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PubMed1976

Estimation of tryptophyl and tyrosyl exposure in tryptophan-rich proteins by ultraviolet difference spectrophotometry. Lysozyme and Chymotrypsinogen.

Ultraviolet difference absorption spectra produced by ethylene glycol were measured for hen lysozyme [EC 3.2.1.17] and bovine chymotrypsinogen. N-Acetyl-L-tryptophanamide and N-acetyl-L-tyrosinamide were employed as model compounds for tryptophyl and tyrosyl residues, respectively, and their ultraviolet difference spectra were also measured as a function of ethylene glycol concentration. By comparison of the slopes of plots of molar difference extinction coefficients (delta epsilon) versus ethylene glycol concentration for the proteins with those of the model compounds at peak positions (291-293 and 284-287 nm) in the difference spectra, the average number of tyrosyl as well as tryptophyl residues in exposed states could be estimated. The results gave 2.7 tryptophyl and 1.9 tyrosyl residues exposed for lysozyme at pH 2.1 and 2.6 tryptophyl and 3.4 tyrosyl residues exposed for chymotrypsinogen at pH 5.4. The somewhat higher tyrosyl exposure of chymotrypsinogen, compared with the findings from spectrophotometric titration and chemical modification, was not unexpected, because delta epsilon285 was larger than delta epsilon292, and the situation is discussed with reference to preferential interaction of ethylene glycol with the tyrosyl residues and/or side chains in the vicinity of the chromophore in the protein. The procedure employed in the present work seems to be suitable for estimation of the average number of exposed tryptophyl and tyrosyl residues in tryptophan-rich proteins. The effects of ethylene glycol on the circular dichroism spectra of lysozyme at pH 2.1 and chymotrypsinogen at pH 5.4 were also investigated. At high ethylene glycol concentrations, both proteins were found to undergo conformational changes in the direction of more ordered structures, presumably more helical for lysozyme and more beta-structured for chymotrypsinogen.

Animals↗
PubMed1978

Cholesterol oxidase: thermochemical studies and the influence of hydroorganic solvents on enzyme activity.

Thermal and binary cosolvent studies of the cholesterol oxidase (cholesterol: oxygen oxidoreductase, EC 1.1.3.6) reaction have been carried out using batch microcalorimetry and ultraviolet spectrophotometry respectively. Heat conduction measurements are shown to provide the basis for a serum cholesterol assay yielding results comparable to conventional automated clinical assay. The enthalpy of the reaction for cholesterol oxidation, measured with different sources of the enzyme in the presence and absence of catalase is -113 +/- 7.2 mJ/mumol. The value is agreement with calculated estimates based on bond energies, enthalpies of formation and trigonal additivity contribution calculations. From this heat of reaction the deltaHf0 of cholestenone (c) is calculated to be -490 kJ . mol-1. No evidence for the reverse reaction could be adduced. Enzyme activation with detergent (Surfal) is attributed to the formation of mixed micelles of cholesterol with detergent molecules. The detergent concentration at which the enzyme is half activated corresponds to the critical micelle concentration of Surfal. The enhanced enzyme activity found when ethanol, acetonitrile and dioxane were examined as binary cosolvents with water is ascribed to a conformational change in the enzyme mediated through the altered structuredness of water. This cosolvent effect is abolished in the presence of 0.18% Surfal due to the formation of inverted mixed micelles of detergent with cholesterol.

3-Hydroxysteroid Dehydrogenases↗
PubMed1976

A modified ultraviolet spectrophotometric method for the determination of theophylline in serum in the presence of barbiturates.

Ultraviolet spectrophotometry is the most commonly used technique for the determination of theophylline levels for therapeutic monitoring. Common interferences in most methods are barbiturates and xanthines. A modified method which eliminates interferences from barbiturates and most xanthines is presented. Theophylline is extracted from serum with chloroform/isopropanol at pH 7.4 back extracted into dilute hydrochloric acid and then the solution is made alkaline with sodium hydroxide. Barbiturate interferences are eliminated. Interference from caffeine, uric acid, 7-(2,3-dihydroxypropyl)theophylline, xanthine, and hypoxanthine are not observed. Theobromine, and the metabolite 3-methylxanthine interfere. Interference is not observed from quinidine, diazepam, salicylate, glutethimide, methylprylon, propranolol, methaqualone, dilatin and ethchlorvynol. Sulfanilamide, procainamide and chlordiazepoxide interfere.

Barbiturates↗
PubMed1979

An ultraviolet spectrophotometric procedure for the routine determination of naproxen.

1. A procedure for the routine assay of Naproxen in serum by ultraviolet spectrophotometry is presented. The absorption coefficient (A1 1% cm) of Naproxen in methanol at 261 nm was found to be 216. 2. The Naproxen procedure appears to be relatively free from interferences of commonly coadministered medications. Salicylate interference is eliminated by a modified procedure. 3. Concentrations of Naproxen in a limited number (40) of clinical samples ranged from non-detected to 12.5 mg/dl. The therapeutic concentration appears to be 4 to 6 mg/dl (174-260 mumol/L).

Humans↗
PubMed1978

Identification of 5,8-oxyretinoic acid isolated from small intestine of vitamin A-deficient rats dosed with retinoic acid.

A retinoid was isolated by a multistep procedure from the small intestines of vitamin A-deficient rats given a single dose of retinoic acid. The compound, designated 8II, was pure, as demonstrated by four high-pressure liquid chromatographic procedures. It was positively identified as 5,8-oxyretinoic acid by ultraviolet spectrophotometry, mass spectrometry, and spectral and chromatographic comparison to known compounds. It is probable that 5,8-oxyretinoic acid was produced from 5,6-epoxyretinoic acid under the acidic conditions used in the isolation. It is highly probable, therefore, that the natural product is 5,6-epoxyretinoic acid.

Animals↗
PubMed1976

Ultraviolet spectrophotometric analysis of barbiturates. Current assessment as an emergency screening procedure.

Several laboratory methods are currently available for the detection of barbiturates, but few are suitable for emergency use, The authors evaluated use of an ultraviolet spectrophotometric method as a screening procedure and compared the results with results obtained by other barbiturate methods (colorimetric, gas-liquid chromatographic, and immunochemical), Procedure time was approximately 40 minutes, sensitivity 0.2 mg/dl, precision (coefficient of variation) 5.4%, recovery 97.8% for phenobarbital, and there was good correlation with results obtained by gas-liquid chromatography and the EMIT system for phenobarbital. The authors recommend ultraviolet spectrophotometry as an excellent screening procedure for assessing the severity of barbiturate intoxication.

Barbiturates↗
PubMed1975

Cyclization of uridine monophosphate by diethyl pyrocarbonate.

Reaction between diethyl pyrocarbonate and uridine 2'-phosphate or uridine 3'-phosphate leads to the formation in high yields of uridine 2':3'-cyclic phosphate. This reaction product was identified in experiments involving (a) ultraviolet spectrophotometry, (b) paper chromatography, (c) high voltage paper electrophoresis at both pH 3.5 and 7.4, (d) acid hydrolysis, and (e) digestion with pancreatic ribonuclease.

Alkaline Phosphatase↗
PubMed1979

Monitoring of serum theophylline concentrations by a fully mechanized enzyme immunoassay (EMIT).

An evaluation of an enzyme immunoassay for the determination of theophylline in serum (EMIT, Syva Corp.) is presented. The EMIT assay was fully mechanized by the use of an Eppendorf analyzer 5010. The precision of this enzyme immunoassay was satisfactory (coefficients of variation from day to day ranged from 5--6%). The EMIT assay proved to be specific, rapid and easy to perform. A comparison of the results obtained by EMIT and ultraviolet spectrophotometry in a series of 100 patients showed a good correlation between both methods.

Cross Reactions↗
PubMed1975

[Biological and physicochemical properties of the alpha- and beta-forms of cephaloridine].

Alfa- and delta forms of cephalosporin were compared. Biological activity, lantimicrobial spectrum and innocuousness of the forms and their physico-chemical properties were investigated with the methods of infrared and ultraviolet spectrophotometry and derivatography. No significant differences in the biological properties of alfa- and delta forms of cephalosporin were found. However, the ingrared spectrum of the delta-form was indicative of higher stability of its molecule, which was confirmed by higher thermostability of delta-cephalosporin.

Animals↗
PubMed1976

Collaborative study of a column chromatographic method for bendroflumethizide and cyclothiazide.

Bendroflumethiazide and cyclothiazide are eluted from a sodium carbonate column with chloroform-acetic acid (98+2) and are measured directly by ultraviolet spectrophotometry. The method was collaboratively studied by 8 analysts. The average per cent recovery and standard deviations for simulated mixes of bendroflumethiazide and cyclothiazide were 99.61+/-0.78 and 99.3+/-1.97. The method has been adopted as official first action for the determination of bendroflumethiazide.

Bendroflumethiazide↗
PubMed1992

[Studies and preparations of fufang danshen granules].

With the preparation of borneol-beta-cyclodextrin inclusion compound, the stability of the product was improved. In the studies on quality standards, TLC method was used in the identification of three medicinal ingredients and TLC-ultraviolet spectrophotometry was used in the content determination of the chemical constituent tanshinone II A in Salvia miltiorrhiza.

Abietanes↗
PubMed1977

The determination of anticonvulsants in biological samples by use of high-pressure liquid chromatography.

