Colorimetric Micromethod Research Articles

Surveillance and characterization of drug-resistant Mycobacterium tuberculosis isolated in a reference hospital from Argentina during 8 years' period.

Argentina is considered a country with a middle tuberculosis (TB) incidence. However, according to the last national epidemiological report released in 2018, since 2013, the trends are steadily increasing. The aims of this study were to determine the drug-resistance (DR), multi-DR and extensively DR (MDR/XDR-TB), and rifampicin resistance (RIF-R) burden as a part of the local TB diagnosis (June 2010-August 2018); to detect the mutations associated to isoniazid (INH) and RIF-R and their geographical distribution; and to analyze the lineage relationship among the genetic patterns of the isolates circulating in the community. Respiratory and extrapulmonary specimens were processed by Ziehl-Neelsen stain and cultured on specific media. Drug-susceptibility testing of isolates was performed by the MGIT 960 and a colorimetric micro-method. Mutations conferring DR were detected by Genotype and DNA sequencing. The study showed a DR-TB prevalence of approximately 20% of the isolated strains, while M/XDR-TB-and particularly RIF-R-affected more than 5.0% of the total amount of cases. DR geographical distribution revealed isolates carrying mutations in the inhA gene promoter region only constrained to three districts where it was also registered two same family relatives' cases with the infrequent rpoB S522 L/Q mutation. The fact that most DR/MDR-TB isolates were not grouped in genetic clusters suggested that these cases may mostly have occurred due to endogenous reactivation rather than recently transmission. According to the obtained results, it would be convenient, in highly MDR-TB suspected individuals, to confirm phenotypically, the INH and RIF susceptibility detected by molecular tests.

Micro-method for the determination of glutathione in human blood

A new procedure is described for the visible-range spectrophotometric analysis of glutathione (GSH) in microvolumes of blood (as low as 0.5 μL) collected by fingerstick. Samples are diluted 1 to 300 (v/v) in a stabilizing solution, followed by determination of haemoglobin concentration and by acid deproteination. GSH is then measured in the clear supernatant by colorimetry using DTNB, i.e., 5,5'-dithio-bis(2-nitrobenzoic acid), in aqueous solution at pH 7.8. The DTNB reagent is prepared and kept at pH 6.2 until just prior its addition, thus avoiding spontaneous decomposition of the reagent. The assay is rapid, easy to adapt to large-scale studies and it avoids artefactual oxidation of GSH, a common methodological shortcoming. The method is precise with 1.7 to 3.4% intra-day relative standard deviation (RSD) and 2.2 to 4.2% inter-day RSD, and accurate with -1.4% to 2.3% intra-day relative error (RE) and -2.8% to 1.6% inter-day RE. GSH is recovered by 97.5 to 100% at all tested concentrations. The new colorimetric micro-method was validated by a reliable previously reported HPLC method. The procedure is suitable for minimally invasive investigation of oxidative stress in peripheral blood.

A Colorimetric Micromethod for the Determination of Serum Cholinesterase Activity

A Colorimetric Micromethod for the Determination of Serum Cholinesterase Activity

Comparison of the Performances of Two In-House Rapid Methods for Antitubercular Drug Susceptibility Testing

Resistance to rifampin (rifampicin), isoniazid, and streptomycin of 69 Mycobacterium tuberculosis isolates was analyzed by an in-house method based on mycobacteriophage D29 and a colorimetric micromethod. Both methods showed sensitivity and specificity values ranging from 93% to 100%. These simple methods offer an option for drug resistance assessment of M. tuberculosis.

Evaluation of antiviral activity of fractionated extracts of sage Salvia officinalis L. (Lamiaceae)

In the present study, we examined cytotoxicity and extracellular and intracellular antiviral activity of frac?tionated extracts of wild and cultivated sage Salvia officinalis L. (Lamiaceae) in vitro using the WISH-VSV model system. Extracts were obtained by fractionating depigmented ethanol extracts of sage plants with supercritical CO2 at different pressures. Cytotoxicity was determined by examining cellular morphology in situ with the aid of a colorimetric micromethod and by cell staining with trypan blue. The fraction of distilled cultivated sage obtained at CO2 pressure of 300 bars and temperature of 60?C (149/3) was the most cytotoxic, with CTD10 44 ?g/ml. That of non-distilled cultivated sage obtained at CO2 pressure of 500 bars and temperature of 100?C (144/5) was the least toxic (CTD10 199 ?g/ml). Moreover, 144/5 had an antiviral effect at the intracellular level: when added 5 hours before VSV infection, it caused 100% reduction of CPE at concentrations of 99.5 and 199.0 ?g/ml; when added after virus penetration had occurred, the same concentrations caused 35 and 60% reduction, respectively. The obtained results indicate that antiviral activity of 144/5 involves inhibition of the early steps of the virus infective cycle without a direct virucidal effect. Abbreviations: WISH - human amnion epithelial cells, VSV - vesicular stomatitis virus, HSV - herpes simplex virus, CPE - cytopathic effect, IS - selectivity index, TCID50 - tissue culture infective dose, CTD10 - 10% cytotoxic concentrations.

