Determination of Vitamin B6 vitamers in human and arthritic animals by High-Performance Liquid Chromatography Assay

Abstract

維生素B6是一種水溶性維生素。維生素B6具有六種不同的型式：比哆醛、比哆醇、比哆胺、磷酸比哆醛、磷酸比哆醇和磷酸比哆胺，代謝最終產物為比哆酸由尿液排除。磷酸比哆醛以輔助酵素的型式參與體內許多重要的代謝反應，如胺基酸代謝、脂質代謝、神經傳導物質的合成及賀爾蒙的調節等。目前常見之分析維生素B6的方法，包括螢光、酵素和微生物等方法以測量磷酸比哆醛和其它形式之維生素B6。臨床上紅血球天門冬胺酸轉胺酶活化作用亦廣泛應用於檢測維生素B6 缺乏，為一間接評估維生素B6狀態之功能性檢測方法。本研究希望建立一簡單迅速之逆相高效能液相層析法同時測量血漿、紅血球和組織中不同形式的維生素B6，並將此方法應用於臨床與動物試驗之檢體。另外我們建立二種不同之大鼠關節炎動物模型，希望研究活體中關節炎對維生素B6 代謝的影響。 高效能液相層析法之結果顯示，我們成功的確認管柱前衍生作用及逆相高效能液相層析法之偵測分析條件。磷酸比哆胺、磷酸比哆醛、比哆酸和比哆醛之最大線性範圍至少為1000 nmol/L。同日間與異日間精確度之變異在磷酸比哆胺、磷酸比哆醛、比哆酸和比哆醛皆少於10%。血漿、紅血球及肝臟中磷酸比哆醛、比哆酸和比哆醛回收率分別大於96%、90%及94%。在關節炎動物模式中發現發炎反應會造成脾臟腫大及肌肉耗損，但不影響血液及肝臟中維生素B6的代謝。在我們的研究中關節炎大鼠的發炎反應可能是輕微的。

Vitamin B6 is a water-soluble vitamin. There are six forms of vitamin B6: pyridoxal (PL), pyridoxine (PN), pyridoxamine (PM), and their phosphate derivatives including pyridoxal 5''-phosphate (PLP), pyridoxine 5''-phosphate (PNP), and pyridoxamine 5''-phosphate (PMP). Vitamin B6 is excreted mainly in urine in the form of 4-pyridoxic acid (4-PA), an end product of vitamin B-6 metabolism. PLP acts as a coenzyme in many biochemical processes that include amino acid metabolism, lipids metabolism, neurotransmitters synthesis and hormones modulation. Fluorometric, enzymatic, and microbiological methods have been used for determining PLP and other B6 vitamers. Clinical vitamin B6 analysis is accomplished at present by a variety of methods. The most widely used method to detect vitamin B6 deficiency is the indirect measurement of PLP in erythrocytes with the erythrocyte aspartate aminotransferase activation assay. In the present study we described the validation of a simple, robust reverse phase HPLC method with pre-column dramatization using semicarbazide that is suitable for the simultaneous measurement of vitamin B6 vitamers in plasma, red blood cells and tissue. Pre-column dramatization, reverse phase chromatography and detection procedures were optimized. We applied the present method in measuring vitamin B6 vitamers of plasma and red blood cells in healthy subjects and vitamin B6 supplement users, as well as in the tissues of rats fed normal diet or vitamin B6 deplete diet. We also investigated the effects of inflammation on vitamin B6 metabolism using 2 different arthritic models in rats. The HPLC method described was found to be suitable for the routine measurement of PMP, PLP, 4-PA and PL in plasma, red blood cells and liver. The linearity was maintained up to at least 1000 nmol/l for PMP, PLP, 4-PA and PL. The intraday and interday imprecision for PMP, PLP, 4-PA and PL were less than 10%. The recoveries of PLP, 4-PA and PL in plasma, red blood cells and liver were greater than 96%, 90% and 94%, respectively. In the arthritis animal models, mild inflammation increased the weight of spleen, decreased the weight of the gastrocnemius muscle, but it did not alter vitamin B6 in blood or liver. The inflammation of the arthritis rats in our study was probably slighter.