Biological characterization of the isoforms of urinary human follicle-stimulating hormone contained in a purified commercial preparation

The main physicochemical and biological properties of the several isoforms of urinary follicle-stimulating hormone (uFSH) present in a commercially available uFSH preparation were analysed. Purified urinary FSH was submitted to chromatofocusing and several immunoactive forms of uFSH with isoelectric points (pl) ranging from 5.5 to 3.8 were identified. An additional isoform was detected after passing through the chromatofocusing column a 1.0 M NaCI solution (salt peak). Each uFSH isoform or pool of neighbouring isoforms (pl value 5.5–5.1, pool I, 3.8 ± 1.0% of total immunoactivity recovered; pl value 5.0−4.6, pool II, 18.4 ± 3.6% of total; pl value 4.5–4.3, pool III, 14.9 ± 1.5% of total; pl value 4.1, pool IV, 8.2 ± 1.4% of total; salt peak, pool V, 51.1 ± 6.4% of total) eluted as single FSH peaks after Sephadex G-100 exclusion chromatography (apparent Mr 60 000). Even though FSH present within each pool was recognized by a receptor preparation, the receptor binding activity expressed as the radioreceptor assay/radioimmunoassay (RRA/RIA) activity ratio varied with the pl value of the particular uFSH isoform tested; starting from a pl value of 5.5, the receptor binding activity of FSH decreased from 5.9 ± 0.39 to 2.4 ± 0.19, as the pl value of the corresponding isoform declined. A similar trend was observed when the potency of each isoform was assessed by an in-vitro bioassay. Isoform V exhibited the shortest plasma clearance rate (0.115 ± 0.01 ml/min) and longest mean residence time (184.6 ± 4.2 min) and half-life (123.5 ± 2.8 min) whereas pools I and IV showed plasma clearance rates, mean residence times and half-lives ranging from 0.589 ± 0.06 to 0.349 ± 0.02 ml/min, 78.4 ± 4.2 to 113.5 ± 10.6 min and 56.6 ± 3.5 to 77.1 ± 7.2 min, respectively. These results demonstrate that this widely used commercial preparation of uFSH contains a significant number of FSH isoforms which may be separated by their electrical properties. For the majority of the uFSH isoforms isolated by chromatofocusing, there is a close relationship between their physicochemical properties and their biological attributes. Even though some of the biological features exhibited by these uFSH isoforms are very similar to those presented by their intrapituitary analogues, there are marked differences between the pattern of charge heterogeneity of the FSH contained in this preparation and those previously reported for the molecules stored and released from the anterior pituitary gland, particularly during the midcycle. The overall data indicate that although natural uFSH is effective for the induction of follicular maturation, it is still far from mimicking the naturally occurring type of pituitary FSH signal to the ovary.