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At least 163 records · Page 9Linked to original sources

Automated determination of urinary creatinine without sample dilution: theory and practice.

The rate of the Jaffé reaction depends on the concentration of sodium hydroxide; the pseudo-first-order rate constant of the reaction, at 37 degrees C in 10 mmol/L picrate solution, is 0.004 mmol/L. We formulated an automated method to determine urinary creatinine directly without manual sample dilution. The conditions are as follows: 10 mmol/L picrate and 60 mmol/L sodium hydroxide (final concentrations); ratio of sample to final volume, 1:41; temperature, 37 degrees C; wavelengths of measurement, 500 or 510 nm; interval of measurement, 30 to 90 s; and mode of measurement, kinetic. Determinations of creatinine in patients' samples by the new method compared favorably with those obtained with the AutoAnalyzer and aca. The run-to-run CVs were 3.6% or less, and the method was accurate for concentrations of creatinine up to 3000 mg/L. We recommend this method as a good replacement for the AutoAnalyzer or aca methods.

Autoanalysis↗

Gas chromatographic determination of dimethylamine and trimethylamine in seafoods.

The volatile amines dimethylamine (DMA) and trimethylamine (TMA) are common degradation products of TMA-oxide in marine fish. Both compounds are used as important indicators of quality in seafoods. DMA is produced along with an equimolar quantity of formaldehyde by action of an endogenous enzyme found primarily in gadoid fish. TMA is produced in fresh, but not frozen seafoods by a bacterial enzyme. The current AOAC method for determination of TMA in fish is based on the colorimetric estimation of TMA as a picrate salt. This method is not specific for TMA; ammonia, monomethylamine, and DMA also form corresponding picrate salts. Gas chromatography provides a means of separating and quantitating the individual volatile amines. A simple perchloric acid extract of fish is neutralized with potassium hydroxide and extracted with benzene. The amines are then separated by gas chromatography on a porous polymer packed column and detected by using a nitrogen-phosphorus-specific flame ionization detector. The method provides simple, rapid simultaneous quantitation of DMA and TMA, and is applicable to a wide variety of seafoods.

Animals↗

Bilirubin interference in a peroxidase-coupled procedure for creatinine eliminated by bilirubin oxidase.

A colorimetric peroxidase-coupled procedure for determination of creatinine in human serum and urine is described. A 30-s sample pre-treatment with bilirubin oxidase eliminates interference from endogenous bilirubin. The 4-aminoantipyrine-2-hydroxy-3,5-dichlorobenzenesulfonate chromogen system of this method is about fourfold more sensitive than current procedures that involve monitoring NAD(P)H and nearly fivefold more sensitive than the traditional picrate procedures. Incorporation of a sample blank eliminates positive interference from endogenous creatine. Results of the proposed procedure are somewhat lower than those of the common kinetic or equilibrium picrate techniques, as would be expected because of the effects of the well-known interferences in the latter methods.

Autoanalysis↗

Falsely high serum creatinine concentration associated with severe methanol intoxication.

A case of severe methanol intoxication (1300 mg/L) was associated with markedly increased serum creatinine (490 mg/L) despite normal urea values and the absence of any other signs of renal disease. These values declined progressively to normal, and the patient recovered with no visual impairment. Additional laboratory experimentation suggested that the high creatinine value was probably ascribable to some unknown foreign material(s) in the patient's blood that reacted with the alkaline picrate used in the measurement of creatinine. One of the presumed metabolites of methanol, formaldehyde, reacts with creatinine but the product does not react with picrate. We believe that the foreign material was derived from either commercial preparations of methanol or contaminants in the patient's drinking water.

Alcoholic Intoxication↗

Automated development of a kinetic method for the continuous-flow determination of creatinine.

A "stopped-flow" method for the kinetic Jaffé determination of creatinine was developed, with the use of a computer-controlled continuous-flow system. Simplex optimization was used to find conditions of hydroxide and picrate giving maximum sensitivity for creatinine. We used a modified central composite experimental design to evaluate creatinine sensitivity and albumin, glucose, and acetone interferences as functions of hydroxide and picrate concentrations. More importantly, this work illustrates that the automated development of clinical chemical methods offers an efficient means of obtaining optimized, well-understood analytical procedures for subsequent routine use in the clinical chemistry laboratory.

