Understanding Elevated Vitamin D Measurements to Uncover Hypercalcemia Etiology

A 69-year-old man presented to a tertiary care center with several weeks of polyuria, polydipsia, blurred vision, and confusion. His initial investigations showed an elevated serum calcium 15.4 (reference interval: 8.4 to 10.4) mg/dL (3.86 [2.10 to 2.60] mmol/L), ionized calcium 7.9 (4.6 to 5.4) mg/dL (1.97 [1.15 to 1.35] mmol/L), and phosphate 5.26 (2.17 to 4.65) mg/dL (1.70 [0.70 to 1.50] mmol/L) with a concurrent acute kidney injury, the creatinine rising from a baseline of 1.13 to 5.73 (0.57 to 1.36) mg/dL (100 to 507 [50 to 120] µmol/L). The patient had known hypertension, treated with an angiotensin receptor blocker, heterozygous hereditary hemochromatosis (C282Y/H63D) managed with phlebotomy, and had previously recovered from hepatitis virus C with therapy. He had also been using an unknown amount of 4 unregulated health supplements, with package labeling for one suggesting a vitamin D content of 1000 IU/drop (labeling was not available for the other 3). The patient was hospitalized for 20 days, during which he was medically managed for hypercalcemia, first with intravenous fluids and empiric pamidronate, then diuretics. The initial parathyroid hormone (PTH) level was suppressed at 6 (7 to 37) pg/mL, confirming this to be non-PTH mediated hypercalcemia. Other causes such as PTH-related peptide (<2.0 [<4.2)] pmol/L), hyperthyroidism (thyroid stimulating hormone [TSH], 1.63 [0.20 to 4.00] mIU/L), myeloma (unremarkable skeletal survey and serum protein electrophoresis), and overt solid organ malignancy (based on computed tomography [CT] of the chest, abdomen, and pelvis) were excluded. After some initial mild improvement in hypercalcemia during the first week, the serum calcium rose to >14 mg/dL (>3.5 mmol/L). The working diagnosis of vitamin D toxicity was determined based on total 25-hydroxyvitamin D (25(OH)D) of 599 (32 to 80) ng/mL (1495 [80 to 200] nmol/L) by immunoassay (DIASORIN® Liaison XL). Concomitant 1,25-dihydroxyvitamin D (1,25(OH)2D) by immunoassay (DIASORIN Liaison XL) was extremely high at 1000 (25 to 87) pg/mL (2400 [60 to 208] pmol/L) with an angiotensin-converting enzyme level of 27 (13 to 57) U/L. In the absence of imaging evidence for sarcoidosis or other granulomatous disease, the working diagnosis continued to be vitamin D intoxication. The patient's supplements were discontinued at admission, and empiric glucocorticoid treatment was administered until hospital discharge, with minimal effect upon calcium levels. Over serial observations, serum calcium slowly decreased, along with 25(OH)D and 1,25(OH)2D levels (Fig. 1). Laboratory profile of a patient with non-PTH–mediated hypercalcemia ranging from initial presentation to duration of follow-up. The dashed vertical lines depict the patient's resumption of over-the-counter supplements. 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D were measured via DIASORIN XL assay. Unit conversions: serum calcium mmol/L to mg/dL: ×4; serum phosphate mmol/L to mg/dL: ×3.1; creatinine µmol/L to mg/dL: ×0.01; 25-hydroxyvitamin D nmol/L to ng/mL: ×0.40; 1,25-dihydroxyvitamin D pmol/L to pg/mL: ×0.42. During outpatient follow-up, 3 months later, a marked elevation in calcium and 25(OH)D once again was noted, the patient sharing that he had resumed use of his supplements. Hypervitaminosis D as the cause of hypercalcemia was queried, based on a persistently elevated 1,25(OH)2D of 510.4 (25 to 86.7) pg/mL (1225 [60 to 208] pmol/L), in addition to 25(OH)D of 351 (32 to 80) ng/mL (876 [80 to 200] nmol/L) (Fig. 1). Along with supplement discontinuation, treatment with ketoconazole was utilized for nearly 3 months during which time the calcium level normalized (1). A hypothesis of possible overactivation of cytochrome P450 family 27 subfamily B member 1 (CYP27B1, encoding for 1-α-hydroxylase) was considered, alongside disorders of vitamin D metabolism, such as a defect in the catabolic enzyme cytochrome P450 family 24 subfamily A member 1 (CYP24A1, encoding for 24-hydroxylase) (2), which principally makes 24,25-dihydroxyvitamin D3 (24,25(OH)2D3). By measuring 24,25(OH)2D using liquid chromatography–tandem mass spectrometry (LC-MS/MS) (Mayo Clinic Laboratories), we were able to rule out a defect in catabolism based on a normal, 25(OH)D:24,25(OH)2D, vitamin D metabolite ratio (VMR) of 19.1 (<25) (3). Simultaneously, testing via LC-MS/MS confirmed that the 25(OH)D3 was truly elevated at 359.6 ng/mL (897.5 nmol/L) (>80.1 ng/mL [>200 nmol/L], indicating toxicity possible). Repeat chest CT to follow up incidental pulmonary nodules showed a possible small granuloma; however, this was not hypermetabolic on subsequent positron emission tomography (PET). Overall, no hypermetabolic activity to suggest sarcoid was found on a full body PET/CT. What possible etiologies should be considered when investigating non-PTH mediated hypercalcemia? What