Abstract
Chronic alcohol consumption is a major reason for several human diseases, and alcoholism has been associated with a variety of societal problems. Changes in fatty acid metabolism in alcoholics and its effects leading to membrane damage are largely unknown. Therefore, we aimed to investigate the fatty acid composition of erythrocyte membrane phospholipids in relation with plasma lipid profile and other plasma metabolites in chronic alcoholics in comparison with controls. We systematically measured the levels of glucose, lactate and pyruvate in the blood and free amino acids, free fatty acids, mucoproteins and glycolipids, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), very-low-density lipoprotein (VLDL) cholesterol and triglycerides (TG) in plasma of chronic alcoholics and controls. Furthermore, we measured fatty acid composition by gas chromatographic analysis. The fatty acid composition clearly revealed certain changes in chronic alcoholic erythrocyte membrane, chiefly increments in C16:0 and a decrease in C22:4 and C22:6 fatty acids besides the presence of unidentified fatty acids, probably C-24 or C-26 fatty acids. In addition, a significant increase in blood lactate, decrease in blood pyruvate and increased levels of free amino acids and free fatty acids, mucoproteins, VLDL cholesterol, TG and HDL-C in chronic alcoholics were observed with no significant change in plasma TC, LDL-C and glycolipids when compared with controls. Alcohol-induced alterations in plasma and erythrocyte membranes of chronic alcoholics in the present study might be an adaptive response to counteract the deleterious effects of alcohol. The implications of our findings warrant further investigation and needs further in-depth study to explore the mechanisms of alcohol-induced membrane changes.
Introduction
Alcoholism is a disabling addictive disorder and has been associated with a host of societal problems. 1,2 Alcoholism and drug addiction are harmful not only for the individual but also for his or her family. Besides, uncontrolled or chronic alcohol consumption ranks among world’s major health problems and accounts for 4% of the global burden of human diseases. 3 Problems related to alcohol consumption were among the world’s major public health concerns and constituted serious hazards for human health, welfare and life. 4
Alcohol is cleared rapidly from circulation. When consumed in large quantities for longer periods, plasma, blood and in particular erythrocytes (erythrocyte, erythrocyte membrane and constituents) are chronically exposed to alcohol. It is well known that blood plays an important role in integration of metabolism as it supplies and receives various intermediates of cellular metabolism. It also acts as a reservoir for the storage of certain key metabolites, substrates and hormones. In fact, erythrocytes are unable to carry out de novo lipid biosynthesis but can incorporate and turn over plasma lipids.
Hence, blood analysis provides essential information regarding various physiological and biochemical processes. Further, as blood sampling is easier and accessible, the present experimentation was designed on erythrocytes, erythrocyte membrane and plasma. Moreover, red cell membrane has long been used as a convenient model for various studies on membranes and red cell biology. 2,5 –7
In biological membranes, the membrane fluidity is affected by several factors, especially by the fatty acid composition and the cholesterol content of membranes to counteract the continued presence of alcohol and to resist perturbations by ethanol. 2,5 –7 The most abundant lipids in all cell membranes are phospholipids that vary in type, fatty acid composition and location, thus contributing to the membrane asymmetry. Fatty acids are part of the building blocks for glycerol phospholipids including lysophospholipids. The functions of the structural and functional membrane proteins such as receptors, enzymes and transporters are also highly associated with membrane fluidity. 2 By contrast, very little is known about the effects of chronic ethanol consumption on human erythrocyte membrane fatty acids. Alcohol is known to promote esterification products of fatty acids, which could be mediators of end organ damage. 8 Fatty acid metabolism is also modified in alcoholics leading to changes in fatty acid composition in several tissues, which appear to be complex, and several investigators postulated that these changes contribute to organ damage. 8 Therefore, we aimed to investigate the fatty acid composition of phospholipids in correlation with plasma lipid profile and other plasma metabolites in chronic alcoholics in comparison with controls.
Methods
Experimental design
A total of 24 male volunteers in each group, aged 44 ± 6 years, residing in Anantapur, India, with similar dietary habits were divided into two groups, namely nonalcoholic controls and chronic alcoholics. Chronic alcoholics were those who had consumed ∼125 g/d of alcohol at least five times a week for the past 10–12 years. All volunteers were informed about the experimentation and their consent was obtained; subjects using any drugs (tranquilizers, analgesics, etc.) or with any disease or disorder were excluded. Institutional ethical committee approved this study.
