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Summary Objective Macroprolactin, which comprises immunoglobulin G (IgG) or anti-prolactin (PRL) autoantibody-bound PRL, is one of the causes of hyperprolactinaemia. This study evaluated the composition and stability of macroprolactin and determined whether the condition was long lasting. We also investigated whether we could predict the composition of macroprolactin based on the ratio of polyethylene glycol (PEG)-precipitable PRL. Patients Two hundred and ninety-two patients with hyperprolactinaemia (252 women and 40 men). Design We used PEG precipitation to screen for macroprolactinaemia in 625 serum samples from 292 patients with hyperprolactinaemia visiting a city hospital over the last 5·5 years. Macroprolactinaemia was defined by PEG-precipitable PRL ratio greater than 60%. IgG-bound PRL and anti-PRL autoantibodies were measured by protein G and 125I-hPRL binding studies, respectively. We also examined the autoantibody specificity and binding characteristics. Results In 39 patients with macroprolactinaemia, IgG-bound PRL was present in 87% and anti-PRL autoantibodies in 67%. A non-IgG-bound form of macroprolactin was found mainly in sera with marginally elevated PEG-precipitable PRL. The higher the PEG-precipitable PRL ratio, the greater the likelihood that autoantibodies were involved in the composition of macroprolactin. The autoantibodies were IgG type and specific to human PRL and had a low affinity and high capacity. Long-term follow-up (2–17 years) revealed that the ratios of PEG-precipitable PRL, IgG-bound PRL and anti-PRL autoantibody-bound PRL were relatively stable. Conclusions This study showed that higher PEG-precipitable PRL ratio in macroprolactinaemic sera might preferentially indicate the presence of anti-PRL autoantibodies and that macroprolactinaemia might be a long-lasting condition.

Summary Objective Macroprolactinaemia is one of the causes of hyperprolactinaemia and often leads to misdiagnosis and inappropriate treatment, but the aetiologies are unclear. The objective of the study was to determine the prevalence of macroprolactinaemia in a healthy population and to investigate the mechanisms underlying the development of macroprolactin. Design Observational study of the prevalence, causes, mechanisms and diagnosis of disease using excess sera after hepatitis B virus screening tests at a hospital in Japan. Participants A total of 1330 hospital workers (1010 women and 320 men) participated in this study. Measurements Macroprolactinaemia was screened using the polyethylene glycol (PEG) precipitation method. Sera containing macroprolactin were further examined by binding studies for possible aetiologies. Results Of the 1330 subjects, 49 (3·68%) were diagnosed with macroprolactinaemia. The frequency of hyperprolactinaemia in untreated samples in subjects with macroprolactinaemia (30·6%) was significantly higher than in individuals without macroprolactinaemia (2·26%). Of the 49 subjects with macroprolactinaemia, all had a normal monomeric PRL concentration following PEG removal of macroprolactin. Of 44 hyperprolactinaemias found, 15 (34·1%) had macroprolactinaemia. IgG-bound prolactin was detected in all sera containing macroprolactin. The levels of IgG-bound prolactin positively correlated with those of macroprolactin, suggesting that IgG-bound prolactin forms macroprolactin. Approximately three quarters of the subjects with macroprolactinaemia had anti-prolactin autoantibodies. Glycosylation, aggregation and covalent/noncovalent binding were also involved in the formation of macroprolactin. Conclusions Macroprolactinaemia is a common disorder and causes hyperprolactinaemia in a healthy population. The major aetiology of macroprolactin in our subjects was complexes of prolactin–IgG comprising mainly anti-prolactin autoantibodies, and other minor complex prolactin species.

