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Urinary and plasma testosterone glucosiduronate measurement by a simple ria method
Journal of Steroid Pergsmon Press
Biochemistry.
Vol.
Ltd1979. Printed
I I, pp. 143 to 146 in Great Britain
URINARY AND PLASMA TESTOSTERONE GLUCOSIDURONATE MEASUREMENT BY A SIMPLE RIA METHOD J. A. F. TRESGUERRES* and J. TAMMt *C%tedra de Endocrinologia Exptal., Facultad de Medicina Universidad Complutense, Madrid, Spain and, tI1 Med. Klinik Universitiits Krankenhaus Eppendorf, Hamburg, Germany SUMMARY
A simple, specific and rapid radioimmunoassay (RIA) for the determination of testosterone-17/?-glucosiduronate (TG) in urine and plasma is described. Antibodies against TG were raised in rabbits after injection of an immunogenic complex obtained by condensation of Cohn’s fraction IV-4 (CF) and TG. This complex revealed higher titer developing than those obtained by simultaneous immunization with another TG complex using bovine serum albumin as carrier protein. To separate free and bound fractions, three methods were compared: Charcoal adsorption, Polyethyleneglycol precipitation and Hydroxyapatite separation. Hydroxyapatite showed a good coefficient of variation (intra-assay 60/ and inter-assay 11%) together with the possibility of working at room temperature and reducing counting cost. TG was estimated in small aliquots of male and female urine after evaporating overnight at 50°C in order to eliminate interfering material. Plasma samples were pre-extracted with diethylether to remove non-conjugated steroids and extracted again with ethanol. The mean excretion of TG in the urine of 90 healthy men amounted to 178 f 46 &24 h and of 50 women to 31 f 11 pg/24 h. In 56 women suffering from idiopathic hirsutism a mean TG of 68 & 27 pg/24 h and in 40 men with hypogonadism 58 + 27 leg/24 h were found. In order to avoid 24 h urine collections, a TG/creatinine index in early morning urine was tested. This showed good correlation (r = 0.8) in both sexes with the correspondent daily excretion. Plasma TG concentrations were found to be 2.1 f 0.45 ng/ml in 20 normal males, 0.47 * 0.18 ng/ml in 20 normal females, 1.08 + 0.3 in 9 hypodogonadal males and 1.36 f 0.68 in 28 hirsute females. TG concentrations in testicular vein was 4.02 f 1.4 ng/ml in 3 normal males (basal) and 5 f 2.7 ng/ml 30 minutes after 500 IU HCG + 66 IU FSH, 65 IU LH (HMG) infusion. The simultaneous cubital vein values were 3 f 1.3 ng/ml and 3.6 f 2.3 ng/ml respectively. The method makes possible the determination of 300 urine samples or 120 plasma samples per week by a well trained technician.
INTRODUCTION
The measurement of testosterone glucosiduronate (TG) in urine is currently used in many laboratories, as an index of the androgenic status [l-3]. Usual
methods involve urine hydrolysis, extraction, purification and determination by gas-chromatography, colorimetry or Radioimmunoassay (RIA). Kellie[4] reported in 1972 the raising of an antibody against TG and one year later Hennam et aI.[YJ developed a RIA method using Kellie’s antibody and showing the possibility of estimating this steroid conjugate directly in small amounts of urine. There is great interest in direct TG determination for two reasons: the remarkable simplification of the method and also the possibility it raises of studying the physiology of this compound in more detail than has previously been possible.
METHOD
TG was used as hapten to prepare immunogenic complexes, linked through its COOH group to Bovine Serum Albumin (BSA) or Cohn’s fraction IV-4 (CF) from human plasma as carrier proteins. Six rabbits were injected intradermally with the BSA-TG complex and another group of four rabbits with LB.
