Precise cytochemical measurement of neotetrazolium formazan by scanning and integrating microdensitometry

Summary The extinction values of the formazan of neotetrazolium, deposited in tissue sections, have been measured using a Vickers M 85 scanning and integrating microdensitometer with a scanning spot of 0.25 diameter. These values have been correlated with the amount of formazan in the same sections as determined by elution and spectrophotometry. The extinction at 520 nm did not increase linearly with increasing amounts of formazan in sections incubated for different times. This has been shown to be due to the presence of two formazan components which have different absorption characteristics; a red diffuse colour with a molar extinction coefficient of 20580 and a more granular purple product with a coefficient of 7370. However, at 585 nm, the isosbestic wavelength, the extinction was directly proportional to the amount of formazan in the section, irrespective of the nature of the end product; at this wavelength the molar extinction coefficient was 7200.These results have shown that scanning and integrating microdensitometry can be used for the precise measurement of the amount of neotetrazolium formazan, in picograms, in a single cell of group or cells in a tissue section.     

The purification and some properties of neotetrazolium chloride and its chief monotetrazolium salt contaminant

Synopsis A simple method which does not involve chromatography is described for the purification of neotetrazolium chloride (NT). Commercial samples of NT were shown to contain variable amounts of a major monotetrazolium salt contaminant, which was isolated and identified as 2-(4-biphenyl-)-3,5-diphenyltetrazolium chloride (BDTC). A qualitative method for checking the purity of NT and BDTC by thin-layer chromatography is described; with this technique the presence of traces of other tetrazolium salt contaminants was detected. The preparation of pure samples of formazan corresponding to NT and BDTC is described; molar extinction coefficients are given with sufficient other information to enable estimation of the amounts of NT and BDTC in mixtures of the two to be made.     

Dehydrogenase activity and loss of formazan from tissue sections

Summary Formazans can be lost from tissue sections when the incubation medium is removed at the end of a dehydrogenase reaction. The loss is not uniform and will clearly affect the interpretation of results. This suggests that both the qualitative evaluation of the reaction and the measurement of enzyme activity should be made at the time of incubation.     

The quantification of formazans in tissue sections by microdensitometry. I. The use of neotetrazolium chloride

Synopsis This article describes the use of a microdensitometer for the measurement of formazan deposits in tissue sections. Some examples are given to illustrate the various applications of this technique in the assessment of glucose-6-phosphate dehydrogenase activity. These are (1) the separate measurement of the red half-formazan intermediate and purple diformazan of neotetrazolium, and the effect of incubation time on their production, (2) the measurement of activities in different regions of the liver lobule, and the selective effect of phenobarbitone, and (3) the measurement of enzyme activity in individual cartilage cells in normal and osteoarthrosis-prone animals. All activities can be expressed in absolute units as nmol hydrogen/mm³/hr, and thus compared with standard biochemical data. The activities obtained all fall within the range of published values for biochemical systems.Paper given at the Royal Microscopical Society's European Histochemistry Meeting at Nottingham in September 1975.     

Localization of MTT formazan in lipid droplets. An alternative hypothesis about the nature of formazan granules and aggregates

MTT (3-(4, 5-dimethyl-2-thiazolyl)-2, 5-dihphenyltetrazolium bromide) assay is a widely used method to assess cell viability and proliferation. MTT is readily taken up by cells and enzymatically reduced to formazan, a dark compound which accumulates in cytoplasmic granules. Formazan is later eliminated by the cell by a mechanisms often indicated as exocytosis, that produces characteristic needle-like aggregates on the cell surface. The shape of formazan aggregates and the rate of exocytosis change in the presence of bioactive amyloid beta peptides (Abeta) and cholesterol. Though the cellular mechanisms involved in MTT reduction have been extensively investigated, the exact nature of formazan granules and the process of exocytosis are still obscure. Using Nile Red, which stains differentially neutral and polar lipids, and a fluorescent analog of cholesterol (NBD-cholesterol), we found that formazan localized in lipid droplets, consistent with the lipophilic nature of formazan. However, formazan granules and aggregates were also found to form after killing cells with paraformaldehyde fixation. Moreover, formazan aggregates were also obtained in cell-free media, using ascorbic acid to reduce MTT. The density and shape of formazan aggregates obtained in cell-free media was sensitive to cholesterol and Abeta. In cells, electron microscopy failed to detect the presence of secretory vesicles, but revealed unusual fibers of 50 nm of diameter extending throughout the cytoplasm. Taken together, these findings suggest that formazan efflux is driven by physico-chemical interactions at molecular level without involving higher cytological mechanisms.     

