From Cholesterogenesis to Steroidogenesis: Role of Riboflavin and Flavoenzymes in the Biosynthesis of Vitamin D^1,2

Mitochondrial (type I) cytochrome P450 system.

The type I mitochondrial enzyme system consists of 3 separate components, namely CYPs, ferredoxin, and ferredoxin reductase. Ferredoxin reductase (also called adrenodoxin reductase) is a 50-kDa flavoprotein located in the mitochondrial inner membrane. NAD(P)H generated within the matrix first interacts with adrenodoxin reductase, which passes electrons to adrenodoxin (or ferredoxin), a 12–14-kDa iron sulfur protein located within the mitochondrial matrix. Adrenodoxin is the electron shuttle between adrenodoxin reductase and CYPs. The pattern of electron shuttle involves NAD(P)H to FAD-adrenodoxin reductase, FAD-adrenodoxin reductase to adrenodoxin, and adrenodoxin to heme protein–associated exchanges and is shown in Fig. 3.

Thus, mitochondrial CYPs receive electrons from NAD(P)H, which is generated by isocitrate dehydrogenase and glucose-6-phosphate dehydrogenase located within the mitochondrial matrix (26). Through the intermediate flavoprotein, adrenodoxin reductase, a reducing equivalence of NAD(P)H is relayed to adrenodoxin and subsequently to the substrate to form a hydroxylated product. The diversity of the P450 redox system and the types of protein-protein arrangements with mono- and diflavin oxidoreductases required for electron transfers have recently been reviewed elsewhere (27).

Type I electron transfers exhibit 1 FAD-binding domain, whereas type II electron transfers with NAD(P)H-cytochrome P450 reductase (CPR) [see NAD(P)H cytochrome P450 oxidoreductase (CPR) below] use both FAD and FMN. Type II transfers are described below (see NADPH-cytochrome P450 reductase, CPR) and differ from type I in that CPR has evolved an FMN-binding domain that functions exclusively within the microsomal compartment. Some type II reactions will also recruit cytochrome b5 for electron transfers but only after the first electron has been delivered by the FMN moiety of CPR.

Microsomal (type II) cytochrome P450 system.

A small but biologically important subfamily of flavoenzymes contains both FAD and FMN coenzymes and constitutes the family of diflavin reductases. The crystallographic and amino acid structures of the diflavin reductases suggest that these proteins evolved by a fusion of 2 ancestral genes that encoded the N-terminal portion of the protein, an FMN-containing flavodoxin, and the C-terminal portion, an FAD-containing ferredoxin-NADP(H)-reductase (28). The prototypic members of mammalian diflavin reductases are NO synthases (EC 1.14.13.39), methionine synthase reductase (EC 1.16.1.8), NAD(P)H sulfite reductase (EC 1.8.1.2), cytoplasmic NR1 protein, and CPR (9, 29, 30). It is the last member of these diflavoproteins that is a membrane-bound component of the endoplasmic reticulum. CPR is the obligate partner protein for all microsomal P450 enzymes involved in metabolism of cholesterol, steroids, and vitamin D.

NAD(P)H cytochrome P450 oxidoreductase (CPR).

CPR was the first member of the diflavin family to be isolated and the most extensively characterized. In mammals, a single CPR is responsible for the electron transport to a diverse array of P450s that are involved in the synthesis of biologically important endogenous compounds such as steroids, FAs, and prostaglandins. Thus, knockout mouse models are embryonically lethal (31). In addition to its physiologic functions, CPR plays a vital role in the reductive degradation and sometimes activation of therapeutic drugs and xenobiotic substances. CPR has been extensively studied (32). It is an 82-kDa, membrane-bound protein that serves as an integral component of the microsomal monooxygenase system that transfers electrons from NAD(P)H to ~57 CYPs via its FAD and FMN component coenzymes.

Studies of the CPR active site show that the proton status of a highly conserved γ-carboxylic acid moiety from glutamate favors binding of NAD(P)H over that of NADP⁺ (33). The resident flavin moieties are located in domains distinct from each other and are separated by a flexible hinge region. After transfer of a hydride ion carrying an electron pair from the reduced nicotinamide ring of NAD(P)H, FAD aligns its isoalloxazine ring with that of FMN and discharges its cargo electrons. The redox environment of FMN allows sequential electron flow to a variety of partner proteins that include both heme and other flavoproteins (34). The electron transfer usually follows the pathway:

Thus, each catalytic cycle of CPR restores the reduced status of CYP with an electron flow from NAD(P)H to the flavin electron acceptors before reacting with its target acceptor proteins.

Two factors that markedly influence the activity of CPR and thus affect cholesterogenesis are dietary riboflavin status and genetic mutations. Studies in riboflavin-deficient animals showed marked decreases in the concentrations of CPR and cytochrome b5 proteins. Subsequent repletion of animals with riboflavin resulted in complete recovery (35, 36). In microsomes of riboflavin-deficient rats, the amount of FMN bound to CPR is lower than that of FAD. A 1:1 ratio of FMN to FAD is critical for maximum activity of CPR (37). The altered FMN:FAD ratio is consistent with the observation that riboflavin deficiency causes a decrease in flavokinase activity and an increase in FMN pyrophosphorylase (FAD synthetase) activity, the enzymes that synthesize FMN and FAD, respectively (38). The addition of FAD to liver microsomes isolated from riboflavin-deficient animals failed to replete enzymatic activity, which illustrates the selectivity for the flavin moieties; however, the FAD that binds to the FMN domain can be readily replaced by FMN, which restores CPR activity. Thus, CPR activity illustrates the critical need to maintain correct balance of the flavin nucleotides and proper coenzymic form within the flavin-binding domains.

