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HDL-cholesterol, apolipoproteins A1 and B
85
Atherosclerosis, 31 (1978) 85-91 @ Elsevier/North-Holland Scientific Publishers, Ltd.
HDL-CHOLESTEROL,
APOLIPOPROTEINS
Al AND B
Age and Index Body Weight
P. AVOGARO,
G. CAZZOLATO,
G. BITTOLO BON, G.B. QUINCI and M. CHINELLO
Unit for Atherosclerosis, Hyperlipaemias Nazionale delle Ricerche, Venice (Italy)
and Diabetes, Regional Hospital, Unit of Consiglio
(Received 3 April, 1978) (Revised, received 5 June, 1978) (Accepted 5 June, 1978)
summary
Some data of previous literature have emphasized a negative correlation between plasma apo Al, HDL cholesterol and coronary heart disease. The present paper stresses a high negative correlation existing both in females and males between values of index body weight (IBW) and plasma levels of HDL cholesterol and apo Al. No correlation has been found between age and, respectively, HDL cholesterol, apo A, and apo B. Key words:
Apolipoproteins
-Body
weight - HDL-cholesterol
Introduction
No data are available concerning the levels of the major apolipoproteins according to age. Few data have stressed some differences existing between the sexes [1,2]. In the course of a study performed in some groups of normal people to ascertain the plasma levels of apo B and apo Al according to sex and age, an inverse relationship between the major apolipoprotein of HDL, namely apo Al, and IBW (index body weight) has been observed both in males and females.
Abbrevations: IBW = index body weight; BFS = blood fasting sugar; BUN = blood urea nitrogen; C = cholesterol; TG = triglyceride; HDL = high density lipoproteins (d > 1.063); ape = apolipoprotein. Requests for reprints should be addressed to P. Avogaro.
86
Materials and Methods Subjects included in this study are both males and females with an age kept between 20 and 60 years. They are part of a population freely submitting to our Unit in a Health Survey Program. An extensive clinical examination was completely negative for all of them. Both the blood pressure and an electrocardiographic record were within normal limits. At a first examination the BFS, the BUN, plasma cholesterol and triglycerides were recorded. From all the people showing normal values of BFS and BUN and values of cholesterol and triglycerides below the 95th percentile for our population [3] one subject was selected at random out of four. The selection was limited to subjects with an IBW between 0.8 and 1.2 and with an age kept between 20 and 60 years. The subjects selected and included in the second phase were 60 males with a mean age of 40.9 yr and an IBW of 1.027 and 60 females with a mean age of 39.6 yr and an IBW of 1.013 (Table 1). The relative weight of these subjects was calculated by dividing the actual weight by the ideal weight according to the tables of Metropolitan Life Insurance Co. [ 41. After a fasting of 12 h a new blood sample was collected in EDTA (1 mg/ 1 ml). The new series of analysis included the determination of cholesterol [ 51, triglyceride [ 61, and HDL cholesterol [ 71. For the determination of HDL cho-
TABLE 1 MEAN VALUES AND CORRELATIONS LIPIDS AND APOLIPOPROTEINS
Age
m
(YY)
iii
IBW Total chol. Total TG HDL
(m&-W (mf6k-W (mg/dl)
iii iii iii
APO A1 APO B
OwAW
iTi iii
IBW IBW IBW IBW IBW Age Ape Age A&
(mg/dl) “8 vs vs vs vs
(yr) (yr) (yr) (YX)
Total chol. TG WDL chol. Total chol. TG
“8
HDL chol APO At Total chol. TG APO B
VS
IBW HDL chol.
“8 vs
APO A1 APO B
vs vs VS “8 VS
APO A1 APO Al APO A1 HDL chol. HDL chol.
OF INDEX
BODY
WEIGHT
AND PLASMA
LEVELS
d(n=60)
?(n=60)
40.98 f 12.6 (range 20-60) 1.027 f 0.11 (range 0.80-1.20)
39.62 f 11.9 (range 20-60) 1.013 f 0.10 (range 0.80-1.20)
212.53 92.26 53.65 128.45 137.69
204.63 2 31.5 82.72 * 25.2 57.93 t 9.7 133.59 + 19.9 138.08 i 27.2
a.73 -0.79 -0.10 -0.01 a.06 0.08 0.07 0.07 0.13 0.25 0.10 0.66 0.16 a.15
f f f k f
31.5 31.0 9.8 21.56 26.1 P < 0.01 P
-0.66 -0.70 0.05 0.20 0.08
P c 0.01 P
n.s. n.s. n.s. n.s.
