19
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 Dehydrogenases is the preferred name for the group of enzymes which belong to the oxidoreductase class of enzymes in which the substrate oxidized is regarded as a hydrogen donor and the usual accep- tor is nicotinamide-adenine dinucleotide (NAD +) or nicotinamide-adenine dinu- clesotide phosphate (NADP + ). Lactate dehydrogenase (LD: EC 1.1.1.27) (table I) is the most important dehydrogenase from the clinical viewpoint. Others mea- sured much less frequently include mal- ate dehydrogenase (MD), glutamate dehydrogenase (GLDH), isocitrate dehy- drogenase (ICD), and sorbitol dehydro- genase (SDH). Lactate dehydrogenase (LD) has a molecular weight of about 140,000 dal- tons and is cytoplasmic in its cellular location.36 It is composed of four peptide chains of two types, H and/or M sub- units, thereby creating isoenzymes. LD 1, the heart type isoenzyme, consists of four H subunits, whereas LD 5, the mus- cle/liver type isoenzyme, consists of four M subunits with the intermediary forms being combinations of subunits. Lactate dehydrogenase catalyzes the reversible reaction: L-lactate + NAD+ ^ pyruvate + NADH. The bidirectional reaction is monitored spectrophotometrically by measuring 13 0091-7370/85/0100-0013 $02.00 © Institute for Clinical Science, Inc.

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A N NALS O F CLINICA L A N D LABORATORY SC IE N C E , VOL. 15, NO. 1 Copyright © 1985, Institute for Clinical Science, Inc.

M easurem ent o f Total Lactate Dehydrogenase Activity

RAYMOND E. VA NDERLINDE, Ph.D .

Departments of Pathology, Laboratory Medicine, ir Biological Chemistry,Hahnemann University School o f Medicine

Philadelphia, PA 19102

ABSTRACT

Lactate dehydrogenase (LD: EC 1.1.1.27) is the most im portant clini­cally of several dehydrogenases occurring in human serum. Lactate dehy­drogenase is cytoplasmic in its cellular location and in any one tissue is composed of one or two of five possible isoenzymes. W hile many of its clinical applications involve quantification of one or more specific serum isoenzymes, an estim ate of total LD is required usually.

Lactate dehydrogenase catalyzes the reversible reaction: L-lactate + N A D + pyruvate + NADH. The bidirectional reaction is m onitored spectrophotom etrically by m easuring 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 m easurem ent of the reaction system in both directions are comprehensively reviewed as well as the standardization efforts proposed to date.

Introduction

Dehydrogenases is the preferred name for the group of enzymes which belong to the oxidoreductase class of enzymes in which the substrate oxidized is regarded as a hydrogen donor and the usual accep­tor is nicotinam ide-adenine dinucleotide (NAD + ) or n ico tinam ide-adenine dinu- clesotide p hospha te (N A D P + ). L acta te dehydrogenase (LD: EC 1.1.1.27) (table I) is the most im portant dehydrogenase from the clinical viewpoint. O thers m ea­sured much less frequently include mal- a te dehy d ro g en ase (M D), g lu tam ate dehydrogenase (GLDH), isocitrate dehy­drogenase (ICD), and sorbitol dehydro­genase (SDH).

L acta te dehy d ro g en ase (LD) has a molecular weight of about 140,000 dal- tons and is cy top lasm ic in its ce llu la r location.36 It is composed of four peptide chains of two types, H and /o r M su b ­units, thereby creating isoenzymes. LD1, the heart type isoenzyme, consists of four H subunits, whereas LD 5, the m us­cle/liver type isoenzyme, consists of four M subunits with the interm ediary forms being combinations of subunits.

Lactate dehydrogenase catalyzes the reversible reaction:

L-lactate + N A D + ^ pyruvate + NADH.

The bidirectional reaction is m onitored sp ec tro p h o to m etrica lly by m easu ring

130091-7370/85/0100-0013 $02.00 © Institute for Clinical Science, Inc.

14 VANDERLINDE

pyruvate and react with o ther substrates. O ne of th ese su b s tra te s , 2 -oxobutyric acid, has been used “preferentially” to d e te rm in e serum L D 1 (heart-type) activity. T he reac tion , know n as B- hydroxybutyric dehydrogenase (HBDH), involves: 2-oxobutyric acid + NADH ^ 2-hydroxybutyric acid + N A D + and is m onitored by m easuring the decrease in abso rbance of N A D H at 340 nm . The ev idence ind ica tes L D is th e enzym e involved in the reduction of glyoxalate which has been found to be enzymati­cally reduced by hum an serum faster at 37°C than either pyruvate or 2-oxobutyr- a te .108

H ow ever, the m easu rem en t of total LD activity in serum or o ther body fluids usually uses either lactate or pyruvate as substrate and is attributable to a mixture of isoenzymes which varies with an indi­vidual’s physiological and pathological conditions.

Pyruvate-to-Lactate M ethodologies

The conditions for the prim ary contin-

TABLE I I

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

P y r u v a t e NADH B u f f e r / c o n c .M e th o d * T ( ° C ) ( m m o l/1 ) (m m o l/1 ) (m m o l/1 ) pH

1 0 7W r o b l e w s k i a n d L aD u e (1 9 5 5 ) 2 5 - 2 7 0 . 7 4 0 . 1 2 P i f 7 . 4 ( 7 . 2 ! - 7 . 6 )H e n r y , C h i a m o r i , G o lu b , a n d B e rk m a n ( 1 9 6 0 ) ^ 32 0 . 6 0 . 1 8 7 P i 1 0 0 7 . 4B o w e rs ( 1 9 6 3 ) 1 5 3 7 1 . 2 0 . 2 1 T r i s 1 0 0 7 . 3 5 a t 3 7°C l[G a y , M cC om b, a n d B o w e rs ( 1 9 6 8 ) ^ 3 0 0 . 9 $ 0 . 2 2 T r i s 1 0 0 7 . 3 0 a t 3 0 ° c HG e rm a n S o c i e t y ( 1 9 7 2 ) ^ 2 5 0 . 6 0 . 1 8 P i 50 7 . 5M cQ u een ( 1 9 7 2 ) 6 9 3 7 2 . 5 0 . 1 2 P i 6 7 7 . 1 8 a t 3 7 °C liS c a n d . S o c . f o r C l i n . C hem .

a n d C l i n . P h y s i o l . ( 1 9 7 4 § ) 29 3 7 1 . 2 0 . 1 5 T r i s 50A 7 . 4 0 a t 3 7 ° C ÏB u h l , J a c k s o n , a n d G r a f f u n d e r ( 1 9 7 8 ) 20 2 5 $ 1 . 0 0 . 1 5 TEA 1 0 0 7 . 0 a t 2 5 " d l

3 0 1 . 5 0 . 1 5 TEA 100 7 . 0 a t 3 0 ° d t

S o c i é t é F r a n c e d e B i o l o g i e C l i n i q u e ( 1 9 8 1 )37 1 . 5 0 . 2 2 TEA 1 0 0 7 .0 a t 3 7 ° c j[30 1 . 6 0 . 2 0 T r i s

N a C l80

2 0 07 . 2 a t 3 0 ° d l

* S a m p le v o lu m e f r a c t i o n o f 1 / 3 0 e x c e p t a s n o t e d . f P i d e s i g n a t e s p h o s p h a t e .$ U s e d 0 . 9 m m o l/1 i n t h e i r s t u d y b u t f o u n d 1 . 2 m m o l /1 t o b e o p t i m a l f o r m ix e d i s o e n z y m e s a m p l e .§ S a m p le v o lu m e f r a c t i o n 1 / 6 0tfpH a d j u s t m e n t a t t e m p e r a t u r e d e s i g n a t e d .A B u f f e r c o n t a i n e d 5 m m o l/1 e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d (E D T A ).^ R e c o m m e n d e d 2 5 ° C b u t o p t i m i z e d a l s o a t 3 0 ° C a n d 3 7 ° C .

