Institute of Laboratory Medicine Department of Medical Neurochemistry, Lund University, Lund, Sweden
* Author to whom correspondence should be addressed at: Institute of Laboratory Medicine Department of Medical Neurochemistry, Lund University Hospital, S-221 85 Lund, Sweden. Fax: +46 46 149870; E-mail: steina.aradottir{at}neurokemi.lu.se
(Received 2 June 2003; first review notified 3 July 2003; in revised form 13 August 2003; accepted 21 August 2003)
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ABSTRACT |
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INTRODUCTION |
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MATERIALS AND METHODS |
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Human subjects
Human blood from 11 healthy donors, (six males aged 3550 and five females aged 3357 years), was collected in sodium-heparin tubes for the incubation with ethanol and subsequent PEth analysis. Blood was also collected in EDTA tubes for haematological analyses. All tubes were of Vacutainer® brand (Becton Dickinson Vacutainer System Europe, France). Informed consent was obtained from all participants. The study was approved by the ethical committee of Lund University.
Animals
All experiments were carried out in accordance with the European Council Directive regarding care and animals for experimental procedures (86/609/EEC). The Ethical Committee for animal experiments at Lund University, Sweden, approved all experiments.
Male SpragueDawley rats ranging in body weight from 208 to 225 g (Beekay, Sweden) at the start of the experiment were used. Animals were individually housed and fed Lieber-DeCarli liquid diet (Dyets, Bethlehem, PA, USA) (Lieber and DeCarli, 1982). The ethanol-fed animals (n = 7) were acclimatized to their diet over a 6-day period by giving ethanol as 3, 4.5 and 6% for 2 days each and finally 9% ethanol v/v for 30 days. Control rats (n = 7) were pair fed, that is, the mean amount of calories consumed by the ethanol-fed rats was given to control animals the next day. All animals had free access to water during the whole experiment. A 12 h dark12 h light cycle and a temperature of 25°C were maintained. All animals were killed by decapitation under sodium pentobarbital (Abbott Laboratories, IL) anaesthesia (100 mg/kg intraperitoneally).
During anaesthesia of the rats, just prior to decapitation, blood was collected with a heparinized syringe directly from the heart. One aliquot was used to measure blood-alcohol concentration (BAC). Another aliquot was immediately extracted for PEth analysis. The remaining blood was incubated in the presence of ethanol to study PEth formation in vitro. Organs were dissected, frozen in liquid nitrogen and stored at 70°C until extraction.
Porcine (Sus domesticus) blood was collected under anaesthesia from the jugular vein; blood from ferret (Mustela putorius furo) was collected from the aorta as the animal was killed.
Preparation of extracts
Two different volumes of blood were used for extraction, 1 ml and 300 µl respectively. When 1 ml was used, the whole blood was centrifuged at 2500g for 10 min and the plasma was discharged. The pellet containing red blood cells (450 µl) was then regarded as tissue and extracted with 24 volumes of hexane:propan-2-ol (3:2 v/v). Whole blood (300 µl) was extracted with 33 volumes of hexane:propan-2-ol (Radin, 1981
). Comparison was done on the recovery of PEth and it was found that the blood volume did not affect the results. All blood samples were extracted by first adding 4 ml of propan-2-ol under agitation on a mixer and then adding 6 ml of hexane in the same way (hexane:propan-2-ol; 3:2 v/v). After 10 min at room temperature and mixing, the samples were centrifuged at 1500 g for 10 min and the supernatants were transferred to new tubes.
Parts from frozen organs, 0.3 g, were homogenized in 18 ml of hexane:propan-2-ol (3:2 v/v) per gram of tissue using a IKA T24 homogeniser (IKA, Staufen, Germany) at 24 000 r.p.m. (Radin, 1981
). The homogenates were centrifuged at 1500 g for 10 min and the supernatants were transferred to new tubes. The pellets were re-extracted with 6 ml of the same solvent per gram of original tissue, following centrifugation and pooling supernatants from each sample. Tissue extracts (containing 42 mg of tissue/ml) were kept at 20°C until analysis.
Sample incubation
All incubations were made with 1 ml of whole blood and 50 or 100 mmol/l ethanol. Sterile 5-ml polypropylene tubes with tight lids were used to prevent evaporation of ethanol. Reactions were carried out with shaking at 37°C in a water bath. Ethanol concentrations were checked during and after incubations and no losses were observed.
To study the rate of PEth degradation, 1 ml of whole blood was incubated with 50 mmol/l ethanol for 20 h; subsequently, the blood was centrifuged for 10 min at 500 g and the upper phase aspired. The cells in the pellet were washed twice with 0.9% (w/v) sodium chloride solution and at this point samples were taken to determine control levels of PEth formation. For further incubation, the pellet was suspended in cell- and platelet-free plasma of A-blood type and incubated at 37°C for a further 6, 24, 36 or 48 h.
