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Lecithin: Cholesterol acyl transfer rate and high density lipoprotein level in coronary artery disease
155
Atherosclerosis, 41( 1982) 155-165 0 Elsevier/North-Holland Scientific
Publishers,
Ltd.
LECITHIN:CHOLESTEROL ACYL TRANSFER RATE AND HIGH DENSITY LIPOPROTEIN LEVEL IN CORONARY ARTERY DISEASE
LARS
WALLENTIN
Department (Sweden) (Received (Accepted
and BIRGITTA
of Internal
Medicine,
MOBERG
Linkijping
University
Medical
School,
S-581
85 LinkGping
26 May, 1981) 1 June, 1981)
Summary The lecithin :cholesterol acyl transfer (LCAT) reaction occurs in connection with the high density lipoproteins (HDL) and might have importance for the development of atheromatosis. The LCAT rate and the lipoprotein concentrations in plasma were therefore determined in 82 patients, 40-60 years old, with incapacitating angina pectoris and significant lesions at coronary angiography. Thirty-eight cases were normolipidemic and 22 had type IV h-yperlipidemia while the others had different types of hypercholesterolemia (n = 7) or were pharmacologically treated for previously diagnosed hyperlipidemia (n = 11) or diabetes mellitus. The normolipidemic group and the type IV group with coronary artery disease were separately compared to one normolipidemic (n = 44) and one type IV hyperlipidemic (n = 29) control group of healthy subjects of about the same age. The fractional LCAT rate (5%* h-l) was lower in patients compared to controls both regarding the normolipidemic and type IV subjects. The molar LCAT rate (Fmol * 1-l * h-l) did not differ between normolipidemic cases and controls while it was lower in type IV patients with coronary artery disease than in the type IV controls. The HDL total cholesterol (TC) concentration and the HDL-TC/TC ratio were lower in normolipidemic cases than in normolipidemic controls while both cases and controls with type IV hyperlipidemia showed equally low levels of these parameters. In conclusion, both a reduced fractional LCAT rate and a decreased HDLTC/TC ratio might be indicators of disturbances of the cholesterol ester metabolism and might contribute to the development of coronary atheromatosis.
This
study
was
financially
supported
by
the
Swedish
Medical
Research
Council
(Projekt
No.
19X-
4529).
0021-9150/82/0000-0000/$02.75
0 1982
Elsevier/North-Holland
Scientific
Publishers,
Ltd.
156
Key words:
Angina pectoris - Cholesterol - Coronary emia - Lecithin:cholesterol acyl transferase
angiography -Lipoproteins
-
HyperlipoproteinTriglycerides
Introduction Disturbances of the lipoprotein metabolism are often found in patients with coronary atheromatosis [ 1,2]. An elevated plasma concentration of low density lipoproteins (LDL) or of very low density lipoproteins (VLDL) increases while an elevated level of high density lipoproteins (HDL) decreases the accumulation of cholesteryl esters (CE) in the arterial wall [l-4]. Most of the cholesterol in the arterial wall is considered to be derived from the LDL while removal of cholesterol from the arterial wall seems to occur by transfer of unesterified cholesterol (UC) to the HDL [ 5-81. Most UC entering the HDL is esterified by the plasma enzyme lecithin:cholesterol acyl transferase (EC 2.3.1.43). The 1ecithin:cholesterol acyl transfer (LCAT) takes place in connection with the HDL and seems to be important for the transfer of cholesterol between tissues and lipoproteins [9,10]. In order to gain further knowledge of the role of LCAT in the development of atheromatosis, we have studied the LCAT rate and its relation to plasma lipoprotein levels in patients with coronary artery disease (CAD). Material and Methods Material
During 2 periods 82 consecutive patients, 40-61 years of age, with incapacitating angina pectoris referred for evaluation by.coronary angiography were studied regarding plasma lipoprotein levels and the rate of LCAT in plasma. Only subjects with at least 70% stenosis or occlusion of at least one major coronary artery and without valvular heart disease were included. Patients treated with lipid-lowering drugs and those with a history or signs of diabetes mellitus, thyroid, liver or renal disease were excluded. Out of the 82 patients entering the study 15 were excluded - 11 cases because of clofibrate treatment of previously diagnosed hyperlipidemia, 3 cases because of diabetes mellitus and one patient because of signs of liver and renal dysfunction. Of the remaining group 42 patients had suffered from at least one myocardial infarction, all investigated more than 3 months after that event. Coronary by .pass operation had been performed in 12 cases and the investigation was carried out at least one year after the operation. A healthy survey in the present geographical district showed that in 994 50year-old men with normal relative weight [weight kg/height cm - 100) < 1.101 the upper 95th percentiles of total cholesterol (TC) and triglyceride (TG) concentrations were 8.0 and 2.3 mmol/l, respectively. Based on these upper reference levels the present material of patients with coronary artery disease was divided into one normolipidemic (n = 38) and one hyperlipidemic part. According to WHO criteria [ll] with LDL-TC < 5.7 mmol/l and VLDL-TC < 0.8 mmol/l as upper reference levels [13,14], 22 cases had type IV hyperlipopro-
157
teinemia and the remaining 7 cases had type IIa, IIb or type III hyperlipoproteinemia. The normolipidemic group and the group with type IV hyperlipoproteinemia were separately compared to two reference groups of apparently healthy subjects, one with normolipidemic and the other with type IV hyperlipoproteinemic subjects. The few subjects with different types of hypercholesterolemia were not included in any statistical comparisons. The 38 normolipidemic men with CAD had a mean age of 52.1 (range 4160), mean body weight 79.0 f 1.74 (SEM) and mean relative weight 1.05 ? 0.02. Nitroglycerin was used by almost all and beta-blocking agents by 26 cases while a few also were treated with cardiac glycosides and/or thiazide diuretics. Supine resting blood pressure was above 160/95 in 10 patients and 17 were smokers. The normolipidemic reference group consisted of healthy 40-60-year-old men without a history or symptoms of coronary artery disease. Subjects with clinical or laboratory signs of diabetes mellitus, thyroid, liver or renal disease were excluded. The subjects were recruited from hospital employees, military personnel and blood donors. The group consisted of 44 men with mean age 50.1, mean body weight 79.2 + 1.63 and relative weight 1.0 * 0.02. Casual blood pressure above 160/95 was found in 2 subjects and 16 were smokers. The 22 men with coronary artery disease with type IV hyperlipoproteinemia had a mean age 54.5 (range 45--61), mean body weight 84.0 f 1.68 and relative body weight 1.12 + 0.02. Most men used organic nitrates and 19 cases also had beta blocking drugs and a few cardiac glycosides and/or thiazide diuretics. Blood pressure exceeding 160/95 was found in 5 and 6 were smokers. The type IV reference group consisted of 29 men with plasma TG > 2.3, LDL-TC < 5.7, VLDL-TC > 0.8 and no chylomicrons in the standing plasma test [12]. Only those without history or symptoms of CAD at clinical and laboratory investigations were included. These men were mainly referred from a health survey in the same geographical district. The mean age was 52.6 (range 41-61) years, mean body weight 81.8 2 1.78 kg and relative weight 1.09 +_ 0.02. Three of these men used hydralazine and hydralazine-spironolacton. Seven men had a supine resting blood pressure exceeding 160/95 and 16 men were smokers. Methods
After an overnight fast venous blood was drawn into evacuated tubes with 1.2 mg K3EDTA/ml blood. Sampling was done with the patient in the sitting position. The samples were immediately cooled and plasma isolated by centrifugation at +4”C. A part of the plasma was stored at this temperature until ultracentrifugation was started within 4 days. The determination of LCAT rate was performed in plasma stored frozen at -70°C for a maximal time of 2 months and thawed immediately before analysis. The fractional LCAT rate was measured by incubation of plasma for 20 min in vitro after the addition of [3H]cholesterol. The fractional esterification of the label was taken as the fractional LCAT rate assuming complete equilibration of the added [3H]cholesterol and UC in plasma lipoproteins. The UC concentration in plasma before incubation was determined by a gas-liquid chromatographic method. The molar LCAT rate (pm01 - 1-l - h-l) was calculated as
158
the product of the fractional esterification rate (% - h-l) and the UC concentration (mmol *1-l). The details and precision of these methods have previously been presented [15,16]. Lipoproteins were separated according to the Lipid Research Clinics Program [12]. VLDL were isolated by ultracentrifugation of plasma for 18 h at hydrated density 1.006. HDL were separated in the infranatant by precipitation of LDL with heparin-MnClz [12]. The TC and TG concentrations in plasma and the lipoprotein fractions were determined by enzymatic Boehringer Mannheim Tests [ 17-191. Routine laboratory tests of thyroid, renal and liver functions and blood glucose concentrations were performed at the Department of Clinical Chemistry, Linkiiping University. Statistical calculations of means, SD and SEM were performed according to standard methods. Because of the skew distribution of most lipoprotein parameters, the significance of differences between groups was assessed by nonparametric rank sum tests [20] and P < 0.05 was taken as significant. Correlations were calculated according to the least squares method [20] and correlation coefficients (r) different from 0 by P < 0.05 were considered significant. LCAT
%,h-'
LCAT %.h-' p< 0.001
p= 0.05
.
10.0 0
. .
a
9.0
t
so 8.0
.
7.0
: .. .
‘f
6.0
At* ...
T
I-
9.0
:I.
8.0
.I.
7.0
?? ?? : 6.0 ..
.* .
5.0
10.0
5.0
4.0
4.0
3.0
3.0
2.0
2.0
1.0
1.0
0
0 Ref.group
CAD
CAD
typeIV
TypeIV
Normolipidemia Fig. 1. Fractional LCAT rates in normollpidemic cases and controls and in cases and controls with type IV hyperlipidemia. Circles denote controls and triangles cases with coronary artery disease (CAD). Open symbols symbolize normolipidemic subjects and closed symbols the type IV subjects. The lines with hooks illustrate means f SEM. Differences between groups were tested by nonparametric rank sum tests and the significance levels are shown in the figure.
159
Results The individual results of the fractional and molar LCAT rates in normolipidemic and type IV cases and controls are shown in Figs. 1 and 2. The fractional LCAT rate was lower in patients compared to controls with respect to both normolipidemic and type IV subjects. Of the normolipidemic subjects with coronary artery disease 12/38 (= 32%) had fractional LCAT rates below 5.0% - h-l, which was the 7th percentile of the distribution of the normolipidemic controls. In the type IV groups 17/22 (= 77%) of the patients with coronary artery disease had fractional LCAT rates below 6.6% * h-l, which was the 14th percentile of the type IV reference group. There were no differences in molar LCAT rate between normolipidemic cases and controls. The patients with coronary artery disease and with type IV had lower molar LCAT rates than their type IV controls. The lipoprotein lipids in the 4 groups have been detailed in a separate report
LCAT
flmol.lK’.h-’
LCAT
t
t
p = 0.001
n.s.
1
260
pmol.l-‘.h-’
.
I ; 260
240
240
220 :
220
. . 200
: I
.I
200
18Oj
180
160
16.i I
80
60
Ref.fJroup Normolipidemia
CAD
CAD type IV
Type IV
Hyperlipidemia
Fig. 2. Molar LCAT rates in the normolipidemlc hyperlipidemia. Signs and symbols as in Fig. 1.
cases and controls
and in cases and controls
with
type IV
160 TABLE
1
LIPID
AND
AND
TYPE
LIPOPROTEIN IV
LIPID
CORONARY
CONCENTRATIONS
ARTERY
DISEASE
(mmol/l)
(CAD)
IN PLASMA
PATIENTS
AND
OF
Normolipidemia
(TC)
5.32
6.09 b
6.51
0.14
0.17
44 1.29
0.94a
0.78
0.04
0.05
(TG)
3.93
4.42
SEM
0.13
0.11
= high
SEM
= standard
density
Significant b P < 0.01,
0.45
SEM
0.04
0.03
TABLE
4.06
0.12
0.20
b
35
29
1.22
1.68
0.12
0.21
21
29
0.82
0.92
1.99
2.68
SEM
0.07
0.05
0.23
0.31
24
35
21
Mean
1.16
1.71
SEM
0.07
0.08
44 LDL
= low
a
39
density
lipoprotein.
29
3.25
3.55
0.23
0.38
22 VLDL
= very
low
29 density
lipoprotein,
n = number.
between
cases
and
controls
by
rank
sum
tests
are
symbolized
a P < 0.001,
c P < 0.05.
2
LECITHIN
: CHOLESTEROL
NORMOLIPIDEMIC TROL
AND
ACYL TYPE
IV
TRANSFER CORONARY
(LCAT)
RATES
ARTERY
AND
DISEASE
HDL
RATIOS
(CAD)
IN PLASMA
PATIENTS
AND
SUBJECTS NormoIipidemia Controls
LCAT
?‘o . h-’
~mol
. 1-l . h-’
Type CAD
Mean
6.15
5.55
SEM
0.18
0.20
n LCAT
Mean SEM n
22
3.08
0.35
0.22
0.02
0.01
0.24
0.16
SEM
0.01
0.01
as in Table
1.
38
22 a
38
MeaIl n
and symbols
38
4.90
38
Mean
TC
7.64 0.22
133.3
SEM n
6.24a
38
2.87
38
29 a
167.9 6.58 29
0.18
0.21
0.01
0.01
22 a
Controls
0.24
102.6
LDL-TC
HDL-TC
c
44
44
IV
CAD
100.9
HDL-TC
Signs
4.54
Mean
of the mean,
differences
0.03 29
22
0.33
lipoprotein,
error
b
38
Mean
24
0.79
22
Mean
n HDL
38
38
0.17 29
0.06 38
6.61
22
SEM
n Triglycerides
38
Mean
n VLDL-TG
Controls
0.11
n VLDL-TC
CAD
SEM
n LDL-TC
CAD
IV
Mean n
HDL-TC
SUBJECTS
Type
Controls Cholesterol
NORMOLIPIDEMIC
CONTROL
0.12 0.01 22
29 0.12 0.01 29
OF CON-
161
TABLE
3
CORRELATION
COEFFICIENTS
LIPOPROTEIN
LIPID
BETWEEN
LEVELS
FRACTIONAL
Type
Normolipidemia
CAD
Controls (n = 29)
-o.35
-o.45
-0.21
b
LDL-TC
a.41
=
VLDL-TC
0.58
VLDL-TG
0.38
TG
0.47 0.49
c P <
TABLE
significance 0.05.
Other
c
c
OR
-o.17
-o.35
0.04
-O.66
-0.24
-o.o5
0.15
0.49
b
0.26
0.49
b
-O.O6
c
0.19
0.50
c
-o.o5
c
0.10
0.33
b
levels signs
LIPID
IV
(n = 20)
c
denote
AND
CAD
4.57
0.01,
h-l)
(n = 36)
-O.42
a9b.c
(% .
Controls
TC
weight
RATE
(n = 24)
HDL-TC
Body
LCAT
IN PLASMA
and
of
correlation
symbols
coefficients
as in Tables
1 and
(r)
0.01
different
from
0
0.38
’
and
a P <
’h-l)
AND
0.001,
b P <
2.
4
CORRELATION LIPOPROTEIN
COEFFICIENTS LIPID
BETWEEN
LEVELS
MOLAR
Normolipidemia
Type CAD
Controls
(n = 20)
(n = 29)
LDL-TC
0.12 0.79
a
0.26
VLDL-TG
0.53
b
0.48
TG
0.76
a
0.50
0.58
b
0.07
as in Table
0.32
0.20
AND
0.38
-o.15
4.01
VLDL-TC
symbols
LIPID
IV
(n = 36)
-o.58b
and
.1-l
CAD
-o.o5
Signs
(~mol
Controls
HDL-TC
weight
RATE
(n = 24) TC
Body
LCAT
IN PLASMA
a.36
-O.16
0.18
-0.16
0.88
a
0.59
b
0.73
a
0.47
b
b
0.84
a
0.47
b
0.44
a
0.01
a
3.
