J. Endocrinol. Invest. 14: 907-912,1991
Variations in the molecular forms of prolactin during the menstrual cycle, pregnancy and lactation M.E. Fonseca, R. Ochoa, C. Moran, and A. Zarate Endocrine Research Unit, Instituto Mexicano del Seguro Social and Universidad Nacional Aut6noma de Mexico, Mexico, O.F., Mexico as progression of gestation. The polymeric PRL forms were found in substantially less amount as gestation progressed, After parturition, in nursing mothers the 22K form remained prominent and in greater concentrations than the 26K monomeric variant. Large and low mol wt PRL forms were constantly detected in sera from women during the lactation period. From these data we confirmed that PRL circulates at various molecular forms and the relative proportion of these molecular variants exhibit changes according to the physiological state, In our study the prodominant form was the 22K PRL (nonglycosylated) and it was of interest to discover the presence of a low mol wt PRL which elutes in the 16K area. The significance of this latter finding is not clear at the present.
ABSTRACT. Size heterogeneity of immunoreactive prolactin (PRL) was studied in serum samples obtained from eight normoprolactinemic women during the menstrual cycle and five additional patients at pregnancy and lactation. Gel filtration of sera from women with normal ovarian function tested at day 1012th of their menstrual cycle showed two predominant PRL forms, approximately 22K and 26K mol wt. In addition two polymeric variants, 50K ("big" PRL) and 100K ("big-big" PRL) were found in less proportion,accounting for approximately 34% of the total PRL immunoreactivity detected in the sera. It was also noted a low mol wt form eluting around the region of 16K mol wt. In pregnant women the major PRL form was the 22K and its proportion showed a gradual increase
INTRODUCTION
Therefore it has not been possible to establish a clinical-biochemical correlation at the present. The physiological significance of the multiple PRL forms as well as the relative proportion and variations during gestation and lactation are still unclear, although there may exist some correlation with specific biological actions of these molecular forms. On the basis of these observations, we studied a group of women with normal ovarian function during the menstrual cycle, pregnancy and postpartum in order to establish the chromatographic profile of serum PRL by gel filtration analyses. Elucidation of the precise mechanisms that ensure correct and differential proportion of molecular weight variants of PRL should enhance the understanding of physiology and be of clinical relevance as well.
It is now well recognized that prolactin (PRL) may exist as variants of different mol wt in blood, cerebrospinal fluid, fetal serum, ammiotic fluid and pituitary extracts (1-5). It is also known that the polymeric and larger mol wt forms, generally denominated as "big-big" PRL and "big" PRL, approximately 100 K and SOK respectively, have reduced biological activity due to diminished binding to PRL receptors and lesser mitogenic activity (6-7). In normal women two monomeric molecules of PRL, 22K and 26K, have been demonstrated and they account for approximately 80 per cent of the total circulating PRL (8-10). It has been demonstrated that the 26K PRL form corresponds to a glycosylated molecule and this form seems to constitute the predominant circulating variant in both normal women and men (8-9). Studies carried out in pregnant and lactating women have shown different results on the proportion of the circulating forms of PRL (10-15).
MATERIALS AND METHODS Subjects The study was approved by the Human Ethical Investigation Committee of the Instituto Mexicano del Seguro Social. Blood samples were obtained from eight women ranging in age from 20-32 yr, with normal menses and normal baseline serum PRL concentrations. Sera were collected between day 10-14th of their menstrual cycle. Serum sam-
Key-words: Prolactin, gel chromatography, molecular PRL forms, pregnancy, lactation, concanavalin-A, pituitary, glycosylated PRL. Correspondence: Dr. Arturo Zarate, Apdo. Postal 107-115, 06760 Mexico, D.F. Mexico. Received October 1, 1990; accepted May 13, 1991.