-0.05 0.01 0.06 0.20
n.s. n.s. n.s. n.s.
P < 0.05 xl.*. P
0.29 0.07 0.63 -0.14 -Q.20
P
< 0.01 n.s. P co.01 as. as.
OF
lesterol the procedure followed was that based upon precipitation with 4% phosphotungstic acid in 1 M NaOH and 2 M MgCl*. Apolipoproteins A, and B were determined in whole plasma adapting the technique of Laurell [8] as described by Curry et al. [ 91. For this purpose we used an LKB 2177 water-coiled Multiphor electrophoresis plate on which 21 samples can be run simultaneously. All the runs were done at 15” at 15 V/cm, in Tris-glycine buffer, pH 8.6. Into the wells was seeded 5 ~1 of suitably diluted standard apo B, of graduated dilutions of apo A1 or of the sample under study. The precipitation peak reached its maximum height in 12 h. For the standardization of the electroimmunoassay of apolipoprotein B the 1040 > d < 1050 fraction was isolated from a pool of plasmas obtained from 20 normolipemic subjects by means of successive ultracentrifugations. One ml aliquots of these fractions were stored in sealed vials containing EDTA and sodium azide at -20°C after testing with anti-apo B, Al, AZ, C and ARP (supplied by Dr. G. Kostner, Graz) and anti-albumin (Boehringer); there was a positive reaction only with anti-apo serum B. The protein concentration of the antigen thus obtained was 0.3 mg/ml. The stability of the concentration of the stored antigen permitted reproducible results through the period of the study. Calibration curves were thus plotted by assaying stepped concentrations of the antigen (from 50 to 200 ,ug of apo B/5 ~1) against a fixed quantity of antiserum dispersed in 1% agarose in Tris-glycine buffer, pH 8.6. The quantity of antiserum was such that at the highest concentration no excess of antigen was detectable. Coefficient of variations for apo B was in the range of 5%. At every
Fig. 1. Rockets of apolipoproteins obtained by the technique of LaurelJ as modified by Curry et al. WI. s = examples of rockets from samples of total plasma; 1, 2, 3 = three rockets from a calibration curve obtained with pure ape Al.
examination 3 points of the calibration curve were interspersed among the unknowns. After staining the agarose with Coomassie brilliant blue, the areas of the rockets obtained with the standard and with the samples under study were calculated by multiplying the height of the peak from the seeding point to the top by the mid-height width. As far as the determination by electroimmunoassay of apo AI is concerned the serum was totally delipidated with a mixture of butanol and di-isopropylether at a ratio of 40:60 according to Cham and Knowles [lo]. The clear lipidfree serum was diluted by 100 volumes by adding 0.05 M Tris-HCl, pH 8.0, containing 8 M urea. Laurel1 electrophoresis was performed in 1% agarose gels containing specific antiserum to apo AI supplied by Dr. Kostner (Graz, Austria) (Fig. 1). A standard highly purified apoprotein Al was kindly obtained by Dr. Alaupovic (Oklahoma). The experiment was carried out in quadruplicate on 5 different plates each. The apo A, value of the controls was obtained from 20 normolipemic subjects. The coefficient variation of apo Al has been always less than 3%. Specific antisera were obtained from rabbits injected with highly purified peptides AI and B. The obtained antisera were monospecific as they did not react with any other proteins and/or peptide from the specific antigen. The statistical significance of differences were analyzed using Student’s t-test. Pearson’s correlation coefficient, r, was used to show the degree of linear association among the different variables [ 111. Results Data obtained are given separately for males and females in Tables 1 and 2. Lower levels of HDL cholesterol and of apo AI were found in men than in females; this difference however was not significant. A high negative correlation
TABLE2 VALUES OF HDLCHOLESTEROL ANDFEMALES IBW
HDLchol.
