This study aimed to explore how PEth and other commonly used alcohol biomarkers (CDT, AST, ALT, and GGT) respond to regular consumption of what has been generally considered to correspond to low to moderate amounts of alcohol over a 2-week period. A total of 21 voluntary participants (aged 31-69 years) took part in a 2-week drinking study. Group 1 (n = 11) consumed one glass of wine daily (16 g of alcohol), close to the present Swedish limit for hazardous alcohol consumption, while Group 2 (n = 10) consumed two glasses daily (32 g of alcohol). Alcohol biomarkers were measured at baseline and at three further occasions during the study. After 1 week of alcohol consumption, all participants had measurable concentrations (> 0.005 μmol/L, ≈3.5 ng/mL) of both PEth-homologues (PEth 16:0/18:1 and PEth 16:0/18:2). After 1- and 2-week periods, significant differences in PEth levels were observed between Group 1 and Group 2. The correlation between the two PEth-homologues was strong and increased as the study progressed. In contrast, other biomarkers showed little to no change during the study period. Both PEth-homologues appear capable of identifying hazardous alcohol consumption. The current Swedish reporting threshold for PEth 16:0/18:1 (0.05 μmol/L, ≈35 ng/mL) demonstrates high specificity but low sensitivity in identifying hazardous alcohol consumption involving regular/daily intake. The sensitivity of the other biomarkers is insufficient for detecting alcohol consumption at this level.

BackgroundAlcohol use disorders are a major but often unrecognized health problem. Alcohol markers can therefore be of great value for diagnosis, follow‐up, and treatment evaluation. Phosphatidylethanol in blood (B‐PEth) is an alcohol biomarker with higher clinical sensitivity and specificity than commonly used alcohol markers but has shown a considerable interindividual variation in relation to reported consumption.MethodsAn in vitro system was used to investigate factors, which may affect the formation rate of PEth or which may give rise to interindividual variation in the rate of formation. In this system, isolated erythrocytes from 31 individuals were incubated in the presence of various concentrations of ethanol (EtOH). The concentration of PEth and phosphatidylcholine (PC), the parent molecule of PEth, was determined by chromatographic methods.ResultsTime, EtOH, and PC concentration were major factors determining the amount of PEth formed. The interindividual variation in PEth formation rate, calculated at an EtOH concentration of 50 mmol/l, showed a coefficient of variation (CV) from 23 to 31% for the different PEth forms studied (PEth 16:0/18:2, total PEth and PEth 16:0/18:1). The concentration of PC was found to be an important determinant of this variation. The formation rate for PEth 16:0/18:2 was somewhat higher than for PEth 16:0/18:1. The formation of PEth 16:0/18:1 but not PEth 16:0/18:2 showed a positive correlation to the concentration of PEth at baseline (endogenous PEth). Calculation of enzyme kinetics for the reaction resulting in the formation of PEth 16:0/18:1 or PEth 16:0/18:2 showed an apparent Km (Michaelis constant) of approximately 160 to 170 mmol/l.ConclusionsInterindividual variation in the formation rate of PEth appears to be a significant but relatively modest source of variation in the relation between B‐PEth and reported consumption. Correction for interindividual variation in PC concentrations might substantially reduce the interindividual variability in PEth formation and consequently in B‐PEth.

Background Phosphatidylethanol (PEth) is an alcohol use biomarker with higher clinical sensitivity and specificity than commonly used alcohol markers. Since its introduction as a clinical alcohol-marker in 2006, the number of samples sent to our laboratory for the determination of PEth has shown a strong annual increase. This has prompted the need to develop a cost-effective and reliable analytical procedure with high capacity. Methods An LC-MS/MS method for the determination of PEth 16:0/18:1 with a short turnaround time (3 min) has been evaluated with respect to accuracy, sensitivity, and precision. We compared this method with a previously used HPLC method, as well as a manual and a simplified automated method for sample workup, and investigated potential causes of analytic and preanalytic errors. Results The method shows limits of detection and quantification of 0.0075 μmol/L (5.2 ng/mL) and <0.05 μmol/L (<35 ng/mL), respectively. During a 2.1-year period, the method has shown a total CV < 8% for control samples (n = 2808) in the range of 0.10 (70) to 3.5 μmol/L (2461 ng/mL). The simplified automated method for sample preparation works equally well as the manual one. No specific and clinically significant causes of preanalytic errors were found. Conclusions This LC-MS/MS method with automated sample workup is well suited for a clinical laboratory with LC-MS/MS experience and has the capability, proven from several years of use, to produce reliable PEth results in a high-volume laboratory (>50000 clinical samples/year).

