Talwar D, Davidson H, Cooney J, St JO'Reilly D
Department of Clinical Biochemistry, Macewen Building, Royal Infirmary, Glasgow G4 0SF, United Kingdom.
Clin Chem. 2000 May;46(5):704-10.
The concentration of thiamin diphosphate (TDP) in erythrocytes is a useful index of thiamin status. We describe an HPLC method for TDP and its results in patients at risk of thiamin deficiency.
We used reversed-phase HPLC with postcolumn derivatization with alkaline potassium ferricyanide and fluorescence detection. Samples were deproteinized and injected directly onto a C(18) column. TDP concentrations in erythrocytes were compared with those in whole blood. Reference intervals for erythrocyte TDP (n = 147; 79 males and 68 females; mean age, 54 years) and whole blood TDP (n = 124; 68 males and 56 females; mean age, 54 years) were determined in an apparently healthy population. We compared erythrocyte TDP with results of the erythrocyte transketolase activation test in 63 patients who were considered at risk of thiamin deficiency.
The method was linear to at least 200 microgram/L. The between-run CV was <8%. The lower limit of quantification for both whole blood and packed erythrocytes was 300 pg on column with a detection limit of 130 pg on column. Recovery of TDP from blood samples was >90%. TDP in erythrocytes correlated strongly with that in whole blood (r = 0.97). Reference intervals for erythrocyte and whole blood TDP were 280-590 ng/g hemoglobin and 275-675 ng/g hemoglobin, respectively. Of the 63 patients suspected of thiamin deficiency, 46 were normal by both TDP and activation tests, 13 were deficient by both tests, 1 was deficient by the activation test but had normal erythrocyte TDP concentrations, and 4 were normal by the activation test but had low TDP.
The HPLC method is precise and yields results similar to the erythrocyte activation assay.
红细胞中硫胺素二磷酸（TDP）的浓度是硫胺素状态的一个有用指标。我们描述了一种用于测定TDP的高效液相色谱法及其在硫胺素缺乏风险患者中的检测结果。
我们采用反相高效液相色谱法，柱后用碱性铁氰化钾衍生化并进行荧光检测。样品经去蛋白处理后直接注入C(18)柱。比较了红细胞和全血中TDP的浓度。在一个看似健康的人群中确定了红细胞TDP（n = 147；79名男性和68名女性；平均年龄54岁）和全血TDP（n = 124；68名男性和56名女性；平均年龄54岁）的参考区间。我们在63名被认为有硫胺素缺乏风险的患者中比较了红细胞TDP与红细胞转酮醇酶活性试验的结果。
该方法至少在200微克/升范围内呈线性。批间变异系数<8%。全血和压积红细胞的定量下限在柱上均为300皮克，检测限在柱上为130皮克。血样中TDP的回收率>90%。红细胞中的TDP与全血中的TDP高度相关（r = 0.97）。红细胞和全血TDP的参考区间分别为280 - 590纳克/克血红蛋白和275 - 675纳克/克血红蛋白。在63名疑似硫胺素缺乏的患者中，46名患者的TDP和活性试验结果均正常，13名患者两项试验均显示缺乏，1名患者活性试验显示缺乏但红细胞TDP浓度正常，4名患者活性试验正常但TDP较低。
高效液相色谱法精确，其结果与红细胞活性测定法相似。
