Vitamin D has been demonstrated to alter cellular proliferation through multiple mechanisms in highly cell‐specific manners. One of the most important mechanisms was the slowing of cell cycle progression induced by vitamin D, typically due to inhibition of advancement from the G1 to the S phase of the cell cycle. Many key regulators, which influence gene transcrip‐ tion and protein stability, including p21waf1, p27kip1, cyclin D1, and others, are taken effect on by vitamin D. The ligand‐activated VDR directly or indirectly influences the cell cycle, apoptosis, and/or differentiation by interacting with important transcriptional regulators or

Anti‐proliferative effects of vitamin D signaling are often, but not always, linked to the pro‐ motion of cellular differentiation. Vitamin D and the VDR also have important interactions with other transcriptional regulators and cell‐signaling systems. In many cell lines includ‐

receptor type I protein or androgen receptors, and downregulate the expressions of estrogen receptors or IGF (insulin‐like growth factor I). Vitamin D analogs also induce the expression of E‐cadherin, promoting translocation of β‐catenin from the nucleus to the cell membrane, to control cell growth and differentiation [19]. In our study, we found that VDR could be down‐ regulated by high glucose and take part in the epithelial‐epithelial‐mesenchymal transition of

physiologic conditions. Additionally, the vitamin D receptor, which binds to, and mediates

not only for the maintenance of normal calcium and phosphorus homeostasis but also for the activation of vitamin D. There is close relationship among vitamin D, VDR, and the kidney**.** Any problem of kidney may lead to the vitamin D‐signaling deficiency, and vitamin D‐signal‐

It was reported that when the glomerular filtration rate declines below 60 mL/min, chronic kidney disease (CKD) was always associated with increased cardiovascular events and mortality. One of the most important reasons was that the reduction/absence of kidney α1‐

lead to the vitamin D deficiency in CKD patients. Vitamin D deficiency causes parathyroid

hydroxylase, which mediates the final hydroxylation step of 25(OH)D to 1,25‐(OH)2

D3 and vitamin D analogs upregulate the expressions of TGF‐β

D3, is widely distributed in the kidney. Thus, the kidney is essential

D3, the active form of vitamin D, under

D, could

**2.2. The effect of vitamin D in cell proliferation and differentiation**

of immune responses and inflammation.

78 A Critical Evaluation of Vitamin D - Basic Overview

cell‐signaling systems [19, 20].

ing cancer cells, 1,25(OH)2

podocyte in mice [21].

the activity of 1, 25‐(OH)2

*3.1.1. Chronic kidney disease*

**3. Vitamin D and diseases**

**3.1. Vitamin D and kidney diseases**

Kidney is the major site of the synthesis of 1, 25‐(OH)2

ing deficiency always causes the abnormal function of kidney [22].

Diabetic nephropathy (DN) is the most common renal complication of diabetes mellitus and a leading cause of end‐stage renal disease (ESRD). Intervention of DN remains a medical chal‐ lenge despite some success.

In recent years, renin‐angiotensin system (RAS) inhibitors have been used as the mainstay treatment for DN because the renin‐angiotensin system (RAS) was considered as a major mediator of progressive renal injury in DN. But the compensatory renin increase caused by the disruption of renin feedback inhibition becomes one major problem which limit the effi‐ cacy use of the RAS inhibitors. These most recent data demonstrate that vitamin D and its analogs have renoprotective and therapeutic potentials in DN through targeting the RAS. Vitamin D negatively regulates the RAS by suppressing renin expression and thus plays a renoprotective role in DN [26, 29]. The molecular mechanism underlying this regulation is that 1,25(OH)2D3 disrupted the cAMP‐signaling pathway, a major regulatory pathway involved in renin biosynthesis [30]. It was reported that vitamin D analog therapy could increase long‐ term survival, reduces heart and kidney weights, and prevents overt renal tissue damage in mouse models. Combination therapy with a RAS inhibitor and a vitamin D analog was found to markedly improve renal injuries in the diabetic vitamin D receptor null‐mutant mice which developed more severe renal injuries because of more robust RAS activation [30]. Another study found that combined therapy with losartan and paricalcitol completely reduced pro‐ teinuria in a model of experimental diabetic nephropathy, suggesting that the combination of an ACE inhibitor or an ANG II receptor blocker plus a VDRA may be a good therapeutic option [23, 24].

Results from two clinical trials have provided some initial insight into the benefit of vita‐ min D therapy in diabetic nephropathy patients. Selective vitamin D receptor activation with paricalcitol for the reduction of albuminuria in patients with type 2 diabetes (VITAL study) investigated an active vitamin D analog exclusively in diabetic nephropathy patients. These patients were all the type 2 diabetics with a stable dose of angiotensin‐converting enzyme (ACE) inhibitors or angiotensin receptor blockers (ARBs). The 281 patients were randomized into three groups: a single dose of placebo, 1 μg paricalcitol or 2 μg paricalcitol each day in a double‐blind fashion. After 4 months of treatment, a significantly greater reduction in 24‐h albumin excretion (measured by percent change from baseline) was found in the 2‐μg pari‐ calcitol group compared to placebo patients [31]. The second study about the measurement of the impact of vitamin D repletion with cholecalciferol on urinary albumin, MCP‐1, and TGF‐β1 was recently published by Kim and colleagues measured [32].

#### *3.1.3. Immunoglobulin A nephropathy*

The renoprotective effect of vitamin D was also investigated in immunoglobulin A (IgA) nephropathy. An open‐label, non‐placebo‐controlled, randomized study about the effect of vitamin D and urinary protein excretion in 50 IgA nephropathy patients was operated on by using calcitriol. Patients who receive two doses (0.5 mg) of calcitriol per week or no treat‐ ment for 48 weeks were randomly assigned (1:1). The urinary protein excretion >0.8 g/d after renin‐angiotensin system‐inhibitor treatment for at least 3 months was considered as the main criterion for inclusion. The changes of 24‐h urinary protein excretion from baseline to last measurement during treatment were compared and considered as the primary end point. The results showed that there was a significant decrease in proteinuria in the calcitriol‐treated group compared with the control group (difference between groups, 41%; 95% confidence interval (CI), 5–79%; *P* = 0.03) the primary end point. At least a 15% decrease in proteinuria was set as the secondary end point. And the result were 7 of 24 (29%) controls and 17 of 26 (65%) of those treated with calcitriol (*P* = 0.02). These two groups showed the similar inci‐ dence of recorded adverse events, but had no significant differences in the decrease of esti‐ mated glomerular filtration rate and change in blood pressure. The conclusion was that the addition of calcitriol to a renin‐angiotensin system inhibitor resulted in a safe decrease in proteinuria in patients with IgA nephropathy [33].

#### *3.1.4. Other renal diseases*

Vitamin D also play renoprotective role in other renal diseases. For example, cholecalcif‐ erol (vitamin D analog) treatment significantly increased serum 25‐hydroxy vitamin D and decreased parathyroid hormone levels with no adverse effects in 25‐hydroxy vitamin D‐defi‐ cient renal‐transplant patients [34]. Vitamin D receptor agonist doxercalciferol modulates dietary fat‐induced renal disease and renal lipid metabolism [35].

Most of these studies have been reviewed recently and support the idea that vitamin D has therapeutic potential in slowing the progression of nephropathy. But these researches have their own limitations. The protection and mechanism of vitamin D need further study.

#### **3.2. Vitamin D and cardiovascular disease**

Cardiovascular disease (CVD) is the leading cause of death among patients with chronic kidney disease, with left ventricular hypertrophy (LVH) being a strong, independent risk factor. LVH is also a major risk factor for coronary ischemia, congestive heart failure, and cardiac arrhythmias [24].

There is a growing body of evidence linking vitamin D signaling and CVD in both experi‐ mental animals and humans. For instance, animals lacking the VDR or having vitamin D deficiency always show cardiovascular abnormalities, such as hypertension and LVH [36]. Cardiomyocytes isolated from VDR knockout mice developed contractile abnormalities, such as accelerated contraction and relaxation rates [37]. In humans, studies found that vitamin D deficiency is prevalent in neonates with congenital cardiac defects, and lower postopera‐ tive 25(OH)D levels are associated with the need for increased inotropic support in neonates undergoing cardiac operations. Low serum 25(OH)D level could also increase the incidence of sudden cardiac death both in healthy people and in hemodialysis patients. Some research showed that vitamin D deficiency may play a role in myocardial injury and postoperative recovery. Furthermore, two studies demonstrated that individuals with lower levels of active vitamin D were at a higher risk of developing hypertension [38, 39]. Vitamin D also was found to modulate the growth, hypertrophy, and differentiation of cardiomyocytes, point‐ ing to a direct role for VDR agonists (VDRA) in cardiac physiology. These findings support that vitamin D deficiency is associated with poor cardiovascular outcomes in experimental animals and humans.

There were some reports about the improved cardiac function, which could be found in experimental animals or patients with administration of vitamin D or VDRAs. For example, after the treatment of paricalcitol, hypertensive rats showed a prevention of LVH and LV dysfunction accompanied with lower levels of brain natriuretic peptide and atrial natriuretic factor. VDRA therapy revealed similar results in another experimental model of LVH, the Cp/ rat model. Moreover, vitamin D is able to modulate contractility of cardiomyocytes in vitro by changing the distribution of the myosin chains and modulating Ca entry into cardiac muscle cells. In patients, the use of VDRA has been associated with improvements in left ventricular function and reductions in LVH [24].

Results from interventional trials, using either nutritional vitamin D or VDR agonists, sup‐ ported the idea that VDR activation was beneficial for improving the underlying factors of CVD such as hypertension, endothelial dysfunction, atherosclerosis, vascular calcification, and cardiac hypertrophy.

#### **3.3. Vitamin D and inflammatory bowel disease**

investigated an active vitamin D analog exclusively in diabetic nephropathy patients. These patients were all the type 2 diabetics with a stable dose of angiotensin‐converting enzyme (ACE) inhibitors or angiotensin receptor blockers (ARBs). The 281 patients were randomized into three groups: a single dose of placebo, 1 μg paricalcitol or 2 μg paricalcitol each day in a double‐blind fashion. After 4 months of treatment, a significantly greater reduction in 24‐h albumin excretion (measured by percent change from baseline) was found in the 2‐μg pari‐ calcitol group compared to placebo patients [31]. The second study about the measurement of the impact of vitamin D repletion with cholecalciferol on urinary albumin, MCP‐1, and

The renoprotective effect of vitamin D was also investigated in immunoglobulin A (IgA) nephropathy. An open‐label, non‐placebo‐controlled, randomized study about the effect of vitamin D and urinary protein excretion in 50 IgA nephropathy patients was operated on by using calcitriol. Patients who receive two doses (0.5 mg) of calcitriol per week or no treat‐ ment for 48 weeks were randomly assigned (1:1). The urinary protein excretion >0.8 g/d after renin‐angiotensin system‐inhibitor treatment for at least 3 months was considered as the main criterion for inclusion. The changes of 24‐h urinary protein excretion from baseline to last measurement during treatment were compared and considered as the primary end point. The results showed that there was a significant decrease in proteinuria in the calcitriol‐treated group compared with the control group (difference between groups, 41%; 95% confidence interval (CI), 5–79%; *P* = 0.03) the primary end point. At least a 15% decrease in proteinuria was set as the secondary end point. And the result were 7 of 24 (29%) controls and 17 of 26 (65%) of those treated with calcitriol (*P* = 0.02). These two groups showed the similar inci‐ dence of recorded adverse events, but had no significant differences in the decrease of esti‐ mated glomerular filtration rate and change in blood pressure. The conclusion was that the addition of calcitriol to a renin‐angiotensin system inhibitor resulted in a safe decrease in

Vitamin D also play renoprotective role in other renal diseases. For example, cholecalcif‐ erol (vitamin D analog) treatment significantly increased serum 25‐hydroxy vitamin D and decreased parathyroid hormone levels with no adverse effects in 25‐hydroxy vitamin D‐defi‐ cient renal‐transplant patients [34]. Vitamin D receptor agonist doxercalciferol modulates

Most of these studies have been reviewed recently and support the idea that vitamin D has therapeutic potential in slowing the progression of nephropathy. But these researches have their own limitations. The protection and mechanism of vitamin D need further study.

Cardiovascular disease (CVD) is the leading cause of death among patients with chronic kidney disease, with left ventricular hypertrophy (LVH) being a strong, independent risk

TGF‐β1 was recently published by Kim and colleagues measured [32].

*3.1.3. Immunoglobulin A nephropathy*

80 A Critical Evaluation of Vitamin D - Basic Overview

proteinuria in patients with IgA nephropathy [33].

**3.2. Vitamin D and cardiovascular disease**

dietary fat‐induced renal disease and renal lipid metabolism [35].

*3.1.4. Other renal diseases*

Immune‐mediated diseases such as inflammatory bowel diseases (IBDs) have increased in developed countries over the last 50 years. Scientists did much research to explain the increased incidence of IBD. They found that changing vitamin D status especially in prenatal as well as childhood could affect the development of the resultant immune response and the development of IBD.

There are reasons to believe that vitamin D could be an external factor that may play a role in the development of IBD. Animal models of experimental IBD had been used to explore the rela‐ tionship between vitamin D signaling and IBD. The results showed that in IL‐10 KO mice, the development of IBD symptoms could be accelerated by vitamin D deficiency [40]. Additionally, a fulminating form of IBD resulted in ulceration of the intestine and the premature mortality of the double VDR and IL‐10 KO mice within a very short time frame (3–5 weeks of age).

