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Impairment of pyruvate dehydrogenase activity by acetaldehyde
Alcohol 25 (2001) 1 – 8
Impairment of pyruvate dehydrogenase activity by acetaldehyde Marjie L. Harda,c, Sandeep Rahab,d, Michael Spinoc,e, Brian H. Robinsonb,d, Gideon Korena,c,* a
Division of Clinical Pharmacology and Toxicology, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8 b Division of Metabolism Program, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8 c Department of Pharmaceutical Science, The University of Toronto, Toronto, Ontario, Canada M5S 2S2 d Department of Biochemistry and Pediatrics, The University of Toronto, Toronto, Ontario, Canada M5S 1A8 e Apotex Inc., Weston, Ontario, Canada M9L 1T9 Received 15 November 2000; received in revised form 16 April 2001; accepted 21 April 2001
Abstract The facial features that are characteristic of fetal alcohol syndrome (FAS) are strikingly similar to those seen in pyruvate dehydrogenase (PDH) deficiency. Furthermore, alcohol-induced central nervous system insult results in midline anomalies such as agenesis of the corpus callosum, which has also been described in several metabolic diseases, including PDH deficiency. The purpose of this work was to examine the effect of acetaldehyde on PDH in vitro. The activity of PDH was measured in the presence of acetaldehyde (10 mM – 1 mM) by measuring the formation of the reduced form of nicotinamide-adenine dinucleotide at 340 nm. Pyruvate dehydrogenase was separated by using the sodium dodecyl sulfate – polyacrylamide gel electrophoresis technique after incubation with [1,2-14C]-acetaldehyde to detect the formation of covalent adducts autoradiographically. The effect of acetaldehyde on the phosphorylation of the complex was also determined autoradiographically after incubating of PDH with 32P-adenosine triphosphate. The results of this study show that acetaldehyde impairs PDH activity by a mixed inhibition type mechanism (Kic = 62.4 ± 25.7 mM, Kiu = 225 ± 68 mM), which is not a result of the formation of covalent adducts with PDH, nor of a stimulation of phosphorylation or inactivation of the complex. Because PDH levels are low throughout development and that the competition between pyruvate and acetaldehyde may be enhanced due to ethanol-induced lowering of ambient pyruvate concentrations, we conclude that impairment of PDH may have a significant effect on the developing fetus. D 2001 Elsevier Science Inc. All rights reserved. Keywords: Fetotoxicity; Acetaldehyde; Pyruvate dehydrogenase; Alcohol; Fetal alcohol syndrome
1. Introduction Since Jones et al. (1973) initiated the recognition of the deleterious effects of alcohol on the developing embryo/ fetus, much work has been done to elucidate the mechanism leading to the fetotoxic effects resulting from maternal alcohol consumption. A number of mechanisms have been proposed, to include oxidative stress (Henderson et al., 1999), retinoic acid deficiency (Zachman & Grummer, 1998), and fetal hypoxia (Mukherjee & Hodgen, 1982). Possible contributions from more than one pathway seems
* Corresponding author. Division of Clinical Pharmacology and Toxicology, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8. Tel.: +1-416-813-5781; fax: +1-416813-7562. E-mail address: [email protected] (B.H. Robinson). Editor: T.R. Jerrells
to indicate that alcohol-related birth defects (ARBD) may be a multifactorial manifestation. The relative contribution of ethanol and its first proximate metabolite, acetaldehyde, to the fetotoxicity still is not known, but both have been shown to be teratogens in animals (Sanchis et al., 1987; Sreenathan et al., 1982; Webster et al., 1983). Under normal ethanol-oxidizing conditions, acetaldehyde is not detectable in peripheral or arterial blood, but its levels may become elevated under certain conditions that include genotypic aldehyde dehydrogenase (ALDH) deficiency, treatment with an ALDH inhibitor, and chronic alcoholism (Eriksson, 1983; Eriksson & Fukunaga, 1993). High acetaldehyde levels may occur in chronic alcoholism due to enhanced ethanol oxidation resulting from induction of cytochrome P-450 2E1 (CYP 2E1) and a concomitant reduction in ALDH activity. Findings obtained from a small study showed that alcoholic women who gave birth to children with fetal alcohol syndrome (FAS) had higher acetaldehyde concentrations than did alcoholic women who
0741-8329/01/$ – see front matter D 2001 Elsevier Science Inc. All rights reserved. PII: S 0 7 4 1 - 8 3 2 9 ( 0 1 ) 0 0 1 5 6 - 2
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M.L. Hard et al. / Alcohol 25 (2001) 1–8
had healthy children (Veghelyi, 1983). It has also been shown that hemoglobin – acetaldehyde adducts were significantly elevated in alcoholic women who delivered children with fetal alcohol effects when compared with findings for alcoholic women who delivered healthy children (Niemela et al., 1991). Because only about 4% of alcoholic women produce children with the full syndrome (Abel, 1995), it may be the subpopulation of alcoholics who produce high acetaldehyde levels that is at risk for having an affected child. The characteristic facial features present in those affected with the full-blown syndrome are not unique to FAS. The presence of epicanthal folds, wide nasal bridge, short nose, undefined philtrum, and thin upper lip is also common to pyruvate dehydrogenase (PDH) deficiency, a mitochondrial disorder affecting energy metabolism (Robinson, 1995). Pyruvate dehydrogenase is a multi-enzyme complex that is responsible for the metabolism of pyruvate to acetyl coenzyme A (CoA), which consists of pyruvate decarboxylase (E1), dihydrolipoyltransacetylase (E2), and dihydrolipoyl dehydrogenase (E3). Another component, protein X, mediates an interaction between E2 and E3. The impaired ability to convert pyruvate to acetyl CoA that occurs in PDH deficiency results in secondary lactic acidosis and thus has a significant effect on development. A wide range of brain anomalies have been described in individuals with FAS, such as agenesis of the corpus callosum (Riley et al., 1995). Abnormal corpus callosum development has also been considered a marker of inborn errors of metabolism (Kolodny, 1989) and has been found in association with PDH deficiency (Shevell et al., 1994). Since Robinson et al. (1987) noted the similar facies of FAS and PDH deficiency, it was found that PDH activity was low and had an altered response to insulin in circulating lymphocytes of an 8-day-old FAS infant, which was no longer seen at 2 months of life (Ferraris et al., 1996). Because of the similarities between FAS and PDH deficiency and the variability of acetaldehyde levels among alcoholics, it is hypothesized that impairment of PDH occurs in the presence of acetaldehyde and contributes to the pathogenesis of ARBD. In this study, we report the kinetics of inhibition of PDH by acetaldehyde with the use of a commercially available purified preparation. We found that covalent adducts are formed between PDH and acetaldehyde, but these adducts do not participate in the inhibition, nor is the inhibition a result of enhanced phosphorylation or inactivation of the complex.
2. Methods 2.1. Chemicals All chemicals, unless specified otherwise, were obtained from Sigma Chemical Co. (St. Louis, MO).
