A Rapid Quantitative Analysis of Native Antioxidants in Natural Rubber (Hevea brasiliensis) During Maturation

Natural rubber (NR) obtained from H. brasiliensis is known to be susceptible to oxidative degradation according to the amount of double bonds in the structure of the polymer, i.e. poly (cis-1,4-isoprene) [1]. However NR has been reported to contain native antioxidants such as phytosterols, phospholipids and tocotrienols [2]. Among those, γ-tocotrienol, present in hevea latex was reported to exhibit “in-vitro” antioxidant activity [3]. However the direct involvement of γ-tocotrienol or other antioxidants naturally present in NR in the protection of NR against oxidation is difficult to assess as the works were carried out with different of rubber types and in different conditions [4,5]. In the framework of a work on the dynamics of γ-tocotrienols in NR samples obtained from coagula maturated during several durations (0 to 15 days) in parallel with measurement of standard properties such as P0 and PRI, methodological development of the analysis of tocotrienols and derivatives is presented in this study.HPLC-MS has been shown to be an accurate technique for lipid analysis in NR [6]. However, this is a time-consuming technique especially with large number of samples due to a necessary step of saponification. Moreover, the structures of γ-tocotrienol and its dimers were found to be affected by the strong alkali condition of saponification (data not shown). Therefore a rapid quantitative method for γ-tocotrienol from NR using high performance thin layer chromatography (HP-TLC) has been developed. Lipid extracts from NR samples could be simply analyzed by HP-TLC without any derivatization and the detectable quantity could be in nanogram range. Statistical analysis of the data showed that the method is precise, accurate, reproducible and sensitive. Thus the proposed HP-TLC method can be successfully used for the quantification of γ-tocotrienol from NR samples. This technique will be useful to conduct further experiments on antioxidant activity of NR lipids and to relate the results with physical properties of NR, especially those linked to resistance to oxidation.

Role of myoglobin as a scavenger of cellular NO in myocardium

Recent studies have detected a 1H nuclear magnetic resonance (NMR) reporter signal of metmyoglobin (metMb) during bradykinin stimulation of an isolated mouse heart. The observation has led to the hypothesis that Mb reacts with cellular nitric oxide (NO). However, the hypothesis depends on an unequivocal detection of metMb signals in vivo. In solution, nitrite oxidization of Mb produces a characteristic set of paramagnetically shifted 1H NMR signals. In the upfield spectral region, MbO2 and MbCO exhibit the γCH3 Val E11 signals at –2.8 and –2.4 ppm, respectively. In the same spectral region, nitrite oxidation of Mb produces a set of signals at –3.7 and –4.7 ppm at 35°C. Previous studies have confirmed the visibility of metMb signals in perfused rat myocardium. With bradykinin infusion, perfusion pressure and rate-pressure product decrease, consistent with endogenous NO formation. However, neither myocardial O2 consumption nor high-energy phosphate levels, as reflected in the 31P NMR signals, show any significant change. Bradykinin still triggers a similar physiological response even in the presence of CO that is sufficient to inhibit 86% Mb. In all cases, the 1H NMR spectra from perfused rat myocardium reveal no metMb signals. The results suggest that bradykinin-induced NO does not interact significantly with cellular Mb to produce an NMR-detectable quantity of metMb in the perfused rat myocardium. As a consequence, the experiments cannot confirm the intriguing proposal that Mb acts as a cellular NO scavenger.

Staphylococcal Cell Wall: Morphogenesis and Fatal Variations in the Presence of Penicillin

SUMMARY The primary goal of this review is to provide a compilation of the complex architectural features of staphylococcal cell walls and of some of their unusual morphogenetic traits including the utilization of murosomes and two different mechanisms of cell separation. Knowledge of these electron microscopic findings may serve as a prerequisite for a better understanding of the sophisticated events which lead to penicillin-induced death. For more than 50 years there have been controversial disputes about the mechanisms by which penicillin kills bacteria. Many hypotheses have tried to explain this fatal event biochemically and mainly via bacteriolysis. However, indications that penicillin-induced death of staphylococci results from overall biochemical defects or from a fatal attack of bacterial cell walls by bacteriolytic murein hydrolases were not been found. Rather, penicillin, claimed to trigger the activity of murein hydrolases, impaired autolytic wall enzymes of staphylococci. Electron microscopic investigations have meanwhile shown that penicillin-mediated induction of seemingly minute cross wall mistakes is the very reason for this killing. Such “morphogenetic death” taking place at predictable cross wall sites and at a predictable time is based on the initiation of normal cell separations in those staphylococci in which the completion of cross walls had been prevented by local penicillin-mediated impairment of the distribution of newly synthesized peptidoglycan; this death occurs because the high internal pressure of the protoplast abruptly kills such cells via ejection of some cytoplasm during attempted cell separation. An analogous fatal onset of cell partition is considered to take place without involvement of a detectable quantity of autolytic wall enzymes (“mechanical cell separation”). The most prominent feature of penicillin, the disintegration of bacterial cells via bacteriolysis, is shown to represent only a postmortem process resulting from shrinkage of dead cells and perturbation of the cytoplasmic membrane. Several schematic drawings have been included in this review to facilitate an understanding of the complex morphogenetic events.

Determination of Vitamin K1 in Powdered Infant Formulas, Using Supercritical Fluid Extraction and Liquid Chromatography with Electrochemical Detection

Vitamin K, (phylloquinone) is extracted from commercial soy proteinbased and milk-based powdered infant formulas by using supercritical fluid extraction with C02 at 8000 psi and 60°C. Quantitative extraction requires only 15 min, and does not suffer from the problems associated with conventional solvent extraction of lipophilic materials from media such as formulas. Vitamin K, is determined in the extracts by using reverse-phase liquid chromatography (LC) with reductive mode electrochemical detection at a silver electrode polarized at —1.1 V vs SCE. LC run time is 9 min. The minimum detectable quantity is 80 pg, and response is linear over at least 5 orders of magnitude. Recovery of vitamin K, from a milk-based powdered formula was 95.6% with RSD of 7.4%, and from a soy protein-based product, 94.4% recovery with RSD of 6.5%.

Thin Layer Chromatographic Confirmation of Aflatoxin Mi Extracted from Milk

Aflatoxin M1 can be confirmed directly on a thin layer plate by reacting the toxin with a mixture of reagents containing p-anisaldehyde. This confirmatory procedure requires only 2 elutions in the same direction using 2 different solvents. The mixture containing p-anisaldehyde is overspotted on Mi after the plate has been developed in toluene-ethyl acetate-ethyl ether-formic acid (25 + 35 + 40 + 5). The plate is heated at 110°C for 10 min and then developed in hexanc-acetonechloroform (15 + 50 + 35). The Rf value of the green fluorescent derivative is less than that of the M1 standard. This confirmatory procedure requires only one-dimensional TLC, so several sample extracts and the standard can be run simultaneously. The minimum detectable quantity of aflatoxin M1 on the TLC plate with this test is 0.3 ng. p- Anisaldehyde reagent solution may also be used as a spray reagent for the confirmation of aflatoxin M1. The procedures described were satisfactory for confirming the mycotoxin in spiked samples of powdered and liquid milk.

Quantitation of hemoglobins within individual red cells: asynchronous biosynthesis of fetal and adult hemoglobin during erythroid maturation in normal subjects

We outline a method for estimating either HbF or HbA content in single erythrocytes and their precursors. Our method depends on microphotometric assay of darkfield reflectance arising from individual pericellular immunoprecipitates developed with anti-HbF or anti-HbA. When uniform-diameter latex microspheres were used to normalize comparisons between preparations, mean coefficient of variation for HbF reflectance among separate preparations of the same sample was < 3%. Reflectance is a faithful (r = 0.99) linear function of the logarithm of picograms per cell in samples with known HbF or HbA content. The following features emerged from such analyses. First, despite the use of antigenically-specific antihemoglobins from different sources, the least detectable quantity of HbF (3.2 pg) and HbA (14.8 pg) remained invariant. Second, these detection thresholds depends on antihemoglobin affinity constants but are little influenced by antibody concentration. Third, our procedure is equally valid for persons with normal HbF constant (mean +/- SD = 4.4 +/- 0.3 pg per cell, 15 subjects) and for those with much higher levels. Fourth, like the percentage of HbF- bearing cells, HbF content is usually unchanging in serial samples. Fifth, the utility of the method is evidenced in bone marrow analyses of five hematologically normal persons in whom HbF content, unlike HbA content, remained constant throughout maturation from erythroblasts to erythrocytes. In vivo HbF biosynthesis is thus normally completed long before HbA production.

Quantitation of hemoglobins within individual red cells: asynchronous biosynthesis of fetal and adult hemoglobin during erythroid maturation in normal subjects

We outline a method for estimating either HbF or HbA content in single erythrocytes and their precursors. Our method depends on microphotometric assay of darkfield reflectance arising from individual pericellular immunoprecipitates developed with anti-HbF or anti-HbA. When uniform-diameter latex microspheres were used to normalize comparisons between preparations, mean coefficient of variation for HbF reflectance among separate preparations of the same sample was < 3%. Reflectance is a faithful (r = 0.99) linear function of the logarithm of picograms per cell in samples with known HbF or HbA content. The following features emerged from such analyses. First, despite the use of antigenically-specific antihemoglobins from different sources, the least detectable quantity of HbF (3.2 pg) and HbA (14.8 pg) remained invariant. Second, these detection thresholds depends on antihemoglobin affinity constants but are little influenced by antibody concentration. Third, our procedure is equally valid for persons with normal HbF constant (mean +/- SD = 4.4 +/- 0.3 pg per cell, 15 subjects) and for those with much higher levels. Fourth, like the percentage of HbF- bearing cells, HbF content is usually unchanging in serial samples. Fifth, the utility of the method is evidenced in bone marrow analyses of five hematologically normal persons in whom HbF content, unlike HbA content, remained constant throughout maturation from erythroblasts to erythrocytes. In vivo HbF biosynthesis is thus normally completed long before HbA production.