907
M. E. Fonseca, R. Ochoa, C. Moran, et al.
Table 1 - Different molecular weight forms of prolactin in women with normal ovarian function during the menstrual cycle at midfollicular phase. Low mol wt (16 K)
"Big-big" (100 K)
Big (50 K)
Glycosylated (26 K)
Nonglycosylated (22 K)
23-26
28-3 1
35-38
39-42
43-48
Kav
0.02
0.20
040
048
0.6-0.7
PM (K)
100
66-40
28-25
23-22
18-1 6
Ve (ml)
~~--~~
Partition coefficients calculated by the formula Kav= ,where Ve represents the peak tube of hormone fraction , Vo the peak tube of the void volume fraction, and Vt the peak tube of the 125 1sodium iodine fraction.
recovery of immunoreactive PRL from the column ranged from 90-98%. Serum samples of 3.0 ml (1 .0 ml of sera diluted to 3.0 ml with water,was used for pregnant and lactating women) were applied to the column. The flow was 4.5 ml/h and fractions of 1.0 ml were collected by the use of an automatic fraction collector (Multiracc 2111 , LKB , Bromma, Finland). All fractions were kept at 4 C until assayed for PRL concentrations. The major molecular forms of PRL determined by the gel filtration chromatography were identified on the basis of their elution volume and the partition constant (Kav). The molecular weight was calculated upon the calibration curve (Table 1). The distribution of serumPRL immunoreactivity was calculated as percent of total PRL immunoreactivity.
pies were also obtained from five pregnant women at the second and third trimester as well as during the postpartum period . Blood samples were collected between 08 :00 and 09:00 and stored at -20 C until assayed for the gel filtration analyses .
Gel chromatography Sephadex G-100 superfine (Pharmacia Fine Chemical , Uppsala, Sweden) was equilibrated with 0.1 M phosphate buffer containing 0.1 % BSA, pH 7.4 at 4 C, and a column of 60 x 1 cm was set up. The column was calibrated with the following molecular size markers : blue dextran (mol wt=200 K) , bovine albumin (mol wt=65 K) ovoalbumin (mol wt=45K) , carbonic anhydrase (mol wt=29 K) , cytochrome C (mol wt= 12 K), 1 25 1 PRL (mol wt=22 K), and 125 I sodium iodide. The column was recalibrated at regular intervals and the
vo
-
oo ~
BSA
•
Ovo
12S
I - PRL
Concanavalin A-affinity chromatography A noncompetitive system of affinity chromatogra-
.
CYTOCHROME C
~
Vt
•
2.
..
!:: > 2 .0 j::
----. ~
32 y .o.
ct
O---
I-
U
« UJ II::
0
2
z
:::>
~
~ ~
l-
u
I
«
...J
0 II:: Q.
0
20
25
30
35
40
45
50
55
60
ELUTION VOLUME(mLl
Fig. 4 - Gel chromatography profile form serum samples collected from a woman at different stages of her pregnancy and lactation showing the variations of PRL mol wt forms. Arrows indicate the peak elution of mol wt markers (Vo=void volume or blue dextran, Vt=total volume, PRL= 125 1 PRL).
40 22 K
30
ing had no effect on the relative distribution of mol
wt variants of PRL in blood samples obtained at two
~
!:
...u~ c
11K
III IE
0
z
weeks postpartum, before and after 30 min of continuous infant suckling . Figure 4 illustrates the gel chromatography profile of PRL immunoreactivity from serum samples collected from one patient during the last part of pregnancy and two weeks postpartum. It is obvious the change in the relative proportion of the mol wt variants.
ZIK
20
10
;:)
::I ::I ~
a: A.
...0
25
III g
20
...z
15
Concanavalin-A-chromatography The percentage of PRL immunoreactivity bound to concanavalin-A is shown in Table 3 in order to distinguish the 22K PRL from the 26K PRL. It is clear that these two variants are completely dissociated by gel filtration suggesting that 26K PRL corresponds to the glycosylated molecule of PRL identified by the use of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (18).
c
III
u
IE III Q.
50K 10 lOOK
5
Me
2nd
lrd
PP
Table 3 - Percentage of PRL immunoreactivity following concanavalin-A affinity chromatography.
