Headspace gas chromatography with capillary column for urine alcohol determination

A headspace gas chromatographic method using a fused-silica capillary column Poraplot Q has been developed and validated for the detection and quantification of ethanol in urine. Under optimized conditions, ethanol was properly separated from acetaldehyde, acetone, isopropanol, methanol and n-propanol. Limits of detection (LODs) and quantification (LOQs) were 0.008 and 0.010 g/l, respectively. The precision studies within-run and between-run, using spiked urine samples (0.08, 0.8 and 2.0 g/l) showed maximum coefficients of variation 5.9 and 6.5%, respectively. Results of ethanol recovery varied from 91.6+/-0.8 to 103.3+/-1.8% over the concentration range from 0.01 to 3.20 g/l. The method was appropriate for the detection of ethanol in urine samples. This matrix can be used for monitoring alcohol abuse in the workplace and used in alcohol rehabilitation programs.     

An unusual oxygen-sensitive, iron- and zinc-containing alcohol dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus

Pyrococcus furiosus is a hyperthermophilic archaeon that grows optimally at 100 degreesC by the fermentation of peptides and carbohydrates to produce acetate, CO2, and H2, together with minor amounts of ethanol. The organism also generates H2S in the presence of elemental sulfur (S0). Cell extracts contained NADP-dependent alcohol dehydrogenase activity (0.2 to 0.5 U/mg) with ethanol as the substrate, the specific activity of which was comparable in cells grown with and without S0. The enzyme was purified by multistep column chromatography. It has a subunit molecular weight of 48,000 +/- 1,000, appears to be a homohexamer, and contains iron ( approximately 1.0 g-atom/subunit) and zinc ( approximately 1.0 g-atom/subunit) as determined by chemical analysis and plasma emission spectroscopy. Neither other metals nor acid-labile sulfur was detected. Analysis using electron paramagnetic resonance spectroscopy indicated that the iron was present as low-spin Fe(II). The enzyme is oxygen sensitive and has a half-life in air of about 1 h at 23 degreesC. It is stable under anaerobic conditions even at high temperature, with half-lives at 85 and 95 degreesC of 160 and 7 h, respectively. The optimum pH for ethanol oxidation was between 9. 4 and 10.2 (at 80 degreesC), and the apparent Kms (at 80 degreesC) for ethanol, acetaldehyde, NADP, and NAD were 29.4, 0.17, 0.071, and 20 mM, respectively. P. furiosus alcohol dehydrogenase utilizes a range of alcohols and aldehydes, including ethanol, 2-phenylethanol, tryptophol, 1,3-propanediol, acetaldehyde, phenylacetaldehyde, and methyl glyoxal. Kinetic analyses indicated a marked preference for catalyzing aldehyde reduction with NADPH as the electron donor. Accordingly, the proposed physiological role of this unusual alcohol dehydrogenase is in the production of alcohols. This reaction simultaneously disposes of excess reducing equivalents and removes toxic aldehydes, both of which are products of fermentation.     

Obesity and high-density lipoprotein cholesterol in black and white 9- and 10-year-old girls: The National Heart, Lung, and Blood Institute Growth and Health Study

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 microM (mean +/- SE) after intragastric and 344 +/- 83 microM 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.     

Dehydrogenation of 3-phenoxybenzyl alcohol in isolated perfused rabbit skin, skin homogenate and purified dehydrogenases