(mg/dl)
APOAI
buz/dl)
AND
APO
A1 ACCORDINGTOQUARTILESOFIBWINMALES
1st
2nd
3rd
4th
0.80-0.90
0.91-1.00
1.01-1.10
1.11-1.20
63.38 60.00 152.00 157.93
60.21 61.76 136.21 143.00
55.14 i 5.0 56.96 f 6.7 122.95 k 13.2 138.52 f 10.3
44.6 47.20 100.20 113.7
f 6.1 i6.0 t 8.0 * 2.2
HDLchol.
ApoA1
s
9
6
1"s 2 = 1"s 3 = 10s 4 = 2 vs 3 = 2vs4 = 3vs4 =
1"s 2 = p.s. 1"s 3 = n.s. 1 “II 4 =P < 0.01 2vs3 = n.s. 2vs 4 = as. 3 vs 4 = ms.
1"s 2 = l”8 3 = 1 “II 4 = 2 vs 3 = 2vs4= 3 vs 4 =
p.s. p.s. P < 0.01 n.s. p.s. p.s.
* 6.4 f 6.5 i8.8 f 7.0
Q p.s. P < 0.01 P < 0.01
n.s. P < 0.01
n.s.
1 "S 2 1 "S 3 1 u* 4 2vs 3 2 "I 4 3 v!34
= = = = = =
as. n.s. P GO.01 n.s. P < 0.01 n.s.
? 7.1 + 7.3 f 9.2 t16.4
89 HD,L-C
(mgldl)
60.
r= -0.66 k-0.73
1 Ap;-A
I (mg/dl)
160.
rt -0.70 k-0.79 0.80
0.90
1.00
1.10
1.20
IBW
Fig. 2. The straight lines represent the regression coefficient. Dashed lines refer to females and solid lines to males. The 4 steps give mean values for quartiles from the lowest (to the left) to the highest (to the right).
has been found between IBW, HDL cholesterol and apo A1 both in males and females (Table 1 and Fig. 2). A positive significant correlation was also recorded between total cholesterol and apo A, (Table 1). No correlation has been found between IBW and apo B as well as between age, IBW, HDL cholesterol and apo Al. If values of HDL cholesterol and apo A1 are given for quartiles of IBW, meaningful variations are found in both sexes among the lowest and the highest (Fig. 2 and Table 2). Discussion The protein moiety of lipoproteins is the most probable determinant of the compositional and structural stability of the lipoprotein molecule [ 121. Despite this, scanty information is available on apolipoproteins. This is mostly due to the technical difficulties met in providing good methods which might be used in studies of large population samples. In our experience this has been achieved with the electroimmunoassay introduced by Curry et al. [9]. The method is rather simple, not expensive and not time-consuming. The results obtained with the various calibration curves as the study of the coefficient of variation make this method an excellent one for application in epidemiological studies. Information on the plasma level of the major apolipoproteins in normal subjects is very inadequate [1,2,13,14]. The data are even less meaningful as they were obtained with different methods such as radioimmunoassay [13,14], radioimmunodiffusion [1,15] and electroimmunoassay [2]. Only two of the
90
previous studies have looked for a difference between sexes [1,2]. These studies have offered contradictory results. According to Albers et al. [l] females have higher values of apo A1 than males (135 f 26 mg/dl vs 120 + 20). Alaupovic et al. [2] have observed that women tend to have higher values of apo A and lower levels of apo B than men; these differences, however, have not been statistically significant. Our data agree with the findings of Alaupovic et al. [2]. Actually, even though women show higher levels of apo A, and HDL cholesterol than men neither of these two differences is significant. The most relevant finding of our study is the high negative correlation existing between the IBW and the plasma levels of apo A1 as well as of HDL cholesterol. This correlation was recorded in both females and males. These data are even more significant as they were obtained from people with an IBW kept within normal limits with exclusion of obese and very lean subjects. A high positive correlation in both sexes has been found also between HDL cholesterol and apo A1 and between total cholesterol and apo Al. The plasma level of apo B has no relationship with the relative body weight. In recent