L
'rim .. '"
PRL mol wt form
Fig. 3 - Percentage of PRL immunoreactivity at the middle of the menstrual cycle (PS), during the second and the third trimester of pregnancy, postpartum (pp), and lactation (L). The upper panel depicts the distribution of the two PRL monomeric forms as well as the low mol wt PRL variant, and the lower panel shows the relative proportion of the two polymeric variants of PRL.
910
Concanavalin A - Sepharose Eluated (%)
Bound (%)
26 k
36
64
22 k
65
35
Molecular forms of PRL
DISCUSSION It is now generally accepted that PRL circulates as different molecular size forms in nonpregnant women and during gestation as well as in postpartum. The proportions of these PRL variants are changing constantly according to the physiological condition and pathological states as well (1-4). Another interesting observation is the fact that some normal women may present underlying severe hyperprolactinemia due to the fact that the predominant PRL forms are the polymeric variants which are supposed to possess less biological activity (2,4,6,7, 14). On the other hand the two monomeric 22K and 26K PRL forms are the predominant molecules and account for the majority of total circulating PRL present in normal conditions (8-9). The mechanism which determines the relative proportion and distribution of PRL forms is not yet known. In conclusion our data confirm the existence of multiple isomorphic forms of PRL of different molecular weight in the serum of women with normal ovarian function and during pregnancy and lactation as well. We also documented changes in the relative proportion of these PRL variants during gestation. A decrease in the large molecular forms and the glycosylated monomeric variant as well as a corresponding increase in the concentration of non glycosylated PRL towards term was evident. No apparent changes in the amount of the low molecular weight PRL were observed during pregnancy. The physiological significance of these small PRL variants is not fully understood and they may represent only fragments of the monomeric PRL molecules. Regulation of the multiple forms of different size of PRL is of vital importance to hormone function, therefore it may be subjected to endocrine control. Likewise regulation of the relative proportion of heterogeneous molecular PRL secretion by the endocrine milieu could contribute to adjust the hormone function by secreting variable forms of PRL.
2. Guyda H.J. Heterogeneity of human growth hormone and prolactin secreted in vitro: immunoassay and radioreceptor assay correlations. J. Clin. Endocrinol. Metab. 41: 953,1975. 3. Kiefer KA, Malarkey W.B. Size heterogeneity of human prolactin in CSF and serum: experimental conditions that alter gel filtration patterns. J. Clin. Endocrinol. Metab. 46: 119, 1978 4. Anderson AN., Pedersen H., Ojursing H., Anderson
B.N., Friesen H.G. Bioactivity of prolactin in a woman with an excess of large molecular size prolactin, persistent hyperprolactinemia and spontaneous conception. Fertil. Steril. 33: 625, 1982. 5. Pankov YA, Butnev V.Y.
Multiple forms of pituitary prolactin, a glycosylated form of porcine prolactin with enhanced biological activity. Int. J. Peptide Res. 28: 113,1986. 6. Garnier P.E., Aubert M.L., Kaplan S.L., Grumbach
M.M. 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: 1273, 1978. 7. Farkouh N.H., Packer M.G., Frantz H.G. 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: 1026,1979. 8. Markoff E, Lee OW. Glycosylated prolactin is a major circulating variant in human serum. J. Clin. Endocrinol. Metab. 65: 1102, 1987. 9. Markoff E., Lee OW., Hollinghsworth O.R. Glycosylated and nonglycosylated prolactin in serum during pregnancy. J. Clin. Endocrinol. Metab. 67: 519,1988. 10. Markoff E., Sigel M.B., Lacour N., Seavey B.K.,
Friesen H.G., Lewis U.J. Glycosylation selectively alters the biological activity of prolactin. Endocrinology 123: 1303,1988.
ACKNOWLEDGMENTS This work was partially supported by the Consejo Nacional de Ciencia y Tecnologfa (Grant-880122). We thank Ms. Paty Hernandez and Guadalupe Sanchez for their skillful preparation of the manuscript.
11. Whitaker MD., Klee G.G, Kao P.C., Randall RV,
Heser OW. Demonstration of biological activity of prolactin molecular weight variants in human sera. J. Clin. Endocrinol. Metab. 58: 826, 1984. 12. Larrea F, Villanueva C., Cravioto M.C., Escorza A,
REFERENCES
Del RealO. Further evidence that "big big" prolactin is preferentially secreted in women with hyperprolactinemia and normal ovarian function. Fertil. Steril. 44: 25,1985.