The formation of 3-phenoxybenzoic acid from 3-phenoxybenzyl alcohol was determined in (a) rabbit ears, single-pass perfused with a protein-free buffer, pH 7.4; (b) the microsomal fraction and its supernatant from homogenized rabbit skin; and (c) purified alcohol dehydrogenase from horse liver and baker's yeast. The inhibition of product formation in (a) was about 60% by various 4-methylpyrazole concentrations, but metyrapone had no effect. Following ultracentrifugation, only the supernatant of homogenized skin showed product formation (apparent Vmay: 32 pmol/min per cm2 skin; apparent Km: 64 microM). 3-Phenoxybenzyl alcohol and ethanol dehydrogenation was similar by alcohol dehydrogenase from horse liver (apparent Km: 0.7 vs. 0.4 mM; apparent Vmax: 0.3 vs. 0.2 U/ microg protein). In baker's yeast, the apparent Km of 3-phenoxybenzoic acid formation was several times larger than that for ethanol dehydrogenation. The KI of 4-methylpyrazole for alcohol dehydrogenase from horse liver was 0.6 (3-phenoxybenzyl alcohol) vs. 0.04 microM (ethanol). The KI for ethanol in baker's yeast was 470 microM. In conclusion dehydrogenation is an important metabolic pathway in the skin for xenobiotics with an aliphatic alcohol at a side chain.     

Chemical pathways of peptide degradation: IX. Metal-catalyzed oxidation of histidine in model peptides

PURPOSE: To elucidate the nature of the reactive oxygen species (i.e., superoxide anion radical, hydroxyl radical, and hydrogen peroxide) involved in the metal-catalyzed oxidation of histidine (His) in two model peptides. METHODS: The degradation of AcAla-His-ValNH2 (Ala-peptide) and AcCysNH2-S-S-AcCys-His-VaNH2 (Cys-peptide) was investigated at pH 5.3 and 7.4 in an ascorbate/cupric chloride/oxygen (ascorbate/ Cu(II)/O2) system, both in the absence and presence of selective scavengers (i.e., catalase, superoxide dismutase, mannitol, sodium formate, isopropanol, and thiourea) of the reactive oxygen species. All reactions were monitored by HPLC. The major degradation products were characterized by electrospray mass spectrometry. RESULTS: The Cys-peptide was more stable than the Ala-peptide at pH 5.3 and 7.4. Both peptides displayed greater stability at pH 5.3 than at 7.4. At pH 5.3, 35 +/- 0.7% of the Cys-peptide and 18 +/- 1% of the Ala-peptide remained after 7 hours, whereas at pH 7.4, 16 +/- 3% of the Cys-peptide and 4 +/- 1% of the Ala-peptide remained. Catalase, thiourea, bicinchoninic acid, and ethylenediaminetetraacetate were effective at stabilizing both peptides toward oxidation, while superoxide dismutase, mannitol, isopropanol, and sodium formate were ineffective. The main degradation products of the Ala- and Cys-peptides at pH 7.4 appeared to be AcAla-2-oxo-His-ValNH2 and AcCysNH2-S-S-AcCys-2-oxo-His-ValNH2, respectively. CONCLUSIONS: Hydrogen peroxide, Cu(I), and superoxide anion radical were deduced to be intermediates involved in the oxidation of the Ala- and Cys-peptides. Hydrogen peroxide degradation to secondary reactive oxygen species may have led to the oxidation of the peptides. The degradation of hydrogen peroxide by a Fenton-type reaction was speculated to form a complexed form of hydroxyl radical that reacts with the peptide before diffusion into the bulk solution.     

Fibrinolytic components in nasal mucosa and nasal secretion

Effects of a newly synthesized antiulcer agent, YJA20379-4, on gastric proton pump (H+/K+-ATPase) activity, Helicobacter pylori (H. pylori) growth, gastric acid secretion, and gastro-duodenal lesions, were examined in comparison with those of omeprazole. YJA20379-4 markedly inhibited the H+/K+-ATPase activity in a concentration-dependent manner and the inhibitory effect was increased under a weak acidic condition; the IC50 values were 32 and 81 microM at pH 6.4 and 7.4, respectively. The inhibition was completely antagonized by 0.5 mM dithiothreitol (DTT). In addition, YJA20379-4 showed a significant anti-H. pylori activity determined by the agar dilution method. The value of minimum inhibitory concentration (MIC, 3.9-11.7 microg/ml) was at least 3 times more potent than that of omeprazole. In pylorus ligated rats, YJA20379-4 inhibited basal gastric acid secretion when administered by the intraduodenal route (ED50: 23.6 mg/kg). In experimental ulcer models, YJA20379-4 administered by the oral route dose-dependently prevented the development of gastro-duodenal lesions in rats. Moreover, repeated administration of YJA20379-4 promoted the healing of gastric ulcers induced by acetic acid. On the basis of the data obtained, it is suggested that YJA20379-4 has a wide spectrum of antiulcer activities, and its mode of antiulcer actions is dependent on the inhibition of H+/K+-ATPase activity and H. pylori growth and the enhancement of a mucosal defense. Thus, YJA20379-4 might prove to be a beneficial therapy for gastritis and peptic ulcer diseases.     

