- Email: [email protected]
Special uses of a gas chromatograph-mass spectrometer in endocrinology
J. steroid Bjoch~m. Vo l. 19. No. I. pp. 20.1- 207. 1983
Copyright
Printed in Gre at Britain. All right s reserved
o
0022- 47 3 Ij8 3 )3.00 + 0.00 1983 Perg amon Pr ess Ltd
SPECIAL USES OF A GAS CHROMATOGRAPH-MASS SPECTROMETER IN ENDOCRINOLOGY D. W. JOHNSON, T. J. BROOM, L. W. COX, C. D. MATTHEWS, G. PHILLlPOU and R. F. SEAMARK Department of Obstetrics and Gynaecology, University of Adelaide, G.P.O. Box 498, Adelaide, South Australia 500 1 and The Endocrine Laboratory, The Queen Elizabeth Hospital, Woodville Rd. Woodville, South Australia 5011 SUMMARY
Based on the isotope dilution principle, and using deuterium-labelled steroids as tracers combined with GC-MS analysis, clinically acceptable procedures for the measurement ofdaily urinary production rates (UPR's) and metabolic clearance rates (MCR's). of hormonal steroids, have been developed. In clinical tests. using progesterone and androstenedione as models the UPR's and MCR's obtained compared favourably with values obtained using radioisotope-labelled tracers. The heavy isotope procedure proved, however. to be superior to the radioisotope methods in accuracy, speed and patient safety.
INTRODU CTION
Plasma steroid hormone concentrations have been extensively quantitated during the human menstrual cycle and during pregnancy as an aid to understanding the hormonal factors which regulate the human female reproductive cycle. The use of plasma hormone content to provide this index of gland function is only valid if the MCR remains constant. For many steroids (e.g. progesterone [I], estradiol [2] and androstenedione [3]), serial MCR measurements of the menstrual cycle of normal women showed no significant variations. There are reports of changes in MCR'S of steroids during pregnancy and higher than normal progesterone MCR's were measured in pregnant women who had diabetes or failed glucose tolerance tests [4]. The significance of these reported changes is unclear partly due to the high coefficient of variation of the radioisotope tracer, constant infusion method employed. However. in a recent study of androstenedione metabolism in late pregnancy [5] , a 40% difference in the MCR's of pregnant and nonpregnant women was reported. Clearly, further studies of MeR's during pregnancy are warranted but the use of radioisotope tracers in pregnancy is now precluded on ethical grounds following the Februar y 1981 ban by the U.S. Department of Health and Human Services [5]. Accordingly we have explored the development of procedures for measuring MCR's using deuterium-labelled tracers followed by quantitation on a GC-MS . A number of advantages over previous methods were foreseen: (I ) greater accuracy and reproducability affo rded by the combined resolution of the gas chromatograph and mass spectrometer; (2) reduced analysis time as there is no requirement to recrystallize the steroid hormone 5.a, 19-I (AI-{)
to constant activity; (3)no radiation risk, during pregnancy. to the fetus. Progesterone, an index of both corpus luteum and placental function and androstenedione, a putative estrogen precursor in pregnancy were chosen as model steroids to develop the MCR procedures. Heavy isotope tracers [7,7,16· 2H 3]-progesterone and [7,7,15-2H3]- androstenedione were prepared by methods previously described [6. 7]. Two approaches were employed, one based on the measurement of steroid production rates in urine, which from the basic equation, MCR = PR/c, where c = plasma concentration of the steroid, indirectly affords the MeR and secondly, a more direct method for measuring MCR's by constant infusion. MATERIALS AND METHODS
and [7,7,15·2H 3} [7,7,16.2H3}progesterone androstenedione were prepared by a combination of the methods of Seamark et al.[6] and Blair et al.[7]. Mass spectral analysis indicated that both were > 96% triply deuterium-labelled and <0.4 % unlabelled. Column chromatography was performed on Lipidex-5000 (Packard, Downers Grove) and high pressure liquid chromatography on a Waters Assocs M6000A pump, U6K injector, model 450 u.v. detector and a 30 em, lO.um C-18 .u-Bondapak column. For mass spectral analysis, a Hewlett-Packard 59928 (jet separator option fitted) was used in the "single ion monitoring" mode. The system was equipped with a 3 m x 2 mm i.d, glass packed column on Gaschrom Q containing 2% Ov-Iul (120-140mesh).
