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Serum apolipoprotein levels in relation to acute myocardial infarction and its risk factors Determination of apolipoprotein D
631
Atherosclerosis, 37 (1980) 631-636 0 Elsevier/North-Holland Scientific Publishers, Ltd.
Preliminary
Note
SERUM APOLIPOPROTEIN LEVELS IN RELATION TO ACUTE MYOCARDIAL INFARCTION AND ITS RISK FACTORS Determination
of Apolipoprotein
D
OLLE WIKLUND, GUNNAR FAGER, SVEN-OLOF OLOFSSON, CLAES WILHELMSSON and GORAN BONDJERS Arterial Biology Group, Department Preventive Cardiology, Department Gijteborg (Sweden)
of Medicine of Medicine,
Land Medical Biochemistry Ostra sjukhuset, University
and Section of Gbteborg,
of
(Received 5 March, 1980) (Revised, received 10 June, 1980) (Accepted 11 June, 1980)
Summary Apolipoprotein A-I, A-II and apoD are all primarily found in the density region d > 1.063 g/ml. In the present study the serum apoD level was determined by electroimmunoassay in a random population sample of middle-aged men (n = 76). The mean level was 0.075 g/l with a standard deviation of 0.017. The apoD level was also determined in a group of patients, in the same age range, with sustained acute myocardial infarction (n = 25). The patients were compared with the random population sample and with a control group matched to the patients with regard to age, serum cholesterol level and body weight index. There was no difference in apoD level between patients and either control group. This is in contrast with the earlier reported low apoA-I, A-II as well as alphalipoprotein cholesterol levels in the same patient group. Key words:
Apolipoprotein
D - Electroimmunoassay
- Myocardial
This study was supported by grants from the Swedish Medical the Bank of Sweden Tercentenary Fund, the Swedish Association Swedish Oleo-Margarine Foundation for Nutritional Research, the University of GGteborg, Sweden. Address for correspondence: OIIe WikIund M.D., Department of 45 GSteborg, Sweden.
infarction
Research Council (project no. 4531). against Heart and Chest Diseases, the Medical Society of Gijteborg and the Medicine I. Sahlgren’s Hospital, S-413
632
Introduction We have previously reported the serum apoA-I and A-II levels in randomly selected middle-aged men as well as in survivors of acute myocardial infarction [l-3]. In this study, serum apoD levels have been determined by electroimmunoassay; The study was confined to the same clinical groups as the apoA-I [ 1,2] and A-II [ 31 studies and to aliquots of exactly the same serum samples. Material and Methods Isolation
of apolipoprotein
D (apoD)
The method used was a modification of that described by McConathy and Alaupovic [4]. HDL was isolated and delipidized as described previously [ 11, and resolubilized in 0.001 M phosphate buffer, pH 8.0, containing 8 M urea. Prior to chromatography, the solution was diluted with phosphate buffer to a final urea concentration of 2 M. The diluted sample was applied to a hydroxylapatite (Bio Rad, Richmond, CA, U.S.A.) column (25 X 1.5 cm) equilibrated with the phosphate buffer. The unretained fraction was recovered and rechromatographed on hydroxylapatite under the same conditions. The final unretained fraction was desalted on Sephadex G25 (Pharmacia Fine Chemicals, Sweden) equilibrated in 2 M acetic acid and eluted in the same buffer. After lyophilization it was resolubilized in 0.001 M phosphate buffer, pH 8.0, containing 8 M urea and chromatographed on Bio Gel P 100 (Bio Rad, Richmond, CA, U.S.A.) equilibrated with the same buffer. The apoD fraction from this chomatography was again desalted on Sephadex G25 and lyophilized. The purified polypeptide showed one band on basic and acidic polyacrylamide gel electrophoresis [ 5,6]. Its migration on basic polyacrylamide gel electrophoresis was similar to that reported for apoD [4,7]. This migration is shared by other polypeptides within the lipoprotein system, mainly apoF [8] and the A-II monomer. However, the polypeptide reacted in double immunodiffusion with its own antiserum but failed to react with antisera to other apolipoproteins, including apoF. The preparation also reacted with antiserum to apoD, obtained by the courtesy of Dr. P. Alaupovic, Oklahoma City. Preparation
