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Use of ORAC to assess antioxidant capacity of human milk
Journal of Food Composition and Analysis 22 (2009) 694–698
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Original Article
Use of ORAC to assess antioxidant capacity of human milk Alexandra Tijerina Sa´enz a, Ingrid Elisia a, Sheila M. Innis b, James K. Friel c, David D. Kitts a,* a
Food Nutrition and Health, University of British Columbia, Vancouver, B.C. V6T 1Z4, Canada Department of Paediatrics, University of British Columbia, 950 West 28th Avenue, Vancouver, B.C. V5Z 4H4, Canada c Department of Human Nutritional Sciences, University of Manitoba, H511 Duff Roblin Building, Winnipeg, MB R3T 2N2, Canada b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 July 2008 Received in revised form 20 January 2009 Accepted 26 January 2009
The oxygen radical absorbance capacity (ORAC–FL) assay, a useful measure of the antioxidant capacity (AC) reported for some biological samples, supplements, and food samples, was standardized and validated for measuring AC of human milk. Limits of linearity, precision and accuracy of the ORAC–FL assay were made by constructing a Trolox Calibrator which included the addition of human milk as the sample matrix. AC assay results indicated excellent linearity (R2 = 0.990 0.005), precision (2.2%) and accuracy on recovery (94.8 3.2%) over a wide range of Trolox concentrations. To validate the assay further, mature milk samples were collected from 100 lactating mothers in Vancouver and Winnipeg, and were measured for vitamin E isomers by HPLC and AC using the standardized ORAC–FL assay. Alpha-tocopherol (a-Toc) was the major vitamin E isomer detected in human milk, established using both ultra-violet and fluorescent detection methods. Milk a-Toc concentrations were found to correlate significantly (P < 0.01) with ORAC–FL assay values in milk obtained from mothers in both Vancouver (R = 0.439, n = 60) and Winnipeg (R = 0.408, n = 40). Although milk is a complex matrix with multiple components possessing potential antioxidant activity, our results indicated that the ORAC–FL assay is a very useful indicator for assessing the antioxidant capacity of human milk. ß 2009 Elsevier Inc. All rights reserved.
Keywords: Human milk Antioxidant capacity (AC) ORAC–FL assay Assay validation with human milk Complex food matrix analysis Food analysis Food composition
1. Introduction Determining the antioxidant capacity (AC) of human milk involves measuring the collective antioxidant activity of numerous components present in milk, and these provide oxidative stability and retard the initiation and propagation of reactive oxygen species that can culminate in the deterioration of milk quality. The complex free radical mechanisms involving oxygen, transition metal ions and oxidizing agents and the deficient antioxidant mechanism present in milk can lead to peroxidation reactions which in turn are the underlying cause for generation hydrolytic off-flavors and loss of important nutrients and bioactive agents that promote neonatal health (Zoeren-Grobben et al., 1993). Antioxidant mechanisms of milk components that involve the transfer of electrons to quench free radicals are based either on the ionization potential constant (e.g. single electron transfer (SET)), or on the dissociation energy constant of the antioxidant hydrogen donor group (e.g. hydrogen atom transfer (HAT)) (Mayer, 2004). Milk AC has been measured using the colorimetric ABTS reducing assay to evaluate the antioxidant content of milk from African women; this method models the SET reaction and yields data describing the affinity of milk constituents to reduce the radical
* Corresponding author. Tel.: +1 604 822 5560; fax: +1 604 822 5143. E-mail address: [email protected] (D.D. Kitts). 0889-1575/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jfca.2009.01.021
cation ABTS, measured in units of Trolox equivalent antioxidant capacity (TEAC) (VanderJagt et al., 2001). The same procedure has been used to evaluate the effect of storage on human milk antioxidant activity (Hanna et al., 2004). However, the TEAC procedure is limited by the fact that ABTS represents a nonphysiological radical and moreover, a compound can only reduce ABTS if it has a redox potential lower than that of ABTS (Phipps et al., 2007). Thus, although the TEAC procedure is relatively simple and rapid, and compatible with both organic and aqueous solvents, it may underestimate some antioxidant components that exhibit a relatively slow reaction mechanism (Arts et al., 2004; Phipps et al., 2007). On the other hand, the oxygen radical absorbance capacity (ORAC) assay is a widely used procedure for measuring the total antioxidant capacity of biological samples, supplements and food samples (Prior et al., 2003; Davalos et al., 2004; Wu et al., 2004; Ferna´ndez-Pacho´n et al., 2005). The radical initiator component of this assay is 2,20 -azobis (2 amidinopropane) dihydrochloride (AAPH), a water-soluble free radical initiator which spontaneously decomposes at 37 8C to form two carbon-centered radicals which react with oxygen to generate peroxyl radical, a common radical in human biology. Fluorescent probes used in this assay decompose in a pattern that is consistent with the HAT mechanism of action when exposed to peroxyl radical (Prior et al., 2005). The fluorescent probe b-phycoerythrin (b-PE) has been employed in the ORAC assay (ORACb-PE) to measure the antioxidant capacity of colostrum, transitional and mature human milk in one study with Italian
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women (Alberti-Fidanza et al., 2002); details of the standardization or validation of the assay with human milk have not been reported. The b-PE probe is also relatively photosensitive and subject to nonspecific protein binding which can lead to underestimation of antioxidant capacity. Alternatively, employing fluorescein (FL) as the fluorescent probe in the ORAC procedure (ORAC–FL) offers advantages due to greater sensitivity and photostability and the specificity for antioxidant activity against peroxyl radicals (Ou et al., 2001). The aim of this study was to standardize and validate the ORAC– FL assay for assessing the oxidative stability of human milk. Experiments were conducted to determine the limits of linearity and precision of the standard calibrator and to determine the accuracy of the method through recovery experiments with known concentrations of Trolox added to human milk. Human milk samples from mothers living in two different geographic locations and at a similar mature stage of lactation were also collected and assayed for AC using the ORAC–FL assay. The results were validated by correlating with the vitamin E content of milk measured by HPLC-UV and fluorescent detection. 2. Materials and methods 2.1. Chemicals Radical cation 2,20 -azinobis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), fluorescein sodium salt (FSS), peroxyl radical initiator 2,20 -azobis (2-amidinopropane) dihydrochloride (AAPH), vitamin E analogue Trolox, and tocopherol (Toc) standards (a-Toc; d-Toc; and g-Toc) were all purchased from Sigma–Aldrich (St. Louis, MO, USA). Phosphate buffer (PB) was prepared from solutions of 0.75 M K2HPO4: 0.75 M NaH2PO4 to 61:39 (v/v). PB was diluted to a final concentration of 75 mM (pH = 7). Working concentrations of FSS were diluted to a final concentration of 200 nM. AAPH was dissolved to a final concentration of 60 mM immediately before use. Trolox at 20 mM was used for the Trolox Calibrator standards and were made fresh prior to use. Tocopherol standards were prepared in ethanol, kept at 20 8C and made fresh every week. HPLC grade solvents included hexane, ethanol, methanol, pyrogallol were purchased from Fisher Scientific (Ontario, Canada). Pyrogallol was dissolved in ethanol to 12% prior to use. 2.2. Human milk for Trolox equivalent antioxidant capacity assay (TEAC) Initial standardization experiments to establish TEAC in human milk were performed from whole human milk (100 mL) collected from the British Columbia (B.C.) Women’s Milk Bank (Vancouver, B.C.). Milk was collected and pooled from 10 different healthy women that were all in a mature stage of lactation. Aliquots of milk sample were taken and stored frozen at 80 8C until used. The TEAC assay was used to characterize the free radical scavenging capacity of milk as an example of the SET mechanism. This procedure is based on a discoloration reaction involving ABTS radical cations that are generated by adding 88 mL of a 140 nM potassium persulphate solution to 5 mL of a 7 mM ABTS solution and incubated at room temperature under light control (VallsBelles et al., 2006). The blue-green species of the generated ABTS was detected at 734 nm in a 96-well assay plate using a microplate spectrophotometer (Multiskan Spectrum, ThemoLabsystem) (Hu et al., 2005). The capacity of antioxidants present in milk to scavenge the radical and inhibit the color production is proportional to the antioxidant capacity of the human milk sample, and is expressed as an equivalent of the micromole concentration of Trolox per milliliter of milk (mmol TE/mL milk).