Vitamin D receptor knockout or vitamin D‐deficient mice always have T cells deficient that have been implicated in the pathology of IBD. Actually, the active form of vitamin D directly and indirectly suppresses the function of these pathogenic T cells while inducing several reg‐ ulatory T cells that could suppress IBD progressing [18].

People found that 1, 25(OH)2 D and analogs of 1,25(OH)2 D could suppress IBD in the IL‐10 KO mice which required adequate dietary calcium, suggesting that 1,25(OH)2 D may directly and indirectly control immune function through calcium regulation in vivo. Many evidences from multiple different animal models suggested that the increased susceptibility of mice to experimental IBD could be caused by vitamin D and VDR deficiency, which could be sup‐ pressed by 1,25(OH)2 D [41].

Although at present there has not been a causal relationship between vitamin D status and IBD established in the clinic, there have been some clinic reports that show the influence of vitamin D in IBD. They found most of children and young adult patients in IBD had low vitamin D status (serum 25(OH)D concentration ≤15 ng/mL). Additionally, a randomized, controlled clinical trial from November 2007 to June 2010 at the Clinical and Translational Study Unit of Children's Hospital in Boston showed that most of the 71 patients (age 5–21) in IBD had low vitamin D status in serum (less than 20 ng/mL). A treatment of oral doses of 2000 IU vitamin D3 daily or 50,000 IU vitamin D2 weekly for 6 weeks could raise serum 25(OH)D concentration obviously and the basic conditions of children and adolescents with IBD could be improved. The change in serum PTH concentration did not differ [42].

Overall, the available data do suggest that vitamin D status and analogs would be useful for normalizing T cell function and in the prevention and treatment of human IBD. But there are still many problems that need to be studied, such as which mechanism of the vitamin D medi‐ ated occur in human cells and IBD patients.

#### **3.4. Vitamin D and cancer**

Numerous studies have suggested that lower vitamin D level is a risk factor for human can‐ cers, and vitamin D deficiency linked with the incidence and mortality of many types of can‐ cers, including breast, colon, and prostate cancers [43, 44].

Beneficial effects of vitamin D have been observed in experimental cancers induced by various chemical carcinogens or genetic mutations, and in studies using human cancer cells implanted in nude mice. The anticancer effects of vitamin D included alterations in cell growth, angio‐ genesis, tumor invasion and metastatic potential, and immune surveillance. Many studies have demonstrated that vitamin D signaling inhibited the growth of many cancer‐derived cell lines in vitro, by inhibiting progression through the cell cycle, inducing apoptosis, and driv‐ ing the cells to a more differentiated phenotype. VDR also played important role in antican‐ cer effect. Some preliminary data suggested that VDR polymorphisms were more frequently associated with tumor genes which are Fok1, Bsm1, Taq1, Apa1, EcoRV, and Cdx2 [43–45]. And another study showed that vitamin D receptor expression is linked to potential markers of human thyroid papillary carcinoma [46].

Trials in clinic also have shown the potential therapeutic effects of vitamin D in different kinds of cancers. For example, in a pilot study of patients with recurrent prostate cancer, oral calcitriol (starting with 0.5 mg/day and escalating the maximum dose to 2.5 mg/day) for 6–15 months resulted in a significant decrease in the rate of prostate‐specific antigen (PSA) rise during ther‐ apy (in comparison to PSA increase before therapy) in six out of seven patients [43]. In another research, scientists provided evidence indicating that vitamin D signaling protected the skin from cancer formation by controlling keratinocyte proliferation and differentiation, facilitating DNA repair, and suppressing activation of the hedgehog (Hh) pathway following UVR exposure [47].

Although the anticancer effect of vitamin D signaling has been discovered, there are still prob‐ lems that need to be discussed both in experimental and in clinical trials. In vitro and in vivo studies have clearly demonstrated the antitumor effects of vitamin D. But the mechanisms of antigrowth effects of vitamin D are quite variable in different cell types, and even in different cell lines derived from the same type of cancer. In clinic, the results of vitamin D and cancer mortality were inconsistent and even oppositive associations. The majority of studies in can‐ cer patients showed that patients with higher vitamin D levels had a decreased risk of mortal‐ ity. But there were some other voice against this. In conclusion, the relationship of vitamin D status and anticancer effect is still unclear and warrants further studies.

#### **3.5. Other diseases**

development of IBD symptoms could be accelerated by vitamin D deficiency [40]. Additionally, a fulminating form of IBD resulted in ulceration of the intestine and the premature mortality of

Vitamin D receptor knockout or vitamin D‐deficient mice always have T cells deficient that have been implicated in the pathology of IBD. Actually, the active form of vitamin D directly and indirectly suppresses the function of these pathogenic T cells while inducing several reg‐

and indirectly control immune function through calcium regulation in vivo. Many evidences from multiple different animal models suggested that the increased susceptibility of mice to experimental IBD could be caused by vitamin D and VDR deficiency, which could be sup‐

Although at present there has not been a causal relationship between vitamin D status and IBD established in the clinic, there have been some clinic reports that show the influence of vitamin D in IBD. They found most of children and young adult patients in IBD had low vitamin D status (serum 25(OH)D concentration ≤15 ng/mL). Additionally, a randomized, controlled clinical trial from November 2007 to June 2010 at the Clinical and Translational Study Unit of Children's Hospital in Boston showed that most of the 71 patients (age 5–21) in IBD had low vitamin D status in serum (less than 20 ng/mL). A treatment of oral doses of 2000 IU vitamin D3 daily or 50,000 IU vitamin D2 weekly for 6 weeks could raise serum 25(OH)D concentration obviously and the basic conditions of children and adolescents with IBD could

Overall, the available data do suggest that vitamin D status and analogs would be useful for normalizing T cell function and in the prevention and treatment of human IBD. But there are still many problems that need to be studied, such as which mechanism of the vitamin D medi‐

Numerous studies have suggested that lower vitamin D level is a risk factor for human can‐ cers, and vitamin D deficiency linked with the incidence and mortality of many types of can‐

Beneficial effects of vitamin D have been observed in experimental cancers induced by various chemical carcinogens or genetic mutations, and in studies using human cancer cells implanted in nude mice. The anticancer effects of vitamin D included alterations in cell growth, angio‐ genesis, tumor invasion and metastatic potential, and immune surveillance. Many studies have demonstrated that vitamin D signaling inhibited the growth of many cancer‐derived cell lines in vitro, by inhibiting progression through the cell cycle, inducing apoptosis, and driv‐ ing the cells to a more differentiated phenotype. VDR also played important role in antican‐ cer effect. Some preliminary data suggested that VDR polymorphisms were more frequently associated with tumor genes which are Fok1, Bsm1, Taq1, Apa1, EcoRV, and Cdx2 [43–45].

D could suppress IBD in the IL‐10

D may directly

the double VDR and IL‐10 KO mice within a very short time frame (3–5 weeks of age).

D and analogs of 1,25(OH)2

KO mice which required adequate dietary calcium, suggesting that 1,25(OH)2

be improved. The change in serum PTH concentration did not differ [42].

ulatory T cells that could suppress IBD progressing [18].

D [41].

ated occur in human cells and IBD patients.

cers, including breast, colon, and prostate cancers [43, 44].

**3.4. Vitamin D and cancer**

People found that 1, 25(OH)2

82 A Critical Evaluation of Vitamin D - Basic Overview

pressed by 1,25(OH)2

There are many more reports and studies about vitamin D and its receptor which were found to play crucial role in many fields, such as vitamin D and obstructive sleep apnea in polycystic ovary syndrome (PCOS) patients [44], serum 25‐hydroxy vitamin D levels in middle‐aged women in relationship to adiposity and height trajectories [48], and vitamin D deficiency impairs skeletal muscle function in a smoking mouse model [49]. In engender, vitamin D deficiency and low ionized calcium are linked with semen quality and sex steroid levels in infertile men [50]. The same result was also found in subfertile women [51]. In cognitive behavior, low vitamin D levels were frequent in depression patients, especially 25‐OH D lev‐ els <50 nmol/L were associated with cognitive/affective‐depressive symptoms, and anhedonia symptoms in particular [52]. Vitamin D supplementation could prevent depression and poor physical function in older adults [53]. Some reports found that the concentration of vitamin D in the plasma of patients with metabolic syndrome was significantly lower than its recom‐ mendations [54], and a relationship was detected between vitamin D concentration and expo‐ nents of metabolic syndrome [55, 56]. But a randomized controlled trial for 1 year in a Chinese population found that the correction of hypovitaminosis D did not improve the metabolic syndrome risk profile [57].

## **4. Conclusion**

It is now well recognized that vitamin D is involved not only in the control of bone health and mineral metabolism but also in many other physical progresses, such as the control of immune responses, responses to infectious agents, and cell‐proliferative mechanisms, particularly in cancer cells. The vitamin D signaling also exhibits multiple effects, such as anti‐inflammation effects, anticancer effect, and cardiovascular‐ and kidney‐protective effects. From a practical point of view, vitamin D deficiency participates in many pathological progression and dis‐ eases. In some diseases, the administration of vitamin D or VDRA could rescue the clinical symptoms and improve outcomes. In conclusion, vitamin D has many potential therapeutic effects and multiple roles in different diseases, physical and pathological progressions, which are all worth to be studied further.

## **Author details**

Jia Guo1, 2, 3 and Zhangsuo Liu1, 2, 3\*

#### **References**

responses, responses to infectious agents, and cell‐proliferative mechanisms, particularly in cancer cells. The vitamin D signaling also exhibits multiple effects, such as anti‐inflammation effects, anticancer effect, and cardiovascular‐ and kidney‐protective effects. From a practical point of view, vitamin D deficiency participates in many pathological progression and dis‐ eases. In some diseases, the administration of vitamin D or VDRA could rescue the clinical symptoms and improve outcomes. In conclusion, vitamin D has many potential therapeutic effects and multiple roles in different diseases, physical and pathological progressions, which

1 Department of Nephrology, The First Affiliated Hospital of Zhengzhou University,

2 Nephropathy Research Institutes of Zhengzhou University, Zhengzhou, Henan, China 3 Institute of Clinical Medicine, The First Affiliated Hospital of Zhengzhou University,

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Zhengzhou, Henan, China

Zhengzhou, Henan, China

S0960‐0760(16)30022‐X.

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\*Address all correspondence to<EMAIL_ADDRESS>
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88 A Critical Evaluation of Vitamin D - Basic Overview

## **Clinical and Biochemical Features of Patients with** *CYP24A1* **Mutations**

Fay Joanne Hill and John A. Sayer

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64503

#### **Abstract**

The *CYP24A1* gene encodes 1,25-hydroxyvitamin-D3-24-hydroxylase, a key enzyme responsible for the catabolism of active vitamin D (1,25-dihydroxyvitamin D3). Loss-offunction mutations in *CYP24A1* lead to increased levels of active vitamin D metabolites. Clinically,twodistinctphenotypeshavebeenrecognisedfromthis:infantswith*CYP24A1* mutations present with infantile idiopathic hypercalcaemia, often precipitated by prophylactic vitamin D supplementation. A separate phenotype of nephrolithiasis, hypercalciuria and nephrocalcinosis often presents in adulthood. *CYP24A1* mutations should be suspected when a classical biochemical profile of high active vitamin D metabolites, high or normal serum calcium, high urine calcium and low parathyroid hormone isdetected. Successfultreatmentwithfluconazole, aP450 enzyme inhibitor,has been shown to be effective in individuals with *CYP24A1* mutations. Although *CYP24A1* mutationsarerare,earlyrecognitioncanpromptdefinitivediagnosisandensuretreatment is commenced.

**Keywords:** *CYP24A1*, vitamin D, hypercalcaemia, idiopathic infantile hypercalcaemia, nephrolithiasis

#### **1. Introduction**

The supplementation of formula milk with vitamin D3 (cholecalciferol) prompted a rise in infants presenting with symptomatic hypercalcaemia in the United Kingdom during the 1950s [1]. While this public health initiative was proving highly successful in preventing rickets, for the small cohort of infants presenting with failure to thrive, dehydration and nephrocalcinosis, the consequences of their hypercalcaemia were at times fatal. A diagnosis of idiopathic infantile hypercalcaemia was given to many in this cohort. The apparent

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

increased susceptibility of this minority group to vitamin D toxicity prompted research into a genetic predisposition. Fifty-nine years later, *CYP24A1* mutations were identified demon‐ strating loss-of-function mutations encoding 1,25-hydroxyvitamin D3 24-hydroxylase, an enzyme with a key role in vitamin D metabolism [2].

More recently, *CYP24A1* mutations have been recognised in an adult population of patients presenting with calcium-containing renal stones. On investigation, these patients typically displayed hypercalciuria, nephrocalcinosis and occasionally chronic kidney impairment. Vitamin D supplementation was not a feature in all cases [3], demonstrating a clinically significant phenotype manifesting from normal dietary vitamin D intake. Importantly, some patients had been symptomatic for many years, undergoing extensive investigations before a diagnosis was made. A continuing focus on preventative medicine, including oral vitamin D supplementation for maintenance of bone health and during pregnancy, is likely to continue to risk triggering manifestations of vitamin D toxicity in individuals carrying biallelic muta‐ tions in *CYP24A1*. As diagnostic tests and successful treatments are starting to emerge, it is important to recognise clinical presentations which should prompt screening for *CYP24A1* deficiency [4–6].

## **2.** *CYP24A1* **and the vitamin D pathway**

The crucial role of vitamin D in calcium and phosphate homeostasis means excessive levels of its active form can precipitate symptomatic hypercalcaemia. The activation of vitamin D takes place in two stages. The first stage takes place in the liver: vitamin D3 is converted to 25 hydroxyvitamin D3, a reaction catalysed by 25-hydroxylase (*CYP2R1*). The second stage occurs in the kidney, when 25-hydroxyvitamin D3 is hydroxylated to 1,25-dihydroxyvitamin D3, the active form. This stage is catalysed by 1α-hydroxylase, an enzyme encoded by the *CYP27B1* [2].

