GENERAL AND COMPARATIVE ENDOCRINOLOGY 83, 1.5%163 (1991)

ln Vitro Metabolism of Progesterone by Peripheral Blood of Rock Hyrax (Procavia capensis)

D. W. MAKAWITI, J. OSASO,* AND S. GOMBE*

Department of Biochemistry and *Reproductive Biology Unit, University of Nairobi, P.O. Box 30197, Nairobi, Kenya

Accepted September 4, 1990

In vitro metabolism of progesterone by hyrax whole blood, erythrocytes, or plasma in the presence or absence of NADPH was investigated. In the presence of NADPH, whole blood metabolized progesterone to So-pregnanedione and SB-pregnanedione, but in the absence of NADPH, only SB-pregnanedione was produced. Erythrocytes in the presence and the ab- sence of NADPH produced only 58-pregnanedione. The plasma component of blood me- tabolized progesterone to give So-pregnanedione, but only in the presence of NADPH. These results suggest the presence of two types of steroid reductases found in plasma and erythrocytes. The extent of progesterone metabolism by the blood of animals in differ- ent reproductive states is in the order of pregnant females > nonpregnant females > male. 0 1991 Academic Press. Inc

The rock hyrax (Procaviu capens&) be- longs to the order Hyracoidea. At 2-3 kg adult female body mass, it has an unusually long gestation period of 7.5 months. Whereas many studies have been carried out on the reproduction of the male (Neaves, 1973, 1979; Millar and Glover, 1973), there is very little information about the female endocrinology.

The plasma concentration of progester- one, one of the main hormones during preg- nancy, has been reported to be low (Heap et al., 1975). The maximum concentration at any one stage of the reproductive cycle (including gestation) does not exceed 4.6 &ml. The levels of progesterone decrease rapidly in freshly collected blood samples (Heap et al., 1975), leading to the sugges- tion that the hyrax utilizes blood to metab- olize progesterone, thereby keeping the levels low. However, there are no reports on in vitro clearance of progesterone in the peripheral circulation.

Progesterone metabolism by erythro- cytes has been reported in fetal lamb, calf, and kid (Nancarrow and Seamark, 1968;

Seamark et al., 1970). In these animals, 20ol-dihydroprogesterone is the main me- tabolite. This study was, therefore, de- signed to assess the nature of initial metab- elites of progesterone upon incubation with whole blood, washed erythrocytes, and plasma.

METHOD

Animals. Adult rock hyraxes (P. capensis) were trapped from the Lukenya area of the Eastern Prov- ince of Kenya. The animals were fed on grass and a mixture of vegetables with water ad libitum. Blood samples (1.0 ml) were collected by cardiac puncture into heparinized syringes while the animals were under chloroform anesthesia. The blood samples were either used immediately for incubation studies or centrifuged (2000g; 10 min) to separate the plasma from the blood cells to be used in further experiments.

Incubation. Tritium-labeled progesterone (Amer- sham International; 85 Wmmol) was purified by thin- layer chromatography (TLC) using a solvent mixture recommended by the manufacturer. Solutions were prepared in phosphate-buffered saline (pH 7.2) con- taining NADPH (1.58 mg/ml) and progesterone (10 ng/ ml) mixed with tritiated progesterone at approximately 300,000 cpm. To an incubation vial, 0.5 ml of 2.0 mM NADPH, 0.5 ml containing 10 ng of progesterone plus tritiated progesterone (approximately 300,000 cpm),

159 0016~6480/91 $1.50 Copyright Q 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

160 MAKAWITI. OSASO, AND GOMBE

and 1.0 ml of whole blood, plasma, or erythrocytes (washed three times and resuspended in 0.154 M NaCI) were added. The contents of the vial were in- cubated in a shaking water bath at 37” for 30 min. The vials were uncapped during incubation. At the end of incubation, 10 vol redistilled diethyl ether was added to each vial containing plasma and shaken vigorously for 10 min. The vials incubated with whole blood or erythrocytes were first centrifuged at 10,OOOg for 5 min at 4” and the supematant was then extracted with 10 vol of ether. The aqueous phase was frozen while the ether phase was decanted into clean tubes and evapo- rated in a vacuum at 37”. The contents of the tube were dissolved in 0.5 ml of absolute ethanol. Incubations with normal saline served as controls.

Thin-layer chromatography. The metabolism cx- tracts reconstituted in ethanol were applied on pre- coated silica gel TLC plates (20 x 20 cm; Macherey- Nagel. Duren, Germany). The plates were then placed in a chromatography tank containing hexane: ethyl ac- etate (5:2). The solvent was allowed to run the full length of the plate twice, each time being removed and dried before replacing back in the tank. The plate was then cut into l-cm strips along the direction of the solvent front. The strips were eluted separately with chloroform into scintillation vials. The chloroform was evaporated to dryness, scintillation fluid (Scitillation Cocktail T; Hopkin and Williams, Essex, UK) was added to each tube, and the amount of radioactivity was determined. Larger amounts of unlabeled proges- terone, 5a-pregnan-3,20-dione, and 5a-pregnan-3,20- dione were separately applied on the plate as stan- dards. After running the solvent, the location of pro- gesterone was identified under a uv lamp, whereas 5a-pregnan-3.20-dione and SB-pregnan-3-dione were charred when sprayed with concentrated sulfuric acid.

The extent of progesterone metabolism was calcu- lated as the counts of the metabohte(s) peak expressed as a percentage of the total counts recovered from the TLC plate.

RESULTS

The red blood cell (RBC) count was 6.3 + 0.5 million/ml (SD; n = 20) of blood and did not vary with sex or reproductive status.

Reproducibility of Incubations

To ascertain the reproducibility of the re- sults obtained from the metabolic studies, five l.O-ml aliquots of blood from one fe- male hyrax were incubated with progester- one in the presence of NADPH. The mean

* SD percentage progesterone metabolism in 30 min was 46.74 + 0.55.

Extent of Progesterone Metabolism by Blood from Hyraxes in Different Reproductive States

Aliquots of blood (1.0 ml) from male, pregnant, and nonpregnant hyraxes were incubated with progesterone in the pres- ence of NADPH for 30 min. The extent of progesterone metabolism in different ani- mals is shown in Table 1. After subjecting the results to paired Student t test it was observed that blood from pregnant animals metabolized progesterone to a greater ex- tent than blood from nonpregnant females (P < 0.05) which in turn metabolized pro- gesterone faster than male blood (P < 0.05). Comparatively, the blood of pregnant hyraxes metabolized progesterone to a much greater extent than the blood of males (P < 0.002).

Plasma, washed erythrocytes, and whole blood (1 .O ml) of pregnant hyrax were incu- bated with progesterone in the presence of NADPH. The incubation mixture was ex- tracted with ether and the extracts were ap- plied to TLC plates. The distribution of counts recovered from the plate is shown in Fig. 1.

Role of NADPH in Progesterone Metabolism

Whole blood, washed erythrocytes, and plasma were separately incubated with pro- gesterone in the absence of NADPH. The

TABLE 1 PERCENTAGE OF PROGESTERONE METABOLIZED BY

WHOLE BLOOD OF HYRAXES IN DIFFERENT

REPRODUCTIVE STATES

Hyrax

Nonpregnant female Pregnant female Male

a Mean 2 SD.

Percentage metabolism

30.7 t 3.8” (n = 6) 43.4 * 4.8 (n = 9) 20.6 + 4.1 (n = 6)

PROGESTERONE METABOLISM BY HYRAX BLOOD 161

Distance from origin (cm 1

FIG. 1. Differential metabolism of progesterone by FIG. 2. Progesterone metabolism by whole blood, whole blood, plasma, and erythrocytes in the presence plasma, and erythrocytes in the absence of NADPH. of NADPH. A, progesterone (unmetabolized); B, A, progesterone (unmetabolized); B, Sa-preg- 5a-pregnan-3,20-dione; C, SP-pregnan-3,20-dione. nan-3,20-dione; C, SP-pregnan-3,20-dione.

ether extracts of incubations were sepa- nol, and ethanol. Similarly, the two metab- rated on the TLC plate. The distribution of olites were recrystallized with SP-preg- counts recovered from the plate is shown in nan-3,20-dione. The results are given in Ta- Fig. 2. ble 2. Whereas metabolite B corecrys-

Control I I

Normal saline (0.154 A4 NaCl) was incu- 100 bated with progesterone (10 rig/ml) mixed ; with triated progesterone at approximately 6 On CON’RoL 300,000 cpm. The results are shown in Fig. 5 6. 3. It was concluded that incubation per se in 5 the absence of hyrax blood did not alter 3 4 progesterone. 2

I @m

Recrystallization to Specific Activity

Metabolites B and C were recrystallized 8 9 10 II 12 13 14 15 16 17

’ ’ 2 ’ ’ uyS,:n,: from origin ,cm ) twice to constant specific activity with FIG. 3. Incubation of progesterone with normal -a- 5a-pregnan-3,20-dione in acetone, metha- line.

