The effect of progestins in combined oral contraceptives on serum lipids with special reference to high-density lipoproteins

The effect of progestins in combined oral contraceptives on serum lipids with special reference to high-density lipoproteins V. WYl\':\1, M.D., F.R.C.P., F.R.C.PATH. R. :'>JITHTHYANA!'\THAN, M.Sc., PH.D.(Lond.), C.CHEM., M.R.S.C. London, England Fasting serum lipids were measured in a group of 293 women desirous of using oral contraceptives (OCs) and 536 women who had been taking five varieties of OCs for many months before being tested. Parameters studied were serwm cholesterol, triglyceride, high-density lipoprotein (HDL), the two main subtractions of HDL, namely, HDL. and HDL3 , and the ra1io of HDL2 chOlesterol to low-density lipoprotein (LDL) cholesterol. Studies of these five groups of women enabled us to compare the effect of varying amounts of levonorgestrel with norethindrone and to study the effects of estrogen combined With these progestins. The levonorgestrel-containing pills and the progestin-only OC significantly depressed HDL2 cholesterol levels and the ratio of HDL. to LDL cholesterol. (AM. J. 0BSTET. GYNECOL. 142:766, 1982.)

OF ALL THE METAB.OLIC effects of ora) contraceptives (OCs), the disturbance in lipid levels and lipid transport mechanisms causes greatest concern, primarily because of the association between the use of OCs and an increased incidence of arterial thrombosis and thromboem holism including myocardial infarction and stroke.I··• Although most studies show that the risk is associated with the current use of the pill and is greatest in predisposed individuals (for example, women beyond age 35 years, heavy cigarette smokers. and women with hyperlipidemia, diabetes, or hypertension), nevertheless, since susceptibility to thrombosis often cannot be predicted, it is essential that a drug designed for widespread use by healthy women and with minimum medical supervision be formulated so that risks are minimized. Moreover, a study by Slone and co-workersH has shown for the first time that the increased risk of myocardial infarction in past users of oral contraceptives who are in the age group 40 and 4~ years was related to the duration of use. There was no increased risk for a duration of use of less than 5 years, From The Alexander Simpson Laboratory jm· Metabolic RPsearch, St. Mary's Hospital. Reprint requests: V. Wynn, M.D., Univet:~ity of London, The Alexander Simpson Laboratory for Metabolic Research, St. Mary's Hospital, London, W2 JNY, England.

766

but between 5 and 9 years the risk increased to 1.6 times and for lO years of use, to 2.5 times compared to the risk in nonusers. This trend was statistically significant (p < 0.0 l). The study demonstrates the potential atherogenicity of the pill in long-term users, but confirmation is needed. While the mechanism(s) for the unwanted eflects on the cardiovascular system of OC users has not been fully elucidated, it is reasonable to suppose that changes in serum lipids might well be implicated. Lipids, using the term in its broadest context to include the prostaglandin series, are deeply involved in thrombosis and atherosclerosis, and the intricate interplay between coagulation mechanisms and endothelial and intimal changes should urge caution in the use of drugs that negatively affect these areas. A useful review of the relationship among lipoproteins, hemostasis, athero· sclerosis, and coronary heart disease and their complex interreaction has recently been published." The idea that estrogen-progestin combined OCs could hr.ve an untoward effect on serum lipids and lipoproteins is not new. In 1966, it was shown 10 that women who used combined pills demonstrated increases in serum cholesterol, triglyceride, very lowdensity (VLDL), and low-density lipoproteins (LDL), and an increase in the atherogenic index. The lipid pattern of these young women resembled that of men 0002-9378/82/060766+07$00.70/0 © 1982 The C. V. Mosby Co.

OC progestins, serum lipids, and HDLs

Volume 142 Number 6, Part 2

767

Table I. Composition of oral contraceptives Pill group

Type of progestin

Proprietary name in U.K.