2.2. Assay for pyruvate dehydrogenase complex A standard spectrophotometric method was carried out by monitoring the formation of the reduced form of nicotinamide-adenine dinucleotide (NADH) at 340 nm at 37C for a commercial preparation of porcine heart PDH (3.2 mg/ml). The reaction mixture used contained 2.5 mM nicotinamide adenine-dinucleotide (NAD), 0.2 mM thiamin pyrophosphate (TPP), 0.1 mM CoA, 0.3 mM 1,4-dithiothreitol (DTT), 1 mM MgCl2, 1 mg/ml of bovine serum albumin (BSA), 0.05 mM potassium phosphate buffer (pH 7.4), PDH (final concentration, 13 mg/ml), and pyruvate (3 –7 mM) and acetaldehyde at indicated concentrations (10 mM– 1 mM). The effect of ethanol on PDH was also tested in place of acetaldehyde at concentrations ranging from 1 to 100 mM. Spectrophotometric assays were performed with a PYEUNICAM UV4 double-beam UV visible spectrophotometer equipped with temperature-controlled sample compartments. Reference cuvettes were prepared containing the entire reaction mixture except for pyruvate. The reaction was initiated by adding pyruvate alone or a mixture of pyruvate and acetaldehyde. The data were fit to equations that describe models of inhibition by using Leonora (Cornish-Bowden, 1995), and the model selection was determined with the use of an F test (Cornish-Bowden, 1995; Ludden et al., 1994; Motulsky & Ransnas, 1987). 2.3. Assay for lipoamide dehydrogenase A similar spectrophotometric method was carried out as described above for a commercial preparation of porcine heart E3 (14.7 mg/ml) with the exception of the reaction mixture. The reaction mixture used contained 2.5 mM NAD, 1 mM MgCl2, 1 mg/ml of BSA, 0.05 mM potassium phosphate buffer (pH 7.4), E3 (final concentration, 15 – 74 mg/ml), and reduced thioctic acid (0.625 mg/ml). Thioctic acid was used to initiate the reaction. 2.4. Covalent adduct analysis [1,2-14C]-Acetaldehyde (10 mCi/mmol), purchased from American Radiolabeled Chemicals Inc. (St. Louis, MO), was received as a frozen aqueous solution (0.21 mCi/ml) and stored at 4C. Pyruvate dehydrogenase (0.85 mg/ml) was incubated with acetaldehyde (10 mM, 100 mM, and 1 mM) in 0.05 M potassium phosphate buffer (pH 7.4) containing 1 mM MgCl2 and 1 mg/ml of BSA at 37C for 2 h and 1, 2, and 3 days. Some reaction mixtures included 1 mM cysteine, which was added to the solution before the addition of acetaldehyde. Pyruvate dehydrogenase was substituted for commercial preparations of porcine heart 2-oxoglutarate dehydrogenase (2OGDH) (1.94 mg/ml), E3 (2.94 mg/ml), or BSA (1 mg/ml) in some reactions. After incubation, reaction mixtures were incubated for another 10 min with and without NaBH4 (final concentra-
M.L. Hard et al. / Alcohol 25 (2001) 1–8
tion, 5 mM –0.1 M) made in 0.1 mM NaOH (pH 9.5). The sodium dodecyl sulfate –polyacrylamide gel electrophoresis technique (SDS-PAGE) was performed in prepoured slab gels containing 16% (wt./vol.) acrylamide in a Tris-glycine buffer system (pH 8) according to the method of Laemmli (1970). Optimal separation of PDH was obtained by running the gel at constant voltage: 85 V for 1 h, followed by 75 V for 4 h. 14C-labeled bands were detected by autoradiography. Molecular weights were located by using a prestained standard, purchased from Helixx Technologies Inc. (Scarborough, ON, Canada), containing myosin (250 kDa), BSA (98 kDa), glutamic dehydrogenase (64 kDa), alcohol dehydrogenase (50 kDa), carbonic anhydrase (36 kDa), myoglobin (30 kDa), lysozyme (16 kDa), aprotinin (6 kDa), and insulin, B chain (4 kDa). Stable acetaldehyde adducts were also measured directly as protein-bound radioactivity after acid precipitation. The enzyme was precipitated with two volumes of 15% trichloroacetic acid containing 1.5% phosphotungstic acid, according to the method of Donohue et al. (1983b). The pellet was suspended in scintillation fluid, and radioactivity was determined by using a Beckman LS 6500 scintillation counter. Radioactivity measurements (disintegrations per
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minute per milligram of protein) were converted to molar quantities and expressed as picomoles of acetaldehyde bound per milligram of protein. 2.5. Phosphorylation of pyruvate dehydrogenase complex 32
P-adenosine triphosphate (ATP) (3,000 Ci/mmol) was purchased from Amersham Pharmacia Biotech (Baie d’Urfe´, PQ, Canada). Phosphorylation of PDH was detected by incubating PDH (0.51 mg/ml) with 32P-ATP (4.4 Ci/mmol) from 0 to 30 min at 37C with and without acetaldehyde (1 mM). Other substrates were added as indicated, which included 10 mM dichloroacetate (DCA), 10 mM adenosine diphosphate (ADP), and 250 mM acetyl CoA. The SDSPAGE was performed in prepoured slab gels containing 16% (wt./vol.) acrylamide, as described above, and 32 P-labeled bands were detected by autoradiography.
3. Results Addition of acetaldehyde to a preparation of porcine heart PDH impairs its activity at 37C. As presented in
Fig. 1. Fractional velocity (v) (ratio of the velocity of the enzyme in the presence of inhibitor to the velocity without inhibitor) of porcine heart pyruvate dehydrogenase at 37C. The inhibitor, acetaldehyde, was used at concentrations of 10 mM and 0.1, 0.4, 0.7, and 1.0 mM in the presence of 3, 5, and 7 mM of the substrate, pyruvate. The velocity of the enzyme was determined by measuring the formation of the reduced form of nicotinamide-adenine dinucleotide spectrophotometrically at 340 nm. The Dixon plot (top inset; inverse velocity [1/v] vs. inhibitor concentration) and the Cornish-Bowden plot (bottom inset; ratio of the substrate concentration to the velocity of the enzyme [s/v] vs. inhibitor concentration) are also shown.
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M.L. Hard et al. / Alcohol 25 (2001) 1–8
Table 1 Comparing models of inhibition for pyruvate dehydrogenase by acetaldehydea Model
SS
df
MIb CI UCI NCI
0.022458 0.04561 0.05595 0.045587
11 12 12 12
a
Enzyme kinetic data obtained for pyruvate dehydrogenase in the presence of acetaldehyde were fit to four models of inhibition. By comparing models with the same number of parameters, one can determine the best fit by the lowest sum of squares (SS). An F test was used to compare the model of mixed inhibition (MI) to the remaining models due to the inclusion of an extra parameter. This analysis allows us to reject the hypothesis that competitive inhibition (CI), uncompetitive inhibition (UCI), and noncompetitive inhibition (NCI) are true models of inhibition and conclude that the data obtained were best fit to a model of MI. b The equation describing mixed inhibition is v = Vxs/(Km(1 + i/ Kic) + s(1 + i/Kiu)), where V is the maximal velocity; Km, the Michaelis – Menten constant; s, the substrate concentration; i, the inhibitor concentration; Kic, the dissociation constant of the enzyme – substrate complex; and Kiu, the dissociation constant of the enzyme – substrate – inhibitor complex. The estimates for the parameters were V = 2.38 ± 0.15 mmol/mg/min, Km = 1.40 ± 0.39 mM, Kic = 62.4 ± 25.7 mM, and Kiu = 225 ± 68 mM.
Fig. 1, the degree of inhibition increases with increasing acetaldehyde concentration. To determine the nature of inhibition, the results were fit to equations that describe four types of inhibition: noncompetitive (NCI), uncompetitive (UCI), competitive (CI), and mixed (MI) by using Leonora (Cornish-Bowden, 1995), a program written specifically for fitting enzyme kinetic data. Predicted values of the velocity in the presence of inhibitor for each model were obtained, and the sum of squares (SS) was calculated to determine the goodness of fit (Table 1). The SS was found to be lowest for MI. However, because of the extra parameter present in the equation that describes MI, the F ratios
were calculated to determine whether the extra parameter had any more than a chance effect on the fit. The results presented in Table 1 show that there was not a significant difference between fitting the data to CI, UCI, or NCI models. For MI, however, there was a significant difference in how close the fit came to the experimental data in comparison with other models [CI: F(1,11) = 11.39, P < .0 1 ; U C I : F(1,11) = 16.40, P < .01; NCI : F(1,11) = 11.39, P < .01]. Therefore, by fitting the data only to a model of MI we can reject the hypothesis that any remaining models are true models of inhibition. The Dixon (top inset to Fig. 1) and Cornish-Bowden (bottom inset to Fig. 1) plots of our data are also consistent with MI (Cornish-Bowden, 1974). The inhibition constants, Kic and Kiu, obtained for MI were calculated by using Leonora as 62.4 ± 25.7 and 225 ± 68 mM, respectively. The kinetics of PDH was examined in the presence of 1 –100 mM ethanol. No inhibition was observed. Incubation of [1, 2-14C]-acetaldehyde with porcine heart PDH resulted in the formation of covalent adducts. As shown in Fig. 2a, the amount of binding increased between 2 h and 1 day of incubation, with no further increases up to 3 days of incubation (results not shown). An increase of the acetaldehyde concentration from 10 mM to 1 mM resulted in an increase in the degree of formation of acetaldehyde –PDH adducts (Figs. 2b and 3). The most intense band that occurs at approximately 54 kDa is likely to correspond to either E3 or protein X, which typically run at 55 and 52 kDa, respectively. Less intense bands also appear in the range that corresponds to E2, E1a, and E1b, which run at 74, 41, and 36 kDa, respectively. The formation of acetaldehyde – protein adducts has been attributed to the formation of an unstable Shiff base that could be reduced, and thus stabilized, in the presence of a reducing agent such as NaBH4 (Tuma et al., 1991). In this study, enhanced formation of stable acetaldehyde –protein adducts was not seen at low
Fig. 2. Autoradiographs of porcine heart pyruvate dehydrogenase (PDH) separated by using the sodium dodecyl sulfate – polyacrylamide gel electrophoresis technique after incubation at 37C with a) 1 mM [1,2-14C] acetaldehyde for 2 h and 1 day and b) 10 mM – 1 mM [1,2-14C] acetaldehyde for 1 day. Reaction mixtures were mixed with (+) and without ( ) NaBH4 (5 mM). Formation of acetaldehyde – PDH adducts increased in a time-dependent fashion and with increasing acetaldehyde concentration. Addition of 5 mM NaBH4 had no effect on the number of covalent adducts formed. Addition of 1 mM cysteine (C) prevented the formation of PDH – acetaldehyde adducts.
