Revised vitamin D copy
Sources and forms of vitamin D
Vitamin D, also termed calciferol, is a fat-soluble secosteroid compound that is an essential regulatory factor for calcium and phosphate metabolism in humans and animals. Its biological functions involve a physiological action in bone formation and mineralization, muscle contraction, nerve signal modulation and transmission as well as many cellular metabolic effects in various organs. There are two forms of vitamin D that are metabolically important; vitamin D2 or ergocalciferol and vitamin D3 or cholecalciferol. The nutritional sources of both forms are limited to certain types of foods that naturally contain vitamin D and therefore it is added to some foods as a supplement. 1.1.1- Exogenous (Diet) Both forms of vitamin D (D2 and D3) are exogenously obtained in low quantities from some types of food in the diet. Vitamin D2 is rare as it is produced from fungal and plant sources such as mushrooms and cereals, as a result of irradiation, by ultraviolet photons, of the plant sterol ergosterol. When these foods are ingested, ergocalciferol is absorbed into the blood. Vitamin D3 , on the other hand, is available in very low amounts from animal sources including oily fish such as salmon and mackerel; other sources include meat, liver, cheese, cod liver oil, eggs and fortified foods such as margarine and milk (Holick, 2006; Engelsen et al., 2005; Nowson et al., 2004). Farmed salmon, for example, contains only 25% of the vitamin D levels present in wild salmon, however, the amount of vitamin D in canned food may affected by modern processing methods (Chen et al., 2007). 1.1.2- Endogenous In humans the principal precursor of vitamin D3 is cholesterol which is obtained from the diet. Cholesterol is initially converted to 7-dehydrocholesterol, provitamin D3, through the action of enzymes termed the mucosal dehydrogenase complex, present in the small intestine. Provitamin D3, is then incorporated within chylomicrons and transported to the skin where temperature dependent photoisomerisation processing of 7-dehydrocholesterol takes place in epidermal cells resulting in the production of D3. Within the epidermal cells, vitamin D3 undergoes photocoversion to its isomers 5,6-transvitamin D3 and suprasterol, a process which relies on the amount of ultraviolet radiation absorbed; inadequate sunlight exposure compromises this process (Holick, 2003; Iqbal, 1994). Sunlight exposure is therefore a crucial element in the regulation and enhancement of endogenous cholecalciferol production (Dusso, et al., 2005; Iqbal, 1994; Reichel, et al., 1989; Smith, 1988). Once photoconversion is completed, cholecalciferol binds to Vitamin D Binding Protein (VDBP) and transported to the liver for further metabolic processing.
Vitamin D metabolism
Both forms of vitamin D (D2 and D3) undergo similar metabolic activation in the liver and kidney respectively to produce the physiologically active form 1,25-dihydroxyvitamin D3. 1.2.1- Skin The skin is characterized by two layers, the outer epidermal region, consisting of several strata, and the inner dermal layer. Skin exposure to UVB rays in sunlight, characterized by a wavelength of 290 nm to 315 nm, allows the initial steps of vitamin D synthesis to occur using the substrate 7-dehydrocholesterol (7-DHC) as illustrated in step 1 of Figure 1. UVB absorption by 7-DHC is thought to occur actively in the stratum basale and stratum spinosum regions of the epidermal layer. The substrate 7-DHC is an important intermediate of cholesteryl ester biosynthesis from squalene. During the reaction, 7-DHC forms procholecalciferol through B ring opening of the steroid structure. This transition state is relatively unstable and can further undergo photocatalyzed reactions to form lumisterol and tachysterol (Wolpowitz and Gilchrest, 2006). Lumisterol and tachysterol have been shown to prevent vitamin D reaching intoxicating levels and do not have any direct vitamin D effects (Bouillon et al., 1998). In addition to this protective mechanism, previtamin D poisoning is also prevented because this is an equilibrium reaction that allows cholecalciferol to revert back to 7-DHC (Webb, 2006). Cholecalciferol (previtamin D3) is produced upon double bond rearrangement of procholecalciferol and remains in the extracellular space where it becomes bound to the ubiquitous VDBP (Holick, 2005). Figure1. Sources and steps of vitamin D synthesis in the three major sites: skin, liver and kidney (Figure obtained from Wolpowitz and Gilchrest, 2006). 1.2.2- Liver Cholecalciferol that has been transported to the liver undergoes the first step of its bioactivation, the hydroxylation of carbon 25 (Dusso, et al., 2005) by two hepatic enzymes; the microsomal and mitochomdrial 25-hydroxylases (Deluca et al., 1990). In hepatic cellular microsomes and mitochondria, vitamin D3 is hydroxylated at carbon 25 and transformed to 25-hydroxyvitamin D3 by both 25-hydroxylase enzymes. This enzyme complex requires the presence of essential catalytic cofactors including nicotinamide adenine dinucleotide phosphate (NAPDH), flavin adenine dinucleotide (FAD), ferredoxin and molecular oxygen for this reaction to proceed (Sahota and Hosking, 1999; Ohyama et al., 1997; Kumar, 1990). Recently, large numbers of hepatic cytochrome P-450 enzymes exhibiting 25-hydroxylase action have been identified in vitamin D activation pathways; these enzymes include CYP27A1, CYP3A4, CYP2D25 and CYP2R1 (Dusso, et al., 2005; Cheng et al., 2003; Sawada et al., 2000). However, CYP2R1 is believed to be the principal enzyme in the hepatic pathway and the presence of a genetic mutation in its gene may compromise the outcome of this process; both CYP27A1 and CYP2D25 demonstrate high capacity and low affinity features, therefore, their activity is considered insignificant in this pathway (Dusso, et al., 2005; Cheng et al., 2003; Sawada et al., 2000). This metabolic step is inefficiently regulated, i.e. the levels of 25-hydroxy vitamin D are elevated as dietary intake of vitamin D increases. Consequently, over 95% of 25-hydroxyvitamin D in serum circulates as 25-hydroxyvitamin D3 which has a half-life of approximately three weeks, and is therefore used in the assessment of vitamin D status (Dusso, et al., 2005; Reichel et al., 1989). The metabolically inert 25-hydroxyvitamin D3 is then transported to the kidney for the second step of its bioactivation. 1.2.3- Kidney The second step of vitamin D3 bioactivation takes place at the proximal convoluted tubule of the kidney. Hydroxylation occurs at C-1 of 25-hydroxyvitamin D3 whereby the highly active 25-hydroxyvitamin D3 1-?-hydroxylase (CYP27B1) incorporates a hydroxyl group to Carbon-1 of the first ring to form the biologically active metabolite 1,25-dihydroxyvitamin D3 (Holick,2006; Dusso, et al., 2005; Deluca et al, 1990; Reichel, et al., 1989). The high activity of 1-?-hydroxylase (CYP27B1) present in kidney is not unique to this organ and can also be found in some other organs (Bouillon, 1998). The renal hydroxylation of 25-hydroxyvitamin D3 is the rate-limiting step in the production of 1,25-dihydroxyvitamin D3 and is well regulated. An alternative pathway of hydroxylation of 25-hydroxyvitamin D3 within renal mitochondria takes place at Carbon-24 to form 24,25-dihydroxyvitamin D3 which is metabolically inert. This process is catalyzed by renal 24-?-hydroxylase in response to 1-?-hydroxylase suppression. However, 24-?-hydroxylase not only initiates the attachment of the hydroxyl group at Carbon-24 but also enhances the dehydrogenation of 24,25-dihydroxyvitamin D3 and hydroxylation at Carbon 23 and 26 (Sahota and Hosking, 1999; Bouillon, 1998; Reichel, et al., 1989). Renal hydroxylases require the presence of catalytic cofactors that enhance their synthetic activities during this process. Figure 2 shows the details of vitamin synthesis including the enzymes and cofactors required for each step. Figure2. Enzymes, cofactor and intermediates compounds of vitamin D metabolism (Bouillon et al. 1998) 1.2.4- Regulation of vitamin D metabolism Numbers of factors have been demonstrated to be important in the regulation of vitamin D metabolism; particularly significant its regulation through renal production. The factors involved in this regulation comprise parathyroid hormone (PTH), calcitonin, dietary calcium and phosphate, insulin and insulin-like growth factor and 1,25-dihydroxyvitamin D3 itself (Holick,2006; Deluca, 2004; Sahota and Hosking, 1999). Key interactions of vitamin D with its receptor are known to initiate gene regulation. These mechanisms have been studied using vitamin D analogues which have revealed the mechanism of assembly of transcriptions factors and promotion of gene regulation by this molecule (Cheng et al., 2004; Wu et al., 2002). Figure 3 shows the effect of various regulators on vitamin D metabolism. Figure 3: Alternate pathway for vitamin D3 under different metabolic conditions of low mineral Ca and P levels, PTH concentration and secretion of GH / IGH (Figure obtained from Gomez, 2006). 220.127.116.11- Parathyroid Hormone Parathyroid hormone (PTH) is the primary regulator of renal 1,25-dihydroxyvitamin D3 formation (Holick, 2006; Dusso et al., 2005; Bouillon et al., 1998; Issa et al., 1998). PTH regulates 1,25-dihydroxyvitamin D3 production directly through enhancing 1-?-hydroxylase activity within kidney cells and increasing the genetic transcription rate of renal proximal tubular 1-?-hydroxylase both of which result in an increase in the renal 1,25-dihydroxyvitamin D3 production rate. High levels of 1,25-dihydroxyvitamin D3 suppress the enzyme transcription activity and PTH concentration. Thus, renal 1,25-dihydroxyvitamin D3 has a negative feedback response on PTH secretion, providing an efficient regulatory control of renal 1,25-dihydroxyvitamin D3 homeostasis (Dusso, et al., 2005; Holick,2003; Sahota and Hosking, 1999; Reichel, et al., 1989; Iqbal, 1994). 18.104.22.168- Calcium Dietary calcium exhibits a direct regulatory influence on renal 1-?-hydroxylase activity via fluctuating serum calcium concentration and indirectly via its effect on serum PTH concentration. Calcium exerts its effect through calcium-sensing receptor (CaR) activation within the parathyroid gland and renal proximal tubules cells in response to low calcium concentration. Thus, the low intracellular calcium levels lead to increased production of 1,25-dihydroxyvitamin D3 within renal cells (Ramasamy, 2006; Bland et al., 1999; Chattopadhyay et al., 1996). On the other hand, it has been shown that high calcium concentrations markedly impair renal 1,25-dihydroxyvitamin D3 formation in human nephrotic cell cultures and in parathyroidectomised animals (Bland et al., 1999; Chattopadhyay et al., 1996). An increase in extracellular calcium indirectly suppresses 1,25-dihydroxyvitamin D3 production at the proximal convoluted tubule by inhibiting PTH release (Deluca, 2004; Carpenter, 1990). However, the detailed mechanism of calcium-sensing receptors (CaR) activation is not yet fully understood (Dusso, et al., 2005; Hewison, et al., 2000). 