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Prolactin-secreting pituitary adenomas: Serum and tissue prolactin levels with ultrastructural correlation
Prolactin~secreting
pituitary adenomas: Serum and tissue prolactin levels with ultrastructural correlation DAVID F. ARCHER, M.D. HERNANDO SALAZAR, M.D. JOSEPH C. MAROON, M.D. LINDA J. HOUGH, B.S.
Pittsburgh, Pennsylvania Adenomatous tissue was obtained from 10 women with evidence of prolactin-secreting pituitary adenoma at the time of transsphenoidal removal of the tumor. Tissue levels of prolactin (hPRL) were estimated after extraction and were compared to preoperative peripheral serum levels of hPRL No correlation was found between the tissue and serum concentrations of hPRL, although high levels of hPRL were found in the tissue extracts. Electron microscopy confirmed active secretion of the individual cells, and the ultrastructural findings were compatible with prolactin-secreting adenomas. Associated with the active secretion was extensive exocytosis of the secretory granules away from the capillary waiL These findings suggest that, although active secretion of hPRL is occurring, large amounts of secreted hPRL are not available lor immediate uptake into the capillary plexus; thus a slow absorption of hPRL could be taking place from the adenomatous tissue. These data could explain the failure of provocative tests of hPRL stimulation and inhibition to unequivocally characterize an adenoma in an individal with elevated serum levels of hPRL. (AM. J. 0BSTET. GYNECOL. 137:646, 1980.)
PROLACTIN-SECRETING pituitary adenomas are no longer considered to be uncommon entities. Diagnosis has been facilitated by the development of a specific radioimmunoassay for prolactin (hPRL) and the availability of hypocycloidal polytomography .1• 2 Diagnostic studies involving stimulation and suppression of secretion of hPRL have not yielded any consistent response pattern that would unequivocally discriminate between those individuals with x-ray evidence of pituitary microadenoma or macroadenoma and those without obvious roentgenographic evidence of sellar or pituitary changes. Stimulation of hPRL secretion by thyrotropin-releasing hormone (TRH) in individuals with pituitary adenoma has, in some instances, resulted in a blunted hPRL response, as
From the Departments of Obstetrics and Gynecology, Pathology, and Neurosurgery, University of Pittsburgh School of Medicine. Received for publication August 6, 1979. Revised November 16, 1979. Accepted November 30, 1979. Reprint requests: David F. Archer, M.D., Depanment of Obstetrics and Gynecology, University of Pittsburgh School of Medicine, Magee-Women's Hospital, Forbes Ave. and Halket St., Pittsburgh, Pennsylvania 15213.
646
compared to that in controls. Other studies have documented a delayed but exaggerated response of hPRL to this stimulus.H Inhibition of hPRL after the administration of either L-dopa or bromergocryptine to women with hPRL-secreting adenomas has been variable and comparable to the responses of women with hyperprolactinemia without roentgenographic evidence of pituitary adenoma. 6• 7 A correlation between the level of serum hPRL and the volume of the sella turcica has been reported. However, there does not appear to be any correlation between absolute levels of serum hPRL and the pres• ence or absence of pituitary adenoma. 8 This study was designed to compare preoperative serum levels of hPRL to the concentration of hPRL in the adenomatous tissue, as well as to assess ultrastructural evidence for the secretory activity of the individual cells. The rationale was that the information obtained might provide a reason for the lack of correlation between serum levels of hPRL, dynamic hPRL test results, and roentgenographic presence of a pituitary adenoma. Material and methods Patient material. Ten women with elevated serum levels of hPRL and documented evidence of pituitary 0002-93781801140646+07$00.7010 © 1980 The C. V. Mosby Co.
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Table I. Tissue weight and prolactin content in patients with prolactin-secreting pituitary adenoma Total prolactin (JAg) Patient
Tissue weight (mg)
Supernatant
D. 1\f. L. G.
·12.5 27.4 15.6 57.4 58.2 17.4 77.8 57.1 41.3 38.1
51.20 42.00 25.05 36.90 56.40 96.00 56.85 57.30 7.95 24.90
J.
F.
P. S. S. J. E. H. P.M. N. P. E. S. S. S.
I
Serum hPRL (ng/ml)
Pellet
JAg hPRL/mg tissue
1.63 0.69 1.85 2.20 4.63 1.65 3.93 1.67 0.08 0.49
1.24 1.56 1.72 0.68 1.05 5.61 0.78 1.01 0.19 0.67
Preop
I
Postop
190
5 67 63 890 25 235
HO 340 2,250
JIO 480 600 420 164 225
:wo 14 51 12
Table II. Tissue protein and DNA content of prolactin-secreting pituitary adenoma Patient
Protein* ( JAglmg tissue)
DNA t ( JAglmg tissue)
hPRLiproteint (JAg I p.g Img tissue)
D.M. L. G. J. F. P. S.