We described two euthyroid patients with normally functioning goiters, but with persistently undetectable and non-stimulatable TSH levels. Subject 1 was a 64-year-old woman with a large diffuse goiter who has been clinically and biochemically euthyroid without any medication for at least 19 years. Subject 2 was a 31-year-old woman with a small diffuse goiter who has been euthyroid for 4 years. Both patients had persistently undetectable levels of serum TSH, TSH receptor antibodies (TRAb) and thyroid stimulating antibodies (TSAb). Their basal TSH levels were very low and their T3 responses to TRH were very diminished or absent. In contrast, the basal levels of the other pituitary hormones and their responses to LHRH, GRH and CRH stimulation were all within normal limits in both patients. MRI images of pituitary glands, 123I thyroid uptake, and thyroid scans were normal. Ectopic thyroids were not detected on (99m)TcO4- and 123I total body scans. Factors interfering with the measurement of TSH were excluded by recovery studies. In subject 1 a T3-suppression test was positive and a perchlorate discharge test was negative. In subject 2 a T3-suppression test was negative. Euthyroid Graves' disease, subclinical hyperthyroidism, destructive thyroiditis, thyrotoxicosis of extrathyroid origin, central hypothyroidism, and nonthyroidal illness were all ruled out by these observations. These results suggest that an unknown factor, such as thyrostimulin, but not TSH or TSAb, stimulates the thyroid and maintains euthyroidism, and may have a role in the regulation of the hypothalamus-pituitary-thyroid axis.

We determined serum growth hormone-binding protein (GHBP), insulin-like growth factor-I (IGF-I), and growth hormone (GH) levels in patients with cirrhosis and in age-matched control subjects, and investigated their relationships. Serum GHBP levels in cirrhotic patients (14.6% +/- 3.9%) (means +/- SD) were significantly lower than those in normal subjects (20.4% +/- 4.7%). GHBP levels had positive correlations with cholinesterase (r = .58, P less than .001) and Normotest (r = .66, P less than .001), both of which represent liver function in cirrhotic patients. Basal GH levels in cirrhotic patients (range, 0.35 to 13.0 micrograms/L; median, 3.9 micrograms/L) were significantly higher than those in normal subjects (0.015 to 6.0 micrograms/L; 0.19 microgram/L). GHBP levels in cirrhotic patients correlated positively with IGF-I levels (r = .39, P less than .01), and negatively with GH levels (r = -.33, P less than .01). These results may indicate that the serum GHBP level reflects the number of hepatic GH receptors, and that the high basal GH level observed in cirrhotic patients is, at least in part, attributable to decreased clearance of GH by these receptors.

Gonadotropin secreting pituitary adenomas have been reported with increasing frequency in men, but they are still rarely recognized in women. We report a 52-year-old postmenopausal woman with LH- and FSH-secreting pituitary adenoma. She had increased LH (37.0 +/- 13.7 IU/l) (mean +/- SD) and FSH (109.9 +/- 26.7 IU/l) but these concentrations were within normal ranges in 80 postmenopausal women (LH: 29.7 +/- 18.3 IU/l, FSH: 104.0 +/- 43.9 IU/l). The administration of GnRH and conjugated estrogen resulted in normal response of LH and FSH. No abnormal response of gonadotropin to TRH and bromocriptine was observed. After transsphenoidal adenomectomy both LH and FSH decreased (LH: 11.1 +/- 4.2 IU/l, FSH: 37.0 +/- 9.6 IU/l). An immunocytochemical study revealed that the adenoma cells synthesize both LH and FSH. The rarity of gonadotropin secreting pituitary adenomas in women could be the result of greater difficulty in recognition due to an increase in serum gonadotropin in postmenopausal women.