M.L. Hard et al. / Alcohol 25 (2001) 1–8
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Fig. 3. Amount of acetaldehyde bound per milligram of pyruvate dehydrogenase (PDH). Pyruvate dehydrogenase was precipitated after incubation with [1,2-14C] acetaldehyde for 1 day with (+) and without ( ) 5 mM NaBH4. The amount of acetaldehyde bound was determined by measuring radioactivity of the pellet, and it ranged from 3.5 to 25 pmol/mg of PDH for acetaldehyde concentrations ranging from 1 mM to 1 mM.
NaBH4 concentrations (Fig. 2a and b), but it was seen at high NaBH4 concentrations (Fig. 4). Addition of 1 mM cysteine to a 1 mM acetaldehyde reaction mixture at the beginning of the incubation prevented the formation of covalent adducts (Fig. 2b), which was expected because cysteine is known to form stable complexes with acetaldehyde. Because the formation of covalent adducts was time dependent, the residual activity of PDH was measured spectrophotometrically after incubation at various times.
Fig. 4. Autoradiograph of porcine heart pyruvate dehydrogenase (PDH), 2-oxoglutarate dehydrogenase (2OGDH), and dihydrolipoyl dehydrogenase (E3) separated by using the sodium docecyl sulfate – polyacrylamide gel electrophoresis techique after 1 day of incubation at 37C with 1 mM [1,2-14C] acetaldehyde with (+) and without ( ) NaBH4 (0.1 M). Acetaldehyde formed covalent adducts with all subunits common to PDH and 2OGDH. The addition of 0.1 M NaBH4 increased the number of covalent adducts formed.
After acetaldehyde was mixed with PDH, under the same conditions as those used to follow covalent adduct formation, no difference in activity was measured up to 4 h of incubation (results not shown). Incubation of the reaction mixture for 24 h resulted in enzyme degradation, but this was attributed to the presence of proteases in the commercial preparation, rather than to acetaldehyde. Therefore, increasing the degree of adducts formed has no more effect on the activity than does instantaneous exposure of PDH to acetaldehyde. Covalent binding of acetaldehyde to PDH occurs primarily on a subunit just above 50 kDa (Fig. 2a and b), which is in a range consistent with that for the E3 or protein X subunit of PDH. The activity of E3 in the presence of acetaldehyde was examined, but no inhibition was observed. Thus, if acetaldehyde were primarily attaching to E3, the activity of the enzyme would not be affected. The results of the timedependent kinetic studies seem to indicate that acetaldehyde inhibition may arise solely from the interaction of acetaldehyde with a catalytic site on PDH, possibly lipoic acid. Lipoic acid is present on both the E2 subunit (74 kDa) and protein X (55 kDa). 2-Oxoglutarate dehydrogenase is almost identical in structure to PDH, but it lacks protein X. To determine whether the prominent band above 50 kDa was E3 or protein X, the binding of acetaldehyde to 2OGDH and E3 was examined and compared with the binding of acetaldehyde to PDH. As presented in Fig. 4, the band patterns of PDH and 2OGDH after addition of acetaldehyde are identical. Furthermore, in the experiment done with E3 alone it is clear that acetaldehyde is binding with the E3 subunit on both PDH and 2OGDH. These results however, do not allow us to rule out the possibility that acetaldehyde is also binding to protein X.
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M.L. Hard et al. / Alcohol 25 (2001) 1–8
Fig. 5. Autoradiographs of porcine heart pyruvate dehydrogenase separated by using the sodium docecyl sulfate – polyacrylamide gel electrophoresis technique after incubation at 37C with 32P-adenosine triphosphate (4.4 Ci/mmol) at 37C a) with (+) and without ( ) 1 mM acetaldehyde from 0 to 30 min and b) for 5 min with (+) and without ( ) 1 mM acetaldehyde (A) with co-addition of substrates that affect kinase activity that include 10 mM dichloroacetate (B), 10 mM adenosine diphosphate (C), and 250 mM acetyl CoA (D). The degree of phosphorylation increases with time. No effect on phosphorylation by acetaldehyde was detected. Acetaldehyde did not inhibit PDH by enhancing the degree of phosphorylation by means of an interaction with the kinase protein.
Findings obtained from incubating PDH with ATP in the presence of acetaldehyde show that acetaldehyde has no effect on the phosphorylation of PDH (Fig. 5a). In addition, in the presence of two kinase inhibitors, DCA and ADP, and a kinase stimulator, acetyl CoA, no effect of acetaldehyde on phosphorylation is detected (Fig. 5b).
4. Discussion In this study, we have examined the effect of acetaldehyde on a commercial preparation of purified porcine heart PDH. Because all mammalian PDHs are kinetically similar and because of the same protein isoenzyme components in every tissue, it is reasonable to assume that the demonstrated effects on heart PDH would be applicable to other tissues and could be extrapolated to the human fetus. The mental retardation resulting from prenatal alcohol exposure is by far the most debilitating manifestation of FAS. Thus, the vulnerability of the brain to the toxic effects of ethanol and acetaldehyde is of primary interest. Ethanol is capable of crossing the human placenta. Because of the presence of CYP 2E1 (Brzezinski et al., 1999) and a currently unidentified cystolic oxidase (Person et al., 2000) in the human fetal brain, it is possible that acetaldehyde may be produced in the target organ of interest. The brain, which relies on glucose as its sole source of fuel, would therefore be quite vulnerable to the potential effects of acetaldehyde on PDH. We fit our kinetic data to equations that describe four types of inhibition: UCI, NCI, CI, and MI. By using Leonora (Cornish-Bowden, 1995), results showed that inhibition of PDH by acetaldehyde occurs by a MI type mechanism (Kic = 62.4 ± 25.7 mM, Kiu = 225 ± 68 mM). The Dixon (top inset to Fig. 1) and Cornish-Bowden (bottom inset to Fig. 1) plots are characteristic of MI (Cornish-Bowden, 1974). Because it still is not known whether it is ethanol or
acetaldehyde that is causing the toxicity resulting in ARBD, we examined the effects of ethanol on PDH. No inhibition was observed. From these observations we can conclude that if inhibition of PDH is contributing to the pathogenesis of ARBD, acetaldehyde, rather than ethanol, is the toxic species. These observations may be significant in the developing fetus because PDH levels are low throughout development and do not reach adult levels until term. Therefore, even a small degree of inhibition may have severe consequences (Robinson et al., 1977). Two previous studies describe the inhibition of PDH by acetaldehyde by both noncompetitive (Ki = 130 ± 30 mM) (Blass & Lewis, 1973) and uncompetitive (Ki = 150 ± 20 mM) (Alkonyi et al., 1978) mechanisms with respect to pyruvate, CoA, TPP, and NAD. The results of these studies were drawn from graphical methods alone (Alkonyi et al., 1978) and did not allow the investigators to reach definite conclusions about the nature of inhibition (Blass & Lewis, 1973). Blass and Lewis (1973) have shown that the oxidation of pyruvate in the absence of CoA or NAD was not inhibited by acetaldehyde, which seems to indicate that inhibition of PDH is not a result of an interaction of acetaldehyde with the E1 subunit. Conversely, Alkonyi et al. (1978) concluded that inhibition was occurring at E1 after examining the effects of acetaldehyde on the activity of E1, E2, and E3. The results of the work presented in the current study are in agreement with those of Alkonyi et al. (1978) because we concluded that inhibition of PDH by acetaldehyde occurs by a combination of CI and UCI. The competitive component implies that acetaldehyde is competing with pyruvate for its catalytic site on E1. The competition of acetaldehyde with pyruvate for PDH may have significance in vivo because it would be further enhanced in the presence of ethanol, which causes a decrease in ambient pyruvate concentrations. We further examined the role of covalent binding and the effect of acetaldehyde on phosphorylation and inactivation of PDH, which has not been previously investigated.
