiser (200) reviewed quantitation in elemental ana1y.i- under the subjects: description of scientific facts by numbers, informing power of analytical methods, interpretation and application, amount of information required by analytical problem, relation between analytical problem and analytical procedure, pre-information and joint in-


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formation. Kaiser (21D) also covered subjects: analytical calibration function, numerical description of analytical results, semiquantitative analyses, use of statistics for compression of data, concepts of population, probability and consequences, correction of misconceptions, estimation of errors, remarks on statistical confidence, significance and distributions, special role of normal distribution, limit of detection, and guarantee of purity. Barnett (6D) reported t h a t the Standards Committee of the College of American Pathologists presented a statement on medical laboratory accuracy, relative medical significance, state of the art, achievements, and proficiency testing of laboratories to serve as a basis for scientific consideration of the entire problem of laboratory performance. Dybkaer (140) reported that the “Recommendation 1966 of the Commission of Clinical Chemistry of the International Union of Pure and Applied Chemistry” was to reduce the number of usages for presentation of clinical chemical data and to close the existing gap between the nomenclature in clinical chemistry and that of allied fields of science. Dybkaer (130, 15D) also reported for the “Commission on Quantities and Units in Clinical Chemistry and the International Federation for Clinical Chemistry,” that their “1966 Recommendation” consisted of a systematic and thorough discussion of the basic and derived kinds of quantities and their appropriate units of main interest to the clinical chemist. Astrup ( 4 0 ) cited the need for international acceptance of standardization of quantities and units in clinical chemistry. The Public Health Service ( 3 0 ) presented regulations under the “Clinical Laboratories Improvement Act of 1967” which included quality control, yroficiency testing, and procedures in clinical chemistry to assure consistent performance of accurate laboratory procedures and services. Radin, Boutwell, and Hall (290) reported that “The National Reference Laboratory Network” (XRLK) has been organized and is being evaluated to provide intercorrectioii between laboratories to assure that a t least one reference laboratory is common to all separate proficiency testing programs. Rand (300) reported that “The Subcommittee on Spectrophotometry of the Standards Committee of the American Association of Cliiiical Chemists” presented a discussion of spectrophotometric standards for use in the routine clinical chemistry laboratory. Prachenska, Kourilek, and Kyral (28D) reviewed the control of methods used in clinical biochemical laboratories. Barnard, Foy, and hlichelotti ( 5 0 ) delineated the characteristics of various standards such as calcium and lithium carbonate, choles20 R

terol, creatinine, dextrose, glycine, magnesium acetate, potassium monobasic phosphate, sodium and potassium chloride, urea and uric acid. Berry (9D) measured glucose, urea N, Na, K, total protein, and transaminase in both lyophilized and frozen serum pools and found that lyophilized pools offered no advantage. Francis and Sobel (160) presented methods for testing the suitability of models for interval estimate of the ratio of a n unknown to a standard. Reed (31D) reported that more statistical research and further modifications are needed before the average-of-normal method will be useful for quality control in the clinical laboratory. Suggestions were made for reducing the problems of “abnormal” normal in the screening of patients and the “unsatisfactory” satisfactory in proficiency testing of laboratories by Schoen and Brooks (389). Amador and Bartholomew (2D) examined the accuracy of normal ranges (estimate) by the following methods: normal probability graphs, truncated normal graphs, Pryce’s convention, average of normals, unadjusted and adjusted procedures, and composite normal distributions. O’Kell and Elliott (260) developed normal values for use in multitest biochemical screening sera, by screening the sera of over 8000 patients. O’Halloran, StudleyRuxton, and Wellby ( 2 5 0 ) observed marked differences between the inpatient population “normal range” and those derived from either normal healthy subjects or non-renal outpatients. Gindler (18 D ) calculated normal ranges by methods used for resolution of overlapping Gaussian distributions. Becktel ( 8 0 ) presented a simple method for estimating the mean and standard deviation of a Gaussian distribution in the presence of one sided iicontamiiiation.’J Best et al. (10D) recommended the Becktel procedure as a valid and useful method of setting normal limits in the laboratory. Hanson (19D) suggested definitives for clinical laboratory standards and reference methods. Borner, Fabricius, and Marowski (110) preferred bovine serum to pooled human serum as control material for chemical assay of 16 serum constituents because of stability, low cost, and absence of hepatitis. Louderbach and Mealy (220) developed a new clinical chemistry control serum in which the values of 23 components were varied randomly with respect to each other. Gilbert (17D) analyzed the results of the “1969 Comprehensive Chemistry Survey of the College of the American Pathologists” and reported that many methods gave mean values that differ from those of other methods, which is not a constant relationship, and interaction also occurs between constituents. Skendzel (33D)outlined a system of data analysis and proficiency evaluation for the


“College of American Pathologists Survey Program” which included statistical techniques used. Skendzel and Youden (340)designed a graph to indicate which laboratories are having systematic errors or random errors as well as which are reporting acceptable values in a n interlaboratory test survey. Barnett and Youden ( 7 0 ) developed a revised and simplified scheme for the comparison of quantitative methods. A procedure for calculating the so-called total error of a method was developed by McFarren, Lishka, and Parker (240). Padmore and Gatt (87D) examined statistically “between bottle variation” as a source of error in quality control sera and found reconstitution was less than the analytical error of the methods used. Skendzel and Youden (35D) made a statistical study of the frequency of errors in the “College of American Pathologists Survey” and found that systematic errors prevailed over random errors. Allen et al. ( I D ) after a study of analytical bias in a quality control scheme, concluded that only through the evaluation of blind control samples tested a t random times can a reliable measure of the proficiency of the laboratory be achieved. Warren and Scott (36D) surveyed 14 spot test and color reactions selected for their potential applicability for identifying urinary components separated by a high resolution analytical system and found lack of specificity in many tests. Christian (12D) tabulated in a review, the alterations known to occur in blood chemistry determinations following the use of drugs. Two procedures were described for processing analytical data with digital computers without need for preliminary plotting by Margoshes and Rasberry (23D). AMINO ACIDS

Niederwieser and Curtius (26E) reviewed the most important methods currently used for qualitative and semiquantitative determination of amino acids in biological material. Levy et al. (21E, 22E) described screening methods for free amino acids in whole blood and urine. Soda (32E) described the micro determination of D-amino acids with D-amino acid oxidase in the presence of catalase and the spectrophotometric determination of the resultant alpha keto acids with 3-methyl2-benzo-thiazolone hydrazone HC1. Moore (%$E)substituted dimethyl sulfoxide as a better solvent than methyl cellosolve for the reduced form of ninhydrin reagent. A colorimetric method for the detection of trace amounts of w-amino acids utilizing a modification of the Berthelot reaction was described by Kitaoka and Nakano (16E). Peters and Berridge (%’E) reviewed the determination of amino acids in plasma and

urine by ion-exchange chromatography. A single column procedure using type UR-30 resin for the study of amino acids of serum in amiiioacidopathies was described by Uenson, Cormick, and Patterson ( 6 E ) . Wells (@E) described a simple rapid method for the determination of uriiiary alpha amino acids without use of ion exchange resin by cupric ion chelation and quantitation with the yellow complex of 2,9-dimethyl-1 ,lophenanthroliiie. -4 modified columnchromatographic method for adequate separation in one run of the important common and uncommoii amino acids in biological fluids was described by Samyn, Carton, and Hooft (SOE). Ersser (10E) described a simple rapid chromatographic method for detection of aminoacidopathies using prepared thin layers of cellulose on aluminum foil. Tager and Zand (%E) described the use of tartrate buffers for the preparative elution of amino acids from ion exchange columns. Spinella ( S S E ) developed an inexpensive two-dimensional paper chromatographic method for screening children for amino-aciduria. A convenient rapid, high resolution method for two-dimensional amino acid chromatography on micro scale (5 x 5 cm) was described by Wadman, de Jonge, aiid de Bree (38E). Using gasliquid chromatogritphy, Roach and Gehrke (28E) obtained excellent separation of the N-trifluoracetyl-n-butyl esters of 17 amino acids using acid washed Chromosorb as support material. Heathcote and Haworth (14E) determined amino acids on cellulose thinlayer chromatograms by densitometry. Culley (8E) described a simple and rapid thin-layer chromatographic method using serum directly for screening of inborn errors of amino acid metabolism. Bremer e t al. (7E) described methods for thin-layer chromatography of uriiiary amino acids which permitted the diagnosis of most metabolic disorders associated with abnormal amino acid excretion. Pasieka e t al. ( M E ) described how most serum and urine amino acids can be separated semiquantitatively by high voltage electrophoresis. Testaferrata, Zammarchi, and Pierro (S7E) used high voltage electrophoresis for the identification of amino acids in phenylketonuria and cysteinuria. A screening method for the detection of specific aminoacidemias in which whole blood dried on small sheets of filter paper was examined b y one-dimensional ascending paper chromatography developed in two subsequent solvents was described by Szeinberg, Saeinberg, aiid Cohen (S5E). Merck (2SE) patented a new procedure for diagnosis of phenylketonuria and other amino acid disorders in which serum or blood was analyzed by thin-Iayer chromatography on Avicel R layers with 80y0 EtOH. Stott (%$E)

described a chromatographic technique for the detection and confirmation of phenylketonuria. Kojima and Wacker (19E) reported a method for the measurement of asparagine in serum and urine with Escherichia coli L-asparaginase. A procedure using a column of Dowex-50 resin at p H 2 for the absorption of cystein from urine for colorimetric determination with 1,2-naphthoquinone-4-sulfonatewas described by Haux and Natelsoii (13E). Sardesai and Provido ( S I E ) described a procedure for the fluorometric determination of glycine in biological fluids in which glycine is degraded in proteinfree filtrates to formaldehyde by Chloramine T. Klasoii (17E) reported a convenient and practical fluorometric method for the determination of histamine in blood. Hassall (12E) described a new method for L-histidine in biological fluids in which spectrophotometric determination of urocanate formed from histidine by histidine ammonia-lyase was made. Gerber (11E) and Ambrose e t al. ( 2 E ) described fluorometric methods for the measurement of histidine in serum based on the condensation of histamine with 0-phthaldialdehyde in NaOH. A method for the determination of hydroxyproline in collagen-like proteins of plasma and in urine was described by Krel and Furtseva (,$WE). Koevoet and Baars (18E) compared three methods for the determination of hydroxyproline in urine to check the inhibitory influence of some urinary constituents on analysis. hiold, Hvidberg, and Rasmussen (5E) developed a procedure for the separation of hydroxyproline containing fractions in blood serum by Sephadex G-200. A simple screening method for detecting isovaleryl-glycine in the urine of neonatyls was described by Ando and Nyhan (4E). Ambrose (1E) improved and shortened the fluorometric determination of phenylalanine by increasing the initial incubation from 60 to 85 "C and decreasing the reaction time from 2 hours to 16 minutes. Jellum e t al. (15E) determined serum phenylalanine by a gas-liquid chromatographic method based on conversion of the amino acid to the volatile neopentylideiie methyl ester derivative by esterification and condensation with 2,2-dimethyl-propanol. Robins (29E) reviewed the application of experimental methods to clinical studies in the measurement of phenylalanine and tyrosine in blood. Wapnir and Stevenson (%E) determined plasma tryptophan by extraction of spots on filter paper with dilute ethanol and spectrofluorimetry in alkaline medium. Ambrose e t al. ( S E ) modified the fluorometric method for the determination of serum tyrosine to eliminate ethylene dichloride extraction and improve the stability of the reaction mixture by the use of phosphoric acid

reagent. A simplified method for column chromatographic quantitation of tyrosine and phenylalanine in physiological fluids was presented by Efroii (9E) for study of inborn errors of metabolism. BLOOD CLOTTING FACTORS, GASOMETRIC ANALYSIS, A N D pH

Rosenfeld (12F) determined plasma fibrinogen spectrophotometrically by control of the physicochemical parameters affecting clot absorbance and fibrin mass. Phillips, Jenkins, and Hardaway (10F) compared fibrinogen determination by thrombin coagulation and sodium sulfite precipitation and reported that the latter method gave consistently higher results. Jesenovec and Ramsak ( 6 F ) described a fast method for the determination of plasma fibrinogen in which thrombin solution of fibrinogen was changed to fibrin a t 37 "C and the fibrin coagulum dissolved a t 60 "C in biuret reagent. Schroer (15F) reviewed the determination of prothrombin coilsumption and factors V, VI, -I, and X. Oberhardt (8F)determined prothrombin time by use of a patented apparatus consisting of a closed chamber containing a heat source and a removable top for inserting test tubes of plasma with a light source to measure clotting as i t proceeds with a photocell. Standardization procedures for determination of prothrombin time were described by Zuker e t al. (2OF). Hamilton ( S F ) reviewed the chemistry, physics and measurement of blood gases; methods of respiratory gas analysis including mass spectrophotometers, infrared absorption, emission radiation, and various gas analyzers. Burton ( I F ) reviewed the chemical and physical methods of analytical measurement of inspired and expired oxygen aiid COS. A gas chromatographic method for determination of COz, OS, and N z in blood was described by Efuni and Sametskaya ( 2 F ) . Sanz and Staunton ( 1 S F ) described a disposable transfusion cell for the determination of pC02 and pOz values in 15-p1 blood samples. Hill (48') patented a COS analyzer which consisted of a plastic or glass reaction tube having Cas04 supported by glass wool plug a t the bottom and packed with rolled filter paper which has been soaked in saturated oxalic acid in MeOH and dried. Peterson (9F) described a rapid infrared micro method for the determination of carbonate in 50 pl of serum. Sastry, Hamm, and Pool ( l 4 F ) investigated the use of trans-1,2-diaminocyclohexane tetra-acetate complex of Mn(II1) for the determination of dissolved oxygen in water. Siest et al. (16F) compared spectrophotometric, reflectometric and manometric methods for measuring 0 saturation of blood.


21 R

Webb and Troutman (18F) devised a n instrument, the “Metabolic Rate Monitor” for the continuous measurement of oxygen consumption in man. Pita ( 1 1 F ) described an electrometric microcell and a thin layer of oil combined in two assemblies to permit the determination of p C 0 ~and p H in biological samples. Zimmerman and Breithaupt (19F) determined pCOz of blood directly with glass electrode and indirectly by manometric and colorimetric methods and studied the source of error in the determination of acid-base equilibrium. Smith and Hahn (17F) reviewed electrodes for the measurement of oxygen and COZtensions. Holmes, Green, and Lopez-hIajano (6F) evaluated methods for calibration of 02 and COz electrodes. Neff and Sambucetti ( 7 F ) patented a n apparatus for measuring hydrogen ion concentration which consisted of an indicating electrode, a reference electrode and a detecting voltmeter for measuring the potential difference generated between the two electrodes. CARBOHYDRATES

BeMiller (3G) reviewed enzymic methods for the determination of carbohydrates. Guilbault, Sadar, and Peres (14G) described fluorometric methods for the determination of lactose, maltose, fructose, and glycogen. Young and Jackson (S4G) after comparing ten procedures for thin-layer chromatography of carbohydrates in urine, and finding considerable differences in sensitivity and ability to separate sugars, developed a procedure for identification of several sugars without prior desalting or concentration. Jolley et al. (17G) evaluated a high resolution column chromatographic system for the determination of carbohydrates in urine and serum. Glucose. Martinek (23G) made a broad review of glucose methods. Ware (33G) and Aw ( I G ) used iodine and molybdate to develop cromagens in the glucose oxidase glucose method. Levee (22G) suggested a p H of 1.5-1.7 to maintain greatest final color stability in the glucose oxidase-peroxidase-0 - dianisidine method. Lenz and Passannante (22G ) described a rapid glucose oxidaseperoxidase ultramicro method for the determination of blood glucose. Kristal and Pickman (20G) and Boehringer (5G) patented blood and urine glucose methods based on the glucose oxidaseperoxidase method. Trinder (SOG) described manual and automated methods for blood glucose using an oxidaseperoxidase system and dl-adrenaline, a noncarcinogen, as 0 detector. Van der Heiden (32G) studied the influence of Brij-35, Tween-2O-Sterox, and Peristone on color formation in the enzymic assay of glucose. A new polarographic method for the determination of glucose 22 R

by measurement of 0 consumption after the addition of glucose oxidase was developed by Okuda and Okuda (25G) and Kadish and Sternberg (18G). Ujvarosi and Ruse (31G) compared the 0-toluidine and enzymic methods for the determination of glucose in cerebrospinal fluid. Carroll, Smith, and Babson (6G) developed a colorimetric serum glucose assay utilizing the hexokinase, glucose-6-phosphate dehydrogenase reaction with the reduction of a tetrazolium salt. Deutsch (9G) patented a serum glucose method utilizing hexokinase in a reagent tablet. Sitzmann and Eschler (27G) determined glucose in blood with a modified enzymic hexokinase method without deproteinization. Ceriotti and de Nadai (7G),and Ceriotti and Frank (8G) improved the 0toluidine colorimetric glucose reagent by incorporating 0.25% thiourea and 35% water. Goodwin (13G) made direct simultaneous estimation of glucose and xylose in serum by intensifying their reaction to 0-toluidine with borate. Bierens de Haan and Roth (4G) used a solution of glycolic acid in a mixture of benzyl alcohol and hexamethyl phosphoric triamide to replace acetic acid in the glucose 0-toluidine reagent. Haertel and Lang (16G) substituted hydroxycarbolic acid for acetic acid in the 0-toluidine glucose reagent. Golikov (l2G) made a detailed evaluation of the 0-toluidine sugar method and found interference from mannose, galactose, fructose, sucrose, and lactose. Thompson (29G) suggested the use of noncarcinogenic reagent diethyl-p-phenylenediamine for 0-tolidine in the glucose reagent. Klein and Lucas (19G) developed a new procedure for the determination of serum glucose based on a coupled ferricyanide redox reaction with the formation of the violet blue Fe(II)-5-pyridyl- benzodiazepin-2 -one chelate. Barnett and Cash ( I G ) evaluated “Kits” used for clinical chemistry analysis of glucose, and found only 5 of 17 “Kits” acceptable. Miscellaneous. Fraser (10G) patented a test paper for the determination of galactose which was impregnated with galactose oxidase, a peroxidase, and a color indicator. A method for the enzymic determination of galactose in 10 pl of whole bood without deproteinization was described by Stork et al. (28G). A simple sensitive specific assay method for erythrocyte, galactose-1-phosphate based on the liberation of galactose by alkaline phosphatase and its oxidation by galactose dehydrogenase was described by Gitzelmann (11G). Mier and Wood (24G) developed a method for the isolation, fractionation, and assay of tissue and mucopolysaccharide. Pennock, Mott, and Batstone (26G) selected a simple turbidity test using cetylpyridinium


chloride in citrate buffer as a method for screening for excess mucopolysaccharide in children’s urine. Harris (16G) described a method for the determination of D-xylose in urine based on the specific absorption of the pentose 0-toluidine complex. CATIONS AND ANIONS


