1 Service dEndocrinologie and 2 Service de Neurochirurgie, Hôpital Bellevue, 42055 Saint-Etienne Cedex 2, France, 3 Laboratoire dHistologie Embryologie moléculaires and INSERM U433, Faculté de Médecine LyonRTH Laennec, 69372 Lyon Cedex 08, France and 4 Service de Radiopharmacie et de Radioanalyses, Hôpital Neurologique et Cardiologique, 69394 Lyon Cedex 03, France
5 To whom correspondence should be addressed. e-mail: lhem{at}laennec.univ-lyon1.fr
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Abstract |
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Key words: hyperprolactinaemia/macroprolactinaemia/pituitary tumours/prolactinomas
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Introduction |
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In the 1980s, a new type of hyperprolactinaemia, termed macroprolactinaemia, was identified (Whittaker et al., 1981; Andersen et al., 1982
; Andino et al., 1985
; Jackson et al., 1985
; Larrea et al., 1985
; Corenblum, 1990
; Carlson et al., 1992
; Hattori et al., 1992a
) and found to occur in 825% of patients with hyperprolactinaemia (Hattori et al., 1992a
; Bjoro et al., 1995
; Hattori, 1996
; Olukoga and Kane, 1999
; Leslie et al., 2001
; Valette-Kasic et al., 2002
). This entity was defined by the bbPRL isoform being the only, or the predominant, form, and was claimed to be poorly symptomatic and idiopathic. Although the nature of these large forms is still under debate (reviewed in Fraser and Lun, 1990
), a tumoral origin has been suggested (Rogol and Rosen, 1974
; Ohnami et al., 1987
).
The identification of a prolactinoma in three hyperprolactinaemic patients with predominantly the bbPRL isoform prompted us to study these large forms in patients with tumoral hyperprolactinaemia and to evaluate the clinical, biological and histological characteristics of these patients.
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Subjects and methods |
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Six women presented with amenorrhoea associated with galactorrhoea; three were taking a combined estrogenprogestogen contraceptive pill and the symptoms persisted despite treatment being halted. Of the remaining five women, three presented with amenorrhoea that had developed over 6 months after 2 years of spaniomenorrhoea, while the other two presented with galactorrhoea alone. None of the 11 women were on antidepressive drugs. There were no clinical, hormonal, and/or ultrasonographic features of polycystic ovarian syndrome and none had hypothyroidism. All had normal body weights (body mass index >20 kg/m2).
One man presented with gynaecomastia and the other with infertility associated with oligoasthenozoospermia on the spermogram.
All patients were treated with bromocriptine; because of side-effects, eight (six women and two men) underwent transphenoidal surgery.
Prolactin studies
Prolactin assay
Blood samples, collected between 10:00 and 12:00, were centrifuged and the plasma stored at 20°C until assayed. Plasma PRL concentrations were measured by radioimmunoassay using a rabbit anti-human PRL antibody prepared in our laboratory (Claustrat et al., 1994). The results were expressed as µg/l by comparison with standard MRC 75/504.
Chromatography
Plasma PRL heterogeneity was studied by gel filtration chromatography. Plasma samples (<2 ml, depending on the PRL concentration) were applied to a Sephadex G100 column (120 cmx2 cm) and eluted using 0.05 mol/l phosphate buffer, pH 7.5, containing 0.1% bovine serum albumin (BSA). The column was calibrated with Blue dextran, free 125I, and [125I]rat PRL (rat PRL does not react with the antihuman PRL antiserum). Thirty fractions were collected, their PRL concentrations determined by radioimmunoassay, and the percentage of each form calculated.