Characterization of a sialate pyruvate-lyase in the cytosol of human erythrocytes

Sialate pyruvate-lyases, also known as sialate aldolases (EC 4.1.3.3), reversibly catalyse the cleavage of free N-acetylneuraminic acids to form pyruvate and N-acetylmannosamine. These enzymes are widely distributed and are present in numerous pro- and eukaryotic cells, in which they are localized only in the cytosol. They play an important role in the regulation of sialic acid metabolism by controlling the intracellular concentration of sialic acids of biosynthetic or exogenous origin, thus preventing the accumulation of toxic levels of this sugar. Application of an original colorimetric micromethod for N-acetylmannosamine determination, as well as the use of [4,5,6,7,8,9- 14C] N-acetylneuraminic acid, led us to evidence a cytosolic neuraminate aldolase activity in human red blood cells (RBCs) and then to define the main characteristics of this enzyme: Michaelis–Menten type, K m: 1.4 ± 0.05 mM, optimal pH: 7.6 ± 0.2, optimal temperature: 70 ± 2 °C, inhibition by heavy metals: Ag + and Hg ++. These enzyme parameters are close to those of the bacterial and mammalian aldolases described up to now. At the moment, the presence of sialate pyruvate-lyase in the cytosol of red blood cells remains an enigma.

Response to oxidative stress as a welfare parameter in swine

In pigs, the genetic selection for lean, large muscle blocks and fast growth has been linked to an increased prevalence of metabolic diseases such as porcine stress syndrome and mulberry heart disease. These diseases are associated with cardiovascular inadequacy, which may lead to oxidative stress. In the present study, reactive oxygen metabolites (ROMs) and the anti-oxidant power (OXY) in sera of different swine groups were investigated. The following groups were selected (each around 80 kg body weight): wild boars (WB), Cinta Senese (CS), and Landrace × Large White (LxLW), the latter as both specific pathogen-free (SPF) and intensively farmed animals. In addition, a group of LxLW agonic sows (AS) was also investigated; this group is known to be under oxidative stress. Two colorimetric micro-methods were used to measure ROMs and OXY; ROMs were expressed as mM H2O2 and OXY as μM HOCl neutralised. Between groups, average ROM and OXY values were found to be significantly different by one-way ANOVA (P < 0.001). ROM levels were lower in WB (13.41 ± 1.85) and CS (19.27 ± 1.68), and highest in LxLW (42.00 ± 1.36). OXY values ranged from 260.10 ± 22.13 (WB) to 396.90 ± 9.83 (LxLW). Only one swine group (the CS group) showed a significant, positive correlation between ROM and OXY values. The AS group even showed a negative correlation between ROM and OXY values. These results imply satisfactory environmental coping occurred only within the CS group. Results are discussed in the light of animal welfare legislation, food safety and consumers' protection.

Kinetic parameters of N-acetylglucosaminidase in adult femaleNippostrongylus brasiliensisby a quantitative colorimetric micromethod

Chitin, the 1,4-β-linked homopolymer of N-acetylglucosamine, is a component of Nematode eggshell. Chitin is hydrolyzed to its constituent monomer by the binary chitinase enzyme system. Chitinase (EC 3.2.1.14) hydrolyses first the polymer to N,N'- diacelylchitobiose which is then split to N-acetylglucosamine by N-acetylglucosaminidase (EC 3.2.1.30). N-acetylglucosaminidase is also able to release N-acetylglucosamine monomers directly from chitin. This study reports the identification of N-acetylglucosaminidase in adult female Nippostrongylus brasiliensis. A quantitative colorimetric micromethod has been developed to detect N-acetylglucosaminidase activity from N. brasiliensis using p-nitrophenyl-N-acetylglucosaminide as substrate. The assay conditions were optimized as follows: 50 μl of protein extract were incubated at 37 °C for 2 h in the presence of 50 μl of 19 mM substrate solution and 50 μl of 0.2 M potassium phosphate buffer pH 6.0. The reaction was stopped by the addition of 50 μl 0.1 N NaOH and the absorbance was measured on a Multiskan MS spectrophotometer at 405 nm within 30 min. after the reaction was stopped. The optimal pH was 6.0. Specific activity of protein extract was found at 94.4 ± 5.6 pmol/min/mg protein (n = 4). Vmax app. and Km app. for p- nitrophenyl-N-acetylglucosaminide were respectively 280 ± 11 pmol/min/mg protein (n = 3) and 1.8 ± 0.2 mM (n = 3). This test can now be proposed as a tool for screening anthelmintic compounds in the field of new inhibitors of chitin metabolism.