M.L. Hard et al. / Alcohol 25 (2001) 1–8
Acetaldehyde is capable of forming covalent adducts with a variety of proteins that include hepatic proteins (Donohue et al., 1983b; Medina et al., 1985), albumin (Donohue et al., 1983a), hemoglobin (San George & Hoberman, 1986), tublin (Tuma et al., 1987), and certain enzymes (Mauch et al., 1986). The functional consequences of adduct formation have been shown in a number of proteins (Mauch et al., 1986; Tuma & Sorrell, 1987). For example, the activity of the lysine-dependent enzymes glucose-6-phosphate dehydrogenase and RNAse was inhibited in the presence of acetaldehyde, and the degree of inhibition correlated with total adduct formation (Mauch et al., 1986). In our study, we found that acetaldehyde forms covalent adducts with PDH, and the degree of adduct formation follows a concentration and time-dependent increase. The formation of acetaldehyde – protein adducts has been attributed to the formation of a Shiff base intermediate, which can be reduced, and thus stabilized, on addition of NaBH4 (Tuma & Sorrell, 1987; Tuma et al., 1991). To increase the number of stable acetaldehyde – PDH adducts, it was necessary to use concentrations of NaBH4 that were much higher (0.1 M) than previously reported in the literature (5 mM) (Donohue et al., 1983a, 1983b; Mauch et al., 1986; Medina et al., 1985). Addition of cysteine (1 mM), which is capable of complexing acetaldehyde, prevents the formation of PDH – acetaldehyde adducts (Fig. 2b) by effectively removing free acetaldehyde from solution. In our kinetic experiments, we attempted to add cysteine to prevent inhibition, but this was ineffective. In the presence of cysteine, the activity of PDH decreased even further. When the inhibitor was removed from solution there was still a reduction in PDH activity when cysteine was added. Because cysteine is known to be reactive with a-keto acids, we suspect that cysteine may be capable of removing pyruvate from solution, accounting for the observed reduction in activity. Because prolonged incubation times had an effect on the number of covalent adducts formed, we further investigated the effect of incubation time on inhibition kinetics. The activity of PDH incubated with acetaldehyde was measured at a number of time points. Over a period of 4 h there were no changes in the amount of inhibition. Because we observed a time-dependent increase in the number of PDH –acetaldehyde adducts formed, which is not associated with a time-dependent increase in inhibition, we conclude that covalent binding of acetaldehyde to PDH does not contribute to the loss of enzymatic activity. In addition, it may be the non-covalent interaction of acetaldehyde with a catalytic site of PDH that is responsible for the inhibition of PDH. Lipoic acid, located on E2, and protein X are essential for PDH function. The reduced form of lipoate consists of two sulfhydry groups that are capable of nucleophilic attack of the electrophilic carbonyl group in acetaldehyde. This resultant acetylation of lipoic acid would effectively reduce the
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catalytic function of PDH. To determine whether acetaldehyde is interacting with the lipoate, we compared the binding sites of acetaldehyde with those of PDH and 2OGDH. 2-Oxoglutarate dehydrogenase is almost identical in subunit structure to PDH except that it lacks protein X. Protein X has a molecular weight that is similar to that for E3. Thus, to distinguish between the binding of acetaldehyde to protein X and E3, we repeated our labeling experiments with 2OGDH and isolated E3. The PDH and 2OGDH lanes in Fig. 4 are indistinguishable, which is indicative of the formation of acetaldehyde adducts with all subunits common to PDH and 2OGDH. In theory, if acetaldehyde were preferentially binding to protein X, rather than to E3, we would not have seen a band above 50 kDa in 2OGDH. Furthermore, use of isolated E3 seems to indicate that the band above 50 kDa is a result of acetaldehyde –E3 adduct formation, but we cannot rule out the possibility that acetaldehyde is also binding to protein X. We also tested the effect of acetaldehyde on E3 activity and found no differences in activity in the presence of acetaldehyde as was previously demonstrated (Alkonyi et al., 1978). Because we have observed that the majority of binding seems to occur at E3, the activity of E3 is not affected in the presence of acetaldehyde, and the degree of inhibition does not increase with increasing adduct formation, we can conclude that the inhibition of PDH by acetaldehyde is due to a non-covalent phenomenon. Protein kinase is responsible for phosphorylation or inactivation of the complex, where the phospho group is obtained from ATP. When we incubated PDH with 32P-ATP, the intensity of the bands increased over time, indicating an increase in the amount of phosphorylation and thus inactivation of the complex. If acetaldehyde inhibited the complex by an interaction with kinase, it would be expected that acetaldehyde would have enhanced the degree of phosphorylation, which would have resulted in an increase in the intensity of the bands shown in Fig. 5a where acetaldehyde was present. However, this was not observed. Our final conclusion is that acetaldehyde will give MI of PDH in the physiological range, which may be made worse by ethanol-induced lowering of pyruvate concentrations. The inhibition is not influenced by covalent adduct formation between E3 and acetaldehyde, is not a result of an effect on phosphorylation of the complex, and is quite reversible. Our results support the hypothesis that inhibition of PDH by acetaldehyde has implications in the developing fetus.
Acknowledgments This work was supported by a grant from the Canadian Institute for Health Research. Gideon Koren is a Senior Scientist of the Canadian Institute of Health Research. Brian Robinson is a holder of a Millenium Canada Research Chair. Marjie Hard is a recipient of a graduate studentship from The Research Institute, The Hospital for Sick Children.
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References Abel, E. L. (1995). An update on incidence of FAS: FAS is not an equal opportunity birth defect. Neurotoxicol Teratol 17, 437 – 443. Alkonyi, I., Bolygo, E., Gyocsi, L., & Sumegi, B. (1978). Kinetic studies of the inhibitory effect of acetaldehyde and D( )acetoin on the pyruvate dehydrogenase complex from pig heart. Acta Biochim Biophys Acad Sci Hung 13, 143 – 152. Blass, J. P., & Lewis, C. A. (1973). Inhibition by acetaldehyde of the pyruvate dehydrogenase complex from ox brain and ox kidney. Biochem J 131, 415 – 416. Brzezinski, M. R., Boutelet-Bochan, H., Person, R. E., Fantel, A. G., & Juchau, M. R. (1999). Catalytic activity and quantitation of cytochrome P-450 2E1 in prenatal human brain. J Pharmacol Exp Ther 289, 1648 – 1653. Cornish-Bowden, A. (1974). A simple graphical method for determining the inhibition constants of mixed, uncompetitive and non-competitive inhibitors. Biochem J 137, 143 – 144. Cornish-Bowden, A. (1995). Analysis of Enzyme Kinetic Data. New York: Oxford University Press. Donohue, T. M., Tuma, D. J., & Sorrell, M. F. (1983a). Acetaldehyde adducts with proteins: binding of [14C]acetaldehyde to serum albumin. Arch Biochem Biophys 220, 239 – 246. Donohue, T. M., Tuma, D. J., & Sorrell, M. F. (1983b). Binding of metabolically derived acetaldehyde to hepatic proteins in vitro. Lab Invest 49, 226 – 229. Eriksson, C. J. (1983). Human blood acetaldehyde concentration during ethanol oxidation (update 1982). Pharmacol Biochem Behav 18 (Suppl 1), 141 – 150. Eriksson, C. J., & Fukunaga, T. (1993). Human blood acetaldehyde (update 1992). Alcohol Alcohol Suppl 2, 9 – 25. Ferraris, S., Mostert, M., Rabbone, I., Cerutti, F., Borgione, S., Curto, M., Mioletti, S., Ponzone, A., Silvestro, L., & Rinaudo, M. T. (1996). Derangement of pyruvate dehydrogenase activity in circulating lymphocytes of a newborn with fetal alcohol syndrome [letter]. Acta Paediatr 85, 640. Henderson, G. I., Chen, J. J., & Schenker, S. (1999). Ethanol, oxidative stress, reactive aldehydes, and the fetus. Front Biosci 4, D541 – D550. Jones, K. L., Smith, D. W., Ulleland, C. N., & Streissguth, P. (1973). Pattern of malformation in offspring of chronic alcoholic mothers. Lancet 1, 1267 – 1271. Kolodny, E. H. (1989). Agenesis of the corpus callosum: a marker for inherited metabolic disease? Neurology 39, 847 – 848. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 – 685. Ludden, T. M., Beal, S. L., & Sheiner, L. B. (1994). Comparison of the Akaike Information Criterion, the Schwarz criterion and the F test as guides to model selection. J Pharmacokinet Biopharm 22, 431 – 445. Mauch, T. J., Donohue, T. M., Zetterman, R. K., Sorrell, M. F., & Tuma, D. J. (1986). Covalent binding of acetaldehyde selectively inhibits the catalytic activity of lysine-dependent enzymes. Hepatology 6, 263 – 269. Medina, V. A., Donohue, T. M., Sorrell, M. F., & Tuma, D. J. (1985). Covalent binding of acetaldehyde to hepatic proteins during ethanol oxidation. J Lab Clin Med 105, 5 – 10.
