- Email: [email protected]
Lipid and lipoprotein changes in women taking low-dose, triphasic oral contraceptives: A controlled, comparative, 12-month clinical trial
Lipid and lipoprotein changes in women taking low-dose, triphasic oral contraceptives: A controlled, comparative, 12-month clinical trial Morris Notelovitz, MD, PhD: Elaine B. Feldman, MD," Marjorie Gillespy, MD: and Jack Gudat, PhDc Gainesville, Florida, and Augusta, Georgia Effects on lipid/lipoprotein metabolism of two triphasic oral contraceptives, Triphasil (ethinyl estradiol/levonorgestrel) and Ortho-Novum 7/7/7 (ethinyl estradiol/norethindrone) were compared in a 12-month controlled, prospective clinical trial. The data indicate that use of both estrogen-progestin preparations were accompanied by increases in cholesterol, low-density lipoprotein and high-density lipoprotein 3 cholesterol, apolipoproteins A, and B, and triglycerides. Also observed were a decline in high-density lipoprotein cholesterol and greater decreases in high-density lipoprotein 2 cholesterol levels; the latter were below the lower limits of laboratory's reference range. All other changes remained within clinically acceptable limits. There were no statistically significant differences between the tes! preparations, suggesting that the impact on lipid metabolism of the triphasic preparations Triphasil and Ortho-Novum 7/7/7 are similar and, given the dynamic balance between the various fractions, are unlikely to impart an adverse cardiovascular risk. (AM J OSSTET GVNECOL 1989;160:1269-80.)
Key words: Atherogenesis, ethinyl estradiol, levonorgestrel. lipid metabolism. lipoprotein, norethindrone, triphasic oral contraceptives
Premenopausal women are relatively resistant to atherogenic disease compared with men, an advantage that is greatly diminished in the postmenopausal years. Although the pathogenesis of this change has not been definitively established, the decrease in ovarian estrogen steroidogenesis and consequent changes in lipids and lipoproteins are thought to playa major causative role. Evidence is increasing that therapy with exogenous estrogen in menopausal women may reverse this trend and that women who have taken long-term estrogen therapy actually have a lower incidence of cardiovascular disease than their untreated peers. I This experience is contrary to that observed with the early use of exogenous sex steroids for oral contraception in younger women. The reported increase in oral contraceptive-related cardiovascular risk was attributed to an estrogen-dependent alteration in the coagulation/anticoagulation profile, predisposing women to an increased risk of thrombosis and thromboembolic phenomena." More recently, progestins have been associated with changes in lipid and lipoprotein metabolism that theoretically could accelerate atherogenesis. 3
From the Center for Climactenc Studies and Women's MedIcal and Dzagnostlc Center," Georgza Institute of Human NutntlOn, MedIcal College of Georgza, Augusta,' and the Department of Pathology, University of Flonda College of Medlcme' Repnnt requests: Morns Notelovltz, MD, PhD. Women's Medical and Diagno;tlc Center. 222 S. W. 36th Terrace, SUite C. GainesVille. FL 32607.
Although there is no clinical evidence that atherogenic disease in women has generally increased since the introduction of oral contraceptives in 1960, efforts have been taken to ensure that the premenopausal protection against cardiovascular disease is not compromised by these agents. The quantities of both estrogen and progestin have gradually been reduced and their ratios altered. This has culminated in the development of the triphasic oral contraceptives. Because of the relative paucity of well-controlled, prospective studies evaluating the metabolic effects of long-term use of the triphasic preparations, a study was initiated to compare the effects of two such regimens over a 12-month period. The trial formulations contained the same estrogen-ethinyl estradiol, but different progestins-Ievonorgestrel (Triphasil, Wyeth Laboratories, Philadelphia, Pa.) or norethindrone (Ortho-Novum 7/7/7, Ortho Pharmaceuticals, Raritan. N.J.). This article compares alterations in plasma lipids and lipoproteins after use of both contraceptive agents to observations in a group of non treated control subjects. It also attempts to place observed biochemical alterations in clinical perspective.
Material and methods Subjects. Eighty volunteers, ranging in age from 21 to 35 years. were enrolled in the study. Subjects were in good physical health, within 10% of desirable body weight, and at least 90 days' postpregnancy. They had
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Table I. Trial formulations Triphasil Phase 1 Phase 2 Phase 3 Ortho-Novum 71717 Phase I Phase 2 Phase 3
No. of days
Plogeltlll
6
5 10
Levonorgestrel Levonorge~t rei Levonorgestrel
7 7 7
Noretlundrone Norethindrone Norethmdrone
regular menstrual cycles, limited alcohol consumption, and smoking, and no contraindication to oral contraceptive usage. Contraindications included thrombophlebitis or thromboembolic disorders (past or present), cerebral vascular or coronary artery disease, and previous estrogen-dependent tumors. All women were screened before they entered the study and had a complete history, full physical examination, and blood pressure recording, a Papanicolaou smear, and hematologic and biochemical profiles. The laboratory tests included renal and liver function tests, serum electrolytes (SMA-25), hemoglobin, hematocrit, and white blood cell count. Lipid parameters were measured at pretreatment and after six and 12 cycles of treatment. Participants again received a full physical examination, Papanicolaou smear, and blood chemistry analysis 4 weeks after the twelfth cycle of therapy. Thirty-eight women were randomized into group I and received the levonorgestrel-containing oral contraceptive (Triphasil), and 42 were assigned to group II and received the norethindrone-containing formulation (Ortho-Novum 71717). The control group was made up of 14 female volunteers who met the same inclusion criteria and who were either surgically &terile with intact ovaries or used a nonhormonal contraceptive method. Laboratory personnel were blinded to the subjects' medication assignment. Material. The formulations of the two oral contraceptives are shown in Table I. Participants were instructed to take the contraceptive pills in cycles of 3 weeks on and 1 week off for 12 months. Treatment was started after blood chemistries and definitive serum lipid and lipoprotein studies were performed. Each subject was given a diary card on which to record medication, withdrawal and intermenstrual bleeding, and side effects. Care was taken to ensure that concomitant medicatiom such as aspirin-containing drugs, antiprostaglandins, and antibiotics were not taken during the study without the knowledge of the investigator. Life-style and other lipid influences on metabolism were reduced
E.ltrogen
JLg
50 75 125
Ethinyl estradiol Ethinyl estradiol Ethinyl estradiol
30 40 30
500 750 1000
Ethinyl estradiol Ethinyl estradiol Ethinyl estradiol
35 35 35
to a minimum and were considered equivalent in all three groups. Laboratory methods. Baseline and subsequent blood samples were obtained between days 20 to 25 (luteal phase) of the pretreatment cycle and treatment cycles. Subjects were instructed to fast for 12 to 24 hours overnight and had a 30 ml venous sample of blood taken between 8:00 and 9:00 AM the following morning. The sample was divided into two aliquots and allowed to clot at room temperature for 1 hour. The portion used for assay of cholesterol, triglycerides, low-density lipoprotein, and high-density lipoprotein was placed in tubes containing ethylenediaminetetra-acetic acid and centrifuged at 2000 rpm for 15 minutes. It was then frozen at 4° C and shipped for analysis the same day via air express. The aliquot used for apolipoprotein analysis was centrifuged at 1000 rpm for 10 minutes; the serum was separated from the clot and transported on dry ice to the lipid laboratories where the sample was stored at - 20° C until analysis. Total cholesterol; triglycerides,' and high-density lipoprotein cholesterol6 levels were determined by automated enzymatic procedures. The results were reported as percentiles according to the Lipid Research Clinic's reference values for women of the same age group.' High-density lipoprotein cholesterol was determined in the supernatant after precipitation of very low-density lipoprotein and low-density lipoprotein." Low-density lipoprotein cholesterol was calculated by means of the Friedewald equation. 9 The high-density lipoprotein 2 and high-density lipoprotein, subfraction separations were carried out by a modification of the procedure described by Grow and Fried. lo Apolipoproteins AI and B were measured by means of an immunonephelometric assay with diagnostic kits provided by Hyland Diagnostics (Deerfield, Ill.). Statistical analysis. Statistically significant (p < 0.05) within-group changes from baseline were determined by the paired t test. Statistically significant (P < 0.05) differences between the treatment groups were determined by analysis of covariance, with the baseline level taken as the covariate.
Lipid/lipoprotein changes with the use of triphasic oes
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Table II. Demographic data of the treatment and control groups No. Age (yr) Parity Height (inches) Weight (pounds) Baseline 12 mo Blood pressure (mm Hg) Baseline 12 mo Cigarettes (per day) Baseline 12 mo Alcohol (drinks/wk) Baseline 12 mo
Group I. EE/LNg
Group II. EE/NE
ControLI
29 25.4 ± 0.7 0.9 ± 0.2 62.6 ± 2.1
33 25.7 ± 0.7 0.9 ± 0.2 65.7 ± 0.6
II 26.9 ± 1.2 1.4 ± 0.5 65.2 ± 0.7
129.9 ± 3.2 131.0 ± 3.2
132.7 ± 3.0 133.7 ± 3.1
134.0 ± 3.7 134.1 ± 3.1
107 ± 1.7/65 ± 1.4 105 ± 2.0/63 ± 1.7
106 ± 1.4/64 ± 1.4 107 ± 1.6/67 ± 1.6
105 ± 2.4/66 ± 1.7 104 ± 2.9/66 ± 2.9
0.4 ± 0.4 1.0 ± 0.6
0.5 ± 0.4 0.9 ± 0.6
o o
1.7 ± 0.3 1.9 ± 0.3
1.6 ± 0.4 1.9 ± 0.4
0.7 ± 0.3 0.6 ± 0.2
Values expressed as means ± SEM. EE / LNg, Ethinvl estradiolllevonorgestrel (Triphasil); EE / NE, ethinyl estradiolllevonorgestrel (Ortho-Novum 7/7/7).
Table III. Change in serum cholesterol and triglycerides in normal control subjects and women on two low-dose, triphasic contraceptives Total cholesterol (mg/dl) No. Baseline 6 mo Mean change % change No. Baseline 12 mo Mean change % change Triglycerides (mg/dl) No. Baseline 6 mo Mean change % change No. Baseline 12 mo Mean change % change
Control
Grout) I, EE/LNg
Group II, EE / NE
II 167.3 ± 10.4 164.1 ± 7.2 -3.2 ± 6.1 -2 Il 167.3 ± 10.4 162.5 ± 7.7 -4.7 ± 8.3 -3
29 159.3 ± 4.7 177.7 ± 5.4** 18.4 ± 5.0 + II 27 159.0 ± 5.0 178.0 ± 6.2*** 18.9 ± 4.9 + II
33 167.1 ± 4.6 182.0 ± 5.0* 14.9 ± 5.6 +8 30 166.9 ± 5.0 181.8 ± 6.0* 14.9:t 6.1 +8
29 85.6 ± 7.1 112.2 ± 7.7* 26.6 ± 10.5 +24 27 87.4 :t 7.5 98.3 :t 5.7t 11.0 ± 6.8 +11
33 86.2 :t 5.4 113.4 :t 7.5*** 27.2 ± 6.6 +24 30 86.8 :t 5.9 103.8 :t 6.1 tt* 17.0 ± 7.4 +16
II 68.8 ± 4.1 81.9 :t 11.6 13.1 ± ILl + 16 11
68.8 ± 4.1 65.5 :t 5.3 -3.4 ± 4.6 -5
Mean (:t SEM) pretreatment values compared with mean (:t SEM) values at 6 and 12 months. EE / LNg, Ethinyl estradiolllevonorgestrel (Triphasil); EE / NE, ethinyl estradiollnorethindrone (Ortho-Novum 7/7/7).
* **
*** = t, tt, ttt =
p < 0.05, < 0.01, < 0.001 when compared with baseline values in the same P < 0.05, < 0.01, < 0.001 when compared with the control group.
group.
Results Twenty-nine women in group I, 33 in group II, and II control subjects completed the study (Table II). Because of incomplete data, two subjects from group I and three from group II had to be dropped from the 12-month analysis. Demographic data shown in Table II demonstrated no significant differences among the study groups at baseline.
