AJCN 19th International Congress of Nutrition
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shin, K.
Right arrow Articles by Lönnerdal, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shin, K.
Right arrow Articles by Lönnerdal, B.
Agricola
Right arrow Articles by Shin, K.
Right arrow Articles by Lönnerdal, B.
American Journal of Clinical Nutrition, Vol. 73, No. 5, 984-989, May 2001
© 2001 American Society for Clinical Nutrition

Original Research Communication

Purification and quantification of lactoperoxidase in human milk with use of immunoadsorbents with antibodies against recombinant human lactoperoxidase1,2

Kouichirou Shin, Hirotoshi Hayasawa and Bo Lönnerdal

1 From the Nutritional Science Laboratory, Morinaga Milk Industry Co Ltd, Zama, Kanagawa, Japan, and the Department of Nutrition, University of California, Davis.

2 Address reprint requests to B Lönnerdal, Department of Nutrition, University of California, One Shields Ave, Davis, CA 95616. E-mail: bllonnerdal{at}ucdavis.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Two heme-containing peroxidases, secretory lactoperoxidase and leukocyte-derived myeloperoxidase, which play host defense roles through antimicrobial activity, were previously identified in human colostrum. Within several days after the start of lactation, the relative contribution of myeloperoxidase to the peroxidase activity in milk was shown to decline as the number of milk leukocytes decreased.

Objective: Our knowledge of lactoperoxidase in human milk is still limited. The objective of this study was to use specific antibodies as a means of simplifying the purification and quantification of lactoperoxidase.

Design: Polyclonal antibodies were raised against recombinant human lactoperoxidase. Immunoglobulin G (IgG) was isolated by means of a protein A column and was characterized by immunoblotting. For the purification of lactoperoxidase from whey, a cation-exchange column and an immunoaffinity column with coupled IgG were used. The concentration of lactoperoxidase was determined by a sandwich enzyme-linked immunosorbent assay by using purified native lactoperoxidase as a standard. Native and biotinylated IgG were used as capture and detector antibodies, respectively.

Results: Two bands with molecular masses of {approx}80 and 100 kDa were detected in an immunoblot of human whey. Similar heterogeneity was observed in the sodium dodecyl sulfate–polyacrylamide gel electophoresis profile of purified lactoperoxidase. The mean (±SD) concentration of lactoperoxidase in 26 whey samples was estimated to be 0.77 ± 0.38 mg/L. The concentrations were positively correlated with the peroxidase activity detected in these samples.

Conclusion: Lactoperoxidase is commonly present in human milk throughout the lactation period and is likely to contribute to the protective effects of milk.

Key Words: Lactoperoxidase • myeloperoxidase • human milk • recombinant human lactoperoxidase • immunoaffinity chromatography • immunoblotting • enzyme-linked immunosorbent assay • ELISA • SDS-PAGE


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
There is much evidence that breast-feeding protects infants against infectious diseases (1, 2). Breast milk contains a variety of immunologic components, such as immunoglobulins, immune cells, and immunomodulatory components, and nonspecific antimicrobial factors, such as lactoferrin, lysozyme, and peroxidases (3, 4). These components are likely to contribute to the protective effects of breast milk. Two heme-containing peroxidases (EC 1.11.1.7), lactoperoxidase secreted from the mammary gland and myeloperoxidase derived from leukocytes, were identified in human colostrum (5–8). Lactoperoxidase is a monomeric glycoprotein with a molecular mass of {approx}80 kDa that is also found in other secretions, such as saliva and tears (9). Myeloperoxidase is a tetrameric glycoprotein consisting of two 59-kDa heavy chains and two 13.5-kDa light chains in its mature form; it is found in the azurophilic granules of neutrophils (10). Products of oxidation of certain halides and thiocyanate, generated in reactions catalyzed by these peroxidases, have broad-spectrum antimicrobial activity (9, 10).

