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Aldehyde dehydrogenase and monoamine oxidase in rat liver mitochondria
Alcohol. Vol. 6, pp. 461-464 © Pergamon Press plc, 1989. Printed in the U.S.A.
0741-8329/89 $3.00 + .00
Aldehyde Dehydrogenase and Monoamine Oxidase in Rat Liver Mitochondria S A F F I Y A C A T O V I C T U R A N , P R I T E S H C. S H A H A N D R E G I N A P I E T R U S Z K O I
Center of Alcohol Studies, Rutgers University, Piscataway, NJ 08855-0969 R e c e i v e d 24 October 1988; A c c e p t e d 11 July 1989
TURAN, S. C., P. C. SHAH AND R. PIETRUSZKO. Aldehydedehydrogenaseand monoamineoxidase in rat liver mitochondria. ALCOHOL 6(6) 461-464, 1989.--Monoamine oxidase (EC 1.4.3.4) and aldehyde dehydrogenase (EC 1.2.1.3) activities were compared in the liver mitochondria of male and female rats. Monoamine oxidase activity using benzylamine as a substrate was significantly higher in males as compared with females: 1.45 versus 0.74 txmoles/mg mitochondrial protein/hr, respectively. Monoamine oxidase activity using tyramine as a substrate and aldehyde dehydrogenase activity were the same in males and females. Monoamine oxidase-tyramine and aldehyde dehydrogenase activities did not vary with the different phases of the estrous cycle in the female but the activity of monoamine oxidase-benzylamine did; rats in the proestrous phase had the highest activity and those in the estrous phase had the lowest. Aldehyde dehydrogenase
Monoamine oxidase
Liver mitochondria
Male
Female
Rats
METHOD
IN the preceding paper (17) we have shown that aldehyde dehydrogenase (E.C. 1.2.1.3) within intact rat liver mitochondria can be inactivated by aerobic incubation with dopamine. This inactivation appears to occur with the simultaneous incorporation of label from J4C dopamine. The mechanism is at present unknown but the results obtained are consistent with the involvement of o-quinones. With the exception of melanogenic tissues (4), in the mammalian organism there is no known enzymic mechanism for quinone formation directly from dopamine. Quinones can be formed directly from dopamine by air oxidation, but the process is slow. The only known pathway is via monoamine oxidase (16) (E.C.1.4.3.4) which results in conversion of the amine into an aldehyde which then readily forms quinones nonenzymically. Such quinones would bear aldehyde groups and as such may be recognized as substrates by aldehyde dehydrogenase (11) and thus would have a potential to damage aldehyde dehydrogenase in a selective manner. Although inhibition of monoamine oxidase (17) only partially abolished aldehyde dehydrogenase inactivation, determination of monam~ne oxidase and aldehyde dehydrogenase levels was attempted to obtain a better understanding of the role of monoamine oxidase in aldehyde dehydrogenase inactivation. We have also demonstrated (17) that inactivation of aldehyde dehydrogenase proceeded to a greater extent and with greater consistency in the mitochondria from female than from male animals; for this reason monoamine oxidase levels in both male and female rat liver mitochondria were determined. Since inactivation of aldehyde dehydrogenase appeared to vary with the estrous cycle an attempt was made to determine levels of monoamine oxidase activity in different phases of the estrous cycle. Aldehyde dehydrogenase levels have also been determined in the mitochondria of the same animals.
Male and female Sprague-Dawley rats (Charles River) 80-105 days old were used; the males weighed between 350-400 g and the females between 215-300 g. Prior to experiments the rats were kept for at least 20 days under conditions of 12 hours of light per day and given Purina laboratory chow and water ad lib. Vaginal smears were taken daily before 10 a.m. to determine the phase of the estrous cycle (20). Females were used only if they had been following a regular estrous cycle for at least 20 days. Both the male and female rats were killed by decapitation before 10 a.m.
Preparation of Mitochondria Mitochondria were isolated according to the fractionation procedure of Hogeboom (6). Immediately after the rat was decapitated the liver was excised and 1 g was homogenized in 9 ml of 0.25 M sucrose using a Potter homogenizer followed by centrifugation at 600 x g for 10 minutes at 4°C in a refrigerated Sorvall RC-5B centrifuge. The supernatant was centrifuged at 5000 x g for 10 minutes. The resulting mitochondrial pellet was then washed with ca. 3 ml of 0.25 M sucrose and centrifuged again at 5 0 0 0 x g for 10 minutes. The mitochondrial pellet was then prepared for estimation of monoamine oxidase activity: the pellet was brought up to 1 g with 0.03 M phosphate buffer, pH 6 containing 1 mM EDTA. This mitochondrial suspension (0.1 ml) was then lysed in 20 ml of 0.05 M phosphate buffer, pH 7.4 containing 3 mM sodium azide and aliquots of this mitochondrial preparation based on the original wet weight of liver were used for monoamine oxidase activity assay. An aliquot of the original
IRequests for reprints should be addressed to Dr. R. Pietruszko.