Alcohol tolerance in patients with non-insulin-dependent diabetes (type 2) treated orally with drugs--derivatives of sulphonylurea

The oral ethanol loading test (0.5 g per kg b.m. given as 40% solution) was carried out in 5 groups, each of 10 patients with non-insulin-dependent (type 2) diabetes before and after 10 days of treatment with one of the following sulphonylurea derivatives: tolbutamide 0.5 t.i.d., chlorpropamide 0.5 once daily morning, glibornuride 0.025 t.i.d, glibenclamide 0.005 t.i.d. and glipizide 0.005 t.i.d. The response to alcohol (facial flush, heart rate, blood pressure) were compared, and blood concentration of ethanol, acetaldehyde, pyruvate, lactate, carbonates as well as blood pH, pO2 and pCO2 were determined in fasting state and during 6 hours after alcohol ingestion. In all patients the family history of diabetes and the presence and degree of vascular complications were registered. Evident flushing phenomenon was observed in 6 patients treated with chlorpropamide, in 3 treated with tolbutamide, in 2 treated with glibenclamide, in one receiving glibornuride and in none treated with glipizide. All drugs caused a greater rise of blood ethanol and acetaldehyde levels in relation to the control tests, but the difference reached statistical significance only in the group receiving chlorpropamide. Moreover, patients (pooled) with positive thermographic response had also significantly higher blood levels of ethanol and acetaldehyde during the second test. The ratio of acetaldehyde to ethanol concentration in blood (mumol:mmol) was not significantly changed in any group indicating parallel impairment of both steps of ethanol metabolism. All studied drugs intensified to a similar degree the alcohol-induced hypoglycaemia, but had no significant effect on the decrease of blood pyruvate level neither on the increase of blood lactate level. They didn't change the post-alcohol decrease of blood bicarbonate and pH, and didn't modify the behaviour of partial gas pressure. There was also no difference between pooled groups of patients with positive and negative thermographic reaction with respect to family history of diabetes and frequency and intensity of vascular complications. It is concluded that in patients with non-insulin-dependent (type 2) diabetes the second generation sulphonylurea derivatives are associated with lower risk of alcohol intolerance in case of its incidental ingestion in small amounts. The hypothesis of association of positive thermographic reaction to alcohol during treatment with sulphonylurea derivatives with more frequent occurrence of diabetes in family members and lower tendency to vascular complications was not confirmed.     

Inhibition of melatonin secretion by ethanol in man

To determine whether ethanol inhibits nocturnal melatonin (MT) secretion, three experiments (A, B, and C) were performed in seven normal subjects. In A, ethanol at a dose of 0.34 g/kg was administered orally at 6:00, 8:00, and 10:00 PM. Each dose was increased to 0.52 g/kg in B. In C, water was substituted for ethanol. Blood samples for determination of serum MT levels were drawn every second hour between 6:00 PM and 8:00 AM. Urinary excretion of MT during the night was also determined. In A, serum ethanol reached a maximal level of 13 +/- 1 mmol/L at 12 midnight. In B, the corresponding maximum was 25 +/- 1 mmol/L. The higher alcohol dose inhibited nocturnal MT secretion by 20% +/- 5% (P < .01), whereas the lower dose lacked such effect. Urinary excretion of MT was left unaffected by alcohol at both doses. Five additional normal subjects were given alcohol as described above at a dose of 0.52 g/kg (experiment D). This induced mild nocturnal hypoglycemia as evidenced by a glucose decremental area (5.9 +/- 1.8 mmol/L.h) that differed significantly from zero (P < .05). To determine whether a reduced glucose delivery to pinealocytes might contribute to the decreased MT secretion in alcohol-intoxicated subjects, two experiments (E and F) were performed in eight healthy individuals. In E, ethanol was given orally as in B; three small oral doses of glucose were also given at 8:00 PM, 10:00 PM, and 12 midnight. In F, water was substituted for ethanol and glucose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Disparity between blood pressure and PRA inhibition after administration of a renin inhibitor to anesthetized dogs: methodological considerations

