- Email: [email protected]
Arterial cholesterol and DNA contents in relation to serum lipids and apolipoproteins
241
Atherosclerosis, 41 (1982) 247-253 0 Elsevier/North-Holland Scientific Publishers, Ltd.
ARTERIAL CHOLESTEROL AND DNA CONTENTS IN RELATION TO SERUM LIPIDS AND APOLIPOPROTEINS
Studies on Cystic Artery Biopsies
OLLE WIKLUND, JOHN G. KRAL, LEIF LINDBLAD, SVEN-OLOF OLEFSSON, TORE SCHERSTEN, LARS SJdSTRGM and G6RAN BONDJERS Arterial Biology and Department
Group, Department of Medicine of Surgery, Sahlgren’s Hospital,
I and Department of Medical Biochemistry University of Giiteborg, Gijteborg (Sweden)
(Received 31 March, 1981) (Revised, received 18 June, 1981) (Accepted 18 June, 1981)
Summary Though various relationships between serum lipoprotein levels and risk for atherosclerotic disease have been shown there are only a few studies on the relationships between serum lipoprotein levels and the lipid contents of the arterial wall. This study presents cholesterol and DNA contents of arterial tissue from biopsies of the cystic artery in 23 patients with uncomplicated cholecystolithiasis. Sm7lrn L.,--ee.
!p~&
of
ch~!&ero],
~rigj;ly~er&-Jesj
alnhnlinonrotein -l____= -=______
&~!&er&
apoA-I, A-II and B were determined, and the relationships between artery and serum variables were calculated. There was a positive correlation between serum apoB and the arterial cholesterol, normalized to the DNA contents (r = 0.43, P < 0.05). There was a tendency towards an inverse correlation between the (Y-lipoprotein cholesterol levels and the arterial cholesterol (r = -0.39, P < 0.10). There were no significant correlations between serum apoA-I or A-II and the arterial cholesterol contents. These data indicate that deposition of cholesterol in the arterial wall is related to the serum level of apoB, with higher levels of arterial cholesterol at
This study was supported by grants from the Swedish Medical Research Council (Project No. 4531). the Swedish Association against Heart and Chest Disease, the Swedish Oleo-Margarine Foundation for Nutritional Research, the Medical Society of GGteborg and the University of Giiteborg. Findus AB. Bjuv, Sweden contributed with TV-dinners ? Correspondence: Giiran Bond&s, Department of Medicine I, Sahlgren’s Hospital, S-413 45 Gateborg, Sweden.
0021-9150/82/0000-0000/$02.75
0 1982 Elsevier/North-Holland
Scientific Publishers, Ltd.
248
higher serum levels of apoB. Earlier observations of an inverse correlation between alphalipoprotein cholesterol and arterial cholesterol could, however, not be conclusively confirmed. Key words:
Apolipoproteins
-Arterial
cholesterol
-Atherosclerosis
-Lipoproteins
Introduction The relationship between serum levels of lipids and lipoproteins and the clinical manifestations of atherosclerosis has been extensively studied. Thus, high levels of serum cholesterol and low density lipoproteins (LDL) are risk factors for coronary heart disease (CHD). The major protein component of LDL, apolipoprotein B (apoB), is also high in patients with CHD [ 1,2]. Serum high density lipoproteins (HDL), on the other hand have been suggested as a protective factor against CHD, and HDL or alphalipoprotein cholesterol [3] as well as the major protein components of HDL (apoA-I and A-II) are low in patients with CHD [ 31. Although several relations between the serum lipoprotein levels and the risk for ciinical manifestations of atlleroscierosis have been shown, oniy a few studies are available on the relation between the serum lipoprotein level and the lipid content of the arterial wall. A positive correlation between serum cholesterol and arterial tissue LDL content [4] as well as a positive correlation between serum LDL and arterial tissue cholesterol [5] has been reported. On the other hand an inverse correlation was found between alphalipoprotein cholesterol and arterial cholesterol [ 5,6]. The previous studies on correlations between serum lipoproteins and arterial wall cholesterol have been restricted to small groups of patients with specific diseases. The general significance of these observations may therefore be questioned. In the present study these correlations were analyzed in another group of patients, and with a different site of the arterial biopsy. In addition, not only serum lipids were determined, but also serum levels of apoA-I, A-II and apoB and their correlations to the arterial cholesterol and DNA content analyzed. Material and Methods Patients The patients were admitted to the hospital for operation of uncomplicated cholecystolithiasis. The following exclusion criteria were used: (1) Symptoms of acute cholecystitis, (2) medication with lipid-lowering drugs or oral contraceptives during the last 6 months, (3) cholestasis, (4) neoplastic disease during the last 10 years and (5) diabetes mellitus. Twenty-three subjects, 12 men and 11 women, were included in the study (mean age 38 years, range 31-70). The patients were admitted to the hospital 5 days before operation. During
