Ortwerth B J, Speaker J A, Prabhakaram M, Lopez M G, Li E Y, Feather M S
Mason Institute of Ophthalmology, University of Missouri, Columbia 65212.
Exp Eye Res. 1994 Jun;58(6):665-74. doi: 10.1006/exer.1994.1064.
L-Threose is a significant degradation product of ascorbic acid at pH 7.0 in the presence of oxygen. When compared to several other ascorbate-derived degradation products, it had the greatest ability to glycate and crosslink lens proteins in vitro. To determine whether L-threose was formed in the lens, the sugars in a TCA-soluble extract from human lenses were reduced to polyols with NaBH4, acetylated and analysed by gas-liquid chromatography. The threitol levels measured were 3.4 +/- 0.8 micrograms per lens (n = 4). GC-MS measurements made after reduction with NaBD4 indicated that threitol, but little or no threose, was originally present in the human lens. Rat lenses were incubated with [1-13C]D-threose for 24 hr, and considerable D-threitol formation was seen by NMR spectroscopy. Analysis of the lenses after medium removal showed that only [1-13C]threitol was present within the lenses indicating a rapid reduction of threose within the lens, presumably by aldose reductase. Assays with human recombinant aldose reductase and with human lens cortical and nuclear extracts all exhibited sorbinil-inhibitable aldose reductase activity with L-threose as substrate. This was confirmed by incubating a preparation of [1-14C]L-tetrose (a mixture of 40% L-threose and 45% L-erythrose) with both the pure aldose reductase and crude lens extracts followed by the subsequent identification of the [1-14C]L-threitol formed by thin layer chromatography. L-Threose degrades very slowly in 0.1 M phosphate buffer at pH 7.0, but the addition of a four-fold excess of N alpha-acetyl-L-lysine accelerated the rate of disappearance of threose 30-fold, indicating a rapid glycation reaction. When [1-14C]L-tetrose was incubated with a complete bovine lens homogenate, a linear incorporation into protein was observed over a 24 hr period. Increasing levels of lens extract exhibited increasing incorporation into protein. These data confirm a rapid reactivity of L-threose with lens protein and argue that glycation would occur in vivo in spite of the presence of aldose reductase.
L-苏糖是抗坏血酸在pH 7.0且有氧存在的条件下的一种重要降解产物。与其他几种抗坏血酸衍生的降解产物相比，它在体外使晶状体蛋白糖化和交联的能力最强。为了确定晶状体中是否会形成L-苏糖，用NaBH4将人晶状体三氯乙酸可溶提取物中的糖类还原为多元醇，进行乙酰化处理后通过气液色谱法进行分析。测得的每只晶状体中苏糖醇水平为3.4±0.8微克（n = 4）。用NaBD4还原后进行的气相色谱-质谱测量表明，人晶状体中原本存在苏糖醇，但几乎没有或不存在苏糖。将大鼠晶状体与[1-13C]D-苏糖孵育24小时，通过核磁共振光谱法可观察到大量D-苏糖醇的形成。去除培养基后对晶状体进行分析表明，晶状体中仅存在[1-13C]苏糖醇，这表明晶状体中的苏糖迅速被还原，推测是由醛糖还原酶催化。用人重组醛糖还原酶以及人晶状体皮质和核提取物进行的测定均显示，以L-苏糖为底物时具有索比尼尔可抑制的醛糖还原酶活性。用纯醛糖还原酶和晶状体粗提物孵育[1-14C]L-丁糖（40% L-苏糖和45% L-赤藓糖的混合物），随后通过薄层色谱法鉴定形成的[1-14C]L-苏糖醇，证实了这一点。L-苏糖在pH 7.0的0.1 M磷酸盐缓冲液中降解非常缓慢，但加入四倍过量的Nα-乙酰-L-赖氨酸可使苏糖消失速率加快30倍，表明发生了快速的糖化反应。当[1-14C]L-丁糖与完整的牛晶状体匀浆孵育时，在24小时内观察到其线性掺入蛋白质中。晶状体提取物水平的增加表现为掺入蛋白质中的量增加。这些数据证实了L-苏糖与晶状体蛋白的快速反应性，并表明尽管存在醛糖还原酶，糖化反应仍会在体内发生。
