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Biochem.J. (1967)105,599Printed in GreatBritain

The Determination of Lactate Dehydrogenase Isoenzymesin Normal Human Muscle and Other Tissues

By A. E. H. EMERYUriver8ity Department of Medical

Genetica,The Royal Infirmary, Manchester

(Received31 March 1967)

1. A technique has been developed, based on preferentialinhibitionby urea, fo rdetermining the amounts and proportions of the M and H sub-units of lactatedehydrogenase (referredto as LDH-Mand LDH-H respectively)in human tissues,including muscle. 2. There was good agreement between the resultsobtained withurea inhibition and those obtained with starch-gel electrophoresis.3. Withincreasing age there was a significantdecrease in the total amount of lactatedehydrogenase and the amount of LDH-M in skeletalmuscle. This could not beaccounted forby the replacement of functioning muscle tissueby fibrous connectivetissue. 4. The proportion of LDH-M was less in certain muscles (e.g. soleusand

extra-ocular)than in other muscles (e.g. gastrocnemius and rectus abdominis).5. The proportions QfLDH-M and LDH-H did not differ significantlyin differentsuperficiallimb muscles and were not significantlyaffectedby eitherage or sex.6. Specimens of muscle from 86 different -individuals(all Europeans) have beensubjected to electrophoresis,bu t no variants of lactatedehydrogenase isoenzymeshave been found.

The enzyme LDH* is composed of two types ofsub-units referred to as A and B by Markert (1962)and as M and by Cahn, Kaplan, Levine &Zwilling (1962),since M sub-units predominate inskeletal muscle and H sub-units predominate in

heart muscle. These types of sub-units are believedto combine in various tetrameric associations toproduce five isoenzymes such that LDH-1, themost rapidly migrating isoenzyme, has thecomposition HHHH ; LDH-2, HHHM; LDH-3,[HMM; LDH-4, HMMM; and LDH-5, MMMM.Evidence in support of this hypothesis has comefrom several sources (Vesell, 1965). The M and Hsub-units of LDH are referred to below as LDH-Mand LDH-H respectively.

Several investigators have demonstrated thatboth LDH-5 and LDH-1 are inhibited by urea,

though the former is inhibited more than thelatter (Plurumer, Wilkinson & Withycombe, 1963;Richterich & Burger, 1963; Brody, 1964; Brody &Engel, 1964; Emerson & Wilkinson, 1965; Hardy,1965; Konttinen & Lindy, 1965; Withycombe,Plummer & Wilkinson, 1965; Lindy & Konttinen,1966).The present work shows that under certainexperimental conditions in the presence of ureathere is an increase in th e activity of LDH-1 anda decrease in the activity of LDH-5. These findingshave led to the development of a technique fordetermining the relative proportions of LDH-Hand LDR-M in human muscle and other tissues.

*Abbreviation: LDH, lactate dehydrogenase.

No previous study appears to have been made ofLDHisoenzymesinhumantissuesbythist.eohnique,and no systematic study has been made of theeffectsof age and sex on the amounts and propor-tions of LDH-M and LDH-H in fresh biopsy

specimens of normal human skeletalmuscle.

EXPERIMENTALTiWue8. All specimens of muscle (300-500mg.) were

removed at operations conducted under general anaesthesia'from individuals with no history of any neuromusculardisorder. Some specimens of tissueother than muscle wereobtained at autopsy. After excisionany adherent fat orconnective tissue was r emoved and the specimens werethen immediately washed in 0-9% NaCI, to remove anycontaminating erythrocytes, blotted dry; weighed (wetweight) and then homogenized in about 5 vol. of water in:a Potter-Elvehjem homogenizer, the tube being kept coldby surrounding it with ice. Enzyme assays, nitrogendeterminations and electrophoresiswere carriedout on thewhole suspension suitablydiluted with water. In none ofthe specimens of muscle did the blood content, estimatedby the method described by Holzer, Sedlmayr & Kiese(1956),exceed 5% and in most cases -the blood contentwas less than 2%, which agrees well with the findingsofothers (Gitlin & Janeway, 1954; Pennington, 1963).Differences in enzyme content in different specimens ofmuscle cannot therefore be due to differencesin the amountof blood present in these specimens.

Chemicals. NAD, p-nitro-bluetetrazolium and phena-zine methosulphate were obtained from Sigma Chemical

Co. (London) Ltd., London, S.W.6.

