Protease digestion, shown earlier to increase the capacity of breast milk to resist death from exogenously added FFA (5), decreased either the cell death or the number of samples that were cytotoxic in stored milk (Table 1). This did not appear to be because of digestion of endogenous or exogenous lipase because lipase activity was unchanged with protease addition (1259 ± 585 fluorescent units vs 1196 ± 487 fluorescent units in the 7-day, 4°C, milk with lipase vs lipase + protease groups). Lipase- and protease-only controls were not cytotoxic to neutrophils (not shown).
The postnatal age of the child at the time of milk donation (range 22–507 days) did not correlate with the cytotoxicity of milk stored 7 days at 4°C (PBS digestion, correlation coefficient 0.15, P = 0.32) or initial lipase activity in the sample (correlation coefficient −0.01, P = 0.49). Mother's age at time of expression was unavailable for 2 samples, but we did not find a correlation between mother's age and cytotoxicity of milk stored 7 days at 4°C (PBS digestion, correlation coefficient −0.17, P = 0.32) or initial lipase activity in the sample (correlation coefficient 0.13, P = 0.35) in the remaining 10 samples. Mothers’ race was primarily white (7 whites/non-Hispanics, 1 black, 1 Asian, 1 half-Asian/half-white, 2 unavailable), so no effects of race are determinable for these data.
FFA Cytotoxic Mechanism
We measured lipase activity in samples stored for 7 days at 4°C or −20°C, or long term at −80°C, normalized by their activities when fresh, and that of raw and pasteurized DM (Fig. 2A). Fresh milk alone had 3.1 ± 1.4 times the activity as 1 mg/mL of exogenous pancreatic lipase (not shown). Pasteurized DM had no detectable lipase activity, in agreement with earlier studies (26), but all other samples had activity. Activity was significantly elevated with freezing (−20°C) compared with refrigeration (4°C). Raw DM and −80°C stored milk also trended toward increased activity compared with 4°C (P = 0.046 for raw DM; P = 0.076 for −80°C stored milk). This agrees with earlier studies showing lipase activation by cooling (2°C) cow's milk (27) and by freezing human milk (BSSL normally needs bile salt to become active, but this requirement is lost after freezing) (28). No correlation was seen between cytotoxicity of the “milk with PBS” after 7-day storage at 4°C and the initial (correlation coefficient −0.04, P = 0.45) or final lipase activity (correlation coefficient −0.35, P = 0.16), suggesting that variations in cytotoxicity were more the result of differences in substrate (eg, milk fat globules) between samples than differences in lipase activity.
Despite a relatively short 5-minute exposure, milk stored at 4°C for 7 days killed 41% of IECs, as compared with 5% from milk stored at −80°C (Fig. 2B). Exogenous lipase digestion increased both of these values a small but significant (by paired t test) amount (see inset in Fig. 2 for raw data of 4°C groups). Cytotoxicity was prevented when lipase was inhibited with orlistat before storage at 4°C. Thus, as with digested formula (5), FFAs created by lipase digestion in stored milk are cytotoxic to intestinal cells. We confirmed that lipase inhibition would also prevent cytotoxicity to neutrophils (Fig. 2C) and that passage of cytotoxic stored milk through glass fiber filters, which bind and remove unbound, but not bound, FFAs (5), significantly reduced cytotoxicity (Fig. 2D).
We measured total and unbound FFAs in aliquots of the same defatted solutions that were tested earlier for cytotoxicity on human neutrophils (Table 1) and stored at −80°C until FFA analysis. We detected little to no FFAs in fresh milk incubated with PBS or proteases (Fig. 3). Pancreatic lipase digestion significantly increased the total and unbound FFAs in fresh breast milk. Milk stored for 7 days at 4°C had significantly higher unbound and total FFA levels compared with fresh milk in all cases, except for unbound FFAs in the “milk with lipase” groups that approached, but did not reach, significance (P = 0.02). Lipase digestion of stored milk increased neither the total nor the unbound concentrations of FFAs compared with the “stored milk with PBS” control unless protease digestion also occurred, in agreement with the cytotoxicity findings (Table 1). It is possible that fat digestion or FFA aqueous solubility in breast milk is near its maximal level in the stored milk already, minimizing the effect of exogenous lipase unless proteases are present to increase the binding of FFA to proteins (see below), removing them from solution and making room for additional FFA (in vivo, solubility is increased via FFA incorporation into bile acid micelles (29)). Protease digestion decreased total FFA levels in all cases. Overall, unbound FFA concentration correlated significantly with cytotoxicity (correlation coefficient 0.706, P < 0.001).
