Use of enzymes in analytical chemistry - ACS Publications

in Clinical Pathology” in Stefanini's book on Progress in Clinical Pathology. Both of these contain valuable pro- cedures for enzyme analysis. Guil-...
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Use of Enzymes in Analytical Chemistry George G. Guilbault, Chemistry Department, Louisiana State University in New Orleans, New Orleans, l a .

T

surveys the literature and developments from January 1966 to January 1968. HIS REVIEW

BOOKS AND REVIEWS

Ruyssen and Vandenriessche (271) have authored a book on “Enyzmes in Clinical Chemistry,” and Phillips and Elevitch (2%) have written a chapter on “Fluorometric Techniques in Clinical Pathology” in Stefanini’s book on Progress in Clilzical Pathology. Both of these contain valuable procedures for enzyme analysis. Guilbault (97) has authored a chapter on fluoronietric methods of analysis of enzymes, substrates, activators, and inhibitors in his book, “Fluorescence. Theory, Instrumentation and Practice.” Passwater’s book, (233) “Guide to the Fluorescence Literature,” contains a valuable bibliography of most of the literature on fluorescence and phosphorescence, covering the years 19501964, and contains references to the fluorometric measurement of enzyme activity. Bergmeyer’s “.\lethods of Enzymatic rinalysis” has now been published in a new, revised edition, and contains practical details of a vast range of enzymic methods (25). Reviews on enzymes have been published by Tampion (320), Okazaki (219), and Bodansky (M),and techniques of enzymic analysis in clinical chemistry and in food chemistry have been written by Kingsley (149) and Borker, Slonian, and Foltz (38). Clinical procedures for the assay of enzymes and substrates by fluorometric methods have been described in a manual published by the Turner Instrument Co. (338). SOURCES OF REAGENTS

One of the often cited objections to enzyniic methods has been the instability and unavailability of enzymes. However, today there are several coiiipanies that sell good enzyme products. These include Uoehringer 3Iannheim (Germany), Sigma (St. Louis) , Calbiochem Co. (Los Angeles), l l a n n Laboratories (Sew York) , Korthington Biochemical (Freehold, N. J.), S u t r i tional Biochemical (Cleveland), and General Biocheinicals (Chagrin Falls, Ohio). Substrates are available from Xldrich (Milwaukee), Eastinan (Rochester, N. Y,), Calbiochein, Sigma, blann, Worthington, I< and I< Labs (Plainview, N. Y , ) , Fisher Scientific, and Pierce Chemical (Rockford, Ill.).

METHODOLOGY

Because the enzyme is a catalyst, and as such affects the rate and not the equilibrium of a reaction, its activity must be measured b y a kinetic (or rate) method or by a direct titration of the active site ( 7 1 ) . Liken-ise activators and inhibitors that affect the enzyme’s catalytic effect can be measured only by a rate method. The substrate can however be measured either via a total change or a kinetic method. The former frees the technician from continuous measurements; rate methods, however, are faqter-becau+e the initial reaction can be measuied without waiting for etjuilibriuni t o be established. The accuracy and precision of both methods are comparable (97), and it is no longer true that equilibrium methods are more reliable than rate methods. In fact, iecent work (99, 100, 228) has indicated that v i t h reasonable care, preci4on and accuracy better than 1% can be obtained. Noreover, some of the difficulties encountered because of 4 d e reactions ai e eliminated in rate methods. K i t h the automated equipment now available for perf arming rate methods, the author believes such techniques will be the ones of choice in the future. Filmer, Cannon, and Reiss (76) have described a new method for determining the rate constants in enzyme reactions that should piove useful in analytical techniques. Bower. and rielley (3Q) made precision estimates in and concluded n in clinical laboratories reported in past enzyme surveys may be related to problems in the test specimen as well as to human, instrumental, and procedui a1 inadequacies. Cheii (48)has publi*hed an article on the practical aspects of the calibration of the Aminco Uowiian -1)ectrophotofluorometer that should be of benefit to researchers uiing fluororescence methods. The effect of solvent on enzyme activity has been noted in reports by Skujins and l l c l a r e n (299) and Ramiiiler (251). The former authors studied enzyme activity a t limited water concentration, and found that the ureaurease reaction proceeds only above 60% relative humidity. The activity of urease follorved the water vapor absorption isotherm of urease. Rainniler observed that DMSO activated several enzymes at l o n concentrations (0 to

7 0 7 22

20 or 30%);

higher concentrations caused an inhibition of the activity, however. DETERMINATION OF ENZYMES

Fluorometric Methods. Fluorometric methods for the assay of enzymes have been extensively used in past years, a n d offer t h e advantages of sensitivity and eliniinat,ion of many interferences. T w o general approaches have been followed: the use of synthetic fluorogenic substrates, themselves nonfluorescent, which yield highly fluorescent products upon enzymic action; and the nieasurement of the pyridine nucleotide coenzj-mes, K,iD and SXIIP, the reduced fornis of which (S.iDH and S . i D P H ) are highly fluorescent. HYDROLYTIC E r z r m s . Esterification of the amino group of naphthylamine or the hydroxy group of naphthol, resorufin, fluorescein, or umbelliferone reduces the fluorescence of the:.e coinpounds. Enzymatic cleavagc of the nonfluorescent ester then regenerates the fluorescent compound. The production of the fluorescence can be folloived kinetically and equated to enzyme activity. Prince (246) was able to detect as little as 0.0003 unit of acetylcholinesterase using the substrate l-niethyl-7acetosy quinolinium iodide. The corresponding i-hj-droxy compound is produced, nhich is highly fluorescent. Siegel, Lehrer, and Silides (292) adapted the procedure of Guilbault and Iiramer (104) to the fluorometric assay of cholinesterase. The substrates l-naphthy1 acetate and butyrate were used, and the fluorescence of 1-naphthol measured. Guilbault, Glazer, Sadar and Skou (101) have described new fluorogenic substrates for cholinesterase that offer excellent stability and sensitivity. -1thorough evaluation of all available fluorogenic substrates revealed 5methyl indosyl acetate and butyrate to be ideal substrates. The hydrolysis product, S-methyl indoxyl, is highly fluorescent and as little as 5 x loT6unit of enzyine is detectable. Guilbault and Heyn (102) have described a fluoronietric procedure for the assay of cellulase, based upon the hydrolysis of the nonfluorescent resorufin acetate by the enzyme t o give the highly fluorescent resorufin (Aez = 540 nip; A,, = 580 nip). -1s little as unit, of enzyme can be assayed. VOL. 40, NO. 5, APRIL 1968

459 R

I'inbrllifei one (7-hydroxycoumarin) and 4-inetliyl-umbelliferone are highly fluoi escent compounds which have been modified to form nonfluorescent substrates for the enzymes glucuronidase, .V-acetyl-0-glucosaminiglucosidase, dase, and 6-galactosidase. Robinson (269) used 4-methylumbelliferone-p-~glucoside as a substrate for 8-glucosidase. The substrate is split specifically by this enzyme, thus permitting the separate identification of p-glucosidase and 6-glucuronidase in paper electrophoresis. Woolen and Turner (358, 569) assayed p-glucuronidase in plasma using 4-methyl umbelliferylp-glucuronide as substrate, and Woolen and Valker (360), proposed 4-methyl unibelliferone-S-acetyl -p-n-glucosaniinide and 4-methyl umbelliferone-pD-galactoside as substrates for .%'-acetylp-D-glucosaniinidase and p-galactosidase. Likewise, Leaback (169) described conditions for the continuous fluorometric assay of !Y-acrtyl-p-Dglucosaminidase using the same substrate, and Veritz, Gambell, and Brown (336) proposed 1-naphthyl-2-acetamido2-deoxy-/3-~-glucopyranosideas a substrate for this enzyme. The production of 1-naphthol was proportional to the concentration of enzyme. Greenberg (96) assayed 0-glucuronidase, using the commercially available 2-naphthyl-6-D-glucuronide as substrate. The reaction could be followed kinetically a t a p H of 5.3, or more sensitively by alkalinization (pH 13) prior to nieasurement of the fluorescence. Guilbault, Kramer, and Hackley (106) described indoxyl acetate as a substrate for hyaluronidase. On cleavage of the acetate the highly fluorescent indigo white is produced. As little as 10-4 unit of hyaluronidase could be detected and distinguished from esterase activity. Jacks and Kircher (133) synthesized and tested the butyryl, hexanoyl, heptanoyl, nonanoyl, palniitoyl, and oleoyl esters of 4-methylumbelliferone as substrates for five preparations of lipase. Enzymatic hydrolysis releases the intensely fluorescent 4-niethylumbelliferone. The highest rate of hydrolysis was obtained with the hexanoyl ester for steapsin, the heptanoyl ester for wheat germ and peanut lipases, the octanoyl ester for caster bean lipase, and the nonanoyl ester for porcine pancrease. I t was claimed that the rate of hydrolysis of these esters proceeds a t a higher rate than dibutyryl fluorescein. Sapiia and Shapiro (273) used fluorescein dibutyrate for the assay of hormone insensitive lipase of rate adipose tissue, and found no interference from common esterases. The use of higher esters of fluoresceiii for the development of methods for the assay of human sera lipase has been suggested 1237). Takeuchi and Kogami (318) used 460 R

