Soluble alpha-Klotho is a pleiotropic protein that act as a paracrine and endocrine hormonal factor in multiple organs. Decreased alpha-Klotho levels have been associated with early aging symptoms in animal models of aging. Determination of Klotho concentrations was performed on frozen NHANES 2013-2014 samples that were received and tested during the period 2019-2020. Analyses were performed by a commercially available ELISA kit produced by IBL International, Japan.
Available pristine serum samples from 40-79 year old participants in NHANES 2013-2014 cycles, who gave consent for their samples to be used in future research, were analyzed with the IBL ELISA method.
The Northwest Lipid Metabolism and Diabetes Research Laboratories, Division of Metabolism, Endocrinology, and Nutrition, University of Washington, performed analyses on all but four fresh-frozen (pristine) samples received from the Centers for Disease Control and Prevention.
Prior to the start of the study, an extensive validation of the IBL ELISA method for measurement of Klotho concentration in human samples was performed and the results of the Validation provided to the study investigators.
In summary, 1) the assay standard curves and the relative signals of the calibrator concentrations were consistently within the criteria specified by the manufacturer, 2) the obtained assay sensitivity was calculated to be 4.33 pg/mL while the manufacturer claimed sensitivity was 6.15 pg/mL, 3) two samples with very high and high Klotho concentrations were used at different dilution to evaluate the assay linearity. Plots of the expected vs. the obtained values, demonstrated an excellent linearity in the assay measurement range (R2 = 0.998 and 0.997, respectively), 4) the intra-assay precision obtained on two recombinant Klotho samples and two human samples exhibited a coefficient of variation of 3.2% and 3.9% for the recombinant and 2.3% and 3.3% for the human samples, 5) The same samples analyzed in duplicate over 4 different days, showed an inter-assay CV of 2.8% and 3.5% for the recombinant samples and 3.8% and 3.4% for the human samples, 5) the reference ranges were evaluated in 114 samples from apparently healthy donors and the alpha-Klotho levels ranged from 285.8 to 1638.6 pg/mL with a mean of 698.0 pg/mL.
All sample analyses were performed in duplicate according to the manufacturer’s protocol and all the results were checked to meet the laboratory’s standardized criteria for acceptability prior to being released for reporting.
Samples were received on dry ice and each package was inspected by the laboratory’s receiving area personnel. Samples were scanned, data compared with those on the received electronic manifest and entered into the laboratory information system. All samples were stored at -80◦C until predefined batches of samples were daily provided to the technicians for analyses.
Samples were analyzed in duplicate and the average of the two values used to calculate the final value. Two quality control samples with low and high concentration of Klotho were also analyzed in duplicate in each ELISA plate. Results of analyses were automatically transmitted from the instrument to the laboratory Oracle Management System and results evaluated by the area supervisor. Samples with duplicate results exceeding 10% were flagged for analyses to be repeated. If the value of a quality control sample was not within the 2SD of the assigned value, the entire analytical run was rejected, and sample analyses repeated.
Demographic and Other Related Variables
The analysis of NHANES 2013-2014 laboratory data must be conducted using the appropriate survey design and demographic variables. The NHANES Demographic Files contain demographic data, health indicators, and other related information collected during household interviews as well as the sample design variables. The recommended procedure for variance estimation requires use of stratum and PSU variables (SDMVSTRA and SDMVPSU, respectively) in the demographic data file.
Detection Limits
The assay sensitivity was 6 pg/mL. There are no Klotho specific cut-points to be used as indicators of biological age. The reference ranges evaluated in 114 samples from apparently healthy donors indicated the alpha-Klotho levels ranged from 285.8 to 1638.6 pg/mL with a mean of 698.0 pg/mL.
Exam sample weights should be used for analyses. Please refer to the NHANES Analytic Guidelines and the on-line NHANES Tutorial for further details on the use of sample weights and other analytic issues.
| Code or Value | Value Description | Count | Cumulative | Skip to Item |
|---|---|---|---|---|
| 151.3 to 2605.1 | Range of Values | 2767 | 2767 | |
| . | Missing | 0 | 2767 |