TABLE I

Abbreviation of Enzymes with Classification Numbers*

D e h y d r o g e n a s e s

L a c t a t e d e h y d r o g e n a s e (LD) EC 1 . 1 . 1 . 2 7M a l a t e d e h y d r o g e n a s e (MD) EC 1 . 1 . 1 . 3 7G l u t a m a t e d e h y d r o g e n a s e (GLDH) EC 1 . 4 . 1 . 3I s o c i t r a t e d e h y d r o g e n a s e (IC D ) EC 1 . 1 . 1 . 4 2S o r b i t o l d e h y d r o g e n a s e EC 1 . 1 . 1 . 1 4

O t h e r s

A l k a l i n e p h o s p h a t a s e (ALP) EC 3 . 1 . 3 . 1A s p a r t a t e a m i n o t r a n s f e r a s e (AST) EC 2 . 6 . 1 . 1

♦E nzym e N o m e n c la t u r e R e c o m m e n d a tio n s (1 9 7 8 ) o f t h e I n t e r n a t i o n a l U n io n o f P u r e a n d A p p l i e d C h e m i s t r y a n d t h e I n t e r n a t i o n a l U n io n o f B i o c h e m i s t r y , New Y o r k , A c a d e m ic P r e s s , 1 9 7 9 .

either the increase in reduced nicotin- am ide-adenine dinucleotide (NADH) at 340 nm produced in the lactate-to-pyru- vate reaction (L —> P) or by the decrease in N A D H at 340 nm p ro d u ced in the pyruvate-to-lactate (P —» L) reaction. The enzyme acts only on L-lactate and only N A D + can function as th e coenzym e. Two of the five LD isoenzymes (LD 1 and LD 2) are not specific for lactate or

MEASUREMENT OF TOTAL LACTATE DEHYDROGENASE ACTIVITY 15

uously m onitored rate (kinetic) m easure­m ent of LD in the pyruvate-to-lactate (P —» L) direction are summ arized in table II. In 1955, W roblewski and L aD ue107 developed the first continuously m oni­tored spectrophotom etric (kinetic) assay m ethod for LD using pyruvate, reduced N A D H and serum in p h o sp h a te (Pi) buffer at pH 7.4. The reaction does not have to be forced because the equilib­rium lies in the direction of the formation of lac ta te . In 1960 H enry , C hiam ori, Golub, and Berkm an,51 in their classical p u b lica tio n on rev ised sp e c tro p h o to ­m etric m ethods, took a major stride by examining and “revising” the conditions for th re e m ajor clin ical labo ra to ry enzym e m easurem ents including LD in the P —» L direction. They were the first to: optimize substrates, emphasize tem ­perature control, and stress pH control.

A very com plete investigation of LD assay conditions was carried out by Gay, McComb, and Bowers42 in 1968 utilizing crudely extracted preparations of hum an heart and liver LD for investigation of the optimal conditions for both the P —» L and lactate-to-pyruvate (L —» P) reac­tions. For the P —» L reaction Gay et al recom m ended Tris (hydroxmethyl) ami- nom ethane (Tris) buffer which system has been adopted with modifications by the Scandinavian Society for Clinical C hem ­istry and Clinical Physiology.29 On the o ther hand, the G erm an Society for Clin­ical C hem istry recom m endations34 are for a m odification of th e H en ry e t al m e th o d 51 w hich u tilizes p h o sp h a te buffer. W hile B ow ers15 re p o rte d th a t phosphate buffer inh ib ited LD activity assayed in the P —» L system, Buhl, Jack­son, and Grafifunder20 found phosphate up to 125 mmol per 1 and, to a small extent, Tris, were stimulatory for hum an LD 1 and 5 at pH values less than 7.5.

Krause and Lott59 have dem onstrated the use of the simplex m ethod to opti­m ize analy tical cond itions in clin ical chem istry by applying it to the P —» L

reaction for LD. Optimization of pH and su b stra te s was ca rried ou t in o rd e r to achieve the maximum rate for Versatol- E.* No application to clinical specimens was included.

In 1972 M cQueen developed an opti­mal assay for the P —» L reaction at 37°C based upon normal and pathological sera as a source of L D activ ity .69 S u b se­quently, Tuckerm an and H en d erso n 100 evaluated the H enry assay51 carried out at 37°C instead of 32°C, the M cQueen assay69 and an op tim ized com m ercial assay kit, all P —> L assays at 37°C. The M cQueen assay, which uses a high pyru­vate concentration, gave significantly less LD activity owing to substra te inh ib i­tion, than the o ther two m ethods which appeared to be equivalent.

Szasz98 investigated the effect of tem ­perature on enzyme activity for the var­ious enzyme m ethods recom m ended by the G erm an Society for Clinical Chem ­istry.34 He showed both “fast” LD (LD 1 and 2) and “slow” LD (LD 5) to deviate from linearity w hen m easured at tem ­peratures greater than 30°C under the G erm an op tim ized cond itions reco m ­m ended for 25°C. Szasz concluded that almost all of the kinetic factors relevant to optimization are highly dependen t on assay tem p e ra tu re . T hus, M cQ ueen70 docum ented the true Arrhenius relation­ships of hum an LD 1 and 5 extracts in the P —* L reaction at 25°, 30°, 35°, 40°, 45°, and 50° and showed clearly for the first tim e the increasing pyruvate and N A D H co n cen tra tio n s re q u ire d at h ig h er te m p e ra tu re s w ith d iffe ren t isoenzymes.

In 1978 Buhl e t al,20 in a follow up to earlier LD studies,21,22 investigated the pyruvate-to-lactate reaction for optimal reaction conditions at 25, 30, and 37°C. Nine buffers were investigated as well as th e n o n -lin earity of th e reac tion response . H ighly p u rified L D 1 from

* General Diagnostics Co., Morris Plains, NJ.

16 VANDERLINDE

hum an e ry th ro cy te s and L D 5 from hum an liver were utilized as the sources of enzym e activity. O p tim al su b s tra te concentrations for the m easu rem en t of the P —* L reaction in imidazole, TEA (trie thano lam ine) or TES (N -tris (hy- droxymethyl)-methyl-2-aminoethanesul- fonic acid) at pH 7.0 are shown in tableII. Imidazole, TEA, and TES were cho­sen as suitable reaction buffers because their pKa’s are near the recom m ended reac tion pH of 7 .0 and b ecause they ex e rted no s tim u la to ry or in h ib ito ry effects. Tris was not evaluated thoroughly bu t was found to be stimulatory, as p re­viously reported by Bowers.15 Also, Buhl found phosphate to be stimulatory for the hum an LD 1 and LD 5 isoenzymes in the P —* L direction only. His findings that buffer effects are pH and substrate dependen t suggest a source of variation in com m ercial rea g e n t k it system s. Buhl20 has recom m ended that all pyru- vate-to-lactate m easurem ents be m ade as soon as possible after the reaction is ini­tiated and be perform ed at 25°C or, if necessary, 30°C because at 37°C there is a different rate of decrease in absorbance for LD 1 and LD 5. His studies suggest that the m easuring tim e interval at each tem perature should be standardized rel­ative both to the tim e after the reaction is initiated and to its duration. The activ­ity of hum an L D 1 and LD 5 assays, based on the P —> L reaction, w ere not affected by the m anner of reaction initi­ation since trig g e rin g w ith p y ruvate , enzym e or N A D H gave e q u iv a len t results.22

Volume 9 of Selected M ethods in Clin­ical Chem istry entitled Selected M ethods f o r the Sm all C h em istry L abora to ry includes the m easurem ent of LD by the P —» L reac tio n in im idazole bu ffer at 37°C by the NADH to N A D + change at 340 nm .59 This kinetic m ethod is felt to be achievable in small laboratories by the author and the editors.

