Long chain polyunsaturated fatty acids (LCP) are essential for early human growth and development (1–3). Large amounts of ω-6 and ω-3 LCP, predominantly arachidonic and docosahexaenoic acids, are deposited in the developing brain and retina during prenatal and early postnatal growth (4–6). In utero, the fetus is supplied with significant amounts of preformed LCP by placental transfer (7). After birth, human milk lipids provide a relatively stable supply of ω-3 and ω-6 LCPs (8,9).
The incorporation of LCP into formulas for low birthweight infants has been recommended in Europe (10). Nowadays, there is good evidence that diets for preterm infants should contain preformed LCPs to provide a sufficient substrate supply for optimal growth and development (11). Therefore, efficient and safe forms of LCP enrichment of infant formulas are needed for preterm infants who do not receive human milk. However, it is still unclear what the preferable form and composition of such enrichments should be, since side effects have been reported in infants who receive these supplements (12–14).
Since 1935, it has been known that absorption of fat is related to the chain length and the degree of “unsaturation” of the fatty acid moieties of dietary triglycerides (15). In human infants, calcium and other mineral retention likewise appear to be related to the type of fat ingested and related to fat absorption, as shown by several investigators (16–19). Therefore, we designed a study to investigate whether or not there are differences in the absorption of calcium, phosphorus, magnesium, zinc, and copper in preterm infants fed low birth-weight infant formula with and without an LCP supplement whose composition is similar to that of human milk.
MATERIALS AND METHODS
A prospective study was conducted on 40 very low birth–weight-infants consecutively selected from patients admitted to the nursery of Hospital das Clinicas de Ribeirão Preto, São Paulo, Brazil. Twenty infants in each group is enough to detect differences of one standard deviation with a power of 0.8 and α = 0.05. To be included in the study, infants had be on full enteral feeding for two days before the beginning of the study and had to meet the following criteria: gestational age between 28 and 34 weeks (determined from the date of the last menstrual period); birth weight between 900 g and 1500 g; mother with no milk; absence of congenital anomalies; and absence of any clinical problems such as sepsis, hyaline membrane disease, patent ductus arteriosus, or the need for ventilatory support or oxygen supplementation.
None of the infants had any evidence of intestinal, hepatic, or metabolic diseases or any other any medical problem at the time of the study. Informed consent was obtained from the mothers of all infants enrolled in the study. The protocol was approved by the hospital Ethics Committee.
The infants received parenteral nutrition from admission until the day when oral feeding had reached 160 ml/kg/d. For all infants, as soon as they were stable, feeding was initiated with pasteurized-pooled human milk or mother's milk. Once the full enteral diet had been reached and well tolerated for two days, if the mother had milk and HIV-negative serology, the infant was not included in the study. If the mother had no milk, the infant were randomized to receive, in a double-blind manner, one of two batches of a commercial LBW infant formula (Prematil, Milupa, Friedrichsdorf, Germany), equal in nutrient contents except for a different fat composition. The formula used contained (per100 kcal): 2.9 g of whey-predominant cow's milk protein; 10.8 g of carbohydrates (7.0 g lactose and 3.8 g dextrinmaltose); and 5.0 g of fat derived from blended vegetable oils, milk fat, and, in the case of the LCP-enriched formula, egg lipid extracts (0.9 g) and evening primrose oil. No medium-chain triglyceride oil was used in the production of the formula. The essential fatty acid composition of the formula has been previously published (20). The energy content of the two formulas was identical (70 kcal per 100 ml). After reconstitution, both formulas showed the same content of minerals (per 100 ml): 86.2 mg of calcium, 44.1 mg of phosphorus, 6.4 mg of magnesium, 0.5 mg of zinc, and 68.8 μg of copper (results of analysis performed in our laboratory). The amount of milk offered was 160 ml/kg/d, with a similar mean protein supply of 3.01 g/kg/d. The 20 infants of each group were followed on their diets for 30 days. One week after the beginning of study, the infants were submitted to a three-day nutritional balance.
The nutritional balance consisted of precise measurements of intake and excreta over a 72-hour period. Carmine (100 mg) was added to the first feeding of the balance and charcoal to the last to delimit the period of feces collection. During the balance period, infants lay in a metabolic bed placed within an incubator as reported previously (21). The total amount given by gavage was precisely measured by weighing the syringe containing the milk volume on a precision scale before and after the feeding. If emesis or regurgitations occurred, the nutritional balance was suspended. Urine and stools were collected separately and stored at −20°C until analysis. Carmine red was used to demarcate the fecal collection. Mineral intake was calculated as concentration of minerals in the diet times the volume ingested divided by the infant weight and by 3, since data were collected over a period of three days. The final result was expressed as mg/kg/d. The same principle was used to calculate minerals in feces and urine. The balance (net retention) of each nutrient was calculated as the difference between intake and the sum of urine and fecal losses during the 72-hour interval. Percentage of fecal loss was calculated as the percentage of the ingested mineral found in feces [Fecal losses*100/Ingestion].
