Qualitative and quantitative differences in the isoelectrofocusing profile of biologically active lutropin in the blood of normally menstruating and post-menopausal women

Abstract

Abstract. A single bolus of 100 μg of gonadoliberin (LRH) was administered intravenously to 8 post-menopausal and 9 normally menstruating women and blood was withdrawn before and 30, 60, 120 and 240 min after LRH stimulation. The plasma samples obtained at different time intervals from women showing a sufficiently high response to LRH (menopausal: 8, menstruating: 3) were combined and 2 ml samples of each pool were fractionated in triplicate by electrofocusing on sucrose density gradient. In addition, two plasma pools, obtained 30 min following LRH stimulation, one from 4 normally menstruating women (exhibiting a relatively low LH-response) and the other from 2 normally menstruating women aged 40, were analyzed in the same way in duplicate electrofocusing experiments. The hLH activity was determined in each electrofocusing fraction by an in vitro bioassay method following elution and purification by gel filtration. The LH activity was distributed as four major peaks at pI values of 7.10 ± 0.05, 7.58 ± 0.06, 8.10 ± 0.04 and 8.54 ± 0.05 and a broad area of activity comprising a number of peaks in the pH range of 8.69–9.50. The analysis of the data revealed marked differences in the relative distribution of the various molecular species present in the blood of menopausal women and of normally menstruating women. A molecular species exhibiting a pI value of 7.10 was invariably present (10 – 15% of the total) in all samples of post-menopausal plasma (PMP) but was consistently absent from all samples of midcycle plasma (MCP). The amount of relatively 'less alkaline' material (eluted from pH range 7.37–8.32) was significantly higher (P < 0.001) in the PMP samples compared to MCP samples. On the other hand, in the MCP samples the amount of relatively 'more alkaline' material eluted from the pH range 8.33–9.50 was significantly (P < 0.001) higher (about 60% of the total recovered activity) compared to the PMP samples (about 30% of the total). Following LRH stimulation significant temporal changes were observed in the relative contribution of various molecular species to the hLH profile. A gradual increase, up to 60 min, in the material eluted in the pH range 6.87–7.36 in the post-menopausal plasma samples was accompanied by a gradual decrease in the material eluted in the pH range 7.84–8.32. Two hours after LRH stimulation a significant drop was found in the material collected from pH range 8.33–8.68, with a concomitant rise in the material eluted in the pH range 8.69–9.50. This last mentioned shift was also observed in the plasma of normally menstruating women. It is concluded that major differences exist in the composition of biologically active hLH species present in the peripheral blood of post-menopausal and normally menstruating women. Moreover, significant temporal changes occur in the composition of circulating hLH species following stimulation by LRH both in post-menopausal and in normally menstruating women.