[ 211 and are summarized in Tables 1 and 2. In the normolipidemic subjects the most prominent differences were the lower HDL-TC concentration and the lower HDL-TC/TC and HDL-TC/LDL-TC ratios in cases compared to controls. There were also somewhat higher LDL and VLDL concentrations in normolipidemic patients compared to their control group. In the type IV subjects there were no major differences in lipoprotein concentrations and the HDLTC/TC and HDL-TC/LDL-TC ratios were similar in cases and controls. The correlation coefficients between the LCAT rates and the levels of lipoprotein lipids are shown in Tables 3 and 4. The body weight was positively correlated to the fractional and molar LCAT rates in both control groups, but not in any of the case groups. The molar LCAT rate was positively correlated to the TG and VLDL concentration in all 4 groups. Discussion The LCAT rate, determined with the present method, is dependent both on the concentration of the enzyme and the concentration and composition of its
162
substrates, products and cofactors [ 10,22-241. Therefore the rate-limiting factors for the esterification rate cannot be elucidated. Besides the enzyme concentration many other factors in plasma have been suggested to influence the cholesterol esterification rate e.g. the availability of the enzyme cofactors apo AI and apo C [25,26], the HDLJHDLz ratio [9,10,27], the phospholipids [28], the accumulation of CE in product lipoproteins and the availability of the CE transferring protein [ 29-321. In the present study the fractional LCAT rate was reduced in relation to the TG and VLDL levels in both normolipidemic and type IV patients with coronary atheromatosis. Two previous studies have reported LCAT rates and concentrations of lipids and lipoproteins in subjects with coronary artery disease i.e. survivors of myocardial infarction. Soloff et al. [33] found reduced fractional LCAT rates and elevated TC and TG levels in a series of patients compared to normolipidemic healthy controls. In a comparison between young myocardial infarction survivors and controls matched for plasma TC levels Wiklund et al. [34] found no differences in fractional or molar LCAT rates or the HDL-TC levels. In the latter study around half of the subjects were hypercholesterolemic according to age-adjusted reference levels. In both these groups the fractional LCAT rates (means 4.9% * h-l) were lower than the fractional LCAT rates of the normolipidemic patients with coronary artery disease in the present study. Furthermore, the young myocardial infarction survivors had elevated TG levels and in relation to TG levels the fractional LCAT rates might have been reduced also in this study. The findings of the reduced fractional LCAT rates in coronary artery disease give rise to some speculations about possible mechanisms. The UC entering the LCAT reaction and HDL is mainly derived from surface constituents of catabolized VLDL or from cellular membranes [9,10]. The uptake of UC from cellular membranes into HDL might be facilitated by cholesterol esterification and thereby the LCAT reaction might be of importance for the removal of cholesterol from tissues e.g. the arterial wall [7,9,10,35]. However, most cholesterol entering the reaction is derived from the catabolism of TG rich lipoproteins [9, 10,36-381. Therefore a high molar LCAT rate does not implicate a large uptake of cholesterol from the arterial wall but rather a high input of cholesterol into plasma via TG-rich lipoproteins [36,37]. Furthermore, the CE produced by LCAT do not remain in the HDL but seem to be distributed to all the plasma lipoproteins [39-411, via a recently discovered CE-transferring protein [29-321. The regulation of the distribution of CE among the lipoproteins is so far unknown. A high LCAT rate might lead to an enhanced formation of CE in VLDL and LDL [9,10]. As the cholesterol esterified by LCAT might be derived both from the VLDL catabolism and from the tissues e.g. arterial walls, and as the CE produced by LCAT are distributed to all the lipoprotein fractions, the interpretation of the LCAT rates in relation to the lipoproteins is complicated. Based on the present results and on previous findings in normolipidemic and hyperlipidemic subjects [42-471, the plasma LCAT rate should be evaluated in relation to the plasma levels of lipids and lipoproteins and to the body weight. An elevated input of VLDL cholesterol into plasma e.g. in obesity [48, 491 should be associated with an enhanced molar LCAT rate [42,44,46]. If
163
the LCAT rate is not elevated in relation to an increased input of UC in the TGrich lipoproteins, the content of UC in plasma lipoproteins and cellular membranes might become enhanced [9,10]. The removal of UC from peripheral tissues to HDL might also be retarded because of saturation of the LCAT reaction and of HDL by UC from the VLDL catabolism in accordance with recently reported in vitro experiments [ 501. Thus, a fractional LCAT rate that is low in relation to the TG level or the body weight might indicate an increased flux of cholesterol to the tissues and/or a reduced removal of cholesterol from the tissues to HDL. The present study demonstrates that most subjects with coronary artery disease had low HDL-TC levels and low HDL-TC/LDL-TC or HDL-TC/TC ratios and that the mean fractional LCAT rate was reduced in coronary artery disease. In type IV hyperlipoproteinemia the HDL-TC level and the HDL-TC/LDL-TC and HDL-TC/TC ratios were equally low in patients and controls, while the fractional LCAT rate was reduced in the patients with coronary artery disease. Both the decreased HDL-TC/TC ratio and the reduced fractional LCAT rate might be markers of disturbances of the CE metabolism, contributing to the development of coronary atheromatosis. Most cases in the present study were treated with beta-blocking drugs and probably led a more sedentary life than their controls. Both these factors tend to change the lipoprotein metabolism i.e. reduce the HDL and elevate the VLDL level [51]. The possible influences of these factors on the LCAT rates are unknown. In the present study the cases with beta-blocking drugs did not differ from untreated cases regarding LCAT rates or HDL-TC levels. However, the results of the present study do not allow any firm conclusions regarding the effects of the disease and its treatment. Acknowledgments The skilful technical assistance roth is gratefully acknowledged.
of MS Ylva Svensson
and MS Gunnel
Alm-
References Castelli, W.P., Doyle, J.I., Gordon. T.. Hames, C.G., Hjortland, M.C.. Hulley, S.B., Kamn. A. and Zukel, W.J., HDL cholesterol and other lipids in coronary heart disease. Circulation. 55 (1977) 767. Connor. W.E., The relationship of hyperlipoproteinemia to atherosclerosis - The decisive role of dietary cholesterol and fat. In: A.M. Scand. R.W. Wissler and G.S. Getz (Eds.), The Biochemistry of Atherosclerosis. Marcel Dekker, New York, NY, 1979, pp. 371418. Miller, N.E.. The evidence for the antiatherogenicity of high density lipoprotein in man, Lipids, 13 (1978) 914. Miller. G.J., High density lipoproteins and atherosclerosis, Ann. Rev. Med.. 31 (1980) 97. Goldstein, J.L. and Brown, M.S., Atherosclerosis - The low density lipoprotein receptor hypothesis, Metabolism, 26 (1977) 1257. Small, D.M.. Cellular mechanisms for lipid deposition in atherosclerosis. N. Engl. J. Med., 297 (1977) 873 and 924. Bates. S.R., Cholesterol sccumulation in arterial cells and in extracellular spaces, Artery, 5 (1979) 362. Stein, Y. and Stein, 0.. Interaction between serum lipoproteins and cellular components of the arterial wall. In: A.M. Scam& R.W. Wissler and G.S. Getz (Eds.), The Biochemistry of Atherosclerosis. Marcel Dekker. New York, NY, 1979. pp. 313-344.