22.214.171.124- Phosphate Dietary phosphate intake and serum phosphate concentrations exhibit regulatory effects on 1,25-dihydroxyvitamin D3 production in proximal renal tubules. This effect has been demonstrated in several studies which showed that a decrease in dietary phosphate accelerated renal formation of 1,25-dihydroxyvitamin D3, but did not directly affect 1, 25-dihydroxyvitamin D3 catabolism. Conversely, elevated serum phosphate and increased phosphate intake led to decreased production of 1, 25-dihydroxyvitamin D3 (Carpenter, 1989; Reichel et al., 1989). Several studies have shown that inorganic phosphate levels have no significant direct effect on mitochondrial 1-?-hydroxylase activity in cultured renal cells in the short term, suggesting that the action of inorganic phosphate is not mediated via changes in PTH and Calcium concentrations and is possibly inducted by other hormones such as growth hormone, insulin and insulin-like growth factor (Khanal et al., 2006; Dusso et al., 2005; Carpenter, 1989). In recent studies, fibroblast growth factor 23 (FGF-23), frizzled-related protein 4 (FRP-4) and matrix extracellular phosphoglycoprotein (MEPE) have all been identified as potent and key regulatory factors of 1-?-hydroxylase activity in renal cells. These factors act through a biphasic mechanism on renal phosphate homeostasis and modulate the circulating levels of 1, 25-dihydroxyvitamin D3 produced by proximal renal tubules (Dusso et al., 2005; Inoue et al., 2005; Mirams et al., 2004). 126.96.36.199- Calcitonin Calcitonin belongs to a family of calcium regulating hormones that is produced in the parafollicular cells of the thyroid gland, also known as C cells. It is a short and linear polypeptide with a molecular weight of only 3.7 kD. It is characterized by 32 amino acids and a disulfide bridge in the N terminal portion of the peptide. Calcitonin is secreted in response to increased free Ca2+ in blood and acts on osteoclasts, the bone resorbing cells, as a suppressor of bone dissolution. Although calcitonin decreases Ca+2 and inorganic phosphate in blood, it also has the ability to recruit phosphorus into other cells. In addition to these metabolic functions, it is also involved in the upregualtion of CYP27B hydroxylase through the protein kinase C pathway (Yoshida et al., 1999) via a phosphorylation cascade that activates cAMP and induces the expression of hydroxylase thereby activating the transformation of 25(OH) D3 to 1,25(OH)2 D3. In addition to the significant role as a calcium regulating hormone, calcitonin is also known to stimulate the production of vitamin D in tandem with PTH (Yoshida et al., 1999; Wongsurawat and Armbrecht, 1991). Previous studies revealed that 1-?-hydroxylase mRNA expression, 1-?-hydroxylase activity and the production of 25(OH)D and 1,25(OH)2D3 all increased in rat kidney cells following the administration of calcitonin (Yoshida et al., 1999; Galante et al., 1972; Rasmussent et al., 1972). However, in cases of diabetes, it is postulated that the kidney becomes immune to the effect of this hormone in diabetic rats which lead to increase vitamin D production (Wongsurawat and Ambrecht, 1991). 188.8.131.52- Growth hormone, Insulin and Insulin-like growth factor-1 Growth hormone (GH) has many regulatory actions in various metabolic processes in humans and mammals and its effect on mineral homeostasis in target organs such as bone and renal cells is well documented. While the regulatory effects of GH on dietary calcium and phosphate metabolism in different tissues have been established, its effect on vitamin D metabolism remains controversial. However, many studies have shown that GH increases the expression of 1-?-hydroxylase and 1, 25-dihydroxyvitamin D3 in cultured cells and experimental animals (Gomez, 2006). Wu and colleagues reported that serum1, 25-dihydroxyvitamin D3 increases after GH administration in hypophysectomized rats fed with a phosphate depleted diet. Short-term studies in healthy humans have shown that GH raises 1-?-hydroxylase enzyme activity and promotes 1, 25-dihydroxyvitamin D3 synthesis without changes in PTH, calcium and phosphate concentrations, suggesting that the increasing circulating levels of 1, 25-dihydroxyvitamin D3 following GH administration is not mediated by PTH action (Wu et al., 1997; Bianda et al., 1997; Wright et al., 1996). GH has also been shown to lead to increased production and serum concentration of 1, 25-dihydroxyvitamin D3 in pigs and in renal impaired prepubescent children. These are thought to be a result of the direct and indirect effects of GH on 1-?-hydroxylase expression, and on calcium and inorganic phosphate homeostasis in renal tubules cells (Strife and Hug, 1996; Denis et al., 1995). However, the action of GH on vitamin D metabolism in vitro remains uncertain and may involve other regulatory factors such as PTH and Insulin-like growth factor-1 (IGF-1). It has been shown that GH does not raise 1, 25-dihydroxyvitamin D3 levels directly in cultured cells obtained from aged-rats; yet it stimulates calcium absorption and the expression of calcium binding proteins in vitro indicating that the effect of GH is mediated through the action of other factors such as IGF-1 (Fleet et al., 1991). Insulin is another key factor with a role in vitamin D homeostasis. Insulin significantly decreases renal hydroxylase activity and renal synthetic capacity of 1, 25-dihydroxyvitamin D3 in insulin deficient patients or those receiving insulin therapy (Armbrecht et al., 1996). However, a study of different routes of therapeutic insulin administration in human diabetic subjects concluded that insulin induces the hepatic hydroxylation of 25-hydroxyvitamin D3. This effect is related to the fact that insulin is a potent inducer of the vast majority of liver hydroxylases enzymes (Colette et al., 1989). This study also showed that there was no significant difference in circulating levels of 1,25-dihydroxyvitamin D3 between different methods of insulin administration. Serum 1,25-dihydroxyvitamin D3 is maintained at normal concentrations in those subjects on long term insulin therapy; however, continuous intraperitioneal infusion procedure (CPII) may augment hepatic 25-hydroxlase activity (Colette et al., 1989). Similarly insulin has shown a significant effect on stimulating 1,25-dihydroxyvitamin D3 production through 1,25-dihydroxyvitamin D3 and PTH stimulation with no concomitant action on 24-hydroxylase expression in rat osteoblast cells when these cells were cultured with known concentrations of 1,25-dihydroxyvitamin D3 and PTH (Armbrecht et al., 1996). Insulin-like growth factor-1 (IGF-1) is a relatively small peptide that is primarily expressed in hepatic cells and to a lesser extent in some other cells and tissues. It has been identified as one of the potent regulatory components of mineral metabolism in humans and mammals. Recent studies on the metabolic effect of IGF-1 revealed that the administration of IGF-1 to aged laboratory animals, fed on a calcium- and phosphate- deficient diet, can restore 1-?-hydroxylase activity and enhance the production of 1,25-dihydroxyvitamin D3. In contrast, there was no significant effect of IGF-1 on enzyme activity and 1,25-dihydroxyvitamin D3 levels in adolescent or elderly rats fed on a calcium and phosphate fortified diet concluding that the expression of IGF-1 is not age related but related to the dietary calcium and phosphorus status. (Gomez, 2006; Wong et al., 1997; Wu et al., 1997). In healthy human subjects, a significant effect of IGF-1 on renal 1,25-dihydroxyvitamin D3 synthesis was observed after short term infusion with IGF-1. There was no noticeable alteration of the levels of circulating calcium, phosphate and PTH highlighting the role of IGF-1 in stimulating renal expression of 1-?-hydroxylase and 1,25-dihydroxyvitamin D3 formation in conjunction with GH, independently from PTH (Bianda et al., 1997). In vitro studies have shown that IGF-1 influences the expression of 1-?-hydroxylase and 1,25-dihydroxyvitamin D3 synthesis in cells cultured from non renal human tissues. Halhali and colleagues demonstrated that IGF-1 noticeably elevates both the enzyme activity and 1,25-dihydroxyvitamin D3 levels when added into cultured syncytiotrophoblast cells obtained from human placental sources. This study demonstrated that IGF-1 strongly enhances the ability of non renal cells to produce 1,25-dihydroxyvitamin D3 without involvement of GH and PTH (Halhali et al., 1997). 184.108.40.206- 1, 25-dihyroxy vitamin D3 The circulating levels of 1,25-dihydroxyvitamin D3 modulate its production by renal cells through an indirect negative feedback mechanism. This mechanism appears to reduce the likelihood of vitamin D toxicity by inhibiting 1,25-dihydroxyvitamin D3 synthesis by an indirect mechanism that controls the 1-?-hydroxylase gene expression at the molecular level rather than inhibiting 1,25-dihydroxyvitamin D3 synthesis directly. However, the exact mechanism is not yet fully understood (Dusso et al., 2005; Deluca et al., 1990). A recent study examined the effect of 1,25-dihydroxyvitamin D3 on 1-?-hydroxylase production by cultured human keratinocytes. Keratinocytes were cultured with labeled 25-hydroxyvitamin D3 and different concentrations of 1-?-hydroxylase mRNA and 24-hydroxylase- suppressed proteins. The 1,25-dihydroxyvitamin D3 did not suppress either the 1-?-hydroxylase activity or the rate of gene transcription. The study implied that metabolic regulation of 1,25-dihydroxyvitamin D3 is related to the molecules biodegradation in response to augmented 24-hydroxylase activity rather than 1,25-dihydroxyvitamin D3 formation by 1-?-hydroxylase (Xie et al., 2002). In addition, Wu and colleagues demonstrated a possible alternative mechanism of 1,25-dihydroxyvitamin D3 synthesis linked to the fact that both 24-hydroxylase and 1-?-hydroxylase enzymes share equivalent metabolic capability and they proposed the possibility of protein- protein interaction between intracellular vitamin D binding protein and 1-?-hydroxylase (Wu et al., 2002). 1.2.5- Vitamin D Transport, receptors and mechanism of action Vitamin D receptor (VDR), also known as calcitriol receptor, is a member of the steroid family and belongs to the nuclear receptor superfamily (NHR). Human VDR until recently was thought to comprises four functional units with a total of 427 amino acids residues with an estimated molecular weight of about 48 kDa. These units are the DNA binding domain (DBD) or C domain, the D domain and the ligand binding domain (LBD) or E domain. More recently, a carboxy-group with undefined function, known as the F region has been identified (Christakos et al., 2003; Aranda and Pascual, 2001; Rastinejad et al., 2000). These units as, shown in figure 4, are also known as A/B domain. The A/B region of VDR contains a low number of amino acids that participates in essential ligand-independent receptor stimulation (Aranda and Pascual, 2001; Issa et al., 1998). It is not yet clear if the deletion of A/B domain from VDR will compromise ligand binding, DNA binding or its transactivation features (Issa et al., 1998). In contrast, the structure of the DNA binding domain or C region among NHRs comprises 40% unique amino acids sequences and a domain of more than 67 resemble amino acids residues (Rastinejad et al., 2000). Moreover, the core structure of DBD comprises between 22 and 114 amino acid residues, nine of them are cysteines. Eight of cysteine residues orchestrate with zinc atoms in tetrahedral fashion to form a dual “zinc-like finger” DNA binding configurations containing approximately 70 amino acids with a carboxy-terminal extension (CTE). This encloses T and A boxes in a dual helix molecule in which one helix is essential for definitive interaction with the main domain on DNA while the second helix takes a part in receptor’s structural properties (i.e. receptor dimerization) (Aranda and Pascual, 2001; Issa et al., 1998). However, the integration of the structural amino acids of the DBD ?-helix one, at the site of the first zinc atom, determines the selectivity and specificity of recognition of DBD and forms an area known as the “P Box”. Similarly; the integration of amino acids at the position of the second zinc atom modulates the formation of a configuration termed the “D Box” which forms a dimerization interface zone (Aranda and Pascual, 2001; Rastinejad et al., 2000; Issa et al., 1998). Furthermore the vast majority of DBD amino acid units are basic amino acids which enhance the non-covalent binding of the DNA helix at the negatively charged phosphate group (Issa et al., 1998). The ligand binding domain (LBD) or E domain has a spherical configuration with many functional regions composed of 12 cohered helix anchors defined as H1 to H12. LBD itself comprises a net of 427 amino acids which contribute to homodimerization and heterodimerization and the interaction of hormones and costimulaotors by a crucial transactivational mechanism (Aranda and Pascual, 2001; Weatherman et al., 2000; Issa et al., 1998). Crystallographic studies show that LBD have two cohered and integrated domains, the Ti or “signature motif” and the carboxy or C terminal AF-2 providing the self-ligand transcriptional properties; hence a higher degree of attraction of 1,25 dihydroxyvitamin D3 binding is observed at 382 to 402 of LBD amino acid sequence and any genetic aberration at this particular amino acids sequence will diminish the interaction capability of LBD (Aranda and Pascual, 2001; Issa et al., 1998). Figure 4: The primary structure of the vitamin D receptor (VDR) and the binding of retinoid X receptor (RXR)-VDR heterodimers to vitamin D response elements (VDREs) in the form of DR3 and ER6 motifs. (Figure from Lin and White, 2003) 1,25-dihydroxyvitamin D3, has been identified as steroid hormone with a mechanism of action similar to other steroid hormones, causing new protein expression in various target organs. Based on the nuclear receptors structural studies, calcitriol is known to exert its biological action through binding with VDR in the cell nucleus to mediate a cascade of transcriptional and translational processes resulting in either the regulation or inhibition of new protein expression in target tissues or the binding to plasma membrane receptors without stimulating new protein synthesis (Nezbedova and Brtko, 2004; Reichel and Norman, 1989). Two different receptors for 1,25-dihydroxyvitamin D3 have been recognized in different target cells; identified as genomic VDRnuc and typical VDRmem .These receptors provide the best dynamical conformational forms for calcitriol interaction and to evoke its genomic and non-genomic effects (Norman et al., 2002). The binding of 1,25-dihydroxyvitamin D3 to VDRnuc enhances the interaction with an undistinguished protein known as the nuclear accessory factor (NAF) and to the caroxy-terminal of VDR. This interaction leads to a structural conversion pattern of the C-terminal of VDR allowing the AF-2 domain to attach with other transcriptional elements such as SCR-1, calcium binding protein (CBP) and P300. This promotes the binding of the heterodimer molecule with DNA at the vitamin D response sites (VDRE) and directs its transcriptional gene activity (Jones et al., 1998; Iqbal, 1994). In addition, these coactivators play a role in DNA configurational changes through histone acetyl transferase activation pathway of the core components of histones. This results in mechanical instability of the DNA structure and enhances the net binding capacity of the coactivators with their corresponding receptors at nucleosomal histone level and leads to the upregulation of these transcriptional coactivators which in trun, accelerate the net gene transcriptional rate to promote the synthesis of the analogous protein (Lipkin and Lamprech, 2006; Jones et al., 1998). Conversely, the non-genomic or classical effect of 1,25-dihydroxyvitamin D3 is modulated through its binding with the surface cellular membrane receptor known as mVDR which initiates an immediate response in various target tissues with no genomic transcriptional activity. Many studies demonstrate the rapid effect of calcitriol in rapidly increasing both the level of circulating calcium and its absorption rate in animal intestines, evoking phosphoinoisitide bioactivation, cyclic guanosine monophosphate (cGMP) elevation, activation of protein kinase C and triggering the mitogen activated protein kinase pathways and involving the chloride gates action potential in different organs (Dusso et al., 2005; Nezbedova and Brtko, 2004; Boyan and Schwartz, 2004; Norman et al., 2002). The entire mechanism, as shown in figure 5, for the rapid effect of calcitriol remains doubtful, however; the proposed mechanism is mediated through the interaction with mVDR leading to a series of intracellular signaling events. Signaling is orchestrated by the activation of various metabolic pathways involving different transportation mechanisms of certain mineral components of target organs. (Pedrozo et al., 1999; Norman et al., 1999; Revelli et al., 1998). However, other studies reveal that the genomic effect of 1,25-dihydroxyvitamin D3 is independent of its non-genomic mechanism (Dusso et al., 2005). Figure 5: Cellular mechanism of action of 1,25(OH)2D3 (Figure from Horst et al., 1997) 1.3- Biological actions of Vitamin D on target tissues and Systems The active form of vitamin D, 1,25-dihydroxyvitamin D3 is well recognized as a member of steroid hormones that mediates several metabolic and non-metabolic processes in various organs in human and animals as shown in figure 6. 1.3.1- Intestine Mineral absorption in the intestines is increased in the presence of the hormone 1,25(OH) vitamin D. However without this, only 10 to 15% of dietary calcium and 60% of phosphorus is absorbed from the diet (De Luca, 2004). Ca2+ and HPO42- are also absorbed when intestinal cells interact with the vitamin D- VDR- RXR complex. The latter enhances the expression of the epithelial calcium channel and calcium-binding protein which recruits calcium and phosphorus (Holick, 2007). Knock out mice experiments studying the effect of VDR gene deletions also show that the size of the small intestines is related to the levels of calcitriol and dietary calcium availability. Vitamin D deficient mice fed with diets low in calcium exhibited the largest small intestine to large intestine ratio (Cantorna et al., 2004). VDR knock-out mice experiments also aid in the discovery of calcium channels, the route for Ca absorption, in the intestine (Peng et al., 1999). Calbindin is a potent calcium transporter in mammals which characterized by a high affinity for calcium ions. Therefore, the binding of vitamin D to VDR and RXR signals an increased production of calbindin which facilitates systemic Ca2+ ions transportation and prevent the occurrence of calcium toxicity in the intestines. Figure 6: Schematic diagram of the effects of Vitamin D on different tissues and organs (Figure from Holick, 2007). 1.3.2- Bone Takeda et al. (1999) studied the role of vitamin D and VDR in bone cells using knock out mice experiments. Their results showed that bone cells formation triggering mechanisms such as cell to cell interaction between osteoblast and osteoclast progenitors and stromal cells induced by 1,25(OH)2 vitamin D3 and provoke the formation of osteoclasts. In their capacity as bone resorbing cells, osteoclasts can be triggered by low serum calcium levels, to break down bone and free calcium back in to the blood thus redistributing calcium throughout the body. However, this does not occur without the expression of VDR and without vitamin D complexing with its receptor. This study emphasizes the important role of recognition sites on the VDR and the structural implications that the receptor-ligand binding has on VDRE’s and transcription initiation. Although the effects of PTH can still induce the action of osteoclasts independently of receptor binding, it also negates non genomic signaling as a proposed pathway for vitamin D enhancement of osteoclast formation (De Boland and Boland, 1994). The effects of 1,25(OH)2D3 in bone formation is also mediated by the production of calcium binding proteins such as calbindin, osteocalcin and osteopontin. The mechanism of calbindin has already been discussed previously; however, osteocalcin and osteopontin promote bone growth by enhancing the absorption of calcium (Ihara et al., 2001; Price and Baukol, 1980). In addition, 1,25(OH)2 vitamin D3 can also affect the production of transcription factors that enhance bone cell differentiation (Drissi et al., 2002). 1.3.3- Kidney 1-?-hydroxylase, the enzyme required for the activation and formation of vitamin D is found in the kidneys. An increased concentration of 1,25-dihydroxyvitamin D3 triggers bone cells to synthesize fibroblast growth factor 23 (FGF-23) which causes the sodium-phosphate co transporter to be internalized by the cells of the kidney and small intestine which results in decreased synthesis of calcitriol (Holick, 2007). Conversely, an increase in PTH levels induces the kidneys to produce calcitriol (Dusso et al., 2005; DeLuca, 2004). In addition, the 1,25(OH)2 vitamin D3 produced in the kidneys can cause reduced rennin production in the same organ and stimulate the secretion of insulin in the beta islet cells of the pancreas (Holick, 2007). Although the synthesis of calcitriol is tightly regulated in the kidneys, the action of the same metabolite is induced in other organs and regulates the dynamic balance of calcium and phosphate metabolism. 