ANALYTICAL CHEMISTRY

riboflavin-5'-phosphate as substrate in the histochemical measurement of tissue phosphatase. Land and Jackim (166) found flavone-3-diphosphate to be a stable, versatile, and sensitive substrate for assaying both acid and alkaline phosphatase. The fluorescence of 3hydroxyflavone is measured at 510 mp. Ultrahigh sensitivity ITas achieved by forming the fluorescent metal chelate of 3-hydroxyflavone with aluminum ions. The authors found this substrate to be more sensitive than naphthyl phosphate and more stable than 30-methyl fluorescein phosphate. Ramirex-Martinez and NcLaren (250) used p-naphthyl phosphate as a substrate for phosphatase and Phillips and Elevitch (236) have described fluorescence kinetic methods for acid and alkaline phosphatase in serum using a-naphthyl phosphate as substrate. Fernley and Walker (76) investigated 4-methylumbelliferyl phosphate as a substrate for calf intestinal alkaline phosphatase. Elevitch et al. have described a film badge technique for phosphatase isoenzymes using p-naphthylphosphate or 3-0-methyl fluorescein phosphate as substrate (68, 68a). Guilbault et al. ( 1 0 7 ~ after ) ~ a thorough evaluation of substrates for acid and alkaline phosphatase, concluded that umbelliferone phosphate was the optimum substrate. As little as units of enzyme could be determined with an accuracy of 1.5y0. Sardesai and Provido (276) used p-tosyl arginine methyl ester (T.AME) as a substrate for plasma trypsin. The methanol released is measured by a fluorometric method previously developed by the authors (274). Roth prepared derivatives of the fluorescent P-naphthylamine as substrates for trypsin and leucine aminopeptidase (266). He reported the procedure for trypsin using S-(p-benzoylarginine)-p-naphthylaniide to be 200-fold more sensitive than any other method. ~-Leucyl-2naphthylamide was used for the assay of leucine aminopeptidase. Pappenhagen, Koppel, and Olivin (227) nieasured fibrinolytic activity using fluorescein-labeled fibrin, and Hidaka, Sagatsu, and Yagi (120a) described a rapid and simple fluorometric assay of serotonin sulfokinase activity. OXIDATIVEEszrucs. Guilbault, Kramer, and Hackley (107) have shown that homovanillic acid is an excellent substrate for the determination of peroxide and peroxidase. Upon oxidation homovanillic acid is converted to a highly fluorescent compound, which has a A, of 315 mp and a A,, of 425 mp. The rate of production of oxidized HVA is proportional to the concentration of peroxide (10-11-10-7 mole per ml) and peroxidase (10-3-10-1 unit per ml). Guilbault, Brignac, and Zimmer (99) extended the HVA procedure to the determination of glucose, hypoxanthine,

and xanthine in blood and urine, and to the determination of glucose oxidase and xanthine oxidase. Guilbault and Hieserman described a procedure for the assay of the oxidative enzymes Dand L-amino acid oxidase in concentrations of 0.00009-0.025 unit. The HVA reaction is used to monitor the peroxide produced in the amino acid oxidase reaction (103). I n attempting to develop methods for galactose oxidase and invertase, Guilbault, Brignac, and Juneau (100) tried 25 different substrates as possible replacements for HVA. p Hydroxylphenylacetic acid was chosen as the best substrate for measuring oxidative enzymes because of its stability, low cost, and the high fluorescence coefficient of its oxidized form (100). Krajl (161) adapted the relatively insensitive spectrophotometric method of Reissbach to the analysis of low activities of monoamine oxidase. Instead of measuring the loss of absorbance of kynuramine, the fluorescence of the product of enzyme action, 4hydroxyquinoline, is measured. Adach and Ilalpen (4)described a sensitive fluorometric assay of tyrosinase in as little as 10-50 p g of sample using tyrosine and dopa. Aifter oxidation with ferricyanide, fluorescence mas measured a t 360 and 490 mp. Haining and Legan (111) used 2-amino-4-hydroxypteridine ( d H P ) in a fluorometric assay of xanthine oxidase. The method was quantitative only if high concentrations of ,IHP were used and the crude tissue was dialyzed overnight. DEHYDROGESASES. Morrison and Brock (201) described a procedure for the quantitative measurement of alcohol dehydrogenase in the lobule of normal livers. The alkaline induced fluorescence of the NAD formed from NADH was measured. An automated fluorometric niethod for lactic dehydrogenase (LDH) ITas described by Passen and Gennaro (231). The fluorescence of KADH \vas measured, and the authors proposed the simultaneous determination of L D H and serum glutamic oxalacetic transaminase. Bergerman ( 2 4 developed a method for the determination of L D H isoenzymes (different enzyme proteins which have the same specificity), and Elevitch et al. (68, 68a) have described a fluorogenic reagent film technique for L D H isoenzymes. Sobral (301) assayed malic dehydrogenase in rat liver; the fluorescent S A D P H formed from S A D P is measured. Benson and Benedict (23) described a rapid kinetic method for the determination of serum a-hydroxybutyrate dehydrogenase, again using the fluorescence of KADH, and Elevitch et al. (68, 68a) measured the isoenzymes of malic dehydrogenase and a-hydroxybutyric dehydrogenase using Elevitch's fluorogenic reagent film technique.

Pitts, Quick, and Robins (240) have coupled the NAD linked succinic semialdehyde dehydrogenase reaction with a transaminase reaction to measure yaminobutyric-a-oxoglutaric transaminase, The X X D H produced is measured fluorometrically. Levine and Hill (171) described the fluorometric determination of serum glutamic-oxalacetic and glutamic-pyruvic transaminases. Helman (119) measured the fluorescence of S A D in a method for glutamic-pyruvic transaminase activity in microdissected rat pancreas, and hlathai and Beutler (186) assayed galactose-1-phosphate uridyltransferase in a coupled enzyme system using glucose-6-phosphate dehydrogenase and ?;ADP. Methods for creatine kinase were described by Sax and hIoore who measured the creatine liberated either by the formation of a fluorophor with ninhydrin in alkaline solution (277) or with the rlTP--IDP system (276). Conn and Anido (58) applied the fluorescent ninhydrin method of Sax and Moore to the determination of creatine phosphokinase, with essentially the same values, and Sherwin, Siber, and Elhilai (288) developed a fluorometric technique for creatine phosphokinase isoenzymes using ADP, hexokinase, and glucose-6-phosphate dehydrogenase : Creatine Phosphate

+ ADP

Kinase A

Creatine Glucose

+ ATP

Hexokinase

Glucose-6-Phosphate

+ ADP

-

Glucose-&Phosphate NADP

+ ATP

+

Dehydrogenase

KADH.

The fluorescence of S X D H was a measure of the creatine phosphokinase activity. Electrochemical and Radiochemical Methods. A summary of the use of the oxygen electrode in enzyme analysis has been prepared by Walker (339), and Andreev, Razengort, and Torubarov (9) have described high frequency procedures for recording enzyme reactions. The H F methods, which work in ionic as well as molecular solutions provided changes in viscosity take place in chemical transformations of the substrate, Ivere applied to the enzymes amylase, serum cholinesterase, and hyaluronidase (9). Nuclear magnetic resonance methods were used by Spotswood, Evans, and Richards (304) to study enzyme systems. The changes in the acetyl I H or 19F atoms of the substrate were noted in studies of the chymotrypsin catalyzed hydrolysis of h’-acetyl-DL-m-fluorophenylalanille and N-acetyl-D and L-phenylalanine. Updike and Hicks (353, 33%) de-

scribed the use of electrochemical sensors for glucose using glucose oxidase immobilized on an acrylamide gel and Katz and Cowans (143) described a direct potentiometric method for urease by continuous recording of the NH4+ produced from urea. Guilbault and Kramer used a spontaneous electrolysis procedure to measure glucosidase (105). The cyanide liberated from amygdalin when glucosidase was added was measured by a silver electrode. The rate of change in potential with time, AElmin, was found to be proportional to glucosidase and amygdalin. hlayer, Harel, and Ben-Shaul (188) have reviewed all methods for the determination of catechol oxidase, and concluded that the polarographic method was best. Erlanger et al. (71) developed a coulometric-amperometric procedure for the determination of chymotrypsin. The specific inactivator, N,N-diphenylcarbamyl chloride is used which releases one equivalent of chloride for every equivalent of chymotrypsin inactivated. Bartik and X c h n o v a described a polarographic method for the determination of oxytocinase (18). hIany sensitive techniques for enzyme assay have been developed based on radiometric methods. Procedures for acetylcholinesterase have been proposed by Reed, Goto, and Wang (25.4) and by Potter (244). The methods employ a ~ e t y l - 1 - C choline ~~ as the substrate, and a preferential extraction of the acetic a ~ i d - 1 - C ’ ~formed by enzymic activity after unhydrolyzed substrate is removed by ion exchange. Enzyme from eel and brain cortex (244) and from rat blood and whole blood (254) was assayed. Street0 and Reddy (313) described a method for measuring adenyl cyclase based on the conversion of 14C-adenosine 5’-triphosphate to 14Cadenosine, 3’,5’-monophosphate in the presence of ATP, pyruvate kinase, caffein, and phosphoenolpyruvic acid. The 14C-adenosine, 3’,5’-monophosphate was isolated by paper chromatography and measured in a liquid scintillation counter. Gabay and George (88) described a radioassay procedure for aromatic aminotransferase using L-glutamic acid14C. iilpert, Kisliuk, and Shuster ( 7 ) and Fonnuni (81) proposed radiochemical methods for choline acetyltransferase. The former authors (7) assayed the acetyl-Co.4 for radioactivity, whereas Fonnum (81) measured the formation of acetyl-14C choline from the labeled acetate. Hutton, Tappel, and Udenfriend (130) developed a rapid assay for collagen proline hydroxylase. The tritiated water formed from 3,CT-~-proline is separated by vacuum distillation and measured. Scott (285) used radioactive substrates to measure esterase activity in cheddar cheese, and Rothen-

berg (668) measured folic acid reductase with isotopically labeled folic acid. Ng, Bergren, and Donne11 (218) described an improved procedure for galactose-1-phosphate uridyl transferase using Cl4 labeled galactose-1-phosphate, and Newsholme, Robinson, and Taylor (217) assayed hexokinase and glycerol kinase by a radiochemical procedure using glucose 14C as substrate. Bengtsson (22) determined oxalic decarboxylase using oxalic acid C14. The C1402 produced was measured in a liquid scintillation counter. Fuller and Hunt assayed phenethanolamine Nmethyl transferase (86),and Guroff and Abramowitz (108) developed a simple radioisotope assay for phenylalanine hydroxylase using p-tritio-1-phenylalanine. Appropriate modifications of this assay were made by Guroff, Rhoads, and Abramowitz (109) which allow measurements in crude tissue extracts. Kammen (141) modified the procedure of Smith and Greenberg (300) for thymidylate synthetase. The tritium atom of deoxyuridylic a ~ i d - 5 - ~ is H released and measured. Hakanson and Hoffman (112) used 14C-labeled tryptophan as a substrate for the radioassay of tryptophan-5-hydroxylase. An isotopic method was developed for tyrosine transaminase by Weinstein, Xedes, and Litwack ( 3 4 4 , based on the use of a radioactive amino acid substrate, and 8-1%-xanthine was used as a substrate for xanthine oxidase in blood serum and tissue (6). Other Techniques. X a t s u z a w a and Katunuma (187) proposed a rapid and accurate method for alanine transaminase and lactic dehydrogenase in serum and tissue using a diazonium salt and coupling enzymes. Searcy, Wilding, and Berk (286) surveyed all methods for the determination of amylase in serum and found saccharogenic techniques to possess the greatest validity. Gage (90) reviewed methods for cholinesterase assay and discussed factors such as natural variations of blood activity, the nature of individual variations, signs of poisoning, etc. Lorenz, Kusche, and Werle (17’5) described a new method for the determination of diamine oxidase. The ammonia released from the amine substrate was measured with the aid of glutamate dehydrogenase and NL4DH. Hillcoat, Nixon, and Blakley (123) studied the effect of substrate decomposition on the spectrophotometric assay of dihydrofolate reductase. Both dihydrofolate and K A D P H were found to decompose a t significant rates a t p H values below 7 , X-IDPH being the more unstable. Christensen (60) reported on a methodological study of the enzymic determination of blood sugar using glucose oxidase. On the basis of results of this study, methods were suggested for making the procedure conVOL 40, NO. 5 , APRIL 1968

