Acetaldehyde-derived modifications on cytosolic human carbonic anhydrases

Acetaldehyde can generate modifications in several proteins, such as carbonic anhydrase (CA) II. In this study, we extended in vitro investigations on acetaldehyde adduct formation by focusing on the other human cytosolic CA enzymes I, III, VII, and XIII. High-resolution mass spectrometric analysis indicated that acetaldehyde most efficiently formed covalent adducts with CA II and XIII. The binding of up to 19 acetaldehydes in CA II is probably attributable to the high number of lysine residues (n?=?24) located mainly on the surface of the enzyme molecule. CA XIII formed more adducts (up to 25) than it contains lysine residues (n?=?16) in its primary structure. Acetaldehyde treatment induced only minor changes in CA catalytic activity in most cases. The present study provides the first evidence that acetaldehyde can bind to several cytosolic CA isozymes. The functional consequences of such modifications will be further investigated in vivo by using animal models.     

Reaction of acetaldehyde with hemoglobin

Acetaldehyde reacted with hemoglobin at neutral pH and 37 degrees C to form adducts that were stable to dialysis and that were not reduced by sodium borohydride. Hemoglobin tetramers having 2, 3, and probably 4 molar eq of bound aldehyde were isolated by cation exchange chromatography. The sites of attachment of the aldehyde were the free amino groups of the N-terminal valine residues of the alpha and beta chains of hemoglobin. Derivatization of the beta chains caused a greater increase in the acidity of the hemoglobin than did derivatization of the alpha chains. Derivatization of the beta chains was also preferred over that of the alpha chains. Acetaldehyde derivatives of the N-terminal octapeptide of hemoglobin S (beta sT-1 peptide), Val-Gly-Gly, and tetraglycine were formed readily, contained 1 M eq of acetaldehyde/mol of peptide, and were not reduced by sodium borohydride. In contrast, Ala-Pro-Gly failed to form a 1:1 adduct with acetaldehyde. 13C NMR analysis of the peptide adducts formed with [1,2-13C]acetaldehyde indicated that tetrahedral diastereomeric derivatives were produced. The 13C chemical shifts of the adducts formed between hemoglobin and [1,2-13C]acetaldehyde were identical to those of the peptide adducts although resonances from the individual diastereomeric adducts at each hemoglobin site could not be resolved. The results cited above as well as other evidence indicate that acetaldehyde reacts with the amino termini of hemoglobin to form stable cyclic imidazolidinone derivatives. An exchange of acetaldehyde residues between peptides was also documented.     

Acetaldehyde adducts with proteins: Binding of [14C]acetaldehyde to serum albumin

Acetaldehyde, the immediate oxidation product of ethanol metabolism, was assessed for its ability to bind covalently to a purified protein in solution. Bovine serum albumin (BSA)² was used as the model protein incubated in the presence of 0.2 mm [¹⁴C]acetaldehyde at pH 7.4 and at 37 °C. Acetaldehyde formed both stable and unstable adducts with serum albumin. Unstable adducts were identified following stabilization with the reducing agent sodium borohydride. We examined both types of binding using trichloroacetic acid precipitation, gel filtration, and dialysis as means to separate bound from free acetaldehyde. All three methods of analysis gave comparable results except that the number of stable acetaldehyde adducts with albumin were significantly lower following separation by dialysis. The effects of l-cysteine, l-lysine, and reduced glutathione were assessed for their abilities as competitive reagents to decrease binding of [¹⁴C]acetaldehyde to BSA. Addition of cysteine caused a rather dramatic concentration-dependent reduction in [¹⁴C]acetaldehyde binding to BSA when compared to that caused by lysine which displayed a relatively mild effect on covalent binding. The presence of glutathione caused a concentration-dependent decrease in protein-bound radioactivity that was stronger than that by lysine but not as effective as cysteine. The ability of each reagent to reverse the formation of preformed acetaldehyde adducts with BSA was also examined. Only l-cysteine effectively decreased the number of unstable acetaldehyde adducts with BSA while stable binding of acetaldehyde remained essentially unaffected by any of the three reagents. These results indicate that acetaldehyde can covalently bind to protein and form unstable as well as stable adducts.     

