Vitamin D, is mainly produced in the skin by sun exposure. On exposure to ultraviolet B (∼290–315 nm) sun-rays, 7-dehydrocholesterol (pro-vitamin D3) in the skin is converted to pre-vitamin D3 and then thermally isomerizes to vitamin D3 to enter the circulation. Vitamin D3 binds with vitamin D binding protein in circulation and is transported to the liver where it is converted into 25-hydroxyvitamin D (25OHD). Hydroxylation at position 25 of vitamin D is mainly carried out by microsomal 25-hydroxylase coded primarily by CYP2R1 [1]. 25OHD is the most abundant circulating form of vitamin D and also, the most reliable marker of vitamin D status [2]. 25OHD is converted later in the kidney to 1,25(OH)2 vitamin D [1,25(OH)2D] (the most active form of vitamin D) by 1α-hydroxylase coded by CYP27B1. Hydroxylation at position 24 of 25OHD and 1,25(OH)2D to 24,25(OH)2D and 1,24,25(OH)3D, respectively, is the major pathway for the inactivation of vitamin D and is mediated by 24-hydroxylase coded by CYP24A1. A minor role in vitamin D inactivation is played by CYP3A4 which converts 25OHD and 1,25(OH)2D to 4β,25(OH)2D and 1,23,25(OH)3D, respectively [3] (Fig. 1). 1,25(OH)2D acts on VDR, a receptor that belongs to the steroid/thyroid/retinoid nuclear hormone receptor family and has two functional domains: ligand-binding domain (LBD) and DNA-binding domain (DBD) [4]. Ligand binding facilitates its heterodimerization with RXR which translocates to the nucleus and binds to the vitamin D response element (VDRE), a specific DNA sequence on the promoter region of vitamin D regulated genes [5].

Dietary phosphorus is absorbed in the small intestine, mostly duodenum and jejunum (Fig. 2). Intestinal phosphate absorption is mediated by at least two distinct pathways: transcellular and paracellular. Passive paracellular phosphate transport via Na+ /H+ exchanger 3 (NHE3, SLC9A3) contributes for the majority (65–80%) of intestinal phosphate absorption whereas transcellular phosphate absorption contributes to the rest and mediated by type II sodium dependent phosphate transporter 2b (NaPi-IIb, SLC34A2). The latter mechanism is regulated by vitamin D which makes ∼30% of phosphate absorption vitamin D dependent. Low serum phosphorus level leads to increased synthesis of 1,25 dihydroxy vitamin D, independent of PTH levels, by transcriptional up regulation of the 1α-hydroxylase gene [6], [7]. It appears that the stimulating impact of phosphorus on 1,25(OH)2D production depends on an intact GH/IGF-1 axis [7]. Interestingly, calcium-phosphate regulation by vitamin D is mostly via its genomic actions. However, there are a few evidences to support its nongenomic action in intestinal phosphorus absorption via protein disulfide isomerase family A member 3 (PDIA3) [8].

Vitamin D does not seem to have a direct role in the regulation of renal phosphorus handling. However, it may have an indirect effect via increase in FGF23 which may reduce phosphate reabsorption that may slightly offset positive effect of vitamin D on serum phosphorus levels [9], *[10](Fig. 2). 1,25(OH)2D may also a play role in phosphate reabsorption from the bone [11]. 1,25(OH)2D primarily acts on osteoblasts which in-turn stimulate the osteoclasts to bring about bone resorption[12]. This increased osteoclastic activity is also observed in thyro-parathyroidectomised rats suggesting a PTH-independent role for 1,25(OH)2D in bone resorption [6].

Vitamin D deficiency is associated with increased PTH levels which in turn causes phosphaturia and hypophosphatemia. Reduced renal phosphate reabsorption in the presence of elevated PTH is mediated by down regulation of NaPi-IIa at the brush border membrane of epithelial cells of proximal convoluted tubule. The mechanisms of PTH-induced down regulation of NaPi-IIa is depicted in Fig. 3. Apical expression of NaPi-IIa is dependent on its three carboxy-terminal amino acids (tryptophan, arginine, leucine, TRL). The TRL sequence is essential to interact with PSD-95, Discs-large and ZO-1 (PDZ) proteins like Na+/H+ exchanger regulatory factors NHERF1 (EBP50) and NHERF2 (E3KARP) etc. PTH binds to its apical and basolateral receptors in the proximal tubule. Stimulation of apical receptors preferentially activates the phospholipase C/protein kinase C (PKC) pathway, whereas that of basolateral receptors activates cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signalling. The differential action on apical and basolateral PTH receptors may be due to presence of NHERF in the former. Nevertheless, activation of both receptors uses common downstream effectors viz. extracellular signal regulated protein kinase (ERK) 1/2. ERK signalling leads to internalisation of NaPi-IIa in clathrin-coated pits and subsequent transportation to endosomes in clathrin-coated vesicles. Endocytosed NaPi-IIa are degraded in lysosomes via a microtubule-dependent step [10].

