Clustering of PCOS-like traits in naturally hyperandrogenic female rhesus monkeys.

Study question: Do naturally occurring, hyperandrogenic (≥1 SD of population mean testosterone, T) female rhesus monkeys exhibit traits typical of women with polycystic ovary syndrome (PCOS)? Summary answer: Hyperandrogenic female monkeys exhibited significantly increased serum levels of androstenedione (A4), 17-hydroxyprogesterone (17-OHP), estradiol (E2), LH, antimullerian hormone (AMH), cortisol, 11-deoxycortisol and corticosterone, as well as increased uterine endometrial thickness and evidence of reduced fertility, all traits associated with PCOS. What is known already: Progress in treating women with PCOS is limited by incomplete knowledge of its pathogenesis and the absence of naturally occurring PCOS in animal models. A female macaque monkey, however, with naturally occurring hyperandrogenism, anovulation and polyfollicular ovaries, accompanied by insulin resistance, increased adiposity and endometrial hyperplasia, suggests naturally occurring origins for PCOS in nonhuman primates. Study design, size, duration: As part of a larger study, circulating serum concentrations of selected pituitary, ovarian and adrenal hormones, together with fasted insulin and glucose levels, were determined in a single, morning blood sample obtained from 120 apparently healthy, ovary-intact, adult female rhesus monkeys (Macaca mulatta) while not pregnant or nursing. The monkeys were then sedated for somatometric and ultrasonographic measurements. Participants/materials, setting, methods: Female monkeys were of prime reproductive age (7.2 ± 0.1 years, mean ± SEM) and represented a typical spectrum of adult body weight (7.4 ± 0.2 kg; maximum 12.5, minimum 4.6 kg). Females were defined as having normal (n = 99) or high T levels (n = 21; ≥1 SD above the overall mean, 0.31 ng/ml). Electronic health records provided menstrual and fecundity histories. Steroid hormones were determined by tandem LC-MS-MS; AMH was measured by enzymeimmunoassay; LH, FSH and insulin were determined by radioimmunoassay; and glucose was read by glucose meter. Most analyses were limited to 80 females (60 normal T, 20 high T) in the follicular phase of a menstrual cycle or anovulatory period (serum progesterone <1 ng/ml). Main results and the role of chance: Of 80 monkeys, 15% (n = 12) exhibited classifiable PCOS-like phenotypes. High T females demonstrated elevations in serum levels of LH (P < 0.036), AMH (P < 0.021), A4 (P < 0.0001), 17-OHP (P < 0.008), E2 (P < 0.023), glucocorticoids (P < 0.02-0.0001), the serum T/E2 ratio (P < 0.03) and uterine endometrial thickness (P < 0.014) compared to normal T females. Within the high T group alone, anogenital distance, a biomarker for fetal T exposure, positively correlated (P < 0.015) with serum A4 levels, while clitoral volume, a biomarker for prior T exposure, positively correlated (P < 0.002) with postnatal age. Only high T females demonstrated positive correlations between serum LH, and both T and A4. Five of six (83%) high T females with serum T ≥2 SD above T mean (0.41 ng/ml) did not produce live offspring. Large scale data: N/A. Limitations, reasons for caution: This is an initial study of a single laboratory population in a single nonhuman primate species. While two biomarkers suggest lifelong hyperandrogenism, phenotypic expression during gestation, prepuberty, adolescence, mid-to-late reproductive years and postmenopause has yet to be determined. Wider implications of the findings: Characterizing adult female monkeys with naturally occurring hyperandrogenism has identified individuals with high LH and AMH combined with infertility, suggesting developmental linkage among traits with endemic origins beyond humans. PCOS may thus be an ancient phenotype, as previously proposed, with a definable pathogenic mechanism(s). Study funding/competing interest(s): Funded by competitive supplement to P51 OD011106 (PI: Mallick), by P50 HD028934 (PI: Marshall) and by P50 HD044405 (PI: Dunaif). The authors have no potential conflicts of interest.

Retinoid metabolism in the prostate: effects of administration of the synthetic retinoid N-(4-hydroxyphenyl)retinamide.

We have carried out a series of complementary in vivo and in vitro studies to better understand the metabolism of vitamin A by the prostate gland. Male Sprague-Dawley rats were fed either a control diet sufficient in vitamin A [CON group; 0.8 microg retinol equivalents (RE)/g diet] or a CON diet supplemented with the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR; CON+/-4HPR group; 1,173 )microg of 4-HPR/g diet). After an i.v. injection of a physiological radiolabeled dose of retinol, the vitamin A content and radioactivity of plasma and a number of tissues, including the prostate glands, were monitored for time periods ranging between 30 min and 41 days. On the basis of the results of these vitamin A turnover studies, we developed tissue subsystem models to describe vitamin A dynamics in the prostates of both the CON and CON+4HPR groups. There was a gradual decrease in the vitamin A content of the prostates of the 4-HPR-treated group as compared with the control, such that by the end of the study period, the CON+4HPR group averaged 0.166 +/- 0.0827 (mean +/- SD) REs, whereas the CON group was 0.732 +/- 0.190 REs. The fraction of vitamin A exiting the prostate each day was not significantly different in the CON as compared with the CON+4HPR group [0.149 +/- 0.103 versus 0.155 +/- 0.191 h(-1) (mean +/- FSD), respectively]; however, the average amount of vitamin A turning over from the CON+4HPR group prostates (0.0885 /microg/day) was nearly three times less than that of the CON group (0.243 microg/day). To obtain more detailed information on the mechanisms that might be involved in the changes in vitamin A kinetics observed in our in vivo studies, we used both a normal human prostate cell line (PrEC) and a human prostate adenocarcinoma cell line (LNCaP) to monitor in vitro retinol and 4-HPR dynamics. Cells were treated with 4-HPR for different time periods up to 48 h (PrEC) or 96 h (LNCaP). Retinol in the media was taken up readily by both PrEC and LNCaP cells, and there was conversion of retinol to the major storage esters of vitamin A, retinyl palmitate and retinyl stearate, as well as several minor retinyl esters, in a pattern indicative of normal retinoid esterification activity. Although 4-HPR was taken up readily and over time accumulated in both cell lines, conversion of 4-HPR to its major metabolite, N-[4-methoxyphenyl]retinamide, as well as several other metabolites of 4-HPR was apparent only in the LNCaP cells. Our findings would suggest that a study design that includes appropriately designed complementary in vivo and in vitro experimental systems represents a useful approach to better understanding possible mechanisms involved in basic retinoid functioning and interactions in the prostate as well as in other organs and related tissue culture systems.

Effects of chronic administration of N-(4-hydroxyphenyl)retinamide (4-HPR) in rats on vitamin A metabolism in the eye.

The retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) effectively inhibits cancer in a variety of tissues. In contrast to many other retinoids, the toxicity problems associated with administration of 4-HPR have been found to be minimal or absent. However, the effects of 4-HPR upon normal metabolism of native physiological forms of vitamin A in vivo have not been adequately investigated. To understand better the interaction between 4-HPR and the native physiological forms of vitamin A, the present study examines the effects of long-term administration of 4-HPR upon normal vitamin A metabolism in the eyes. Male Sprague-Dawley rats were fed either a control diet sufficient in vitamin A (CON group; 0.8 retinol equivalents [RE]/g diet; n = 28) or a CON diet supplemented with 4-HPR (CON + 4-HPR group; 1173 micrograms 4-HPR/g diet; n = 28). Following an i.v. dose of physiologically radiolabelled retinol, associated with its normal plasma transport complex, the vitamin A content and radioactivity of the plasma and eyes were examined at different times over a 41 day period. Mean plasma retinol levels measured during the study period were significantly reduced in the CON + 4-HPR group as compared with the CON group (23.5 +/- 7.0 and 50.3 +/- 5.3 [mean +/- S.D.] micrograms/dl, respectively). From approximately 7 days post-dosing, vitamin A levels in the eyes of the 4-HPR-treated group steadily decreased such that by the end of the study, they were only approximately one-fifth those of the CON group (0.098 +/- 0.075 and 0.50 +/- 0.053 RE, respectively). Kinetic analysis of vitamin A turnover in the eyes indicated that there was no apparent down-regulation of the fraction of vitamin A leaving this tissue on a daily basis; these values were found to be similar in both groups, averaging 0.104 +/- 0.0393 and 0.113 +/- 0.0373 per day (mean +/- fractional standard deviation [F.S.D.]) for the CON and CON + 4-HPR groups, respectively. At the same time, the flow of vitamin A through the eyes was significantly decreased in the CON + 4-HPR group eyes (0.0162 +/- 0.101 microgram/day) as compared with the CON group (0.0604 +/- 0.0672 micrograms/day). Our results suggest that compensatory mechanisms that would normally function to conserve depleting ocular vitamin A stores may be blocked in the 4-HPR-treated animals and further, that the 4-HPR itself appears to be interfering with the normal uptake and/or metabolism of vitamin A in the eye. These findings may help to provide at least a partial explanation for the visual impairment problems that have been reported in human trials that include long-term administration of 4-HPR.

Effects of N-(4-hydroxyphenyl)retinamide supplementation on vitamin A metabolism.

The efficacy of the retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) has been demonstrated in the inhibition of cancers in a variety of tissues. Moreover, toxicity effects following administration of 4-HPR have been found to be reduced or absent when compared to other retinoids. Pharmacokinetic studies in both animals and humans have focused on the metabolism of 4-HPR and its metabolites, and relatively little information has been published detailing the effects of long-term administration of 4-HPR upon normal endogenous vitamin A metabolism. Thus, the present study was carried out to examine the effects of long-term administration of 4-HPR upon plasma and tissue vitamin A kinetics. Male Sprague-Dawley rats were fed either a control diet sufficient in vitamin A [CON group; 1.0 retinol (ROH) equivalents/g diet] or a CON diet supplemented with 4-HPR (CON+4HPR group; 1173 micrograms 4-HPR/g diet). Following i.v. injection of a physiologically radiolabeled dose of ROH, ROH tracer and tracee kinetics were monitored in plasma and tissues over a 41-day period. Kinetic parameters were determined using the SAAM/CONSAM computer modeling programs to carry out graphical analysis of the tracer concentration curves. Mean plasma ROH levels measured for the CON+4HPR group were reduced to one-third of those of the CON group. Most of the kinetic parameters calculated were found to be significantly altered by the inclusion of 4-HPR in the diet. The fraction of the plasma ROH being catabolized per day (fractional catabolic rate) was nearly twice as high in the CON+4HPR treated group (3.61 +/- 0.49 day-1; mean +/- SD) as compared to the CON group (2.00 +/- 0.68 day-1). The amount of time that vitamin A molecules spent in the body before being lost irreversibly from the system (system residence time) was decreased by half in the CON+4HPR group (19.20 +/- 7.13 days) versus the CON group (38.63 +/- 9.62 days). Despite the increased catabolic rates and decreased system residence times measured for the CON+4HPR group, the estimated vitamin A use in these animals (11.01 +/- 3.10 micrograms/day) was 33% less than that used by the CON group (16.31 +/- 2.47 micrograms/day). Studies investigating the mechanisms by which 4-HPR alters vitamin A kinetics are presently under way in our laboratory. Nevertheless, these results suggest that long-term administration of 4-HPR markedly perturbs normal vitamin A metabolism in rats. Whether 4-HPR similarly alters human vitamin A metabolism with untoward clinical consequences deserves careful evaluation.

Relationship between age and serum vitamin A in children aged 4-11 y.

The association between age and serum vitamin A concentrations in children was examined by using total serum vitamin A values from the second National Health and Nutrition Examination Survey (NHANES II) and serum retinol values for Mexican Americans from the Hispanic HANES. Analyses included multivariate strategies to identify confounders of serum vitamin A. After the effect of the use of vitamin-mineral supplements on total serum vitamin A values was controlled for, the data indicated that younger children (aged 4-5 y) have lower serum vitamin A concentrations than do older children (aged 9-11 y) regardless of whether the measure was total serum vitamin A or serum retinol. This relationship was systematic across the distribution of values and suggested that the difference may be due to normal physiological events. A different interpretive criterion may be needed for younger and older children when serum vitamin A is used to indicate vitamin A status.

Response of plasma levels of vitamin A to a dose of vitamin A as an indicator of hepatic vitamin A reserves in rats.

Rats were fed diets deficient [-A] or sufficient [+A] (3 mg retinol equivalents/kg) in vitamin A, and without [-RA] or with [+RA] (12 mg/kg) retinoic acid supplementation for up to 33 days. Rats with plasma vitamin A levels ranging from 7 to 62 micrograms/dl were studied at intervals during progressive depletion of liver stores of vitamin A (expt. 2) and when liver stores were nearly exhausted (less than 10 micrograms/g) or replete (up to 100 micrograms/g) with vitamin A (expt. 1). A dose of retinyl acetate in corn oil (20 micrograms retinol equivalents) was administered by intubation directly into the stomach. The relative dose response (RDR), expressed as a percentage and defined as the absolute magnitude of the rise in plasma vitamin A levels 5 hours after the dose of retinyl acetate, divided by the plasma level of vitamin A attained after 5 hours, was determined for each rat and correlated over a wide range of vitamin A plasma and liver levels. An RDR above 50% invariably was associated with low plasma levels (10 to 30 micrograms/dl) and low liver stores (less than 10 micrograms/g) of vitamin A, whereas an RDR of less than 40% was associated with plasma levels above 30 micrograms/dl and liver stores ranging from 3 to 100 micrograms/g.

Effects of dietary vitamin A deficiency, retinoic acid and protein quantity and quality on serially obtained plasma and liver levels of vitamin A in rats.

Rats were fed vitamin A-deficient diets either alone, supplemented with retinoic acid (RA), or of limited protein quality or quantity (7%rice or 7% casein protein); one group was fed 7% rice protein supplemented with vitamin A. Plasma and liver levels of vitamin A were determined serially. Plasma levels in rats fed otherwise adequate vitamin A-deficient diets remained above 30 micrograms/dl until liver reserves were below 10 micrograms/g tissue, at which point plasma levels decreased in some but not all rats while liver levels continued to decline (at a slower rate) to levels as low as 3 micrograms/g. Supplementation with RA caused an immediate and sustained reduction of 15 to 20 micrograms/dl in circulating vitamin A. At 7% dietary protein, plasma levels of vitamin A remained above 30 micrograms/dl when casein protein was fed or when the rice protein diet was supplemented with dietary vitamin A, but not when the rice protein diet was fed without an exogenous source of the vitamin. A scheme is proposed suggesting possible regulatory mechanisms that might control homeostatic levels of plasma vitamin A.