Human placental aldehyde dehydrogenase. Subcellular distribution and properties

Freshly obtained human term placentae were subjected to subcellular fractionation to study the localization of NAD-dependent aldehyde dehydrogenases. Optimal conditions for the cross-contamination-free subcellular fractionation were standardized as judged by the presence or the absence of appropriate marker enzymes. Two distinct isozymes, aldehyde dehydrogenase I and II, were detected in placental extracts after isoelectric focusing on polyacrylamide gels. Based on a placental wet weight, about 80% of the total aldehyde dehydrogenase activity was found in the cytosolic acid and about 10% in the mitochondrial fraction. The soluble fraction (cytosol) contained predominantly aldehyde dehydrogenase II which has a relatively high Km (9 mmol/l) for acetaldehyde and is strongly inhibited by disulfiram. The results indicate that cytosol is the main site for acetaldehyde oxidation, but the enzyme activity is too slow to prevent the placental passage of normal concentrations of blood acetaldehyde (less than 1 mumol/l) produced by maternal ethanol metabolism.     

An enzymatic model for rat liver alcohol dehydrogenase in the presence of low-Km aldehyde dehydrogenase

Four isoenzymes of aldehyde dehydrogenase were partially purified from rat liver mitochondria by hydroxylapatite chromatography and gel filtration. While three forms display low affinity for acetaldehyde, the fourth is active at extremely low aldehyde concentrations (Km less than or equal to 2 microM) and allows the oxidation of the acetaldehyde formed by catalysis of alcohol dehydrogenase at pH 7.4. Different models of alcohol dehydrogenase have been examined by analysis of progress curves of ethanol oxidation obtained in the presence of low-km aldehyde dehydrogenase. According to the only acceptable model, when the acetaldehyde concentration is kept low by the action of aldehyde dehydrogenase, NADH no longer binds to alcohol dehydrogenase, but acetaldehyde still competes with ethanol for the active site of the enzyme. The seven kinetic parameters of the two enzymes (four for alcohol dehydrogenase and three for aldehyde dehydrogenase) and the equilibrium constant of the reaction catalyzed by alcohol dehydrogenase have been determined by applying a new fitting procedure here described.     

The action of cytoplasmic aldehyde dehydrogenase on methyl p-nitrophenyl carbonate and p-nitrophenyl dimethylcarbamate.

Cytoplasmic aldehyde dehydrogenase catalyses the hydrolysis of methyl p-nitrophenyl (PNP) carbonate at an appreciable rate that is markedly stimualted by NAD+ or NADH. The nuleotides accelerate the rate-limiting hydrolysis of the acyl-enzyme intermediate while slowing the observed burst of p-nitrophenoxide production. With PNP dimethylcarbamate the enzyme catalyses the slow release of approx. 1 molecule of p-nitrophenoxide per tetrameric enzyme molecule; during the reaction the enzyme becomes effectively inactivated, as the rate of hydrolysis of the acyl-enzyme is virtually zero. The presence of NAD+, NADH, propionaldehyde, chloral hydrate, diethylstilboestrol or disulfiram slows the reaction of enzyme with PNP dimethylcarbamate. The reaction appears to be dependent on a group of pKa 7.6, possibly a cysteine residue. The effect of PNP dimethylcarbamate on the dehydrogenase activity of the enzyme is consistent with there being a single type of active site for the enzyme's dehydrogenase and esterase activities. Steric and electronic factors that govern reaction of the enzyme with PNP substrates are discussed.     

Interaction of sheep liver cytosolic aldehyde dehydrogenase with quercetin, resveratrol and diethylstilbestrol

The effects of quercetin and resveratrol (substances found in red wine) on the activity of cytosolic aldehyde dehydrogenase in vitro are compared with those of the synthetic hormone diethylstilbestrol. It is proposed that quercetin inhibits the enzyme by binding competitively in both the aldehyde substrate binding-pocket and the NAD(+)-binding site, whereas resveratrol and diethylstilbestrol can only bind in the aldehyde site. When inhibition is overcome by high aldehyde and NAD(+) concentrations (1 mM of each), the modifiers enhance the activity of the enzyme; we hypothesise that this occurs through binding to the enzyme-NADH complex and consequent acceleration of the rate of dissociation of NADH. The proposed ability of quercetin to bind in both enzyme sites is supported by gel filtration experiments with and without NAD(+), by studies of the esterase activity of the enzyme, and by modelling the quercetin molecule into the known three-dimensional structure of the enzyme. The possibility that interaction between aldehyde dehydrogenase and quercetin may be of physiological significance is discussed.     