The global median (95% CI) prevalence rates of serum 25OHD level of < 30, < 50, and < 75 nmol/L are 15.7% (13.7–17.8), 47.9% (44.9–50.9), and 76·6% (74.0–79.1), respectively. Although a slight decrease in the prevalence of hypovitaminosis D was noted during 2011–2022 than that during 2000–2012, the prevalence still remains high [13]. The prevalence of hypovitaminosis D is further higher during winter-spring, in females, and people living in high-altitude areas, lower-middle income countries and Eastern-Mediterranean countries[13]. Common causes and predisposing factors for vitamin D deficiency are listed in Table 1.

A study including children from United Kingdom showed a significant association of low 25OHD with serum phosphate (p = 0.004)[14]. In contrast, a large out-patient based study (n = 23,134) from Germany (r = 0.01, p = 0.529) [15] and a hospital registry-based study from Turkey (r = 0.016, p-0.62)[16] reported no significant correlation between serum phosphorus and 25OHD levels. We also found no significant correlation between serum phosphorus and 25OHD levels (r = −0.006, p = 0.83) in the extended analysis of a previously published community-based study from India by our group [17]. In a study from Iran, positive correlation with serum phosphorus and 25OHD was limited to patients with stroke who had significantly lower serum 25OHD and serum phosphorus than the control group [18]. This may indicate the positive association between serum phosphorus and 25OHD levels only in population with relative vitamin D deficiency [18]. Intriguingly, in patients with chronic kidney disease, a negative association between serum phosphorus and 25OHD levels is more consistently reported [19], [20], [21].

The nutritional rickets/osteomalacia evolves gradually over a few months to years. The stage I is characterised by a transient decrease in serum calcium associated with modest elevation of serum alkaline phosphatase (Table 2). Mostly, this phase is unrecognised and progresses to stage II which is the most common stage at presentation of nutritional rickets/osteomalacia. Stage II and stage IIII are characterised by hypophosphatemia which plays a major role in the defective mineralization in nutritional rickets.

A greater role for phosphorus in defective mineralization was evidenced by its occurrence even in patients with phosphopenic disorders (X-linked hypophosphatemic rickets). Interestingly, calcium deprivation during neonatal period and early infancy usually does not manifest as rickets as prolonged hypophosphatemia due to secondary hyperparathyroidism needs to prevail before radiological signs of rickets become apparent [22]. Bone mineralization in the growth plate is initiated by a cascade of events triggered by apoptosis of hypertrophic chondrocytes. Availability of phosphate is required for the caspase 9-mediated apoptosis of hypertrophic chondrocytes; therefore, hypophosphatemia forms the basis of both phosphopenic and calcipenic rickets [23], [24].

Hypophosphatemia in vitamin D deficiency can be more severe than primary hyperparathyroidism, as reduced intestinal phosphorus absorption also contributes to hypophosphatemia in the former [9]. Notably, serum phosphorus may be normal in 1/4th to 1/3rd of children with nutritional rickets from different countries[14], [25], *[26]. Interestingly, hypophosphatemia is further less pronounced in children with calcium deficiency rickets from Nigeria [27], *[28]. A subset of individuals with vitamin D deficiency may have hyperphosphatemia, mostly in association with renal insufficiency. However, when hyperphosphatemia is noted in individuals with normal renal function, it may result from vitamin D deficiency-related resistance to the action of PTH (pseudohypoparathyroidism type 2).