Staroh was suppliedby Connaught Medical Research Laboratories, Toronto,

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Canada. PurifiedLDH-1 (pigheart)and purifiedLDH-5(rabbitmuscle) were obtained from C. F. Boehringer Corp.(London) Ltd., London, W. 5. Al l other chemicalsweresupplied by BritishDrug Houses Ltd.,Poole,Dorset.LDH e8timation.LDH activitywas determined spectro-

photometrically with lactate as substrate. The methodused is based on that of Wacker, Ulmer & Vallee(1956)with certainmodifications (Boutwell,1961).The reactionmixture consistedof 1-5ml.of 50mM-tris-HCl buffer,pH9-0, 1-0ml.of 0 25M-sodium lactate, pH9-0, 0-3ml. of50mm-NAD and 0-2m]. of suitablydilutedhomogenate.The NAD solutionwas freshlyprepared beforeeach experi-ment. Activity was calculatedfrom the rate of increaseofE' * during the 5 min. periodafter the additionof thehomogenate, at 25.The concentration of each homogenatewas so adjusted (usually1:330,w/v, dilutionin water) thatthe change in extinction was approx. 0-020-0-040/min.Under theseconditions the reactionrate is linearand directlyproportional to the amount of enzyme present. The effectsof urea were studied by incorporatingurea in the tris-HClbuffer; in this case, after the addition of the homogenateto the reaction mixture, time (1-2min.)was allowed forequilibration,and then the rate of increase in Ej49

*

wasdetermined over the next 5min. Allenzyme determinationswere carriedout within 0.5-1hr. after the tissueshad beenexcised. Enzyme activites are expressedas ,umolesofsubstrate converted/min. at 250and pH9 0.Non-collagen nitrogene8timation.The amount of non-

collagennitrogen in muscle homogenates was determinedby digestingthe homogenate (1: 33,w/v, dilutionin water)overnight at 20 with 9vol. of 0-05N-NaOH (Lilienthal,Zierler,Folk,Buka & Riley,1950),centrifugingthe digestand then determining the amount of nitrogenin the super-natant fluid by a micro-Kjeldahlprocedure.Electrophore8i8.Homogenates were subjectedto vertical

starch-gelelectrophoresis(Smithies,1959) at 40 and 4-0v/cm. for approx. 18hr. (Emery, Sherbourne & Pusch, 1965).The starch gelswere buffered at pH8-5 with mM-EDTA-25mM-boric acid-45mM-tris (final concns.)as describedbyBoyer, Fainer & Naughton (1963). The concentration ofeach homogenate was so adjusted that the volume (30,ul.)of material subjectedto electrophoresisalways containedabout the same amount of LDH activity(85-95mpmoles/min.). After electrophoresis,LDH activitywa s locatedbythe method of Dewey& Conklin (1960)with certain modi-fications (Blanco & Zinkham, 1963). The proportions ofthe individual isoenzymes were determined with a Chromo-scan recordingand integratingdensitometer (Joyce-LoeblCo. Ltd.). Electrophoresiswas carriedout the same daythat a biopsyspecimen was excised and only on freshlyprepared homogenates.For each specimen of tissue the amount of enzyme

activity, the amount of non-collagennitrogen and thepercentages of the various isoenzymes on electrophoresisare the mean values of duplicatedeterminations on thesame homogenate.

RESULTSThe effectsof differentconcentrations of urea on

the activities of purifiedLDH-1 (from pig heart)and purified LDH-5 (from rabbit muscle) werefound to be quite different(Fig. 1). With 2M-urea(final concentration in the reaction mixture) there

150

100

50

0 2 3 4Final conen. of urea (M)

Fig. 1. Percentage activity of purifiedLDH-1 (e ) andLDH-5 (0) in the presence of urea with lactate (finalconcn. 83mM)as substrate at pH9-0 and 25. Each pointis the mean of three experiments, an d the vertical linesrepresent 1 S.E.M.on either sideof the mean.

was almost complete inhibition of LDH-5, whereasthe activity of LDH-1 was approx. 1-5 times theactivity in the absence of urea. Very similar resultswere obtained when homogenates of human heartand liverwere used.