**Figure 1.** Vitamin D metabolism pathway. Activation of Vitamin D: 1. Stage 1 occurs in the liver. Vitamin D3 is convert‐ ed to 25-hydroxyvitamin D3 by the enzyme 25-hydroxylase. The *CYP2R1* gene encodes 25-hydroxylase. 2. Stage 2 oc‐ curs in the kidney. 25-hydroxyvitamin D3 is converted to 1,25-dihydroxyvitamin D3 by the enzyme 1α-hydroxylase. The *CYP27B1* gene encodes 1α-hydroxylase. 1,25-dihydroxyvitamin D3 is the physiologically most active form of vita‐ min D3 which binds to the vitamin D receptor. Inactivation of Vitamin D: Several hydroxylation steps occur in the ca‐ tabolism of 1,25-dihydroxyvitamin D3 to calcitroic acid. The first of these steps is catalysed by the enzyme 1,25 hydroxyvitamin-D3-24-hydroxylase, which is encoded by the *CYP24A1* gene.

The inactivation of vitamin D metabolites relies upon two pathways which both include steps catalysed by 1,25-hydroxyvitamin-D3-24-hydroxylase; *CYP24A1* encodes this mitochondrial enzyme which is part of the cytochrome P450 system [6]. The enzyme is present in vitamin D target cells, predominantly located in the intestine and kidneys (**Figure 1**) [5].

#### **2.1. Phenotypes**

increased susceptibility of this minority group to vitamin D toxicity prompted research into a genetic predisposition. Fifty-nine years later, *CYP24A1* mutations were identified demon‐ strating loss-of-function mutations encoding 1,25-hydroxyvitamin D3 24-hydroxylase, an

More recently, *CYP24A1* mutations have been recognised in an adult population of patients presenting with calcium-containing renal stones. On investigation, these patients typically displayed hypercalciuria, nephrocalcinosis and occasionally chronic kidney impairment. Vitamin D supplementation was not a feature in all cases [3], demonstrating a clinically significant phenotype manifesting from normal dietary vitamin D intake. Importantly, some patients had been symptomatic for many years, undergoing extensive investigations before a diagnosis was made. A continuing focus on preventative medicine, including oral vitamin D supplementation for maintenance of bone health and during pregnancy, is likely to continue to risk triggering manifestations of vitamin D toxicity in individuals carrying biallelic muta‐ tions in *CYP24A1*. As diagnostic tests and successful treatments are starting to emerge, it is important to recognise clinical presentations which should prompt screening for *CYP24A1*

The crucial role of vitamin D in calcium and phosphate homeostasis means excessive levels of its active form can precipitate symptomatic hypercalcaemia. The activation of vitamin D takes place in two stages. The first stage takes place in the liver: vitamin D3 is converted to 25 hydroxyvitamin D3, a reaction catalysed by 25-hydroxylase (*CYP2R1*). The second stage occurs in the kidney, when 25-hydroxyvitamin D3 is hydroxylated to 1,25-dihydroxyvitamin D3, the active form. This stage is catalysed by 1α-hydroxylase, an enzyme encoded by the *CYP27B1* [2].

**Figure 1.** Vitamin D metabolism pathway. Activation of Vitamin D: 1. Stage 1 occurs in the liver. Vitamin D3 is convert‐ ed to 25-hydroxyvitamin D3 by the enzyme 25-hydroxylase. The *CYP2R1* gene encodes 25-hydroxylase. 2. Stage 2 oc‐ curs in the kidney. 25-hydroxyvitamin D3 is converted to 1,25-dihydroxyvitamin D3 by the enzyme 1α-hydroxylase. The *CYP27B1* gene encodes 1α-hydroxylase. 1,25-dihydroxyvitamin D3 is the physiologically most active form of vita‐ min D3 which binds to the vitamin D receptor. Inactivation of Vitamin D: Several hydroxylation steps occur in the ca‐ tabolism of 1,25-dihydroxyvitamin D3 to calcitroic acid. The first of these steps is catalysed by the enzyme 1,25-

hydroxyvitamin-D3-24-hydroxylase, which is encoded by the *CYP24A1* gene.

enzyme with a key role in vitamin D metabolism [2].

92 A Critical Evaluation of Vitamin D - Basic Overview

**2.** *CYP24A1* **and the vitamin D pathway**

deficiency [4–6].

#### *2.1.1. Idiopathic infantile hypercalcaemia*

The first recognised phenotype of *CYP24A1* mutations was in infants diagnosed with idio‐ pathic infantile hypercalcaemia. These individuals presented with vomiting, dehydration, fevers and failure to thrive. On investigation, a typical biochemical profile of high serum calcium and suppressed parathyroid hormone levels emerged. Renal ultrasound often demonstrated nephrocalcinosis, deposition of calcium salts within the kidney. It was not initially known whether the underlying pathophysiology of idiopathic infantile hypercalcae‐ mia (IIH) was due to excess production of vitamin D metabolites, or an inability to inactivate vitamin D. A candidate gene approach was used to investigate families with typical presen‐ tations of idiopathic infantile hypercalcaemia. This research revealed a recessive loss-offunction mutation, in which patients with *CYP24A1* mutations were unable to inactivate vitamin D as they were deficient in the enzyme catalysing this pathway (1,25-hydroxyvitamin-D3-24-hydroxylase). Affected children presented either after sustained low-dose vitamin D prophylaxis or directly following bolus doses of vitamin D. One sibling in which vitamin D prophylaxis was avoided was proven to carry the same mutation but had remained clinically silent. This supported evidence directly linking exogenous vitamin D supplementation with precipitation of symptomatic hypercalcaemia [2].

#### *2.1.2. Adult nephrolithiasis*

Hypercalciuria is the most common cause of calcium-containing kidney stones. The recogni‐ tion that 40–45% of patients with idiopathic hypercalciuria have at least one relative with nephrolithiasis implicates a genetic predisposition in many cases [4]. *CYP24A1* mutations have now been proven in a cohort of adults presenting with nephrolithiasis, hypercalciuria, nephrocalcinosis and intermittent hypercalcaemia [4]. These patients had undergone exten‐ sive investigations before the cause of their nephrolithiasis was known, and multiple stone episodes and nephrocalcinosis may lead to progressive chronic kidney disease (CKD) [7]. This is important in highlighting the potential clinical spectrum of the phenotype, which may manifest without the trigger of vitamin D exposure. A typical biochemistry profile was found within this phenotype group, with normal/high serum calcium levels, suppressed parathyroid hormone, high levels of active vitamin D metabolites (25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3) and low levels of inactivated vitamin D (24,25-dihydroxyvi‐ tamin D3). A recent study screening patients with known calcium nephrolithiasis for *CYP24A1* mutations did not identify any biallelic variants in a cohort of 166 patients, sug‐ gesting *CYP24A1* mutations are a rare cause of idiopathic nephrolithiasis [8]. However, giv‐ en our increased understanding of this phenotype, it is imperative that recognition of the

typical biochemical pattern (suppressed PTH, hypercalcaemia, hypercalciuria) in any pa‐ tients with nephrolithiasis prompts investigation for *CYP24A1* mutations [4, 6, 8]. Establish‐ ing a molecular diagnosis in this small cohort of patients can facilitate correct treatment and lifestyle modification (**Table 1**) [9].

**Table 1.** Key features of *CYP24A1* mutation phenotypes.

#### *2.1.3. Investigation*

In patients with *CYP24A1* mutations, an elevation in total vitamin D levels is typically seen. In particular, 1,25-dihydroxyvitamin D3 levels are increased, but this assay is not routinely performed in many laboratories. Conversely, serum 24,24-dihydroxyvitamin-D3 levels are sometimes low or undetectable in patients with *CYP24A1* mutations. A blood test that calculates the ratio between vitamin D metabolites could be utilised in future clinical practice as a screening tool for *CYP24A1* mutations in those patients presenting with a typical bio‐ chemical profile. In the first study of this, Molin et al. used liquid chromatography–tandem mass spectrometry to calculate the ratio of active to inactive vitamin D metabolites: Molar ratio (R) of 25-hydroxyvitamin-D3: 24,25-dihydroxyvitamin D3. A large increase in the ratio of active to inactive vitamin D metabolites, usually R > 80, was demonstrated in subjects who had biallelic mutations resulting in loss of function of *CYP24A1*. Importantly, through use of a ratio calculation, this test can avoid misleading results in patients who might have low 24,24 dihydroxyvitamin-D3 levels due to vitamin D deficiency [6].

#### **2.2.** *CYP24A1* **variants**

Several different loss-of-function mutations have now been identified within the *CYP24A1* gene. The mutations are reported to be inherited in an autosomal recessive pattern, although it is not yet clear whether partial penetrance or environmental factors may alter manifesta‐ tion of a recognised phenotype. One study showed individuals with biallelic mutations pre‐ sented with the clinically recognised phenotype and that heterozygous carriers were not sufficient to manifest clinical disease. However, it was hypothesised that infants with hap‐ loinsufficiency/heterozygous variants may be more sensitive to hypercalcaemia during childhood while the kidney is still developing, and this could become relevant in consider‐ ing additional vitamin D supplementation which might overwhelm the 1,25-hydroxyvita‐ min-D3-24-hydroxylase enzyme pathway in this cohort (**Table 2**) [4, 6].

typical biochemical pattern (suppressed PTH, hypercalcaemia, hypercalciuria) in any pa‐ tients with nephrolithiasis prompts investigation for *CYP24A1* mutations [4, 6, 8]. Establish‐ ing a molecular diagnosis in this small cohort of patients can facilitate correct treatment and

In patients with *CYP24A1* mutations, an elevation in total vitamin D levels is typically seen. In particular, 1,25-dihydroxyvitamin D3 levels are increased, but this assay is not routinely performed in many laboratories. Conversely, serum 24,24-dihydroxyvitamin-D3 levels are sometimes low or undetectable in patients with *CYP24A1* mutations. A blood test that calculates the ratio between vitamin D metabolites could be utilised in future clinical practice as a screening tool for *CYP24A1* mutations in those patients presenting with a typical bio‐ chemical profile. In the first study of this, Molin et al. used liquid chromatography–tandem mass spectrometry to calculate the ratio of active to inactive vitamin D metabolites: Molar ratio (R) of 25-hydroxyvitamin-D3: 24,25-dihydroxyvitamin D3. A large increase in the ratio of active to inactive vitamin D metabolites, usually R > 80, was demonstrated in subjects who had biallelic mutations resulting in loss of function of *CYP24A1*. Importantly, through use of a ratio calculation, this test can avoid misleading results in patients who might have low 24,24-

Several different loss-of-function mutations have now been identified within the *CYP24A1* gene. The mutations are reported to be inherited in an autosomal recessive pattern, although it is not yet clear whether partial penetrance or environmental factors may alter manifesta‐ tion of a recognised phenotype. One study showed individuals with biallelic mutations pre‐ sented with the clinically recognised phenotype and that heterozygous carriers were not

**•** ↑ 25-hydroxyvitamin D<sup>3</sup> **•** ↑ 1,25-dihydroxyvitamin D<sup>3</sup> **•** ↓ 24,25-dihydroxyvitamin D<sup>3</sup> **•** ↑ or high normal serum calcium

**•** ↑ urine calcium

**•** ↓ parathyroid hormone

lifestyle modification (**Table 1**) [9].

94 A Critical Evaluation of Vitamin D - Basic Overview

**Table 1.** Key features of *CYP24A1* mutation phenotypes.

dihydroxyvitamin-D3 levels due to vitamin D deficiency [6].

Idiopathic infantile hypercalcaemia:

**•** Vomiting **•** Dehydration **•** Failure to thrive

**•** Fever

**•** Adult presentation:

*2.1.3. Investigation*

**2.2.** *CYP24A1* **variants**

**•** Nephrolithiasis **•** Nephrocalcinosis

**Clinical features Biochemical profile**

CKD, chronic kidney disease; IIH, idiopathic infantile hypercalcaemia; PTH, parathyroid hormone.