Blood sampling
Blood samples were obtained after the subjects had fasted overnight. Venous blood was drawn into heparin-treated evacuated tubes and centrifuged immediately at 1500 r/min for 10 min at 4°C to separate plasma and red cells.
Measurement of plasma lipid profile levels
Plasma total cholesterol (TC), triglycerides (TG) and high-density lipoprotein cholesterol (HDL-C) were determined by enzymatic colorimetric methods that were performed using appropriate kits (Span diagnostics, Surat, India) and according to the manufacturer’s instructions. The concentration of low-density lipoprotein cholesterol (LDL-C) was calculated according to the formula:
Measurement of blood glucose, pyruvate and lactate
Blood was deproteinized with 0.3 N HClO4 and centrifuged. Blood glucose was estimated by the method of Hugget and Nixon. 9 To an aliquot of supernatant, glucose oxidase reagent (125 mg of glucose oxidase, 4 mg of peroxidise in 100 ml of 0.5 M phosphate buffer, pH 7 and 0.5 ml of 0.01% w/v O-dianisidine in 95% ethanol) was added and the color developed was measured at 440 nm and the results were expressed as milligrams per deciliter.
Blood was deproteinized with cold 10% trichloroacetic acid (TCA) and centrifuged, and the supernatant was used for pyruvate and lactate analysis. Blood pyruvic acid was estimated by the method of Friedemann and Gaugen. 10 The concentration of pyruvate was expressed as milligrams per deciliter. Blood lactic acid was estimated by the method of Barker and Summerson, 11 and the results were expressed as milligram per deciliter.
Determination of plasma amino acids and free fatty acids
Plasma amino acids were estimated by the method of Moore and Stein. 12 Plasma was deproteinized by mixing with 4 volumes of 10% of TCA. To 1.0 ml of TCA filtrate, 1.0 ml of ninhydrin-buffer reagent (equal volumes of 0.16% stannous chloride in 0.2 M citrate buffer, pH 5 and 4% (w/v) ninhydrin in methyl cellosolve) was added and heated for 15 min at 100°C, cooled and diluted with 5 ml of 50% (v/v) aqueous isopropanol. The intensity of the color was read at 570 nm. A standard curve was made using glycine. Results were expressed as milligram per deciliter.
Plasma free fatty acids were estimated by the method of Falholt et al. 13 To 0.1 ml of plasma, 1.0 ml of phosphate buffer, 6 ml of extraction solvent and 2.5 ml of copper reagent (10 ml of copper solution was mixed with 10 ml of triethanolamine and 6 ml of sodium hydroxide and diluted to 100 ml, then 33 g of sodium chloride was added and the pH was adjusted to 8.1) were added. All the tubes were shaken vigorously. Activated silicic acid of 200 mg was added and left aside for 30 min. The tubes were centrifuged and 3 ml of the copper layer wastransferred to another tube containing 0.5 ml of diphenylcarbazide (1.5 M in ethanol) and mixed carefully. A solution containing 0.08–0.4 µEq of palmitic acid in chloroform was used as the standard. The absorbance was read at 550 nm immediately. Results were expressed as microequivalents per liter.
Glycolipids
Glycolipids were estimated by the method described by Roughan and Batt. 14 Plasma samples were digested with 2 ml of 2 N H2 SO4 for 2 h. After hydrolysis, 4 ml of chloroform was added and the mixture was centrifuged. From the top aqueous layer, 1 ml was taken out separately. To this, 50 ml of 80% phenol was added followed by 4 ml of concentrated H2 SO4 and the orange color developed was measured at 480 nm. The concentration of the galactose part of the lipid was calculated from the standard curve, where different concentrations of (20–200 mg) galactose standards were run simultaneously under similar conditions. Then the value was multiplied by 4.45 to estimate glycolipids and results were expressed as milligrams per 100 ml.