Plasma total cholesterol (Table III) increased at 6 months in response to treatment with both oral contraceptives and remained about the same over the next 6 months. Although the plasma cholesterol increases in users of both oral contraceptives was statisticallv significant when compared with baseline values, the levels were well within the normal range for our laboratory
1272 Notelovitz et al.
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Table IV. Change in serum lipoproteins in normal control subjects and in women on two low-dose, oral triphasic contraceptives Control Total HDL cholesterol (mg/dl) No. Baseline 6 mo Mean change % change No. Baseline 12 mo Mean change % change HDL2 cholesterol (mg/dl) No. Baseline 6 mo Mean change % change No. Baseline 12 mo Mean change % change HDL, cholesterol (mg/dl) No. Baseline 6 mo Mean change % change No. Baseline 12 mo Mean change % change LDL cholesterol (mg/dl) No. Baseline 6 mo Mean change % change No. Baseline 12 mo Mean change % change
11 53.6 ± 3.9 56.9 ± 3.5 3.3 ± 2.2 +6
Group I, EE 1LNg 29 52.2 ± 2.3 51.7 ± 2.0 -0.5 ± 2.6
Group II, EE 1NE
II
27 52.4 ± 2.5 49.0 ± 2.0 -3.4 ± 2.5 -6
33 56.2 ± 2.4 57.0 ± 2.9 0.8 ± 3.0 +1 30 55.7 ± 2.4 53.4 ± 3.2 -2.3 ± 2.9 -4
II
29 18.8 ± 1.8 15.0 ± J.l -3.9 ± 2.0 -20 27 18.8 ± 1.8 10.0 ± 0.7ttt*** -8.8 ± 1.5 -47
33 21.4 ± 2.2 18.3 ± 1.9 -3.0 ± 2.9 -14 30 20.7 ± 2.2 12.4 ± l.4tt** -8.3 ± 2.4 -40
29 34.4 ± 1.6 36.7 ± 1.5 2.3 ± 1.7 +6 27 34.4 ± 1.7 39.0 ± Ui* 4.5 ± 2.0 + 12
33 34.8 ± 1.5 38.6 ± 1.6** 3.8 ± 1.3 +10 30 35.0 ± 1.6 41.0 ± 2.2** 6.0 ± 1.8 + 15
53.6 ± 3.9 55.1 ± 3.9 1.5 ± 3.0 +3 18.6 ± 3.0 21.8 ± 2.7 3.2 ± 3.5 + 15 11 18.6 ± 3.0 18.2 ± 2.8 -0.5 ± 2.7 -2 11
35.0 ± 2.9 35.1 ± 2.6 0.09 ± 3.4 NC 11 35.0 ± 2.9 36.9 ± 2.0 1.9 ± 2.7 +5 11 105.5 ± 9.7 97.4 ± 8.4 -8.2 ± 6.8 -8 II
105.5 ± 9.7 99.0 ± 6.4 -6.5 ± 8.6 -6
-I
29 97.6 ± 4.2 112.7 ± 5.3** 15.1 ± 4.2 + 13 27 97.0 ± 4.5 115.4 ± 6.0** 18.4 ± 5.4 + 16
33 101.3 ± 4.3 110.0 ± 4.2 8.7 ± 5.4 +8 30 101.7 ± 4.7 114.4 ± 4.9* 12.7 ± 5.8 +11
Mean (± SEM) pretreatment values compared with mean (± SEM) values at 6 and 12 months. NC, No change; EEILNg, ethinyl estradioi/levonorgestrel (Triphasil); EEINE, ethinyl estradiol/norethindrone (Ortho-Novum 7/717); HDL, high-density lipoprotein; LDL, low-density lipoprotein. * **. *** = p < 0.05, < 0.01, < 0.001 when compared with baseline values in the same group. t. tt. ttt = P < 0.05. < 0.01. < 0.001 when compared with the control group.
«200 mg/dl*). Moreover, they did not differ significantly from those of the control women at each test point, remaining within two standard errors of the control mean. There was no statistical or clinically significant difference between the two treatment groups. Plasma triglycerides (Table III) were predictably affected by the levonorgestrel- and norethindrone-based
oral contraceptives, which demonstrated very similar increases with both regimens at 6 months. These statistically significant alterations declined to lower levels at the end of 1 year but were still elevated over baseline values and were significantly greater than those found in the (ontrol group. However, the absolute concentrations of triglycerides in all study groups remained well within the normal range. .
*Unless otherwise noted. reference values are given as the seventy-fifth percentile of normal.
Total plasma high-density lipoprotein cholesterol levels (Table IV) were unaffected at 6 months and showed no significant decreases in either treatment
Lipid/lipoprotein changes with tKe use of triphasic OCs
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Table V. Change in apolipoprotein AI and apolipoprotein B in normal control subjects and women receiving two low-dose, oral triphasic contraceptives Control
Apolipoprotein AI (mg/dl) No. Baseline 6 mo Mean change % change No. Baseline 12 mo Mean change % change Apolipoprotein B (mg/dl) No. Baseline 6 mo Mean change % change No. Baseline 12 mo Mean change % change
Group /, EE/LNg
Group II, EE / NE
10
28 134.5 ± 4.9 166.5 ± 5.8t*** 32.0 ± 6.0 +19 26 133.2 ± 5.2 144.8 ± 8.4 11.6 ± 9.1 +8
32 140.8 ± 4.5 176.8 ± 6.7tt*** 35.9 ± 7.2 +20 30 140.2 ± 4.5 152.0 ± 8.3 11.9 ± 8.5 +8
10
28 77.6 ± 4.0 89.6 ± 4.4t* 12.0 ± 5.4 + 13 26 77.9 ± 4.3 97.2 ± 5.9tt** 19.3 ± 6.2 +20
32 75.6 ± 3.3 94.3 ± 3.6tt** 18.6 ± 5.1 +20 30 74.9 ± 3.4 87.1 ± 4.2t* 12.3 ± 4.8 +14
138.0 ± 10.7 142.9 ± 9.0 4.9 ± 9.0 +4 10 138.0 ± 10.7 130.3 ± 7.6 -7.7 ± 15.2 -6 73.3 ± 10.4 69.6 ± 5.7 -3.7 ± 10.3 -5 10 73.3 ± 10.4 65.2 ± 5.5 -8.1 ± 9.6 -ll
Mean (±SEM) pretreatment values compared with mean (±SEM) values at 6 and 12 months. EE / LNg, Ethinyl estradioillevonorgestrel (Triphasil); EE / NE, ethinyl estradiollnorethindrone (Ortho-Novum 71717).
* **. *** = p < 0.05, < 0.01, < 0.001 when compared with baseline values in the same group. t, tt, ttt = P < 0.05, < 0.01, < 0.001 when compared with the control group.
group at 12 months. These values did not differ statistically from pretreatment levels within each group or compared with values in the other treatment or control groups. By contrast, the high-density lipoprotein subfractions (Table IV) exhibited a significant and possibly clinically meaningful change. For high-density lipoprotein", the ethinyl estradiollievonorgestrel oral contraceptive users had a 20% and 47% reduction, respectively, in mean 6- and 12-month values compared with baseline values. The ethinyl estradiol/norethindrone-treated subjects had a fall of 14% and 40%, respectively. The 12-month values were significantly different both within groups (p < 0.001 and p < 0.0l) and compared with control subjects (p < 0.001 and p < 0.01) but were not significantly different between treatment groups. For high-density lipoprotein." these values were higher than pretreatment levels and slightly higher than the control group's levels, but the difference was not statistically significant. Paralleling the change in total cholesterol, the low-density lipoprotein cholesterol fraction (Table IV) increased at 6 months and maintained these elevated levels thereafter. Again, all the lipoprotein concentrations, with the exception of high-density lipoprotein" at 12 months, were within the clinically acceptable ranges (total highdensity lipoprotein cholesterol = 37 to 74 mg/dl;* *Tenth to seventieth percentiles.
high-density lipoprotein" cholesterol = 15 to 60 mg/ dl; high-density lipoprotein, cholesterol = 14 to 65 mg/dl; low-density lipoprotein cholesterol = <130 mg/dl). Apolipoprotein AI, the m~or protein component of high-density lipoprotein cholesterol, showed a significant increase at 6 months in both treated groups, with a subsequent decline in serum levels when assessed 6 months later (Table V). Values at 6 months in the treated women rose to the laboratory's upper range of normal (97 to 174 mg/dl) and in some instances exceeded it. These changes were highly significant (p < 0.001) both within groups and compared with the control group (p < 0.05 and <0.01). Apolipoprotein B, the apoprotein carrier for lowdensity lipoprotein cholesterol, showed similar elevations in both groups at six months. A further increase in the ethinyl estradiollievonorgestrel group (from 13% up to 20%) and a slight decline in the ethinyl estradiollnorethindrone group (from 20% down to 14%) were noted at 12 months. Although the net increases were statistically significant within groups and when compared with control subjects, these changes did not exceed the normal range (46 to 120 mg/dl). As with all the other lipid and lipoprotein parameters, there was no statistically significant difference between the two treatment groups. It is now customary to refer to ratios between the
1274 Notelovitz et al.
May 1989 Am J Obstet Gynecol
Table VI. Alteration in ratios of lipids and lipoproteins in normal controls and in women on two low-dose oral triphasic contraceptives Total cholesterollHDL cholesterol (mg/dl) Baseline 6 mo 12 mo HDLlLDL cholesterol Baseline 6 mo 12 mo Apolipoprotein AI/apolipoprotein B Baseline 6 mo 12 mo
Control
Group I, EE/LNg
3.2 ± 0.2 3.0 ± 0.2 3.1 ± 0.2
3.2 ± 0.1 3.6 ± 0.2* 3.8 ± 0.2t
3.1 ± 0.1 3.4 ± 0.2 3.6 ± 0.2*
0.6 ± 0.07 0.6 ± 0.08 0.6 ± 0.06
0.6 ± 0.04 0.5 ± 0.05 0.5 ± 0.06
0.6 ± 0.04 0.5 ± 0.03 0.5 ± 0.03
2.3 ± 0.4 2.2 ± 0.3 2.1 ± 0.1
1.9 ± 0.1 2.0 ± 0.1 1.6 ± 0.1 *
2.0 ± 0.1 1.9 ± 0.1 1.8 ± 0.1
Group II, EE / NE
Mean (±SEM) pretreatment values compared with mean (±SEM) values at 6 and 12 months. EE / LNg, Ethinyl estradiol/levonorgestrel (Triphasil); EE / NE, ethinyl estradiol/norethindrone (Ortho-Novum 7/7/7); HDL, high-density lipoprotein; LDL, low-density lipoprotein. *, t, * = P < 0.01. < 0.001, < 0.05 when compared with baseline values in the same group.
lipid, lipoprotein, and apoprotein factors, as reflected in Table VI. The ratio of total cholesterol to highdensity lipoprotein cholesterol, probably the most commonly used lipid ratio in clinical practice, underwent a progre~sive and statistically significant increase from baseline at both evaluation intervals in both treatment groups. The calculated ratio of high-density lipoprotein/low-density lipoprotein, cholesterol was unaffected over the course of therapy in either treatment group when baseline, 6- and 12-month values were compared and no statistically significant differences from control values were found. Results were similar with the protein ratios except that the decline in apolipoprotein AIIB ratio by 12 months found in all groups was statistically significant in the ethinyl estradiollievonorgestrel group, but not in the other two groups. No clinically significant changes in blood pressure were observed during the study, nor were statistically significant alterations in weight noted. Side effects were of the type usually associated with oral contraceptive use and similar for each treatment group. As reflected in Table VII, nine patients withdrew from each treatment group and three dropped out of the control group before completion of the study. One pregnancy occurred in the ethinyl estradiollievonorgestrel group because of method failure.
Comment Much confusion surrounds the interpretation of lipid and lipoprotein levels. The principles governing the transport and metabolism of these substances are complex and interdependent. In addition, laboratory values are often extrapolated to predict biologic significance without proper consideration of the complex factors
and unknowns involved. A brief review of these concepts may help place the present findings into a clearer practical perspective. Lipidllipoprotein metabolism: A brief overview. Lipids are essential components of cell membranes; they are basic to steroid synthesis and constitute a transportable metabolic energy pool. Lipoproteins serve to transport lipids and regulate their metabolism. The interaction between these substances is modulated by lipid enzymes. The "good" (antiatherogenic) and "bad" (atherogenic) lipid and lipoproteins are summarized in Fig. 1. The three major interacting pathways involved in their metabolism are shown in Fig. 2. Both the exogenous and endogenous pathways result in a net lipogenic effect. Exogenously, dietary fats are absorbed as chylomicrons and are catabolized via lipoprotein lipase, producing free fatty acids used for energy or stored in fat cells. Although chylomicron particles are not atherogenic, their partially catabolized remnants are. Endogenously, very low-density lipoprotein, the major lipoprotein produced in the liver, is hydrolyzed also via lipoprotein lipase into intermediate-density lipoprotein. Although a portion of intermediate-density lipoprotein is cleared by the liver, the remainder is transformed into the denser and more highly atherogenic fraction, low-density lipoprotein. Raised levels of intermediate-density lipoprotein cholesterol fractions are atherogenic, since they are rich in both cholesterol and triglycerides. Low-density lipoprotein cholesterol transports about two thirds of circulating cholesterol and thus is considered the most atherogenic of the lipoprotein particles. Counterbalancing the lipogenic effects of the exogenous and endogenous pathways is the antiatherogenic effect of the reverse cholesterol transport system. Na-
lipid/lipoprotein changes with the use of triphasic oes
Volume 160 I\"umber 5, Part 2
Table VII. Reasons for early withdrawal from study
THE "GOOD" Group II, EE/NE
Side effects of DC Breast discomfort Spotting Nausea Other contraception Headaches Medical reasons Asthma Incidental surgery Chronic vaginal infection Lost to follow-up/moved Miscellaneous Pregnancy Excessive smoking Religious reasons Total ~C,
AND
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THE "BAD" Cholesterol
HDL·C HDL2
LDL·C
APO·A,
APO·S
HDL3
Tnglycendes
~ 2
o
I
2 I I
8
Fig. 1. Lipid/ltpoprotein balance.