Colorimetric Micromethod for Protein Determination with Erythrosin B

Reaction of erythrosin B with proteins results in a stable, highly colored chromophore with an absorbance maximum at 545 nm. This is the basis for a new quantitative determination method of proteins in solution. The assay can be performed at room temperature, but is faster and more sensitive at 90-95°C. The erythrosin assay is characterized by (i) stable dye-protein color, (ii) high sensitivity (2-14 μg/ml protein), (iii) short reaction time (1.5-2 min at 90-95°C), (iv) good reproducibility, (v) limited interference by common reagents, and (vi) low protein-to-protein variability. Thus, the erythrosin assay can be useful for routine analytical purposes and may overcome some of the limitations of other currently employed assays.

Properties of damaged starch granules. I. Comparison of a micromethod for the enzymic determination of damaged starch with the standard AACC and Farrand methods

An enzymic colorimetric micromethod for the determination of damaged starch in samples weighing 50 mg was used. Oligosaccharides in filtered fungal α-amylase digests of damaged granules were hydrolysed with amyloglucosidase to yield glucose which was measured with a glucose oxidase—peroxidase—chromogen reagent. Sixteen manual determinations were performed in 3 h, or 32 determinations in 3 h if automated flow injection analysis of the digests was used. A comparative study of the American Association of Cereal Chemists (AACC) and the Farrand methods, both of which determine damaged starch as reducing sugars by non-specific methods, provided evidence that the AACC method overestimates damaged and gelatinized starch by about 30 % (α-glucan basis), while the Farrand method gives corresponding values which are about three times greater. These methods quantify, as ‘maltose’, only 64–70 % of the starch digested by α-amylase, and substantial correction factors are then used to calculate the total damaged starch content. Since amylose contplexed with endogenous lipids of cereal starches is not digested by fungal α-amylase, a small correction factor should be used for damaged or gelatinized cereal starches; no correction is required for the lipid-free waxy cereal starches and non-cereal starches. The micromethod can also be used to quantify gelatinized starch in processed cereal foods.

Colorimetric micromethods for glutaraldehyde determination by means of phenol and sulfuric acid or phenol and perchloric acid

Aqueous micromolar solutions of glutaraldehyde produce a yellow color when treated with 20% phenol in ethanol followed by concentrated sulfuric acid or with phenol in 70% perchloric acid. The absorbance at 482 or 479 nm, respectively, is linearly related to the glutaraldehyde concentration. Of the two methods developed, the sulfuric acid-phenol assay gives a higher sensitivity and the perchloric acid-phenol assay allows the determination of glutaraldehyde in the presence of sugars and proteins.

27] Rapid colorimetric micromethod for the quantitation of complexed iron in biological samples

Publisher Summary This chapter discusses the rapid colorimetric micro-method for the quantitation of complexed iron in biological samples. The array of methods available for quantitating iron in an experimental sample often bewilders the investigator occasionally faced with the need for such quantitation. However, because of the development of more sensitive chromophoric chelators and simpler techniques, new methodologies continually evolve. This chapter describes one of these more recent methodologies for the quantitative determination of iron with ease, sensitivity, and simplicity. Methods commonly employed for the quantitation of complexed iron in biological samples rely on an initial treatment, which releases the complexed iron for its subsequent quantitative determination. Dry ashing in a furnace or wet ashing with hot concentrated acid releases the iron, but both procedures take time and present hazards, particularly for the researcher with only an occasional need to determine the iron content of a biological sample. This chapter explains that the simplicity of the assay procedure easily accommodates a preliminary investigation to examine for possible interference or contamination by any buffer system under consideration. In conclusion, the procedure detailed in this chapter offers the investigator without access to more sophisticated instrumentation, a simple and sensitive colorimetric method for the quantitation of iron in biological material.

