Glutathione reductase assay

Darby, W. J. (1972) Application of the erythrocyte glutathione reductase assay in evaluating riboflavin nutritional status in a high school student population. Am. J. Clin. Nutr. 

Saubcriich, H. E., Judd, J. H., Nichoalds, C. F.., Broquist, H. P, and Darby, W. J, (1972). Application of the erythrocyte glutathione reductase assay in evaluating riboflavin status in a high school student population. Am. /. Cfjri. Nutr, 25, 756-762. 

Although methods have been devised for the measurement of GSH utilization by the enzyme, and of H202 consumption, these are difficult and have now largely been replaced by an assay based on that of Paglia and Valentine (1967) in which the product GSSG is used to drive the oxidation of NADPH+H+ catalysed by glutathione reductase ... 

The three enzymes are quite specific for their respective pyridine nucleotide substrates. Under conditions normally used for assay, lipoamide dehydrogenase is less than % as active with NADPH as with NADH IS) and thioredoxin reductase is less than 1% as active with NADH as with NADPH 36, Sff). Lipoamide dehydrogenase can transfer electrons to a number of NAD analogs 37). Yeast glutathione reductase is quite specific for NADPH 60), but the erythrocyte enzyme is 20% as active with NADH as with NADPH under the conditions of the standard assay 30,40,61). 

Flavins are lost from the body as intael riboflavin, rather than as a breakdown product of riboflavin. Hence, vitamin status may be assessed by measuring the level of urinary riboflavin. Generally, the loss of 30 ig of riboflavin/g creatinine or less per day indicates a deficiency. This metht>d of assessment is not preferred because it is influenced by a number of factors unrelated to vitamin status. Another problem with this method is its great sensitivity to a short-term deficiency thus, it does not necessarily reflect the true concentrations of FAD and FMN in tissues. The most reliable way to assess riboflavin status is by a functional test. The test involves the assay of glutathione reductase, using red blood cells as the source of... 

Glutathione is discussed further in the section on selenium and glutathione in Chapter 10. The enzyme assay is conducted using glutathione reductase extracted from red blood cells with and without added FAD. Chmnic consumption of a diet deficient in riboflavin allows the continued synthesis of a variety of flavoproteins, but results in the accumulation of apoenzyme without its conversion to holoen-zyme. Addition of chemically pure FAD to a biological fluid containing apoenzyme results In the stimulation of enzyme activity because of the formation of the holoenzyme. It is this stimulation of enzyme activity that is used to determine vitamin status in humans. 

The most reliable way to assess riboAavin status is by a funcAonal test. The test involves the assay of glutathione reductase, using red blood cells as the source of... 

Riboflavin status is assessed by (1) determination of urine riboflavin excretion, (2) a functional assay using the activation coefficient of stimulation of the enzyme glutathione reductase by FAD, or (3) direct measurement of riboflavin or its metabolites in plasma or erythrocytes. The advantages and disadvantages of functional or direct methods have been discussed in the section on thiamine. 

Glutathione peroxidases (Se-dependent and -independent enzymes) activities toward cumene hydroperoxide and H2O2 as substrates were determined by the coupled enzyme method of Lawrence and Burk (1976) with minor modifications. The standard assay contained 100 mM sodium phosphate buffer (pH 7.5), 1 mM EDTA, 0.12 mM NADPH, 1 mM NaN3 (for H2O2 assay), 2 mM GSH, 0.8 mM cumene hydroperoxide or 0.4 mM H2O2, 1 unit glutathione reductase and sample. The oxidation of NADPH was followed at 340 nm e — —6.22 mM cm ). 

In the GST assay, to the supernatant fluid, a cocktail containing 0.1-M potassium phosphate (pH 6.5), 30-mM l-chloro-2,4-dinitrobenzene and 20-mM glutathione are added and measured immediately at 340 nm for 4 min [14]. In the GPX assay, to the supernatant fluid, a cocktail of 0.1-mM potassium phosphate buffer (pH 7), glutathione reductase (4ng/pL hnal), 10-mM glutathione, 30-mM EDTA, and 1-mM NADPH is added. The reaction is initated by adding cumene hydroperoxide and measured immediately at 340nm for 3 min. 

Because of substrate instability it is impossible to perform the assay at temperatures higher than 30°. The protein can also utilize glutathione, glutathione reductase, and NADPH to reduce disulfide substrates, and this demonstrates the thioltransferase activity of the enzyme. The maximal rate of activity with different concentrations of GSH and L-cystine is obtained with 10 vaM GSH and 2.5 mM L-cystine higher concentrations led to inhibition. The thioltransferase activity of the P. furiosus protein shows a linear dependence on the protein concentration up to 0.12 tiM, and inhibition is detected at higher protein concentrations. 

Fig. lAy IB. Percentage changes in the specific activity of ascorbate peroxidasey onodehydroascorbate reductase, and glutathione reductase in leaf extracts from chill-exposed Z. diploperennis and /. mays assayed at 25 C. 

A more specific type of chemical assay is based on enzymatic measurement of vitamin co-enzyme activity. This approach is designed to detect a vitamin deficiency in tissues, and is only feasible for those vitamins that serve as co-enzymes. For instance, thiamin depletion in a subject can be diagnosed by measuring the transketolase activity in red blood cells with and without the addition of thiamin pyrophosphate (TPP) in vitro. If TPP increases the activity by more than a given amount, thiamin deficiency is indicated. Similarly, a subnormal level of riboflavin is indicated in tissues if the activity of erythrocyte glutathione reductase is increased after the addition of flavin adenine dinucleotide (FAD). Erythrocyte transaminase activation by pyridoxal-5 -phosphate (PLP) can be measured to establish a deficiency of vitamin B . 

When rats are administered with bacoside A (10 mg/kg), levels of glutathimie, vitamin C, vitamin E, and vitamin A were reduced. The activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase were also assayed. Copper, iron, zinc, and selenium levels in brain and serum ceruloplasntin activity were also measured. Administration of bacoside A improved the antioxidant status and maintained the levels of trace elements [16]. 

The coenzyme activity of Alg-NADP was determined enzymatically by the glutathione reductase system (38). The assay mixture consisted of 250 pmol Tris-HCl buffer (pH 8.0), 2.7 [xmol EDTA, 13.2 pmol GSSG, an appropriate amount of Alg-NADP", and 20 units of GR in a total volume of 2.87 ml. The reaction was started by the addition of the enzyme and the reaction mixture was incubated at 30°C. Absorbance at 340 nm was measured with a double beam spectrophotometer. The reference contained all components except for Alg-NADP. In the calculation of the amount of Alg-NADPH produced, a molar absorption coefficient for NADPH of 6.22 X 10 l-mol -cm was used. 

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