Detection of autoantibody-bound PRL
This was performed as described by Hattori et al. (1992b). Briefly, plasma samples (100 µl) and [125I]PRL (100 µl, 10 000 cpm) diluted in phosphate-buffered saline (0.05 mol/l, pH 7.4) containing 0.5% BSA were incubated overnight at 4°C, then 200 µl of 25% (w/w) polyethylene glycol (PEG 6000; Merck, France) was added, the mixture centrifuged, and the radioactivity in the precipitate measured in a
-counter. Non-specific binding was determined using 20 plasma samples from different normal subjects as controls in each series. A plasma was considered positive when the amount of radioactivity precipitated differed significantly from that precipitated by the controls, using Students t-test (confidence interval of 95%).
Tumour studies
Eight PRL adenomas were studied by light microscopy and immunocytochemistry. For light microscopy, pieces of tumour tissue were immersed in BouinHollande fixative for 4 days, then embedded in paraffin. Sections (5 µm thick) were prepared and stained using the Herlants tetrachrome and periodic acidSchifforange G methods. Amyloïd deposits were detected using Congo red.
The tumour type was identified immunocytochemically. Serial sections were processed by the indirect immunoperoxidase method using a streptavidinbiotin complex (Dako A/S, Denmark), as previously described (Trouillas and Girod, 1996). Mouse monoclonal antibodies against human proteins (indicated by h), obtained from Immunotech, Marseille, France [anti-hPRL (164-22-12), anti-
hFSH (300-10-E-14-3), and anti-
-subunit (326-2-1; lot f 1079)] and from Dako A/S, Copenhagen, Denmark (anti-
hTSH) and rabbit polyclonal antibodies (anti-hGH and anti-
hLH; both kindly donated by Dr A.F.Parlow, National Institute of Arthritis, Diabetes, Digestive and Kidney Disease, Bethesda MD, USA) were used at dilutions of 1/200 to 1/10 000.
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Results |
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PRL chromatographic profiles
In eight patients (seven women and one man), the predominant isoform was mPRL (Figure 1A), while in five (four women and one man), the bbPRL isoform (Figure 1B) predominated. No evidence for anti-PRL autoantibodies was found in the large PRL group.
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Total PRL levels were high in both the monomeric and large PRL groups (100 µg/l). mPRL levels were high in all patients, except for one case in the large PRL group, a woman with a galactorrhoea without menstrual disturbances and with a low mPRL level (2 µg/l).
Histological features of prolactinomas
Histology and immunocytochemistry confirmed the presence of a PRL adenoma in all eight patients tested (four women and one man from the mPRL group and two women and one man from the large PRL group).
In all five patients with predominantly the mPRL isoform (nos. 8, 9, 10, 11 and 12) and in the male patient with predominantly the large PRL isoform (no. 2), the pituitary tumour exhibited the typical features of a sparsely granulated prolactinoma with a solid architectural pattern and numerous vessels. On immunocytochemical testing, all the cells were strongly stained with anti-hPRL antibodies. Bound antibody was located near the nucleus in a dot-like pattern, corresponding to the Golgi complex (Figure 2A). In the two women in the large PRL isoform group, a typical prolactinoma with irregular limits surrounded by non-tumoral pituitary with PRL cell hyperplasia was seen in one (no. 4), the large cells, arranged in wide rows, being strongly and diffusely PRL-positive, while, in the other (no. 5), the PRL immunoreactivity was unusual, with irregular aggregates of PRL deposits scattered in the cytoplasm of many cells, giving them a clotted appearance (Figure 2B). In all tumours, all other antibodies tested gave negative results and no amyloïd deposits were observed.
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Discussion |
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We found that the occurrence of a prolactinoma may be associated with the presence of macroprolactinaemia. In this small series, the frequency was high (five out of 13 patients, or 40%). As far as we are aware, no systematic screening for macroprolactinaemia has been performed in patients with prolactinoma, most studies having evaluated the occurrence of prolactinoma in a population with macroprolactinaemia. In the series of 106 patients with macroprolactinaemia studied by Valette-Kasic et al. (2002), four cases of PRL adenoma (4%) were identified and proven by histology. Three adenomas were found by pituitary imaging in another series (Olukoga and Kane, 1999
), but none was proven by histology.
