Helmuth Sippel

The distribution and metabolism of acetaldehyde in rats during ethanol oxidation-I. The distribution of acetaldehyde in liver, brain, blood and breath.

Abstract An in vivo investigation was made of the distribution of acetaldehyde (AcH) during ethanol metabolism. Different doses of ethanol were administered orally to male and female Sprague-Dawley rats ( n = 96) and AcH measured at various times thereafter in the liver, blood, brain and breath. The results showed that the liver was the primary site for the oxidation of the ethanol-derived AeH. Only a small amount of the total AcH formed in this organ escaped into the rest of the body, but this amount increased with increased hepatic ethanol concentration. The hepatic AcH level was higher in male rats than in females with the same hepatic ethanol concentration. The extrahepatic AcH levels in arterial cerebral and in peripheral tail blood correlated well with the corresponding hepatic AcH levels. The bulk of the hepatic AcH output was eliminated extrahepatically, thus drastically changing the AcH level from that initially leaving the liver. Sex differences also appeared in the extrahepatic blood AcH levels, with the female rats displaying higher AcH levels, as a result of their less efficient extrahepatic AcH elimination. The peripheral tail blood AcH was found to be similar to the AcH level of the venous blood before the hepatic blood AcH is added to it. Regardless of the AcH levels in the liver and blood, no AcH was found in the brain. Less than 5 per cent of the hepatic AcH output was exhaled. Pentobarbital anaesthesia strongly depressed the amount of AcH exhaled. The AcH in the breath did not reflect the hepatic AcH as well as the blood AcH levels did.

Combined glucose and lactate values in vitreous humour for postmortem diagnosis of diabetes mellitus

The combined glucose and lactate values in vitreous humour were examined in 52 autopsy cases without diabetes and 10 cases with diabetes, and the significance thresholds for the distribution of the combined values were calculated. The use of these values in the diagnosis of antemortem hyperglycaemia, lactic acidosis or hypoglycaemia is discussed.

Kupffer cell inactivation alleviates ethanol-induced steatosis and CYP2E1 induction but not inflammatory responses in rat liver

BACKGROUND/AIMS Gadolinium chloride inactivates Kupffer cells and alleviates alcohol-induced liver lesions. We investigated the mechanism of gadolinium chloride protection after oral ethanol feeding. METHODS Rats were maintained ethanol-intoxicated for 6 weeks by feeding ethanol in a low-carbohydrate/high-fat liquid diet. Macrophages were inactivated by intravenous administrations of gadolinium chloride. At termination, liver samples and cell lysates obtained from the periportal and perivenous region were analyzed for histopathology, mRNA expression of endotoxin-associated parameters and cytokines and for enzymes involved in oxidative stress. RESULTS Ethanol treatment alone caused marked microvesicular/macrovacuolar steatosis and focal inflammation. Gadolinium significantly alleviated pathology, by reducing steatosis but not inflammation. Gadolinium treatment eliminated ED2 immunopositive Kupffer cells, which were larger and more frequent periportally. Ethanol significantly increased the mRNA expression of the endotoxin (LPS) receptor CD14 and the LPS binding protein LBP, but not that of the pro-inflammatory cytokines TNF-alpha and IL-1beta. The mRNA of CD14 was found to be expressed preferentially in the perivenous region, but gadolinium treatment had no significant effect on the expression or the distribution. However, gadolinium significantly moderated the ethanol induction of CYP2E1 and this effect correlated to the degree of steatosis. Ethanol increased glutathione transferase and reduced glutathione peroxidase activity, but these changes persisted after gadolinium treatment. CONCLUSIONS Our results suggest that gadolinium chloride reduces symptoms of ALD mainly by counteracting steatosis, and that CD14-positive Kupffer cell populations are not involved in gadolinium protection. The strong correlation between pathology and CYP2E1 induction might suggest a steatopathogenic role for this enzyme.

Acinar distribution of glutathione-dependent detoxifying enzymes. Low glutathione peroxidase activity in perivenous hepatocytes.

The acinar distribution of glutathione S-transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G-6-PDH) was examined by analyzing periportal (p.p.) and perivenous (p.v.) rat hepatocytes selectively isolated by the digitonin-collagenase perfusion. The cytosolic GST activity was higher in p.v. cells, but the microsomal GST and cytosolic GR were found to be evenly distributed in the acinus. In contrast, the activity of both the Se-dependent GPx and the microsomal (Se-independent) GPx, as well as G-6-PDH, was much lower in the p.v. than in the p.p. cells. The heterogeneous distribution of GST, GPx and G-6-PDH was confirmed by analyzing liver perfusion effluents collected after ante- or retrograde digitonin infusion. The relatively low activities of GPx and G-6-PDH in the p.v. cells could partly explain the susceptibility of this region to chemical injury.

The antiestrogen toremifene protects against alcoholic liver injury in female rats

BACKGROUND/AIMS Females are generally considered to be more susceptible to alcohol-induced liver injury than males. To elucidate whether gonadal hormones are involved, female rats were chronically treated with ethanol and with an antiestrogen. METHODS Ethanol was administered in a low-carbohydrate liquid diet. Estrogen action was blocked by daily intubation of toremifene, a non-hepatotoxic second generation estrogen receptor antagonist. RESULTS The female rats consuming intoxicating amounts of ethanol diet for 6 weeks developed massive microvesicular/macrovesicular steatosis, frequent inflammatory foci and spotty necrosis. Serum alanine aminotransferase increased 7-fold. Toremifene treatment did not affect steatosis, but significantly reduced inflammation and necrosis. Ethanol increased the expression of CD14 and tumor necrosis factor- (TNF) alpha mRNA and also the production of TNF-alpha by isolated Kupffer cells, but toremifene had no significant counteracting effect. However, toremifene significantly alleviated both ethanol induction of the pro-oxidant enzyme CYP2E1 and ethanol reduction of the oxidant-protective enzyme Se-glutathione peroxidase. CONCLUSIONS The partial protection by toremifene against ethanol-induced liver lesions suggests a pathogenic contribution of estrogens, possibly associated with an oxygen radical mediated mechanism.

Aryl hydrocarbon receptor-associated genes in rat liver: Regional coinduction of aldehyde dehydrogenase 3 and glutathione transferase Ya

The tumor-associated aldehyde dehydrogenase 3 (ALDH3) and the glutathione transferase (GST)Ya form are coded by members of the Ah (aryl hydrocarbon) battery group of genes activated in the liver by polycyclic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The physiological role of the Ah receptor (AHR), its gene-activating mechanism and its endogenous ligands are still poorly clarified. We had previously observed that 3-methylcholanthrene (3MC) and beta-naphthoflavone (betaNF) induced the AHR-associated CYP1A1/1A2 pair in different liver regions, an effect not explained by the acinar distribution of the AHR protein. Here, we investigated AHR-associated regional induction by comparing the expression patterns of ALDH3 and GSTYa. Analysis of samples from periportal and perivenous cell lysates from 3MC-treated animals revealed that ALDH3 mRNA, protein and benzaldehyde-NADP associated activity were all confined to the perivenous region. In contrast, such regio-specific induction was not seen after beta-NF induction. Immunohistochemically, a peculiar mono- or oligocellular induction pattern of ALDH3 was seen, consistently surrounding terminal hepatic veins after 3MC but mainly in the midzonal region after betaNF. A ligand-specific difference in regional induction of GSTYa1 mRNA was also observed: The constitutive perivenous dominance was preserved after 3MC while induction by betaNF was mainly periportal. A 3MC-betaNF difference was also seen by immunohistochemistry and at the GSTYa protein level, in contrast to that of the AHR-unassociated GSTYb protein. However, experiments with hepatocytes isolated from the periportal or perivenous region to replicate these inducer-specific induction responses in vitro were unsuccessful. These data demonstrate that the different acinar induction patterns by 3MC and betaNF previously observed for CYP1A1 and CYP1A2 are seen also for two other Ah battery genes, GSTYa1 and ALDH3, but in a modified, gene-specific form. We hypothesize that unknown protein(s) operating in vivo and modifying the Ah-mediated response at the common XRE element located upstream of these genes is affected zonespecifically by 3MC and betaNF.

Long-lived photoproducts of rhodopsin in the retina of the frog.

Abstract We find that Baumanns (1972) model with appropriate values for the rate constants rather well describes the spectral changes observed after irradiation of frog retinas at varying temperatures (0–25°C) and pH-values (6·0–7·9). The rates of both reactions leading to retinal, i.e. the decay of metarhodopsin III and the direct conversion of metarhodopsin II into retinal, decrease with increasing pH, which explains the accumulation of large amounts of M III in slightly alkaline retinas

Positive prostate-specific antigen (PSA) reaction in post-mortem rectal swabs: A cautionary note

Prostate-specific antigen (PSA) tests are considered a valuable screening method for the forensic examination of semen in vaginal and rectal swabs of alleged victims of sexual abuse. Although these membrane tests have been also applied to autopsy specimens no study has assessed their reliability when performed on post-mortem (PM) rectal swabs from decomposed cadavers. The present study describes the results obtained with the Seratec PSA Semiquant Kit test on 39 male and 10 female adult cadavers with no history of sexual assault and with a PM interval up to 136 days. Overall 64% of the 39 male cadavers tested positive for the PSA, the positive PSA reaction being more frequent in the 20 males with advanced decomposition than in the 19 males with no putrefaction signs (70% vs. 58%). The Phosphatesmo KM Paper Test for detection of acid phosphatase (AP) gave a positive color reaction with 60% of the rectal swabs obtained from decomposed male cadavers. Both the PSA-test and the Phosphatesmo KM paper-test gave a negative result in each of the rectal samples from female cadavers. Y STR multiplex revealed no DNA other than that of the subject tested in the rectal swab positive for PSA. The results of the present study show that PSA membrane tests are unreliable and can be misleading when derived from male rectal samples obtained at autopsy.

Effect of ethanol on the microsomal glutathione S-transferase activity in glutathione-depleted rat liver

Depletion of hepatic glutathione in male rats by starvation caused a significant increase in microsomal glutathione S-transferase activity, which was not affected by acute ethanol pretreatment. An additional depletion in fasted rats by diethylmaleate (0.5 g/kg) caused a further increase in the enzyme activity, but this increase was delayed in ethanol intoxicated rats. Although ethanol caused a small increase in hepatic microsomal lipid peroxidation in control animals, this effect of ethanol was not observed in diethylmaleate treated rats and thus was apparently not responsible for the delay in enzyme activation. It is suggested that the activation of microsomal glutathione S-transferase activity towards 1-chloro-2,4-dinitrobenzene in glutathione-depleted rat liver may be produced by changes in thiol/disulfid ratio and/or some reactive oxygen species.

Inefficacy of oxygenated drinking water in accelerating ethanol elimination in humans

Abstract In our earlier report it was found that after intravenous infusion of ethanol into monkeys the drinking of oxygenated water accelerated their elimination rate of ethanol 60% on the average. The acceleration was assumed to be due to increased hepatic oxygen tension through the portal blood flow. Now we have repeated similar experiments using humans as subjects. In humans the elimination rate of ethanol was not changed by oxygenated drinks. We are not able to explain the species difference, because the mechanism by which oxygenated water increased ethanol elimination in monkeys is unsettled.