Although several polymorphisms and missense mutations have been identified in the CPR gene, 2 missense mutations, in particular V492E and R457H, result in instability and misfolding of CPR that involve FAD binding (39). The binding affinity of V492E is lower than that of R457H for FAD; however, it does cause the protein to have an activity as an electron donor that is more easily reconstituted with the addition of exogenous FAD. V492E is located at the base of the β-flap and influences stacking and arrangement of the molecules between the FAD/NAD(P)H domain and the connecting domain. By contrast, the amino acid substitution, R457H, displays a more tightly bound FAD, but reestablishment of activity is somewhat more resistant to FAD reconstitution. Regions of polypeptide unfolding within CPR differ among these CPR variants. V492E is less stable due to its low binding affinity for FAD and demonstrates a gradual, localized unfolding in the absence of FAD as demonstrated by partial trypsin digestion (40). R457H, on the other hand, binds FAD more tightly, but unfolding is observed more globally throughout the CPR variant. Both mutants are stable to partial trypsinization in the presence of FAD (40). The presence of FAD markedly stabilizes CPR function and suggests a preventive role for riboflavin supplementation in women whose fetuses may harbor these mutations. Mutations in CPR manifest in utero as ambiguous genitalia, abnormal patterns of steroid hormones, and/or skeletal deformities. Thus, patients identified prenatally or with less severe cases of CYP reductase deficiency may be rescued postnatally with supplemental riboflavin (40).

Given the obligatory role of CPR as the primary electron donor for a variety of CYP enzymes, deficits in flavin availability other than those caused by dietary deficiency would also have far-reaching metabolic and developmental consequences. Riboflavin deficiencies can be caused by endocrine abnormalities such as adrenal or thyroid hormone insufficiencies, overexposure to certain tri- and tetracyclic drugs, or excessive ethanol consumption. These deficits in riboflavin would manifest with deformities similar to those occurring in patients with CPR missense variants, V492E and R457H. Such abnormalities would impair riboflavin status by blocking conversion of the vitamin to its physiologically active forms, by interfering with intestinal absorption and transport, and by enhancing renal excretion. Reviews of factors that adversely affect riboflavin metabolism have been published elsewhere (1, 35, 38). The teratogenic effects of specific drugs that interfere with flavin metabolism will be discussed later in this review. In particular, tri- and tetracyclic drugs that bear structural relations with riboflavin and interfere with its metabolism can cause abnormalities similar to those in individuals with defects in FAD-dependent oxidoreductase, 24-dehydrocholesterol reductase (DHCR24), CYP51, and the microsomal 25-hydroxylase, CYP2R1. The pattern of abnormalities observed in individuals with mutations in these enzymes is similar to the teratogenic effects observed with drugs that induce alterations in flavin metabolism. Many of these abnormalities include cleft palate formation, hydrocephalus, and skeletal deformities as well as shortening of distal extremities of fetuses.

Squalene monooxygenase.

Squalene monooxygenase (EC 1.14.99.7), an FAD-dependent enzyme, is a potential regulatory site in cholesterogenesis that relies on CPR for reducing equivalents. This 90-kDa enzyme catalyzes oxidation of squalene to squalene-2,3-epoxide [(3S)-2,3-oxidosqualene]. This epoxide undergoes a rapid cyclization catalyzed by lanosterol synthase to form lanosterol, the first C30 steroidal intermediate (46). In this remarkable reaction, a series of 1,2-methyl group and hydride transfers occur along the squalene chain to cause ring closures and formation of the tetracyclic structure of cholesterol, the cyclopentanophenanthrene ring (47). This process is considered one of the most complex single enzymatic reactions thus far identified for an enzyme of such a small molecular size (48). FAD binding to the protein occurs within a moderately hydrophobic pocket bearing a conserved amino acid sequence motif, GxGxxG, characteristic of Rossmann folds, which recognize the ADP moiety of FAD (49). The isoalloxazine ring of FAD interacts with conserved aromatic amino acid residues, thus forming π-π interacting ring structures, which help stabilize the enzyme (50).

