Effects of Diphenylhydantoin, Phenobarbital, and Diazepam on the Metabolism of Methylprednisolone and Its Sodium Succinate MELVIN R. STJERNHOLM1 AND FRED H. KATZ Division of Endocrinology, Department of Medicine, University of Colorado School of Medicine and Veterans Administration Hospital, Denver, Colorado ABSTRACT. The metabolic clearance rates (MCR) of methylprednisolone (MP) (no. = 13) and methylprednisolone-21-Na-hemisuccinate (MPHS) (no. = 6) were studied in normal humans using tritiumlabeled steroids. The cumulative appearance of the labeled steroid was examined for the whole urine and for three major urinary fractions. The MCR, half-life, and volume of distribution were, respectively, 383 ± 72 (SD) liters/day, 165 ± 49 minutes, and 61 ± 12 liters for MP, and 234 ± 37, 160 ± 19, and 41 ± 6 for MPHS. Diphenylhydantoin (DPH) administered to 4 subjects increased the MCR of MP from 424 ± 71 to 977 ± 132 (P < 0.01), and decreased the half-life from 149 ± 44 to 69 ± 7 (P < 0.001). Similar effects were found with phenobarbital (PB). Diazepam
(DZP) had no effect. Major increases in urinary metabolites after DPH and PB were in the unconjugated ethyl acetate fraction, and this suggests that MP metabolism is significantly altered by hepatic microsomal hydroxylation enzyme induction by DPH and PB, but not DZP. This could occur with the formation of a 6/3-hydroxy derivative which could be readily cleared by the kidney. The urinary pattern of excretion for MPHS was similar to that of MP. The MCR of MPHS was affected to a lesser extent by DPH and PB than was the MCR of MP (P < 0.01). Therefore, the use of hepatic microsomal hydroxylase inducers should be taken into consideration in clinical states in which MP is being used. (J Clin Endocrinol Metab 41: 887, 1975)
S
YNTHETIC corticosteroids are metabolized differently in the liver than the naturally occurring corticosteroids, cortisol and cortisone (1,2). Reductases play a major role in the metabolism of cortisol and cortisone and, following their action, conjugation to glucuronic acid occurs. However, the 1-2 double bond in the steroid nucleus of synthetic corticosteroids causes resistance to A-ring reduction and appears
to enhance metabolism by hepatic microsomal hydroxylation, resulting in a prolonged half-life. The activity of the liver cytochrome P-450 hydroxylase enzyme (3) is markedly enhanced following the administration of diphenylhydantoin (DPH) and phenobarbital (PB). This enzyme is responsible for the introduction of a hydroxyl group at the 6/3 position of the steroid nucleus (4). The clinical significance of increased corticosteroid hydroxylation has Received September 26, 1974. been suggested by Jubiz et al. (5) in their Supported by NIH Training Grant No. T01AM5101; studies showing poor dexamethasone supGrant RR00051 from the General Clinical Research pression following DPH, due to enhanced Center Program, Division of Research Resources, USPHS; Veterans Administration Project 4844-01; a dexamethasone metabolism, and also by grant from The Population Council, New York; and a Brooks et al. (6) in their studies showing grant from The Upjohn Company, Kalamazoo, Michi- increased bronchospasm following the adgan. ministration of PB to prednisone-depend1 Present address: 2750 Broadway, Boulder, Col- ent asthmatics. orado. Reprints: Dr. Fred H. Katz, University of Colorado Medical Center, Denver, CO 80220. Non-proprietary names and trademark of drugs: 6a-methylprednisolone, Medrol (Upjohn); 6amethylprednisolone-hemisuccinate, Solu-medrol (Upjohn); Diphenylhydantoin, Dilantin (Parke-Davis); Diazepam, Valium (Roche).
