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Letters |
Randox Laboratories Ltd., Ardmore, Diamond Rd., Crumlin BT29 4QY N. Ireland, UK
a Author for correspondence.
To the Editor:
Schofield and Braganza recently questioned [1] the accuracy and suitability of a Total Antioxidant Status (TAS) kit (Randox Labs., Crumlin, UK) for measuring total antioxidant capacity. They described data produced under reaction conditions not recommended by the manufacturer and reached incorrect conclusions based on the data obtained.
The changes in calculated TAS with reading time demonstrated by Schofield and Braganza (1) are supported by our own studies and are the reason why a 3-min time to reading is recommended in all analyzer applications. Observations made at alternative reading times are therefore not relevant when a standardized 3-min reading time is adhered to. Dilution of samples, which results in an increase in TAS, although not ideal, in practice is not necessary, given that we have never obtained a serum or plasma TAS value >2.5 mmol/L (linear limit of method). The lower recovery obtained by the authors is not in keeping with our own recovery studies, which have consistently achieved recoveries of >95%.
In all diagnostic tests, precise assay conditions must be selected and adhered to; with the availability of automated analyzers, these conditions can be easily achieved. The Randox TAS kit, therefore, allows many laboratories to perform TAS analyses under the same analytical conditions, leading to valid comparison of results between research groups.
Our data support the findings of Schofield and Braganza of different reaction kinetics for ascorbate, uric acid, and albumin. We believe, however, that the authors' key requirement that all antioxidants contribute to the total antioxidant capacity in an analogous manner is neither realistic nor necessary; antioxidants do not have the same reaction kinetics in vivo and may also interact with one another. It may be more meaningful, therefore, to measure the total antioxidants, given that the antioxidant system is composed of a number of elements that exert their actions in different ways (2)(3). Thus, the TAS of a sample is a quantitative measurement of the state of balance of these various components under specified reaction conditions.
Our own studies have demonstrated that TAS can be correlated with
biochemical analytes other than ascorbate, uric acid, and albumin.
Although the highest correlations were obtained with albumin and uric
acid, correlations between TAS and other analytes, including bilirubin,
creatinine, and glutathione reductase, were also significant
(P <0.05). A study with >200 individual subjects showed
significant correlations between TAS and several different analytes
(Table 1
). These results suggest that TAS represents the total
contribution from a wide range of components. Indeed, the total
antioxidant status of these subjects cannot be accounted for by the
main antioxidants uric acid and albumin. The combined antioxidant
activity of uric acid and albumin accounts for only 51% of the total,
the remaining activity being a measure of the combined activity of
other plasma antioxidants.
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Two separate vitamin supplementation studies we carried out (unpublished) with volunteers taking daily one tablet containing selenium (10 µg), vitamin A (450 µg), vitamin C (100 mg), vitamin E (91 mg), and bioflavonoids (50 mg) also suggest that antioxidants other than uric acid and albumin contribute to TAS. In the first study, involving 22 subjects, blood samples were taken before supplementation and at 28 days after starting supplementation. In the second study, 16 subjects took supplements for 120 days, and blood samples were taken before supplementation and at 60 and 120 days after. After 28 days in the first study, TAS increased significantly (P <0.005), to 1.60 mmol/L from the mean presupplementation concentration of 1.48 mmol/L. In the second study, we observed a significant increase (P <0.039) in TAS after 60 days, the mean TAS concentration being 1.62 mmol/L after supplementation compared with 1.56 mmol/L before supplementation.
We do not question the need to measure individual antioxidants when their identity is known, but we believe that measuring TAS can provide information on an individual's overall antioxidant status, which may include those antioxidants not yet recognized or not easily measured.
References
Pancreato-Biliary Service, Manchester Royal Infirmary, Oxford Rd., Manchester M13 9WL, UK
a Author for correspondence.
To the Editor:
We note the support of the authors from Randox Labs. for our findings (1) regarding the variation in calculated plasma total antioxidant capacity with run time, and the effect of dilution of samples, when determined with their kit for Total Antioxidant Status (TAS).