Long-Term Milk Storage at −20°C
To explore the effects of long-term milk storage at −20°C on IEC cytotoxicity, we stored fresh milk at −20°C for 0, 4, 8, or 12 weeks, transferring the aliquots to −80°C until simultaneous digestion and testing on IECs (total storage = 20.7 ± 8.3 weeks, maximum 37.6 weeks). Every group, including the 0-week group that was kept at −80°C for the duration, increased their cytotoxicity with lipase digestion (Fig. 4, also apparent in Fig. 2B). This suggests that frozen milk, even at −80°C, loses some of the resistance to lipolysis present in fresh milk, subject to variations between donors and/or assay conditions. After 8 weeks, however, we saw an “additional” cytotoxic effect of storage at −20°C. This increase was seen even in the absence of exogenous pancreatic lipase digestion.
To determine whether the secondary increase in cytotoxicity during storage at −20°C is because of retention of residual lipase activity even while frozen, we tested the effect of lipase inhibition on milk before storage. Milk from 7 individual donors was stored 28 days or approximately 2 months (60.1 ± 3.0 days) at −20°C with or without 0.25 mg/mL orlistat, and then moved to −80°C before thawing and immediate defatting and testing for cytotoxicity to neutrophils without additional digestion or incubation at 37°C (total storage time 33.1 ± 3.0 and 61.1 ± 3.0 days, respectively). Aliquots of the defatted milk were immediately returned to −80°C until testing for FFA concentration (total storage time 38.1 ± 3.0 and 68.1 ± 3.0 days, respectively). We found no appreciable cell death at either time point (3% ± 1% with or without orlistat at 28-day time point, 2% ± 1% with or without orlistat at 2-month time point). At 28 days we found approximately the same low concentration of FFAs as we observed in the fresh milk group (Fig. 3) in both groups (0.13 ± 0.12 mmol/L FFA without orlistat and 0.10 ± 0.04 mmol/L FFA with orlistat). At the 2-month time point, however, FFAs were doubled (0.22 ± 0.11 mmol/L FFA), and this increase was prevented by pretreatment with orlistat (0.10 ± 0.04 mmol/L FFA; P < 0.011).
Milk From Donor Bank
Given that 8 weeks of storage at −20°C can increase FFAs and cytotoxicity and that milk banks allow donors to store milk for longer periods before shipping to their facilities, we determined the cytotoxicity of DM, with and without exogenous lipase digestion. We found that 2 of 6 DM samples were initially cytotoxic to neutrophils, but after lipase digestion all 6 samples were cytotoxic (Fig. 5, left). The IEC assay gave similar results for the control and lipase-digested DM, although by that method, 4 of the samples were considered cytotoxic to start with, increasing to 5 of 6 with lipase digestion (Fig. 5, right). The addition of proteases to the digestion of DM resulted in a decrease in cytotoxicity by IEC assay, but increased cytotoxicity as measured by the neutrophil assay, because of the higher protease concentration and extra approximately 1.5 hours of digestion (55 minutes longer incubation with neutrophils plus approximately 30 minutes for defatting step) in that assay (see below).
FFA Binding Capacity as Measured by Cytotoxicity Response to Exogenous FFA
Because FFAs are likely to insert themselves into milk fat globule membranes, the fat layer itself may provide some protection from cytotoxicity. We therefore compared the ability of defatted and whole milk to lower the cytotoxicity of exogenous oleic acid (eg, via binding the FFA) and determined how those capacities are affected by protease digestion. Similar to earlier results with milk (5), digestion by proteases significantly improved the ability of milk to lower the cytotoxicity of exogenous FFA, whether or not the milk was defatted before oleic acid addition (Fig. 6). This suggests that protease digestion increases FFA binding capacity, not just in whole milk but also in the components that remain in defatted milk. There was also a significant protection against cytotoxicity if oleic acid was added to milk before, as opposed to after, defatting. This suggests that the fat layer and/or pellet also have a significant FFA binding capacity. There was a significant interaction by analysis of variance between defatting and digestion, implying that the binding capacity of the fat layer/pellet is also increased by protease digestion. This agrees with the FFA measurement (Fig. 3) showing a decrease in “total” FFA concentration in milk digested by proteases before defatting, despite no decrease in lipase activity.
