Biochemical analysis - ACS Publications

Leningrad. Univ.: Leningrad, USSR. (297) Tserkovnitskaya, I. A., Grigor'eva,. M. F., Probl. Sovrem. Khim. Koord. Soedin., 1970, 234. (298) Tserkovnits...
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(296) Tserkovnitskaya, I. A., Borovaya, N . S., Ankudinova, M. M.,Prima. Org. Reagentov Anal. Khim. 1969, 180;

Ed., Tserkovnitskaya, I. A., Izd. Leningrad. Univ.: Leningrad, USSR. (297) Tserkovnitskaya, I. A., Grigor’eva, M. F., Probl. Sovrem. Khim. Koord. Soedin., 1970,234. (298) Tserkovnitskaya, I. A., Shipunova, L. G., Primen. Org. Reagentov Anal. Khim., 1969,173, Ed., Tserkovnitskaya, I. A,, Izd. Leningrad. Univ.: Leningrad, USSR. (299) Tutundzic, P. S., Stojkovic, L).J., Glas. Hem. Drus., Beograd, 33, 245 (1968 ).

(3i)O) Uiatenko, Yu. I., I>anilenko, E. F., Zavod. Lab., 36, 915 (1970).

(301) TJsatenko, Yu. I., Garus, Z. F., Tulvupa. Khim. Tekhnol.. Tulyupa, F. M., Tekhnol., 1968. 1968, No. 14, 23. No.-14, (302) Usatenko, Yu. I., Suprunovich, V. I.,

Kulikovskaya, Zh. B., Zh. Anal. Khim.,

25, 1890 (1970). (303) Usatenko, Yu. I., Tulyupa, F. M., Garus, Z. F., Khim. Tekhnol., 1968, No. 14, 26. (304) Usvyatsov, A . A., Solomatin, V. T., Zavod. Lab., 36, 1.54 (1970). (30.5) Usvyatsov, A . A., Sudakov, A . It., Krylov, Yu. A., Fronchek, E. V., ibid., 37, 130 (1971). (306) Vajgand, V. J., Jaredic, M. I)., Glas. Hem. Drus., Ijeograd., 34, 223 ( 196:)) . (307) Vajgand, V. J., Pastor, T. J., Bjelica, L. J., ibid., 35, 343 (1970). (308) Vahilevskis, J., Olson, I). C., Loos, K., Chem. Commun., 1970, No. 24, 17. (309) Vesheva, L. \‘., lieishakhrit, L. S., Shcherbakova, S. AT., Primen. Org. Keagenlov Anal. Khim., 1969, 218; Ed,, Tserkovnitskaya, I. A . , Iad.

Leningrad. Tjniv.. Leningrad, USSli.

(310) Vitkina, XI. A . , Hekleshova, G. E., Khim. Il’ekhnol. H e s p . Mezhved. ,Vauch.Il’ekh. Sb., 1969,KO. 15, 100.

(311) Vogt, W., Fresaius’ Z . Anal. Chem., 251, 92 (1970). (312) Voloshina, V. V., Usatenko, Yu. I., Arishkevich, A. M., Zavod. Lab., 36 ,530 (1970). (313) Vorlic‘ek, J., Acta. Geol. Geogr. Univ. Comenianae, Geol., 15, 121 (1968). (314) Vorlicek, J., Fara, )I., Vydra, F., Fresenzus’ Z. Anal. Chem., 241, 314 (lF)fIR\ \ - I _ _

(315) VFestal, J., Kotrly, S., Talanta, 17, 1.51 (1970). (316) Vydra, F., Petak, P., J . Electro-

anal. Chem. Interfacial Electrochem., 24, 379 (1970). (317) Vydra, F., Stulik, K., Acta Geol. Geogr. Univ. Comenianae, Geol., 15, 87 (1968). (318) Wasilewska, L., Acta Pol. Pharm., 27, .i. (1970). % (319) Wasilewska, L., Szyszko, E., Diss. Pharm. Pharmacol., 21, 591 (1969). (320) Yoshimori, T., Ishiwari, S., Talanta, 17, 349 (1970). (321) Yoshimori, T., Natsubara, I., Bull. Chem. SOC.Jap., 43, 2800 (1970). (322) Yoshimori, T., AIatsubara, I., Hiro-

sawa, K., Tanaka, T., Bunseki Kagaku,

19, 681 (1970). (323) Yoshimori, T., Matsubara, I., Tan-

aka, T., Yoshida, K., Tanaka, K., Tanabe, T., Bull. Chem. SOC.Jap., 44,

734 (1971). (324) Zaia, P., Peruzzo, V., Lazxogna, G., Anal. Chim. Acta, 51, 317 (1970). (323) Zakharov, V. A., Songina, 0. A., Bessarabova, I. M.,Lebedeva, L. N., Zh. Anal. Khim., 25, 879 (1970). (326) Zakharov, V. A., Songina, 0. A.,

Bessarabova, I. M., llakhimshanov, P., 1T.Y.S.R. 280,049 (Cl. G Oln, C O l b ) , 26 Aug. 1970, A pl. 12 May 1969. (327) Zakharov, $: A., Songina, 0. A., Chulturova. V. Sh.. Mambetkaziev. E. A,, Zh. A n d . Khim:, 24, 1401 (1969’). (328) Zakharov, V. A., Songina, 0. A,, Klyueva, 11. I., Zavod. Lab., 35, 1309 ( 1969 ).

(329) Zhdanov, A. K., Akent’eva, N. A., Utb. Khim. Zh., 13 (6), 3 (1969). (330) Zhdanov, A. K., Akent’eva, N. A.,

Luk’yanenko, I. L., Dokl. Akad. Nauk Uzb. SSR, 27 ( 8 ) , 37 (1970). (331) Zhdanov, A. K., Akent’eva, N. A,, Kapitsa, N. V., Tr. Tashkent. Gos. Univ., 1968,No.323, 157. (332) Zhdanov, A. K., Akent’eva, N. A , , Luk’yanenko, I. L., Dokl. Akad. iVauk Uzb. SSR, 27 (lo), 42 (1970). (333) Ibid., (12), 28. (334) Zhdanov, A. K., Akhmedov, G., Uzb. Khim. Zh., 13 (4), 29 (1969). (333) Zhdanov, A. K., Akhmedov, G., Izv. Vyssh. Ucheb. Zaved., Khim. Khim. Tekhnol., 13, 1720 (1970). (336) Zhdanov, A. K., Akhmedov, G., Zh. Prikl. Khim. (Leningrad), 44, 660 (1971). (337) Zhdanov, A . K., Akhmedov, G., Luk’yanova, T. V., Uzb. Khim. Zh. 13 (5), 12 (1969). (338) Zhdanov, A. K., Jatrudakis, S., Tr. Tashkent, Gos. Univ., 1968,No.323, 161. (339 j Ibid., p 167. (340) Zbid., p 191. (341) Zhdanov, A. K., Kapitsa, N. V., ibid., p 139. (342) Zhdanov, A. K., Kapitsa, N. V., Dokl. Akad. ,Vauk Uzb. SSR, 26 ( l ) , 29 (1969). (343) Zbid., (9), 26. (344) Zhdanov,’ A. K., Kapitsa, N. V., Akent’eva, N. A., Zh. Anal. Khim., 26, 83.5 (1971). (34.5) Zhdanov, A. K., Markhabaev, I. A., Lenchenko, T. A., Izv. Vyssh. Ucheb. Zaved., Khim. Khim. Tekhnol., 14, 355 (1971). (346) Zhdanov, A. K., Markhabaev, I. A , ,

Lenchenko, T. A., Zh. Prikl. Khim. (Leningrad), 44, 4.56 (1971). (347) Ziemba, S., Chem. Anal. (Warsaw), 15, 829 (1970).

Biochemical Analysis Morton K. Schwartz, Department of Biochemistry, Memorial Hospital for Cancer and Allied Diseases; and Division of Biochemistry, Sloan-Kettering Institute for Cancer Research, New York, N. Y. 7 002 7

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H I S Rl,:VIl,;JV WILL COVh;R developments ill the area of biochemical annlyais during the period from January 1968 through December 1971. It would be presumptuous for any reviewer to claim that he intends to completely cover all of the publications in this dynamic area. As pointed out by the last reviewer in this series ( I n ) , the large volume of pertinent reports encompasses journals in chemistry, biology, physics, electronics, and engineering. Today journals in medicine and computer and nuclear science, as well as in instrumentation must be included. There are literally thousands of references that have appeared during the four-year period of this report. The enormity of the assignment is empha-

sized by the fact that each of the bimonthly Biochemical Methods Sections o j Chemical Abstracts contains over 100 entries and the Journal of Lipid Research, which maintains a cumulative monthly listing of pertinent references to lipid methods, had accumulated 346 references in this limited area through the first eleven months of 1971 ( 5 A ) . It is mandatory that this review be selective rather than comprehensive and precludes incorporation of routine applications or minor modifications of accepted techniques or methods. This reviewer will attempt to cover the subgroupings included in previous reviews of this same title and to emphasize new developments in biochemical analysis with par-

ticular reference to those areas of interest to the writer. Limited reference will be made to the areas of biochemical methodology considered in other reviews in this series. These include clinical chemistry (SA, 4 A ) , fluorescent analysis (6=1), chromatography ( 7 A ) , and the use of enzymes in analytical chemistry (,%?A). NEW BOOKS AND JOURNALS

Of general interest is a Handbook of Biochemistry containing a vast amount of biochemical data (126B). There have been numerous volumes published in continuing series, each devoted to specific areas of biochemistry. The Methods of Biochemical Analysis series continues to present excellent and thor-

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ough reviews of biochemical analysis including chemical, physical, microbiological, and animal assays, as well as basic technique and instrumentation for the determination of enzymes, vitamins, hormones, lipids, carbohydrates, proteins and their products, minerals, metabolites, etc. In 1968 isotope derivative methods, biluminescence assay, automation of enzyme kinetics, enzymatic synthesis and hydrolysis of cholesterol esters, determination of histidine decarboxylase activity, estimation of catecholamines and automation of analysis of absorbent and fluorescent substances on paper &ips were presented (55B). The 1969 volume included chapters devoted to oxygen electrode measurements, separation and determination of bile pigments liquid scintillation counting, gas chromatography, fluorometric assay of enzymes, assay of blood phenylalanine and tyrosine, determination of urea, ammonia, and urease, and separation, identification, and estimation of prostaglandins (56B). The topics in 1970 included gel filtration, free zone electrophoresis, optical rotary dispersion and circular dichroism, automated peptide chromatography, use of the dansyl reaction, and steroid analysis by gas chromatography (57B). The two volumes in 1971 included chapters reviewing isoelectric focusing, mass spectrometry, gas chromatography of carbohydrates, activation analysis and polarography, analysis of cyclic 3’5’-AMP and 3‘5‘GMP, studies of conformational forms of nucleic acid, assay of phytate and inosital phosphate, glutamic and aspartic acids and their amides, hydrogen isotope exchange in globular proteins, and the temperature-jump method for measuring the rate of fast reactions (58B, 59B). There was also a supplemental volume devoted entirely to the analysis of biogenic amines and their related enzymes (60B). The Methods in Medical Research series also contains reviews of interest to the biochemist. For example, the latest (1770) issue (10SB) contains sections devoted to adsorption chromatography, gas-liquid chromatography, ion-exchange chromatography, electrochromatography, and the use of the techniques in the isolation and characteristics of a variety of metabolites in man. The Methods of Enzymology Series with Sidney P. Colowick and Nathan 0. Kaplan as Editors-in-Chief, has continued to present reviews and step procedures for a variety of biochemical methods. These have included the components and enzymes of the citric acid cycle (84B), lipids (85B), steroids and terpenoids (S6B), fast reactions (79B), amino acids and amines (128B), vitamins and coenzymes (91B), proteolytic enzymes (112B), nucleic acids 10R

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and protein synthesis (61B, 96B), enzyme purifications (69B) and photosynthesis (117B). Reviews in other specialized fields have been considered in volumes of annual series. During the period of this review these have included general biochemistry (22B, l24B, 125B), carbohydrates (ldOB), nutritional biochemistry ( l B ) , medicinal chemistry (47B), vitamins (65B-67B), hormones (rB-lOB), microbiology (ISlB-lSCB), virology (86B,87B, 123B) cell physiology (llSB-l15B), proteins (4B-6B1 97B, 136B), immunology (1S9B),enzymes (S5B, 92B, 99B-IOdB), lipids (6.93, 105B-108B) , nucleic acids (37B-SSB) , steroids (24B), catalysis (4SB-46B), chromatography (52% 54B), cancer research (26B-S8B, 74B7 7 4 , clinical chemistry (17B-ZOB), neurochemistry (51B), molecular biology (64B), the biology of surfaces (S6B), and biomedical engineering (7lB). The Essays in Biochemistry series has continued to present reviews aimed a t overall views of biochemical subjects (29B-32B). These reviews present in depth the origin, present status, and likely developments in areas of biochemistry, particularly adaptation, differentiation, ’biosynthesis, and metabolic control. A new series of Critical Reviews has appeared during the period of this report with the stated objective of providing reviews that organize, evaluate, and present what is known about a subject. The first volume in biochemistry has considered interferon, blood coagulation, steroid receptors, chymotrypsin, structural predicting from amino acid sequence, insulin, and asparaginase (68B). Other texts in this series of particular interest to the biochemist are those in analytical chemistry (9SB, 94B), clinical laboratory science (&B, 49B), and bioengineering (50B). The Automation in Analytical Chemistry series, presents a compilation of papers describing automated biochemical analysis by continuous flow technique ( 4 l B , 4ZB, 119B). Each of these volumes contains approximately 200 papers of automation methodology in the areas of biochemistry, industrial chemistry, and other fields of biology. Numerous papers related to biochemical methodology are collected in the Protides of Biological Fluids series. Each volume contains papers from three different areas of protein chemistry. During the period of this report the following have been considered: genetics and antibody response, molecular variation, and electrofocusing ( l l I B ) , conformation and structure of protein molecules, proteins of bodily fluids (llOB), membranes, complement activity, and separation methods (109B). In a series devoted to cytochemistry, the subject was con-

ANALYTICAL CHEMISTRY, VOL. 44, NO. 5, APRIL 1972

sidered from the standpoint of instrumentation, techniques and errors (138B) Emphasis was placed on the interpretation of quantitative staining and fluorescence techniques. Many texts have appeared during the four years of this report. The subjects of these books have been general laboratory techniques ( l d l B ) , physical methods in biology (lo@), spectral analysis (15B),optical methods (122B), gas chromatography (7OB, 8SB, ISOB, l29B), membranes ( S S B ) , enzymatic methods of analysis (6SB), fluorescence assay (lSOB,1S7B), protein sequence analysis (16B), centrifugation techniques ( l S B , l 4 B , ,UB), X-ray analysis (I27B), activation analysis (78B),spectrochemical emission analysis (12lB), chromatography techniques (SB, 7SB, 88B), and electrophoresis (S4B, 90B). There have also been books that have considered techniques in protein chemistry (dB, 81B, 98B), isoenzymes @SI?), microcalorimetry (25B), and a detailed consideration of cyclic AMP (116B). New journals appearing during this review period have been devoted to chemical instrumentation ( I l B ) , chromatography @OB),steroids (89B),computers (8SB, 118B), bioenergics (12B), chemico-biological reactions (1S5B) and general biochemistry (72B). Other new journals have been concerned with clinical investigation (96B), cellular immunology (80B), and general topics in the field (72B). CENTRIFUGATION, DIALYSIS, AHD FILTRATION

Centrifugation] dialysis, and ultrafiltration have continued to be powerful tools in separation of substances suspended in solutions and in determining the physical properties of these materials. There have been several reviews of ultracentrifugal techniques and sedimentation theory (182, 2lC, 26C, 44c, 7 w . Homogenizers have been described for large scale production of brain homogenate (60C) and for the continuous isolation of cell constituents (SSC). Ehrlich ascites tumor cells have been totally disintegrated in 6 seconds in a disintegrator which uses glass beads

(IC).