are the clinical explanations for excessive 25-hydroxyvitamin D compared to excessive 1,25-dihydroxyvitamin D? How can one explain vitamin D intoxication when the vitamin D supplement indicates a cholecalciferol content of just 1000 IU/drop? How might glucocorticoids or ketoconazole be used to treat severe hypercalcemia from various kinds of vitamin D excess disorders? What are the potential advantages of vitamin D or steroid compound measurements by LC-MS/MS compared to immunoassay? Clinical decision-making in hypercalcemic disorders is heavily dependent on biochemical data. Understanding how the data is derived and its possible limitations can inform the diagnostic workup. In otherwise healthy individuals, vitamin D toxicity is believed to occur at 25(OH)D levels >150 ng/mL (>375 nmol/L), as endorsed in statements by the Institute of Medicine and the Endocrine Society (1). In this case of non-PTH mediated hypercalcemia, where a history of supplement use was present and 25(OH)D markedly elevated, hypervitaminosis D had been the unifying diagnosis until a seemingly elevated measurement of 1,25(OH)2D was found (Fig. 1). This led to diagnostic confusion, additional investigations, and a medication trial with ketoconazole. By blocking cytochrome P450/ CYP27B1, ketoconazole has an inhibitory effect on vitamin D metabolism (1). Additional evaluation of the patient's vitamin D metabolome was performed in collaboration with a research facility. The research LC-MS/MS analysis demonstrated normal levels of 1,25(OH)2D, suggesting that previously reported high results were likely due to immunoassay interference. The erroneous result for 1,25(OH)2D by immunoassay was confirmed by a nonlinear result on serial dilution. Interference by heterophile antibody was ruled out using a scantibody blocking tube. The DIASORIN Liaison XL assay for 1,25(OH)2D is a 3-step immunoassay which includes complex formation of analyte with a recombinant vitamin D binding protein followed by antibody recognition. Both the 25(OH)D and 1,25(OH)2D assays were validated for clinical use according to local regulatory guidelines from the College of Physicians and Surgeons of Alberta (CPSA). The manufacturer stated cross-reactivity of <0.1% of various other vitamin D metabolites, including the 25-hydroxy, 24,25-dihydroxy, and non-hydroxylated forms. Through the research assay, elevation of products 24,25(OH)2D3, 23,25,26-trihydroxyvitamin D3 (23,25,26(OH)3D3), 25-hydroxyvitamin D3-26,23-lactone (25(OH)D3-26,23-lactone), and 1,24,25-trihydroxyvitamin D3 (1,24,25(OH)3D3) were observed (Table 1). The spurious 1,25(OH)2D result was therefore attributed to the presence of 1,24,25(OH)3D3 or other unmeasured metabolites. Vitamin D metabolites measured using 2 different assays from an October 2021 patient sample. Em dashes (—) indicate not tested with immunoassay. aResearch lab of Drs. Jones and Kaufmann at Queen's University. bAlberta Precision Laboratories, DIASORIN Chemiluminescent Immunoassay. Vitamin D metabolites measured using 2 different assays from an October 2021 patient sample. Em dashes (—) indicate not tested with immunoassay. aResearch lab of Drs. Jones and Kaufmann at Queen's University. bAlberta Precision Laboratories, DIASORIN Chemiluminescent Immunoassay. When no granulomatous disease was uncovered, re-evaluating the initial immunoassay-derived data using LC-MS/MS showed that 1,25(OH)2D was in fact not elevated. This supported and confirmed the initial diagnosis of vitamin D intoxication. Evaluation of the vitamin D metabolome found all the measurable metabolites to be elevated (Table 1). 1,24,25(OH)3D3 was increased more than 7 times the upper reference limit and interacts with the vitamin D receptor (VDR) approximately 50% as much as 1,25(OH)2D. The persistent effect seen over months can be attributed to vitamin D being lipophilic and its half-life in adipose tissue of about 2 months (4). The discrepancies observed with antibody-based methods for 25(OH)D and 1,25(OH)2D have been observed previously. DEQAS, the global vitamin D external quality assessment scheme, has documented interference of elevated dihydroxyvitamin D metabolites, particularly 24,25(OH)2D, in routine 25(OH)D assays and highlighted the need for adequate chromatographic separation for accuracy (5). Furthermore, Hawkes et al. (6) have reported that 25(OH)D at high concentration interferes in the most common 1,25(OH)2D immunoassay but not in LC-MS/MS assays (7). Consequently, for this case, we place much more emphasis on the results generated by LC-MS/MS, which chromatographically separates the individual vitamin D metabolites and also assesses differences in molecular mass and fragmentation properties. LC-MS/MS enables simultaneous measurement of various metabolites in vitamin D biosynthetic and catabolic pathways, which can aid in identifying the cause of vitamin D-related hypercalcemia. Although elevated 25(OH)D may be sufficient to