The recognition of 25-hydroxyvitamin D2 and D3 by a new binding protein based 25-hydroxyvitamin D assay

Zengliu Su, Brian R. Slay, Randall Carr, Yusheng Zhu

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Background: The circulating form of vitamin D, 25-Hydroxyvitamin D (25-OHD), is commonly measured in clinical labs to evaluate vitamin D status of an individual. Vitamin D exists in 2 major forms: vitamin D3 and D2. Current methodologies for 25-OHD testing include antibody or vitamin D binding protein based assays, HPLC, and LC-MS. Although most current antibody or vitamin D binding protein based assays claim that they measure 25-OHD3 and 25-OHD2 equally, bias may still exist in the measurement of total 25-OHD when high concentration of 25-OHD2 is present. LC-MS/MS can measure 25-OHD3 and 25-OHD2 separately. We determined the ability of a new competitive protein binding assay (CPBA) to recognize 25-OHD2 and 25-OHD3 by comparing it with an LC-MS/MS method. Methods: Seventy-one serum samples were collected and divided into 3 groups according to 25-OHD2 and 25-OHD3 concentrations determined by LC-MS/MS: group A containing only 25-OHD3 (25-OHD2 < 3. ng/ml), group B containing both 25-OHD3 and 25-OHD2 (25-OHD2. ≥. 3. ng/ml, 25-OHD3. ≥. 10. ng/ml), and group C containing mainly 25-OHD2 (25-OHD3 < 10. ng/ml). The correlation between total 25-OHD concentrations measured by LC-MS/MS and CPBA was analyzed with linear regression analysis and the difference between the two methods was analyzed with paired, 2-tailed Student's t-Test. The biases between the two methods were also calculated. Results: The correlation analysis between LC-MS/MS (x) and CPBA (y) in the 3 groups are as follows: y=1.13x-1.01 (R2=0.65, n=25) in group A; y=0.68x+4.02 (R2=0.76, n=31) in group B, and y=0.49x+6.53 (R2=0.38, n=15) in group C. In group A, the mean total 25-OHD measured with CPBA was higher than that with LC-MS/MS, while in groups B, CPBA results were lower, but the differences were not statistically significant (p=0.476 for group A and 0.059 for group B). In group C, the mean 25-OHD measured with CPBA was lower than that with LC-MS/MS and the difference was statistically significant (p=0.002). The average biases in total 25-OHD concentrations between LC-MS/MS and CPBA were 10.8%, -23.6%, and -38.4% for groups A, B, and C, respectively. Conclusions: CPBA has a better correlation with LC-MS/MS for samples containing both 25-OHD2 and 25-OHD3 as compared to samples with mainly 25-OHD2 or 25-OHD3 only. However, it measures 25-OHD3 more accurately than 25-OHD2. Compared to LC-MS/MS, CPBA overestimates 25-OHD3, but underestimates 25-OHD2.

Original languageEnglish (US)
Pages (from-to)62-66
Number of pages5
JournalClinica Chimica Acta
Volume417
DOIs
StatePublished - Feb 8 2013

Fingerprint

25-Hydroxyvitamin D 2
Vitamin D-Binding Protein
Calcifediol
Competitive Binding
Protein Binding
Assays
Carrier Proteins
Vitamin D
Ergocalciferols
25-hydroxyvitamin D
Antibodies
Cholecalciferol
Linear Models
Linear regression
Regression analysis

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Clinical Biochemistry
  • Biochemistry, medical