Plasma mucoproteins
Plasma mucoproteins were estimated by the method described by Varley. 15 To 0.5 ml of serum, 4.5 ml of 0.85% saline followed by 2.5 ml 1.8 M perchloric acid were added, mixed and filtered through Whatman No. 50 filter paper. To 5 ml of filtrate,1 ml of 5% phosphotungstic acid was added, mixed and then centrifuged at 2000 r/min for 10 min and supernatant was decanted. The precipitate was washed with the 0.6 M phosphotungsic acid in perchloric acid and supernatant fluid was decanted and precipitate was dissolved in 1 ml of 20% sodium carbonate solution. To that, 3.5 ml of water was added followed by 0.5 ml of phenol reagent and was kept in boiling water bath at 37°C for 15 min and absorbance was read at 680 nm. Tyrosine standards ranging 20–100 mg were treated similarly along with the test and the value obtained was multiplied by 23.8 to get mucoproteins. Results were expressed as milligram per 100 ml.
Extraction of membrane lipids
Erythrocyte membrane was prepared as described previously. 2 Erythrocyte membrane lipids were extracted. To the lysed membrane preparations, 5 ml of methanol was added followed by chloroform. After 30 min, the same was filtered to collect filtrate and the residue was again subjected to same step and filtered again. The filtrates were pooled up and used for lipid analysis.
Methyl esterification
The extracted lipids were methyl esterified by the method of Stoffel et al. 16 The extracted lipids were dissolved in 4 ml of 5% hydrochloric acid in super dry methanol and 0.5 ml of dry benzene in a 15-ml microsublimation tube to which a condenser with a calcium chloride moisture trap was connected. The mixture was refluxed in a silicone bath at 90°C for 2 h, with frequent shaking at the start to dissolve the lipid mixture. After cooling to room temperature, 2 volumes of water were added, and the methyl esters were extracted three times with 3 ml of petroleum ether. The pooled extracts were simultaneously neutralized and dried over sodium sulfate–sodium bicarbonate mixture for 1 h. The esters were then quantitatively transferred with petroleum ether to a second microsublimation tube and the solvent was evaporated to dryness at reduced pressure in a water bath at 40°C. After the microsublimation tube was fitted to the cold finger, a vacuum of 0.2 mm of mercury was produced. The tube was then lowered into a silicone bath at 60°C for 60 min. The assembly was disconnected after cooling, and the sublimated methyl esters were rinsed off with petroleum ether into a glass stoppered tube. After evaporation of solvent, the preparation was subjected to gas chromatographic analysis as described by Hrelia et al. 17 The gas chromatographic peaks were identified on the basis of their retention times ratio’s relative to predetermined authentic samples.
Gas chromatographic analysis of fatty acid composition
Fatty acid methyl esters were chromatographed both on a capillary and traditionally packed column to determine fatty acid composition. Gas chromatographic analysis was carried out using Hawlett Packard gas chromatograph model 6890 equipped with gas capillary column (10 m × 0.32 mm ID) coated with a thermostable stationary phase with programmed temperature of 60–320°C with a gradient of 100°C/min. The analysis on the packed column was carried out with a glass column (2 m × 1/4 in ID) at 2000°C with helium as carrier gas at a flow rate of 40 ml/min. The gas chromatographic peaks were identified on the basis of their retention time ratios relative to methyl stearate and other predetermined authentic samples.
Statistical analysis
Student’s t test was used for statistical significance between controls and chronic alcoholics (*p < 0.05, **p < 0.01 and ***p < 0.001). Values represent mean ± SEM of 24 subjects in each group. Correlations between variables were assessed with Pearson’s correlation coefficients (r).
Results
Metabolic changes in blood
Unlike general anesthetics, alcohol is consumed in large quantities over longer durations. As a consequence, it continuously and readily enters the circulation exposing the blood (plasma, red blood cell (RBC), white blood cell, platelets, etc.) and also elevating blood alcohol (ethanol) levels thereby affecting the constituents of plasma and blood cells. Measurement of blood metabolites of intermediary metabolism and analysis of blood cells and their constituents provide valuable information related to the effects of alcohol and metabolic status of the subject. Hence in the present study, metabolic profile of blood such as glucose, pyruvate, lactate and plasma free amino acids and free fatty acids were determined. Data presented in Figure 1 show that alcohol consumption did not change blood glucose levels of chronic alcoholics (who are the experimental subjects in the study) when compared with teetotalers (controls). In addition, a significant increase in blood lactate (62%) and a significant decrease in blood pyruvate (64%) in experimental subjects were observed when compared with controls. Furthermore, increased levels of free amino acids (56%) and free fatty acid (84%) were also found in this study when compared with controls (Figure 1).
