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Departments of Medical Sciences, Clinical Chemistry (L.W., K.E.), and Women’s and Children’s Health, Obstetrics and Gynaecology (T.N., I.S.-P.), University Hospital, SE 751 85 Uppsala, Sweden
Address all correspondence and requests for reprints to: Leif Wide, M.D., Ph.D., Department of Clinical Chemistry, University Hospital, SE 751 85 Uppsala, Sweden. E-mail: leif.wide{at}medsci.uu.se.
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Abstract |
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Objective: The objective of the study was to determine the contributions of sialic acid and sulfonated N-acetylgalactosamine and sialic acid on LH and FSH to the observed alterations in charge.
Design/Participants: Serum samples were obtained from 59 young women with regular cycles, nine postmenopausal women, 12 women with PCOS, and 40 young men.
Main Outcome Measures: The number of sulfonated N-acetylgalactosamine and sialic acid residues per LH and FSH molecule in serum and the distributions of molecules with 0-1-2-3-4 sulfonated residues were determined by electrophoretic analyses before and after removal of sialic acid.
Results: Considerably decreased sulfonation of LH was found at midcycle and in women with PCOS concomitant with slightly increased sialylation. The sulfonation of LH increased in the luteal phase, and the sialylation was highest after menopause for both hormones. The frequencies of sulfonated LH and FSH isoforms were directly related (P < 0.01) to body mass index in women with PCOS.
Conclusion: The observed variations in sialic acid and sulfonated residues on serum gonadotropins are suggested to reflect alterations in the isoform composition of the hormones secreted by the pituitary, resulting in modulations of their biological properties, such as half-life in blood.
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Introduction |
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Increased number of sialic acid increases the half-life of human FSH in the circulation (5, 6) and can reduce the bioactivity of the molecule at the target organ (7, 8). LH molecules with increased number of sulfonated N-acetylgalactosamine (GalNAc) disappear more rapidly from the circulation due to binding to a receptor in the liver specific for the SO3-4GalNAcß1–4GlcNAc terminus (9). Hitherto, estimations of the number of sialic acid and sulfonated GalNAc residues on human FSH, LH, and TSH have been restricted to hormones purified from very large pools of pituitaries (3).
The isoforms of both FSH and LH circulating in blood have been reported to be less negatively charged at midcycle, compared with the follicular and luteal phase of the normal menstrual cycle (10, 11, 12). Both gonadotropins become more negatively charged at menopause when the serum levels increase (13, 14, 15). It is not known whether these variations in charge are due to changes in sialic acid and/or sulfonate groups on the serum gonadotropins. To investigate this, we developed a method for estimation of the average number of sialic acid and sulfonated GalNAc residues per FSH and LH molecule in individual human serum samples (about 1 ml). This method is based on neuraminidase treatment, electrophoresis, and immunofluorometric hormone assays.
The GalNAc-4-SO3 receptor is dimeric and binds bovine LH with a high affinity only when there are two or more sulfonated GalNAc residues present on multiple oligosaccharides (16). Because human LH has a similar structure to bovine LH, the distribution of molecules with zero to four sulfonated GalNAc residues per molecule was determined for each sample.
All sera contain a large number of isoforms of the gonadotropins, and the isoform composition varies within and between individuals. We describe here the changes during the normal menstrual cycle and after menopause in the average number of sialic acid and sulfonated GalNAc residues per LH and FSH molecule and in the distributions of isoforms with zero to four sulfonated residues. For comparison, analyses of sera from 40 men were also included.
A preponderance of more alkaline LH isoforms accompanied with raised serum LH levels has been found in both adolescents and adults with polycystic ovarian syndrome (PCOS) (17, 18). An inverse relationship was observed between the half-life of LH and body mass index (BMI) in PCOS patients (19), but it has not been definitely settled whether there is a prolonged or a shorter half-life of LH in the circulation in this syndrome, compared with fertile women (18, 19). More basic isoforms due to a decrease in sulfonated GalNAc is expected to increase the half-life, whereas a shorter half-life is expected of a decrease in the sialic acid content. In this study the degrees of sulfonation and sialylation of serum LH and FSH were investigated in a group of 12 adult women with PCOS.