Cell-line cultivation and ethanol exposure
All cells were cultured in 60-mm diameter plastic dishes in a humidified atmosphere with 5% CO2 at 37°C. Human HepG2 cells were cultured in RPMI 1640 medium supplemented with 10% FCS. Cells were seeded at 200 000 cells/ml. Rat C6 glioma cells were cultured in 4 ml DMEM supplemented with 5% FCS. Cells were seeded at 100 000 cells/ml. Culture media for both cell-lines were supplemented with 100 IU/ml penicillin and 100 µg/ml streptomycin. The cell culture medium was changed every 23 days. At the time of the experiments, 5 days after seeding, the cells had reached confluence. Upon the start of the experiment, the culture medium was changed to a medium containing 2% FCS, 100 nmol/l TPA and 50 mmol/l ethanol. The culture dishes were placed in polystyrene boxes saturated with 50 mmol/l ethanol to maintain constant ethanol concentration during the exposure (Caron et al., 2000). Following a 2 h incubation, some dishes were harvested. The remaining dishes were washed twice with HBSS and incubated for 30, 60, 120 or 240 min in medium containing 2% FCS. Enzyme inhibitors were added at different concentrations. Incubations were interrupted by washing twice with HBSS and once with ice-cold water and then cells were harvested in 1 ml ice-cold water. Aliquots were taken for protein assays (Bradford, 1976
). Lipids were extracted essentially according to Bligh and Dyer (1959)
by adding 3.75 ml chloroform/methanol (1:2 v/v). After mixing, phase separation was achieved by adding 1.25 ml chloroform and 1.25 ml water. The organic lower phase was removed and analysed by HPLC.
PEth analysis by HPLC
The lipid extracts were evaporated under nitrogen and dissolved in 150 µl hexane:propan-2-ol (3:2 v/v).
PEth was analysed by HPLC (Waters Alliance 2690) using an evaporative light-scattering detector (Alltech 500 ELSD). A 250 x 4 mm, Licrosphere 100 DIOL, 5 µm particle size column (Merck, Germany) was used with a tertiary gradient of hexane (A), propan-1-ol:water (17:3 v/v) (B) and propan-1-ol:acetic acid:triethylamine (316:16:1 v/v) (C). The gradient was run as described earlier (Varga et al., 2000). The detection limit was 0.2 nmol PEth.
Haematological analyses
The haematocrit, RBC count and mean corpuscular volume (MCV) were determined by automated techniques on Sysmex® SE-9000 instruments (Toa, Japan). The analyses were performed at the Clinical Chemistry Laboratory at the Lund University Hospital, Sweden.
Analysis of BAC
Ethanol content of sera was analysed by a standard enzymatic colourimetric method (Roche diagnostic).
Data presentation
Wilcoxon Signed Rank test was used to assess the significances of the time and concentration dependencies of PEth formation in blood. Statistical analyses on cell culture experiments were performed using Student's t-test.
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RESULTS |
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Human blood from 10 healthy donors was incubated with 50 or 100 mM ethanol for 20 and 40 h. The formation of PEth was significantly higher with 100 mM than with 50mM ethanol (P < 0.01 for both 20 and 40 h) and the formation of PEth at 40 h was significantly higher than at 20 h (P < 0.01 at both concentrations). The PEth formation was found to be proportional to the exposure time, and increased with ethanol concentration (Fig. 1). No relation could be found between RBC count, MCV or hematocrit and PEth formation. At the time of sampling, four of the ten donors had measurable although low PEth levels (0.13, 0.30, 0.35 and 1.53 µmol/l). These samples did not show any difference in the rate of PEth formation as compared with the other six donors. The PEth amounts at time zero were subtracted from the values at 20 and 40 h given in Fig. 1. Since the formation rate was found to be linear, the PEth formation/h for each individual and ethanol concentration could be calculated and was found to be 46.9nmol/l/h (ranging from 23.9 to 106.2) at 50 mM ethanol and 74.4 nmol/l/h (ranging from 39.1 to 166.2) at 100 mM ethanol. There was no evidence for gender differences in PEth formation rate.
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PEth formation in rat, pig and ferret blood in vitro
Whole blood from alcohol naive rat (n = 7), pig (n = 1) and ferret (n = 2) was incubated for 24 h with 50 mmol/l ethanol with or without 100 nmol/l TPA. Blood from the alcohol-treated rats (n = 7) was incubated with an additional 50 mmol/l ethanol. At the time of sampling, the BAC levels in alcohol-fed rats were on average 44 mmol/l (range: 3566 mmol/l), which increased the concentration of ethanol in the incubation to between 85 and 116 mmol/l. These samples were also incubated with or without 100 nmol/l TPA. No formation of PEth could be detected in the blood following any of these 24 h incubations.
Accumulation and degradation of PEth in HepG2- and C6-cells
In C6 cells basal net accumulation (formation minus degradation) of PEth in the presence of 50 mM ethanol during 2 h was 0.037 ± 0.003 nmol/mg of protein whereas in HepG2 cells no basal net accumulation could be detected. Net accumulation of PEth in cells exposed to 50 mM ethanol and 100 nM TPA for 2 h was 2.00 ± 0.12 and 2.64 ± 0.24 nmol PEth/mg of protein in C6 and the HepG2 cells, respectively.