2

Gonane Gonane

Eugynon 30 Ovranette Microgynon Micronor Neogest Norinyl-1 Orthonovin l :50 Minovlar Loestrin

4

Estrane Gonane Estrane

5

Estrane

3

No. of subjects

97 206 (26) (16) 42 39 76

Estrogen component

Progestin component

30 J.Lg EO 30 ~-tg EO

250 J.Lgd-NG

50 ~-tg ME 50 J.Lg ME 50 J.Lg EO 20 Mg EO

150~-tgd-NG

350 ~-tg NE 35 J.Lg d-NG 1 mg NE 1 mg NE 1 mg NEA I mg NEA

Legend: EO ethinyl estradiol: ME mestranol: NE = norethindrone; NEA norethindrone acetate; NG levonorgestrel. (In the United States norgestrel is an equal mixture of the active d form, levonorgestrel, and the inactive l form; 250 Mg of d-NG is equivalent to 500 Mg of NG.)

and postmenopausal women; both groups, of course, had a much greater risk of developing thrombotic cardiovascular disease than premenopausal women. Further studies confirmed these findings. 11 Concern was expressed at the nature of these changes and attention drawn to the importance of modifying OC formulations and, in particular, reducing the high estrogen content and progestin contents of various pills in use at that time.'~ A more recent study has shown the importance of the effect of estrogen-progestin combinations on serum lipid levels."l The clear demonstration of a strong inverse relationship between the level of high-density lipoprotein (HDL) and the subsequent development of atherosclerosis and myocardial infarction in both men and women has introduced a new emphasis. 14 - 16 The lower the HDL level, the greater the apparent risk of developing atherosclerosis. This association was first described in I 95I, 14 but modern research has drawn attention to it again. Attempts have been made to explain the apparent protective effect of a high HDL level. Glomset 17 has suggested that HDL represents macromolecules in which cholesterol released from the tissues is being transported elsewhere for disposal. The biochemistry of this is quite complex and involves the plasma enzvme, lecithin cholesterol acyltransferase. Both in vitro and in vivo evidence indicates that such a mechanism may be important, but much still needs to be learned about the way cholesterol may be removed. We need to know more about the protective effect of the HDL. HDL is heterogenous and can be divided into two main subfractions, HDL 2 and HDL 3 , the former being. less dense than the latter. It appears that compared to total HDL there is a stronger association between low HDL 2 levels and atherogenesis, as judged by coronary angiographic studies, 16 and that HDL3 levels do not

add any further significant information. Our study focused on serum lipids, especially serum cholesterol. triglyceride, total serum HDL, and the subfractions HDL 2 and HDL 3 • It has been suggested that for any given level of serum cholesterol, the ratio of HDL to LDL determines risk rather than the absolute levels of each. 18 For this reason, we also studied the ratio of HDL to cholesterol minus HDL, the ratio of HDL 2 cholesterol to LDL cholesterol, and the influence of various OC formulations on these parameters.

Material and patients Oral contraceptives tested (Table I). Our study began in I 978 when the importance of HDL and its subfractions had become apparent and suitable methods for these estimations attainable. At that time, we had available for observation three main groups of subjects taking OCs, namdy, women taking pills containing 30 ~-tg of estrogen and either 250 or 150 ~-tg of Ievonorgestrel and a third group of women taking 20 p,g of estrogen and I mg of norethindrone acetate. A small group took 50 ~-tg of estrogen and l mg of norethindrone or norethindrone acetate, and a fifth group took a progestin-only pill, either norethindrone or levonorgestrel. These pill groups have been arranged in a series. Group l took the high-dose combined norgestrel-containing pill, Group 2 the low-dose combined norgestrel-containing pill, and Group 3 the progestin-only pill. Group 4 consisted of those women taking the medium estrogen (50 ~-tg)-norethindrone (acetate) pill and Group 5 the low estrogen (20 p,g)norethindrone acetate pill. Table I shows the composition of the OCs used and the number- of subjects in each group. Group 3 consisted of only 42 subjects of whom 26 were taking an estrane and 16 a gonane progestin for contraceptive purposes. A separate analysis of the results for these two groups showed no signifi-

768

Wynn and Niththyananthan _\m.

cant differences in any of the parameters tested, and they have been combined into one group. We intend, however, to enlarge this group as the opportunity arises because we realize that the numbers of subjects are still too small for certainty in the analytical results. Subjects. Women Jess than age 45 vears were studied. Two hundred and ninety-three controls wished to start OC use. and 536 women had been taking five varieties of OCs for many months before being tested (Groups 1 to 5). The women were apparently healthy and were not taking drugs other than OCs. None of the controls had been pregnant within 6 months of inclusion in this study. Women who weighed more than l50o/c of their ideal body weight as judged by body weight tables (Documentia Geigy) were excluded. The women were oflow parity (mostly 0 to 2). Cigarette smoking, alcohol consumption, and levels of physical activity were determined, and controls and pill takers were not significantly different in these respects. Methods

