Effect of Added Calcium, Phosphorus, and Infant

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mendations in NEOFAX (13), a nutritional guide used commonly in. NICUs. The guidelines for different nutrition fortifiers supply different amounts of calcium and ...
R. S. Cohen, MD

ORIGINAL ARTICLE: HEPATOLOGY

AND

NUTRITION

Effect of Added Calcium, Phosphorus, and Infant Formula on Calcium and Phosphorus Dialyzability in Preterm Donor Human Milk 

April D. Fogleman, yRonald S. Cohen, zPauline Sakamoto, and §Jonathan C. Allen

ABSTRACT Objectives: We studied the effect of preparing donor human milk (DHM) with commonly used nutritional additives on the dialyzability of calcium and phosphate. We hypothesized that the additives to DHM would decrease the dialyzability of calcium and phosphate when prepared according to hospital protocols. Methods: An in vitro system simulating premature infant digestion was developed to measure dialyzability of calcium and phosphate in DHM. Dialyzable calcium and phosphate were measured after in vitro digestion in DHM before and after preparation of DHM with the following additives according to hospital protocols: calcium glubionate, sodium potassium phosphate, calcium glubionate and sodium potassium phosphate added together, Similac human milk fortifier, Similac NeoSure, or Enfamil Enfacare. Results: The percentage of dialyzable calcium in DHM with added calcium and calcium and phosphate together was greater than the percentage of dialyzable calcium in DHM with added powdered infant formulas (P < 0.0001). Dialyzable calcium was greater in DHM with added calcium and with added calcium and phosphate than in all other treatment groups (P < 0.0001). Dialyzable calcium in DHM without additives was not different from dialyzable calcium in DHM with added phosphate or with added powdered infant formulas. Dialyzable phosphate did not differ between the treatment groups. Conclusions: The addition of calcium alone or calcium and phosphate together increased calcium dialyzability in DHM significantly, whereas the addition of powdered human milk fortifier or formulas did not. The addition of calcium or calcium with phosphate together to DHM may provide the most dialyzable calcium. Key Words: calcium, dialyzability, donor human milk, fortification, infant formula, phosphate

Received November 14, 2011; accepted March 9, 2012. From the North Carolina State University, Raleigh, NC, the yDepartment of Pediatrics, Lucile Packard Children’s Hospital, Stanford University, Palo Alto, CA, the zMothers’ Milk Bank, San Jose, CA, and the §North Carolina State University, Raleigh, NC. Address correspondence and reprint requests to April D. Fogleman, PhD, IBCLC, RD, Assistant Professor of Nutrition, North Carolina State University, Schaub Food Science Building 218-B, Box 7624, NCSU Campus, Raleigh, NC 27695 (e-mail: [email protected]). Funding for this research was provided by the Mothers’ Milk Bank of San Jose, CA. The authors are not affiliated with the manufacturers of the nutritional additives studied in this article. The authors report no conflicts of interest. Copyright # 2012 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition DOI: 10.1097/MPG.0b013e318254ec07

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alcium is the most abundant mineral in the human body, with 99% of adult body calcium located in bone and the remaining 1% located in soft tissues and extracellular fluid. The full-term newborn has about 30 g of body calcium, whereas a 24-week preterm infant has only 10% to 15% of this value at 3.0 to 4.5 g body calcium (1). From 28 to 40 weeks of gestation, fetal calcium content quadruples due to increased bone mineralization (2). Approximately, 85% of body phosphorus is located in bone and 15% in soft tissues and extracellular fluid (3). The full-term newborn body contains about 16 g of phosphorus (3). Approximately, 80% of calcium and phosphorus accretion occurs during the third trimester, between 24 and 40 weeks of gestation (3), and infants born preterm miss this period of calcium and phosphorus accretion. Failure of the preterm infant to meet the mineral requirements may lead to metabolic bone disease, also called rickets or osteopenia of prematurity (3). To optimize bone mineralization and prevent metabolic bone disease, the American Academy of Pediatrics recommendations for preterm infant formulas are 140 to 160 mg of calcium per 100 kcal and 95 to 108 mg of phosphate per 100 kcal (4). The European Society for Pediatric Gastroenterology, Hepatology, and Nutrition recommendations for preterm infant formulas are 70 to 140 mg calcium per 100 kcal and 50 to 87 mg phosphate per 100 kcal (4). These recommendations are meant to serve as guidelines to ensure adequate calcium and phosphorus intake for preterm infants; however, they do not take into account the variable absorption of calcium from different sources, which depend upon many factors other than the amount of calcium provided. For example, these recommendations do not take into account the type of calcium salt, the amount or type of fat in the diet, the type of milk, and the processes used for infant formula manufacturing (5). It is important to understand the bioavailability of calcium and phosphate in donor human milk (DHM) with nutritional additives because premature infants are often given this type of feeding in the neonatal intensive care unit (NICU) and are at risk for metabolic bone disease. Ideally, bioavailability should be studied in humans; however, these studies are time-consuming, expensive, and may not be practical to perform in premature infants (6). As alternative to measuring bioavailability, in vitro methods have been used consisting of a gastrointestinal digestion followed by measurement of mineral dialyzability through a membrane (6–12). Measuring mineral dialyzability estimates the fraction of the mineral soluble in the gastrointestinal tract and available for in vivo absorption (12). Although dialyzability estimates calcium and phosphate that is soluble in gastrointestinal fluids, sometimes referred to as bioaccessibility, it may provide a good estimate of the potential bioavailability of the minerals to preterm infants

