Klinische Wochenschrift

Klin. Wochenschr. 56 (SuppL I), 169-172 (1978)

© Springer-Verlag 1978

Compounds in the Urinary pH 1 Extract Reacting with Corticosteroid and Tetrahydrocorticosteroid Antisera. Separation of a Nonpolar Substance with Affinity to a Tetrahydroaldosterone Antiserum K. Lichtwald, T.M. Connolly, K.-H. Kohl, and P. Vecsei Department of Pharmacology, University of Heidelberg

Urininhaltstoffe des pH 1 Extraktes mit Immunoreaktivitiit gegen Corticoidantisera. Separierung einer unpolaren Verbindung mit Affinit/it zu einem Tetrahydroaldosteronantiserum Zusammenfassung. Im p H 1 Extrakt aus Harn, vorbereitet zur Aldosteron-18-Glucuronid Bestimmung, wurden Substanzen gefunden, die nicht nur mit A1dosteronantiserum, sondern auch mit unterschiedlichen Corticosteroid- und Tetrahydrocorticosteroidantik6rpern kreuzreagierten. Aldosteron wurde sowohl vor als auch nach chromatographischer Reinigung bestimmt. Zur weiteren Charakterisierung der immunoreaktiven Stoffe wurde der Extrakt chromatographisch getrennt und mit der I m m u n o g r a m m technik untersucht. Nach A C T H - , Dexamethason-, Metopirongabel sowie w/ihrend der Schwangerschaft konnte eine deutliche Exkretionsfinderung von nicht identifierten antigenen Aquivalenten festgestellt werden. Uber die Separierung der immunoreaktiven Substanzen mit anschlieBender Anwendung der GaschromatographieMassenspektrometrie (GC-MS) wird berichtet. Sehliisselwiirter: Aldosteronglucuronid - Steroidantiseren - I m m u n o g r a m m a n a l y s e - Gaschromatographie - Massenspektrometrie. Summary. In h u m a n urinary p H 1 extracts prepared for the aldosterone- 18-glucuronide estimation, several other substances are present, crossreacting not only with aldosterone antisera, but also with various corticosteroid and tetrahydrocorticosteroid antisera. A1dosterone was measured before and after chromatographic purification. Further characterization of the non-aldosterone immunoreactive material was made 1

Metyrapon

Offprint requests to: K. Lichtwald, M.D. (address see p. 172)

by immunological analysis of paper c h r o m a t o g r a m eluats. Pregnancy, and administration of A C T H , dexamethasone, and metopirone led to a change of excretion in the antigenic equivalents. A method for the separation of the antigenic material is described. For structural elucidation the gaschromatography-mass spectrometry (GC-MS) method was applied.

Key words: Aldosterone glucuronide - Steroid antisera - Immonogramanalysis - G a s c h r o m a t o g r a p h y - Mass spectrometry.

It has been suggested that the urinary p H 1 extracts prepared for the estimation of aldosterone-18-glucoronide include unidentified materials which may bind with aldosterone antibodies (Connolly et al. in this issue of Klin. Wochenschr.). Radioimmunoassays using antibodies raised against different corticosteroids and corticosteroid metabolites were carried out on the p H 1 extracts to characterize these materials. Additionally, i m m u n o g r a m s were made with antibodies directed against the same corticosteroids and corticosteroid metabolites to obtain qualitative information on the identity of the materials. Furthermore, an attempt was made to isolate the unidentified immunoreactive substance in one fraction which crossreacted with tetrahydroatdosterone antibody to better elucidate its characteristics.

Methods Human urinary samples were prepared for atdosterone-18-glucuronide estimation as described [6]. They were pre-extracted with dichlormethane to remove the free steroids, and then hydrolyzed with sulfuric acid at pH 1. Aldosterone concentrations were determined, with and without chromatography in the resulting pH 1

170

K. Lichtwatd et al. : Unidentified Steroids in the pH 1 Urinary Extract

Table 1. Total equivalents of corticosteroid and tetrahydrocorticosteroid immunoreactive substances in the urinary pH 1 extract and their changes in pregnancy and following administration of various drugs ALD (with Chr.) ~tg/24 h

"ALD . . . . (without Chr.) gg/24 h

B. . gg/24 h

.