164 9 Glomset, J.A., Lecithin:cholesterol awl transferase - An exercise in comparative biology. In: S. Eisenberg (Ed.), Progress in Biochemical Pharmacology, Vol. 15 (Lipoprotein Metabolism), Karger, Bawl, New York. NY, 1979. pp. 41-66. acyl transferase. In: A.M. Scanu. R.W. Wissler and G.S. Getz 10 Glomset, J.A., Lecithin:cholesterol (Eds.), The Biochemistry of Atherosclerosis. Marcel Dekker, New York, NY, 1979, PP. 247-274. 11 Beaumont, J.-L.. Carlson, L.A., Cooper. G.R., Fejfar, Z.. Fredricson, D.S. and Strasser, T.. Classification of hyperlipidaemias and hyperlipoproteinaemias, Bull. Wld Hlth Org., 43 (1970) 891. 12 Bachorik. P.S. and Wood, P.D.S., Laboratory considerations in the diagnosis and management of hyperlipoproteinemia. In: B.M. Rifkind and R.I. Levy (Eds.), Hyperlipidemia - Diagnosis and Therapy, Grune & Stratton, New York, NY, 1977, PP. 41-69. 13 Olsson, A.G. and Carlson. L.A., Studies in asymptomatic primary hyperlipidemia, Acta Med. Stand., Suppl. 580 (1975). 14 Carlson, L.A. and Ericsson. M., Quantitative and qualitative serum lipoprotein analysis, Part 1 (Studies in healthy men). Atherosclerosis, 21 (1975) 417. 15 Stokke, K.T. and Norum, K.R.. Determination of 1ecithin:cholesterol awl transfer in human blood plasma, &and. J. Clin. Lab. Invest., 27 (1971) 21. 16 Wallentin, L. and Vikrot, O., Evaluation of an in vitro assay of 1ecithin:cholesterol awl transfer rate in plasma, Stand. J. Clin. Lab. invest.. 35 (1975) 661. 17 18 19 20 21 22
23 24
25
26 2,7 28 29 30 31
32
33 34
35
Riischlau, P., Bernt, E. and Gruber. W.. Enzymatische Bestimmung des Gesamt-Cholesterins im Serum. J. Clin. Chem. Clin. Biochem., 12 (1974) 403. Eggstein, M., Eine neue Bestimmung der Neutralfette im Blutserum und Gewebe, Klin. Wschr., 44 (1966) 267. Schmidt, F.H. and van Dahl, K., Zur Methode der enzymatischen Neutralfett-Bestimmung in biologischem Material, J. Clin. Chem. Clin. Biochem., 6 (1968) 156. Snedecor, G.W. and Cochran, W.G.. Statistical Methods. 6th edition. The Iowa State University Press. Ames, IA, 1967. Moberg, B. and Wallentin, L.. HDL and other lipoproteins in normolipidaemic and hypertriglyceridaemic (type IV) men with coronary artery disease. Europ. J. Clin. Invest., In press. Sodhi. H.S., Esterification of plasma free cholesterol. In: D. Kritchevsky and O.J. Pollak (Eds.), Clinical Methods in Study of Cholesterol Metabolism (Monographs of Atherosclerosis, Vol. 19). Karger. Basel. New York. NY, 1979, pp. 87-93. Lacko. A.G., On the interpretation and potential diagnostic value of the measurements related to 1ecithin:cholesterol acyltransferase activity, Clin. Biochem., 9 (1976) 212. Thanabalasingham, S. Thompson, G.R., Trayner, I.. Myant, N.B. and Soutar, A.K., Effect of lipoprotein concentration and 1ecithin:cholesterol acyltransferase activity on cholesterol esterification in human plasma after plasma exchange, Europ. J. Clin. Invest., 10 (1980) 45. Soutar. A.K.. Garner, C.W., Baker. N., Sparrow, J.T., Jackson, R.I., Gotto, Jr., A.M. and Smith, L.C., Effect of human plasma apolipoproteins and phosphatidylcholine acyl donor on the activity of 1ecithin:cholesterol acyltransferase, Biochemistry. 14 (1975) 3057. Albers, J.J.. Lin, J. and Roberts, B.P., Effect of human plasma apolipoproteins on the activity of purified 1ecithin:cholesterol acyltransferase, Artery, 5 (1979) 61. Pinon. J.C., Bridoux, .A.M. and Laudat. M.H., Initial rate of cholesterol esterification associated with high density lipoproteins in human plasma, J. Lip. Res., 21 (1980) 406. Drevon, C.A. and Norum, K.R., Effect of lipid emulsions on the plasma 1ecithin:cholesterol acyl transfer in guinea pigs, Nutr. Metab., 19 (1975) 180. Chajek, T. and Fielding, C.J., Isolation and characterization of a human serum cholesteryl ester transfer protein, Proc. Nat. Acad. Sci. (USA). 75 (1978) 3445. Zilversmit, B.B., Hughes, L.B. and Balmer. J., Stimulation of cholesterol ester exchange by lipoprotein free rabbit plasma, Biochim. Biophys. Acta, 409 (1976) 393. Sniderman, A., Teng, B.. Vezina. C. and Marcel, Y.L., Cholesterol exchange between human Plasma high and low density lipoproteins mediated by a plasma protein factor, Atherosclerosis, 31 (1978) 327. Marcel. Y.L.. Vezina, C., Teng, B. and Sniderman. A., Transfer of cholesterol esters between human high density lipoproteins and triglyceride-rich lipoproteins controlled by a plasma protein factor. Atherosclerosis, 35 (1980) 127. Soloff, L.A., Rutenberg. H.L. and Lacko, A.G., Serum cholesterol esterification in patients with corenary heart disease. Amer. Heart J., 85 (1973) 153. Wiklund. 0. and Gustafson. A., Lecithin:cholesterol acyl transfer (LCAT) and fatty acid composition of lecithin and cholesterol esters in young male myocardial infarction survivors, Atherosclerosis, 33 (1979) 1. Ray, E., Bellini. F., Stoudt, G., Hempedy. S., and Rothblat, G., Influence of 1ecithin:cholesterol acyltransferase on cholesterol metabolism in hepatoma cells and hepatocytes, Biochim. Biophys. Acta, 617 (1980) 318.
36 37
38 39 40 41 42 43 44 45 46
47 48 49 50
51
Kudchodkar. B.J. and Sodhi. H.S.. Turnover of plasma cholesteryl esters and its relationship to other parameters of lipid metabolism in man. Europ. J. Clin. Invest., 6 (1976) 285. Angelin. B.. Einarsson, K., Leijd. B. and Wallentin, L., Plasma cholesterol esterlfication rate in type IV hyperllpoproteinemia - Relation to bile acid kinetics and triglyceride metabolism, J. Lab. Clin. Med., In press. Stein, Y. and Stein, 0.. Metabolism of plasma lipoproteins. In: A.M. Gotto, Jr., L.C. Smith and B. Allen (Eds.). Atherosclerosis V, SpringerVerlag, Berlin, 1980, pp. 653-665. Nichols, A.V. and Smith, L., Effect of very low-density lipoproteins on lipid transfer in incubated serum. J. Lipid Res., 6 (1965) 206. Lallu, J.I. and Barter, P.I.. The in viva metabolism of esterified cholesterol in the plasma high density lipoproteins of rabbits, J. Lab. Clin. Med., 93 (1979) 570. Nestel, P.J., Reardon. M. and Blllington. I.. In viva transfer of cholesterol esters from high density lipoproteins to very low density lipoproteins in man, Biochim. Biophys. Acta, 573 (1979) 403. Akanuma, Y., Kuzuya. T., Hayashi, M., Ide, I. and Kuzuya, N., Positive correlation of serum lecithin: cholesterol acyl transferase activity with relative body weight. Europ. J. Clin. Invest.. 3 (1973) 136. Wallentin. L. and Vikrot. 0.. Lecithin:cholesterol acyl transfer in plasma of normal persons in relation to lipid and lipoprotein concentration. Stand. J. Clin. Lab. Invest., 35 (1975) 669. Rose, H.G. and Juliano, J., Regulation of plasma 1ecithin:cholesterol acyltransferase in man. Part 1 (Increased activity in primary hypertriglyceridemia), J. Lab. Clin. Med., 88 (1976) 29. Wallentin, L.. Lecithin:cholesterol acyl transfer rate in plasma and its relation to lipid and lipoprotein concentrations in primary hyperllpidemia, Atherosclerosis, 26 (1977) 233. Sutherland, W.H.F., Temple, W.A., Nye. E.R. and Herbison, P.G., Lecithin:cholesterol acyltransferase activity, plasma and lipoprotein lipids and obesity in men and women, Atherosclerosis, 34 (1979) 319. Wallentin, L. and SkGldstam, L., Lipoproteins and cholesterol esterification rate in plasma during a 10 day modified fast in man. Amer. J. Clin. Nutr., 33 (1980) 1925. Nestel, P.J.. Schreibman, P.H. and Ahrens. Jr.. E.H., Cholesterol metabolism in human obesity, J. Clin. Invest., 52 (1973) 2389. Miettinen, T.A., Cholesterol production in obesity, Circulation, 44 (1971) 842. Fielding, P.E. and Fielding, C.J.. Competition between cellular and plasma free cholesterol to supply substrate for 1ecithin:cholesterol acyltransferase and transfer protein activities in human plasma. In: Council on Arteriosclerosis of the American Heart Association. Abstracts of the 34th annual meeting, Miami Beach, American Heart Association. New York, NY, 1980. Leren. P., Foss, P.O.. Helgeland. A., Hjermann, I.. Holme, nolo1 and prazosin on blood lipids, Lancet, ii (1980) 4.