461
462
TURAN. SHAH AND PIETRUSZKO
mitochondrial suspension was saved for protein determination by the Lowry method (10) using bovine serum albumin as the standard and the rest was sonicated to release aldehyde dehydrogenase.
TABLE 1 MONOAMINE OXIDASE ACTIVITY IN LIVER MITOCHONDRIA FROM MALE AND FEMALE RATS Substrate
Measurement of Monoamine Oxidase Activity Monoamine oxidase activity was determined by a modified procedure of Heikkila et al. (5) using hydrogen peroxide as the primary standard. The molarity of hydrogen peroxide was determined by titration with potassium permanganate (14). Incubation volumes (1 ml) were set up containing a 5 mg equivalent of liver weight and the substrates, benzylamine (1 mM) or tryamine (1 mM). Tissue banks without substrate were also used. The above samples were incubated at 37°C for 1 hour. At the end of the incubation time, 0.5 ml of glucostat-peroxidase-chromogen color reagent and 0.6 ml of 5% Triton X-100 were added and the samples were read at 520 nm. The amount of hydrogen peroxide produced was determined by a comparison to a known standard. All values were corrected with the tissue blank (no substrate).
Measurement of Aldehyde Dehydrogenase Activity The enzyme was assayed spectrophotometrically in a system containing 0.1 M sodium pyrophosphate buffer, pH 9.0, 500 Ixm NAD and 1.0 mM propionaldehyde. The assay was done at 25°C and 1 unit of enzyme is defined as the amount that produces 1 ixmole NADH/minute at 340 nm. An extinction coefficient of 6.22 m M - 1 cm 1 was used for NADH.
Sources of Compounds Propionaldehyde, redistilled before use, was from J. T. Baker Chemical Co. NAD and glucostat-peroxidase-chromogen color reagent were from Boehringer-Mannheim. Benzylamine and tyraine were from Sigma Chemical Company. Hydrogen peroxide was from Fisher Scientific. RESULTS The activities of two enzymes were assayed: monoamine oxidase, located in the mitochondrial membranes, and aldehyde dehydrogenase, located in the mitochondrial matrix. The results are presented in Tables 1 and 2. Nine males and twenty-three females (twenty in the case of aldehyde dehydrogenase) were used in the experiment since the females were further subdivided into four groups based on the different phases of the estrous cycle-proestrus, estrus, metestrus and diestrus. When comparing males with females the most striking difference was in the monoamine oxidase-benzylamine activity which was almost double p < 0 . 0 0 1 using the Student's t-test) in males than in females. Monoamine oxidase-tyramine and aldehyde dehydrogenase activities were the same in males and females when expressed per mg of mitochondrial protein. During the different phases of the estrous cycle in the female rat, there were no significant differences in monoamine oxidasetyramine activity (Table 1). Monoamine oxidase-benzylamine activity, however, was highest in the proestrous phase of the cycle, the activity in the proestrous phase was almost double that in the estrous phase (p<0.05 using the Student's t-test). Aldehyde dehydrogenase activity appeared to be higher in the proestrous and diestrous phases but according to the Student's t-test these differences were not significant. DISCUSSION
There have been a number of studies done indicating sex and
Tyramin<'
Benzylamine
txmoles,mg Mitochondriai Protein/hr
ixmoies/mg Mitochondrial Protei n/hr
Subjects
(n)
Mean
~ SEM)
Mean
( ± SEM)
Males Females
(9) (231
2.06 2.20
: (!.25) :~:(! 27)
1.45 0.74
~~ 0.14)* ( m0.08)*
(5) (6) (7) (5)
2.06
:n 0.45) _:.(t.40 ~ z 0,66) ±0.685
0.94 0.51 0.72 0.85
( '-0< 13ti ( ±0.13)+ ( ±0. 171 ( ± 0 17t
Cycle of Females Proestrus Estrus Metestms Diestrus
1.96
2.55 2.13
n = Number of subjects. SEM = Standard error of the mean. *p<0.001 (two-tailed). tp<0.05 (two-tailed).
