James A. Willis

Longitudinal (T1) relaxation times of phosphorus metabolites in the bovine and rabbit lens

Longitudinal (T1) magnetic relaxation times for the major phosphorus-containing metabolites present in the bovine and rabbit lens under organ culture conditions and in the bovine and rabbit globe have been determined. Significant differences in T1 for the major phosphorus metabolites in each case are observed, as well as for the same metabolite in the two species examined. Species-dependent lens hydration may account, in part, for these differences. Because of the requirement for rapid repetitive pulsing for the attainment of optimum signal collection efficiency by the Fourier transform nuclear magnetic resonance method, significant differential saturation of metabolite resonance intensities occurs in circumstances where appreciable differences in T1 relaxation times are present, which, unless corrected, leads to erroneous determinations of relative metabolite levels. The net effect of assessing relative metabolite levels in terms of the percentage of total phosphate signal, without a correction for T1 discrimination, is to underestimate metabolites with a long T1 (sugar phosphates) and overestimate those metabolites with a short T1 (ATP). Individual metabolite T1 discrimination factors are calculated from integrated areas of spectra acquired using short and long repetition times as well as from metabolite T1 values. They are then employed, for the first time, for the correction of 31P-NMR spectra of bovine and rabbit lenses. Corrected spectra provide relative metabolite levels for lenticular ATP which are in excellent agreement with values determined by chemical and enzymatic assays.

Measurement of longitudinal relaxation times using surface coils

Abstract The poor B 1 field homogeneity associated with surface coils reduces the effectiveness of inversion-recovery techniques for determination of T 1 relaxation times. This paper presents a variation of the saturation-recovery T 1 experiment which introduces periodic phase shifts in the saturating irradiation to achieve rapid and effective saturation of the sample magnetization, thereby avoiding the complications of B 1 field inhomogeneity. Comparison of the presented technique with the inversion-recovery technique utilizing a composite inverting pulse and alternating phase acquisition is provided. A discussion of the relative merits of each technique is presented.

Oxidative-stress induced protein glutathione mixed-disulfide formation in the ocular lens

The biochemistry of protein-glutathione mixed disulfide formation in the ocular lens was examined by 13C-NMR spectroscopic measurements of glutathione oxidative metabolism in intact rabbit lenses maintained in organ culture. Lenticular amino acid uptake and glutathione biosynthetic mechanisms were employed to facilitate the incorporation of L-[3-13C]cysteine from the incubation medium into the cysteinyl residue of glutathione. Subsequent exposure to increasing levels of oxidative stress induced by tert-butylhydroperoxide resulted in decreased levels of ([3-13C]cysteinyl)-glutathione and a loss of 13C NMR resonance intensity, a reflection of protein-glutathione mixed disulfide formation. The rate of ([3-13C]cysteinyl)-glutathione loss depended on the concentration of tert-butylhydroperoxide; 13C-labeled oxidized glutathione was observed only at the highest concentration (2 mM) of oxidant tested. Removal of the oxidative stress led to a partial recovery of ([3-13C]cysteinyl)-glutathione levels and 13C resonance intensity. Recovery was significantly enhanced by the addition of 2-mercaptoethanol. The mechanism of protein-glutathione adduct formation was further characterized by the in vitro monitoring of the reaction of oxidized glutathione with bovine lens gamma-II crystallin protein using proton NMR spectroscopy. These experiments provided insight into the role of the cellular glutathione redox-couple, [GSH]/[GSSG], in maintaining reduced protein thiol groups, and suggested that protein-glutathione adduct formation may function as a mechanism for modulating the glutathione redox buffer under conditions of oxidative stress in ocular tissue. In addition, the results demonstrate the feasibility of direct chemical reduction of protein-glutathione disulfide bonds in vivo which may reflect a mechanism for the inhibition of disulfide-linked light scattering protein aggregate formation.

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

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)

Effects of Hypoxia and Toxicant Exposure on Arginine Kinase Function as Measured by 31P-NMR Magnetization Transfer in Living Abalone

Abstract The activity of arginine kinase (AK) was evaluated by saturation transfer NMR in red abalone (Haliotis rufescens) in response to hypoxia, sodium azide (NaN3; an inhibitor of cytochrome c oxidase), or pentachlorophenol (PCP; an uncoupler of oxidative phosphorylation) exposure. Pseudo-first order rate constants (Kfor) in the forward (ATP forming) reaction direction showed maximal increases from basal values of 0.025 s−1 to 0.095, 0.114, 0.126 s−1 for NaN3 hypoxia, and PCP exposures, respectively. Increases in Kfor were inversely correlated (r2 = 1.00) to declines in ATP concentration in all exposed animals. Flux (the product of Kfor and phosphoarginine concentration) appeared to converge on a common value, from basal flux values of 0.257 mM PA s−1 to 0.703, 0.770, and 0.627 mM PA s−1 for NaN3, hypoxia, and PCP exposures, respectively. It seems likely that all three stresses were equally effective at inhibiting mitochondrial ATP formation, which may account for the similarity in flux increase, possibly to maximal rates of AK-mediated ATP formation. Differences in Kfor are related to declines in ATP concentrations, which appear to be stress specific, and likely indicate additional mechanisms of toxicity for NaN3 and PCP.

