Background: The purpose of these studies was to evaluate the changes in osmolality which occur in human breast milk or formula after two modifications; these changes are sometimes used in preparing these milks for consumption by premature infants, namely, the addition of fortifiers and the addition of exogenous lactase enzyme.
Methods: The osmolality of expressed, previously frozen human breast milk, breast milk fortified with commercial fortifiers, liquid formulas, powdered formulas, or glucose polymers was measured. Osmolality was measured before and after warming (15 minutes at 37°C) or after refrigeration at 4°C for 12 hours with subsequent warming. In a second group of experiments, the osmolality of expressed breast milk and three lactose-containing formulas was measured before and after incubation with lactase (Lactaid ®) at 4°C for 2, 6, and 24 hours.
Results: Warming of breast milk mixed with some of the additives was associated with a significant increase in osmolality. The additives which increased osmolality included liquid glucose polymers, two commercially available powdered human milk fortifiers, and three formulas which contain glucose polymers (a protein hydrolysate infant formula powder, powdered lactose free formula, and a powdered preterm formula). Maximum increase in osmolality of breast milk occurred with the addition of 20 ml/100ml liquid glucose polymers (Polycose, Ross Laboratories) which resulted in a 21% increase in osmolality after refrigeration and warming. The addition of liquid glucose polymers and of powdered preterm formulas containing glucose polymers (Enfacare, Mead Johnson, 9.5 g/100ml and Nutramigen, Mead Johnson, 6 g/100ml) resulted in a final osmolality of over 425 mOsmol/L. The addition of lactase and subsequent incubation under refrigeration resulted in significant increases in osmolality that ranged from 25 to 66% in fortified breast milks and lactose-containing formulas. Incubation of these milks at 37° for 15 minutes produced about 50% greater increase in osmolality than observed after 2 hours of incubation under refrigeration.
Conclusions: Routine warming of breast milk with glucose polymer containing additives, or the addition of lactase enzyme to lactose containing feedings, can increase osmolality to levels that exceed current guidelines for premature infant feedings.
Breast milk is the first choice for feeding the preterm infant due to its nutritional and immunologic superiority. However, the nutritional needs of the tiny preterm infant are such that supplementation of breast milk with protein, calcium, phosphorus, and other nutrients may be necessary to achieve nutrient accretion and growth at the intrauterine rate (1,2). Commercial products have been developed to increase the nutrient concentrations of breast milk for these patients. One concern about modified or fortified breast milk for preterm infants is its osmolality (3). Necrotizing enterocolitis has been associated with feedings of high osmolality (4,5). Breast milk fortifiers increase the osmolality of the product, and routine preparation and storage has been documented to result in even further increases in osmolality (6). The increase in osmolality of fortified breast milk during storage is presumed to be due to the action of endogenous breast milk amylase on the glucose polymers contained in the fortifiers (6).
Lactase is the last of the major intestinal disaccharidases to develop in preterm infants. Lactase activity is only 30% of its full-term level by 26 to 34 weeks gestation (7). Thus, preterm infants may have relative lactose malabsorption (8,9). Some studies have indicated that feeding tolerance in preterm infants may improve when the lactose in feedings is replaced with another carbohydrate of equivalent osmolality (10). A commercial lactase (beta-Galactosidase) preparation can be used to hydrolyze lactose in formulas and breast milk; however, the resulting changes in osmolality from hydrolysis of lactose in breast milk have not been documented (11).
In light of the possible increased risk of necrotizing enterocolitis from hyperosmolar feedings, the recommendation has been made that feedings for premature neonates have an osmolality no greater than 425 mOsm/kg (12). The impact on osmolality of routine hospital-employed techniques for storage and handling of breast milk, its fortification, and its treatment with lactase have not been documented. Thus, the purpose of this study was to measure the effect of routine handling, fortification, and lactase enzyme treatment on the osmolality of breast milk. For comparison purposes we evaluated commercially available premature and term infant formulas under the same conditions.
MATERIALS AND METHODS
Frozen breast milk was collected from seven mothers who were expressing milk for their infants in the Neonatal Intensive Care Unit of Foothills Medical Center, Calgary, Alberta. All breast milk donors had delivered preterm infants and the milk was frozen immediately after collection. Before the experiments, the milk was thawed at room temperature. The expressed milk of three mothers was combined for each experiment. Two samples of each mixture were prepared and duplicate osmolality measurements were made on each sample. A third measurement was made if the first two differed by more than 3 mOsm/kg.