Total peroxidase activity in human milk is highest in colostrum and declines rapidly during the first several days of lactation (11). This phenomenon appears to be associated with a decrease in the number of milk leukocytes and of milk-leukocyte-derived myeloperoxidase activity during this period (5, 12). Recently, we partially purified the enzyme responsible for the peroxidase activity in mature milk and identified it as lactoperoxidase (13). We also cloned lactoperoxidase complementary DNA from the human mammary gland and expressed the gene in a baculovirus–insect cell system (14). Recombinant human lactoperoxidase was purified and characterized in terms of its physical and catalytic properties (14). However, our knowledge of lactoperoxidase in human milk is still limited. Detailed studies of the characteristics and distribution of lactoperoxidase are essential for elucidating the importance of this enzyme in human milk. In the present study, we purified lactoperoxidase from human milk by immunoaffinity chromatography and determined the concentration of lactoperoxidase in milk samples by a sandwich enzyme-linked immunosorbent assay (ELISA) by using antibodies raised against recombinant human lactoperoxidase.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Columns for purification (1 mL HiTrap rProtein A, 5 mL HiTrap SP, and 1 mL HiTrap NHS-activated), PD-10 desalting columns, and enhanced chemifluorescence substrate were purchased from Amersham Pharmacia Biotech (Uppsala, Sweden). Nylon membrane filter cartridges with a 0.45-µm pore size were obtained from Gelman Sciences (Ann Arbor, MI). We used a centrifugal membrane concentrator (Centricon 30) from Amicon (Beverly, MA). Lactoperoxidase purified from bovine milk (bovine lactoperoxidase; A412/A280 = 0.93) and 3,3',5,5'-tetramethylbenzidine (TMB) were from Sigma Chemical Co, St Louis. Myeloperoxidase purified from human leukocytes (A430/A280 = 0.73) was from Athens Research and Technology (Athens, GA). Alkaline phosphatase–conjugated goat anti-rabbit immunoglobulin G (IgG) was from Life Technologies (Gaithersburg, MD). Alkaline phosphatase–conjugated streptavidin and N-hydroxysulfosuccinimidobiotin were from Pierce Chemical Co (Rockford, IL). The other chemicals we used were of the highest grade available.

Samples
Human milk samples were donated by healthy mothers at various times of lactation and were kept frozen at –20°C until used. Each milk sample was ultracentrifuged at 264000 x g for 1 h at 4°C to separate the lipid and casein micelles from the whey. The lipid layer was removed and the supernatant fluid (human whey) was filtered through a membrane cartridge. Human saliva samples were collected from healthy adults by expectoration and were centrifuged at 26000 x g for 15 min at 4°C to remove particles. The protocol was approved by the Human Subjects Review Committee at the University of California, Davis.

The bovine milk samples were obtained from Holstein cows at the University of California Dairy Barn. Bovine milk was centrifuged at 4000 x g for 30 min at 4°C to remove the fat. The skim milk was adjusted to a pH of 4.5 with 2 mol HCl/L and incubated for 1 h at 4°C. Bovine whey was obtained by centrifuging the skim milk at 26000 x g for 30 min at 4°C and was adjusted to pH 6.5 with 2 mol NaOH/L.

