Minerals and Trace Elements

Partial Sequence of Human Plasma Glutathione Peroxidase and Immunologie Identification of Milk Glutathione Peroxidase as the Plasma Enzyme1'2 NELLY AV1SSAR,3 J. RANDALL SLEMMON, IVAN S. PALMER* AND HARVEY J. COHEN University of Rochester School of Medicine and Dentistry, Department of Pediatrics, Biochemistry and Cancer Center, Rochester, NY 14642 and *South Dakota State University, Section Biochemistry, Brookings, SD 57607 As shown previously, there are two distinct forms of Se-dependent GSHPx in mammals, a cellular form (c-GSHPx) and an extracellular or plasma form (pGSHPx) (11-15). The two enzymes have different af finities for glutathione (GSH) and hydroperoxides. The plasma form is more heat stable, has a slightly higher subunit molecular weight and has a lower specific activity than c-GSHPx. It contains in tramolecular disulfide bridges and carbohydrate,- cGSHPx does not (11-15). Polyclonal antibodies directed against one enzyme do not recognize the other (11, 12). Using these mutually non-crossreactive antibodies, p-GSHPx was shown to be syn thesized for secretion by a human hepatic cell une, whereas c-GSHPx remained within all cells tested (15). The differences between the two enzymes could be due to two different gene products or to a variation in the processing of a protein product of one gene to adapt it to its specific function and compartment, i.e., cellular vs. extracellular environment. To determine if the two enzymes are products of two genes, we partially sequenced p-GSHPx. All the peroxidase activity in human plasma can be attributed to p-GSHPx (16). Glutathione peroxidase

ABSTRACT Plasma glutathione peroxidase (p-GSHPx) is a unique selenoglycoprotein. A hepatic cell line syn thesizes both this extracellular form for secretion and the cellular form that remains within the cells. Because the two forms could be a result of post-translational modifications of a product of a single gene, we partially sequenced p-GSHPx. Purified p-GSHPx was trypsin digested, and three of the peptides were sequenced. Only one of the peptide sequences was partially homol ogous to a sequence found in human cellular glutathione peroxidase. Because p-GSHPx is a secreted enzyme, we determined whether GSHPx in milk (another extracel lular fluid) is due to this form of the enzyme. Ninety percent of human milk GSHPx activity could be precipi tated by antî-p-GSHPx-immunoglobulin G. Thus, most, if not all, GSHPx activity in milk is due to the plasma selenoprotein form of the enzyme. In milk of two North American women, 3.6% and 14.3% of selenium was associated with GSHPx. J. Nutr. 121: 1243-1249, 1991. INDEXING KEY WORDS:

•human plasma •human milk •selenium •glutathione peroxidase •amino add sequence

Selenium is an essential micronutrient as judged by symptoms accompanying Se deficiency in humans and animals (1, 2), but high levels of Se are toxic (1-3). Selenium is specifically incorporated into pro teins as selenocysteine in response to the opal codon UGA (4-6). But it also can be incorporated as selenomethionine instead of methionine, and this form is increased in methionine deprivation (7). Selenocys teine is part of the active site of glutathione perox idase (GSHPx)4 (GSHcHzOj oxidoreductase, EC 1.11.1.9) and this is its only known function in mammals (4-6). This enzyme plays a pivotal role in the defense mechanisms of the body against oxidative damage (8-10).

'A preliminary report of the sequence data was presented at the Annual Meeting of the Federation of American Societies for Experi mental Biology, Washington, DC, April 1-5, 1990 [Avissar, N., Slemmon, J. R. &.Cohen, H. J. (1990) Partial sequencing of plasma glutathione peroxide. FASEBJ. 4: A1061 (abs. 4614)]. Supported in part by U. S. Public Health Service grant R01DK33231, and the University of Rochester Cancer Center Core grant NIH 5-P30 CA11198-21. 'To whom correspondence should be addressed. 'Abbreviations used: c-GSHPx, cellular glutathione peroxidase; DTT, dithiotreitol; GSH, glutathione,- IgG, immunoglobulin G; pGSHPx, plasma glutathione peroxidase; t-BuOOH, tert-butyl hydroperoxide; TPCK, L-l-tosylamide-2-phenyl-ethylchloromethyl ketone.

