Inhibition of ethanol production by Saccharomyces cerevisiae in human blood by sodium fluoride

Production of ethanol in antemortem blood samples inoculated with an efficient ethanol-producing microorganism and incubated at various temperatures is discussed. Whole blood samples inoculated with Saccharomyces cerevisiae were incubated in gray stoppered Venoject tubes (approximate draw volume 7 mL) containing sodium fluoride (17.5 mg) and potassium oxalate (14.0 mg) at 4 degrees C, 25 degrees C, and 37 degrees C for 0, 24, 96, 192, and 408 h. No volatile substances (such as ethanol, methanol, isopropanol, acetone, or acetaldehyde) (< 0.010 g/dL) were produced in any of the samples at 4 or 25 degrees C. At 24 h incubation a trace amount (< 0.018 g/dL) of ethanol was detected at 37 degrees C.     

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.     

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.     

Influence of pentobarbital sodium anesthesia on hematologic values in the dog

The effects of intravenously administered commercial pentobarbital sodium, pentobarbital sodium in saline solution, 40% propylene glycol in saline solution, 10% ethanol in saline solution, and saline solution on erythrocyte fragility and blood coagulation variables were studied in the dog. The pentobarbital solution and the 40% propylene glycol caused erythrocyte lysis and obvious hemoglobin release into the plasma. They also caused a shortening of the whole blood clotting time and partial thromboplastin time tests, evidence of increased procoagulant activity involving the intrinsic coagulation system. Alterations in these variables were not noticed after pentobarbital sodium in saline solution, 10% ethanol in saline solution, and saline solution injections. Changes were not noticed in the extrinsic coagulation system or in platelet function.     

Fetal hemoglobin levels in sudden infant death syndrome

OBJECTIVE: The aim of this study was to determine and compare fetal hemoglobin levels from infants dying of the sudden infant death syndrome (SIDS) with aged-matched control infants dying of other causes. Similar previous studies have reported both elevated and normal levels of fetal hemoglobin in whole blood samples from infants dying of SIDS. DESIGN: Triton-acid-urea gel electrophoresis and densitometry were used to determine fetal hemoglobin levels in postmortem whole blood samples from infants dying of SIDS and from appropriately age-matched control infants. Whole blood samples were analyzed blindly and matched for postgestational age. Infant ages at death ranged from birth to less than 1 year. MAIN OUTCOME MEASURES: Fetal hemoglobin in whole blood from infants dying of SIDS and control infants. RESULTS: During the period of postnatal development most associated with SIDS cases (2 to 6 months after birth), fetal hemoglobin levels were found to be significantly elevated in postmortem whole blood samples from SIDS infants compared with gestational age-matched control infants dying of causes other than SIDS. CONCLUSION: We conclude that levels of fetal hemoglobin are elevated in postmortem whole blood of SIDS infants compared with controls. Furthermore, the apparent conflict in the literature regarding fetal hemoglobin levels in SIDS infants and controls is most likely due to variability in the control data of some studies.     

Effects of methylprednisolone on the physical properties of the human red cell

Prompted by evidence suggesting preserved red cell deformability in cardiac surgical patients pretreated with pharmacologic dosages of methylprednisolone, we performed in vitro experiments to examine the ability of similar levels of methylprednisolone and hydrocortisone to modify erythrocyte membrane changes produced by metabolic depletion or membrane-active compounds. Variables measured included cell morphology, blood biscosity, membrane deformability, osmotic fragility, red cell cholesterol, and glycolytic intermediates. In incubated samples, methylprednisolone partially prevented the transition of discs to echinocytes, the rise in whole blood viscosity, the decrease in membrane deformability, and the loss of red cell cholesterol which accompany ATP depletion, but it had no apparent effect on red cell glycolysis. The drug also inhibited esterification of cholesterol in cell-free serum. In unimcubated samples to which lysolecithin was added, methylprednisolone partially prevented and reversed morphologic and rheologic responses without affecting membrane cholesterol. Hydrocortisone demonstrated similar properties. Possible mechanisms for these actions are discussed. The concept is advanced that preserved blood fluidity may contribute to the beneficial responses to these drugs in certain clinical conditions.     