Very recently, Vassault, Maire, Seville,

Bozon, and Lalegerie101 have proposed an official reco m m en d a tio n by the Société Française de Biologie Clinique entitled A n Im provem ent fo r the D eter­mination o f Total Lactate D ehydrogen­ase C a ta ly tic A c tiv ity W h a te v er the Isoenzym es. I t is w ell know n th a t the reaction catalyzed by LD H is inhibited in th e p re se n c e of h igh levels of su b ­s tra te .36 This inh ib ition is observed in both directions and with both types of enzym es, a lthough th e in h ib itio n is m uch m ore pronounced with LD 1 than with LD 5 and with pyruvate than lac­tate. It has been shown to be due to the formation of an abortive ternary complex or adduct of LD -N A D +-pyruvate with in h ib its s trong ly th e P —* L reaction resulting in a non-linear reaction ra te .36 R ecently , R ivedal and S anner86 have found that an increase in ionic strength can modify the stability of the ternary abortive complex and decreases inhibi­tion. Thus, Vassault et al101 have shown that the addition of N a+ and C l- ions to the P —> L reaction mixture modifies the stability of the inhibitory ternary com­plex. The proposed m ethod includes the addition of 200 mmol per 1 NaCl to the Tris buffer reaction m ixture (table II). The m ethod is stated to show the follow­ing advantages: minimizing of differences for op tim al co n cen tra tio n o f py ruvate betw een LD 1 and LD 5, im provem ent in the linearity of the kinetics, and an increase in the upper limits of linearity.

Many P —» L procedures initiate the reaction with pyruvate (table II), in order to p rov ide a p re in cu b a tio n p e rio d to remove endogenous substrates reacting with NADH.

Lactate-to-Pyruvate M ethodologies

The conditions for the prim ary m eth­ods for the continuously m onitored rate (kinetic) m easurem ent of LD in the lac- tate-to-pyruvate (L —* P) d irection are sum m arized in tab le I II . W acker,

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

MEASUREMENT OF TOTAL LACTATE DEHYDROGENASE ACTIVITY

TABLE I I I

17

M e th o d *B u f f e r / c o n c .

r (°c) ( r n n o l/1 ) ( m m o l/1 ) ( im n o l/1 ) pH

2 5 5 3 (DL) 5 P i t 50 8 . 825 7 7 (DL) 5 . 2 5 P P i t 50 8 . 62 5 1 7 5 (DL) 1 G l y c i n e 53 1 0 . 0

N a C l 5 3

3 7 . 5 82 (DL) 7 . 5 AMP§ 1 8 8 9 . 030 7 7 ..5 (DL) 5 . 2 5 P P i $ 50 8 . 5 5 a t 3 0 ° d f30 9 0 (DL) 5 . 5 AMP§ 6 0 0 9 . 0

2 5 4 0 ( L i s a l t ) A 3 DEA o r 2 0 0 8 . 7 a t 2 5 ° C30 50 ( L i s a l t ) A 5 A M P d io l 2 0 0 8 . 7 a t 30 °C37 70 ( L i s a l t ) * 7 B u f f e r 2 0 0 8 . 7 a t 3 7 ° C

W a c k e r , U lm e n , a n d V a l l e e ( 1 9 5 6 ) A m a d o r , D o r f m a n , a n d W a c k e r ( 1 9 6 3 ) ^ K i n g ( 1 9 6 5 ) 5 8

,4 2

M o r g e n s t e r n , F l o r , K e s s l e r , a n d K l e i n ( 1 9 6 5 ) 7 3 ' 9 9

G a y , M cCom b, a n d B o w e rs ( 1 9 6 8 ) ‘ D e m e t r i o u , D r e w e s , a n d G in ( 1 9 7 4 ) ' B u h l , J a c k s o n , L u b i n s k i ,

a n d V a n d e r l i n d e ( 1 9 7 7 ) 2 3

* S a m p le v o lu m e f r a c t i o n 1 / 1 5 . f P h o s p h a t e b u f f e r - P i .^ P y r o p h o s p h a t e b u f f e r - P P i .§AMP i s 2 - a m i n o - 2 - m e t h y 1 - 1 - p r o p a n o l .I fU s e d pH 8 . 7 5 i n t h e i r s t u d y b u t f o u n d 8 . 5 5 o p t i m a l f o r a m ix e d i s o e n z y m e s a m p l e .* L “ l a c t a t e .

Ulmen, and Vallee103 in 1956 were the first group to m easure LD kinetically in the L —* P direction for clinical labora­tory purposes. Subsequently , Amador, Dorfman, and Wacker3 found phosphate to be an inadequate buffer at a pH of 8 .8 , su b s titu te d py ro p h o sp h a te , and increased the lactate and N A D + concen­tra tio n s . T hese changes re su lte d in a markedly im proved assay. King58 intro­duced glycine with added sodium chlo­ride at pH 10.0 as a buffer system. W hile widely used, especially in G reat Britain, the high pH is incompatible with the use of NAD + , w hich decom poses at very alkaline p H ’s.31,38 In addition, Fendley, Jacobs, D unn , and F rin g s39 found the King assay at pH 10 to underestim ate grossly LD 5 and have modified the pH to 8.8, which alleviates both problems.

W hile M o rg en ste rn , F lor, K essler, and Klein73 in 1965 developed an L ^ P m eth o d for th e A uto A nalyzer* u tiliz ­ing AM P (2-am ino-m ethyl-l-propanol) buffer with both colorimetric and kinetic (340 nm) versions, the 340 nm spectro-

* Technicon Instruments Corp., Tarrytown, NY.

photom etric m easuring system was not m ade availab le as p a rt o f Technicon equ ipm en t until the 1970’s " A sim ilar m anual k inetic m eth o d u tiliz in g AM P buffer has been recom m ended by D em e­triou, Drewes, and Gin in a well recog­nized textbook32 (table III).

An early very com plete investigation of LD assay conditions was carried out by Gay, McComb, and Bowers.42 It u ti­lized [crudely] extracted preparations of hum an heart and liver LD for investiga­tion of the optimal conditions for the P —> L reaction in addition to the L —» P assay. However, this carefully executed study did not include the evaluation of a large num ber of buffers. The finalized optimal conditions for the L —» P reaction by Gay, M cC om b, and B ow ers42 are m inor modifications of the Amador e t al pyrophosphate m ethod3 (see table III). W hile they used pH 8.75 in their study, 8.55 was found optimal for a mixed isoen­zyme specimen. In addition, they advo­cated proper tem perature control as well as a d ju s tm en t of th e final pH of the buffer at the recom m ended assay tem ­perature of 30°C. Both Amador e t al3 and

18 VANDERLINDE

Gay, M cCom b, and Bowers42 in itia ted the reaction with enzyme whereas King’s m ethod58 for the L —» P assay specifies in itia tio n w ith NAD + . B uhl, Jackson, L ub insk i, and V anderlinde22 found hum an LD 1 and LD 5 activity, when assayed L —» P, is adversely affected if the enzyme is preincubated w ith NAD + and the reaction is initiated with lactate. The am ount of inactivation depends on the buffer used; activities are decreased more in sodium pyrophosphate than in Tris. On the o ther hand, N A D + has been reported to have a stabilizing effect on L D ac tiv ity .8,33 B ecause th is effect is m ore pronounced in phosphate buffers than in Tris buffer,63,106 it has been sug­g es ted th a t p h o sp h a te ac tivates the N A D + binding sites.63

