Tissue lactate dehydrogenase isoenzyme patterns in rats fed a hypercholesterolaemic diet

Atherosclerosis

Elsevier Publishing

TISSUE FED

Company,

LACTATE

Amsterdam

DEHYDROGENASE

ISOENZYME

A HYPERCHOLESTEROLAEMIC

P. SEETHANATHAN

AND

Division of Biochemistry, (Received

393

- Printed in The Netherlands

PATTERNS

IN RATS

DIET

P. A. KURUP*

University

of Kerala,

Triuandrum-1

(India)

April Znd, 1970)

SUMMARY

LDH isoenzyme

changes in various tissues in rats fed a high fat-high

diet have been studied:

an attempt

enzyme changes are correlated.

In the liver and aorta, where maximum

lation occurs, the predominant

decrease is in LDH-1

muscle, where lipid accumulation

cholesterol

was made to see if lipid accumulation

and iso-

lipid accumu-

and 2. In the heart and skeletal

is much less, the maximum

decrease is in LDH-4

and 5. In tissue like the kidney and lungs, where fat accumulation

is moderate, LDH-4

and 5 increase while LDH-1 and 2 usually remain unaffected or show a small decrease. Thus, decreased LDH-1

and 2 appear to be associated

lation, while decreased LDH-4

Key words:

Atherosclerosis

with maximum

lipid accumu-

and 5 with less lipid accumulation.

-

Hypercholesterolaemic

isoenzymes - Lipid accumulation

diet

-

Lactic

dehydrogenase

- Tissue enzymes

INTRODUCTION

Lactate fractionated

dehydrogenase

(r.-1actate:NAD

oxidoreductase,

into five well defined isoenzymes,

LDH-4 and LDH-5.

designated

LDH-2,

LDH-3,

Separation of these fractions was first achieved by electrophoresis

on starch block1 and on papers. All the five &enzymes fluids and the relative distribution of predominance

E.C. 1.1.1.27) has been

LDH-1,

of the isoenzymes

1 & 2 + 3; lung: 3 B 4 and 5 >

occur in most tissues and body

varies from tissue to tissue. The following in different

tissues has been reporteda.

order Heart:

1, and 2; liver: 5 & 4 and 3 B 2 and 1; kidney:

2 and 1 9 3 and 4; spleen: 3 and 4 9 2 and 1 9 5; brain: 3 > 2 and 1 > 4 $ 5;

* Reprints may be requested from the senior author (P.A.K.). Atherosclerosis,

1970, 12: 393-399

394

P.SEETHANATHAN,P.A.KURUP

skeletal

muscle:

5 3 4 and 3 & 2 and 1; skin: 5 and 4, 9 3; erythrocytes:

2 and 1

~33;leucocytes:2and3>4and5,>1;serum:2>1~3~4>5. Thus there LDH-5

is undoubtedly

in liver

LDH-1

and skeletal

a predominance

muscle

of LDH-1

and LDH-3

in heart

in the lungs,

muscle,

brain

of

and spleen.

and 2 are present in almost equal amounts in the serum, kidney and red cells. Serum

lactate

atherosclerosis isoenzyme

dehydrogenase

pattern

in human

has been studied by a number

pattern

polyacrylamide

of the serum

in patients

gel electrophoresis

a reduced LDH-B/LDH-1

with

heat stability fractions

ratio, but no concomitant

technique

and observed

on the 3rd post-infarction

A concomitant

atherosclerosis

elevation

infarction

significantly

other investigations

of total

patients

elevated

day, which attained

rise in total LDH-2,

days. Several

coronary

infarction

using

of LDH-I,

serum

LDH.

by the differential

total

LDH

and LDH-1

a peak value by the 5th day.

3 and 4 was also observed have reported

and

et al.4 studied the

CHAZOV

and found an increased relative content

et al.5 studied the serum of myocardial

RAMDEO

myocardial

of workers.

increased

on the 3rd and 5th

LDH-1

after myocardial

infarction6y7. However, atherosclerosis, tissues

no reports except

are available

for the aorta.

was therefore

undertaken

about

A study

tissue

LDH

isoenzymes

of the isoenzyme

in rats fed for a long period

cholesterol

diet: the results are reported

MATERIALS

AND

pattern

pattern

in

in various

on a high fat-high

in this communication.

