ANNALS OF CLINICAL AND LABORATORY SCIENCE, VOL. 15, NO. 1 Copyright © 1985, Institute for Clinical Science, Inc.

Measurement of Total Lactate Dehydrogenase Activity

RAYMOND E. VANDERLINDE, Ph.D.

Departments of Pathology, Laboratory Medicine, ir Biological Chemistry, Hahnemann University School of Medicine Philadelphia, PA 19102

ABSTRACT Lactate dehydrogenase (LD: EC 1.1.1.27) is the most important clini­ cally of several dehydrogenases occurring in human serum. Lactate dehy­ drogenase is cytoplasmic in its cellular location and in any one tissue is composed of one or two of five possible isoenzymes. While many of its clinical applications involve quantification of one or more specific serum isoenzymes, an estimate of total LD is required usually. Lactate dehydrogenase catalyzes the reversible reaction: L-lactate + NAD+ pyruvate + NADH. The bidirectional reaction is monitored spectrophotometrically by measuring either the increase in NADH at 340 nm produced in the lactate-to-pyruvate reaction (L —» P) or by the decrease in NADH at 340 nm produced in the pyruvate-to-lactate (P —» L) reaction. Kinetic assay systems for the measurement of the reaction system in both directions are comprehensively reviewed as well as the standardization efforts proposed to date.

Introduction Lactate dehydrogenase (LD) has a molecular weight of about 140,000 dal- Dehydrogenases is the preferred name tons and is cytoplasmic in its cellular for the group of enzymes which belong location.36 It is composed of four peptide to the oxidoreductase class of enzymes in chains of two types, H and/or M sub­ which the substrate oxidized is regarded units, thereby creating isoenzymes. LD as a hydrogen donor and the usual accep­ 1, the heart type isoenzyme, consists of tor is nicotinamide-adenine dinucleotide four H subunits, whereas LD 5, the mus­ (NAD + ) or nicotinamide-adenine dinu- cle/liver type isoenzyme, consists of four clesotide phosphate (NADP + ). Lactate M subunits with the intermediary forms dehydrogenase (LD: EC 1.1.1.27) (table being combinations of subunits. I) is the most important dehydrogenase Lactate dehydrogenase catalyzes the from the clinical viewpoint. Others mea­ reversible reaction: sured much less frequently include mal- L-lactate + NAD+ ^ pyruvate ate dehydrogenase (MD), glutamate + NADH. dehydrogenase (GLDH), isocitrate dehy­ drogenase (ICD), and sorbitol dehydro­ The bidirectional reaction is monitored genase (SDH). spectrophotometrically by measuring 13 0091-7370/85/0100-0013 $02.00 © Institute for Clinical Science, Inc. 14 VANDERLINDE

TABLE I pyruvate and react with other substrates. Abbreviation of Enzymes with One of these substrates, 2-oxobutyric Classification Numbers* acid, has been used “preferentially” to determine serum LD 1 (heart-type) Dehydrogenases activity. The reaction, known as B- Lactate dehydrogenase (LD) EC 1.1.1.27 hydroxybutyric dehydrogenase (HBDH), M a l a t e d e h y d r o g e n a s e (MD) EC 1 . 1 . 1 . 3 7 Glutam ate dehydrogenase (GLDH) EC 1 . 4 . 1 . 3 involves: 2-oxobutyric acid + NADH ^ Isocitrate dehydrogenase (ICD) EC 1.1.1.42 2-hydroxybutyric acid + NAD+ and is Sorbitol dehydrogenase EC 1.1.1.14 monitored by measuring the decrease in O t h e r s absorbance of NADH at 340 nm. The A lkaline phosphatase (ALP) EC 3 . 1 . 3 . 1 evidence indicates LD is the enzyme A spartate am inotransferase (AST) EC 2 . 6 . 1 . 1 involved in the reduction of glyoxalate which has been found to be enzymati­ ♦Enzyme Nomenclature Recommendations (1978) of the International Union of Pure and Applied cally reduced by human serum faster at Chemistry and the International Union of 37°C than either pyruvate or 2-oxobutyr- Biochem istry, New York, Academic Press, 1979. ate.108 However, the measurement of total either the increase in reduced nicotin- LD activity in serum or other body fluids amide-adenine dinucleotide (NADH) at usually uses either lactate or pyruvate as 340 nm produced in the lactate-to-pyru- substrate and is attributable to a mixture vate reaction (L —> P) or by the decrease of isoenzymes which varies with an indi­ in NADH at 340 nm produced in the vidual’s physiological and pathological pyruvate-to-lactate (P—» L) reaction. The conditions. enzyme acts only on L-lactate and only NAD+ can function as the coenzyme. Pyruvate-to-Lactate Methodologies Two of the five LD isoenzymes (LD 1 and LD 2) are not specific for lactate or The conditions for the primary contin-

TABLE I I

Lactate Dehydrogenase Kinetic Assay Methods (Pyruvate-to-Lactate)

P y r u v a t e NADH Buffer/conc. M e th o d * T (°C) (mmol/1) (mmol/1) (m m o l/1 ) pH

1 0 7 Wroblewski and LaDue (1955) 25-27 0.74 0.12 P i f 7.4 (7.2!-7.6) Henry, Chiamori, Golub, and Berkman (1960)^ 32 0 . 6 0 . 1 8 7 P i 100 7 . 4 Bowers (1963)15 3 7 1 . 2 0 . 2 1 T r i s 100 7.35 at 37°Cl[ Gay, McComb, and Bowers (1968)^ 30 0 . 9 $ 0.22 Tris 100 7.30 at 3 0 °c H German Society (1972)^ 2 5 0 . 6 0 . 1 8 P i 50 7 . 5 McQueen (1972)69 3 7 2.5 0.12 Pi 67 7.18 a t 3 7 °C li Scand. Soc. for Clin. Chem. and Clin. Physiol. (1974§)29 3 7 1.2 0.15 Tris 50A 7.40 at 37°CÏ Buhl, Jackson, and Graffunder (1978)20 2 5 $ 1 . 0 0.15 TEA 100 7.0 a t 2 5 " d l 30 1 . 5 0 . 1 5 TEA 100 7 . 0 a t 3 0 ° d t 37 1 . 5 0 . 2 2 TEA 100 7 .0 at 37°cj[ Société France de Biologie Clinique (1981) 30 1 . 6 0 . 2 0 T r i s 80 7 . 2 a t 3 0 ° d l N aC l 2 0 0

