Alcohol. Vol. 7, pp. 397401. v PergamonPress plc, 1990. Printed in the U.S.A.
I)741-8329/90 $3.00 + .00
Methodological Aspects of Aldehyde Dehydrogenase Assay by SpectrophotometricTechnique S. C. G U R U A N D K. T A R A N A T H
SHETTY l
Department of Neurochemistry National Institute of Mental Health and Neurosciences Bangalore 560 029, India R e c e i v e d 12 O c t o b e r 1989; A c c e p t e d 19 F e b r u a r y 1990
GURU, S. C. AND K. TARANATH SHETTY. Methodologicalaspects of aldehyde dehydrogenase assay by 3pectrophotometric technique. ALCOHOL 7(5) 397-401, 1990.--Aldehyde dehydrogenase (ALDH) activity was assayed spectrophotometrically by measuring the increase in AA at 340 nm, as a criteria of NAD conversion to NADH in the presence of propionaldehyde. The effect of pH and substrate(s) concentration of nonenzymatic increase in absorbance at 340 nm was studied. Results indicate that the increase in absorbance at 340 nm is not entirely due to NAD conversion to NADH. It was observed that nonenzymatic interaction of NAD and aldehyde could as well result in increase in absorbance at 340 nm. The magnitude of the nonenzymatic contribution towards increase in absorbance at 340 nm is found to be pH, substrate(s) conc., and time dependent. Further, the observed nonenzymatic reaction product was found to be different from that of NADH as confirmed by u.v. spectral characteristics (h max. 346 nm) and its inability to activate NADH/NADPH-dependent glutathione reductase. Based on these findings, a final assay method comprising a substrate blank consisting of NAD and aldehyde, and the assay pH of 7.4 is recommended for measuring the ALDH activity. Further, under these experimental conditions the Km value of human RBC ALDH was found to be 0.59 mM for propionaldehyde substrate. Aldehyde dehydrogenase
Nonenzymatic reaction
Enzyme/substrate blank
ALDEHYDE dehydrogenase (ALDH: EC. 1.2.1.3) is a ubiquitous enzyme present in almost all mammalian organs and tissues including red blood cells (RBC's) (7-10). This enzyme is involved in the conversion of aldehyde metabolites, including those arising from oxidative deamination of biogenic amines, to their corresponding acids. Recently, considerable interest has been focussed on this enzyme, with reference to its role in metabolism of acetaldehyde. Biochemical studies on ALDH have revealed the existence of enzyme polymorphism in different ethnic groups leading to the theory for a biological basis of alcoholism and alcohol-related health problems. Further, the pharmacological basis of antabuse (disulfiram) used as a therapeutic deterrent to alcohol abuse is attributed to inhibition of ALDH (3,19). Reports about the decreased ALDH activity in RBC's of alcoholics (2) and its reverting back to normal levels following abstinence (1,11) have raised the possibility of using this parameter as a " s t a t e " marker of alcoholism, hence its utility to monitor patient compliance in deaddiction services (2,18). The most commonly used method for ALDH quantification by spectrophotometric method is based on the measurement of increase in AA at 340 nm as a criteria of NAD conversion to NADH by the enzyme in the presence of aldehyde substrate. However, it is observed from literature that
different workers have used different substrates at varying concentrations, and different conditions of pH, temperature in the assay system (4, 5, 13, 16, 18). This has made comparison of results difficult due to variation in conditions of the ALDH assay system. Added to this, the nature and composition of the blank employed also has an impact on the final value of enzyme activity. Depending upon the reactivity of the substrate(s) such as carbonyl compounds, in the present context aldehyde(s), the possible effect of experimental conditions such as pH, and temperature on the overall reaction of the assay system has to be taken into consideration. The present study is aimed at looking into the effects of some of the factors which could possibly affect ALDH assay system particularly the pH and the nature of the blank that would affect the final values of enzyme activity. Based on the findings of this study, the assay conditions most suitable for ALDH assay are described. METHOD Propionaldehyde (Fluka A.G.), acetaldehyde (Loba Chemie), pyrazole, nicotinamide adenine dinucleotide (NAD), glutathione reductase (GR) and 5,5' dithiobis [2-nitrobenzoic acid (DTNB)],
~Requests for reprints should be addressed to K. Taranath Shetty, Department of Neurochemistry, NIMHANS, Bangalore, 560 029, India.