of antiserum
Antiserum was prepared in New Zealand cedure described by Fager et al. [ 11. Protein
white rabbits according
to the pro-
and lipid analyses
Methods for determination of protein [9], serum cholesterol [lo], serum triglycerides [ 111 and alphalipoprotein cholesterol [ 121 have been described in a previous publication [ 121, as have the electroimmunoassays of apoA-I and A-II [ 1,3]. Electroimmunoassay
of apoD
Electroimmunoassay [13] was performed in l-5% agarose gel (Indubiose A 37, L’industrie Biologique Francaise, France) containing 5% Dextran T 10 (Pharmacia Fine Chemicals, Sweden) in 0.05 M barbital buffer, ionic strength
633
I
I
0.0025
CONCENTRATION
I
0.0050 OF APO Cl (g/l)
I
0.0075
L__I__I_---_-L-_-_-----I
1180 1140
DILUTION
l/20
OF SERUM
‘110
Fig. 1. Standardization of the reference serum against purified apoD. Regression between rocket height (y) and apoD content (z) of the apoD preparation ( 0-o) (y = 16762 + 0.25, r = 0.99, n = 24) and between rocket height (Y) and dilution of reference serum (X) (X- - - - - -X) (y = 174x + 1.55, r = 1.00, n = 28). Inset shows electroimmunoassay of apoD. a: Dilution series of purified apoD (0.0090. 0.0045, 0.0023 and 0.0012 g/l); b: Dilution series of the reference serum (1710, l/20, l/40 and l/80); c: Dilution series of HDL (0.36, 0.18 and 0.09 g HDL protein/l).
0.005, pH 8.5. Electrophoresis was carried out at 7 V/cm for 7 h. Unless otherwise stated, serum samples were run in dilution l/20. Purified and lyophilized apoD was solubilized in a small volume of 0.1 M ammonium carbonate and diluted with barbital buffer. A reference serum was [l] run on all plates in a dilution series for the construction of a standard curve as described before [l] (Fig. 1). Serum was obtained after a 12 h fast, and stored at -85°C in rubber-stoppered glass tubes. All analyses were carried out 6 months after sampling and within a period of 2 weeks. Study population Selection and characteristics of the study groups have been described in detail elsewhere [12]. The patients with acute myocardial infarction (AMI) comprised men who suffered their first myocardial infarction at 40-44 years of age between January 1,1975 and December 31,1977. Two control groups were selected from a random sample of the male population of Goteborg, Sweden, born between 1930 and 1937 (population size 18,612). One control group was selected at random and will be referred to as the reference group (n = 76) (RG). In the other control group, the matched control group (MC), two subjects were allotted for each AM1 patient and matched for serum cholesterol, age and body weight index (n = 47, three subjects from different control pairs had to be excluded [ 121). Basic characteristics of the groups have been given elsewhere [ 121. Data on serum lipids and lipoproteins are given in Table 1.
634 TABLE
1
LIPOPROTEIN AND
Serum
MATCHED
IN
CONTROL
(MC)
cholesterol
cholesterol
triglycerides A-I
ApoIipoprotein
A-II
Apolipoprotein
D (g/l)
when
GROUPS
(mmol/l)
(mmol/l)
ApoIipoprotein
Pvalues
REFERENCE
(mmol/l)
Alphalipoprotein Serum
VARIABLES
(g/l) (g/l)
compared
with
AMI:
*
(RG), (mean
ACUTE
MYOCARDIAL
INFARCTION
(AMI)
f SD)
RG
AMI
MC
(n = 76)
(n = 25)
(n = 47)
5.77
+ 0.89
6.36
f 1.37
6.43
f 0.93
1.52
? 0.30
***
1.26
+ 0.23
1.41
+ 0.26
1.50
f 1.07
**
2.56
f 1.93
1.87
+ 1.14
*
1.75
f 0.20
**
1.56
+ 0.27
1.66
f 0.29
*
0.46
+ 0.10
***
0.37
+ 0.10
0.44
+ 0.14
**
0.015
f 0.017
0.071
f 0.012
0.010
+ 0.017
P < 0.05.