METHODS

Young male albino rats were obtained from the King Institute,

Guindy,

Madras

(average weight 120 g) and were divided into 2 groups of 10 rats each. One group was fed a hypercholesterolaemic gram (Cicer arietinum) and Wakeman)

diet of the following composition:

30%,

sucrose 25%,

whole milk powder 16%, salt mixture

4%, yeast tablets

I%,

(Hubbel,

shark liver oil 2%, hydrogenated

BengalMendal

ground nut

oil 10% and cholesterol

5%. The other group was given the normal laboratory

(Hind

supplied

Lever

rat feed),

cereals, oilcakes, tables.

by Hindustan

Lever

bran and milk solids; it contains

The animals were maintained

Ltd.

Its ingredients

24% protein and 4% ether extrac-

on the respective

diet for 6 months.

of this period the animals in both groups were fasted overnight on the head. Various estimating

LDH

were determined

Estimation After specimens.

tissues were immediately

isoenzymes.

Total

cholesterol,

in the tissues by conventional

At the end

and killed by a blow

removed into ice-cold containers free cholesterol

for

and phospholipids

methodss.

of LDH isoenzymes excision,

any adherent

The tissues

any contaminating

fat or connective

were then immediately

erythrocytes.

and then homogenized

tissue

was removed

washed with 0.9%

They were then blotted

1970, 12: 393-399

it with ice. The homogenate

from all

NaCl to remove

dry, quickly

in 5 vol. of distilled water in a Potter-Elvehjem

the tube was kept cold by surrounding Atherosclerosis,

diet

include

weighed wet homogenizer;

was centrifuged

LDH

ISOENZYMES

TABLE

RATS

395

1 LDH

TISSUE

IN HYPERCHOLESTEROLAEMIC

ISOENZYME

PATTERNS

IN

RATS

FED

HIGH

FAT-HIGH

CHOLESTEROL

DIET

The reaction system contained 0.02 ml of the enzyme extract + 1 ml of 0.1 M Sorenson glycine buffer, containing 0.476% Na-lactate (pH lO.O), 0.2 ml of 0.57’ NAD solution and incubated at 37°C for 15 min. The reaction was stopped by the addition of 1 ml of DNP solution and leaving for 15 min. The o.d. after adding 10 ml of 0.4 N NaOH was read at 440 mp. The total LDH and the enzyme stable to 15 min incubation at 55 and 60°C respectively were determined. The enzyme activity is expressed in terms of ,ug of pyruvate formed per mg of protein of the enzyme extract. a, Rats fed normal diet. b, Rats fed high fat-high cholesterol diet. Total LDH LDH-4 and 5 LDH-3 (pg of pyruvate formed per mg of protein) 1. Liver

a b

2. Lungs ; 3. Kidney ; 4. Pancreas : 5. Heart : 6. Skeletal muscle a b 7. Serum F? 8. Aorta* :

21.1 14.03 31.00 28.79 70.67 86.38 28.79 28.34 246.13 179.90 85.3 71.7 1.51 5.91 23.03 20.73

5 * & & + & * + & & * + f * + f

1.20 0.80 1.1 0.7 1.5 1.01 0.41 0.38 1.25 1.60 0.56 0.74 0.05 0.09 1.40 0.90

17.19 11.51 10.33 22.55 18.32 25.91 7.19 12.40 105.49 26.9 40.2 1.98 0.15 0.96 14.80 11.51

+ f f + + * & & f k & f + * & *

1.4 1.2 0.61 1.11 0.71 0.68 0.26 0.34 0.60 0.58 0.43 0.21 0.02 0.03 0.98 0.74

1.35 1.61 14.76 1.44 5.23 5.75 3.59 12.40 17.58 8.96 26.73 57.59 0.38 1.60 3.70 7.67

LDH-1

& 0.26 * 0.30 * 0.70 * 0.35 += 0.20 & 0.11 * 0.19 += 0.26 + 0.26 & 0.18 & 1.01 * 1.03 & 0.01 + 0.02 & 0.19 & 0.20