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Subjects and Methods |
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As part of the training in clinical chemistry for medical students at Uppsala University Hospital, blood samples were taken from about 120 students each year during the period 2000–2006. The samples were taken on an ambulatory basis in the morning, after an overnight fast and analyzed for a large number of different compounds. All students who agreed to participate gave a health declaration with information about diseases, medications, alcohol intake, and recent physical activity. In women, information was given about the menstrual bleedings (first day of last menstruation, menstrual cycle length, and regularity over the last year) and hormonal contraceptives. Serum samples from different phases of the cycle were obtained from 59 women. These women were selected on the criteria that they were apparently healthy, had regular menstrual cycles (mean 28 d, range 25–31), and did not use any hormonal contraception. The cycle day was adjusted to a 28-d menstrual cycle. The midcycle phase group included 15 samples from d 12–16 plus two samples with LH levels above 20 IU/liter, taken on d 11 and 17. The number of women in the follicular and luteal phases were 22 and 20, respectively.
Serum samples from 40 male medical students were selected with the only criterion that they were apparently healthy.
Serum specimens were also obtained from nine apparently healthy postmenopausal women, residents in the Uppsala community, without a history of estrogen replacement therapy.
Serum samples from a group of 12 women with the diagnosis of PCOS were obtained before therapy was initiated. PCOS was defined according to the Rotterdam criteria (20). The following three features were present in all patients: 1) oligoamenorrhea with eight or fewer menstruations in the previous 12 months or amenorrhea, 2) clinical and/or biochemical signs of hyperandrogenism, 3) polycystic ovaries on ultrasound examination. The patients had no other identifiable diseases and normal fasting glucose levels. The LH to FSH ratio was in nine of the 12 women with PCOS above the upper 95% limit (1.64) of the follicular phase group. Six PCOS patients were nonobese with BMIs ranging from 21.1 to 25.6 kg/m2 and six with BMIs from 28.3 to 45.9 kg/m2 were obese, defined by a BMI above 27 kg/m2 (21).
Immunoassays for gonadotropins
The concentrations of LH and FSH in serum samples and 200-µl fractions eluted after agarose suspension electrophoresis were measured using sandwich fluoroimmunoassays (Delfia; PerkinElmer-Wallac Oy, Turku, Finland), as previously described (22). Gonadotropin values were expressed in international units per liter using the International Standards for pituitary LH (80/552) and FSH (94/632) as reference standards. The detection limits in serum were 0.02 IU/liter and the interassay coefficient of variation was less than 3% for both hormones.
There is a common (about 17% of 816 medical students in Uppsala) genetic variant form of LH that has two mutations (Trp8Arg and Ile15Thr) on the ß-subunit with an extra glycosylation consensus site (Asn13-Ala-Thr), compared with the wild-type LH (23). All sera were initially tested to exclude from the present study all individuals with this variant form of LH. This was made by determination of the ratio of two LH immunoassay measurements, one detecting only wild type of LH, the other both the wild-type and variant form of LH using the procedure previously described (23, 24). Reagents for the immunoassay that detects only the wild type of LH was kindly supplied by Dr. Kim Pettersson (Department of Biotechnology, University of Turku, Turku, Finland).
Neuraminidase treatment of serum
Each serum (500 µl) was desalted and buffer exchanged using an NAP-5 column (GE Healthcare, Uppsala, Sweden) equilibrated with 10 ml 0.2 M sodium acetate buffer (pH 5.6). The proteins were eluted with 1 ml acetate buffer and then mixed and incubated for 24 h at 37 C with 70 mU neuraminidase (from Arthrobacter ureafaciens, EC.3.2.1.18; MP Biomedicals, Solon, OH) in 25 µl phosphate buffer. The mixture was then kept at –20 C until analyzed (900 µl) by electrophoresis. The mean ± SEM recovery of FSH activity after neuraminidase treatment of 56 sera was 109.6 ± 1.4% and the corresponding figures for LH were 100.3 ± 1.6%.