47.43 79.83 99.55 38.72 77.85 72.13 49.83 72.21 46.37 20.21
0.082 0.080 0.160 0.!31 0.112 0.072 0.206 0.060 0.034 0.021
0.026 0.019 0.017 0.017 0.013 0.077 0.016 0.014 0.004 0.033
s. J.
E. H. P.M. N. P. E. S. S. S.
hPRLIDNAt ( p.gl p.glmg tissue)
Pi.16 19.48 .10.78
5.20 9.36 79.94 3.79 I fi. 78 fJ.7[
:11.74
*p < 0.02. tp
microadenoma by hypocycloidal polytomography were investigated. All patients underwent transsphenoidal partial hypophysectomy. Postoperative levels of hPRL were determined in all patients between 1 and 7 days after removal of the adenoma and confirmed by repetitive sampling 2 months or more postoperatively. All postoperative hPRL values are the means of two or more samples obtained randomly on each patient between 7 days and 3 months after operation. Serum and tissue analysis. Peripheral levels of hPRL were determined by a double antibody radioimmunoassay as previously reported. 5* Tissue levels of hPRL were assayed by the same radioimmunoassay system after extraction by the following technique: A sample of the adenomatous tissue removed at the time of operation was immediately placed on ice in a O.lM sodium carbonate buffer, pH 10.0, and transported to the laboratory. The tissue was weighed on a Roller Smith milligram balance after blotting to remove excess fluid, replaced in the buffer, and homogenized with a *The hPRL standard was obtained from the National Institute of Arthritis, Metabolism, and Digestive Diseases, and the hPRL antiserum was a gift from Dr. Henry Friesen.
sonifier cell disruptor for 30 seconds at 50 watts. The homogenate was centrifuged at 1,088 x g for 10 minutes to separate a "supernatant" from a "pellet." The "pellet" was resuspended in 1.0 ml of buffer, and the content of hPRL was then determined for both of the fractions. The protein content was estimated with aliquots of the "supernatant" and "pellet" fractions by the method of Lowry and associates 9 ; the fmaltotal protein was the addition of these two determinations. The tissue DNA content was analyzed by Burton's method as modified by Tedesco and Mellman 10 on the recombined "supernatant" and "pellet." fractions. Previous experiments in our laboratory demonstrated a recovery rate of standard DNA between 95% and 99% by this technique. Tissue hPRL, protein, and DNA content; were analyzed and compared to the weight of the tissue sample removed. The total weight of the adenoma was not available because of the need for histologic examination, as well as the fragmentary nature of the removal of the adenoma. Electron microscopy. At the time of operation, portions of the removed adenoma were immediately fixed in 2.5% glutaraldehvde in Millonig's phosphate buffer,
648
Archer et al. :\111.
I
Jul-. 1.-,, 1\ IH\ 1 ()IJ>rt• \ . (;\llt'IOI
Fig. 1. Prolactin-secreting adenoma formed by largely degranulated cells with abundant cvwplasm rich in organelles. Note scant granules of variable size in Golgi regions and the periphery of the n:~lls. (X 5,860.)
pH 7 .35. The tissue to be examined under the electron microscope was then further processed by standard procedures. Results
Large amounts of hPRL were present in the tumor tissue of all 10 patients. Extraction was performed at pH 10 in order to remove hPRL bound to the cell membrane. 11 With the use of this technique, more than 92% of the total tissue hPRL was present in the "supernatant" fraction, compared to that present in the ''pellet" (Table 1). The total hPRL present in the "supernatant" ranged from 7.95 to 96.0 Mg. When calculated as micrograms of hPRL per milligram of tissue, a wide range of values was observed (Table 1). The surgical technique was such that total weight and size of the adenomatous tissue removed could not be ascertained; therefore, a total hPRL content of the adenoma could not be determined. There was no correlation between the preoperative serum level of hPRL and the tissue concentrations of hPRL. A positive correlation was found between the preoperative and the postoperative levels of serum hPRL (r = 0.9345) (Table I). Protein and DNA contents of the tumor were also found to vary widely when expressed per milligram of
tissue (Table 11). Despite this, a po~itive correlation was found between total DNA contem to total tissue ·weight (r = 0.8004, p < 0.0 I) and total protein to total tissue weight (r = 0.7457, p < 0.02). Converseh·. tissue hPRL was not found to correlate with DNA, tissue protein concentration, or tissue weight. The fine structure of the prolanin-producing adenoma cells revealed highly degranulated cytoplasm but with high secretory activity, as evidenced by welldeveloped, rough endoplasmic reticulum cisternae either dilated or arranged in parallel, prominent Golgi complexes, free ribosomes, and numerous mitochondria (Figs. l to 3). The scant secretory granules varied in size from 45 to 750 nanometers, had rounded or irregular outlines, and were located chieHy in the (~olgi region (Fig. 2) at the periphery of the cells dose to the plasma membrane (Figs. l. 3, and 4) and also in inter~ cellular spaces (Figs. :3 and 4). Exocytosis of secretory granules occurred not only at the capillary side of the secretory cells, but also in an ectopic manner at the cell borders adjacent to neighboring cells (Figs. 3 and 4). With minor variability from case to case . all the adenomas showed ultrastructural evidence of high secretory activity but small numbers of secretory granules within and without the cytoplasm.
