Standard Colorimetric Assay Research Articles

Effect of FIO2 on Oxidative Stress during Interval Training at Moderate Altitude

To evaluate the effect of different fractions of inspired oxygen (FIO2) on oxidative stress during a high-intensity interval workout in trained endurance athletes residing at altitude. Subjects (N = 19) were trained male cyclists who were residents of moderate altitude (1800-1900 m). Testing was conducted at 1860 m (PB 610-612 torr, PIO2 approximately 128 torr). Subjects performed three randomized, single-blind trials consisting of a standardized interval workout (6 x 100 kJ) while inspiring a medical-grade gas with FIO2 0.21 (PIO2 approximately 128 torr), FIO2 0.26 (PIO2 approximately 159 torr), and FIO2 0.60 (PIO2 approximately 366 torr). Serum lipid hydroperoxides (LOOH) and whole-blood reduced glutathione (GSH) were measured 60 min preexercise and immediately postexercise, and analyzed using standard colorimetric assays. Urinary malondialdehyde (MDA) and 8-hydroxydeoxyguanosine (8-OHdG) were measured 24 h preexercise and 24 h postexercise, and analyzed via HPLC and ELISA, respectively. Compared with the control trial (FIO2 0.21), total time (min:s) for the 100-kJ work interval was faster (5% in FIO2 0.26; 8% in FIO2 0.60 (P < 0.05)) and power output (W) was higher (5% in FIO2 0.26, 8% in FIO2 0.60 (P < 0.05)) in the supplemental oxygen trials. There was a significant pre- versus postexercise main effect (P < 0.05) for LOOH and GSH; however, there were no significant differences in LOOH or GSH between the FIO2 trials. MDA and 8-OHdG were unaffected by either the interval training session or FIO2. Supplemental oxygen used in conjunction with high-intensity interval training at altitude ("live high + train low via supplemental O2" (LH + TLO2)) results in a significant improvement in exercise performance without inducing additional free radical oxidative stress as reflected in hematological and urinary biomarkers.

A simplified method for HPLC determination of creatinine in mouse serum.

Mouse models are frequently used to study renal function. However, mouse serum contains chromagens that interfere with standard picric acid-based assays for serum creatinine. Several alternative methods exist for serum creatinine measurements, including assay by high-performance liquid chromatography (HPLC), but only one has been adapted to mouse serum. Creatinine was measured in serum by acetonitrile deproteinization, followed by isocratic, cation exchange HPLC. The HPLC method was compared with a standard alkaline picrate colorimetric assay, using serum from animals with low-to-moderate renal injury. Acidification of acetonitrile with HCl in the deproteinization step produced variable results, including an extra peak that interfered with integration of the creatinine peak or loss of the creatinine peak. Deproteinizing with acetonitrile alone resulted in a more reliable measurement of serum creatinine, which was validated by a series of known additions of creatinine standard. The HPLC assay was reproducible with coefficients of variation from 1.6 to 5.1%. The picric acid assay overestimated serum creatinine, when directly compared with the HPLC assay. The extent of overestimation, up to sixfold, was greatest at normal (0.1 to 0.2 mg/dl) to moderately elevated (0.5 mg/dl) serum creatinine levels. Mouse serum contains substances that interfere with standard picric acid assays for creatinine. Our new HPLC assay can accurately detect creatinine from 5 microl of mouse serum. These results support the widespread adoption of HPLC to accurately measure serum creatinine in mouse models of renal injury.

Measuring β-Galactosidase Activity in Bacteria: Cell Growth, Permeabilization, and Enzyme Assays in 96-Well Arrays

We describe a high-throughput procedure for measuring β-galactosidase activity in bacteria. This procedure is unique because all manipulations, including bacterial growth and cell permeabilization, are performed in a 96-well format. Cells are permeabilized by chloroform/SDS treatment directly in the 96-well blocks and then transferred to 96-well microplates for standard colorimetric assay of β-galactosidase activity as described by Miller [J. H. Miller (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY]. Absorbance data are collected with a microplate reader and analyzed using a Microsoft Excel spreadsheet. The β-galactosidase specific activity values obtained with the high-throughput procedure are identical to those obtained by the traditional single-tube method of Miller. Thus, values obtained with this procedure may be expressed as Miller units and compared directly to Miller units reported in the literature. The 96-well format for permeabilization and assay of enzyme specific activity together with the use of 12-channel and repeater pipettors enables efficient processing of hundreds of samples in an 8-h day.