**P
<
0.01,
***
P < 0.001
Statistical methods Non-parametric statistics were used for correlation analyses and for significance analysis between groups [ 12,141. Results Characterization of antiserum to human apoD On immunodouble diffusion, rabbit antiserum to human precipitation arcs to purified apoD, HDL and whole serum, tion with purified apoA-I, A-II, C-I, C-II, C-III, apoE or LpB. electrophoresis of human serum against anti-apoD gave only line.
apoD gave single but no precipitaCrossed immunoone precipitation
Elec troimmunoassay of apoD At electroimmunoassay of apoD, whole serum gave single and well defined precipitations (Fig. 1). When purified apoD and serum were run in the same electroimmunoassay system, the precipitates differed slightly in appearance with a somewhat broader precipitation line (Fig. 1). Dilution series of purified apoD and serum gave a linear relationship to rocket height within the analyzed range (0.001-0.12 g/l) (Fig. 1). The apoD concentration of the reference serum calculated from two different apoD preparations was equivalent to 0.097 g/l (0.095 and 0.098 g/l, respectively) of lipidfree apoD. The coefficient of variation for samples run in duplicate on the same plate (n = 84). Coefficient of variation for the rocket heights of different was 2.8% dilutions of the reference serum from 5 plates was 3.4% (5.8% in dilution l/40 and 2.1% in dilution l/5). Serum apoD levels in RG The mean value of the apoD levels in RG was 0.075 g/l with a standard deviation of 0.017 (median value = 0.070 g/l) (Table 1). Values were unequally distributed about the mean (coefficient of skewness = 1.27, P < 0.01). Kurtosis significantly deviated from normal distribution (coefficient of kurtosis = 5.3, p < 0.01). There was an inverse correlation between apoD levels and smoking scores
635
(r = -0.24, P< 0.05) and between apoD and serum triglyceride levels (r = -0.30, P< 0.05). The apoD levels were positively correlated to alphalipoprotein cholesterol (r = 0.53, P< O.OOl), apoA-I (r = 0.37, P < 0.01) and A-II (r = 0.38, P < 0.01) levels. ApoD
level in AMI compared
with controls
The apoD level in AM1 was 0.071 g/l (SD = 0.012; median = 0.074) was not significantly different from any of the control groups.
which
Discussion In the present study, electroimmunoassay was performed as described by [15]. A reference serum was used as a Laurel1 [ 131 with some modifications standard. To obtain units that may be comparable to those of other investigators, the reference serum was standardized against purified apoD. Similar results were obtained with two different preparations of the apolipoprotein. There was a small difference in the appearance of the rockets between serum and apoD. Therefore, the results of the apoD determinations in serum should be regarded as relative to apoD rather than as absolute concentrations. Bearing these considerations in mind we found the serum concentration of apoD in middle-aged men to be 0.075 g/l. This is lower than the levels reported by Curry et al. (0.10 g/l) [15]. The discrepancy in apoD levels may be explained by differences in the populations studied as well as in the assay systems and in the standardization procedure. ApoA-I, A-II and apoD are all primarily found in the density region d > 1.063 g/ml [ 151. However, apoD seems to form its own lipoprotein family [ 151. ApoA-I, A-II as well as alphalipoprotein cholesterol levels have been found to be low in the patients with sustained myocardial infarction [2,3,12]. In contrast, no difference could be observed in apoD levels between patients and controls. This emphasizes the importance of regarding HDL as heterogeneous with regard to the lipoprotein families. References 1 Fager. G., Wiklund, 0.. Olofsson, S.-O., Norfeldt, P.-I., Vedin, A. and Bondiers. G., Quantitation of human serum apolipoprotein A-I by electroimmunoassay - Studies on some techniques for standardization of the assay and determination of serum apolipoprotein A-I levels in a random population of middle-aged men. Stand. J. Clin. Lab. Invest.. 40 (1980) 451. 2 Fager, G., Wiklund, 0.. Olofsson. S.-O., Wilhelmsson. C. and Bondjers, G., Serum apolipoprotein levels in relation to acute myocardial infarction and its risk factors - Apolipoprotein A-I levels in male survivors of myocardial infarction, Atherosclerosis, 36 (1980) 67. 3 Fager, G., Wiklund, O., Olofsson. S.-O.. Wilhelmsen, L. and Bond&s, G., Serum apolipoprotein levels in relation to acute myocardial infarction and its risk factors - Determination of polypeptides A-II, Artery, 6 (1980) 188. 4 McConathy, W.J. and Alaupovic. P., Isolation and partial characterization of apolipoprotein D - A new protein moiety of the human plasma lipoprotein system, FEBS L&t.. 37 (1973) 178. 5 Davis, B.J., Disc electrophoresis. Part 2 (Method and application to human serum proteins). Ann. N.Y. Acad. Sci., 121 (1964) 404. 6 Reisfeld, R.A., Lewis, V.J. and Williams. D.E., Disc electrophoresis of basic proteins and peptides on polyacrylamide gels, Nature (Land.), 195 (1962) 281. 7 Olofsson, S.-O. and Gustafson, A., Studies on human serum high-density lipoproteins, Part 2 (Isolation of subfraction in the cold), Stand. J. Clin. Lab. Invest., 34 (1974) 257.