8
Oral contraceptives.
scent (new) high-density lipoprotein, which is synthesized in the liver and intestine in the form of high-density lipoproteinj, is converted into relatively cholesterol-rich high-density lipoprotein,. This in turn is reconverted into cholesterol-poor high-density lipoprotein., by hepatic lipase. The high-density lipoprotein lipoproteins, especially high-density lipoprotein~, are antiatherogenic and are involved in the transport of cholesterol from the peripheral tissues to the liver for excretion and conversion to bile acids. The protein components apolipoprotein AI and apolipoprotein A2 also participate in this antilipogenic process as activators of the enzymes lecithin-cholesterol acyltransferase and hepatic lipase, respectively. Apolipoproteins also identify cell receptor sites and bind to them and have emerged as important discriminators of the presence or absence of atherogenic disease. For example, in a recent studyll plasma concentrations of apolipoprotein B were predictive of 87% of patients with ischemic heart disease and 75% of control subjects compared with other accepted risk factors (family history, serum cholesterol, serum triglycerides, and high-density lipoprotein cholesterol), which correlated with only 61 % of patients and 69% of control subjects. When apolipoprotein B was combined with apolipoprotein AI and family history, identification was correct in 79% of patients and 83% of control subjects. Another interrelated and central factor in the lipidlipoprotein balance is the low-density lipoprotein receptor. It is known that high intracellular levels of cholesterol suppress the hepatic synthesis of low-density lipoprotein receptors. This in turn elevates low-density lipoprotein both through diminished uptake (and metabolism) and enhanced conversion of very low-density lipoprotein to low-density lipoprotein. 12 The significance of this vast multifactorial network
makes it unfeasible to assess atherogenic potential based on the evaluation of a single metabolic parameter. Epidemiologic studies have shown that ratios of proand antiatherogenic lipidsllipoproteins are better predictors of atherogenic disease. An imbalance or perturbation among the pro- and antiatherogenic factors may produce vascular intimal damage, which leads eventually to formation of an atheromatous plaque. Thus in the context of oral contraceptive-induced lipid changes, the clinician must assess the import of an increase in one atherogenic factor (e.g., raised plasma cholesterol) if a simultaneous antiatherogenic change (e.g., increased apolipoprotein AI) may provide "balance" in the overall drug-induced event. A shift in favor of the antiatherogenic or "good" side of the lipid equation is probably desirable, but the untoward or beneficial long-term effects of such changes induced by low-dose estrogenprogestin combination preparations are unknown. Oral contraceptive-induced changes in lipids and lipoproteins. A "perfect" oral contraceptive would neither increase levels of cholesterol and low-density lipoprotein cholesterol nor lower high-density lipoprotein cholesterol. In addition, the protein carriers (and their activity) would be unaffected. As reflected in a recent clinical trial and review article,13 II such an absence of effect is unlikely, since all estrogen- and progestin-containing formulations marketed in the United States have some influence on lipid and lipoprotein metabolism. Most recently, Burkman et al. Ll compared the effect of four low-dose oral contraceptives (ethinyl estradiol, 35 J..Lg + ethynodiol diacetate, 1 mg; ethinyl estradiol, 30 J..Lg + levonorgestrel, 0.05 mg: ethinyl estradiol, 35 J..Lg + norethindrone, 1 mg; and ethinyl estradiol, 35 J..Lg + norethindrone, 0.5 and 1 mg) over a 6-month period and found that total cholesterol increased 5.9% to 9.1 % above baseline values; triglyceride levels increased 37.6% to 45.3%; lowdensity lipoprotein cholesterol increased 10% to 15.6%; and (with the exception of the group using the ethynodiol diacetate-containing oral contraceptive) highdensity lipoprotein decreased 4.5% to 8.7%. The lipid protein carriers were also altered. Apolipoprotein B
1276
Notelovitz et al.
May 1989 Am J Obstct Gynccol
I-~DLSYNTHESIS --l
PATHWAY
Liver
Chylomicron
VLDL
LIPOPROTEIN COMPONENTS
Tnglycendes Cholesterol + APO 848 APOCII & III APOE
Tnglycendes Cholesterol + APO 8100, APO CII, III APOE
ENZYMES
LIPoprotleln lipase
LIPID
+
,
J
DISPOSITION
Energy + Fat cell storage
HD~
I AP~~II LJAT ,Lipoprotein
i'
---...r~;;-L;~----~.------- ~_~ ___ L ___ -1 I
Chylomicron Chylomicron + remnant FFA
Cholesterol APO 8100
r Liver
,
Penpheral cholesterol accumulation
I
IiP1se
,
Liver
I--
I
I
I
AP2'
'
~I,
l ~I~~~C
I I ,
!
I 1
1
I I
,HDL2
Cholesterol Tnglycendes APO 8100 + APO E
L!L
1
1
~4---~~
:
I
1
I
Lipoprotein lipase
I 1
Intestine Liver (nascent)
,
IDL
METABOLIC PRODUCT
AND REVERSE CHOLESTEROL TRANSPORT
11
t
+
..
I I
Intestine Absorbed dietary fats
SITE
I
ENDOGENOUS
EXOGENOUS
II
Ii
, ,
I :,1
1
I 1
Penpheral Liver cholesterol A removal-J-
1 I
L ________ -.J
Fig. 2. Pathways of lipid/lipoprotein metabolism. APO, Apolipoprotein; VLDL, very low-density lipoprotein; FFA, free fatty acids; IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein; HDL, high-density lipoprotein; LCAT, lecithin-cholesterol acyltransferase.
values increased 24.8% to 29.9%. Apolipoprotein Al was also elevated, with the greatest increase occurring with the ethynodiol diacetate preparation (19.3%) and the least with the levonorgestrel preparation (3.3%). The ratios of apolipoprotein B/apolipoprotein AI and high-density lipoproteinltotal cholesterol differed from baseline ratios, but as with most of the other parameters, did not differ among the various treatment groups. These results are very similar to those of the present study (Tables III and VI). The proatherogenic changes, elevation of lowdensity lipoprotein cholesterol and lowering of the high-density lipoprotein component, are said to be caused by progestins. 14 Dorflinger" and Powell et al. 6 have suggested that levonorgestrel, which is a more "potent" progestin, has a greater potential for decreasing high-density lipoprotein cholesterol than does norethindrone or ethynodiol diacetate. This suggestion does not take into account the fact that the clinical dose of levonorgestrel is one tenth that of norethindrone. Thus the presumed greater effect on high-density lipoprotein was not confirmed in our study. On the contrary, both preparations displayed a similar trend in all
of the factors measured, and there was no significant difference between the two. Powell et al. 16 compared five oral contraceptives (100 I-lg mestranol + 0.50 mg ethynodiol diacetate; 100 I-lg mestranol + 1.0 mg ethynodiol diacetate; 50 I-lg ethinyl estradiol + 1 mg ethynodiol diacetate; 30 I-lg ethinyl estradiol + 2 mg ethynodiol diacetate; and 30 I-lg ethinyl estradiol + 0.15 mg levonorgestrel). The study, characteristic of many others in the literature, was of only 3 months' duration. The investigators found that whereas all the trial formulations had similar effects on total triglycerides, total cholesterol, and LDL cholesterol, high-density lipoprotein cholesterol decreased 6.3% with an ethinyl estradiollethynodiol diacetate preparation versus 12% with the ethinyl estradiol/levonorgestrel-containing regimen. However, examination of the study data suggests possible flaws in both data interpretation and patient selection. The actual 3-month high-density lipoprotein values were 57.6 ± 1.7 and 54.0 ± 1.6 mg/dl, respectively, a difference unlikely to have a meaningful clinical impact. When individual values are compared, decreases occurred in 20 women in each treatment group composed of a total
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Lipid /lipoprotein changes with the use of triphasic OCs
of 28 subjects taking ethynodiol diacetate and 26 patients taking levonorgestrel. Furthermore. eight of the levonorgestrel-treated women had baseline highdensity lipoprotein values below the fiftieth percentile compared for age versus none in the ethynodiol diacetate-treated group. When our data are examined. the levonorgestrel oral contraceptive could similarly be considered potentially more atherogenic insofar as low-density lipoprotein cholesterol increased 16% at 1 year versus 11 % with the norethindrone preparation. However, the I-year values were 115.4 ± 6.0 mg/ dl (Ievonorgestrel) versus 114.4 ± 4.9 mg/dl (norethindrone). with respective increases over baseline of 18.4 ± 5.4 versus 12.7 ± 5.8 mg/dl. Given the complex interplay between lipids, lipoproteins, and their protein components and lipolytic enzymes, caution is appropriate in attempting to extrapolate a statistical change to a potential adverse clinical event. As noted with sex steroid changes in the coagulation/anticoagulation profile, alterations in lipid values should be interpreted within the context of a normal reference range. This issue was addressed by Kraus et al.,17 who noted that a 7-week course of 30 f.lg ethinyl estradiol + 0.3 mg norgestrel lowered highdensity lipoprotein cholesterol from 46 to 44 mg/dl, whereas 35 f.lg ethinyl estradiol + 0.4 mg norethindrone increased high-density lipoprotein cholesterol from 46 to 51 mg/dl. A similar result was seen in the high-density lipoprotein" subclass: values decreased with the norgestrel oral contraceptive (106 to 98 mg/dl) and increased with the norethindrone preparation (119 to 123 mg/dl) (reference range for the laboratory, 33 to 109 mg/dl for high-density lipoprotein cholesterol; 79 to 165 mg / dl for the high-density lipoprotein"" subfraction). Commenting on the potential atherogenic risk of the norgestrel contraceptive, the authors noted that "none of the resultant values were outside of the reference ranges. and none were sufficiently high to result in a higher than average risk for coronary disease on the basis of existing epidemiologic data."17
The Framingham study identified high-density lipoprotein cholesterol as having a strong inverse relationship with coronary heart disease l"; the higher the high-density lipoprotein cholesterol level. the lower the incidence of coronary heart disease independent of low-density lipoprotein and total cholesterol. Examination of the various subfractions of high-density lipoprotein cholesterol suggests that high-density lipoprotein 2 is considered mainly responsible for this cardioprotective effect. Therefore the significant and progressive decrease in high-density lipoprotein" cholesterol levels in both our treated populations (Table IV) was discouraging. Although there was no statistically significant difference between the two groups, the high-density lipoprotein" decreased to a greater extent in the ethinyl estradioillevonorgestrel users (47% versus 40%). The actual 12-month values recorded (10.7 ± 0.7 mg/dl for the ethinyl estradiollievonorgestrel group and 12.4 ± 1.4 mg/dl for those treated with ethinyl estradiollnorethindrone), were below our laboratory's reference range and significantly different from the values for the control group. Conversely, the high-density lipoprotein cholesterol values were increased both at the 6- and 12-month intervals. The reasons for these changes are not known, but they may be the result of the effect of progestins on hepatic lipase activity, inhibiting the resynthesis of high-density lipoprotein" to high-density lipoprotein,. This effect appears to be mitigated by lower doses of progestin, whereas the use of estrogen alone usually results in an increase in high-density lipoprotein". This premise was illustrated by our evaluation of the 6-month effect of a low-dose combination oral contraceptive (35 f.lg ethinyl estradiol, 0.4 mg norethindrone) in healthy, .older premenopausal women (mean age, 38 yean). Using a similar lipid/lipoprotein analysis, we found no change in total high-density lipoprotein cholesterol levels, but a 12% decrease in the high-density lipoprotein2 subfraction. The baseline and 6-month high-density lipoprotein cholesterol values were 52.7 ± 12.0 and 52.7 ± 11.4 mg/dl. The respective values for higndensity lipoprotein 2" were 45.6 ± 17.3 and 40.5 ± 16.0 mg-/dl. 20 An unexpected change in these patients'lipid profile was an increase in the apolipoprotein Al and B values, also observed by others. 13 "I This change raises the issue of qualitative versus quantitative changes in the various lipid fractions and how closely these measured parameters may reflect events in the vascular intima. Thus the increase in apolipoprotein Al could be indicative of a more "efficient" transport system that compensates for the reduced high-density lipoprotein" lipoprotein levels, with little net change in overall effect. The reasons for the increase in apolipoprotein B levels and their biologic impact are uncertain. However, the values attained at both 6 and 12 months were significantly
A useful guide to the interpretation of the significance of oral contraceptive-induced lipid changes is the population-based lipidllipoprotein reference study published by Knopp et al. I" According to their data for young women, the presence and severity of hyperlipidemia, with high risk of atherosclerosis, are determined by total cholesterol and low-density lipoprotein cholesterol values in excess of the ninetieth to ninetyfifth percentile (212 to 215 and 139 to 151 mg / dl, respectively) and by plasma high-density lipoprotein cholesterol levels below the fifth to tenth percentile (35 to 38 mg/dl). These hyperlipidemic values may be indicators of an underlying genetic predisposition. The values obtained in our study were all well within the suggested normal reference ranges.
