Clinical And Animal Studies Reinterpretation In Light Of Molecular Data

Studies of clinical populations and animals have indicated that iron deficiency alters folate-dependent one-carbon metabolism. Specifically, iron deficiency can influence all of the biochemical indicators listed in Fig. 2. However, marked discrepancies exist within the literature, primarily resulting from different patient populations, indicators of folate status or metabolism, experimental protocols, as well as insufficient measurements and lack of experimental controls (70). These discrepancies led one author in 1966 to conclude that ".. .the causal relationship of folate and iron deficiencies cannot be satisfactorily resolved on the basis of data available today" (71). Although this statement is still accurate at the present time, new avenues for investigation are now emerging. Recent mechanistic studies suggest that iron metabolism is capable of influencing cellular folate metabolism by two distinct mechanisms: by altering the expression of cSHMT and by influencing folate catabolism leading to frank folate deficiency (52,58). These effects will be considered individually next, and potential biochemical mechanisms that account for these effects will be postulated based on the aforementioned biochemical studies of iron deficiency conducted in cell culture models.

Effects of Iron on Folate-Dependent DNA Synthesis

Interpretations of clinical data that support or dismiss an iron-folate relationship must be viewed with caution considering what is known about this relationship at the molecular level. HCF-mediated increases in cSHMT ex pression can affect DNA precursor synthesis in two opposing ways: It can facilitate DNA synthesis by increasing the rate of de novo thymidylate biosynthesis, and it can impair DNA synthesis by sequestering intracellular folate as 5-methyltetrahydrofolate, thereby creating a "functional" folate deficiency. This functional folate deficiency is the result of the depletion of other folate cofactor forms that results from 5-methyltetrahydrofolate accumulation, as occurs during vitamin B12 deficiency (27). These two opposing effects of cSHMT expression on DNA synthesis might account for the discrepancies that exist within the clinical literature.

Iron Deficiency Induces Functional Folate Deficiency

Megaloblastosis, which occurs during folate deficiency, has been hypothesized to occur as a result of thymidylate "starvation" (69,72). Folate deficiency induced by gastrointestinal disorders (73) results in neutrophil hypersegmentation, elevated urinary FIGLU, and giant metamyelocytes in bone marrow despite normal or elevated RBC folate. Studies have demonstrated that iron therapy alone can improve the symptoms of folate deficiency (73). Similar observations of apparent folate deficiency were seen in patients with intestinal parasitism (74). These patients presented with mega-loblastic anemia and elevated neutrophil lobe count without evidence for inadequate folate intake or absorption. For these patients, iron repletion alone also reversed the symptoms of folate deficiency. A more recent case-control study supports these observations that iron deficiency can inhibit DNA synthesis by inducing a symptomatic and functional folate deficiency (75). In this study, all patients who had evidence for vitamin B12 or folate deficiency, infection, or who were undergoing chemotherapy were excluded because these factors are known to promote neutrophil hypersegmentation by impairing folate-dependent DNA synthesis. Fifty patients with iron-deficiency anemia were matched with 50 control patients. The authors found that 62% of patients with iron-deficiency anemia exhibited neutrophil hypersegmentation compared to only 4% of control patients. The symptomatic folate deficiency in this study occurred despite patients displaying significantly increased RBC folate concentrations relative to control patients, and this elevation in RBC folate during iron-deficiency anemia is consistent with findings from other studies (76,77). The clinical observation that iron deficiency causes symptoms of folate deficiency, which are caused by impaired DNA synthesis, is consistent with cell culture models. Specifically, decreases in the regulatory iron pool increase cellular levels of cSHMT and result in trapping of cellular folate as 5-methyltetrahydrofolate (58). Furthermore, the response of the hypersegmentation to iron supplementa tion suggests that iron therapy rescues a cSHMT-induced methyl trap of folate cofactors (Fig. 1). Therefore, cSHMT joins vitamin B12 as a factor that can induce a folate methyl trap.

