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AIMS: To determine the changes in serum zinc concentration and the extent of urinary zinc excretion in patients with iron overload receiving the oral iron chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1) or desferrioxamine (DFX), and to correlate these results with blood glucose concentration. METHODS: Serum zinc and ferritin concentrations, urinary zinc and iron excretion were regularly assayed in 39 patients and the glucose tolerance test (GTT) was performed in each patient. Patients were segregated according to their GTT into normal, diabetic, and those with an abnormal GTT. The mean of L1- or DFX associated urinary zinc excretion for each group was determined and compared with the other two groups and with normal value. L1 associated urinary zinc excretion was also compared with L1 dose, serum ferritin values, and urinary iron excretion. RESULTS: Both DFX and L1 were associated with a significantly increased urinary zinc excretion (15.1 (7.3) mumol/24 hours, 11.1 (6.0) mumol/24 hours, respectively) compared with normal subjects. In patients receiving DFX this increase only occurred in patients with diabetes mellitus. Both diabetic and non-diabetic patients receiving L1 treatment excreted more zinc than normal. Diabetic patients receiving L1 or DFX excreted more zinc than non-diabetics receiving the same treatment. No correlation was found between urinary zinc excretion and L1 dose or patients' serum ferritin concentrations. In seven patients receiving long term L1 treatment a fall in serum zinc was observed from an initial 13.6 (1.6) mumol/l to a final 9.6 (0.8) mumol/l. In one patient this was associated with symptoms of dry skin and itchy skin patches requiring treatment with oral zinc sulphate. CONCLUSIONS: In contrast to DFX, L1 treatment is associated with increased zinc loss. This, however, is modest and does not lead in most patients to subnormal serum zinc concentrations. In a few patients whose negative zinc balance may give rise to symptoms, zinc supplementation rapidly corrects the deficit.

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The method based on high-performance liquid chromatography currently used to determine the lipid levels of octadeca-9(Z),11(E)-dienoic acid in human serum fails to distinguish between this acid and octadeca-10(E),12(Z)-dienoic acid. Adducts with the fluorescent dienophile 4-[3-(1-propenyl)propyl]-1,2,4-triazoline-3,5-dione permit the separation of these two isomeric acids as does gas-liquid chromatography of their methyl esters, but these alternatives produce equivocal results when applied to biological samples.

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1. Oxygen-derived free radicals have been implicated in reperfusion injury after thrombolytic therapy in acute myocardial infarction. To test the hypothesis that diabetic patients may have increased oxidative stress which may lead to increased reperfusion damage and thereby contribute to a poorer outcome in these patients, we measured two indices of free-radical activity, diene conjugate molar ratios as an index of lipid isomerization and thiobarbituric acid-reactive material as an index of lipid peroxidation, in 66 non-diabetic and 26 diabetic patients admitted with acute myocardial infarction who received thrombolytic therapy and in whom reperfusion was assessed using early time to peak creatine kinase-MB isoenzyme release. 2. Baseline diene conjugate molar ratios or thiobarbituric acid-reactivity did not differ significantly between diabetic and non-diabetic patients (1.97 +/- 0.98 versus 2.16 +/- 1.34; not significant and 2.10 +/- 0.60 versus 1.99 +/- 0.73 mumol/l; not significant). In patients with enzymic evidence of reperfusion (i.e. time to peak enzyme release < or = 12 h) diene conjugate molar ratios peaked at 6 h compared with 12h in those with unsuccessful reperfusion (i.e. time to peak enzyme release > 12 h). In patients with unstable angina the maximum increase in the diene conjugate molar ratios was significantly less than in patients with acute myocardial infarction (6.80 +/- 12.3 versus 15.82 +/- 22.55%; P = 0.035). There was a significant fall in thiobarbituric acid-reactivity at 24 h in patients with enzymic evidence of reperfusion (P = 0.017). There were no major differences in these rises and falls between diabetic and non-diabetic patients.(ABSTRACT TRUNCATED AT 250 WORDS)