Methods
Recombinant human lactoperoxidase was expressed in a baculovirus–insect cell system and purified according to the procedure described by Shin et al (14). Expression of catalytically active recombinant human lactoperoxidase was carried out by using Tricoplusia ni cells cultured in a serum-free culture medium supplemented with the heme precursor {delta}-aminolevulinic acid. The purification procedure involved fractionation of the culture supernatant fluid by cation-exchange chromatography followed by hydrophobic interaction chromatography. Our previous deglycosylation study using peptide-N-glycosidase F showed that recombinant human lactoperoxidase is glycosylated to an extent similar to that of bovine lactoperoxidase (14). We also observed that native human lactoperoxidase is glycosylated to an extent similar to that of bovine lactoperoxidase (13). However, the full structures of the glycans in the case of both recombinant and native human lactoperoxidase are still unknown. Polyclonal antibodies against recombinant human lactoperoxidase were produced by Antibodies, Inc (Davis, CA). A standard immunization protocol was used to immunize female New Zealand white rabbits. Purified recombinant human lactoperoxidase (100 µg) was injected 5 times at intervals of 2 wk. For the isolation of IgG, 3 mL of the antiserum was applied to the HiTrap rProtein A column, which had been equilibrated with 20 mmol sodium phosphate buffer/L, pH 7.0. After the column had been washed with the same buffer, the IgG was eluted with 0.1 mmol sodium citrate buffer/L, pH 3.0, and immediately neutralized with 1 mol tris-HCl buffer/L, pH 9.0. The purified IgG was desalted and, through buffer exchange using a PD-10 column, the buffer was replaced with phosphate-buffered saline (PBS; 138 mmol NaCl/L, 2.7 mmol KCl/L, and 10 mmol phosphate buffer/L, pH = 7.4). The IgG concentration was calculated on the basis of A280 = 13.8 for a 1% solution. A portion of the purified IgG was biotinylated by incubation with a 20-fold molar excess of N-hydroxysulfosuccinimidobiotin in PBS on ice for 2 h. To remove unreacted biotin, the reaction mixture was applied to a PD-10 column.

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed by using the method of Laemmli (15). The acrylamide concentration of the gels we used was 8% throughout the investigation. To detect the proteins, the gels were stained with Coomassie Brilliant Blue R (Sigma) and destained with a mixture of 25% ethanol and 10% acetic acid.

The proteins separated by SDS-PAGE were electrotransferred to a polyvinylidene difluoride membrane by using the method of Towbin et al (16) and a Hoefer (San Francisco) model TE22 transfer apparatus. After electrotransfer, the blots were blocked at room temperature for 1 h with 5% bovine serum albumin (BSA) in PBS containing 0.1% Tween 20 (Sigma; PBS-Tween). The blots were then washed 3 times in PBS-Tween for 10 min and incubated for 1 h with 5 mg anti-recombinant human lactoperoxidase IgG/L in PBS-Tween. After 3 washes with PBS-Tween for 10 min, the blots were incubated for 1 h in a solution of alkaline phosphatase–conjugated goat anti-rabbit IgG at a dilution of 1:10000 in PBS-Tween. After 3 washes with PBS-Tween for 10 min, the blots were incubated with enhanced chemifluorescence reagent at room temperature for 10 min and visualized by means of a Storm 860 fluorescence scanning system (Molecular Dynamics, Sunnyvale, CA).

For purification of native human lactoperoxidase, 50 mL whey was diluted 2-fold with 50 mmol sodium phosphate buffer/L (pH 6.4) and applied to the HiTrap SP column, which had been equilibrated with the same buffer. The column was washed until the absorbance at 280 nm returned to the baseline level. Then, a linear gradient of 0–1 mol NaCl/L was applied by using a fast-protein liquid chromatography system that was programmed for 60 min at a flow rate of 5 mL/min. Fractions were collected every 1 min and the peroxidase activity in each fraction was assayed. Fractions showing activity were pooled and neutralized with 0.2 mol dibasic sodium phosphate/L. A portion of the purified anti-recombinant human lactoperoxidase IgG was coupled to a HiTrap NHS–activated column according to the manufacturer's instructions. The column was washed with 0.1 mol acetic acid/L and then equilibrated with 25 mmol sodium phosphate buffer/L (pH 7.0) containing 0.5 mol NaCl/L. The active fraction from cation-exchange chromatography was applied at a flow rate of 1.0 mL/min. The column was washed until the absorbance at 280 nm returned to the baseline level. The protein was eluted by lowering the pH with 0.1 mol acetic acid/L (pH 2.8) at a flow rate of 1.0 mL/min. Fractions were collected every 1 min and adjusted to pH 5.0 with 0.5 mol sodium acetate/L. The fractions showing absorbance at 280 nm were pooled. Through buffer exchange with a centrifugal concentrator, the buffer was replaced with 10 mmol sodium acetate buffer/L (pH 5.4) containing 0.1 mol sodium sulfate/L, and the sample was then concentrated down to 72 µL.