These observations and the fact that the L —» P reaction is an ordered sequen­tial reaction, with N A D + binding first,92 shou ld lead to en h an ced L D activ ity when the enzym e is preincubated with NAD + . The contrary experim ental find­ings m ay be th e re su lt of in h ib ito rs , which have been found in N A D + prep ­arations from various sources.4 Also, a red u c tio n of LD activ ity w ould be expected if the inhibitor-enzym e disso­ciation constant is less than the NAD + - enzym e d issociation c o n s ta n t.22 Buhl in te rp re te d the results to suggest also th a t lac ta te affects enzym e b in d in g of NAD + . Further, both LD 1 and 5 have higher L —» P activity when assayed in Tris than when assayed in sodium pyro­phosphate buffer.22 Although the latter system contains lower concentrations of N A D + and lactate, this probably does no t account for th e low er activ ity observed because the N A D + and lactate co n cen tra tio n s used are s a tu ra tin g .42 B uh l’s resu lts d em o n stra te also th a t buffer is an im portant factor in the LD reaction and that the buffer may interact with the enzyme and affect the binding of the substrates. The very complex issue of su b s tra te s , in h ib ito rs and reac tion

mechanisms of the LD reaction has been ably reviewed by Everse and Kaplan.36

T he lack of a g re e m e n t in choice of buffer, the finding of inhibition by the pyrophosphate buffer, and the finding of discrepant results in assays of both puri­fied isoenzymes and sera under various buffer cond itions led Buhl e t al21 to investigate seventeen buffers at a variety of p H ’s in the assay using highly purified hum an L D 1 and LD 5. T he buffers se lec ted in c lu d ed the four m ost com ­m only used (table III): pyrophosphate (PPi), Tris, 2-am ino-2-m ethyl-l-propanol (AMP) and glycine and an additional 13 buffers with pKa values greater than 7.9.

Of the four most commonly used buff­ers, th ree proved unsatisfactory because of direct effects on LD. Glycine and PPi were found unsatisfactory because they inhibit LD activity with increasing con­centration. W ith AMP the pH optimum for LD 1 is not compatible with the use of NAD + , and the pH optim a for LD 1 and LD 5 are vastly different. The con­cen tra tio n of AM P also g rea tly affects total LD activity; hence large amounts of AMP m ust be used to achieve maximal LD activity. Currently a concentration of 600 m m ol/lite r is u sed by T echn icon" and recom m ended by D em etriou et al.32 Additional disadvantages of AMP buffer are th a t LD activ ity is d e p e n d e n t on the m ethod of preparation, the buffer has an unpleasant odor, and a gas is visibly re leased d u rin g p H ad ju stm en t. Although ethanolam ine and dimethlyam- inoethanol (DAE) w ere satisfactory buff­ers by som e c rite r ia , e thano lam ine exerted a stim ulating effect on LD activ­ity and DAE showed potential undesir­able chem ical reac tions as w ell as an obnoxious odor.

The three buffers Buhl e t al21 found to have no detrim ental effects on LD ac­tivity are Tris, d iethanolam ine (DEA), and 2-am ino-2-m ethyl 1 ,3-propanediol (AMPdiol). He felt Tris may not be com­pletely satisfactory because its pH opti-

MEASUREMENT OF TOTAL LACTATE DEHYDROGENASE ACTIVITY 19

mum for LD 1 is greater than 8.5, while its Pk3 at 30°C is 8.0. Thus, Tris has lim­ited bu ffe ring capacity at pH 8.5, the optimal reaction pH for mixtures of LD 1 and LD 5.

O f the 17 buffers examined, DEA and AMPdiol at concentrations of 200 mmol per 1 and pH 8.7 w ere recom m ended as the buffers o f choice for the m easure­m ent of LD activity in the L —» P direc­tion. However, DEA shows a significant cost advantage over AMPdiol. In a sub­se q u en t in v estig a tio n B uhl, Jackson, Lubinski, and Vanderlinde23 established the optimal substrate conditions for the assay of the hum an LD isoenzymes 1 and 5 L —» P in these two buffers at 25, 30, and 37°C (table III).

Choosing the optimal concentration of lactate for each tem perature was difficult because excess lac ta te in h ib its LD 1, w hereas L D 5 requ ires large am ounts (relative to LD 1) of lactate for saturation. Krieg, Gorton, and H enry60 found crude hum an ex tracts of LD 1 and LD 5 to show m ore nearly the same velocity/sub- strate concentration profiles at 37°C than at 25°C as observed earlier by Vesell.102 However, the source, purity and type of lactate w ere not stated, and Buhl et al22 have dem onstrated the purity and form of lac ta te to b e ex trem ely im portan t. H ence , lith iu m -L ( + )-lac ta te of high purity was used by Buhl and his associ­ates in th e ir su b seq u e n t investiga­tions.21,23 The lack of linearity in product formation (constant AA per min) has sev­era l co m ponen ts , w hich have been observed b u t not separated in previous studies.68,93 The initial rise (<20 s), Buhl suggested, is probably due to experim en­tal design or deficiencies in instrum ent response while the decrease after 20 to 30 s has m ore th an one com ponent, including substrate depletion, and prob­ably involves p roduct inhibition. Sub­strate depletion can be overcome simply by adding additional su b s tra te .68 H ow ­ever, a tte m p ts to rem ove the p ro d u ct

fo rm ed w ith sem icarbazide have m et w ith m ixed su ccess ,93'94 p robab ly b ecause of th e com plexity of th e su b ­strate inter-relationships and biochem i­cals of the LD reaction.36

T he a p p a re n t K m ’s for lac ta te and N A D + for nonhum an LD increase with tem p e ra tu re .43,49,77 Buhl’s findings that increased amounts of lactate and NAD + are required for saturation of hum an LD 1 and LD 5 with increasing tem perature is consistent with the earlier observations on animal isoenzymes.

Reaction conditions have been estab­lished which m easure LD 1 and LD 5 with equal efficiency. Assuming that the o th e r th re e LD isoenzym es are m ea­su red equally w ell, th ese cond itions should be appropria te for de term in ing total LD activity in normal and patho­logic sera, as well as individual isoen­zyme determ inations from column pro­cedures.

Buhl recom m ended that all L —» P LD m easurem ents be m ade within 60 s after the reaction is initiated. If this is not fea­sib le b ecause of de layed in s tru m e n t response tim e, the m easurem ent should be made as quickly as possible.

H e also reco m m en d ed th a t L —» P assays be p e rfo rm ed at 25°C or 30°C because at the lower tem peratures the reaction response was linear for a longer tim e, less reag en ts w ere n e e d ed , and less interlaboratory and intralaboratory variation would be encountered on sam­ples m easured with a variety of instru­m ents and on different isoenzymic mix­tures having similar total LD activity.

Although it would seem m ore precise to have higher assay values, his results showed this not to be true. Because of an appreciably different decrease in AA per min for LD 1 and LD 5 at 37°C but not at 30°C or 25°C, it is more difficult to m easure each isoenzyme at 37°C with equal efficiency, and the d ifference in efficiency was ob se rv ed to becom e greater with tim e after the reaction is ini­

20 VANDERLINDE

tiated. The consequence is that any assay of an isoenym ic m ixture w ith elevated LD activity will have a large in terinstru­m ent variability. Also, more intrainstru­m ent variability would be encountered at 37°C than at 30°C or 25°C in sam ples with the same total LD activity but dif­fering isoenzymic mixtures. The single­instrum ent variation will be greatest for instrum ents which m easure LD activity one min or m ore after initiation.