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who depend primarily on passive calcium absorption through the leaky tight junctions of the intestinal epithelium. We developed a simulated premature infant digestion system to measure the dialyzability of calcium and phosphate in DHM with and without common nutritional additives. We hypothesized that adding these minerals, HMF, and the 2 powdered infant formulas to DHM would decrease the dialyzability of calcium and phosphate when prepared according to present hospital protocols.

METHODS Preparation and In Vitro Digestion of DHM Samples Preterm DHM was shipped from the San Jose´ Mother’s Milk Bank (San Jose´, CA) to our laboratory. Mothers who donated the milk gave signed consent that their milk may be used for research purposes, as is policy for all donations to milk banks of the Human Milk Banking Association of North America. The institutional review board at North Carolina State University approved the study. Five batches of preterm DHM were used in the study and treated according to standard fortification protocols used in NICUs (13). It is standard protocol for Human Milk Banking Association of North America milk banks to pool milk from 4 to 6 mothers to reduce variability of the nutritional composition. Therefore, milk from 20 to 30 mothers was included in the analyses. Each sample was studied 7 ways: no additives (control); addition of 0.15 mL calcium glubionate (Rugby, Duluth, GA) per 1 mL milk; 0.23 mL sodium potassium phosphate (sodium phosphate and potassium phosphate) per 1 mL milk; 0.15 mL calcium glubionate and 0.23 mL sodium potassium phosphate per 1 mL milk; 0.064 g Enfamil Enfacare (Mead Johnson, Glenview, IL) per 1 mL milk; 0.1563 g Similac Human Milk Fortifier (Abbott Nutrition, Columbus, OH) per 1 mL milk; and 0.072 g Similac NeoSure (Abbott Nutrition) per 1 mL milk. The amounts of calcium and phosphate content added to each treatment are listed in Table 1. The quantities of the infant formula added were based upon recommendations in NEOFAX (13), a nutritional guide used commonly in NICUs. The guidelines for different nutrition fortifiers supply different amounts of calcium and phosphorus to premature infants. Quantities of calcium glubionate and sodium potassium phosphate were based on protocols used at Lucile Packard Children’s Hospital at Stanford University.

In Vitro Digestion Protocol An in vitro digestion model was developed to simulate the gastrointestinal tract of the premature infant. The model was

