CHARACTERISTICS OF ALDEHYDE DEHYDROGENASES OF CERTAIN AEROBIC BACTERIA REPRESENTING HUMAN COLONIC FLORA

We have proposed the existence of a bacteriocolonic pathwayfor ethanol oxidation resulting in high intracolonic levelsof toxic and carcinogenic acetaldehyde. This study was aimedat determining the ability of the aldehyde dehydrogenases (ALDH)of aerobic bacteria representing human colonic flora to metabolizeintracolonically derived acetaldehyde. The apparent Michaelisconstant (K_m) values for acetaldehyde were determined in crudeextracts of five aerobic bacterial strains, alcohol dehydrogenase(ADH) and ALDH activities of these bacteria at conditions prevailingin the human large intestine after moderate drinking were thencompared. The effect of cyanamide, a potent inhibitor of mammalianALDH, on bacterial ALDH activity was also studied. The apparentK_m for acetaldehyde varied from 6.8 (NADP⁺ -linked ALDH of Escherichiacoli IH 13369) to 205 µM (NAD⁺ -linked ALDH of Pseudomonasaeruginosa IH 35342), and maximal velocity varied from 6 nmol/min/mg(NAD⁺ -linked ALDH of Klebsiella pneumoniae IH 35385) to 39nmol/min/mg (NAD⁺ -linked ALDH of Pseudomonas aeruginosa IH35342). At pH 7.4, and at ethanol and acetaldehyde concentrationsthat may be prevalent in the human colon after moderate drinking,ADH activity in four out of five bacterial strains were 10–50times higher than their ALDH activity. Cyanamide inhibited onlyNAD⁺ -linked ALDH activity of Pseudomonas aeruginosa IH 35342at concentrations starting from 0.1 mM. We conclude that ALDHsof the colonic aerobic bacteria are able to metabolize endogenicacetaldehyde. However, the ability of ALDHs to metabolize intracolonicacetaldehyde levels associated with alcohol drinking is ratherlow. Large differences between ADH and ALDH activities of thebacteria found in this study may contribute to the accumulationof acetaldehyde in the large intestine after moderate drinking.ALDH activities of colonic bacteria were poorly inhibited bycyanamide. This study supports the crucial role of intestinalbacteria in the accumulation of intracolonic acetaldehyde afterdrinking alcohol. Individual variations in human colonic floramay contribute to the risk of alcohol-related gastrointestinalmorbidity.     

ALDEHYDE DEHYDROGENASE ACTIVITY AND ACETATE PRODUCTION BY AEROBIC BACTERIA REPRESENTING THE NORMAL FLORA OF HUMAN LARGE INTESTINE

We have recently proposed the existence of a bacteriocolonicpathway for ethanol oxidation, i.e ethanol is oxidized by alcoholdehydrogenase of intestinal bacteria resulting in high intracoloniclevels of reactive and toxic acetaldehyde. This study was aimedto examine aldehyde dehydrogenase (ALDH) activity, acetaldehydeconsumption and production of acetate by aerobic bacteria (n=27),representing the normal human colonic flora. Most bacterialstrains did not show any membrane-associated aldehyde dehydrogenase,but possessed marked cytosolic NADP⁺- and NAD⁺-dependent aldehydedehydrogenase activity, ranging from 155 nmol of NAD(P)H produced/min/mgof protein to zero with acetaldehyde as substrate. NADP⁺-linkedALDH activity was significantly higher than NAD⁺-linked activityin most of the tested bacteria. In addition, aerobic bacteriametabolized acetaldehyde effectively in vitro and this couldbe inhibited by cyanamide in nearly half of the tested strains.Production of acetate from acetaldehyde ranged from 2420 nmol/10⁹colony-forming units to almost negligible. In conclusion, manyhuman aerobic colonic bacteria possess significant aldehydedehydrogenase activity and can, consequently, produce acetatefrom acetaldehyde in vitro at least under the partially aerobicconditions proposed to prevail on the colonic mucosal surface.Individual variation in the capability of colonic flora to removetoxic acetaldehyde may be one factor regulating intracolonicacetaldehyde levels, as well as the rate of bacteriocolonicpathway for ethanol oxidation.     

HIGH BLOOD ENDOTOXIN DOES NOT AFFECT ETHANOL ELIMINATION IN RATS

Gut-derived endotoxins have been proposed as mediators of theenhancement of ethanol elimination after chronic alcohol administration.We investigated whether chronically elevated blood-endotoxinlevels affect the rate of ethanol elimination in a study whereendotoxin was administered chronically from an osmotic minipumpto rats fed ethanol in a liquid diet. As expected, an acutedose of ethanol (1.2 g/kg body wt, i.p.) was eliminated significantlyfaster (329±11 mg/kg/h) by chronically ethanol-fed animalsthan by pair-fed controls (285 ± 9 mg/kg/h). However,although endotoxin administration significantly elevated blood-endotoxinlevels, the rate of ethanol elimination in endotoxin-treatedgroups was almost identical when compared either to controls(289 vs 285) or to ethanol-fed rats (328 vs 329). We concludethat chronic endotoxin exposure at levels that only resultedin mild hepatic changes, had no effect on the rate of ethanolelimination and that it is unlikely that endotoxins are involvedin the induction of the ethanol elimination rate following chronicalcohol administration.     

A new general solid-phase method for the synthesis of backbone-to-backbone cyclized peptides

A model peptide with the sequences Ala-Pro-Lys(2ClZ)-Tyr(2BrZ) was synthesized on a 4-methylbenzhydryl amine (MBHA) polystyrene resin using conventional Boc/benzyl protective group strategy. The amino acid aldehyde Boc-valinal was coupled by reductive alkylation with NaCNBH₃ in acidified DMF for 1 h. The secondary amine in the peptide-resin Boc-Valψ[CH₂NH]Ala-Pro-Lys(2CIZ)-Tyr(2BrZ)-MBHA was reductively alkylated by 3(4-methylbenzylthio)-propanal at 40 °C for 6 h, resulting the peptide-resin Boc-Valψ[CH₂N(CH₂CH²CH₂-S-pMeBzl)]Ala-Pro-Lys(2ClZ)-Tyr(2BrZ)-MBHA. After the removal of the Boc group the synthesis was continued employing the above-mentioned methods, which led to the resin-bound peptide Leuψ[CH₂N(CH₂CH₂CH₂S-pMeBzl)]Ser-Pro-Gly-Lys(2ClZ)-Valψ[CH₂N(CH₂CH₂CH₂-S-pMeBzl)]Ala-Pro-Lys(2ClZ)-Tyr(2BrZ)-MBHA. The peptide was cleaved from the resin with hydrogen fluoride. Reversed-phase HPLC and plasma desorbtion mass spectrometry analysis showed that the expected peptide Leuψ[CHIN(CH₂CH₂CH₂SH)ISer-Pro-Gly-Lys-Valψ[CH₂N(CH₂CH₂CH₂-SH)]Ala-Pro-Lys-Tyr-NH₂ was obtained as the major product with low levels of side products. Intramolecular oxidation of the thiols gave the backbone to backbone cyclized peptide Leuψ[CH₂N(CH₂CH₂CH₂S)]Ser-Pro-Gly-Lys-Valψ[CH₂N(CH₂CH₂CH₂-S)]A1a-Pro-Lys-Tyr-NH₂.     

HEPATOTOXICITY AND ABSORPTION OF EXTRAHEPATIC ACETALDEHYDE IN RATS

Acetaldehyde, the first metabolite of ethanol oxidation, has been proposed as a major initiating factor in ethanol-induced liver injury. The aims of this study were to examine whether acetaldehyde is absorbable from the digestive tract and whether, when delivered chronically in drinking water, it is capable of inducing liver injury in rats. Acetaldehyde concentrations in the rat portal and peripheral blood were measured by head space gas chromatography after intragastric (5 ml) and intracolonic (3 ml) administration of 20 mM acetaldehyde solution. In the hepatotoxicity study, rats were exposed to acetaldehyde (20 and 120 mM ) delivered in drinking water for 11 weeks and histopathological changes in the liver were morphometrically assessed. Peak blood acetaldehyde levels were found at 5 min after acetaldehyde infusion and were 235±11 μμ (mean±SE) after intragastric and 344±83 μμ after intracolonic infusion of 20 mM acetaldehyde solution. The exposure of rats to 120 mM acetaldehyde solution for 11 weeks resulted in the development of fatty liver and inflammatory changes. Morphometric analysis showed significantly more fat accumulation in rats receiving 120 mM acetaldehyde solution (85±2 per cent of hepatocytes occupied by fat) than in rats receiving 20 mM acetaldehyde solution (38±11 per cent) or in controls (36±10 per cent). The dose of extrahepatic acetaldehyde (500 mg/kg per day) producing liver injury corresponds to only around 3 per cent of that derived from hepatic ethanol oxidation in animals receiving an ethanol-containing totally liquid diet (15 g/kg per day). These results indicate that acetaldehyde delivered via the digestive tract can reach the liver by the portal circulation and that acetaldehyde of extrahepatic origin appears to be more hepatotoxic than acetaldehyde formed during ethanol oxidation within the liver.     

Group IIA phospholipase A2 as a prognostic marker in prostate cancer: relevance to clinicopathological variables and disease‐specific mortality

Group IIA Phospholipase A₂ (PLA2‐IIA), a key enzyme in arachidonic acid and eicosanoid metabolism, participates in a variety of inflammatory processes but possibly also plays a role in tumor progression in vivo. Our aim was to determine the mRNA and protein expression of PLA2‐IIA during prostate cancer progression in localized and metastatic prostate tumors. We evaluated the prognostic significance of PLA2‐IIA expression in biochemical recurrence, clinical recurrence and disease‐specific survival after surgical treatment. The expression of PLA2‐IIA was examined by immunohistochemistry and chromogenic in situ hybridization in tissue microarrays of radical prostatectomy specimens and advanced/metastatic carcinomas. The expression data were analyzed in conjunction with clinical follow‐up information and clinicopathological variables. The mRNA and protein expression of PLA2‐IIA was significantly increased in Gleason pattern grade 2–4 carcinomas compared with benign prostate (p‐values 0.042–0.001). In metastases, the expression was significantly lower than in local cancers (p=0.001). The PLA2‐IIA expression correlated positively with Ki‐67 and α‐methylacyl CoA racemase (AMACR) expression. The prognostic evaluation revealed decreased PLA2‐IIA protein expression among patients who had died of prostate cancer. In conclusion, PLA2‐IIA expression is increased in carcinoma when compared with benign prostate. However, metastatic carcinoma showed decreased expression of PLA2‐IIA when compared with primary carcinomas. PLA2‐IIA may serve as a marker for highly proliferating, possibly poorly differentiated prostate carcinomas. The protein expression of PLA2‐IIA may be diminished in patients who consequently die of prostate cancer.