Within the endoplasmic reticulum, sterol carrier protein 1 [also known as supernatant protein factor (SPF)] (51) binds squalene in the presence of phosphatidylserine, FAD, NAD(P)H, and oxygen and promotes squalene epoxidation (52). In the squalene monooxygenase reaction, NAD(P)H forms a transient complex with the enzyme and dissociates immediately after transferring its hydride equivalence to FAD (53). Purified SPF stimulates squalene monooxygenase and squalene-2,3-oxide lanosterol cyclase [also known as lanosterol synthase (LSS)]. The latter enzyme also conducts a side reaction with squalene-2,3-epoxide to produce a potentially useful 2,3;22,23-squalene dioxide, a reaction that has an absolute requirement for exogenous FAD (54, 55). These studies show that 2,3;22,23-squalene dioxide has a half-maximal inhibitory concentration (IC₅₀) of 16 μmol/L for liver oxidosqualene cyclase (LSS), the enzyme that generates lanosterol, the tetracyclic precursor of cholesterol. Partial inhibition of LSS by squalene dioxide favors synthesis of 24(S),25-epoxycholesterol over that of cholesterol. 24(S),25-Epoxycholesterol has been shown to repress 3-hydroxy-3-methylglutaryl–coenzyme A (HMG-CoA) reductase activity and enhance its degradation. Thus, supplemental dietary riboflavin may have the potential to favor formation of 24(S),25-epoxycholesterol. Overproduction of this cholesterol derivative would result in the generation of a negative regulatory feedback loop with HMG-CoA reductase that would limit cholesterol biosynthesis. Epoxidations of the terminal double bonds of squalene are shown in Fig. 4.

Squalene monooxygenase appears to play a central role in overall regulation of cholesterol biosynthesis. Studies show that addition of cholesterol to hepatocytes in culture suppresses mRNA levels of squalene monooxygenase, subsequently diminishing the activity of squalene monooxygenase and resulting in accumulation of squalene (56). Thus, squalene monooxygenase is an attractive therapeutic target for drugs to lower serum cholesterol (57). In view of its flavin requirement, the apparent Michaelis constant (K_m) of 0.3 μmol/L is consistent with a freely dissociable FAD moiety, and the K_m for its protein partner, CPR, the primary reductant flavoprotein, is in the low nmol/L range (46). The associated K_m of squalene monooxygenase with CPR is lower than that compared with most of the other CPR client proteins (including heme partner enzymes). In view of the high-affinity protein-protein interactions, targeting CPR may potentially limit monooxygenase activity (58). A blockade at the site of squalene monooxygenase would result in accumulation of squalene, which is considered nontoxic and thus would be expected to spare needed isoprenyl units for other vital cell metabolic processes (57). By contrast, inhibitors of enzymes involved in the initial phase of cholesterogenesis, such as HMG-CoA reductase, would limit production of dolichol, farnesyl, and geranylgeranyl, key isoprenoid metabolites essential for protein signaling and cell proliferation.

Several dietary constituents have been shown to interfere with squalene monooxygenase, which may explain their marginal effects on lowering serum cholesterol and improving cardiovascular function. Of interest are the green tea polyphenolics (epigallocatechin-3-O-gallates), red wine stilbenoids (resveratrol, trans-3,4′,5-trihydroxystilbene), and allylsulfides (including allylcysteines) from garlic. These derivatives act as reversible, noncompetitive inhibitors by scavenging the reactive oxygen species formed at the active site of the flavin moiety, namely the flavin C4a hydroperoxide (59, 60). A review of nonstatin strategies for treatment of hypercholesterolemia has been published elsewhere and offers a perspective for the use of inhibitors that start at the site of squalene synthase in the cholesterol biosynthetic pathway (61).

Cytochrome P450, family 51, subfamily A, polypeptide 1.

After formation of lanosterol, the first sterol in the cholesterogenic pathway, the 14α-methyl moiety is oxidatively removed by lanosterol 14α-demethylase to produce 4,4-dimethyl-5α-cholest-8(9),14,24-triene-3β-ol. This enzyme also demethylates 24,25-dihydrolanosterol, which is the reaction product of the flavoenzyme, sterol-Δ²⁴-reductase, on lanosterol (see 24-dehydrocholesterol reductase below). Removal of the C14-methyl moiety is critical because sterols with a methyl in this position cannot replace membrane cholesterol or any of the steroid hormones (61). Lanosterol 14α-demethylase is a 53-kDa CYP51A1 protein located in the endoplasmic reticulum that catalyzes a 3-step reaction each requiring 1 molecule of oxygen and 2 NAD(P)H-derived reducing equivalents supplied by CPR (62). Figure 5 shows the transfer of reducing equivalence required in 3 consecutive reactions resulting in the C14-demethylation of lanosterol to 4,4-dimethyl-5α-cholest-8(9),14,24-triene-3β-ol.

CPR plays a critical role in this reaction in that a mutation in FAD binding in this flavoenzyme or a diminished association with CYP51A1 is linked to a skeletal dysmorphology syndrome (Antley-Bixler syndrome) (63, 64). Interference in the ability of these 2 partner proteins to interact appears to be the major underlying mechanism that leads to Antley-Bixler syndrome. This view is supported by the fact that, to date, no genetic mutation has been identified that directly disrupts or abolishes CYP51A1 enzymic activity (65). However, a V13E amino acid variant in the N-terminal portion of CYP51A1 has been reported to affect its ability to associate with the endoplasmic membrane and perhaps the membrane-bound CPR (42, 62). As mentioned previously, mutations identified in CPR could also account for decreased CYP51A1. A decrease in the activity of this enzyme is observed in patients with Antley-Bixler syndrome.

