help button home button Endocrine Society JCEM JCEM Call for Nominations for EIC
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Vihma, V.
Right arrow Articles by Tikkanen, M. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vihma, V.
Right arrow Articles by Tikkanen, M. J.
The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 6 2552-2555
Copyright © 2003 by The Endocrine Society

Quantitative Determination of Estradiol Fatty Acid Esters in Lipoprotein Fractions in Human Blood

Veera Vihma, Aila Tiitinen, Olavi Ylikorkala and Matti J. Tikkanen

Departments of Medicine (V.V., M.J.T.) and Obstetrics and Gynecology (A.T., O.Y.), University of Helsinki, 00290 Helsinki, Finland

Address all correspondence and requests for reprints to: Matti J. Tikkanen, M.D., Department of Medicine, Helsinki University Central Hospital, PB 340, 00290 Helsinki, Finland. E-mail: matti.tikkanen{at}hus.fi.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
According to experimental studies, 17ß-estradiol associates with lipoproteins in blood in the form of fatty acid esters. However, concentrations of endogenous estradiol esters in human lipoprotein fractions have not been previously reported. We investigated the distribution of estradiol fatty acid esters between plasma lipoproteins in 10 healthy women during late pregnancy. Following extraction from serum and ultracentrifugally isolated, gel-filtered lipoproteins, estradiol esters were separated from nonesterified estradiol by column chromatography. After saponification and chromatographic purification of the estradiol ester fraction, the concentration of hydrolyzed esters was determined by estradiol time-resolved fluoroimmunoassay. Of total serum estradiol, a mean of 0.7% (549 pmol/liter, n = 10) was in the form of fatty acid esters. Estradiol fatty acid ester concentrations measured in serum and lipoprotein fraction correlated positively (n = 10; r = 0.98; P < 0.001). The majority of lipoprotein estradiol esters, 54%, was recovered in high-density lipoprotein, and 28% in low-density lipoprotein fraction. Most lipoprotein fractions contained undetectable amounts of nonesterified estradiol (< 36 pmol/liter). In conclusion, our results indicate that estradiol fatty acid esters are mostly bound by lipoproteins in blood in vivo.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THE PRINCIPAL ESTROGEN of fertile women, 17ß- estradiol, circulates in human blood mainly bound by SHBG and albumin (1). Several experimental studies suggest, however, that estradiol also associates with plasma lipoproteins in the form of lipophilic fatty acid esters (2, 3, 4, 5, 6, 7, 8). According to studies in vitro, fatty acid esters of steroids are synthesized in blood by a high-density lipoprotein (HDL)-associated enzyme, lecithin:cholesterol acyltransferase (4, 8, 9, 10, 11). From HDL, steroid fatty acid esters may be transferred to very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) particles (7, 12). The metabolic function of esterified steroids is not known, but incorporation in lipoproteins might provide an alternative means of steroid transport and delivery to cells (13). Estradiol-17-fatty acid esters are metabolically stable and could conceivably serve as a lipophilic storage form of active estradiol in tissues (14). In addition to their prolonged estrogenic action, estradiol fatty acid esters are of interest because of their antioxidant potential (15, 16).

Only a few studies have measured concentrations endogenous estradiol fatty acid esters in humans. In premenopausal women, plasma estradiol fatty acid ester levels constitute 2–22% of total serum estradiol levels (17), and their concentrations increase in women undergoing ovarian stimulation (17) and during pregnancy (18). The distribution of endogenous estradiol fatty acid esters in human plasma is, however, not known. In this study, we set out to measure estradiol fatty acid ester and nonesterified estradiol concentrations in human lipoproteins in late pregnancy.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Isolation of serum lipoproteins

Blood was drawn from 10 healthy women at 37–40 wk of normal gestation who used no medication. The study was approved by the ethics committee of the Department of Obstetrics and Gynecology in Helsinki University Central Hospital, and informed consent was obtained from subjects. Serum was prepared by centrifugation at 1200 x g 10 min. Lipoproteins (VLDL, LDL, HDL subclass 2 [HDL2], and HDL subclass 3 [HDL3]) were isolated from 3 ml serum by sequential ultracentrifugation as previously described (8). In addition to individual lipoprotein fractions, a total lipoprotein fraction (containing VLDL, LDL, and HDL fractions) was isolated from 2 ml serum by a single ultracentrifugation at density (d) less than 1.21 g/ml.