*Sample volume fraction of 1/30 except as noted. fPi designates phosphate. $Used 0.9 mmol/1 in th eir study but found 1.2 mmol/1 to be optimal for mixed isoenzyme sample. §Sample volume fraction 1/60 tfpH adjustm ent at tem perature designated. ABuffer contained 5 mmol/1 ethylenediam inetetraacetic acid (EDTA). ^Recommended 25°C but optim ized also at 30°C and 37°C. MEASUREMENT OF TOTAL LACTATE DEHYDROGENASE ACTIVITY 15 uously monitored rate (kinetic) measure­ reaction for LD. Optimization of pH and ment of LD in the pyruvate-to-lactate (P substrates was carried out in order to —» L) direction are summarized in table achieve the maximum rate for Versatol- II. In 1955, Wroblewski and LaDue107 E.* No application to clinical specimens developed the first continuously moni­ was included. tored spectrophotometric (kinetic) assay In 1972 McQueen developed an opti­ method for LD using pyruvate, reduced mal assay for the P —» L reaction at 37°C NADH and serum in phosphate (Pi) based upon normal and pathological sera buffer at pH 7.4. The reaction does not as a source of LD activity.69 Subse­ have to be forced because the equilib­ quently, Tuckerman and Henderson100 rium lies in the direction of the formation evaluated the Henry assay51 carried out of lactate. In 1960 Henry, Chiamori, at 37°C instead of 32°C, the McQueen Golub, and Berkman,51 in their classical assay69 and an optimized commercial publication on revised spectrophoto­ assay kit, all P —> L assays at 37°C. The metric methods, took a major stride by McQueen assay, which uses a high pyru­ examining and “revising” the conditions vate concentration, gave significantly less for three major clinical laboratory LD activity owing to substrate inhibi­ enzyme measurements including LD in tion, than the other two methods which the P —» L direction. They were the first appeared to be equivalent. to: optimize substrates, emphasize tem­ Szasz98 investigated the effect of tem­ perature control, and stress pH control. perature on enzyme activity for the var­ A very complete investigation of LD ious enzyme methods recommended by assay conditions was carried out by Gay, the German Society for Clinical Chem­ McComb, and Bowers42 in 1968 utilizing istry.34 He showed both “fast” LD (LD 1 crudely extracted preparations of human and 2) and “slow” LD (LD 5) to deviate heart and liver LD for investigation of from linearity when measured at tem­ the optimal conditions for both the P —» peratures greater than 30°C under the L and lactate-to-pyruvate (L —» P) reac­ German optimized conditions recom­ tions. For the P —» L reaction Gay et al mended for 25°C. Szasz concluded that recommended Tris (hydroxmethyl) ami- almost all of the kinetic factors relevant nomethane (Tris) buffer which system has to optimization are highly dependent on been adopted with modifications by the assay temperature. Thus, McQueen70 Scandinavian Society for Clinical Chem­ documented the true Arrhenius relation­ istry and Clinical Physiology.29 On the ships of human LD 1 and 5 extracts in other hand, the German Society for Clin­ the P —* L reaction at 25°, 30°, 35°, 40°, ical Chemistry recommendations34 are 45°, and 50° and showed clearly for the for a modification of the Henry et al first time the increasing pyruvate and method51 which utilizes phosphate NADH concentrations required at buffer. While Bowers15 reported that higher temperatures with different phosphate buffer inhibited LD activity isoenzymes. assayed in the P —» L system, Buhl, Jack­ In 1978 Buhl et al,20 in a follow up to son, and Grafifunder20 found phosphate earlier LD studies,21,22 investigated the up to 125 mmol per 1 and, to a small pyruvate-to-lactate reaction for optimal extent, Tris, were stimulatory for human reaction conditions at 25, 30, and 37°C. LD 1 and 5 at pH values less than 7.5. Nine buffers were investigated as well as Krause and Lott59 have demonstrated the non-linearity of the reaction the use of the simplex method to opti­ response. Highly purified LD 1 from mize analytical conditions in clinical chemistry by applying it to the P —» L * General Diagnostics Co., Morris Plains, NJ. 16 VANDERLINDE human erythrocytes and LD 5 from Bozon, and Lalegerie101 have proposed human liver were utilized as the sources an official recommendation by the of enzyme activity. Optimal substrate Société Française de Biologie Clinique concentrations for the measurement of entitled An Improvement for the Deter­ the P —* L reaction in imidazole, TEA mination of Total Lactate Dehydrogen­ (triethanolamine) or TES (N-tris (hy- ase Catalytic Activity Whatever the droxymethyl)-methyl-2-aminoethanesul- Isoenzymes. It is well known that the fonic acid) at pH 7.0 are shown in table reaction catalyzed by LDH is inhibited II. Imidazole, TEA, and TES were cho­ in the presence of high levels of sub­ sen as suitable reaction buffers because strate.36 This inhibition is observed in their pKa’s are near the recommended both directions and with both types of reaction pH of 7.0 and because they enzymes, although the inhibition is exerted no stimulatory or inhibitory much more pronounced with LD 1 than effects. Tris was not evaluated thoroughly with LD 5 and with pyruvate than lac­ but was found to be stimulatory, as pre­ tate. It has been shown to be due to the viously reported by Bowers.15 Also, Buhl formation of an abortive ternary complex found phosphate to be stimulatory for the or adduct of LD-NAD +-pyruvate with human LD 1 and LD 5 isoenzymes in inhibits strongly the P —* L reaction the P —* L direction only. His findings resulting in a non-linear reaction rate.36 that buffer effects are pH and substrate Recently, Rivedal and Sanner86 have dependent suggest a source of variation found that an increase in ionic strength in commercial reagent kit systems. can modify the stability of the ternary Buhl20 has recommended that all pyru- abortive complex and decreases inhibi­ vate-to-lactate measurements be made as tion. Thus, Vassault et al101 have shown soon as possible after the reaction is ini­ that the addition of Na+ and Cl- ions to tiated and be performed at 25°C or, if the P —> L reaction mixture modifies the necessary, 30°C because at 37°C there is stability of the inhibitory ternary com­ a different rate of decrease in absorbance plex. The proposed method includes the for LD 1 and LD 5. His studies suggest addition of 200 mmol per 1 NaCl to the that the measuring time interval at each Tris buffer reaction mixture (table II). temperature should be standardized rel­ The method is stated to show the follow­ ative both to the time after the reaction ing advantages: minimizing of differences is initiated and to its duration. The activ­ for optimal concentration of pyruvate ity of human LD 1 and LD 5 assays, between LD 1 and LD 5, improvement based on the P —> L reaction, were not in the linearity of the kinetics, and an affected by the manner of reaction initi­ increase in the upper limits of linearity. ation since triggering with pyruvate, Many P —» L procedures initiate the enzyme or NADH gave equivalent reaction with pyruvate (table II), in order results.22 to provide a preincubation period to Volume 9 of Selected Methods in Clin­ remove endogenous substrates reacting ical Chemistry entitled Selected Methods with NADH. for the Small Chemistry Laboratory includes the measurement of LD by the Lactate-to-Pyruvate Methodologies P —» L reaction in imidazole buffer at 37°C by the NADH to NAD+ change at The conditions for the primary meth­ 340 nm.59 This kinetic method is felt to ods for the continuously monitored rate be achievable in small laboratories by the (kinetic) measurement of LD in the lac- author and the editors. tate-to-pyruvate (L —* P) direction are Very recently, Vassault, Maire, Seville, summarized in table III. Wacker, MEASUREMENT OF TOTAL LACTATE DEHYDROGENASE ACTIVITY 17

TABLE I I I

Lactate Dehydrogenase Kinetic Assay Methods (Lactate-to-Pyruvate)

Buffer/conc. M e th o d * r (°c) (rnnol/1) (mmol/1) (imnol/1) pH

Wacker, Ulmen, and Vallee ( 1 9 5 6 ) 25 5 3 (DL) 5 P i t 50 8 . 8 Amador, Dorfman, and Wacker (1963)^ 25 7 7 (DL) 5 . 2 5 P P i t 50 8 . 6 King (1965)58 2 5 1 7 5 (DL) 1 Glycine 53 10.0 N a C l 53 M orgenstern, Flor, K essler, and Klein (1965)73'99 3 7 . 5 82 (DL) 7 . 5 AMP§ 1 8 8 9 . 0 ,4 2 Gay, McComb, and Bowers (1968)‘ 30 7 7 ..5 (DL) 5 . 2 5 P P i $ 50 8 . 5 5 a t 3 0 ° d f Demetriou, Drewes, and Gin (1974)' 30 9 0 (DL) 5 . 5 AMP§ 6 0 0 9 . 0 Buhl, Jackson, Lubinski, and Vanderlinde (1977)23 2 5 4 0 ( L i s a l t ) A 3 DEA o r 2 0 0 8 . 7 a t 2 5 °C 30 50 ( L i s a l t ) A 5 A M P d io l 2 0 0 8 . 7 a t 30 °C 37 70 ( L i s a l t ) * 7 Buffer 200 8.7 a t 3 7 ° C

*Sample volume fraction 1/15. fPhosphate buffer - Pi. ^Pyrophosphate buffer - PPi. §AMP is 2-am ino-2-m ethy1-1-propanol. IfUsed pH 8.75 in th eir study but found 8.55 optim al for a mixed isoenzyme sample. *L “ lactate.