The wells of microtiter plates (Nalge Nunc International, Rochester, NY) were coated with 100-µL aliquots of 5 mg anti-recombinant human lactoperoxidase IgG/L in 20 mmol sodium carbonate buffer/L (pH 9.6) and the plates were incubated overnight at 4°C. The wells were washed 3 times with 200 µL washing buffer (PBS containing 0.05% Tween 20) and saturated by incubation with 200 µL dilution buffer (1% BSA in PBS containing 0.05% Tween 20) at 37°C for 1 h. The wells were then washed 3 times with washing buffer. Solutions of purified recombinant human lactoperoxidase, native human lactoperoxidase, bovine lactoperoxidase, and myeloperoxidase were prepared in the dilution buffer at concentrations ranging from 0.49 to 1000 µg/L. Whey samples were diluted 100-fold in the same buffer. Aliquots of each of these solutions were added to the washed wells in triplicate at 100 µL/well and the plates were incubated at 37°C for 2 h. After the wells had been washed 3 times with washing buffer, 100-µL aliquots of 20 mg biotinylated anti-recombinant human lactoperoxidase IgG/L in the dilution buffer was added and the plates were incubated at 37°C for 2 h. The wells were washed 3 times with washing buffer. Then, 100-µL aliquots of 2 mg alkaline phosphatase–conjugated streptavidin/L in dilution buffer was added to the washed wells and the plates were incubated in the dark at room temperature for 30 min. After the wells had been washed 3 times with washing buffer, 100 µL phosphatase substrate (0.5 g p-nitrophenylphosphate/L in 1.0 mol/L diethanolamine buffer, pH 9.8) was added to the wells and the plates were incubated at 37°C for 1 h. The absorbance at 405 nm was measured by using a Multiskan Ascent plate reader (Labsystems Inc, Franklin, MA). The data were analyzed by means of ASCENT (version 2.3; Labsystems Oy, Helsinki, Finland) and a 4-variable logistic model.

Peroxidase activity was assayed by measuring hydrogen peroxide–dependent oxidation of TMB as described by Thomas et al (17) with slight modifications (13). Protein content was determined by the method of Bradford (18) with use of BSA as the standard.

Data analysis
Least-squares linear regression analysis was performed with use of KALEIDA GRAPH for WINDOWS (version 3.09; Synergy Software, Reading, PA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of anti-recombinant human lactoperoxidase IgG
Immunoblotting was performed to characterize the anti- recombinant human lactoperoxidase IgG. Two proteins with molecular masses of {approx}80 and 100 kDa were detected in human whey (Figure 1Go). Two bands of proteins with similar molecular masses were also observed in analysis of human saliva. Purified bovine and recombinant human lactoperoxidase showed a band at a position corresponding to a mass of 80 kDa. Because the amount of each purified protein used in this experiment was 10 ng, it seemed that the reactivity of anti-recombinant human lactoperoxidase IgG against bovine lactoperoxidase was weaker than that against recombinant human lactoperoxidase. The molecular mass of the immunoreactive protein detected in analysis of purified bovine lactoperoxidase was smaller than that of the one detected in analysis of bovine whey, suggesting that proteolysis may have occurred during the preparation of this enzyme. Unlike lactoperoxidase, myeloperoxidase was not detected by the immunoblotting. These results indicate that anti-recombinant human lactoperoxidase IgG can specifically recognize lactoperoxidase in human whey and saliva.



View larger version (93K):
[in this window]
[in a new window]
  		FIGURE 1. . Immunoblot using anti-recombinant human lactoperoxidase immunoglobulin G. Samples of human saliva and whey, recombinant human lactoperoxidase, bovine whey, lactoperoxidase purified from bovine milk (bovine lactoperoxidase), and human myeloperoxidase were subjected to electrophoresis on an 8% acrylamide gel and transferred to a polyvinylidene difluoride membrane. The amounts used were 10 µL in the case of the saliva and whey samples and 10 ng in the case of the purified proteins. The membrane was incubated with recombinant human lactoperoxidase immunoglobulin G and alkaline phosphatase–conjugated anti-rabbit immunoglobulin G. The proteins were visualized with use of enhanced chemifluorescence substrate and the Storm system (see Materials and methods).

 
Purification of lactoperoxidase from human milk
The active fraction obtained after fractionation of whey by cation-exchange chromatography was further purified by immunoaffinity chromatography. In a typical round of purification, lactoperoxidase was purified 3397-fold from human whey; the recovery was 7.6% (Table 1Go). The specific activity of the purified lactoperoxidase was 625 units/mg. As shown in the SDS-PAGE profile in Figure 2Go, the purified lactoperoxidase appeared as 2 bands at positions corresponding to molecular masses of {approx}80 and 100 kDa. These values correspond well with the molecular mass estimates obtained in our analysis of lactoperoxidase in human whey by immunoblotting, and this lactoperoxidase preparation did not contain any other proteins.


View this table:
[in this window]
[in a new window]
  		TABLE 1.. Purification of human lactoperoxidase1
 


View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 2. . Sodium dodecyl sulfate–polyacrylamide gel elecrophoresis profile of lactoperoxidase purified from human whey. Molecular weight markers (left lane) and purified human lactoperoxidase (1.2 µg; right lane) were subjected to electrophoresis on an 8% acrylamide gel and stained with Coomassie Brilliant Blue R.

 
Standard curve for ELISA and cross-reactivity
To develop a sandwich ELISA system for human lactoperoxidase, biotinylated anti-recombinant human lactoperoxidase IgG was used as the detector antibody, which could be recognized by alkaline phosphatase–conjugated streptavidin. Typical standard curves for the ELISA are shown in Figure 3Go. In the ELISA, native human lactoperoxidase showed slightly lower absorbance than did recombinant human lactoperoxidase. Therefore, we chose purified native human lactoperoxidase as the standard in the following quantification of lactoperoxidase. In the case of both recombinant human and native lactoperoxidase, the lower and upper limits of detection were {approx}0.5 and 1000 µg/L, respectively. No substantial cross-reaction was observed against either human myeloperoxidase or bovine lactoperoxidase at concentrations <=1000 µg/L, indicating high specificity of the ELISA system.



View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 3. . Typical standard curves for enzyme-linked immunosorbent assay. Purified recombinant human lactoperoxidase ({circ}), human lactoperoxidase (•), lactoperoxidase purified from bovine milk ({square}), and human myeloperoxidase ({blacktriangleup}) were prepared in the range of 0.49 to 1000 µg/L in 1% bovine serum albumin and 0.05% Tween 20 in phosphate-buffered saline solution (see Materials and methods). Values represent means of quadruplicate assays in the case of recombinant and native human lactoperoxidase and means of duplicate assays in the case of bovine lactoperoxidase and human myeloperoxidase.

 
Quantification of lactoperoxidase in human whey
Quantification of lactoperoxidase in mature human whey was carried out by ELISA, with use of native human lactoperoxidase as the standard in 26 samples collected at 1–5 mo postpartum from 13 mothers. The mean (±SD) concentration of lactoperoxidase was calculated to be 0.77 ± 0.38 mg/L. We also compared the concentration of lactoperoxidase in each sample with the peroxidase activity detected on the basis of hydrogen peroxide– dependent oxidation of TMB as shown in Figure 4Go. A correlation between the concentrations determined by ELISA (y) and the levels of peroxidase activity (x) was found by linear regression analysis (y = 0.21x + 0.47, r = 0.84).



View larger version (11K):
[in this window]
[in a new window]
  		FIGURE 4. . Correlation between concentrations of lactoperoxidase as determined by enzyme-linked immunosorbent assay and the levels of peroxidase activity measured on the basis of the oxidation of 3,3',5,5'-tetramethylbenzidine in whey prepared from mature human milk. Values represent means of duplicate assays with these 2 methods. The correlation was calculated as y = 0.21x + 0.47, r = 0.84, by linear regression analysis.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study showed for the first time the quantification of lactoperoxidase in human milk at the protein level. We produced polyclonal antibodies against recombinant human lactoperoxidase and tested their usefulness in the detection, quantification, and purification of human lactoperoxidase. During the course of our previous purification study, we noticed that the low concentrations of lactoperoxidase in human milk make the purification of this enzyme extremely difficult (13). Indeed, complete purification of lactoperoxidase from human milk was not achieved in any previous study carried out by our group or others using conventional column chromatography procedures (7, 8, 13). Our system for expression of recombinant human lactoperoxidase (14) is reproducible and can be used to obtain sufficient amounts of antigen to raise specific antibodies. The antibodies reacted with 2 proteins with molecular masses of 80 and 100 kDa in the analysis of human whey samples by immunoblotting. These 2 bands were also detected in the SDS-PAGE profile of human lactoperoxidase purified by cation-exchange and immunoaffinity chromatography. In a previous study, we observed similar molecular heterogeneity in a preparation of partially purified human lactoperoxidase, which was identified by immunoblotting with antibody raised against bovine lactoperoxidase, and the heterogeneity was further confirmed by amino acid sequencing (13). Thus, it is reasonable to conclude that both the 80-kDa and the 100-kDa proteins observed in this study are lactoperoxidase.

Because lactoperoxidase and myeloperoxidase catalyze the oxidation of a wide variety of substrates and have similar specificity, identification of the species of peroxidase in human milk cannot be identified simply by measuring the peroxidase activity. In addition, endogenous thiocyanate, iodide, and other materials in milk could compete in the peroxidase assay and make quantification of the enzyme difficult. Our data indicate that the ELISA procedure has advantages for both identification and quantification of lactoperoxidase in human milk. An immunometric assay was reported for the quantification of a secretory peroxidase (salivary peroxidase; SPO) in human saliva (19, 20). This assay with anti-bovine lactoperoxidase, together with purified bovine lactoperoxidase as the standard, relies largely on the cross-reactivity of the anti-bovine lactoperoxidase antibodies against SPO (19, 20). Genetic data from previous studies suggested that SPO is another product of the single chromosomal gene for lactoperoxidase (14, 21–23). However, other investigators reported low cross-reactivity of anti-bovine lactoperoxidase antibody against SPO (17, 24). We produced anti-recombinant human lactoperoxidase antibodies, which showed strong immunoreactivity with native human lactoperoxidase and no cross-reactivity in the ELISA. This characteristic of the antibodies is useful to make the ELISA system specific for human lactoperoxidase. We used purified native human lactoperoxidase as the standard in the ELISA for accurate quantification. The ELISA detects human lactoperoxidase at concentrations as low as 0.5 µg/L (Figure 3Go) and is much more sensitive than is the conventional peroxidase assay. The average concentration of lactoperoxidase in the 26 samples of whey prepared from mature human milk as determined by the ELISA was 0.77 ± 0.38 mg/L. There was a positive correlation between the concentrations of lactoperoxidase and the levels of peroxidase activity in these samples (Figure 4Go). These results provide evidence that lactoperoxidase is commonly present in human milk. In addition to the positive but relatively poor correlation, we observed a high value for the intercept. Our ELISA might have detected a variable amount of nonheme lactoperoxidase, which has been found in the milk of several species, including cows and humans, in a catalytically inactive form (25). The specific activity of human lactoperoxidase is estimated to be 2.52 units/µg on the basis of the correlation determined in Figure 4Go. Because purified lactoperoxidase from human milk showed a lower specific activity (0.625 units/µg), this preparation might have been partially inactivated during purification.

The antimicrobial activity of lactoperoxidase of bovine origin has been well characterized in vitro (26). Thiocyanate in animal secretions was identified as the physiologic substrate associated with the antimicrobial activity of lactoperoxidase (9, 26). Therefore, it is likely that lactoperoxidase contributes to some extent to the protective effects of human milk. However, further study of lactoperoxidase in terms of its protective effects is needed at the physiologic concentration present in human milk. Besides its antimicrobial activity, lactoperoxidase catalyzes the oxidation of several types of organic molecules such as thiols, phenols, and aromatic amines (9). Products of oxidation of halides and thiocyanate, generated in reactions catalyzed by lactoperoxidase, can also chemically modify several compounds. Through these catalytic activities, lactoperoxidase may play some physiologic roles in the lactating mammary gland and in the gastrointestinal tract of infants. One possible function of lactoperoxidase that affects the biochemical and nutritional properties of human milk is iodination of proteins (27, 28). A close correlation between the intracellular distribution of iodinated milk proteins and that of endogenous peroxidase activity was shown in the lactating mammary gland of rats (29). Continuing human studies will yield further insight into the physiologic roles of lactoperoxidase.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Cunningham AS, Jelliffe DB, Jelliffe EFP. Breast-feeding and health in the 1980s: a global epidemiologic review. J Pediatr 1991;118: 659–66.[Medline]
  2. Dewey KG, Heinig MJ, Nommsen-Rivers LA. Differences in morbidity between breast-fed and formula-fed infants. J Pediatr 1995;126: 696–702.[Medline]
  3. Lönnerdal B. Biochemistry and physiological function of human milk proteins. Am J Clin Nutr 1985;42:1299–317.[Abstract/Free Full Text]
  4. Hamosh M. Protective function of proteins and lipids in human milk. Biol Neonate 1998;74:163–76.[Medline]
  5. Moldoveanu Z, Tenovuo J, Mestecky J, Pruitt KM. Human milk peroxidase is derived from milk leukocytes. Biochim Biophys Acta 1982;718:103–8.[Medline]
  6. Hashinaka K, Yamada M. Identification of myeloperoxidase in human colostrum. Arch Biochem Biophys 1986;247:91–6.[Medline]
  7. Langbakk B, Flatmark T. Lactoperoxidase from human colostrum. Biochem J 1989;259:627–31.[Medline]
  8. Pruitt KM, Rahemtulla F, Månsson-Rahemtulla B, Baldone DC, Laven GT. Peroxidases in human milk. Adv Exp Med Biol 1991; 310:137–44.[Medline]
  9. Thomas EL, Bozeman PM, Learn DB. Lactoperoxidase: structure and catalytic properties. In: Everse J, Everse KE, Grisham MB, eds. Peroxidases in chemistry and biology. Vol 1. Boca Raton, FL: CRC Press, 1991:123–42.
  10. Klebanoff SJ. Myeloperoxidase: occurrence and biological function. In: Everse J, Everse KE, Grisham MB, eds. Peroxidases in chemistry and biology. Vol 1. Boca Raton, FL: CRC Press, 1991:1–35.
  11. Gothefors L, Marklund S. Lactoperoxidase activity in human milk and in saliva of newborn infants. Infect Immun 1975;11:1210–5.[Abstract/Free Full Text]
  12. Blanc B. Biochemical aspects of human milk—comparison with bovine milk. World Rev Nutr Diet 1981;36:1–89.[Medline]
  13. Shin K, Tomita M, Lönnerdal B. Identification of lactoperoxidase in mature human milk. J Nutr Biochem 2000;11:94–102.[Medline]
  14. Shin K, Hayasawa H, Lönnerdal B. PCR cloning and baculovirus expression of human lactoperoxidase and myeloperoxidase. Biochem Biophys Res Commun 2000;271:831–6.[Medline]
  15. Laemmli UK. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 1970;227:680–5.[Medline]
  16. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 1979;76:4350–4.[Abstract/Free Full Text]
  17. Thomas EL, Jefferson MM, Joyner RE, Cook GS, King CC. Leukocyte myeloperoxidase and salivary lactoperoxidase: identification and quantitation in human mixed saliva. J Dent Res 1994;73:544–55.[Abstract/Free Full Text]
  18. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–54.[Medline]
  19. Vilja P, Lumikari M, Tenovuo J, Sievers G, Tuohimaa P. Sensitive immunometric assays for secretory peroxidase and myeloperoxidase in human saliva. J Immunol Methods 1991;141:277–84.[Medline]
  20. Lenander-Lumikari M, Laurikainen K, Kuusisto P, Vilja P. Stimulated salivary flow rate and composition in asthmatic and non-asthmatic adults. Arch Oral Biol 1998;43:151–6.[Medline]
  21. Kiser C, Caterina J, Engler JA, Rahemtulla B, Rahemtulla F. Cloning and sequence analysis of the human salivary peroxidase-encoding cDNA. Gene 1996;173:261–4.[Medline]
  22. Dull TJ, Uyeda C, Strosberg AD, Nedwin G, Seilhamer JJ. Molecular cloning of cDNA encoding bovine and human lactoperoxidase. DNA Cell Biol 1990;9:499–509.[Medline]
  23. Ueda T, Sakamaki K, Kuroki T, Yano I, Nagata S. Molecular cloning and characterization of the chromosomal gene for human lactoperoxidase. Eur J Biochem 1997;243:32–41.[Medline]
  24. Månsson-Rahemtulla B, Rahemtulla F, Humphreys-Beher MG. Human salivary peroxidase and bovine lactoperoxidase are cross-reactive. J Dent Res 1990;69:1839–46.[Abstract/Free Full Text]
  25. Dumontet C, Rousset B. Identification, purification, and characterization of a non-heme lactoperoxidase in bovine milk. J Biol Chem 1983;258:14166–72.[Abstract/Free Full Text]
  26. Reiter B, Perraudin J-P. Lactoperoxidase: biological functions. In: Everse J, Everse KE, Grisham MB, eds. Peroxidases in chemistry and biology. Vol 1. Boca Raton, FL: CRC Press, 1991:143–80.
  27. Huber RE, Edwards LA, Crane TJ. Studies on the mechanism of the iodination of tyrosine by lactoperoxidase. J Biol Chem 1989;264: 1381–6.[Abstract/Free Full Text]
  28. Etling N, Gehin-Fouque F. Iodinated compounds and thyroxine binding to albumin in human breast milk. Pediatr Res 1984;18:901–3.[Medline]
  29. Strum JM. Site of iodination in rat mammary gland. Anat Rec 1978;192:235–44.[Medline]
Received for publication May 11, 2000. Accepted for publication September 25, 2000.




This article has been cited by other articles:


Home page
 Clin Med ResHome page
N. Leon-Sicairos, F. Lopez-Soto, M. Reyes-Lopez, D. Godinez-Vargas, C. Ordaz-Pichardo, and M. de la Garza
Amoebicidal Activity of Milk, Apo-lactoferrin, sIgA and Lysozyme.
Clin. Med. Res., June 1, 2006; 4(2): 106 - 113.
[Abstract] [Full Text] [PDF]

Home page
 J DAIRY SCIHome page
W. Noppe, K. Vanhoorelbeke, I. Y. Galaev, B. Mattiasson, and H. Deckmyn
A Probe for Capture and Fe3+-Induced Conformational Change of Lactoferrin Selected from Phage Displayed Peptide Libraries
J Dairy Sci, October 1, 2004; 87(10): 3247 - 3255.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Clin. Nutr.Home page
B. Lonnerdal
Nutritional and physiologic significance of human milk proteins
Am. J. Clinical Nutrition, June 1, 2003; 77 (6): 1537S - 1543S.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
S. El-Chemaly, M. Salathe, S. Baier, G. E. Conner, and R. Forteza
Hydrogen Peroxide-Scavenging Properties of Normal Human Airway Secretions
Am. J. Respir. Crit. Care Med., February 1, 2003; 167(3): 425 - 430.
[Abstract] [Full Text] [PDF]

Home page
 J Med MicrobiolHome page
K. SHIN, K. YAMAUCHI, S. TERAGUCHI, H. HAYASAWA, and I. IMOTO
Susceptibility of Helicobacter pylori and its urease activity to the peroxidase-hydrogen peroxide-thiocyanate antimicrobial system
J. Med. Microbiol., March 1, 2002; 51(3): 231 - 237.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shin, K.
Right arrow Articles by Lönnerdal, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shin, K.
Right arrow Articles by Lönnerdal, B.
Agricola
Right arrow Articles by Shin, K.
Right arrow Articles by Lönnerdal, B.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS