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Metabolism of Progesterone by Human Lymphocytes: Production of Neuroactive Steroids¹

Departments of Medicine, Obstetrics & Gynecology, and Psychiatry, McGill University, Montreal, Canada H3G 1A4

Address correspondence and requests for reprints to: Dr. Bev Pearson Murphy, Montreal General Hospital, 1650 Cedar Avenue, Montreal, Canada H3G 1A4.

	Abstract

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Although it has long been recognized that lymphocytes have the capacity to reduce cortisol at the C3, C5, and C20 positions, the specificity and the physiological variation of these reactions have received little attention. We have shown that such reactions also occur with progesterone. Lymphocytes were isolated from whole blood using Percoll density gradient centrifugation. The cells were incubated for 20 h with tritiated progesterone as radioactive tracer. After extractions into ethyl acetate, the residue was subjected to high performance liquid chromatography, and the radioactivities of the separated compounds were determined. Without cells, 95–97% of the tracer added was recovered in the progesterone peak, while in the presence of 4 x l0⁶ lymphocytes, this was reduced to 45–90%. The metabolites obtained included at least 10 different compounds, including those corresponding in their retention times to the neuroactive 5 and 5ß dihydroprogesterones and their 3- and 3ß- tetrahydroprogesterone derivatives. The conversion decreased with the addition of other steroids such as testosterone, cortisol, and corticosterone, suggesting that these steroids are metabolized by the same enzymes. When the cells from two pregnant patients were combined and incubated with tracer, and with and without nonradioactive progesterone, no peaks were detected by two progesterone radioimmunoassays in the absence of added nonradioactive progesterone, while in its presence three peaks corresponding to 5-dihydroprogesterone, 3-hydroxy-5-pregnane-20-dione and 3ß-hydroxy-5-pregnane-20-dione eluted before the P peak. Their identities were confirmed using the two different progesterone radioassays that cross-reacted with these metabolites. The highest mean conversion (44.7% ± 3.2 SE) was found with the lymphocytes of pregnant women and with that of one lactating woman (50%). Conversions by lymphocytes of women in the follicular phase (29.3% ± 1.3 SE) were significantly lower than those in pregnancy (P = 0.014) but did not differ significantly (P 0.05) from those of women in the luteal phase (22.2% ± 3.4 SE), those of postmenopausal women (23.5% ± 4.9 SE), or of men (22.5% ± 2.4 SE). Lymphocytes appear to provide a hitherto unrecognized but possibly important source of neuroactive steroids.

	Introduction

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METABOLISM of cortisol by lymphocytes was shown in the early 1960’s (1, 2) and was studied in normal and leukemic human lymphocytes by Jenkins and Kemp (3) in 1969. They found more marked metabolism (reduction at C3,C5,C20) in leukemic than in normal lymphocytes and noted that the pattern in both differed from that seen in other extrahepatic tissues by the presence of ring A reduction and by the absence of oxidation of the 11ß-hydroxyl group. Similar results were found by Klein et al. (4).

Testosterone was shown to be metabolized by lymphocytes to androst-4-enedione and to 5-dihydrotestosterone and its 3- and 3ß- tetrahydro derivatives by Clair et al. (5) in 1984.

To our knowledge the metabolism of progesterone by lymphocytes has not been investigated, although conversion of progesterone to 20-dihydroprogesterone in blood was shown by van der Molen and Groen (6) in 1968.

There are six metabolites obtained by reduction of the A-ring of progesterone at C3 and C5, and a further 14 metabolites by reduction at C20 as well. While many of these have been shown to have some anesthetic activity (7), the most potent include 5ß-dihydroprogesterone, 3,5-tetrahydroprogesterone, 3,5ß-tetrahydroprogesterone, and 3,5ß,20-heptahydroprogesterone (8, 9). Because of their extreme insolubility in water, application to anesthesia has been limited. However these substances are known to occur naturally in the human, although little is known about their physiological significance (9). At low doses in rats, the 5ß-dihydroprogesterone was shown to decrease motor activity; however the 5-dihydroprogesterone isomer increased it (10). With the demonstration of binding of some ring-A-reduced progesterone metabolites to the -aminobutyric acidA (GABA) receptor (11), there has been increasing recent interest in these steroids (12). We present evidence that lymphocytes may be a hitherto unrecognized source of some of these compounds (13).

	Materials and Methods

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Experimental methods

Subjects were healthy hospital personnel who gave informed consent.