Autoanalysis↗

Depletion of 18O from C18O2 in erythrocyte suspensions. The permeability of the erythrocyte membrane to CO2.

The depletion of 18O from CO2, caused by the exchange of oxygen between CO2 and water during the hydration-dehydration cycle, is catalyzed by carbonic anhydrase. This depletion process at chemical equilibrium in the presence of erythrocytes is biphasic, exhibiting a very rapid depletion rate immediately following the addition of cells to an isotonic solution containing 18O-enriched CO2, followed by a much slower depletion rate. It is hypothesized that these depletion characteristics are caused by the diffusion of labeled CO2 into erythrocytes where depletion occurs rapidly due to the large intracellular carbonic anhydrase content. Kinetic equations which describe this hypothesis are solved and a rate constant is obtained which represents the depletion of 18O in CO2 caused by the presence of red cells. These are equilibrium experiments with no net uptake or loss of CO2 in the cells. Consequently, depletion processes are not limited in rate by bicarbonate-chloride exchange or proton distribution across the membrane. The purpose of these measurements is to determine whether the rate of 18O depletion in red cell suspensions is determined by carbonic anhydrase activity in the cell or by the diffusion process by which CO2 enters the cell. This goal is achieved by partially inhibiting carbonic anhydrase with acetazolamide. The rate constant representing 18O depletion caused by the presence of red cells is unchanged, even though up to 90% of carbonic anhydrase is inhibited. From this rate constant the permeability constant of the membrane of rat erythrocytes to CO2 at 25 degrees and pH 7.4 is determined to be (7.6 +/- 1.2) X 10(-3) cm s-1 in the presence of 3.2 mM picrate, a passive anion diffusion inhibitor intended to block HCO3 -flux across the membrane. Using no picrate and allowing HCO3-flux to introduce an error in the measurements, the permeability constant is (1.6 +/- 0.4) X 10(-2) cm s-1. The permeability constants measured by this technique include the diffusion barrier to CO2 not only of the red cell membrane but also of a portion of the intracellular medium.

Animals↗

Are picro-dye reactions for collagens quantitative? Chemical and histochemical considerations.

Previous studies of picro-dye reactions demonstrated wide variations in the binding of different dyes. Picro-Sirius Red F3BA was recommended because it colors all collagens intensely and is suitable for polarization microscopy. Recent publications on quantitative uses of this stain were surprising. To obtain further information on the chemical mechanisms of dye binding by proteins, 94 sulfonated azo dyes were tested under the conditions of the picro-Sirius Red F3BA reaction. Reaction patterns varied widely, from failure to compete successfully with picrate ions for binding sites to strong coloration of all tissue structures. Only a few dyes stained collagen, reticulum fibers and basement membranes intensely and selectively. The reactivity of dyes was determined by their molecular configuration and the nature and position of substituents. Correlation with physico-chemical data showed that dye binding is due to non-ionic interactions, i.e. van der Waals and dispersion forces and hydrophobic bonding. Coulomb forces do not impart affinity - increasing sulfonation actually decreases dye uptake - but draw dyes within reach of non-ionic sites. Bound dyes form aggregates with additional dye ions; the aggregation number can range from 2 to many powers of 10. Clearly, dye binding by proteins is not stoichiometric.

Azo Compounds↗

Non-mediated zero voltage conductance of hydrophobic ions through bilayer lipid membranes.

A charge pulse technique applied to the study of charge transfer at metal-solution interfaces has been used to determine the capacity and the conductance of a membrane bilayer at both zero time and zero voltage. The transport of hydrophobic ions across a glycerol-monooleate bilayer (tetraphenyl borate, picrate, dipicrylamine and tetraphenyl arsonium) has been investigated by this method. A theoretical approach to the problem has been proposed based on one analogous to that used for the compact double layer at metallic electrodes.

Arsenicals↗

Evaluation of serum creatinine measurement by the direct acidification method for errors contributed by non-creatinine chromogens.

Serum creatinine determination by the direct acidification method was found to be non-specific. Chromogens other than creatinine could react with alkaline picrate and their developed color was also destroyed by acid. Only elevated levels of bilirubin and acetoacetate caused a significant interfering effect. The degree of this interference depended on: (1) the amount of the interfering substance; (2) the reaction time allowed for color development; and (3) the final acidity for decolorization.

Acetoacetates↗

Determination of potassium in pharmaceutical formulations by means of 18-crown-6: extraction studies.