I I/IA---n
143
CF-TG in order to compare antibody production of each. The increase in antibody titers was found to be greater in animals injected with TG-CF compared with those immunized with TG-BSA [6]. This is probably due to the fact that CF consist partially or carbohydrate moieties. It Seems likely that higher titers would have been obtained after a longer period of immunization of ihe animals, but for technical reasons, rabbits had to be killed after 13 weeks of treatment. Serum K-74, showing a titer of 1: 8.000, K, of 2.2 x lo9 l/mol and exhibiting significant cross reactions only with free testosterone (T) (27%) and Sa-dihydrotestosterone-glucosiduronate (DHT-G) (207;) was chosen. Using this antiserum a RIA method has been developed, with [‘HI-TG as tracer and 0.1% BSA in saline as buffer, which allows the measurement of urinary [6] and plasma TG [A. At the beginning of our study a conventional charcoal-dextran suspension was used to separate bound and free fractions [6]. Due to the “stripping” effect of charcoal, care had to be taken in maintaining the same incubation time for all the tubes of the assay with the charcoal suspension. Also this separation step had to be performed at 4°C. A polythyleneglycol (PEG) separation method was also tested after-
144
.I. A. F. TREWJERRESand J. TAMM Table 1. Testosterone glucosiduronate
values in urine
x f SD 178 + 30 f 58 + 68 +
Normal men Normal women Men with hypogonadism Hirsute women
46 j&24 10 &24 27 &24 27 &24
h (n h (n h (n h (n
= = = =
90) 50) 40) 56)
interfering material leading to overestimation when crude urine was used [S, 61. A standard curve was
I
2!l
se
loo
250 500
1500PJGT
Fig. 1. Standard curves of TG performed using charcoal (2 + SEM n = 18) or hydroxyapatite (2 + SEM n = 12) as separation methods.
wards [7]. This system selectively precipitated antibody bound activity, with no stripping effect at all; so was independent of incubation time. But 20-25x PEG in saline was however a viscous solution, that
significantly increased pipetting time. Also, expensive “y” globulin had to be used in the assay to allow a good separation of bound and free fractions. Finally an Hydroxyapatite (HA) suspension has been used in this step, allowing a time independent, temperature independant separation method, with the highest precision. This system first described by Trafford et nI.[8] precipitates the bound fraction without the need of additional “7” globulin. Hydroxyapatite suspension in saline (150 jd of a 0.1 g/ml suspension) is added to all tubes and well mixed. After centrifugation, the supernatant is discarded, the pellet is washed with saline and centrifuged again. After discarding the saline, 200~1 of a 0.1 M Buffer phosphate are added and the tube well shaken to eluate the antibody bound [“HI-TG from the HA. Finally 2 ml of a water miscible scintillation cocktail (InstagelR, PCSR or Bray’s) are poured into the assay tube, the tube is fitted with a plastic cap, well shaken and put into a counting vial. By this method it is possible to save 8 ml of scintillation fluid, the counting vial, and the vial cap. Figure 1 shows standard curves using charcoal and HA separation methods, with very similar results. Sensitivity is 25 pg of TG and the standard curve can be used up to 1200 pg. The intra-assay coefficient of variation (CV) was found to be 6% in 25 measurements of the same urine pool and the interassay CV was 11% in 18 different assays.
prepared in the same way by evaporating corresponding amounts of the ethanolic TG solution to obtain a range between 25 and 1600pg. All measurements were carried out in triplicate. Antibody and C3H]-TG were dissolved in physiologic saline with 0.1% BSA and the incubation time was 2 h at 4°C. A very good linear response was observed from increasing volumes of heated urine. Experiments showed a 92 f 6% recovery of non-labelled TG added to male or female urine. Over a wide range endogenous TG did not influence the recovery. Results obtained with this direct method have been correlated with those obtained by a conventional RIA following /I-glucuronidase hydrolysis. In a double blind experiment a coefficient of correlation of 0.89 was found [6]. The mean urinary excretion of TG in normal individuals and patients with hypogonadism or hirsutism can be seen in Table 1. Using the 24 hour urinary TG excretion as index, testicular function tests were performed in primary and secondary hypogonadism. After two control 24 h
.m
/ati
TG
1
TG in urine
The total volume of 24 h urine was measured, and 0.5 ml diluted 1: 10 with .double distilled water. Aliquots of diluted urine (20 ~1) in normal male subjects and 50 to 100 ~1 in normal females and patients with expected low concentrations of TG were transferred to RIA glass tubes (1 x 5.5 cm) and evaporated overnight in an oven. This was done in order to eliminate
E
a
I
*
,
.
,
e
I
Fig. 2. Effect of daily administration 2000 IU HCG i.m. after two basal 24 h urine collections on primary (A) and secondary (B) male hypogonadism. Arrow indicates the beginning of the treatment. Control subjects are expressed in open circles (0-Q X f SD (n = 5).
