Long-term longitudinal measurements of plasma dehydroepiandrosterone sulfate in normal men.

Dehydroepiandrosterone sulfate (DS) was measured by direct tritium RIA in longitudinal plasma specimens from 97 normal healthy male participants in the Baltimore Longitudinal Study of Aging. Fasting blood was collected at regular visits (approximately 1.5 yr apart) over an average 13 yr of adulthood (cumulative age range: 32-83 yr). DS was measured in 3-4 widely spaced specimens from each subject. A decline in DS was found in 65 (67%) subjects, 13 subjects (13%) showed no change, and increases were found in the 19 remaining subjects during the study period. A plot of individual data points revealed the same pattern we had obtained previously from a cross-sectional study of a different normal male population. A plot of DS values vs. age among subjects whose DS increased during the study also revealed an age-related decline. Thus, the longitudinal decrease in circulating DS, long inferred from cross-sectional data, is confirmed for normal men in the present study. A more detailed study of every specimen collected during the study period from 12 of the Baltimore Longitudinal Study of Aging subjects (4 whose values tended to be low, 4 whose values tended to be high, and 4 whose values were near the mean) failed to reveal any patterns of variation that could be correlated with changes in life circumstances, health status, or any other discernible factors. Hence, the wide variability seen in DS among individuals within normal populations remains unexplained.

Esterase activity in human breast cyst fluid: associations with steroid sulfates and cations.

Human breast cyst fluid (BCF) contains an esterase that on the basis of electrophoretic mobility and response to inhibitors differs from those found in the plasma. From a total of 384 BCF samples analyzed for esterase using p-nitrophenyl hexanoate as substrate, 149 (39%) showed significant activity. The samples had been analyzed for the concentrations of the sulfates of estrone, estriol, dehydroepiandrosterone, as well as the potassium and sodium cations (K+/Na+). The data were submitted to statistical analysis using the Spearman rank order test. The esterase-positive samples exhibited a significant positive association with each of the steroid sulfates and the K+/Na+ ratios. Except for protein concentration, there was no significant correlation between the esterase-positive and esterase-negative cysts. These observations may have physiological significance in that high K+/Na+ ratio cysts have been related to the histological status of the cyst.

Relationship between the concentrations of estriol sulfate and estrone sulfate in human breast cyst fluid.

Estriol-3-sulfate (E3S) is present in human breast cyst fluid (BCF) in median levels of 8.7-10.4 nmol/L, yet is barely detectable in the serum (less than 0.034 nmol/L). The source of this huge concentration of E3S is unknown. It may accumulate from blood by active transport or be synthesized and concentrated within the cyst. Since estrone sulfate (E1S) and its possible precursor, dehydroepiandrosterone sulfate (DHEAS) are elevated in BCF, E3S may originate via 16 alpha-hydroxylation of E1S. The present study examined the correlations between the levels of DHEAS and E1S with those of E3S in BCF. The sodium and potassium ions were also quantified and related to the steroid concentrations. By linear regression analysis of log-normalized data there was a highly significant correlation between the concentrations of E1S and E3S (n = 355, r = 0.690, P less than 0.001) and between DHEAS and E3S (n = 361, r = 0.577, P less than 0.001). The BCF were classified according to their K/Na ion ratios: type 1, greater than 1.0, type II, less than 0.25, and type III, 0.25-1.0. By Student's t test, the concentrations of E3S differed between each BCF Type (P less than 0.002). This was also true for E1S and DHEAS. Type 1 cysts were associated with the highest estrogen sulfate levels and type II with the lowest levels. The possible physiological importance of this observation resides in reports that the BCF type expressing the highest steroid concentrations has been related to an aporcine-like epithelial lining of the cyst wall and a somewhat higher risk for developing breast cancer. The results suggest that E3S in BCF may originate from E1S, but alternate mechanisms are not precluded.

Elevated serum dehydroepiandrosterone sulfate levels in practitioners of the Transcendental Meditation (TM) and TM-Sidhi programs.