A colorimetric method has been developed for the determination of potassium in pharmaceutical formulations. The method is based on the formation of a complex of potassium with 18-crown-6, followed by conversion to an ion-pair with picrate anion; the ion-pair is extracted with either methylene chloride or toluene-methylene chloride (80:20, v/v) and determined colorimetrically.

Colorimetry↗

On the mechanism of nitrobenzene liquid membrane oscillators containing hexadecyltrimethylammonium bromide.

The oscillatory behaviour of a liquid membrane oscillator was investigated in order to contribute to the oscillation mechanism at the molecular level. The chosen system involved nitrobenzene as liquid membrane containing a constant amount of picric acid. The aqueous donor phase contained the cationic surfactant, hexadecyltrimethylammonium bromide, and ethanol. The aqueous acceptor phase was made up by sucrose solution. It was established that the oscillations take place exclusively at the aqueous acceptor phase/membrane interface. Three stages mechanism of the oscillation (I--induction period, II--first peak formation, III--creation of the first peak) together with appropriate processes were proposed. The molecular events provoking the oscillations of electric potential difference between the two aqueous phases concern: 1) the diffusion of hexadecyltrimethylammonium bromide and ion pairs formed by cation of the surfactant and the picrate anion to the vicinity of the membrane/acceptor phase interface, 2) sudden adsorption of these ion-pairs at this interface in non-catalytic and autocatalytic steps, 3) desorption of ion pairs from the a/m interface to the acceptor phase. It is shown by numerical simulations that the proposed mechanism may account for the observed oscillations and for the species distribution throughout the system as found experimentally.

Cetrimonium↗

Increasing the efficiency of in-capillary electrophoretically mediated microanalysis reactions via rapid polarity switching.

We report herein a new approach to enhance the sensitivity or speed of CE-based methods that involve in-line reactions. Rapid polarity switching (RPS) is used as a novel means for in-line mixing of two reactant solutions via rapid (1-5 s) and sequential switching of the applied potential field. By employing the RPS approach with a model chemical reaction, that between creatinine and alkaline picrate, significant enhancement in sensitivity (or a decrease in analysis time) is realized. Both increased convection and electrophoretic stacking of the ionic reagent appear to contribute to the rise in apparent reaction rate. When coupled with in-line chemistry of the Jaffe method for creatinine, the RPS methodology allows for 3-fold faster determination of creatinine in the concentration range needed for the analysis of clinical blood serum specimens. The new approach also allows the analysis to be performed without the need for the cumbersome and problematic enhanced sensitivity cell.

Creatinine↗

Lipophilic G-quadruplexes are self-assembled ion pair receptors, and the bound anion modulates the kinetic stability of these complexes.

With an eye toward the eventual selective modification of noncovalent structures, we used ESI-MS, X-ray crystallography, and NMR spectroscopy to study the anion's influence on the structure and dynamics of self-assembled ion pair receptors formed from guanosine G 1. We compared five complexes of formula (G 1)(16).2Ba(2+).4A(-) containing different organic anions: 2,4,6-trinitrophenolate (2), 2,6-dinitrophenolate (3), 4-methyl-2,6-dinitrophenolate (4), 4-methoxy-2,6-dinitrophenolate (5), and 2,5-dinitrophenolate (6). Crystallography reveals that anion-nucleobase hydrogen bond geometry is sensitive to both phenolate basicity and structure. For the 2,6-substituted anions 2-5, progressive shortening of anion-nucleobase hydrogen bonds is correlated with increased phenolate basicity. Lipophilic G-quadruplexes with different anions also have much different kinetic stabilities in CD(2)Cl(2) solution. Proton NMR shows that free 6 exchanges faster with G-quadruplex-bound anion than do the 2,6-dinitrophenolates 2-5. The increased lability of 6 is probably because, unlike the 2,6-dinitrophenolates, this anion cannot effectively chelate separate G(8).M(2+) octamers via anion-nucleobase hydrogen bonds. In addition to these structural effects, the anion's basicity modulates the anion exchange rate between its free and bound states. 2D EXSY NMR shows that 3 and 5 exchange about 7 times slower than the less basic picrate (2). The use of 3, a relatively basic dinitrophenolate that hydrogen bonds with the amino groups of the two "inner" G(4)-quartets, resulted in extraordinary kinetic stabilization of the G-quadruplex in CD(2)Cl(2). Thus, no isomerization product (G 1)(8).Ba(2+).(G 1)(8).Sr(2+).4(3) was observed even 2 months after the separate G-quadruplexes (G 1)(16).2Ba(2+).4(3) and (G 1)(16).2Sr(2+).4(3) were combined in CD(2)Cl(2). In sharp contrast, G-quadruplexes containing the isomeric 6 anion have isomerization half-lives of approximately t(1/2) = 30 min under identical conditions. All the evidence indicates that the structure and electronics of the organic anions, bound to the assembly's periphery, are crucial for controlling the kinetic stability of these cation-filled G-quadruplexes.