Dynamic assessment of hexose monophosphate shunt activity in the intact rabbit lens by proton NMR spectroscopy

A proton nuclear magnetic resonance technique is demonstrated for ascertaining the real-time contribution of the hexose monophosphate shunt to glucose metabolism in the intact incubated rabbit lens. This measurement requires incubation of the tissue in medium supplemented with [1-13C]glucose, and depends on the presence of the 13C label in the methyl position of lactate which creates satellite resonances by way of 13C - 1H spin-spin scalar coupling. The assumptions required to make the measurement are presented. For lenses maintained under control conditions, a basal level corresponding to 5% hexose monophosphate shunt activity was determined. An eight-fold increase in activity was observed under conditions known to stimulate the shunt.

Surface coil phosphorus-31 nuclear magnetic resonance studies of the intact eye

The feasibility of employing the surface coil probe technique for the non-invasive study of ocular tissue metabolism by phosphorus-31 nuclear magnetic resonance spectroscopy (31P NMR) in enucleated bovine, rabbit, human and rat globes is demonstrated. An assessment of individual phosphorus-metabolite contributions from ocular tissues, including the cornea, lens and iris, to the overall 31P NMR spectrum (NMR spectral acquisition parameters optimized for the lens region of the globe) was accomplished through the combination of surgical ablation and difference spectroscopy. The NMR measurements also provided tissue pH values for the lens and cornea. The strengths and limitations of the surface coil NMR method, which is particularly appropriate for in vivo metabolic studies of ocular tissues such as the lens, are discussed.

13C NMR spectroscopic measurement of glutathione synthesis and antioxidant metabolism in the intact ocular lens

A 13C NMR spectroscopic method for non-invasive, time-resolved measurements of glutathione function in the intact ocular lens maintained in organ culture is described. L-[beta-13C]cysteine (1 mM) included in the incubation medium is incorporated, by way of lenticular amino acid uptake and glutathione biosynthetic mechanisms, into the cysteinyl residue of intralenticular glutathione. 13C-NMR chemical shift measurements facilitate analysis of glutathione synthesis and anti-oxidant reactions in the intact tissue. The results of this preliminary study demonstrate the viability of a rapid non-invasive method for monitoring the multiple aspects of glutathione biosynthesis, metabolism, and function in intact tissue.

Phosphorus and proton magnetic resonance spectroscopic studies on the relationship between transparency and glucose metabolism in the rabbit lens

We report the results of a series of nuclear magnetic resonance (NMR) experiments designed to investigate the relationship between particular aspects of glucose metabolism and cataract formation in the rabbit lens. The glucose metabolism of the rabbit lens incubated in TC-199 medium containing 5.5 mM glucose, in glucose-deficient medium, and in modified Earles medium containing 5.5 mM glucose devoid of NaCl, is examined in conjunction with the assessment of lens transparency. Significant age-dependent differences in the phosphorus metabolite profile and in hexosemonophosphate shunt flux, as measured by NMR, were observed in lenses incubated in TC-199 medium containing 5.5 mM glucose. Incubation in glucose-deficient medium for 8 hr results in significant increases in the levels of inorganic phosphate and phosphomonoesters, and decreases in ATP and L-alpha-glycerolphosphate. These levels regain near-normal values after 24 hr incubation in control medium containing 5.5 mM glucose. By contrast, shunt flux is three times the basal level during the recovery period. Lens clarity, as assessed by slit lamp micrography, was maintained throughout the duration of the experiment. Incubation of adult and juvenile lenses for 18 hr in Earles medium (pH 7.4 or 9.2) containing 5.5 mM glucose, and no NaCl, results in uniform lenticular opacification within 18 hr and changes in ultrastructure of the epithelial and cortical lens fiber cells. No statistically significant change in the NMR visible phosphorus metabolite profile or intralenticular pH is observed for the adult rabbit lens relative to a lens incubated under control conditions. For the juvenile rabbit lens, small, but statistically significant differences in the levels of dinucleotide and uridinediphosphoglucose were observed. Shunt flux, in contrast, is increased two-fold. These results demonstrate that the NMR visible phosphorus metabolite profile of the lens does not necessarily correlate with transparency, and that hexosemonophosphate shunt activity provides a sensitive measure of prior or current lenticular stress.

13C-NMR spectroscopic studies of 2-mercaptoethanol-stimulated glutathione synthesis in the intact ocular lens

13C-NMR spectroscopy was employed non-invasively for the real-time measurement of the rates of glutathione synthesis in intact rabbit lenses supported in organ culture containing L-[3-13C]cysteine. Supplementation of the organ culture medium with 2-mercaptoethanol resulted in a dose- dependent enhancement of lenticular glutathione synthesis rates (dose for 50% maximal effect = 125 microM). At the most effective concentration (400 microM) 2-mercaptoethanol increased the rate of glutathione synthesis 163% relative to the rate observed under control conditions. The mechanism of action for this effect was investigated in intact lenses using antagonists of specific amino acid uptake systems. These experiments demonstrated that the enhanced rates of glutathione synthesis observed in the presence of 2-mercaptoethanol were due to the affinity of the mixed-disulfide formed between cysteine and 2-mercaptoethanol for L system amino acid uptake, thereby providing a mechanism for increasing intracellular cysteine levels by circumventing normal cellular cysteine uptake pathways in the lens. Because of the role of cysteine as the rate limiting substrate in lenticular glutathione biosynthesis, these results suggest a potential strategy to prevent tissue opacification associated with depleted glutathione levels.