- Email: [email protected]
Metabolism of pregnenolone by the human placenta
Volume 104 :\'umber 4
REFERENCES
I. Lundin, F. E., Erickson, C. C., and Sprunt, D. H.: Socioeconomic distribution of cervical cancer in relation to early marriage and pregnancy, Public Health Monograph No. 73, Washington, D. C., 1964, U. S. Government Printing Office, p. 30. 2. Figge, D. C., and Bennington, J. L.: AM. J. OasT. & GYNEC. 98:516, 1967. 1520 Farnell Court Decatur, Georgia 30033
Metabolism of pregnenolone by the human placenta KRISHNA B. SINGH, M.D., M.S.* Department of Obstetrics and Gynecology, University of Kansas Medical Center, Kansas City, Kansas
ALTHouGH a number of investigators have demonstrated that pregnenolone is readily converted to progesterone indicating an active pregnenolone-3,8-ol-dehydrogenase enzyme system in the human placental tissue,!• 2 • 3· 5 studies on the steroid 17,20-desmolase are limited to few observations. Sobrevilla, Hagerman, and Villee/ using homogenates of term placentas incubated with 14 C-labeled pregnenolone and 17a-hydroxyprogesterone, and Pion and associates, 1 on in situ perfusion of midterm placentas with 3Hlabeled pregnenolone and 17a-hydroxypregnenolone, did not find any significant 17,20-desmolase activity. The present study was undertaken to see if 17 ,20-desmolase could be demonstrated in the term human placenta with 7a-sH-pregnenolone as the substrate. From each of two fresh term placentas 10 Gm. portions of blotted tissue free of fetal membranes were obtained. These were separately homogenized in 40 ml. of a O.OSM K phosphate buffer with 0.04M nicotinamide and 0.004 magnesium chloride at pH 7.4. As a control, a I 0 Gm. portion of the first placenta was similarly homogenized and boiled for 10 minutes. The homogenates were transferred to separate Erlenmeyer flasks, to each flask was added 40 ,umoles of ATP, 23.8 p.moles of NADPH, and 12 p.moles of NADP and 167 p.moles of glucose. After this addition, 14 p.g of 7a-3H-pregnenolone (representing a total radioactivity of 5 p.c) was added to each flask. This mixture *Present address: Department of Gynecology and Obstetrics, St. Louis University School of Medicine, 1325 South Grand Boulevard. St. Louis, Missouri 63104.
Communications
1n
brief
605
was incubated at 37° C. for one-half hour. After that initial incubation 13.6 p.moles of DPN was added to each flask and the incubation was continued for 2 additional hours. The incubations were stopped by adding 5 volumes of acetone to each flask and 400 p.g each of testosterone, androstenedione, dehydroisoandrosterone, and progesterone were added as carrier. Protein was removed by filtration, the acetone fraction taken to the aqueous, and steroids were extracted with ethyl acetate, partitioned between equal volumes of Skellysolve B and 90 per cent methanol, and finally the methanol fraction was taken up in ether and repeatedly washed with sodium hydroxide. The neutral fractions were then chromatographed separately on Whatman No. 2 paper using various Zaffaroni systems. After the initial series of chromatograms it was noted that while almost all the radioactivity in the control incubation remained in the form of pregnenolone only very detectable amounts of pregnenolone remained in the incubations made with viable placenta. Thus the placenta had almost completely metabolized this pregnenolone, and the major product of metabolism was progesterone itself. Although a peak of radioactivity was originally associated with the testosterone carrier following acetylation and rehydrolysis of subsequent chromatography, after the formation of each derivative the carrier was essentially free of radioactivity. The carrier androstenedione fraction contained less than 0.001 per cent of the added radioactivity and was not studied further. The dehydroisoandrosterone fraction after chromatography in the Zaffaroni and Bush systems contained approximately O.Dl per cent of the added radioactivity and attempt was made to crystallize this compound to constant specific activity after the addition of 30 mg. of DHIA carrier. But with each subsequent crystallization specific activity fell and identity could not be established. These data demonstrate that the major metabolite of pregnenolone and homogenized whole placenta is certainly progesterone. The incubation was purposely conducted with holding DPN in the original cofactor addition to see if desmolase activity on pregnenolone could be demonstrated. It may be said that no significant conversion of pregnenolone to 19-carbon compounds identifiable as androstenedione, testosterone, or dehydroisoandrosterone occurred. Thus, with pregnenolone as substrate the 17,20desmolase activity of the human placenta is
606
Communications
1n
negligible. Although the placenta may contain minute amounts of steroid 17 ,20-desmolase, 6 this is probably not an enzyme of biologic importance and biosynthesis of 19-carbon compounds which might potentially serve as estrogen precursors from progesterone and pregnenolone obviously does not occur to any significant extent in this organ." REFERENCES
I. Pion, R., Jaffe, R., Eriksson, G., Wiqvist, N.,
2. 3. 4. 5. 6.
