Abnormal plasma cholesterol metabolism in cigarette smokers

Abnormal

Plasma

Cholesterol

Metabolism

Laic de Parscau and Christopher

in Cigarette

Smokers

J. Fielding

Plasma cholesterol metabolism was studied in young, nonobese. normolipidemic men with a moderate level of cigarette smoking (24 + 5 d-‘1 and in a comparable nonsmoking normal control group. The smokers showed a decreased cholesterol net transport from cell membranes into plasma (P < 0.001) and a decreased ratio of cholesteryl ester transfer to low and very low density lipoprotein, relative to lecithin:cholesterol acyltransferase (P < 0.05). Apoprotein E was increased in smokers’ plasma (P < 0.05) whereas apoprotein A-l, the major apoprotein of HDL, was decreased (P < 0.05). This pattern of abnormalities has been previously observed in several other groups of subjects at increased risk for atherosclerotic vascular disease (diabetics, dysbetalipoproteinemics, and hyperbetalipoproteinemics). These data suggest a deleterious effect of smoking on plasma lipoprotein metabolism significant even in young smokers, which could partly explain the later incidence of atherosclerotic vascular disease in this group. B 1986 by Grune & Stratton, Inc.

A

CLEAR CORRELATION between smoking and atherosclerotic vascular disease is now well-established.‘** Smokers, like others at risk for atherosclerotic vascular disease, have usually been found to have a low concentration of high density lipoprotein (HDL)3d or an increased ratio of low density lipoprotein (LDL) to HDL.‘,’ In addition to these lipoprotein alterations, a consistent pattern of abnormalities of plasma cholesterol metabolism has been reported in other patients at risk, such as noninsulin-dependent diabeticsg3” and those with one of several well-defined genetic hyperlipidemias.” The plasma of all these groups showed a reversed net transport of free cholesterol from cell membranes to plasma and a decreased transfer of cholesteryl esters from HDL to low and very low density lipoprotein (VLDL). The purpose of this study was to investigate plasma cholesterol metabolism in young, normolipidemic cigarette smokers without any other known risk factor for atherosclerotic vascular disease. This study shows significant abnormalities of plasma cholesterol metabolism in this group, similar to those found in the other groups at risk. MATERIALS

AND METHODS

Subject Selection The study population consisted of 20 volunteers recruited among the staff and students of the medical center, ten who smoked more than 20 cigarettes per day (mean 24 * 5) for an average of nine years and ten nonsmokers of comparable age, physical activity, dietary habit, and low alcohol intake. All were healthy men, between 20- and 40-years-old and had no familial history of atherosclerotic vascular disease. Both smoking and nonsmoking groups consisted of white donors, none of whom took part in regular exercise. All consumed a normal diet. Alcohol consumption was moderate and comparable in both groups (on average less than 5 mL ethanol/d).

From the Cardiovascular Research Institute and the Department of Physiology, University of California, San Francisco. Supported by grants from the National Institute of Health. Arteriosclerosis Specialized Center of Research (HL 14237) and HL 23738. Address reprint requests to Christopher J. Fielding, PhD. Cardiovascular Research Institute. M-1315, University of California, San Francisco, CA 94143. Q 1986 by Grune & Stratton, Inc. 0026-0495/86/351 I-oOl4sO3.00/0

1070

Blood was drawn after an overnight fast into l/20 vol of 0.2 mol/L sodium citrate. Plasma was obtained by centrifugation (1,000 g, 30 minutes at 4 “C) and used immediately for the assays described below. A OS-mL plasma sample was frozen and later used to determine thiocyanate, an index of the intake of smoke. Thiocyanate was measured by a colorimetic method.” Mean concentrations of thiocyanate (*SD) were 207 f 74 nmol/mL in smokers, Y 91 + 35 nmol/mL in controls. Such a doubling of thiocyanate concentration is consistent with the stated cigarette intake of the smoking group.

