Androgen and estrogen dynamics in the female baboon (Papio anubis)

Androgen and estrogen dynamics were studied in 5 female baboons (Papio anubis) using constant infusions of [3H]androstenedione/[14C]estrone and [3H]testosterone/[14C]estradiol. Blood samples were obtained prior to the infusions and both blood and plasma was used for measurements of androstenedione (A), testosterone (T), dihydrotestosterone (DHT), estrone (E1), estradiol (E2). Plasma was used for measurements of sex-hormone binding globulin (SHBG), and the percents of T and E2 free, bound to SHBG, and to albumin. Blood samples obtained during the infusions were analyzed for radioactivity as purified androgens and estrogens. Metabolic clearance rates (MCR), and transfer factors ([rho]BB; fraction of steroid infused which is converted to and measured in blood as product) and blood production rates were calculated from whole blood data. All urine was collected for 96 h and an aliquot analyzed for radioactivity as the glucuronides of estrone and estradiol and the % peripheral aromatization calculated. The MCR's, calculated in whole blood, of A, E1, E2 and T were 53 +/- 6 1/day/kg, 39.3 +/- 3 1/day/kg, 29.9 +/- 5.2 1/day/kg and 10.1 +/- 2.3 1/day/kg, respectively. Each MCR was different (P less than 0.05) from the others. The PB of E1 was 15 +/- 2 micrograms/day and was not different from that of E2 (12 +/- 3 micrograms/day). The PB of A, 231 +/- 55 micrograms/day, was greater than that of T, 13 +/- 5 micrograms/day. The interconversions of both the androgens (18.9 +/- 3.4% vs 3.9 +/- 1.0%) and the estrogens (48.8 +/- 10.7% vs 4.0 +/- 0.8%) favored the oxidative pathway, i.e. conversion of 17-OH to 17-oxo steroids. The conversion ratio of A to DHT was greater than that of T to DHT (16.4 +/- 2.1% vs 5.3 +/- 0.7%), and A is a more important source of DHT than is T. The percent of T bound to SHBG (80.7 +/- 0.9%) was greater than percent of E2 (36.9 +/- 9.8%) and inversely the percents of T bound to albumin and free (17.5 +/- 0.8% and 1.65 +/- 0.16%) were less than the respective percents for estradiol (60.5 +/- 9.5% and 2.40 +/- 0.27%). The mean SHBG concentration was 54 +/- 6 nM. The peripheral aromatization of androstenedione, 1.36 +/- 0.05%, was greater than of testosterone, 0.18 +/- 0.02%. This difference is, in part, due to the lack of SHBG-binding of androstenedione. The general pattern of androgen and estrogen dynamics is similar to that in women. This similarity is due, in part, to the presence of SHBG in both baboons and women.     

The effects of l-thyroxine and dexamethasone on steroid dynamics in male cynomologous monkeys

Male cynomologous monkeys (M. fascicularis) were infused with [3H]androgens, [14C]estrogens and [3H]cortisol before and after the administration of l-thyroxine, (l-T4) 150 micrograms/day for 6 wk, dexamethasone 8 mg every 8 h for 3 doses and dexamethasone 1.0 mg/day for 8 days. Blood samples were obtained before each of the infusions and analyzed for endogenous T, A, E1, E2 and F concentrations, % free T and % free E2, sex hormone-binding globulin (SHBG) and cortisol binding globulin (CBG) capacity. When l-T4 was being administered, T4 and triiodothyronine (T3) concentrations were also measured. Blood samples were obtained during the infusions and analyzed for radioactivity as testosterone (T), androstenedione (A), dihydrotestosterone (DHT), estradiol (E1), estrone (E2), and cortisol (F). All urine was collected for 96 h and an aliquot of the pooled urine was analyzed for radioactivity as estrone and estradiol glucuronide. The administration of l-T4 for 6 wk to 3 monkeys resulted in a marked rise in T4 and T3 levels, from 4.8 +/- 0.4 micrograms/dl and from 136 +/- 6 to 515 +/- 71 ng/dl, respectively. MCRT, MCRE2 and MCRE1 did not change, but MCRA values increased slightly and MCRF increased 2-3 fold. [rho]T.E2 did not change but [rho]A.E1BM showed a slight but significant increase. The inter-conversions between the androgens and between the estrogens were not altered. There was a 2-3-fold increase in SHBG and a decrease in %FT but no change in %FE2 or CBG. The concentrations of T, A and DHT rose but there was no trend in the levels of the estrogens. The administration of dexamethasone 8 mg every 8 h for 3 doses or 1 mg/day for 8 days caused no changes in the MCRs for T, A, E1 and E2 but did cause a significant decrease in MCRF. Measurement of splanchnic and peripheral tissue extractions before and after acute dexamethasone administration in 1 monkey showed that the decrease in MCRF was the result of a marked decrease, 11-2%, in splanchnic extraction of F. The extractions of T and E2 were relatively unaffected. The concentrations of T and F fell but E2 remained the same. % FT and % FE2 rose slightly and the concentrations of SHBG and CBG were unchanged. The androgen interconversions and estrogen interconversions were not affected but [rho]T,E2BM and [rho]A,E1BM showed slight decreases.(ABSTRACT TRUNCATED AT 400 WORDS)     

Estrogen dynamics in the female rhesus monkey

The metabolic clearance rates (MCR) and interconversions [( rho]BB) values for estrone (E1) and estradiol (E2) in female rhesus (Macaca mulatta) monkeys on Days 9, 14, and 23 of the menstrual cycle were measured using constant infusions of [3H] estradiol and [14C] estrone. The menstrual cycles in these monkeys were reproduced by using Silastic capsules of E2 and progesterone after bilateral ovariectomy. The serum levels of E2 and progesterone were measured by radioimmunoassay and were similar to those for the intact menstrual cycle. The MCR of E2 on Day 14 (52.8 +/- 6.8 l/day/kg) was significantly greater (p less than 0.05) than that measured on Day 9 (31.1 +/- 3.6 l/day/kg) or Day 23 (35.4 +/- 2.1 l/day/kg). The MCR of E1 was also different (p less than 0.05) on Day 14 (77.6 +/- 14.9 l/day/kg) compared to the values on Days 9 and 23 (50.2 +/- 4.9 and 48.2 +/- 3.9 l/day/kg, respectively. There was no change in percentage of free E2, percentage of albumin-bound E2, or sex hormone-binding globulin levels on those 3 days of the cycle. The interconversions between E2 and E1 were not influenced by the day of the cycle. We conclude that the high levels of E2 occurring at the time of the E2 peak result in increases in the MCRs of both E2 and E1 that are not associated with changes in the pattern of protein-binding or in the activity of the 17 beta-hydroxy steroid dehydrogenase.     

Steroid dynamics in the rabbit

Male rabbits were infused at a constant rate with 3H-androstenedione/14C-estrone (n = 5) or 3H-testosterone/14C-estradiol-17 beta (n = 3) for 3 1/2 hr and blood samples were obtained over the last hour and analyzed for radioactivity as androstenedione (A), testosterone (T), estrone (E1), estradiol-17 beta (E2 beta) and estradiol-17 alpha (E2 alpha). The mean value for the metabolic clearance rate of androstenedione (MCRA) was 85 +/- 10 l/day/kg, which was significantly greater than the mean MCRE1 59 +/- 10 l/day/kg. MCRT, 42 +/- 8 l/day/kg, and MCRE2 beta, 45 +/- 9 l/day/kg were not different. The conversion ratio of androstenedione to testosterone (CRA,T) was greater than CRT,A but for the estrogens, CRE2 beta, E1 was greater than CRE1,E2 beta. CRE2 beta, E2 alpha was greater than CRE1,E2 alpha. The overall aromatization of androstenedione to estrone, the fraction of 3H-androstenedione infused into the blood and measured as 3H-estrone in blood [( rho]A,E1BB) was 0.0005 +/- 0.0001 and for [rho]T,E2 beta BB was 0.0012 +/- 0.0006. In the rabbit both sex hormone binding globulin (SHBG) and albumin binding may effect the MCRs, and peripheral aromatization of androgens occurs to a far lesser degree than in humans and primates.     

Androgen and estrogen dynamics: stability over a two year interval in peri-menopausal women

As part of a study on hormones and bone density in peri-menopausal women, metabolic clearance rates (MCR), and interconversions of androgens and estrogens and the peripheral aromatization of androgens were measured twice 2 yr apart. Measurements of clearance rates and interconversions were made from blood samples obtained during constant infusions of [3H]androgens and [14C]estrogens. Measurements of peripheral aromatization were made from the estrogen glucuronides in a pooled 4-day urine collection timed from the start of the infusions. The women were divided into 3 groups: Group A (n = 15) were having menstrual cycles throughout the 2 yr interval; Group B (n = 11) were having menstrual cycles at the time of Study 1 but had been amenorrheic for at least 1 yr at the time of Study 2; Group C (n = 28) were amenorrheic for at least 1 yr at the time of Study 1 and had remained amenorrheic through Study 2. The MCRs for testosterone, androstenedione, estrone and estradiol were not different for Study 1 and Study 2 in any of the groups. The interconversions of the androgens were similar in both studies for all groups. The conversion of estrone to estradiol decreased in Group A, otherwise the interconversions of the estrogens did not vary between the studies for the other groups. The peripheral aromatization of androstenedione, but not of testosterone, was significantly greater at study 2 compared to Study 1 for all groups. We conclude that the MCRs and interconversions of androgens and of estrogens are stable over time, but that the peripheral aromatization of androstenedione increases over a 2 yr interval. This increase may be menopausal and/or age related.     

Estrogen interconversions in the induced cycle in female rhesus monkeys

To determine the extractions and interconversions of estrone and estradiol across and within the uterus, [3H]estradiol and [14C]estrone were infused at a constant rate in six ovariectomized female rhesus (Macaca mulatta) monkeys. Studies were done on Days 9, 14, and 23 of artificial menstrual cycles induced by the timed insertion and removal of Silastic capsules of estradiol and progesterone. Measurements of estrogen radioactivity were made from peripheral arterial blood and uterine venous blood as well as from endometrial biopsy samples. A significant increase occurred in the conversion of estradiol to estrone measured within the uterus on Day 23 compared to Days 9 and 14. The conversion of estrone to estradiol, measured within the uterus, fell progressively from Day 9 to Day 23, but this decrease was not significant. The extractions and interconversions across the uterus, and the overall interconversions of estrone and estradiol were not significantly different on Days 9, 14, or 23 of the cycle. Thus, we have been able to confirm in vivo the increase in the activity of the 17 beta-hydroxysteroid dehydrogenase, the enzyme responsible for estradiol to estrone interconversions, shown earlier by studies done in vitro. However, the increase in 17 beta-hydroxysteroid activity in the uterus is not reflected in the overall interconversions of estrone and estradiol as reflected by measurements in peripheral arterial blood.     

The sexually differentiated metabolism of [6,7-3H]17 beta-oestradiol in rats: male-specific 15 alpha- and male-selective 16 alpha-hydroxylation and female-selective catechol formation.

The oxygenated-metabolite profiles of exogenous 17 beta-oestradiol (E2) in adult male and female Wistar rats have been characterized and major sex-dependent biotransformations observed which correlate with the regioselectivities of known sexually differentiated hepatic P450. [6,7-3H]E2 (27 micrograms/kg) was given i.v. The metabolites of E2 were rapidly and extensively excreted in bile (46 and 78% of the dose over 1 and 6 h, respectively). Female rats metabolized E2 by one major pathway: oxidation to oestrone (E1) followed by C-2 hydroxylation and O-methylation; the principal aglycones (0-1 h bile collections) were E1 (14%), 2-hydroxyE1 (2-OHE1) (42%) and 2-methoxyE1 (24%). Male rats metabolized E2 principally by two parallel composite pathways of E1 hydroxylation which yielded a complex mixture of mono- and di-oxygenated compounds: 15 alpha-OHE1 (33%), 2,15 alpha-diOHE1 (7%), and 2-methoxy-15 alpha OHE1 (14%); 16 alpha-OHE1 (13%), 2,16 alpha-diOHE1 (4%) and 2-methoxy-16 alpha-OHE1 (2%). 15 alpha-Hydroxylation was unique to males. The balance of aromatic and alkyl hydroxylation in males was dose-dependent: at 3 mg/kg, 15 alpha-hydroxylation was decreased approx. 50% in favour of 2-hydroxylation whilst 16 alpha-hydroxylation was largely unaffected. The male-specific 15 alpha-hydroxylation and male-predominant 16 alpha-hydroxylation of E1 derived from E2 in vivo may be ascribable to the male-specific isoforms P450IIC13 and P450IIC11, respectively.     

Serum and urinary estrone sulfate during the menstrual cycle, measured by a direct radioimmunoassay, and fate of exogenously injected estrone sulfate

Serum and early-morning urinary levels of estrone sulfate during the menstrual cycle were measured by a direct radioimmunoassay without hydrolysis. These levels were high and showed prominent peaks [serum, 2.67 +/- 0.37 ng/ml (mean +/- SE); urine, 5.82 +/- 2.3 micrograms/l] around the day of the preovulatory estradiol-17 beta peak, and increased again during the luteal phase. Following intravenous injection of estrone sulfate, serum estrone sulfate, estrone and estradiol-17 beta were measured. The conversion of estrone sulfate to estrone and/or estradiol-17 beta was very small during their transit in the general circulation.     

Differences in aromatase activity between Leydig cells from the scrotal and abdominal testis in the naturally unilateral-cryptorchid boar

In an earlier study, estrogen production was much lower in Leydig cells from the abdominal than from the scrotal testis in naturally occurring unilateral cryptorchidism in the boar. A more direct assessment of aromatase activity was made in thirty-two mature male pigs to examine this observation further, using nonradioactive androstenedione (delta 4A 1.0 x 10(-6) M - 1.5 x 10(-5) M) and [1 beta, 2 beta-3H] delta 4A as substrates. Purified Leydig cells were prepared from normal boars and from unilaterally and bilaterally cryptorchid animals. Combined estrone sulfate (E1S) and estrone (E1) formation from delta 4A were measured by radioimmunoassay. Little or no estrogen secretion was seen with cells from the abdominal testis in unilaterally cryptorchid boars (n = 7), and E1S formation from delta 4A was 6- to 14-fold higher for scrotal cells (n = 6). Aromatase activity as reflected in percent conversion of substrate to [3H]-labeled water was clearly lower in cells from the abdominal testis (1.10 +/- 0.08 and 11.22 +/- 0.7%, respectively, p less than 0.01, n = 6). No marked reduction was noted for unilaterally cryptorchid boars with an inguinally located testis (10.18 +/- 0.27 and 13.09 +/- 0.58% for inguinal and scrotal testes, respectively, n = 3). Concentrations of E1S in testicular arterial and venous blood (n = 9) gave additional evidence of lower estrogen production by the undescended testis of the cryptorchid boar. It was concluded that lower aromatase activity is present in Leydig cells of the abdominal testis.     

Steroid metabolism in the cat. Biliary and urinary excretion of metabolites of [4-14C]testosterone

1. [4-(14)C]Testosterone was administered intravenously to anaesthetized male cats as a single injection or as a 45-60min. infusion. 2. Most of the administered radioactivity was excreted in the bile (70-80%); only 2.9-5.5% of the dose was excreted in the urine. 3. Bile and urine samples were hydrolysed successively to yield glucuronide, ;cold-acid-hydrolysed' and ;hot-acid-hydrolysed' fractions. 4. The proportion of glucuronides in bile decreased in successive samples, but cold-and hot-acid-hydrolysed metabolites showed no consistent change. 5. After hydrolysis most of the radioactivity in both bile and urine could not be extracted by ether from neutral aqueous solution.     

Metabolism of selenocyanate in the rat

Rats injected subcutaneously with 2 mg Se/kg body weight of [75Se]selenocyanate or [14C, 75Se]selenocyanate excreted dimethylselenide (DMSe) in the breath and trimethyl-selenonium ion (TMSe) in the urine. The 24-h respiratory DMSe and urinary TMSe excretions were 26.8 +/- 8.1 and 14.5 +/- 5.1% of the dose, respectively. Tissue concentrations of 75Se were highest in the kidneys (1.89 +/- 0.2% dose/g), liver (1.46 +/- 0.2% dose/g), and blood (0.50 +/- 0.05% dose/ml), and lower (greater than 0.3% dose/g) in the other tissues. Trimethyl-selenonium was the major form (61%) of selenium in urine. Approximately 2% of the dose of doubly labeled SeCN- was excreted unchanged in urine (about 12% of urinary Se). 14C from doubly labeled SeCN- was not present in the methylated selenium metabolites, but a major 14C urinary metabolite was identified as thiocyanate. These results indicate that a substantial part of selenocyanate in the body undergoes metabolism and Se is excreted in methylated forms following scission of the C-Se bond.     

Within-person variability of the ratios of urinary 2-hydroxyestrone to 16alpha-hydroxyestrone in Caucasian women

The ratio of urinary 2-hydroxyestrone (2-OHE1) to 16alpha-hydroxyestrone (16alpha-OHE1) has been suggested as a potential biomarker for breast cancer risk. We evaluated within-person variability of this biomarker in ten healthy Caucasian women aged 23-58 years. Each study participant was asked to provide an overnight fasting morning urine sample once a week for an average of 8 weeks. These urine samples were assayed for 2-OHE1 and 16alpha-OHE1 by using competitive enzyme immunoassay kits purchased from the ImmunaCare Corporation. The coefficients of variation for urinary 2-OHE1/16alpha-OHE1 over the study period ranged from 13.7 to 59.6% (mean, 33.3%) in our study participants. There was a good correlation between the level of the urinary 2-OHE1/16alpha-OHE1 ratio in any single urine sample and the average ratio over the 8-week study period from the same woman, with the mean correlation coefficient of 0.85. These results indicated that the within-person variation of the 2-OHE1 to 16alpha-OHE1 ratio for most women was moderate and the level of this ratio in a single urine sample, in general, reflects reasonably well the level of this biomarker over a 2-month period.     

Monitoring ovarian function in marmosets and tamarins by the measurement of urinary estrogen metabolites

Practical aspects of urinary estrogen analysis were considered with regard to establishing simple and reliable methods for monitoring ovarian function in marmosets and tamarins. Changes in the hormone:creatinine ratio in small volumes of urine from the common marmoset were significantly correlated with changes in 24-h excretion. Comparison of the metabolism and excretion of estrogens during the ovarian cycle in the common marmoset and cottontop tamarin revealed interesting species differences. High concentrations of conjugated estrone were measured in marmoset plasma, but estradiol 17β was the predominant estrogen in urine. In contrast, estrone was the most abundant estrogen measured in tamarin urine. Both species excreted very little estriol. Sulfates and glucuronides were present in urine in similar proportions before ovulation in the marmoset, although after ovulation sulfates were the more abundant. Conversely, most of the estrogens in tamarin urine appeared to be conjugated as glucuronides. Direct assay for estrone sulfate was applied to the measurement of urinary estrogen excretion during the ovarian cycle in a marmoset. The results compared well with those for total estradiol 17β after hydrolysis and ether extraction. The use of direct assays for conjugated estrogens in small volumes of urine is suggested as a practical method for monitoring ovarian function in marmosets and tamarins.     

Identification of a new kinin in human urine

The types of kinins excreted in fresh urine of dogs, rats, and humans were compared. Urinary kinins were separated by reverse-phase (C18) high performance liquid chromatography and quantitated by radioimmunoassay using an antibody directed against the COOH-terminal region of the peptide. Kinins were found in the following proportions: 53 +/- 3% bradykinin, 23 +/- 4% Lys-bradykinin, and 13 +/- 7% des-Arg1-bradykinin in dog urine; 67 +/- 6% bradykinin, 6 +/- 3% Lys-bradykinin, and 10 +/- 3% des-Arg1-bradykinin in rat urine; and 12 +/- 4% bradykinin, 30 +/- 3% Lys-bradykinin, 2 +/- 1% des-Arg1-bradykinin, and 41 +/- 3% unknown kinin in human urine. The unknown kinin was purified from a pool of human urine. Amino acid sequencing revealed a structure similar to Lys-bradykinin except that proline in position 4 was replaced by alanine ([Ala3]Lys-bradykinin). Synthetic and endogenous [Ala3]Lys-bradykinins had similar high performance liquid chromotography elution volumes and both had vasodilator activity and contracted the rat uterus. Human urinary kallikrein incubated with semipurified human low molecular weight kininogen released 76% of the total kinins as Lys-bradykinin, 7% as bradykinin, and 17% as [Ala3]Lys-bradykinin. In contrast, rat urinary kallikrein released 86% bradykinin, 18% Lys-bradykinin, and negligible amounts of [Ala3]Lys-bradykinin. The study revealed the presence of a new kinin, [Ala3]Lys-bradykinin, in human urine and it also proves that the types of kinins generated intrarenally are species-dependent.     

Development of a liquid chromatography-tandem mass spectrometry method for the determination of 23 endogenous steroids in small quantities of primate urine

A quantitative method using liquid chromatography-tandem mass spectrometry (LC-MS-MS) was developed for the simultaneous determination of 23 endogenous steroids in primate urine. The introduced method includes estrone, pregnandiol, cortisol, testosterone and several human urinary glucocorticoid and androgen metabolites. As the method is intended for the analysis of steroid hormones in behavioral studies on wild-living primates, it was adapted for a sample volume of 200microL urine. The sample preparation consisted of an enzymatic hydrolysis of steroid glucuronides using beta-glucuronidase from E. coli followed by a solvolytic cleavage of steroid sulfates employing sulfuric acid/ethyl acetate. The extraction of steroids from urine was optimized with respect to pH during extraction, type of ether and the amount of enzyme necessary for complete hydrolysis of glucuronides. The recovery of steroids spiked into urine before hydrolysis was 58.9-103.7% with an intra-day precision of 2.7-14.3% and an inter-day precision of 2.9-14.8%. Detection limits ranged from 0.1-0.5ng/mL. The reproducibility of the whole sample preparation process was also demonstrated for unspiked urine (CV 1.2-16.5%). The proportion of steroid hormone excreted as sulfate was determined for 21 steroids in chimpanzee urine. The solvolysis proved to be essential for all investigated steroids except for pregnandiol, tetrahydrocortisol and tetrahydrocortisone, which were found to be less then 10% in the solvolysis fraction.     

Utilization of circulating androstenedione and testosterone for estradiol production during gestation in the rat

The placenta provides androgen precursors for ovarian estradiol (E2) production during the second half of gestation in the rat. However, no studies have measured E2 synthesis in vivo from circulating testosterone (T) or androstenedione (A) before or after Day 12 of gestation. In addition, it is not known whether the placenta near term continues to serve as the major source of androgens. Therefore, we measured the ovarian conversion of circulating T and A to E2 in vivo on Days 11, 16, and 21 of gestation (term = Day 23). Rats (N = 6-8/group) were anesthetized with pentobarbital and a constant infusion of [3H]T or [3H]A initiated via a jugular vein. After isotopic equilibrium was achieved at 60 min, blood samples were obtained from the contralateral jugular (J) vein and a uterine-ovarian (UO) vein, and the ovaries were removed. In a second group of rats on Day 16 of gestation, either the gravid uterus or both ovaries were removed after initiation of isotope infusion, and blood samples obtained 60 min later. Radiolabeled T, A, and E2 were isolated and purified by sequential paper chromatography. The concentration of [3H]E2 following infusion of either androgen was greater in the UO vein than in the J vein on Days 16 and 21 (p less than 0.02), but not on Day 11, of gestation. In animals infused with [3H]T, [3H]E2 (cpm/ml) in UO vein increased (p less than 0.001) from 84 +/- 33 (mean +/- SE) on Day 11 to 357 +/- 30 and 312 +/- 46 on Days 16 and 21, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential metabolic fate of the carbon skeleton and amino-N of [13C]alanine and [15N]alanine ingested during prolonged exercise.

The decarboxylation/oxidation and the deamination of 13C- and [15N]alanine ingested (1 g/kg or 73.7 +/- 2 g) during prolonged exercise at low workload (180 min at 53 +/- 2% maximal O2 uptake) was measured in six healthy male subjects from V13CO2 at the mouth and [15N]urea excretion in urine and sweat. Over the exercise period, 50.6 +/- 3.5 g of exogenous alanine were oxidized (68.7 +/- 4.5% of the load), providing 10.0 +/- 0.6% of the energy yield vs. 4.8 +/- 0.4, 47.6 +/- 4.3, and 37.4 +/- 4.7% for endogenous proteins, glucose, and lipids, respectively. Alanine could have been oxidized after conversion into glucose in the liver and/or directly in peripheral tissues. In contrast, only 13.0 +/- 3.2 mmol of [(15)N]urea were excreted in urine and sweat (10.6 +/- 0.4 and 2.4 +/- 0.5 mmol, respectively), corresponding to the deamination of 2.3 +/- 0.3 g of exogenous alanine (3.1 +/- 0.4% of the load). These results confirm that the metabolic fate of the carbon skeleton and the amino-N moiety of exogenous alanine ingested during prolonged exercise at low workload are markedly different. The large positive nitrogen balance (8.5 +/- 0.3 g) suggests that in this situation protein synthesis could be increased when a large amount of a single amino acid is ingested.     

Brain-to-blood efflux transport of estrone-3-sulfate at the blood-brain barrier in rats

Efflux transport of estrogens such as estrone-3-sulfate (E1S), and estrone (E1) across the blood-brain barrier (BBB) was evaluated using the Brain Efflux Index (BEI) method. The apparent BBB efflux rate constant (Keff) of [3H]E1S, and [3H]E1 was 6.63 x 10(-2) +/- 0.77 x 10(-2) min(-1), and 6.91 x 10(-2) +/- 1.23 x 10(-2) min(-1), respectively. The efflux transport of [3H]E1S from brain across the BBB was a saturable process with Michaelis constant (Km) of 96.0 +/- 34.4 microM and 93.4 +/- 22.0 microM estimated by two different methods. By determining [3H]E1S metabolites using high performance liquid chromatography (HPLC) after intracerebral injection, significant amounts of [3H]E1S were found in the jugular venous plasma, providing direct evidence that most of [3H]E1S is transported from brain across the BBB in intact form. To compare the apparent efflux clearance across the BBB of E1S with that of E1, the brain distribution volume of E1S and E1 was estimated using the brain slice uptake method. The apparent efflux clearance of [3H]E1S was determined to be 74.9 +/- 3.8 microl/(min x g brain) due to the distribution volume of 1.13 +/- 0.06 ml/g brain. By contrast, the apparent efflux clearance of E1 was more than 227 +/- 3 microl/(min x g brain), since the distribution volume of [3H]E1 at 60 min was 3.28 +/- 0.13 ml/g. The E1S efflux transport process was inhibited by more than 40% by coadministration of bile acids (taurocholate, and cholate), and organic anions (sulfobromophthalein, and probenecid), whereas other organic anions did not affect the E1S efflux transport. The [3H]E1S efflux was significantly reduced by 48.6% after preadministration of 5 mM dehydroepiandrosterone sulfate. These results suggest that E1S is transported from brain to the circulating blood across the BBB via a carrier-mediated efflux transport system.     

Metabolism and excretion of peptide leukotrienes in the anesthetized rat

The metabolism and excretion of the peptide leukotrienes C4, D4, E4 and N-acetylleukotriene E4 have been studied in the anesthetized rat. The intravenous administration of [3H]leukotriene C4 (2.6 X 10(-11) mol/kg) showed a rapid clearance of radioactivity from the blood and a time-related biliary excretion, recovering 69 +/- 1.6% (n = 6) over 60 min. Less than 1% of total radioactivity was recovered in the urine over the same time period. Similarly, the intravenous administration of [3H]leukotriene D4 (2.5 X 10(-11) mol/kg), [3H]leukotriene E4 (2.5 X 10(-11) mol/kg) and N-acetyl[3H]leukotriene E4 (2.1 X 10(-11) mol/kg) showed a 62 +/- 7.5% (n = 4), 52 +/- 1.5% (n = 4) and 37 +/- 4.6% (n = 5) biliary recovery of radioactivity, respectively, after 60 min. Examination of bile identified leukotriene D4 and N-acetylleukotriene E4 as the main products, although substantial radioactivity, which probably represents unidentified polar products, was present at the solvent fronts of the reverse-phase HPLC. Time course studies indicated a relatively rapid conversion of leukotriene C4 to leukotriene D4, while leukotriene D4 metabolism appeared to be much slower. Leukotriene E4 was a minor product, suggesting that the N-acetylation process is rapid. Incubation of [3H]leukotriene C4 in rat plasma and whole blood in vitro resulted in a slow conversion of leukotriene C4 to leukotriene D4 and leukotriene E4 only. These data suggest that the majority of the leukotriene metabolism and excretion in vivo in the anesthetized rat occurs predominantly in the hepatic system. We conclude that this model is suitable for the measurement of in vivo production of peptide leukotrienes.     

Estrone sulfatase activity in the human brain and estrone sulfate levels in the normal menstrual cycle

When the plasma concentrations of estrone sulfate (E1S) were measured in five menstrual cycles, the highest concentrations were found on the day of LH peak (14.25 nmol/l +/- 2.94 [SE]). Peak levels of E1S were 20 times higher than the highest E2 levels measured (0.769 +/- 0.276 nmol/l). To determine whether E1S can be metabolized by adult and fetal tissues we examined estrone (E1) sulfatase activity in brain and other tissues. E1 Sulfatase activity was present in all tissues studied including adult endometrium, fat and skin. When the rate of sulfatase activity was measured in homogenates of fetal hypothalamus, frontal cortex and pituitary (n = 4), the hypothalamic activity (306.0 +/- 39.1 [SE] pmol/min/mg protein) was significantly higher than that of the frontal cortex (127.4 +/- 19.4, P less than 0.002) or pituitary (193.7 +/- 43.3, P less than 0.03). This was not apparent in the adult (n = 2) where the enzyme activity was similar in the hypothalamus (413.9 +/- 27.3) and frontal cortex (446.3 +/- 82.2) and lower in the pituitary (98.2 +/- 19.2). The Km for E1 sulfatase in the fetal frontal cortex was 28.9 microM. The high E1 sulfatase activity in estrogen responsive target tissues, particularly fetal hypothalamus, accompanied by a large circulating reservoir of E1S, suggest that this enzyme could possibly have a regulatory role in controlling the level of intracellular estrogens and in modulating their intracellular function.