Agarose suspension electrophoresis
The serum samples were analyzed before and after neuraminidase treatment with a modification of the electrophoretic technique previously described (25), using a suspension of 0.10% agarose (Agarose IEF; GE Healthcare) in 75 mmol/liter sodium veronal buffer (pH 8.7). The temperature of the column was 12–13 C. Each serum sample (500 µl) was run on a 1.3 x 67 cm column for 18 h at 52 mA. The start zone was identified by adding 10 µl of a lipid solution (Intralipid; Fresenius KABI, Uppsala, Sweden) to the serum and the eluted fractions were read at 340 nm. The zone with albumin was read at 280 nm. Human serum albumin was used as an internal standard. The charge was expressed as electrophoretic mobility in albumin mobility units (AMU); 1 AMU = 1 cm2 x S–1 x V–1 x 104. Fractions eluted from the column were measured for hormone activity and the relationship between mobility and hormone activity was plotted.
Estimation of number of sulfonated GalNAc and sialic acid residues per molecule
The mobility of human FSH without sulfonated GalNAc and sialic acid residues was estimated to 132 mAMU, determined with desialylated recombinant human FSH [Gonadal-F; Ares-Serono (Europe Ltd.), London, UK] added to serum. The increase in mobility per charged group on FSH was estimated to 61.6 mAMU using for calibration a highly purified pituitary human FSH preparation, Roos Ca 54 containing eight sialic acid groups per FSH molecule (26). The mobility of LH without sulfonate and sialic acid groups was minus 49 mAMU and the increase in mobility per charged group on LH was 81 mAMU. These figures were determined from analyses of the electrophoretic LH peak patterns of 48 serum samples.
The area of hormone activity plotted in relation to mobility was resolved into peaks at the positions determined for different numbers of charged groups on FSH and LH, respectively. This is illustrated in Fig. 1
for LH in neuraminidase-treated sera from two women, one with PCOS and one in the luteal phase. The percentage of the total activity eluted at each charge position was multiplied with the number of charged groups at the position. The sum of the products represented the mean number of charged groups per FSH or LH molecule in the serum sample. Before neuraminidase treatment this sum represented the mean number of negatively charged groups (sulfonated GalNAc plus sialic acid) per molecule and after the treatment the mean number of sulfonated GalNAc residues per molecule. The difference between the two mean numbers represents the mean number of sialic acid groups per molecule.
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Statistical methods
The data were expressed as mean ± SEM if normally distributed or as median and percentiles if data were skewed. Normality was checked by Kolmogorov-Smirnov testing. Relationships were identified by calculating Pearson r and differences between groups were examined by two-tailed t tests when the data were normally distributed. The Mann-Whitney rank-sum test was used if the data were skewed. Statistical significance was accepted at P < 0.05.
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Results |
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The mean values of sulfonated GalNAc and sialic acid residues per LH and FSH molecule are presented in Table 1 for the six groups of individuals: follicular phase, midcycle phase, luteal phase, postmenopausal, women with PCOS, and men. The distributions of isoforms of LH and FSH with 0-1-2-3-4 sulfonated GalNAc residues per molecule in the serum samples, expressed as percent of total, are also given in Table 1.
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Because both sialic acid and SO3–4GalNAc groups can modify the half-life of the gonadotropins, the relationships between the LH and FSH serum levels and the contents of sialic acid and sulfonate groups were plotted and are shown as group mean values with 95% confidence limits of mean in Fig. 2(upper and middle panels). This figure (lower panels) also illustrates the relationships between the contents of sialic acid and sulfonated GalNAc residues per molecule on LH and FSH as group mean values with 95% confidence limits of mean.
