Acidic dissociation constants of folic acid, dihydrofolic acid, and methotrexate.

The acidic dissociation constants in the range HO--1.5 to pH 7 of folic acid, dihydrofolic acid, methopterin (N(10)methylfolic acid), and methotrexate have been measured by potentiometric and spectrophotometric titrations. Assignment of these dissociations was made by comparison to model compounds, by proton magnetic resonance measurements, and by examination of associated ultraviolet absorbance changes. For folic acid, the dissociation constants are as follows: N(1), pK' 2.35; N(10), pK' 0.20; N(5), pK' greater than -1.5. For dihydrofolic acid: N(5), pK' 3.84; N(1), pK' 1.38; N(10), pK' 0.28. For methotrexate: N(1), pK' 5.71; gamma-carboxyl, pK' 4.70; alpha-carboxyl, pK' 3.36; N(10), pK' 0.50; N(5), boxyl, pK' 4.70; alpha-carboxyl, pK' 3.36; N(10), pK' 0.50; N(5) pK' greater than -1.5. For methopterin: acidic ionization of amide, pK' 7.68; gamma-carboxyl, pK' 4.62; N(1), pK' 2.40; N(10), pK; 0.36; N(5), pK' greater than -1.5. The pK' values were determined directly for the four compounds at 25 degrees near 0.1 ionic strength, or in 0.1 to 4 M HCl for pK ln 0.1 M NaCl.     

Enzymatic reduction of dehydrocholic acid to 12-ketochenodeoxycholic acid with NADH regeneration

12-Ketochenodeoxycholic acid, an essential intermediate in the synthesis of chenodeoxycholic acid, has been enzymatically prepared from dehydrocholic acid. The specific reduction of dehydrocholic with NADH was catalysed by 3α-hydroxysteroid dehydrogenase (3α-hydroxysteroid: NAD(P)⁺ oxidoreductase, EC 1.1.1.50) and 7α-hydroxysteroid dehydrogenase (7α-hydroxysteroid:NAD⁺ 7-oxidoreductase, EC 1.1.1.159). Cofactor regeneration was obtained through the formate dehydrogenase (formate:NAD⁺ oxidoreductase, EC 1.2.1.2) catalysed oxidation of formate. Complete transformation of dehydrocholic acid to the 12-keto derivative was achieved with a coenzyme turnover number up to 1200. No steroid by-products were detected by high performance liquid chromatography and thin layer chromatography. The process yielded 9 g product l^?1 in 66–84 h. The high purity of the enzymatically prepared 12-ketochenodeoxycholic acid should drastically reduce the formation of the toxic by-product lithocholic acid, which occurs in the synthesis of chenodeoxycholic acid when using chemical methods alone.     

Photobiochemistry of folates: A photochemical reduction of folic acid

Exposure of deaerated folic acid solutions containing an electron donor to UV radiation (310–390 nm, I = 0.4 W m⁻²) induced formation of dihydrofolic acid (DHFA), a photoexcitation which gave tetrahydrofolic acid (THFA). Only DHFA was formed in the presence of EDTA (E′_o = +0.40 V), while the presence of stronger reductants—NADH (E′_o = −0.32 V) and boron hydride (E′_o = −0.48 V)—induced photoreduction to THFA. It was demonstrated that UV radiation had no effect on the THFA formylation, giving the coenzyme 5,10-methenyltetrahydrofolic acid and its transformation into another coenzyme, 5-formyltetrahydrofolic acid.     

Biosynthesis of folic acid

Summary The influence of various vitamins on the biogenesis of folic acid has been studied in microorganisms requiring these as growth factors. In L. arabinosus, the folic acid synthesised was directly proportional to the availability of both riboflavin and pantothenic acid. The influence of cyanocobalamin on folic synthesis varied radically in different organisms. In case of the B₁₂/methionine auxotroph of E. coli there was an inverse relationship of vitamin B₁₂ to folic acid synthesis, while in Euglena the folic acid elaborated was in proportion to cyanocobalamin supplied. Synthesis of both folic acid and vitamin B₁₂ was depressed when thymine supply was adequate in the nutrition of E. coli 15 T ^-, a thymine auxotroph.     

Photogenotoxicity of folic acid

Folic acid (FA), also named vitamin B9, is an essential cofactor for the synthesis of DNA bases and other biomolecules after bioactivation by dihydrofolate reductase (DHFR). FA is photoreactive and has been shown to generate DNA modifications when irradiated with UVA (360 nm) in the presence of DNA under cell-free conditions. To investigate the relevance of this reaction for cells and tissues, we irradiated three different cell lines (KB nasopharyngeal carcinoma cells, HaCaT keratinocytes, and a melanoma cell line) in the presence of FA and quantified cytotoxicity and DNA damage generation. The results indicate that FA is phototoxic and photogenotoxic by two different mechanisms. First, extracellular photodecomposition of FA gives rise to the generation of H₂O₂, which causes mostly DNA strand breaks. If this is prevented, e.g., by the presence of catalase, DNA damage generated by intracellular FA becomes evident. The damage spectrum in this case consists predominantly of oxidatively generated purine modifications sensitive to the repair glycosylase Fpg, as characteristic for type I photoreactions, and is associated with the formation of micronuclei. In KB cells, the DNA damage is strongly enhanced after pretreatment with the DHFR inhibitor methotrexate, which prevents the loss of the chromophore associated with the intracellular reduction of FA by DHFR. The results indicate that FA is photoreactive in cells and gives rise to nuclear DNA damage under irradiation.