* 461 R

siderably more accurate. Prodanov, Venkov, and Mavlov (247) modified the assay of glucose-6-phosphate isomerase, shifting the equilibrium t o the side of fructose-6-phosphate production. Richterich and Danwalder (258) proposed phenolphthalein glucuronide as substrate for @-glucuronidase, and Szasz (311) compared p-nitrophenyl glucuronide and phenolphthalein glucuronide as substrates for the assay of @glucuronidase. Caraway (45) used the colorimetric hypochlorite reaction to measure the ammonia produced in the deamination of guanine in the determination of guanase. Bartels (17) used Sephadex columns and dialysis t o remove inhibitors prior to a colorimetric assay of lactic dehydrogenase. Ipata (132) described a coupled optical assay for 5’-nucleotidase that was based on the spectrophotometric determination of adenosine released from .AM P. Zugaza and Hidalgo (366) used infrared to measure the kinetics of the pencillinpencillinase reaction. The disappearance of the 1770 cm-’ band (corresponding to the CO group of the @-lactam ring) is measured. Several substrates have been proposed for the assay of acid and alkaline phosphatase. Coleman ( 5 5 ) used thymolphthalein monophosphate as substrate, and Kilkinson and Vodden (364) and Fischl, Segal, and Rabiah (77) deicribed a simple, rapid method for phosphatase using phenolphthalein monophosphate. Keumann and Van Vreedendaal (226) and Hausman et al. (117) used p-nitrophenyl phosphate as substrate. The absorbance of p-nitrophenol a t 405 mp was measured. Moss (202) proposed the use of the orthophosphates of 1- and 2-naphthol as substrates for alkaline phosphatase. H e found that contrary to the fluorometric assay, 1-naphthol is more absorbing than 2-naphthol. Pon and Bondar (242) described a direct spectrophotometric assay for pyruvate kinase. The disappearance of phosphoenolpyruvate is followed a t 230 mp. Ambellan and Hollander (8) proposed a simplified assay for RNase in crude tissue extracts based on the precipitation of unhydrolyzed RNA with a buffered lanthanum-magnesium ethanol reagent. A rapid, highly sensitive assay for tyrosine transaminase was suggested by Diamondstone (60) based on the conversion of p-hydroxyphenylpyruvic acid to p-hydroxybenzaldehyde in strong alkali. A 2-4 fold increase in sensitivity over other currently available methods is realized. A colorimetric method for the determination of xanthine oxidase that is 8 times more sensitive than the photometric assay wai described by Fried (83) using a tetrazolium salt. 462 R

0

ANALYTICAL CHEMISTRY

DETERMINATION OF SUBSTRATES

Carbohydrates. As in the past, glucose has undoubtedly received the most attention from analysts during t h e last two years. Pardue, Burke, and Jones (228) described a practical, kinetic experiment for the undergraduate laboratory using glucose and glucose oxidase Glucose

+ O2 + 21- + 2H+ e Acid + HPO + IP Oxidase

The course of the reaction is monitored continuously by following the electrolysis current a t a rotating P t electrode. Ware and Marbach (542) utilized this same reaction for glucose analysis but measured iodine photometrically. Guilbault, Brignac, and Zimmer (99) proposed a fluorometric method for the sensitive determination of glucose in biological samples like blood and urine. The peroxide produced upon enzymic action is monitored with homovanillic acid (HVA), which is oxidized to a highly fluorescent product. Phillips and Elevitch (238) used the HVA procedure for the assay of glucose in plasma. As little as 1 p1 of sample was needed. Several chromogenic dyes have been used to monitor glucose by the conventional reaction: Glucose

-

+ O2 + Red Dye Oxidase Ox. Dye + Gluconic Acid

The rate of formation of the oxidized form of the dye, which is colored, is then proportional to the concentration of glucose. I n the conventional Glucostat test, o-dianisidine is used. Johnson and Fusaro (136) used this method for the analysis of the glucose and oligoglucoside content of liver homogenates in an investigation of the in vivo and in vitro liver carbohydate metabolism. Martinsson (184) observed that a freeze dried Glucostat reagent could be used to identify 1-10 pg of glucose on chromatograms. h’o interference was noted from other sugars. Putter and Struffe (249) found that analytical methods for glucose based on the oxidation of peroxide by peroxidase and o-dianisidine could be improved by the addition of polyvinylpyrrolidone preparations. Thompson used a o-tolidine-iodide-glucose oxidase reagent to determine glucose in blood (330). The peroxide formed oxidized iodide to iodine in the presence of a molybdate catalyst and the iodine then oxidized o-tolidine to a colored product. Kawerau (146) used o-tolidine and peroxidase to monitor the peroxide produced in the glucose oxidase assay of blood sugar. For the in vivo determination, the patient was con-

nected directly to the Autoiinalyzer by an indwelling intravenous catheter. I n a comparative study of various enzymic and colorimetric methods, Lorentz and Leudemann (174) and Hinterberger (124) concluded that the glucose oxidase-peroxidase-o-dianisidine method was the procedure of choice. Fyowa (87) used a glucose oxidase-catalase-@-diketone reagent to determine 20-100 pg of glucose in fermentation products, and Jerabek added sorbitol to improve the o-toluidine procedure (134). Schulthess (281) found that sodium selenite improved the assay of glucose in whole blood by removing the interference from reduced glutathione. Mueller (206) found that if glutathione is first oxidized to disulfide no interference is observed in the glucose determination. Reatherburn and Logan (344) used a mixed bed resin to separate interferences prior t o a determination of glucose in urine. Saganna, Rajamma, and Rao (210), Kotas (160), Kutter (165), and Hollister, Helmke, and Wright (127) evaluated glucose oxidase test papers for the determination of glucose. Kotas noted a precision of about 1.32% but Naganna, Rajamma, and Rao noted that Clinistix strips failed to detect glucose in the presence of bilirubin, ascorbic acid, adrenaline, hydroquinone, and alkaptonuric acid. These substances presumably interfere with the peroxidase reaction. Hollister, Helmke, and Wright (127) compared the blood glucose determined by an enzyme strip test and with the Autohnalyzer in 542 cases, and found that the strip method has limited usefulness and should only be used where other methods are not available. Kline et al. (156) utilized a continuous blood sugar analysis as the input and constructed a feed back system which maintains the blood sugar in animals or humans within a few per cent of a preset level. In comparing enzymic and chemical methods for glucose, Gaffrini, Regolisti, and Veggetti (89) concluded that the values determined by a n enzymic technique are always lower by a difference which is proportional to the amount of monosaccharide in the blood. Barletta, Scolari, and Testi (16) likewise observed that the enzyme method gave lower blood sugar values in some cases, but concluded this technique is suitable for routine work. Leske and hfayersbach (170) removed reducing substances in urine with perchloric acid prior to a glucose determination. Kadish and Hall (138) and Alakino and Koono (179) measured glucose in blood by measuring oxygen uptake. lfakino and Koono used an oxygen electrode whereas Kadish and Hall utilized a polarographic oxygen analyzer. Bergmeyer and Noellering ( 2 7 ) reported the enzymatic determination of

glucose with acyl phosphate and Dglucose -6-phosphotransferase and found the method more specific than the hexokinase procedure. Keller (146) utilized the enzymes hexokinase and glucose-6-phosphate dehydrogenase in a new method to determine urinary glucose by a NADPH measurement a t 340 mg. Kamel, Hart, and Anderson (160) found this method to be ultraspecific and sensitive for the determination of glucose in the presence of Dmannose, D-fructose, 2-deoxy-~-glucose, and maltose. Boehringer and Soehne ($7) used glucose transferase, acylphosphate, and glucose-6-phosphate dehydrogenase to measure glucose in blackberries and blood. Again the NADH formed is measured and equated to glucose concentration. Joergensen and Joergensen (135) used glucose oxidase to determine which glucose anomer was produced in the ,%glucosidase degradation of maltose, and Rohwer, Henschel, and Engel (262) assayed glucose in starch hydrolysates, corn syrups, and sugar solutions. Moehler and Looser (195) determined glucose and fructose in urine and Weichel proposed enzymic methods for the assay of fructose (347). White measured glucose in honey by a photometric procedure in which contaminant invertase was inhibited by tris buffer (350), and Tengstrom (328) determined both glucose and galactose in urine. Kleinbaum (155) proposed a single method for the determination of galactose in biological fluids, and Hjelm (126) used a galactose oxidase-peroxidase-o-dianisidine reagent for the determination of galactose in whole blood, plasma, and erythrocytes. Guilbault, Brignac, and Juneau (200) have described a sensitive fluorometric procedure for the determination of single sugars and mixtures of glucose, galactose, and sucrose using the enzymes glucose oxidase, galactose oxidase, and invertase. In each case the peroxide produced is determined by a peroxidasep-hydroxyphenylacetic acid coupled reaction. The rate of production of fluorescence is equated to the concentration of the sugar.

