Background: alpha-Klotho (alpha Kl) regulates mineral metabolism such as calcium ion (Ca2+) and inorganic phosphate (Pi) in circulation. Defects in mice result in clinical features resembling disorders found in human aging. Although the importance of transmembrane-type alpha Kl has been demonstrated, less is known regarding the physiological importance of soluble-type alpha Kl (s alpha Kl) in circulation. Objectives: The aims of this study were: (1) to establish a sandwich ELISA system enabling detection of circulating serum s alpha Kl, and (2) to determine reference values for s alpha Kl serum levels and relationship to indices of renal function, mineral metabolism, age and sex in healthy subjects. Results: We successively developed an ELISA to measure serum s alpha Kl in healthy volunteers (n = 142, males 66) of ages (61.1 +/- 18.5 year). The levels (mean +/- SD) in these healthy control adults were as follows: total calcium (Ca; 9.46 +/- 0.41 mg/dL), Pi (3.63 +/- 0.51 mg/dL), blood urea nitrogen (BUN; 15.7 +/- 4.3 mg/dL), creatinine (Cre; 0.69 +/- 0.14 mg/dL), 1,25 dihydroxyvitamin D (1,25(OH)(2)D; 54.8 +/-17.7 pg/mL), intact parathyroid hormone (iPTH: 49.2 +/- 20.6 pg/mL), calcitonin (26.0 +/- 12.3 pg/mL) and intact fibroblast growth factor (FGF23; 43.8 +/- 17.6 pg/mL). Results: Serum levels of s alpha Kl ranged from 239 to 1266 pg/mL (mean SD; 562 146 pg/mL) in normal adults. Although soda levels were not modified by gender or indices of mineral metabolism, s alpha Kl levels were inversely related to Cre and age. However, s alpha Kl levels in normal children (n = 39, males 23, mean SD; 7.1 +/- 4.8 years) were significantly higher (mean +/- SD; 952 +/- 282 pg/mL) than those in adults (mean +/- SD; 562 +/- 146, P < 0.001). A multivariate linear regression analysis including children and adults in this study demonstrated that s alpha Kl correlated negatively with age and Ca, and positively with Pi. Finally, we measured a serum s alpha Kl from a patient with severe tumoral calcinosis derived from a homozygous missense mutation of a-klotho gene. In this patient, s alpha Kl level was notably lower than those of age-matched controls. Conclusion: We established a detection system to measure human serum s alpha Kl for the first time. Age, Ca and Pi seem to influence serum s alpha Kl levels in a normal population. This detection system should bean excellent tool for investigating s alpha Kl functions in mineral metabolism. (C) 2010 Elsevier Inc. All rights reserved.