Conformational variability of different sulfonamide inhibitors with thienyl-acetamido moieties attributes to differential binding in the active site of cytosolic human carbonic anhydrase isoforms

The X-ray crystal structures of the adducts of human carbonic anhydrase (hCA, EC 4.2.1.1) II complexed with two aromatic sulfonamides incorporating 2-thienylacetamido moieties are reported here. Although, the two inhibitors only differ by the presence of an additional 3-fluoro substituent on the 4-amino-benzenesulfonamide scaffold, their inhibition profiles against the cytosolic isoforms hCA I, II, III, VII and XIII are quite different. These differences were rationalized based on the obtained X-ray crystal structures, and their comparison with other sulfonamide CA inhibitors with clinical applications, such as acetazolamide, methazolamide and dichlorophenamide. The conformations of the 2-thienylacetamido tails in the hCA II adducts of the two sulfonamides were highly different, although the benzenesulfonamide parts were superimposable. Specific interactions between structurally different inhibitors and amino acid residues present only in some considered isoforms have thus been evidenced. These findings can explain the high affinity of the 2-thienylacetamido benzenesulfonamides for some pharmacologically relevant CAs (i.e., isoforms II and VII) being also useful to design high affinity, more selective sulfonamide inhibitors of various CAs.     

Covalent binding of acetaldehyde to tubulin: evidence for preferential binding to the alpha-chain

The covalent binding of [14C]acetaldehyde to purified beef brain tubulin was characterized. As we have found for several other proteins, tubulin bound acetaldehyde to form both stable and unstable adducts. Unstable adducts (Schiff bases) were stabilized, and rendered detectable, by treating incubated reaction mixtures with the reducing agent sodium borohydride. In short-term incubations, the majority of the adducts formed were unstable, but the percentage of total adducts that were stable gradually increased with time. Stable adduct formation was greatly increased by the inclusion of sodium cyanoborohydride in reaction mixtures (reductive ethylation). When reaction mixtures were submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis to separate the alpha- and beta-chains of the heterodimeric tubulin molecule, the alpha-chain of free tubulin, but not intact microtubules, was the preferential site of stable adduct formation under both reductive and nonreductive conditions. Denaturation studies showed that the native tubulin conformation was necessary for the alpha-chain to show enhanced reactivity toward acetaldehyde. Competition binding studies showed that alpha-tubulin could effectively compete with beta-tubulin and bovine serum albumin for a limited amount of acetaldehyde. Unstable acetaldehyde adducts with free tubulin or microtubules did not exhibit alpha-chain selectivity. Analysis of reaction mixtures indicates that lysine residues are the major group of the protein participating in adduct formation. These data indicate that the alpha-chain of free tubulin is the preferential site of stable acetaldehyde-tubulin adduct formation. Further, these data raise the possibility that alpha-tubulin may be a selective target for acetaldehyde adduct formation in cellular systems.     

Cytosolic lysine residues enhance anterograde transport and activation of the erythropoietin receptor

Lysine residues are key residues in many cellular processes, in part due to their ability to accept a wide variety of post-translational modifications. In the present study, we identify the EPO-R [EPO (erythropoietin) receptor] cytosolic lysine residues as enhancers of receptor function. EPO-R drives survival, proliferation and differentiation of erythroid progenitor cells via binding of its ligand EPO. We mutated the five EPO-R cytosolic lysine residues to arginine residues (5KR EPO-R), eliminating putative lysine-dependent modifications. Overexpressed 5KR EPO-R displayed impaired ubiquitination and improved stability compared with wt (wild-type) EPO-R. Unexpectedly, fusion proteins consisting of VSVGtsO45 (vesicular stomatitis virus glycoprotein temperature-sensitive folding mutant) with wt or 5KR EPO-R cytosolic domains demonstrated delayed glycan maturation kinetics upon substitution of the lysine residues. Moreover, VSVG-wt EPO-R, but not VSVG-5KR EPO-R, displayed endoplasmic reticulum-associated ubiquitination. Despite similar cell-surface EPO-binding levels of both receptors and the lack of EPO-induced ubiquitination by 5KR EPO-R, the lysine-less mutant produced weaker receptor activation and signalling than the wt receptor. We thus propose that EPO-R cytosolic lysine residues enhance receptor function, most probably through ubiquitination and/or other post-translational modifications.     

