Determination of glutathione peroxidase activity inhuman milk

Angeles Torres1, Rosaura Farr�1, Mar�a Jesffls Lagarda1 and Javier Monle�n2

1 Introduction

Glutathione peroxidase (GPx) (E.C.1.11.1.9) is a selenium-containing enzyme that contributes to protect cells againstlipid peroxidation induced damage [1]. Cellular GPx was thefirst enzyme recognized as a selenoprotein, the selenium com-ponent being present in the form of selenocysteine. GPx is atetrameric protein composed of four apparently identical subu-nits, each containing one atom of selenium. The plasmaenzyme (p-GPx) likewise presents a tetrameric form with iden-tical subunits and with one selenium atom per subunit. how-ever, it is a glycosylated protein, and is kinetically, structurallyand antigenically distinct from the cellular enzyme (c-GPx).Both p-GPx and c-GPx are the products of two different genes[2, 3]. Milk is another extracellular fluid known to containGPx activity [4–8]. 90% of human milk GPx activity can beprecipitated by anti-p-GPx-immunoglobulin G (IgG). Thus,most if not all GPx activity in milk is due to the plasma seleno-protein form of the enzyme. The latter may be secreted by theliver and transported into breast milk via the blood stream, oralternatively it could be secreted by the mammary gland [2].

GPx catalyses the decomposition of hydrogen peroxide(H2O2) and organic hydroperoxides (R1OOH) by glutathione(GSH) according to the following reaction:

R1OOH + 2GSH gggsGPx

R1OH + GSSG

Two types of assays are used to measure GPx activity. Bothare based on a glutathione reductase NADPH-coupled reactionwhere glutathione reductase (GSH-R) is used to regenerateglutathione (GSH) from oxidized glutathione (GSSG) as fol-lows:

GSSG + NADPH ggggsGSH-R

2GSH + NADP+ + H+

In the first assay, the amount of remaining reduced glu-tathione (GSH) is measured at specific time intervals, in thepresence of small amounts of peroxides, while in the secondassay measurements are made of the decrease in NADPH con-tent occurring when oxidized glutathione (GSSG) is convertedto glutathione (GSH) [11]. Most methods used to measure GPxactivities in blood and human milk are based on the secondtype of assay, and mainly comprise modifications of themethod proposed by Paglia and Valentine [12]. These modifi-cations may affect the concentration of the assay components,the composition and pH value of the buffer, and the tempera-ture of reaction. In GPx activity determinations the most rele-vant problems are a lack of standardization of the availablemethods that makes it impossible to compare the valuesobtained in different laboratories, and the variability in theunits used to express the activities [11].

We were aware of these problems in our study on seleniumnutritional status during pregnancy and lactation, and requireda standardized method to measure GPx activity in human milk.Thus, the main objective of our study was to adapt to humanmilk the standardized analytical method for the determinationof GPx activity in whole blood [13]. Following validation, themethod was applied to milk from 11 lactating women living inValencia (Spain), with the aim of assessing its suitability forthis purpose.

2 Materials and methods

2.1 Apparatus, reagents and samples

An ultracentrifuge (Dupont model Sorvall RC 28S) with anHS-4 rotor, a UV-V spectrophotometer (Perkin-Elmer modelLambda 2) fitted with a thermostatic bath (08–1008C, sensi-tivity 0.18C) (Selecta Digiterm 200), and a water deionizationsystem (Millipore-Milli Q) were used. Dithiothreitol (DTT)(Sigma D5545); b-NADPH (Sigma N7505); glutathione perox-idase: lyophilized powder from bovine erythrocytes (SigmaG6137); glutathione reductase (Sigma G4751); reduced glu-tathione (Sigma G4251); t-butyl hydroperoxide (t-BHP)(Sigma B2633); Millipore Milli-Q water, K2HPO4 and KH2PO4

(Panreac p.a.), Na2 EDTA (Probus p.a.) and sodium azide(Merck p.a.) were used. Dilution solvents: 0.1 M DTT was pre-pared by dissolving 700 mg DTT in 50 mL water; potassiumphosphate buffer 0.25 M phosphate/0.025 M EDTA (pH 7.0)was prepared by mixing 100 mL of 0.25 M K2HPO4/ 0.025 M