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2.3. Standardization of oxygen radical absorbance capacity assay (ORAC–FL) 2.3.1. Human milk for standardization of ORAC–FL assay Subsamples of human milk collected from the BC Women’s Milk Bank (Vancouver, B.C.), previously described in Section 2.2, were taken and stored frozen at 80 8C until used for experiments on linearity, precision and accuracy of the assay. 2.3.2. The ORAC–FL assay The ORAC–FL assay was standardized for human milk based on the methodology described by Ou et al. (2001) and by Kitts and Hu (2005). This assay is based on the capacity of antioxidants in a sample to quench peroxyl radicals that are generated from the thermal decomposition of AAPH. Concentrations of Trolox standard (0.0–4.0 mM), a water-soluble vitamin E analogue, were used to construct the Trolox Calibrator. The ORAC–FL assays standardized for specific dilutions of milk were conducted in 96well black microplate assay plates (BioRad, Cat. No. 353241) incubated at 37 8C for all concentrations of Trolox Calibrator. AAPH was added to each well of the plate, except for the control and blank. The final volume of the assay was 200 mL. The microplate was shaken for 10 s, and fluorescence was read every minute for 60 min in a Fluoroskan Ascent FL (Labsystems) fluorometer at excitation of 485 nm and emission of 527 nm, respectively. Data integration and calculations were performed according to Davalos et al. (2004). Area under the curve (AUC) values were calculated following the formula: AUC ¼ 1 þ
t i ¼60min X
Ai =A0
t0 ¼60min
where A0 is the initial fluorescence reading at 0 min and Ai is the fluorescence reading at time i. Milk samples and Trolox Calibrator net AUC values were obtained by subtracting the AUC value of the blank from that of milk sample or Trolox Calibrator. Sample and Trolox Calibrator net AUC values were plotted versus concentration. Linear regression analyses were performed to obtain the slope of the regression equation. The ORAC–FL value of a milk sample was calculated by dividing the slope of the sample (mS) by the slope of Trolox Calibrator (mTC). The final ORAC–FL values for human milk were expressed as an equivalent of the micromole concentration of Trolox standard solution per milliliter of milk (mmol TE/mL milk). All analyses were performed in triplicate. 2.3.3. Linearity, precision and accuracy of the human milk ORAC–FL assay The linearity and precision of the ORAC–FL assay was calculated using ten independent runs of Trolox Calibrator based on the coefficient of determination (R2) and the coefficient of variation (CV), respectively. These data were obtained from the equations derived in linear regression analysis of Trolox Calibrator curves. The feasibility of the ORAC–FL assay to measure the AC of human milk was also observed by comparing the standard curve of Trolox Calibrator to the curve generated from the addition of human milk to Trolox Calibrator. A milk volume of 15 mL was added to all concentrations in the Trolox Calibrator. An increase in the AUC values would reflect the capacity of human milk to scavenge peroxyl radicals. In addition, known concentrations of Trolox standard were added to human milk to determine Trolox recovery and to establish the accuracy of the ORAC–FL assay for measuring the oxidative stability of milk. Concentrations of 0.5, 1.0, 2.0 and 3.0 mM Trolox were added to a milk volume of 10 mL to produce a Trolox spiked-milk sample. Milk samples were analyzed for antioxidant capacity (AC) following the procedure of the ORAC–FL assay as described above. Accuracy
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was determined by subtracting the net AUC value of the non-spiked milk sample (AUCNS) from the net AUC values of the spiked sample (AUCS). The resulting values corresponded to the experimental net AUC values of Trolox standard added to milk (AUCTE). Net AUCTE values were compared to net AUC values of Trolox Calibrator (AUCTC) and percentage of accuracy on recovery was calculated. Accuracy on recovery of Trolox standard was analyzed in triplicate. 2.4. Validation of ORAC–FL Assay 2.4.1. Human milk for validation of ORAC–FL assay Validation of the ORAC–FL assay on human milk was done by correlating milk AC values, established from ORAC–FL assay with the vitamin E content in the same milk sample. This experiment was achieved by collecting human milk samples from two distinct populations of lactating women from two major Canadian cities, namely Vancouver (n = 60) and Winnipeg (n = 40). All milk samples were collected from women at a mature stage of lactation. Milk was divided into subsamples for ORAC–FL testing and vitamin E analysis measured by HPLC, and these were stored frozen at 80 8C until used. 2.4.2. Vitamin E analysis For the vitamin E analysis, milk was pre-treated by fat extraction according to the procedure of Rodas Mendoza et al. (2003). Briefly, 12% pyrogallol was added to the samples and vortexed. Ethanol, and water, and hexane were added to the mixture and samples were centrifuged (2000 g for 10 min) before collecting the organic layer, and the extraction was repeated twice. The organic layer was evaporated to dryness under nitrogen gas at ambient temperature and reconstituted in methanol for HPLC analysis. Vitamin E isomers, namely alpha tocopherol (a-Toc), delta tocopherol (d-Toc), and gamma tocopherol (g-Toc) were quantified in human milk using an Agilent 1100 HPLC system (Agilent Technology 1100 series, Palo Alto, CA) equipped with a 3 mm Sphereclone column (150 mm 4.6 mm, Phenomenex, Torrance, CA) with both diode-array detector (DAD) and fluorescent detector. Vitamin E isomers were detected at 292 nm for a-Toc and at 298 nm for both d-Toc and g-Toc. The fluorescence detection was made at excitation and emission wavelengths of 292 and 330 nm, respectively. Recoveries of added tocopherol isomers in mature human milk were 99.7 1.9% for a-Toc, 100.5 2.3% for d-Toc and 100.1 2.2% for g-Toc. Quantitation of tocopherol isomers in human milk was achieved using a standard curve constructed with an external standard (a-Toc 2.5–50 mg/mL, dToc and g-Toc 0.5–25 mg/mL) for each isomer with linearity R2 > 0.990.