Aldehyde dehydrogenases of the rat colon: comparison with other tissues of the alimentary tract and the liver

Intracolonic bacteria have previously been shown to produce substantial amounts of acetaldehyde during ethanol oxidation, and it has been suggested that this acetaldehyde might be associated with alcohol-related colonic disorders, as well as other alcohol-induced organ injuries. The capacity of colonic mucosa to remove this bacterial acetaldehyde by aldehyde dehydrogenase (ALDH) is, however, poorly known. We therefore measured ALDH activities and determined ALDH isoenzyme profiles from different subcellular fractions of rat colonic mucosa. For comparison, hepatic, gastric, and small intestinal samples were studied similarly. Alcohol dehydrogenase (ADH) activities were also measured from all of these tissues. Rat colonic mucosa was found to possess detectable amounts of ALDH activity with both micromolar and millimolar acetaldehyde concentrations and in all subcellular fractions. The ALDH activities of colonic mucosa were, however, generally low when compared with the liver and stomach, and they also tended to be lower than in small intestine. Mitochondrial low K(m) ALDH2 and cytosolic ALDH with low K(m) for acetaldehyde were expressed in the colonic mucosa, whereas some cytosolic high K(m) isoenzymes found in the small intestine and stomach were not detectable in colonic samples. Cytosolic ADH activity corresponded well to ALDH activity in different tissues: in colonic mucosa, it was approximately 6 times lower than in the liver and about one-half of gastric ADH activity. ALDH activity of the colonic mucosa should, thus, be sufficient for the removal of acetaldehyde produced by colonic mucosal ADH during ethanol oxidation. It may, however, be insufficient for the removal of the acetaldehyde produced by intracolonic bacteria. This may lead to the accumulation of acetaldehyde in the colon and colonic mucosa after ingestion of ethanol that might, at least after chronic heavy alcohol consumption, contribute to the development of alcohol-related colonic morbidity, diarrhea, and cancer.     

Biochemical markers of alcohol use and abuse: experiences from the Pilot Study of the WHO/ISBRA Collaborative Project on state and trait markers of alcohol. International Society for Biomedical Research on Alcoholism

The effects of acute ethanol and acetaldehyde treatment on cell proliferation, cell adhesion capacity, neutral red incorporation into lysosomes, glutathione content, protein sulfhydryl compounds, lipid peroxidation, inner mitochondrial membrane integrity (MTT test), lactate dehydrogenase activity (LDH) and ultrastructural alterations were investigated in a human fetal hepatic cell line (WRL-68 cells). WRL-68 cells were used, due to the fact that, although this cell line expresses some hepatic characteristics, it does not express alcohol dehydrogenase or cytochrome P450 activity, so it could be a good model to study the effect of the toxic agents per se. Cells were exposed during 120 min with 200 mM ethanol or 10 mM acetaldehyde. Under these conditions, cells presented 100% viability and no morphological alteration was observed by light microscopy. Acetaldehyde-treated cells reduced their proliferative capacity drastically while the ethanol-treated ones presented no difference with control cells. Cell adhesion to substrate, measured as time required to adhere to the substrate and time required to detach from the substrate, was diminished in acetaldehyde WRL-68-treated cells. Cytotoxicity measures as neutral red and MTT test showed that acetaldehyde-treated cells presented more damage than ethanol-treated ones. Cellular respiratory capacity was compromised by acetaldehyde treatment due to 40% less oxygen consumption than control cells. Lipid peroxidation values, measured as malondialdehyde production, were higher in ethanol-treated WRL-68 cells (127%) than in acetaldehyde-treated ones (60%) to control cell values. Lactate dehydrogenase activity (LDH) in extracellular media of ethanol-treated cells presented the highest values. GSH content was reduced 95% and thiol protein content was diminished severely in acetaldehyde-treated cells. Transmission electron microscopy showed more ultrastructural alterations in cells treated with acetaldehyde. The results indicate that acetaldehyde, like ethanol, produced damage at cellular level, although more damage could be observed in acetaldehyde WRL-68-treated cells.     