A zonal centrifuge rotor has been developed to permit gradient recovery from the rotor center (light end of the gradient) or from the rotor edge (heavy end of the gradient) (SC). Another rotor and centrifuge permits continuous sample flow for large scale virus isolation (7C, 9C). The rotor can hold a 3000-ml gradient and has a 700-ml stream volume. It is capable of a centrifugal force of 83,440 X g. A most useful application of ultracentrifugation has been the development of a microanalytical system. The system is independent of surface tension, air bubbles, or viscosity and has a re-

producibility well below 1% (SC). Sample and reagents are brought together in cuvettes in the rotor by centrifugal force. The rotor containing the many cuvettes spins past a beam of light and the signal is displayed on an oscilloscope. A peak is observed continuously for each sample (6C). In an early version of the system, 15 analyses for biuret protein determinations are completed in as little as 30 seconds after the rotor is started (6C). The cuvettes are emptied and rinsed during rotation allowing time between runs of less than two minutes. Methods are being developed to permit analysis of simple substances, active groups, or enzyme activity (8'2). Along similar lines a path rotor has been described for the precipitation of proteins and removal of the pellets. The samples and precipitating reagents are in separate chambers and are brought together in a sedimentation chamber by centrifugal force during rotation. The supernatant then decants itself when the rotor comes to rest (6C). A method has been described for studying sedimentation in angle-head centrifuges using a titanium rotor, polycarbonate tubes and an electronic integrator (42). A high capacity programmed gradient pump has been described which permits preparation for zonal centrifugation of 10 density gradient tubes in 5 minutes with a reproducibility of between 3-5% (66C). A device has been developed to prepare reproducible density gradients with a Technicon proportioning pump ( 6 I C ) . Sucrose gradients have been stored at -60 "C and used after a 90-minute thaw a t room temperature (64C). Devices and techniques for the control, accurate collection, fractionation, and analysis of cesium chloride or sucrose gradients have been described (16C, 3OC, 43C). The usefulness of isokinetic gradients and their preparation for zonal rate centrifugation has been described (4OC, 6OC, SSC), as have flow through cuvettes for the evaluation of density gradients (34C, 66C). Density gradient studies have been carried out in polyglucose ( 4 I C ) , ficoll (68C, 63C), and cesium chloride ( S I C ) . Solid cesium chloride has been used to create an internal standard for the determination of buoyant density (R4C). It was shown that the stability of brain fractions was improved by substituting in the gradient iso-osmotic ficoll-sucrose for hyperosmotic sucrose (26C). Immunoprecipitation has been applied to identification of antigens following centrifugation in an acrylamidesucrose linear gradient and photopolymerization (46C). Automated analysis of protein and semiautomated analysis of enzyme markers has also been used

following density gradient centrifugation (74C) to identify fractions. Centrifugation techniques have been described for the preparation of a Golgi rich fraction (SRC), brain mitochondria in an 0.4M to 1.7M discontinuous sucrose gradient (67C), inner membrane vesicles from rat liver mitochondria by digitonin precipitation (SSC), serum lipoproteins after precipitation of low density lipoproteins with polyanions such as heparin and divalent cations (17c),ribosomes in a 0-1M linear lithium chloride gradient ( I S C ) , microbial lipids in a column of ultrafine silica (67C), adrenal chromaffin granules (37C, YOC), rat small intestine brush borders @IC), and synchronous cell cultures (63C). Sedimentation equilibrium studies have been described for the determination of molecular weights and specific volumes of aldolase and DNAase (69C), and acetylcholinesterase (SSC). A technique was described for molecular weight determinations without reaching equilibrium (4%) in an analytical ultracentrifuge. The equations for molecular weight determinations by sedimentation technique have been considered (28C, 46C, 47C, 6SC) and computer treatment of velocity-sedimentation data described (SSC). Papers were published concerning the alignment of Schlieren and Rayleigh optical systems in the ultracentrifuge (68C, 69C) and modifications of the photoelectric scanner (56C). A scanning densitometer for ultracentrifuge films, based on the Gilford linear transport and recording spectrophotometer was described (64C) as was a magnetic densitometer useful in protein molecular weight determinations by ultracentrifugal techniques (36C). Interference optics were considered from the standpoint of protein measurement by use of the centrifuge as a differential refractometer ( I S C ) . The labeling of fringes (SSC), and equations for determining initial concentrations between 0.2 and 1.0 mg per ml by an overspeeding procedure either a t the beginning or a t the end of a low-speed ultracentrifuge equilibration run (39C) were described as was a new 3-channel ultracentrifuge cell with increased optical reliability (lOC). Optical rotation has been applied as an optical detection system for the analytical ultracentrifuge (SSC). Protein fractionation and particle separation has been accomplished by use of membrane-filtration techniques. This technique has been used to measure 260-nm absorbing substances from bacterial cells (16C). Cell supernatant of rat liver homogenates has been collected by passage under suction through a Millipore filter that retains nuclei, mitochondria, and some of the endoplasmic reticulum (S7C). Separation was accomplished within two minutes

of the death of the rat. Charge-mosaic membranes were used for dialytic s e p arations of KC1 from low-weight nonelectrolytes and neutral amino acids (7%). The problems involved in the use of protein-retentive membranes in the partition of mixtures of macrosolutes has been pointed out (14.3, and four systems of commercially available membranes for concentration of biological membranes have been compared (19C). A rapid dialyzer has been used in place of trichloroacetic acid precipitation and washing in the aminoacylation assay of tRNA (ZOC). A diffusion capsule has been designed which permits constant passage of solute into surrounding solution until about 65% of the solute has diffused out of the capsule. The capsule is a substitute for a pump (49C). Microdiffusion cells for the formation of protein crystals have also been designed and applied to the crystallization of enzymes (?'IC, 76C). Techniques for continuous flow dialysis have been described ( I I C , 2@), as well as a technique for multiple small sample ultrafiltration using dry cellophane tubes suspended in glass tubes (48C). ELECTROPHORESIS

During the period of this review, the majority of electrophoresis reports have been concerned with particle separation on polyacrylamide tubes or slabs. A vertical gel apparatus for polyacrylamide was developed that permits easy temperature regulation and no electrical leakage (5D). Devices have been described for the slicing of polyacrylamide gel slabs (17D, ZYD, 46D). An apparatus was described to permit preparation of sigmoid concentration gradients of gel and a technique developed for two-dimensional electrophoresis (6'7D). A column device for preparative polyacrylamide gel electrophoresis maintained a stable gel column and permitted electronically controlled discontinuous elution. High protein recoveries with minimal protein denaturation were observed (69D). A system for preparative electrophoresis on polyacrylamide gel slabs has also been described ( 8 0 , S l D ) . A method has been described for the concentration and recovery of charged hydrophilic substances following fractionation by polyacrylamide electroas has a procedure for phoresis (,!?OD) the purification and recovery of enterotoxin A (61D ) . The factors affecting resolution and the theoretical considerations of polyacrylamide gel electrophoresis have been considered and the relationships between band width and gel concentrations and band dispersion and electrophoretic mobility discussed (4OD). A mixing and layering device has been proposed for the preparation of polyacryl-

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amide gel slabs in batches for electrophoresis in a continuous gradient ( 4 3 0 ) . It was suggested that better resolutions of serum proteins were obtained by electrophoresis in a gradient than in a homogenous gel. The molecular sieving effect in polyacrylamide gel was considered from the point of view of the relationship between the properties of the protein and the concentration and degree of cross-linking of the gel. Equations were derived to permit rapid determination of molecular weights (620). The detection of radioactive components of polyacrylamide disks was accomplished by mechanical fractionation, automated mixing with eluent carrier, and passage through a flow cell in a scintillation spectrometer

(m. A gel

electrophoresis device was described that prevented curvature of the protein band as it migrated (310). Polyacrylamide electrophoresis has been used to determine as little a t 10-9 gram of protein (260), acidic mucopolysacchar ides (18 0 ) , glycoproteins (18 0 , 2 2 0 , B D ) ,DNA polymerase (630), acid phosphatase (410), RNAase ( 6 8 0 ) , pepsin and pepinogen (11D), and gonadotropins from rat pituitaries (210). The technique has been used to prepare brain membrane proteins ( 4 9 0 , 6 4 0 ) , to detect and quantitate as little as lo-* gram of chondroitin sulfate (380), and to determine sulfated nucleotides as well as to separate 3’ from 5’ adenosine sulfates ( 5 4 0 )and to follow binding of transfer RNA to polyribosomes and single ribosomes (29D). Gels containing 15% acrylamide have been found to have a unique resolving power for electrophoretic separation of low-molecular weight RNA fragments ( 1 5 0 ) . Oligonucleotides of equal size or chain length were separated into their isomers by polyacrylamide electrophoresis (40). The use of polyacrylamide gel concentration gradients for determining molecular properties of macromolecule was introduced and called “Pore Limit” electrophoresis (55D). This technique was used to determine molecular weights of proteins by virtue of the fact that electrophoresis can be carried out to an “end point” of complete arrest of protein at a particular concentration of gel (350, 4 3 0 , 5 6 0 ) . This methodology has been used with a 2.5% to 12% polyacrylamide gel gradient to separate all RNA’s from 45 to 285 ( 9 0 ) . Various modifications of this procedure have been described for molecular weight determinations and the influences of gel density, protein charge, and protein shape on this technique have been considered ( 6 0 ) . Using a 3 to 20’% linear gel gradient, a linear relationship was obtained between the log of molecular weights and the distance of migration ( 5 5 0 ) . A similar procedure has been described using a 4-701, 12 R

gel gradient at p H 4.0 in 9M urea (47D). Molecular weights between 12,000 and 138,000could be determined within 5% of those previously reported ( 4 7 0 ) . Polyacrylamide gel electrophoresis has been used to determine the retardation coefficients, molecular radii, free mobility, and valence of various macromolecules @ I D ) ,and molecular weights of oligopeptides (60D) and acidic mucopolysaccharides (19D) have been estimated by this form of electrophoresis. Equations and computer programs for the definition of distance migrated and velocity of migration have been described ( 5 1 0 ) . The sulfate content of acidic glycosamineoglycans has been determined based on the fact that the electrophoretic mobility in 0.1M HC1 is proportional to the sulfate content of polysaccharide (660). Electrophoresis in polyacrylamide disks has also been successfully used for identification of proteins (320) and the determination of their molecular weights. Proteins separated in polyacrylamide gels in 5 m m precision-bore round quartz tubes were directly scanned a t 280 nm in a special carriage in a Gilford model 2000 spectrophotometer ( 6 5 0 ) . An agarose-acrylamide gel has been used to separate high molecular weight RNA’s ( 5 0 0 ) . When E. Coli RXA and rat liver RNA were mixed, it was possible to separate 125, 235, 185, and 16s RNA’s from each other. Agarose electrophoresis has been used to separate serum lipoproteins (250). Cellulose acetate has also been widely used for electrophoretic studies. With this technique serum glycoproteins (10) and crystalline insulin preparations (140) have been evaluated quantitatively and human 8-glycosidases have been separated and identified by the fluorescence of the aglycon hydrolyzed from 4-methylumbelliferyl-3-~-galactopyranoside (130). High voltage electrophoresis (500 to 4000 V) has been used to separate organic and inorganic phosphates. The order of migration for organic phosphates is ATP, ADP, AMP, cocarboxylase, and thiamine monophosphate (100). A similar technique a t different pH has been used to separate amino acids in urine (240). a-Aminolevulink acid has been determined by electrophoresis on Eastman 6064 cellulose chromogram in a high voltage electrophoresis unit a t 60 V/cm ( 5 3 0 ) and the kinin peptides (KQ, Klo, and KI1) were studied by high voltage paper electrophoresis and their isoelectric points approximated ( 3 7 0 ) . A polymethacrylate device has been described for preparative column electrophoresis using cellulose powder as the supporting medium ( 6 9 0 ) . Better than 95% pure fractions of serum proteins can be obtained with this tech-

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nique. Electrophoresis on a thin layer of Sephadex G-25 was used to evaluate urinary amino acids and peptides (JSD). Electrophoresis in a Teflon (Du Pont) tube containing a sucrose-density gradient has been used to determine electrophoretic mobilities of viruses. The virus zone was determined with an ultraviolet flow cell (7D). A capsule (a column with removable top and bottom caps) containing a liquid gradient column has been placed in a Utube and used for electrophoresis of viruses ( 5 8 0 ) . After electrophoresis, the gradient is pumped out through a monitor and into a fraction collector. A simple J-shaped apparatus for liquid density gradient electrophoresis has also been described ( 4 8 0 ) . Moving boundary electrophoresis was considered and a technique described to permit determinations in an impure mixture of mobilities of very low concentrations of material (30). Equations relating to the electrophoretic transport of materials in boundary gradients, as well as to the shape of the curves have been presented (440, 4 5 0 ) . Agar has been used as a supporting medium for electrophoresis of pepsin and pepsinogen (SOD). Electrophoresis in agar containing antibodies and subsequent autoradiography has been used to determine nanogram concentrations of serum proteins (160). Agarose containing antibodies has been used in the electrophoretic detection of components of heterogeneous proteins (280, 3/30), as little as 100 microunits of insulin immunoreactive material ( $ 9 0 ) and for the direct determination of 8-lipoprotein and albumin in human aorta intima ( 5 7 0 ) . An immunoelectrophoresis technique has been described for assay of water insoluble erythrocyte membrane proteins ( 1 2 0 ) . Immunoelectrophoresis has been carried out in disks of polyacrylamide gel. When the gel was formed, a Plexiglas rod was placed in the middle of the tube and removed just before use. The created orifice was filled with an appropriate antiserum mixed with molten agar (420). Serum a-fetoprotein has been detected in agar by a cross-over technique in which antibody was placed a t the anode and the antigen in serum placed a t the cathode ( 3 4 0 ) . The reactants migrated toward each other. The assay can be completed in 1.5 t o 2 hours with a 5-fold increase in sensitivity compared to immunodiffusion techniques. ISOELECTRIC FOCUSING

Isoelectric focusing has become an important method for the separation and characterization of charged particles. This technique is based on ieoelectric points and depends on electrophoresis in a gradient of varying pH. In such a gradient, the lowest pH is always at the anode and higher pH

value a t the cathode. Proteins will migrate to the p H area corresponding t o their isoelectric point, where their net charge is zero and the protein is “focused”. Although the principle of the technique was known for many years, the problem in its use was the preparation of stable usable p H gradients. Recently, there has become available carrier material with properties necessary for quantitative isoelectric focusing and maintenance of a stable p H gradient. The carrier material was a series of alphatic aminocarboxyl acids (ampholytes) (&‘E, 443). Natural gradients from pH 1-3 were obtained with electrolytes of acids and commerically available ampholytes ( W E ) . The topic of electrofocusing has been the subject of several reviews (19E, % E , S6E, 4SE, 45E, 48E). Isoelectric focusing procedures were originally carried out in columns of buffer. Recently, techniques have been developed for focusing in polyacrylamide gel ( 8 E , 12E, S6E, 46E, 47E). I n one such technique, disk electrophoresis apparatus is used and fractionation takes up to three hours. Proteins are visualized by trichloroacetic acid precipitation within the gel or with protein dyes after removal of carrier ampholytes (12E). In another procedure ( 8 E ) , the aliphatic aminocarboxylic acids used as ampholyte carrier were modified to narrow the pH ranges and the gradient was stabilized in a 0.4-cm column of polyacrylamide gel. Using this technique, human serum was found to have more than 40 bands. It has been shown that during isoelectric focusing on polyacrylamide, a uniform conductivity is not present throughout the gradient and concentration zones of the carrier material may be formed (16E). Miniaturization of polyacrylamide electrofocusing equipment has permitted study of very small amounts of protein with a precision greater than 0.1 p H unit. The microtechnique has been used in studies of hemoglobin and in combination with immunochemical methods in studies of proteins in human sweat (21E , 24E). Isoelectric focusing has been combined with acrylamide gel gradient electrophoresis to yield a t the same time, information concerning the molecular weight and the isoelectric point of a protein (ZSE). T o prevent protein precipitation during electrofocusing the nonionic detergent Brij 35 has been used (17E). The possibility of protein precipitation has also been minimized by using carrier ampholytes as buffer ( @ E ) . Several spectrophotometer attachments have been described for direct scanning of proteins after isoelectric fractionation (5.73, 6 E , 1SE). Zone convection electrofocusing has been described (41E). This technique uses a series of communicating U-tubes,

and the proteins “focus” at the bottom of the tubes. This procedure does not require a stabilizing medium and may permit better resolution and ease of operation. Focusing has been carried out in 6 t o 24 hours on thin layers of Sephadex G-75 in ampholyte (pH 3-10) carrier solution (thin-layer isoelectric focusing). At the end of the run, a sheet of filter paper is placed over the gel, then dried, ampholyte washed out, and the protein stained. Using this system, both metmyoglobin and oxymyoglobin were shown to focus into more than 20 bands (SSE). A novel approach to the preparation of a p H gradient is the use of a temperature dependent buffer (Tris buffer in a sucrose density gradient) in a column maintained a t a temperature gradient between 0 to 50 “C. In this system, a gradient of 1 p H unit is obtained rapidly and fractionation can be completed in 15 minutes (SOE). Hemoglobin A and S were separated from each other in 16 minutes. Immunochemical techniques have been used in conjunction with electrofocusing for identification of numerous proteins. It has been shown that electrofocusing in a column between pH 4 to 6 did not affect immunological properties of human serum albumin ( S E ) . After isoelectric focusing in polyacrylamide plates or tubes, immunoglobulins G, A, and ill were identified by immunodiffusion with specific antiserums ( 4 E ) . After focusing in 5% acrylamide containing 8 M urea, the gels were frozen, cut into segments, and extracted with buffer. The focused materials were determined directly on the extracts by complement fixation, passive hemolysis, or hemagglutination (18E). The effect of urea (higher isoelectric points) on characterization of proteins by isoelectric focusing has been pointed out (ZZE). A system has been described for elution of sucrose gradients with maintained electrical fields during electrofocusing (S9E). Gradient electrofocusing has been used as a final step in the purification of glycoproteins from human plasma (Z9E) and in studying the properties of pituitary gonadotropins (S4E). The technique was modified to permit study of particulate matter in the p H range 3-5, as well as pH 1-3 and was used to determine the isoelectric points of membranes of erythrocytes, platelets, and lymphocytes (BOE). Serum lipoproteins have been studied in a medium containing 44y0 ethylene glycol using either a 5y0 polyacrylamide gel or a sucrose gradient (25E). Isoelectric focusing has been used to separate urinary proteins ( V E ) ,muscle and liver lactic dehydrogenase ( T E ) , hemoglobins (11E, l 4 E ) , chymotrypsinogen ( Z E ) , red cell oxidoreductases ( S S E ) ,

insulins ( B E , S l E , ) eye lens proteins (1.0, myosin (16E), and IgG, IgA, and myeloma proteins (Z7E). A 2-dimensional system has been described in which after acrylamide electrofocusing, the gel is imbedded in a polyacrylamide slab and subjected to electrophoresis in the second dimension (9E, IOE). This system was stated to be capable of separating IRA from IgG. THIN-LAYER CHROMATOGRAPHY