Hence, Buhl23 recom m ended the m ea­suring tim e interval be standardized and assays of LD m easured L —» P be m ea­sured at 25°C or 30°C as they will show less variation attributable to instrum ent and isoenzyme content.

Other Methodologies

W hile clin ical lab o ra to ries u tilize widely continously m onitored rate reac­tions at 340 nm involving the absorbancy change in N A D + —NADH, with lactate or pyruvate as substrate, o ther m ethod­ological approaches for the m easurem ent of LD have been advanced. Thus, Bab- son and B abson5 d e sc rib ed in 1973 a k inetic colorim etric assay p rocedure in which the reduction of NAD + is coupled to the red u c tio n of a te trazo liu m salt, 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride (INT), w ith phen- azine methosulfate serving as an inter­m ediate electron carrier. The kinetic col­orim etric m ethod co rre la ted well with 340 nm k inetic m ethods b u t d id not requ ire an u ltrav io let spectrophotom e­ter. A sim ilar rapid single step kinetic colorimetric assay for LD in serum was d ev e lo p ed by A llain, H enson , N adel, and Knoblesdorff2 but used diaphorase as the interm ediate electron carrier.

The feasibility of microcalorimetry for th e d e te rm in a tio n o f th e k inetics of enzyme catalyzed reactions, specifically LD and uricase, was in v estig a ted by Rehak, E verse , Kaplan, and B erger.82

The rela tionship betw een activity and concentration of both soluble and immo­b ilized ch icken LD 1 was d e te rm in e d using th e ra te of h e a t p ro d u ctio n in a batch-type calorim eter. A nother novel approach to th e m ea su rem e n t of LD was m ade by N ikolelis, Pain ton , and Mottola76 who found the air oxidation of NADH, catalyzed by peroxidase, to pro­vide a useful “indicator reaction” for the determ ination of e ither serum LD or of NADH. They described application of th is in d ica to r reac tio n to re p e titiv e determ inations by sample injection into a continuously c ircu lated reagent mix­ture through the m onitoring of oxygen depletion with an am perom etric sensor.

Curry, Pardue, Mieling, and Santini30 have designed a filter fluorom eter that incorporates a photon-counting detector and ev a lu a ted its app licab ility in the f lu o ro m e tric d e te rm in a tio n of serum LD. W hile reflectance spectroscopy has not been widely used in the clinical lab­oratory, Zipp, W atson, and G reyson110 have developed a solid state matrix sys­tem for th e d e te rm in a tio n of LD in serum in which the quantitation is car­ried ou t by re f le c ta n c e spectroscopy. T hey claim th a t th is approach can be used to m easure total LD in serum with the convenience and simplicity of a test strip without compromising the quanti­tative nature of present wet methods. In addition, the dry film based technology of the Eastm an Kodak Company’s new Ektachem 400 System utilizes the prin­ciple of reflectance spectroscopy,96 which is well established in the photographic field . H ow ever, w hile th e ir am ylase methodology is available, other enzymes including LD have ju s t been released recently.

A vidicon spectrophotom etric ap­proach to th e m easu rem en t s im ulta­neously of two enzymes, exemplified by serum LD and alkaline phosphatase (EC3 .1 .3 .1 ), has b e e n d e m o n stra ted by M ilano and P a rd u e 72 to give enzym e

MEASUREMENT OF TOTAL LACTATE DEHYDROGENASE ACTIVITY 21

activities equivalent to those in common use.

NADH and N A D + Inhibitors

As early as 1961, the Kaplan group38 recognized the presence of an inhibitor in NADH which can strongly inhibit LD; some clinical chemists were aware of the p ro b lem 52,97 in th e m idd le sixties. In 1968 McComb and Gay67 published their findings on four commercial sources of NADH. Subsequently, Berry, Lott, and Grannis12 showed lot to lot variation in the am oun t of th e in h ib ito r and sug­gested a p ro ce d u re for assessing the quality of com m ercial NA DH p rep ara ­tions. H ow ever, th e p rob lem was no t unique to NADH and the pyruvate-to- lactate continuously m onitored rate reac­tion as D alz ie l31 in 1963 had d e m o n ­s tra te d N A D + to con ta in n u c leo tid e im purities which interfered with the lac- ta te -to -p y ru v a te reac tio n . B abson and A rndt4 confirm ed D alziel’s observation by showing that commercial preparations of NA D+ contained various amounts of one or m ore LD inhibitors bu t not the inhibitor described for NADH. Thus, in the early seventies, the quality, storage, and refreezing characteristics of NADH and/or NAD + , utilized in clinical labo­ratory kinetic m easurem ents of LD in e ith e r d irec tio n , becam e o f concern . This concern stim ulated efforts in several additional fronts: namely, by the profes­sional societies, by the National Bureau of Standards, and by the m anufacturers, among others.

S ince a d e n o s in e d ip h o s p h o r ib o s e (ADPR), adenosine diphosphate (ADP), and adenosine m onophosphate (AMP) are poor inhibitors for the lactate dehy­drogenases,45 the inhibitors w ere p re ­sumably entirely different structures.

G erhardt e t al44 under the auspices of the Scandanavian Society for C lin ical Chem istry and Clinical Physiology eval­uated practical m ethods for the detection

of inhibitors in NADH and for m onitor­ing its quality and described specifica­tions for both reference and commercial preparations. The U.S. National Bureau of Standards group, under Margolis and Schaffer, has spent several years identi­fying im purities in NADH and develop­ing specifications for reference quality NADH. The molar absorbtivity of high purity NADH was evaluated in 1976 by M cC om b e t al66 in the U n ited S tates and by Ziegenhorn et al109 in Europe. M cComb’s group61 as well as the NBS group55,64,65 stud ied the form ation and properties of the LD inhibitors in NADH by column chrom atography and/or high perfo rm ance liq u id ch rom atography (HPLC). Loshon et al61 described at least two in h ib ito ry com ponen ts w hich can form in concentrated NADH solutions, only one of which can be separated on DEAE-cellulose or DEAE-Sephadex.

The second inhibitor, which separated on the trailing edge of the NADH peak in chrom atography on DEAE-cellulose, was resolved from the NADH by Shaf­fer’s group by HPLC on u Bondapak C 18. Because this new inhibitor had ultravi­olet p ro p e rtie s sim ilar to those of NADH, a ratio of absorbance at 260/340 nm of less than 2.32 could not be used as an indication of an inhibitor free p rep ­aration and HPLC chrom atography was deem ed necessary to ensure its absence in preparations of NADH used for clini­cal assay.64 W hile Loshon et al61 showed the presence of N A D + to influence the rate of inhibitor production from unbuff­e re d aqueous N A D H so lu tions, no inhibitor was produced by N A D + incu­bated alone under the same conditions. H ow ever, G alla ti40,41 has p re p a re d an in h ib ito r of LD by the tre a tm e n t of N A D + and phosphate at alkaline pH and Johnson and M orrison as early as 1970 postulated NAD to undergo ring opening