.

S. . gg/24 h

.

.

F. . gg/24 h

.

.

18-OH-DOC" gg/24 h

Control (n=6)

3.7 + 9.5 5.8--.0.9

6.4 + 19.9 13.7± 2.1

20.8 - 55.9 39.7+- 4.8

7.6 - 17.7 10.2± 1.5

25.9 -73.7 48.0+- 7.1

2.0 -6.6 4.9±0.6

ACTH (n=6)

12.6--42.7 21.7+_ 4.55

39.0--210.5 101.1+- 26.6

I73 -506 341.9+ 55.9

22.3-58.3" 38.2+- 6.4

283.7--1579 484.3+- 96.7

11.8-28.1 18.5+- 2.6

2.3-- 9.2 5.8+- 1.1

6.8--15.5 11.9± 1.4

17.2--51.3 26.3± 5.4

2.3-10.4 4.9± 1.2

3.9--i2.9 5.8+ 1.5

1.6--7.7 3.4+-0.9

119 -465 270.9± 41.5

28.6-71.5 45.9++ 4.8

92.0-301 142.9+- 16.9

13.I -.27.5 18.3++ 1.6

Dexamethasone (n=6) Pregnancy (n=ll) Metopirone (n=3)

53.1 - 145.7 84.5+- 30.6 "DOC . . . . gg/24 h

Control (n=6)

5 . 3 - 7.2 6.4+_ 0.3

ACTH (n=6)

10.6 -47.4 20.1+_ 5.6

Dexamethasone (n=6) Pregnancy (n=ll) Metopirone (n = 3) a b

TH-ALD . . . . ~tg/24 h

TH-B . . . . gg/24 h

TH-DOC . . . . TH-F . . . . ~tg/24 h gg/24 h

TH-S" gg/24 h

33 -84.8 58.8+ 9.0

46 -142 107.2+_ 16.2

57.6-187.6 t03.9± 20.4

48.3-204 129.2+_ 22. i

20.2-29.1 25.2± 1.5

51.5-114.6 88.8± 1 0 . 2

195 - 8 7 4 a 423.7++_I16.7

84 -372 191.6+ 42.9

302 -1035 487.5+ 1 1 4 . 9

2 . 8 - 7.3 4.9+ 0.7

29.2-73.4 51.5++ 8.0

51.0-119 78.4± 10.2

74 -388 161.2± 48.8

18.8-85.2 39.9± 5.9

123.0-352 229.4+_ 25.5

171. -I821 757.1++_ 1 5 3 . 2

475 -3000 861.9+_226.3

l 9. I - 274 116.0 + 79.5

525.3 +

98.9

16.3-46.2 27.9+ 4.2 19.1-258.9 b 104.1+-23.0

52.0 -321 146.5± 41.8 10.4-35.8 17.0+ 3.9 54.0-370.0 102.5++_27.7 1439.3 +-265.4

rZ=5 n=lO

extract. The crude extract was also used to estimate the radioimmunological response with specific antisera [2-5, 7] against cortisol, corticosterone, ll-deoxycorticosterone, 18-hydroxy-ll-deoxycorticosterone, 11-deoxycortisol, tetrahydroaldosterone, tetrahydrocorticosterone, tetrahydrocortisol, tetrahydro-ll-deoxycorticosterone, and tetrahydro-11-deoxycortisol. After adding 3H-labeled corticosteroid tracers to the samples, a radioimmunological paper chromatographic analysis was made. In order to obtain a larger amount of reacting material, 10 liters of urine from women of late pregnancy were worked up as described above. The dried residue was chromatographed on neutral aluminia (activity III/IV) by stepwise increasing the polarity (benzene-chloroform-methanol). Each 10 ml fraction was tested with various antisera. Fractions with immunoreactivity were examined for their purity by silica thin layer chromatography (TLC) with development in at least two solvent systems (benzene-methanol 90/10; chloroform-methanol 95/5). After localization, elution, and drying, the separated material was silylated with N,N-bis-trimethylsilyltrifluoroacetamide in preparation for the analysis by the GC-MS technique.