Ulmen, and Vallee103 in 1956 were the photometric measuring system was not first group to measure LD kinetically in made available as part of Technicon the L —* P direction for clinical labora­ equipment until the 1970’s" A similar tory purposes. Subsequently, Amador, manual kinetic method utilizing AMP Dorfman, and Wacker3 found phosphate buffer has been recommended by Deme­ to be an inadequate buffer at a pH of triou, Drewes, and Gin in a well recog­ 8.8, substituted pyrophosphate, and nized textbook32 (table III). increased the lactate and NAD+ concen­ An early very complete investigation trations. These changes resulted in a of LD assay conditions was carried out markedly improved assay. King58 intro­ by Gay, McComb, and Bowers.42 It uti­ duced glycine with added sodium chlo­ lized [crudely] extracted preparations of ride at pH 10.0 as a buffer system. While human heart and liver LD for investiga­ widely used, especially in Great Britain, tion of the optimal conditions for the P the high pH is incompatible with the use —> L reaction in addition to the L —» P of NAD + , which decomposes at very assay. However, this carefully executed alkaline pH’s.31,38 In addition, Fendley, study did not include the evaluation of a Jacobs, Dunn, and Frings39 found the large number of buffers. The finalized King assay at pH 10 to underestimate optimal conditions for the L —» P reaction grossly LD 5 and have modified the pH by Gay, McComb, and Bowers42 are to 8.8, which alleviates both problems. minor modifications of the Amador et al While Morgenstern, Flor, Kessler, pyrophosphate method3 (see table III). and Klein73 in 1965 developed an L ^ P While they used pH 8.75 in their study, method for the Auto Analyzer* utiliz­ 8.55 was found optimal for a mixed isoen­ ing AMP (2-amino-methyl-l-propanol) zyme specimen. In addition, they advo­ buffer with both colorimetric and kinetic cated proper temperature control as well (340 nm) versions, the 340 nm spectro- as adjustment of the final pH of the buffer at the recommended assay tem­ * Technicon Instruments Corp., Tarrytown, NY. perature of 30°C. Both Amador et al3 and 18 VANDERLINDE

