JOSEPH W. GOLDZIEHER, M.D.t C. BRANDON CHENAULT, M.D. ARMANDO DE LA PENA, M.S. TAZEWELL S. DOZIER DUANE C. KRAEMER, D.V.M., PH.D.

Transcription

1 FERTILITY AND STERILITY Copyright ' 1978 The America Fertility Society Vol. 3, No. 5, November 1978 Prited i U.S A. COMPARATIVE STUDIES OF THE ETHYNYL ESTROGENS USED IN ORAL CONTRACEPTIVES. VII. EFFECTS WITH AND WITHOUT PROGESTATIONAL AGENTS ON ULTRACENTRIFUGALLY FRACTIONATED PLASMA LIPOPROTEINS IN HUMANS, BABOONS, AND BEAGLES* JOSEPH W. GOLDZIEHER, M.D.t C. BRANDON CHENAULT, M.D. ARMANDO DE LA PENA, M.S. TAZEWELL S. DOZIER DUANE C. KRAEMER, D.V.M., PH.D.:j: Southwest Foudatio for Research ad Educatio, Sa Atoio, Texas Ethyyestradiol ad mestraol, i doses ragig from 5 to 1 pg!day, were give to wome i 21-day cycles; baboos ad beagle dogs received 1 ad 4 pg/kg/day i a similar regime. After a umber of such cycles, megestrol acetate, orethidroe acetate, or dl-orgestrel was give cocomitatly. Protei, cholesterol, triglyceride, ad phospholipid levels were determied i total plasma ad i ultracetrifugally separated lipoprotei fractios. Over the dosage rage studied, the effects of the two kids of estroge were idistiguishable. Except for huma total plasma triglyceride, o dose-related differeces were observed. The lowerig of serum protei ad the icrease i cholesterol iduced by estroge were more proouced i baboos ad beagles tha i huma subjects. The cholesterol-depressig effect of progestatioal compouds observed i humas was very proouced i baboos but abset i beagles. I all three species, estroge icreased the lipoprotei fractio cholesterol, except for huma low-desity lipoprotei cholesterol, which was decreased. Huma plasma triglyceride ad phospholipid icreased o estroge admiistratio ad were decreased by the progestis; i the two aimal species, triglyceride is ormally very low ad the estroge-iduced chages were egligible; the phospholipid rose with estroge but was uaffected by progestis. I sum, the two aimal species show may similarities to, as well as importat differeces from, the huma respose of plasma lipids to various cotraceptive steroids. Fertil Steril3:522, 1978 Received April 24, 1978; revised May 3, 1978; accepted July 1, *Cliical studies supported by Cotract csd/2821, Office of Populatio, Uited States Agecy for Iteratioal Developmet; aimal studies supported by Cotract HD , Ceter for Populatio Research, Natioal Istitute of Child Health ad Huma Developmet, Uited States Public Health Service. treprit requests: Joseph W. Goldieher, M.D., Southwest Foudatio for Research ad Educatio, P.O. Box 28147,8848 West Commerce Street, Sa Atoio, Tex :J:Preset address: Texas A & M Uiversity College of Veteriary Medicie, Departmet of Veteriary Physiology ad Pharmacology, ad Istitute of Comparative Medicie, College Statio, Tex The relatioship of sex hormoes to plasma lipids, i the cotext of atherogeic vascular disease, has bee uder ivestigatio for a very log time. With the advet of steroid cotraceptives, a substatial part of the adult female populatio became exposed to a hormoal eviromet whose impact o lipid metabolism, ad ultimately o the vascular system, required ivestigatio. As with other aspects of steroid cotraceptive use, a volumious literature quickly developed. The effects o plasma cholesterol, triglycerides, ad phospholipids have bee reviewed. 1-3 Most studies report that total triglycerides are elevated durig

2 Vol. 3, No.5 COMPARATIVE STUDIES OF ETHYNYL ESTROGENS 523 oral cotraceptive use, although some foud o chage. 3 The effects o cholesterol are also cotroversial4 Phospholipids appear to be quatitatively ad qualitatively altered. 1 These ivestigatios have geerally ivolved wome usig commercial steroid cotraceptives. With the variety of formulatios available, it is extremely difficult to sort out the respective effects of the estroges ad progestis ad to examie their iteractios. There are a umber of studies o the effect of the separate costituets, 4 " 7 but differeces i protocol make comparisos difficult. Aftergood et alb have suggested that the effects of mestraol o plasma cholesterol are greater tha those of ethyylestradiol, but Stokes ad Wy 9 cocluded that they were equivalet. Yeug 1 oted o effects with low doses oforethisteroe, but a ehacemet of the estroge-iduced alteratio with high doses. Brody et al 11 cocluded that pills cotaiig ethyodiol diacetate or megestrol acetate were differet from those which cotaied orethisteroe acetate, especially with respect to phospholipid effects. Gershberg et al.4 believed that combiatio agets with orethyodrel or orethisteroe were comparable. Aalyses of plasma lipoproteis have also yielded icosistet results. While va der Steeg ad Prok 3 foud o chage i the chylomicro fractio, Furma et al.l 2 metio a possible icrease i its cholesterol ad phospholipid cotet. Two cross-sectioal