EDTA with a sufficient amount of 0.25 M KH2PO4/0.25 M

EDTA to obtain pH 7, and containing also 0.1% azide. Initia-tor: 25 nM t-BHP. Reaction mixture: Solutions A, B, C and D

430 Nahrung/Food 47 (2003) No. 6, pp. 430– 433 i 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

An analytical method for determining glutathione peroxidase (GPx)(EC 1.11.1.9) activity in whole blood has been adapted to human milksamples. The values obtained for precision (relative standard deviation:8.4%), linearity and accuracy (recovery: 90.4%) indicate the adequacyof the method for the mentioned purpose. The method was applied to 11

human milk samples obtained in the range from 6 to 135 days post par-tum. The resulting GPx activities (83.3 l 23.6 U/L) did not correlate tothe selenium content of the samples, though a reciprocal correlation wasfound between the duration of lactation and milk GPx activity.

Correspondence: Rosaura Farr�, Nutrition and Food Chemistry Fa-culty of Pharmacy, Valencia University, E-46100 Burjassot, Valencia,SpainE-mail: rosaura.farre@uv.esFax: +34-9635449541Nutrition and Food Chemistry Faculty of Pharmacy, Valencia Univer-sity, Burjassot, Valencia, Spain2Department of Obstetrics and Gynecology, “La Fe” University Hospi-tal, Valencia, Spain

Abbreviations: DTT, dithiothreitol; GPx, glutathione peroxidase;t-BHP, t-butyl hydroperoxide

Keywords: Glutathione peroxidase activity / Human milk / Seleniumcontent /

Glutathione peroxidase activity in human milk

i 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Nahrung/Food 47 (2003) No. 6, pp. 430 – 433 431

in 4 :2 :1 :1 v/v/v/v proportion. Solution A: potassium phos-phate buffer (pH 7.0); solution B: 5 U/mL glutathione reduc-tase diluted in 3.6 M (NH4)2SO4, dilution being carried out tak-ing into account the information on the label, since the glu-tathione reductase content can vary among batches; solution C:50 nM reduced glutathione; solution D: 2.5 nM b-NADPH.Solutions B, C, D and 0.1 M DTT were freshly prepared on theday of assay; stock buffer reagents and initiator solution couldbe prepared in advance and stored at +2 to +88C. Milk wasobtained from 11 apparently healthy lactating women in theperiod comprised between post partum days 6 and 135. Sam-ples were collected in the “La Fe” University Hospital inValencia (Spain) using a manual breast pump, in acid-washedcontainers, followed by immediate transport to the laboratoryin an icebox.

2.2 GPx activity determination

2.2.1 Sample treatment

Fat was completely removed by centrifuging the milk at10000 g for 1.5 h, at 48C. For blank preparation we used heat-denatured and defatted milk. To inactivate GPx heating at758C was carried out, and different time points (15, 30 and45 min) were assayed. No statistically significant differences(p a 0.05) were observed in blank absorbance with the differ-ent heating times. Then, blanks were prepared with defattedmilk by heating at 758C for 15 min, followed by cooling toroom temperature for 30 min. Blanks and defatted sampleswere frozen until analysis.

2.2.2 Preliminary assays

Blanks and samples were diluted and potassium phosphatebuffer and 0.1 M DTT were assayed as solvents. The procedurewas applied and the absorbances at 340 nm against three milkdilutions (1 :2, 1 :3, 1 :4) were measured, The curves, regres-sion equations and coefficients are shown in Fig. 1. No differ-ences between the slopes of the curves were found, thoughwhen potassium phosphate buffer was used the straight lineintercept did not significantly differ from zero, and the correla-tion coefficient was better than with DTT. Accordingly,defatted samples and blanks (heated defatted milk) werediluted 1:2 with potassium phosphate buffer.