Motulsky, H. J., & Ransnas, L. A. (1987). Fitting curves to data using nonlinear regression: a practical and nonmathematical review. FASEB J 1, 365 – 374. Mukherjee, A. B., & Hodgen, G. D. (1982). Maternal ethanol exposure induces transient impairment of umbilical circulation and fetal hypoxia in monkeys. Science 218, 700 – 702. Niemela, O., Halmesmaki, E., & Ylikorkala, O. (1991). Hemoglobin – acetaldehyde adducts are elevated in women carrying alcohol-damaged fetuses. Alcohol Clin Exp Res 15, 1007 – 1010. Person, R. E., Chen, H., Fantel, A. G., & Juchau, M. R. (2000). Enzymic catalysis of the accumulation of acetaldehyde from ethanol in human prenatal cephalic tissues: evaluation of the relative contributions of CYP2E1, alcohol dehydrogenase, and catalase/peroxidases. Alcohol Clin Exp Res 24, 1433 – 1442. Riley, E. P., Mattson, S. N., Sowell, E. R., Jernigan, T. L., Sobel, D. F., & Jones, K. L. (1995). Abnormalities of the corpus callosum in children prenatally exposed to alcohol. Alcohol Clin Exp Res 19, 1198 – 1202. Robinson, B. H. (1995). Mitochondrial defects: an overview of inborn errors associated with lacticacidemia. Int Pediatr 10, 82 – 88. Robinson, B. H., De Meirleir, L., Glerum, M., Sherwood, G., & Becker, L. (1987). Clinical presentation of mitochondrial respiratory chain defects in NADH-coenzyme Q reductase and cytochrome oxidase: clues to pathogenesis of Leigh disease. J Pediatr 110, 216 – 222. Robinson, B. H., Sherwood, W. G., & Oei, J. (1977). The development of pyruvate dehydrogenase in the subhuman primate Macaca mulatta. Biol Neonate 32, 154 – 157. San George, R. C., & Hoberman, H. D. (1986). Reaction of acetaldehyde with hemoglobin. J Biol Chem 261, 6811 – 6821. Sanchis, R., Sancho-Tello, M., Chirivella, M., & Guerri, C. (1987). The role of maternal alcohol damage on ethanol teratogenicity in the rat. Teratology 36, 199 – 208. Shevell, M. I., Matthews, P. M., Scriver, C. R., Brown, R. M., Otero L. J., Legris, M., Brown G. K., & Arnold, D. L. (1994). Cerebral dysgenesis and lactic acidemia: an MRI/MRS phenotype associated with pyruvate dehydrogenase deficiency. Pediatr Neurol 11, 224 – 229. Sreenathan, R. N., Padmanabhan, R., & Singh, S. (1982). Teratogenic effects of acetaldehyde in the rat. Drug Alcohol Depend 9, 339 – 350. Tuma, D. J., Hoffman, T., & Sorrell, M. F. (1991). The chemistry of acetaldehyde – protein adducts. Alcohol Alcohol Suppl 1, 271 – 276. Tuma, D. J., Jennett, R. B., & Sorrell, M. F. (1987). The interaction of acetaldehyde with tubulin. Ann N Y Acad Sci 492, 277 – 286. Tuma, D. J., & Sorrell, M. F. (1987). Functional consequences of acetaldehyde binding to proteins. Alcohol Alcohol Suppl 1, 61 – 66. Veghelyi, P. V. (1983). Fetal abnormality and maternal ethanol metabolism [letter]. Lancet 2, 53 – 54. Webster, W. S., Walsh, D. A., McEwen, S. E., & Lipson, A. H. (1983). Some teratogenic properties of ethanol and acetaldehyde in C57BL/6J mice: implications for the study of the fetal alcohol syndrome. Teratology 27, 231 – 243. Zachman, R. D., & Grummer, M. A. (1998). The interaction of ethanol and vitamin A as a potential mechanism for the pathogenesis of Fetal Alcohol syndrome. Alcohol Clin Exp Res 22, 1544 – 1556.
Impairment of pyruvate dehydrogenase activity by acetaldehyde Marjie L. Harda,c, Sandeep Rahab,d, Michael Spinoc,e, Brian H. Robinsonb,d, Gideon Korena,c,* a
Division of Clinical Pharmacology and Toxicology, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8 b Division of Metabolism Program, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8 c Department of Pharmaceutical Science, The University of Toronto, Toronto, Ontario, Canada M5S 2S2 d Department of Biochemistry and Pediatrics, The University of Toronto, Toronto, Ontario, Canada M5S 1A8 e Apotex Inc., Weston, Ontario, Canada M9L 1T9 Received 15 November 2000; received in revised form 16 April 2001; accepted 21 April 2001
Abstract The facial features that are characteristic of fetal alcohol syndrome (FAS) are strikingly similar to those seen in pyruvate dehydrogenase (PDH) deficiency. Furthermore, alcohol-induced central nervous system insult results in midline anomalies such as agenesis of the corpus callosum, which has also been described in several metabolic diseases, including PDH deficiency. The purpose of this work was to examine the effect of acetaldehyde on PDH in vitro. The activity of PDH was measured in the presence of acetaldehyde (10 mM – 1 mM) by measuring the formation of the reduced form of nicotinamide-adenine dinucleotide at 340 nm. Pyruvate dehydrogenase was separated by using the sodium dodecyl sulfate – polyacrylamide gel electrophoresis technique after incubation with [1,2-14C]-acetaldehyde to detect the formation of covalent adducts autoradiographically. The effect of acetaldehyde on the phosphorylation of the complex was also determined autoradiographically after incubating of PDH with 32P-adenosine triphosphate. The results of this study show that acetaldehyde impairs PDH activity by a mixed inhibition type mechanism (Kic = 62.4 ± 25.7 mM, Kiu = 225 ± 68 mM), which is not a result of the formation of covalent adducts with PDH, nor of a stimulation of phosphorylation or inactivation of the complex. Because PDH levels are low throughout development and that the competition between pyruvate and acetaldehyde may be enhanced due to ethanol-induced lowering of ambient pyruvate concentrations, we conclude that impairment of PDH may have a significant effect on the developing fetus. D 2001 Elsevier Science Inc. All rights reserved. Keywords: Fetotoxicity; Acetaldehyde; Pyruvate dehydrogenase; Alcohol; Fetal alcohol syndrome
1. Introduction Since Jones et al. (1973) initiated the recognition of the deleterious effects of alcohol on the developing embryo/ fetus, much work has been done to elucidate the mechanism leading to the fetotoxic effects resulting from maternal alcohol consumption. A number of mechanisms have been proposed, to include oxidative stress (Henderson et al., 1999), retinoic acid deficiency (Zachman & Grummer, 1998), and fetal hypoxia (Mukherjee & Hodgen, 1982). Possible contributions from more than one pathway seems
* Corresponding author. Division of Clinical Pharmacology and Toxicology, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8. Tel.: +1-416-813-5781; fax: +1-416813-7562. E-mail address: [email protected] (B.H. Robinson). Editor: T.R. Jerrells
to indicate that alcohol-related birth defects (ARBD) may be a multifactorial manifestation. The relative contribution of ethanol and its first proximate metabolite, acetaldehyde, to the fetotoxicity still is not known, but both have been shown to be teratogens in animals (Sanchis et al., 1987; Sreenathan et al., 1982; Webster et al., 1983). Under normal ethanol-oxidizing conditions, acetaldehyde is not detectable in peripheral or arterial blood, but its levels may become elevated under certain conditions that include genotypic aldehyde dehydrogenase (ALDH) deficiency, treatment with an ALDH inhibitor, and chronic alcoholism (Eriksson, 1983; Eriksson & Fukunaga, 1993). High acetaldehyde levels may occur in chronic alcoholism due to enhanced ethanol oxidation resulting from induction of cytochrome P-450 2E1 (CYP 2E1) and a concomitant reduction in ALDH activity. Findings obtained from a small study showed that alcoholic women who gave birth to children with fetal alcohol syndrome (FAS) had higher acetaldehyde concentrations than did alcoholic women who
0741-8329/01/$ – see front matter D 2001 Elsevier Science Inc. All rights reserved. PII: S 0 7 4 1 - 8 3 2 9 ( 0 1 ) 0 0 1 5 6 - 2
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had healthy children (Veghelyi, 1983). It has also been shown that hemoglobin – acetaldehyde adducts were significantly elevated in alcoholic women who delivered children with fetal alcohol effects when compared with findings for alcoholic women who delivered healthy children (Niemela et al., 1991). Because only about 4% of alcoholic women produce children with the full syndrome (Abel, 1995), it may be the subpopulation of alcoholics who produce high acetaldehyde levels that is at risk for having an affected child. The characteristic facial features present in those affected with the full-blown syndrome are not unique to FAS. The presence of epicanthal folds, wide nasal bridge, short nose, undefined philtrum, and thin upper lip is also common to pyruvate dehydrogenase (PDH) deficiency, a mitochondrial disorder affecting energy metabolism (Robinson, 1995). Pyruvate dehydrogenase is a multi-enzyme complex that is responsible for the metabolism of pyruvate to acetyl coenzyme A (CoA), which consists of pyruvate decarboxylase (E1), dihydrolipoyltransacetylase (E2), and dihydrolipoyl dehydrogenase (E3). Another component, protein X, mediates an interaction between E2 and E3. The impaired ability to convert pyruvate to acetyl CoA that occurs in PDH deficiency results in secondary lactic acidosis and thus has a significant effect on development. A wide range of brain anomalies have been described in individuals with FAS, such as agenesis of the corpus callosum (Riley et al., 1995). Abnormal corpus callosum development has also been considered a marker of inborn errors of metabolism (Kolodny, 1989) and has been found in association with PDH deficiency (Shevell et al., 1994). Since Robinson et al. (1987) noted the similar facies of FAS and PDH deficiency, it was found that PDH activity was low and had an altered response to insulin in circulating lymphocytes of an 8-day-old FAS infant, which was no longer seen at 2 months of life (Ferraris et al., 1996). Because of the similarities between FAS and PDH deficiency and the variability of acetaldehyde levels among alcoholics, it is hypothesized that impairment of PDH occurs in the presence of acetaldehyde and contributes to the pathogenesis of ARBD. In this study, we report the kinetics of inhibition of PDH by acetaldehyde with the use of a commercially available purified preparation. We found that covalent adducts are formed between PDH and acetaldehyde, but these adducts do not participate in the inhibition, nor is the inhibition a result of enhanced phosphorylation or inactivation of the complex.
