R.E. Basford

The Critical Relationship Between Free Radicals and Degrees of Ischemia: Evidence for Tissue Intolerance of Marginal Perfusion

UNLABELLED Skin-flap ischemia has been associated with the presence of free radicals. In this study, two enzyme systems involved in free-radical metabolism were used to compare a distal skin flap to a skin graft. Forty-two rats were divided into several test groups. A 10 X 3 cm dorsal rat flap was used, and tissue biopsies for xanthine oxidase and malonyldialdehyde (MDA) were obtained 2.5, 5.5, and 8.5 cm from the base of the flap at the hours given. In group I (control), the flap was outlined but not elevated, and biopsies were obtained. In group II, the flap was elevated, and biopsies were obtained at 6 hours. In group III, the flap was elevated, the distal 4 X 3 cm was amputated and replaced as a full-thickness skin graft, and biopsies were obtained at 6 hours. In group IV, the flap was elevated, and biopsies were obtained at 12 hours. In group V, the flap was treated as in group III, and biopsies were obtained at 12 hours. In group VI, the flap was elevated, and biopsies were obtained at 24 hours. In group VII, the flap was treated as in group III, and biopsies were obtained at 24 hours. RESULTS Xanthine oxidase was significantly higher in all distal biopsies compared to proximal biopsies. Xanthine oxidase also increased with time. Malonyldialdehyde increased over time as well as with distance from the flap base. Distal flap biopsies at 24 hours had greatly increased levels of malonyldialdehyde compared to skin grafts (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Deferoxamine increases skin flap survival: additional evidence of free radical involvement in ischaemic flap surgery

This study presents further evidence of free radical involvement in skin flap necrosis in a dorsal rat flap model. Rats receiving deferoxamine, a free radical scavenger and iron chelator had significantly less necrosis (p less than 0.001) than saline treated rats. In a separate experiment, tissue determinations for malonyldialdehyde (MDA) were consistent with the survival results in showing a significant decrease in MDA in all biopsy sites (p less than 0.05 or less), indicative of reduced lipoperoxidation in the deferoxamine treated rats.

Leishmania donovani: Surface membrane acid phosphatase blocks neutrophil oxidative metabolite production

We show that a purified preparation of the prominent tartrate-resistant acid phosphatase (E.C.3.1.3.2), isolated from the external surface of the intracellular parasite Leishmania donovani (promastigote form), inhibits toxic oxidative metabolite production of neutrophils. Preincubation of a neutrophil suspension (2.5 X 10(6) cells/ml) for 15 min at 37 C with 250 units (1 unit equals 1 nmole of 4-methylumbelliferyl phosphate cleaved per hr at pH 5.5) of the acid phosphatase in Krebs-Ringer phosphate buffer (pH 7.4) decreased O2 consumption, O2- production, and H2O2 production of N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe)-stimulated neutrophils to 15-25% of control values. The acid phosphatase also affected concanavalin A-stimulated O2-production by neutrophils, but had no effect on the rate of phorbol myristic acetate-stimulated O2- production, chemotactic peptide binding, degranulation, or membrane depolarization. Addition of an acid phosphatase inhibitor (Complex E; (NH4)6[P2Mo18O62] X 9H2O) to suspensions of opsonized promastigotes and neutrophils resulted in a threefold or greater enhancement of O2- production. These results suggest a possible pathophysiologic role for the acid phosphatase of L. donovani promastigotes.

Platelets and ATP prime neutrophils for enhanced O2- generation at low concentrations but inhibit O2- generation at high concentration

Platelets are currently thought to play a role in tissue injury and inflammatory states both directly and indirectly through their action on neutrophils (PMNs). Both stimulation and inhibition of PMN superoxide anion (O2 ‐) production by platelets has been reported. To clarify these discrepant observations, we investigated the effects of wide ranges of platelet to PMN ratios as well as concentrations of ATP and ADP on human PMN O2 ‐ production. Platelets, at low platelet‐to‐PMN ratios (1:1 and 5:1), primed PMNs which were stimulated with either FMLP or PMA. However, at higher platelet‐to‐PMN ratios (25:1, 50:1, and 100:1), inhibition of O2 ‐ production was seen. ATP also had a biphasic effect on O2 ‐ production: low concentrations of ATP (1 x 10‐6 to 3.2 x 10‐4 M) increased O2 production and high concentrations of ATP (6.4 x 10‐4 M and above) inhibited O2 production. ADP, when added to stimulatory concentrations of ATP, also caused inhibition of O2 ‐ production. The incubation time for platelet‐neutrophil interactions in vitro was also crucial. Short incubation periods lead to priming, whereas longer periods (> 5 min) lead to inhibition. We believe that these studies help to resolve the controversy over the effects of platelets upon the production of O2 ‐ by human PMNs and lend further support to the notion that platelets may modulate injury resulting from neutrophil activation.

Increased plasma chemoattractant in Sweet's syndrome

The neutrophil function and plasma leukotactic activity of a patient with Sweets syndrome and cystonodular acne were evaluated during a 2 1/2-year period. These studies demonstrated that chemotaxis was frequently slightly increased, especially during an exacerbation of Sweets syndrome, but showed some decrease during isotretinoin therapy. Other functions, such as phagocytosis, metabolic activation, and bacterial killing, also were slightly increased. In addition, the patients serum contained a heat-stable, nonlipid chemoattractant that was present at all times except during a course of isotretinoin. Although his symptoms responded to aspirin, the plasma continued to show this chemoattractant. These findings are consistent with the hypothesis that excess chemoattractant in Sweets syndrome attracts neutrophils, which then mediate an inflammatory response. In addition, aspirin may be used to control Sweets syndrome symptoms, although it does not suppress the plasma chemoattractant.

Augmentation of skin flap survival with allopurinol.

Although allopurinol is primarily known as an effective medication for gout, it has been shown to enhance tissue survival in a wide range of ischemic conditions. The study reported here investigated the effects of allopurinol on flap survival in a dorsal rat model. In a preliminary study, animals were given varying doses of allopurinol (0 to 400 mg per kilogram). A clinically efficacious dose was established upon conclusion of the test period by laboratory determinations and necropsy data. Other animals were divided into 3 groups: 1 (saline control), N = 11; 2 (50 mg per kilogram of allopurinol daily), N = 10; 3 (100 mg per kilogram of allopurinol qd), N = 11. Flaps were raised and necrosis assessed at 8 days. Flaps treated with allopurinol 100 mg per kilogram had significantly better survival than the controls (p < 0.001) and 50 mg per kilogram (p < 0.01). Allopurinol 50 mg per kilogram had no effect on flap survival.

Neutrophil Function in Chronic Neutrophilic Leukemia: Defective Respiratory Burst in Response to Phorbol Esters

Functional analyses were performed on neutrophils isolated from 6 patients from two institutions who displayed features of chronic neutrophilic leukemia (CNL). These neutrophils demonstrated a consistent deficiency (44 +/- 8% of control values) in superoxide anion (O2-) production in response to the phorbol ester, phorbol myristate acetate (PMA). O2- production in response to chemotactic peptides was near normal (82.3 +/- 10.7% of control values). Bacterial killing was normal in the two patients studied, and chemotaxis was diminished in response to zymosan-activated plasma and to high concentrations of chemotactic peptides in the patients studied. Cytosolic C kinase activity was decreased in one of the two patients studied. These results suggest that a deficient O2- release in response to PMA is a hallmark of neutrophils in CNL and may provide a diagnostic indicator of this condition.

Can lipid peroxidation damage the amputated digit prior to replantation

The free radical induced lipid peroxidation has often been implicated in cellular damage (1). One of the requirements for this process to occur is the presence of molecular oxygen (2). When this principle is applied to the discussion of amputated digits before replantation, one can ask the question whether or not lipid peroxidation is involved in causing damage. However, one has to be careful in answering this question because the net perfusion of the amputated digit is zero and hence the oxygen content is negligible. In this situation, can lipid peroxidation occur and, if so, is it due to the diffusion of oxygen from the atmosphere into the digit? Also, one has to be concerned about the fate of the Superoxide ion that is formed.