基于HPLC-MS/MS诊断新生儿维生素A缺乏症新方法的建立与应用研究

Vitamin A Deficiency (VAD) is a public nutritional problem in the world，and is one of the most common micronutrient disorders in developing countries, including China. So it has been paid much attention already. Previous studies in our group found that adequate vitamin A at delivery or infants had beneficial influence on neonatal birth outcomes and children's neurodevelopment in later childhood and repair to brain injury during pregnancy as well. It is thus very important for early diagnosis of VAD. Nevertheless, there is no standard to diagnose neonatal VAD. This study, based on our early success on the detection of retinol and its precursors in the dried blood spot from neonates using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), is aimed to measure retinol, its metabolites and related coenzymes, investigate the value of them to diagnose VAD and the most appropriate indictor by multivariate regression analysis, analyse its 95% normal reference range, and diagnose the newborns suffered from pneumonia and brain injury, who are at higher risk of VAD according to our previous finding. The end of this project was to establish a simple, feasibility and high-throughput method and the diagnostic standard for neonatal VAD detection and screening, to provide the basis for treatment, and a new direction to improve the conditions undertaken in newborn screening programs in China, and ultimately to reduce birth defects and improve the overall quality of population.

维生素A缺乏症 （VAD）是发展中国家儿童最常见的微量营养素缺乏症之一，已得到国内外的高度重视，本课题组前期研究发现新生儿体内足量VA有益于其儿童期神经发育，早期营养干预对孕期开始的VAD子代脑损伤修复具有重要作用，因此早期诊断VAD极为重要。但目前尚缺乏诊断新生儿VAD的标准。在前期高效液相-串联质谱技术（HPLC-MS/MS）成功检测新生儿微量血滤纸片VA及其前体的基础上，我们拟完善分析微量血滤纸片中VA及其代谢物与相关辅酶的分析方法，通过多元回归分析探讨VA及其代谢物与VAD诊断的相关性和价值，获得新生儿VAD诊断的最佳指标及其95%正常参考区间，用于诊断前期研究中已发现的新生儿肺炎、脑损伤等高危VAD患儿，从而建立一种简便、易行、高通量的诊断与筛查新生儿VAD的新方法，为其早期治疗提供实验依据和新策略。本研究有助于拓宽我国新生儿筛查病种，为降低出生缺陷和提高人口整体素质奠定基础。

维生素A缺乏症（VAD）是发展中国家儿童最常见的微量营养素缺乏症之一，目前尚缺乏诊断新生儿VAD的方法和标准。本项目建立了基于HPLC-MS/MS检测新生儿微量血中VA及其前体的新方法，并进行方法学验证，与传统HPLC法的相关性好（R2=0.83，P<0.05），诊断6-70月儿童VAD切值为0.68μmol/L，灵敏度95.7%，特异度性91.4%。应用新方法分析258例重庆市主城区正常新生儿生后3-5天足跟微量血，VA的95%参考区间为0.22μmol/L-0.63μmol/L，小样本验证该参考区间是适合的；新生儿血视黄醇乙酸酯及棕榈酰酯、β-胡萝卜素水平极低；孕母血视黄醇水平为新生儿血视黄醇、β-胡萝卜素水平的独立影响因素（相关系数r=0.310，0.307；P=0.005，0.005）；因此视黄醇作为诊断新生儿VAD的指标是适合的。参照此参考区间，188例高危新生儿（患有肺炎住院）VA水平0.149±0.075 μmol/L，有29.3%位于其参考区间的下限，诊断为VAD。从而建立了一种简便、易行、高通量诊断与筛查新生儿VAD的新方法，获得重庆市主城区新生儿VA的95%正常参考区间，诊断前期研究中已发现的高危新生儿VAD患儿，为治疗提供依据和新视角，拓宽我国新生儿筛查病种，为降低出生缺陷和提高人口整体素质奠定了基础。