I., and Lund-Larsen,
P.G., Effect
of propra-
Atherosclerosis, 41( 1982) 155-165 0 Elsevier/North-Holland Scientific
Publishers,
Ltd.
LECITHIN:CHOLESTEROL ACYL TRANSFER RATE AND HIGH DENSITY LIPOPROTEIN LEVEL IN CORONARY ARTERY DISEASE
LARS
WALLENTIN
Department (Sweden) (Received (Accepted
and BIRGITTA
of Internal
Medicine,
MOBERG
Linkijping
University
Medical
School,
S-581
85 LinkGping
26 May, 1981) 1 June, 1981)
Summary The lecithin :cholesterol acyl transfer (LCAT) reaction occurs in connection with the high density lipoproteins (HDL) and might have importance for the development of atheromatosis. The LCAT rate and the lipoprotein concentrations in plasma were therefore determined in 82 patients, 40-60 years old, with incapacitating angina pectoris and significant lesions at coronary angiography. Thirty-eight cases were normolipidemic and 22 had type IV h-yperlipidemia while the others had different types of hypercholesterolemia (n = 7) or were pharmacologically treated for previously diagnosed hyperlipidemia (n = 11) or diabetes mellitus. The normolipidemic group and the type IV group with coronary artery disease were separately compared to one normolipidemic (n = 44) and one type IV hyperlipidemic (n = 29) control group of healthy subjects of about the same age. The fractional LCAT rate (5%* h-l) was lower in patients compared to controls both regarding the normolipidemic and type IV subjects. The molar LCAT rate (Fmol * 1-l * h-l) did not differ between normolipidemic cases and controls while it was lower in type IV patients with coronary artery disease than in the type IV controls. The HDL total cholesterol (TC) concentration and the HDL-TC/TC ratio were lower in normolipidemic cases than in normolipidemic controls while both cases and controls with type IV hyperlipidemia showed equally low levels of these parameters. In conclusion, both a reduced fractional LCAT rate and a decreased HDLTC/TC ratio might be indicators of disturbances of the cholesterol ester metabolism and might contribute to the development of coronary atheromatosis.
This
study
was
financially
supported
by
the
Swedish
Medical
Research
Council
(Projekt
No.
19X-
4529).
0021-9150/82/0000-0000/$02.75
0 1982
Elsevier/North-Holland
Scientific
Publishers,
Ltd.
156
Key words:
Angina pectoris - Cholesterol - Coronary emia - Lecithin:cholesterol acyl transferase
angiography -Lipoproteins
-
HyperlipoproteinTriglycerides
Introduction Disturbances of the lipoprotein metabolism are often found in patients with coronary atheromatosis [ 1,2]. An elevated plasma concentration of low density lipoproteins (LDL) or of very low density lipoproteins (VLDL) increases while an elevated level of high density lipoproteins (HDL) decreases the accumulation of cholesteryl esters (CE) in the arterial wall [l-4]. Most of the cholesterol in the arterial wall is considered to be derived from the LDL while removal of cholesterol from the arterial wall seems to occur by transfer of unesterified cholesterol (UC) to the HDL [ 5-81. Most UC entering the HDL is esterified by the plasma enzyme lecithin:cholesterol acyl transferase (EC 2.3.1.43). The 1ecithin:cholesterol acyl transfer (LCAT) takes place in connection with the HDL and seems to be important for the transfer of cholesterol between tissues and lipoproteins [9,10]. In order to gain further knowledge of the role of LCAT in the development of atheromatosis, we have studied the LCAT rate and its relation to plasma lipoprotein levels in patients with coronary artery disease (CAD). Material and Methods Material
During 2 periods 82 consecutive patients, 40-61 years of age, with incapacitating angina pectoris referred for evaluation by.coronary angiography were studied regarding plasma lipoprotein levels and the rate of LCAT in plasma. Only subjects with at least 70% stenosis or occlusion of at least one major coronary artery and without valvular heart disease were included. Patients treated with lipid-lowering drugs and those with a history or signs of diabetes mellitus, thyroid, liver or renal disease were excluded. Out of the 82 patients entering the study 15 were excluded - 11 cases because of clofibrate treatment of previously diagnosed hyperlipidemia, 3 cases because of diabetes mellitus and one patient because of signs of liver and renal dysfunction. Of the remaining group 42 patients had suffered from at least one myocardial infarction, all investigated more than 3 months after that event. Coronary by .pass operation had been performed in 12 cases and the investigation was carried out at least one year after the operation. A healthy survey in the present geographical district showed that in 994 50year-old men with normal relative weight [weight kg/height cm - 100) < 1.101 the upper 95th percentiles of total cholesterol (TC) and triglyceride (TG) concentrations were 8.0 and 2.3 mmol/l, respectively. Based on these upper reference levels the present material of patients with coronary artery disease was divided into one normolipidemic (n = 38) and one hyperlipidemic part. According to WHO criteria [ll] with LDL-TC < 5.7 mmol/l and VLDL-TC < 0.8 mmol/l as upper reference levels [13,14], 22 cases had type IV hyperlipopro-
157
teinemia and the remaining 7 cases had type IIa, IIb or type III hyperlipoproteinemia. The normolipidemic group and the group with type IV hyperlipoproteinemia were separately compared to two reference groups of apparently healthy subjects, one with normolipidemic and the other with type IV hyperlipoproteinemic subjects. The few subjects with different types of hypercholesterolemia were not included in any statistical comparisons. The 38 normolipidemic men with CAD had a mean age of 52.1 (range 4160), mean body weight 79.0 f 1.74 (SEM) and mean relative weight 1.05 ? 0.02. Nitroglycerin was used by almost all and beta-blocking agents by 26 cases while a few also were treated with cardiac glycosides and/or thiazide diuretics. Supine resting blood pressure was above 160/95 in 10 patients and 17 were smokers. The normolipidemic reference group consisted of healthy 40-60-year-old men without a history or symptoms of coronary artery disease. Subjects with clinical or laboratory signs of diabetes mellitus, thyroid, liver or renal disease were excluded. The subjects were recruited from hospital employees, military personnel and blood donors. The group consisted of 44 men with mean age 50.1, mean body weight 79.2 + 1.63 and relative weight 1.0 * 0.02. Casual blood pressure above 160/95 was found in 2 subjects and 16 were smokers. The 22 men with coronary artery disease with type IV hyperlipoproteinemia had a mean age 54.5 (range 45--61), mean body weight 84.0 f 1.68 and relative body weight 1.12 + 0.02. Most men used organic nitrates and 19 cases also had beta blocking drugs and a few cardiac glycosides and/or thiazide diuretics. Blood pressure exceeding 160/95 was found in 5 and 6 were smokers. The type IV reference group consisted of 29 men with plasma TG > 2.3, LDL-TC < 5.7, VLDL-TC > 0.8 and no chylomicrons in the standing plasma test [12]. Only those without history or symptoms of CAD at clinical and laboratory investigations were included. These men were mainly referred from a health survey in the same geographical district. The mean age was 52.6 (range 41-61) years, mean body weight 81.8 2 1.78 kg and relative weight 1.09 +_ 0.02. Three of these men used hydralazine and hydralazine-spironolacton. Seven men had a supine resting blood pressure exceeding 160/95 and 16 men were smokers. Methods
After an overnight fast venous blood was drawn into evacuated tubes with 1.2 mg K3EDTA/ml blood. Sampling was done with the patient in the sitting position. The samples were immediately cooled and plasma isolated by centrifugation at +4”C. A part of the plasma was stored at this temperature until ultracentrifugation was started within 4 days. The determination of LCAT rate was performed in plasma stored frozen at -70°C for a maximal time of 2 months and thawed immediately before analysis. The fractional LCAT rate was measured by incubation of plasma for 20 min in vitro after the addition of [3H]cholesterol. The fractional esterification of the label was taken as the fractional LCAT rate assuming complete equilibration of the added [3H]cholesterol and UC in plasma lipoproteins. The UC concentration in plasma before incubation was determined by a gas-liquid chromatographic method. The molar LCAT rate (pm01 - 1-l - h-l) was calculated as
158
the product of the fractional esterification rate (% - h-l) and the UC concentration (mmol *1-l). The details and precision of these methods have previously been presented [15,16]. Lipoproteins were separated according to the Lipid Research Clinics Program [12]. VLDL were isolated by ultracentrifugation of plasma for 18 h at hydrated density 1.006. HDL were separated in the infranatant by precipitation of LDL with heparin-MnClz [12]. The TC and TG concentrations in plasma and the lipoprotein fractions were determined by enzymatic Boehringer Mannheim Tests [ 17-191. Routine laboratory tests of thyroid, renal and liver functions and blood glucose concentrations were performed at the Department of Clinical Chemistry, Linkiiping University. Statistical calculations of means, SD and SEM were performed according to standard methods. Because of the skew distribution of most lipoprotein parameters, the significance of differences between groups was assessed by nonparametric rank sum tests [20] and P < 0.05 was taken as significant. Correlations were calculated according to the least squares method [20] and correlation coefficients (r) different from 0 by P < 0.05 were considered significant. LCAT
%,h-'
LCAT %.h-' p< 0.001
p= 0.05
.