2.7 0.89 5.90 4.79 47.10 54.70 17.95 3.54 123.06 143.9 12.2 26.9 0.98 3.36 4.25 1.53

ad

2

f 0.26 * 0.17 * 0.52 * 0.40 * 1.15 * 1.20 & 0.23 + 0.19 i_ 0.56 & 0.69 * 0.16 ~_t0.24 * 0.02 & 0.01 + 0.18 * 0.07

* The results in the case of aorta are expressed as ,ng of pyruvate formed per 10 mg of wet tissue. TABLE LDH

2

ISOENZYME

PATTERN

land P values. a, Normal diet. b, high fat-high cholesterol

1. Liver 2. Lungs 3. Kidney 4. Pancreas 5. Heart 6. Muscle 7. Serum 8. Aorta

t’s P t’s P t’s P t’s P t,s P t’s P t’s P t’s P

IN

TISSUES

FED

THE

HIGH

FAT-HIGH

CHOLESTEROL

DIET

diet.

between a and b between a and b between a and b between a and b between a and b between a and b between a and b between a and b

Total LDH

LDH4 and 5

LDH-3

LDH1 and 2

24.5 1 O.OOl 163.09
15.40
3.27 0.01 >O.OOl 85.10
29.14
Atherosclerosis,

1970, 12: 393-399

3

E

El

.s

;;

r: 2. _*

:: z

P %

b

3

1. 2. 3. 4. 5. 6. 7.

_

Serum Liver Heart Lungs Kidney Muscle Aorta

Normal diet. b, High fat-high

a,

diet.

HIGH

FAT-HIGH

& 12.50

f 16.50 * 12.10 f 8.90 ~_t 11.50 * 8.60 5 16.50

50.5

378.75 207.64 315.62 369.3 70.10 499.4

a

15.56 10.50 12.60 10.70 9.10 18.70

16.40

DIET

+ & & * f + f

21.5 296.29 105.26 260.41 309.52 52.8 375.0

a 3.40 12.70 11.60 7.40 11.90 10.50 11.60

219.8 4173.91 122.13 470.58 408.75 54.00 1120.0

b

Free cholesterol ;yg,;,q;nl OY100 g wet tissue)

CHOLESTEROL

h 5 * & * &

542.87 * 5928.20 240.0 519.85 467.59 81.18 1633.87

b

Total cholesterol $;g/l,;,;zl or 1OOg wet tissue)

cholesterol

TISSUE LIPID LEVELS IN RATS FEIJ THE

TABLE

f + f + f + &

12.50 18.7 12.8 10.90 13.1 11.6 12.80 96.0 2506.6 2223.3 2665.9 2615.4 1241.4 1935.4

a + & * * + * &

10.80 14.60 19.10 16.6 16.80 15.60 21.50

344.0 5175.9 2818.8 2702.70 3066.5 1330.3 3157.9

b

Phospholipids ;;gn,yq;nl OY1OOg wet tissue)

5 i f& & * f

12.50 17.80 15.0 18.4 17.10 21.60 18.90

LDH

ISOENZYMES

TABLE

IN HYPERCHOLESTEROLAEMIC

P

397

4

LIPID ,_.E”ELS OF THE DIFFERENT TISSUES IN RATS AND

RATS

FED

THE

HIGH

FAT-HIGH

CHOLESTEROL

DIET

VALUES

Normal diet. b, High fat-high

a,

cholesterol

diet. Total cholesterol

t’s between P t’s between P t’s between P t’s between P t’s between P t’s between P t’s between P

1. Serum

2. Liver 3. Heart 4. Lungs 5. Kidney ,6. Muscle 7. Aorta

.at 0°C at 1500

.and calorimetric content

LOWRY

1.98 0.95 > 0.9 101.72

34.23 (0.001 383.56 0.05 35.67 0.1 96.43

33.57 0.001 42.08



53.39 (0.001

546.46

a and b


a and b

4.52 0.01

a and b

> 0.001

29.60
a and b


a and b a and b

x g; the supernatant,

.cedure described by

.of

Phospholipids

a and b

enzyme assay. Separation

protein

Free cholesterol

after suitably

and estimation

ROMAN

estimation

diluting with water, was used for

of the enzyme was carried out by the pro-

et al.9, based on the heat stability of the pyruvate

of the enzyme

extract

et al. after trichloroacetic

formed

of the various fractions

in the enzyme

in each case was estimated

reaction.

The

by the method

acid precipitationlo.

