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Within-subject and between-subject biological variation of first morning void urine amino acids in 12 healthy subjects

Hamit Hakan Alp EMAIL logo , Halil İbrahim Akbay , Erdem Çokluk , Zubeyir Huyut , Sıddık Keskin and Mehmet Ramazan Şekeroğlu



Urine amino acid analysis is used for the assessment of various diseases. The aim of this study was to estimate the valid biological variation (BV) components (within- and between-subjects) required for the safe clinical application of free urine amino acids.


First morning void urine samples were taken from 12 healthy subjects (five females, seven males) once a week for 10 consecutive weeks, and amino acid analysis was performed using an Agilent 6470 triple quadrupole tandem mass spectrometer instrument. The obtained data were subjected to normality, outlier and variance homogeneity analyses prior to coefficient of variation (CV) analysis. Within- and between-subject BV values (CVI and CVG) of 39 amino acids were determined for all subjects. In addition, the index of individuality (II), reference change value (RCV), imprecision, bias and total error were estimated using BV data obtained from our study.


The CVI values ranged from 8.9 (histidine) to 36.8% (trans-4-hydroxyprolin), while the CVG values ranged from 25.0 (1-methyl-L-histidine) to 63.3% (phenylalanine). The II value of most amino acids was less than 0.6 and ranged between 0.21 and 0.88. The imprecision, bias and total error ranged between 4.45 and 16.6, between 7.69 and 16.6, and between 18.4 and 43.2, respectively.


This study, designed according to a rigorous protocol, has the feature of being the first to give information about BV data of urine amino acids. We believe that the reference intervals have a limitation in the evaluation of consecutive results from an individual, so the use of RCV would be more appropriate.

Corresponding author: Assoc. Prof. Hamit Hakan Alp, Van Yuzuncu Yil University, Medical Faculty, Department of Biochemistry, Van, Turkey, Phone: +905333799702


We would like to thank all volunteers involved in the study and Gökçe Göksu (Jasem R&D Manager) for technical assistance.

  1. Research funding: This study was funded by the Research Fund of the Van Yüzüncü Yıl University, Project number: TSA-2018-6777.

  2. Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  3. Competing interests: Authors state no conflict of interest.

  4. Informed consent: Informed consent was obtained from all individuals included in this study.

  5. Ethical approval: Ethical approval was obtained from the local Ethics Committee (degree no: 03-2017).


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Supplementary Material

The online version of this article offers supplementary material (

Received: 2019-11-12
Accepted: 2020-03-27
Published Online: 2020-04-22
Published in Print: 2020-10-25

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