Acetaldehyde binding increases the catabolism of rat serum low-density lipoproteins

Acetaldehyde was found to form adducts with rat serum lipoproteins. The binding of [14C]acetaldehyde to lipoproteins was studied at low concentrations which are known to exist during ethanol oxidation. The amount of lipoprotein adducts was a linear function of acetaldehyde concentration up to 250 microM. Incubation of rat plasma low-density lipoproteins (LDL) with 200 microM acetaldehyde increased the disappearance rate of the 3H-label from the cholesterol ester moiety of LDL injected into normal rats. The data show that even low concentrations of acetaldehyde are capable of affecting LDL metabolism. These findings may provide an explanation for the low concentrations of serum LDL in alcoholics.     

Carbonic anhydrase inhibitors. Inhibition of the newly isolated murine isozyme XIII with anions

The inhibition of the newly discovered cytosolic carbonic anhydrase (CA, EC 4.2.1.1) isozyme XIII of murine origin (mCA XIII) has been investigated with a series of anions, such as the physiological ones (bicarbonate, chloride), or the metal complexing anions (cyanate, cyanide, azide, hydrogen sulfide, etc), nitrate, nitrite, sulfate, sulfamate, sulfamide as well as with phenylboronic and phenylarsonic acids. The best mCA XIII inhibitors were cyanate, thiocyanate, cyanide and sulfamide, with K(I)-s in the range of 0.25microM-0.74 mM, whereas fluoride, iodide, azide, carbonate and hydrogen sulfide were less effective (K(I)-s in the range of 3.0-5.5mM). The least effective inhibitors were sulfate, chloride and bicarbonate (K(I)-s in the range of 138-267 mM). The affinity of mCA XIII for anions is very different from that of the other cytosolic isozymes (hCA I and II) or the mitochondrial isozyme hCA V. This resistance to inhibition by the physiological anions bicarbonate and chloride suggests an evolutionary adaptation of CA XIII to the presence of high concentrations of such anions (e.g., in the reproductive tract of both female and male), and the possible participation of this isozyme (similarly to CA II, CA IV and CA V) in metabolons with proteins involved in the anion exchange and transport, such as the anion exchangers (AE1-3) or the sodium bicarbonate co-transporter (NBC1 and NBC3) proteins, which remain to be identified.     

Carbonic anhydrase inhibitors. Inhibition of cytosolic isozyme XIII with aromatic and heterocyclic sulfonamides: a novel target for the drug design

The inhibition of the newly discovered cytosolic carbonic anhydrase isozyme XIII (CA XIII) has been investigated with a series of aromatic and heterocyclic sulfonamides, including some of the clinically used derivatives, such as acetazolamide, methazolamide, dichlorophenamide, dorzolamide, and valdecoxib. Inhibition data for the physiologically relevant isozymes I and II (cytosolic forms) and the tumor associated isozyme IX (transmembrane) were also provided for comparison. A very interesting and unusual inhibition profile against CA XIII with these sulfonamides has been observed. The clinically used compounds (except valdecoxib, which was a weak CA XIII inhibitor) potently inhibit CA XIII, with Ki's in the range of 17-23 nM, whereas sulfanilamide, halogenated sulfanilamides, homosulfanilamide, 4-aminoethylbenzenesulfonamide, and orthanilamide were slightly less effective, with Ki's in the range of 32-56 nM. Several low nanomolar (Ki's in the range of 1.3-2.4 nM) CA XIII inhibitors have also been detected, all of them belonging to the sulfanilyl-sulfonamide type of inhibitors, of which aminobenzolamide is the best known representative. Because CA XIII is an active isozyme predominantly expressed in salivary glands, kidney, brain, lung, gut, uterus, and testis, where it probably plays an important role in pH regulation, its inhibition by sulfonamides may lead to novel therapeutic applications for this class of pharmacological agents.     