Warming and Storage Effects on Fortified Breast Milk
Samples of thawed, pooled breast milk were either warmed for 15 minutes at 37°C, or were stored for 12 hours in a refrigerator at 4°C and then warmed for 15 minutes at 37°C. These conditions were selected to replicate our routine hospital nursery procedures. The following fortifiers were added to breast milk after thawing:
1. Enfamil human milk fortifier (Mead Johnson, Ottawa, Canada), added at 3.24 g/100 ml.
2. Similac human milk fortifier (Ross, Montreal, Canada), added at 3.6 g/100ml. Each of these human milk fortifiers was added at a dose of 4 pkg/100ml.
3. Special Care 24 (Ross, Montreal, Canada), a preterm liquid formula tested alone and in a 1:1 ratio with breast milk.
4. A post discharge preterm powdered formula—Enfacare (Mead Johnson, Evansville, IN), added 1½ packed teaspoon/l.5 oz (13) = 9.5 g/dl
5. A liquid glucose polymer solution, Polycose (Ross, Montreal, Canada), of which 20 ml was added to 100 ml of breast milk. The following powders were added at 6 g per 100ml.
6. A powdered form of a standard 20 Kcal/oz lactose containing formula—Similac Advance (Ross, Montreal, Canada)
7. A powdered form of protein hydrolysate infant formula containing glucose polymer as carbohydrate—Nutramigen (Mead Johnson, Ottawa, Canada)
8. A lactose-free infant formula with glucose polymer as carbohydrate—Enfalac Lactose Free (Mead Johnson, Ottawa, Canada). The estimated carbohydrate content of the mixtures used is reported in Table 1.
For the initial evaluation of the impact of lactase on osmolality, expressed breast milk and three commercially available lactose containing formulas (20 kcal/oz term Similac Advance [Ross, Montreal, Canada] and preterm 20 and 24 kcal/oz Special Care [Ross, Montreal, Canada]) were used. Lactaid ®, a beta-Galactosidase (with 250 neutral lactase units per drop) was added to milk or formula at the recommended dose of 15 drops per liter. Treated milk or formula was stored in the refrigerator at 4°C. Osmolality was tested at 2, 6, and 24 hours.
Lactase and Warming Effect
Breast milk and the same lactose containing formulas were evaluated following simple lactase addition and were tested again after warming to 37°C for 15 minutes.
Osmolality was determined by vapor pressure using a Wescor vapor pressure Osmometer, model 5500 (Wescor, Inc, Logan, UT). The instrument was calibrated in milliosmoles (mOsm) per kg with aqueous solutions of sodium chloride (100, 290, and 1000 mOsm/kg). Accuracy of this instrument at the range of osmolalities measured in this study is + 3 mOsm/kg.
The Student's t test was used to compare the significance of the difference between two milk samples. A probability of P < 0.05 was considered statistically significant.
Warming and Storage Effects
Warming of the milk mixtures to 37°C was associated with increases in osmolality which ranged from insignificant for breast milk with no additives to 15% (60 mOsmol/Kg) increase for breast milk with added glucose polymer solution (Table 2). Increases for the fortified human milk ranged from 18 to 35 mOsm/kg were found after fortification of breast milk with other powders or liquid formulas containing glucose polymer.
Storage of thawed and fortified milks for 12 hours in the refrigerator followed by 15-minute warming brought about greater increases in osmolality over those noted after warming alone. The osmolality of unfortified breast milk was unchanged by refrigeration and rewarming (Table 3). The increase in osmolality after refrigeration and warming was highest in breast milk fortified with Polycose solution (82 mOsm/Kg, 21%). The osmolality of mixtures of breast milk with the post-discharge preterm formula powder (Enfacare), with glucose polymers (Polycose), and with the protein hydrolysate powdered formula (Nutramigen) exceeded the suggested maximum for preterm feedings of 425 mOsm/kg after storage and warming.