There are several published cases of vitamin D deficiency mimicking or misdiagnosed as pseudohypoparathyroidism type 2. Most of these had hypocalcemia with one or more of the hypocalcemic manifestations, elevated serum phosphate and PTH with severe vitamin D deficiency. Interestingly, vitamin D treatment normalised serum calcium and reduced both serum phosphorus and PTH levels in the majority*[29], [30], [31], [32]. Hence, a trial of vitamin D supplementation is prudent in patients with severe vitamin D deficiency but biochemical features of pseudohypoparathyroidism.

Except for X-linked hypophosphatemic osteomalacia, muscle weakness is a common feature of most forms of osteomalacia including nutritional osteomalacia and more common than in primary hyperparathyroidism [33]. Indeed, nutritional osteomalacia is still a common cause of proximal myopathy in several countries [34], [35]. All living cells use phosphate as a key substrate for their metabolism. In skeletal muscle, phosphate is stored as organic phosphorus, primarily in the form of adenosine triphosphate (ATP) and phosphoryl creatinine. The Pit1 and Pit2 transporters control the free intracellular inorganic phosphorus (Pi), which is approximately 1–2 mg/dL (3–5 mmol) in myocytes [36]. This amount of phosphorus in muscle cells is necessary to maintain creatinine phosphate stores and the effectiveness of ATP as an energy source for muscular mechanical action. Low blood Pi concentrations may lead to impaired muscle ATP generation [37], which may explain some of the muscle weakness and myopathy observed in hypophosphatemic rickets patients. Notably, some patients with nutritional osteomalacia have normal serum phosphorus [38], [39], [40]. This may suggest either individualised serum phosphate thresholds to cause nutritional osteomalacia or a role of factors other than serum phosphorus like direct effects of vitamin D deficiency and PTH [41], [42], [43], [44]. Elevated PTH impairs the activity of mitochondrial and myofibrillar creatinine phosphokinase and ATPases leading to impaired energy production, transfer, and utilisation [41]. Several studies have reported an association of elevated PTH, independent of vitamin D status, with adverse effects on muscle strength, and postural stability; however, analyses whether the association is independent of serum phosphorus are unavailable [42], [45], [46]. A direct role for vitamin D in myopathy has also been recently proposed as vitamin D deficiency may lead to oxidative stress and muscle atrophy [44], [47].

Treatment with vitamin D, either daily or stoss regimen, increases serum phosphorus by 4–8 days with peak levels between 2–4 weeks and concomitant increase in TMP/GFR and decrease in PTH [48], [49], [50]. Interestingly, rise in serum phosphorus levels after vitamin D replacement is most often exaggerated that may be sustained for up to 4–6 months [50].

Increase in serum phosphorus is considered the earliest biochemical response to stoss therapy [51]. Some authors suggest that lack of normalisation of serum phosphorus by a week after vitamin D replacement may suggest nonresponse to vitamin D [48]. However, in a recent study from India, stoss therapy failed to normalise serum phosphorus in 17% and 9% of rachitic children by 3 and 6 weeks after therapy, respectively, but normalised serum phosphorus in all by 3 months [25]. Also, the peak serum phosphorus levels attained later i.e., at 3–6 months that sustained till 12 months [25]. In another study from Qatar, stoss therapy normalised serum phosphorus in all rachitic children by 1 month. Change in serum phosphorus after stoss therapy correlated positively with change in serum calcium (r = 0.256) and negatively with changes in serum alkaline phosphatase (r = 0.276) and PTH (r = 0.366). Surprisingly, change in serum phosphorus did not correlate with change in serum 25OHD (r = 0.067) [26].

The biological effect of vitamin D is mediated mainly by 1,25(OH)2D (calcitriol). Conversion of 25OHD to 1,25(OH)2D is catalysed by 1α-hydroxylase (CYP27B1) in the kidney. Biallelic mutations in the gene encoding 1α-hydroxylase (CYP27B1, mapped on chromosome 12q13) cause deficiency of 1,25(OH)2D and result in VDDR1A (pseudo-vitamin D deficiency). VDDR1A is inherited as an autosomal recessive condition. The common presenting symptoms are deformities, delayed motor development, hypotonia, failure to thrive, or hypocalcemic seizures [52], *[53], [54]. Typical laboratory results include hypocalcaemia, hypophosphatemia, increased serum levels of alkaline phosphatase (ALP) and PTH, and low or low-normal levels of 1,25(OH)2D [55] (Table 3). In a recent systematic review by our group, the median serum phosphorus level in VDDR1A probands was 3.8 (2.4–4.99) mg/dl, hypophosphatemia being observed in only ∼three-fourth of patients [53]. However, it is not clear whether presence of hypophosphatemia indicates a more severe disease. In the systematic review, serum phosphorus levels did not differ between VDDR1A probands patients with truncating and nontruncating variants [53]. However, a later study reported milder hypophosphatemia [− 2.4 (−3.5 to −1.5) vs. − 3.6 (−7.3 to −1.1) in patients with milder disease (p.Aal129Thr), though statistically insignificant [56].