145
RIA for testosterone glucosiduronate 3r 0
2’6
0
Fig. 3. Correlation between 24 h TG excretion and TG/Creatinine index in 42 male and female individuals.
urine collections were completed, 2000ILJof HCG were injected daily for 5 days. Figure 2 shows the good group separation obtained between patients with primary and secondary hypogonadism. In order to avoid 24 hour urine collections which can be very difficult in some patients a TG/creatinine index has been tested. Results shown in Fig. 3 show a good correlation (r = 0.8) between 24 h collection versus TG/creatinine index. This simple index is potentially useful for clinical screening in a manner analogous to that already in use for urinary oestrogens [9, lo].
TG
in plasma
Four millilitre plasma samples of male or female subjects with an internal standard of 2200d.p.m. [1,2’H]-TG were extracted twice with 40 ml of diethylether. The organic phase was used to measure non-conjugated T, DHT, androstane-3a,l7fl-diol and estradiol. The remaining plasma was extracted again with 8 ml ethanol and left at room temperature for 4 h. After removal of proteins by centrifugation the ethanolic phase was evaporated to dryness under nitrogen in a water bath at 50°C. Residue was redis-
I
solved in 2ml dd. water. Fifty ~1 were used in triplicate assays and 200~1 for recovery control. Addition of increasing amounts of TG to a plasma pool or stepwise enlargement of plasma volumes results in a linear response in TG determination. In 30 plasma samples TG was estimated by direct RIA ‘and after fl-glucuronidase hydrolysis followed by extraction and purification by TLC. The ratio TG/T (1,77 + 0.30) was found to be close to the molecular weight ratio of TG/T (1.64) and the coefficient of correlation of r = 0.92 as shown in Fig. 4. Plasma TG values are compiled in Table 2. Plasma TG has been measured in the spermatic vein (SV) [ 1l] of 3 normal males during intravenous infusions of 5OOIU HCG plus HMG (66 IU FSH, 65 IU LH). The blood-was drawn by an indwelling catheter every 5 minutes for a total period of 30min. Results can be seen in Table 3. Simultaneous determination of testosterone showed a marked increase up to 1800/ whereas in TG no significant increase occurred. On the other hand TG levels in S.V. were found to be higher than in simultaneous peripheral vein determination indicating a possible contribution of the human testis to TG formations.
CORRELAM BETWEENMEASUREMNT AND T AFTER HYDROL~S
TG
IO
t
Fig. 4. Correlation between measurement of TG directly in plasma and T after hydrolysis.
J. A. F.
146
TREKWERRES
and J.
TAMM
Table 2. Testosterone (T) and testosterone-glucosiduronate
T Normal men Normal women Men with hypogonadism Women with idiopathic hirsutism Women with male pattern alopecia
6.32 f 0.52 + 1.82 + 0.90 k 0.37 f
(TG) levels in plasma TG
2.27 q/ml 0.18 q/ml 0.68 ng/ml 0.48 ng/ml 0.2 ng/ml
2.1 + 0.45 ng/ml (n = 20) 0.48 k 0.20 ng/ml (n = 20) 1.08 f 0.3 ng/ml (n = 9) 1.44 & 0.56 ng/ml (n = 28) 0.93 k 0.16 ng/ml (n = 6)
Table 3. Effect on plasma TG concentrations of 5OOIU HCG plus HMG infused intravenously during 30 min in three normal male subjects I + SD
Spermatic vein Cubital vein
Basal
15 min
30 min
4.02 k 1.4 q/ml 3 f 1.3 ng/ml
5.6 f 2.8 @ml 3.12 + 1.5 q/ml
5.1 f 2.7 ng/ml 3.6 + 2.3 ng/ml
CONCLUSlONS
The results presented show that Cohn’s fraction IV-4 used as carrier protein with TG as hapten gives rise to a higher titer of antibodies compared with the BSA-TG Complex. The use of hyclroxyapatite to separate bound and free fractions in the RIA demonstrate very good reproducibility and precision in the results’ working at room temp, together with a marked reduction in counting costs. Direct urinary TG measurement can be used for the clinical diagnosis of the androgenic status and for testicular stimulation tests. Also the TG/creatinine index may be of interest in clinical screening and when urine collection over a 24 h period is not possible, as has been shown earlier for urinary oestrogens [9, lo]. Plasma TG values in normal males and females show less marked differences than those of the corresponding T levels [7]. This is probably due to the fact that in the female the androstenedione and dehydroepiandrosterone which are converted to TG, does not enter the plasma pool of unconjugated T. Despite this statement, plasma TG is more significantly elevated in hirsute women than the corresponding aglycone, which is of interest for diagnostic purposes. The higher TG values found in the SV can be explained by a potential contribution of the testis or by the epididymis as shown by Djeseland in the rat [12]. Recent studies on human seminal plasma [13] yielded TG concentrations 69 times above those found in blood plasma. This could be compatible with T glucuronidation in the seminiferous tubules or male accesory glands 1141. Acknowledgements-This work has been supported by a grant of the Deutsche For.schungsgemeinschaft (SFB-34, Endokrinologie). Cohn’s fraction IV-4 from human plasma has been generously supplied by the American National Red Cross, National Fractionation Center (Dr. Hao), Bethesda, Maryland. The authors wish to thank Dr. L. Perez Mendez and Mrs. U. Volkwein for their helpful assistance.