The technique of thin-layer chromatography has remained a powerful tool in biochemistry and has been the subject of several texts published during the period of this report (68F, 7BF). A method has been described for chromatography on thin layers of liquid on solid surfaces ( 1 7 F ) . With this technique, nonvolatile substances can be separated in 10 to 100 seconds. Rough surfaced films of Indium oxide or etched anodized aluminum have been used to determine as little a t gram of fluoresceiit material (17 F ) . Hydrocarbons have been separated on a mixture of particulat’e polytetrafluorethylene and a microcrystalline nylon and identified by ultraviolet luminescence under liquid nitrogen (77 “IC) (61F). Thin-layer chromatography was carried out directly on tissue sections with the ascendent solvent extracting substances from the tissue ( S 5 F ) . Frozen tissue sections were applied directly to silica gel plates and lipids separated and determined by direct spraying of the plates with chromic-sulfuric acid (1BF). Absorbent, supports, and developing units for thin-layer chromatography have been compared with the conclusion that reliable dat.a with silica gel can be obtained regardless of the support or the equipment used (S9F). Amino acids have been determined quantitatively after chromatography on cellulose powder supported on thin sheets of polyethylene terephthalate ( l 4 F ) . Protein terminal amino acids have been determined by silica gel chromatography: reaction with phenylthiocyanate, and infrared analysis (52F). N-Formyl arid S-acetyl groups in polypeptides have been detected after cleavage with hydrazine to form an acetylhydrazine and then 2-dimensional chromatography of the dansyl derivatives (668’). Cellulose thin layer has been used to separate peptides of G-actin and polyamide thin layers have been used to separate the corresponding dansyl-peptides (Z5F). Pteroylglutamines have been separated and identified on thin layers of XG 5OW-X4-M?; cellulose G plates ( 1 6 F ) . Amino acids in urine have been determined by thin-layer chromatography (SZF, 5QF). In one study, the amino acids after chromatography were visualized as yellow trinitrophenyl derivatives and the intensity of the colors was read densitometri-

ANALYTICAL CHEMISTRY, VOL. 44, NO. 5, APRIL 1972

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cally (69F). Proline and hydroxyproline are not visualized by this technique. An apparatus has been described for direct quantitation of dansyl(1dimethylamino - 1 - naphthalene 5 - sulfonyl) derivatives of amino acids (71F). As little as lo-" mole of material can be measured by luminescence of the derivative after excitation by ultraviolet light (71F). The N-dansyl derivatives and the N,O-didansyl derivatives of seryltyrosine have been separated on silica gel (47F). Picogram amounts of dansyl derivatives of serotonin and 7-amino bytyric acid have been measured following chromatography on polyamide layers and then identification by fluorescence after irradiation a t 248 nm (55F). Twc-dimensional chromatography on precoated cellulose plates and reaction with diazctized p-nitroaniline allows separation and identification of epinephrine, norepinephrine, 3,Pdihydroxyphenalanine, dopamine, 3-methoxytryamine1 metanephrine, normetanephrine, 3,4dihydroxymandelic acid, 3-methoxy-4hydroxymandelic acid, homovanillic acid, 3,4-dihydroxyphenylglycol, 3methoxy-4hydroxyphenylglyco1, tyrosine, and tyramine ( 2 1 F ) . A similar technique has been described with identification on the same cellulose plate of the norepinephrine group by p-nitroaniline and the tryptophan-serotonin metabolites (indoles) by visualization with p-dimethyl aminocinnamaldehyde ( 4 F ) . Epinephrine, norepinephrine, and dopamine have been extracted from urine on AlnOacolumns, converted to O,O,N-triacetyl derivatives and then separated on silica gel and determined by fluorescence measurements after reaction with methanolic potassium ferricyanide-ethylenediamine (248'). By using silica gel, acetyl derivatives of metabolites of serotonin-14C have been separated and detected by fluorescence and then scraped off the plate for liquid scintillation counting (44F). The nucleosides from enzymatically hydrolyzed RNA have been separated and then quantitated after elution from thin layers of microcrystallized cellulose (33F). Following 2-dimensional chromatography on cellulose plates, hypoxanthine, uridine, uracil, inosine, and thymine were detected under ultraviolet light by in situ reflectance spectrophotometry ( 2 S F ) . Nucleotides and nucleosides have also been separated on polyethyleneine cellulose (d9F, ?OF). Complete separation of the deoxyribonucleic acid components, thymidine, deoxycytidine, deoxyadenosine, and deoxyguanosine was achieved by 2-dimensional chromatography on the borate form of polyethylenimine cellulose ( 7 F ) . Differentiation has been made between ribonucleotides and deoxyribonucleotides on thin layers of cellulose (8SF). As little as 0.0001 pmole of

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ATP or adenine separated on microcrystallized cellulose has been identified by dipping the plate in acidified ethanol containing 3% vanillin, heating a t 90 OC and heating with AgNOa bromphenol blue (88F). Cyclic adenosine 3l-5' phosphate can be separated from other adenosine-containing compounds by chromatography on thin layers of silica gel impregnated with sodium tetraborate (79F). The area in which thin-layer chromatography has achieved its greatest importance is in lipid chemistry. After separation on silicic acid, lipids have been identified by color formation after treatment with sodium iodate and then Schiff reagent (1% pararosaniline-HC1 decolorized by saturation with sulfur dioxide) (67F). Gangliosides in human peripheral nerve have been separated by thin-layer chromatography (60F) as have phospholipids from their phospholipid analogs (4OF). Aliphatic non-esterified fatty acids from meat have been separated on cellulose layers on Kieselguhr-silica gel and detected by reaction with benzidine-sodium iodate (7%'). Ninety-five per cent recoveries of phospholipids have been achieved on silica gel using chloroformmethanol-water as the solvents (OF). Phospholipids have been detected on gels by direct charring and then color reaction with the freed inorganic phosphate (38F). Free fatty acids have been separated directly from crude extracts of tissue by chromatography on silica gel plates with linearly decreasing thickness from 1000 ,U at the upper end (5F). The technique of linearly decreasing gel thickness is stated to be useful in isolation of trace amounts of lipids ( 5 F ) . Phospholipids from yeast cells (d6F), animal tissue cells (brain and mitochondrial membranes) (64F), and human blood plasma (78F) have been separated by 2-dimensional thin-layer chromatography and quantitated by inorganic phosphate analyses. A microanalysis of phospholipids in blue-green algae, Hela cells, and pig liver nuclear membranes has been carried out on silica gel-Cas04 plates with detection of the lipids by spraying with Rhodamine B-Tinopal reagent (45F). Following silica gel chromatography, phospholipids were identified by infrared spectroscopy (26F) or spraying the plates with ammonium bisulfate until translucent, charring for 1 hour a t 170 "C and then automatically scanning with an integrating densitometer ( I F ) . Fluorescence after reaction with rhodamine incorporated into silica plates (6.") or spraying (66F) with material has been used to detect and quantitate lipid fractions. Layers of magnesium oxide or absorbents containing magnesium oxide have been used to separate lipids. The pat-

ANALYTICAL CHEMISTRY, VOL. 44, NO. 5, APRIL 1972

terns were different than those observed on silica gel or florid (19F). Thinlayer chromatography has also been used in the detection, isolation, and characterization of menaquinones (48F), ceramides (41F), liver biopsy triglycerides (37F), erythrocyte lipids (77F), glycolipids (64F), and xanthones (8F). Thin-layer chromatography is useful in studies of hormones and vitamins. Water soluble vitamins have been determined and R , values of l l vitamins estimated on a 2 : 1 polyamide-silica gel plate (1329. Pyridoxal, pyridoxine, pyridoxamine, and Ppyridoxic acid were separated from chick embryo liver and quantitated fluorometrically (69F). About 40-picogram amounts of estradiol were determined on silica gel (18F). Fluorimetric assay after chromatography on silica gel impregnated glass fibers sheets permitted detection of nanogram quantities of plasma estradiol (d7F). Urinary estrogens have been separated by thin-layer chromatography and quantitated by gas chromatography (76F). Isomers of ergosterol were detected by spraying with 20% antimony trichloride in chloroform and heating (51F). Prostaglandins in seminal fluids were separated by thinlayer chromatography, extracted with ethanol, and determined a t 238 nm (10F). Hexadecane - impregnated Kieselguhr thin plates have been used to separate analogs of vitamin KP @OF). Steroids have been separated on Adsorbosil and identified by spraying with ethanolic phosphomolybdic acid and then heating (53F); chromatography on polyamines and detection as nitrophenyl hydrazones (588'); on silica gel and measurement of fluorescence quenching (46F); and on digitoninimpregnated silica gel thin layers (76F). Silica gels impregnated with 0.50/, triethanolamine have been used t o separate dansyl derivatives of ketosteroids (andosterone, etiocholanolone, dehydroepiandrosterone, and keto-estrogens) (11F). The thin-layer determination of bile alcohols and bile acids has been reviewed (6OF) and procedures described for the quantitative determination of individual bile acids in serum (57F). Detection of bile acids on chromatograms has been accomplished by spraying with MnC12-H2S04 ( d 8 F ) . A NAD+ linked 3-hydroxysteroid dehydrogenase has been used in the quantitative determination of 3-hydroxy bile acids ( 8 F ) . Twenty-two solvent systems were evaluated for silica gel chromatography of saponins, and chromatography with chloroform - methanol - water indicated that soybean saponins were divided into more than 10 fractions (818'). A1208 was used to separate soybean isoflavones from their 5-hydroxy derivatives (80F). Acid or alkaline silica plates were used for the separation and identification of

isomers of chondroitin and keratin sulfates (49F). Thin-layer chromatography is also useful in studies of carbohydrates (36F). Ten procedures for thin-layer chromatography of carbohydrates in urine have been evaluated and urinary carbohydrates separated on celite and identified with anisaldehyde (84F). Monosaccharides separated on silica gel were visualized and the plate photographed on Polaroid film and scanned in an integrating recording densitometer (4%'). Kieselguhr plates have been used for separation of fructooligosaccharides (15F). Chromatography on talc plates has been used for separation of various prophyrins (6F). Silica gel has been used to identify barbiturates and glutethimide by spraying the dried plates with diphenylcarbazone and mercuric nitrate (6%'). Lysergide (LSD) has been determined by chromatography on silica gel with 1 :9 morpholine-toluene and identified by ultraviolet irradiation and treatment with methanolic 4 - dimethylaminobenzaldehyde ( S F ) . Mixed layers of silica and A1203 have been shown to be the best material for separating hypoglycemic sulfanylurea and biguanide derivatives (65F). Following thin-layer separation, streptomycins have been identified by covering the chromatograms with dried paper on agar plates seeded with spores of bacillus subtilis. hfter incubation, the streptomycins were indicated by clear zones of inhibition (34F). Semiquantitative determination of Znz+ on cellulose powder plates and Fe3+, CuZ+,and Niz+, on A1203 plates has been described (31F). The metal ions are identified by use of a variety of spray solutions. Polyphosphates have been separated on microcrystalline cellulose and identified as spots of molybdenum blue (74F). By varying solvent systems, organphosphorus compounds including phosphines, phosphites, phosphates, phosphonates, phosphinates, and phosphine oxides have been separated (48F). Silica has been separated from phosphate in extracts of biological material by thin-layer chromatography on ECTEOLA cellulose coated glass plates (60F). GAS CHROMATOGRAPHY

Gas chromatography has been extensively used in the detection and identification of compounds of biochemical interest. The technique has been of particular use in studies of steroids, lipids, and in the analysis of drugs and their metabolites. The subject has been considered in several reviews (26G, 62G, 61G, 91G, 111G, 128G). Although the major usefulness of gas chromatography has been in studies of organic compounds, the technique has

also been used in studies of inorganic materials. Chromium has been determined by chelation and extraction with trifluoracetyl acetone in benzene and chromatography on a column impregnated with 5% QF-1 (116G, ll7'G) or by reaction with 1,ll-trifluoro-2,3pentanedione in hexane and then determination of the chromium trifluoroacetyl acetonate using an electron capture detector with a tritium source (68G). Rat liver aluminum has been determined as a trifluoroacetylacetonate using a W i electron capture detector (95G). As little as 2.95 X 10-9 gram of BeZ+ has been determined in biological fluids by electron capture gasliquid chromatography of a trifluoroacetylacetone complex (13OG) and a similar procedure has been used to determine Be2+ in blood and tissue homogenates (129G). Sulfate in glycosamino-glycans (chondroitin 4-sulfate) was determined by exchange of sulfate to a Dowex 50 resin (H+ form), elution with water, treatment with butylamide to form butyl ammonium sulfate and then alkalinization and gas chromatography with a hydrogen flame ionization detector to estimate the stoichiometrically liberated butyl amide (126G). Aminothiols, and disulfides can be determined by gasliquid chromatography by formation of volatile neopentyledene derivatives after reaction with 2,2-dimethylpropanol (67G). The method was used to determine as little as 8 X mole cysteamine and cystamine/ml serum. A nitrogen specific and nitrogen selective version of a thermionic detector has been used for the determination of nitrogen-containing drugs in the ppm range (SBG). Gas chromatography has been used for quantitative determination of gases including 02, COz, NzO (SOG), Hz, Nz, 02, CH4, COZ, Kr (18G), as well as for the assay of the anesthesia fluothane (halothane) in blood (l4G). Nanomolar concentrations of amino acids have been determined by gas chromatography of their butyl N-trifluoroacetyl esters (82G, 89G, 94G, 103G, 109G, IClG, l48G). I n one such technique, amino acids were isolated and purified on cation and anion exchange resins and an optimal 50:l mole ratio of trifluoroacetic anhydride/amino acid was used to acetylate the amino acids. The solution was then concentrated and chromatography carried out on an ethylene glycol adipate chromasorb G column with a hydrogen flame ionization detector (14G). N-Hepta-fluorobutyryl *propyl derivatives have also been used for separation of 20 amino acids (97G). Amino. acids have also been determined by formation of dry amino acid hydrochlorides, then trimethylsilyl derivatives, and chromatography on col-

umns of phenylmethyl siloxane coated on chromosorb G (45G). A temperature program system (180 to 310 "C) has been used to detect trimethylsilylated phenylthiohydantoin amino acids (54G). Methylthiohydantoins have also been used (126G). Trimethylsilyl derivatives were used to detect phenylalanine (59G),tyrosine and tryptophane derivatives (4G) as well as glycopeptides (12G). A rapid four-hour hydrolysis method for proteins was developed and quantitation of 20 amino acids as N-trifluoroacetyl-n-butyl esters or trimethylsilyl derivatives demonstrated (47G). A single step method was described for formation of trimethylsilyl derivatives of all 20 amino acids and then separation on a single column. Methionine derivatives were determined as methyl thiocyanate formed after treatment with cyanobromide (S7G), and as little as gram of phenylalanine was determined after conversion to the volatile neopentylidene methyl ester, by treatment with 2,2-dimethylpropanol (68G). Gas chromatography is widely used in lipid biochemistry. Radiochromatography techniques have been described for studies of methyl esters of fatty acids (85G) and natural triglycerides (15G). Monomethyl branched fatty acids have been differentiated by oxidative degradation and chromatography with a hydrogen flame ionization detector (102G) and by use of an argon detector and a dimethyl-dichlorosilane treated polyethyleneglycol adipate column (l4OG). Accuracy of the determination of methyl esters of palmitic, stearic, oleic, linoleic, and linolenic acid was stated to be improved to *lye by the use of an argon ionizing detector (76G). A procedure was described for the qualitative differentiation of unsaturated and saturated fatty acids by direct hydrogenation with a palladium-celite catalyst on a siliconized column (79G). Adipose tissue triglycerides and fatty acids have been determined by gas chromatography (IOG), as have trimethylsilyl derivatives of linoleic acid and hydroperoxides (4OG), and 1,2 and 1,3 diglyceride isomers of the same carbon length (107G). A temperature programmed system (90 to 240 "C) has been used to detect and quantitate propylene glycol, glycerol, and triethylene glycol in methanolic extracts of tobacco (137G). Alkyl a-glycerol ethers have been determined in amounts as low as 0.01 pmole by conversion to alkoxy aldehydes by periodate oxidation, thin-layer chromatography, and then gas-liquid chromatography on polar and nonpolar phase columns (48G). Dimethyl hydrazones of alcohols of long-chain aldehydes have been determined by chromatography on a semipolar column (72G). Less than 1 pg ethanol per ml of tissue homogenate has been deter-