Another synthetic corticosteroid, 6amethylprednisolone (MP) is presently used in a wide range of clinical states (7-9). Therefore, we decided to investigate the extent to which the widely used drugs DPH, PB, and diazepam (DZP) affect the
887
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888
STJERNHOLM AND KATZ
metabolism of MP and its 21-sodium hemisuccinate (MPHS). Materials and Methods Nineteen healthy, paid volunteers were studied. The study had the prior approval of the University Human Research Committee and the subjects gave their informed consent. The volunteers included medical students, teachers, and medical technologists between the ages of 19 and 32. None of the subjects had been receiving other drugs for a period of at least 2 months prior to study. Control MCR of MP was measured in 13 subjects, 5 females (F) and 8 males (M). Four subjects were restudied after DPH, 300 nig per day; four after PB, 120 mg per day; and four after DZP, 20 mg per day. The drugs were given in 3 or 4 divided doses daily for 3 weeks. Control MCR determinations using MPHS were performed in 6 subjects, (3 M, 3 F). These studies were repeated after DPH in 2 subjects and after PB in 2 subjects. Administration of tritium-labeled steroids. MP (10 /nCi/75 fig) and MPHS (10 /xCi/100 /tig) generally labeled with 3H were prepared and purified by New England Nuclear Corporation, Boston. Mobility on thin-layer chromatography (TLC) was identical to that of the unlabeled steroids used. The labeled steroids were stored in crystalline form and dissolved in pure ethanol for MP and ethanol with 2% deionized water for MPHS, prior to use. One ml (approximately 10 fid) was measured with a glass tuberculin syringe and transferred with sterile technique to a 25 ml glass vial containing sterile isotonic saline. The total radioactivity injected was determined by removing 100 fx\ of the solution and counting in a liquid scintillation spectrometer. The remainder of the 26 ml was then injected, over 60-90 seconds, into the antecubital vein of the recumbent subjects who had been fasting overnight. Several minutes of lightheadedness following the injection, presumably a consequence of the bolus of ethanol, occurred in most subjects. The MCR's were started between 8 and 9 AM. Venous blood was sampled through an indwelling 21 gauge Mini-Cath (Deseret Pharm. Co., Sandy, Utah) using a 3-way heparin lock. Heparinized plasma was obtained before injection of labeled steroid and at 5, 10, 20, 30, 60, 90, 120, 150, and 180 minutes after injection of the labeled MPHS, and, in addition,
JCE & M • 1975 Vol 41 • No 5
at 210 and 240 minutes after injection of MP. The samples were kept at 4 C and either extracted immediately after completion of the MCR or stored at — 20 C until extraction procedures could be performed. The plasma concentrations of the labeled steroids were determined and the MCR computed by the method of Peterson and Wyngaarden (10) using a 2-compartment model (11) and computer program as previously described (12). Recovery of MP from plasma. Two ml aliquots of plasma were extracted with 10 ml of freshly distilled, nanograde, dichloromethane (DCM) similar to the method of Kozower and coworkers (13). Eight ml aliquots of the DCM were then dried in counting vials with air at 37 C. Ten ml of a liquid scintillation fluid made up by mixing Biosolv BBS-3 (Beckman Co.) 350 ml, toluene 3500 ml and Omnifluor (New England Nuclear) 14 g, was added to the dry residue for counting. Quench correction was determined with an external barium standard. Recovery of the tritiated MP from plasma averaged 85 ± 4.9 (SD)% by this method. Recovery of MPHS from plasma. Only 6.0 ± 0.2% of MPHS added to plasma was extractable with DCM or ethyl acetate without prior acidfication. Therefore, the pH of 2 ml aliquots of plasma was adjusted to between 1.5 and 2.0 with 3N H2SO4, using pH paper. The plasma was extracted twice with 10 ml of freshly distilled nanograde ethyl acetate. Eight ml aliquots of each 10 ml aliquot of ethyl acetate were combined and dried for counting. Recoveries of a known amount of 3H-MPHS added to 2 ml of the baseline plasma from individual subjects extracted by this procedure averaged 75 ± 7.5%. Larger concentrations of acid did not increase the yield. The residual counts remained in the aqueous phase rather than being found in the precipitated proteins. This is not surprising in view of the water solubility of MPHS. Identification of the radioactive material in plasma. Two ml aliquots of plasma taken from each subject at 150 and 180 minutes for MPHS and at 210 and 240 minutes for MP were extracted as described above. Several samples from different subjects were pooled to increase counts and, thereby, the accuracy of recovery calculations. The residues were transferred to
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DIPHENYLHYDANTOIN AND METHYLPREDNISOLONE thin-layer chroinatography (TLC) plates, activated by heating to 110 C for one h, using ethyl acetate:ethanol:H2O (50:50:1) for spotting. The plates were run by ascending technique in ethyl acetate:ethanol:NH4OH (5:5:1). Non-radioactive MP and MPHS were added to the respective samples to allow localization of the steroids by UV absorption. Known quantities of the tritiated steroids were individually added to control plasma, extracted, and chromatographed as reference standards and as controls for the recoveries of the injected labeled steroids. The origin, MP, and MPHS areas were identified and the silica gel transferred by scraping all sections of the chromatograms in 2.5 cm divisions into counting vials. One ml of tetrahydrofuran:H2O (1:1) was added, the vials were mixed, and then scintillation fluid added. Recovery of radioactivity applied directly to thinla\ CM- plates was 73.6 ± 13.6% for MPHS.