1.3.4- Immune System The role of vitamin D in the promotion of immunological activities in the body is attributed to the ability of 1,25(OH)2D to reduce the effects of cytokines such as IL- 2 and IL- 12 (Nagpol et al., 2005; Griffin et al., 2003; Penna and Adorini, 2000; D’Ambrosio et al., 1998). These lymphokines are produced in the event of infection and signal the initiation of inflammation reactions. The presence of cytokines has also been shown to induce the formation of calcitriol in cells outside the liver. Because of this, 1,25(OH)2D3 has been implicated in the regulation of cell differentiation (Peterlik and Cross, 2005). The immunological origin of cytokines has also resulted in studies determining the relationships between vitamin D and the onset of auto-immune diseases such as multiple sclerosis and auto-immune encephalomyelitis. For example, studies showed that subjects with low levels of vitamin D are susceptible for multiple sclerosis as the expression of cytokine transforming growth factor ?1is reduced and high levels of this cytokine is essential to maintain low risk of multiple sclerosis (Holick, 2006). In addition, patients who contracted diabetes and the autoimmune disease multiple sclerosis have also been identified to be deficient in vitamin D (Wolpowitz and Gilchrest, 2006). Because of the expanding functions of vitamin D and its implications in the realm of immune functions and malfunctions, it has been postulated to play roles in the prevention of diseases such as osteoarthritis, diabetes, autoimmune diseases, cardiovascular diseases and tuberculosis (Uitterlinden et al., 2004). Dendritic cells express both the enzymes required for vitamin D3 synthesis and receptors that mediate cellular protection and immunity. Therefore, 1,25(OH)2 vitamin D3 levels, for instance, induce the formation the cellular adhesion molecules such as selectins protein (E and P selectins), CCR10 and CCR4 chemokine receptors that enhances the transport of lymphocytes to the skin (Mebius, 2007). 1,25(OH)2 vitamin D3, on the hand, has stimulatory and inhibitory effect on IL-12 expression. Several studies have concluded that 1,25(OH)2 vitamin D3 inhibits the expression of IL-12 produced by myeloid dendritic cells while recent studies showed that 1,25(OH)2 vitamin D3 noticeably increases IL-12 production, an essential cytokine for T-cells activation, from myeloid dendritic cells and stimulates the transcription and expression of keratinocytes T-lymphocytes associated chemokine receptor CCR10 (Sigmundsdottir et al., 2007; D’Ambrosio et al., 1998). The immune effects of 1,25(OH)2 vitamin D3 is also governed by its ability to bind with VDR and its effect on VDREs. VDR, as a testament to its ubiquity, is expressed in both innate and adaptive immune cells, monocytes, macrophages, dendritic cells, B and T cells, as well as NK cells. In addition, it possesses immunosuppressive and immunomodulatory functions by affecting the expression of oncogenes and cell surface antigens. In fact, VDRE, for example, enhances the synthesis of c-fos and c-fms oncogenes and suppresses the production of c-myc (Bunce et al., 1997). The VDR complex of 1,25(OH)2 vitamin D3 can also give rise to the immunosuppression of T cells by inhibiting the differentiation of dendritic cells into T cells. In contrast, VDR has been observed to enhance the activity of macrophages (Pena and Adorini, 2000) and induce the production of cathelicidin (CD) in the macrophage. The compound CD is a cationic peptide with antimicrobial effects that is produced in macrophages, triggered by the presence of bacteria, viruses and fungi. This molecule can also cause the destruction of infective agents including M. tuberculosis (Holick et al., 2006). This may contribute to the increased risk of tuberculosis seen in African Americans whose high density of melanin in the skin prevents the absorption of ample UVB for vitamin D synthesis (Gombart et al., 2005). These actions of vitamin D are being actively researched because of their therapeutic clinical applications in the prevention and treatment of medical conditions and inflammatory diseases such as rheumatism and arthritis, cancers and autoimmune diseases. 1.4 Factors influence Vitamin D levels Several factors have been demonstrated to influence vitamin D level in humans by playing a part in its absorption and transformation. These factors identified as biological and physical factors and exert their effects on various organs by different mechanisms. 1.4.1- Biological Factors The human body has to maintain a certain level of circulating vitamin D to sustain its functional effect in VDR expressing cells (Barger-Lux et al, 1998). Despite comparable abilities of healthy individuals to produce cholecalciferol in the skin, other factors govern the serum levels of vitamin D produced. These factors include skin color (race), age and liver and kidney diseases; therefore, they are important determinants and may be used to predict which populations are at risk for vitamin D deficiency. 220.127.116.11- Skin Color and Age Vitamin D is essential for maintaining calcium metabolism and physiological bone formation and remodeling during life. As UVB absorption in the skin is the key step for photocatalysis reactions to generate cholecalciferol, the amount of UVB absorbed is crucial for vitamin D production. Skin pigment components are very effective filters of UVB and have been shown to protect dark-skinned individuals from skin cancer (Chen et al., 2007). However, it also prevents them from absorbing adequate UVB for vitamin D synthesis. Although healthy individuals may have the same capacity to synthesize vitamin D, pigmented skin is characterized by high concentrations of melanin and requires longer exposures to UVB for vitamin D synthesis (Wolpowitz and Gilchrest, 2006). Melanin also acts as a UV absorbing species that competes with 7-dehydroxycholesterol, 7-DHC, (Webb, 2006). Figure 6 shows the relative amounts of vitamin D and rate of synthesis in individuals of different skin pigmentation. Type V, associated with higher pigmentation, shows lower rates of vitamin D synthesis due to the presence of melanin whereas type I exhibits higher increase in serum levels of vitamin D upon constant sun exposure (Chen et al., 2007). High amounts of melanin in the skin have also been viewed as a mechanism for preventing excessive vitamin D concentration (Clemens et al., 1982) and also as a protective factor for locals in regions near the equator (Webb, 2006). On the contrary, excess cholecalciferol production is controlled by equilibrium mechanisms and the production of other intermediates from procholecalciferol that has little effect on vitamin D mediated processes, thus, maintaining the balance of this intermediate (Holick, 2003). Analysis of vitamin D synthesis on different skin types (II and V) show that at 5 minutes of sun exposure, type V skin is still devoid of vitamin D3 while type II skin cells have already initiated synthesis (Chen et al., 2007). Figure 6: Serum 25-hydroxyvitamin D levels in volunteers with different skin types after weekly exposure to simulated sunlight for 12 weeks (Figure and caption from Chen et al., 2007) The effect of skin color on vitamin D production was also investigated in a comparative study on African American subjects that concluded that African Americans have a high risk for vitamin D deficiency. Research on vitamin D levels in African American women, aged 15 to 49 years, showed that their serum levels are <25 nmol/ L (Nesby- O’Dell et al., 2002). African American as well as Hispanic children (Looker et al., 2002) have a higher risk for vitamin deficiency. Aging is also a factor that affects the status of vitamin D (Salamone, 1993). The decline of the body’s ability with age to synthesize or absorb vitamin D is attributed to several factors. One of the most obvious effects of aging is the lifestyle change, which result from the decreased capabilities and resiliency of the human body as aging progresses. Studies have shown that 7-dehydrocholesterol status in the skin declines or decreases as a person ages; for instance, a 70- year- old person has 25% of the capability to generate cholecalciferol as compared with a healthy young adult (Webb, 2006; Matsuoka & Wortsman, 1989; McLaughlin & Holick, 1985). This decline may likely be due to the decreased rate of metabolic pathways that produce this substance, which necessitates the use vitamin D supplements (Rapuri and Gallagher, 2004) especially to maintain bone density and resist muscle weakness that can lead to falls and fractures (Jackson et al., 2006). In addition, in line with healthy young adults, the status of vitamin D may reach 42%, utilizing a cut-off point of 50 nmol/L for serum 25-OHD (Gordon et al, 2004). Part of aging is the slowing of body metabolism that may be associated with obesity. Obese individuals have inefficient cholecalciferol storage because the non-polar nature of vitamin D metabolites allows its storage in adipocytes. Thus, available cholecalciferol during seasons of low sunlight exposure is depleted, leading to decreased cholecalciferol synthesis. However, in cases where the adipocytes form a thick mass, especially in obese individuals, cholecalciferol becomes difficult to mobilize causing a 50% decrease in recovered vitamin D compared with normal patients (Wortsman, 2000). As the amount of IGF-1 decreases with age, the effects of IGF-1 on vitamin D metabolism is reduced (Halhali et al., 1999). 18.104.22.168- Liver Diseases Liver diseases such as hepatic failure can cause a drastic change in the rate of 25-(OH) vitamin D3 synthesis. Liver problems can be categorized as an acute or mild to moderate dysfunction. Mild liver failure results in reduced absorption of vitamin D; however, in such cases, the production of 25-(OH) vitamin D3 is still possible (Holick, 2007). Acute liver failure, on the other hand, results in the inability to synthesize sufficient 25-(OH) vitamin D3 (Holick, 2006). The administration of activated vitamin D such as paricalcitol and calcitriol to improve vascular functions in patients with renal failure may also pose some difficulties due to the varied effects of the vitamin D form on PTH, calcium, and phosphate ions levels (Wolf and Thadhani, 2007). Furthermore, smoking and high alcohol intake also impairs hepatic capacity to synthesize 25-(OH) vitamin D3 as a result of hepatocellular damage induced by liver alcoholic cirrhosis. In addition, long term administration of some drugs like corticosteroids and heparin may also compromise hepatic ability to produce 25-(OH) vitamin D3, resulting in secondary osteoporosis (Christodoulou and Cooper, 2003). 