Background Plasmalogens are critical membrane structural components that are mainly generated by de novo synthesis starting in peroxisomes. Hence, patients with defects in peroxisome biogenesis (PBD) exhibit markedly reduced plasmalogen levels. Plasmalogen ratios are traditionally measured by gas chromatography-mass spectrometry (GC-MS); however, this method entails a lengthy sample extraction and derivatization and does not report concentrations of individual plasmalogen species. We have developed a robust and easy-to-implement liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify the 18 most abundant ethanolamine plasmalogen (PlsEtn) species in packed red blood cells (RBCs). However, no reference intervals have been published for individual PlsEtn. Here, we describe the establishment of age-specific reference intervals for individual PlsEtn species and their total values (16:0, 18:0, 18:1 species, and total plasmalogens). Methods Plasmalogens were extracted using methanol containing two labeled internal standards, shaking for one hour at room temperature. Chromatographic separation was performed using an Acquity Premier BEH C18 UPLC column with a binary gradient of 5 mM ammonium acetate in water:methanol (15:85) and 5 mM ammonium acetate in methanol. Analysis was performed using a XEVO TQ-XS Mass Spectrometer with Ultra-High Performance Liquid Chromatography (Waters) in multiple reaction monitoring mode. Eighteen PlsEtn species were quantified using four commercially available standards; additionally, totals were calculated for 16:0, 18:0 or 18:1 species, and for total plasmalogens. Reference intervals were established using 376 RBCs from self-reported healthy volunteers and de-identified clinical samples referred for unrelated testing (182 females and 194 males; range 0 to 88 years). Data was analyzed using the R programming language. The study was approved by the Institutional Review Board of the University of Utah. Results Initial age groups were identified using a model-based clustering algorithm followed by iterative Harris-Boyd analysis. Finally, the adjacent groups were merged if their means differed by less than 10%. Once the final age groups were partitioned, data in each individual age group were analyzed using parametric or non-parametric statistics to determine reference intervals (95%, with 90%confidence intervals). PlsEtn species displayed the lowest concentration in the first few months of life, which increased in childhood until adolescence or adulthood (depending on PlsEtn). For most species, the concentrations increased over time reaching a plateau between 18 and 48 years of age, and then starting to decrease. The total values followed the same trend, with neonates showing significantly lower values compared to other age groups. Conclusions We applied a novel statistical approach to identify age groups and determine age-specific reference intervals for 18 individual PlsEtn in RBC and their totals. Lacking previously published data, this study is critical for supporting test implementation in clinical laboratories.

中文翻译：

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B-170 使用液相色谱串联质谱法测定红细胞中乙醇胺缩醛磷脂种类的年龄特异性参考区间


背景缩醛磷脂是关键的膜结构成分，主要通过从过氧化物酶体开始的从头合成产生。因此，过氧化物酶体生物发生（PBD）缺陷的患者表现出缩醛磷脂水平显着降低。缩醛磷脂比率传统上通过气相色谱-质谱法 (GC-MS) 测量；然而，该方法需要长时间的样品提取和衍生化，并且不报告单个缩醛磷脂种类的浓度。我们开发了一种稳健且易于实施的液相色谱-串联质谱 (LC-MS/MS) 方法，用于定量浓缩红细胞 (RBC) 中 18 种最丰富的乙醇胺缩醛磷脂 (PlsEtn)。然而，尚未公布单个 PlsEtn 的参考区间。在这里，我们描述了各个 PlsEtn 物种及其总值（16:0、18:0、18:1 物种和总缩醛磷脂）的年龄特定参考区间的建立。方法使用含有两个标记内标的甲醇提取缩醛磷脂，并在室温下摇动一小时。使用 Acquity Premier BEH C18 UPLC 柱进行色谱分离，采用 5 mM 乙酸铵水:甲醇 (15:85) 溶液和 5 mM 乙酸铵甲醇溶液二元梯度。使用配备超高效液相色谱 (Waters) 的 XEVO TQ-XS 质谱仪在多反应监测模式下进行分析。使用四种市售标准对 18 种 PlsEtn 物种进行了定量；此外，还计算了 16:0、18:0 或 18:1 物种以及总缩醛磷脂的总数。 使用来自自我报告的健康志愿者的 376 个红细胞和转介进行无关测试的去识别化临床样本（182 名女性和 194 名男性；范围 0 至 88 岁）建立参考区间。使用 R 编程语言分析数据。该研究得到了犹他大学机构审查委员会的批准。结果 使用基于模型的聚类算法确定初始年龄组，然后进行迭代 Harris-Boyd 分析。最后，如果相邻组的平均值相差小于 10%，则将其合并。一旦划分了最终的年龄组，就使用参数或非参数统计分析每个年龄组的数据，以确定参考区间（95％，90％置信区间）。 PlsEtn 物种在生命的最初几个月表现出最低浓度，在儿童期直至青春期或成年期（取决于 PlsEtn）增加。对于大多数物种来说，浓度随着时间的推移而增加，在 18 岁至 48 岁之间达到稳定水平，然后开始下降。总数值遵循相同的趋势，新生儿的数值明显低于其他年龄组。结论 我们应用了一种新颖的统计方法来识别年龄组并确定 18 个个体 PlsEtn 的 RBC 及其总数的年龄特定参考区间。由于缺乏先前发表的数据，这项研究对于支持临床实验室的测试实施至关重要。