Fig. 1. Time-dependent changes in ABTS radical scavenging by milk when present in different amounts in the TEAC assay. Milk volumes added are: A = 0 mL, B = 0.05 mL, C = 0.1 mL; D = 0.15 mL; E = 0.2 mL to final volume of 100 mL. Values represent single measurements taken every minute over 60 min. Absorbance values were taken at 720 nm.
transformed data from the decay curve, and it shows the net area under the curve (AUC) and the concentration of Trolox standard. Results of regression analyses of multiple independent runs of Trolox Calibrator showed good assay linearity (R2 = 0.990) and precision (CV = 2.2%) of the ORAC–FL assay. A step-wise addition of a known volume of pooled human milk to Trolox Calibrator produced relatively higher net AUC values than those of Trolox Calibrator without milk added (Fig. 3). This result pointed to the antioxidant capacity of milk that produced the increase in net AUC values and the feasibility of the ORAC–FL assay to measure AC of human milk. Experimental net AUC values of Trolox added to mature human milk (AUCTE) were calculated from the difference of the average net AUC values of non-spiked milk sample (AUCNS = 13.7) from the net AUC values of the spiked sample (AUCS), as presented in Table 1. AUCTE values were compared to the net AUC values of Trolox Calibrator (AUCTC), and Trolox recovery was calculated. The accuracy of recovery of Trolox standard ranged from 91.7 to 97.9% with an average value of 94.8 3.2%. The major vitamin E isomer found in human milk from both city locations was a-Toc, which ranged in concentration from 0.66 to 5.02 mg/mL (average = 2.32 0.88 mg/mL) for Vancouver mothers
3. Results Fig. 1 shows the results of the TEAC assay where ABTS radical was scavenged by milk components over time. The addition of milk samples to the assay, ranging from 0.05 to 0.2 mL, produced a volume-dependent downward shift in absorbance values that were also time-dependent. This result illustrates the potential for inaccuracy of using fixed-time intervals for the TEAC assay for testing the antioxidant capacity of human milk samples. Different rates of absorbance decreases over time can be attributed to the quenching of ABTS radical by relative volumes of milk in the assay and the available reducing power required to reduce ABTS. A typical ORAC–FL decay curve for Trolox Calibrator over a range of Trolox standard concentrations is presented in Fig. 2. With increasing concentration of Trolox, a longer time is required for the fluorescence intensity to decay and approach zero. The resulting curve of Trolox Calibrator after data integration represents the
Fig. 2. Effect of concentration of Trolox standard on Trolox Calibrator decay curve. Measured by changes in fluorescein (FL) intensity from time 0 to 60 min. Trolox concentrations are: (^) blank, (&) 0.5 mM, (~) 1.0 mM, (—) 2.0 mM, (X) 3.0 mM, (*) 4.0 mM.
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Fig. 3. Increase on average net area under the curve (AUC) values while adding a known volume of human milk to Trolox Calibrator standard curve. Dashed line and (*) is Trolox with milk added; straight line and (^) is Trolox only. Table 1 Accuracy on experimental calculation of recovery of Trolox added to human milk. Trolox added (mM)a
Average AUCS
Average AUCTEb
Average AUCTCc
Accuracy (%)d
0.5 1.0 2.0 3.0 Average
31.1 0.1 35.1 0.2 43.4 0.2 49.2 0.6
17.3 0.1 21.4 0.2 29.6 0.2 35.5 0.6
17.9 0.2 21.8 0.3 32.1 0.2 38.7 0.4
97.1 0.7 97.9 0.8 92.3 0.5 91.7 1.5 94.8 3.2
AUCS: area under the curve of spiked milk (Average AUC of non-spiked milk = 13.7); AUCTE: calculated area under the curve of Trolox experimentally recovered; AUCTC = area under the curve of Trolox Calibrator. a Trolox added as final concentration in mM. b AUCTE = AUCS—13.7. c AUTTC from Trolox Calibrator 0.0005–0.0030 mmol Trolox/mL. d Values are in average percentage SD. Table 2 ORAC–FL values and corresponding alpha tocopherol (a-Toc) concentration of human milk from Vancouver and Winnipeg. Citya
ORAC–FLb
a-Tocc
R-valued
Vancouver Winnipeg
3.41 0.53 (2.26–4.90) 2.46 0.28 (1.87–3.14)
2.32 0.88 (0.66–5.02) 2.11 0.66 (1.21–4.62)
0.439 (P < 0.01) 0.408 (P < 0.01)
a
Mothers in mature stage of lactation; Vancouver n = 60 and Winnipeg n = 40. Average SD (range of values); units are mmol TE/mL. Average SD (range of values); units are mg/mL. d Coefficient of correlation between milk ORAC–FL value and a-Toc concentration. b c
and 1.21 to 4.62 mg/mL (average = 2.11 0.66 mg/mL) for Winnipeg mothers, respectively. In both groups of lactating women, a-Toc was the principle vitamin E isomer (accounting for between 70–90% of the total vitamin E). The AC of mature milk measured using the standardized ORAC–FL assay was slightly higher for Vancouver mothers (2.26–4.90 mmol TE/mL milk) compared to Winnipeg mothers (1.87–3.14 mmol TE/mL milk). Milk samples collected had ORAC–FL values that were positively correlated to the milk a-Toc content for mothers sampled from both Vancouver (R = 0.439; n = 60; P < 0.01) and Winnipeg (R = 0.408; n = 40; P < 0.01) (Table 2). There was no correlation between milk AC and other vitamin E isomers. 4. Discussion The TEAC method, albeit a relatively simple and rapid method, produced inconsistent results over a wide range of milk volumes tested. Almost 20% lower absorbance of the ABTS radical in samples that contained the greatest total solids content indicated that this assay was not suitable for antioxidant testing in human milk. Moreover, the apparent instability of the reaction over time is
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a clear indication that a potential underestimation of the antioxidant components in milk can occur using the TEAC method. In this study, the ORAC–FL assay was standardized and validated for human milk and the resulting ORAC–FL values, expressed as mmol TE/mL milk, were used to assess the antioxidant capacity of human milk in terms of a scavenging affinity against a thermal-induced peroxyl radical. Unlike the ABTS radical, peroxyl free radicals are common radicals in human biology and therefore provide some additional relevance to the antioxidant assay. The ORAC–FL assay for human milk, standardized in our laboratory, gave excellent linearity (a coefficient R2 > 0.990) and a precision (e.g. 2.2%) that was actually lower than the 15% established by Ou et al. (2001) over a wide range of Trolox concentrations. Davalos et al. (2004) reported similar precision results (1.9, 2.9 and 1.7%) for three Trolox Calibrator curves in their study. An increase in the net AUC values in the ORAC–FL assay reflects a greater activity of chain-breaking antioxidants to prevent potential AAPH-induced peroxyl radical damage (Cao et al., 1993; Ou et al., 2001; Prior et al., 2003; Davalos et al., 2004). Increased net ORAC–FL–AUC values were obtained after adding a known volume of human milk to Trolox Calibrator, thus reflecting a potential antioxidant capacity of natural milk chain-breaking antioxidants such as the vitamin E isomer a-Toc. Using fluorescein (FL) as the fluorescent probe for evaluating or measuring antioxidant activity makes the ORAC–FL assay more advantageous than other probes. FL is photostable, inexpensive, sensitive, and specific (Ou et al., 2001). ORAC–FL values are based on the standard curve of Trolox, a water-soluble vitamin E analogue. The ORAC–FL assay has been widely used to assess antioxidant capacity of food, dietary supplements, juices and wines, and biological samples such as plasma and urine (Prior et al., 2003; Davalos et al., 2004; Wu et al., 2004; Ferna´ndez-Pacho´n et al., 2005). Only one study has published the use of the ORAC assay to assess the antioxidant capacity of human milk (AlbertiFidanza et al., 2002); however, this report used a different fluorescent probe, b-phycoerythrin (b-PE), in the procedure. No data were given on the standardization and validation of the ORACPE assay in human milk, and results published in the paper (average 1.01 0.37 mmol Trolox/mL) were markedly lower than the values reported herein for mature milk using the ORAC–FL assay. The