A continuous fluorescent assay for the determination of plasma and tissue angiotensin I-converting enzyme activity

A continuous assay using internally quenched fluorescent peptides with the general sequence Abz-peptidyl-(Dnp)P-OH (Abz = ortho-aminobenzoic acid; Dnp = 2,4-dinitrophenyl) was optimized for the measurement of angiotensin I-converting enzyme (ACE) in human plasma and rat tissues. Abz-FRK(Dnp)P-OH, which was cleaved at the Arg-Lys bond by ACE, was used for the enzyme evaluation in human plasma. Enzymatic activity was monitored by continuous recording of the fluorescence (lambdaex = 320 nm and lambdaem = 420 nm) at 37ºC, in 0.1 M Tris-HCl buffer, pH 7.0, with 50 mM NaCl and 10 µM ZnCl2. The assays can be performed directly in the cuvette of the fluorimeter and the hydrolysis followed for 5 to 10 min. ACE measurements in the plasma of 80 healthy patients with Hip-His-Leu and with Abz-FRK(Dnp)P-OH correlated closely (r = 0.90, P < 0.001). The specificity of the assay was demonstrated by the complete inhibition of hydrolysis by 0.5 µM lisinopril or captopril. Abz-FRK(Dnp)P-OH cleavage by ACE was monitored in rat lung, kidney, heart, and liver homogenates in the presence of a cocktail of inhibitors containing trans-epoxy-succinyl-L-leucylamido-(4-guanido)-butene, pepstatin, phenyl-methylsulfonyl fluoride, N-tosyl-L-phenylalanyl-chloromethyl ketone, and N-tosyl-lysyl-chloromethyl ketone to prevent undesirable hydrolysis. ACE activity in lung, heart and kidney homogenates, but not in liver homogenates, was completely abolished by 0.5 µM lisinopril or captopril. The advantages of the method are the procedural simplicity and the high sensitivity providing a rapid assay for ACE determinations.

Determination of renal function in long-term heart transplant patients by measurement of urinary retinol-binding protein levels

Significant improvements have been noted in heart transplantation with the advent of cyclosporine. However, cyclosporine use is associated with significant side effects, such as chronic renal failure. We were interested in evaluating the incidence of long-term renal dysfunction in heart transplant recipients. Fifty-three heart transplant recipients were enrolled in the study. Forty-three patients completed the entire evaluation and follow-up. Glomerular (serum creatinine, creatinine clearance measured, and creatinine clearance calculated) and tubular functions (urinary retinol-binding protein, uRBP) were re-analyzed after 18 months. At the enrollment time, the prevalence of renal failure ranged from 37.7 to 54% according to criteria used to define it (serum creatinine > or = 1.5 mg/dL and creatinine clearance <60 mL/min). Mean serum creatinine was 1.61 ± 1.31 mg/dL (range 0.7 to 9.8 mg/dL) and calculated and measured creatinine clearances were 67.7 ± 25.9 and 61.18 ± 25.04 mL min-1 (1.73 m²)-1, respectively. Sixteen of the 43 patients who completed the follow-up (37.2%) had tubular dysfunction detected by increased levels of uRBP (median 1.06, 0.412-6.396 mg/dL). Eleven of the 16 patients (68.7%) with elevated uRBP had poorer renal function after 18 months of follow-up, compared with only eight of the 27 patients (29.6%) with normal uRBP (RR = 3.47, P = 0.0095). Interestingly, cyclosporine trough levels were not different between patients with or without tubular and glomerular dysfunction. Renal function impairment is common after heart transplantation. Tubular dysfunction, assessed by uRBP, correlates with a worsening of glomerular filtration and can be a useful tool for early detection of renal dysfunction.

Determination of low tetanus or diphtheria antitoxin titers in sera by a toxin neutralization assay and a modified toxin-binding inhibition test

