Scand. J . din. Lab. Invest. 43, 313-315, 1983.

On the rationale of the a-hydroxybutyrate dehydrogenase (HBD) determination

K A A R I N A O J A L A & T . W E B E R

Aurora Hospital, Clinical Laboratory, Helsinki, Finland

Ojala, K. & Weber, T. On the rationale of the a-hydroxybutyrate dehydrogenase (HBD) determination. Scand. J . din. Lab. Invest. 43, 313-315, 1983.

The discriminating power of HBD, LD and of the HBDlLD ratio in comparison with LDl/LD2 and LD51LD4 ratios in acute myocardial infarction and hepatic disease is evaluated. The results demonstrate that there are no clinical reasons for the determination of the HBD when a method using the conditions of the Committee of Enzymes of the Scandinavian Society for Clinical Chemistry (SCE) is used.

Key-words: LD; LD isoenzymes; myocardial infarction

Kaarina Ojala, Aurora Hospital, Clinical Laboratory, Nordenskioldinkatu 20, SF-00250 Helsinki 25, Finland

Rosalki and Wilkinson [4] first demonstrated that a-hydroxybutyrate dehydrogenase (HBD, E.C. 1.1.1.30) can be used as a convenient pro- cedure for the preferential measurement of the cardiac (anodic) lactate dehydrogenase (LD) isoenzymes. In a preliminary report (March 1973, not published) of the SCE a method for the determination of HBD catalytic activity was presented. However, according to this report ‘the relevance of this parameter is still uncertain’ and ‘the method is nearly insensitive to increase in H-subunits’. Thus, any recommended method for HBD determination was never published by SCE, but, in spite of that, the determination of HBD is widely used in Scandinavian laboratories.

The purpose of the present study was to evaluate the discriminating power of HBD and LD and of the HBD/LD ratio in comparison with LDl/LD2 and LD5/LD4 ratios in acute myocardial infarction and hepatic disease.

0036-5513/83/060C-0313 $02.00 @ 1983 Medisinsk Fysiologisk Forenings Forlag

M A T E R I A L A N D M E T H O D S

Reagents. Commercial reagents, manufactured by Medix Labs Ltd. (Helsinki, Finland) were used for the enzyme determinations. The methods are according to the recommendations of the Scandinavian Society for Clinical Chemistry, except the HBD assay, where the 2-oxobutyrate concentration is 9.7 mmoVl and the other reagents are as in the LD assay. LD isoenzyme estimation was performed by gel electrophoresis using the universal agarose film (Corning-ACI, Medfield, MA 02052, USA) in barbital buffer (50 mmoV1, pH 8.6). The frac- tions were localized by the NADH fluorescence and quantitated by densitometry.

Subjects. The reference subjects (n =20) were supposedly healthy volunteers with no signs or symptoms of heart or liver disease. The patient groups (n = 16 and 25) consisted of patients with acute myocardial infarction or hepatic disease, respectively. The diagnosis of myocardial infarc- tion was confirmed by a history of acute chest pain and by positive electrocardiographic

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314 K . Ojala & T. Weber

HBDlLD

changes. Blood samples were collected at admis- sion or in the following morning. The patients in the liver disease group were chosen by means of the clinical picture and elevated values of liver enzymes: ASATIALAT >40 Ull 37°C a n d o r y-glutamyltransferase >50 U/l 37°C. Signifi- cance was tested for by the Student’s test.

LD 1 I L D 2

table, LD and H B D behave in a very similar way in hepatic disease and myocardial infarction, the correlation being closest in liver diseases. O n the other hand the LDlILD2 and LD5ILD4 ratios show highly significant differences in the same conditions.

HD

_ _ - _ - -- . .. .... .... .. .. . ..... . . - - _ _ _ _ _

.

