Immunological detection of in vitro formed phosphatidylethanol--an alcohol biomarker--with monoclonal antibodies.

BACKGROUND: Phosphatidylethanol (PEth) is a promising new marker for detecting long-term alcohol abuse with excellent sensitivity and specificity. Current methods are based on the high performance liquid chromatography-mass spectrometric method and therefore require high levels of expertise and expensive instrumentation. This study was designed to generate PEth-specific monoclonal antibodies for PEth immunoassay development. METHODS: C57/BL6 mice were immunized with PEth in 3 different carriers, mouse serum albumin, mouse high-density lipoproteins, and human low-density lipoprotein (LDL). Mouse splenocytes were fused with a mouse myeloma cell line using the hybridoma technique. Mouse IgM-producing cell lines were selected by limiting dilutions. Binding characteristics of the anti-PEth antibodies were studied using luminometric immunoassays and sequence analysis of the variable region mRNA sequences of the antibodies. Produced antibodies were purified by chromatographic methods. PEth was detected with these antibodies in fluorescence immunoassay and flow cytometric analysis. RESULTS: We generated monoclonal cell lines (2B1 and 2E9) that produce IgM antibodies binding specifically to PEth but not to structurally or chemically similar phospholipids, such as phosphatidylcholine, phosphatidic acid, and cardiolipin. We show here that these anti-PEth antibodies can be used to detect PEth in a fluorescent PEth assay and FACS analysis of human red blood cell samples spiked with PEth. CONCLUSIONS: The present study shows that PEth-specific monoclonal antibodies can be generated using traditional hybridoma technique. Immunogenicity of PEth was enhanced using human LDL as an immunization carrier. The generated monoclonal anti-PEth antibodies, 2B1 and 2E9 bind to PEth in fluid phase and in biological membranes.

Biochemical markers of alcoholism.

Alcohol and alcohol-related diseases have become a major cause of death in Western countries. The most sensitive and specific of the commonly used biomarkers of alcohol intake are carbohydrate-deficient transferrin (CDT), and the combination of gamma-glutamyltransferase (GGT) and CDT. Other widely used laboratory markers are GGT, mean corpuscular volume of erythrocytes and the ratio of aspartate aminotransferase to alanine aminotransferase. Blood ethanol levels reveal recent alcohol use. However, more specific and sensitive biomarkers to improve the detection of excessive alcohol use at an early stage are needed. New biomarkers, not yet used in routine clinical work, include phosphatidylethanol, fatty acid ethyl esters, ethyl glucuronide, sialic acid, and acetaldehyde adducts.

Effects of phosphatidylethanol on mouse adipocyte differentiation and expression of stearoyl-CoA desaturase 1.

BACKGROUND: Phosphatidylethanol (PEth) is an aberrant phospholipid formed in vivo only in the presence of ethanol. In circulation PEth is associated with lipoproteins and is transferred from one lipoprotein to another. Lipoprotein-associated PEth affects endothelial and smooth muscle cells of blood vessels, but its effects on other cell types have not been explored. Adipocytes have a central role in metabolic syndrome and obesity. In this study we tested whether lipoprotein-associated PEth affects stearoyl-CoA desaturase 1 (SCD1) which plays a major role in lipid-mediated signaling in the differentiation of adipocytes. METHODS: Mouse 3T3-L1 preadipocytes were differentiated to adipocytes in the presence of high-density lipoproteins (HDL) isolated from the plasma of healthy volunteers or PEth-containing HDL modified in vitro. After incubation, fat accumulation, SCD1 mRNA expression, SCD1 protein content, and fatty acid composition of adipocytes were determined. RESULTS: Phosphatidylethanol-containing HDL particles inhibited adipocyte differentiation and decreased the 18:1/18:0 ratio of cellular fatty acids by 28% compared with native HDL particles. Moreover, PEth-containing HDL reduced the SCD1 protein content by 39%. CONCLUSIONS: Lipoprotein-associated PEth may mediate the effects of ethanol on SCD1 and differentiation of preadipocytes to adipocytes.

Phosphatidylethanol in high density lipoproteins increases the vascular endothelial growth factor in smooth muscle cells.

To study whether qualitative changes in high density lipoprotein (HDL) phospholipids mediate part of the advantageous effects of ethanol on atherosclerosis, we investigated whether HDL associated phosphatidylethanol (PEth) affects the secretion of vascular endothelial growth factor (VEGF) from cultured human smooth muscle cells. Serum-starved human umbilical vein HUVS-112D smooth muscle cells were incubated in the presence of PEth-HDL, HDL, or buffer. The phosphorylation of protein kinase C (PKC) and mitogen activated protein kinase (p44/42 MAPK) was determined by specific antibodies against phosphorylated and total proteins. VEGF concentrations were measured from cell culture medium of the cells. PEth increased the secretion of VEGF into the culture medium of HUVS cells. PEth-HDL increased the PKC phosphorylation by 2.1-fold and p44/42 MAPK phosphorylation by 3.3-fold compared with HDL, indicating that PEth-containing HDL particles influence vascular smooth muscle cells by PKC and p44/42 MAPK signalling. This may mediate the effects of ethanol on vascular wall by increasing the VEGF secretion from smooth muscle cells. The secreted VEGF may inhibit the formation of neointima and in doing so helps prevent atherosclerosis.

Transfer of phosphatidylethanol between lipoproteins.