Effect of alcohol on blood metabolite levels in chronic alcoholics. Values represent mean ± SEM of 24 male volunteers in each group. *Significant difference from controls. *p < 0.05; **p < 0.01; ***p < 0.001.
Alcohol-induced plasma lipid changes
To evaluate the alcohol-induced plasma lipid changes, we analyzed the plasma lipid profile of these two groups. Increased plasma levels of very-low-density lipoprotein (VLDL) cholesterol (40%), TG (40%) and HDL-C (30%) in chronic alcoholics with no significant change in other parameters such as plasma TC and LDL-C when compared with controls are obvious from the present study (Figure 2). A marked increase in plasma mucoproteins (92%) with no significant change in the plasma glycolipid moiety in alcoholics is evident from this study (Table 1).
Alcohol-induced plasma lipid changes. Values represent mean ± SEM of 24 male volunteers in each group. *Significant difference from controls. *p < 0.05; **p < 0.01; ***p < 0.001.
Changes in carbohydrate moieties in controls and chronic alcoholics a
aValues represent mean ± SEM of each group.
bSignificant difference from controls (p < 0.001).
Alcohol-induced adaptive changes in erythrocyte membranes
As membranes are known targets for the action of alcohol, studies were carried out using gas chromatography to identify changes in free fatty acid composition of erythrocyte membrane in chronic alcoholics. Gas chromatographic analysis of fatty acid composition clearly revealed certain changes in the fatty acids of phospholipids of chronic alcoholic erythrocyte membrane, chiefly increments in C16:0 and a decrease in C22:4 and C22:6 fatty acids besides the presence of unidentified fatty acids probably C-24 or C-26 fatty acids in small quantities in chronic alcoholic membranes (Table 2). The presence of very long chain fatty acids in alcoholics is an important finding in the present study. Fatty acid composition of phospholipid moiety suggests no significant alterations in myristic acid (14:0), palmitoleic acid (16:1), linoleic acid (18:2), oleic acid (18:1), arachidonic acid (20: 4) and eicosanoic acid (20:3), with a significant decrease in decosa tetranoic acid (22: 4) and decosa hexanoic acid (22: 6).
Total fatty acid composition of erythrocyte membrane phospholipids in control and chronic alcoholic subjects a
aValues represent mean ± SEM of each group.
bSignificant difference from controls (p < 0.05).
cSignificant difference from controls (p < 0.001).
To ascertain the possible relationship between plasma fatty acids and erythrocyte membrane free fatty acid composition, we performed correlation analysis in these parameters. The relationships between the levels of plasma free fatty acids and erythrocyte membrane altered free fatty acids such as C16:0, C22:4 and C22:6 in chronic alcoholics, as presented in Figure 3. The plasma free fatty acid levels were positively correlated with erythrocyte membrane free fatty acid C16:0 (r = 0.7505, p < 0.001) and negatively correlated with C22:4 (r = −0.6076, p < 0.01) and C22:6 (r = −0.6909, p < 0.01) in chronic alcoholics. But no significant correlation was observed in other parameters.
Correlation analysis between (a) plasma free fatty acids and erythrocyte membrane C16:0 fatty acid, (b) plasma free fatty acids and erythrocyte membrane C22:4 fatty acid and (c) plasma free fatty acids and erythrocyte membrane C22:6 fatty acid in chronic alcoholics. Pearson’s correlation analysis was performed to determine the correlation coefficient (r). Values represent mean ± SEM of eight male volunteers in each group. *Significant difference from controls. *p < 0.05; **p < 0.01; ***p < 0.001.
Discussion
Results of the present study indicated that there is no change in blood glucose levels of chronic alcoholics when compared with teetotalers (controls), indicating a well-maintained glucose homeostatic mechanism. In general, alcohol possesses the ability to induce hypoglycemia in normal fasting subjects mainly because it effectively inhibits gluconeogenesis in liver. 18 The metabolic effects of alcohol (ethanol) are due to a direct action of ethanol or its metabolites, changes in redox state occurring during its metabolism. Maintenance of stable levels of blood glucose is one of the finely regulated homeostatic mechanisms in which various tissues, hormones, enzymes and other factors play a role. In addition, alcohol seems to be interfering with insulin action by modulating the sensitivity of insulin receptor. 19 Besides the above mentioned, the intracellular metabolic adjustments such as inhibition of gluconeogenesis and glycogenesis together are responsible for the observed normoglycemic effect in the present study as glucose homeostatis is a complex process.