A dissociation between changes in blood pressure (BP) and plasma renin activity (PRA) has been noted after administration of renin inhibitors. In the present study, the renin inhibitor PD 132002 was given to salt-deplete, anesthetized dogs. PRA was measured at pH 6.0 by a conventional angiotensin I (ANG I) RIA method (PRA-C) and by an ANG I antibody-trapping RIA method (PRA-AT) performed at pH 7.4. PD 132002 at 0.01, 0.1, 1, and 10 mg/kg IV, reduced BP by 3 +/- 2, 9 +/- 2, 24 +/- 4, and 39 +/- 4 mm Hg, respectively, (baseline of 136 +/- 8 mm Hg, N = 5), when infused IV over 30 minutes with a 30 minute recovery between doses. The BP response at 10 mg/kg equaled that of saralasin (20 micrograms/kg/min IV). PRA-AT (baseline of 20 +/- 6 ng ANG l/ml/hr, N = 4) was inhibited by 0%, 28% +/- 12%, 75% +/- 10%, and 97% +/- 1% at 0.01, 0.1, 1, and 10 mg/kg, respectively. Plasma concentrations of immunoreactive ANG II were also reduced dose-dependently and paralleled changes in BP. In contrast, PRA-C (baseline of 13 +/- 4 ng ANG l/ml/hr, N = 4) was inhibited by 82% +/- 8% at 0.01 mg/kg and by > 98% at higher doses. After a single dose of PD 132002 at 10 mg/kg infused over 30 minutes, BP recovery paralleled changes in immunoreactive ANG II and PRA-AT, yet PRA-C inhibition showed no recovery over the same time course. Our data support the conclusion that BP relates better to PRA-AT than PRA-C. Thus the dissociation sometimes observed in studies with renin inhibitors between changes in BP and PRA may be attributed to the assay used to determine PRA.     

Effects of ethanol intake on retinol concentration in the milk of lactating rats

The effect of the consumption of ethanol (5%) on retinol concentration in milk was studied in the rat on day 12 after delivery, together with the evolution of dam body weight and pup growth rate. Female Wistar rats receiving alcohol (5%) in drinking water during lactation (N = 7) were compared to normal controls fed ad libitum (N = 6). The mean maternal alcohol intake was 3.96 +/- 0.23 g/kg body weight per day. To determine retinol levels in milk we used the Bessey and Lowry method, modified by Araújo and Flores ((1978) Clinical Chemistry, 24:386-392). The pups were separated from dams for a 2-4-h period, after which the dams were injected intraperitoneally with anesthetic and oxytocin. The concentration of retinol in milk was 162.88 +/- 10.60 micrograms/dl in the control group and 60.02 +/- 8.22 micrograms/dl in the ethanol group (P < 0.05). The ethanol group consumed less food than the controls and lost a significant amount of weight during lactation. On days 8, 10 and 12, the body weight of the pups from rats given ethanol (13.46 +/- 0.43, 16.12 +/- 0.48 and 18.60 +/- 0.91 g, respectively) were significantly lower (P < 0.05) than the weight of pups from controls (15.2 +/- 0.44, 18.36 +/- 0.54, 20.77 +/- 0.81 g). These data show that ethanol intake during the suckling period, even at low concentrations, decreases the amount of retinol in milk and, therefore, the amount available to the pups.     

