O’Neill, Edward F.; Radmacher, Paula G.; Sparks, Blake; Adamkin, David H.
Human milk (HM) is the preferred method of enteral feeding for all infants, including the very-low-birth-weight (VLBW) infant (1–12). HM provides preterm infants important protection against infection (6,13,14) and necrotizing enterocolitis (2,7,15); however, HM may have an insufficient concentration of protein, energy, and minerals to support the rapid growth of the VLBW infant (16–21). Consequently, most HM fed to VLBW infants is fortified with either powder or liquid products. This fortification is based on general assumptions of energy and protein content of the milk (22,23). One method to estimate the energy content of an individual milk sample was first published by Lucas et al in 1978 called the “creamatocrit” (24) and also reported by Lemons et al (25). Much like the familiar hematocrit, this method uses the percentage of cream to total volume, which is then substituted into an equation to yield energy (kcal/oz). The equation is based on assumptions of constant protein and carbohydrate content, with fat being the only variable nutrient. The contributions of protein and carbohydrate are assumed based on values from the literature (26–28). Meier (29) and Valentine (30) showed that the creamatocrit (CMCT) could be used by nursing staff in the clinical setting to take advantage of the natural variations within a milk expression to engineer the milk a mother provided for her infant to contain a higher fat content and, consequently, higher energy. Mothers, too, could perform the measurement to participate in “lactoengineering” their own milk.
Although inexpensive and easy to do, the CMCT-derived energy calculation is directly related to fat content only; it cannot provide information about nonfat nutrients. This limitation may be less important for the term newborn but could have important ramifications for the VLBW infant, whose mother's milk changes significantly during the first weeks of lactation and who has increased growth requirements (17,31–35).
Infrared (IR) spectroscopy has long been used in the dairy industry to analyze bovine milk quality (36). This technology has been adapted and calibrated to measure fat, protein, and lactose in HM (37), and there are now multiple devices that use mid- or near-IR wavelengths for analysis (37–40). Concentrations of these macronutrients (g/dL) are then used to calculate energy content (kcal/oz). The present study compares fat and energy results in discrete HM samples by CMCT and human milk analyzer (HMA).
Aliquots of HM (n = 51) left over from daily preparation were obtained. All had been previously frozen and thawed on the same day as analyzed. No mother's milk was sampled more than once. All milk was prewarmed to 40°C for 10 minutes before analysis and was analyzed within minutes.
CMCT was performed in triplicate, according to the method of Lemons et al (25). Well-mixed milk samples were drawn into a standard capillary tube, sealed at 1 end with clay, then centrifuged for 15 minutes at 3500 rpm. The cream layer and the total volume were each measured to the nearest 1 mm; the percentage of the total volume of the sample represented by the lipid layer was calculated. The CMCT (percent fat) was then substituted into the following equation: kcal/dL = 38.7 + (5.9 × CMCT [%])/3.33 to estimate the energy content (kcal/oz).
The HMA (Calais Human Milk Analyzer, Metron Instruments, Solon, OH) uses mid-IR spectroscopy (36). IR energy with wavelength 3 to 10 μm radiates from an incandescent source lamp and passes through 6 filters mounted on a disk. This allows readings at 6 wavelengths (3 for fat, 1 each for protein, lactose, and solids). Absorption at each wavelength is most sensitive to a particular component in the sample to determine the amount of protein, fat, and carbohydrate based on the absorption of IR energy by specific chemical bonds: CH groups in fatty acid chains of fat molecules (3.48 μm), carbonyl groups in ester linkages of fat molecules (5.723 μm), peptide linkages between amino acids of protein molecules (6.465 μm), and OH groups in lactose molecules (9.610 μm). A detector converts the IR energy into an electrical signal processed by a computer to generate a value for each macronutrient.
The HMA device has been adapted from use in the dairy industry and is specifically calibrated to HM with a series of 6 pooled HM samples with varying concentrations of each compound. Nutrient content of the calibrators was assayed by a certified reference laboratory (DQCI, Mounds View, MN) by the following methods: protein (Kjeldahl) (41), carbohydrate (high-performance liquid chromatography) (42), and fat (Mojonnier) (43). Those values are then entered into a computer model, which was used to derive the results of subsequent samples. The energy content is a mathematical function, multiplying the protein and carbohydrates by 4 kcal/g and the fat by 9 kcal/g. A control milk was included in daily runs as an additional check on instrument performance. This is a pooled HM sample that has been analyzed similarly to the calibration samples and comes with an established range of expected results for each compound. Additionally, 10 random samples of milk were analyzed locally by both methods and then sent to the reference laboratory for analysis.