Gay, McComb, and Bowers42 initiated mechanisms of the LD reaction has been the reaction with enzyme whereas King’s ably reviewed by Everse and Kaplan.36 method58 for the L —» P assay specifies The lack of agreement in choice of initiation with NAD + . Buhl, Jackson, buffer, the finding of inhibition by the Lubinski, and Vanderlinde22 found pyrophosphate buffer, and the finding of human LD 1 and LD 5 activity, when discrepant results in assays of both puri­ assayed L —» P, is adversely affected if fied isoenzymes and sera under various the enzyme is preincubated with NAD + buffer conditions led Buhl et al21 to and the reaction is initiated with lactate. investigate seventeen buffers at a variety The amount of inactivation depends on of pH’s in the assay using highly purified the buffer used; activities are decreased human LD 1 and LD 5. The buffers more in sodium pyrophosphate than in selected included the four most com­ Tris. On the other hand, NAD+ has been monly used (table III): pyrophosphate reported to have a stabilizing effect on (PPi), Tris, 2-amino-2-methyl-l-propanol LD activity.8,33 Because this effect is (AMP) and glycine and an additional 13 more pronounced in phosphate buffers buffers with pKa values greater than 7.9. than in Tris buffer,63,106 it has been sug­ Of the four most commonly used buff­ gested that phosphate activates the ers, three proved unsatisfactory because NAD+ binding sites.63 of direct effects on LD. Glycine and PPi These observations and the fact that were found unsatisfactory because they the L —» P reaction is an ordered sequen­ inhibit LD activity with increasing con­ tial reaction, with NAD+ binding first,92 centration. With AMP the pH optimum should lead to enhanced LD activity for LD 1 is not compatible with the use when the enzyme is preincubated with of NAD + , and the pH optima for LD 1 NAD + . The contrary experimental find­ and LD 5 are vastly different. The con­ ings may be the result of inhibitors, centration of AMP also greatly affects which have been found in NAD+ prep­ total LD activity; hence large amounts of arations from various sources.4 Also, a AMP must be used to achieve maximal reduction of LD activity would be LD activity. Currently a concentration of expected if the inhibitor-enzyme disso­ 600 mmol/liter is used by Technicon" ciation constant is less than the NAD + - and recommended by Demetriou et al.32 enzyme dissociation constant.22 Buhl Additional disadvantages of AMP buffer interpreted the results to suggest also are that LD activity is dependent on that lactate affects enzyme binding of the method of preparation, the buffer has NAD + . Further, both LD 1 and 5 have an unpleasant odor, and a gas is visibly higher L —» P activity when assayed in released during pH adjustment. Tris than when assayed in sodium pyro­ Although ethanolamine and dimethlyam- phosphate buffer.22 Although the latter inoethanol (DAE) were satisfactory buff­ system contains lower concentrations of ers by some criteria, ethanolamine NAD+ and lactate, this probably does exerted a stimulating effect on LD activ­ not account for the lower activity ity and DAE showed potential undesir­ observed because the NAD+ and lactate able chemical reactions as well as an concentrations used are saturating.42 obnoxious odor. Buhl’s results demonstrate also that The three buffers Buhl et al21 found to buffer is an important factor in the LD have no detrimental effects on LD ac­ reaction and that the buffer may interact tivity are Tris, diethanolamine (DEA), with the enzyme and affect the binding and 2-amino-2-methyl 1,3-propanediol of the substrates. The very complex issue (AMPdiol). He felt Tris may not be com­ of substrates, inhibitors and reaction pletely satisfactory because its pH opti- MEASUREMENT OF TOTAL LACTATE DEHYDROGENASE ACTIVITY 19 mum for LD 1 is greater than 8.5, while formed with semicarbazide have met its Pk3 at 30°C is 8.0. Thus, Tris has lim­ with mixed success,93'94 probably ited buffering capacity at pH 8.5, the because of the complexity of the sub­ optimal reaction pH for mixtures of LD strate inter-relationships and biochemi­ 1 and LD 5. cals of the LD reaction.36 Of the 17 buffers examined, DEA and The apparent Km’s for lactate and AMPdiol at concentrations of 200 mmol NAD+ for nonhuman LD increase with per 1 and pH 8.7 were recommended as tem perature.43,49,77 Buhl’s findings that the buffers of choice for the measure­ increased amounts of lactate and NAD + ment of LD activity in the L —» P direc­ are required for saturation of human LD tion. However, DEA shows a significant 1 and LD 5 with increasing temperature cost advantage over AMPdiol. In a sub­ is consistent with the earlier observations sequent investigation Buhl, Jackson, on animal isoenzymes. Lubinski, and Vanderlinde23 established Reaction conditions have been estab­ the optimal substrate conditions for the lished which measure LD 1 and LD 5 assay of the human LD isoenzymes 1 and with equal efficiency. Assuming that the 5 L —» P in these two buffers at 25, 30, other three LD isoenzymes are mea­ and 37°C (table III). sured equally well, these conditions Choosing the optimal concentration of should be appropriate for determining lactate for each temperature was difficult total LD activity in normal and patho­ because excess lactate inhibits LD 1, logic sera, as well as individual isoen­ whereas LD 5 requires large amounts zyme determinations from column pro­ (relative to LD 1) of lactate for saturation. cedures. Krieg, Gorton, and Henry60 found crude Buhl recommended that all L —» P LD human extracts of LD 1 and LD 5 to measurements be made within 60 s after show more nearly the same velocity/sub- the reaction is initiated. If this is not fea­ strate concentration profiles at 37°C than sible because of delayed instrument at 25°C as observed earlier by Vesell.102 response time, the measurement should However, the source, purity and type of be made as quickly as possible. lactate were not stated, and Buhl et al22 He also recommended that L —» P have demonstrated the purity and form assays be performed at 25°C or 30°C of lactate to be extremely important. because at the lower temperatures the Hence, lithium-L( +)-lactate of high reaction response was linear for a longer purity was used by Buhl and his associ­ time, less reagents were needed, and ates in their subsequent investiga­ less interlaboratory and intralaboratory tions.21,23 The lack of linearity in product variation would be encountered on sam­ formation (constant AA per min) has sev­ ples measured with a variety of instru­ eral components, which have been ments and on different isoenzymic mix­ observed but not separated in previous tures having similar total LD activity. studies.68,93 The initial rise (<20 s), Buhl Although it would seem more precise suggested, is probably due to experimen­ to have higher assay values, his results tal design or deficiencies in instrument showed this not to be true. Because of response while the decrease after 20 to an appreciably different decrease in AA 30 s has more than one component, per min for LD 1 and LD 5 at 37°C but including substrate depletion, and prob­ not at 30°C or 25°C, it is more difficult ably involves product inhibition. Sub­ to measure each isoenzyme at 37°C with strate depletion can be overcome simply equal efficiency, and the difference in by adding additional substrate.68 How­ efficiency was observed to become ever, attempts to remove the product greater with time after the reaction is ini­ 20 VANDERLINDE tiated. The consequence is that any assay The relationship between activity and of an isoenymic mixture with elevated concentration of both soluble and immo­ LD activity will have a large interinstru­ bilized chicken LD 1 was determined ment variability. Also, more intrainstru­ using the rate of heat production in a ment variability would be encountered at batch-type calorimeter. Another novel 37°C than at 30°C or 25°C in samples approach to the measurement of LD with the same total LD activity but dif­ was made by Nikolelis, Painton, and fering isoenzymic mixtures. The single­ Mottola76 who found the air oxidation of instrument variation will be greatest for NADH, catalyzed by peroxidase, to pro­ instruments which measure LD activity vide a useful “indicator reaction” for the one min or more after initiation. determination of either serum LD or of Hence, Buhl23 recommended the mea­ NADH. They described application of suring time interval be standardized and this indicator reaction to repetitive assays of LD measured L —» P be mea­ determinations by sample injection into sured at 25°C or 30°C as they will show a continuously circulated reagent mix­ less variation attributable to instrument ture through the monitoring of oxygen and isoenzyme content. depletion with an amperometric sensor. Curry, Pardue, Mieling, and Santini30 have designed a filter fluorometer that Other Methodologies incorporates a photon-counting detector and evaluated its applicability in the While clinical laboratories utilize fluorometric determination of serum widely continously monitored rate reac­ LD. While reflectance spectroscopy has tions at 340 nm involving the absorbancy not been widely used in the clinical lab­ change in NAD+—NADH, with lactate oratory, Zipp, Watson, and Greyson110 or pyruvate as substrate, other method­ have developed a solid state matrix sys­ ological approaches for the measurement tem for the determination of LD in of LD have been advanced. Thus, Bab- serum in which the quantitation is car­ son and Babson5 described in 1973 a ried out by reflectance spectroscopy. kinetic colorimetric assay procedure in They claim that this approach can be which the reduction of NAD + is coupled used to measure total LD in serum with to the reduction of a tetrazolium salt, the convenience and simplicity of a test 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl strip without compromising the quanti­ tetrazolium chloride (INT), with phen- tative nature of present wet methods. In azine methosulfate serving as an inter­ addition, the dry film based technology mediate electron carrier. The kinetic col­ of the Eastman Kodak Company’s new orimetric method correlated well with Ektachem 400 System utilizes the prin­ 340 nm kinetic methods but did not ciple of reflectance spectroscopy,96 which require an ultraviolet spectrophotome­ is well established in the photographic ter. A similar rapid single step kinetic field. However, while their amylase colorimetric assay for LD in serum was methodology is available, other enzymes developed by Allain, Henson, Nadel, including LD have just been released and Knoblesdorff2 but used diaphorase recently. as the intermediate electron carrier. A vidicon spectrophotometric ap­ The feasibility of microcalorimetry for proach to the measurement simulta­ the determination of the kinetics of neously of two enzymes, exemplified by enzyme catalyzed reactions, specifically serum LD and alkaline phosphatase (EC LD and uricase, was investigated by 3.1.3.1), has been demonstrated by Rehak, Everse, Kaplan, and Berger.82 Milano and Pardue72 to give enzyme MEASUREMENT OF TOTAL LACTATE DEHYDROGENASE ACTIVITY 21 activities equivalent to those in common of inhibitors in NADH and for monitor­ use. ing its quality and described specifica­ tions for both reference and commercial NADH and NAD+ Inhibitors preparations. The U.S. National Bureau of Standards group, under Margolis and As early as 1961, the Kaplan group38 Schaffer, has spent several years identi­ recognized the presence of an inhibitor fying impurities in NADH and develop­ in NADH which can strongly inhibit LD; ing specifications for reference quality some clinical chemists were aware of the NADH. The molar absorbtivity of high problem52,97 in the middle sixties. In purity NADH was evaluated in 1976 by 1968 McComb and Gay67 published their McComb et al66 in the United States findings on four commercial sources of and by Ziegenhorn et al109 in Europe. NADH. Subsequently, Berry, Lott, and McComb’s group61 as well as the NBS Grannis12 showed lot to lot variation in group55,64,65 studied the formation and the amount of the inhibitor and sug­ properties of the LD inhibitors in NADH gested a procedure for assessing the by column chromatography and/or high quality of commercial NADH prepara­ performance liquid chromatography tions. However, the problem was not (HPLC). Loshon et al61 described at least unique to NADH and the pyruvate-to- two inhibitory components which can lactate continuously monitored rate reac­ form in concentrated NADH solutions, tion as Dalziel31 in 1963 had demon­ only one of which can be separated on strated NAD+ to contain nucleotide DEAE-cellulose or DEAE-Sephadex. impurities which interfered with the lac- The second inhibitor, which separated tate-to-pyruvate reaction. Babson and on the trailing edge of the NADH peak Arndt4 confirmed Dalziel’s observation in chromatography on DEAE-cellulose, by showing that commercial preparations was resolved from the NADH by Shaf­ of NAD+ contained various amounts of fer’s group by HPLC on u Bondapak C18. one or more LD inhibitors but not the Because this new inhibitor had ultravi­ inhibitor described for NADH. Thus, in olet properties similar to those of the early seventies, the quality, storage, NADH, a ratio of absorbance at 260/340 and refreezing characteristics of NADH nm of less than 2.32 could not be used and/or NAD + , utilized in clinical labo­ as an indication of an inhibitor free prep­ ratory kinetic measurements of LD in aration and HPLC chromatography was either direction, became of concern. deemed necessary to ensure its absence This concern stimulated efforts in several in preparations of NADH used for clini­ additional fronts: namely, by the profes­ cal assay.64 While Loshon et al61 showed sional societies, by the National Bureau the presence of NAD+ to influence the of Standards, and by the manufacturers, rate of inhibitor production from unbuff­ among others. ered aqueous NADH solutions, no Since adenosine diphosphoribose inhibitor was produced by NAD+ incu­ (ADPR), adenosine diphosphate (ADP), bated alone under the same conditions. and adenosine monophosphate (AMP) However, Gallati40,41 has prepared an are poor inhibitors for the lactate dehy­ inhibitor of LD by the treatment of drogenases,45 the inhibitors were pre­ NAD+ and phosphate at alkaline pH and sumably entirely different structures. Johnson and Morrison as early as 1970 Gerhardt et al44 under the auspices of postulated NAD to undergo ring opening the Scandanavian Society for Clinical 'of the nicotinamide ring in strongly alka­ Chemistry and Clinical Physiology eval­ line solutions.57 uated practical methods for the detection More recently, Guilbert and Johnson48 22 VANDERLINDE have demonstrated the pseudobase fredsen and Ottesen46 showed NADH hydroxide ring addition adduct of under humid conditions to form 1,6-di- NAD + , i|/NAD-OH, to be reversibly hydro-NAD, a potent inhibitor of LD formed and, subsequently, to undergo with a Kj of 2.5 x 10_7M. They report ring opening at the number six carbon of their 1,6-dihydro-NAD inhibitor to have the nicotinamide ring to form open ring spectroscopic properties at 340 nm sim­ NAD designated as ONAD. ONAD has ilar to those of the dimer of NAD + iden­ pKa values at 1.9 and 11.2 and absorbs tified by Biellmann14 and present evi­ maximally at 350 nm in its acidic form, dence to support Biellmann’s compound at 372 nm in its neutral form, and at 340 most likely being 1,6-dihydro-NAD. nm in its anionic form. The hydrolysis of Furthermore, Godtfredsen et al47 ONAD leads to the formation of 2-car- present evidence to show that this com­ boxyamideglutacondialdehyde and aden­ pound is probably the major inhibitor osine diphosphate ribosylamine with the formed in moist NADH at pH 8.8 and former breaking down further to the base have presented experimental data to sup­ fluorescent compound, 2-hydroxynico- port their hypothesis46 that the 1,6-di­ tinaldehyde. Thus, NAD+ in alkaline hydro-NAD is generated in NADH prep­ solution can form at least 5 intermediate arations by a bimolecular reaction end products. More recently, a cooper­ between NADH and the small quantities ative group formed between the Louis of NAD+ which are usually present in Pasteur University at Strasbourg and samples of NADH. One major implica­ Boehringer Mannheim GmbH13 and a tion of this finding is that no or very little second group in Copenhagen under inhibitor formation should take place in Godtfredsen46’47 have made great strides NADH preparations completely free of in the structural elucidation and inter­ NAD + . Thus it would seem fairly well relationships between the various LD established that the major two inhibitors inhibitors occurring in both NADH and in N A D H are: (a) l,6 -d ih y d ro -N A D + NAD + . Biellmann et al13 found NADH which absorbs at 340 nm similar to on storage in a moist atmosphere to NADH and cochromatographs on DEAE result in three inhibitory compounds, cellulose or Sephadex with NADH and only two of which were inhibitors. The (b) 4-phosphoryloxy-1,4-dihydro-NAD +, structure of one inhibitor was not eluci­ a phosphate adduct of NAD+ . dated owing to its instability; another Also, the pH of the preparation and inhibitor was demonstrated not to origi­ the nature of the inorganic salts present, nate directly from NADH but from con­ particularly phosphate, alter which taminating NAD + . It was shown to be inhibitors occur in various NADH and phosphate-NAD+ adduct formed in NAD+ preparations. small amounts by the addition of a phos­ Wenz et al104 showed in 1976 various phate ion to the 4-position of the nicotin- commercial preparations of NADH to amidinium ring of the contaminating yield up to 12 different compounds NAD + . This 4-phosphoryloxy-l, 4-dihy- which can act as LD inhibitors. All man­ dro-NAD adduct was shown to be a ufacturers have been aware of the in­ potent competitive inhibitor of LD with hibitor problem with NAD+ and partic­ an inhibitor constant (Kj) of 2 x 10_7M ularly NADH for many years. Conse­ and to be identical with the inhibitor pre­ quently, many of the manufacturers offer pared by Gallati40,41 from NAD+ plus good routine service quality NADH and phosphate in alkaline medium. The third NAD+ preparations as well as chroma- inhibitor product isolated from NADH topure or ultrapure preparations of both was identified by Biellmann et al13 as a NADH and NAD+ at 50 to 100 percent dimer of NAD + . Subsequently, Godt- increased cost. Buhl, Jackson, and MEASUREMENT OF TOTAL LACTATE DEHYDROGENASE ACTIVITY 23

