Karin Eriksson

Serum Half-Life of Pituitary Gonadotropins Is Decreased by Sulfonation and Increased by Sialylation in Women

CONTEXT The gonadotropins are secreted from the human pituitary as spectra of isoforms with different degrees of sulfonation and sialylation of the oligosaccharides, modifications suspected to determine their half-lives in the circulation. OBJECTIVES Our objectives were to determine the isoform composition of the serum gonadotropins during GnRH receptor blockade, and to estimate the half-lives in circulation of isoforms with 0-1-2-3 sulfonated N-acetylgalactosamine (SO(3)-GalNAc) residues. DESIGN/PARTICIPANTS Serum samples were collected in seven healthy women before and up to 20 h after administration of the NAL-GLU GnRH antagonist. MAIN OUTCOME MEASURES The number of sialic acid and SO(3)-GalNAc residues per LH and FSH molecule and the distribution of molecules with 0-1-2-3 sulfonated residues were measured. The half-lives were estimated by monoexponential decay. RESULTS More sialylated and less sulfonated gonadotropin isoforms remain longer in circulation during GnRH receptor blockade. LH isoforms with two and three sulfonated residues per molecule had shorter half-lives compared with those with zero and one (109 and 80 vs. 196 and 188 min; P < 0.01). FSH isoforms with one and two sulfonated residues had shorter half-lives than those with zero (485 and 358 vs. 988 min; P < 0.01). CONCLUSIONS The decline in LH and FSH during GnRH receptor blockade is associated with a decrease in sulfonated and increase in sialylated residues. The rapid disappearance of LH isoforms with two and three SO(3)-GalNAc residues suggests their removal by hepatic SO(3)-GalNAc-receptors similar to those in rodents. Episodical secretion of spectra of isoforms with different half-lives is expected to lead to continuous changes in gonadotropin isoform compositions in blood.

The common genetic variant of luteinizing hormone has a longer serum half-life than the wild type in heterozygous women.

CONTEXT The common genetic variant of human LH has two mutations and an extra N-linked oligosaccharide chain, a modification expected to affect the half-life in the circulation. OBJECTIVES Our objectives were to determine the half-lives of variant and wild-type forms of LH during GnRH receptor blockade in heterozygous women and to determine the time-related changes in isoform composition. DESIGN AND PARTICIPANTS Serum samples were obtained from three healthy women heterozygous for variant LH before and up to 20 h after administration of the NAL-GLU GnRH antagonist. MAIN OUTCOME MEASURES The half-lives were estimated by monoexponential decay. The number of sialic acid and sulfonated N-acetylgalactosamine residues per wild-type and variant LH molecule and the distribution of molecules with zero, one, two, or three sulfonated residues were measured. RESULTS The variant LH had a half-life that was approximately 40% longer than the corresponding forms of wild-type LH (148 vs. 108 min; P < 0.001). Variant LH had more sialic acid residues per molecule than wild type (3.6 vs. 2.4; P < 0.05), whereas the number of sulfonated residues was similar (1.0 vs. 0.98). The decline in the variant LH during GnRH receptor blockade was associated with a decrease in sulfonated and an increase in sialic acid residues similar to that for in wild-type LH. Isoforms of either variant or wild-type LH with two to three sulfonate groups per molecule had the shortest half-life. CONCLUSION Variant LH remains longer in circulation than wild type during GnRH receptor blockade in heterozygous women, in accord with its higher content of sialic acid.

Dynamic changes in glycosylation and glycan composition of serum FSH and LH during natural ovarian stimulation.