Descriptive statistics are provided. The results of fat and energy analysis were compared by paired t test and 1-way analysis of variance. Statistical significance was set at P < 0.05.
Table 1 displays the mean ± SD values and ranges for fat content and energy by the 2 methods for both discrete samples and controls. CMCT results exceeded HMA in all analyses. Overall, mean fat content of HM samples was, on average, 80% higher by the CMCT method compared with the HMA. Mean energy content of those samples was, on average, 26% higher by the CMCT method when compared with the HMA. When comparing fat content results from the control milks, the mean CMCT method exceeded the HMA results by 46% and the energy content by 16%. Three strata of CMCT values were created posthoc and the CMCT was compared with the HMA value (Fig. 1). There was a significant difference between the ratio for <4% stratum and the >6% stratum for both fat and energy. The mean protein content in the HM samples, which cannot be determined from the CMCT method, was 1.42 ± 0.41 g/dL as measured by the HMA.
Table 2 displays results for 10 random milk samples, which were analyzed locally by HMA and CMCT and then sent to the certified reference laboratory for analysis. Laboratory results for fat and energy showed no statistically significant difference from HMA. CMCT results for fat were significantly higher than laboratory or HMA (P < 0.001) and, on average, 74% to 76% higher. Energy results from the CMCT calculation were significantly higher than laboratory or HMA (P = 0.002) and, on average, 23% to 24% higher.
Extrauterine growth failure is common in the ELBW (extremely-low-birth-weight; <1000 g birth weight) infant, and may be associated with long-term growth failure and neurodevelopmental deficit (4,44–46). HM is often not sufficient on its own to provide the nutrients needed for adequate growth by the ELBW infant, and therefore must be fortified to increase the energy, protein, and minerals. HM is routinely fortified with the assumption it has 20 kcal/oz of energy and 2.1 g/100 kcal of protein. As our study shows, this is not always the case. The macronutrient content is known to vary from mother to mother, within an expression period, and from week to week in lactation (26–28). Individualized fortification for the extremely small premature infant based on an accurate assessment of macronutrient content in HM allows for standardized intakes of macronutrients.
Point of care analysis of HM has been largely limited to a method called the CMCT, developed in the late 1970s by Lucas et al (24) and subsequently used by others in clinical settings (29,30,47,48). This is a simple and inexpensive tool to estimate energy content based on measurement of the percent fat in an HM sample, but assuming constant amounts of protein and lactose. The lipid component in HM has the highest degree of variability within a single feeding/expression among the major macronutrients, and therefore, is the major component in the milk energy calculation by the CMCT method. When planning fortification of milk for the VLBW infants, the provision of adequate protein is one of the most important considerations. The CMCT method cannot provide this information.
We compared fat and energy content as measured by the CMCT and HMA on discrete HM samples and “control” milks. The CMCT method is an extremely gross measurement based on the gravimetric differences between lipid and nonlipid components in the milk sample and visual quantification. The HMA employs measurements that are specific to the chemical bonds of the lipid (as well as other components) and are independent of human estimation. We demonstrated an increasing deviation between the methods for fat and energy as the CMCT increased, sometimes as much as 2-fold. This resulted in much higher estimates of energy content by CMCT when compared to the HMA and laboratory-based results. The use of an external control milk in our protocol checked proper instrument performance. The laboratory analysis confirmed the accuracy and reliability of the HMA results.
Although it was not the purpose of the present study to include measurement of other nutrients, the HMA analyzer provides information about protein content, which is often the limiting nutrient in postnatal growth for this population. Having this information in real time contributes to the ongoing nutritional management of these infants.
The mid-infrared HMA is an excellent option for real-time analysis of the lipid, protein, and carbohydrate content in HM. It is easy to use, and has a process for calibration and instrument reliability. It provides information that can be used to individualize HM fortification for infants at high risk for extrauterine growth failure. The present study demonstrated its greater accuracy versus the CMCT, which overestimates fat and energy content of HM.
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