GrafFunder20 obtained about 10 percent shift from end-point to kinetic assays by higher activities with the pure human many smaller laboratories. LD 1 and LD 5 isoenzymes with a more The College of American Pathologists pure commercial B-NADH preparation (CAP) Enzyme Chemistry Program but found no difference in substrate employs sets of linearly related enzyme requirements. However, equally impor­ specimens including LD which are sent tant are the numerous factors such as the to participants quarterly.26 The 1982 humidity and storage conditions, freez­ Enzyme Survey V-A Summary Report ing and thawing of solutions, the nature provides valuable participant information of the contaminants, which salts are pres­ on the instruments/reagents and number ent, and pH of the preparation which of laboratories utilizing each methodol­ effect the stability of NADH and NAD + ogy which are summarized in table IV. maintained within the user’s labora­ All methods utilized by ten or more lab­ tory.36,47 Furthermore, research and oratories involve the L —» P reaction, and standardization involving dehydrogen­ only a minor number of participants use ases such as LD and alcohol dehydrogen­ P —» L procedures. ase (EC 1.1.1.1) measurements which Howell, McCune, and Shaffer56 in are especially sensitive to inhibitors may 1979 re-examined the L —> P and P —*■ L require highly purified NAD+ and assays for LD and state, that the P —> L NADH sources whereas the less expen­ assay can now yield linearity equal to or sive commercial preparations may suffice better than that obtained by the L P for routine clinical laboratory measure­ assay. In addition, they state there are ments. significant advantages to the P —* L reac­ tion, namely: (a) a greater change in absorbance per unit of time, which Choice of Methodologies