Previous investigators have shown that, withlactate as substrate, LDH-1 is inhibited after ithas been incubated with 2m-urea alone for about15min. before the reaction rate is determined(Withycombe et al. 1965). This findinghas beenconfirmed in the present investigation.However,when the enzyme is added to urea in the presenceof NAD and substrate there is increased enzymeactivity. Under these conditions the percentageincrease in activity with 2M-u re a gradually de-creases from about 150% after 5-6min., to about145% after lOmin. and 140% after 20min.Since in the presence of 2M-urea the activityof

LDH-1, which is composed entirelyof H sub-units,is increased 1-5 times, and the activityof LDH-5,which is composed entirely of M sub-units, isalmost completely inhibited,it is presumed that inany mixture of isoenzymes with 2M-urea theactivityof all the H sub-units would be increased1-5 times and the activity of all the M sub-unitswould be almost completely inhibited. It shouldtherefore be possibleto measure the amounts andproportions of H and M sub-units in any particulartissue by determining the enzyme activity in thepresence and absence of 2M-urea:

If activityin the absence of urea = Tand activityin the presence of 2M-urea = U

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LACTATE DEHYDROGENASEISOENZYMESthen activity due to LDH-H only in

the absence of urea = U/1-5

and the percentageof LDH-M = T-U/1 5 x 100

n this way the amounts and proportions ofDH-M and LDH-H have been determined in

queous homogenates of various tissues removedautopsy (Table 1). The results agree in generalith those obtained by other methods such asensitometry of isoenzyme bands after electro-horesis (Wr6blewski & Gregory, 1961), elutionrom the supporting medium after electrophoresisVesell& Beam, 1961), column chromatography onDEAE-Sephadex (Richterich,Schafroth & Aebi,963) or the use of coenzyme analogues (Dawson,

Goodfriend & Kaplan, 1964).Further evidence that the method of urea

nhibition,as used in this investigation, is probably

reliablemeans ofassessing the relative proportionsf M and H sub-units in human tissuescomes fromhe close agreement between the observed andredicted relationships between the percentage ofDH-5 on electrophoresis and that of M sub-units determined by urea inhibition. Assuming thathe various isoenzymes of LDH are tetramersormed by random combinations of the two typesf sub-units (M and H), and that the proportionsf the various isoenzymes depend on the relativeroportions of the two sub-units, it is possible toalculate from the binomial expansion the per-entage of LDH-5 for d iffe rentproportions of M

sub-units. The relative proportions of the fiveisoenzymes are given by the terms ofthe expansion:

(H+M)n = n!HrMn-r/(n-r)!r!

where M and H represent the relativeproportionsof M and H sub-units (e.g. 1/10 and 9/10),r is the

number of H sub-units contributing to any par-ticular isoenzyme (e.g. in LDH-1, r is 4) and n isthe total number of sub-units in each isoenzyme(i.e. 4). The predicted relationship between thepercentage of LDH-5 and the percentage of Msub-units (Table 2) is in fairlycloseagreement withthe observed relationship(Fig. 2). The observedrelationship was obtained by determining thepercentage of LDH-5 from the results of electro-phoresis and that of LDH-M from the results ofurea inhibition.

The activities and proportions of LDH-M and

LDH-H in various skeletal muscles fromindividuals

with no neuromuscular disease are given in Table3. There was considerable variation in the totalactivity of LDH (LDH-M and LDH-H) evenwithin the same musele, though there was clearlyless enzyme in soleus and extra-ocular muscles thanin the other skeletal muscles that have beenstudied. There was a slight bu t significantregres-sion on age for the total amount of LDH and fo rthe amount of LDH-M (Table 4). The regressionson age for males and females were both significant,but the differencebetween them was not significant.There was slightlymore enzyme in muscles from

Table 1. Activitiesand proportions of LDH-M and LDH-H in varioustissues

Fo r detailsseethe text.

Enzymeactivity(,umoles/min./g.) Enzymeactivity (% of total)

Tissue

ver

pleen

ung

hyroid

ymph node

idney medulla

idney cortex

rain

III

IIIIIIIIIIVIIII

II

I

II

I

II

I

Total

21-626-3

13*517-317-48-43*66-313-412-113-810*817-438-015-120-69*810-3

H1-61-3

3*14*07-52-61.92*15.94.95-35-27-2

20-713-218-08*49-6

M20-025-0

10*413-39.95-81-74- 27-57-28-55-610-217-31.92-61-40-7

H7-45*0

23-023-143-131-052-833-344-140-538-448-241*454-587-487*485*793*2

M92*695077-076-956*969-047-266-755.959-561-651-858-645-512-612-614-36-8

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A. E. H. EMERY

Table 2. Predicted percentageofLDH-5 for differentproportions of H and M aub-unit8

For details of the caloulationsee the text.

Europeans) with no neuromuscular disease havebeen subjected to electrophoresis, bu t no variantsof LDH isoenzymes have been found.

