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“α-Hydroxybutyrate dehydrogenase” activity of human tissue homogenates
CLINICA CHIMICA ACTA
“c+HYDROXYBUTYRATE OF HUMAN
415
DEHYDROGENASE” TISSUE HOMOGENATES
ACTIVITY
SUMMARY
Observations on human tissue extracts have shown that L.D. : H.B.D. ratios and Km u-ketobutyrate decrease with increasing proportions of fast-moving L.D. isoenzymes. The significance of this finding is discussed.
The enzymic reduction by human serum of ~-ketobutyrate (serum “a-hydroxybutyrate dehydrogenase”, H.B.D. activity) has been shown to differ from the reduction of pyrnvate (serum lactate dehydrogenase, L.D. activity) in respect of temperature coefficients, Michaelis constants and PH optimal. Electrophoretic separation of sera showed L.D. and H.B.D. activity in the al, a,, @ and y globulin fractions, but whereas L.D. activity was only slightly greater than H.B.D. activity in the fastmoving a fractions, in the slow-moving ,6 and y fractions it was two to three times as great. It was therefore suggested that the differences in the action of serum towards the two substrates might indicate a difference in the properties of the electrophoretic components (isoenzymes) of lactate dehydrogenase, or that there exists in serum an enzyme other than lactate dehydrogenase capable of reducing a-ketobutyrtate. The present observations on human tissue extracts are in support of the former view. MATERIAL
AND METHODS
Post-mortem tissues were obtained within 24 hours of death from bodies kept refrigerated until necropsy. Placenta was obtained within four hours of delivery. Diseased tissues were not examined. Tissue fragments were well washed in ice-cold 0.067 M Stirensen phosphate buffer, PH 7.4, blotted dry on filter paper, weighed and homogenised in 20 ml buffer using an Ultra-Turrax vertical drive homogeniser. Homogenates were filtered through Whatman No. I filter paper and suitably diluted. Fresh red cell haemolysates were also examined. Lactate dehydrogenase (L.D.) and a-hydroxybutyrate dehydrogenase (H.B.D.) activities and Michaelis constants (Km ~-ketobut~ate) were determined as described elsewhere”*2. In addition, tissue extracts and haemolysates diluted to yield total L.D. activities between 250 and 500 International Units per litre were separated by electrophoresis on cellulose acetate. Using an enzyme staining technique3 a visual assessment was made of the L.D. isoenzyme patterns following separation. CL&.Chim.Acta,
8 (~963) 415-417
416
S. B. ROSALKI TABLE
Average L.D. activity in International units per kg wet tissue. (Range in brackets)
Number Tissue
Heart Kidney Red-cells
of extracts examined
6 4 2
Placenta
3
Lung
3
I40,OOO (100,000-190,000) 1~0,000 (110,000--210,000) 110,000* ( 54,000~160,000) 24,000 ( 20,000-26,000) 48,000
(
Muscle
4
Liver
6
44,ooo57,000) I 60,000 ( 95,000-190,000) 130,000 (~Io,ooo-180,000)
I
Average H.B.D. activity in International units per kg wet tissue. (Range in brackets)
110,000 (80,000~150,000) 100,000 (76,000-140,000) 71,000* (36,000-100,000) r5,ooo (13,000-16,000) 20,000 (17,000~22,000) 48,000 (41,00062,000) 36,000 (27.00053,000)
Average L.D: H.B.D. ratios (Range in brackets)
Average Km u-ketobutyrate in IO-~M (Range in brackets)
I.27 (1.19-1.43) 1.50 (1.30-1.57) I.55 (1.50-1.60) 1.60 (1.54-1.66) 2.40 (2.20-2.62) 3.33 (2.26-4.11) 3.60 (3.23-4.55)
( (
(
Predominalzt I>.D. isoenzyme fractiow (in order of staining intensity)
5.6 5.0-6.0)
5.4
6.3 6.0-b.6) 6.6
5.4
(-) 7.5 7.0-8.0)
9.3 9.0-9.5) 13.0 (IZ.O-16.0)
4,5 4.5 4.3,5 3.4,2
(
13.3 (13.0-13.5)
3,2,I I,2
* per kg haemoglobin RESULTS
L.D. and H.B.D. activities, L.D.: H.B.D. ratios, Km cr-ketobutyrate and the predominant L.D. isoenzyme fractions are shown in Table I. L.D. isoenzymes are numbered using the nomenclature whereby Fraction 5 refers to the fastest-moving L.D. fraction4. Tissues are arranged in order, such that the greater the proportion of faster-moving L.D. isoenzymes the nearer is the tissue to the top of the table. From Table I it can be seen that the greater the proportion of faster-moving L.D. isoenzymes the smaller is the L.D.: H.B.D. ratio and the smaller is the Km ccketobutyrate. DISCUSSION