REFERENCES
1. Hllbl W. and Schollberg K.: Die fluorimetrsche Bestimmung von Testosteron Epitestosteron und Androstendion nach dilnnschicht-chromatographischer Isolierung aus dem Ham. Acta endoer., Copenh. 58 (1968) 353363. 2. Abraham G. E.: Radioimmunoassay of steroids in biological materials. Acta endocr., Cope&. 75 Suppl 183 (1974) 7-42. 3. Watson J. T.: A simplified determination of urinary testosterone utilizing column and gas-liquid chromatography. J. Chromatog. 43 (1969) 339-349. 4. Kellie A. E., Samuel V. K., Riley W. J. and Robertson D. M.: Steroid glucuronide-BSA complexes as antigens, the radio-immunoassay of steroid conjugates. J. steroid Biochem 3 (1972) 275-288. 5. Hennam J. F., Collins W. P. and Sommerville I. F.: Radioimmunoassay of urinary testosterone .glucuronoside. Steroids 21 (1973) 285299. 6. Tresguerres J. A. F., Lisboa B. P. and Tamm J.: A simple radioimmunoassay for the measurement of testosterone glucosiduronate in unextracted urine. Steroids Zs (1976) 1323. 7. Tresguerres J. A. F., Volkwein V. and Tamm J.: Studies on testosterone 1‘IS-glucosiduronate in human plasma. J. steroid Biochem 8 (1977) 1071-1075. 8. Trafford D. J. H, Ward P. R., Ying-Foo A. and Makin H. L. J.: Hydroxyapatite-a reagent for the separation of free and antibody-bound steroid during radioimmunoassay. Steroids 27 (1976) 405-422. 9. Hainsworth I. R. and Hall P. E.: Simple automated method for the measurement of oestrogens in the urine of pregnant women. C/in. Chim. Acta 35 (1971) 201-208. 10. Salvatori B., Vadora E. and Coppola F.: Diurnal variations in oestrogens excretion in pregnancy. J. Obstet. Gynecol. Brit. Commun. 79 (1972) 625-628.
11. Tamm J., Bticheler E., Volkwein V. and Mischke W.: Acute effects of intravenously infused HCG and HCG/HMG on steroid concentrations in the human spermatic vein. Acta ena’ocr., Copenh. (In press). 12. Djoseland 0.: Androgen metabolism by rat epididymis. Steroids 27 (1976) 617-636. 13. Purvis K., Saksena S. K., Landgren B. M., Cakan Z. and Diczfalusy E.: Steroid conjugates in human seminal plasma. C/in. endocr. 5 (1976) 253261. 14. Chung L. W. K. and Coffey D. S.: Androgen glucuronides. Direct formation in rat accessory sex organs. Steroids 30 (1977) 223-243.
Biochemistry.
Vol.