Anions↗

Estimation of renal function in subjects with normal serum creatinine levels: influence of age and body mass index.

BACKGROUND: The Cockcroft-Gault (CG) and simplified Modification of Diet in Renal Disease (MDRD) formulas are the most widely used estimates of renal function. The influence of age and body mass index (BMI) on the performance of these equations was analyzed in 850 subjects with serum creatinine levels less than 1.5 mg/dL (<133 micromol/L). METHODS: Glomerular filtration rate (GFR) was measured as urinary clearance of continuously infused technetium Tc 99m-labeled diethylene triaminopentaacetic acid. Performance was assessed as bias, precision, and accuracy. RESULTS: In the total population, the CG and MDRD calculations based on enzymatic measurement of serum creatinine (which is constantly less than that obtained by using the alkaline picrate [Jaffé] method) significantly underestimated GFR by 4.9 and 12.4 mL/min/1.73 m2 (0.08 and 0.21 mL/s/1.73 m2), respectively. In patients 65 years and older, underestimation by means of the CG formula was enhanced, whereas that by means of the MDRD formula was blunted, compared with the group younger than 65 years (-11.3 versus -3.7 mL/min/1.73 m2 [-0.19 versus -0.06 mL/s/1.73 m2] for CG and -3.7 versus -14.0 mL/min/1.73 m2 [-0.06 versus -0.23 mL/s/1.73 m2] for MDRD). GFR was underestimated to a large extent by means of the MDRD equation irrespective of BMI. Conversely, the underestimation by means of the CG formula found in lean people (-13.0 mL/min/1.73 m2 [-0.22 mL/s/1.73 m2]) was blunted in overweight people (BMI, 25 to 30 kg/m2) and reversed to overestimation (+10.1 mL/min/1.73 m2 [+0.17 mL/s/1.73 m2]) in obese subjects (BMI > 30 kg/m2). CONCLUSION: As suggested by estimations obtained using enzymatic serum creatinine measurement, the MDRD equation may be the estimation of choice in elderly patients, whereas the CG estimate is preferable in subjects younger than 65 years. Nevertheless, when obesity is present, no reliable estimation can be obtained by using the CG or MDRD formula.

Adolescent↗

Enzymes as reagents in peptide synthesis: enzymatic removal of amine protecting groups.

A model system is described for the enzymatic deprotection of suitably masked amino groups during stepwise peptide synthesis. Nitrophenyl esters of amino acids, N-protected with trypsin-labile benzyloxycarbonylarginyl groups, were prepared as crystalline, analytically pure picrate salts in a standardized procedure. These intermediates were shown to react with amino compounds to form the expected peptide linkages. A pair of diasteriomeric peptides prepared in this way and featuring benzyloxycarbonylarginyl-L-, AND -D-glutaminyl sequences, respectively, were subjected to tryptic digestion. In both cases, a specific cleavage of the arginyl bond was achieved; however, the peptide containing the L-glutaminyl residue was deprotected much more rapidly than its diasteriomer containing the D-glutaminyl residue. The hydrolysis of the former isomer was not noticeably impeded by the presence of the latter. The results of these studies suggest that C-activated amino-acid derivatives, N-protected with trypsin-labile groups, are readily prepared in convenient form and that the peptide derivatives prepared from these intermediates are readily freed of their amino-protecting groups under mild, aqueous conditions with a potentially useful degree of stereospecificity. Theoretical implications of this first enzyme-catalyzed step in the repetitive cycle of peptide elaboration are discussed along with the procedural advantages implicit in the alternation of strongly and weakly basic groups in the protected and unprotected peptide intermediates, respectively.