To determine whether the differential response to protease digestion shown in Figure 5 could be the result of differences in the degree of protein digestion, we kinetically tested the effect of protease digestion on FFA binding capacity in DM. A total of 5 mmol/L exogenous oleic acid was added to defatted DM, which was then digested for 0, 0.5, 1, 2, or 4 hours with 1 mg/mL trypsin and chymotrypsin before inhibiting protease activity with 1 mmol/L phenylmethanesulfonyl fluoride and incubating with neutrophils. Unlike in Figure 5 (left), we saw a significant decrease in neutrophil cytotoxicity compared with 0-hour digestion (59% ± 11%) at 0.5-hour (47% ± 11%, P < 0.0001) and 1-hour (46% ± 11%, P < 0.015) digestion (N = 6). This was, however, followed by a near-significant “increase” in cytotoxicity at the 2-hour time point (55% ± 21%, P = 0.052 vs 1-hour time point, with 1 sample having a 10% increase in cytotoxicity compared with its “0-hour” time point) and a second significant decrease at 4 hours (33% ± 11%, P < 0.01 vs 2-hour time point). The complexity of the kinetics suggests that >1 protein is likely to be involved in this process.
The present results indicate that stored human milk is capable of killing IECs with as little as 5-minute exposure; 3-day storage at either 4°C or −20°C will cause milk to become cytotoxic after lipase digestion similar to that in the infant intestine; and some milk from donor banks is already cytotoxic on distribution and, unlike fresh milk, gains significant cytotoxicity after lipase digestion.
Cytotoxicity could be prevented by pretreatment with the lipase inhibitor, orlistat, before milk storage, confirming that the products of lipolysis are the cause of cell death. Cytotoxicity could be decreased by protease digestion, and by filtering through glass fiber prefilters, a procedure that removes unbound FFAs but not FFAs bound to proteins such as albumin (5,7). Both total and unbound FFAs are increased in stored milk, and unbound FFA concentration correlates with levels of cell death. These findings support the hypothesis that unbound FFAs are the main cytotoxic mediator in stored milk, although monoglycerides may also contribute.
We observed membrane bleb formation and rupture and eventually total cell destruction, similar to that observed with other FFA sources (7), and consistent with the idea of physical disruption of the bilipid membrane. Cell destruction by FFAs is cell type independent (eg, epithelial cells (5), endothelial cells (5), leukocytes (6), red cells (6)). Although neutrophils were used here primarily as a convenient measure of general cytotoxicity, neutrophils are present in intestinal tissue and, like other cell types in the intestine that remain to be investigated, may be affected by FFA cytotoxicity when the mucosal barrier is breached. In our earlier work, the cell sources were human, rat, and bovine, all of which gave qualitatively similar results (5), making it unlikely that there are major species differences with regard to response of cells exposed to FFAs.
Freezer stored milk (−20°C) caused cell death that appears to increase in a biphasic fashion. In the first phase, we saw that freezing for 3 or more days resulted in increased cytotoxicity after subsequent exogenous lipase digestion. We saw no increase in cytotoxicity, and levels of FFA that approximated that of fresh milk, in milk stored for 4 weeks at −20°C “without” subsequent digestion, suggesting that no appreciable fat digestion occurs while milk is frozen for <4 weeks. Therefore, the increased cytotoxicity versus fresh milk we observed at 3 and 7 days (Table 1) after exogenous lipase digestion is likely because of an increase in the susceptibility of the milk fat to lipolysis (eg, disruption of the milk fat globule membrane). Alternatively, BSSL activated by freezing (28) (Fig. 2A) may interact synergistically with the exogenous lipase to increase lipolysis.
The secondary increase in cytotoxicity of milk stored for 8 and 12 weeks at −20°C but not milk kept at −80°C “is” likely the result of lipase activity while the milk is frozen, because FFA concentration was doubled in milk stored at −20°C for 8 weeks without orlistat. Partial digestion of milk fat or the milk fat globule membrane may also prime the fat for increased lipolysis after thawing. The non-negligible death in milk stored for long term at −80°C (Fig. 4, “0-week control” group) and the small but significant increase in cytotoxicity with exogenous lipase digestion after short-term storage at −80°C (Fig. 2B) suggest that the same changes to milk that occur at −20°C may also occur at −80°C, but to a lesser extent that varies between individuals.
Two-thirds of the milk samples obtained from the donor bank were cytotoxic to IECs without additional lipid digestion, increasing to five-sixths after exogenous lipase digestion. This is in line with our findings of increased cytotoxicity compared with fresh milk after long-term storage at −20°C.
