A carbon 21 steroidal metabolite from progestin, 20ß-hydroxy-5α-dihydroprogesterone, stimulates the androgen receptor in prostate cancer cells.

BACKGROUND: Clarifying the mechanisms underlying prostate cancer (PC) progression and resistance to androgen deprivation therapy (ADT) is an urgent clinical issue. ADT influences steroidal metabolism in patients with PC and promotes the accumulation of carbon 21 steroids (C21s), such as progestin. Because the enzymes responsible for C21s metabolism are similar to those for androgen metabolism, PC cells may be able to metabolize C21s intracellularly. Therefore, there is a possibility that intracrine C21s are implicated in PC progression and resistance to ADT, and the influence of C21s on PC cells is yet to be elucidated. In the present study, we focused on 20ß-hydroxy-5α-dihydroprogesterone (20ß-OHDHP), a C21s metabolized from progestin, and showed that 20ß-OHDHP is synthesized in PC cells and is able to directly stimulate the androgen receptor (AR). METHODS: LNCaP, VCaP, and DU145 cells, which express a mutant AR (mAR), wild-type AR (wAR), and glucocorticoid receptor (GR), respectively, were incubated in the presence of several agents. After incubation, cell growth was determined by the MTS assay. PSA levels were determined by an enzyme immunoassay, and C21s and androgen levels were measured using liquid chromatography-mass spectrometry. Gene expression was analyzed by quantitative real-time polymerase chain reaction, and steroidal-receptor-related signaling was determined by a reporter assay. RESULTS: We affirmed that 20ß-OHDHP was synthesized from pregnenolone intracellularly in LNCaP and VCaP cells, and 20ß-OHDHP significantly promoted the growth of both cell lines without androgen conversion. 20ß-OHDHP directly stimulated both mAR and wAR. The presence of 20ß-OHDHP was sufficient for the proliferation and survival of LNCaP or VCaP cells, and 20ß-OHDHP promoted cell growth even in the presence of abiraterone. Using DU145 cells, we affirmed that 20ß-OHDHP did not stimulate GR, which has a potential to bypass AR signaling in PC cells promote PC cell growth. CONCLUSIONS: Under ADT, 20ß-OHDHP synthesized intracellularly from accumulated progestin in PC cells may accelerate cell growth via stimulation of both wAR and mAR. This pathway may represent an interesting candidate for targeted therapy.

Evaluation of the therapeutic effects of AGA drugs by measuring finasteride, dutasteride, and dihydrotestosterone in hair.

BACKGROUND: Androgenetic alopecia (AGA) is treated by 5α-reductase inhibitors (5ARI) such as finasteride and dutasteride, which are widely used as therapeutic agents. However, their pharmacokinetics in target organs (scalp and hair follicles) have not yet been investigated. PURPOSE: To confirm the effective action of finasteride and dutasteride in the hair follicle tissues, we developed a method to measure these concentrations in hair. RESULTS: Compared to the non-detection (N.D.) group, the dihydrotestosterone (DHT) concentrations decreased significantly in both the finasteride and dutasteride groups. The dutasteride group showed significantly lower DHT concentrations among all groups. CONCLUSIONS: Measurement of finasteride, dutasteride, and DHT concentrations in hair would aid in evaluating the drug pharmacokinetics and its therapeutic effects on AGA patients.

Serum diiodotyrosine - a biomarker to differentiate destructive thyroiditis from Graves' disease.

OBJECTIVE: Conventional diagnostic methods are limited in their ability to differentiate destructive thyroiditis from Graves' disease. We hypothesised that serum diiodotyrosine (DIT) and monoiodotyrosine (MIT) levels could be biomarkers for differentiating destructive thyroiditis from Graves' disease. DESIGN: Patients with destructive thyroiditis (n = 13) and Graves' disease (n = 22) were enrolled in this cross-sectional study. METHODS: We assayed the serum DIT and MIT levels using liquid chromatography-tandem mass spectrometry. A receiver operating characteristic (ROC) curve analysis was used to determine the sensitivity and specificity of the serum DIT and MIT levels as biomarkers for differentiating destructive thyroiditis from Graves' disease. RESULTS: The serum DIT and MIT levels were significantly higher in patients with destructive thyroiditis than in those with Graves' disease. The ROC curve analysis showed that the serum DIT levels (≥359.9 pg/mL) differentiated destructive thyroiditis from Graves' disease, significantly, with 100.0% sensitivity and 95.5% specificity (P < 0.001). The diagnostic accuracy of the serum MIT levels (≥119.4 pg/mL) was not as high as that of the serum DIT levels (sensitivity, 84.6%; specificity, 77.3%; P = 0.001). CONCLUSIONS: The serum DIT levels may serve as a novel diagnostic biomarker for differentiating destructive thyroiditis from Graves' disease.