Sugar

H202

+

Enzyme 02

H202

p-hydroxyphenylacetic acid

Peroxidase

Fluorescent Product

From 0.1-150 pg of D-glUCOSe, 2deoxy-D-glucose, D-galactose, stachyose, sucrose, D-raffinose, D-galactosamine, N acetyl+-galactosamine, p-D-melibiose, and methyl-P-D-galactopyranoside were determined with an accuracy and precision of 1-2%, Rognoni and Ronchini (261) determined glucose-l-phosphate based on its conversion to glucose-6-phosphate with phosphoglucomutase. Hankin and Wickroski used an

enzymic approach for the analysis of lactose in meat products (115) and for the determination of maize syrup in prepared meat products (116). Taufel, Behnke, and Wersuhn (327) determined sucrose in beet molasses using glucose oxidase after invertase inversion, and DeSouza and Panek (59) assayed the hydrolysis products of starch after enzymatic cleavage. Ruttloff, Friese, and Taufel (270) determined the saccharide constituents of dietetic foods, yeast fermentation, glucose oxidase, and invertase. A one step ultramicro method for the assay of disaccharidases such as maltase, isomaltase, sucrase, lactase, and cellibiase was described by Messer and Dahlqvist (193). Suzuki studied polysaccharide structures by enzyme analysis (315) and Palmquist and Baldwin (226) used enzymic techniques for measuring pathways of carbohydrate utilization in rumen. Passonneau et al. (232) described an enzymic method for glycogen based on the measurement of NADPH produced from the glycogen phosphate, glucose6-phosphate dehydrogenase, K A D P reaction. Glycogen in as little as 30 pg of brain or 0.3 pg of liver can be specifically determined. Rerup and Lundquist (256) used the glucose oxidase, peroxidase, and o-tolidine reaction to meazure glycogen. Rosa and Johnson utilized an enzymic method for the cytochemical demonstration of glycogen (264), and Abdullah, Lee, and Whelan proposed an enzymatic procedure for the microdetermination of the average unit chain length of glycogen and amylopectin (1). Amines and Amino Acids. Beyerman and Knoll (31) proposed an enzymatic microdetermination of arginine, histidine, and lysine. After enzymic decarboxylation the conductance of the C o n liberated was measured and related to the concentration of the amino acids in the 1-20 pg region. Graham and Aprison (93) adapted to fluorescence the conventional enzymatic method for amino acids (coupled dehydrogenase-SAD reaction that yields the fluorescent XADH), The precision was excellent, and lo-" to 10-9 mole of aspartate, a-aminobutyrate and glutamate were determined. Young and Lowry (364) described a procedure for the histochemical determination of alanine, glutamate, and glutamine in pg quantities of brain tissue, and Sowerby and Ottaway (302) used the glutamate dehydrogenase reaction coupled to the phenazine methosulfate catalyzed reduction of tetrazolium salts for the determination of 0-0.3 pmole of glutamate and glutamine. Preuss, Bise, and Schreiner (245) described a method for the determination of glutamine in plasma and urine in which the ammonia produced upon

addition of glutaminase is a measure of the amino acid present. Owens and Belcher (225) assayed glutathione in tissue by a colorimetric micromethod using glutathione reductase and 5,5'dithiobis-(2-nitrobenzoate). Kotaki, Napi, and Okamura (159) determined D-lysine using D-amino acid oxidase, and Blauch (34) used tyrosinase and UV irradiation to measure tyrosine; the melanine formed is measured at 550

.

Mella, Volz, and Pfleiderer used aprotease from Crotalus atrox verrum for the determination of amino acid sequences (191), and White and Gauger (351) described a procedure for the simultaneous determination of L-lysine and total amino acids in seed hydrolysates using enzymatic decarboxylation and the ninhydrin reaction. .A procedure for the resolution of a-amino acids by asymmetric hydrolysis of acylamino acids with acylases ad-jorbed on an anion exchange resin has been described (322),and Artis (11) used paper strips impregnated with L-amino acid oxidase, peroxidase, and o-toluidine for the detection of amino acids in urine. Bachrach and Reches (13) a-sayed spermine and spermidine, using an amine oxidase catalyzed oxidation and NcEqen and Sober (177) studied the interaction of primary, aliphatic amines with highly purified rabbit serum monoamine oxidase. An enzymatic method for the determination of the racemization rate of hyoscyamine and scopolamine was described by Werner and Seiler (349). Guilbault and Hieserman proposed a fluorometric method for the assay of D- and L-amino acids using the amino acid oxidase-peroxidase-hornovanillic acid (HVA) system. The initial rate of formation of the fluorescent oxidized product of HVX is measured and related to the concentration of D- and Larginine, -leucine, -methionine, -phenylalanine, -proline, -tryptophan, and -tyrosine in the concentration range 0.0150 pg. An increase in sensitivity of several orders of magnitude over other available procedures is realized (103). Organic Acids. Stegink (305) used acetyl-Coh synthetase and malic dehydrogenase to determine acetyl groups in proteins and peptides; after hydrolysis the acetic acid liberated was measured. Bergmeyer a n d Rloellering (28) measured acetate in animal tissues, serum, and food with acetate kinase, phosphotransacetylase, and citrate synthase, and Wierzbicka, Legocki, and Pawelkiewicz (353) used a stable preparation of propionate (acetate) kinase for the determination of acetate and/or propionate in biological material. Aloellering and Gruber (196), Mayer and Pause (189)) and Saruse, Cheng, and Woelsch (613) have proposed enzymic methods for the deVOL. 40, NO. 5, APRIL 1968

0

463 R

teiwination of citrate using citrate lyase. In the method of hloellering and Grubcr (196) coupled reactions using oxalacetic decarboxylase, pyruvate decarboxylase, and alcohol dehydrogenase are employed. The rate of disappearance of NADH is measured : Citrate

-Citrate Lyase

oxaloacetate

oxalacetic

decarbox,.lase pyruvate

pyruvate

Pyruvate

NADH

acetaldehyde

ethanol

dehydrogenase

Alternatively malic dehydrogenase and S A D H were used; again the disappearance of KADH is measured (f89, 196) : NADH

Oxaloacetate

dehydrogenase

malate

+ N-AD

Saruse, Cheng, and Roelsch (213) used aconitase and isocitric dehydrogenase with citrate lyase to determine as little as 10-9 mole of citric acid in nervous tissue. The X=IDH produced is measured : Citrate

lyase __t

D-Isocitrate

aconitase

cis-aconitate D-isocitrate

+ NAD dehrdrogenase a-ketoglutarate + NADH isocitric

Bergmeyer and Bernt (26) described enzymic methods for the quantitative determination of acetoacetate and D(-)3 hydroxybutyrate in blood, and Schievelbein and Buchfink (278) proposed a method for the assay of 3-hydroxyanthranilic acid using 3-hydroxyanthranilic acid oxidase. Nagel and Hasegawa (2f2)used an isomerase and dehydrogenase from Erivinia carotovora and from E. coli for the enzymic determination of D-galacturonic acid. Stoner and Evans (310) used a-aminobutyric acid transaminase-succinic semialdehyde dehydrogenase for the enzymatic determination of a-ketoglutaric acid. Young and Renold (363) fluorometrically determined acids and ketone bodies, such as 8-hydroxybutyrate and acetoacetate, in small quantities of blood using D-8-hydroxybutyrate dehydrogenase. The assay of lactic acid in liquids containing sucrose was the subject of a review by Schmidt-Berg and Bourzutschky (280). Mohme-Lundholm, Svedmyr, and Vamos determined lactic acid in fingertip blood (197),and Clarke and Podmore (54) also used lactic dehydrogenase to measure lactate in suspected glycosidase deficiency. Engel and Wulff ( Y O ) , Parijs and Barbier 464 R

ANALYTICAL CHEMISTRY

(229), and Hiyama, Koga and Fukui (125) all used the lactate dehydro-

genase system for the assay of lactic acid in blood. The first two groups measured the absorbance of S A D H , but the latter used a partially purified enyzme from Lactobacillus mesenteroides which is non-NAD dependent, coupled with the ferric-o-phenanthroline complex. The production of ferroin a t 510 mp was indicative of the lactate concentration. Simple, rapid colorimetric methods for the assay of serum lactic acid employing tetrazolium salts were described by Babson and Phillips (12), Chapman and Boutwell (47) and Briere, Preston, and Batsakis (40). Rosenberg and Rush measured both lactate and pyruvate in blood by an enzymic-spectrophotometric LDHNAD procedure (265), and hlarbach and Weil (182) used metaphosphoric acid to prepare a protein-free filtrate prior to the assay of lactate and pyruvate in blood. dntonis, Clark, and Pilkington (10) used specific dehydrogenases linked with pyridine nucleotide coenzymes in a semiautomated fluorometric method for pyruvate, lactate, acetoacetate, and 6-hydroxybutyrate levels in plasma. I n the cases of acetoacetate and pyruvate the loss of S A D H was measured; whereas, for lactate and 6-hydroxybutyrate, the formation of the fluorescent NADH mas measured. Kretovich and Stepanovich (163) proposed the use of an enzyme extracted from parsley leaves that catalyzes the reductive amination of hydroxypyruvate to serine for the assay of this acid. Pyruvic acid in urine was determined by direct spectrophotometry after enzymic reduction by Rankine (252) and optical enzymic methods were described by Blouin (36) for the determination of trace amounts of intermediate metabolites (such as oxo acids). S n automatic continuous method for estimation of pyruvic and lactic acids was described by Freud (82), and succinic dehydrogenase from Ascaris lumbricoides was used for the determination of 0.0030.055 pmolejml of succinic acid (168). Simmonds (293), Friedel (84), and Sen and Smith (287) described procedures for the analysis of uric acid in urine, serum, and foods, respectively. I n all three methods the decrease in absorbance of uric acid after the addition of uricase was measured at 293 mp. A coupled enzyme system, in which the peroxide produced in the uric acid-uricase reaction is measured with peroxidase and o-dianisidine, was proposed by Marymount and London (185) and by Lorentz and Berndt ( f 7 3 ) . Care must be used in this technique for uric acid is also a substrate for peroxidase; an incubation period is commonly used (173). Caraway and RIarable (46) found good agreement between the carbonate and uricase-carbonate

serum uric acid methods. Automated uric acid methods were described by Klein, Flor, and Kaufman (152) and Barron and Bouley ( f 6 ) , and Wachter patented a method for the detection and determination of uric acid in body fluids using an enzyme reaction system and a peroxide detecting system (538). Guilbault (98) has proposed the use of five enzyme systems for the detection and determination of 20 organic acids. Using lactate, malate, glutamate, and a-hydroxy and P-hydroxybutyrate dehydrogenases, coupled with NAD, phenazine methosulfate, and resazurin, procedures were developed for 0.1-100 pg of the following acids: lactic, glutamic, malic, formic, acetic, chloroacetic, glutaric, adipic, malonic, succinic, tartaric, a-hydroxy- and P-hydroxybutyric, oxalic, phthalic, glycolic, glyceric, citric, isocitric, and mandelic. The rate of production of the highly fluorescent compound, resorufin, is equated to the concentration of the acid.