0022-3166/91 $3.00 ©1991 American Institute of Nutrition. Received 2 July 1990. Accepted 3 January 1991. 1243 Downloaded from https://academic.oup.com/jn/article-abstract/121/8/1243/4754570 by University of Glasgow user on 06 August 2018

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activity has been found in another extracellular fluid, milk (17-23). We hypothesized that the form of GSHPx in milk, an extracellular fluid, was the extra cellular form also present in plasma. To test this hypothesis, we used the specific antibodies described here.

MATERIALS AND METHODS Material*. Reduced glutathione, glutathione reductase, NADPH, tert-butyl hydroperoxide (t-BuOOH), HjOi, protein A Sepharose, DEAE-cellulose DE-52, Sephadex G-200 and 2,3-diaminonaphthalene were purchased from Sigma Chemical (St. Louis, MO). Vydac C4 column was from Alltech Associates (Deerfield, IL). Electrophoretic reagents were obtained from Schwartz Mann Biotech (Cleveland, OH). All the rea gents for amino acid sequencing were obtained from Applied Biosystems (Foster City, CA). All other rea gents were of analytical grade or better. Milk samples. Milk was obtained 1-8 mo postpartum from 10 apparently healthy lactating women, aged 25-35 y, living in Rochester, NY. Milk samples were collected by mechanical expression and immedi ately defatted by centrifugation for 1.5 h at 10,000 x g at 4°C.The defatted mille was stored frozen at -70°C and thawed in a water ice bath just before analysis. Blood samples. Heparinized blood (2 mL) collected from the lactating women and their age-matched con trols was centrifuged at 900 x g for 5 min at 4"C. Plasma was removed, and the RBC were washed three times in buffered saline. Hemolysates were prepared by adding 0.1 mL of washed RBC to 1.9 mL of 1 mmol/L potassium phosphate buffer (pH 7.4). The lysed cells were centrifuged at 10,000 x g for 30 min at 4°C;the resulting supernatant was frozen at -70°C and thawed in a water ice bath just before analysis. The use of human subjects in this investigation has been reviewed annually and approved by the Institu tional Review Board of the University of Rochester. Purification of p-GSHPx and RBC GSHPx. The pGSHPx was purified by combining our previously described method (11) with the procedure described by Maddi pati and Mamett (14). The supernatant from 20% saturated ammonium sulfate-treated freshly frozen human plasma was adjusted to 35% saturated ammonium sulfate,- the precipitate was applied to and eluted from phenyl Sepharose essentially as described (14). This was followed by DEAE-cellulose and Sephadex G-200 gel filtration chromatographies as described previously (11). These modifications im proved the yield (10-fold), and the protein obtained was pure as analyzed by SDS-PAGE. The RBC GSHPx was purified according to Awasthi et al. as previously described (11, 24). Preparation of polyclonal antibodies. Polyclonal antibodies against either p-GSHPx or RBC GSHPx Downloaded from https://academic.oup.com/jn/article-abstract/121/8/1243/4754570 by University of Glasgow user on 06 August 2018