22.214.171.124- Renal Diseases Chronic renal diseases can result in decreased synthesis of 1,25(OH)2 vitamin D3. Decreased synthesis also occurs in patients who had their parathyroid glands removed because of hyperparathyroidism, or in cases of hypoparathyroidism and hyperphosphatemia. Hyperphosphatemia results from decreased excretion of phosphate ions and causes an increase in the concentration of fibroblast growth factor 23 (FGF23) from bone cells (Dusso, 2005; Shimada, 2004). As a result, 1?-hydroxylase activity decreases and eventually leads to low levels of synthesized 1,25(OH) vitamin D3. Consequently, hypocalcemia, secondary hyperparathyroidism, and renal bone disease develop (Shimada, 2004). 1.4.2- Physical Factors Physical factors affect absorption of UVB from the sun include barriers to sunshine exposure such as sunscreens, protective clothing, indoor lifestyle, geographic locations, and weather. 126.96.36.199- Sun protective agents An important contributing factor to vitamin D deficiency is insufficient exposure to sunlight. UV irradiation causes random mutations in nucleic acids and contributes to the risks for skin cancer and melanoma; therefore, a solution to prevent exposure to UV radiation includes the use of sunscreens and other sun protective agents. As a result, these agents decrease vitamin D production in the skin (Gartner and Greer, 2003). The efficiency of sunscreens is characterized by the chemical’s ability to protect the skin from solar radiation. Sun protection factor (SPF) refers to the additional amount of time a person be exposed to sunlight without experiencing its harmful effects (Holick, 2002). It is also defined as the effect of the chemical when applied at 2 mg/cm2 of the skin surface (Jeffrey, 2002). Sunscreens depend on uniform and frequent application to provide protection. Even sunscreens with a relatively low SPF can contribute to vitamin D deficiency. Studies demonstrate that using a SPF 8 sunscreen, for example, can inhibit greater than 95% of vitamin D production in the skin (Chen et al., 2007; Holick, 2002). Increasing events of skin cancer in Australia have led to a campaign of increasing sunscreen use; however, these programs have led to increased populations with vitamin D deficiency in Australia and New Zealand (Benson and Skull, 2007). Cases of vitamin D deficiencies are rampant in Australian refugees who are mostly Muslims from the Middle East (Benson and Skull, 2007). Their conservative clothing and general tendency to avoid going outdoors contribute to their vitamin D deficiency (Benson and Skull, 2007). In addition, people who are also used to an indoor lifestyle are at risk of vitamin D deficiency (Gartner and Greer, 2003). Furthermore, a Scandinavian study reported that women from Arabic and Muslim cultures wear clothing that minimizes sun exposure can affect vitamin D synthesis, this effect can be relieved by vitamin D injection and oral supplements (Glerup et al., 2000). However, without UVB, the transformation of the inactive form of vitamin D to the active form cannot occur. This dilemma prompted several studies to weigh the risks of sun exposure against its known potential benefits. Recent studies show that exposure to UV may be correlated to decreased susceptibility to upper respiratory tract infection, liver cirrhosis, heart disease, and eclampsia and it may be used to treat anemia, increase mental functions, and improve skin tone (Wolpowitz and Gilchrest, 2006). 188.8.131.52- Latitude (Geographical) Since the amount of UVB absorbed by the skin is a key determinant in the synthesis of active vitamin D metabolites, individuals from countries who have low sun exposure are at risk for vitamin D deficiency without proper supplementation. Thus, vitamin D levels in populations are dependent on geographic locations. The quality and quantity of UV that reaches different regions of the earth depends on the distance and latitude of a particular region. In higher latitude areas, the amount of the type of UV helpful for the synthesis of vitamin D is lower, causing greater risks of exposure to harmful UV. Thus, the amount of UVB absorbed by skin is affected by the presence of particulates in the atmosphere and the solar zenith angle (SZA), which is the measured angle of the Earth’s surface between the sun and the zenith that is exposed to UVB during the day (Webb, 2006). The SZA, illustrated in Figure 7 below, can best be interpreted as the route of sunlight through the atmospheric divide. In regions of low SZ angles, the sunrays hit strongly and directly due to the shorter distance of the sun from the surface while the contrary happens at larger SZs (Webb, 2006). Thus, regions of larger SZAs may require longer times of sun exposure to absorb enough vitamin D synthesizing UV, although, a lower SZA may also mean greater risks of exposure to erythemal UV. In Australia, for example, where the amount of sun exposure should pose little problem to vitamin D production, problems regarding the prevalence of skin cancer arise (Benson and Skull, 2007) which may have been augmented by the skin pigmentation of Australians. In addition, the time of the day, seasons, and latitude differences also reflect changes in SZA, which affect UV absorption (Webb, 2006). The quality of the atmosphere also poses difficulties as air pollution, particles, smog, and clouds, which are clear obstacles for UV and tend to scatter or reflect the light as it traverses the atmosphere (Webb et al., 2000). Thus individuals in urban areas tend to be vitamin D deficient (Agarwal et al., 2002). Figure 7. Solar zenith angle is the angle between the local vertical and the direct solar beam. At a large SZA, the radiation traverses a longer path length through the atmosphere, which can greatly attenuate shorter wavelengths, and the attenuated power is spread over a larger surface area (Figure and caption from Webb, 2006). 184.108.40.206- Seasonal Variations (Geographical) Climate changes such as winter, onset of cloudy periods and shade, and even air pollution have been identified as reducers of vitamin D synthesis (Gartner and Greer, 2003; Rapuri and Gallagher, 2004). Since the type of UV light absorbed can greatly impact the potential health benefits of UV absorption for vitamin D synthesis, exposure to both types of UV radiation (visible light 280-320 nm and UV light 300-400 nm) should be cautioned. Especially since exposure to the sun means exposure to a broad spectrum of light and the portion of the light spectrum causing greater impact on vitamin D synthesis (280-320 nm) overlaps with the type of UV that can cause erythemal response (300-400 nm) (Mckinlay and Diffey, 1987). Weather can indirectly affect the amount of UVB absorbed for vitamin D synthesis. At higher latitudes and during the winter, exposure to the sun is furthermore decreased because of the phenomenon of longer night hours resulting in reduced daylight hours. However, during the winter, protective clothing, which limits the amount of UVB absorbed by skin, is widely used. In addition, the cold weather limits outdoor activities, thereby causing a large decline in the amount of UVB absorbed. A study by Webb et al. (1988) revealed that virtually no vitamin D synthesis occurred during the winter season in Boston and Edmonton. Thus, the region’s physical setting and surfaces, which absorb or reflect UVB, can also affect its absorption levels in the skin. Examples include amount of vegetation, presence of snow, soil or rocks, shaded areas and buildings (Webb et al., 2000; Feister and Grewe, 1995). On the contrary, a study by Kimlin et al., (2007) showed that seasonal changes in seasons might not be affecting vitamin D synthesis as expected during the winter seasons in lower latitude regions because the human body can recognize the times and locations at which epidermal production of vitamin D does not take place during exposure to UV radiation; therefore, the amount of the type of UV helpful for the synthesis of vitamin D is equal to the amount during summer (Kimlin et al., 2007; Webb et al., 1988). 1.5-Vitamin D Deficiency 1.5.1- Definition of vitamin D deficiency Vitamin D deficiency is defined in terms of the 25-(OH) vitamin D concentrations, instead of the active metabolite concentration. Holick (2007) explained that the mechanisms for the synthesis of active vitamin D, including the strict regulatory mechanisms in the pathways involved, produce varying and inconsistent values for 1,25(OH)2 vitamin D3. Thus, clinicians must recognize that vitamin D levels in the form of 1,25(OH)2 vitamin D3 do not necessarily mirror the true situation . Although the definition of vitamin D deficiency differs, generally a person is deficient if the concentration of 25-(OH) vitamin D is less than 50 nmol/ L where the serum level of parathyroid hormone becomes 3.2 to 4.2 pmol/L (Holick, 2006; Thomas and Demay, 2000; Fouda, 1999; Malabanan et al., 1998). Despite the ubiquitous source of driving energy for the synthesis of vitamin D in the body, several factors delimit the availability of this vitamin for many important processes in different tissues. Vitamin D assays are used to screen vitamin D deficiency. Serum level between 50 and 100 nmol/L of 25-(OH) vitamin D is considered to be normal. However, at levels from 25 /L to 50 nmol/ L, which indicate insufficient vitamin D concentration, mitigation in this case is necessary and should require administration of oral supplements at 100,000 to 150,000 IU where increase in dosage may also be achieved by daily intake of 500 to 1000 IU of vitamin D supplements, this dosage is also applicable to pregnant women and infants (Grant and Holick, 2005; Holick, 2005; Thomas and Demay, 2000). Very low levels of serum 25-(OH) vitamin D at less than 25 nmol/L may involve other complications and result in low bone density and fractures in adults or rickets in children with noticeable changes in their psychological status (Holick, 2005). 1.5.2 Criteria for Vitamin D deficiency Various criteria for vitamin D deficiency are proposed. Wolpowitz and Gilchrest ( 2006), provide an extensive review of the required amounts of vitamin D necessary for normal functions, the sources of these compounds, and the conditions that may result if the bodily vitamin D needs are unmet. Vitamin D deficiency is characterized in terms of serum levels of 25-(OH) vitamin D. Thus, based on pooled research from Dawson-Hughes et al. (1997), Gloth et al. (1995), Kinyamu et al. (1998), Malabanan et al. (1998), Ooms et al. (1995), Thomas et al. (1998), Nesby-O’Dell et al. (2002), Parfitt et al. (1982), Tangpricha et al. (2002) and Webb et al. (1990), the following criteria of vitamin D deficiency are suggested. These criteria characterize vitamin D status as deficient, insufficient, and sufficient. In a deficient state, serum levels of 25-(OH) vitamin D below 20 or 25 nmol/ L where the PTH levels are boosted to higher than 6.8 pmol/ L and are observed in cases of rickets or osteomalacia. On the other hand, insufficient levels of serum vitamin D range from concentration of 37.5 to 50 nmol/ L, the wide range being caused by several factors affecting vitamin D metabolism. Another qualifier is the level of PTH, which is also variable, but should be within the range of 1.3-6.8 pmol/ L. Optimal or sufficient levels of serum vitamin D are characterized by 25-(OH) vitamin D serum levels from 50 to 100 nmol/L. In some studies where the PTH levels are also characteristically less than 6.8 pmol/L; individuals in this group are phenotypically devoid of bone disease such as osteomalacia. 