889
whole urine. Urinary creatinine was measured to ensure completeness of collection. Data is expressed as % of administered dose ± SD. Paired t tests, comparing values before and after DPH, PB, or DZP, were used to determine statistical significance. Apparent volume of distribution (V) is represented by the volume of the inner pool occurring after equilibrium and the half-life refers to half-life of the exponential slope (/3) following equilibration (11). Results
Steroid characterization in plasma and control values. As was true for prednisolone in the study of Kozower et al. (13), recovery of MP from plasma by thinlayer chromatography was very efficient. As much as 98% of the radioactivity plated could be recovered from the plates and Radioactivity in urine. Urine was collected in 95% of this migrated at the R of authentic f aliquots at 4, 8, and 24 h after the administration MP. of tritiated MP and at 3, 6V2, 10&, and 24 h after MPHS offered not only the difficulty of the administration of tritiated MPHS. Radioacextraction from plasma mentioned above, tivity was determined by a technique quite similar to the method of Haque et al. (4). Thus but was also more poorly recovered from radioactivity measurements were obtained for TLC plates. Table 1 illustrates the results the whole urine and for four constituent frac- of chromatography experiments. Twotions: DCM, free steroids soluble in dichloro- thirds of the radioactivity applied to the methane; EA, polar free steroids soluble in plates was recovered after elution, for ethyl acetate after "salting out," usually re- reasons that are not understood. However, lated to 6/3-hydroxylation (14); DCM conjuas can be seen in Table 1, 70% of the gates, steroids soluble in dichloromethane after /3-glucuronidase hydrolysis; and steroids soluble recovered radioactivity migrated as MPHS. only in ethyl acetate after glucuronidase treat- It is possible that the finding of some MP ment. The latter group is negligible for syn- on the chromatograms is due to hydrolysis thetic glucocorticoids and will not be mentioned of the MPHS salt in plasma or in the subsequently. The 4 fractions together ac- strongly alkaline medium of the chrocounted for 85 ± 4.4% of the radioactivity in the matography solvents, since similar re-
TABLE 1. TLC of plasma extracts containing MPHS % recovery of total counts plated
% of recovered counts at origin
% of recovered counts at MP position R, = 0.67
% of recovered counts at MPHS position R, = 0.26
Control plasma with 3 H-MPHS added in vitro
59 ± 1.1
9 ±2.3
15 ± 2.3
71 ± 4.7
Pooled plasma at 150 and 180 minutes during MCR
66 ± 2.8
15 ± 3.5
16 ± 10.1
69 ± 12.9
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890
STJERNHOLM AND KATZ
JCE & M • 1975 Vol 4 1 . No 5
TABLE 2. Control steroid data (±S.D.)
no.
