Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine, Kyoto 6028566, Japan
1 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kamigyo-ku, Kyoto 6028566, Japan. e-mail: kitawaki{at}koto.kpu-m.ac.jp
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Abstract |
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Key words: bound leptin/controlled ovarian hyperstimulation/free leptin/IVF/soluble leptin receptor
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Introduction |
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In serum, leptin circulates as free and protein-bound forms (Sinha et al., 1996). A soluble LEPR (SLEPR) has been shown to account for the majority of the serum leptin binding activity (Lammert et al., 2001
; Maamra et al., 2001
). SLEPR consists entirely of the extracellular ligand-binding domain and lacks the transmembrane residues and intracellular domain responsible for signal transduction (Lee et al., 1996
). SLEPR plays a role in the regulation of the biological activity of leptin.
Recent studies have shown a reverse relationship between the serum SLEPR level and the body mass index (BMI) (Chan et al., 2002; Lahlou et al., 2002
; Shimizu et al., 2002
; Van Dielen et al., 2002
; Wu et al., 2002
). However, the relationships between the serum levels of SLEPR, and total, free and bound leptin have not been fully clarified. In the present study, we investigated these serum levels in non-pregnant Japanese women of reproductive age. In addition, the serum level of total leptin has been shown to be elevated under conditions of ovarian hyperstimulation (Mannucci et al., 1998
; Messinis et al., 1998
; Strowitzki et al., 1998
; Butzow et al., 1999
; Stock et al., 1999
; Lindheim et al., 2000
; Unkila-Kallio et al., 2001
). We investigated the change in the serum level of SLEPR during an IVF cycle.
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Materials and methods |
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Blood was drawn in the morning after an overnight fast. For the patients undergoing IVF, blood samples were collected at the following five points during the treatment: (i) the mid-luteal phase before the start of buserelin acetate (cycle day 21.6 ± 3.3); (ii) in the presence of ovarian suppression but before gonadotrophin administration; (iii) during maximal ovarian stimulation 12 days before the hCG injection; (iv) at the time of oocyte retrieval; and (v) 14 days after oocyte retrieval. Sera were separated by centrifugation (1700 g for 10 min) and kept frozen at 20°C until analysis. Serum concentration of estradiol was assayed using radioimmunoassay (Diagnostic Products Co, Los Angeles, CA, USA).
Assay of total, bound and free leptin in serum
Total leptin concentrations were initially measured by enzyme-linked immunosorbent assay (R & D Systems, Minneapolis, MN, USA). The detection limit was 7.8 pg/ml, and the intra- and inter-assay coefficients of variation were 3.03.3% at 64.5621 pg/ml and 3.55.4% at 65.7581 pg/ml respectively. Each concentration was determined by duplicate assays followed by averaging. Bound and free portions of leptin were subsequently separated by gel filtration chromatography as described by Sinha et al. (1996) with modifications. All experiments were performed at 4°C. A 2.0 ml aliquot of the serum sample was pre-incubated with [125I]-leptin (PerkinElmer Life Sciences Inc., Boston, MA, USA,
36 000 cpm, 0.27 ng) for 24 h, loaded onto a Sephadex G-100 column (1.5x40 cm) and then eluted with 25 mmol/l phosphate-buffered saline, pH 7.4, containing 0.01% sodium azide. The radioactivity in 50 fractions (1.0 ml each) formed two peaks. The first peak eluted in the void volume and represented the bound fraction, and the second peak represented the free fraction. The recovery of leptin from the column was 93.4 ± 3.0%. The percentages of bound and free leptin to total leptin were calculated by dividing by the total [125I]-leptin eluted. The absolute concentrations of bound and free leptin were calculated by multiplying the percentage of bound and free leptin, respectively, by the total leptin concentration and dividing by 100.
Assay of the soluble form of LEPR (SLEPR) in serum
The SLEPR concentration in serum was measured by enzyme-linked immunosorbent assay (BioVendor Laboratory Medicine Inc., Brno, Czech Republic). The detection limit was 0.4 unit/ml, and the intra- and inter-assay coefficients of variation were 5.68.2% at 8.4522.73 units/ml and 3.95.1% at 16.6535.15 units/ml respectively. Since the assay data were not influenced by the addition of up to 100 units/ml leptin to the serum, standards and controls, the kit appeared to measure the total SLEPR, either bound or free. Each concentration was determined by duplicate assays followed by averaging.