-

+ NAD Dehydrogenase NADH Phenazine XADH + Resazurin Methosulfate Acid

__f

Resorufin

Alcohols and Esters. .An enzymatic procedure was used t o determine low amounts of ethanol in beverages a n d aromatic distillates (323), and Miller (194) described a fluorometric assay of ethanol in blood and serum using alcohol dehydrogenase and NAD. Syed (316) automated this fluorometric assay using the XutoAnalyzer and was able to determine 0.0005% ethyl alcohol in whole blood. A study of the substrate specificity and stereospecificity of alcohol dehydrogenases was made by Dickinson and Dalziel (61). Frings and Pardue (85) have developed an automatic spectrophotometric method for the enzymatic determination of glycerol. The rate of disappearance of the colored dye species was measured a t 600 mp, Glycerol

+ XAD NADH

KADH

dehydrogenase

-

+ dihydroxyacetone

+ dye-ox(b1ue) S A D + dye-red(color1ess) diaphorase

+

Glycerol in wine and must has been assayed by Drawert and Kupfer using a series of enzymic conversion steps (64), and Laurel1 and Tibbling (168) developed a simple and rapid method for plasma and whole blood glycerol by a fluorometric adaptation of the enzymic conversion of glycerol and NAD to NADH. Kaufmann and Wessels (144) suggested the use of chromatography and enzyme hydrolysis for the selective determination of triglyceride structure, and Eggstein and Kreutz (66) and

Spinella and Mager (303) used a modified enzymatic method for the assay of blood and plasma neutral fats. The glycerol and triglycerides are determined using the glycerokinase, and a-glycerophosphate dehydrogenase systems in the presence of the coenzymes KAD and ATP. Methods for the assay of glycerol, dihydroxy acetone, and glyceraldehyde were described by Pinter, Hayashi, and Watson (239),and Kessler and Lederer (147) and Eggstein and Kreutz (67) described a procedure for the enzymic determination of neutral fats in blood serum. As little as 0.01 pmole of glycerol was detectable. The selective hydrolysis of glycerides with pancreatic lipase has been used by Vela et al. (334, 336) t o detect esterified oils in olive oil. Drawert, Gebbing, and Ziegler (63) used o-diphenol oxidase for the detection of phenols on thin layer plates. The enzyme, prepared from cultured mushrooms and dandelion roots, was successfully applied for the assay of pyrocatechol, pyrogallol, 3,4-dihydroxyphenylacetic acid, caffeic acid, gallic acid, catechol, and other phenols. Miscellaneous Methods. Bacterial or enzymic spoilage of food has been determined by Purr (248) using a test paper for the detection of esterases, and Leybold, Rieper, and Weissbecker (172) determined the cortisol binding capacity of plasma by an enzyme fluorometric determination. Creatine phosphokinase was used for the deterniination of creatine in blood by Kibrick and Ililhorat (148). Reythar and Palecek (257) described the determination of denaturated DNA in the presence of native DKA, and a semi-automated filter paper disk technique for the determination of transfer ribonucleic acids was proposed by Goldstein, Maddox, and Rubin (92). A procedure for indole-3-acetic acid was described by RIendt and Gaines; following a peroxidase oxidation, the products were determined colorimetrically (192). Sorepinephrine in brain tissue was determined enzymatically by Saelens, Schoen, and Kovacsics (272), and the decrease in absorbance of orotic acid in the presence of Jiicrococcus glutamicus was used by Kagano et al. (211) for the assay of 5-phosphoribosylpyrophosphate. Walsh (S40,341), determined plasma pyridoxyl phosphate with wheat germ glutamic-aspartic apotransaminase. A method using tyrosine decarboxylase for the determination of pyridoxyl phosphate in plasma and other biological samples mas described by Hamfelt ( l l S , 114). The reaction rate was calculated from the amount of tyramine I4C produced. Yamoda, Saito, and Tamura (361) determined pyridoxyl-5-phosphate and pyridoxyl in biological material, and the fluorescence of P-naphthylamine

formed was used by Roth and Reinharz (267) for the determination of renin. Several enzymatic methods have been proposed for the analysis of urea. I n most of the methods the ammonia liberated is assayed by a colorimetric procedure. Kaftalm, Whitaker, and Stephens (209) used the absorbance change a t 660 mp after addition of hypobromite to measure the ammonia produced from urea in blood. Wil5on (366) described an automatic method for the determination or urea using urease, hypochlorite, and alkaline phenol that offers advantages of speed and precision over the conventional Xes4er’s reagent. Cirje and Sandru assayed urea in blood and urine using this improved method (52), and a comparison of the ‘C‘rastat method with the xanthydrol hypobromite micromethod for urea in blood by Xanzini (181) showed the former had greater simplicity, speed, and reliability. The use of potassium phosphate buffer rather than an EDTA buffer was shown by Watson (343) to effect a more rapid hydrolysis of urea by urease. An enzyme chromatographic method (Urastat strip test) was used for the determination of blood urea nitrogen levels in ox, horse, pig, and sheep by Parmense (230). X coupled optical enzyme assay for urease was developed by Kaltwasser and Schlegel (139). The N A i D H dependent glutamate dehydrogenase was used; the rate of ammonia production from urease was calculated from the rate of K A D H oxidation. This same method was used by Roch-Ramel (260), except that the NAD+ formed from Y X D H was measured fluorometrically. From 2 x to mole of urea or 4 X 10-11 to 2 X equivalent of S H 4 +was determined. Procedures for the determination of xanthine and hypoxanthine in biological fluids were described by Simmonds and Wilson (294). Specificity \vas obtained by the use of uricase in combination with the nonspecific xanthine oxidase. Petersen, Joerni, and Joergensen (235) and Klineberg et al. (157) described an enzymatic spectrophotometric method for the separate determination of hypoxanthine and xanthine in urine and blood. The decrease in uric acid at 292 mp was measured. Jones, Murray, and Burt (137) determined hyppxanthine in tissues by perchloric acid extraction and automation of the xanthine oxidase spectrophotometric assay, and Talsky and Fink (319) studied the oxidation of aldehydes by bovine milk xanthine oxidase, with methylene blue as acceptor. Guilbault, Brignac and Zimmer (99) have described a fluorometric method for the assay of nanogram quantities of xanthine and hypoxanthine in biological samples. The homovanillic acid-peroxidase fluorescence indicator reaction

was used to monitor the peroxide formed from the xanthine oxida5e catalyzed oxidation of hypoxanthine and xanthine. Inorganic Substances. Fluorometric and colorimetric methods for ammonia using t h e X d D H coupled glutamate dehydrogenase reaction were described by Roch-Ramel (260) and Schmidt and Schwartz (279). The decrease in N.IDH is measured and equated to ammonia concentration. Dunnil and Foirden (66) and Flosb, Hadwiger, and Conn (80) described procedures for cyanide based on it3 injection into organic molecules a5 catalyzed by E. coli and Lotus teams seedlings. The binding of cyanide to SAD in the presence of pig heart lactate dehydrogenase %as discussed by Geilach, Rfleiderer, and Holbrook (91). Weetall and Keliky (346) described an enzyme impregnated paper prepared by chemically coupling horse radish peroxidase to carboxymethylcellulose paper strips in the presence of S,S,dicyclohexylcarbodiimide, for the detection of small amounts of peroxide. Guilbault et al. (99, 107) u5ed homovanillic acid (HVA) for the determination of as little as lo-” mole of peroxide. The rate of production of the fluorescence of oxidized HT’h ib measured and related to peroxide concentration. Putter and Stiufe added polyvinylpyrrolidine compounds for stabilization of color in the peroxidase-o-dianisidine procedure for peroxidase (249). Schulz, Passonneau, and Low1y (282) have described a fluorometric enzymic method for the measurement of inorganic phosphate based on the following enzyme sequence. Glycogen

-

+ Phosphate phosphorylase a

Glucose-l-l-’ho+hate phosphoglucomutase

Glucose-1-Phosphate > Glucose-6-Phosphate

-

Glucose-6-phosphate

XanP

+

dehydrogenase

6-Phosphogluconolactone

+ K;.IDPH

S A D P H is fluorescent, and the rate of its formation indicates the phosphate present at concentration5 of 0.025 X mole. Faway, Koth, and Faway (74) assayed inorganic phosphate in tissue and serum using thi, same reaction sequence, except for a spectrophotometric measurement of KADPH, and Mozersky) I’ettinati, and Kolman (204) described an improved method for orthophosphate by a modification of the method of Martin and Doty (183). COENZYMES AND ACTIVATORS

.In enzyme activator converts an inactive enzyme into an active biological catalyst, generally a t very low VOL. 40, NO. 5, APRIL 1968

465 R

concentrations. B y their mode of action coenzymes can be classified as activators of enzymes, because in their absence the enzyme cannot function. The firefly reaction has been elucidated, and the luminescent reaction has been performed in Vivo by mixing cellfree extracts and even pure reactants. The reaction requires .4TP and hIg2+ in addition to luciferin, luciferase, and oxygen. Luciferin

