a Department of Medical Sciences , Clinical Chemistry, University Hospital, Uppsala University , Sweden.
Ups J Med Sci. 2017 Nov;122(4):217-223. doi: 10.1080/03009734.2017.1412373. Epub 2018 Jan 4.
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.
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.
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.
The methods can be used for identification and characterization of glycoforms of circulating FSH, LH, and TSH in physiological and clinical studies.
FSH、LH 和 TSH 是脑垂体分泌的糖蛋白激素，以完全和低天冬酰胺糖基化的形式分泌。这些激素的糖型以大量的同工型存在，其糖链末端阴离子单糖（AMS），即唾液酸（SA）和磺化 N-乙酰半乳糖胺（SU）的含量不同。由于其巨大的异质性和血清中的低浓度，开发可靠的分析方法来测量和表征这些激素的循环糖型一直是一个挑战。
根据每个分子的 AMS 含量，用校准的 0.1%琼脂糖悬浮电泳对激素进行分离。然后，通过 180 校准的葡聚糖 G-100 凝胶过滤法，根据大小对分离的馏分中的糖型进行分析。用时间分辨夹心荧光免疫分析法测量激素。所有分离和测定均在 pH8.7 的硼酸盐缓冲液中进行。还在去除末端 SA 后分析血清和馏分。
除了完全糖基化的 FSH、LH 和 TSH 外，还可以在血清样本中鉴定出三糖基化的 FSH 和二糖基化的 LH 和 TSH 形式。低和完全糖基化的激素在大小和每个分子的 AMS 中位数方面都有所不同。基于电泳分布的算法，为每种激素开发了用于估计血清中低糖基化形式百分比的算法。使用脱唾液酸后获得的结果估计每个糖型分子中 SA 和 SU 的中位数数量。
该方法可用于鉴定和表征生理和临床研究中循环 FSH、LH 和 TSH 的糖型。