Pfordte and Ponsold

(34H) compared the serum calcium

methods: oximetric, flame photometer, and two colorimetric methods: chloranilic acid and Eriochrome Blue SE, and found the latter easy and rapid. King and Buchanan ( 8 1 H ) compared a manual titrimetric EDTA-calcium, an automated fluorometric EDTA-calcium, and the atomic absorption technique with the Clark-Collip method for the determination of urinary calcium and found the atomic absorption best. Robertson ( 4 1 H ) described three methods for the determination of ionized calcium in plasma ultrafiltrates and urine, involving spectrophotometry, potentiometry, and a computer calculation procedure. Savory, Wiggins, and Heintges (46H), Muranaka et al. (29H), and Pybus (37H) described improved methods for the determination of calcium and magnesium in serum and urine by atomic absorption spectrophotometry. Sideman, Murphy, and Wilson (47H) studied inter-laboratory variability in the determination of serum calcium by atomic absorption spectrometry and noted a need for further methodologic standardization. A new electrochemical method using a calcium-specific electrode for the anaerobic determination of serum ionized calcium was presented by Hattner e t al. ( 1 5 H ) . Sach e t al. (43II) determined Ca2+ electrometrically in a flow-through system by use of a liquid membrane electrode and a cation exchange resin highly selective for calcium. Farese, Mager, and Blatt ( 8 H ) described a rapid simple procedure for separating diffusible calcium from protein bound serum calcium by centrifuging through high flux, ultrafiltrate membranes. DeWitt and Parsons ( 6 H ) , Lewin, Wills, and Baron (26H), and hfoser and Gerarde (2811) described improved micro methods for the fluorimetric determination of serum calcium. Puchtler, Meloan, and Terry (36H) described the history and mechanism of alizarin and Alizarin Red S stains for calcium. H a m (14H) modified the chloranilic acid calcium method of Ferro and H a m by changing both sodium chloranilate and the wash reagent. Alonso, Tumilasci, and Nikonov ( 2 H ) increased the sensitivity of the Kingsley-Robnett Plasmo-Corinth B serum calcium method by increasing the concentration of alkaline dye solution. Fraguda and Le Beau ( 9 H ) patented a sodium rhodizonate reagent

which reacts specifically and quantitatively with serum Ca2+ to produce a tightly colored compound. Halogens. R u p e (@H) patented a dip and read test strip chloride ion indicator. Sakaguchi and Taguchi (44H)developed a new method for the microspectrophotometric determination of chloride ion with 4,4’-bis (dimethylamino) thiobenzophenone. Kupke and Sauer (22H) described the spectrophotometric microdetermination of chloride in serum as a hexachloroferrate (111) complex. Singer, Armstrong, and Neefus, Cholak, and SaltzVogel (48H), man (SOH), Armstrong ( S H ) , and Ke, Regier, and Power (2OH) determined fluoride in biological fluids with fluoride-specific ion electrodes. Ionescu ( 1 9 H ) described a modified variant of the colorimetric method with Zr-Eriochrome Cyanine R for the determination of fluorine in urine. Paletta and Panzenbeck ( S 2 H ) tested the applicability of an iodide ion sensitive electrode for the specific determination of iodine in certain organic substances in biological material. Lithium and Magnesium. Pybus and Bowers (38H) determined lithium by atomic absorption in 1:lO serum dilutions using standards and blanks containing physiological amounts of sodium and potassium. Levy and Katz ( 2 4 H ) reported that flame photometry was more sensitive, slightly less precise, but easier to use, than atomic absorption spectrophotometry for serum lithium determination. Alcock ( 1 H ) reviewed the development of methods for the determination of magnesium in biological fluids. Hunt and Manery ( 1 8 H ) used Dowex 50 resin to prepare erythrocytes for magnesium determination. Phosphorous. Martinek (26H) reviewed methods for the determination of inorganic phosphorous in biologic materials. A specific assay method for inorganic pyrophosphate in which a coupled enzyme system catalyzes the oxidation of N A D H equivalent to the added pyrophosphate was developed by Reeves and Malin (4OH). Parekh and Jung ( S S H ) described a new reducing agent, p phenylenediamine-2 HC1 for the determination of inorganic P in serum. Hokl and Minks ( 1 7 H ) proposed a modified method for the determination of inorganic P in serum by carrying out the reduction of phosphomolybdate with ascorbic acid in the presence of Sb ions. A rapid simple microprocedure for the determination of both pyrophosphate and orthophosphate was developed by Grindey and Nichol ( I S H ) . Goodwin ( I I H ) devised a direct method for the determination of phosphorous in biologic fluids by forming phosphomolybdate in the presence of borate and reduction with ascorbic acid. Gindler and Ishizaki (11H) determined inor-

ganic serum P without removal of protein by the use of a mixture of Bion NE9 (9 ethylene-oxide unit adduct of p nonylphenol), p-methylammonium phenol sulfate, and sodium bisulfite. A new, more sensitive method for the determination of P as molybdophosphate using Triton X-100 instead of reducing agents was described by Eibl and Lands ( 7 H ) . Potassium and Sodium. Wise, Kurey, and Baum (49H), Pioda et al. (35H),and Frant and Ross (1OH)determined serum potassium with liquid membrane ion exchange electrodes. Lazarou ((ZSH) determined K in serum by a colorimetric method based on the precipitation of K as the complex salt, K2Me2+ Cu(NOz)e (Me2+ may be Ca2+, Srzf, Ba2+, or Pb2+). Bugyi et al. ( 4 H ) described a method for measurement of Na and K in erythrocytes and whole blood to obtain greater precision in order to increase the chemical significance of these analyses. Sanui and Pace (45H) determined corrective measures to permit accurate atomic absorption measurement of Na, K, rubidium, and cesium. Hieftje and Malmstadt (16H) used flame spectrometry with isolated droplets for direct analysis of serum Ca and Na using simple water dilution of sample. Ramirez-Munoz (S9H) determined the limiting interference ratios, and established some recommendations in making atomic absorption measurements in the presence of high concentrations of Na. Coetzee and Rohwer ( 6 H ) described the separation of Na and K by means of ammonium molybdophosphate cation-exchange columns for flame spectropho’tometric determination. The preparation of an ultra-micro open end capillary glass electrode for N a + determination in biological fluids was described by Ohara and Newton (S1H). Miyada, Kazuko, and Matsuyama ( 2 7 H ) developed a potentiometric electrode system for the direct measurement of K and Na concentrations in biologic fluids. LIPIDS

Ameiita ( S J ) presented a rapid analytical system for the quantification of total lipid and lypid fractions in blood and feces. Adkiaenssens et al. ( I J ) described a simple mass screening technique for hereditary metabolic diseases using blood collected on filter paper. Firings and Dunn ( 9 J ) determined serum total lipids by a colorimetric method based on the sulfophospho-vanillin reaction. Cheek and Wease ( 8 4 made quantitation of total lipids from cholesterol, phospholipid, and triglyceride determinations and compared it to the phenol turbidity and gravimetric methods. Wildgrube, Erb, and Boehle ( I I J ) determined serum phospholipids triglycerides, mono-

glycerides, free fatty acids, and free cholesterol esters by quantitative onedimensional thin-layer chromatography. Buckley et al. ( 6 J ) reported that direct thin-layer chromatography of serum was the most practical method for rapid visual quantitation of triglycerides and free and cholesterol esters in hyperlipidemia screening. Kanno and Hirabayashi ( I I J ) extracted serum lipids with C H C k M e O H and separated them by thin-layer chromatography for the densiometric determination of color spots developed by dichromate. New methods for analyzing blood and plasma lipids by chromatography and infrared spectrophotometer were described by Nelson ( 1 4 4 . Itaya and Kadowaki (1OJ)described an improved colorimetric method for the determination of free fatty acids of serum in which wavelength was changed from 440 mp to 600 n ~ pby use of bicyclo-hesanoneoxalyldihydrazone instead of diethyldithiocarbamate for the color development reagent. A simple gas chromatographic technique for the quantitation of free fatty acids (6 carbon atoms or less) in biologic fluids was described by Perky et al. ( 1 5 4 . Caster ( 7 4 made a critical evaluation of the gas chromatographic technique for identification and determination of fatty acid esters. Mlytz and Methfessel (12 4 determined serum nonesterified fatty acids by using Cuprotest as a color complex forming agent. An improved procedure for the quantitative determination of phospho-lipids in serum by combined thin-layer chromatography and phosphorous analysis was described by Williams, Kuchmak, and Witter (WWJ). Adams and Sallee ( 1 J ) separated serum phospholipids by thin-layer chromatography on Adsorbosil-5 silica gel. Rosenthal and Han ( I N ) determined glycero phosphatide phosphorous in blood ether-methanol estracts by nondigestion using sulfuric-periodic acid reagent. Berner and Guhr ( 4 4 determined free and bound glycerol in preparations of water soluble, finely emulsified, and water insoluble monoglycerides by both an enzymic and chemical assay methods. Seitz and Tarnowski ( 1 7 4 made enzymic determinations of glycerol in serum and organ estracts. Witter et al. (23J)found most commercial lyophilized preparations unsatisfactory as secondary serum cholesterol and triglyceride standards. Timms et al. (2OJ) modified the Loflands colorimetric semi-automated serum triglyceride method and assessed it by a new specific enzymic method. Stole, Rost, and Honigmann (19J) described a micro serum triglyceride method in which serum phosphat,ides are separated on Kieselgel, the triglycerides saponified, the glycerol oxidized to H COOH with N a I 0 4 , and the HCHO transferred into diacetyldihydrolutidine with acetyl


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acetone and NH3. An improved, simplified technique for direct determination of triglyceride concentrations in serum was presented by Smernoff, Murphy, and Kameda (185). Zoellner, Wolfram, and Wolfram (245) analyzed glycerol liberated from plasma by the 3-step glycerokinase-pyruvate kinaselactate dehydrogenase system. Matsumiya, Okishio, and Omori ( 1 S J ) determined serum triglyceride by an enzymic method based on measurement of the decrease in N A D H due to the enzymic reduction of pyruvate derived from glycerol liberated from triglycerides by glyceral and pyruvate kinases. Brady ( 5 5 ) recommended the use of radiolabeled lipids for the detection of nine known sphingolipid storage diseases. ENZYMES

Guilbault (20K) reviewed the use of enzymes in analytical chemistry under the titles; books and reviews, detection of enzymes, determinat,ion of substrates, coenzymes and activators, inhibitors, immobilization of enzymes, and automation of enzyme systems. Wilkinsoii (6'7K)discussed the clinical significances of enzyme measurement in diagnosing myocardial infarction, liver disease, enzymic release into circulation and removal from circulation, and precision of enzyme tests. Wacker and Coombs (64K) reviewed enzymatic methodology used in the study of genetic diseases with known enzymatic defects. Monk and Wadso (43K) determined enzyme activities of glucose oxidase, cholinesterase, alkaline phosphates, L D H , and ATPase activity in tissue homogenates by flow microcalorimetry. Bergmeyer, Bernt, and Rey ( 9 K ) pat.ented enzyme assay reagents. Moss (44K) cited the need of systematic and sustained research in the standardization of enzyme assays. Darrow and Amador (16K) evaluated eight brands of commercial sera with normal and high enzymic activities and found most of the stated activities of the sera were not reliable for standardization or quality control. Posen (48K) reviewed the dynamic equilibrium and turnover of circulating plasma enzymes of interest to bhe clinical chemist. Roth (54K) made a detailed review of the fluorimetric assay of enzymes including instrumeutation, principle of assay, and the assay of many enzymes. Amylase. Ceska, Birath, and Brown (12K) described a new, rapid method for the det'ermiiiation of alphaamylase in biological fluids based on the breakdown of the blue starch polymer by amylase. A new serum amylase substrate which was prepared by coupling Reactone Red 2 B to amylopectin in alkaline solution was described by Babson , Tenney, and Megraw ( 6 K ) . Lorentz and Oltmanns (S8K) 24R

determined serum (0.02 ml) amylase activity by measuring the glucose and maltose hydrolyzed from soluble starch b y measuring colorimetrically the reduction of colorless triphenyltetrazolium chloride to a red formazan. A new serum chromogenic amylase substrate, Cibachron Blue F 3 GA-Amylose, was investigated by Dalal and Winsten (14K), Klein, Foreman, and Searcy (SOK, SZK) and Take, Berk, and Fridhandler (68K). Klein, Foreman, and Searcy (S1K) synthesized a new chromogenic amylase substrate which was prepared by treating starch with a blue chlorotriazine dye in alkaline solution and developed a simple rapid procedure for serum amylase activity. Hall et al. (ZSK) and Pragay, Chilcote, and Least (49K) described a n improved serum amylase method using a new starch substrate, Remazolbrilliant blue starch. Jamieson, Pruitt, and Caldwell (27K) improved the accuracy and sensitivity of the Bernfeld's method for amylase assay b y reducing the concentration of 3,5-dinitrosalicylic acid in the color reagent, increasing the incubation temperature, and making other changes. Fridhandler and Berk (19K) adapted the saccharogenic serum amylase assay method, in which amylopectin b a s used as substrate, to an automated procedure. Other Enzymes. Pinto, Van Dreal, and Kaplan (47K) made a critical evaluation of the Sigma and Boehringer reagent kits for measuring aldolase activity b y a colorimetric method and found the original procedure more satisfactory, and also made a critical study (46K) of two commercial aldolase kits employing a UV method and found neither satisfactory. An optimal substrate for the determination of serum and tissue alanine aminotransferase was developed by Arvan and Coyle ( S K ) . Loeb and Stuhlman (S7K) demonstrated arginase activity b y determining the decrease in arginine concentration in a reaction mixture by the method of Sakaguchi. Campanini et al. (11K) reported that the measurement of serum argininosuccinate lyase was a more specific and sensitive test for disease of liver parenchyma than commonly used liver function tests. A simple highly sensitive fluorimetric method for the determination of serum arylsulfatases in which 4-methylumbelliferone sulfate was used as substrate was described b y Rinderknecht et al. (6SK). Van Munster et al. (6SK) developed a new method for the determination of serum carnosinase activity using L-carnosine(IW) p-alanyl as substrate. Redalieu et al. (50K) described two new assay methods which eliminate the saponification step for the quantitative extraction of coenzyme Qlo(CoQlo) from human blood. Wilkinson and Steciw (69K) evaluated a single tablet in which all reagents were compressed for mea-


suring serum creatine kinase activity (CPK). Koedam (SSK) modified a serum creatine phosphokinase fluorometric method. Menache and Gaist (41K) used alkaline E D T A and AgN03 to eliminate side reactions of the -SH group of the activator and those of the heavy metals in the determination of serum CPK. Crowley and Alton (1SK) compared three colorimetric and one spectrophotometric CPK methods and found the latter provided greater precision and consistency with clinical data. Ressler (51K)described a method for assay of deoxycytidylate deaminase activity based on the measurement of ammonia formation by means of the Berthelot reaction. Guilbault and Hieserman (21K) described fluorescent methods for assay of D- and L-amino acid oxidase based on the conversion of the nonfluorescent homovanillic acid to the highly fluorescent 2,2'-dihydroxy3,3'-dimethoxybisphenyl- 5,5' - diacetic acid. Dror et al. (17K) presented a rapid, sensitive spectrophotometric method for the determination of the activity of glucose-phosphate dehydrogenase in liver and blood. A simple screening test for glutathione peroxidase activity in red cells based upon the defluorescence of NADPH was devised by Boguslawska-Jaworska and Kaplan (10K). Russell, Tougas, and Taylor (56K) described a rapid assay method for the glycogen cycle enzymes, glycogen synthetase, and alpha-glucan phosphorylase in muscle. Al-Khalidi et al. ( 1 K ) described a method for the determination of guanase in biological fluids based on the measurement of radioactive uric acid produced upon incubation of the sample with radioactive guanine and an excess xanthine oxidase. Kulhanek (36K) patented a diagnostic agent for the determination of total activity of lactic acid dehydrogenase (LDH) isoenzymes in serum. Homer, Yott, and Lim (26K) described a more satisfactory method for the separation and demonstration of L D H isoenzymes utilizing Beckmaii Instrument's Microzone equipment. Wilkinson (66K) reviewed the structure of isoenzymes, properties of L D H isoenzymes, L D H isoenzymes in diagnosis, alkaline phosphatase isoenzymes, and other isoenzymes. A more sensitive fluorometric method for measuring leucine aminopeptidase activity in RBC, serum, or tissue in which p-naphthylamide was used was described by Uete, Tsuchikura, and Ninomiya (60K). Ayavou (4K) described a colorimetric method for the determination of leucine amino peptidase activity in serum. Gilbault and Hieserman ( d 2 K ) described a new fluorometric substrate for assay of lipase and sulfatase in which N-methyl iridoxyl myristate was used for the former and 4-methyl umbelliferone sulfate and 6naphthol sulfate was used for the latter.

Mahadevan, Dillard, and Tappel (39K) modified the Duncombe method for the measurement of free fatty acids for lipase assay by substitution of diphenyl carbohydrazide for diethyldithiocarbamate color complexiiig agent to increase sensitivity. Reider and Otero (52K) described a simple procedure for assay of serum 5'-nucleotidase which eliminated the need for removal of protein. ii sensitive colorinietric assay for 5'nucleotidase activity in which adenosine formed by hydrolysis of 5'-adenylic acid is deaminated enzymatically and the ammonia determined by the Berthelot reaction was described by Belfield, Ellis, and Goldberg ( 7 K ) . Whitaker (65K) described two color reactions for the determination of N'ADH, a colored formazan and a colored F e z + dipyridyl complex. Kupfer and hlunsell (34K) determined NADH and N A D P H by a reaction of the reduced pyridine nucleotide with p-dimethyl aminobenzaldehyde in weak acid solution which produced a color for spectrophotometric measurement a t 510 mp. hliller and Ashe (42K) reported that fluorometric activities of NADH and N A D P H are invalidated by the presence of significant quantities of bilirubin and temperature stabilization is critical. A specific assay for N A D P H in which the change in fluorescence due to the addition of a N A D P H specific enzyme was followed was described by Ben-Hayim, GrometElhanan, and Avron ( 8 K ) . Kaplan, Shore, and Beutler (29K) described a rapid fluorescent method for the estimation of triosephosphate isomerase. Hines, Love, and Peart (24K) used purified rabbit skeletal muscle apophosphorylase b in a new UV assay technique for the determination of serum and blood pyridoxal phosphate. Uete, Wasa, and Shimogami (61K) carried out a simplified method for the determination of pepsinogen in blood and urine using the patient's own serum protein as a substrate a t pH 2. Szasz (67K) described a kinetic photometric method for serum gamma-glutamyl transpeptidase. Uete, Aqahara, and Tsuchikura (59K) measured fluorimetrically the activity of crystalline trypsin and trypsin-like amidase activity and the activity of trypsin inhibitors in serum with alpha-benzoyl-Larginine-p-naphthylamide as substrate. Van Handel (62K) presented a method for the evaluation of serum trehalase which depends on the determination of glucose produced from trehalose after incubation with serum. Phosphatase. Wolf, Dinwoodie, and Morgan (71K) measured alkaline phosphatase activity of liver, bone, intestine, and bile extracts by the pnitrophenylphosphate, phenolphthalein, monophosphate, @-naphthyl phosphate, and phenyl phosphate methods and found the latter had equal sensitivity

for all tissues. Wilkinson, Boutwell, and Winsten (68K) presented data from three laboratories in the study of a new systematized, kinetic method for the determination of alkaline phosphatase. The use of sodium thymolphthalein monophosphate for measuring alkaline phosphatase activity in serum was described by Roy (55K). An investigation by Amador, Price, and Marshall ( 2 K ) indicated that the alphanaphthyl phosphate substrate for prostatic acid phosphatase was not specific for this enzyme. Nagode, Koestner, and Steinmeyer (45K) identified organ specific alkaline phosphatase isoenzymes from liver, bone, kidney, and intestinal mucosa of dogs by polyacrylamide gel disk electrophoresis in combination with L-phenylalanine inhibition, and heat (56 "C) and 6M urea inactivation tests. Johnson (28K) developed a new fluorometric method for the estimation of total and fractional alkaline phosphatase. Fitzgerald, Fennelly, and McGeeney (18K) used a rapid standardized heat inactivation procedure to determine whether an elevated serum alkaline phosphatase was of hapatobiliary or skeletal origin. Maruna (,$OK) described a new' method for urine phosphatase isoenzyme fractionation by thin-layer starch gel electrophoresis. Transaminase. Winsten, Wilkinson, and Boutwell (7OK) evaluated a new system for the kinetic measurement of serum glutamic oxaloacetic transaminase (SGOT). Lippi and Guidi (36K) presented a new ultramicro method for colorimetric determination of SGOT and serum glutamic pyruvic transaminase (SGPT) activity based on the use of glutamate dehydrogenase for enzymatic est,imation of glutamate formed in which a diazonium salt is reduced. Itoh and Srere (26K) described a new method for measurement of GOT activity by condensing oxaloacetate formed with acetyl CoA to form citrate and CoA in a system coupled with citrate synthase and the CoASH formed was measured by its reaction with 5,5' - dithiobis - (2 -nitrobenzoic acid). Doumas and Biggs (16K) described a method for the determination of SGOT in which oxalacetate formed is quantitated by reaction with 6-benzamido-4-metlioxy-m-toluidine diazonium chloride. Babson, Amdt, and Sharkey ( 5 K ) critically evaluated the colorimetric SGOT methods based on the reaction of oxalacetic acid with diazonium salts and revised the procedure to obtain a simple and more specific method. FUNCTION TESTS

