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Biomedical subjects

A Helander

Publications and source records attributed to A Helander.

At least 73 records · Page 4Linked to original sources
PubMed1993

Inhibition of aldehyde dehydrogenases by methylene blue.

The effect of the redox dye methylene blue on the stability of NADH and on the activity of the enzyme aldehyde dehydrogenase (ALDH; EC 1.2.1.3) was examined. NADH was measured by HPLC with fluorometric or spectrophotometric detection. The ALDH activity assays were carried out by following the formation of 3,4-dihydroxyphenylacetic acid (DOPAC) from 3,4-dihydroxyphenylacetaldehyde (DOPAL) using HPLC and electrochemical detection. Incubation of NADH solutions in the presence of methylene blue resulted in a time-dependent direct oxidation of NADH. Methylene blue inhibited the human erythrocyte and leukocyte ALDHs and the rat liver mitochondrial low-Km ALDH in a concentration-dependent manner. The inactivation was reversible by dilution, and kinetic analysis indicated that methylene blue inhibits the rat liver mitochondrial low-Km and human erythrocyte ALDHs competitively with respect to DOPAL, while no effect of the NAD+ concentration was apparent. For the rat liver low-Km ALDH, a Ki of 8.4 +/- 2.8 microM (mean +/- SD; N = 5) was calculated. The inhibition of ALDH and the resulting decrease in the redox effect on the NAD system bound to alcohol dehydrogenase (EC 1.1.1.1) could explain the protective effect of methylene blue against metabolic redox effects of ethanol.

Aldehyde Dehydrogenase↗
PubMed1993

Time course of ethanol-induced changes in serotonin metabolism.

The effect of acute ethanol consumption on serotonin metabolism was examined in healthy volunteers in the fasted and fed state by determination of plasma and urinary levels of the serotonin metabolites 5-hydroxyindole-3-acetic acid (5-HIAA) and 5-hydroxytryptophol (5-HTOL). The plasma and urinary levels of 5-HIAA were reduced by about 40% and 25%, while the 5-HTOL levels were increased on an average 7-fold and 50-fold, respectively, after oral intake of ethanol (0.8 g/kg) over 30 min in a fasted state. The maximal effect on both 5-HIAA and 5-HTOL levels was found 4-6 h after starting drinking. Urinary 5-HTOL and the 5-HTOL/5-HIAA ratio did not return to baseline until 19 h after the start of the administration (i.e., about 10 h after ethanol reached zero level). The mean 24-h excretion of 5-HTOL was increased 15-fold by the ethanol intake, while the 5-HIAA excretion was not significantly different. A clear dose dependent effect was observed in one individual who also ingested a lower amount of ethanol (0.5 g/kg). When ethanol (0.8 g/kg) was ingested over 3 h together with food, the urinary level of 5-HTOL and the 5-HTOL/5-HIAA ratio did not return to baseline until after 20-22 h. In other subjects who had unlimited access to ethanol and ingested between 1.3-2.3 g/kg together with food, the time to reach baseline 5-HTOL/5-HIAA ratio in urine ranged from 20 h to over 26 h.

Adult↗
PubMed1993

Distribution of acetaldehyde in human blood: effects of ethanol and treatment with disulfiram.

The distribution of free and bound acetaldehyde in human blood was studied. Fresh whole blood was precipitated with a perchloric acid (PCA) in saline solution and an aliquot of the crude sample was taken for determination of 'total' acetaldehyde. The remaining sample was centrifuged and the clear supernatant taken for analysis of 'soluble' acetaldehyde. 'Bound' acetaldehyde was calculated by subtracting soluble from total amounts. In samples collected from healthy control subjects, the acetaldehyde level in separated plasma was usually below the limit of detection of the method (0.2 microM), while much higher concentrations (> 2.5 microM) were detected when analyses were carried out on whole blood. In whole blood, about 70% was recovered as bound (i.e. PCA-insoluble) acetaldehyde. The soluble (i.e. free + PCA-soluble) level was higher than that found in separated plasma, suggesting that some acetaldehyde was liberated from the blood cells by PCA treatment. In blood spiked with ethanol, a spontaneous formation of acetaldehyde occurred during the analytical procedure. The artefactual formation increased only the soluble amount, while the bound level remained unchanged. Likewise, in samples drawn from intoxicated subjects, artefactual formation of acetaldehyde was observed in the soluble fraction, while the bound amount was not significantly increased. No significant differences in acetaldehyde levels were found between males and females, nor between healthy control subjects and alcoholic patients undergoing treatment with the aldehyde dehydrogenase inhibitor disulfiram (Antabuse). However, some of the Antabuse patients possessed elevated levels of bound acetaldehyde.