Cite this

@article{cf8a95e536af45d8a081003089000339,
title = "The recognition of 25-hydroxyvitamin D2 and D3 by a new binding protein based 25-hydroxyvitamin D assay",
abstract = "Background: The circulating form of vitamin D, 25-Hydroxyvitamin D (25-OHD), is commonly measured in clinical labs to evaluate vitamin D status of an individual. Vitamin D exists in 2 major forms: vitamin D3 and D2. Current methodologies for 25-OHD testing include antibody or vitamin D binding protein based assays, HPLC, and LC-MS. Although most current antibody or vitamin D binding protein based assays claim that they measure 25-OHD3 and 25-OHD2 equally, bias may still exist in the measurement of total 25-OHD when high concentration of 25-OHD2 is present. LC-MS/MS can measure 25-OHD3 and 25-OHD2 separately. We determined the ability of a new competitive protein binding assay (CPBA) to recognize 25-OHD2 and 25-OHD3 by comparing it with an LC-MS/MS method. Methods: Seventy-one serum samples were collected and divided into 3 groups according to 25-OHD2 and 25-OHD3 concentrations determined by LC-MS/MS: group A containing only 25-OHD3 (25-OHD2 < 3. ng/ml), group B containing both 25-OHD3 and 25-OHD2 (25-OHD2. ≥. 3. ng/ml, 25-OHD3. ≥. 10. ng/ml), and group C containing mainly 25-OHD2 (25-OHD3 < 10. ng/ml). The correlation between total 25-OHD concentrations measured by LC-MS/MS and CPBA was analyzed with linear regression analysis and the difference between the two methods was analyzed with paired, 2-tailed Student's t-Test. The biases between the two methods were also calculated. Results: The correlation analysis between LC-MS/MS (x) and CPBA (y) in the 3 groups are as follows: y=1.13x-1.01 (R2=0.65, n=25) in group A; y=0.68x+4.02 (R2=0.76, n=31) in group B, and y=0.49x+6.53 (R2=0.38, n=15) in group C. In group A, the mean total 25-OHD measured with CPBA was higher than that with LC-MS/MS, while in groups B, CPBA results were lower, but the differences were not statistically significant (p=0.476 for group A and 0.059 for group B). In group C, the mean 25-OHD measured with CPBA was lower than that with LC-MS/MS and the difference was statistically significant (p=0.002). The average biases in total 25-OHD concentrations between LC-MS/MS and CPBA were 10.8{\%}, -23.6{\%}, and -38.4{\%} for groups A, B, and C, respectively. Conclusions: CPBA has a better correlation with LC-MS/MS for samples containing both 25-OHD2 and 25-OHD3 as compared to samples with mainly 25-OHD2 or 25-OHD3 only. However, it measures 25-OHD3 more accurately than 25-OHD2. Compared to LC-MS/MS, CPBA overestimates 25-OHD3, but underestimates 25-OHD2.",
author = "Zengliu Su and Slay, {Brian R.} and Randall Carr and Yusheng Zhu",
year = "2013",
month = "2",
day = "8",
doi = "10.1016/j.cca.2012.12.018",
language = "English (US)",
volume = "417",
pages = "62--66",
journal = "Clinica Chimica Acta",
issn = "0009-8981",
publisher = "Elsevier",

}

The recognition of 25-hydroxyvitamin D2 and D3 by a new binding protein based 25-hydroxyvitamin D assay. / Su, Zengliu; Slay, Brian R.; Carr, Randall; Zhu, Yusheng.

In: Clinica Chimica Acta, Vol. 417, 08.02.2013, p. 62-66.

Research output: Contribution to journalArticle

TY - JOUR

T1 - The recognition of 25-hydroxyvitamin D2 and D3 by a new binding protein based 25-hydroxyvitamin D assay

AU - Su, Zengliu

AU - Slay, Brian R.