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greater than those in the control group, with greater increases in ethinyl estradioillevonorgestrei-treated women. Unfortunately, we were unable to allocate the apolipoprotein B into its B I 00 and B48 components. This could be important, since apolipoprotein B 100 plays a major role in the cell surface receptor binding and endocytosis of low-density lipoprotein cholesterol. Epidemiologic studies have shown that ratios of proatherogenic and antiatherogenic lipidsllipoproteins are better predictors of atherogenic disease than individual lipid parameters. As with alterations in the coagulation/anticoagulation cascade, the "dynamic balance" among these factors may allow the clinician a clearer understanding of the actual impact of these changes on the cardiovascular system. Three ratios were calculated in the present study. Although the cholesterollhigh-density lipoprotein ratio showed a progressive increase in both oral contraceptive groups. the end point ratios (3.8-levonorgestrel group and 3.6norethindrone group) were still within the normal reference range (2.5 to 5.6 Ib ) and were not significantly different from those for the control group, and they did not differ significantly between groups. The pattern was the same for the high-density lipoproteinllowdensity lipoprotein ratio. Since the apolipoprotein B levels increased to a greater extent in the ethinyl estradiollievonorgestrel users, the apolipoprotein Al apolipoprotein B ratio in this group was significantly reduced at 1 year from the baseline value; however, once again, the ratio did not differ significantly from the control group. nor was it significantly different between groups. The significance of the minimal changes in these dynamic ratios is unclear. but they appear to reflect less of a clinical impact than might have been inferred from some of the greater changes observed in the individual lipid and lipoprotein fractions. Most studies evaluating combination oral contraceptives have demonstrated some "unfavorable" lipid Ilipoprotein metabolic effect. Yet after almost 30 years of oral contraceptive use, only one published study-a study conducted with preparations containing estrogen and progestin dosages three to four times higher than those in current use-suggests a possible atherogenic potential resulting from the long-term use of these agents. 22 How then can one reconcile the findings of such studies as the Bogalusa Heart study, which correlated changes in lipid and lipoprotein levels in children, to later atherosclerotic disease or the numerous epidemiologic studies indicating that even a minimal reduction of cholesterol levels can significantly decrease the morbidity and mortality from coronary heart disease and stroke? The difference may lie in the underlying cause of the hyperlipidemia. its degree. and the presence (or absence) of other compensatory (and negating) cardioprotective mechanisms. For example. Adams et al./ I evaluating the effect of contraceptive
May 1989
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J Ob.tct Gvnecol
steroids on coronary atherosclerosis in cynomolgus macaques demonstrated that oral contraceptives containing ethinyl estradiollnorgestrel resulted in marked reductions in total high-density lipoprotein cholesterol and its high-density lipoprotein, subfractions (similar to those reported in this article). However. the prevalence of coronary atherosclerosis was unaffected in one study group, whereas in another the degree of plaque formation was actually reduced. By contrast, 17-f3-estradiol and norgestrel administered by intravaginal ring were associated with an increased extent of coronary artery disease (plaque size). These studies suggest that the more potent oral estrogen induces an environment in the vascular intima that protects against the atherogenic effect of progestininduced alterations in the lipidllipoprotein milieu. In this broad context the alterations in the plasma, lipids, and lipoproteins demonstrated in this and other studies should be interpreted by the clinician. Other factors must also be taken into account, including inherent individual potential for atherogenic disease (family history, life-style, diet, and exercise. and smoking habits) and the variable individual metabolic response to oral contraceptives. 2I Moreover, a clearer distinction must be drawn between the risk for myocardial infarction caused by lipogenic mechanisms and those caused by thrombotic mechanisms and current risk versus that resulting from the use of preparations no longer available to the American public. New progestins with less potential for lipid alteration are now available in some countries2 ; and may be used by women with a higher risk for cardiovascular disease. Irrespective of the preparations used, high-risk subjects should be screened and monitored with routinely available lipid and lipoprotein assays, including plasma cholesterol. triglycerides, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol (calculated). Apart from absolute values, the ratios of cholesterollhighdensity lipoprotein cholesterol and high-density lipoprotein/low-density lipoprotein cholesterol should be interpreted and used as a clinical guide. Although some of the lipid and lipoprotein changes that resulted from the long-term use of the two tested triphasic oral contraceptives cannot be explained. these two preparations appear to have a similar effect on lipid metabolism. Neither induced significant changes that could enhance the long-term development of atherogenic disease. Parallel studies conducted on carbohydrate metabolism (glucose tolerance and plasma insulin response). coagulation and anticoagulation (prothrombin time, partial thromboplastin time. fibrinogen. antithrombin III antigen and activity, plasminogen antigen and activity, and 0:2 antiplasmin activitv), and blood pressure and body weight revealed minimal changes in the two treatment groups, equivalent to changes noted in the control group (data to be pub-
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lished separately). The findings of the present studv thus endorse the relative cardiovascular safety of the two oral contraceptive preparations tested at least in normolipidemic women. In conclusion, much of todav's concern about the impact of oral contraceptives on lipids and lipoproteins has emerged from studies conducted with higher dose preparations and interpretation of these data must be viewed with caution. Some investigators have failed to control for the length of exposure to the oral contraceptive being studied or to standardize for the time of blood sampling. Many studies have been cross sectional and involved relatively few subjects. Onlv a few studies have included a parallel group of matched women not taking oral contraceptives. In addition, earlier investigations based on the use of high- or medium-dose estrogen/progestin preparations are no longer applicable. The present study is the first to evaluate the effects of two commonly used, low-dose triphasic formulations on lipid patterns in a prospective, comparative, controlled fashion. The trial preparations contained comparable dosages of the same estrogen, ethinyl estradiol, but different progestins. The subjects were assigned in a randomized manner to either treatment group and were seen longitudinally for 1 year. We standardized the time of blood sampling and took measures to minimize differences in life-style and other influences on lipid metabolism among the three groups. Laboratory personnel were blinded to the subjects' identities and treatment. Because of the complexity of lipid/lipoprotein metabolism, numerous proatherogenic and antiatherogenic parameters were assayed in an attempt to prevent the formulation of clinical conclusions on the basis of one or two potentially adverse metabolic changes. For this reason, emphasis was placed on the importance of the dynamic balance between the measured lipid/lipoprotein assays and the clinical interpretation of lipid values against the template of a normal reference range. Results of the study demonstrate that both trial preparations (Triphasil and Ortho-Novum 71717) altered the lipid and lipoprotein milieu of otherwise normal women. However, effects of the two formulations appear to be similar and in the context described probably of minimal clinical significance. Any contribution to increased atherogenesis by either formulation is highly unlikely. Obviously, at least 30 vears of follow-up will be needed before a definitive conclusion can be reached. Since the individual metabolic response to an oral contraceptive varies and the long-term effect of oral contraceptives regarding atherogenesis are still to be defined, high-risk women taking oral contraceptives (including the low-dose, triphasic oral contraceptives) should be monitored with annual lipid and lipoprotein profiles.
We thank Yvonne Suggs, RN, l'\ancv Packard, RN, and Betty Russell, for their assistance.
REFERENCES I. Henderson BE. Ross RK. Paganini-Hill A. :\1ack T:\1. Estrog-en use and cardiovascular disease. A~(.J OIl~TET C;\NECOI 1986: 154: 1181. 2. Ro} al College of General Practltioner~. Further analvsi, of mortalit} in oral contraceptive users. Lancet 1981: i:541. 3. Bradlev DD, Wingerd .J. PetittI DB. Krauss RM. Ramcharan S. Serum hig-h-density-lipoprotein cholesterol in women using- oral contraceptives. e,trogens dnd progestins. N Engl J Med 1978:229: 17. 4. Allain CC. Poon LS, Chan CSG. Richmond W. Fu Pc. Enzymatic determination of total serum cholesterol. Ciin Chern 1974;20:470. 5. Wahlefeld AW. Methods of enzymatic analysis. In: Berg-meyer HV, ed. Triglvcerides. Gilford svstems. New York: Academic Press. 1974:1831. 6. Finley PR, Schlfman RB. Williams RI. Lichti DA. HDL. Gilford Systems. Clin Chern 1978:24:931. 7. American Heart Association special report. Recommendations for treatment of hyperlipidemia in adults. AJomt statement of the Nutntion Committee and the Council on Artenosclerosis. Prepared bv the Ad Hoc Committee to Design a Dietary Treatment of Hvperlipoproteinemia. Circulation 1984;69: 1065A-90A. 8. Kostner GM. Enzvmatlc determination of cholesterol in high-density-lipoprotein fractions prepared by polvanion precipitation [Letter]. ClIn Chem 1976:22: 695. 9. Friedewald W. Levv R. FredlICl<.son D. Estimation of the concentration of I~w-density-lipoprotem cholesterol in plasma without the use of the preparative ultracentrifuge. Clin Chem 1972:18:499. 10. Grow TE. Fried M. Interchange of apolipoprotein components between the human plasma high-densItvlipoprotein subclasses HDL, and HDL, in vitamins . .J Bioi Chem 1978:253:8034. II. Durrington PN. Ishola M, Hunt L. et al. Apolipoprotein (a). AI. and B and parental histoq in men with early onset ischaemic heart disease. Lancet 1988:t:1070. 12. Brown MS, Goldstein JL. How LDL receptor5 influence cholesterol and atherosclerosis. Sci Am 1984:251 :58. 13. Burkman RT. Robinson C. Kruszon-:'.1oran D. et al. Lipid and lipoprotem changes associated with oral contraceptive use: a randomized clinical trial Ob,tet Gvnecol 1988: 71:33. 14. Fotherby K. Oral contraceptives. lipids and cardiovascular disease [Review]. Contraception 1985:31 :367. 15. Dorflinger LJ. Relative potency of progestim used in oral contraceptives. Contraception 1985:31 :557. 16. Powell MG, Hedlin A. Cerskus I. et al. Effects of oral contraceptives on lipoprotein lIpids: a prospective study. Obstet Gvnecol 1984;63:764. 17. Krauss R'M. Rov S. Mlshell DR, et al. Effects of two lowdose oral contniceptives on serum lipids and lipoproteins: differential changes in high-density-lipoprotein subclasses. A~( .I OBSTET (;\,:--ECOL 1983: 145:446. 18. Knopp RH. Bergelin RO. Wahl PW, et al. Population based lipoprotein lipid reference values for pregnant women compared to non-pregnant women classified b\ sex hormone usage. A~( J OIl~rE'l C;\':\[COI. 1982:14:l: 620. 19. Castelli WI', Garrison RJ. Wilson PWF. et al. Incidence of coronary heart disease and lipoprotein cholesterol levels: the Frammgham Studv. .lAMA 1986:256:2835 20. Notelovitz M Cnpublished data. 21. Lipson A, Sto\ DB, LaRosaJC, et al. Progestins and oralcontraceptive-induced lipoprotein changes: a prospectIve study. Contraception 1986:34: 121.
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22. Slone D, Shapiro S, Kaufman DW, et al. Risk of myocardial infarction in relation to current and discontinued use of oral contraceptives. N Engl J Med 1981 ;305:420. 23. Adams MR, Clarkson TB, Koritnik DR, et al. Contraceptive steroids and coronary atherosclerosis in cynomolgus macaques. Fertil Steril 1987;47:1010. 24. Goldzieher JW, Chenault CB. Scientific exhibit. Pre-
May 1989 Am J Obstet Gynecol
sented at the Annual Meeting of the American College of Obstetricians and Gynecologists, April 24-30, 1987, Las Vegas, Nevada. 25. Marz W, Gross W, Gahn G, et al. A randomized crossover comparison of two low-dose contraceptives: effects on serum lipids and lipoproteins. AM J OBSTET GYNECOL 1985; 153:287-93.
From menarche to menopause: Coronary artery atherosclerosis and protection in cynomolgus monkeys Thomas B. Clarkson, DVM, Michael R. Adams, DVM, Jay R. Kaplan, PhD, Carol A. Shively, PhD, and Donald R. Koritnik, PhD Winston-Salem, North Carolina The effects on atherogenesis of stress, pregnancy, and oral contraceptive therapy were studied in a nonhuman primate model. The stress of social subordination was associated with ovarian dysfunction, unfavorable lipoprotein changes, and increased coronary artery atherosclerosis compared with nonstressed (socially dominant) or normal monkeys. Although pregnant animals exhibited lower high-density lipoprotein cholesterol concentrations, they had only one half as much diet-induced coronary artery atherosclerosis as their nonpregnant counterparts. Monkeys treated with an Ovral-like regimen also exhibited adverse lipoprotein changes. Nevertheless, prevalence and extent of coronary artery plaques decreased. We conclude that estrogen is an important factor in the animals' "female protection" against diet-induced atherosclerosis. We also suggest that the lowering of high-density lipoproteins by the progestin component of higher-dose contraceptives is not necessarily atherogenic if a sufficiently potent exogenous estrogen is administered concomitantly. (AM J OBSTET GVNECOL 1989;160:1280-5.)
Key words: Atherosclerosis, cholesterol, estrogen, stress, oral contraceptives, Macaca Jascicularis
In certain races and societies, premenopausal women enjoy a degree of protection from coronary artery atherosclerosis relative to their male counterparts. Explanations suggested for this male-female difference include gender differences in plasma lipoprotein concentrations (particularly in high-density lipoprotein cholesterol), a protective effect of estrogens, and the possibility that females are spared the stress and pathophysiologic effects of the competitive and sometimes hostile behavior of males. Progress in understanding this relative protection of female human beings has been slow to develop because until recently there was not a suitable animal model for research. About a decade ago, we developed such a nonhuman primate From the Arteriosderosls Re.learch Center, Bowman Gray School of Medlcme, Wake Fore.lt Unwenltv. Supported In pari Ii.v SCOR lIZ Arle";OIderosls Grant HL-14164 jrom the National Heart, Lung, and Blood lmtitute and Conllact No. NOI-HD-32800 from the Nat1Onal/n.ltltute of Child Health and Human Development Reprmt request:,: Thomas B. ClarklOn, DVM, Departmmt oj ComparatIVe Medlcllle. Bowman Gray School oj Medilllle. 300 S. Hawthorne Rd .. Wln.lton-Salem, NC 27103.
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model in which a series of investigations was conducted on the atherogenic and/ or protective effects of stress, pregnancy, and treatment with contraceptive steroids.