were obtained in rabbits and the immunoglobulin G fractions were isolated as previously described (11, 12). The antibodies were found to be noninhibitory and to specifically precipitate the corresponding pu rified enzyme protein and activity (11, 12). Sequencing of p-GSHPx. Purified p-GSHPx (0.44 nmol) in a final volume of 0.5 ml, was denaturateci by 6 mol/L guanidine hydrochloride in Tris-HCl (pH 8.5), reduced by incubating 4 h at 37'C with 0.25 mg of dithiothreitol (DTT) and alkylated by incubating for 20 min at room temperature with 5 mg of iodoacetic acid. The enzyme was exhaustively dialyzed against 0.1 mol/L Tris base, neutralized to pH 8 and subjected to trypsinization. L-l-Tosylamide-2-phenyl-ethylchloromethyl ketone (TPCK)-treated trypsin (2 mg) was purified by reverse-phase HPLC using a Vydac C4 column (0.46 x 25 cm) in 8.7 mmol/L trifluoroacetic acid with an acetonitrile gradient. The reduced, alkylated p-GSHPx was incubated at 35°Cfor 24 h with 25 \iL of the purified TPCK-trypsin. The re sulting peptides were diluted 1:5 in 8.7 mmol/L trifluoroacetic acid and purified using reverse-phase HPLC chromatography as described by Slemmon (25). The isolated peptides were sequenced using an Ap plied Biosystems 477A/120 Protein Sequencer (Ap plied Biosystems, Foster City, CA) as described by Slemmon (25). Glutathione peroxidase activity. The standard re action mixture for the determination of GSHPx ac tivity in the presence of t-BuOOH was composed of the following: 0.1 mmol/L Tris-HCl (pH 8), 0.2 mmol/ L NADPH, 0.5 mmol/L EDTA, 2 mmol/L GSH, 0.0167 ukat of glutathione reductase and an appro priate amount of milk sample. Reaction was started by the addition of 70 umol/L of t-BuOOH. For mea suring the activity with H^O^, the reaction mixture was about the same except that 0.1 mol/L sodium phosphate buffer (pH 7) was used instead of Tris buffer; 1 mmol/L NaN3 was added and the reaction was started by the addition of 15 umol/L of liyO2. In the reference cuvette, either defatted milk or heatinactivated defatted milk and all the reagents except the peroxides were present. An additional blank con taining all components except the milk was deter mined to correct for nonenzymatic oxidation of NADPH by the peroxides. The oxidation of NADPH was followed at 340 nm at 37'C. One ukat of GSHPx activity is defined as the oxidation of 1 umol of NADPH per s at 37'C (11, 26). Immunoprecipitation of milk glutathione perox idase activity. One milligram of each of the IgG was incubated for 30 min with 20 mg of protein-ASepharose. Thawed, freshly frozen, defatted milk (1.75 mL) containing 0.33-0.83 nkat of GSHPx was added, the volume was brought to 3.7 mL with buffered saline and the mixture was further incubated for 3 h at 4"C. The GSHPx activity was determined either with t-BuOOH or HaOz in the supernatant after cen-

PLASMA GLUTATHIONE PEROXIDASE IN HUMAN MILK

trifugation for 20 min at 2000 x g (l mg of specific antibody is enough to precipitate more than 1.67 nkat of p-GSHPx or c-GSHPx). Determination of the amount of selenium asso ciated with glutathione peroxidase in milk. The Se associated with GSHPx was determined in milk of two women in separate experiments. Either nonimmune or anti-p-GSHPx-IgG (7.5 mg) was preincubated with 100 mg of protein-A Sepharose for 30 min; 7.5 mL and 8.75 mL of thawed, freshly frozen, defatted milk containing approximately 3.30 and 6.67 nkat of GSHPx activity, respectively, were added, the volume was brought up to 10 and 11.25 mL, respec tively, with buffered saline and the mixture was further incubated for 4 h at 4°C.The mixture was centrifuged for 30 min at 2000 x g, and the GSHPx activity with HjOî was determined in the superna tant; Se was determined in the supernatant and pre cipitate (1 mg of specific antibody is enough to precip itate more than 1.67 nkat of p-GSHPx or c-GSHPx). Selenium determination. The Se content was deter mined flourimetrically using 2,3-diaminonaphthalene after digestion with nitric acid and perchloric acid (27). Statistical analysis. Student's t test was used to analyze the results. The probability level at which differences were considered significant was P < 0.05.

-100 0.4- 80 - 60 0.3- 40

CD

I

- 20

1245

RESULTS To obtain sequence information for p-GSHPx, 175 pmol of the protein was subjected to terminal amino acid sequence analysis. No sequence analysis could be obtained, which suggests that the amino terminal residue of p-GSHPx is blocked. Therefore, we gen erated internal peptides by trypsinization of the en zyme. Because there is evidence that p-GSHPx con tains internal disulfide bridges (15), the protein was reduced and alkylated prior to trypsinization. To ensure full digestion of the protein, excess trypsin was used for 24 h at 37"C. A representative Chromato graph of the peptides after reverse-phase HPLC sepa ration is presented in Figure 1. All the fractions con taining peptides were manually collected, and eight were subjected to amino acid sequencing. We were able to obtain sequence information from the three peptides marked in Figure 1 as peaks 1, 2 and 3. The sequences determined are shown in Figure 2. There was no full homology of any of the three peptides to sequences of human, rat, mouse and bovine c-GSHPx (5, 6, 28, 29). Eight of the nine amino acids of peptide 3 were found to be homologous to amino acid residues 165-171 and 173 of the human cGSHPx and to residues 164-171 and 172 of the rat, mouse and bovine enzyme as deduced from either their cDNA or amino acid sequences (5, 6, 28, 29). Another homology could be found between three se quential amino acids in peptide 1 (Thr-Phe-Leu) and rat and mouse c-GSHPx amino acids at positions 124—126(5, 28). A combination of two amino acid residues (Asp-fle; He-Leu) is the largest combination in peptide 2 that could be found in the amino acid sequence of all known c-GSHPx, but in more than one location. Thus, at this point, a possible location for peptide 2 along known c-GSHPx sequences could not be determined. The sequences obtained demon strate that p-GSHPx is a product of a separate gene. We examined human milk to determine the source