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Fig. 2. Portion of lactotroph from an adenoma with secretory granules of variable size and shape being "packed" within vesicles of the Golgi complex. Note vesicles and cisternae mntaining fin e particulate material without granule formation. ( x 44,000.)
Comment The tissue samples were found to contain large concentrations of prolactin, but there was no correlation with the serum levels of hPRL (Table I). It appears that the content of hPRL per milligram of tissue in these adenomas hv this extraction technique is significantly increased over the hPRL content reported in normal pituitary glands (44 to 220 J..tg of hPRL per total pituitary gland). 11 Our calculations, estimating a pituitary weight of approximately 600 mg, determine a maximal normal concentration of pituitary hPRL of 0.37 J..tg/mg. There was one exception to rhis, Patient E. S.. in whom the hPRL content was 0.19 J..tg/mg (Table I). ll would appeat that 1he increased tissue hPRL content should reHect an increased number of cells present in the adenoma; however, we were unable to find any correlation between tissue hPRL and either total protein or DNA content. Since the total protein and DNA concentrations were correlated with the tissue w'eight, it appears that tissue concentrations of hPRL represent accumulation of the hPRL within the adenoma . The exact reason for this latter finding is unclear at the presenL time . A possible explanation for this Jack of correlation is the increased exocytosis of secretoq· granules around the periphe ry of the cell margins
(Figs. :{and 4). This phenomenon has been described as misplaced exucytosis and has been ob~tTved in pituitary adenomas. 12 Since this appears to have on:urred in om cases, the increased quantity of. tissu e hPRL that was present may have been mainh tc'xtracellular and not readily removed by the intrapituit~ll} capillary plexus. This phenomenon could also explain the lack of correlation between preoperative se1um values of hPRL and the tissue levels of hPRL (I abk l ). The small number of secretory granules, both imr
650
Archer et al.
juh 15, 191\(1 Am . J. Ohstt-t . Gvn e rul.
Fig. 3. Portions of lactotroph from hPRL-secreting adenoma illustrating rich vesiculated cytoplasm with secretory granules of variable size and shape located 'in the hyperplastic Golgi regions (G), at the periphery of the cells close to the plasma membrane, and in the intercellular spaces (arrows). {X7,150.)
ergocryptine. "1 These data indicate that the cell membrane receptors for stimulation and inhibition are present even in adenomatous cells. Only one report of a human pituitary adenoma maintained in tissue culture could be found, but in this study somatomedin did decrease the release of hPRL in two of three cases studied. 14 Therefore, the failure of acutely administered L-dopa or bromoergocryptine to completely suppress peripheral levels of hPRL in individuals with hPRL-secreting adenomas may be due to the gradual absorbance of the intercellular hPRL. Chronic administration of bromoergocryptine to individuals with pituitary adenoma does result in normalization of the peripheral serum levels of hPRL within several days. 15 The abnormal responses to TRH, therefore, could be due to an increased secretion of hormone into the intercellular space, with a resultant delay in reaching the peripheral circulation. Another possibility is that, with maximum secretion, there is no readily releasable intracellular pool of hPRL to respond to stimulation. The neurosurgical removal of the microadenoma included in-st-illation of alcohol into the resultant defect in the pituitary gland. Despite this, a surgical "cure," i.e ., the restoration of normal serum levels of hPRL
postoperatively, was obtained in only four patients. The reasons for this poor success rate are not fully understood at the present time. To date, no evidence exists for multiple microadenomas of the pituitary, although hyperplasia of pituitary lactotrophs has been described in patients with chronic diseases who had elevated serum levels of hPRL, 16 and a recent report has demonstrated lactotrophic hyperplasia in the normal pituitary tissue surrounding the pituitary microadenoma. 17 The positive correlation found between preoperative and postoperative serum levels of hPRL confirms other reports of high levels of hPRL associated with a decreased incidence of normalization of the postoperative hPRL.ts. t9 Whether hPRL-secreting adenomas are due to a lack of hypothalamic regulation is moot at present. Preliminary evidence suggests that a hypothalamic deficienc.;y of dopamine or the enzymes necessary for normal catecholamine metabolism does exist in individuals with hPRL-secreting adenomas. 20 It is conceivable that prolonged lack of suppression could result in !actotrophic hyperplasia and/or adenoma formation. This could serve to explain the poor results in terms of sur-
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651
Fig. 4. Momage of three examples of "misplaced" exocytosis from hPRL-secreting adenomas. A. Small secretory granules lined up along plasma membrane. Note intercellular space (is). ( x 4 7,000.) 8, Large secretory granule in the process of being released into the intercellular space. Note rich parallel cisternae of rough endoplasmic reticulum and other secretory granules. ( X27,580.) C, L<~rg;c secretory granules of various sizes and shapes. One has been released into the intercellular space (X 105,500.)
gical "cure," arguing for long-term medical therapy with dopamine agonists. Our findings of high tissue levels of hPRL and active secretion documented by electron microscopy indicate that hPRL-secreting pituitary adenomas have marked hormonal activity. The inability to correlate peripheral levels of hPRL with the presence or absence of pituitary microadenoma by roentgenographic means is possibly due to the phenomenon of misplaced exocytosis in these adenomas or their inability to form and release
secretory granules in a normal fashion . ·1 hese findings may also explain the differences in h PRL response to the acute administration of TRH or dopamine agonists. We wish to express our appreciation to 1\-lr. Charles Kritko for his assistance with the prolactin assays, Mr. Robert Florida for assistance with the electron microscopy, and to Ms. Kathy Caprara for her diligence in the preparation of the manuscript.