age differences in monoamine oxidase activities in rat and man. In humans, platelet and brain monoamine oxidase activity was higher in the females as compared to the males (2,15). In the rat. the monoamme oxidase activity in the hypothalamus of adult females tended to be more active than in males with tyramine as a substrate (8) but when 13-phenylethylamine was used as a substrate, there was no difference in monoamine oxidase activity between males and females (18). In the cerebral cortex and amygdala of the rat. males had higher monoamine oxidase activity than females (8/. Age seemed to be another important factor affecting monoamine oxidase levels. In humans, platelet monoamine oxidase activity in older females and males was higher than in younger females and males, respectively (2). In most regions of the human and rat brain monoamine oxidase-B activity seemed to increase with age while monoamine oxidase-A activity did not seem to change significantly (1, 3. 9). In the female rat there have been variations in
TABLE 2 ALDEHYDE DEHYDROGENASE ACTIVITYIN LIVER MITOCHONDRIA FROM MALE AND FEMALE RATS txmoleshng Mitochondrial Protein/hr Subjects
(n)
Mean
( - SEM)
Males Females
(9) (20)
2.52 2.54
(_+0.23) ( ± 0.19)
(4) (6) (5) (5)
3.10 I91 2.58 2~81
( + 0.26) (±0.33) (±0.4!) ( ± 0:373
Cycle of Females Proestrus Estrus Metestrus Diestrus
SEM = standard error of the mean. n = number of subjects.
MITOCHONDRIAL ALDH AND MAO
463
monoamine oxidase activity in different phases of the estrous cycle. These variations in monoamine oxidase activity occurred in the uterus, ovaries, adrenal glands and in the hypothalamic, limbic and midbrain regions (7,8). The studies done to date have primarily dealt with comparing monoamine oxidase activity in different brain regions of males and females. In a previous study (12) it was also demonstrated that the activity of alcohol dehydrogenase in rat liver showed clear sex differences. The alcohol dehydrogenase activity of the total liver was 1.6 times higher in females than in males. However, when aldehyde dehydrogenase activity was measured (13), the two substrates, acetaldehyde and propionaldehyde, yielded similar values in both males and females. With both alcohol dehydrogenase and aldehyde dehydrogenase, however, the intraacinar distribution profiles showed sex differences. The rats employed during this investigation were young adults chosen to assure reliable cycling of females. In this age group (80-105 days) there was no difference between males and females in monoamine oxidase-tyramine or aldehyde dehydrogenase activities but monoamine oxidase-benzylamine activity was almost double in the males as compared to the females (p<0.001 according to the Student's t-test). Monoamine oxidase plays an important role in metabolism of both dietary amines and neurotransmitters. Two activities in the membrance-bound enzyme can be distinguished: a serotonin oxidizing activity which is inhibited by clorgyline (Type A) and benzylamine oxidizing activity which is inhibited by deprenyl (Type B): Tyramine, tryptamine and dopamine are deaminated by both systems; it is at present not clear if one or two isozymes are involved in catalyzing the activities (19). Tyramine used as a substrate in this investigation is not exclusive to Type A, but benzylamine is almost exclusive to Type B. Thus, our results indicate that there was more Type B activity in male mitochondria. The importance of this difference in monoamine oxidase-benzylamine activity is at present unknown. The activities of monoamine oxidase and aldehyde dehydrogenase were also analyzed during the different phases of the estrous cycle in the female rat. As the results indicate, there was no significant difference in monoamine oxidase-tyramine and aide-
hyde dehydrogenase activities in the different phases of the cycle. There appeared to be some cycling of monoamine oxidasebenzylamine activity (Type B); the highest activity occurred in the proestrous phase and the lowest occurred in the estrous phase. In conclusion, monoamine oxidase-benzylamine activity (Type B) was significantly higher in the males as compared to the females and there seemed to be some differences in monoamine oxidase-benzylamine activity in the female rat, depending on the phase of the estrous cycle; the highest activity occurred in the prOestrous phase and the lowest in the estrous phase. Aldehyde dehydrogenase activity appeared to be the same in male and female mitochondria. Some variation noted in the different phases of the estrous cycle were not significant (according to the Student two-tailed t-test using p-<0.01 as the null hypothesis). Inactivation of aldehyde dehydrogenase by dopamine was considerably faster in female than in male mitochondria (17). Thus, if monoamine oxidase activity was important in aldehyde dehydrogenase inactivation more activity would be expected in female than in the male animals. Levels of monoamine oxidase determined in the mitochondria of male and female animals during this investigation were opposite from expected in that activity of monoamine oxidase was in fact somewhat higher in males than females. This distribution of monoamine oxidase strongly suggests that monoamine oxidase activity may not be primarily responsible for aldehyde dehydrogenase inactivation. In fact, it appears more than likely that some other system exists in the mitochondria capable of producing quinone radicals and melanin from dopamine directly. Current experiments are designed to test the possibility if catalase could convert dopamine to o-quinone and thus be involved in this process. ACKNOWLEDGEMENTS We wish to express our gratitude to Alcohol Beverage Medical Research Foundation and the Charles and Johanna Busch Memorial Fund for financial support. We would also like to thank Pat LaSasso for the typing of this manuscript and Beth-Anne Sieber who helped us with the monoamine oxidase assay.
REFERENCES 1. Arai, Y.; Kinemuchi, H. Differences between monoamine oxidase concentrations in striatum and forebrain of aged and young rats. J. Neural Transm. 72:99-105; 1988. 2. Bridge, T. P.; Soldo, B. J.; Phelps, B. H.; Wise, C. D.; Francak, M. J.; Wyatt, R. J. Platelet monoamine oxidase activity: Demographic characteristics contribute to enzyme activity variability. J. Gerontol. 40:23-28; 1985. 3. Fowler, C. J.; Wiberg, A.; Oreland, L.; Marcusson, J.; Winblad, B. The effect of age on the activity and molecular properties of human brain monoamine oxidase. J. Neural Transm. 49:1-20; 1980. 4. Hearing, V. J.; Jimenez, M. Mammalian tyrosinase--the critical regulatory control point in melanocyte pigmentation. Int. J. Biochem. 19:1141-1147; 1987. 5. Heikkila, R. E.; Manzino, L.; Cabbat, F. S.; Randall, R. J. Studies on the oxidation of the dopaminergic neurotoxin 1-methyl-4-phenyl1,2,5,6-tetrahydropyridine by monoamine oxidase B. J. Neurochem. 45:1049-1054; 1985. 6. Hogeboom, G. D. Fractionation of cell components of animals tissues. Methods Enzymol. 1:16-19; 1955. 7. Holzbauer, M.; Youdim, M. B. H. The oestrous cycle and monoamine oxidase activity. Br. J. Pharmacol. 48:600--608; 1973. 8. Kamberi, I. A.; Kobayashi, Y. Monoamine oxidase activity in the hypothalamus and various other brain areas and in some endocrine glands of the rat during the estrous cycle. J. Neurochem. 17:261-268; 1970. 9. Leung, T. K. C.; Lai, J. C. K.; Lim, L. The regional distribution of monoamine oxidase activities towards different substrates: Effects in
10. 11.
12. 13. 14. 15.
16. 17.
rat brain of chronic administration of manganese chloride and of ageing. J. Neurochem. 36:2037-2043; 1981. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with folin phenol reagent. J. Biol. Chem. 193:265-275; 1951. MacKerell, A. D., Jr.; Blatter, E. E.; Pietruszko, R. Human aldehyde dehydrogenase: Kinetic identification of the isozyme for which biogenic aldehydes and acetaldehyde compete. Alcohol.: Clin. Exp. Res. 10:266-270; 1986. Maly, I. P.; Sasse, D. Microquantitative determination of the distribution patterns of alcohol dehydrogenase activity in the liver of rat, guinea-pig and horse. Histochemistry 83:431-436; 1985. Maly, I. P.; Sasse, D. Microquantitative determination of intra-acinar distribution profiles of low-K m and high-Km aldehyde dehydrogenase activity in rat liver. Eur. J. Biochem. 170:173-178; 1987. Pierce, W. C.; Haenisch, E. L. Quantitative analysis. New York: John Wiley & Sons, Inc.; 1940:182-187. Robinson, D. S.; Davis, J. M.; Nies, A.; Ravaris, C. L.; Sylwester, D. Relation of sex and aging to monoamine oxidase activity of human brain, plasma, and platelets. Arch. Gen. Psychiatry 24:536-539; 1971. Singer, T. P.; von Kroff, R. W.; Murphy, D. L. Monoamine oxidase: Structure, function and altered functions. London: Academic Press; 1979. Turan, S. C.; Shah, P. C.; Pietruszko, R. Inactivation of aldehyde dehydrogenase in intact rat liver mitochondria by dopamine. Alcohol this issue 6: 455-460; 1989.
464
18. Vaccari, A.; Caviglia, A.; Sparatore, A.; Biassoni, R. Gonadal influences on the sexual differentiation of monoamine oxidase type A and B activities in the rat brain. J. Neurochem. 37:640--648; 1981. 19. Weyler, W.; Salach, J.l. Purification and properties of mitochondrial monoamine oxidase type A from human placenta. J. Biol. Chem.
TURAN. SHAH AND PIETRUSZKO
260:13199-13207; 1985. 20. Zarrow, M. X.; Yahim, J. M.; McCarthy, J. L. In: Experimental endocrinology, A. Source book of basic techniques. New York: Academic Press; 1964:19-39.
