Volatile emissions of plant tissue cultures

Summary The low-molecular-weight volatiles released by a variety of plant tissue cultures were examined by gas chromatography. Callus cultures invariably produced carbon dioxide, ethylene, acetaldehyde and ethanol. In cultures with developed shoots, ethanol was absent and acetaldehyde was detected only rarely.     

Analysis of reactive carbonyls in the expired air of transgenic mice.

Methods for the determination of trace levels of volatile carbonyl compounds in air expired from mice were developed and validated. Tumor bearing transgenic mice or nontransgenic control mice were placed into a glass chamber through which air was passed continuously at 90 ml/min for 1 h. The effluent gas stream was bubbled into an aqueous cysteamine solution or an aqueous methylhydrazine solution. Formaldehyde, acetaldehyde, and acetone in expired air were derivatized to thiazolidine with cysteamine and malonaldehyde was derivatized to 1-methyl-2-pyrazole with methylhydrazine. The derivatized compounds were analyzed by capillary gas chromatography with flame photometric or nitrogen-phosphorous-specific detection. The lowest level quantitated was 4 micrograms/ml thiazolidine, equivalent to 1.35 micrograms/ml formaldehyde. Formaldehyde was recovered at a level of 1356 +/- 234 nmol/kg0.75 (mean +/- SD) from mice with tumors and 898 +/- 97 nmol/kg0.75 from mice without tumors, suggesting that tumor bearing transgenic mice expired significantly more formaldehyde than did tumor free controls. Amounts of expired acetaldehyde and acetone were not different among mice. Malonaldehyde was not detected in either group of mice.     

Determination of acetaldehyde in biological samples by gas chromatography with electron-capture detection

A simple specific assay was developed for the determination of acetaldehyde in biological samples. Acetaldehyde was derivatized to 2,4-dinitrophenylhydrazone, which was determined by gas chromatography with electron-capture detection. The use of this detection method is an important device to which no one drew notice. This procedure was very simple and so sensitive that as little as 500 fmol of acetaldehyde could be measured in aqueous solution. The calibration curve of acetaldehyde was linear at least up to 40 μM. Its recoveries from human plasma and rat liver homogenate were 96.5 and 95.7%, respectively.     

Transport and intracellular accumulation of acetaldehyde in saccharomyces cerevisiae

The rate of acetaldehyde efflux from yeast cells and its intracellular concentration were studied in the light of recent suggestions that acetaldehyde inhibition may be an important factor in yeast ethanol fermentations. When the medium surrounding cells containing ethanol and acetaldehyde was suddenly diluted, the rate of efflux of acetaldehyde was slow relative to the rate of ethanol efflux, suggesting that acetaldehyde, unlike ethanol, may accumulate intracellularly. Intracellular acetaldehyde concentrations were measured during high cell density fermentations, using direct injection gas chromatography to avoid the need to concentrate or disrupt the cells. Intracellular acetaldehyde concentrations substantially exceeded the extracellular concentrations throughout fermentation and were generally much higher than the acetaldehyde concentrations normally recorded in the culture broth in ethanol fermentations. The technique used was sensitive to the time taken to cool and freeze the samples. Measured intracellular acetaldehyde concentrations fell rapidly as the time taken to freeze the suspensions was extended beyond 2 s. The results add weight to recent claims that acetaldehyde toxicity is responsible for some of the effects previously ascribed to ethanol in alcohol fermentations, especially Zymomonas fermentations. Further work is required to confirm the importance of acetaldehyde toxicity under other culture conditions. (c) 1993 John Wiley & Sons, Inc.     

Harman in human platelets.

The β-carbolines present in human platelets have been extracted with diethyl ether, isolated by liquid column chromatography and thin-layer chromatography (TLC), and identified by ultraviolet fluorimetry, gas liquid chromatography (GC) and mass spectrometry (MS). Harman (1-methyl-β-carboline) was the only β-carboline unequivocally identified in platelet samples with these techniques. Since harman is thought to be biosynthesized by the condensation of tryptamine and acetaldehyde, its formation may be of importance in the metabolism and the pharmacological-toxicological actions of alcohol.     