Liver. Martinek (17L) reviewed methods for the determination of bilirubin in biologic fluids. Hargreaves

(7L) obtained complete coupling of bilirubin and diazobenzene-p-sulfonic acid in neonates with high serum bilirubin when half of the recommended amount of serum was used. Sudmanii (27L) described a spectrophotometric micromethod for bilirubin in serum of newborn in which hemolysis is corrected by subtracting absorption a t 575 mp from that of bilirubin a t 455 mp. Four methods for the determination of bilirubinoids in amniotic fluid were compared by Kulenda and Prochazka (11L). Mallikarjuneswara, Clemetson, and Carr (16L) developed a new, simple and efficient method for the determination of bilirubin in amniotic fluid. Lalive et al. (15L) determined serum bilirubin by an ultramicro system requiring only 0.02 ml serum sample. Fried and Hoeflmayr (6L) using a patented method determined body fluid bilirubin by comparing the diazosulfanilic derivative of bilirubin in alkaline medium with an aqueous solution of 0.2-1.0 mg yo Evans blue dye. Green ( 6 L )patented a reagent for the determination of bilirubin which contained the borofluoride salt of p-diazonium benzenesulfonic acid. Kulhanek and Appelt (12L) patented a reagent containing diazotized 4-chloro-2-aminoanisol for bilirubin determination. Thompson (28L) avoided higher results for conjugated bilirubin than for total bilirubin in the method of Michaelsson by adding the accelerator diphylline, 4 minutes after the diazo reagent is added. Iwata ( 9 L ) prepared a reagent by diazotization of sulfanilamide for the rapid determination of serum total bilirubin. Bartels and Boehmer (1L) determined bilirubin colormetrically with a stabilized 2chloro-4-nitroaniline as the diazo component a t p H 2 using ultravone as a stabilizing agent. Dybkaer and Hertz (3L) enriched human serum by using bilirubin in cyanide-formamide t o prepare a reference for bilirubin determination. Perrelli and Watson (21L) compared the Weber-Schalm with the Ducci-Watson method for the determination of conjugated and nonconjugated serum bilirubin. Kurashige (13L) improved the solvent-partition method for the measurement of free and conjugated bilirubin in serum. Winsten and Cehelyk (29L) evaluated a new commercial reagent for the rapid determination of total bilirubin which consisted of a tablet containing diazotized sulfanilic acid and a liquid reagent containing 30% dimethyl sulfoxide. Shioda, Wood, and Kinsell (25L) developed a method for the determination of 6 major conjugated bile acids in bile. Jablonski and Owen (1OL) reviewed the clinical chemistry of bromsulfophthalein and other cholephilic dyes for the diagnosis of liver disease. Fischl et al. (41,) described a rapid


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method of electrophoresis on a membrane filter for separating porphyrin fractions for subsequent spectrophotometric determination. Mundschenk (2OL) analyzed complex porphyrin mixtures by solvent-solvent extraction in combination with thin-layer chromatography and absorbance spectra measurements. Miles Laboratories (19L) obtained a patent on a filter paper color test for detection of porphyrins in biological fluids. Hawkins (8L) preferred blood to urine in performing the pediatric xylose absorption test. Kidney. Bartels and Cikes (WL) showed that several currently used routine creatinine methods are in error in that a high portion of “chromagens” found by some methods actually consist of protons. Seelig and Wuest (RdL) reported that special care is required to obtain the same final NaOH concentration in the blank, standard, and sample in determining creatinine by the Jaff6 reaction. Because of the presence of chromogenic substances in serum, Kushiro et al. (14L) reported the determination of serum creatinine content by the Jaff6 reaction gave 10% higher values a t 490 mp than a t 540 mp. A new procedure based on the separation of creatinine from other urinary components by Sephadex gel filtration and subsequent measurement a t 235 mp against a phosphate buffer blank which showed no interference was described by Sinha and Gabrieli (86L). Scheuerbrandt and Helger (2%) substituted LiOH for NaOH in the alkaline picrate reagent for the determination of serum creatinine. Merck (18L) patented a new color reagent for the photometric determination of serum creatinine which is composed of 1 part of 10% LiOH solution and 5 parts of lithium picrate. Ramakrishnan (22L) described a method for the quantitative determination of creatine in urine using the reagents: 10% NaOH, 1%alpha-naphthol in ethyl alcohol and alkaline NaBrO prepared by dissolving 1 ml of bromine in 225 ml of N NaOH. HEMOGLOBIN

Martinek (5il.I) reviewed methods for the determination of hemoglobin (Hb) in human blood. Schrumpf ( 9 M ) reviewed the method recommended by the European and International Societies of Hematology for the determination of Hb in blood and discussed and stressed some practical points. Matsubara and Susumu ( 6 M ) evaluated the Internationally Standardized Method for Haemoglobinometry and found it satisfactory. Frazini (ZM) prepared a concentrated standard of azide-methemoglobin of H b determination which was stable if stored cold. Van Assendelft, Van Kampen, and Zijlstra ( 1 O M ) presented data in which hemi26R

globin azide (HiN3) and hemiglobin cyanide (HiCN) methods were compared for the determination of hemoglobin and reported that HiCN could not be used because of its narrow absorption band. Weichselbaum ( I 1 M )patented a hemolyzing reagent for performing hemolysis of blood cells. Lee and Ling (3M) colorimetrically microdetermined the H b content of serum or plasma by utilizing the peroxidase reaction of H b with chlorpromazine. Maas, Hamelink, and de Leeuw ( 4 M ) evaluated the spectrophotometric determination of HbOz, HbCO, and H b in blood with the GOoximeter I L 182. Nalbandian et al. ( 7 M ) presented data for a simple inexpensive specific test for detecting S-Hb based on a unique molecular anomaly of S-Hb. Roy et al. ( 8 M ) observed that haptoglobin protects H b from acid denaturation and used this as a basis for a method for quantitative estimation of haptoglobin. Affonso ( I M ) used plaster of paris strips to separate electrophoretically Hb (1)haptoglobin (11) complex from free H b in serum. METALS

Tompsett (46N) determined lithium, strontium, barium, and gold by atomic absorption spectrophotometry. Sachdev and West (S5N) presented a procedure for the preconcentration and determination of Ag+, Cda+,Coz+, Cuz+, NiZ+, and P b 2 + by simultaneous extraction of all these ions with dithizone in ethyl propionate for the atomic absorption method. Leddicotte (WON) discussed activation analysis techniques and methodology for the determination of trace elements. A survey method for trace element analysis of human hair by spark source mass spectrometry was developed by Yurachek, Clemena, and Harrison ( M N ) . Szilagyi and Pahoki (44N) made a critical review of serum iron and copper methods. Copper. Ichida and Nobuoka (17N) described a diluent for serum copper determination by atomic absorption which contained ethylene glycol and histidine-HC1. Zlatkis, Bruening, and Bayer (66N) investigated the use of the chelating polymer, poly-triaminophenol-glyoxal in the microanalysis of copper by column chromatography followed by atomic absorption. MikacDevic (28N) degeloped a microspectrophotometric method for the determination of copper in serum and urine employing 1,5-diphenyl-carbohydrazide.A method for the determination of serum and urinary copper with N , N , N ’ , N ’ tetra-ethylthiuram disulfide was described by Matsuba and Takahashi (26N). Liplakk and Ushkova (22”) determined copper spectrophotometrically with sodium diethyl dithiocarbamate in the presence of trilon B to


mask all interfering cations. Gre. gorowicz, Kwapulinska, and Piwowarska (16N) described the colorimetric determination of copper in serum with 2,2‘bicinchoninic acid. Foster and Trusell (1SN) improved the sensitivity of the spectrophotometric determination of copper with 6,7-dimethyl-2,3-di(2-pyridyl) quinoxaline. Blomfield and MacMahon, ( S N ) described the microdetermination of plasma copper by complexing with ammonium pyrroledinodithiocarbamate for atomic absorption determination. Sunderman and Nomot0 (48N) evaluated the optimum reaction conditions for the assay of serum ceruloplasmin by the measurement of its p-phenylenediamine oxidase activity in which a purple oxidation product is produced. Iron. Uny, Brule, and Spitz (48N) applied ultrasonic pulverization for nebulization and atomization of serum sample for the determination of iron by atomic absorption. Good agreement was demonstrated between atomic absorption and spectrophotometric serum iron methods by Tavenier and Hellendoorn (46N). Schmidt ( 3 8 N ) determined serum iron and copper by atomic absorption without protein precipitation by adding Lac13 to sample solutions. Ramirez-Munoz and Roth (SWN) estimated iron in blood by atomic absorption spectrometry without preliminary extraction. Olson and Hamlin (SON) described a new atomic absorption spectrophotometric method for serum iron and iron binding capacity which utilizes treatment of serum with 20% trichloracetic acid and heating a t 90 “ C for 15 minutes. Dawes and Park ( 8 N ) made microdeterminations of total serum iron by releasing iron with NaClO solution followed by colorimetric estimation with NHlSCN in methoxyethanol. Lorentz and Flatter (ZSN) determined F e a + spectrophotometrically by reaction with 2-methyl3-hydroxy-4-pyrone. Schilt and Taylor (37“) determined iron and copper by a new spectrophotometric method by complexing with 3-(2-pyridyl)-5,6-diphenyl1,2,3,4-triazine. Stookey (CON) proposed the use of a new spectrophotometric reagent for iron, the disodium salt of 3-(2-pyridyl)-5,6-bis(Cphenyl sulfuric acid)-l,2,4-triazine (Ferrozine). Sabatino et al. (34N) and Klein, Lucas, and Searcy (18N) determined serum iron with a new ligand, a 5-pyridyl-benzodiazepin-2-one derivative. Klein et al. (19N) presented a new and simple procedure for the determination of iron in which iron was separated from H b with 2.5% sodium hypochlorite solution, chelated with 7-bromo-l,3-dihydro-(3-dimethylamino propyl-5-(2 pyridyl)-2H-l,4-benzodiazepin-2- one - HC1 and its absorbance measured. This iron reagent was patented by Evans and Searcy (11N). Fink, Pivnichny, and

Ohnesorge (12 N ) applied the quenching of the intense luminescence emitted by the ligand of 2,2',2''-terpyridine to the determination of iron in aqueous solutions. Baginski et al. (WN)and Zak et uE. (64N) recommended the use of the sensitive reagent, 2,6-bis(4-phenyl-2 pyridyl) -4-phenyl pyridine (terosite) and its sulfonate for the determination of serum iron. A new method for the determination of serum iron and ironbinding capacity with haematoxylin as a specific agent was described by MikacDevic (27N). Lehmann and Kaplan (21N) described a rapid column chromatographic procedure for the removal of excess unbound Fe in serum for total iron binding capacity (TIBC) determination. Goodwin and Williams (14N) described a method for the direct estimation of serum iron and unsaturated iron-binding capacity in a single aliquot. Colenbrander and Vink ( 7 N ) improved the preparation of Teepol for use in the determination of serum iron and TIBC. Brozovich and Copestake ( 6 N ) determined the unsaturated ironbinding capacity in 0.1 ml of serum by using radioactive Fe amd MgC03 as a n adsorbing substance. Bouda ( 6 N ) described a simple bathophenanthroline method to determine iron-binding capacity in a single serum sample, both blank and test without heating. O'Malley et al. ( S I N ) eliminated adsorbing agents to permit the use of only one tube for the ambient measurement of iron and T I B C in which the color reagent, 2,4,6-tri-pyridyl-S-triazine was used. Manganese. Ajemian and Whitman ( I N ) described the preparation of urine for the determination of manganese by atomic absorption spectrophotometry. Mahoney et al. (24N) performed manganese determination on serum diluted 1:l with double distilled water with the Perkin-Elmer 303 atomic absorption spectrophotometer. Voskian, Rousselet, and Girard (50N) described atomic absorption techniques to measure manganese in biological media. Srivastava, Pandya, and Zaidi ( S 9 N ) determined manganese in blood and other tissue by a method based on the Mn-catalyzed oxidation of N , N diethylaniline. Nickel. Sunderman and Nomoto (29N, 4 I N ) , and Schaller, Kuehner, and Lehnert (S6N) complexed nickel in serum with ammonium pyrrolidine dithiocarbaminate and extracted with methylisobutyl ketone or isobutylalcoho1 for atomic absorption determination. Zinc. Dawson and Walker (9N) determined zinc in whole blood, plasma, and urine after a 1 to 20 dilution in 0.1N HC1 with the 303 Perkin-Elmer atomic absorption spectrometer. Roth and Ramirez-Munoz (SSN) determined Zn in serum and whole blood by simple

water dilution for atomic absorption flame photometry. Mikac-Devic (26N) determined Zn in 0.5 ml of serum b y use of the specific reagent di-b-napthylthiocarbazone in CC1,. Boiteau, Bliaux, and Gelot ( 4 N ) determined zinc in biological material by X-ray fluorescence spectrometry. Other Metals. Deguchi (1ON) determined 0.1 pg of cadmium with 2,2'-bisbenzothiaeolein I. Groenewald (16N) determined gold by the use of tertiary and quaternary amines for the qualitative extraction of gold complex ions into diisobutyl ketone for atomic absorption measurement. Walton et al. (61N ) developed a simple rapid spectrophotometric method for the determination of low levels of gold3+ with di-2-pyridylketoxine in plasma and urine. Woods et al. (62N) determined lithium in biological fluids by diluting 1:l with 0.040/, Sterox S E and applying an atomic absorption procedure. Vengerskaya and Salikhodzhaev (49N) determined molybdenum (Mo) in blood and urine by a colorimetric method based on the reaction of Mo with methyl violet in acidic medium after ashing of specimen and dissolving in HCl. An atomic absorption spectrophotometric method for measurement of rubidium in serum, plasma, whole blood, and urine was evaluated by Sutter, Platman, and Fieve (4SN). Tszyu, Bocharova, and Men'Kov (47N) measured scandium in biological fluids by colorimetric determination with its xylene orange complex. NITROGEN COMPOUNDS

Meijers and Rutten (22P) determined total nitrogen in urine by digestion with 70% HCIOl a t 200 "C and colorimetric determination of the formed ammonia salts with phenol reagent. Nakagawa (23P) determined N in Kjeldahl digests with a reagent based on the combined action of hypochlorite and alpha-naphthol on ammonia. Hall, Peyton, and Wilson (IOP) described an indirect method for the determination of alpha-amino N in urine in which Cu was solubilized from copper phosphate by complexing with alpha-amino groups at slight alkaline p H and the soluble Cu in filtrate measured by atomic absorption. Ammonia. Kaplan ( I 7 P ) reviewed the determination of ammonia and urea N in body fluids. Weichselbaum, Hagerty, and Mark (SOP) determined NH3 and blood urea N by a reaction rate method based on the pentacyanonitrosyloferrate catalyzed Berthelot reaction. Beecher and Whitten ( I P ) determined the effect of reagent modification and interfering compounds on the Weatherburn biological fluid ammonia procedure. Gerok and Pausch (6P) compared three methods for the determina-

tion of blood N H a ; the direct phenol hypochlorite method, the cation exchange resin and determination of N H a in the eluate with Berthelot method, and the glutamate dehydrogenase reaction. The last was preferred. The use of glutamic dehydrogenase as a specific NH, reagent was described by Levitski (19P). Oreskes, Hirsch, and Kupfer (24P) determined NH3 in protein-free filtrates with a reagent containing P-NADH, alpha-keto-glutarate, ADP, and phosphate. Gilardoni ( 7 P ) presented a whole blood ammonia method using a deproteinized solution free from "$-generating compounds. Frazer et al. ( 4 P ) determined microgram amounts of NH3 using distillation, flow spectrophotometry, and data acquisition by computer. Urea. Martinek ( 2 I P ) reviewed methods for the determination of Urea N in biologic fluids. Kano et al. (16P) compared three commercial kits for the routine clinical determination of serum urea N with an automated diacetyl monoxime method and found good correlation. Puech and Genevrier (25P), Geiger ( 5 P ) , and Fonty ( S P ) determined urea in biological fluids with the p-dimethyl amino benzaldehyde method. Manoukian and Fawaz (2OP) described a direct' enzymatic urea micromethod in which converted NHs is estimated by its reaction with glutamic dehydrogenase. Reardon (26P) patented a reagent for ammonia nitrogen determination in which dichloro-S-triazinetrione was substituted for hypochlorite and salicylate for phenol in the Uerthelot method. Hughes ( I S P ) patented a combination of reagents used in the Berthelot method for the determination of urea in biological fluids. Humbel, Idoux, and Ragenard ( I @ ) found satisfactory agreement when comparing the AZO stix strip method and the Berthelot method for blood urea determination. Harvill and Shrawder ( I I P ) detected urea with a patented indicator paper impregnated with urease and a bromothymol blue indicator. Guilbault and Montalvo (8P, 9 P ) developed several types of urea transducer or enzyme electrodes suitable for rapid continuous detection of urea. Other N Compounds. Tietze (28P) described a method for analysis of nanogram quantities of blood and urine glutathione based on the catalytic action of oxidized or reduced glutathione in the reduction of Ellman reagent by a mixture of T P N H and yeast glutathione reductase. Tan and Lee (27P) developed a new simple, sensitive method for the determination of glutathione in erythrocytes by the addition of reduced glutathione to a predetermined excess of palladium chlorpromazine complex which results in the formation of a stoichiometric amount of light yellow palladium glutathione complex. Wata-