RESULTS

The LDH isoenzyme pattern in the heart, liver, lungs, kidney, pancreas, muscle, aorta and serum of the rats fed the high fat-high with the corresponding ‘Table

1. Tissue total

and free cholesterol

and phospholipid

cholesterol

diet varied

pattern

the predominant

LDH-4

and 5 and LDH-3

decreased,

LDH-1

and 2 slightly

5 are the major decreased where

fractions,

considerably,

normally


LDH-4

total

fractions,

the former

In the skeletal LDH

while LDH-3

in different

from tissue

and 2 are normally

increased.

3.

activity

In the

heart,

total LDH activity

where

decreased.

much more than the latter, muscle, where normally slightly

and LDH-1

decreased,

tissues in the rats fed

to tissue.

LDH-3

decreased,

increased,

Atherosclerosis,

and

and 5

In the liver,

total LDH activity

slightly

while

LDH-4

LDH-4

and 2 increased.

and 5 are the major fractions,

and 5 slightly

are set out in Table

2 and 4.

The changes in the LDH isoenzyme LDH-1

skeletal

diet, as compared

levels in the tissues of rats fed the normal diet are shown in

‘The t and P values are given in Tables .a high fat-high

cholesterol

slightly

while LDH-1

1970, 12: 393-399

P. SEETHANATHAN, P. A. KURUP

398 and 2 considerably major

fraction,

increased, LDH

total

LDH-3

decrease.

decreased. LDH

while LDH-3

activity

was not affected,

LDH-4

increased,

In the serum,

total

LDH

the increase

increased,

where normally

activity

increased,

in LDH-1

and 5 a small

and 2 are the major fractions,

increased,

total

in-

LDH

while LDH-1

and 2

was only slightly decreased,

while LDH-1

LDH-1

LDH-3,

total

and 2 slightly

In the pancreas,

and 5 and LDH-3 LDH-3

is the

LDH-4

and 2 showed

and 5 and LDH-1

In the aorta, total LDH activity

and 5 slightly decreased,

isoenzymes,

LDH-1

raised, LDH-4

LDH-4

affected,

and LDH-1

was more or less unaffected.

decreased.

ably decreased.

was not appreciably decreased

where normally

was only slightly

creased,

considerably

activity

was considerably

In the kidney,

activity

In the lungs, where in the normal rat LDH-3

and 2 was consider-

and 2 are the predominant

4 and 5 and LDH-1

and 2 all

and 2 being the most prominent.

DISCUSSION The serum should reflect the overall increase or decrease in the individual isoenzymes

taking

agree with this.

Increased

has been reported of workers.

place in the tissues and the pattern total

LDH

activity

in human atherosclerosis

The results now reported

observed

in the serum

and myocardial

(particulary

infarction

is the aorta.

activity

in homogenates

sclerotic

portions

activity

Kirk

for the serum in rats fed a high fat-high

et al .11 found no significant

of human coronary

with age in bovine aortas.

normal rat aorta.

catalyses

seems to be available dehydrogenase

the reaction: necrotic

aorta,

L-lactate

lipid accumulation

the predominant

In the kidney LDH isoenzyme tion.

Decreased

decreased

LDH-4

Atherosclerosis,

pattern LDH-1

decrease

similar results

in rabbits.

and, as such, are similar

the changes

in other

NAD-linked

glycolytic

+ NADHs.

tissues,

enzyme

cholesterol

which

Determination

to be one of the most reliable methods

of

for de-

lipid accumulation

diet were the liver and aorta, while

was in LDH-1 was in LDH-4

where

and 2. In the heart

and 5, while LDH-1

fat accumulation

and 2 remained

unaffected

can be roughly correlated and 2 are associated

and 5 concomitant

1970, 12: 393-399

in extracts

than that in the

occurred in the heart and muscle. In both the liver and

decrease

and lungs,

while LDH-1

LDH

in the literature.