studies foud a higher proportio of lipoproteis i the very low-desity lipoprotei (VLDL) fractio i combiatio oral cotraceptive users tha i ousers. I a logitudial study of 26 wome receivig ethyylestradiol-orgestrel for 2 moths, va der Steeg ad Prok 3 failed to fid a sigificat icrease; Rosser et al., 15 usig a.1-mg mestraolchlormadioe acetate combiatio, however, foud a sigificat icrease i cholesterol, triglyceride, ad phospholipid i the VLDL. With respect to the low-desity lipoprotei (LDL) fractio, Barr 16 ad his collaborators foud a decrease; Wy et al. 13 ad Gad-El-Mawla et al. 17 foud o chage; ad va der Steeg ad Prok, 3 Kuku ad Akiyaju, 14 Osma et al., 2 ad Castelli et al. 1 B observed a icrease. I a aalysis of the costituets of this fractio, Aurell et au 9 foud a icrease i the cholesterol, Rosser et al. 15 foud o chage, ad Furma et al. 12 foud o chage or possibly a decrease. A icrease i triglyceride was observed by Aurell et al. 19 ad by Rosser et al., 15 together with a icrease i phospholipid. A icrease i the cholesterol cotet of the high-desity lipoprotei (HDL) fractio with estroge therapy was first reported by Barr 16 ad subsequetly cofirmed by Furma et al. 12 ad Wy et al. 13 However, Rosser et al. 15 ad va der Steeg ad Prok 3 oted o chage, while Kuku ad Akiyaju 14 ad Aurell et al. 19 reported a decrease. Triglyceride was geerally reported as icreased, although Aftergood et alb foud a decrease. Phospholipids were reported either as uchaged or icreased I view of the variety of estroges ad progestis used, as well as differeces i dosage ad duratio of treatmet, such diversity is ot etirely uexpected. MATERIALS AND METHODS Details of the ivestigatioal desig have bee described Briefly, 163 iformed ormal adult female voluteers were assiged radomly (as far as possible) to treatmet with oral doses of 5 or 8 J.Lg of ethyylestradiol (EE) or 5, 8, or 1 J.Lg of mestraol (ME) daily i the usual 21-day cotraceptive regime. The wome takig the lowest estroge dose had to be usig itrauterie devices (IUDs), which upset radom assigmet to this group. After six cycles of therapy, the subjects i each of the five treatmet groups were separated ito three subsets ad received, i additio to the same dose of estroge, either orethidroe acetate (2.5 mg/day), megestrol acetate (2 mg/day), or dl-orgestrel (.5 mg/day) for a further six cycles. Blood samplig for various edocrie ad metabolic studies was always carried out toward the ed of certai treatmet cycles. Thirty-four wome usig oly IUDs were sampled i the same way, as a compariso populatio. Their age distributio differed slightly from that of the steroid users. 22 Ibred female beagles of a average weight of 6.9 kg were assiged radomly to oral treatmet regimes cosistig of 1 or 4 J.Lg/kg/day of ethyylestradiol or mestraol for 21 days, followed by 7 days without drug. This program was used for four cycles, after which each of the four groups was subdivided ito three, ad oe of the three progestatioal agets (orethidroe acetate, 1 mg/day; megestrol acetate, 1 mg/day; or dl-orgestrel,.5 mg/day) was give together with the estroge for a further four cycles. The the treatmet was discotiued, ad the tests were repeated 3 moths later. Blood samples were obtaied before treatmet, durig the last week of treatmet cycles 1, 2, 4, 5, 6, ad 8, ad 3 moths

3 524 GOLDZIEHER ET AL. November 1978 after the ed of treatmet. A similar desig was used for the study of adult female baboos weighig 13.6 kg o the average. A total of 6 beagles ad 6 baboos was used. There were two 3- aimal replicatios of each protocol i each species to radomie time- ad seaso-depedet biases. I additio, etry of the 3 aimals ito each replicate experimet was staggered for logistical reasos. This further radomied time-depedet biases. The aimals were assiged to the various drug-dose groups by computer-geerated radom allocatio. Pills cotaiig the various dose levels of ethyylestradiol, mestraol, orethidroe acetate, megestrol acetate, or dl-orgestrel were prepared by Wyeth Laboratories, Philadelphia, Pa., with precise quality cotrol to esure uiform bioavailability. All blood samples were obtaied i the morig. Baboos were uder miimal Seryla traquiliatio; o sedatio was required for the beagles. The plasma was deep-froe immediately. Ultracetrifugatio separatio of the lipoprotei fractios was performed by the method of Havel et al. 23 Cholesterol was determied by the method of Chiamori ad Hery, 24 phospholipids by the method of Suderma ad Suderma, 25 triglycerides by the method of Fletcher, 26 ad total protei by the Weichselbaum methodp Cetrifugatio for 1 hour yielded the "chylomicro" fractio with desity of <1.6; cetrifugatio for 18 hours yielded the fractio which cotais the VLDL. The desity fractio is desigated as LDL, ad that with a desity ofl as the HDL fractio. No special procedures