203
204
D. W. Urinary
JOHNSON
Production Rate Determination
A solution of deuterated steroid CO.8mg/ml> in propylene glycol/water< 3: 1) patient's
was injPcted into the
antecubital vein. 2
Urine C sao ml J extract with hexane( 250 ml l dry and evaporate hexane
1 Lipidex-5000 chromatography elute with hexane(3mll
1 HPLC purification (10/J C18 bondapak ) elute
with 65 CH 3CN . 35 H20
Oer i vat iz e
05
pentafluoropropionate
(CH3CN, acetone, PFPA (1:1:1»)
Fig. I. Flow diagram of the injection procedure and the sample purification employed for the UPR determinations of progesterone and androstenedione. Solutions for injection were filtered through 0.2 Jlm Millipore filters under sterile conditions. URINARY PRODUCTION RATE METHOD
Experimental The procedure, which will be described in detail elsewhere [8], is outlined in Fig. I. Two purification steps were necessary to achieve clean mass spectral traces and the procedure, with minor modifications, was used to obtain PR's on both progesterone and androstenedione. The pentafluoropropionyl enol derivatives were chosen for measuring the ratio of protonium to deuterium-labelled steroid because of their excellent molecular ion yield during electron impact mass spectrometry. For progesterone, the SIM channels were set to mjz 460.3 (protonium) and 463.3 (trideuterated) and for androstenedione mlz 432.3 (protonium) and 435.3 (trideuterated). The UPR can be calculated from the dose of deuterium-labelled steroid administered multiplied by the peak height ratio of the molecular ions of unlabelled to labelled steroid (corrected for the contribution by natural abundance isotopes).
Results and discussion
et al.
(mean UPR = 96 Jlmolf24 h measured in luteal phase) and 15.5% (mean UPR = 1165Jlmolf24 h measured in late pregnancy) were obtained on pooled 24 h urine samples from women who had been injected with deuterated progesterone. Progesterone UPR's were then measured on women at different stages of the menstrual cycle and the results compared with those previously published using radioisotope tracer methods [9]. There was good agreement. On day 7 of the follicular phase a mean UPR (n = 5) of 5 Jlmol/24 h (Ref. [9] 7 ttmol/24 h) was obtained and on day 7 of the luteal phase the mean UPR (n = 7) was 75 Jlmolf24 h (Ref. [9] 80 /lmol/24 h). In addition, plasma progesterone levels were measured prior to the UPR determinations on the women on day 7 of their luteal phase. The derived MCR was 2051 ± 2751f24h. Androstenedione UPR determinations were made on a women whose adrenals had been surgically removed. A mean UPR (n = 2) of 10ttmol/24 h compares closely with a published PR [10] of 11 ttmol/24 h. Unfortunately, the UPR approach cannot be used on normal women because the dehydroepiandrosterone formed in the adrenals is converted to androstenedione and cleared in the urine [3, II] without entering the blood thus diluting the labelled androstenedione and affording a much higher PR than that obtained in the blood. To demonstrate the value of this procedure for clinical use, a large number (n > 50) of progesterone UPR's were determined on women at various stages of pregnancy . Included in this study were women with a history of previous miscarriage, interuterine growth retardation and diabetes, who had noticeably lower UPR's than normal women at the same stage of pregnancy (Fig. 2). Sequential determinations of progesterone UPR's and plasma progesterone concentrations were. made on an adrenalectomized woman (Fig. 3). Changes in the derived MCR's were evident, being very low during the follicular phase of the cycle but normal during 1600
a
'000 ~
e
-e
...
0
•
NORMAL
0
TWINS
•
TRIPLETS
00
••
THREAT MISCARRIAGE
•
DIABETE S
•
IUGR
0 0
o •• 0
00
e
•• •
0
e
"" ;;;
..