249
the first day clinical examination was performed and the patients were interviewed about smoking habits, earlier diseases and medication. During hospitalization, the energy intake was adjusted to a value 40% above basal metabolic rate (BMR). BMR was estimated from age! sex and body surface using the measurements of Bothby and Du Bois [7], Exactly the same food was given each day; it consisted of Findus pre-prepared TV-dinners @ (Findus AB, Bjuv, Sweden): meat-balls, potatoes and vegetables for lunch and beef with onions and pommes frittes for dinner. Together with 7 pieces of Swedish Crisp Bread (totally 84 g) (Wasa knlckebrijd @),Wasa Brad AB, Filipstad, Sweden), 4 g butter and 1 dl of skim milk these two TV-dinners contained 4970 kJoules (1190 kcal) and 16 joule-% protein, 39 joule-% fat and 45 joule-% carbohydrate. A gruel with exactly the same composition with respect to protein, fat and carbohydrate was used as breakfast and extra evening meal in order to adjust the energy intake to the estimated individual need. Thus, irrespective of energy intake all subjects obtained the same food composition. Blood samples were obtained after 12 h fasting on the morning of the 4th day. Cholecystectomy and arterial biopsy were performed on the 6th day. Biochemical methods Cholesterol [ 81, triglycerides [ 91 and alphalipoprotein cholesterol were determined on fresh serum. The alphalipoproteins were isolated as the supernatant after precipitation with MnClz and heparin [lo]. Before apolipoprotein analyses serum samples were stored at -80°C in aliquots, in rubber-stoppered glass tubes. The apolipoproteins were determined with electroimmunoassay [2]. For each apolipoprotein all samples were analyzed at the same time, utilizing the same batch of antiserum. Arterial biopsy Routine anesthetic procedures were used during the operation. The cystic artery was dissected free during cholecystectomy and a segment was removed with great care to avoid stretching and bending of the artery. The vessel was dissected free from other tissue and was frozen in Ringer’s solution at -80°C until analyzed. After thawing the vessels were cut in 0.5-mm segments with a McIlvain tissue chopper. The adventitia was removed under a dissecting microscope and the intima-media preparations were analyzed for cholesterol [ll] and DNA [ 121 contents. All specimens were analyzed at the same time. Statistical methods The conventional statistical methods were used for the determination of means, standard deviations and correlation coefficients. To test if there was any significant deviation from the normal, distribution coefficients of skewness and curtosis were calculated [ 131. In the correlation analyses log-transformed data were used for those variables that showed a significant deviation from normal distribution. Log-transformed data did not significantly deviate from the normal distribution.
250
Results Mean values, standard deviations and ranges for serum cholesterol, triglycerides, alphalipoprotein cholesterol, apoA-I, A-II and apol3 as well as for arterial cholesterol and DNA contents are given in Tables 1 and 2. Two subjects had serum cholesterol levels above 7.5 mmol/l (7.9 and 9.1, respectively) and 4 had serum triglycerides above 2.1 mmol/l. The correlation coefficients between serum lipids or lipoproteins and arterial cholesterol or DNA contents are given in Table 2. There were no significant correlations between serum cholesterol or triglycerides and the arterial variables. There was a tendency towards an inverse correlation between alphalipoprotein cholesterol and the arterial cholesterol contents (r = -0.30, P = 0.066) (Fig. 1). There were, however, no significant correlations between the serum apoA-I or A-II levels and the arterial cholesterol or DNA. Serum apoB was positively correlated to the cholesterol content of the arterial wall expressed per pg DNA (r = 0.43, P = 0.041) (Fig. 2).
TABLE
1
MEANS,
STANDARD
CHOLESTEROL (A-CH).
SERUM
INCLUDED
A-I IN THE
(yr)
Weight
index
Iwdeht(keb/hdeht. L ._D___\___,,_______
S-CH
S-TG
(mmol/l)
(mmol/l)
A-CH
ApoA-I
ApoA-II
AP~B
DEVIATIONS
APOLIPOPROTEIN
PATIENTS
Age
(S-CH).
(mmol/I)
(g/I)
(g/l)
(g/I)
IrmJ \_.__,
-1001 ___l
(SD)
AND
RANGES
TRIGLYCERIDES (apoA-I).