A method for the screening of tetanus and diphtheria antibodies in serum using anatoxin (inactivated toxin) instead of toxin was developed as an alternative to the in vivo toxin neutralization assay based on the toxin-binding inhibition test (TOBI test). In this study, the serum titers (values between 1.0 and 19.5 IU) measured by a modified TOBI test (Modi-TOBI test) and toxin neutralization assays were correlated (P < 0.0001). Titers of tetanus or diphtheria antibodies were evaluated in serum samples from guinea pigs immunized with tetanus toxoid, diphtheria-tetanus or triple vaccine. For the Modi-TOBI test, after blocking the microtiter plates, standard tetanus or diphtheria antitoxin and different concentrations of guinea pig sera were incubated with the respective anatoxin. Twelve hours later, these samples were transferred to a plate previously coated with tetanus or diphtheria antitoxin to bind the remaining anatoxin. The anatoxin was then detected using a peroxidase-labeled tetanus or diphtheria antitoxin. Serum titers were calculated using a linear regression plot of the results for the corresponding standard antitoxin. For the toxin neutralization assay, L+/10/50 doses of either toxin combined with different concentrations of serum samples were inoculated into mice for anti-tetanus detection, or in guinea pigs for anti-diphtheria detection. Both assays were suitable for determining wide ranges of antitoxin levels. The linear regression plots showed high correlation coefficients for tetanus (r² = 0.95, P < 0.0001) and for diphtheria (r² = 0.93, P < 0.0001) between the in vitro and the in vivo assays. The standardized method is appropriate for evaluating titers of neutralizing antibodies, thus permitting the in vitro control of serum antitoxin levels.

Determination of myocardial infarction size in rats by echocardiography and tetrazolium staining: correlation, agreements, and simplifications

Triphenyltetrazolium chloride (TTC) staining and echocardiography (ECHO) are methods used to determine experimental myocardial infarction (MI) size, whose practical applicability should be expanded. Our objectives were to analyze the accuracy of ECHO in determining infarction size in rats during the first days following coronary occlusion and to test whether a simplified single measurement by TTC correctly indicates MI size, as determined by the average value for multiple slices. Infarction was induced in female Wistar rats by coronary artery occlusion and MI size analysis was performed after the acute (7th day) and chronic periods (after 4 weeks) by ECHO matched with TTC. ECHO and TTC showed similar values of MI size (% of left ventricle perimeter) in acute (ECHO: 33 ± 11, TTC: 35 ± 14) and chronic (ECHO: 38 ± 14, TTC: 39 ± 13 periods), and also presented an excellent correlation (r = 0.92, P < 0.001). Although measurements from different heart planes showed discrepancies, a single measurement acquired from the mid-ventricular level by TTC was a good estimate of MI size calculated by the average of multiple planes, with minimal disagreement (Bland-Altman test with mean ratio bias of 0.99 ± 0.07) and close to an ideal correlation (r = 0.99, P < 0.001). In the present study, ECHO was confirmed as a useful method for the determination of MI size even in the acute phase. Also, the single measure of a mid-ventricular section proposed as a simplification of the TTC method is a satisfactory prediction of average MI extension.

Determination of CYP2D6 *3, *4, and *10 frequency in women with breast cancer in São Luís, Brazil, and its association with prognostic factors and disease-free survival

The CYP2D6 enzyme is crucial for the metabolism of tamoxifen. The CYP2D6 gene is highly polymorphic, and individuals can be extensive, intermediate, or poor tamoxifen metabolizers. The aim of this study was to determine the frequencies of the CYP2D6 *3, *4, and *10 alleles in women with breast cancer who were treated with tamoxifen and analyze the association of enzyme activity with prognostic factors and disease-free survival. We observed a high frequency of CYP2D6 *10, with an allelic frequency of 0.14 (14.4%). The *3 allele was not present in the studied population, and *4 had an allelic frequency of 0.13 (13.8%). We conclude that patients with reduced CYP2D6 activity did not present worse tumor characteristics or decreased disease-free survival than women with normal enzyme activity, as the difference was not statistically significant. We also observed a high frequency of CYP2D6 *10, which had not been previously described in this specific population. This study is the first in north-northeastern Brazil that aimed to contribute to the knowledge of the Brazilian regional profile for CYP2D6 polymorphisms and their phenotypes. These findings add to the knowledge of the distribution of different polymorphic CYP2D6 alleles and the potential role of CYP2D6 genotyping in clinical practice prior to choosing therapeutic protocols.

ENZYMATIC DETERMINATION OF STARCH IN DOCE DE LEITE USING DIALYSIS

The importance of starch for the food industry makes it necessary to develop new, fast, economic and accurate methodologies for its quantification. In the present paper starch hydrolysis using commercial enzymes of industrial grade are studied aiming to develop an easy and cheap analysis, available to a greater number of industries and technicians. The proposed method is simple, divided in a first step where soluble sugars are eliminated from the samples by using dialysis, followed by starch hydrolysis of the retained fraction with a thermoresistent bacterial alfa-amylase (Termamyl 120L®) and an amyloglucosidase (AMG 300L®). The hydrolysis conditions were those suggested by the enzyme producer. After the hydrolysis step the material was dialysed again for the extraction of glucose that was quantified by the glucose-oxidase colorimetric reactant. The results allowed the construction of calibration equations for starch determination on the analyzed samples. These samples were produced on a laboratory scale and native and acid-modified corn starches were added in known concentrations. By considering the final dilutions employed for glucose determination on the samples, it was possible to confirm that they were identical to that of the glucose-oxidase reactant calibration.