Results Table 1 shows the results of radioimmunoassays performed with the antibodies against various steroids on the crude pH 1 extract. The values are expressed

as e q u i v a l e n t s o f t h e a n t i g e n steroids. U r i n e s c o l l e c t e d after administration of ACTH, dexamethasone, and metopirone, and from subjects of late pregnancy, contained different amounts of crossreacting materials t h a n f o u n d in u r i n e s f r o m n o r m a l subjects. Figure 1 shows a typical immunogram made from t h e p H 1 e x t r a c t . T h i s a n d all o t h e r i m m u n o g r a m s have shown that only a part of the immunoactivity a p p e a r e d in a r e a s o f c h r o m a t o g r a m strips c o r r e sponding to the corticosteroid and tetrahydrometabolites. O t h e r part(s) o f t h e i m m u n o a c t i v e m a t e r i a l ( s ) w e r e f o u n d at u n r e l a t e d l o c a t i o n s . T h e r e s i d u e f r o m t h e 10 1 o f p r e g n a n t u r i n e , c h r o m a t o g r a p h e d o n aluminia, consisted of several fractions with immunoactivity. O n e f r a c t i o n (110 rag) s h o w e d a s t r o n g r e a c t i o n w i t h t h e t e t r a h y d r o a l d o s t e r o n e a n t i b o d y , a n d was i d e n t i c a l w i t h t h e n o n p o l a r m a t e r i a l , s h o w n in F i g u r e 2. R e p e a t e d c o l u m n c h r o m a t o g r a p h y by t h e s a m e a b s o r b e n t w i t h n - h e x a n e - b e n z e n e split t h e m a t e r i a l into two tetrahydroaldosterone-active fractions. F r o m t h e m o r e p o l a r f r a c t i o n r e s i d u e 5/Lpregnane-3c~, 2 0 e - d i o l c o u l d b e i s o l a t e d by c r i s t a l l i z a t i o n f r o m

K. Lichtwa|d et al. : Unidentified Steroids in the pH 1 Urinary Extract

171

~ ' ', TH-ALD-IMMUNOGRAM ....

[3HJ- TH-ALD

[pgllOO/ull [1300

A ~ l

[cpm] 100

1000

5(:

500

o. j '""

'~I~__._,.____~ ~ X

=0.11 =0.11

//

_ ~ / / /

=0.75

100

10

1 ;el

2

3

4

5

6

7

8

9

10 11 12 13 14 15 16 17 18 19

i ALDI

X

PAPER - OHROMATOGRAM SOLVENT-SYSTEM: n - butyl acetate--ethylene glycol Fig. 1. Chromatographic analysis of p H i urine extract by radioimmunoassay with tetrahydroaldosterone antibody and 3H-tetrahydroaldosterone tracer

2000" 1500TLC. (2A1)

1000-

IMMUNOGRAM

500"

SOLVENT-SYSTEM

TH - A L D - ANTI S E R U M

BENZENE/METHANOL (90/101

H3P0 ~ {50°10)

t

START

SOLVENT- SYSTEM CHLOROFORM/METHANOL

(955)

i

~ 0-'~"~:"~'~'~)'

SPOT

UV

~ , 254 ram, 365nm

UV

~

FRONT

H~POj,(50%1

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400.

V~0LET

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254 rim,

366nm

V I O L E T SPOT

TIN- ALD - ANTISERUM

IMMUNOORA•

Boo4 1200'

1600" [pgl Fig. 2. TLC of one of the tetrahydroaldosterone-tike substances in two solvent systems and comparison of UV, phosphoric acid, and immunoreaction detection methods

172 methanol. After TLC of the mother liquid containing the immunoreactivity 0.7 ug tetrahydroaldosterone equivalents were obtained. Neither UV nor phosphoric acid colouration was sufficiently sensitive to detect this substance. Location was only possible by immunoreaction (Fig. 2). The mass-spectra of the derived material by the GC-MS method showed a characteristic steroidal fragmentation pattern. The interpreation of the spectral data is still in progress.

K. Lichtwald et al. : Unidentified Steroids in the pH 1 Urinary Extract ticoid-like substance shown in Figure 1, seems to be applicable for other cases of materials with presumed biological significance. Because of its reaction with a specific tetrahydroaldosterone antibody, we expect that the compound separated from the pH 1 extract has to some extent structural similarity to tetrahydroaldosterone.

References Discussion In evaluating the estimation of urinary aldosterone without chromatography, immunoreactions of the pH 1 extracts with different corticosteroid and tetrahydrocorticosteroid antibodies were studied. An unexpectedly large amount of material which reacted with different corticosteroid and corticosteroid metabolite antibodies was found. This has not been previously reported except in the study by Cope and Loizou, who observed D O C immunoreactivity in the pH 1 extract [1]. With the exception of aldosterone the immunograms revealed that a large part of these materials are not identical with the steroids and metabolites against which the antibodies were raised. They are probably steroids, steroid metabolites, or their conjugates, whose identity and biological role are unknown. The relationship of these materials to the adrenal cortex is indicated by the fact that amounts found in the pH 1 extract, changed according to functional alterations of the adrenals, e.g. the amounts were higher after A C T H , lower after dexamethasone, and higher after metopirone administration. These findings indicate the biological and also possible diagnostic significance of these materials. The outlined method for separation of the nonpolar tetrahydrocor-

1. Cope, C.L., Loizou, S. : Solvolyzabledeoxycorticosteroneconjugates in human urine. J. Endocr. 68, 273-281 (1976) 2. Hornung, J., Gless, K-H., Abdelhamid, S., Vielhauer, W., Vecsei, P. : Radioimmunoassayof free urinary 18-hydroxydeoxycorticosterone (18-OH-DOC) in patients with essential hypertension. Clin. Chim. Acta 67, 181-187 (1978) 3. KohI, K-H., Gless, K-H., Abdelhamid, S., Penke, B., Vecsei, P. : Radioimmunoassay of tetrahydrocorticosterone (THB) in human urine. Acta Endocr. (Kbh) 88, 139-148 (1978) 4. Mok, M., Gless, K-H., Vecsei, P. : Tetrahydrodeoxycorticosterone radioimmunoassay in human urine after chromatography. Acta Endocr. (Kbh) 84, 115-116 (1977), Suppl. 208 5. Vecsei, P. : Glucocorticoidsand their metabolites. In: Methods of Hormone Radioimmunoassay, Eds. B. Jaffe and H. Behrman, pp. 767 769. New York and London: Academic Press 1978 6. Vetter, W., Vetter, H., Siegenthaler, W.: Radioimmunoassay for aldosterone without chromatography. 1. Determination of urinary 18-glucuronide excretion. Acta Endocr. (Kbh) 74, 548-557 (1973) 7. Will, H., Adeljan, R., Winkter, Th., Penke, B., Vecsei, P. : Radioimmunoassay of tetrahydrocortisone and tetrahydrocortisol in human urine. Acta Endocr. (Kbh) 86, 369-379 (1977) Received August 1, 1978 Dr. K. Lichtwald Department of Pharmacology University of Heidelberg Im Neuenheimer Feld 366 D-6900 Heidelberg Federal Republic of Germany