Lactase addition resulted in a significant increase in osmolality during refrigeration over 24 hours in all of the lactose containing milks tested (P < 0.001;Table 4). The changes in osmolality were most rapid in the first 2 hours of incubation and increased further in the subsequent tests at 6 and 24 hours. By 24 hours the osmolality of the lactase treated milks increased by 66% in breastmilk (from 279 to 464 mOsm/kg), by 44 and 49% in the preterm formulas Special Care 20 and Special Care 24, respectively, and by 65% (from 298 to 492 mOsm/kg) in the term formula, Similac Advance. For all tested milks, except for the 20 Kcal/oz preterm formula (Special Care, Ross), the osmolality of the lactase-treated milks exceeded 425 mOsm/kg after 24 hours of incubation.
Lactase and Warming
During the 15-minute warming of milks treated with lactase, the osmolality increased significantly, by 87 to 122 mOsm/kg (P < 0.001;Table 5). In all milks, the increases in osmolality were greater in the samples warmed for 15 minutes than in those seen in milks incubated for 2 hours in the refrigerator (P < 0.05). Samples of breast milk combined with powdered fortifiers, glucose polymer solution, or 24 Kcal/oz preterm formula Special Care had osmolalities greater than 425 mOsm/kg after 15 minutes of warming with lactase.
Two common manipulations of preterm infant feedings—fortification and lactase treatment—can bring about increases in osmolality to levels that exceed current guidelines. After routine storage and warming, breast milk with fortifiers that included glucose polymers had significantly increased osmolality. The increase in osmolality is presumed to be due to amylase (6), an enzyme in breast milk that hydrolyses glucose polymers, which are products of partial hydrolysis of starch, at neutral and acidic pH (6,14). Two previous studies have evaluated osmotic changes in breast milk with added fortifiers and have found increases in osmolality following initial mixing (6) and with refrigerator storage (15). Our study is the first to assess the impact of warming.
Treatment with lactase enzyme also produced significant increases in osmolality in both breast milk and lactose-containing formulas, in some cases to levels above the range considered safe for premature feeding. The addition of both glucose polymer containing additives and lactase to breast milk provides two mechanisms by which osmolality of the final product can be increased.
In one study in which infant formulas were treated with lactase, the disappearance of lactose correlated well with increases in osmolality (11). The recommended technique for lactose digestion suggested by the manufacturer of the commercially available lactase (Lactaid ®) is to store milk with added enzyme for 24 hours in the refrigerator before use. This lengthy incubation, which results in near elimination of lactose, is probably not necessary for infant feedings since some malabsorption of lactose is normal and probably harmless in infancy (8,9). We found that simply incubating the milk with lactase for 15 minutes at 37° was sufficient to accomplish over half of the 24-hour digestion. We expect that this level of hydrolysis is sufficient for the purpose of improving feeding tolerance.
Our findings suggest that the use of lactase may be inappropriate for preterm infants given the clear increase in osmolality produced. Although some of the osmolalities achieved during the warming were acceptable, there would be no guarantee that lactose hydrolysis would be halted at an acceptable level in a clinical situation. For the breast-fed preterm infant with suspected lactose malabsorption, it may be reasonable to accept some degree of malabsorption and loose stools, and continue to feed breast milk to gain the benefits of breast milk.
There is evidence that feeding hypertonic solutions to newborns places them at a potentially higher risk for the development of necrotizing enterocolitis (4,5). Due to the potential risk for harm from hyperosmolar feedings, recommendations have been made that neonate feedings have osmolalities no greater than 425 mOsm/kg (12). This value is similar to the osmolarity of 400 mOsm/liter recommended by the American Academy of Pediatrics (16). Feedings administered to low-birth-weight infants can remain in the stomach and small bowel as long as 2 to 3 hours (17) resulting in lengthy exposure of the mucosa to the hypertonic solution. Hypertonic solutions can then induce bowel ischemia (18,19,20) by a mechanism involving a shift of fluid into the intestine to dilute the feeding (21), and a concomitant decrease in blood flow, which may be due to a direct effect on the microcirculation (21).
Although the use of additives in breast milk may have nutritional benefits, and lactase-treated milks may improve tolerance in some infants, the potential adverse effects of some mixtures with an increased osmolality should be balanced against the potential benefits.
The authors thank Stacy Delisle, RD, for her interest and enthusiasm in the early phases of this project development.
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