Groups that contain a single-carbon atom can be transferred from one compound to another. These carbon atoms may be in a number of different oxidation states. The most oxidized form, CO2, is transferred by biotin. One-carbon groups at lower levels of oxidation than CO2 are transferred by reactions involving tetrahydrofolate (FH4), vitamin B12, and S-adenosylmethionine (SAM).
Tetrahydrofolate. Tetrahydrofolate, which is produced from the vitamin folate, is the primary one-carbon carrier in the body. This vitamin obtains one-carbon units from serine, glycine, histidine, formaldehyde, and formate (Fig. 38.1). While these carbons are attached to FH4, they can be either oxidized or reduced. As a result, folate can exist in a variety of chemical forms. Once a carbon has been reduced to the methyl level (methyl-FH4); however, it cannot be reoxidized. Collectively, these one-carbon groups attached to their carrier FH4 are known as the one-carbon pool. The term “folate” is used to represent a water-soluble B-complex vitamin that functions in transferring single-carbon groups at various stages of oxidation.
FIGURE 38.1 Overview of the one-carbon pool. FH4 • C indicates tetrahydrofolate (FH4) containing a one-carbon unit that is at the formyl, methylene, or methyl level of oxidation (see Fig. 38.3). The origin of the carbons is indicated, as are the final products after a one-carbon transfer. dTMP, deoxythymidine monophosphate.
The one-carbon groups carried by FH4 are used for many biosynthetic reactions. For example, one-carbon units are transferred to the pyrimidine base of deoxyuridine monophosphate (dUMP) to form deoxythymidine monophosphate (dTMP), to the amino acid glycine to form serine, to precursors of the purine bases to produce carbons C2 and C8 of the purine ring, and to vitamin B12.
Vitamin B12. Vitamin B12 is involved in two reactions in the body. It participates in the rearrangement of the methyl group of L-methylmalonyl coenzyme A (L-methylmalonyl CoA) to form succinyl CoA, and it transfers a methyl group, obtained from FH4, to homocysteine, forming methionine.
S-adenosylmethionine. SAM, produced from methionine and adenosine triphosphate (ATP), transfers the methyl group to precursors that form a number of compounds, including creatine, phosphatidylcholine, epinephrine, melatonin, methylated nucleotides, methylated histones, and methylated DNA.
Methionine metabolism is very dependent on both FH4 and vitamin B12. Homocysteine is derived from methionine metabolism and can be converted back into methionine by using both methyl-FH4 and vitamin B12. This is the only reaction in which methyl-FH4 can donate the methyl group. If the enzyme that catalyzes this reaction is defective, or if vitamin B12 or FH4 levels are insufficient, homocysteine will accumulate. Elevated homocysteine levels have been linked to cardiovascular and neurological disease. A vitamin B12 deficiency can be brought about by the lack of intrinsic factor, a gastric protein required for the absorption of dietary B12. A consequence of vitamin B12 deficiency is the accumulation of methyl-FH4 and a decrease in other folate derivatives. This is known as the methyl-trap hypothesis, in which, because of the B12 deficiency, most of the carbons in the FH4 pool are trapped in the methyl-FH4 form, which is the most stable. The carbons cannot be released from the folate because the one reaction in which they participate cannot occur because of the B12 deficiency. This leads to a functional folate deficiency, even though total levels of folate are normal. A folate deficiency (whether functional or actual) leads to megaloblastic anemia caused by an inability of blood cell precursors to synthesize DNA and, therefore, to divide. This leads to large, partially replicated cells being released into the blood to attempt to replenish the cells that have died. Folate deficiencies also have been linked to an increased incidence of neural tube defects, such as spina bifida, in mothers who become pregnant while folate deficient.