397
398
GURU AND TARANATH SHETTY
CH3CH]CHC
CON(" 1 0 ~ M
15 A
B
-
it~
12
/
C)
x 9 E c
c) '4'
./
i W /
F
I)741-8329/90 $3.00 + .00
Methodological Aspects of Aldehyde Dehydrogenase Assay by SpectrophotometricTechnique S. C. G U R U A N D K. T A R A N A T H
SHETTY l
Department of Neurochemistry National Institute of Mental Health and Neurosciences Bangalore 560 029, India R e c e i v e d 12 O c t o b e r 1989; A c c e p t e d 19 F e b r u a r y 1990
GURU, S. C. AND K. TARANATH SHETTY. Methodologicalaspects of aldehyde dehydrogenase assay by 3pectrophotometric technique. ALCOHOL 7(5) 397-401, 1990.--Aldehyde dehydrogenase (ALDH) activity was assayed spectrophotometrically by measuring the increase in AA at 340 nm, as a criteria of NAD conversion to NADH in the presence of propionaldehyde. The effect of pH and substrate(s) concentration of nonenzymatic increase in absorbance at 340 nm was studied. Results indicate that the increase in absorbance at 340 nm is not entirely due to NAD conversion to NADH. It was observed that nonenzymatic interaction of NAD and aldehyde could as well result in increase in absorbance at 340 nm. The magnitude of the nonenzymatic contribution towards increase in absorbance at 340 nm is found to be pH, substrate(s) conc., and time dependent. Further, the observed nonenzymatic reaction product was found to be different from that of NADH as confirmed by u.v. spectral characteristics (h max. 346 nm) and its inability to activate NADH/NADPH-dependent glutathione reductase. Based on these findings, a final assay method comprising a substrate blank consisting of NAD and aldehyde, and the assay pH of 7.4 is recommended for measuring the ALDH activity. Further, under these experimental conditions the Km value of human RBC ALDH was found to be 0.59 mM for propionaldehyde substrate. Aldehyde dehydrogenase
Nonenzymatic reaction
Enzyme/substrate blank
ALDEHYDE dehydrogenase (ALDH: EC. 1.2.1.3) is a ubiquitous enzyme present in almost all mammalian organs and tissues including red blood cells (RBC's) (7-10). This enzyme is involved in the conversion of aldehyde metabolites, including those arising from oxidative deamination of biogenic amines, to their corresponding acids. Recently, considerable interest has been focussed on this enzyme, with reference to its role in metabolism of acetaldehyde. Biochemical studies on ALDH have revealed the existence of enzyme polymorphism in different ethnic groups leading to the theory for a biological basis of alcoholism and alcohol-related health problems. Further, the pharmacological basis of antabuse (disulfiram) used as a therapeutic deterrent to alcohol abuse is attributed to inhibition of ALDH (3,19). Reports about the decreased ALDH activity in RBC's of alcoholics (2) and its reverting back to normal levels following abstinence (1,11) have raised the possibility of using this parameter as a " s t a t e " marker of alcoholism, hence its utility to monitor patient compliance in deaddiction services (2,18). The most commonly used method for ALDH quantification by spectrophotometric method is based on the measurement of increase in AA at 340 nm as a criteria of NAD conversion to NADH by the enzyme in the presence of aldehyde substrate. However, it is observed from literature that
different workers have used different substrates at varying concentrations, and different conditions of pH, temperature in the assay system (4, 5, 13, 16, 18). This has made comparison of results difficult due to variation in conditions of the ALDH assay system. Added to this, the nature and composition of the blank employed also has an impact on the final value of enzyme activity. Depending upon the reactivity of the substrate(s) such as carbonyl compounds, in the present context aldehyde(s), the possible effect of experimental conditions such as pH, and temperature on the overall reaction of the assay system has to be taken into consideration. The present study is aimed at looking into the effects of some of the factors which could possibly affect ALDH assay system particularly the pH and the nature of the blank that would affect the final values of enzyme activity. Based on the findings of this study, the assay conditions most suitable for ALDH assay are described. METHOD Propionaldehyde (Fluka A.G.), acetaldehyde (Loba Chemie), pyrazole, nicotinamide adenine dinucleotide (NAD), glutathione reductase (GR) and 5,5' dithiobis [2-nitrobenzoic acid (DTNB)],
~Requests for reprints should be addressed to K. Taranath Shetty, Department of Neurochemistry, NIMHANS, Bangalore, 560 029, India.
397
398
GURU AND TARANATH SHETTY
CH3CH]CHC
CON(" 1 0 ~ M
15 A
B
-
it~
12
/
C)
x 9 E c
c) '4'
./
i W /
F
