FSH isoform pattern in classic galactosemia

Blood samples from five galactosemia patients (aged between 21 and 26 years) and two PMM2-CDG patients (both aged 33 years) with hypergonadotropic hypogonadism and five postmenopausal women (aged 45–68 years) were obtained under standard conditions, and serum was kept frozen at –80°C until assayed. All women were diagnosed with ovarian insufficiency by FSH measurements (>30 IU/l) and clinical features (amenorrhea). The women used no medication at the time of blood withdrawal (hormone replacement therapy, if used, was stopped for at least 7 consecutive days before blood withdrawal). Because both galactosemia patients and PMM2-CDG patients with POI have biochemical (i.e. hypergonadotropic- hypoestrogenic) postmenopausal-like features, we decided to use serum of postmenopausal women as controls. The institutional review board of Maastricht University Medical Center approved the protocol as part of a larger study, and the protocol is in accordance with the Declaration of Helsinki. After oral and written information about the study design and protocol, the patients signed an informed consent for this study.

We studied chromatofocusing isoform distribution patterns of FSH according to its net charge. The analytical procedure and the consecutive statistical analysis was described in detail by Thomas et al. (2009). Instead of 2.5 mL serum, we used 0.5 mL serum for the present procedure. Samples and buffers were prepared as described previously, and an Äkta FPLC system (GE Healthcare) was equipped with a 4-mL Mono-P 5/200 GL column and equilibrated with 40 mL of startbuffer (i.e., bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane; bis-tris, 7.14 mM, pH 7.3-7.4). A pH gradient was generated by introduction of elution buffer (Polybuffer 74; GE Healthcare Biosciences, Uppsala, Sweden) used at a dilution of 1:35, pH 3.2 at a flow rate of 1.0 mL per min. After collection of 70 consecutive 2-mL effluent fractions in tubes containing 50 μL of 0.8 M phosphate buffer in 0.15 M NaCl with 0.2% NaN3 (pH 7.4), the pH was 3.2. In every fifth tube not containing phosphate buffer, the pH was measured manually with a pH meter (Metrohm 744; Metrohm, Herisau, Switserland). To increase sensitivity, all the collected fractions were concentrated 10-fold by evaporating them under nitrogen gas at 30°C overnight. Subsequently, the dried fractions were reconstituted in 0.2 ml ultra pure water (Elga Ultra Pure Water System, High Wycombe, UK) and the FSH concentrations in all individual effluent fractions were determined with the random access analyser Architect (Abbott, Chicago, IL, USA). The measured FSH concentrations in all collected effluent pH fractions were corrected for procedural losses by taking the quantity of FSH loaded onto the Mono-P column as 100%. From these corrected data, we calculated the relative amounts of FSH at fixed pH values of pH 7.0, 6.9, 6.8, …, to pH 3.0 by interpolation. In order to test the largest discriminatory capacity between the five galactosemia patients and the two PMM2-CDG patients with hypergonadotropic hypogonadism and the five postmenopausal women, we determined with Student’s t tests in Excel 2007 for which pH interval (i.e., the sum of the relative amounts of FSH present in several consecutive fixed pH fractions), the difference between the sums of the FSH amounts in the various groups, was largest. In addition, we calculated the mean, median and peak values (defined as the pH interval in which the highest percentage of FSH was found) for each group from the experimental values and compared the obtained values using SPSS 16.0.