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Myocardial and pulmonary impairment after cardiopulmonary bypass may be caused by oxygen free radicals produced by reperfusion and by activated neutrophils. Free radical activity was assessed by assays for lipid peroxidation (thiobarbituric acid-reactive material) and phospholipid-esterified diene conjugation (18:2[9,11]/18:2[9,12] molar ratio) in 25 patients during coronary artery operations. Arterial blood samples were obtained before, during the ischemic period, and for 24 hours thereafter. There were no significant changes in free radical indices during the ischemic periods, but after cessation of bypass they increased significantly. Ten minutes after bypass thiobarbituric acid-reactive material rose from 96 (median; range 65 to 145) nmol/gm albumin to 138 (85 to 200) nmol/gm albumin (p < 0.001), and molar ratio rose from 2.23% (0.45% to 7.70%) to 2.51% (0.39% to 7.93%) (p < 0.02). Values of thiobarbituric acid-reactive material subsequently decreased, but molar ratio reached a peak at 4 hours after bypass, 2.64% (0.55% to 10.08%) (p < 0.001), thereafter returning to baseline. The postoperative increases in thiobarbituric acid-reactive material and in molar ratio were correlated (r = +0.53; p = 0.006). These increases in thiobarbituric acid-reactive material and in molar ratio were not related to age, preoperative left ventricular function, or the number of grafts performed. Increase in thiobarbituric acid-reactive material correlated with the duration of cardiopulmonary bypass (r = +0.43; p = 0.03). In 10 patients in whom cardiopulmonary bypass was performed using a bubble oxygenator, the increases in thiobarbituric acid-reactive material were significantly greater than in the 15 in whom a membrane oxygenator was used (p < 0.02). These data show that after apparently uncomplicated coronary operations with bypass there is an increase in lipid peroxidation and diene conjugation, indicating increased free radical activity. This increase varies between patients and does not relate to patient or surgical factors but may depend on the type of oxygenator employed during bypass.

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BACKGROUND: Experimental data suggest that reperfusion injury involving free radicals contributes to the impairment of left ventricular function after successful thrombolysis. METHODS: In 72 patients presenting with acute myocardial infarction, markers of free radical activity were measured before streptokinase and two hours later. Thiobarbituric acid reactive material (TBA-RM) reflects lipid peroxidation by free radicals, and the concentration of plasma total thiols (34 patients) reflects oxidative stress. Coronary arteriography was performed at 18-72 hours after thrombolysis to determine coronary patency, and left ventricular function was assessed by ventriculography and from QRS scoring of the electrocardiogram. RESULTS: The infarct related artery was patent (Thrombolysis In Myocardial Infarction Trial grade 2 or better) in 60 (83%) and occluded in 12. In the 60 with a patent artery, the concentration of TBA-RM increased after streptokinase by (mean (SD)) 9.2 (14.0) nmol/g albumin, whereas in the 12 with an occluded artery TBA-RM decreased by 7.0 (11.3) nmol/g albumin (p < 0.01 between groups). In those with a patent artery the rise in TBA-RM associated with thrombolysis correlated with left ventricular ejection fraction (R = -0.41, p < 0.002), and with the QRS score (R = +0.38, p = 0.003). Plasma total thiol concentrations decreased by 12.7 (31.1) mumol/l in those with a patent artery, and this decrease associated with thrombolysis correlated with left ventricular ejection fraction (R = +0.39, p < 0.02) but not with the QRS score (R = -0.2, NS). CONCLUSIONS: These findings suggest that reperfusion injury mediated by free radicals may be of clinical importance in humans.

