Impact of preanalytical conditions and biological variables on urinary concentrations of soluble alpha klotho, a novel biomarker of growth hormone activity
A. Gagliardoa (Ms), M. Haenelta (Mr), J. Rol Schweizer*a (Dr), D. Teupserb (Prof), M. Bidlingmaiera (Dr), K. Schilbacha (Prof)
a Medizinische Klinik und Poliklinik IV, LMU Klinikum, Munich, GERMANY ; b Institut für Laboratoriumsmedizin, LMU Klinikum, Munich, GERMANY
INTRODUCTION: Pragmatic screening parameters, that are robust against influencing factors for diagnosing acromegaly are desirable. Soluble alpha klotho (saKL) is a peptide that has been shown to be elevated in serum of patients with growth hormone (GH) excess. In contrast to insulin-like growth factor (IGF)-I, it seems to be less influenced by biological factors such as BMI, age and sex. SaKL in spontaneous urine may potentially also function as a biomarker for acromegaly. We assessed the preanalytical stability of urine saKL (usaKL) and investigated correlations with serum saKL, anthropometric data, and urine parameters potentially suitable for standardization
METHODS: Urine and serum samples were collected along with anthropometric data from 20 healthy subjects. SaKL concentrations in serum and urine were measured by ELISA-IBL. In order to assess preanalytical stability, different storage conditions (room-temperature, 4°C, -20°C, -80°C), protease inhibitor addition, centrifugation, linearity and freeze-thaw-cycles were tested.
RESULTS: UsaKL is stable in different preanalytical conditions except when stored at -20°C where saKL concentration dropped significantly. In 19 of 20 healthy participants, usaKL was measurable; in those 19, usaKL fell below the Limit of Quantification in 47.4% when stored at -20°C. Protease inhibitor or centrifugation do not prevent this decrease. No correlations were found between absolute saKL concentrations and anthropometric data or traditional GH biomarkers. Serum saKL concentrations did not correlate with absolute saKL concentrations in urine, which was also true after normalization for parameters such as creatinine, urea, uric acid and osmolality.
DISCUSSION: SaKL is measurable in spontaneous urine and is stable at room-temperature, 4°C and -80°C, and is resistant to repeated freezing and thawing. However, storage of urine for saKL measurement should be avoided at -20°C, as this is likely a eutectic temperature for urine, in which the tertiary structure of proteins is at risk. As expected and seen for serum saKL, usaKL was not influenced by biological factors. In the presented healthy cohort, we did not find correlations between urine saKL and GH-dependent parameters in serum. It might be possible that correlations can only be detected with substantially higher saKL concentrations as seen in GH excess.
The author has declared no conflict of interest.