In recent years, laboratories in the TABLE IV United States have increasingly shifted Current LD Enzyme Methodologies* from colorimetric end-point enzyme measurements to continuously moni­ Number o f tored rate (kinetic) reactions and to the I n s tr u m e n t Reagents./ Laboratories f measurement of LD by the L —» P rather Abbott ABA-100 A b b o tt 36 than the P —» L reaction. For example, A b b o tt VP A b b o tt 45 the clinical laboratories in New York American M onitor KDA American Monitor 64 D uP ont ACA D uP ont 272 State, outside of New York City, ENA G em ini Electro-Nucleonics 25 ENI Gemsaec Smith-Kline 11 increased their utilization of kinetic rate Gilford-Various Systems W orthington 33 methods for LD from 21.7 percent of all Gilford-Various Systems G i l f o r d 15 Hycel Super or Mark 17 H y c e l 11 laboratories in 1971 to 61.0 percent in Technicon SMA 12/60 Fisher Scientific 10 Technicon SMA 12/60 Technicon 11 1975.18 In addition, the percentage uti­ Technicon SMA 12/60 W orthington 10 lizing an L —* P method instead of P —> Technicon SMAC Technicon 100 Technicon SMA II T e c h n ic o n 35 L jumped from 43.4 percent in 1971 to Centrifi-Chem (Baker) Baker Diagnostics 19 81.0 percent in 1975. The major reason Centrifi-Chem (Baker) Union Carbide 20 Chemetrics II Worthington 11 for this shift was the increased use of automated enzyme analyzers utilizing *Data taken from College of American Pathologists the L -* P reaction at 340 Yim, especially Enzyme Survey V-A Summary Report of June 1982, by permission and courtesy of the College of continuous flow 340 nm based kinetic American Pathologists, flnstrum ents/reagents shown only for 10 or more analyzers.* A secondary reason was the participating laboratories. All involve L—^P methods but utilize varying buffers (PPi, Tris, AMP), tem peratures (25°C , 30°C, 37°C) and * Technicon Instruments Co., Tarrytown, NY. substrate concentrations. 24 VANDERLINDE

itors.” A current review of representative allows a more accurate spectrophoto- biochemical reagent catalogs showed metric readout; (b) lower reactant con­ many of the manufacturers to offer good centrations which thereby reduce costs; routine service laboratory quality NADH (c) solid reagents are used to prepare the and NAD+ preparations as well as chro- assay solution; and (d) the reagent solu­ matopure or ultrapure preparations of tions are more stable. They point out that NADH and NAD+ at 50 to 100 percent inhibitors in commercial NADH prepa­ increased cost (See section on NADH rations may substantially effect LD activ­ and NAD+ inhibitors). ities measured P —» L and state that a It is not possible to assess at this time Standard Reference Material for NADH new findings such as the Société Fran­ is being developed for issuance by the çaise de Biologie Clinique recommenda­ NBS. However, for a number of reasons tion101 of a new more linear L —» P assay this project has been postponed.80 (see L —» P section). On the other hand, Buhl and Jack­ son,19 who have compared serum LD Sources of variation catalysis under optimal conditions devel­ oped for the pure human isoenzymes in P r e f e r r e d B l o o d S a m p l e both the L —» P and P —» L reactions at 25°C, 30°C, and 37°C, found the results Serum is the preferred sample for all from the L —» P reaction to be more reli­ enzyme activity measurements including able for interinstrument and interlabo­ LD. Haymond and Knight50 found the ratory comparisons. Interconversions of LD activities of capillary serum and cap­ results between the bidirection reactions illary plasma were significantly greater were not practicable. Measurements of than in simultaneously assayed venous LD in either direction at 25°C, 30°C, or serum, the greatest difference being 37°C were stated to be equally reliable if between capillary and venous serum. the volume fraction and the resulting AA Although some difference is attributable per min is small. However, they recom­ to tissue fluid, most of the difference was mended that the P —» L reaction be mea­ attributed to platelets. They recommend sured as soon as possible after start of the that when capillary blood is to be used reaction, the measuring interval be rea­ for enzyme assay, it should be processed sonably short (<10 s) and that the L —> as plasma. Subsequently, Tothwell, Jen- P reaction be measured within the first drzejczak, Becker, and Doumas88 inves­ 40 s. Furthermore, they suggested that tigated the LD activity of venous serum any future reference method or selected versus venous plasma and demonstrated method include a recommendation for platelets to be the source of up to a four the time and duration of the measuring fold increase in plasma LD. More re­ interval after the reaction is initiated. cently, Bais, Prior, and Edwards7 have While they found neither the P —» L nor shown platelets to increase LD as mea­ the L —» P response to be linear, they sured by the SMAC continuous flow ana­ report that at equal AA per min the activ­ lyzer* and the detergent in the detection ity of the L —* P reaction does not buffer to further enhance the effect. decrease as rapidly and is therefore more Hence, if plasma must be used as the reliable. Buhl and Jackson19 have source of specimen, the sample must be reported, “Some available preparations centrifuged such as to provide a platelet- and improperly stored 3-NADH contain free plasma. inhibitors of LD, whereas many (3- NAD+ preparations are free of LD inhib­ * Technicon Instruments Corp., Tarrytown, NY. MEASUREMENT OF TOTAL LACTATE DEHYDROGENASE ACTIVITY 25

Various anticoagulants such as oxalate protein-bound sulphydryl groups are and fluoride are inhibitory to LD while responsible. Recently, Bails and Ed­ heparin was reported by Batsakis and wards6 have shown that alcohol dehydro­ Henry to be erratic.9 More recently, genase can be present in pathological Robertson, Chesler, and Elin87 found sera, can be identified as a sixth LD serum collected with the antiglycolytic band, and shows considerable activity at agent, lithium iodoacetate to be unsuit­ pH 9 in buffers used to measure LD in able for the measurement of LD. the L —» P direction including DEA, AMPdiol, and AMP.

H e m o l y s i s Howell54 has shown that Zn+ + can be an inhibitor for the LD catalyzed reduc­ The high intra-erythrocytic concen­ tion of pyruvate-to-lactate at even the tration of LD 1 isoenzyme requires rapid upper normal limit of serum Zn++, but separation of serum from the clot.107 The inhibition is greatly reduced by pyruvate calculated error per gram of hemoglobin concentrations in excess of 1 mmol per 1 for LD activity is 463 percent, while the which is usual in the P —» L assay. observed error per gram of hemoglobin The diuretic, triamterene (2,4,7-tri- was 550 percent.17 Hence, hemolysis in amino-6-phenyIpteridine), has been sera for LD measurements must strin­ found to induce spurious elevations in gently be avoided. serum LD as measured by an automated fluorometric assay but not spectropho-

S t a b il it y o f S e r u m tometrically.27 A serum inactivator to the M subunit Schmidt and Schmidt91 report the of LD isoenzymes which migrated elec- average loss of serum LD activity on stor­ trophoretically between the beta and age at 4°C to be 0 percent at 24 hours, gamma globulins was reported by Naga- four percent at 48 hours, eight percent mine.74 Also, Acheampong-Mensah1 has at three days, nine percent at five days reported a persistent increase in serum and 12 percent for seven days, whereas LD activity to be related to an enzyme- at 25°C the corresponding losses are 0, IgG-Lambda immunoglobin complex. 1, 2, 10, and 15 percent, respectively. Several LD-immunoglobulin complexes However, because LD 1 is relatively sta­ in human serum have been described ble whereas LD 5 is thermolabile, sta­ previously by Biewanga and Feltkamp.14 bility may vary with the isoenzyme com­ position of individual sera. In general, S o u r c e s o f E r r o r freezing is to be avoided. The sources of error in measuring LD