Impaired Fat Utilization in Parenterally Fed Low‐Birth‐Weight Infants Suffering from Sepsis

Lipid infusion in low-birth-weight infants suffering from sepsis is still controversial. Consequently, we investigated the fat tolerance in six low-birth-weight infants with sepsis and 15 low-birth-weight infants without sepsis. For measurement of fat clearance, we assayed the serum concentrations of triglycerides enzymatically, and of the free fatty acids by colorimetric micromethod. The fatty acid oxidation was analyzed with the [13C]triolein breath test by means of ratio-mass spectrometry. The infants were maintained on continuous parenteral nutrition with various amounts of soybean oil emulsion (1 g, 2 g, and 3 g fat/kg body weight per day). Comparing the lipid infusion of 1 and 2 g fat/kg body weight per day between the two groups, we found triglyceride and free fatty acid values in both groups to be in the normal range. At a dose of 3 g of fat/kg body weight per day, septic low-birth-weight infants showed a significantly higher concentration of triglycerides (2.02 +/- 0.46 mmol/liter) and of free fatty acids (2.06 +/- 0.45 mmol/liter) than the nonseptic low-birth-weight infants (triglycerides: 1.09 +/- 0.43 mmol/liter; free fatty acids: 1.05 +/- 0.41 mmol/liter). The low-birth-weight infants with sepsis showed a reduced fat oxidation rate of 16.0 +/- 1.5% in contrast to that of the low-birth-weight infants without sepsis, whose rate was 38.4 +/- 1.8%. Accordingly, we apply dosages not exceeding 2 g of fat/kg body weight per day to septic low-birth-weight infants.

The interaction of sialic acids with immobilized 5-hydroxytryptamine

The interaction of N-acetylneuraminic acid (NeuAc) with 5-liydroxytryptamine ( S A T ) has been described in several biologically important systems. These include sialic acidcontaining glycolipids and glycoproteins in synaptosomal membranes (Dette & Wesemann, 1978) and platelets (Tamir et al., 1980), where receptor-type interactions have been described. The high affinity of these interactions has prompted the development of an affinity chromatographic technique with immobilized 5-HT (Sturgeon & Sturgeon, 1982). This work demonstrated the possibility of fractionating sialic acids and sialoglycoconjugates on such a matrix and identified several features of the sialic acid molecule as being important for binding. We have investigated the nature of the sialic acid binding t o immobilized 5-HT by using a range of sialic acids, some derivatives and analogues, and also with compounds related to 5-HT in immobilized or free form. Sialic acids, derivatives and sialoglycoconjugates were prepared o r obtained as before (Schauer, 1978, 1982; Corfield et al., 1979). 5-HT and its analogues were immobilized on Sepharose C L-4B by the procedure described by Corfield et al. (1979). Columns ( 1 nil) of immobilized 5-HT were used t o evaluate the binding of sialic acids and derivatives (0.20.3pmol in 1 nil). Columns were washed with water (5 ml) and eluted with SO niM-Tris/HCl. pH 7.3, or 25 mMammonium formate, pH 5.0 (5 nil). Fractions ( 1 nil) were collected and analyscd for radioactivity or by colorimetric micromethods (Schauer. 1978). The NeuAc-binding capacity of the gel was proportional to the amount of 5-HT immobilized over the range 0.95.8pmol of S-HT/ml of gel and the mole ratio of binding was 0.5-0.7pmol of NeuAc/pmol of immobilized 5-HT. Five separate preparations gave the same ratio. Tryptamine, tryptophan, 5-hydroxytryptophan and S-methoxytryptaniine were ininiobilized by the same technique. but did not bind [ 14C] NeuAc (Amershani) in the radioactive binding assay. When free 5-HT o r its analogues were used t o displace [ I4C] NeuAc from immobilized 5-HT columns, NeuAc was eluted by SO pM-5-HT, 50pM-5-methoxytryptamine or SO p M 4ryptamine; 100 pM-N-acetyl-5-hydroxytryptaniine; 250pM-5-hydroxylindole acetic acid; > 1 mM5-hydroxytryptophan or 1 mM-tryptophan. These findings implicate an involvement of the amino group of 5-HT in optimal binding of NeuAc and that the presence of a carboxyl group on the a-carbon inhibits the binding. The binding of 0.2-0.3 pmol of sialic acids, derivatives or analogues to immobilized 5-HT under the above conditions is shown in Table 1 . Only two sialic acid derivatives did not bind to immobilized 5-HT. These were the 0methylglycoside of neuraminic acid, which has a free amino group, and the N-acetylnonulosamine derivative of NeuAc, in which a primary alcohol replaces the carboxyl group at C-l . This indicates the importance of an N-acylated

Colorimetric micromethod for the determination of protein in solutions with silver ions and dithizone

A method of determination of protein based on the measurement of complexed Ag + and AgCl absorbed on the macromolecules is described. This reaction is carried out in the presence of chloride ions and detergents (Triton X-100 or SDS) in alkaline solution. The silver ions are detected with dithizone. With this method it is possible to estimate 0.1 μg bovine serum albumin in 0.5 ml sample with a coefficient of variation of 16%, and 1.5 μg/sample with a coefficient of variation of 3.6%. The method is simple, rapid, inexpensive, and very sensitive.

A simplified, colorimetric micromethod for xylose in serum or urine, with phloroglucinol.

We have developed a simplified xylose assay procedure that requires only 10 min and requires 50 microL of serum or 5 microL of urine. The reaction with phloroglucinol is more sensitive than the classic p-bromaniline color reaction, and requires only 4 min of heating for color development. A single reagent is mixed with the specimen directly, without prior protein precipitation. Analytical recovery of xylose added to serum was quantitative; precision studies resulted in a between-day coefficient of variation of 5.2%. Glucose, which has significant potential for interference in most other xylose procedures, reacts under the test conditions only to the extent of 70 mumol of apparent xylose per liter for a 5.5 mmol/L solution of glucose. The new procedure has been valuable in the assessment of malabsorption, especially in children and infants, where serum xylose is the preferred measurement.

Emergency determination of acetaminophen.

I describe a senstive colorimetric micromethod in which a pH 11.0 carbonate buffer is used to induce acetaminophen to react with Folin-Ciocalteau reagent at room temperature. These reagents and a simple solvent extraction favor formation of a stable indophenol dye with acetaminophen, giving this procedure a good degree of specificity. Results correlate well with those by liquid-chromatographic procedures, and day-to-day precision is less than 8%.

A simplified colorimetric micromethod for determination of serum cholesterol.

A simple and rapid micromethod is described for quantitation of cholesterol in 5-10 microliter of serum using uranyl acetate to deproteinize the serum and o-phthalaldehyde reagent to develop color. Independent analyses of serum samples by the present method and by two reference methods, Abell-Kendall and automated colorimetric method, indicated accuracy of the micromethod (r = 0.99). Use of capillary blood specimens makes this procedure applicable for screening infants and small children.

ATP-ase activity in the odontoblastic layer of rat incisor. Determination with a radiochemical and a colorimetric method.

The ATP-splitting enzyme activity in odontoblasts isolated from rat incisors has been studied by means of a radiochemical and a colorimetric micromethod. The results with the two methods were virtually identical. The reaction was linear with time for at least 45 min. The pH optimum was found to be 9.8 independently of the ATP concentration. Maximal substrate saturation occurred at a total ATP concentration of 3 mM. Ca2+ and Mg2+ ions activated ATP degradation. F-ions did not affect the activity at low concentrations, whereas higher concentrations were inhibitory. Na+ and ions were slightly inhibitory. Urea inhibited the enzyme activity at concentrations above 1.5 M, while EDTA and EGTA were strong inhibitors at very low concentrations. When incubating in the presence of low concentrations of specific inhibitors for nonspecific alkaline phosphatase, levamisole and R8231, about 20% ATP degrading enzyme activity remained. In conclusion it is suggested that there are at least two ATP degrading phosphatases active at alkaline pH.

An improved automated method for the measurement of red cell 2,3-diphosphoglycerate.

A modified automated colorimetric micromethod for the determination of red cell 2,3-diphosphoglycerate (2,3-DPG) adapted from that of Grisolia et al (1969) is described. In the modified method, ethylenediaminetetra-acetic acid (EDTA) is not used and consequently concentrations of several reagents are changed. During the development of the method it was found that the presence of EDTA, either in the blood or in reagents, consistently reduced the measured value of 2,3-DPG by 15%. This effect of EDTA, not previously recognized, is independent of the EDTA concentration within the range of 5 to 50 mmol/1 and is at present unexplianed. In normal subjects (41 men and 30 women) the mean red cell 2,3-DPG was 14-5 mol/g haemoglobin (range 12-1-18-1 mol/g haemoglobin). There was no significant difference in 2,3-DPG concentrations between male and female subjects.