June !J, J%!1
brief
and Diczfalusy, E.: Acta endocrinol. 48: 234, 1965. Pion, R. J., Conrad, S. H., and Wolf, B. J.: J. Clin. Endocrinol. 26: 225, 1966. Ryan, K. ]., Meigs, R., and Petro, Z.: AM. J. 0BST. & GYNEC. 96: 676, 1966. Sobrevilla, L., Hagerman, D., and Villee, C. A.: Biochim. Biophys. Acta 93: 665, 1964. Solomon, S.: J. Clin. Endocrinol. 26: 762, 1966. Warren, J. C., and Cheatum, S. C.: Canad. J. Biochem. 42: 143, 1964.
Cinecystourethrography in the diagnosis of urethral diverticula of women
\w .
.1. ()h,t.
& Gvner
bladder neck suspension. Later the caUSL' was found to be a urethral diverticulum. The physical examination is often unrevealing. Spraitz and Welch 5 report that in their series of 94 patients 68 per cent presented no findings clearly indicative of a urethral diverticulum. If present, the finding might be a tender, palpable mass posterior to the urethra from which a purulent material may be expressed. Cystoscopy was considered a useful diagnostic tool. However, Hoffman and Adams 3 found that the reliance upon the endoscope to confirm the diagnosis or to establish the number of openings was of doubtful value. Even though the orifice of a diverticulum may be seen, a radiographic examination defining the size, position, and number is of assistance to the surgeon. Other radiographic diagnostic techniques have fwen used. The diverticular orifice may be injected directly with contrast media. This method depends upon prior cystoscopic diagnosis, hm\f~Ver. Second, the diverticulum has been shown on a single film exposed while a patient \\as voiding contrast media. Here one relies upon a single view takl'n at a random phase of voiding. The diverticulum may not fill during this para
FRANK H. GRUBER, M.D. Department of Radiology, Medical College of South Carolina, Charleston, South Carolina
T H E I M P o R T A N c E of this lesion lies not in its prevalence, although it is not rare, but rather in the fact that it is usually overlooked. This is regrettable as the persistent aggravating symptoms of this cryptic abnormality are relieved by diverticulectomy. Since the symptoms of urethral diverticula are much the same as those we encounter so often with urinary tract infections and cystoceles, the delay in diagnosis is understandable. The diverticulum is usually infected, causing persistent dysuria, frequency, and pyuria which arc attributed to a urinary tract infection for which the typical patient is repeatedly treated. Being chronically and locally infected, the diverticulum may act as an abscess, and as such may result in pelvic pain or pressure and dyspareunia. In 2 of our patients the presenting complaint was gross hematuria. Another group of symptoms may he attributed to a coexisting cystocele or ureterocele. These are incontinence and postvoiding dribbling caused by the trapping of urine below the main urethral sphincter. Butler1 cites a case in which a patient continued to leak urine after
Fig. 1. The presenting complaints of this 35-ycarold female were dyspareunia and vaginal pain, leakage of urine unrelated to stress, urethral bleeding. and the usual symptoms of cystitis. Repeated cystoscopies failed tn demonstrate this diverticulum. v. vesick neck; 11_, urethra; arrows. divertic~u· hnn.