Active site of human liver aldehyde dehydrogenase

Bromoacetophenone (2-bromo-1-phenylethanone) functions as an affinity reagent for human aldehyde dehydrogenase (EC 1.2.1.3) and has been found specifically to label a unique tryptic peptide in the enzyme. Amino-terminal sequence analysis of the labeled peptide after purification by two different procedures revealed the following sequence: Val-Thr-Leu-Glu-Leu-Gly-Gly-Lys. Radioactivity was found to be associated with the glutamate residue, which was identified as Glu-268 by reference to the known amino acid sequence. This paper constitutes the first identification of an active site of aldehyde dehydrogenase.     

Differences in nucleotide specificity and catalytic mechanism between Vibrio harveyi aldehyde dehydrogenase and other members of the aldehyde dehydrogenase superfamily

The fatty aldehyde dehydrogenase (Vh-ALDH) isolated from the luminescent bacterium, Vibrio harveyi, differs from other aldehyde dehydrogenases in its high affinity for NADP(+). The binding of NADP(+) appears to arise from the interaction of the 2'-phosphate of the adenosine moiety of NADP(+) with a threonine (T175) in the nucleotide recognition site just after the beta(B) strand as well as with an arginine (R210) that pi stacks over the adenosine moiety. The active site of Vh-ALDH contains the usual suspects of a cysteine (C289), two glutamates (E253 and E377) and an asparagine (N147) involved in the aldehyde dehydrogenase mechanism. However, Vh-ALDH has one polar residue in the active site that distinguishes it from other ALDHs; a histidine (H450) is in close contact with the cysteine nucleophile. As a glutamate has been implicated in promoting the nucleophilicity of the active site cysteine residue in ALDHs, the close contact of a histidine with the cysteine nucleophile in Vh-ALDH raises the possibility of alternate routes to increase the reactivity of the cysteine nucleophile. The effects of mutation of these residues on the different functions catalyzed by Vh-ALDH including acylation, (thio)esterase, reductase and dehydrogenase activities should help define the specific roles of the residues in the active site of ALDHs.     

Characterization of aldehyde dehydrogenase from HTC rat hepatoma cells

We have proposed developing rat hepatoma cell lines as an in vitro model for studying the regulation of changes in aldehyde dehydrogenase activity occurring duringhepatocarcinogenesis. Aldehyde dehydrogenase purified in a single step from HTC rat hepatoma cells is identical to the aldehyde dehydrogenase isolated from rat hepatocellular carcinomas. HTC aldehyde dehydrogenase is a 110 kDa dimer composed of 54-kDa subunits, prefers NADP⁺ as coenzyme, and preferentially oxidizes benzaldehyde-like aromatic aldehydes but not phenylacetaldehyde. The substrate and coenzyme specificity, effects of disulfiram, pH profile and isoelectric point of HTC aldehyde dehydrogenase are also identical to these same properties of the tumor aldehyde dehydrogenase. In immunodiffusions, both isozymes are recognized with complete identity by anti-HTC aldehyde dehydrogenase antibodies. Having established that HTC aldehyde dehydrogenase is very similar, if not identical, to the aldehyde dehydrogenase found in hepatocellular carcinomas, simplifies the development of molecular probes for examination of the regulation of tumor aldehyde dehydrogenase activity in vivo and in vitro.     

Characterization of aldehyde dehydrogenase from HTC rat hepatoma cells

We have proposed developing rat hepatoma cell lines as an in vitro model for studying the regulation of changes in aldehyde dehydrogenase activity occurring during hepatocarcinogenesis. Aldehyde dehydrogenase purified in a single step from HTC rat hepatoma cells is identical to the aldehyde dehydrogenase isolated from rat hepatocellular carcinomas. HTC aldehyde dehydrogenase is a 100 kDa dimer composed of 54-kDa subunits, prefers NADP+ as coenzyme, and preferentially oxidizes benzaldehyde-like aromatic aldehydes but not phenylacetaldehyde. The substrate and coenzyme specificity, effects of disulfiram, pH profile and isoelectric point of HTC aldehyde dehydrogenase are also identical to these same properties of the tumor aldehyde dehydrogenase. In immunodiffusion, both isozymes are recognized with complete identity by anti-HTC aldehyde dehydrogenase antibodies. Having established that HTC aldehyde dehydrogenase is very similar, if not identical, to the aldehyde dehydrogenase found in hepatocellular carcinomas, simplifies the development of molecular probes for examination of the regulation of tumor aldehyde dehydrogenase activity in vivo and in vitro.     

