Effects of continuous conjugated estrogen and micronized progesterone therapy upon lipoprotein metabolism in postmenopausal women

Transcription

1 Effects of continuous conjugted estrogen nd micronized progesterone therpy upon lipoprotein metbolism in postmenopusl women Bernrd M. Wolfe,, * P. Hugh R. Brrett, Lisnne Lurier,* nd Murry W. Huff* Deprtment of Medicine,* The University of Western Ontrio, London, Ontrio, Cnd, N6A 5A5 nd Deprtment of Medicine, University of Western Austrli, Perth, WA 6000, Austrli Abstrct The effects of continuously dministering both conjugted equine estrogens (CEE) nd micronized progesterone (MP) on the concentrtion, composition, production nd ctbolism of very low density (VLDL) nd low density lipoproteins (LDL) hve not previously been reported. The mechnism of the hormonlly induced reductions of plsm LDL cholesterol of S f 0 20 (men 6%, P 0.005) nd LDL pob (men 6%, P 0.025) were investigted by studying the kinetics of VLDL nd LDL polipoprotein (po) B turnover fter injecting utologous I-lbeled VLDL nd 25 I-lbeled LDL into ech of the 6 modertely hypercholesterolemic postmenopusl subjects under control conditions nd gin in the fourth week of 7-week course of therpy (0.625 mg/d of CEE 200 mg/d of MP). The combined hormones significntly lowered plsm LDL pob by incresing the men frctionl ctbolic rte of LDL pob by 20% (0.2 vs pools/d, P 0.0). Tretment lso induced significnt increse in IDL production (6. vs..7 mg/kg/d, P 0.028). However, this did not result in n increse in LDL production becuse of n increse in IDL pob direct ctbolism (men 02%, P 0.0). VLDL kinetic prmeters were unchnged nd the concentrtions of plsm totl triglycerides (TG), VLDL- TG, VLDL-poB did not rise s often seen with estrogen lone. Plsm HDL-cholesterol rose significntly (P 0.02). Our mjor conclusion is tht incresed frctionl ctbolism of LDL underlies the LDL-lowering effect of the combined hormones. Wolfe, B. M., P. H. R. Brrett, L. Lurier, nd M. W. Huff. Effects of continuous conjugted estrogen nd micronized progesterone therpy upon lipoprotein metbolism in postmenopusl women. J. Lipid Res : Supplementry key words cholesterol estrogen progesterone VLDL LDL including coronry bypss surgery found no overll crdiovsculr benefit of HRT (4), observtionl studies of HRT or replcement of estrogen lone (ERT) hve reported reductions in both ll-cuse nd CHD mortlity in other PMP women (5, 6). Elevtions of plsm cholesterol (7, 8) nd triglycerides (7) hve been reported in high proportion of PMP women nd re thought to contribute to the ssocited increse in CHD (, 9 ). Estrogen replcement by HRT or ERT hs the potentil to reduce CHD risk through fvorble effects on both lipoproteins nd the rteril wll (2). Increses in both plsm triglycerides nd HDL cholesterol hve been reported in response to ERT in PMP women (). However, triglyceride responses to HRT involving continuous conjugted estrogen (CEE, mg/d) nd medroxyprogesterone cette (MPA, 2.5 mg/d) hve been inconsistent. Significnt increses in triglycerides hve been reported in some, but not ll, studies of 2 months or more in durtion (4 7), but no significnt increse in plsm triglycerides hs been reported in shorter studies of.7 to 9 months in durtion (8 2). MPA hs lso been reported to significntly reduce the CEE-induced increse in plsm triglycerides (5). Furthermore, estrogen-induced increments in HDL cholesterol hve tended to be blunted more by concomitnt dministrtion of MPA thn by nturl ovrin progestin dministered s micronized progesterone (MP 4, 24). Becuse prolonged estrogen therpy tht is unopposed by progestin is ssocited with risk of endometril neoplsi, there is need for progestin (4, 25, 26). Despite clinicl usge of MP together with CEE by PMP women (4, 24), there hs been no informtion bout the Ovrin hormone replcement therpy, defined s tretment with estrogen nd progestin (HRT), provides effective relief of vsomotor symptoms (), prevents osteoporosis (2) nd my reduce coronry hert disese (CHD, ). Although recent rndomized clinicl tril in older postmenopusl (PMP) women with dvnced CHD Abbrevitions: CHD, coronry hert disese; CEE, conjugted equine estrogen; MP, micronized progesterone; FCR, frctionl ctbolic rte; HRT, hormonl replcement therpy; IDL, intermedite density lipoproteins; MPA, medroxyprogesterone cette; Lp[], lipoprotein []. To whom correspondence should be ddressed. 68 Journl of Lipid Reserch Volume 4, 2000