Statistics
Correlations between variables were examined by Pearsons correlation coefficients. Simple regression lines were made in which the maximal coefficient of determination (R2) was obtained. Variables during the IVF cycles were expressed as the mean ± SEM, and analysed using one-way ANOVA followed by multiple comparisons using Bonferroni/Dunns procedure. A P-value of <0.05 was considered significant.
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Results |
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Interestingly, although there was a positive correlation between the absolute concentrations of bound leptin and the BMI (r = 0.701, P < 0.0001) (Figure 2E), and a negative correlation between SLEPR and the absolute concentrations of bound leptin (r = 0.433, P < 0.0001) (Figure 2F), little variation was observed in the absolute concentrations of bound leptin compared with the free leptin levels, regardless of the BMI or SLEPR concentration. The mean concentration of bound leptin in all serum specimens was 2.5 ± 1.3 ng/ml.
The variations in the total leptin and SLEPR concentration in the serum during the IVF cycle are summarized in Figure 3. The total leptin levels at the time of maximal ovarian stimulation (group c) were significantly higher than the levels in the presence of ovarian suppression (group b) (P < 0.05) (Figure 3B) in accordance with the increase in serum estradiol levels (Figure 3A). In contrast, there was no significant difference in the serum level of SLEPR among the five different points measured (Figure 3D). There was no significant difference when the patients were divided into three groups according to the BMI. Accordingly, the absolute concentration of bound leptin was almost constant during the IVF cycle, while the absolute concentration of free leptin was higher at the time of maximal ovarian stimulation (group c) than in the presence of ovarian suppression (group b) (P < 0.05) (Figure 3C).
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Discussion |
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Our results show that the approximate serum levels of free leptin can be calculated simply by subtracting the constant value of the bound leptin level (2.5 ± 1.3 ng/ml) from the total leptin level. This amplifies the difference in free leptin levels between lean and obese women. Furthermore, our results show that, while the total serum leptin level increases with the BMI, the serum SLEPR levels are minimal in women with a BMI of 30 kg/m2 or higher, indicating that only the increase of free leptin accounts for the increase of total leptin in obese women. This suggests that the extremely high levels of leptin in obese women are over the programmed range within which the serum leptin activity can be regulated by SLEPR.
In the present study, the gel chromatographic separation of the serum specimens after incubation with radiolabelled leptin resulted in two major peaks, the bound and free peaks. This suggests that SLEPR is the major leptin binding component in human serum. The proportions of free and bound leptin in human serum were analysed by other groups using gel filtration chromatography (Sinha et al., 1996; McConway et al., 2000
), and were comparable with ours. Landt et al. (2000
) and Wu et al. (2002
) reported similar results using a different methodology to measure the bound form.
The present results show little variation in the serum level of SLEPR during the IVF cycle. In contrast, the serum level of total leptin is elevated during ovarian hyperstimulation, being consistent with previous reports by other groups (Butzow et al., 1997; Mannucci et al., 1998; Messinis et al., 1998
; Strowitzki et al., 1998; Stock et al., 1999; Lindheim et al., 2000; Unkila-Kallio et al., 2001). Although the exact mechanism is still unknown, these authors have postulated that the rise in the estrogen level is involved in the elevation of the leptin level. Again, our data show that the elevation of free leptin accounts for increases in total leptin and that the level of bound leptin is almost constant. Lewandowski et al. (1999
) measured the serum levels of SLEPR in pregnant women after 20 weeks gestation and showed no significant change, although the levels were
3-fold higher than our non-pregnant subjects. In contrast, the mRNA expression of the long form of LEPR in the endometrium was reduced during the secretory phase (Kitawaki et al., 2000
) and inhibited by the addition of progesterone in vitro (Koshiba et al., 2001
).
In conclusion, the serum level of SLEPR is inversely correlated with both the serum level of total leptin and the BMI in non-pregnant women of reproductive age. We have also demonstrated that the absolute level of bound leptin is almost constant, regardless of the leptin levels or the BMI. The serum SLEPR level does not change under the conditions of ovarian hyperstimulation during IVF treatment. These results demonstrate a skillful mechanism where a change in the serum SLEPR level regulates, in part, the biological activity of leptin in the circulation.
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Submitted on August 29, 2002; resubmitted on November 27, 2002; accepted on January 9, 2003.