Observed increase in blood lactate in the present study is in agreement with earlier reports. It is well known that alcohol oxidation increases the ratio of reduced Nicótinamide adenine dinucletide (NADH) to Nicotinamide adenine dinucleotide (NAD); hence, there is an increased production of lactate in the plasma and in the peripheral tissues. 20 Moreover, increased operation of Cori’s cycle and hypoxic condition 21 might have contributed for the observed lactate accumulation, because lactate utilization via gluconeogenesis is reported to be reduced in chronic alcoholics. 18 The possibility of lactate formation from methyl glyoxalate might also have contributed for the same. 22 There was a significant decrease in the blood pyruvate in the present study, suggesting an active utilization of pyruvate or conversion of pyruvate by the action of acetaldehyde to form acetoin, making pyruvate unavailable for energy purposes. 23
Enhanced levels of plasma free amino acids pointed to either decreased utilization of amino acids for protein synthesis and/or enhanced protein catabolism as both are reported by earlier investigators. 24 Besides, alcohol-induced inhibition of gluconeogenic process by which amino acids are used up is well established, 18,24 and this might be contributing for the observed increase in plasma free amino acids in the present study.
A twofold increase in plasma free fatty acids of chronic alcoholics when compared with controls suggests inhibition of oxidation of free fatty acids representing their placement by ethanol as an obligatory mitochondrial fuel. Furthermore, the generated H+ equivalents as by-products, which lead to the stimulation of de novo fatty acid synthesis, may partly contribute for the observed hike in plasma free fatty acids. There are diversified opinions and conflicting results on the plasma free fatty acids and fatty acid mobilization from tissues in alcoholic subjects and animals. Ethanol induces fatty acid synthesis pathways by the activation of sterol regulatory element-binding protein in different cell lines 25 and a hike in the levels of fatty acids by alcohol consumption have also been reported in mice, 1 which may be attributed to increased lipolysis and ketogenesis probably mediated by alcohol-induced insulin resistance 8 . In the present study, the observed hike in free fatty acid levels may be due to a combined/mixed effect resulting from adipose lipolysis and/or inhibition of fatty acid oxidation and/or stimulation of de novo fatty acid synthesis and/or modulation in rates of esterification and triacyl glycerol formation. Moreover, inhibition of gluconeogensis also contributes for the reported effect.
It is interesting to note from the present study that there was no significant change in the plasma TC content in chronic alcoholics. However, from this study, it is pertinent to note a significant hike in HDL-C with no significant alteration in LDL-C, indicating cardioprotective effect in chronic alcoholics. These findings are in confirmatory with the results of some earlier studies. 26
Alcohol consumption is associated with reduced risk for coronary heart disease. 27,28 Epidemiological and physiological data indicate a causal relationship. Various mechanisms of action have been proposed for the protective action of alcohol consumption and more than 50% of the protective action of alcohol is associated with the increase in HDL-C. In addition, it may be involved in hemostatic and vascular wall functioning. One of the other protective mechanisms is by increased serum paraoxonase activity. Paraoxonase is an HDL-associated enzyme that may protect LDL against oxidation. Further, HDL is important in reverse cholesterol transport. In conclusion, alcohol consumption stimulates cellular cholesterol efflux and its esterification in plasma. These effects are mostly independent of the kind of alcohol beverage.
LDL is the major cholesterol carrying particle and is derived from progressive depletion of VLDL and is taken up by high-affinity receptors. 29 Observed increase in plasma TG associated with raised VLDL cholesterol with no change in LDL cholesterol in the present study appear to be inconsistent with changes in other lipoprotein particles in chronic alcoholics. Such changes were reported by earlier investigators among abusive drinkers. 30,31 When taken with food, alcohol also seems to stimulate the transfer of cholesterol ester and TG to TG-rich lipoproteins and HDL, respectively. 32,33 Increased fatty acid mobilization in chronic alcoholics in the present study might also be responsible for increased TG biosynthesis, which results in elevated TG.