A microplate fluorimetric assay for the study of enzyme diversity in soils

A fluorimetric microplate enzyme assay has been developed to study enzyme diversity in soil as an approach to understanding functional diversity. The microplate assay allows a large number of soil samples and/or enzymes to be analysed in a short time. The substrates used are conjugates of the highly fluorescent compounds 4-methylumbelliferone (MUB) and 7-amino-4-methyl coumarin (AMC). The main advantage of using fluorimetrically-labelled substrates is that product formation can be measured directly in the microplate without previous extraction and purification of the product. A detailed protocol for this new technology is presented and some potential applications are outlined. A comparative study was carried out between the new microplate fluorimetric assay and a standard colorimetric enzyme assay based on p-nitrophenyl substrates. The kinetics of β-glucosidase and phosphatase activities were investigated in soils with different fertilizer backgrounds. Both methods generated similar values for V max (maximum rate of activity) whereas the affinity of β-glucosidase and phosphatase for their respective substrates (as indicated by K m values, Michaelis–Menten constant) was up to two orders of magnitude greater for the 4-methylumbelliferyl substrates compared to the p-nitrophenyl substrates.

The Use of Para-Aminobenzoic Acid (PABA) as a Compliance Measure for Large, Long-Term Feeding Studies

LEARNING OUTCOME: To analyze the use of para-aminobenzoic acid for large long-term feeding studies as a measure of compliance. Metabolic diets are precisely designed to meet each feeding studies’ unique protocol. Valid results are solely dependent upon the total consumption of each prescribed diet. Therefore, compliance is a major concern. Para-aminobenzoic acid (PABA) is a non-metabolized marker that can be incorporated easily into many foods and is then excreted in the urine. The specific aims of this study were to measure the long-term effects of freezing PABA containing products and to identify the percent PABA recovery in known compliant participants subjected to realistic study control indicative of long-term feeding studies. The effects of frozen storage was measured by placing PABA containing muffins into a freezer (−20C) at week 10, 9, 8, 7, 6, 5, 4, 3, 2, and 1. At week 0 they were removed from the freezer and analyzed for % PABA recovery. There was no significant difference in PABA recovery, thus indicating that frozen storage had no effect on PABA. A 3-day feeding study was then conducted with 10 subjects that work at Pennington Biomedical Research Center (PBRC) and know the importance of compliance. All meals were provided by the PBRC metabolic kitchen. Breakfast and lunch were consumed on site and they were given a take home dinner. Each participant consumed 288 mg of PABA each day that was incorporated into different foods evenly throughout the day. Food production, storage, packaging, and serving was done in the same manner used in large, long-term feeding studies. A baseline urine collection was done prior to the start of the study to measure any natural occurring PABA. Then, 24-hour urine collections were made each day of the study. PABA recovery was measured using a standard colorimetric assay and only approximately 10% was recovered from each subject each day. The reason for such low recovery is not known at this time. The results indicate that PABA may not be an effective compliance marker for long-term out-patient feeding studies.

Comparison of total and bone-specific alkaline phosphatase in patients with nonskeletal disorder or metabolic bone diseases

To evaluate the diagnostic validity of new assays for bone-specific alkaline phosphatase (BAP), we compared measurements of total alkaline phosphatase (TAP) in serum with results for three different assays of serum BAP in healthy adults (n = 119), patients with chronic nonskeletal disorders (n = 123), and patients with metabolic bone diseases (n = 113). Serum TAP was determined by a standard colorimetric assay, BAP by the methods of lectin precipitation (L-BAP), enzyme immunoassay (E-BAP), and immunoradiometric assay (I-BAP). Impairment of liver function resulted in significant increases of all alkaline phosphatase (AP) measurements, with the smallest changes being exhibited by E-BAP. Compared with the results by TAP, diagnostic sensitivity (i.e., of values exceeding the reference interval) was not improved by BAP, but receiver-operating characteristic (ROC) curve analyses revealed improved discrimination for primary hyperparathyroidism by E-BAP. These results indicate that, in the presence of liver disease, the specificity of AP measurements is improved by measuring BAP. In most other clinical situations, serum TAP appears to provide sufficient clinical information; however, the cross-sectional study design used here allows no statement about the usefulness of BAP in serial measurements.