Acylation↗

Atomic resolution structures and solution behavior of enzyme-substrate complexes of Enterobacter cloacae PB2 pentaerythritol tetranitrate reductase. Multiple conformational states and implications for the mechanism of nitroaromatic explosive degradation.

The structure of pentaerythritol tetranitrate (PETN) reductase in complex with the nitroaromatic substrate picric acid determined previously at 1.55 A resolution indicated additional electron density between the indole ring of residue Trp-102 and the nitro group at C-6 of picrate. The data suggested the presence of an unusual bond between substrate and the tryptophan side chain. Herein, we have extended the resolution of the PETN reductase-picric acid complex to 0.9 A. This high-resolution analysis indicates that the active site is partially occupied with picric acid and that the anomalous density seen in the original study is attributed to the population of multiple conformational states of Trp-102 and not a formal covalent bond between the indole ring of Trp-102 and picric acid. The significance of any interaction between Trp-102 and nitroaromatic substrates was probed further in solution and crystal complexes with wild-type and mutant (W102Y and W102F) enzymes. Unlike with wild-type enzyme, in the crystalline form picric acid was bound at full occupancy in the mutant enzymes, and there was no evidence for multiple conformations of active site residues. Solution studies indicate tighter binding of picric acid in the active sites of the W102Y and W102F enzymes. Mutation of Trp-102 does not impair significantly enzyme reduction by NADPH, but the kinetics of decay of the hydride-Meisenheimer complex are accelerated in the mutant enzymes. The data reveal that decay of the hydride-Meisenheimer complex is enzyme catalyzed and that the final distribution of reaction products for the mutant enzymes is substantially different from wild-type enzyme. Implications for the mechanism of high explosive degradation by PETN reductase are discussed.

2,4-Dinitrophenol↗

Proton and multinuclear N.M.R. studies of cyclogentiotetraose peracetate-alkali cation complexation.

Complexation of alkali cation picrates with cyclogentiotetraose peracetate (CGD4Ac) have been studied by 1H-N.M.R. spectroscopy in acetone d6 and nitromethane d3. We determined the stability constants directly from the observed change of the chemical shifts of H-4 and H-6 pro S protons of CGD4Ac at constant ligand concentration with increasing amounts of alkali salt. The stability constants have also been determined by multinuclear n.m.r. spectroscopies, from the observed change of the chemical shifts of Lithium-7, Sodium-23, Potassium-39, Rubidium-87 and Cesium-133 at constant alkali salt concentration with increasing amount of CGD4Ac. The stabilities of the complexes varied in the order Cs+ greater than Rb+ greater than K+ greater than Na+ greater than Li+. The complexation of CGD4Ac with Cs+ induced conformational change, the gg conformer being predominant at the complexed state. In most cases the cationic exchanges between the free and complexed sites were rapid. However in the CsPic-CGD4Ac-Acetone system the exchange was slow enough to observe below 288 K two 133Cs+ resonances.

Carbohydrates↗

Interference of creatinine measurement in CAPD fluid is dependent on glucose and creatinine concentrations.

BACKGROUND: High glucose concentration in CAPD fluid is known to interfere with creatinine measurement, which is required for assessment of peritoneal membrane permeability and adequacy of dialysis. Correction formulae have been proposed but they may be method/analyser-dependent. We studied such interference in detail. METHODS: CAPD fluid was diluted to prepare six specimens with glucose concentrations ranging from 9.1 to 154.4 mmol/l. To each specimen, creatinine standard was added to give five different concentrations from 50 to 800 mumol/l. The 30 specimens were assayed for creatinine with six routine clinical chemistry analysers (Hitachi 911 and 747, Technicon RAXT and SMAC3, Beckman CX7, and Kodak Ektachem 700). Creatinine interference was calculated by subtracting the apparent creatinine concentration with corresponding baseline creatinine concentration (measured at glucose = 9.1 mmol/l) in the same series. RESULTS: At constant creatinine concentration, interference increased with increasing glucose concentration to varying extents (up to 200%) amongst the six analysers. At constant glucose concentration, interference decreased with increasing creatinine concentration in analysers using the alkaline picrate reaction but increased in the Kodak analyser using enzymatic assay. CONCLUSION: Interference of creatinine measurement in CAPD fluid is dependent on glucose and creatinine concentrations, and each centre should derive specific correction formulae for its analytical system.

Ascitic Fluid↗