Clinical chemistry - American Chemical Society


Clinical chemistry - American Chemical Societypubs.acs.org/doi/pdf/10.1021/ac60355a005Similaremphasis of this review is...

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Clinical Chemistry Nathan Gochman Veterans Administration Hospital, San Diego, CA 92 16 1

Donald S. Young National lnstitutes of Health, Bethesda, MD 200 14

We (20A)presented a selective review of developments in clinical chemistry which covered the period from December 1970 to November 1972. This report continues the review to cover the scientific literature from December 1972 to November 1974. This interval has seen the introduction of new automated instrumentation, including computercontrolled systems, the greatly increased use of radioisotope-based analyses, frequently combined with immunochemical techniques, and the increased utilization of enzymes as reagents to improve the specificity of measurements of biological constituents. As before, the primary emphasis of this review is on the development and evaluation of the analytical methodology of clinical chemistry, with only limited references to the extensive role of this specialty in clinical diagnosis, patient therapy, and biomedical research.

BOOKS AND REVIEWS Henry, Cannon, and Winkelman (23A)edited the second



edition of “Clinical Chemistry: Principles and Technics,” which is a comprehensive review of methodology, normal values, and interpretation of laboratory tests used in the biochemical evaluation of disease. Bodansky and Latner edited Volume 15 (IOA) and Volume 16 (11A)of “Advances in Clinical Chemistry”, each of which contains five in-depth review articles. Individual chapters from both of these volumes will be cited under appropriate categories. Biggs and Woodson (8A)authored a useful text, “Clinical Biochemistry”, which emphasizes the molecular structures and reactions which are pertinent to human physiology. Baron’s (7A)paperback, “A Short Textbook of Clinical Biochemistry”, provides information on present-day chemical pathology for medical students and clinicians. “Practical Clinical Enzymology and Biochemical Profiling: Techniques and Interpretations” by Wolf, Williams, and Von der Muehll (41A)provides helpful information in a rapidly advancing field. In their text, “Biochemistry: A Case-Oriented Approach”, Montgomery, Dryer, Conway, and Spector (29A)have emphasized the presentation of case studies of diseases that have a biochemical basis. Zilva and Pannall (45A)wrote a handbook for the interpretation of clinical chemistry procedures, with specific guidelines for tests used to confirm the diagnosis. In his book, “Fluorometric Techniques in Clinical Chemistry”, Elevitch (18A)presented many principles and procedures for the application of fluorometry to the detection of substances in biological specimens. Nelson (30A)edited a comprehensive volume on “Blood Lipids and Lipoproteins”, which contains contributions by 21 authors on analytical methods, composition, and metabolism. In his book, “Drug Interactions”, Hansten (21A)has detailed the interAuthors have not been supplied with free reprints for dlstributlon. Extra copies of the review issue may be obtained from Special issues Sales, ACS, 1155 16th St., N.W., Washington, DC 20036. Remit $4 for domestlc U S . orders; add $0.50 for additional postage for foreign destinatlons.

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actions of drugs with each other in the patient, and also lists the effects of numerous drugs upon tests commonly performed in the clinical laboratory. Nerenberg (31A)covered instrumentation, methodology, and clinical applications in the book, “Electrophoretic Screening Procedures”. In his book, “Clinical Aspects of the Plasma Proteins”, Kawai (24A)reports much useful information on the interpretation of protein electrophoretic patterns from a variety of body fluids. Scriver and Rosenberg (35A)have provided a comprehensive book on “Amino Acid Metabolism and Its Disorders”. Rose, Milgrom, and van Oss (33A) edited “Principles of Immunology”, a multi-author review of fundamentals and clinical applications of immunology. In his book, “Electronic Instrumentation in the Clinical Laboratory”, Ackerman (6A)provides a description of the electrooptical principles used, and specific examples of common instrumental systems. Volume 37 of “Advances in Enzymology”, edited by Meister (27A)contains review articles on lactate dehydrogenase, succinate dehydrogenase, and threonine deaminases, while Volume 39 (28A)includes discussions of the hexokinases, rhodanase, and amidotransferases, and glutamate dehydrogenase. Skelley et al. (38A)reviewed the principles of radioimmunoassay (RIA), normal values, and techniques for separation of bound and free antigen, and listed commercial suppliers of reagents for the analysis of numerous blood and urine substances. Zettner (43A)reviewed equilibrium techniques for competitive binding assays, and Zettner and Duly (44A)discussed the advantages and disadvantages of sequential saturation techniques for binding assays. Hawker (22A)and Conway and Muller-Eberhard (16A) reviewed the principles of RIA and applications to the measurement of biological constituents. Yalow (42A)discussed problems encountered in RIA applications, and Challand et al. (14A)reviewed the organization and practice of RIA as a diagnostic procedure in the clinical laboratory. Bloom (9A)reviewed the application of RIA to measurement of the hormones of the gastrointestinal tract. Recent developments in the enzymatic and immunological determination of myocardial infarction were reviewed by Cohen and Morgan (15A). Dubowski (17A) reviewed the applications of breath analysis in clinical chemistry including measurement of gases and volatile drugs, and gave examples of analytical techniques and instrumentation which have been used. Free and Free (19A)reviewed the methodology of urinalysis and discussed the clinical information yielded by urine studies. Sauberlich et al. (34A)reviewed currently available biochemical tests, including vitamins and minerals, for the assessment of human nutritional status. The application of digital computers in the clinical laboratory, with a listing of commercially-available systems, was discussed by Laessig and Schwartz (25A).Seitz and Neary (36A)reviewed principles, instrumentation, and applications of chemi- and bioluminescence in chemical analysis. The production and function of catecholamines during various nor-

Nathan Gochman is Chief, Clinical Chemistry Section at the Veterans Administration Hospital, San Diego, CA, and Associate Professor In Residence of Chemistry and Pathology at the University of California, San Diego. Following receipt of his BS from Brooklyn College in 1955 and his PhD in biochemistry from Northwestern University in 1958, he was employed as a biochemist at G. D. Searle & Co. in Chicago. From 1962 to 1968 he served as research chemist and director of research for Technicon Instruments Corp., Tarrytown, NY. Through 1972 he was Assistant Chief of the Clinical Chemistry Service at the Clinical Center of the National Institutes of Health in Bethesda. His research interests are primarily concerned with the development of automated analytical methods and instrumentation for the clinical laboratory. Dr. Gochman is actively involved in the promotion of improved quality of clinical laboratory performance and currently serves as President of the National Committee for Clinical Laboratory Standards. He is also an active member of the American Association of Clinical Chemists, having served on various local and national committees and as Chairman of the Capital Section. He is a member of the editorial board of Clinical Chemistry. Among his other organizational memberships are the American Chemical Society, AAAS, Sigma Xi, the N.Y. Academy of Sciences, and the Association of Clinical Scientists.

Donald S.Young is Chief, Clinical Chemistry Service, Clinical Pathology Department, Clinical Center, National Institutes of Health, Bethesda, MD. He has a medical degree from the University of Aberdeen and a PhD in chemical pathology from the Universitv of London He was trained King and I D P Wootton at the under E Royal Postgraduate Medical School In London Dr. Young was a visiting scientist at the National Institutes of Health until appointed to his present position in 1967 He serves on the Editorial Boards of Analytlcal Letters and Clincal Chemistry His research interests include laboratory automation and high-resolution analytical techniaues in clinical chemistry

mal and abnormal conditions were described by von Euler (39A).Winkel (40A)reviewed the types of multivariate approaches which can be used for interpreting clinical chemistry results, including recognition of abnormal patterns and clusters. Caraway (12A) traced the historical origins and scientific development of clinical chemistry up to 1948. The procedure of immunoisoelectric focusing was reviewed by Catsimpoolas ( 1 3 A ) , and Latner (26A) discussed the clinical and biochemical applications of isoelectric focusing techniques. Rodbard (32A) reviewed the various types of electrophoresis. Siest (37A) edited part of the proceedings of a 1972 conference, “Reference Values in Human Chemistry”, which is concerned with the derivation of normal ranges and the factors which influence them. The proceedings ( 1 A ) of the Technicon International Congress, 1972, were reported in nine volumes, and include numerous articles on automated, continuous-flow methods, quality control, and biochemical profiling. The June 1973 ( 3 A ) and July 1974 ( 4 A ) issues of Clinical Chemistry, respectively, contain the abstracts of the papers presented at the 25th and 26th National Meetings of the American Association of Clinical Chemists. These abstracts have not been cited individually; however, they include over 400 papers, many of which appear as published articles in later issues. Clinical Chemistry published the proceedings of the 5th ( 2 A ) and 6th ( 5 A ) annual “Symposium on Advanced Analytical Concepts for the Clinical Laboratory” held in Oak Ridge, 1973 and 1974, respectively.

APPARATUS AND EQUIPMENT Automation. Gochman (15B) identified recent trends relating the development and application of various technologies to the automation of clinical laboratory analyses. An overview of automation and its relationship to clinical chemistry was presented by Martin (24B). Tiffany (41B) reviewed instrumentation features and clinical applications of the centrifugal fast analyzers. The adaptation of the centrifugal analyzer system to fluorescence measurements and its application to phosphatase assays with fluorogenic substrates was described by Tiffany et al. (43B).Tiffany et al. (42%) reported the development of a dynamic, multicuvette fluorometer-spectrophotometer based on the centrifugal analyzer principle. Burtis et al. ( 7 B ) ,Scott and Burtis (35B),and Scott et al. (36B)reported various aspects of the design and development of a miniaturized version of the centrifugal analyzer. Renoe et al. (29B) developed a minidisc module for the centrifugal analyzer which extends the range of radiation sources and wavelength selectors available to the system. Burtis et al. (8B) reported improvements and analytical applications of the technique of dynamic introduction of liquids into centrifugal analyzers. An all-digital measurement system for use in centrifugal analyzers was developed by Avery et al. (2B).Several variables in instrument performance, including cuvette pathlength, temperature, wavelength accuracy, etc., and their effects on reaction-rate enzyme assays were noted by Maclin et al. (22B). Schwartz et al. (33B) reported an extensive chemical and clinical evaluation of the new Technicon computer-controlled, multichannel automated continuous-flow analyzer (SMAC). An assessment of the Vickers M-300 multichannel analyzer for the determination of eleven serum constituents, including three enzymes, was reported by Bick et al. ( 4 B ) . Taylor and Bell (40B) reported an evaluation of the Instrumentation Laboratory Clinicard Analyzer. Campbell ( 9 B ) described the Abbott Bichromatic Analyzer 100, and Alexander ( 1 B ) discussed the DuPont Automatic Clinical Analyzer. Horowitz and Stewart (19B) evaluated the Hycel Mark XVII multichannel analyzer, and Baba and Fujita ( 3 B ) discussed the methodology of the Hycel Mark X. Lustgarten et al. (21B) evaluated an automated, selectiveion electrolyte analyzer, the Technicon STAT/ION, for the simultaneous determination of serum sodium, potassium, and chloride. Morel1 et al. (26B) modified the Gilford 3400 Automatic Enzyme Analyzer to increase sample-throughput of reaction-rate analyses. Horn et al. (18B) reported modifications in the AutoAnalyzer I1 system to correct for sensitivity changes due to temperature variation. The Technicon flow-through colorimeter was modified by Borst et al. ( 6 B ) so that absorbances at two different wavelengths could be measured simultaneously, and which can be used for automatic blank subtraction. Neeley et al. (28B) designed a signal comparator for the colorimeter-recording system of continuous-flow analyzers, which permits air bubble passage through the flowcell and increases rates of analysis. The design and performance features of a miniaturized, high-speed continuous-flow analyzer, assembled partly from commercially available components, was described by Neeley et al. (27B). Toren et al. (44B, 45B) developed an automated, spectrophotometric analyzer which is computer-controlled to perform additional biochemical tests on a sequential basis depending on results obtained on initial testing. Spencer et al. (39B) reported the design of an automated, flowcell polarization fluorometer, and its application to the enzyme-inhibitor assay of antitrypsin and the antigen-antibody assay ANALYTICAL CHEMISTRY, VOL. 4 7 , NO. 5, APRIL 1975

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of insulin. Dessy and Titus (13B) reviewed electronic devices for computer interfacing of analytical instruments. Scott (34B) reviewed the high-pressure, ion-exchange chromatography systems available for analyzing body fluids. Recent developments in the application of high resolution liquid chromatography to the separation of complex biological mixtures were discussed by Scott et al. (37B). Scott and Pitt (38B) described a differential highpressure liquid chromatograph with dual parallel columns. Robinson et al. (30B) described an automated apparatus for the quantitative analysis of volatile compounds in urine. An instrumental detection system based on light scattering, which is applicable to agglutination-based reactions, was reported by Blume et al. (5B). Den Boer et al. (12B) designed an automated, titrimetric apparatus for the measurement of plasma bicarbonate. Miscellaneous. Rose and Steadman ( 3 I B ) designed a portable, solid-state hemoglobinometer for use with the cyanmethemoglobin method. An electrochemical detection system suitable for monitoring the eluate from high resolution liquid chromatographs for trace amounts of organic constituents was described by Kissinger et al. (20B). Hinckley ( I 7 B ) discussed systems and applications for two related high resolution, displacement electrophoretic techniques, transphoresis, and isotachophoresis. Helman and Ting (16B) described a procedure for counting of gamma emitters involved in clinical radiochemical assays in liquid scintillation counters, which uses a second sample support vial containing a scintillator fluid. The design and evaluation of a filter fluorometer in which a photomultiplier detector is used in the photon-counting mode was reported by Curry et al. ( J I B ) .Cook et al. (IOB)described the design of a silicon-target, vidicon tube, flame spectrometer and its application to the simultaneous determination of serum sodium and potassium. Rowe et al. (32B) designed a compact apparatus for the routine performance of electrophoretic separations in vertical acrylamide gel slabs for up to forty samples simultaneously. The potential errors in the use of piston-activated sampling pipets, including temperature-volume effects, were discussed by Ellis ( 1 4 B ) . Wenk and Lustgarten (46B) evaluated the precision and accuracy of delivery of manually operated sampling pipets from six manufacturers. Mavrodineanu and Lazar (25B) described the design and production of 10-mm light path quartz cuvettes for high-accuracy spectrophotometry, which are available as Standard Reference Materials from the National Bureau of Standards, The design of high precision thermometers at the National Bureau of Standards (SRM 933 and 934) for use in the clinical laboratory and calibrated at temperatures of 0,25,30, and 37 OC were reported by Mangum (23B).