ANALYTICAL CHEMISTRY, VOL. 44, NO. 5, APRIL 1972

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15 R

mined by this technique after increasing the volatility by conversion of the alcohol to a nitrate (49G). Ceramides (sphingosine and sphingamine) have been separated by gas-liquid chromatography of their trimethylsilyl derivatives (114G) as have cerebrosides and phospholipids (64G). Phospholipids have been determined by phosphorolysis conversion to 1,2 and 1,3 diglycerides, formation of trimethylsilyl derivatives, and then gas chromatography (20G). The technique has been used in studies of egg lecithin, cephalin and cardiolipin, and human plasma sphingomyelin. The sialic acid component of gangliosides from brain of several species has been assayed by conversion of the sialic acid component to the methyl ketoside and chromatography as the trimethylsilyl derivative (IS9G). Derivatives of choline and its esters were determined by directly sweeping them in a nitrogen stream into the gas chromatography column (127G). Brain choline esters were extracted and then reacted with ammonium reineckate and sodium benzenethiolate to form tertiary amines for gas chromatography (57G). The subject of choline ester analysis has been reviewed (7OG). Plasma lipids have been determined quantitatively (22G, 81G) and in micro concentrations by gas chromatography (57G, 70G, 7 l G , I27G). In one technique, acetycholine is converted with dimethylaminoethyl acetate, purified, and extracted into chloroform before chromatography on a silanized column containing 1% phenyl diethanolamine succinate in a hydrogen flame ionization detector (7IG). Methods have been described for the determiiiation of urinary cholesterol (234G), serum cholesterol and cholesterol ester.+ (SSG, 65G), as well as LL programmed temperature system for the simultaneous analysis of fatty acids, cholesterol, and bile acids (28G). Perhaps the most potent tool for the modern steroid chemist is gas chromatography. Its use has permitted separation and quaiititation of the multitude of steroids and their derivatives. The subject has been reviewed from the standpoint of electron capture detection (ISSG), sterol precursors (44G), bile acids (80G), adrenal steroids (90G), and a general consideration of gas phase chromatography of steroid metabolites (60G, 62G), corticosteroids (8G), estrogens (SG), and androgens (35G). Numerous methods have been described for determination of estrogens in pregnancy urine (2G, 4SG, 8SG, 87G, 115G) and plasma (42G, 99G). Estrogens have been determined in nonpregnancy urine by electron capture technique (78G) and by concentration on XAD-2 resin prior to gas-liquid chromatography (2SG). Formation of 16R

the heptafluorobutyrate esters of estrogens permitted analysis a t the 200-pg level with electron capture technique (IIOG). Methods have also been described for pregnanediol, (9SG, 105G, 112G) and pregnanetriol in urine and progesterone in plasma (77G). Aldosterone has been determined by oxidation to a y-lactone, purification on thin-layer chromatography and then gas chromatography of the hepatofluorobutyrate derivative (51G). Electron capture technique has also been used to determine aldosterone and as little as 20 ng of 18-hydroxydeoxy corticosterone (108G). It has been pointed out that success of gas-liquid chromatography of corticosteroids depends on the initial preparation of volatile derivatives (104G), and the problems of derivative formation were studied (62G). (2-19 and C-21 steroids have been determined as trimethylsilyl ethers (S9G) and 0-methyl oximes as heptafluorobutyrates (88G). Gas chromatographic methods for determination of androgens have been compared to competitive protein binding procedures (135G) and plasma testosterone has been determined by 6aNi electron capture analysis of a heptafluorobutyrate derivative (26G) or as the iodomethyldimethylsilyl ether derivative (S4G, 131G). Androgens have also been determined by separations of 11-deoxy- and 11-hydroxy-17ketosteroids on alumina columns and then gas chromatography of the benzene-ethanol eluate on a column of chromasorb G ( I C ) . Steroids in the urine of human infants have been determined by hydrolysis of the steroids, formation of trimethylsilyl ethers, and gas chromatography in a 1% SE-30 or OV-1 column (63G). Bile acids and their derivatives have been determined as trimethylsilyl ethers on OV columns (38G) and as methyl trifluoroacetylated esters on saNi electron capture detectors (74G). This derivative was also used for the determination of hyocholic acid (98G). Phytosterols (cholesterol, campesterol, stigmasterol, and @-sitosterol)in tobacco have been determined quantitatively on a glass column packed with Anakrom coated with silicones (OV-101) (5SG). As much as a 3.5 times increase in sensitivity in steroid analysis has been achieved by using pure oxygen instead of air to support the combustion of hydrogen in a flame ionization detector (138G). Gas chromatography on glass capillary columns has been shown to be more sensitive for the separation and detection of steroids than gas chromatography on conventional columns (1S6G, 106G). With this technique urinary steroids have been separated without purification (I36G). Capillary columns coated with polyimid were

ANALYTICAL CHEMISTRY, VOL. 44,

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superior to those coated with silicones for the separation of trimethylsilyl derivatives of steroids (106G). The use of gas chromatography in the determination of carbohydrates has been the subject of a review (24G). Trimethylsilyl ethers of glucose-6-phosphate, aldose-1-phosphates and their sugar nucleotides (36G) and of a variety of carbohydrates with structural similarity have been separated on columns of ethyleneglycol succinate or 1,4butanediol succinate (50G) or with polar and nonpolar silicone liquid phases (56G). Gas chromatography has been used to determine myoinositol (123G), oxalic acid (,%'IC), hexoseamines (11G, 184G), acetyl groups in mucopolysaccharides (101G) and Krebs cycle intermediates (92G). Polybasic and hydroxyacids (succinic, adipic, malic, mevalonic, citric, and 3-hydroxy-% methylglutaric acids) were determined as diazomethane esters with a hydrogen flame ionization detector (100G). Phenolic acids and alcohols have been determined on open tubular capillary (75G). Saccharin, when columns methylated, forms a volatile derivative that can be determined by either a flame ionization or an electron capture detector (29G). Cyclohexylamine in solutions of sodium cyclamate has been determined in amounts as low as 10-11 gram by formation of a N-2,4-dinitrophenyl derivative by electron capture technique (I22G). Tritnethylsilyl derivatives of purine and pyrimidine bases and nucleosides and nucleotides have been determined in the 3-5 ng range with a hydrogen flame ionization detector (46G). Trimethylsilyloxymethoxy-phene thylamine-pentifluorobenzaldehyde derivatives have been used in gas chromatography to separate and detect primary amines of the catecholamine series. It has been possible to detect 10 pg of these compounds (96G). Normetanephrine and metanephrine have been detected as trifluoroacetyl derivatives (9G), and 3-methoxy-4-hydroxyphenylglycol has been oxidized to vanillin and determined as methoximetrimethylsilyl ether derivatives (7SG). Metanephrine and vanilmandelic acid are separated by extraction with ethyl acetate and each determined by sodium iodate oxidation to vanillin and then gas chromatography as trimethylsilyl ethers (132G). Trytophane-niacin metabolites have been detected as trimethylsilyl derivatives (5G). A gas chromatography method for the simultaneous determination of trimethylsilyl ethers of catecholamines, morphine, normorphine , codeine, and other metabolites of these compounds (19G) has been described, as well as methodology for quantitative determination of tocopherol (84G), nicotine (I7G), melatonin (SIG), and prostaglandin E ( I I S G ) .

Methods have also been described for the estimation of tetrahydrocannabinolic acid (dl G), acetyldopamine (YG), digitogenin/gitogenin ratio (27G), putrescine (121G), and quinic and shikimic acid ( l S G ) ,and gossypol (90G). A combined technique has been developed in which components are separated by gas chromatography and identified by mass spectrometry. These techniques have been used for studies of steroids (16G, 118G, 119G), bile acids (120G), thiamine metabolites (6G), pterins (86G), and hesahydroubiquinone-4 (55G). The combined technique has been used for identification of organic compounds in human breath (66G), and in studies of metabolic disorders with identification of the gasliquid chromatography peaks by computer matching of unknown mass spectra against a library file of 7600 compounds (69G). ADSORBENT TECHNIQUES

Chromatography is a basic biochemical tool for the separation and characterization of many substances. ‘l’he subject can be subdivided into the following distinct areas: iori exchaiige chromatography; column adsorption chromatogralihy; gel filtration; hydrosyapatite and calcium phosphate gel chromatography; affinity chromatography; arid liquid-liquid partition chromatography. Chromatography has beell a titled subject it1 this series of reviews ( 7 A ) and has recently been the subject of a coiisiderable portioii of a volume of Methods in Ihzymology (69B). Gel filtration 011 a Sephadex columii duriiig contiiiuous applicatioii of current (electroretardation filtration) was used to separate serum proteins into 13 fractions ( 7 H ) , and a column for electrochroinatography was described to allow direct withdrawal on elutioii of specific segments of chromatography material ( 4 7 H ) . New ion-eschange materials, carbosymethylagarose and cross-linked carbosymethyl agarose, allowed combinatioii of molecular sieving and ion eschange ( S 2 H ) . Dipolar adsorbents prepared by coupling glycine, P-alanine and t-aminocaproic acid to Sephadex G-75 were insensitive to pH change but highly sensitive to ionic strength (51H ) . Benzoylated DEAE was used to separate single from double stranded DNA, and RNA (57H). Transfer RNA’s were separated by reverse-phase chromatography on a new plastic support, polychlorotrifluoroethylene (48H) and lipophilic ion-exchange resins were prepared by binding saturated and unsaturated aliphatic amines of varying chain length to polyacrylic acid resins (58H). Lipophilic Sephades has been useful in studies of lipids, steroids, steroid conjugates, and trimethylsilyl ethers of carbohydrates (ii6H). Sephadex LH-20

has been useful in chromatography of mixtures of polar and nonpolar lipids by virtue of its combined adsorptive and molecular sieving prbperties (24H). A hydroxyalkoxypropyl derivative of Sephadex LH-20 has been used to resolve compounds differing by only one methylene unit (2SZ-I). The elution volume required for a homologous series of compounds was related to the logarithm of the molecular weight ( 5 H ) . However, iodoamino acids eluted in a sequence unrelated to their molecular weights indicating that elution was governed by Sephadex adsorptive properties and not sieving effects (60H). Sephades LH-20 has been used to fractionate glycerol and diol lipids ( S H ), tristearin, tributyrin, stearic, capric, butyric, and acetic acids (25H), and neutral and phenolic steroids (9H). This technique with methyl alcohol as the mobile phase has been used to separate carcinogenic nitrosoarnines (26H) and with chloroform in petroleum ether, vitamin from its more polar metabolites (S6H). An interesting development in chromatography during the period of this report has been the use of “affinity chromatography ” in the purification of macromolecules. In this technique, a substance capable of entering into a specific reaction is bound to an insoluble polymer or gel. Nonreactirig material passes through the column while specific reactants are adsorbed until conditions are changed to permit elution (22H). A variety of inert materials have been used as matrix, including agarose (18H) and polyacrylamides (19H, 2OH, SSH). Polyacrylamide has been stated to be inferior to agarose for this purpose (56H). Agarose was used by preparing a W aminoalkyl-Sepharose and then preparing bromacetyl alkylated, carboxyl and diazonium-Sepharose derivatives capable of binding to a variety of proteins (18H). Ligands are attached covalently through their amino, carboxyl, phenolic, or imidazole groups and are susceptible to various chemical reagents which separate the intact protein-ligand complex from the matrix (19H). Successful application of affinity chromatography may depend on placing the ligand a t a considerable distance from the matrix backbone

(,%‘OH). L-Tyrosine bound to agarose has been used to separate %deoxy-~-arabinohepttulosonate 7-phosphate from a crude protein mixture (16H) and thyroxin bound agarose used in the purification of serum thyroxin binding protein (49H). Ribonuclease has been purified with an agarose-column bound with a uridine derivative, ribonuclease inhibitor (59H). Insulin bound to agarose has been used to isolate antiinsulin antibodies (17 H ) . Avidin has ANALYTICAL

been purified using a biotin derivative (e-N-biotinyl-L-lysine) bound to Sepharose ( 2 l H ) and Avidin coupled to Sepharose 4B has been used in separating biotin or biotin-containing peptides (SN). Mercurated dextran was used to fractionate nucleotides (54H), and thiol-containing peptides from insulin digests were isolated on an organomercurial polymer of ethylene and maleic acid (4OH). Peptides containing modified residues have been isolated on Sepharose columns bound with specific antibodies (SSH). DNA has been bound to nitrocellulose filters (29H) and to cellulose columns (4SH), but it binds to agarose 50-fold more effectively than to cellulose (50H). A protamine assay has been described using DNA-agarose. The sample containing protamine is layered on the DNA-agarose and the diameter of the resulting precipitation ring is proportional to the protamine (2H). Kieselguhr columns bound with polyamino acids have been used for separation o f nucleic acids (SQH, 4 l H , 42H, 52H). I n one such system, polyornithine, polyarginine, or polylysine on Kieselguhr were used and e-labeled aminoacetyl transfer RNA completely adsorbed on the polyarginine column (S9H). RNA from chick embryo cells was adsorbed on a polylysine-coated Kieselguhr and then eluted with an increasing pH gradient into 4 components ( 4 H ) . The sequence of elution of nucleic acids depends on the polyamino acid used (42H). Methyl albumin-Kieselguhr columns have been used to isolate aminoacetyl transfer RNA with improved elution with magnesium-EDTA in the gradient ( S l H ) . Hyflosupercel substituted for Kieselguhr allowed a 3-fold increase in elution rate (44H). DNA has been fractionated on columns of wood cortical cell protein (SOH). Entrapment of macromolecules in polyacrylamide gel has been used for a variety of biochemical studies. Hexokinase, phosphoglucoisomerase, phosphofructokinase, and aldolase in a polyacrylamide gel column were used to measure the kinetics of glycolysis (6H). DNA in polyacrylamide has been used to purify DKA polymerase (1SH) and trypsin in acrylamide (45H) and hexokinase and glucose-6-phosphate in Sepharose4B and acrylamide-acrylic acid copolymers have been used in metabolite studies (46H). Homopolynucleotides have been prepared by using polynucleotide phosphorylase covalently bound to cellulose (S5H), and glucose has been analyzed by passage of solutions through a polystyrene coil coated with immobilized glucose oxidase (37 H ) . Adsorbent techniques have achieved great popularity in immunochemical

CHEMISTRY, VOL. 44,

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and radiounmune assay techniques.