Acetaldehyde-stimulated PKC activity in airway epithelial cells treated with smoke extract from normal and smokeless cigarettes

Previously, we have found that acetaldehyde, a volatile component of cigarette smoke, stimulates the protein kinase C (PKC) pathway and inhibits ciliary motility. A "smokeless" cigarette (Eclipse) now exists in which most of the tobacco is not burned, reducing the pyrolyzed components in the extract. We hypothesized that acetaldehyde is a component of cigarette smoke that activates PKC in the airway epithelial cell, and therefore the Eclipse cigarette would not activate epithelial cell PKC. In this study, bovine bronchial epithelial cells (BBEC) were incubated with cigarette smoke extract (CSE) or Eclipse smoke extract (ESE). We found that PKC activity was significantly higher in cells exposed to 5% CSE than cells exposed to 5% ESE or media. When acetaldehyde levels of both extracts were measured by gas chromatography, CSE was found to have 15-20 times greater concentration (microM) of acetaldehyde than ESE. When BBEC were treated with 5% CSE, ciliary beating was further decreased from baseline levels. This decrease in ciliary beating was not observed in cells treated with ESE, suggesting that acetaldehyde contained in CSE slows cilia. These results suggest that volatile components such as acetaldehyde in cigarette smoke may inhibit ciliary motility via a PKC-dependent mechanism.     

Decomposition of BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) in aqueous solution

BCNU, labelled with ¹⁴C in the chloroethyl groups, decomposes in neutral aqueous solution to release half of its radioactivity as volatile products. These have been identified by a combination of gas chromatography and mass spectrometry as vinyl chloride, acetaldehyde, dichloroethane, and chloroethanol. This set of products is consistent with the existence of chloroethylcarbonium ions as reactive intermediates which would produce the previously described substituted nucleotides.     

CARBONYL COMPOUNDS PRODUCED BY CERATOCYSTIS FAGACEARUM

Ceratocystis fagacearum produces both in nature and in laboratory culture a characteristic fruity aroma. It is assumed that the odor may serve as an attractant for insects which are vectors for the disease as well as important agents in the fertilization mechanism. A number of carbonyl compounds were identified from liquid cultures of C. fagacearum and included: acetaldehyde, furfuraldehyde, acetone, 2-hexenal and 2-methylbutanal. The 2,4-dinitrophenylhydrazones of the compounds were identified by means of thin-layer chromatography, melting-point determination, gas chromatography and infrared spectroscopy.     

Pyrolysis of Cellulose

Comparative study of acetaldehyde, furfural and 5-hydroxymethyl furfural from celluloses which differed in crystallinity was made by pyrolytic gas chromatography.

Pyrolysis of tobacco cellulose at 200~300°C resulted in rapid increase in the yields of furfurals from the amorphous regions in comparison with that from the crystalline regions. At 500°C, however, acetaldehyde was obtained in higher yields from microcrystalline cellulose than that from tobacco cellulose under the same condition.

In thermogravimetric analysis, the threshold temperature for the pyrolysis of tobacco cellulose was lower than that of microcrystylline cellulose. These results showed that the yields of the volatile compounds from pyrolysis of cellulose depended on temperature and crystallinity.     

Structure of Xanthocidin

A burnt flavoring compound, which imparts to aged sake its characteristic and dominant flavor, was isolated by Diaion HP–20, silicic acid and Dowex 1–X8 (CH₃COO^?) column chromatography and chloroform extraction. Based on thin-layer and gas liquid chromatography, UV and GC–MS spectral data, it was identified as 3-hydroxy-4,5-dimethyl-2 (5H)-furanone and its structure was also confirmed by synthesis. It was suggested that this compound was formed by the condensation of a-ketobutyric acid with acetaldehyde which occurred from degradation of threonine.     

Specific determination of threonine in biological samples by gas chromatography with electron capture detection

Threonine was oxidized into acetaldehyde at 0 degrees C for 30 min with periodic acid. The acetaldehyde formed was converted to a hydrazone with 2,4-dinitrophenyhydrazine. The hydrazone was extracted with n-heptane and quantified by gas liquid chromatography with electron capture detection. An internal standard, 2-amino-3-hydroxyhexanoic acid, was used. The calibration curve of threonine was linear up to 200 nmol in 200 microl sample solution and the determination limit of threonine was 1 nmol in 200 microl sample solution. The recoveries were 100.0, 94.0 and 100.0% from homogenates of octopus tentacles and blood plasma and rat livers, respectively. This method was applied to the determination of threonine in tissues of rats given threonine and starved octopuses. This threonine determination method has been used for studies on the metabolism of d-lactate.     

