Establishment of sandwich ELISA for soluble alpha-Klotho measurement: Age-dependent change of soluble alpha-Klotho levels in healthy subjects

Abstract

Background: Alpha-Klotho (alphaKl) regulates mineral metabolism such as calcium ion (Ca(2+)) 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 alphaKl has been demonstrated, less is known regarding the physiological importance of soluble-type alphaKl (salphaKl) in circulation.

Objectives: The aims of this study were: (1) to establish a sandwich ELISA system enabling detection of circulating serum salphaKl, and (2) to determine reference values for salphaKl 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 salphaKl in healthy volunteers (n=142, males 66) of ages (61.1+/-18.5year). The levels (mean+/-SD) in these healthy control adults were as follows: total calcium (Ca; 9.46+/-0.41mg/dL), Pi (3.63+/-0.51mg/dL), blood urea nitrogen (BUN; 15.7+/-4.3mg/dL), creatinine (Cre; 0.69+/-0.14mg/dL), 1,25 dihydroxyvitamin D (1,25(OH)(2)D; 54.8+/-17.7pg/mL), intact parathyroid hormone (iPTH; 49.2+/-20.6pg/mL), calcitonin (26.0+/-12.3pg/mL) and intact fibroblast growth factor (FGF23; 43.8+/-17.6pg/mL). Serum levels of salphaKl ranged from 239 to 1266pg/mL (mean+/-SD; 562+/-146pg/mL) in normal adults. Although salphaKl levels were not modified by gender or indices of mineral metabolism, salphaKl levels were inversely related to Cre and age. However, salphaKl levels in normal children (n=39, males 23, mean+/-SD; 7.1+/-4.8years) were significantly higher (mean+/-SD; 952+/-282pg/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 salphaKl correlated negatively with age and Ca, and positively with Pi. Finally, we measured a serum salphaKl from a patient with severe tumoral calcinosis derived from a homozygous missense mutation of alpha-klotho gene. In this patient, salphaKl level was notably lower than those of age-matched controls.

Conclusion: We established a detection system to measure human serum salphaKl for the first time. Age, Ca and Pi seem to influence serum salphaKl levels in a normal population. This detection system should be an excellent tool for investigating salphaKl functions in mineral metabolism.

Fig.1

Enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation analysis by two useful anti-sαKl monoclonal antibodies. (A) Typical dose-response curve of recombinant human sαKl (rhsαKl). Sandwich ELISA for sαKl was performed by 67G3 as a capture antibody and 91F1 as a detection antibody. The results are expressed as the mean ± standard deviation (SD) of triplicate measurements. (B) Immunoprecipitation of plasma sαKl by 67G3 and 91F1 antibodies. Conditioned medium from CHO cells stably expressing FGF23 (rhsαKl) and plasma from healthy volunteers (plasma 1, plasma 2) were immunoprecipitated anti-Klotho antibodies (67G3 and 91F1) and control mouse IgG1 (cont. Ab.), and analyzed by Western blot with anti-human Klotho rabbit polyclonal antibodies, N116 and C939.

Fig.2

Serum sαKl levels (A) Comparison of serum sαKl levels between healthy adults and children. Serum sαKl levels of healthy adults (n=142, age: >20 years) and children (n=39, age: <17years) were measured. Results represent mean ± standard deviation (SD). Statistical analysis was performed by student's t-test. P values less than 0.05 were taken as statistically significant. Correlation of serum sαKl with (B) age (C) Pi (D) BUN (E) Cre and (F) FGF23 levels in simple regression analysis in healthy subjects. The open circles and triangles represent male and female subjects, respectively. Statistical analysis was performed by Pearson correlation. P values less than 0.05 were taken as statistically significant. (G) Comparison of serum sαKl levels between a patient with klotho missense mutation and age-matched healthy subjects. Serum sαKl levels from 11 healthy subjects (left, age: from 11.2 to 16.6 years) and a patient with klotho homozygous gene mutation (right, age: 14 years) were shown. An error bar along the values for healthy controls represents mean ± 2 fold of standard deviation (2SD) in order to indicate a reference range.