The removal of cytosolic-type aldehyde dehydrogenase from preparations of sheep liver mitochondrial aldehyde dehydrogenase and the unusual properties of the purified mitochondrial enzyme in assays.

The pI approximately 5.2 isoenzymes of mitochondrial aldehyde dehydrogenase were separated from the other isoenzymes by pH-gradient chromatography on DEAE-Sephacel. The pI approximately 5.2 material is immunologically identical with cytosolic aldehyde dehydrogenase. It also shows sensitivity to 20 microM-disulfiram and insensitivity to 4M-urea in assays. These and other criteria seem to establish that the material is identical with the cytosolic enzyme. Mitochondrial enzyme that had been purified to remove pI approximately 5.2 isoenzymes shows concentration-dependent lag phases in assays. These effects are possibly due to the slow establishment of equilibrium between tetramer and either dimers or monomers, with the dissociated species being intrinsically more active than the tetramer.     

Role of reduced lipoic acid in the redox regulation of mitochondrial aldehyde dehydrogenase (ALDH-2) activity. Implications for mitochondrial oxidative stress and nitrate tolerance

Chronic therapy with nitroglycerin results in a rapid development of nitrate tolerance, which is associated with an increased production of reactive oxygen species. We have recently shown that mitochondria are an important source of nitroglycerin-induced oxidants and that the nitroglycerin-bioactivating mitochondrial aldehyde dehydrogenase is oxidatively inactivated in the setting of tolerance. Here we investigated the effect of various oxidants on aldehyde dehydrogenase activity and its restoration by dihydrolipoic acid. In vivo tolerance in Wistar rats was induced by infusion of nitroglycerin (6.6 microg/kg/min, 4 days). Vascular reactivity was measured by isometric tension studies of isolated aortic rings in response to nitroglycerin. Chronic nitroglycerin infusion lead to impaired vascular responses to nitroglycerin and decreased dehydrogenase activity, which was corrected by dihydrolipoic acid co-incubation. Superoxide, peroxynitrite, and nitroglycerin itself were highly efficient in inhibiting mitochondrial and yeast aldehyde dehydrogenase activity, which was restored by dithiol compounds such as dihydrolipoic acid and dithiothreitol. Hydrogen peroxide and nitric oxide were rather insensitive inhibitors. Our observations indicate that mitochondrial oxidative stress (especially superoxide and peroxynitrite) in response to organic nitrate treatment may inactivate aldehyde dehydrogenase thereby leading to nitrate tolerance. Glutathionylation obviously amplifies oxidative inactivation of the enzyme providing another regulatory pathway. Furthermore, the present data demonstrate that the mitochondrial dithiol compound dihydrolipoic acid restores mitochondrial aldehyde dehydrogenase activity via reduction of a disulfide at the active site and thereby improves nitrate tolerance.     

Effects of tungstate on the growth of Desulfovibrio gigas NCIMB 9332 and other sulfate-reducing bacteria with ethanol as a substrate

Growth of Desulfovibrio gigas NCIMB 9332 in mineral, vitamin-supplemented media with ethanol as substrate was strongly stimulated by the addition of tungstate (optimal level approximately 10^-7 M). At suboptimal tungstate concentrations, up to 1.0 mM acetaldehyde was detected in the culture supernatant and growth was slow. Omission of both tungstate and molybdate from the media prevented growth and ethanol utilization. Tungstate-deprived cultures that were grown on lactate had much lower aldehyde dehydrogenase (benzylviologen as acceptor; BV-AIDH) levels than tungstate-supplemented cultures. These data suggest that tungstate is required for the synthesis of active BV-AIDH. The characteristics of the enzyme activities in cell-free extracts show that the BV-AIDH activity present in tungstate-supplemented cultures is not due to the recently characterized molybdenum-containing aldehyde dehydrogenase of D. gigas. Out of 13 other strains of ethanol-oxidizing, gram-negative, sulfate-reducing bacteria tested, most strains grew well with either tungstate or molybdate supplementation. In contrast to a recent report, good growth on ethanol of two D. baculatus (Desulfomicrobium) strains (DSM 1741 and DSM 1743) was observed.Abbreviations BV-AIDH Benzylviologen-linked aldehyde dehydrogenase - DCPIP-AIDH 2,6-dichlorophenolindophenol-linked aldehyde dehydrogenase - DTT dithiothreitol     

Chemical modification of aldehyde dehydrogenase by a vinyl ketone analogue of an insect pheromone.

