Vitamin D Assays Flawed, but Improving

MINNEAPOLIS – Some day physicians will be able to order detailed vitamin D assay panels much like the lipid panels used today to assess cholesterol.

We’re just not there yet.

“Where we’re at today with 25-hydroxyvitamin D measurement is where we were at 50 or 60 years ago with lipid measurement ... but I believe vitamin D assay panels are coming,” said Dr. Neil C. Binkley of the Institute on Aging at the University of Wisconsin, Madison.

Patrice Wendling/IMNG Medical Media

Part of the problem with 25-hydroxyvitamin D [25(OH)D] measurement is that it has not yet been standardized, and there is no agreement on which cut points should be used by laboratories and assay manufacturers.

In its 2011 report that set for the first time Recommended Dietary Allowances for calcium and vitamin D, the Institute of Medicine declared an “urgent clinical and public health need” to reassess laboratory ranges for 25(OH)D to avoid problems of overtreatment and undertreatment.

The IOM committee concluded that the prevalence of vitamin D deficiency in North Americans has been overestimated by some because of the use of “inappropriate cut points” that greatly exceed levels identified in the report (J. Clin. Endocrinol. Metab. 2011;96:53-8).

Substantial progress has been made in the past decade, thanks in large part to the work of Dr. Graham Carter and DEQAS, the Vitamin D External Quality Assessment Scheme, Dr. Binkley said at the annual meeting of the American Society for Bone and Mineral Research. DEQAS provides serum samples on a quarterly basis to laboratories that wish to check the accuracy of their assays, and statistically trims the data to produce an all-laboratory trimmed mean (ALTM), standard deviation, and coefficient of variation for all major methodologies. Recent DEQAS data show, however, that ALTMs can vary substantially, even among groups striving to improve the quality of their program, he said.

In 2010, the National Institutes of Health Office of Dietary Supplements also launched the international vitamin D standardization program dedicated to standardizing 25(OH)D measurement to the National Institute of Standards and Technology (NIST) reference procedures. The program recently began quantitating DEQAS materials with real values assigned by NIST, and is also working on standardizing measurements for 25(OH)D2 and D3, the C-3 epimer of 25(OH)D2 and D3, and 24,25(OH)2D, he said.

“This, I think, is a substantial positive development; however, despite these efforts some scatter is going to persist in 25(OH)D measurement,” Dr. Binkley said.
The reason for this is that analyses will pick up components of a sample other than the analyte – a phenomenon known as the matrix effect. Given the number of vitamin D metabolites that exist, it’s not surprising that assays have trouble identifying one analyte from other similar analytes, he explained.

One of those confounders is the C-3 epimer (3-epi) of 25(OH)D3, which was previously thought to exist in infants only and, as such, wasn’t of much concern, but is now known to be present at variable, albeit low, levels in virtually all human serum (J. Clin. Endocrinol. Metab. 2012;97:163-8). Since 3-epi has the same molecular structure and molecular weight as 25(OH)D, it’s not surprising that it can add “noise” to your 25(OH)D result, Dr. Binkley said.

Another confounder is 24,25(OH)2D3, which has historically been considered as simply an inactivating step in the 25(OH)D degradation pathway on the way to calcitroic acid. As such, many feel it isn’t worth measuring, he said. Recent studies, however, have found that 24,25(OH)2D3 possesses physiologic effects on cartilage and bone, and that once again, it is present at variable levels in adults up to about 10% of the 25(OH)D level.

“Three of our current immunoassays that I know of have 100% cross-reactivity with 24,25-dihydroxyvitamin D3, so when you’re measuring 25-hydroxyvitamin D, you are also measuring 24,25-dihydroxyvitamin D3,” he said.
A more recent study reports that patients with high levels of serum 24,25-dihydroxyvitamin D3 may have a less robust response to vitamin D3 (J. Steroid. Biochem. Mol. Biol. 2011;126:72-7).

Finally, two additional studies find that vitamin D3 is more potent at increasing 25(OH)D than is vitamin D2, but what may be more important is whether current assays can measure the two equally, Dr. Binkley said. Indeed, unpublished data he presented clearly show that a newer immunoassay was not up to the task.

“By prescribing ergocalciferol, we clinicians are needlessly making life more difficult for our laboratory colleagues” and unnecessarily confounding assay methodologies, he said.