Serum dehydroepiandrosterone sulfate (DHEA-S) levels were measured in 270 men and 153 women who were experienced practitioners of the Transcendental Meditation (TM) and TM-Sidhi programs, mental techniques practiced twice daily, sitting quietly with the eyes closed. These were compared according to sex and 5-year age grouping to 799 male and 453 female nonmeditators. The mean DHEA-S levels in the TM group were higher in all 11 of the age groups measured in women and in 6 of 7 5-year age groups over 40 in men. There were no systematic differences in younger men. Simple regression using TM-group data revealed that this effect was independent of diet, body mass index, and exercise. The mean TM-group levels measured in all women and in the older men were generally comparable to those of nonmeditator groups 5 to 10 years younger. These findings suggest that some characteristics of TM practitioners are modifying the age-related deterioration in DHEA-S secretion by the adrenal cortex.

Direct radioimmunoassay of androstenediol-3-sulfate in the serum of normal men.

A commercially available antidihydrotestosterone antiserum was used for the direct radioimmunoassay of androstenediol-3-sulfate (ADS) in human serum. Aliquots of 1 or 2 microliter male serum (mean age of 40 subjects, 38.2 +/- 5.0 years) were diluted and extracted with ethanol for assay. The tracer, [7-3H]ADS, was prepared by sodium borohydride reduction of [7-3H]dehydroepiandrosterone sulfate (DS). Significantly cross-reacting steroids were testosterone, DS, androsterone sulfate, and epiandrosterone sulfate, which combined to produce a mean overestimation of ADS of 4.3 micrograms/dl in male serum. Mean serum ADS was 23.6 +/- 10.0 micrograms/dl (SD) in 20 fresh-frozen sera versus 28.4 +/- 9.7 micrograms/dl (SD) in 20 long-term (24.4 +/- 1.2 years) frozen specimens, showing stability on long-term frozen storage. Androstenediol-3-sulfate also showed a strong correlation with serum DS (r = 0.75). The possible physiologic significance of ADS is discussed, particularly in terms of the known estrogenicity of unconjugated androstenediol.

Radioimmunoassay of estrone sulfate in the serum of normal men after a non-chromatographic procedure that eliminates interference from dehydroepiandrosterone sulfate.

Duplicate aliquots of 20 fresh-frozen normal human male sera were prepared for estrone sulfate (ES) radioimmunoassay (RIA) by each of three different methods: the thin-layer chromatography (TLC) procedure we previously reported, a new procedure including overnight heating (100 C) of an ethanol extract reconstituted in dilute acetate buffer, and the new procedure with the hot incubation omitted. The purpose of the 100 C incubation was the selective thermal solvolysis of dehydroepiandrosterone sulfate (DS), the only steroid conjugate present in serum in high enough concentrations to interfere with a high-specificity ES RIA. Dehydroepiandrosterone released by solvolysis and endogenous unconjugated steroids were extracted from the samples with ether before RIA. Estrone sulfate values obtained after the thermal solvolysis preparation averaged 854 +/- 501 pg/ml (SD) versus 826 +/- 474 pg/ml (SD) after the TLC method, with excellent correlation between the two (r = 0.97). Samples prepared by the new method but with thermal solvolysis omitted averaged a 33.8% elevation of measured ES level, an elevation significantly correlated (P less than 0.02) with DS levels obtained from the same specimens. In addition, a single specimen showed no elevation after preparation by the thermal solvolysis method when up to 8 micrograms/ml authentic DS as added before extraction. Compared with the TLC method, the new method also provides substantial savings in specimen volume requirements and sample processing time.

Radioimmunoassay of estrone sulfate in the serum of normal men after a chromatographic procedure that eliminates dehydroepiandrosterone sulfate interference.

Fifty fresh-frozen normal male sera containing tritiated estrone sulfate (ES) and dehydroepiandrosterone sulfate (DS) were extracted with ethanol after ether extraction of unconjugated steroids. Washed extracts were defatted and chromatographed on polyamide-coated plates by reversed phase paired ion TLC. Plates were scanned for radioactivity, and ES peaks were cut, eluted and assayed by direct RIA with a commercially available antiserum. Mean ES values were 445 +/- 209 pg/mL (SD), in agreement with the three lowest of the seven laboratories which had previously reported normal male ES values. No differences were observed in ES values when samples were rechromatographed prior to assay, or when up to 4 micrograms/mL unlabeled DS was added to serum before extraction. These data confirm the absence of interference by DS in the current study and suggest that previously reported high (716-1194 pg/mL) mean normal male ES values reflect DS interference. The present study also demonstrates the the stability of ES in sera stored frozen at -40 C for an average of 17 years (mean: 406 +/- 258 pg/mL; [SD]; n = 41).

A new reversed phase, paired ion thin-layer chromatographic method for steroid sulfate separations.

The sulfoconjugated steroids estrone sulfate (ES) and dehydroepiandrosterone sulfate (DS) were separated in the reversed phase mode on polyamide-coated TLC plates. Baseline resolution was obtained between tritiated ES and DS standards when run with a mobile phase of 20% acetonitrile in 5mM aqueous triethylamine, triethanolamine, tris-hydroxymethylaminomethane, tributylamine or ammonia. ES and DS showed no mobility in the absence of an ion-pair reagent. The radioactive peaks were detected and integrated non-destructively by scanning. Quantitation was confirmed by elution of cut-out peak areas and liquid scintillation counting. Similar results were obtained with washed ethanol extracts of serum labeled with tritiated ES and DS. The extracts were defatted on the plate with hexane: ethyl acetate (1:1) prior to the reversed phase development.

Serum retinol and retinol-binding protein levels do not predict subsequent lung cancer.

Retinol and retinol-binding protein levels were measured in sera previously obtained, and stored in the frozen state, at multiphasic health checkups from 151 persons subsequently found to have lung cancer (cases) and 302 persons who remained free of cancer (controls). Two controls were matched to each case for sex, skin color, age, date of multiphasic health checkup, and aspects of the smoking habit. Mean levels in cases and controls were, respectively, retinol: 82.17 and 82.37 micrograms/dl (p = 0.93), and retinol-binding protein: 6.04 and 6.00 mg/dl (p = 0.81). Mean differences between cases and controls were, retinol: 0.195 micrograms/dl with 95% confidence limits, -3.91 and 4.30 micrograms/dl; retinol-binding protein: -0.033 mg/dl with 95% confidence limits, -0.31 and 0.24 mg/dl. No significant trend in relative risk of lung cancer was observed when the retinol or retinol-binding protein distribution was divided into quintiles. No significant associations were observed in subgroups based on age, sex, histologic type of cancer, cigarette consumption, or interval between blood drawing and cancer diagnosis. In this large study, retinol and retinol-binding protein levels were not useful in predicting the subsequent development of lung cancer.

Analysis of ear sebum of the Syrian hamster (Mesocricetus auratus) reveals pronounced sexual dimorphism.

External ear of male and female Syrian hamsters (Mesocricetus auratus) were extracted with hexane and separated by class on thin-layer chromatography (TLC). Separated lipid classes were eluted and saponified, and non-saponifiable lipids further characterized by TLC, gas-liquid chromatography (GLC), UV and IR spectroscopy and functional group analyses. Many sex differences were observed, most notably the presence of sex-specific sterols of males and females. Mature animals were found to have greater quantities of ear sebum, but the characteristic qualitative lipid profiles of each sex were apparent in immature animals.

A new partition thin-layer chromatographic method for steroid separations.

TLC plates with a 25 mu thick polyamide stationary phase were modified for the separation of neutral steroids by impregnation with propylene glycol. A mixture of tritiated 5 alpha-androstan-3 alpha,17 beta-diol, testosterone, 17 beta-hydroxy-5 alpha-androstan-3-one and 4-androstene-3,17-dione was applied to the plate and developed in a toluene mobile phase to a height of 13.6 cm. This resulted in complete resolution of the 4 compounds as detected by a gas flow scanner or imaging analyzer. Cutting and elution of peak areas with methanol resulted in quantitative recovery of all four steroids. The thinness of the layer also permitted a 3-5% counting efficiency on scanning, resulting in good quantitation of recovery without liquid scintillation counting. The high sorptive capacity of the polyamide layer also enabled extracts of normal human serum to be defatted on the TLC plate by development with pure hexane prior to the toluene step. The new method thus offers several advantages over existing methods for steroid separations and should be adaptable to separations of other relatively non-polar compounds.

Age changes and sex differences in serum dehydroepiandrosterone sulfate concentrations throughout adulthood.

In a cross-sectional study, serum dehydroepiandrosterone sulfate (DS) concentrations were measured in 981 men and 481 women, aged 11-89, yr. The resulting data were asymetrically distributed and were normalized by logarithmic transformation and analyzed by 5-yr age grouping (e.g. 15-19 yr, 20-24 yr, etc.). The DS concentration peaked at age 20-24 yr in men (logarithmic mean, 3470 ng/ml) and at age 15-19 yr in women (log mean, 2470 ng/ml). Mean values then declined steadily in both sexes (log mean at greater than 70 yr of age, 670 ng/ml in men and 450 ng/ml in women) and were significantly higher in men than women at ages from 20-69 yr. Analysis of 517 randomly selected sera (from women) which had been stored frozen for 10-15 yr gave results indistinguishable from values obtained from fresh specimens. In a supplementary study, a longitudinal analysis of weekly specimens from 4 normal men, aged 36-59 yr, revealed individual variability (mean coefficient of variation, 19%) and failed to demonstrate any monthly, seasonal, or annual rhythmicity. Based on the above analyses, a table of normal serum DS ranges for adult men and women is presented for use as a clinical reference.

In-vitro testosterone metabolism in the mouse preputial gland: intercellular co-operation and changes with cell maturation.

In-vitro [14C]testosterone metabolism was investigated in isolated cells of adult male mouse preputial sebaceous glands. Labelled steroids were extracted and chromatographed after a 2-h incubation, and were identified as 5 alpha-dihydrotestosterone, androstenedione, 5 alpha-androstane-3,17-dione, 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol, androsterone and 3-epiandrosterone. In cells separated according to state of maturity (lipid content) by isopycnic centrifugation in a metrizamide gradient, maximal testosterone metabolism occurred in large, nearly mature cells. In this population, mean hydroxysteroid 5 alpha-reductase and 17 beta-hydroxysteroid dehydrogenase activities were 3.8 and 2.3 nmol/10(6) cells per 2h respectively, more than 100-fold greater than in the densest population, comprised of undifferentiated and early differentiating cells. It was also found that the profile of testosterone metabolites was dependent on the proportion of the label metabolized. The metabolite index (MI), i.e. the average number of enzymatic steps undergone per molecule of metabolite, increased with increasing substrate utilization. Metrizamide showed reversible, non-specific inhibition of testosterone metabolism and reduction of the MI. Thus, it was postulated that testosterone is metabolized sequentially by different cells, with metrizamide inhibiting cellular uptake and intercellular substrate transport. This suggested that most of the metabolites would be found in the medium, rather than in the cellular compartment. Further, in incubations run without cell disaggregation, efficient substrate cycling among cells should result in a high MI, independent of metrizamide concentration and substrate utilization. These predictions were all confirmed, providing strong evidence that testosterone metabolism is a co-operative effort among several cells in this tissue.

Sebaceous gland differentiation: III. The uses and limitations of freshly isolated mouse preputial gland cells for the in vitro study of hormone and drug action.

The effects of selected hormones and drugs on freshly isolated mouse preputial gland cells have been studied. The steroid hormones, testosterone, DHT, androstanediol, androsterone and androstanedione all failed to stimulate, and estradiol failed to inhibit, either DNA or lipid synthesis under the conditions studied. Thyroxine and insulin had no effect on lipogenesis but epinephrine and PGE2 caused significant stimulation as did Bt2cAMP. The antilipemic drugs clofibrate, nicotinic acid and hydroxycitrate were all able to inhibit lipogenesis. Of the anti-acne drugs only L-DOPA was able to inhibit lipogenesis, neither tetracycline nor trans-retinoic acid showed any effect. Pyridoxine was unable to inhibit lipogenesis but DMSO caused dramatic stimulation though it was without effect on DNA synthesis. Evidence is presented which suggests that the lack of response to steroid hormones is not due to the inability of the cells to take up and metabolize the steroids but is due to the fact that the time-span of exposure is not long enough to elicit a cellular response. It is concluded that these freshly isolated cells are suitable for the study of those effects of hormones and drugs which occur within the first 3 hr after exposure to the compound.