0741-8329/89 $3.00 + .00
Aldehyde Dehydrogenase and Monoamine Oxidase in Rat Liver Mitochondria S A F F I Y A C A T O V I C T U R A N , P R I T E S H C. S H A H A N D R E G I N A P I E T R U S Z K O I
Center of Alcohol Studies, Rutgers University, Piscataway, NJ 08855-0969 R e c e i v e d 24 October 1988; A c c e p t e d 11 July 1989
TURAN, S. C., P. C. SHAH AND R. PIETRUSZKO. Aldehydedehydrogenaseand monoamineoxidase in rat liver mitochondria. ALCOHOL 6(6) 461-464, 1989.--Monoamine oxidase (EC 1.4.3.4) and aldehyde dehydrogenase (EC 1.2.1.3) activities were compared in the liver mitochondria of male and female rats. Monoamine oxidase activity using benzylamine as a substrate was significantly higher in males as compared with females: 1.45 versus 0.74 txmoles/mg mitochondrial protein/hr, respectively. Monoamine oxidase activity using tyramine as a substrate and aldehyde dehydrogenase activity were the same in males and females. Monoamine oxidase-tyramine and aldehyde dehydrogenase activities did not vary with the different phases of the estrous cycle in the female but the activity of monoamine oxidase-benzylamine did; rats in the proestrous phase had the highest activity and those in the estrous phase had the lowest. Aldehyde dehydrogenase
Monoamine oxidase
Liver mitochondria
Male
Female
Rats
METHOD
IN the preceding paper (17) we have shown that aldehyde dehydrogenase (E.C. 1.2.1.3) within intact rat liver mitochondria can be inactivated by aerobic incubation with dopamine. This inactivation appears to occur with the simultaneous incorporation of label from J4C dopamine. The mechanism is at present unknown but the results obtained are consistent with the involvement of o-quinones. With the exception of melanogenic tissues (4), in the mammalian organism there is no known enzymic mechanism for quinone formation directly from dopamine. Quinones can be formed directly from dopamine by air oxidation, but the process is slow. The only known pathway is via monoamine oxidase (16) (E.C.1.4.3.4) which results in conversion of the amine into an aldehyde which then readily forms quinones nonenzymically. Such quinones would bear aldehyde groups and as such may be recognized as substrates by aldehyde dehydrogenase (11) and thus would have a potential to damage aldehyde dehydrogenase in a selective manner. Although inhibition of monoamine oxidase (17) only partially abolished aldehyde dehydrogenase inactivation, determination of monam~ne oxidase and aldehyde dehydrogenase levels was attempted to obtain a better understanding of the role of monoamine oxidase in aldehyde dehydrogenase inactivation. We have also demonstrated (17) that inactivation of aldehyde dehydrogenase proceeded to a greater extent and with greater consistency in the mitochondria from female than from male animals; for this reason monoamine oxidase levels in both male and female rat liver mitochondria were determined. Since inactivation of aldehyde dehydrogenase appeared to vary with the estrous cycle an attempt was made to determine levels of monoamine oxidase activity in different phases of the estrous cycle. Aldehyde dehydrogenase levels have also been determined in the mitochondria of the same animals.
Male and female Sprague-Dawley rats (Charles River) 80-105 days old were used; the males weighed between 350-400 g and the females between 215-300 g. Prior to experiments the rats were kept for at least 20 days under conditions of 12 hours of light per day and given Purina laboratory chow and water ad lib. Vaginal smears were taken daily before 10 a.m. to determine the phase of the estrous cycle (20). Females were used only if they had been following a regular estrous cycle for at least 20 days. Both the male and female rats were killed by decapitation before 10 a.m.
Preparation of Mitochondria Mitochondria were isolated according to the fractionation procedure of Hogeboom (6). Immediately after the rat was decapitated the liver was excised and 1 g was homogenized in 9 ml of 0.25 M sucrose using a Potter homogenizer followed by centrifugation at 600 x g for 10 minutes at 4°C in a refrigerated Sorvall RC-5B centrifuge. The supernatant was centrifuged at 5000 x g for 10 minutes. The resulting mitochondrial pellet was then washed with ca. 3 ml of 0.25 M sucrose and centrifuged again at 5 0 0 0 x g for 10 minutes. The mitochondrial pellet was then prepared for estimation of monoamine oxidase activity: the pellet was brought up to 1 g with 0.03 M phosphate buffer, pH 6 containing 1 mM EDTA. This mitochondrial suspension (0.1 ml) was then lysed in 20 ml of 0.05 M phosphate buffer, pH 7.4 containing 3 mM sodium azide and aliquots of this mitochondrial preparation based on the original wet weight of liver were used for monoamine oxidase activity assay. An aliquot of the original
IRequests for reprints should be addressed to Dr. R. Pietruszko.
