Metabolism Clinical and Experimental VOL.

XXV,

NO. 1

JANUARY

1976

Lactate Dehydrogenase and Isoenzyme Changes in Rats With Experimental Thiamine Deficiency Yuzo

Hirota,

Edward

M.

Heart and liver lactate dehydrogenase (LDH) and isoenzyme distributions were studied in rats with experimental thiamine deficiency. No change in total heart LDH activity was observed on days 24 and 40 of a thiamine deficient diet. At the time of symptoms (average 53 days on diet), total heart muscle LDH was slightly decreased in thiamine deficient rats (248 f 12 U/g tissue, Mn l SE) compared to ad lib fed controls (290 f 12, p < O.OS), but did not differ from pair fed controls (273 f 15). Heart muscle LDH isoenzyme distribution showed a relative increase in the H, band and a decrease in the H,M, and HM, bands in thiamine deficiency on day 24. This pattern became more prominent by day 40. Calculated total M-LDH activity of heart muscle in thiamine deficient rats was significantly decreased at

Cohen,

and

Oscar

H. L. Bing

day 40 (44 l 3) compared to pair fed controls (57 + 5, p < 0.05) and ad lib fed controls (85 f 7, p < 0.001). Further changes were not observed in the symptomatic stage. Total H-LDH in heart muscle did not change during the period of study, while total liver LDH activity was reduced in thiamine deficient rats at the symptomatic stage ( 137 & 15) compared with pair fed controls ( 193 l 12, p < 0.05) and ad lib fed controls (407 ~50, p < 0.01). liver isoenzyme electrophoresis showed an almost 100% distribution to the M, band. The present investigation demonstmtes a decrease in cardiac muscle M-LDH in experimental thiamine deficiency. DiRerences in l.DH isoenzyme distribution in chronic hypoxia and thiamine deficiency are discussed.

T

HIAMINE IS one of the vitamins essential for the metabolism of glucose since it is a coenzyme of pyruvate dehydrogenase, alpha ketoglutarate dehydrogenase, and transketolase. The metabolic blockade which results from

From the Thorndike Memorial Laboratory and the Tufts Hospital, and the Department of Medicine. Harvard Medical of Medicine, Boston, Mass. Received for publication February IO. 197s. Supported in part by Grant 940 from the Massachusetts 10539. HL 5244. and 5 SOI RR5569 06 from the National Health Service. Reprint requests should be addressed to Oscar H.L. Bing. Hospital, 330 Brookline Avenue, Boston, Mass. 02215. 0 1976 by Gtune & Stratton, Inc. Metabolism,

Vol. 25, No. 1 (January),

1976

Circulation Laboratory. Boston City School and the Tufts University School

Heart Heart

Association. and Lung

M.D..

Cardiovascular

and by Grants HL Institute. U.S. Public Unit.

Beth Israel

1

2

HIROTA,

COHEN,

AND

BING

thiamine deficiency might explain the accumulation of blood, tissue pyruvate, and lactate.‘J On the other hand, the cause for the disproportionate increase in pyruvate concentration3*4 and reduced lactate/pyruvate ratio5 is unclear. Lactate dehydrogenases (LDH) are NAD-dependent enzymes which regulate the reversible reaction between lactate and pyruvate.6 It would seem that this system might play a role in explaining some of the metabolic findings of thiamine deficiency. Lactate dehydrogenases are tetramers of two different parent polypeptides, i.e., H and M subunits, and five different LDH isoenzymes can be differentiated by electrophoresis6 In this experiment we measured heart muscle LDH activity and isoenzyme distribution as well as liver LDH activity in rats with experimental thiamine deficiency. MATERIALS