+ 02 + A T P ---= Oxyluciferin + A D P + P i Luciferase

This reaction forms the basis for the most sensitive method known for ATP. Picogram quantities of A T P were determined by Lyman and devincenzo (1767, and Yokoyama and Nose (366) assayed A T P in erythrocytes. Procedures for ATP determination in red cells were proposed by Beutler and Baluda (29), and Medort, Weed, and Troup (5) found that the light emission of the luciferin-luciferase reaction is inhibited by increasing the ionic strength of the medium. This decrease was proportional to the concentration of some cations as Li+, K+,and Rb+, over the range 10-100 mM. A detailed study of the assay of A T P and A D P was made by Holmsen, Holmsen, and Bernhardsen (168). The ATP is determined as above; the A D P is converted to ATP with a pyruvate kinase system, which gives practically a 100% yield and is independent of the prescnce of AMP. A comparison of the normal red cell A T P levels as measured by the firefly system and the hexokinase system was made by Beutler and Mathai (30). They found that both techniques gave the same results on measuring ATP content of trichloroacetic acid blood filtrates, but that protein stimulated the light output when lyophilized firefly extract was used. Thus, when using the firefly method for ATP determination, protein should be added to the standard. A kinetic method for the microdetermination of nucleoside triphosphates using the glucose-1-phosphate kinase, phosphoglucomutase, and glucose-6-phosphate dehydrogenase systems has been described (72). Concentrations of A T P as low as 10-15-lf can be detected. A radioenzymatic method was described for the determination of A T P by hleerov (190). The A T P is converted to ADP and glucose-6phosphate I4C which is precipitated as the barium salt and counted. Another sample is treated with hexokinase as well as adenylate kinase simultaneously, giving the total of ilTP ADP. Procedures for the enzymic synthesis of nucleoside di- and tri-phosphate were described by Maley, Maley, and hfcGarrahan (180). Kratochvil, Boyer, and Hicks (162) found that hln(II), Mg(II), and Co(I1)

+

466 R

ANALYTICAL CHEMISTRY

activate the isocitric dehydrogenase enzyme system. Trace amounts of these metals were determined either by measurement of reaction rates in the presence of a nonlimiting excess of reactants, or, for all but magnesium, by titration with EDTA. I n a study of the effect of various cations and anions on the oxidative decarboxylation of keto acids, Hayakawa (118) found that Mg(11) and Mn(I1) activate heart pyruvic dehydrogenase and bacterial 2-oxyglutarate dehydrogenase. Both Ca(I1) and Mg(1I) activate the latter enzyme also. Mg(I1) and Mn(I1) have an activating effect upon the DNA-DNase enzyme system and could be determined (815).

INHIBITORS

Because enzymes are inhibited specifically by very low concentrations of chemical substances, they are good analytical reagents for the detection and analysis of these materials. Baker (14) and Bendetskii (61) have summarized the theories of interaction of enzymes and inhibitors from a research viewpoint. Lactic dehydrogenase, dihydrofolic reductase, thymidine phosphorylase, guanase, and xanthine oxidase are covered in detail. Catalase is inhibited by ascorbic acid in concentrations as low a s 2 X 10-6Ji (221), and the rate of inhibition is increased by catalytic amounts of Cu(I1) which does not inhibit alone. Thomas and Aldiidge (329) concluded from a study of 10 enzyme systems that only two of them, alkaline phosphatase and phosphoglucomutase, are inhibited by beryllium. The inhibitory effect of D-cycloserine on catalase and peroxidase activity in plant and animal materials was studied by Sicho and Kas (289), and the inhibition of &glucuronidase by cholesterol and retinol was assessed by Tappel and Dillard (326). As usual, cholinesterase has received the most attention from researchers. Abou-Donia and Menzel (2) studied the inhibition of fish brain cholinesterase by carbamates and developed an automatic method for the assay of this pesticide. Winteringham and Fowler (357), in a survey of the carbamate inhibition of acetylcholinesterase suggested that the Sevin inhibition is caused by an enzymic destruction of the carbamates. Cimasoni (51) reported the determination of 15-25 &' quantities of fluoride based on its inhibition of horse serum cholinesterase; 10 times as much fluoride was required to inhibit acetylcholinesterase (51, 164). The enzyme liver esterase was used to detect nanogram quantities of fluoride in tooth enamel by McGaughey and Stowell (178). No interference from ZrOz or S n 2 +was observed.

Beynon and Stoydin (33) reported a rapid screening test for cholinesterase inhibition by pesticides making use of agar-agar plates. As little as 10-3 fig of D D V P and other inhibitors could be detected. Several workers described the automation of cholinesterase-inhibition determinations using the Autohalyzer. Voss (357) and Ott and Gunther (223) proposed procedures that required prior extraction and cleaning, and Ott and Gunther in a later publication (664) used the spots scraped off a TLC pla,te as input sample for the ButoAnalyzer. X test for the detection of organophosphorus pesticides on TLC plates, using cholinesterase and either 2-azobenzene-1-naphthy1 acetate or indoxyl acetate, was described by Ortloff and Franz (222). Lau (167) used cholinesterase inhibition for the determination of both Bildrin and hzodrin in crops with a sensitivity of 0.1 ~ g .Procedures were described for the separation of these t'wo compounds from each other and from other pesticides. I3eynon et al. (32) reported an analysis of diethyl-l(2,4-dichlolphenyl)-2-chlorovinyl phosphate (compound 4072) in soil and crops. The insecticide was determined by cholinesterase inhibition after extraction of the sample and cleanup on a Florisil column. Ackermanii (3) used silica gel TLC plates for the semiquantitative determination of organophosphorus and carbamate pesticides, and Beam and Hankinson (19) reported a procedure for the estimation of compounds and carb on choliiie3terase inhibition. El-Refai and Ilopkins (69) described the use of plates coated with a cellulose powder containing 10% CaSO., as binder for the separation of organophosphorus liesticides and their oxons. Four spray reagents were used, including one based on cholinesterase inhibition that was able to detect l o p 3 to 2 nanograms of the various pesticides. Zadrozinska (365) used p a l m chroniatography for the estimation of organophosphorus pesticides in crops. Following extraction and paper chromatographic separation, fluorometric enzyme procedures were used for the detection of the pesticides. A comprehensive review of the enzymic hydrolysis of organophosphorus compoiinds was prepared by IlousteMazy (66). Reactions involving hydrolysis of ester linkages by nonspecific and specific enzymes are presented, and the hydrolysis of acid anhydride linkages by ATPase and acylphosphatases are discussed. Tanabe, Futai, and Tanabe (321) described the purification and properties of an enzyme monofluoroacetate fluorohydrolase that catalyses the defluorination of monofluoroacetate.

Peroxylacetyl nitrate was determined in air pollution studies by an inactivation of enzymes caused by an oxidation of enzyme sulfhydryl groups and by inhibition of the incorporation of acetate into fatty acids (205). Senov (214) described a procedure for the determination of Sevin in food products by a micro enzymic method, and Pavlic (234) reported an inhibitory effect of tris (hydroxymethyl) amino methane on the activity of acetyl- and butyrylcholinesterases. Kratochvil, Boyer, and Hicks (162) studied the effect of various metal ions on the enzyme isocitric dehydrogenase. A number of ions were found to inhibit ( 9 a + , Be2+, Ca2+, In3+, Te+, Sr2+, Ea2+, M 3 + , Ce3+, J l 0 0 ~ ~Fe3+, - , Xi2+, Cu2+, Ag+, Cd2+, Hg2+, Pb2+, T h 4 + , and VOi2+), and could be determined. Inhibiting metals that form complexes with a chelating agent more stable than those of activating metals can be determined by E D T A titrations. Neske (215) reported that cattle pancreas DNase was inhibited by Cu2+, and Guilbault, Kramer, and Hackley (106) described procedures for the determination of Fez+, Cu2+,and CIS- based on their inhibition of the enzyme hyaluronidase. I n a study of the inhibition of peroxidase, Guilbault, Brignac and Zimmer (99) reported that CN-, S2-, Cr207?-, Cu2+, Fez+, Fe3+, Xn2+, PbZ+,Cot+, Cd2+, and hydroxylamine inhibit this enzyme and can be determined a t submicrogram concentrations. Guilbault and Kramer (105) proposed a method for the selective determination of H g 2 +over a concentration range of to 5 X 10-7JI using the enzyme glucosidase and a spontaneous electrolysis cell. I n all the above-mentioned procedures, the specific determination of one metal ion in the presence of others is impossible unless a prior separation or masking procedure is used. Stehl, Margerum, and Latterell (306) have discussed the masking of metal ions in selective rate methods, and the use of making reagents (such as &Os2- for Ag+ or Hg2+; C K - and chlorohydrate for Xi2+, Co2+,Fe3+, etc.) will allow the researcher to develop specific procedures for very low concentrations of the metal ions of interest. IMMOBILIZATION OF ENZYMES

As was mentioned in the last review article, a prime objection to the use of enzymes as analytical reagents lies in the high cost of using large quantities of these materials. The immobilization or insolubilization of the enzyme would eliminate this problem, for the enzyme could be used over and over again. A review on the preparation of insoluble enzymes has been prepared by

Chibata and Tosa (49). The techniques of combining active enzymes with some insoluble carrier with covalent bonds, ionic combination or physical adsorption are discussed. Hicks and Updike (120) have described the preparation of a stable, lyophilized polyacrylamide enzyme gel of uniform particle size. The immobilized enzyme was found to behave similarly to soluble enzymes, and has the advantages of convenience, economy, and easy adaptability to automation. Glucose oxidase and lactic dehydrogenase, immobilized in the gel, were found to retain their activity over long periods of time. This same immobilization technique was used by Updike and Hicks (333, 333a) in the development of electrochemical sensors for the in vitro measurement of glucose in biological solutions and tissues. Wieland, Determan, and Buennig (352) also prepared insoluble enzymes in polyacrylamide gels. Alcohol dehydrogenase, trypsin, and lactate dehydrogenase gels were described. Habeeb (110) manufactured water insoluble derivatives of trypsin, using glutaraldehyde to conjugate trypsin to aminoethyl cellulose. Katchalski (142) prepared water insoluble derivatives of papain, by adsorption of the papain on a collodion matrix. R7eetall and Weliky (345)have described the synthesis and continual operation of a carboxymethylcellulose enzyme column and the preparation of a similar enzyme paper preparation which still retains its activity after 2 months storage without refrigeration (346). Reese and Mandels (255) described a method of obtaining an essentially continuous enzyme reaction on a twophase column utilizing partition chromatography. The enzyme was retained as the stationary phase on a column of the hydrophilic solid, cellulose. AUTOMATED METHODS