QUALITY ASSURANCE Laessig et al. (14C) reported on the improvement of interlaboratory precision of automated multichannel analyzers in a state-wide survey program in which a common lot of serum calibration material was used by all participants. Georges (5C) discussed the relative error contribution of volumetric and colorimetric stages in analytical procedures. Laessig et al. ( I 3 C ) compared instrument manufacturers’ label values for aspartate aminotransferase activity in calibration sera with reference method values. The sources of error and criteria necessary for evaluation of photometers of analytical systems in clinical laboratories were reviewed by Richterich et al. (19C).Beeler (2C) surveyed the performance of spectrophotometers in over one hundred different clinical laboratories. A detailed analysis of the effects of various types of photometric errors in equilibrium and rate 18R

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analyses based on absorption spectroscopy. was presented by Pardue et al. (16C). Aronsson et al. ( I C ) discussed the application of computer simulation techniques to assess the factors which influence the quality of analytical methods in clinical chemistry. Krause and Lott ( I I C ) presented examples of the application of the simplex method to optimization of analytical conditions for clinical chemistry methods and instrumentation. Silex optimization of analytical methods was discussed by Morgan and Deming (15C), with use of the measurement of serum cholesterol as an example. Harris (7C) discussed some of the problems involved in estimation of biological constituent correlations from single-sample measurements, and recommended multiple, independent samples. The use of patient data for calculation of the indirect normal range and for quality control was reported by Glick ( 6 0 . Winkelman et al. (29C)reported on a survey of serum chemical values determined by the SMA 12/60, and the influence of climate, sex, weight, etc., on the normal ranges found. Methods for estimating normal ranges, including an evaluation of a gaussian transformation method, were discussed by Reed and Wu ( I 8 C ) . Statland et al. (24C) examined the within-day variation of the serum concentration of 22 constituents in healthy subjects. The effects of exercise and diet (physiological and methodological) on the intraindividual variation of these measurements was also reported by Statland et al. (25C). Westgard and Hunt (27C)described the application and limitations of commonly used statistical tests in method comparison studies, with examples obtained from glucose method studies. Criteria for judging the precision and accuracy of clinical chemistry methods were formulated by Westgard et al. (26C). Cali (3C) and Schaffer (21C) discussed programs of the National Bureau of Standards for reference materials and methods. Payne (17C) applied the standard deviation, calculated from successive duplicates, to the assessment of quality control data. A laboratory computer program for the monitoring and calculation of reaction-rate data from enzyme assays performed by automated spectrophotometer systems was reported by Hicks et al. (IOC). Heilbron et al. (8C) examined quantitative warning methods based on the measurement of serum controls to signal day-to-day displacements in serum calcium determinations. Elion-Gerritzen ( 4 C ) used a combination of the absorbance value of a standard and the analyzed value of a control serum for quality control of photometric methods. The various methods available for quality control and processing of RIA data were reviewed by Rodbard (20C). Schwartz (22C) extensively discussed the influence of specimen quality and the effects of drug and non-drug interferences on diagnostic biochemical procedures. Significant differences were observed by Ladenson et al. (12C)between plasma and serum values for calcium, glucose, inorganic phosphorus, potassium, and total protein. Helman et al. (9C) discussed methods of eliminating the errors caused by hemolysis and bilirubin-induced color quenching in RIA. The 42-day stability of 18 chemical constituents in serum, under several defined conditions of storage, was reported by Wilson et al. (28C). Simon et al. (23C) reported on the prevalence of hepatitis-associated antigen in 36 commercially prepared control sera.

ANIONS, CATIONS, TRACE ELEMENTS Anions. Feldkamp et al. ( 1 1 0 ) critically studied the factors that cause positive interference with the mercuric thiocyanate, continuous-flow method for chloride determination. Mahner et al. ( 2 6 0 ) developed an automated method

for measuring chloride in serum, urine, and sweat, based on the formation of an iron(II1)-chlorine complex in the presence of perchloric acid. A direct determination of chloride in sweat with an ion-selective electrode, was described by Szabo et al. ( 3 7 0 ) . Hall et al. ( 1 7 0 ) reported a potentiometric method for the determination of total ionic fluoride in plasma using an ion-selective electrode. The measurement of serum bromide was modified by Torrance ( 3 8 0 ) . Garry et al. ( 1 5 0 ) described a continuous-flow colorimetric method for the determination of dialyzable iodine in urine, based on the ceric-arsenite reaction. Davies et al. ( 8 0 ) compared vanadate and stannous fluoride as reducing compounds in the continuous-flow method for the determination of inorganic phosphate. Cartier and Thuillier ( 7 0 ) used the UDPG-pyrophosphorylase reaction and fluorometric measurement of NADPH to determine inorganic pyrophosphate in plasma. A three-stage, coupled enzymatic method for the determination of inorganic phosphate in serum or urine with the CentrifiChem centrifugal analyzer was developed by Pesce et al. ( 3 1 0 ) . Leskovar and Weidmann ( 2 4 0 ) precipitated serum sulfate with a known amount of barium chloride, and then measured the residual free barium by flame photometry. Cations. Anand et al. ( 2 0 ) noted that small errors in serum potassium values were due to the effect of varying sodium/potassium ratios, and recommended methanol as a diluent to eliminate this interference. Robertson et al. ( 3 3 0 ) compared atomic absorption spectrophotometry and flame emission for the determination of therapeutic concentrations of lithium in blood and urine. Pickup et al. ( 3 2 0 ) evaluated, and suggested protocol revisions in, the atomic absorption “reference” method for the determination of total serum calcium. A colorimetric micromethod for the determination of serum calcium, in which dimethyl sulfoxide is used to obviate protein precipitation, was reported by Baginski et al. ( 3 0 ) . Morin ( 3 0 0 ) developed a single-reagent, cresolphthalein complexone procedure for the colorimetric determination of serum calcium. The anaerobic storage of serum in disposable plastic syringes for the ion-selective electrode determination of ionized calcium, was recommended by Subryan et al. ( 3 5 0 ) .Ladenson and Bowers ( 2 2 0 ) evaluated the Orion ion-selective electrode system for the measurement of ionic calcium in serum, and determined normal values for a population of healthy adults. The ion-selective electrode technique was applied by Ladenson and Bowers ( 2 3 0 ) to investigate the correlation of ionic calcium levels with total calcium in normal subjects and those with various disease states. Zief et al. ( 4 7 0 ) investigated the properties of magnesium gluconate as a potential high purity standard reference material for the measurement of magnesium by atomic absorption. Trace Elements. Kniseley et al. ( 2 0 0 ) described a multichannel emission spectrometric method for the determination of numerous trace elements in microliter samples of biological fluids. Bearse et al. ( 4 0 ) used proton-induced X-ray emission to perform multielemental analysis (Fe, Cu, Zn, Se, Rb) of whole blood. Haeckel et al. ( 1 6 0 ) compared eight colorimetric methods for the measurement of unsaturated iron-binding capacity (IBC), with a continuous-flow immunoprecipitin method for the direct determination of transferrin. A non-dialysis, continuous-flow method for the determination of serum iron with TPTZ was reported by Jones and Deadman ( 1 9 0 ) .Fraser ( 1 2 0 ) improved a widely used automated procedure for the measurement of serum total IBC. Megraw et al. ( 2 9 0 ) described a singletube technique for the measurement of both serum iron and total IBC. A micromethod for the determination of serum iron and total IBC by flameless atomic absorption

spectrophotometry, with use of a protein-free filtrate, was developed by Yeh and Zee ( 4 5 0 ) . McClean and Purdy ( 2 8 0 ) determined iron in serum by a coulometric procedure. Yee and Goodwin ( 4 4 0 ) described a colorimetric method for the sequential measurement of serum copper and iron in a single aliquot of protein-free filtrate, in which the bis(1piperidylthiocarbony1)disulfide and ferrozine complexes are measured. A procedure for the simultaneous colorimetric determination of copper and iron in serum was reported by Fried and Hoeflmayr ( 1 3 0 ) .Ward et al. ( 4 2 0 )described a microsampling cup procedure for the rapid atomic absorption determination of serum copper. Flameless atomic absorption was used by Kurz et al. ( 2 1 0 )to measure zinc in microliter quantities of serum and urine. Carter ( 6 0 ) described a colorimetric micromethod for the determination of serum zinc with 1-(2-pyridylazo)-2-naphthol.A multichannel atomic absorption spectrometer for the simultaneous analysis of zinc, copper, and cadmium in biological fluids was developed by Falchuk et al. ( 1 0 0 ) . Ediger and Coleman ( 9 0 ) used the Delves cup technique to measure cadmium in blood by atomic absorption. Van Ormer and Purdy ( 3 9 0 ) reported an atomic absorption method for the determination of manganese in urine. The analysis of manganese in biological materials by longpath atomic absorption was described by Suzuki and Wacker ( 3 6 0 ) . Versieck et al. ( 4 1 0 ) utilized neutron activation analysis for the determination of manganese, copper, and zinc. The concentration of these trace elements in the serum and packed erythrocytes of subjects with acute and chronic hepatitis, and post-hepatitis cirrhosis were reported by Versieck et al. ( 4 0 0 ) .Galenkamp et al. ( 1 4 0 ) reported the determination of aluminum in blood by neutron activation analysis. Hohnadel et al. ( 1 8 0 ) determined the concentrations of nickel, copper, zinc, and lead by atomic absorption, in the sweat of healthy subjects undergoing dry heat exposure. The measurement of gold in serum by atomic absorption was reported by Aggett ( I D ) . Maessen et al. ( 2 5 0 ) described a flameless atomic absorption method for the direct determination of gold, cobalt, and lithium in plasma, with use of a carbon rod atomizer. The determination of arsenic in urine by X-ray spectroscopy was reported by Mathies ( 2 7 0 ) . Young and Christian ( 4 6 0 ) used GLC for the measurement of selenium in body fluids. Cardenas and Mortenson ( 5 0 ) reported a colorimetric method for the determination of molybdenum and tungsten in the presence of each other in biological fluids, with use of selective extraction and 4-methyl-1,2-dimercaptobenzene.The measurement of chromium in urine by flameless atomic absorption was reported by Schaller et al. ( 3 4 0 ) .Widau et al. ( 4 3 0 ) determined uranium in urine by a fluorometric method.

CARBOHYDRATES Glucose. Cooper ( 8 E )presented a comprehensive review of methods for the determination of glucose in blood. Carey et al. ( 5 E ) evaluated the Gochman and Schmitz automated glucose oxidase method for serum glucose as adapted to the SMA 12/60. Several continuous-flow methods for the determination of serum glucose, which are based on glucose oxidase, were compared by Romano (26E) for performance and specificity characteristics. Seiter et al. (28E) developed a continuous-flow, fluorometric micromethod for the determination of glucose in whole blood, in which the peroxide formed in the glucose oxidase reaction oxidizes homovanillic acid to a fluorophore. Chinh ( 6 E ) noted that negative interference of uric acid is due to bleaching of the chromophore in the glucose oxidase-peroxidase method for glucose A N A L Y T I C A L C H E M I S T R Y , VOL. 47, NO. 5, APRIL 1975

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in which azinodiethylbenzthiazolinonesulfonic acid (ABTS) acts as the chromogen. Kunz and Stastny ( 1 4 E ) described an analytical system for the measurement of serum glucose, in which glucose oxidase immobilized on controlled-pore glass acts as an on-stream reagent. The ferricyanide-luminol system was used by Bostick and Hercules ( 4 E ) to measure the hydrogen peroxide formed through the action of immobilized glucose oxidase on glucose in blood. Llenado and Rechnitz ( 17 E ) reported a continuousflow method for the determination of glucose based on measurement of iodide released by oxidation with an ionselective electrode. Nagy et al. (21E) described the use of an iodide ion-selective electrode system for the measurement of glucose. Coburn and Carroll ( 7 E ) described a manual and a continuous-flow colorimetric determination of serum glucose based on coupling the hexokinase/glucose-6-phosphatedehydrogenase/NADP reaction to diaphorase and a tetrazolium (INT) indicator. The hexokinase/G-6-PD reaction was adapted by Hasson et al. (13E) to the determination of serum glucose with the Rotochem centrifugal analyzer. Richterich et al. (25E) described the measurement of glucose by the hexokinase/G-6-PD reaction with the Greiner Electronic Selective Analyzer. The use of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides, which functions with NAD as a coenzyme, in the hexokinase-coupled determination of serum glucose was reported by Bondar and Mead ( 3 E ) .Lever et al. (16E) compared the specificity of 4-hydroxybenzoic acid hydrazide (HBAH) with other reagents for the determination of glucose. The HBAH colorimetric reaction was adapted to the continuous-flow measurement of serum glucose by Fingerhut (IOE). Amador ( I E ) used the dinitrosalicylic acid reaction for the continuous-flow determination of urinary glucose. Yee and Goodwin (30E) evaluated the factors affecting the o -toluidine procedure for measuring blood glucose. Seven current continuous-flow methods for the determination of serum glucose, including three glucose oxidase-based procedures, were evaluated by Pennock et al. (24E). Miscellaneous. Mopper and Gindler (19E) described a tetrazolium blue color reagent for the quantitation of sugars which are chromatographically separated. Maurer and Christophis (18E) used sorbitol dehydrogenase and NAD for the determination of xylitol in blood and urine. Manual and continuous-flow colorimetric methods for the determination of urine hexoses and pentoses, based on their reaction with o-anisidine, were reported by Goodwin and Yee (12E).Sabater and Asensio (27E)used fructokinase for the measurement of fructose in serum. Paper chromatography was used to separate isomaltose and lactose in urine by Vitek and Vitek (29E). Niessner and Beutler (22E) described procedures for the fluorometric determination of intermediates in the glycolytic and citric acid pathways in blood platelets. Gerbaut et al. (11E) presented an improved continuousflow, colorimetric method for the determination of N acetylneuraminic acid in serum, based on the thiobarbituric acid reaction with the oxidation product of the hydrolyzed amino sugar. A continuous-flow procedure for the colorimetric determination of sialic acids and 2-deoxyribose in blood and tissue hydrolyzates was reported by Engen et al. ( 9 E ) .O’Brien et al. (23E)used GLC for the determination of monosaccharides obtained by depolymerization of glycosaminoglycans. Ecteola cellulose and Dowex AG-1 X 2 resin were used for chromatographic separation of urinary glycosaminoglycans, and hexuronic acid-containing oligosaccharides by Lakatos and Di Ferrante (15E). Murphy et al. (20E) described a GLC method for the mea20R

surement of the acetylated derivatives of glucosamine and galactosamine for studies of urinary glycosaminoglycans. Blumenkrantz and Asboe-Hansen ( 2 E ) used m-hydroxybiphenyl for the measurement of uronic acids in urine. A method for the measurement of submicrogram quantities of heparin in plasma was reported by Yin et al. (31E).