27 R

nabe, Watanabe, and Okada (19P) developed a new fluorimetric assay for the determination of urinary 3-hydroxykynurenine based on its reaction with p-toluene-sulfonyl chloride in basic condition. An improved method for the determination of 5-hydroxytryptamine in human urine was described by Korf (f 8P). Chalmers and Watts (ZP) determined xanthine and hypoxanthine by measuring the extinction a t 280 mp that occurred when oxypurine was oxidized to uric acid by xanthine oxidase. A method utilizing 14Clabelled oxypurines to measure blood plasma levels of hypoxanthine, xanthine, and uric acid was devised by Hayashi and Gilling (IZP). Kamali and Manhouri (16P) described a modified Schneider orcinol method for pentose estimation in which ferric chloride is replaced with cupric acetate in glacial acetic acid as catalyst to make the test more sensitive and less DNA-reactive for R N A spectrophotometric determination. HORMONES

Thyroxine. Leeper (1QQ) reviewed recent advances in biochemistry of thyroid regulation and assay of thyrotropin-releasing factor, thyroid stimulating hormone, and thyroid binding protein. Veselsky, Nedbalek, and Suschny (S5Q) reviewed convent,ional P B I methods and described a method for P B I determination by activation analysis. Zaroda (S7Q) converted organically bound blood iodine to inorganic state by burning dried sample in a Schoniger oxygen flask and subjecting the extract to conventional cericarsenate assay. Hordynsky et al. (14Q) described an ultramicro ashing method for serum (25 gl) PUI. Hoch and Lewallen ( I @ ) determined iodine in 12.3 pl of serum by wet ashing and t.he use of the catalyzed cerate-arsenite reaction. Benotti, Pino, and Grimaldi ( 2 9 ) described a simplified 17-hour dialysis method for the determination of free thyroxine T4 in serum. Fang and Selenkow ( 7 Q ) report'ed the equilibrium dialysis of thyroxine T4 in diluted serum at low temperatures gave consistent and reproducible values for free serum thyroxine. Hathaway, Sannella, and (12Q) described a relatively rapid continuous spectrophotometric method for the determination of serum thyroxine in which standards and specimens are passed through anionexchange resin columns to obtain free thyroxine in eluates from which iodide is freed with bromine or reduction with ceric ammonium sulfate. Hanok et al. (If&) studied the factors affecting the precision and accuracy of the chromatographic and analytical schemes for the determination of serum T4 levels by column chromatography aiid bromina28R

tion of the column eluates. A simple and rapid method for the determination of serum thyroxine based on replacement of 1251-labeledthyroxine attached to thryoxine-binding globulin (TBG) b y patients' thyroxine was described by Learnes et al. (18Q). Roberts and Nikolai (27Q) assayed 315 normal subjects for thyroxine-binding globulin (TBG) by the polyacrylamide electrophoresis method of Kikolai and Seal. Roberts and Nikolai (289) developed a simplified procedure utilizing dextrancoated charcoal for the determination of serum thyroxine-binding globulin which did not require prior electrophoretic separation of the three serum thyroxinebinding proteins. Nobel and Barnhart (234) adapted the Murphy-Jachan serum thyroxine method for routine use in which mixing, resin addition, and temperature control was modified to give greater precision and simplicity. Pain and Oldfield (26Q) surveyed six T3 published methods and concluded that the method of choice was the TB-charcoal-hemoglobin assay method. Leonards (ZOQ) presented a new T3 test based on the competition between serum-binding proteins and Sephadex G-25 for lZ5I-triiodothyronine. Free and Irwin (8Q) described a 20-minute T3 test employing 0.05 ml of serum which used Sephadex t o separate radioactive T3bound to plasma proteins from unbound radioactive T3. Catechol Amines. Manger et al. (229) reported that blood collected for cat,echolamine determination should be cooled with ice water and treated with sodium thiosulfate to reduce the association of catecholamines wit'h the formed elements. Valori el al. (34Q)described an improved method for the formation of epinephrine and norepinephrine fluorophors by the trihydroxyindole reaction. Bannow and Routh ( I & ) developed a screening test for L-dopa and its metabolites in urine. Spiegel and Tonchen ( S I Q ) described a semiautomated fluorometric method for plasma analysis of dopa [3-(3,4-dihydroxyphenyl)- alanine] which is separated from interfering catecholamines by adsorption on alumina, eluted with 0.1N HCI and then adsorbed on and eluted from a cation exchange column. Oberman, Chayen, and Herzberg (24Q) determined dopamine in urine by treating directly with l-dimet,hyl aminonaphthalene-5-sulfonyl chloride in borate buffer, p H 9 to form a fluorescent derivative. Sato and DeQuatt'ro (Z9Q) measured 3,4-dihydroxymandelic acid in biological fluids with a seiisit,ive and specific enzymic assay using rat liver catechol-0-methyltransferase. Sprinkle et al. ( 3 2 9 ) developed a gas chromatographic method for the determination of urinary homovanilic acid and vanilmandelic acid. Imai and Tamura ( I S Q ) determined urinary vanillyl-


mandelic acid by a selective, sensitive method employing chromatography, elution, and gas chromatography. Yuwiler et al. (S6Q) described a rapid accurate fluorometric procedure for the determination of serotonin in blood. Kulinskii and Kostyukovskaya (I7Q) determined serotonin in blood by a fluorometric method in which the ninhydrin condensation product was measured. Steroid Hormones. Horton (16Q) reviewed laboratory determination of aldosterone and the clinical significance of the methodologies. Golikov (9Q) estimated transcortin by means of filtering plasma through biogel P-10, and fluorometric estimation of corticosterone by means of alcohol-sulfuric acid reagent. Smith and Muehlbaecher (300) described a fluorometric method for the determination of cortisol and transcortin in 0.3-1.0 ml of plasma after preliminary CC14 fractionation. A simple, rapid fluorimetric serum cortisol method in which cortisol and corticosterone are oxidized to their corresponding 17 P-carboxylic acids with metaperiodate was described by Clark and Rubin (SQ). Dixon (SQ) determined plasma cortisol by a method based on a microcolumn procedure. Grannis and Dickey (fOQ)developed a simplified procedure for the determination of estrogen in pregnancy urine which only requires extraction of estrogen and color development. A rapid quantitative method for the estimation of oestrogens in pregnancy urine which eliminates acid hydrolysis and solvent extraction by use of the absorption characteristics of Sephadex G-10 was devised b y Contractor and Jacoby (4Q). Lurie and Patterson (21Q) designed a simple method for the assay of progesterone in nonpregnancy plasma in which chromatography was not used. Pieterse, Scholtis, and Schmidt (269) described a method involving enzymatic hydrolysis, extraction with dichloromethane, and purification and fractionation on silica gel for the separation and determination of hydro-lldesoxycortisol, tetrahydrocortisone, and tetrahydrocortisol. Uettwiller (SSQ) and Demetriou and Austin (59) described improved methods for the determination of plasma testosterone based on the principle of competitive protein binding technique. ORGANIC ACIDS AND COMPOUNDS

Zaura and Metcoff (29R) quantified biologically significant concentrations of pyruvate, lactate, fumarate, succinate, malate, alpha-ketoglutarate, and citrate in urine by gas-liquid chromatography. Hautala and Weaver (8R) separated and determined quantitatively: lactic, pyruvic, fumaric, succinic, malic, and citric acids by gas chromatography.

Harmon and Doelle (7R) investigated gas chromatographic separation and determination of micro quantities of the esters of tricarboxylic acid cycle acids and related compounds. Goeschke (4R) described a simplified method for the determination of ketone bodies in biological fluids b y converting acetoacetate and 3-hydroxy butyrate to acetone and determined the latter colorimetrically as acet,one-2,4-dinitrophenylhydrazone. Van Stekelenburg and de Bruyn (27R) described a simple head gas sampling procedure for the chromatographic determination of acetone and betaketobutyric acid in 0.2 ml of serum. Persson (23R) and Wildenhoff (28R) described micromethods for the determination of acetoacetate and 3-hydrosybutyrate in biological fluids. Zender, de Torrente, and Schneider ($OR), O’Neill and Sakamoto (21R) and Costello and O’Neill (3R) described enzymatic procedures for the determination of citrate in biological specimens. Kang, Gerald, and Whealan (15R) developed a new method for the early detection of alcaptonuria by separating homogentisic acid from urine by ascending chromatography and semiquantitative determination of creatinine with Jaff6 reagent. Collombel et al. (2R) measured the urinary excretion of homogentisic acid by thin-layer chromatography. Pryce (24R) modified the Barker-Summerson lactic acid method by increasing copper sulfate in precipitating agent and adding H3P04 to increase sensitivity. Green (6R) patented a reagent for the determination of lactic acid based on the lactate dehydrogenase catalyzed reaction. Hodgkinson (9R) critically compared various methods of oxylate analysis in biological material. Sandler (25R) reviewed methodology for determination of hydroxyindoles by biological assay, fluorimetry, spectrophosphorimetry, infrared spectro-photometry, immunoassay, colorimetry, and paper thin-layer, gas-liquid, and column chromatography. Korf and Valkenburgh-Sikkema (17R) determined 5hydroxyindole-acetic acid in urine and spinal fluid by a simple fluorimetric method based on fluorophore formation with 0-phthalaldehyde. Gutteridge (6R) used thin-layer chromatography for visual detection of pathological amounts of urinary phenolic acids. A new method for the quantitative determination of free acid bound phenol in biological fluids based on a modified diazo method using p-dihydroxy phenylacetic acid for standardization was described by hliieting, Keller, and Kraus (2OR). Mellinger and Hvidberg (19R) analyzed urine for 3,4-dihydroxyphenyl acetic acid by ether extraction and subsequent separations by paper chromatography to eliminate interfering

substances. Humbel (12R) developed a thin-layer chromatographic method for the estimation of O-hydroxyphenylacetic acid in urine for the diagnosis of phenylketonuria. Hoffman and Gooding (IOR)described a rapid accurate gas chromatographic method for the determination of 0-hydroxy phenylacetic acid, 0-phenyllactic acid, and phenylpyruvic acid in phenylketonuric urine. Uric Acid. Martinek (18R) reviewed methodology for the determination of uric acid in biological fluids and made recommendations for the routine clinical laboratory. Parekh and Jung (22R) incorporated Na3P04 and triethanolamine in an alkaline phosphotungstic acid for the determination of serum uric acid. Jung and Parekh (13R) improved the serum uric acid method by destroying serum chromogens with Na3P04, elimination of turbidity by the use of phosphotungstic acid for tungstate, and addition of triethanolamine to carbonate-urea reagent to increase stability of color. Caraway (1R) reported t h a t colorimetric methods for the determination of uric acid in serum are sufficiently specific if alkali treatment is employed prior to color development to destroy ascorbic acid. Hughes (11R) patented a phospholithotungstic acid method for the determination of uric acid in blood. A method for the determination of urate binding to plasma proteins by means of equilibrium dialysis was described by Klinenberg and Kippen (16%). Simkin (26R) determined serum uric acid by a method based on the affinity of purines for polyacrylamide resin. Kageyama (14R) described the colorimetric determination of uric acid using uricasecatalase and acetyl acetone to develop diacetyldehydrolutidine for color measurement. PROTEINS

Martinek (35s)reviewed methodology of protein determination in biological fluids and made recommendations for the routine clinical laboratory. Tsvetanova (51s) compared the turbidimetric, biuret, Lowry’s, and spectrophotometric at 220 mp, methods with the micro Kjeldahl method for spinal fluid total protein determinations. Trifonov and Aleksandrov (508) compared the Folin phenol method with the Gornall and Kingsley biuret methods for protein determination. Peters (428) presented a discussion of the “Standards Committee of the American Association of Clinical Chemists” on the nature of total serum proteins and the problems associated with its determination. Ackers (IS)reviewed analytical gel chromatography of proteins. Ternovoi and Strigin (49s) reported t h a t ultra fine Sephadex was suitable for sepai ation of proteins by thin-layer

chromatography. A comparative evaluation of paper and cellulose acetate electrophoresis was made with an electronic scanner by Schmidtmann, Kaempffer, and Reschke (47s). Luxton (34s) critically evaluated the Micro Zone system for quantitative electrophoresis of serum proteins. Abraham et al. ( I S ) fractionated serum proteins into more than 20 separate components by continuous electrophoresis in polyacrylamids. A procedure for the twodimensional separation of serum proteins b y isoelectric focusing and diskgel electrophoresis was described by Domschke, Seyde, and Domagk (128). Martinek (368) reviewed methods for removing or solubilizing protein in biological fluids. Firestone and Aronson (16s) described a quantitative 2-dimensional agarose-gel immunoelectrophoretic method capable of separating 30 proteins in three to four hours, Becker, Schwick, and Storiko (68) determined ten additional plasma proteins found in low concentration by applying radial immunodiffusion using highly purified physicochemically well-defined proteins as standards. Weeke (628) compared immunochemical protein determination with paper electrophoretic fractions in normal and pathologic serum and found good agreement. Iammarino (268) applied a clinically useful system of immunoelectrophoresis to the study of spinal fluid and urinary proteins by concentration of sample. Clarke and Freeman (108) used a modification of Laurell’s antigen-antibody crossed electrophoresis using the specificity of the antigen-antibody reaction to characterize different serum proteins for quantitative measurement. Kindmark (28s) measured C-reactive protein quantitatively and rapidly by electrophoresis of sera in agarose gel containing anti-C-reactive protein bodies. Grannis (228) compared the determination of fibrinogen as precipitable protein and as clottable protein and evaluated the reliability of the latter procedure. A method for direct analysis of total serum globulin based on a purple color formation when heated (100 “C) a i t h a dye was reported by Goldenberg and Drewes (20s). Losza, Kereszti, and Berencsi (338) described the electrophoretic determination of serum glycoproteins. Arai and Wallace (38) described a staining method for the determination of glycoproteins separated by electrophoresis on cleared cellulose acetate membrane. de Goldman, Ballivian, and Melgar (11s)determined the serum content of macroglobulins (19s fraction) by thin-layer gel filtration. Hirs (25s) reviewed chemical methods for the detection of peptides. Bramhall et al. (98) described the use of xylene brilliant cyanine G for measuring 10-200 pg of protein spotted on paper.


NO. 5, APRIL 1971

29 R

Konrad (30s) patented the use of Naphthol Red S in a reagent for the determination of protein in biological fluids. Goodwin and Choi (21s) quantified protein in dilute solutions by complexing with trinitrobenzenesulfonic acid in the presence of sulfite to obtain a highly colored product for spectrophotometric measurement a t 420 mp. Atkinson and Fader (4s) patented a reagent system for detection of proteins in biological fluids using a base and tetrabromophenol blue. O’Malley and O’Doherty (40s) applied direct fluorometry (254 pm) t o the measurement of spinal fluid protein. Mayer and Miller (37s) found the quantitative measurement of peptides and proteins a t 191-194 mp to be more sensitive than at 280 mp. Hatcher and Anderson (248) described a new microanalytical system (4 pl serum) (GeMSAEC) for the determination of serum protein during the first few seconds of the biuret reaction. A simple quantitative method for the determination of protein in sputum by the use of the biuret reaction was described by Fields and Chodosh (158). Bode, Goebell, and Harald (88) used K C N to eliminate the blue color of the biuret complex and errors caused by lipid turbidity and hemoglobin in the protein determination. Stewart, Thomas, and Hull (488) reported that Tris buffer interfered with the biuret reaction in the determination of proteins. Klungsoeyr (29s) estimated protein by passing the protein treated with a modified biuret reagent through columns of Sephadex G-25 and determining the protein bound copper colorimetrically in the basic eluate. Lipoproteins. H a t c h and Lees (238) discussed five relatively simple economical procedures for the analysis of plasma lipoprotein. Gebott (19s) presented two new procedures for lipoprotein electrophoresis: lipid oxidation and staining with a modified Schiff reagent and the other, using a conventional lipid stain. Prellwitz and Koettgen (448) compared three different methods for the determination of serum lipoproteins. Lewis (32s) compared three methods for the evaluation of serum lipoprotein abnormalities. Bertrand et al. (78)described a gel cellulose acetate microelectrophoretic method for the identification of lipoprotein in 1.25 p1 of serum within 20 minutes. Beckering and Ellefson ( 5 s )used cellulose acetate as support medium for rapid serum lipoprotein electrophoresis with improved resolution. Farber et al. ( 1 4 s ) compared cellulose acetate and paper techniques for the measurement of alpha, pre-beta-, and beta-lipoproteins. Fletcher and Styliou (17s)described a simple rapid method for separating serum lipoproteins into clear discrete and reproducible bands by electrophoresis on cellulose acetate. Winkel30R


man, Wybenga, and Ibbott (66s) reported that the stability of serum specimens for cellulose acetate electrophoresis of lipoproteins was good for 3 days a t room temperature and 14 days a t freezing temperature. Moinuddin and Taylor (38s) described a paper electrophoretic method in which four serum lipoprotein components were separated without the use of albumin in buffer. Kostner, Albert, and Holasek (31s) separated serum lipoproteins after prior staining with Sudan Black into 7 to 8 distinct fractions by isoelectric focusing into a medium containing 44% ethylene glycol. Fried and Hoeflmayr (18s) patented a method for the colorimetric determination of serum beta-lipoproteins. Werner et al. (63s) evaluated the nephelometry of pre-beta-lipoproteins and chylomicrons after separation by ultrafiltration for their separate estimation in serum. Winkelman, Wybenga, and Ibbott (55s) quantitated by microdensitometry the serum beta-, pre-beta-, and alpha-lipoprotein bands separated by electrophoresis on cellulose acetate in each of the five recognized hyperlipoproteinemia phenotypes. Winkelman et al. ( 5 4 s )reported studies on the phenotyping of hyperlipoproteinemias, evaluation of cellulose acetate technique, and comparison with paper electrophoresis. Ross and Brown (458) fractionated lipoproteins into chylomicrons, beta, pre-beta, and alpha bands b y electrophoresis using cellulose acetate membrane and oil Red 0 staining. Dyerberg and Hjoerne (13s) reported that agarose gel electrophoresis was a quantitative method for plasma lipoprotein estimation and Kahlke (278) recommended agarose gel electrophoresis for best separation of serum lipoproteins. A method for the separation of serum lipoproteins by electrophoresis in an agarose-agar gel mixture on a thin polyester photographic film strip was developed by Noble (39s). Papadopoulos and Kintzios (41s)described an electrophoretic procedure for serum lipoprotein determination utilizing agarose gel as supporting medium and oil Red 0 dye for staining which was rapid and sensitive. Zoellner et al. (678) obtained good separation of five serum lipoprotein fractions using 0.8% agarose with added albumin. Sata et al. (46s) compared the fractionation of lipoproteins of normal and hyperlipemic patients on a column of 2% agarose with ultracentrifugation and paper electrophoresis procedures. Pratt and Dangerfield (438) described the preparation of polyacrylamide gel which gave lipoprotein resolution superior to conventional electrophoretic and ultracentrifuge methods. STEROLS