AU - Carr, Randall

AU - Zhu, Yusheng

PY - 2013/2/8

Y1 - 2013/2/8

N2 - Background: The circulating form of vitamin D, 25-Hydroxyvitamin D (25-OHD), is commonly measured in clinical labs to evaluate vitamin D status of an individual. Vitamin D exists in 2 major forms: vitamin D3 and D2. Current methodologies for 25-OHD testing include antibody or vitamin D binding protein based assays, HPLC, and LC-MS. Although most current antibody or vitamin D binding protein based assays claim that they measure 25-OHD3 and 25-OHD2 equally, bias may still exist in the measurement of total 25-OHD when high concentration of 25-OHD2 is present. LC-MS/MS can measure 25-OHD3 and 25-OHD2 separately. We determined the ability of a new competitive protein binding assay (CPBA) to recognize 25-OHD2 and 25-OHD3 by comparing it with an LC-MS/MS method. Methods: Seventy-one serum samples were collected and divided into 3 groups according to 25-OHD2 and 25-OHD3 concentrations determined by LC-MS/MS: group A containing only 25-OHD3 (25-OHD2 < 3. ng/ml), group B containing both 25-OHD3 and 25-OHD2 (25-OHD2. ≥. 3. ng/ml, 25-OHD3. ≥. 10. ng/ml), and group C containing mainly 25-OHD2 (25-OHD3 < 10. ng/ml). The correlation between total 25-OHD concentrations measured by LC-MS/MS and CPBA was analyzed with linear regression analysis and the difference between the two methods was analyzed with paired, 2-tailed Student's t-Test. The biases between the two methods were also calculated. Results: The correlation analysis between LC-MS/MS (x) and CPBA (y) in the 3 groups are as follows: y=1.13x-1.01 (R2=0.65, n=25) in group A; y=0.68x+4.02 (R2=0.76, n=31) in group B, and y=0.49x+6.53 (R2=0.38, n=15) in group C. In group A, the mean total 25-OHD measured with CPBA was higher than that with LC-MS/MS, while in groups B, CPBA results were lower, but the differences were not statistically significant (p=0.476 for group A and 0.059 for group B). In group C, the mean 25-OHD measured with CPBA was lower than that with LC-MS/MS and the difference was statistically significant (p=0.002). The average biases in total 25-OHD concentrations between LC-MS/MS and CPBA were 10.8%, -23.6%, and -38.4% for groups A, B, and C, respectively. Conclusions: CPBA has a better correlation with LC-MS/MS for samples containing both 25-OHD2 and 25-OHD3 as compared to samples with mainly 25-OHD2 or 25-OHD3 only. However, it measures 25-OHD3 more accurately than 25-OHD2. Compared to LC-MS/MS, CPBA overestimates 25-OHD3, but underestimates 25-OHD2.

AB - Background: The circulating form of vitamin D, 25-Hydroxyvitamin D (25-OHD), is commonly measured in clinical labs to evaluate vitamin D status of an individual. Vitamin D exists in 2 major forms: vitamin D3 and D2. Current methodologies for 25-OHD testing include antibody or vitamin D binding protein based assays, HPLC, and LC-MS. Although most current antibody or vitamin D binding protein based assays claim that they measure 25-OHD3 and 25-OHD2 equally, bias may still exist in the measurement of total 25-OHD when high concentration of 25-OHD2 is present. LC-MS/MS can measure 25-OHD3 and 25-OHD2 separately. We determined the ability of a new competitive protein binding assay (CPBA) to recognize 25-OHD2 and 25-OHD3 by comparing it with an LC-MS/MS method. Methods: Seventy-one serum samples were collected and divided into 3 groups according to 25-OHD2 and 25-OHD3 concentrations determined by LC-MS/MS: group A containing only 25-OHD3 (25-OHD2 < 3. ng/ml), group B containing both 25-OHD3 and 25-OHD2 (25-OHD2. ≥. 3. ng/ml, 25-OHD3. ≥. 10. ng/ml), and group C containing mainly 25-OHD2 (25-OHD3 < 10. ng/ml). The correlation between total 25-OHD concentrations measured by LC-MS/MS and CPBA was analyzed with linear regression analysis and the difference between the two methods was analyzed with paired, 2-tailed Student's t-Test. The biases between the two methods were also calculated. Results: The correlation analysis between LC-MS/MS (x) and CPBA (y) in the 3 groups are as follows: y=1.13x-1.01 (R2=0.65, n=25) in group A; y=0.68x+4.02 (R2=0.76, n=31) in group B, and y=0.49x+6.53 (R2=0.38, n=15) in group C. In group A, the mean total 25-OHD measured with CPBA was higher than that with LC-MS/MS, while in groups B, CPBA results were lower, but the differences were not statistically significant (p=0.476 for group A and 0.059 for group B). In group C, the mean 25-OHD measured with CPBA was lower than that with LC-MS/MS and the difference was statistically significant (p=0.002). The average biases in total 25-OHD concentrations between LC-MS/MS and CPBA were 10.8%, -23.6%, and -38.4% for groups A, B, and C, respectively. Conclusions: CPBA has a better correlation with LC-MS/MS for samples containing both 25-OHD2 and 25-OHD3 as compared to samples with mainly 25-OHD2 or 25-OHD3 only. However, it measures 25-OHD3 more accurately than 25-OHD2. Compared to LC-MS/MS, CPBA overestimates 25-OHD3, but underestimates 25-OHD2.

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