Of the 14 cytochrome proteins involved in cholesterol homeostasis, CYP51A1 is the only P450 enzyme that participates in cholesterol biosynthesis. Thirteen other CYPs are involved solely in cholesterol degradation, subsequent steroidogenesis, and bile acid formation (66). Thus, interfering with the ability of the CYPs to interact with CPR or disrupting the stability of protein-protein complexes may provide a viable target to control cholesterogenesis. A blockade of CYP51A1 (lanosterol demethylase) activity results in accumulation of lanosterol and 24-dihydrolanosterol (67). In studies using CPR-null mouse livers, the accumulation of these methylated sterols stimulated ubiquitination of HMG-CoA reductase causing degradation of the enzyme and blocking the initial phase of cholesterogenesis (68).

24-Dehydrocholesterol reductase.

The FAD-dependent oxidoreductase DHCR24 (EC 1.3.1.72) catalyzes reduction of the Δ²⁴ double bond in the side chain of several obligatory intermediates in the cholesterol pathway. Due to the broad substrate specificity of this flavoenzyme for metabolites between lanosterol and cholesterol, the precise sequence of reactions that lead directly to cholesterol appears to follow a circuitous route. For convenience, the postsqualene pathway is divided into the Bloch and the Kandutsch-Russell pathways. In the Kandutsch-Russell division, DHCR24 acts initially on lanosterol. Thus, subsequent metabolites from 24,25-dihydrolanosterol to 7-dehydrocholesterol exhibit a saturated side chain. Among the other key precholesterol metabolites in the Bloch division, zymosterol and desmosterol are excellent substrates for DHCR24, whereas 24-dehydrolathosterol exhibits the highest affinity for DHCR24 and is the most reactive substrate (69). In contrast to metabolites in the Kandutsch-Russell pathway, Bloch division metabolites from lanosterol to desmosterol exhibit the Δ²⁴ double bond. Figure 6 shows the separate but interactive pathways of postlanosterol metabolites possessing a fully saturated side chain (Kandutsch-Russell pathway) and a Δ²⁴ double bond (Bloch pathway). The molar ratios of the sister metabolites in each pathway are governed by the catalytic capacity of the flavoenzyme, DHCR24, the bioavailability of dietary riboflavin, or drugs known to block conversion of riboflavin to its physiologically active coenzymes.

Desmosterolosis, a rare disorder of cholesterol biosynthesis, is caused by mutations in the gene that encodes for DHCR24 (70). To date, desmosterolosis has been described in only 3 patients (71). DHCR24 contains a leader sequence that directs it to the endoplasmic reticulum membrane. A missense mutation in this flavoprotein causes desmosterolosis, which presents biochemically with elevated desmosterol and phenotypically with spasticity, microcephaly, and micrognathia in affected patients (72). In 1 patient, the mutation of R94H, for which the moiety R94 resides at the FAD binding site, resulted in desmosterolosis (71).

The syndrome resulting from genetic mutations of DHCR24 is similar to those observed in animals during severe maternal riboflavin deficiency, namely micrognathia, cleft palate, hydrocephalus, and skeletal anomalies as well as reduction-type defects in the distal extremities of fetuses (73, 74). It is also intriguing to consider that drugs that bear structural similarities to those of riboflavin [e.g., tricyclic antidepressants and phenothiazine derivatives (75–77)] and interfere with its conversion to FMN and FAD exhibit comparable teratogenic manifestations (78).

Enzyme analyses of DHCR24 that follow the reduction of the Δ²⁴ double bond in desmosterol to form cholesterol show 2-fold activity increases in the presence of exogenous FAD. This indicates the responsiveness of the conserved domain to exogenous flavins, which is characteristic of the FAD-dependent oxidoreductases found in the DHCR24 amino acid sequence (72). Conversely, in constructs containing mutant DHCR24 alleles from patients with desmosterolosis, the conversion from desmosterol into cholesterol is absent or markedly reduced (72).

7-Dehydrocholesterol reductase.

7-Dehydrocholesterol reductase (DHCR7) catalyzes the terminal reaction in cholesterol biosynthesis, which involves reducing 7-dehydrocholesterol to form cholesterol. Functional DHCR7 is a 55.5-kDa NAD(P)H-requiring protein located in the microsomal membrane. Previous studies had suggested that DHCR7 required CPR for catalyzing the reduction of 7-dehydrocholesterol (98). However, recent studies show that the activity of DHCR7 is not enhanced in the presence of FAD, nor does it form a partner complex with the flavoprotein CPR (99). Further studies showed that in microsomes from livers of CPR-null mice, a modest increase in levels of DHCR7 mRNA occurs.

It is important to consider here that 7-dehydrocholesterol is the immediate precursor of both cholesterol and vitamin D₃ (cholecalciferol) and thus represents a critical branch point in the cholesterol biosynthetic pathway (56, 100). Most important, cholesterol cannot be oxidized back to 7-dehydrocholesterol and therefore cholesterol is not a precursor of vitamin D₃. Rather, both vitamin D₃ and cholesterol share 7-dehydrocholesterol as a common precursor (see Fig. 7).