REFERENCES l. Hwang, P. , Guyda, H. , and Friesen, H.: A radioimmunoassay for human prolactin, Proc. Nat!. Acad . Sci. U.S.A. 68: 1902' 1971. 2. Vezina, J. L., and Sutton, T. J.: Prolactin-secreting pituitary microadenomas: Roentgenographic diagnosis, Am. J. Roentgenol. Radium Ther. Nucl. Med. 120:46, 1974. 3. Snyder, P. J., jacobs, L. S., Rabello, M. M., Sterling, F. H. , Shore, R.N ., Utiger, R. D., and Daughaday, W. H .: Diagnostic value of thyrotropin-releasing hormone in pituitary and hypothalamic disease, Ann . Intern. Med . 81:751. 1974. 4. Archer, D. F., Nankin, H . R .. Gabos, P. F. , Maroon, J..
Nosetz, S., Wadhwa, S. R., and Josimovich. J. B.: Serum prolactin in patients with inappropriate lactation, AM. J. 0BSTE'r. GYNECOL. 119:466, 1974. 5. Lowden, E. A.., Ratcliffe,]. G., Thomson, J. A., MacPherson, P., Doyle, D., and Teasdale, G. M. : Tests of prolactin secretin in diagnosis of prolactinomas, Lancet I: 1155. 1979. 6. Tolis, G., Somma, M., VanCampenhout, ]., and Friesen , H .: Prolactin secretion in sixty-five patients with galaciOrrhea, AM. J. 0BSTET. GYNECOL. 118:91 , 1974. 7. Guitelman, A., Aparicio, N.J., Mancini, A.M., and Debeljuk, L.: Serum levels of prolactin in basal conditions and
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Archer et al. An>.
8. 9. 10.
II.
12. 13.
14.
after administration ot 2-bromo-5a-ergocryptme in normal and galactorrheic subjects, Fertil. Steril. 29:26, 1978. Child, D. F., Nader, S.. Mashiter, K., Kjeld, M., Banks, L., and Russell-11raser, T.: Prolactin studies in "functionless" pituitary tumors, Br. Med. J. 1:604, 1975. Lowry, 0. H., Rosebrough, N.J., Farr, A. L., and Randall, R. J .: Protein measurement with the folin phenol reagent,]. Bioi. Chern. 193:265, 1951. Tedesco, T. A., and Mellman, W. ].: Desoxyribonucleic acid assay as a measure of cell number in preparations from monolayer cell cultures and blood leukocytes, Exp. Cell. Res. 45:230. 1967. Zimmerman, E. A., Defendini, R., and Frantz, A. G.: Prolactin and growth hormone in patients with pituitary adenomas: A correlative study of hormone in tumor and plasma by immunoperoxidase technique and radioimmunoassay,]. Clin. Endocrinol. Metab. 38:577, 1974. Horvath, E., and Kovacs, K.: Misplaced exocytosis, distinct ultrastructural feature in some pituitary adenomas, Arch. Pathol. Lab. Med. 97:221, 1974. Hill-Samli, M., and MacLeod. R. M.: Interaction of thyrotropin-releasing hormone and dopamine on the release of prolactin from the rat anterior pituitary in vitro, Endocrinology 95:1189, 1974. Peillon, F., Cesselin, F., Garnier, P. E., Brandi, A. M.,
15. 16.
17.
18. 19.
20.
J.
.Jull 1.-•. l~t\IJ Ob"ct. (;,,,.,.,1
Donnadieu, M., L'Hermite, M., and DuBois. M. P.: Ptolactin secretion and synthesis in short- and long-term organ culture of pituitary tumors from acromegalic p<~ tients, Acta Endocrinol. (Copenh) 87:701, 197R. Archer, D. F.: Unpublished observations. McKeel, D. W., Jr., and Jacobs, L S.: Non-adenomatom pituitary mammotroph hyperplasia in patients with pathologic hyperprolactinemia. Abstract No. 136, Presented at the 59th Annual Meeting of the Endocrine Society. June 8, 1977, Chicago, Illinois. Landolt, A. M., and Yasargil, M. C.: Possible causes of failure in prolactinoma surgery. Paper No. 26, Presented at the Annual Meeting of the American Association oi Neurological Surgeons, April 22, I 979. Los Angeles. California. Hardy,].: The transsphenoidal surgical approach to the pituitary, Hosp. Pract. 14:81. 1979. Keye. W. R., Jr., Chang, R. J., Monroe, S. E., Wilson, C. B., and Jaffe, R. B.: Prolactin-secreting pituitary adenomas in women, AM. ]. OasTET. CYNECOL. 134:360, 1979. Fine, S. A., and Frohman, L. A.: Loss of central nervous system component of dopaminergic inhibition of prolactin secretion in patients with prolactin-secreting pituitary tumors,]. Clin. Invest. 61:973, 1978.