were used to purify the chylomicro fractio further. After completio of the ivestigatios, all data were trasferred to computer tapes ad verified. The baselie data for each variable were the examied for the uderlyig distributio of the values ad it was foud that they coformed most closely to a logormal distributio. Therefore, all values were log-trasformed prior to statistical aalysis, which cosisted of comparig by t-test each experimetal poit (species, cycle, dose) for the two estroges, to see whether the type of estroge (i.e., EE or ME), at equal dosage, produced ay differece. The a similar set of aalyses was performed to compare differet doses of the same estroge, by species ad by cycle. Where o differeces were observed betwee types ad/or doses of estroge, it was cosidered legitimate to combie treatmet groups, thereby icreasig sample sie. Next, a three-way aalysis of variace examied the effect of estroge dose, cycle of use, effect of the added progestatioal compoud, iteractio betwee estroge dose ad cycle umber, betwee estroge dose ad type of progestatioal aget, ad iteractio of estroge, cycle umber ad progestatioal aget. RESULTS I all three species, statistical aalysis showed that the effects of the two estroges, mestraol ad ethyylestradiol, were idistiguishable. Moreover, with mior exceptios, there was o differece i effect over the dosage rage examied. Aalysis of variace showed o differeces betwee the progestatioal compouds except as specifically idicated i the text. O this basis, it was cosidered legitimate to combie the data from the various treatmet groups, by cycle, ad to perform statistical comparisos o these relatively large samples. The data for whole plasma ad for three of the ultra cetrifugal fractios, by species, are show i the followig graphs ad tables. The chylomicro fractio should be composed largely of triglyceride; it was foud to cotai oly 14%, idicatig that it was impure, as is ordiarily the case with this ultracetrifugal fractio. The HDL fractio cotaied HDL protei as well as the remaiig serum proteis; chages i its protei cotet were therefore also ot aalyed statistically. Protei I huma subjects, there was o effect of steroid treatmet o total protei. I baboos (Fig. 1), the average value of 6.9 gm/dl showed a highly sigificat decrease (P <.1) to 6.6 gm/dl i the first cycle, ad this decrease did ot level out util the fourth cycle of estroge treatmet. The additio of the progestatioal compouds produced o further chage. Protei i the VLDL fractio also showed a dowward tred durig estroge treatmet ad remaied depressed eve i the post-treatmet sample (P <.1). I beagles, a slight decrease durig estroge treatmet was ot statistically sigificat. However, whe the progestatioal compouds were added, there was a sharp, highly sigificat, icrease (P <.1) from 5.6 to 6. gm/dl which fell toward ormal but was still sigificatly elevated (P <.2) at the post-treatmet samplig. No sigificat chages were observed i the protei of the other ultracetrifugal fractios.

4 Vol. 3, No.5 COMPARATIVE STUDIES OF ETHYNYL ESTROGENS 525 BABOON BEAGLE 18 '!:? 16 "' E "' E '!:? 6.6 E "' ' 5.7 ' E "' / l \!. / 'f hv... I It I II FIG. 1. Serum protei levels, by cycle, i baboos ad beagles. Total protei (lower sectio) is i grams per deciliter, ultracetrifugal fractios (upper sectio) are i milligrams per deciliter. o--- -o, LDL; --, VLDL; o--o, HDL; O O, whole plasma. Cholesterol (Fig. 2) Huma Subjects. I huma subjects, pretreatmet total cholesterol averaged 166 mg/dl (95% cofidece limits = 156 to 177) i the IUD users (N = 34) ad 165 mg/dl (159 to 172) i the wome who were to receive steroids (N = 161). The level i the IUD users did ot vary sigificatly i the course of the experimetal observatio period. I the estroge-treated group, there was a gradual rise, which, however, did ot become statistically sigificat util the sixth cycle of treatmet, whe the level averaged 181 mg/dl (]> <.1). There was o differece betwee the estroge dosage groups (Table 1). The progestatioal compouds produced a sigificat(]> <.2) lowerig of the level to 166 mg/dl by the 12th cycle. Megestrol did ot lower the values as much as the other two progestis, but the differeces were ot statistically sigificat. The level of VLDL cholesterol was low, averagig 13.3 mg/dl i the IUD group ad 14.8 mg/dl i the steroid group. There was o chage i the IUD group over time. With estroge treatmet, however, VLDL cholesterol rose to 19.1 mg/dl i the first estroge cycle (P <.1) ad remaied at this level for the duratio of estroge treatmet. The progestatioal agets lowered this level sigificatly (P <.1) to 16.1 mg/dl; there was o differece betwee the three agets. The baselie LDL cholesterol of74.1 mg/dl fell slightly to 65.8 mg/dl i the third cycle, the rose to 72.5 by ed of estroge treatmet; the progestatioal agets produced o further chage. Baselie HDL cholesterol averaged 65.6 mg/dl i the IUD cotrols ad 62.9 i the steroid group. There was o chage i time i the IUD users. The cholesterol level rose sigificatly i the estroge users i the first cycle (P <.1) to 7.6 mg/dl ad remaied at this level durig estroge treatmet. Progestatioal therapy caused it to decrease to baselie values (average, 58.5 mg/dl; P <.1). Megestrol was sigificatly more depressat tha were equipotet uder these experimetal cir- dl-orgestrel or orethidroe acetate which ' cumstaces. Baboos. I baboos, pretreatmet total cholesterol levels averaged 115 mg/dl (19 to 121). With estroge treatmet there was a immediate, pro-

