Figure presents the time-course of appearance of DHEA label and DHEA-sulfate in the plasma of rats given a single oral dose of tritium-labelled oleoyl-DHEA or DHEA. Total DHEA label in plasma was statistically indistinguishable for both sexes and gavages. No differences were found, either, in the patterns (anova) of plasma DHEA-sulfate for both gavages. In female rats, the height of DHEA-sulfate peak was more marked than in males, both under DHEA and oleoyl-DHEA gavages.
Figure 1 Appearance of DHEA-derived label (left) and levels of DHEA-sulfate (right) in plasma, after the administration of a gavage of tritium-labelled oleoyl-DHEA (upper row) or DHEA (lower row) to male and female Wistar rats. Males: white circles, solid lines, (more ...)
However, in both sexes, a difference was observed in the pattern of appearance of DHEA-sulfate between the DHEA and oleoyl-DHEA gavages: in DHEA rats, the 30-min data were higher than those at 1 h, whereas in those receiving oleoyl-DHEA, the values were lower at 30 min than half hour later. This delay may be explained by the timing of oleoyl-DHEA hydrolysis to DHEA and its incorporation into DHEA-sulfate.
In females, the peak of plasma radioactivity, at 1 h after gavage, was coincident with that of DHEA sulfate, both label and the sulfate levels decaying rapidly, with low values from 6 h onwards. The presence of label at 24 h was maintained, albeit very low. In spite of the different systems of calculation, the similarity of the pattern and the closeness of the concentrations involved suggest that most of the label recovered in the first hours in plasma corresponds to DHEA-sulfate at least in females. In males, plasma label DHEA equivalents were higher than the corresponding levels of DHEA-sulfate, irrespective of the gavage being DHEA or oleoyl-DHEA.
The distribution of label in plasma (Figure ) fractions was practically identical for all four groups of animals. In general, a small and variable part of the label (7–15 %) in plasma was found in the intermediate TLC area, and may be attributed to DHEA or to similarly soluble androgen- or estrogen-derived hormones. Most of the label, however could be found in the hydrophilic zone, coherent with a large presence of DHEA-sulfate, but may contain other hydrophilic esters (i.e. glucuronates or sulfates) of estrogens or androgens. Only a small fraction of the label could be found in the form of highly lipophilic compounds (up to 13 % at 24 h in female rats).
Figure 2 Percentual distribution along time of the oleoyl-DHEA derived tritium label in plasma of Wistar rats (male: left column, female: right column) receiving a single gavage of oleoyl-DHEA (upper row) or DHEA (lower row). Black area: "lipophilic" zone of distribution (more ...)
Table shows the distribution of oleoyl-DHEA-derived label in selected tissues of the rat 24 h after the gavage. A large part of the label was found in the liver and kidneys, suggesting that their presence was related with direct elimination. The distribution of label (in %; H = hydrophilic; I = intermediate and L = lipophilic) was: liver: males H: 97 ± 1; I: 3 ± 1; L: 0 ± 0, and females H: 97 ± 1; I: 2 ± 1; L: 1 ± 1; kidneys: males H: 95 ± 1; I:4 ± 1; L: 1 ± 1, and females H: 82 ± 2; I: 18 ± 2; L: 0 ± 0; adrenal glands: males H: 96 ± 2; I: 4 ± 2; L: 0 ± 0, and females H: 74 ± 1; I: 15 ± 1; L: 11 ± 3; mesenteric WAT: males H: 88 ± 7; I: 10 ± 4; L: 3 ± 4, and females H: 57 ± 8; I: 25 ± 7; L: 18 ± 7. Skeletal muscle showed much lower label content and a pattern of distribution similar those of liver and kidneys. In all, the tissues analysed accounted for more than 3/4 of the rat weight, but contained, 24 h after the gavage, less than 0.5 % of the initial label.
Tissue and organ distribution of DHEA label, 24 h after the administration of a single gavage of 35 nmol/g of tritium DHEA or oleoyl-DHEA to Wistar rats
The distribution of nonesterified DHEA-derived label in the same tissues showed, in general, a higher label content, but the distribution patterns between the tissues were similar to those obtained with oleoyl-DHEA (no statistically significant differences were found). The presence of lipophilic fractions in the tissues analysed followed, again, a pattern very close to that described for oleoyl-DHEA.
The more marked differences in the distribution of label were found when comparing male and female rats. Liver, heart and brain levels were similar for both genders; males showed higher accumulation of oleoyl-DHEA-derived label in the kidneys, spleen and adrenal glands, while females accumulated more label in the adipose tissues. The pattern obtained from DHEA gavages was similar: no gender differences in liver, kidneys, adrenal glands and heart, higher male levels in spleen and increased label presence in adipose tissues in females.
The intestinal oleoyl-DHEA esterase activity at three different pH is shown in Figure . Most of the esterase activity was concentrated in the small intestine, with relatively lower levels of activity in the jejunum, and much lower in the stomach and the caecum. The distal part of the intestine shows a esterase capability lower than that of its median part, especially at pH 8. The patterns of esterase activity for male and female rats were fairly similar, both in activity and distribution along the digestive tract. The main differences (not significant) can be found in the relatively lower duodenum activity of males at pH 7 and 8, and the higher large intestine esterase of males at pH 5 and females at pH 7. These patterns are not consistent with the existence of a single esterase activity, and may be more easily justifiable with at least two different enzymes, one working at a more acidic pH and the other with maximal activity at pH 8 or beyond, both presenting different distribution patterns along the alimentary canal.
Figure 3 Distribution of the oleoyl-DHEA esterase activity along the alimentary canal in male and female Wistar rats. The measurements were done at three different pH and are expressed in pkat/g of protein; N = 5 for each pH group. The data are plotted against (more ...)
The liver oleoyl-DHEA esterase activity was similar, but lower, to those of the intestine: 215 ± 77 pkat/g prot at pH 5.0, 362 ± 104 pkat/g prot at pH 7.0, and 596 ± 158 pkat/g prot at pH 8.0 for males and 140 ± 26 pkat/g prot at pH 5.0, 309 ± 48 pkat/g prot at pH 7.0, and 351 ± 71 pkat/g prot at pH 8.0 for females. The large mass of the liver and the obliged transit of the portal blood flow enhance its overall ability to break up the oleoyl-DHEA not hydrolysed by the intestine.
Pure pancreatic cholesterol esterase acts effectively hydrolysing oleoyl-DHEA. The effects on the DHEA and the cholesterol ester (Figure ) were slightly different, with higher yields (for the same amounts of enzyme and substrate) in the breakup of cholesterol at pH 5 and 7, but similar at pH 8. In both cases, the presence of taurocholate was essential for the enzyme to work, and the presence of the inhibitor diethyl-umbeliferyl phosphate resulted in a deep inhibition of the esterase for both substrates, an effect more marked at pH 5. The presence of taurocholate was also essential for the breakup of oleoyl-cholesterol by the intestinal homogenate. However, this same homogenate was able to hydrolyse a significant proportion of oleoyl-DHEA in the absence of taurocholate; the proportion of substrate hydrolysed was not different in the absence of taurocholate than in its presence. The inhibition of the homogenate esterase by diethyl-umbeliferyl phosphate was as effective on this preparation as was for the pure enzyme.
Figure 4 Comparison of the esterase activity of pure hog cholesterol esterase and a female rat duodenum homogenate at three different pH and in the presence of taurocholate (TC) and diethyl-umbeliferyl phosphate (DEUP). The data correspond to the same female rat (more ...)