Determination to Plasma

of Cholesterol

Net Transport From Cells

Cholesterol net transport from cell membranes to plasma was measured by incubation of plasma from smokers or control subjects with cultured normal human skin fibroblasts. The method has been previously detai1ed.13 It compares the consumption of plasma free cholesterol by lecithin:cholesterol acyltransferase (LCAT) in the presence or absence of cell membrane cholesterol. For each plasma sample, fibrinogen was first removed by antihuman fibrinogen immunoaffinity chromatography using a column (I x 10 cm) of antibody covalently linked to agarose, equilibrated with 0.15 mol/L NaCI, 1 mmol/L disodium EDTA (pH 7.4). The nonadsorbed eluate was pooled and diluted to 1.2% v/v relative to plasma protein concentration in phosphate-buffered saline solution. Normal skin fibroblasts were cultured in 10% human serum in Dulbecco’s modified Eagle’s medium. When the cells approached confluence (cell cholesterol content 8 to 10 bug/dish), the medium was removed and the dishes washed eight times with phosphatebuffered saline solution. Fibrinogen-free plasma medium (3 mL) was then added to five dishes of washed cells and to five empty dishes. An initial I-mL sample of medium was taken for analysis of free cholesterol, then the remaining medium in the ten dishes incubated for 60 minutes at 37 “C. At the end of the incubation, a second 1 mL sample of medium was taken from all dishes for analysis of free cholesterol content. Free and esterified cholesterol were determined fluorimetrically.‘3 As the generation of cholesteryl esters by LCAT was similar in the presence and absence of the cells (ratio 0.99 + 0.13) cholesterol net transport is given as the decrease in plasma free cholesterol consumed in the presence of cells when cell membranes were also available to donate cholesterol for esterification: Cholesterol net transport = [(decrease in plasma free cholesterol in the absence of cells) - (decrease in plasma free cholesterol in the presence of cells)] Net transport from cell membranes to plasma has a positive sign, whereas net uptake of cholesterol from plasma by cells has a negative sign.

Metabolism, Vol35,

No 11 (November). 1986: pp 1070-1073

1071

PLASMA CHOLESTEROL METABOLISM IN SMOKERS

Table 1. Characteristics of the Study Population Age Group

Cholesterol

Triglycerides

(mg/dL)

(mg/dL)

img/dL1

BMI’

iW)

Phospholiplds

GlUCOSL?

(ma/dL)

Smokers

30.6

k 3.5

23.6

2 1.4t

173 + 26

84 + 48

186 t 14

78 f 14

Controls

31.9

& 5.4

21.6

i- 1.7

169 f 20

65 ? 31

197 r 34

82t

10

Values are given as mean + SD. *BMI (body mass index) = weight/lheight)’ tP

as kg/m*.

< 0.02.

Determination

of LCAT Activity

in Plasma

Statistical

LCAT activity was determined in terms of the rate of decrease in plasma free cholesterol mass during incubation for one hour at 37 OC.‘j Five aliquots were taken before and at the end of the incubation period for the enzymatic determination of free cholester01.‘~ Decrease in free cholesterol was linear over the one-hour incubation; it was inhibited >95% by 2.3 mmol/L 5J’dithiobis (2 nitro-benzoic acid) (DTNB), an inhibitor of LCAT.14

Determination in Plasma

of Cholesteryl

Results are expressed as mean + SD. Two sample Student t-test (for normal distribution) or Mann Whitney test (for skewed distribution) were used for comparison of smokers’ and controls’ data. Comparisons requiring adjustments of related variables were evaluated by analysis of covariance. Specifically, this was carried out to correct for effects of the slight weight differences between smoking and nonsmoking groups (see below) on plasma metabolic factors, apoproteins, and lipids.

Ester Transfer Rate RESULTS

Cholesteryl ester transfer was measured as the rate of decrease in HDL-cholesteryl ester during incubation for one hour after inhibition of the LCAT with DTNB.” This assay measures the rate of transfer of cholesteryl ester from HDL to LDL and VLDL in conditions in which plasma cholesteryl ester is held constant.

Lipoprotein

Analysis

Anal_vsis

Lipoproteins were initially fractionated by affinity chromatography on heparin agarose in 0.15 mol/L NaCI, 1 mmol/L Na, EDTA. Under these conditions, LDL together with VLDL containing apoprotein E are adsorbed to the support, while HDL (including HDL containing apoprotein E) and a fraction of VLDL lacking apoprotein E. are recovered in the nonadsorbed eluate.” Adsorbed VLDL and LDL were eluted with 3 mol/L NaCl 1 mmol/L Na, EDTA and then separated by preparative centrifugation at d 1.Ol9 g/mL. Nonadsorbed VLDL was separated from bulk nonadsorbed proteins using the same conditions. The lipid composition of isolated plasma lipoproteins was determined after extraction into chloroform/methanol. Free cholesterol and cholesteryl ester were assayed by an enzymatic fluorimetric method.” Triglycerides were separated from phospholipids by thin layer chromatography, and triglyceride glycerol was determined after periodate oxidation with chromotropic acid.” Lipoprotein phospholipid was determined as lipid phosphorus.”

Other Assays in Plasma Total cholesterol, triglycerides, and phospholipids were measured by the same methods as for lipoprotem composition. Plasma glucose was assayed by a glucose oxidase method (Sigma, St Louis). Plasma apoproteins were measured by specific radial immunodiffusion as previously described.9,17

The characteristics of the subject groups are shown in Table 1. There was no significant difference in plasma glucose, total cholesterol, triglycerides, and phospholipids between the two groups. The smokers were slightly heavier than the controls as indicated by the body mass index. Their mean relative body weight was 106.6 + 5.1% of their ideal body weight for height v 100.1 c 6.0 for the controls (Metropolitan Life Insurance Tables 1959). Smokers’ plasma showed an abnormal pattern of apoproteins with a decreased apoprotein A-I and increased apoproteins B and E (Table 2). There was no significant difference in the other apoproteins measured. The mean net transport of free cholesterol from cell membranes to plasma was significantly decreased in smokers (0.06 + 0.26 v 0.44 * 0.08 pg/h/dish in controls, P < 0.001). In fiveof ten, net transport was reversed (Fig I). This means that influx from plasma to cells exceeded total e&x. Two smokers showed net transport values within the normal range. Low net transport was not related to a defect of free cholesterol demand by LCAT, which in contrast tended to be greater in smokers (24.4 + 8.2 1118.8 + 5.2 pg sterol esterified/mL/h in controls, 0.05 < P < 0.1) (Fig 2). Transfer of cholesteryl esters from HDL to LDL and VLDL was not significantly different in smokers and controls (respectively, 10.5 + 7.3 and 12.4 t 4.8 pg sterol transferred/ mL/h). However, the proportion of total LCAT-derived cholesteryl esters transferred to LDL and VLDL was significantly decreased in smokers (0.44 + 0.20 v 0.65 k 0.26 in controls, 0.02 < P < 0.05). in spite of the fact that LDL

Table 2. Apoprotein Concentrations in Plasma Al

Smokers P

Controls

1.09 + 0.21 0.02 < P < 0.05 1.29 t 0.17

Values are given as mg/mL.

A II

B

c Ill

D

0.38 + 0.08

0.94 t 0.20 P = 0.05

0.068

* 0.021 -

0.045

* 0.009

0.36 + 0.06

0.76 + 0.19

0.057

+ 0.019

0.046

t 0.008

E

0.060 t 0.011 0.02 < P < 0.05 0.050 i 0.015

1072

DE PARSCAU AND FIELDING

icant (P < 0.01). Excess weight appears to be an independent factor for atherosclerotic vascular disease,* The relationship between body mass index and cholesterol net transport provides further support for the hypothesis that net transport reflects the risk of atherosclerotic vascular disease. Two of the smokers showed values of net transport within the normal range. This suggests that some individuals might be resistant to the effect of smoking on plasma cholesterol metabolism. In these two subjects, the only difference with the other smokers was a relatively low plasma apoprotein E (0.050 and 0.055 mg/mL). Smokers also had a significantly increased ratio of apoprotein B to apoprotein A-I and higher levels of apo E. Neither of them was significantly related to body mass index, and the differences between the smoking and nonsmoking groups remained significant when the increased body mass index of the smoking group was corrected for.

0.6C f

4/ ’

19

a

I

22

DISCUSSION

25

In normal plasma, LCAT acts to balance cellular cholesterol content increased by de novo synthesis and endocytosis. Cell membranes and plasma lipoproteins are two competitive sources of free cholesterol for LCAT.18 When net transport of free cholesterol from cell membranes is decreased as in smokers, plasma lipoproteins are the major source of free cholesterol. This explains the contrast between low net transport and maintained or increased LCAT activity. The ratio between cholesteryl ester transfer to VLDL + LDL, and total cholesteryl ester synthesis by LCAT, was not related to body mass index, and represents a second, significant abnormality in the plasma cholesterol metabolism of smokers. This pattern of apoprotein abnormalities has been found in several groups of subjects at increased risk of atherosclerotic vascular disease, including hyperbetalipoproteinemics,‘9 dysbetalipoproteinemics,*’ and those with noninsulin-dependent (type II) diabetes mellitus? The same groups also show reduced or reversed cholesterol transport and a reduced ratio of transfer of cholesteryl ester to LDL and VLDL relative to LCAT.9-” The abnormality in smokers is generally milder. However, it should be emphasized that the hyperbetalipoproteinemic, dysbetalipoproteinemic, and diabetic groups in the earlier studies were of middle-aged or elderly subjects with severe and longstanding metabolic abnormalities, while the

BODY MASS INDEX ( Kg/m*) Fig 1. Relationship between net transport of free cholesterol from cell membranes to plasma and body mass index in smokers (A) and controls (0).

protein (apoprotein B) was increased and HDL protein (apoprotein A-I) was decreased in smokers’ plasma (Fig 2). The most striking abnormality of cholesterol metabolism in the plasma of smokers is the very low or negative net transport of cholesterol from cell membranes in response to LCAT activity in plasma. The groups of smokers and controls were comparable in terms of age, dietary habit, physical activity, and plasma concentrations of glucose, cholesterol, triglycerides, and phospholipids. However, smokers were slightly heavier than controls (Table l), and an inverse linear relationship between net transport of cholesterol in plasma and body mass index, was found in the control group (P < 0.01) (Fig 1). As the coefficients for the regression slope in the two groups were not significantly different, the difference in net transport values between the smoking and control groups was tested after correction for the effect of body mass index (difference between the elevations of the two regression lines). This difference remained highly signif-

c

6

L W iii z I i . T : .

8

8 ;.

i .

0.5

2 0 t a

.

.

: . .

I S

C

Fig 2. Individual data and means of LCAT (AI, cholesteryl ester transfer (6). and ratio of cholesteryl ester transfer to LCAT (C) in plasma of smokers (S) and controls ((3.

PLASMA

CHOLESTEROL METABOLISM

1073

IN SMOKERS

measurements in the plasma of smokers in this study were made in young, normolipidemic, normoglycemic individuals in excellent general physical health. Smoking therefore, is the major factor in the subjects of this study that could explain their abnormal plasma cholesterol metabolism. The qualitative similarity between this pattern in smokers and the other groups at risk for atherosclerotic vascular disease suggests that abnormal cholesterol metabolism partly

explains the high incidence smokers and that cholesterol cator of this risk.

of coronary net transport

heart disease in is an early indi-

ACKNOWLEDGMENT

We thank Dr Nancy Phillips for expert statistical advice. We acknowledge the technical assistance of Mercy de1 Rosario, Elaine Hoye, and Mary Fisher.

REFERENCES

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Study. Cambridge

Mass, Har-

3. Garrison RJ, Kannel WB, Feinleib M, et al: Cigarette smoking and HDL cholesterol. The Framingham offspring study. Atherosclerosis 30: 17725. 1978 4. Berg K, Borresen AL, Dahlen G: Effect of smoking on serum levels of HDL apoproteins. Atherosclerosis 34:339-343, 1979 5. Criqui MH, Wallace RB, Heiss G, et al: Cigarette smoking and plasma high density lipoprotein cholesterol. The lipid research clinics program prevalence study. Circulation 62:70-6, 1982 (suppl 4) 6. Stubbe I, Eskilsson J, Nilsson Ehle P: High density lipoprotein concentrations increase after stopping smoking. Br Med J 284: 15I l1513, 1982 7. Phillips NR, Have1 RJ, Kane JP: Levels and interrelationships of serum and lipoprotein cholesterol and triglycerides. Association with adiposity and the consumption of ethanol, tobacco, and beverages containing caffeine. Arteriosclerosis 1:13-24, 1981 8. Augustin J, Beedgen B, Buchholz L, et al: The influence of smoking on plasma lipoproteins, in Schettler G, Gotto AM, Middelhoff G, et al (eds): Atherosclerosis VI. Berlin, Springer Verlag, 1983 pp 878-87 9. Fielding CJ, Reaven GM, Fielding PE: Human noninsulindependent diabetes: identification of a defect in plasma cholesterol transport normalized in viva by insulin and in vitro by selective immunoadsorption of apolipoprotein E. Proc Natl Acad Sci USA 79:6365-6369,1982 10. Fielding CJ, Reaven GM, Liu G, et al: Increased free cholesterol in plasma low and very low density lipoproteins in noninsulin-dependent diabetes mellitus: Its role in the inhibition of

cholesteryl ester transfer. Proc Nat1 Acad Sci USA 8 I:2512-25 16, 1984 11. Fielding PE, Fielding CJ, Have1 RJ, et al: Cholesterol net transport, esterification, and transfer in human hyperlipidemic plasma. J Clin Invest 7 1:449-460, 1983 12. Pettigrew AR, Fell GS: Simplified calorimetric determination of thiocyanate in biological fluids, and its application to investigation of the toxic amblyopias. Clin Chem 18:996-1000, 1972 13. Fielding CJ: Lecithin:cholesterol acyltransferase and cholesterol transport. Methods Enzymol 111:267-274, 1985 14. Stokke KT, Norum KR: Determination of lecithin:cholesterol acyltransfer in human blood plasma. Stand J Clin Lab Invest 27:21-27,1972 15. Carlson LA: Determination of serum triglycerides. J Atherosclerosis Res 3:334-336, 1963 16. Bartlett GR: Phosphorus assay in column chromatography. J Biol Chem 234:466-468, 1959 17. Fielding CJ, Fielding PE: A cholesteryl ester transfer complex in human plasma. Proc Nat1 Acad Sci USA 77:3327-3330, 1980 18. Fielding PE, Fielding CJ: Competition between cellular and plasma free cholesterol to supply substrate for lecithin cholesterol acyltransferase and transfer protein activities in human plasma. Circulation 62:264, 1980 19. Blum CB, Aron L, Sciacca R: Radioimmunoassay studies of human apolipoprotein E. J Clin Invest 66:1240-1250, 1980 20. Have1 RJ, Kotite L, Vigne J-L, et al: Radioimmunoassay of human arginine-rich apolipoprotein, apoprotein E. J Clin Invest 66:1351-1361, 1980 21. Fielding CJ: The origin and properties of free cholesterol potential gradients in plasma and their relation to atherogenesis. J Lipid Res 25:1624-1628, 1984