Time-course of free radical activity during coronary artery operations with cardiopulmonary bypass.

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.

Free radical activity and left ventricular function after thrombolysis for acute infarction.

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.

Platelet function and conjugated diene concentrations in diabetic and non-diabetic survivors of acute myocardial infarction.

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.

Systemic pattern of free radical generation during coronary bypass surgery.

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.

Studies on cervical intraepithelial neoplasia: the level of octadeca-9,11-dienoic acid and measurement of free radical content by electron spin resonance spectroscopy.

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."

Lipid peroxidation associated with successful thrombolysis.

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.

Aberrant free radical activity in cystic fibrosis.

It has been suggested that the molar ratio of octadeca 9,11 dienoic acid to linoleic acid in biological material provides an index of activity along the non-peroxide pathway of a free radical attack on polyunsaturated fatty acids. In 17 adults with cystic fibrosis the 'molar ratio' in nasal epithelial cells--a recognised target of the disease--exceeded that in 20 controls (median 2.09%, range 1.70-3.01% versus 1.56, 0.92-2.23%, p = 0.0002). The difference was also apparent, although less stark, upon analysis of serum in a further 22 CF patients (2.48%, 1.60-5.24%) and 25 controls (1.96%, 0.81-3.90%, p = 0.0348). There was no correlation between the 'molar ratio' and blood white cell count or erythrocyte sedimentation rate, severity of lung or liver disease, indicating that the raised values are a primary feature, rather than reflecting disease severity. Aberrant free radical activity may underlie cellular dysfunction in cystic fibrosis.

In praise of peroxidation.

Free-radical-mediated lipid peroxidation has become closely associated with destructive biochemical processes and, more recently, with disease. Its potential survival value may be overlooked.

Effect of cardiopulmonary bypass on circulating concentrations of leucocyte elastase and free radical activity.

Circulating concentrations of leucocyte elastase and free radical activity were measured in 11 adults undergoing cardiopulmonary bypass. In all patients the bypass procedure was associated with pronounced changes in plasma elastase concentrations, and peak enzyme concentrations correlated closely with the duration of bypass (r = 0.91, p less than 0.001). Serial measurement of octadeca-9, 11-dienoic acid, a non-peroxide marker of free radical activity, showed significant changes only in the plasma free fatty acid fraction, suggesting a direct relation to the action of heparin rather than to the bypass procedure as such. These studies support the hypothesis that neutrophil activation plays a central role in the organ dysfunction that may complicate cardiopulmonary bypass and suggest that elastase release rather than free radical generation may be the appropriate marker of the event.

Neutropenia and plasma free radical reaction products during haemodialysis.

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.

The experimental and clinical pathology of diene conjugation.

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.

Macrocytic anaemia due to copper deficiency in a patient with late onset hypogammaglobulinaemia.

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.

Diagnosis of cervical neoplasia by the estimation of octadeca-9,11-dienoic acid.

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.

Recognition of cervical neoplasia by the estimation of a free-radical reaction product (octadeca-9,11-dienoic acid) in biopsy material.

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.

Recognition of cervical neoplasia by the estimation of a free-radical reaction product (octadeca-9,11-dienoic acid) in exfoliated cells.

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.