•
500
I•
::>
I
•
••
':• ••.... .....-: •••
'-0
Excretion of 95% of the detectable, labelled progesterone after injection of a volunteer with trideuterated progesterone, was demonstrated to occur in the first 24 h. Coefficients of variation of between 5.3%
•
0 0
0°
,S
20
25
30
3s
• • (0
GESTATION (WEEKS)
Fig. 2. Scatter plot of UPR's of progesterone measured in pregnant women by the deuterium-labelled tracer GC-MS method.
Special uses of a GC-MS in endocrinology 0.1
~ ro.
plasma conc n.
~mol.l-l)
205 1
\J
.../
I 16 0.1
o o
o
o
0
o
urinary production rate o 0 Cmmol.d- 1)
o
o
o
o
o
o
o
o
? 9 oi
16
16
2000
2000
.... f
1000
1000
clearance rate
.
o
iii
16
doy of cycle normal
adrenalectomized
Fig. 3. Scatter plots of (a) plasma concentrations (b) UPR's and (c) derived MCR's of progesterone in a normal woman and an adrenalectomized woman across the menstrual cycle. The plasma concentrations were measured by an isotope dilution GC-MS method and the UPR's by the method outlined in Fig. 1.
the luteal phase. The physiological basis of the cyclical variation in MCR is unknown but it may be a product of an enzyme deficiency or relate to estrogen promoted changes in plasma binding protein [12]. The case provides an excellent example of the usefulness of steroid PR determinations to supplement measurements of plasma steroid concentrations. METABOLIC CLEARANCE RATE METHOD
Experimental The procedure based on previous studies using radioisotope-labelled tracer [3], is outlined in Fig. 4. In trial experiments with a constant infusion of either deuterium-labelled progesterone or androstenedione, blood concentrations of the labelled steroid had reached a steady-state within 2 h. Accordingly, infusion was continued for at least 2.5h and blood samples were taken at 2 and 2.5 h. For quantitation, a plasma fraction was prepared by centrifugation and stored frozen at - lOOC and prior to analysis internal standards (IS) were added to the samples of plasma and the infusion mixture. l6-IX-Methylprogesterone was the IS used for progesterone and 19-norandrostenedione was the IS used for androstenedione. The samples containing the IS were extracted with hexane, derivatized under the conditions for pentafluoropropionyl enol formation and analysed on the GC-MS. For progesterone MCR determinations, SIM channels were set to mjz 460.3 (progesterone), 463.3 (trideuterated progesterone) and 474.3 (16-IX-methylprogesterone). The corresponding channels for androstenedione were mlz 418.3 (19-norandrostenedione), 432.3 (androstenedione) and 435.3 (trideuterated androstenedione). Quantitation of the labelled and
endogenous steroid is achieved by substitution into a linear regression equation generated from the relative mass spectral responses of mixtures of derivatized steroid and IS. The MCR is calculated from the product of the M.C.R. by Constant Infusion Solution A: Oeuterated steroid CO.5mg/ml) in propylene glycol/ water( 3: 1I. Solution B: Sal ineC 70mll and Saln. AC 2 mI).
Infuse soln. Be20ml/ hrl through tef Ion catheter into antecubital vein.
1 (allect blood samples at 2h and 2.5h. Centrifuge _plasma
1 Collect sample of infusion mixture when catheter removed from vein.
1 Add internal standard to plasma and infusion mixture and extract.
1 Oerivatize and quantitate on
GC-MS
Fig. 4. Flow diagram of the constant infusion procedure employed for measuring MCR's of progesterone and androstenedione.