REFERENCES
I. Lundin, F. E., Erickson, C. C., and Sprunt, D. H.: Socioeconomic distribution of cervical cancer in relation to early marriage and pregnancy, Public Health Monograph No. 73, Washington, D. C., 1964, U. S. Government Printing Office, p. 30. 2. Figge, D. C., and Bennington, J. L.: AM. J. OasT. & GYNEC. 98:516, 1967. 1520 Farnell Court Decatur, Georgia 30033
Metabolism of pregnenolone by the human placenta KRISHNA B. SINGH, M.D., M.S.* Department of Obstetrics and Gynecology, University of Kansas Medical Center, Kansas City, Kansas
ALTHouGH a number of investigators have demonstrated that pregnenolone is readily converted to progesterone indicating an active pregnenolone-3,8-ol-dehydrogenase enzyme system in the human placental tissue,!• 2 • 3· 5 studies on the steroid 17,20-desmolase are limited to few observations. Sobrevilla, Hagerman, and Villee/ using homogenates of term placentas incubated with 14 C-labeled pregnenolone and 17a-hydroxyprogesterone, and Pion and associates, 1 on in situ perfusion of midterm placentas with 3Hlabeled pregnenolone and 17a-hydroxypregnenolone, did not find any significant 17,20-desmolase activity. The present study was undertaken to see if 17 ,20-desmolase could be demonstrated in the term human placenta with 7a-sH-pregnenolone as the substrate. From each of two fresh term placentas 10 Gm. portions of blotted tissue free of fetal membranes were obtained. These were separately homogenized in 40 ml. of a O.OSM K phosphate buffer with 0.04M nicotinamide and 0.004 magnesium chloride at pH 7.4. As a control, a I 0 Gm. portion of the first placenta was similarly homogenized and boiled for 10 minutes. The homogenates were transferred to separate Erlenmeyer flasks, to each flask was added 40 ,umoles of ATP, 23.8 p.moles of NADPH, and 12 p.moles of NADP and 167 p.moles of glucose. After this addition, 14 p.g of 7a-3H-pregnenolone (representing a total radioactivity of 5 p.c) was added to each flask. This mixture *Present address: Department of Gynecology and Obstetrics, St. Louis University School of Medicine, 1325 South Grand Boulevard. St. Louis, Missouri 63104.
Communications
1n
brief
605
was incubated at 37° C. for one-half hour. After that initial incubation 13.6 p.moles of DPN was added to each flask and the incubation was continued for 2 additional hours. The incubations were stopped by adding 5 volumes of acetone to each flask and 400 p.g each of testosterone, androstenedione, dehydroisoandrosterone, and progesterone were added as carrier. Protein was removed by filtration, the acetone fraction taken to the aqueous, and steroids were extracted with ethyl acetate, partitioned between equal volumes of Skellysolve B and 90 per cent methanol, and finally the methanol fraction was taken up in ether and repeatedly washed with sodium hydroxide. The neutral fractions were then chromatographed separately on Whatman No. 2 paper using various Zaffaroni systems. After the initial series of chromatograms it was noted that while almost all the radioactivity in the control incubation remained in the form of pregnenolone only very detectable amounts of pregnenolone remained in the incubations made with viable placenta. Thus the placenta had almost completely metabolized this pregnenolone, and the major product of metabolism was progesterone itself. Although a peak of radioactivity was originally associated with the testosterone carrier following acetylation and rehydrolysis of subsequent chromatography, after the formation of each derivative the carrier was essentially free of radioactivity. The carrier androstenedione fraction contained less than 0.001 per cent of the added radioactivity and was not studied further. The dehydroisoandrosterone fraction after chromatography in the Zaffaroni and Bush systems contained approximately O.Dl per cent of the added radioactivity and attempt was made to crystallize this compound to constant specific activity after the addition of 30 mg. of DHIA carrier. But with each subsequent crystallization specific activity fell and identity could not be established. These data demonstrate that the major metabolite of pregnenolone and homogenized whole placenta is certainly progesterone. The incubation was purposely conducted with holding DPN in the original cofactor addition to see if desmolase activity on pregnenolone could be demonstrated. It may be said that no significant conversion of pregnenolone to 19-carbon compounds identifiable as androstenedione, testosterone, or dehydroisoandrosterone occurred. Thus, with pregnenolone as substrate the 17,20desmolase activity of the human placenta is
606
Communications
1n
negligible. Although the placenta may contain minute amounts of steroid 17 ,20-desmolase, 6 this is probably not an enzyme of biologic importance and biosynthesis of 19-carbon compounds which might potentially serve as estrogen precursors from progesterone and pregnenolone obviously does not occur to any significant extent in this organ." REFERENCES
I. Pion, R., Jaffe, R., Eriksson, G., Wiqvist, N.,
2. 3. 4. 5. 6.