Added Nutrients and Mineral Dialyzability in Donor Human Milk modified from those described previously (14,15). In the gastric phase, 0.2 g pepsin (Sigma, St Louis, MO) was dissolved in 5 mL of 0.1 N HCl and 0.25 mL was added to each 4-mL sample of DHM. Additionally, 1.7 g lipase with similar specificity as human milk lipase (16) (Sigma) was dissolved into 15 mL 0.1 N HCl and 1.5 mL was added to each DHM sample. Lipase was added in the gastric phase because it has been shown that there is a high degree of gastric lipolysis in premature infants (17). The low pH optimum (2.5–6.5), the absence of requirements for cofactors or bile salts, and resistance to pepsin digestion enable lipase to remain active in the infant’s stomach and contribute significantly to fat digestion (16,17). The pH of each nutritional additive was adjusted to 5 before addition to the milk. All of the DHM samples were adjusted to pH 5.0 by addition of HCl or NaOH and then placed in a shaking water bath at 378C for 2 hours. They then were placed on ice for 10 minutes to stop digestion. In the intestinal phase, 0.05 g pancreatin (Sigma) and 0.3 g bile extract (Sigma) were dissolved in 25 mL of 0.1 mol/L NaHCO3 and 1.25 mL of this solution was added to each DHM sample. To add 17.2 mU of lactase (Sigma) to each DHM sample, 0.25 g of lactase was dissolved in 200 mL H2O, and 2 mL was added to each DHM sample. DHM samples were adjusted to a pH of 7.0 by 1 mol/L NaHCO3 and to a final volume of 10 mL by addition of cell culture grade water (Sigma). The DHM samples were placed in a shaking water bath at 378C for 2 hours. The DHM samples were placed on ice for 10 minutes to stop digestion and they were adjusted to pH 7.0. After the gastric and intestinal phases, the samples were centrifuged at 3500g for 1 hour at 48C. Aliquots of the supernatant were transferred to tubes and stored at 208C until further analysis, unless samples were analyzed within 24 hours, in which case they were stored at 48C. Dialysis was completed using Spectra/Por Float-A-Lyzer G2 (Model G235067, Spectrum Labs, Rancho Dominguez, CA) dialysis tubing with a molecular weight cutoff of 8000 to 10,000 Da. The Spectra/Por Float-A-Lyzer was submerged and allowed to soak in deionized water for 15 to 30 minutes. The hydrated membrane was not allowed to dry out. Using a pipette, 10 mL of the previously digested sample was added to the inside of the membrane. The cap was replaced and the membrane was placed inside a glass tube that contained 25 mL of either a solution of 0.9% NaCl with 1% albumin or 0.9% NaCl, pH 7. In this experiment, all of the samples were dialyzed with and without albumin in the buffer to determine the effects of having a protein with moderate calcium binding in the dialyzate on the final distribution of calcium and phosphate.

TABLE 1. Hospital protocols and calcium and phosphate content of additives

Treatment group Donor milk Donor milk þ calcium Donor milk þ phosphate Donor milk þ calcium and phosphate Donor milk þ Similac human milk fortifier Donor milk þ Similac Neosure Donor milk þ Enfamil EnfaCare

Preparation of donor milk samples according to hospital protocols

Calcium added, mg/mL

Phosphate added, mg/mL

No additives 3.5 mL/kg body weight/day 0.23 mL sodium potassium phosphate per 1 mL donor milk 3.5 mL/kg body weight/day and 0.23 mL sodium potassium phosphate per 1 mL donor milk 1 pack/25 mL donor milk

0 3392.5 0

0 0 725

3392.5

725

1133

649

312 322

182 162

1 tsp level powder þ 40 mL donor milk 1 tsp level powder þ 45 mL donor milk

Hospital protocols and calcium and phosphate content of additives (13).

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Fogleman et al

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The solutions dialyzed for 24 hours, except in the case of the time course experiments, after which time the volumes in the inside and outside of the membranes were measured. The contents on the inside and outside of the dialysis membrane were removed and analyzed for total calcium concentration. In a separate experiment to determine the rate and kinetics of calcium and phosphate equilibration across the dialysis membrane, total calcium and phosphate concentrations were measured at 1, 4, 8, 12, and 24 hours during dialysis with and without albumin in the buffer. The initial conditions placed the digested mixtures inside the dialysis tubing, and either 0.9% NaCl with 1% albumin or 0.9% NaCl as dialyzate on the outside. Additionally, total calcium and phosphate concentrations were measured after days 1, 2, 3, 4, and 5 of dialysis with albumin in the buffer to look for long-term changes in the redistribution of calcium and phosphate in the dialysis system.



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Electron Corp, Vantaa, Finland) was used to measure absorbance at 650 nm. A standard curve was created by plotting absorbance at 650 nm as a function of phosphate concentration. The standard curve was used to determine the phosphate concentration of each unknown sample. First, 0 to 200 mL of each standard and sample was added to the wells of the 96-well microplate and the volume of each well was adjusted to 200 mL with distilled water. Next, 30 mL of the phosphate reagent was added to all of the standard and sample wells. The samples were mixed for 30 seconds on a plate shaker and incubated for 30 minutes at room temperature. After the 30-minute incubation period, the absorbance of each sample was measured at 650 nm in triplicate. The equation resulting from the standard curve was used to determine the phosphate concentration of each sample. The equation is as follows: Phosphate concentration ¼

Biochemical Assays

  absorbance  intercept  ðDFÞ; slope

Calcium Analysis After dialysis, total dialyzable calcium was measured by analyzing the calcium content of the dialyzate using an atomic absorption spectrophotometer (Perkin Elmer Model 3100, Norwalk, CT). Calcium standards were made at calcium concentrations of 0.5 to 10 ppm in buffer containing 0.01 N HCl with 0.5% lanthanum oxide. The digested samples were diluted in buffer containing 0.01 N HCl with 0.5% lanthanum oxide so they could be measured within the range of the calcium standards.