Preprandial blood samples (2 ml) were obtained three hours after the previous meal at the beginning and at the end of the 30 days of the study for Ca, P, Mg, Zn, and Cu measurement. Plasma, urine, and feces mineral contents were measured by atomic absorption spectrophotometry (Perkin-Elmer 380). Alkaline phosphatase was measured using the Cera-Pak kit (Miles do Brasil, Ltda).
Body weight was measured at the same time each day using electronic scales. Adequacy of weight to gestational age was based on an international growth chart (22). Crown-to-heel length was measured using a measuring board with fixed head and sidepieces and containing a built-in millimeter ruler.
Statistical analyses were performed by unpaired t test and Fisher's exact test. The significance level was set at 5%.
As shown in Table 1, 20 infants were fed conventional formula without LCP (F) and 20 infants were fed LCP-enriched formula (F+LCP). The only difference between groups was the chronological age at study start, when the Formula group was as mean, 6 days old. The two groups did not differ regarding number of males and females, birth weight, gestational age, corrected gestational age, or adequacy for gestational age. Two thirds of the infants in each group were small for gestational age. During the 30-day study period, the two groups had comparable milk intake and reached similar and satisfactory weight gains and longitudinal growth.
Within each group, there was no change in plasma mineral concentrations over the course of the study, and there were no differences at each time point between groups. Zinc plasma levels felt during the study, but as can be seen in Table 2 all values were within the normal range for age.
As presented in Table 3, the only difference between the F and F+LCP groups detected during the balances was the higher urine phosphorus loss in the F+LCP group. No differences in mineral balance were detected between F and F+LCP groups, with both groups demonstrating comparable intake, net retention and fecal losses of each mineral.
The Committee on Nutrition of the European Society of Pediatric Gastroenterology and Nutrition (ESPGHAN) states that: “enrichment of metabolites of both linoleic and alpha-linolenic acid approximating levels typical for human milk lipids (ω-6 LCP 1%, ω-3 LCP 0.5% of total fatty acids) is desirable for formulas for low birth weight infants” (10).
It is important to ensure that, when adding a different nutrient to formula or milk, this would not interfere with the delicate equilibrium between nutrients of those complex solutions. This study was designed to determine whether the addition of an LCP blend containing ω-3 and ω-6 fatty acids to preterm formula might alter the balance of Ca, P, Mg, Zn, and Cu.
Except for chronological age at study start, the study groups had similar main characteristics at the beginning of the study and after the 30-day follow-up. The fact that the F group was 6 days older than the F+LCP was not enough to reflect any difference on corrected gestational age. All infants presented adequate weight and longitudinal growth. Regarding minerals plasma levels, no alterations were detected. At the beginning of the study and after the 30 days of the study, plasma levels were adequate in infants of the two study groups (23,24). Alkaline phosphatase, an indicator of bone mineralization, also was similar between groups and within the normal range (25).
On the other hand, the three-day nutritional balance revealed some interesting findings. The mean Ca ingestion by the two groups was similar (Table 3), but lower than the 200–250 mg/kg/d recommended by the AAP (26), or the 180 mg/kg/d recommended by ESPGHAN (10). Net calcium retention was similar between groups, but did not reach the lower level of the 120–130 mg/kg/d intrauterine estimated aggregation during the last trimester of gestation (27). If any difference between the formula groups had been found, it would have been assumed to be caused by the differences in absorption related to the fat composition. Therefore, stool losses are a very important component of the nutritional balance. As can be seen in Table 3, the percentage of the ingested minerals lost in feces was similar between formulas, indicating no reduction of absorption due to LCP addition.
Phosphorus ingestion was adequate according to ESPGHAN recommendations of 50 to 90 mg/100 kcal (10), but lower than the 110–125 mg/kg/d range indicated by the AAP (26). Similar to the calcium analyses, phosphorus net retention was inferior to intrauterine estimated aggregation during the last trimester of gestation of 65–70 mg/kg/d (27). There was a tendency to lower phosphorus net retention in the F+LCP group as compared to the F group, but the difference did not reach statistical significance. The main reason for this tendency was the higher urine loss. If LCP had been involved, we would have expected a higher fecal loss, which did not occur.
The amounts of Mg, Zn, and Cu ingested and retained by the groups were similar, adequate, and higher than the intrauterine estimated aggregation during the last trimester of gestation (28–31).
The search for the best way of feeding a preterm infant still continues. Fortified human milk has systematically been shown to be superior to formula for the well being of preterm infants (32). When human milk is not available, adequate formula should be provided. Increased nutritional knowledge has made it possible to continuously improve formulas for preterm feeding. If any nutrient is to be added to a formula, it is important to thoroughly evaluate the new formula due to the possible interference of the new component with the nutrient balance. In the present study, the contents of the LCP blend added to the preterm formula were similar to those of human milk and caused no disturbance in mineral balance.
Careful attention to the fatty acid composition of formulas is indicated to assure good mineral retention in these small infants.
The authors thank Isabel Machado de Souza for laboratory assistance.
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