suspect hypervitaminosis D, simultaneous measurement of 24,25(OH)2D enabled us to exclude a CYP24A1 mutation. Furthermore, a normal LC-MS/MS 1,25(OH)2D concentration helped rule out sarcoidosis, or other causes of excess 1,25(OH)2D, as the cause of the hypercalcemia. The research platform also found elevated 24,25(OH)2D3, 23,25,26(OH)3D3 and 25(OH)D3-26,23-lactone, which are catabolites of 25(OH)D formed by CYP24A1 and consistent with hypervitaminosis D, as observed in other literature reports (8). The metabolite 1,24,25(OH)3D3 was elevated and, while it is the first step in the catabolism of 1,25(OH)2D3, it can also be derived from 24,25(OH)2D3 by the action of CYP27B1. This is the likely dominant pathway in hypervitaminosis D given the vast excess of 24,25(OH)2D3. The finding of elevated 1,24,25(OH)3D3 is noteworthy in this case as it highlights alternative pathways of vitamin D metabolism that can occur in hypervitaminosis D, that this metabolite can transactivate the VDR and contribute to the hypercalcemia, and that 1,24,25(OH)3D3 may cross-react with immunoassays contributing to the aberrant 1,25(OH)2D values on initial workup. One remaining question pertains to how excessive vitamin D intake results in hypercalcemia without causing an elevation in the active form, 1,25(OH)2D. This has been debated over the last few decades and was reviewed by Jones in 2008 (4). It is still unclear which is the hypercalcemia-inducing form of vitamin D, whether it is 25(OH)D itself or one of its catabolites. Initial observations that 1,25(OH)2D is not elevated have been reinforced by many studies since 2008, which report suppression of CYP27B1 expression (the renal 1-α-hydroxylase) and PTH in hypercalcemia. A further dose escalation experiment using the CYP27B1-null mouse showed that excessive dietary vitamin D intake results in hypercalcemia in CYP27B1-null and normal wild-type littermates at exactly the same dose, despite the null mice lacking any ability to synthesize 1,25(OH)2D (9). Accordingly, it appears that the hypercalcemia of hypervitaminosis D is not caused by free or total 1,25(OH)2D displaced from vitamin D binding protein (4). Likewise, it is not the result of the catabolite 1,24,25(OH)3D in the CYP27B1-null mouse. Thus, we are left to conclude that either the greatly elevated 25(OH)D itself, or one of its other catabolites can bind to the cellular VDR, in vitamin D target cells, and transduce effects on vitamin D-dependent genes, including those involved in raising serum calcium and phosphate (10). For our patient, a third upward trend in calcium and vitamin D was seen again 6 months later and, once more, it was discovered that the patient had resumed taking his supplements. Seeing this third recurrence of vitamin D toxicity, the patient was convinced to permanently stop the supplements and all biochemistry normalized in subsequent follow-up. When elevated 1,25(OH)2D levels are not explained by clinically apparent conditions such as granulomatous disease, consider issues with immunoassay interference. Serial dilutions can be used to infer the presence of interfering substances in immunoassay measurements. Measurement of vitamin D upstream and downstream metabolites can be used to investigate the presence or function of important enzymes in vitamin D catabolism. Hypercalcemia of hypervitaminosis D is likely caused by the action of 25(OH)D itself, and/ or a catabolite binding with the vitamin D receptor. The amount of vitamin D present in unregulated health supplements may not be accurate compared to the actual vitamin D content, therefore 25(OH)D and 1,25(OH)2D levels are important to include in the investigation of PTH-independent hypercalcemia. PTH, parathyroid hormone; 25(OH)D, 25-hydroxyvitamin D; 1,25(OH)2D, 1,25-dihydroxyvitamin D; 24,25(OH)2D3, 24,25-dihydroxyvitamin D3; 1,24,25(OH)3D3, 1,24,25-trihydroxyvitamin D3. The corresponding author takes full responsibility that all authors on this publication have met the following required criteria of eligibility for authorship: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. Nobody who qualifies for authorship has been omitted from the list. Umair Sajid (Conceptualization-Lead, Data curation-Equal, Formal analysis-Equal, Investigation-Equal, Methodology-Supporting, Writing—original draft-Lead), Dennis Orton (Conceptualization-Equal, Data curation-Equal, Formal analysis-Equal, Investigation-Equal, Methodology-Equal, Writing—review & editing-Equal), Martin Kaufmann (Formal analysis-Equal, Investigation-Equal, Writing—review & editing-Equal), Glenville Jones (Formal analysis-Equal, Investigation-Equal, Methodology-Equal, Writing—review & editing-Equal), and Gregory Kline (Conceptualization-Lead, Formal analysis-Equal, Investigation-Equal, Methodology-Equal, Supervision-Lead, Writing—review & editing-Lead) No authors declared any potential conflicts of interest.

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