An innovative LC-MS/MS-based method for determining CYP 17 and CYP 19 activity in the adipose tissue of pre- and postmenopausal and ovariectomized women using 13C-labeled steroid substrates.
BACKGROUND
Does adipose tissue produce steroid hormones like an endocrine organ?


OBJECTIVE
To clarify whether adipose tissue produces sex steroid hormone like an endocrine organ, we estimated several key steroid hormone levels, as well as CYP17 and CYP19 activity, in ovariectomized, pre- and postmenopausal women by liquid chromatography-tandem mass spectrometry (LC-MS/MS).


METHODS
The subjects were 19 premenopausal (n = 12), postmenopausal (n = 4), and ovariectomized women (n = 3) aged 27-68 years. Serum, visceral adipose and sc adipose samples were taken from these subjects and stored at -70°C. The levels of cortisol, cortisone, progesterone (Prog), androstenedione, dehydroepiandrosterone, estrone, estradiol (E2), and T in serum and adipose tissue were estimated simultaneously by LC-MS/MS. CYP17 and CYP19 activity in tissues were assayed with the use of (13)C-labeled steroid precursors and LC-MS/MS-based estimation of the metabolites.


RESULTS
E2 and Prog levels in the sera of postmenopausal or ovariectomized women were less than 10% of those in premenopausal women. No marked variations were seen in other hormones. Estrone, androstenedione, dehydroepiandrosterone, and Prog levels in the visceral and sc tissues of postmenopausal and ovariectomized women were 9-60 times higher than those in serum, whereas E2 and T levels were 3- to 7-fold higher than those in serum, and cortisol and cortisone levels were 20% of those found for serum. CYP17 in adipose tissue was found to have 17-hydroxylase and 20,17-lyase activity, with each catalytic activity being essentially equal. Therefore, CYP17 in adipose tissue is of the testicular/ovarian type but not adrenal type, which has 17-hydroxylase activity dominant. The presence of CYP19 activity in adipose tissue was approximately 3% of CYP17.


CONCLUSIONS
Our findings suggest that adipose tissue acts as an endocrine organ, with CYP17 and CYP19 activity playing an essential role in sex steroid hormone biosynthesis.
Department of Obstetrics and Gynecology (T.K., A.T., N.T.), Toho University Medical Center Sakura Hospital, Chiba 285-8741, Japan; Department of Analytical Research (S.H., Y.W., H.M., M.O.), ASKA Pharma Medical Co Ltd, Kawasaki 213-8522, Japan; Department of Urology (Y.S.), Gunma University Graduate School of Medicine, Maebashi 371-8511, Japan; and Department of Clinical Analysis (K.Y.), Tohoku Pharmaceutical University, Sendai 981-8558, Japan.
Toshihiko
T
Kinoshita
Seijiro
S
Honma
Yasuhiro
Y
Shibata
Kouwa
K
Yamashita
Yoko
Y
Watanabe
Hitoe
H
Maekubo
Mitsunobu
M
Okuyama
Akiko
A
Takashima
Naoki
N
Takeshita
D000273:Adipose Tissue / Q000737:chemistry* / Q000201:enzymology; D000328:Adult; D000368:Aged; D001141:Aromatase / Q000032:analysis* / Q000378:metabolism; D002247:Carbon Isotopes / Q000032:analysis; D002853:Chromatography, Liquid; D005260:Female; D006801:Humans; D008875:Middle Aged; D010052:Ovariectomy; D017698:Postmenopause / Q000378:metabolism; D017697:Premenopause / Q000378:metabolism; D013254:Steroid 17-alpha-Hydroxylase / Q000032:analysis* / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*
D002247:Carbon Isotopes; D001141:Aromatase; D013254:Steroid 17-alpha-Hydroxylase