Analysis of hair steroid hormone concentrations at different parts of the head by liquid chromatography-tandem mass spectrometry.

BACKGROUND: Steroid hormones are known to be associated with diseases like androgenetic alopecia (AGA) resulting in hair loss. The lack of a detailed analysis of the local concentration of steroids in different parts of the head underlies the rationale and purpose of this study. METHODS: To evaluate the concentration distributions of steroid hormones in hair in different parts of the head, hair samples of 8 healthy men from 9 point-areas covering the frontal, parietal, and occipital regions were collected. Eight steroid hormones were measured by using the LC-MS/MS and region-wise comparison for different hormones was done using the mean z-score and Tukey's HSD. RESULTS: Five of the 8 hormones had a high concentration in the parietal region, with dihydrotestosterone (DHT) showing a peak in the central parietal region (z = 1.59) suggesting a correlation with AGA's clinical presentation. Whereas, no significant differences were observed for testosterone and cortisol between the parietal and occipital regions. Higher DHT levels at the parietal region were also verified with a small group of AGA patients. CONCLUSIONS: This research expands upon the role of steroid hormones in hair follicle tissue elucidating their relationship with disease, thus contributing to disease management.

Quantification of androgens and their precursors in full-term human placenta.

INTRODUCTION: The two major androgens in humans are testosterone (T) and dihydrotestosterone (DHT). DHT is produced via the classical, backdoor, and alternative steroidogenic pathways. In addition, recent studies have identified C11-oxy C19 steroids as novel human androgens. Although the placenta is known to be involved in steroid metabolism, androgen levels in full-term placentas have poorly been investigated. SUBJECTS AND METHODS: Ten placentas of healthy full-term neonates (five males and five females) were examined. We quantified progesterone, androstenedione (A4), T, allopregnanolone, androsterone, and estradiol, as well as four C11-oxy androgens (11ß-hydroxyandrostenedione, 11ß-hydroxytestosterone, 11-ketoandrostenedione (11KA4), and 11-ketotestosterone (11KT)), using liquid chromatography-tandem mass spectrometry. RESULTS: In all samples, levels of the ten steroids were above the detection limit. Progesterone was by far most abundant, while levels of T and androsterone were relatively low. Levels of 11KT and 11KA4 were higher than those of T and A4, respectively. There were no differences in steroid levels between male and female samples. DISCUSSION: This study demonstrates that full-term placentas contain several steroids in the classical, backdoor, and alternative pathways. Placentas are likely to function as the supplier of progesterone to other steroidogenic tissues. More importantly, we found that placentas comprise relatively large amounts of 11KA4 and 11KT, which may be produced through steroid transfer from the adrenal gland or from the maternal circulation. These results indicate that the placenta participates in a feto-maternal multi-organ network for androgen biosynthesis.

Sex differences in serum levels of 5α-androstane-3ß, 17ß-diol, and androstenediol in the young adults: A liquid chromatography-tandem mass spectrometry study.