A major component of the sex pheromone from the tobacco budworm moth Heliothis virescens is a C16 straight-chain aldehyde with a single unsaturation at the eleventh position. The sex pheromones are inactivated when metabolized to their corresponding acids by insect aldehyde dehydrogenase. During this investigation it was demonstrated that the C16 aldehyde is a good substrate for human aldehyde dehydrogenase (EC 1.2.1.3) isoenzymes E1 and E2 with Km and Kcat. values at pH 7.0 of 2 microM and 0.4 mumol of NADH/min per mg and of 0.6 microM and 0.24 mumol of NADH/min per mg respectively. A vinyl ketone analogue of the pheromone inhibited insect pheromone metabolism; it also inactivated human aldehyde dehydrogenase. Total inactivation of both isoenzymes was achieved at stoichiometric (equal or less than the subunit number) concentrations of vinyl ketone, incorporating 2.1-2.6 molecules/molecule of enzyme. Substrate protection was observed in the presence of the parent aldehyde and 5'-AMP. Peptide maps of tryptic digests of the E2 isoenzyme modified with 3H-labelled vinyl ketone showed that incorporation occurred into a single peptide peak. The labelled peptide of E2 isoenzyme was further purified on h.p.l.c. and sequenced. The label was incorporated into cysteine-302 in the primary structure of E2 isoenzyme, thus indicating that cysteine-302 is located in the aldehyde substrate area of the active site of aldehyde dehydrogenase. Affinity labelling of aldehyde dehydrogenase with vinyl ketones may prove to be of general utility in biochemical studies of these enzymes.     

Interaction of sheep liver cytosolic aldehyde dehydrogenase with quercetin,resveratrol and diethylstilbestrol

The effects of quercetin and resveratrol (substances found in red wine) on the activity of cytosolic aldehyde dehydrogenase in vitro are compared with those of the synthetic hormone diethylstilbestrol. It is proposed that quercetin inhibits the enzyme by binding competitively in both the aldehyde substrate binding-pocket and the NAD⁺-binding site, whereas resveratrol and diethylstilbestrol can only bind in the aldehyde site. When inhibition is overcome by high aldehyde and NAD⁺ concentrations (1 mM of each), the modifiers enhance the activity of the enzyme; we hypothesise that this occurs through binding to the enzyme-NADH complex and consequent acceleration of the rate of dissociation of NADH. The proposed ability of quercetin to bind in both enzyme sites is supported by gel filtration experiments with and without NAD⁺, by studies of the esterase activity of the enzyme, and by modelling the quercetin molecule into the known three-dimensional structure of the enzyme. The possibility that interaction between aldehyde dehydrogenase and quercetin may be of physiological significance is discussed.     

Differences in nucleotide specificity and catalytic mechanism between Vibrio harveyi aldehyde dehydrogenase and other members of the aldehyde dehydrogenase superfamily

The fatty aldehyde dehydrogenase (Vh-ALDH) isolated from the luminescent bacterium, Vibrio harveyi, differs from other aldehyde dehydrogenases in its high affinity for NADP⁺. The binding of NADP⁺ appears to arise from the interaction of the 2′-phosphate of the adenosine moiety of NADP⁺ with a threonine (T175) in the nucleotide recognition site just after the β_B strand as well as with an arginine (R210) that pi stacks over the adenosine moiety. The active site of Vh-ALDH contains the usual suspects of a cysteine (C289), two glutamates (E253 and E377) and an asparagine (N147) involved in the aldehyde dehydrogenase mechanism. However, Vh-ALDH has one polar residue in the active site that distinguishes it from other ALDHs; a histidine (H450) is in close contact with the cysteine nucleophile. As a glutamate has been implicated in promoting the nucleophilicity of the active site cysteine residue in ALDHs, the close contact of a histidine with the cysteine nucleophile in Vh-ALDH raises the possibility of alternate routes to increase the reactivity of the cysteine nucleophile. The effects of mutation of these residues on the different functions catalyzed by Vh-ALDH including acylation, (thio)esterase, reductase and dehydrogenase activities should help define the specific roles of the residues in the active site of ALDHs.     