ENZYMES Schwartz (49F) reviewed the role of various serum enzyme and isoenzyme measurements in the diagnosis of cancer. Wilkinson and Robinson (66F) described the effects of high-energy phosphate compounds on the release of intracellular enzymes. Methods of preparation and analytical applications of immobilized enzymes were discussed by Weetall (64F).Forrester et al. (17F)reported the use of immobilized enzymes in the microcalorimetric assay of substrates. A computer-interfaced centrifugal analyzer was applied by Tiffany et al. (60F) to the evaluation of steadystate, enzyme kinetic parameters. Szasz (57F) reported the effects of assay temperature on the activities of several clinically important enzymes. Semi-automated, reactionrate methods and reference values for lactate and malate dehydrogenases, and aspartate aminotransferase in human cerebrospinal fluid were reported by Sharpe et al. (50F). Moss (34F) discussed the advantages of reaction-rate monitoring over fixed-incubation time methods for enzyme assay. Howell et al. (24F) observed that the residual fluorescence after reaction of NADH with acetaldehyde and alcohol dehydrogenase was a useful criterion for purity of the NADH preparation. Specifications for enzyme reference materials with regard to their application, e.g., calibration, intra-laboratory control, etc., were discussed by Fasce et al. (15F).Belfiore et al. ( 3 F )reviewed several categories of enzymes which undergo changed activity levels in serum in subjects with diabetes mellitus. Phosphatases. Proksch et al. (39F) described a continuous-flow adaptation of the colorimetric determination of alkaline phosphatase, with use of sodium thymolphthalein monophosphate as substrate. The use of ammonium thymolphthalein monophosphate was proposed by Morin (33F) as a substrate for alkaline and acid phosphatase determinations in serum, and he noted increased stability of reagents in comparison to the sodium salt. Smith and Fogg (51F) presented evidence that the observed increase in alkaline phosphatase activity of lyophilized serum control material may be due to the breakdown of an enzyme-lipoprotein complex. Several human and non-human tissue sources of alkaline phosphatase isoenzymes were evaluated by Compton et al. (12F)for potential use in reference sera. Statland et al. (55F) described the use of the centrifugal analyzer in conjunction with chemical inhibitors for the fractionation of serum alkaline phosphatase isoenzymes. The association of various hepatobiliary disorders in patients with their serum alkaline phosphatase isoenzyme patterns, as determined by cellulose acetate electrophoresis, was reported by Rhone et al. (42F).Forman et al. (16F) used polyacrylamide gel electrophoresis and heat inactivation to differentiate alkaline phosphatase of hepatobiliary from osteoblastic origin. Agar gel electrophoresis was used by Sundblad et al. (56F) for the separation of alkaline phosphatase isoenzymes, in which sera from patients with obstructive jaundice served as markers. Bethune et al. ( 5 F ) described an AutoAnalyzer I1 method for the determination of 5’-nucleotidase activity in serum, based on measurement of liberated ammonia with phenol-hypochlorite. Milisauskas and Rose (31F) prepared a monkey anti-serum to human prostatic acid phosphatase, and used this material for immunochemical quantitation of

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the isoenzyme. Sample collection techniques for use with acid phosphatase determinations were investigated by Modder (32F),and it was concluded that acid-citrate-dextrose plasma is preferred to serum or heparinized plasma. Aminotransferases. Smith and Taylor (52F) compared reaction-rate methods for aminotransferases with endpoint colorimetric determinations on the AutoAnalyzer 11, and concluded that there is little difference in precision or operating costs. Rej et al. (40F)recommended the inclusion of pyridoxal phosphate in the reagents for aspartate aminotransferase (AsAT) assay because of the increased activity noted. An extensive investigation was reported by Rej and Vanderlinde (41F) of the effects of serum proteins on the determination of AsAT activity. Rodgerson and Osberg (44F) documented sources of error, particularly involving side reactions, in spectrophotometric reaction-rate methods for the measurement of aminotransferase activities in serum. Dehydrogenases. Berry et al. (4F) reported on the variability of NADH preparations with respect to lactate dehydrogenase (LDH) inhibitor, and the effect on the precision of analyses of LDH in serum. Nathan et al. (35F) discussed the utility of measuring the slowest-moving isoenzyme of LDH (LD-5) with the automated ACA method as an index of hepatic disease. Reaction-rate methods for the determination of total, heat-stable, and urea-stable LDH activity in serum with the GEMSAEC centrifugal analyzer were reported by Hanson and Freier (22F).Burd et al. (IOF) described the preparation and characterization of an anti-M LDH anti-serum for the immunochemical assay of LDH isoenzymes in human serum. Several techniques were investigated by Brydon and Smith ( 9 F ) , including heat and urea-stability, for the determination of the heart isoenzyme of LDH. Nicholson et al. (36F) presented methods for the reaction-rate determination of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities in erythrocytes with the CentrifiChem centrifugal analyzer. Creatine Kinase. Phornphutkul et al. (38F) noted several causes for increased creatine kinase (CK) activity in the serum of patients following surgery, including the administration of anesthetic agents. Lau and Guilbault (27F) described a solid-surface fluorescence method for the rapid estimation of CK activity in which micro serum samples are added to a silicone rubber pad impregnated with lyophilized reagent. The direct and indirect coupled enzyme methods for the determination of CK activity in serum were investigated by Dinovo et al. (14F), and problems of high activity underestimation in some coupled systems were noted. Armstrong et al. ( I F ) adapted the fluorometric ninhydrin procedure for the determination of CK activity in serum to continuous-flow automation. Mercer (30F) reported an ion-exchange chromatographic procedure for the separation of tissue and serum CK isoenzymes, which includes quantitation of the fractions with the Rosalki method. Amylase and Lipase. Zinterhofer et al. (67F) reported a reaction-rate, nephelometric method for the determination of amylase activity in serum. Takeuchi et al. (58F) described the electrophoretic separation of amylase isoenzymes in serum on cellulose actate, and densitometric measurement after visualization on a blue starch-agar gel plate. A method for identifying serum amylase isoenzymes after electrophoresis was presented by Spiekerman et al. (54F), in which thin-film plates of agar impregnated with a chromogenic starch substrate are used. Rosalki and Tarlow (45F) compared three insoluble substrate procedures for the measurement of amylase activity in serum with a sac-

charogenic procedure and the Caraway method. A saccharogenic amylase method was compared with four commercial dye-substrate methods by Melnychuk (29F).Mazzuchin et al. (28F) adapted the Phadebas chromogenic substrate amylase procedure to continuous-flow automation. A gel chromatographic technique for isolation and measurement of serum and urine amylase isoenzymes was described by Fridhandler et al. (19F). Zinterhofer et al. (68F)reported a nephelometric method for the determination of lipase activity in serum, in which the rate of decrease in light-scattering is continuously monitored. Tietz and Repique (59F) described a titrimetric method for the measurement of lipase activity in serum, in which the initial rate of fatty acid production is monitored in a pH-stat instrument and recorded. Peptidases. Pate1 and O’Gorman (37F) separated seven zones of y-glutamyl transpeptidase (GGT) activity in serum samples by electrophoresis on Cellogel. The inclusion of excess glutamate in the assay of GGT activity in serum was recommended by Bondar and Moss (7F) to avoid variable enhancement of the reaction. Rosalki and Tarlow (46F) described the adaptation of the reaction-rate determination of GGT activity in serum to the LKB 8600. Tovey et al. (61F) reported a colorimetric reaction-rate method for the determination of cystine aminopeptidase activity in serum, based on measurement of liberated nitroaniline from S-benzyl-~-cysteine-4’-nitroanilide as substrate. Sensitive fluorometric and spectrophotometric methods for enterokinase (enteropeptidase) activity determination, suitable for routine clinical use, were described by Rinderknecht et al. (43F).Geokas et al. (20F) developed a sensitive and specific radioimmunoassay for pancreatic carboxypeptidase B activity in serum. Proteases. Goldberg and Spierto (21F) compared two commercially available RIA kits for the measurement of plasma renin activity, and noted that mean levels were 35% lower by the Squibb vs. the Schwartz-Mann procedure. A selected method for the determination of renin activity in plasma, based on RIA of the liberated angiotensin I, was presented by Lash and Fleischer (26F).Vandermeers et al. (63F) reported a continuous-flow, colorimetric method for the assay of trypsin and chymotrypsin in duodenal fluid, with use of amino acid ester substrates. A colorimetric method for the assay of chymotrypsin, in which a TCA-soluble diazotizable amine is liberated from the substrate N benzoyl-L-tryosyl-p-aminobenzoic acid, was described by Imondi et al. (25F). Wenger and Sundy (65F) developed a continuous-flow, fluorometric method for the determination of functional serum protease-inhibitors, with use of chymotrypsin and casein. Plasminogen was measured by a radiochemical method described by Schmer and Krys (48F), in which 14C and lZ5Iare released from labeled casein because of enzymatic activity. Miscellaneous. Dietz et al. (13F) reported a proposed selected method for the determination of cholinesterase activity in serum, based on the colorimetric measurement of liberated thiocholine from propionylthiocholine with dithiobis(nitrobenzoic acid). Smith (53F) compared six methods for the assay of serum cholinesterase activity in the presence of a number of anti-cholinesterase insecticides. Saifer and Perle (47F) adapted the pH-inactivation of hexosaminidase A to the continuous-flow determination of total enzyme and hexosaminidase B activity, in the serum of suspected Tay-Sachs disease heterozygotes. An automated micromethod for the assay of galactose-l-phosphate uridyltransferase in blood samples was developed by Frazier and Summer (18F), in which the production of NADPH is measured fluorometrically. Beutler and Matsu-

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mot0 ( 6 F ) described a technique for performing many galactokinase assays in whole blood samples, with the use of labeled galactose. A colorimetric method for the determination of tpansketolase activity in as little as 50 ~1 of blood was reported by Basu et al. (2F). Valentine and Frankenfeld (62F)described a spectrophotometric method for the assay of 3-mercaptopyruvate sulfurtransferase activity in erythrocytes, based on measurement of the pyruvate formed with an LDH-coupled reaction. An automated method for the determination of porphobilinogen synthetase activity.was reported by Bourbon et al. (8F).Burlina and Galzigna (11F) described the fractionation of arylesterase into isoenzymes on cellulose acetate. Monoamine oxidase activity was measured in liver and brain samples by a fluorometric method described by Harada and Nagatsu (23F).

HORMONES Thyroxine and Related Compounds. The number of thyroid function tests which can be performed readily in the clinical laboratory continues to expand, with emphasis on radioisotope-based procedures. Competitive proteinbinding (CPB) radioassays for total thyroxine (T4) and triiodothyronine (Ts)-uptake have been supplemented by radioimmunoassay methods for free and total T4, TB,and thyroxine-binding globulin (TBG). Sterling (110G) reviewed biochemical tests for the assessment of thyroid function. The accuracy of protein-binding and RIA methods for thyroid hormones was reviewed by Burke and Eastman (18G). Jacobs et al. (62G)measured the free fractions of T4 and T3 in thyroid storm and recurrent hyperthyroidism. T4 and T3 concentrations in urine were assayed by Chan (24G) to assess thyroid function. Dunn and Foster (39G)described a method for the RIA of serum T4 which is more sensitive and specific than CPB methods. Watson and Lees (121G) pointed out that differences in the results of Tq measurements may be due to differences in standards used in various kit methods. An improved CPB procedure for the measurement of serum T4, in which small Sephadex columns are used and regenerated, was reported by Alexander and Jennings (4G). Meinhold and Wenzel (83G) reported a method for the RIA of T4 in unextracted serum. Wilson et al. (123G) developed a method for the determination of free T4, with use of diluted serum labeled with 1251-T4which is subjected to dialysis and activated charcoal purification of the dialysate. Hoffenberg (56G) reviewed chemical and immunological procedures for the measurement of T3 in serum and urine. The Sterling method for T3 was evaluated and modified by Wahner (119G) by the introduction of a more sensitive binding assay. Siege1 et al. (105G) described a solid-phase procedure for the RIA of T3 in serum. A method for the RIA of T3 in serum was developed by Sekadde et al. (104G) which uses a rabbit antiserum as binding agent and polyethylene glycol for separation of bound and unbound T3. Pate1 et al. (91C) extracted serum with ethanol and then measured T3 in the supernatant by RIA. Huefner and Hesch (59G) used ion-exchange chromatography followed by RIA for the measurement of serum Ts. A convenient method for the RIA of serum T3 was reported by Alexander and Jennings ( 5 G ) which incorporates small, re-usable Sephadex G-25 columns. Fang and Refetoff (43G) evaluated several techniques to control interferences from binding proteins in the RIA of serum T3. A method for the RIA of reverse T3 (3,3’,5’,-T3)was developed by Chopra (28G). Van Herle et al. (116G) developed a double antibody method for the RIA of serum TBG. A solid-phase radioassay for the measurement of circulating TBG was reported 22R