2 effects of continuously dministering CEE nd MP (CEE cont / MP cont ) on the concentrtions, production, ctbolism nd/or composition of the mjor polipoprotein (po) B- contining lipoproteins [very low density (VLDL), intermedite density (IDL) nd low density lipoproteins (LDL)] in PMP women. The present study ws undertken to test the hypothesis tht HRT with CEE cont /MP cont would lower LDL-cholesterol by enhncing its frctionl ctbolism nd would blunt the increses in VLDL production. SUBJECTS AND METHODS Subjects Six prticipnts, who hd experienced typicl menopusl symptoms nd were menorrheic for t lest 6 months (serum follicle stimulting hormone 70 U/L) were recruited from London Helth Sciences Centre University Cmpus out-ptient clinics (Tble ). Bseline fsting plsm cholesterol concentrtions during Phse I Americn Hert Assocition diets prior to entry into the study exceeded 5.2 mmol/l, the cut-point of eligibility for dietry tretment recommended by the Ntionl Cholesterol Eduction Progrm (27). Two subjects hd fsting levels of plsm HDL cholesterol levels which were below the recommended cut-point of 0.9 mmol/l (27), 2 hd plsm triglycerides which exceeded the desirble upper limit of 2. mmol/l (28), nd 2 hd fsting levels of lipoprotein [] (Lp[]) which were slightly bove the 95th percentile (29). One subject hd primry hypothyroidism nd ws treted throughout the control nd experimentl periods with l-thyroxine 0.5 mg/d, long with 50 mg of cptopril nd 50 mg of hydrochlorothizide dily for hypertension. Although hydrochlorothizide cn rise serum cholesterol (0), ny potentil effect on lipid metbolism ws minimized by mintining the sme dosge level during both control nd tretment periods of the experiment. Subjects received no other medictions known to ffect lipid metbolism. Intke of ethnol ws less thn 0 ml/d for the month before ech study nd ws disllowed during the turnover studies. Subjects were instructed to mintin their low ft diets nd their regulr physicl ctivity for the durtion of the study, nd body weight ws monitored on eight occsions during ech control nd tretment period of their studies. Thus, men vlues for body weight (Tble ) nd body mss index (2.7. vs. 2.6.) were similr during the control nd tretment periods (P 0.5). All subjects hd norml fsting concentrtions of serum glucose, thyroxine, cretinine, clcium, lbumin, sodium, potssium, chloride, nd bicrbonte. Hemtologicl nd urine nlyses were norml. The experimentl protocol pproved by The Stnding Committee on Humn Reserch of The University of Western Ontrio ws explined to ech subject nd informed consent ws obtined in writing. Subjects were mintined on modertely low ft, low cholesterol diets contining pproximtely 29% of energy from ft (men rtio of polyunsturted:monounsturted:sturted ft c 0.8:.:.0, 200 mg cholesterol per dy), 9% of energy from protein, nd 52% of energy from crbohydrte. During dmissions to hospitl for the first dy only of ech turnover study the diet ws unchnged, except tht the crbohydrte content of the diet ws incresed t the expense of ft to mintin cloric intke. Ft intke ws reduced to 5 g per dy to essentilly eliminte the contribution of intestinl lipoproteins to plsm triglyceride-rich lipoproteins (). Smples of venous blood for determintion of plsm totl cholesterol, LDL cholesterol (S f 0 20) nd HDL cholesterol, totl triglycerides, nd Lp[] concentrtions were obtined from forerm vein (fter 2-h fst) from ech subject on four occsions t pproximtely weekly intervls ) during the initil -week control period before strting hormonl replcement nd b) during weeks to 6 of the 7-week course of tretment with CEE (0.625 mg/d) nd MP (200 mg/d), together referred to s CEE cont /MP cont. Subjects underwent studies of the kinetics of VLDL nd LDL pob turnover during the control period nd gin during the lst 6 dys of hormone replcement therpy (HRT) with CEE cont /MP cont. All subjects received 00 mg/d potssium iodide for dys before nd dys fter the reinjection of their rdiolbeled lipoproteins. Preprtion of lbeled lipoproteins nd kinetic studies The procedures for the lipoprotein turnover studies, including the isoltion nd preprtion of lbeled lipoproteins nd their reinjection, hve previously been described, long with the smpling nd frctiontion of VLDL (S f ), intermedite density lipoprotein (IDL, S f 2 60), nd LDL (S f 0 2) nd the isoltion of pob from ech lipoprotein frction by isopropnol precipittion nd determintion of its specfic ctivity (2, 2 5). The bolus injection of I-lbeled VLDL ws immeditely followed by the 25 I-lbeled LDL. Isotopic crossover ws corrected throughout the studies. Lipids were mesured in these lipoprotein frctions s described below. TABLE. Chrcteristics of postmenopusl subjects Weight Cholesterol Triglycerides HDL-cholesterol Subject Age Height Control Treted Control Treted Control Treted Control Treted yr cm kg mmol/l mmol/l mg/dl Men b SE Vlues for ech subject re the men of four fsting blood plsm smples obtined over weeks during low ft, low cholesterol diet for ech of the control nd tretment periods. Significntly different from control, P 0.0. b Significntly different from control, P Wolfe et l. Conjugted estrogen nd progesterone nd lipids 69