Abstract Background. Glycosylation and glycan composition are of fundamental importance for the biological properties of FSH and LH. The aim of this study was to determine the glycosylation, sialylation, and sulfonation of serum FSH and LH throughout the normal menstrual cycle. Methods. Serum samples were collected from 79 healthy women with regular menstrual cycles. The mean numbers of anionic monosaccharide (AMS), sialic acid (SA), and sulfonated N-acetylgalactosamine (SU) residues per FSH and LH molecule were estimated for all sera with methods based on electrophoreses, neuraminidase treatments, and fluoroimmunoassays of the gonadotrophins. Results. Di-glycosylated glycoforms (FSHdi, LHdi) were detected in serum in addition to tetra-glycosylated FSH (FSHtetra) and tri-glycosylated LH (LHtri). FSHdi exhibited two peaks: one on day 5 to 7 and one, more pronounced, at midcycle. FSHtetra plateaued at a high concentration from day 5 to 15, without a midcycle peak. There were lower concentrations of LHdi than LHtri, except at midcycle when the opposite occurred. The mean numbers of SA and SU residues per molecule of FSH and LH in serum showed four different patterns during the cycle, all with highly significant (P < 0.0001) differences between levels at different phases of the cycle. The pattern of SA residues on FSH was ‘M’-shaped, and that of SU on LH ‘V’-shaped. Conclusion. Serum FSH and LH governing the natural ovarian stimulation process exhibited dynamic changes of glycosylation and glycan composition. This new information on the FSH and LH molecular structures may lead to more successful mono-ovulatory treatment regimens for ovulation induction in anovulatory women.

A New Principle Suggested for Detection of Darbepoetin-α (NESP) Doping

Doping with darbepoetin-α, also termed novel erythropoiesis stimulating protein (NESP), a hypersialylated, very effective analogue of erythropoietin, is a serious threat in sport. We report here on a new principle for the detection of darbepoetin-α in serum based upon increase in immunoactivity after desialylation with neuraminidase. The method is evaluated on sera from patients taken 2-14 days after last injection of darbepoetin-α. Thirty-two venous blood samples and 3 capillary samples taken from finger tips were obtained from 13 patients with end stage renal disease treated with intravenous or subcutaneous injections of Aranesp, 0.45 to 2.60 μg*kg-1. Blood samples from 37 individuals with endogenous erythropoietin were used as controls. The sera were diluted 1:2 with acetate buffer pH 5.6 with or without neuraminidase and incubated at 37°C for 1 or 24 h before immunoassay. The erythropoietin immunoactivity in serum volumes of 12.5-50 μL was measured with ELISA-kits from R&D Systems Inc and medac GmbH. The relative increase in immunoactivity after desialylation was in all cases higher for the darbepoetin-α samples than for any of the control samples assayed in parallel, varying incubation time with the enzyme, serum volumes and batches of both ELISA-kits. The mean relative increase in immunoactivity of endogenous erythropoietin after neuraminidase was 42% with the medac-kit and 117% with R&D-kit while the corresponding figures for darbepoetin-α were 282% and 231% with 1 h and 299% and 256% with 24 h enzyme incubation, respectively. Endogenous and recombinant human erythropoietin showed similar relative increase after desialylation. The method to detect darbepoetin-α in serum is simple to perform, robust, sensitive and requires a small amount of blood. The drug was detected in all patient sera taken 2-14 days after last injection. We suggest that the method should be evaluated for the detection of darbepoetin-α doping in sport.

Time-related effects of a progestogen on the isoforms of serum gonadotrophins in 17β-oestradiol treated post-menopausal women

OBJECTIVE It has previously been shown that 17β‐oestradiol (E2) implants counteract the formation of more acidic isoforms of the gonadotrophins in post‐menopausal women. A much lesser effect was observed on the charge of the gonadotrophin isoforms in women with chronic oral daily therapy with 2 mg E2 combined with a progestogen, 1 mg norethisterone acetate (NETA), in spite of similar serum levels of E2 and SHBG. The presence of the progestogen in the latter study may explain the difference observed. The present study investigated the effect of the progestogen NETA on the charge and concentration of serum FSH and LH in E2 implant treated women.

Effects of 17β-oestradiol and norethisterone acetate on sulfonation and sialylation of gonadotrophins in post-menopausal women