**Table 2.** Identified mutations in *CYP24A1*.

#### **2.3. Treatment**

**Year mutation reported**

**Age at presentation**

96 A Critical Evaluation of Vitamin D - Basic Overview

2013 3 months IIH followed by adult

2014 ~20 years Nephrolithiasis,

2015 10 years Nephrolithiasis,

2015 45 years Nephrocalcinosis,

2015 32 years Nephrolithiasis,

presentation with nephrocalcinosis, CKD, hypercalcaemia and hypercalciuria

hypercalcaemia, hypercalciuria

hypercalcaemia, hypercalciuria

hypercalcaemia, hypercalciuria

nephrocalcinosis, hypercalcaemia, hypercalciuria

2015 6 months IIH Homozygous L409S Figueres et al. [19]

2015 1 day Hypercalcaemia, apnoea Heterozygous M374T Molin et al. [6]

suppressed PTH

hypercalcaemia, suppressed PTH

hypercalcaemia, suppressed PTH

hypercalcaemia, suppressed PTH

2015 28 days IIH Compound

2015 2 months IIH Compound

2015 2 months IIH Compound

2015 6 months IIH Compound

2015 3 days Infection, hypercalcaemia,

2015 13 days Small for gestational age,

2015 11 days Prematurity,

2015 4 days Prematurity,

**Phenotype** *CYP24A1* **mutation Reference**

Compound heterozygous G469Afs\*22 and P21R

Compound

heterozygous R157W and M374T

heterozygous L409S and R396W

heterozygous R396W and R396G

heterozygous delE143 and L409S

Heterozygous M374T Molin et al. [6]

Heterozygous G322A Molin et al. [6]

Heterozygous R439C Molin et al. [6]

Heterozygous M374T Molin et al. [6]

R157W

heterozygous L409S and

Homozygous W210R Meusburger et al. [14]

Homozygous delE143 Jacobs et al. [15]

Homozygous delE143 Sayers et al. [7]

Figueres et al. [19]

Figueres et al. [19]

Figueres et al. [19]

Figueres et al. [19]

Figueres et al. [19]

Figueres et al. [19]

*CYP24A1* mutations lead to calcium stone formation, and conventional treatments for calcium stones are recommended. These would include maintaining a high fluid intake and avoiding excess dietary sodium. Specific measures would include avoiding dietary vitamin D supple‐ ments (in foods and drinks) and avoidance of excessive sunlight exposure [7]. Ketoconazole was first demonstrated as an effective treatment for reducing the effects of vitamin D toxicity in patients with *CYP24A1* mutations. As a non-specific P450 enzyme inhibitor ketoconazole inhibits the enzyme catalysing production of 1,25-dihydroxyvitamin D3, (1α-hydroxylase), thereby decreasing levels of active vitamin D3. However, *CYP24A1*-deficient individuals require lifelong treatment as they will always lack the enzyme to inactivate vitamin D, and the side-effect profile of ketoconazole, which includes hepatotoxicity, hypogonadism and adrenal insufficiency, makes it unsuitable for this purpose [4]. More recently, low-dose fluconazole, also acting as a P450 enzyme inhibitor, has been shown to reduce serum calcium levels and urinary calcium excretion in a patient with *CYP24A1* mutation. It is likely that this drug, alongside lifestyle modifications such as avoiding excess sun exposure and following a low calcium and oxalate diet, will become the main treatment offered to patients diagnosed with *CYP24A1* mutations (**Figure 2**) [7, 18, 19].

**Figure 2.** Chemical structures of ketoconazole, an imidazole antifungal agent, and fluconazole, a triazole antifungal agent. Azole agents are cytochrome inhibitors primarily used as antifungal agents. They are heterocyclic ring com‐ pounds and are generally classified as either imidazoles (e.g. ketoconazole) or triazoles (e.g. fluconazole), containing two or three nitrogen atoms, respectively, in the azole ring. They exhibit their antifungal action through inhibition of lanosterol 14-α demethylase, a cytochrome P450 enzyme important for the synthesis of a fungal plasma membrane constituent.

#### **2.4. Evidence for genetic heterogeneity of idiopathic infantile hypercalcaemia**

Since the discovery of *CYP24A1* mutations underlying idiopathic infantile hypercalcaemia (IIH) in 2011, a cohort of IIH patients has been identified without *CYP24A1* mutations. In 2015, a new loss-of-function mutation in *SLC34A1*, which encodes the renal sodium–phosphate cotransporter 2A (NaPi-IIa), was recognised in this group [20]. These patients presented with a classical IIH phenotype, with symptoms of hypercalcaemia. Importantly, however, their symptoms did not resolve with removal of vitamin D supplementation. Instead, their hyper‐ calcaemia corrected rapidly after commencing phosphate replacement, highlighting the different mechanism driving the hypercalcaemia. In patients with *SLC34A1* mutations, renal phosphate wasting leads of inappropriately high levels of 1,25-dihydroxyvitamin-D3, which in turn causes hypercalcaemia. It is crucial to distinguish between patients carrying mutations in *CYP24A1* versus *SLC43A1*, as different intervention is required to successfully treat their hypercalcaemia [20]. As *SLC34A1* mutations have also been identified as a cause of nephroli‐ thiasis, there is overlap between *SLC34A1* and *CYP24A1* mutation phenotypes in both paediatric and adult presentations [21].

## **3. Conclusions**

urinary calcium excretion in a patient with *CYP24A1* mutation. It is likely that this drug, alongside lifestyle modifications such as avoiding excess sun exposure and following a low calcium and oxalate diet, will become the main treatment offered to patients diagnosed with

**Figure 2.** Chemical structures of ketoconazole, an imidazole antifungal agent, and fluconazole, a triazole antifungal agent. Azole agents are cytochrome inhibitors primarily used as antifungal agents. They are heterocyclic ring com‐ pounds and are generally classified as either imidazoles (e.g. ketoconazole) or triazoles (e.g. fluconazole), containing two or three nitrogen atoms, respectively, in the azole ring. They exhibit their antifungal action through inhibition of lanosterol 14-α demethylase, a cytochrome P450 enzyme important for the synthesis of a fungal plasma membrane

Since the discovery of *CYP24A1* mutations underlying idiopathic infantile hypercalcaemia (IIH) in 2011, a cohort of IIH patients has been identified without *CYP24A1* mutations. In 2015, a new loss-of-function mutation in *SLC34A1*, which encodes the renal sodium–phosphate cotransporter 2A (NaPi-IIa), was recognised in this group [20]. These patients presented with a classical IIH phenotype, with symptoms of hypercalcaemia. Importantly, however, their symptoms did not resolve with removal of vitamin D supplementation. Instead, their hyper‐ calcaemia corrected rapidly after commencing phosphate replacement, highlighting the different mechanism driving the hypercalcaemia. In patients with *SLC34A1* mutations, renal phosphate wasting leads of inappropriately high levels of 1,25-dihydroxyvitamin-D3, which in turn causes hypercalcaemia. It is crucial to distinguish between patients carrying mutations in *CYP24A1* versus *SLC43A1*, as different intervention is required to successfully treat their hypercalcaemia [20]. As *SLC34A1* mutations have also been identified as a cause of nephroli‐ thiasis, there is overlap between *SLC34A1* and *CYP24A1* mutation phenotypes in both

**2.4. Evidence for genetic heterogeneity of idiopathic infantile hypercalcaemia**

*CYP24A1* mutations (**Figure 2**) [7, 18, 19].

98 A Critical Evaluation of Vitamin D - Basic Overview

paediatric and adult presentations [21].

constituent.

Overall, *CYP24A1* mutations are rare and account for a small proportion of symptomatic hypercalcaemia or nephrolithiasis cases. However, a greater awareness of their phenotypes will increase clinical suspicion in patients presenting with a typical biochemical profile. Testing for mutations in *CYP24A1* can establish a definitive diagnosis, avoiding protracted further investigations and allowing treatment to commence. Alongside dietary and lifestyle advice, aimed at minimising vitamin D intake, fluconazole is proving a promising lifelong treatment to prevent effects of vitamin D toxicity.

## **Acknowledgements**

JAS is supported by the Northern Counties Kidney Research Fund.

## **Author details**

Fay Joanne Hill1 and John A. Sayer1,2\*

\*Address all correspondence to<EMAIL_ADDRESS>
1 Renal Services, Newcastle upon Tyne NHS Foundation Trust Hospitals, Newcastle upon Tyne, United Kingdom

2 Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, United King‐ dom

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100 A Critical Evaluation of Vitamin D - Basic Overview

**Synthesis of Low Abundant Vitamin D Metabolites and Assaying Their Distribution in Human Serum by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) as a New Tool for Diagnosis and Risk Prediction of Vitamin D-Related Diseases Synthesis of Low Abundant Vitamin D Metabolites and Assaying their Distribution in Human Serum by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) as a New Tool for Diagnosis and Risk Prediction of Vitamin D-Related Diseases**

Lars Kattner and Dietrich A. Volmer Lars Kattner and Dietrich A. Volmer

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/ 10.5772/64518

#### **Abstract**

This chapter provides an overview of versatile and efficient chemical syntheses of vitamin D derivatives by application of either linear or convergent synthesis ap‐ proaches. Synthesis of the most relevant naturally occurring vitamin D metabolites and their deuterated counterparts to use as calibration and reference standards in LC‐ MS/MS assays is also shown. The chapter then summarizes the most important mass spectrometric approaches to quantify important vitamin D metabolites in human biofluids. In addition, new developments are described that are aimed at the pathobio‐ logical interpretation of the measured vitamin D metabolite distributions in various human diseases.

**Keywords:** vitamin D deficiency, biomarkers, low abundant vitamin D metabolites, assay development, LC‐MS/MS, diagnosis, disease risk prediction

## **1. Introduction**

Vitamin D is mostly known for its role in the regulation of calcium and phosphorous homeo‐ stasis [1–3]. Consequently, vitamin D deficiency may cause various disorders related to bone mineralization [4]. Drugs based on vitamin D analogs are commonly used to treat bone diseases (osteoporosis, osteomalacia, and rickets) or psoriasis. More recently, it has been

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

suggested that vitamin D deficiency is also connected to a wide range of other diseases beyond bone mineralization, such as diabetes, autoimmune diseases, cardiovascular diseases, and cancer, as various clinical and epidemiological studies have shown [5–9]. However, the development of drugs for treatment of these diseases based on appropriate vitamin D analogs has mostly failed, either due to their rapid metabolic clearance or their calcemic effects. Vitamin D analogs are usually hormonally active compounds with pleiotropic functions and their levels in the body are strictly regulated by the hormonal system. In cases of oversupply, they are enzymatically degraded to avoid harmful effects such as calcemia, leading mainly to inactivation and conversion into water soluble degradation products suitable for renal clearance. Consequently, prevention of vitamin D deficiency rather than therapy of a vitamin D‐related disease is a promising approach. Due to low concentration and short half‐life of the active metabolite, 1α,25‐dihydroxyvitamin D3 (1,25(OH)2D3, calcitriol) (**4**) in serum, the biosynthetic precursor 25‐hydroxyvitamin D3 (25(OH)D3) (**3**) is usually measured as a marker of vitamin D status. Additionally, more than 50 naturally occurring but low abundant vitamin D metabolites of mostly unknown physiological function have been identified. Common assays, particularly ELISA and radio‐immunoassays (RIA), are often restricted to 25(OH)D3 because of sensitivity and specificity limitations [10–12]. Fortunately, recent mass spectrometry advances have permitted reaching deeper into the metabolic cascade of vitamin D, including low abundant species [13–17]. Consequently, a wide variety of naturally occurring metabolites of potential biological activity can be analyzed simultaneously by liquid chromatography‐ tandem mass spectrometry (LC‐MS/MS), which is currently considered the "gold standard" technique. However, this method has suffered in the past from the limited availability of the required vitamin D metabolites needed as calibration and reference standards. Consequently, as a prerequisite, all metabolites of interest have to be available for chemical synthesis, as described in this chapter.

#### **2. Metabolism of vitamin D**

Vitamin D3 (**2**) is mainly synthesized in the skin from 7‐dehydrocholesterol (**1**) by UV irradi‐ ation and subsequent thermal isomerization of the intermediate product previtamin D3. Vitamin D3 is then hydroxylated in the liver to 25(OH)D3 (**3**), followed by further oxidation in the kidneys to 1,25(OH)2D3 (calcitriol) (**4**), which is usually considered the biologically active metabolite (**Scheme 1**). Calcitriol **4** is metabolized via CYP24 to other oxidative products, mainly 1α,24(R),25‐trihydroxyvitamin D3 (1,24,25(OH)3D3) (**5**), followed by subsequent enzymatic oxidation and degradation of the carbon side chain to water soluble calcitroic acid [1, 18] for final clearance from the body. 25(OH)D3 (**3**) is degraded in an analogous manner via 24(R),25‐dihydroxyvitamin D3 (24,25(OH)2D3) **6**. Alternatively, 25(OH)D3 (**3**) and 1,25(OH)2D3 (**4**) can be metabolized to their corresponding 26,23‐lactones (**7,8**) [19]. Vitamin D metabolites also have the potential to be metabolized through a C‐3 epimerization pathway, leading to C‐3‐ *epi*‐metabolites such as **9**‐**12**, with an inversion of the stereogenic center at position C‐3 [20, 21]. These reactions lead to products that are believed to be inactive. Since high concentrations of 3‐*epi*‐25‐hydroxyvitamin D3 (3‐*epi*‐25(OH)D3) (**10**) were found in infants [22], it was initially assumed that this pathway is only a consequence of an immature vitamin D metabolism. It has also been shown, however, that the epimerization pathway is favored if, for any reason, side chain degradation by CYP24 is inhibited [23].