Theoretical mechanism of the formation of cholesteryl chloride from cholesterol and thionyl chloride

This work describes a theoretical approach to the substitution reaction mechanism involving the conversion of cholesterol to cholesteryl chloride. Two chlorosulfite ester molecules were formed as intermediates. An iso-steroid was found as the transition state. The final product was cholesteryl chloride and the side products were HCl and SO₂. Calculations were carried out at high level Hartree–Fock theory, using the 6–31G* basis set. From the electronic structure of the reactants, the most important physicochemical properties involved in the reaction pathway were used. Thus, to determine the participation of each molecule and to explain the mechanism of reaction; the total energy, HOMO and LUMO, atomic orbital contribution to frontier orbitals formation, electrostatic potentials, atomic charges, hardness and dipole moment were used. Characterization of intermediates and transition state was supported by their respective energy minima, fundamental frequencies and equilibrium geometry.Figure Synopsis of the reaction pathway. The reaction starts when the lone pair of the Ch oxygen interacts with the sulfur atom, releasing a chloride ion. As a result, the first intermediate is formed. Next, in the first intermediate the nucleophilic chloride ion bonds the electrophilic hydrogen atom, releasing HCl and yielding the second intermediate. In the second intermediate, the electrophilic H-atom from HCl bonds with the lone pair of the Cl atom adjacent to the sulfur atom, restoring HCl. Concurrently, SO₂ is liberated and causes the formation of the C3-C5 partial bond and breaking of the C5-C6 -bond leading to the transition state. In the transition state, the electrophilic H from HCl bonds with the Cl lone pair at C6-Cl, forming HCl again and leaving the C6 atom electron-deficient, which restores the C5-C6 -bond and breaks the C3-C5 partial bond. Finally, the electrophilic C3 atom and the nucleophilic Cl atom form a bond, yielding cholesteryl chloride. HCl and SO₂ are also formed as side products. The arrows show the rearrangement of electrons.     

Oxygen cost of chloride transport in perfused rectal gland ofSqualus acanthias

Summary In the stimulated state, with glucose as substrate, oxygen uptake by the isolated perfused rectal gland is directly related to the rate of chloride secretion. Lactate production is negligible under aerobic conditions in the stimulated gland. A stoichiometric relationship exists between chloride transport and oxygen consumption, with a Cl/O₂ ratio of about 301, resembling that reported for sodium in mammalian kidneys. This ratio remains constant under varying degrees and modes of stimulation. The ratio does not change when the gland is induced to secrete chloride against varying electrochemical gradients by altering the concentration of urea in the perfusate.Established Investigator of the American Heart Association.     

Colorimetric microtiter plate assay of glucose and fructose by enzyme-linked formazan production: applicability to the measurement of fructosyl transferase activity in higher plants

A rapid, enzyme-linked colorimetric assay, for the sequential determination of nanomole quantities of glucose and fructose in the same sample, has been developed for the measurement of fructosyl transferase activity in plant extracts. The assay extends the conventional dehydrogenase-linked assay for these sugars by utilizing the intermediary electron carrier, phenazine methosulfate, to couple NADP reduction to the production of a formazan dye from the tetrazolium salt, thiazolyl blue, in a form suitable for measurement using a microtiter plate reader. When the microtiter plate assay was used to measure the activities of yeast invertase and sucrose:sucrose fructosyl transferase from Lolium temulentum, results obtained were very similar to results obtained using the conventional procedure. The rapidity, small scale, and ease of execution of the method offers considerable advantages over the conventional hexose assay and is particularly suitable for screening of large numbers of small samples, exploiting both the speed of the microtiter plate reader and the facility of for microcomputer processing of data. The potential of this method for use with other enzyme systems and other metabolites is discussed.     

Lithium chloride increases the production of amyloid-beta peptide independently from its inhibition of glycogen synthase kinase 3

Glycogen synthase kinase 3 (GSK3) is able to phosphorylate tau at many sites that are found to be phosphorylated in paired helical filaments in Alzheimer disease. Lithium chloride (LiCl) efficiently inhibits GSK3 and was recently reported to also decrease the production of amyloid-beta peptide (Abeta) from its precursor, the amyloid precursor protein. Therefore, lithium has been proposed as a combined therapeutic agent, inhibiting both the hyperphosphorylation of tau and the production of Abeta. Here, we demonstrate that the inhibition of GSK3 by LiCl induced the nuclear translocation of beta-catenin in Chinese hamster ovary cells and rat cultured neurons, in which a decrease in tau phosphorylation was observed. In both cellular models, a nontoxic concentration of LiCl increased the production of Abeta by increasing the beta-cleavage of amyloid precursor protein, generating more substrate for an unmodified gamma-secretase activity. SB415286, another GSK3 inhibitor, induced the nuclear translocation of beta-catenin and slightly decreased Abeta production. It is concluded that the LiCl-mediated increase in Abeta production is not related to GSK3 inhibition.