In view of the regulatory function that several flavoenzymes have within the postsqualene section of the cholesterol biosynthetic pathway, riboflavin status potentially would have an impact on controlling vitamin D₃ formation from 7-dehydrocholesterol. Studies show that the intracellular concentrations of cholesterol per se influence vitamin D₃ status at 2 sites controlled by FAD-dependent enzymes, namely at squalene monooxygenase and CYP51A1 (101). In studies using cell culture models for cholesterogenesis, both the expression of mRNA and the activity of squalene monooxygenase were suppressed by the addition of cholesterol to the media (68, 102). This inhibition at the squalene level would decrease formation of 7-dehydrocholesterol and limit available substrate for the photosynthesis of vitamin D₃. At the second site, inhibition of the CYP51 (lanosterol demethylase) would result in accumulation of lanosterol and 24-dihydrolanosterol, both of which block 7-dehydrocholesterol formation by accelerating ubiquitination and proteasomal degradation of HMG-CoA reductase (67, 103). Thus, decreased dermal formation of 7-dehydrocholesterol would be expected to diminish its conversion to cholecalciferol (previtamin D₃) when exposed to UV-B sunlight. By contrast, in patients with Smith-Lemli-Opitz syndrome (104, 105), which is characterized by marked increases in 7-dehydrocholesterol, cutaneous synthesis of vitamin D and circulating concentrations of vitamin D metabolites were not found to differ from normal controls who were matched for age and season of blood collection (106).

Formation of 25-hydroxycholecalciferol: role of type II cytochrome P450 heme proteins.

Cholecalciferol, either biosynthesized in skin or derived from dietary sources, is converted to 25(OH)D in a variety of tissues. This vitamin D metabolite is the major circulating form of vitamin D and a key biomarker of vitamin D status. The formation of 25(OH)D is catalyzed by 5 microsomal CYP hydroxylases (CYP2R1, CYP2J2/3, CYP3A4, CYP2D25, and CYP2C11) (type II) and 1 mitochondrial hydroxylase (CYP27A1) (type I) (116, 117). Each of the CYP proteins exhibits different catalytic properties that are not exclusive in their formation of 25(OH)D from cholecalciferol because they also hydroxylate other vitamin D analogs, other sterols, and bile acid metabolites. An in-depth summary of vitamin D 25-hydroxylases covering 4 decades of research has been published (117).

Cytochrome P450 2R1.

Of the microsomal 25-hydroxylases, CYP2R1 appears to play a prominent role in maintaining physiologic concentrations of 25(OH)D in plasma (118). Unlike the other microsomal 25-hydroxylases, kinetic studies using recombinant Cyp2R1 indicate that the enzyme can conduct 25-hydroxylation by using physiologic concentrations of cholecalciferol (vitamin D₃) and ergocalciferol (vitamin D₂) as well as with their respective 1α(OH)-derivatives (119). To further suggest that CYP2R1 plays a physiologically important role in the vitamin D 25-hydroxylation in humans, genetic defects in the other microsomal enzymes show no or only marginal decreases in circulating 25(OH)D and present minimal risk for formation of rickets or osteoporotic lesions. By contrast, studies using Cyp2R1^−/− mice indicate that serum concentrations of 25(OH)D were reduced by 50% of those in control animals, whereas 1α,25-dihydroxyvitamin D [1α,25(OH)₂D] concentrations in serum remained unchanged (120). Nonetheless, the importance of the enzymatic redundancy and promiscuity among the 25-hydroxylases for sterol metabolites requires further study (117).

As discussed previously with the cholesterogenic (type II) microsomal enzymes, vitamin D 25-hydroxylases require reducing equivalence from the FAD/FMN oxidoreductase, CPR, which is tethered to the membrane of the endoplasmic reticulum. CPR is the exclusive flavoprotein that donates electrons to all 5 of the microsomal hydroxylases, and thus mutations in CPR would be expected to present clinically with overlapping symptoms such as those observed with apparent pregnene hydroxylation deficiency, Antley-Bixler skeletal malformation syndrome, Shprintzen-Goldberg syndrome, and DHCR7 deficiency, which manifests phenotypically as Smith-Lemli-Opitz syndrome (121–126).

In similar fashion, deficits in riboflavin availability or imbalances in the intracellular ratio of FAD and FMN, particularly during fetal development, would be expected to elicit clinical manifestations similar to those caused by mutations of CPR. This flavoenzyme exhibits a commonality of function with all microsomal CYP enzymes. Early studies of acute riboflavin deficiency during embryonic development suggested that specific pathways governed by the action of flavoenzymes were essential for normal differentiation. According to this concept, if the flavin concentration of the embryo, or within certain areas of the embryo, falls below a critical nadir, anomalies will result (127). Subsequent investigations showed that the total riboflavin and FAD concentrations of differentiating rat pup embryos can be reduced by as much as 30% without ill effects. By contrast, a 60% reduction in FAD is teratogenic (128).

Congenital malformations due to riboflavin deficiency and those associated with the genetic alteration of CPR and vitamin D deficiencies manifest as cleft palate, micrognathia, micromelia, absent or fused vertebrae, short ribs, and failed closure of the neurotube (craniorachischisis). As mentioned previously, these same teratogenic manifestations have also been related to use of phenothiazine drugs, which bear structural analogies to the isoalloxazine moiety of flavins. An increased risk of congenital malformations in humans occurs when the human fetus is exposed to phenothiazines during weeks 4 to 10 of gestation (129). In addition, the side chain of tricyclic compounds plays an important role in their inhibitory action on flavoenzymes and conversion of riboflavin to FMN and FAD (130). Accordingly, in rats, riboflavin administration can lower prenatal death rates caused by single doses of phenothiazines given on the 14th day of gestation and can provide varying degrees of protection against certain other drug-induced teratogenicities (131). The experimental congenital malformations induced in the offspring of pregnant riboflavin-deficient rats and in those administered drugs structurally similar to riboflavin have features in common with those induced by defects in vitamin D and sterol metabolism. The commonality in these defects appears to converge on a central alteration associated with diminished activity of CPR, due possibly to decreased riboflavin availability, displacement of the FMN and FAD coenzymes from their respective binding sites, or alterations in the FMN:FAD ratio of the enzyme within the endoplasmic reticulum (121).