1.5.3 Populations at risk for Vitamin D deficiency Vitamin D deficiency affects different people from infancy to adulthood and is related to the body’s ability to produce a sufficient amount of vitamin D to maintain its physiological effect on different tissues. 220.127.116.11 Infants and children Levels of vitamin D in infants and children are associated with bone specific alkaline phosphatase and osteocalcin levels (Johansen et al., 1988) and can serve as markers for bone mineral density studies (Fares et al., 2003). Children with vitamin D deficiency are prescribed 200 IU vitamin D daily dose throughout their infancy and childhood and should achieve 25(OH) vitamin D levels of 27.5 nmol/L to prevent the onset of rickets (Holick, 2007; Cashman, 2006). Breast milk has a low concentration of vitamin D, approximately 25 IU/ L (Gartner and Greer, 2003), that fails to meet required amounts for normal bodily functions during childhood (Kreiter et al, 2000). In addition, the dangers of sun exposure are more prevalent in children and the American Academy of Pediatrics has warned that infants less than 6 month old should avoid exposure to UV radiation (Holick, 1998). Thus, to meet the demands of growth and to prevent rickets in infants and children, sun exposure and breastfeeding with vitamin D and milk supplements should be combined (Gartner and Greer, 2003). 18.104.22.168- Adolescents Observations of decreased levels of vitamin D in adolescents in Europe (El-Hajj Fuleihan et al., 2001; Lehtonen- Veromaa et al., 1999) and the Middle East (Fares et al., 2003) have been associated with high events of bone turnover during this period of growth spurt when bones lengthen at fast rates. The decline in vitamin D levels during this stage is also greater in girls, leading to a lower peak bone mass as a result of low calcium intake and vitamin D level. Fares et al.,2003) demonstrated that gender differences in vitamin D levels of adolescents are key indicators of bone remodeling events, which suggest important applications of vitamin D in bone formation, calcification, and turnover. The effective biomarkers for bone density in female adolescents include urine determination of N-telopeptide (NTX), serum detection of C-telopeptide-1 to helix (ICTP), urine C-telopeptide- 2 of type I collagen cross links, and serum C-terminal telopeptide of type I collagen cross links (S-CTX) (Lehtonen-Veromaa et al., 2000). Bone resorption markers are also observed to increase their levels in adolescent males and females (Johansen et al., 1988). Fares et al (2003) noted that bone formation markers are higher in boys than in girls at Tanner stages IV and V and are independent of body mass and weight. These support the observation that peak bone mass in males is higher than that of females (Cadogan et al., 1998). The marker levels also indicate that female adolescents require longer periods of bone remodeling that may extend through Tanner stage V during this active growth stage (Fares et al., 2003). Femoral measurements of bone mineral density also show higher values in males and support the hypothesis that bone mineral density is gender specific, as hormonal changes among males and females differ during puberty (Szulc et al., 2000). 22.214.171.124- The Elderly Studies show that the bone mass density in older people may be predicted by conditions occurring during their infancy. This association has been studied by relating the polymorphic regions in the VDR gene with birth weight and weight at one year (Arden et al., 2005; Calvo et al., 2005; Pal et al., 2003). Arden et al., 2005 proposed that common genes may control birth weight and 1,25(OH)2 vitamin D3 level in the serum of older individuals, as the level of vitamin D is a crucial moderator of the intrauterine and early postnatal environmental which, in turn, affects adult bone mineral density (Arden et al., 2005). Aside from their inherent genes, the elderly are also at risk for vitamin D deficiency due to their decreased ability to produce, transport, and convert 7-DHC to vitamin D precursor in keratinocytes (Lanske and Razzaque, 2007). However, other factors involved are substrate and enzyme levels , sedentary lifestyles, reduced metabolic rates, common increase in body mass during aging, kidney malfunction, other hormonal changes, and less exposure to sunlight (Holick, 2006; Webb, 2006; Dusso et al., 2005; Lips, 2001); however, Vitamin D2 supplements can reverse the deficiency (Rapuri and Gallagher, 2004). 126.96.36.199- Pregnancy and lactation Levels of vitamin D and calcium are decreased in lactating animals because calcium is used for the demands of the growing fetus as well as in milk formation (Horst et al., 1994). A lactating dairy cow for example may lose 80-100 g of calcium daily to sustain the calcium levels for its milk, rat models secrete a range of 120-200 mg of calcium daily while the pregnant humans women lose 220-340 g of calcium daily (Horst et al., 1994). In lactating individuals, aside from 1,25(OH)2 vitamin D3, PTH and calcitonin, the levels of calcium are highly mediated by other hormones such as the PTHrp (PTH related peptide), estrogen, and prolactin (Horst et al., 1997). In severe cases in animal models including rats, dogs, and cows, and excessive decline in serum calcium levels to sustain the amount required for milk production causes death (Horst et al., 1994). Vitamin D mediates calcium absorption in the intestines and bones. When calcium is low, PTH is recruited as a signal for 1,25(OH)2 vitamin D3 synthesis, which, in turn, triggers bone dissolution and increased intestinal absorption of calcium. During lactation, 1,25(OH)2 vitamin D3 synthesis in the liver increases through the action of PTH, prolactin, and a PTH related peptide. In addition, levels of expressed VDR are higher in lactating cows than in non-lactating cows (Reinhardt et al., 1980). In humans, high levels of IGF-1 and 1,25(OH)2 vitamin D3 in the serum and liver of pregnant women indicate a strong association in the regulatory reactions involving these two metabolites (Gomez, 2006). However, during the later stages, after three months of lactation, plasma levels of 1,25(OH)2 vitamin D3 is decreased due to the established levels of calcium during the periods of gestation where calcium absorptive mechanisms are active. Lactating women experience vitamin D independent bone loss to remedy the decreased calcium levels (Kent et al., 1993). This event is postulated to be mediated by PTHrp which is characterized by PTH-like effects on bone (Thiede and Rodan, 1988) and low plasma estrogen concentration, which is observed during early lactation periods. Thus, decreased levels of this hormone favor high activity of the osteoclasts (Manolagas et al., 1993). In addition, the synthesis of calcitriol can also exist in human placental cells other than the liver as induced by IGF-1. These cells may be potential sources of 1,25(OH)2 vitamin D3 and may explain observations of high levels of vitamin D in pregnant women, confirming that a different process of vitamin D regulation may be in place during pregnancy (Halhali et al., 1999). 1.6- Disorders of Vitamin D Metabolism The pathways of vitamin D metabolism involve a network of mechanisms and regulatory steps that start from the DNA to transcription factors, hormones, and allosteric effects in enzymes and minerals. Thus, points of control are rigidly structured. However, because of these control points, systems that undergo changes or mutations can easily affect the mechanisms in place. In addition, because of the many functions that vitamin D plays in the body, disorders in metabolism affect many systems that could be crucial for continued existence and should be detected right away even if phenotypic signs are not observed. Disorders of the metabolic pathways involved in vitamin D synthesis include rickets, osteoporosis, osteomalacia along with vitamin D toxicity and resistance. These metabolic disorders can typically be treated by increased dosage of vitamin D at 50,000 IU once a week for 8 weeks while maintenance can be achieved by giving 50,000 IU of vitamin D once every two weeks (Malabanan et al., 1998). However, vitamin D levels should be regulated to prevent vitamin D toxicity. 1.6.1- Rickets Rickets is a childhood bone disease that occurs because the body cannot absorb calcium and phosphate effectively. It may also result from abnormalities in the enzymes catalyzing vitamin D synthesis or from malfunctions of the vitamin D receptor. Vitamin D- dependent rickets, or Type I rickets, results a mutation in the CYP27B1 gene that produces dysfunctional 1-alpha-hydroxylase responsible for the conversion of 25(OH) vitamin D3 to 1,25(OH)2 vitamin D3 (Holick, 2006), resulting in reduced or no synthesis of the active vitamin D form. Experiments using knock-out mice show that metabolic processes involving vitamin D as well as calcium uptake in the bones require interactions between 1,25(OH)2 vitamin D3, VDR, PTH, calcium, and 1-alpha-hydroxylase enzymes (Panda et al., 2004). This form of rickets can be treated with increased calcium doses. Treatment is normally achieved by administration of the activated vitamin D 1,25(OH)2D3. Type II and type III rickets result in vitamin D resistance and accumulation. 188.8.131.52. Phosphatemic Rickets The autosomal dominant form of phosphatemic rickets results from mutations in the fibroblast growth factor 23 (FGF23) genes and causes decreased intestinal absorption of phosphorus, resulting in low calcitriol levels. A mutation causes resistance of the FGF23 protein to metabolic breakdown resulting in an increase in its concentration in the system. The X-linked phosphatemic rickets also arises from a mutation in FGF23 (Shimada et al., 2004). 