Abstract Background. The number of terminal sialic acid and sulfonated N-acetylgalactosamine (SO3-GalNAc) on gonadotrophins in serum varies during the menstrual cycle and changes at menopause, suggesting that gonadal steroids modify their oligosaccharide synthesis. Our objective was to determine the effects of 17β-oestradiol (E2) and a progestogen, norethisterone acetate (NETA), on the sulfonation and sialylation of gonadotrophins in post-menopausal women. Methods. Serum samples were obtained from eight post-menopausal women treated with 20 mg E2 implants every 6 months, from four women who in addition were treated daily with 5 mg NETA orally for a 2-week period, and from four women who got this NETA treatment during a 4-week period. Sera from 11 non-treated post-menopausal women served as a reference group. The gonadotrophin serum concentrations, the number of SO3-GalNAc and sialic acid residues per serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) molecule, and the distributions of molecules with 0-1-2-3-4 sulfonated residues were measured. Results. The E2-treated post-menopausal women had considerably less (P < 0.001) sialic acid and slightly more (P < 0.01) SO3-GalNAc per serum LH and FSH molecule than the non-treated. Two weeks of NETA treatment increased the sulfonation of LH (P < 0.01) and FSH (P < 0.05) concomitantly with decreased (P < 0.05) sialylation of LH. Conclusion. The primary effect of E2 treatment was a decrease in sialylation and, due to competition for the same substrate, a secondary and consequentially minor increase in sulfonation of LH and FSH. The primary effect of the NETA therapy was an increase in the sulfonation of LH and FSH concomitantly with secondary and consequentially decreases in sialylation of LH.

Molecular size and charge as dimensions to identify and characterize circulating glycoforms of human FSH, LH and TSH

Abstract Background: FSH, LH, and TSH are glycoprotein hormones secreted from the pituitary as fully and low-asparagine-glycosylated hormones. These glycoforms of the hormones exist as a large number of isoforms varying in their glycan contents of terminal anionic monosaccharides (AMS), i.e. sialic acid (SA) and sulfonated N-acetylgalactosamine (SU). Due to the immense heterogeneity and the low concentrations in serum it has been a challenge to develop reliable analytical methods to measure and characterize the circulating glycoforms of these hormones. Methods: The hormones were separated with respect to AMS content per molecule by calibrated 0.1% agarose suspension electrophoreses. Glycoforms in separated fractions were then analyzed with respect to size by 180 calibrated Sephadex G-100 gel filtrations. The hormones were measured with time-resolved sandwich fluoroimmunoassays. All separations and assays were performed in veronal buffer at pH 8.7. Sera and fractions were also analyzed after removal of terminal SA. Results: In addition to the fully glycosylated FSH, LH, and TSH, also tri-glycosylated FSH and di-glycosylated LH and TSH forms could be identified in serum samples. The low- and fully glycosylated hormones differed both with respect to size and to median number of AMS per molecule. Algorithms, based on the distributions by electrophoreses, were developed for each hormone to estimate percent low-glycosylated forms in serum. The median numbers of SA and SU per glycoform molecule were estimated using results obtained after desialylation. Conclusion: The methods can be used for identification and characterization of glycoforms of circulating FSH, LH, and TSH in physiological and clinical studies.

Low-glycosylated forms of both FSH and LH play major roles in the natural ovarian stimulation

Abstract Background: The natural ovarian stimulation is mediated by four gonadotrophin glycoforms: FSHtri with three, FSHtetra with four, LHdi with two, and LHtri with three N-glycans. The aim of the study was to determine the serum concentrations of the four glycoforms and their contents of anionic monosaccharides (AMS), i.e. sialic acid (SA) and sulfonated N-acetylgalactosamine (SU) residues throughout the menstrual cycle. Methods: Serum samples were collected from 78 healthy women with regular menstrual cycles. The serum glycoform molecules were identified by their distributions at electrophoreses. Analyses were also performed after removal of terminal SA. The hormones were measured with time-resolved sandwich fluoroimmunoassays. Results: The concentration profiles of the four glycoforms were markedly different. FSHtri, which had a 3-fold higher biopotency than FSHtetra, had peak levels on cycle day 5 and at midcycle and nadirs on cycle days 9 and 21–23. FSHtetra had a raised level on cycle days 5–12, followed by a decrease. LHdi and LHtri had similar patterns, but the peak/nadir ratio was much more pronounced for LHdi than for LHtri, 18 versus 4. The numbers of SA residues per molecule were at a maximum around midcycle when the corresponding numbers of SU were at a minimum. The SU/SA ratio was at a minimum on cycle day 12. Conclusion: The results indicate that the LHdi and the FSHtri molecules play major roles in the natural ovarian stimulation. The SU/SA ratios per molecule favoured a prolonged circulatory half-life of all glycoforms at the midcycle phase. The observations may lead to more successful inductions of ovulation in anovulatory women.