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by Castro and Ugarte (23G). Nelson et al. (88G) used a multiple ligand-binding method for the RIA of diiodotyrosine in plasma. Steroids and Prostaglandins. Abraham ( I G ) reviewed methods for the RIA of steroids in biological fluids. The determination of steroids by saturation analysis and radioimmunoassay were reviewed by James and Jeffcoate (63G). Wotiz and Chattoraj (126G) reviewed the application of GLC to steroid hormone analysis. A computer-coupled GLC-mass spectrometer system was used by Baty and Wade (11G) for the identification of steroids in biological fluids. Luyten and Rutten (76G) applied high-resolution GLC to the determination of individual steroids in urine. Stillwell et al. (111G) used ammonium carbonate to saturate plasma or urine, followed by solvent extraction prior to steroid determinations by GLC or other techniques. Methods for the determination of steroids by TLC were reviewed by Touchstone and Murawec (113G). Bravo (16C) evaluated GLC for the measurement of aldosterone in urine. Grose et al. (52G) used ion-exchange and paper chromatography to isolate urinary metabolites of aldosterone, including their glucuronides and sulfates. Methods for the determination of plasma aldosterone by RIA, without prior chromatographic separation, were presented by Martin and Nugent (77G),Varsano-Aharon and Ulick (117G),and Farmer et al. (44G). Drewes et al. (38G) reported a method for the RIA of aldosterone in urine, in which dichloromethane extraction was used for purification. A solid-phase RIA of plasma aldosterone was developed by Bizollon et al. (14G).Klumpp et al. (71G) reported a method for the determination of plasma aldosterone by RIA. A procedure for the simultaneous measurement of plasma aldosterone and deoxycorticosterone by RIA was developed by Castro et al. (21G). Castro et al. (20G) reported a method for the determination of deoxycorticosterone in plasma by RIA, which compared favorably with CPB or isotope dilution techniques. Ficher et al. (48G)recommended the use of horse serum as a source of assay protein to improve the sensitivity of plasma and urine corticosteroid determination by CPB radioassay. The specificity of a simplified fluorometric method for the determination of plasma ll-hydroxycorticosteroids was investigated by Mejer and Blanchard (84G).Dela Pena and Goldzieher (34G) presented a selected method for the determination of plasma cortisol by CPB radioassay. Farmer and Pierce (45G) compared RIA and CPB methods for the determination of plasma cortisol. A singleantibody technique for the RIA of plasma cortisol, in which extraction is avoided and heat denaturation of binding proteins is applied, was reported by Foster and Dunn (49G). Juselius and Barnhart (66G) presented a selected method for the determination of urinary free cortisol by CPB radioassay. A simultaneous CPB radioassay for plasma cortisol, cortisone, and prednisolone, which uses TLC for preliminary separation, was reported by Turner et al. (114G). Colburn and Buller (31G) described the measurement of prednisolone and its major metabolite in serum by means of RIA. A combined fluorometric and radioassay method for the simultaneous analysis of cortisol and corticosterone in plasma was reported by Painter et al. (90G).Rad0 (98G) investigated the falsely increased fluorescence of samples containing carbamazepine in the fluorometric determination of cortisol. A proposed, selected method for the fluorometric determination of free cortisol in urine was presented by Ratliff and Hall (99G). Bruton et al. ( 1 7 G ) compared four methods for the determination of plasma 17-hydroxycorticosteroids, including Porter-Silber, fluorometric, double-isotope derivative, and CPB procedures. A more sensi-

tive reagent for the determination of 17-hydroxycorticosteroids in urine, p-hydrazinobenzene sulfonic acid in phosphoric acid, was discussed by Sanghvi et al. (102G). Bailey (1OC) developed a GLC procedure for the group analysis of 11-keto-17-hydroxycorticosteroidsin urine. Jiang- et al. (64G) described a method for the RIA of serum estrogens (sum of estrone and estradiol), suitable for a rapid and sensitive clinical procedure. A rapid RIA procedure for estrogens in plasma was developed by Wu et al. (127G). Powell and Stevens (96G) reported a method for the RIA of five selected ovarian steroids in a single serum sample, which includes a semi-automatic column chromatographic separation. A RIA procedure for 17P-estradiol in serum, without initial chromatography, was developed by England et al. (42G). Doerr (36G) reported improvements in the reliability of the hapten-RIA procedure for plasma estradiol. Measurement of estrone and [email protected] serum by RIA, without prior chromatography, was reported by Kushinsky and Anderson (72G). Gelbke ( 5 1 G ) described a chemical procedure for the determination of 2hydroxyestrone in pregnancy urine. A GLC method for the determination of 2-hydroxyestriol in human late-pregnancy urine was reported by Gelbke and Knuppen (50G). Dolphin (37G) used high-resolution liquid chromatography for the analysis of estrogenic steroids in urine. The determination of up to 1 2 estrogens in microliter quantities of pregnancy urine by GLC was described by Aldercreutz and Hunneman (3G). Pirke (95G) compared CPB and RIA methods, with and without chromatography, for the measurement of plasma testosterone. Solvent partition followed by CPB was used by Patterson and Lurie (93G) for a simple and specific assay of plasma testosterone. Castro et al. (22G) developed a simplified procedure for the RIA of plasma testosterone without column chromatography. Horgan and Riley (57G) described a CPB method for the determination of plasma testosterone, in which an ether extract is purified by column chromatography. A method for the RIA of circulating testosterone in plasma was presented by Lox et al. (74G). Strickland et al. (112G) used aluminum oxide, TLC, and CPB to measure serum testosterone and androstanediol. Sanghvi et al. (103G) described a GLC procedure for estimating the major urinary 17-ketosteroids, pregnanediol, pregnanetriol, and pregnanetriolone without derivative formation. A CPB method for the assay of dehydroepiandrosterone and its sulfate in plasma was reported by Andre and James ( 8 G ) . Littmann and Gerdes (73G) compared GLC and CPB methods for the measurement of urinary testosterone. Cameron and Scarisbrick (19G) described a proposed, selected method for the determination of plasma progesterone by RIA, in which 3H-labeled progesterone and antiprogesterone-lla-hemisuccinylbovineserum albumin are used. A substantial increase in sensitivity and specificity was accomplished by Bodley et al. (15G) in the RIA of progesterone following purification of the sheep antibodies on QAE-Sephadex A-50. A procedure for the RIA of 16a-hydroxyprogesterone in plasma was reported by Abraham et al. (2G). Metcalf (86G) used GLC to analyze for the progesterone metabolites, pregnanediol and pregnanolone in urine. Anderson and Leovey (7G) separated and measured prostaglandins by high-pressure liquid chromatography. A GLC procedure for the determination of E prostaglandins in amniotic fluid was described by Keirse and Turnbull (69G). Kelly (70G) measured prostaglandin F P in~ biological fluids by GLC-mass spectrometry. Belliveau and Bachur (12G) reported a method for the determination of prostaglandins in plasma, in which prostaglandins A and E

are converted to B, and B is measured by RIA. A sensitive procedure for the RIA of endogenous prostaglandin Fzo, in plasma was described by Patron0 (92G). Youssefnejadian et al. (128G) reported a method for the determination of prostaglandin Fzo, in 0.1 ml serum, without prior extraction.

Catecholamines and Related Compounds. Peyrin and Cottet-Emard (94G) developed a continuous-flow method for the specific determination of epinephrine and norepinephrine in the presence of each other. Anderson et al. ( 6 G ) reported an improved fluorometric continuous-flow method for urinary catecholamines based on the trihydroxyindole reaction. A sensitive GLC procedure to measure catecholamines, 3-methoxytyramine, normetanephrine, and metanephrine in plasma was described by Wang et al. (120G). Wong et al. (124G) developed a GLC procedure with electron capture detection for the measurement of epinephrine norepinephrine, and dopamine in urine as their pentafluoropropionyl derivatives. Merzhaeuser (85G) reported the determination of epinephrine and norepinephrine by high-pressure liquid chromatography. A twocolumn purification system for the measurement of plasma catecholamines was described by Jiang et al. (6%). Mc‘Donald and Murphy (81G) separated epinephrine, norepinephrine, dihydroxyphenylalanine and dopamine in 6 hours by acid elution through Sephadex G-10. A sensitive GLC method for the determination of 3-methoxy-4-hydroxyphenylethylene glycol (MHPG), the principal metabolite of norepinephrine, in cerebrospinal fluid and urine, was reported by O’Keefe and Brooksbank (89G). Karoum et al. (67G) described a GLC procedure for the determination of the urinary excretion of free and conjugated Speigel MHPG and 3-methoxy-4-hydroxy-phenylethanol. and Christian (109G) devised a semi-automated colorimetric method for the analysis of dihydroxphenylacetic acid in the presence of other catechols. A sensitive double-isotope derivative technique for the assay of dopamine in plasma was developed by Christensen (29G). Feldman et al. ( 4 6 G ) noted negative interference with the measurement of urinary 3-methoxy-4-hydroxymandelic acid and 5-hydroxyindoleacetic acid due to reducing metabolites of aspirin and L-DOPA. Mass fragmentography was employed by Anggard et al. (9G) to quantify homovanillic acid (HVA) in serum. Prasad and Fahn (97G) determined HVA in cerebrospinal fluid in the presence of other catecholamine metabolites by fluorometry. Dziedzic and Gitlow (40G) measured the hexafluoroisopropyl esters of trifluoroacetylated HVA and 3-hydroxy-4-methoxy-phenylaceticacid (isoHVA). Peptide Hormones. Jacobs and Lawton (61G) reviewed methods for the determination of pituitary and glycopeptide hormones based on immunological assay. Coffey et al. (30G) used zirconyl phosphate gel as the solid phase in the RIA of insulin in plasma. An automated procedure for the RIA of insulin was described by Ingrand et al. (60G). Feldman and Chapman (47G) noted that certain individuals show significant differences between the insulin concentrations of their serum and plasma as measured by RIA. A simplified approach to RIA methods, in which a solidphase antibody-gel binding reagent is placed in disposable columns, was described by Updike et al. (115G) and applied to the determination of angiotensin I and insulin. Velasco et al. (118G) presented a selected method for the RIA of serum insulin, in which the insulin antibodies are covalently bonded to Sephadex. A procedure for measuring insulin in the presence of insulin antibodies in serum was developed by Martin and Russell (78G). Dixon (3%’) described a CPB radioassay for the determination of antiA N A L Y T I C A L C H E M I S T R Y , VOL. 47,

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bodies to insulin in serum. A simple method for the extraction of insulin from urine was reported by Crossley (33G). Luyckx (75G) reviewed the techniques for RIA of glucagon in plasma. Sperling et al. (108G) developed a sensitive method for the RIA of plasma glucagon, and investigated the specificity and applications of the procedure. Habener et al. (53G) found that dioxane precipitation is a satisfactory method for RIA of calcitonin, while dextrancharcoal adsorption provides the most reliable results in the parathyroid hormone (PTH) assay. Methods for the RIA of serum P T H were described by McCann et al. (80G), Conaway and Anast (32G), and Woo and Singer (125G). May and Donabedian (79G) reported a procedure for the RIA of thyrotropin (thyroid-stimulating hormone, TSH) releasing factor, pyroglutamyl-histidyl-proline amide. Wick chromatography was used by Weeke and Oerskov (122G) for the immunoassay of serum TSH. Sluiter et al. (107G) described a procedure for the RIA of serum TSH which employs a solid-phase second antibody and a purified globulin preparation for standardization of non-specific protein interactions. Procedures for the RIA of plasma calcitonin were reported by Bieler et al. (13G),Samaan et al. (101G), Heynen and Franchimont (55G),and Silva et al. (106G). Reeder et al. (IOOG) measured the endogenous circulating cholecystokinin by RIA. A procedure for the RIA of cholecystokinin sensitive to 5 pg/ml and without cross reactivity from gastrin, secretin, or glucagon, was developed by Harvey et al. (54G). Kat0 and Torigoe (68G) reported a specific and sensitive procedure for the RIA of oxytocin in plasma. Chard and Martin (26G) reviewed techniques for the RIA of oxytocin, vasopressin, and neurophysins. Procedures and problems in the RIA of oxytocin and vasopressin were reviewed by Chard (25G).Horgan and Riley (58G)reported an improved method for the RIA of plasma corticotorpin, in which fullers earth is used for extraction. A procedure for the RIA of serum prolactin was described by McNeilly (82G).Moser and Hollingsworth (87G) reported a double-antibody method for the RIA of human chorionic somatomammotropin in serum, amniotic fluid, and urine. A procedure for the RIA of serum neurophysin was devised by Cheng and Friesen (27G). Emanuel et al. (41G) described double-antibody techniques for the RIA of angiotensin I-like activity and angiotensin 11.

LIPIDS Cholesterol. Richmond (39H)described the preparation and properties of a cholesterol oxidase from Nocardia cells, and applied it to the enzymatic determination of cholesterol in saponified extracts of serum, based on colorimetric measurement of liberated hydrogen peroxide. The use of a bacterial cholesterol dehydrogenase to measure serum cholesterol was reported by Flegg (14H).Allain et al. ( I H ) described an enzymatic method for the determination of total cholesterol in serum, which includes enzymatic hydrolysis, oxidation of free cholesterol with cholesterol oxidase, and colorimetric measurement of liberated hydrogen peroxide. Witte et al. ( 5 1 H ) compared a cholesterol oxidase method for the determination of serum cholesterol, in an automated version on the ABA-100, with colorimetric methods based on the Liebermann-Burchard reaction. The kinetics of the Liebermann-Burchard cholesterol reaction were investigated by Hewitt and Pardue ( 2 0 H ) ,and a reactionrate method applicable to serum was described. Velapoldi et al. (48H) presented spectral data and kinetic studies of the iron-sulfuric acid reaction with cholesterol. The nonextraction serum cholesterol method of Wybenga was adapted by Slickers et al. (44H)to a reaction-rate determination with the Centrifichem centrifugal analyzer. Klein et 24R

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al. (24H) synthesized several inorganic and organic salts of cholesterol hemisuccinate, and evaluated the morpholine salt for use in an aqueous cholesterol standard solution. The concentration of free and total cholesterol in cerebrospinal fluid was determined by Pedersen (34H). GLC methods for the determination of serum cholesterol were reported by Blomhoff ( 3 H ) , MacGee et al. (30H), Harris and Harris (17H),and Ishikawa et al. (22H). Triglycerides. Bucolo and David ( 4 H ) described a completely enzymatic method for the determination of serum triglycerides, which iiicludes hydrolysis and coupling of the liberated glycerol to the photometric measurement of NADH decrease. An enzymatic method for the determination of glycerol was adapted to the GEMSAEC centrifugal analyzer by Olson and Brennan (32H), and used for measurement of serum triglycerides after alkaline hydrolysis. Tiffany et al. (47H) presented an instrumental and clinical evaluation of an enzymatic method for serum triglycerides as used on the centrifugal analyzer. The enzymatic method of Bucolo and David for serum triglycerides was adapted to the GEMSAEC centrifugal analyzer by Chong-Kit and McLaughlin ( 7 H ) ,with use of an aqueous triolein standard. Sone et al. (46H)described a colorimetric procedure for the determination of serum triglycerides which does not use adsorbents. Lipoproteins. Harlan and Shaw (16H) reviewed the methodology for the measurement and the interpretation of hyperlipidemias. The properties and significance of the proteins associated with plasma lipoproteins were reviewed by Scanu and Ritter (40H).Berenson et al. ( 2 H )reported a primary screening procedure for the detection of hyperlipidemias based on turbidimetric estimations of P-lipoproteins in serum. An automated screening procedure for hyperlipoproteinemias, by measurement of total cholesterol and 6- and pre-P-lipoprotein cholesterol, was developed by Lopez et al. (29H).Naito et al. (31H) described a screening procedure for serum lipoprotein abnormalities based on polyacrylamide gel disc electrophoresis, and compared it with other analytical methods. A rapid method for the indication of Type I11 hyperlipoproteinemia, based on selective visualization of very low density lipoproteins in agarose gel after electrophoresis, was reported by Wieland and Seidel (50H).Heiberg (19H) found that agarose gel electrophoresis was preferable to paper or cellulose acetate for classification of hereditary lipoproteinemias. The measurement of serum cholesterol and triglycerides was used by Hatch et al. ( 1 8 H ) for standardization of agarose separation of lipoproteins. Fidge (13H) reviewed methods and metabolic studies associated with the radioiodination of lipoproteins. Tetramethylurea was used to delipidate serum high-density lipoproteins by Kane ( 2 3 H ) ,in order to study the apoproteins. Petek et al. (37H) discussed methods for the immunological determination of lipoprotein-Xi. An improved method for the immunochemical quantitation of serum lipoproteinX, in which other lipoproteins are removed by immunoprecipitation, was reported by Kostner et al. (25H). Peeters et al. (36H) applied automated nephelometry to the analysis of low-density lipoproteins. An automated nephelometric technique for the determination of P- and pre-P-lipoproteins was reported by Canal et al. ( 5 H ) . Schonfeld et al. (41H) used a double-antibody procedure for the RIA of apolipoprotein B in plasma. Phospholipids. Raheja et al. (38H) devised a colorimetric procedure for the determination of phospholipids in serum, without acid digestion. TLC was used by Pedersen (35H) for the isolation of total and fractionated phospholipids in cerebrospinal fluid. Warren et al. (49H) described