Disks of poly(tetrduoroethy1ene-g-isothiocyanatostyrene) (11H ) or polypropylene (16H) coated with antibody prepared with human chorionic gonadotropin have been used in the radioimmune assay of human chorionic gonadotropin and leuteinizing hormone. Insulin and gonadotropins have been determined in plastic tubes coated with antiinsulin antibodies 10H, l 4 H ) and gonadotropins with polymerized antibodies (23H). Plastic tubes coated with intrinsic factor have been used for assay of Vitamin B12 (65H); with antiestrogen antibodies in the assay of 17b-estradiol ( 1 H ) ; and coated with antihuman growth hormone antibody in radioimmune assay of growth hormone (12H). Enzyme purifications have been aided by use of specific substrate elution from chromatographic materials. As examples of this technique, ribonuclease has been eluted from phosphocellulose with RNA (d7H), glucose-6-phosphate dehydrogenase from carboxymethyl Sephadex with glucose-6-phosphate (48H) and fructose l16-diphosphatase from carboxymethyl cellulose with fructose 1,6-diphosphate (64H). ELECTROCHEMICAL METHODS

The application of ion-specific electrodes has continued to be a developing area of biochemical analysis. Glass ion-specific microelectrodes have been the subject of a book (311) and several reviews (61, 111, 291, 611). The use of immobilized enzymes in electrodes has also been reviewed (211). Electrodes were miniaturized (661). A microglass capillary electrode was described for the measurement of pH or sodium concentration in a 0.1-nl sample (631). A rapid mixing, continuous flow system capable of electrochemical measurements under turbulent flow conditions in times as short as 10 microseconds was used to determine rates of complex formation of calcium, magnesium, or beryllium with ligands (141). An apparatus was described to permit pH measurements in 0.003 second with a precision of 0.005 pH unit (661). Intramitochondrial pH was measured with an error of A0.016 pH unit by use of the distribution of an inert weak acid, 5,5dimethyl-2,4-oxazoledinedione between the intra- and extramitochondrial compartments (11). A technique was described for measurement with glass electrodes of ion-exchange processes involving sodium, potassium, and hydrogen (161). An electrode made from a valinomycin liquid membrane was selective for potassium in the presence of ammonia and divalent ions (161) and had a reproducibility of 0.07 millequivalent potassium per liter (621). A liquid ionexchange electrode was described with a 93 to 1 selectivity of potassium to 18 R

rn

sodium (681). Ionized calcium was measured with calcium selective electrodes using flow through systems (541,671). Fluoride in hydrochloric acid solutions of bone ash (601) or in perchloric acid digests of tooth enamel, dentine, or bone (591) was measured using a fluoride selective electrode. Aluminum was found to interfere significantly with this technique (391). Fluoride specfic electrodes have been used to measure fluoride in urine (461, 611), serum (171),and water (261). Fluoride covalently bound in carbohydrates was determined directly by alkaline digestion and use of a specific electrode (691). As little as 0.2 pg of fluoride in vegetation was determined by oxygen flask combustion in a Schoeniger flask and the application of the specific electrode (521) or directly after acid extraction of pulverized plant tissue (271). A silver/sulfur specific ion electrode has been used to determine thiols (201) and disulfide groups in proteins (241). Mercury and large amounts of halides interfere in the use of this electrode (201). Cyanide (10-6 to lO-'M) liberated during acid digestion of plants has been measured with a tubular gold electrode (121). An ion-specific liquid membrane electrode has been designed to measure 10-1 to 10-6M acetylcholine with high selectivity over sodium, potassium, and ammonium (SI). Selective nitrate and chloride electrodes have been used to assay these ions in plant tissue (61). Electrodes have been devised to determine enzyme activity and substrate concentration. Urease activity has been measured with an ammonium ion sensitive electrode (411, 421). Urea has been determined by placing a film of urease immobilized in acrylamide gel over the surface of an ammonium ion responsive electrode. The steady-state potential developed a t the electrode surface during urea hydrolysis is proportional to the urea concentration (221, 231). An electrode for measurement of amygdalin has been developed by coating a cyanide sensing electrode with b-glucosidase immobilized in acrylamide gel (561, 651). Cyanide formed during the hydrolysis of amygdalin gives rise to an electrometric response. Cholinesterase activity has been measured using a liquid membrane electrode with a high selectivity for acetylcholine compared to choline and thereby permitting electrode monitoring of acetylcholine hydrolysis (41). 1Monamine oxidase activity in flowing streams has been measured based on differential amperometric measurement with tubular carbon electrodes (381). As little as 0.03 micromole of cu-chymotrypsin has been measured with a fluoride-sensitive electrode. The enzyme is reacted with diphenylcarbamoyl fluoride which inactivates chymotrypsin

ANALYTICAL CHEMISTRY, VOL. 44, NO. 5, APRIL 1972

to yield 1 mole of fluoride per mole of active enzyme (181). Both lactate and glucose have been measured using either lactic dehydrogenase or glucose oxidase in a jellied layer over a platinum electrode and recording the electroxidation of benzoquinone in the case of glucose and ferricyanide in the case of lactate (661). Polarography has had wide biological application. An apparatus for simultaneous spectrophotometric and polarographic measurements has been developed (661). Direct measurement of oxygen consumption in a variety of liquids and gases has been made with a membrane covered polarographic oxygen electrode (101) and the development of polarographic electrodes for assays in living tissues has been discussed (101). An electrode system was constructed to measure rates of photosynthesis or respiratory oxygen exchange a t hydrostatic pressure up to 1400 atmosphere (621). Polarographic methods have been used to measure ferricyanide reduction in respiration and photosynthesis (71). Polarography has been used to determine lead (181, 461), zinc (681, 691), cadmium (371), copper (91), and protein bound sulfur (91) in biological material. Glucose has been determined by polarographic measurement of oxygen consumption after treatment with glucose oxidase (261, 281). I n one procedure, the reaction was recorded on a polarographic oxygen analyzer in 60 seconds and could measure as little as 0.5 pg of glucose using a 0.01-ml sample (491). D-Glucose anomers were determined after attainment of mutarotational equilibration using an oxygen electrode and an absolutely specific p-D-glucose oxidase (471,481).

As little as 5 ng of serum albumin was determined by pulse polarography in ammonia buffer containing a cobalt salt (601) and quantitative analysis of the Brdicka double wave. Pulse polarography has also been used in studies of conformation of nucleic acids (641) and of peptide hormones (191). Polarography has been used to assay cholinesterase activity using thiocholine perchlorate as the substrate (381). Devices have been described for recording electrochemical changes during biochemical reactions. These have included an instrument used in fermentation and enzyme studies to record changes in conductivity in one solution compared to a control solution (21); a coulometric pH stat with numerical readout used for cholinesterase assays (361); a six-channel conductivity recording instrument applicable to enzyme analysis with ionic substrates (301); and a redoxstat with electrolytically generated oxidant or reductant to maintain equilibrium (671).

Kjeldahl nitrogen determinations have been carried out by coulometric titration with an accuiacy of 0.2Q/, (81). Allylic barbituric derivatives were analyzed amperometrically by titration with electrogenerated bromine (401) and amylase w w determined by titrating formed reducing sugars with bromine (4%). Cholinesterase in 2040 pl samples was determined by conductivity measurements during acetylcholine hydrolysis (541). Maltose was determined in a coupled systeni in which added maltose converted the maltose to glucose which in turn was oxidized by added glucose oxidase to gluconic acid and hydrogen peroxide. In the presence of molybdenium ion, the formed hydrogen peroxide oxidized iodide to iodine which reacted with excess thiosulfate. The remaining thiosulfate was subsequently determined by titration with generated iodine (441). RADIO AND RADIOIMMUNE ASSAY

Radioactive isotopes are widely used in biochemical analysis. Of particular importance is the continuing development of radioimmune assays both for peptide and nonpeptide materials. Radioimmune assays are based on the ability of an antigen to inhibit the binding of an isotopically-labeled antigen by antibody. The extent of inhibition of binding and displacement of the tagged antigen is related to the concentration of the unknown antigen. The subject has been extensively reviewed ( S S J , 38J, 107J) and has been the subject of published proceedings of symposia (5SJ,6 2 4 . A comparison of laboratories performing radioimmune assays of insulin indicated that the laboratories agreed on ranking of samples according to insulin content, but the absolute values were likely to range from about 0.5 to twice the interlaboratory mean values ( 1 7 4 . Insulin has been determined by separation of free from bound antigen with ethanol ( 6 1 4 , dextran-coated charcoal ( 7 J ) , anti-globulin antibody (double antibody technique) ( 5 1 4 , and insulin antibody bound to a polystyrene test tube (185). Proinsulin has been determined by radioimmune assay before and after treatment of the sample with an insulin degrading enzyme (54J). Glucagon has been determined by a double antibody technique (4SJ) and with antiserum to both pancreatic and gut hormone using IZ5I-taggedantigen and ethanol separation of free and bound material (45J). Human growth hormone assay methods have been discussed and the hormone determined using anti-globulin antibody separation (67J, ?SJ, 9OJ). Free '*5I-labeled human growth hormone has been degraded by gluthionineactivated ficin and then the remaining antibody bound-hormone precipitated

with trichloroacetic acid (794. Dextran-coated charcoal has been used for separation of free from antibody bound hormone (66J,111J) and in one assay the entire reaction was carried out in a single test tube ( I I I J ) . A combined double antibody assay of growth hormone and insulin in a single serum sample permits detection of 1 punit of insulin and 0.06 ng growth hormone per ml ( 4 9 4 . The problems involved in the radioimmune assay of follicle stimulating and luteinizing hormones have been disSJ, 14J, S 1 J ) . In assays cussed ( M , of follicle stimulating hormone, separation of free from bound antigens was accomplished by absorption on bentonite ( I 1J ) ; chromatoelectrophoresis (94J) and double antibody technique (80J, 9SJ). A committee of the Medical Research Council of Canada recommended a radioimmune assay for FSH (87J). Close agreement has been observed between radioimmune FSH assays and bioassay techniques (f.t?J, 26J ). In the radioimmune assay of luteinizing hormone 1251 human gonadotropinantibody was separated by adsorption on amberlite CG-400 resin ( 5 6 4 ; by double antibody technique ( 7 4 4 ; dioxane (109J), or alcohol precipitation ( 6 2 4 or antibody fixation on polystyrene tubes ( 3 5 4 . I n a novel system, antihuman chorionic gonadotropin serum polymerized with ethyl chloroformate or glutaraldehyde was used as an insoluble immunoadsorbent (46J). Human urinary chorionic gonadotropin was purified by electrophoresis and used in an assay system in which the free hormone was separated from antibody-bound hormone by salting out with ammonium sulfate ( 6 J ) . The method gave good correlation with the bioassay method, but there was cross reactivity with luteinizing hormone. A seven-hour human chorionic gonadotropin assay has been described using rabbit anti-gonadotropin and goat antigamma globulin to separate the hormone antibody complex ( 4 2 4 . Serum chorionic gonadotropins has been determined using charcoal-dextran (11OJ). Prolactin has been determined by radioimmune assay with a sensitivity equal to 5.9 ng of prolactin/ml plasma when chromatoelectrophoresis was used to separate bound from unbound material or 0.2 ng of prolactin/ml of tissue extract with charcoal separation (1OJ). A one-tube system has been described with antibody bound to the test tube ( 5 7 4 as well as double antibody techniques (48J,7 7 J ) . Thyrocalcitonin was determined using porcine thyrocalcitonin, 1311-labeledhormone and separation of free from bound material by chromatoelectrophoresis or charcoal ( Z I J , 104J, 1055). As little

&s 15 X gram of material could be detected (w1.J) and normal human Serum contained 0.02 to 0.4 ng per ml (f0bJ). The preparation of calcitonin and its assay have been the subject of a symposium (106J). Amounts of calcitonin as low as l pg/ml were determined in a method in which an insoluble immunoadsorbent was prepared b y coupling a cellulose derivative to porcine calcitonin by diazotization (19J). Radioimmune assays have been described for parathyroid hormone ( 6 J , 9 6 4 , antidiuretic hormone (68J), oxytoxin (16J), angiotensin (S2J, 37J, 8 1 J ) , renin (34J, 82J, 8 5 J ) , vasopressin (84, cholecystokinin-pancreozymin (S6J, 1 1 7 4 , gastrin (79J, IOOJ, 1 2 5 4 , and ACTH (9J). The radioimmune assay of gastrin has been reported to yield values 2.2 times those obtained by bioassay ( 6 4 4 . Bradykinin (lZ5Ilabel on 8-tyrosine) was linked to ovalbumin and used to prepare rabbit serum antibody for a radioimmune assay sensitive in the 0.1 mpg range (IOU). Dextran-coated charcoal was used to separate free from bound hormone. Radioimmune assays have also been carried out for nonpeptide materials and have been used in studies of steroids (70J). Antibodies to estradiol have been produced by linking it to bovine serum albumin. For the radioimmune assay, tritiated estradiol was used and (NH&S04 used to separate free and bound hormone ( 4 1 4 . Methods with tritiated hormone as a hapten bound to albumin for antibody formation have been described for plasma estrogens (11.44, 2-hydroxy-estrone (116J), aldosterone ( 6 5 4 , and progesterone ( I J , 97J). Radioimmune assays have been described for urinary albumins ( 7 1 4 , F prostaglandins ( 1 3 4 , triiodothyronine ( S S J ) , and cyclic nucleotides (101J). A recent interesting development has been the use of radioimmune assays for the measurement of enzymes. This technique has been used for trypsin and chymotrypsin (108J), fructose 1,6diphosphatase ( S 9 J ) , and a method capable of detecting quantitatively a minimum of 0.025 fig of red cell carbonic anhydrase I and 0.005 pg of the genetically distinct form, carbonic anhydrase 11(44J). I n addition to radioimmune assays, radiochemical analysis in biochemistry can be divided in the following broad areas: competitive protein and nonprotein binding technique; double isotope procedures; and enzymes assays using radiochemical substrates. An incomplete list of radiometric enzyme assays reported during the period of this report includes aryl hydrolase (2OJ), arginase (15J), uridine phosphorylase (6OJ), acetyl- and cholinesterase (255, 5 5 4 , nucleoside kinase and nucleotidase

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(47J), enzymes of the ornithine urea cycle ( 8 8 4 , choline acetyl transferase (90J), elastase (854,acetate thiokinase (98J), pyruvate kinase (76J), renin (69J), ATP-citrate lyase (99J), 3',5'phosphodiesterase ( 4 0 4, chymotrypsin (68J), lipoprotein and triglyceride lipase (605, 9 6 J ) , and p-hydroxyphenylpyruvate hydroxylase (69J). Radioprotein binding techniques are widely used in steroid assays and have been the subject of a published symposium ( 2 3 4 . Competitive protein binding depends on the competition of radioactive and nonradioactive ligands for a protein with a high degree of specific binding power (767). The technique has been used for the assay of androsterone ( 8 6 4 , aldosterone ( 8 4 4 , progesterone (4J, 8 9 4 , prednisolone (91J), cyclic AMP (68J, llZJ), vitamin B12 ( 8 7 J ) , 17P-estradiol (Z443), thyroxine (29J), cortersteroids (78J, Q S J ) , and testosterol (22J, 102J, 113J). ACTIVATION ANALYSIS

Activation analysis is a useful tool in the analysis of trace elements in biological material. The basis of the technique is nuclear bombardment of the element to convert it to a detectable radioisotope. The subject has been reviewed (18K, Z I K , 36K). An automated method for the neutron activity analysis of trace elements has been described (32K). Iodine in tissue has been directly analyzed ( S K , 4 K ) . Thyroid hormone iodine has been determined by Sephadex separation and then as I28Iafter neutron activation (3:3K, 37K). Neutron activated iodide in biological materials has been detected rapidly by using a semiautomated ion exchange system ( 1 4 K ) , or a resin which specifically separates the lz8I from all other radioactive components (16K). Iodine and bromine were determined in the same sample (88K) as was iodine in insulin ( 6 K ) . Iodine was determined by neutron activation of the naturally occurring isotope In (16K). I Whole body calcium has been determined by 49Ca induction (6K, 8 K ) . Total body sodium, chloride, nitrogen, and phosphorus have been determined ( 7 K , M K , 41K). By y-spectrometry and irradiation of bone ash a t a flux neutron/cm-* sec level of 5.2 X calcium, magnesium, sodium, and chloride were determined simultaneously ( Q K ) . Calcium determined in arterial tissue by neutron activation compared well with values obtained by atomic absorption spectroscopy (2SK). Copper has been determined in plants ( l K , SOK, 43K) and in bone, liver, serum, fingernails, and hair by irradiation in a thermal neutron flux and separation of the induced 64Cu by electroplating (ZK). 20R

