Quantitative verification of the existence of high molecular weight protein aggregates in the intact normal human lens by light-scattering spectroscopy.

The method of quasi-elastic light-scattering spectroscopy was used to establish quantitatively the concentration of high mmolecular weight (HMW) aggregates present in the normal human intact lens as a function of age. The concentration of HMW proteins increases monotonically with age. HMW proteins are absent in the infant lens, but represent 3% of the total soluble lens protein at age 60 years. The percent concentration of HMW proteins measured in intact lenses of various ages by quasi-elastic light scattering is in striking agreement with values determined biochemically.

Identification of heavy-molecular-weight soluble protein in aqueous humor in human phacolytic glaucoma.

Aqueous humor was obtained by paracentesis at the time of cataract surgery from six patients with phacolytic glaucoma, diagnosed on the basis of acute unilateral open-angle glaucoma associated with an apparently leaking hypermature or mature cataract, and from six control patients with immature cataracts. Three of the latter had primary open-angle glaucoma. Quantities of heavy-molecular-weight (HMW) protein (MW greater than 150 X 10(6)) sufficient to obstruct aqueous outflow were identified in all six phacolytic aqueous humor specimens but in none of the controls. Three of the hypermature cataractous lenses from the cases of phacolytic glaucoma were also examined and were found to have 14-fold greater quantities of HMW protein in their liquefying cortex than were present in the cortex of immature cataractous lenses. These findings, correlated with experimental HMW protein perfusion studies in excised human eyes that we have already reported, strongly suggest that direct obstruction of the aqueous outflow channels by liberated HMW soluble lens protein may be a significant and previously unappreciated factor in the pathogenesis of phacolytic glaucoma.

Obstruction of aqueous outflow by lens particles and by heavy-molecular-weight soluble lens proteins.

Enucleated human eyes were perfused via the anterior chamber at 25 mm Hg pressure with lens particles (whole lens homogenates) in one series of experiments and with soluble lens proteins from human cataractous lenses in another series. Adding 1% of a homogenate of a single cataractous lens to the anterior chamber induced a 68% decrease in outflow. Perfusion with HMW soluble lens proteins (1 mg/ml; MW more than 150 million) caused a 60% decrease in outflow in 1 hr. In neither series was the obstruction to outflow relieved by subsequent irrigation of the anterior chamber with balanced salt solution or alpha-chymotrypsin. The results show that both lens particles and soluble lens proteins can directly obstruct the aqueous outflow pathways of human eyes. Such obstruction may be a significant factor in certain lens-induced glaucomas.

The sorbitol pathway in the human lens: aldose reductase and polyol dehydrogenase.

The sorbitol pathway in human lenses is evaluated on the enzymic level. Adult lenses, normal and nondiabetic as well as diabetic cataracts, are found to contain limited levels of aldose reductase (AR) and high levels of polyol dehydrogenase (PD) relative to the animal lens. AR is confined primarily to the lens epithelium and is two to three times higher in juvenile lenses than in the adult lens. The level of AR in the epithelium of juvenile lenses is sufficient to cause significant osmotic stress. The Km of glucose of AR is roughly 200 mM, whereas the Km for NADPH is 0.06 mM. NADP inhibits human lens AR noncompetitively and has a Ki equivalent to the Km for NADPH. PD occurs in both the lens epithelium and cortex, remains persistently high with age, and decreases with increased cortical involvement. The Km of sorbitol for PD is 1.4 mM and for NAD is 0.06 mM. NADH (Ki 0.002 mM) competitively inhibits PD in the forward direction. PD purified 100-fold from diabetic and nondiabetic cataracts and normal lenses exhibit similar kinetic constants. PD has an extremely high Vmax in the fructose-to-sorbitol direction. The Km of fructose is 40 mM and for NADH is 0.02 mM. At high enough concentration, alrestatin also inhibits PD. The added activities of AR and PD in producing sorbitol and fructose in combination with decreased hexokinase with age may account for diabetic cataract formation in human lenses exposed to a high glucose stress. Nucleotide levels are reported for senile cataractous lenses.

Human lens enzyme alterations with age and cataract: glyceraldehyde-3-P dehydrogenase and triose phosphate isomerase.

The isoelectric point distribution of G-3-P DH and TPI from human lenses was examined as a function of age and cataract formation. Both enzymes exhibited progressive heterogeneity with age and a shift towards an acidic charge. Little qualitative differences in the pI profiles of G-3-P DH and TPI were found to distinguish mixed cataracts from age comparable normal lenses. While the most alkaline form of G-3-P DH required less HAsO4= for optimal activity, no other kinetic property, i.e. Km substrate, cofactor and inhibitors distinguished any of the charge forms of G-3-P DH. All metaor isozyme forms of TPI had the same Km substrate in the forward and reverse reaction direction. The most acidic forms of G-3-P DH and TPI were less stable to increased temperatures than their more alkaline counterparts suggesting a decreased stability.