pituitary adenomas: Serum and tissue prolactin levels with ultrastructural correlation DAVID F. ARCHER, M.D. HERNANDO SALAZAR, M.D. JOSEPH C. MAROON, M.D. LINDA J. HOUGH, B.S.
Pittsburgh, Pennsylvania Adenomatous tissue was obtained from 10 women with evidence of prolactin-secreting pituitary adenoma at the time of transsphenoidal removal of the tumor. Tissue levels of prolactin (hPRL) were estimated after extraction and were compared to preoperative peripheral serum levels of hPRL No correlation was found between the tissue and serum concentrations of hPRL, although high levels of hPRL were found in the tissue extracts. Electron microscopy confirmed active secretion of the individual cells, and the ultrastructural findings were compatible with prolactin-secreting adenomas. Associated with the active secretion was extensive exocytosis of the secretory granules away from the capillary waiL These findings suggest that, although active secretion of hPRL is occurring, large amounts of secreted hPRL are not available lor immediate uptake into the capillary plexus; thus a slow absorption of hPRL could be taking place from the adenomatous tissue. These data could explain the failure of provocative tests of hPRL stimulation and inhibition to unequivocally characterize an adenoma in an individal with elevated serum levels of hPRL. (AM. J. 0BSTET. GYNECOL. 137:646, 1980.)
PROLACTIN-SECRETING pituitary adenomas are no longer considered to be uncommon entities. Diagnosis has been facilitated by the development of a specific radioimmunoassay for prolactin (hPRL) and the availability of hypocycloidal polytomography .1• 2 Diagnostic studies involving stimulation and suppression of secretion of hPRL have not yielded any consistent response pattern that would unequivocally discriminate between those individuals with x-ray evidence of pituitary microadenoma or macroadenoma and those without obvious roentgenographic evidence of sellar or pituitary changes. Stimulation of hPRL secretion by thyrotropin-releasing hormone (TRH) in individuals with pituitary adenoma has, in some instances, resulted in a blunted hPRL response, as
From the Departments of Obstetrics and Gynecology, Pathology, and Neurosurgery, University of Pittsburgh School of Medicine. Received for publication August 6, 1979. Revised November 16, 1979. Accepted November 30, 1979. Reprint requests: David F. Archer, M.D., Depanment of Obstetrics and Gynecology, University of Pittsburgh School of Medicine, Magee-Women's Hospital, Forbes Ave. and Halket St., Pittsburgh, Pennsylvania 15213.
646
compared to that in controls. Other studies have documented a delayed but exaggerated response of hPRL to this stimulus.H Inhibition of hPRL after the administration of either L-dopa or bromergocryptine to women with hPRL-secreting adenomas has been variable and comparable to the responses of women with hyperprolactinemia without roentgenographic evidence of pituitary adenoma. 6• 7 A correlation between the level of serum hPRL and the volume of the sella turcica has been reported. However, there does not appear to be any correlation between absolute levels of serum hPRL and the pres• ence or absence of pituitary adenoma. 8 This study was designed to compare preoperative serum levels of hPRL to the concentration of hPRL in the adenomatous tissue, as well as to assess ultrastructural evidence for the secretory activity of the individual cells. The rationale was that the information obtained might provide a reason for the lack of correlation between serum levels of hPRL, dynamic hPRL test results, and roentgenographic presence of a pituitary adenoma. Material and methods Patient material. Ten women with elevated serum levels of hPRL and documented evidence of pituitary 0002-93781801140646+07$00.7010 © 1980 The C. V. Mosby Co.
hPAL-secreting pituitary adenomas
Volume l'l7 Number ti
647
Table I. Tissue weight and prolactin content in patients with prolactin-secreting pituitary adenoma Total prolactin (JAg) Patient
Tissue weight (mg)
Supernatant
D. 1\f. L. G.
·12.5 27.4 15.6 57.4 58.2 17.4 77.8 57.1 41.3 38.1
51.20 42.00 25.05 36.90 56.40 96.00 56.85 57.30 7.95 24.90
J.
F.
P. S. S. J. E. H. P.M. N. P. E. S. S. S.
I
Serum hPRL (ng/ml)
Pellet
JAg hPRL/mg tissue
1.63 0.69 1.85 2.20 4.63 1.65 3.93 1.67 0.08 0.49
1.24 1.56 1.72 0.68 1.05 5.61 0.78 1.01 0.19 0.67
Preop
I
Postop
190
5 67 63 890 25 235
HO 340 2,250
JIO 480 600 420 164 225
:wo 14 51 12
Table II. Tissue protein and DNA content of prolactin-secreting pituitary adenoma Patient
Protein* ( JAglmg tissue)
DNA t ( JAglmg tissue)
hPRLiproteint (JAg I p.g Img tissue)
D.M. L. G. J. F. P. S.