Our study was performed entirely in accordance with the application notes supplied by Randox Labs. for the Cobas Bio automated analyzer, with a single modification, namely, to monitor the individual cuvette absorbances at 10-s intervals so as to follow the kinetics of the reaction, rather than simply rely on the instrument to calculate plasma TAS at 3 min.
In view of our findings of variation of measured TAS with run time (1), as well as earlier reports that the ABTS method is strongly influenced by temperature (2), clearly a precise analytical protocol would have to be defined to permit valid comparison of results between laboratories. Nevertheless, the fact remains that the specified run time of 3 min appears arbitrary, which, together with the increase in apparent TAS observed on dilution of plasma, must cast doubt on the accuracy of any data obtained through use of the kit.
As is well recognized in assessing the contribution of individual antioxidants to the total antioxidant activity of a physiological sample, the observed "hierarchy" of those antioxidants depends on the specific chemical method used for determining that activity (3). For example, in this study ascorbic acid is a potent antioxidant, but in the ox-brain homogenate model (4) it acts as a prooxidant instead. The evidence from the performance of this kit is that even slight changes in reaction conditions have marked effects on the apparent contributions of individual antioxidants, notably albumin; therefore, any inference regarding possible contributions to total antioxidant activity in vivo must be drawn with even greater caution.
The presence of significant correlations between concentrations
of some individual plasma constituents and TAS does not imply that a
causal relationship exists, because both may be similarly influenced by
further factors. This may explain the apparent paradox that glucose and
creatinine are reported to be unreactive at concentrations as great as
10 mmol/L in the original methodology (5) on which this
kit is based. Furthermore, straightforward calculations show that
-glutamyltransferase, which is present at submicromolar
concentrations in plasma, would require a Trolox Equivalent Antioxidant
Capacity value in excess of 100 to make even a 1% contribution to TAS.
Our interest in investigating the performance of the kit was to augment the range of measurements of individual antioxidants we currently carry out to ensure both the efficacy and safety of micronutrient antioxidant supplementation in pancreatitis (6). The vitamin supplementation data reported above by Lamont et al. reinforce our reservations regarding the usefulness of the kit in monitoring antioxidant therapy in specific patients in the clinical situation. Their 28-day study shows a mean increase in TAS of only 8%, whereas the increase in the longer-term investigation is <4%. In view of the interassay CV of 2.4%, quoted in the promotional literature from Randox Labs., such an increase is unlikely to be detected in individual patients—indeed, we note that the increase in the longer-term study is smaller than the difference in mean baseline TAS between the two groups of volunteers! Furthermore, the presence of a normal or even a high TAS value in a jaundiced and (or) uremic patient could hardly be taken as being reassuring, and may be frankly misleading. We thus reiterate our recommendation that the monitoring of individual antioxidants in physiological samples remains essential.
References
,
a Author for correspondence.
To the Editor:
A standardized protocol is easily achieved with modern automated analyzers: Use of a specific run time should not "cast doubt on" the accuracy of the data. Moreover, the choice of a 3-min run time was not arbitrary; rather, this timing produced absorbances in the linear range of most analyzers, could be used by many analyzers, and minimized assay time. Dilution of plasma is required only when TAS exceeds 2.5 mmol/L, a value we have never found in practice.
The change in apparent TAS upon sample dilution reflects the effect on TAS of multiple components, some of which (e.g., uric acid, albumin, bilirubin) are clearly part of the antioxidant system and others (e.g., glutathione reductase) that are associated with the system but are not antioxidants. Although in certain pathological conditions a normal or increased TAS may be observed, monitoring TAS provides information in some clinical conditions (e.g., ()).
Given that not all components of the antioxidant system (or their interactions) are known, a method that measures the contributions of a variety of antioxidants (e.g., TAS) is an attractive alternative to measuring the values for a range of individual antioxidants.
References
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