The disappearance rate of PEth was well approximated by exponential decay functions in both cell lines. The half-life of PEth was 0.67 and 0.81 h for C6 and HepG2 cells, respectively (Fig. 2). The phospatidylinositol-phospholipase C (PI-PLC) inhibitor U73122 and the phospholipase A2 (PLA2) inhibitor PACOCF3 failed to block the breakdown of PEth. The phosphatidylcholine phospholipase C (PC-PLC) inhibitor D609 and the phosphatidic acid phosphohydrolase (PPH) inhibitor propranolol both inhibited the degradation of PEth, however at a different degree in the cell lines (Fig. 4). Propranolol prominently attenuated PEth degradation in both C6 and HepG2 cells. A dose-dependent decrease in the degradation of PEth was elicited in the cell lines when exposed to propranolol (100500µM), whereas D609 (30150µM) only showed dose-dependency in the HepG2-cells (results not shown).
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DISCUSSION |
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Although rats synthesized and accumulated PEth in their organs, no PEth was found in their blood. In a previous study, rats given 20 or 6% ethanol in water had the highest PEth levels in the stomach (Aradottir et al., 2002). The animals in this particular experiment accumulated much more PEth in intestines, kidneys and lungs than in the stomach, maybe reflecting faster gastric emptying when fed ethanol in a liquid diet. In spleen, heart and liver, no pronounced difference in PEth concentration depending on the type of exposition was observed (Aradottir et al., 2002
). PEth was not found in testis and pancreas, which is in accordance with an earlier report (Aradottir et al., 2002
). It is assumed that both the differences in diets and in ethanol concentrations are of importance for PEth formation among the organs.
Surprisingly, in vitro incubations with ethanol and blood from rat, pig or ferret showed no detectable PEth formation, in contrast to human blood treated in the same way. Furthermore, efforts to stimulate PLD activity through PKC activation with TPA gave no measurable PEth in blood from any of these animals. Other common laboratory animals needs to be tested before it can be concluded that the human blood is unique in its ability to form PEth. Although no PEth was found in the non-human blood in the present study, this finding does not exclude the presence of PLD in the erythrocytes of these mammals. PLD activity has earlier been demonstrated in rabbit erythrocytes (Ochi et al., 1996) by labelling newly synthesized PEth with [3H]ethanol. Labelling experiments are sensitive enough to reveal very low enzyme activity. Anyway, the abundance or activity of PLD is most likely much lower than in human erythrocytes.
The method for PEth assay used in this study (HPLC with evaporative light-scattering detection), has the potential to be used in routine clinical chemistry laboratories to measure PEth as a marker of alcohol abuse (Gunnarsson et al., 1998; Varga et al., 1998
). The present study has revealed that human blood is particular in its ability to form PEth in sufficient amounts to be detected by the HPLC method. It was also demonstrated that in human whole blood in vitro, PEth formation is linear with time and dependent on ethanol concentration. However, there were individual variations in PEth formation in vitro, but moderate previous consumption of ethanol did not affect the formation rate. PEth as a marker of ethanol consumption has high specificity. The present study indicates that the formation rate is individual, therefore it may not be possible to correlate PEth in blood exactly to the absolute amount of ingested ethanol. This holds true also for other biological markers used as tests to monitor alcohol consumption.
The degradation of PEth was studied in the human HepG2- and the rat C6-cell-lines since no degradation of PEth could be observed in human blood in vitro (Fig. 2). In both cell lines degradation of PEth occurred. The C6 cells displayed a slightly faster rate of degradation than HepG2 cells. Pai et al. (1987) showed that isolated PLA2 released arachidonic and palmitic acid from PEth in vitro. The PLA2 inhibitor PACOCF3, however, had no effect on the PEth degradation in either cell line. Inhibition of PEth metabolism by propranolol has earlier been reported in intact pancreatic islets (Metz and Dunlop, 1991
). In this study, the PPH-inhibitor propranolol as well as the PC-PLC inhibitor D609 inhibited PEth-degradation. In both cell-lines propranolol was a more effective inhibitor of PEth degradation than D609 (Fig. 4). Both pathways would hypothetically convert PEth to diacylglycerol (DAG) and ethylphosphate. Further studies are needed to elucidate the pathways of PEth degradation. It has been suggested that human RBC lack PC-PLC-activity (Selle et al., 1992
) which could partly explain why PEth is not degraded in human blood.
To conclude, the rat is not suitable as a model for investigations on PEth toxicity in organs as related to blood PEth levels due to absence of PEth formation in blood. Human blood seems to be particular in its ability to synthesize PEth and maintain a stable level of PEth due to the lack of PEth degradation.
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ACKNOWLEDGEMENTS |
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