b-PE probe has an affinity to form non-specific protein-binding complexes (Cao and Prior, 1999), in addition to being more photolabile than fluorescein (Ou et al., 2001). These differences likely explain the markedly lower AC values of human milk reported using the ORACPE assay compared to ORAC–FL, thus explaining an underestimation of the antioxidant capacity of human milk with the former approach. To compare our human milk AC values to those of reported adult North American dietary intakes, we refer to the example given by Wu et al. (2004), who estimated a total daily antioxidant intake from 2.5 servings of fruit, fruit juices and vegetables to approximate 2200 mmol TE, measured using the ORAC–FL assay. This result is similar to values of 2510 mmol TE/g dietary supplement (Wu et al., 2004) or 1300–2030 mmol TE/g vitamin E formulation (Naguib et al., 2003). Average milk volumes consumed by 1–6-month-old infants, who are exclusively breast-fed by well-nourished mothers, is approximately 750– 850 g/day (Butte et al., 1991; Dewey et al., 1991), an intake that is mostly controlled by infant demand (Dewey et al., 1991) and the caloric density of the mother’s milk (Butte et al., 1991). It is difficult to give accurate volume intakes of breast milk among partially breast-fed infants (Neville, 1995). Based on our results from the two populations of lactating women studied, the daily milk intake of exclusively breast-fed infants (800 g/day, or 825 mL/day, based on a specific gravity of mature human milk of 1.031 g/mL (Pao et al., 1980)) would provide about 2030–2813 mmol TE. Thus, we
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conclude that the daily antioxidant intake from human milk by the breast-fed infant is similar to the estimated antioxidant intakes of adults. The estimate of AC in human milk accounts for activity offered by non-enzymatic antioxidants such as lactoferrin, casein phosphopeptide, vitamins A, C, E and carotenoids, as well as by enzymatic antioxidants that are also present; some examples include catalase, superoxide dismutase, and glutathione peroxidase (L’Abbe and Friel, 2000; Lindmark-Mansson and Akesson, 2000; Hamosh, 2001; Kasapovic et al., 2005; Kitts, 2005; Wong et al., 2006). Our findings that mother’s milk ORAC–FL values showed similar correlations with a-Toc content from two distinct populations of lactating Canadian women, validates this assay as a useful measure for assessing the antioxidant capacity of human milk. Notwithstanding this result, the tremendous inter-individual variation observed for both human milk a-Toc content and ORAC–FL values from mothers sampled in this study did not preclude obtaining similar associations between the two measured variables. In conclusion, the ORAC–FL assay was standardized for human milk through linearity, precision, and accuracy to enable the measurement of antioxidant capacity (AC) against thermalinduced peroxyl radical attack. Potential antioxidant protection of human milk was identified; this protection may be the result of natural milk components showing antioxidant protection, thus reflecting the oxidative stability of milk. Further research will help to identify the specific components in human milk that provide protection against free radical damage. Acknowledgements The authors are grateful to Steven Tomiuk for his technical assistance and the B.C. Women’s Milk Bank, and mothers in Vancouver and Manitoba, respectively, for their donation of human milk samples. References Alberti-Fidanza, A., Burini, G., Perriello, G., 2002. Total antioxidant capacity of colostrum, and transitional and mature human milk. The Journal of Maternal-Fetal and Neonatal Medicine 11, 275–279. Arts, M.J.T.J., Dallinga, J.S., Voss, H.-P., Haenen, G.R.M.M., Bast, Aalt., 2004. A new approach to assess the total antioxidant capacity using the TEAC assay. Food Chemistry 88, 567–570. Butte, N.F., Wong, W.W., Klein, P.D., Garza, C., 1991. Measurement of milk intake: tracer-to-infant deuterium dilution method. British Journal of Nutrition 65, 3–14. Cao, G., Prior, R.L., 1999. The measurement of oxygen radical absorbance capacity in biological samples. Methods in Enzymology 299, 50–62. Cao, G., Alessio, H.M., Cutler, R.G., 1993. Oxygen radical absorbance capacity assay for antioxidants. Free Radical Biology & Medicine 14, 303–311. Davalos, A., Gomez-Cordoves, C., Bartolome, B., 2004. Extending applicability of the oxygen radical absorbance capacity (ORAC-FLuorescein) assay. Journal of Agriculture and Food Chemistry 52, 48–54. Dewey, K.G., Heining, M.J., Nommsen, L.A., Lonnerdal, B., 1991. Maternal versus infant factors related to breast milk intake and residual milk volume. The DARLING study. Pediatrics 87, 829–837.
˜ o, D., Troncoso, A.M., Garcı´a-Parrilla, M.C., 2005. Ferna´ndez-Pacho´n, M.S., Villan Antioxidant capacity of plasma after red wine intake in human volunteers. Journal of Agricultural and Food Chemistry 53, 5024–5029. Hamosh, M., 2001. Bioactive factors in human milk. Pediatric Clinics of North America 48, 69–86. Hanna, N., Ahmed, K., Anwar, M., Petrova, A., Hiatt, M., Hegyi, T., 2004. Effect of storage on breast milk antioxidant activity. Archives of Disease in Childhood: Fetal and Neonatal Edition 89, F518–F520. Hu, C., Kwok, B.H.L., Kitts, D.D., 2005. Saskatoon berries (Amelanchier alnifolia nutt.) scavenge free radicals and inhibit intracellular oxidation. Food Research International 38, 1079–1085. Kasapovic, J., Pejic, S., Mladenovic, M., Radlovic, N., Pajovic, S.B., 2005. Superoxide dismutase activity in colostrum, transitional and mature human milk. The Turkish Journal of Pediatrics 47, 343–347. Kitts, D.D., 2005. Antioxidant properties of casein-phosphopeptides. Trends in Food Science & Technology 16, 549–554. Kitts, D.D., Hu, C., 2005. Biological and chemical assessment of antioxidant activity of sugar-lysine model maillard reaction products. Annals of the New York Academy of Sciences 1043, 501–512. L’Abbe, M.R., Friel, J.K., 2000. Superoxide dismutase and glutathione peroxidase content of human milk from mothers of premature and full-term infants during the first 3 months of lactation. Journal of Pediatric Gastroenterology and Nutrition 31, 270–274. Lindmark-Mansson, H., Akesson, B., 2000. Antioxidative factors in milk. British Journal of Nutrition 84, S103–S110. Mayer, J.M., 2004. Proton-coupled electron transfer: a reaction chemist’s view. Annual Review of Physical Chemistry 55, 363–390. Naguib, Y.M.A., Hari, S.P., Passwater, R., Huang, D., 2003. Antioxidant activities of natural vitamin E formulations. Journal of Nutritional Science and Vitaminology 49 (4), 217–220. Neville, M., 1995. Volume and caloric density of human milk. In: Jensen, R.G. (Ed.), Handbook of Milk Consumption. Academic Press, San Diego, California, pp. 99– 113. Ou, B., Hampsch-Woodill, M., Prior, R.L., 2001. Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe. Journal of Agricultural and Food Chemistry 49, 4619– 4626. Pao, E.M., Himes, J.M., Roche, A.F., 1980. Milk intakes and feeding patterns of breastfed infants. Journal of American Dietetic Association 77, 540–545. Phipps, S.M., Sharaf, M.H.M., Butterweck, V., July–August 2007. Antioxidant activity in botanicals and other dietary supplements. Pharmacopeial Forum 33 (4), 810– 814. Prior, R.L., Hoang, H., Gu, L., Wu, X., et al., 2003. Assays for hydrophilic and lipophilic antioxidant capacity (oxygen radical absorbance capacity (ORACFL) of plasma and other biological and food samples. Journal of Agricultural and Food Chemistry 51, 3273–3279. Prior, L.R., Wu, X., Schaich, K., 2005. Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. Journal of Agricultural Food Chemistry 53, 4290–4302. Rodas Mendoza, B., Morera Pons, S., Castellote Bargallo, A.I., Lopez-Sabater, M.C., 2003. Rapid determination by reverse-phase high performance liquid chromatography of vitamins A and E in infant formulas. Journal of Chromatography 1018, 197–202. ˜ iz, P., Beltran, S., Martinez-A´lvarez, J.R., Codon ˜ erValls-Belles, V., Torres, M.C., Mun Franch, P., 2006. Defatted milled grape seed protects adriamycin-treated hepatocytes against oxidative damage. European Journal of Nutrition 45, 251– 258. VanderJagt, D.J., Ojolo, S.N., Constanza, A., Blackwell, W., Glew, R.H., 2001. Antioxidant content of the milk of nigerian women and the sera of their exclusively breast-fed infants. Nutrition Research 21, 121–128. Wong, P.Y.Y., Nakamura, S., Kitts, D.D., 2006. Functional and biological activities of casein glycomacropeptide as influenced by lipophilization with medium and long chain fatty acid. Food Chemistry 97, 310–317. Wu, X., Gu, L., Holden, J., Haytowitz, D.B., Gebhardt, S.E., Beecher, G., Prior, R.L., 2004. Development of a database for total antioxidant capacity in foods: a preliminary study. Journal of Food Composition and Analysis 17, 407–422. Zoeren-Grobben, D.V., Moison, R.M.W., Berger, H.M., 1993. Lipid peroxidation in human milk and infant formula: effect of storage, tube feeding and exposure to phototherapy. Acta Paediatrica 82, 645–649.
