- Email: [email protected]
Serum apolipoprotein A-II, a biochemical indicator of alcohol abuse
CIinica &mica Elsevier
Acta, 185 (1984) 185-189
185
CCA 03036
Serum apolipoprotein A-II, a biochemical indicator of alcohol abuse Pascal Puchois a, Michel Fontan ‘, Jean-Louis Gentilini b, Philippe Gelez a, Jean-Charles Fruchart a,* a Departement Lipides, Lipoproreines, Normolipoproteinemiants et C. T. B. INSERM, Faculte de Pharmacie, 3, rue du Professeur Laguesse, 50945 Lille b Luboratoire de Biologie du Centre des Examens de Sante, Institut Pasteur de Lille, 15, rue Camille Guerin, 59019 Lille and’ Service de Physchiatrie Gets&ale, Unites de Soins Normalisees ‘B’, Centre Hospitalier Regional, 6, rue du Professeur Luguesse, 59037 Lille (France) (Received February 27th, 1984; revision June 13th, 1984) Key words: Alcohol abuse; High density lipoprotein; Apoprotein A- II
Summary
The possible use of high density lipoprotein (HDL) cholesterol, HDL phospholipids, apolipoproteins (APO) A-I and A-II as markers of alcohol abuse was studied in 78 intemperate drinkers. The mean value for each of these parameters was higher in drinkers than in control subjects. The most significant increase was observed in the plasma apo A-II levels (+ 45%). A composite index of gammaglutamyltranspeptidase (GGTP) and apo A-II was superior to GGTP alone in discriminating drinkers ( + 14%). Moreover, apo A-II assay is simple to perform.
Introduction
Due to the medicosocial impact of alcohol abuse, early detection of intemperate drinkers represents the best approach to the prevention of alcoholism. Recently, evaluation of high density lipoprotein (HDL) cholesterol has been proposed as a new index of alcohol consumption [1,2]. Although the exact mechanisms by which alcohol exerts its effects on plasma HDL are not well understood, nevertheless, the alcohol induced increase in the HDL concentration [3] could be a useful marker, and it has been suggested that the measurement of HDL cholesterol should be used in conjunction with other tests in order to confirm the suspected diagnosis of excessive alcohol consumption [4]. In the present study, we have determined, in addition to the HDL cholesterol, * To whom correspondence
0009-8981/84/$03.00
should be addressed.
0 1984 Elsevier Science Publishers B.V.
186
HDL phospholipids and plasma apolipoprotein A-I and A-II levels in intemperate drinkers. We have studied the sensitivity of each parameter as a biochemical indicator of alcohol abuse, separately and in combination with evaluation of plasma gammaglutamyltranspeptidase (GGTP). Materials and methods Subjects The study was carried out at the Institute Pasteur (Lille, France) where people
come for a health control examination. All subjects received a full clinical and biochemical investigation. None of the subjects retained in our study exhibited diseases such as hyperlipidaemia, obesity, diabetes, liver disease, etc., which could affect metabolism of lipoproteins. Subjects who were taking hypolipidaemic drugs were also excluded. Criteria for a subject’s acceptance into the ‘drinker’s’ population included a minimal alcohol consumption of 450 g weekly. The consumption data were obtained during a standardized interview. Subjects taking medications known to affect GGTP levels were excluded. Thus, our ‘drinker’s’ population was composed of 79 subjects, 53 males with a mean age of 35 + 10 yr (mean f SD) and 26 females, mean age 39 f 7 yr. Among these 79 subjects, 41 were scoring > 4 in the Le Go screening test