Protein interactions with particulate teflon: Implications for the foreign body response

Purpose: This study examined the nature of protein interactions with particulate polytetrafluoroethylene (PTFE, Teflon) to elucidate possible mechanisms involved in the foreign body response directed against failed Proplast/Teflon implants. Materials and Methods: Fifty milligrams PTFE prepared to particle sizes ranging from < 32 μm to > 300 μm was incubated with newborn bovine serum. The total amount of protein adsorbed to the PTFE particles was determined using a standard colorimetric assay. The structural and functional integrity of the proteins adsorbed to PTFE was also examined. For these studies, xanthine oxidase was substituted for serum, and the enzymatic activity of xanthine oxidase adsorbed to PTFE was determined. Finally, primary interactions between protein and PTFE particles were assessed in experiments using water, 2 or 8 mol/L urea, 1 mol/L Nacl, or 1% sodium dodecyl sulfate in an attempt to dissociate bound protein from the surfaces of PTFE particles. Results: Serum proteins bind almost instantly to the surface of PTFE particles. The effective surface area of PTFE increases dramatically with reduction of the material to small particles, as does the total amount of protein adsorbed by the particulate PTFE. Proteins bind to PTFE principally by hydrophobic interactions, and their three-dimensional structure is significantly perturbed by this interaction. In the case of xanthine oxidase, adsorption to PTFE distorts protein structure to the extent that biologic activity is eliminated. Conclusions: The amount of serum protein adsorbed to PTFE particles varies inversely with particle size for a constant mass of material. It is believed that the foreign body response directed against this material is related to the amount and relative distortion of proteins adsorbed to its surface. If so, it appears that reduction of an implant to small particles (typically 50 μm or less) will dramatically increase the biologic signal to local cell populations. Thus, the severity of the biologic response to PTFE debris may be dependent largely on the size of the debris particles.

An in situ electrosynthesized amperometric biosensor based on lactate oxidase immobilized in a poly-o-phenylenediamine film: determination of lactate in serum by flow injection analysis

The electrochemical immobilization of lactate oxidase in a poly-o-phenylenediamine film permits the one-step and all-chemical construction of a lactate amperometric biosensor. The sensor was prepared in situ i.e. in the flow injection analysis (FIA) system by simply injecting a plug of a solution containing the monomer and the enzyme. At a flow rate of 50 μL/min linearity was observed up to 0·2 mM lactate and detection limits of about 2μM could be easily achieved. Faradaic interferences caused by ascorbate, urate, cysteine and acetaminophen were sufficiently minimized to permit lactate determination in diluted serum by FIA. Results obtained by FIA-amperometric detection compared well (according to a proper t-test at a 95% confidence level) with those obtained by a standard enzymatic colorimetric assay. At a flow rate of 1 ml/min a sample throughput higher than 70 sample h −1 was achieved. After one week of continuous use in the FIA system a 75% decrease in biosensor sensitivity was observed.

Histochemical quantitation of gamma-glutamyl transferase in human leukemia cell lines.

The enzyme gamma-glutamyl transferase (gamma-GT) is involved in many biochemical systems, including the signal transduction of hematopoietic growth factors. Standard colorimetric gamma-GT assays require larger cell numbers than may be obtainable in many cases, such as with highly purified stem-cell populations. To study gamma-GT expression in limited populations, we used a histochemical stain to analyze gamma-GT semiquantitatively in cells of hematopoietic origin. Several human leukemic cell lines, including one with inducible increases in gamma-GT, were stained for gamma-GT and graded 0 through 4+ for the amount of positive granules. The gamma-GT activity demonstrated by this stain was found to be directly proportional to the gamma-GT activity obtained with a colorimetric assay and could be used to calculate approximate gamma-GT activity. This stain therefore provides a useful method for determining gamma-GT activity when limited cell numbers are available.