In the past, the gold standard for determining if someone was B12 deficient was the Schilling test. This test consists of the patient ingesting radioactive (57Co) crystalline vitamin B12, after which a 24-hour urine sample was collected. The radioactivity in the urine sample is compared with the input radioactivity, and the difference represents the amount of B12 absorbed through the digestive tract. Such tests could distinguish between problems in removing B12 from bound dietary proteins or if the deficiency is due to a lack of intrinsic factor, or other proteins involved in transporting B12 throughout the body. Another method to determine if intrinsic factor activity is reduced is to determine the levels of anti-intrinsic factor antibodies in the blood. Autoantibodies toward intrinsic factor commonly develop in individuals with lack of intrinsic factor activity, and the levels of such antibodies can be determined in an ELISA using recombinant human intrinsic factor bound to plastic wells as the antigen. The Schilling test has been replaced by a competitive binding luminescent assay. This new test has recently been criticized for being unreliable, particularly if a patient exhibits anti-intrinsic factor antibodies. Suspected B12 deficiencies can also be determined by assaying blood levels of methylmalonic acid or homocysteine.
THE WAITING ROOM 
After resection of the cancer in his large intestine and completion of a course of postoperative chemotherapy with 5-fluorouracil (5-FU) and oxaliplatin, Clark T. returned to his gastroenterologist for a routine follow-up colonoscopy. His colon was completely normal, with excellent healing at the site of the anastomosis. His physician expressed great optimism about a possible cure of Clark T.’s previous malignancy but cautioned him about the need for regular colonoscopies and surveillance CT scans over the next few years.
Beatrice T., a 75-year-old woman, went to see her physician because of numbness and tingling in her legs. A diet history indicated a normal and healthy diet, but she was not taking any supplemental vitamin pills. Laboratory results indicated a low level of serum B12.
The initial laboratory profile, determined when Jean T. first presented to her physician with evidence of early alcohol-induced hepatitis, included a hematologic analysis that showed that Jean T. was anemic. Her hemoglobin was 11.0 g/dL (reference range, 12 to 16 g/dL for an adult woman). The erythrocyte (red blood cell) count was 3.6 million cells/mm3 (reference range, 4.0 to 5.2 million cells/mm3 for an adult woman). The average volume of her red blood cells (mean corpuscular volume [MCV]) was 108 fL (reference range = 80 to 100 fL; 1 fL = 10–12 mL), and the hematology laboratory reported a striking variation in the size and shape of the red blood cells in a smear of her peripheral blood (see Chapter 42). The nuclei of the circulating granulocytic leukocytes had increased nuclear segmentation (polysegmented neutrophils). Because these findings are suggestive of a macrocytic anemia (in which blood cells are larger than normal), measurements of serum folate and vitamin B12 (cobalamin) levels were ordered.
Folate deficiencies occur frequently in people with chronic alcohol use disorder. Several factors are involved: inadequate dietary intake of folate; direct damage to intestinal cells and brush border enzymes, which interferes with absorption of dietary folate; a defect in the enterohepatic circulation, which reduces the absorption of folate; liver damage that causes decreased hepatic production of plasma proteins; and interference with kidney reabsorption of folate.
Hematopoietic precursor cells, when exposed to too little folate and/or vitamin B12, show slowed cell division, but cytoplasmic development occurs at a normal rate. Hence, the megaloblastic cells tend to be large, with an increased ratio of RNA to DNA. Megaloblastic erythroid progenitors are usually destroyed in the bone marrow (although some reach the circulation). Thus, marrow cellularity is often increased but production of red blood cells is decreased, a condition called ineffective erythropoiesis.
Therefore, Jean T. has megaloblastic anemia, characteristic of folate or B12 deficiency.
I. Tetrahydrofolate (FH4)
A. Structure and Forms of FH4
Folates exist in many chemical forms. The coenzyme form that functions in accepting one-carbon groups is tetrahydrofolate polyglutamate (Fig. 38.2), generally referred to as just tetrahydrofolate or FH4. It has three major structural components, a bicyclic pteridine ring, para-aminobenzoic acid, and a polyglutamate tail consisting of several glutamate residues joined in amide linkage. The one-carbon group that is accepted by the coenzyme and then transferred to another compound is bound to N5, to N10, or both.