Development of model and methods for quantifying coronary artery atherosclerosis Study 1. We first investigated the cynomolgus macaque (Macaca Jascicularzs) because females of that species have menstrual cycles like those of women."" A 16month study comparing the effects of a moderately atherogenic diet on these animals subsequently demonstrated male-female differences similar to those of human beings. I Although both male and premenopausal female cynomolgus monkeys developed comparable total plasma cholesterol concentrations in response to the cholesterol-containing diet, the females, like human females, exhibited higher high-density lipoprotein cholesterol concentrations than did males. Thus there were important gender differences in the ratio of total plasma cholesterol and high-density lipoprotein cholesterol concentrations. Most important was our finding that coronary artery atherosclerosis was
Key words: Atherogenesis, ethinyl estradiol, levonorgestrel. lipid metabolism. lipoprotein, norethindrone, triphasic oral contraceptives
Premenopausal women are relatively resistant to atherogenic disease compared with men, an advantage that is greatly diminished in the postmenopausal years. Although the pathogenesis of this change has not been definitively established, the decrease in ovarian estrogen steroidogenesis and consequent changes in lipids and lipoproteins are thought to playa major causative role. Evidence is increasing that therapy with exogenous estrogen in menopausal women may reverse this trend and that women who have taken long-term estrogen therapy actually have a lower incidence of cardiovascular disease than their untreated peers. I This experience is contrary to that observed with the early use of exogenous sex steroids for oral contraception in younger women. The reported increase in oral contraceptive-related cardiovascular risk was attributed to an estrogen-dependent alteration in the coagulation/anticoagulation profile, predisposing women to an increased risk of thrombosis and thromboembolic phenomena." More recently, progestins have been associated with changes in lipid and lipoprotein metabolism that theoretically could accelerate atherogenesis. 3
From the Center for Climactenc Studies and Women's MedIcal and Dzagnostlc Center," Georgza Institute of Human NutntlOn, MedIcal College of Georgza, Augusta,' and the Department of Pathology, University of Flonda College of Medlcme' Repnnt requests: Morns Notelovltz, MD, PhD. Women's Medical and Diagno;tlc Center. 222 S. W. 36th Terrace, SUite C. GainesVille. FL 32607.
Although there is no clinical evidence that atherogenic disease in women has generally increased since the introduction of oral contraceptives in 1960, efforts have been taken to ensure that the premenopausal protection against cardiovascular disease is not compromised by these agents. The quantities of both estrogen and progestin have gradually been reduced and their ratios altered. This has culminated in the development of the triphasic oral contraceptives. Because of the relative paucity of well-controlled, prospective studies evaluating the metabolic effects of long-term use of the triphasic preparations, a study was initiated to compare the effects of two such regimens over a 12-month period. The trial formulations contained the same estrogen-ethinyl estradiol, but different progestins-Ievonorgestrel (Triphasil, Wyeth Laboratories, Philadelphia, Pa.) or norethindrone (Ortho-Novum 7/7/7, Ortho Pharmaceuticals, Raritan. N.J.). This article compares alterations in plasma lipids and lipoproteins after use of both contraceptive agents to observations in a group of non treated control subjects. It also attempts to place observed biochemical alterations in clinical perspective.
Material and methods Subjects. Eighty volunteers, ranging in age from 21 to 35 years. were enrolled in the study. Subjects were in good physical health, within 10% of desirable body weight, and at least 90 days' postpregnancy. They had
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Table I. Trial formulations Triphasil Phase 1 Phase 2 Phase 3 Ortho-Novum 71717 Phase I Phase 2 Phase 3
No. of days
Plogeltlll
6
5 10
Levonorgestrel Levonorge~t rei Levonorgestrel
7 7 7
Noretlundrone Norethindrone Norethmdrone
regular menstrual cycles, limited alcohol consumption, and smoking, and no contraindication to oral contraceptive usage. Contraindications included thrombophlebitis or thromboembolic disorders (past or present), cerebral vascular or coronary artery disease, and previous estrogen-dependent tumors. All women were screened before they entered the study and had a complete history, full physical examination, and blood pressure recording, a Papanicolaou smear, and hematologic and biochemical profiles. The laboratory tests included renal and liver function tests, serum electrolytes (SMA-25), hemoglobin, hematocrit, and white blood cell count. Lipid parameters were measured at pretreatment and after six and 12 cycles of treatment. Participants again received a full physical examination, Papanicolaou smear, and blood chemistry analysis 4 weeks after the twelfth cycle of therapy. Thirty-eight women were randomized into group I and received the levonorgestrel-containing oral contraceptive (Triphasil), and 42 were assigned to group II and received the norethindrone-containing formulation (Ortho-Novum 71717). The control group was made up of 14 female volunteers who met the same inclusion criteria and who were either surgically &terile with intact ovaries or used a nonhormonal contraceptive method. Laboratory personnel were blinded to the subjects' medication assignment. Material. The formulations of the two oral contraceptives are shown in Table I. Participants were instructed to take the contraceptive pills in cycles of 3 weeks on and 1 week off for 12 months. Treatment was started after blood chemistries and definitive serum lipid and lipoprotein studies were performed. Each subject was given a diary card on which to record medication, withdrawal and intermenstrual bleeding, and side effects. Care was taken to ensure that concomitant medicatiom such as aspirin-containing drugs, antiprostaglandins, and antibiotics were not taken during the study without the knowledge of the investigator. Life-style and other lipid influences on metabolism were reduced
E.ltrogen
JLg
50 75 125
Ethinyl estradiol Ethinyl estradiol Ethinyl estradiol
30 40 30
500 750 1000
Ethinyl estradiol Ethinyl estradiol Ethinyl estradiol
35 35 35
to a minimum and were considered equivalent in all three groups. Laboratory methods. Baseline and subsequent blood samples were obtained between days 20 to 25 (luteal phase) of the pretreatment cycle and treatment cycles. Subjects were instructed to fast for 12 to 24 hours overnight and had a 30 ml venous sample of blood taken between 8:00 and 9:00 AM the following morning. The sample was divided into two aliquots and allowed to clot at room temperature for 1 hour. The portion used for assay of cholesterol, triglycerides, low-density lipoprotein, and high-density lipoprotein was placed in tubes containing ethylenediaminetetra-acetic acid and centrifuged at 2000 rpm for 15 minutes. It was then frozen at 4° C and shipped for analysis the same day via air express. The aliquot used for apolipoprotein analysis was centrifuged at 1000 rpm for 10 minutes; the serum was separated from the clot and transported on dry ice to the lipid laboratories where the sample was stored at - 20° C until analysis. Total cholesterol; triglycerides,' and high-density lipoprotein cholesterol6 levels were determined by automated enzymatic procedures. The results were reported as percentiles according to the Lipid Research Clinic's reference values for women of the same age group.' High-density lipoprotein cholesterol was determined in the supernatant after precipitation of very low-density lipoprotein and low-density lipoprotein." Low-density lipoprotein cholesterol was calculated by means of the Friedewald equation. 9 The high-density lipoprotein 2 and high-density lipoprotein, subfraction separations were carried out by a modification of the procedure described by Grow and Fried. lo Apolipoproteins AI and B were measured by means of an immunonephelometric assay with diagnostic kits provided by Hyland Diagnostics (Deerfield, Ill.). Statistical analysis. Statistically significant (p < 0.05) within-group changes from baseline were determined by the paired t test. Statistically significant (P < 0.05) differences between the treatment groups were determined by analysis of covariance, with the baseline level taken as the covariate.
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Table II. Demographic data of the treatment and control groups No. Age (yr) Parity Height (inches) Weight (pounds) Baseline 12 mo Blood pressure (mm Hg) Baseline 12 mo Cigarettes (per day) Baseline 12 mo Alcohol (drinks/wk) Baseline 12 mo
Group I. EE/LNg
Group II. EE/NE
ControLI
29 25.4 ± 0.7 0.9 ± 0.2 62.6 ± 2.1
33 25.7 ± 0.7 0.9 ± 0.2 65.7 ± 0.6
II 26.9 ± 1.2 1.4 ± 0.5 65.2 ± 0.7
129.9 ± 3.2 131.0 ± 3.2
132.7 ± 3.0 133.7 ± 3.1
134.0 ± 3.7 134.1 ± 3.1
107 ± 1.7/65 ± 1.4 105 ± 2.0/63 ± 1.7
106 ± 1.4/64 ± 1.4 107 ± 1.6/67 ± 1.6
105 ± 2.4/66 ± 1.7 104 ± 2.9/66 ± 2.9
0.4 ± 0.4 1.0 ± 0.6
0.5 ± 0.4 0.9 ± 0.6
o o
1.7 ± 0.3 1.9 ± 0.3
1.6 ± 0.4 1.9 ± 0.4
0.7 ± 0.3 0.6 ± 0.2
Values expressed as means ± SEM. EE / LNg, Ethinvl estradiolllevonorgestrel (Triphasil); EE / NE, ethinyl estradiolllevonorgestrel (Ortho-Novum 7/7/7).
Table III. Change in serum cholesterol and triglycerides in normal control subjects and women on two low-dose, triphasic contraceptives Total cholesterol (mg/dl) No. Baseline 6 mo Mean change % change No. Baseline 12 mo Mean change % change Triglycerides (mg/dl) No. Baseline 6 mo Mean change % change No. Baseline 12 mo Mean change % change
Control
Grout) I, EE/LNg
Group II, EE / NE
II 167.3 ± 10.4 164.1 ± 7.2 -3.2 ± 6.1 -2 Il 167.3 ± 10.4 162.5 ± 7.7 -4.7 ± 8.3 -3
29 159.3 ± 4.7 177.7 ± 5.4** 18.4 ± 5.0 + II 27 159.0 ± 5.0 178.0 ± 6.2*** 18.9 ± 4.9 + II
33 167.1 ± 4.6 182.0 ± 5.0* 14.9 ± 5.6 +8 30 166.9 ± 5.0 181.8 ± 6.0* 14.9:t 6.1 +8
29 85.6 ± 7.1 112.2 ± 7.7* 26.6 ± 10.5 +24 27 87.4 :t 7.5 98.3 :t 5.7t 11.0 ± 6.8 +11
33 86.2 :t 5.4 113.4 :t 7.5*** 27.2 ± 6.6 +24 30 86.8 :t 5.9 103.8 :t 6.1 tt* 17.0 ± 7.4 +16
II 68.8 ± 4.1 81.9 :t 11.6 13.1 ± ILl + 16 11
68.8 ± 4.1 65.5 :t 5.3 -3.4 ± 4.6 -5
Mean (:t SEM) pretreatment values compared with mean (:t SEM) values at 6 and 12 months. EE / LNg, Ethinyl estradiolllevonorgestrel (Triphasil); EE / NE, ethinyl estradiollnorethindrone (Ortho-Novum 7/7/7).
* **
*** = t, tt, ttt =
p < 0.05, < 0.01, < 0.001 when compared with baseline values in the same P < 0.05, < 0.01, < 0.001 when compared with the control group.
group.
Results Twenty-nine women in group I, 33 in group II, and II control subjects completed the study (Table II). Because of incomplete data, two subjects from group I and three from group II had to be dropped from the 12-month analysis. Demographic data shown in Table II demonstrated no significant differences among the study groups at baseline.
Plasma total cholesterol (Table III) increased at 6 months in response to treatment with both oral contraceptives and remained about the same over the next 6 months. Although the plasma cholesterol increases in users of both oral contraceptives was statisticallv significant when compared with baseline values, the levels were well within the normal range for our laboratory
1272 Notelovitz et al.
May 1989 Am J Obstet Gynecol
Table IV. Change in serum lipoproteins in normal control subjects and in women on two low-dose, oral triphasic contraceptives Control Total HDL cholesterol (mg/dl) No. Baseline 6 mo Mean change % change No. Baseline 12 mo Mean change % change HDL2 cholesterol (mg/dl) No. Baseline 6 mo Mean change % change No. Baseline 12 mo Mean change % change HDL, cholesterol (mg/dl) No. Baseline 6 mo Mean change % change No. Baseline 12 mo Mean change % change LDL cholesterol (mg/dl) No. Baseline 6 mo Mean change % change No. Baseline 12 mo Mean change % change
11 53.6 ± 3.9 56.9 ± 3.5 3.3 ± 2.2 +6
Group I, EE 1LNg 29 52.2 ± 2.3 51.7 ± 2.0 -0.5 ± 2.6
Group II, EE 1NE
II
27 52.4 ± 2.5 49.0 ± 2.0 -3.4 ± 2.5 -6
33 56.2 ± 2.4 57.0 ± 2.9 0.8 ± 3.0 +1 30 55.7 ± 2.4 53.4 ± 3.2 -2.3 ± 2.9 -4
II
29 18.8 ± 1.8 15.0 ± J.l -3.9 ± 2.0 -20 27 18.8 ± 1.8 10.0 ± 0.7ttt*** -8.8 ± 1.5 -47
33 21.4 ± 2.2 18.3 ± 1.9 -3.0 ± 2.9 -14 30 20.7 ± 2.2 12.4 ± l.4tt** -8.3 ± 2.4 -40
29 34.4 ± 1.6 36.7 ± 1.5 2.3 ± 1.7 +6 27 34.4 ± 1.7 39.0 ± Ui* 4.5 ± 2.0 + 12
33 34.8 ± 1.5 38.6 ± 1.6** 3.8 ± 1.3 +10 30 35.0 ± 1.6 41.0 ± 2.2** 6.0 ± 1.8 + 15
53.6 ± 3.9 55.1 ± 3.9 1.5 ± 3.0 +3 18.6 ± 3.0 21.8 ± 2.7 3.2 ± 3.5 + 15 11 18.6 ± 3.0 18.2 ± 2.8 -0.5 ± 2.7 -2 11
35.0 ± 2.9 35.1 ± 2.6 0.09 ± 3.4 NC 11 35.0 ± 2.9 36.9 ± 2.0 1.9 ± 2.7 +5 11 105.5 ± 9.7 97.4 ± 8.4 -8.2 ± 6.8 -8 II
105.5 ± 9.7 99.0 ± 6.4 -6.5 ± 8.6 -6
-I
29 97.6 ± 4.2 112.7 ± 5.3** 15.1 ± 4.2 + 13 27 97.0 ± 4.5 115.4 ± 6.0** 18.4 ± 5.4 + 16
33 101.3 ± 4.3 110.0 ± 4.2 8.7 ± 5.4 +8 30 101.7 ± 4.7 114.4 ± 4.9* 12.7 ± 5.8 +11
Mean (± SEM) pretreatment values compared with mean (± SEM) values at 6 and 12 months. NC, No change; EEILNg, ethinyl estradioi/levonorgestrel (Triphasil); EEINE, ethinyl estradiol/norethindrone (Ortho-Novum 7/717); HDL, high-density lipoprotein; LDL, low-density lipoprotein. * **. *** = p < 0.05, < 0.01, < 0.001 when compared with baseline values in the same group. t. tt. ttt = P < 0.05. < 0.01. < 0.001 when compared with the control group.