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Original Article
Use of ORAC to assess antioxidant capacity of human milk Alexandra Tijerina Sa´enz a, Ingrid Elisia a, Sheila M. Innis b, James K. Friel c, David D. Kitts a,* a
Food Nutrition and Health, University of British Columbia, Vancouver, B.C. V6T 1Z4, Canada Department of Paediatrics, University of British Columbia, 950 West 28th Avenue, Vancouver, B.C. V5Z 4H4, Canada c Department of Human Nutritional Sciences, University of Manitoba, H511 Duff Roblin Building, Winnipeg, MB R3T 2N2, Canada b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 July 2008 Received in revised form 20 January 2009 Accepted 26 January 2009
The oxygen radical absorbance capacity (ORAC–FL) assay, a useful measure of the antioxidant capacity (AC) reported for some biological samples, supplements, and food samples, was standardized and validated for measuring AC of human milk. Limits of linearity, precision and accuracy of the ORAC–FL assay were made by constructing a Trolox Calibrator which included the addition of human milk as the sample matrix. AC assay results indicated excellent linearity (R2 = 0.990 0.005), precision (2.2%) and accuracy on recovery (94.8 3.2%) over a wide range of Trolox concentrations. To validate the assay further, mature milk samples were collected from 100 lactating mothers in Vancouver and Winnipeg, and were measured for vitamin E isomers by HPLC and AC using the standardized ORAC–FL assay. Alpha-tocopherol (a-Toc) was the major vitamin E isomer detected in human milk, established using both ultra-violet and fluorescent detection methods. Milk a-Toc concentrations were found to correlate significantly (P < 0.01) with ORAC–FL assay values in milk obtained from mothers in both Vancouver (R = 0.439, n = 60) and Winnipeg (R = 0.408, n = 40). Although milk is a complex matrix with multiple components possessing potential antioxidant activity, our results indicated that the ORAC–FL assay is a very useful indicator for assessing the antioxidant capacity of human milk. ß 2009 Elsevier Inc. All rights reserved.
Keywords: Human milk Antioxidant capacity (AC) ORAC–FL assay Assay validation with human milk Complex food matrix analysis Food analysis Food composition
1. Introduction Determining the antioxidant capacity (AC) of human milk involves measuring the collective antioxidant activity of numerous components present in milk, and these provide oxidative stability and retard the initiation and propagation of reactive oxygen species that can culminate in the deterioration of milk quality. The complex free radical mechanisms involving oxygen, transition metal ions and oxidizing agents and the deficient antioxidant mechanism present in milk can lead to peroxidation reactions which in turn are the underlying cause for generation hydrolytic off-flavors and loss of important nutrients and bioactive agents that promote neonatal health (Zoeren-Grobben et al., 1993). Antioxidant mechanisms of milk components that involve the transfer of electrons to quench free radicals are based either on the ionization potential constant (e.g. single electron transfer (SET)), or on the dissociation energy constant of the antioxidant hydrogen donor group (e.g. hydrogen atom transfer (HAT)) (Mayer, 2004). Milk AC has been measured using the colorimetric ABTS reducing assay to evaluate the antioxidant content of milk from African women; this method models the SET reaction and yields data describing the affinity of milk constituents to reduce the radical
* Corresponding author. Tel.: +1 604 822 5560; fax: +1 604 822 5143. E-mail address: [email protected] (D.D. Kitts). 0889-1575/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jfca.2009.01.021
cation ABTS, measured in units of Trolox equivalent antioxidant capacity (TEAC) (VanderJagt et al., 2001). The same procedure has been used to evaluate the effect of storage on human milk antioxidant activity (Hanna et al., 2004). However, the TEAC procedure is limited by the fact that ABTS represents a nonphysiological radical and moreover, a compound can only reduce ABTS if it has a redox potential lower than that of ABTS (Phipps et al., 2007). Thus, although the TEAC procedure is relatively simple and rapid, and compatible with both organic and aqueous solvents, it may underestimate some antioxidant components that exhibit a relatively slow reaction mechanism (Arts et al., 2004; Phipps et al., 2007). On the other hand, the oxygen radical absorbance capacity (ORAC) assay is a widely used procedure for measuring the total antioxidant capacity of biological samples, supplements and food samples (Prior et al., 2003; Davalos et al., 2004; Wu et al., 2004; Ferna´ndez-Pacho´n et al., 2005). The radical initiator component of this assay is 2,20 -azobis (2 amidinopropane) dihydrochloride (AAPH), a water-soluble free radical initiator which spontaneously decomposes at 37 8C to form two carbon-centered radicals which react with oxygen to generate peroxyl radical, a common radical in human biology. Fluorescent probes used in this assay decompose in a pattern that is consistent with the HAT mechanism of action when exposed to peroxyl radical (Prior et al., 2005). The fluorescent probe b-phycoerythrin (b-PE) has been employed in the ORAC assay (ORACb-PE) to measure the antioxidant capacity of colostrum, transitional and mature human milk in one study with Italian
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women (Alberti-Fidanza et al., 2002); details of the standardization or validation of the assay with human milk have not been reported. The b-PE probe is also relatively photosensitive and subject to nonspecific protein binding which can lead to underestimation of antioxidant capacity. Alternatively, employing fluorescein (FL) as the fluorescent probe in the ORAC procedure (ORAC–FL) offers advantages due to greater sensitivity and photostability and the specificity for antioxidant activity against peroxyl radicals (Ou et al., 2001). The aim of this study was to standardize and validate the ORAC– FL assay for assessing the oxidative stability of human milk. Experiments were conducted to determine the limits of linearity and precision of the standard calibrator and to determine the accuracy of the method through recovery experiments with known concentrations of Trolox added to human milk. Human milk samples from mothers living in two different geographic locations and at a similar mature stage of lactation were also collected and assayed for AC using the ORAC–FL assay. The results were validated by correlating with the vitamin E content of milk measured by HPLC-UV and fluorescent detection. 