**Scheme 1.** Metabolic pathways of vitamin D.

suggested that vitamin D deficiency is also connected to a wide range of other diseases beyond bone mineralization, such as diabetes, autoimmune diseases, cardiovascular diseases, and cancer, as various clinical and epidemiological studies have shown [5–9]. However, the development of drugs for treatment of these diseases based on appropriate vitamin D analogs has mostly failed, either due to their rapid metabolic clearance or their calcemic effects. Vitamin D analogs are usually hormonally active compounds with pleiotropic functions and their levels in the body are strictly regulated by the hormonal system. In cases of oversupply, they are enzymatically degraded to avoid harmful effects such as calcemia, leading mainly to inactivation and conversion into water soluble degradation products suitable for renal clearance. Consequently, prevention of vitamin D deficiency rather than therapy of a vitamin D‐related disease is a promising approach. Due to low concentration and short half‐life of the active metabolite, 1α,25‐dihydroxyvitamin D3 (1,25(OH)2D3, calcitriol) (**4**) in serum, the biosynthetic precursor 25‐hydroxyvitamin D3 (25(OH)D3) (**3**) is usually measured as a marker of vitamin D status. Additionally, more than 50 naturally occurring but low abundant vitamin D metabolites of mostly unknown physiological function have been identified. Common assays, particularly ELISA and radio‐immunoassays (RIA), are often restricted to 25(OH)D3 because of sensitivity and specificity limitations [10–12]. Fortunately, recent mass spectrometry advances have permitted reaching deeper into the metabolic cascade of vitamin D, including low abundant species [13–17]. Consequently, a wide variety of naturally occurring metabolites of potential biological activity can be analyzed simultaneously by liquid chromatography‐ tandem mass spectrometry (LC‐MS/MS), which is currently considered the "gold standard" technique. However, this method has suffered in the past from the limited availability of the required vitamin D metabolites needed as calibration and reference standards. Consequently, as a prerequisite, all metabolites of interest have to be available for chemical synthesis, as

Vitamin D3 (**2**) is mainly synthesized in the skin from 7‐dehydrocholesterol (**1**) by UV irradi‐ ation and subsequent thermal isomerization of the intermediate product previtamin D3. Vitamin D3 is then hydroxylated in the liver to 25(OH)D3 (**3**), followed by further oxidation in the kidneys to 1,25(OH)2D3 (calcitriol) (**4**), which is usually considered the biologically active metabolite (**Scheme 1**). Calcitriol **4** is metabolized via CYP24 to other oxidative products, mainly 1α,24(R),25‐trihydroxyvitamin D3 (1,24,25(OH)3D3) (**5**), followed by subsequent enzymatic oxidation and degradation of the carbon side chain to water soluble calcitroic acid [1, 18] for final clearance from the body. 25(OH)D3 (**3**) is degraded in an analogous manner via 24(R),25‐dihydroxyvitamin D3 (24,25(OH)2D3) **6**. Alternatively, 25(OH)D3 (**3**) and 1,25(OH)2D3 (**4**) can be metabolized to their corresponding 26,23‐lactones (**7,8**) [19]. Vitamin D metabolites also have the potential to be metabolized through a C‐3 epimerization pathway, leading to C‐3‐ *epi*‐metabolites such as **9**‐**12**, with an inversion of the stereogenic center at position C‐3 [20, 21]. These reactions lead to products that are believed to be inactive. Since high concentrations of 3‐*epi*‐25‐hydroxyvitamin D3 (3‐*epi*‐25(OH)D3) (**10**) were found in infants [22], it was initially

described in this chapter.

**2. Metabolism of vitamin D**

104 A Critical Evaluation of Vitamin D - Basic Overview

Finally, the corresponding metabolites of vitamin D2 (**13**) also have to be considered [18], because food from plant origin (in particular mushrooms) and food supplements may contain vitamin D2. Vitamin D2 metabolites have similar physiological function as compared to their corresponding vitamin D3 analogs, although their potency is apparently lower [24]. A detailed comparison of the metabolism of vitamin D2 and D3 [18] reveals that 25(OH)D3 (**3**) and 1,25(OH)2D3 (**4**) are preferentially metabolized at their side chains, particularly at C‐23, C‐24, and C‐26, leading to metabolites susceptible to further oxidation, which is similar for vitamin D2, even though there are slight differences as a consequence of the C‐22/23 carbon double bond and a methyl group at C‐24. Thus, C‐24 hydroxylation of vitamin D2 may occur initially, leading to 24‐hydroxyvitamin D2, following by C‐1 hydroxylation in the kidneys, giving 1,24‐ dihydroxyvitamin D2, which is subsequently oxidized in the side chain. Furthermore, 1,25‐ dihydroxyvitamin D2, formed in an analogous manner to its D3 counterpart, may be metabolized by side chain oxidation either to C‐1,24,25 or C‐1,25,26 trihydroxylated vitamin D2, again followed by further side chain oxidation. Each of these both pathways again leads to calcitroic acid as final metabolite.

## **3. Synthesis of low abundant vitamin D metabolites**

The commercial unavailability of many relevant vitamin D3 and D2 species has limited the scope of LC‐MS/MS assays in the past. For use as reference standards, vitamin D3 and D2 metabolites, as well as their corresponding stable isotopes (labeled with 2 H (D) or 13C), have to be synthesized by application of a versatile and cost‐effective methodology. A large variety of chemical syntheses of vitamin D derivatives has been developed in the last few decades by several academic and commercial groups [1, 25–27], mainly with the aim of synthesizing new analogs for drug discovery and development purposes. These efforts have resulted in a large number of more than 3000 synthesized compounds [28]. Critical evaluation of these methods reveals, however, that only a few of them are suitable to reproducibly generate metabolites of interest in gram quantities at reasonable costs within short time frames. In this chapter, we will review some of the more suitable strategies that have been successfully applied and optimized in our laboratory.

The synthesis of deuterated vitamin D3 and D2 metabolites is mostly accomplished using the same procedures as those developed for nondeuterated metabolites.

The biosynthetic and technical synthesis (starting from an appropriate steroid precursor (an‐ alog to **1**) is not favorable, due to low yields reached in the photochemical ring‐opening re‐ action. It usually fails if a 1‐hydroxy substituent is present. By far the most suitable methods for synthesis of vitamin D3 and D2 metabolites start with readily available Vitamin D2 (**13**). Two alternative strategies are applied, either by keeping the vitamin D skeleton intact dur‐ ing the course of the reaction sequence (linear synthesis) (**Scheme 2**), or by first cleaving the molecule to obtain the A‐ring and the CD‐ring building block, and subsequently reconnect‐ ing both molecules after separate appropriate chemical modifications (convergent synthesis) (**Scheme 3**) [1, 26, 27, 29, 30].

#### **3.1. Linear synthesis of vitamin D metabolites**

Following a linear synthesis (**Scheme 2**), the *cis*‐diene moiety of vitamin D2 (**13**) is protected by sulfur dioxide after silylation of the hydroxyl group at C‐3 as a *tert*‐butyldimethylsilyl ether, leading to the SO2 adduct **14**. Labeling with deuterium at C‐6,19,19 can be carried out conven‐ iently by treatment with D2O or deuterated alcohol (e.g., isopropanol). Finally, thermal removal of SO2 and photochemical isomerization leads to threefold deuterated vitamin D2 (d‐**13**). This simple and convenient method to obtain C‐6,19,19 deuterated metabolites [31] can also be applied to a wide variety of other vitamin D3 and D2 derivatives. In order to obtain metabolites of the corresponding C‐3‐*epi* series of vitamin D3 or D2 metabolites, the configuration of the C‐3 OH group of vitamin D2 (**13**) can be epimerized under the so‐called "Mitsunobu conditions" dihydroxyvitamin D2, formed in an analogous manner to its D3 counterpart, may be metabolized by side chain oxidation either to C‐1,24,25 or C‐1,25,26 trihydroxylated vitamin D2, again followed by further side chain oxidation. Each of these both pathways again leads

The commercial unavailability of many relevant vitamin D3 and D2 species has limited the scope of LC‐MS/MS assays in the past. For use as reference standards, vitamin D3 and D2

to be synthesized by application of a versatile and cost‐effective methodology. A large variety of chemical syntheses of vitamin D derivatives has been developed in the last few decades by several academic and commercial groups [1, 25–27], mainly with the aim of synthesizing new analogs for drug discovery and development purposes. These efforts have resulted in a large number of more than 3000 synthesized compounds [28]. Critical evaluation of these methods reveals, however, that only a few of them are suitable to reproducibly generate metabolites of interest in gram quantities at reasonable costs within short time frames. In this chapter, we will review some of the more suitable strategies that have been successfully applied and optimized

The synthesis of deuterated vitamin D3 and D2 metabolites is mostly accomplished using the

The biosynthetic and technical synthesis (starting from an appropriate steroid precursor (an‐ alog to **1**) is not favorable, due to low yields reached in the photochemical ring‐opening re‐ action. It usually fails if a 1‐hydroxy substituent is present. By far the most suitable methods for synthesis of vitamin D3 and D2 metabolites start with readily available Vitamin D2 (**13**). Two alternative strategies are applied, either by keeping the vitamin D skeleton intact dur‐ ing the course of the reaction sequence (linear synthesis) (**Scheme 2**), or by first cleaving the molecule to obtain the A‐ring and the CD‐ring building block, and subsequently reconnect‐ ing both molecules after separate appropriate chemical modifications (convergent synthesis)

Following a linear synthesis (**Scheme 2**), the *cis*‐diene moiety of vitamin D2 (**13**) is protected by sulfur dioxide after silylation of the hydroxyl group at C‐3 as a *tert*‐butyldimethylsilyl ether, leading to the SO2 adduct **14**. Labeling with deuterium at C‐6,19,19 can be carried out conven‐ iently by treatment with D2O or deuterated alcohol (e.g., isopropanol). Finally, thermal removal of SO2 and photochemical isomerization leads to threefold deuterated vitamin D2 (d‐**13**). This simple and convenient method to obtain C‐6,19,19 deuterated metabolites [31] can also be applied to a wide variety of other vitamin D3 and D2 derivatives. In order to obtain metabolites of the corresponding C‐3‐*epi* series of vitamin D3 or D2 metabolites, the configuration of the C‐3 OH group of vitamin D2 (**13**) can be epimerized under the so‐called "Mitsunobu conditions"

H (D) or 13C), have

to calcitroic acid as final metabolite.

106 A Critical Evaluation of Vitamin D - Basic Overview

in our laboratory.

(**Scheme 3**) [1, 26, 27, 29, 30].

**3.1. Linear synthesis of vitamin D metabolites**

**3. Synthesis of low abundant vitamin D metabolites**

metabolites, as well as their corresponding stable isotopes (labeled with 2

same procedures as those developed for nondeuterated metabolites.

[32], by treatment with an aromatic acid and an azodicarboxylate, resulting in the correspond‐ ing ester **15** with concomitant inversion of the stereogenic center at C‐3 (orientation of the substituent has changed from β to α). The ester is finally cleaved by saponification with potassium hydroxide or by reduction with lithium aluminum hydride to afford C‐3‐*epi* vitamin D2 (**16**), which can be used to synthesize a wide variety of C‐3 epimers, including 3‐ *epi*‐25‐hydroxyvitamin D3 (3‐*epi*‐25(OH)D3) (**10**, d‐**10**) and 3‐*epi*‐1α,25‐dihydroxyvitamin D3 (3‐ *epi*‐1,25(OH)2D3) (**11**, d‐**11**). Yields are only moderate due to the favored formation of a C‐3,4‐5,6‐7,8 all‐*trans* triene system by elimination. To further proceed with the synthesis, the C‐22/23 double bond in the side chain of SO2 adduct **14** is cleaved by ozonolysis, leading to alcohol **17** after reductive workup with sodium boronhydride, which can be used to add a wide variety of modified side chains as appropriate. For instance, **17** can be converted to an iodide, which is coupled in a nickel‐zinc‐mediated reaction with ethyl acrylate after prior removal of the SO2 protective group to afford **18**. At this stage, an allylic oxidation by selenium dioxide can be carried out, leading to a 1α‐hydroxy series of metabolites, such as **19** as appropriate precursor. Silylation of the newly generated hydroxyl group, followed by Grignard reaction with methylmagnesium bromide, leads to 1α,25‐dihydroxy metabolites. If a deuterated Grignard reagent, labeled with 3 deuterium atoms at their methyl groups, is employed, the corresponding sixfold deuterium labeled metabolite is obtained, containing 3 deuterium atoms at C‐26 and C‐27, respectively. Finally, the C‐5/6 double bond has to be isomerized photochemically, and the silyl protective groups have to be removed, ending up with nondeuterated or deuterated 25(OH)D3 (**3**, d‐**3**) or 1,25(OH)2D3 (**4**, d‐**4**) [33], as shown in **Scheme 2**. If 3‐*epi*‐vitamin D2 (**16**) is employed in this sequence, 3‐*epi*‐25(OH)2D3 (**10**, d‐**10**) is obtained. 3‐*epi*‐1,25(OH)2D3 (**11**, d‐**11**) is obtained, if 1α‐hydroxylation by selenium dioxide was carried out, and the resulting C‐1‐OH‐group is epimerized by oxidation and final reduction.

Vitamin D2 metabolites are synthesized by application of analogous strategies as applied for their corresponding D3 counterparts, although they are more challenging to synthesize, for two reasons: vitamin D2 metabolites contain an olefinic double bond in C‐22/23‐position, which has to be arranged in *trans* geometry during the course of a suitable coupling reaction of a CD‐ ring precursor and an alternated side chain building block by olefination. Most synthetic olefination methods yield a mixture of *cis* and *trans* double bonds, leading to product mixtures that are difficult to separate due to their similar polarity. If the reaction conditions are too basic, C‐20 epimerization can also occur, leading to even more complex product mixtures. Addi‐ tionally, vitamin D2 metabolites contain a chiral methyl group in C‐24 position that has to be ranged in a defined configuration. When a suitable building block bearing this methyl group is employed in the synthesis, epimerization of the chiral center may occur under basic reaction conditions, along with the risk for epimerization at C‐20 in the coupling reaction. Thus, alcohol **17** is converted to its corresponding phenylsulfone, which is coupled by olefination with aldehyde **20**, a method originally invented by Julia [34]. Note that **20** can be synthesized in a few routine steps from commercially available (S)‐(+)‐3‐hydroxyisobutyric acid methyl ester, which is submitted to a Grignard reaction with methyl‐magnesium bromide. Analogously to the synthesis of the other sixfold deuterated metabolites labeled at C‐26/27, the addition of deuterated Grignard reagent in the course of the reaction leads to sixfold deuterated vitamin D2 metabolites. Finally, the double bond at C‐5/6 of **21** has to be photochemically isomerized and the silyl protective groups have to be removed, leading to nondeuterated or deuterated 25(OH)D2 (**22**, d‐**22**) [35].

**Scheme 2.** Linear synthesis of vitamin D metabolites. TBDMSCl: *tert*‐butyldimethylsilyl chloride, SO2: sulfur dioxide, O3: ozone, NaBH4: sodium borohydride, *i*PrOD: deuterated isopropanol, Bu4NF: tetra‐*n*‐butylammonium fluoride, NaHCO3: sodium hydrogen carbonate, TsCl: *p*‐toluenesulfonyl chloride, PhSH: thiophenol, MCPBA: 3‐chloroperben‐ zoic acid, I2: iodine, PCC: pyridinium chlorochromate, D3CMgBr: deuterated methylmagnesium bromide, SeO2: seleni‐ um dioxide, DIAD: diisopropyl azodicarboxylate, PPh3: triphenylphosphine, KOH: potassium hydroxide, LiAlH4: lithium aluminium hydride.

#### **3.2. Convergent synthesis of vitamin D metabolites**

Alternatively, a convergent approach can be applied (**Scheme 3**), which is more versatile than a linear synthesis and allows for more harsh reaction conditions and wider scope of suitable substrates and reagents. In a classical and widely applied strategy, vitamin D2 (**13**) is submitted to ozonolysis, leading to Inhoffen‐Lythgoe diol **23** and allylic alcohol **24** after reductive workup with sodium boronhydride. After appropriate chemical modification of the CD‐ring precursor, this compound is coupled with an A‐ring building block. Two strategies can be distinguished. In the most common approach, invented by Lythgoe and further developed by the Hoffmann‐ La Roche group [36], allylic alcohol **24** is converted via an allylic chloride **25** in diphenylphos‐ phine oxide **26**, which is coupled with a C‐8 ketone by a Wittig‐Horner olefination. The A‐ring diphenylphosphine oxide **26** can be coupled before or after appropriate alteration of the side chain at the CD‐ring building block. To employ an A‐ring diphenylphosphine oxide such as **26** is favourable, if no further substituents except a C‐3 OH group and a C‐10 *exo*‐methylene group in the A‐ring are needed, as it is already present in vitamin D2 (**13**). As example for an efficient application of this strategy, the synthesis of 24,25(OH)2D3 (**6**) and its deuterated counterpart (d‐**6**), their 3‐*epi*‐analogs (**9**/d‐**9**) and 1α‐analogs (**5**/d‐**5**), is shown in **Scheme 3** [37]. Here, diol **23** is converted to its corresponding iodide, which is coupled with enone **27 (**obtained in a few routine steps from (R)‐(‐)‐lactic acid using a zinc‐copper‐catalyzed reaction mediated by ultrasound, leading to **28**. By Grignard reaction of **28** with nondeuterated or deuterated methyl‐magnesium bromide, **29** or d‐**29** are obtained. The 1,2‐diol moiety is protected as isopropylidene ether, followed by oxidation with pyridinium chlorochromate to obtain ketone **30**/d‐**30**, which can be coupled with diphenylphosphine oxide **26** to obtain d‐24,25(OH)2D3 (d‐**6**) after removal of silyl‐ and isopropylidene protective groups. To access the corresponding C‐3‐*epi*‐analog, vitamin D2 is first bis‐hydroxylated at C‐7/8 using potassium permanganate. Inversion of the stereogenic center at C‐3 under "Mitsunobu‐conditions" using picolinic acid leads to the corresponding C‐3‐*epi*‐triol **31**. The yields of this reaction are much higher as compared to the synthesis of **16** used in the linear synthesis, because an elimination reaction leading to a favored C‐3,4‐5,6‐7,8‐all‐*trans* system is avoided [38]. Silylation and cleavage of the diol moiety of triol **31** by lead tetraacetate and reductive workup with sodium boronhy‐ dride lead to the CD‐ring fragment **32** and C‐3‐*epi* allylic alcohol **33**, which can be used after conversion to the corresponding diphenylphosphine oxide **34**. Note that **34** is coupled to d‐**30** to obtain d‐3‐*epi*‐24,25(OH)2D3 (d‐**9**) or its corresponding nondeuterated counterpart **9**. 3‐*epi*‐ diphenylphosphine oxide **34** can be used as a versatile A‐ring building block to be employed

D2 metabolites. Finally, the double bond at C‐5/6 of **21** has to be photochemically isomerized and the silyl protective groups have to be removed, leading to nondeuterated or deuterated

**Scheme 2.** Linear synthesis of vitamin D metabolites. TBDMSCl: *tert*‐butyldimethylsilyl chloride, SO2: sulfur dioxide, O3: ozone, NaBH4: sodium borohydride, *i*PrOD: deuterated isopropanol, Bu4NF: tetra‐*n*‐butylammonium fluoride, NaHCO3: sodium hydrogen carbonate, TsCl: *p*‐toluenesulfonyl chloride, PhSH: thiophenol, MCPBA: 3‐chloroperben‐ zoic acid, I2: iodine, PCC: pyridinium chlorochromate, D3CMgBr: deuterated methylmagnesium bromide, SeO2: seleni‐ um dioxide, DIAD: diisopropyl azodicarboxylate, PPh3: triphenylphosphine, KOH: potassium hydroxide, LiAlH4:

Alternatively, a convergent approach can be applied (**Scheme 3**), which is more versatile than a linear synthesis and allows for more harsh reaction conditions and wider scope of suitable substrates and reagents. In a classical and widely applied strategy, vitamin D2 (**13**) is submitted to ozonolysis, leading to Inhoffen‐Lythgoe diol **23** and allylic alcohol **24** after reductive workup with sodium boronhydride. After appropriate chemical modification of the CD‐ring precursor, this compound is coupled with an A‐ring building block. Two strategies can be distinguished. In the most common approach, invented by Lythgoe and further developed by the Hoffmann‐ La Roche group [36], allylic alcohol **24** is converted via an allylic chloride **25** in diphenylphos‐ phine oxide **26**, which is coupled with a C‐8 ketone by a Wittig‐Horner olefination. The A‐ring diphenylphosphine oxide **26** can be coupled before or after appropriate alteration of the side

25(OH)D2 (**22**, d‐**22**) [35].

108 A Critical Evaluation of Vitamin D - Basic Overview

lithium aluminium hydride.

**3.2. Convergent synthesis of vitamin D metabolites**

**Scheme 3.** Convergent synthesis of 24,25(OH)xD metabolites. TBDMSCl: *tert*‐butyldimethylsilyl chloride, O3: ozone, NaBH4: sodium borohydride, I2: iodine, Cu: copper, Zn: zinc, D3CMgBr: deuterated methylmagnesium bromide, Me2CO: acetone, PCC: pyridinium chlorochromate, KMnO4: potassium permanganate, DIAD: diisopropyl azodicar‐ boxylate, NCS: N‐chlorosuccinimide, LiPPh2: lithium diphenylphosphide, H2O2: hydrogen peroxide. BuLi: *n*‐butyllithi‐ um, Bu4NF: tetra‐*n*‐butylammonium fluoride, Pb(OAc)4: lead tetraacetate, SeO2: selenium dioxide.

in a Wittig-Horner reaction for the synthesis of a wide variety of 3-*epi* vitamin D3 and D2 metabolites, by connection with appropriate CD-ring building blocks with modified side chain, as shown for the synthesis of 3-*epi*-24,25(OH)2D3 (**9**, d-**9**) as representative example. Finally, permanganate oxidation of vitamin D2 (**13**) leads to triol **35**, which is converted in a few routine steps, including a stereoselective 1α-allylic oxidation with SeO2 via **36** to diphenylphosphine oxide **37**, which is then coupled with d-**30** to afford 1,24,25(OH)3D3 (**5**/d-**5**).

Two other low abundant metabolites, 23(S),25(R)-dihydroxyvitamin D3 26,23-lactone **7** and 1α-1,23(S),25(R)-trihydroxyvitamin D3 26,23-lactone **8**, are synthesized in an analogous manner [29] (**Scheme 4**), by coupling a CD-ring ketone carrying an appropriate side chain with diphenylphosphine oxide **26** or **37**. Here CD-ring-building block **32** obtained from vitamin D2 **13** is used, which is converted to aldehyde **38**. In order to prepare an appropriate modified side chain, isopentenol **39** is converted using a few routine steps to sulfone **40**, which is then coupled with **38** by a Julia olefination to afford **41**. Removal of the isopropylidene protective group, oxidation to an unsaturated α-hydroxy carboxylic acid and subsequent iodolactonization leads to ketone **44** via **42** and **43**, which is coupled with diphenylphosphine oxide **26** or **37**, respectively, to give **7** and **8**.

**Scheme 4.** Convergent synthesis of 23,26(OH)x-lactone metabolites. TBDMSCl: *tert*-butyldimethylsilyl chloride, O3: ozone, LDA: lithium diisopropylamide, Na: sodium, Hg: mercury, PPTS: pyridinium *p*-toluenesulfonate, DMSO: dimethyl sulfoxide, Py: pyridine, SO2: sulfur dioxide, I2: iodine, KOH: potassium hydroxide, Bu3SnH: tributyltin hydride, Bu4NF: tetra-*n*-butylammonium fluoride, PCC: pyridinium chlorochromate, BuLi: *n*-butyllithium.

#### **3.3. Convergent Palladium-catalyzed synthesis of vitamin D metabolites**

The A-ring building block is preferably synthesized *de novo*, if further substituents apart from a C-3-OH group and a C-10 exo methylene group in the A-ring (already present in vitamin D2) are needed. This is particularly valuable for synthetic analogs containing substituents at C-2, which were developed in the past as new drugs [30]. However, natural metabolites may also contain additional substituents in the A‐ring, such as 4β,25‐dihydroxy vitamin D3 [39]. In these cases, Pd catalyzed tandem reactions can be applied (**Scheme 5**). Initially invented by Trost [40], an acyclic enyne **46** is coupled with a CD‐ring vinyl bromide **47** obtained by Peterson olefination from the corresponding ketone. Closure of the A‐ring and its connection with CD‐ ring is preferably carried out in one pot. Alternatively, the corresponding triflate **48** can be employed in a Zn‐mediated reaction, which was invented by Negishi [41]. Additionally, CD‐ ring alkene boranates **49** can be used via an analogous coupling method originally invented by Suzuki [42]. Finally, a cyclic A‐ring enynes, such as **50**, can be coupled with CD‐ring triflate **50**, a coupling method invented by Sonogashira [43]. In all these cases, the CD‐ring vinyl bromide **47**, boranate **49**, or triflate **51** are usually carrying the modified side chain already, before coupling with the acyclic or cyclic A‐ring enyne. A major drawback of these methods is the fact that synthesis of acyclic or cyclic enynes or other A‐ring building blocks not derived from vitamin D2 involves many synthesis steps, and in some cases separation of diastereomeric mixtures is also necessary to obtain enantiomerically and diastereomerically pure product. However, the suitable starting materials can usually be obtained from the natural chiral pool (i.e., terpenes such as carvone [44], malic acid [45], quinic acid [43], or carbon hydrates such as D‐glucose [46] or D‐xylose [47]), which are usually readily available. A wide range of substrates are tolerated. Consequently, these advanced strategies involving Pd catalyzed reactions have widely been applied in recent years and are by now well established.

in a Wittig-Horner reaction for the synthesis of a wide variety of 3-*epi* vitamin D3 and D2 metabolites, by connection with appropriate CD-ring building blocks with modified side chain, as shown for the synthesis of 3-*epi*-24,25(OH)2D3 (**9**, d-**9**) as representative example. Finally, permanganate oxidation of vitamin D2 (**13**) leads to triol **35**, which is converted in a few routine steps, including a stereoselective 1α-allylic oxidation with SeO2 via **36** to diphenylphosphine

Two other low abundant metabolites, 23(S),25(R)-dihydroxyvitamin D3 26,23-lactone **7** and 1α-1,23(S),25(R)-trihydroxyvitamin D3 26,23-lactone **8**, are synthesized in an analogous manner [29] (**Scheme 4**), by coupling a CD-ring ketone carrying an appropriate side chain with diphenylphosphine oxide **26** or **37**. Here CD-ring-building block **32** obtained from vitamin D2 **13** is used, which is converted to aldehyde **38**. In order to prepare an appropriate modified side chain, isopentenol **39** is converted using a few routine steps to sulfone **40**, which is then coupled with **38** by a Julia olefination to afford **41**. Removal of the isopropylidene protective group, oxidation to an unsaturated α-hydroxy carboxylic acid and subsequent iodolactonization leads to ketone **44** via **42** and **43**, which is coupled with diphenylphosphine oxide **26** or

**Scheme 4.** Convergent synthesis of 23,26(OH)x-lactone metabolites. TBDMSCl: *tert*-butyldimethylsilyl chloride, O3: ozone, LDA: lithium diisopropylamide, Na: sodium, Hg: mercury, PPTS: pyridinium *p*-toluenesulfonate, DMSO: dimethyl sulfoxide, Py: pyridine, SO2: sulfur dioxide, I2: iodine, KOH: potassium hydroxide, Bu3SnH: tributyltin hydride,

The A-ring building block is preferably synthesized *de novo*, if further substituents apart from a C-3-OH group and a C-10 exo methylene group in the A-ring (already present in vitamin D2) are needed. This is particularly valuable for synthetic analogs containing substituents at C-2, which were developed in the past as new drugs [30]. However, natural metabolites may

Bu4NF: tetra-*n*-butylammonium fluoride, PCC: pyridinium chlorochromate, BuLi: *n*-butyllithium.

**3.3. Convergent Palladium-catalyzed synthesis of vitamin D metabolites**

oxide **37**, which is then coupled with d-**30** to afford 1,24,25(OH)3D3 (**5**/d-**5**).