Monitoring of volatiles in alcoholic fermentations on molasses via a gas membrane sensor

Summary Gas membrane sensors, connected to a gas chromatography, have been tested in fermentation on complex media of industrial interest. Selectivity, sensitivity and reliability have been assessed: the calibration on fermentation medium is recommended. The use of such a sensor is demonstrated in the study of the kinetics of batch and fed-batch alcoholic fermentations on beet molasses. The kinetics of acetaldehyde are especially pointed out in relation to ethanol, fusel alcohols and carbon dioxide production.Offprint requests to: M.-N. Pons     

Bioelectronic sniffers for ethanol and acetaldehyde in breath air after drinking

Two kinds of bioelectronic gas sensors (bio-sniffer) incorporating alcohol oxidase (AOD) and aldehyde dehydrogenase (ALDH) were developed for the convenient analysis of ethanol and acetaldehyde in expired gas, respectively. The sniffer devices for gaseous ethanol and acetaldehyde were constructed by immobilizing enzyme on electrodes covered with filter paper and hydrophilic PTFE membrane, respectively. The AOD and ALDH sniffers were used in the gas phase to measure ethanol vapor from 1.0 to 500 ppm, and acetaldehyde from 0.11 to 10 ppm covering the concentration range encountered in breath after alcohol consumption. Both bio-sniffers displayed good gas selectivity which was attributed to the substrate specificity of the relevant enzymes (AOD and ALDH) as gas recognition material. From the results of physiological application, the bio-sniffers could monitor the concentration changes in breath ethanol and acetaldehyde after drinking. The ethanol and acetaldehyde concentrations in expired air from ALDH2 [-] (aldehyde dehydrogenase type 2 negative) subjects were higher than that of the ALDH2 [+] (positive) subjects. The results indicated that the lower activity of ALDH2 induced an adverse effect on ethanol metabolism, leading to ethanol and acetaldehyde remaining in the human body, even human expired air.     

Floral scents of Bartsia alpina (Scrophulariaceae): chemical composition and variation between individual plants

Volatile compounds were collected from the inflorescences of individual Bartsia alpina plants in sub-alpine and alpine populations, and analysed by gas chromatography and mass spectrometry. The compounds detected were: α-pinene, Z-3-hexenol, Z-3-hexenyl acetate, phenyl acetaldehyde and phenyl acetonitrile. A significantly larger proportion of Z-3-hexenol and Z-3-hexenyl acetate were collected from the sub-alpine population than from the alpine population but the differences between the amounts of the other compounds were slight.     

Reaction of acetaldehyde with hemoglobin

Acetaldehyde reacted with hemoglobin at neutral pH and 37 degrees C to form adducts that were stable to dialysis and that were not reduced by sodium borohydride. Hemoglobin tetramers having 2, 3, and probably 4 molar eq of bound aldehyde were isolated by cation exchange chromatography. The sites of attachment of the aldehyde were the free amino groups of the N-terminal valine residues of the alpha and beta chains of hemoglobin. Derivatization of the beta chains caused a greater increase in the acidity of the hemoglobin than did derivatization of the alpha chains. Derivatization of the beta chains was also preferred over that of the alpha chains. Acetaldehyde derivatives of the N-terminal octapeptide of hemoglobin S (beta sT-1 peptide), Val-Gly-Gly, and tetraglycine were formed readily, contained 1 M eq of acetaldehyde/mol of peptide, and were not reduced by sodium borohydride. In contrast, Ala-Pro-Gly failed to form a 1:1 adduct with acetaldehyde. 13C NMR analysis of the peptide adducts formed with [1,2-13C]acetaldehyde indicated that tetrahedral diastereomeric derivatives were produced. The 13C chemical shifts of the adducts formed between hemoglobin and [1,2-13C]acetaldehyde were identical to those of the peptide adducts although resonances from the individual diastereomeric adducts at each hemoglobin site could not be resolved. The results cited above as well as other evidence indicate that acetaldehyde reacts with the amino termini of hemoglobin to form stable cyclic imidazolidinone derivatives. An exchange of acetaldehyde residues between peptides was also documented.     