AND

METHODS

Male Sprague-Dawley rats were divided into three groups. A thiamine deficient group (Group I) was fed a commercially available diet deficient in thiamine yet otherwise nutritionally complete ad libitum. A pair fed control group (Group 2) was given the same diet in amounts just sufficient to maintain body weight equal to Group I, but 0.20 mg thiamine hydrochloride was injected subcutaneously twice a week. Finally, an ad lib fed control group (Group 3) was fed ad libitum a commercial diet similar in composition to the Bt deficient diet but not lacking thiamine (General Biochemicals, Chagrin Falls, Ohio). In addition, biweekly subcutaneous thiamine hydrochloride injections were administered in Group 3. The rats were housed individually in wire bottomed cages, and tap water was given ad libitum. On days 24 and 40 of the experimental diet, animals from the three groups were sacrificed by decapitation, and left ventricular muscle LDH activity and isoenzyme distribution were measured. The remaining animals were allowed to live until Group 1 rats showed clinical signs of thiamine deficiency. The average time for the appearance of life threatening symptoms was 53 days. At the time of appearance of symptoms in Group I rats, animals from all three groups were sacrificed and studied. The mechanical properties of papillary and trabecular muscles from the left ventricles of these animals were studied and have been reported.’ Thiamine deficiency was documented by a decrease in red blood cell transketolase activity and increased thiamine pyrophosphate adding effect.’ Analysis of total heart muscle and liver LDH activity was carried out by the method of Kornberg’ which was modified as previously reported from this laboratory.’ Tissue for the measurement of LDH activity was minced with fine scissors and homogenized with five times its weight of 0.01 M tris-acetate buffer pH 8.2 in a virtus homogenizer for 5 min at medium speed. The homogenate was then centrifuged at 6000 RPM for 30 mitt, decanted and recentrifuged for IS min and the supernatant was used for further study. The whole procedure was carried out at a temperature of O-4” C. LDH activity was determined by reading the change in absorbance of NADH at 340 rnp at room temperature using a Beckman DU spectrophotometer with a Gilford linear scanning attachment. The assay mixture consisted of 0.1 ml of 2 mg/ml NADH, 0.1 ml of 0.3 M Na pyruvate, and 2.78 ml of 0.01 M tris-acetate buffer, pH 8.2. The reaction was started by using an appropriate dilution of homogenate to give a linear rate of reaction for the initial period and was carried out for 21 min. The final volume of the reaction mixture was 3 ml. Total heart muscle LDH activity was also measured in symptomatic rats using low substrate concentrations in the assay mixture. The assay mixture contained 0.1 ml of O.IM Na pyruvate and the procedure was carried out identical to that as described above. LDH isoenzymes were separated by electrophoresis at pH 8.8 using high resolution buffer (Gelman) and cellulose acetate electrophoresis strips. The electrophoresis was carried out for a period of I hr at 4°C with a current of 1.5 mA per strip. Separated isoenzymes were stained with nitroblue tetrazolium. Cellulose acetate strips were cleared using methanol-acetic acid mixture and scanned on a Gilford spectrophotometer with a linear transport scanning attachment.

LACTATE

DEHYDROGENASE

1

I

AND

ISOENZYME

3

CHANGES

24 DAYS f+

MniSEM

q Thiomine Pair IAd

Deficiency

Fed Control

(n=l6) (n =15)

Lib Fed Control

In=141

25 A--

40 DAYS n=tO n=9 m

n=9

s -ii

O

50

SWPTOtMATK

STAGE n=7 n=tO

25

n=13 A--

0

Hq (LDH,)

H3M(L44

H2M2(LDH3) HM3(LDHq)

Mq(LDH5)

Fig. 1 Heart muscle LDH isoenzyme distribution in mh with thiamine deficiency to controls. An increase in the H, band is seen at 40 days and in the symptomatic amine-deficient mts. A decease in the H,M, and HM, band is also present.

as compared stage in thi-

The LDH isoenzymes were calculated according to the formula by Everse and Kaplan6: %H-LDH = %H,LDH + %H,MLDH x 9 + %H,M,LDH x 4 + %M,LDH x 4, %M-LDH = 100 - % H-LDH. Absolute H-LDH = total LDH x ‘A H-LDH and Absolute M-LDH = total LDH x % M-LDH. RESULTS

After approximately fourteen days on the thiamine deficient diet, Group 1 rats developed anorexia, and subsequent gradual weight loss was observed. However, clinical symptoms of thiamine deficiency, i.e., peripheral neuropathy initially manifested by paralysis of the lower extremities and ataxia followed by tonic-clonic convulsions, were not observed until an average of 53 days. The body weights of Group 1 rats were 90% on day 24, 70% on day 40, and 56% at the symptomatic stage, of initial weight. The distribution of heart muscle LDH isoenzymes is shown in Fig. 1. A relative increase in the H, (LDH,) band and decrease in the H,M,(LDH,) and HM,(LDH,) bands were observed on day 40 and in symptomatic rats. The absolute value of H-LDH did not change during the experiment, however (Fig. 2). The absolute value of M-LDH in Group 1 animals was 72 f 5 U/g on day 24 and less than Group 3 rats (89 f 5) U/g, p < 0.05) but did not differ from group 2 animals (82 + 5 U/g). On day 40 it was 44 + 3 U/g in Group 1,

4

HIROTA,

I

COHEN,

AND

BING

LDH (%I 24 DAYS

q

Thiamine

Deficiency

Pair Fed Control MAd

Lib

(n=l6)

(n=t5)

Fed Control

(n=t4)

200 100

40 DAYS

SYMPTOMAT/C STAGE 100

0

M LDH Fig. 2 Relative and ficiency. No significant was significantly lower Table 1).