Wotiz and Clark (25T) reviewed newer developments in the analysis of


steroids b y gas chromatography and covered detectors, mass spectrophotometric, quantitative analytical steroid derivatives, methodology, and applications. Voellmin (232’) separated urinary steroids as trimethylsilyl ether derivatives on glass capillary columns in a combination gas chromatographmass spectrometer. Muehlbaecher and Smith (142’) examined three hydrolysis methods for 17-ketosteroid sulfate determination and compared them by colorimetric and gas-liquid chromatographic analysis. Gorog (8T)presented a new method for the determination of ketosteroids which utilized a diethyl oxalate reagent in alkaline solution in which Claisen condensation takes place transforming ketosteroids quantitatively to glyoxalyl derivatives which have intense light absorption. Cholesterol. Martinek (13T) and Mueller (16T)reviewed methods for the determination of cholesterol and cholesterol esters in serum. Makino et al. (1.”) evaluated serum cholesterol methods for micro and ultramicro estimation. Eberhagen ( 5 T ) reviewed the principles involved in the determination of total cholesterol and sources of interferences a t each stage of analysis. Williams, Kuchmak, and Witter (242’) evaluated the purity of cholesterol primary standards. A new method for serum cholesterol determination using ferric acetate-uranium acetate in acetic acid was evaluated with sulfuric acidferrous sulfate reagent by Parekh and Jung (175”). Zlatkis and Zak (272’) described a new cholesterol method in which 0.1 ml of specimen was treated with 2 ml of 0-phthalaldehyde in glacial acetic acid and 1 ml of concentrated H2S04 layered in and mixed. Kegrin (16T) described a new spectrophotometric method for the determination of cholesterol in tissues based on the reduction of the yellow aqueous solution of phospho-6-tungstic-12-molybdic acid by a solution of cholesterol in concentrated sulfuric acid. Ushikoshi (21T ) studied the reliability and reproducibility of a modification of the turbidimetric cholesterol method of Kingsley and Robnett. Kishi et al. (10T) evaluated the reliability and reproducibility of six commercial “kits” for serum cholesterol determination. Jordan and Knoblock (92’) proposed a micromethod for serum total cholesterol determination which eliminated interference by high bilirubin concentrations. Ferreira and Bandeira de Mello ( 6 T ) described several colorimetric serum cholesterol methods which did not require proteinfree filtrates. Solow and Freeman (202’) adapted fluorometry to the ferric chloride method to measure 1 pg of cholesterol in serum and spinal fluid. A method for the determination of free and esterified cholesterol in serum by thin-

layer chromatography was described by Beukers, Veltkamp, and Hooghwinkel (IT). Young and Hall (262') studied the side chain cleavage of cholesterol and cholesterol sulfate by enzymes from bovine adrenocortical mitochondria. Estrogens. Bush (ST) reviewed the determination of estrogens, androgens, and progesterone in plasma and urine, assessed methods, advances in techniques, and presented detailed procedures. -4 systematic study of the ammonium sulfate precipitation of conjugated estrogens in pregnancy urine was made by Pinkus and Pinkus (192'). Pieterse, Scholtis, and Schmidt (182') described a simple and accurate method for the assay of oestrone, oestradiol-17 beta, and oestriol in low titer urines, following enzymes hydrolysis and gel filtration. Goebelsmann (72') described a reliable method for estimation of urinary estriol based upon acid hydrolysis, solvent partition, methylation, and chromatography. Dit0 and Shelly (4T) described a rapid (4-hour) combined chromatographic fluorometric method for urinary estriol in pregnancy. Baynton and Campbell (1T ) determined urinary estriol, pregnanediol, and pregnanolone by enzymatic hydrolysis, extraction, conversion of steroids to triinethylsilyl ethers, and measurement by gas-liquid chromatography. Van de Calseyde et al. (222') described a simple gas chromatographic method for assaying urinary pregnandiol and pregnanetriol using either the acetate or silyl ether derivatives. Levelle and Cottam (117') modified the Klopper method for more rapid determination of pregnanediol in urine. TOXICOLOGY

Lubran (S9U) reviewed drugs affecting clinical laboratory results and their causes. Pippenger, Scott, and Gillen ( S 5 U ) described a rapid semiquantitative method for the determination of anticonvulsant drugs (dilantin, phenobarbital, mysoline, phenylethyl-malondiamide, messantoin and nirvanol) in blood and urine by thin-layer chromatography. A rapid quantitative method for estimation of anticonvulsant drugs in biological fluids in which a Jarrell-Ash flame ionization gas chromatograph was used was described by Pippenger and Gillen (S4U). Amphetamine. Toseland and Scott (49U) and Schweitzer and Friedhoff ( 4 S U ) determined amphetamines in biological liquids by gas-liquid chromatography. Lebish, Finkle, and Brackett (2424') determined amphetamine, methamphetamine, and related amines in blood and urine by gas chromatography with hydrogen-flame ionization detector. Bruce and Maynard ( 4 U ) described a method for the determination of amphetamine and

related amines based on the formation of heptafluorobutyryl derivatives of their amines and their subsequent determination by gas-liquid chromatography using an electron capture detector. Barbiturates. Morselli (31U ) described an improved technique for routine determination of diphenylhydantoin in plasma and tissues. Sabih and Sabih (4OU) described a gas chromatographic method for the determination of diphenylhydantoin directly in blood in 1 hour without preliminary chemical treatment. Wallace (62U) converted diphenylhydantoin after chloroform extraction to benzophenone by permanganate oxidation for its determination in biologic specimens. Sine et al. (46U) presented a method for the rapid gas-liquid chromatographic determination of barbiturates and glutethimide in serum. Grove and Toseland (15 U ) determined hydroxyamylobarbitone in plasma and urine by gasliquid chromatography by a novel approach. A simple rapid one-hour quantitative method for the simultaneous analysis of barbiturates and glutethimide was developed by Dain and Trainer (6U).Kupferberg (ISU) developed a gas-liquid chromatographic method for the simultaneous determination of phenobarbital, primidone, and diphenylhydantoin in plasma. Van Meter, Buckmaster, and Shelley (51 U ) determined phenobarbital and diphenylhydantoin in plasma by vapor phase chromatography after simple solvent partition and solid sample injection. Tompsett (48U) investigated the interference of other substances in the determination of barbiturates in biological materials. Foster and Frings ( 1 4 U ) determined diazepam (Valium) by injecting a concentrated serum chloroform extract into a gas-liquid chromatograph. Ethanol. Hancock, Mill, and Miles (17 U ) compared blood alcohol methods: gas liquid chromatography (GLC) , redox back titration, and enzymatic oxidation with D P N , and found the GLC method most specific, and redox titration most reproducible. Morales and Greene (SOU) compared t,hree ethanol methods: dichromate titration, colorimetric dichromate, and spectrophotometric enzymic oxidation for biological specimen analysis, and preferred the more specific enzymic method. Anders ( I U ) determined blood alcohol by gas chromatography using double columns. Brower and Woodbridge ( S U ) described a rapid determination of blood ethanol by oxidation of ethanol t o acetaldehyde, and the reduction of NAD to NADH by alcohol dehydrogenase and measurement, of the latter by a coupling reaction which produces equi-molecular amounts of formazan. Leric, Kaplan, and Brown (25U) described a new colorimetric blood alcohol method in which a blood filtrate is in-

cubated with alcohol dehydrogenase and NAD in the presence of p-iodonitrotetrazolium violet and phenazine methosulfate. Jones, Gerber, and Drell(2OU) described a single mixed reagent employing alcohol dehydrogenase for the rapid determination of ethanol directly in 1 ml of serum. Weidemann (5SU) described a rapid enzymic method for ethanol in capillary blood using alcohol dehydrogenase and NAD. Reanal (S7U) described a patented detector tube for indicating the alcohol content of exhaled air. Organic Toxicants. Lowe ( S 7 U ) reviewed the determination of volatile organic anesthetics in gases, blood, and tissues by gas chromatography. Schlunegger (42U) described a gas chromatographic method for the direct quaiititation of organic solvents in blood. Rodkey and Collison ( M U ) reported that indirect spectrophotometric procedures for the determination of blood CO such as COHb saturation are not sensitive enough for measurement of normal or very low CO concentration in blood. Routh et al. (SQU) modified the Axelrod method for the determination of caffein in serum and urine by employing ether-chloroform extraction to avoid spectral interference of benzene and applying a differential absorbance method. A simple rapid method for t.he simultaneous determination of conjugated and unconjugated chlorpromazine metabolites in urine was described by Turner, Turano, and March (SOU). Dymond and Russell ( I O U ) described a spectrophotometric method for the determination of isonicotinic acid hydrazide in whole blood by reaction with 2,4,6trinitrobenzenesulfonic acid. Haux and Natelson (18U) described a practical specific method for the determination of phenothiazine in blood and urine. Burston (5U) described a rapid reliable, and simple method for the determination of salicylate in plasma. Kelly, Peets, and Hoyt (21U ) described a new fluorimetric method for the determination of low concentrations of tetracycline in biologic samples in which anhydrotetracycline is extracted from protein free filtrates and then treated in chloroform extract with ethanolic aluminum chloride to form a highly fluorescent chelate. Tompsett (47U) described a distillation procedure followed by ultraviolet spectrophotomet'ry t'o determine basic and other drugs in urine. A simple sensitive method for the detection of basic organic drugs and their metabolites in urine in which ethanol extraction followed by ether extraction of the latter and evaporation followed by application to thin-layer chromatography was described by Bastos et al. (SU). Nakaniura and Meuron (S2U) applied ion pair elution as a simple and rapid procedure for


31 R

separating heroin from illicit mixtures for determination by UV absorption. Ono, Engelke, and Fulton, ( S S U ) identified morphine, codeine, methadone, and other drugs in urine b y extraction and separation by thin-layer chromatography. Lower, Murphy, and Bryan (28U) described a colorimetric determination of p-aminophenyl glucuronide derivative in urine by a method based upon preliminary urine fractionation on a cationexchange resin. Lead. Sun, St’ein, and Gruen (46U) utilized a single ion-exchange column instead of two ion-exchange columns for the determination of urinary delta-aminolaevulinic acid in screening for lead posioning. HaegerAronsen (16U) evaluated two methods for measuring delta-aminolaevulinic acid in urine. Zurlo, Griffini, and Colombo (54U) determined lead in urine by atomic absorption spectrometry after coprecipitation with thorium. Segal (44U)reported that various urine salts and organic compounds contributed t o the absorbancy a t 217 nm which could be corrected for by subtracting .Azz0 from A Z 1 7 when determining urinary lead by atomic absorption. Devoto (8U)described a urine acid digestion method for the preparation of sample for the determination of lead by atomic absorption. Farelly and Pybus ( I S U ) determined lead in R B C b y an extraction method which avoids acid digestion and protein precipitation. Elser, Savory, and Mushak (12U) developed an atomic absorption method for the measurement of lead in blood which involves preliminary wet ashing, chelation of the lead, and extraction into an organic solvent. Einarsson and Lindstedt ( 1 1 U ) described a routine method for atomic absorption analysis of lead in blood filtrates which does not require solvent extraction of the metal. Delves ( 7 U ) described a microsampling (10 pl) method for the rapid determination of lead in whole blood by atomic absorpt’ion spectrophotometry. Other Metals. Lieberman and Kramer (26U) described a rapid postirradiation chemical procedure for the determination of cadmium. A method for measuring serum and urine chromium by formation of a volatile chelate chromium trifluoroacetylacetonate and its detection by gas chromatography was developed by Savory, hlushak, and Sunderman (41 U ) . Dietz and Rubin ( 9 U ) described an atomic absorption spectrophotometric method for the determination of serum gold. Johansen and Steinlies (19U) described a simple neutron act,ivation method based on 65 hour Ig7Hg for the determination of mercury (Hg) in biological material. A rapid and sensitive method for the determination of mercury in urine which was based on the formation of a 32 R

colored H g complex with cupric iodide was described by Krylova and Rubtsov (22U). Rathje ( M U ) described a method for the determination of subgamma amounts of H g in urine based on the absorption of the 253.7 nm Hg resonance line b y H g vapor using a Hg vapor detector. VITAMINS

Selvaraj and Susheela (9V) employed a simple spectrofluorometric method for the assay of vitamin A in 50 p1 of serum in which t’he vitamin was released from proteins by hot KOH hydrolysis and separated by xylene extraction. A new procedure in which silicic acid column chromatography and fluorometry were used to assay plasmavitamin A was investigated by Carry, Pollack, and Owen ( 4 V ) . Hansen and Warwick (5V) described a fluorimetric micromethod for vitamin A and free and total vitamin E. Tibbling (1OV) studied a radioisotopic method for the determination of serum vitamin BIZ concentration based on the principle of saturation analysis. An inproved and simplified method for the measurement of vitamin Bl2 in serum using intrinsic factor 6iCo1312and coated charcoal was described by Raven et al. (8V). Friedner, Josephson, and Levin (bV) determined serum vitamin BlZby a new procedure using the competition for intrinsic factor with ( W O )cobalamine and radioisotope dilution and ultra filtration. Anderson, Peart, and Fulford-Jones (1V) measured serum pyridoxal by a microbiological assay using Lactobacillus casei. Hubmann, hfonnier, and Rot,h ( 6 V )made rapid accurate determination of ascorbic acid in plasma metaphosphoric acid filtrates by its reduction of nonfluorescent 1,2-naphthoquinone-4sulfonic acid to fluorescent lJ2-dihydroxy naphthalene-4-sulfonic acid. Karlin et al. ( 7 V ) determined folate levels in serum, whole blood, and erythrocytes by three different methods employing Lactobacillus casei. Fujita, Tokuhisa, and Michinaka (SV) made quantitative determination of vitamin D by alumina or silica gel thin-layer chromatography in the range of 5-50 rg * SUPPLEMENT

The 6th volume of “Standard Methods of Clinical Chemistry” (Roderick P. MacDonald, Ed.) was not received in time to be included in the main body of this review. The methodologies presented in the 6th vol. of “Standard Methods of Clinical Chemistry” are covered in the following report. Remp (1) presented a uricase uric acid method which avoided turbidity and provided greater specificity. Kaplan (2) described the separation and


quantitation of proteins in serum and body fluids by cellulose acetate electrophoresis. Bowers and McComb (3) reviewed the standardization and conversion of enzyme activity units to the international unit and its definition, Klein (4) used column chromatographic separation of hydroxyproline in complex biological fluid and tissue as a preliminary step for the most specific technique for its analysis. Fernandez and Jacobs (5) described refined procedures for the determination of porphyrin, porphobilinogen, and aminolevulinic acid in urine based on the methods of Schwartz, Zieve, and Watson, and others. Nobel (6) outlined a guide to the clinical laboratory for the development of a program to handle toxicologic problems. Goodwin (7) determined spectrophotometrically plasma and urinary amino nitrogen with fluorodinitrobenzene by a method based on that of Dubin. Sunderman (8) presented a 36-step method for the colorimetric determination of vanilmandelic acid in urine based on the method of Sunderman, Cleveland, Law, and Sunderman. Jacobs and Fernandez (9) modified the method of Vanzetti and Valente for the determination of hemoglobin in plasma in which sensitivity, removal of interfering substances and development of a reproducible and stable color was achieved. Kingsley and Tager ( I O ) described an ion-exchange method for tho determination of plasma ammonia nitrogen with the Berthelot reaction and described techniques for stabilizing ammonia in specimens prior to analysis. Klein and Cooper (11) determined serum lipoproteins by prestaining with Sudan Black B prior to electrophoretic staining. Lanchant,in (12) determined total seromucoid by a method based on its hexose composition as originally outlined by Winzler and modified b y Weimer and Moshin and others. Sax and Moore (13) presented a modified colorimetric serum glutamic oxalacetic transaminase method employing coupling with the diazonium salt, Fast Ponceau L. Cooper and McDaniel (14) presented filtrate and direct procedures for the determination of glucose with the ortho-toluidine reagent. Gambino (15) described the measurement of the partial pressure of oxygen (Po2) of blood with the polarographic oxygen electrode. McNair (16) presented the iodometric measurement of amylase by Caraway as a standard method. Pybus and Bowers (17) outlined a technique for the determination of serum lithium b y atomic absorption spectrometry. Reiner, Cheung, and Thomas (18) described reagents and techniques for the “Manual” chemical analysis of components of human calculi. Meites (19) submitted a fluorometric calcine serum calcium method based on the method of Kepner

and Hercules a s modified by Phillips. Rice (20) determined serum triglyceride (‘[neutral fat”) b y a method based on t h a t of Van Handel a s modified b y Kawade and Nicolaysen and Nygaard. Dybkaer (21) cited the need for international cooperation in the standardization of the nomenclature for quantities and units and suggested specific recommendations. Fleischer (22) submitted enzymatic methods for the determination of blood lactic and pyruvic acids based on the method of Scholz et al. as modified by Hohorst and Bucher et al. LITERATURE CITED Reviews

(4B) Auphan, M., Perilhou, J., (to U.S. Phillips Corp.), U.S. Patent, 3,490,876 (Cl. 23-253 G Oln), Jan. 20, 1970, Fr., (Appl. April 5, 1966; 4 pp); CA, 72, 87107 (1970). (5B) Blackburn, W. E., Hamilton, D. A., Inners, L. A., Reid, G. C., (to Xerox Corp.), U.S. Patent, 3,479,320 (Cl. 23230; G O h ) , Feb. 24, 1970, (Appl. Dec. 15,1966; 19pp); CA, 72,129353 (1970). (6B) Boehringer, Mannheim G.m.b.H., Brit. 1,159,627 (Cl. G Oln) July 30, 1969, (Ger. Appl. Nov. 22, 1966; 5 pp); CA, 72, 19034 (1970). (7B) Bowers, W. F., Haschemeyer, R. H., Anal. Biochem., 25, 549 (1968). (8B) Brownstone, A. D., ibid., 27, 25 (1969). (9B) Burtis, C. A., Goldstein, G., Scott, C. D., Clin. Chem., 16,201 (1970). llOB) Crouch. S. R.. ANAL.CHEM.. , 41., 880 (1969). ‘ (1lB) Csizmas, L. L., Patel, V., (to Miles Laboratories, Inc.), U.S. Patent, 3,480,400 (Cl. 23-259; B Oll), NOV.25, 1969, (Appl. Mar. 21, 1966; 5 pp); CA 72, 28809 (1970). (12B) Derr. Derr, D. B.. B., Neff. Neff, G. W.. W., Sam’ bucetti. C. J. (to International Business Machines Corp.), Ger. Offen., 1,993,302 (Cl. G Oln), Jan. 22, 1970, (US Appl. Jul. 15, 1968; 3 pp); CA, 72, 129361 (1970). (13B) Durst, R. A. Ed., Ion-Selective Electrodes, Proceedings of a Symposium Nut. Bur. Std.,(US.), Spec. Pub. 314, pp 480, Washington, D.C., 1969. (14B) Edwards, W. R., (to Johnson & Johnson), U.S.Patent, 3,449,080 (Cl. 23-230; G Oln), Jun. 10, 1969, (Appl. Oct. 29, 1964; 5 pp); CA, 71, 36346 (1969). (15B) Ertin hausen, G., Adler, H. J., Reichler, S., J . Chromatogr. 42, 355 (1969). (16B) Farese, G., Mager, M., Clin. Chem., 16,281 (1970). (17B) Glick, D., Ann. N . Y . Acad. Sci., 157, 265 (1969). (18B) Haljamae, H., Larsson, S.,Chem. Instrum., 1, 131 (1968). (19B) Hardy, S. bl., Fresenius 2. Anal. Chem. 243, 713 (1968); CA, 70, 93744 ~

(1A) Boltz, D. F., hlellon, M. G., ANAL. CHEM.,42, l521t (1970). (2A) Crummett, W., Hummel, R., ibid., p 23911. (3A) Fahr, E., Rohlfing, W.,Fresenius’ Z . Anal. Chem., 243,43 (1968); CA, 70, 54661 _ _(1969). _ . ~ (4A) Franke, ’R., Thiele, K:, “Physicochemical Methods in Clinical Laboratories,” Volk und Gesundheit (Berlin), I, p 200, 11, p 232 (1969); CA, 71, 88367 (1969). (5A) Henry, J. B., Beeler, M.F., Copeland, B. E., Wert, E. B., Amer. J . Clin. Pathol., 52, 296 (1969). (6A) Juvet, It. S., Jr., Cram, S. P., ANAL. CHEM.,42, llt (1970). (7A) Kingsley, G. R., ibid., 41, 1411-351% (1969). (8A) Margoshes, LI., Scribner, B. F., ibid., 42, 3981t (1970). (9A) Purdy, W. C., Melville, 11. S., ibid. (81, P 32.4. (10A) Reynolds, hI. D., “Clinical Chemis; try for the Small Hospital Laboratory, C. C Thomas, Springfield, Ill., 1969, p 196. (11A) Scott, R. M., “Clinical Analysis by Thin-Layer Chromatography Techniques,” Ann Arbor Humphrey Science Publishers, Inc., Ann Arbor, Mich., 1970, p 227. (12A) Searcy! R. L., “Diagnostic Biochemistry, RIcGraw-Hill Book Co., New York, N. Y., 1969, p 660. (13A) Street, H. U., Advan. Clin. Chem., 12. 217 (1969). (14A) Strickland, 11. D., ANAL. CHEM., 42.3212 119701 \--. --I

(15A) Thalmank, K., Z . Med. Labortech., 10, 270 (1969); CA, 72, 118273 (1970). (16A) Tietz, N . W.. Ed. “Fundamentals of Clinical Chemistry,” W. B. Saunders Co., Philadelphia, Pa. 1970, p 981. (17A) Varlev. H.. Ed.. “Practical Clinical Biochemi&y,’” John Wile & Sons, Inc., New York, K. Y., 4 t i ed., 1967, p 802. (18A) Weygand, F., Fresenius’ 2. Anal. Chem., 243, 2 (1968); CA, 70, 54746 (1969). (19A) White, C. E., Weissler, A., ANAL. CHEM.,42, 57R (1970). (20A) Winefordner, J. P., Vickers, T. J., ibid., p 206R. ~


(1B) Abadi, D. M.,Clin. Chem., 15, 35 (1969). (2B) Anderson, N . G., Anal. Biochem., 28,545 (1969). (3B) Arthur, E. P., Carlsen, E. N., Stevenson. G. W.. (to Beckman Instruments, Inc.), U.’S.~ Patent, 3,398,079 (C1204-195), Aug. 20,1968, (AppL June 4, 1964; 3 pp). Continuation-in-part of U.S. Patent, 3,147,081; CA, 61, 12318 (1964); CA, 70, 17519 (1969).