FIGURE 38.2 Reduction of folate to tetrahydrofolate (FH4). The same enzyme, dihydrofolate reductase, catalyzes both reactions. Multiple glutamate residues are added within cells (n ~ 5). Plants can synthesize folate, but humans cannot. Therefore, folate is a dietary requirement. R is the portion of the folate molecule shown to the right of N10. The different precursors of FH4 are indicated in the figure. NADP, nicotinamide adenine dinucleotide phosphate; PABA, para-aminobenzoic acid.
Different forms of folate may differ in the oxidation state of the one-carbon group, in the number of glutamate residues attached, or in the degree of oxidation of the pteridine ring. When the term “folate” or “folic acid” is applied to a specific chemical form, it is the most oxidized form of the pteridine ring (see Fig. 38.2). Folate is reduced to dihydrofolate and then to tetrahydrofolate by dihydrofolate reductase present in cells. Reduction is the favored direction of the reaction; therefore, most of the folate present in the body is present as the reduced coenzyme form, FH4.
B. The Vitamin Folate
Folates are synthesized in bacteria and higher plants and ingested in green leafy vegetables, fruits, and legumes in the diet. The vitamin was named for its presence in green, leafy vegetables (foliage). Most of the dietary folate derived from natural food sources is present in the reduced coenzyme form. However, vitamin supplements and fortified foods contain principally the oxidized form of the pteridine ring.
Jean T.’s serum folic acid level was 3.1 ng/mL (reference range, 6 to 15 ng/mL), and her serum B12 level was 210 ng/L (reference range, 180 to 914 ng/L). Her serum iron level was normal. It was clear, therefore, that her megaloblastic anemia was caused by a folate deficiency (although her B12 levels were in the low range of normal). The management of a pure folate deficiency in a patient with alcohol use disorder includes cessation of alcohol intake and a diet that is rich in folate.
Sulfa drugs, which are used to treat certain bacterial infections, are analogs of para-aminobenzoic acid. They prevent growth and cell division in bacteria by interfering with the synthesis of folate. Because we cannot synthesize folate, sulfa drugs do not affect human cells in this way.
The current U.S. Recommended Dietary Allowance (RDA) for folate equivalents is approximately 400 μg for adult men and women. In addition to being prevalent in green leafy vegetables, other good sources of this vitamin are liver, yeast, legumes, and some fruits. Protracted cooking of these foods, however, can destroy up to 90% of their folate content. A standard U.S. diet provides 50 to 500 μg of absorbable folate each day. Folate deficiency in pregnant women, especially during the month before conception and the month after, increases the risk of neural tube defects, such as spina bifida, in the fetus. To reduce the potential risk of neural tube defects for women capable of becoming pregnant, the recommendation is to take 400 μg of folic acid daily in a multivitamin pill (some organizations recommend 400 to 800 μg daily). If the woman has a history of having a child with a neural tube defect, this amount is increased to 4,000 μg/day for the month before and the month after conception. Flour-containing products in the United States are now supplemented with folate to reduce the risk of neural tube defects in newborns.
As dietary folates pass into the proximal third of the small intestine, folate conjugases in the brush border of the lumen cleave off glutamate residues to produce the monoglutamate form of folate, which is then absorbed (see Fig. 38.2, upper structure, when n = 1). Within the intestinal cells, folate is converted principally to N5-methyl-FH4, which enters the portal vein and goes to the liver. Smaller amounts of other forms of folate also follow this route.
The liver, which stores half of the body’s folate, takes up much of the folate from the portal circulation; uptake may be through active transport or receptor-mediated endocytosis. Within the liver, FH4 is reconjugated to the polyglutamate form before being used in reactions. A small amount of the folate is partially degraded, and the components enter the urine. A relatively large portion of the folate enters the bile and is subsequently reabsorbed (very similar to the fate of bile salts in the enterohepatic circulation).
N5-Methyl-FH4, the major form of folate in the blood, is loosely bound to plasma proteins, particularly serum albumin.
C. Oxidation and Reduction of the One-Carbon Groups of Tetrahydrofolate
One-carbon groups transferred by FH4 are attached either to N5 or N10, or they form a bridge between N5 and N10. The collection of one-carbon groups attached to FH4 is known as the one-carbon pool. While they are attached to FH4, these one-carbon units can be oxidized and reduced (Fig. 38.3). Thus, reactions that require a carbon in a particular oxidation state may use carbon from the one-carbon pool that was donated in a different oxidation state.