Lipoprotein purification

Ultracentrifugally isolated lipoprotein fractions were purified from unbound estrogens and low molecular weight contaminants by size exclusion gel filtration using Sephadex G-25 column (column dimensions 1 x 20 cm; Pharmacia Biotech, Uppsala, Sweden). Lipoproteins were eluted with PBS (pH 7.4) in the void volume (3–6 ml) and stored at -80 C. The protein concentration in each lipoprotein fraction was determined before and after gel filtration (19). Total lipoprotein fractions (n = 5) isolated by single ultracentrifugation at d less than 1.21 g/ml contained no detectable albumin (<0.1 µg albumin/10 µg protein) as determined by SDS-PAGE with Coomassie blue staining.

Measurement of serum and lipoprotein estradiol and estradiol fatty acid ester concentrations

Estradiol fatty acid ester and estradiol concentrations in serum and gel-filtered lipoprotein fractions were determined as previously described (18). In short, tritiated internal standard (3H-estradiol-3,17ß-dioleate) was added to serum (1 ml), lipoprotein fraction (3–6 ml) obtained after gel filtration on Sephadex G-25, and estradiol-17ß-stearate containing control serum (1 ml) samples, followed by extraction with diethylether:ethyl acetate. After separation of estradiol fatty acid esters from nonesterified estradiol by Sephadex LH-20 column chromatography (Amersham Pharmacia Biotech AB, Uppsala, Sweden), the estradiol ester fraction was saponified in methanolic KOH, followed by washing in Sep-Pak C18 column (Waters Corp., Milford, MA) and removing of cholesterol and other lipid impurities by Lipidex 5000 (Packard Bioscience B.V., Gröningen, The Netherlands) reversed-phase and Sephadex LH-20 column chromatography. The dry residues of the hydrolyzed estradiol ester fraction and the nonesterified estradiol fraction were dissolved in buffer, and the concentration of estradiol in both fractions was analyzed in duplicate by estradiol time-resolved fluoroimmunoassay (Wallac Oy, Turku, Finland) as previously described (18). The concentration of estradiol fatty acid esters in serum and lipoprotein fractions was calculated by correcting for recovery of the tritiated internal estradiol ester standard, as measured by liquid scintillation counting. Estrogen concentrations in total lipoprotein fraction were also corrected according to recovery of lipids in lipoprotein ultracentrifugation (mean recovery of cholesterol, 97%), and recovery of proteins in gel filtration on Sephadex G-25 (mean recovery of protein, 95%). The concentration of estradiol fatty acid esters is expressed as picomoles of estradiol per liter. According to the coefficients of variation of the low, medium, and high control samples, the interassay imprecision of the quantitative estradiol ester method in this study was 6%, 7%, and 13% in seven assays, respectively.

Other measurements

Serum and lipoprotein cholesterol (ABX Diagnostics, Montpellier, France) and triglyceride concentrations (ABX Diagnostics) were measured by enzymatic methods.

Statistical analyses

Data are expressed as the mean (SEM) unless otherwise stated. Correlation analyses were performed using Spearman’s nonparametric correlation coefficient.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Serum lipids in late pregnancy

All subjects had pregnancy-associated physiologic hyperlipidemia. The mean concentration of serum cholesterol was 7.2 (0.4) mmol/liter, triglycerides 3.5 (0.4) mmol/liter, and LDL cholesterol 4.3 (0.3) mmol/liter (n = 10).

Endogenous estradiol fatty acid esters and nonesterified estradiol in pregnancy serum and lipoproteins

Estradiol fatty acid ester and nonesterified estradiol concentrations in serum and gel-filtered total lipoprotein fraction (isolated by single ultracentrifugation at d < 1.21 g/ml) are shown in Table 1Go. Of total estradiol in late pregnancy serum, a mean of 0.7% was in the form of fatty acid esters [95% confidence interval (CI), 0.5–1.0%]. The concentration of estradiol fatty acid esters in serum correlated positively with the concentration of total nonesterified estradiol in serum (n = 10, r = 0.81, P = 0.005). The majority of serum estradiol fatty acid esters, 75% (95% CI, 69–82%), was recovered in the isolated total lipoprotein fraction, indicating that hydrophobic estradiol esters bind preferentially to lipoprotein particles in blood. As shown in Fig. 1Go, a strong positive correlation existed between estradiol fatty acid ester concentrations measured in serum and in total lipoprotein fraction (n = 10; r = 0.98; P < 0.001). Serum estradiol fatty acid ester concentration also significantly correlated with estradiol ester concentration in LDL fraction (n = 10; r = 0.88; P = 0.001).