A human whole blood assay for clinical evaluation of biochemical efficacy of cyclooxygenase inhibitors

In this study, PGE2 levels in lipopolysaccharide (LPS)-challenged human whole blood and TxB2 levels following blood coagulation were measured as biochemical index for cyclooxygenase (Cox)-2 and Cox-1 activity respectively. Incubation of human mononuclear cells isolated from whole blood with LPS (100 mu g/mL) induced a time-dependent increase in the expression of Cox-2 protein (>100 fold at 24 hr). This is associated with increases in PGE2 production and free arachidonate release in the plasma. Cox-1 protein was detected in the human mononuclear cells at time zero but was not induced by either LPS or PBS. Most non-steroidal anti-inflammatory drugs (NSAIDs) are more potent at inhibiting Cox-1 than Cox-2. Five experimental compounds CGP-28238, Dup-697, NS-398, SC-58125 and L-745,337, have a greater selectivity for Cox-2. Indomethacin at a single oral dose (25 mg) inhibited approximately 90% the whole blood Cox-2 and Cox-1 activities ex vivo in healthy subjects. These results support the use of this assay to assess the biochemical efficacy of selective Cox-2 inhibitors in clinical trials.     

Stimulation of newborn-like hemoglobin synthesis in adult rats by recombinant human erythropoietin and suppression by aspirin

A study was carried out to determine whether recombinant human erythropoietin can induce newborn-like hemoglobin synthesis in adult rats. A fixed dose of recombinant erythropoietin was administered each time intravenously in each rat for altogether 5 weeks. Blood samples drawn at 7-day intervals were analyzed by DEAE-cellulose chromatography. Hematological parameters like red blood cell counts, hematocrit values and reticulocyte counts were evaluated and compared. A significant changing pattern for certain hemoglobin components in red cells of erythropoietin-treated rats was measured compared to their baseline values. However, aspirin (a prostaglandin synthesis inhibitor) intake along with recombinant erythropoietin administration totally abolished the reversion of hemoglobin proportions toward newborn values, but not the increase in hemoglobin synthesis. These data reveal that concurrent prostaglandin synthesis is needed for reversing hemoglobin proportions in adult rats, but not for hemoglobin synthesis per se.     

Prevalence of ethanol consumption may be higher in women than men in a university health service population as determined by a biochemical marker: whole blood-associated acetaldehyde above the 99th percentile for teetotalers

To estimate ethanol consumption by university students attending a student health facility, a biochemical marker of alcohol intake [whole blood associated acetaldehyde (WBAA)] was quantified by fluorimetric HPLC. Over a two year period we studied blood samples, coded by date and sex, from 645 females and 332 males, and compared the results to previously established reference ranges for teetotalers by sex. Men had higher absolute values for WBAA than women (9.9 versus 9.5 microM in the present study). However, significantly greater numbers of women (74%) than men (44%) had WBAA levels above the 99th percentile for teetotalers. Variations occurred during the academic year, with significant elevations occurring in the late fall and winter months. Testing of WBAA levels in a student health service may be important especially for women to facilitate counseling on the dangers of alcohol abuse.     

Oxygen consumption by hepatic tissue after transfusion of a stroma-free haemoglobin solution

The ability of stroma-free haemoglobin solutions to transport oxygen was investigated by determining the oxygen consumption by the liver using Warburg's microrespirator. 50 ml of blood were removed from the rabbit under general anaesthesia and replaced by an identical volume of a non-oxygenated or oxygenated haemoglobin solution. The control rabbits received no transfusions. It was found that arterial hypotension produced by blood-letting caused a significant rise in oxygen consumption by the hepatic tissue. The rise was increased even more when the rabbits received no transfusions. Transfusion of non-oxygenated haemoglobin solutions caused likewise a rise in oxygen consumption immediately after transfusion. This rise was, however, not so significant as in the control group. On the other hand, transfusion of oxygenated haemoglobin solutions produced no rise in the oxygen consumption by the liver as compared to its value after blood-letting.     