PI. A control group of non-alcoholic subjects selected by Siest and Petit Clerc’s method [6], was also studied. The control group had a similar distribution of sex, mean ages, relative weights, dietary and cigarette consumption and physical activity as the ‘drinkers’. Laboratory
analytic methods
Blood samples, obtained in the morning after a 12-h overnight fast, were allowed to clot at room temperature. Lipoprotein fractions were separated by selective precipitation with sodium phosphotungstate magnesium at pH 6.15, and cholesterol and phospholipid concentrations were determined by enzymatic methods [7,8]. Apolipoprotein A-I was measured by an electroimmunoassay on prepared plates (apofilm A-I, Sebia, Issy les Moulineaux, France) as described previously [9]. Quantification of apolipoprotein A-II was performed by electroimmunoassay. The monospecific antisera to A-II was prepared by immunizing rabbits with the peptide A-II isolated according to a procedure described recently [lo]. This electroimmunoassay was also performed on plates ready to use (apofilm A-II, Sebia, Issy les Moulineaux, France). Plates were kept and rehydrated 30 min and buffered in a phosphate buffer, pH 9.2, just before using. Within and between assay precisions were excellent for this assay [lo]. Results Effects of alcohol consumption on plasma HDL and apolipoprotein A-I and A-II concentration The plasma value for HDL cholesterol and phospholipid contents and plasma
187
apolipoproteins A-I and A-II levels of intemperate drinkers compared to control subjects are shown in Table I. There was a significant increase of 21 and 16% in the levels of HDL cholesterol and phospholipids, respectively, in drinkers. A small, but significant, increase was observed in the plasma apo A-I concentration of drinkers. The most predominant effect of alcohol consumption was in the plasma apo A-II concentration which showed a 45% increase in test subjects compared to the control group. Sensitivity of determination HDL levels for the detection of intemperate drinkers If two standard deviations above the mean of the controls are considered as the arbitrary upper limit of normal, results (Table II) indicate that each test allows detection of a number of intemperate drinkers. The results obtained for the different parameters in decreasing order for success were: GGTP, apo A-II, apo A-I, HDL phospholipid, HDL cholesterol. Whereas, only 19% of the drinkers had HDL cholesterol levels above 0.92 g/l or > 2 SD above the normal mean, 42% had abnormally high apo A-II levels. Discriminant analysis was conducted in order to determine the effectiveness of utilization of the various biochemical indices to detect intemperate drinkers. As TABLE I High density lipoprotein cholesterol, phospho~pids and plasma a~tipoproteins control subjects and in ‘drinkers’
Control (n =lOO) ‘ Drinkers’ (n = 79) Mean increase
A-I and A-II levels in
HDL cholesterol
HDL phospholipids
Apoprotein A-I
Apoprotein A-II
(mmol/l)
(mmol/l)
(g/l)
(g/l)
1.50 + 0.44
1.28+0.27
1.38 + 0.12
1.81+ 0.57 *
1.48 “t 0.41 *
1.47+0.29
+21%
The results are expressed subjects.
i SD.
** p
*+
i
0.71+0.2!3
*
+45%
+6.5%
+ 16%
as mean
0.49+0.11
0.01; * p c 0.001 for differences from values of control
TABLE II Percentage of drinkers with abnormally high levels for the different parameters GGTP (lop9 Katal/l) X’ %
633 59
HDL cholesterol
HDL phospholipids
Apoprotein A-I
Apoprotein A-II
(mmol/l)
(mmol/l)
(g/l)
(g/l)
1.82 24
1.62 28
0.71 42
2.38 19
x ’ = mean of the controls plus 2
SD.