Animal experiments have consistently shown that estrogen receptor ß (ERß)-selective ligands have antidepressant and anxiolytic effects. In humans, endogenous ligands for ERß include 5α-androstane-3ß, 17ß-diol (3ßAdiol) and androstenediol (Δ5-diol). We determined, for the first time, the exact serum levels of 3ßAdiol and Δ5-diol in young healthy volunteers using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We investigated the effect of the menstrual cycle on the levels of these steroids in women; then, we performed a gender comparison. Blood samples were collected from 48 subjects: 23 women (mean age = 28.4±7.8 years) and 25 men (mean age = 31.4±7.8 years). We collected the blood samples of women at three time-points in the menstrual cycle: the early follicular phase, ovulatory or mid-cycle phase, and mid-luteal phase. A total of 92 blood samples were analyzed using LC-MS/MS. The levels of two well-studied steroids, namely dehydroepiandrosterone (DHEA) and 17ß-estradiol (E2), were simultaneously measured. Depression rating scale (Hamilton Rating Scale for Depression, Beck Depression Inventory-II and Quick Inventory of Depressive Symptomatology) scores were also recorded at the time of blood sampling. Significant differences in the levels of 3ßAdiol and E2 and in the depression rating scale scores were observed over the duration of the menstrual cycle of the women. The levels of 3ßAdiol and Δ5-diol were significantly lower in women than in men. E2 levels were higher in women than in men, and DHEA levels did not differ significantly between men and women. Further, women had higher scores than men on the Hamilton Rating Scale for Depression. Sex differences in depressive symptoms can be explained by 3ßAdiol and Δ5-diol levels, and the effect of the menstrual cycle on mood can be explained by 3ßAdiol and E2 levels, not by Δ5-diol level.

Analyses of Molecular Characteristics and Enzymatic Activities of Ovine HSD17B3.

17ß-hydroxysteroid dehydrogenase type 3 (HSD17B3) converts androstenedione (A4) into testosterone (T), which regulates sex steroid production. Because various mutations of the HSD17B3 gene cause disorder of sex differentiation (DSD) in multiple mammalian species, it is very important to reveal the molecular characteristics of this gene in various species. Here, we revealed the open reading frame of the ovine HSD17B3 gene. Enzymatic activities of ovine HSD17B3 and HSD17B1 for converting A4 to T were detected using ovine androgen receptor-mediated transactivation in reporter assays. Although HSD17B3 also converted estrone to estradiol, this activity was much weaker than those of HSD17B1. Although ovine HSD17B3 has an amino acid sequence that is conserved compared with other mammalian species, it possesses two amino acid substitutions that are consistent with the reported variants of human HSD17B3. Substitutions of these amino acids in ovine HSD17B3 for those in human did not affect the enzymatic activities. However, enzymatic activities declined upon missense mutations of the HSD17B3 gene associated with 46,XY DSD, affecting amino acids that are conserved between these two species. The present study provides basic information and tools to investigate the molecular mechanisms behind DSD not only in ovine, but also in various mammalian species.

Profiles of 5α-Reduced Androgens in Humans and Eels: 5α-Dihydrotestosterone and 11-Ketodihydrotestosterone Are Active Androgens Produced in Eel Gonads.

Although 11-ketotestosterone (11KT) and testosterone (T) are major androgens in both teleosts and humans, their 5α-reduced derivatives produced by steroid 5α-reductase (SRD5A/srd5a), i.e., 11-ketodihydrotestosterone (11KDHT) and 5α-dihydrotestosterone (DHT), remains poorly characterized, especially in teleosts. In this study, we compared the presence and production of DHT and 11KDHT in Japanese eels and humans. Plasma 11KT concentrations were similar in both male and female eels, whereas T levels were much higher in females. In accordance with the levels of their precursors, 11KDHT levels did not show sexual dimorphism, whereas DHT levels were much higher in females. It is noteworthy that plasma DHT levels in female eels were higher than those in men. In addition, plasma 11KDHT was undetectable in both sexes in humans, despite the presence of 11KT. Three srd5a genes (srd5a1, srd5a2a and srd5a2b) were cloned from eel gonads. All three srd5a genes were expressed in the ovary, whereas only both srd5a2 genes were expressed in the testis. Human SRD5A1 was expressed in testis, ovary and adrenal, whereas SRD5A2 was expressed only in testis. Human SRD5A1, SRD5A2 and both eel srd5a2 isoforms catalyzed the conversion of T and 11KT into DHT and 11KDHT, respectively, whereas only eel srd5a1 converted T into DHT. DHT and 11KDHT activated eel androgen receptor (ar)α-mediated transactivation as similar fashion to T and 11KT. In contrast, human AR and eel arß were activated by DHT and11KDHT more strongly than T and 11KT. These results indicate that in teleosts, DHT and 11KDHT may be important 5α-reduced androgens produced in the gonads. In contrast, DHT is the only major 5α-reduced androgens in healthy humans.