FIGURE 38.3 One-carbon units attached to FH4. A. The active form of FH4. For definition of R, see Figure 38.2. B. Interconversions of one-carbon units of FH4. Only the portion of FH4 from N5 to N10 is shown, which is indicated by the green box in A. After a formyl group forms a bridge between N5 and N10, two reductions can occur. Note that N5-methyl-FH4 cannot be reoxidized. The most oxidized form of FH4 is at the top of the figure, whereas the most reduced form is at the bottom. ADP, adenosine diphosphate; ATP, adenosine triphosphate; NAD, nicotinamide adenine dinucleotide; Pi, inorganic phosphate.
Hereditary folate malabsorption is a rare disease due to a mutation in a proton-coupled folate transporter (PCFT, gene SLC46A1). Loss of PCFT activity in the intestinal proximal jejunum and duodenum lead to systemic folate deficiency. Newborns begin to exhibit symptoms of folate deficiency after a few months of life, after the folate obtained from the mother has been excreted. The children develop anemia, diarrhea, and become immunocompromised (due to the reduction in blood cell differentiation). High-dose oral folate can be used to treat the patients, as another folate transporter (the reduced folate carrier, gene SLC19A1) can absorb sufficient folate when folate is present at high concentrations.
A deficiency of folate results in the accumulation of FIGLU, which is excreted in the urine. A histidine load test can be used to detect folate deficiency. Patients are given a test dose of histidine (a histidine load), and the amount of FIGLU that appears in the urine is measured and compared to normal values. Values greater than normal indicate folate deficiency.
The concentration of folate in serum can also be determined by a microbiological test. Certain strains of bacteria require folate for growth, and if these bacteria are plated on a medium that lacks folate, they will not grow. Folate levels can be quantitated by preparing a standard curve using plates containing different levels of folate and determining the level of growth of the bacteria at that folate level. The unknown sample is then tested for bacterial growth and the extent of growth compared to the standard curve, to determine the amount of folate in the unknown sample. Other tests for folate include measuring the binding of folate to certain proteins, and use of immunologic reagents that specifically bind to and identify folate.
The individual steps for the reduction of the one-carbon group are shown in Figure 38.3. The most oxidized form is N10-formyl-FH4. The most reduced form is N5-methyl-FH4. Once the methyl group is formed, it is not readily reoxidized back to N5,N10-methylene-FH4, and thus N5-methyl-FH4 tends to accumulate in the cell.
D. Sources of One-Carbon Groups Carried by FH4
Carbon sources for the one-carbon pool include serine, glycine, formaldehyde, histidine, and formate (Fig. 38.4). These donors transfer the carbons to folate in different oxidation states. Serine is the major carbon source of one-carbon groups in the human. Its hydroxymethyl group is transferred to FH4 in a reversible reaction, catalyzed by the enzyme serine hydroxymethyltransferase. This reaction produces glycine and N5,N10-methylene-FH4. Because serine can be synthesized from 3-phosphoglycerate, an intermediate of glycolysis, dietary carbohydrate can serve as a source of carbon for the one-carbon pool. The glycine that is produced may be further degraded by the donation of a carbon to folate. Additional donors that form N5,N10-methylene-FH4 are listed in Table 38.1.
TABLE 38.1 One-Carbon Pool: Sources and Recipients of Carbon
| SOURCEa | FORM OF ONE-CARBON DONOR PRODUCEDb | RECIPIENT | FINAL PRODUCT |
| Formate | N10-formyl-FH4 | Purine precursor | Purine (C2 and C8) |
| Serine | N5,N10-methylene-FH4 | dUMP | dTMP |
| Glycine | Glycine | Serine | |
| Formaldehyde | |||
| N5,N10-methylene-FH4 | N5-methyl FH4 | Vitamin B12 | Methylcobalamin |
| Histidine | N5-formimino-FH4 is converted to N5,N10-methenyl-FH4 | ||
| Choline | Betaine | Homocysteine | Methionine and dimethylglycine |
| Methionine | S-adenosylmethionine (SAM) | Glycine (there are many others; see Fig. 38.9B) | N-methylglycine (sarcosine) |
dUMP, deoxyuridine monophosphate; dTMP, deoxythymidine monophosphate; FH4, tetrahydrofolate; SAM, S-adenosylmethionine.
aThe major source of carbon is serine.
bThe carbon unit attached to FH4 can be oxidized and reduced (see Fig. 38.3). At the methyl level, reoxidation does not occur.