10.0 0
. .
a
9.0
t
so 8.0
.
7.0
: .. .
‘f
6.0
At* ...
T
I-
9.0
:I.
8.0
.I.
7.0
?? ?? : 6.0 ..
.* .
5.0
10.0
5.0
4.0
4.0
3.0
3.0
2.0
2.0
1.0
1.0
0
0 Ref.group
CAD
CAD
typeIV
TypeIV
Normolipidemia Fig. 1. Fractional LCAT rates in normollpidemic cases and controls and in cases and controls with type IV hyperlipidemia. Circles denote controls and triangles cases with coronary artery disease (CAD). Open symbols symbolize normolipidemic subjects and closed symbols the type IV subjects. The lines with hooks illustrate means f SEM. Differences between groups were tested by nonparametric rank sum tests and the significance levels are shown in the figure.
159
Results The individual results of the fractional and molar LCAT rates in normolipidemic and type IV cases and controls are shown in Figs. 1 and 2. The fractional LCAT rate was lower in patients compared to controls with respect to both normolipidemic and type IV subjects. Of the normolipidemic subjects with coronary artery disease 12/38 (= 32%) had fractional LCAT rates below 5.0% - h-l, which was the 7th percentile of the distribution of the normolipidemic controls. In the type IV groups 17/22 (= 77%) of the patients with coronary artery disease had fractional LCAT rates below 6.6% * h-l, which was the 14th percentile of the type IV reference group. There were no differences in molar LCAT rate between normolipidemic cases and controls. The patients with coronary artery disease and with type IV had lower molar LCAT rates than their type IV controls. The lipoprotein lipids in the 4 groups have been detailed in a separate report
LCAT
flmol.lK’.h-’
LCAT
t
t
p = 0.001
n.s.
1
260
pmol.l-‘.h-’
.
I ; 260
240
240
220 :
220
. . 200
: I
.I
200
18Oj
180
160
16.i I
80
60
Ref.fJroup Normolipidemia
CAD
CAD type IV
Type IV
Hyperlipidemia
Fig. 2. Molar LCAT rates in the normolipidemlc hyperlipidemia. Signs and symbols as in Fig. 1.
cases and controls
and in cases and controls
with
type IV
160 TABLE
1
LIPID
AND
AND
TYPE
LIPOPROTEIN IV
LIPID
CORONARY
CONCENTRATIONS
ARTERY
DISEASE
(mmol/l)
(CAD)
IN PLASMA
PATIENTS
AND
OF
Normolipidemia
(TC)
5.32
6.09 b
6.51
0.14
0.17
44 1.29
0.94a
0.78
0.04
0.05
(TG)
3.93
4.42
SEM
0.13
0.11
= high
SEM
= standard
density
Significant b P < 0.01,
0.45
SEM
0.04
0.03
TABLE
4.06
0.12
0.20
b
35
29
1.22
1.68
0.12
0.21
21
29
0.82
0.92
1.99
2.68
SEM
0.07
0.05
0.23
0.31
24
35
21
Mean
1.16
1.71
SEM
0.07
0.08
44 LDL
= low
a
39
density
lipoprotein.
29
3.25
3.55
0.23
0.38
22 VLDL
= very
low
29 density
lipoprotein,
n = number.
between
cases
and
controls
by
rank
sum
tests
are
symbolized
a P < 0.001,
c P < 0.05.
2
LECITHIN
: CHOLESTEROL
NORMOLIPIDEMIC TROL
AND
ACYL TYPE
IV
TRANSFER CORONARY
(LCAT)
RATES
ARTERY
AND
DISEASE
HDL
RATIOS
(CAD)
IN PLASMA
PATIENTS
AND
SUBJECTS NormoIipidemia Controls
LCAT
?‘o . h-’
~mol
. 1-l . h-’
Type CAD
Mean
6.15
5.55
SEM
0.18
0.20
n LCAT
Mean SEM n
22
3.08
0.35
0.22
0.02
0.01
0.24
0.16
SEM
0.01
0.01
as in Table
1.
38
22 a
38
MeaIl n
and symbols
38
4.90
38
Mean
TC
7.64 0.22
133.3
SEM n
6.24a
38
2.87
38
29 a
167.9 6.58 29
0.18
0.21
0.01
0.01
22 a
Controls
0.24
102.6
LDL-TC
HDL-TC
c
44
44
IV
CAD
100.9
HDL-TC
Signs
4.54
Mean
of the mean,
differences
0.03 29
22
0.33
lipoprotein,
error
b
38
Mean
24
0.79
22
Mean
n HDL
38
38
0.17 29
0.06 38
6.61
22
SEM
n Triglycerides
38
Mean
n VLDL-TG
Controls
0.11
n VLDL-TC
CAD
SEM
n LDL-TC
CAD
IV
Mean n
HDL-TC
SUBJECTS
Type
Controls Cholesterol
NORMOLIPIDEMIC
CONTROL
0.12 0.01 22
29 0.12 0.01 29
OF CON-
161
TABLE
3
CORRELATION
COEFFICIENTS
LIPOPROTEIN
LIPID
BETWEEN
LEVELS
FRACTIONAL
Type
Normolipidemia
CAD
Controls (n = 29)
-o.35
-o.45
-0.21
b
LDL-TC
a.41
=
VLDL-TC
0.58
VLDL-TG
0.38
TG
0.47 0.49
c P <
TABLE
significance 0.05.
Other
c
c
OR
-o.17
-o.35
0.04
-O.66
-0.24
-o.o5
0.15
0.49
b
0.26
0.49
b
-O.O6
c
0.19
0.50
c
-o.o5
c
0.10
0.33
b
levels signs
LIPID
IV
(n = 20)
c
denote
AND
CAD
4.57
0.01,
h-l)
(n = 36)
-O.42
a9b.c
(% .
Controls
TC
weight
RATE
(n = 24)
HDL-TC
Body
LCAT
IN PLASMA
and
of
correlation
symbols
coefficients
as in Tables
1 and
(r)
0.01
different
from
0
0.38
’
and
a P <
’h-l)
AND
0.001,
b P <
2.
4
CORRELATION LIPOPROTEIN
COEFFICIENTS LIPID
BETWEEN
LEVELS
MOLAR
Normolipidemia
Type CAD
Controls
(n = 20)
(n = 29)
LDL-TC
0.12 0.79
a
0.26
VLDL-TG
0.53
b
0.48
TG
0.76
a
0.50
0.58
b
0.07
as in Table
0.32
0.20
AND
0.38
-o.15
4.01
VLDL-TC
symbols
LIPID
IV
(n = 36)
-o.58b
and
.1-l
CAD
-o.o5
Signs
(~mol
Controls
HDL-TC
weight
RATE
(n = 24) TC
Body
LCAT
IN PLASMA
a.36
-O.16
0.18
-0.16
0.88
a
0.59
b
0.73
a
0.47
b
b
0.84
a
0.47
b
0.44
a
0.01
a
3.