Evidence for mitochondrial localization of betaine aldehyde dehydrogenase in rat liver: purification, characterization, and comparison with human cytoplasmic E3 isozyme.

Betaine aldehyde dehydrogenase has been purified to homogeneity from rat liver mitochondria. The properties of betaine aldehyde dehydrogenase were similar to those of human cytoplasmic E3 isozyme in substrate specificity and kinetic constants for substrates. The primary structure of four tryptic peptides was also similar; only two substitutions, at most, per peptide were observed. Thus, betaine aldehyde dehydrogenase is not a specific enzyme, as formerly believed; activity with betaine aldehyde is a property of aldehyde dehydrogenase (EC 1.2.1.3), which has broad substrate specificity. Up to the present time the enzyme was thought to be cytoplasmic in mammals. This report establishes, for the first time, mitochondrial subcellular localization for aldehyde dehydrogenase, which dehydrogenates betaine aldehyde, and its colocalization with choline dehydrogenase. Betaine aldehyde dehydrogenation is an important function in the metabolism of choline to betaine, a major osmolyte. Betaine is also important in mammalian organisms as a major methyl group donor and nitrogen source. This is the first purification and characterization of mitochondrial betaine aldehyde dehydrogenase from any mammalian species.     

Interaction of human aldehyde dehydrogenase with aromatic substrates and ligands

The substrate benzaldehyde (but not propionaldehyde) could elute aldehyde dehydrogenase from a p-hydroxyacetophenone-affinity column, and inhibit the esterase activity (K(i)=47 microM), indicating that this simple aromatic aldehyde binds to the free enzyme and possibly in the substrate-binding site. Thus, the kinetic mechanism for aldehyde dehydrogenase might be dependent upon which aldehyde is used in the reaction. Chloramphenicol which also elutes the enzyme from the affinity column, shows a discriminatory effect by inhibiting the ALDH1 oxidation of benzaldehyde and activating that of propionaldehyde while showing no effect when assayed with hexanal or cyclohexane-carboxaldehyde. Chloramphenicol is an uncompetitive inhibitor against NAD when benzaldehyde is the substrate. We propose that this drug might interact with both the benzaldehyde and NAD binding sites.     

Haloenol lactones as inactivators and substrates of aldehyde dehydrogenase

Human aldehyde dehydrogenase (EC 1.2.1.3) isozymes E1 and E2 were irreversibly inactivated by stoichiometric concentrations of the haloenol lactones 3-isopropyl-6(E)-bromomethylene tetrahydro-pyran-2-one and 3-phenyl-6(E)-bromomethylene tetrahydro-pyran-2-one. No inactivation occurred with the corresponding nonhalogenated enol lactones. Both the dehydrogenase and esterase activities were abolished. Activity was not regained on dialysis or treatment with 2-mercaptoethanol. The inactivation was subject to substrate protection: NAD afforded protection which increased in the presence of the aldehyde-substrate competitive inhibitor chloral. Saturation kinetics gave positivey-axis intercepts, allowing the determination of binding constants. Inactivation stiochiometry determined with¹⁴C-labeled 3-(1-naphthyl)-6(E)-iodomethylene tetrahydropyran-2-one was found to correspond to the active-site number. The nonhalogenated lactone, 3-(1-naphthyl)-6(E)-methylene tetrahydropyran-1-one was shown to be a substrate for aldehyde dehydrogenase via its esterase function. Inactivation and enzymatic hydrolysis occurred within a similar time frame. Opening of the lactone ring to form enzyme-acyl intermediate with active site cysteine appears to be a necessary prerequisite to inactivation, since halogen in the lactone ring is nonreactive. Thus, the inactivation of aldehyde dehydrogenase by haloenol lactones is mechanism-based. Inactivation by haloenol lactones occurs in a manner analogous to that of chymotrypsin with which aldehyde dehydrogenase shares esterase activity and binding of haloenol lactones at the active site.     

