J. Lunec

HYPOXIC-REPERFUSION INJURY IN THE INFLAMED HUMAN JOINT

A series of experiments showed that, on exercise of the inflamed human knee, intra-articular pressure rises above synovial capillary perfusion pressure, causing intra-articular hypoxia; and that, on cessation of exercise, there is oxidative damage to lipids and IgG within the joint. These findings are consistent with the hypothesis that persistence of synovial inflammation can be due to exercise-induced hypoxic-reperfusion injury mediated by reactive oxygen species.

Increased diene conjugates in diabetic subjects with microangiopathy

Free radicals are highly reactive unstable chemical species which have been implicated in the vascular damage associated with several disease states. Diene conjugates, probable products of free radical activity on lipids, were measured spectrophotometrically in 26 diabetic patients with microangiopathy, 36 uncomplicated diabetic patients, and 36 healthy controls. Total diene conjugates and diene conjugate to triglyceride ratios were signficantly elevated in diabetic patients with microangiopathy (0.57±0.08 and 0.31±0.14 OD units/ml respectively; mean ±SD) when compared with patients without complications (0.32±0.10, p<0.001, and 0.17±0.06, OD units/ml, p<0.011). There was no difference in total diene conjugation and their ratio to triglycerides between healthy controls and uncomplicated diabetic patients and the results were not influenced by the type of diabetes or level of diabetic control. Diene conjugate ratios repeated in 14 patients on two occasions, 6 weeks apart, with stable metabolic control were consistent. Increased diene conjugation suggesting increased free radical activity is associated with microangiopathy and this may have pathogenetic implications.

Raised cerebrospinal-fluid copper concentration in Parkinson's disease.

The cerebrospinal-fluid copper concentration, measured by electrothermal atomisation/atomic absorption spectrophotometry, was significantly higher in 24 patients with untreated, idiopathic Parkinsons disease than in a control population of 34 patients (p less than 0.001). The difference in the in-vitro capacity of copper to damage DNA, measured by the phenanthroline assay was even greater. The high phenanthroline-copper concentration correlated with disease severity (p = 0.02) and with the rate of progression of disease (p less than 0.05). A possible role is suggested for copper-catalysed oxidative mechanisms in the pathogenesis of Parkinsons disease.

Low Plasma Ascorbate Levels in Patients with Type 2 Diabetes Mellitus Consuming Adequate Dietary Vitamin C

Low ascorbate concentrations in diabetes may be secondary to inadequate dietary vitamin C intake or may relate to the varied metabolic roles of the vitamin. To determine whether inadequate dietary intake is a factor we calculated daily vitamin C intakes using both a vitamin C questionnaire and a 4‐day food diary in a group of 30 patients with Type 2 diabetes (mean age 68.8 ± 6.9 yr, 17M/13F) and in 30 community controls (mean age 68.0 ± 5.5 yr, 12M/18F)). Measures of plasma glucose, serum fructosamine, and plasma ascorbic and dehydroascorbic acid were obtained from 20 subjects in each group. There was no significant difference in daily vitamin C intake between the two groups using both methods: food diary, 61.4 ± 28.3 (patients) vs 69.5 ± 33.4 (controls) mg; questionnaire, 54.0 ± 28.9 (patients) vs 65.0 ± 30.9 (controls) mg. Vitamin C intake derived from both methods was significantly correlated (p < 0.001). Plasma ascorbate (30.4 ± 19.1 μol l−1) and dehydroascorbate (27.6 ± 6.4 μmol l−1) levels were significantly lower in patients vs in controls (68.8 ± 36.0 and 31.8 ± 4.8 μmol l−1, respectively), p < 0.0001 and p < 0.01. Plasma ascorbate levels were significantly correlated with vitamin C intake derived from the food diary (p < 0.01) and questionnaire (p < 0.01) methods in the diabetic group only. Low ascorbate levels in diabetes appears to be a consequence of the disease itself and not due to inadequate dietary intake of vitamin C. A short vitamin C questionnaire is a convenient and reliable estimate of vitamin C intake. Vitamin C supplementation of the diabetic diet deserves further consideration.

REDUCED SUSCEPTIBILITY TO LIPID-PEROXIDATION IN COLD ISCHEMIC RABBIT KIDNEYS AFTER ADDITION OF DESFERRIOXAMINE, MANNITOL, OR URIC-ACID TO THE FLUSH SOLUTION

Rabbit kidneys were stored for 24 hr at 0 degree C after single passage arterial flush with 30 ml of cold isotonic 0.9% sodium chloride (saline) solution alone or saline to which was added 12, 30, or 60 mM desferrioxamine, 1 or 3 mM uric acid, or 100 mM mannitol. They were then subjected to in vitro biochemical assay for evidence of free radical damage immediately after storage. Results were compared to those obtained with fresh, unstored kidneys. Levels of Schiff base fluorescence, diene conjugates, and thiobarbituric acid-reactive material were each significantly elevated in kidneys stored for 24 hr after flush with saline alone. These levels were in turn each significantly reduced by the addition of 60 mM desferrioxamine, 3 mM uric acid, and 100 mM mannitol to the flush solution. Likewise, glutathione redox activity fell in those flushed with saline alone, presumably in line with increased lipid peroxidation, but was restored to control levels by inclusion of the three scavenging agents.

Oxidative damage to lipids within the inflamed human joint provides evidence of radical-mediated hypoxic-reperfusion injury.

Previous work has established the existence of a pathophysiological environment within the inflamed human joint, capable of sustaining a hypoxic-reperfusion event. Using four different assay systems (two standard and two novel) applied to synovial fluid for the assessment of lipid peroxidation, a series of studies demonstrate that exercise of the inflamed human knee promotes radical-mediated lipid peroxidation within the joint. The implication for novel antioxidant therapeutic approaches to inflammatory joint disease is discussed.

Reactive oxygen species induce antigenic changes in DNA

Reactive oxygen species (ROS) are released at sites of inflammation during the respiratory burst which accompanies the phagocytic process. Using an in vitro system to simulate this process we have shown that ROS induce antigenic changes in DNA. More specifically, results of experiments using ROS scavengers have shown that hydroxyl radicals produced in close proximity to DNA‐bound metal ions play a predominant role. ROS‐mediated attack resulted in increased binding of anti‐DNA antibodies to the denatured DNA. These changes were detected using IgG, IgA and IgM isotype binding to antibodies in systemic lupus erythematosus sera. Of these the IgA isotype was most discriminating in its detection of hydroxyl radical‐induced damage.

Endothelial cell cytotoxicity in inflammatory vascular diseases--the possible role of oxidised lipoproteins.

One of the proposed mechanisms of vascular damage in connective tissue disease is the direct action of a cytotoxic serum factor inducing endothelial cell damage. The nature of this serum factor is unclear, but has been suggested to be a lipoprotein. Sera from patients with (1) systemic necrotising arteritis (polyarteritis nodosa, Wegeners granulomatosis, and necrotising arteritis associated with rheumatoid synovitis), (2) systemic or joint restricted rheumatoid disease, and (3) large vessel/giant cell arteritis have been examined for cytotoxicity to human cultured endothelial cells and azide-resistant ferroxidase-like activity (indicative of the oxidised lipoprotein content). Stored sera from patients with necrotising arteritis showed a significantly enhanced tendency to develop oxidised lipoprotein, which correlated closely with human endothelial cell cytotoxicity. Fresh sera also contained this factor, but to a lesser extent. It is argued that the cytotoxic factor detected in previous clinical studies is in part an in-vitro artefact, although its accelerated development in certain patient groups might suggest an excess of pro-oxidants that have developed in vivo.

In Vitro Screening of Iron Chelators Using Models of Free Radical Damage

The effect of chelators on free radical damage to deoxyribose induced by iron, on IgG by UV irradiation, and on mouse muscle by homogenisation has been studied using the Thiobarbituric acid method and the increase in fluorescence in IgG mixtures. Although there were some variations on the effects of the chelators in the three models, it was shown that most of the chelators could inhibit the noxious effects of the free radicals and some which are orally effective in increasing iron excretion in animals could be potentially useful in preventing iron toxicity in related pathogenic diseases.

Desferrioxamine reduces susceptibility to lipid peroxidation in rabbit kidneys subjected to ward ischaemia and reperfusion

Rabbit kidneys were clamped and subjected to warm ischaemia for 60 or 120 min then reperfused with blood for 60 min or for 24 hr. Treated rabbits received desferrioxamine at 15 or 50 mg/kg i.v. 15 min before reperfusion. Their kidneys were then removed and assayed for phospholipid Schiff base fluorescence (ex. 360 nm, em. 435 nm), diene and triene conjugates by UV spectrophotometry (240 nm and 268 nm respectively), for superoxide dismutase and for reduced and oxidised glutathione to provide an index of glutathione redox activity. All indices of lipid peroxidation were significantly elevated in untreated rabbits and glutathione redox activity was reduced. Treatment with desferrioxamine however effectively prevented these deviations and in many cases maintained them at the levels in fresh rabbit kidneys. These data provide further evidence that lipid peroxidation occurring during the reperfusion period is superimposed on the damage set up during warm ischaemia and may be preventable by administration of suitable therapeutic agents.