Ltd1979. Printed
I I, pp. 143 to 146 in Great Britain
URINARY AND PLASMA TESTOSTERONE GLUCOSIDURONATE MEASUREMENT BY A SIMPLE RIA METHOD J. A. F. TRESGUERRES* and J. TAMMt *C%tedra de Endocrinologia Exptal., Facultad de Medicina Universidad Complutense, Madrid, Spain and, tI1 Med. Klinik Universitiits Krankenhaus Eppendorf, Hamburg, Germany SUMMARY
A simple, specific and rapid radioimmunoassay (RIA) for the determination of testosterone-17/?-glucosiduronate (TG) in urine and plasma is described. Antibodies against TG were raised in rabbits after injection of an immunogenic complex obtained by condensation of Cohn’s fraction IV-4 (CF) and TG. This complex revealed higher titer developing than those obtained by simultaneous immunization with another TG complex using bovine serum albumin as carrier protein. To separate free and bound fractions, three methods were compared: Charcoal adsorption, Polyethyleneglycol precipitation and Hydroxyapatite separation. Hydroxyapatite showed a good coefficient of variation (intra-assay 60/ and inter-assay 11%) together with the possibility of working at room temperature and reducing counting cost. TG was estimated in small aliquots of male and female urine after evaporating overnight at 50°C in order to eliminate interfering material. Plasma samples were pre-extracted with diethylether to remove non-conjugated steroids and extracted again with ethanol. The mean excretion of TG in the urine of 90 healthy men amounted to 178 f 46 &24 h and of 50 women to 31 f 11 pg/24 h. In 56 women suffering from idiopathic hirsutism a mean TG of 68 & 27 pg/24 h and in 40 men with hypogonadism 58 + 27 leg/24 h were found. In order to avoid 24 h urine collections, a TG/creatinine index in early morning urine was tested. This showed good correlation (r = 0.8) in both sexes with the correspondent daily excretion. Plasma TG concentrations were found to be 2.1 f 0.45 ng/ml in 20 normal males, 0.47 * 0.18 ng/ml in 20 normal females, 1.08 + 0.3 in 9 hypodogonadal males and 1.36 f 0.68 in 28 hirsute females. TG concentrations in testicular vein was 4.02 f 1.4 ng/ml in 3 normal males (basal) and 5 f 2.7 ng/ml 30 minutes after 500 IU HCG + 66 IU FSH, 65 IU LH (HMG) infusion. The simultaneous cubital vein values were 3 f 1.3 ng/ml and 3.6 f 2.3 ng/ml respectively. The method makes possible the determination of 300 urine samples or 120 plasma samples per week by a well trained technician.
INTRODUCTION
The measurement of testosterone glucosiduronate (TG) in urine is currently used in many laboratories, as an index of the androgenic status [l-3]. Usual
methods involve urine hydrolysis, extraction, purification and determination by gas-chromatography, colorimetry or Radioimmunoassay (RIA). Kellie[4] reported in 1972 the raising of an antibody against TG and one year later Hennam et aI.[YJ developed a RIA method using Kellie’s antibody and showing the possibility of estimating this steroid conjugate directly in small amounts of urine. There is great interest in direct TG determination for two reasons: the remarkable simplification of the method and also the possibility it raises of studying the physiology of this compound in more detail than has previously been possible.
METHOD
TG was used as hapten to prepare immunogenic complexes, linked through its COOH group to Bovine Serum Albumin (BSA) or Cohn’s fraction IV-4 (CF) from human plasma as carrier proteins. Six rabbits were injected intradermally with the BSA-TG complex and another group of four rabbits with LB.
I I/IA---n
143
CF-TG in order to compare antibody production of each. The increase in antibody titers was found to be greater in animals injected with TG-CF compared with those immunized with TG-BSA [6]. This is probably due to the fact that CF consist partially or carbohydrate moieties. It Seems likely that higher titers would have been obtained after a longer period of immunization of ihe animals, but for technical reasons, rabbits had to be killed after 13 weeks of treatment. Serum K-74, showing a titer of 1: 8.000, K, of 2.2 x lo9 l/mol and exhibiting significant cross reactions only with free testosterone (T) (27%) and Sa-dihydrotestosterone-glucosiduronate (DHT-G) (207;) was chosen. Using this antiserum a RIA method has been developed, with [‘HI-TG as tracer and 0.1% BSA in saline as buffer, which allows the measurement of urinary [6] and plasma TG [A. At the beginning of our study a conventional charcoal-dextran suspension was used to separate bound and free fractions [6]. Due to the “stripping” effect of charcoal, care had to be taken in maintaining the same incubation time for all the tubes of the assay with the charcoal suspension. Also this separation step had to be performed at 4°C. A polythyleneglycol (PEG) separation method was also tested after-
144
.I. A. F. TREWJERRESand J. TAMM Table 1. Testosterone glucosiduronate
values in urine
x f SD 178 + 30 f 58 + 68 +
Normal men Normal women Men with hypogonadism Hirsute women
46 j&24 10 &24 27 &24 27 &24
h (n h (n h (n h (n
= = = =
90) 50) 40) 56)
interfering material leading to overestimation when crude urine was used [S, 61. A standard curve was
I
2!l
se
loo
250 500
1500PJGT
Fig. 1. Standard curves of TG performed using charcoal (2 + SEM n = 18) or hydroxyapatite (2 + SEM n = 12) as separation methods.