Sphingosine 1-phosphate (S1P) is a signalling sphingolipid affecting multiple cellular functions of vascular and immune systems. It circulates at submicromolar levels bound to HDL-associated apolipoprotein M (apoM) or to albumin. S1P in blood is mainly produced by platelets and erythrocytes, making blood sampling for S1P quantification delicate. Standardisation of sampling is thereby of great importance to obtain robust data. By optimising and characterising the extraction procedure and the LC-MS/MS analysis, we have developed and validated a highly specific and sensitive method for S1P quantification. Blood was collected from healthy individuals ( n = 15) to evaluate the effects of differential blood sampling on S1P levels. To evaluate correlation between S1P and apoM in different types of plasma and serum, apoM was measured by ELISA. The method showed good accuracy and precision in the range of 0.011 to 0.9 μM with less than 0.07 % carryover. We found that the methanol precipitation used to extract S1P co-extracted apoM and several other HDL-proteins from plasma. The platelet-associated S1P was released during coagulation, thus increasing the S1P concentration to double in serum as compared to that in plasma. Gel filtration chromatography revealed that the platelet-released S1P was mainly bound to albumin. This explains why the strong correlation between S1P and apoM levels in plasma is lost upon the clotting process and hence not observed in serum. We have developed, characterised and validated an efficient, highly sensitive and specific method for the quantification of S1P in biological material.

BACKGROUND:In clinical practice as well as research situations, it is of great importance to get reliable information about a patient's alcohol consumption. The aim of the study was to investigate the correlation of alcohol biomarkers (phosphatidylethanol [PEth], carbohydrate-deficient transferrin [CDT], γ-glutamyltransferase, aspartate aminotransferase, and alanine aminotransferase) to retrospective as well as diary-based alcohol self-reports and to examine whether it is possible to correlate a biomarker result to a more precise level of alcohol consumption.METHODS:One hundred and sixty alcohol-dependent patients were included in a randomized, placebo-controlled clinical trial of pharmacotherapy for alcohol dependence, of which 115 (76 men and 39 women) completed the study. Retrospective alcohol consumption data were collected at baseline, and alcohol diaries were used during the study. Blood samples for determination of alcohol biomarkers were collected on 5 occasions during the study.RESULTS:PEth and CDT showed a better correlation with alcohol consumption documented in the diary (PEth rs = 0.56 and CDT rs = 0.35) than with retrospective consumption data (PEth rs = 0.23 and CDT rs = 0.22). An even higher correlation (rs = 0.63) was seen between the 2 alcohol biomarkers PEth and CDT. At all consumption levels, PEth had the highest sensitivity of all biomarkers studied.CONCLUSIONS:PEth was the biomarker with the best correlation to self-reported alcohol consumption. PEth was superior to CDT owing to its substantially higher sensitivity but also due to its closer correlation to self-report. PEth values can be translated into an approximate level of alcohol consumption and PEth appears to be a more reliable measure of alcohol consumption than self-reports.

Various phosphatidylethanol (PEth) derivatives, the corresponding reversed positional isomers (RPI-PEths), lyso-PEth-16:0, and penta-deuterium-labeled PEth analogs (d5-PEths), were synthesized by enzyme-independent synthetic routes. A general solvent system consisting of a mixture of acetone-d6 and methanol-d4 (97:3; v/v) was found to provide a good solubilizing capacity and excellent hydrogen-1 NMR ((1)H-NMR) peak resolution of various PEth homologues. Analytical differentiation of PEth from the corresponding RPI-PEth by carbon-13 NMR ((13)C-NMR) was demonstrated by comparison of the (13)C-NMR signals of the carbonyl groups, the allylic positions, and of the β-carbons. An exemplary stable long-term room temperature, DMSO-d6-based, and proton-sensitive quantitative nuclear magnetic resonance ((1)H-qNMR) independently quantified calibrator comprising PEth-16:0/18:1 for liquid chromatography (tandem) mass spectrometry (LC-MS/MS) analytical applications were prepared by employment of sodium dodecyl sulfate (SDS) as a solubilizing additive. In summary, novel hypothetically occurring PEth derivatives, e.g., RPI-PEths, have been independently synthesized with regio- and stereochemical control. Use of polar organic solvents, e.g., mixtures of acetone-d6 and methanol-d4 or DMSO-d6, improves spectral line shapes as compared to traditional hydrophobic solvents and allow for analytical differentiation between closely related PEth derivatives, as well as LC-MS/MS-independent concentration determination of dissolved single species by employment of (1)H-qNMR.