a GLC procedure for the measurement of palmitic acid as a means of assessing amniotic fluid lecithin-concentrations. An X-ray fluorescence method for the determination of total serum phospholipids was reported by Leyden et al. (28H).Hodge (21H) used TLC to quantify lecithin in amniotic fluid. A modified TLC procedure for the rapid estimation of the lecithin/sphingomyelin (L/S) ratio in amniotic fluid samples was reported by Coch et al. (IOH). Olson and Graven (33H) compared six different methods of TLC plate visualization for the determination of the L/S ratio in amniotic fluid. Coch et al. ( 9 H ) presented a selected method for the determination of the LIS ratio in amniotic fluid based on the TLC procedure of Gluck. Miscellaneous. Seifert (4323) developed lipid profiles from 0.1-ml blood samples by TLC. Schwertner and Friedman (42H) discussed the changes in serum lipid and lipoprotein values that can occur due to bacterial contamination of the samples. A procedure for the quantitative extraction and determination of non-esterified plasma lipids was reported by Duncombe and Rising ( I I H ) . Soloni and Sardina (45H) described a modified colorimetric method for the determination of serum non-esterified fatty acids (NEFA), based on the extraction of copper soaps, and complexation of the copper with cuprizone. A micromethod for the analysis of NEFA by atomic absorption was developed by Lehane and Werner (27H).Labadie et al. (26H) extracted NEFA from plasma with hexane-isopropanol, then used TLC and GLC for quantitation. Gruenert and Baessler ( 1 5 H ) , and Falholt et al. ( 1 2 H ) reported colorimetric micromethods for the measurement of plasma NEFA. Christophe et al. ( 8 H ) described methods for the quantitative and qualitative determination of NEFA in urine. A radiochemical procedure for the determination of serum NEFA, based on formation of the 6oCo-fatty acid complexes, was reported by Chlouverakis and Hojnicki (623).

NITROGEN COMPOUNDS Amino Acids and Amines. Scriver et al. (695) reviewed the range of methods available for the diagnosis of aminoacidopathies. The factors determining the validity of plasma amino acid analyses were discussed by Armstrong and Stave ( 3 5 ) .Oddy (625) described a continuous-flow, ninhydrin colorimetric method for the determination of a-amino nitrogen in protein-free filtrates of plasma. The urinary aamino nitrogen values in infants were determined and reported by Dhondt et al. ( 1 9 5 ) . Lorentz (485) used benzoquinone to determine amino acids in biological fluids. Roth and Hampai (685) described a fluorometric method for the detection of urinary amino acids separated by column chromatography, with o-phthalaldehyde and 2-mercaptoethano1 as reagents. An automated short-column chromatographic system for the high-speed routine analysis of urinary amino acids was developed by Barlow and Miles ( 4 5 ) . Klosse et al. ( 4 3 4 described an automated- ion-exchange chromatographic system for the combined analysis of urinary peptides and amino acids. A system of ligand-exchange chromatography to separate peptides from amino acids was devised by Bellinger and Buist ( 6 5 ) . Lou and Hamilton (495) described improvements in a chromatographic system for the separation of amino acids, peptides, and proteins in normal urine. Significant contamination of distilled water, hydrochloric acid, and ammonium hydroxide with amino acids and proteins from airborne microbes was reported by Hamilton and Myoda ( 3 4 5 ) .Ma and Chan ( 5 1 4 studied the barium precipitation of sulfate in urine to derive a measure of the endogenous production of acid from sulfur-containing amino acids. A rapid procedure for

the chromatography of free, basic amino acids in blood or serum was reported by Vratny and Zbrozek ( 7 6 5 ) .McGregor et al. (545) described a GLC procedure for the measurement of amino acids in hydrolysates of urine as their n-propyl-N-acetyl esters. Goulle and Broun (295) used Lamino acid oxidase to rapidly determine the L-amino acids in blood. Grunbaum and Pace (305) described a simplified procedure for the determination of hydroxyproline in urine. Fluorescamine was used by Felix and Terkelson (235) to measure hydroxyproline. Blumenkrantz and Asboe-Hansen (85)developed an automated procedure for the measurement of urinary hydroxyproline based on reaction with Ehrlich's reagent. Three methods for the measurement of urinary hydroxyproline, including a continuous-flow approach, were compared by Burkhardt et al. ( 9 5 ) .Seymour and Jackson (705) developed an ion-exchange column chromatography procedure for the specific determination of urinary hydroxyproline, in which the eluate is measured continuously with the p -dimethylaminobenzaldehyde reaction. Guilbault and Froelich (315) described a sensitive method for the determination of serum tryptophan, in which the decrease in UV-excited fluorescence due to formaldehyde addition is measured. An ultrafiltration method for extracting free tryptophan from plasma and its measurement by fluorescence was reported by Eccleston (215).Flentge et al. (245) described an automated procedure for the determination of tryptophan in cerebrospinal fluid, and also measured the free and bound forms in serum. A selected method for the fluorometric determination of tyrosine in serum, based on its reaction with 1-nitroso-2-naphthol,was presented by Ambrose ( 2 5 ) .Shihabi and Summer (715) reported a GLC method for the determination of phenylalanine in serum and urine, which involves its oxidation to phenyllactic acid and trimethylsilylation. A colorimetric method for the determination of glycine in serum and urine was evaluated and modified by Goodwin and Stampwala ( 2 8 5 ) ,and revised data on the concentration of free glycine in serum were presented. Proelss and Wright (67J) developed a method for the determination of whole blood ammonia, in which a perchloric acid filtrate is alkalinized and the liberated ammonia is measured by an ion-selective electrode. A flow-through ammonia electrode was used by Park and Fenton (635) for the measurement of plasma ammonia. Jacobs and Olthius (385) adapted an enzymatic reaction-rate method, employing glutamic dehydrogenase, to the determination of plasma ammonia with the LKB 8600. A procedure for the determination of plasma ammonia without deproteinization was reported by Da Fonseca-Wollheim (175). Cunarro and Weiner (165) compared techniques for the measurement of blood ammonia. A procedure for the automated simultaneous determination of titratable acidity and ammonia in urine was described by Bennett ( 7 5 ) . Evans et al. ( 2 2 5 ) ,Martin and Harrison ( 5 2 5 ) ,and Siraganian (725) reported automated fluorometric procedures for the assay of histamine in body fluids. Veening et al. (755) developed an ion-exchange chromatographic procedure for the separation and fluorometric determination of individual urinary polyamines. An automated amino acid analyzer was applied by Marton et al. (535) to the determination of putrescine, spermidine, and spermine in urine and serum. Gehrke et al. (255) discussed GLC methods for the measurement of polyamines in urine. GLC methods for the analysis of short-chain, aliphatic polyamines in urine, and their application to the study of cancer patients was discussed by Denton et al. (185). Abramson et al. (15)used

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mass spectrometry for the measurement of femtomole quantities of biogenic amines and amino acids. Humbel and Marsault (375) separated the urinary metabolites of kynurenine by TLC, following tryptophan loading of the subject. Kynurenine in urine was measured by GLC in a report by Naruse et al. (605). Buxton and Guilbault (115) described a fluorometric method for the determination of the tryptophan metabolite, N’- formylkynurenine in plasma and urine. Menichini et al. ( 5 5 4 reported a procedure for the measurement of serum guanidine and applied it to uremic patients. Stein and Micklus (735) studied uremic patients with an ion-exchange, colorimetric method for the determination of serum and urine guanidine, and its monomethyl and dimethyl derivatives. Creatinine and Urea. Yatzidis (785) reported a modified Jaffe reaction procedure, with alkaline picrate reagents a t two different pH values, for the colorimetric determination of “true” creatinine in serum. The Jaffe reaction was modified by Heinegard and Tiderstrom (355) by the addition of sodium dodecyl sulfate and borate, to permit serum creatinine determination without deproteinization. Kirbeger and Keller ( 4 1 5 ) discussed the errors that may occur in serum creatinine results due to storage of the sample. The enzyme creatininase was used by Thompson and Rechnitz (745) with a membrane electrode for ammonia to measure creatinine. Bartels and Boehmer (55) discussed the determination of creatinine by reaction-rate measurement with the LKB 8600 analyzer. Kammermeier (405) employed paper or TLC to isolate creatine, and a fluorescent ninhydrin reaction to quantitate the compound. Lau and Guilbault (455) reported a reaction-rate enzymatic method for the determination of creatine in urine, based on measurement of the fluorescence decrease of NADH. Guilbault and Nagy ( 3 3 4 described an improved electrode for the measurement of urea in biological fluids. An automated system with an ammonia electrode detector was used by Llenado and Rechnitz (475) for the determination of urea in serum. Wenk et al. (775) evaluated commercially available kits, based on diacetyl monoxime or urease methods, for the determination of urea. Uric Acid, Purines, and Pyrimidines. Meites et al. (565) compared two uricase-based procedures for ultramicro serum uric acid determination, the Beckman oxygenrate sensing analyzer and a colorimetric peroxide indicator reaction. Lum and Gambino (50J) reported that a uricase, reaction-rate method for the determination of serum uric acid with the DuPont ACA gave comparable results to a manual UV-uricase end-point method, and two reducing methods were less satisfactory. A sensitive fluorometric method for serum uric acid determination was reported by Godicke and Godicke (26J), in which uricase and peroxito a non-fluodase oxidize 3,5-diacetyl-1,4-dihydrolutidine rescent compound. Nanjo and Guilbault (595) described a direct reaction-rate method for the determination of uric acid, based on measurement of oxygen uptake with a platinum/immobilized uricase electrode. The use of immobilized uricase for the measurement of uric acid in biological fluids was described by Dritschilo and Weibel (205).Pesce et al. (645) reported a uricase-based, UV-spectrophotometric method with the CentrifiChem centrifugal analyzer for the determination of serum or urinary uric acid. Klein and Lucas (425) reported manual and continuous-flow colorimetric methods for uric acid determination, based on ferricyanide oxidation and measurement of the resulting ferrous ion with a 5-pyridyl-benzodiazepin-2-one derivative. A method for the measurement of serum uric acid, based on reduction of ferric phenanthroline, was described by Morin and Prox (585).Morin (575) reported two methods for the determination of serum uric acid based on oxidation by al26R

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kaline ferricyanide, one of which uses the decrease in absorbance at 293 nm. Jones (395) reviewed methods for the measurement of urinary purines by TLC. Endogenous plasma purines were separated by high-pressure liquid chromatography on silicic acid by Pfadenhauer (665). Layzell (465) described an enzymatic, spectrophotometric method for the determination of low levels of the urinary oxypurines, xanthine and hypoxanthine. Butts et al. (205)developed a GLC procedure for the assay of diphylline in serum, that is free from interference by other methylxanthines. Ion-exchange chromatography was employed by Byrne and Chapman (125) to isolate and quantitate deoxycytidine from urine. Chang et al. (135) used GLC to measure methylated nucleosides and pseudouridine in urine. Chilcote and Mrochek ( 1 4 4 developed a high-resolution anion-exchange system which was used for the analysis of nucleosides and bases in physiological fluids. Hughes et al. (365) described a method for the RIA of thymidine in serum. Indoles and Related Compounds. Peskar and Spector (655) described a procedure for the radioassay of serotonin (5-hydroxytryptamine). The ninhydrin reaction was applied by Nathenes et al. (615) to the spectrofluorometric assay of plasma serotonin. Korf et al. (445) developed a continuous-flow method for the measurement of serotonin and 5-hydroxyindoleacetic acid (5-HIAA), based on their fluorescence with cysteine and o-phthaldialdehyde. Guilbault and Froehlich (325) described a cation-exchange, fluorometric procedure for the determination of the tryptophan metabolites, serotonin, 5-HIAA, and 5-hydroxytryptophan, in serum. An improved colorimetric method for the determination of 5-HIAA in urine, based on the nitrosonaphthol reaction, was presented by Goldenberg (275). Cole and Crank (155) developed a fluorometric procedure to assay melatonin ( N -acetyl-5-methoxytryptamine) in serum.