«200 mg/dl*). Moreover, they did not differ significantly from those of the control women at each test point, remaining within two standard errors of the control mean. There was no statistical or clinically significant difference between the two treatment groups. Plasma triglycerides (Table III) were predictably affected by the levonorgestrel- and norethindrone-based
oral contraceptives, which demonstrated very similar increases with both regimens at 6 months. These statistically significant alterations declined to lower levels at the end of 1 year but were still elevated over baseline values and were significantly greater than those found in the (ontrol group. However, the absolute concentrations of triglycerides in all study groups remained well within the normal range. .
*Unless otherwise noted. reference values are given as the seventy-fifth percentile of normal.
Total plasma high-density lipoprotein cholesterol levels (Table IV) were unaffected at 6 months and showed no significant decreases in either treatment
Lipid/lipoprotein changes with tKe use of triphasic OCs
Volume 160 Number 5, Part 2
1273
Table V. Change in apolipoprotein AI and apolipoprotein B in normal control subjects and women receiving two low-dose, oral triphasic contraceptives Control
Apolipoprotein AI (mg/dl) No. Baseline 6 mo Mean change % change No. Baseline 12 mo Mean change % change Apolipoprotein B (mg/dl) No. Baseline 6 mo Mean change % change No. Baseline 12 mo Mean change % change
Group /, EE/LNg
Group II, EE / NE
10
28 134.5 ± 4.9 166.5 ± 5.8t*** 32.0 ± 6.0 +19 26 133.2 ± 5.2 144.8 ± 8.4 11.6 ± 9.1 +8
32 140.8 ± 4.5 176.8 ± 6.7tt*** 35.9 ± 7.2 +20 30 140.2 ± 4.5 152.0 ± 8.3 11.9 ± 8.5 +8
10
28 77.6 ± 4.0 89.6 ± 4.4t* 12.0 ± 5.4 + 13 26 77.9 ± 4.3 97.2 ± 5.9tt** 19.3 ± 6.2 +20
32 75.6 ± 3.3 94.3 ± 3.6tt** 18.6 ± 5.1 +20 30 74.9 ± 3.4 87.1 ± 4.2t* 12.3 ± 4.8 +14
138.0 ± 10.7 142.9 ± 9.0 4.9 ± 9.0 +4 10 138.0 ± 10.7 130.3 ± 7.6 -7.7 ± 15.2 -6 73.3 ± 10.4 69.6 ± 5.7 -3.7 ± 10.3 -5 10 73.3 ± 10.4 65.2 ± 5.5 -8.1 ± 9.6 -ll
Mean (±SEM) pretreatment values compared with mean (±SEM) values at 6 and 12 months. EE / LNg, Ethinyl estradioillevonorgestrel (Triphasil); EE / NE, ethinyl estradiollnorethindrone (Ortho-Novum 71717).
* **. *** = p < 0.05, < 0.01, < 0.001 when compared with baseline values in the same group. t, tt, ttt = P < 0.05, < 0.01, < 0.001 when compared with the control group.
group at 12 months. These values did not differ statistically from pretreatment levels within each group or compared with values in the other treatment or control groups. By contrast, the high-density lipoprotein subfractions (Table IV) exhibited a significant and possibly clinically meaningful change. For high-density lipoprotein", the ethinyl estradiollievonorgestrel oral contraceptive users had a 20% and 47% reduction, respectively, in mean 6- and 12-month values compared with baseline values. The ethinyl estradiol/norethindrone-treated subjects had a fall of 14% and 40%, respectively. The 12-month values were significantly different both within groups (p < 0.001 and p < 0.0l) and compared with control subjects (p < 0.001 and p < 0.01) but were not significantly different between treatment groups. For high-density lipoprotein." these values were higher than pretreatment levels and slightly higher than the control group's levels, but the difference was not statistically significant. Paralleling the change in total cholesterol, the low-density lipoprotein cholesterol fraction (Table IV) increased at 6 months and maintained these elevated levels thereafter. Again, all the lipoprotein concentrations, with the exception of high-density lipoprotein" at 12 months, were within the clinically acceptable ranges (total highdensity lipoprotein cholesterol = 37 to 74 mg/dl;* *Tenth to seventieth percentiles.
high-density lipoprotein" cholesterol = 15 to 60 mg/ dl; high-density lipoprotein, cholesterol = 14 to 65 mg/dl; low-density lipoprotein cholesterol = <130 mg/dl). Apolipoprotein AI, the m~or protein component of high-density lipoprotein cholesterol, showed a significant increase at 6 months in both treated groups, with a subsequent decline in serum levels when assessed 6 months later (Table V). Values at 6 months in the treated women rose to the laboratory's upper range of normal (97 to 174 mg/dl) and in some instances exceeded it. These changes were highly significant (p < 0.001) both within groups and compared with the control group (p < 0.05 and <0.01). Apolipoprotein B, the apoprotein carrier for lowdensity lipoprotein cholesterol, showed similar elevations in both groups at six months. A further increase in the ethinyl estradiollievonorgestrel group (from 13% up to 20%) and a slight decline in the ethinyl estradiollnorethindrone group (from 20% down to 14%) were noted at 12 months. Although the net increases were statistically significant within groups and when compared with control subjects, these changes did not exceed the normal range (46 to 120 mg/dl). As with all the other lipid and lipoprotein parameters, there was no statistically significant difference between the two treatment groups. It is now customary to refer to ratios between the
1274 Notelovitz et al.
May 1989 Am J Obstet Gynecol
Table VI. Alteration in ratios of lipids and lipoproteins in normal controls and in women on two low-dose oral triphasic contraceptives Total cholesterollHDL cholesterol (mg/dl) Baseline 6 mo 12 mo HDLlLDL cholesterol Baseline 6 mo 12 mo Apolipoprotein AI/apolipoprotein B Baseline 6 mo 12 mo
Control
Group I, EE/LNg
3.2 ± 0.2 3.0 ± 0.2 3.1 ± 0.2
3.2 ± 0.1 3.6 ± 0.2* 3.8 ± 0.2t
3.1 ± 0.1 3.4 ± 0.2 3.6 ± 0.2*
0.6 ± 0.07 0.6 ± 0.08 0.6 ± 0.06
0.6 ± 0.04 0.5 ± 0.05 0.5 ± 0.06
0.6 ± 0.04 0.5 ± 0.03 0.5 ± 0.03
2.3 ± 0.4 2.2 ± 0.3 2.1 ± 0.1
1.9 ± 0.1 2.0 ± 0.1 1.6 ± 0.1 *
2.0 ± 0.1 1.9 ± 0.1 1.8 ± 0.1
Group II, EE / NE
Mean (±SEM) pretreatment values compared with mean (±SEM) values at 6 and 12 months. EE / LNg, Ethinyl estradiol/levonorgestrel (Triphasil); EE / NE, ethinyl estradiol/norethindrone (Ortho-Novum 7/7/7); HDL, high-density lipoprotein; LDL, low-density lipoprotein. *, t, * = P < 0.01. < 0.001, < 0.05 when compared with baseline values in the same group.
lipid, lipoprotein, and apoprotein factors, as reflected in Table VI. The ratio of total cholesterol to highdensity lipoprotein cholesterol, probably the most commonly used lipid ratio in clinical practice, underwent a progre~sive and statistically significant increase from baseline at both evaluation intervals in both treatment groups. The calculated ratio of high-density lipoprotein/low-density lipoprotein, cholesterol was unaffected over the course of therapy in either treatment group when baseline, 6- and 12-month values were compared and no statistically significant differences from control values were found. Results were similar with the protein ratios except that the decline in apolipoprotein AIIB ratio by 12 months found in all groups was statistically significant in the ethinyl estradiollievonorgestrel group, but not in the other two groups. No clinically significant changes in blood pressure were observed during the study, nor were statistically significant alterations in weight noted. Side effects were of the type usually associated with oral contraceptive use and similar for each treatment group. As reflected in Table VII, nine patients withdrew from each treatment group and three dropped out of the control group before completion of the study. One pregnancy occurred in the ethinyl estradiollievonorgestrel group because of method failure.
Comment Much confusion surrounds the interpretation of lipid and lipoprotein levels. The principles governing the transport and metabolism of these substances are complex and interdependent. In addition, laboratory values are often extrapolated to predict biologic significance without proper consideration of the complex factors
and unknowns involved. A brief review of these concepts may help place the present findings into a clearer practical perspective. Lipidllipoprotein metabolism: A brief overview. Lipids are essential components of cell membranes; they are basic to steroid synthesis and constitute a transportable metabolic energy pool. Lipoproteins serve to transport lipids and regulate their metabolism. The interaction between these substances is modulated by lipid enzymes. The "good" (antiatherogenic) and "bad" (atherogenic) lipid and lipoproteins are summarized in Fig. 1. The three major interacting pathways involved in their metabolism are shown in Fig. 2. Both the exogenous and endogenous pathways result in a net lipogenic effect. Exogenously, dietary fats are absorbed as chylomicrons and are catabolized via lipoprotein lipase, producing free fatty acids used for energy or stored in fat cells. Although chylomicron particles are not atherogenic, their partially catabolized remnants are. Endogenously, very low-density lipoprotein, the major lipoprotein produced in the liver, is hydrolyzed also via lipoprotein lipase into intermediate-density lipoprotein. Although a portion of intermediate-density lipoprotein is cleared by the liver, the remainder is transformed into the denser and more highly atherogenic fraction, low-density lipoprotein. Raised levels of intermediate-density lipoprotein cholesterol fractions are atherogenic, since they are rich in both cholesterol and triglycerides. Low-density lipoprotein cholesterol transports about two thirds of circulating cholesterol and thus is considered the most atherogenic of the lipoprotein particles. Counterbalancing the lipogenic effects of the exogenous and endogenous pathways is the antiatherogenic effect of the reverse cholesterol transport system. Na-
lipid/lipoprotein changes with the use of triphasic oes
Volume 160 I\"umber 5, Part 2
Table VII. Reasons for early withdrawal from study
THE "GOOD" Group II, EE/NE
Side effects of DC Breast discomfort Spotting Nausea Other contraception Headaches Medical reasons Asthma Incidental surgery Chronic vaginal infection Lost to follow-up/moved Miscellaneous Pregnancy Excessive smoking Religious reasons Total ~C,
AND
1275
THE "BAD" Cholesterol
HDL·C HDL2
LDL·C
APO·A,
APO·S
HDL3
Tnglycendes
~ 2
o
I
2 I I
8
Fig. 1. Lipid/ltpoprotein balance.
8
Oral contraceptives.
scent (new) high-density lipoprotein, which is synthesized in the liver and intestine in the form of high-density lipoproteinj, is converted into relatively cholesterol-rich high-density lipoprotein,. This in turn is reconverted into cholesterol-poor high-density lipoprotein., by hepatic lipase. The high-density lipoprotein lipoproteins, especially high-density lipoprotein~, are antiatherogenic and are involved in the transport of cholesterol from the peripheral tissues to the liver for excretion and conversion to bile acids. The protein components apolipoprotein AI and apolipoprotein A2 also participate in this antilipogenic process as activators of the enzymes lecithin-cholesterol acyltransferase and hepatic lipase, respectively. Apolipoproteins also identify cell receptor sites and bind to them and have emerged as important discriminators of the presence or absence of atherogenic disease. For example, in a recent studyll plasma concentrations of apolipoprotein B were predictive of 87% of patients with ischemic heart disease and 75% of control subjects compared with other accepted risk factors (family history, serum cholesterol, serum triglycerides, and high-density lipoprotein cholesterol), which correlated with only 61 % of patients and 69% of control subjects. When apolipoprotein B was combined with apolipoprotein AI and family history, identification was correct in 79% of patients and 83% of control subjects. Another interrelated and central factor in the lipidlipoprotein balance is the low-density lipoprotein receptor. It is known that high intracellular levels of cholesterol suppress the hepatic synthesis of low-density lipoprotein receptors. This in turn elevates low-density lipoprotein both through diminished uptake (and metabolism) and enhanced conversion of very low-density lipoprotein to low-density lipoprotein. 12 The significance of this vast multifactorial network
makes it unfeasible to assess atherogenic potential based on the evaluation of a single metabolic parameter. Epidemiologic studies have shown that ratios of proand antiatherogenic lipidsllipoproteins are better predictors of atherogenic disease. An imbalance or perturbation among the pro- and antiatherogenic factors may produce vascular intimal damage, which leads eventually to formation of an atheromatous plaque. Thus in the context of oral contraceptive-induced lipid changes, the clinician must assess the import of an increase in one atherogenic factor (e.g., raised plasma cholesterol) if a simultaneous antiatherogenic change (e.g., increased apolipoprotein AI) may provide "balance" in the overall drug-induced event. A shift in favor of the antiatherogenic or "good" side of the lipid equation is probably desirable, but the untoward or beneficial long-term effects of such changes induced by low-dose estrogenprogestin combination preparations are unknown. Oral contraceptive-induced changes in lipids and lipoproteins. A "perfect" oral contraceptive would neither increase levels of cholesterol and low-density lipoprotein cholesterol nor lower high-density lipoprotein cholesterol. In addition, the protein carriers (and their activity) would be unaffected. As reflected in a recent clinical trial and review article,13 II such an absence of effect is unlikely, since all estrogen- and progestin-containing formulations marketed in the United States have some influence on lipid and lipoprotein metabolism. Most recently, Burkman et al. Ll compared the effect of four low-dose oral contraceptives (ethinyl estradiol, 35 J..Lg + ethynodiol diacetate, 1 mg; ethinyl estradiol, 30 J..Lg + levonorgestrel, 0.05 mg: ethinyl estradiol, 35 J..Lg + norethindrone, 1 mg; and ethinyl estradiol, 35 J..Lg + norethindrone, 0.5 and 1 mg) over a 6-month period and found that total cholesterol increased 5.9% to 9.1 % above baseline values; triglyceride levels increased 37.6% to 45.3%; lowdensity lipoprotein cholesterol increased 10% to 15.6%; and (with the exception of the group using the ethynodiol diacetate-containing oral contraceptive) highdensity lipoprotein decreased 4.5% to 8.7%. The lipid protein carriers were also altered. Apolipoprotein B
1276
Notelovitz et al.
May 1989 Am J Obstct Gynccol
I-~DLSYNTHESIS --l
PATHWAY
Liver
Chylomicron
VLDL
LIPOPROTEIN COMPONENTS
Tnglycendes Cholesterol + APO 848 APOCII & III APOE
Tnglycendes Cholesterol + APO 8100, APO CII, III APOE
ENZYMES
LIPoprotleln lipase
LIPID
+
,
J
DISPOSITION
Energy + Fat cell storage
HD~
I AP~~II LJAT ,Lipoprotein
i'
---...r~;;-L;~----~.------- ~_~ ___ L ___ -1 I
Chylomicron Chylomicron + remnant FFA
Cholesterol APO 8100
r Liver
,
Penpheral cholesterol accumulation
I
IiP1se
,
Liver
I--
I
I
I
AP2'
'
~I,
l ~I~~~C
I I ,
!
I 1
1
I I
,HDL2
Cholesterol Tnglycendes APO 8100 + APO E
L!L
1
1
~4---~~
:
I
1
I
Lipoprotein lipase
I 1
Intestine Liver (nascent)
,
IDL
METABOLIC PRODUCT
AND REVERSE CHOLESTEROL TRANSPORT
11
t
+
..
I I
Intestine Absorbed dietary fats
SITE
I
ENDOGENOUS
EXOGENOUS
II
Ii
, ,
I :,1
1
I 1
Penpheral Liver cholesterol A removal-J-
1 I
L ________ -.J
Fig. 2. Pathways of lipid/lipoprotein metabolism. APO, Apolipoprotein; VLDL, very low-density lipoprotein; FFA, free fatty acids; IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein; HDL, high-density lipoprotein; LCAT, lecithin-cholesterol acyltransferase.
values increased 24.8% to 29.9%. Apolipoprotein Al was also elevated, with the greatest increase occurring with the ethynodiol diacetate preparation (19.3%) and the least with the levonorgestrel preparation (3.3%). The ratios of apolipoprotein B/apolipoprotein AI and high-density lipoproteinltotal cholesterol differed from baseline ratios, but as with most of the other parameters, did not differ among the various treatment groups. These results are very similar to those of the present study (Tables III and VI). The proatherogenic changes, elevation of lowdensity lipoprotein cholesterol and lowering of the high-density lipoprotein component, are said to be caused by progestins. 14 Dorflinger" and Powell et al. 6 have suggested that levonorgestrel, which is a more "potent" progestin, has a greater potential for decreasing high-density lipoprotein cholesterol than does norethindrone or ethynodiol diacetate. This suggestion does not take into account the fact that the clinical dose of levonorgestrel is one tenth that of norethindrone. Thus the presumed greater effect on high-density lipoprotein was not confirmed in our study. On the contrary, both preparations displayed a similar trend in all
of the factors measured, and there was no significant difference between the two. Powell et al. 16 compared five oral contraceptives (100 I-lg mestranol + 0.50 mg ethynodiol diacetate; 100 I-lg mestranol + 1.0 mg ethynodiol diacetate; 50 I-lg ethinyl estradiol + 1 mg ethynodiol diacetate; 30 I-lg ethinyl estradiol + 2 mg ethynodiol diacetate; and 30 I-lg ethinyl estradiol + 0.15 mg levonorgestrel). The study, characteristic of many others in the literature, was of only 3 months' duration. The investigators found that whereas all the trial formulations had similar effects on total triglycerides, total cholesterol, and LDL cholesterol, high-density lipoprotein cholesterol decreased 6.3% with an ethinyl estradiollethynodiol diacetate preparation versus 12% with the ethinyl estradiol/levonorgestrel-containing regimen. However, examination of the study data suggests possible flaws in both data interpretation and patient selection. The actual 3-month high-density lipoprotein values were 57.6 ± 1.7 and 54.0 ± 1.6 mg/dl, respectively, a difference unlikely to have a meaningful clinical impact. When individual values are compared, decreases occurred in 20 women in each treatment group composed of a total
Volume 160 "'umber 5. Part 2
Lipid /lipoprotein changes with the use of triphasic OCs
of 28 subjects taking ethynodiol diacetate and 26 patients taking levonorgestrel. Furthermore. eight of the levonorgestrel-treated women had baseline highdensity lipoprotein values below the fiftieth percentile compared for age versus none in the ethynodiol diacetate-treated group. When our data are examined. the levonorgestrel oral contraceptive could similarly be considered potentially more atherogenic insofar as low-density lipoprotein cholesterol increased 16% at 1 year versus 11 % with the norethindrone preparation. However, the I-year values were 115.4 ± 6.0 mg/ dl (Ievonorgestrel) versus 114.4 ± 4.9 mg/dl (norethindrone). with respective increases over baseline of 18.4 ± 5.4 versus 12.7 ± 5.8 mg/dl. Given the complex interplay between lipids, lipoproteins, and their protein components and lipolytic enzymes, caution is appropriate in attempting to extrapolate a statistical change to a potential adverse clinical event. As noted with sex steroid changes in the coagulation/anticoagulation profile, alterations in lipid values should be interpreted within the context of a normal reference range. This issue was addressed by Kraus et al.,17 who noted that a 7-week course of 30 f.lg ethinyl estradiol + 0.3 mg norgestrel lowered highdensity lipoprotein cholesterol from 46 to 44 mg/dl, whereas 35 f.lg ethinyl estradiol + 0.4 mg norethindrone increased high-density lipoprotein cholesterol from 46 to 51 mg/dl. A similar result was seen in the high-density lipoprotein" subclass: values decreased with the norgestrel oral contraceptive (106 to 98 mg/dl) and increased with the norethindrone preparation (119 to 123 mg/dl) (reference range for the laboratory, 33 to 109 mg/dl for high-density lipoprotein cholesterol; 79 to 165 mg / dl for the high-density lipoprotein"" subfraction). Commenting on the potential atherogenic risk of the norgestrel contraceptive, the authors noted that "none of the resultant values were outside of the reference ranges. and none were sufficiently high to result in a higher than average risk for coronary disease on the basis of existing epidemiologic data."17
The Framingham study identified high-density lipoprotein cholesterol as having a strong inverse relationship with coronary heart disease l"; the higher the high-density lipoprotein cholesterol level. the lower the incidence of coronary heart disease independent of low-density lipoprotein and total cholesterol. Examination of the various subfractions of high-density lipoprotein cholesterol suggests that high-density lipoprotein 2 is considered mainly responsible for this cardioprotective effect. Therefore the significant and progressive decrease in high-density lipoprotein" cholesterol levels in both our treated populations (Table IV) was discouraging. Although there was no statistically significant difference between the two groups, the high-density lipoprotein" decreased to a greater extent in the ethinyl estradioillevonorgestrel users (47% versus 40%). The actual 12-month values recorded (10.7 ± 0.7 mg/dl for the ethinyl estradiollievonorgestrel group and 12.4 ± 1.4 mg/dl for those treated with ethinyl estradiollnorethindrone), were below our laboratory's reference range and significantly different from the values for the control group. Conversely, the high-density lipoprotein cholesterol values were increased both at the 6- and 12-month intervals. The reasons for these changes are not known, but they may be the result of the effect of progestins on hepatic lipase activity, inhibiting the resynthesis of high-density lipoprotein" to high-density lipoprotein,. This effect appears to be mitigated by lower doses of progestin, whereas the use of estrogen alone usually results in an increase in high-density lipoprotein". This premise was illustrated by our evaluation of the 6-month effect of a low-dose combination oral contraceptive (35 f.lg ethinyl estradiol, 0.4 mg norethindrone) in healthy, .older premenopausal women (mean age, 38 yean). Using a similar lipid/lipoprotein analysis, we found no change in total high-density lipoprotein cholesterol levels, but a 12% decrease in the high-density lipoprotein2 subfraction. The baseline and 6-month high-density lipoprotein cholesterol values were 52.7 ± 12.0 and 52.7 ± 11.4 mg/dl. The respective values for higndensity lipoprotein 2" were 45.6 ± 17.3 and 40.5 ± 16.0 mg-/dl. 20 An unexpected change in these patients'lipid profile was an increase in the apolipoprotein Al and B values, also observed by others. 13 "I This change raises the issue of qualitative versus quantitative changes in the various lipid fractions and how closely these measured parameters may reflect events in the vascular intima. Thus the increase in apolipoprotein Al could be indicative of a more "efficient" transport system that compensates for the reduced high-density lipoprotein" lipoprotein levels, with little net change in overall effect. The reasons for the increase in apolipoprotein B levels and their biologic impact are uncertain. However, the values attained at both 6 and 12 months were significantly
A useful guide to the interpretation of the significance of oral contraceptive-induced lipid changes is the population-based lipidllipoprotein reference study published by Knopp et al. I" According to their data for young women, the presence and severity of hyperlipidemia, with high risk of atherosclerosis, are determined by total cholesterol and low-density lipoprotein cholesterol values in excess of the ninetieth to ninetyfifth percentile (212 to 215 and 139 to 151 mg / dl, respectively) and by plasma high-density lipoprotein cholesterol levels below the fifth to tenth percentile (35 to 38 mg/dl). These hyperlipidemic values may be indicators of an underlying genetic predisposition. The values obtained in our study were all well within the suggested normal reference ranges.
j
1277
1278
Notelovitz et al.