2. Materials and methods 2.1. Chemicals Radical cation 2,20 -azinobis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), fluorescein sodium salt (FSS), peroxyl radical initiator 2,20 -azobis (2-amidinopropane) dihydrochloride (AAPH), vitamin E analogue Trolox, and tocopherol (Toc) standards (a-Toc; d-Toc; and g-Toc) were all purchased from Sigma–Aldrich (St. Louis, MO, USA). Phosphate buffer (PB) was prepared from solutions of 0.75 M K2HPO4: 0.75 M NaH2PO4 to 61:39 (v/v). PB was diluted to a final concentration of 75 mM (pH = 7). Working concentrations of FSS were diluted to a final concentration of 200 nM. AAPH was dissolved to a final concentration of 60 mM immediately before use. Trolox at 20 mM was used for the Trolox Calibrator standards and were made fresh prior to use. Tocopherol standards were prepared in ethanol, kept at 20 8C and made fresh every week. HPLC grade solvents included hexane, ethanol, methanol, pyrogallol were purchased from Fisher Scientific (Ontario, Canada). Pyrogallol was dissolved in ethanol to 12% prior to use. 2.2. Human milk for Trolox equivalent antioxidant capacity assay (TEAC) Initial standardization experiments to establish TEAC in human milk were performed from whole human milk (100 mL) collected from the British Columbia (B.C.) Women’s Milk Bank (Vancouver, B.C.). Milk was collected and pooled from 10 different healthy women that were all in a mature stage of lactation. Aliquots of milk sample were taken and stored frozen at 80 8C until used. The TEAC assay was used to characterize the free radical scavenging capacity of milk as an example of the SET mechanism. This procedure is based on a discoloration reaction involving ABTS radical cations that are generated by adding 88 mL of a 140 nM potassium persulphate solution to 5 mL of a 7 mM ABTS solution and incubated at room temperature under light control (VallsBelles et al., 2006). The blue-green species of the generated ABTS was detected at 734 nm in a 96-well assay plate using a microplate spectrophotometer (Multiskan Spectrum, ThemoLabsystem) (Hu et al., 2005). The capacity of antioxidants present in milk to scavenge the radical and inhibit the color production is proportional to the antioxidant capacity of the human milk sample, and is expressed as an equivalent of the micromole concentration of Trolox per milliliter of milk (mmol TE/mL milk).
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2.3. Standardization of oxygen radical absorbance capacity assay (ORAC–FL) 2.3.1. Human milk for standardization of ORAC–FL assay Subsamples of human milk collected from the BC Women’s Milk Bank (Vancouver, B.C.), previously described in Section 2.2, were taken and stored frozen at 80 8C until used for experiments on linearity, precision and accuracy of the assay. 2.3.2. The ORAC–FL assay The ORAC–FL assay was standardized for human milk based on the methodology described by Ou et al. (2001) and by Kitts and Hu (2005). This assay is based on the capacity of antioxidants in a sample to quench peroxyl radicals that are generated from the thermal decomposition of AAPH. Concentrations of Trolox standard (0.0–4.0 mM), a water-soluble vitamin E analogue, were used to construct the Trolox Calibrator. The ORAC–FL assays standardized for specific dilutions of milk were conducted in 96well black microplate assay plates (BioRad, Cat. No. 353241) incubated at 37 8C for all concentrations of Trolox Calibrator. AAPH was added to each well of the plate, except for the control and blank. The final volume of the assay was 200 mL. The microplate was shaken for 10 s, and fluorescence was read every minute for 60 min in a Fluoroskan Ascent FL (Labsystems) fluorometer at excitation of 485 nm and emission of 527 nm, respectively. Data integration and calculations were performed according to Davalos et al. (2004). Area under the curve (AUC) values were calculated following the formula: AUC ¼ 1 þ
t i ¼60min X
Ai =A0
t0 ¼60min
where A0 is the initial fluorescence reading at 0 min and Ai is the fluorescence reading at time i. Milk samples and Trolox Calibrator net AUC values were obtained by subtracting the AUC value of the blank from that of milk sample or Trolox Calibrator. Sample and Trolox Calibrator net AUC values were plotted versus concentration. Linear regression analyses were performed to obtain the slope of the regression equation. The ORAC–FL value of a milk sample was calculated by dividing the slope of the sample (mS) by the slope of Trolox Calibrator (mTC). The final ORAC–FL values for human milk were expressed as an equivalent of the micromole concentration of Trolox standard solution per milliliter of milk (mmol TE/mL milk). All analyses were performed in triplicate. 2.3.3. Linearity, precision and accuracy of the human milk ORAC–FL assay The linearity and precision of the ORAC–FL assay was calculated using ten independent runs of Trolox Calibrator based on the coefficient of determination (R2) and the coefficient of variation (CV), respectively. These data were obtained from the equations derived in linear regression analysis of Trolox Calibrator curves. The feasibility of the ORAC–FL assay to measure the AC of human milk was also observed by comparing the standard curve of Trolox Calibrator to the curve generated from the addition of human milk to Trolox Calibrator. A milk volume of 15 mL was added to all concentrations in the Trolox Calibrator. An increase in the AUC values would reflect the capacity of human milk to scavenge peroxyl radicals. In addition, known concentrations of Trolox standard were added to human milk to determine Trolox recovery and to establish the accuracy of the ORAC–FL assay for measuring the oxidative stability of milk. Concentrations of 0.5, 1.0, 2.0 and 3.0 mM Trolox were added to a milk volume of 10 mL to produce a Trolox spiked-milk sample. Milk samples were analyzed for antioxidant capacity (AC) following the procedure of the ORAC–FL assay as described above. Accuracy
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was determined by subtracting the net AUC value of the non-spiked milk sample (AUCNS) from the net AUC values of the spiked sample (AUCS). The resulting values corresponded to the experimental net AUC values of Trolox standard added to milk (AUCTE). Net AUCTE values were compared to net AUC values of Trolox Calibrator (AUCTC) and percentage of accuracy on recovery was calculated. Accuracy on recovery of Trolox standard was analyzed in triplicate. 2.4. Validation of ORAC–FL Assay 2.4.1. Human milk for validation of ORAC–FL assay Validation of the ORAC–FL assay on human milk was done by correlating milk AC values, established from ORAC–FL assay with the vitamin E content in the same milk sample. This experiment was achieved by collecting human milk samples from two distinct populations of lactating women from two major Canadian cities, namely Vancouver (n = 60) and Winnipeg (n = 40). All milk samples were collected from women at a mature stage of lactation. Milk was divided into subsamples for ORAC–FL testing and vitamin E analysis measured by HPLC, and these were stored frozen at 80 8C until used. 2.4.2. Vitamin E analysis For the vitamin E analysis, milk was pre-treated by fat extraction according to the procedure of Rodas Mendoza et al. (2003). Briefly, 12% pyrogallol was added to the samples and vortexed. Ethanol, and water, and hexane were added to the mixture and samples were centrifuged (2000 g for 10 min) before collecting the organic layer, and the extraction was repeated twice. The organic layer was evaporated to dryness under nitrogen gas at ambient temperature and reconstituted in methanol for HPLC analysis. Vitamin E isomers, namely alpha tocopherol (a-Toc), delta tocopherol (d-Toc), and gamma tocopherol (g-Toc) were quantified in human milk using an Agilent 1100 HPLC system (Agilent Technology 1100 series, Palo Alto, CA) equipped with a 3 mm Sphereclone column (150 mm 4.6 mm, Phenomenex, Torrance, CA) with both diode-array detector (DAD) and fluorescent detector. Vitamin E isomers were detected at 292 nm for a-Toc and at 298 nm for both d-Toc and g-Toc. The fluorescence detection was made at excitation and emission wavelengths of 292 and 330 nm, respectively. Recoveries of added tocopherol isomers in mature human milk were 99.7 1.9% for a-Toc, 100.5 2.3% for d-Toc and 100.1 2.2% for g-Toc. Quantitation of tocopherol isomers in human milk was achieved using a standard curve constructed with an external standard (a-Toc 2.5–50 mg/mL, dToc and g-Toc 0.5–25 mg/mL) for each isomer with linearity R2 > 0.990.
