Metabolic phenotype of breast-fed infants, and infants fed standard formula or bovine MFGM supplemented formula: a randomized controlled trial

Formula-fed (FF) infants exhibit a different metabolic profile than breast-fed (BF) infants. Two potential mechanisms are the higher protein level in formula compared with breast milk and the removal of the milk fat and associated milk fat globule membranes (MFGM) during production of infant formula. To determine whether MFGM may impact metabolism, formula-fed infants were randomly assigned to receive either an MFGM isolate-supplemented experimental formula (EF) or a standard formula (SF) from 2 until 6 months and compared with a BF reference group. Infants consuming EF had higher levels of fatty acid oxidation products compared to infants consuming SF. Although the protein level in the study formula was approximately 12 g/L (lower than most commercial formulas), a metabolic difference between FF and BF remained such that FF infants had higher levels of amino acid catabolism by-products and a low efficiency of amino acid clearance (preference for protein metabolism). BF infants had higher levels of fatty acid oxidation products (preference for fat metabolism). These unique, energy substrate-driven metabolic outcomes did not persist after diet was shifted to weaning foods and appeared to be disrupted by complementary feeding. Our results suggest that MFGM may have a role in directing infant metabolism.

(orange). The 95% confidence interval was estimated using the normal distribution.

Supplementary Methods
Quantitative NMR-based metabolomics assessment Sample preparation. Serum samples were filtered through a 3 kDa molecular weight ultracentrifugal filter (Amicon ultracentrifugal filter, Millipore, Billerica, MA) to remove insoluble lipid particles and proteins.
The volume of each filtrate was carefully measured and the appropriate amount of Mili-Q water (Milipore, Billerica, MI) was added to each filtrate to ensure a final volume of 199 µL. 8 µL of potassium phosphate buffer (1M, pH 6.1) was added, and the sample pH was adjusted to 6.80 ± 0.08 (average ± standard deviation). 23 µL of an internal standard (5 mM DSS-d6) containing 0.2% NaN3 in 99.8% D2O was added to inhibit microbial growth and ensure NMR locking respectively. 180 µL aliquots were subsequently transferred to 3 mm Bruker NMR tubes (Bruker, Brillerica, MA) and stored at 4 °C until spectral acquisition.
NMR acquisition, data processing and quantification. 1 H NMR spectra were acquired at 298K using the NOESY 1 H presaturation experiment ('noesypr1d') on a Bruker Avance 600 MHz NMR spectrometer (Bruker BioSpin, Germany) equipped with a SampleJet autosampler (Bruker BioSpin, Germany). Data were acquired using 32 transients and 8 dummy scans over a spectral width of 12 ppm with a total acquisition time of 2.5 s. Water saturation was applied during relaxation delay (2.5 s) and mixing time (100 ms). The resulting spectra were Fourier transformed with zero filling to 128 k data points and the Free Induction Decays (FIDs) were transformed with an exponential apodization function corresponding to a linebroadening of 0.5 Hz. Spectra were manually phased and baseline corrected using Chenomx NMR Suite v8.3 (Chenomx Inc, Edmonton, Alberta, Canada). Quantification of each metabolite was assigned manually using Chenomx Profiler based on the established method of targeted profiling 1 .

Untargeted MS-based metabolomics assessment
Plasma from infants at 6 months of age were analyzed for the presence and relative quantity of metabolites using GC-MS and LC-MS at the Swedish Metabolomics Centre, Umeå, Sweden. After data acquisition, all non-processed MS-files were exported from ChromaTOF software (LECO) into MATLAB R2016a (Mathworks, Natick, MA, US). Baseline correction, chromatogram alignment, data compression and multivariate curve resolution were performed using custom scripts developed at the Swedish Metabolomics Centre and run on Matlab. Peak detection and compound identification was performed by comparing retention index and mass spectra with reference spectra 3 using the NIST spectral search program 2.0 (NIST/EPA/NIH Mass Spectral Library). Annotation of mass spectra was based on reverse and forward searches in the library. Masses and ratio between masses indicative of a derivatized metabolite were noted, and if the mass spectrum indicated a high probability for a particular metabolite and the retention index between the sample and the library for the suggested metabolite was ± 5, the deconvoluted peak was identified.

LC-MS analysis: data acquisition and processing. LC-MS analysis was performed on an Agilent 1290
Infinity UHPLC-system (Agilent Technologies, Waldbronn, Germany) interfaced with an Agilent 6540 Q-TOF mass spectrometer equipped with a jet stream electrospray ion source, operating in positive or negative ion mode.
2 µL of sample was applied onto an Acquity UPLC HSS T3 column (2.1 x 50 mm, 1.8 µm particle) in combination with a Van Guard precolumn (2.1 mm x 5mm, 1.8 µm particle, Waters Corporation, Milford, MA, USA) at 40 °C. The chromatographic separation was performed using 0.1% formic acid in water (mobile phase A) and 0.1 % formic acid in acetonitrile-isopropanol (75:25, v/v) (mobile phase B) under linear gradient condition. Under a flow rate of 500 µL/min, the compounds were eluted with a linear gradient consisting of 0.1-10% B over 2 min. B was then increased to 99% over 5 min and held at 99% for 2 min. The column was then returned to the initial conditions (0.1% B) for 0.3 min. To reduce reequilibration time, the flow-rate was increased to 800 µL/min for 0.5 min, and held at this rate for 0.9 min, after which the flow-rate was reduced to 500 µL/min for 0.1 min before the next injection.
Analysis was performed in both polarities, positive and negative modes, in order to determine as many compounds as possible that have either basic or acidic characteristics. The settings of both modes were kept identical, with the exception of the capillary voltage. The instrument was operated at +4000 V capillary voltage in positive ion mode and -4000 V in negative ion mode, 40 psig nebulizer pressure, 300 °C gas temperature, 8 L/min drying gas flow, 0 V collision energy, 350°C sheath gas temperature and 11 L/min sheath gas flow. The nozzle, fragmentor, skimmer and octopole voltages were 0 V, 100 V, 45 V and 750 V respectively. Mass spectra were recorded across the measured range from 70 to 1700 m/z in centroid mode at 4 scans/sec. Blanks, diluted samples, and quality control samples were analyzed at the middle and the end of the sequence to control system stability.