636 8 Olofsson, S.-O., McConathy. W.J. and Alaupovic. P.. Isolation and partial characterization of a new acidic apollpoprotein (apolipoprotein F) from high density lipoproteins of human plasma, Biochemistry, 17 (19’78) 1032. 9 Lowry, O.H.. Rosebrough. N.J. Farr, A.L. and Randall, R.J.. Protein measurements with the Folin phenol reagent, J. Biol. Chem., 193 (1951) ‘265. 10 Bomer, K. and Klose, S., Enzymatic Bestimmung des Gesamtcholesterins mit dem Greiner Selective Analyzer (GSA-II), J. Clin. Chem. Clin. Biochem., 15 (1977) 121. 11 Wahlefeld, A.W.. Triglyceride - Bestimmung nach enzymatischer Verseifung. In: H.K. Bergmeyer (Ed.), Methods of Enzymatic Analysis, 2nd edition, Academic Press. New York, London, 1974, p. 1831. 12 Wiklund, O., Fager. G., Craig, I.H., Wilhelmsson. C.-E.. Vedin. A., Olofsson, A.-O., Bondjers, G. and Wilhelmsen, L.. Alphalipoprotein cholesterol levels in relation to acute myocardial infarction and its risk factors, Stand. J. Clin. Lab. Invest., 40 (1980) 239. 13 Laurel& C.-B., Electroimmunoassay. Stand. J. CLin. Lab. Invest., 29, Suppl. 124 (1972) 21. 14 Siegel. S., Nonparametric Statistics for Behavioural Sciences. McGraw-Hill, New York, 1956. P. 202. 15 Curry, M.D., McConathy, W.J. and Alaupovic. P.. Quantitative determination of human aPoliPoprotein D by electroimmunoassay and radial immunodiffuslon, Biochim. Biophys. Acta, 491 (1977) 232.
Atherosclerosis, 37 (1980) 631-636 0 Elsevier/North-Holland Scientific Publishers, Ltd.
Preliminary
Note
SERUM APOLIPOPROTEIN LEVELS IN RELATION TO ACUTE MYOCARDIAL INFARCTION AND ITS RISK FACTORS Determination
of Apolipoprotein
D
OLLE WIKLUND, GUNNAR FAGER, SVEN-OLOF OLOFSSON, CLAES WILHELMSSON and GORAN BONDJERS Arterial Biology Group, Department Preventive Cardiology, Department Gijteborg (Sweden)
of Medicine of Medicine,
Land Medical Biochemistry Ostra sjukhuset, University
and Section of Gbteborg,
of
(Received 5 March, 1980) (Revised, received 10 June, 1980) (Accepted 11 June, 1980)
Summary Apolipoprotein A-I, A-II and apoD are all primarily found in the density region d > 1.063 g/ml. In the present study the serum apoD level was determined by electroimmunoassay in a random population sample of middle-aged men (n = 76). The mean level was 0.075 g/l with a standard deviation of 0.017. The apoD level was also determined in a group of patients, in the same age range, with sustained acute myocardial infarction (n = 25). The patients were compared with the random population sample and with a control group matched to the patients with regard to age, serum cholesterol level and body weight index. There was no difference in apoD level between patients and either control group. This is in contrast with the earlier reported low apoA-I, A-II as well as alphalipoprotein cholesterol levels in the same patient group. Key words:
Apolipoprotein
D - Electroimmunoassay
- Myocardial
This study was supported by grants from the Swedish Medical the Bank of Sweden Tercentenary Fund, the Swedish Association Swedish Oleo-Margarine Foundation for Nutritional Research, the University of GGteborg, Sweden. Address for correspondence: OIIe WikIund M.D., Department of 45 GSteborg, Sweden.
