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Assessment of ethanol intake. Current tests and new assays on the horizon.

The number of tests used for the detection of ethanol ingestion is increasing. The field is rapidly moving beyond ethanol alone as a marker of ethanol intake. The combined measurement of carbohydrate deficient transferrin, FAEEs, 5-HTOL/5-HIAA, acetaldehyde adducts, and phosphatidylethanol may one day be used to approximate the time and amount of ethanol ingestion. The ultimate configuration of a panel of tests for monitoring ethanol intake awaits the results of studies that identify the clinical usefulness of each marker.

Acetaldehyde↗

Storage of specimens at 4 degrees C or addition of sodium fluoride (1%) prevents formation of ethanol in urine inoculated with Candida albicans.

The microbial synthesis of ethanol was investigated in urine specimens containing 0.5% or 1.0% (w/v) glucose and inoculated with the yeast Candida albicans (100 cfu/mL). Aliquots (10 mL) of urine were dispensed into plastic tubes containing enough sodium fluoride to give final concentrations of 0.1%, 0.25%, 0.5%, 0.75%, 1%, and 2% (w/v), and C. albicans was added. The tubes were tightly stoppered and allowed to stand either at room temperature (22 degrees C) or in a refrigerator (4 degrees C) for up to 34 days before concentrations of ethanol were determined by headspace gas chromatography. Urine samples stored at 22 degrees C without sodium fluoride produced 0.25 g/L ethanol after two days, and the concentration increased to 2.10 g/L and 4.50 g/L after eight days for specimens containing 0.5% (w/v) and 1% (w/v) glucose, respectively. The ratio of the serotonin metabolites 5-hydroxytryptophol/5-hydroxyindoleacetic acid (5HTOL/5HIAA) in urine remained within the reference range (< 15 pmol/nmol) despite high concentrations of ethanol being produced. Urine samples kept at 4 degrees C did not produce any ethanol (< 0.01 g/L) even without sodium fluoride present as a preservative. The production of ethanol by C. albicans was stopped completely by adding 1% or 2% (w/v) sodium fluoride but not by concentrations of 0.75% (w/v) or less. The microbial synthesis of ethanol in urine samples initially stored at room temperature without sodium fluoride was slowed down considerably by moving them into a refrigerator at 4 degrees C. In conclusion, the production of ethanol in urine by C. albicans can be prevented by storage of samples in a refrigerator at 4 degrees C or by adding sodium fluoride > or = 1% (w/v). Measuring the ratio of 5HTOL/5HIAA can help to distinguish postsampling production of ethanol from metabolism and excretion processes.

Candida albicans↗

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 development of reliable diagnostic tools for assessing alcoholism and harmful alcohol consumption is an utmost necessity for the success of efforts to prevent and treat alcohol-induced damage to both individuals and to society. A multinational study is underway to aid in the development of biological screening tools (state markers) which can, with good sensitivity and specificity, identify problem drinkers. To attain this goal information needs to be available on an individuals's drinking history and habits and related factors. A detailed instrument has been developed to obtain this information. The second goal of the study was to begin to develop diagnostic 'trait markers' which provide biological information on genetically determined predisposing and protective factors in the development of alcoholism. The developed questionnaire also provides background information on subject characteristics necessary for the development of trait markers. Centres will assay the obtained biological samples for 'traditional' and newly identified state markers of excessive alcohol consumption. These will include methanol measurements, gamma-glutamyltransferase, aspartate aminotransferase, carbohydrate-deficient transferrin, serotonin metabolite ratios, and erythrocyte aldehyde dehydrogenase. DNA obtained from the lymphocytes of subjects will be assayed for polymorphisms of alcohol- and aldehyde-metabolizing enzymes and dopamine receptor polymorphisms which can provide insights into protective and predisposing factors in alcoholism. The platelet enzymes, monoamine oxidase and adenylyl cyclase, will be assayed to assess the relationships between these putative trait markers and the genetic and environmental factors contributing to the aetiology of alcoholism. The current report is meant to introduce the study design and present a portion of the preliminary data gathered in the process of establishing this research programme.

Adolescent↗

Total 5-hydroxyindoles in blood related to migraine attacks.

The total 5-hydroxyindoles (5HI) in whole blood were measured in 20 migraine patients during spontaneous migraine attacks and in headache-free periods. A statistically significant fall in blood 5-HI was found during headache in 17 patients suffering from classical and common migraine. In one patient with complicated migraine no change was found, and in two patients, one with common migraine and one with migraine and associated symptoms, there was a rise in total blood 5-HI during migraine attacks. The results are compared with previous findings in this field, and it is suggested that during migraine attacks there might be a rise in the plasma 5-HI. The possibility of using the 5-HI fall during spontaneous migraine attacks as a simple test for the diagnosis of migraine is discussed.

Adult↗

Effect of selective monoamine oxidase inhibitors on rat pineal melatonin synthesis in vitro.

Freshly cultured pineal glands respond to the monoamine oxidase A (MAO A) inhibitor clorgyline and to high concentrations of the MAO B inhibitor, deprenyl, with an increase in serotonin N-acetyltransferase activity and N-acetylated indoles and a fall in 5-hydroxylated serotonin degradation products. Glands cultured for 48 hours before challenge respond less. Response is absent in glands cultured for 72 hours and in glands from ganglionectomized animals cultured for 48 hours before challenge. These data are consistent with the hypothesis that these MAO inhibitors stimulate melatonin synthesis by protecting norepinephrine from degradation.