The biological activity of these large forms seems to depend on the experimental model used. A lower biological activity of the large forms has been suggested as the reason for the lack of typical symptoms of hyperprolactinaemia; this is supported by in-vitro studies using the radioreceptor assay (Garnier et al., 1978; Farkouh et al., 1979
) and, in some cases, using the N2b lymphoma model (Jackson et al., 1985
; Larrea et al., 1989
). Other studies using the N2b lymphoma model showed the in-vitro bioactivity of the small and large forms to be similar (Andersen et al., 1982
; Rowe et al., 1983
; Whitaker et al., 1984
); in this case, the lack of symptoms cannot be explained in terms of lower bioactivity of the large forms. It is likely that, because of their high molecular weight, the large forms do not readily cross the capillary walls (Andersen et al., 1982
; Larrea et al., 1985
). All 13 patients reported here presented with clinical symptoms of PRL excess. The high plasma mPRL levels may explain the appearance of symptoms in both the large and monomeric PRL groups, as suggested by other authors (Jeske et al., 2002
). In our study, one patient in the large PRL group did not fit this hypothesis, but the PRL adenoma was not confirmed by histology. The biological characteristics of the PRL adenomas associated with macroprolactinaemia were the same as those in patients with a predominance of the mPRL isoform. Levels of total PRL were high (
100 µg/l), suggesting a tumoral hyperprolactinaemia.
The large forms seem to be heterogeneous in terms of aetiology, and the origin of these large forms remains controversial. For a long time, they were thought to be aggregates of mPRL. More recently, Hattori et al. (1992a, 1994) claimed that bbPRL was an anti-PRL autoantibody, but we did not find any evidence for autoantibodies in our patients with exclusively bbPRL. Two studies have reported an association between a PRL adenoma and macroprolactinaemia (Rogol and Rosen, 1974
; Ohnami et al., 1987
). In our study, this association was confirmed by histology in all three patients tested. The unusual immunocytochemical feature of deposits of PRL aggregates in one of these (patient no. 5) may suggest the tumoral origin of the large forms of PRL. This finding is in agreement with those of Ohnami et al. (1987
), who, on the basis of the similarity of the chromatographic PRL patterns of tumoral extracts and serum samples from eight patients, concluded that these large forms are possibly secreted by the tumour.
Our results suggest that PRL adenoma may be associated with macroprolactinaemia. The clinical and biological characteristics of prolactinomas associated or not associated with macroprolactinaemia are similar. The unusual immunocytochemical features seen in one case may suggest the tumoral origin of these large forms. These results should be confirmed by a study of a larger cohort with tumoral hyperprolactinaemia and a chromatographic PRL study on both tumoral extracts and serum samples.
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Acknowledgements |
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References |
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Andersen, A.N., Pedersen, H., Djursing, H., Andersen, B.N. and Friesen, H.G. (1982) Bioactivity of prolactin in a woman with an excess of large molecular size prolactin, persistent hyperprolactinemia and spontaneous conception. Fertil. Steril., 38, 625628.[ISI][Medline]
Andino, N.A., Bidot, C., Valdés, M. and Machado, A.J. (1985) Chromatographic pattern of circulating prolactin in ovulatory hyperprolactinemia. Fertil. Steril., 44, 600605.[ISI][Medline]
Bjoro, T., Morkrid, L., Wergeland, R., Turter, A., Kvisborg, A., Sand, T. and Torjesen, P. (1995) Frequency of hyperprolactinemia due to large molecular weight prolactin. Scand. J. Clin. Lab. Invest., 55, 139147.[ISI][Medline]
Blacker, C., Feinstein, M.C., Avoine, D. and Gervasi, G. (1994) Hétérogénéité de la prolactine: conséquences cliniques. Gynécologie, 2, 352357.