VDDR1A is a well-known condition to be misdiagnosed initially, most commonly as nutritional rickets but also as distal renal tubular acidosis, or hypophosphatemic rickets. Recently, misdiagnosis of VDDR1A as normocalcemic primary hyperparathyroidism was also reported [57]. More interestingly, despite hypophosphatemia being a usual finding, serum phosphorus may sometimes be elevated leading to misdiagnosis as pseudohypoparathyroidism [58], [59]. A low 1,25(OH)2D in patients with calcipenic (elevated PTH) osteomalacia/rickets that is nonresponsive to standard doses of vitamin D, particularly in the presence of norma-high serum 25OHD is characteristic of VDDR1A. However, in some patients, 1,25(OH)2D may be normal or even modestly elevated. The latter observation is more often observed in patients with high-normal to elevated 25OHD. In such scenarios, a lower ratio of 1,25(OH)2D to 25OHD (0.31) may enhance the diagnostic sensitivity. However, a normal serum 1,25(OH)2D level was also noted in a few VDDR1A patients with hypovitaminosis D *[53], [55], [59] which cautions a careful interpretation and consideration of genetic testing to attain the accurate diagnosis.

The usual dose of calcitriol is ∼45 [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49], [50], [51], [52], *[53], [54], [55], [56], [57], [58], [59], [60] ng/kg/day [53]. Latency periods of ∼5 months and ∼1 year for normalisation of serum calcium and alkaline phosphatase, respectively, were observed in the recent French study [56], whereas in another study from China, serum ALP and PTH normalised by 3 and 6 months after therapy [60]. There are limited studies that emphasise on the normalisation of serum phosphorus with therapy in VDDR1A. Serum phosphorus was 0.87 ± 0.23 mmol/L at diagnosis (2.1 ± 0.8 years of age) and increased after therapy to 1.29 ± 0.30 at the last visit (8.2 ± 4.7 years of age). Notably, serum phosphorus remained low in ∼half of the patients which was associated with persistent elevation of PTH indicating inadequate therapy in them [61].

Vitamin D has short half-life (<1–2 days) whereas 25OHD (calcifediol) has a longer half-life (2–3 weeks) which makes it is the most abundant circulating for of vitamin D. 25-hydroxylation of vitamin D is predominantly mediated by CYP2R1 and inactivating mutations in CYP2R1 that reduce the function of 25-hydroxylase, are the major cause of VDDR1B. It is a relatively rare disorder with only around 50 cases reported to date. However, the condition is probably underdiagnosed as it mimics the nutritional vitamin D deficiency (pseudo nutritional vitamin D deficiency). This is an autosomal semidominant condition with the disease manifesting both in monoallelic and biallelic variants, albeit with less severe manifestations among those with monoallelic variants [62].

The biochemical evaluation mimics vitamin D deficiency characterised by decreased serum 25OHD, hypocalcemia, hypophosphatemia and elevated PTH. However, reduced responsiveness to standard vitamin D dosages distinguishes the condition from vitamin D deficiency. A high index of suspicion is required to diagnose the condition. Patients requiring continuous therapy with high doses of vitamin D to maintain 25OHD levels should be considered for genetic testing of CYP2R1.

VDDR1B can be managed in multiple ways. Physiological dosages of calcitriol or pharmacological doses of ergocalciferol or cholecalciferol combined with additional calcium is a typical strategy. Calcifediol is available in some countries and is an improved method to treat VDDR1B as it avoids the 25-hydroxylation flaw [67].