View this table:
[in this window]
[in a new window]
  		Table 1. Concentrations of endogenous estradiol (E2) fatty acid esters and nonesterified E2 in serum and lipoprotein fraction in 10 women in late pregnancy

 


View larger version (14K):
[in this window]
[in a new window]
  		Figure 1. Correlation between estradiol fatty acid ester concentration in total lipoprotein fraction and estradiol fatty acid ester concentration in serum in human late pregnancy (n = 10, r = 0.98, P < 0.001). Total lipoprotein fraction was isolated by single ultracentrifugation at d less than 1.21 g/ml followed by gel filtration by Sephadex G-25, and serum and lipoprotein estradiol ester concentrations were determined as described in Subjects and Methods. E2 ester, Estradiol fatty acid ester (expressed as picomoles per liter estradiol).

 
All of the individual lipoprotein fractions studied (VLDL, LDL, HDL2, and HDL3) contained quantifiable amounts of estradiol fatty acid esters. The HDL3 subfraction contained estradiol only in esterified form in every subject, and its estradiol ester concentration correlated positively with estradiol ester concentrations in other lipoprotein fractions: VLDL (n = 10, r = 0.76, P = 0.011), HDL2 (n = 10, r = 0.65, P = 0.043) and most significantly with that in LDL fraction (n = 10, r = 0.88, P = 0.001). The mean estradiol fatty acid ester concentration in VLDL was 89 (18) pmol/liter, LDL 143 (27) pmol/liter, HDL2 148 (20) pmol/liter, and HDL3 137 (45) pmol/liter. As illustrated in Fig. 2AGo, most of the lipoprotein estradiol fatty acid esters were recovered in HDL and LDL fractions. The estradiol ester to cholesterol ratio (picomoles per millimole) was greatest in the HDL fraction (HDL2 + HDL3) (Fig. 2BGo).



View larger version (12K):
[in this window]
[in a new window]
  		Figure 2. The mean (SD) distribution of estradiol fatty acid esters in isolated and purified lipoprotein fractions (A) and the mean (SD) distribution of estradiol fatty acid esters in lipoprotein fractions relative to lipoprotein cholesterol content (picomoles per millimole) (B) in human late pregnancy (n = 10). The results are expressed as percentage of total estradiol ester concentration in lipoproteins (A) and total estradiol ester to cholesterol ratio in lipoproteins (B).

 
Low concentrations of nonesterified estradiol constituting on the average 0.2% of serum estradiol levels (95% CI, 0.1–0.3%) were detected in all subjects in the total lipoprotein fraction (Table 1Go). In most of the subjects, however, nonesterified estradiol was not detectable in isolated individual lipoprotein fractions (VLDL, LDL, and HDL subfractions; data not shown).


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
We determined the concentrations of estradiol fatty acid esters in various lipoprotein fractions during human late pregnancy when serum concentrations of free and derivatized estrogens are increased. This is the first study to quantify endogenous nonpolar derivatives of estradiol in human lipoprotein fractions. The majority of lipoprotein estradiol fatty acid esters was associated with HDL (54%) and LDL particles (28%). In contrast to estradiol fatty acid esters, nonesterified estradiol showed low binding to serum lipoproteins. Serum estradiol fatty acid ester concentration correlated positively with serum total estradiol concentration.

The quantitative determination of estrogen concentrations in lipoprotein fractions was methodologically more complicated than in serum because of the many isolation and purification steps needed. Our results suggest that about 75% of serum estradiol fatty acid esters are carried in lipoproteins. Considering the extreme nonpolarity of esterified estradiol, it was unexpected that not all of it could be detected in the total lipoprotein fraction. This finding was probably not due to methodological losses because we performed careful corrections for recovery. First, procedural losses during ultracentrifugation of the total lipoprotein fraction were estimated according to recovery of cholesterol. Second, following isolation, lipoprotein fractions were subjected to purification by gel filtration and recovery was assessed according to the recovery of protein. Finally, radioactive estradiol ester was added to gel-filtered lipoprotein fraction and used as an internal standard.

The subjects in this study had pregnancy-associated hyperlipidemia that may, in theory, influence the association of steroid hormones with plasma lipoproteins (20). Alterations in the size and composition of lipoproteins during pregnancy (21, 22) could also affect esterification of estradiol in HDL. Thus, it is not known whether the present results can be extrapolated to normolipidemic or nonpregnant subjects. A smaller proportion of total serum estradiol appears to be esterified with fatty acids in pregnant women, compared with nonpregnant and especially with postmenopausal subjects (23). This is in line with the suggested role of estradiol fatty acid esters as a storage form of active hormone (17).