H LDH

M LDH

H LDH

absolute values of H and M-LDH in rat heart muscle with thiamine dechanges of the absolute values for H-LDH were present. M-LDH activity in the deficient mts at 40 days and at the symptomatic stage (see text and

57 f 5 U/g in Group 2, and 85 f 7 U/g in Group 3 (Group 1 versus Group 2; p < 0.05, Group 1 versus Group 3; p < 0.001, Group 2 versus Group 3; p < 0.025). Similar evidence of a decreased M-LDH in heart muscle was again seen in symptomatic rats (Table 1). Total LDH activity in heart muscle from thiamine deficient (Group 1) animals on days 24 and 40 did not differ from either of the control groups. In the symptomatic stage it was significantly decreased as compared with ad lib fed (Group 3) rats (p < 0.05) but did not differ significantly from pair fed (Group 2) animals (Table 1). It is known that a high pyruvate concentration inhibits LDH activity especially the H subunit. WOTherefore total heart muscle LDH was measured with 0.1 M pyruvate in the assay mixture. Animals were studied at the symptomatic stage of thiamine deficiency and the effects of substrate inhibition of H-LDH were examined. As compared to 0.3 M pyruvate in the assay mixture, similar results were obtained using 0.1 M pyruvate (Table 2). Total liver LDH activity and enzyme electrophoresis was carried out in symptomatic rats (Table 3). Electrophoresis showed that essentially all LDH activity was in the M,LDH fraction. Total liver LDH activity in Group 1 was

1 = thiamine

Group

to Group

&I < 0.025

deficiency

Heart

Group

89+

72 f 82 f

3.

Ii-LDH

248 f

10

10 8

LDH and

263~~ 268zt

Muscle

compared

to Group

2 = pair fed control

5

5’ 5

M-LDH

24 Days

Total

3. 2 and p < 0.001

11

10 11

331 f

352 f 337*

to Group to Group

compared

1.

Total LDH

*p < 0.05 compared tp < 0.05 compared

14

3

Group

16 15

1 2

No

Group Group

Days on Diet

Table

3.

Group

9

10 9

NO

(Units/gm

tissue,

19

15 13

f

With

201~

223 f 225zt

Ii-LDH

13

13 11

0.3 M Pyruvate SE)

3t

57+55 85 f 7

44 f

M-LDH

3 = ad lib fed control

286+

267 f 281 f

MN 40 Days

Measured

Total LDH

heart

Its Comwition

13

7 10

No

in Thiamine

29Ozt

248+ 273 f

12

12’ 15

Total LDH

Stage

59 f 75 f

Q 5

37

M-LDH 46 f

Symptomatic

Deficiency

214* 205 f

11 12

201 * 10

H-LDH

z ZI

1

2 2

Z

5 i!

r 0

5 ? 2 x 2 z 8 5! !i

6

HIROTA,

Table

2.

Total

Heart

Muscle LDH and Its Composition at Symptomatic Stage of Thiamine (Units/gm NO

lp tp

Group

1

7

Group

2

Group

3

< 0.01 compared c 0.001 compared

tissue,

Mn

f

Measumd Deficiency

0.1

AND

M Pyruvate

H-LDH

M-LDH

f

15

43*

3*

204+

14

10

257 f

13

56 f

37

201 f

11

14

275 f

8

74 f

2

203 f

7

to Group 2. p < 0.001 to Group 3.

compared

EilNG

SE)

Total LDH 247

with

COHEN,

to Group

3.

significantly lower than either Group 2 or Group 3 (Group p < 0.05, Group 1 versus Group 3p < 0.01).

1 versus Group

2

DISCUSSION

Several studies have examined the LDH activity and isoenzyme distribution in thiamine deficiency. Van Eys” and Gubler” found a decrease in liver LDH in thiamine deficiency as well as in rats treated with a thiamine antagonist. In Gubler’s study, however, no change in LDH activity was seen in heart, kidney, or brain tissue where aerobic metabolism is predominant. A decrease in liver LDH and unchanged heart LDH were also reported by Park et al.4 who in addition found a shift in isoenzyme distribution such that a decrease of LDHj (H,M,) and LDH4(HM3) and increase in LDH*(M,H,) and LDH,(H,) was observed in hearts of thiamine deprived rats. Only relative changes were measured, however, and Park et al. interpreted their data as indicating an increase in H subunits. Although a similar relative change in isoenzyme distribution was found in the present study, an absolute decrease in M-type LDH and unchanged H-type LDH is demonstrated. The interpretation of the findings of the present study is based in part on the theory advanced by Everse and Kaplan.6 Here H-LDH is thought to be responsible for the conversion of lactate to pyruvate which is distributed in tissues with predominantly aerobic metabolism, whereas the M-type subunit which converts pyruvate to lactate is more prevalent in anaerobic tissues. In tissues such as the heart where a mixture of the two subunits is present, adaptive changes to environmental hypoxia have been reported. Accordingly, anemic ratsI as well as rats adapted to high altitude’* have a greater tolerance to hypoxia, and M-type LDH is increased in heart muscle from anemic rats15 as well as high altitude adapted rats.16 In thiamine deficiency, Krebs cycle activity is disturbed at the level of acetyl CoA and succinyl CoA formation because of the decreased availability of thiamine pyrophosphate as a coenzyme of pyruvate dehydrogenase and alpha Table

3.

Total

LDH Activities Thiamine

in liver

Deficient

at Symptomatic

Diet (Mn

f

NO

of

Unit/gm

tissue

Group

1

4

137*

15’

Group

2

3

193 f

127

Group

3

4

407 4z 50

‘p

< 0.05 compared

tp

i

0.025

Stage

SE)

compared

to Group to Group

2;

p