D I S C U S S I O N R E S U L T S

The different patient groups are compared in Table I and in Fig. 1. As can be seen from the

In the H B D assay by Rosalki & Wilkinson [4], the 2-oxoglutarate concentration was 3.3 mmoVl and temperature 25°C. According to Ellis &

TABLE 1. Results of LD, HBD and LD isoenzyme determinations. The results are expressed as arithmetic mean 2 SEM

Reference Acute myocardial Liver disease group (n = 20) infarction (n=16) (n = 25)

LD U/L 3292 10 18092 4101 445251$ HBD U/l 1952 6 10362 177T 2482 24$ r (HBD: LD) 0.85 0.92 0.99 HBD/LD ratio 0.602 0.004 0.642 0.023* 0.56-tO.0921 LDlILD2 ratio 0.63?0.016 0.93+0.060$ 0.572 0.030 LD5/LD4 ratio 0.732 0.042 1.422 0.328 2.292 0.5321

*P<0.05 tPi0.005 SP< 0.001

0 . 5 t I

1.2

0.8

0 . 4

HD

.

. ---_-

i I

i .. .. .. --w- .. . .

I LD51LD4

AM1

FIG. 1. HBDILD, LDllLD2 and LD51LD4ratios in different groups ofpatients. Dotted linesshow the reference limits (mean + 2SD). REF = Reference group, AM1 = Acute myocardial infarction, HD = Liver disease.

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a-hydroxybutyrate dehydrogenase 3 15

Goldberg [ 11, the optimal substrate concentra- tion is about 1 5 mmoV1 at 37°C. h u n g & Hen- derson [3] found that a method using a subopti- mal 2-oxobutyrate concentration gives the best and a method using an optimal concentration gives the worst relationship with LD1 isoen- zyme. All three methods, which they tested, showed a less marked relationship with LD5 activity.

In the H B D method we used, a suboptimal substrate concentration 9.7 mmol/l at 37°C was therefore chosen. According to the manu- facturer’s information, this concentration gives the best separation between the reference and the acute myocardial infarction groups. How- ever, the calculated HBD/L ratios give no clear separation between the groups studied. Both in the myocardial infarction and in the liver disease group about half of the values fall within the normal reference limits. Statistically the myocardial infarction group differs even less significantly from the normals than does the liver disease group.

The correlation coefficients between the HBD and L D values in clinical groups (Table I) also indicate that determination of H B D cannot give any additional information to that obtained from total LD, i.e. if L D is elevated, so is HBD, too.

Leung & Henderson [2] showed in their studies that the LDl /LD2 ratio has a very high specificity and sensitivity for the diagnosis of acute myocardial infarction. Our results almost exactly reproduce theirs. The excellent diagnos- tic specificity and sensitivity of LD5/LD4 ratio

for the diagnosis of liver disease, shown in Fig. 1, tends also to add weight to the clinical usefulness of L D isoenzyme electrophoresis. We conclude that there are no clinical reasons for the deter- mination of H B D at 37°C in the diagnosis of acute myocardial infarction or liver disease. Instead, the determination of LD isoenzymes or other more organ specific enzymes is highly advisable.

A C K N O W L E D G M E N T S

Supported by grants from the Sigrid Juselius Foundation, Helsinki, Finland.

R E F E R E N C E S

1 Ellis, G. & Goldberg, D.M. Serum a-hydroxy- butyrate dehydrogenase activity: An improved method. Amer. J . din. Pathol. 56, 627, 1971.

2 Leung. F.Y. & Henderson, A.R. Thin-layer agarose electrophoresis of lactate dehydrogenase isoenzymes in serum: a note on the method of reporting and on the lactate dehydrogenase isoenzyme-t/isoenzyme-Z ratio in acute myocar- dial infarction. Clin. Chern. 25, 209, 1979.

3 Leung, F.Y. & Henderson, A.R. Comparison of three serum a-hydroxybutyrate dehydrogenase procedures for lactate dehydrogenase isoenzyme discrimination. C h . chim. Acfa 115, 145, 1981.

4 Rosalki, S.B. & Wilkinson, J.H. Reduction of a-ketobutyrate by human serum. Nature 188, 1110,1960.

Received 11 November 1982 Accepted 25 February 1983

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