Blood samples were taken at about 9:00 A.M. from subjects who had been fasting for 12 hours. They had been ingesting a diet containing at least 200 gm of carbohydrate a day for 3 days before the test, according to written instructions. This was done because the same women were subjected to oral glucose tolerance tests as described in a separate communication. 19 In order to minimize the influence of posture and venous stasis on serum lipid values, blood was drawn from an antecubital vein after the women had been lying supine for a minimum of 30 minutes. 20 Minimal venous occlusion wa' used in taking the blood. Blood samples were placed into plastic tubes without anticoagulant but containing Plas-Aid (plastic pellets which promote clotting). After allowing I hour for the blood to clot, the serum was separated by spinning the samples at 1,000 x g for 5 to 10 minutes. The serum was stored at 4° C, and the analyses were completed within 2 to 4 days. Total cholesterol was measured on these samples by the cholesterol oxidase enzymatic method (Boehringer-Manheim, catalogue no. 148 393). Serum triglyceride was measured enzymatically (BoehringerManheim, catalogue no. 166 448). Measurements of HDL, HDL 2 , and HDL 3 cholesterol. HDL was separated by precipitating the other lipoproteins with heparin and Mn 2 + by a modification 21 of the method of Burstein and colleagues 22 • 23 and the cholesterol was measured enzymatically in the supernatant. The HDL3 subfraction was separated by the method

J.

\larch 15. l9H2 Obstet. Gvnecol.

of Gidez and co-workers. 24 A 50 ILl volume of 1.21 ';i dextran sulfate (Sochibo, France, molecular mass = 15,000) was added to 0.5 ml of the heparin-Mn 2 " supernatant. After mixing in the \ortex. samples were left to stand for 30 minutes at room temperature and then spun at l ,600 x g for 30 minutes. The supernatant, HDL3 , was separated and the cholesterol value determined immediately. The value for the HDL 2 cholesterol was obtained by subtracting HDLa cholesterol from total HDL cholesterol. As a check, correlations were obtained between the precipitation method and preparative ultracentrifugation as described by HaYcl and co-workers. 23 VLDL, LDL, and HDL were isolated at conventional density gradients. HDL 2 and HDL,, were isolated at densities 1.125 gm/ml and 1.125 to 1.210 gm/ml, respectively. Fifty-two samples were analyzed: the correlation coefficient f(w HDL was 0.92, for HDL 2 0.94, and for HDL 3 0.91. In a separate study of precision, HDL subclasses were measured in a further 20 samples in duplicate by the precipitation method. Correlation coefficients for plots of duplicate values were: HDL = 0.98, HDL 2 = 0.97. and HDL,3 = 0.97. Statistics. For parameters listed in Table ll, Student's t test for unpaired data was used. As the trigln·eride values were positively skewed, logarithmic transformation was used to normalize 1he data and the log transformed data were used in the statistical analysis.

. vana . bles. Th e --: --:--::---"H..::.D~L_c:=::·h:c:.o::.c:le..::.stc:..::e..::.ro~l---= Ra tio cholesteroi-HDL cholesterol ratio was examined as follows: We have accepted 0.3 as the lower limit of normal for this ratio. 26 The proportion in the five pill groups was compared with the conHDL cholesterol . . trois of cases with greater cholesterol- HDL cholesterol than 0.3 as determined by Yates corrected XF statistics. A comparison was also made between Group I and Group 2 by use of the same test. . HDL 2 The ratio-- cholesterol was calculated by means LDL of the following formula 27 : LDL cholesterol = total cholesterol HDL cholesterol _ serum triglyceride 5 In this context LDL cholesterol refers ro the density range I.O 19 to 1.063. As the distribution of this ratio variable was positivelv skewed, a logarithmic transformation was made to normalize the data. An analysis of variance on all six groups was followed, by Student's t test, between selected means by use of the error mean square from the analysis.