461
462
TURAN. SHAH AND PIETRUSZKO
mitochondrial suspension was saved for protein determination by the Lowry method (10) using bovine serum albumin as the standard and the rest was sonicated to release aldehyde dehydrogenase.
TABLE 1 MONOAMINE OXIDASE ACTIVITY IN LIVER MITOCHONDRIA FROM MALE AND FEMALE RATS Substrate
Measurement of Monoamine Oxidase Activity Monoamine oxidase activity was determined by a modified procedure of Heikkila et al. (5) using hydrogen peroxide as the primary standard. The molarity of hydrogen peroxide was determined by titration with potassium permanganate (14). Incubation volumes (1 ml) were set up containing a 5 mg equivalent of liver weight and the substrates, benzylamine (1 mM) or tryamine (1 mM). Tissue banks without substrate were also used. The above samples were incubated at 37°C for 1 hour. At the end of the incubation time, 0.5 ml of glucostat-peroxidase-chromogen color reagent and 0.6 ml of 5% Triton X-100 were added and the samples were read at 520 nm. The amount of hydrogen peroxide produced was determined by a comparison to a known standard. All values were corrected with the tissue blank (no substrate).
Measurement of Aldehyde Dehydrogenase Activity The enzyme was assayed spectrophotometrically in a system containing 0.1 M sodium pyrophosphate buffer, pH 9.0, 500 Ixm NAD and 1.0 mM propionaldehyde. The assay was done at 25°C and 1 unit of enzyme is defined as the amount that produces 1 ixmole NADH/minute at 340 nm. An extinction coefficient of 6.22 m M - 1 cm 1 was used for NADH.
Sources of Compounds Propionaldehyde, redistilled before use, was from J. T. Baker Chemical Co. NAD and glucostat-peroxidase-chromogen color reagent were from Boehringer-Mannheim. Benzylamine and tyraine were from Sigma Chemical Company. Hydrogen peroxide was from Fisher Scientific. RESULTS The activities of two enzymes were assayed: monoamine oxidase, located in the mitochondrial membranes, and aldehyde dehydrogenase, located in the mitochondrial matrix. The results are presented in Tables 1 and 2. Nine males and twenty-three females (twenty in the case of aldehyde dehydrogenase) were used in the experiment since the females were further subdivided into four groups based on the different phases of the estrous cycle-proestrus, estrus, metestrus and diestrus. When comparing males with females the most striking difference was in the monoamine oxidase-benzylamine activity which was almost double p < 0 . 0 0 1 using the Student's t-test) in males than in females. Monoamine oxidase-tyramine and aldehyde dehydrogenase activities were the same in males and females when expressed per mg of mitochondrial protein. During the different phases of the estrous cycle in the female rat, there were no significant differences in monoamine oxidasetyramine activity (Table 1). Monoamine oxidase-benzylamine activity, however, was highest in the proestrous phase of the cycle, the activity in the proestrous phase was almost double that in the estrous phase (p<0.05 using the Student's t-test). Aldehyde dehydrogenase activity appeared to be higher in the proestrous and diestrous phases but according to the Student's t-test these differences were not significant. DISCUSSION
There have been a number of studies done indicating sex and
Tyramin<'
Benzylamine
txmoles,mg Mitochondriai Protein/hr
ixmoies/mg Mitochondrial Protei n/hr
Subjects
(n)
Mean
~ SEM)
Mean
( ± SEM)
Males Females
(9) (231
2.06 2.20
: (!.25) :~:(! 27)
1.45 0.74
~~ 0.14)* ( m0.08)*
(5) (6) (7) (5)
2.06
:n 0.45) _:.(t.40 ~ z 0,66) ±0.685
0.94 0.51 0.72 0.85
( '-0< 13ti ( ±0.13)+ ( ±0. 171 ( ± 0 17t
Cycle of Females Proestrus Estrus Metestms Diestrus
1.96
2.55 2.13
n = Number of subjects. SEM = Standard error of the mean. *p<0.001 (two-tailed). tp<0.05 (two-tailed).