5 526 GOLDZIEHER ET AL. November 1978 HUMAN BABOON BEAGLE 3!--- l!' !ii 23 g 22 g I I FIG. 2. Cholesterol levels i whole plasma ad i various plasma lipoprotei fractios obtaied ultracetrifugally i humas, baboos, ad beagles. The absolute values o the ordiates are broke ito segmets because of differeces i cocetratio betwee species ad betwee fractios, but the scale of uits at each level is the same for all species. Huma subjects received estroge for six cycles, estroge-progesti combiatios for six cycles. Beagles ad baboos were exposed to each type of regime for four cycles, followed by a post-treatmet evaluatio 3 moths later. The treatmet regimes are show as bars o the abscissa, with the clear bars idicatig estroge, the black bars idicatig progesti treatmet. o-- -o, LDL; - VLDL; o-o, HDL, o... o, whole plasma. II gressive icrease which was statistically sigificat by the ed of the first cycle (138 mg/dl; P <.1). The cholesterol reached a level of 163 mg/dl at the ed of estroge treatmet, ad dropped precipitately (P <.1) to 144 mg/dl after the first cycle of added progestatioal agets. The decrease leveled off after that, but remaied sigificatly <P <.1) elevated; it retured to baselie values by 3 moths post-treatmet. The level of cholesterol i the VLDL fractio was very low (4.8 mg/dl). Nevertheless, it showed a statistically sigificat (P <.1) icrease to a maximum of 6.6 mg/dl i the fourth cycle. Progestatioal agets produced a sigificat (P <.1) decrease which disappeared by cycle 8, at which time the level was agai sigificatly (P <.1) above baselie; it remaied elevated posttreatmet. The LDL cholesterol averaged 37 mg/dl i the baselie cycle. It also rose sigificatly <P <.1) durig estroge treatmet to a peak of 6 mg/dl, showed a trasiet declie <P <.1) durig progestatioal treatmet, ad retured to baselie values post-treatmet. HDL cholesterol icreased sigificatly (P <.1) from a baselie value of 68 mg/dl to 82 mg/dl by the ed of the first estroge cycle. Progestatioal compouds iduced a immediate ad sigificat (P <.1) decrease, although the level remaied sigificatly elevated (P <.1) above baselie. It approached baselie values by the ed of cycle 11. Beagles. I beagles, pretreatmet total cholesterol averaged 138 mg/dl (13 to 146). Upo admiistratio of estroge, there was a immediate ad cotiuous icrease to levels of 182 mg/dl (172 to 193) by the fourth cycle of estroge treatmet (P <.1). After additio of the progestatioal compoud, the cholesterol level cotiued to icrease for oe cycle, reachig a mea value of 197 mg/dl, ad remaied costat thereafter. It decreased post-treatmet, but the level at cycle 11 was still sigificatly <P <.1) above

6 Vol. 3, No.5 COMPARATIVE STUDIES OF ETHYNYL ESTROGENS 527 TABLE 1. Total Cholesterol-Huma Subjects" Cycle IUD Mea 95% CH Estroge, 5 f.lg Mea Estroge, 8 f.lg Mea Estroge, 1 f.lg Mea All drugs Mea mgldl a Total plasma cholesterol, by cycle, i huma subjects usig a IUD or receivig various doses of ethyyl estroge i a 21-day cotraceptive regime for six cycles, followed by aother six cycles of the same estroge dosage combied with a progestatioal compoud. The data from users of ethyylestradiol or mestraol have bee pooled, as have the data with the differet progestatioal compouds. b CL, Cofidece limits. cotrol. The VLDL fractio cotaied extremely low levels of cholesterol: the baselie mea value was 2.9 mg/dl. However, a statistically sigificat icrease (P <.1) was detected durig estroge treatmet. There was o further chage with progestatioal treatmet, ad the level remaied sigificatly elevated at cycle 11 (P <.1). The LDL cholesterol showed a icrease durig estroge therapy which was statistically sigificat (P <.1) after oe cycle ad the stabilied; it showed a further gradual icremet (P <.5) whe the progesti was added. The posttreatmet level was still above baselie (P <.2). HDL cholesterol rose substatially (P <.1) uder estroge treatmet from a baselie of 114 mg/dl to 14 7 mg/dl by cycle 4 ad to 16 mg/dl after oe cycle of additioal progesti. (This latter icremet was ot statistically sigificat.) The value remaied statioary throughout combied treatmet (the apparet decrease is ot statistically