2.2.3 Procedure and analytical parameters

800 lL of reaction mixture and 100 lL of diluted sample orblank were transferred to a 1 mL cell (1 cm light path), wellmixed and allowed to stand at 378C for 5 min. Then, 100 lLof 25 nM t-BHP solution was added and quickly mixed, fol-lowed by measurement of the absorbances at 340 nm for2 min, using a thermostated spectrophotometer at 378C. Thechanges in absorbance per minute (Da/t) in both blanks andsamples were calculated. Oxidized a-NADPH nmol/min/mL insamples were calculated as follows:

Das/t – Dab/t)6 [(16109)/6.226106)]6 (Vr/cell light path in cm]6

6 [Ds/Vs in mL] = (Das/t – Dab/t) 10a62d6160.8c

where Das/t is the change in absorbance per minute of sample,Dab/t is the change in absorbance per minute of blank, Vr is thevolume of reagent, Ds is the sample dilution, Vs is the samplevolume, 10a is the dilution factor in the cell, 2d is the milkdilution, and 160.8c is the factor for converting oxidized nmolof (b-NADPH/min/mL of initial sample, the molar extinctioncoefficient of (b-NADPH being 6.226103 L Nmol–1 cm–1. Inorder to validate the quality of the method, estimations weremade of its linearity, precision and accuracy.

3 Results

The linearity range, regression equation and coefficient areshown in Fig. 1. The linearity range includes the GPx activitiesof the diluted samples (as mentioned in the procedure). Preci-sion was estimated from the GPx values (86.8; 88.4; 91.7;75.6; 88.4; 80.4; 101.3; 98.1; 90.0; 90.0 U/L) obtained fromthe analysis of 10 aliquots of a milk sample; for a mean GPxactivity of 89.1 U/L, a relative standard deviation of 8.4% wasobtained. To estimate accuracy it was necessary to determinethe GPx activity of the reconstituted lyophilized powder frombovine erythrocytes; known amounts of this equivalent to 53.0U/L were then added to a milk sample (47.8 U/L). Accuracy,estimated by three recovery assays, was 90.4 l 4.7%. Thevalues obtained for GPx activities in human milk samples areindicated in Table 1. The previously measured selenium con-tents of these milks [14] are also included. No correlation wasfound between GPx activities and selenium contents, though areciprocal correlation was found between the duration of lacta-tion and GPx activity (r = 0.569; p a 0.05) (see Fig. 2).

Figure 1. GPx activity determination: absorbance data vs. milk dilu-tions with phosphate buffer and DTT.

Table 1. Milk samples from 11 women: selenium content and glu-tathione peroxidase GPx activities

Post partum day Se (lg/L) GPx (U/L)

6 9.4 67.59 13.5 113.4

11 10.7 90.818 11.6 112.619 11.1 73.723 12.6 84.835 4.6 90.038 5.8 83.643 7.1 91.6

120 8.7 25.7135 3.0 83.6

Torres et al.

432 Nahrung/Food 47 (2003) No. 6, pp. 430– 433 i 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

4 Discussion

The values obtained for linearity, precision and accuracy con-firm the adequacy of the standardized method for determiningGPx activity in human milk. Two types of peroxidases canexpress glutathione-dependent activity: glutathione S-trans-ferases, which use only lipid hydroperoxides, and the selenium-

dependent forms (GPx), which decompose both lipid hydroper-oxides and hydrogen peroxide. Therefore, in the presence ofadequate amounts of t-BHP, both GPx activities would berevealed, whereas in the presence of hydrogen peroxide (H2O2)only the selenium-dependent activity would be detected [2].The standardized procedure uses t-BHP; it is therefore unable todiscriminate between selenium-dependent GPx activity andselenium independent GPx activity, and thus measures totalGPx activity. Some authors [5–7] have reported that only one-third of the total GPx activity in milk can be ascribed to sele-nium-dependent GPx activity. This proportion is based on thedifferences between the enzyme activity measured using t-BHPand H2O2 as substrate. In contrast, other authors [2, 15] havereported no differences in GPx activity in milk when H2O2

replaced t-BHP as substrate, thus suggesting the milk enzyme tobe the selenium-dependent form (see Table 2).