The sera of patients with myocardial infarction possess raised H.B.D. activity and show low L.D.: H.B.D. ratios. Significant elevation of L.D.: H.B.D. ratios (often with normal H.B.D. levels) occurs in the sera from patients with liver disease&. Since myocardial infarction and liver disease are known to be associated with increase in the fastest and slowest-moving serum L.D. isoenzymes respectively4, these observations are in accord with the differences in the L.D.: H.B.D. ratios of the serum L.D. isoenzymes previously noted’. Low L.D. : H.B.D. ratios have been observed in the fast-moving L.D. isoenzymes of rabbit cardiac tissue extracts and high ratios in the more slowly moving isoenzymes of liver and muscle extracts. This, together with the previous findings on L.D. : B.H.D. ratios of pathological sera, has led to the conclusion that comparative measurements of serum L.D. and H.B.D. activities provides a means of assessing the various amounts of fast and slow-moving L.D. isoenzymes in serums. The observations reported here show that for human tissue extracts also, comClin. Chim. Acta, 8 (1963) 4I5-4I7
cc-HBD ACTIVITY OF HUMANTISSUE HOMOGENATES
4’7
parative measurement of L.D. and H.B.D. activities provides a means of assessing the proportion of fast and slow-moving L.D. isoenzymes, for, in the tissues examined, the geater the proportion of fast-moving L.D. components the lower is the L.D.: H.B.D. ratio. The parallel decrease in Km a-ketobutyrate is of particular interest. WR~BLEWSKI ANDGREGORY’ have shown that for rabbit L.D. isoenzymes, the greater the electrophoretic mobility of the isoenzyme, the lower is the isoenzymeKm pyruvate. It would therefore appear that Km a-ketobutyrate, Km pyruvate and L.D. : H.B.D. ratios decrease in parallel with increasing electrophoretic mobility of the L.D. isoenzymes. It may be that they are all dependent on L.D. isoenzyme molecular size. The results suggest that H.B.D. cannot be an enzyme separate from L.D., for it would be necessary to postulate that the enzyme has different properties varying with its tissue of origin, and it would be difficult to explain the apparent dependence of H.B.D. activity and Michaelis constants on the electrophoretic mobility of L.D. The fact that H.B.D. activity has never been detected unaccompanied by L.D. activity is also evidence against its identity as a separate enzyme. As a consequence of the high Km cr-ketobutyrate values of liver and muscle extracts, the standard spectrophotometric assay of H.B.D.112 is normally carried out at too low a substrate concentration for the detection of optimal activity derived from these tissues. Whilst doubling the substrate concentration in the assay of normal sera or sera from patients with myocardial infarction is not associated with increase of H.B.D. activity, an increase was observed in sera from some patients with liver disease. The observations on the L.D.: H.B.D. ratios of various tissues indicate that H.B.D. activity will less frequently be raised than L.D. activity in sera from patients with muscle disorders or pulmonary infarction, and that such sera will show high L.D. : H.B.D. ratios. This has been confirmed in a small number of cases. ACKNOWLEDGEMENTS I wish to thank Dr. B. W. MEADE and Dr. J. H. WILKINSON for helpful advice and criticism, Drs. M. E. A. POWELL AND E. A. FRENCH for provision of necropsy material and Mr. J. M. DALY for assistance with the electrophoretic separations. REFERENCES 1 S. B.
ROSALKI AND J. H. WILKINSON, Nature, 188 (1960) IIIO. * S. B. ROSALKI, M. D. Thesis, University of London, 1961. B H. BARNETT. personal communication. 4 F. WR~BLEW~KI, C. Ross AND K. F. GREGORY, New Engl. J. Med., 263 (1960) 531. 6 B. A. ELLIOTT AND 1. H. WILKINSON, Lalzcet, i (1961) 698. 6 J. N. WILKINSON, By A.ELLIOTT, K. B. COOKE A& D: T: PLUMMER, Proc. Assoc. Cl&. Biochem.. 2 (1962) I. ’ F. WR~BLEWSKI AND K. F. GREGORY, Ann. N. Y. Acad. Sci., 94 (1961) 912.