sigificat), ad was still at 148 mg/dl post-treatmet. Triglyceride (Fig. 3) Huma Subjects. I huma subjects, pretreatmet plasma triglyceride levels averaged 98 mg/dl (82 to 116) i the IUD group ad 115 mg/dl (89 to 149) i the steroid group. There was o chage i level i the IUD users with time. With estroge treatmet (Table 2), the level rose sigificatly (P <.1) to 161 mg/dl i the first cycle ad showed a slight, gradual, further icrease with treatmet. There appeared to be some relatioship to dose (Fig. 4), but its statistical sigificace was equivocal. The additio of the progestatioal compouds depressed the level (P <.5) to baselie (131 mg/dl); megestrol did ot seem to be quite as effective i this regard as the other two progestis. The VLDL level i the IUD users (36.7 mg/dl) did ot chage with time. I the steroid group, the level of 4.6 mg/dl icreased to 56.4 mg/dl i the first cycle (P <.1) ad remaied at this level for the duratio of estroge treatmet. The additio of progestis produced a sigificat (P <.1) fall to a average level of 43.7 mg/dl; there was o differece betwee the progestatioal compouds. I the LDL fractio, the small amout of triglyceride (19.9 mg/dl baselie level) rose o estroge treatmet to stabilie at 29 mg/dl (P <.1). The progestatioal agets did ot lower this level sigificatly. HDL triglyceride remaied stable i the IUD group. I the estroge users, the baselie level of 24.5 mg/dl rose to 43.7 i the first cycle ad to 5.9 after six cycles of estroge treatmet (P <.1). The progestatioal agets decreased the level to a average of 33.3 mg/dl. This was ot statistically sigificat; however, the decrease with megestrol was less tha the decrease with the other progestis.

7 528 GOLDZIEHER ET AL. November HUMAN BABOON BEAGLE 24 2 : E "' 12,!;; 8 a::, 7 w 6 u 5 -r--t - ---, 1 l l u t,.f , A... - f t T 3...! : I I =9..., - II I II 2 1 FIG. 3. Triglyceride levels i whole plasma ad i various plasma lipoprotei fractios obtaied ultracetrifugally, i humas, baboos, ad beagles. The absolute values o the ordiates are broke ito segmets because of differeces i cocetratio betwee species ad betwee fractios, but the scale of uits at each level is the same for all species. Huma subjects received estroge for six cycles, estroge-progesti combiatios for six cycles. Beagles ad baboos were exposed to each type of regime for four cycles, followed by a post-treatmet evaluatio 3 moths later. The treatmet regimes are show as bars o the abscissa, with the clear bars idicatig estroge, black bars idicatig progesti treatmet. o-- -o, LDL; -, VLDL; o-o, HDL; o... o, whole plasma. 2 Baboos. Whole plasma triglyceride levels i baboos are comparatively low, averagig 39 mg/dl i the baselie cycle. The triglyceride level rose immediately to 53 mg/dl (P <.1) after oe cycle of estroge treatmet ad remaied stable thereafter. The itroductio ofprogestatioal compouds lowered this level sigificatly (P <.1) to 46 mg/dl, ad it was still at 44 mg/dl 3 moths after the ed of treatmet. The triglycerides i the VLDL fractio showed erratic, osigificat fluctuatios of a relatively low baselie level (8.1 mg/dl). However, the baselie value of triglyceride i the LDL fractio (11 mg/dl) rose promptly ad sigificatly (P <.1) to a plateau at about 17 mg/dl uder estroge treatmet. There was a sigificat (P <.1) fall i the first two cycles of progestatioal treatmet, ad the a highly sigificat (P <.1) reboud durig the last two progesti cycles. After discotiuatio of steroids, the level lapsed to baselie values. HDL triglyceride averaged 13 mg/dl i the baselie cycle ad also showed a cotiuig icrease (P <.1) durig estroge treatmet. No chage took place o the additio of the progestatioal compouds. The level declied oly miimally 3 moths post-treatmet. Beagles. Baselie plasma triglyceride is very low i beagles, averagig 31.2 mg/dl (3 to 33). Because of the large umber of aimals tested, the icrease to 37.1 mg/dl i the fourth cycle of estroge treatmet was statistically sigificat (P <.1). Whether or ot such a small chage has biologic sigificace is aother matter. A further icrease occurred oly i cycle 6, the secod cycle of combied estroge-progesti treatmet. The level of 34.9 mg/dl post-treatmet was still sigificatly higher (P <.1) tha the baselie value. The baselie triglyceride i the VLDL fractio was 7.6 mg/dl. It dropped slightly durig the first two cycles of estroge treatmet, the showed a sigificat (P <.1) icrease i the ext two cycles ad o further chage thereafter. It retured to baselie levels by cycle 11. The LDL triglyceride is also very low (mea baselie level 7.6 mg/dl). It showed a prompt rise (P <.1) i treatmet cycle 1 to 1.1 mg/dl ad remaied essetially costat thereafter. It did ot retur to baselie by cycle 11. The triglyceride

8 Vol. 3, No.5 COMPARATIVE STUDIES OF ETHYNYL ESTROGENS 529 TABLE 2. Total Plasma Triglyceride-Huma Subjects" Cycle IUD Mea 95% cu Estroge, 5 )Lg Mea Estroge, 8 p.g Mea Estroge, 1 p.g Mea All drugs Mea ' : mgidl : ' a Total plasma triglyceride, by cycle, i huma subjects usig a IUD or receivig various doses of ethyyl estroge i a 21-day cotraceptive regime for six cycles, followed by aother six cycles of the same estroge dosage combied with a progestatioal compoud. The data from users of ethyylestradiol or mestraol have bee pooled, as have the data with the differet progestatioal compouds. bcl, Cofidece limits. of the HDL fractio showed a slight upward tred, but oe of the chages were statistically sigificat. Phospholipids (Fig. 5) Huma Subjects. I huma subjects, pretreatmet phospholipid averaged 211 mg/dl i the IUD group ad 215 (28 to 221) i the steroid group (Table 3). The level remaied costat over time i the IUD subjects. Uder estroge treatmet there was a sigificat rise (P <.1) to 256 mg/dl i the first cycle ad a plateau at about 272 mg/dl by the third cycle. The progestatioal agets produced a fall to 228 mg/dl; orgestrel ad orethidroe acetate were equivalet i their actio, ad they were more effective tha megestrol. All of the phospholipid fractios were stable i the IUD users. I the steroid-treated group, the VLDL phospholipid rose from 22.4 mg/dl to 28.5 (P <.1) i the first estroge cycle ad remaied at that level. It was depressed sigificatly (P <.1) to baselie levels by all three progestis. LDL phospholipid averaged 64.1 mg/dl at the outset, ad did ot chage with either estroge or progesti exposure. O the other had, HDL phospholipid rose from 15 to 14 mg/dl i the first estroge cycle ad to 154 mg/dl (p <.1) after six cycles. The 19-or progestatioal agets had a. sigificat (P <.1) ad equal lowerig actio, while megestrol had o effect uder the experimetal coditios, Baboos. The whole plasma phospholipid level i utreated baboos averaged 188 mg/dl (178 to 199). Estroge treatmet sigificatly (P <.1) elevated this value to 2 mg/dl i the first cycle ad it stabilied.at 229 mg/dl by the secod cycle. It was ot sigificatly affected by the progestatioal compouds. There was a sigificat (P <.1) fall after termiatio of treatmet, but the value was still sigificatly (P <.1) above 2 18 o> E 16!;i a:: w 12 u I IUD FIG. 4. Whole plasma triglyceride i huma subjects, by estroge dose, by cycle. Oly the mea values are show; variaces are give i Table 2. 12

9 53 GOLDZIEHER ET AL. November 1978 HUMAN BABOON BEAGLE o> 18 E 14 1 :: 1- w u u a:: :J I a. (f) I a t f 1! ! 12 :t J t l.'-...! T J. - : II 24 2 ::! " 2?.. '!'.c? : II FIG. 5. Phospholipid levels i whole plasma ad i various plasma lipoprotei fractios obtaied ultracetrifugally i humas, baboos, ad beagles. The absolute values o the ordiates are broke ito segmets because of differeces i cocetratio betwee species ad betwee fractios, but the scale of uits at each level is the same for all species. Huma subjects received estroge for six cycles, estroge-progesti combiatios for six cycles. Beagles ad baboos were exposed to each type of regime for four cycles, followed by a post-treatmet evaluatio 3 moths later. The treatmet regimes are show as bars o the absicca, with the clear bars idicatig estroge, the black bars idicatig progesti treatmet. o-- -o, LDL; -, VLDL; o-o, HDL; o... o, whole plasma. baselie. The VLDL fractio cotais very little phospholipid (baselie values averaged 8.3 mg/dl; it showed erratic chages durig treatmet. Possibly there was some depressio of the mea level durig estroge admiistratio ad some rise (P <.5) durig progesti treatmet. The LDL fractio averaged 36 mg/dl i the baselie cycle, rose to 46 mg/dl (P <.1) i the first estroge cycle ad remaied at about this level thereafter. Durig progestatioal treatmet there was a trasiet but statistically sigificat (P <.3) fall at cycle six, ad a retur to elevated values (P <.1) by cycle eight. Discotiuatio of treatmet caused the level to revert to baselie values. The HDL phospholipid averaged 125 mg/dl at the outset, rose to 159 i the first cycle (P <.1) ad to 171 mg/dl by the ed of cycle 4. There was o further chage upo itroductio of the progestatioal agets, ad a partial but icomplete fall toward baselie after the ed of treatmet. Beagles. I beagles, the baselie phospholipid level of 272 mg/dl rose sharply ad cotiuously uder estroge admiistratio, ad did ot level off util cycle 5 (oe cycle of combied hormoe treatmet). The level reached at the ed of estroge treatmet (333 mg/dl was sigificatly (P <.1) elevated. There was further elevatio durig progestatioal treatmet: the level of 366 mg/dl i cycle 8 was sigificatly (P <.5) higher tha the level at the ed of estroge treatmet. There was a slight fall to 346 mg/dl 3 moths after termiatio of treatmet, but this was still sigificatly higher (P <.2) tha the baselie level. The baselie VLDL phospholipid was very low, averagig 8.1 