Tritiated progesterone ([1,2,6,7-³H(N)]-progesterone, NET 381, 101 Ci/mmol, New England Nuclear Corp., Boston, MA) was diluted in ethanol to 50 µCi/mL. Nonradioactive steroids were obtained from Steraloids (Wilton, NH) or Sigma Chemical Co. (St. Louis, MO).

Percoll (Sigma Chemical Co.) was diluted to give 400 mL of stock solution containing 40 mL of 2.5 mol/L NaCl to 360 mL Percoll. The stock solution was diluted with 0.15 mol/L NaCl 208 mL to 325 mL Percoll stock solution to give a working solution with specific gravity 1.077.

Ham’s F10 culture medium was obtained from Sigma Chemical Co. Phosphate buffer (0.075 mol/L, pH 7.4) (PBS) was filter-sterilized.

Lymphocyte preparation

Blood (20 mL) was drawn at 0800–1000 h into heparinized tubes, centrifuged, the buffy coat generously removed, diluted 1:1 with PBS, and mixed well by drawing in and out with the pipette. The final volume was adjusted to a multiple of 3 mL. Three milliliters of diluted cells was layered carefully over 3 mL Percoll working solution and centrifuged at 400 g for 30 min. The band of mononuclear cells was collected into several tubes using a Pasteur pipette. The cells were washed 3 times with 5 mL PBS and centrifuged for 15 min at 100 g after each wash. After the final wash, the supernatant was decanted and the cell button resuspended in 1.0 mL culture medium.

The cells were counted and aliquots of 4 x 10⁶ cells were added to sterile centrifuge tubes containing dried tracer (0.25 µCi per tube) and 100 µL Ham’s F10 culture medium. The tubes were incubated overnight (17 h) at 37 C in a Dubnoff bath set at speed 3.

Extraction

The tube contents were extracted with ethyl acetate 5 volumes x 2 and the extract dried under air and stored in ethanol at -20 C until chromatographed.

High performance liquid chromatography (HPLC)

An aliquot of each ethanolic residue was transferred to an injection vial, dried, and redissolved in 40 µL methylene dichloride. Of this, 25 µL was injected into an HPLC column as modified after Purdy et al. (13) and eluted at a rate of 1 mL/min using various concentrations of methylene dichloride (A) and 10% ethanol in hexane (B) as follows: 4% B for 30 min, rising to 20% B over the next 20 min, falling to 4% B over 20 min, and remaining at 4% B for a further 40 min (total 100 min). Fifty fractions were collected (6 mL x 1, 1 mL x 34, 2 mL x 10, 8 mL x 5) in counting vials and evaporated to dryness under air. Scintillator was added, and the vials were counted to 10,000 counts or for 10 min. Metabolism was expressed as 100 - % added counts in the progesterone peak. The counts in each peak were calculated as a percentage of the total amount added.

Incubations with added nonradioactive steroids.

Studies were done in the same fashion but with various amounts of steroids added to the incubation medium: 10,20,50,100, or 250 ng of progesterone, cortisol, corticosterone, or testosterone. In one experiment, 6 x 10⁶ cells were incubated in the presence and absence of nonradioactive progesterone 70 ng/mL (500 ng in 7 mL), and the metabolites were determinad by two different radioassays (see below).

Competitive binding assays of progesterone metabolites

The radioimmunoassay (RIA) employed an antiserum raised in our laboratory to progesterone-3-carboxymethyloxime-bovine serum albumin. At a dilution of 1/10,000, the sensitivity was 15 pg progesterone. The radiotransinassay (RTA) used the progesterone-binding globulin of pregnant guinea pig serum (14) at a dilution of 1/20,000, with a sensitivity of 23 pg progesterone. The relative cross-reactivities of some metabolites as compared with progesterone are shown in Table 1; the particular guinea pig serum used was exceptionally sensitive to 5-dihydroprogesterone. All the assays were carried out as described previously for cortisol and other steroids (15). Protein-tracer solutions were made up using 0.01 mol/L phosphate buffer, pH 7.0 with gelatin 5g/L (16), and sodium azide 0.01%. Incubation was 2 h at 4 C. Adsorption was done using dextran-coated charcoal. Intraassay coefficients of variation (CV) were 7%, and interassay CVs were 12%.