Cfin. Chim. Acta, 8 (1963) 415-417
“c+HYDROXYBUTYRATE OF HUMAN
415
DEHYDROGENASE” TISSUE HOMOGENATES
ACTIVITY
SUMMARY
Observations on human tissue extracts have shown that L.D. : H.B.D. ratios and Km u-ketobutyrate decrease with increasing proportions of fast-moving L.D. isoenzymes. The significance of this finding is discussed.
The enzymic reduction by human serum of ~-ketobutyrate (serum “a-hydroxybutyrate dehydrogenase”, H.B.D. activity) has been shown to differ from the reduction of pyrnvate (serum lactate dehydrogenase, L.D. activity) in respect of temperature coefficients, Michaelis constants and PH optimal. Electrophoretic separation of sera showed L.D. and H.B.D. activity in the al, a,, @ and y globulin fractions, but whereas L.D. activity was only slightly greater than H.B.D. activity in the fastmoving a fractions, in the slow-moving ,6 and y fractions it was two to three times as great. It was therefore suggested that the differences in the action of serum towards the two substrates might indicate a difference in the properties of the electrophoretic components (isoenzymes) of lactate dehydrogenase, or that there exists in serum an enzyme other than lactate dehydrogenase capable of reducing a-ketobutyrtate. The present observations on human tissue extracts are in support of the former view. MATERIAL
AND METHODS
Post-mortem tissues were obtained within 24 hours of death from bodies kept refrigerated until necropsy. Placenta was obtained within four hours of delivery. Diseased tissues were not examined. Tissue fragments were well washed in ice-cold 0.067 M Stirensen phosphate buffer, PH 7.4, blotted dry on filter paper, weighed and homogenised in 20 ml buffer using an Ultra-Turrax vertical drive homogeniser. Homogenates were filtered through Whatman No. I filter paper and suitably diluted. Fresh red cell haemolysates were also examined. Lactate dehydrogenase (L.D.) and a-hydroxybutyrate dehydrogenase (H.B.D.) activities and Michaelis constants (Km ~-ketobut~ate) were determined as described elsewhere”*2. In addition, tissue extracts and haemolysates diluted to yield total L.D. activities between 250 and 500 International Units per litre were separated by electrophoresis on cellulose acetate. Using an enzyme staining technique3 a visual assessment was made of the L.D. isoenzyme patterns following separation. CL&.Chim.Acta,
8 (~963) 415-417
416
S. B. ROSALKI TABLE
Average L.D. activity in International units per kg wet tissue. (Range in brackets)
Number Tissue
Heart Kidney Red-cells
of extracts examined
6 4 2
Placenta
3
Lung
3
I40,OOO (100,000-190,000) 1~0,000 (110,000--210,000) 110,000* ( 54,000~160,000) 24,000 ( 20,000-26,000) 48,000
(
Muscle
4
Liver
6
44,ooo57,000) I 60,000 ( 95,000-190,000) 130,000 (~Io,ooo-180,000)
I
Average H.B.D. activity in International units per kg wet tissue. (Range in brackets)
110,000 (80,000~150,000) 100,000 (76,000-140,000) 71,000* (36,000-100,000) r5,ooo (13,000-16,000) 20,000 (17,000~22,000) 48,000 (41,00062,000) 36,000 (27.00053,000)
Average L.D: H.B.D. ratios (Range in brackets)
Average Km u-ketobutyrate in IO-~M (Range in brackets)
I.27 (1.19-1.43) 1.50 (1.30-1.57) I.55 (1.50-1.60) 1.60 (1.54-1.66) 2.40 (2.20-2.62) 3.33 (2.26-4.11) 3.60 (3.23-4.55)
( (
(
Predominalzt I>.D. isoenzyme fractiow (in order of staining intensity)
5.6 5.0-6.0)
5.4
6.3 6.0-b.6) 6.6
5.4
(-) 7.5 7.0-8.0)
9.3 9.0-9.5) 13.0 (IZ.O-16.0)
4,5 4.5 4.3,5 3.4,2
(
13.3 (13.0-13.5)
3,2,I I,2
* per kg haemoglobin RESULTS
L.D. and H.B.D. activities, L.D.: H.B.D. ratios, Km cr-ketobutyrate and the predominant L.D. isoenzyme fractions are shown in Table I. L.D. isoenzymes are numbered using the nomenclature whereby Fraction 5 refers to the fastest-moving L.D. fraction4. Tissues are arranged in order, such that the greater the proportion of faster-moving L.D. isoenzymes the nearer is the tissue to the top of the table. From Table I it can be seen that the greater the proportion of faster-moving L.D. isoenzymes the smaller is the L.D.: H.B.D. ratio and the smaller is the Km ccketobutyrate. DISCUSSION
The sera of patients with myocardial infarction possess raised H.B.D. activity and show low L.D.: H.B.D. ratios. Significant elevation of L.D.: H.B.D. ratios (often with normal H.B.D. levels) occurs in the sera from patients with liver disease&. Since myocardial infarction and liver disease are known to be associated with increase in the fastest and slowest-moving serum L.D. isoenzymes respectively4, these observations are in accord with the differences in the L.D.: H.B.D. ratios of the serum L.D. isoenzymes previously noted’. Low L.D. : H.B.D. ratios have been observed in the fast-moving L.D. isoenzymes of rabbit cardiac tissue extracts and high ratios in the more slowly moving isoenzymes of liver and muscle extracts. This, together with the previous findings on L.D. : B.H.D. ratios of pathological sera, has led to the conclusion that comparative measurements of serum L.D. and H.B.D. activities provides a means of assessing the various amounts of fast and slow-moving L.D. isoenzymes in serums. The observations reported here show that for human tissue extracts also, comClin. Chim. Acta, 8 (1963) 4I5-4I7
cc-HBD ACTIVITY OF HUMANTISSUE HOMOGENATES
4’7
parative measurement of L.D. and H.B.D. activities provides a means of assessing the proportion of fast and slow-moving L.D. isoenzymes, for, in the tissues examined, the geater the proportion of fast-moving L.D. components the lower is the L.D.: H.B.D. ratio. The parallel decrease in Km a-ketobutyrate is of particular interest. WR~BLEWSKI ANDGREGORY’ have shown that for rabbit L.D. isoenzymes, the greater the electrophoretic mobility of the isoenzyme, the lower is the isoenzymeKm pyruvate. It would therefore appear that Km a-ketobutyrate, Km pyruvate and L.D. : H.B.D. ratios decrease in parallel with increasing electrophoretic mobility of the L.D. isoenzymes. It may be that they are all dependent on L.D. isoenzyme molecular size. The results suggest that H.B.D. cannot be an enzyme separate from L.D., for it would be necessary to postulate that the enzyme has different properties varying with its tissue of origin, and it would be difficult to explain the apparent dependence of H.B.D. activity and Michaelis constants on the electrophoretic mobility of L.D. The fact that H.B.D. activity has never been detected unaccompanied by L.D. activity is also evidence against its identity as a separate enzyme. As a consequence of the high Km cr-ketobutyrate values of liver and muscle extracts, the standard spectrophotometric assay of H.B.D.112 is normally carried out at too low a substrate concentration for the detection of optimal activity derived from these tissues. Whilst doubling the substrate concentration in the assay of normal sera or sera from patients with myocardial infarction is not associated with increase of H.B.D. activity, an increase was observed in sera from some patients with liver disease. The observations on the L.D.: H.B.D. ratios of various tissues indicate that H.B.D. activity will less frequently be raised than L.D. activity in sera from patients with muscle disorders or pulmonary infarction, and that such sera will show high L.D. : H.B.D. ratios. This has been confirmed in a small number of cases. ACKNOWLEDGEMENTS I wish to thank Dr. B. W. MEADE and Dr. J. H. WILKINSON for helpful advice and criticism, Drs. M. E. A. POWELL AND E. A. FRENCH for provision of necropsy material and Mr. J. M. DALY for assistance with the electrophoretic separations. REFERENCES 1 S. B.
ROSALKI AND J. H. WILKINSON, Nature, 188 (1960) IIIO. * S. B. ROSALKI, M. D. Thesis, University of London, 1961. B H. BARNETT. personal communication. 4 F. WR~BLEW~KI, C. Ross AND K. F. GREGORY, New Engl. J. Med., 263 (1960) 531. 6 B. A. ELLIOTT AND 1. H. WILKINSON, Lalzcet, i (1961) 698. 6 J. N. WILKINSON, By A.ELLIOTT, K. B. COOKE A& D: T: PLUMMER, Proc. Assoc. Cl&. Biochem.. 2 (1962) I. ’ F. WR~BLEWSKI AND K. F. GREGORY, Ann. N. Y. Acad. Sci., 94 (1961) 912.
Cfin. Chim. Acta, 8 (1963) 415-417