Effect of chronic anemia on the endothelium-dependent dilatation of the brachial artery

The effect of changes in main determinants of whole blood viscosity after red blood cell transfusion on endethel dependent dilatation of brachial artery was studied in patients treated with transfusion because of symptoms of chronic anaemia. PATIENTS AND METHODS: 10 patients were involved, 8 females 2 males, mean age 50.9 +/- 16.6 years. Following blood tests were performed at hospital admission: hemoglobin, red blood cell count, heamatocrit, white blood cell count, platelet count, plasma total protein, fibrinogen, plasma viscosity, blood urea nitrogen, creatinine, cholesterol, triglicerides. Flow mediated dilatation of brachial artery was determined, too. Blood tests and flow mediated dilatation study were repeated after transfusion. RESULTS: The main determinants of whole blood viscosity increased after transfusion. The increase of hemoglobin, red blood cell count, hematocrit were highly significant. The central flow velocity in brachial artery decreased at rest and during hyperemia as well. The flow mediated dilatation of brachial artery wasn't significantly changed by transfusion. CONCLUSIONS: Change of determinants of whole blood viscosity caused by transfusion didn't change the flow mediated dilatation of brachial artery. The probable reason for this that the increase of whole blood viscosity in associated with the decrease of central flow velocity. These two counteracting changes probably equal each other.     

Volume and dose parameters for survival of non-small cell lung cancer patients

The study was performed on 4 groups of male Wistar rats, receiving p.o. through 3 months every day: 1) distilled. water (control group); 2) sodium nitrite in dose 30 mg/kg b.w. x day (20% LD50); 3) lead acetate in dose 10 mg/kg b.w. x day (6.7% LD50); 4) lead acetate and sodium nitrite in amounts as above. The methemoglobin and hemoglobin were determined in whole blood, tryptophan--in plasma and free sulfhydryl groups--in erythrocytes. There was shown methemoglobin creative effects by nitrite (4.17%) and lead (3.02%) after 90-days intoxication. Both nitrite and lead significantly decrease free sulfhydryl groups and tryptophan levels in blood. There was also observed that lead administrated together with sodium nitrite does not increase methemoglobin concentration.     

In vitro pharmacological properties of KRH-594, a novel angiotensin II type 1 receptor antagonist

Although most people are thought to receive their highest acute exposures to gasoline while refueling, relatively little is actually known about personal, nonoccupational exposures to gasoline during refueling activities. This study was designed to measure exposures associated with the use of an oxygenated fuel under cold conditions in Fairbanks, Alaska. We compared concentrations of gasoline components in the blood and in the personal breathing zone (PBZ) of people who pumped regular unleaded gasoline (referred to as regular gasoline) with concentrations in the blood of those who pumped an oxygenated fuel that was 10% ethanol (E-10). A subset of participants in a wintertime engine performance study provided blood samples before and after pumping gasoline (30 using regular gasoline and 30 using E-10). The biological and environmental samples were analyzed for selected aromatic volatile organic compounds (VOCs) found in gasoline (benzene, ethylbenzene, toluene, m-/p-xylene, and o-xylene); the biological samples were also analyzed for three chemicals not found in gasoline (1,4-dichlorobenzene, chloroform, and styrene). People in our study had significantly higher levels of gasoline components in their blood after pumping gasoline than they had before pumping gasoline. The changes in VOC levels in blood were similar whether the individuals pumped regular gasoline or the E-10 blend. The analysis of PBZ samples indicated that there were also measurable levels of gasoline components in the air during refueling. The VOC levels in PBZ air were similar for the two groups. In this study, we demonstrate that people are briefly exposed to low (ppm and sub-ppm) levels of known carcinogens and other potentially toxic compounds while pumping gasoline, regardless of the type of gasoline used.     

Expression and loss of the transferrin receptor in growing and differentiating HD3 cells

During induced differentiation and maturation of HD3 cells (a chicken erythroblast cell line infected with a temperature-sensitive mutant of the avian erythroblastosis virus), the levels of transferrin receptor (TFR) and nucleoside transporter increase. Both these activities increase before elevated levels of hemoglobin are detected. Shortly after induction, as cellular TFR levels rise, a native-size TFR is detected in the cell-free culture medium, associated with an exosome fraction (100,000 xg pellet). Nucleoside transporter (measured as NBMPR-binding activity) is not increased in this pellet with induction. Previous studies have suggested that exosome formation in peripheral reticulocytes may be a significant route for loss of specific membrane proteins (Johnstone et al., 1991). Although the present experiments in HD3 cells do not address the quantitative importance of exosome formation, the studies suggest that exosome formation is an early event in commitment to the red cell lineage and is not a phenomenon restricted to the terminal stages of red cell maturation.     

