Determination of macromolecular exchange and PO2 in the microcirculation: a simple system for in vivo fluorescence and phosphorescence videomicroscopy

We have developed a system with two epi-illumination sources, a DC-regulated lamp for transillumination and mechanical switches for rapid shift of illumination and detection of defined areas (250-750 µm²) by fluorescence and phosphorescence videomicroscopy. The system permits investigation of standard microvascular parameters, vascular permeability as well as intra- and extravascular PO2 by phosphorescence quenching of Pd-meso-tetra (4-carboxyphenyl) porphine (PORPH). A Pechan prism was used to position a defined region over the photomultiplier and TV camera. In order to validate the system for in vivo use, in vitro tests were performed with probes at concentrations that can be found in microvascular studies. Extensive in vitro evaluations were performed by filling glass capillaries with solutions of various concentrations of FITC-dextran (diluted in blood and in saline) mixed with different amounts of PORPH. Fluorescence intensity and phosphorescence decay were determined for each mixture. FITC-dextran solutions without PORPH and PORPH solutions without FITC-dextran were used as references. Phosphorescence decay curves were relatively unaffected by the presence of FITC-dextran at all concentrations tested (0.1 µg/ml to 5 mg/ml). Likewise, fluorescence determinations were performed in the presence of PORPH (0.05 to 0.5 mg/ml). The system was successfully used to study macromolecular extravasation and PO2 in the rat mesentery circulation under controlled conditions and during ischemia-reperfusion.

Immunophenotype of normal and leukemic bone marrow B-precursors in a Brazilian population. A comparative analysis by quantitative fluorescence cytometry

The distinction between normal and leukemic bone marrow (BM) B-precursors is essential for the diagnosis and treatment monitoring of acute lymphoblastic leukemia (ALL). In order to evaluate the potential use of quantitative fluorescence cytometry (QFC) for this distinction, we studied 21 normal individuals and 40 patients with CD10+ ALL. We characterized the age-related changes of the CD10, CD19, TdT, CD34 and CD79a densities in normal and leukemic BM. Compared to normal adults, the B-precursors from normal children expressed significantly lower values of CD34-specific antibody binding capacity (SABC) (median value of 86.6 vs 160.2 arbitrary units (a.u.) in children and adults, respectively). No significant age-related difference was observed in the expression of the other markers in the normal BM, or in any of the markers in the leukemic BM. Based on the literature, we set the cut-off value for the normal CD10 expression at 45 x 10³ a.u. for both age groups. For the remaining markers we established the cut-off values based on the minimum-maximum values in the normal BM in each age group. The expression of CD10 was higher than the cut-off in 30 ALL cases and in 18 of them there was a concomitant aberrant expression of other markers. In 9 of the 10 CD10+ ALL with normal CD10 SABC values, the expression of at least one other marker was aberrant. In conclusion, the distinction between normal and leukemic cells by QFC was possible in 38/40 CD10+ ALL cases.

Quenching of the intrinsic fluorescence of bovine serum albumin by chlorpromazine and hemin

The binding of chlorpromazine (CPZ) and hemin to bovine serum albumin was studied by the fluorescence quenching technique. CPZ is a widely used anti-psychotic drug that interacts with blood components, influences bioavailability, and affects function of several biomolecules. Hemin is an important ferric residue of hemoglobin that binds within the hydrophobic region of albumin with high specificity. Quenching of the intrinsic fluorescence of bovine serum albumin (BSA) was observed by selectively exciting tryptophan residues at 290 nm. Emission spectra were recorded in the range from 300 to 450 nm for each quencher addition. Stern-Volmer graphs were plotted, and the quenching constant estimated for BSA solution titrated with hemin at 25ºC was 1.44 (± 0.05) x 10(5) M-1. Results showed that bovine albumin tryptophans are not equally accessible to CPZ, in agreement with the idea that polar or charged quenchers have more affinity for amino acid residues on the outer wall of the protein. Hemin added to albumin solution at a molar ratio of 1:1 quenched about 25% of their fluorescence. The quenching effect of CPZ on albumin-hemin solution was stronger than on pure BSA. This increase can be the result of combined conformational changes in the structure of albumin caused firstly by hemin and then by CPZ. Our results suggest that the primary binding site for hemin on bovine albumin may be located asymmetrically between the two tryptophans along the sequence formed by subdomains IB and IIA, closer to tryptophan residue 212.