0.2O

1)

Phe-Tyr-Thr-Phe-Leu-Lys

2)

Met-Asp-Ile-Leu-Ser-Tyr-Met-Arg

0.1-

30

60

90

Time (min)

FIGURE 1 Separation by reverse-phase HPLC of peptides formed by trypsin digestion of purified human plasma glutathione peroxidase (p-GSHPx). Buffer B: 8.7 mmol/L trifluoroacetic acid and 19.5 mol/L acetonitrile. Peaks 1, 2 and 3 correspond to fractions from which sequence infor mation could be obtained. A parallel chromatogram was obtained from a second trypsin-digested p-GSHPx prepara tion. Downloaded from https://academic.oup.com/jn/article-abstract/121/8/1243/4754570 by University of Glasgow user on 06 August 2018

3)

Phe-Leu-Val-Gly-Pro-Asp-Gly-Ile-Pro

c-GSHPx

Phe-Leu-Val-Gly-Pro-Asp-Gly-Val-Pro

165 166 167 168 169 170 171 172 173

FIGURE 2 Amino acid sequences of human plasma glutathione peroxidase (p-GSHPx) peptides formed by trypsin digestion and isolated by reverse-phase HPLC. Se quences of peptides 1, 2 and 3 are the results of sequencing the collected fractions 1, 2 and 3. Approximately 100 pmol of each peptide was subjected to 10 cycles of Edman degra dation. Identical sequences were obtained from a duplicate preparation.

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120 TABLE 1 Glutathione peroxidase activity in plasma, hemolysate milk of lactatÃ-ng women and matched controls'

and c o

SubjectControlLactatingSubstratet-BuOOHH202t-BuOOHHjOzPlasmapJcat/L5.0 Hb23.2 u 9 1.36.7 ± 5.841.9 ± 1.35.7 ± 17.430.3 ± 1.46.6 ± 0.230.50 ± 9.342.6 ± ± 9.0Milkjikat/Z.——0.48 ±0.22 ±1.6Hemolysate\tkat/aunol 'Values are means ±so. Five lactating and five age-matched nonlactating women were tested for hemolysate and plasma glutathione peroxidase (GSHPx) activity, and eight lactating Anti p-GSHPx Anti RBC GSHPx Control women were tested for milk GSHPx activity. No significant dif IgG ference was found between lactating women and controls in GSHPx activity of hemolysate and plasma using either tert-butyl hydroperoxide (t-BuOOH) or H^Oj as substrates (Student's t test, P FIGURE 3 Immunoprecipitation of human milk glutathione peroxidase (GSHPx) activity by anti-RBC > 0.2).

for the extracellular GSHPx. There are two types of peroxidases that can express GSH-dependent activity: glutathione S-transferases, which utilize only lipid hydroperoxides, and the Se-dependent forms (GSHPx), which decompose both lipid hydroperoxides and hydrogen peroxide (1, 2, 30). Therefore, in the presence of adequate concentrations of t-BuOOH, both GSH peroxidase activities would be revealed, whereas in the presence of HjO^, only the Se-de pendent activity would be detected. The difference between the activity in the presence of t-BuOOH vs. HjC^ was used in the literature to measure nonSe-dependent GSHPx activity. However, the relative amount of GSHPx activity with t-BuOOH depends on the latter concentration, on the isoform distribution of GSH S-transferase in a particular tissue or fluid and on the affinity of the different enzymes for the two substrates (30). Defatted milk was used for all the following experi ments. The activity of milk GSHPx in the presence of peroxides when measured against a reference cuvette containing heat-inactivated milk (but not peroxide) was two to three times higher than the activity mea sured against a reference cuvette of freshly frozen milk. This indicated that freshly frozen, defatted milk had a nonspecific NADPH oxidizing activity as well as GSHPx. To subtract this nonspecific oxidation of NADPH, all measurements reported in this work were performed using freshly frozen, defatted milk in the reference cuvette. Table 1 summarizes the activity of GSHPx tested with t-BuOOH and HjOj in plasma, RBC hemolysate and milk of lactating women in comparison with agematched, nonlactating control women. In this small sample of lactating women, the activity of GSHPx in plasma and RBC did not differ significantly from the activity of controls (Student's t test, P > 0.2). HowDownloaded from https://academic.oup.com/jn/article-abstract/121/8/1243/4754570 by University of Glasgow user on 06 August 2018