Measurement of steroid hormones by liquid chromatography-tandem mass spectrometry with small amounts of hair.

Steroid hormone levels in hair reflect the integrated values (average values) of hormone secretion over the past few months. We have used a method to evaluate diseases and chronic stress, discrimination of banned drug use, and so on. In contrast, the hair analysis methods reported so far required at least 10 mg (about 50 to 100 hair strands) of hair to analyze multiple steroid hormones from the same sample. Here, we developed a new method for measuring steroid hormones in hair by liquid chromatography-tandem mass spectrometry, which identifies multiple steroid hormones from 5 to 10 (about 1 mg) hair strands. Ten steroid hormones (cortisol, cortisone, testosterone, dihydrotestosterone, dehydroepiandrosterone, androstenedione, progesterone, pregnenolone, androstenediol and estradiol) covering from sex hormones to stress hormones were derivatized and measured by four different measuring systems. The method showed good linearity for all steroids with correlation coefficients of 0.999 or more. The accuracy and precision of intra- and inter-assay ranged from 96.0 to 106.4% and 4.8 to 8.1% for intra-assay, and from 96.9 to 104.9% and 6.9 and 10.6% for inter-assay, respectively. A mixed solution containing 0.1 M trifluoroacetic acid and 50% acetonitrile was used to extract hair and to enhance the cortisol extraction efficiency approximately twice compared to the previously reported extraction with methanol. This method has the potential to clarify the relationship between steroid hormone levels and diseases that show alopecia such as chronic stress and androgenetic alopecia.

Dietary soybean oil, canola oil and partially-hydrogenated soybean oil affect testicular tissue and steroid hormone levels differently in the miniature pig.

The present study was conducted to examine the influence of dietary canola oil (CAN) and partially-hydrogenated soybean oil (HSO) compared to soybean oil (SOY, control) on the morphology and function of testes using miniature pigs as the test subject. Male miniature pigs were fed a diet containing 10%SOY, 9%CAN+1%SOY, or 9%HSO+1%SOY for 18 months. The scheduled autopsies revealed no abnormalities in histopathological examination of the major organs, except the testes. Atrophy of the seminiferous tubules and hyperplasia in the Leydig cells were found in the SOY and CAN groups. DNA microarray analysis indicated downregulation in the CAN and the HSO groups of genes encoding for gonadotropins in the pituitary gland and of enzymes and proteins involved in steroid hormone metabolism in the testes, compared to the SOY group. Plasma levels of sex hormones in the CAN and HSO groups tended to be higher and testosterone and dihydrotestosteorne in the HSO group were significantly higher than in the SOY group. These results demonstrate that testes are morphologically and functionally affected by the dietary oils, while the plasma steroid hormone levels do not necessarily reflect the gene expression, probably owing to feedback regulation via the gonadal hormones in the hypothalamus-pituitary-gonadal axis.

Characterization of in vivo metabolites of toad venom using liquid chromatography-mass spectrometry.

The structures of in vivo metabolites of marinobufotoxin (marinobufagin 3-suberoylarginine ester), marinobufagin, or bufalin which are typical components of toad venom widely used as the traditional Chinese drug, Ch'an Su, are confirmed using authentic samples based on their liquid chromatography-mass spectrometric behavior. A rat is orally administered 2 mg of the previously mentioned components of toad venom, the serum is collected 30 min after the administration, extracted, and then characterized. Marinobufotoxin is hydrolyzed and further epimerized into 3-epimarinobufagin, but marinobufagin 3-hemisuberate is not detected. After the administration of marinobufagin, 3-epimarinobufagin is detected in both the male and female rats, but marinobufagin 3-sulfate is formed only in the female rats. Bufalin is metabolized to 3-epibufalin, which is found to undergo further conjugation resulting in its 3-glucuronide. Furthermore, 3-epibufalin 3-sulfate is formed only in the female rats.