The procedure for anticonvulsant analysis represent use of a relatively new technology. The procedures compare favorably in accuracy and precision with accepted methodology using gas chromatography [14]. It is possible to determine a greater number of anticonvulsants simultaneously with liquid chromatography than with reported gas-chromatographic procedures. This is because gas chromatography necessitates manipulation of the sample to be analyzed, the various components of which may have completely different manipulation requirements. Liquid chromatography is able to effect the analysis without extensive manipulation of the sample. The columns recommended in Sect. III are currently available standard columns. Column technology is developing at an accelerating rate. It is now anticipated that new developments in microparticulate packings and improvements in packing techniques will give the analyst greater column efficiency. This may be used either to shorten overall analysis time or to include more drugs in a simultaneous analysis. There is not yet available to the analyst a variety of different detectors suitable for routine use. This situation will change. In the case of the successful use of ultraviolet spectrophotometric detectors, the technique is not being fully exploited. Ultraviolet spectrophotometry is firmly established in practice, but as yet it is not routine to apply dual-wavelength spectroscopy to the column effluent. Application of this technique may be expected to improve selectivity and quantitation besides permitting optical resolution of some components that are not separated by the column. In the procedures given in this chapter there was no detailed discussion on gradient elution. This technique is potentially of great utility for simultaneous analyses of many compounds. It should be used only where isocratic elution is out of the question because it calls for somewhat stricter requirements of equipment in control of chromatography conditions. An additional time for equilibration between analyses may be considered too high a price to pay for any advantage gradient elution may give the analyst. The problem of fully automatic operation of liquid chromatographs for the type of analysis discussed here remains to be solved. Automatic operation would advance the use of liquid chromatography in the clinical laboratory probably more than any other single improvement. However, regardless of refinements of this type, liquid chromatography in its modern manifestation has versatility and sensitivity in abundance for analyzing compounds of toxicological and clinical interest. It will therefore assume greater importance in these areas.

Anticonvulsants↗
PubMed1979

Ultraviolet spectrophotometric determination of benzoic acid in soy sauce.

Proteins and other interfering substances are precipitated from soy sauce, using sodium tungstate under acidic conditions. After centrifugation, the supernate is successively extracted with ethyl ether to isolate the benzoic acid in the organic solvent. The ethyl ether extract is washed with dilute HCl. Benzoic acid is then quantitatively determined by ultraviolet spectrophotometry. Recovery of sodium benzoate added to soy sauce ranged from 94 to 104%.

Benzoates↗
PubMed1978

Determination of drugs in plasma by high-performance thin-layer chromatography.

High-performance thin-layer chromatography was used to determine chlorpromazine, amitriptyline, nortriptyline, imipramine, desipramine, phenobarbital, and phenytoin in plasma, to demonstrate the utility of this technique for routine analysis. We quantitated the separated components by use of ultraviolet reflectance spectrometry with detection limits as low as 1 microgram/liter. Regressions of psychoactive agents extracted from plasma were linear over the range of 0 to 300 microgram/liter. The anti-convulsant drugs, phenobarbital and phenytoin, were determined over a range of 0 to 50 mg/liter. Analyses were rapid, reproducible, and well-suited to large-scale programs. Separated components also can be identified in situ by ultraviolet spectrophotometry.

Amitriptyline↗
PubMed1977

Measurement of plasma contrast levels during excretory urography.

Ultraviolet spectrophotometry permits easy measurement of plasma levels of intravenously injected contrast media. The initial results are described from the determination of plasma methylglucamine iothalamate (Conray 60) levels during routine excretory urography in 10 normal patients. This simple procedure has many potential applications in the investigation of contrast media physiology.

Humans↗
PubMed1976

Radioimmunoassay, enzyme immunoassay, spectrophotometry, and gas-liquid chromatography compared for determination of phenobarbital and diphenylhydantoin.

Sera from epileptic patients were assayed for phenobarbital and diphenylhydantoin by four different analytical procedures. Quantitative results obtained by radioimmunoassay (I) and enzyme immunoassay (II) were compared to each other and to the results obtained on aliquots of the same sample by gas-liquid chromatography (III) and ultraviolet spectrophotometry (IV). For phenobarbital the correlation coefficients were I vs. II, 0.909; I vs. III, 0.947; II vs. III, 0.917; I vs. IV, 0.950; II vs. IV, 0.953. For diphenylhydantoin the correlation coefficients were I vs. II, 0.953; I vs. III, 0.951; II vs. III, 0.957; I vs. IV, 0.862; II vs. IV, 0.898. The immunoassays can be substituted for liquid chromatography or ultraviolet spectrophotometry without changing the resulting clinical interpretations.

Chromatography, Gas↗