Clinical investigations that examine metabolism of micronutrients often find that deficiency symptoms of vitamins tend to be multiple. The impact of a single vitamin deficiency on the efficacy of another is best examined by using animal models where pair-feeding regiments as well as eucaloric and control diets can be maintained. Aside from rare genetic defects in riboflavin transport such as the Brown-Vialetto-Van Laere syndrome, which is characterized by progressive sensorineuropathies, paralysis, and eventual respiratory failure, isolated deficiencies of riboflavin are not widely prevalent in the general population.

Several physical and clinical symptoms that manifest during riboflavin deficiency are not pathognomonic or exclusive to deficits in riboflavin. In this regard, only 1 study was found in the literature that addressed the impact of riboflavin deficiency directly on vitamin D metabolism (132). In this animal study, riboflavin deficiency caused a moderate decline in serum calcium as well as a decrease in the active transport of calcium through the small intestine. Measurements of 25(OH)D in the blood serum, the formation of 24,25-dihydroxyvitamin D [24,25(OH)₂D] in kidney slices and the content of nuclear receptors for 1α,25(OH)₂D in intestinal mucosa were significantly lower than those of control animals. Administration of vitamin D to riboflavin-deficient rats 6 d before death restored calcium homeostasis.

In conclusion, the 5 microsomal P450 isoforms capable of synthesizing 25(OH)D have different catalytic properties toward sterols and secosterols, exhibit different tissue distributions, and contribute diversely to sterol homeostasis. Despite their apparent variability as well as redundancy toward substrates, microsomal P450 enzymes draw electrons from the same diflavin oxidoreductase (CPR) within the endoplasmic reticulum and thus can be influenced by the extent of riboflavin trafficking and the concentration and ratio of flavin coenzymes within subcellular compartments.

Formation of 25(OH)D, 1α,25(OH)₂D, and their 24-hydroxy metabolites: role of type I oxidoreductases.

The other enzymes reliant on flavoproteins and critical for vitamin D metabolism are located exclusively in mitochondria. Accordingly, CYP27A1, CYP27B1, and CYP24 thus rely on type I oxidoreductase reactions within the mitochondrial matrix.

CYP27A1 (sterol 27-hydroxylase).

CYP27A1 is the only ubiquitously expressed mitochondrial enzyme that conducts a 25-hydroxylation with cholecalciferol. Thus, as a mitochondrial enzyme, its electron transfer partners are FAD-dependent adrenodoxin reductase and 2Fe-2S-adrenodoxin (133). CYP27A1 is located primarily in the liver, kidney, intestine, ovary, lung, and skin and the human CYP27A1 gene is under modest regulatory control by growth hormone, insulin-like growth factor-1, thyroid hormones, and dexamethasone (134).

Human CYP27A1 can hydroxylate many metabolites of cholecalciferol (but not ergocalciferol) to form C-25, C-26, and C-27 monohydroxy and C-24,25, C-1α,25 and C-25,26 dihydroxy derivatives (135, 136). Human CYP27A1 can also catalyze multiple reactions with cholesterol and bile acids (137). In addition, hydroxylations of vitamin D metabolites exhibit smaller maximum velocity (V_max):K_m ratio value (i.e., catalytic efficiency) than those of cholesterol metabolites (138). Thus, although CYP27A1 has the ability to activate vitamin D, it nonetheless lacks specificity and catalyzes multiple hydroxylations in addition to that of a 25-hydroxylase, a property it shares with the microsomal CYP hydroxylase CYP2D25 (139).

CYP27A1 exhibits a greater affinity toward cholesterol metabolites than toward vitamin D metabolites. Thus, high concentrations of cholesterol can diminish 25(OH)D synthesis. This effect is compatible with the observations that vitamin D insufficiency is often associated with increased blood cholesterol concentrations (140) and that obese children and adults with hypercholesterolemia usually express low circulating concentrations of 25(OH)D (141–143). More detailed analysis showed that the low concentrations of circulating 25(OH)D correlate with high concentrations of total cholesterol, LDL cholesterol, and TGs, whereas the low concentrations of active 1α,25(OH)₂D are associated with low HDL cholesterol (144).

Genetic mutations in CYP27A1 can completely inactivate the enzyme and lead to a reduced production of bile acids, in particular chenodeoxycholic acid, with a concomitant increase in bile acid alcohols. To date, >50 genetic mutations have been implicated that affect the amino acid domains critical for enzymic activity. These changes in amino acid profile of the enzyme markedly affect substrate binding, heme binding, interfacing with its mitochondrial redox partner (ferredoxin/adrenodoxin), membrane binding, and protein folding (41).