In Vivo Intestinal Environment
Although we tried to match factors such as digestion time and temperature, enzyme concentrations, and cell exposure time, our in vitro protocols are not, of course, a perfect representation of digestion as it occurs in an infant in vivo. For example, lipase profiles change during early development and in response to weaning (30). For the present study, we chose to use pancreatic lipase so our findings would be comparable with our earlier study comparing formula and fresh milk (5). This is likely to match the conditions in infants who have begun to wean but are consuming some human milk, but may not match conditions in infants on a milk-only diet, because before weaning most of the lipase activity in a neonatal intestine is from pancreatic lipase–related protein 2 (PLRP2) or BSSL (30). PLRP2 may be less efficient at lipid digestion in milk than pancreatic lipase (31), which could lead to lower FFA levels and cytotoxicity in the intestine than demonstrated in the present experiments. In contrast, lingual and gastric lipases, also not included in the present study, may prime milk fats for easier digestion in the intestine, leading to higher FFA levels and greater cytotoxicity. Interestingly, because BSSL and PLRP2 have a lower affinity for large saturated fatty acids such as palmitic acid (32,33), which is almost exclusively located at the sn-2 position in human milk triglycerides and is found there >3 times as often as any other fatty acid (34), and because pancreatic lipase also avoids cleaving the sn-2 triglyceride position, it is unlikely that the “composition” of FFAs will differ substantially regardless of which lipase dominates. Although we clearly demonstrate here that subsequent lipase digestion may cause noncytotoxic stored milk to become cytotoxic (Table 1), testing cytotoxicity in infant intestinal aspirates will be necessary to resolve the question of the “degree” to which digestion increases stored milk cytotoxicity in vivo.
Likewise, we did not include bile acids in our digestions because their concentration is low and variable in neonates (24). Because bile acids are small, amphiphilic molecules such as FFAs, and are themselves cytotoxic (35), however, with the in vivo purposes of activating BSSL (28), emulsifying fats, and increasing FFA solubility and transport of FFAs across the mucin barrier by incorporating them into bile acid micelles (36), it is likely that bile acids would only increase overall cytotoxicity from stored milk.
Lastly, although milk supplied to donor banks is expected to come from later in lactation and although older infants could also be affected by milk stored later in lactation, it is possible that colostrum obtained in the first or second week may develop cytotoxicity either faster or slower with storage than the milk used for the present study.
Protease Digestion of Stored Milk
Similar to our findings with fresh milk and a few infant formulas (5), cytotoxicity of stored milk also decreased significantly with protease digestion, which increases the FFA-binding capacity of the milk. This is opposite to the effects of protease digestion on FFA-binding proteins we have studied earlier (eg, albumin and the binding proteins in intestinal wall homogenates) (7,25), suggesting the involvement of a specialized protein. One possible candidate is the casein micelle, which is disrupted on protease digestion (37) to expose hydrophobic portions of the individual casein molecules and which is known to bind to hydrophobic lipid after disruption (31,38). Alternatively, partial digestion could induce the conformational change in α-lactalbumin that allows it to bind FFAs after the removal of its calcium ion (39).
This putative specialized protein is likely not the only protein involved in this process, however, because Figure 4 indicates that milk has the capacity to bind millmole per liter quantities of FFA even without protease digestion. For example, serum albumin is also present in human milk, and, as mentioned above, is capable of binding FFAs unless first digested with protease (7). A simultaneous decrease in binding capacity of some proteins and increase in binding capacity of other proteins with protease digestion could easily explain our findings in Figure 5 and the kinetic experiment on DM. We have preliminary data suggesting that the protection provided by protease digestion of milk eventually disappears altogether as protease digestion continues. It is possible that the pasteurization step that occurs during the preparation of DM may accelerate the rate at which the protective protein is degraded compared with the unpasteurized milk in Table 1. The effect of protease on the fat layer may also have an effect.
Cytotoxicity Reduction Strategies
Our findings suggest a number of possible strategies to limit cytotoxicity in stored breast milk. Storage of breast milk at −80°C instead of −20°C or above, or earlier pasteurization for DM to denature the lipase, may reduce the potential production of cytotoxic mediators before and during digestion of the milk. Because freezing activates milk lipases, more limited storage of thawed milk before consumption or pasteurization may also serve as a preventive measure. If pancreatic insufficiency is present, pretreatment of breast milk with trypsin and/or chymotrypsin may decrease cytotoxicity. Guidelines for milk storage times at 4°C or −20°C could be more limited. If long-term storage is required and colder storage is unavailable, the efficacy and safety of glass fiber filtration or addition of a lipase inhibitor could be considered.
We determined that milk stored according to present practices, whether obtained from a fresh source or from a donor bank, can become cytotoxic, although to a lesser degree than nearly every infant formula we tested earlier (5). It remains to be examined to what degree the cytotoxicity is increased during digestion in vivo; however, the incidence of cytotoxicity in stored milk may explain why NEC can occur in milk-fed infants and why DM has not always performed better than formula in preventing NEC.
We thank Tiffany Lai for help in recruiting milk donors, Lynn Han and Leena Kurre for help in analysis of IEC death, Parth Chokshi for assistance with IEC culturing, and Dr Emily Blumenthal for statistical assistance.
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Keywords:© 2014 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology,
breast milk donor banks; intestinal cell damage; lipase; necrosis; necrotizing enterocolitis