Substrate-Specific Reduction of Tetrazolium Salts by Isolated Mitochondria, Tissues, and Leukocytes.

Tetrazolium salts are commonly used in cytochemical and biochemical studies as indicators of metabolic activity of cells. Formazans, formed by reduction of tetrazolium salts, behave as pseudo-solutions during initial incubation, which allows monitoring their optical density throughout incubation. The criteria and conditions for measuring oxidative activity of mitochondria and dehydrogenase activity in reduction of nitroblue tetrazolium (NBT) and methyl thiazolyl tetrazolium (MTT) in suspensions of isolated mitochondria, tissue homogenates, and leukocytes were investigated in this work. We found that the reduction of these two acceptors depended on the oxidized substrate - NBT was reduced more readily during succinate oxidation, while MTT - during oxidation of NAD-dependent substrates. Reduction of both acceptors was more sensitive to dehydrogenase inhibitors that to respiratory chain inhibitors. The reduction of NBT in isolated mitochondria, in leukocytes in the presence of digitonin, and in liver and kidney homogenates was completely blocked by succinate dehydrogenase inhibitors - malonate and TTFA. Based on these criteria, activation of succinate oxidation was revealed from the increase in malonate-sensitive fraction of the reduced NBT under physiological stress. The effect of progesterone and its synthetic analogs on oxidation of NAD-dependent substrates by mitochondria was investigated using MTT. Both acceptors are also reduced by superoxide anion; the impact of this reaction is negligible or completely absent under physiological conditions, but can become detectable on generation of superoxide induced by inhibitors of individual enzyme complexes or in the case of mitochondrial dysfunction. The results indicate that the recording of optical density of reduced NBT and MTT is a highly sensitive method for evaluation of metabolic activity of mitochondria applicable for different incubation conditions, it offers certain advantages in comparison with other methods (simultaneous incubation of a large set of probes in spectral cuvettes or plates); moreover, it allows determination of activity of separate redox-dependent enzymes when selective inhibitors are available.

Effect of surfactant on nitroblue tetrazolium reduction of polymorphonuclear leucocytes stimulated with type Ia group B streptococci.

Activation of polymorphonuclear leucocytes (PMN) was investigated after incubation of adult human PMN and group B streptococci (GBS) type Ia with a type-specific polyclonal antiserum and a modified porcine surfactant (Curosurf). The level of oxidative metabolism of PMN was studied using a micromethod modification of the nitroblue tetrazolium (NBT) reduction test. GBS alone did not stimulate significant oxygen metabolite release from PMN, and incubation of PMN with surfactant alone resulted in decreased NBT reduction. After opsonization of GBS with a specific antibody, PMN were activated and the increased oxygen metabolite release was not suppressed when surfactant was added to the system. We conclude that the encapsulated GBS strain investigated needs opsonization with specific antibody to increase oxidative metabolism of PMN, and that incubation of PMN and opsonized GBS with surfactant does not interfere with NBT reduction.

Actions of adenosine on nitro blue tetrazolium deposition and surface pH during intestinal reperfusion injury.

Mesenteric arteries supplying an intestinal segment were occluded for 5 minutes and then released. During reperfusion, two series of measurements were made with various substances topically applied to the extraluminal surface. In the first series, reduced nitro blue tetrazolium (NBT) was extracted from tissue and measured spectrophotometrically, as an index of oxidative damage. In the second series, mucosal and serosal surface pH was measured as an index of the functional ability to maintain ion gradients. In control conditions, NBT deposition averaged 55-63 micrograms/g tissue. After 60 and 120 minutes of reperfusion, NBT was elevated to 446-479 micrograms/g, which was approximately half as large as the NBT increment (846 micrograms/g) produced by a 15-minute application of xanthine plus xanthine oxidase to well-perfused tissue. As expected, NBT levels were significantly lower (299 micrograms/g) in tissue that was continuously suffused with superoxide dismutase (SOD) plus catalase (CAT) before occlusion and during reperfusion. Similar NBT levels (274 micrograms/g) were observed after reperfusion in animals that were fed a diet supplemented with the antioxidant vitamin E for 4-6 weeks. These observations affirm that some, but not all, NBT deposition after reperfusion can be attributed to oxyradicals. However, with exogenous adenosine (ADO) applied for the first 30 minutes after occlusion, NBT was elevated to 174 micrograms/g after 60 minutes, which was only half as large as the increment with SOD plus CAT, even though those substances were continuously applied. The opposite effect was produced by an ADO receptor antagonist, 8-phenyltheophylline; NBT was increased to 516 micrograms/g.(ABSTRACT TRUNCATED AT 250 WORDS)

[The activation of the oxygen-dependent bactericidal system of human and rabbit monocytes by dust with different degrees of fibrogenicity]. / Issledovanie aktivatsii kislorodzavisimoi bakteritsidnoi sistemy monotsitov cheloveka i krolika pyl'iu s razlichnoi stepen'iu fibrogennosti.

Phagocytosis of quartz dusts by the monocytes of man and rabbit goes together with the stimulation of the substrate-free reduction of nitroblue tetrasolium, which reflects the activation of the cells' oxygen-depending bactericidal system. The quantity of the formasan produced increases, as compared to the control group, depending on the volume of the added dust. A less fibrogenic and cytotoxic dust of aluminum oxide causes only significant formation of formasan at the reduction of nitroblue tetrasolium. No extracellular generation of superoxide radicals and nitrogen peroxide by the cells were traced in the phagocytosis of quartz dusts by human and rabbit monocytes. The data obtained reveals a close similarity of the biochemical processes in the action of alveolar macrophages and monocytes with mineral dusts, and confirms a possibility of using the substrate-free reduction of nitroblue tetrasolium in the assessment of the hazardousness of industrial dusts.