Our results provide evidence that estradiol fatty acid esters are transported in human plasma mainly by HDL particles by which they are synthesized. The presence of endogenous estradiol esters also in VLDL and LDL fractions suggests transfer of these derivatives from HDL, in analogy with cholesterol esters. This is in agreement with our previous study that showed that estradiol fatty acid esters may be transferred from HDL to LDL by a cholesterol ester transfer protein-dependent mechanism (7).

Shwaery et al. (15) have shown that esterified estradiol may act as antioxidant protecting lipoproteins against oxidation. In their study, LDL that was isolated after incubating plasma with 1.0–10 nmol/liter labeled estradiol, contained label corresponding to 0.04–0.38 pmol esterified estradiol per milligram of LDL protein. This resulted in an increased resistance of LDL to copper-mediated oxidation in vitro. We measured similar concentrations of endogenous esterified estradiol in pregnancy LDL (0.05–0.38 pmol/mg LDL protein).

Some previous studies have found relatively high concentrations of nonesterified estradiol in lipoproteins in fertile women, 5–10% (24, 25) or even higher percentages (26) of serum total estradiol. In the present study, however, most of the individual lipoprotein fractions analyzed did not contain detectable amounts of nonesterified estradiol despite the high serum estradiol concentration in late pregnancy. Several studies in which estradiol has been incubated with human plasma or isolated lipoprotein fractions (2, 7, 16) have also indicated that nonesterified estradiol does not significantly bind to lipoproteins.

In conclusion, we report that three fourths of endogenous estradiol fatty acid esters are transported by lipoproteins in vivo in human late pregnancy. Further studies are needed to clarify the physiological role of lipoprotein-bound estrogen derivatives in pregnant and nonpregnant subjects.


   Acknowledgments
 
Kirsti Räsänen and Terhi Hakala are acknowledged for expert technical assistance.


   Footnotes
 
This work was supported by grants from the Sigrid Juselius Foundation, the Päivikki and Sakari Sohlberg Foundation, and Erityisvaltionosuus (EVO) Grants TYH 0337 and 1241.

Abbreviations: CI, Confidence interval; d, density; HDL, high-density lipoprotein; HDL2, HDL subclass 2; HDL3, HDL subclass 3; LDL, low-density lipoprotein; VLDL, very low-density lipoprotein.

Received December 3, 2002.

