What Is Known
- Neonates are prone to oxidative stress-related conditions.
- Fresh human milk shows a unique antioxidative profile.
- The main nonenzymatic antioxidants in human milk are ascorbate and urate.
- Pasteurization and freezing/storage may alter antioxidative properties of human milk.
What Is New
- Total nonenzymatic antioxidative capacity is not significantly affected by pasteurization and freezing/storage for 30 days.
- The processing shifts the load of antioxidative activity from ascorbate to urate.
- Pasteurization and freezing/storage cause a significant drop in the activity of superoxide dismutase and glutathione peroxidase.
- Colostrum and milk contain different antioxidative systems.
American Academy of Pediatrics strongly recommends breast-feeding to be the preferred feeding for infants, in particular during the first 4 months of life. If breast-feeding is not possible, human milk is collected and fed to the babies, or infant formulas are used (1). According to UK guidelines, individually matched mother's milk or donated milk represent a more appropriate choice than formula feeding, especially for preterm infants (2). Nutrition is essential for protection against oxidative stress/damage, to which preterm neonates appear to be particularly prone due to inefficient endogenous antioxidative system and increased sensitivity of proliferating cells to oxidative damage (3). In addition, preterm infants are at increased risk of exposure to oxidative stress because of oxygenation, infections, and injuries related to invasive clinical procedures (4). Numerous disorders in early infancy, such as sepsis, necrotizing enterocolitis, retinopathy, bronchopulmonary dysplasia, and periventricular leukomalacia, have been linked to oxidative damage (3). Antioxidant-rich infant feeds are considered to be of great benefit as a preventive measure against these diseases (5). Fresh human milk shows different antioxidative profile and better antioxidant performance compared with infant formulas (6,7). Redox properties, however, may be affected by milk processing (4,8–10). Donor milk is usually pasteurized (30 minutes at 62.5°C) and stored at −20°C to mitigate the contamination with pathogenic agents. Alternatively, raw milk can be stored at −20°C for up to 3 months before being fed to babies (2). Available data on the effects of pasteurization and freezing/storage on redox properties of human milk are limited.
Here, we examined the effects of pasteurization and storage (7 and 30 days at −20°C) on the main antioxidative parameters of mature human milk. Antioxidative properties of human milk refer to the sum of activities derived from active antioxidative enzymes (superoxide dismutase (SOD), glutathione (GSH) system–glutathione peroxidase (GPx), and reductase (GR)), nonenzymatic antioxidants (such as ascorbate), and other bioactive factors (eg, urate (UA)) (11). In addition, a comparative analysis of antioxidative properties of fresh colostrum and mature milk was conducted.
Milk was collected from 10 healthy mothers of preterm infants (gestational age 28–36 weeks; birth weight 900–2470 g), within the first 4 days postpartum (colostrum) and 6 weeks after the delivery (mature milk). The mothers were asked to express milk between 8:00 and 10:00 AM. Milk was aliquoted and examined prior (0 days) and after Holder pasteurization (62.5°C for 30 minutes) and/or storage at −20°C (for 7 or 30 days). This study was approved by the Ethics Committee of the Institute for Neonatology, N°2401/4 (April 18th, 2014). Informed consent was obtained from all participants.
Nonenzymatic Antioxidative Capacity Parameters
Total nonenzymatic antioxidative capacity was determined using oxygen radical absorbance capacity (ORAC) assay. ORAC represents the measure of the ability of agents in biological samples to donate hydrogen atom and is closely related to their ability to act as chain-breaking antioxidants. ORAC was performed as described previously (12), with slight modifications. Fluorescein (stock solution prepared in water) was dissolved in the working solution (75 mM Na3PO4; pH 7.4) at the concentration of 1.7 nM. The solution (150 μL) was incubated at 37°C for 20 minutes and then mixed with samples (25 μL) and incubated for another 10 minutes. After this, the assay was initiated by the addition of 25 μL of 2,2′-azobis (2-amidinopropane) dihydrochloride solution (9.2 mM; made fresh each day). Fluorescence (excitation at 485 nm and emission at 511 nm) was read every minute for 1 hour. ORAC values are expressed as equivalents of Trolox.
Static oxidation-reduction potential (ORP) is an integrated, comprehensive measure of the balance between pro-oxidative and antioxidative components in biological system. Lower ORP stands for higher reducing capacity. ORP was measured at room temperature using RedoxSYS Analyzer (Aytu BioScience, Inc, Englewood, CO).
Ascorbate concentration was determined by reflectometric method using Reflectoquant ascorbic acid test and reflectometer RQflex (Merck, Darmstadt, Germany), according to the manufacturer's protocol.
The Activity of Antioxidative Enzymes
SOD activity was assayed via superoxide radical anion-mediated oxidation of adrenaline to adrenochrome at pH 10.2 (13). One unit (U) of SOD was defined as the amount of protein causing 50% inhibition of adrenaline autoxidation at 26°C. Results are expressed in U/mL/mg of proteins. GPx activity was measured according to previously described method that measures nicotinamide adenine dinucleotide phosphate consumption (14). GR activity was assayed as described previously (15). One unit of activity was defined as the amount of enzyme required to transform 1 mM of substrate per minute. All of the chemicals were of analytical grade and were obtained from Sigma-Aldrich (St Louis, MO) or Merck. All of the solutions and buffers were prepared using bidistilled deionised ultrapure (18 MΩ) water.