mg/dl. It showed a highly sigificat fall (P <.1) i the first cycle of estroge treatmet to 4.5 mg/dl, but rose thereafter durig the rest of estroge treatmet, almost reachig baselie levels (6.6 mg/dl); it did ot chage appreciably thereafter. The baselie level of phospholipid i the LDL fractio averaged 23 mg/dl. It showed a slight, osigificat upward tred durig estroge therapy ad a sigificat elevatio (P <.1) betwee cycle 4 ad cycle 6 to 34 mg/dl. There was o further chage, ad the cycle 11 value was still sigificatly (P <.1) above the baselie value. The HDL phospholipid level of218 mg/dl rose sigificatly (P <.1) to 251 mg/dl after oe cycle of estroge treatmet ad reached a plateau by cycle 5 at 32 mg/dl. The further rise durig progestatioal treatmet was sigificat (P <.2) ad the level

10 Vol. 3, No.5 COMPARATIVE STUDIES OF ETHYNYL ESTROGENS 531 TABLE 3. Total Phospholipid-Huma Subjects" IUD Mea 95% CIP Estroge, 5 J.Lg Mea Estroge, 8 J.Lg Mea Estroge, 1 J.Lg Mea All drugs Mea Cycle mgldl "Total plasma phospholipids, by cycle, i huma subjects usig a IUD or receivig various doses of ethyyl estroge i a 21-day cotraceptive regime for six cycles, followed by aother six cycles of the same estroge dosage combied with a progestatioal COII_J.poud. The data from users of ethyylestradiol or mestraol have bee pooled, as have the data with the differet progestatioal compouds. "CL, Cofidece limits. of 298 mg/dl i cycle 11 was still sigificatly (P <.1) higher tha the baselie level. DISCUSSION Several aspects of the desig of these studies deserve metio. Their prospective ature permitted observatio of the effects of the ethyyl estroges themselves for a period of time sufficiet for a steady state to be reached; subsequet to this, the additioal effect of several chemical types of progestatioal aget was observed. Care was take to esure uiform bioavailability of the steroids-a problem which complicates evaluatio of studies i which a variety of commercial preparatios are used. The two estroges produced equivalet effects, ad the dosage rage did ot produce sigificat quatitative differeces. This allowed the experimetal groups to be combied, ad these elarged sets were the available for statistical evaluatio after log trasformatio of the data. The log time over which both huma ad aimal subjects were itroduced ito the experimetal protocol served to eutralie time-depedet ad methodologic biases. I the cliical studies, data from the IUD users provided further assurace agaist such problems. All these precautios take together provide a cosiderable degreee of cofidece eve i small (absolute) chages i some of the variables, ad this is documeted by the relatively arrow 95% cofidece limits. Oe of the major purposes of this study was to compare the respose of humas, beagles, ad baboos to estroge ad estroge-progesti exposure i order to detect similarities ad differeces i respose, ad to evaluate the two experimetal aimals as presumptive biologic models for ma. I all three species, ethyylestradiol ad mestraol were equipotet. This is cosistet with our cocurret observatios o a umber of other variables. There was essetially o dosage effect perceptible over the rage studied except i the case of huma plasma triglyceride, which raises questios as to the putative beefits of decreasig the cliical estroge dosage to 5 p,g/day. At the time our studies were iitiated, still lower estroge dosage was ot uder active ivestigatio. I huma subjects, the slight lowerig of total protei durig estroge treatmet was ot statistically sigificat. However, i both beagles ad baboos, it was ad it was more proouced i the latter species. O the other had, oly the beagle showed a rise i total protei i respose to all three progestatioal compouds, ad 3 moths after treatmet a effect was still perceptible. The gradual, slight rise i huma total cholesterol iduced by estroge was exaggerated i both

11 532 GOLDZJEHER ET AL. November 1978 baboos ad beagles; the cholesterol-depressig effect of progestatioal compouds see i huma subjects was very proouced i baboos but was ot foud i the beagle. I all three species, there was a rise i the cholesterol level of the ul tracetrifugal fractios except for LDL cholesterol i humas, which fell. Progestatioal compouds produced a rise i huma ad beagle LDL cholesterol; chages i baboos were erratic. The estroge-iduced rise i HDL-cholesterol was more proouced i both aimal species tha i ma; the progestatioal agets had a very proouced effect i humas ad baboos, oe i beagles. The steroid effect appeared to persist loger i beagles tha i baboos. I huma subjects, plasma triglyceride showed a sigificat elevatio o estroge admiistratio, ad a decrease o the additio ofprogestis. Triglyceride levels i the two aimal species are extremely low by compariso, ad ay chages see were egligible. I humas, triglyceride i the