184.108.40.206 Hereditary Vitamin D resistant rickets (HVDRR) Hereditary vitamin D resistant rickets (HVDRR) is a rare disorder characterized by early contraction of rickets and resistance to 1,25(OH)2 vitamin D3. This hereditary disorder results from a mutation in the VDR gene but can be treated by analogs that bind to the mutated VDR (Gardezi et al., 2001). The mutation leads to the overproduction of vitamin D response element binding proteins (VDRE-BP ) that compete with the VDR and retinoid X receptor (RXR) complex to vitamin D-responsive elements (VDREs) in the gene (Chen et al., 2000) which, in turn, prevents transcription of vitamin D related genes. The resistance originates from the expression of intracellular vitamin D binding protein (IVDBP) belonging to a family of heat shock proteins, which bind hydroxylated vitamin D metabolites and prevent their activities in the cell (Wu et al., 2000). Individuals with vitamin D deficient rickets exhibit normal serum calcium levels due to the action of the PTH on the osteoclasts but they experience low normal or low fasting serum phosphorus levels. The enzyme alkaline phosphatase levels also increase but 25-(OH) vitamin D3 levels (<37.5nmol/L) decline due to the effect of PTH to stimulate production of 1,25(OH)2 vitamin D3; thus, calcitriol levels can increase and its detection may provide erroneous diagnosis (;Hollick, 2006; Christakos, 2003). The dissolution of the bone and depletion of calcium results in hypocalcemia, growth retardation, bone deformities in the legs and ribcage, bending of the spine, and weak muscles. Observed cases of severe anemia and conditions associated with vitamin D resistant rickets have been reported where the development of CD3 and CD34+ progenitor cells is remarkably impaired (Yetgin and Yalcin, 2004). A patient usually manifests these symptoms between 4 to 12 months of age (Hollick, 2007) or, in some cases, up to 18 months. 1.6.2- Vitamin D Resistance Vitamin D resistant rickets, or Type II rickets, results from abnormalities in the vitamin D receptor, usually caused by a genetic mutation in the VDR gene. This causes partial or complete resistance to 1,25(OH)2 vitamin D3 action and leads to accumulation of this metabolite. Type III rickets is also vitamin D dependent but causes excessive production of hormone responsive element binding proteins. These molecules deter the action of the VDR- RXR- vitamin D complex in activating transcription and cause resistance as well as elevated levels of 1,25(OH)2 vitamin D3 (Holick, 2006). Using VDR gene polymorphisms in the candidate gene approach and knock-out mice to study the functions of the transcription products (Lander, 1994) has also revealed lethal mutations that can occur in this DNA region and cause vitamin D (1, 25(OH)2D3) resistant rickets. This field has garnered much attention and research has focused on the analogs and hydroxylated products of vitamin D that may be potential drugs for vitamin D disorders due to VDR mutations (Makishima et al., 2002; Akutsu et al., 2001). Diseases such as Crohn’s disease, Whipple’s disease, cystic fibrosis, and sprue (Bengoa et al., 1984) cause vitamin D deficiency because of inability of these patients to absorb the fat-soluble vitamin D into the chylomicrons. Crohn’s disease, a type of inflammatory bowel disease (IBD), along with ulcerative colitis is a condition characterized by vitamin D deficiency with abnormal responses to bacteria in the lumen and severe inflammation when the immune system is activated (Cantorna and Mahon, 2004). This condition was improved by administration of 1,25(OH)2 vitamin D3 in mice models (Froicu and Cantorna, 2007), supporting the regulatory immune functions of vitamin D. Patients with diseases that cause vitamin D deficiencies due to abnormalities in fat absorption can increase their levels of vitamin D by exposing themselves to ample amounts of sunshine or inducing synthesis or cholecalciferol by using UV lamps or tanning beds (Tangpricha et al., 2004) which allow synthesis of vitamin D, although it cannot be stored as cholecalciferol in fat cells. In fact, patients with Crohn’s disease were observed to increase their serum levels of 25(OH) vitamin D3 by 700% after using a tanning bed and being exposed to UVB thrice weekly for three months (Koutkia et al., 2001). 1.6.3- Osteomalacia Osteomalacia is a type of rickets commonly observed in adults with vitamin D deficiency. Symptoms include fatigue, bone pain, and muscle weakness (Holick, 2006). It results from demineralization of formed bones or excessive production of osteoid cells that can occur when the bone mineral hydroxyapatite cannot fully be developed due to lack of calcium (calcipenic osteomalacia) and phosphorus (phosphopenic osteomalacia). Calcipenic osteomalacia often results from insufficient vitamin D synthesis or intake and results in the imbalance in calcium absorption in the intestines. Hypophosphatemia, on the other hand, signals increased activity for CYP27A1 to produce 1,25(OH)2D3 and it is linked to increased intakes of antacid aluminum hydroxide. It can also be hereditary such as in X-linked hypophosphatemic rickets or due to ion toxins, such as lead or cadmium (Horner, 2006). Osteomalacia is also characterized by trabecular tissues and osteoid cells that are virtually devoid of calcium, unlike osteoporosis where low quantities of osteoid are found. Osteomalacia and its relationship with vitamin D levels have been established using knock- out mice experiments (Lips, 2006). These studies illustrated that bone mineralization requires the interaction of osteoblasts and osteoclasts as well as the presence of 1,25(OH)2D3 and VDR. VDR complexed with vitamin D acts as an activator of gene expression which results in increased calcium absorption, In addition, low levels of vitamin D signal PTH secretion which in turn signal bone remodeling and bone loss that may eventually lead to osteoporosis (Lips, 2001). 1.6.4- Osteoporosis Bone structure is mainly composed of type I collagen fibers, bone cells in the form of osteoblasts (which initiate bone development), osteoclasts (which dissolve the skeleton) and osteocytes (cells lining the bone surface), and the calcium mineral in the form of hydroxyapatite (Horner, 2006). The bones undergo remodeling or turnover depending on factors such as vitamin D levels, PTH secretions, mineral levels of calcium and phosphate, mechanical stress, signals from other hormones, and other molecules such as cytokines, prostaglandins, and growth factors. During aging, the rate of bone remodeling increases and turnover becomes more frequent as evidenced in menopausal women who undergo high bone turnover. Studies have shown that bone mineral density is related to the polymorphisms in the vitamin D receptor gene as well as in other genes, including IL-6 and growth factors (Calvo and Whiting, 2005; Pal et al., 2003; Yamada et al., 2003). Osteoporosis is the major metabolic bone disorder and is indicative of reduced mineral density due to low calcium levels. Thus, onset of osteoporosis is dictated by several factors including age. Another factor the risk for osteoporosis includes administration of corticosteroids since these decrease calcium absorption and bone cell functions. The measurement of bone density is done through X ray absorptiometry and lower values of deviation from young adult mean measurements indicate higher bone mineral density. The normal measure of bone mineral density by dual energy X-ray absorptiometry is 1. In 1994, the World Health Organization (WHO) proposed that bone mineral density measurements 2.5 standard deviations below the mean value for a young adult is characteristic of osteoporosis (Horner, 2006). Bone mineral density standard deviation for normal individuals should not exceed 1. In addition, the young adult population is used as a standard since it is a stage of highest bone mineral density and bone mass (Horner, 2006). Osteoporosis may be treated using pharmaceuticals such as bisphosphonates, which prevent bone dissolution by osteoclasts. Hormone replacement therapy such as oestrogen receptor modulator therapy reduces the incidents of fracture (Grady et al., 1992) and has been shown to lower the risk of endometrial cancer when administered with progestagens. The effect, however, is reversible and bone demineralization and loss can return when treatment is stopped. Prolonged use of oestrogen, on the other hand, may lead to breast cancer (Colditz et al., 1995), thus requiring its controlled use. The administration of calcitonin can also help to inhibit osteoclast synthesis and prevent bone turnover (Chestnut et al., 2000). Parathyroid hormone injections can induce bone formation and enhance bone strength (Delmas et al., 2002) while simultaneous intakes of calcium and vitamin D2 supplements can increase bone mineral density (Elders et al., 1994). Increased doses of calcitriol can trigger the natural effects of vitamin D but controlled doses should be used to prevent hypercalcemia. 1.6.5- Vitamin D Toxicity Vitamin D toxicity occurs rarely but may result from the excessive use of injectable vitamin D drugs (Holick, 2007) usually administered to patients with hepatic failure. It is also associated with hypercalcemia and hyperphosphatemia (Wolf and Thadhani, 2007). The excessive levels of calcium and phosphates are associated with 25-(OH) vitamin D concentrations of >374 nmol/L and may result from daily vitamin D supplementation of 50,000 IU. Daily doses of up to 10,000 IU do not cause vitamin D toxicity (Vieth, 2004). Other individuals are more sensitive to 25-(OH) vitamin D increase, e.g. >75nmol/L. For example, in cases of granulomatous disorders, macrophages also synthesize 1,25(OH)2D3. Normal individuals on the other hand, require 50-75 nmol/L (Holick et al., 2006). The rare cases of vitamin D intoxication can be diagnosed by measuring 25-(OH) vitamin D levels (Hollis, 1996), although excessive vitamin D synthesis in the body has a very low likelihood due to the strict regulatory mechanisms and the equilibrium reactions involved in the metabolic pathways. 1.7-Assay Methods for Vitamin D and its Metabolites (Historical Background) Detection and quantification methods of vitamin D are extremely important in clinical practice due to their applications in the characterization of vitamin D associated diseases and corresponding therapeutic advances (Mata-Granados et al., 2004). Currently used methods measure the amounts of several forms of vitamin D, but limitations in the instrumentation as well as time constraints can restrict a researcher or clinician to few analyses. A suitable method would entail short preparative steps and extraction protocols, high specificity, fast analysis, high recovery with unimpaired sensitivity and accuracy, and safe, hazard-free use. Levels of 25-(OH) vitamin D3 are a diagnostic tool for several clinical conditions, including vitamin D-dependent rickets, type I and II rickets, and hypercalcemia since these conditions are associated with increased levels of the metabolite. 