Despite the growing evidence that elevated total homocysteine (tHcy) in plasma is a cardiovascular risk factor, the mechanism underlying the vascular injury is still unknown. Studies are difficult due to the fact that little is known about the formation of different homocysteine species in vivo. In the present study we have investigated the different fractions of tHcy in 21 patients with acute coronary syndromes and elevated concentration of plasma tHcy. A subgroup of the patients (n=16) was investigated before and after a 3 months study period with or without vitamin supplementation (folic acid 5 mg, pyridoxine 40 mg and cyanocobalamin 1 mg once daily). A major finding is that these patients had a lowered ratio (0.95%) between the concentration of reduced homocysteine (HcyH) and tHcy compared to controls (1.38%). A low ratio HcyH/tHcy in plasma in combination with elevated plasma tHcy concentrations might reflect increased oxidative activity or decreased reducing capacity in plasma from the patients. Another main finding in the present study is that, although vitamin supplementation of these patients normalized plasma tHcy, the ratio between HcyH and tHcy did not normalize. Since substantial evidence indicates that progression of arteriosclerosis is related to enhanced oxidant activity, the premature vascular disease associated with increased plasma tHcy concentration might be due to increased oxidative activity and the elevated plasma tHcy concentration may only reflect the increased oxidative stress.

It has been suggested that hyperhomocysteinemia observed in patients with occlusive vascular disease is caused by reduced renal function secondary to renovascular disease. We have therefore used serum cystatin C, a new sensitive marker for glomerular filtration, in 59 patients with acute coronary syndromes and high plasma homocysteine (tHcy) concentration to measure renal function. Samples were also obtained from 34 patients with low-normal plasma tHcy and 50 control subjects. The patients with low-normal plasma tHcy concentration showed decreased concentrations of serum cystatin C and serum creatinine and increased concentrations of blood folate and serum cobalamin compared to the controls and to the patients with high plasma tHcy. There was a large overlap in cystatin C concentrations between patients with high and low-normal plasma tHcy. None of the parameters investigated except plasma tHcy were significantly different in the group of patients with high plasma tHcy concentration compared to the control group. In order to further demonstrate the importance of renal impairment, a subgroup of the patients with high plasma tHcy was supplemented daily with folic acid 5 mg, pyridoxine 40 mg and cyancobalamin 1 mg for 3 months. Vitamin therapy reduced plasma tHcy from 18.3+/-4.6 pmol/l to 9.6+/-2.2 pmol/l (p<0.0001). However, vitamin treatment did not strengthen the correlation between cystatin C and plasma tHcy concentrations. These findings do not support the hypothesis that subtle renal dysfunction is an important cause of high plasma tHcy concentration in patients with acute coronary syndromes.

Glutathione, the most abundant low-molecular weight thiol in the skin, has been shown to protect the skin from both photobiological and chemical injury The thiols, glutathione in particulars have also been shown to be crucially involved in defence against contact allergens. Since the levels of extracellular thiol concentrations are important determinants of intracellular thiol status, we have compared the normal concentrations and the redox status of the main low-molecular weight thiol components in the extracellular fluid at the dermo-epidermal junction with the corresponding plasma levels. In their sulfhydryl form, all three thiols, i.e. glutathione, cysteine and homocysteine, were more abundant in experimental skin blister fluid than in plasma, as were the free disulfides of glutathione and homocysteine, whereas the free disulfides of cysteine were about the same in blister fluid and in plasma. Protein mixed disulfide levels were higher in plasma than in blister fluid. The present results provide information concerning the extracellular defence in the skin.

Thiols are known to influence the metabolism of glutathione. In a previous study (Toxicology 156 (2001) 93) dithiothreitol (DTT) did not show any effect on intra- or extracellular glutathione concentrations in HeLa cell cultures but increased the effects of mercury ions on glutathione concentrations, whereas monothiols such as N-acetylcysteine (NAC) or glutathione did not. In the present study, we have investigated the effects of thiols as well as the interaction between thiols and mercury ions in cultures of both HeLa and hepatoma cells. Furthermore, we have added α-lipoic acid (LA) to the previously used test panel of thiols, since it is metabolised intracellularly to a dithiol (dihydrolipoate). The present study shows that LA increased intra- and extracellular concentrations of glutathione in both HeLa and hepatoma cell cultures. In contrast to results for HeLa cells, the presence of DTT increased the intracellular glutathione concentration in hepatoma cells. No increase of glutathione concentrations was observed in hepatoma cell cultures in the presence of the monothiols (NAC, homocysteine or glutathione) tested, in agreement with previous findings in HeLa cell cultures. The presence of dithiols, either DTT or dihydrolipoate (the metabolite of LA), increased the effects of mercury ions on glutathione concentrations in hepatoma cells, whereas monothiols such as NAC or glutathione did not, in agreement with previous findings in HeLa cells. Thus, metabolic effects of mercury ions were observed in hepatoma cells as well as in HeLa cells at a lower concentration than the supposed toxicity threshold for mercury in blood.