The production of intrinsically labeled milk protein provides a functional tool for human nutrition research

Abstract

Oral or intravenous administration of labeled, free amino acids does not allow the direct assessment of protein digestion and absorption kinetics following dietary protein intake. Consequently, dietary protein sources with labeled amino acids incorporated within the protein are required. The aim of this study was to produce milk proteins intrinsically labeled with l-[1-(13)C]phenylalanine that would allow the assessment of protein digestion and absorption kinetics and the subsequent muscle protein synthetic response to dietary protein intake in vivo in humans. Two Holstein cows (body weight of 726 +/- 38 kg) were continuously infused with l-[1-(13)C]phenylalanine at 402 micromol/min for 44 to 48 h, during and after which plasma samples and milk were collected. After milk protein separation, casein was used in a subsequent human proof-of-principle experiment. Two healthy males (aged 61 +/- 1 yr; body mass index of 22.4 +/- 0.1 kg/m(2)) ingested 35 g of casein highly enriched with [1-(13)C] phenylalanine. Plasma samples were collected at regular intervals, and skeletal muscle biopsies were collected before and 6 h after casein ingestion. In the initial experiment, a total of 5.83 kg of l-[1-(13)C]phenylalanine-enriched milk protein (casein enrichment was 29.4 molar percent excess) was collected during stable isotope infusion in the cows. In the proof-of-principle study, ingestion of 35 g of intrinsically labeled casein resulted in peak plasma l-[1-(13)C]phenylalanine enrichments within 90 min after protein ingestion (9.75 +/- 1.47 molar percent excess). Skeletal muscle protein synthesis rates calculated over the entire 6-h period averaged 0.058 +/- 0.012%/h. The production of intrinsically labeled milk protein is feasible and provides dietary protein that can be used to investigate protein digestion and absorption and the subsequent muscle protein synthetic response in vivo in humans.

The muscle protein synthetic response to the combined ingestion of protein and carbohydrate is not impaired in healthy older men

“…13 C]phenylalanine-labeled protein, specifically produced by the infusion of large amounts of tracer in a lactating Holstein cow (Pennings et al 2011c;van Loon et al 2009b), we are able to detect the appearance of dietary protein-derived phenylalanine in the circulation (Fig.3) and assess its subsequent incorporation in skeletal muscle protein Koopman et al 2009b;Pennings et al 2011a;Pennings et al 2011b;. Two hours after ingestion of the protein, skeletal muscle protein already showed a substantial increase in [1-13 C]phenylalanine enrichment (0.0071±0.0016 and 0.0072±0.0013 MPE in the young and older group, respectively; P=0.97).…”

Food ingestion in an upright sitting position increases postprandial amino acid availability when compared with food ingestion in a lying down position

“…To simultaneously assess gastric emptying rates and protein digestion and amino acid absorption, subjects were administered 20 g intrinsically L-[1-13 C]-phenylalanine-labeled milk protein (van Loon et al 2009) mixed with 1.5 g paracetamol (acetaminophen) (van Can et al 2014, Panahi et al 2014, Holwerda et al 2016). This study extends upon our previous work (Holwerda et al 2016) and shows that protein ingestion in an upright, sitting body position accelerates gastric emptying rate when compared to protein ingestion in a lying down body position, resulting in more rapid protein digestion and amino acid absorption.…”

Good, Bad, or Risky? : intestinal permeability to protein: human and in vitro studies

“…Caco-2 cells are the most frequently used cellular models to study intestinal epithelial permeability [66,67]. Caco-2 cells can spontaneously differentiate into polarised intestinal cells in culture [68,69]. Even though they are derived from a colon adenocarcinoma, upon differentiation they show many characteristics of small intestinal enterocytes [68,69].…”

“…Caco-2 cells can spontaneously differentiate into polarised intestinal cells in culture [68,69]. Even though they are derived from a colon adenocarcinoma, upon differentiation they show many characteristics of small intestinal enterocytes [68,69]. They have the ability to differentiate into a polarized monolayer on microporous filters after which they form domes, and present tight junctions and an apical brush border with its associated hydrolases, such as sucrase-isomaltase.…”

“…Detection methods can then be developed that specifically target such peptides, as was reported here for Ara h 6 in chapter 4. If mass spectrometry is applied, the use of intrinsically labelled proteins could support the detection of small peptides with higher specificity in humans [68].…”