*

Manganese and copper have been determined simultaneously ( P K ) as were magnesium and manganese which were separated after neutron irradiation by chromatography on a Chelex-100 cation exchange resin column (S8K). Manganese, zinc, and copper have been determined in tissues of rats (4OK); manganese and copper in bacterial cells (19K); zinc in human skin (Z6K); strontium, zinc, copper, and other trace elements in teeth ( S I K ) , and protein bound trace metals in human serum ( I Z K ) . Neutron activation has been used to determine iron in bone marrow (17K), and in a variety of biological materials; cadmium (BZK), vanadium @OK), selenium ($4410, mercury (11K , Z9K), thallium (SQK), thorium (36K), uranium (B7K),and chromium (34K). Manganese, strontium, and barium have been determined simultaneously by neutron activation and an ion-exchange procedure which separated these elements from those with atomic numbers less than 84 (13K). Neutron activation and Kjeldahl nitrogen analysis were compared (1OK). SPECTRAL AND ELECTRON PROBE ANALYSIS

X-ray and electron probe spectrography have been the subject of several reviews (6L, 14L, 19L) as has laser microprobe-emission spectroscopy (1OL). X-ray spectrophotometry of lung tissue has been used to determine antimony a t levels of 2 mg/cm2 (3L) and beryllium (13L) has been analyzed in lung tissue with a quartz spectrograph. X-ray fluorescence has been used to determine replacement of thymine in DNA by halogenated or sulfated compounds by determining the ratios of halogen or sulfur to phosphorus (27L). This technique has been used to measure as little as 0.024 mg of zinc and 0.020 mg of calcium per gram of hair (28L), chromium in urine (dL), magnesium, aluminum, silicon, phosphorus, sulfur, potassium, and calcium in plant and fecal materials (8L), bromine in blood and urine (QL), and total sulfur in serum, urine, and feces (26L) and in pasture samples (18L). X-ray diffraction has been used in mineral studies of bone (16%) and kidney stones (21L) and emission spectroscopy has been used to assay numerous elements in tissues, fecal material, bone, and plants ( I L , ZOL). The use of an argon atmosphere improved the precision and permitted analysis of 1 to 7 ng of chromium in a 0.2-ml aliquot of serum ( I d L ) . Cobalt has been determined by ion exchange separation from interfering elements and then (16L) copper spark spectroscopy. Electron probe analysis has been useful in histochemistry. The electron beam from the probe excites y-ray

ANALYTICAL CHEMISTRY, VOL. 44, NO. 5, APRIL 1972

emission from elements present in the mmple. These rays have a specific wavelength for each element, This technique has been used to study EDTA-activated ATPase in 10-micron slices of skeletal muscle (YL) and in skeletal muscle mitochondria (11L). @-Glucuronidase activity in rat liver was determined by virtue of the electron opaque properties of the diazo product formed during a reaction with naptholglucuronide as the substrate and hexazonium pararosaniline as the diazo reagent (17L). Acid phosphatase and glucose-6-phosphate dehydrogenase were demonstrated in preparations of smooth endoplasmic reticulum (d4L). Leucyl-naphthylamidase was detected by electron opacity of a copper chelate of the enzyme reaction product (2SL). Serum lipoproteins have been examined a t 256,000 magnifications after negative staining with sodium phosphotungstate (QL)and negative staining techniques have been used with protein free particle suspensions (ZZL). A technique has been described for the direct measurement of molecular weight of single stranded DNA by electron microscopy (6L). Audioradiography electron niicroscopy with tritium has been described (26L). MASS SPECTROMETRY, MAGNETIC RESONANCE, LASERS

Mass spectrometry has been used to a limited estent in biochemical analysis. The use of this technique in the study of structure of sugar-containing substances has been reviewed (18M). Spectroscopy with a spark source has been used for trace element analysis ( I S M , 34.11). In one report, 50 or more trace elements were stated to be amenable to simultaneous analysis a t ppb levels in serum, kidney, tumor, lung tissue, bone, and plant leaves (13iM). Mass spectroscopy has been used in the sequence analysis of proteins (2M, S M , 4 O M ) , for the identification of phenylthiohydantoins from the Edman degradation of peptides ( I M , 4 1 M ) , and dansyl derivatives of amino acid (32M). The problems of mass spectroscopy of sulfur-containing amino acids have been overcome by reductive desulfuration, isobutosycarbonylation, and permethylation (36M). Dansyl derivatives of biogenic amines have been studied (8M) and in one report 1-dimethyl amino-5napthalenesulfonyl derivatives of 79 biogenic amines were subjected to low resolution mass spectrometry I39M). As little as 10-9 mole was needed for identification (39M). Serotonin and eight of its hydrosyindole derivatives were identified by direct introduction into a mass spectrometer (24M). Many 2,4,7-trinitro9-fluorenone derivatives of indoles re-

lated to indole-3-acetic acid were d e termined (83M) and spectra for ribonucleosides were described (IOM). Mass spectrometry has been used for the determination of molecular weight distribution of triglyceride mixtures ( I l M ) and to determine microgram quantities of trimethylsilyl derivatives of glycosphinolipids ( 9 M ) . Methods have been described for mass spectrometric structural analysis of cardenolides ( 4 M ) and in determining mescaline and tetrahydroisoquinoline precursors (SOM). In studies of the condensation phase of cigarette smoke, 56 ions were identified in the spectrum (15M). A quantitative mass spectrophotometric system has been described in which ion current a t a particular mle value is recorded (5144). With this system, as little as gram of p tyramine in rat brain has been measured. Resonance techniques are being used with increasing frequency in biochemical analysis. A low frequency nuclear magnetic resonance (NMR) spectrometer has been described capable of holding samples up to 1.25 liters or the whole body of a small animal (25;M). A compact and inexpensive fixed frequency pulsed NMK. spectrometer has been described (18iM). NMR and ESR (electron spin resonance) have been chapter subjects in a text (43111) and NMR analysis of proteins (38M) and enzyme-substrate complexes (6M) has been reviewed. NMR spectral data for trimethylsilyl derivatives of aromatic acids have been described (22M) and NMR has been used in studies of polypeptides (19M). The NMR procedure has been used to study enzyme interactions including z i n c 4 Coli alkaline phosphatase (7M) and atropine and its analogs-acetyl cholinesterase (26M). The lanthanide cations were reported to be excellent NMR probes of loosely and lightly bound ligands of lysozyme (33M). The technique has also been used in iron protein (36M) and azidoferricyNMR tochrome c studies (17M). spectroscopy has been used to separate and determine biological phosphonates from phosphates (16M). Proton magnetic resonance (PMR) has been used in studies of oligosaccharide configuration (42M). Electron magnetic resonance has been used in studies of heme containing materials (10M, 1 l A l ) . The laser beam has been used in a variety of biochemical techniques. It has been combined with emission spectrography in studies of trace elements such as cadmium or zinc in tissues (44M) and in quantitative histochemistry of lead in tissue (87M), as well as for phosphatase assays by analysis of the lead precipitated a t the site of the enzyme

reaction ( H M , 29M). The laser has been used in temperature jump reiaxation studies ( d 7 M ) of cells or organelles and in conjunction with Raman spectroscopy in studies of glycoproteins (14M) and in determining the constituent amino acids of lipozyme (%M). ATOMIC ABSORPTION

The technique of atomic absorption has been useful in biochemical analysis and has become an indispensible tool in clinical chemistry. There has been a text devoted to the subject (88N), and the application of the technique to plant tissue analysis has been reviewed ( S N )* A flameless, electrically heated graphite tube furnace with temperatures up to 2700 "C has been described (88149 as well as atomizers and burners resistant to corrosion, erosion, and heat (16N). The flameless carbon rod method has been applied to analysis of iron, copper, zinc, and lead ( I N , 15N). A hollow cathode tube has been described which permits water cooling, high emission intensity, discharge shielding, and ease of operation (1SN). A method for electronic activated oxygen ashing of specimens has been described (83iV)and a method for using spike height absorbance measurements to detect as little as 0.2 to 0.4 ppm of copper in 90 to 100 p1 of solution (5N). Copper in chlorophyllins and iron in cytochrome c were detected in water solutions of these materials using an air-methane flame (86N), and chromium was detected in animal feed and excreta by a method in which added known amounts of CaZ+ as Cas(PO)c overcame ion interference ( 2 N ) . A dual, double beam atomic absorption spectrophotometer was described which used strontium as an internal reference ( 2 l N ) . The precision of the instrument for calcium analysis was 0.3670. Automated colorimetric calcium determinations with cresolphthalein were compared with atomic absorption analysis (19N). A microsystem for sodium, potassium, calcium, and magnesium determination in 100 pl of sample was described with lanthanum and cesium added to suppress intefering ions ( 6 N ) . Calcium and phosphate were determined in small samples by adding excess molybdenum and then extracting the formed phosphomolybdenum complex with isobutyl methyl ketone and analyzing it and the calcium remaining in the aqueous extract by atomic absorption spectroscopy (Ish). Mercury in biological materials has been determined after wet digestion ( 8 N , 14N, 8 7 N ) . The sensitivity of mercury detection was increased to less than microgram amounts by extraction in an ammonium pyrolidine dithiocarbamate/methyl isobutyl ketone system (18N). Atomic absorption methods

have been described for analysis of gold ( l l N ) , magnesium in muscle and bone ( 7 N ) , cadmium (26N),boron (17 N ), cobalt ( Q N ) ,copper ( I 4 N ) , iron (4N), and lithium (lON, SON). SPECTROPHOTOMETRY, COLORIMETRY

Spectrophotometric and colorimetric analysis are perhaps the most widely used techniques in biochemistry. The spectrophotometry methods used in studies of photosynthesis have been reviewed (100) as have the applications of spectrophotometry in biology (2.90). A double beam scanning spectrophotometer has been described (80) &s has a low temperature device (liquid nitrogen temperatures) for spectrophotometry (210, 250). A titration assembly for anaerobic titration in a spectrophotometer has been used ( 1 1 0 ) . Steroids have been determined by reflectance spectroscopy @lo),as have hemoprotein (200) and ammonia by derivative spectrometry (17 0 ) . Spectrophotometric techniques have been used for the quantitative analysis of elements. Palladium in tissues has been determined by reaction with afuril dioxime (370); gold by reaction with di-2-pyridylketoxime (400); boron by conversion to boron tetrafluoride and then formatioii of a colored complex with Azure-C ( 4 1 0 ) ; copper by complexing with iV,RT,N1,11.'1-tetraethylthiuran disulfide (280); iron with sulfonated ferroin (ferrozine) (60); fluoride by complexing with cerric alizarin ( 1 9 0 ) ; and sulfur by the stoichiometric combination with mercury in the presence of dithizone indicator (40). Sulfide has been analyzed by incorporation of the ion into methylene blue in the presence of N,N-dimethyl-p-phenylene-diamine and ferric chloride (150). Disulfide was analyzed by reduction with dithioerythritol and reaction 5,5-dithiobis (2-nitrobenzoic acid) in the presence of aresenite (430). This reagent has also been used to determine total protein-bound and noliprotein sulfhydryl groups (330) and reduced coenzymes (160). Sulfhydral groups in intact proteins have been determined by disulfide eschange with 4,4dipyridine disulfide (10) and reaction with 2-vinylquinoline to form a complex with an extinction coefficient of 10,000 a t 318 nm (240). Sulfolipids have been determined by formation of a colored complex with the cationic dye Azure A (2.20). Tryptophane in intact proteins was determined a t 390 nm after treatment mith ninhydrin in HC02H and hydrochloric acid (1.20). Glycine has been determined in the presence of other amino acids by reaction with 2,4,6trichloro-btriazine (350). Proline reacted with ninhydrin to form a red pigment which was stabilized by sodium chloride (SO). Taurine was deter-

ANALYTICAL CHEMISTRY, VOL. 44, NO. 5, APRIL 1972

0

21 R

mined by reaction with o-phthalaldehyde in the presence of urea and phosphate ion (130). Hydroxyproline was determined by periodate oxidation and reaction with pdimethylaminobenraldehyde (60). Free glutamine in brain has been determined by acid extraction and direct spectrophotometric analysis (70).Carbonyl groups of proteins have been determined by reaction with 2,4dinitrophenylhydrazine and analysis a t 370 nm (90) and free amino groups with fluorodinitrobenzene (360). Interferences in the Lowry protein technique have been described. These interfering materials have included sulfhydryl reagents (380), hexoseamine (20), and sucrose (140). Tris, glutamine, and phosphate were shown to inhibit the indophenol ammonia reaction (290). Light scattering errors in spectrophotometric protein determinations have been discussed (420). Copper or iron was shown to enhance color development in the indole reaction for the determination of DNA (340). Amino sugars have been determined by chlorination of the amine or amide and reaction with amylose-potassium iodide to produce a blue amylosetriiodide complex (380). A method for inositol assay involves chromic acid oxidation and analysis of the formed inone (270). Skeletal muscle glycogen has been determined by use of a phenolsulfuric acid reaction which yields a colored complex with an extinction coefficient of 12,000 a t 490 nm (260); glyoxalates (allantoin, allantoate, ureidoglycolate, and glyoxylate) have been differentiated by phenylhydrazone formation (390). Diphenylamine has been shown to form a colored complex with RNA (300),and a method described for the direct assay of 5,6-dihydrouridine in RNA (180). There have also been numerous spectrophotometer and colorimetric procedures described for steroid analysis. BIOLUMINESCENCE AND FLUORESCENCE

Bioluminescence and fluorescence techniques have wide application in biochemical microanalysis. Bioluminescence techniques are based on stimulation or inhibition of luciferinluciferase light production. Bioluminescent reactions have been reviewed (14P). The technique has been widely used in ATP analysis and instruments have been devised to quantitate the luciferase-light production. Luciferase has been purified from firefly tails by gel filtration (63P). Light produced in the 30-second period after mixing of sample and enzyme was integrated in a system with a linear reaction over a 1000-fold range of ATP (67P). The luciferase reaction has been used in the assay of pyridine and flavin nucleotides 22R

(FMN+, NADH) (MP)and by use of a liquid scintillation spectrometer, as little as lo-" mole of NADH or FMN were measured (68P). As little as 7.2 X 10-9 mole of adenosine-3',5'phosphate (cyclic AMP) has been measured in 100-pl samples by luciferase luminescence (33P). After extraction of cyclic AMP from tissue and conversion to ATP by an added phosphodiesterase-myokinase-pyruvate kinase system, 2.7 to 4.2 pmole/mg wet weight of tissue have been measured (19P). The luciferin-luciferase assay has also been applied to the detection of ATP sulfurylase (6P). It has been stated that the luciferase assay system can be utilized in all pyridine nucleotide reactions (11P ) . Fluorescence analysis has been the subject of several reviews (16P, 61P, 70P). An instrument and technique for stopped flow fluorimetry was described ( I I P ) , as were techniques for the use of fluorescence for the study of enzyme kinetics and detecting metabolites in the intact living cell (36P38P). Fluorescent methods have been described for studies of cell membranes using as probes various fatty acid analogs (74P1 76P). The possible instrumental errors in fluorometric analysis have been considered (51P). Brownian relaxation time has been studied by use of trypsin bound to a fluorochrome (66P). N-terminal groups of peptides and proteins have been determined fluorometrically after reaction with 2-fluoro3-nitropyridine or 2-fluoro-5-nitropyridine (64P) or 2-p-chlorosulfophenyl-3phenylindone ( S I P ) . The advantages and limitations of albumin analysis by binding with fluorimetric agents have been discussed (66P). The fluorescence of dansyl derivatives of amino acids has been used to assay as little as 10-11- mole of proline, serine, glycine, or leucine (67P). Arginine has been determined in tissue after reaction with ninhydrin and excitation a t 367 nm with maximum fluorescence at 510 nm (1OP). Aromatic amino acids are fluorescent after treatr ment with mercury (1SP) and glycine has been determined by deamination to formaldehyde and formation of a fluorescent compound by reaction with acetylacetone and ammonia (69P). Histidine and o-phthaldialdehyde condense quantitatively to form a fluorescent product (22P). Primary aliphatic amines react with 9-isothiocyanatoacridine to form fluorescent thiourea derivatives (66P) and with ninhydrin and phenylacetaldehyde to form highly fluorescent ternary products (68P). A review of methods for histamine anaIysis has appeared (63P) as have several references for the fluorometric analysis of this compound by condensa-

