Concerted derivatization and concentration method with dispersive liquid-liquid microextraction for liquid chromatographic analysis of 5-hydroxyindoles in human serum.

We developed a concerted derivatization and concentration method based on dispersive liquid-liquid microextraction (DLLME) for the liquid chromatography (LC) determination of 5-hydroxyindoles (5-HIs; serotonin, 5-hydroxyindole-3-acetic acid, N-acetylserotonin, and 5-hydroxytryptohol). Concerted derivatization and concentration could be affected by adding a mixture of an ionic liquid (1-hexyl-3-methylimidazolium hexafluorophosphate, extraction solvent), methanol (disperser), and water containing fluorescence derivatization reagents [benzylamine and potassium hexacyanoferrate(III)] into the sample. The resulting sedimented phase was injected into a reversed-phase LC column using a mixture of acetonitrile and 250 mM acetate buffer (pH 4.3) as the mobile phase for gradient elution, and the derivatives obtained were fluorometrically detected at excitation and emission wavelengths of 345 nm and 452 nm, respectively. The derivatization (reagent concentrations and pH) and extraction (extraction and disperser solvent type) conditions were optimized simultaneously. The limits of detection of the 5-HIs were in the range of 0.08-0.33 nM. The method was validated for 10 and 50 pmol/mL human serum levels, and the recovery of 5-HIs was between 66% and 98%, within a relative standard deviation of 9.5%. The proposed method is well suited for the highly sensitive analysis of trace amounts of 5-HIs in human serum samples.

HPLC determination of serotonin and its metabolites from human platelet-rich plasma; shift to 5-hydroxytryptophol formation following alcohol consumption.

A sensitive, simple, and reliable high-performance liquid chromatographic method with electrochemical detection is developed for the measurement of four natural products, the serotonin-related indols from human platelet-rich plasma (PRP) using N-methylserotonin as internal standard. Separation of serotonin (5HT), 5-hydroxytryptophan (5HTP), 5-hydroxytryptophol (5HTOL), and 5-hydroxyindole-acetic acid (5HIAA) is carried out on Supelcosil LC-18DB stationary phase. A mixture of 48 mM citric acid, 28 mM sodium phosphate dibasic, 0.027 mM Na2EDTA, and 3% methanol (pH 3.18) serves as the mobile phase. Measurements are carried out at 25 degrees C at Eox=0.65 V. The calibration curves are linear through the range of 10-200 pg/mL. Method validation is performed according to internationally accepted criteria. Blood is collected from healthy controls and schizophrenic subjects. Significantly higher PRP serotonin is measured in schizophrenics; patients with recent alcohol consumption could be characterized with significantly elevated 5HTOL/5HIAA ratio.

Prevalence of high alcohol and benzodiazepine consumption in sleep apnea patients studied with blood and urine tests.

OBJECTIVE: To evaluate the prevalence of alcoholism and benzodiazepine abuse among patients with obstructive sleep apnea syndrome (OSAS). Such abuse may aggravate the tendency to apneas, especially in patients with OSAS. MATERIAL AND METHODS: The study included 98 consecutive OSAS patients. Two patients dropped out; blood samples could not be obtained from two other patients and a urine sample could not be obtained from one. Blood and urine samples were examined for carbohydrate-deficient transferrin (CDT) and 5-hydroxytryptophol (5-HTOL), markers of excess alcohol intake, and urine-benzodiazepines (u-Benz), a marker of drug abuse. Patients with positive screening tests were offered therapy for their abuse. RESULTS: The CDT test was positive in 8/94 patients (8.5%), the 5-HTOL test in 6/95 (6.3%) and the u-Benz test in 3/95 (3.2%). CONCLUSIONS: Our findings correlate well with current views concerning alcohol and drug abuse in Sweden, and do not indicate that the frequency of such abuse is higher among OSAS patients. It should be noted that none of the patients who screened positive in the laboratory tests admitted to being alcohol or drug abusers when they consulted their physician. We recommend screening all OSAS patients for alcohol abuse using not only a questionnaire but also a laboratory test such as the CDT test.

[5-HTOL--a new biochemical alcohol marker with forensic applications]. / 5-HTOL--ny biokemisk alkoholmarkör med rättsmedicinska tillämpningar.

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.

[Peripheral markers, future perspectives]. / Marqueurs périphériques, perspectives d'avenir.

Four of the recently described peripheral markers of alcohol abuse have been reviewed. The acetaldehyde adducts allow to detect an alcohol abuse lasting for several weeks, even after a recent alcohol withdrawal. Inversely, 5-hydroxytryptophol (5-HTOL) reflects the alcohol consumption of the last 24 hours. Its detection is possible after the blood alcohol concentration has disappeared. Its measurement is run in urine samples, thus without invasive sampling. The hyaluronic acid and the activity of beta-hexosaminidase are markers of hepatobiliary alcohol induced disorders more than direct markers of alcohol intake. Acetaldehyde adducts could be used as markers of long term alcohol abuse, CDT as a marker of the recent alcohol abuse, and 5-HTOL the detection of alcohol abuse of the past day.