age differences in monoamine oxidase activities in rat and man. In humans, platelet and brain monoamine oxidase activity was higher in the females as compared to the males (2,15). In the rat. the monoamme oxidase activity in the hypothalamus of adult females tended to be more active than in males with tyramine as a substrate (8) but when 13-phenylethylamine was used as a substrate, there was no difference in monoamine oxidase activity between males and females (18). In the cerebral cortex and amygdala of the rat. males had higher monoamine oxidase activity than females (8/. Age seemed to be another important factor affecting monoamine oxidase levels. In humans, platelet monoamine oxidase activity in older females and males was higher than in younger females and males, respectively (2). In most regions of the human and rat brain monoamine oxidase-B activity seemed to increase with age while monoamine oxidase-A activity did not seem to change significantly (1, 3. 9). In the female rat there have been variations in
TABLE 2 ALDEHYDE DEHYDROGENASE ACTIVITYIN LIVER MITOCHONDRIA FROM MALE AND FEMALE RATS txmoleshng Mitochondrial Protein/hr Subjects
(n)
Mean
( - SEM)
Males Females
(9) (20)
2.52 2.54
(_+0.23) ( ± 0.19)
(4) (6) (5) (5)
3.10 I91 2.58 2~81
( + 0.26) (±0.33) (±0.4!) ( ± 0:373
Cycle of Females Proestrus Estrus Metestrus Diestrus
SEM = standard error of the mean. n = number of subjects.
MITOCHONDRIAL ALDH AND MAO
463
monoamine oxidase activity in different phases of the estrous cycle. These variations in monoamine oxidase activity occurred in the uterus, ovaries, adrenal glands and in the hypothalamic, limbic and midbrain regions (7,8). The studies done to date have primarily dealt with comparing monoamine oxidase activity in different brain regions of males and females. In a previous study (12) it was also demonstrated that the activity of alcohol dehydrogenase in rat liver showed clear sex differences. The alcohol dehydrogenase activity of the total liver was 1.6 times higher in females than in males. However, when aldehyde dehydrogenase activity was measured (13), the two substrates, acetaldehyde and propionaldehyde, yielded similar values in both males and females. With both alcohol dehydrogenase and aldehyde dehydrogenase, however, the intraacinar distribution profiles showed sex differences. The rats employed during this investigation were young adults chosen to assure reliable cycling of females. In this age group (80-105 days) there was no difference between males and females in monoamine oxidase-tyramine or aldehyde dehydrogenase activities but monoamine oxidase-benzylamine activity was almost double in the males as compared to the females (p<0.001 according to the Student's t-test). Monoamine oxidase plays an important role in metabolism of both dietary amines and neurotransmitters. Two activities in the membrance-bound enzyme can be distinguished: a serotonin oxidizing activity which is inhibited by clorgyline (Type A) and benzylamine oxidizing activity which is inhibited by deprenyl (Type B): Tyramine, tryptamine and dopamine are deaminated by both systems; it is at present not clear if one or two isozymes are involved in catalyzing the activities (19). Tyramine used as a substrate in this investigation is not exclusive to Type A, but benzylamine is almost exclusive to Type B. Thus, our results indicate that there was more Type B activity in male mitochondria. The importance of this difference in monoamine oxidase-benzylamine activity is at present unknown. The activities of monoamine oxidase and aldehyde dehydrogenase were also analyzed during the different phases of the estrous cycle in the female rat. As the results indicate, there was no significant difference in monoamine oxidase-tyramine and aide-
hyde dehydrogenase activities in the different phases of the cycle. There appeared to be some cycling of monoamine oxidasebenzylamine activity (Type B); the highest activity occurred in the proestrous phase and the lowest occurred in the estrous phase. In conclusion, monoamine oxidase-benzylamine activity (Type B) was significantly higher in the males as compared to the females and there seemed to be some differences in monoamine oxidase-benzylamine activity in the female rat, depending on the phase of the estrous cycle; the highest activity occurred in the prOestrous phase and the lowest in the estrous phase. Aldehyde dehydrogenase activity appeared to be the same in male and female mitochondria. Some variation noted in the different phases of the estrous cycle were not significant (according to the Student two-tailed t-test using p-<0.01 as the null hypothesis). Inactivation of aldehyde dehydrogenase by dopamine was considerably faster in female than in male mitochondria (17). Thus, if monoamine oxidase activity was important in aldehyde dehydrogenase inactivation more activity would be expected in female than in the male animals. Levels of monoamine oxidase determined in the mitochondria of male and female animals during this investigation were opposite from expected in that activity of monoamine oxidase was in fact somewhat higher in males than females. This distribution of monoamine oxidase strongly suggests that monoamine oxidase activity may not be primarily responsible for aldehyde dehydrogenase inactivation. In fact, it appears more than likely that some other system exists in the mitochondria capable of producing quinone radicals and melanin from dopamine directly. Current experiments are designed to test the possibility if catalase could convert dopamine to o-quinone and thus be involved in this process. ACKNOWLEDGEMENTS We wish to express our gratitude to Alcohol Beverage Medical Research Foundation and the Charles and Johanna Busch Memorial Fund for financial support. We would also like to thank Pat LaSasso for the typing of this manuscript and Beth-Anne Sieber who helped us with the monoamine oxidase assay.