ANALYTICAL CHEMISTRY, VOL. 44, NO. 5, APRIL 1972

tion with o-phthalaldehyde (4Pl 3OP, 60P, W P ) . Acetylcholine has been determined as a fluorescent salicylaldehyde derivative (81P ) . Fluorometric procedures for the analysis of tissue DNA have been described (29P, Y I P ) and acridine orange fluorescence used in studies of polyanions in mast cells (41P). Impulse fluorometry has been used in an automated system to measure DNA in Ehrlich ascites tumor cells (26P) and the fluorescence of ethidium bound to circular DNA has permitted studies of breakage and joining of DNA (62P). ?-Amino butyric acid was measured fluorometrically by condensation with 1(dimethy1amino)naphthalene - 5 - sulfonyl chloride (68P). Malic acid condenses with resorcinol to form fluorescent compounds allowing measurement of less than microgram quantities of (Isubstituted malic acids (69P). 4'Hydrazino-2-stilbazole was shown to be a specific selective, and sensitive fluorometric reagent for (I-oxy acids (43P)* A sensitive method for the assay of alloxan in tissue was based on the formation of a fluorescent compound after treatment with l12-phenylenediamine ( 7 P ) . An automated spectrofluorometer was used to determine as little as 0.22 pg/ml of morphine and 0.10 pg/ml of quinine in biological materials after acid extraction, alkalinization, and heating (44P). Selenium was determined fluorimetrically after wet digestion and reaction with 2,3-diaminonaphthalene (20P). The fluorescent phenylhydrazone of pyridoxal phosphate was used in its assay (1P ) , and tissue tocopherol determined by direct fluorometry of hexane extracts of tissue (28P). Fluorescence is widely used in studies of catecholamines and their metabolites (76P),and methods have been described for the fluorometric analysis of epinephrine and norepinephrine in plasma (26P, 48P, 64P) and for acid and alcohol metabolites of these compounds (72P). In tissues, dopamine has been differentiated from norepinephrine by microspectrofluorimetry ( 8 P ) . A semiautomated fluorometric method for d o p amine with ferricyanide as oxidant has been described (66P) as well as one in which the dansyl derivative is formed (47P). Homovanilic acid, the chief metabolite of dopamine, has been determined fluorometrically after Sephadex extraction (39P). Serotonin was determined by direct fluoroscopy after extraction (18P) and by condensation with o-phthaldialdehyde (73P). Fluorescent techniques have also been used in the assay of 5 hydroxyindole acetic acid and other indole derivatives (6P, 17P, 34P, 4 2 p ) . Fluorometric techniques have been widely used in steroid analysis. Aldo-

sterone has been determined by oxidation and condensation of the resulting formaldehyde with acetyl acetone to form a highly fluorescent compound (I4P), Cortisol has been determined using phase-separating filter paper (S6P) Many substances have been analyzed by virtue of the fact that they are substrates in enzymatic reactions involving conversion of noduorescent pyridine nucleotide to highly fluorescent reduced pyridine nucleotide. Such materials include, among others, acetyl-coenzyme A (IP, 4OP), acetoacetate, @-hydroxybutyrate, pyruvate, and lactate (49P); prostaglandins (SP); cyclic AMP (SIP) ; acetylcholine (60P) ; bile acids (&P) ; and glutamine and asparagine (46P). Thiamine has been determined by enzymatic conversion to fluorescent thiochrome (16P);carbohydrates by enzymatic methods using fluorescent resazarin-resorufin indicators (IYP); and picomole concentrations of uric acid following uricase treatment (9P). MISCELLANEOUS

During the period of the review, there have been many papers published concerning the adaptation of manup1 methods for automated analysis. The great majority of these have been for continuous flow analysis with the AutoAnalzyer and many of these papers have appeared in the proceedings of the Technicon Symposia on Advances in Automated Analysis. The majority of the automation articles published during this period relate to clinical chemistry or enzyme analysis and therefore will not be reviewed here. Increasing use is being made of enzymatic and non-enzymatic catalytic properties in the analysis of inorganic and organic substances of biochemical interest. The use of bound enzymes for these purposes has been the subject of several reviews. Although gradually being replaced by immunological and radioimmune assays, bioassays still remain a useful tool for many biochemical studies particularly in area of hormone analysis. A subject not covered here is the use in biochemistry of histochemical techniques including autoradiography. There are also biochemical assays using countercurrent distribution, infrared spectroscopy, circular dichroism, and optical rotary dispersion, light scattering and electron field-jump relaxation, and thermal analysis. It has not been possible to include these subjects in this review. LITERATURE CITED

Introduction (1A) D’Eustachio, A. J., ANAL. CHEM., 40,19R (1968). (2A) Guilbault, G. G., ibid., 42, 334R f 1970). (3A)-Kingsley, G. R., ibid., 41, 14R (1969).

(4A) -Zbid.. 43. 15R (1971). (5A) NeA-a;, H. A.-I.,‘Braun, P. E., J. Lipzd Res., 12,781 (1971). (6A) White, C. E., ANAL. CHEM., 42, 57R (1970). (7A) Zweig, G., Moore, R. B., ibid., 349R. \---,

New Books and Journals (1B) Albanese, A. A., Ed., Newer Methods of Nutritional BiochemiPtry: With Applications and Inter retations, Vol. 4, Academic Press, ?few York, N.Y., 1970. (2B) Al:xander, P., Lundgren, H. P., Ed., A Laborator Manual of Analytical Methods of Jrotein Chemistry,” Vol. 5, Pergamon Press, New York, N.Y., 1969. (3B) Altgelt, K. H., Segal, L., Ed., “Gel Permeation Chromatography,” Marcel Dekker, New York, N.Y., 1971. (4B) Anfinsen, G. B., Jr., Edsall, J. T., Richards, F. M., Ed., Advances m Protein Chemistry, Vol. 23, Academic Press, New York, N.Y. 1969. (5B) Ibid., Vol. 24, 1970. (6B) Ibid., Vol. 25, 1971. (7B) Astwood, E. B., Ed., Recent Progress in Hormone Research, Vol. 24, Academic Press, New York, N.Y. 1968. (8B) Zbid., 1’01.25, 1969. (9B) Zbid.. Vol. 26. 1970. (lOB) Zbid., Vol. 27 1971. (11B) Auerbach, d., Ed. Chemical Znstrumentation, Vol. 1, ko. 1, Marcel Dekker, New York, N.Y., July 1968. (12B) Avery, J. Ed., Journal of Bioenergetzcs, voi. 11, NO. 1, Plenum Press, London, Jun; 1970. (13B) Berner, D. A., Principles of Chemical Sedimentology,” McGraw Hill, New York, N.Y., 1971. (14B) Birnie. G. D.. Fox. S. M.. “Subcellular Cdmponents. Preparation and Fractionation,” Plenum Press, New York N.Y., 1969. (15B) blackburn, J. A,, “Spectral Analysis: Methods and Techniques,” Marcel Dekker, New York, N;Y., 1970. (16B) Blackburn, S., Protein Sequence Determinations: Methods and Techniques,” Marcel Dekker, New York, N.Y ., 1970. (17B) Bodansky, O., Stewart, C. P., Ed., Advances in Clinical Chemistry, Vol. 11, Academic Press, New York, N.Y., 1968. (18B) Ibid., Vol. 12, 1969. (19B) Zbid., Vol. 13, 1970. (20B) Zbid., Vol. 14, 1971. (21B) Bowen T. J., “An Introduction of Ultracentri!ugation,” Interscience, New York, N.Y., 1970. (22B) Boyer, P. D., Ed., Annual Review of Bzochemistry, Vol. 37, Annual Reviews, Palo Alto, zalif., 1968. (23B) Brewer, G. J., Isoenzyme Techniaues.” Academic Press. New York. N.k., 1970. (24B) Briggs, M. H., Ed., Advances in Steroid Biochemiatry and Pharmacology, Vol. 1, Academic Press, New York, N.Y. 1970. (25B) Brown, H. D., Ed., “Biochemical Microcalorimetry,” Academic Press, New York, N.Y., 1960. (26B) Busch, H., Ed., Methods in Cancer Research, Vol. 3, Academic Press, New York, N.Y., 1968. (27B) Zbid., Vol. 4,1969. (28B) Zbid., Vol. 5,1970. (29B) Campbell, P. N., Dic$yns, F., ed., “Essays in Biochemistry, Vol. 4, Academic Press, New York, N.Y., 196s. (30B) Zbid., Vol. 5, 1969. (31B) Zbid., Vol. 6, 1970. (32B) Zbid., Vol. 7, 1971. (33B) Cerejido, M., Rotunno, C. A., “Introduction to the Study of Bio~

logical Membranes,” Gordon and Breach, New York, N.Y. 1970. (34B) Chin, H. P., “Celhose Acetate Electrophoresis Techniques and A p plications,” Ann Arbor-Humphrey, Ann Arbor, Mich., 1970. (35B) Clayton, R. B., Ed., Methods in Enzymology, Vol. 15, Academic Press, New York, N.Y., 1969. (36B) Danielli, J. A., Riddiford, A. C., Rosenberg, M. D., Ed., Recent Progress in Surface Science, Vol. 13, Academic Press, New York, N.Y., 1970. (37B) Davidson, J. N., Cohn, W. E., Ed., Progress in Nucleic Aczd Research and Molecular Biology, Vol. 8, Academic Press, New York, N.Y., 1968. (38B) Zbid., Vol. 9, 1969. (39B) Zbid., Vol. 10, 1970. (40B) Deans, D. R., Ed., Chromatographia Vol. 1, No. 1, Pergamon Press, oxford, February 1968. (41B) Editorial Board, “Advances in Automated Analysis,” Technicon International Congress, 1969. Vol. 1, 2, 3, Mediad, White Plains, N.Y., 1970. (42B) Editorial Board, “Advances in Automated Analysis,” Technicon International Congress, 1970, Vol. 1, 2, 3, Thurman Associates, Miami, Fla., 1471

(4iB)-Eley, D. D., Pines, H., Weizs, P. B., Ed., Advances in Catalysis, Vol. 18, Academic Press, New York, N.Y., 1968. (44B) Zbid., Vol. 19, 1969. (45B) Zbid., Vol. 20, 1969. (46B) Ibid., Vol. 21, 1970. (47B) Ellis, G. P West G. B., Ed., Progress in Medicinal &hemistry, Vol. 6, Plenum Press, New York, N.Y., 1969. (48B) Faulkner, W., R., King, J. W., Ed., Critical Reviews an Clinical Laboratory Sciences, Vol. 1, CRC Press, Cleveland, Ohio, 1970. (49B) Ibid., Vol. 2, 1971. (50B) Fleming, D. G., Ed., Critical Reviews in Bioengineering, Vol. 1, CRC Press, Cleveland, Ohio, 1971. (51B) Fried, R., Ed., Methods of Neurochemistry, Vol. 1, Marcel Dekker, New York, N.Y., 1971. (52B) Giddings, J. C., Keller, R. A., Ed., Advances in Chromatography, Vol. 7, Marcel Dekker, New York, N.Y., 1968. (53B) Ibid., Vol. 8, 1969. (54B) Zbid., Vol. 9, 1970. (55B) Glick, D., Ed., Methods of Biochemical Analysis, Vol. 16, Interscience, New York, N.Y., 1968. (56B) Zbid., Vol. 17, 1969. (57B) Zbid., Vol. 18, 1970. 158B) Ibid.. Vnl. 19., ~1971. (59N) Ibid.: Vol. 20, 1971. (60B) Ibid., Supplemental Volume, 1971, (61B) Grossman, L., Moldane, K., Ed., Methods in Enzymology, Vol. 12, Part A. B: Vol. 21. Part D. Academic Press. N’ew York, N.Y., 1968; 1971;< (62B) Guilbault, G. G., Enzymatic Methods of Analysis,” Pergamon Press, New York, N.Y., 1970. (63B) Gunstone, F. D., Ed., Topics in Lipid Chemistry, Vol. 1, Wiley-Interscience, New York, N.Y., 1970. (64B) Hahn, F. E., Ed., Progress in Molecular and Subcellular Biology, Vol. 1, Springer-Verlag, New York, N.Y., 1969. (65B) Harris, R. S., Wool, I. G., Loraine, K. V., Ed., Vitamins and Hormones, Vol. 26. Academic Press. New York. \ - - - I

~

~

Hormones, Vol.

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23R

mones, Vol. 28, Academic Press, New York, N.Y., 1970. (68B) Harte, R. A., Ed., Critical Reviews in Biochemistry, Vol. 1, CRC Press, Cleveland. Ohio. 1971.

(99B) Nord. F. F.. Ed.. Advances in ’ Enzymology, Vol,’ 30,’ Interscience, New York, N.Y., 1968. (100B) Zbid., Vol. 31,1968. (101B) Ibid.. Vol. 32. 1969. (102Bj Ibid.; Vol. 33; 1970. (103B) Olson. R. E.. Ed.. Methods in -~ Medical Risearch, Vol. 12, Year Book Medical Publishers, ‘Yhicago, Ill. 1970. (104B) Oster, G., Ed., Physical Methods in Biological Research; Vol. 1, Part A, Optical Techniques,” Academic Press, New York, N.Y., 1971. (105B) Paoletti, R., Kritchevsky, D., Ed., Advances in Lipid Research, Vol. 6, Academic Press, New York, N.Y., 1968. (106B) Ibid., Vol. 7,1969. 1107BI Ibid.. Vol. 8. 1970. (io8Bj Ibid.: V O ~9; . 1971. (109B) Peeters, H., Ed., Protides of the Biological Fluids. Vol. 15, Elsevier, Amstkrdam, New Pork, 1968; (llOB) Zbid., Vol. 16,1969. (111B) Ibid., Vol. 17,1970. (112B) Perlmann, G. E., Lorand, L., Ed., Methods in Enzymology, Vol. 19, Academic Press, New York, N.Y., 1970. 913B) Prescott, D. E., Ed., Methods in Cell Physiology, Vol. 3, Acadermc Press, New York. N.Y.. 1968. (114B) ?bid.: Vol. 4,1970. (115B) Ibzd., Vol. 5,1971. (116B) Robison, G. A., Butcher, R. W., Sutherland, E. W., “Cyclic AMP,” Academic Press, New York, N.Y., 1971. (117B) San Pietro, A., Ed., Mqthods in Enzymology, Vol. 23, Academic Press, New York, N.Y., 1971. (118B) Schneider, W., Ed., Computer Proarams zn Bio-Medicine. Vol. 1. No. 1, 3 o r t h Holland, Amst’erdam, ’ January 1970. (119B) Sco;a, N. B., Secretary Editorial Board, Automation in Analytical Chernjstry ; Technicon Sym osia, 1967, Vol. 1, 2, Mediad, &hite Plains, N.Y ., 1968. (120B) Simpson, L., “Gas Chromatography. Laboratory Instruments and Techniques Series, Barnes and Noble, New York, N.Y., ‘?YO. (121B) Slavin, N., Emission Spectrochemical Analysis,” Vol. 34 in Chemical Analysis Series, Wiley-Interscience, New York, N.Y., 1971i1 (122B) Slayter, E. M., Optical Methods in Biology,” Wiley-Interscience, New York, N.Y., 1970. (123B) Smith, K. M., Lauffer, M. A., Bang, F. B., Ed., Advances in Virus Research, Vol. 15, Academic Press, NewYork, N.Y., 1969. (124B) Snell, E. E., Ed., Annual Review o f Biochemistrv. Vol. 38. Annual ReGiews, Palo AlG, Calif., 1969. (125B) Ibid., Vol. 39,1970. (126B) Sober, H. A., Ed., “Handbook of Biochemistry Selected Data for Molecular Biology,” 2nd Ed., CRC, Cleveland, Ohio. 1970. (1?7B) Stout, G. H., Jensen, L. H., X-ray Structure Determination; A Practical Guide,” Macmillan, New York, N.Y., 1968. (128B) Tabor, H., Tabor, C. W., Ed., Methods in Enzymology, Vol. 17, Academic Press, New York, N.Y;: 1971, (129B) Tranchant, J., Ed., Practictf Manual of Gas Chromatography, American Elsevier, New York, N.Y., 1969. (130B) .Udertfriend, S., “Fluorescence Assay in Biolo y and Medicine,” Vol. 2, Academic f’ress, New York, N.Y., 1969. (131B) Umbreit, . W. W., Perlman, D., Ed., Advances zn Applied Microbiology, Vol. 10, Academic Press, New York, N.Y., 1969. (132B) Ibid., Vol. 11,1969. \ -