Patients with inborn errors in CYP27A1 manifest with cerebrotendinous xanthomatosis (CTX), a rare lipid-storage disease that is characterized by bile acid deficiency, neurologic dysfunctions, premature atherosclerosis, and sometimes osteoporosis. The clinical manifestations of CYP27A1 mutations also mimic features of amyotrophic lateral sclerosis (ALS) with progressive upper and lower motor neuron loss (145). It is interesting to consider that a relation between CTX and Brown-Vialetto-Van Laere syndrome may exist in that both neurodegenerative disorders are characterized by progressive multiple neurologic abnormalities in combination with ALS. Brown-Vialetto-Van Laere syndrome has been identified with mutations in 2 riboflavin transporter genes (146). An extension of these studies to ALS patients in general revealed decreases in expression of FAD synthetase, riboflavin kinase, cytochrome c1, and succinate dehydrogenase complex subunit B, a mitochondrial enzyme bearing a covalently linked FAD moiety (147). These results suggest that specific electron transport chain abnormalities associated with mitochondrial riboflavin metabolism or interference with a flavoenzyme may underlie or contribute to these neurodegenerative disorders.

In a similar fashion, interference with mitochondrial riboflavin metabolism after alcohol exposure in utero may underlie the cardiac pathologies and other deformities associated with the fetal alcohol syndrome. Ethanol markedly decreases bioavailability of dietary flavins (148) and has been shown to reduce activities of 2 mitochondrial inner membrane enzymes, cytochrome c oxidase and succinate dehydrogenase (149). When flavin-linked substrates were used to assess mitochondrial function in prenatal mice, respiration was depressed. By contrast, ADP-stimulated respiration was not compromised (149).

Earlier investigations had considered that CYP27A1 is a major contributor to the circulating pool of 25(OH)D (138). However, knockout mouse models of CYP27A1 and monoclonal antibodies raised against CYP27A1 did not show reductions in 25-hydroxylation of cholecalciferol (150). Even patients with CTX demonstrate only modest depressions in serum concentrations of 25(OH)D and exhibit only marginal risk of development of bone lesions (151). In view of the importance of CYP27A1 in the synthesis of bile acids, determinants that affect development of osteoporosis are probably associated more with malabsorption of fat-soluble vitamin D coupled with secondary production deficits in bile acids rather than with reduced formation of 25(OH)D (152). In this regard, it is important to consider that the gene promoter region of CYP27A1 does not exhibit a vitamin D–response element but is controlled by bile acids. In addition, in terms of activation of vitamin D metabolites and its utility for vitamin D metabolism, CYP27A1 exhibits specificity for cholecalciferol (vitamin D₃) and not ergocalciferol (vitamin D₂). This selectivity for vitamin D analogs is in contrast to that of the microsomal CYP2R1, which hydroxylates both vitamin D₂ and vitamin D₃ (135).

CYP27B1 (sterol 1α-hydroxylase).

The conversion of 25(OH)D to the active hormone 1α,25(OH)₂D or calcitriol is under strict regulatory control by CYP27B1, which occurs primarily within kidney mitochondria (153). In addition to renal production of calcitriol, real-time PCR and specific immunoprecipitation reactions have confirmed the extrarenal presence of 1α-hydroxylase (CYP27B1). Thus, mitochondria in placenta, macrophages, astrocytes, skin, intestine, as well as breast and prostate cells can also synthesize calcitriol (154). The activity of CYP27B1 in kidney is highly regulated. Renal CYP27B1 is upregulated by parathyroid hormone and downregulated by calcitonin, major hormones of the mammalian calciotropic homeostatic loop. In addition, renal CYP27B1 is downregulated by fibroblast growth factor-23 (FGF-23), a component of the phosphotropic homeostatic loop. Renal mitochondria are important for systemic control of calcitriol formation. By contrast, 1α-hydroxylase activity in extrarenal mitochondria functions in a paracrine/autocrine manner and is influenced by cytokines such as interleukins and interferon-γ.

CYP24A1 hydroxylase (sterol 24-hydroxylase).

Another critical mitochondrial enzyme that controls the vitamin D metabolic machinery is CYP24A1 hydroxylase. This enzyme is responsible for converting 25(OH)D and 1α,25(OH)₂D to less bioactive metabolites, 24R,25(OH)₂D and 1α,24,25(OH)₃D, respectively, and is responsible for the 26-hydroxylation of these metabolites (155). The former metabolite, 24R,25(OH)₂D, has been reported to counteract the hypercalcemic activity of calcitriol and regulate calcium homeostasis for bone formation, whereas the latter trihydroxy metabolite is further catabolized via a 6-step monooxygenation to form calcitroic acid, a urinary metabolite of vitamin D (156). An overview of the microsomal and mitochondrial CYP enzymes involved in vitamin D metabolism is shown in Fig. 8.

With regard to understanding the role of riboflavin and the importance of flavoenzymes in mitochondrial vitamin D metabolism, consideration must be made of the bioavailability and delivery of riboflavin into mitochondria and the activation of the nascent apoflavoenzyme within the matrix. In this regard, CYP27A1, CYP27B1, and CYP24A1 are dependent on the reducing equivalents delivered by adrenodoxin reductase and adrenodoxin to the P450 heme complex. Thus, a coordination of protein import and cofactor supply to the mitochondrial matrix is indispensable for mitochondrial function. Although more in-depth information is required, recent studies have focused attention on the subcellular distribution of riboflavin and its coenzymic forms between the cytosolic, mitochondrial, and peroxisomal compartments (157).