1. Suh H.K., Frantz AG.
Size heterogeneity of human prolactin in plasma and pituitary extracts. J. Clin. Endocrinol. Metab. 39: 929, 1974.
911
ME Fonseca, R. Ochoa, C. Moran, et a/.
13. Pansini F, Bergamini C.M, Malfaccini M., Cocilovo G., Linciano M., Jacobs M., Bagni B. Multiple molecular forms of prolactin during pregnancy. J. Endocrinology 106: 81, 1985. 14. Fraser I.S., Lun Z.G., Zhou J.P., Herington A.C., McCarron G., Caterson I., Tan K., Markham R. Detailed assessment of big-big prolactin in women with hyperprolactinemia and normal ovarian function J. Clin. Endocrinol. Metab. 69: 585, 1989. 15. Jackson RD., Wortsman J., Malarkey W.B. Persistence of large molecular weight prolactin secretion during pregnancy in women with macroprolactinemia and its presence in fetal cord blood. J. Clin. Endocrinol. Metab. 68: 1046, 1989. 16. Matsuura S., Chen H-C A simple and effective solvent system for elution of
912
gonadotropins from concanavalin A affinity chromatography. Analyt. Biochem. 106: 402,1980. 17. Zarate A., Moran C., Miranda R., Loyo M, Medina M., Fonseca M.E. Long-acting bromocriptine for the acute treatment of large macroprolactinomas. J. Endocrinol. Invest. 10: 233, 1987. 18. Champier J., Claustrat B., Sasso las G., Berger M. Detection and enzymatic deglycosylation of a glycosylated variant of prolactin in human plasma. FEBS Lett. 212: 220,1987. 19. Whitaker R.G., Wilcox T., Lind T. Maintained fertility in patient with hyperprolactinemia due to big big prolactin. J. Clin. Endocrinol. Metab. 53: 863, 1981.
Variations in the molecular forms of prolactin during the menstrual cycle, pregnancy and lactation M.E. Fonseca, R. Ochoa, C. Moran, and A. Zarate Endocrine Research Unit, Instituto Mexicano del Seguro Social and Universidad Nacional Aut6noma de Mexico, Mexico, O.F., Mexico as progression of gestation. The polymeric PRL forms were found in substantially less amount as gestation progressed, After parturition, in nursing mothers the 22K form remained prominent and in greater concentrations than the 26K monomeric variant. Large and low mol wt PRL forms were constantly detected in sera from women during the lactation period. From these data we confirmed that PRL circulates at various molecular forms and the relative proportion of these molecular variants exhibit changes according to the physiological state, In our study the prodominant form was the 22K PRL (nonglycosylated) and it was of interest to discover the presence of a low mol wt PRL which elutes in the 16K area. The significance of this latter finding is not clear at the present.
ABSTRACT. Size heterogeneity of immunoreactive prolactin (PRL) was studied in serum samples obtained from eight normoprolactinemic women during the menstrual cycle and five additional patients at pregnancy and lactation. Gel filtration of sera from women with normal ovarian function tested at day 1012th of their menstrual cycle showed two predominant PRL forms, approximately 22K and 26K mol wt. In addition two polymeric variants, 50K ("big" PRL) and 100K ("big-big" PRL) were found in less proportion,accounting for approximately 34% of the total PRL immunoreactivity detected in the sera. It was also noted a low mol wt form eluting around the region of 16K mol wt. In pregnant women the major PRL form was the 22K and its proportion showed a gradual increase
INTRODUCTION
Therefore it has not been possible to establish a clinical-biochemical correlation at the present. The physiological significance of the multiple PRL forms as well as the relative proportion and variations during gestation and lactation are still unclear, although there may exist some correlation with specific biological actions of these molecular forms. On the basis of these observations, we studied a group of women with normal ovarian function during the menstrual cycle, pregnancy and postpartum in order to establish the chromatographic profile of serum PRL by gel filtration analyses. Elucidation of the precise mechanisms that ensure correct and differential proportion of molecular weight variants of PRL should enhance the understanding of physiology and be of clinical relevance as well.
It is now well recognized that prolactin (PRL) may exist as variants of different mol wt in blood, cerebrospinal fluid, fetal serum, ammiotic fluid and pituitary extracts (1-5). It is also known that the polymeric and larger mol wt forms, generally denominated as "big-big" PRL and "big" PRL, approximately 100 K and SOK respectively, have reduced biological activity due to diminished binding to PRL receptors and lesser mitogenic activity (6-7). In normal women two monomeric molecules of PRL, 22K and 26K, have been demonstrated and they account for approximately 80 per cent of the total circulating PRL (8-10). It has been demonstrated that the 26K PRL form corresponds to a glycosylated molecule and this form seems to constitute the predominant circulating variant in both normal women and men (8-9). Studies carried out in pregnant and lactating women have shown different results on the proportion of the circulating forms of PRL (10-15).
MATERIALS AND METHODS Subjects The study was approved by the Human Ethical Investigation Committee of the Instituto Mexicano del Seguro Social. Blood samples were obtained from eight women ranging in age from 20-32 yr, with normal menses and normal baseline serum PRL concentrations. Sera were collected between day 10-14th of their menstrual cycle. Serum sam-
Key-words: Prolactin, gel chromatography, molecular PRL forms, pregnancy, lactation, concanavalin-A, pituitary, glycosylated PRL. Correspondence: Dr. Arturo Zarate, Apdo. Postal 107-115, 06760 Mexico, D.F. Mexico. Received October 1, 1990; accepted May 13, 1991.
907
M. E. Fonseca, R. Ochoa, C. Moran, et al.
Table 1 - Different molecular weight forms of prolactin in women with normal ovarian function during the menstrual cycle at midfollicular phase. Low mol wt (16 K)
"Big-big" (100 K)
Big (50 K)
Glycosylated (26 K)
Nonglycosylated (22 K)
23-26
28-3 1
35-38
39-42
43-48
Kav
0.02
0.20
040
048
0.6-0.7
PM (K)
100
66-40
28-25
23-22
18-1 6
Ve (ml)
~~--~~
Partition coefficients calculated by the formula Kav= ,where Ve represents the peak tube of hormone fraction , Vo the peak tube of the void volume fraction, and Vt the peak tube of the 125 1sodium iodine fraction.
recovery of immunoreactive PRL from the column ranged from 90-98%. Serum samples of 3.0 ml (1 .0 ml of sera diluted to 3.0 ml with water,was used for pregnant and lactating women) were applied to the column. The flow was 4.5 ml/h and fractions of 1.0 ml were collected by the use of an automatic fraction collector (Multiracc 2111 , LKB , Bromma, Finland). All fractions were kept at 4 C until assayed for PRL concentrations. The major molecular forms of PRL determined by the gel filtration chromatography were identified on the basis of their elution volume and the partition constant (Kav). The molecular weight was calculated upon the calibration curve (Table 1). The distribution of serumPRL immunoreactivity was calculated as percent of total PRL immunoreactivity.
pies were also obtained from five pregnant women at the second and third trimester as well as during the postpartum period . Blood samples were collected between 08 :00 and 09:00 and stored at -20 C until assayed for the gel filtration analyses .
Gel chromatography Sephadex G-100 superfine (Pharmacia Fine Chemical , Uppsala, Sweden) was equilibrated with 0.1 M phosphate buffer containing 0.1 % BSA, pH 7.4 at 4 C, and a column of 60 x 1 cm was set up. The column was calibrated with the following molecular size markers : blue dextran (mol wt=200 K) , bovine albumin (mol wt=65 K) ovoalbumin (mol wt=45K) , carbonic anhydrase (mol wt=29 K) , cytochrome C (mol wt= 12 K), 1 25 1 PRL (mol wt=22 K), and 125 I sodium iodide. The column was recalibrated at regular intervals and the
vo
-
oo ~
BSA
•
Ovo
12S
I - PRL
Concanavalin A-affinity chromatography A noncompetitive system of affinity chromatogra-
.
CYTOCHROME C
~
Vt
•
2.
..
!:: > 2 .0 j::
----. ~
32 y .o.
ct
O---
I-
U
« UJ II::
0
2
z
:::>
~
~ ~
l-
u
I
«
...J
0 II:: Q.
0
20
25
30
35
40
45
50
55
60
ELUTION VOLUME(mLl
Fig. 4 - Gel chromatography profile form serum samples collected from a woman at different stages of her pregnancy and lactation showing the variations of PRL mol wt forms. Arrows indicate the peak elution of mol wt markers (Vo=void volume or blue dextran, Vt=total volume, PRL= 125 1 PRL).
40 22 K
30
ing had no effect on the relative distribution of mol
wt variants of PRL in blood samples obtained at two
~
!:
...u~ c
11K
III IE
0
z
weeks postpartum, before and after 30 min of continuous infant suckling . Figure 4 illustrates the gel chromatography profile of PRL immunoreactivity from serum samples collected from one patient during the last part of pregnancy and two weeks postpartum. It is obvious the change in the relative proportion of the mol wt variants.
ZIK
20
10
;:)
::I ::I ~
a: A.
...0
25
III g
20
...z
15
Concanavalin-A-chromatography The percentage of PRL immunoreactivity bound to concanavalin-A is shown in Table 3 in order to distinguish the 22K PRL from the 26K PRL. It is clear that these two variants are completely dissociated by gel filtration suggesting that 26K PRL corresponds to the glycosylated molecule of PRL identified by the use of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (18).
c
III
u
IE III Q.
50K 10 lOOK
5
Me
2nd
lrd
PP
Table 3 - Percentage of PRL immunoreactivity following concanavalin-A affinity chromatography.
L
'rim .. '"
PRL mol wt form
Fig. 3 - Percentage of PRL immunoreactivity at the middle of the menstrual cycle (PS), during the second and the third trimester of pregnancy, postpartum (pp), and lactation (L). The upper panel depicts the distribution of the two PRL monomeric forms as well as the low mol wt PRL variant, and the lower panel shows the relative proportion of the two polymeric variants of PRL.
910
Concanavalin A - Sepharose Eluated (%)
Bound (%)
26 k
36
64
22 k
65
35
Molecular forms of PRL
DISCUSSION It is now generally accepted that PRL circulates as different molecular size forms in nonpregnant women and during gestation as well as in postpartum. The proportions of these PRL variants are changing constantly according to the physiological condition and pathological states as well (1-4). Another interesting observation is the fact that some normal women may present underlying severe hyperprolactinemia due to the fact that the predominant PRL forms are the polymeric variants which are supposed to possess less biological activity (2,4,6,7, 14). On the other hand the two monomeric 22K and 26K PRL forms are the predominant molecules and account for the majority of total circulating PRL present in normal conditions (8-9). The mechanism which determines the relative proportion and distribution of PRL forms is not yet known. In conclusion our data confirm the existence of multiple isomorphic forms of PRL of different molecular weight in the serum of women with normal ovarian function and during pregnancy and lactation as well. We also documented changes in the relative proportion of these PRL variants during gestation. A decrease in the large molecular forms and the glycosylated monomeric variant as well as a corresponding increase in the concentration of non glycosylated PRL towards term was evident. No apparent changes in the amount of the low molecular weight PRL were observed during pregnancy. The physiological significance of these small PRL variants is not fully understood and they may represent only fragments of the monomeric PRL molecules. Regulation of the multiple forms of different size of PRL is of vital importance to hormone function, therefore it may be subjected to endocrine control. Likewise regulation of the relative proportion of heterogeneous molecular PRL secretion by the endocrine milieu could contribute to adjust the hormone function by secreting variable forms of PRL.
2. Guyda H.J. Heterogeneity of human growth hormone and prolactin secreted in vitro: immunoassay and radioreceptor assay correlations. J. Clin. Endocrinol. Metab. 41: 953,1975. 3. Kiefer KA, Malarkey W.B. Size heterogeneity of human prolactin in CSF and serum: experimental conditions that alter gel filtration patterns. J. Clin. Endocrinol. Metab. 46: 119, 1978 4. Anderson AN., Pedersen H., Ojursing H., Anderson
B.N., Friesen H.G. Bioactivity of prolactin in a woman with an excess of large molecular size prolactin, persistent hyperprolactinemia and spontaneous conception. Fertil. Steril. 33: 625, 1982. 5. Pankov YA, Butnev V.Y.
Multiple forms of pituitary prolactin, a glycosylated form of porcine prolactin with enhanced biological activity. Int. J. Peptide Res. 28: 113,1986. 6. Garnier P.E., Aubert M.L., Kaplan S.L., Grumbach
M.M. 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: 1273, 1978. 7. Farkouh N.H., Packer M.G., Frantz H.G. 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: 1026,1979. 8. Markoff E, Lee OW. Glycosylated prolactin is a major circulating variant in human serum. J. Clin. Endocrinol. Metab. 65: 1102, 1987. 9. Markoff E., Lee OW., Hollinghsworth O.R. Glycosylated and nonglycosylated prolactin in serum during pregnancy. J. Clin. Endocrinol. Metab. 67: 519,1988. 10. Markoff E., Sigel M.B., Lacour N., Seavey B.K.,
Friesen H.G., Lewis U.J. Glycosylation selectively alters the biological activity of prolactin. Endocrinology 123: 1303,1988.
ACKNOWLEDGMENTS This work was partially supported by the Consejo Nacional de Ciencia y Tecnologfa (Grant-880122). We thank Ms. Paty Hernandez and Guadalupe Sanchez for their skillful preparation of the manuscript.
11. Whitaker MD., Klee G.G, Kao P.C., Randall RV,
Heser OW. Demonstration of biological activity of prolactin molecular weight variants in human sera. J. Clin. Endocrinol. Metab. 58: 826, 1984. 12. Larrea F, Villanueva C., Cravioto M.C., Escorza A,
REFERENCES
Del RealO. Further evidence that "big big" prolactin is preferentially secreted in women with hyperprolactinemia and normal ovarian function. Fertil. Steril. 44: 25,1985.
1. Suh H.K., Frantz AG.
Size heterogeneity of human prolactin in plasma and pituitary extracts. J. Clin. Endocrinol. Metab. 39: 929, 1974.
911
ME Fonseca, R. Ochoa, C. Moran, et a/.
13. Pansini F, Bergamini C.M, Malfaccini M., Cocilovo G., Linciano M., Jacobs M., Bagni B. Multiple molecular forms of prolactin during pregnancy. J. Endocrinology 106: 81, 1985. 14. Fraser I.S., Lun Z.G., Zhou J.P., Herington A.C., McCarron G., Caterson I., Tan K., Markham R. Detailed assessment of big-big prolactin in women with hyperprolactinemia and normal ovarian function J. Clin. Endocrinol. Metab. 69: 585, 1989. 15. Jackson RD., Wortsman J., Malarkey W.B. Persistence of large molecular weight prolactin secretion during pregnancy in women with macroprolactinemia and its presence in fetal cord blood. J. Clin. Endocrinol. Metab. 68: 1046, 1989. 16. Matsuura S., Chen H-C A simple and effective solvent system for elution of
912
gonadotropins from concanavalin A affinity chromatography. Analyt. Biochem. 106: 402,1980. 17. Zarate A., Moran C., Miranda R., Loyo M, Medina M., Fonseca M.E. Long-acting bromocriptine for the acute treatment of large macroprolactinomas. J. Endocrinol. Invest. 10: 233, 1987. 18. Champier J., Claustrat B., Sasso las G., Berger M. Detection and enzymatic deglycosylation of a glycosylated variant of prolactin in human plasma. FEBS Lett. 212: 220,1987. 19. Whitaker R.G., Wilcox T., Lind T. Maintained fertility in patient with hyperprolactinemia due to big big prolactin. J. Clin. Endocrinol. Metab. 53: 863, 1981.