2. Methods 2.1. Chemicals All chemicals, unless specified otherwise, were obtained from Sigma Chemical Co. (St. Louis, MO).
2.2. Assay for pyruvate dehydrogenase complex A standard spectrophotometric method was carried out by monitoring the formation of the reduced form of nicotinamide-adenine dinucleotide (NADH) at 340 nm at 37C for a commercial preparation of porcine heart PDH (3.2 mg/ml). The reaction mixture used contained 2.5 mM nicotinamide adenine-dinucleotide (NAD), 0.2 mM thiamin pyrophosphate (TPP), 0.1 mM CoA, 0.3 mM 1,4-dithiothreitol (DTT), 1 mM MgCl2, 1 mg/ml of bovine serum albumin (BSA), 0.05 mM potassium phosphate buffer (pH 7.4), PDH (final concentration, 13 mg/ml), and pyruvate (3 –7 mM) and acetaldehyde at indicated concentrations (10 mM– 1 mM). The effect of ethanol on PDH was also tested in place of acetaldehyde at concentrations ranging from 1 to 100 mM. Spectrophotometric assays were performed with a PYEUNICAM UV4 double-beam UV visible spectrophotometer equipped with temperature-controlled sample compartments. Reference cuvettes were prepared containing the entire reaction mixture except for pyruvate. The reaction was initiated by adding pyruvate alone or a mixture of pyruvate and acetaldehyde. The data were fit to equations that describe models of inhibition by using Leonora (Cornish-Bowden, 1995), and the model selection was determined with the use of an F test (Cornish-Bowden, 1995; Ludden et al., 1994; Motulsky & Ransnas, 1987). 2.3. Assay for lipoamide dehydrogenase A similar spectrophotometric method was carried out as described above for a commercial preparation of porcine heart E3 (14.7 mg/ml) with the exception of the reaction mixture. The reaction mixture used contained 2.5 mM NAD, 1 mM MgCl2, 1 mg/ml of BSA, 0.05 mM potassium phosphate buffer (pH 7.4), E3 (final concentration, 15 – 74 mg/ml), and reduced thioctic acid (0.625 mg/ml). Thioctic acid was used to initiate the reaction. 2.4. Covalent adduct analysis [1,2-14C]-Acetaldehyde (10 mCi/mmol), purchased from American Radiolabeled Chemicals Inc. (St. Louis, MO), was received as a frozen aqueous solution (0.21 mCi/ml) and stored at 4C. Pyruvate dehydrogenase (0.85 mg/ml) was incubated with acetaldehyde (10 mM, 100 mM, and 1 mM) in 0.05 M potassium phosphate buffer (pH 7.4) containing 1 mM MgCl2 and 1 mg/ml of BSA at 37C for 2 h and 1, 2, and 3 days. Some reaction mixtures included 1 mM cysteine, which was added to the solution before the addition of acetaldehyde. Pyruvate dehydrogenase was substituted for commercial preparations of porcine heart 2-oxoglutarate dehydrogenase (2OGDH) (1.94 mg/ml), E3 (2.94 mg/ml), or BSA (1 mg/ml) in some reactions. After incubation, reaction mixtures were incubated for another 10 min with and without NaBH4 (final concentra-
M.L. Hard et al. / Alcohol 25 (2001) 1–8
tion, 5 mM –0.1 M) made in 0.1 mM NaOH (pH 9.5). The sodium dodecyl sulfate –polyacrylamide gel electrophoresis technique (SDS-PAGE) was performed in prepoured slab gels containing 16% (wt./vol.) acrylamide in a Tris-glycine buffer system (pH 8) according to the method of Laemmli (1970). Optimal separation of PDH was obtained by running the gel at constant voltage: 85 V for 1 h, followed by 75 V for 4 h. 14C-labeled bands were detected by autoradiography. Molecular weights were located by using a prestained standard, purchased from Helixx Technologies Inc. (Scarborough, ON, Canada), containing myosin (250 kDa), BSA (98 kDa), glutamic dehydrogenase (64 kDa), alcohol dehydrogenase (50 kDa), carbonic anhydrase (36 kDa), myoglobin (30 kDa), lysozyme (16 kDa), aprotinin (6 kDa), and insulin, B chain (4 kDa). Stable acetaldehyde adducts were also measured directly as protein-bound radioactivity after acid precipitation. The enzyme was precipitated with two volumes of 15% trichloroacetic acid containing 1.5% phosphotungstic acid, according to the method of Donohue et al. (1983b). The pellet was suspended in scintillation fluid, and radioactivity was determined by using a Beckman LS 6500 scintillation counter. Radioactivity measurements (disintegrations per
3
minute per milligram of protein) were converted to molar quantities and expressed as picomoles of acetaldehyde bound per milligram of protein. 2.5. Phosphorylation of pyruvate dehydrogenase complex 32
P-adenosine triphosphate (ATP) (3,000 Ci/mmol) was purchased from Amersham Pharmacia Biotech (Baie d’Urfe´, PQ, Canada). Phosphorylation of PDH was detected by incubating PDH (0.51 mg/ml) with 32P-ATP (4.4 Ci/mmol) from 0 to 30 min at 37C with and without acetaldehyde (1 mM). Other substrates were added as indicated, which included 10 mM dichloroacetate (DCA), 10 mM adenosine diphosphate (ADP), and 250 mM acetyl CoA. The SDSPAGE was performed in prepoured slab gels containing 16% (wt./vol.) acrylamide, as described above, and 32 P-labeled bands were detected by autoradiography.
3. Results Addition of acetaldehyde to a preparation of porcine heart PDH impairs its activity at 37C. As presented in
Fig. 1. Fractional velocity (v) (ratio of the velocity of the enzyme in the presence of inhibitor to the velocity without inhibitor) of porcine heart pyruvate dehydrogenase at 37C. The inhibitor, acetaldehyde, was used at concentrations of 10 mM and 0.1, 0.4, 0.7, and 1.0 mM in the presence of 3, 5, and 7 mM of the substrate, pyruvate. The velocity of the enzyme was determined by measuring the formation of the reduced form of nicotinamide-adenine dinucleotide spectrophotometrically at 340 nm. The Dixon plot (top inset; inverse velocity [1/v] vs. inhibitor concentration) and the Cornish-Bowden plot (bottom inset; ratio of the substrate concentration to the velocity of the enzyme [s/v] vs. inhibitor concentration) are also shown.