A c t iv a t o r s , I n h ib it o r s are failure to maintain the reaction at a n d I nterferences constant temperature, use of poor quality reagents, e.g., NADH or NAD + , hemo- The “nothing-dehydrogenase” reac­ lyzed samples, and deviation from the tion while mainly associated as an artifact prescribed procedure. Increasing or in serum isoenzyme analysis based on decreasing the temperature will change tetrazolium methods may occur also in the LD result approximately seven per­ the serum where its origin is unknown.95 cent per degree.51 Various sources of Sometimes it is said to represent LD NADH and NAD+ contain inhibitors of activity but since a correction with serum the LD reaction4'12> 13’40’41'44_48’52'55’57’61’64_ proteins and particularly their sulphydryl 67,97,104,109 an(j d ifferen t com m ercial groups can be made, Somer believes that sources of lactic acid also affect the 26 VANDERLINDE amount of LD measured.22 Erythrocytes buffer, measurement at 37°C, and a contain large amounts of LD, and any pyruvate substrate concentration two hemolysis will give abnormally high fold that which was recommended by the results.17 Changes in the procedure — German Society for 25°C. such as substitution of buffer, change of While the British Association of Clin­ pH, or change in the order of addition of ical Biochemistry-Working Party has reagents to the reaction mixture— will made method recommendations for also alter the amount of LD mea­ three enzymes, LD was not included.105 sured.20’21’22’23 Very recently, the Société Française de Biologie Clinique (S.F.B.C.) has de­

R e f e r e n c e R a n g e s scribed a P —> L method for total LD at 30° with the goal of measuring each Laboratories using different reagents isoenzyme with optimal conditions.101 and/or different procedures will have dif­ They have found that the addition of Na+ ferent normal ranges and should use val­ and Cl" ions to the reaction mixture ues established preferably by their own modifies the stability of the ternary com­ laboratory35 or as documented by the plex between enzyme, coenzyme, and manufacturer of their kit or reagent substrate which usually inhibits the P —*■ instrumental system. However, it is not L reaction. They report the method to widely recognized that pediatric LD val­ show the following advantages: minimi­ ues decrease with maturity28 and older zation of the differences in the optimal individuals show significantly higher LD concentration of pyruvate required for activities.71 LD 1 and LD 5, improved linearity, and an increase in the upper limits of linear­ LD Standardization Efforts ity. The 1982 College of American Pathol­ Most clinical chemists accept the need ogists (CAP) Conference on Analytical for national and international standard­ Goals in Clinical Enzymology at Aspen, ization of the conditions under which Colorado included a paper on “The Mea­ enzymes are measured. surement of Lactic Dehydrogenase” by The German Society for Clinical Dr. Robert McComb. Papers were pre­ Chemistry was the first national society sented for discussion emphasizing all to put forth a recommendation for the aspects of the analytical process in clini­ standardization of a method for the esti­ cal enzymology, including reagent qual­ mation of serum lactate dehydrogen­ ity, recommended methods for measur­ ase.34 Their “standard method” recom­ ing various enzymes, quality control, and mended in 1972 is a modification of the the relationship of analytical process to Wroblewski and LaDue107 P —> L m ethod the clinical usefulness of enzyme data.53 carried out at 25°C. They also included The Expert Panel on Enzymes of the a “standard method” for a-HBDH activ­ International Federation of Clinical ity. Chemistry (IFCC) has made Provisional The Committee on Enzymes of the Recommendation (1974) on “IFCC Scandinavian Society for Clinical Chem­ Methods for the Measurement of Cata­ istry and Clinical Physiology published lytic Concentration of Enzymes,”16 “Part Recommended Methods for the Deter­ 2: Aspartate aminotransferase. Measure­ mination of Four Enzymes in Blood ment of the catalytic concentration in which included LD in the P —» L direc­ human serum”,10 and “Part 3: Revised tion.29 However, the Scandinavian Soci­ IFCC method for aspartate aminotrans­ ety opted for Tris instead of phosphate ferase (AST)”.11 However, no information MEASUREMENT OF TOTAL LACTATE DEHYDROGENASE ACTIVITY 27 has been requested or issued for lactate T-Tentative Guidelines to Kinetic Anal­ dehydrogenase. ysis of Enzyme Reactions.”75 While sev­ The National Bureau of Standards in eral scientists and organizations have February of 1982 issued Standard Ref­ suggested requirements for enzyme ref­ erence Material (SRM) 909, Human erence materials,37,84 to date only a mate­ Serum (Enzymes Values Determined).79 rial for aspartate aminotransferase (E.C. SRM 909, a Human Serum Standard Ref­ 2.6.1.1) has been prepared, character­ erence Material, has been available for ized and validated25’83,85’89 as a proposed use in assessing the accuracy of clinical biological validation and transfer material methods for six analytes: namely, glu­ (BVTM).90 The availability of a BVTM for cose, calcium, chloride, lithium, potas­ aspartate aminotransferase in conjunc­ sium, and uric acid, for calibrating instru­ tion with the IFCC proposed reference mentation used for these analytes, and method for this enzyme11 provides a for validating in-house or commercially model system for the standardization of produced quality control materials. The other enzymes of clinical laboratory revised certificate now provides uncer­ interest including LD. Some of the tified values, for information only, of the developmental work for an analogous catalytic concentrations of seven material for lactate dehydrogenase has enzymes including lactate dehydrogen­ been accomplished in that a highly puri­ ase. The enzymes were determined fied LD 1 has been used as a reference cooperatively by teams of experts using material in the New York State Profi­ the “best available” methodology. Where ciency Testing Program.18 Also, the inter­ possible, methods of the IFCC or the converting of results for serum samples American Association for Clinical Chem­ and for highly purified LD isoenzymes 1 istry were used. The certificate provides and 5 assayed L —» P in pyrophosphate, outlines of the methods and the final Tris and AMP has been investigated.24 assay reaction conditions, and gives lit­ erature references. The Bowers15 proce­ Acknowledgments dure for P -» L was utilized except for The author wishes to thank Dr. Robert McComb temperature where a reaction tempera­ of the Hartford Hospital, Hartford, CT for reviewing ture of 29.77°C was used, verified by a the manuscript and making some helpful suggestions. Gallium Melting Point Cell, SRM 1968. Sodium pyruvate is available also from References the National Bureau of Standards as SRM 910. 1. A c h e a m p o n c -M e n s a h , D.: Persistent increase of serum lactate dehydrogenase activity related A hierarchy of definitive method, ref­ to enzyme-IgG (lambda) immunoglobulin com­ erence method, and Standard Reference plex. Clin. Chem. 26:789-790, 1980. Material (SRM) can be developed to pro­ 2 . A l l a i n , C. C., H e n s o n , C. P., N a d e l , M. K . , and K noblesdorff , A . F.: Rapid single-step vide an accuracy base for many of the kinetic colorimetric assay for lactate dehydro­ inorganic, and biochemical analytes in genase in serum. Clin. Chem. 19:2 2 3 - 2 2 7 , human serum measured routinely by 1 9 7 3 . 3 . A m a d o r , E., D o r f m a n , L. E., and W a c k e r , clinical laboratories.78,81 In the area of W. E.: Serum lactic dehydrogenase activity: An clinical enzymology, standardization analytical assessment of current assays. Clin. efforts have centered primarily on the Chem. 9:391-399, 1963. 4. B a b s o n , A . L. and A r n d t , E. G.: Lactic dehy­ development of reference and selected drogenase inhibitors in NAD. Clin. Chem. methods rather than on enzyme materi­ 16:254-255, 1970. als. However, the National Committee 5. B a b s o n , A. L. and B a b s o n , S. R .: Kinetic col­ orimetric measurement of serum lactate dehy­ on Clinical Laboratory Standards has drogenase activity. Clin. Chem. i9:766-769, recently published a report entitled “C7- 1973. 28 VANDERLINDE