FIGURE 38.4 Sources of carbon (reactions 1 to 4) for the FH4 pool and the recipients of carbon (reactions 5 to 8) from the pool. See Figure 38.3 to view the FH4 derivatives involved in each reaction. ATP, adenosine triphosphate; dTMP, deoxythymidine monophosphate; dUMP, deoxyuridine monophosphate; NAD, nicotinamide adenine dinucleotide; Pi, inorganic phosphate; PPi, pyrophosphate.
Histidine and formate provide examples of compounds that donate carbon in different oxidation levels (see Fig. 38.4). Degradation of histidine produces formiminoglutamate (FIGLU), which reacts with FH4 to donate a carbon and nitrogen (generating N5-formimino-FH4), thereby releasing glutamate. Formate, produced from tryptophan oxidation, can react with FH4 and generate N10-formyl-FH4, the most oxidized folate derivative.
E. Recipients of One-Carbon Groups
The one-carbon groups on FH4 may be oxidized or reduced (see Fig. 38.3) and then transferred to other compounds (see Fig. 38.4 and Table 38.1). Transfers of this sort are involved in the synthesis of glycine from serine, the synthesis of the base thymine required for DNA synthesis, the purine bases required for both DNA and RNA synthesis, and the transfer of methyl groups to vitamin B12.
FH4 is required for the synthesis of deoxythymidine monophosphate and the purine bases used to produce the precursors for DNA replication. Therefore, FH4 is required for cell division. Blockage of the synthesis of thymine and the purine bases, either by a dietary deficiency of folate or by drugs that interfere with folate metabolism, results in a decreased rate of cell division and growth.
Because the conversion of serine to glycine is readily reversible, glycine can be converted to serine by drawing carbon from the one-carbon pool.
The nucleotide deoxythymidine monophosphate (dTMP) is produced from deoxyuridine monophosphate (dUMP) by a reaction in which dUMP is methylated to form dTMP (Fig. 38.5). The source of carbon is N5,N10-methylene-FH4. Two hydrogen atoms from FH4 are used to reduce the donated carbon to the methyl level. Consequently, dihydrofolate (FH2) is produced. Reduction of FH2 by NADPH in a reaction catalyzed by dihydrofolate reductase (DHFR) regenerates FH4. This is the only reaction involving FH4 in which the folate group is oxidized as the one-carbon group is donated to the recipient. Recall that DHFR is also required to reduce the oxidized form of the vitamin, which is obtained from the diet (see Fig. 38.2). Thus, DHFR is essential for regenerating FH4 both in the tissues and from the diet. These reactions contribute to the effect of folate deficiency on DNA synthesis because dTMP is required only for the synthesis of DNA.
FIGURE 38.5 Transfer of a one-carbon unit from N5,N10-methylene-FH4 to deoxyuridine monophosphate (dUMP) to form deoxythymidine monophosphate (dTMP). Tetrahydrofolate (FH4) is oxidized to dihydrofolate [FH2] in this reaction. FH2 is reduced to FH4 by dihydrofolate reductase and FH4 is converted to N5,N10-methylene FH4 using serine as a carbon donor. Shaded bars indicate the steps at which the antimetabolites 5-fluorouracil (5-FU) and methotrexate act. 5-Flurouracil (5-FU) inhibits thymidylate synthase. Methotrexate inhibits dihydrofolate reductase. Deoxyribose-P, deoxyribose phosphate; NADP, nicotinamide adenine dinucleotide phosphate.
Individuals with non-Hodgkin lymphoma receive a number of drugs to treat the tumor, including methotrexate. The structure of methotrexate is shown below.
What compound does methotrexate resemble?
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