[ 211 and are summarized in Tables 1 and 2. In the normolipidemic subjects the most prominent differences were the lower HDL-TC concentration and the lower HDL-TC/TC and HDL-TC/LDL-TC ratios in cases compared to controls. There were also somewhat higher LDL and VLDL concentrations in normolipidemic patients compared to their control group. In the type IV subjects there were no major differences in lipoprotein concentrations and the HDLTC/TC and HDL-TC/LDL-TC ratios were similar in cases and controls. The correlation coefficients between the LCAT rates and the levels of lipoprotein lipids are shown in Tables 3 and 4. The body weight was positively correlated to the fractional and molar LCAT rates in both control groups, but not in any of the case groups. The molar LCAT rate was positively correlated to the TG and VLDL concentration in all 4 groups. Discussion The LCAT rate, determined with the present method, is dependent both on the concentration of the enzyme and the concentration and composition of its
162
substrates, products and cofactors [ 10,22-241. Therefore the rate-limiting factors for the esterification rate cannot be elucidated. Besides the enzyme concentration many other factors in plasma have been suggested to influence the cholesterol esterification rate e.g. the availability of the enzyme cofactors apo AI and apo C [25,26], the HDLJHDLz ratio [9,10,27], the phospholipids [28], the accumulation of CE in product lipoproteins and the availability of the CE transferring protein [ 29-321. In the present study the fractional LCAT rate was reduced in relation to the TG and VLDL levels in both normolipidemic and type IV patients with coronary atheromatosis. Two previous studies have reported LCAT rates and concentrations of lipids and lipoproteins in subjects with coronary artery disease i.e. survivors of myocardial infarction. Soloff et al. [33] found reduced fractional LCAT rates and elevated TC and TG levels in a series of patients compared to normolipidemic healthy controls. In a comparison between young myocardial infarction survivors and controls matched for plasma TC levels Wiklund et al. [34] found no differences in fractional or molar LCAT rates or the HDL-TC levels. In the latter study around half of the subjects were hypercholesterolemic according to age-adjusted reference levels. In both these groups the fractional LCAT rates (means 4.9% * h-l) were lower than the fractional LCAT rates of the normolipidemic patients with coronary artery disease in the present study. Furthermore, the young myocardial infarction survivors had elevated TG levels and in relation to TG levels the fractional LCAT rates might have been reduced also in this study. The findings of the reduced fractional LCAT rates in coronary artery disease give rise to some speculations about possible mechanisms. The UC entering the LCAT reaction and HDL is mainly derived from surface constituents of catabolized VLDL or from cellular membranes [9,10]. The uptake of UC from cellular membranes into HDL might be facilitated by cholesterol esterification and thereby the LCAT reaction might be of importance for the removal of cholesterol from tissues e.g. the arterial wall [7,9,10,35]. However, most cholesterol entering the reaction is derived from the catabolism of TG rich lipoproteins [9, 10,36-381. Therefore a high molar LCAT rate does not implicate a large uptake of cholesterol from the arterial wall but rather a high input of cholesterol into plasma via TG-rich lipoproteins [36,37]. Furthermore, the CE produced by LCAT do not remain in the HDL but seem to be distributed to all the plasma lipoproteins [39-411, via a recently discovered CE-transferring protein [29-321. The regulation of the distribution of CE among the lipoproteins is so far unknown. A high LCAT rate might lead to an enhanced formation of CE in VLDL and LDL [9,10]. As the cholesterol esterified by LCAT might be derived both from the VLDL catabolism and from the tissues e.g. arterial walls, and as the CE produced by LCAT are distributed to all the lipoprotein fractions, the interpretation of the LCAT rates in relation to the lipoproteins is complicated. Based on the present results and on previous findings in normolipidemic and hyperlipidemic subjects [42-471, the plasma LCAT rate should be evaluated in relation to the plasma levels of lipids and lipoproteins and to the body weight. An elevated input of VLDL cholesterol into plasma e.g. in obesity [48, 491 should be associated with an enhanced molar LCAT rate [42,44,46]. If
163
the LCAT rate is not elevated in relation to an increased input of UC in the TGrich lipoproteins, the content of UC in plasma lipoproteins and cellular membranes might become enhanced [9,10]. The removal of UC from peripheral tissues to HDL might also be retarded because of saturation of the LCAT reaction and of HDL by UC from the VLDL catabolism in accordance with recently reported in vitro experiments [ 501. Thus, a fractional LCAT rate that is low in relation to the TG level or the body weight might indicate an increased flux of cholesterol to the tissues and/or a reduced removal of cholesterol from the tissues to HDL. The present study demonstrates that most subjects with coronary artery disease had low HDL-TC levels and low HDL-TC/LDL-TC or HDL-TC/TC ratios and that the mean fractional LCAT rate was reduced in coronary artery disease. In type IV hyperlipoproteinemia the HDL-TC level and the HDL-TC/LDL-TC and HDL-TC/TC ratios were equally low in patients and controls, while the fractional LCAT rate was reduced in the patients with coronary artery disease. Both the decreased HDL-TC/TC ratio and the reduced fractional LCAT rate might be markers of disturbances of the CE metabolism, contributing to the development of coronary atheromatosis. Most cases in the present study were treated with beta-blocking drugs and probably led a more sedentary life than their controls. Both these factors tend to change the lipoprotein metabolism i.e. reduce the HDL and elevate the VLDL level [51]. The possible influences of these factors on the LCAT rates are unknown. In the present study the cases with beta-blocking drugs did not differ from untreated cases regarding LCAT rates or HDL-TC levels. However, the results of the present study do not allow any firm conclusions regarding the effects of the disease and its treatment. Acknowledgments The skilful technical assistance roth is gratefully acknowledged.
of MS Ylva Svensson
and MS Gunnel
Alm-
References Castelli, W.P., Doyle, J.I., Gordon. T.. Hames, C.G., Hjortland, M.C.. Hulley, S.B., Kamn. A. and Zukel, W.J., HDL cholesterol and other lipids in coronary heart disease. Circulation. 55 (1977) 767. Connor. W.E., The relationship of hyperlipoproteinemia to atherosclerosis - The decisive role of dietary cholesterol and fat. In: A.M. Scand. R.W. Wissler and G.S. Getz (Eds.), The Biochemistry of Atherosclerosis. Marcel Dekker, New York, NY, 1979, pp. 371418. Miller, N.E.. The evidence for the antiatherogenicity of high density lipoprotein in man, Lipids, 13 (1978) 914. Miller. G.J., High density lipoproteins and atherosclerosis, Ann. Rev. Med.. 31 (1980) 97. Goldstein, J.L. and Brown, M.S., Atherosclerosis - The low density lipoprotein receptor hypothesis, Metabolism, 26 (1977) 1257. Small, D.M.. Cellular mechanisms for lipid deposition in atherosclerosis. N. Engl. J. Med., 297 (1977) 873 and 924. Bates. S.R., Cholesterol sccumulation in arterial cells and in extracellular spaces, Artery, 5 (1979) 362. Stein, Y. and Stein, 0.. Interaction between serum lipoproteins and cellular components of the arterial wall. In: A.M. Scam& R.W. Wissler and G.S. Getz (Eds.), The Biochemistry of Atherosclerosis. Marcel Dekker. New York, NY, 1979. pp. 313-344.
