Ngkeekwong F C, Ng L L
Department of Medicine and Therapeutics, Clinical Sciences Building, Leicester Royal Infirmary, Leicester LE2 7LX, U.K.
Biochem J. 1997 May 15;324 ( Pt 1)(Pt 1):225-30. doi: 10.1042/bj3240225.
In diabetes, a major cause of mortality is from cardiovascular causes, and low levels of antioxidants such as vitamin C have been associated with such complications. Leucocyte ascorbic acid status can reflect total body stores but the mechanisms that mediate the uptake of ascorbic acid (AA) or dehydroascorbic acid (DHA) in human lymphoid cells are undefined. We have investigated the uptake of AA and DHA with mass assays in human lymphoblasts by using HPLC, with precautions to prevent the oxidation of AA and to take into account the instability of DHA in buffers. Human lymphoblasts exhibit distinct uptake mechanisms for both AA and DHA, with Vmax values of 1.35+/-0.14 and 29.0+/-5.8 nmol/h per 10(6) cells and Km values of 23.5+/-6 and 104+/-84 microM respectively. The AA uptake was Na+-dependent and inhibitable with ouabain, whereas DHA uptake was independent of Na+ and ouabain-insensitive. Both uptake mechanisms were inhibited by phloretin or cytochalasin B. AA uptake was decreased significantly (by 13+/-2%) only at extracellular glucose concentrations of 20 mM (P<0.05). In contrast, glucose competitively inhibited DHA uptake with a Ki of 2.2 mM so that DHA uptake was decreased by glucose even in the physiological range. Phorbol esters stimulated AA but not DHA uptake; this was abolished in the presence of extracellular reductant, indicating that AA was converted to DHA before uptake occurred. Prolonged increased glucose levels (20 mM) led to a decrease in the Vmax of DHA uptake. At concentrations of plasma AA or DHA, the AA uptake mechanism might be nearly half-saturated but the DHA mechanism has enormous spare capacity. This allows for cellular uptake and regeneration of AA from DHA derived from oxidative stress. In diabetes, high glucose levels might impair DHA uptake acutely by competitive inhibition or by down-regulation of uptake with chronic glucose exposure, leading to an impaired ability to store and recycle oxidized AA.
在糖尿病中，主要死因是心血管疾病，而低水平的抗氧化剂如维生素C与这类并发症有关。白细胞中的抗坏血酸状态可反映全身储备，但介导人体淋巴细胞摄取抗坏血酸（AA）或脱氢抗坏血酸（DHA）的机制尚不清楚。我们通过高效液相色谱法（HPLC），采用预防措施防止AA氧化并考虑DHA在缓冲液中的不稳定性，利用质量分析法研究了人淋巴母细胞对AA和DHA的摄取。人淋巴母细胞对AA和DHA表现出不同的摄取机制，每10^6个细胞的Vmax值分别为1.35±0.14和29.0±5.8 nmol/h，Km值分别为23.5±6和104±84 μM。AA摄取依赖于Na+，可被哇巴因抑制，而DHA摄取不依赖于Na+且对哇巴因不敏感。两种摄取机制均被根皮素或细胞松弛素B抑制。仅在细胞外葡萄糖浓度为20 mM时，AA摄取显著降低（降低13±2%）（P<0.05）。相反，葡萄糖竞争性抑制DHA摄取，Ki为2.2 mM，因此即使在生理范围内，DHA摄取也会因葡萄糖而降低。佛波酯刺激AA摄取但不刺激DHA摄取；在细胞外还原剂存在的情况下，这种刺激作用消失，表明AA在摄取前被转化为DHA。长时间升高的葡萄糖水平（20 mM）导致DHA摄取的Vmax降低。在血浆AA或DHA浓度下，AA摄取机制可能接近半饱和，但DHA机制具有巨大的备用能力。这使得细胞能够摄取并从氧化应激产生的DHA中再生AA。在糖尿病中，高血糖水平可能通过竞争性抑制或长期暴露于葡萄糖导致摄取下调而急性损害DHA摄取，从而导致储存和循环氧化AA的能力受损。