206
D. W. JOHNSON et al.
Table l. MCR's of progesterone and androstenedione, in women, measured by the deuterium-labelled tracer, constant infusion method
Progesterone Early luteal I Early luteal 2 Androstenedione Follicular Hirsute I Hirsute 2 Hirsute 3 Hirsute 4
Plasma concn
MCR (1/day)
(nmol/l)
PR (jlmol/day)
1939 2102
17.9 17.9
34.7 37.6
1866 2225 2264 2484 2568
2.1 7.5 2.8 4.2
4.0 16.7 6.3
11.2
28.8
lOA
pump flow rate and the concentration of deuterated steroid in the plasma (average value of 2 and 2.5 h) divided by the concentration of deuterated steroid in the infusion mixture. Since the endogenous steroid level is also determined the PR is easily calculated. Result s and Discussion
The minimum plasma concentration of deuteriumlabelled steroid required to achieve accurate quantitation was 3-6 Jlmol/l therefore an infusion mixture containing at least 30 Jlmol/l of deuterated steroid, infused at 20 ml/h, was required. Administration of progesterone at these doses was shown not to affect endogenous levels and the values obtained using this procedure compare favourably with published values for studies using radioisotope-labelled tracer techniques [9J, and with the MCR values derived from the UPR measurements, as shown in Table 1. Determinations of MCR's of Androstenedione in a normal woman (during follicular phase) and in women with diagnosed hirsutism (see Table I.) yielded similar values to those reported by Kirshner et al.,[13J (normal-mean = 20701/24 h ; hirsutemean = 19601/24 h). CONCLUSIONS
The two methods described in this paper complement those of Pinkus et al.[14, 15J, Taylor et al.[16J and Baillie et al.[ 17J to further demonstrate the usefulness of deuterium-labelled steroids for in vivo studies of human steroid hormone pharmacokinetics. The PR measurement in urine, although less imposing on the patient , is potentially prone to greater error because it relies on the patient collecting a complete 24 h urine specimen. The constant infusion MCR method is capable of good precision because the mass spectrometric quantitations of the deuterium-labelled steroid in the plasma sample and the infusion mixture are both relative to the same internal standard. In addition to the advantage of the deuteriumlabelled tracer methods outlined in the Introduction there is the potential to measure PR's and MCR's of two or more steroids simultaneously. Where the
steroids studied are interconvertable deuteriumlabelled steroids with different numbers of deuterium atoms incorporated will be required. Acknowledgements-The authors would like to thank Drs C. Kirby lind L. Poole for their assistance in the clinical
studies. We also thank the National Health and Medical Research Council of Australia for the award of a Senior Research Fellowship (D.W.J) and support of this work. REFERENCES
I. Lin T. 1., Billiar R. B. and Little B.: Metabolic clearance rates of progesterone in the menstrual cycle. J. d in. Endocr. Metab.35 (1972) 879-886.
2. Longcope C. D., Layne D. S. and Tait J. F.: Metabolic clearance rates and interconversions of estrone and 17P estradiol in normal males and females. J. din. [nvest. 47 (1968) 93. 3. Horton R. and Tait 1. F.: Androstenedione production and interconversion rates measured in peripheral blood and studieson the possible site of its conversion to testosterone. J. din. lnoest. 45 (1966) 301-313. 4. Lin T. J., Lin S. c, Erlenmeyer F., Kline I. T., Underwood R., Billiar R. B. and Little B.: Progesterone production rates during the third trimester of pregnancy in normal women, diabetic women and women with abnormal glucose tolerance. J. din. Endocr. Metab. 34 (1972) 287-297. 5. Edman C. D., Toofanian A., MacDonald P. C. and Gant N. F.: Placental clearance rate of maternal plasma androstenedione through placental estradiol formation: An indirect method of assessing uteropiacental blood flow. Am. J. Obstet. Gynec. 141 (1981) 1029-1037. 6. Seamark R. F., Phillipou G. and McIntosh J. E. A.: Ovarian steroidogenesis studied by mass fragmentography. J. steroid Biochem. 8 (1977) 885-891. 7. Blair I. A., Phillipou G. and Seaborn C. 1.: Synthesis of C7-2H2 steroidsfor human metabolism studies. J. Lab. Camp. 15 (1978) 645-655. 8. Broom T. 1., Johnson D. W., Cox L. W., Phillipou G. and Seamark R. F.: Applications of heavy isotope tracers to clinical studies: Progesterone urinary production rate determinations in the menstrual cycle and pregnancy. Submitted to J. din. Endocr. Metab. 9. Little B.and Billiar R. B.: Progesterone production. In Progress in Endocrinology (Edited by C. Gual). Exerpta Medica Foundation, Amsterdam (1969) pp. 871-879. 10. Baird D. T., Horton R., Longcope C. and Tait J. F.: Steroid prehormones. Persp. Bioi. Med. 3 (1968) 384-391. 11. Vande Wiele R, L., MacDonald P. c, Gurpide E. and Lieberman S.: Studies on the secretion and interconversion of the androgens. Recent Proq. Harm. Res. 19 (1963) 275-310. 12. Angel A., Frajria R., Bisbacci D. and Ceresa F.: Temporal changes in plasmatranscortin (CBG) binding capacity during the menstrual cycle. J. interdiscipl. Cycie Res. 8 (1977) 237-242. 13. Kirshner M. A., Zucker I. R. and Jespersen D.: Idiopathic hirsutism-an ovarian abnormality. New Enql. J. Med. 294(1976) 637-640. 14. Pinkus 1. L., Charles D. and Chattoraj S. C: Deuterium-labelled steroids for study in humans I. Estrogen production rates in normal pregnancy. J. bioi. Chem. 246(1971) 63J-636. 15. Pinkus J. L., Charles D. and Chattoraj S. c.: Deuterium-labelled steroids for study in humans II. Preliminary studies on estrogen production rates in pre- and post-menopausal women. Hormone Res. 10 (1979) 44-56.
Special uses of a GC-MS in endocrinology 16. Taylor E. S., Hagerman D. D., Albrecht B. H. and Penney L. L. : Estriol production rate measurement studies in pregnant women. Am. J. Obstet. Gynec. 120 (1974) 752-758.
207
17. Baillie T. A., Curstedt T., Sjovall K. and Sjovall J.: Production rates and metabolism of sulphates of 3p-hydrox y-5:x-pregnane derivatives in pregnant women. J . steroid Biochem. 13(1980) 1473-1488 .
Copyright
Printed in Gre at Britain. All right s reserved
o
0022- 47 3 Ij8 3 )3.00 + 0.00 1983 Perg amon Pr ess Ltd
SPECIAL USES OF A GAS CHROMATOGRAPH-MASS SPECTROMETER IN ENDOCRINOLOGY D. W. JOHNSON, T. J. BROOM, L. W. COX, C. D. MATTHEWS, G. PHILLlPOU and R. F. SEAMARK Department of Obstetrics and Gynaecology, University of Adelaide, G.P.O. Box 498, Adelaide, South Australia 500 1 and The Endocrine Laboratory, The Queen Elizabeth Hospital, Woodville Rd. Woodville, South Australia 5011 SUMMARY
Based on the isotope dilution principle, and using deuterium-labelled steroids as tracers combined with GC-MS analysis, clinically acceptable procedures for the measurement ofdaily urinary production rates (UPR's) and metabolic clearance rates (MCR's). of hormonal steroids, have been developed. In clinical tests. using progesterone and androstenedione as models the UPR's and MCR's obtained compared favourably with values obtained using radioisotope-labelled tracers. The heavy isotope procedure proved, however. to be superior to the radioisotope methods in accuracy, speed and patient safety.
INTRODU CTION
Plasma steroid hormone concentrations have been extensively quantitated during the human menstrual cycle and during pregnancy as an aid to understanding the hormonal factors which regulate the human female reproductive cycle. The use of plasma hormone content to provide this index of gland function is only valid if the MCR remains constant. For many steroids (e.g. progesterone [I], estradiol [2] and androstenedione [3]), serial MCR measurements of the menstrual cycle of normal women showed no significant variations. There are reports of changes in MCR'S of steroids during pregnancy and higher than normal progesterone MCR's were measured in pregnant women who had diabetes or failed glucose tolerance tests [4]. The significance of these reported changes is unclear partly due to the high coefficient of variation of the radioisotope tracer, constant infusion method employed. However. in a recent study of androstenedione metabolism in late pregnancy [5] , a 40% difference in the MCR's of pregnant and nonpregnant women was reported. Clearly, further studies of MeR's during pregnancy are warranted but the use of radioisotope tracers in pregnancy is now precluded on ethical grounds following the Februar y 1981 ban by the U.S. Department of Health and Human Services [5]. Accordingly we have explored the development of procedures for measuring MCR's using deuterium-labelled tracers followed by quantitation on a GC-MS . A number of advantages over previous methods were foreseen: (I ) greater accuracy and reproducability affo rded by the combined resolution of the gas chromatograph and mass spectrometer; (2) reduced analysis time as there is no requirement to recrystallize the steroid hormone 5.a, 19-I (AI-{)
to constant activity; (3)no radiation risk, during pregnancy. to the fetus. Progesterone, an index of both corpus luteum and placental function and androstenedione, a putative estrogen precursor in pregnancy were chosen as model steroids to develop the MCR procedures. Heavy isotope tracers [7,7,16· 2H 3]-progesterone and [7,7,15-2H3]- androstenedione were prepared by methods previously described [6. 7]. Two approaches were employed, one based on the measurement of steroid production rates in urine, which from the basic equation, MCR = PR/c, where c = plasma concentration of the steroid, indirectly affords the MeR and secondly, a more direct method for measuring MCR's by constant infusion. MATERIALS AND METHODS
and [7,7,15·2H 3} [7,7,16.2H3}progesterone androstenedione were prepared by a combination of the methods of Seamark et al.[6] and Blair et al.[7]. Mass spectral analysis indicated that both were > 96% triply deuterium-labelled and <0.4 % unlabelled. Column chromatography was performed on Lipidex-5000 (Packard, Downers Grove) and high pressure liquid chromatography on a Waters Assocs M6000A pump, U6K injector, model 450 u.v. detector and a 30 em, lO.um C-18 .u-Bondapak column. For mass spectral analysis, a Hewlett-Packard 59928 (jet separator option fitted) was used in the "single ion monitoring" mode. The system was equipped with a 3 m x 2 mm i.d, glass packed column on Gaschrom Q containing 2% Ov-Iul (120-140mesh).
203
204
D. W. Urinary
JOHNSON
Production Rate Determination
A solution of deuterated steroid CO.8mg/ml> in propylene glycol/water< 3: 1) patient's
was injPcted into the
antecubital vein. 2
Urine C sao ml J extract with hexane( 250 ml l dry and evaporate hexane
1 Lipidex-5000 chromatography elute with hexane(3mll
1 HPLC purification (10/J C18 bondapak ) elute
with 65 CH 3CN . 35 H20
Oer i vat iz e
05
pentafluoropropionate
(CH3CN, acetone, PFPA (1:1:1»)
Fig. I. Flow diagram of the injection procedure and the sample purification employed for the UPR determinations of progesterone and androstenedione. Solutions for injection were filtered through 0.2 Jlm Millipore filters under sterile conditions. URINARY PRODUCTION RATE METHOD
Experimental The procedure, which will be described in detail elsewhere [8], is outlined in Fig. I. Two purification steps were necessary to achieve clean mass spectral traces and the procedure, with minor modifications, was used to obtain PR's on both progesterone and androstenedione. The pentafluoropropionyl enol derivatives were chosen for measuring the ratio of protonium to deuterium-labelled steroid because of their excellent molecular ion yield during electron impact mass spectrometry. For progesterone, the SIM channels were set to mjz 460.3 (protonium) and 463.3 (trideuterated) and for androstenedione mlz 432.3 (protonium) and 435.3 (trideuterated). The UPR can be calculated from the dose of deuterium-labelled steroid administered multiplied by the peak height ratio of the molecular ions of unlabelled to labelled steroid (corrected for the contribution by natural abundance isotopes).
Results and discussion
et al.
(mean UPR = 96 Jlmolf24 h measured in luteal phase) and 15.5% (mean UPR = 1165Jlmolf24 h measured in late pregnancy) were obtained on pooled 24 h urine samples from women who had been injected with deuterated progesterone. Progesterone UPR's were then measured on women at different stages of the menstrual cycle and the results compared with those previously published using radioisotope tracer methods [9]. There was good agreement. On day 7 of the follicular phase a mean UPR (n = 5) of 5 Jlmol/24 h (Ref. [9] 7 ttmol/24 h) was obtained and on day 7 of the luteal phase the mean UPR (n = 7) was 75 Jlmolf24 h (Ref. [9] 80 /lmol/24 h). In addition, plasma progesterone levels were measured prior to the UPR determinations on the women on day 7 of their luteal phase. The derived MCR was 2051 ± 2751f24h. Androstenedione UPR determinations were made on a women whose adrenals had been surgically removed. A mean UPR (n = 2) of 10ttmol/24 h compares closely with a published PR [10] of 11 ttmol/24 h. Unfortunately, the UPR approach cannot be used on normal women because the dehydroepiandrosterone formed in the adrenals is converted to androstenedione and cleared in the urine [3, II] without entering the blood thus diluting the labelled androstenedione and affording a much higher PR than that obtained in the blood. To demonstrate the value of this procedure for clinical use, a large number (n > 50) of progesterone UPR's were determined on women at various stages of pregnancy . Included in this study were women with a history of previous miscarriage, interuterine growth retardation and diabetes, who had noticeably lower UPR's than normal women at the same stage of pregnancy (Fig. 2). Sequential determinations of progesterone UPR's and plasma progesterone concentrations were. made on an adrenalectomized woman (Fig. 3). Changes in the derived MCR's were evident, being very low during the follicular phase of the cycle but normal during 1600
a
'000 ~
e
-e
...
0
•
NORMAL
0
TWINS
•
TRIPLETS
00
••
THREAT MISCARRIAGE
•
DIABETE S
•
IUGR
0 0
o •• 0
00
e
•• •
0
e
"" ;;;
..
•
500
I•
::>
I
•
••
':• ••.... .....-: •••
'-0
Excretion of 95% of the detectable, labelled progesterone after injection of a volunteer with trideuterated progesterone, was demonstrated to occur in the first 24 h. Coefficients of variation of between 5.3%
•
0 0
0°
,S
20
25
30
3s
• • (0
GESTATION (WEEKS)
Fig. 2. Scatter plot of UPR's of progesterone measured in pregnant women by the deuterium-labelled tracer GC-MS method.
Special uses of a GC-MS in endocrinology 0.1
~ ro.
plasma conc n.
~mol.l-l)
205 1
\J
.../
I 16 0.1
o o
o
o
0
o
urinary production rate o 0 Cmmol.d- 1)
o
o
o
o
o
o
o
o
? 9 oi
16
16
2000
2000
.... f
1000
1000
clearance rate
.
o
iii
16
doy of cycle normal
adrenalectomized
Fig. 3. Scatter plots of (a) plasma concentrations (b) UPR's and (c) derived MCR's of progesterone in a normal woman and an adrenalectomized woman across the menstrual cycle. The plasma concentrations were measured by an isotope dilution GC-MS method and the UPR's by the method outlined in Fig. 1.
the luteal phase. The physiological basis of the cyclical variation in MCR is unknown but it may be a product of an enzyme deficiency or relate to estrogen promoted changes in plasma binding protein [12]. The case provides an excellent example of the usefulness of steroid PR determinations to supplement measurements of plasma steroid concentrations. METABOLIC CLEARANCE RATE METHOD
Experimental The procedure based on previous studies using radioisotope-labelled tracer [3], is outlined in Fig. 4. In trial experiments with a constant infusion of either deuterium-labelled progesterone or androstenedione, blood concentrations of the labelled steroid had reached a steady-state within 2 h. Accordingly, infusion was continued for at least 2.5h and blood samples were taken at 2 and 2.5 h. For quantitation, a plasma fraction was prepared by centrifugation and stored frozen at - lOOC and prior to analysis internal standards (IS) were added to the samples of plasma and the infusion mixture. l6-IX-Methylprogesterone was the IS used for progesterone and 19-norandrostenedione was the IS used for androstenedione. The samples containing the IS were extracted with hexane, derivatized under the conditions for pentafluoropropionyl enol formation and analysed on the GC-MS. For progesterone MCR determinations, SIM channels were set to mjz 460.3 (progesterone), 463.3 (trideuterated progesterone) and 474.3 (16-IX-methylprogesterone). The corresponding channels for androstenedione were mlz 418.3 (19-norandrostenedione), 432.3 (androstenedione) and 435.3 (trideuterated androstenedione). Quantitation of the labelled and
endogenous steroid is achieved by substitution into a linear regression equation generated from the relative mass spectral responses of mixtures of derivatized steroid and IS. The MCR is calculated from the product of the M.C.R. by Constant Infusion Solution A: Oeuterated steroid CO.5mg/ml) in propylene glycol/ water( 3: 1I. Solution B: Sal ineC 70mll and Saln. AC 2 mI).
Infuse soln. Be20ml/ hrl through tef Ion catheter into antecubital vein.
1 (allect blood samples at 2h and 2.5h. Centrifuge _plasma
1 Collect sample of infusion mixture when catheter removed from vein.
1 Add internal standard to plasma and infusion mixture and extract.
1 Oerivatize and quantitate on
GC-MS
Fig. 4. Flow diagram of the constant infusion procedure employed for measuring MCR's of progesterone and androstenedione.
206
D. W. JOHNSON et al.
Table l. MCR's of progesterone and androstenedione, in women, measured by the deuterium-labelled tracer, constant infusion method
Progesterone Early luteal I Early luteal 2 Androstenedione Follicular Hirsute I Hirsute 2 Hirsute 3 Hirsute 4
Plasma concn
MCR (1/day)
(nmol/l)
PR (jlmol/day)
1939 2102
17.9 17.9
34.7 37.6
1866 2225 2264 2484 2568
2.1 7.5 2.8 4.2
4.0 16.7 6.3
11.2
28.8
lOA
pump flow rate and the concentration of deuterated steroid in the plasma (average value of 2 and 2.5 h) divided by the concentration of deuterated steroid in the infusion mixture. Since the endogenous steroid level is also determined the PR is easily calculated. Result s and Discussion
The minimum plasma concentration of deuteriumlabelled steroid required to achieve accurate quantitation was 3-6 Jlmol/l therefore an infusion mixture containing at least 30 Jlmol/l of deuterated steroid, infused at 20 ml/h, was required. Administration of progesterone at these doses was shown not to affect endogenous levels and the values obtained using this procedure compare favourably with published values for studies using radioisotope-labelled tracer techniques [9J, and with the MCR values derived from the UPR measurements, as shown in Table 1. Determinations of MCR's of Androstenedione in a normal woman (during follicular phase) and in women with diagnosed hirsutism (see Table I.) yielded similar values to those reported by Kirshner et al.,[13J (normal-mean = 20701/24 h ; hirsutemean = 19601/24 h). CONCLUSIONS
The two methods described in this paper complement those of Pinkus et al.[14, 15J, Taylor et al.[16J and Baillie et al.[ 17J to further demonstrate the usefulness of deuterium-labelled steroids for in vivo studies of human steroid hormone pharmacokinetics. The PR measurement in urine, although less imposing on the patient , is potentially prone to greater error because it relies on the patient collecting a complete 24 h urine specimen. The constant infusion MCR method is capable of good precision because the mass spectrometric quantitations of the deuterium-labelled steroid in the plasma sample and the infusion mixture are both relative to the same internal standard. In addition to the advantage of the deuteriumlabelled tracer methods outlined in the Introduction there is the potential to measure PR's and MCR's of two or more steroids simultaneously. Where the
steroids studied are interconvertable deuteriumlabelled steroids with different numbers of deuterium atoms incorporated will be required. Acknowledgements-The authors would like to thank Drs C. Kirby lind L. Poole for their assistance in the clinical
studies. We also thank the National Health and Medical Research Council of Australia for the award of a Senior Research Fellowship (D.W.J) and support of this work. REFERENCES
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