Folates have been used with cytotoxic agents for decades and today they are used in hundreds of thousands of patients annually. Folate metabolism is complex. In the treatment of cancer with 5-fluorouracil, the administration of folates mechanistically leads to the formation of [6R]-5,10-methylene-tetrahydrofolate, and the increased concentration of this molecule leads to stabilization of the ternary complex comprising thymidylate synthase, 2'-deoxy-uridine-5'-monophosphate, and [6R]-5,10-methylene-tetrahydrofolate. The latter is the only natural folate that can bind directly in the ternary complex, with other folates requiring metabolic activation. Modulation of thymidylate synthase activity became central in the study of folate/cytotoxic combinations and, despite wide use, research into the folate component was neglected, leaving important questions unanswered. This article revisits the mechanisms of action of folates and evaluates commercially available folate derivatives in the light of current research. Better genomic insight and availability of new analytical techniques and stable folate compounds may open new avenues of research and therapy, ultimately bringing increased clinical benefit to patients.

BACKGROUND:In Nordic countries, the standard treatment of colorectal cancer (CRC) in the adjuvant setting is bolus 5-fluorouracil (5-FU) plus leucovorin alone or in combination with oxaliplatin. 5-FU competes with the natural occurring pyrimidine uracil (Ura) as a substrate for dihydropyrimidine dehydrogenase (DPD; enzyme commission number 1.3.1.2). Low DPD activity is associated with toxicity during treatment. Pretherapeutic detection of DPD deficiency could prevent severe toxicity otherwise limiting drug administration. Assays showing that DPD deficiency impairs breakdown of Ura to dihydrouracil (UH 2 ) seem promising for clinical use. METHODS: Urine was collected from 56 untreated volunteers and 143 patients with CRC before adjuvant treatment. Ura and UH 2 were analyzed using a column-switching high-performance liquid chromatography method that incorporates reversed-phase and cationexchange columns. Ura, UH 2 , and UH 2 /Ura levels were related to toxicity. RESULTS: Ura and UH 2 in patients were not different from controls. UH 2 was significantly higher in women compared with men. The UH 2 /Ura ratio, however, did not differ according to sex. Low UH 2 and UH 2 /Ura levels were associated with diarrhea in men. Women experiencing thrombocytopenia had significantly higher Ura compared with women with no thrombocytopenia. The UH 2 /Ura ratio correlated negatively with total toxicity score in men (r ¼ À0.39, P ¼ .020). CONCLUSION: Pretherapeutic Ura and UH 2 levels per se may be related to risk of side effects during adjuvant 5-FU-based treatment, whereas the UH 2 /Ura ratio may not always reveal such a risk. Sex is a strong risk factor for toxicity, showing the importance of evaluating male and female patients separately.

A liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS) method has been developed for the determination of 5,10-methylenetetrahydrofolate (methyleneTHF), tetrahydrofolate (THF) and 5-methyltetrahydrofolate (methylTHF) in colorectal mucosa and tumor tissues. The folate extraction method includes homogenization, heat and folate conjugase treatment to hydrolyze polyglutamyl folate to monoglutamyl folate. Before analysis on LC-MS/MS, simple and fast sample purification with ultrafiltration (molecular weight cut-off membrane, 10 kDa) was performed. Folates were detected and quantified using positive electrospray. The method described in the present paper was successfully applied to determine the level of three folate monoglutamates in mucosa and tumor samples from 77 colorectal cancer patients, starting from a limited amount of tissue. The results showed that the LC-MS/MS method has a great advantage over other previously used methods because of its high sensitivity and selectivity. Significantly higher levels of methyleneTHF and THF were found in tumor compared with matched mucosa tissues. Folate levels in adjacent mucosa were associated with tumor location, age and gender. The correlation between folate levels and tumor site further strengthens the fact that development of right- and left-sided tumors follows different pathways.

Colorectal cancer is commonly treated with 5-fluorouracil and 5-formyltetrahydrofolate (leucovorin). Metabolic action of leucovorin requires several enzymatic steps that are dependent on expression of corresponding coding genes. To identify folate pathway genes with possible impact on leucovorin metabolism, a retrospective study was performed on 193 patients with stage III colorectal cancer. Relative expression of 22 genes putatively involved in leucovorin transport, polyglutamation and metabolism was determined in tumor and mucosa samples using quantitative real-time polymerase chain reaction. After surgery, patients received adjuvant 5-fluorouracil-based bolus chemotherapy with leucovorin during six months, and were followed for 3 to 5 years. Cox regression analysis showed that high tumoral expression of the genes SLC46A1/PCFT (proton-coupled folate transporter) and SLC19A1/RFC-1 (reduced folate carrier 1) correlated significantly (p < 0.001 and p < 0.01, respectively) with a decreased risk of recurrent disease, measured as disease-free survival (DFS). These two genes are involved in the transport of folates into the cells and each functions optimally at a different pH. We conclude that SLC46A1/PCFT and SLC19A1/RFC-1 are associated with DFS of patients with colorectal cancer and hypothesize that poor response to 5-fluorouracil plus leucovorin therapy in some patients may be linked to low expression of these genes. Such patients might need a more intensified therapeutic approach than those with high gene expression. Future prospective studies will determine if the expression of any of these genes can be used to predict response to leucovorin.