Lipid-Based Nutrient Supplementation Increases High-Density Lipoprotein (HDL) Cholesterol Efflux Capacity and Is Associated with Changes in the HDL Glycoproteome in Children

Prenatal plus postnatal small-quantity lipid-based nutrient supplements (SQ-LNS) improved child growth at 18 months in the International Lipid-Based Nutrient Supplements DYAD trial in Ghana. In this secondary outcome analysis, we determined whether SQ-LNS versus prenatal iron and folic acid (IFA) supplementation improves the cholesterol efflux capacity (CEC) of high-density lipoprotein (HDL) particles and alters their lipidomic, proteomic, or glycoproteomic composition in a subset of 80 children at 18 months of age. HDL CEC was higher among children in the SQ-LNS versus IFA group (20.9 ± 4.1 vs 19.4 ± 3.3%; one-tailed p = 0.038). There were no differences in HDL lipidomic or proteomic composition between groups. Twelve glycopeptides out of the 163 analyzed were significantly altered by SQ-LNS, but none of the group differences remained significant after correction for multiple testing. Exploratory analysis showed that 6 out of the 33 HDL-associated proteins monitored differed in glycopeptide enrichment between intervention groups, and 6 out of the 163 glycopeptides were correlated with CEC. We conclude that prenatal plus postnatal SQ-LNS may modify HDL protein glycoprofiles and improve the CEC of HDL particles in children, which may have implications for subsequent child health outcomes. This trial was registered at clinicaltrials.gov as NCT00970866.


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The enrichment analysis is done using the hypergeometric distribution. N is the total number of glycopeptides; m is the total number of glycopeptides of a particular protein; Number Glycopeptide Increase in SQ-LNS is the number of glycopeptides that are increased in the SQ-LNS group; Number Glycopeptide Decrease in SQ-LNS is the total number of glycopeptides that are decreased in SQ-LNS group (higher in the IFA group); P value Increased can be interpreted as the probability that having x number of glycopeptides increased in the SQ-LNS group is by chance; P value Decreased can be interpreted as the probability that having x number of glycopeptides decreased in the SQ-LNS group is by chance. SQ-LNS, small-quantity lipid-based nutrient supplements; IFA, iron and folic acid.  Figure S1. Flowchart of study profile downloaded from Uniprot.org. We used in-house libraries for N-glycan and O-glycan compositions. (Glyco)peptides were identified based on the accurate mass of the precursor ions with tolerance set at 10 ppm and by matching MS/MS fragmentation with theoretical MS/MS spectra generated from in silico digestion of the provided protein database.
A total of 33 HDL-associated proteins were monitored in this study including

Targeted glycoproteomics analysis
Tryptic digestion of the HDL samples was done in a 96-well format to facilitate batch processing. Samples were randomized before plating. All reagents were freshly prepared in a buffer of 50 mM ammonium bicarbonate. A sample volume of 10 μL purified HDL was used for tryptic digestion. After every 20 samples, 10 μL of commercially available human serum (Sigma-Aldrich) was also digested to serve as sample preparation controls. Protein standards (APOA1, APOC1, APOD, APOE, CLUS; all from Sigma-Aldrich) were mixed in known amounts (250,250,125,200, and 125 μg/mL, respectively) and digested with the batch to serve as calibration standards. Serial dilution of the digested protein mixture provided the calibration curve for absolute quantitation of the 5 proteins. The digested mixture was diluted by factors of 160,80,40,20,16,8,4,2, and 1 to obtain 9 calibration standards, from which calibration curves spanning 4 orders of magnitude were calculated.
After pipetting the samples, controls, and standards onto the 96-well plate, 10 μL of 100 mM dithiothreitol was added to each well to reduce the protein disulfide bonds. Protein denaturation was continued by heating in a water bath for 1 h at 65°C. Samples were then alkylated with 5 μL of 360 mM iodoacetamide for 30 min at room temperature away from light.
Excess iodoacetamide was quenched with 5 μL of 100 mM dithiothreitol. Proteins were digested with 10 μl of 200 μg/mL sequencing grade trypsin for 18 h at 37°C. The digestion was stopped by acidifying the solution with 5 μL 10% (v/v) formic acid (Fluka). To account for batch variability and possible run-order effects, 5 μL of a 1 μg/mL synthetic peptide with sequence RPAIAINNPYVPR (Bionexus, Oakland, CA) was added as an internal standard. The final volume of the HDL digest is 50 μL, a 5-fold dilution of the purified HDL solution. The samples were injected into the LC-MS instrument without further cleanup.
(Glyco)peptides were quantified on an Agilent 1290 Infinity II LC system coupled to an Agilent 6495B Triple Quadrupole MS. Injection volumes were set at 5 μL for protein standards S37 and HDL samples, and 1 μL for serum digests. A pooled sample of the digested serum was run after every 10 HDL samples to serve as quality control (QC). They were used to monitor the stability of the instrument and the reproducibility of the batch analysis.
The HPLC was equipped with a 150 mm Agilent Zorbax Eclipse Plus C18 column with 1.8 μm particle size. A C18 column guard was used to protect the column from the buildup of lipids and other hydrophobic substances in the sample. A binary gradient of (A) 3% acetonitrile with 0.1% formic acid in water and (B) 90% acetonitrile with 0.1% formic acid in water was set at a flow rate of 0.5 mL/min. The HPLC pump parameters were programmed to ramp from 0% to 20% B in 20 min, 30% at 40 min, 44% at 47 min, and 100% at 48 min followed by 12 min column flushing cycle with 100% B and 7 min equilibration at 100% A. The Electrospray Ionization (ESI) voltage was set to 3500 V in the positive mode.