improved antioxidant essay

8/9/2019 Improved Antioxidant Essay

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INDIAN J. BIOCHEM. BIOPHYS., VOL. 46, FEBRUARY 2009128

correlation (ICC) between the two methods was 0.82,again suggesting a good correlation. Individual valuesfor 30 samples analyzed by the two methods are givenin Table 1. The intra and inter assay coefficient ofvariation was 2.5% and 3.95% by the original kit

method, whereas it was 2.98% and 4.19%respectively by microplate method.For the study on total antioxidant levels in

industrial workers, 161 subjects were recruited, ofwhich 84 were males and 77 females. Among them,44 males and 37 females were of < 40 yrs of age,

whereas 40 males and 40 females were of age ≥ 40yrs. The mean total antioxidant status in age category< 40 yrs was 0.987 ± 0.175 and 0.855 ± 0.187 mmol/lfor males and females, respectively. The observeddifference was statistically significant (p<0.001). In

the age group ≥ 40 yrs, the mean antioxidant was0.895 ± 0.25 and 0.996 ± 0.227 mmol/l for males andfemales, respectively. However, the difference wasnot statistically significant (p = 0.137) (Fig. 2).

Microplate assays are commonly used in themicrobiological assays and in food, drug and pharmaceutical industries, because of low sampleavailability. The assay provides the advantages of

reducing labor time, material cost and sample volume.Some of the assays have already been adapted formore convenient mass screening8,9, quantitativespectrophotometer assays10 as well as in agricultureand food industry11. A number of assays have beendescribed for estimation of total antioxidant status inserum, which are based on either of the techniques —spectrophotometric, fluorimetric or chemilumine-scence. Although adaptation of antioxidant assay onmicroplate has been reported and commercialized byCAYMAN, the method does not incorporate blankingof individual samples, which could result in

imprecision. Our method has a provision of blankingfor each and every set of samples analyzed.

The incubation time is very critical for theantioxidant assay and our microplate assay does notdeviate from the original protocol in terms ofincubation time. The microplate adaptation has also been reported by Wong et al12. The authors reported ahigh intra (4.3%) and inter (14.1%) assay. Theanalysis has been carried out with 2.5 µl serum bythese authors which could have resulted in the highvariation. The intra and inter assay coefficient of

Table 1—Total antioxidant status of samples (30) analyzed byRANDOX and microplate method

Serial no RANDOX method(mmol/l)

Microplate method(mmol/l)

1 0.676 0.772

2 0.734 0.743

3 0.792 0.861

4 0.871 0.842

5 0.800 0.711

6 0.705 0.801

7 0.672 0.651

8 0.600 0.563

9 0.631 0.744

10 0.722 0.715

11 0.681 0.782

12 0.753 0.843

13 0.782 0.851

14 0.813 0.785

15 0.870 0.860

16 0.700 0.730

17 0.672 0.653

18 0.740 0.730

19 0.682 0.662

20 0.733 0.741

21 0.874 0.841

22 0.601 0.562

23 0.722 0.713

24 0.630 0.744

25 0.675 0.77126 0.840 0.830

27 0.633 0.742

28 0.671 0.652

29 0.722 0.714

30 0.801 0.712

Fig. 2 ⎯ Distribution of total antioxidant levels in <40 years

(n = 44 males, 37 females) and ≥ 40 years (n = 40 males,40 females) age group



SHORT COMMUNICATION 129

variation reported in our method compares well withthe original method.

The microplate method offers advantage over the

original protocol method. It reduces the assay timesignificantly. Sixty samples can be analyzed by thismethod in an hour as against only 18 samples by theoriginal method. Reduction of cost to one-fourthmaking it more amenable for research, is anotheradvantage that the plate method offers. The high cost per sample of the original RANDOX method makes itless amenable to widespread use.

Very little information on total antioxidant status isavailable from Indian population. Total antioxidantstatus in an industrial population of Baroda wasreported by Desai et al13 using ABTS method. Thereported mean total antioxidants were 1.8 ± 0.2mmol/l in healthy individuals. The mean valuesreported by our method are much lower, but since wehave not evaluated the micronutrient intake in oursubjects, we cannot comment on the values.

In conclusion, microplate adaptation of the ABTSmethod described here is cost-effective and would beuseful in epidemiological studies, where largenumbers of samples are to be analyzed.

References1 Wayner D D M, Burton G W, Ingold K U & Locke S (1985)

FEBS Lett 187, 33-372 Benzie I F F & Strain J J (1996) Anal Biochem 239, 70-76

3

Glazer A N (1990) Methods Enzymol 186, 161-1684 Lewis S E, Boyle P M, Mckinney K A, Young I S &

Thompson W (1995) Fertil Steril 64, 868-8705 Cao G & Prior R L (1998) Clin Chem 44, 1309-13156 Miller N J, Rice-Evans C, Davies M J, Gopinathan V &

Milner A (1993) Clin Sci 84, 407-4127 Reddy K S, Prabhakaran D, Chaturvedi V, Jeemon P,

Thankappan K R, Ramakrishnan L, Mohan B V M, PandavC S, Ahmed F U, Joshi P P, Meera R, Amin R B, Ahuja R C,Das M S & Jaison T M on behalf of the Sentinel SurveillanceSystem for Indian Industrial Population Study Group (2006)

Bull World Hlth Organ 84, 461-4698 Akizawa T, Uratani T, Matsukawa M, Kunimatsu A, Ito Y,

Itoh M, Oshiba Y, Yamada M & Seiki M (1999) Ann N Y

Acad Sci 878, 622-624

9

Gerasimova N S, Steldova I V & Tuuminen T (1989) ClinChem 35, 2112-2115

10 Erickson J A, Cousin R, Wu J T, & Ashwood E R (2003) Clin Chem 49, 970-972

11 Vermeir S, Nicolai B M, Jans K, Maes G & Lammertyn J(2007) J Agric Food Chem 55, 3240-3248

12 Wong W R, Stephens J W, Acharya J, Hurel H J, HumphriesS E & Talmud P J (2004) J Lipid Res 45, 1565-1571

13 Desai S A, Mani U V & Iyer U M (2002) Int J Diab Dev

Ctries 22, 91-99

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