wards [7]. This system selectively precipitated antibody bound activity, with no stripping effect at all; so was independent of incubation time. But 20-25x PEG in saline was however a viscous solution, that
significantly increased pipetting time. Also, expensive “y” globulin had to be used in the assay to allow a good separation of bound and free fractions. Finally an Hydroxyapatite (HA) suspension has been used in this step, allowing a time independent, temperature independant separation method, with the highest precision. This system first described by Trafford et nI.[8] precipitates the bound fraction without the need of additional “7” globulin. Hydroxyapatite suspension in saline (150 jd of a 0.1 g/ml suspension) is added to all tubes and well mixed. After centrifugation, the supernatant is discarded, the pellet is washed with saline and centrifuged again. After discarding the saline, 200~1 of a 0.1 M Buffer phosphate are added and the tube well shaken to eluate the antibody bound [“HI-TG from the HA. Finally 2 ml of a water miscible scintillation cocktail (InstagelR, PCSR or Bray’s) are poured into the assay tube, the tube is fitted with a plastic cap, well shaken and put into a counting vial. By this method it is possible to save 8 ml of scintillation fluid, the counting vial, and the vial cap. Figure 1 shows standard curves using charcoal and HA separation methods, with very similar results. Sensitivity is 25 pg of TG and the standard curve can be used up to 1200 pg. The intra-assay coefficient of variation (CV) was found to be 6% in 25 measurements of the same urine pool and the interassay CV was 11% in 18 different assays.
prepared in the same way by evaporating corresponding amounts of the ethanolic TG solution to obtain a range between 25 and 1600pg. All measurements were carried out in triplicate. Antibody and C3H]-TG were dissolved in physiologic saline with 0.1% BSA and the incubation time was 2 h at 4°C. A very good linear response was observed from increasing volumes of heated urine. Experiments showed a 92 f 6% recovery of non-labelled TG added to male or female urine. Over a wide range endogenous TG did not influence the recovery. Results obtained with this direct method have been correlated with those obtained by a conventional RIA following /I-glucuronidase hydrolysis. In a double blind experiment a coefficient of correlation of 0.89 was found [6]. The mean urinary excretion of TG in normal individuals and patients with hypogonadism or hirsutism can be seen in Table 1. Using the 24 hour urinary TG excretion as index, testicular function tests were performed in primary and secondary hypogonadism. After two control 24 h
.m
/ati
TG
1
TG in urine
The total volume of 24 h urine was measured, and 0.5 ml diluted 1: 10 with .double distilled water. Aliquots of diluted urine (20 ~1) in normal male subjects and 50 to 100 ~1 in normal females and patients with expected low concentrations of TG were transferred to RIA glass tubes (1 x 5.5 cm) and evaporated overnight in an oven. This was done in order to eliminate
E
a
I
*
,
.
,
e
I
Fig. 2. Effect of daily administration 2000 IU HCG i.m. after two basal 24 h urine collections on primary (A) and secondary (B) male hypogonadism. Arrow indicates the beginning of the treatment. Control subjects are expressed in open circles (0-Q X f SD (n = 5).
145
RIA for testosterone glucosiduronate 3r 0
2’6
0
Fig. 3. Correlation between 24 h TG excretion and TG/Creatinine index in 42 male and female individuals.
urine collections were completed, 2000ILJof HCG were injected daily for 5 days. Figure 2 shows the good group separation obtained between patients with primary and secondary hypogonadism. In order to avoid 24 hour urine collections which can be very difficult in some patients a TG/creatinine index has been tested. Results shown in Fig. 3 show a good correlation (r = 0.8) between 24 h collection versus TG/creatinine index. This simple index is potentially useful for clinical screening in a manner analogous to that already in use for urinary oestrogens [9, lo].