Age (yr) MCR (I/Day) MCR/M2 SA* Half-life (min) Vol (1)
MP
MPHS
13 25 ± 5 383 ± 72 215 ± 4 1 165 ± 49 61 ± 12
6 26 ± 5 234 ± 37 130 ± 13 160 ± 19 41 ± 6
3JO
CONTROL ....DPH 1.0
0.5
* Surface area.
suits were found for MPHS added to control plasma in vitro (Table 1). At any rate, it seems quite likely that about 70% of the radioactivity in the plasmas collected during the MCR determination was in the form of MPHS because of a parallel experiment, testing the extractability of the labeled steroid. Aliquots of the 30, 60, 90, and 120-minute specimens obtained during 4 control MCR studies of MPHS were extracted with dichloromethane. An average of 26.5 ± 8.8% of the total radioactivity in the plasma was extracted. Since only 6% at most of MPHS can be extracted from plasma with this solvent, this suggests that over 70% of the radioactivity was not exti'actable and was likely to be in the form of MPHS or a more polar metabolite. Table 2 shows the comparative values of the MCR, half-life, and V for MP and MPHS in the baseline studies. Effect of drugs on steroid metabolism. In Table 3 are shown the effects of DPH, PB, and DZP on the MCR, half-life and V for MP. Figure 1 shows the disappearance curves of labeled MP before and after DPH. The significant change with DPH is reflected in the marked increase in the MCR (+130%) and decrease in half-life (-56%). Figure 2 shows that a major increase in the excretion of products of TABLE
MCR (I/Day) Half-life (min) Vol (1)
0.1
30
60
120
180
240
TIME- MINUTES
FIG. 1. Disappearance curves for MP in 4 subjects before and after the administration of DPH. Values represent the mean ± SD. (P < 0.01 after 60 minutes).
labeled MP in the urine after DPH was due to a significant increase in the polar unconjugated ethyl acetate fraction. A significant, but small, decrease occurred in the conjugated DCM fraction. There was no change in the extraction of the free steroid in the unconjugated DCM fraction. After PB the MCR of MP increased by 90%, which was slightly less than after DPH, and similarly there was a smaller but still significant increase (P < 0.01 at 24 h) in the unconjugated ethyl acetate urinary fraction after PB. DZP had no effect on the disappearance curves of labeled MP. The urinary fractions showed only a slight, but statistically significant, increase in the unconjugated ethyl acetate fraction at 4 and 24 hours. The results of the MCR studies with MPHS are shown in Table 4. Because of the similar effects of DPH and PB on the MCR of MPHS, the results for the two drugs were combined for graphic presentation in Fig. 3. It must be emphasized that
3. Effect of DPH, PB, and DZP on the MCR of MP in 4 subjects
Pre
Post DPH
P
Pre
Post PB
P
Pre
Post DZP
P
424 ± 71 149 ± 44 63 ± 1 1
977 ± 132 69 ± 7 85 ± 15
(DZP) had no effect. Major increases in urinary metabolites after DPH and PB were in the unconjugated ethyl acetate fraction, and this suggests that MP metabolism is significantly altered by hepatic microsomal hydroxylation enzyme induction by DPH and PB, but not DZP. This could occur with the formation of a 6/3-hydroxy derivative which could be readily cleared by the kidney. The urinary pattern of excretion for MPHS was similar to that of MP. The MCR of MPHS was affected to a lesser extent by DPH and PB than was the MCR of MP (P < 0.01). Therefore, the use of hepatic microsomal hydroxylase inducers should be taken into consideration in clinical states in which MP is being used. (J Clin Endocrinol Metab 41: 887, 1975)
S
YNTHETIC corticosteroids are metabolized differently in the liver than the naturally occurring corticosteroids, cortisol and cortisone (1,2). Reductases play a major role in the metabolism of cortisol and cortisone and, following their action, conjugation to glucuronic acid occurs. However, the 1-2 double bond in the steroid nucleus of synthetic corticosteroids causes resistance to A-ring reduction and appears
to enhance metabolism by hepatic microsomal hydroxylation, resulting in a prolonged half-life. The activity of the liver cytochrome P-450 hydroxylase enzyme (3) is markedly enhanced following the administration of diphenylhydantoin (DPH) and phenobarbital (PB). This enzyme is responsible for the introduction of a hydroxyl group at the 6/3 position of the steroid nucleus (4). The clinical significance of increased corticosteroid hydroxylation has Received September 26, 1974. been suggested by Jubiz et al. (5) in their Supported by NIH Training Grant No. T01AM5101; studies showing poor dexamethasone supGrant RR00051 from the General Clinical Research pression following DPH, due to enhanced Center Program, Division of Research Resources, USPHS; Veterans Administration Project 4844-01; a dexamethasone metabolism, and also by grant from The Population Council, New York; and a Brooks et al. (6) in their studies showing grant from The Upjohn Company, Kalamazoo, Michi- increased bronchospasm following the adgan. ministration of PB to prednisone-depend1 Present address: 2750 Broadway, Boulder, Col- ent asthmatics. orado. Reprints: Dr. Fred H. Katz, University of Colorado Medical Center, Denver, CO 80220. Non-proprietary names and trademark of drugs: 6a-methylprednisolone, Medrol (Upjohn); 6amethylprednisolone-hemisuccinate, Solu-medrol (Upjohn); Diphenylhydantoin, Dilantin (Parke-Davis); Diazepam, Valium (Roche).