various ultracetrifugal fractios teded to parallel that i whole plasma, with the smallest chages i the LDL fractio. I the baboo, oly HDL triglyceride rose durig estroge treatmet. Plasma phospholipid i humas rose i respose to estroges ad declied whe progestatioal compouds were added. Baboo phospholipid also rose ad quickly reached a plateau which was uaffected by progestis. Beagles showed a progressive rise i respose to estroge, with o depressat effect from progestis. VLDL phospholipid i humas paralleled the total phospholipid; i baboos it showed o chage, ad i beagles it was actually depressed by estroges. LDL phospholipid showed o chage i humas ad beagles, but a icrease occurred i baboos uder estroge treatmet. HDL phospholipid followed total phospholipid chages closely i all three species. It is evidet that either aimal species reproduces exactly the chages iduced i huma plasma lipids ad lipoproteis by these steroids; i some respects they show a relatively exaggerated respose, while i the case of other costituets which are preset i very low cocetratios, they fail to show the chages see i huma subjects. The two 19-or progestis altered certai estroge-iduced resposes i huma subjects, while megestrol seemed to have very little effect; i the experimetal aimals the progestatioal compouds were idistiguishable from oe aother. It is clear that much ca be leared about the effects of these hormoal agets o lipid metabolism by the use of these aimal models, but it is equally clear that extrapolatios to huma lipid metabolism or pathophysiology must be guarded. The preset ivestigatios do ot justify speculatios about their relatioship to atherogeic cardiovascular disease: much more fudametal ivestigatios of the effects of steroid hormoes o lipid metabolism will be required. Nevertheless, there has already bee a great deal of extrapolatio from fidigs i lower aimals such as rodets ad birds, without eve as much validatio of the qualitative ad quatitative similarity of their hormoe-iduced resposes to those of humas as we have tried to obtai i the baboo ad beagle. A good deal of cautio i drawig parallels ad makig causal ifereces would appear to be justified. There has also bee a great deal of simplistic iterpretatio of epidemiologic data, which provide idicatios of positive or egative cardiovascular risk associated with measured levels of various blood lipid compoets. The most recet report of the Framigham study 28 o idividuals 49 to 82 years of age showed that total cholesterol was ot associated with the risk of coroary heart disease; that HDL cholesterol was the major potet egative risk factor, eve whe other lipids ad stadard risk factors for coroary heart disease were take ito cosideratio. A weak positive risk associatio was observed for LDL cholesterol. I this light, oe ca speculate about the possible sigificace of the effects of estrogeic ad progestatioal compouds o these two plasma costituets i a much youger age group, but i ay evet it is ot permissible to equate the risk-idicator role of these costituets with causatio. Equally oversimplified ifereces have bee draw regardig triglyceride levels. I the Framigham study, 28 triglycerides were associated with the icidece of coroary heart disease oly i wome, ad the oly whe the level of other lipids was ot take ito accout. Cocomitat lipid disorders markedly affect the relatioship of hypertriglyceridemia to myocardial ifarctio. These associatios also do ot imply that the elevatio oftriglycerides itself causes the disease-it may merely be a idicator of some uderlyig pathologic process. Furthermore, oe caot assume the all causes of elevated plasma triglyceride possess a idetical potetial for cardiovascular harm. Just as all elevatios of blood glucose do ot carry with them the cardiovascular implicatios of diabetes mellitus, 29 so it is equally possible that all causes of elevated plasma tri-

12 Vol. 3, No.5 COMPARATIVE STUDIES OF ETHYNYL ESTROGENS 533 glyceride do ot automatically imply the same-or perhaps ay-haard of cardiovascular damage. Cardiovascular disease is clearly oe of the major complicatios of our cotemporary life-style, ad it is exceedigly difficult to refrai from traslatig experimetal or epidemiologic fidigs ito practical suggestios. However, the last decade has show us what a eormous ad very possibly useless displacemet of huma livig habits ca be brought about by premature coversio of highly circumscribed iformatio ito programmatic or regulatory actio. Ackowledgmets. We wish to express our appreciatio for the techical assistace of Mr. C. Celaya ad Mr. D. Garg, ad for the quality-cotrolled tablets of the various drugs prepared by Wyeth Laboratories. We are also most grateful for the helpful suggestios ad commets of Dr. Arthur Kruski. REFERENCES 1. 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