47.43 79.83 99.55 38.72 77.85 72.13 49.83 72.21 46.37 20.21
0.082 0.080 0.160 0.!31 0.112 0.072 0.206 0.060 0.034 0.021
0.026 0.019 0.017 0.017 0.013 0.077 0.016 0.014 0.004 0.033
s. J.
E. H. P.M. N. P. E. S. S. S.
hPRLIDNAt ( p.gl p.glmg tissue)
Pi.16 19.48 .10.78
5.20 9.36 79.94 3.79 I fi. 78 fJ.7[
:11.74
*p < 0.02. tp
microadenoma by hypocycloidal polytomography were investigated. All patients underwent transsphenoidal partial hypophysectomy. Postoperative levels of hPRL were determined in all patients between 1 and 7 days after removal of the adenoma and confirmed by repetitive sampling 2 months or more postoperatively. All postoperative hPRL values are the means of two or more samples obtained randomly on each patient between 7 days and 3 months after operation. Serum and tissue analysis. Peripheral levels of hPRL were determined by a double antibody radioimmunoassay as previously reported. 5* Tissue levels of hPRL were assayed by the same radioimmunoassay system after extraction by the following technique: A sample of the adenomatous tissue removed at the time of operation was immediately placed on ice in a O.lM sodium carbonate buffer, pH 10.0, and transported to the laboratory. The tissue was weighed on a Roller Smith milligram balance after blotting to remove excess fluid, replaced in the buffer, and homogenized with a *The hPRL standard was obtained from the National Institute of Arthritis, Metabolism, and Digestive Diseases, and the hPRL antiserum was a gift from Dr. Henry Friesen.
sonifier cell disruptor for 30 seconds at 50 watts. The homogenate was centrifuged at 1,088 x g for 10 minutes to separate a "supernatant" from a "pellet." The "pellet" was resuspended in 1.0 ml of buffer, and the content of hPRL was then determined for both of the fractions. The protein content was estimated with aliquots of the "supernatant" and "pellet" fractions by the method of Lowry and associates 9 ; the fmaltotal protein was the addition of these two determinations. The tissue DNA content was analyzed by Burton's method as modified by Tedesco and Mellman 10 on the recombined "supernatant" and "pellet." fractions. Previous experiments in our laboratory demonstrated a recovery rate of standard DNA between 95% and 99% by this technique. Tissue hPRL, protein, and DNA content; were analyzed and compared to the weight of the tissue sample removed. The total weight of the adenoma was not available because of the need for histologic examination, as well as the fragmentary nature of the removal of the adenoma. Electron microscopy. At the time of operation, portions of the removed adenoma were immediately fixed in 2.5% glutaraldehvde in Millonig's phosphate buffer,
648
Archer et al. :\111.
I
Jul-. 1.-,, 1\ IH\ 1 ()IJ>rt• \ . (;\llt'IOI
Fig. 1. Prolactin-secreting adenoma formed by largely degranulated cells with abundant cvwplasm rich in organelles. Note scant granules of variable size in Golgi regions and the periphery of the n:~lls. (X 5,860.)
pH 7 .35. The tissue to be examined under the electron microscope was then further processed by standard procedures. Results
Large amounts of hPRL were present in the tumor tissue of all 10 patients. Extraction was performed at pH 10 in order to remove hPRL bound to the cell membrane. 11 With the use of this technique, more than 92% of the total tissue hPRL was present in the "supernatant" fraction, compared to that present in the ''pellet" (Table 1). The total hPRL present in the "supernatant" ranged from 7.95 to 96.0 Mg. When calculated as micrograms of hPRL per milligram of tissue, a wide range of values was observed (Table 1). The surgical technique was such that total weight and size of the adenomatous tissue removed could not be ascertained; therefore, a total hPRL content of the adenoma could not be determined. There was no correlation between the preoperative serum level of hPRL and the tissue concentrations of hPRL. A positive correlation was found between the preoperative and the postoperative levels of serum hPRL (r = 0.9345) (Table I). Protein and DNA contents of the tumor were also found to vary widely when expressed per milligram of
tissue (Table 11). Despite this, a po~itive correlation was found between total DNA contem to total tissue ·weight (r = 0.8004, p < 0.0 I) and total protein to total tissue weight (r = 0.7457, p < 0.02). Converseh·. tissue hPRL was not found to correlate with DNA, tissue protein concentration, or tissue weight. The fine structure of the prolanin-producing adenoma cells revealed highly degranulated cytoplasm but with high secretory activity, as evidenced by welldeveloped, rough endoplasmic reticulum cisternae either dilated or arranged in parallel, prominent Golgi complexes, free ribosomes, and numerous mitochondria (Figs. l to 3). The scant secretory granules varied in size from 45 to 750 nanometers, had rounded or irregular outlines, and were located chieHy in the (~olgi region (Fig. 2) at the periphery of the cells dose to the plasma membrane (Figs. l. 3, and 4) and also in inter~ cellular spaces (Figs. :3 and 4). Exocytosis of secretory granules occurred not only at the capillary side of the secretory cells, but also in an ectopic manner at the cell borders adjacent to neighboring cells (Figs. 3 and 4). With minor variability from case to case . all the adenomas showed ultrastructural evidence of high secretory activity but small numbers of secretory granules within and without the cytoplasm.