188
TABLE III Percentage of drinkers with normal GGTP and abnormally A-I and A-II HDL Cholesterol 6.3
!%
HDL Phospholipids 7.6
high levels for lipids of HDL and apoprotein
Apoprotein A-I 10.1
Apoprotein A-II 13.9
shown in Table III a more detailed examination of the data indicated that 13.9% of subjects had abnormal plasma apo A-II levels and normal GGTP. Therefore, if evaluation of GGTP alone allows detection of 59% of drinkers, simultaneous determination of GGTP and apo A-II will increase the level of detection to 72.9%. Discussion
In the present study, we have observed that in addition to HDL cholesterol levels, [ll] alcohol consumption caused an increase in HDL phospholipids and apolipoproteins A-I and A-II. The exact mechanism by which alcohol influences the HDL levels remains poorly understood [12,13]. Nevertheless our results indicated that determination of plasma apo A-II is a more sensitive indicator of a change in the HDL levels in drinkers (Table I) which could reflect an increased production of lipoprotein particles containing mainly apo A-II [14]. The significance of determination of apo A-II levels is further substantiated by our observation that a significant rise in HDL cholesterol levels was detected in only 19% of subjects, whereas 42% had abnormal apo A-II levels (Table II). Moreover, a detailed examination of our data showed that many subjects, who had a normal GGTP, had abnormal levels of other HDL components (Table III). Therefore, combination of both tests, namely GGTP and HDL, allows detection of a greater number of drinkers, than using either test alone. This is especially apparent for the index GGTP-apo A-II, a combination which allows detection of 72.9% of subjects as opposed to the 59% of population of drinkers detected by the GGTP test alone. References Gordon T, Ernst N, Fischer M, Fifkind BM. Alcohol and high density lipoprotein cholesterol. Circulation 1981; 64: 652. Jaqueson A, Richard JL, Ducimetiere P, Wamet JM, Claude JR. High density lipoprotein cholesterol and alcoholic consumption in a french male population. Atherosclerosis 1983; 48: 131-138. Fraser GE, Anderson JT, Foster N, Golberg R, Jacobs D, Blackbum H. The effect of alcohol on serum high density lipoprotein. Atherosclerosis 1983; 46: 275-286. Sanchez-Graig M, Annis HM. Gamma-glutamyltranspeptida.se and high density lipoproteins cholesterol in male problem drinkers: advantages of a composite index for prediction alcohol consumption. Alcoholism Clin Exp Res 1981; 5: 540-544. Le Go JM. Le depistage pr&oce de l’ethylisme. Presse Med 1968; 76: 579-580.
189 6 Siest G, Petit Clerc C. Selection of people for the production of population reference values. Ann Biol Clin 1978; 36: 217-220. 7 Fruchart JC, Beucler I, Blaton V, Canal J, Douste Blazy L. Methode stlectionn& pour le dosage du cholesterol des lipopromines non prkipitables par le phosphotungstate de sodium en presence de chlorure de magn6sium. ISB 1982; 2: 69-72. 8 Puchois P, Duthilleul P, Daunizeau A, Gentilini JL, Fruchart JC. Determination semi-automatique des phospholipides des HDL. Indr~t clinique. Acta Pharmacol Biol 1981; 1: 69-72. 9 Fruchart JC, Kora I, Cachera C, Clavey V, Moschetto Y. Simultaneous measurement of plasma apolipoproteins A-I and B by electroimmunoassay. Clin Chem 1982; 28: 59-62. 10 Kora I, Vanhoutte G, Parra H, Bermann MC, Cachera C, Clavey V, Puchois P, Fruchart JC. Dosage de l’apolipoproteine A-II serique par electroimmunodiffusion sur plaques pretes a l’emploi. Ann Biol Clin (submitted for publication). 11 Hartung GH, Foreyt JP, Mitchell RE, Mitchell JG, Reeves RS, Gotto AM. Effect of alcohol intake on high density lipoprotein cholesterol levels in runners and inactive men. J Am Med Assoc 1983; 246: 747-750. 12 Taskinen MR, Valimaki M, Nikkila EA, Kujusi T, Ehnolm C, YlikaIui R. High density lipoprotein sufractions and postheparin plasma lipases in alcoholic men before and after ethanol withdrawal. Metabolism 1982; 31: 1168-1174. 13 Devenyi P, Robinson G, Roncari DAK. Alcohol and high density lipoproteins. J Can Med Assoc 1980; 123: 981-984. 14 Suenram A, McConathy WJ, Alaupovic P. Evidence for the lipoprotein heterogeneity of human plasma HDL isolated by three different procedures. Lipids 1979; 14: 505-510.