Muller (207) has reviewed autoniation in clinical chemistry, especially the SMA-12 AutoAnalyzer, and Skeggs (295) has surveyed the principles of colorimetric analysis as performed by the AutoAnalyzer and described its modules and multiple analytical systems. Annual Technicon Symposia on automated methods were held in October 1966 and 1967 in New York. Papers from these symposia were published in “Automation in Analytical Chemistry” (1966) (297) and (1967) (298). The need of automated systems for clinical serum analysis was emphasized by Skeggs (296). A completely automated system for the determination of enzyme activity by the measurement of initial reaction rates has been described by Schwartz (283), Posen et al. ( 2 4 3 , and N u r r a y and Harmon (208). Schwartz (284)has pre-

sented a survey of automation of enzyme methods, arid has discussed the problems of converting manual enzyme procedures to the AutoAnalyzer. Tappel and Beck have described an automatic multiple enzyme monitor for column chromatography which is applicable to a wide variety of hydrolytic enzymes for which nitrophenyl esters are substrates (325), and have proposed methods for obtaining continuous enzyme data under gradient conditions (324). Beck and Tappel (20) developed an automated multiple enzyme monitor for column chromatography; the effluent of a chromatographed soluble enzyme factor of rat liver lysosomes from a carboxymethylcellulose column was monitored by a single flow path to give protein fluorescence and to show the distribution of five lysosomal enzymes by way of chromogenic substrates. Brown and Ebner (42) have devised a continuous flow analysis procedure for the kinetic analysis of multiple enzyme systems. A procedure for the simultaneous automated determination of L-lysine and total amino acids in seed hydrolysates was described by White and Gauger (351) and Strumeger and Romano (314) automated the assay of submicrogram levels of amylase by a reducing sugar procedure. Mounter, Groff, and Sim (203) used a multichannel analytical system for continuous monitoring of blood cholinesterase, and Stein and Lewis (307) proposed an automatic continuous colorimetric method for acetylcholinesterase using the Autohalyzer. An electrometric method for the determination of cholinesterase using automatic recording was described by Rozengart, Shmeleva, and Shcherbak (269), and Humiston and Wright (129) automated the determination of cholinesterase by reaction of cholinesterase with acetylthiocholine to release thiocholine, which reacts with bis-dithio-nitrobenzoate to form a yellow color. A sensitive automated fluorometric assay of creatine phosphokinase was described by Willis, Kosal, and King (355),and Siege1 and Cohen (290, 291) developed a colorimetric method for this enzyme using the AutoAnalyzer. Mercaptoethanol was used as a source of sulfhydryl groups and the color was developed with I-naphthol and diacetyl. Fleisher (78, 79) automated the determination of serum creatine kinase activity by reactivation of creatine kinase with cysteine, followed by treatment with A D P and phosphocreatine. -4n automated method for ornithine carbamoyl transferase was proposed by Strandjard and Clayson (312). The estimation of alcohol by an enzymatic fluorometric technique on the Technicon AutoAnalyzer was described by Syed (316). Utilizing a continuous blood sugar analysis as the input, Kline VOL 40, NO. 5, APRIL 1968

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et al. (156) constructed a feed back system to maintain the level of blood sugar within a few per cent of a present level. The glucose oxidase-peroxidase-o-tolidine method was automated by Faulkner (73) for the deterniination of glucose in blood. An automated spectrophotometric method for the enzymatic determination of glycerol was described by Frings and Pardue (85). The rate of disappearance of the blue dye in the glycerol dehydrogenase, NL4D,diaphorase, dye coupled reaction is automatically measured and related to the concentration of glycerol. Automated methods for lactate dehydrogenase were described by Rlorgenstern, Flor, and Kessler (198) using the cupric-neocuproine reduction with XADH; by Plaut (241) with a single colorimetric method; by Brooks and Olken (41) who used a fluorometric method; and by Capps et al. (44) who adapted a tetrazolium dye-phenazine methosulfate reaction. Strandjard and Clayson (311) developed a n automated method for determining total L D H and heat stable L D H 1, and Oldenwurtel (220) described a highly specific automated analysis of pyruvic or lactic acids in biological fluids, employing the fluorescence of S A D H . An automated system for the simultaneous fluorometric determination of serum lactate dehydrogenase and glutamic oxaloacetic transaminase was presented by Passen and Gennaro (231). An automated method for the assay of lysozyme was proposed by Burrows (437, and an automatic fluorometric analysis of serum leucine-aminopeptidase was described by Ratliff, Thrasher, and Gochman (253). Automated techniques for acid phosphatase using the Robot Chemist and Autodnalyzer were described by Klein, Oklander, and Norgenstern (154), Klein and Auerbach (150), Roos (263), and Klein, Auerbach, and Morgenstern (151). All used either 1-naphthyl phosphate or phenyl phosphate as the substrate. An automated procedure for the determination of serum alkaline phosphatase using phenolphthalein monophosphate as substrate was proposed by Klein and Kaufman (153). Automated methods for alkaline phosphatase were proposed by Coleman and Stroje (56) who used thymolphthalein monophosphate, by Xorgenstern et al. (199) and Sterling et al. (308) who adapted the p nitrophenyl phosphate procedure, and by Hviid (1311, Tietz (331) and Comfort and Campbell (57) who utilized phenolphthalein phosphate. Green, Giovanniello, and Fishman (94) developed an automated differential analysis of several serum phosphatase isoenzymes and also proposed an automated procedure for the determination of total and L-tartrate sensitive serum phenyl and naphthyl acid phosphatases (95). 468

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Morgenstern and coworkers ($00) automated the glutamic-oxaloacetic transaminase procedure employing Fast Ponceau L to measure the oxaloacetic acid liberated. Levine and Hill (171) described an automated fluorometric method for the determination of serum glutamic oxalacetic transaminase and glutamic pyruvic transaminase. Resazurin was used which is reduced by KADH in the presence of diaphorase to the highly fluorescent resorufin. An automated procedure employing the AutoAnalyzer for the estimation of serum 5’-nucleotidase was described by Hill and Sammons ( I % ) , and Goldstein, Maddox, and Rubin (92) developed a semiautomatic radiochemical paper disk method for the determination of transfer ribonucleic acids. Stevens, Sauberlich, and Long (309) automated the trans-ketolase determination, and the Technicon AutoAnalyzer was used by Clarke and Nicklas (53) for the automated assay of trans-glutaminase. Hill and Cowart (121) automated the mutarotase assay, by measuring the p-Dglucose produced from a-D-glucose with glucose oxidase, peroxidase, and guaiac. CONCLUSIONS

Because of the extreme sensitivity and specificity of enzymes, these substances possess a great potential usefulness in analytical chemistry. With the new, more sensitive techniques available for assay of enzymes, the advent of the immobilized enzyme which has alleviated the problems of cost and supply, and the progress that has been made in automation of enzyme systcins for rapid, accurate analysis, enzymes are becoming common, acceptable reagents by analysts. This is clearly indicated by the increase in the number of papers reviewed in the last two year period. LITERATURE CITED

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\_.__,.

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(54) Clarke, A. D., Podmore, D. A., Clin. Chim. Acta 13, 725 (1966). (55) Coleman, C. M., Zbid. p 401. (56) Coleman, C. M., Stroje, R. C., Clin. Chem. 11, 815 (1963). (57) Comfort, D., Campbell, D. J., Clin. Chim. Acta 14, 263 (1966). (58) Conn, R. B., Anido, V., Am. J . Clin. Pathol. 42, 177 (1966). (59) DeSouza, K. O., Panek, A., J . Chromatog. 15, 103 (1964). (60) Diamondstone, T., Anal. Biochem. 16.z 395 - ~ (1966). - --, (Si? nickinson, F. M., Dalziel, K., Nature 214, 31 (1967). (62) Douste-Blazy, L., Colloq. Nationauz Centre, Sat. Rech. . Sci., Paris 1965, 333. (63) Drawert, F., Gebbing, H., Ziegler, A., J . Chromatogr. 30, 259 (1967). (64) Drawert, F., Kupfer, G., Z. Lebensm. Untersuch. Forsch. 123, 211 (1963). (65) Dunnil, P. M.,Fowden, L., A’ature 208, 1206 (1965). (66) Eggstein, M., Kreutz, F. H., Ergeb. Laboratoriumsmed. 2, 99 (1965). (67) Eggstein, >I., Kreutz, F. H., Klin. Wochsch. 44, 262 (1966). (68) Elevitch, F. R., Aronson, S., Feichtmeir, T. V., Enterline, M. L., 113th Annual Meeting, American Medical Association, San Francisco, June 22-25, 1964. ( 6 8 4 Zbid., Tech. Bull. Reg. LMed. Tech, 36, 282 (1966). (69) El-ltefai, A., Hopkins, T. L., J . Agr. Food Chem. 13, 477 (1965). (70) Engel, S., Wulff, V., Beckman Rep. 1965 (3-4)) 17-18. (71) Erlanger, B. F., Buxbaum, S. N., Sack, 13. A., Cooper, -4. G., Anal. Biochem. 19, 542 (1967). (72) Eyer, P., Pette, D., Enzymol. Biol. Clin. 8 , 189 (1967). (73) FFulkner, D. E., Analyst 90, 736 (1963). (74) Faway, E., Roth, L., Faway, G., Biochem. 2. 344, 212 (1966). ( 7 5 ) Fernley, H. N., Walker,’ P. G.. Biochem. J . 97, 95 (1965). (76) Filmer, D. L., Cannon, J. R., Reiss, N.. J . Theoret. Biol. 16. 280 (19671. ( 7 7 ) ’Fischl, N., Segal, ’S.,Rabiah, S., CEin. Chem. 13, 941 (1967). ( 7 8 ) Fleisher, G. A., Zbid., 12, 532 (1966). (79) Fleisher, G. A., Zbzd., 13,233 (1967). ( 8 0 ) Floss, H., Hadwiger, L., Conn, E. E,, Kature 208. 1207 (1965). (81) Fonnum; F., Biochem. J . 100, 479 (1966). (82) Freud, H., Ann. Biol. Clin. Paris 25, 421 (1967) (Fr.). (83) Fried, It., Anal. Biochem. 16, 427 (1966). (84) Friedel, W., Z. Med. Labortech. 5, 301 - - (1964). ~- - ~ (85) Frings,’C. S., Pardue, H. L., Anal. Chim. Acta 34, 225 (1966). (86) Fuller, R. W., Hunt, J. hl.. Anal. Biochem. 16, 349 (1966); (87) Fyowa Fermentation Industry, French Patent 1,410,747 (1967). (88) Gabay, S., George, H., Anal. Biochem. 21, 111 (1967). (89) Gaffrini, &I.,Ilegolisti, A., Veggetti, G. P., Pathologica 58, 261 (1966). (90) Gage, J. C., Residue Rev. 18, 159 (1967). (91) Gerlach, D., Rfleiderer, G., Holbrook J., Biochem. 2 . 3 4 3 , 3 5 4 (1965). (92) Goidstein, G., hladdox, W. . L., Rubin, I. B., Technicon Symposium on Automation in Analytical Chemistry, New York, Sept. 1967, Paper 10. (93) Graham, L. T., Aprison. lL H.. Anal. Biochem. 15, 487 11966)’. (94) Green, S., Giovanniello, T., Fishman, W., Technicon Symposium on Automation in Analytical Chemistry, New York, Sept. 1966, Paper 97. \ - -