time a major emphasis has been placed on HDL as an anti-risk factor [ 161. There is an agreement about a decreased level of HDL cholesterol in atherosclerotic patients [17] with the only exceptions being data obtained from a personal series [ 181. With the data presented in this paper it is obvious that this problem has to be considered since no attention has been previously paid to the index body weight of patients. Also some decrease of HDL recorded in hypertriglyceridemic patients and during carbohydrate feeding [ 191 may be entirely due to the high relative body weight present in these two situations. References 1 Alaupovic. P., Curry, M.D. and McConathy. W.J.. Electroimmunoassay of serum apollpoproteins in normolipidemic subjects and patients with primary hypcrlipoproteinemias. In: G. Schettler. Y. Goto. Y. Hata and G. Klose (Eds.), Atherosclerosis IV. Springer-Verlag. Berlin, 1977. p. 220. 2 Albers, J.J.. Wahl, P.W., Cabana, G.V., Hazzard, W.H. and Hoover, J.J., Quantitation of apolipoprotein Al of human plasma high density lipoprotein, Metabolism, 25 (19761 633. 3 Avogaro. P., Cazzolato, G., Capri. C., Pais. M. and Trabulo, G.F., Livelli lipidemici, fenotipi lipoprotelnemici e analisi delle classi lipoproteiche in una popolazione italiana, Giorn. It. Csrdiol.. 4 (19741 237. 4 Statistical Bulletin of the Metropolitan Life Insurance Co., No. 40. Nov.-Dec., 1959. In: Documenta Geigy Tables Scientifisues, 6th edition, BasIe, 1963. p. 634. 5 Watson, D.. A simple method for the determination of serum cholesterol, Clin. Chlm. Acta. 5 (1960) 637. 6 Eggstein. M. and Kreutz. F.H.. Eine neue Bestimmung der Neutralfette in Blutserum and Gewebe. 1 (Princip, Durchfilhrung und Besprechung der Methode). Klin. Wschr.. 44 (1966) 262. 7 Lopes-Virella. M.F., Stone, P., Ellis. S. and Colwell. J.A., Cholesterol determination in high density lipoproteins separated by three different methods Clln. Chem.. 23 (1977) 882. 8 Laurell, C.B.. Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies, Anal. Biochem.. 45 (1966) 1966. 9 Curry, M.D., Alaupovic, P. and Suenram. C.A., Determination of apolipoprotein A and its constitutive A-I and A-II polvpeptides by separate electroimmunoassays, Clin. Chem.. 22 (1976) 315. 10 Cham. B.E. and Knowles, R.B., A solvent system for delipidation of plasma or serum without protein precipitation, J. Lipid Res., 17 (1976) 176. 11 Snedecor. G.W. and Cochran. W.G., Statistical Methods, 6th edition, Iowa State University Press, Ames, Iowa. 1967. 12 13
AkuPovib. Schonfeld, lipoprotein
P., Apolipoprotelns and lipoproteins, Atherosclerosis, 13 (1971) 141. G. and Pfleger, B.. The structure of human high density lipoprotein and the levels of apeA-l in plasma as determined by radioimmunoassay. J. Clln. Invest., 54 (1974) 236.
91. 14 15 16 17 18 19
Schonfeld, G., Lees. R.S., George, P.K. and Pfleger. B., Assay of total plasma apohpoprotein B concentration in human subjects, J. CIin. Invest., 63 (1974) 1468. Fainaru, M.. Gelangeaud. M.C. and Eisenberg. S.. Radioimmunoassay of human high density Iipoprotein apoprotein A-l, Biochem. Biophys. Acta. 386 (1975) 432. Avogaro, P. and Cazzolato, G., FamiIiaI hyperHDL-(cu)-cholesterolemia. Atherosclerosis, 22 (1975) 63. Miller, C.J. and Miller, N.E.. Plasma-high-density lipoprotein concentration and development of ischaemic heart disease, Lancet, 1 (1975) 16. Avogaro, P., Cazzolato. G., Taroni, G. and Belussi, F., Chemical composition of ultracentrifugal fractions in different patterns of human atherosclerosis, Atherosclerosis, 26 (1977) 163. Assmann, G., High density lipoproteins associated with disease. In: G. Schettler. Y. Goto, Y. Hata and G. Klose (Eds.), Atherosclerosis IV, Springer-Verlag. Berlin, 1977, P. 238.
Atherosclerosis, 31 (1978) 85-91 @ Elsevier/North-Holland Scientific Publishers, Ltd.
HDL-CHOLESTEROL,
APOLIPOPROTEINS
Al AND B
Age and Index Body Weight
P. AVOGARO,
G. CAZZOLATO,
G. BITTOLO BON, G.B. QUINCI and M. CHINELLO
Unit for Atherosclerosis, Hyperlipaemias Nazionale delle Ricerche, Venice (Italy)
and Diabetes, Regional Hospital, Unit of Consiglio
(Received 3 April, 1978) (Revised, received 5 June, 1978) (Accepted 5 June, 1978)
summary
Some data of previous literature have emphasized a negative correlation between plasma apo Al, HDL cholesterol and coronary heart disease. The present paper stresses a high negative correlation existing both in females and males between values of index body weight (IBW) and plasma levels of HDL cholesterol and apo Al. No correlation has been found between age and, respectively, HDL cholesterol, apo A, and apo B. Key words:
Apolipoproteins
-Body
weight - HDL-cholesterol
Introduction
No data are available concerning the levels of the major apolipoproteins according to age. Few data have stressed some differences existing between the sexes [1,2]. In the course of a study performed in some groups of normal people to ascertain the plasma levels of apo B and apo Al according to sex and age, an inverse relationship between the major apolipoprotein of HDL, namely apo Al, and IBW (index body weight) has been observed both in males and females.
Abbrevations: IBW = index body weight; BFS = blood fasting sugar; BUN = blood urea nitrogen; C = cholesterol; TG = triglyceride; HDL = high density lipoproteins (d > 1.063); ape = apolipoprotein. Requests for reprints should be addressed to P. Avogaro.
86
Materials and Methods Subjects included in this study are both males and females with an age kept between 20 and 60 years. They are part of a population freely submitting to our Unit in a Health Survey Program. An extensive clinical examination was completely negative for all of them. Both the blood pressure and an electrocardiographic record were within normal limits. At a first examination the BFS, the BUN, plasma cholesterol and triglycerides were recorded. From all the people showing normal values of BFS and BUN and values of cholesterol and triglycerides below the 95th percentile for our population [3] one subject was selected at random out of four. The selection was limited to subjects with an IBW between 0.8 and 1.2 and with an age kept between 20 and 60 years. The subjects selected and included in the second phase were 60 males with a mean age of 40.9 yr and an IBW of 1.027 and 60 females with a mean age of 39.6 yr and an IBW of 1.013 (Table 1). The relative weight of these subjects was calculated by dividing the actual weight by the ideal weight according to the tables of Metropolitan Life Insurance Co. [ 41. After a fasting of 12 h a new blood sample was collected in EDTA (1 mg/ 1 ml). The new series of analysis included the determination of cholesterol [ 51, triglyceride [ 61, and HDL cholesterol [ 71. For the determination of HDL cho-
TABLE 1 MEAN VALUES AND CORRELATIONS LIPIDS AND APOLIPOPROTEINS
Age
m
(YY)
iii
IBW Total chol. Total TG HDL
(m&-W (mf6k-W (mg/dl)
iii iii iii
APO A1 APO B
OwAW
iTi iii
IBW IBW IBW IBW IBW Age Ape Age A&
(mg/dl) “8 vs vs vs vs
(yr) (yr) (yr) (YX)
Total chol. TG WDL chol. Total chol. TG
“8
HDL chol APO At Total chol. TG APO B
VS
IBW HDL chol.
“8 vs
APO A1 APO B
vs vs VS “8 VS
APO A1 APO Al APO A1 HDL chol. HDL chol.
OF INDEX
BODY
WEIGHT
AND PLASMA
LEVELS
d(n=60)
?(n=60)
40.98 f 12.6 (range 20-60) 1.027 f 0.11 (range 0.80-1.20)
39.62 f 11.9 (range 20-60) 1.013 f 0.10 (range 0.80-1.20)
212.53 92.26 53.65 128.45 137.69
204.63 2 31.5 82.72 * 25.2 57.93 t 9.7 133.59 + 19.9 138.08 i 27.2
a.73 -0.79 -0.10 -0.01 a.06 0.08 0.07 0.07 0.13 0.25 0.10 0.66 0.16 a.15
f f f k f
31.5 31.0 9.8 21.56 26.1 P < 0.01 P
-0.66 -0.70 0.05 0.20 0.08
P c 0.01 P
n.s. n.s. n.s. n.s.