Carlson, H.E., Markoff, E. and Lee, D.W. (1992) On the nature of serum prolactin in two patients with macroprolactinemia. Fertil. Steril., 58, 7887.[ISI][Medline]
Claustrat, B., Brun, J., Champier, J. and Jolly-Pharaboz, M.O. (1994) Heterogeneity of human prolactin: influence on immunoassays. Nucl. Med. Biol., 21, 331335.[CrossRef][ISI][Medline]
Corenblum, B. (1990) Asymptomatic hyperprolactinemia resulting from macroprolactinemia. Fertil. Steril., 53, 165167.[ISI][Medline]
Fahie-Wilson, M.N. and Soule, S.G. (1997) Macroprolactinemia: contribution to hyperprolactinemia in a district general hospital and evaluation of a screening test based on precipitation with polyethylene glycol. Ann. Clin. Biochem., 34, 252258.[ISI][Medline]
Fahie-Wilson, M.N. (1999) Polyethylene glycol precipitation as a screening method for macroprolactinemia. Clin. Chem., 45, 436437.
Fang, V.S. and Refetoff, S. (1978) Heterogenous human prolactin from giant pituitary tumor in a patient with panhypopituitarism. J. Clin. Endocrinol. Metab., 47, 780787.[Abstract]
Farkouh, N.H., Packer, M.G. and Frantz, A.G. (1979) Large molecular size prolactin with reduced receptor activity in human serum: high proportion in basal state and reduction after thyrotropin releasing hormone. J. Clin. Endocrinol. Metab., 48, 10261032.[ISI][Medline]
Fonseca, M.E., Ochea, R., Moron, C. and Zarate, A. (1991) Variations in the molecular forms of prolactin during the menstrual cycle, pregnancy and lactation. J. Endocrinol. Invest., 14, 907912.[ISI][Medline]
Fraser, I.S. and Lun, Z.G. (1990) Polymers of prolactin and their clinical significance. Obstet. Gynecol. Surv., 45, 515520.[Medline]
Garnier, P.E., Aubert, M.L., Kaplan, S.L. and Grumbach, M.M. (1978) Heterogeneity of pituitary and plasma prolactin in man: decreased affinity of big prolactin in a radioreceptor assay and evidence for its secretion. J. Clin. Endocrinol. Metab., 47, 12731281.[ISI][Medline]
Hattori, N. (1996) The frequency of macroprolactinemia in pregnant women and the heterogeneity of its etiologies. J. Clin. Endocrinol. Metab., 81, 586559.[Abstract]
Hattori, N., Ikekubo, K., Ishihara, T., Moridera, K., Hino, M. and Kurahachi, H. (1992a) A normal ovulatory woman with hyperprolactinemia: presence of anti-prolactin autoantibody and the regulation of prolactin secretion. Acta Endocrinol. (Copenh.), 126, 497500.
Hattori, N., Ishihara, T., Ikekubo, K., Moridera, K., Hino, M. and Kurahachi, H. (1992b) Autoantibody to human prolactin in patients with idiopathic hyperprolactinemia. J. Clin. Endocrinol. Metab., 75, 12261229.[Abstract]
Hattori, N., Ikekubo, K., Ishihara, T., Moridera, K., Hino, M. and Kurahachi, H. (1994) Correlation of the antibody titers with serum prolactin levels and their clinical course in patients with anti-prolactin autoantibody. Eur. J. Endocrinol., 130, 438445.[ISI][Medline]
Jackson, R.D., Wortsman, J. and Malarkey, W.B. (1985) Characterization of a large molecular weight prolactin in women with idiopathic hyperprolactinemia and normal menses. J. Clin. Endocrinol. Metab., 61, 258264.[Abstract]
Jeske, W., Zgliczynski, W. and Zdunowski, P. (2002) Letter to the Editor regarding "Laboratory and clinical experience in 55 patients with macroprolactinemia identified by a simple Polyethylene Glycol Precipitation Method". J. Clin. Endocrinol. Metab., 87, 1909.