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Table 1 Comparing models of inhibition for pyruvate dehydrogenase by acetaldehydea Model
SS
df
MIb CI UCI NCI
0.022458 0.04561 0.05595 0.045587
11 12 12 12
a
Enzyme kinetic data obtained for pyruvate dehydrogenase in the presence of acetaldehyde were fit to four models of inhibition. By comparing models with the same number of parameters, one can determine the best fit by the lowest sum of squares (SS). An F test was used to compare the model of mixed inhibition (MI) to the remaining models due to the inclusion of an extra parameter. This analysis allows us to reject the hypothesis that competitive inhibition (CI), uncompetitive inhibition (UCI), and noncompetitive inhibition (NCI) are true models of inhibition and conclude that the data obtained were best fit to a model of MI. b The equation describing mixed inhibition is v = Vxs/(Km(1 + i/ Kic) + s(1 + i/Kiu)), where V is the maximal velocity; Km, the Michaelis – Menten constant; s, the substrate concentration; i, the inhibitor concentration; Kic, the dissociation constant of the enzyme – substrate complex; and Kiu, the dissociation constant of the enzyme – substrate – inhibitor complex. The estimates for the parameters were V = 2.38 ± 0.15 mmol/mg/min, Km = 1.40 ± 0.39 mM, Kic = 62.4 ± 25.7 mM, and Kiu = 225 ± 68 mM.
Fig. 1, the degree of inhibition increases with increasing acetaldehyde concentration. To determine the nature of inhibition, the results were fit to equations that describe four types of inhibition: noncompetitive (NCI), uncompetitive (UCI), competitive (CI), and mixed (MI) by using Leonora (Cornish-Bowden, 1995), a program written specifically for fitting enzyme kinetic data. Predicted values of the velocity in the presence of inhibitor for each model were obtained, and the sum of squares (SS) was calculated to determine the goodness of fit (Table 1). The SS was found to be lowest for MI. However, because of the extra parameter present in the equation that describes MI, the F ratios
were calculated to determine whether the extra parameter had any more than a chance effect on the fit. The results presented in Table 1 show that there was not a significant difference between fitting the data to CI, UCI, or NCI models. For MI, however, there was a significant difference in how close the fit came to the experimental data in comparison with other models [CI: F(1,11) = 11.39, P < .0 1 ; U C I : F(1,11) = 16.40, P < .01; NCI : F(1,11) = 11.39, P < .01]. Therefore, by fitting the data only to a model of MI we can reject the hypothesis that any remaining models are true models of inhibition. The Dixon (top inset to Fig. 1) and Cornish-Bowden (bottom inset to Fig. 1) plots of our data are also consistent with MI (Cornish-Bowden, 1974). The inhibition constants, Kic and Kiu, obtained for MI were calculated by using Leonora as 62.4 ± 25.7 and 225 ± 68 mM, respectively. The kinetics of PDH was examined in the presence of 1 –100 mM ethanol. No inhibition was observed. Incubation of [1, 2-14C]-acetaldehyde with porcine heart PDH resulted in the formation of covalent adducts. As shown in Fig. 2a, the amount of binding increased between 2 h and 1 day of incubation, with no further increases up to 3 days of incubation (results not shown). An increase of the acetaldehyde concentration from 10 mM to 1 mM resulted in an increase in the degree of formation of acetaldehyde –PDH adducts (Figs. 2b and 3). The most intense band that occurs at approximately 54 kDa is likely to correspond to either E3 or protein X, which typically run at 55 and 52 kDa, respectively. Less intense bands also appear in the range that corresponds to E2, E1a, and E1b, which run at 74, 41, and 36 kDa, respectively. The formation of acetaldehyde – protein adducts has been attributed to the formation of an unstable Shiff base that could be reduced, and thus stabilized, in the presence of a reducing agent such as NaBH4 (Tuma et al., 1991). In this study, enhanced formation of stable acetaldehyde –protein adducts was not seen at low
Fig. 2. Autoradiographs of porcine heart pyruvate dehydrogenase (PDH) separated by using the sodium dodecyl sulfate – polyacrylamide gel electrophoresis technique after incubation at 37C with a) 1 mM [1,2-14C] acetaldehyde for 2 h and 1 day and b) 10 mM – 1 mM [1,2-14C] acetaldehyde for 1 day. Reaction mixtures were mixed with (+) and without ( ) NaBH4 (5 mM). Formation of acetaldehyde – PDH adducts increased in a time-dependent fashion and with increasing acetaldehyde concentration. Addition of 5 mM NaBH4 had no effect on the number of covalent adducts formed. Addition of 1 mM cysteine (C) prevented the formation of PDH – acetaldehyde adducts.
M.L. Hard et al. / Alcohol 25 (2001) 1–8
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Fig. 3. Amount of acetaldehyde bound per milligram of pyruvate dehydrogenase (PDH). Pyruvate dehydrogenase was precipitated after incubation with [1,2-14C] acetaldehyde for 1 day with (+) and without ( ) 5 mM NaBH4. The amount of acetaldehyde bound was determined by measuring radioactivity of the pellet, and it ranged from 3.5 to 25 pmol/mg of PDH for acetaldehyde concentrations ranging from 1 mM to 1 mM.
NaBH4 concentrations (Fig. 2a and b), but it was seen at high NaBH4 concentrations (Fig. 4). Addition of 1 mM cysteine to a 1 mM acetaldehyde reaction mixture at the beginning of the incubation prevented the formation of covalent adducts (Fig. 2b), which was expected because cysteine is known to form stable complexes with acetaldehyde. Because the formation of covalent adducts was time dependent, the residual activity of PDH was measured spectrophotometrically after incubation at various times.
Fig. 4. Autoradiograph of porcine heart pyruvate dehydrogenase (PDH), 2-oxoglutarate dehydrogenase (2OGDH), and dihydrolipoyl dehydrogenase (E3) separated by using the sodium docecyl sulfate – polyacrylamide gel electrophoresis techique after 1 day of incubation at 37C with 1 mM [1,2-14C] acetaldehyde with (+) and without ( ) NaBH4 (0.1 M). Acetaldehyde formed covalent adducts with all subunits common to PDH and 2OGDH. The addition of 0.1 M NaBH4 increased the number of covalent adducts formed.
After acetaldehyde was mixed with PDH, under the same conditions as those used to follow covalent adduct formation, no difference in activity was measured up to 4 h of incubation (results not shown). Incubation of the reaction mixture for 24 h resulted in enzyme degradation, but this was attributed to the presence of proteases in the commercial preparation, rather than to acetaldehyde. Therefore, increasing the degree of adducts formed has no more effect on the activity than does instantaneous exposure of PDH to acetaldehyde. Covalent binding of acetaldehyde to PDH occurs primarily on a subunit just above 50 kDa (Fig. 2a and b), which is in a range consistent with that for the E3 or protein X subunit of PDH. The activity of E3 in the presence of acetaldehyde was examined, but no inhibition was observed. Thus, if acetaldehyde were primarily attaching to E3, the activity of the enzyme would not be affected. The results of the timedependent kinetic studies seem to indicate that acetaldehyde inhibition may arise solely from the interaction of acetaldehyde with a catalytic site on PDH, possibly lipoic acid. Lipoic acid is present on both the E2 subunit (74 kDa) and protein X (55 kDa). 2-Oxoglutarate dehydrogenase is almost identical in structure to PDH, but it lacks protein X. To determine whether the prominent band above 50 kDa was E3 or protein X, the binding of acetaldehyde to 2OGDH and E3 was examined and compared with the binding of acetaldehyde to PDH. As presented in Fig. 4, the band patterns of PDH and 2OGDH after addition of acetaldehyde are identical. Furthermore, in the experiment done with E3 alone it is clear that acetaldehyde is binding with the E3 subunit on both PDH and 2OGDH. These results however, do not allow us to rule out the possibility that acetaldehyde is also binding to protein X.
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Fig. 5. Autoradiographs of porcine heart pyruvate dehydrogenase separated by using the sodium docecyl sulfate – polyacrylamide gel electrophoresis technique after incubation at 37C with 32P-adenosine triphosphate (4.4 Ci/mmol) at 37C a) with (+) and without ( ) 1 mM acetaldehyde from 0 to 30 min and b) for 5 min with (+) and without ( ) 1 mM acetaldehyde (A) with co-addition of substrates that affect kinase activity that include 10 mM dichloroacetate (B), 10 mM adenosine diphosphate (C), and 250 mM acetyl CoA (D). The degree of phosphorylation increases with time. No effect on phosphorylation by acetaldehyde was detected. Acetaldehyde did not inhibit PDH by enhancing the degree of phosphorylation by means of an interaction with the kinase protein.
Findings obtained from incubating PDH with ATP in the presence of acetaldehyde show that acetaldehyde has no effect on the phosphorylation of PDH (Fig. 5a). In addition, in the presence of two kinase inhibitors, DCA and ADP, and a kinase stimulator, acetyl CoA, no effect of acetaldehyde on phosphorylation is detected (Fig. 5b).
