Biomedical subjects
Publications and source records attributed to J A Willis.
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Genetic predisposition to haemochromatosis may be an important aetiological factor in some cases of Type 2 diabetes. Our aim was therefore to test the hypothesis that the haemochromatosis gene mutations Cys282Tyr and His63Asp are more prevalent in Type 2 diabetic patients compared with the Canterbury, New Zealand general population. We studied 230 consecutive patients referred to the Diabetes Services with age > or = 30 years and considered to have Type 2 diabetes. DNA was extracted from whole blood and amplified by polymerase chain reaction prior to restriction fragment length polymorphism analysis. The frequency of the mutations was compared with that observed previously in 1064 subjects from the Canterbury general population by chi2 testing. Iron was measured by a colorimetric method, transferrin by rate nephelometry and ferritin by immunoassay. There were 2/230 (0.8%) Cys282Tyr homozygous subjects in the diabetic group compared with 5/1064 (0.5%) NS in the general population. Although there was a trend to lower incidence of Cys282Tyr heterozygosity in the diabetic group, there was no significant difference for any of the six genotype frequencies between the two groups. Haemochromatosis gene mutations Cys282Tyr and His63Asp are therefore not increased in Type 2 diabetics compared with the general population. Transferrin saturation was a sensitive marker (100%) of genetic haemochromatosis, although ferritin had low specificity (77.8%). Genetic susceptibility to haemochromatosis is not an important aetiological factor for diabetes, and targeted screening of diabetic patients for haemochromatosis is not indicated.
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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.
This study aimed to determine the prevalence of antibodies against glutamic acid decarboxylase (anti-GAD) and islet cell antibodies (ICA) in relation to beta-cell function in adults newly-diagnosed with diabetes mellitus. beta-cell function was assessed in adults aged 25-70 years newly-diagnosed with diabetes mellitus (n = 84) and control subjects (n = 34) using a 1.6 MJ mixed meal test procedure. beta-cell function was evaluated by the true insulin (defined as immunoreactive insulin minus proinsulin) response to the mixed meal test. Subjects were classified on the basis of the area under the true insulin curve (normal 16830-107700 pmol min/I) and the sum of the 30 and 60 min incremental response (normal 285-3295 pmol/I). The prevalence of anti-GAD and ICA was determined using radioimmunoprecipitation and indirect immunofluorescence, respectively. Twelve (14%) of the study cohort were insulin deficient showing little or no true insulin release. Of the insulin deficient individuals, seven (58%) subjects were anti-GAD antibody positive, compared with eleven (15%) of the subjects without insulin deficiency (P < 0.001). Seven (58%) insulin deficient subjects were ICA positive, whereas only two (3%) non-insulin deficient subjects were ICA positive (P < 0.001). Eight (67%) of the insulin deficient individuals had anti-GAD or ICA, compared with twelve (17%) of those who were not insulin deficient (P < 0.001). The positive predictive values for insulin deficiency of anti-GAD and ICA were 39 and 78% respectively. The sensitivity of both antibodies for detecting insulin deficiency was 50%. The specificity for detecting insulin deficiency was 85% for anti-GAD and 97% for ICA. Positivity for both anti-GAD and ICA gave a specificity and positive predictive value for insulin deficiency of 99%, and a sensitivity of 50%. Nearly one in seven adults presenting with diabetes mellitus as a new diagnosis are insulin deficient using our criteria. Loss of beta-cell function in two thirds of individuals who are insulin deficient can be identified by anti-GAD and ICA. Early detection of these immune markers of beta-cell damage creates the potential for immune modulation to limit such damage.
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.
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.
The therapeutic effectiveness of calcium channel antagonists (CCA) in hypertension and angina are well established. More recently, CCAs have also been demonstrated to ameliorate neurologic dysfunction that often accompanies ischemia associated with subarachnoid hemorrhage and stroke. We have hypothesized that retinal degeneration associated with ischemia may also result from the accumulation of calcium intracellularly, so-called "Ca++ overload". To test this hypothesis, a rat model of acute retinal ischemia, produced by direct occlusion of posterior ciliary and central retinal arteries, was developed. The extent of retinal dysfunction induced by ischemia was evaluated by electroretinograms (ERGs). Occlusion of the retinal arteries resulted in the disappearance of both a- and b-waves during the occlusion period (30 minutes) in vehicle-treated rats. Total retinal ischemia did not produce any significant change in magnitude of ERG a-wave amplitude during three-hours of reperfusion. However, ERG b-waves amplitudes were significantly reduced by more than 60%. In rats, pretreatment with nifedipine (0.33 to 3.3 mg/kg, i.p.) 30 minutes prior to the occlusion of the retinal vessels produced a significant dose-dependent increase in the recovery of b-wave amplitude when compared to vehicle-treated rats. These data support the idea that "Ca++ overload", resulting from the deregulation of intracellular Ca++ homeostasis, is a primary factor involved in ischemic retinal degeneration and that CCAs can protect the retina from ischemic damage.
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Surface coil 13C nuclear magnetic resonance (NMR) spectroscopy was used to investigate the in vivo carbohydrate metabolism of rat C6 gliomas during and after infusion with [1-13C] glucose. In vivo 1H-decoupled 13C NMR spectra of the glioma following infusion with [1-13C]glucose revealed the direct production of [3-13C]lactic acid, [1-13C]glycogen, and [4-13C], [3-13C], and [2-13C]glutamate/glutamine. Lactate levels of in vivo gliomas increased and reached steady state levels during [1-13C]glucose infusion, and decreased following termination of infusion. Complementary in vitro studies using supernatant media collected from C6 glioma cells incubated with media containing [1-13C] or [6-13C]glucose and glutamine were examined by 1H NMR spectroscopy. The [3-(13C/12C)]lactate ratios obtained from 1H spectra of supernatant media containing [1-13C]glucose revealed the percentage of glucose metabolized through the hexose monophosphate shunt to be 10.01 +/- 0.85% (n = 3), while similar measurements of media containing [6-13C]glucose and glutamine showed that glutaminolysis contributed 9.0 +/- 1.0% of total lactate production under these conditions. Enzymatic analysis of media determined lactate production to be 139 +/- 9 nmol per 10(6) cells per h (n = 4). These measurements demonstrate the ability of NMR to monitor brain tumor carbohydrate metabolism both in vitro and in vivo.
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 Earle's 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 Earle's 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.
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.
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)
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.
Since cytosolic malate dehydrogenase has been shown to play a role in the regulation of liver cytosolic [NAD+]/[NADH] redox state during ethanol metabolism, it is possible that differences in this enzyme could cause differences in response to ethanol. The present study demonstrates that the isozyme pattern of this cytosolic enzyme in whole blood samples is the same as that in liver and that the pattern does not differ in alcoholic and control subjects. A marginally significant elevation of activity of malate dehydrogenase in blood from alcoholic subjects is reported. Further studies are needed to confirm this latter finding and to assess fully its possible significance.
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.