**37**, respectively, to give **7** and **8**.

110 A Critical Evaluation of Vitamin D - Basic Overview

**Scheme 5.** Convergent Pd‐catalyzed synthesis of vitamin D metabolites via *de novo*‐A‐ring synthesis. Pd: palladium, ZnCl2: zinc chloride.

## **4. LC-MS/MS assays for vitamin D metabolites**

In the second part of this chapter, an overview of the mass spectrometric analysis of vitamin D metabolites in biological samples is presented. Because of the chemical nature of these secosteroidal molecules, liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) currently provides the optimum analytical platform for analysis of vitamin D metabolites. While mass spectrometry assays are often not as rugged and more expensive than most non‐ mass spectrometric assays (in particular immunoassays), they provide the ability to capture multiple metabolites simultaneously at very low concentration levels. Immunoassays measure the 25(OH)D3 and 25(OH)D2 metabolites only and have limitations with respect to detection sensitivity as well as selectivity and specificity issues.

In most published assays, the metabolites are determined from serum or plasma matrices. Other biological matrices have rarely been used, although vitamin D metabolites have successfully been quantified in saliva [48] and various soft tissues [49]. In the following sections, we discuss the requirements and characteristics of LC‐MS/MS of vitamin D metabo‐ lites; that is, chromatographic separation, ionization, *m/z* analysis, interferences and accuracy issues, multimetabolite screening applications, and the clinical role of vitamin D fingerprinting methods.

#### **4.1. Liquid chromatographic separation of vitamin D metabolites**

Due to the hydrophobic structure, vitamin D metabolites are generally easily separated on reversed‐phase liquid chromatography stationary phases (e.g., octadecyl C18) materials utilizing hydrophobic interactions. The vitamin D metabolites generally elute in the order trihydroxylated < dihydroxylated < monohydroxylated metabolites, that is, for vitamin D3‐ related molecules, the order of chromatographic retention is 1,25(OH)2D3 < 25(OH)D3 < D3. Between corresponding D2 and D3 analogs, the D2 metabolites elute marginally later than the D3 versions. For the two important isomers of dihydroxylated vitamin D metabolites, 24,25(OH)2D3 and 1,25(OH)2D3, the order of retention is 24,25(OH)2D3 < 1,25(OH)2D3.

Possibly more interesting—from a chromatography point of view—are the biochemically formed isomers after stereochemical reversal (β→α) at C‐3; for example, the epimers 25(OH)D3 and 3‐*epi*‐25(OH)D3. The subtle diastereomeric differences between these species can be distinguished using specialized columns, such as combined C18/chiral [50], pentafluor‐ ophenyl (PFP) [51–53], and cyano (CN) [54–57].

#### **4.2. Ionization of vitamin D metabolites for mass spectrometric analysis**

Mass spectrometric determination of vitamin D metabolites using liquid chromatography‐ mass spectrometry (LC‐MS) is not trivial because of the structural limitations that the analytes provide with respect to attaching a charge to the molecules. Gas chromatography‐mass spectrometry techniques have been used in the past for qualitative analysis and structure determinations, but these methods have been almost completely replaced by modern LC‐MS

methods. While analysis of transformation products such as vitamin D sulfates is relatively easy by LC‐MS as the metabolites can be simply analyzed as deprotonated molecules, ionizing the relatively nonpolar vitamin D metabolites is not straightforward. For LC‐MS, electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) are the most common ionization techniques, which usually result in the formation of [M+H]+ ions for most biological molecules. APCI has been successfully applied to steroids, as gas‐phase chemical ionization often efficiently transfers a proton to these types of molecules. ESI usually relies on a charging mechanism in the liquid phase and one would therefore expect ESI to be less efficient than APCI for vitamin D metabolites. In reality this is not the case, however, and both techniques have equally been applied to vitamin D analysis, with the analytical figures of merit being quite similar between the two ionization techniques. There appears to be a slight trend toward application of ESI rather than APCI in recent years.

**4. LC-MS/MS assays for vitamin D metabolites**

112 A Critical Evaluation of Vitamin D - Basic Overview

sensitivity as well as selectivity and specificity issues.

ophenyl (PFP) [51–53], and cyano (CN) [54–57].

**4.1. Liquid chromatographic separation of vitamin D metabolites**

methods.

In the second part of this chapter, an overview of the mass spectrometric analysis of vitamin D metabolites in biological samples is presented. Because of the chemical nature of these secosteroidal molecules, liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) currently provides the optimum analytical platform for analysis of vitamin D metabolites. While mass spectrometry assays are often not as rugged and more expensive than most non‐ mass spectrometric assays (in particular immunoassays), they provide the ability to capture multiple metabolites simultaneously at very low concentration levels. Immunoassays measure the 25(OH)D3 and 25(OH)D2 metabolites only and have limitations with respect to detection

In most published assays, the metabolites are determined from serum or plasma matrices. Other biological matrices have rarely been used, although vitamin D metabolites have successfully been quantified in saliva [48] and various soft tissues [49]. In the following sections, we discuss the requirements and characteristics of LC‐MS/MS of vitamin D metabo‐ lites; that is, chromatographic separation, ionization, *m/z* analysis, interferences and accuracy issues, multimetabolite screening applications, and the clinical role of vitamin D fingerprinting

Due to the hydrophobic structure, vitamin D metabolites are generally easily separated on reversed‐phase liquid chromatography stationary phases (e.g., octadecyl C18) materials utilizing hydrophobic interactions. The vitamin D metabolites generally elute in the order trihydroxylated < dihydroxylated < monohydroxylated metabolites, that is, for vitamin D3‐ related molecules, the order of chromatographic retention is 1,25(OH)2D3 < 25(OH)D3 < D3. Between corresponding D2 and D3 analogs, the D2 metabolites elute marginally later than the D3 versions. For the two important isomers of dihydroxylated vitamin D metabolites,

24,25(OH)2D3 and 1,25(OH)2D3, the order of retention is 24,25(OH)2D3 < 1,25(OH)2D3.

**4.2. Ionization of vitamin D metabolites for mass spectrometric analysis**

Possibly more interesting—from a chromatography point of view—are the biochemically formed isomers after stereochemical reversal (β→α) at C‐3; for example, the epimers 25(OH)D3 and 3‐*epi*‐25(OH)D3. The subtle diastereomeric differences between these species can be distinguished using specialized columns, such as combined C18/chiral [50], pentafluor‐

Mass spectrometric determination of vitamin D metabolites using liquid chromatography‐ mass spectrometry (LC‐MS) is not trivial because of the structural limitations that the analytes provide with respect to attaching a charge to the molecules. Gas chromatography‐mass spectrometry techniques have been used in the past for qualitative analysis and structure determinations, but these methods have been almost completely replaced by modern LC‐MS An important, often neglected consideration in the choice of ionization source is the (usually detrimental) impact of coionized sample components that coelute with vitamin D metabolites from the LC column. Here, ionization suppression effects caused by these coeluting molecules may impact the outcome even stronger than the differences seen in ionization efficiencies between ESI and APCI [58, 59]. However, no systematic comparison of ionization efficiency/ ionization suppression effects between ESI and APCI has been performed yet. Furthermore, coionization of coeluting components also leads to the formation of isobaric interferences when sample matrices such as plasma or serum are analyzed, which will be discussed in more detail below.

Another useful ionization technique is atmospheric pressure photoionization (APPI). This technique is very common in fields such as petroleum analysis, but surprisingly, it is virtually unknown in the clinical community. To our knowledge, only one study has systematically compared APPI with APCI and shown that APPI generated significantly higher ion currents for 25(OH)D3 than APCI [60]. A second study applied APPI to quantification of 25(OH)D3 without comparison to ESI or APCI [61].

Finally, another option to overcome problems of ionization efficiency and resulting problems with detection sensitivity is derivatization of vitamin D metabolites, to convert them into better responding transformation products. Such procedures are now quite common in the vitamin D analytical field; they comprise introduction of a chemical group that is readily ionized or is permanently charged. Cookson‐type triazoline‐diones and triazoline‐dione‐related reagents (e.g., 4‐phenyl‐1,2,4‐triazoline‐3,5‐dione [PTAD]) are most often applied, which utilize the reaction of the reagent's dienophile with the *cis*‐diene group at C‐5/C‐6 and C‐10/C‐19 of vitamin D (Diels‐Alder [4+2] reaction) (**Scheme 6**).

Several promising PTAD assays have been described in the literature [39, 49, 62–64]. The advantage of using the *cis*‐diene moiety is that all vitamin D metabolites contained in the sample are converted when the reagent is present at sufficient concentrations (i.e., in large excess quantities) as all relevant vitamin D metabolites possess this structural motif. Ionization efficiency of 25(OH)D‐PTAD has been reported to be 100‐fold higher than the nonderivatized molecules in ESI mode [65]. A structural variation in PTAD includes a permanently charged quaternary ammonium moiety, which enhances detection sensitivity even further [66]. This reagent is commercially available. A recent chemotyping assay for multiple vitamin D metabolites has shown detection limits in the fg/mL range for vitamin D metabolites from human serum (see discussion below).

**Scheme 6.** Diels‐Alder [4+2] derivatization of vitamin D metabolites using PTAD.

#### **4.3 Mass analyzers for vitamin D analysis**

Most clinical applications for vitamin D utilize quadrupole‐based mass analyzers with low resolving powers. Because these mass spectrometers measure *m/z* signals at "unit mass resolution," they cannot be operated in full scan mode for the accurate quantification of vitamin metabolites when complex sample matrices such as serum are analyzed. Serum contains multiple components of identical nominal molecular mass to vitamin D metabolites, several of which have been shown to coelute during High Performance Liquid Chromatography (HPLC) [67]. The coeluting metabolites then generate so‐called "isobaric" ions in the mass spectra after ESI, which cannot be distinguished from the vitamin D target molecules using low resolution MS. Therefore, tandem mass spectrometry (MS/MS) is implemented, which activates the ionized species further and forces them to undergo collision‐induced dissociation (CID) for generating structure‐specific product ions. The metabolite‐specific product ions are then measured and their response used for quantification of the target metabolites.

By far the most common low resolution tandem mass spectrometer for vitamin D analysis in clinical laboratories is the triple quadrupole (QqQ) instrument. In a QqQ mass spectrometer, the [M+H]+ ions of vitamin D metabolites are selected in the first quadrupole, fragmented in a quadrupole collision cell, and the products analyzed in the final quadrupole mass analyzer. The acquisition mode that is almost always applied is the so‐called selected reaction monitor‐ ing (SRM) mode. In this mode, the [M+H]+ precursor ion is isolated in Q1 (e.g., *m/z* 401 for 25(OH)D3), dissociated in q2, and one specific product ion selected in Q3 (e.g., *m/z* 383). The quality of the precursor/product ion selection determines the selectivity of analysis; that is, the product ion structure should ideally directly reflect the chemical structure of the chosen precursor ion for maximum specificity. For vitamin D metabolites, the selection of an appro‐ priate product ion is surprisingly difficult because of some inherent structural limitation of the precursor structure, leading primarily to simple dehydration reactions at low collision energies, which turn into complex mass spectra at higher collision energies (**Figure 1**) [68]. Unfortunately, because of multiple, overlapping series of fragmentation reactions, leading to "picket fence" type product ion spectra, fragment ions for vitamin D metabolites in the diagnostic *m/z* range often resemble each other.

reagent is commercially available. A recent chemotyping assay for multiple vitamin D metabolites has shown detection limits in the fg/mL range for vitamin D metabolites from

Most clinical applications for vitamin D utilize quadrupole‐based mass analyzers with low resolving powers. Because these mass spectrometers measure *m/z* signals at "unit mass resolution," they cannot be operated in full scan mode for the accurate quantification of vitamin metabolites when complex sample matrices such as serum are analyzed. Serum contains multiple components of identical nominal molecular mass to vitamin D metabolites, several of which have been shown to coelute during High Performance Liquid Chromatography (HPLC) [67]. The coeluting metabolites then generate so‐called "isobaric" ions in the mass spectra after ESI, which cannot be distinguished from the vitamin D target molecules using low resolution MS. Therefore, tandem mass spectrometry (MS/MS) is implemented, which activates the ionized species further and forces them to undergo collision‐induced dissociation (CID) for generating structure‐specific product ions. The metabolite‐specific product ions are

then measured and their response used for quantification of the target metabolites.

By far the most common low resolution tandem mass spectrometer for vitamin D analysis in clinical laboratories is the triple quadrupole (QqQ) instrument. In a QqQ mass spectrometer,

quadrupole collision cell, and the products analyzed in the final quadrupole mass analyzer. The acquisition mode that is almost always applied is the so‐called selected reaction monitor‐

25(OH)D3), dissociated in q2, and one specific product ion selected in Q3 (e.g., *m/z* 383). The quality of the precursor/product ion selection determines the selectivity of analysis; that is, the product ion structure should ideally directly reflect the chemical structure of the chosen precursor ion for maximum specificity. For vitamin D metabolites, the selection of an appro‐ priate product ion is surprisingly difficult because of some inherent structural limitation of the precursor structure, leading primarily to simple dehydration reactions at low collision energies, which turn into complex mass spectra at higher collision energies (**Figure 1**) [68].

ions of vitamin D metabolites are selected in the first quadrupole, fragmented in a

precursor ion is isolated in Q1 (e.g., *m/z* 401 for

human serum (see discussion below).