Acetaldehyde↗
PubMed1992

Mechanisms for plasma-mediated activation of human blood cell aldehyde dehydrogenase.

Aldehyde dehydrogenase (ALDH; EC 1.2.1.3) activity assays were carried out on isolated human blood cells in phosphate-buffered saline (PBS) and in PBS mixed with human plasma. In assays with intact erythrocytes or sonicated leukocytes, the presence of 50% (v/v) or greater of plasma in the reaction mixtures produced a 2-fold increase in the rate of aldehyde oxidation. In corresponding assays with sonicated erythrocyte samples, the ALDH activity was enhanced on an average 1.5-fold, whereas a slight decrease was observed in assays with intact leukocytes. The ALDH inhibitor disulfiram almost completely abolished the enzyme activity both in the absence and presence of plasma. In assays with sonicated leukocytes, the activation effect could be antagonized by EDTA, indicating that it was caused largely by divalent cations. With sonicated erythrocytes, a significantly reduced ALDH activity was found only with the highest concentration of EDTA tested, and since a similar reduction was obtained also when plasma was omitted, the plasma-mediated activation of erythrocyte ALDH was suggested to be due to a different mechanism. After separation of plasma by gel filtration, an active fraction was identified by GC-MS and 1H-NMR to contain pyruvic acid, lactic acid and glucose. When tested at physiological plasma concentrations, pyruvic acid caused an increase in erythrocyte ALDH activity similar to that obtained with plasma, while lactic acid and glucose did not. Pyruvic acid did not activate the leukocyte ALDH. Based on these results, it is indicated that the plasma-mediated activation of erythrocyte ALDH is due to pyruvic acid, which reoxidizes NADH via lactate dehydrogenase (EC 1.1.1.27) and, thereby, increases the rate of dissociation of NADH from the terminal enzyme-NADH complex, the rate-limiting step in the ALDH pathway.

Aldehyde Dehydrogenase↗
PubMed1992

Determination of urinary 5-hydroxytryptophol by high-performance liquid chromatography with electrochemical detection.

A high-performance liquid chromatographic method for the routine determination of elevated urinary levels of the serotonin metabolite 5-hydroxytryptophol (5-HTOL) is described. Urine samples were treated with beta-glucuronidase, and 5-HTOL was isolated by solid-phase extraction on a small Sephadex G-10 column prior to injection onto an isocratically eluted C18 reversed-phase column. Detection of 5-HTOL was performed electrochemically at +0.60 V vs. Ag/AgCl. The limit of detection was ca. 0.05 microM, and the intra-assay coefficients of variation were below 6% with urine samples containing 0.2 and 2.1 microM 5-HTOL and a standard solution of 2.0 microM (n = 5). The recovery of 5-HTOL after the sample clean-up procedure was close to 100%. A good correlation (r2 = 0.97; n = 12) was obtained between the present method and a sensitive and specific gas chromatographic-mass spectrometric method. The total (free plus conjugated) 5-HTOL levels in urine were normally below 0.2 microM, but after an acute dose of alcohol they increased to 0.5-15 microM.