infarction
Research Council (project no. 4531). against Heart and Chest Diseases, the Medical Society of Gijteborg and the Medicine I. Sahlgren’s Hospital, S-413
632
Introduction We have previously reported the serum apoA-I and A-II levels in randomly selected middle-aged men as well as in survivors of acute myocardial infarction [l-3]. In this study, serum apoD levels have been determined by electroimmunoassay; The study was confined to the same clinical groups as the apoA-I [ 1,2] and A-II [ 31 studies and to aliquots of exactly the same serum samples. Material and Methods Isolation
of apolipoprotein
D (apoD)
The method used was a modification of that described by McConathy and Alaupovic [4]. HDL was isolated and delipidized as described previously [ 11, and resolubilized in 0.001 M phosphate buffer, pH 8.0, containing 8 M urea. Prior to chromatography, the solution was diluted with phosphate buffer to a final urea concentration of 2 M. The diluted sample was applied to a hydroxylapatite (Bio Rad, Richmond, CA, U.S.A.) column (25 X 1.5 cm) equilibrated with the phosphate buffer. The unretained fraction was recovered and rechromatographed on hydroxylapatite under the same conditions. The final unretained fraction was desalted on Sephadex G25 (Pharmacia Fine Chemicals, Sweden) equilibrated in 2 M acetic acid and eluted in the same buffer. After lyophilization it was resolubilized in 0.001 M phosphate buffer, pH 8.0, containing 8 M urea and chromatographed on Bio Gel P 100 (Bio Rad, Richmond, CA, U.S.A.) equilibrated with the same buffer. The apoD fraction from this chomatography was again desalted on Sephadex G25 and lyophilized. The purified polypeptide showed one band on basic and acidic polyacrylamide gel electrophoresis [ 5,6]. Its migration on basic polyacrylamide gel electrophoresis was similar to that reported for apoD [4,7]. This migration is shared by other polypeptides within the lipoprotein system, mainly apoF [8] and the A-II monomer. However, the polypeptide reacted in double immunodiffusion with its own antiserum but failed to react with antisera to other apolipoproteins, including apoF. The preparation also reacted with antiserum to apoD, obtained by the courtesy of Dr. P. Alaupovic, Oklahoma City. Preparation
of antiserum
Antiserum was prepared in New Zealand cedure described by Fager et al. [ 11. Protein
white rabbits according
to the pro-
and lipid analyses
Methods for determination of protein [9], serum cholesterol [lo], serum triglycerides [ 111 and alphalipoprotein cholesterol [ 121 have been described in a previous publication [ 121, as have the electroimmunoassays of apoA-I and A-II [ 1,3]. Electroimmunoassay
of apoD
Electroimmunoassay [13] was performed in l-5% agarose gel (Indubiose A 37, L’industrie Biologique Francaise, France) containing 5% Dextran T 10 (Pharmacia Fine Chemicals, Sweden) in 0.05 M barbital buffer, ionic strength
633
I
I
0.0025
CONCENTRATION
I
0.0050 OF APO Cl (g/l)
I
0.0075
L__I__I_---_-L-_-_-----I
1180 1140
DILUTION
l/20
OF SERUM
‘110
Fig. 1. Standardization of the reference serum against purified apoD. Regression between rocket height (y) and apoD content (z) of the apoD preparation ( 0-o) (y = 16762 + 0.25, r = 0.99, n = 24) and between rocket height (Y) and dilution of reference serum (X) (X- - - - - -X) (y = 174x + 1.55, r = 1.00, n = 28). Inset shows electroimmunoassay of apoD. a: Dilution series of purified apoD (0.0090. 0.0045, 0.0023 and 0.0012 g/l); b: Dilution series of the reference serum (1710, l/20, l/40 and l/80); c: Dilution series of HDL (0.36, 0.18 and 0.09 g HDL protein/l).
0.005, pH 8.5. Electrophoresis was carried out at 7 V/cm for 7 h. Unless otherwise stated, serum samples were run in dilution l/20. Purified and lyophilized apoD was solubilized in a small volume of 0.1 M ammonium carbonate and diluted with barbital buffer. A reference serum was [l] run on all plates in a dilution series for the construction of a standard curve as described before [l] (Fig. 1). Serum was obtained after a 12 h fast, and stored at -85°C in rubber-stoppered glass tubes. All analyses were carried out 6 months after sampling and within a period of 2 weeks. Study population Selection and characteristics of the study groups have been described in detail elsewhere [12]. The patients with acute myocardial infarction (AMI) comprised men who suffered their first myocardial infarction at 40-44 years of age between January 1,1975 and December 31,1977. Two control groups were selected from a random sample of the male population of Goteborg, Sweden, born between 1930 and 1937 (population size 18,612). One control group was selected at random and will be referred to as the reference group (n = 76) (RG). In the other control group, the matched control group (MC), two subjects were allotted for each AM1 patient and matched for serum cholesterol, age and body weight index (n = 47, three subjects from different control pairs had to be excluded [ 121). Basic characteristics of the groups have been given elsewhere [ 121. Data on serum lipids and lipoproteins are given in Table 1.