114 A Critical Evaluation of Vitamin D - Basic Overview

**4.3 Mass analyzers for vitamin D analysis**

ing (SRM) mode. In this mode, the [M+H]+

the [M+H]+

**Scheme 6.** Diels‐Alder [4+2] derivatization of vitamin D metabolites using PTAD.

**Figure 1.** Collision‐induced dissociation (CID) spectrum of the [M+H]+ ion of 1,25(OH)2D3 (spectrum after fully com‐ pleted dehydration reactions after electrospray ionization and CID; *m/z* analysis by Fourier‐transform ion cyclotron resonance, FTICR).

A few studies also report the use of a low resolution quadrupole ion trap (IT) [69, 70] in MS/MS mode for 25(OH)D3 and 25(OH)D2, but these instruments are rarely implemented in clinical environments. IT instruments are often not fit for purpose for quantitation of low abundant biological molecules, because of the large detrimental contributions of acquisition time overhead to the duty cycle, thus limiting detection sensitivity. There is also a general decline of this instrument type in modern mass spectrometry labs.

The most anticipated future development for vitamin D analysis is the introduction of high resolution mass spectrometry (HRMS), which is now firmly established in many other fields of modern mass spectrometry, in particular, in pharmaceutical applications and the proteomics field [71]. This trend is mostly due to the availability of robust quadrupole‐quadrupole‐time‐ of‐flight (QqTOF) and orbitrap mass analyzers in recent years, which have transformed many analytical approaches to mass spectrometry. The use of mass defect as metabolite‐specific property, for example, is now an integral part of many metabolite identification routines for drug metabolites [72]. While the number of applications of HRMS in the vitamin D field is still limited, the existing work has clearly demonstrated the potential of HRMS for vitamin D analysis. For example, orbitrap mass spectrometers in full scan [73, 74] and MS/MS [75, 76] modes have been applied successfully to the analysis of 25(OH)D3 in human serum and analytical performance has been shown to be equivalent or better than triple quadrupole and immunoassays. The important role of HRMS in the separation of isobaric interferences will be shown below.

#### **4.4. Interferences during LC-MS/MS**

The LC‐MS/MS analysis of vitamin D metabolites is affected by various sources of error, which can affect both precision and accuracy. As with any other LC‐MS/MS analysis from biological samples, ion suppression by coeluting sample components or chemical modifiers from the sample preparation or chromatography can lead to reduced analyte signals. There are several options to assess whether or not ion suppression is present, which have been summarized in many review articles, e.g., by Matuszewski et al. [77]. Stable isotope standards of vitamin D metabolites (mostly deuterated analogs) can usually correct for accuracy errors from ion suppression effects, as long as it is guaranteed that protein binding for the isotope standard is the same as for the endogenous analyte, which requires implementation of a careful incubation routine [78]. Importantly, deuterated isotope standards are commercially available for most of the relevant vitamin D metabolites but unfortunately not for all.

A second important source of analytical error originates from isobaric noise; that is, endoge‐ nous or exogenous metabolites that coelute with the vitamin D analytes and erroneously contribute to the analytical signal, if unspecific ions are used for mass spectral analysis. This has recently been described in detail by Qi et al. [67], who clearly demonstrated the presence of multiple isobars of 25(OH)D3 in human serum. The isobars have to be carefully removed in low resolution mass spectrometry, by application of appropriate MS/MS or ion mobility spectrometry routines [67]. A number of exogenous and endogenous molecules have been identified as relevant metabolites in serum samples [50, 67, 79].

Many of these interferences can be eliminated by application of high resolution mass spec‐ trometry using sufficiently high resolving powers; for example, through implementation of orbitrap or Fourier‐transform ion cyclotron resonance (FTICR) mass spectrometers. For example, Liebisch and Matysik demonstrated that the orbitrap MS instrument in their study was able to separate an isobaric interference of 25(OH)D3 in the MS/MS domain; this interfer‐ ence was caused by fragmentation of the d6‐25(OH)D2 isotope standard [75].

Importantly, the issues relating to isobaric noise have only been studied for the 25(OH)D3 analyte; other metabolites will likely be affected by similar interferences, the impact of which during quantification, in particular, in multimetabolite assays (see below), remains unknown.

#### **4.5. Method accuracy and certified reference materials**

The vitamin D analytical community is supported through the Vitamin D External Quality Assessment Scheme (DEQAS), a nonprofit organization that evaluates the performance of analytical assays of member laboratories for 25(OH)D3 and 1,25(OH)2D3 [80] through round‐ robin analyses. DEQAS has clearly demonstrated that analytical performance of vitamin D analysis has greatly improved over the years between 1994 and 2009 [81].

The United States National Institute of Standards and Technology (NIST) provides certified standard solutions for 25(OH)D3 and 25(OH)D2. Furthermore, the NIST and the National Institutes of Health (NIH) Office of Dietary Supplements (ODS) have established the Vitamin D Metabolites Quality Assurance Program (VitDQAP), for interlaboratory comparison of measurement of 25(OH)D2, 25(OH)D3, and 3‐*epi*‐25(OH)D3 in serum and plasma and provide method‐appropriate control materials.

A number of commercial reference, calibration, and quality control materials are available from several companies that allow rapid implementation and validation of vitamin D analytical methodologies.

#### **4.6. Vitamin D multimetabolite assays**

modes have been applied successfully to the analysis of 25(OH)D3 in human serum and analytical performance has been shown to be equivalent or better than triple quadrupole and immunoassays. The important role of HRMS in the separation of isobaric interferences will be

The LC‐MS/MS analysis of vitamin D metabolites is affected by various sources of error, which can affect both precision and accuracy. As with any other LC‐MS/MS analysis from biological samples, ion suppression by coeluting sample components or chemical modifiers from the sample preparation or chromatography can lead to reduced analyte signals. There are several options to assess whether or not ion suppression is present, which have been summarized in many review articles, e.g., by Matuszewski et al. [77]. Stable isotope standards of vitamin D metabolites (mostly deuterated analogs) can usually correct for accuracy errors from ion suppression effects, as long as it is guaranteed that protein binding for the isotope standard is the same as for the endogenous analyte, which requires implementation of a careful incubation routine [78]. Importantly, deuterated isotope standards are commercially available for most of

A second important source of analytical error originates from isobaric noise; that is, endoge‐ nous or exogenous metabolites that coelute with the vitamin D analytes and erroneously contribute to the analytical signal, if unspecific ions are used for mass spectral analysis. This has recently been described in detail by Qi et al. [67], who clearly demonstrated the presence of multiple isobars of 25(OH)D3 in human serum. The isobars have to be carefully removed in low resolution mass spectrometry, by application of appropriate MS/MS or ion mobility spectrometry routines [67]. A number of exogenous and endogenous molecules have been

Many of these interferences can be eliminated by application of high resolution mass spec‐ trometry using sufficiently high resolving powers; for example, through implementation of orbitrap or Fourier‐transform ion cyclotron resonance (FTICR) mass spectrometers. For example, Liebisch and Matysik demonstrated that the orbitrap MS instrument in their study was able to separate an isobaric interference of 25(OH)D3 in the MS/MS domain; this interfer‐

Importantly, the issues relating to isobaric noise have only been studied for the 25(OH)D3 analyte; other metabolites will likely be affected by similar interferences, the impact of which during quantification, in particular, in multimetabolite assays (see below), remains unknown.

The vitamin D analytical community is supported through the Vitamin D External Quality Assessment Scheme (DEQAS), a nonprofit organization that evaluates the performance of analytical assays of member laboratories for 25(OH)D3 and 1,25(OH)2D3 [80] through round‐ robin analyses. DEQAS has clearly demonstrated that analytical performance of vitamin D

shown below.

**4.4. Interferences during LC-MS/MS**

116 A Critical Evaluation of Vitamin D - Basic Overview

the relevant vitamin D metabolites but unfortunately not for all.

identified as relevant metabolites in serum samples [50, 67, 79].

**4.5. Method accuracy and certified reference materials**

ence was caused by fragmentation of the d6‐25(OH)D2 isotope standard [75].

analysis has greatly improved over the years between 1994 and 2009 [81].

One of the most important advantages of LC‐MS/MS assays over clinical immunoassays is the ability to determine multiple vitamin D species independently and simultaneously. As a result, there are now several very capable LC‐MS/MS assays described in the literature that provide the capability for profiling the most relevant vitamin D metabolites at the same time, within a single analytical run, and with sufficient dynamic range to allow measuring the required physiological levels, down to the picomolar range. The topic has recently been reviewed in detail and the interested reader is referred to Ref. [82]. Briefly, most of these multimetabolite assays utilize derivatization techniques that transform all vitamin D species into better responding analogs (vide supra). This procedure in turn permits analysis of both high and low abundant vitamin D species with similar analytical figures of merit [62, 83].

The simultaneous acquisition of all important metabolite levels then provides the possibility of using these vitamin D metabolite distributions ("chemotypes") as complex diagnostic or prognostic biomarkers for correlation with disease phenotype or clinical outcome of treatment. A few examples for such correlations are summarized in the last section.

## **5. Vitamin D fingerprints (chemotypes) in clinical applications**

A number of recent studies have gone beyond the usual determination of 25(OH)D3—as marker for vitamin D status [84]—and 1,25(OH)2D3—for diagnosis of renal diseases, hyper‐ calcemic syndromes, and disorders of 25(OH)D3 metabolism [85]. These broader profiling techniques are aimed at discovering dynamic effects of metabolites and catabolites, which are located further downstream the 25(OH)D3 metabolic cascade. Current studies highlight 24,25(OH)2D3 as important diagnostic marker, which was previously only considered a clearance product of vitamin D without activity. In fact, it has been shown that 24,25(OH)2D3 has crucial roles in bone metabolism [86] and renal diseases [87].

Capturing multiple vitamin D species and their dynamic changes allows for a better under‐ standing of interindividual variations after vitamin D supplementation. Müller et al. recently demonstrated an inverse linear correlation between baseline 25(OH)D3 and response to supplementation for patients with chronic liver disease [83]. The study also showed that lower baseline 24,25(OH)2D3 levels were linked to larger changes of 25(OH)D3 levels, and that those patients who exhibited greater response to vitamin D supplementation had lower levels of 3‐ *epi*‐25(OH)D3 (**Figure 2**).

Berg et al. implemented the ratio of 24,25(OH)2D3 and 25(OH)D3 as a novel status marker for vitamin D [88]. Wagner et al. used the same ratio and demonstrated that it was predictive of 25(OH)D3 response to supplementation [89]. Binkley et al. measured multiple vitamin D species and developed a model to describe interindividual variation of 25(OH)D3 levels after supplementation [90]; the authors highlighted the role of absorption (as measured by the nonmetabolized vitamin D3 species) and degradation (via the 24,25(OH)2D3 species) and presented a treat‐to‐target regime for tailored serum levels of 25(OH)D3 [90].

**Figure 2.** Nonparametric correlations between baseline vitamin D metabolites and response to vitamin D supplementa‐ tion for patients with chronic liver diseases after 6‐month treatment: (a) the relative change in serum 25(OH)D3 (in re‐ sponse to vitamin D supplementation) correlated inversely with baseline 25(OH)D3 concentrations; (b) similarly, an inverse correlation between relative change in serum 25(OH)D3 and baseline 24,25(OH)2D3 was observed; (c) baseline 24,25(OH)2D3 concentrations were nonsignificantly higher in women as compared to men; (d) patients with lower 3‐ epi‐25(OH)D3 concentrations at baseline tended to have a larger response to vitamin D supplementation (reprinted with permission from Ref. [83]).

Other important studies include the work by Bosworth et al. [87] and Stubs et al. [91], who utilized multimetabolite LC‐MS/MS methods to characterize chronic kidney disease (CKD). Similarly, Duan et al. [64] studied patients with multiple sclerosis and observed comparable levels of 25(OH)D3 in the healthy control subjects and the patients; however, serum levels for 1,25(OH)2D3 and 24,25(OH)2D3 were lower in patients than controls.

## **6. Conclusions**

baseline 24,25(OH)2D3 levels were linked to larger changes of 25(OH)D3 levels, and that those patients who exhibited greater response to vitamin D supplementation had lower levels of 3‐

Berg et al. implemented the ratio of 24,25(OH)2D3 and 25(OH)D3 as a novel status marker for vitamin D [88]. Wagner et al. used the same ratio and demonstrated that it was predictive of 25(OH)D3 response to supplementation [89]. Binkley et al. measured multiple vitamin D species and developed a model to describe interindividual variation of 25(OH)D3 levels after supplementation [90]; the authors highlighted the role of absorption (as measured by the nonmetabolized vitamin D3 species) and degradation (via the 24,25(OH)2D3 species) and

**Figure 2.** Nonparametric correlations between baseline vitamin D metabolites and response to vitamin D supplementa‐ tion for patients with chronic liver diseases after 6‐month treatment: (a) the relative change in serum 25(OH)D3 (in re‐ sponse to vitamin D supplementation) correlated inversely with baseline 25(OH)D3 concentrations; (b) similarly, an inverse correlation between relative change in serum 25(OH)D3 and baseline 24,25(OH)2D3 was observed; (c) baseline 24,25(OH)2D3 concentrations were nonsignificantly higher in women as compared to men; (d) patients with lower 3‐ epi‐25(OH)D3 concentrations at baseline tended to have a larger response to vitamin D supplementation (reprinted

Other important studies include the work by Bosworth et al. [87] and Stubs et al. [91], who utilized multimetabolite LC‐MS/MS methods to characterize chronic kidney disease (CKD). Similarly, Duan et al. [64] studied patients with multiple sclerosis and observed comparable

presented a treat‐to‐target regime for tailored serum levels of 25(OH)D3 [90].

*epi*‐25(OH)D3 (**Figure 2**).

118 A Critical Evaluation of Vitamin D - Basic Overview

with permission from Ref. [83]).