'o f the nicotinam ide ring in strongly alka­line solutions.57

More recently, G uilbert and Johnson48

22 VANDERLINDE

have d e m o n s tra ted th e p seudobase hydrox ide ring add ition ad d u c t of NAD + , i|/NAD-OH, to be rev ers ib ly form ed and, subsequently , to undergo ring opening at the num ber six carbon of the nicotinamide ring to form open ring NAD designated as ONAD. ONAD has pKa values at 1.9 and 11.2 and absorbs maximally at 350 nm in its acidic form, at 372 nm in its neutral form, and at 340 nm in its anionic form. The hydrolysis of ONAD leads to the formation of 2-car- boxyamideglutacondialdehyde and aden­osine diphosphate ribosylamine with the form er breaking down further to the base fluo rescen t com pound, 2-hydroxynico- tin a ld eh y d e . T hus, N A D + in alkaline solution can form at least 5 interm ediate end products. More recently, a cooper­ative group formed betw een the Louis P as teu r U n iversity at S trasbourg and B oeh ringer M annheim G m b H 13 and a second g roup in C openhagen u n d e r Godtfredsen46’47 have made great strides in the structural elucidation and in te r­relationships betw een the various LD inhibitors occurring in both NADH and NAD + . Biellmann et al13 found NADH on sto rage in a m oist a tm o sp h ere to re su lt in th re e in h ib ito ry com pounds, only two of which were inhibitors. The structure of one inhibitor was not eluci­d a ted ow ing to its instab ility ; an o th e r inhibitor was dem onstrated not to origi­nate directly from NADH b u t from con­tam inating NAD + . It was shown to be p h o sp h a te -N A D + adduct fo rm ed in small amounts by the addition of a phos­phate ion to the 4-position of the nicotin- am id in ium ring of th e con tam inating NAD + . This 4-phosphoryloxy-l, 4-dihy- dro-N A D ad d u c t was shown to be a potent com petitive inhibitor of LD with an inhibitor constant (Kj) of 2 x 10_7M and to be identical with the inhibitor p re­p a re d by G alla ti40,41 from N A D + plus phosphate in alkaline medium. The third inh ib ito r p roduct isolated from NADH was identified by Biellmann e t al13 as a d im er of NAD + . Subsequently, Godt-

fred sen and O tte se n 46 show ed N A D H under hum id conditions to form 1,6-di- hydro-N A D , a p o te n t in h ib ito r o f LD with a Kj of 2.5 x 10_7M. They report their 1,6-dihydro-NAD inhibitor to have spectroscopic properties at 340 nm sim­ilar to those of the dim er of NAD + iden­tified by B iellm ann14 and p resen t evi­dence to support Biellmann’s compound m ost likely being 1,6-dihydro-NAD.

F u rth e rm o re , G o d tfred sen e t al47 p resent evidence to show that this com­pound is probably the m ajor inh ib ito r formed in moist NADH at pH 8.8 and have presented experim ental data to sup­po rt the ir hypothesis46 tha t the 1 ,6-di­hydro-NAD is generated in NADH p rep ­ara tions by a b im o lecu lar reac tion betw een NADH and the small quantities of N A D + which are usually p resen t in samples of NADH. One major implica­tion of this finding is that no or very little inhibitor formation should take place in NADH preparations completely free of NAD + . Thus it would seem fairly well established that the major two inhibitors in N A D H are: (a) l,6 -d ih y d ro -N A D + w hich absorbs at 340 nm sim ilar to NADH and cochromatographs on DEAE cellulose or Sephadex with NADH and(b) 4-phosphoryloxy-1,4-dihydro-NAD +, a phosphate adduct of N A D + .

Also, the pH of the preparation and the nature of the inorganic salts present, pa rticu la rly p hospha te , a lte r w hich inhibitors occur in various NADH and N A D + preparations.

W enz e t al104 showed in 1976 various com m ercial p rep a ra tio n s of N A D H to y ield up to 12 d iffe ren t com pounds which can act as LD inhibitors. All m an­u fac tu re rs have b een aw are o f th e in ­hibitor problem with N A D+ and partic­u larly N A D H for m any years. C o n se ­quently, many of the manufacturers offer good routine service quality NADH and N A D+ preparations as well as chroma- topure or ultrapure preparations of both NADH and N A D+ at 50 to 100 percent increased cost. B uhl, Jackson, and

MEASUREMENT OF TOTAL LACTATE DEHYDROGENASE ACTIVITY 23

GrafFunder20 obtained about 10 percent h igher activities w ith the pu re hum an LD 1 and LD 5 isoenzymes with a more pure commercial B-NADH preparation b u t found no d iffe rence in su b s tra te requirem ents. However, equally im por­tant are the num erous factors such as the hum idity and storage conditions, freez­ing and thawing of solutions, the nature of the contaminants, which salts are pres­e n t, and p H of th e p rep a ra tio n w hich effect the stability of NADH and NAD + m ain ta in ed w ith in th e u s e r ’s lab o ra ­to ry .36,47 F u rth e rm o re , re sea rch and standardization involving dehydrogen­ases such as LD and alcohol dehydrogen­ase (EC 1.1.1.1) m easurem ents which are especially sensitive to inhibitors may re q u ire h igh ly p u rif ied N A D + and NADH sources whereas the less expen­sive commercial preparations may suffice for routine clinical laboratory m easure­ments.

Choice of Methodologies

In re c e n t years, lab o ra to ries in the U nited States have increasingly shifted from co lo rim etric en d -p o in t enzym e m easu rem en ts to con tinuously m oni­tored rate (kinetic) reactions and to the m easurem ent of LD by the L —» P rather than the P —» L reaction. For example, the clin ical labo ra to ries in N ew York S ta te , o u tsid e of N ew York City, increased their utilization of kinetic rate m ethods for LD from 21.7 percent of all laboratories in 1971 to 61.0 percent in 1975.18 In addition, the percentage u ti­lizing an L —* P m ethod instead of P —> L jum ped from 43.4 percent in 1971 to 81.0 percent in 1975. The major reason for th is shift was th e in creased use of au to m ated enzym e analyzers u tiliz ing the L -* P reaction at 340 Yim, especially continuous flow 340 nm based kinetic analyzers.* A secondary reason was the

* Technicon Instruments Co., Tarrytown, NY.

shift from end-point to kinetic assays by many smaller laboratories.

The College of American Pathologists (CAP) E nzym e C h em istry P rogram employs sets of linearly related enzyme specimens including LD which are sent to p a rtic ip an ts q u a rte rly .26 T he 1982 Enzym e Survey V-A Sum m ary R eport provides valuable participant information on the instrum ents/reagents and num ber of laboratories utilizing each m ethodol­ogy which are summ arized in table IV. All m ethods utilized by ten or more lab­oratories involve the L —» P reaction, and only a m inor num ber of participants use P —» L procedures.

H ow ell, M cC une, and Shaffer56 in 1979 re-exam ined the L —> P and P —*■ L assays for LD and state, that the P —> L assay can now yield linearity equal to or b e tter than that obtained by the L P assay. In addition, they state there are significant advantages to the P —* L reac­tion , nam ely: (a) a g re a te r change in abso rbance p e r u n it of tim e , w hich

TABLE IV

Current LD Enzyme Methodologies*

I n s tr u m e n tNumber o f

R e a g e n ts ./ L a b o r a t o r i e s f

A b b o t t ABA-100 A b b o t t 36A b b o t t VP A b b o t t 45A m e ric a n M o n ito r KDA A m e ric a n M o n ito r 64D u P o n t ACA D u P o n t 272ENA G em in i E l e c t r o - N u c l e o n i c s 25ENI G em saec S m i th - K l in e 11G i l f o r d - V a r i o u s S y s te m s W o r th in g to n 33G i l f o r d - V a r i o u s S y s te m s G i l f o r d 15H y c e l S u p e r o r M ark 17 H y c e l 11T e c h n ic o n SMA 1 2 /6 0 F i s h e r S c i e n t i f i c 10T e c h n ic o n SMA 1 2 /6 0 T e c h n ic o n 11T e c h n ic o n SMA 1 2 /6 0 W o r th in g to n 10T e c h n ic o n SMAC T e c h n ic o n 100T e c h n ic o n SMA I I T e c h n ic o n 35C e n t r i f i - C h e m (B a k e r) B a k e r D i a g n o s t i c s 19C e n t r i f i - C h e m (B a k e r) U n io n C a r b id e 20C h e m e tr ic s I I W o r th in g to n 11