Fig. 1. Time-dependent changes in ABTS radical scavenging by milk when present in different amounts in the TEAC assay. Milk volumes added are: A = 0 mL, B = 0.05 mL, C = 0.1 mL; D = 0.15 mL; E = 0.2 mL to final volume of 100 mL. Values represent single measurements taken every minute over 60 min. Absorbance values were taken at 720 nm.
transformed data from the decay curve, and it shows the net area under the curve (AUC) and the concentration of Trolox standard. Results of regression analyses of multiple independent runs of Trolox Calibrator showed good assay linearity (R2 = 0.990) and precision (CV = 2.2%) of the ORAC–FL assay. A step-wise addition of a known volume of pooled human milk to Trolox Calibrator produced relatively higher net AUC values than those of Trolox Calibrator without milk added (Fig. 3). This result pointed to the antioxidant capacity of milk that produced the increase in net AUC values and the feasibility of the ORAC–FL assay to measure AC of human milk. Experimental net AUC values of Trolox added to mature human milk (AUCTE) were calculated from the difference of the average net AUC values of non-spiked milk sample (AUCNS = 13.7) from the net AUC values of the spiked sample (AUCS), as presented in Table 1. AUCTE values were compared to the net AUC values of Trolox Calibrator (AUCTC), and Trolox recovery was calculated. The accuracy of recovery of Trolox standard ranged from 91.7 to 97.9% with an average value of 94.8 3.2%. The major vitamin E isomer found in human milk from both city locations was a-Toc, which ranged in concentration from 0.66 to 5.02 mg/mL (average = 2.32 0.88 mg/mL) for Vancouver mothers
3. Results Fig. 1 shows the results of the TEAC assay where ABTS radical was scavenged by milk components over time. The addition of milk samples to the assay, ranging from 0.05 to 0.2 mL, produced a volume-dependent downward shift in absorbance values that were also time-dependent. This result illustrates the potential for inaccuracy of using fixed-time intervals for the TEAC assay for testing the antioxidant capacity of human milk samples. Different rates of absorbance decreases over time can be attributed to the quenching of ABTS radical by relative volumes of milk in the assay and the available reducing power required to reduce ABTS. A typical ORAC–FL decay curve for Trolox Calibrator over a range of Trolox standard concentrations is presented in Fig. 2. With increasing concentration of Trolox, a longer time is required for the fluorescence intensity to decay and approach zero. The resulting curve of Trolox Calibrator after data integration represents the
Fig. 2. Effect of concentration of Trolox standard on Trolox Calibrator decay curve. Measured by changes in fluorescein (FL) intensity from time 0 to 60 min. Trolox concentrations are: (^) blank, (&) 0.5 mM, (~) 1.0 mM, (—) 2.0 mM, (X) 3.0 mM, (*) 4.0 mM.
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Fig. 3. Increase on average net area under the curve (AUC) values while adding a known volume of human milk to Trolox Calibrator standard curve. Dashed line and (*) is Trolox with milk added; straight line and (^) is Trolox only. Table 1 Accuracy on experimental calculation of recovery of Trolox added to human milk. Trolox added (mM)a
Average AUCS
Average AUCTEb
Average AUCTCc
Accuracy (%)d
0.5 1.0 2.0 3.0 Average
31.1 0.1 35.1 0.2 43.4 0.2 49.2 0.6
17.3 0.1 21.4 0.2 29.6 0.2 35.5 0.6
17.9 0.2 21.8 0.3 32.1 0.2 38.7 0.4
97.1 0.7 97.9 0.8 92.3 0.5 91.7 1.5 94.8 3.2
AUCS: area under the curve of spiked milk (Average AUC of non-spiked milk = 13.7); AUCTE: calculated area under the curve of Trolox experimentally recovered; AUCTC = area under the curve of Trolox Calibrator. a Trolox added as final concentration in mM. b AUCTE = AUCS—13.7. c AUTTC from Trolox Calibrator 0.0005–0.0030 mmol Trolox/mL. d Values are in average percentage SD. Table 2 ORAC–FL values and corresponding alpha tocopherol (a-Toc) concentration of human milk from Vancouver and Winnipeg. Citya
ORAC–FLb
a-Tocc
R-valued
Vancouver Winnipeg
3.41 0.53 (2.26–4.90) 2.46 0.28 (1.87–3.14)
2.32 0.88 (0.66–5.02) 2.11 0.66 (1.21–4.62)
0.439 (P < 0.01) 0.408 (P < 0.01)
a
Mothers in mature stage of lactation; Vancouver n = 60 and Winnipeg n = 40. Average SD (range of values); units are mmol TE/mL. Average SD (range of values); units are mg/mL. d Coefficient of correlation between milk ORAC–FL value and a-Toc concentration. b c
and 1.21 to 4.62 mg/mL (average = 2.11 0.66 mg/mL) for Winnipeg mothers, respectively. In both groups of lactating women, a-Toc was the principle vitamin E isomer (accounting for between 70–90% of the total vitamin E). The AC of mature milk measured using the standardized ORAC–FL assay was slightly higher for Vancouver mothers (2.26–4.90 mmol TE/mL milk) compared to Winnipeg mothers (1.87–3.14 mmol TE/mL milk). Milk samples collected had ORAC–FL values that were positively correlated to the milk a-Toc content for mothers sampled from both Vancouver (R = 0.439; n = 60; P < 0.01) and Winnipeg (R = 0.408; n = 40; P < 0.01) (Table 2). There was no correlation between milk AC and other vitamin E isomers. 4. Discussion The TEAC method, albeit a relatively simple and rapid method, produced inconsistent results over a wide range of milk volumes tested. Almost 20% lower absorbance of the ABTS radical in samples that contained the greatest total solids content indicated that this assay was not suitable for antioxidant testing in human milk. Moreover, the apparent instability of the reaction over time is