Proportion ofsub-units (% )

H M100 070 3050 5040 6030 7020 8010 900 100

LDH-5 (%)0-000-816-2512-9624-0140-9665-61100-00

100 r

50 F

0

0 a

/

*,

50

LDH-M(%)

0

Fig. 2. Observed (0 ) and predicted (----) ribetween the proportion of LDH-5 and the prLDH-M in various skeletalmuscles.

males compared with females but thewas not significant.

The proportion of LDH-M was greatermuscles (e.g. gastrocnemius and rectus athan in other muscles (e.g. soleus Andextibu t did not differ significantlyin differificial limb muscles. There was no significlation between age and the proportions c(correlation coefficient0-17) and LDH-Ition coefficient 0-16). The proportionwas slightlygreater in males (89-5 + 3-80females (86-8 + 4-5/), and that of LDslightly greater in females (13-2+450*OAmales (10-5+338%), bu t the differences

significant(these values ar e means + s.imens of skeletal muscle from 86 indiv

DISCUSSION

In the present investigation LDH activity was

determined with lactate as substrate. The rate ofreaction is greater with pyruvate as substrate,but there are several theoretical as well as prac-tical reasons for preferring the reverse reaction(Snodgrass, Wacker, Eppinger & Vallee, 1959;Fawcett, Ciotti &Kaplan, 1961; Amador, Dorfman& Wacker, 1963; Amador, Reinstein & Benotti,1965).Under the experimental conditions used in this

investigation it has been found that with 2M-ureathere was almost complete inhibition of LDH-5whereas the activity of LDH-1 was approx. 1-5

times the activity in the absence of urea. Theshapes of the curves for the effectsof various con-% t centrations of urea on LDH-1 and LDH-5 are

comparable with those obtained by Fritz (1965)for the effect of oxaloacetate on the activities of

/* these isoenzymes. Fritz (1965) reported that withb/I low concentrations of oxaloacetate LDH-1 was!00 inhibited, bu t the activity of LDH-5 was increased

to about 120% of the activity in the absence ofoxaloacetate. This effectwas ascribed to allostery;oxaloacetate probably brings about a molecularalteration in LDH-5 that leads to th-e exposure ofnew substrate sites and therefore to increasedenzyme activity. Possibly urea in low concentra-- t-i tions has a similar effecton LDH-1.

100 Several previous investigators have studied theLDH content of human skeletalmuscle, bu t without

elationshis exceptionall have used pyruvateas substrate.oportionof (Ronzoni,Berg& Landau, 1960; Schmidt &

Schmidt, 1960; Tada, Watanabe & Chikaoka,1961; Heyck, Laudahn & Luders, 1963; Kar &Pearson, 1963; Richterich et al. 1963; Dawson &Kaplan, 1965; Monckton & Nihei,1965; Johnston,

difference Wilkinson, Withycombe & Raymond, 1966; Kleine& Chlond, 1967). Those investigatorswho have

in certain studied the enzyme content of different skeletal6bdominis) muscles and have used fresh biopsy material ratherra-ocular), than autopsy material, in which there is some lossent super- of enzyme activity, have failed to demonstrate any-ant corre- significant difference in the amount of LDH)f LDH-M activity in different superficial limb musclesI (correla- (Laudahn & Heyck, 1963). However, some recent)f LDH-M findings suggest that the enzyme content of redVO)than in muscle may be less than that of white muscle)H-H was (Kleine & Chlond, 1967), and in the present investi-D)than in gation less enzyme was found in soleus (red)were not than in adjacent gastrocnemius (white) muscle.

i.). Speci- Monckton & Nihei (1965) have found that theiduals (all LDH content of limb muscles is significantly

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LACTATE DEHYDROGENASE ISOENZYMES

Table 3. Activitie8and proportionmof LDH-M and LDH-H in variou8 8keletalmu8clesFor detailsseethe text.

Enzyme activityAge (Qmolesfmin./g.)

Muscle Sex (years) AH M

ectus F 78 1-5 12-6abdominis M 58 3-2 20-7M 61 4-2 32-6M 60 5-5 28-2M 69 3-7 23-9