VDDR2A, also called hereditary Vitamin D resistant rickets (HVDRR), is a rare disorder caused by end-organ resistance to the action of vitamin D and is inherited by autosomal recessive pattern. VDDR2A is usually due to biallelic pathogenic variants in the VDR gene that encodes for vitamin D receptor (VDR) and is located on chromosome 12q13.11. Patients with VDDR2A typically present with calcipenic rickets in early life with disease onset at 19 [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24] months of age. Around half of the patients may have growth failure at presentation whereas dental abnormalities (enamel hypoplasia) and recurrent pneumonia may have been in 10–15% of patients. Alopecia is a characteristic feature of HVDDR and observed in four-fifths of patients.[63] Alopecia was considered a marker of poor response to oral calcium and calcitriol [68]. However, in a recent review, we have reported no association between alopecia and response to therapy [63].

Typical biochemical characteristics of HVDDR include hypocalcemia, hypophosphatemia, elevated ALP, high PTH, normal/low levels of 25OHD and elevated 1,25(OH)2D (Table 3). The latter parameter differentiates it from VDDR1A. In the absence of alopecia, VDDR2A closely mimics calcium deficiency rickets that is common in some underdeveloped and developing countries. Similar to the above-discussed forms of VDDR, hypophosphatemia may be absent in a proportion of patients. In a recent systematic review, serum phosphorus in VDDR2A probands was 03 (2.4–3.6) mg/dl with hypophosphatemia in only ∼58% of patients. Serum phosphorus levels did not differ in VDDR2A probands with and without alopecia [3.3(2.5–3.8) vs. 2.7 (2.3–3.8) mg/dl] or among patients with ligand binding domain (LBD)-truncating [2.9 (2.5–3.4) mg/dl], LBD-non truncating [3.2 (2.5–3.7) mg/dl], DNA binding domain (DBD)-truncating [2.7 (2.2–3.5) mg/dl] and DBD-non truncating [3.3 (2.5–3.9) mg/dl] variants [63].

The usual treatment regimen is high doses of oral calcium and calcitriol. In refractory cases, intravenous calcium infusion for a few weeks to achieve radiological healing of rickets is helpful [63]. There is limited data on the serum phosphorus response to therapy in VDDR2A. Serum phosphorus levels in VDDR2A patients with and without compliance to therapy were comparable, though PTH was significantly higher in patients with poor compliance [23].

This is a less well-defined condition which mimics clinical and biochemical characteristics of VDDR type 2 but without recognizable abnormalities in the VDR gene. In 1993, a patient with typical clinical and biochemical features (including hypophosphatemia) was reported without molecular abnormalities in the VDR gene. The VDR from this patient was able to bind calcitriol but failed to show nuclear localization and hence, no functional response to calcitriol. In expression studies, the patient’s VDR showed normal transactivation in the presence of calcitriol suggesting a functionally normal VDR [69]. Later, an extended study by the group demonstrated interference by the constitutively overexpressed heterogeneous nuclear ribonucleoprotein (hnRNP) with binding of a normally functioning VDR-retinoid X receptor (RXR) dimer to the vitamin D response element (VDRE) [70]. Giraldo et al. reported more than 200 rachitic children with biochemical characteristics of VDDR2 but normal VDR sequence who were diagnosed to have VDDR2B [64]. These rachitic children had higher phosphorus levels than normal children despite higher PTH and was associated with lower urinary phosphorus excretion whereas therapy with high dose calcitriol and calcium phosphate resulted in no change in serum phosphorus levels but increased urinary phosphorus excretion. The phosphorus metabolism characteristics in these children were intriguing and further studies that replicate them are lacking.

VDDR3 is an autosomal dominant condition where vitamin D deficiency is caused by accelerated vitamin D metabolism. It is caused due to a gain-of-function mutation in CYP3A4, which encodes a P450 enzyme. This is a very rare condition with only three cases being reported to date. Interestingly, all the three patients were from unrelated families but had the same mutation (c .902 T > C, p.Iso301Thr) in homozygous state. Notably, the mutated protein with this variant has no impact on the catabolism of other substrates. The condition is characterised by early-onset rickets, decreased serum levels of calcium, phosphorus, 25OHD, 1,25(OH)2D and inadequate responsiveness to both 25OHD and calcitriol [66]. Elevation of both 1,25(OH)2D and 25OHD on day 3 after vitamin D administration (150,000 IU) in VDDR3 is comparable to that in children with nutritional rickets but levels become significantly lower by 7 days [65]. The disease can be managed with supraphysiological doses of either vitamin D (10,000–50,000 IU per day) or calcitriol *[65], [66]. Serum phosphorus normalisation is reported after adequate therapy [66].