3 Fig.. Multicomprtmentl model for pob metbolism. See Methods: Kinetic Anlysis for detils. Kinetic nlysis A multicomprtmentl model ws used to describe VLDL, IDL, nd LDL-poB trcer dt. In multicomprtmentl modeling, ech comprtment or pool represents group of kineticlly homogeneous prticles. In this study the SAAM II progrm (SAAM Inst., Settle, WA) ws used to fit the model to the observed trcer dt. ApoB metbolic prmeters re subsequently derived from the model prmeters giving the best fit. All trcer dt ws fit simultneously using the comprtmentl model (Fig. ). Comprtments through 5 re used to describe the kinetics of pob in the VLDL frction. It ws ssumed tht ll VLDL pob enters plsm vi comprtment. Comprtments though 4 represent delipidtion chin or cscde s originlly described by Phir et l. (6). It is ssumed tht the residence time of prticles in ech comprtment of the chin is equl. In ddition, the frction of ech comprtment in the cscde converted to the slowly turning over VLDL comprtment, comprtment 5, is the sme. VLDL prticles in comprtment 4 cn be converted to IDL or cn be removed directly from plsm. The IDL section of the model includes comprtments 6 nd 7, rpidly nd slowly turning over pool of IDL prticles, respectively. Prticles in comprtment 6 cn be converted to the slow IDL comprtment, to LDL, or cn be removed directly from plsm. Becuse lbeled IDL ws not injected, it ws ssumed tht ll IDL ws derived from VLDL. The LDL section of the model is chrcterized by plsm comprtment, comprtment 8, nd n extrvsculr exchnge comprtment, comprtment 9. This model ssumes tht LDL pob is kineticlly homogeneous; however, the isoltion of LDL subfrctions nd/or the collection of urine rdioctivity, fter the injection of lbeled LDL, my provide evidence of kinetic heterogeneity within the LDL frction. Exmintion of the rw dt reveled tht significnt input of cold or unlbeled LDL must occur, s seen by observing the reltionship between the I-lbeled IDL nd LDL pob specific rdioctivity curves. The present model puts quntittive vlues on these observtions. The model tht ws fit to the experimentl dt ws the simplest model consistent with the dt. Errors of prmeters were low ( 20%) nd the residuls were rndomly llocted round the model fits. Anlyses of metbolites Plsm VLDL nd IDL pob concentrtions were mesured immunoturbidimetriclly using Tin-qunt polipoprotein B Kit obtined from Boehringer Mnnheim Cnd, Lvl, Quebec. The ssy ws stndrdized to LDL isolted by ultrcentrifugtion (d ) in which it ws determined tht pob ws the only protein present. The protein ws determined by the modified Lowry procedure (7). Lp[] ws mesured using Mcr Lp[] Kit obtined from Terumo Medicl Corp., Elkton, MD. The intr-ssy coefficients of vrition for pob nd Lp[] were.8 nd 4.7%, respectively. Fsting venous blood smples were lso obtined t weekly intervls from weeks 6 for mesurement of lipids, including LDL cholesterol of S f 0 20, which ws determined t these time points by subtrction of the cholesterol content of VLDL of S f (8) plus HDL cholesterol from tht of whole plsm. Concentrtions of cholesterol nd triglycerides in the chloroform methnol extrct of whole plsm were determined s previously described (9), except tht dignostic kits from Boehringer Mnnheim GmbH Dignostic, Montrel, Quebec (C-system Kit for cholesterol nd Test Combintion Kit for triglycerides) were used to mesure cholesterol content of fsting plsm VLDL, IDL, nd LDL nd triglyceride content of VLDL during the lst 6 dys the turnover studies becuse of the smll mounts of mteril vilble for ssy. HDL cholesterol ws mesured fter precipittion with heprin mngnese chloride (40). Vlues from control nd tretment periods were compred using Student s pired two-tiled t-test (4) or the Wilcoxon signed rnk test (42), s specified. Vrince ws expressed s stndrd error of the men. RESULTS Concentrtions of lipoprotein lipids nd pob During weeks to 6 of the dministrtion of CEE cont / MP cont, there were significnt decreses of 8 2% in fsting plsm totl cholesterol ( vs mmol/l, P 0.0, Tble ) nd 6 2% in the concentrtion of cholesterol of LDL of S f 0 20 ( vs mmol/l, P 0.005), wheres HDL cholesterol rose by 0 9% (P 0.025, Tble ). The hormones induced 29 % decrese in the men vlue for the rtio of totl cholesterol to HDL cholesterol versus control ( vs , P 0.02) nd 5 2% decrese in the vlue of the rtio of LDL (S f 0 20) cholesterol to HDL cholesterol (. 