634 TABLE
1
LIPOPROTEIN AND
Serum
MATCHED
IN
CONTROL
(MC)
cholesterol
cholesterol
triglycerides A-I
ApoIipoprotein
A-II
Apolipoprotein
D (g/l)
when
GROUPS
(mmol/l)
(mmol/l)
ApoIipoprotein
Pvalues
REFERENCE
(mmol/l)
Alphalipoprotein Serum
VARIABLES
(g/l) (g/l)
compared
with
AMI:
*
(RG), (mean
ACUTE
MYOCARDIAL
INFARCTION
(AMI)
f SD)
RG
AMI
MC
(n = 76)
(n = 25)
(n = 47)
5.77
+ 0.89
6.36
f 1.37
6.43
f 0.93
1.52
? 0.30
***
1.26
+ 0.23
1.41
+ 0.26
1.50
f 1.07
**
2.56
f 1.93
1.87
+ 1.14
*
1.75
f 0.20
**
1.56
+ 0.27
1.66
f 0.29
*
0.46
+ 0.10
***
0.37
+ 0.10
0.44
+ 0.14
**
0.015
f 0.017
0.071
f 0.012
0.010
+ 0.017
P < 0.05.
**P
<
0.01,
***
P < 0.001
Statistical methods Non-parametric statistics were used for correlation analyses and for significance analysis between groups [ 12,141. Results Characterization of antiserum to human apoD On immunodouble diffusion, rabbit antiserum to human precipitation arcs to purified apoD, HDL and whole serum, tion with purified apoA-I, A-II, C-I, C-II, C-III, apoE or LpB. electrophoresis of human serum against anti-apoD gave only line.
apoD gave single but no precipitaCrossed immunoone precipitation
Elec troimmunoassay of apoD At electroimmunoassay of apoD, whole serum gave single and well defined precipitations (Fig. 1). When purified apoD and serum were run in the same electroimmunoassay system, the precipitates differed slightly in appearance with a somewhat broader precipitation line (Fig. 1). Dilution series of purified apoD and serum gave a linear relationship to rocket height within the analyzed range (0.001-0.12 g/l) (Fig. 1). The apoD concentration of the reference serum calculated from two different apoD preparations was equivalent to 0.097 g/l (0.095 and 0.098 g/l, respectively) of lipidfree apoD. The coefficient of variation for samples run in duplicate on the same plate (n = 84). Coefficient of variation for the rocket heights of different was 2.8% dilutions of the reference serum from 5 plates was 3.4% (5.8% in dilution l/40 and 2.1% in dilution l/5). Serum apoD levels in RG The mean value of the apoD levels in RG was 0.075 g/l with a standard deviation of 0.017 (median value = 0.070 g/l) (Table 1). Values were unequally distributed about the mean (coefficient of skewness = 1.27, P < 0.01). Kurtosis significantly deviated from normal distribution (coefficient of kurtosis = 5.3, p < 0.01). There was an inverse correlation between apoD levels and smoking scores
635
(r = -0.24, P< 0.05) and between apoD and serum triglyceride levels (r = -0.30, P< 0.05). The apoD levels were positively correlated to alphalipoprotein cholesterol (r = 0.53, P< O.OOl), apoA-I (r = 0.37, P < 0.01) and A-II (r = 0.38, P < 0.01) levels. ApoD
level in AMI compared
with controls
The apoD level in AM1 was 0.071 g/l (SD = 0.012; median = 0.074) was not significantly different from any of the control groups.