Secreted alkaline phosphatase: an internal standard for expression of injected mRNAs in the Xenopus oocyte

The Xenopus oocyte is widely used to study the various aspects of eukaryotic cell structure and function. It is also being used increasingly in expression cloning of cDNAs encoding proteins for which there are no structural data. One of the drawbacks of the Xenopus oocyte system is that individual oocytes taken at the same time from the same frog vary considerably in the amount of protein synthesized from the same amount of injected mRNA. In this report we describe the preparation and use of the mRNA for a secreted mutant form of human placental alkaline phosphatase as an internal, coinjected standard to monitor translation in oocytes. Secreted alkaline phosphatase can be readily determined in the medium of cultured oocytes by using a standard colorimetric assay. The amounts of alkaline phosphatase secreted into the medium were shown to parallel the level of expression of two membrane proteins. This permits rapid identification and selection of those oocytes that efficiently express injected mRNAs. The procedure yields more precise data and results in an enormous saving of time and expense, especially in investigations that involve complex measurements on individual oocytes.

Nitrate analysis by capillary gas chromatography

A microanalytical gas chromatographic (GC) method for the analysis of nitrate in rat urine is described. The method involves the conversion of nitrate to nitromesitylene and quantitation using 3,4-dimethylnitrobenzene as an internal standard. The nitroaromatics were separated on a wide-bore capillary column and detected with a nitrogen-phosphorus thermionic detector. The method exhibited linearity over the range of 1.0 to 1000 μ m nitrate, and the detection limit was 0.5 μ m nitrate (200-μl sample). The coefficient of variation (CV) range for intra-day precision was 2.2 to 5.8% (20 μ m level) and 3.1 to 6.5% (200 μ m level). Inter-day CVs ranged from 2.0 to 6.1% for the samples tested. The average recovery was 77% (20 μ m level) and 80% (20 μ m level). The accuracy of the GC method compared favorably with results obtained from a standard colorimetric nitrate assay. Interference by urinary chloride was eliminated by pretreatment of samples with saturated silver acetate. Both processed and unprocessed samples were stable for at least 60 days at −15°C. The procedure was used to measure urinary nitrate in rats fed a custom low-nitrate diet.

Germination of Saccharomyces cerevisiae ascospores without trehalose mobilization as revealed by in vivo 13C nuclear magnetic resonance spectroscopy.

Saccharomyces cerevisiae ascospores germinate in the presence of acetate without any detectable trehalose degradation, as revealed by high-resolution nuclear magnetic resonance spectroscopy and by a standard colorimetric assay. The results presented here substantiate the hypothesis that in S. cerevisiae trehalose supplies energy during dormancy of the spores and not during the germination process.

A fluorometric assay for determining cell growth in lymphocyte proliferation and lymphokine assays

A microplate method for assessing cell growth and viability based on the hydrolysis of fluorogenic substrates by cell esterases has been investigated. Living cells incubated with fluorescein diacetate or 4-methylumbelliferyl heptanoate generate a fluorescent product which is proportional to the number of cells. This can be used as a simple and economical readout for various bioassays such as, for example, the assessment of IL-2 and IL-3 on factor-dependent cell lines, and on antigen- and mitogen-stimulated proliferation of lymphocytes. This fluoremetric assay has similar sensitivity to the measurement of [ 3H]thymidine uptake and greater sensitivity than standard colorimetric assays. Incubation with MUH for a period of 30–60 min at 22°C is adequate.

Determination of Cholesterol in Egg Yolk by High Performance TLC with Densitometry

Abstract A simple quantitative TLC determination of cholesterol in egg yolk is described. Cholesterol in the yolk is extracted with chloroform-methanol (2:1) and the extract filtered through glass wool. After dilution to a known volume, samples and standards are developed on a high performance preadsorbent silica gel layer, cholesterol is detected with cupric acetate-H3-PO4 reagent, and zones are scanned and compared. Precision of the method, evaluated by replicate analyses, was 2.2%, and recovery from a sample spiked at a concentration level of 14.3 mg/g was 104%. Analysis of 20 eggs gave cholesterol values ranging from 9.7–26.2 mg/g, and the method was also applied to butter and cream samples. Because of the selectivity of the TLC method, lower results were obtained compared to a standard colorimetric assay. Fractions representing sterols, diacylglycerols, triacylglycerols, stearyl esters, and hydrocarbons were identified from egg yolk, and the sterol fraction was found to contain only cholesterol by ar...