REFERENCES 1. Arai, Y.; Kinemuchi, H. Differences between monoamine oxidase concentrations in striatum and forebrain of aged and young rats. J. Neural Transm. 72:99-105; 1988. 2. Bridge, T. P.; Soldo, B. J.; Phelps, B. H.; Wise, C. D.; Francak, M. J.; Wyatt, R. J. Platelet monoamine oxidase activity: Demographic characteristics contribute to enzyme activity variability. J. Gerontol. 40:23-28; 1985. 3. Fowler, C. J.; Wiberg, A.; Oreland, L.; Marcusson, J.; Winblad, B. The effect of age on the activity and molecular properties of human brain monoamine oxidase. J. Neural Transm. 49:1-20; 1980. 4. Hearing, V. J.; Jimenez, M. Mammalian tyrosinase--the critical regulatory control point in melanocyte pigmentation. Int. J. Biochem. 19:1141-1147; 1987. 5. Heikkila, R. E.; Manzino, L.; Cabbat, F. S.; Randall, R. J. Studies on the oxidation of the dopaminergic neurotoxin 1-methyl-4-phenyl1,2,5,6-tetrahydropyridine by monoamine oxidase B. J. Neurochem. 45:1049-1054; 1985. 6. Hogeboom, G. D. Fractionation of cell components of animals tissues. Methods Enzymol. 1:16-19; 1955. 7. Holzbauer, M.; Youdim, M. B. H. The oestrous cycle and monoamine oxidase activity. Br. J. Pharmacol. 48:600--608; 1973. 8. Kamberi, I. A.; Kobayashi, Y. Monoamine oxidase activity in the hypothalamus and various other brain areas and in some endocrine glands of the rat during the estrous cycle. J. Neurochem. 17:261-268; 1970. 9. Leung, T. K. C.; Lai, J. C. K.; Lim, L. The regional distribution of monoamine oxidase activities towards different substrates: Effects in
10. 11.
12. 13. 14. 15.
16. 17.
rat brain of chronic administration of manganese chloride and of ageing. J. Neurochem. 36:2037-2043; 1981. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with folin phenol reagent. J. Biol. Chem. 193:265-275; 1951. MacKerell, A. D., Jr.; Blatter, E. E.; Pietruszko, R. Human aldehyde dehydrogenase: Kinetic identification of the isozyme for which biogenic aldehydes and acetaldehyde compete. Alcohol.: Clin. Exp. Res. 10:266-270; 1986. Maly, I. P.; Sasse, D. Microquantitative determination of the distribution patterns of alcohol dehydrogenase activity in the liver of rat, guinea-pig and horse. Histochemistry 83:431-436; 1985. Maly, I. P.; Sasse, D. Microquantitative determination of intra-acinar distribution profiles of low-K m and high-Km aldehyde dehydrogenase activity in rat liver. Eur. J. Biochem. 170:173-178; 1987. Pierce, W. C.; Haenisch, E. L. Quantitative analysis. New York: John Wiley & Sons, Inc.; 1940:182-187. Robinson, D. S.; Davis, J. M.; Nies, A.; Ravaris, C. L.; Sylwester, D. Relation of sex and aging to monoamine oxidase activity of human brain, plasma, and platelets. Arch. Gen. Psychiatry 24:536-539; 1971. Singer, T. P.; von Kroff, R. W.; Murphy, D. L. Monoamine oxidase: Structure, function and altered functions. London: Academic Press; 1979. Turan, S. C.; Shah, P. C.; Pietruszko, R. Inactivation of aldehyde dehydrogenase in intact rat liver mitochondria by dopamine. Alcohol this issue 6: 455-460; 1989.
464
18. Vaccari, A.; Caviglia, A.; Sparatore, A.; Biassoni, R. Gonadal influences on the sexual differentiation of monoamine oxidase type A and B activities in the rat brain. J. Neurochem. 37:640--648; 1981. 19. Weyler, W.; Salach, J.l. Purification and properties of mitochondrial monoamine oxidase type A from human placenta. J. Biol. Chem.
TURAN. SHAH AND PIETRUSZKO
260:13199-13207; 1985. 20. Zarrow, M. X.; Yahim, J. M.; McCarthy, J. L. In: Experimental endocrinology, A. Source book of basic techniques. New York: Academic Press; 1964:19-39.