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The concentrations of the gonadotropins and the mean contents of sulfonated GalNAc and sialic acid residues per LH and FSH molecule in 59 individual sera were plotted in relation to a 28-d menstrual cycle in Fig. 3.
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). The values then returned during the luteal phase to follicular phase levels.
The distribution patterns of isoforms of LH and FSH with zero to four sulfonated GalNAc residues per molecule are shown in Fig. 4. The figure illustrates the large differences in the patterns for LH, compared with FSH. The LH molecules had similar distribution patterns during follicular and luteal phases, whereas the pattern at midcycle was different with a larger frequency with 0 and fewer with two to four sulfonated GalNAc residues per isoform. The highest frequency of LH isoforms with two to four sulfonated residues was found in the luteal phase, and this value, 36.7%, was significantly higher than those of all other groups studied (Table 1).
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). During the follicular phase, the cycle day exhibited a positive correlation with sialylation (r = 0.52; P = 0.012) and a negative correlation with sulfonation of FSH (r = –0.46; P = 0.032). The reverse was observed during the luteal phase when day of cycle exhibited a negative correlation with sialylation (r = –0.44; P = 0.050) and positive correlation with sulfonation of FSH (r = 0.62; P = 0.004). Postmenopausal women
The isoforms of both LH and FSH were significantly more acidic in the group of postmenopausal women than in all other groups of this study (Table 1). The sialic acid contents of both LH and FSH in the postmenopausal women were the highest of all groups (Fig. 2). The contents of sulfonated GalNAc per LH and FSH were significantly lower in these women, compared with the follicular and luteal phase values. The distribution pattern of LH isoforms with different number of sulfonated GalNAc was similar with that at the midcycle phase (Fig. 4).
PCOS
The group of women with PCOS had a mean serum LH concentration significantly raised above the mean level in the follicular phase (Table 1). The sulfonation of the LH molecules was significantly decreased and the sialylation increased in PCOS patients, compared with follicular and luteal phase values (Table 1).
The distribution pattern of isoforms with zero to four sulfonated GalNAc residues per LH molecule in the PCOS women was different from the other groups as shown in Fig. 4. The women with PCOS had a high frequency of LH isoforms with no sulfonated GalNAc residues (41%). The frequencies of LH isoforms with two (9.5%) and two to four sulfonated GalNAc residuals (15.9%) was significantly lower than in all other groups (Table 1).
There were significantly positive correlations between BMI and the frequency of sulfonated isoforms of LH (r = 0.74; P = 0.008) and FSH (r = 0.84; P < 0.001) in serum of the PCOS patients (Fig. 5). For comparison, the corresponding individual values for the 22 healthy women in follicular phase are shown in Fig. 5.
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When compared with the follicular phase values, the sulfonation of LH in the nonobese PCOS patients had decreased more (–0.500 ± 0.045 residues; P < 0.001) than the sialylation had increased (+0.382 ± 0.056 residues; P < 0.001), resulting in more basic isoforms (–9.52 ± 3.74 mAMU; P = 0.017) in this PCOS group.
The FSH isoforms were more sialylated and less sulfonated in the PCOS patients than in the women during the normal menstrual cycle (Table 1). The women with PCOS had a high frequency of FSH isoforms with no sulfonated GalNAc residues (92%), and two serum samples from nonobese PCOS patients contained less than 0.01 sulfonated GalNAc residues on the FSH isoforms.
Men
The LH and FSH isoform compositions in the young healthy men differed from those of the young healthy women (Fig. 2). Both LH and FSH were significantly more sialylated in men than women during the normal menstrual cycle. The mean number of sulfonated GalNAc residues per LH molecule and the frequency of two to four sulfonated residues per LH molecule in men was significantly lower than in women at the follicular and luteal phases and higher than at the midcycle phase. The sulfonation of FSH in men was lower than in women during the menstrual cycle (Table 1). Three men had less than 0.05 sulfonated residues per FSH molecule.