Objective Macroprolactinaemia, mainly caused by anti-prolactin (PRL) autoantibodies, is frequently found in patients with hyperprolactinaemia. Characteristically, these patients lack clinical symptoms of hyperprolactinaemia, but the serum bioactive PRL concentrations in vitro measured by the Nb2 bioassay are usually high. In this study, we investigated the causes of the discrepancy and the true biological features of macroprolactin. Subjects and methods Sixteen patients with macroprolactinaemia due to anti-PRL autoantibodies were studied. PRL bioactivity was determined by the phosphorylation of signal transducer and activator of transcription (Stat)5 in T47D human breast cancer cells and the proliferation of Nb2 rat lymphoma cells. Results PRL bioactivity by the T47D bioassay, expressed as the density of the band of phosphorylated Stat5/immunoreactive PRL, was significantly lower in sera containing anti-PRL autoantibodies (2.4 +/- 1.1) than in control sera (7.2 +/- 3.1). Dissociation of PRL from the autoantibodies by acidification resulted in an increase in phosphorylated Stat5. PRL bioactivity by the Nb2 bioassay was not significantly different between sera with and without anti-PRL autoantibodies, and free PRL in the medium gradually increased during the incubation in a time-dependent manner in sera containing anti-PRL autoantibodies. Conclusions We conclude that the level of bioactivity of macroprolactin in the Nb2 bioassay is normal due to dissociation of PRL from the autoantibodies as a result of the longer incubation and more dilute assay conditions than in the T47D bioassay. The bioactivity of macroprolactin is low in vivo due to anti-PRL autoantibodies, but monomeric PRL dissociated from the autoantibodies retains full biological activity in patients with macroprolactinaemia.

The aim of this study was to develop an assay to selectively detect high-affinity components among TRAb. Using an rhTSHR-coated tube system, a 1 step TRAb assay method was developed that included 1) co-incubation with (125)I-bTSH, 2) a 50 microl serum sample, 3) an increased incubation volume (450 microl), and 4) a 1 hour incubation time. Sixty-one TRAb positive Graves' sera were studied. When the regular TRAb assay (Reg) results were quantitatively compared to the 1 step assay (1 step) results, certain dispersions and overestimations using the latter were seen. Further, some 1 step positive results were observed in the low Reg range. Overestimations were considered mostly due to the differences between TRAb standard and patients' serum TRAb in the binding competition against co-incubated (125)I-bTSH, which was shown from a modified assay mimicking the 1 step conditions. Therefore, the 1 step results were decided to be expressed by % inhibition against (125)I-bTSH. As for data dispersions, TRAb absorptions during the regular 1st incubation were studied. Individually, the absorption rates varied from 11 to 69%, and higher absorptions were observed in lower Reg range, especially in those negative by the 1 step. Observed 1 step positive results in the low Reg range were of interest, and 1 step/Reg ratios were calculated. The ratios with 1 step negative samples were significantly lower than those of 1 step positive samples. In conclusion, the 1 step assay was proved to detect a particular and biologically active TRAb, especially in those with low TRAb. The clinical significance of the 1 step results should be of future interest.

We analyzed TSH receptor (TSHR) effects, both binding and thyroid-stimulation, of TSH and Graves' IgG. A new TRAb assay system utilizes rhTSHR coated tubes and is comprised of two step incubation, the first incubation with patient serum followed by a second incubation with 125I-bTSH. We called TRAb measured by this method as hTRAb. 125I-bTSH binding capacity of the tube was found close to saturation at 1 hr with 200 microl of 125I-bTSH. Up to 5 hr of first incubation for hTRAb assay revealed significant increases in all hTRAb activities. hTRAb was not affected by second incubation time or dose of 125I-bTSH. When 1 step incubation with 125I-bTSH and Graves' serum was performed, hTRAb again increased significantly with time. A simple competitive equilibrium model could not be applied to these ligands. Second, Graves' IgG and bTSH were compared for in vitro thyroid-stimulation sequentially up to 24 hr, measuring cAMP generation from cultured porcine thyrocytes. While bTSH yielded peak cAMP generation by 8 hr, TSAb revealed more cAMP generation by 24 hr than at 8 hr. We concluded that individual Graves' sera contain heterogeneous TRAb of variable avidities, and that slow-acting TRAb, which may lack biological activity, can be detected by prolonged incubation.