6. B a i l s , R . and E d w a r d s , J. B . : Alcohol dehy­ tate at 25, 30 and 37°C. Clin. Chem. 24:2 6 1 - drogenase interference in lactate dehydrogen­ 266, 1978. ase assay. Clin. Chem. 26:525-526, 1980. 21. B u h l , S. N ., J a c k s o n , K . Y., L u b i n s k i , R ., and 7. B a i l s , R., P r i o r , M. P., and E d w a r d s , J. B . : V a n d e r l i n d e , R. E.: A search for the best Plasma lactate dehydrogenase activities will be buffer to assay human lactate dehydrogenase increased if detergent and platelets are present. lactate-to-pyruvate. Clin. Chem. 22:1872— Clin. Chem. 23:1056-1058, 1977. 1875, 1976. 8. B a r t h a l m e s , P., D u r c h s c h l a g , H ., and J a e n - 22. Buhl, S. N., Jackson, K . Y., Lubinski, R., and i c k e , R.: Molecular properties of lactic dehy­ V a n d e r l i n d e , R. E. : Effects of reaction initiator drogenase under the conditions of the enzy­ on human lactate dehydrogenase assay. Clin. matic test. Eur. J. Biochem. 39:101-108, 1973. Chem. 22:1098-1099, 1976. 9. B a t s a k i s , J. G . and H e n r y , J. B . : Lactic dehy­ 23. B u h l , S. N ., J a c k s o n , K . Y., L u b i n s k i , R ., and drogenase. ASCP Check Sample Program, V a n d e r l i n d e , R. E.: Optimal Conditions for Clinical Chemistry No. CC-80, 20 pgs., 1973. assaying human lactate dehydrogenase by the 10. B e r g m e y e r , H . U ., H o r d e r , M., and Moss, lactate-to-pyruvate reaction: Arrhenius relation­ D. W.: Provisional recommendations on IFCC ships for lactate dehydrogenase isoenzymes 1 methods for measurement of catalytic concen­ and 5. Clin. Chem. 23:1289-1295, 1977. trations of enzymes. Part 2. IFCC method for 24. B u h l , S. N ., R i c h a r d s , A. H . , J a c k s o n , K . Y., aspartate aminotransferase. Clin. Chem. L u b i n s k i , R . , and V a n d e r l i n d e , R . E.: Inter­ 23:887-893, 1977. converting measurements of human lactate 11. B e r g m e y e r , H . U ., H o r d e r , M., and Moss, dehydrogenase activities in three buffers. Clin. D. W.: Provisional recommendations on IFCC Chem. 23:200-204, 1977. methods for measurement of catalytic concen­ 25. B u r t i s , C. A., S a m p s o n , E. J., B a y s e , D. D ., trations of enzymes. Part 3. Revised IFCC M c K n e a l l y , S . S . , and W h i t n e r , V. S.: An method for aspartate aminotransferase. Clin. interlaboratory study of measurement of aspar­ Chim. Acta 80:F21-F22, 1977. tate aminotransferase activity with use of puri­ 12. B e r r y , A. J., Loir, J. A., and G r a n n i s , G . F.: fied enzyme materials. Clin. Chem. 24:916— NADH preparations as they effect reliability of 926, 1978. serum lactate dehydrogenase determinations. 26. Cap Enzyme Chemistry Program for 1982. Col­ Clin. Chem. 79:1255-1258, 1973. lege of American Pathologists, 7400 Skokie 13. B i e l l m a n n , J. F., L a p i n t e , C., H a i d , E ., and Blvd., Skokie, IL 60077. W e i s m a n n , G.: Structure of lactate dehydro- 27. C h a i n u v a t i , T., H a r i n a s u t a , U ., and Z i m m e r ­ gense inhibitor generated from coenzyme. Bio­ m a n , H . J . : Spurious elevation of apparent lac­ chemistry 18:1212—1217, 1979. tate dehydrogenase activity caused by triamter­ 14. B i e w e n g a , J. and F e l t k a m p , T. E. W .: Lactate ene. Clin. Chem. 79:1202-1204, 1973. dehydrogenase (LDH)-IgG immunoglobulin 28. C h e n g , M. H ., L i p s e y , A. I., B l a n c o , V ., complexes in human serum. Clin. Chim. Acta W o n g , H. T., and S p i r o , S . H.: Microchemical 64:101-106, 1975. analysis for 13 constituents of plasma from 15. B o w e r s , G. N.: Lactic dehydrogenase. Std. healthy children. Clin. Chem. 25:692—698, Meth. Clin. Chem. 4:163-172, 1963. 1979. 16. B o w e r s , G. N ., B e r g m e y e r , H. U., and Moss, 29. Committee on Enzymes of the Scandinavian D. W.: Provisional recommendation (1974) on Society for Clinical Chemistry and Clinical IFCC methods for the measurement of catalytic Physiology: Recommended methods for the concentration of enzymes. Part 1. General con­ determination of four enzymes in blood. Scand. siderations. Clin. Chim. Acta 65:F11-F24, J. Clin. Lab. Invest. 33:291-306, 1974. 1974. 30. C u r r y , R. E., P a r d u e , H . L . , M i e l i n g , G . E., 17. B r y d o n , W. G. and R o b e r t s , L. B . : The effect and S a n t i n i , R. E.: Design and evaluation of of haemolysis on the determination of plasma filter fluorometer that incorporates a photon- constituents. Clin. Chim. Acta 47:435—438, counting detector. Clin. Chem. 19:1259-1264, 1972. 1973. 18. B u h l , S. N ., C o p e l a n d , W. H ., F a s c e , C . F ., 31. D a l z i e l , K . : The purification of nicotinamide K o w a l s k i , P., R i c h a r d s , A. H., and V a n d e r - adenine dinucleotide and the kinetic effects of l i n d e , R . E.: Proficiency testing: Purification of nucleotide impurities. J. Biol. Chem. lactate dehydrogenase 1 and results of its use as 238:1538-1543, 1963. a reference material in the New York State Pro­ 32. D e m e t r i o u s , J. A., D r e w e s , P. A., and G i n , gram. Clin. Chem. 23:1000-1011, 1977. J. B . : Enzymes. Determination of lactic dehy­ 19. B u h l , S. N. and J a c k s o n , K . Y.: Optimal con­ drogenase. Enzymes in Clinical Chemistry — ditions and comparison of lactate dehydrogen­ Principles and Techniques. Henry, R. J., Can­ ase catalysis of the lactate-to-pyruvate and pyru- non, D. C., and Winkelman, J. W., eds. Hag­ vate-to-lactate reactions in human serum at 25, erstown, MD, Harper and Row, 1974, p. 824. 30, and 37°C. Clin. Chem. 24:828-831, 1978. 33. D e S a b a t o , G. and K a p l a n , N. O.: The déna­ 20. B u h l , S. N ., J a c k s o n , K . Y., and G r a f f u n d e r , turation of lactic dehydrogenase. J. Biol. Chem. 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