25-(OH) vitamin D2 is usually measured as well and a combination of the two metabolites is used for confirmatory results (Hollis, 1996). The assays are rigorous due to the unique properties of vitamin D, its polarity and its binding properties. Around 85% of 25-(OH) vitamin D3 is DBP bound, while 15% is complexed with albumin and 0.03% remains free (Bikle et al., 1986). This natural complex requires purification during analysis to avoid inaccurate signals, which is generally achieved using chromatographic separations (Wootton, 2005). The quantitative method initially used is the competitive protein binding assay but recent developments have already introduced more advanced methods such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), and liquid chromatography coupled with mass spectrometry (LC-MS) (Hollis and Horst, 2007). 1.7.1- Competitive Protein Binding Assay Competitive protein binding assays (CPBA) for the quantification of vitamin D through concentration of its metabolites were developed in the early 1970s. These commonly used methods require careful handling because of the use of tritium and the difficulty in sample preparation, which includes organic extraction, solvent drying under nitrogen gas and chromatography prior to analysis. Competitive protein binding assays have been developed for vitamin D metabolites 25-(OH) vitamin D3 (Haddad and Chyu, 1971) and 1,25-(OH)2 vitamin D3 (Brumbaugh et al., 1974). The method measures ligand binding of the vitamin D forms to DBP or chick intestinal receptor and is detected by tritium labeled ligand. In addition, the steroid’s hydrophobic nature poses critical problems during the assay and the time for analysis and sample preparation restricts the use of this technique in clinical practice (Hollis and Horst, 2007). The CPBA method was later modified to incorporate chemiluminescence detection and it is still used as a method to detect and quantify vitamin D levels (Roth et al., 2001). 1.7.2- Enzyme Linked Immunosorbent Assay Enzyme linked immunosorbent assay (ELISA) is an immunological technique that uses antibodies to measure several biochemical markers such vitamin D which acts as an antigen. Both ELISA and radioimmunoassay (RIA) are the method of choice to measure biochemical markers for bone remodeling studies (Arden et al., 2005). The method uses color reactions by complexing the antibodies with enzymes that catalyze color forming reactions and allow detection. However, the method for vitamin D detection on ELISA uses chemiluminescent dyes as labeling agents. Despite the simplicity of the technique, this method requires expensive antibodies and a special instrument for detecting the dye (Hollis and Horst, 2007). Tedious work may also involve designing antibodies that will recognize a specific metabolite or other forms of vitamin D as well as analogs. Current commercial methods employing this technique include Immundiagnostik AG, which uses an enzyme immunoassay for vitamin D detection, and the Biomedica Gruppe (Lensmeyer et al., 2006). 1.7.3- Radioimmunoassay Radioimmunoassay (RIA) for vitamin D uses antibodies specific for both 25-(OH) vitamin D2 and 25-(OH) vitamin D3 and employs acetonitrile for the quantification of total serum levels of 25-(OH) vitamin D. This method was later improved by using iodine-125 to act as a labeled tracer. Other commercial methods use selective antibodies such as in the Diasorin and IDS Ltd. methods where I125 is also used (Hollis and Napoli, 1985; Hollis et al., 1993; Lenmeyer et al., 2006). Recent chemiluminescent assays have also been used in RIA methods. These modifications have used DBP (Nichols Institute Diagnostics, 2004) or antibodies for selective binding (Clive et al., 2002). Diasorin RIA is a commercial kit based on the Hollis assay that uses organic solvents such as acetonitrile for initial extraction of the vitamin D metabolites. Then, a competitive radioimmunoassay step follows, which is tagged by I125 labelled 25-(OH) vitamin D and antibody to 25-(OH) vitamin D that is detected by a secondary antibody (Hollis et al., 1993). 1.7.4- Chemiluminescence Protein Binding Assay The chemiluminescence protein binding assay ( CBPA ) is a modification of the process flowered by the elimination of the preparative chromatography step (Belsey et al., 1974) but the currently adapted CBPA uses the Nichols advantage automated chemiluminescent method for 25-(OH) vitamin D and no longer uses tritium as a label (Roth et al., 2001). However, some reports indicate that this method is not as accurate as HPLC methods (Glendenning et al., 2006), because it cannot distinguish D2 and D3 co-contamination and favors detection of 25-(OH) vitamin D3 (Carter et al., 2004), and results in low metabolite recovery (Hollis and Horst, 2007). Easy separation of 25-(OH) vitamin D is achieved using denaturing agents to act on the protein and detection methods employ chemiluminescence that uses acridinium-ester (Nichols Institute Diagnostics Manual, 2004). 1.7.5- High Performance Liquid Chromatography High performance liquid chromatography (HPLC) is a fast and direct method for quantification of vitamin D and it has the ability to distinguish between C-25 hydroxylation derivatives of vitamin D2 or D3 (Hollis and Horst, 2007; Lensmeyer et al., 2006) or other forms, such as 25-(OH) vitamin D3, 1,25(OH) 2 vitamin D3, and 24,25(OH) 2 vitamin D (Wootton, 2005). For example, HPLC uses preparative methanol/ hexane extraction, silica Sep-Pak purification, differential dissolution, filtration, and improved sample injection methods. In 1989, Kukasziewicz and colleagues improved HPLC detection and quantification methods by using fully mechanized schemes that reduce assay difficulties. The technique, employed by Mata-Granados et al., 2004 has efficiently combined a solvent solid-phase extraction method with HPLC as a purification tool. Using this method, analysis times are reduced and standard results are statistically sound (Mata-Granados et al., 2004). This method allowed subsequent analyses of vitamin D3 at sensitivity values of 25 nmol/L and some other fat -soluble vitamins in human serum. In addition, despite varying levels of 25-(OH) vitamin D and 1,25(OH)2 vitamin D3, Brunetto et al. (2004), employed reverse phase HPLC to simultaneously detect the levels of these two forms of vitamin D in human plasma. They achieved detection of the compounds at 265 nm in a broad range of 7.5-80.0 nmol/L. The limit of detection was also at 7.5 nmol/L and the time analysis was relatively short at only 16 minutes, yet characterized by high precision as compared with other detection methods. In addition, Hollis and Horst (2007) stressed that for non-routine vitamin D assays, the use of HPLC provides real time analysis and easier troubleshooting but they do not recommend this type of analysis for a fast-paced, high capacity clinical environment. Nonetheless, HPLC is an easier and more efficient method than some commercially available kits (Binkley et al., 2004). 1.7.6- Liquid Chromatography-Tandem Mass Spectrometry Liquid chromatography-tandem mass spectrometry (LC-MS) is a fairly recent method used to analyze vitamin D concentration and provides solutions to many problematic areas in other assays such as detection limits and metabolite polarity dilemma (Hollis and Horst, 2007). However, the machine is costly and has difficulties in distinguishing other metabolite forms of vitamin D. This method utilizes a Technologies Aria™ TX series HTLC™ coupled with a mass spectrometer system for analysis. However, samples still require initial separation analysis and small samples are injected to the LC-MS system gradient was achieved within 7 minutes (Chen et al., 2007). Other modern modifications to the LC-MS tandem include the coupling of the method to other advanced accessories such as data analysis software. This achieves fast reaction times by allowing analysis of crude extracts. The LC-MS/ MS methods, for example, include fast atom bombardment LC-MS/MS with Cookson-type reagents (Yeung et al., 1992). Another trio involves an isotope-dilution electrospray LC-MS/MS ( Maunsell et al, 2005; Tsugawa et al., 2005; Vogeser et al., 2004). 1.7.7- Limitations of Vitamin D Assays Although synthesized in the skin, the amount of Vitamin D is determined by measuring the concentration of the metabolite in the circulatory system (Webb, 2005). However, instead of the active metabolite, 25-(OH) vitamin D levels are measured. The levels of 1,25-(OH)2 vitamin D3 may provide an erroneous assessment of an individual’s vitamin D intake or synthesis as levels are affected by many molecular signals in the cell. 1,25-(OH)2 vitamin D3 levels have been observed to be 1000 times lower than that of 25-(OH) vitamin D. This variability poses difficulties in the assays currently used. In addition, secondary hyperparathyroidism also increases the production of 1,25-(OH)2 vitamin D3 in the kidneys (Hollick, 2002). Because 25-(OH) vitamin D3 has a long half-life (Wootton, 2005), it is detected easily at constant values and the fluctuation of vitamin D levels in different individuals can be seen from the following values. In a study of determining vitamin D levels in 46 volunteer females age 50 to 94 years, Brunetto et al. (2004) observed that cholecalciferol levels ranges from 12 to 90 nmol/L while 25-(OH) vitamin D3 plasma levels ranges from 10 to 36.52 nmol/L. Thus, current methods measure both derivatives of vitamin D but the wide concentration differences of each in the sample also complicate the analysis. The type of direct analysis such as UV spectroscopy detection that can detect the minute quantities of 1,25(OH)2 vitamin D3 (occurring at picomole quantities in the sample) has also been employed and scientists choose methods that can determine the metabolite’s quantity within their limits of detection. On the other hand, 25-(OH) vitamin D, exists in nanomole quantities such that co-detection is problematic (Hollis and Horst, 2007). Adding to the variability is the difference in the sensitivity of the methods that which can easily confuse researchers and physicians (Lensmeyer et al., 2006). Under- or overestimation is also a pressing problem in vitamin D assays and it stems from differences in binding affinities of the antibody or binding proteins employed (Hollis, 2000). Temperature inconsistencies during antigen-antibody or protein binding hybridization might play a role. Intermolecular forces of attraction and variable recognition of the 25-(OH) vitamin D2 and 25-(OH) vitamin D3 can also account for differences (Wootton, 2005). The advent of kits has made the assays easier, but kits are generally expensive and can sometimes be wasteful, thus, some experts believe that commercial kits yield inaccurate results (Wootton, 2005). Nonetheless, these methods are undergoing further modification and improvement. New methods are also evolving to modify, simplify, and improve the assay for vitamin D to advance our current knowledge on the potentials and health benefits of this vital vitamin and hormone.