Acta, 185 (1984) 185-189
185
CCA 03036
Serum apolipoprotein A-II, a biochemical indicator of alcohol abuse Pascal Puchois a, Michel Fontan ‘, Jean-Louis Gentilini b, Philippe Gelez a, Jean-Charles Fruchart a,* a Departement Lipides, Lipoproreines, Normolipoproteinemiants et C. T. B. INSERM, Faculte de Pharmacie, 3, rue du Professeur Laguesse, 50945 Lille b Luboratoire de Biologie du Centre des Examens de Sante, Institut Pasteur de Lille, 15, rue Camille Guerin, 59019 Lille and’ Service de Physchiatrie Gets&ale, Unites de Soins Normalisees ‘B’, Centre Hospitalier Regional, 6, rue du Professeur Luguesse, 59037 Lille (France) (Received February 27th, 1984; revision June 13th, 1984) Key words: Alcohol abuse; High density lipoprotein; Apoprotein A- II
Summary
The possible use of high density lipoprotein (HDL) cholesterol, HDL phospholipids, apolipoproteins (APO) A-I and A-II as markers of alcohol abuse was studied in 78 intemperate drinkers. The mean value for each of these parameters was higher in drinkers than in control subjects. The most significant increase was observed in the plasma apo A-II levels (+ 45%). A composite index of gammaglutamyltranspeptidase (GGTP) and apo A-II was superior to GGTP alone in discriminating drinkers ( + 14%). Moreover, apo A-II assay is simple to perform.
Introduction
Due to the medicosocial impact of alcohol abuse, early detection of intemperate drinkers represents the best approach to the prevention of alcoholism. Recently, evaluation of high density lipoprotein (HDL) cholesterol has been proposed as a new index of alcohol consumption [1,2]. Although the exact mechanisms by which alcohol exerts its effects on plasma HDL are not well understood, nevertheless, the alcohol induced increase in the HDL concentration [3] could be a useful marker, and it has been suggested that the measurement of HDL cholesterol should be used in conjunction with other tests in order to confirm the suspected diagnosis of excessive alcohol consumption [4]. In the present study, we have determined, in addition to the HDL cholesterol, * To whom correspondence
0009-8981/84/$03.00
should be addressed.
0 1984 Elsevier Science Publishers B.V.
186
HDL phospholipids and plasma apolipoprotein A-I and A-II levels in intemperate drinkers. We have studied the sensitivity of each parameter as a biochemical indicator of alcohol abuse, separately and in combination with evaluation of plasma gammaglutamyltranspeptidase (GGTP). Materials and methods Subjects The study was carried out at the Institute Pasteur (Lille, France) where people
come for a health control examination. All subjects received a full clinical and biochemical investigation. None of the subjects retained in our study exhibited diseases such as hyperlipidaemia, obesity, diabetes, liver disease, etc., which could affect metabolism of lipoproteins. Subjects who were taking hypolipidaemic drugs were also excluded. Criteria for a subject’s acceptance into the ‘drinker’s’ population included a minimal alcohol consumption of 450 g weekly. The consumption data were obtained during a standardized interview. Subjects taking medications known to affect GGTP levels were excluded. Thus, our ‘drinker’s’ population was composed of 79 subjects, 53 males with a mean age of 35 + 10 yr (mean f SD) and 26 females, mean age 39 f 7 yr. Among these 79 subjects, 41 were scoring > 4 in the Le Go screening test