Quantitation of picomole levels of N-acetyl- and N-glycolylneuraminic acids by a HPLC-adaptation of the thiobarbituric acid assay

A simple HPLC adaptation of the periodate-TBA assay for free N-acetyl- and N-glycolylneuraminic acids greatly extends the sensitivity and increases the specificity of this standard colorimetric assay. The method, employing a C 18 reverse-phase column eluted isocratically with a phosphoric acid-MeOH buffer, is linear over a range of 2 pmol to 20 nmol. Analyses can be performed directly on cell lysates and digests without prior purification of released sialic acids from contaminating salts and biological materials. Interference from 2-deoxysugars is completely eliminated as the chromophore from these compounds is completely resolved from that derived from sialic acids. The application of the technique to quantify cell-surface and total cellular TBA-reactive sialic acids on the surfaces of a variety of tumor cells is described. Additionally, the extent of desialylation of erythrocytes necessary to expose the T antigen is determined.

Reactivity of key metabolic sterols in standard colorimetric assays for cholesterol

Reactivity of key metabolic sterols in standard colorimetric assays for cholesterol

Reactivity of key metabolic sterols in standard colorimetric assays for cholesterol.

The reaction of lanosterol, desmosterol and 7-dehydrocholesterol, key intermediates in cholesterol biosynthesis, were-compared with cholesterol in 3 standard colorimetric assays for cholesterol based on formation of chomogens with acetic anhydride, ferric chloride and ferrous sulfate. Marked differences in the reaction of the sterols in the different assays were due both to formation of chomogens with qualitatively similar spectral patterns but with greatly different extinctions and to formation of chromogens with clearly different absorption maxima. For example, in all assays, cholesterol and desmosterol formed chromogens with very similar absorption spectra but with varying extinctions, whereas the lanosterol chromogen in all assays was different from cholesterol's in both absorption maxima and in extinctions. The findings show that attempts to measure tissue sterol levels by colorimetric methods can result in greater errors when cholesterol is not the sole sterol. Also, the unique spectral properties of the lanosterol chromogen formed in the Liebermann-Burchard reaction (a sharp absorption peak at 450 nm) suggests the possible use of this method as a qualitative test for lanosterol.

Sensitive fluorometric assay for leghemoglobin

A sensitive spectrofluorometric assay for leghemoglobin is based upon the action of hot saturated oxalic acid on heme proteins. The assay will detect 200 ng of leghemoglobin per milliliter and is specific enough to permit estimation in single nodules or extracts of whole roots. The leghemoglobin concentration measured fluorometrically shows a correlation with nitrogenase [C 2H 2] activity, even during nodule senescence, when standard colorimetric assays may overestimate leghemoglobin.

Submicrogram quantitation of an acidic polysaccharide by rocket immunoelectrophoresis and rocket affinoelectrophoresis

Rocket immunoelectrophoresis has been used to quantitate an acidic polysaccharide (a succinylated lipomannan) isolated from the membranes of the bacterium, Micrococcus lysodeikticus. This procedure is superior in sensitivity to standard colorimetric assays for carbohydrate and allows the detection of as little as 10 ng of mannan. The observed relationships between rocket height and both antibody and antigen concentrations follow those described by Weeke for protein antigens [ Weeke, B. (1973) Scand. J. Immunol. 2, Suppl. 1, 37–46]. Furthermore, the lectin concanavalin A could be used as an alternative to immune serum in the quantitation of this polysaccharide by rocket electrophoresis. However, this modification (rocket affinoelectrophoresis) appeared to be less sensitive than rocket immunoelectrophoresis, allowing the detection of about 25 ng of mannan.

The effect of streptozotocin diabetes on the levels of glycolate and lactate excreted in rat urine

The concentrations of glycolate (hydroxyacetate) and lactate are significantly elevated above control values in urines from streptozotocin-diabetic rats, regardless of whether data are expressed in terms of μg/ml urine or μg/day. The same levels of oxalate and glyoxylate are excreted in 24 h in the urines from normal and diabetic rats. Lactate levels are elevated above control values in serum from streptozotocin-diabetic rats. The elevation of glycolate levels in diabetic rat urine compared to control values occurs regardless of diet and regardless of whether rats were fed or fasted during the 24 h urine collection period. Rat liver glycolate oxidase may be used to assay glycolate concentrations in the presence of up to 500 μg/ml l-lactate when pH 8.6 Tris-Cl is used as buffer. Results obtained with this assay compare qualitatively with the standard colorimetric assay using 2,7-dihydroxynaphthlene for glycolate determination. Beef liver glycolate oxidase is not effective for use in glycolate assays. The identity of urinary glycolate was confirmed by gas-liquid and by paper chromatography.