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Amino Acids

pp 1–19 | Cite as

Development and validation of GC–MS methods for the comprehensive analysis of amino acids in plasma and urine and applications to the HELLP syndrome and pediatric kidney transplantation: evidence of altered methylation, transamidination, and arginase activity

  • Erik Hanff
  • Stephan Ruben
  • Martin Kreuzer
  • Alexander Bollenbach
  • Arslan Arinc Kayacelebi
  • Anibh Martin Das
  • Frauke von Versen-Höynck
  • Constantin von Kaisenberg
  • Dieter Haffner
  • Stefan Ückert
  • Dimitrios TsikasEmail author
Original Article

Abstract

We developed and validated gas chromatography–mass spectrometry (GC–MS) methods for the simultaneous measurement of amino acids and their metabolites in 10-µL aliquots of human plasma and urine. De novo synthesized trideutero-methyl esters were used as internal standards. Plasma proteins were precipitated by acidified methanol and removed by centrifugation. Supernatants and native urine were evaporated to dryness. Amino acids were first esterified using 2 M HCl in methanol and then amidated using pentafluoropropionic anhydride for electron-capture negative-ion chemical ionization. Time programmes were used for the gas chromatograph oven and the selected-ion monitoring of specific anions. The GC–MS methods were applied in clinical studies on the HELLP syndrome and pediatric kidney transplantation (KTx) focusing on l-arginine-related pathways. We found lower sarcosine (N-methylglycine) and higher asymmetric dimethylarginine (ADMA) plasma concentrations in HELLP syndrome women (n = 7) compared to healthy pregnant women (n = 5) indicating altered methylation. In plasma of pediatric KTx patients, lower guanidinoacetate and homoarginine concentrations were found in plasma but not in urine samples of patients treated with standard mycophenolate mofetil-based immunosuppression (MMF; n = 22) in comparison to matched patients treated with MMF-free immunosuppression (n = 22). On average, the global arginine bioavailability ratio was by about 40% lower in the MMF group compared to the EVR group (P = 0.004). Mycophenolate, the major pharmacologically active metabolite of MMF, is likely to inhibit the arginine:glycine amidinotransferase (AGAT), and to enhance arginase activity in leukocytes and other types of cell of MMF-treated children.

Keywords

AGAT Amino acids Derivatization GAMT GC–MS Immunosuppression Quantification Stable isotopes 

Abbreviations

AA

Amino acid

ADMA

Asymmetric dimethylarginine

AGAT

Arginine:glycine amidinotransferase

CKD

Chronic kidney disease

CNI

Calcineurin inhibitor

DDAH

Dimethylarginine dimethylaminohydrolase

DMA

Dimethylamine

ECNICI

Electron-capture negative-ion chemical ionization

EVR

Everolimus

GAA

Guanidinoacetate

GABR

Global arginine bioavailability ratio

GAMT

Guanidinoacetate N-methyltransferase

GATM

Glycine:arginine transamidinase

GC–MS

Gas chromatography–mass spectrometry

GNMT

Glycine N-methyltransferase

hArg

Homoarginine

HELLP

Hemolysis, elevated liver enzymes, low platelet

IQR

Interquartile range

IS

Internal standard

KTx

Kidney transplantation

LOD

Limit of detection

LOQ

Limit of quantification

Me

Methyl

MeOH

Methanol

MMF

Mycophenolate mofetil

MPA

Mycophenolic acid

m/z

Mass-to-charge ratio

NMDAR

N-Methyl-d-aspartate glutamate receptor

NO

Nitric oxide

NOS

Nitric oxide synthase

OH-Pro

Hydroxyproline

PFP

Pentafluoropropionyl

PFPA

Pentafluoropropionic anhydride

PRMT

Protein-arginine methyltransferase

QC

Quality control

rLLOQ

Relative low limit of quantification

Sar

Sarcosine

SDMA

Symmetric dimethylarginine

SIM

Selected-ion monitoring

S/N or SN

Signal-to-noise ratio

TIC

Total ion current

UNOxR

Urinary nitrate-to-nitrite molar ratio

WoG

Week of gestation

Notes

Compliance with ethical standards

Conflict of interest

The authors declare no competing financial interest.

Ethical approval

Both studies were conducted in accordance with the ethical principles of Good Clinical Practice (GCP) that have their origins in the Declaration of Helsinki.

Supplementary material

726_2018_2688_MOESM1_ESM.pdf (1.5 mb)
Supplementary material 1 (PDF 1504 kb)

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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  • Erik Hanff
    • 1
  • Stephan Ruben
    • 2
  • Martin Kreuzer
    • 2
  • Alexander Bollenbach
    • 1
  • Arslan Arinc Kayacelebi
    • 1
  • Anibh Martin Das
    • 3
  • Frauke von Versen-Höynck
    • 4
  • Constantin von Kaisenberg
    • 4
  • Dieter Haffner
    • 2
  • Stefan Ückert
    • 5
  • Dimitrios Tsikas
    • 1
    Email author
  1. 1.Core Unit Proteomics, Hannover Medical SchoolInstitute of ToxicologyHannoverGermany
  2. 2.Department of Pediatric Kidney, Liver and Metabolic DiseasesHannover Medical SchoolHannoverGermany
  3. 3.Department of PediatricsHannover Medical SchoolHannoverGermany
  4. 4.Department of Obstetrics and GynecologyHannover Medical SchoolHannoverGermany
  5. 5.Department of Urology and Urological OncologyHannover Medical SchoolHannoverGermany

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