Availability of FAD within the mitochondrial matrix.

Recent studies in trafficking of riboflavin between subcellular compartments that include mitochondria and peroxisomes have focused on the existence of cytosolic and mitochondrial isoforms of FAD synthetase within eukaryotic cells (172, 173). In the cytosol, FMN is pyrophosphorylated with ATP to form FAD by FAD synthetase (FADS; ATP:FMN adenylyl transferase, FMNAT; or FAD pyrophosphorylase, EC 2.7.7.2) (174). The mitochondrial isoform is distinct in size and intracellular location from that in the cytosol (175). The mitochondrial isoform is referred to as FAD synthetase-1, whereas the cytosolic form is referred to as FAD synthetase-2. Mitochondrial FAD synthetase-1 displays an additional 97-amino acid domain at the N-terminus portion and localizes to the mitochondrial matrix. Isoform-1 also displays a 17-amino acid region, which represents a cleavable mitochondrial targeting sequence similar to that found in other proteins synthesized via nuclear DNA (176).

Investigations of the 2 isoforms have called special attention to a riboflavin/FAD cycle between the cytosolic compartment and mitochondria (177). The formation of intramitochondrial FAD requires transport of riboflavin from the cytosol into mitochondria and subsequent phosphorylation to FMN via a putative mitochondrial flavokinase (178). FAD synthetase-1 transforms FMN into FAD, which then combines with imported client apoflavoproteins inside the mitochondrial matrix. In yeast mitochondria, unbound FAD is quickly exported from the mitochondria to the cytosol via a flavin exchange protein (FLX1), which belongs to a family of mitochondrial transport proteins (179). In yeast, FLX1 plays a crucial role in maintaining normal levels of FAD-binding enzyme activity within the mitochondria and serves as a key regulator for unbound FAD within the mitochondria (180). Because FAD itself does not traverse membranes, FAD requires degradation back to riboflavin. Mitochondria display a pair of FAD dephosphorylating enzymes that differ from the cytosolic isoforms. Namely, mitochondrial FAD pyrophosphatase and FMN phosphohydrolase differ from the extramitochondrial enzymes in their pH maxima and sensitivity to inhibitors (178). Thus, in view of the complete complement of both flavin biosynthetic and dephosphorylating enzymes, as well as the presence of a flavin exchange protein, FLX1, it has been postulated that a riboflavin-FAD cycle occurs within mitochondria and further that it plays a major role in controlling mitochondrial flavoprotein turnover.

On the basis of sequence similarities to FLX1, a human candidate gene described as a mitochondrial folate transporter (MFT) was able to functionally complement the yeast FLX1 (181). In addition to the MFT, the ABCG2 transporter previously described to export riboflavin from the cell has also been detected on the inner membrane of the mitochondria (182). It is interesting to speculate whether MFT and ABCG2 may share a dual function because they both export riboflavin and folate from the mitochondria. Thus, FAD unaffiliated with client proteins is dephosphorylated to its parent form, riboflavin. Riboflavin is quickly “escorted” from the mitochondrial matrix by either the MFT or ABCG2 to prevent unscheduled electron transfer and avert formation of reactive oxygen species. Understanding the homeostatic control and turnover of the flavin coenzymes within the mitochondrial compartment is crucial to clarifying the regulatory functions of flavoenzymes in maintenance of normal cellular metabolism and bioenergetics.

It is important to note that the mitochondrial CYP system in the presence of unbound FAD or in the presence of excess riboflavin can function as a nonproductive electron transport system and react with molecular oxygen (183). As noted previously, unscheduled electron transfer via P450s and molecular oxygen can produce superoxide and other reactive oxygen species (184). Thus, a coordination among imported proteins, coenzymes, and cofactors within the mitochondrial matrix is indispensable for “correct” CYP function. Flavins unaffiliated with their client proteins or not confined within their proper domains in the electron transport chain may force unscheduled electron shunting within the chain and result in inappropriate formation of reactive oxygen species (185). Leakage of electrons within the matrix is the major source of reactive oxygen in the mitochondria. The generation of superoxide occurs at both the matrix side of the inner mitochondrial membrane as well as the cytosolic side (186). Riboflavin transported into mitochondria requires immediate transformation to FMN and FAD by the mitochondrial flavin biosynthetic enzymes.

A compromised reduction in activities of the flavin biosynthetic enzymes (flavokinase and FAD synthetase-1), an altered FMN:FAD ratio, or a defect in an export protein could result in accumulation of free riboflavin that may contribute to formation of excessive reactive oxygen species (187). FMN and FAD serve as coenzymes and are stabilized against photoreactivity and electron transfer while buried within their protein domains. Unlike its coenzymic derivatives, FMN and FAD, the parent compound, riboflavin, does not share the same extent of privileged binding domains and thus can interfere with scheduled electron transfers. Human motor neurons are particularly susceptible to injury if marked changes occur in components involved with mitochondria electron transfers (188). Thus, unattended riboflavin present within the inner compartments of mitochondrial membranes may short circuit the tightly controlled flow of electrons through heme transport and would be expected to interfere with vitamin D metabolism.