Another synthetic corticosteroid, 6amethylprednisolone (MP) is presently used in a wide range of clinical states (7-9). Therefore, we decided to investigate the extent to which the widely used drugs DPH, PB, and diazepam (DZP) affect the
887
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888
STJERNHOLM AND KATZ
metabolism of MP and its 21-sodium hemisuccinate (MPHS). Materials and Methods Nineteen healthy, paid volunteers were studied. The study had the prior approval of the University Human Research Committee and the subjects gave their informed consent. The volunteers included medical students, teachers, and medical technologists between the ages of 19 and 32. None of the subjects had been receiving other drugs for a period of at least 2 months prior to study. Control MCR of MP was measured in 13 subjects, 5 females (F) and 8 males (M). Four subjects were restudied after DPH, 300 nig per day; four after PB, 120 mg per day; and four after DZP, 20 mg per day. The drugs were given in 3 or 4 divided doses daily for 3 weeks. Control MCR determinations using MPHS were performed in 6 subjects, (3 M, 3 F). These studies were repeated after DPH in 2 subjects and after PB in 2 subjects. Administration of tritium-labeled steroids. MP (10 /nCi/75 fig) and MPHS (10 /xCi/100 /tig) generally labeled with 3H were prepared and purified by New England Nuclear Corporation, Boston. Mobility on thin-layer chromatography (TLC) was identical to that of the unlabeled steroids used. The labeled steroids were stored in crystalline form and dissolved in pure ethanol for MP and ethanol with 2% deionized water for MPHS, prior to use. One ml (approximately 10 fid) was measured with a glass tuberculin syringe and transferred with sterile technique to a 25 ml glass vial containing sterile isotonic saline. The total radioactivity injected was determined by removing 100 fx\ of the solution and counting in a liquid scintillation spectrometer. The remainder of the 26 ml was then injected, over 60-90 seconds, into the antecubital vein of the recumbent subjects who had been fasting overnight. Several minutes of lightheadedness following the injection, presumably a consequence of the bolus of ethanol, occurred in most subjects. The MCR's were started between 8 and 9 AM. Venous blood was sampled through an indwelling 21 gauge Mini-Cath (Deseret Pharm. Co., Sandy, Utah) using a 3-way heparin lock. Heparinized plasma was obtained before injection of labeled steroid and at 5, 10, 20, 30, 60, 90, 120, 150, and 180 minutes after injection of the labeled MPHS, and, in addition,
JCE & M • 1975 Vol 41 • No 5
at 210 and 240 minutes after injection of MP. The samples were kept at 4 C and either extracted immediately after completion of the MCR or stored at — 20 C until extraction procedures could be performed. The plasma concentrations of the labeled steroids were determined and the MCR computed by the method of Peterson and Wyngaarden (10) using a 2-compartment model (11) and computer program as previously described (12). Recovery of MP from plasma. Two ml aliquots of plasma were extracted with 10 ml of freshly distilled, nanograde, dichloromethane (DCM) similar to the method of Kozower and coworkers (13). Eight ml aliquots of the DCM were then dried in counting vials with air at 37 C. Ten ml of a liquid scintillation fluid made up by mixing Biosolv BBS-3 (Beckman Co.) 350 ml, toluene 3500 ml and Omnifluor (New England Nuclear) 14 g, was added to the dry residue for counting. Quench correction was determined with an external barium standard. Recovery of the tritiated MP from plasma averaged 85 ± 4.9 (SD)% by this method. Recovery of MPHS from plasma. Only 6.0 ± 0.2% of MPHS added to plasma was extractable with DCM or ethyl acetate without prior acidfication. Therefore, the pH of 2 ml aliquots of plasma was adjusted to between 1.5 and 2.0 with 3N H2SO4, using pH paper. The plasma was extracted twice with 10 ml of freshly distilled nanograde ethyl acetate. Eight ml aliquots of each 10 ml aliquot of ethyl acetate were combined and dried for counting. Recoveries of a known amount of 3H-MPHS added to 2 ml of the baseline plasma from individual subjects extracted by this procedure averaged 75 ± 7.5%. Larger concentrations of acid did not increase the yield. The residual counts remained in the aqueous phase rather than being found in the precipitated proteins. This is not surprising in view of the water solubility of MPHS. Identification of the radioactive material in plasma. Two ml aliquots of plasma taken from each subject at 150 and 180 minutes for MPHS and at 210 and 240 minutes for MP were extracted as described above. Several samples from different subjects were pooled to increase counts and, thereby, the accuracy of recovery calculations. The residues were transferred to