Chromatography, High Pressure Liquid↗
PubMed1992

Synthesis and conformational and NMR studies of alpha-D-mannopyranosyl and alpha-D-mannopyranosyl-(1----2)-alpha-D-mannopyranosyl linked to L-serine and L-threonine.

alpha-D-Mannopyranosyl and alpha-D-mannopyranosyl-(1----2)-alpha-D- mannopyranosyl linked to L-serine and L-threonine have been synthesised as model substances for the linkage region in certain O-linked glycoproteins. Metropolis Monte Carlo simulations were performed with a modified version of the GESA program, to yield theoretical NOEs and interatomic distances as ensemble-average values, and these were compared with results from steady-state NOE experiments. The NOEs were determined as ensemble-average and as global minimum values. NMR chemical shift differences, obtained for signals of the glycopeptides relative to those of the respective monomers, were interpreted in terms of short inter-residue atomic distances as found within the global minima, and on the basis of averaged distances derived from Monte Carlo simulations.

Carbohydrate Conformation↗
PubMed1992

Urinary excretion of 5-hydroxyindole-3-acetic acid and 5-hydroxytryptophol after oral loading with serotonin.

The urinary excretion patterns of the serotonin (5-hydroxytryptamine; 5-HT) metabolites 5-hydroxyindole-3-acetic acid (5-HIAA) and 5-hydroxytryptophol (5-HTOL) were examined after ingestion of bananas, a food rich in 5-HT. The bananas contained on an average 25 micrograms 5-HT/g pulp. Both urinary 5-HIAA and 5-HTOL increased markedly (15- to 30-fold) shortly after eating 3-4 bananas, with the highest concentrations found in urine specimens collected after 2-4 h, and did not return to normal until after 8-10 h. The excretion of 5-HIAA increased from a control mean value of 3.9 mg/24 h to 12.7 mg/24 h, when conventional diets were supplemented with 3-4 bananas. The corresponding results for 5-HTOL were 16.8 micrograms/24 h and 60.7 micrograms/24 h, respectively. Of the banana-derived 5-HT ingested, 60-80% was recovered in the urine as 5-HIAA and only 0.3-0.5% as 5-HTOL. However, since both the time-course and relative increase in 5-HTOL was similar to that of 5-HIAA, there was no effect on the urinary 5-HTOL to 5-HIAA ratio. By contrast, acute alcohol consumption produced a considerable elevation of this ratio.

Administration, Oral↗
PubMed1991

Determination of urinary 5-hydroxyindole-3-acetic acid by high-performance liquid chromatography with electrochemical detection and direct sample injection.

A method for the routine quantitative determination of the major serotonin metabolite 5-hydroxyindole-3-acetic acid (5-HIAA) in urine is described. 5-HIAA was analyzed without prior sample cleanup, using an automated high-performance liquid chromatography system with isocratic elution and electrochemical detection (+0.60 V versus a Ag/AgCl reference electrode). The urine samples were mixed with a solution of the internal standard (5-hydroxyindole-3-propionic acid) and centrifuged. The supernatant was transferred to sealed glass vials, and a 2-microliters aliquot was injected directly onto a C18 reversed-phase analytical column, using an automatic sample injector. Samples of urine could be stored for several months at -80 or at +7 degrees C for 2 days without loss of 5-HIAA. However, a gradual decline with time occurred in crude samples stored at room temperature or above, as well as in urine samples diluted with the mobile phase. The detector response was linear in the range of 0-65 mumol/l 5-HIAA, and the intra- and interassay coefficients of variation were about 5 and 7%, respectively (n = 10).

Chromatography, High Pressure Liquid↗
PubMed1991

Inhibition of human blood aldehyde dehydrogenase activity by cigarette-smoke condensate.

The effect of a cigarette-smoke condensate (CSC) and three CSC subfractions on the aldehyde dehydrogenase (ALDH; EC 1.2.1.3) activity in human blood cells was examined under physiological conditions in vitro. Incubation of intact or sonicated cells with different concentrations of crude CSC resulted in a dose-dependent reduction of the ALDH activity. The inactivation was only restored in part after extensive washing of the cells, indicating that the inhibition observed was mainly irreversible. The nonvolatile (NV) subfraction of the CSC caused a reduction in ALDH activity similar to that obtained with crude CSC, while the semivolatile (SV80) and volatile (SV20) subfractions did not significantly affect ALDH. The present results, showing that the human blood cell ALDH is inactivated by constituents of cigarette smoke in vitro, suggest that the blood ALDH activity reduction found in habitual smokers is also caused by components formed during the combustion of tobacco.