which
Discussion In the present study, electroimmunoassay was performed as described by [15]. A reference serum was used as a Laurel1 [ 131 with some modifications standard. To obtain units that may be comparable to those of other investigators, the reference serum was standardized against purified apoD. Similar results were obtained with two different preparations of the apolipoprotein. There was a small difference in the appearance of the rockets between serum and apoD. Therefore, the results of the apoD determinations in serum should be regarded as relative to apoD rather than as absolute concentrations. Bearing these considerations in mind we found the serum concentration of apoD in middle-aged men to be 0.075 g/l. This is lower than the levels reported by Curry et al. (0.10 g/l) [15]. The discrepancy in apoD levels may be explained by differences in the populations studied as well as in the assay systems and in the standardization procedure. ApoA-I, A-II and apoD are all primarily found in the density region d > 1.063 g/ml [ 151. However, apoD seems to form its own lipoprotein family [ 151. ApoA-I, A-II as well as alphalipoprotein cholesterol levels have been found to be low in the patients with sustained myocardial infarction [2,3,12]. In contrast, no difference could be observed in apoD levels between patients and controls. This emphasizes the importance of regarding HDL as heterogeneous with regard to the lipoprotein families. References 1 Fager. G., Wiklund, 0.. Olofsson, S.-O., Norfeldt, P.-I., Vedin, A. and Bondiers. G., Quantitation of human serum apolipoprotein A-I by electroimmunoassay - Studies on some techniques for standardization of the assay and determination of serum apolipoprotein A-I levels in a random population of middle-aged men. Stand. J. Clin. Lab. Invest.. 40 (1980) 451. 2 Fager, G., Wiklund, 0.. Olofsson. S.-O., Wilhelmsson. C. and Bondjers, G., Serum apolipoprotein levels in relation to acute myocardial infarction and its risk factors - Apolipoprotein A-I levels in male survivors of myocardial infarction, Atherosclerosis, 36 (1980) 67. 3 Fager, G., Wiklund, O., Olofsson. S.-O.. Wilhelmsen, L. and Bond&s, G., Serum apolipoprotein levels in relation to acute myocardial infarction and its risk factors - Determination of polypeptides A-II, Artery, 6 (1980) 188. 4 McConathy, W.J. and Alaupovic. P., Isolation and partial characterization of apolipoprotein D - A new protein moiety of the human plasma lipoprotein system, FEBS L&t.. 37 (1973) 178. 5 Davis, B.J., Disc electrophoresis. Part 2 (Method and application to human serum proteins). Ann. N.Y. Acad. Sci., 121 (1964) 404. 6 Reisfeld, R.A., Lewis, V.J. and Williams. D.E., Disc electrophoresis of basic proteins and peptides on polyacrylamide gels, Nature (Land.), 195 (1962) 281. 7 Olofsson, S.-O. and Gustafson, A., Studies on human serum high-density lipoproteins, Part 2 (Isolation of subfraction in the cold), Stand. J. Clin. Lab. Invest., 34 (1974) 257.
636 8 Olofsson, S.-O., McConathy. W.J. and Alaupovic. P.. Isolation and partial characterization of a new acidic apollpoprotein (apolipoprotein F) from high density lipoproteins of human plasma, Biochemistry, 17 (19’78) 1032. 9 Lowry, O.H.. Rosebrough. N.J. Farr, A.L. and Randall, R.J.. Protein measurements with the Folin phenol reagent, J. Biol. Chem., 193 (1951) ‘265. 10 Bomer, K. and Klose, S., Enzymatic Bestimmung des Gesamtcholesterins mit dem Greiner Selective Analyzer (GSA-II), J. Clin. Chem. Clin. Biochem., 15 (1977) 121. 11 Wahlefeld, A.W.. Triglyceride - Bestimmung nach enzymatischer Verseifung. In: H.K. Bergmeyer (Ed.), Methods of Enzymatic Analysis, 2nd edition, Academic Press. New York, London, 1974, p. 1831. 12 Wiklund, O., Fager. G., Craig, I.H., Wilhelmsson. C.-E.. Vedin. A., Olofsson, A.-O., Bondjers, G. and Wilhelmsen, L.. Alphalipoprotein cholesterol levels in relation to acute myocardial infarction and its risk factors, Stand. J. Clin. Lab. Invest., 40 (1980) 239. 13 Laurel& C.-B., Electroimmunoassay. Stand. J. CLin. Lab. Invest., 29, Suppl. 124 (1972) 21. 14 Siegel. S., Nonparametric Statistics for Behavioural Sciences. McGraw-Hill, New York, 1956. P. 202. 15 Curry, M.D., McConathy, W.J. and Alaupovic. P.. Quantitative determination of human aPoliPoprotein D by electroimmunoassay and radial immunodiffuslon, Biochim. Biophys. Acta, 491 (1977) 232.