es, R. A,, “An Introduction to /71B) Kenedi: R. M.. Ed.. Advances in Biomedical ’Engineeiing, ‘Vol. 1, Academic Press, New York, N.Y., 1971. (72B) Kerkut, G. A., Ed., The Interndional Journal of ‘Biochemistry, Vol. 1, No. 1, Scientechnica, Bristol, England, February 1970. (73B) Kirkland,. J.. J., Ed., “Moder; Practice of Liquid Chromatography, Wiley-Interscience, New York, 1971. (74B) Klein, G., Weinhouse, S., Ed., Advances i n Cancer Research, Vol. 11, Academic Press, New York, N.Y., 1968. (75B) Ibid., Vol. 12, 1969. (76B) Ibid., Vol. 13, 1970. (77B) Ibid., Vol. 14, 1:71: (78B) Krueer. P.. Princides of Ac‘ tivktion Xialysik,” Wiley:Interscience, New York, N.Y., 1971. (79B) Kustin, K., Ed., Methods in Enzymology, Vol. 16, Academic Press, New York. N.Y.. 1969. (80B) Lawrence, H. S., Ed., Cellular Immunology, Vol. 1, KO.1, Academic Press, New York, N.Y., May 1970. (81B) Leach, S. J., Ed., “Physical Principles and Techniques of Protein. Part B,” Academic Press, New York, N.Y., 1970. (82B) Ledley, R. S., Ed., Computers i n Biology and Medicine, Vol. 1, No. 1, Pergamon, New York, N.Y., August 1970. (83B) Littlewood, A. B., “Gas Chromatography: Principles, Techniques and Applications,” Academic Press, New York, N.Y., 1970. (84B) Lowenstein, J. M., Ed., Methods i n Enzymology, Vol. 13, Academic Press, Kew York, N.Y., 1968. (85B) Zbzd., Vol. 14,1969. (86B) Maramorosch, K., Koprowsky, H., Ed., Methods i n Virology, Vol. 3, Academic Press, New York, N.Y., 1968. (87B) Ibid., Vol. 4,1968. (88B) Marinette, G. V., ,E!., “Lipid Chromatographic Analysis, Vol. 2, Marcel Dekker, New York, N.Y., 1969. (89B) Marois, M., Ed., Steroidologia, Vol. 1, No. 1, Karger, Bazel, January 1970. (90B) Maurer, H. R., Disc Electrophoresis and Related Techniques of Polyacrylamide Gel Electrophoresis,” 2nd Ed., deGruyter, NewYork, N.Y., 1971. (91B) McCormick, P. B., Wright, L. D., Ed., Methods i n Enzymology, Vol. 18, Academic Press. New York. N.Y., 1971. (92B) Meister, A., Ed., Advances in Enzymology, Vol. 35, Interscience, New York, N.Y., 1971. (93B) Meites, L., Ed., Critical Reviews i n Analytical Chemistry, Vol. 1, CRC Press, Cleveland, Ohio, 1970. (94B) Ibid., Vol. 2, 1971. (95B) Moldane, K., Grossman, L., Ed., Methods in Enzymology, Vol. 20, Part C, Academic Press, New York, N.Y., 1Q71 -. (96B) Muller, A. F., Reinold, A. E., Ed., European Journal of Clinical Investigation, Vol. 1, No. 1, Springer-Verlag, Berlin, March 1970. (97B) Neurath, H., Ed., TheProteins, Vol. 5, Academic Press, New York, N.Y., 1970. (98B) Niederwieser, A., Pataki, G., “New Techniques in Amino Acid, Peptide, and Protein Analysis,” Ann ArborHumphrey, Ann Arbor, Mich., 1970. %

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(133B) Ibid., Vol. 12,1960. (134B) Ibid., Vol. 13,1970. (135B) Warwick, G. P., Ed., Chemic& Biological Interactions, Vol. 1, No. 1, Elsevier, Amsterdam, Octobr 1969. (136B) Weinstein, B., Ed., Chemistry and Biochemistry of Amino Acid, Peptides and Proteins,” Vol. 1, Marcel Dekker, New York, N.Y., 1971. (137B) White, C. E., Argavere, R . . J., “Fluorescye Analysis: A Practical Approach, Marcel Dekker, New York, N.Y., 1970. (138B) Wied, G. L., Bahr! G. F., Ed., “Introduction to Quantitative Cytochemistry,” Vol. 11, Academic Press, New York, N.Y., 1970. (139B) Williams, C. A., Chase, M. W., Ed., “Methods in Immunology and Inimuno-chemistry,” Vol. 111, Academic Press, New York, N.Y., 1971. (140B) Wolfrom, bl. L., Tyson, R. S., Ed.. Advances i n Carbohudrate Chemistri, vo Vol. 24, Academic” Press, New York, N.Y., 1969. (141B) Work, T. S., Work, E:, E., Ed., “Laboratory Techniques in Biochemistry and Molecular Biology,” Vol. I, Wdey-Intersceince, New York, N.Y., 1969. Centrifugation Dialysis and Filtration (IC) Andersen, B. L., Christiansen, E. N., Kvamme, E., Acta Chem. Scand., 24, 2511 (1970). (2C) Anderson, N., Rankin, C. T., Brown, D. H.. Nunley, C. E., Hsu, H. W., Anal. Biochem., 26,415 (1968). (3C) Anderson, N. G., ibid., 23, 207 (1968). (4C) Ibid., p 72. (5C) Ibid., 28,545 (1969). (6C) Ibid., 31,272 (1969). (7C) Anderson, N. G., Waters, D. A., Nunley, C. E., Gibson, R. F., Schilling, R . M., Denny, E. C., Cline, G. B., Babelay, E. F., Perardi, T. E., ibid., 32,460 (1969). (8C) Anderson, N. G., Amer. J. Clin. Pathol., 53,778 (1970). (9C) Anderson, N. G., Xunley, C. E., Rankin, C. T., Jr., Anal. Biochem., 31,255 (1971). (1OC) Anseven, T. T., Roark, D. E., Yphantis, D. A., Anal. Biochem., 34, 237 (1970). (1lC) Arreguin, L. B., Taboada, J., Bol. Inst. Quim. Univ. Nac. Auton. Mez., 20,95 (1968). (12C) Babul, J., Stellwagen, E., Anal. Biochem., 28,216 (1969). (13C) Bielka, H., Grummt, F., Schneider, I., Acta. Biol. M e d . Geo., 24,705 (1970). (14C) Blatt, W. S., J . Agr. Food Chem., 19,589 (1971). (15C) Brown, M. R. W., Farwell, J. A., Rosenbluth, S. A., Anal. Baochem., 27, 484 (1969). 116C) Bubel. H. C.. Riley, ” . B. P., ibid., 22,’335 (lG68). ‘ (17C) Burstein, M., Scholnick, H. R., Morfin, R., J . Lipid Res., 11, 583 (1970). (18C) Casciato, R. J., ANAL.CHEM.,41, (13), 99A, 102A, 106A (1969). (19C) Charm, S. E., Lai, C. J., Biotechnol. Bioeng., 13,185 (1971). (20C) Chen, H., O’Keal, C. H., Craig, L. L. ANAL.CHEM.,43, 1017 (1971). (21C) boates, J. H., in “Phys. Principles Tech. Protein Chem.,” Leach, S. J., Ed., Academic Press, ?Sew York, N.Y., ’

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e.,

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Adsorbent Techniques (1H) Abraham, G. E., Odell, W. D., Edwards, It., Purdy, J. M., Acta Endocrino!. (Copenhagen). Suppl., 147, 332 (1970). (2H) Bauer, K., J . Chromatogr., 59, 200 ( 1971~ I . (3Kj Bodanszky, A., Bodanszky, M , , Expericntia, 26,327 (1970). 14H) Bromlev. P. A.. Barrv. R. D.. Anal. Biochem., 36, 278 11970;.‘ (5Hj Brooks, C. J. W., Keats, R. A. B., J. Chromalogr., 44,569 (1969). 16H) Brown, H. D., Patel, A. B., Chattopadhyay, S. J., ibid., 35, 103 (1968). (7H) Bundschuh, G., ibid., 56,241 (1971). IXH) Calderon. M . . Baumann. W. J . . Biochim. Biobhys.’Acta, 210, 7’(1970).- ’ (9H) Carr, B. R.,SIikhail, G., Flickinger, G. L., J. Clin. Endusrin. Me!.. 33. 358 (ig7ij. ilOH) Catt. K. J.. Acta Endocrid. ‘ (Copenhasen),Sup’l., 142,222 (1969). (11H) Catt, K. J., Jiall, H. D., Tregear, G. W., Burger, H. G., J. Clan. Endocrinol. Metab:, 28, 121 (1968). (12H) Catt, X. J., Tregar, G. W., Burger, H. K., Skermer, C., Clin. Chim. Acta, 27,267 (1970). (13H) Cavalieri, L. E., Carroll, E., Proc. Nat. Acad. Sci., 67,807 (1970). i14H) Ceska, M., Grossmueller, F., Lundkvist, U., Acta Endocrinol., 64, 111 11970). (l.I Bocchini, ., V., Becker, AM.,Biochemzstry, 10, 2828 11970). (34H) Grueriwedel, D. W., Fu, J. C. C., Proc. Xat. Acad. Sc7. U.S., 68, 2002 (1971). (35H) Hoffman, C. H., Harris, E., Chodroff, S., hlichelson, S., Rothrock, J. W., Peterson, E., Reuter, W., Biochem. Biophys. Res. Commun., 41, 710 (1970). (36H) Holick, M. F., DeLuca. H. F., J . Lzpad Rcs , 12,460 (1971). (37Hj Hornby, W. E., Filippssun, H., McDonald, A,, FEBS (Fed. Eur. Baochsm. SOC.) Lett., 9 , 8 (1970). (38H) Inman, J. K., Dintzis, H. M., Bzochemistry, 8,4024 (1969). 139H) Jarvis, D., Loeser, R.. Herrlich, P., Roeschenthaler, R., J . Chromatogr., 52. 153 11970). (40”) Liener, I. E., Chao, L., Anal. Baochem., 25,317 (1968). (41H) Lin H. J., Bzochzm. BzovSus. . ” Acta. zi7,232’(1970j. 142H) Loeser. R.. Roeschenthaler. R.. ‘ Herrlich, P., Biochemistry, 9 , ’2364 i1970). (43H) Merriam, E. V., Dumas, L. B., Sinsheimer, R. L., Anal. Biochem., 36, 389 (1970j. (44H) Modak, M. J., Modak, S., Venkaibid., 34, 284 (1970). taraman, -4., (45H) Mosbach, K., Acta Chem. Scand., 24,2034 (1970). (46H) Moebach, K., Mattiasson, B., $$,82093. erenberg, S. T., J . Lob. Clin. M e d . , 77,517 (1971). (48H) Pearson, R. L., Weiss, J. F., Kelrner;, A. D., Bwchink. Bzophys. Acta, 228,770 (19711. (43H) Pensky, J., Marshall, J. S., Arch. Biochem. Biophys., 135, 304 (1969). (5OH) Pconian, M. S., Schalabach, A. J., Weissbach, A., Biochemistry, 10, 424 (1971). 151H) Porath, J., Fryklund, I.., -liaturt, 226,1169 (1970). (52H) Rattazzi, M. L., Biochim. Biophys. Acta. 181. l(19691. (53H) ’Rubiu, J. R., Zn Vitro Proced. Radioisotop. Med. Proc. Symp., Vienna, Austria Sept. 8-12, 1969. (54H) Serngadharan, M. G., Watanabe, A., Pogell, B. SI., J . Biol. Chem., 245, 1926 (1970). (55H) Sjovall, J., Nystrorn, E., Haahti, E., Advan. Chromatogr.,6 , 119 (1968). (56Hj Steers, E., Cuatrecasas, P., Pollard, H., J . Biol. Chem., 246, 196 (1971). I

k.,

I

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(57H) Udvardy, A,, Venetianer, P., Acta Bwchim. Bw hys., 6,27 (1971). (58HI Weiss. Buecher., T.., Eur. J. . Bidchem., 17,561 (1970). (59H) Wilchek, M., Gorecki, M., ibid., 11,491 (1969). (6OH) Williams, A. D., Freeman, D. E., Florsheim, W. H., J. Chromatogr., 45, 371 (1971).

%..

Electrochemical Methods (11) Addanki, S., Cahill, F. D., Sotos, J. F., Anal. Bwchm., 25, 17 (1968). (21) Andeev. V. S.. Bashtanov. A. V.. -2avod. Lad., 34,1546 (1968). ’ (31) Baurn, G., Anal. Lett., 3, 105 (1970). (41) Baum.. G.,, A n d . Bzochem., 39. 65 I

,

(1971). (51) Cantliffe, D. J., MacDonald, G. E., Peck, N. H., 1Y.Y. Food Life Sci. Bull., 3 , 7 p p (1970). (61) Carr, W. C., Ann. N . Y . Acad. Sci., 148,180 (1968). (71) Caswell, A. H., Pressman, B. C., Anal. Biochem., 32,396 (1969). (81) Cedergren, A., Johansson, G., Sci. Tools, 16,19 (1969). (91) Chernyi, V. A., Povkhan, M. F., Galushko, S. V., Peterfalvi, A,, Fizioi. Biokhim. Kzd’t. Rust., 2, 652 (1970). ( l O I j Clark, L. C., Jr., Sachs, Q., Ann. N . Y . Acad. Sci., 148, 133,(1968). (111) Covineton, A. K., ibzd., (121) Easty, D. B., B1aecfeY.U’. J., Anderson, L., ANAL. CHXM.,43, 509 (1971). (131) Erlanger, B. F., Sack, R. A., Anal. Biochem., 33,318 (1970). (141) Fleet,, B., Itechnitx, G., ANAL. CHI:M.,42,690 (1970). (151) Frant, M. S.,Koss, J . W., Jr., Scicncs, 167,987 (1370). (161) Friedman, S. M., Palaty, V., Nakashima, M., Anal. Biochem., 29, 107 (1969). (171) Fry, 8 . W., Taves, D. R., J . Lab. Clin. Med., 75, 1020 (1970). (181) Girard, M. L., Dreux, C., Paolaggi, F., Delattre, J., Ann. Riol. Clin., 26, 40). (196s). (191) Goldstein, R., Konigsbuch, hl., Zsr. J . Chem., 8,65 (1970). (201) Gruen, L. C., Harrap, B. S., Anal. Biochtm.. 42.377 (1971). (211) Guilbauk, ,.^_.G. G., Pkre Appl. Chem., 25, l z i !1!?11). (221) Guilbault, G. G., Hrabankova, E., Anal. Chim. Acta, 52. 287 11970). (231) Guilbault, G.’ G.,’ Montalvo; J. G., Jr., J . Amtr. Chem. SOC., 92, 2533 11370). (241) Hirrup, €3. S., Gruen, L. C., Anal. Biochem., 42,398 (1971). (251) Homolka, J., Clin. Chent., 16, 155 (1970). (261) Izuka, Y., Akiba, C., Nakayama, Y., Buli. Tokyo Dent. C O X , 11, 155 (1970 ,. (27i) Jacobson, J. S., Heller, L. I., Envzyon. Lett., 1,43 (1971). (281) Jemmali, Rf., Rodriguez-Kabana, R.,Anal.. Biochem., 37, 253 (1970). (291) Khuri, R. N., Nut. Bur. Staqd. (U.S.) Sp6.c. PzLbl., 314, 287 (1969). (301) Lawrence, A. J., Eur. J . Bwchem., 18,221 (1971). (311) Lavally, M., Ed., “Glass Microalect?odes, John Wiley, New York, N.Y.. 1969. 1321) Levaggi, 14. A., Oyung, W., Feldstein, M., J . Air Pollut. Contr. Ass., 21, 255 (19711. (331) Lhug