ORGANIC ACIDS AND OTHER METABOLITES Healy et al. (14K) developed a computer program for the quantitation of acidic metabolites separated by GLC. A TLC procedure for the measurement of Krebs cycle acids was described by Kraiker and Burch (21K). Von Nicolal and Zilliken ( 4 1 K ) used GLC to separate oxalic, malonic, and succinic acids in biological samples. A GLC method for the determination of methylmalonic acid in urine and serum, in which the ether extract is derivatized with BSTFA, was reported by Schiller and Summer ( 3 1 K ) .Mee and Stanley ( 2 6 K ) described a GLC procedure for the measurement of oxalic acid in biological fluids. Chambers and Russell ( 3 K ) reported a method for the specific assay of oxalic acid in plasma. A GLC procedure for lactic acid analysis, in which the acid is converted to acetaldehyde, was described by Owen and Lechocki ( 2 8 K ) . Zivin and Snarr (43K) developed a sensitive enzymatic method for the assay of 3-hydroxybutyric acid in plasma. Paper electrophoresis ‘and fluorometry were employed by Hosoda et a]. (18K) to detect a-keto acids in urine. Mamer and Gibbs (25K) described a simplified GLC method for the analysis of the trimethylsilyl esters of shortchain fatty acids in serum and urine. Gibbs et al. (12K) presented a GLC technique for the quantitation of propionic acid and other volatile fatty acids in urine. A rapid GLC procedure for the determination of short-chain fatty acids in microliter quantities of serum was reported by Van den Berg and Hommes ( 3 9 K ) .Collin and McCormick ( 5 K ) reported a titrimetric method for the determination of short-chain fatty acids in stool ultrafiltrate and urine, in which ion-exchange treatment is used to remove interfer-

ences. Katz et al. (20K)developed a sensitive ion-exchange chromatography procedure for the assay of oxidizable aromatic acids, based on measurement of the UV fluorescence of cerium(II1). Feldman and Bowman ( I I K ) described a method for the qualitative and quantitative determination of urinary homogentisic acid, which involves extraction, TLC, and colorimetry with Folin’s phenol reagent. Urinary hippuric acid and methylhippuric acid were quantitated Evans and Niwith GLC by Buchet and Lauwerys (2K). cholls (9K)used GLC to measure l-methylimidazole-&acetic acid GLC in urine. Teunissen et al. (37K) compared five methods, one colorimetric, two enzymatic by reaction-rate, and two enzymatic end-point, for the determination of 2,3-diphosphoglycerate (2,3-DPG) in blood. A continuous-flow colorimetric method for the determination of 2,3-DPG in blood was reported Dyce and Bessman ( 8 K )developed by Lappin et al. (23K). a rapid, nonenzymatic assay procedure for 2,3-DPG in multiple specimens of blood. Parkinson and Medley (29K)estimated ATP, ADP, and AMP in human plasma by use of luciferin/luciferase and a scintillation counter. A CPB procedure for the radioassay of cyclic AMP, with use of thyroid cytosol, was developed by Orgiazzi et al. (27K). Doizaki and Zieve (6K)described a GLC procedure for quantitation of individual mercaptans in whole blood. Bile Acids. Subbiah (36K)reviewed methods available for determining bile acids in bile and feces. Simmonds et al. (33K)described a method for the RIA of conjugated cholyl bile acids in serum. A fluorometric method for the determination of individual bile acids in plasma, in which TLC is used to isolate and purify the acids, was reported by Feher et al. (IOK). Schwartz et al. (32K)described a simplified fluorometric method for the quantitation of serum bile acids, in which the acids are adsorbed with Amberlite XAD-2 ion-exchange resin. TLC was employed by Makino to measure sulfated and non-sulfated bile acids et al. (24K) in serum and urine. Van Berge Henegouwen et al. (40K) described a GLC procedure for the determination of individual bile acids in serum and bile, and reported results obtained with fasting healthy subjects. Bilirubin and Related Compounds. Doumas et al. (7K) evaluated the sources of error in serum bilirubin determinations by several methods, and indicated a need for improved standardization and control sera. A procedure for the determination of direct-reacting bilirubin with the SMA 12/60 was reported by Sterling and Matthews (35K). Pearlman and Lee (30K)described a diazo method for the measurement of total bilirubin in serum, in which the anionic surfactant, Duponol, is used as a solubilizing agent to promote coupling of non-conjugated bilirun. The non-albumin-bound bilirubin in the serum of newborns was deterJamined with Sephadex columns by Trivin et al. (38K). cobsen and Wennberg (I9K)described an enzymatic method for the determination of unbound, non-conjugated bilirubin in the serum of newborns, based on oxidation in the presence of peroxidase. Grahnen et al. (13K)devised a spectropolarimetric method for the quantitation of nonconjugated bilirubin in serum, which depends on extrinsic Cotton effects associated with albumin binding. A discussion of the properties of bilirubin UDP-glycosyltransferase and clinical applications of the enzyme assay was presented by Heirwegh et al. (16K). Lamon et al. (22K)examined the Hoesch version of the p-dimethylaminobenzaldehyde reaction for urinary porphobilinogen, and listed several advantages over the commonly used Watson-Schwartz procedure. Stercobilin and urobilin in urine were measured colorimetrically as calcium salts of their oxides by Hoeflmayr and Fried (I7K). Bailen-

ger et al. ( I K ) determined fecal stercobilin by measurement of the fluorescence of the zinc complex. Walters (42K)employed DEAE-cellulose, hydrochloric acid elution, and spectrophotometry to quantitate urinary porphyrins. A fluorometric micromethod for the assay of protoporphyrin in acidified, acetone extracts of blood was reported by ChiSobel et al. (34K) described a fluorometric solm et al. (4K). method for the determination of urinary coproporphyrin and uroporphyrin, in which an anion-exchange resin is used for separation of the compounds.

PR 0TEIN S Koch et al. (27L)reported the application of the biuret reaction to the reaction-rate determination of serum total protein with the CentrifiChem analyzer. A specific and sensitive colorimetric method for the determination of urinary total protein was developed by Doetsch and Gadsden ( I I L ) ,in which interferences are removed by gel filtration and the copper bound to peptide bonds is measured with diethyldithiocarbamate. Buergi and Kaufmann ( 5 L ) studied the interference by myoinositol in the UV measurem.ent of the biuret reaction applied to protein determination in cerebrospinal fluid. The use of the synthetic peptide, glycylglycylglycine as a standard for the colorimetric measurement of serum protein by the biuret reaction was suggested by Klein (26L).Pesce and Strande (37L)described a new colorimetric micromethod for the quantitation of cerebrospinal fluid and urinary protein, based on precipitation of the sample with trichloroacetic acid containing Ponceau S dye. Doetsch et al. (12L)employed Sephadex G 50-80 and treatment of the effluent with the biuret reaction to determine protein in cerebrospinal fluid. The bromcresol green dye-binding method for the determination of albumin in serum was adapted by Gyure and Raisys (19L)to the SMA 12/60. Alexander and Rechnitz ( I L ) described a continuous-flow method for the determination of protein, based on detection of sulfur with a silver sulfide crystal membrane electrode. Smith and Carr ( 5 I L ) determined serum protein by a thermometric titration system. The separation of urinary proteins by ion-exchange chromatography was reported by Takita et al. (55L). Laurel1 (26%) discussed the relative value of electrophoresis and specific protein assays for the measurement and interpretation of plasma protein concentrations. Isoelectric focusing (IEF) in thin-layer polyacrylamide gel was applied by Vesterberg and Nise ( 6 I L ) to the study of urinary proteins in subjects exposed to cadmium. Wellner and Hayes (62L)reviewed the technique of IEF of proteins in polyacrylamide gels. Scheil (44L)employed IEF, followed by gel electrophoresis, to develop protein maps from the serum of normal and pathological subjects. Infrared absorption spectrometry was used by Wenzel and Hoffman (63L)to detect protein on unstained electrophoretic separations with cellulose acetate. Rabin et al. (39L)developed a rapid technique for the electrophoresis of protein in unconcentrated urine on agarose gel. Driscoll (13L) reported normal ranges for many individual proteins in serum as determined by rocket immunoelectrophoresis (IEP). The technique of electroradioimmuin which the noassay was described by Matzku et al. (32L), double-antibody RIA is modified so that separation of free and bound antigen is achieved by electroimmunodiffusion in agarose gel. Stephan (52L)compared methods for the quantitation of proteins in serum by IEP. Versey et al. (60L) described a semi-automated apparatus for performing two-dimensional IEP, and Versey and Slater (59L)reported a simplified area calculation for quantitation of proteins separated by two-dimensional IEP. Wright et al.

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(65L) described modifications to the technique of two-dimensional IEP, which decrease the time required to obtain the serum protein pattern and improve identification and quantitation. The procedure of immunocore electrophoresis was applied by Zeineh et al. (66L) to the measurement of urinary albumins. Ritchie et al. (40L) described the complete automation of specific protein assays by immunological reactions with use of the AutoAnalyzer system. A turbidimetric, continuous-flow method for the determination of individual serum proteins by immunochemical reaction and measurement with a high-sensitivity photometer, was reported by Blom and Sorenson ( 4 L ) .Heintges et al. (20L) determined total protein in 25 pl of cerebrospinal fluid by a nephelometric method. Killingsworth and Savory (25L) employed immunochemical reactions and nephelometry for the determination of albumin, transferrin, and an-macroglobulin in urine. Lizana and Hellsing (29L) modified the continuous-flow, immuno-nephelometric method for the quantitation of urinary albumin, by addition of polyethylene glycol to enhance the precipitin reaction. Polyethylene glycol was employed by Lizana and Hellsing (30L) to improve the manual, immuno-nephelometric determination of plasma albumin and fibrinogen. Rochefort et al. (42L) compared automated immuno-nephelometric analysis of urinary proteins with single and double immunodiffusion methods, and found the procedure to be suitable for the quantitation of differential protein clearance. Roberts et al. (41L) employed mercuric potassium thiocyanate to specifically precipitate fibrinogen and to enable its measurement independent of clotting. Three different methods for plasma fibrinogen assay were compared by Stevens and Sanfelippo ( 5 3 L ) , and the clottable protein assay was found to be least affected by extraneous factors. Hirai et al. (22L) developed a double-antibody method for the RIA of a-fetoprotein in serum. Silver et al. (49L) and Masseyeff et al. (31L) reported methods for the RIA of serum a-fetoprotein. Ferritin was assayed by Miles et al. (34L) with a two-site immunoradiometric procedure. Dietz et al. (10L) presented selected methods for the determination of al-antitrypsin in serum by immunodiffusion and proteolytic assay. Grappel (17L) combined IEP with agar gel casein precipitation on a single medium to detect and identify protease inhibitors in serum. Several alternative isotope counting procedures for the RIA of hepatitis-associated antigen were devised and evaluated by Jordan et al. (23L). Immunoglobulins. Cawley et al. (7L) reported a solidphase technique for the RIA of IgG. The measurement and interpretation of IgG and albumin in cerebrospinal fluid was discussed by Ganrot and Laurel1 (16L). Perry et al. (35L) compared the accuracy, sensitivity, and simplicity of electroimmunodiffusion and radial immunodiffusion for the determination of IgG in cerebrospinal fluid. Several methods for quantitating immunoglobulins by radial immunodiffusion were compared by Berne ( 2 L ) ,with particular regard to equations used for calculation. Killingsworth and Savory (24L) explored analytical factors of a model system for specific protein measurements, by use of lightscattering measurements of the precipitin reaction of IgGantiIgG. Tiffany et al. (57L) devised a rapid light-scattering method for immunoglobulin measurements with a miniature centrifugal analyzer. A laser-equipped Rotochem analyzer was employed by Buffone et al. ( 6 L )for the reactionrate determination of serum IgG by light-scattering measurements. Heremans and Masson (21L) recommended routine testing of sera for immunoglobulin disorders with a combination of agarose electrophoresis and immunochemi28R

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cal quantitation of IgG, IgA, and IgM. Agarose gel IEP was employed by Gale et al. (15L) for the detection of “heavychain” disease proteins. Skrede et al. (50L) investigated serum immunoglobulin concentrations as an index of liver damage. Data on serum immunoglobulin concentrations of normal children, obtained by quantitation against an international reference preparation, were presented by Cejka et al. ( 8 L ) . McFarlane (33L) described factors which influence serum immunoglobulin levels in selected populations. Electroimmunodiffusion and immunofixation were employed by Propp et al. (38L) for the determination of the third component of complement in unconcentrated cerebrospinal fluid. Pesce et al. (36L) developed a solid-phase adsorbent, enzyme-linked technique for the immunoassay of serum anti-DNA antibody. An indirect counter-IEP procedure was employed by Tripodi et al. (58L) for the rapid detection of the antibody to hepatitis-associated antigen. Hemoglobin and Related Compounds. Schneider et al. (48L) reported on the use of Mylar-backed cellulose acetate plates impregnated with citrate agar to improve this type of electrophoresis of hemoglobins. Similar hemoglobins were differentiated electrophoretically by Schneider (47L), by liberation and electrophoresis of the globin chains. Schmidt et al. (46L) edited the reports of an international meeting on standardization of laboratory reagents and methods for the detection of hemoglobinopathies. An evaluation of commercially available kits for the detection of abnormal hemoglobins by electrophoresis was reported by Schmidt and Holland (45L).Wilson and Schmidt (64L) evaluated two commercially available procedures for the detection of hemoglobin S: the “Sicklequik” solubility test and an instrument, the “SCAT Screener”. A radial immunodiffusion technique was described by Chudwin and Rucknagel ( 9 L ) for the measurement of hemoglobins F and An. Groff et al. (18L) developed a continuous-flow method for the simultaneous colorimetric determination of methemoglobin and total hemoglobin. Rodkey and O’Neal (43L) improved the Evelyn and Malloy procedure for the determination of methemoglobin in blood. A continuous-flow method for the automated determination of myoglobin in urine was reported by Sudaka et al. (54L). Blessing and Gebele ( 3 L ) employed thin-layer filtration with dextran gels to separate hemoglobin from myoglobin in serum. A new method for the immunological determination of fetal hemoglobin was described by Tamachi (56L).Furlan and Bucher (14L) applied differential spectrophotometry to the determination of plasma hematin in the presence of free hemoglobin.