* D a ta t a k e n f ro m C o l le g e o f A m e ric a n P a t h o l o g i s t s Enzym e S u r v e y V-A Sum m ary R e p o r t o f J u n e 1 9 8 2 , b y p e r m i s s i o n a n d c o u r t e s y o f t h e C o l le g e o f A m e ric a n P a t h o l o g i s t s ,

f l n s t r u m e n t s / r e a g e n t s sh o w n o n l y f o r 10 o r m o re p a r t i c i p a t i n g l a b o r a t o r i e s . A l l i n v o l v e L —^ P m e th o d s b u t u t i l i z e v a r y i n g b u f f e r s ( P P i , T r i s , AMP), t e m p e r a t u r e s ( 2 5 ° C , 3 0 ° C , 37°C ) a n d s u b s t r a t e c o n c e n t r a t i o n s .

24 VANDERLINDE

allows a m ore accu ra te sp ec tro p h o to - m etric readout; (b) lower reactant con­centrations which thereby reduce costs;(c) solid reagents are used to prepare the assay solution; and (d) the reagent solu­tions are m ore stable. They point out that inhibitors in commercial NADH prepa­rations may substantially effect LD activ­ities m easured P —» L and state that a Standard Reference M aterial for NADH is being developed for issuance by the NBS. However, for a num ber of reasons this project has been postponed.80

On the o th e r hand , B uhl and Jack­so n ,19 who have com pared serum LD catalysis under optimal conditions devel­oped for the pure hum an isoenzymes in both the L —» P and P —» L reactions at 25°C, 30°C, and 37°C, found the results from the L —» P reaction to be more reli­able for in te rin s tru m en t and in te rlabo­ratory comparisons. Interconversions of results betw een the bidirection reactions were not practicable. M easurem ents of LD in e ither direction at 25°C, 30°C, or 37°C w ere stated to be equally reliable if the volume fraction and the resulting AA per min is small. However, they recom ­m ended that the P —» L reaction be mea­sured as soon as possible after start of the reaction, the m easuring interval be rea­sonably short (<10 s) and that the L —> P reaction be m easured within the first 40 s. Furtherm ore, they suggested that any future reference m ethod or selected m ethod include a recom m endation for the time and duration of the m easuring in terval after the reaction is in itiated . W hile they found neither the P —» L nor the L —» P response to be linear, they report that at equal AA per min the activ­ity of the L —* P reac tion does not decrease as rapidly and is therefore more re liab le . Buhl and Jackson19 have reported, “Some available preparations and im properly stored 3-NADH contain in h ib ito rs of LD , w hereas m any (3- N A D+ preparations are free of LD inhib­

itors.” A curren t review of representative b iochem ical rea g e n t catalogs show ed many of the m anufacturers to offer good routine service laboratory quality NADH and N A D + preparations as well as chro- m atopure or u ltrapu re preparations of NADH and N A D + at 50 to 100 percent in creased cost (See section on N A D H and N A D + inhibitors).

It is not possible to assess at this time new findings such as the Société Fran­çaise de Biologie Clinique recom m enda­tion101 of a new m ore linear L —» P assay (see L —» P section).

Sources of variation

P r e f e r r e d B l o o d S a m p l e

Serum is the preferred sample for all enzyme activity m easurem ents including LD. Haymond and Knight50 found the LD activities of capillary serum and cap­illary plasm a w ere significantly g rea ter than in sim ultaneously assayed venous serum , the g rea te s t d iffe rence be ing b e tw een cap illa ry and venous serum . Although some difference is attributable to tissue fluid, most of the difference was attribu ted to platelets. They recom m end that when capillary blood is to be used for enzyme assay, it should be processed as plasma. Subsequently, Tothwell, Jen- drzejczak, Becker, and Doum as88 inves­tigated the LD activity of venous serum versus venous plasma and dem onstrated platelets to be the source of up to a four fold increase in p lasm a LD . M ore r e ­cently, Bais, Prior, and Edw ards7 have shown platelets to increase LD as mea­sured by the SMAC continuous flow ana­lyzer* and the detergent in the detection bu ffer to fu rth e r enhance th e effect. Hence, if plasma m ust be used as the source of specimen, the sample m ust be centrifuged such as to provide a platelet- free plasma.

* Technicon Instruments Corp., Tarrytown, NY.

MEASUREMENT OF TOTAL LACTATE DEHYDROGENASE ACTIVITY 25

Various anticoagulants such as oxalate and fluoride are inhibitory to LD while h ep arin was re p o r te d by Batsakis and H en ry to be e r ra tic .9 M ore recen tly , R obertson , C hesler, and E lin 87 found serum collected w ith the antiglycolytic agent, lithium iodoacetate to be unsuit­able for the m easurem ent of LD.

H e m o l y s i s

The high in tra-ery throcytic concen­tration of LD 1 isoenzyme requires rapid separation of serum from the clo t.107 The calculated error per gram of hemoglobin for LD activity is 463 percent, while the observed error per gram of hemoglobin was 550 pe rcen t.17 H ence, hemolysis in sera for LD m easurem ents m ust strin­gently be avoided.

S t a b il it y o f S e r u m

Schm idt and S chm id t91 re p o r t the average loss of serum LD activity on stor­age at 4°C to be 0 percent at 24 hours, four percent at 48 hours, eight percent at three days, nine percent at five days and 12 percent for seven days, whereas at 25°C the corresponding losses are 0,1, 2, 10, and 15 percent, respectively. However, because LD 1 is relatively sta­ble whereas LD 5 is therm olabile, sta­bility may vary with the isoenzyme com­position of individual sera. In general, freezing is to be avoided.

A c t iv a t o r s , I n h ib it o r s

a n d I n t e r f e r e n c e s

The “no th ing-dehydrogenase” reac­tion while mainly associated as an artifact in serum isoenzym e analysis based on tetrazolium m ethods may occur also in the serum w here its origin is unknown.95 S om etim es it is said to re p re s e n t LD activity but since a correction with serum proteins and particularly their sulphydryl groups can be made, Somer believes that

p ro te in -b o u n d su lphyd ry l g roups are responsible. Recently, Bails and E d ­wards6 have shown that alcohol dehydro­genase can be p re s e n t in pathological sera, can be id en tif ied as a sixth LD band, and shows considerable activity at pH 9 in buffers used to m easure LD in th e L —» P d irec tio n includ ing D EA , AMPdiol, and AMP.

Howell54 has shown that Z n + + can be an inhibitor for the LD catalyzed reduc­tion of pyruvate-to-lactate at even the upper normal lim it of serum Z n ++, but inhibition is greatly reduced by pyruvate concentrations in excess of 1 mmol per 1 which is usual in the P —» L assay.