Comparison of methods by TLC and HPTLC for determination of aflatoxin M1 in milk and B1 in eggs

Milk and egg matrixes were assayed for aflatoxin M1 (AFM1) and B1 (AFB1) respectively, by AOAC official and modified methods with detection and quantification by thin layer chromatography (TLC) and high performance thin layer chromatography (HPTLC). The modified methods: Blanc followed by Romer, showed to be most appropriate for AFM1 analysis in milk. Both methods reduced emulsion formation, produced cleaner extracts, no streaking spots, precision and accuracy improved, especially when quantification was performed by HPTLC. The use of ternary mixture in the Blanc Method was advantageous as the solvent could extract AFM1 directly from the first stage (extraction), leaving other compounds in the binary mixture layer, avoiding emulsion formation, thus reducing toxin loss. The relative standard deviation (RSD%) values were low, 16 and 7% when TLC and HPTLC were used, with a mean recovery of 94 and 97%, respectively. As far as egg matrix and final extract are concerned, both methods evaluated for AFB1 need further studies. Although that matrix leads to emulsion with consequent loss of toxin, the Romer modified presented a reasonable clean extract (mean recovery of 92 and 96% for TLC and HPTLC, respectively). Most of the methods studied did not performed as expected mainly due to the matrixes high content of triglicerides (rich on saturated fatty acids), cholesterol, carotene and proteins. Although nowadays most methodology for AFM1 is based on HPLC, TLC determination (Blanc and Romer modified) for AFM1 and AFB1 is particularly recommended to those, inexperienced in food and feed mycotoxins analysis and especially who cannot afford to purchase sophisticated (HPLC,HPTLC) instrumentation.

Ochratoxin A determination in beer by immunoaffinity column clean-up and high-performance liquid chromatography

Analyses of ochratoxin A (OTA) in domestic and imported beers were perfomed by immunoaffinity column and high - perfomance liquid chromatography (HPLC) using a fluorescence detector. Recoveries of OTA from beer samples spiked at levels from 8.0 to 800pg/mL ranged from 81.2% to 95.0%, with coefficient of variation between 0% e 11.0%. Detection limit and quantification limit were 2.0pg/mL and 8.0pg/mL, respectively. Of the total of 26 samples produced in Brazil only 6 (23%), contained trace amounts of OTA. Of the 4 imported beers, in 2, Ireland and Germany, were detected OTA at levels of 25pg/mL and 82pg/mL, respectively.

Determination of trans-10-hydroxy-2-decenoic acid (10-HDA) in royal jelly from São Paulo State, Brazil

The 10-HDA content in Brazilian samples (São Paulo State) of royal jelly (RJ) was analyzed using an HPLC method based on the work by BLOODWORTH et al. [2]. The chromatographic conditions were: isocratic system, reversed phase C18-H column, auto sampler, diode array UV-VIS detector adjusted to 225nm, mobile phase composed by methanol/water (45:55) at pH= 2.5 adjusted with phosphoric acid; a-naphtol was used as internal standard, and the running time was 30min. By statistical analysis of the results, the 10-HDA contents of the samples analyzed seem to have two ranges: 1.8% and 3% (w/w), that would be useful to qualify the RJ. This is the first data regarding 10-HDA content of Brazilian RJ.

A method for fast determination of epigallocatechin gallate (EGCG), epicatechin (EC), catechin (C) and caffeine (CAF) in green tea using HPLC

Tea has been considered a medicine and a healthy beverage since ancient times, but recently it has received a great deal of attention because of its antioxidant properties. Green tea polyphenols have demonstrated to be an effective chemopreventive agent. Recently, investigators have found that EGCG, one of the green tea catechins, could have anti-HIV effects when bound to CD4 receptor. Many factors can constitute important influences on the composition of tea, such as species, season, age of the leaf, climate, and horticultural practices (soil, water, minerals, fertilizers). This paper presents an HPLC analytical methodology development, using column RP-18 and mobile phase composed by water, acetonitrile, methanol, ethyl acetate, glacial acetic acid (89:6:1:3:1 v/v/v/v/v) for simultaneous determination and quantification of caffeine (CAF), catechin (C), epicatechin (EC) and epigallocatechin gallate (EGCG) in samples of Camellia sinensis (green tea) grown in Brazil and harvested in spring, in summer and in autumn, in comparison to Brazilian black tea, to samples of Japanese and Chinese green tea and to two standardized dry extracts of green tea. The method has been statistically evaluated and has proved to be adequate to qualitative and quantitative determination of the samples.

Determination of thermobacteriological parameters and size of Bacillus stearothermophilus ATCC 7953 spores

In order to determine thermobacteriological parameters for B. stearothermophilus spores, they were diluted in a saline solution medium and in ground corn-soybean mix, distributed in TDT tube, and submitted to heat for a specific period of time. The D value (time to reduce 1 log cycle of microbial count under a certain temperature) and z value (variation of temperature to cause 10-fold change in D value) were estimated. To estimate their dimensions, the spores were visualized by using a scanning electron microscope. D121.1 ºC and z values for these spores, as determined in the saline solution, were 8.8 minutes and 12.8 ºC, respectively. D121,1 ºC and z values determined in the corn-soy mix were 14.2 minutes and 23.7 ºC, respectively. The micrographs indicated that the spores have homogeneous shape and size, with length and diameter of 2 and 1 µm, respectively. These results confirm that the spore is highly thermal-resistant, and it is a good biological indicator to evaluate the extrusion process as a feed sterilizer.

(E)-2-Nonenal determination in brazilian beers using headspace solid-phase microextraction and gas chromatographic coupled mass spectrometry (HS-SPME-GC-MS)

(E)-2-nonenal is considered an important off-flavor of beer, related to the flavor of beer staling. In this study, a new method for determination of (E)-2-nonenal in beer using headspace solid-phase microextraction and gas chromatographic coupled mass spectrometry (HS-SPME-GC-MS) was developed and applied in Brazilian beer samples. The extractions were carried out in CAR-PDMS (carboxen-polydimethylsiloxane) fiber and the best results were found with 15 minutes of equilibrium and 90 minutes of extraction at 50 °C. The method was linear in the range from 0.02 to 4.0 μg.L-1 with correlation coefficient of 0.9994. The limits of detection and quantification were 0.01 and 0.02 μg.L-1, respectively. 96.5% of recovery and 4% precision (RSD) were obtained in the fortification of beer samples with 2.0 μg.L-1 of (E)-2-nonenal. The developed method proved to be simple, efficient and highly sensitive to the determination of this analyte being easily applied in the quality control of the brewery. (E)-2-nonenal was found in all beer samples analyzed with levels between 0.17 and 0.42 μg.L-1.

Psychophysical estimation of acid intensity and determination of sweet-acid interaction in a fish paste containing glycerol

In a fish paste made with cooked Brazilian flathead (Percophis brasiliensis), glycerol (17%), sodium chloride (1.5%) and potassium sorbate (0.1%) the following acid percentages: 0.2; 0.4; 0.6; 0.8, 1 and 1.5% w/w were incorporated to determine the relationship between added acetic acid and the sensorially perceived intensity, and the effects of the combination of sweet-acid tastes. Tests for paired comparison, ranking and structured verbal scales for sweet and acid attributes and psychophysical test were carried out. There was a perceptible difference among samples for differences of 0.4 units of acid concentration. Samples indicated as sweeter by 89.47% of the judges were those containing a lesser acid concentration. A reduction in glycerol sweetness when increasing acid levels was observed. Acetic acid reduced the sweetness of glycerol and inversely glycerol reduced the acidity of acetic acid. The data obtained with the magnitude estimation test agree with Steven's law.

Comparison of methodologies for moisture determination on dried bee pollen samples

Bee pollen moisture value is one of the quality parameters for this product. Some countries such as Argentina, Brazil, Bulgaria, Poland and Switzerland have bee pollen regulations on quality parameters, but these are not clear regarding which method should be used for moisture determination. The aim of this paper was to compare six methods of moisture determination in dried bee pollen samples. The methods were: conventional oven at 100 °C, vacuum oven at 70 °C, desiccator with sulfuric acid, drying out process with infrared light at 85 °C, lyophilization and Karl Fisher's method. Based on the results, the best methods for moisture determination of bee pollen were the drying process with infrared and the lyophilization, since these have shown lower moisture values.