In the present study, there is a significant increase in the carbohydrate moiety bound to proteins in chronic alcoholic plasma when compared with controls, suggesting an increased glycosylation of the proteins. Earlier studies 34 have shown an enhanced ethanol-induced glycosylation of proteins in alcohol cirrhotic patients. Furthermore, enhanced glycosylation was attributed to decreased albumin turnover. Chronic alcohol intake decreased liver membrane sialyl transferase and increased plasma sialidase in rats and humans. 35 Such changes in enzymes may impair the integrity of cell surface molecules in many tissues and the immune system. Chronic exposure to alcohol impairs glycoproteins, gangliosides and glycolipids in the cell surface membranes of erythrocyte, liver and brain. 36 Hemolysis in chronic alcoholics as reported by some earlier investigators 37 might be contributing to the reported elevation of glycoprotein/glycoconjugates in plasma in the present study. Although certain enzymes related to the glycoprotein synthesis and degradation and glycoconjugates appear to serve as convenient indirect markers of alcohol ingestion, very limited work was carried out on alcohol’s impact on glycobiology. More information is needed to identify how many impaired glycoconjugates occur during chronic alcohol intake. Furthermore, it is also felt that more research is needed on various aspects related to the mechanisms by which chronic exposure to alcohol impairs glycoproteins. 35
Furthermore, in the present study, gas chromatographic analysis of fatty acid composition was performed to find out the role of altered composition in the observed alterations in membrane fluidity. With all the earlier published data 2,5,6,38 from our group, it clearly points to the decreased membrane fluidization leading to the rigidification of the membrane in chronic alcoholics, which appears to be an adaptive response in chronic alcoholics. Although our study was restricted to small population of Indian men and findings may not be generalized with other ethnicities. Probably, diet might be a factor for the observed alterations. Furthermore, fatty acid analysis clearly revealed certain changes in the fatty acids of phospholipids of chronic alcoholic erythrocyte membrane, chiefly increments in C16:0 and a decrease in C22:4 and C22:6 fatty acids besides the presence of unidentified fatty acids, probably C-24 or C-26 fatty acids in small quantities in chronic alcoholic membranes, which suggest the role of these fatty acids for the observed decrease in fluidity. Ethanol ingestion decreased fatty acid 22:6 percentage in microvillus membranes of mice which are also fed with the fish oil, 39 suggesting the type of dietary fat taken along with alcohol, which would also influence the fatty acid composition. It has been reported that fatty acid composition of RBC fatty acid ethyl esters changes over time following ethanol ingestion; these alterations in membrane structure may lead to pathologic changes in RBC membrane function. 40 In contrary to this, increased 22:6 fatty acids were reported in the serum of mice that are fed with diet in which ethanol was added to account for 27.5% of total calories. 1 The changes detected by us in chronic erythrocyte membranes especially palmitic acid (C16) is very much important in determining certain biophysical properties of membranes such as membrane fluidity. It has been reported that 41 increased C16:0 fatty acids were positively correlated with LDL-C, which was found to be increased in chronic alcoholic subjects in the present study. In addition, C16:0 fatty acids were positively correlated with plasma free fatty acid levels suggesting relative contribution of plasma free fatty acids in the incorporation of chronic alcoholic erythrocyte membranes, thereby making the membrane more rigid. In contrary, both C22:4 and C22:6 membrane fatty acids were negatively correlated with plasma free fatty acid levels. It is clearly known that saturated fatty acids pack together significantly tighter than unsaturated fatty acids because the lack of double bonds makes them more linear and less kinky. The kinks in the unsaturated fatty acids reduce their ability to pack together. The increase in C16:0 fatty acids in the RBC membranes in chronic alcoholics may be due to preferential uptake of palmitic acid in relation to other fatty acids into the RBC membranes 40 and is an adaptive response in chronic alcoholics when compared with controls. This clearly suggests that the role of C16:0 may be a discriminative determinant of erythrocyte membrane protection in chronic alcoholics in the present study. However, the results of our study should be viewed as the starting points for future studies on the mechanism of these alterations and their potential pathologic functions.
Footnotes
Funding
This work was supported in part by the University Grants Commission (grant no. F-3-11/97), New Delhi, India.
Declaration of conflict of interests
The authors declared no conflicts of interest.