Aldehyde Dehydrogenase↗
PubMed1991

Increased frequency of acetaldehyde-induced sister-chromatid exchanges in human lymphocytes treated with an aldehyde dehydrogenase inhibitor.

Acetaldehyde, the first metabolite of ethanol oxidation, in concentrations ranging from 100 microM to 400 microM caused a dose-dependent linear increase in the frequency of sister-chromatid exchanges (SCE) in cultured human peripheral lymphocytes. The SCE frequency was on an average 2-fold higher when the cells were exposed to the acetaldehyde after 24 h incubation instead of at the time of mitogen stimulation (0 h). When acetaldehyde was added together with the potent aldehyde dehydrogenase inhibitor 1-aminocyclopropanol (0.1 mM), the SCE response was significantly (p less than 0.05) increased. The present results indicate that acetaldehyde is metabolized within human lymphocytes, and, moreover, that alcohol consumption during treatment with drugs that inactivate aldehyde dehydrogenase may cause a further increased incidence of acetaldehyde-induced SCE and concomitant lesions.

Acetaldehyde↗
PubMed1991

Comparison of blood aldehyde dehydrogenase activities in moist snuff users, cigarette smokers and nontobacco users.

The aldehyde dehydrogenase (ALDH; EC 1.2.1.3) activity was determined in samples of whole blood and in isolated erythrocytes and leukocytes from users of Swedish moist snuff, cigarette smokers and non-tobacco-using controls. The mean whole blood ALDH activity of the smokers was reduced by 21% (p less than 0.001) when compared to the controls, while that of the snuff users was reduced by only 8% (not significant). Similar but somewhat less pronounced differences were obtained both in the assays with erythrocytes and leukocytes. In the cigarette smokers, the whole blood activity correlated significantly (r = -0.79, p less than 0.001) with the plasma concentration of cotinine, the major metabolite of nicotine, whereas no correlation was observed for the users of moist snuff. Similar plasma nicotine and cotinine levels were found in smokers and snuff users, which indicates that the reduced blood ALDH activity in smokers is not caused by nicotine or any of its metabolites, but more likely, by components formed during combustion of tobacco. Since a reduced blood ALDH activity has previously been suggested as an indicator of excessive alcohol consumption, the present results show that, in future studies on blood ALDH, the smoking habits should also be taken into account.

Adult↗
PubMed1990

Use of leukocyte aldehyde dehydrogenase activity to monitor inhibitory effect of disulfiram treatment.

Aldehyde dehydrogenase (ALDH; EC 1.2.1.3) activity was determined in leukocytes and erythrocytes from alcoholic patients during different stages of disulfiram (Antabuse) treatment. Assays were performed by incubating intact isolated blood cells in phosphate-buffered saline (pH 7.4, 37 degrees C), using 3,4-dihydroxyphenylacetaldehyde (DOPAL), the biogenic aldehyde derived from dopamine, as substrate. The ALDH activity was assessed from the amount of 3,4-dihydroxyphenylacetic acid (DOPAC) formed, as analysed by high-performance liquid chromatography with electrochemical detection. The leukocyte ALDH, which is similar to the liver "mitochondrial" low-Km ALDH isozyme, was maximally inhibited (about 40-60%) within 2 to 3 days after the initial disulfiram administration (dosage 200 or 400 mg/day orally). The time to reach maximum inhibition (about 95%) of the erythrocyte ALDH, which closely resembles the liver "cytosolic" high-Km isozyme, varied from 3 to more than 6 days. When medication was completed, the leukocyte ALDH activity remained unaltered for the first 2 days, and did not revert to normal levels until about 6 to 7 days after terminating treatment. The erythrocyte ALDH was still inhibited by about 90% 1 week after the last disulfiram administration. These results suggest that the leukocyte ALDH activity might provide an easily accessible marker for monitoring effect and time course of ALDH inhibition during disulfiram treatment.

