Correction for relative interferences in a cholesterol determination

The detrimental influences of thiouracil, propylthiouracil, and bromide on the ferric perchlorate reaction for cholesterol have been previously reported (2, 3). On the basis of recovery studies it can be shown that their role in the reaction results in a relative rather than an absolute error of cholesterol measurement. Because a relative error causes a linear fanning effect on increasing concentrations of analyte which depends for its slope on both the concentration of the interference as well as on the concentration of constituent being measured, the method of standard additions can be used to correct a matrix effect on the reaction. A system in which the standard is used internally can eliminate the need to pretreat the sample in order to separate the interfering compound from the analyte. Therefore, both the interference and its obviation by this method of internal standardization are discussed here in a procedure which results in a means for simpler rescue of a contaminated sample than is afforded by the more laborious and time consuming pretreatment of previous workers.     

A procedure for the direct determination of micromolar quantities of lecithin employing enzymes as reagents

A procedure has been proposed for the determination of micromolar concentrations of lecithin. This procedure, which utilizes enzymes as reagents, is relatively quick, simple, and inexpensive, and involves no extractions. Clinical studies of the applicability of this procedure for the determination of lecithin concentrations in amniotic fluid have, as yet, not been concluded.The synthesis of sodium 2-hydroxy-3, 5-dichlorobenzenesulfonate was described. This material in purified form has proven to be very convenient for routine use in a peroxidase-catalyzed coupling to 4-aminoantipyrine.     

The simultaneous determination of cysteine and cystine using N-bromosuccinimide: Application in an enzymatic system—cystine reductase

Summary A new, rapid, convenient, and accurate method for the simultaneous determination of cysteine and cystine has been developed for use in pure solutions as well as in the enzymatic system, cystine reductase. N-Bromosuccinimide is the only standard solution employed in these determinations. Cysteine was determined at p_H 7 by displacement of iodine from potassium iodide with N-bromosuccinimide. The total cysteine plus cystine was determined by N-bromosuccinimide using Bordeaux Red as an indicator. A comparative study to determine cysteine using N-bromosuccinimide and potassium iodate was performed.It has been found that the values obtained with potassium iodate in the enzymatic system are relatively high. N-Bromosuccinimide gives more accurate results as shown by determining the residual amount of cystine after a particular time of incubation. When N-bromosuccinimide is used as a titrant for cysteine and/or cystine in pure solutions the error is less than ±1%. When N-bromosuccinimide is used as a titrant in the enzymatic system for the determination of cystine, the method is more precise and more accurate when compared to the use of potassium iodate.

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Zusammenfassung Ein Verfahren zur gleichzeitigen Bestimmung von Cystein und Cystin sowohl in reinen Lösungen wie im enzymatischen System der Cystinreduktase wurde ausgearbeitet. N-Bromsuccinimid (I) dient als einzige Standardlösung für solche Bestimmungen. Cystein wurde bei p_H 7 auf der Basis der Jodfreisetzung aus Kaliumjodid mit I.bestimmt. Die Gesamtmenge Cystein + Cystin wurde mit I gegen Bordeaux-Rot als Indikator titriert.Vergleichsweise wurde die Cysteinbestimmung mit I und mit Kaliumjodat durchgeführt. Im enzymatischen System ergaben sich mit Jodat relativ hohe Werte. I gibt genauere Resultate, wie sich durch Bestimmung der Restmenge Cystin nach einer bestimmten Inkubationszeit zeigen läßt. Wird I als Maßlösung zu Cystein und/oder Cystin in reinen Lösungen verwendet, so ist der Fehler kleiner als ±1 %. Verwendet man I zur Titration von Cystin im enzymatischen System, so erzielt man größere Genauigkeit als mit Kaliumjodat.

     

Bilirubin and hemoglobin interferences in direct colorimetric cholesterol reactions using enzyme reagents

The interferences of bilirubin and hemoglobin were tested in two cholesterol procedures in which enzymes were used as chemical reagents. Both procedures used similar approaches with cholesterol esterase to free cholesterol from its esters and cholesterol oxidase to generate hydrogen peroxide from the total free cholesterol resulting. From that common start, one procedure then used catalase to generate formaldehyde from methanol and the peroxide produced from cholesterol, and the formaldehyde was then reacted with acetylacetone to produce a yellow chromogen, while the other procedure used peroxidase to catalyze a reaction directly between peroxide and 4-aminoantipyrine plus phenol to generate a pink chromogen. Bilirubin and hemoglobin were shown to produce some interference by reacting competitively with peroxide in both systems and by contributing residual absorbance at the wavelengths of measurement of each of the chromogens. Since bilirubin showed a spectral change, static blanking with sample blanks caused overcorrections. However, the elimination of a sample blank for either procedure could result in a favorable compensating error because the residual color of bilirubin could substitute in part at least for the lost reactivity of the peroxide used up in reaction with the bilirubin of the sample.     

Spectrophotometric study of both the Zimmermann reaction and the application of a corrective measure for irrelevant absorption

A spectrophotometric study of the Zimmermann reaction carried out in several solvent matrices has been described. The advantages of an aqueous system over those containing pyridine are detailed. Purification devices such as batch chromatography and color extraction used commonly in 17KS procedures have been examined, and the results obtained are shown as evidence that they are not always effective if the actions and reactions of the interference mimic those of 17KS. Finally, the study of mathematical corrections for irrelevant absorption resulted in values that indicated that the concept of such corrective action was not always easily applicable to the determination.     

Polarographic investigation of the Zimmermann reaction mechanism

Polarographic examination of the Zimmermann reaction adds further experimental evidence to the proposed theory of methylene group attack to the para position of m-dinitrobenzene. It also supports the theory that nitro anion formation can occur at high concentrations of 3β-hydroxyandrost-5-en-17-one compared to m-dinitrobenzene. Furthermore, polarographic analysis shows that the purple chromogen measured in the determination of steroids by the Zimmermann reaction does not contain a nitro anion.     

Spectrophotometric study of influences on the direct ferric perchlorate method for the determination of serum cholesterol

A spectrophotometric study of several influences on a direct colorimetric determination of serum cholesterol has been described. Interferences of in vitro and in vivo types were considered, and it was found that certain compounds such as bromide, uracils, and bilirubin could exert both positive and negative interfering influences on the reaction of ferric perchlorate with cholesterol in an ethyl acetate-ethanol-sulfuric acid medium. Some interferences such as bilirubin are noncompeting side reactions which are absolute in their interference capabilities, while others such as the uracils and bromide have an entirely different influence. In the latter circumstance, the fine structures of spectra are altered by a nonadditive phenomenon of the reactant and hyperchromic (bromide), and hypochromic (uracils) effects take place which appear to result in relative rather than absolute errors.