Course of transformation of benzaldehyde bySaccharomyces cerevisiae

The course of fermentation of benzaldehyde bySaccharomyces cerevisiae simultaneously fermenting a sugar substrate was studied by a polarographic method and gas chromatography. In single-dose experiments, 0.2% benzaldehyde was fermented. It was fermented within 90–120 minutes, giving rise to 23.1 to 39.2% phenylacetyl-carbinol and 40.5 to 24% benzyl alcohol. The addition of acetaldehyde to the fermentation medium retarded the transformation of benzaldehyde, lowered the formation of benzyl alcohol and raised the yield of phenyl-acetylcarbinol. In four-dose experiments the utilization of the individual doses of benzaldehyde for the formation of phenyl-acetylcarbinol is described.     

Chromatographic methods for blood alcohol determination

This review is focused on the different chromatographic strategies for blood alcohol determination which can be adopted for clinical and/or forensic purposes. Particular attention is paid to gas chromatography and to high-performance liquid chromatography. However, other analytical techniques in common use, such as chemical and enzymic methods, are also briefly presented, together with some, at present unusual or experimental, approaches, such as enzymic reactors and catalytic electrodes, which are suitable for application in column liquid chromatography. Finally, mention is made of the methods for the determination of acetaldehyde, the major ethanol metabolite, and of some proposed markers of chronic alcohol abuse, such as acetaldehyde—protein adducts and carbohydrate-deficient transferrin. In order to give the background of knowledge for the rational choice of an analytical strategy, an updated outline of ethanol metabolism and toxicology is presented, together with basic information for the interpretation of the results. Problems concerning blood sampling and storage are also discussed.     

Alcohol and acetaldehyde metabolism in Caucasians,Chinese and Amerinds.

Ethanol (0.4 to 0.8 g/kg in 30 minutes) was given by mouth to 102 healthy young volunteers (37 Caucasian men, 21 Caucasian women, 20 Chinese men and 24 Ojibwa men). Venous blood concentrations of ethanol and acetaldehyde 60, 90, 120 and 150 minutes after the end of drinking were measured by gas chromatography. The calculated rates of ethanol metabolism in the Caucasian men and women did not differ, but the overall group means for subgroups of Caucasians (103.6 mg/kg-h), Chinese (136.6 mg/kg-h) and Ojibwa (182.7 mg/kg-h) with decreasing postabsorption values differed significantly from each other. Mean acetaldehyde values paralleled the rates of ethanol metabolism: Ojibwa, 14.6 mug/ml; Chinese, 10.0 mug/ml; and Caucasians, 9.4 mug/ml. The high rate of ethanol metabolism in Amerind subjects differs from previous findings. Habitual level of alcohol consumption, proportion of body fat and genetic factors appear to account for most of the group differences.     

Covalent binding of acetaldehyde to tubulin: evidence for preferential binding to the alpha-chain

The covalent binding of [14C]acetaldehyde to purified beef brain tubulin was characterized. As we have found for several other proteins, tubulin bound acetaldehyde to form both stable and unstable adducts. Unstable adducts (Schiff bases) were stabilized, and rendered detectable, by treating incubated reaction mixtures with the reducing agent sodium borohydride. In short-term incubations, the majority of the adducts formed were unstable, but the percentage of total adducts that were stable gradually increased with time. Stable adduct formation was greatly increased by the inclusion of sodium cyanoborohydride in reaction mixtures (reductive ethylation). When reaction mixtures were submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis to separate the alpha- and beta-chains of the heterodimeric tubulin molecule, the alpha-chain of free tubulin, but not intact microtubules, was the preferential site of stable adduct formation under both reductive and nonreductive conditions. Denaturation studies showed that the native tubulin conformation was necessary for the alpha-chain to show enhanced reactivity toward acetaldehyde. Competition binding studies showed that alpha-tubulin could effectively compete with beta-tubulin and bovine serum albumin for a limited amount of acetaldehyde. Unstable acetaldehyde adducts with free tubulin or microtubules did not exhibit alpha-chain selectivity. Analysis of reaction mixtures indicates that lysine residues are the major group of the protein participating in adduct formation. These data indicate that the alpha-chain of free tubulin is the preferential site of stable acetaldehyde-tubulin adduct formation. Further, these data raise the possibility that alpha-tubulin may be a selective target for acetaldehyde adduct formation in cellular systems.