June !J, J%!1
brief
and Diczfalusy, E.: Acta endocrinol. 48: 234, 1965. Pion, R. J., Conrad, S. H., and Wolf, B. J.: J. Clin. Endocrinol. 26: 225, 1966. Ryan, K. ]., Meigs, R., and Petro, Z.: AM. J. 0BST. & GYNEC. 96: 676, 1966. Sobrevilla, L., Hagerman, D., and Villee, C. A.: Biochim. Biophys. Acta 93: 665, 1964. Solomon, S.: J. Clin. Endocrinol. 26: 762, 1966. Warren, J. C., and Cheatum, S. C.: Canad. J. Biochem. 42: 143, 1964.
Cinecystourethrography in the diagnosis of urethral diverticula of women
\w .
.1. ()h,t.
& Gvner
bladder neck suspension. Later the caUSL' was found to be a urethral diverticulum. The physical examination is often unrevealing. Spraitz and Welch 5 report that in their series of 94 patients 68 per cent presented no findings clearly indicative of a urethral diverticulum. If present, the finding might be a tender, palpable mass posterior to the urethra from which a purulent material may be expressed. Cystoscopy was considered a useful diagnostic tool. However, Hoffman and Adams 3 found that the reliance upon the endoscope to confirm the diagnosis or to establish the number of openings was of doubtful value. Even though the orifice of a diverticulum may be seen, a radiographic examination defining the size, position, and number is of assistance to the surgeon. Other radiographic diagnostic techniques have fwen used. The diverticular orifice may be injected directly with contrast media. This method depends upon prior cystoscopic diagnosis, hm\f~Ver. Second, the diverticulum has been shown on a single film exposed while a patient \\as voiding contrast media. Here one relies upon a single view takl'n at a random phase of voiding. The diverticulum may not fill during this para
FRANK H. GRUBER, M.D. Department of Radiology, Medical College of South Carolina, Charleston, South Carolina
T H E I M P o R T A N c E of this lesion lies not in its prevalence, although it is not rare, but rather in the fact that it is usually overlooked. This is regrettable as the persistent aggravating symptoms of this cryptic abnormality are relieved by diverticulectomy. Since the symptoms of urethral diverticula are much the same as those we encounter so often with urinary tract infections and cystoceles, the delay in diagnosis is understandable. The diverticulum is usually infected, causing persistent dysuria, frequency, and pyuria which arc attributed to a urinary tract infection for which the typical patient is repeatedly treated. Being chronically and locally infected, the diverticulum may act as an abscess, and as such may result in pelvic pain or pressure and dyspareunia. In 2 of our patients the presenting complaint was gross hematuria. Another group of symptoms may he attributed to a coexisting cystocele or ureterocele. These are incontinence and postvoiding dribbling caused by the trapping of urine below the main urethral sphincter. Butler1 cites a case in which a patient continued to leak urine after
Fig. 1. The presenting complaints of this 35-ycarold female were dyspareunia and vaginal pain, leakage of urine unrelated to stress, urethral bleeding. and the usual symptoms of cystitis. Repeated cystoscopies failed tn demonstrate this diverticulum. v. vesick neck; 11_, urethra; arrows. divertic~u· hnn.