Larrea, F., Escorza, A., Valero, A., Hernandez, L., Cravioto, M.C. and Diaz-Sanchez, V. (1989) Heterogeneity of serum prolactin throughout the menstrual cycle and pregnancy in hyperprolactinemic women with normal ovarian function. J. Clin. Endocrinol. Metab., 68, 982987.[Abstract]
Larrea, F., Villanueva, C., Cravioto, M.C., Escorza, A. and del Real, O. (1985) Further evidence that big big prolactin is preferentially secreted in women with hyperprolactinemia and normal ovarian function. Fertil. Steril., 44, 2530.[ISI][Medline]
Leslie, H., Courtney, C.H., Bell, P.M., Hadden, D.R., McCance, D.R., Ellis, P.K., Sheridan, B. and Atkinson, A.B. (2001) Laboratory and clinical experience in 55 patients with macroprolactinemia identified by a simple polyethylene glycol precipitation method. J. Clin. Endocrinol. Metab., 86, 27432746.
Malarkey, W.B., Jackson, R. and Wortsman, J. (1988) Long-term assessment of patients with macroprolactinemia. Fertil. Steril., 3, 413418.
Ohnami, S., Eto, S., Ohnami, S., Soejima, T. and Nakata, H. (1987) Characterization of "big big prolactin" in serum and tumor extract in patients with PRL-secreting tumor. Endocrinol. Jpn., 34, 325334.[Medline]
Olukoga, A.O. and Kane, J.W. (1999) Macroprolactinaemia: validation and application of the polyethylene glycol precipitation test and clinical characterization of the condition. Clin. Endocrinol., 51, 119126.[CrossRef][ISI][Medline]
Rogol, A.D. and Rosen, S.W. (1974) Prolactin of apparent large molecular size: the major immunoreactive prolactin component in plasma of a patient with pituitary tumor. J. Clin. Endocrinol. Metab., 38, 714717.[ISI][Medline]
Rowe, R.C., Cowden, E.A., Faiman, C. and Friesen, H.G. (1983) Correlation of Nb2 bioassay and radioimmunoassay values for human serum prolactin. J. Clin. Endocrinol. Metab., 57, 942945.[Abstract]
Sinha, Y.N., Gilligan, TA. and Lee, D.W. (1984) Detection of a high molecular weight variant of prolactin in human plasma by a combination of electrophoretic and immunologic techniques. J. Clin. Endocrinol. Metab., 58, 752754.[Abstract]
Suh, H.K. and Frantz, A.G. (1974) Size heterogeneity of human prolactin in plasma and pituitary extracts. J. Clin. Endocrinol. Metab., 39, 928935.[ISI][Medline]
Trouillas, J. and Girod, C. (1996) Pathology of pituitary adenomas. In Landolt, A.M., Vance, M.L. and Reilly, P.L. (ed.), Pituitary Adenomas. Churchill Livingstone, New York, pp. 2746.
Valette-Kasic, S., Morange-Ramos, I., Selim, A., Gunz, G., Morange, S., Enjalbert, A. et al. (2002) Macroprolactinemia revisited: a study on 106 patients. J. Clin. Endocrinol. Metab., 87, 581588.
Viera, J.G., Tachibana, T.T., Obara, L.H. and Maciel, R.M. (1998) Extensive experience and validation of polyethylene glycol precipitation as a screening method for macroprolactinemia. Clin. Chem., 44, 17581759.
Whitaker, M.D., Klee, G.G., Kao, P.C., Randall, R.V. and Heser, D.W. (1984) Demonstration of biological activity of prolactin molecular weight variants in human sera. J. Clin. Endocrinol. Metab., 58, 826830.[Abstract]
Whittaker, P.G., Wilcox, T. and Lind, T. (1981) Maintained fertility in a patient with hyperprolactinemia due to big big prolactin. J. Clin. Endocrinol. Metab., 53, 863866.[Abstract]
Submitted on May 27, 2002; resubmitted on October 22, 2002; accepted on January 6, 2003.