-0.05 0.01 0.06 0.20
n.s. n.s. n.s. n.s.
P < 0.05 xl.*. P
0.29 0.07 0.63 -0.14 -Q.20
P
< 0.01 n.s. P co.01 as. as.
OF
lesterol the procedure followed was that based upon precipitation with 4% phosphotungstic acid in 1 M NaOH and 2 M MgCl*. Apolipoproteins A, and B were determined in whole plasma adapting the technique of Laurell [8] as described by Curry et al. [ 91. For this purpose we used an LKB 2177 water-coiled Multiphor electrophoresis plate on which 21 samples can be run simultaneously. All the runs were done at 15” at 15 V/cm, in Tris-glycine buffer, pH 8.6. Into the wells was seeded 5 ~1 of suitably diluted standard apo B, of graduated dilutions of apo A1 or of the sample under study. The precipitation peak reached its maximum height in 12 h. For the standardization of the electroimmunoassay of apolipoprotein B the 1040 > d < 1050 fraction was isolated from a pool of plasmas obtained from 20 normolipemic subjects by means of successive ultracentrifugations. One ml aliquots of these fractions were stored in sealed vials containing EDTA and sodium azide at -20°C after testing with anti-apo B, Al, AZ, C and ARP (supplied by Dr. G. Kostner, Graz) and anti-albumin (Boehringer); there was a positive reaction only with anti-apo serum B. The protein concentration of the antigen thus obtained was 0.3 mg/ml. The stability of the concentration of the stored antigen permitted reproducible results through the period of the study. Calibration curves were thus plotted by assaying stepped concentrations of the antigen (from 50 to 200 ,ug of apo B/5 ~1) against a fixed quantity of antiserum dispersed in 1% agarose in Tris-glycine buffer, pH 8.6. The quantity of antiserum was such that at the highest concentration no excess of antigen was detectable. Coefficient of variations for apo B was in the range of 5%. At every
Fig. 1. Rockets of apolipoproteins obtained by the technique of LaurelJ as modified by Curry et al. WI. s = examples of rockets from samples of total plasma; 1, 2, 3 = three rockets from a calibration curve obtained with pure ape Al.
examination 3 points of the calibration curve were interspersed among the unknowns. After staining the agarose with Coomassie brilliant blue, the areas of the rockets obtained with the standard and with the samples under study were calculated by multiplying the height of the peak from the seeding point to the top by the mid-height width. As far as the determination by electroimmunoassay of apo AI is concerned the serum was totally delipidated with a mixture of butanol and di-isopropylether at a ratio of 40:60 according to Cham and Knowles [lo]. The clear lipidfree serum was diluted by 100 volumes by adding 0.05 M Tris-HCl, pH 8.0, containing 8 M urea. Laurel1 electrophoresis was performed in 1% agarose gels containing specific antiserum to apo AI supplied by Dr. Kostner (Graz, Austria) (Fig. 1). A standard highly purified apoprotein Al was kindly obtained by Dr. Alaupovic (Oklahoma). The experiment was carried out in quadruplicate on 5 different plates each. The apo A, value of the controls was obtained from 20 normolipemic subjects. The coefficient variation of apo Al has been always less than 3%. Specific antisera were obtained from rabbits injected with highly purified peptides AI and B. The obtained antisera were monospecific as they did not react with any other proteins and/or peptide from the specific antigen. The statistical significance of differences were analyzed using Student’s t-test. Pearson’s correlation coefficient, r, was used to show the degree of linear association among the different variables [ 111. Results Data obtained are given separately for males and females in Tables 1 and 2. Lower levels of HDL cholesterol and of apo AI were found in men than in females; this difference however was not significant. A high negative correlation
TABLE2 VALUES OF HDLCHOLESTEROL ANDFEMALES IBW
HDLchol.