Accepted March 6, 2003.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Dunn JF, Nisula BC, Rodbard D 1981 Transport of steroid hormones: binding of 21 endogenous steroids to both testosterone-binding globulin and corticosteroid-binding globulin in human plasma. J Clin Endocrinol Metab 53:58–68[Abstract]
  2. Larner JM, Rosner W, Hochberg RB 1987 Binding of estradiol-17-fatty acid esters to plasma proteins. Endocrinology 121:738–744[Abstract]
  3. Leszczynski DE, Schafer RM 1990 Nonspecific and metabolic interactions between steroid hormones and human plasma lipoproteins. Lipids 25:711–718[Medline]
  4. Leszczynski DE, Schafer RM 1991 Metabolic conversion of six steroid hormones by human plasma high-density lipoprotein. Biochim Biophys Acta 1083:18–28[Medline]
  5. Meng QH, Höckerstedt A, Heinonen S, Wähälä K, Adlercreutz H, Tikkanen MJ 1999 Antioxidant protection of lipoproteins containing estrogens: in vitro evidence for low- and high-density lipoproteins as estrogen carriers. Biochim Biophys Acta 1439:331–340[Medline]
  6. Abplanalp W, Scheiber MD, Moon K, Kessel B, Liu JH, Subbiah MT 2000 Evidence for the role of high density lipoproteins in mediating the antioxidant effect of estrogens. Eur J Endocrinol 142:79–83[Abstract]
  7. Helisten H, Höckerstedt A, Wähälä K, Tiitinen A, Adlercreutz H, Jauhiainen M, Tikkanen MJ 2001 Accumulation of high-density lipoprotein-derived estradiol-17ß fatty acid esters in low-density lipoprotein particles. J Clin Endocrinol Metab 86:1294–1300[Abstract/Free Full Text]
  8. Höckerstedt A, Tikkanen MJ, Jauhiainen M 2002 LCAT facilitates transacylation of 17ß-estradiol in the presence of HDL3 subfraction. J Lipid Res 43:392–397[Abstract/Free Full Text]
  9. Jones DL, James VH 1985 The identification, quantification and possible origin of non-polar conjugates in human plasma. J Steroid Biochem 22:243–247[CrossRef][Medline]
  10. Pahuja SL, Hochberg RB 1995 A comparison of the esterification of steroids by rat lecithin:cholesterol acyltransferase and acyl coenzyme A:cholesterol acyltransferase. Endocrinology 136:180–186[Abstract]
  11. Kanji SS, Kuohung W, Labaree DC, Hochberg RB 1999 Regiospecific esterification of estrogens by lecithin:cholesterol acyltransferase. J Clin Endocrinol Metab 84:2481–2488[Abstract/Free Full Text]
  12. Provost PR, Lavallée B, Bélanger A 1997 Transfer of dehydroepiandrosterone- and pregnenolone-fatty acid esters between human lipoproteins. J Clin Endocrinol Metab 82:182–187[Abstract/Free Full Text]
  13. Provencher PH, Roy R, Bélanger A 1992 Pregnenolone fatty acid esters incorporated into lipoproteins: substrates in adrenal steroidogenesis. Endocrinology 130:2717–2724[Abstract]
  14. Hochberg RB 1998 Biological esterification of steroids. Endocr Rev 19:331–348[Abstract/Free Full Text]
  15. Shwaery GT, Vita JA, Keaney Jr JF 1997 Antioxidant protection of LDL by physiological concentrations of 17ß-estradiol. Requirement for estradiol modification. Circulation 95:1378–1385[Abstract/Free Full Text]
  16. Shwaery GT, Vita JA, Keaney Jr JF 1998 Antioxidant protection of LDL by physiologic concentrations of estrogens is specific for 17-beta-estradiol. Atherosclerosis 138:255–262[CrossRef][Medline]
  17. Larner JM, Shackleton CH, Roitman E, Schwartz PE, Hochberg RB 1992 Measurement of estradiol-17-fatty acid esters in human tissues. J Clin Endocrinol Metab 75:195–200[Abstract]
  18. Vihma V, Adlercreutz H, Tiitinen A, Kiuru P, Wähälä K, Tikkanen MJ 2001 Quantitative determination of estradiol fatty acid esters in human pregnancy serum and ovarian follicular fluid. Clin Chem 47:1256–1262[Abstract/Free Full Text]
  19. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ 1951 Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275[Free Full Text]
  20. Leszczynski DE, Schafer RM 1989 Characterization of steroid hormone association with human plasma lipoproteins. Steroids 54:37–53[CrossRef][Medline]
  21. Skryten A, Johnson P, Gustafson A 1980 Studies in normal pregnancy—serum lipids, lipoproteins, and uric acid (II). Acta Obstet Gynecol Scand 59:1–5[Medline]
  22. Winkler K, Wetzka B, Hoffmann MM, Friedrich I, Kinner M, Baumstark MW, Wieland H, März W, Zahradnik HP 2000 Low density lipoprotein (LDL) subfractions during pregnancy: accumulation of buoyant LDL with advancing gestation. J Clin Endocrinol Metab 85:4543–4550[Abstract/Free Full Text]
  23. Vihma V, Vehkavaara S, Yki-Järvinen H, Hohtari H, Tikkanen MJ 2003 Differential effects of oral and transdermal estradiol treatment on circulating estradiol fatty acid ester concentrations in postmenopausal women. J Clin Endocrinol Metab 88:588–593[Abstract/Free Full Text]
  24. Leszczynski DE, Schafer RM, Kummerow FA 1987 Interactions of plasma steroid hormones with lipoproteins. Proc Symposium on the Analysis of Steroids, Sopron, Hungary, pp 75–80
  25. Tang M, Abplanalp W, Subbiah MT 1997 Association of estrogens with human plasma lipoproteins: studies using estradiol-17ß and its hydrophobic derivative. J Lab Clin Med 129:447–452[CrossRef][Medline]
  26. Santanam N, Shern-Brewer R, McClatchey R, Castellano PZ, Murphy AA, Voelkel S, Parthasarathy S 1998 Estradiol as an antioxidant: incompatible with its physiological concentrations and function. J Lipid Res 39:2111–2118[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Clin. Endocrinol. Metab.Home page
M. Badeau, V. Vihma, T. S. Mikkola, A. Tiitinen, and M. J. Tikkanen
Estradiol Fatty Acid Esters in Adipose Tissue and Serum of Pregnant and Pre- and Postmenopausal Women
J. Clin. Endocrinol. Metab., November 1, 2007; 92(11): 4327 - 4331.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
A. Hockerstedt, M. Jauhiainen, and M. J. Tikkanen
Lecithin/Cholesterol Acyltransferase Induces Estradiol Esterification in High-Density Lipoprotein, Increasing Its Antioxidant Potential
J. Clin. Endocrinol. Metab., October 1, 2004; 89(10): 5088 - 5093.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Vihma, V.
Right arrow Articles by Tikkanen, M. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vihma, V.
Right arrow Articles by Tikkanen, M. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals