Determination of Infused (13C)Progesterone in Ovarian Arterial Blood by Selected Ion Monitoring

John M. Halkett and Freimut Leidenberger Institute for Hormonal and Fertility Disorders, Osterstr. 86, D-2000 Hamburg 20, Federal Republic of Germany

Niels EinerJensen Department of Physiology, University of Odense, Campusvej 55, DK-5230 Odense, Denmark

Arne Bendz Department of Obstetrics and Gynaecology, University of Gothenburg, S-41345 Gothenberg, Sweden

Gas chromatography mass spectrometry (GUMS) with selected ion monitoring (SIM) is employed to detect and approximate the levels of ('3C)progesterone in small btood samples obtained at intervals from ipsilateral and contralateral ovarian arteries and a peripheral vein before, during and after infusion into the utero-ovarian vein (five women during hysterectomy). The internal standard was 16a-methylprogesterone. The method is rapid and allows the time course of the infused hormone )o be plotted in spite of large and rapidly fluctuating concentrations of endogenous progesterone. The results show for the first time the local in viuo transfer of a steroid hormone from the uterine vein to the adjacent ovary in humans.

INTRODUCTION

A close anatomical relationship has been shown to exist between the utero-ovarian vein and the ovarian artery in several mammalian species.'-' Experiments with tritium-labelled prostaglandin Fzu in sheep3 and the unlabelled prostaglandin in cattle6 provided evidence supporting the counter-current transfer of PGFzu from the utero-ovarian vein to the ovarian artery in these species giving rise to the large local concentrations found in the ovary.3 In the human, radioactive pr~gesterone,~ methylantipyrine' and PGF,,' have been shown to pass from the utero-ovarian vein to the ovarian artery in vitro. Further, radioactive krypton has been shown to reach the ovary when injected into the utero-ovarian vein in vivo.'

The aim of the present study was to determine if progesterone is locally transferred in the human under in vivo conditions, avoiding radioactive exposure of the test subjects. This was accomplished by infusing ('3C)progesterone and employing GC/MS to detect and measure this compound in the ovarian blood after cor- recting for the isotopic contributions from endogenous progesterone. The results demonstrate the power of the method in allowing observation of the time course of an infused hormone in vivo in the presence of large and fluctuating levels of the endogenous compound.

t Author to whom correspondence should be addressed. Present address: Department of Chemical Pathology, Queen Charlotte's Maternity Hospital, Goldhawk Road, London W6 OXG, UK

EXPERIMENTAL

Materials

(3,4-13C2)Progesterone (90 at% I3C) was obtained from Amersham International, Amersham, UK and 16a- methylprogesterone was obtained from Sigma Chemical Co., St Louis, USA. Progesterone (p.A. 99%) and hepta- fluorobutyric anhydride were purchased from Serva Feinbiochemica, Heidelberg, FRG. Solvents were obtained from E. Merck, Darmstadt, FRG and were of analytical grade. A radioimmunoassay kit (double anti- body method) for progesterone was purchased from RSL, Carson, California USA.

Infusion solutions

Infusion solutions were prepared by dissolving 5 mg of labelled progesterone in 2.5 ml of ethanol. Saline (47.5 ml) was added immediately before use.

Patients

The five patients had been admitted to hospital for hysterectomy: patient 1, age 46, follicular phase ; patient 2, age 36, luteal phase; patient 3, age 49, luteal phase; patient 4, age 38, follicular phase; patient 5, age 36, follicular phase.

@ Wiley Heyden Ltd, 1985

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BIOMEDICAL MASS SPECTROMETRY, VOL. 12, NO. 8, 1985 429

J. M. HALKET, F. LEIDENBERGER, N. EINER-JENSEN AND A. BENDZ

< 10

( ' 3 ~ ) ~ r o g e s t e r o n e ng mL-' serum

I 4

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%:

(13C) PROGESTERONE

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- 1

100

10

1.0

0 1

:

- - -

L 3

3 x c 'u

h F

m 2

10000

1000

100

10000

1000

0 a K 1

t-

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0 0 4 0 8 0 120 160 2 0 0

"g

Figure 1. Calibration curve for the determination of ('3C)progesterone in small ovarian blood samples as its hepta- fluorobutyrate derivative. The internal standard was 16a-methyl- progesterone (400 ng ml-').

< 10

10000 , 1 2 3 L 5 6

1000 Operative procedure

During surgery a uterine vein running towards the ovarian pedicle was punctured and 20ml of infusion solution ( 2 mg of labelled steroid) were infused over 5 min in patient 1 and 50 ml (5 mg of labelled steroid) in patients 2-5. Full details of the operative and sampling procedure are described elsewhere." Incisions were made in the stroma of both ipsilateral and contralateral ovaries close to the hilus region avoiding the larger follicles and corpus luteum. The ovarian blood was collected before (sample l) , during (sample 2 ) and after (samples 3-6) the infusion at 2-5 min intervals for sample 1 and 5 min for the others. Parallel blood samples were taken from a peripheral vein. The samples were kept on ice until centrifugation yielding supernatants ranging in volume from 140 pl to 2 ml which were stored at -80 "C until analysis.

100

- 1 2 3 4 5 6

1 Ratio;

1 2 3 4 5 6 I

10000

1000

too Extraction and derivatization

Since the amounts of ('3C)progesterone to be expected, if any, were completely unknown before these experi- ments, a rough attempt was made to guess the best amount of internal standard to be added to each tube by measuring the total progesterone concentration in aliquots of a cross section of the larger samples by radioimmunoassay, after dilution where necessary, and the amount of internal standard (l6a-methyl- progesterone) to be added to each sample was chosen to correspond to 400 ng m1-I.

Aliquots (200 pl, or total sample where <200 pl) of serum were added to tubes together with 80 ng of internal standard. After equilibration at 37 "C for 1 h, the samples were subjected to extraction and derivatization accord- ing to a published procedure," except that no chromato- graphic pre-purification step was employed: after extrac- tion with 5 ml n-hexane, drying with sodium sulphate, evaporation under N2 and formation of heptafluorobu- tyrates, the derivatives were dissolved in 20 p1 of ethyl

1 2 3 6 5 6 1 2 3 4 5 6

Sample no

I ipsilateral ovarian artery rn C contralateral ovarian artery

A V peripheral vein

Figure 2. ('3C)Progesterone was infused during 5 min into a uterine vein in five women patients. Parallel blood samples were obtained at intervals from the ipsilateral and contralateral ovaries and from a peripheral vein. A higher concentration of labelled steroid was found in the ipsilateral samples than in contralateral in four cases, indicating a transfer from the uterine vein to the ipsilateral ovarian artery.

430 BIOMEDICAL MASS SPECTROMETRY, VOL. 12, NO. 8, 1985

DETERMINATION OF INFUSED ("C)PROGESTERONE BY SELECTED ION MONITORING

acetate. Microlitre aliquots were then subjected to gas chromatographic/mass spectral analysis.

Gas chromatography/mass spectrometry

A Finnigan MAT44S gas chromatographic/mass spec- trometric system was employed in the SIM mode monitoring ions at m / z 512 for the (3,4- "C,)progesterone derivative molecular ion, m / z 524 for the 16a-methylprogesterone internal standard and m / z 510 for unlabelled progesterone." The ionizing energy was 70eV and the source and interface tem- peratures were maintained at 200 and 250 "C, respec- tively.

A 25 m X0.32 mm i.d. glass capillary column coated with SE-52 was employed isothermally at 250 "C and a falling-needle injector was used for sample introduc- tion.', Under these conditions both the progesterone and internal standard had almost identical retention times (230 and 225 s, respectively).

The areas of the single ion curves were output by the MAT44S system so that the ratios of m/ z 512 to m/ z 524 could be calculated. Corrections for possible significant isotopic contributions from .large amounts of endogenous progesterone to the signal at m / z 512 were carried out according to a formula similar to that employed in isotopic dilution ana lys i~ '~ and utilizing the ratios obtained by measuring peak areas of m / z 512 and m / z 510 in (I3C)progesterone and unlabelled pro- gesterone (standard curve data, fixed amount of internal standard). The application of such correction equations has been discussed in detailI4 and is essential to the present work where large amounts of endogenous pro- gesterone were found in some samples.

~

RESULTS AND DISCUSSION

The calibration curve obtained for (I3C)progesterone is shown in Fig. 1 ( y = 5 3 . 4 ~ - 2.4; r = 0.9999).

Amounts found in samples having total volumes <200 pI were corrected. Concentrations of samples above 800 ng ml-' (160 ng/tube) were calculated by linear regression including the 1600 ng sample ( y = 4 7 . 9 ~ + 4.5) and a few samples found to have even higher concentrations were estimated by this regression.

REFERE

1. S. Barret, M. A. de Blockey, J. M. Brown, I. A. Cumming, J. R. Goding,B. J. Moleand J. M. 0bst.J. Reprod. Fertil.24.136 (1971).

2. J. A. McCracken, in Prostaglandins in Fertility Control, edited by S. Bergstrom, K. Green and B. Sarnuelsson, p. 234 WHO, Karolinska Institutet, Stockholm (1972).

3. J. A. McCracken, J. C. Carlsson, M. E. Glew, J. R. Goding, D. T. Baird, K. Green and B. Sarnuelsson, Nature, New Biol. 225, 600 (1 972).

4. C. H. Del Campo and 0 J. Ginther, Am. J. Vet. Res. 33,2561 (1972). 5. B. Vollmerhaus, Zentralbl. Veterinarmed. 11, 596 (1964). 6. J. E. Hixon and W. Hansel, Biol. Reprod. 11, 543 (1974). 7. A. Bendz, 0. Lundgren and L. Hamberger, Acta Physiol. Scand.

114, 611 (1982). 8. A. Bendz, Thesis. Countercurrent Exchange in the Human

Female Reproductive Tract. GOTAB, Goteborg, Sweden (1 982).

The time courses of the ('3C)progesterone concentra- tions are shown in Fig. 2 for all five patients.

In patients 2-5 the infusion was found to cause a larger concentration of labelled progesterone in serum from the ipsilateral ovary compared with the contra- lateral ovary during infusion and during the following five minute collection period. The labelled progesterone found in the brachial vein serum was equal to or lower than the concentrations in the contralateral ovary serum.

Patients 2 and 3 (luteal phase) showed larger con- centration differences between ipsilateral and contra- lateral ovaries than the patients in the follicular phase. This could indicate that the efficiency of the counter- current system may change during the ovulatory cycle.

In patient 1 (follicular phase) the concentrations of labelled progesterone were increased in both ipsilateral and contralateral ovaries. This case is discussed more fully elsewhere;'" a similar observation has been made with 85krypton.9

The use of a non-radioactively labelled compound has two advantages. Firstly, the patients are not exposed to radiation. Secondly, the gas chromatographic/ mass spectrometric method allowed the estimation of the (I3C)progesterone in the presence of very large and fluctuating levels of endogenous progesterone which had hindered previous studies based on infusion of unlabelled hormone. The simultaneous monitoring of the ion at m / z 510 allowed the estimation of the endogenous hormone at the same time in each sample. The concentrations found ranged from < 10 ng ml-' (majority of brachial vein samples) to approximately 10 pg ml-' (patient 1 , ipsilateral sample). No significant pattern could be discerned. The distinction between infused and endogenous progesterone is therefore of the utmost importance in these experiments.

The present results support the theory of counter- current exchange between the blood vessels of the human uterine adnexa based on similar experiments with 85krypton9 and further demonstrate the potential of stable isotopically labelled steroids in endocrinology as already reviewed and discussed for deuterium- labelled compound^.'^

Acknowledgements

The authors wish to thank W. Janssen and W. Meldrum for technical assistance.

:NCES

9. A. Bendz. N. Einer-Jensen, 0. Lundgren and P. 0. Janson, J.

10. A. Bendz, N. Einer-Jensen, J. M. Halket and F. Leidenberger, J.

11. I. Bjorkhern, R. Blomstrand and 0. Lantto, Clin. Chim. Acfa 65,

12. P. M. Van den Berg and Th. P. H. Cox, Chromatographia 5,301

13. L. Siekrnann, J. Steroid Biochem. 11, 117 (1979). 14. B. N. Colby and M. W. McCaman, Biomed. Mass Spectrom. 6,

15. D. W. Johnson, G. Phillipou and R. F. Seamark, J. Sferoid

Reprod. fertil. 57, 137 (1 979).

Reprod. F e d . submitted (1 985).

345 (1975).

(1 972).

225 (1 979).

Biochem. 14, 793 (1981).

Received 23 July 1984; accepted (revised) 18 March 1985

BIOMEDICAL MASS SPECTROMETRY, VOL. 12, NO. 8, 1985 431