Dr. Binkley reported that he had no relevant financial disclosures.

MINNEAPOLIS – Some day physicians will be able to order detailed vitamin D assay panels much like the lipid panels used today to assess cholesterol.

We’re just not there yet.

“Where we’re at today with 25-hydroxyvitamin D measurement is where we were at 50 or 60 years ago with lipid measurement ... but I believe vitamin D assay panels are coming,” said Dr. Neil C. Binkley of the Institute on Aging at the University of Wisconsin, Madison.

Patrice Wendling/IMNG Medical Media

Part of the problem with 25-hydroxyvitamin D [25(OH)D] measurement is that it has not yet been standardized, and there is no agreement on which cut points should be used by laboratories and assay manufacturers.

In its 2011 report that set for the first time Recommended Dietary Allowances for calcium and vitamin D, the Institute of Medicine declared an “urgent clinical and public health need” to reassess laboratory ranges for 25(OH)D to avoid problems of overtreatment and undertreatment.

The IOM committee concluded that the prevalence of vitamin D deficiency in North Americans has been overestimated by some because of the use of “inappropriate cut points” that greatly exceed levels identified in the report (J. Clin. Endocrinol. Metab. 2011;96:53-8).

Substantial progress has been made in the past decade, thanks in large part to the work of Dr. Graham Carter and DEQAS, the Vitamin D External Quality Assessment Scheme, Dr. Binkley said at the annual meeting of the American Society for Bone and Mineral Research. DEQAS provides serum samples on a quarterly basis to laboratories that wish to check the accuracy of their assays, and statistically trims the data to produce an all-laboratory trimmed mean (ALTM), standard deviation, and coefficient of variation for all major methodologies. Recent DEQAS data show, however, that ALTMs can vary substantially, even among groups striving to improve the quality of their program, he said.

In 2010, the National Institutes of Health Office of Dietary Supplements also launched the international vitamin D standardization program dedicated to standardizing 25(OH)D measurement to the National Institute of Standards and Technology (NIST) reference procedures. The program recently began quantitating DEQAS materials with real values assigned by NIST, and is also working on standardizing measurements for 25(OH)D2 and D3, the C-3 epimer of 25(OH)D2 and D3, and 24,25(OH)2D, he said.

“This, I think, is a substantial positive development; however, despite these efforts some scatter is going to persist in 25(OH)D measurement,” Dr. Binkley said.
The reason for this is that analyses will pick up components of a sample other than the analyte – a phenomenon known as the matrix effect. Given the number of vitamin D metabolites that exist, it’s not surprising that assays have trouble identifying one analyte from other similar analytes, he explained.

One of those confounders is the C-3 epimer (3-epi) of 25(OH)D3, which was previously thought to exist in infants only and, as such, wasn’t of much concern, but is now known to be present at variable, albeit low, levels in virtually all human serum (J. Clin. Endocrinol. Metab. 2012;97:163-8). Since 3-epi has the same molecular structure and molecular weight as 25(OH)D, it’s not surprising that it can add “noise” to your 25(OH)D result, Dr. Binkley said.

Another confounder is 24,25(OH)2D3, which has historically been considered as simply an inactivating step in the 25(OH)D degradation pathway on the way to calcitroic acid. As such, many feel it isn’t worth measuring, he said. Recent studies, however, have found that 24,25(OH)2D3 possesses physiologic effects on cartilage and bone, and that once again, it is present at variable levels in adults up to about 10% of the 25(OH)D level.

“Three of our current immunoassays that I know of have 100% cross-reactivity with 24,25-dihydroxyvitamin D3, so when you’re measuring 25-hydroxyvitamin D, you are also measuring 24,25-dihydroxyvitamin D3,” he said.
A more recent study reports that patients with high levels of serum 24,25-dihydroxyvitamin D3 may have a less robust response to vitamin D3 (J. Steroid. Biochem. Mol. Biol. 2011;126:72-7).

Finally, two additional studies find that vitamin D3 is more potent at increasing 25(OH)D than is vitamin D2, but what may be more important is whether current assays can measure the two equally, Dr. Binkley said. Indeed, unpublished data he presented clearly show that a newer immunoassay was not up to the task.

“By prescribing ergocalciferol, we clinicians are needlessly making life more difficult for our laboratory colleagues” and unnecessarily confounding assay methodologies, he said.

Dr. Binkley reported that he had no relevant financial disclosures.