4. Discussion In this study, we have examined the effect of acetaldehyde on a commercial preparation of purified porcine heart PDH. Because all mammalian PDHs are kinetically similar and because of the same protein isoenzyme components in every tissue, it is reasonable to assume that the demonstrated effects on heart PDH would be applicable to other tissues and could be extrapolated to the human fetus. The mental retardation resulting from prenatal alcohol exposure is by far the most debilitating manifestation of FAS. Thus, the vulnerability of the brain to the toxic effects of ethanol and acetaldehyde is of primary interest. Ethanol is capable of crossing the human placenta. Because of the presence of CYP 2E1 (Brzezinski et al., 1999) and a currently unidentified cystolic oxidase (Person et al., 2000) in the human fetal brain, it is possible that acetaldehyde may be produced in the target organ of interest. The brain, which relies on glucose as its sole source of fuel, would therefore be quite vulnerable to the potential effects of acetaldehyde on PDH. We fit our kinetic data to equations that describe four types of inhibition: UCI, NCI, CI, and MI. By using Leonora (Cornish-Bowden, 1995), results showed that inhibition of PDH by acetaldehyde occurs by a MI type mechanism (Kic = 62.4 ± 25.7 mM, Kiu = 225 ± 68 mM). The Dixon (top inset to Fig. 1) and Cornish-Bowden (bottom inset to Fig. 1) plots are characteristic of MI (Cornish-Bowden, 1974). Because it still is not known whether it is ethanol or
acetaldehyde that is causing the toxicity resulting in ARBD, we examined the effects of ethanol on PDH. No inhibition was observed. From these observations we can conclude that if inhibition of PDH is contributing to the pathogenesis of ARBD, acetaldehyde, rather than ethanol, is the toxic species. These observations may be significant in the developing fetus because PDH levels are low throughout development and do not reach adult levels until term. Therefore, even a small degree of inhibition may have severe consequences (Robinson et al., 1977). Two previous studies describe the inhibition of PDH by acetaldehyde by both noncompetitive (Ki = 130 ± 30 mM) (Blass & Lewis, 1973) and uncompetitive (Ki = 150 ± 20 mM) (Alkonyi et al., 1978) mechanisms with respect to pyruvate, CoA, TPP, and NAD. The results of these studies were drawn from graphical methods alone (Alkonyi et al., 1978) and did not allow the investigators to reach definite conclusions about the nature of inhibition (Blass & Lewis, 1973). Blass and Lewis (1973) have shown that the oxidation of pyruvate in the absence of CoA or NAD was not inhibited by acetaldehyde, which seems to indicate that inhibition of PDH is not a result of an interaction of acetaldehyde with the E1 subunit. Conversely, Alkonyi et al. (1978) concluded that inhibition was occurring at E1 after examining the effects of acetaldehyde on the activity of E1, E2, and E3. The results of the work presented in the current study are in agreement with those of Alkonyi et al. (1978) because we concluded that inhibition of PDH by acetaldehyde occurs by a combination of CI and UCI. The competitive component implies that acetaldehyde is competing with pyruvate for its catalytic site on E1. The competition of acetaldehyde with pyruvate for PDH may have significance in vivo because it would be further enhanced in the presence of ethanol, which causes a decrease in ambient pyruvate concentrations. We further examined the role of covalent binding and the effect of acetaldehyde on phosphorylation and inactivation of PDH, which has not been previously investigated.
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Acetaldehyde is capable of forming covalent adducts with a variety of proteins that include hepatic proteins (Donohue et al., 1983b; Medina et al., 1985), albumin (Donohue et al., 1983a), hemoglobin (San George & Hoberman, 1986), tublin (Tuma et al., 1987), and certain enzymes (Mauch et al., 1986). The functional consequences of adduct formation have been shown in a number of proteins (Mauch et al., 1986; Tuma & Sorrell, 1987). For example, the activity of the lysine-dependent enzymes glucose-6-phosphate dehydrogenase and RNAse was inhibited in the presence of acetaldehyde, and the degree of inhibition correlated with total adduct formation (Mauch et al., 1986). In our study, we found that acetaldehyde forms covalent adducts with PDH, and the degree of adduct formation follows a concentration and time-dependent increase. The formation of acetaldehyde – protein adducts has been attributed to the formation of a Shiff base intermediate, which can be reduced, and thus stabilized, on addition of NaBH4 (Tuma & Sorrell, 1987; Tuma et al., 1991). To increase the number of stable acetaldehyde – PDH adducts, it was necessary to use concentrations of NaBH4 that were much higher (0.1 M) than previously reported in the literature (5 mM) (Donohue et al., 1983a, 1983b; Mauch et al., 1986; Medina et al., 1985). Addition of cysteine (1 mM), which is capable of complexing acetaldehyde, prevents the formation of PDH – acetaldehyde adducts (Fig. 2b) by effectively removing free acetaldehyde from solution. In our kinetic experiments, we attempted to add cysteine to prevent inhibition, but this was ineffective. In the presence of cysteine, the activity of PDH decreased even further. When the inhibitor was removed from solution there was still a reduction in PDH activity when cysteine was added. Because cysteine is known to be reactive with a-keto acids, we suspect that cysteine may be capable of removing pyruvate from solution, accounting for the observed reduction in activity. Because prolonged incubation times had an effect on the number of covalent adducts formed, we further investigated the effect of incubation time on inhibition kinetics. The activity of PDH incubated with acetaldehyde was measured at a number of time points. Over a period of 4 h there were no changes in the amount of inhibition. Because we observed a time-dependent increase in the number of PDH –acetaldehyde adducts formed, which is not associated with a time-dependent increase in inhibition, we conclude that covalent binding of acetaldehyde to PDH does not contribute to the loss of enzymatic activity. In addition, it may be the non-covalent interaction of acetaldehyde with a catalytic site of PDH that is responsible for the inhibition of PDH. Lipoic acid, located on E2, and protein X are essential for PDH function. The reduced form of lipoate consists of two sulfhydry groups that are capable of nucleophilic attack of the electrophilic carbonyl group in acetaldehyde. This resultant acetylation of lipoic acid would effectively reduce the
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catalytic function of PDH. To determine whether acetaldehyde is interacting with the lipoate, we compared the binding sites of acetaldehyde with those of PDH and 2OGDH. 2-Oxoglutarate dehydrogenase is almost identical in subunit structure to PDH except that it lacks protein X. Protein X has a molecular weight that is similar to that for E3. Thus, to distinguish between the binding of acetaldehyde to protein X and E3, we repeated our labeling experiments with 2OGDH and isolated E3. The PDH and 2OGDH lanes in Fig. 4 are indistinguishable, which is indicative of the formation of acetaldehyde adducts with all subunits common to PDH and 2OGDH. In theory, if acetaldehyde were preferentially binding to protein X, rather than to E3, we would not have seen a band above 50 kDa in 2OGDH. Furthermore, use of isolated E3 seems to indicate that the band above 50 kDa is a result of acetaldehyde –E3 adduct formation, but we cannot rule out the possibility that acetaldehyde is also binding to protein X. We also tested the effect of acetaldehyde on E3 activity and found no differences in activity in the presence of acetaldehyde as was previously demonstrated (Alkonyi et al., 1978). Because we have observed that the majority of binding seems to occur at E3, the activity of E3 is not affected in the presence of acetaldehyde, and the degree of inhibition does not increase with increasing adduct formation, we can conclude that the inhibition of PDH by acetaldehyde is due to a non-covalent phenomenon. Protein kinase is responsible for phosphorylation or inactivation of the complex, where the phospho group is obtained from ATP. When we incubated PDH with 32P-ATP, the intensity of the bands increased over time, indicating an increase in the amount of phosphorylation and thus inactivation of the complex. If acetaldehyde inhibited the complex by an interaction with kinase, it would be expected that acetaldehyde would have enhanced the degree of phosphorylation, which would have resulted in an increase in the intensity of the bands shown in Fig. 5a where acetaldehyde was present. However, this was not observed. Our final conclusion is that acetaldehyde will give MI of PDH in the physiological range, which may be made worse by ethanol-induced lowering of pyruvate concentrations. The inhibition is not influenced by covalent adduct formation between E3 and acetaldehyde, is not a result of an effect on phosphorylation of the complex, and is quite reversible. Our results support the hypothesis that inhibition of PDH by acetaldehyde has implications in the developing fetus.
Acknowledgments This work was supported by a grant from the Canadian Institute for Health Research. Gideon Koren is a Senior Scientist of the Canadian Institute of Health Research. Brian Robinson is a holder of a Millenium Canada Research Chair. Marjie Hard is a recipient of a graduate studentship from The Research Institute, The Hospital for Sick Children.
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