The effect of prolonged elevated glucose levels on the phosphate metabolism of the rabbit lens in perfused organ culture

Abstract

13C and 31P nuclear magnetic resonance (NMR) techniques were used to monitor phosphate metabolite longitudinal (T1) relaxation times and metabolism, the sorbitol pathway, and related bioenergetic processes in cultured rabbit lenses through 9 days of incubation with constant perfusion. Lenses were incubated in a modified TC-199 medium containing either 5.5- or 35.5 mM [1-13C]-enriched glucose. The NMR studies were augmented by biochemical and cation analyses, and by the visual assessment of lens clarity. In the hyperglycemic rabbit lens, relative to normal values, longitudinal (T1) relaxation times for phosphorus metabolites increased from 33- to 50% [with the exception of inorganic phosphate (Pi)]. This provides the first documentation that a pathophysiological insult to the lens can alter phosphorus metabolite T1 values. The determination of steady state levels for the NMR visible phosphorus metabolites present in the lens was obtained after correction for T1 differential saturation effects, and normalization to reflect the content of phosphorus equivalents in each metabolite. Relative to control lenses (i.e. incubated in 5.5 mM glucose-containing medium) the NMR visible phosphate metabolite pool of rabbit lenses subjected to a hyperglycemic stress for an extended period of time (greater than 72 hr) is characterized by the following statistically significant differences: a 23% decrease in the mean level of ATP; a 51% increase in the mean level of alpha-glycerolphosphate (alpha GP); a 56% decrease in the mean level of Pi; the appearance of an unidentified resonance at 6.2 ppm after 115- to 120-hr incubation; and lenticular acidification of 0.10 to 0.17 pH units. No statistically significant differences in the mean levels of ADP, dinucleotides (predominantly NAD+-NADH), and phosphomonoesters (other than alpha GP) were evident. Parallel 13C NMR measurements provided a real-time confirmation of sorbitol production and accumulation in rabbit lenses incubated in 35.5 mM glucose-containing medium. In agreement with classical biochemical analysis (Chylack and Kinoshita, 1969) sorbitol production attained a plateau level after ca. 3 days of incubation. Cation determinations performed at the conclusion of the 9-day incubation indicated that the lenses incubated under the two conditions have similar Na+ and K+ levels. Rabbit lenses incubated in normal glucose medium remained clear for at least 8 days. By contrast, for the rabbit lenses incubated in elevated glucose medium, equatorial opacification became evident by day 5; by day 8 extensive bleb formation and opacification was evident.(ABSTRACT TRUNCATED AT 400 WORDS)