Ascorbic Acid Glycation: the Reactions of l-Threose in Lens Tissue

• Published in: Experimental eye research Vol. 58; no. 6; pp. 665 - 674
• Main Authors: Ortwerth, B.J., Speaker, Jane Anne, Prabhakaram, Malladi, Lopez, Mercedes G., Li, Elaine Yinan, Feather, Milton S.
• Format: Journal Article
• Language: English
• Published: London Elsevier Ltd 01.06.1994; Elsevier
• Subjects: Aldehyde Reductase - metabolism; aldose reductase; Animals; ascorbic acid; Ascorbic Acid - metabolism; Biological and medical sciences; Cattle; Chromatography, Gas; Crystallins - metabolism; Culture Techniques; Eye and associated structures. Visual pathways and centers. Vision; Fundamental and applied biological sciences. Psychology; glycation; Glycosylation; l-threose; lens; Lens, Crystalline - drug effects; Lens, Crystalline - metabolism; Lysine - analogs & derivatives; Lysine - pharmacology; oxidation; senile cataract; Sugar Alcohols - metabolism; Tetroses - metabolism; threitol; Vertebrates: nervous system and sense organs; glycation; ascorbic acid; threitol; oxidation; aldose reductase; senile cataract; lens; l-threose; Eye; Human; Proteins; Visual system; Ascorbic acid; Crystallin; Enzyme; Metabolite; Aldehyde reductase; Lens; Oxidation; Oxidoreductases
• ISSN: 0014-4835; 1096-0007
• 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 μg 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 Nα-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 glyeation would occur in vivo in spite of the presence of aldose redactuse.