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Non-transferrin-bound iron (NTBI) in plasma is toxic due to its ability to participate in free radical formation with resultant peroxidation and damage to cell membranes and other biomolecules. NTBI concentration was determined in serum in 12 normal volunteers and in 52 patients with beta-thalassaemia major by a modification of the method described by Singh et al (1990). There was no detectable NTBI in normal individuals. In the patients NTBI values ranged from -1.5 to 9.0 mumol/l (mean +/- SD: 3.6 +/- 2.3). The patients' serum ferritin concentrations ranged from 207 to 11,400 micrograms/l (2674 +/- 2538), total serum iron from 20 to 61 mumol/l (39.5 +/- 9.6) and transferrin saturation from 44 to 110% (84.5 +/- 13.8). The NTBI correlated significantly with serum ferritin (r = 0.467, P < 0.001), total serum iron (r = 0.608, P < 0.001) and transferrin saturation (r = 0.481, P < 0.005). When patients were grouped according to their compliance with desferrioxamine (DFX) therapy, the good compliers had significantly lower NTBI concentrations compared to the poor compliers (poor: 5.4 +/- 1.8 mumol/l v good: 2.7 +/- 1.7 mumol/l, P < 0.001). There was also a significant difference between the level of NTBI and whether or not the patients had complications of iron overload (5.2 +/- 1.7 mumol/l v 2.9 +/- 1.6 mumol/l, P < 0.001). During this study 10 patients were entered into a trial of the oral iron chelator 1,2- dimethyl-3-hydroxypyrid-4-one (L1). Their NTBI values were observed during the first 6 months of the trial and showed a significant fall (paired t-test: P = 0.007). These results suggest that the level of NTBI may prove helpful in assessing the efficiency of chelation in patients with transfusion dependent anaemia and help to predict organ damage.

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Eleven patients with beta thalassemia major were entered into the trial of the oral chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1). Their ages ranged from 17 to 26 years (mean +/- SD, 22.3 +/- 2.7). Six were male and five were female. L1 was administered at an initial daily dose of 42.5 to 60 mg/kg as a single dose. After 4 weeks, the dose was increased to 85 to 119 (102 +/- 10.7) mg/kg for 191 to 352 days divided into either two or four doses daily, except for one patient who developed agranulocytosis after 11 weeks and was taken off the trial. Initial serum ferritin values in the remaining 10 patients ranged between 1,000 and 9,580 (5,549 +/- 3,333) micrograms/L and at end of the trial their mean serum ferritin was significantly lower (4,126 +/- 2,278; P less than .05 using the paired t-test). Urinary iron excretion at a daily dose of 85 to 119 mg/kg administered as two divided doses ranged between 0.14 and 0.82 (0.44 +/- 0.26) mg/kg/24 h. In three patients, the four doses per day schedule caused substantially more iron excretion than the same total dose divided into two. During the course of the trial, several possible adverse effects have been encountered. One patient (female, aged 20) developed agranulocytosis 11 weeks after starting treatment and 6 weeks after beginning treatment with a daily dose of 105 mg/kg. This patient's neutrophil count recovered spontaneously 7 weeks after the discontinuation of L1. A decrease in serum zinc levels to subnormal levels was observed in four patients with symptoms of dry skin, with an itchy scaly rash in two that was associated with low serum zinc levels that responded to zinc therapy. Urinary zinc levels ranged from 4.7 to 23.4 (13 +/- 5.5) mumol/24 h and were above 9 mumol/24 h (upper limit of normal) in eight patients. Mild nausea occurred in three patients and transient diarrhea in a fourth. Mild musculoskeletal symptoms occurred in three patients but settled without discontinuation of L1 therapy in two and with temporary discontinuation of L1 in the third. A transient increase in serum aspartate transaminase was also noted in five patients, but serum aspartate transaminase levels subsequently decreased in all of them. No cardiovascular, neurologic, renal, or retinal toxicities were demonstrable. These results confirm that L1 is an effective oral iron chelator. Further clinical trials are needed to determine the incidence and severity of adverse effects.

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STUDY OBJECTIVE: The aim was to compare platelet function in diabetic and non-diabetic survivors of acute myocardial infarction and to relate it to an index of free radical activity in these patients. DESIGN: In vivo and in vitro indices of platelet function and diene conjugate molar ratios were measured in diabetic and non-diabetic infarct survivors on admission to hospital and sequentially for 72 h. PATIENTS: The patients were 17 diabetics (age 61.9 years, range 32-74) and 12 non-diabetics (60.8 years, range 39-75) admitted to hospital with acute myocardial infarction confirmed according to WHO criteria. MEASUREMENTS AND MAIN RESULTS: Agonist induced platelet aggregation, beta thromboglobulin levels, and linoleic acid 18:2(9,11)/18:2(9,12) molar ratios did not differ between diabetic and non-diabetic patients on admission. Concentrations of adrenaline producing 50% maximum platelet aggregation (EC50) in whole blood on admission were lower than non-infarct controls in both patient groups. The EC50 values in platelet rich plasma in both groups fell during the 72 h following admission (increases in platelet sensitivity). beta Thromboglobulin concentrations fell following admission in the diabetic group. Diene conjugate molar ratios were higher at 12 h and 24 h compared to admission in diabetic patients. Increases in diene conjugate ratios between admission and 24 h correlated with peak aspartate transaminase levels in both patient groups. No correlations were observed between platelet aggregation, beta thromboglobulin levels, or diene conjugate molar ratios. CONCLUSIONS: During 72 h following myocardial infarction there is a reduction in platelet activation in vivo and an increase in platelet sensitivity to exogenous agonists in vitro. Free radical generated isomers of linoleic acid increase in relation to infarct size, but are unrelated to platelet function. There were no differences in platelet function between diabetic and non-diabetic subjects.

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Diffuse impairment of ventricular function after cardiac surgery may be related to the generation during reperfusion of the myocardium of free radicals derived from oxygen. Fifteen patients undergoing elective coronary bypass surgery were studied by previously described assays for peroxidised lipids and for isomerised lipids which were used as indices of free radical activity. Serial blood samples were obtained from systemic arterial, mixed venous, and coronary sinus catheters before, during, and after the ischaemic period. The patients underwent coronary artery surgery on cardiopulmonary bypass with a membrane oxygenator, relative hypothermia 30-34 degrees C, and intermittent cross-clamping of the aorta. During the ischaemic periods there were no significant changes in the indices of free radical activity. During the reperfusion phase there was a significant increase in free radical indices in arterial and mixed venous blood. A small rise in free radical indices in coronary venous blood was not statistically significant. These data indicate that free radical activity is increased in patients shortly after the cessation of cardiopulmonary bypass. The pattern of distribution between the different sampling sites suggests that much of the observed increase in isomerised and peroxidised lipids originates from tissues other than the myocardium.

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In 50 patients receiving thrombolytic treatment for acute myocardial infarction, peripheral venous blood samples were taken before streptokinase and 2 h later for assay of markers of free radical activity. Coronary arteriography was carried out within 72 h of thrombolysis. In the 42 patients with patent arteries after thrombolysis, the levels of thiobarbituric-acid-reactive material (TBA-RM), which reflects lipid peroxidation by free radicals, rose after streptokinase by 10.5 (SD 9.6) [corrected] nmol/g albumin, whereas in the 8 whose arteries remained occluded TBA-RM fell by 14.7 (8.0) [corrected] nmol/g albumin. There was no significant change in the 18:2 (9,11)/18:2 (9,12) molar ratio, an indicator of lipid isomerisation, either between the groups or after streptokinase. Thus, after successful thrombolysis there is a rise in lipid peroxidation, not seen in patients whose arteries remain occluded. This finding suggests free-radical-mediated damage at the time of reperfusion, and provides indirect evidence of reperfusion injury in man. This study provides evidence associating indicators of free radical activity with documented myocardial reperfusion in man.

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The efficacy of electron spin resonance spectroscopy (a technique that measures free radicals) in detecting cervical intraepithelial neoplasia was evaluated in 68 women. In addition, electron spin resonance spectroscopic data were correlated with the phospholipid-esterified octadeca-9,11-dienoic acid (18:2(9,11]/octadeca-9,12-dienoic acid (18:2(9,12] molar ratio, a suggested marker of free radical damage to linoleic acid. Incubation of cervical epithelial cells with the spin-trap N-tert-butyl-alpha-phenylnitrone and subsequent examination by electron spin resonance spectroscopy gave signals from a nitroxide radical, which has been tentatively assigned to an adduct from either a carbon-centered lipid radical or a lipid alkoxyl radical; no significant differences in either the type of radical detected or the intensity of the signals was found between patients with cervical intraepithelial neoplasia and normal control subjects. There was no significant correlation between the intensity of the electron spin resonance signal and the phospholipid-esterified 18:2(9,11)/18:2(9,12) molar ratio. This finding provides no support for the classification of 18:2(9,11) as a "free radical product."

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To determine whether haemodialysis produces an increase in free radical activity, plasma phospholipid plus free fatty acid (PL+FFA) octadeca-9,11-dienoic acid (18:2(9,11)) and plasma thiobarbituric acid reactivity (TBAR) were measured serially during dialysis with cuprophan and polycarbonate membranes. Plasma TBAR did not change significantly during dialysis with either membrane. There was however, an increase in PL + FFA 18:2(9,11) and in the molar ratio of 18:2(9,11) to its parent compound, linoleic acid (18:2(9,12)), with both membranes, although this was most significant with cuprophan membranes. The administration of heparin to normal controls without dialysis produces a comparable rise in PL + FFA 18:2(9,11). Haemodialysis with prostacyclin anticoagulation resulted in no significant change in PL+FFA 18:2(9,11). The data indicates that the increase in PL+FFA 18:2(9,11) during haemodialysis is due to a heparin-induced rise in FFA 18:2(9,11) and is not a consequence of increased free radical activity. In contrast to animal models of systemic complement activation, we were unable to demonstrate an increase in plasma free radical activity during haemodialysis. However, this does not preclude a role for free radicals, released by activated neutrophils, in the pathogenesis of pulmonary dysfunction during haemodialysis.

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The simple spectroscopic measurement of diene conjugation has long been an established but somewhat problematic marker of free-radical activity in biological systems. The main diene-conjugated compounds in human tissues and tissue fluids have now been identified as esters of octadeca-9,11-dienoic acid (18:2(9,11)), a non-peroxide isomer of linoleic acid (18:2(9,12)); and a range of high-performance liquid chromatographic methods has been developed for their detection and measurement. Significant abnormalities of phospholipid-esterified 18:2(9,11) have been found in the serum of chronic alcoholics and in paraquat poisoning and of non-esterified 18:2(9,11) in lipolytic states. The phospholipid-esterified 18:2(9,11) is increased in the bile of patients with pancreatic disease. In exfoliated cells from the cervix uteri an abnormal molar ratio between phospholipid-esterified 18:2(9,11) and 18:2(9,12) may prove to be the most sensitive biochemical marker of precancerous change.

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Linoleic acid (18:2(9,12)) and its diene-conjugated isomer (18:2(9,11)) were measured in 65 cervical biopsy samples. Both the 18:2(9,11) concentration and the 18:2(9,11)/18:2(9,12) molar ratio showed highly significant differences between the normal and precancerous groups. Both showed a further significant increase in 4 invasive carcinomas. The findings in histologically normal areas from organs with precancer correlated significantly with the results in the precancerous lesions.

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The molar ratio between a diene-conjugated linoleic-acid isomer (18:2(9,11)) and the parent linoleic acid (18:2(9,12)), both esterified as phospholipids, was significantly different in exfoliated cells from normal cervices and from cervices with colposcopic and cytological evidence of precancer. The measurement may provide a simple and perhaps improved alternative to cytological screening.

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Very few cases of acquired severe copper deficiency have been described. The principal effects are haematological, but the precise abnormalities are uncertain due to the possible association of other deficiencies. A case of isolated severe copper deficiency associated with late onset hypogammaglobulinaemia is reported in which the chief findings were macrocytic anaemia, neutropenia and a decrease in mean platelet volume. All these abnormalities resolved when copper therapy was instituted and recurred when the medication was stopped.

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Exfoliated cells from the precancerous cervix have an increased concentration of octadeca-9,11-dienoic acid, a diene-conjugated isomer of linoleic acid. A high-performance liquid chromatographic method which measures both has been automated. It may form the basis of a sensitive diagnostic test.

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Free-radical activity was studied in patients on maintenance haemodialysis by measuring plasma octadeca-9, 11-dienoic acid (9,11-LA'), a diene-conjugated derivative of linoleic acid. Baseline values of 9,11-LA' (esterified as phospholipids and as free fatty acids) in 51 haemodialysis patients were similar to that of normal control subjects. However, during haemodialysis there was a highly significant (P less than 0.001) increase in 9,11-LA' in all 13 patients studied, which reached a peak 30 min after haemodialysis was started and then declined. The rise in plasma 9,11-LA' may be due to free radicals generated by activated neutrophils. Abnormal free-radical activity may be partly responsible for some haemodialysis-related complications, including pulmonary dysfunction in early haemodialysis.

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