Acetaldehyde↗
PubMed1989

Isolation and characterization of a glycosylated form of human insulin-like growth factor I produced in Saccharomyces cerevisiae.

Expression and secretion of human insulin-like growth factor-I (IGF-I) in Saccharomyces cerevisiae was achieved by linking an actin (ACT) promoter to an MF alpha 1 prepro leader peptide/IGF-I gene fusion. Purified human IGF-I from yeast culture media was found to contain, in addition to the native form, also a glycosylated variant. Structural studies showed that both IGF-I forms were processed identically, resulting in 70-amino-acid long polypeptides, with intact N-terminal and C-terminal residues of glycine and alanine, respectively. The glycosylation site was determined to threonine-29 (Thr29), by 1H NMR spectroscopy and protein sequence analysis of an isolated tryptic peptide(22-36). No other glycosylation sites were found. Only mannose was detected in the sugar analysis, with an estimated content of 4.5% w/w corresponding to 2 mannose residues per molecule of IGF-I. The carbohydrate structure, determined by 1H and 13C NMR spectroscopy, was found to be alpha-D-Manp(1----2)alpha-D-Manp(1----3)Thr corresponding to an O-linked glycoprotein structure. No other post-translational modifications could be identified in the glycosylated IGF-I form. Furthermore, this form was highly active, comparable to native IGF-I, exhibiting a specific activity of 20,500 units/mg, as determined by a radio-receptor assay.

Actins↗
PubMed1989

Inhibition of human erythrocyte and leukocyte aldehyde dehydrogenase activities by diethylthiocarbamic acid methyl ester. An in vivo metabolite of disulfiram.

The inhibitory effects of diethylthiocarbamic acid methyl ester (DTC-Me), an in vivo metabolite of disulfiram (Antabuse), on the aldehyde dehydrogenase (ALDH; EC 1.2.1.3) activities in human erythrocytes and leukocytes were studied. ALDH assays were performed by incubating intact isolated blood cells in the presence of different concentrations of DTC-Me, using 3,4-dihydroxy-phenylacetaldehyde, the aldehyde derived from dopamine, as the substrate. DTC-Me was more selective as inhibitor of the leukocyte ALDH activity (which resembles the liver "mitochondrial" low Km ALDH), whereas both disulfiram and diethyldithiocarbamic acid, the reduced monomer of disulfiram, were more selective for the erythrocyte ALDH (which is similar to the "cytosolic" high-Km ALDH). Diethylthiocarbamic acid, the free acid of DTC-Me, was less potent than DTC-Me, and caused similar inactivation of the erythrocyte and leukocyte ALDH activities. The inhibition of ALDH by DTC-Me could not be completely restored by extensive dilution of intact or sonicated blood cell samples, which indicated that ALDH was irreversibly inhibited. Since the inhibition patterns with DTC-Me agrees with the previously reported patterns of inhibition of the high-Km and low-Km isozymes after the administration of disulfiram, the results suggest that DTC-Me might be the active in vivo inhibitory metabolite of disulfiram.

Aldehyde Dehydrogenase↗
PubMed1989

Aldehyde dehydrogenase activity in blood from various vertebrates.

1. Aldehyde dehydrogenase activity was determined in whole blood samples from 17 selected vertebrates of 5 classes, using 3,4-dihydroxyphenylacetaldehyde (the aldehyde derived from dopamine) as substrate. 2. Aldehyde dehydrogenase activity in blood was widely but unevenly distributed among the species studied. 3. Mean aldehyde dehydrogenase activities in the range of 40-140 nmol/min.ml blood (measured at 37 degrees C, pH 8.8) were found in blood from man, monkey, rabbit, guinea pig and mouse (C57BL and NMRI strains), with the highest activity in rabbit blood. 4. Much lower aldehyde dehydrogenase activities (0.5-7.5 nmol/min.ml blood) were found in blood from Sprague-Dawley and Wistar rat, dog, cat, horse, pig, chicken, caiman, frog and rainbow trout, whereas the activities in blood from DBA mouse, cow, sheep and crucian carp were close to the detection limit.

Aldehyde Dehydrogenase↗