The selenium content and GPx activities of the analyzed milksamples are indicated in Table 1, while Table 2 shows our meanvalues together with the values for GPx activity in human milkgiven by different authors (all of them from non-Europeancountries). The variability in the reported values is high, rangingfrom 27.5 to 186; such variability can be ascribed to thereported differences in the substrate used (t-BHP or H2O2), and/or the temperature (25 or 378C), and perhaps to the lactatingperiod involved. Our mean values (83.3 l 23.6) are included inthis wide range. We found that GPx activity in human milk didnot correlate with the selenium content, this observation beingin agreement with some reports [8, 16], and in discrepancy withothers [4, 7, 9, 17].

In the analyzed human milk samples, GPx activity decreasedwith an increasing duration of the lactation period, in agree-ment with several others studies [4, 8, 16, 18]. GPx activity in

Table 2. Milk GPx activity (U/L) reported by different authors

Country Type of human milk (n) Substrate (temperature) GPx activity (U/L) Ref.

USA 1–8 m (10) t-BHP (37 8C)H2O2 (37 8C)

29.4 l 13.828.8 l 13.2

[2]

USA (10) t-BHP (25 8C)H2O2 (25 8C)

125.1 l 4.136.0 l 3.7

[5]

USA (5) t-BHP (25 8C)H2O2 (25 8C)

107.1 l 12.343.2 l 5.3

[6]

USA 1–2 m foremilk (18)1–2 m hindmilk (18)

t-BHP (25 8C) 68.1 l 3.590.4 l 3.3

[7]

USA 1–3 m (15) H2O2 (25 8C) 31.3 l 1.8 [8]USA Vegetarians (26)

Nonvegetarians (12)Vegetarians (26)Nonvegetarians (12)

t-BHP (25 8C)

H2O2 (25 8C)

109.3 l 3.4118.7 l 5.740.1 l 1.527.5 l 1.2

[9]

USA 3 d (9)7 d (6)21 d (21)42 d (11)

H2O2 (25 8C) 28.2 l 4.128.2 l 4.422.9 l 2.729.5 l 5.5

[10]

Japan 4 d (5)7–8 d (4)36–86 d (13)

H2O2 66.0 l 8.646.0 l 3.338.8 l 5.9

[4]

New Zealand 29–35 d (10) H2O2 = t-BHP (25 8C) 31.0 l 8.0 [14]Gambia 1–19 m (10) H2O2 (37 8C)

t-BHP (37 8C)45.9 l 3.6

122.0 l 6.6[16]

Kuwait 0–6 m (17)6–12 m (17)12–18 m (17)

(37 8C) 182 l 16136 l 10128 l 9.0

[18]

Spain 6–135 d (11) t-BHP (37 8C) 83.3 l 23.6 Present study

n, samples number; U/L, number of micromoles of NADPH oxidized per minute; d, days; m, months

Figure 2. Correlation between milk GPx activity and the duration ofthe lactation period.

Glutathione peroxidase activity in human milk

i 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Nahrung/Food 47 (2003) No. 6, pp. 430 – 433 433

human milk appears to be affected directly by the selenium sta-tus of the mother [7]. Supplementation with organic seleniumin lactating women for three months almost doubled GPxactivity in human milk, though no changes were observedwhen inorganic selenium was used [19]. It has also beenreported that selenium intake does not affect the GPx activityof early milk, but only of mature milk samples [17].

The presence of GPx in human, goat and cow milk suggeststhat this enzyme is of functional relevance for the breastfedneonate and/or reflects physiological events occurring in themammary gland during lactation [5]. No GPx activity wasdetected either in market-pasteurized milk or in infant formu-las; this is probably a result of the heating processes involvedin these products [20]. The presence of a sufficient amount ofGPx in milk may protect lipids from oxidation [8]. We agreewith Al Awadi and Srikumar [18] that further studies areneeded to determine how GPx would contribute to the bio-availability or antioxidant property of selenium in infants, par-ticularly in the first six months of life.

This study is part of a project financed by FISS (Fondo de Investigaci�nSanitaria de la Seguridad Social) grant 94/0467. Thanks are due toGuadalupe Garc�a for her helpful collaboration.

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Received May 15, 2003Accepted August 18, 2003