(mg/dl)
APOAI
buz/dl)
AND
APO
A1 ACCORDINGTOQUARTILESOFIBWINMALES
1st
2nd
3rd
4th
0.80-0.90
0.91-1.00
1.01-1.10
1.11-1.20
63.38 60.00 152.00 157.93
60.21 61.76 136.21 143.00
55.14 i 5.0 56.96 f 6.7 122.95 k 13.2 138.52 f 10.3
44.6 47.20 100.20 113.7
f 6.1 i6.0 t 8.0 * 2.2
HDLchol.
ApoA1
s
9
6
1"s 2 = 1"s 3 = 10s 4 = 2 vs 3 = 2vs4 = 3vs4 =
1"s 2 = p.s. 1"s 3 = n.s. 1 “II 4 =P < 0.01 2vs3 = n.s. 2vs 4 = as. 3 vs 4 = ms.
1"s 2 = l”8 3 = 1 “II 4 = 2 vs 3 = 2vs4= 3 vs 4 =
p.s. p.s. P < 0.01 n.s. p.s. p.s.
* 6.4 f 6.5 i8.8 f 7.0
Q p.s. P < 0.01 P < 0.01
n.s. P < 0.01
n.s.
1 "S 2 1 "S 3 1 u* 4 2vs 3 2 "I 4 3 v!34
= = = = = =
as. n.s. P GO.01 n.s. P < 0.01 n.s.
? 7.1 + 7.3 f 9.2 t16.4
89 HD,L-C
(mgldl)
60.
r= -0.66 k-0.73
1 Ap;-A
I (mg/dl)
160.
rt -0.70 k-0.79 0.80
0.90
1.00
1.10
1.20
IBW
Fig. 2. The straight lines represent the regression coefficient. Dashed lines refer to females and solid lines to males. The 4 steps give mean values for quartiles from the lowest (to the left) to the highest (to the right).
has been found between IBW, HDL cholesterol and apo A1 both in males and females (Table 1 and Fig. 2). A positive significant correlation was also recorded between total cholesterol and apo A, (Table 1). No correlation has been found between IBW and apo B as well as between age, IBW, HDL cholesterol and apo Al. If values of HDL cholesterol and apo A1 are given for quartiles of IBW, meaningful variations are found in both sexes among the lowest and the highest (Fig. 2 and Table 2). Discussion The protein moiety of lipoproteins is the most probable determinant of the compositional and structural stability of the lipoprotein molecule [ 121. Despite this, scanty information is available on apolipoproteins. This is mostly due to the technical difficulties met in providing good methods which might be used in studies of large population samples. In our experience this has been achieved with the electroimmunoassay introduced by Curry et al. [9]. The method is rather simple, not expensive and not time-consuming. The results obtained with the various calibration curves as the study of the coefficient of variation make this method an excellent one for application in epidemiological studies. Information on the plasma level of the major apolipoproteins in normal subjects is very inadequate [1,2,13,14]. The data are even less meaningful as they were obtained with different methods such as radioimmunoassay [13,14], radioimmunodiffusion [1,15] and electroimmunoassay [2]. Only two of the
90
previous studies have looked for a difference between sexes [1,2]. These studies have offered contradictory results. According to Albers et al. [l] females have higher values of apo A1 than males (135 f 26 mg/dl vs 120 + 20). Alaupovic et al. [2] have observed that women tend to have higher values of apo A and lower levels of apo B than men; these differences, however, have not been statistically significant. Our data agree with the findings of Alaupovic et al. [2]. Actually, even though women show higher levels of apo A, and HDL cholesterol than men neither of these two differences is significant. The most relevant finding of our study is the high negative correlation existing between the IBW and the plasma levels of apo A1 as well as of HDL cholesterol. This correlation was recorded in both females and males. These data are even more significant as they were obtained from people with an IBW kept within normal limits with exclusion of obese and very lean subjects. A high positive correlation in both sexes has been found also between HDL cholesterol and apo A1 and between total cholesterol and apo Al. The plasma level of apo B has no relationship with the relative body weight. In recent time a major emphasis has