Dihydrotestosterone synthesis pathways from inactive androgen 5α-androstane-3ß,17ß-diol in prostate cancer cells: Inhibition of intratumoural 3ß-hydroxysteroid dehydrogenase activities by abiraterone.

Intratumoural dihydrotestosterone (DHT) synthesis could be an explanation for castration resistance in prostate cancer (PC). By using liquid chromatography-mass spectrometry, we evaluated the intratumoral DHT synthesis from 5α-androstane-3ß,17ß-diol (3ß-diol), which is inactive androgen metabolized from DHT. 3ß-diol had biochemical potential to be converted to DHT via three metabolic pathways and could stimulate PC cell growth. Especially, 3ß-diol was not only converted back to upstream androgens such as dehydroepiandrosterone (DHEA) or Δ5-androstenediol but also converted directly to DHT which is the main pathway from 3ß-diol to DHT. Abiraterone had a significant influence on the metabolism of DHEA, epiandrosterone and 3ß-diol, by the inhibition of the intratumoural 3ß-hydroxysteroid dehydrogenase (3ß-HSD) activities which is one of key catalysts in androgen metabolic pathway. The direct-conversion of 3ß-diol to DHT was catalysed by 3ß-HSD and abiraterone could inhibit this activity of 3ß-HSD. These results suggest that PC had a mechanism of intratumoural androgen metabolism to return inactive androgen to active androgen and intratumoural DHT synthesis from 3ß-diol is important as one of the mechanisms of castration resistance in PC. Additionally, the inhibition of intratumoural 3ß-HSD activity could be a new approach to castration-resistant prostate cancer treatment.

Sex differences in the serum level of endogenous ligands for estrogen receptor ß in the elderly population.

Animal studies suggest that estrogen receptor ß (ERß)-agonists, but not ERα-agonists, are antidepressants. Several endogenous ligands for ERß have been proposed, including 5α-androstane-3ß, 17ß-diol (3ßAdiol), Androstenediol (Δ5-diol), and 7α-hydroxydehydroepiandrosterone (7α-OH-DHEA). The aim of this study was to determine the serum and salivary levels of natural ERß ligands in men and women with and without past depressive episodes in the elderly population. DHEA (a precursor of 3ßAdiol, Δ5-diol, and 7α-OH-DHEA), 17ß-estradiol (E2), and cortisol (F) were also measured. Samples were collected from 51 subjects and liquid chromatography tandem mass spectrometry was used for measurement. Comparisons were made between groups based on sex and depression history. E2, 3ßAdiol, and Δ5-diol levels were significantly lower in women than in men regardless of depression history. There were no significant differences between men and women in DHEA or 7α-OH-DHEA levels. DHEA was significantly lower in women with depression than in women without depression. Reduced DHEA levels may be related to depression vulnerability in women. Further studies are needed to determine the mechanism underlying sex differences in the prevalence of depression and increased risk of depression during menopause. Not only E2 but also two other estrogenic steroids (3ßAdiol and Δ5-diol) should be involved in these studies.

Steroidogenic pathways involved in androgen biosynthesis in eumenorrheic women and patients with polycystic ovary syndrome.

The conventional Δ5 and Δ4 steroidogenic pathways mediate androgen production in females. While multiple non-conventional pathways to dihydrotestosterone (DHT) have recently been postulated in humans, the functional significance of these pathways remains to be elucidated. The aim of this study was to clarify the origin of androgens in healthy women and in patients with polycystic ovary syndrome (PCOS), a multifactorial disorder characterized by androgen overproduction. We measured 13 steroids in blood samples of 31 eumenorrheic females and 28 PCOS patients using liquid chromatography-tandem mass spectrometry and chemiluminescent enzyme immunoassay. We found that 17-hydroxy (17-OH) progesterone (17-OHP), androstenedione (Δ4A), testosterone, androstanedione, androsterone, and androstanediol levels were higher in the patient group than in the eumenorrheic group, while levels of other steroids were comparable between the two groups. In the eumenorrheic group, DHT levels were correlated with testosterone, androstanedione, and androstanediol. Quantitative correlations were also observed among 17-OH allopregnanolone, androsterone, androstanediol, and DHT, and among Δ4A, androstanedione, androsterone, and androstanediol. In the patient group, DHT levels were correlated with testosterone levels, but not with androstanedione or androstanediol levels. Δ4A and testosterone paralleled 17-OHP. Androstanedione, androsterone, androstanediol, and 17-OH allopregnanolone were quantitatively correlated. In both groups, multivariable linear regression analyses suggested relationships between androsterone and androstanedione, as well as between androsterone and 17-OH allopregnanolone. These results indicate that multiple androgen biosynthesis pathways are operating in eumenorrheic females and PCOS patients. In PCOS patients, excessive androgens are produced primarily via the conventional pathways, while two alternative pathways; i.e., an androstanedione-mediated pathway and a so-called backdoor pathway, likely serve as sources of a weak androgen and potential precursors of DHT.