greater than those in the control group, with greater increases in ethinyl estradioillevonorgestrei-treated women. Unfortunately, we were unable to allocate the apolipoprotein B into its B I 00 and B48 components. This could be important, since apolipoprotein B 100 plays a major role in the cell surface receptor binding and endocytosis of low-density lipoprotein cholesterol. Epidemiologic studies have shown that ratios of proatherogenic and antiatherogenic lipidsllipoproteins are better predictors of atherogenic disease than individual lipid parameters. As with alterations in the coagulation/anticoagulation cascade, the "dynamic balance" among these factors may allow the clinician a clearer understanding of the actual impact of these changes on the cardiovascular system. Three ratios were calculated in the present study. Although the cholesterollhigh-density lipoprotein ratio showed a progressive increase in both oral contraceptive groups. the end point ratios (3.8-levonorgestrel group and 3.6norethindrone group) were still within the normal reference range (2.5 to 5.6 Ib ) and were not significantly different from those for the control group, and they did not differ significantly between groups. The pattern was the same for the high-density lipoproteinllowdensity lipoprotein ratio. Since the apolipoprotein B levels increased to a greater extent in the ethinyl estradiollievonorgestrel users, the apolipoprotein Al apolipoprotein B ratio in this group was significantly reduced at 1 year from the baseline value; however, once again, the ratio did not differ significantly from the control group. nor was it significantly different between groups. The significance of the minimal changes in these dynamic ratios is unclear. but they appear to reflect less of a clinical impact than might have been inferred from some of the greater changes observed in the individual lipid and lipoprotein fractions. Most studies evaluating combination oral contraceptives have demonstrated some "unfavorable" lipid Ilipoprotein metabolic effect. Yet after almost 30 years of oral contraceptive use, only one published study-a study conducted with preparations containing estrogen and progestin dosages three to four times higher than those in current use-suggests a possible atherogenic potential resulting from the long-term use of these agents. 22 How then can one reconcile the findings of such studies as the Bogalusa Heart study, which correlated changes in lipid and lipoprotein levels in children, to later atherosclerotic disease or the numerous epidemiologic studies indicating that even a minimal reduction of cholesterol levels can significantly decrease the morbidity and mortality from coronary heart disease and stroke? The difference may lie in the underlying cause of the hyperlipidemia. its degree. and the presence (or absence) of other compensatory (and negating) cardioprotective mechanisms. For example. Adams et al./ I evaluating the effect of contraceptive
May 1989
Am
J Ob.tct Gvnecol
steroids on coronary atherosclerosis in cynomolgus macaques demonstrated that oral contraceptives containing ethinyl estradiollnorgestrel resulted in marked reductions in total high-density lipoprotein cholesterol and its high-density lipoprotein, subfractions (similar to those reported in this article). However. the prevalence of coronary atherosclerosis was unaffected in one study group, whereas in another the degree of plaque formation was actually reduced. By contrast, 17-f3-estradiol and norgestrel administered by intravaginal ring were associated with an increased extent of coronary artery disease (plaque size). These studies suggest that the more potent oral estrogen induces an environment in the vascular intima that protects against the atherogenic effect of progestininduced alterations in the lipidllipoprotein milieu. In this broad context the alterations in the plasma, lipids, and lipoproteins demonstrated in this and other studies should be interpreted by the clinician. Other factors must also be taken into account, including inherent individual potential for atherogenic disease (family history, life-style, diet, and exercise. and smoking habits) and the variable individual metabolic response to oral contraceptives. 2I Moreover, a clearer distinction must be drawn between the risk for myocardial infarction caused by lipogenic mechanisms and those caused by thrombotic mechanisms and current risk versus that resulting from the use of preparations no longer available to the American public. New progestins with less potential for lipid alteration are now available in some countries2 ; and may be used by women with a higher risk for cardiovascular disease. Irrespective of the preparations used, high-risk subjects should be screened and monitored with routinely available lipid and lipoprotein assays, including plasma cholesterol. triglycerides, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol (calculated). Apart from absolute values, the ratios of cholesterollhighdensity lipoprotein cholesterol and high-density lipoprotein/low-density lipoprotein cholesterol should be interpreted and used as a clinical guide. Although some of the lipid and lipoprotein changes that resulted from the long-term use of the two tested triphasic oral contraceptives cannot be explained. these two preparations appear to have a similar effect on lipid metabolism. Neither induced significant changes that could enhance the long-term development of atherogenic disease. Parallel studies conducted on carbohydrate metabolism (glucose tolerance and plasma insulin response). coagulation and anticoagulation (prothrombin time, partial thromboplastin time. fibrinogen. antithrombin III antigen and activity, plasminogen antigen and activity, and 0:2 antiplasmin activitv), and blood pressure and body weight revealed minimal changes in the two treatment groups, equivalent to changes noted in the control group (data to be pub-
Volume 160 Number 5. Part 2
Lipid / lipoprotein changes with the use of triphasic OCs
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lished separately). The findings of the present studv thus endorse the relative cardiovascular safety of the two oral contraceptive preparations tested at least in normolipidemic women. In conclusion, much of todav's concern about the impact of oral contraceptives on lipids and lipoproteins has emerged from studies conducted with higher dose preparations and interpretation of these data must be viewed with caution. Some investigators have failed to control for the length of exposure to the oral contraceptive being studied or to standardize for the time of blood sampling. Many studies have been cross sectional and involved relatively few subjects. Onlv a few studies have included a parallel group of matched women not taking oral contraceptives. In addition, earlier investigations based on the use of high- or medium-dose estrogen/progestin preparations are no longer applicable. The present study is the first to evaluate the effects of two commonly used, low-dose triphasic formulations on lipid patterns in a prospective, comparative, controlled fashion. The trial preparations contained comparable dosages of the same estrogen, ethinyl estradiol, but different progestins. The subjects were assigned in a randomized manner to either treatment group and were seen longitudinally for 1 year. We standardized the time of blood sampling and took measures to minimize differences in life-style and other influences on lipid metabolism among the three groups. Laboratory personnel were blinded to the subjects' identities and treatment. Because of the complexity of lipid/lipoprotein metabolism, numerous proatherogenic and antiatherogenic parameters were assayed in an attempt to prevent the formulation of clinical conclusions on the basis of one or two potentially adverse metabolic changes. For this reason, emphasis was placed on the importance of the dynamic balance between the measured lipid/lipoprotein assays and the clinical interpretation of lipid values against the template of a normal reference range. Results of the study demonstrate that both trial preparations (Triphasil and Ortho-Novum 71717) altered the lipid and lipoprotein milieu of otherwise normal women. However, effects of the two formulations appear to be similar and in the context described probably of minimal clinical significance. Any contribution to increased atherogenesis by either formulation is highly unlikely. Obviously, at least 30 vears of follow-up will be needed before a definitive conclusion can be reached. Since the individual metabolic response to an oral contraceptive varies and the long-term effect of oral contraceptives regarding atherogenesis are still to be defined, high-risk women taking oral contraceptives (including the low-dose, triphasic oral contraceptives) should be monitored with annual lipid and lipoprotein profiles.
We thank Yvonne Suggs, RN, l'\ancv Packard, RN, and Betty Russell, for their assistance.
REFERENCES I. Henderson BE. Ross RK. Paganini-Hill A. :\1ack T:\1. Estrog-en use and cardiovascular disease. A~(.J OIl~TET C;\NECOI 1986: 154: 1181. 2. Ro} al College of General Practltioner~. Further analvsi, of mortalit} in oral contraceptive users. Lancet 1981: i:541. 3. Bradlev DD, Wingerd .J. PetittI DB. Krauss RM. Ramcharan S. Serum hig-h-density-lipoprotein cholesterol in women using- oral contraceptives. e,trogens dnd progestins. N Engl J Med 1978:229: 17. 4. Allain CC. Poon LS, Chan CSG. Richmond W. Fu Pc. Enzymatic determination of total serum cholesterol. Ciin Chern 1974;20:470. 5. Wahlefeld AW. Methods of enzymatic analysis. In: Berg-meyer HV, ed. Triglvcerides. Gilford svstems. New York: Academic Press. 1974:1831. 6. Finley PR, Schlfman RB. Williams RI. Lichti DA. HDL. Gilford Systems. Clin Chern 1978:24:931. 7. American Heart Association special report. Recommendations for treatment of hyperlipidemia in adults. AJomt statement of the Nutntion Committee and the Council on Artenosclerosis. Prepared bv the Ad Hoc Committee to Design a Dietary Treatment of Hvperlipoproteinemia. Circulation 1984;69: 1065A-90A. 8. Kostner GM. Enzvmatlc determination of cholesterol in high-density-lipoprotein fractions prepared by polvanion precipitation [Letter]. ClIn Chem 1976:22: 695. 9. Friedewald W. Levv R. FredlICl<.son D. Estimation of the concentration of I~w-density-lipoprotem cholesterol in plasma without the use of the preparative ultracentrifuge. Clin Chem 1972:18:499. 10. Grow TE. Fried M. Interchange of apolipoprotein components between the human plasma high-densItvlipoprotein subclasses HDL, and HDL, in vitamins . .J Bioi Chem 1978:253:8034. II. Durrington PN. Ishola M, Hunt L. et al. Apolipoprotein (a). AI. and B and parental histoq in men with early onset ischaemic heart disease. Lancet 1988:t:1070. 12. Brown MS, Goldstein JL. How LDL receptor5 influence cholesterol and atherosclerosis. Sci Am 1984:251 :58. 13. Burkman RT. Robinson C. Kruszon-:'.1oran D. et al. Lipid and lipoprotem changes associated with oral contraceptive use: a randomized clinical trial Ob,tet Gvnecol 1988: 71:33. 14. Fotherby K. Oral contraceptives. lipids and cardiovascular disease [Review]. Contraception 1985:31 :367. 15. Dorflinger LJ. Relative potency of progestim used in oral contraceptives. Contraception 1985:31 :557. 16. Powell MG, Hedlin A. Cerskus I. et al. Effects of oral contraceptives on lipoprotein lIpids: a prospective study. Obstet Gvnecol 1984;63:764. 17. Krauss R'M. Rov S. Mlshell DR, et al. Effects of two lowdose oral contniceptives on serum lipids and lipoproteins: differential changes in high-density-lipoprotein subclasses. A~( .I OBSTET (;\,:--ECOL 1983: 145:446. 18. Knopp RH. Bergelin RO. Wahl PW, et al. Population based lipoprotein lipid reference values for pregnant women compared to non-pregnant women classified b\ sex hormone usage. A~( J OIl~rE'l C;\':\[COI. 1982:14:l: 620. 19. Castelli WI', Garrison RJ. Wilson PWF. et al. Incidence of coronary heart disease and lipoprotein cholesterol levels: the Frammgham Studv. .lAMA 1986:256:2835 20. Notelovitz M Cnpublished data. 21. Lipson A, Sto\ DB, LaRosaJC, et al. Progestins and oralcontraceptive-induced lipoprotein changes: a prospectIve study. Contraception 1986:34: 121.
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22. Slone D, Shapiro S, Kaufman DW, et al. Risk of myocardial infarction in relation to current and discontinued use of oral contraceptives. N Engl J Med 1981 ;305:420. 23. Adams MR, Clarkson TB, Koritnik DR, et al. Contraceptive steroids and coronary atherosclerosis in cynomolgus macaques. Fertil Steril 1987;47:1010. 24. Goldzieher JW, Chenault CB. Scientific exhibit. Pre-
May 1989 Am J Obstet Gynecol
sented at the Annual Meeting of the American College of Obstetricians and Gynecologists, April 24-30, 1987, Las Vegas, Nevada. 25. Marz W, Gross W, Gahn G, et al. A randomized crossover comparison of two low-dose contraceptives: effects on serum lipids and lipoproteins. AM J OBSTET GYNECOL 1985; 153:287-93.
From menarche to menopause: Coronary artery atherosclerosis and protection in cynomolgus monkeys Thomas B. Clarkson, DVM, Michael R. Adams, DVM, Jay R. Kaplan, PhD, Carol A. Shively, PhD, and Donald R. Koritnik, PhD Winston-Salem, North Carolina The effects on atherogenesis of stress, pregnancy, and oral contraceptive therapy were studied in a nonhuman primate model. The stress of social subordination was associated with ovarian dysfunction, unfavorable lipoprotein changes, and increased coronary artery atherosclerosis compared with nonstressed (socially dominant) or normal monkeys. Although pregnant animals exhibited lower high-density lipoprotein cholesterol concentrations, they had only one half as much diet-induced coronary artery atherosclerosis as their nonpregnant counterparts. Monkeys treated with an Ovral-like regimen also exhibited adverse lipoprotein changes. Nevertheless, prevalence and extent of coronary artery plaques decreased. We conclude that estrogen is an important factor in the animals' "female protection" against diet-induced atherosclerosis. We also suggest that the lowering of high-density lipoproteins by the progestin component of higher-dose contraceptives is not necessarily atherogenic if a sufficiently potent exogenous estrogen is administered concomitantly. (AM J OBSTET GVNECOL 1989;160:1280-5.)
Key words: Atherosclerosis, cholesterol, estrogen, stress, oral contraceptives, Macaca Jascicularis
In certain races and societies, premenopausal women enjoy a degree of protection from coronary artery atherosclerosis relative to their male counterparts. Explanations suggested for this male-female difference include gender differences in plasma lipoprotein concentrations (particularly in high-density lipoprotein cholesterol), a protective effect of estrogens, and the possibility that females are spared the stress and pathophysiologic effects of the competitive and sometimes hostile behavior of males. Progress in understanding this relative protection of female human beings has been slow to develop because until recently there was not a suitable animal model for research. About a decade ago, we developed such a nonhuman primate From the Arteriosderosls Re.learch Center, Bowman Gray School of Medlcme, Wake Fore.lt Unwenltv. Supported In pari Ii.v SCOR lIZ Arle";OIderosls Grant HL-14164 jrom the National Heart, Lung, and Blood lmtitute and Conllact No. NOI-HD-32800 from the Nat1Onal/n.ltltute of Child Health and Human Development Reprmt request:,: Thomas B. ClarklOn, DVM, Departmmt oj ComparatIVe Medlcllle. Bowman Gray School oj Medilllle. 300 S. Hawthorne Rd .. Wln.lton-Salem, NC 27103.
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model in which a series of investigations was conducted on the atherogenic and/ or protective effects of stress, pregnancy, and treatment with contraceptive steroids.
Development of model and methods for quantifying coronary artery atherosclerosis Study 1. We first investigated the cynomolgus macaque (Macaca Jascicularzs) because females of that species have menstrual cycles like those of women."" A 16month study comparing the effects of a moderately atherogenic diet on these animals subsequently demonstrated male-female differences similar to those of human beings. I Although both male and premenopausal female cynomolgus monkeys developed comparable total plasma cholesterol concentrations in response to the cholesterol-containing diet, the females, like human females, exhibited higher high-density lipoprotein cholesterol concentrations than did males. Thus there were important gender differences in the ratio of total plasma cholesterol and high-density lipoprotein cholesterol concentrations. Most important was our finding that coronary artery atherosclerosis was