Igci Nasit, Sharafi Parisa, Demiralp Duygu Ozel, Demiralp Cemil Ozerk, Yuce Aysel, Emre Serap Dokmeci
Department of Molecular Biology and Genetics, Faculty of Sciences and Arts, Nevsehir Haci Bektas Veli University, Turkey.
Medical Biology Department, Faculty of Medicine, Hacettepe University, Ankara, Turkey.
Adv Clin Exp Med. 2017 Oct;26(7):1053-1061. doi: 10.17219/acem/65784.
Gaucher disease (GD) is defined as an autosomal recessive disorder resulting from the deficiency of glucocerebrosidase (E.C. 3.2.1.45). Glucocerebrosidase is responsible for the degradation of glucosylceramide into ceramide and glucose. The deficiency of this enzyme results in the accumulation of undegraded glucosylceramide, almost exclusively in macrophages. With Fourier transform infrared (FTIR) spectroscopy, the complete molecular diversity of the samples can be studied comparatively and the amount of the particular materials can be determined. Also, the secondary structure ratios of proteins can be determined by analysing the amide peaks.
The primary aim of this study is to introduce FTIR-ATR spectroscopy technique to GD research for the first time in the literature and to assess its potential as a new molecular method.
Primary fibroblast cell cultures obtained from biopsy samples were used, since this material is widely used for the diagnosis of GD. Intact cells were placed onto a FTIR-ATR crystal and dried by purging nitrogen gas. Spectra were recorded in the mid-infrared region between 4500-850 cm-1 wavenumbers. Each peak in the spectra was assigned to as organic biomolecules according to their chemical bond information. A quantitative analysis was performed using peak areas and we also used a hierarchical cluster analysis as a multivariate spectral analysis.
We obtained FTIR spectra of fibroblast samples and assigned the biomolecule origins of the peaks. We observed individual heterogeneity in FTIR spectra of GD fibroblast samples, confirming the well-known phenotypic heterogeneity in GD at the molecular level. Significant alterations in protein, lipid and carbohydrate levels related to the enzyme replacement therapy were also observed, which is also supported by cluster analysis.
Our results showed that the application of FTIR spectroscopy to GD research deserves more attention and detailed studies with an increased sample size in order to evaluate its potential in the diagnosis and follow-up of GD patients.
戈谢病（GD）被定义为一种常染色体隐性疾病，由葡萄糖脑苷脂酶（E.C. 3.2.1.45）缺乏所致。葡萄糖脑苷脂酶负责将葡糖神经酰胺降解为神经酰胺和葡萄糖。该酶的缺乏导致未降解的葡糖神经酰胺积累，几乎全部在巨噬细胞中。通过傅里叶变换红外（FTIR）光谱，可以比较研究样品的完整分子多样性，并确定特定物质的含量。此外，通过分析酰胺峰可以确定蛋白质的二级结构比例。
本研究的主要目的是首次在文献中将FTIR-ATR光谱技术引入戈谢病研究，并评估其作为一种新的分子方法的潜力。
使用从活检样本中获得的原代成纤维细胞培养物，因为这种材料广泛用于戈谢病的诊断。将完整细胞置于FTIR-ATR晶体上，通过吹扫氮气进行干燥。在4500 - 850 cm-1波数的中红外区域记录光谱。根据光谱中每个峰的化学键信息将其指定为有机生物分子。使用峰面积进行定量分析，并且我们还使用层次聚类分析作为多变量光谱分析。
我们获得了成纤维细胞样本的FTIR光谱，并确定了峰的生物分子来源。我们在戈谢病成纤维细胞样本的FTIR光谱中观察到个体异质性，在分子水平上证实了戈谢病中众所周知的表型异质性。还观察到与酶替代疗法相关的蛋白质、脂质和碳水化合物水平的显著变化，聚类分析也支持这一点。
我们的结果表明，将FTIR光谱应用于戈谢病研究值得更多关注，并进行样本量增加的详细研究，以评估其在戈谢病患者诊断和随访中的潜力。
