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Modified throughput ninhydrin method for the qualitative assessment of dietary protein absorption in pig plasma

Pierzynowska, Kateryna LU orcid ; Zaworski, Kamil ; Wychowanski, Piotr ; Donaldson, Janine ; Wolinski, Jarosław LU ; Borowitz, Drucy ; Gallotto, Robert and Pierzynowski, Stefan LU (2024) In Biology Methods and Protocols 9(1).
Abstract

Protein maldigestion and malabsorption lead to malnutrition and are a feature of exocrine pancreatic insufficiency (EPI). Although it is the current standard, measurement of nitrogen in stool to assess protease activity is indirect. Up to 80% of hydrolysed proteins appear in blood in the form of peptides, so we developed a method to measure peptide-derived amino acids in plasma as a relevant measure of proteolysis, verified its accuracy, precision, and linearity, and validated it in a porcine model. We modified a ninhydrin method. Large proteins were eliminated from plasma with 10 kDa-cut-off centrifugal filters. Free and total amino acids were measured in permeate before and after its hydrolysis. Peptide-derived amino acids were... (More)

Protein maldigestion and malabsorption lead to malnutrition and are a feature of exocrine pancreatic insufficiency (EPI). Although it is the current standard, measurement of nitrogen in stool to assess protease activity is indirect. Up to 80% of hydrolysed proteins appear in blood in the form of peptides, so we developed a method to measure peptide-derived amino acids in plasma as a relevant measure of proteolysis, verified its accuracy, precision, and linearity, and validated it in a porcine model. We modified a ninhydrin method. Large proteins were eliminated from plasma with 10 kDa-cut-off centrifugal filters. Free and total amino acids were measured in permeate before and after its hydrolysis. Peptide-derived amino acids were quantified by subtracting free amino acids from total amino acids. We verified the method in vitro and by comparing results in healthy and EPI pigs. The accuracy of the analysis was close to 100%, with excellent precision (mean relative standard deviation for low, medium, and high amino acid levels ¼ 0.88%) and with stringent linearity (r2 ¼ 0.986, %RE ¼ 5.23). The high-throughput ninhydrin method detected levels of peptide-derived amino acids in vivo with maximal changes seen approximately 2 hours postprandially in young pigs. The AUC and Cmax were significantly higher in healthy compared to EPI pigs (P ¼ .0026 and P ¼ .0037, respectively). The high-throughput ninhydrin method is a sensitive, reliable, and practical method for the estimation of dietary peptide-derived amino acids. This assay endpoint could serve as a direct biomarker of protein digestion and absorption.

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author
; ; ; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
EPI pigs, healthy pigs, method, peptide absorption, validation
in
Biology Methods and Protocols
volume
9
issue
1
article number
bpae078
publisher
Oxford University Press
external identifiers
  • pmid:39512855
  • scopus:85208814319
ISSN
2396-8923
DOI
10.1093/biomethods/bpae078
language
English
LU publication?
yes
additional info
Publisher Copyright: © The Author(s) 2024. Published by Oxford University Press.
id
6285b569-f20a-4669-9026-7a9fe6dc118e
date added to LUP
2025-01-23 08:32:25
date last changed
2025-07-10 22:10:12
@article{6285b569-f20a-4669-9026-7a9fe6dc118e,
  abstract     = {{<p>Protein maldigestion and malabsorption lead to malnutrition and are a feature of exocrine pancreatic insufficiency (EPI). Although it is the current standard, measurement of nitrogen in stool to assess protease activity is indirect. Up to 80% of hydrolysed proteins appear in blood in the form of peptides, so we developed a method to measure peptide-derived amino acids in plasma as a relevant measure of proteolysis, verified its accuracy, precision, and linearity, and validated it in a porcine model. We modified a ninhydrin method. Large proteins were eliminated from plasma with 10 kDa-cut-off centrifugal filters. Free and total amino acids were measured in permeate before and after its hydrolysis. Peptide-derived amino acids were quantified by subtracting free amino acids from total amino acids. We verified the method in vitro and by comparing results in healthy and EPI pigs. The accuracy of the analysis was close to 100%, with excellent precision (mean relative standard deviation for low, medium, and high amino acid levels ¼ 0.88%) and with stringent linearity (r<sup>2</sup> ¼ 0.986, %RE ¼ 5.23). The high-throughput ninhydrin method detected levels of peptide-derived amino acids in vivo with maximal changes seen approximately 2 hours postprandially in young pigs. The AUC and Cmax were significantly higher in healthy compared to EPI pigs (P ¼ .0026 and P ¼ .0037, respectively). The high-throughput ninhydrin method is a sensitive, reliable, and practical method for the estimation of dietary peptide-derived amino acids. This assay endpoint could serve as a direct biomarker of protein digestion and absorption.</p>}},
  author       = {{Pierzynowska, Kateryna and Zaworski, Kamil and Wychowanski, Piotr and Donaldson, Janine and Wolinski, Jarosław and Borowitz, Drucy and Gallotto, Robert and Pierzynowski, Stefan}},
  issn         = {{2396-8923}},
  keywords     = {{EPI pigs; healthy pigs; method; peptide absorption; validation}},
  language     = {{eng}},
  number       = {{1}},
  publisher    = {{Oxford University Press}},
  series       = {{Biology Methods and Protocols}},
  title        = {{Modified throughput ninhydrin method for the qualitative assessment of dietary protein absorption in pig plasma}},
  url          = {{http://dx.doi.org/10.1093/biomethods/bpae078}},
  doi          = {{10.1093/biomethods/bpae078}},
  volume       = {{9}},
  year         = {{2024}},
}