MINNEAPOLIS – Some day physicians will be able to order detailed vitamin D assay panels much like the lipid panels used today to assess cholesterol.

We’re just not there yet.

“Where we’re at today with 25-hydroxyvitamin D measurement is where we were at 50 or 60 years ago with lipid measurement ... but I believe vitamin D assay panels are coming,” said Dr. Neil C. Binkley of the Institute on Aging at the University of Wisconsin, Madison.

Patrice Wendling/IMNG Medical Media

Part of the problem with 25-hydroxyvitamin D [25(OH)D] measurement is that it has not yet been standardized, and there is no agreement on which cut points should be used by laboratories and assay manufacturers.

In its 2011 report that set for the first time Recommended Dietary Allowances for calcium and vitamin D, the Institute of Medicine declared an “urgent clinical and public health need” to reassess laboratory ranges for 25(OH)D to avoid problems of overtreatment and undertreatment.

The IOM committee concluded that the prevalence of vitamin D deficiency in North Americans has been overestimated by some because of the use of “inappropriate cut points” that greatly exceed levels identified in the report (J. Clin. Endocrinol. Metab. 2011;96:53-8).

Substantial progress has been made in the past decade, thanks in large part to the work of Dr. Graham Carter and DEQAS, the Vitamin D External Quality Assessment Scheme, Dr. Binkley said at the annual meeting of the American Society for Bone and Mineral Research. DEQAS provides serum samples on a quarterly basis to laboratories that wish to check the accuracy of their assays, and statistically trims the data to produce an all-laboratory trimmed mean (ALTM), standard deviation, and coefficient of variation for all major methodologies. Recent DEQAS data show, however, that ALTMs can vary substantially, even among groups striving to improve the quality of their program, he said.

In 2010, the National Institutes of Health Office of Dietary Supplements also launched the international vitamin D standardization program dedicated to standardizing 25(OH)D measurement to the National Institute of Standards and Technology (NIST) reference procedures. The program recently began quantitating DEQAS materials with real values assigned by NIST, and is also working on standardizing measurements for 25(OH)D2 and D3, the C-3 epimer of 25(OH)D2 and D3, and 24,25(OH)2D, he said.

“This, I think, is a substantial positive development; however, despite these efforts some scatter is going to persist in 25(OH)D measurement,” Dr. Binkley said.
The reason for this is that analyses will pick up components of a sample other than the analyte – a phenomenon known as the matrix effect. Given the number of vitamin D metabolites that exist, it’s not surprising that assays have trouble identifying one analyte from other similar analytes, he explained.

One of those confounders is the C-3 epimer (3-epi) of 25(OH)D3, which was previously thought to exist in infants only and, as such, wasn’t of much concern, but is now known to be present at variable, albeit low, levels in virtually all human serum (J. Clin. Endocrinol. Metab. 2012;97:163-8). Since 3-epi has the same molecular structure and molecular weight as 25(OH)D, it’s not surprising that it can add “noise” to your 25(OH)D result, Dr. Binkley said.

Another confounder is 24,25(OH)2D3, which has historically been considered as simply an inactivating step in the 25(OH)D degradation pathway on the way to calcitroic acid. As such, many feel it isn’t worth measuring, he said. Recent studies, however, have found that 24,25(OH)2D3 possesses physiologic effects on cartilage and bone, and that once again, it is present at variable levels in adults up to about 10% of the 25(OH)D level.

“Three of our current immunoassays that I know of have 100% cross-reactivity with 24,25-dihydroxyvitamin D3, so when you’re measuring 25-hydroxyvitamin D, you are also measuring 24,25-dihydroxyvitamin D3,” he said.
A more recent study reports that patients with high levels of serum 24,25-dihydroxyvitamin D3 may have a less robust response to vitamin D3 (J. Steroid. Biochem. Mol. Biol. 2011;126:72-7).

Finally, two additional studies find that vitamin D3 is more potent at increasing 25(OH)D than is vitamin D2, but what may be more important is whether current assays can measure the two equally, Dr. Binkley said. Indeed, unpublished data he presented clearly show that a newer immunoassay was not up to the task.

“By prescribing ergocalciferol, we clinicians are needlessly making life more difficult for our laboratory colleagues” and unnecessarily confounding assay methodologies, he said.

Dr. Binkley reported that he had no relevant financial disclosures.