Electron Paramagnetic Resonance Spectroscopy
Electron paramagnetic resonance (EPR) spin-trapping spectroscopy was applied to examine the ability of human milk to scavenge hydroxyl radical (HO•) and to establish reactive products of oxidation of milk constituents. The majority of free radicals that are produced in biological and biochemical systems show rather short lifetimes that prevent their direct detection. Spin-trap reacts with radicals to generate a long-living EPR-active products (spin adducts) that show characteristic spectra for different radicals (16). HO• radicals were generated via Fenton reaction (Fe2+ + H2O2 → Fe3+ + OH− + HO•), which was performed by combining 0.6 mM FeSO4 (Merck) and 3 mM H2O2 (Carlo Erba Reagents, Milano, Italy). Spin-trap DEPMPO (5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide; Plymouth Meeting, PA) was applied at the final concentration of 4 mM before the initiation of the reaction. Samples were also examined without the spin-trap to obtain clear signal of ascorbyl radical (Asc•), which is more stable and can be detected directly. All of the solutions were prepared in deionized water and contributed to the final volume of the sample by only 6% (milk 94%). Samples were drawn into 10 cm long gas-permeable Teflon tubes (Zeus industries, Raritan, NJ) that were packed into quartz capillaries. Incubation (1 minute) and measurements were performed at 20°C. EPR spectra were recorded using a Varian E104-A EPR spectrometer operating at X-band (9.56 GHz) with EW software (Scientific Software, Bloomington, IL), and with the following settings: modulation amplitude, 2 G; modulation frequency, 100 kHz; microwave power, 20 mW; time constant, 32 milliseconds; scanning time, 2 minutes. EPR signal identification and intensity estimation (in arbitrary units–AU) were conducted via simulation and double integration using WINEPR SimFonia computer program (Bruker Analytische Messtechnik GmbH, Darmstadt, Germany). Parameters of simulation were: DEPMPO/OH (spin adduct of HO•): aN = 14.0 G, aH = 13.0 G, aHγ = 2.7 G (3H), aP = 47.3 G; DEPMPO/UA (spin adduct of UA radical): aN = 14.8 G, aH = 20.7 G, aP = 47.8 G; Asc•: aH = 2.3 G (17,18).
Statistical analysis was conducted using STATISTICA 8.0 (StatSoft Inc, Tulsa, OK). Results are presented as means ± standard deviation. P values were determined using 2-way analysis of variance with post hoc Duncan test or using nonparametric 2-tailed Mann-Whitney U test. Results were considered to be statistically significant if P < 0.05.
Colostrum showed significantly higher ORAC values and slightly lower ORP (indicating better antioxidative performance) compared with mature milk (Fig. 1A and B). Pasteurization and storage of milk did not affect total nonenzymatic antioxidative capacity and ORP. Colostrum showed significantly higher ascorbate level and almost doubled GR activity compared with mature milk. Milk pasteurization caused a significant drop in the level of ascorbate (Fig. 1C), and in the activities of SOD (Fig. 1D) and GPx (Fig. 1E). The effects of pasteurization on GR activity were less pronounced and became evident (significant) after 7 days of storage (Fig. 1F). Storage at −20°C did not further promote the effects of pasteurization on other parameters. Storage of raw milk induced a decrease in ascorbate concentration and SOD activity. GPx activity was slightly affected at 30 days of storage (nonsignificant), whereas GR activity was not changed.
Colostrum and (un)treated milk showed significant HO• scavenging capacity (Fig. 2). EPR signal intensities in all examined milk samples were at least three-fold lower than in the control system (Fig. 2A). Colostrum showed superior antioxidative activity compared with milk. Only weak signals of Asc• and/or DEPMPO/UA were detected (Fig. 2B). The exposure of milk to HO• generating-system resulted in the production of UA radical and Asc• (Fig. 2C, D). The signal of DEPMPO/UA adduct was significantly higher, whereas Asc• was lower in pasteurized milk compared with fresh milk (Fig. 2C–F). Storage did not induce further changes in pasteurized milk, but it affected the properties of raw milk. Namely, signals of DEPMPO/UA and Asc• increased and decreased, respectively, during 30 days. The effects of 30 days of storage on raw milk practically equalled the immediate effects of pasteurization.
According to ORAC and ORP values, pasteurization and 30 days of storage at −20°C did not affect total nonenzymatic antioxidative capacity and total reducing capacity of milk. On the one hand, our results are in line with the previous study that used ORAC to examine the effects of pasteurization on human milk (19). On the other hand, Silvestre and co-workers applied CUPRAC assay (measures the ability of samples to reduce copper (20)) to show that Holder pasteurization induce a decrease in the reducing capacity of human milk (9). Their results may reflect pasteurization-induced degradation of ascorbate, which represents the main reducing agent in the human milk when it comes to transition metals, such as copper. The level of ascorbate was also affected by freezing/storage of raw milk, which is in line with previous findings (21,22). Two studies have applied ABTS assay to show that the storage of raw colostrum and mature milk at −20°C for 7 or 14 days, causes a drop in the total antioxidative capacity (4,10). The difference between ORAC and ABTS assays may explain the discrepancy between our and previous findings. ORAC is run to completion of antioxidative activity and takes into account both fast- and slow-reacting antioxidants. ABTS is run for a fixed time and, generally, is more sensitive to fast-reacting antioxidants, such as ascorbate (20).
It appears that pasteurization- and storage-related loss in the antioxidative activity of ascorbate is compensated by UA. This is confirmed here by increased level of UA radical that is paralleled by decrease in Asc• level in the processed milk exposed to oxidation. We found a strong negative correlation (Pearson correlation coefficient r = −0.780, P = 0.002) between UA radical and Asc• in raw milk. It has been shown that antioxidative activities of ascorbate and UA are coupled in bovine milk. Ascorbate reacts with UA radical to give rise to UA and Asc•(23). This implies that the level of UA radical increases with pasteurization and storage of raw milk at least partially because of the loss of ascorbate, which is the next in line electron donor. UA represents a powerful HO• scavenger and repairer of oxidized proteins (17,24), whereas UA radical has been shown to scavenge superoxide (25). Specific physiological functions of ascorbate, however, cannot be replaced by UA. For example, ascorbate reduces Fe3+ to Fe2+ to enhance iron absorption in the gut (26). UA has the reducing potential of 260 mV and therefore cannot reduce Fe3+ (110 mV at pH 7) (27). It is noteworthy that preterm neonates are at increased risk of iron deficiency (28).
Nonenzymatic antioxidative systems in colostrum and mature milk appear to be different. Although the level of ascorbate was higher in colostrum by only ∼20% compared with milk (which is in accordance with previous results (29)), colostrum almost completely annihilated HO• from the Fenton system and showed significantly higher total antioxidative capacity. The latter is in line with previous studies that compared colostrum and mature milk via ORAC and other assays (11,30). Colostrum showed almost two-fold higher GR activity than mature milk. In accordance with this, Ankrah et al (31) have found a significantly higher level of GSH in colostrum than in mature milk; this may contribute to superior antioxidative performance of colostrum. Furthermore, colostrum contains up to 5 times more human milk oligosaccharides than mature milk (32). It is tempting to speculate that in addition to their prebiotic and immunological functions, human milk oligosaccharides may serve as antioxidants. Pertinent to this, we have shown previously that some mono- and polysaccharides are potent HO• scavengers (33,34).
Pasteurization decreased SOD and GPx activity by about three-fold, but had modest effects on GR. It has been reported that human extracellular SOD (copper-zinc SOD) is inactivated by heating at 65°C (35). The results obtained on GPx and GR are in accordance with their respective lability/stability at increased temperatures (36). It has been shown previously that Holder pasteurization inactivates GPx in human milk (9). Storage of raw milk for 7 days at −20°C exerted drastic inactivating effects on SOD. This enzyme is resilient to freezing in water/buffers, so a decrease in SOD activity in frozen milk is most likely based on the effects of some agent that can inhibit SOD. It should be noted that UA radical has the ability to inhibit some enzymes (37). Previous reports have found that the storage of raw human milk at −20°C has negative effect on GPx activity (38,39). We noted a similar (although nonsignificant) trend.
In the early infancy, particularly in premature infants, gastrointestinal tract is not fully developed, which results in incomplete or slow protein digestion (40). In addition, different proteins may show resistance to proteolysis. For example, it has been found that GPx is not degraded by trypsin or chymotrypsin (protein degradation in small intestine) but it is prone to pepsin (stomach degradation) (41). Hence, at least some amounts of active antioxidative enzymes from the milk may reach infant's intestines (40,42). Pertinent to this, SOD and GSH-related enzymes from the milk may be involved in the formation of healthy redox conditions in gut and in the early development of microbiota profile that is known to be sensitive to redox settings (43). Other physiological functions of these enzymes in intestine, such as the relaxing effects of SOD on smooth musculature, have been proposed (7).
Our results imply that spiking of processed human milk with ascorbate before being fed to the babies should be considered. Pertinent to this, 2 trials on infants (3–12 months) and 1 on children (12–30 months) have shown that the fortification of powdered cow milk with ascorbate and iron is effective at reducing the rates of anemia and iron deficiency (44–46). Alternatively, the level of ascorbate can be increased in preprocessed milk either by spiking or via dietary supplementation of ascorbate to donors (47). Processing of fortified milk, however, may result in the accumulation of ascorbate oxidation/decomposition products, which can exert unwanted effects. For example, dehydroascorbate is transported via glucose transporters into intestinal cells (48), where it may cause depletion of reduced nicotinamide adenine dinucleotide phosphate and glutathione (49). Finally, the processing affects enzymatic antioxidative component (SOD in particular), which is hard to compensate, so other methods of antimicrobial treatment and storage (eg, at −80°C) of human milk should be taken into consideration.
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