R. D. Abeysinghe

Plasma Lactoferrin as a Marker of White Cell Degranulation in Venous Disease

Objective: To measure plasma lactoferrin as a marker of neutrophil degranulation in groups of patients with varying severity of venous disease and compare with age- and sex-matched control subjects. Design: Prospective study of patients with varicose veins compared with a group of control subjects with no history or clinical findings of varicose veins. Setting: The Middlesex Hospital Vascular Laboratory, Mortimer Street, London WIN 8AA, UK. Patients: Patients referred to the Middlesex Hospital Vascular Laboratory for investigation of venous disease. Control subjects were obtained from within the laboratory and hospital staff, and from a group of Patients attending the London Foot Hospital for routine chiropody. Neither group had arterial disease nor any other illness or medication known to alter white cell activity. Interventions: 10 ml of blood taken from an arm vein into EDTA for a neutrophil count and measurement of Plasma lactoferrin using an ELISA. Results: Significantly raised plasma lactoferrin was found in all four groups of patients compared with their controls (p = 0.0156 for uncomplicated varicose veins, P = 0.01 for lipodermatosclerosis, p = 0.0413 for active venous ulceration, and p = 0.0005 for healed ulcers, Mann-Whitney U-test). Differences between medians (95% confidence interval) for the four groups were 269 (62–603), 199 (60–314), 133 (44–218) and 215 (98–349) ng/ml respectively. There was no difference in the neutrophil count between the patient and control groups, and correcting plasma lactoferrin for the neutrophil count did not remove significance in any group. Conclusions: This study shows evidence of increased neutrophil activation as shown by increased degranulation in patients with venous disease.

Secretion of neutrophil secondary granules occurs during granulocyte‐macrophage colony stimulating factor induced margination

The effects of recombinant human granulocytemacrophage colony‐stimulating factor (rhGM‐CSF) on neutrophil lactoferrin (LF) and transcobalamin (TC) 1 and 3 secretion were determined in vitro and during in vivo administration in humans. In whole blood, in vitro incubation with GM‐CSF reproducibly produced a rise in plasma LF concentration (P<0.05) whereas in purified neutrophils the results were variable. Exposure of whole blood to GM‐CSF also resulted in a significant rise in plasma TC 1 and 3 (190.60%, P<0.05). The response was dose dependent with maximal effect at GM‐CSF concentrations of 10 ng/ml and above. rhGM‐CSF was administered on seven occasions to six patients with malignant disease prior to chemotherapy. Plasma LF and unsaturated TC 1 and 3 levels rose significantly in each patient studied and the rise coincided with the initial neutropenia due to margination that occurs during infusions of rhGM‐CSF. Patients receiving rhGM‐CSF may therefore have hypofunctional neutrophils due to secondary granule depletion.

Neutrophil Activation in Experimental Venous Hypertension

Objective: To investigate the white cell trapping hypothesis of venous ulceration by measuring plasma lactoferrin as a marker of neutrophil degranulation in normal volunteers in two experimental models of venous hypertension. Design: A prospective study of volunteers with no history or clinical evidence of venous disease. Setting: The Middlesex Hospital Vascular Laboratory, Mortimer Street, London WIN 8AA, UK. Patients: Volunteers within the Middlesex Hospital Vascular Laboratory with no history or clinical findings of venous or arterial disease, no other systemic disease, on no medication known to alter white cell activity, and with no recent infection. Interventions: Venous blood was taken from cannulae in both feet and the right arm for a neutrophil count and Plasma lactoferrin, measured using an ELISA, during application of a tourniquet to 80 mmHG for 30 min to the right leg while supine, 5 min after release of tourniquet, and then during a 30 min period of standing. Results: During application of a tourniquet to the right leg there was a significant rise in plasma lactoferrin and in lactoferrin corrected for the neutrophil count (p < 0.05, Wilcoxon). In the unoccluded leg, although Plasma lactoferrin rose, this was not significant when corrected for the rise in neutrophil count. After standing for 30 min, the lactoferrin and neutrophil count increased in all three limbs; corrected lactoferrin showed a significant increase in the legs (p < 0.02), though not in the arm. Conclusion: Increased neutrolphil degranulation occurs during periods of short-term venous hypertension in normal volunteers, in keeping with the white cell trapping hypothesis.

The use of skin Fe levels as a surrogate marker for organ Fe levels, to monitor treatment in cases of iron overload

A system based on the detection of K-shell x-ray fluorescence (XRF) has been used to investigate whether a correlation exists between the concentration of iron in the skin and the concentration of iron in the liver, as the degree of iron loading increases. The motivation behind this work is to develop a non-invasive method of determining the extent of the bodys iron stores via measurements on the skin, in order to monitor the efficacy of chelation therapy administered to patients with beta-thalassaemia. Sprague-Dawley rats were iron loaded via injections of iron dextran and subsequently treated with the iron chelator CP94. The non-haem iron concentrations of the liver, heart and spleen were determined using bathophenanthroline sulphonate as the chromogen reagent. Samples of abdominal skin were taken and the iron concentrations determined using XRF. A strong correlation between the skin iron concentration and the liver iron concentration has been demonstrated (R2 = 0.86). Similar correlations exist for the heart and the spleen. These results show that this method holds great potential as a tool in the diagnosis and treatment of hereditary haemochromatosis and beta-thalassaemia.

6-Alkoxymethyl-3-hydroxy-4H-pyranones: potential ligands for cell-labelling with indium

Abstract. We have identified ligands for cell labelling with indium-111: 3-hydroxy-6-propoxymethyl-4H-pyran-4-one and 6-butoxymethyl-3-hydroxy-4H-pyran-4-one. The leucocyte labelling efficiencies of 111In complexes of these ligands were higher and label stabilities were found to be similar compared with those obtained using 111In-tropolonate. High labelling efficiencies of neutrophils and lymphocytes were achieved with 111In complexes of pyranones. Tropolone was found to have a greater inhibitory effect on metalloenzymes and to cause greater impairment of platelet function than 3-hydroxy-6-propoxymethyl-4H-pyran-4-one. Thus 6-alkoxymethyl-3-hydroxy-4H-pyran-4-ones may have advantages over current ligands used in cell labelling with 111In.

Platelet labelling with indium-hydroxypyridinone and indium-hydroxypyranone complexes

In order to identify new compounds which label platelets without affecting their function, three classes of metal chelating agents have been compared with oxine for their efficiency of indium-113m platelet labelling and for their short- and long-term effects on platelet function. The 3-hydroxypyridinones (both 2-ones and 4-ones) and 3-hydroxypyranones are bidentate chelators of trivalent metal ions that are neutrally charged in the metal-complexed form and hence gain access to cells readily. The hydroxypyranone ethylmaltol has been compared with the 3-hydroxypyridin-4-one CP94 and to its structurally related lipophilic analogue CP25 as well as with the 3-hydroxypyridin-2-one, CP02. The platelet labelling efficiencies with these ligands were between 75% and 95% of that obtained with oxine, following a 12-min incubation in saline. The optimal concentration for the hydroxypyridin-2-ones and hydroxypyridin-4-ones was approximately 10 μM compared with 100 μM for the hydroxypyranone ethylmaltol and 60 μM for oxine. Oxine and tropolone were found to produce significant inhibition of platelet aggregation to collagen in short-term experiments (10 min) or in longer term (18 and 42 h) ex vivo platelet cultures respectively. By contrast, ethylmaltol had no such inhibitory effects at either time interval. The relatively hydrophilic hydroxypyridin-4-one CP94 showed no inhibitory effects on collagen-induced aggregation in short-term studies, unlike the more lipid-soluble derivative CP25. These results suggest that ethylmaltol and related pyranones may have advantages over oxine and tropolone as indium platelet labelling agents where it is important not to damage platelets by the labelling process itself.