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a clear indication that a potential underestimation of the antioxidant components in milk can occur using the TEAC method. In this study, the ORAC–FL assay was standardized and validated for human milk and the resulting ORAC–FL values, expressed as mmol TE/mL milk, were used to assess the antioxidant capacity of human milk in terms of a scavenging affinity against a thermal-induced peroxyl radical. Unlike the ABTS radical, peroxyl free radicals are common radicals in human biology and therefore provide some additional relevance to the antioxidant assay. The ORAC–FL assay for human milk, standardized in our laboratory, gave excellent linearity (a coefficient R2 > 0.990) and a precision (e.g. 2.2%) that was actually lower than the 15% established by Ou et al. (2001) over a wide range of Trolox concentrations. Davalos et al. (2004) reported similar precision results (1.9, 2.9 and 1.7%) for three Trolox Calibrator curves in their study. An increase in the net AUC values in the ORAC–FL assay reflects a greater activity of chain-breaking antioxidants to prevent potential AAPH-induced peroxyl radical damage (Cao et al., 1993; Ou et al., 2001; Prior et al., 2003; Davalos et al., 2004). Increased net ORAC–FL–AUC values were obtained after adding a known volume of human milk to Trolox Calibrator, thus reflecting a potential antioxidant capacity of natural milk chain-breaking antioxidants such as the vitamin E isomer a-Toc. Using fluorescein (FL) as the fluorescent probe for evaluating or measuring antioxidant activity makes the ORAC–FL assay more advantageous than other probes. FL is photostable, inexpensive, sensitive, and specific (Ou et al., 2001). ORAC–FL values are based on the standard curve of Trolox, a water-soluble vitamin E analogue. The ORAC–FL assay has been widely used to assess antioxidant capacity of food, dietary supplements, juices and wines, and biological samples such as plasma and urine (Prior et al., 2003; Davalos et al., 2004; Wu et al., 2004; Ferna´ndez-Pacho´n et al., 2005). Only one study has published the use of the ORAC assay to assess the antioxidant capacity of human milk (AlbertiFidanza et al., 2002); however, this report used a different fluorescent probe, b-phycoerythrin (b-PE), in the procedure. No data were given on the standardization and validation of the ORACPE assay in human milk, and results published in the paper (average 1.01 0.37 mmol Trolox/mL) were markedly lower than the values reported herein for mature milk using the ORAC–FL assay. The b-PE probe has an affinity to form non-specific protein-binding complexes (Cao and Prior, 1999), in addition to being more photolabile than fluorescein (Ou et al., 2001). These differences likely explain the markedly lower AC values of human milk reported using the ORACPE assay compared to ORAC–FL, thus explaining an underestimation of the antioxidant capacity of human milk with the former approach. To compare our human milk AC values to those of reported adult North American dietary intakes, we refer to the example given by Wu et al. (2004), who estimated a total daily antioxidant intake from 2.5 servings of fruit, fruit juices and vegetables to approximate 2200 mmol TE, measured using the ORAC–FL assay. This result is similar to values of 2510 mmol TE/g dietary supplement (Wu et al., 2004) or 1300–2030 mmol TE/g vitamin E formulation (Naguib et al., 2003). Average milk volumes consumed by 1–6-month-old infants, who are exclusively breast-fed by well-nourished mothers, is approximately 750– 850 g/day (Butte et al., 1991; Dewey et al., 1991), an intake that is mostly controlled by infant demand (Dewey et al., 1991) and the caloric density of the mother’s milk (Butte et al., 1991). It is difficult to give accurate volume intakes of breast milk among partially breast-fed infants (Neville, 1995). Based on our results from the two populations of lactating women studied, the daily milk intake of exclusively breast-fed infants (800 g/day, or 825 mL/day, based on a specific gravity of mature human milk of 1.031 g/mL (Pao et al., 1980)) would provide about 2030–2813 mmol TE. Thus, we
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conclude that the daily antioxidant intake from human milk by the breast-fed infant is similar to the estimated antioxidant intakes of adults. The estimate of AC in human milk accounts for activity offered by non-enzymatic antioxidants such as lactoferrin, casein phosphopeptide, vitamins A, C, E and carotenoids, as well as by enzymatic antioxidants that are also present; some examples include catalase, superoxide dismutase, and glutathione peroxidase (L’Abbe and Friel, 2000; Lindmark-Mansson and Akesson, 2000; Hamosh, 2001; Kasapovic et al., 2005; Kitts, 2005; Wong et al., 2006). Our findings that mother’s milk ORAC–FL values showed similar correlations with a-Toc content from two distinct populations of lactating Canadian women, validates this assay as a useful measure for assessing the antioxidant capacity of human milk. Notwithstanding this result, the tremendous inter-individual variation observed for both human milk a-Toc content and ORAC–FL values from mothers sampled in this study did not preclude obtaining similar associations between the two measured variables. In conclusion, the ORAC–FL assay was standardized for human milk through linearity, precision, and accuracy to enable the measurement of antioxidant capacity (AC) against thermalinduced peroxyl radical attack. Potential antioxidant protection of human milk was identified; this protection may be the result of natural milk components showing antioxidant protection, thus reflecting the oxidative stability of milk. Further research will help to identify the specific components in human milk that provide protection against free radical damage. Acknowledgements The authors are grateful to Steven Tomiuk for his technical assistance and the B.C. Women’s Milk Bank, and mothers in Vancouver and Manitoba, respectively, for their donation of human milk samples. References Alberti-Fidanza, A., Burini, G., Perriello, G., 2002. Total antioxidant capacity of colostrum, and transitional and mature human milk. The Journal of Maternal-Fetal and Neonatal Medicine 11, 275–279. Arts, M.J.T.J., Dallinga, J.S., Voss, H.-P., Haenen, G.R.M.M., Bast, Aalt., 2004. A new approach to assess the total antioxidant capacity using the TEAC assay. Food Chemistry 88, 567–570. Butte, N.F., Wong, W.W., Klein, P.D., Garza, C., 1991. Measurement of milk intake: tracer-to-infant deuterium dilution method. British Journal of Nutrition 65, 3–14. Cao, G., Prior, R.L., 1999. The measurement of oxygen radical absorbance capacity in biological samples. Methods in Enzymology 299, 50–62. Cao, G., Alessio, H.M., Cutler, R.G., 1993. Oxygen radical absorbance capacity assay for antioxidants. Free Radical Biology & Medicine 14, 303–311. Davalos, A., Gomez-Cordoves, C., Bartolome, B., 2004. Extending applicability of the oxygen radical absorbance capacity (ORAC-FLuorescein) assay. Journal of Agriculture and Food Chemistry 52, 48–54. Dewey, K.G., Heining, M.J., Nommsen, L.A., Lonnerdal, B., 1991. Maternal versus infant factors related to breast milk intake and residual milk volume. The DARLING study. Pediatrics 87, 829–837.
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