Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays

Hydroperoxide metabolism in mammalian organs.

Publisher Summary This chapter discusses the role of free radicals and catalytic metal ions in human disease. The importance of transition metal ions in mediating oxidant damage naturally leads to the question as to what forms of such ions might be available to catalyze radical reactions in vivo . The chapter discusses the metabolism of transition metals, such as iron and copper. It also discusses the chelation therapy that is an approach to site-specific antioxidant protection. The detection and measurement of lipid peroxidation is the evidence most frequently cited to support the involvement of free radical reactions in toxicology and in human disease. A wide range of techniques is available to measure the rate of this process, but none is applicable to all circumstances. The two most popular are the measurement of diene conjugation and the thiobarbituric acid (TBA) test, but they are both subject to pitfalls, especially when applied to human samples. The chapter also discusses the essential principles of the peroxidation process. When discussing lipid peroxidation, it is essential to use clear terminology for the sequence of events involved; an imprecise use of terms such as initiation has caused considerable confusion in the literature. In a completely peroxide-free lipid system, first chain initiation of a peroxidation sequence in a membrane or polyunsaturated fatty acid refers to the attack of any species that has sufficient reactivity to abstract a hydrogen atom from a methylene group.

Role of free radicals and catalytic metal ions in human disease: an overview.

(First of Two Parts) THE part played by phagocytes in defense against invading pathogens has been recognized since 1883. In that year, Metschnikoff, a Russian zoologist, reported that foreign particles injected into metazoans (in Metschnikoff's experiments, starfish larvae) were taken up by a population of "wandering mesodermal cells" that resided in interstitial tissues.1 He postulated a crucial role in host defense for these wandering cells, which he named "phagocytes." Since Metschnikoff's discovery, hundreds of scientists studying dozens of species have reported thousands of studies on these cells, perhaps the most widely recognized of which are those of the eminent English . . .

Oxygen-dependent microbial killing by phagocytes (second of two parts).

Neutrophilic polymorphonuclear leukocytes (neutrophils) are highly specialized for their primary function, the phagocytosis and destruction of microorganisms. When coated with opsonins (generally complement and/or antibody), microorganisms bind to specific receptors on the surface of the phagocyte and invagination of the cell membrane occurs with the incorporation of the microorganism into an intracellular phagosome. There follows a burst of oxygen consumption, and much, if not all, of the extra oxygen consumed is converted to highly reactive oxygen species. In addition, the cytoplasmic granules discharge their contents into the phagosome, and death of the ingested microorganism soon follows. Among the antimicrobial systems formed in the phagosome is one consisting of myeloperoxidase (MPO), released into the phagosome during the degranulation process, hydrogen peroxide (H2O2), formed by the respiratory burst and a halide, particularly chloride. The initial product of the MPO-H2O2-chloride system is hypochlorous acid, and subsequent formation of chlorine, chloramines, hydroxyl radicals, singlet oxygen, and ozone has been proposed. These same toxic agents can be released to the outside of the cell, where they may attack normal tissue and thus contribute to the pathogenesis of disease. This review will consider the potential sources of H2O2 for the MPO-H2O2-halide system; the toxic products of the MPO system; the evidence for MPO involvement in the microbicidal activity of neutrophils; the involvement of MPO-independent antimicrobial systems; and the role of the MPO system in tissue injury. It is concluded that the MPO system plays an important role in the microbicidal activity of phagocytes.

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Myeloperoxidase: friend and foe

Chronic granulomatous disease is an X-linked defect in the killing of certain bacteria by peripheral blood granulocytes and may be detected with the nitroblue tetrazolium (NBT) test. The r...

https://www.nejm.org/doi/pdf/10.1056/NEJM196805022781801?listPDF=true

Quantitative Nitroblue Tetrazolium Test in Chronic Granulomatous Disease

The biochemical basis of nitroblue tetrazolium reduction in normal human and chronic granulomatous disease polymorphonuclear leukocytes

Because ascorbate potentiates chemotaxis of normal leukocytes, we examined the effect of ascorbate on polymorphonuclear leukocytes from a patient with the Chediak-Higashi syndrome. Chemotactic migration was 104±16 leukocytes per 10 fields (mean±S.D.) initially and 258±44 (P<0.001) after ascorbate, as compared to 182±10 in controls. There was no bactericidal activity by 40 minutes in the patient's untreated leukocytes. After ascorbate bactericidal activity of patient and untreated control cells was the same. The addition of ascorbate reduced cAMP levels in the patient's cells from a mean of 34.5 pmoles per 107 polymorphonuclear leukocytes to 5.9, as compared to a control value of 3.1 ±1.4. The association of elevated cAMP and impaired function in the polymorphonuclear leukocytes of patients with the Chediak-Higashi syndrome may be related to abnormal microtubular assembly. (N Engl J Med 295:1041–1045, 1976)

https://www.nejm.org/doi/pdf/10.1056/NEJM197611042951904?listPDF=true

Correction of Leukocyte Function in Chediak-Higashi Syndrome by Ascorbate

Autooxidation as a basis for altered function by polymorphonuclear leukocytes

Retrospective evaluation of the occurrence of septicemia and meningitis in 200 children who had staging laparotomy iwth splenectomy for Hodgkin's disease revealed 20 episodes occurring in 18 children. Symptoms were usually fulminant; only 10 of these patients survived their episode. Infections occurred eight days to three years after splenectomy. Adolescents, as well as younger children, were affected; half were older than 10 years of age. Leukopenia was not a major factor in onset or survival since the average white-cell count was 12,000 in both survivors and children who died. Pneumonococcus accounted for 50 per cent, and streptococcus for 15 per cent of infections; there was one episode each of Haemophilus influenzae and meningococcus; in 25 per cent, no organism was isolated. Predominance of penicillin-sensitive organisms and high mortality suggest that penicillin prophylaxis and the protection offered by bacterial vaccines should be evaluated in children with Hodgkin's disease whose staging laparotomy includes splenectomy.

Robert L. Baehner

Papers

Quantitative Nitroblue Tetrazolium Test in Chronic Granulomatous Disease

The biochemical basis of nitroblue tetrazolium reduction in normal human and chronic granulomatous disease polymorphonuclear leukocytes

Correction of Leukocyte Function in Chediak-Higashi Syndrome by Ascorbate

Autooxidation as a basis for altered function by polymorphonuclear leukocytes

Septicemia and meningitis in children splenectomized for hodgkin's disease.