Gastrocnemius M 35 3-7 31-9M 44 4-8 29-3F 25 4-3 23-6

Deltoid M 23 1-4 22-0M 31 1-6 53-8

ectoralis F 50 4-8 16-2major F 40 3-6 28-2

M 33 3.5 32-1F 45 2-6 22-3F 39 3-1 29-1M 51 3-2 32-8

Quadriceps F 18 3-6 31-5terno F 20 5-4 25-2mastoidartorius M 52 1-4 16-4

Diaphragm F 17 3-4 28 0atissimus M 70 3-5 24-0dorsiSoleus M 81 6-4 3-6

M 70 7-3 3-1Ocular 3-0 1-4muscles* 1-7 1-2

Enzyme activity(,umoles/min./ Enzyme activity100mg. of non-collagenN) (%)

rotal H M Total H M

14-1 4-6 39-0 43-6 10-6 89-423-9 8-2 53-8 62-0 13-2 86-836-8 14-1 108-3 122-4 11-5 88-533.7 16-8 86-2 103-0 16-3 83-727-6 20-4 131-9 152-3 13-4 86-635-6 11-3 97-8 109-1 10-4 89-634-1 15-2 93-4 108-6 14-0 86-027-9 17-9 97-8 115-7 15-5 84-523-4 8-7 138-8 147-5 5-9 94-155-4 7-0 236-4 243-4 2-9 97-121-0 21-2 72-3 93-5 22-7 77-331-8 15-8 124-0 139-8 11-3 88-735-6 17-0 157-2 174-2 9-8 90-224-9 13-8 116-2 130-0 10-6 89-432-2 16-8 160-4 177-2 9-5 90-536-0 12-3 127-7 140-0 8- 8 91-235-1 18-7 161-5 180-2 10-4 89-630-6 27-0 126-1 153-1 17-6 82-4

17-8 6-3 74-9 81-2 7-8 92-231-4 12-5 103-7 116-2 10-8 89-227-5 13-4 92-7 106-1 12-6 87-4

10-0 -10-444 - -2-9 -

36-0 64-029-8 70-2

- 68-2 31-8- 58-6 41-4

* In each experiment the material from three patientswas combined.

Table 4. ReIation8hip between age (X) and amount of enzyme activity(Y) in variou88keletalmU8c1e8Total LDH (1) is based on the 21 resultsgiven in Table 2. Total LDH (2) is based on the resultsof 50 fresh

biopsy specimens of various skeletalmuscles in which theamounts of LDH-H and LDH-Mwere not determined.LDH activities are given as means + s.D. The resultson ocularand soleusmuscleshave been excluded. N.S.,Not significant.

LDH activity(umoles/min./

No. of 100mg. ofnon-results collagenN)21 14-2+ 5-521 114-3+43-321 128-5+44-450 121-5+50-224 124-0+46-326 119-1+54-4

Regression lineY= 17-8-0-08XY= 168-9- 1-24XY= 186-7-1-33XY= 199-1-1-67XY=200-3-1-58XY= 198-7-1-77X

reater than that of trunk muscles. Dawson & were not given and their observations were confinedKaplan (1965) considered th e possible effects of age to autopsy material. In th e present study it wasnd sex on th e LDH content of skeletal muscle. found that there is a small but significant decreaseThey noted that '. . . younger individuals, particu- in LDH content with increasing age. Since thisarly men, tended to have ... higher levels of total effect is seen when the amount of LDH is expressedLDH' in their muscles, bu t results at different ages in terms of the amount of non-collagen nitrogen,

Vol. 105 603

LDH-HLDH-MTotal LDH (1)Total LDH (2)MalesFemales

Significanceofregressioncoefficient

N.S.P

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604 A. E. H. EMERY 1967

the effect of age cannot be du e merely to gradualreplacement of functioning muscle tissueby fibrousconnective tissue. No significantdifferencewasfound in the LDH content of muscles from malesand females.By using the technique of urea inhibition it was

found that certain muscles (e.g. soleus and extra-

ocular) have a greater proportion of LDH-H thanother muscles (e.g. gastrocnemius and rectusabdominis). These findings are in agreement withthose of Kar & Pearson (1963),who used agar-gelelectrophoresis,and Dawson & Kaplan (1965),whoused coenzyme analogues. Muscles situated deeperin the limb (e.g. soleus)and concerned more withsupport than with phasic contraction have a greaterproportion of the more rapidly migrating iso-enzymes than neighbouring superficialmuscles suchas the gastrocnemius. These observations suggestthat the isoenzyme pattem of a particular skeletal

muscle may be determined by the proport ion ofred and white fibresin that muscle. By an ingenioustechnique involving the determination of iso-enzyme patterns in isolated muscle fibres, vanWijhe, Blanchaer & St George-Stubbs (1964) havepresented evidence that supports this idea.Gratefulthanks are due to Dr A. H. Gowenlock formuch

helpfuladvice and to the Muscular Dystrophy Group ofGreat Britain for a research grant. Miss V. Hodson andMiss C. Mold gave valuable technicalassistance.

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