Kinetic determination of malate dehydrogenase activity eliminating problems due to spontaneous conversion of oxaloacetate to pyruvate

Abstract An automated system for the sensitive, reproducible determination of malate dehydrogenase activity in serum is described and evaluated. After incubation of serum at 25°C and double dialysis, fluorometry is used to measure the disappearance of NADH during reaction. Certain precautions are necessary to maintain the stability of NADH and oxaloacetate and to assure washout of adsorbed serum components from the system. Control serum (Enza-trol) is used as an enzyme reference material. Results from the automated method agree well with those from an independent manual method. Replicability is excellent, and results from samples stored at 4° or -20°C are essentially the same as those from fresh samples. A mean value of 36.6 ± 8.5 U of malate dehydrogenase per liter was obtained on sera from 34 healthy adults, a value that agrees well with normal values obtained by others.

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Kinetic determination of serum sorbitol dehydrogenase activity with a centrifugal analyzer.

Clinical Chemistry ◽

10.1093/clinchem/25.12.2026 ◽

1979 ◽

Vol 25 (12) ◽

pp. 2026-2029 ◽

Cited By ~ 23

Author(s):

J F Dooley ◽

L J Turnquist ◽

L Racich

Keyword(s):

Dehydrogenase Activity ◽

Sorbitol Dehydrogenase ◽

Kinetic Determination ◽

Standard Curve ◽

Simultaneous Oxidation ◽

Safety Studies ◽

The Common ◽

The Mean ◽

Apparently Healthy

Abstract We describe a mechanized method for centrifugal analyzer determination of sorbitol dehydrogenase in serum, based on conversion of D-fructose to sorbitol with simultaneous oxidation of NADH, in triethanolamine buffer at pH 7.4 and 30 degrees C. The standard curve for this assay is linear to 200 U of activity per liter of serum. The mean within-run precision (CV) of the assay is 0.8%. Results correlate well with those by a spectrophotometric method. In sera from 20 apparently healthy adult humans, sorbitol dehydrogenase activity averaged 1.7 (SD +/- 0.8; range, 1-3) U/L. The mean activity (U/L) for a group of 30 rats was 4.4 (SD, +/- 0.2; range, 3-6); for 20 dogs, 5.8 (SD, +/- 0.7; range 3-9); and for 30 mice, 26.8 (SD +/- 2.1; range, 22-34). To determine the utility of measuring this enzyme in the serum of rats for assessment of hepatotoxicity in drug-safety studies, we compared sorbitol dehydrogenase activity with that of alkaline phosphatase, aspartate aminotransferase, and alanine aminotranferase in the sera of rats treated with thioacetamide or in which the common bile duct has been ligated.

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Gluconeogenesis in the kidney cortex. Flow of malate between compartments

Biochemical Journal ◽

10.1042/bj1160493 ◽

1970 ◽

Vol 116 (3) ◽

pp. 493-502 ◽

Cited By ~ 9

Author(s):

R. Rognstad

Keyword(s):

Dehydrogenase Activity ◽

Malate Dehydrogenase ◽

Paper Chromatography ◽

Analytical Methods ◽

Specific Radioactivity ◽

Kidney Cortex ◽

Cortex Slices ◽

Glucose Formation ◽

Kidney Cortex Slices

1. Kidney-cortex slices from starved rats were incubated with l-[U-14C]lactate or l-[U-14C]malate plus unlabelled acetate and the specific radioactivity of the glucose formed was determined. In parallel experiments the specific radioactivity of the glucose formed from [1-14C]acetate plus unlabelled l-lactate and l-malate was determined. 2. By analytical methods the major products formed from the substrates were measured. The glucose formed was purified by paper chromatography for determination of specific radioactivity. 3. The specific radioactivity of the glucose formed from l-[U-14C]lactate agrees with predictions of a model based on interaction of the gluconeogenic and the oxidative pathways. 4. The specific radioactivity of the glucose formed from l-[U-14C]malate agrees with the predicted value if rapid malate exchange between the cytosol and mitochondria is assumed. 5. The rate of malate exchange between compartments was estimated to be rapid and at least several times the rate of glucose formation. 6. The specific radioactivity of the glucose formed from [1-14C]acetate plus unlabelled l-lactate or l-malate agrees with the predictions from the model, again assuming rapid malate exchange between compartments. 7. Malate exchange between compartments together with reversible malate dehydrogenase activity in the mitochondria and cytosol also tends to equilibrate isotopically the NADH pool in these compartments. 3H from compounds such as l-[2-3H]lactate, which form NAD3H in the cytosol, appears in part in water; and 3H from dl-β-hydroxy[3-3H]butyrate, which forms NAD3H in the mitochondria, appears in part in glucose, largely on C-4.

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Automatic determination of malate dehydrogenase activity with a flow-injection multidetection system

Journal of Automatic Chemistry ◽

10.1155/s1463924690000062 ◽

1990 ◽

Vol 12 (2) ◽

pp. 49-52

Author(s):

J. M. Fernández-Romero ◽

M. D. Luque de Castro

Keyword(s):

Human Serum ◽

Dehydrogenase Activity ◽

Malate Dehydrogenase ◽

Flow Injection ◽

Sampling Rate ◽

Flow Cell ◽

Automatic Determination ◽

Serum Samples ◽

Different Types

An automatic flow-injection method for the determination of malate dehydrogenase activity is proposed. The manifold used includes a selecting valve for closing the circuit along which the reacting plug is continuously circulated, and passed through the flow-cell of a conventional spectrophotometer, inserted into the closed circuit. A multipeak recording is obtained, each peak corresponding to one passage of the plug through the flow-cell. This recording allows the sensitivity to be modified, as required, using different types of measurements (for example, absorbance of the maxima or minima and absorbance differences between maxima or minima or their sums). The activity of the analyte can be determined in the range 0.02-2.00 U/l, with an r.s.d, of less than 0.90% at a sampling rate between 11 and 65 samples/h. The applicability of the method was checked by applying it to human serum samples. Analyte recoveries between 96 and 103% were obtained.

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