been placed on HDL as an anti-risk factor [ 161. There is an agreement about a decreased level of HDL cholesterol in atherosclerotic patients [17] with the only exceptions being data obtained from a personal series [ 181. With the data presented in this paper it is obvious that this problem has to be considered since no attention has been previously paid to the index body weight of patients. Also some decrease of HDL recorded in hypertriglyceridemic patients and during carbohydrate feeding [ 191 may be entirely due to the high relative body weight present in these two situations. References 1 Alaupovic. P., Curry, M.D. and McConathy. W.J.. Electroimmunoassay of serum apollpoproteins in normolipidemic subjects and patients with primary hypcrlipoproteinemias. In: G. Schettler. Y. Goto. Y. Hata and G. Klose (Eds.), Atherosclerosis IV. Springer-Verlag. Berlin, 1977. p. 220. 2 Albers, J.J.. Wahl, P.W., Cabana, G.V., Hazzard, W.H. and Hoover, J.J., Quantitation of apolipoprotein Al of human plasma high density lipoprotein, Metabolism, 25 (19761 633. 3 Avogaro. P., Cazzolato, G., Capri. C., Pais. M. and Trabulo, G.F., Livelli lipidemici, fenotipi lipoprotelnemici e analisi delle classi lipoproteiche in una popolazione italiana, Giorn. It. Csrdiol.. 4 (19741 237. 4 Statistical Bulletin of the Metropolitan Life Insurance Co., No. 40. Nov.-Dec., 1959. In: Documenta Geigy Tables Scientifisues, 6th edition, BasIe, 1963. p. 634. 5 Watson, D.. A simple method for the determination of serum cholesterol, Clin. Chlm. Acta. 5 (1960) 637. 6 Eggstein. M. and Kreutz. F.H.. Eine neue Bestimmung der Neutralfette in Blutserum and Gewebe. 1 (Princip, Durchfilhrung und Besprechung der Methode). Klin. Wschr.. 44 (1966) 262. 7 Lopes-Virella. M.F., Stone, P., Ellis. S. and Colwell. J.A., Cholesterol determination in high density lipoproteins separated by three different methods Clln. Chem.. 23 (1977) 882. 8 Laurell, C.B.. Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies, Anal. Biochem.. 45 (1966) 1966. 9 Curry, M.D., Alaupovic, P. and Suenram. C.A., Determination of apolipoprotein A and its constitutive A-I and A-II polvpeptides by separate electroimmunoassays, Clin. Chem.. 22 (1976) 315. 10 Cham. B.E. and Knowles, R.B., A solvent system for delipidation of plasma or serum without protein precipitation, J. Lipid Res., 17 (1976) 176. 11 Snedecor. G.W. and Cochran. W.G., Statistical Methods, 6th edition, Iowa State University Press, Ames, Iowa. 1967. 12 13
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P., Apolipoprotelns and lipoproteins, Atherosclerosis, 13 (1971) 141. G. and Pfleger, B.. The structure of human high density lipoprotein and the levels of apeA-l in plasma as determined by radioimmunoassay. J. Clln. Invest., 54 (1974) 236.
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Schonfeld, G., Lees. R.S., George, P.K. and Pfleger. B., Assay of total plasma apohpoprotein B concentration in human subjects, J. CIin. Invest., 63 (1974) 1468. Fainaru, M.. Gelangeaud. M.C. and Eisenberg. S.. Radioimmunoassay of human high density Iipoprotein apoprotein A-l, Biochem. Biophys. Acta. 386 (1975) 432. Avogaro, P. and Cazzolato, G., FamiIiaI hyperHDL-(cu)-cholesterolemia. Atherosclerosis, 22 (1975) 63. Miller, C.J. and Miller, N.E.. Plasma-high-density lipoprotein concentration and development of ischaemic heart disease, Lancet, 1 (1975) 16. Avogaro, P., Cazzolato. G., Taroni, G. and Belussi, F., Chemical composition of ultracentrifugal fractions in different patterns of human atherosclerosis, Atherosclerosis, 26 (1977) 163. Assmann, G., High density lipoproteins associated with disease. In: G. Schettler. Y. Goto, Y. Hata and G. Klose (Eds.), Atherosclerosis IV, Springer-Verlag. Berlin, 1977, P. 238.