Volume H2 6, Part 2

OC progestins, serum lipids, and HDLs

769

~umber

Table II. Subjects' characteristics, mean duration of pill use (months), and mean serum lipid measurements (in milligrams per 100 ml) (±SD) Pill

No. 293 Age (yr) 27.9 ± 7.2 % IBWt* 100.7 ± 15.1 Duration of use (mo) Cholesterol 171.8 ± 32.0 60 6 + 30.3 Triglyceridet . 20.2 55.9 12.5 HDL cholesterol

97 27.3 ± 6.4 102.9 ± 14.0 42.8 ± 42.7 t164.2 ± 28.3 62 5 + 28.9 . 19.8 §48.1 ± 11.0

group

2

3

4

5

206 27.0 ± 6.1 99.6 ± 13.5 40.8 ± 37.2 167.5 ± 31.4 §71 3 + 32.7 . - 22.4 §52.4 ± 11.3

42 30.8 ± 6.9 101.1 ± 12.1 51.2 ± 41.0 171.5 ± 32.5 66 8 + 29.3 20.4 . 54.0 ± 12.6

39 31.7±7.0 99.4 ± 13.6 81.0 ± 50.3 182.1 ± 38.6

76 26.6 ± 5.7 97.0 ± 10.1 18.7 ± 18.6 180.0 ± 40.3 §77 6 + 36.6 . 24.9 58.6 ± 15.1

§94.5 +

~;:~

56.9 ± 15.1

*Percentage ideal body weight from Documentia Geigy tables. tp < 0.05 compared with controls. tTriglyceride values have been calculated from log transformed data. §p < 0.001 compared with controls.

Table III. Mean HDL2 and HDL 3 cholesterol (in milligrams per 100 ml) (±SD) in controls and OC users

~ill group

I

Controls 162 25.5 ± 13.3 30.6 ± 8.3

1----1---,----2---,-----3---.----4---.,----5---

I

94 *21.1 ± 9.9 *27.2 ± 10.0

125 t20.6 ± 10.7 32.1 ± 9.0

35 t20.2 ± 12.2 32.7 ± 8.7

34 27.9 ± 11.8 29.0 ± ll.8

74 28.2 ± 15.1 30.1 ± 11.8

*p < 0.0 I compared with controls. tp < 0.001 compared with controls. tp < 0.05 compared with controls.

Results Table II shows the mean age and percentage ideal body weight for the five pill groups and controls. Matching for these variables was reasonable. The mean duration ofOC use was 40 months or more in Groups I to 4. and these can therefore be considered long-term pill users. The SO-month duration in Group 4 is not likelv to bias the results, according to our experience in other studies, but the greater age of this group needs to he taken into account because of its effect on serum cholesterol levels (see below). Group 5 had been taking the OC for a mean duration of 18 months, the shortest time of all the groups. Nevertheless, in our experience this duration of OC use is sufficient to produce the main changes seen in lipid metabolism. The mean serum cholesterol level was significantly reduced (by 7.6 mg/ 100 ml) in Group 1 subjects compared with controls, but no other changes in serum cholesterol reached significance. The mean serum cholesterol was highest in Groups 4 and 5, and possible reasons for this will be referred to below. Serum triglyceride was significantly elevated in Groups 2, 4, and 5, with the highest in Group 4. These effects are to be

expected, as it is well established that the elevation of serum triglyceride is related to the estrogen content of the pill modified by the antiestrogenic effect of progestin.12 HDL cholesterol was significantly reduced in Groups I and 2, was unchanged in Groups 3 and 4, and was slightly elevated in Group 5. It is interesting that HDL cholesterol was lower in Group l than in Group 2, this being a progestin effect, but the difference was not significant. The reduction in HDL cholesterol in Group l compared with controls (7.8 mg/100 ml) was the same as the fall in total serum cholesterol already referred to, indicating that the impact of this pill is entirely on the HDL fraction with no change in LDL or VLDL cholesterol. A similar conclusion may be drawn about Group 2, except that with this pill there is probably a slight increase in VLDL. The highest HDL level found in Group 5, although not significant, probably represents the effect of estrogen and the younger age and lower body weight in this group. The raised serum cholesterol in Group 4, while not significant, probably reflects the effect of age [in our women cholesterol rises about 1.4 mg/ 100 ml for every year of age in OC users (unpublished observations)] and increasing VLDL, an

770

Wynn and Niththyananthan _\ut.