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DIPHENYLHYDANTOIN AND METHYLPREDNISOLONE thin-layer chroinatography (TLC) plates, activated by heating to 110 C for one h, using ethyl acetate:ethanol:H2O (50:50:1) for spotting. The plates were run by ascending technique in ethyl acetate:ethanol:NH4OH (5:5:1). Non-radioactive MP and MPHS were added to the respective samples to allow localization of the steroids by UV absorption. Known quantities of the tritiated steroids were individually added to control plasma, extracted, and chromatographed as reference standards and as controls for the recoveries of the injected labeled steroids. The origin, MP, and MPHS areas were identified and the silica gel transferred by scraping all sections of the chromatograms in 2.5 cm divisions into counting vials. One ml of tetrahydrofuran:H2O (1:1) was added, the vials were mixed, and then scintillation fluid added. Recovery of radioactivity applied directly to thinla\ CM- plates was 73.6 ± 13.6% for MPHS.
889
whole urine. Urinary creatinine was measured to ensure completeness of collection. Data is expressed as % of administered dose ± SD. Paired t tests, comparing values before and after DPH, PB, or DZP, were used to determine statistical significance. Apparent volume of distribution (V) is represented by the volume of the inner pool occurring after equilibrium and the half-life refers to half-life of the exponential slope (/3) following equilibration (11). Results
Steroid characterization in plasma and control values. As was true for prednisolone in the study of Kozower et al. (13), recovery of MP from plasma by thinlayer chromatography was very efficient. As much as 98% of the radioactivity plated could be recovered from the plates and Radioactivity in urine. Urine was collected in 95% of this migrated at the R of authentic f aliquots at 4, 8, and 24 h after the administration MP. of tritiated MP and at 3, 6V2, 10&, and 24 h after MPHS offered not only the difficulty of the administration of tritiated MPHS. Radioacextraction from plasma mentioned above, tivity was determined by a technique quite similar to the method of Haque et al. (4). Thus but was also more poorly recovered from radioactivity measurements were obtained for TLC plates. Table 1 illustrates the results the whole urine and for four constituent frac- of chromatography experiments. Twotions: DCM, free steroids soluble in dichloro- thirds of the radioactivity applied to the methane; EA, polar free steroids soluble in plates was recovered after elution, for ethyl acetate after "salting out," usually re- reasons that are not understood. However, lated to 6/3-hydroxylation (14); DCM conjuas can be seen in Table 1, 70% of the gates, steroids soluble in dichloromethane after /3-glucuronidase hydrolysis; and steroids soluble recovered radioactivity migrated as MPHS. only in ethyl acetate after glucuronidase treat- It is possible that the finding of some MP ment. The latter group is negligible for syn- on the chromatograms is due to hydrolysis thetic glucocorticoids and will not be mentioned of the MPHS salt in plasma or in the subsequently. The 4 fractions together ac- strongly alkaline medium of the chrocounted for 85 ± 4.4% of the radioactivity in the matography solvents, since similar re-
TABLE 1. TLC of plasma extracts containing MPHS % recovery of total counts plated
% of recovered counts at origin
% of recovered counts at MP position R, = 0.67
% of recovered counts at MPHS position R, = 0.26
Control plasma with 3 H-MPHS added in vitro
59 ± 1.1
9 ±2.3
15 ± 2.3
71 ± 4.7
Pooled plasma at 150 and 180 minutes during MCR
66 ± 2.8
15 ± 3.5
16 ± 10.1
69 ± 12.9
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890
STJERNHOLM AND KATZ
JCE & M • 1975 Vol 4 1 . No 5
TABLE 2. Control steroid data (±S.D.)