TG
in plasma
Four millilitre plasma samples of male or female subjects with an internal standard of 2200d.p.m. [1,2’H]-TG were extracted twice with 40 ml of diethylether. The organic phase was used to measure non-conjugated T, DHT, androstane-3a,l7fl-diol and estradiol. The remaining plasma was extracted again with 8 ml ethanol and left at room temperature for 4 h. After removal of proteins by centrifugation the ethanolic phase was evaporated to dryness under nitrogen in a water bath at 50°C. Residue was redis-
I
solved in 2ml dd. water. Fifty ~1 were used in triplicate assays and 200~1 for recovery control. Addition of increasing amounts of TG to a plasma pool or stepwise enlargement of plasma volumes results in a linear response in TG determination. In 30 plasma samples TG was estimated by direct RIA ‘and after fl-glucuronidase hydrolysis followed by extraction and purification by TLC. The ratio TG/T (1,77 + 0.30) was found to be close to the molecular weight ratio of TG/T (1.64) and the coefficient of correlation of r = 0.92 as shown in Fig. 4. Plasma TG values are compiled in Table 2. Plasma TG has been measured in the spermatic vein (SV) [ 1l] of 3 normal males during intravenous infusions of 5OOIU HCG plus HMG (66 IU FSH, 65 IU LH). The blood-was drawn by an indwelling catheter every 5 minutes for a total period of 30min. Results can be seen in Table 3. Simultaneous determination of testosterone showed a marked increase up to 1800/ whereas in TG no significant increase occurred. On the other hand TG levels in S.V. were found to be higher than in simultaneous peripheral vein determination indicating a possible contribution of the human testis to TG formations.
CORRELAM BETWEENMEASUREMNT AND T AFTER HYDROL~S
TG
IO
t
Fig. 4. Correlation between measurement of TG directly in plasma and T after hydrolysis.
J. A. F.
146
TREKWERRES
and J.
TAMM
Table 2. Testosterone (T) and testosterone-glucosiduronate
T Normal men Normal women Men with hypogonadism Women with idiopathic hirsutism Women with male pattern alopecia
6.32 f 0.52 + 1.82 + 0.90 k 0.37 f
(TG) levels in plasma TG
2.27 q/ml 0.18 q/ml 0.68 ng/ml 0.48 ng/ml 0.2 ng/ml
2.1 + 0.45 ng/ml (n = 20) 0.48 k 0.20 ng/ml (n = 20) 1.08 f 0.3 ng/ml (n = 9) 1.44 & 0.56 ng/ml (n = 28) 0.93 k 0.16 ng/ml (n = 6)
Table 3. Effect on plasma TG concentrations of 5OOIU HCG plus HMG infused intravenously during 30 min in three normal male subjects I + SD
Spermatic vein Cubital vein
Basal
15 min
30 min
4.02 k 1.4 q/ml 3 f 1.3 ng/ml
5.6 f 2.8 @ml 3.12 + 1.5 q/ml
5.1 f 2.7 ng/ml 3.6 + 2.3 ng/ml
CONCLUSlONS
The results presented show that Cohn’s fraction IV-4 used as carrier protein with TG as hapten gives rise to a higher titer of antibodies compared with the BSA-TG Complex. The use of hyclroxyapatite to separate bound and free fractions in the RIA demonstrate very good reproducibility and precision in the results’ working at room temp, together with a marked reduction in counting costs. Direct urinary TG measurement can be used for the clinical diagnosis of the androgenic status and for testicular stimulation tests. Also the TG/creatinine index may be of interest in clinical screening and when urine collection over a 24 h period is not possible, as has been shown earlier for urinary oestrogens [9, lo]. Plasma TG values in normal males and females show less marked differences than those of the corresponding T levels [7]. This is probably due to the fact that in the female the androstenedione and dehydroepiandrosterone which are converted to TG, does not enter the plasma pool of unconjugated T. Despite this statement, plasma TG is more significantly elevated in hirsute women than the corresponding aglycone, which is of interest for diagnostic purposes. The higher TG values found in the SV can be explained by a potential contribution of the testis or by the epididymis as shown by Djeseland in the rat [12]. Recent studies on human seminal plasma [13] yielded TG concentrations 69 times above those found in blood plasma. This could be compatible with T glucuronidation in the seminiferous tubules or male accesory glands 1141. Acknowledgements-This work has been supported by a grant of the Deutsche For.schungsgemeinschaft (SFB-34, Endokrinologie). Cohn’s fraction IV-4 from human plasma has been generously supplied by the American National Red Cross, National Fractionation Center (Dr. Hao), Bethesda, Maryland. The authors wish to thank Dr. L. Perez Mendez and Mrs. U. Volkwein for their helpful assistance.
REFERENCES
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