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.

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.

Metabolism of platelet 5-hydroxytryptamine in acidified platelet concentrates.

The present study was designed to understand the metabolism of 5-hydroxytryptamine (serotonin, 5HT), a dense granule constituent of platelets, in acidified platelet concentrates (PC) during in vitro storage in polyvinylchloride plastic containers at 22 degrees C. High-performance liquid chromatographic analysis showed that significant amounts of 5HT and its metabolites, 5-hydroxytryptophol and 5-hydroxyindole-3-acetic acid (5HIAA), were detected in the plasma of all PC stored for 3 days with a pH below 6.0, measured at 37 degrees C. PC with a platelet number over 7 x 10(10) cells maintained low PO2 values. Rapid fall in pH occurred in these PC with low PO2 values. The rate of lactate production of platelets increased several fold because of an insufficient oxygen supply to them. In PC with a sufficient supply of oxygen, the adenosine triphosphate (ATP) production rates were estimated to be 8 nmol/min/10(9) platelets. In PC acidified below pH 6.0, the oxidative phosphorylation nearly ceased, and glycolysis was also inhibited: ATP production rates averaged 1 nmol/min/10(9) platelets at 3-day storage. Numerous clusters of platelet aggregates were found in acidified PC, and platelets changed into spherical shapes with accompanying swelling. These platelets did not respond to adenosine triphosphate and failed to produce shrinking when suspended in hypotonic solutions. 5HT was not metabolized in acidified platelet-poor plasma. These data suggest that the metabolic activity of platelets was impaired when the pH of the PC fell below 6.0, leading to the release of platelet 5HT into the plasma. A substantial amount of 5HT might be metabolized by platelet monoamine oxidase into 5HIAA and 5-hydroxytryptophol.

Stress-induced synthesis of melatonin: possible involvement of the endogenous monoamine oxidase inhibitor (tribulin).

Cold-restrained stress increased rat pineal melatonin and N-acetylserotonin content. This effect was partially prevented by lorazepam. Serotonergic turnover (ratio of 5-hydroxyindole acetic acid to serotonin) was significantly decreased in stressed but not in stressed rats pretreated with lorazepam, suggesting stress-induced inhibition of monoamine oxidase (MAO). Literature data indicate that the same type of stress increases the production of the endogenous MAO inhibitor. The implication of stress-induced MAO inhibition on melatonin synthesis in anxiety and drug withdrawal is discussed.

Measurement of 5-hydroxytryptophol and 5-hydroxyindoleacetic acid in human and rat brain and plasma.

The levels of 5-hydroxyindoleacetic acid (5-HIAA) and free and total 5-hydroxytryptophol (5-HTOL) in human and rat brain regions and plasma were determined by a specific capillary column gas chromatographic--mass spectrometric method. The human brains were obtained 2-3 hours post mortem, and the levels of 5-HIAA were in the range of 0.48-31.3 nmoles/g in the regions investigated. The levels of free and total 5-HTOL were 10.9-387 pmoles/g and 14.5-821 pmoles/g, respectively. The ratio of total 5-HTOL to 5-HIAA was in the range of 0.6-5.5%. In human plasma the levels of free and total 5-HTOL were 0.9 +/- 0.3 and 2.9 +/- 0.8 pmoles/ml +/- S.E.M., respectively. In regions of rat brain, the 5-HIAA levels ranged from 0.37-2.84 nmoles/g. Free and total 5-HTOL were in the range of 11.4-56.1 and 16.2-77.1 pmoles/g, respectively. The ratio of total 5-HTOL and 5-HIAA ranged from 2.3-5.1%. Higher levels of 5-HIAA and 5-HTOL occurred in the rat pineal gland. In rat plasma the levels of free and total 5-HTOL were 1.34 +/- 0.06 and 21.6 +/- 1.6 pmoles/ml +/- S.E.M., respectively.

Differential changes of blood and urinary indoleamines following intravenous ethanol administration in alcoholic and non-drinker women.

Total blood serotonin (5-HT) and the urinary excretion of 5-hydroxyindoleacetic acid (5-HIAA) and 5-hydroxytryptophol (5-HTOL) were measured before and after intravenous administration of 0.65 g/kg ethanol to nine chronic alcoholic female patients. Changes were compared to those obtained in twelve age-matched non-drinker neurotic or hysteric women. The two groups showed different pattern of indoleamine response to ethanol: alcoholics reacted by a reduction of 5-HIAA ouput in the urine, while controls showed no change in the 5-HIAA excretion but a marked fall of blood serotonin. Urinary 5-HTOL excretion increased equally in both groups. Despite these biochemical variations, behavioral responses to ethanol were indistinguishable in the two groups. The results indicate that chronic alcoholism leads to permanent changes of the biogenic amine metabolism.

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.