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


OBJECTIVE
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.


CONCLUSIONS
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.
Yamazaki
Yuji
Y
Antibody Research Laboratories, Kyowa Hakko Kirin Co. Ltd., Tokyo 194-8533, Japan. yuji.yamazaki@kyowa-kirin.co.jp
Imura
Akihiro
A
Urakawa
Itaru
I
Shimada
Takashi
T
Murakami
Junko
J
Aono
Yukiko
Y
Hasegawa
Hisashi
H
Yamashita
Takeyoshi
T
Nakatani
Kimihiko
K
Saito
Yoshihiko
Y
Okamoto
Nozomi
N
Kurumatani
Norio
N
Namba
Noriyuki
N
Kitaoka
Taichi
T
Ozono
Keiichi
K
Sakai
Tomoyuki
T
Hataya
Hiroshi
H
Ichikawa
Shoji
S
Imel
Erik A
EA
Econs
Michael J
MJ
Nabeshima
Yo-Ichi
Y
D000328:Adult; D000367:Age Factors; D000368:Aged; D000369:Aged, 80 and over; D000375:Aging / Q000097:blood*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002118:Calcium / Q000097:blood; D002648:Child; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D000089703:Fibroblast Growth Factor-23; D005966:Glucuronidase / Q000097:blood* / Q000235:genetics / Q000276:immunology; D006801:Humans; D000090265:Klotho Proteins; D008297:Male; D051379:Mice; D008875:Middle Aged; D009154:Mutation
D000911:Antibodies, Monoclonal; C000717427:FGF23 protein, human; C000717428:Fgf23 protein, mouse; D000089703:Fibroblast Growth Factor-23; D005966:Glucuronidase; D000090265:Klotho Proteins; D002118:Calcium