164 9 Glomset, J.A., Lecithin:cholesterol awl transferase - An exercise in comparative biology. In: S. Eisenberg (Ed.), Progress in Biochemical Pharmacology, Vol. 15 (Lipoprotein Metabolism), Karger, Bawl, New York. NY, 1979. pp. 41-66. acyl transferase. In: A.M. Scanu. R.W. Wissler and G.S. Getz 10 Glomset, J.A., Lecithin:cholesterol (Eds.), The Biochemistry of Atherosclerosis. Marcel Dekker, New York, NY, 1979, PP. 247-274. 11 Beaumont, J.-L.. Carlson, L.A., Cooper. G.R., Fejfar, Z.. Fredricson, D.S. and Strasser, T.. Classification of hyperlipidaemias and hyperlipoproteinaemias, Bull. Wld Hlth Org., 43 (1970) 891. 12 Bachorik. P.S. and Wood, P.D.S., Laboratory considerations in the diagnosis and management of hyperlipoproteinemia. In: B.M. Rifkind and R.I. Levy (Eds.), Hyperlipidemia - Diagnosis and Therapy, Grune & Stratton, New York, NY, 1977, PP. 41-69. 13 Olsson, A.G. and Carlson. L.A., Studies in asymptomatic primary hyperlipidemia, Acta Med. Stand., Suppl. 580 (1975). 14 Carlson, L.A. and Ericsson. M., Quantitative and qualitative serum lipoprotein analysis, Part 1 (Studies in healthy men). Atherosclerosis, 21 (1975) 417. 15 Stokke, K.T. and Norum, K.R.. Determination of 1ecithin:cholesterol awl transfer in human blood plasma, &and. J. Clin. Lab. Invest., 27 (1971) 21. 16 Wallentin, L. and Vikrot, O., Evaluation of an in vitro assay of 1ecithin:cholesterol awl transfer rate in plasma, Stand. J. Clin. Lab. invest.. 35 (1975) 661. 17 18 19 20 21 22
23 24
25
26 2,7 28 29 30 31
32
33 34
35
Riischlau, P., Bernt, E. and Gruber. W.. Enzymatische Bestimmung des Gesamt-Cholesterins im Serum. J. Clin. Chem. Clin. Biochem., 12 (1974) 403. Eggstein, M., Eine neue Bestimmung der Neutralfette im Blutserum und Gewebe, Klin. Wschr., 44 (1966) 267. Schmidt, F.H. and van Dahl, K., Zur Methode der enzymatischen Neutralfett-Bestimmung in biologischem Material, J. Clin. Chem. Clin. Biochem., 6 (1968) 156. Snedecor, G.W. and Cochran, W.G.. Statistical Methods. 6th edition. The Iowa State University Press. Ames, IA, 1967. Moberg, B. and Wallentin, L.. HDL and other lipoproteins in normolipidaemic and hypertriglyceridaemic (type IV) men with coronary artery disease. Europ. J. Clin. Invest., In press. Sodhi. H.S., Esterification of plasma free cholesterol. In: D. Kritchevsky and O.J. Pollak (Eds.), Clinical Methods in Study of Cholesterol Metabolism (Monographs of Atherosclerosis, Vol. 19). Karger. Basel. New York. NY, 1979, pp. 87-93. Lacko. A.G., On the interpretation and potential diagnostic value of the measurements related to 1ecithin:cholesterol acyltransferase activity, Clin. Biochem., 9 (1976) 212. Thanabalasingham, S. Thompson, G.R., Trayner, I.. Myant, N.B. and Soutar, A.K., Effect of lipoprotein concentration and 1ecithin:cholesterol acyltransferase activity on cholesterol esterification in human plasma after plasma exchange, Europ. J. Clin. Invest., 10 (1980) 45. Soutar. A.K.. Garner, C.W., Baker. N., Sparrow, J.T., Jackson, R.I., Gotto, Jr., A.M. and Smith, L.C., Effect of human plasma apolipoproteins and phosphatidylcholine acyl donor on the activity of 1ecithin:cholesterol acyltransferase, Biochemistry. 14 (1975) 3057. Albers, J.J.. Lin, J. and Roberts, B.P., Effect of human plasma apolipoproteins on the activity of purified 1ecithin:cholesterol acyltransferase, Artery, 5 (1979) 61. Pinon. J.C., Bridoux, .A.M. and Laudat. M.H., Initial rate of cholesterol esterification associated with high density lipoproteins in human plasma, J. Lip. Res., 21 (1980) 406. Drevon, C.A. and Norum, K.R., Effect of lipid emulsions on the plasma 1ecithin:cholesterol acyl transfer in guinea pigs, Nutr. Metab., 19 (1975) 180. Chajek, T. and Fielding, C.J., Isolation and characterization of a human serum cholesteryl ester transfer protein, Proc. Nat. Acad. Sci. (USA). 75 (1978) 3445. Zilversmit, B.B., Hughes, L.B. and Balmer. J., Stimulation of cholesterol ester exchange by lipoprotein free rabbit plasma, Biochim. Biophys. Acta, 409 (1976) 393. Sniderman, A., Teng, B.. Vezina. C. and Marcel, Y.L., Cholesterol exchange between human Plasma high and low density lipoproteins mediated by a plasma protein factor, Atherosclerosis, 31 (1978) 327. Marcel. Y.L.. Vezina, C., Teng, B. and Sniderman. A., Transfer of cholesterol esters between human high density lipoproteins and triglyceride-rich lipoproteins controlled by a plasma protein factor. Atherosclerosis, 35 (1980) 127. Soloff, L.A., Rutenberg. H.L. and Lacko, A.G., Serum cholesterol esterification in patients with corenary heart disease. Amer. Heart J., 85 (1973) 153. Wiklund. 0. and Gustafson. A., Lecithin:cholesterol acyl transfer (LCAT) and fatty acid composition of lecithin and cholesterol esters in young male myocardial infarction survivors, Atherosclerosis, 33 (1979) 1. Ray, E., Bellini. F., Stoudt, G., Hempedy. S., and Rothblat, G., Influence of 1ecithin:cholesterol acyltransferase on cholesterol metabolism in hepatoma cells and hepatocytes, Biochim. Biophys. Acta, 617 (1980) 318.
36 37
38 39 40 41 42 43 44 45 46
47 48 49 50
51
Kudchodkar. B.J. and Sodhi. H.S.. Turnover of plasma cholesteryl esters and its relationship to other parameters of lipid metabolism in man. Europ. J. Clin. Invest., 6 (1976) 285. Angelin. B.. Einarsson, K., Leijd. B. and Wallentin, L., Plasma cholesterol esterlfication rate in type IV hyperllpoproteinemia - Relation to bile acid kinetics and triglyceride metabolism, J. Lab. Clin. Med., In press. Stein, Y. and Stein, 0.. Metabolism of plasma lipoproteins. In: A.M. Gotto, Jr., L.C. Smith and B. Allen (Eds.). Atherosclerosis V, SpringerVerlag, Berlin, 1980, pp. 653-665. Nichols, A.V. and Smith, L., Effect of very low-density lipoproteins on lipid transfer in incubated serum. J. Lipid Res., 6 (1965) 206. Lallu, J.I. and Barter, P.I.. The in viva metabolism of esterified cholesterol in the plasma high density lipoproteins of rabbits, J. Lab. Clin. Med., 93 (1979) 570. Nestel, P.J., Reardon. M. and Blllington. I.. In viva transfer of cholesterol esters from high density lipoproteins to very low density lipoproteins in man, Biochim. Biophys. Acta, 573 (1979) 403. Akanuma, Y., Kuzuya. T., Hayashi, M., Ide, I. and Kuzuya, N., Positive correlation of serum lecithin: cholesterol acyl transferase activity with relative body weight. Europ. J. Clin. Invest.. 3 (1973) 136. Wallentin. L. and Vikrot. 0.. Lecithin:cholesterol acyl transfer in plasma of normal persons in relation to lipid and lipoprotein concentration. Stand. J. Clin. Lab. Invest., 35 (1975) 669. Rose, H.G. and Juliano, J., Regulation of plasma 1ecithin:cholesterol acyltransferase in man. Part 1 (Increased activity in primary hypertriglyceridemia), J. Lab. Clin. Med., 88 (1976) 29. Wallentin, L.. Lecithin:cholesterol acyl transfer rate in plasma and its relation to lipid and lipoprotein concentrations in primary hyperllpidemia, Atherosclerosis, 26 (1977) 233. Sutherland, W.H.F., Temple, W.A., Nye. E.R. and Herbison, P.G., Lecithin:cholesterol acyltransferase activity, plasma and lipoprotein lipids and obesity in men and women, Atherosclerosis, 34 (1979) 319. Wallentin, L. and SkGldstam, L., Lipoproteins and cholesterol esterification rate in plasma during a 10 day modified fast in man. Amer. J. Clin. Nutr., 33 (1980) 1925. Nestel, P.J.. Schreibman, P.H. and Ahrens. Jr.. E.H., Cholesterol metabolism in human obesity, J. Clin. Invest., 52 (1973) 2389. Miettinen, T.A., Cholesterol production in obesity, Circulation, 44 (1971) 842. Fielding, P.E. and Fielding, C.J.. Competition between cellular and plasma free cholesterol to supply substrate for 1ecithin:cholesterol acyltransferase and transfer protein activities in human plasma. In: Council on Arteriosclerosis of the American Heart Association. Abstracts of the 34th annual meeting, Miami Beach, American Heart Association. New York, NY, 1980. Leren. P., Foss, P.O.. Helgeland. A., Hjermann, I.. Holme, nolo1 and prazosin on blood lipids, Lancet, ii (1980) 4.
I., and Lund-Larsen,
P.G., Effect
of propra-