Iron Deficiency Masks Functional Folate Deficiency

Although cSHMT is capable of sequestering or trapping cellular 5-methyltetrahydrofolate, cSHMT has also been demonstrated to enhance thymidylate biosynthesis and thereby support DNA synthesis. The degree to which these two opposing mechanisms affect overall DNA synthesis would be dependent on the magnitude of increase in cSHMT expression and on cellular folate status. High levels of cSHMT would be expected to create a severe methyl trap and inhibit DNA synthesis; this effect would be compounded by folate deficiency. Alternatively, moderate levels of cSHMT expression would not be sufficient to trap folate cofactors but would be expected to enhance thymidylate biosynthesis and thereby promote DNA synthesis (Fig. 1). This suggestion is supported by a study of iron- and folate-deficient patients by Das et al., who were among the first to suggest that iron deficiency can mask apparent folate deficiency (78). In this study, patients were chosen who presented uncomplicated iron-deficiency anemia as evidenced by apparent normal RBC folate concentrations and normal bone marrow dU suppression tests. Iron supplementation alone resulted in the appearance of hypersegmented neutrophils, abnormal dU suppression in the bone marrow, and megaloblastic transformations, all symptoms of folate deficiency. These indicators of folate deficiency emerged only after iron therapy, whereas folate values continued to read in the normal range throughout the entire study.

Emerging mechanisms described earlier may account for this apparent masking of functional folate deficiency by iron deficiency. Moderate increases in cSHMT expression that are observed with increased HCF expression may also occur during iron deficiency (52). Increased cSHMT expression at levels that do not produce a methyl trap would be expected to stimulate thymidylate biosynthesis and thereby mask marginal functional folate deficiency, because folate deficiency normally impairs thymidylate biosynthesis (72). Therefore, increases in cSHMT activity compensate for folate deficiency and thereby yield a normal dU suppression test. According to this model, this stimulation is lost by iron repletion, which is expected to restore cSHMT levels to normal. It is not likely nor is it supported in the literature that iron deficiency can mask severe folate deficiency.

In conclusion, the discrepancies among clinical studies that suggest an iron-folate relationship in DNA synthesis pathways may be the result of subtle differences in their patient populations. These differences include the severity of iron deficiency, which, in turn, may influence the extent of increased cSHMT expression. According to the above model, the level of cSHMT expression will determine if DNA synthesis is stimulated by increasing thymidylate biosynthesis or inhibited by 5-methyltetrahydro-folate trapping. Furthermore, this model indicates that the masking of folate deficiency by iron deficiency will only occur in marginally folate-deficient populations and that megaloblastosis will occur during severe folate deficiency and iron deficiency. In other words, the severity of both iron and folate deficiencies will work in combination to determine the final metabolic effect on DNA synthesis.

Effects of Iron on Folate Status and Folate Catabolism

Changes in cellular folate concentrations independent of dietary folate intake may be influenced by iron status as well as alterations in HCF expression (49). Iron-related changes in cellular folate concentrations can occur by two distinct mechanisms: by increased rates of folate turnover mediated by HCF (49) and by increased sequestration of cellular folates caused by increased expression of cSHMT, a major folate-binding protein in the cytoplasm of some cell types (26,49). In fact, polymorphic variants of cSHMT in human populations significantly influence RBC folate concentrations

(79). Therefore, some reevaluation of the clinical literature that relates iron status to actual folate status is warranted.

Patients with iron-deficiency anemia often display symptomatic folate deficiency yet tend to exhibit significant increases in RBC folate (73,75,76), indicating that a folate methyl trap may be operative. RBC folate concentrations fall markedly following iron repletion, with simultaneous increases in plasma folate (76). However, at least one study failed to see this association

(80). Another study of nonanemic pregnant women noted that subjects with depleted iron reserves tend to be less responsive to folate supplementation, consistent with the occurrence of a folate methyl trap (81). This effect of folate accumulation during iron deficiency is also observed in the liver of rodents (17,82) and is reversed upon iron repletion, although other studies have failed to see this effect (83). These discrepancies in the animal literature have been previously discussed (17). Iron depletion does not influence folate polyglutamate processing or absorption in the intestine, indicating that the effect occurs at the cellular level (17). The decreases in RBC folate and increases in serum folate that occur following iron repletion have been proposed to result from increased demand for folate following iron repletion (73). However, sequestration and trapping of 5-methyltetrahydrofolate resulting from increased cSHMT expression would be an alternative explanation for elevated folate content that can be readily tested experimentally.

