Vitamin D is essential for the metabolism and stable equilibrium of calcium and phosphate—elements that are necessary for the growth and development of bones and teeth. Vitamin D also aids in the normal functioning of the body’s nervous system. Because vitamin D is vital for bone health and improved resistance to certain diseases, there has been significant interest in identifying patients potentially at risk of vitamin D deficiency. However, in this era of increased supplementation, there is growing evidence that individuals may be at risk of vitamin D toxicity as a result of overdosing. The use of multiple products that contain vitamin D supplementation can combine to create potentially toxic levels in the body.
For patients with vitamin D-related disorders, clinicians measure serum concentrations of total 25-hydroxyvitamin D [25(OH)D] or 1,25-dihydroxyvitamin D [1,25(OH)2D]. 25(OH) D serum concentrations have been used as a nutritional marker for assessment of optimum vitamin D supplementation. 25(OH) D and 1,25(OH)2D are converted to inactive metabolites by an enzyme 24 hydroxylase encoded by CYP24A1. 25(OH) D is converted to 24,25(OH)2D and normally the ratio is <25. Inactivating mutations of CYP24A1 (cytochrome P450, family 24, subfamily A, polypeptide 1) cause hypercalcemia (elevated levels of calcium in blood), hypercalciuria (elevated levels of calcium in urine), and increased 1,25(OH)2D concentrations. If CYP24A1 mutations cause reduced or complete loss of 24-hydroxylase function, then the serum concentrations of 24,25(OH)2D may be undetectable.
Loss-of-function mutations in CYP24A1 have been identified as the underlying cause of hypercalcemia. However, the population of frequency of CYP24A1 mutations is currently unknown. A 25(OH)D/24,25(OH)2D value that could identify patients who are candidates for CYP24A1 mutation testing would be beneficial for future testing and diagnosis.
Researchers at Mayo Clinic developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for 24,25(OH)2D quantification, established a 25(OH)D/24,25(OH)2D reference interval, and identified a diagnostic cutoff for 25(OH)D/24,25(OH)2D that can recognize patients in whom genetic testing for CYP24A1 mutations is warranted. The study was published in Clinical Chemistry.
“We developed this new assay because a recent patient had a history of kidney stones, but after various checkups and testing, we couldn’t find anything wrong. We then performed the 24,25(OH)2D quantification test and found that this patient was very deficient in these enzymes,” said Ravinder Singh, Ph.D., Director of the Mayo Clinic Endocrine Laboratory and senior author on this paper.
In reviewing previous studies, one of the first reports describing an LC-MS/MS method for 24,25(OH)2D quantification showed a linear relationship between 25(OH)D and 24,25(OH)2D. Mayo Clinic researchers observed a similar linear relationship in their testing results.
“Based on our results, we found that serum 25(OH)D/24,25(OH)2D is potentially useful for assessing differential diagnosis of hypercalcemia. However, so far its use has been limited to research studies, and its use in routine clinical practice has just started,” said Dr. Singh.
This study validates that vitamin D toxicity is a particular concern, and supplementation should be monitored as needed.
“Our results indicate that hypercalemia and hypercalciuria due to vitamin D toxicity is, in fact, a reality in some patients. Measurement of 25(OH)D/24,25(OH)2D should be considered as part of the clinical workup in patients,” added Dr. Singh. Mayo will be using the following cut offs for the interpretation of the tests with additional cautions.
- Ratio of 25-OH-D/24, 25-(OH) 2-D <25: normal
- Ratio of 25-OH-D/24, 25-(OH) 2-D 25-80: ratios in this range can be seen in patients with low vitamin D or heterozygous CYP24A1 mutation. Molecular testing is recommended.
- Ratio of 25-OH-D/24, 25-(OH) 2-D >80: probable bi-allelic CYP24A1 mutation. Molecular testing is recommended.
Results should be interpreted in the context of other biochemical findings including serum calcium, parathyroid hormone, and 1, 25 dihydroxyvitamin D concentrations.
For more information about vitamin D toxicity, view the Mayo Medical Laboratories Hot Topic video on clinical implications of vitamin D toxicity