Table IV. Ratio of HDL cholesteroll(serum cholesterol-HDL cholesterol): Comparison of the proportion of the groups with ratio >0.3

No. Ratio >0.3 expressed as o/c Ratio <0.3 expressed as o/c

1

2

293 91

97 *77

9

23

3

4

5

206 88

42 88

39 87

76 91

12

12

13

9

\lan!J 15, l91'i:2 Obstet (;\'ne' ul

Table V. HDL 2 cholesterol/LDL cholesterol in controls and pill groups Pill group Controls 1--1--,--_-?-,.--3---r--.~--,--,-

I

Pill group

Controls

J.

Group 1 versus Group 2

t

*p < 0.001 compared with controls. tp < 0.05 compared with controls.

estrogenic effect. In Group 5 the rise in serum cholesterol (not significant) probably reflects the rise in HDL cholesterol and VLDL. Table Ill shows the effect on the HDL subfractions of the various pill formulations. In Groups l, 2, and 3 there was a significant reduction in HDL 2 , whereas in Groups 4 and 5 HDL 2 was increased, although not significantly. These findings show that the gonane progestin used in Groups I and 2 overcomes the effect of estrogen, which is normally to raise HDL 2 levels. The effect of progestin taken alone in lowering HDL 2 levels is well demonstrated in this study. In Groups 4 and 5 the estrogen has overcome the progestin effect, and even 20 J.tg of of estrogen will prevent the lowering of HDL2 levels by 1 mg of norethindrone acetate. Indeed, the highest level of HDL 2 was found with this pilL 1\iorethindrone acetate clearly emerges here as being less antiestrogenic than levonorgestrel as judged by its effect on the HDL 2 level when the progestin is combined with estrogen. When the progestins are used alone, the effect of the estrane and gonane progestins on HDL2 is comparable (unpublished data). HDL3 was significantly reduced in Group l, but no other subjects showed significant changes. We conclude, therefore, that the gonane progestin levonorgestrel significantly reduces HDL 2 levels and, in addition, at a dose of 250 J.tg, significantly reduces HDL 3 levels. The progestin, whether norgestrel or norethindrone alone, lowers HDL 2 levels, although the amount of progestin used was much smaller than is found in the combined pill. Table IV shows that the proportion of subjects in whom the ratio of HDL to cholesterol minus HDL was reduced below 0.3 is significantly increased in Group I compared with controls. No other group shows this effect, however. A significant difference is found between Groups l and 2, showing the importance of the

No. HDL2 LDL

±SD

I

162

94

125

3.'i

:14

74

0.22

*0.19

tO.l8

t0.17

:j:O.:l6

:j:0.:!4

+0.21 -0.11

+0.18 -0.09

+0.16 -0.09

+0.22

t-0.2:3

-t-0.2:">

-0.10

-0.12

-0.12

*p < 0.05, Groups 4 and 5 compared with Group I. tp < 0.05 compared with controls. :j:p < 0.0 1, Groups 4 and 5 compared with Group 2.

amount of the gonane progestin in depressing this ratio. Table V shows that the ratio of HDL 2 cholesterol to LDL cholesterol is significantly reduced in Groups 2 and :l and is also reduced in Group I but not significantly. In Group l a considerable part of the impact was on HDL:l, and this may account for the lack of significance in this present analvsis. In Groups-land 5 the ratio was above the control value although nut markedly so, but there was a highlv significant difference (p < 0.01) between Groups -l and 5 compared with Group 2 and a significant difference (p < 0.05) between Groups 4 and 5 compared to Group I. These data are important because Groups ·land 5 had higher (but not significant) cholesterol levels than controls, and the data show that the estrane progestin combint'C! with estrogen raises HDL 2 cholesterol in relation to LDL cholesterol-the reverse of the finding with the gonane-containing pills.

Comment The relativelv low incidence of coronary heart disease and thrombotic stroke in premenopausal women compared with age-matched men and the increased incidence of these diseases after menopause have been well established by many studies. The mechanism ot the relative immunity of premenopausal women to cardiovascular disease, although not certified, is widely believed to be due to the protective effect of estrogen. Such an effect has not been found with estrogen administered to men at risk of vascular disease28 or when given as treatment for prostatic carcinoma29 ; these men had an increased mortality rate from coronary he
Volume 142 Number 6, Part 2

thrombotic stroke with the use of the combined pill is now supported by many studies and must be considered an adverse effect of the OC-perhaps the most serious disadvantage identified so far. It seems logical to assume, therefore, that the progestin plays an important part in the pathologic process leading to myocardial infarction or stroke, either alone or, more probably, by a combined effect with the estrogen. It is only during the past few years that the importance of the progestin in the etiology of the adverse cardiovascular effects of OCs (including hypertension) has come to be realized, and during this period the effect of progestational steroids in lowering serum HDL levels has been described. We have known for many years that the HDL level (particularly HDL 2 ) is lower in men than in premenopausal women 14 and that androgens administered to women lower their serum HDL and, in particular, HDL 2 levels. 3 ~· :ra Progestational steroids with androgenic activity have the same effect, whereas progesterone alone and progestins deri\ed from 17-hydroxyprogesterone (the pregnanes), such as chlormadinone or megestrol, have little or no effect,'14 -'"' in keeping with their much lower androgenic action.'" It is not surprising, therefore, that

OC progestins, serum lipids, and HDLs

our study has shown that the gonane norgestrel depresses HDL and HDL2 levels when the gonane is combined with 30 ~-tg of estrogen. On the other hand, the estrane norethindrone (acetate), which has a weaker androgenic action than norgestrel, does not lower HDL or HDLz levels when this progestin is combined with as little as 20 ~-tg of estrogen. We conclude, therefore, that if changes in HDL and HDL 2 levels are to be avoided the preferred progestin is the estrane norethindrone and not the gonane norgestrel, at any rate in the doses currently and widely prescribed. Our studies suggest that a combined pill containing less than 50 ~-tg of estrogen (perhaps 30 to 35 ~-tg of ethinyl estradiol) and I mg of norethindrone (acetate), and possibly a little less, should be a satisfactory contraceptive for most women. This combination will not affect HDL, at any rate as measured by the techniques described in this article, but it will moderately increase the serum triglyceride and the VLDL level. Prospective studies in large numbers of women will be needed to establish the safety of this contraceptive, but this applies to any drug administered widely and intended for prolonged use.

REFERENCES I. Vessey, M.P.: Female hormones and vascular diseasean epidemiological overview, Br. J. Fam. Plann. (Suppl.) 6:1, 1980. 2. :vlann,J. l., and Vessey, M.P.: Trends in cardiovascular disease mortality and oral contraceptives, Br. .J. Fam. Plann. 6:99, 1981. 3. Mann, J. 1.: Progestogens in cardiovascular disease: An introduction to the epidemiologic data, AM. J. 0BSTET. GYNECOL. 142:752, 1982. 4. Kay, C. R.: Progestogens and arterial disease-Evidence from the Royal College of General Practitioners' study, AM. j. 0BSTET. GYNECOL. 142:762, 1982. 5. Meade, T. W.: Effects of progestogens on the cardiovascular system, AM . .J. 0BSTET. GYNECOL. 142:776, 1982. 6. Stadel, B. V.: Oral contraceptives and cardiovascular disease. Part I,!'\. Engl. J. Med. 305:612, 1981. 7. Stadel, B. V.: Oral contraceptives and cardiovascular disease. Part II, :'-J". Engl. J. Med. 305:672, !981. 8. Slone, D., Shapiro, S., Kaufman, D. W., et al.: Risk of mvocardial infarction in relation to current and discontir;ued use of oral contraceptives, N. Engl. .J. Med. 305:420, 1981. 9. Miller, N. E., and Lewis, B., editors: Lipoproteins, Atherosclerosis and Coronary Heart Disease, Amsterdam, 1981, Elsevier North Holland. 10. Wynn, V., Doar, J. W. H., and Mills, G. L.: Some effects of oral contraceptives on serum lipid and lipoprotein levels, Lancet 2:720, 1966. 11. Wynn, V., Doar, J. W. H., Mills, G. L., et al.: Fasting serum triglyceride, cholesterol and lipoprotein levels during oral contraceptive therapy, Lancet 2:756, 1969. 12. Stokes, T., and Wynn, V.: Serum-lipids in women on oral contraceptives, Lancet 2:677, 197!. 13. Wynn, V .. Adams, P. W., Godsland, l., et al.: Comparison

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H. 15.

16.

I 7. 18. 19.

20. 21.