Acetaldehyde-induced increase in paracellular permeability in Caco-2 cell monolayer

Evidence indicates that endotoxin-mediated liver injury plays an important role in the pathogenesis of alcoholic liver disease. Elevated plasma endotoxin level in alcoholics is suggested to be caused by enteric bacterial overgrowth and/or increased intestinal permeability to endotoxin. In this study, the effect of ethanol and acetaldehyde on the paracellular permeability was evaluated in Caco-2 cell monolayers. Ethanol was administered into the incubation medium, whereas acetaldehyde was administered by exposing cell monolayers to vapor phase acetaldehyde, or by direct administration of an acetaldehyde generating system (AGS), ethanol + NAD+ + alcohol dehydrogenase. Paracellular permeability was assessed by measuring transepithelial electrical resistance (TER), sodium chloride dilution potential, and unidirectional flux of D-[2-(3)H]mannitol. Administration of ethanol up to 900 mM produced no significant effect on paracellular permeability. Vapor phase acetaldehyde, generated from 5 to 167 mM acetaldehyde solutions in neighboring wells, resulted in a time- and dose-dependent increase in acetaldehyde concentration (99 to 760 microM) in the buffer bathing cell monolayer. Acetaldehyde induced a reduction of TER and dilution potential, and an elevation of mannitol flux in a time and concentration-related manner, without affecting the ability of cells to exclude trypan blue. Removal of acetaldehyde after 1, 2, or 4 hr treatment and subsequent incubation in the absence of acetaldehyde resulted in a time-dependent reversal of TER to baseline values. Administration of AGS also reduced TER and dilution potential, associated with an increase in mannitol flux. This effect of AGS was prevented by 4-methylpyrazole, an alcohol dehydrogenase inhibitor. These results show that acetaldehyde, but not ethanol, reversibly increases the paracellular permeability of Caco-2 cell monolayer.     

Studies of the oxidation of ethanol to acetaldehyde by oxyhemoglobin using fluorigenic high-performance liquid chromatography

We noted a rise in acetaldehyde levels in clinical samples of venous whole blood containing ethanol that did not occur in samples from teetotalers. Experiments were performed to define the mechanism involved in acetaldehyde production. The addition of 0.10% ethanol to whole blood produced an immediate increase in acetaldehyde due to acetaldehyde in the stock solution followed by a subsequent increase that became statistically significant by 48 hr. Separation of blood into components documented that the increase in acetaldehyde was associated with the red cell but not plasma fraction. Incubation of isolated hemoglobin with ethanol produced a rise in acetaldehyde levels. Incubation of oxygenated whole blood with ethanol produced a linear increase in acetaldehyde, whereas nitrogen-exposed blood produced no increase. The rise of acetaldehyde in the presence of ethanol was dependent on the concentration of oxygenated hemoglobin A0. Addition of inhibitors of catalase, alcohol dehydrogenase, and glycolytic enzymes (aminotriazole, azide, pyrazole, sodium fluoride, sodium citrate, and iodoacetate) did not inhibit the rise of acetaldehyde, but addition of the hemoglobin ligand cyanide abolished the rise in acetaldehyde. Kinetic analysis with oxygenated whole blood plus inhibitors revealed a Km of 2.5 mM and Vmax of 1.42 microM/min. We conclude that oxyhemoglobin contributes to the metabolism of ethanol to acetaldehyde. These findings may explain in part the high levels of acetaldehyde found in red cells compared with plasma. The results also have implications for the optimum storage of blood samples for acetaldehyde analysis.     

