Publisher Summary This chapter discusses selected methods for the determination of ascorbic acid in animal cells, tissues, and fluids. Methods for determining ascorbic acid are numerous. In general, chemical analyses for the vitamin are divided into two groups; the determination of the reduced form and the determination of the oxidized form. The former group of analyses is usually based upon the oxidation–reduction properties of ascorbic acid. These are widely used as the fundamental reactions in the measurement of vitamin C. The latter group of analyses is usually based upon the oxidation of the ascorbic acid and the subsequent formation of a hydrazone or a fluorophore. Best results are obtained if samples, especially plasma, are quickly stabilized with either trichloroacetic acid or metaphosphoric acid and immediately analyzed. Prompt stabilization is especially important in the case of plasma or serum. The greater stability of ascorbic acid in acid solution is because of the decreased tendency for the hydrolysis of the lactone ring with decreasing pH.

Selected methods for the determination of ascorbic acid in animal cells, tissues, and fluids

An automated kinetic method to determine creatinine in serum with the Jaffe reaction is described. The method does not require deproteinization or adsorption of creatinine on Fuller9s earth or ion-exchange resin. A maximum of 29 serum samples, standards, and control sera can be assayed in less than 1.5 min. Interferences by most of the known Jaffe-positive serum constituents were studied and minimized by optimal selection of the time interval during which the reaction rate was measured. Results of precision studies and correlation with a manual method requiring adsorption of creatinine on ion-exchange resin are reported. Spectroscopic data suggest that the product of reaction between picrate and creatinine is a 1:1 adduct of the two starting molecules.

Automated Reaction-Rate Method for Determination of Serum Creatinine with the CentrifiChem

(1965). Plasma Creatinine Determination A New and Specific Jaffe Reaction Method. Scandinavian Journal of Clinical and Laboratory Investigation: Vol. 17, No. 4, pp. 381-387.

Plasma Creatinine Determination A New and Specific Jaffe Reaction Method

Vitamin C (Ascorbic Acid), the antiscorbutic vitamin, cannot be synthesized by humans and other primates, and has to be obtained from diet. Ascorbic acid is an electron donor and acts as a cofactor for fifteen mammalian enzymes. Two sodium-dependent transporters are specific for ascorbic acid, and its oxidation product dehydroascorbic acid is transported by glucose transporters. Ascorbic acid is differentially accumulated by most tissues and body fluids. Plasma and tissue vitamin C concentrations are dependent on amount consumed, bioavailability, renal excretion, and utilization. To be biologically meaningful or to be clinically relevant, in vitro and in vivo studies of vitamin C actions have to take into account physiologic concentrations of the vitamin. In this paper, we review vitamin C physiology; the many phenomena involving vitamin C where new knowledge has accrued or where understanding remains limited; raise questions about the vitamin that remain to be answered; and explore lines of investigations that are likely to be fruitful.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/odi.12446

Vitamin C: the known and the unknown and Goldilocks.

Total chromogen, true, and AutoAnalyzer methods of measuring serum and urine creatinine by the Jaffe reaction were investigated. Some factors influencing this reaction were examined. These included wavelength, blank, linearity, and conditions of color development. Modifications of the three methods were made and their precision, recovery, and sample stability determined. The interference of ketones and glucose were measured. Finally, the values obtained by the three methods on the same samples of serum and urine were compared statistically.

Estimation of Creatinine by the Jaffe Reaction A Comparison of Three Methods

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The determination of creatinine in plasma or serum, and in urine; a critical examination.

Methods based on the ability of ascorbic acid to reduce the dye 2:6-dichlorophenolindophenol (indophenol) to a colourless compound (Tillmans, Hirsch & Reinshagen, 1928) have been widely used in the estimation of ascorbic acid. Many other substances, including thiol compounds, thiosulphates, hydrogen sulphide, polyphenols and reductones (Lugg, 1942; Harris & Mapson, 1947) also decolorize indophenol and may therefore interfere with the determination of ascorbic acid. Most of these interfering substances reduce indophenol more slowly than does ascorbic acid (Harris & Mapson, 1947). The presence of substances which act in this way has been demonstrated in whole serum (Bierring, Mathiessen & Nielsen, 1945), and also in extracts of various mammalian tissues, e.g. kidney (Hopkins & Slater, 1935), brain (Young & Mitolo, 1934), whole blood (Butler & Cushman, 1940) and urine (Evelyn, Malloy & Rosen, 1938; Scarborough & Stewart, 1937). Mann & Leone (1953) reported that about 85% of the indophenol-reducing activity of hog semen was due to the presence of ergothioneine. To increase the specificity of the indophenol method for the estimation of ascorbic acid Van Eekelen & Emmerie (1936) proposed the use of mercuric acetate to precipitate some of the interfering substances. An alternative method, based on the formnation of complexes of interfering substances and formaldehyde, was proposed by Lugg (1942). However, methods based on these principles have certain disadvantages, which are discussed below. In an attempt to devise a specific yet simple method for the determination of ascorbic acid in mammalian tissues we have found that p-chloromercuribenzoic acid successfully suppresses the interference due to thiol compounds. The results obtained by using this reagent in the determination of ascorbic acid in human plasma, erythrocytes and urine are presented. Preliminary conmunications of some of the results have already been given (Owen, Iggo & Horn, 1954; Owen & Iggo, 1955).

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The use of p-chloromercuribenzoic acid in the determination of ascorbic acid with 2:6-dichlorophenolindophenol

IN certain circumstances hydrogen sulphide, cysteine and related sulphydryl compounds react with 2 : 6-dichlorophenol-indophenol (indophenol) and thus may interfere with the determination of ascorbic acid using this reagent. In view of the known ability of p-chloromercuribenzoic acid to combine with sulphydryl compounds1, the possibility of using this reagent to suppress such interference has been examined.

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J. A. Owen

Papers

The determination of creatinine in plasma or serum, and in urine; a critical examination.

The use of p-chloromercuribenzoic acid in the determination of ascorbic acid with 2:6-dichlorophenolindophenol

Use of p-Chloromercuribenzoic Acid in the Determination of Ascorbic Acid in the Presence of Sulphydryl Compounds

The determination of vitamin C in normal human plasma and erythrocytes

Serum proteins in myelomatosis; a follow up study.