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Fig. 2. Portion of lactotroph from an adenoma with secretory granules of variable size and shape being "packed" within vesicles of the Golgi complex. Note vesicles and cisternae mntaining fin e particulate material without granule formation. ( x 44,000.)
Comment The tissue samples were found to contain large concentrations of prolactin, but there was no correlation with the serum levels of hPRL (Table I). It appears that the content of hPRL per milligram of tissue in these adenomas hv this extraction technique is significantly increased over the hPRL content reported in normal pituitary glands (44 to 220 J..tg of hPRL per total pituitary gland). 11 Our calculations, estimating a pituitary weight of approximately 600 mg, determine a maximal normal concentration of pituitary hPRL of 0.37 J..tg/mg. There was one exception to rhis, Patient E. S.. in whom the hPRL content was 0.19 J..tg/mg (Table I). ll would appeat that 1he increased tissue hPRL content should reHect an increased number of cells present in the adenoma; however, we were unable to find any correlation between tissue hPRL and either total protein or DNA content. Since the total protein and DNA concentrations were correlated with the tissue w'eight, it appears that tissue concentrations of hPRL represent accumulation of the hPRL within the adenoma . The exact reason for this latter finding is unclear at the presenL time . A possible explanation for this Jack of correlation is the increased exocytosis of secretoq· granules around the periphe ry of the cell margins
(Figs. :{and 4). This phenomenon has been described as misplaced exucytosis and has been ob~tTved in pituitary adenomas. 12 Since this appears to have on:urred in om cases, the increased quantity of. tissu e hPRL that was present may have been mainh tc'xtracellular and not readily removed by the intrapituit~ll} capillary plexus. This phenomenon could also explain the lack of correlation between preoperative se1um values of hPRL and the tissue levels of hPRL (I abk l ). The small number of secretory granules, both imr
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juh 15, 191\(1 Am . J. Ohstt-t . Gvn e rul.
Fig. 3. Portions of lactotroph from hPRL-secreting adenoma illustrating rich vesiculated cytoplasm with secretory granules of variable size and shape located 'in the hyperplastic Golgi regions (G), at the periphery of the cells close to the plasma membrane, and in the intercellular spaces (arrows). {X7,150.)
ergocryptine. "1 These data indicate that the cell membrane receptors for stimulation and inhibition are present even in adenomatous cells. Only one report of a human pituitary adenoma maintained in tissue culture could be found, but in this study somatomedin did decrease the release of hPRL in two of three cases studied. 14 Therefore, the failure of acutely administered L-dopa or bromoergocryptine to completely suppress peripheral levels of hPRL in individuals with hPRL-secreting adenomas may be due to the gradual absorbance of the intercellular hPRL. Chronic administration of bromoergocryptine to individuals with pituitary adenoma does result in normalization of the peripheral serum levels of hPRL within several days. 15 The abnormal responses to TRH, therefore, could be due to an increased secretion of hormone into the intercellular space, with a resultant delay in reaching the peripheral circulation. Another possibility is that, with maximum secretion, there is no readily releasable intracellular pool of hPRL to respond to stimulation. The neurosurgical removal of the microadenoma included in-st-illation of alcohol into the resultant defect in the pituitary gland. Despite this, a surgical "cure," i.e ., the restoration of normal serum levels of hPRL
postoperatively, was obtained in only four patients. The reasons for this poor success rate are not fully understood at the present time. To date, no evidence exists for multiple microadenomas of the pituitary, although hyperplasia of pituitary lactotrophs has been described in patients with chronic diseases who had elevated serum levels of hPRL, 16 and a recent report has demonstrated lactotrophic hyperplasia in the normal pituitary tissue surrounding the pituitary microadenoma. 17 The positive correlation found between preoperative and postoperative serum levels of hPRL confirms other reports of high levels of hPRL associated with a decreased incidence of normalization of the postoperative hPRL.ts. t9 Whether hPRL-secreting adenomas are due to a lack of hypothalamic regulation is moot at present. Preliminary evidence suggests that a hypothalamic deficienc.;y of dopamine or the enzymes necessary for normal catecholamine metabolism does exist in individuals with hPRL-secreting adenomas. 20 It is conceivable that prolonged lack of suppression could result in !actotrophic hyperplasia and/or adenoma formation. This could serve to explain the poor results in terms of sur-
hPAL-secreting pituitary adenomas
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Fig. 4. Momage of three examples of "misplaced" exocytosis from hPRL-secreting adenomas. A. Small secretory granules lined up along plasma membrane. Note intercellular space (is). ( x 4 7,000.) 8, Large secretory granule in the process of being released into the intercellular space. Note rich parallel cisternae of rough endoplasmic reticulum and other secretory granules. ( X27,580.) C, L<~rg;c secretory granules of various sizes and shapes. One has been released into the intercellular space (X 105,500.)