Drosophila alcohol dehydrogenase: acetate-enzyme interactions and novel insights into the effects of electrostatics on catalysis

Drosophila alcohol dehydrogenase (DADH) is an NAD+-dependent enzyme that catalyzes the oxidation of alcohols to aldehydes/ketones and that is also able to further oxidize aldehydes to their corresponding carboxylic acids. The structure of the ternary enzyme-NADH-acetate complex of the slow alleloform of Drosophila melanogaster ADH (DmADH-S) was solved at 1.6 A resolution by X-ray crystallography. The coenzyme stereochemistry of the aldehyde dismutation reaction showed that the obtained enzyme-NADH-acetate complex reflects a productive ternary complex although no enzymatic reaction occurs. The stereochemistry of the acetate binding in the bifurcated substrate-binding site, along with previous stereochemical studies of aldehyde reduction and alcohol oxidation shows that the methyl group of the aldehyde in the reduction reaction binds to the R1 and in the oxidation reaction to the R2 sub-site. NMR studies along with previous kinetic studies show that the formed acetaldehyde intermediate in the oxidation of ethanol to acetate leaves the substrate site prior to the reduced coenzyme, and then binds to the newly formed enzyme-NAD+ complex. Here, we compare the three-dimensional structure of D.melanogaster ADH-S and a previous theoretically built model, evaluate the differences with the crystal structures of five Drosophila lebanonensis ADHs in numerous complexed forms that explain the substrate specificity as well as subtle kinetic differences between these two enzymes based on their crystal structures. We also re-examine the electrostatic influence of charged residues on the surface of the protein on the catalytic efficiency of the enzyme.     

Chemical studies of high-Km aldehyde dehydrogenase from rat liver mitochondria

Studies of pH-dependent kinetics implicate two ionizable groups in the dehydrogenase and esterase reactions catalysed by high-Km aldehyde dehydrogenase from rat liver mitochondria. Sensitized photooxidation completely arrests the bifunctional activities of the dehydrogenase. Carboxamidomethylation abolishes the dehydrogenase activity, whereas acetimidination eliminates the esterase activity. These results suggest that histidine (pKa near 6) and cysteine (pKa near 10) are likely the catalytic residues for the dehydrogenase activity, while the esterase activity is functionally related to histidine (pKa near 7) and a residue with the pKa value of 10-11. The two residues, a carboxyl group and an arginine, that discriminate between NAD+ and NADP+ are present at the coenzyme binding site of the mitochondrial high-Km aldehyde dehydrogenase from rat liver.     

Effect of acetaldehyde and cyanamide on the metabolism of formaldehyde by hepatocytes, mitochondria, and soluble supernatant from rat liver

Formaldehyde can be metabolized primarily by two different pathways, one involving oxidation by the low-Km mitochondrial aldehyde dehydrogenase, the other involving a specific, glutathione-dependent, formaldehyde dehydrogenase. To estimate the roles played by each enzyme in formaldehyde metabolism by rat hepatocytes, experiments with acetaldehyde and cyanamide, a potent inhibitor of the low-Km aldehyde dehydrogenase were carried out. The glutathione-dependent oxidation of formaldehyde by 100,000g rat liver supernatant fractions was not affected by either acetaldehyde or by cyanamide. By contrast, the uptake of formaldehyde by intact mitochondria was inhibited 75 to 90% by cyanamide. Acetaldehyde inhibited the uptake of formaldehyde by mitochondria in a competitive fashion. Formaldehyde was a weak inhibitor of the oxidation of acetaldehyde by mitochondria, suggesting that, relative to formaldehyde, acetaldehyde was a preferred substrate. In isolated hepatocytes, cyanamide, which inhibited the oxidation of acetaldehyde by 75 to 90%, produced only 30 to 50% inhibition of formaldehyde uptake by cells as well as of the production of 14CO2 and of formate from [14C]formaldehyde. The extent of inhibition by cyanamide was the same as that produced by acetaldehyde (30-40%). In the presence of cyanamide, acetaldehyde was no longer inhibitory, suggesting that acetaldehyde and cyanamide may act at the same site(s) and inhibit the same formaldehyde-oxidizing enzyme system. These results suggest that, in rat hepatocytes, formaldehyde is oxidized by cyanamide- and acetaldehyde-sensitive (low-Km aldehyde dehydrogenase) and insensitive (formaldehyde dehydrogenase) reactions, and that both enzymes appear to contribute about equally toward the overall metabolism of formaldehyde.