GSHPx immunoglobulin G (IgG), anti-p-GSHPx-IgG and nonimmune-IgG. Values are expressed as means ±SD. Four women were studied with HjO^ six women were studied with t-BuOOH. Significant difference between specific immunoprecipitation and precipitation with nonimmune-IgG (control) was evident only with the use of anti-p-GSHPxIgG (Student's t test; P < 0.001 for anti-p-GSHPx; P > 0.2 for anti-RBC-GSHPx).

ever, larger groups of lactating and nonlactating women need to be tested to verify this. The GSHPx activity in 1 mL of milk is -10% of the activity in 1 mL of plasma. Activity could be measured using both substrates. Under our assay conditions for measuring the GSHPx with the two substrates, the activity as measured with HjC^ was equal to or higher than the activity measured with t-BuOOH. To identify the forms and quantity of the different GSHPx that might be present in milk, we used spe cific immunoprecipitation assays. The results of the immunoprecipitations by specific antibodies of the two forms of GSHPx are shown in Figure 3. Most of the activity, measured with either t-BuOOH or HjOa, was precipitated by anti-p-GSHPx-IgG. Only negligible amounts were precipitated using anti-RBC GSHPx-IgG compared with precipitation with nonimmune IgG. Student's t test indicated significant specific precipitation only with anti-p-GSHPx-IgG. The precipitation of most of the GSHPx activity in the presence of t-BuOOH by an antibody directed against the Se-dependent GSHPx indicated that there is almost no detectable GSH S-transferase with perox idase activity in milk. However, under our assay con ditions, we might not have detected all the nonSe-dependent GSHPx (30). The fact that almost all the activity was precipitated in the presence of HjOj by anti-p-GSHPx-IgG indicates that most of the Sedependent GSHPx activity in milk is due to the extra cellular form. Frequently, GSHPx activity is used as an indicator of Se status of a specific tissue (1,2). However, tissues

PLASMA GLUTATfflONE

PEROXIDASE IN HUMAN

MILK

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TABLE 2 Analysis

of Se associated

1«GNon-immuneAnti-p-GSHPXExpt.1

21

with glutathione

peroxidase

using anti-p-GSHPx-IgG1

activitynkat3.10 GSHPx

Seng76.4

Sen«14.9

(lOOp (100)0.28 6.30

89.980.3

18.719.5

in GSHPx2%—3.614.3

(9.0)

37.5Se 2Supernatant0.57 (9.1)Supernatant 79.0Precipitate 'Milk samples from two women (Experiments 1, 2) were analyzed individually in duplicate. Se was determined on coded samples. GSHPx, glutathione peroxidase; IgG, immunoglobulin G, p-GSHPx, plasma glutathione peroxidase. aThe percentage of Se in GSHPx - 100 x (Se specifically precipitated)/(Se specifically precipitated + Se in supernatant). 3The percentage of GSHPx activity calculated from the activity of the control (in the presence of nonimmune IgG) is given in parenthesis.

DISCUSSION as well as milk contain other selenoproteins (19, 20). Therefore, it is important to determine the amount of Se associated with GSHPx. Because almost all of the GSHPx activity could be precipitated from milk by anti-p-GSHPx-IgG (Fig. 3), the measured amount of Se in the precipitate corresponds to almost all the Se associated with GSHPx in the original sample, plus the amount of Se associated with the added antibody. The concentrations of Se in milk of three women were 13.3, 13.3 and 12.6 |ig/L. To be within the sensitivity of the method for Se determination when measuring 5% of the foregoing concentrations (to be able to detect as low as 5% of Se associated with GSHPx in the sample), we used 7.5 mL or more of milk. This assay required a large amount of antibody, and was therefore performed on only two milk sam ples. Protein-A-Sepharose was preincubated with either nonimmune rabbit IgG or with anti-p-GSHPx-IgG; milk was then added under conditions that were pre viously shown to precipitate almost all the GSHPx activity from milk (Fig. 3). The mixtures were centrifuged, GSHPx activity was determined in the super natant and the Se content was measured in both the supernatant and the sediment. The results of the ex periment are presented in Table 2. Anti-p-GSHPx-IgG precipitated 90% of milk GSHPx measured with either t-BuOOH or HjOî, whereas with nonimmune IgG no activity could be precipitated from the milk. There was nonspecific precipitation of Se with nonimmune IgG that amounted to 16.3 and 17.2% of total Se in samples 1 and 2, respectively. Taking this nonspecific Se precipitation into account and as suming that the 10% of GSHPx that was not precipi tated is also p-GSHPx, the specific percentage of Se associated with GSHPx was 3.6% and 14.3% for samples 1 and 2, respectively. Downloaded from https://academic.oup.com/jn/article-abstract/121/8/1243/4754570 by University of Glasgow user on 06 August 2018

Both P-GSHPx and c-GSHPx are tetramere. Each has four Se molecules, and both interact with lipid hydroperoxides and H¡O2.However, they are kinetically, structurally and antigenically different (11-14). The enzymes could be products either of the same gene but post-translationally modified, or of separate genes and subject to separate genetic control. In this study we have shown that p-GSHPx and c-GSHPx are the products of two different genes (Fig. 2). These data are consistent with a recently published report of the cDNA for p-GSHPx cloned from human placenta (31). A GSHPx form antigenically corresponding to pGSHPx was found in the extracellular medium of a hepatoma cell Une (15). The liver cell is believed to secrete this form of GSHPx into plasma. Milk is another extracellular fluid known to contain a GSHdependent peroxidase activity (17-23). However, the form, the cell of origin or the function of this perox idase had not been investigated previously. Possibil ities existed that the enzyme could be secreted by the liver and transported into the breast milk via the blood stream, or it could be secreted by the mammary gland. Alternatively, the cellular form could be present in milk as a result of leakage from the mammary cells or other cells present in breast mille Debski et al. (19) and Milner et al. (20) reported that only a third of total GSH-dependent peroxidase activity in milk can be attributed to Se-dependent GSHPx activity. They based this number on the dif ference between the activity of the enzyme in the presence of t-BuOOH and HjOz. In their study, the total GSHPx activity, measured with t-BuOOH, was about 2.08 nkat/L (125 mU/mL) (19, 20), four times higher than we obtained (Table 1). This discrepancy in the measured activity could be explained on the basis of using different methods for determination of

1248

AVISSAR ET AL.

GSH-dependent GSHPx activity. Notable in their study is the high concentration of t-BuOOH (5 mmol/ L) and the use of heat-inactivated milk as a blank (19), both contributing to nonspecific oxidation of NADPH. Therefore, their assay (19) determined the specific and nonspecific oxidation of NADPH. The nonspecific degradation of NADPH, which could be equivalent to part of the activity removed by dialysis, may have contributed to the high GSH-dependent activity found (20). The assay conditions for GSHPx activity using t-BuOOH or iÃ-¡O2 are not equivalent because the K,,, values for the two substrates are different. Therefore, a direct comparison of units using the two substrates is not accurate (11-14, 30). Using gel chromatography separation, Debski et al. (19) showed that a protein with GSHPx activity having a similar molecular weight to GSH S-transferase is also present in milk Immunoprecipitation of the activity by specific an tibodies directed against the different protein forms of the enzyme was our method of choice to reliably identify the forms of GSH-dependent peroxidase ac tivity in human milk. Our studies showed that 90% of the GSH-dependent peroxidase activity in milk is due to the extracellular form of the Se-dependent enzyme, that the amount of Se dependent c-GSHPx is negligible, and that, if there is another GSH-de pendent peroxidase in milk, it does not account for more than 10% of total GSH-dependent peroxidase (unless this peroxidase was not detected under our assay conditions). The activity with HjOz that was not precipitated by anti-p-GSHPx-IgG (10%) could be due to c-GSHPx. However, these small differences could be within the limits of the sensitivity of the assay, and p-GSHPx could be the only GSH-de pendent peroxidase in human milk Milncr et al. (20), using gel filtration separation, found that 15-30% of Se in milk was associated with GSHPx. The values obtained using gel filtration tech nique are based on the assumption that the peak corresponding to GSHPx activity does not overlap with other selenoproteins, and that there is no GSHPx activity associated with other proteins. As previously discussed, this method cannot be used to measure accurately the amount of Se in GSHPx (12). We previously showed (12), using specific immunoprecipitations, that -12% of Se is associated with GSHPx in plasma of two North American women. The ratio of p-GSHPx activity to Se in these experiments was 1 (ikat to 3.23 |ig of Se. Assuming that there are no specific activators or inhibitors of GSHPx in milk that are not present in plasma, the amount of Se due to GSHPx can be calculated on the basis of this ratio. GSHPx activity in the two milk samples was 0.40 ukat/L and 0.72 jikat/L, and Se in each of the samples was 13.3 ng/L. Therefore, the percentage of Se calculated to be associated with GSHPx is 9.5 and 17.4%, respectively. The calculated Downloaded from https://academic.oup.com/jn/article-abstract/121/8/1243/4754570 by University of Glasgow user on 06 August 2018

percentages are somewhat higher than the measured ones, but they are in close agreement. Thus, only a «mallfraction of Se is associated with GSHPx in human milk. Therefore, the reliability of measuring the Se status of milk (as well as plasma and hemolysate Se status) by determining GSHPx activity is questionable. Milk has been reported to contain at least 44 en zymes, including xanthine oxidase, sulfhydryl oxidase and Superoxide dismutase (32, 33). The products of the reaction of these enzymes in milk could be lipid hydroperoxides or H/^. Although not yet estab lished, GSHPx in milk (inside or outside the breast cells) could be responsible for the neutralization of these damaging oxidants. Whether GSHPx is func tionally important to the integrity of mille remaina to be determined.

LITERATURE CITED 1. Oldfield, J. E. (1987) The two faces of selenium. J. Nutr. 117: 2002-2008. 2. Levander, O. A. (1987) A global view of human selenium nutrition. Annu. Rev. Nutr. 7: 227-250. 3. Olson, O. £.(1986) Selenium toxicity in animals with em phasis on man. J. Am. Coll. Toxicol. 5: 45-70. 4. Forstrom, J. W., Zakowski, J. J. &.Tappel, A. L. (1978)Identifi cation of the catalytic site of rat liver glutathione peroxidase as selenocysteine. Biochemistry 17: 2639-2644. 5. Chambers, L, Framptom, J., Goldfarb, P., Affara, N., McBain, W. & Harrison, P. R. (1986) The structure of the mouse glutathione peroxidase gene: the selenocysteine in the active site is encoded by the termination codon TGA EMBO J. 5: 1221-1227. 6. Mullenbach, G. T., Tahrizi, A, Irvine, B. D., Bell, G. I. &. Hallewell, A, R. (1987) Sequence of a cDNA coding for human glutathione peroxidase confirms TGA encodes active site sele nocysteine. Nucleic Acids Res. 15: 5485. 7. McConnell, K. P. &.Hoffman, J. L. (1972) Methionine-selenomethionine parallels in rat liver polypeptide chain synthesis. FEES Lett. 24: 60-62. 8. Halli well, B. (1987) Oxidants and human disease: some new concepts. FASEB J. 1: 358-364. 9. Machlin, L. J. & Bendick, A (1987)Free radical tissue damage: protective role of antioxidant nutrients. FASEBJ. 1: 441-445. 10. Hyslop, P. A, Hinshaw, D. B., Halsey, W. A, Jr., Schraufstatter, I. U, Sauerherber, R. D., Spragg,T. G., Jackson, J. H. & Cochrane, C. G. (1988) Mechanism oÃ-oxidan t mediated cell injury. J. Biol. Chem. 263: 1665-1675. 11. Takahashi, K., Avissar, N., Whitin, J. & Cohen, H. J. (1987) Purification and characterization of human plasma glutathione peroxidase: a selenoglycoprotein distinct from known cellular enzyme. Arch. Biochem. Biophys. 256: 677-686. 12. Avissar, N., Whitin, J. C., Allen, P. Z., Palmer, L S. & Cohen, H. J. (1989) And human plasma glutathione peroxidase anti bodies: immunologie investigations to determine plasma glutathione peroxidase protein and selenium content in plasma. Blood 73: 318-323. 13. Broderick, D. J., Deagan, J. T. &. Whanger, P. O. (1987) Prop erties of glutathione peroxidase isolated from human plasma. J. Ihorg. Biochem. 30: 299-308. 14. Maddipati, K. R. A.Mamett, L. J. (1987)Characterization of the major hydroperoxide reducing activity of human plasma: puri fication and properties of selenium dependent glutathione per-

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oxidase. J. Biol. Chem. 262: 17398-17403. 15. Avissar, N., Whitin, J. C., Allen, P. Z., Wagner, D. D., Liegey, P. & Cohen, H. J. (1989) Plasma selenium dependent glutathione peroxidase: cell of origin and secretion. J. Biol. Chem. 264: 15850-15855. 16. Maddipati, K. R., Casparski, C. & Mamett, L. J. (1987)Charac terization of the hydroperoxide-reducing activity of human plasma. Arch. Biochem. Biophys. 254: 9-17. 17. Hojo, Y. (1987) Selenium and glutathione peroxidase in human saliva and other human body fluids. Sci. Total Envir. 65: 85-94. 18. Hojo, Y. (1986) Sequential study on glutathione peroxidase and selenium contents of human milk Sci. Total Environ. 52: 83-91. 19. Debski, B., Picciano, M. F. &. Milner, J. A. (1987) Selenium content and distribution of human, caw and goat milk. J. Nutr. 117: 1091-1097. 20. Milner, J. A, Sherman, L. Ä.Picciano, M. F. (1987) Distri bution of selenium in human milk. Am. J. Clin Nutr. 45: 617-624. 21. Mannan, J. A. & Picciano, M. F. (1987) Influence of maternal selenium status on human milk selenium concentration and glutathione peroxidase activity. Am. J. Clin. Nutr. 46: 95-100. 22. Debski, B., Finely, D. A., Picciano, M. F., Lönnerdal,B. & Milner, J. (1989) Selenium content and glutathione peroxidase activity of milk from vegetarian and non-vegetarian women. J. Nutr. 119: 215-220. 23. Ellis, L., Picciano, M. F., Smith, A M., Hamosh, M. & Mehta, N. K. (1990) The impact of gestational length on human milk selenium concentration and glutathione peroxidase activity. Pediat. Res. 27: 32-35. 24. Awasthi, Y. C., Beutler, E. & Srivastava, S. K. (1975) Purifi

Downloaded from https://academic.oup.com/jn/article-abstract/121/8/1243/4754570 by University of Glasgow user on 06 August 2018

25. 26. 27. 28.

29.

30.

31.

32. 33.

MILK

1249

cation and properties of human erythrocyte glutathione peroxi dase. J. Biol. Chem. 250: 5144-5149. Slemmon, J. R. (1989) Sequence analysis of a proteolyzed site in Drosophila cholinc acetyltransferase. J. Neurochcm. 52: 1898-1904. Beutler, E. (1984) Red Cell Metabolism: A Manual of Bio chemical Methods, ed. 3, pp. 74-76, Gruñe&. Straton, Phila delphia, PA. Koh, T-S. &.Benson, T. H. (1983)Critical reappraisal of fluorometric method for determination of selenium in biological materials. J. Assoc. Off. Anal. Chem. 66: 918-926. Mullenbach, G. T., Tabrizi, A, Irvine, B. D., Bell, G. L, Trainer, J. A &.Hallewell, R. A (1988)Selenocysteine's mech anism of incorporation and evolution revealed in cDNAs of three glutathione peroxidase. Protein Eng. 2: 239-246. Gunzler, W. A, Steffens, G. J., Grossmann, A, Kim, S-M. A, Otting, F., Wendel, A &. Flohe, L. (1984) The amino acid sequence of bovine glutathione peroxidase. Hoppe-Seyler's Z. Physiol. Chem. 365: 195-212. Lawrence, R. A. &.Burk, R. F. (1978) Non-selenium dependent glutathione peroxidase. In: Functions of Glutathione in Liver and Kidney (Sies, H. &.Wendel, A, eds.) pp. 114-119, SpringerVerlag, New York, NY. Takahashi, K., Akasaka, M., Yamamoto, Y., Kobayashi, C., Mizoguchi, J. & Koyama, J. (1990)Primary structure of human plasma glutathione peroxidase deduced from cDNA sequences. J. Biochem. 108: 145-148. Lawrence, R. A (1989)Breastfeeding: A Guide for the Medical Profession, ed. 3, pp. 106-110, C. V. Mosby, St. Louis, MO. Willinger, L., Rosenfeld, W., Koo, H-C. & Concepción, L. (1990) Superoxide dismutase concentration in human breast milk. Pediat. Res. 27: 120A (abs. 702).