Iron may also influence rates of folate turnover. Increased expression of heavy-chain ferritin occurs in physiological states associated with increased rates of folate catabolism and folate deficiency. Tumor cells exhibit increased rates of folate uptake while displaying cellular folate deficiency (11,84,85). Mice with ascitic tumors have greatly increased concentrations of urinary p-aminobenzoylglutamate, an indicator of increased rates of folate catabolism (41). Similarly, the folate content of neoplastic cells is significantly lower than surrounding normal cells in patients with colorectal adenomas (86). Consistent with studies of cell culture models that demonstrate that increased HCF expression increases rates of folate catabolism, HCF is elevated in most tumors and its expression is markedly increased by the oncogene c-myc (86-91). Therefore, changes in iron metabolism independent of iron status can influence intracellular folate concentrations. Pregnancy is also associated with folate deficiency, and increased rates of folate catabolism have been observed in rodent studies, but such observations have been inconsistent in human studies (24). In rodents, HCF was found to be induced by progesterone, and HCF expression was induced 8- to 10-fold in the endometrial stromal cells of pregnant rats (92). Although definitive whole-animal studies that conclusively demonstrate a role for HCF in catalyzing folate catabolism and regulating intracellular folate concentrations are lacking, in vitro and cell culture studies support a role for alterations in iron metabolism influencing intracellular folate concentrations.

Other studies indicate a reciprocal relationship between iron and folate status. Iron overload occurs in patients with folate deficiency, presumably because iron is preferentially deposited in the parynchemal cells during folate deficiency, predisposing that individual to iron overload (93). Folate deficiency has been observed in hemochromatosis without evidence for defective intestinal malabsorption of folate. The authors of this study concluded that hemochromatosis impairs folate storage in liver (94). However, no study has been reported that systematically investigated the effects of iron overload on folate catabolism in humans or experimental animals.

Maternal Iron Deficiency and Lactation

Maternal iron deficiency in rodent animal models dramatically decreases milk folate content (35-47% reduction from moderate to severe iron depletion), an effect that is independent of maternal folate status (82). However, the folate status of iron-deficient and iron-replete pups is similar at birth, indicating that pups are at risk for folate deficiency only after parturition

(95,96). Pups nursed by folate-sufficient, iron-depleted mothers become folate deficient by d 18 and display growth retardation (96) despite increased milk consumption relative to pups nursed by iron-replete dams (97). Additionally, pups nursed by iron-deficient dams do not have impaired intestinal folate absorption, indicating that the folate-related growth retardation is the result of inadequate folate intake. Similar results are seen in piglet models (98). The decreased milk folate content in iron-deficient dams occurs without differences in maternal RBC or tissue folate concentrations compared to iron-replete dams throughout lactation, indicating that the effect of iron on milk folate may be a mammary-specific effect in this animal model (99). The activities of enzymes associated with folate retention, including methio-nine synthase and formlylpolyglutamate synthetase, are unaffected in the mammary gland of iron-deficient dams, and the content of milk folate-bind-ing protein is not influenced by maternal iron status (100). Therefore, it was proposed that iron deficiency specifically targets mammary tissue in lactat-ing dams and impairs the ability of the mammary epithelial secretory cells to accumulate folate (99,100). Previous biochemical studies have indicated that small-molecule chemical iron chelators, which induce cellular iron deficiency by sequestering both the functional and regulatory iron pools, inhibit cSHMT transcription specifically in mammary carcinoma cells (62). This relationship should be tested in a rodent model. If cSHMT is a major folate-binding protein in the secretory mammary cell, as has been documented in other cells, and iron deficiency inhibits cSHMT transcription, as seen with chemical iron chelators in cultured cells, this would at least partially account for the inability of the mammary cells to accumulate folate.

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