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Future Research on Quality Factors

In responding to its statement of task, the committee identified topics related to the assessment of protein quality and normal physical growth that require further research. This chapter discusses the future research needs for these quality factors.

PROTEIN QUALITY

Human Milk Amino Acid Pattern

While the committee recommended adoption of the human milk amino acid pattern (HMAA) as the reference pattern to assess protein quality in infant formula, it noted that this approach should be refined by research, preferably on a global basis. The potential contributions of geographical region, season, ethnicity, age, nutritional status, and behavioral habits on the pattern of both indispensable and conditionally indispensable amino acids (AA) needs to be assessed.

The use of the standard reference unit mg or µmole AA per g nitrogen needs to be assessed to enable comparison between investigations. In addition, consideration should be given to the nitrogen-to-protein conversion units and whether a conversion factor other than the standard 6.25 should be used for infant formula when novel protein sources are introduced. The committee recognizes that the Stakeholder Program on Infant Formula and Adult Nutrition (SPIFAN), overseen by the International Special Dietary Foods Industry (ISDI) has published standard

method performance factors for AAs in infant formula (AOAC International, 2015).

There is a need to standardize methodology for the AA analysis of human milk. The influence of different infant formula processing time on protein hydrolysis, specific indispensable AAs and conditionally indispensable AAs, and different chemical parameters needs further validation and standardization. Once a broader basis of data has been created by standardized methods and becomes available, a revision of the present HMAA patterns in different jurisdictions of the world should be attempted if considered appropriate.

Assessment of Digestibility

While the committee accepts that the digestibility of protein/AAs in human milk is not of concern, standardized procedures for assessing digestibility of protein sources in infant formula are essential when new protein sources are introduced.

Over the past 25 years, the young, growing pig has emerged as the premier pre-clinical model for the study of pediatric nutrition due to its comparable anatomy, physiology, and metabolism to the human infant and its adaptability to artificial rearing systems (Burrin et al., 2020; Odle et al., 2014). To ensure rigor and reproducibility, standardized procedures for conducting digestibility and growth monitoring studies in piglets should be established. A standard set of metadata should be required including the selection of breed, age, and sex of the animals, housing conditions, mode and timing of feeding, length of study and composition of the control formula.

The committee reviewed several in vitro digestibility assays to assess protein and AA digestibility. No method has been sufficiently tested and validated for infant formulas. There is a need to standardize and harmonize in vitro digestibility methods and validate these methods against in vivo methods. The committee notes that other authoritative bodies that have recommended digestible indispensable amino acid score (DIAAS) be validated as a method of choice for infant formula and was aware that the “Milk and milk products—In vitro digestion protocol for the determination of protein digestibility and in vitro digestible indispensable amino acid score (DIAAS)” assays have been submitted for ISO certification (ISO, 2024); U.S. Food and Drug Administration (FDA) engagement in that effort would advance the use of validated assays for digestibility of protein in infant formulas.

Considerations for Compounds that Influence Digestibility

It is well known that adducts/Maillard reaction products form when milk protein components react with lactose. Research is needed on the impact of the reaction products that are formed due to new or novel protein sources and how they might influence indispensable and specific conditionally indispensable AA digestibility. Furthermore, infant formula processing might impact AA bioavailability. The impact of protein sources on protein quality needs to be quantified to ensure that the “availability” of AAs is not compromised in infant formulas. Thus, the amino acid composition must be analyzed post-processing of infant formula to include all end-products of the hydrolyzed protein.

GROWTH MONITORING STUDIES

The FDA has considered whether to conduct and report an adequate growth monitoring study (GMS) (as noted in Chapter 2) in the case of limited publicly available information on current “alternative study designs.” The committee has identified an evidence gap on what could be considered an alternative study design. To determine alternative study design, information on GMS submissions and an understanding of regulatory requirement interpretation and implementation would be needed to assess how various design features implemented in GMSs are interpreted by FDA. Published guidance on alternative study designs would support regulatory adherence by all infant formula manufacturers with and without prior experience with the FDA process.

In the GMSs that have been submitted with infant formula marketing applications, FDA has a unique dataset representing the growth of formula-fed infants. The pooled data are amenable to meta-analysis, given the required aspects of study design and data collection. FDA could make anonymized data public for additional analysis, which among other things could result in a standard for growth of formula-fed infants and estimate of the bias of formula feeding relative to breastfeeding, as represented by the World Health Organization (WHO)/U.S. Centers for Disease Control and Prevention (CDC) growth standard charts (that is, normal physical growth of solely formula-fed infants compared to exclusively breastfed infants). Furthermore, the combined data on reported AEs experienced by formula-fed infants would constitute a larger and perhaps more detailed representation of AEs in infancy, a reference against which future studies on infant formula could be compared. To support pooling of data on adverse events (AEs), FDA needs to publish a standardized classification of AEs as guidance.

Body Composition as a Quality Factor of Growth

While body composition techniques beyond routine anthropometry are not currently used by FDA in its evaluation of infant formula, these methodologies are an active area of research. Assessment of body composition using air-displacement plethysmography (ADP), dual-energy x-ray absorptiometry (DXA), or multisite skinfold thickness is now widely available, and normal reference standards have been published. Use of deuterium dilution with urine collection can also be used to measure body composition if one has access to mass spectrometry technology. In the few studies identified in the scoping review, some showed significant differences in body fat and lean mass in response to feeding different formulas, and some showed significant differences between feeding formula and human milk (Jaramillo-Ospina et al., 2022; Kouwenhoven et al., 2020, 2021; Liotto et al., 2018; Plaza-Diaz et al., 2022; Putet et al., 2016; Sobik et al., 2021; Totzauer et al., 2018). Population-based reference data for body composition measures of percentage fat or lean mass or fat or lean mass index are being collected and should be considered in developing future measures of quality factor assessment of infant formulas (Gallagher et al., 2020).

Early-onset alterations in body composition may have long-term health consequences for measured outcomes at 6 years (Totzauer et al., 2018). Research is needed to assess measures of total body fat and lean mass as quality factors in assessing the efficacy of experimental formulas.

Biomarkers

In the final FDA rule (79 FR 33037, June 10, 2014), quality factors were defined as “those factors necessary to demonstrate the safety of the infant formula and the bioavailability of its nutrients, as prepared for market and when fed as the sole source of nutrition, to ensure the healthy growth of infants.” The only quality factor required by FDA is normal physical growth, with no specific requirement to assess biomarkers.

In the scoping review, 17 of the 143 identified studies used biomarkers as measures in randomized controlled trials (RCTs) designed to investigate an experimental formula with specific modifications in the content or composition (see Appendix E). The biomarkers in those RCTs included circulating nutritional biomarkers (e.g., amino acids, fatty acids, ferritin), or metabolomic markers (mostly in the scenario of assessing longer term outcomes), and immune markers/gut microbiome. Measures of bone mass were also included. Biomarkers are potentially useful for determining the safety and efficacy of an infant formula or the validity of specific label claims. However, biomarkers were not specifically included in the

statement of task, and the scoping review was not conducted in a way to determine if biomarkers could be used as a complementary measure to anthropometry measures.

It would be valuable to carefully review and develop sensitive and specific biomarkers that can be validated and correlated with current and long-term growth and health outcomes of infants, especially in evaluating the safety, or long-term impact on growth, metabolic, immune, or developmental outcomes of a new formula.

REFERENCES