Small amounts (2–5 ng) of five progesterone metabolites were added to a progesterone tracer (5000 cpm) and chromatographed on HPLC as above. These included 5-pregnane-3,20-dione (5-DHP), 5ß-pregnane-3,20-dione (5ß-DHP), 3ß-hydroxy-5ß-pregnan-20-one (5ß,3ß-THP), 3-hydroxy-5-pregnan-20-one (5,3-THP), and 3ß-hydroxy-5-pregnan-20-one (5,3ß-THP). The eluate was split 30/70, the smaller portion being used to determine the position and recovery of the progesterone tracer,and the remainder assayed by the assays described above. This was repeated every few months or when a new column was used as the exact positions of the metabolites relative to progesterone may vary slightly over time and from column to column.

Determination of endogenous steroids in serum

Progesterone 5000 cpm was added to 1.0 mL of serum, extracted as above, and run in the same HPLC system. The eluate was split so that 30% was collected separately. This 30% aliquot was counted to determine the position and recovery of the progesterone tracer, and the remainder was split and assayed using the assays described above.

	Results

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In the absence of cells, recovery of radioactivity added was 96.2% ± 3.4 (SD) in the progesterone peak. No radioactivity eluted beyond 75 min. An example of a pattern obtained in a mildly diabetic postmenopausal subject (52% metabolism) to which ³H-progesterone had been added and incubated overnight is shown in Fig. 1. (The result for this patient was not included with the other postmenopausal women because she was diabetic). This pattern was chosen because it shows all the peaks seen; while in the figure some of these appear very small, even the smallest (# 9) contained more than 1000 counts, as each vial was counted for 10 min. Not all of the peaks were present in all subjects; there was considerable individual variation. Peaks were tentatively identified according to their retention times relative to that of progesterone, as indicated in Fig. 1.

View larger version (16K): [in this window] [in a new window]   Figure 1. Pattern obtained in a patient with 52% metabolism. Each vial was counted for 10 min so that the number of counts observed was 10 times that shown on the ordinate. Steroids correspond in retention time to the peaks as follows: P = progesterone (pregn-4-ene-3,20-dione); 1 = unidentified; 2 = 5-dihydroprogesterone; 3 = 5ß-dihydroprogesterone; 4 = 3ß,5ß-tetrahydroprogesterone; 5 = 3,5-tetrahydroprogesterone; 6 = 3ß,5-tetrahydroprogesterone; 7 = unidentified; 8 = 3,5ß-tetrahydroprogesterone; 9 = 20ß-dihydroprogesterone; 10 = 20-dihydroprogesterone; 11 = unidentified.

When 100 ng of testosterone or 250 ng progesterone, cortisol, or corticosterone was added to the incubation medium of various samples, marked inhibition was achieved, suggesting that these steroids compete for the same enzyme sites. When smaller amounts were added, 50% inhibition was achieved with 10 ng testosterone or about 50 ng of the other steroids.

Pattern obtained with and without added nonradioactive progesterone

When cells from two women in late pregnancy were combined and incubated in the presence of tritiated progesterone, one small peak and three large peaks preceding the progesterone peak were seen, the three large peaks corresponding in retention times to 5-DHP, 5,3-THP, and 5,3ß-THP (Fig. 2, A) (i.e. to peaks 2, 5, 6 of Fig. 1). When nonradioactive progesterone was present in a concentration of 70 ng/mL, a similar pattern with slightly smaller peaks was seen (Fig. 2, B). In the absence of added nonradioactive progesterone, no peaks were detected by either of the assays (data not shown). However in the presence of progesterone, four peaks were seen by RIA (Fig. 2, C), corresponding to progesterone and the three metabolites, while two clear peaks were seen by RTA, which cross-reacts very strongly with 5-DHP, but poorly with 5,3-THP, and 5,3ß-THP (Fig. 2, D). The positions of the metabolites corresponded exactly to those seen when pure standards were assayed by RIA 1 (Fig. 2, F).

View larger version (18K): [in this window] [in a new window]   Figure 2. Metabolism of progesterone by lymphocytes of women in late pregnancy in the presence and absence of added progesterone: raw data are shown. A, Metabolism of radioactive progesterone (P*) in the absence of nonradioactive progesterone. B, Metabolism of P* in the presence of 70 ng/mL nonradioactive progesterone. C, Metabolism of nonradioactive progesterone as measured by RIA. The peaks correspond to the radioactive peaks seen in A and B. D, Metabolism of nonradioactive progesterone as measured by RTA. E, Pattern detected by RIA in serum of the same patients. F, Positions of standards of progesterone and its metabolites as measured using RIA.In the absence of added nonradioactive progesterone no peaks were seen by either assay.