Osteogenesis imperfecta: the distinction from child abuse and the recognition of a variant form

Inactivation of glutathione peroxidase correlates with the rate of hemoglobin chain oxidation. The enzyme inactivation is mainly present in those conditions where the autoxidation of the oxygenated chains is followed by transformation of the oxidized molecule into a hemichrome. Free hemoglobin chains have been encapsulated in human red blood cells by a dialysis technique that involves transient hypotonic hemolysis followed by isotonic resealing. Chain-loaded erythrocytes represent a good in vitro model of thalassemia. The presence of free human chains in the cell alters the intraerythrocytic glutathione peroxidase activity (alpha chains are more effective in the inactivation of the enzyme with respect to the beta chains).     

Longitudinal sliding of the median nerve during movements of the upper limb

Repeated transfusion of small increments of blood are frequently required for the sick and premature newborn infant to correct endogenous hypovolemia and/or to replace blood obtained for laboratory monitoring purposes. Previously fresh group and type specific whole blood was used. To eliminate waste of fresh whole blood, maintain fresh red blood cell properties, eliminate the hazards of transfusing plasma and to provide a more efficient system, a pediatric frozen red cell pack (PFRCP) has been developed. Units of group O rr red blood cells are glycerolized using a high glycerol method. The glycerolized red blood cells are separated into three equal aliquots and frozen. When needed, the PFRCP are deglycerolized by a modified procedure using the IBM Cell Processor. During a six month period, 71 infants were given 153 separate transfusions of deglycerolized red blood cells using 102 PFRCP prepared from 34 units of red blood cells. Red blood cell recovery, hematocrit, white blood cell removal, residual glycerol, total protein, and supernatant hemoglobin levels were measured. Clinical response was followed and found to be excellent.     

Effect of sodium ortho-iodobenzoate on oxygen transport and erythropoiesis in hypoxemic dogs with a right-to-left cardiac shunt

Eight dogs were made hypoxemic by surgical construction of a right-to-left cardiac shunt; and they were given sodium ortho-iodobenzoate (OISB) before and for 3 months after operation. The P(50) at 50% saturation) rose from 27.2 +/- 0.7 to 31.2 +/- 0.6 mm Hg (p less than 0.001) during OLSB treatment before operation and increased further to 32.2 +/- 0.8 mm Hg 3 months after creation of hypoxemia. The P(50) remained elevated for an additional 3 months after OISB was stopped. Administration of OISB before operation did not alter the red blood cell 2,3-diphosphoglycreate concentration. Hypoxemia caused an increase of this metabolite from 0.91 +/- 0.21 to 1.50 +/- 0.28 moles/moles of hemoglobin (p less than 0.05); the rise was not as great as that observed in hypoxemic dogs without OISB treatment. In spite of significant hypoxemia, hematocrit rose only slightly during the period of OISB infusion. OISB increased P50 and prevented the compensatory polycythemia regularly seen when dogs are made hypoxemic. Altering oxygen transport in this fashion may increase tissue oxygen delivery in patients with conditions which result in tissue hypoxia.     

Inflammatory cytokine production in whole blood modified by liposome-encapsulated hemoglobin

The effect of Neo Red Cells (NRC), liposome-encapsulated hemoglobin, on production of tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) were studied in whole blood preparations ex vivo. Venous blood was collected with heparin and incubated in a CO2 incubator. Treatment of blood samples with NRC reduced the constitutive levels of TNF-alpha and IL-6. Lipopolysaccharide (LPS) treatment for 24 h increased production of TNF-alpha and IL-6 in a dose-dependent manner. Pretreatment with NRC (5%) for 24 h markedly potentiated the LPS-induced TNF-alpha production and, that of IL-6 to a lesser extent. Northern blotting analysis of total RNA in whole blood showed that pretreatment with NRC caused a marked increase in TNF-alpha mRNA expression in response to LPS. It is concluded that NRC potentiates LPS-induced TNF-alpha and IL-6 production in whole blood ex vivo, and that the potentiating effect of NRC on LPS-induced TNF-alpha production can be attributed, at least in part, to an increase in its mRNA expression.