An HPLC-UV method for the measurement of permeability of marker drugs in the Caco-2 cell assay

The Caco-2 cell line has been used as a model to predict the in vitro permeability of the human intestinal barrier. The predictive potential of the assay relies on an appropriate in-house validation of the method. The objective of the present study was to develop a single HPLC-UV method for the identification and quantitation of marker drugs and to determine the suitability of the Caco-2 cell permeability assay. A simple chromatographic method was developed for the simultaneous determination of both passively (propranolol, carbamazepine, acyclovir, and hydrochlorothiazide) and actively transported drugs (vinblastine and verapamil). Separation was achieved on a C18 column with step-gradient elution (acetonitrile and aqueous solution of ammonium acetate, pH 3.0) at a flow rate of 1.0 mL/min and UV detection at 275 nm during the total run time of 35 min. The method was validated and found to be specific, linear, precise, and accurate. This chromatographic system can be readily used on a routine basis and its utilization can be extended to other permeability models. The results obtained in the Caco-2 bi-directional transport experiments confirmed the validity of the assay, given that high and low permeability profiles were identified, and P-glycoprotein functionality was established.

STABILITY OF OILS HEATED BY MICROWAVE: UV - SPECTROPHOTOMETRIC EVALUATION

The effects of microwave heating on the oxidative stability of refined canola, corn and soybean oils were determined by absorptivity in the UV spectrum and by chemical analysis (peroxide and acid values). Samples were heated in a microwave oven (800 W, 2,450 MHz) for 0 to 36 min. Microwave heating produced oxidative degradation in the three oils. Absorptivity at 232 and 270 nm increased gradually with an increase in microwave exposure time (0-36 min) for canola, corn and soybean oils. Values of absorptivity at 232 nm increased from 4.812, 3.568 and 4.183 to 10.579, 12.874 and 15.950 after 36 min of heating canola, corn and soybean oil, respectively. The absorptivity at 232nm, due to the formation of conjugated dienes, was a good index for measuring the degradation of microwaved samples. UV scanning (220 - 320 nm) detected alterations in the spectrum of microwaved samples. Acid value also increased within 36 min of heating for all oils. Peroxide value showed a significant difference (P<0.05) in the initial stage of heating (0-6 min) for all oils. After this period it could not be correlated with absorptivity at 232 nm, due to the instability of hydroperoxides at high temperatures.

Determinação simultânea de amarelo tartrazina e amarelo crepúsculo em alimentos via espectrofotometria UV-VIS e métodos de calibração multivariada

Os corantes sintéticos são usualmente adicionados a alimentos industrializados para conferir e restaurar a cor obtendo-se a qualidade estética desejada. Em função destes aspectos e do potencial toxicológico que alguns corantes podem apresentar, o controle de qualidade destes compostos é de fundamental importância. Neste trabalho, foi estudado o potencial das metodologias matemáticas como o princípio da aditividade, espectrofotometria derivativa e técnica multivariada (Regressão por Mínimos Quadrados Parciais - PLSR) na determinação simultânea de dois corantes alimentícios: Amarelo Crepúsculo (AC) e Amarelo Tartrazina (AT), extraídos com lã natural. Estas metodologias foram avaliadas e comparadas em função das suas capacidades de previsão, sendo que o modelo PLSR otimizado (faixa espectral de 305 a 645 nm, empregando-se 1ª derivada como transformação dos dados e 2 componentes principais) apresentou o menor valor de Raiz Quadrada da Soma dos Erros de Previsão (RMSEP) (AT = 0,191 e AC = 0,102). A faixa de concentração estudada foi de 1,0 a 16,0 mg.L-1 para AC e de 2,0 a 22,0 mg.L-1 para AT. O conjunto de validação externa apresentou erros relativos médios de 1,97% para AC e 1,39% para AT. A aplicação desta metodologia em amostras reais mostrou que em todas as amostras analisadas as concentrações destes corantes estavam de acordo com os limites estabelecidos pela legislação brasileira

Biochemical and color changes of fresh-cut melon (Cucumis melo L. cv. Galia) treated with UV-C

The importance of minimally processed commodities in the retail groceries of most developed countries has been rising continuously during the last decades. Cantaloupe melon is used more than any other fruit in fresh-cut processing. Ultraviolet (UV) light has been extensively used to simulate biological stres in plants and for determining resistance mechanisms of plant tissues. In this study the effect of ultraviolet irradiation on some properties of fresh-cut cantalope melon was determined during storage. Freshly cut cantalope melons cubes treated with ultraviolet irradiation at the doses of 1, 2 or 3 min before storage, and then placed in a cold room at 5±1°C temperature and 85-90% RH. Hue angle values of control group is low compared to UV-C treated samples, whereas L values of is high. EL of UV treated samples higher than those of control group. Total soluble solids of fresh-cut melon samples in UC3 treatment increased during storage. The results indicate that UV-C treatments on fresh-cut cantaloupe melon cubes increased total soluble solids independently from water loss.

Postharvest UV-B treatments increased fructose content of tomato (Solanum lycopersicon L. cv. Tayfun F1) harvested at different ripening stages

Abstract In the postharvest stage, taste and flavor are the key components of the marketability of tomato. Therefore, greater emphasis is now being placed on improving traits such as sugar content. In this study postharvest ultraviolet-B (UV-B) treatments on sugar, total soluble solids, and color of tomatoes harvested at different stages were investigated. Tomatoes harvested at turning, pink, and red stages were treated with two different doses of UV-B irradiation: UVB4 and UVB8. Color L* and hue angle values of tomatoes treated with UV-B were found to be high, which means the red color of tomatoes was improved. UVB4 treatments increased the color a* and saturation index values of tomatoes at pink and red harvest stage, although it did not affect at the turning stage. Additionally, UV-B irradiation treatments had no effect on sucrose content of the tomatoes. Fructose, glucose, and TSS content of tomatoes treated with UVB8 at red harvest stage were found to be high. Hence, the results obtained from this study are of great importance in terms of providing an increase in the amount of sugar without the need for breeding, and also consumer satisfaction.

Use of chlrophyll fluorescence sorting to improve soybean seed quality

The occurrence of green seeded soybeans [Glycine max (L.) Merrill] is a problem closely related to unfavorable climatic conditions, mainly drought, that occurs during the final stages of seed maturation. This problem causes serious losses to soybean seed quality in Brazil. In these seeds, chlorophyll is not properly degraded during maturation, drastically reducing seed quality. Using the chlorophyll fluorescence technique, it is possible to remove green seeds from the seed lot, improving seed quality in several species in which the occurrence of green seeds is also a problem. The objective of this research was to study the use of the chlorophyll fluorescence technique in sorting green seeds from soybean seed samples and its effects on quality. Five seed samples of soybean, cultivar TMG 113 RR, with 0%, 5%, 10%, 15%, and 20% of green seeds were used in this study. Seeds from each sample were sorted into two fractions based on the chlorophyll fluorescence signals and then compared to the control (non-sorted seeds). The sorting process showed great differences between the low and high chlorophyll fluorescence fractions. It was concluded that: green seeds of soybeans present high chlorophyll fluorescence and that this characteristic affects the quality of the seeds; it is possible to improve the quality of soybean seed by removing green seeds using the chlorophyll fluorescence sorting technique.