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23. 24.

of effects of different combined oral contraceptive formulations on carbohydrate and lipid metabo'lism, Lancet 1:1045, 1979. Barr, D. P., Russ, E. M., and Eder, H. A.: Protein-lipids relationships in human plasma. II. In atherosclerosis and related conditions, Am. J. Med. 11:480, 1951. :Vfiller, G. ]., and :Vfiller, N. E.: Plasma-high-densitylipoprotein concentration and development of ischaemic heart disease, Lancet 1: 16, 197 5. Miller. N. E., Hammett, F., Saltissi, S., et al.: Relation of angiographically defined coronary artery disease to plasma lipoprotein subfractions and apolipoproteins, Br. :vted. J. 282:1741, 1981. Glomset. .J. A.: The plasma lecithin: cholesterol acyltransferase reaction,]. Lipid Res. 9:155, 1968. Witztum, .J.. and Schonfeld, G.: High density lipoproteins, Diabetes 28:326, 1979. Wynn, V.: Effect of duration of a low-dose oral contraceptive administration on carbohydrate metabolism, AM.J. 0BSTET. GYNECOL. 142:739, 1982. Stoker, D. J., and Wynn, V.: Effects of posture on the plasma cholesterol level, Br. Med . .J. 1:%6, 1966. Warnick, G. R., and Albers, .J. J.: A comprehensive evaluation of the heparin-manganese precipitation procedure for estimating high density lipoprotein cholesterol.]. Lipid Res. 19:65, 1978. Burstein, M., and Samaille,J.: Sur une nouvelle methode de dosage des ,(3-lipoproteins seriques par l'heparine, C. R. Acad. Sci. [D] (Paris) 243:2185, 1956. Burstein, M., and Scholnick, H. R.: Lipoprotein-polyanion-metal interactions, Adv. Lipid Res. 11:67, 19i3. Gidez, l., Miller, G . .J., Burstein, M., et al.: Analysis of plasma high density lipoprotein subclasses by a precipitation procedure: correlation with preparative and analytical ultracentrifugation, in Lippe!, K., editor: Report of

772

Wynn and Niththyananthan

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the High Densit; Lipoprotein Methodology, Workshop, l'nited States Department of Health, Education and \Veil are. :\IH Puhlie.i. l'lwmpsun. c;. R.: Dicl~tn and pliarmJcct, .f.A.\I.A. 214:1 'iO:l. 1:170. lbiler. J C Thromboembolism and oestrogen ther:qn. Lancet 2:560. 1967. Rosenl>erg·. L .'ilone. ll .. Shapim, S .. el al.: :\oncolltr·an:pti\'t' c~tn1g~n~ and tnyotardial inLtn tion In ~·oung 1\'0il!Cli,.J. ,\ \1. :\. 244::~~19. l9HO. Ross. R. ~ .. Paganini-1-!ill, .\ .. \fack, I \1.. t'l al.: :\Icnu-

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IMll"tl oc'irogen therapy and JH"tt·cti<! athemsclemsis, Circulation 8:6·41, 19'>:1 B~tn _ [) !'.: lnflur·rKf' of sex and,,., j-,,,monc·, "i'"n th,dnclopment oLtthcrosderosi., and''!""' rlw lip<>l~lnteir.s 'd pi~!Ctll~l, J Chronic Dis. 1:li:'•· JL)_-,,; rikkarwn. \I. J '\ikkila, F -\ l-.11!1\l . Ci ;d Rt•dtit lion ot plasm:; high-dcnsit1 lipoprotein, cholntcrol .md tiHTl'lhl' nf postlwp,nin plasma hcpati< lipase actiYit\ dtnin~ jHDg·L·stin tH.'f.\l'stins on plasma lipoprolt'lns .md '"'Jl:llill-rck;;sahk lipa~e:--.. in ).fau\·ais-Jctr\·i~. P .. editor: Pruge~tcnHit' ~ll!d Prng-estins. :'-iew York. R:nen Pres' In pn·ss. Bradln. D. IL Wingcrd, .J. Petitti. U lL t'l aL. '•TUJII ltigh-densin-lipoprotein cholesterol in \,omen '"ing oral comratcplil·es. estrogerh and prog·esrin>. \;. Lngl. I· ;\fed. 299:17, ]')/,'(, Fl ,\lilkillangY. \!. "- .. 1\lttll. \ .. :lltd l ,t~>Tt'll<<'. D. \f. Se~ lt .. nnone hindi11g g·iobulin