0. vs , P 0.0). Dt obtined t time of the turnover studies compring control versus tretment indicted tht CEE cont /MP cont reduced the men plsm concentrtion of cholesterol trnsported in LDL of S f 0 2 by % ( vs mmol/l, P 0.005) nd incresed men HDL cholesterol by 0 8% ( vs mmol/l, P 0.0, Tble 2). Simultneously, the men vlues tended to decline during tretment for ech of plsm totl triglycerides ( vs mmol/l), VLDL triglycerides ( vs mmol/l, Tble 2), IDL triglycerides ( vs mmol/l), nd LDL triglycerides ( vs mmol/l), however, the chnges were not LDL cholesterol of S f 0 20 ws not determined during turnover studies when LDL of S f 0 2 ws mesured. 70 Journl of Lipid Reserch Volume 4, 2000

4 TABLE 2. Effects of CEE cont /MP cont on levels of cholesterol nd triglycerides of plsm lipoproteins on first dy of turnover studies Subject, Period Whole Plsm VLDL Cholesterol Triglycerides Cholesterol Triglycerides IDL Cholesterol LDL Cholesterol HDL Cholesterol mmol/l Control Treted Control Treted Control Treted Control Treted Control Treted Control Treted Men SE Control Treted b c Vlues for ech subject were bsed upon fsting blood plsm smples obtined immeditely prior to injection of utologous lbeled I-lbeled VLDL (S f ) nd 25 I-lbeled LDL (S f 0 2) t the beginning of ech turnover study. Significntly different from control, P b Significntly different from control, P c Significntly different from control, P 0.0. sttisticlly significnt. The chnges in the men vlues for concentrtions of HDL triglycerides ( vs mmol/l) nd Lp[] (7 8 vs mg/dl) lso filed to be sttisticlly significnt. The men vlue for concentrtion of pob in plsm LDL ws significntly lower during hormonl tretment versus control (82 4 vs. 87 mg/dl, P 0.05); however, there ws no significnt chnge in men VLDL pob concentrtion ( vs mg/dl, P 0.2). The men vlue for the rtio of cholesterol to pob in plsm VLDL (S f ) ws 27 4% lower during hormonl tretment versus control ( vs , P 0.02 by Wilcoxon signed rnk test), but there ws no chnge in the rtio of triglycerides to pob (6 6 vs. 7 5, P 0.5). The men vlue for the rtio of cholesterol to pob in plsm IDL (S f 2 60) ws significntly lower (by 7 2%) during CEE cont /MP cont versus control (.8 0. vs , P 0.005), but there ws no chnge in the rtio of triglycerides to pob in IDL ( vs , respectively, P 0.5). There ws no significnt chnge in the men vlue for the rtio of cholesterol to pob of plsm LDL (S f 0 2) during CEE cont /MP cont versus control (.4 0. vs..5 0., respectively, P 0.), nor in the rtio of triglyceride to pob ( vs , P 0.). Consistent with stedy-stte, there were no systemtic chnges during the turnover studies in plsm concentrtions of the most redily quntified indices, nmely totl protein content of LDL (men coefficient of vrition of 0.7%, corresponding to men SD of 9.2 mg/dl, n 6 pired studies) nd totl protein content of VLDL (men coefficient of vrition of 2%, corresponding to men SD of.7 mg/dl, n 6 pired studies). Metbolism of lipoproteins Vlues for the specific ctivities of I-lbeled pob in VLDL, IDL, nd LDL over 72 h fter injection of I- lbeled VLDL during CEE cont /MP cont versus control for representtive subject re shown in Fig. 2. The kinetic prmeters of pob were determined from the simultneous nlysis of ll the specific ctivity dt by using the model shown in Fig.. The kinetic prmeters of VLDL turnover re summrized in Tble. There were nonsignificnt trends during tretment with CEE cont /MP cont towrds increses in production rte of VLDL pob (men vlues.4 2. vs mg/kg/d, P 0.26), in frctionl ctbolic rte of VLDL pob (men vlues vs , P 0.47) nd in pool size of VLDL pob ( 20 vs. 9 mg, P 0.2). Tht the conversion of VLDL to LDL did not chnge (6 6 vs. 6%, P 0.4) despite incresed conversion of VLDL to IDL (62 7 vs. 45 %, P 0.05) is consistent with greter proportion of the IDL pool being clered directly nd not converted to LDL. IDL to LDL conversion decresed during tretment (74 5 vs. 87 5, P 0.0) indicting tht higher proportion of IDL ws clered directly (26 vs. %). Sig- Wolfe et l. Conjugted estrogen nd progesterone nd lipids 7