Animals↗

Pineal ultrastructure and indole profiles spanning the summer rise in arginine vasotocin immunoactivity.

A correlative radioimmunological-biochemical-ultrastructural study of the rat pineal gland was undertaken during the summer months when pineal arginine vasotocin (AVT) immunoactivity increases up to 200-fold. RIA confirmed a rapid rise in AVT activity during mid-August regardless of the time of day sampled. Pineal indoles were separated by HPLC and measured using electrochemical detection. Serotonin (5-HT) and 5-hydroxyindoleacetic acid levels were consistently elevated in daytime samples, and there was a significant trend for increased day and nighttime levels of 5-HT from July to September. Mid-dark levels of melatonin also exhibited a significant increase over the sample period. Nighttime levels of N-acetylserotonin mirrored fluctuations in 5-HT in the preceding photoperiod. Ultrastructural components implicated in peptide/protein and/or indole biosynthesis were quantified by stereological morphometry. The greatest amounts of rough endoplasmic reticulum stacks, lipid droplets, and annulate lamellae-like bodies coincided with peak AVT activity. Dense-cored vesicles and synaptic ribbons were consistently more frequent during the dark period. The number of dense-cored vesicles and nucleolar size tended to be greatest before and after the peak in AVT immunoactivity. These observations are consistent with the hypotheses that endoplasmic reticulum and lipid are functionally related to the synthesis and/or storage of peptide/protein factors and that numerical changes in synaptic ribbons and dense-cored vesicles are more closely related to day/night differences in indole metabolism.

5-Hydroxytryptophan↗

Hydroxyindole-O-methyltransferase catalyses production of methoxyindoles in rat pineal gland dependent on the concentration of hydroxy precursors and their affinity for the enzyme.

Indole metabolites were separated by thin-layer chromatography following organ culture of rat pineal glands with tritiated tryptophan. Methoxyindole production was shown to differ substantially from results obtained when pineal glands were incubated with radiolabelled serotonin. The correlation between hydroxy- and corresponding methoxyindoles was, however, remarkably similar to previous results, and indicated that in the pineal gland, it is probable that production of methoxyindoles is dependent on the concentration of the various hydroxyindoles and their relative affinities for hydroxyindole-O-methyltransferase. It is also probable that although several forms of the enzyme may exist in the pineal gland, the catalytic sites are homogeneous in their activity.

5-Hydroxytryptophan↗

[5-HTOL--a new biochemical alcohol marker with forensic applications].

The concentration of ethanol in blood and urine provides important evidence in criminal and civil litigation when alcohol-related crimes are investigated (e.g., drunk driving). The determination of ethanol in body fluids is a routine procedure at forensic chemistry and toxicology laboratories and when gas chromatographic methods are used accurate and precise results are obtained. However, the risk for artifactual formation of ethanol, especially in postmortem specimens, always needs to be considered. The ratio of 5-HTOL/5-HIAA in urine provides a useful way to distinguish between ethanol produced after death, or generated in vitro after sampling, from the ethanol consumed. This article describes the application of the 5-HTOL/5-HIAA ratio as a biochemical marker for acute alcohol intake in various forensic situations. Examples include suspected drunk drivers, rape victims, and medico-legal autopsies where forensic ethanol analysis is requested.

Accidents, Traffic↗

Specific antagonism of enteric neural serotonin receptors by dipeptides of 5-hydroxytryptophan: evidence that serotonin is a mediator of slow synaptic excitation in the myenteric plexus.

Research on the role of serotonin (5-hydroxytryptamine, 5-HT) in the function of the enteric nervous system has been impeded by the lack of specific inhibitors of the enteric neural actions of 5-HT. Saturable, reversible, high affinity enteric binding sites for 3H-5-HT have recently been characterized and radioautographically located. Affinity for the 3H-5-HT binding site requires an indole ring substituted with a free hydroxyl group. These 3H-5-HT binding sites have been proposed to be enteric neural 5-HT receptors. This hypothesis was tested in the current study by comparing the ability of compounds to inhibit the binding of 3H-5-HT with their electrophysiologically determined actions on myenteric neurons. 5-Methoxytryptamine did not inhibit the binding of 3H-5-HT to enteric membranes and neither mimicked nor antagonized the effects of 5-HT on the membrane potential of myenteric neurons. Two dipeptides of 5-hydroxytryptophan, N-acetyl- and N-hexanoyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP and N-hex-5-HTP-DP) inhibited the binding of 3H-5-HT (K1 = 0.25 microM for 5-HTP-DP and 1.19 microM for N-hex-5-HTP-DP). 5-HTP-DP applied by pressure microejection or superfusion (10 microM) antagonized the slow postsynaptic depolarization of myenteric neurons evoked by microejection of 5-HT. 5-HTP-DP also blocked the 5-HT-induced presynaptic reduction in amplitude of nicotinic fast synaptic potentials; however, 5-HTP-DP itself did not affect these responses. Moreover, 5-HTP-DP also failed to affect responses of myenteric neurons to microejected substance P, their muscarinic response to acetylcholine, or antidromic action potentials. In contrast, both dipeptides blocked the slow synaptic potentials seen in type II/AH neurons following stimulation of fiber tracts in interganglionic connectives. These data support the hypotheses that enteric 3H-5-HT binding sites are enteric neural 5-HT receptors, that dipeptides of 5-hydroxytryptophan are specific antagonists at these receptors, and that 5-HT is one of the mediators of slow synaptic potentials in the myenteric plexus.

5-Hydroxytryptophan↗