(2OB)-Harvey, 11. A., Anal. Biochem., 29, 58 (1969). (21B) Hughes, L. A., (to Electronic Instrument Co.), U.S. Patent, 3,449,081 (C1. 23-253: B Old). Jun. 10.’ 1969. (Appl. Mar.’29, 1965; ’5 pp). (22B) Hughes, L. A,, Tressel, W. J., Flavell, E. R., (to Nuclear-Chicago Corp.), Ger. Offen., 1,948,625 (Cl. G Oln), Apr. 9, 1970, (U.S. Appl. Oct. 1, 1968; 30 pp); CA, 73, 63139 (1970). (23B) Johnson, 1). lt., Nadeau, It. G., Nieuweboer, G., Truett, W. L., (to du Pont de Nemours, E. I. & Co.), Fr. 1,520,338 (Cl. G O h ) , Apr, 5, 1968, ( U S . Appl. Apr. 26, 1966; 10 pp); CA, 71, 27853 (1969). (24B) Jolley, R. L., Pitt, W. W., Jr., Scott, C. D., Anal. Biochem., 28, 300 (1969). (25B) Kaffczyk, F., Helger, R., Lang, H., Clin. Chem., 16, 663 (1970). (26B) Kaffczyk, F., Helger, It., Lang, H., Scheuerbrandt, G., zbzd., 16 (43), 526 (1970). (27B) Kahn, A. R., Spracklen, S. B., (to Beckman Instruments Inc.), U.S. Patent, 3,436,329 (Cl. 204-195; C 23c B Olk), Apr. 1, 1969, (Appl. Ilec. 11, 1964; 5pp). (28B) Kiess, R. W., (to Bio-Dynamics, Inc.), Brit. Patent, 1,151,868 (Cl. G Oln), May 14, 1969, (Appl. May 2, 1966; 10 pp); CA, 71,36345 (1969). (29B) Kuz’min, S. V., Matveev, V. V., Pressman, E. K., Sandakhchiev, L. S.,

Biokhimiya, 34, 706 (1969); CA, 71, Biok

120315 (1969). (30B) Lefar, M. S., Lewis, A. D., ANAL. CHEM.,42 (3), 79A (1970). (31B) Levkoff, A . H., Westphal, Westphal, M.C., Finklea, J. F., Amer. J . Clin. Pathol., Fink 54,562 (1970). 54,5 (32B) Loebl, H., (to Joyce, Loebl and Co., 132B) Ltd.). Brit. Patent. 1.149.383 (C1. G Oln);’ Apr. 23, 1969, ‘(Appl. May 7, 1965; 5 pp); CA, 71, 19485 (1969). (33B) Luckey, M.J., U.S. Patent, 3,437,449 (Cl. 23-254; G Oln), Apr. 8, 1969, (Appl. June 8,1966; 6 pp). (34B) Makin. H. L. J., Warren. P. J., ‘ Clin. Chim.’ Acta, 29, 443, (1970). f35B) Natelson. S.. (to Scientific Indus‘ t i e s , Inc.). U.S. Patent, 3,489,525 (Cl. 23-253; G Oln), Jan. 12, 1970, (Appl. Aug. 25, 1967; 9 pp); C A , 72, 118418 (\1*4711) ”.”,.

(36B) Oliva, W. E., Schultz, C. A., (to Baxter Laboratories, Inc.), US., Patent, 3,407,133 (Cl. 204-299), Oct. 22, 1968, (Appl. Jun. 18, 1965; 7 pp). (37B) Pitt, W. W., Jr., Scott, C. D., Johnson, W. F., Jones, G., Jr., Clzn. Chem., 16, 657 (1970). (38B) Pressman, B. G., Methods Enqmol., 10,714 (1967). (39B) Rait, J. M., U.S.Patent, 3,415,627 (Cl. 23-253), Dec. 10, 1968, (Appl. June 11, 1962-Mar. 29, 1966; 9 pp); CA, 70, 44785 (1969). (40B) Stuart, J. L., NASA Contract, Rep. 1969, NASA-CR-103420, 82 pp. Avail. CFSTI, from Sci. Tech. Aerosp. Ren.. 7. 3296 (1969): CA. 72. 28726 (1970). ’ (41B) Thacker, L. H., Pitt, W. W., Jr., Katz, S., Scott, C. D., Clzn. Chem., 16, 626 (1970). (42B) Timmins, 12. S., DeFilippi, R. P., (to Abcor, Inc.), U.S. Patent, 3,518,982 (Cl. 128-2; A 61b), Jul. 7, 1970, (Appl. Feb. 20, 1968; 10 pp); CA, 73, 73786 (1970). (43B) Toren, E. C., J r . , Eggert, J. A., Sherry, A. E., Hicks, G. P., Clzn. Chem., 16, 215 (1970). (44B) Unicam Instruments, Ltd., Fr. Patent, 1,494,561 (C1. G Oln, A 616), Sept. 8, 1967, (Swiss Appl. Sept. 27, 1965; 12 pp); CA, 71, 120460 (1969). (45B) Vaills, Laurent (Ministere de 1’Agriculture-Service Veterinare). Fr. Patent, 1,514,348 (Cl. G Oln, A Blb), Feb. 23, 1968, (Appl. Jan. 12, 1967; 10 PP). (46B) Vestergaard, P., Clin. Chem., 16, 651 (1970). (47B) Wadso, I., Chem. Weekbl., 65, 9 (1969). (48B) White, D. It., Offerman, J. L., Bio-Med. Eng., 4, 362 (1969); C.4, 71, 120287 (1969). (49B) Williams, D. L., Doig, A. It., Jr., Korosi. A.. ANAL. CHEM.. 42. 118 (1970): ’ (50B) Winkelman, J., Amer. J . Clin. Pathol., 51, 804 (1969). I





(IC) Ahuja, J. N., Basis, M. L., Clin. Chem., 15 (106), 815 (1969). (2C) Ibid., 16 (69), 531 (1970). (3C) Alpert, N. L., ibid., 15, 1198 (1969). (4C) Ambrose, J. A., Advan. Autoinat. Anal., Technicon Int. Congr., 1, 25 (1969). (5C) Anderson, N. G., Clin. Chim. $eta, 25,321 (1969). (6C) Anderson, N. G., Amer. J . Clin. Pathol., 53, 778 (1970). (7C) Azrow., G.., Anal. Biochem.. 28, 130 ‘ (1969). (8C) Baillod, C. R., Boyle, W. C., Environ. Sci. Technol., 3, 1205 (1969); CA, 72, 5816 (1970).




33 R

(9C) Barnard, W. P., Logan, R. W., Clin. Chim. Acta, 29, 401 (1970). (1OC) Bartels, H., Boehmer, M., Advan.

Automat. Anal., Technicon Znt. Congr., 1, 33 (1969); CA, 73,22018 (1970).

(1lC) Belfield, A., Goldberg, D. M., Clin. Chem., 15,931 (1969). (12C) Bennet, A., Gartelmann, D., Mason, J. I., Owen, J. A., Clin. Chim. Acta, 29, 161 (1970). (13C) Bide, R. W., Anal. Biochem., 30, 271 (1969). (14C) Block, W. D., Jarrett, K. J., Jr., Amer. J . Med. Technol., 35, 93 (1969). (15C) Bradley, D. W., Tappel, A. L., Anal. Biochem., 33,400 (1970). (16C) Broughton, P. M. G., Buttolph, M. A., Gowenlock, A. H Neill, D. W., Skentelbery, R. G., J . C i k . Pathol., 22, 278 (1969). (17C) Buckle, A. L. J., Riley, C., (to Vickers Ltd.), U.S. Patent, 3,489,521 (Cl. 23-253; G Oln), Jan. 13, 1970, (Appl. Jun. 20, 1966; 18 pp); CA, 72, 129355 (1970). (18C) Burr, R. G., Clin. Chem., 15, 1191 (1969). (19C) Clarke, D. D., Nicklas, R., Automat. Anal. Chem., Technicon Symp., 3rd 1967, 1, 181 (1968), Mediad. Inc.,

White Plains, N. Y. (20C) Cornish, C. J., Neale, F. C., Posen, S., Amer. J . Clin. Pathol., 53, 68 (1970). (21C) Craig, C. It., Azzaro, A. J., Frame, B. K., Hunt, W. A., Advan. Automat. Anal., Technicon Int. Congr., 2, 189 f1969\.

(22C)-Dahms, H., (to IBM Corp.), Fr. Patent, 1,526,569 (Cl. G O h ) , May 24, 1968, (U.S. Avul. Jul. 15, 1966: 13 DV): -. 1

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(\-.,.-,. iwn)

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I ,

Blood Clotting Factors, Gasometric Analysis, and pH

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35 R

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Cations and Anions

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(15H) Hattner, R. S., Johnson, J. W., Bernstein D. S., Wachman, A., Brackman, J., &in. Chim. Acta, 28,67 (1970). (16H) Hieftje, G. M., Malmstadt, H. V., ANAL.CHEM.,41, 1735 (1969). (17H) Hokl, J., Minks, J., Acta Vet. (Czech.)(Bmo), 38, 479 (1969); CA, 73, 63038 (1970). (18H) Hunt, B. J., Manery, J. F., Clin. Chem., 16,269 (1970). (19H) Ionescu, F., Igiena, 17, 429 (1968); CA, 71, 19441 (1969). (20H) Ke, D. J., Regier, L. W., Power, H. E., ANAL.CHEM.,41, 1081 (1969). (21H) King, J. S., Jr., Buchanan, R., Clin. Chem., 15, 31 (1969). (22H) Kupke, I., Sauer, D., Fresenius’ 2. Anal. Chem., 250, 101 (1970); CA, 73, 42291 (1970). (23H) Lazarou, N., Lab. Delo, 11, 688 (1969); CA, 72, 75499 (1970). (24H) Levy, A. L., Katr, E. M., Clin. Chem., 16, 840 (1970). (25H) Lewin, M. R.,, Wills, M. R., Baron, D. N., J. Clzn. Pathol., 22, 222 (1969). (26H) Martinek, R. G., J . Amer. Med. Technol., 32, 337 (1970). (2713) Miyada, D. S., Kazuko, I., Matsuyama, G., Clin. Chem., 16 (56), 528 (,-i- w . - ,n. ~ (28H) Moser, G. B., Gerarde, H. W., ibid., 15, 376 (1969). (29H) Muranaka, H. Kazui, M., Kanou, T.,Inouye, H., sudo, K., Rinsho Ryori, 17,489 (1969); CA, 72, 75513 (1970). (30H) Neefus, J. D., Cholak, J., Saltzman, B. E., Amer. Ind. H y g . Ass. J., 31, 96 (1970); CA, 73,830 (1970). (31H) Ohara, K., Newton, J. L., J a p . J . Physiol., 18, 632 (1968); CA, 70, 17470 (1969). (32H) Paletta, B., Panzenbeck, K., Clin. Chim. Acta, 26, 11 (1969). (33H) Parekh, A. C., Jung, D. H., ibid., 27, 373 (1970). (34H) Pfordte, K., Ponsold, W., Z. Gesamte Inn. Med. Ihre Grenzgeb, 23, 534 (1968); CA, 70, 34953 (1969). (35H) Pioda, L. A. R., Simon, W., Bosshard, H. R., Curtius, H. C., Clin. Chim. Acta, 29, 289 (1970). (36H) Puchtler, H., Meloan, S.N., Terry, M. S..J . Histochem. Cvtochem.,. 17,. 110 (igsgj. (37H) Pybus, J., Clin. Chim. Acta, 23, 309 (1968). (38H) Pybus, J., Bowers, G. N., Jr., Clin. Chem., 16, 139 (1970). (39H) Ramirez-Munoz, J., ANAL.CHEM., 42,517 (1970). (40H) Reeves, R. E., Malin, L. K., Anal. Biochem., 28, 282 (1969). f41H) Robertson. W. G..’ Clin. Chem.. ‘ A&, 24, 149 (1969). (42H) Rupe, C. O., (to Miles Laboratories, Inc.), U.S. Patent, 3,447,904 IC1. 23-230: G Oln). Jun. 3. 1969, (Appl. Sept.’25, 1964;‘s pp); CA, 71; 36343 (1969). (43H) Sach, C., Bourdeau, A. M., Balsan, S., Presle, Y., Ann. Biol. Clin. (Paris), 27,487 (1969); CA, 72, 28748 (1970). (44H) Sakaguchi, T., Taguchi, K., Bunseki, Kagaku, 19, 200 (1970); CA, 73, 11249 (1970). (45H) Sanui, H., Pace, N., Anal. Biochem., 25, 330 (1968). (46H) Savory, J., Wiggins, J. W., Heintges, M. G., Amer. J . Clin. Pathol., 51,720 (1969). (47H) Sideman, L., Murphy, J. J., Jr., Wilson. D. T.. Clzn. Chem., 16, 597 f 1970).‘ (48H) Singer, L., Armstrong, W. D., Vogel, J. J., J . Lab. Clin. Med., 74, 354 (1969). (49H) Wise, W. M., Kurey, M. J., Baum, G., Clin. Chem., 16, 103 (1970). .



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. . (1968). (18J) Smernoff, R. B., hilurphy, J. D., Kameda, N., Clin. Chem., 16 (86), 534 (1970). (19J) Stolz, P., Rost, G., Honigmann, G., 2. Med. Labortech., 9, 215 (1968). (205) Timms, A. R., Kelly, L. A., Spirito, J. A., Engstrom, R. G., J. Lipid Res., 9, 675 (1968). (215) Wildgrube, J., Erb, W., Boehle, E., 2. Klin. Chem. Klin. Biochem., 7, 514 (1969); CA, 71, 120349 (1969). (225) Williams, J. H., Kuchmak, M., Witter, R. F., Clin. Chim. Acta, 25, 447 (1969). (235) Witter, R. F., Kuchmak, M., Williams, J . H., Whitner, V. S., Winn, C. L., Clin. Chem., 16, 743 (1970). (245) Zoellner, N., Wolfram, H., Wolfram, G., 2. Klin. Chem. Klin. Biochem., 7, 339 (1969); CA, 71,67863 (1969). Enzymes

(1K) Al-Khalidi, U. A. S., Aftimos, S., Musharrafieh, S., Khuri, N. N., Clin. Chim. Acta, 29, 381,(1970). (2K) Amador, E., Price, J. W., Marshall, G., Amer. J . Clin. Pathol., 51, 202 (1969). ~ - -(3K) A;&, D. A,, Coyle, M., Clin. Chem., 15 (50), 785 (1969). (4K) Ayavou, T., Feuill. Biol., 10, 45 (1969); CA, 73, 84506 (1970). (5K) Babson, A. L., Arndt, E. G., Sharkey, L. J., Clin. Chim. Acta, 26, 419 (1969). (6K) Babson, A. L., Tenney, S. A., Megraw, R. E., Clin. Chem., 16, 38 (1970). (7K) Belfield, A,, Ellis, G., Goldberg, D. M., ibid., p 396. (8K) Ben-Hayim, G., Gromet-Elhanan, Z., Avron, M., Anal. Biochem., 28, 6 (1969). (9K) Bergmeyer, H. U., Bernt, E., Rey, H. G. (to Boehringer, C. F., and Solhne

J. C., Clin. Chim. Acta, 26, 459 (1969). (11K) CamDanhi, R. Z..TaDia, R. A,. Sarnat, W., Natelson, S., Clik. Chem.; 16,44 (1970). (12K) Ceska, M., Birath, K., Brown, B., Clin. Chim. Acta, 26, 437 (1969). (13K) Crowley, L. V., Alton, M., Clin. Chem., 15 (8), 760 (1969). (14K) Dalal, F. R., Winsten, S., ibid., 16 (30), 523 (1970). (l5K) Darrow, D. A., Amador, E., Amer. J . Clin. Pathol., 53, 60 (1970). (16K) Doumas, B., Biggs, H. G., Clin. Chim. Acta, 23, 75 (1969). (17K) Dror, Y., Sasson, H. F., Watson, J. J., Johnson, B. C., ibid., 28, 291 (1970). (18K) Fitzgerald, M. Y. &I., Fennelly, J. J., McGeeney, K., Amer. J. Clin. Pathol., 51, 194 (1969). (19K) Fridhandler, L., Berk, J. E., Clin. Chem., 16, 911 (1970). (20K) Guilbault, G. G., ANAL. CHEM., 42,334R (1970). (21K) Guilbault, G. G., Hieserman, J. E., Anal. Biochem., 26, 1 (1968). (22K) Guilbault, G. G., Hieserman, J. E., ANAL.CHEM.,41, 2006 (1969). (23K) Hall, F. F., Culp, T. W., Hayakawa, T., Ratliff, C. R., Hightower, N. C., Amer. J . Clin. Pathol., 53, 627 (1970). (24K) Hines, J. D., Love, D. S., Peart, M. B., J . Lab. Clin. Med., 73, 343 ( 19691. (2;K) Homer, G. M., Yott, B., Lim, J. G., Amer. J. Clin. Pathol., 51, 287 (1969). (26K) Itoh, H., Srere, P. A., Anal. Biochem., 35, 405 (1970). (27K) Jamieson, A. D., Pruitt, K. M., Caldwell, R . C., J. Dent. Res., 48, 483 (1969); CA, 71, 56893 (1969). (28K) Johnson, R. B., Jr., Clin. Chem., 15, 108 (1969). (29K) Kaplan, J. C., Shore, N., Beutler, E., Tech. Bull. Regist. Med. Technol., 38, 274 (1968). (30K) Klein, B., Foreman, J. A., Searcy, R. L., Clin. Chem., 16, 32 (1970). (31K) Zbid., 15 (48), 784 (1969). (32K) Klein, B., Foreman, J. A., Searcy, R. L., Anal. Biochem., 31, 412 (1969). (33K) Koedam, J. C., Clin. Chim. Acta, 23,63 (1969). (34K) Kupfer, D., Munsell, T., Anal. Riochem., 25, 10 (1968); C A , 70, 751 (1969). (3jK) Kulhanek, V., Czech. Patent 132,877 (Cl. G Oln), Jun. 15, 1969, (Appl. Jan. 17, 1968; 2 pp); CA, 73, 84572 (1970). (36K) Lippi, U., Guidi, G., Clin. Chim. Acta, 28, 431 (1970). (37K) Loeb, W. F., Stuhlman, 11. A,, Clin. Chem., 15, 162 (1969). (38K) Lorentz, K., Oltmanns, D., ibid., 16,300 (1970). (39K) Mahadevan, S., Dillard, C. J., Tappel, A. L., Anal. Biochem., 27, 387 (1969). (40K) Maruna. F. L.. Clin. Chim. Acta. 25.'133 (1969'1. (41K') Menache, R., Gaist, L., ibid., 26, ' 5gi (1969). (42K:) Miller, L. A., Ashe, E. G., Clin. Chem., 15 (29), 774 (1969). (43K) Monk, P., Wadso, I., Acta Chem. Sca nd., 23, 29 (1969). (44K '1 Moss, D. W., Clin. Chem., 16, 5001 (1970). (45K') Nagode, L. A., Koestner, A., Steinmeyer, C. L., Clin. Chim. Acta, 26, 45 (1969 i. (46K) Pinto, P. U. C., Kaplan, A., Van Dreal, P. A., Clin. Chem., 15,349 (1969).