+ NAD = pyruvate

considered

in LDH in athero-

decreased

activity

lesions in tissues. The tissues in which maximum

muscle, the maximum increased

Regarding

is an important

occurred in rats fed the high fat-high minimum

age change

activity

with total LDH activity

for the rat aorta.

this enzyme is at present tecting

less enzyme

ALEKSEEVA AND USHKALOV14 observed

to what is now reported Lactate

observed

cho-

is available in

but a 17% reduction

KITTINGER et al.13, estimating

rats,

These reports were only concerned no information

arteries

of these vessels. MANDEL AND KEMPF~~ observed

of aortas from arteriosclerotic

LDH-1)

by a number

lesterol diet are similar. The only other tissue about which information the literature

LDH

in the serum may

was moderate, or slightly

and skeletal

and 2 increased. LDH-4

decreased.

and 5

Thus,

the

with the degree of lipid accumulawith

with increased

lipid LDH-1

accumula.tion,

whereas

and 2 are found when

LDH

ISOENZYMES

IN

HYPERCHOLESTEROLAEMIC

less lipid accumulates.

399

RATS

LDH-1 isoenzyme is known predominantly

to catalyse the

formation of pyruvic acid from lactic acid, thus providing for more complete utilization of glucose via the Krebs cycle than glycolysis. LDH-4 and 5, on the other hand, allow the rapid conversion of pyruvate to lactate. The implications of the different reaction directions catalyzed by these isoenzymes are not at present clear.

REFERENCES 1

2

VESSEL, E. S. AND fractions, Proc. Sot. SAYRE, F. W. AND centration gradient

A.

G. BEARN,

Localisation

of lactic

dehydrogenase

activity

in

serum

Exptl. Biol. Med., 1957, 94: 96. B. R. HILL, Fractionation of serum lactate dehydrogenase by elution and paper electrophoresis, Proc. Sot. Exptl. Biol. Med.,

salt con1959, 96:

69.5. 3

4

5

6

7 8

9

10

11

12

13

14

ROMAN, W., Quantitative estimation of lactate dehydrogenase isoenzymes in serum, Enzymology, 1969, 36: 189. CHAZOV, E. I., V. N. SMIRNOV, A. P. ZISKO AND V. M. STARK, Serum lactic dehydrogenase isoenzyme patterns in coronary atherosclerosis, J. Atheroscler. Res., 1969, 9: 203. RAMDEO, I. N., H. S. ANDLEIGH AND K. C. JOSHI, Serum lactate dehydrogenase and its isoenzymes in myocardial infarction, Ind. J. ,‘k’ed. Res., 1969, 57: 1037. WROBLEWSKI, F. AND K. F. GREGORY, Lactate dehydrogenase isoenzymes and their distribution in normal tissues, plasma and in disease states. Ann. N. Y. Acad. Sk., 1961, 94: 912. WILKINSON, J. T., Isoenzymes, Span, London, 1965, p. 43. ACKERMAN, P. G. AND G. TORO, Blood lipids. In: S. FRANKEL AND S. REITMAN (Eds.), Gradwohl’s Clinical Laboratory Methods and Diagnosis, Mosby, St. Louis, MO., 1963, p. 255. ROMAN, W., R. C. S. OON, R. T. H. GAN AND J. RUGS, Quantitative estimation of lactate dehydrogenase isoenzymes in serum, Part 2 (A simple routine method for the separation of the isoenzymes by heat stability), Enzymology., 1969, 36: 323. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, Protein measurement with Folin phenol reagent, J. Biol. Chem., 1951, 193: 265. KIRK, J. E., J. R. MATZKE, N. BRANDSTRLJP AND I. WANG, The lactic dehydrogenase, malic dehydrogenase and phosphogluco-isomerase activities of coronary artery tissue in individuals of various ages, J. Gerontol., 1958, 13: 24. MANDEL, P. AND E. KEMPF, Enzymic activities of arteries of young and aged bovines; lactate dehydrogenase, malic dehydrogenase and transaminase, Compt. Rend. Sot. Biol. (Paris), 1960, 154: 791. KITTINGER, G. W., B. C. ~‘EXLER AND B. F. MILLER, quoted in T. ZEMPLBNYI, Enzyme Biochemistry of the Arterial Wall as Related to Atherosclerosis, Lloyd-Luke, London, 1968, p. 24. AL~KSEEVA, A. S. AND A. F. USHKALOW, Activity of lactic dehydrogenase of aorta and serum in experimental atherosclerosis, Enzymol. Biol. Clin., 1963, 2: 108.

Atherosclerosis,

1970, 12: 393-399