ABSTRACT

A homologous radioimmunoassay (RIA) system for human growth hormone-releasing factor 1-29NH₂ (hGRF 1-29-NH₂) was developed and applied to the measurement of immunoreactive (IR) concentrations of the peptide in anaesthetized rats to determine some of its pharmacokinetics after i.v. and s.c. administration. Analysis of the biphasic disappearance curve of IR-hGRF-1-29NH₂ from plasma after i.v. injection (10μg) gave values for the half-lives of the initial distribution phase (α) and for the elimination phase (β) of 1.9 ± 0.2 min and 10.4 ± 0.2 min respectively. There was rapid uptake of the peptide from the site of s.c. injection but comparison of areas under the plasma IR-hGRF1-29NH₂/time curves showed that the estimated total amount in the circulation after s.c. injection was only 4% of that obtained after i.v. administration. A large degree of degradation of the peptide is indicated either at the site of injection or during transfer to plasma; this susceptibility to rapid breakdown is reflected in the short half-life of the peptide in the circulation. Therefore the measurement of the above parameters is a prerequisite when assessing potency of GRF analogues in-vivo and when implementing suitable dosage regimens for clinical purposes.

Abstract

FSH is a glycoprotein containing N-linked carbohydrates which exhibit a variety of forms ranging from mono- to multibranched structures. Variation in glycosylation, particularly the degree of terminal sialylation, determines the half-life of the hormone and hence its in vivo bioactivity. The glycoform content of FSH preparations can differ according to the source (e.g. pituitary, urine), cell line (for rDNA-derived material) and selectivity of purification procedures, and may create difficulties in the preparation and characterization of standards and therapeutic products. In order to develop a simple method to detect changes in glycocomposition, an FSH ELISA was modified by the incorporation of lectins of recognized sugar specificity, and used to examine the terminal sugar composition of ampouled preparations of pituitary, urinary and rDNA-derived FSH.

FSH was captured with a specific monoclonal antibody (MAb) and detected with either biotinylated anti-FSH MAb (ELISA) or the sugar-specific lectins (L-ELISA) from Triticum vulgaris (sialic acid, SA), Sambucus nigra (α2,6-linked SA), Maackia amurensis (α2,3-linked SA) or Ricinus communis (free terminal galactose; GAL). Relative estimates of the amounts of terminal SA, its different forms and GAL were derived from the L-ELISA/ELISA data compared with the highly sialylated 1st International Standard for pituitary FSH (IS) 83/575.

All the FSH preparations had less SA than the IS with the ratio of α2,3- and α2,6-linked SA varying between preparations. The amounts of α2,6-linked SA relative to the IS were not significantly different in the urinary and pituitary preparations whereas α2,3-linked SA in all preparations was generally less than that of the standard. The rDNA FSH material, produced in CHO cells, was confirmed as containing only α2,3-linked SA. The degree of asialylation revealed by the Ricinus lectin showed that the more heavily sialylated IS had less exposed GAL than the other preparations. The method provides a simple and rapid technique for assessing changes in terminal sugar composition and allows comparison of the patterns of terminal glycosylation between preparations from different sources and, when assayed against the appropriate standard, between batches of therapeutic products.

Journal of Endocrinology (1995) 145, 527–533

Abstract

Assays have been developed for the isoforms of erythropoietin (EPO) based on their binding to eight different lectins. These assays were used to compare the isoform compositions of two preparations of human urinary EPO (uEPO) and four preparations of recombinant DNA-derived human EPO (rEPO), which had been shown to differ in their biological and immunological properties and in their isoform composition as judged by isoelectric focusing and electrophoresis.

Agarose-bound Ricinus communis agglutinin I (RCA), Erythrina cristagalli agglutinin (ECA), Maackia amurensis leukoagglutinin (MAL), Sambucus nigra agglutinin (SNA), Lycopersicon esculentum agglutinin (LEA), concanavalin A (Con A), Phaseolus vulgaris agglutinin-L4 (L-PHA) and Agaricus bisporus agglutinin (ABA) were used to bind EPO isoforms possessing: N-glycans containing non-sialylated outer Galβ1–4GlcNAc (RCA and ECA), NeuAcα2-3Galβ1–4GlcNAc (MAL), NeuAcα2–6Gal (SNA), or repeating Galβ1–4GlcNAc sequences (LEA); biantennary N-glycans (Con A); tetraantennary and 2,6-branched triantennary N-glycans (L-PHA); and O-glycans containing NeuAcα2–6GalNAc (SNA) and Galβ1–3GalNAc (ABA). Free EPO was measured by mouse spleen cell bioassay or immunoassay. Estimates from most lectin-binding assays were reproducible between assays and batches of lectin-agarose, although batches of MAL- and ABA-agarose, and to a lesser extent LEA-agarose, differed in their EPO-binding.

Lectin-binding assays showed differences between the isoform compositions of all EPOs, including the two Chinese hamster ovary cell-derived rEPOs, with RCA-and ECA-binding assays being the most discriminating. Lectin-binding estimates provided evidence that uEPO differs from these rEPOs in its lower content of isoforms with biantennary N-glycans and higher content of those with multiantennary N-glycans, and in its lower content of isoforms with N-glycans possessing repeating Galβ1–4GlcNAc sequences and of those with O-glycans containing Galβ1–3GalNAc. Lectin-binding estimates also indicated that, contrary to some reports, uEPO possesses Galβ1–3GalNAc-containing O-glycans but not NeuAcα2–6GalNAc-containing O-glycans or NeuAcα2–6Gal-containing N-glycans.

Most groups of lectin-bound EPO isoforms did not differ in their relative bioactivities and immunoreactivities. However, estimates for ABA-bound EPO isoforms suggested that O-glycans might influence the bioactivity of EPO differently to its immunoreactivity. Furthermore, the bioactivities of some ECA-bound EPO isoforms were higher, and those of some of the MAL-bound EPO isoforms lower, than their immunoreactivities, consistent with the reported enhancement of EPO in vitro bioactivity by desialylation.

Journal of Endocrinology (1996) 150, 401–412

ABSTRACT

We have studied the rebound secretion of GH following short-term somatostatin (SS) infusions in conscious rats, using an automatic sampling system for withdrawing frequent microsamples of blood. Intravenous infusions of SS (5–50 μg/h per rat) inhibited spontaneous GH secretion, but when SS was withdrawn there was a large burst of rebound GH secretion. A sub-anaesthetic dose of urethane reduced such rebound bursts of GH, suggesting a hypothalamic involvement in rebound GH secretion. Passive immunization with an antibody against rat GH-releasing factor (GRF) attenuated the rebound GH secretory response to the withdrawal of an SS infusion (GH concentration during rebound secretion was 26±21 μg/l vs 475 ± 127 μg/l (mean ± s.e.m.), after 0·5 ml anti-GRF serum or non-immune serum respectively). The inhibition of GH rebound secretion was related to the dose of anti-GRF serum administered. Intravenous infusions of human GH (20– 100 μg/h per rat) also reduced the size of the rebound GH secretion following SS withdrawal, in both male and female rats. We suggest that the rebound GH secretion that follows SS withdrawal in vivo is caused mainly by a hypothalamic release of GRF. Exogenous GH inhibits SS-induced rebound GH secretion in the conscious rat, possibly by inhibiting hypothalamic GRF release.

J. Endocr. (1988) 119, 397–404