(47K) Pinto, P. U. C., Van Dreal, P. A., KaDlan. A.. ibid.. D 339. (48Kj Po$en,'S., ibbd:, 16, 71 (1970). (49K) Pragay, D. A., Chilcote, M. E., Least, C., ibid., 15 (46), 783 (1969). (5OK) Redalieu, E., Nilsson, I. M., Nilsson. J. L. G., Kjaer-Pedersen. D. L., Folkers; K., Znt. 2. Vitaminforsch, 38; 345 (1968); CA, 70, 17484 (1969). (51K) Ressler, N., Clin. Chem., 15, 575 (1969). (52K) Rieder, S. V., Otero, M., ibid., p 727. (53K) Rinderknecht, H., Geokas, M. C., Carmack. C.. Haverback. B. J.. Clin. Chim. A d a , 29,481 (1970): (54K) Roth, M., Methods Biochem. Anal., 17, 189 (1969). (55K) Roy, A. U., Clin. Chem., 16, 431 (imn) \--.-/. (56K) Russell, J . C., Tougas, D., Taylor, A. W., ibid., p 900. (57K) Szasz, G., ibid., 15, 124 (1969). (58K) Take, S., Berk, J. E., Fridhandler, L., Clin. Chim. Acta, 26, 533 (1969). (59K) Uete, T., Asahara, RI., Tsuchikura, H., Clin. Chem., 16, 322 (1970). (60K) Uete, T., Tsuchikura, H., Ninomiya, K., ibid., p 412. (61K) Uete, T., Wasa, M., Shimogami, A,. ibid.. 15. 42 (1969). (62K) Van Hindel; E., Clin. Chim. Acta, 29,349 (1970). (63K) Van Munster, J. J., Trijbels, J. M. F., Van Heeswijk, P. J., Schut-Jansen, B., Moerkerk, C., ibid., p 243. (64K) Wacker. W. E. C.. Coombs. T. L..' ' Ann. Rev. Bbochem., 38; 539 (1969). (65K) Whitaker, J. F., Clin. Chim. Acta, 24, 23 (1969). (66K) Wilkinson, J. H., Clin. Chem., 16, 733 (1970). (67K) Zbid., p 882. (68K) Wilkinson, J. H., Boutwell, J. H., Winston, S., ibid., 15, 487 (1969). (69K) Wilkinson, J. H., Steciw, B., ibid., 16.370 119701. (70K)- Winsten; S,., , Wilkinson, J. H., Boutwell, J. H., zbzd., 15,.496 (1969). (71K) Wolf, M., Dinwoodie, A., Morgan H. G., Clin. Chim. Acta, 24, 131 (1969): Function Tests

(1L) Bartels, H., Boehmer, M., 2. Klin. Chem. Klin. Biochem., 7, 444 (1969); CA, 71, 109697 (1969). (2L) Bartels, H., Cikes, M., Clin. Chim. Acta, 26, 1 (1969). (3L) Dybkaer, R., Hertz, H., Scand. J . Clin. Lab. Znvest., 25, 151 (1970). (4L) Fischl, J Eichhorn, F., Ruttenberg, A., hilGor, Ch., Clin. Chem., 16, 331 (1970). (5L) Fried, R., Hoeflmayr, J., (to Haury, Dr. H., Chemische Fabrik). Ger. Patent, 1,279,385, (Cl. G Oln), Oct. 3, 1968, (Appl. Mar. 31, 1966; 2 pp); CA, 70, 17521 (1969). (6L) Green, S. E., (to Smith, Kline and French Laboratories), Ger. Offen. 1,801,278, (Cl. G Oln), Jun. 26, 1969, (U.S. Appl. Oct. 6, 1967; 13 pp); CA, 71, 67880 (1969). (7L) Harereaves. T.. Clin. Chim. Acta. 26,331?1969).' ' (8L) Hawkins, K. I., Clin. Chem., 16, 749 (19701. (9L)-Iwata,' J., Bunseki Kagaku, 18, 431 (1969); CA, 71, 46583 (1969). (1OL) Jablonski, P., Owen, J. A..' Advan. Clin. Chem., 12, 309 (1969). (11L) Kulenda, 2.. Prochazka. M.,Getburtsch. Frauenheilk, 29, 812 (1969); CA, 72, 616 (1970). (12L) Kulhanek, V., Appelt, J., Czech. Patent, 128,803 (Cl. G Oln), Aug. 15, 1968. (ADD].Mar. 9. 1967: 2 DD). (13Lj-Kur'ashige, A.,' Nippon E&eikensa Gishikai Zasshi, 18, 822 (1969) (Japan); CA, 72, 129343 (1970). '


37 R

(8N) Dawes, R. L. F., Park, C., J. Med. Lab. Technol., 27, 55 (1970). (9N) Dawson, J. B., Walker, B. E., Clin. Chim. Acta, 26,465 (1969). (10N) Deguchi, M., Bunseki Kagaku, 17, 1124 (1968); CA, 70, 9282 (1969). (11N) Evans, G., Searcy, R., (to Hoffman-LaRoche, Inc.), U.S. Patent, 3,506,404 (Cl. 23-230; G Oln), Apr. 14, 1970, (Appl. Dec. 19, 1967; 6 pp); CA, 73,913 (1970). (12N) Fink, D. W., Pivnichny, J. V., Ohnesorge, W. E., ANAL.CHEM.,41, 833 (1969). (13N) Foster, D., Trusell F. C., Anal. Chim. Acta, 47, 154 (1969): (14N) Goodwin, J. F., Williams, A. L., Jr., Clin. Chem., 15 (60) 791 (1969). (l5N) Gregorowicz, Z., dwapulinska, G., Piwowarska, B., Chem. Anal. (Warsaw), 13, 887 (1969); CA, 70,44717 (1969). (16N) Groenewald., T.., ANAL.CHEM..41. ‘ 1012 (19691. (17N) Ichida, T., Nobuoka, hI., Clin. Chim. Acta, 24,299 (1969). (18N) Klein, B., Lucas, L., Searcy, R. L., ibid., 26, 517 (1969). (19N) Klein, B., Weber, B. K., Luctts, L . , ?yeman, J. A,, Searcy, R. L., ibid., p (20N) Leddicotte, G. W., AEC Symp. Ser., 13, 455 (1968); CA, 71, 8823j II l-Q_6_Q_I ,. .


(1M) Affonso, A., Clin. Chim. Acta, 22, 466 (1968). (2M) Frazini, C., Lab. Diagn. Med., 13, 279 (1968); CA, 71, 88253 (1969). (3M) Lee, K.-T., Ling, A.-M., Mikrochim. Acta, 5, 995 (1969); CA, 71, 120387 ( 1969). (4M)-%iaas,A. H. J., Hamelink, M. L., de Leeuw, R. J. M., Clin. Chim. Acta, 29,303 (1970). (5M) Martinek, R. G., J. Amer. Med. Technol., 32, 37 (1970). (6M) Matsubara, T., Susumu, S., Clin. Chim. Acta, 23,427 (1969). (7M) Nalbandian, R. M., Henry, R. L., Nichols, B. M., Camp, F. R., Jr., Wolf, P. L., Clin. Chem., 16, 945 (1970). (8M) Roy, R. B., Shaw, R. W., Connell, G. E., Doris. L., J . Lab. Clin. Med.. 74. (9M) Schrumpf, A., Tidsskr. h’orske Laegeforen., 89, 407 (1969); CA, 71, 6779 I1969 \. \~._.,

(10M) Van Assendelft, 0. W., Van Kampen, E. J., Zijlstra, W. G., Proc. Kon. Ned. Akad. Wetensch., Ser. C, 72, 249 (1969); CA, 71, 57444 (1969). (1lM) Weichselbaum, T. E., (to Brunswick Corp.), U.S. Patent, 3,446,751 (Cl. 252-408; C OSk, G Oln), May 27, 1969, (Appl. Nov. 29, 1965; 2 pp); CA, 71, 36340 (1969). Metals

(1N) Ajemian, R. S., Whitman, N. E., Amer. Ind. Hyg. Ass. J . 30, 52 (1969). (2N) Baginski, E. S., Epstein, E., Weiner, L. M., Zak, B., Clin. Chem., 15 (61), 791 (1969). (3N) Biomfield, J., MacMahon, R. J . Clin. Pathol., 22, 136 (1969). (4N) Boiteau, H. L., Bliaux, M. Gelot, S., Ann. Biol. Clin., (Paris), 67 (1970); CA, 73, 84481 (1970). (5N) Bouda, J., Clin. Chim. Acta, 51 1 I l Q f i Q I


(21N) Lehmann, H. P., Kaplan, A,, Clin. Chem., 16 (60), 529 (1970). (22N) Liplakk, I. L., Ushkova, L. N., Lab. Delo, 7, 399 (1969). (23N) Lorentz, K., Flatter, B., Mikrochim. Acta, 5, 1023 (1969); CA, 71, 98872 (1969). (24N) Mahoney, J. P., Sargent, K., Greland, M., Small, W., Clin. Chem., 15, 312 (1969). (25N) Matsuba. Y.. Takahashi., Y.., Anal. ‘ Biochem., 36, 182 11970). (26N) Mikac-Devic, D., Clin. Chim. Acta, 23,499 (1969). (27N) Zbid., 24, 293 (1969). (28N) Zbid., 26, 127 (1969); CA, 71, 109687 (1969). (29N) Nomoto, S.,Sunderman, F. W., Jr., Clin. Chem., 16, 477.(1970). (30N) Olson, A. D., Hamlin, W. B., ibid., 15,438 (1969). (31N) O’Malley, J. A., Hassan, A., Shiley, J., Traynor, H., ibid., 16, 92 (1970). (32N) Ramirez-Munoz, J., Roth, N. E., Flame Notes, 5, 38 (1970); CA, 73, 63059 (1970). (33N) Roth, M. E., Ramirez-Munoz, J., ibid., 4, 25 (1969); CA, 72, 18993 (1970). (34N) Sabatino, J. D., Weber, 0.. W., Padmanabhan, G. R., Senkowski, B. Z., ANAL.CHEM.,41, 905 (1969). (35”) Sachdev, S. L., West, P. W., Anal. Chim. Acta, 44, 301 (1969). (36N) Schaller, K. H., Kuehner, A., Lehnert,, G., Blut., 17, 155 (1968); CA, 70, 26276 (1969). (37N) Schilt, A. A., Taylor, P. J., ANAL. CHEM.,42, 220 (1970). (38N) Schmidt. W.. Fresenius’ 2. Anal. ‘ Chem., 243, 198 (1968); CA, 70, 54747 ( 1969). (39N) Srivastava, S. P., Pandya, K. P., Zaidi, S. H., Analyst, (London), 94, 823 (1969); CA, 72,28768 (1970). (40N) Stookey, L. W., ANAL.CHEM.,42, 779 (1970). (41N) Sunderman, F. W., Jr., Nomoto, S.,Clin. Chem., 16 (58), 529 (1970). (42N) Zbid.. D 903. (43N) Suttei, E., Platman, S. R., Fieve, R. R., ibid., p 602. (44N) Szilagyi, L., Pahoki, I., Kiserl. Orvostud, 21, 554 (1969); CA, 72, 97204 (1970).


(45N) Tavenier, P., Hellendoorn, H. B. A,, Clin. Chim. Acta, 23, 47 (1969). (46N) Tompsett, S. L., Proc. Ass. Clin. Biochem., 5, 125 (1968). (47N) Tszyu, N. P., Bocharova, R. I., Men’Kov, A. A., Lab. Delo, 5, 275 (1969); CA, 71, 78048 (1969). (48N) Uny, G., Brule, M., Spitz, J., Ann. Biol. Clin. (Paris), 27, 387 (1969); CA, 71, 98855 (1969). (49N) Vengerskaya, Kh. Ya., Salikhodzhaev, S. S., Nov. Obl. Prom.-Sanit. Khim., p 237 (1969), Murav’eva, S.I., Ed., fzd “Meditsina,” Moscow, U.S.S.R.; CA, 72, 619 (1970). (50N) Voskian, H., Rousselet, F., Girard, M. L., Photom. Absorption, At. Flamme, Colloq. Inform. Sci., 203 (1968); CA, 72, 7.5510 (1970). (51N) Walton, It. J., Thibert, R. J., Bozic, J., Holand, W. J., Can. J. Biochem., 48, 823 (1970); CA, 73,63043 (1970). (52N) Woods, A. E., Crowder, R. D., Coates, J. T., Wittrig, J. J., At. Absorption Newslett., 7, 85 (1968). (53N) Yurachek, J. P., Clemena, G. G., Harrison. W. W., ANAL. CHEM.. . 41,. 1666 (1969). (54N) Zak, B., Baginski, E. S., Epstein, E., Weiner, M., Clin. Chim. Acta, 29, 77 . . (1970). ~ - - .

(55N) Zlatkis, A,, Bruening, W., Bayer, E., ANAL.CHEM.,42, 1201 (1970). Nitrogen Compounds

(1P) Beecher, G. R., Whitten, B. K., Anal. Biochem., 36, 243 (1970). (2P) Chalmers, It. A., Watts, I. H., Parekh, A. C., Clin. Chem., 16, 247 (1970). (14R) Kageyama, K.,Nippon Eiseikensa Gishikai Zasshi, 18, 59 (1969); CA, 71, 27838 119691. (15R) Kang, E. S., Gerald, P. S.,Whealan, D., J.Pediat., 76,939 (1970). (16R) Klinenberg, J. R., Kippen, I., J . Lab. Clin. Med., 75, ,503 (1970). (17K) Korf. J.. Valkenburcrh-Sikkema. T.. Clin. Chi&. Acta, 26, 307 (1969). (18R) Martinek, It. G., J . Amer. Med. Technol.. 32. 233 11970). I


Chem.. 16 (98). 536‘119761. (2312) Persson, ’B., Scand.’J. Clin. Lab. Invest., 25, 9 (1970). (24R) Pryce, J. I)., Analyst (London), 94, 1151 (1969); C A , 72, 75478 (1970). (25R) Sandler, M., Ann. Med. Exp. Biol. Fenn., 46, 479 (1968). (26R) Simkin, P. A., Clin. Chem., 16, 191 (1970). (27It) Van Stekeleiiburg, G. J., de Bruyn, J. W., Clin. Chim. Acta, 28, 233 (1970). (28R) Wildenhoff, K. E., Scand. J . Clin. Lab. Invest., 25, 171 (1970). (2911) Zaura, D. S.,Metcoff, J., ANAL. CHEM., 41, 1781 (1969).

(30R) Zender, R., de Torrente, C., Schneider, U., Clin. Chim. Acta, 24, 335 (1969). Proteins


K., Schuett, K., Mueller, I., Hoffmeister, H., 2. Klin. Chem. Klin. Biochem., 8, 92 (1970); CA, 73, 829 (\-_.-,. IQ~o\.

(25) Ackers, G. K., Advan. Protein Chem., 24, 343. (1970); CA, 73, 52868 (1970). (35) Arai, K., Wallace, H. W., Anal. Biochem., 31, 71 (1969). (4s) Atkinson, R. L., Fader, M. L., (to Miles Laboratories, Inc.), Fr. Patent, 1,515,793 (Cl. G Oln), Mar. 1, 1968, ( U S . Appl. May 5, 1966; 6 pp). ( 5 5 ) Beckering, R. E., Jr., Ellefson, R. D., Amer. J . Clin. Pathol., 53, 84 (1970). (6s) Becker, N., Schwick, H. G., Storiko, K., Clin. Chem., 15, 649 (1969). 17s) Bertrand. F.. Watelet. 34.. Genetet. ‘ F., Nabet, P., Paysant, P., Ann. Biol: Clin. (Paris), 27, 735 (1969); CA, 72, 118322 (1970). (8s)Bode, C., Goebell, H.,. Harald, S. E., 2. Klzn. Chem. Klin. Bzochem.. 6. 418

Clin. Chem.,’l6, 362 (19iO). ’ (185) Fried, It., Hoeflniayr, J., (to Haury, Dr. Heina, Chemische Fabrik), Ger. Patent, 1,277,592 (Cl. G Oln), Sept. 12, 1968, (-4ppl. Mar. 26, 1963; 2 pp); C A , 70, 809 (1969). (19s) Gebott. JI. I>., Czirr. Lab. Pract., 1, 25 (1968). (205) Goldenberg, H., Dreweb, P. A., Clin. Chem., 16 (103), 537 (1970). (21s) Goodwin, J. F., Chol, S-Y., ibid., p 24

(2%) Grannis, G. F., ibid., p 486. (23s) Hatch, F. T., Lees, 11. S., Advan. Lipid Res., 6, 1 (1968). (24s) Hatcher, I). W., Anderson, N . G., Amer. J . Clin.Pathol., 52, 64.5 (1969). (255) Hirs, C. H. W., ,Ilethods Enzymol., 11, 325 (1967). (265) Iammariiio, R. M., Clin. Biochem., 2,447 (1969). (275) Kahlke,. W.,, Aerztl. Lab., 14, 495 (1968); (285) Kindmark, C-O., Clin. Chim. Acta, 26,95 (1969). 1295) Klungsoeyr, L., Anal. Biochem., 27, 91 (1969). (30s) Konrad, H., Ger. (East) Patent, 61,640 (Cl. G Oln), May 5 , 1968, (Appl. June 7, 1967; 5 pp). (315) Kostner, G., Albert, W., Holasek, A., Hoppe-Seiiler’s Z . Physiol. Chem., 350, 1347 (1969); C A , 72, 287.i4 (1970). (325) Lewis, L. A , , Lipids, 4, 60 (1969). (335) Lorza, A., Kereszti, Z., 13erencsi, G., Aerztl. Lab., 14, 292 (196s); CA, 70,9271 (19691 --_-, (34s) Luxtc311, G. C., Can. J . Med. l’echnol., 30, 35 & 83 ( 1968).