PI. A control group of non-alcoholic subjects selected by Siest and Petit Clerc’s method [6], was also studied. The control group had a similar distribution of sex, mean ages, relative weights, dietary and cigarette consumption and physical activity as the ‘drinkers’. Laboratory
analytic methods
Blood samples, obtained in the morning after a 12-h overnight fast, were allowed to clot at room temperature. Lipoprotein fractions were separated by selective precipitation with sodium phosphotungstate magnesium at pH 6.15, and cholesterol and phospholipid concentrations were determined by enzymatic methods [7,8]. Apolipoprotein A-I was measured by an electroimmunoassay on prepared plates (apofilm A-I, Sebia, Issy les Moulineaux, France) as described previously [9]. Quantification of apolipoprotein A-II was performed by electroimmunoassay. The monospecific antisera to A-II was prepared by immunizing rabbits with the peptide A-II isolated according to a procedure described recently [lo]. This electroimmunoassay was also performed on plates ready to use (apofilm A-II, Sebia, Issy les Moulineaux, France). Plates were kept and rehydrated 30 min and buffered in a phosphate buffer, pH 9.2, just before using. Within and between assay precisions were excellent for this assay [lo]. Results Effects of alcohol consumption on plasma HDL and apolipoprotein A-I and A-II concentration The plasma value for HDL cholesterol and phospholipid contents and plasma
187
apolipoproteins A-I and A-II levels of intemperate drinkers compared to control subjects are shown in Table I. There was a significant increase of 21 and 16% in the levels of HDL cholesterol and phospholipids, respectively, in drinkers. A small, but significant, increase was observed in the plasma apo A-I concentration of drinkers. The most predominant effect of alcohol consumption was in the plasma apo A-II concentration which showed a 45% increase in test subjects compared to the control group. Sensitivity of determination HDL levels for the detection of intemperate drinkers If two standard deviations above the mean of the controls are considered as the arbitrary upper limit of normal, results (Table II) indicate that each test allows detection of a number of intemperate drinkers. The results obtained for the different parameters in decreasing order for success were: GGTP, apo A-II, apo A-I, HDL phospholipid, HDL cholesterol. Whereas, only 19% of the drinkers had HDL cholesterol levels above 0.92 g/l or > 2 SD above the normal mean, 42% had abnormally high apo A-II levels. Discriminant analysis was conducted in order to determine the effectiveness of utilization of the various biochemical indices to detect intemperate drinkers. As TABLE I High density lipoprotein cholesterol, phospho~pids and plasma a~tipoproteins control subjects and in ‘drinkers’
Control (n =lOO) ‘ Drinkers’ (n = 79) Mean increase
A-I and A-II levels in
HDL cholesterol
HDL phospholipids
Apoprotein A-I
Apoprotein A-II
(mmol/l)
(mmol/l)
(g/l)
(g/l)
1.50 + 0.44
1.28+0.27
1.38 + 0.12
1.81+ 0.57 *
1.48 “t 0.41 *
1.47+0.29
+21%
The results are expressed subjects.
i SD.
** p
*+
i
0.71+0.2!3
*
+45%
+6.5%
+ 16%
as mean
0.49+0.11
0.01; * p c 0.001 for differences from values of control
TABLE II Percentage of drinkers with abnormally high levels for the different parameters GGTP (lop9 Katal/l) X’ %
633 59
HDL cholesterol
HDL phospholipids
Apoprotein A-I
Apoprotein A-II
(mmol/l)
(mmol/l)
(g/l)
(g/l)
1.82 24
1.62 28
0.71 42
2.38 19
x ’ = mean of the controls plus 2
SD.
