A comparison of preparations of highly purified human pituitary luteinizing hormone: differences in the luteinizing hormone potencies as determined by in vivo bioassays, in vitro bioassay and immunoassay

Abstract

Abstract. The LH potencies of 12 preparations of highly purified human pituitary LH, from 6 laboratories, were estimated by 2 in vivo bioassays and an in vitro bioassay in terms of the International Reference Preparation of Human Pituitary Gonadotrophins (FSH and LH) for Bioassay (coded 69/104); and by immunoassay in terms of the International Reference Preparation of Human Pituitary Luteinizing Hormone for Immunoassay (IRP; coded 68/40). The LH potencies varied between preparations, including the IRP (68/40), from 864 to 5740 IU/mg by seminal vesicle weight gain (SVW) assay; from 1510 to 11500 IU/mg by ovarian ascorbate depletion (OAAD) assay; from 4490 to 14500 IU/mg by in vitro (testicular interstitial-cell testosterone production) bioassay; and from 2030 to 9180 IU/mg by immunoassay. Estimates of protein content were based on the assumption that the absorbance of LH at 280 nm (A 1% 1 cm) was 6.0. The LH potency of most preparations was highest by in vitro bioassay and lowest by SVW assay. The correlation between activities determined by SVW and OAAD assays was more marked than that between estimates by OAAD assay and in vitro bioassay; there was no correlation between estimates by SVW assay and in vitro bioassay. The slopes of the log dose-response curves of preparations in the OAAD assay were positively correlated with their potencies by OAAD assay and negatively correlated with the slopes of their log dose-response curves in the SVW assay. The qualitative differences between preparations are considered to be a reflection of the heterogeneity of LH and of its modification by different purification procedures. The present data, together with the different patterns of heterogeneity found in some of these preparations by isoelectric focusing in a separate study, suggest that the more basic molecular forms of LH, which are preferentially purified during the isolation of LH free from FSH and TSH, have shorter plasma survival times than the more acidic forms. The LH immunoreactivities of all preparations were significantly correlated with their potencies estimated by each of the in vivo bioassays but not with those estimated by in vitro bioassay. The ratios of in vitro bioactivity (in terms of IRP (68/40)): immunoreactivity varied between preparations from 0.53–1.5. The FSH content of each preparation was less than 2% (w/w) by bioassay and immunoassay. Most preparations were more potent by in vitro bioassay than by in vivo bioassay, which contrasted with, and complemented, findings for purified FSH preparations. This indicated that, as in the case of LH, the more basic molecular species of FSH are associated with lower ratios of in vivo: in vitro bioactivity than are the more acidic species. This study provides the most comprehensive comparison available of the activities of purified preparations of LH isolated from frozen and acetone-dried human pituitary glands in different experienced laboratories. These data are needed for selecting material for an international reference preparation of LH for immunoassay on the basis of high LH potency by in vivo bioassay, recommended by the WHO as a criterion for the identity of the hormone and for its freedom from contaminants. The consequences of the heterogeneity of LH are considered for the purification of the reference material and for the suitability of the latter for the various types of specimens which require LH assays.

Epoetin alpha and beta in their erythropoetin isoform compositions and biological properties

“…This is because glycosylation of the polypeptide moiety of a glycoprotein such as EPO is a post-translational process which is influenced by the type of cell in which the EPO is synthesized and the physiological factors, including culture conditions, acting upon this cell (Rademacher et al, 1988;Cumming, 1991). The isoform composition of an EPO preparation can be further influenced by the selectivity of isolation procedures used to purify it, as shown for the glycoprotein LH (Storring et al, 1982). EPOs differing in their isoform composition have also differed in their biological activities and immunoreactivities (Fukuda et al, 1989;Takeuchi et al, 1989;Storring & Gaines Das, 1992).…”

NMR investigations of the N‐linked oligosaccharides at individual glycosylation sites of human lutropin

“…2, fractions A1-6, Az-6 and B-6) were detected at each glycosylation site. This could be a consequence of the use of reverse-phase HPLC for the purification of hLH, as opposed to more commonly employed ion-exchange chromatography; the latter may result in a limited recovery of the more acidic isoforms of hLH, as suggested in [39]. The 'H-NMR spectrum of fraction B-6 indicated mainly sialylated triantennary N-glycans (bifurcated at Man4) with an overall ratio of NeuAca2-3/NeuAcn2-6 approaching 2 : 1 ; this can be inferred from the characteristic Man H-2 chemical shifts at approximately 4.22 ppm (Man3 and Man4) and 4.12 ppm (Man4), and from the relative intensities of the NeuAc H-3e signals at 2.7.59 and 2.670 ppm [26,371.…”

Analytical and clinical significance of peptide hormone heterogeneity with particular reference to growth hormone and luteinizing hormone in serum

“…There is considerable evidence that pituitary extraction and purification of LH can alter its pattern of heterogeneity. For LH there appears to be a preferential selection of more basic isoforms of short halflife during its purification from pituitary extracts (Storring et al, 1982;Zaidi et al, 1982) though this is unlikely to have a major effect on antibody binding. Any alteration of the polypeptide portion (e.g.…”