Characterization of CA XIII, a novel member of the carbonic anhydrase isozyme family

The carbonic anhydrase (CA) gene family has been reported to consist of at least 11 enzymatically active members and a few inactive homologous proteins. Recent analyses of human and mouse databases provided evidence that human and mouse genomes contain genes for still another novel CA isozyme hereby named CA XIII. In the present study, we modeled the structure of human CA XIII. This model revealed a globular molecule with high structural similarity to cytosolic isozymes, CA I, II, and III. Recombinant mouse CA XIII showed catalytic activity similar to those of mitochondrial CA V and cytosolic CA I, with k(cat)/K(m) of 4.3 x 10(7) m(-1) s(-1), and k(cat) of 8.3 x 10(4) s(-1). It is very susceptible to inhibition by sulfonamide and anionic inhibitors, with inhibition constants of 17 nm for acetazolamide, a clinically used sulfonamide, and of 0.25 microm, for cyanate, respectively. Using panels of cDNAs we evaluated human and mouse CA13 gene expression in a number of different tissues. In human tissues, positive signals were identified in the thymus, small intestine, spleen, prostate, ovary, colon, and testis. In mouse, positive tissues included the spleen, lung, kidney, heart, brain, skeletal muscle, and testis. We also investigated the cellular and subcellular localization of CA XIII in human and mouse tissues using an antibody raised against a polypeptide of 14 amino acids common for both human and mouse orthologues. Immunohistochemical staining showed a unique and widespread distribution pattern for CA XIII compared with the other cytosolic CA isozymes. In conclusion, the predicted amino acid sequence, structural model, distribution, and activity data suggest that CA XIII represents a novel enzyme, which may play important physiological roles in several organs.     

Carbonic anhydrases: current state of the art, therapeutic applications and future prospects

Carbonic anhydrases (CAs, EC 4.2.1.1) are wide-spread enzymes, present in mammals in at least 14 different isoforms. Some of these isozymes are cytosolic (CA I, CA II, CA III, CA VII, CA XIII), others are membrane-bound (CA IV, CA IX, CA XII and CA XIV), CA V is mitochondrial and CA VI is secreted in the saliva and milk. Three cytosolic acatalytic forms are also known (CARP VIII, CARP X and CARP XI). The catalytically active isoforms, which play important physiological and patho-physiological functions, are strongly inhibited by aromatic and heterocyclic sulfonamides. The catalytic and inhibition mechanisms of these enzymes are understood in great detail, and this greatly helped the design of potent inhibitors, some of which possess important clinical applications. The use of such CA inhibitors (CAIs) as antiglaucoma drugs are discussed in detail, together with the recent developments that led to isozyme-specific and organ-selective inhibitors. A recent discovery is connected with the involvement of CAs and their sulfonamide inhibitors in cancer: many potent CAIs were shown to inhibit the growth of several tumor cell lines in vitro and in vivo, thus constituting interesting leads for developing novel antitumor therapies. Future prospects for drug design of inhibitors of these ubiquitous enzymes are dealt with. Although activation of CAs has been a controversial issue for some time, recent kinetic, spectroscopic and X-ray crystallographic experiments offered an explanation of this phenomenon, based on the catalytic mechanism. It has been demonstrated recently, that molecules that act as carbonic anhydrase activators (CAAs) bind at the entrance of the enzyme active site participating in facilitated proton transfer processes between the active site and the reaction medium. In addition to CA II-activator adducts, X-ray crystallographic studies have been also reported for ternary complexes of this isozyme with activators and anion (azide) inhibitors. Structure-activity correlations for diverse classes of activators is discussed for the isozymes for which the phenomenon has been studied, i.e., CA I, II, III and IV. The possible physiological relevance of CA activation/inhibition is also addressed, together with recent pharmacological/ biomedical applications of such compounds in different fields of life sciences.     

Review Article

Carbonic anhydrases (CAs, EC 4.2.1.1) are wide-spread enzymes, present in mammals in at least 14 different isoforms. Some of these isozymes are cytosolic (CA I, CA II, CA III, CA VII, CA XIII), others are membrane-bound (CA IV, CA IX, CA XII and CA XIV), CA V is mitochondrial and CA VI is secreted in the saliva and milk. Three cytosolic acatalytic forms are also known (CARP VIII, CARP X and CARP XI). The catalytically active isoforms, which play important physiological and patho-physiological functions, are strongly inhibited by aromatic and heterocyclic sulfonamides. The catalytic and inhibition mechanisms of these enzymes are understood in great detail, and this greatly helped the design of potent inhibitors, some of which possess important clinical applications. The use of such CA inhibitors (CAIs) as antiglaucoma drugs are discussed in detail, together with the recent developments that led to isozyme-specific and organ-selective inhibitors. A recent discovery is connected with the involvement of CAs and their sulfonamide inhibitors in cancer: many potent CAIs were shown to inhibit the growth of several tumor cell lines in vitro and in vivo, thus constituting interesting leads for developing novel antitumor therapies. Future prospects for drug design of inhibitors of these ubiquitous enzymes are dealt with. Although activation of CAs has been a controversial issue for some time, recent kinetic, spectroscopic and X-ray crystallographic experiments offered an explanation of this phenomenon, based on the catalytic mechanism. It has been demonstrated recently, that molecules that act as carbonic anhydrase activators (CAAs) bind at the entrance of the enzyme active site participating in facilitated proton transfer processes between the active site and the reaction medium. In addition to CA II-activator adducts, X-ray crystallographic studies have been also reported for ternary complexes of this isozyme with activators and anion (azide) inhibitors. Structure-activity correlations for diverse classes of activators is discussed for the isozymes for which the phenomenon has been studied, i.e, CA I, II, III and IV. The possible physiological relevance of CA activation/inhibition is also addressed, together with recent pharmacological/biomedical applications of such compounds in different fields of life sciences.     

Metabolic effects of acetaldehyde

Acetaldehyde, the toxic product of ethanol metabolism in the liver, covalently binds to a variety of proteins, thereby altering liver function and structure. Through its binding to tubulin, acetaldehyde decreases the polymerization of microtubules thereby impairing protein secretion and favouring their retention, with associated swelling of hepatocytes. Acetaldehyde adduct formation also impairs some enzyme activities. Either directly or through binding with GSH, acetaldehyde favours lipid peroxidation. Various mitochondrial functions are altered, particularly after chronic ethanol consumption which sensitizes the mitochondria to the toxic effects of acetaldehyde. In cultured myofibroblasts, acetaldehyde stimulates collagen production. The acetaldehyde-protein adducts stimulate the production of antibodies directed against the acetaldehyde epitope. This immune response may contribute to the aggravation or perpetuation of alcohol-induced liver damage. Some acetaldehyde effects, however, could conceivably be considered as beneficial, such as the stimulation of vascular prostacyclin release which may take part in the 'protective' effect of moderate ethanol consumption against some cardiovascular complications.     

Increased covalent binding of acetaldehyde to calmodulin in the presence of calcium

The regulatory protein, calmodulin, undergoes major conformational changes in response to changes in intracellular calcium concentration. Furthermore, calmodulin has been reported to have lysine residues which markedly increase their reactivity toward electrophilic substances in the calcium-loaded state. We found that calmodulin formed two to three times more stable adducts with acetaldehyde in the calcium-loaded state as compared to the calcium-free state. Competition-binding studies showed that calmodulin could preferentially compete with albumin for acetaldehyde in the presence, but not in the absence, of calcium. When calmodulin was in the calcium-loaded state, trifluoperazine, an inhibitor of calmodulin activity, significantly decreased the stable binding of acetaldehyde to the protein, whereas in the calcium-free state, minimal effects on binding were observed. Since calmodulin is involved in regulation of multiple important processes in the cell, it is possible that acetaldehyde-calmodulin adducts could contribute to liver injury by perturbation of calcium-dependent homeostatic mechanisms within the hepatocyte.     

Covalent binding of acetaldehyde to type III collagen

Incubation of neutral salt soluble type III pN-collagen with [14C]acetaldehyde in vitro resulted in the formation of spontaneously stable acetaldehyde-protein adducts. This reaction occurred primarily at lysine residues and it was not affected by 0.2-2 mM concentrations of ascorbate but addition of sodiumcyanoborohydride increased the stable adducts by 3-5-fold. When confluent cultures of human skin fibroblasts were incubated with physiologically relevant concentrations of acetaldehyde, it became covalently bound to type III procollagen secreted into the medium. We propose that acetaldehyde binding to collagen fibrils occurs in vivo following chronic alcohol consumption.     

Carbonic anhydrase inhibitors. Comparison of chlorthalidone,indapamide, trichloromethiazide,and furosemide X-ray crystal structures in adducts with isozyme II,when several water molecules make the difference

Thiazide and high ceiling diuretics were recently shown to inhibit all mammalian isoforms of carbonic anhydrase (CA, EC 4.2.1.1) with a very different profile as compared to classical inhibitors, such as acetazolamide, methazolamide, and ethoxzolamide. Some of these structurally related compounds have a very different behavior against the widespread isozyme CA II, with chlorthalidone, trichloromethiazide, and furosemide being efficient inhibitors against CA II (K_Is of 65–138 nM), whereas indapamide is a much weaker one (K_I of 2520 nM). Furthermore, some of these diuretics are quite efficient (low nanomolar) inhibitors of other isoforms, for example, chlorthalidone against hCA VB, VII, IX, and XIII; indapamide against CA VII, IX, XII, and XIII, trichloromethiazide against CA VII and IX, and furosemide against CA I and XIV. Examining the four X-ray crystal structures of their CA II adducts, we observed several (2–3) active site water molecules interacting with the chlorthalidone, trichloromethiazide, and furosemide scaffolds which may be responsible for this important difference of activity. Indeed, indapamide bound to CA II has no interactions with active site water molecules. Chlorthalidone bound within the CA II active site is in an enolic (lactimic) tautomeric form, with the enolic OH also participating in two strong hydrogen bonds with Asn67 and a water molecule. The newly evidenced binding modes of these diuretics may be exploited for designing better CA II inhibitors as well as compounds with selectivity/affinity for various isoforms with medicinal chemistry applications.     

Differential modification of hemoglobin chains by acetaldehyde

Acetaldehyde-hemoglobin adducts have been suggested as potential markers for alcohol consumption. These adducts were formed in vitro with [14C]acetaldehyde and separated into hemoglobin subunits by cation-exchange chromatography to examine the relative modification of the alpha- and beta-chains. The effect of varying concentrations of acetaldehyde on the relative amounts of polypeptide adducts and on the specific radioactivities of undissociated hemoglobin (Hb) following reaction with hydroxymercurybenzoate (HMB) was also studied. There were linear relationships (P less than 0.05) between increasing levels of [14C]acetaldehyde (0.0, 0.1, 0.2, 0.5 mM) and the radioactivities of the alpha- and one of the two beta-chain adducts (22, 25, 53 dpm/mg Hb and 151, 272, 626 dpm/mg Hb, respectively). Increases in radioactivities of a minor unidentified hemoglobin adduct fraction were also observed. The ratios of specific radioactivities of beta-to alpha-chain (8.8 +/- 1.2 SEM) did not vary with the concentrations of acetaldehyde. Although the amounts of undissociated hemoglobin following reaction with HMB did not increase with increasing concentrations of acetaldehyde, the significant increase of specific radioactivities of this fraction (152, 1967, and 6562 dpm/mg Hb for 0.1, 0.2, and 0.5 mM acetaldehyde, respectively) suggested possible crosslinks within the tetramer or dimer. The amino acid analysis of alpha- and beta-subunit adducts formed with 0.1 and 0.5 mM acetaldehyde showed that unreacted cysteine residues were more often detected at the higher acetaldehyde concentration consistent with the formation of cysteine adducts labile to acid hydrolysis or the shielding of cysteine residues in acetaldehyde-modified Hb against the subunit separation by HMB treatment. Thus acetaldehyde reacts differentially with the alpha- and beta-hemoglobin subunits and with the undissociated hemoglobin molecule.     

Carbonic anhydrase inhibitors. Interaction of 2-N,N-dimethylamino-1,3,4-thiadiazole-5-methanesulfonamide with 12 mammalian isoforms: kinetic and X-ray crystallographic studies

2-N,N-Dimethylamino-1,3,4-thiadiazole-5-methanesulfonamide was tested for its interaction with the 12 catalytically active mammalian carbonic anhydrase (CA, EC 4.2.1.1) isozymes, CA I-XIV. The compound is a potent inhibitor of CA IV, VII, IX, XII, and XIII (K(I)s of 0.61-39 nM), a medium potency inhibitor of CA II and VA (K(I)s of 121-438 nM), and a weak inhibitor against the other isoforms (CA III, VB, VI, and XIV), making it a very interesting candidate for situations in which a strong/selective inhibition of certain isozymes is needed. The crystal structure of the hCA II adduct of this sulfonamide revealed interesting interactions between the inhibitor and the enzyme which are quite different from those observed in the adducts of CA II with the structurally related aliphatic derivatives zonisamide, 2-amino-1,3,4-thiadiazolyl-5-difluoromethanesulfonamide, and 2-dimethylamino-5-[sulfonamido-(aminomethyl)]-1,3,4-thiadiazole reported earlier.     

Atherosclerosis and acetaldehyde metabolism in blood.

Acetaldehyde elimination in blood homogenates and erythrocyte aldehyde dehydrogenase (ALDH) activity were studied in 64 patients operated before the age of 60 years because of symptomatic stenosis of aorta, iliac, or carotid arteries and in 38 healthy controls. The disappearance of acetaldehyde in blood homogenates was biphasic. Patients showed an enhanced elimination of acetaldehyde during the second phase (30-60 min), as compared to controls (T1/2 of acetaldehyde was 103 +/- 47 and 198 +/- 93 min, respectively, P less than 0.001). No correlation was found between ALDH activity and acetaldehyde elimination rate. Acetaldehyde elimination in blood homogenates and [14C]acetaldehyde binding to plasma proteins, hemoglobin, and erythrocyte membranes were studied in 10 patients with atherosclerotic disease and in 12 healthy controls. There was a significant correlation between unstable binding of [14C]acetaldehyde to plasma proteins and the half-life of acetaldehyde in the elimination test (p = 0.74, P less than 0.005). Fractionation of plasma proteins after incubation with [14C]acetaldehyde revealed no difference between patients and controls in the distribution of radioactivity. The binding of [14C]acetaldehyde to hemoglobin or erythrocyte membranes did not differ between patients and controls. These results indicate that patients with angiopathy and an enhanced acetaldehyde elimination in blood have reduced binding of acetaldehyde to plasma proteins. As unstable binding of acetaldehyde to proteins is known to involve free amino groups of amino acid residues, modification of these residues in atherosclerotic disease is conceivable.