188
TABLE III Percentage of drinkers with normal GGTP and abnormally A-I and A-II HDL Cholesterol 6.3
!%
HDL Phospholipids 7.6
high levels for lipids of HDL and apoprotein
Apoprotein A-I 10.1
Apoprotein A-II 13.9
shown in Table III a more detailed examination of the data indicated that 13.9% of subjects had abnormal plasma apo A-II levels and normal GGTP. Therefore, if evaluation of GGTP alone allows detection of 59% of drinkers, simultaneous determination of GGTP and apo A-II will increase the level of detection to 72.9%. Discussion
In the present study, we have observed that in addition to HDL cholesterol levels, [ll] alcohol consumption caused an increase in HDL phospholipids and apolipoproteins A-I and A-II. The exact mechanism by which alcohol influences the HDL levels remains poorly understood [12,13]. Nevertheless our results indicated that determination of plasma apo A-II is a more sensitive indicator of a change in the HDL levels in drinkers (Table I) which could reflect an increased production of lipoprotein particles containing mainly apo A-II [14]. The significance of determination of apo A-II levels is further substantiated by our observation that a significant rise in HDL cholesterol levels was detected in only 19% of subjects, whereas 42% had abnormal apo A-II levels (Table II). Moreover, a detailed examination of our data showed that many subjects, who had a normal GGTP, had abnormal levels of other HDL components (Table III). Therefore, combination of both tests, namely GGTP and HDL, allows detection of a greater number of drinkers, than using either test alone. This is especially apparent for the index GGTP-apo A-II, a combination which allows detection of 72.9% of subjects as opposed to the 59% of population of drinkers detected by the GGTP test alone. References Gordon T, Ernst N, Fischer M, Fifkind BM. Alcohol and high density lipoprotein cholesterol. Circulation 1981; 64: 652. Jaqueson A, Richard JL, Ducimetiere P, Wamet JM, Claude JR. High density lipoprotein cholesterol and alcoholic consumption in a french male population. Atherosclerosis 1983; 48: 131-138. Fraser GE, Anderson JT, Foster N, Golberg R, Jacobs D, Blackbum H. The effect of alcohol on serum high density lipoprotein. Atherosclerosis 1983; 46: 275-286. Sanchez-Graig M, Annis HM. Gamma-glutamyltranspeptida.se and high density lipoproteins cholesterol in male problem drinkers: advantages of a composite index for prediction alcohol consumption. Alcoholism Clin Exp Res 1981; 5: 540-544. Le Go JM. Le depistage pr&oce de l’ethylisme. Presse Med 1968; 76: 579-580.
189 6 Siest G, Petit Clerc C. Selection of people for the production of population reference values. Ann Biol Clin 1978; 36: 217-220. 7 Fruchart JC, Beucler I, Blaton V, Canal J, Douste Blazy L. Methode stlectionn& pour le dosage du cholesterol des lipopromines non prkipitables par le phosphotungstate de sodium en presence de chlorure de magn6sium. ISB 1982; 2: 69-72. 8 Puchois P, Duthilleul P, Daunizeau A, Gentilini JL, Fruchart JC. Determination semi-automatique des phospholipides des HDL. Indr~t clinique. Acta Pharmacol Biol 1981; 1: 69-72. 9 Fruchart JC, Kora I, Cachera C, Clavey V, Moschetto Y. Simultaneous measurement of plasma apolipoproteins A-I and B by electroimmunoassay. Clin Chem 1982; 28: 59-62. 10 Kora I, Vanhoutte G, Parra H, Bermann MC, Cachera C, Clavey V, Puchois P, Fruchart JC. Dosage de l’apolipoproteine A-II serique par electroimmunodiffusion sur plaques pretes a l’emploi. Ann Biol Clin (submitted for publication). 11 Hartung GH, Foreyt JP, Mitchell RE, Mitchell JG, Reeves RS, Gotto AM. Effect of alcohol intake on high density lipoprotein cholesterol levels in runners and inactive men. J Am Med Assoc 1983; 246: 747-750. 12 Taskinen MR, Valimaki M, Nikkila EA, Kujusi T, Ehnolm C, YlikaIui R. High density lipoprotein sufractions and postheparin plasma lipases in alcoholic men before and after ethanol withdrawal. Metabolism 1982; 31: 1168-1174. 13 Devenyi P, Robinson G, Roncari DAK. Alcohol and high density lipoproteins. J Can Med Assoc 1980; 123: 981-984. 14 Suenram A, McConathy WJ, Alaupovic P. Evidence for the lipoprotein heterogeneity of human plasma HDL isolated by three different procedures. Lipids 1979; 14: 505-510.