(355) Martinek, R. G., J . Amer. Med. Technol., 32, 177 (1970). (36s) Ibid., p 345. (375) Mayer, M. M., Miller, J. A., Anal. Biochem., 36,.91 (1970). (38s) Moinuddin, M., Taylor, L., Lipids, 4, 186 (1969). (395) Noble, R. P., J . Lipid Res., 9, 693 (1968). (405) O’Malley, W. E., O’Doherty, D. S., Trans. Amer. Neurol. Ass., 94, 320 (1969); CA, 72, 129299 (1970). (415) Papadopoulos, N. M., Kintzios, J. A., Anal. Biochem., 30,426 (1969). (425) Peters, T., Jr., Clin. Chem., 14, 1147 (1969). (435) Pratt, J. J., Dangerfield, W. G., Clin. Chim. Acta, 23, 189 (1969). (445) Prellwitz, W., Koettgen, E., Aerztl. Lab., 15,20 (1969). (455) Ross, D. L., Brown, K., Amer. J. Med. Technol., 35, 540 (1969). (46s) Sata, T., Estrich, D. L., Wood, P. D. S., Linsell, L. W., J . Lipid Res., 11, 331 (1970); CA, 73, 52886 (1970). (475) Schmidtmann, W., Kaempffer, R., Iteschke, L., Aerztl. Lab., 15, 165 (1969); CA, 71, 88282 (1969). (485) Stewart, L. E., Thomas, J. W., Hull, G. E., Anal. Chim. Acta, 44, 453 (1969). (49s) Ternovoi, A. P., Strigin, V. A., Lab. Delo, 4, 231 (1969); CA, 71, 19420 (1969). (505) Trifonov, St., Aleksandrov, I., Bulg. Akad. h’auk, 13, 239 (1969), (Bulg.); CA, 73, 32206 (1970). (51s) Tsvetanova, E., Nevrol., Psikhiat. n’evrokhir.. 7. 313 (1968). (525) Weeke, B., Ugeskr. Laeger, 131, 1423 (1969); CA, 72, 609 (1970). (535) Werner, hl., hlontgomery, C. K., Jones, A. L., Nussenbaum, S., Clin. Chem., 16, 573 (1970). (54s) Winkelman, J., Ibbott, F. A,, Sobel, C., Wybenga, D. R., Clin. Chim. Acta, 26, 33 (1969). (55s) Winkelman, J., Wybenga, I). It., Ibbott, F. A., ibid., 27, 181 (1970). (56s) Winkelman, J., Wybenga, D. R., Ibbott, F. A., Clin. Chewa., 16, 507 (1970). (57s) Zoellner, N., Groebner, W., Berger, C.. Wolfram. G.. 2. Klin. Chem. Klin. Blochem., 7; 525 (1969); CA, 71, 120364 (1969). Sterols

(1T) Ba nton, R . D., Campbell, D. J., Clin.(?hem., 15, 190 (1969). (2T) Beukers, H., Veltkamp, W. A., Hooghwinkel, G. J. >I., Clin. Chim. Acta, 25, 403 (1969). (3T) Bush, I. E., Advan. Clin. Chem., 12,

57 (1969).

(4T) Ilito, W. It., Shelly, J., Amer. J . Clin. Pathol., 51, 177 (1969). (5T) Eberhagen, I)., Z . Klin. Chem. Klin. Biochem.. 7. 167 11969). hi. P., Bandeira de Mello, (6T) Ferreira; hl. E. J., Hospital (Rio de Janeiro), 75, 1439 (1969); CA, 72, 9765: (1970). (7T) Goebelsmanii, Goebelsmanii, XI., Clzn. Chzm. Acta,

23, 469 (1969). (8T) Gorog, Giirog, S., ANAL.CHEM.,42, 560 (1970). (9T) Jordan. W. J.. Knoblock.’ E. C.. Clzn. Chem., 16, 18 (1970). (10T) Kishi, hI., Nagayama, C., Mizobe, Y.. Ushikoshi. A I . . Tsukamnto. K.. Ogiso, T., Sippon EiGikensa Gishikai Zasshi, 19, 45 (1970); CA, 73, 11267 - I



‘ihIartinek, 11. G., J . Amer. Med. Technol., 32, 64 (1970) 40R

(14T) Muehlbaecher, C. A., Smith, E. K., Clin. Chem., 16, 158 (1970). (15T) Mueller, G., Deut. Gesundheitsw., 24, 1944 (1969); CA, 72, 75407 (1970). (16T) Negrin, A., Clin. Chem., 15, 829 (1969). (17T) Parekh, A. C., Jung, D. H., ANAL. CHEM.,42, 1423 (1970). (18T) Pieterse, E. W. M. G., Scholtis, R. J. H., Schmidt, N. A., Clin. Chim. Acta, 26,111 (1969). (19T) Pinkus, G. S., Pinkus, J. L., Clin. Chem., 16, 824 (1970). (20T) Solow, E. B., Freeman, L. W., ibid., p 472. (21T) Ushikoshi, M., Nippon Eiseikensa Gishikai Zasshi, 19, 49 (1970); CA, 73, 11268 (1970). (22T) Van de Calseyde, J. F., Scholtis, R. J. H., Schmidt, N. A., Kuypers, A. M. J., Clin. Chim. Acta., 27, 139 (1970). (23T) Voellmin, J. A., Chromatographia, 3.238 (1970). (24T) Williams, J . H., Kuchmak, M., Witter, R. F., Clin. Chem., 16, 423 (1970). (25T) Wotiz, H . J., Clark, S. J., Methods Biochem. Anal., 18, 339 (1970). (26T) Young, D. G., Hall, P. F., Biochemistry,.8, 2987 (1969). (27T) Zlatkis, A., Zak, R. G., ANAL. CHEM.,29, 143 (1969). Toxicology

(1U) Anders, H., Fachz. Lab., 12, 783 (1968); CA, 70, 9263 (1969). (2U) Bastos, M. L., Kananen, G. E., Young, It. M., Monforte, J. R., Sunshine, I., Clin. Chem., 16, 931 (1970). (3U) Brower, M. E., Woodbridge, J. E., ibid., (112), p 539. (4U) Bruce, R. B., Maynard, W. R., Jr., ANAL.CHEM.,41, 977 (1969). (5U) Burston, G. R., Scot. Med. J . , 14, 55 (1969); CA, 71, 19449 (1969). (6U) Dain, D. W., Trainer, T. D., Clin. Chem., 16, 318 (1970). (7U) Delves, H. T., Analyst (London), 95,431 (1970); CA, 73, 73712 (1970). (8U) Devoto, G., Rass. Med. Sarda, Suppl., 71, 289 (1968); CA, 73, 11271 (1970).

(96jD;etz, A. A., Rubin, H. M., Clin. Chem., 15 ( 5 3 ) , 787 (1969). (IOU) Dymond, L. C., Russell, D. W., Clin. Chim. Acta, 27, 513 (1970). (11U) Einarsson. 0.. Lindstedt. G.. Scand.

(lifij’Foster, 1,. B., Frings, C. S., ibid.,

16, 177 (1970). (18U) Grove. J.. Toseland. P. A.. Clin. ‘ Chim. Acta; 29; 253 (1970j. (16U) Haeger-Aronsen, B., Scand. J. Clin. Lab. Invest., 25, 19 (1970). (17U) Hancock, J. A., Mill, F. L., Miles, J. R., Clin. Chem., 15 (14), 764 (1969). (18U) Haux, P., Natelson, S., Amer. J . Clin. Pathol., 53, 77 (1970). (19U) Johansen, O., Steinnes, E., Int. J . Appl., Radiat. Zsotop., 20, 751 (1969); CA, 72, 626 (1970). (20U) Jones, D., Gerber, 1,. P., Drell, W., Clin. Chem., 16,402 (1970). (21U) Kelly, R. G., Peets, L. M., Hoyt, K. D., Anal. Biochem., 28, 222 (1969). (22U) Krylova, A. N., Rubtsov, A. F., Vopr. Sud. Med., Alin Zhravookhr. SSSR, 328 (1968); CA, 72, 87102 (1970). (23U) Kupferberg, H. J., Clin. Chim. Acta, 29 283 (1970). (24U) Leblsh, P., Finkle, B. S., Brackett, J. W., Jr., Clin. Chem., 16, 195 (1970). (23U) Leric, H., Kaplan, J-C., Broun, G., Clin.Chim. Acta, 29, 523 (1970)


(26U) Lieberman, K. W., Kramer, H. H., ANAL.CHEM.,42, 266 (1970). (27U) Lowe, H. J., “Theory Appl. Gas Chromatop. Int; ,Med., -Hahnemann S mp., 1st 1966, 194-209, H. S. d o m a n , Ed., Grune and Stratton, New York, N.Y., 1968. (28U) Lower G. M., Jr., Murphy, S. B., Bryan, G. $., Clin. Chim. Acta, 29, 421, (1970). (29U) Lubran, M., Med. Clin. N . Amer., 53, 211 (1969). (30U) Morales, R. C., Greene, G. L., Turrialba, 19, 297 (1969); CA, 71, 120456 (1969). (31U) Morselli, P. L., Clin. Chim. Acta, 28,37 (1970). (32U) Nakamura, G. It., Meuron, H. J., ANAL.CHEM.,41, 1124 (1969). (33U) Ono, M., Engelke, B. F., Fulton, C., Bull. Narcotics, 21, 31 (1969). (34U) Pippenger, C. E., Gillen, H. W., Clin. Chem., 15, 582 (1969). (35U) Pippenger, C. E., Scott, J. E., Gillen, H. W., ibid., p 255. (36U) Rathje, A. O., Amer. Ind. Hyg.Ass. J., 30, 126 (1969). (37U) Reanal, F., Brit. Patent, 1,151,538 (Cl. G Oln), May 7, 1969, (Hung. Appl., Jul. 16, 1965; 4 pp); CA, 71, 36342 (1969). ~~.-., (38U) Rodkey, F. L., Collison, H. A., Clin. Chem., 16, 896 (1970). (39U) Routh, J. I., Shane, N. A., Arredondo. E. G.. Paul, W. D., ibid.,. 15.. 661 (1969). ’ (40U) Sabih, K., Sabih, K., ANAL.CHEM., 41, 1452 (1969). (41U) Savory, J., Mushak, P., Sunderman, F. W., Jr., Clin. Chem., 15, (59), 790 (1969). (42U) Schlunegger, U. P., Minn. Med., 52, 175 (1969); CA, 70, 84838 (1969). (43U) Schweitzer, J. W., Friedhoff, A. J., Clin. Chem., 16, 786 (1970). (44U) Segal, R. J., ibid., 15, 1124 (1969). (45U) Sine, H . E., McKenna, M. J., Rejent, T. A., Murray, M. H., ibid., 16, 587 (1970). (46U) Sun, M-W., Stein, E., Gruen, F. W., ibid., 15, 183 (1969). (47U) Tompsett, S. L., ibid., p 591. (48U) Tompsett, S. L., J . Clin. Pathol., 22, 291 (1969). (49U) Toseland, P. A., Scott, P. H., Clin. Chim. Acta, 25, 75 (1969). (50U) Turner, W. J., Turano, P. A., March, J. E., Clin. Chem., 16, 916 (1970). (51U) Van Meter, J. C., Buckmaster, H. S., Shelle , L. L., ibid., p 135. (52U) Walcce, J. E., ibid., 15, 323 (1969). (53U) Weidemann, G., 2. Klin. Chem. Klin. Biochem., 7, 560 (1969); CA, 71, 120443 (1969). (54U) Zurlo, N., Griffini, A. M., Colombo, G., Anal. Chim. Acta, 47, 203 (1969). Vitamins

(1V) Anderson, B. B., Peart, M. B., Fulford-Jones, C. E., J . Clin. Pathol., 23,232 (1970). (2V) Friedner, S., Josephson, B., Levin, K., Clin. Chim. Acta, 24, 171 (1969). (3V) Fujita, A,, Tokuhisa, S., Michinaka, K., Bitamin, 40, 121 (1969); CA, 71, 120316 (1969). (4V) Garry, P. J., Pollack, J. D., Owen, G. hI., Clin. Chem., 16, 767 (1970). (5V) Hansen, L. G., Warwick, W. J., Amer. J . Clin. Pathol., 51, 538 (1969). (6V) Hubmann, B., Monnier, D., Roth, M., Cli?. Chim. Acta, 25, 161 (1969). (7V) Karlin, R., Hours, C., Bertoye, R., Berry, N., Vallier, C., .Morand, H., C. R. SOC.Biol., 163, 881 (1969); CA, 72, 107737 (1970). (8C) Raven, J. L., Robson, M. B., Walker P. L., Barkhan, P., J . Clin. Pathol., 22, 205 (1969).

(9V) Selvaraj, R. J., Susheela, T. P., Clin. Chim. Acta, 27, 165 (1970). (1OV) Tibbling, G., ibid., 23, 209 (1969). Supplamenl

(1) Remp, D. G., Stand. Methods Clin. Chem., 6, 1 (1970).

(2) Kaplan, A.,Savory, J., ibid., p 13. (3). Bowers, G. N., Jr., McComb, R. B., abad.,D 31. (4) Klein, L., ibid.,p 41.

(5) Fernandea, A. A., Jacobs, S. L., ibid., p 57. (6)Nobel, S., ibid., p 73. (7)Goodwin, J. F., ibid., p 89. (8) Sunderman, F. W., Jr., ibid., p 99. (9) Jacobs, S. L., Fernandez, A. A., ibid., p 107. (10) Kingsley, G. R., Tager, H. S., ibid., p 115. (11) Klein, G. C., Cooper, G. R., ibid., p 127. (12) Lanchantin, G. F., ibid., p 137.

(13) Sax, S. M., Moore, J. J., ibid.,p 149. (14) Cooper, G. R., McDaniel, V., ibid., p

, rn lay.

(15) Gambino, S. R., ibid., p 171. (16) McNair, R. D.,ibid., p 183. (17) Pybus, J., Bowers, G. N., Jr., ibid., n 189.

Coatings M. H. Swann, M. I. Adams, and G. G. Esposito, U.S. Army Coating and Chemical laboratory, Aberdeen Proving Ground, Md.


HIS BIENNIAL REVIEW covers the period from January 1969 through December 1970 and includes the authors’ choice of important contributions t o the analysis of coating materials that have appeared since the previous summary (102). It is hoped that the attempt to be selective has not caused omission of commendable contributions. Other reviews of general (82) and specific nature were made within this period, and a monthly index to coatings literature is published in each issue of Paint Technology from a review of 26 journals on coatings and includes reference to articles on analysis. A number of books on various phases of polymer analysis have been published in this two-year period, most of which are of special interest to the coating analyst. Myers and Long (79) have edited volume 2 of the Treatise on Coatings, in which each chapter treats separate physical techniques for coating characterization. T h e analysis of both raw materials and finished surface coatings are included and the applications of gas chromatography, thermal analysis, microscopy, and spectroscopy are discussed in separate chapters. A 456-page book containing 740 infrared spectra was published (16) b y t h e Chicago Society for Paint Technology. Stevens published a 198-page book (99) on polymer analysis b y gas chromatography, which was intended t o serve as a n up-to-date monograph on the subject. Although the bibliography contains 380 references, the application of gas chromatography to drying oil analysis was omitted. The final chapter of the latest book in the series of monographs on chemical analysis, “The Analytical Chemistry of Nitrogen and its Compounds’’ (101) concerns nitrogen polymers. It contains a number of procedures for analyzing cellulose

nitrate, amino-formaldehydes, acrylonitrile, polyamides, polyurethanes and other synthetic polymers. Other books have appeared on such subjects as X-ray diffraction methods ( I ) , polymer characterization (10), analysis of polyurethanes (22), infrared spectra and structure of long chain polymers (26), adhesive and coating testing (SI), infrared analysis of polymers, resins and additives (47), and the chemical analysis of plastics (61). GENERAL ANALYTICAL SCHEMES

T h e application of thermal methods of analysis to organic coatings was discussed by Holsworth (46). T h e characterization and identification of some film-forming polymers b y differential scanning calorimetry was described (43). Yeagle (122) illustrated the qualitative and quantitative analysis of polyester resins with nuclear magnetic resonance spectra for eleven polyols, nine acids, and eight known polyester systems. Scott (94) published a paper on the application of X-ray diffraction in the paint industry, pointing out its usefulness in quality control of finished products as well as individual pigments. The application of X-ray emission analysis to pigmented paint samples in the form of dried thin films was investigated (70) and several elements such as titanium, lead, and calcium were determined in a single sample preparation. Other applications of X-ray emission analysis were presented (16) in which samples in solid, liquid, and powder form were included. Two foreign publications (6, 57) reported the use of thin-layer chromatography for identifying acids and polyols in some coatings and for determining compositional uniformity.

Valero (110) discussed the analysis of paints in general by a variety of methods and included 102 literature references. A general discussion of the applications of infrared spectrophotometry to coatings was published (10.4). Low and hIark (68) studied a variety of clear and opaque coatings on steel and glass by infrared interference spectroscopy, and discussed (69) the principles involved in examining a number of clear metal coatings by infrared Fourier transform spectroscopy. &‘ithers (120) outlined the application of internal reflection spectroscopy t o coatings. Sherwood (95) explained his technique for combining micro-pyrolysis with infrared spectrophotometry t o identify wire enamels using l/Z-inch lengths of wire. Three foreign publications dealt with compositional analysis b y a combination of pyrolysis with gas chromatography. Hagen (38) used both gas and thinlayer chromatography to examine such polymers as poly(viny1 chloride) , polyurethanes, nitrogen resins, phenolics, and poly(methy1 methacrylate). Sonntag (97) made experimental comparison of three different techniques for pyrolyzing three polymers and Audebert (4) reviewed the progress made in gas chromatographic analysis of polymers for t h e period 1954 to 1967. Another review (86) was made of the applications of gas chromatography in the paint industry and included solvent, oil, resin, plasticizer, polymer, and emulsion analysis. A paper was published (77) on t h e application of trimethylsilylation and gas chromatography to the determination of the composition of polyamide resins. T h e use of gel permeation chromatography for quantitatively analyzing moisture-cure polyurethane coatings was illustrated (59) in which the commercial coatings were


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