Androgen deprivation promotes intratumoral synthesis of dihydrotestosterone from androgen metabolites in prostate cancer.

Intratumoral synthesis of dihydrotestosterone (DHT) from precursors cannot completely explain the castration resistance of prostate cancer. We showed that DHT was intratumorally synthesized from the inactive androgen metabolites 5α-androstane-3α/ß,17ß-diol (3α/ß-diol) in prostate cancer cells via different pathways in a concentration-dependent manner. Additionally, long-term culture in androgen-deprived media increased transcriptomic expression of 17ß-hydroxysteroid dehydrogenase type 6 (HSD17B6), a key enzyme of oxidative 3α-HSD that catalyzes the conversion of 3α-diol to DHT in prostate cancer cells. Correspondingly, the score for HSD17B6 in tissues of 42 prostate cancer patients undergoing androgen deprivation therapy (ADT) was about 2-fold higher than that in tissues of 100 untreated individuals. In men receiving ADT, patients showing biochemical progression had a higher HSD17B6 score than those without progression. These results suggested that 3α/ß-diol also represent potential precursors of DHT, and the back conversion of DHT from androgen derivatives can be a promising target for combination hormone therapy.

Effect of castration monotherapy on the levels of adrenal androgens in cancerous prostatic tissues.

The mechanism accounting for the development of castration-resistant prostate cancer (CRPC) remains unclear. Studies in CRPC tissues suggest that, after androgen deprivation therapy (ADT), the adrenal androgens may be an important source of testosterone (T) and 5-alpha dihydrotestosterone (DHT) in CRPC tissues. To clarify the role of adrenal androgens in the prostatic tissues (prostatic tissue adrenal androgens) during ADT, we developed a high sensitive and specific quantification method for the levels of androgens in prostatic tissue using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Human prostatic tissues were purified using mixed-mode reversed-phase, strong anion exchange Oasis cartridges (Oasis MAX). Analysis of steroids was performed using LC-MS/MS after picolinic acid derivatization. The validation tests showed that our method of quantitative analysis was precise and sensitive enough for the quantification of dehydroepiandrosterone (DHEA), androstenedione, androstenediol, T, and DHT in the prostatic tissue. The levels of adrenal androgens in prostate cancer tissues after ADT were similar to those in untreated PCa. Especially, DHEA was the most existing androgen precursor in PCa tissues after ADT. The levels of DHEA were high in PCa tissues, irrespective of ADT. We assumed that DHEA played a significant role in the synthesis of T and DHT in PCa tissues after ADT.

Trilostane, an inhibitor of 3ß-hydroxysteroid dehydrogenase, has an agonistic activity on androgen receptor in human prostate cancer cells.

The intracellular androgen metabolism and cell activity in prostate cancer cells with mutated (LNCaP-FGC) or wild-type (VCaP) androgen receptors in the presence of trilostane, an inhibitor of 3ß-hydroxysteroid dehydrogenase, were examined. Trilostane suppressed the intracellular production of androstenedione, testosterone, and dihydrotestosterone from dehydroepiandrosterone in LNCaP-FGC cells. In both LNCaP-FGC and VCaP cell types, the prostate-specific antigen (PSA) levels in media were increased by trilostane alone in a concentration-dependent manner. Both cells pretreated with trilostane showed a dose-dependent decrease in PSA production with bicalutamide (P<0.001). Trilostane should be used with particular concern when treating prostate cancer.