The d iuretic , triam terene (2,4,7-tri- am ino -6 -pheny Ip te rid ine), has been found to induce spurious elevations in serum LD as m easured by an autom ated fluorom etric assay b u t not spectropho- tometrically.27

A serum inactivator to the M subunit of LD isoenzymes which m igrated elec- tro p h o re tica lly b e tw een th e b e ta and gamma globulins was reported by Naga- m ine.74 Also, Acheampong-M ensah1 has reported a persistent increase in serum LD activity to be related to an enzyme- IgG -L am bda im m unog lob in com plex. Several LD-imm unoglobulin complexes in hum an serum have been d escrib ed previously by Biewanga and Feltkam p.14

S o u r c e s o f E r r o r

The sources of error in m easuring LD are failu re to m ain ta in th e reac tion at constant tem perature, use of poor quality reagents, e .g ., NADH or NAD + , hemo- lyzed sam ples, and deviation from the p re sc rib e d p ro ce d u re . In creasin g or decreasing the tem perature will change the LD result approximately seven per­cen t p e r d e g re e .51 Various sources of NADH and N A D + contain inhibitors of the LD reaction4'12> 13’40’41'44_48’52'55’57’61’64_ 67,97,104,109 an(j d iffe ren t com m ercial sources of lactic acid also affect th e

26 VANDERLINDE

am ount of LD m easured.22 Erythrocytes contain large amounts of LD , and any hem olysis w ill give abnorm ally h igh re su lts .17 C hanges in the p rocedure — such as substitution of buffer, change of pH , or change in the order of addition of reagents to the reaction m ix tu re— will also a lte r th e am oun t o f LD m ea­sured.20’21’22’23

R e f e r e n c e R a n g e s

Laboratories using different reagents and/or different procedures will have dif­ferent normal ranges and should use val­ues established preferably by their own lab o ra to ry 35 or as d o cu m en ted by the m an u fac tu rer o f th e ir k it or rea g e n t instrum ental system. However, it is not widely recognized that pediatric LD val­ues decrease w ith m aturity28 and older individuals show significantly higher LD activities.71

LD Standardization Efforts

Most clinical chemists accept the need for national and international standard­ization of th e cond itions u n d e r w hich enzymes are measured.

T he G erm an Society for C lin ical Chem istry was the first national society to put forth a recom m endation for the standardization of a m ethod for the esti­m ation of se rum lac ta te d e h y d ro g e n ­ase.34 Their “standard m ethod” recom ­m ended in 1972 is a modification of the W roblewski and LaD ue107 P —> L m ethod carried out at 25°C. They also included a “standard m ethod” for a-H B D H activ­ity.

T he C o m m ittee on E nzym es of the Scandinavian Society for Clinical C hem ­istry and Clinical Physiology published Recom m ended M ethods fo r the D eter­m ina tion o f F our E nzym es in B lood which included LD in the P —» L direc­tion.29 However, the Scandinavian Soci­ety opted for Tris instead of phosphate

buffer, m easu rem en t at 37°C, and a p y ruvate su b s tra te co n c en tra tio n two fold that which was recom m ended by the G erm an Society for 25°C.

W hile the British Association of Clin­ical B iochem istry -W ork ing P arty has m ade m eth o d reco m m en d a tio n s for th ree enzymes, LD was not included.105 Very recently, the Société Française de Biologie Clinique (S.F.B.C.) has de­scribed a P —> L m ethod for total LD at 30° w ith th e goal of m easu rin g each isoenzym e w ith op tim al c o n d itio n s .101 They have found that the addition of N a+ and C l" ions to th e reac tio n m ix ture modifies the stability of the ternary com­plex betw een enzym e, coenzym e, and substrate which usually inhibits the P —*■ L reaction. They report the m ethod to show the following advantages: minimi­zation of the differences in the optimal concentration of pyruvate req u ired for LD 1 and LD 5, im proved linearity, and an increase in the upper limits of linear­ity.

The 1982 College of American Pathol­ogists (CAP) C onference on Analytical Goals in Clinical Enzymology at Aspen, Colorado included a paper on “The M ea­surem ent of Lactic D ehydrogenase” by Dr. Robert McComb. Papers w ere p re­se n te d for d iscussion em phasizing all aspects of the analytical process in clini­cal enzymology, including reagent qual­ity, recom m ended m ethods for m easur­ing various enzymes, quality control, and the relationship of analytical process to the clinical usefulness of enzym e data.53 T he E x p e rt P anel on E nzym es of the In te rn a tio n a l F e d e ra tio n of C lin ical Chem istry (IFCC) has m ade Provisional R ecom m endation (1974) on “ IF C C M ethods for the M easurem ent of Cata­lytic Concentration of Enzym es,”16 “Part 2: Aspartate aminotransferase. M easure­m en t of th e cataly tic co n cen tra tio n in hum an serum ” ,10 and “Part 3: Revised IFC C m ethod for aspartate am inotrans­ferase (AST)”.11 However, no information

MEASUREMENT OF TOTAL LACTATE DEHYDROGENASE ACTIVITY 27

has been requested or issued for lactate dehydrogenase.

The National Bureau of Standards in February of 1982 issued Standard Ref­e re n c e M ateria l (SRM) 909, H um an Serum (Enzymes Values D eterm ined).79 SRM 909, a Hum an Serum Standard Ref­erence Material, has been available for use in assessing the accuracy of clinical m ethods for six analytes: namely, g lu­cose, calcium, chloride, lithium, potas­sium, and uric acid, for calibrating instru­m entation used for these analytes, and for validating in-house or commercially produced quality control materials. The revised certificate now provides uncer­tified values, for information only, of the cata ly tic co n cen tra tio n s of seven enzym es including lactate dehydrogen­ase. T he enzym es w ere d e te rm in e d cooperatively by teams of experts using the “best available” methodology. W here possible, m ethods of the IF C C or the American Association for Clinical C hem ­istry w ere used. The certificate provides ou tlines of th e m ethods and th e final assay reaction conditions, and gives lit­erature references. The Bowers15 proce­dure for P -» L was utilized except for tem perature where a reaction tem pera­ture of 29.77°C was used, verified by a Gallium M elting Point Cell, SRM 1968. Sodium pyruvate is available also from the National Bureau of Standards as SRM 910.

A hierarchy of definitive m ethod, ref­erence m ethod, and Standard Reference M aterial (SRM) can be developed to pro­vide an accuracy base for many of the inorganic, and biochem ical analytes in hum an serum m easu red ro u tin e ly by clinical labora to ries.78,81 In the area of c lin ical enzym ology, s tandard iza tion efforts have cen te red prim arily on the developm ent of reference and selected m ethods rather than on enzyme m ateri­als. However, the National Com m ittee on C lin ical L aborato ry S tandards has recently published a report entitled “C7-

T-Tentative Guidelines to Kinetic Anal­ysis of Enzym e Reactions.”75 W hile sev­era l sc ien tis ts and o rgan izations have suggested requirem ents for enzym e ref­erence m aterials,37,84 to date only a m ate­rial for aspartate aminotransferase (E.C.2.6.1.1) has been p repared , charac te r­ized and validated25’83,85’89 as a proposed biological validation and transfer material (BVTM).90 The availability of a BVTM for aspartate am inotransferase in conjunc­tion with the IFCC proposed reference m ethod for th is en zy m e11 p rov ides a model system for the standardization of o th e r enzym es of clin ical labo ra to ry in te re s t in c lud ing LD . Som e o f th e d ev e lo p m en ta l w ork for an analogous m aterial for lactate dehydrogenase has been accomplished in that a highly puri­fied LD 1 has been used as a reference m ateria l in th e N ew York S ta te P ro fi­ciency Testing Program .18 Also, the in ter­converting of results for serum samples and for highly purified LD isoenzymes 1 and 5 assayed L —» P in pyrophosphate, Tris and AMP has been investigated.24

Acknowledgments

The author wishes to thank Dr. Robert McComb of the Hartford Hospital, Hartford, CT for reviewing the manuscript and making some helpful suggestions.

References

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28 VANDERLINDE

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