5 TABLE. Kinetic prmeters of humn VLDL pob turnover in CEE cont /MP cont -treted postmenopusl subjects Subject, Period VLDL Pool Size Frctionl Ctbolic Rte VLDL Totl Production Conversion of VLDL to IDL Conversion of VLDL to LDL Fig. 2. Specific ctivity time curves of pob of VLDL, ILD, nd LDL fter intrvenous injection of utologous I-lbeled VLDL during control (open symbols) nd conjugted equine estrogen/micronized progesterone tretment (closed symbols) for subject no.. mg pools/d mg/kg/d % production Control Treted Control Treted Control Treted Control Treted Control Treted Control Treted Men SE Control Treted Significntly different from control, P nificntly incresed conversion of VLDL to IDL during hormone dministrtion prtly explins the significnt increse in IDL pob production ( vs mg/kg/d, P 0.028, Tble 4). The incresed IDL production would more thn offset the trend towrds n increse in IDL FCR ( vs pools/d, P 0.086), such tht there ws no significnt chnge in IDL pob pool size ( vs mg, P 0.4). The kinetic prmeters of LDL turnover re summrized in Tble 5. The FCR of LDL pob incresed significntly, by n verge of 20 7%, during hormonl dministrtion versus control ( vs pools/d, P 0.0). This resulted in significnt 6 2% decrese in LDL pob pool size (262 7 vs mg, P 0.05). Although production of LDL pob tended to rise, with increses in 5 of the 6 subjects, the chnge ws not significnt (P 0.0). LDL direct synthesis ws not significntly ltered by the combined hormones (8..5 vs mg/kg/d, P 0.58) nd direct LDL production s percent of totl LDL production ws unchnged by hormonl tretment (67 7 vs. 7 4%, P 0.5). Figure illustrtes the effect of CEE cont /MP cont on the decrese of the 25 I-lbeled LDL pob specific ctivity from the plsm for representtive subject. Dt in Fig. re presented s percent of pek specific ctivity becuse bsolute vlues for pek specific ctivity were different between the tretment nd control. The steeper slope during tretment indictes tht the frctionl ctbolic rte of LDL is more rpid during hormonl tretment thn control. TABLE 4. Kinetic prmeters of humn IDL pob turnover in CEE cont /MP cont -treted postmenopusl subjects Subject, Period IDL Pool Size Frctionl Ctbolic Rte IDL Totl Production mg pools/d mg/kg/d Control Treted Control Treted Control Treted Control Treted Control Treted Control Treted Men SE Control Treted Significntly different from control, P Journl of Lipid Reserch Volume 4, 2000

6 TABLE 5. Kinetic prmeters of humn LDL pob turnover in CEE cont /MP cont -treted postmenopusl subjects Subject, Period LDL Pool Size Frctionl Ctbolic Rte LDL Totl Production LDL Direct Production mg pools/d mg/kg/d mg/kg/d % Control Treted Control Treted Control Treted Control Treted Control Treted Control Treted Men SE Control Treted b Significntly different from control, P b Significntly different from control, P 0.0. DISCUSSION The present studies re the first to determine the effects of the continuously dministered CEE nd MP on the production, ctbolism, nd composition of VLDL, IDL, nd LDL. The combined hormones reduced plsm concentrtions of LDL-cholesterol nd LDL pob significntly by significntly incresing the FCR of LDL (Tble 5). An increse in the FCR of LDL-poB hs lso been observed during continuous dministrtion of CEE MPA (2). However, cyclicl estrdiol continuous norgestrel reduce LDL pool size by inhibiting LDL production (2). Wheres previous study involving the dily dministrtion of 2 mg of estrdiol lone to nine PMP women (2) found significnt 2% increse in LDL pob () production, the present study found only trend towrds smll increse (men 0%, Tble 5) suggesting the possibility tht MP nd/or other progestins could modulte the effects of estrogens on LDL kinetics. Phrmcologicl doses of ethinyl estrdiol in the rbbit hve been shown to increse LDL receptor expression (4) nd the increse in LDL frctionl ctbolism with the present hormones could be explined by estrogen-enhncement of LDL receptor ctivity. Prmeters of LDL metbolism in the present study were determined by multicomprtmentl nlysis of rdioiodinted LDL, which remins reference method for determining LDL-FCR (). The men control vlue for LDL-FCR in the present PMP women of pools/d ws in the sme rnge s tht obtined using similr technology in other groups of estrogen-deficient PMP women (2, 2, 44, 45). Comprisons between the present findings involving exogenous lbeling versus studies involving endogenous lbeling of LDL should tke into ccount the different experimentl methods nd different subject Fig.. Specific rdioctivity of LDL pob expressed s percent of the pek specific ctivity fter intrvenous injection of utologous 25 I-lbeled LDL for subject no. Wolfe et l. Conjugted estrogen nd progesterone nd lipids 7

7 popultions. Vlues for LDL-FCR derived from endogenous lbeling studies cn be overestimted if the experimentl protocol is too short, resulting in insufficient informtion on the LDL trcer dt to support the existence of n LDL exchnge pool. Similrly, overestimtion results if the studies rely upon LDL trcer dt derived from injected VLDL. Thus, somewht higher men vlue for LDL-FCR (i.e., 0.6, rnge ) hs been obtined during endogenous lbeling of LDL pob in helthy young men who hd men levels of LDL cholesterol which were % lower thn the present PMP women (46). Conversely, estrogen replcement increses vlues for LDL-FCR in PMP women, tending to rise them into the sme rnge s those of helthy men with norml to borderline high plsm cholesterol (47, 48). One of the potentil drwbcks of the exogenous pproch is tht VLDL isolted for lbeling nd reinjection my under-represent tht portion of the VLDL popultion tht is converted to LDL vi the delipidtion pthwy. In contrst, endogenous lbeling my llow the experimentlist to see the kinetics of ll VLDL prticles, leding to the conclusion tht lrger proportion of LDL is derived from the VLDL frction tht is seen with exogenous lbeling. However, the men control vlue for direct production of LDL in the present PMP womn (8. mg/kg/d) is similr to tht previously reported in other estrogen-deficient PMP women (2, 44). The 6% reduction in the fsting plsm concentrtion of LDL-cholesterol (S f 0 20) with the CEE cont /MP cont ccords with reductions of 5% observed during cyclicl dministrtion of MP together with continuous or cyclicl CEE (4, 24). The present findings indicte tht CEE cont /MP cont significntly increses IDL production (Tble 4) nd tht this, in turn, is lrgely explined by incresed conversion of VLDL to IDL (Tble ). However, the increse in production of IDL did not result in n increse in LDL production becuse of the increse in direct removl of IDL, which is consistent with up-regultion of LDL receptors by the CEE. Tretment lso significntly ltered the composition of IDL (S f 2 60), s reflected in 7% lower vlue for the rtio of cholesterol to pob in plsm IDL (S f 2 60) nd this explins why IDL cholesterol concentrtion ws unchnged despite the tendency for IDL pob pool size to increse (Tbles 2 nd 4). There hve been no previous reports of the effects on HDL-cholesterol of continuously dminstering both CEE nd MP. However, the reltively high proportionl increse in HDL-cholesterol with the present HRT could be explined, t lest in prt, by the observtion tht subjects with low HDL experience reltively lrger thn verge increses in HDL-cholesterol during ovrin hormone replcement (49). Furthermore, it hs been reported tht dministrtion of continuous CEE nd cyclicl MP tended to increse HDL more thn CEE lone (24). However, other studies found tht MP, like MPA, blunts estrogeninduced increses in HDL-cholesterol, but the reductions in increment re smller thn with MPA (4, 50). CEE lone increses production of VLDL TG (5) nd estrdiol dministered lone increses production of VLDL pob (). However, when CEE ws combined with MP in the present study (Tble 4) or combined with the C- 9 progestin MPA (2), no significnt increse in VLDL production or TG concentrtion ws observed. This suggests tht these progestins blunt the triglyceride-elevting effect of CEE, consistent with previous report tht the C- 9 progestin norethindrone cette inhibits heptic triglyceride secretion in swine model (50). As mentioned bove, numerous short-term nd some long-term studies re consistent with the view tht co-dministrtion of MPA with CEE blunts the expected estrogen-induced increments in plsm TG (5 20). In summry, the CEE cont /MP cont lowered LDL cholesterol nd pob by incresing the frctionl ctbolism of LDL of S f 0 2 nd thereby improved the plsm lipoprotein vsculr risk profile (52, 5). This work is supported by grnts from the Medicl Reserch Council of Cnd (MT4248) nd the Hert nd Stroke Foundtion of Ontrio (T297), nd the Ntionl Institutes of Helth (NHLBI HL490 nd NCRR RR0276). Murry W. Huff is Creer Investigtor of the Hert nd Stroke Foundtion of Ontrio. We thnk Brin Sutherlnd for expert technicl ssistnce. We thnk Mry Cnn for co-ordinting the studies. We re grteful to Jenette Mikulic nd Elene Wolfe for typing the mnuscript, nd Elene Wolfe for the grphic rt. Mnuscript received 26 April 999 nd in revised form 2 November 999. REFERENCES. Greendle, G. A Symptom relief nd side effects of postmenopusl hormones: results from the postmenopusl estrogen/progestin interventions tril. Obstet. & Gynecol. 92: Writing Group for the PEPI Tril Effects of hormone therpy on bone minerl density. J. Am. Med. Assoc. 276: Goldmn, L., nd A. N. Tosteson. 99. Uncertinty bout postmenopusl estrogen. N. Engl. J. Med. 25: Hulley, S., D. Grdy, T. Bush, C. Furberg, D. Herrington, B. Riggs, nd E. Vittinghoff Rndomized tril of estrogen plus progestin for secondry prevention of coronry hert disese in postmenopusl women. J. Am. Med. Assoc. 280: Stmpfer, M. J., G. A. Colditz, W. C. Willett, J. E. Mnson, B. Rosner, F. E. Speizer, nd C. H. Hennekens. 99. Postmenopusl estrogen therpy nd crdiovsculr disese. Ten-yer follow-up from the Nurses Helth Study. N. Engl. J. Med. 25: Grdy, D., S. M. Rubin, D. B. Petitti, C. S. Fox, D. Blck, B. Ettinger, V. L. Ernster, nd S. R. Cummings Hormone therpy to prevent disese nd prolong life in postmenopusl women. Ann. Int. Med. 7: Connelly, P. W., D. R. McLen, L. Horlick, B. O Connor, A. Petrsovits, nd A. Little Plsm lipids nd lipoproteins nd the prevlence of risk for coronry hert disese in Cndin dults. Cndin hert helth surveys: profile of crdiovsculr risk. Cn. Med. Assoc. J. 46 (Specil Supplement): Bengtsson, C., nd O. Lindquist Coronry hert disese during the menopuse. In Coronry Hert Disese in Young Women. M. F. Oliver, editor. Churchill-Livingstone, New York, NY Lucino, A. A Hormone replcement therpy in postmenopusl women. Infert. Reprod. Clin. N. Am. : Mishell, D. R., Jr Steroids from menrche to menopuse. Introduction. Am. J. Obstet. Gynecol. 66: Brrett-Connor, E., nd T. L. Bush. 99. Estrogen nd coronry hert disese in women. J. Am. Med. Assoc. 265: Lobo, R. A. 99. Clinicl review 27: Effects of hormonl replcement on lipids nd lipoproteins in postmenopusl women. J. Clin. Endocrinol. Metb. 7: Journl of Lipid Reserch Volume 4, 2000

8 . Wlsh, B. W., I. Schiff, nd B. Rosner. 99. Effects of postmenopusl estrogen replcement on the concentrtions nd metbolism of plsm lipoproteins. N. Engl. J. Med. 25: The Writing Group for the PEPI Tril Effects of estrogen or estrogen/progestin regimens on hert disese risk fctors in postmenopusl women. The postmenopusl estrogen/progestin interventions (PEPI) Tril. J. Am. Med. Assoc. 27: Lobo, R. A., J. H. Pickr, R. A. Wild, B. Wlsh, nd E. Hirvonen Metbolic impct of dding medroxyprogesterone cette to conjugted estrogen therpy in postmenopusl women. Obstet. Gynecol. 84: Lucino, A. A., M. J. DeSouz, M. P. Roy, M. J. Schoenfeld, J. C. Nulsen, nd C. V. Hlvorson. 99. Evlution of low-dose estrogen nd progestin therpy in postmenopusl women. J. Rep. Med. 8: Weinstein, L., C. Bewtr, nd J. C. Gllgher Evlution of continuous combined low-dose regimen of estrogen-progestin for tretment of the menopusl ptient. Am. J. Obstet. Gynecol. 62: Lucino, A. A., R. N. Turksoy, J. Crleo, nd J. W. Hendrix Clinicl nd metbolic responses of menopusl women to sequentil versus continuous estrogen nd progestin replcement therpy. Obstet. Gynecol. 7: Prough, S. G., S. Aksel, R. H. Wiebe, nd J. Shepherd Continuous estrogen/progestin therpy in menopuse. Am. J. Obstet. Gynecol. 57: Weinstein, L Efficcy of continuous estrogen-progestin regimen in the menopusl ptient. Obstet. Gynecol. 69: Kble, W. T., J. C. Gllgher, L. Nchtigll, nd D. Goldgr Lipid chnges fter hormone replcement therpy for menopuse. J. Rep. Med. 5: McLennn, A. H., A. McLennn, S. Wenzel, H. M. Chmbers, nd K. Eckert. 99. Continuous low-dose oestrogen nd progestogen hormone replcement therpy: rndomised tril. Med. J. Aust. 59: Wolfe, B. M., nd M. W. Huff Effects of continuous low-dosge hormonl replcement therpy on lipoprotein metbolism in postmenopusl women. Metbolism. 44: Moorjni, S., A. Dupont, F. Lbrie, B. DeLignieres, L. Cusn, P. Dupont, J. Milloux, nd P-J. Lupien. 99. Chnges in plsm lipoprotein nd polipoprotein composition in reltion to orl versus percutneous dministrtion of estrogen lone or in cyclic ssocition with Urogestn in menopusl women. J. Clin. Endocrinol. Metb. 7: Lne, G., M. I. Whitehed, nd R. J. B. King Effects of oestrogens nd progestogens on the histology, ultrstructure, nd biochemistry of the postmenopusl endometrium. Int. Med. 2: Gmbrell, R. D., F. M. Msse, T. A. Cstned, A. J. Ugens, C. A. Ricci, nd J. M. Wright Use of the progestogen chllenge test to reduce the risk of endometril cncer. Obstet. Gynecol. 55: Expert Pnel on Detection, Evlution, nd Tretment of High Blood Cholesterol in Adults. Summry of the Second Report of the Ntionl Cholesterol Eduction Progrm (NCEP) Expert Pnel on Detection, Evlution, nd Tretment of High Blood Cholesterol in Adults (Adult Tretment Pnel II). 99. J. Am. Med. Assoc. 269: Cndin Consensus Conference on Cholesterol Finl Report. Cn. Med. Assoc. J. 9 (suppl): Jenner, J. L., J. M. Ordovs, S. Lmon-Fv, M. M. Schefer, P. W. Wilson, W. P. Cstelli, nd E. J. Schefer. 99. Effects of ge, sex, nd menopusl sttus on plsm lipoprotein [] levels. The Frminghm Offspring Study. Circultion. 87: Elliott, W. J Dose-response of serum cholesterol during longterm therpy with thizides. Clin. Phrmcol. Ther. 55: 206 (Abstr.). Hvel, R. J., J. P. Kne, E. O. Blsse, N. Segel, nd L. V. Bsso Splnchnic metbolism of free ftty cids nd production of triglycerides of very low density lipoproteins in normotriglyceridemic nd hypertriglyceridemic humns. J. Clin. Invest. 49: Wolfe, B. M., nd M. W. Huff Effects of combined estrogen nd progestin dministrtion on plsm lipoprotein metbolism in postmenopusl women. J. Clin. Invest. 8: Fidge, N. H., nd P. Poulis, 974. Studies on the rdio-iodintion of very low density lipoproteins obtined from different mmmlin species. Clin. Chim. Act. 52: Rerdon, M. F., N. H. Fidge, nd P. J. Nestel Ctbolism of very low density lipoprotein B poprotein in mn. J. Clin. Invest. 6: Huff, M. W., D. E. Telford, K. Woodcroft, nd W. L. Strong Mevinolin nd cholestyrmine inhibit the direct synthesis of low density lipoprotein polipoprotein B in miniture pigs. J. Lipid Res. 26: Phir, R. D., M. G. Hmmond, J. A. Bowden, M. Fried, W. R. Fisher, nd M. A. Bermn Preliminry model for humn lipoprotein metbolism in hyperlipoproteinemi. Fed. Proc. 4: Lowry, O. H., N. J. Rosebrough, A. L. Frr, nd R. J. Rndll. 95. Protein mesurement with the Folin phenol regent. J. Biol. Chem. 9: Hvel, R. J., H. A. Eder, nd J. H. Brgdon The distribution nd chemicl composition of ultrcentrifuglly seprted lipoproteins in humn serum. J. Clin. Invest. 4: Huff, M. W., P. M. Giovnnetti, nd B. M. Wolfe Turnover of very low-density lipoprotein-poprotein B is incresed by substitution of soyben protein for met nd diry protein in the diets of hypercholesterolemic men. Am. J. Clin. Nutr. 9: Wrnick, G. R., nd J. J. Albers A comprehensive evlution of the heprin-mngnese precipittion procedure for estimting high density lipoprotein cholesterol. J. Lipid Res. 9: Snedecor, G. W., nd W. G. Cochrn The comprison of two smples. In Sttisticl Methods, 6th edition. Iow Stte University Press, Ames, IA ; Kirkwood, B. R Non-prmetric methods. In Essentils of Medicl Sttistics. Blckwell Scientific Publictions, Oxford M, P. T., T. Ymmoto, J. L. Goldstein, nd M. S. Brown Incresed mrna for low density lipoprotein receptor in livers of rbbits treted with 7 -ethinyl estrdiol. Proc. Ntl. Acd. Sci. USA. 8: Wolfe, B. M., nd M. W. Huff. 99. Effects of low dosge progestin-only dministrtion upon plsm triglycerides nd lipoprotein metbolism in postmenopusl women. J. Clin. Invest. 92: Rerdon, M. F., N. H. Fidge, nd P. J. Nestel Ctbolism of very low density lipoprotein B poprotein in mn. J. Clin. Invest. 6: Demnt, T., C. J. Pckrd, H. Demmelmir, P. Stewrt, A. Bedynek, D. Bedford, D. Seidel, nd J. Shepherd Sensitive methods to study humn poliprotein B metbolism using stble isotopelbeled mino cids. Am. J. Physiol. 270: E022 E Simons, L. A., S. Blsubrmnim, nd J. Hollnd. 98. Low density lipoprotein metbolism in the norml to modertely elevted rnge of plsm cholesterol: comprisons with fmilil hypercholesterolemi. J. Lipid Res. 24: Grundy, S. M. 99. Multifctoril etiology of hypercholesterolemi. Arterioscler. Thromb. : Gibbons, W. E., H. L. Judd, A. A. Lucino, L. E. Nchtigll, M. Y. Dwood, K. Burry, H. Ziegl, M. S. LeBoff, G. El-Hij, F. Ogrinc, M. J. Schoenfield, R. P. Buylos, E. Adshi, nd P. Schntz. 99. Comprison of sequentil versus continuous estrogen/progestin replcement therpy on serum lipid ptterns. Am. Soc. Gynecol. Invest. 44: No. 49 (Abstr.) 50. Brrett-Connor, E., S. Slone, G. Greendle, D. Kritz-Silverstein, M. Espelnd, S. R. Johnson, M. Mclwiw, nd S. E. Fineberg The postmenopusl estrogen/progestin interventions study: primry outcomes in dherent women. Mturits. 27: Wolfe, B. M., nd D. M. Grce Norethindrone cette inhibition of splnchnic triglyeride secretion in conscious glucose-fed swine. J. Lipid Res. 20: Mishell, D. R Estrogen replcement therpy: n overview. Am. J. Obstet. Gynecol. 6: Wolf, P. H., J. H. Mdns, F. F. Finucne, M. Higgins, nd J. C. Kleinmn. 99. Reduction of crdiovsculr disese-relted mortlity mong postmenopusl women who use hormones: evidence from ntionl cohort. Am. J. Obstet. Gynecol. 64: Wolfe et l. Conjugted estrogen nd progesterone nd lipids 75