New quantification method for estradiol in the prostatic tissues of benign prostatic hyperplasia using liquid chromatography-tandem mass spectrometry.

Estrogen is suspected to play a role in the pathogenesis of benign prostatic hyperplasia (BPH) and prostate cancer. To clarify the role of estradiol (E2) in the prostatic tissues (prostatic tissue E2) during the development of prostatic disorders, we developed a new sensitive and specific quantification method for prostatic tissue E2 using liquid chromatography-tandem mass spectrometry (LC-MS/MS). For the solid-phase extraction, E2 was purified by anion-exchange through an Oasis MAX cartridge. In addition, after the formation of 3-pentaflurobenzyl-17beta-pyridinium-estradiol derivative (E2-PFBPY), E2-PFBPY was purified by cation-exchange through an Oasis WCX cartridge. These processes in the LC-MS/MS method improved the specificity and sensitivity for prostatic tissue E2 measurement, compared to the radioimmunoassay (RIA) method. The validation tests showed that intra-day and inter-day precisions were both within +/-15% (except for 15.5% of the inter-day precision of the lowest concentration), with the accuracy ranging from 88 to 110%. The quantification limit of this assay was 0.15pg/tube in our method, which was 80-fold more sensitive than that of the RIA method. With the use of our present method, the median E2 levels in the prostatic tissues in patients with BPH (n=20, median age: 71 years) were 12.0pg/g tissue (95% confidence interval=9.1-22.6pg/g tissue). Furthermore, the E2 levels increased significantly with aging. These results showed that our present method would be useful for elucidating the role of prostatic tissue E2 in the development of prostatic disorders with a small amount of tissue samples.

Development of highly sensitive quantification method for testosterone and dihydrotestosterone in human serum and prostate tissue by liquid chromatography-electrospray ionization tandem mass spectrometry.

We developed highly sensitive detection of testosterone (T) and 5alpha-dihydrotestosterone (DHT) by liquid chromatography-electrospray ionization tandem mass spectrometry using high proton affinitive derivatization of 17beta-hydroxyl group of T and DHT with picolinic acid, mobile phase consisting of MeCN-MeOH-H(2)O-formic acid and conventional octadecylsilica (ODS) column. Purification of the derivatives was carried out using solid-phase extraction with ODS cartridge. By this method, T and DHT were determined simultaneously with limits of quantification (LOQs) of 1 pg/0.2 ml in serum, and T and DHT with LOQs of 0.5 pg and 1 pg/3mg in prostate tissue, respectively, under acceptable assay performance (intra-assay and inter-assay accuracy and precision). The present method provides reliable and reproducible results for quantification of T and DHT in small volumes of serum and prostate samples for diagnosis in prostatic disorders and male climacteric.

Characterization of in vitro metabolites of toad venom using high-performance liquid chromatography and liquid chromatography-mass spectrometry.

The characterization of the in vitro metabolites of toad venom, which has been widely used as a traditional Chinese drug, Ch'an Su, has been completed. Toad venom contains bufotoxins (such as marinobufotoxin; marinobufagin 3-suberoylarginine ester) and bufogenins (such as marinobufagin and bufalin) as the main cardiac steroids. An in vitro experiment using the rat or human liver cytosolic fraction disclosed that marinobufotoxin produced marinobufagin, but not its 3-hemisuberate. Marinobufagin was subjected to the enzyme reaction using the rat or human liver microsomal fraction together with NADPH and NAD, which produced 3-dehydromarinobufagin and 3-epimarinobufagin. Marinobufagin produced its 3-sulfate upon treatment with the rat or human liver cytosolic fraction and 3'-phosphoadenosine 5'-phosphosulfate. Bufalin was also subjected to the above enzyme reactions and showed almost the same results except for the result that the hydroxylation occurred at the 5beta-position. On the other hand, small amounts of marinobufagin 3-glucuronide were obtained only by treatment with the human liver microsomal fraction and uridine 5'-diphosphoglucuronic acid. The structures of these metabolites were confirmed using authentic samples regarding their high-performance liquid chromatographic behavior and/or liquid chromatography-mass spectrometry analysis.