Concentration-time profiles of ethanol in arterial and venous blood and end-expired breath during and after intravenous infusion

Ethanol (0.40 g/kg) was administered to 13 healthy men by intravenous (i.v.) infusion at a constant rate for 30 min. The concentrations of ethanol in arterial blood (ABAC), venous blood (VBAC), and end-expired breath (BrAC) were measured at 17 exactly timed intervals. Blood-ethanol was determined by headspace gas chromatography and breath-ethanol was measured with a quantitative infrared analyzer (DataMaster). BrAC was multiplied by 2300 to estimate the concentrations of alcohol in blood. During the infusion of ethanol, ABAC exceeded VBAC by about 10 mg/dL on the average and ABAC was also higher than BrAC x 2300 by about 4 mg/dL on average. When infusion of alcohol ended, ABAC, VBAC, and BrAC were 94.8 +/- 2.06 (+/- SE), 84.7 +/- 1.54, and 89.3 +/- 2.10 mg/dL, respectively. The concentrations of alcohol in blood (ABAC and VBAC) and breath decreased abruptly after the administration of alcohol stopped and by 5 min postinfusion, the A-V differences in concentration of ethanol were small or negligible. The mean apparent half-life of the distribution plunge was 7 to 8 min, being about the same for ABAC, VBAC, and BrAC. The disappearance rate of ethanol was 15.5 +/- 0.55 mg/ dL/h (mean +/- SE) for arterial blood, 15.2 +/- 0.49 mg/dL/h for venous blood, and 16.3 +/- 0.73 mg/230 L/h for breath; no significant differences were noted (p > 0.05). We conclude that A-V differences in the concentration of ethanol exist during the loading phase but are rapidly abolished when the administration of ethanol terminates. In the post-absorptive phase of ethanol kinetics, when alcohol has mixed with the total body water, VBAC exceeds ABAC by about 1-2 mg/100 mL on average.     

Influence of pH on metabolism and urease activity of Helicobacter pylori

BACKGROUND & AIMS: The metabolic and urease responses of Helicobacter pylori to variations in gastric acidity are unknown. The aim of this study was to determine effects of changes of environmental pH on metabolism, urease activity, and survival of H. pylori in an unbuffered environment. METHODS: Bacterial metabolism and urease activity were determined by measuring pH changes in perfused microphysiometer chambers over a pH range from 2.5 to 9.0 with or without urea and survival by restoration of metabolism at pH 7.4. RESULTS: Glucose metabolism by acid-adapted H. pylori occurred at a perfusion pH between 3.5 and 8.6 and was highest between 7.4 and 8.2. Metabolism was irreversibly inhibited at pH <3.5 or >8.6. In the presence of 2.5 mmol/L urea, the chamber pH increased to about 6.2 during perfusion between pH 5.5 and 4.0. At pH 4.0 and below, urease activity increased several-fold without change of chamber pH. Urea in the perfusate enabled retention of metabolism after acid exposure but was toxic at pH 7.4. CONCLUSIONS: The metabolic range of acid-adapted H. pylori is between an environmental pH of 3.5 and 8.6. Extracellular pH-regulated internal urease activity allows metabolism in the pH range between 4.0 and 2. 5 by maintaining periplasmic pH at 6.2. The organism is an acid-tolerant neutralophile due to internal urease activity.     

Age-dependent eradication of Helicobacter pylori with dual therapy

BACKGROUND: Combined treatment using an acid-inhibiting drug with antibiotics can cure Helicobacter pylori infection. However, eradication rates are highly variable, especially if a proton pump inhibitor is used with amoxycillin. Therefore it is important to define factors/predictors of the clinical outcome. METHODS: In a single-blind study, 60 H. pylori-positive patients prospectively matched for diagnosis (erosive gastritis, duodenal and gastric ulcer), age (above and below 50 years) and smoking habits were randomly treated (each group n = 20) for 2 weeks with amoxycillin (1 mg b.d.) and either omeprazole (20 mg b.d.), lansoprazole (30 mg b.d.) or ranitidine (300 mg b.d.). Intragastric pH and plasma levels of the administered drugs were monitored over a dosing interval of 12 h. RESULTS: The overall eradication rates were 45% (intention-to-treat, ITT, 27/60) or 47% (per protocol 27/58); they did not differ (ITT) between omeprazole (50%), lansoprazole (40%) and ranitidine (45%). Median pH and time at which intragastric pH was above 4 was slightly lower for ranitidine (4.0 +/- 1.7; 51 +/- 25%) than for omeprazole (5.4 +/- 1.1: 77 +/- 25%; P < 0.05) or lansoprazole (4.4 +/- 1.6: 68 +/- 32%). Plasma concentrations of amoxycillin were comparable in all three treatment groups. Post-treatment H. pylori status was not dependent on those levels, or the drug-induced extent or duration of increased intragastric pH. However, H. pylori-eradicated patients were significantly (P < 0.05) older (56 +/- 13 years) than patients still H. pylori-positive (47 +/- 14 years). In addition, in patients older than 50 years (n = 33), eradication was higher (P < 0.01) than in patients (n = 25) below 50 years (65 vs. 24%). Eradication rate was highest (75-83%) in subgroups of patients (> 50 years and history of peptic ulcer or smokers). Neither activity/grade of peptic ulcer or erosive gastritis nor initial diagnosis were predictors for clinical outcome. CONCLUSION: The age of patients must be regarded as a major determinant of H. pylori eradication rate and may represent an important factor contributing to the highly variable clinical results.     

Osmotic water permeability of capillaries from the isolated spiral ligament: new in-vitro techniques for the study of vascular permeability and diameter

Perilymph is separated from blood by a barrier called the blood-labyrinth or blood-perilymph barrier in analogy to the blood-brain or blood-cerebrospinal fluid barrier. These barriers consist mainly of vascular endothelial cells. To characterize the blood-labyrinth barrier we developed in vitro techniques for the quantitative determination of the osmotic water permeability and for the determination of changes in the diameter of isolated inner ear capillaries. Both techniques rely on measurement of the velocity of marker red cells trapped in the lumen of capillaries. The velocity of marker red cells is a measure for the capillary permeability when a water flux across the capillary wall is induced by an osmotic gradient or a measure for a change in the capillary diameter. With these techniques the osmotic water permeability coefficient (Pf) and the pH sensitivity of isolated capillaries from the spiral ligament of the inner ear was determined. Pf at 23 degrees C was (1.49 +/- 0.17) 10(-3) cm/s at pH 7.4 and (1.61 +/- 0.23) 10(-3) cm/s at pH 6.8 (n = 12: mean +/- SEM: n = number of tissues). Pf at 37 degrees C was (2.26 +/- 0.23) 10(-3) cm/s at pH 7.4 and (2.35 +/- 0.17) 10(-3) cm/s at pH 6.8 (n = 13). No change in capillary diameter was observed when the pH of the interstitial fluid was lowered from pH 7.4 to 6.8. These data demonstrate that Pf and the capillary diameter of spiral ligament capillaries are pH independent and suggest that water crosses the blood-labyrinth barrier via an aqueous pathway. Further, these data suggest that the relatively low Pf is another characteristic shared by the blood-labyrinth and the blood-brain barrier.     

Inhibition of muscarinic receptor-stimulated glial cell proliferation by ethanol

Acetylcholine and other muscarinic agonists stimulate the proliferation of rat cortical astrocytes and 132 1N1 human astrocytoma cells by activating muscarinic m3 cholinergic receptors. Ethanol was a potent inhibitor of carbachol-stimulated proliferation, measured by [3H]thymidine incorporation, with an IC50 of 10 mM. On the other hand, basal and serum-stimulated proliferation of astrocytes and astrocytoma cells was inhibited by ethanol with lower potency (IC50 = 200-250 mM). Concentration-response experiments with carbachol, in the presence of 10 mM ethanol, suggested that inhibition of proliferation by the alcohol was of the noncompetitive type. Experiments with acetaldehyde and with the alcohol dehydrogenase inhibitor 4-methylpyrazole suggested that the inhibitory effect of alcohol was due to ethanol itself and not to its metabolite acetaldehyde. Proliferation of astrocytoma cells induced by carbachol and the inhibitory effects of ethanol were also confirmed by flow cytometry using the 5-bromodeoxyuridine-Hoechst 33258 method. Ethanol (10 mM) had no effect on proliferation induced by 50 micrograms/ml insulin and 100 ng/ml platelet-derived growth factor BB; on the other hand, the mitogenic effect of 1 mM histamine, 100 U/ml interleukin-1, and 100 ng/ml 12-O-tetradecanoylphorbol 13-acetate were inhibited by approximately 50%. These results indicate that proliferation of glial cells induced by muscarinic agonists is especially sensitive to the inhibitory effect of ethanol. This action of ethanol may be relevant to its developmental neurotoxicity, particularly microencephaly, which is one of the common features of the fetal alcohol syndrome.     

Phosphatidylethanol in blood as a marker of ethanol consumption in healthy volunteers: comparison with other markers

Phosphatidylethanol is a "pathological" phospholipid, formed via the action of phospholipase D only in the presence of ethanol. The present study was made to elucidate how different levels and patterns of alcohol intake affect blood levels of phosphatidylethanol in comparison with other markers of abuse. We used a new HPLC-evaporative light-scattering detection technique for phosphatidylethanol quantitation. This method had a total coefficient of variation of <20% at the detection limit of 0.2 nmol, equaling 0.8 micromol/liter of whole blood. Two groups were studied. (a) Five healthy volunteers were given 32 to 47 g of ethanol in a single dose, to give blood ethanol levels of approximately 25 mmol/liter after 30 to 60 min. Phosphatidylethanol, carbohydrate-deficient transferrin (CDT), and blood ethanol were measured before and after the intake. (b) Twelve student volunteers were studied during a 3 week period of prolonged alcohol consumption (total estimated intake: 1334 +/- 488 g, mean +/- SD) and phosphatidylethanol, serum-CDT, gamma-glutamyltransferase, and blood ethanol were measured at the start of the period (day 1) and twice at the end of the period (days 18 and 21). In group (a), no phosphatidylethanol was detected at any time after ethanol dosage/intake. In group (b), no blood phosphatidylethanol or blood ethanol could be demonstrated at the start, and serum-CDT was below the discrimination limit (1.3%) in all persons. No phosphatidylethanol was detected in those four persons with the lowest intake (742 +/- 150 g). However, the remaining eight persons had detectable levels of phosphatidylethanol (1.0 to 2.1 micromol/liter), and these had a higher total intake (1630 +/- 389 g). There was a statistically significant (p = 0.02) increase in serum CDT for 3 weeks. However, only 3 of 12 persons increased above the discrimination limit. The present results indicate that a substantial alcohol intake is needed to elevate blood phosphatidylethanol. In comparison with serum-CDT, blood phosphatidylethanol appears more sensitive.     

Prognostic factors and rational approach in the treatment of submucosal cancer of the stomach

Metallothionein (MT) mRNA levels were analyzed following exposure of neonatal rat primary astrocyte cultures to physiologic pH (7.4), acidosis (pH 6.5 and 6.0), and dimethyl sulfoxide (DMSO). Treatments were carried out both in the presence and absence of the bioflavonoid, quercetin. Total RNA was probed on northern blots with [alpha32P]dCTP-labeled synthetic cDNA probes specific for rat MT isoform mRNAs. MT-I and MT-II mRNA levels in astrocytes exposed to pH 6.5 or pH 6.0 were increased compared to controls (pH 7.4). Treatment with DMSO in the presence and absence of acidosis, also increased MT-I and MT-II mRNA levels compared to controls (pH 7.4). The DMSO-induced increase in MT mRNA expression was reversed by treatment of astrocytes with quercetin, such that MT-I and MT-II mRNA levels in DMSO plus quercetin-treated astrocytes were indistinguishable from mRNA levels in their respective controls at pH 7.4, pH 6.5, and pH 6.0. These findings suggest that both acidosis and DMSO exposure are associated with increased astrocytic MT synthesis at the mRNA level, and that quercetin, effectively blocks MT mRNA induction by DMSO.