The quantitative data are shown in Table 2. The conversions calculated by assaying the steroids compared well with those observed for the radiotracer in the presence of the added nonradioactive progesterone. The concentrations of the three peaks in serum, corrected for cross-reactivity in the RIA as shown in Table 1, were 26 ng/mL for 5-DHP, 27 ng/mL for 5,3-THP, and 17 ng/mL for 5,3ß-THP.

Extent of metabolism in various groups

The extent of the metabolism of progesterone by lymphocytes of various groups of healthy subjects (mean ± SD) is shown in Fig. 3. The highest metabolism was found in pregnancy (44.7% ± 2.2 SE) and in a lactating woman studied at 8 and 9 weeks post-partum (51%, 49%). Values for cycling women in the follicular phase (29.3% ± 1.3 SE) were slightly but not significantly higher (P = 0.15) than those in the luteal phase (22.2% ± 3.4 SE). Mean values for men (22.5% ± 2.4 SE) and postmenopausal women (23.5% ± 4.9 SE) were similar to those for women in the luteal phase.

View larger version (42K): [in this window] [in a new window]   Figure 3. Metabolism of progesterone in various groups of healthy subjects (Mean ± SD). Numbers of subjects are shown in brackets.

While the extent of metabolism of the diabetic subject shown in Fig. 1 was unusually high, the pattern was otherwise unremarkable.

	Discussion

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The metabolites of progesterone are numerous. In the adrenal, progesterone is a precursor for cortisol, corticosterone, desoxycorticosterone, aldosterone, and their metabolites. To date the conversion of progesterone to its neuroactive metabolites has not been demonstrated in lymphocytes, although this finding is in keeping with previous studies demonstrating the presence in lymphocytes of the 3 and 5 reductases. The strong inhibition of progesterone metabolism by cortisol, corticosterone, and testosterone suggests that the same enzymes are involved.

The detection of three peaks by radioassay, in the presence but not in the absence of nonradioactive progesterone, provides good evidence that the substances detected are indeed progesterone metabolites. The specificities of the radioassays used provide confirmation of the identities of these metabolites. There are few progesterone metabolites that elute earlier than progesterone in this system; most are considerably more polar (and therefore elute later). It remains to confirm the identities of the other peaks detected in the other subjects. Preliminary data have shown that other endogenous peaks detected in serum by radioimmunoassays specific for progesterone and its ring A-reduced metabolites correspond to the other radioactive peaks produced by lymphocytes (Murphy, in preparation). Similar studies of the corresponding desoxycorticosterone (DOC) series of compounds are in progress; these are also of interest, as some of them are also potent anesthetics and bind to the GABA receptor.

Changes in the metabolism of cortisol and testosterone by lymphocytes in pregnancy have not, so far as we know, been studied. Clair et al. (5) found higher metabolism of testosterone in men than in women and decreased metabolism in subjects aged 75 yr or more.

The presence of the 3 and 5 reduced products of progesterone is of particular interest as these steroids are all powerful anesthetics (7); 5,3-THP is known to bind to the GABA receptor complex in the central nervous system (11, 12). If our rough estimate that about 60 µg/day of each of several neuroactive steroids may be produced by lymphocytes, these amounts may well be significant. For comparison, 50 µg per day of estradiol given transdermally is sufficient to provide estrogen replacement therapy in postmenopausal women. While a concentration of 70 ng/mL progesterone in peripheral serum occurs only in preg-nancy, and levels in men and in nonpregnant women are usually 2 ng/mL (6 nmol/L), conversion by lymphocytes in blood coming from the gonads and adrenals might be an important factor in decreasing this concentration. The secretion rate of progesterone by the adrenal was estimated to be 600 µg/day in women (17), but may be more.

It is of interest that the pattern of metabolites seen in the serum removed from the subjects studied in Fig. 2 was similar to that produced by the lymphocytes. This might mean either that the lymphocytes produce a lot of metabolites or that the pattern produced by the lymphocytes is similar to that of other tissues, possibly including the brain. This similarity is being further explored.

	Footnotes

 
¹ Supported in part by the Stairs Foundation, FRSQ (Fonds de la recherche en santé du Québec) and NARSAD (National Alliance for Research in Schizophrenia and Depression). This material was presented in part to the 10th International Congress of Endocrinology, San Francisco, June, 1996.

Received June 19, 1997.

Accepted July 29, 1997.

	References

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