162 MAKAWITI, OSASO, AND GOMBE

tallized with 5ar-pregnan-3,20-dione, metab- olite C corecrystallized with SP-pregnan- 3,20-dione. It was concluded that B is 5a-pregnan-3,20-dione and C is Sp- pregnan-3,20-dione.

DISCUSSION

This study confirms some earlier findings that the adult hyrax blood metabolizes pro- gesterone (Heap et al., 1975). The rate of metabolism is related to the reproductive status. The study has also shown that there is differential metabolism of this steroid by plasma and erythrocytes. Hyrax blood me- tabolized progesterone to two less polar metabolites: 5cw-pregnan-3,20-dione and 5P-pregnan-3,20-dione. The two metabo- lites were each produced by the plasma and erythrocytes, respectively. Whereas the plasma metabolism is dependent of NADPH, the erythrocytes will metabolize progesterone with or without the cofactor. The addition of the cofactor to the erythro- cytes only increases the extent of proges- terone metabolism. The metabolites of pro- gesterone are different from those observed in kid, calf, and lamb (Nancarrow and Sea- mark, 1968; Seamark et al., 1970).

Reductases have been isolated and puri- lied from erythrocytes (Kajita et al., 1969;

TABLE 2 RECRYSTALLIZATION TO CONSTANT SPECIFIC

ACTIVITYOF METABOLITES B ANDC SEPARATELY WITH kx-PREGNAN-3,2@DlONE AND 5p-PREGNAN-3.20-DIONE

Solvent

Incorporation Acetone Ethanol Methanol Acetone Ethanol Methanol

Scr-Pregnan- S$-Pregnan- 3,20-dione 3,20-dione

B C B C

wmh3

9751 1597 13490 5904 8230 11002 9924 6735 8794 6942 7162 6786 8056 4122 560.5 7344 7718 1619 3902 6142 8367 713 1941 6925 7927 163 891 6569

Niethammer and Huennekens, 1971a,b). NADPH is also known to occur in the erythrocytes. This explains why progester- one metabolism proceeds both with and without the addition of this cofactor. En- zymes such as P-glucuronidase, aldolase, lactate dehydrogenase, and alkaline and acid phosphatases exist in plasma. How- ever, there are no reports of the presence of steroid reductase in plasma. The reductase activity in the plasma component may be located in the platelets which are known to contain several metabolic enzymes (Niethammer and Huennekens, 1971a). Further studies are therefore essential to lo- calize the enzyme.

It is suggested at this stage that the hyrax utilizes the blood to metabolize progester- one to pregnanediones which are less po- tent in progestational activity. The preg- nanediones are likely to be intermediates for further reduction to pregnanediols which in turn may be excreted in urine as such or conjugated to glucuronic acid or sulfate and excreted as pregnanediol gluc- uronides or pregnanediol sulfates, respec- tively.

In addition to 20a-hydroxysteroid dehy- drogenase, active levels of 3@hydroxy- steroid dehydrogenase and 6P-steroid hy- droxylase have been detected in sheep fetal RBC (Seamark et al., 1970). In adult ani- mals, metabolism of progesterone by eryth- rocytes has only been observed in sheep (Seamark et al., 1970). Further evidence from this study indicates that the hyrax blood contains progesterone metabolizing enzymes. This phenomenon does not occur in adult human, rat, or chicken (Nancarrow and Seamark, 1968). Experiments in our laboratory with blood from adult male and female goats and rats indicated the absence of progesterone metabolism.

ACKNOWLEDGMENTS

This investigation was partly assisted by funds pro- vided by the World Health Organization, Human Re-

PROGESTERONE METABOLISM BY HYRAX BLOOD 163

productive Programme. We thank Mr. D. Ndegwa for drawing the diagrams and Miss G. Gikonyo for typing the manuscript.

REFERENCES

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Millar, R. P., and Glover. T. D. (1973). Regulation of seasonal sexual activity in an ascrotal mammal, the rock hyrax, Procavia cupensis. J. Reprod. Fertil. Suppl. 19, 203-220.

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