no.
Age (yr) MCR (I/Day) MCR/M2 SA* Half-life (min) Vol (1)
MP
MPHS
13 25 ± 5 383 ± 72 215 ± 4 1 165 ± 49 61 ± 12
6 26 ± 5 234 ± 37 130 ± 13 160 ± 19 41 ± 6
3JO
CONTROL ....DPH 1.0
0.5
* Surface area.
suits were found for MPHS added to control plasma in vitro (Table 1). At any rate, it seems quite likely that about 70% of the radioactivity in the plasmas collected during the MCR determination was in the form of MPHS because of a parallel experiment, testing the extractability of the labeled steroid. Aliquots of the 30, 60, 90, and 120-minute specimens obtained during 4 control MCR studies of MPHS were extracted with dichloromethane. An average of 26.5 ± 8.8% of the total radioactivity in the plasma was extracted. Since only 6% at most of MPHS can be extracted from plasma with this solvent, this suggests that over 70% of the radioactivity was not exti'actable and was likely to be in the form of MPHS or a more polar metabolite. Table 2 shows the comparative values of the MCR, half-life, and V for MP and MPHS in the baseline studies. Effect of drugs on steroid metabolism. In Table 3 are shown the effects of DPH, PB, and DZP on the MCR, half-life and V for MP. Figure 1 shows the disappearance curves of labeled MP before and after DPH. The significant change with DPH is reflected in the marked increase in the MCR (+130%) and decrease in half-life (-56%). Figure 2 shows that a major increase in the excretion of products of TABLE
MCR (I/Day) Half-life (min) Vol (1)
0.1
30
60
120
180
240
TIME- MINUTES
FIG. 1. Disappearance curves for MP in 4 subjects before and after the administration of DPH. Values represent the mean ± SD. (P < 0.01 after 60 minutes).
labeled MP in the urine after DPH was due to a significant increase in the polar unconjugated ethyl acetate fraction. A significant, but small, decrease occurred in the conjugated DCM fraction. There was no change in the extraction of the free steroid in the unconjugated DCM fraction. After PB the MCR of MP increased by 90%, which was slightly less than after DPH, and similarly there was a smaller but still significant increase (P < 0.01 at 24 h) in the unconjugated ethyl acetate urinary fraction after PB. DZP had no effect on the disappearance curves of labeled MP. The urinary fractions showed only a slight, but statistically significant, increase in the unconjugated ethyl acetate fraction at 4 and 24 hours. The results of the MCR studies with MPHS are shown in Table 4. Because of the similar effects of DPH and PB on the MCR of MPHS, the results for the two drugs were combined for graphic presentation in Fig. 3. It must be emphasized that
3. Effect of DPH, PB, and DZP on the MCR of MP in 4 subjects
Pre
Post DPH
P
Pre
Post PB
P
Pre
Post DZP
P
424 ± 71 149 ± 44 63 ± 1 1
977 ± 132 69 ± 7 85 ± 15