Calcium Dialyzability Calcium dialyzability, which is the amount of dialyzable calcium, was estimated by the equilibrium dialysis of calcium in this system and it is the amount of soluble calcium capable of moving through the dialysis tubing during the 24-hour time period of dialysis, when equilibrium was achieved. Calcium dialyzability ¼ Calcium concentration outside dialysis tubing  volume outside dialysis tubing

Percent Calcium Dialyzability Percent calcium dialyzability was calculated according to the following formula: Percent calcium dialyzability ¼

Dialyzable calcium Total calcium content of sample  100

Total Phosphate Assay Phosphate concentration was determined using a phosphate colorimetric assay kit (BioVision K410–500, Mountain View, CA). The assay uses a preparation of malachite green and ammonium molybdate, which forms a chromogenic complex with phosphate ions, resulting in an absorption band around 650 nm. The kit can directly determine phosphate concentrations between 1 mmol/L and 1 mmol/L, with a lower limit of detection of approximately 0.1 nmol. The assay was performed after in vitro digestion on all of the DHM samples. A microplate reader (Multiskan EX, Thermo

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where absorbance ¼ average of the absorbencies of wells for each sample; intercept ¼ y-intercept from the standard curve graph; slope ¼ slope from the standard curve graph; DF ¼ dilution factor used to dilute DHM samples (dilution factor used varied among samples).

Statistical Analysis Experiments were performed in triplicate for analysis of total calcium and phosphate. Statistical analysis was performed using JMP (SAS Inc, Cary, NC). One-way analysis of variance with the Tukey post hoc test to describe the relation between means was used, and P < 0.05 was regarded as statistically significant.

RESULTS Percent Dialyzable Calcium When albumin was included in the dialysis buffer, the percentage of dialyzable calcium in DHM without additives (33.14%  8.3) and with added calcium (47.10%  8.7), phosphate (32.88%  4.9), and calcium and phosphate together (35.25%  7.3) was significantly greater than the percentage of dialyzable calcium in DHM with added Similac human milk fortifier (10.15%  1.4%) (P < 0.0001). Percentage of dialyzable calcium in DHM with added calcium and with calcium and phosphate together was significantly greater than in DHM with added Similac Neosure (14.32%  2.3%), Enfamil Enfacare (14.28%  0.68%), or Similac human milk fortifier (P < 0.0001). Percentage of dialyzable calcium was significantly greater in DHM without additives than in DHM with added Similac human milk fortifier (Table 2). When albumin was not used in the dialysis buffer, the percentage of dialyzable calcium in DHM with added calcium (27.56%  4.0%), phosphate (32.74%  3.7%), and calcium and phosphate together (33.55%  4.8%) was significantly greater than the percentage of dialyzable calcium in DHM with added Enfamil Enfacare (13.43%  1.6)%, with added Similac NeoSure (13.24%  0.75%), and with added Similac human milk fortifier (12.58%  0.5%) (P < 0.0001). When the 2 methods of dialysis are compared, with and without use of albumin in the dialysis buffer, percentage of dialyzable calcium is significantly greater in DHM with added calcium when albumin is included in the dialysis buffer (47.10%  8.7%) than when albumin is not included in the buffer (27.56%  4.0%) (P ¼ 0.0018) (Table 2). www.jpgn.org

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Added Nutrients and Mineral Dialyzability in Donor Human Milk

TABLE 2. Calcium: total, percent dialyzable, and total dialyzable Total dialyzable calcium, mg/mL

Percentage of dialyzable calcium

Samples DHM DHM þ calcium glubionate DHM þ sodium potassium phosphate DHM þ calcium þ phosphate DHM þ Similac human milk fortifier DHM þ Similac Neosure DHM þ Enfamil Enfacare P

Albumin present in dialyzate

Albumin not present in dialyzate

Albumin present in dialyzate

Albumin not present in dialyzate

33.14  8.3a,b  47.10  8.7a a,b 32.88  4.9 35.25  7.3a 10.15  1.4c 14.32  2.3b,c 14.28  0.68b,c