Arthur S. Brecher

Interaction of acetaldehyde with plasma proteins of the renin-angiotensin system

Chronic alcohol abuse may lead to hypertension by stimulating the activity of the renin angiotensin system (RAS). While there are reports on the alcohol associated increase of angiotensin II in rats and increases of plasma renin activity in rats and human alcoholics, the exact mechanisms of stimulation of the RAS activity is not clear. This study provides evidence for a biochemical interaction of acetaldehyde, the primary oxidative metabolite of ethanol, upon bilaterally nephrectomized (NEPEX) rat plasma that contains significant quantities of angiotensinogen and lacks active renin. Rat plasma served as the source of renin in this study. Preincubation of NEPEX plasma with 0.2 M acetaldehyde at 4 degrees C for 2 h resulted in a 21% increase in the angiotensin I (A I) formation by the rat plasma renin and 27% increase in the A I formation by the trypsinized rat plasma renin. When the rat plasma which contains modest quantities of endogenous angiotensinogen in addition to renin was preincubated with 0.2 M acetaldehyde at 4 degrees C for 2 h, the rate of A I formation was increased by 10%. Equivalent amounts of ethanol did not modify the rate of A I generation when added to NEPEX plasma or rat plasma. These results suggest the possibility of a biochemical interaction of acetaldehyde with the renin substrate which may enhance the activity of the RAS cascade, thereby contributing to hypertension in chronic alcoholics.

A perspective on acetaldehyde concentrations and toxicity in man and animals.

Acetaldehyde (AcH) at a concentration of 593 mM lowers the natural fluorescence of commercial human serum by 12%. It also lowers the fluorescence of a beta-naphthylamine standard curve (recovery) in serum by 17%. These results contrast with earlier reports showing that 447 mM AcH had no effect upon fluorescence of serum or a beta-naphthylamine standard curve in serum. Because 447 mM AcH and 593 mM AcH represent 2.5% and 3.3% AcH, it is apparent that there is a narrow window between which AcH may affect fluorescence by adduct formation with blood components and exogenous fluorophores. Nonetheless, serum has the capacity to bind > 2.5% (> 447 mM) AcH without alteration in fluorescence, suggesting that serum has a great carrying capacity for AcH, undoubtedly in the form of adducts to nucleophiles. These results are discussed in the light of toxicity of AcH and ethanol, the probable significance of the approximately 30 microM free AcH that is reported in chronic alcoholics and the planning of in vitro and in vivo studies with AcH.

Coagulation Protein Function. IV. Effect of Acetaldehyde Upon Factor X and Factor Xa, the Proteins at the Gateway to the Common Coagulation Pathway

Acetaldehyde (AcH) (447 mM) exerts an inhibition on Factor Xa, as followed by a clotting assay, but does not inhibit the hydrolysis of the synthetic fluorogenic substrate, N-tBOC-Ile-Glu-Gly-Arg-7-amido-4-methylcoumarin. These data suggest that AcH, although not reacting at the catalytic site of Factor Xa nor at the binding site for the synthetic substrate, does interact with the functional groups on the enzyme that bind to its natural substrate, prothrombin. As a consequence of such interaction, the charge and conformation of Factor Xa is altered, thereby limiting effective activation of prothrombin. Additionally, alkylation of factor Xa may also affect its capacity to associate with Factor Va for the activation of prothrombin. AcH also reacts with Factor X, prolonging clotting times when the zymogen is activated with Russells viper venom (RVV). It also reduces the rate of hydrolysis of the fluorogenic substrate after activation of the alkylated zymogen by RVV. These data lead to the considerations that AcH-modified Factor X is no longer as effectively activated by RVV due to an alteration of its charge/conformation. Additional possibilities include a likely alkylation of the Factor Xa moiety of Factor X by AcH such that the activation product has an altered charge/conformation compared to native Factor Xa, including possible alkylation of its binding site(s) for prothrombin. The reduced rate of hydrolysis of the synthetic fluorogenic substrate for Factor Xa by the alkylated, activated Factor X lends further support to the generation of a modified Factor Xa by RVV, which may have a lower binding or catalytic rate for the fluorogenic substrate. These results support the suggestion that chronic consumption of alcohol may prolong the reported coagulation times as a result of reaction of alcohols primary metabolite, AcH, with clotting factors, thereby reducing their physiological potential.

Coagulation Protein Function. II. Influence of Thiols Upon Acetaldehyde Effects

It has been reported that prolonged prothrombin time may be a result of the interaction of acetaldehyde (AcH) with clotting proteins to form alkylated inactive products. The current investigation focuses on the influence of L-cysteine (CysH), DL-homocysteine (HC), D-penicillamine, N-acetyl-L-cysteine (NAC), L-serine and L-alanine at 0.01 M concentrations, lactalbumin hydrolysate (2 mg/ml), and 1.0 mM dithiothreitol (DTT) on clotting time as well as their interaction with AcH. The sulfhydryl amino acids, as well as DTT prolonged clotting upon preincubation with plasma. Cysteine and NAC, upon addition to plasma prior to the addition of AcH, exhibited a prolongation of clotting time compared to that of AcH alone. On sequential addition of serine, alanine, or lactalbumin hydrolysate to plasma followed by the addition of acetaldehyde, a prolongation of clotting time comparable to that of AcH alone was exhibited. When HC and penicillamine were added to plasma prior to the addition of AcH, a prolonged clotting time was observed, which was significantly less than that of AcH alone. Premixing of serine, alanine, and lactalbumin hydrolysate with AcH for 20 min prior to addition to the plasma reduced the effectiveness of AcH in prolonging clotting time as compared to successive additions of the amino acid and AcH. Since CysH and penicillamine have been reported to form cyclic adducts with AcH, it is suggested that a similar possibility exists for penicillamine and for HC. The reversible cyclic adduct formation reported for CysH may explain why cysteine did not lower the prolonged clotting time induced by AcH.(ABSTRACT TRUNCATED AT 250 WORDS)

Coagulation protein function. III. Effect of acetyldehyde upon the activation of prothrombin

When prothrombin is mixed with acetaldehyde (AcH), the primary metabolite of ethanol, at room temperature for 30 min, dialyzed to remove excess AcH, and then activated with Echis carinatus venom (ECV), a prolonged clotting time is observed. Similarly treated prothrombin, however, readily hydrolyzes the synthetic substrate, benzoyl-DL-arginine-beta-naphthylamide (BANA). These results suggest that AcH does not react with the catalytic site of thrombin, which is protected in the prothrombin molecule. However, it does react with susceptible sites on the prothrombin surface which remain alkylated during extensive dialysis to remove excess AcH and subsequent activation of the molecule by ECV. These alkylated sites on the newly formed thrombin molecule may inhibit or prevent the effective/efficient binding of fibrinogen at its binding sites, causing prolonged clotting times. The binding sites for accommodating fibrinogen on the thrombin molecule are apparently quite different from those that bind BANA. These data further suggest that prolonged clotting times that are reported in chronic alcoholics may be due, in part, to ready interaction of acetaldehyde with circulating prothrombin. Fibrinogen, which has been exposed to AcH and subsequently dialyzed to remove excess AcH, also responds with a prolonged clotting time in the presence of added thrombin, signifying that sites on fibrinogen that are essential for its function/conformation are susceptible to AcH.

A hypothesis linking hypoglycemia, hyperuricemia, lactic acidemia, and reduced gluconeogenesis in alcoholics to inactivation of glucose-6-phosphatase activity by acetaldehyde

Preliminary data have been obtained indicating that glucose-6-phosphatase is inactivated upon preincubation with 447 and 224 mM acetaldehyde for 30 min at room temperature, resulting in a loss of 67% and 33% of the original activity, respectively. The reaction with acetaldehyde is rapid because 44% of the enzymic activity is lost in 5 min. Comparable quantities of ethanol inhibit the enzyme to the extent of 11%, indicating a very slight, statistically insignificant organic solvent effect. Because chronic alcoholics present a clinical picture of hypoglycemia, hyperuricemia, reduced gluconeogenesis, and lactic acidemia, it is hypothesized that glucose-6-phosphatase may be a focal enzyme whose inactivation may be related to each of the disorders. Glucose-6-phosphatase is the terminal key enzyme in the gluconeogenesis pathway leading to increased blood glucose. Inhibition thereof may explain both the alternate reduction of pyruvate with concommittent increased formation of lactic acid, and the increase in the pentose phosphate pathway leading to hyperuricemia (as also observed in von Gierkes disease).

Identification of α1-acid glycoprotein, α2-macroglobulin and antithrombin III as components of normal and malignant human tissues

alpha1-Acid glycoprotein, alpha2-macroglobulin, and antithrombin III have been identified, by immunological means, as components of the 90000 X g supernatant fraction of malignant and adjacent normal human breast, colon, and anal tissues, as well as malignant stomach and ileum. Malignant lung tissue only contained alpha1-acid glycoprotein. These protease inhibitors are immunologically equivalent to those present in human plasma.

Acetaldehyde Inhibits Serum Aminopeptidases

Aminopeptidase A (APA)- and aminopeptidase M (APM)-like activity were assayed in Moni-Trol ES with L-alpha-aspartyl-beta-naphthylamide and L-alanyl-beta-naphthylamide, respectively. Upon preincubation of the serum with 89.4, 223.5, and 447 mM acetaldehyde at room temperature for 30 min, a reduction in 26.8%, 55.3%, and 75.8% aminopeptidase A activity was observed. Similarly, aminopeptidase M activity was reduced by 26.5% and 53.1% upon preincubation with 223.5 and 447 mM acetaldehyde. Ethanol at 84.9, 212.3, and 427.9 mM did not significantly affect the enzymic activity. Because aminopeptidase A and aminopeptidase M also degrade the pressor substance, angiotensin II, it is suggested that inhibition of aminopeptidase A- and aminopeptidase M-like activity by acetaldehyde, the product of ethanol metabolism, may lead to higher levels of circulating angiotensin II and, consequently, hypertension, in alcoholics. The hydrolysis of lysine-p-nitroanilide, an aminopeptidase B substrate, was also inhibited upon addition of acetaldehyde to Moni-Trol ES serum.

Acetaldehyde inhibits the anti-elastase activity of α1-antitrypsin

Abstract There are genetic and exogenous factors responsible for α 1 -antitrypsin ( α 1 -AT) deficiency which may lead to cirrhosis of the liver and emphysema. The present study was initiated on a biochemical level in order to determine whether acetaldehyde, the major product of ethanol metabolism, is capable of influencing the physiological effect of α 1 -AT upon elastase, an enzyme which is capable of inducing emphysema. The effects of acetaldehyde and ethanol upon elastase and α 1 -AT were tested. Acetaldehyde at 0.3-M and 1.2-M concentrations inhibited the anti-elastase activity of α 1 -AT. Acetaldehyde at 0.03-M and 0.07-M concentrations did not affect elastase activity and had a slight effect at 0.12-M levels. Equivalent amounts of ethanol were without influence upon elastase activity or α 1 -AT function. These data provide biochemical support for the possibility that heterozygous males with lower than normal α 1 -AT levels may be at much higher risk to develop liver disease, emphysema, and α 1 -AT deficiency as a consequence of chronic exposure to ethanol and concommitent circulating acetaldehyde levels.

Coagulation Protein Function: Enhancement of the Anticoagulant Effect of Acetaldehyde by Sulfated Glycosaminoglycans

In view of the increased anticoagulant effect of acetaldehyde-treated heparin, other glycosaminoglycans (GAGs) such as chondroitin sulfates A and C, dermatan sulfate (chondroitin sulfate B), heparan sulfate, and hyaluronic acid were tested for anticoagulant activity before and after exposure to acetaldehyde. Clotting times of human plasma Ci-Trol coagulation control, level I (Baxter Healthcare Corp.), were tested in the presence of 1.8, 3.0, 3.6, or 4.5 μg heparin (0.32, 0.54, 0.64, 0.81 units heparin). Additionally, 9, 27, or 90 μg of chondroitin sulfates A, B, or C was utilized in lieu of heparin. The effects of 2 μg heparin (0.36 units), chondroitin sulfates A, B, and C, (20 μg each), 2 μg heparan sulfate, and 2 μg hyaluronic acid, respectively, in the presence of 44.7 mM acetaldehyde on the clotting time of plasma were studied. It was observed that chondroitin sulfate B (dermatan sulfate) prolonged the clotting time of plasma, although to a lesser extent than heparin. Chondroitin sulfates A and C, heparan sulfate, and hyaluronic acid did not prolong clotting time. However, pretreatment of all the sulfated GAGs with acetaldehyde gave products that enhanced the anticoagulant effect of acetaldehyde, notwithstanding the lack of anticoagulant effect of the GAGs. In contrast, hyaluronic acid exhibited no effect upon clotting time nor did its acetaldehyde-treated product. Furthermore, ethanol exhibited no effect upon the clotting times of the GAG–plasma mixtures. These results suggest that sulfated GAGs may be modified by acetaldehyde, a component of plasma in chronic alcoholics, and that the resultant products may contribute to the prolonged clotting times.