\ - - - - , -

\

(95) Green, S., Giovanniello, T., Fishman, W,., Technicon Symposium on Automation in Analytical Chemistry, New York, 1967, Paper 108. (96) Greenberg, L. J., Anal. Biochem. 14, 265 (1966). (97) Guilbault, G. G., in “Fluorescence, Theory, Instrumentation and Practice,” G. Guilbault, Ed., hlarcel Dekker, New York, 1967, pp 297-358. (98) Guilbault, G. G., Presented a t 155th Xational hleeting of American Chemical Society, San Francisco, April 1968. (99) Guilbault, G. G., Brignac, P., Zimmer, hl., ANAL. CHEM. 40, 190 Juneau, SI.,Ibid. in (101) Guilbault. ’

(102) Guilbault, G. G., Heyn, A,, Zbid. 1, 3 (1967). . ) Guilbault, G. G., Hieserman, J., Anal. Biochem., in press. (104) Guilbault, G. G., Kramer, D. X., ANAL.CHEM.37, 1675 (1965). (105) Guilbault, G. G., Kramer, D. N., Anal. Biochem. 18, 313 (1967). (106) Guilbault, G. G., Kramer, D. N., Hacklev, E., Ibid., p 241. (107) Guilbault, G. G., Kramer, D. N., Hackley, E., ANAL. CHEM. 39, 271 (1967). (107a) Guilbault, G. G., Sadar, S., Glazer, R., Haynes, J., Anal. Letters 1, 333 (1968). (108) Guroff, G., Abramowitz, A., Anal. Biochem. 19, 548 (1967). (109). Guroff, G., Rhoads, C.. Abramowltz, A., Ibid. 21, 273 (1967): (110) Habeeb, A., Arch. Biochem. Biophys. 119, 264 (1967). (111) Haining, J. L., Legan, J. S., Anal. Biochem. 21, 337 (196’7). (112) Hakanson, R., Hoffman, G., Biochem. Pharmacol. 16, 1677 (1967). (113) Hamfelt, A., Clin. Chim. Acta 16, 19 (1967). (114) Hamfelt, A,, Scand. J . Clin. Lab. Invest. 20, 1 (1967). (115) Hankin, L., Wickroski, A. F., J . Assoc. Offic. Agr. Chemists’ 47, 695 I1 ~964) ----,. (116) Hankin, L., Wickroski, A. F., Zbid. p 903. (117) Hausman, T. U., Helger, R., Rick, W., Gross, W., Clin. Chim. Acta 15, 241 (1967). (118) Hayakawa, T., Biochem. Biophys. Acta 128, 574 (1966). (119) Helman, B., Acta Physiol. Scand. 65, 337 (1965). (120) Hicks, G. P., Updike, S. J., ANAL. CHEM.38, 726 (1966). (120a) Hidaka, H., Nagatsu, T., Yagi, K., Anal. Biochem. 19, 388 (1967). (121) Hill, J. B., Cowart, D. S., Zbid. 16, 327 (1966). (122) Hill, P. G., Sammons, H. G., Clin. Chim. Acta 13, 739 (1966). (123) Hillcoat, B. L., Nixon, P., Blakley, R., Anal. Biochem. 21, 178 (1967). U., Arzneimittel. (124) Hinterberger, Forsch. 7 (20), 1242 (1966). (125) Hiyama, T., Koga, Y., Fukui, S., J . Gen. Appl. illicrobiol. Tokyo 13, 121 (1967). (126) Hjelm, AT., Clin. Chim. Acta 15, 87 (1967). (127) Hollister, L., Helmke, E., Wright, A., Diabetes 15, 691 (1966). (128) Holmsen, H., Holmsen, I., Bernhardsen, A., Anal. Biochem. 17, 456 (1966). (129) Humiston, C. G., Wright, G. J., Clin. Chem. 11,802 (1965). (130) Hutton, J. J., Tappel, A. L., Udenfriend, S., Anal. Biochem. 16, 384 (1966).

(131) Hviid, K., Clin. Chem. 13, 281 (1967). (132) Ipat>a, P. L., Anal. Biochem. 20, 30 (1967). (133) Jacks, T. J., Kircher, H. W., Zbid. 21. 279 11967). (134) Jerabek, J., Prumysi Potravin 17, 426 (1966); Chem. Abstr. 65, 14088, 1966. (135) Joergensen, B. B., Joergensen, 0. B., Acta Chem. Scand. 20, 437 (1966). (136) Johnson, J., Fusaro, It., Anal. Biochem. 18, 107 (1967). (137) Jones, N. R., hlurray, J., Burt, J. R., J . Food Sci. 30, 791 (1965). (138) Kadish, .4. H., Hall, D. A., Clin. Chem. 9, 869 (1965). (139) Kaltwasser, H., Schlegel, H., Anal. Biochem. 16. 132 (1966). (140) Kamel, 31,Y:, Hart, 11. R., Anderson, It. L., Ibid. 18, 270 (1967). (141) Kammen, .H., Zbid. 17, 533 (1967). (142) Katchalski, E., Technical Report. AFOSli Grant 67-202.5. June 30. 1967. (143) Katz. S.. Cowan;. J.. Bioorhim. .... .. Bioophys. ’Acta 107, 60.i’( 1965). (144) Kaufmann, H. P., Wessels, H., Fette, Seifen, Anstrichmittel 66. 13 (1964). . (145) Kawerau, E., 2. Klin. Chem. 4, 224 (1966). (146) Keller, D. ll., Clin. Chem. 11, 471 (1965). (147) Kessler, G., Lederer, 13.) Technicon Symposium on Automation in Analytical Chemistry, Sew York, Sept. 1965, p 341. (148) Kibrick, A. C., llilhorat, A. T., Clin. Chim. Acta 14, 201 (1966). (149) Kingsley, G. It., ANAL. CHEW.39, 22R (1967). (150) Klein, B., Auerbach, J., Clin. Chem. 12. 289 11966). (151) Klein, B., ‘Auerbach, J., llorgenstern, S.,Clin. Chem. 11, 998 (1963). (152) Klein, B., Flor, ll.,Kaufman, J. H., Zbid. 12, 748 (1966). (153) Klein, B., Kaufman, J., Zbid. 13, 290 i1967). (154) Klein,‘ B., Oklander, AI., llorgenstern, S., Ibid. 12, 226 (1966). (135) Kleinbaum, II., 2. M e d . Labortech. 8,.18 (1967). (156) Kline, N. S., Blair, J. H., Sterns, H., Kohn, X, Shimano, E., Technicon S y m p o h m on ilutomation in Analytical Chemiitry, Sew York, Sept. 1966, Paper 102. (157) Klineberg, J. R., Goldfinger, S., Bradley, K. H., Seegmiller, J., Clin. Chem. 13, 834 (1967). (138) Kmetec, E., Anal. Biochem. 16, 474 (1966). (159) Kotaki, A., Xapi, X, Okamura, K., J . Biochem. Tokyo 60, 236 (1966). (160) Kotas, J., Casopis Lekaru Ceskych 104, 932 (1964); Chem. Abstr. 65, 5861e (1966). (161) Krajl, >I., Biochem. Pharmacol. 14, 1686 (1965). (162) Kratochvil, B., Boyer, S. L., Hicks, G. P., ANAL. CHEX. 39, 45 (1967). (163) Kretovich, V. L., Stepanovich, K. ll., Zzv. Akad. Sauk SSSR, Ser. Biol. 31, 295 (1966). (164) Krupka, It. M., Mol. Pharmacol. 2, 558 (1966). (165) Kutter, D., Znst. Grand-Duval Luz~

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emborg, Sect. Sci. Sat. Phys. Math. Arch. 29, 51 (1962); Chem. Abstr. 64,

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(322) Tanabe Seiyaku Co., British Patent 1,072,876, June 21, 1967. (323) Tanner, H., Brunner, E., Mitt. Gebiete Lebensm. Hyg. 55, 480 (1964). (324) Tappel, A. L., Beck, C., Abstracts of Technicon Symposium on Automation in Analvtical Chemistrv. New York, Sept. 1965, p 559. (325) Ibid., New York, Sept. 1967, Paper .,I

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(339) Walker, J. R., J . New Zealand Znst. Chem. 31, 41 (1967). (340) Walsh, hl. P., Am. J. Clin. Pathol. 46, 282 (1966). ’ (341) Walsh, M. P., Tech. Bull. Registry Med. Technologists 36, 178 (1966). (342) Ware, A. G., Marbach, E. P., Clin. Chem. 11, 792 (1965). (343) Watson, D., Clin. Chim. Acta 14, 571 (1966). (344) Weatherburn, M. W., Logan, J. E., Diabetes 15, 127 (1966). (345) Weetall, H., Weliky, N., Jet Propulsion Laboratory Space Programs Summary N o . 37-86, 4, 160 (1965). (346) Weetall, H., Weliky, N., Anal. Biochem. 14. 160 (1966). (347) Weichei, H. H., Deut. Lebensm. Rundschau, 61, 53 (1965). ~r

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