氘标记的胆固醇-葡聚糖颗粒(d4-CholDex),通过共沉淀法制备,被培养的人皮肤成纤维细胞和HEK293细胞内化。在碘克沙醇梯度上分离来自d4-CholDex处理的HEK293细胞的亚细胞颗粒。梯度中超过60%的d4-胆固醇(d4-UC)与溶酶体标记和NPC1共分级。细胞中d4-胆固醇酯(d4-CE)的形成表明d4-CholDex是溶酶体加工的。根据这些发现,我们通过荧光显微镜观察到负载CholDex的细胞中溶酶体胆固醇含量增加。成纤维细胞培养物,包括13个NPC1缺陷,用d4-CholDex以最终d4-UC浓度为0.05mg/ml(127.98μmol/L)处理四个杂合子和六个对照品系3h,并在不含d4-CholDex的培养基中追逐48h。通过串联质谱法(MS/MS)测量收获的细胞中d4-UC和d4-CE的浓度。与对照组相比,NP-C系的d4-UC/d4-CE比率升高(n=6,平均值=4.36,范围=1.89-8.91),严重NP-C1表型患者的比率最高,青少年/成人型患者的比率最低。NP-C1形式之间存在重叠:早期婴儿(n=1,平均值=48.6),晚期婴儿(n=4,平均值=36.3,范围=20.6-54.0),少年(n=5,平均值=24.7,范围=13.4-38.3),青少年/成人(n=3,平均值=14.5,范围=11.7-19.8)。NP-C1杂合子的比率轻度升高(n=4,平均值=16.4,范围=14.9-17.4),与青少年/成人NP-C1患者相当。该测试可用于评估可疑NP-C患者,其生物标志物或分子测试结果不确定。其优点包括在4°C下具有更长保质期的颗粒的标准化制备,定量结果,对放射性化学品没有要求.
Deuterium-labeled cholesterol-dextran particles (d4-CholDex), prepared by co-precipitation, were internalized by cultured human skin fibroblasts and HEK293 cells. Subcellular particles from d4-CholDex-treated HEK293 cells were fractionated on iodixanol gradients. More than 60% of d4-cholesterol (d4-UC) in the gradient co-fractionated with lysosomal markers and NPC1. This and formation of d4-cholesteryl esters (d4-CE) in the cells suggests that d4-CholDex is lysosomally processed. In accordance with these findings, we observed an increase in lysosomal cholesterol content by fluorescence microscopy in CholDex-loaded cells. Fibroblast cultures including 13 NPC1-deficient, four heterozygous and six control lines were treated with d4-CholDex at final d4-UC concentration of 0.05 mg/ml (127.98 μmol/L) for 3 h and chased for 48 h in medium without d4-CholDex. Concentrations of d4-UC and d4-CE in harvested cells were measured by tandem mass spectrometry (MS/MS). d4-UC/d4-CE ratios were elevated in NP-C lines compared to controls (n = 6, mean = 4.36, range = 1.89-8.91), with the highest ratios in severe NP-C1 phenotypes and the lowest in adolescent/adult type patients. There were overlaps between NP-C1 forms: early infantile (n = 1, mean = 48.6), late infantile (n = 4, mean = 36.3, range = 20.6-54.0), juvenile (n = 5, mean = 24.7, range = 13.4-38.3), adolescent/adult (n = 3, mean = 14.5, range = 11.7-19.8). The ratios in NP-C1 heterozygotes were mildly elevated (n = 4, mean = 16.4, range = 14.9-17.4) and comparable to patients with adolescent/adult NP-C1. The test can be useful in evaluation of suspected NP-C patients with inconclusive results of biomarker or molecular tests. Its advantages include standardized preparation of particles with longer shelf life at 4 °C, quantitative results, and no requirement for radioactive chemicals.