TOXICOLOGY Sunshine ( 4 4 N ) presented a retrospective and prospective view of the toxicology field in a special issue of Clinical Chemistry devoted to this subject. Methods for the qualitative identification of commonly encountered drugs of abuse in urine specimens were reviewed by Frings (15N). Kullberg and Gorodetzky (30N) investigated the detection of abused drugs in urine by adsorption with XAD-2 nonionic resin and organic solvent elution. A simplified drug screening procedure which employs charcoal adsorption of the urine, solvent elution, and TLC was reported by Meola and Vanko ( 3 4 N ) . McBay ( 3 3 N ) discussed toxicological findings associated with fatal poisonings, including opinions concerning therapeutic and toxic concentrations in human tissues for most of the common drug and chemical poisons. Lundberg et al. (31N) reported on the experiences of a clinical toxicology service in a large urban hospital by reviewing one year’s analytical data. The present status of alcohol testing in the United States, including comparative

comments on sampling procedures and methods used, was reviewed by Mason and Dubowski (32N). Boerner et al. ( 6 N )described a direct computer-controlled, mass spectrometric drug analysis of body fluids from acutely poisoned patients. The use of various salt-solvent pairs for the isolation of drugs and metabolites from biological fluids was investigated by Horning et al. (24N).Costello et al. ( I I N )reported the use of GLC-mass spectrometry and a computersearchable file of 300 spectra of drugs, metabolites, and other compounds, for the identification of drugs in the body fluids of overdose victims. A laboratory-based digital computer was applied by Jatlow and Seligson (25N) to the identification of drugs from analytical data obtained by UV spectrophotometry, TLC, and colorimetry. Mule et al. ( 3 5 N ) evaluated several immunoassay methods for the detection of abused drugs in urine, and compared these with fluorometric and TLC techniques for sensitivity and specificity. Berk et al. ( 4 N ) reported rapid, tritium-labeled, double-antibody methods for the RIA of serum digoxin and gentamicin, which are suitable for emergency determinations. A turbidimetric homogeneous, enzyme immunoassay, applicable to the detection of opiates in urine at the level of 0.5 wg/ml, was described by Schneider et al. ( 4 1 N ) . Catlin et al. ( 9 N ) reported a rapid procedure for the RIA of morphine and immunologically related substances in urine and serum. A sensitive and specific method for the quantitation of morphine in urine, with use of a coupled gas chromatograph-chemical ionization quadrapole mass spectrometer, was developed by Clarke and Foltz (ION). Hayes ( 2 2 N ) reported a continuous-flow method for the determination of amphetamine in urine, which includes on-stream extraction and steam distillation of the sample. The use of a spray reagent containing fluorescamine for the detection of amphetamine in urine by TLC, was presented by Klein et al. (26N).Aaron et al. ( I N ) studied the analytical characteristics of nine important hallucinogenic drugs by fluorescence and low-temperature phosphorescence techniques. Derivatization of barbiturates to form butyl compounds was reported by Greeley (19N) to give improved GLC resolution. Griffiths et al. ( 2 0 N ) employed temperature-programmed GLC for the quantitation of many anticonvulsant drugs in a single serum sample. Bailey and Jatlow ( 3 N ) investigated GLC and UV methods for the determination of serum methaqualone, and their correlation with clinical toxicity. A rapid GLC method for the qualitative and quantitative assay of methaqualone in serum, in which propionyl-benzoyl-chloroaniline is used as an internal standard, was reported by Evenson and Lensmeyer ( 1 2 N ) .Bonnichsen et al. ( 7 N ) employed GLCmass spectrometry to identify free and conjugated metabolites of methaqualone in urine, blood, and liver. A rapid spectrophotometric procedure for the determination of ethchlorvynol (Placidyl) in whole blood, serum, or urine, based on oxidation of the drug to a UV-absorbing product, was developed by Wallace et al. (46N). Evenson and Poquette ( 1 4 N ) measured therapeutic and toxic levels of ethchlorvynol in serum by a rapid and specific GLC method. Glutethimide concentrations in the cerebrospinal fluid and serum of overdose patients were investigated by Gold et al. ( 1 7 N ) with a GLC technique. Hansen and Fischer ( 2 1 N ) reported a GLC method for the simultaneous determination of glutethimide and an active hydroxylated metabolite in tissues, plasma and urine. Improved flame-ionization GLC procedures for the qualitative and quantitative assay of the benzodiazepines in whole blood were presented by Greaves (18N).Peterson and Rodgerson (36N) developed a GLC method for the determination of ethylene glycol in serum, in which the dibenzoate ester derivative is

formed, A screening test for acetylaminophenol overdose, which is based on addition of o-cresol to a urine sample, was reported by Simpson and Stewart (43N). Searle et al. (42N) employed anodic stripping voltammetry with a commercially available instrument for the ultramicro determination of lead in blood and urine. The Delves atomic absorption microsampling system was evaluated by Hicks et al. ( 2 3 N ) for blood lead analysis, and this approach was found suitable for pediatric specimens and for screening large numbers of patients. Evenson and Pendergast ( 1 3 N ) described a rapid ultramicro method for the measurement of erythrocyte lead concentration, with use of the graphite-tube furnace and atomic absorption spectrophotometry. The techniques and equipment required to properly obtain a micro-scale capillary blood specimen for subsequent lead analysis were discussed by Bratzel and Reed ( 8 N ) .Anderson et al. ( 2 N ) evaluated eight atomic absorption systems for the determination of lead in capillary blood, with regard to accuracy and suitability for analysis of up to 100 samples per day. Sources of error in several methods of sample preparation for blood lead determination by atomic absorption were presented by Kopito et al. (27N). Kufner and Schlegel ( 2 9 N ) determined &aminolevulinic acid (ALA) in urine by TLC. A one-tube method for the direct measurement of urinary ALA was reported by Satgunasingam et al. (40N).Berlin et al. ( 5 N )presented the results of an interlaboratory survey of methods for the determination of urinary ALA. A comparison of four methods for the quantitation of ALA in urine was reported by Roels et al. (39N).Tomokuni ( 4 5 N ) described a colorimetric procedure for the determination of ALA dehydratase activity in erythrocytes, based on measurement of residual substrate, as an index of lead exposure. A simplified atomic absorption method for the determination of cadmium, lead, and thallium in a single urine sample, which relies on the use of a carbon rod atomizer and solvent extraction, was developed by Kubasik and Volosin (28N). Giovanoli-Jakubczak et al. (16N) presented methods for the quantitation of total and inorganic mercury in hair by flameless atomic absorption, and of methylmercury by GLC. A microdiffusion colorimetric method for the determination of whole blood cyanide was described by Pettigrew and Fell ( 3 7 N ) ,and applied to the study of the conversion of cyanide to thiocyanate. Ramieri et al. (38N) presented a new procedure for the rapid estimation of carboxyhemoglobin, in which a dual-wavelength spectrophotometer is used to null out the absorbance of reduced and oxyhemoglobin.

THERAPEUTIC DRUGS AND VITAMINS Marks et al. (28P) reviewed the basic pharmacokinetic considerations and procedures for the measurement of several commonly used therapeutic agents. Separation and measurement techniques applicable to the quantitative determination of therapeutic drugs in biological materials were reviewed by Maickel (27P). Horning et al. (2OP) reported the analysis of selected sedatives and their metabolites by use of combined GLC-mass spectrometry-computer systems. A solid-phase RIA of serum digoxin, in which anti-digoxin serum is conjugated to agarose or to organofunctional glass beads, was developed by Line et al. (2"). Horgan and Riley (19P) presented a rapid method for the RIA of plasma digoxin, in which an lZ5I-labeleddigoxin derivative is used as the tracer. The results of an interlaboratory comparison of three methods for the RIA of digoxin were reported by Gutcho et al. ( I 4 P ) . Drewes and Pileggi ( 5 P ) developed an improved method for the RIA of serum digoxin, which includes the use of ammonium sulfate fracA N A L Y T I C A L C H E M I S T R Y , VOL. 47, NO. 5 , APRCL 1975

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tionation and individual serum blanks. Low serum albumin concentration may produce falsely low digoxin levels by RIA procedures, according to a study by Holtzman et al. (18P). Wells et al. (51P) developed a GLC method for the determination of methylphenidate (Ritalin), and its metabolite ritalinic acid in urine. A fluorometric procedure for the measurement of propoxyphene (Darvon) in whole blood, urine, or tissue a t therapeutic levels, based on reaction of was reported the drug with chloro-nitrobenzo-oxadiazole, by Valentour et al. (50P). Evenson and Koellner ( 7 P ) determined propoxyphene in serum, at both toxic and therapeutic levels, by a rapid GLC method. GLC was employed by Knowles et al. (24P) to quantitate low levels of meperidine in plasma and urine. Tigelaar et al. (47P) developed a method for the RIA of plasma diphenylhydantoin, and presented a comparison of results with a GLC procedure. A sensitive GLC method for the simultaneous analysis of plasma carbamazepine (Tegretol) and other common anticonvulsants, was reported by Roger et al. (39P). Reider (38P) described a fluorometric procedure for the determination of nitrazepam and its major metabolites. High-pressure liquid chromatography was employed by Thompson et al. (46P) to identify and quantitate theophylline and its metabolites in urine. Moody et al. (30P) described a specific fluorometric method for the measurement of protriptyline in plasma. A pH-dependent solvent extraction system to separate tolbutamide and its major metabolites prior to colorimetric or GLC assay, was developed by Matin and Rowland (29P). Aggarwal and Sunshine ( I P ) described a rapid GLC method for the determination of sulfonylureas and their metabolites in plasma. Hichens and Hogans (16P) developed a method for the RIA of dexamethasone in plasma, which is sufficiently sensitive for monitoring of therapeutic concentrations. Metyrapone and its reduced derivative were determined in plasma with a GLC procedure by Hollands and Johnson ( I 7 P ) . Randolph et al. (37P) described a UV-spectrophotometric procedure for the measurement of doxepin and its metabolites in urine, based on oxidation of the compounds to their ketone form. An improved fluorometric method for the determination of furosemide in serum and urine was developed by Forrey et al. ( 9 P ) ,and applied to the study of the drug's binding to serum proteins. Smith (42P) employed gentamicin: adenine mononucleotide transferase obtained from bacteria, to measure gentamicin and other aminoglycosides in body fluids. The method of Hayes and DuBuy for the determination of oxytetracycline in plasma was modified by Murthy and Goswami (32P). Horowitz and Spector (PIP) devised a procedure for the RIA of d-tubocurarine in serum. Schulman and Young (41P) improved the method of Rubin for the measurement of the anti-malarial, chloroquine in biological samples. Anodic stripping voltammetry, with chronopotentiometric detection, was applied by Schmid and Bolger (40P)to the analysis of gold in therapeutic gold salts and in serum. Owen (34P) described the factors necessary to consider in the analysis of the dye, indocyanine green, used to monitor cardiac output. Uchiyama and Okuda (48P)employed a Sephadex complex as the solid-phase for a RIA of vitamin BIZin serum. A microbiological method for the assay of vitamin B12 was adapted to the Mecholab analyzer by Davis et al. (3P). Frenkel et al. (IOP) found good agreement between the results of a manual CPB radioassay and the Phadebas kit for the determination of vitamin BIZin serum. A CPB radioassay of serum vitamin BIZ,in which the gamma emitter 57C0 was measured by a liquid-scintillation counting technique, was reported by Gutcho et al. (13P).Yonahara et al. (52P) 30R

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described a CPB procedure for the determination of vitamin B12 in blood. A continuous-flow, colorimetric method for the assay of urinary aminoimidazolecarboxamide was described by Harrison et al. (15P), and applied to the investigation of the relationship between this metabolite and serum concentrations of folate and vitamin BIZ. Dunn and Foster (6P) developed a CPB method for the radioassay of serum folate, based on the use of P-lactoglobulin as the protein-binding agent and charcoal separation of the bound and free folate. A radioisotopic procedure for the measurement of serum folate, in which 3 H - p t e r ~ y l m ~ n ~ g l u t a m a t e is used as the tracer, was reported by Kamen and Caston (23P). Zettner and Duly (53P) reported evidence for the presence of a specific binding protein for folate in human serum, and discussed the potential interference of this substance in competitive binding assays. Kahan (22P) reported a fluorometric procedure for the quantitation of vitamin A in serum. A simplified microcolumn adsorption technique for the fluorometric determination of retinol in plasma was presented by Pollack et al. (35P). Vahlquist (49P) determined the ratio of free to esterified retinol in biological fluids. Thompson et al. (45P) described a fluorometric method for the simultaneous assay of vitamins A and E in plasma. A chloramphenicolresistant strain of Lactobacillus casei was used for a semiautomated microbiological assay of pyridoxal in serum by Davis et al. ( 4 P ) ,and Smith (43P). Srivastava and Beutler (44P) devised a fluorometric procedure for the assay of pyridoxal-5-phosphate in body fluids. Nichoalds (33P) presented a selected method for assessment of the status of riboflavin nutriture by assay of erythrocyte glutathione reductase activity. Riboflavin in urine was determined by a CPB technique described by Fazekas et al. ( 8 P ) . Muiruri et al. ( 3 I P ) reported an automated method for the measurement of thiamine in urine. Baker et al. ( 2 P )discussed the interference by ascorbate-2-sulfate in the dinitrophenylhydrazine assay procedure for ascorbic acid. An automated method for the measurement of ascorbic acid in plasma or whole blood was developed by Garry et al. ( I l l ' ) . Preece et al. (36P) described a CPB procedure for the assay of 25-hydroxycholecalciferol and 25-hydroxyergocalciferol. A combined TLC and GLC technique was employed by Lovelady (26P)to measure individual tocopherols in erythrocytes and plasma. Gugler and Dengler (12P) described a fluorometric method for the determination of the flavonoid, quercitin in plasma, based on chelation with tetraphenyldiboroxide.

SCREENING AND PROFILE TECHNIQUES Goldberg et al. (8Q) investigated the effect of demographic factors, such as age, sex, body weight, smoking, etc., on the concentrations of seven commonly analyzed serum constituents from healthy subjects. Several problem areas associated with the application of laboratory tests in health-screening programs, including definition of normalcy and interpretation of results, were reviewed by Sackett (19Q). Schwartz ( 2 I Q ) reviewed the development of continuous-flow analytical instrumentation, and the application of new multichannel systems to biomedical profiling. High-resolution ion exchange chromatography with UVdetection was applied by Mrochek et al. (16Q)to the investigation of urinary metabolite excretion patterns of patients with Parkinsonism and other neurological diseases. Mrochek and Rainey (17Q) studied the excretion of glucuronides in urine by means of GLC-mass spectrometry. A GLC-mass spectrometry-computer identification system was applied by Jellum et al. ( I O Q ) to the analysis of body fluids, and detected 40 of the known inborn errors of me-

tabolism. Zlatkis et al. (29Q) developed profiles of volatile metabolites in urine by GLC-mass spectrometry. The highresolution GLC analysis of the volatile constituents of body fluids, with use of glass capillary columns, was reported by Novotny et al. ( I S Q ) . Streeter et al. (24Q)developed a modification of the continuous-flow dithionite test for hemoglobin S to permit its use with a filter-paper readout unit. The "Sickle-ID" test as a screening procedure for the rapid identification of sickle-cell trait and anemia, was evaluated by Louderback et al. (13Q). Serjeant et al. (22Q) reported results of a screening program for sickle-cell disease in Jamaica, in which 8000 cord-blood samples were tested by electrophoresis for abnormal hemoglobins. A continuous-flow, fluorometric method for screening for erythrocyte glucose-6phosphate dehydrogenase deficiency was described by. Dickson et al. (0). Lowe et al. (15Q) investigated the stability of several erythrocyte enzymes, including glucose-6phosphatedehydrogenase, pyruvate kinase, triosephosphate isomerase, and glutathione reductase, in various preservatives prior to screening by qualitative fluorescent spot tests. A colorimetric continuous-flow method for the assay of acetylgalactosaminidase (hexosaminidase) activity in serum, in which heat-inactivation is used for screening of Tay-Sachs disease carriers, was developed by Lowden et al. (14Q). Delvin et al. (5Q) reported a fluorometric continuous-flow method for the determination of hexosaminidase activity in serum, suitable for Tay-Sachs screening. A continuous-flow fluorometric method for the measurement of galactose, applicable to screening for galactosemia by use of blood samples collected on filter paper, was described by Grenier and Laberge (9Q). Witten et al. (28Q) investigated the urinary organic acid metabolic profiles of 21 healthy young adults on a controlled diet, by combined GLC-mass spectrometry. The effect of ethanol ingestion by the subjects on these profiles was reported by Witten et al. (27Q). Sternowsky et al. (23Q) described a GLC screening procedure for the measurement of 0-keto and phenolic acids in urine and serum. A simple titrimetric method for total organic acids extracted from urine by solvents, which is suitable for screening procedures, was reported by Kesner et al. (118).Aksu et al. ( I Q ) determined the urinary excretion concentrations of 14 non-nitrogenous organic acids from normal children, by automated silicic acid chromatography. Tucker and Molinary (25Q) discussed the utility of GLC determinations of plasma and urinary amino acids for the investigation of pediatric aminoacidurias. A simplified and rapid one-dimensional TLC procedure for screening of LITERATURE CITED Books and Revlews (1A) "Advances in Automated Analysis", Vols. 1-9, Mediad, Inc., Tarrytown, NY, 1973. (2A) Clin. Chem.. 19, 801 (1973). (3A) Clin. Chem., 19, 640 (1973). (4A) Clin. Chem., 20, 854 (1974). (5A) Clin. Chem., 20, 931 (1974). (6A) Ackerman, P. G., "Electronic Instrumentation in the Clinical Laboratory", Little, Brown & Co., Boston, MA, 1972, 349 pp. (7A) Baron, D. N., "A Short Textbook of Clinical Biochemistry", J. 6. Lippincott Co., Phiiadelphia, PA, 1974, 247 pp, (8A) Biggs, H. G., Woodson, G., "Clinical Biochemistry'', Harper & Row, New York, NY. 1973, 283 pp, (9A) Bloom, S. R., Brit. Med. Bull., 30, 62 (1974). (lOA) Bodansky, O., Latner, A. L., Eds., "Advances in Clinical Chemistry", Vol. 15, Academic Press, New York, NY, 1972, 420 pp. (1 1A) Bodansky. O., Latner, A. L., Eds., "Advances in Clinical Chemistry", Vol. 16, Academic Press, New York, NY, 1973, 329 pp.