A-II
(S-TG). (apoA-II)
AND
OF
AGE,
WEIGHT
ALPHALIPOPROTEIN APOLIPOPROTEIN
INDEX,
SERUM
CHOLESTEROL B (apo
B)
IN
STUDY Men
Women
All
n=12
n=
n=
Mean
53
44
SD
11
10
11
Range
32-70
31-62
31-70
11
23
49
Mean
1.03
1.03
1.03
SD
0.15
0.20
0.17
Range
0.87-1.45
0.80-1.45
0.80-1.45
Mean
6.05
4.75
5.43
SD
1.51
0.81
1.37
Range
3.8-9.1
3.2-6.0
3.2-9.1
Mean
1.84
1.14
1.50
SD
0.78
0.25
0.68
Range
0.7-3.4
0.8-l
Mean
1.60
SD
1.43 ^ -1 U.34
.6
0.7-3.4
0.28
1.51 - ^^ U.BZ 1 .o-2.0
Range
1.0-2.0
1.1-2.0
Mean
1.87
1.91
1.89
SD
0.25
0.26
0.25
Range
1.67-2.50
1.48-2.43
1.48-5.50
Mean
0.52
0.47
0.50
SD
0.06
0.05
0.06
Range
0.444.68
0.39-0.55
0.39-0.68
MeaIl
1.48
1.12
1.31
SD
0.36
0.26
0.36
Range
0.90-2.10
0.90-1.65
0.90-2.10
THE
251 TABLE
2
MEANS,
STANDARD
DEVIATIONS
ARTERIAL
DNA
INCLUDED
IN THE
Arterial
CH
AND
(SD)
ARTERIAL
AND
(nmol/mg
dry
Mean
weight)
Arterial
TABLE
(pg/mg
CH/DNA
ARTERIAL
RATIO
CHOLESTEROL
(CH/DNA)
IN THE
(CH), PATIENTS
dry
weight)
(pglpg)
Men
Women
All
n=12
n=
n=
33.99
31.45
11
32.77
3.17
31.0940.01
23
3.31
25.98-36.34
25.9840.01
Mean
7.23
7.17
7.21
SD
1.71
1.04
1.40 4.79-11.05
Range
4.79-11.05
5.10--9.01
Mean
1.89
1.72
1.81
SD
0.38
0.27
0.33
Range
1.36-0.38
1.33-2.21
1.33-2.74
3
CORRELATION
COEFFICIENTS
PROTEINS DNA
OF DNA
3.06
Range DNA
TO
STUDY
SD
Arterial
RANGES
CHOLESTEROL
AND
ARTERIAL
BETWEEN CONTENTS
OF
SERUM
LIPIDS,
CHOLESTEROL
LIPOPROTEINS
OR
DNA
OR
OR
APOLIPO-
CHOLESTEROL
PER
fig
(n = 23)
Log
serum
cholesterol
Log
serum
triglycerides
Alphalipoprotein Log
apoA-I
Log
apoA-II
cholesterol
Arterial
Log
cholesterol
DNA
Log
arterial
cholesterol/DNA
-0.OH
4.36
0.35
0.14
4.23
0.33
-xl.39
*
-0.06
4.24
ApoB
arterial
-0.16
0.04
4.19
0.13
-0.07
0.17
0.18
a.29
0.43
**
* P = 0.066 **
P = 0.041 TISSUE CHOLESTEROL/DNA
TISSUE CHOLESTEROL nmollmg
lJg’c19
o:-
o=
X=5
50 -
2.5 0
40-
2.0 -
30 -
1.5-
20 -
!.D -
Fig.
1.
Tissue
cholesterol
serum,
r = -0.39,
Fig.
The
2.
r = 0.41.
ratio
P < 0.05.
(nmol/mg
dry
X3
x
weight)
in relation
ox
x x
0: m:rXR 8”
x x
to alphalipoprotein
concentration
(mmolil)
in
P < 0.10. tissue
cholesterol
to
DNA
(pg/fig)
in arterial
tissue
in relation
to serum
apoB
(g/l)
levels.
252
Discussion In two earlier reports [5,6] the relationships between serum lipoprotein levels and the arterial cholesterol contents have been studied. One of these studies concerned severely obese patients [5] and the other study was restricted to women undergoing hysterectomy [6] due to uterine myoma. In the present study subjects admitted to the hospital for elective cholecystectomy were investigated. The selection of the patients might have affected the results of these studies. Severely obese subjects are different from a non-obese population in several aspects of lipid metabolism [14] and their alphalipoprotein cholesterol levels are usually low [ 5,14,15]. Patients with gallstones, on the other hand, might have a more general disturbance in the cholesterol metabolism. Not only the selection of patients but also the location of the arterial biopsy has to be considered when the results of the present study are compared with earlier reports. In the present study the cystic artery was analyzed. Atherosclerosis, however, is unevenly distributed within the arterial tree and differences between different arterial regions might explain variations in the results. We do not know if the cystic artery is representative for other arterial regions. In the present study there was a positive correlation between the serum apoB levels and the arterial contents of cholesterol per pg of DNA. ApoB is the major protein component of LDL and a tendency towards a correlation between serum LDL cholesterol and the cholesterol content of the mesenteric artery has earlier been reported [ 51. Deposition of apoB in the atherosclerotic lesions has been reported by several investigators [16,17]. Furthermore, apoB has been suggested to bind to some components of the intercellular substance of the arterial wall [ 18-201. The present data indicate that the deposition of cholesterol is related to the serum level of apoB or LDL, with higher levels of arterial cholesterol at higher serum apoB levels. In two previous studies from our group an inverse correlation between arterial cholesterol and alphalipoprotein cholesterol has been reported [5,6]. This inverse correlation has been taken to support the suggested role of HDL as a cholesterol acceptor in the removal of cholesterol from the tissue [3]. In the present study there were no significant correlations between alphalipoprotein cholesterol, apoA-I or A-II and arterial cholesterol, and this study does not support the hypothesis, although the tendency regarding alphalipoprotein cholesterol is in agreement with earlier observations. Due to discrepancies between the different studies these correlations have to be further analyzed in nther ““._“I
m-n~mw nf nntipntn b-‘ -y” VL y.e’ -u’-“u
and with u1.u ,.A”__ &her
sit.er nf “_“_., .._ thP 1_.V nrtminl -“___-
hinnw “--llJ.