gical "cure," arguing for long-term medical therapy with dopamine agonists. Our findings of high tissue levels of hPRL and active secretion documented by electron microscopy indicate that hPRL-secreting pituitary adenomas have marked hormonal activity. The inability to correlate peripheral levels of hPRL with the presence or absence of pituitary microadenoma by roentgenographic means is possibly due to the phenomenon of misplaced exocytosis in these adenomas or their inability to form and release
secretory granules in a normal fashion . ·1 hese findings may also explain the differences in h PRL response to the acute administration of TRH or dopamine agonists. We wish to express our appreciation to 1\-lr. Charles Kritko for his assistance with the prolactin assays, Mr. Robert Florida for assistance with the electron microscopy, and to Ms. Kathy Caprara for her diligence in the preparation of the manuscript.
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Nosetz, S., Wadhwa, S. R., and Josimovich. J. B.: Serum prolactin in patients with inappropriate lactation, AM. J. 0BSTE'r. GYNECOL. 119:466, 1974. 5. Lowden, E. A.., Ratcliffe,]. G., Thomson, J. A., MacPherson, P., Doyle, D., and Teasdale, G. M. : Tests of prolactin secretin in diagnosis of prolactinomas, Lancet I: 1155. 1979. 6. Tolis, G., Somma, M., VanCampenhout, ]., and Friesen , H .: Prolactin secretion in sixty-five patients with galaciOrrhea, AM. J. 0BSTET. GYNECOL. 118:91 , 1974. 7. Guitelman, A., Aparicio, N.J., Mancini, A.M., and Debeljuk, L.: Serum levels of prolactin in basal conditions and
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after administration ot 2-bromo-5a-ergocryptme in normal and galactorrheic subjects, Fertil. Steril. 29:26, 1978. Child, D. F., Nader, S.. Mashiter, K., Kjeld, M., Banks, L., and Russell-11raser, T.: Prolactin studies in "functionless" pituitary tumors, Br. Med. J. 1:604, 1975. Lowry, 0. H., Rosebrough, N.J., Farr, A. L., and Randall, R. J .: Protein measurement with the folin phenol reagent,]. Bioi. Chern. 193:265, 1951. Tedesco, T. A., and Mellman, W. ].: Desoxyribonucleic acid assay as a measure of cell number in preparations from monolayer cell cultures and blood leukocytes, Exp. Cell. Res. 45:230. 1967. Zimmerman, E. A., Defendini, R., and Frantz, A. G.: Prolactin and growth hormone in patients with pituitary adenomas: A correlative study of hormone in tumor and plasma by immunoperoxidase technique and radioimmunoassay,]. Clin. Endocrinol. Metab. 38:577, 1974. Horvath, E., and Kovacs, K.: Misplaced exocytosis, distinct ultrastructural feature in some pituitary adenomas, Arch. Pathol. Lab. Med. 97:221, 1974. Hill-Samli, M., and MacLeod. R. M.: Interaction of thyrotropin-releasing hormone and dopamine on the release of prolactin from the rat anterior pituitary in vitro, Endocrinology 95:1189, 1974. Peillon, F., Cesselin, F., Garnier, P. E., Brandi, A. M.,
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Donnadieu, M., L'Hermite, M., and DuBois. M. P.: Ptolactin secretion and synthesis in short- and long-term organ culture of pituitary tumors from acromegalic p<~ tients, Acta Endocrinol. (Copenh) 87:701, 197R. Archer, D. F.: Unpublished observations. McKeel, D. W., Jr., and Jacobs, L S.: Non-adenomatom pituitary mammotroph hyperplasia in patients with pathologic hyperprolactinemia. Abstract No. 136, Presented at the 59th Annual Meeting of the Endocrine Society. June 8, 1977, Chicago, Illinois. Landolt, A. M., and Yasargil, M. C.: Possible causes of failure in prolactinoma surgery. Paper No. 26, Presented at the Annual Meeting of the American Association oi Neurological Surgeons, April 22, I 979. Los Angeles. California. Hardy,].: The transsphenoidal surgical approach to the pituitary, Hosp. Pract. 14:81. 1979. Keye. W. R., Jr., Chang, R. J., Monroe, S. E., Wilson, C. B., and Jaffe, R. B.: Prolactin-secreting pituitary adenomas in women, AM. ]. OasTET. CYNECOL. 134:360, 1979. Fine, S. A., and Frohman, L. A.: Loss of central nervous system component of dopaminergic inhibition of prolactin secretion in patients with prolactin-secreting pituitary tumors,]. Clin. Invest. 61:973, 1978.