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Discussion |
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The sulfonation pathway to terminal SO3–4GalNAc is first controlled by a ß1–4GalNAc-transferase activity that modifies the subterminal GlcNAc residues of the biantennary glycan chains (27). This enzymatic effect occurs in competition with a ß1–4 galactosyltransferase in a sialylation pathway leading to terminal sialic acid. A peptide motif, a tripeptide six- to nine-residue aminoterminal of the glycosylation site, on LH increases the catalytic efficiency of the ß1–4GalNAc-transferase and enhances the sulfonation pathway on LH (28). FSH is synthesized within the same pituitary cell as LH (29). The peptide motif on LH is not present on the FSH ß-subunit with the consequence that the sulfonation pathway is expressed to a lesser degree and the sialylation pathway is dominating on FSH. Our results are in agreement with these differences in expression of the sulfonation and the sialylation pathways on LH vs. FSH.
The hormones are secreted in pulses from the pituitary as spectra of isoforms with different half-lives in the circulation. Isoforms with a low number of sialic acid residues (6) and those with two or more sulfonated GalNAc residues are expected to disappear faster than the other (16). These dynamic changes in the isoform composition in the circulation between the pulsatile secretions are not revealed when single samples are taken at random, as in the present study.
The results of this study confirm previously reported alterations in charge of serum FSH and LH in different physiological and pathological conditions (10, 11, 12, 15, 17, 18). The more basic isoforms of LH at midcycle and in nonobese women with PCOS were due to considerably decreased sulfonation of LH concomitant with slightly increased sialylation. Both these changes are expected to increase the survival time in the circulation, raising the serum LH level. This decreased sulfonation of LH may be of physiological importance at midcycle for the induction of ovulation and also an important factor in the pathogenesis of PCOS in some women. The mechanisms behind the decreased sulfonation are not known, but it seems likely that they are mediated via a decreased ß1–4GalNAc-transferase activity. In contrast to LH, the sialylation of FSH decreased at midcycle, leading to in a shorter half-life of FSH (12).
The highest frequency (36.7%) of two to four sulfonated GalNAc residuals on LH was observed in women during the luteal phase of the cycle. For FSH there was a significant correlation between day of luteal phase and the content of sulfonated GalNAc residues per molecule. A possible explanation is that progesterone induces an increase in the ß1–4GalNAc-transferase activity.
After menopause both FSH and LH increase in serum concentrations. The changes of the isoforms to more sialylated and less sulfonated are compatible with a longer half-life, as has been reported for FSH (5, 6). Estrogen given to postmenopausal women has been shown to counteract the charge alterations of FSH and LH seen at menopause (14). The most likely explanation is that estrogen inhibits enzyme activity on the sialylation pathway in the pituitary.
The finding of a positive relationship between BMI and the frequency of sulfonated LH isoforms in serum of women with PCOS is compatible with the negative relationship between BMI and the half-life of LH in PCOS patients observed by Srouji et al. (19). Our results indicate that obesity in women with PCOS is associated with alterations in the isoform compositions of both LH and FSH.
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Footnotes |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online August 21, 2007
Abbreviations: AMU, Albumin mobility unit; BMI, body mass index; GalNAc, N-acetylgalactosamine; PCOS, polycystic ovarian syndrome; SO3-4GalNAc, sulfonated ß1–4-linked N-acetylgalactosamine.
Received June 18, 2007.
Accepted August 13, 2007.
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References |
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that mediates rapid clearance of lutropin. Cell 67:1103–1110[CrossRef][Medline]This article has been cited by other articles:
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  L. Wide, K. Eriksson, P. M. Sluss, and J. E. Hall Serum Half-Life of Pituitary Gonadotropins Is Decreased by Sulfonation and Increased by Sialylation in Women J. Clin. Endocrinol. Metab., March 1, 2009; 94(3): 958 - 964. [Abstract] [Full Text] [PDF]
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, follicular phase (n = 22); 
, midcycle phase (n = 17); 
, luteal phase (n = 20); 
, postmenopausal (n = 9); •, women with PCOS (n = 12); 
, healthy men (n = 40).