References G., Quinci, G.B. and Belussi, G., Plasma levels of apolipoAvogaro, P., Bittolo Bon. G.. Cazzolato, protein A-I and apolipoproteln B in human atherosclerosis, Artery, 4 (1978) 385. Fager. G., Serum Apolipoprotein Levels in Relation to Acute Myocardial Infarction and its Established Risk Factors, Thesis, University of GBteborg, GGteborg. Sweden, 1979. Bondjers, G., Wiklund, 0.. Olofsson, S.-O., Gustafson. A. and BjSrkerud. S.. Relationship between high density lipoproteins and atherosclerosis. In: C.E. Day (Ed.). High Density Lipoproteins, Dekker Inc.. New York, NY, 1981, p. 463-503. Smith, E.B. and Slater, R.S., Relationship between low-density lipoprotein in aortic intima and serumlipid levels. Lancet. i (1972) 463.
253 5 Bondjers, G.. Gustafson, A., Kral, H.. Schersten, T. and Sjiistriim. L., Cholesterol content in arterial tissue in relation to serum lipoproteins in man. Artery, 2 (1976) 200. 6 Bondjers, G., Wiklund, 0. and Olofsson, S-0.. Alphalipoprotein cholesterol and arterial cholesterol in human uterine arteries, Artery, 8 (1980) 305. 7 Documenta Geigy, Scientific Tables, 7th edition, Ed. K. Diem and C. Lentner, J.R. Geigy %A., Basel. 1970, p. 540. 8 Leffler, H.H.. Estimation of cholesterol in serum, Amer. J. Clin. Path., 31 (1959) 310. 9 Carlson, L.A.. Determination of serum glycerides, Acta Sot. Med. Upsalien., 64 (1959) 208. 10 Wlklund, 0.. Fager, G., Craig, I.H., Wilhelmsson, C.E.. Vedin, A., Olofsson, S.-O., Bondjers, G. and Wilhelmsen, L.. Alphalipoprotein levels in relation to acute myocardial infarction and its risk factors, Stand. J. Clin. Lab. Invest., 40 (1980) 239. determination of cholesterol and cholesteryl ester in 11 Bondjers, G. and BjGrkerud. S., Fluorometric tissue on the nanogram levels, Anal. Biochem., 42 (1971) 363. 12 Kissane. J.M. and Robins, E., The fluorometric measurement of deoxyribonucleic acid in animal tissues with special reference to the central nervous system, J. Biol. Chem.. 233 (1958) 184. 13 Armitage, P., Statistical Methods in Medical Research. 3rd printing, Blackwell Scientific Publications, Oxford. 1974. 14 Nestel, P. and Goldrick, B., Obesity - Changes in lipid metabolism and the role of insulin, Clin. Endocrinol. Metab.. 5 (1976) 313. 15 Wilson, D.E. and Lees, R.S.. Metabolic relationships among the plasma lipoproteins - Reciprocal changes of very low and low density lipoproteins in man, J. Clin. Invest.. 51 (1972) 1051. 16 Walton, K.W. and Williamson. N., Histological and immunofluorescent studies on the human atheromatous plaque, Atherosclerosis, 8 (1968) 599. 17 Hoff. H.F., Jackson. R.L.. Mao, J.T. and Gotto, A.M., Localization of low density lipoproteins in arterial lesions from normolipemics employing a purified fluorescent-labeled antibody, Biochim. Biophys. Acta. 351 (1974) 407. 18 Avial, E.M., Lopez. F. and Camejo, G.. Properties of low density lipoproteins related to its interaction with arterial wall components - In vitro and in viva studies, Artery, 4 (1978) 36. 19 Hoff, H.F., Heideman, C.L., Gaubatz. J.W., Titus, J.L. and A.M. Gotto Jr., Quantitation of apoB in human aortic fatty streaks - A comparison with grossly normal intima and fibrous plaques, Atherosclerosis, 30 (1978) 263. 20 Smith. E.B., Biochemical studies on permeability and the interaction between blood constituents and arterial components in atherosclerosis. In: A.M. Gotto, L.C. Smith and B. Allen (Ed%), Atherosclerosis V (Proceedings of the 5th International Symposium), Springer-Verlag. Berlin, 1980, p. 121.
Atherosclerosis, 41 (1982) 247-253 0 Elsevier/North-Holland Scientific Publishers, Ltd.
ARTERIAL CHOLESTEROL AND DNA CONTENTS IN RELATION TO SERUM LIPIDS AND APOLIPOPROTEINS
Studies on Cystic Artery Biopsies
OLLE WIKLUND, JOHN G. KRAL, LEIF LINDBLAD, SVEN-OLOF OLEFSSON, TORE SCHERSTEN, LARS SJdSTRGM and G6RAN BONDJERS Arterial Biology and Department
Group, Department of Medicine of Surgery, Sahlgren’s Hospital,
I and Department of Medical Biochemistry University of Giiteborg, Gijteborg (Sweden)
(Received 31 March, 1981) (Revised, received 18 June, 1981) (Accepted 18 June, 1981)
Summary Though various relationships between serum lipoprotein levels and risk for atherosclerotic disease have been shown there are only a few studies on the relationships between serum lipoprotein levels and the lipid contents of the arterial wall. This study presents cholesterol and DNA contents of arterial tissue from biopsies of the cystic artery in 23 patients with uncomplicated cholecystolithiasis. Sm7lrn L.,--ee.
!p~&
of
ch~!&ero],
~rigj;ly~er&-Jesj
alnhnlinonrotein -l____= -=______
&~!&er&
apoA-I, A-II and B were determined, and the relationships between artery and serum variables were calculated. There was a positive correlation between serum apoB and the arterial cholesterol, normalized to the DNA contents (r = 0.43, P < 0.05). There was a tendency towards an inverse correlation between the (Y-lipoprotein cholesterol levels and the arterial cholesterol (r = -0.39, P < 0.10). There were no significant correlations between serum apoA-I or A-II and the arterial cholesterol contents. These data indicate that deposition of cholesterol in the arterial wall is related to the serum level of apoB, with higher levels of arterial cholesterol at
This study was supported by grants from the Swedish Medical Research Council (Project No. 4531). the Swedish Association against Heart and Chest Disease, the Swedish Oleo-Margarine Foundation for Nutritional Research, the Medical Society of GGteborg and the University of Giiteborg. Findus AB. Bjuv, Sweden contributed with TV-dinners ? Correspondence: Giiran Bond&s, Department of Medicine I, Sahlgren’s Hospital, S-413 45 Gateborg, Sweden.
0021-9150/82/0000-0000/$02.75
0 1982 Elsevier/North-Holland
Scientific Publishers, Ltd.
248
higher serum levels of apoB. Earlier observations of an inverse correlation between alphalipoprotein cholesterol and arterial cholesterol could, however, not be conclusively confirmed. Key words:
Apolipoproteins
-Arterial
cholesterol
-Atherosclerosis
-Lipoproteins
Introduction The relationship between serum levels of lipids and lipoproteins and the clinical manifestations of atherosclerosis has been extensively studied. Thus, high levels of serum cholesterol and low density lipoproteins (LDL) are risk factors for coronary heart disease (CHD). The major protein component of LDL, apolipoprotein B (apoB), is also high in patients with CHD [ 1,2]. Serum high density lipoproteins (HDL), on the other hand have been suggested as a protective factor against CHD, and HDL or alphalipoprotein cholesterol [3] as well as the major protein components of HDL (apoA-I and A-II) are low in patients with CHD [ 31. Although several relations between the serum lipoprotein levels and the risk for ciinical manifestations of atlleroscierosis have been shown, oniy a few studies are available on the relation between the serum lipoprotein level and the lipid content of the arterial wall. A positive correlation between serum cholesterol and arterial tissue LDL content [4] as well as a positive correlation between serum LDL and arterial tissue cholesterol [5] has been reported. On the other hand an inverse correlation was found between alphalipoprotein cholesterol and arterial cholesterol [ 5,6]. The previous studies on correlations between serum lipoproteins and arterial wall cholesterol have been restricted to small groups of patients with specific diseases. The general significance of these observations may therefore be questioned. In the present study these correlations were analyzed in another group of patients, and with a different site of the arterial biopsy. In addition, not only serum lipids were determined, but also serum levels of apoA-I, A-II and apoB and their correlations to the arterial cholesterol and DNA content analyzed. Material and Methods Patients The patients were admitted to the hospital for operation of uncomplicated cholecystolithiasis. The following exclusion criteria were used: (1) Symptoms of acute cholecystitis, (2) medication with lipid-lowering drugs or oral contraceptives during the last 6 months, (3) cholestasis, (4) neoplastic disease during the last 10 years and (5) diabetes mellitus. Twenty-three subjects, 12 men and 11 women, were included in the study (mean age 38 years, range 31-70). The patients were admitted to the hospital 5 days before operation. During
249
the first day clinical examination was performed and the patients were interviewed about smoking habits, earlier diseases and medication. During hospitalization, the energy intake was adjusted to a value 40% above basal metabolic rate (BMR). BMR was estimated from age! sex and body surface using the measurements of Bothby and Du Bois [7], Exactly the same food was given each day; it consisted of Findus pre-prepared TV-dinners @ (Findus AB, Bjuv, Sweden): meat-balls, potatoes and vegetables for lunch and beef with onions and pommes frittes for dinner. Together with 7 pieces of Swedish Crisp Bread (totally 84 g) (Wasa knlckebrijd @),Wasa Brad AB, Filipstad, Sweden), 4 g butter and 1 dl of skim milk these two TV-dinners contained 4970 kJoules (1190 kcal) and 16 joule-% protein, 39 joule-% fat and 45 joule-% carbohydrate. A gruel with exactly the same composition with respect to protein, fat and carbohydrate was used as breakfast and extra evening meal in order to adjust the energy intake to the estimated individual need. Thus, irrespective of energy intake all subjects obtained the same food composition. Blood samples were obtained after 12 h fasting on the morning of the 4th day. Cholecystectomy and arterial biopsy were performed on the 6th day. Biochemical methods Cholesterol [ 81, triglycerides [ 91 and alphalipoprotein cholesterol were determined on fresh serum. The alphalipoproteins were isolated as the supernatant after precipitation with MnClz and heparin [lo]. Before apolipoprotein analyses serum samples were stored at -80°C in aliquots, in rubber-stoppered glass tubes. The apolipoproteins were determined with electroimmunoassay [2]. For each apolipoprotein all samples were analyzed at the same time, utilizing the same batch of antiserum. Arterial biopsy Routine anesthetic procedures were used during the operation. The cystic artery was dissected free during cholecystectomy and a segment was removed with great care to avoid stretching and bending of the artery. The vessel was dissected free from other tissue and was frozen in Ringer’s solution at -80°C until analyzed. After thawing the vessels were cut in 0.5-mm segments with a McIlvain tissue chopper. The adventitia was removed under a dissecting microscope and the intima-media preparations were analyzed for cholesterol [ll] and DNA [ 121 contents. All specimens were analyzed at the same time. Statistical methods The conventional statistical methods were used for the determination of means, standard deviations and correlation coefficients. To test if there was any significant deviation from the normal, distribution coefficients of skewness and curtosis were calculated [ 131. In the correlation analyses log-transformed data were used for those variables that showed a significant deviation from normal distribution. Log-transformed data did not significantly deviate from the normal distribution.
250
Results Mean values, standard deviations and ranges for serum cholesterol, triglycerides, alphalipoprotein cholesterol, apoA-I, A-II and apol3 as well as for arterial cholesterol and DNA contents are given in Tables 1 and 2. Two subjects had serum cholesterol levels above 7.5 mmol/l (7.9 and 9.1, respectively) and 4 had serum triglycerides above 2.1 mmol/l. The correlation coefficients between serum lipids or lipoproteins and arterial cholesterol or DNA contents are given in Table 2. There were no significant correlations between serum cholesterol or triglycerides and the arterial variables. There was a tendency towards an inverse correlation between alphalipoprotein cholesterol and the arterial cholesterol contents (r = -0.30, P = 0.066) (Fig. 1). There were, however, no significant correlations between the serum apoA-I or A-II levels and the arterial cholesterol or DNA. Serum apoB was positively correlated to the cholesterol content of the arterial wall expressed per pg DNA (r = 0.43, P = 0.041) (Fig. 2).
TABLE
1
MEANS,
STANDARD
CHOLESTEROL (A-CH).
SERUM
INCLUDED
A-I IN THE
(yr)
Weight
index
Iwdeht(keb/hdeht. L ._D___\___,,_______
S-CH
S-TG
(mmol/l)
(mmol/l)
A-CH
ApoA-I
ApoA-II
AP~B
DEVIATIONS
APOLIPOPROTEIN
PATIENTS
Age
(S-CH).
(mmol/I)
(g/I)
(g/l)
(g/I)
IrmJ \_.__,
-1001 ___l
(SD)
AND
RANGES
TRIGLYCERIDES (apoA-I).
A-II
(S-TG). (apoA-II)
AND
OF
AGE,
WEIGHT
ALPHALIPOPROTEIN APOLIPOPROTEIN
INDEX,
SERUM
CHOLESTEROL B (apo
B)
IN
STUDY Men
Women
All
n=12
n=
n=
Mean
53
44
SD
11
10
11
Range
32-70
31-62
31-70
11
23
49
Mean
1.03
1.03
1.03
SD
0.15
0.20
0.17
Range
0.87-1.45
0.80-1.45
0.80-1.45
Mean
6.05
4.75
5.43
SD
1.51
0.81
1.37
Range
3.8-9.1
3.2-6.0
3.2-9.1
Mean
1.84
1.14
1.50
SD
0.78
0.25
0.68
Range
0.7-3.4
0.8-l
Mean
1.60
SD
1.43 ^ -1 U.34
.6
0.7-3.4
0.28
1.51 - ^^ U.BZ 1 .o-2.0
Range
1.0-2.0
1.1-2.0
Mean
1.87
1.91
1.89
SD
0.25
0.26
0.25
Range
1.67-2.50
1.48-2.43
1.48-5.50
Mean
0.52
0.47
0.50
SD
0.06
0.05
0.06
Range
0.444.68
0.39-0.55
0.39-0.68
MeaIl
1.48
1.12
1.31
SD
0.36
0.26
0.36
Range
0.90-2.10
0.90-1.65
0.90-2.10
THE
251 TABLE
2
MEANS,
STANDARD
DEVIATIONS
ARTERIAL
DNA
INCLUDED
IN THE
Arterial
CH
AND
(SD)
ARTERIAL
AND
(nmol/mg
dry
Mean
weight)
Arterial
TABLE
(pg/mg
CH/DNA
ARTERIAL
RATIO
CHOLESTEROL
(CH/DNA)
IN THE
(CH), PATIENTS
dry
weight)
(pglpg)
Men
Women
All
n=12
n=
n=
33.99
31.45
11
32.77
3.17
31.0940.01
23
3.31
25.98-36.34
25.9840.01
Mean
7.23
7.17
7.21
SD
1.71
1.04
1.40 4.79-11.05
Range
4.79-11.05
5.10--9.01
Mean
1.89
1.72
1.81
SD
0.38
0.27
0.33
Range
1.36-0.38
1.33-2.21
1.33-2.74
3
CORRELATION
COEFFICIENTS
PROTEINS DNA
OF DNA
3.06
Range DNA
TO
STUDY
SD
Arterial
RANGES
CHOLESTEROL
AND
ARTERIAL
BETWEEN CONTENTS
OF
SERUM
LIPIDS,
CHOLESTEROL
LIPOPROTEINS
OR
DNA
OR
OR
APOLIPO-
CHOLESTEROL
PER
fig
(n = 23)
Log
serum
cholesterol
Log
serum
triglycerides
Alphalipoprotein Log
apoA-I
Log
apoA-II
cholesterol
Arterial
Log
cholesterol
DNA
Log
arterial
cholesterol/DNA
-0.OH
4.36
0.35
0.14
4.23
0.33
-xl.39
*
-0.06
4.24
ApoB
arterial
-0.16
0.04
4.19
0.13
-0.07
0.17
0.18
a.29
0.43
**
* P = 0.066 **
P = 0.041 TISSUE CHOLESTEROL/DNA
TISSUE CHOLESTEROL nmollmg
lJg’c19
o:-
o=
X=5
50 -
2.5 0
40-
2.0 -
30 -
1.5-
20 -
!.D -
Fig.
1.
Tissue
cholesterol
serum,
r = -0.39,
Fig.
The
2.
r = 0.41.
ratio
P < 0.05.
(nmol/mg
dry
X3
x
weight)
in relation
ox
x x
0: m:rXR 8”
x x
to alphalipoprotein
concentration
(mmolil)
in
P < 0.10. tissue
cholesterol
to
DNA
(pg/fig)
in arterial
tissue
in relation
to serum
apoB
(g/l)
levels.
252
Discussion In two earlier reports [5,6] the relationships between serum lipoprotein levels and the arterial cholesterol contents have been studied. One of these studies concerned severely obese patients [5] and the other study was restricted to women undergoing hysterectomy [6] due to uterine myoma. In the present study subjects admitted to the hospital for elective cholecystectomy were investigated. The selection of the patients might have affected the results of these studies. Severely obese subjects are different from a non-obese population in several aspects of lipid metabolism [14] and their alphalipoprotein cholesterol levels are usually low [ 5,14,15]. Patients with gallstones, on the other hand, might have a more general disturbance in the cholesterol metabolism. Not only the selection of patients but also the location of the arterial biopsy has to be considered when the results of the present study are compared with earlier reports. In the present study the cystic artery was analyzed. Atherosclerosis, however, is unevenly distributed within the arterial tree and differences between different arterial regions might explain variations in the results. We do not know if the cystic artery is representative for other arterial regions. In the present study there was a positive correlation between the serum apoB levels and the arterial contents of cholesterol per pg of DNA. ApoB is the major protein component of LDL and a tendency towards a correlation between serum LDL cholesterol and the cholesterol content of the mesenteric artery has earlier been reported [ 51. Deposition of apoB in the atherosclerotic lesions has been reported by several investigators [16,17]. Furthermore, apoB has been suggested to bind to some components of the intercellular substance of the arterial wall [ 18-201. The present data indicate that the deposition of cholesterol is related to the serum level of apoB or LDL, with higher levels of arterial cholesterol at higher serum apoB levels. In two previous studies from our group an inverse correlation between arterial cholesterol and alphalipoprotein cholesterol has been reported [5,6]. This inverse correlation has been taken to support the suggested role of HDL as a cholesterol acceptor in the removal of cholesterol from the tissue [3]. In the present study there were no significant correlations between alphalipoprotein cholesterol, apoA-I or A-II and arterial cholesterol, and this study does not support the hypothesis, although the tendency regarding alphalipoprotein cholesterol is in agreement with earlier observations. Due to discrepancies between the different studies these correlations have to be further analyzed in nther ““._“I
m-n~mw nf nntipntn b-‘ -y” VL y.e’ -u’-“u
and with u1.u ,.A”__ &her
sit.er nf “_“_., .._ thP 1_.V nrtminl -“___-
hinnw “--llJ.
References G., Quinci, G.B. and Belussi, G., Plasma levels of apolipoAvogaro, P., Bittolo Bon. G.. Cazzolato, protein A-I and apolipoproteln B in human atherosclerosis, Artery, 4 (1978) 385. Fager. G., Serum Apolipoprotein Levels in Relation to Acute Myocardial Infarction and its Established Risk Factors, Thesis, University of GBteborg, GGteborg. Sweden, 1979. Bondjers, G., Wiklund, 0.. Olofsson, S.-O., Gustafson. A. and BjSrkerud. S.. Relationship between high density lipoproteins and atherosclerosis. In: C.E. Day (Ed.). High Density Lipoproteins, Dekker Inc.. New York, NY, 1981, p. 463-503. Smith, E.B. and Slater, R.S., Relationship between low-density lipoprotein in aortic intima and serumlipid levels. Lancet. i (1972) 463.
253 5 Bondjers, G.. Gustafson, A., Kral, H.. Schersten, T. and Sjiistriim. L., Cholesterol content in arterial tissue in relation to serum lipoproteins in man. Artery, 2 (1976) 200. 6 Bondjers, G., Wiklund, 0. and Olofsson, S-0.. Alphalipoprotein cholesterol and arterial cholesterol in human uterine arteries, Artery, 8 (1980) 305. 7 Documenta Geigy, Scientific Tables, 7th edition, Ed. K. Diem and C. Lentner, J.R. Geigy %A., Basel. 1970, p. 540. 8 Leffler, H.H.. Estimation of cholesterol in serum, Amer. J. Clin. Path., 31 (1959) 310. 9 Carlson, L.A.. Determination of serum glycerides, Acta Sot. Med. Upsalien., 64 (1959) 208. 10 Wlklund, 0.. Fager, G., Craig, I.H., Wilhelmsson, C.E.. Vedin, A., Olofsson, S.-O., Bondjers, G. and Wilhelmsen, L.. Alphalipoprotein levels in relation to acute myocardial infarction and its risk factors, Stand. J. Clin. Lab. Invest., 40 (1980) 239. determination of cholesterol and cholesteryl ester in 11 Bondjers, G. and BjGrkerud. S., Fluorometric tissue on the nanogram levels, Anal. Biochem., 42 (1971) 363. 12 Kissane. J.M. and Robins, E., The fluorometric measurement of deoxyribonucleic acid in animal tissues with special reference to the central nervous system, J. Biol. Chem.. 233 (1958) 184. 13 Armitage, P., Statistical Methods in Medical Research. 3rd printing, Blackwell Scientific Publications, Oxford. 1974. 14 Nestel, P. and Goldrick, B., Obesity - Changes in lipid metabolism and the role of insulin, Clin. Endocrinol. Metab.. 5 (1976) 313. 15 Wilson, D.E. and Lees, R.S.. Metabolic relationships among the plasma lipoproteins - Reciprocal changes of very low and low density lipoproteins in man, J. Clin. Invest.. 51 (1972) 1051. 16 Walton, K.W. and Williamson. N., Histological and immunofluorescent studies on the human atheromatous plaque, Atherosclerosis, 8 (1968) 599. 17 Hoff. H.F., Jackson. R.L.. Mao, J.T. and Gotto, A.M., Localization of low density lipoproteins in arterial lesions from normolipemics employing a purified fluorescent-labeled antibody, Biochim. Biophys. Acta. 351 (1974) 407. 18 Avial, E.M., Lopez. F. and Camejo, G.. Properties of low density lipoproteins related to its interaction with arterial wall components - In vitro and in viva studies, Artery, 4 (1978) 36. 19 Hoff, H.F., Heideman, C.L., Gaubatz. J.W., Titus, J.L. and A.M. Gotto Jr., Quantitation of apoB in human aortic fatty streaks - A comparison with grossly normal intima and fibrous plaques, Atherosclerosis, 30 (1978) 263. 20 Smith. E.B., Biochemical studies on permeability and the interaction between blood constituents and arterial components in atherosclerosis. In: A.M. Gotto, L.C. Smith and B. Allen (Ed%), Atherosclerosis V (Proceedings of the 5th International Symposium), Springer-Verlag. Berlin, 1980, p. 121.