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Abstract. A highly purified LH preparation, prepared from human pituitaries (NM14) was studied using immunochemical and in vitro biological techniques. Using isoelectric focusing 5 different biologically active components could be detected, 4 of which were located between pH = 7.0 and 8.6, one was located at pH = 4.9. The biological activity in the acidic part of the pH gradient is probably due to the formation of an artefact during storage in solution. The experiments were performed with special emphasis on the occurrence of LH subunits, for which until now no pI-values have been reported.Using specific radioimmunochemical (RIA) systems at least 7 different α-subunit components and 4 different β-subunit components could be detected. The presence of even more components is likely. The α-subunit components, with pI-values of: 4.6, 5.2, 6.0, 7.1, 8.1, 8.8 and 9.7, were located spread over the entire pH-gradient whereas all β-subunit components were located above pH = 7.5, the pI-values being 7.7, 8.4, 8.5 and 10.4.The identification of these components as α- or β-subunit was based on the relative response in the different RIA systems, the absence of biological activity and the response changes during incubation at 37°C.Refocusing of the above mentioned biologically active components individually resulted each time in a single component with a pI-value identical to its corresponding 'parent'. After incubation at 37°C of these components each time the same variety of subunit components was found with discrete pI-values, identical to those above.

Abstract. A large pituitary tumour was discovered in a 20 year old man who came to medical attention bacause of grand-mal seizures. The tumour produced biologically active LH as demonstrated by supranormal plasma LH and plasma testosterone values. Free alpha-subunit values were also elevated. In contrast, plasma FSH was in the lower normal range. Transsphenoidal operation failed to remove all tumour tissue. Detailed studies were carried out in the postoperative period. TRH and GnRH administration were associated with a rise of plasma LH and alpha-subunit, whereas plasma FSH was low and unresponsive. Bromocriptine treatment was ineffective. In contrast, both during and after treatment with SMS 201-995 for 6 weeks, a decrease of basal plasma LH values was observed. Furthermore, the administration of a single dose of SMS 201-995 reproducibly induced a decrease of plasma LH lasting for a period of about 6 h. The study suggests that SMS 201-995 may be useful in the treatment of patients with gonadotrope cell adenomas.

The charge heterogeneity of an LH preparation containing relatively acidic components (LH Type I) was studied. Four biological active components, with pI-values of 5.04, 5.60, 6.06 and 6.57 were detected. A total of four different α-subunits, with pI-values of 4.49, 4.79, 5.16 and 6.02 could be detected after incubation at 37°C. With the exception of the most acidic component all these α-subunits were also present in earlier studied LH Type II preparations. After neuraminidase treatment a strong shift to more basic components was observed, resulting in a population of components similar to the one detected in LH Type II preparations. The β-subunits detected were very different from those observed in Type II preparations. All six components detected had pI-values < 7.5. Upon incubation at 56°C these subunits appeared to be unstable resulting in a shift to more basic pI-values, these pI-values being very similar to those of β-subunits observed before in Type II preparations. After neuraminidase treatment, the pH values of the population of β-subunits became identical to those of the population in LH Type II. From these results it is concluded that the major charge difference between LH Type I and Type II is located in the β-subunits. This difference cannot be explained completely by differences in sialic acid content, but may also be due to heat labile charged groups such as sulphate.

Abstract. By preparative isoelectric focussing of a highly purified LH preparation in a sucrose density gradient, four biologically active LH components were isolated. The effect of neuraminidase treatment of each component on the charge heterogeneity was studied by isoelectric focussing followed by in vitro biological and immunochemical techniques. The number of biologically active components with pI-values > 7 containing varying amounts of sialic acid is at least six. The pI-value of the most basic (= asialo) LH component was 9.26. The two most basic components were not present in our preparation (NM 14) before neuraminidase treatment. It is concluded that the difference between the pI-values of LH components is caused by a difference in sialic acid content. When an intact LH component was incubated with neuraminidase there was detectable dissociation owing to the elevated temperature (37°C) and necessary acidic conditions of the incubation. Under these conditions we found the same subunits as we have described before. The most basic α-subunit had a pI-value of 9.29, whereas two β-subunits with pI > 9 were observed at pI 9.26 and pI 9.9. On the other hand, when an LH component was forced to dissociate by incubation at 56°C prior to neuraminidase digestion, two additional α-subunits were found. From this it is concluded that in the intact LH molecule, some sialic acid residues are poorer substrates for neuraminidase action than in the free subunits.

Abstract. The charge heterogeneity of three international reference preparations containing biologically active human luteinizing hormone (LH) or its free α-or β-subunits, respectively, was investigated with isoelectric focussing in sucrose density gradients. The immunoreactive profiles throughout the pH-gradient were assessed with radioimmunoassay (RIA) systems specific for intact, i.e. undissociated, LH or free α- or β-subunits. For the preparation of intact LH (MRC 68/40), two peaks were found at pH = 7.69 and 8.05; for the α-subunit preparation (NIBSC 78/554), the peak values were 4.76, 5.04, 5.94, 6.70, 6.96, 7.35, 8.02, 8.72 and 9.32; and for the β-subunit preparation (NIBSC 78/556), the values were 7.60, 8.40, 8.55 and 9.61. These results are in excellent agreement with those obtained for a highly purified LH preparation (NM 14) which was prepared by one of us and on which we have reported earlier. In addition, with the abovementioned techniques, the spontaneous dissociation of MRC 68/40 into subunits upon incubation at 37°C in phosphate buffer was clearly demonstrated by the increased immunoreactivity in the profiles assessed with both RIA-subunit systems. It is concluded that charge heterogeneity of LHi, α- and β-subunits, as observed for different preparations, is confined to a limited population of forms. Differences between preparations are only quantitative. A single preparation, therefore, can be used as a general model for the study of human luteinizing hormone.