BACKGROUND: Phosphatidylethanol (PEth) is an abnormal phospholipid formed only in the presence of ethanol. It has been recently shown that lipoprotein-associated PEth may mediate the effects of ethanol on endothelial cells, and this may explain, at least in part, the beneficial effect of ethanol on atherosclerosis. This study was performed to investigate the transfer of PEth between lipoproteins and the effects of PEth on cholesteryl ester transfer protein (CETP) activity in plasma. METHODS: Lipoproteins were isolated from the plasma of healthy male volunteers (n = 16) and male alcoholics (n = 13). The transfer of cholesteryl esters and PEth was determined between labeled low-density lipoprotein (LDL) and unlabeled high-density lipoprotein particles in vitro. The electrophoretic mobility of PEth-modified LDL particles was determined by agarose gel electrophoresis. RESULTS: PEth was transferred from PEth-modified LDL to high-density lipoprotein at an initial rate of 25.9 nmol/ml/hr. Monoclonal antibody (TP2) against the putative lipid-binding domain of CETP inhibited the transfer rate of PEth by approximately 64%, whereas the cholesteryl ester transfer was inhibited by 86%. This indicates that most of PEth was transferred by transfer proteins other than CETP. CONCLUSIONS: The transfer of PEth between lipoproteins enables the redistribution of PEth from lipoprotein fractions with a slow turnover to those with a rapid clearance. Moreover, the PEth-induced change in the electrical charge of lipoproteins may affect the binding of lipoproteins to their receptors and binding proteins. This in turn may alter the metabolism of lipoproteins and lipid-mediated signaling pathways in the cells delineating the vascular wall.

Lipoprotein-associated phosphatidylethanol increases the plasma concentration of vascular endothelial growth factor.

OBJECTIVE: To study whether qualitative changes in high-density lipoprotein (HDL) phospholipids mediate part of the beneficial effects of alcohol on atherosclerosis, we investigated whether phosphatidylethanol (PEth) in HDL particles affects the secretion of vascular endothelial growth factor (VEGF) from endothelial cells. METHODS AND RESULTS: PEth increased the secretion of VEGF into the culture medium of EA.hy 926 endothelial cells. The mitogen-activated protein kinase (MAPK) phosphorylation increased by 3.3-fold and protein kinase C (PKC) by 2.2-fold by PEth-containing HDL. Moreover, we showed that intravenous injection of PEth incorporated into HDL particles increased plasma concentration of VEGF by 2.4-fold in rats in vivo. Similar effect was observed when the rats were injected with HDL particles isolated from alcohol drinkers. CONCLUSIONS: HDL particles containing PEth affect endothelial cells by MAPK and PKC signaling. This may mediate the effects of ethanol on the arterial wall by increasing VEGF secretion from endothelial vascular cells. That may explain, at least in part, the beneficial effect of moderate alcohol consumption on atherosclerosis.

Effects of ethanol on lipids and atherosclerosis.

Moderate alcohol consumption is associated with an increase in plasma high density lipoprotein (HDL) cholesterol concentration and a decrease in low density lipoprotein (LDL) cholesterol concentration. Changes in the concentration and composition of lipoproteins are estimated to account for more than half of alcohol's protective effect for coronary heart disease. Alcohol intake also affects plasma proteins involved in lipoprotein metabolism: cholesteryl ester transfer protein, phospholipid transfer protein, lecithin:cholesterol acyltransferase, lipoprotein lipase, hepatic lipase, and phospholipases. In addition, alcohol intake may result in acetaldehyde modification of apolipoproteins. Furthermore, "abnormal" lipids, phosphatidylethanol and fatty acid ethyl esters are formed in the presence of ethanol and are associated with lipoproteins in plasma. Ethanol and ethanol-induced modifications of lipids may modulate the effects of lipoproteins on the cells in the arterial wall. The molecular mechanisms involved in these processes are complex, requiring further study to better understand the specific effects of ethanol in the pathogenesis of atherosclerosis. This review discusses the effects of ethanol on lipoproteins and lipoprotein metabolism, as well as the novel effects of lipoproteins on vascular wall cells.

Effect of alcohol on lipids and lipoproteins in relation to atherosclerosis.

Several studies indicate that light-to-moderate alcohol consumption is associated with a low prevalence of coronary heart disease. An increase in high-density lipoprotein (HDL) cholesterol is associated with alcohol intake and appears to account for approximately half of alcohol's cardioprotective effect. In addition to changes in the concentration and composition of lipoproteins, alcohol consumption may alter the activities of plasma proteins and enzymes involved in lipoprotein metabolism: cholesteryl ester transfer protein, phospholipid transfer protein, lecithin:cholesterol acyltransferase, lipoprotein lipase, hepatic lipase, paraoxonase-1 and phospholipases. Alcohol intake also results in modifications of lipoprotein particles: low sialic acid content in apolipoprotein components of lipoprotein particles (e.g., HDL apo E and apo J) and acetaldehyde modification of apolipoproteins. In addition, "abnormal" lipids, phosphatidylethanol, and fatty acid ethyl esters formed in the presence of ethanol are associated with lipoproteins in plasma. The effects of lipoproteins on the vascular wall cells (endothelial cells, smooth muscle cells, and monocyte/macrophages) may be modulated by ethanol and the alterations further enhanced by modified lipids. The present review discusses the effects of alcohol on lipoproteins in cholesterol transport, as well as the novel effects of lipoproteins on vascular wall cells.