amino acids in the plasma and urine of newborns was described by Century et al. (3Q). Levy et al. (12Q) screened filter-paper urine samples of newborns for histidinemia by chromatography, and noted that this condition may be a benign metabolic disorder. Gitlitz et al. ( 7 Q )measured the amino acids in sweat, plasma, and urine from 22 healthy subjects by automated ion-exchange chromatography. A continuous-flow colorimetric method for the determination of acetylisoniazid in urine was reported by Varughese et al. (ZSQ),which is suitable for the phenotyping of large numbers of patients for rate of inactivation of isoniazid. Cooke et al. ( 4 4 ) evaluated a micro-scale screening procedure for increased blood lead levels, which involved collection of capillary blood on filter paper and measurement by atomic absorption spectrophotometry. Butts et al. ( 2 Q )developed a continuous-flow colorimetric method for the determination of serum thiocyanate, which may be used to distinguish sm'okers from non-smokers. Schneider and Hesse (20Q) reviewed current procedures for the analysis of urinary calculi.

BLOOD GASES ANDpH Rej and Vanderlinde ( 9 R ) reported the results of an interlaboratory proficiency survey of determinations of pH, pCO2, and total C02 in serum-based samples. Problems associated with the correct analysis of blood gas concentrations were discussed by Dowd and Jenkins ( 2 R ) .Joly et al. ( 4 R ) investigated the influence of different concentrations of heparin on the measurement of blood pH, PO*, and pCO2. Drinker et al. (3R) compared the Corning 165 and Radiometer blood gas analyzers, and reported on the use of a sodium chloride-phosphate buffer for pH standardization with an isotonic NaCl bridge. A quality control system for blood pH and gas measurements, with use of a dailyanalyzed tonometered bicarbonate-chloride solution, was described by Noonan and Burnett (8R).Schmidt and Heise (IOR)presented equations and computer programs for conversion of pCO2 values in blood at different pH values and temperatures, into COz concentrations. The measurement of urinary bicarbonate by a titrimetric procedure was described by Lin and Chan ( 6 R ) . Ladenson et al. ( 5 R ) employed tris buffers for the quality control of blood pH measurement. The use of control sera for monitoring temperature changes during pH and pC0z measurements was discussed by Manning et al. ( 7 R ) . Dahms and Horvath ( I R ) described a technique for the analysis of carbon monoxide in blood, in which vortex extraction is followed by measurement in a gas chromatograph with thermal conductivity detector.

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Life Sci., 12, 327 (1973). (23J) Felix, A. M., Terkelson. G., Anal. Biochem., 58, 610 (1973). (24J) Flentge, F., Venema, K., Korf, J., Biochem. Med., 11, 234 (1974). (25J) Gehrke, C. W., Kuo, K . C., Zumwalt. R. W., Waalkes, T. P., in "Polyamines in Normal and Neoplastic Growth, Proc. Symp. 1972", D. H. Russell, Ed., Raven Press, New York, NY, 1973, p 343. (26J) Godicke, W., Godicke, I., Clin. Chim. Acta, 44, 159 (1973). (27J) Goldenberg. H., Clin. Chem., 19, 38 (1973). (28J) Goodwin. P. F., Stampwala, S.,Clin. Chem., 19, 1010 (1973). (29J) Goulle, J. P.. Broun, G.. Ann. Biol. Clin. 31, 467 (1973). (30J) Grunbaum, B. W., Pace, N.. Microchem. J., 18, 146 (1973). (31J) Guilbault, G. G., Froelich, P. M., Clin. Chem., 19, 1112(1973). (32J) Guilbault, G. G., Froelich, P. M.. Clin. Chem., 20, 812 (1974). (33J) Guilbault, G. G., Nagy, G., Anal. Chem., 45, 417 (1973). (34J) Hamilton, P. B., Myoda. T. T., Clin. Chem., 20, 687 (1974). (35J) Heinegard, D., Tiderstrom, G., Clin. Chim. Acta, 43, 305 (1973). (36J) Hughes, W. L., Christine, M., Stoilar, B. D.. Anal. Biochem.,55, 468 (1973). (37J) Humbel, R., Marsault. C., J. Chromatogr., 79, 347 (1973). (38J) Jacobs, H. A. M., Olthius, F. M. F. G., Clin. Chim. Acta, 43, 81 (1973). (39J) Jones, C. E., in "Quantitative Thin Layer Chromatography", J. C. Touchstone, Ed., Wiley. New York, NY, 1973, p 155. (40J) Kammermder. H., Anal. BiOChem., 58, 341 (1973). (41J) Kirberger, E., Keiier, H., 2.Klin. Chem. Klin. Biochem., 11, 205 (1973). (42J) Klein, E., Lucas. L. B., Clin. Chem., 19, 67 (1973). (43J) Klosse, J. A.. Huistra, D. Y.. De Bree, P. K., Wadman, S. K., Vliegenthart, J. F. G., Clin. Chim. Acta. 42. 409 11972). (44J) Korf, J..~SChutie, H.'H., Venema, K.. Anal. Biochem., 53, 146 (1973). (45J) Lau, H. K. Y., Guilbault, G. G., Clin. Chim. Acta, 53, 209 (1974). (46J) Layzell, D. J., Clin. Chim. Acta, 43, 351 (1973). (47J) Llenado, R. A.. Rechnitz, G. A,. Anal. Chem., 46, 1109 (1974). (48J) Lorentz, K., Fresenius' Z.Anal. Chem., 269, 182 (1974). (49J) Lou. M. F., Hamilton, P. B., Biochem. Med., 8, 485 (1973). (SOJ) Lum. G.. Gambino, S. R.. Clin. Chem., 19. 1184 (1973). (51J) Ma, R. S. W., Chan, J. C. M., Clin. Biochem., 6, 82 (1973). (52J) Martin, L. E., Harrison, C., Biochem. Med., 8. 299 (19731. (53J) Marton: L. J:, Russell, D. H.. Levy, C. C., Clin. Chem., 29, 923 (1973). (54J) McGregor, R. F.. Brittin, G. M., Sharon, M. S.,Clin. Chim. Acta, 48, 65 (1973). (55J) Menichini, G. C., Giovannetti, S..Lupetti, S., Experientia, 29, 506 (1973). (56J) Meites. S.,Thompson, C., Roach, R. W., Clin. Chem., 20, 790 (1973). (57J) Morin, L. G., Clin. Chem., 20, 51 (1974). (58J) Morin, L. G., Prox, J., Am. J. Clin. Pathol., 60, 691 (1973). (59J) Nanjo, M., Guilbault, G. G., Anal. Chem., 46, 1769 (1974). (60J) Naruse, M., Hirano, K., Kawai, S.,Ohno, T., Masada, Y.. Hashimoto, K., J. Chromatogr., 82, 331 (1973). (61J) Nathenes, J., Dexter, J.. Katzman, R., Biochem. Med., 8, 259 (1973). (62J) Oddy, V. H.. Clin. Chim. Acta, 51, 151 (1974). (63J) Park, N. J., Fenton, J. C. B., J. Clin. Pathol., 26, 802 (1973). (64J) Pesce, M. A.. Bodourian, S. H., Nicholson, J. F., Ciin. Chem., 20, 1231 (1974). (65J) Peskar, B., Spector, S.,Science, 179, 1340 (1973). (66J) Pfadenhauer, E. H.. J. Chromatogr., 81, 85 (1973). (67J) Proelss, H. F., Wright, B. W., Clin. Chem., 19, 1162 (1973).

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A N A L Y T I C A L C H E M I S T R Y , VOL. 47, N O . 5, A P R I L 1975

35R

(34K) Sobel, C., Cano, C.. Thiers, R. E., Clin. Chem., 20, 1397 (1974). (35K) Sterling, R . E., Matthews, W. S.,Clin. Biochem., 7, 271 (1974). (36K) Subbiah. M. T. R., Ann. Clin. Lab. Sci., 3, 362 (1973). (37K) Teunissen, A. J., deleeuw, R . J. M., Boink, A. E. T. J., Hamelink, M. L., Mass, A. H. J., Clin. Chem., 20, 649 (1974). (38K) Trivin. F., Levillain, P., Lemonnier. A., Ann. Bid. Clin., 31, 329 (1973). (39K) Van den Berg, H., Hommes, F. A,, Clin. Chim. Acta, 51, 225 (1974). (40K) Van Berge Henegouwen. G. P.. Ruben, A., Brandt, K. H., Clin. Chim. Acta, 54, 249 (1974). (41K) Von Nicolal, H.. Zilliken, F., J. Chromatogr., 92, 431 (1974). (42K) Walters. M. I., Ann. Clin. Lab. Sci., 4, 29 ( 1974). (43K) Zivin. J. A,, Snarr, J. F., Anal. Biochem., 52, 456 (1973).

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(13N) Evenson, M. A,, Pendergast, D. D., Clin. Chem., 20, 163 (1974). (14N) Evenson, M. A,, Poquette, M. A,, Clin. Chem., 20, 212 (1974). (15N) Frings, C. S..Crit. Rev. Clin. Lab. Scb. 4, 357 (1973). (16N) GiovanoliJakubczak, T., Greenwood, M. R.. Smith, J. C., Clarkson, T. W., Clin. Chem., 20, 222 (1974). (17N) Gold, M.. Tassoni, E., Etzi, M., Clln. Chem., 19, 1158 (1973). (18N) Greaves, M. S., Clin. Chem., 20, 141 (1974). (19N) Greeley. R. H., Clln. Chem., 20, 192 (1974). (20N) Griffiths, W. C., Oleksyk, S. K., Dextraze. P., Diamond, I., Ann. Clin. Lab. S c i , 3, 369 (1973). (21N) Hansen. A. R., Fischer. L. J.. Clln. Chem., 20, 236 (1974). (22N) Hayes, T. S., Clin. Chem., 19, 390 (1973). (23N) Hicks, J. M . , Gutierrez, A. N., Worthy, B. E.. Clin. Chem., 19, 322 (1973). (24N) Horning, M. G., Gregory, P., Nowlin. J.. Stafford, M.. Lertratanangkoon, K., Butler, C.. Stillwell, W. G., Hill, R. M., Clin. Chem., 20, 282 (1974). (25N) Jatlow, P., Seligson. D., Ciin. Chim. Acta, 50. 19 (1974). (26N) Kk;in.~B., Sheehan, J. E., Grunberg, E., Clin. Chem., 20, 272 (1974). (27N) Kopito, L. E., Davis, M. A,, Shwachman, H., Clin. Chem., 20, 205 (1974). (28N) Kubasik, N. P., Voiosin, M. T., Clin. Chem., 19, 954 (1973). (29N) Kufner, G., Schlegel, H., J. Chromatogr., 85, 109 (1973). (30N) Kullberg, M . P., Gorodetzky, C. W.. Clin. chem., 20, 177 (1974). (31N) Lundberg, G. D., Walberg. C. E., Pontlik, V. A,, Clin. Chem., 20, 121 (1974). (32N) Mason, M. F., Dubowski, K. M., Clin. Chem.. 20, 126 (1974). (33N) McBay, A. J., Clin. Chem., 19, 361 (1973). (34N) Meola, J. M . , Vanko, M . , Clin. Chem., 20, 184 (1974). (35N) Mule, S. J., Bastos, M. L., Jukofsky, D., Clin. Chem., 20, 243 (1974). (36N) Peterson, R. L., Rodgerson. D. O., Clin. Chem.,'20, 820 (1974). (37N) Pettigrew, A. R., Fell, G. S., Clin. Chem., 19, 466 (1973). (38N) Ramieri. A,. Jatlow, P., Seligson, D., Clin. Chem., 20, 278 (1974). (39N) Roels, H., Lauwerys, R., Buchet, J. P.. Berlin, A., Smeets, J., Clin. Chem., 20, 753 (1974). (40N) Satgunasingam, N., Buttery, J. E., De Win, G. F., J. Clin. Pathob, 28, 800 (1973). (41N) Schneider, R. S.,Lindquist, P., Wong, E. T., Rubenstein, K. E., Ullman, E. F., Clin. Chem., 19, 821 (1973). (42N) Searle, B., Chan, W., Davidow. B.. Clin. Chem., 19, 76 (1973). (43N) Simpson, E., Stewart, M. J., Ann. Clin. Biochem., 10, 171 (1973). (44N) Sunshine, I., Clin. Chem., 20, 112 (1974). (45N) Tomokuni, K., Clin. Chem., 20, 1287 (1974). (46N) Wallace, J. E., Hamilton, H. E., Riloff, J. A,, Blum, K., Clin. Chem., 20, 159 (1974).

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