Preliminary evidence for cell membrane amelioration in children with cystic fibrosis by 5-MTHF and vitamin B12 supplementation: a single arm trial

Abstract

Background: Cystic fibrosis (CF) is one of the most common fatal autosomal recessive disorders in the Caucasian population caused by mutations of gene for the cystic fibrosis transmembrane conductance regulator (CFTR). New experimental therapeutic strategies for CF propose a diet supplementation to affect the plasma membrane fluidity and to modulate amplified inflammatory response. The objective of this study was to evaluate the efficacy of 5-methyltetrahydrofolate (5-MTHF) and vitamin B12 supplementation for ameliorating cell plasma membrane features in pediatric patients with cystic fibrosis.

Methodology and principal findings: A single arm trial was conducted from April 2004 to March 2006 in an Italian CF care centre. 31 children with CF aged from 3 to 8 years old were enrolled. Exclusion criteria were diabetes, chronic infections of the airways and regular antibiotics intake. Children with CF were supplemented for 24 weeks with 5-methyltetrahydrofolate (5-MTHF, 7.5 mg /day) and vitamin B12 (0.5 mg/day). Red blood cells (RBCs) were used to investigate plasma membrane, since RBCs share lipid, protein composition and organization with other cell types. We evaluated RBCs membrane lipid composition, membrane protein oxidative damage, cation content, cation transport pathways, plasma and RBCs folate levels and plasma homocysteine levels at baseline and after 24 weeks of 5-MTHF and vitamin B12 supplementation. In CF children, 5-MTHF and vitamin B12 supplementation (i) increased plasma and RBC folate levels; (ii) decreased plasma homocysteine levels; (iii) modified RBC membrane phospholipid fatty acid composition; (iv) increased RBC K(+) content; (v) reduced RBC membrane oxidative damage and HSP70 membrane association.

Conclusion and significance: 5-MTHF and vitamin B12 supplementation might ameliorate RBC membrane features of children with CF.

Trial registration: ClinicalTrials.gov NCT00730509.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Consort style flowchart of participants through the study.

Figure 2. The intracellular folate metabolism.

(A) Human cells receive exogenous 5-methyltetrahydrofolate (5-MTHF) from the bloodstream and produce endogenous 5-MTHF through the irreversible methylenetetrahydrofolate reductase (MTHFR) reaction that catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. 5-MTHF serves as methyl donor in the remethylation of homocysteine to methionine, which in turn is converted to S-adenosylmethionine (SAM). The transmethylation reaction, which requires vitamin B12 and the methionine synthase enzyme, converted 5-MTHF in tetrahydrofolate (THF). As 5-MTHF is a poor substrate for folylpolyglutamate synthetase, this enzyme adds glutamyl residues to non-methylated folates, guaranteeing the intracellular folate retention. (B) A reduced activity of the methionine synthase activity or a deficit of vitamin B12 causes a reduction of the intracellular folates. In fact, 5-MTHF leaks out of cells because cells cannot accumulate it, being monoglutamised and therefore shorter than other intracellular folates. Conversely, (C) a reduced activity of the methylenetetrahydrofolate reductase (MTHFR) causes the non-methylated folate accumulation, because these folates are polyglutamised by folylpolyglutamate synthase. (D) Immunoblot analysis of methythertrahydrofolate reductase (MTHFR) protein expression in red cells from healthy controls and in CF patients. One representative gel of other 20 with similar results. Graph reporting the immuno-blot analysis for the quantification of MTHFR expression as detected by densitometry in normal controls (black bar) and in CF patients (gray bar); data are presented as percentage of baseline values (n = 20).

Figure 3. Membrane proteins, ion content and cation transport pathways of red blood cells from healthy controls and CF patients.

(A) Red cell membrane proteins were separated by mono-dimensional electrophoresis and labelled with fluorescein-5-maleimide, which labels the proteins with active thiol-groups. Fluorescence was detected by placing the gel on a UV light source, photographed and analyzed. One representative gel of other 10 with similar results. (B) Red cell Na+ and K+ content in healthy controls and CF patients at baseline and after 5-MTHF and vitamin B12 supplementation for 24 weeks. Data are reported as medians (range for controls: RBCs Na+ 28.4–74.3 mmol/Kg Hb, RBCs K+ 320–512 mmol/Kg Hb n = 10; CF at baseline: RBCs Na+ 19.2–41.6 mmol/Kg Hb, RBCs K+ 199.7–335 mmol/Kg Hb n = 20; CF supplemented with 5-MTHF and vitamin B12: RBCs Na+ 15.6–61 mmol/Kg Hb, RBCs K+ 236–447 mmol/Kg Hb, n = 11); * P<0.05 compared to control red cells; ° P<0.05 compared to untreated CF patient red cells. (C) Cation transport pathways of red cells from controls (n = 10) and CF patients at baseline (n = 11) and after supplementation (n = 11). Data are expressed as medians (range for controls: Na-K pump 4.1–6.2 mmol×1013 cells×hour, Na-K-2Cl cotransport 0.98–1.90 mmol×1013 cells×hour, Na-H exchange 0.68–1.2 mmol×1013 cells×hour; CF baseline: Na-K pump 3.28–11 mmol×1013 cells×hour, Na-K-2Cl cotrasnport 0.15–1.70 mmol×1013 cells×hour, Na-H exchange 0.21–1.20; CF supplemented: Na-K pump 3.70–10.0 mmol×1013 cells×hour; Na-K-2Cl cotrasnport 0.49–4.70 mmol×1013 cells×hour; Na-H exchange 1.0–5.0 mmol×1013 cells×hour); * P<0.05 compared to control red cells; ° P<0.05 compared to untreated CF patient red cells.

Figure 4. Effects of 5MTHF and vitamin B12 supplementation on levels of plasma and red cell folate, of plasma vitamin B12 and homocysteine in CF patients.

(A) Plasma folate levels were 6.62 ng/mL (range: 4.99–8.16 ng/mL) at baseline and 52.85 ng/mL (range: 47.95–60.51 ng/mL) after supplementation; (B) red cell folate levels were 451.50 ng/mL (range: 339.00–708.00 ng/mL) at baseline and 4800.27 ng/mL (range: 3624–6249 ng/mL) after supplementation; (C) plasma vitamin B12 levels were 1157 pg/mL (range: 871–1448 pg/mL) at baseline and 2697.09 pg/mL (range: 2463–2963 pg/mL), after supplementation; (D) plasma homocysteine levels were 7.50 µM/L (range: 6.85–8.50 µM/L) at baseline and 5.30 µM/L (range: 4.42–6.21 µM/L) after supplementation.

Figure 5. Effects of 5-MTHF and vitamin B12 supplementation on malondialdehyde content and membrane proteins of red blood cells from CF patients.

(A) Malondialdehyde content of red blood cells at baseline and after supplementation (B) Red cell membrane proteins were separated by mono-dimensional electrophoresis and labelled with fluorescein-5-maleimide, which labels the proteins with active thiol-groups. Fluorescence was detected by placing the gel on a UV light source, photographed and analyzed. CF patients are indicated by numbers from 1 to 11; b: baseline; a: after supplementation with 5-MTHF and vitamin B12. The bands showing different fluorescence are indicated on the colloidal Commassie stained gel with numbers from 1 to 8.

Figure 6. Effects of 5MTHF and vitamin B12 supplementation on heat shock protein 70 associated with red cell membrane from CF patients.

(A) HSP70 associated with red cell membrane from CF patients were separated by mono-dimensional electrophoresis. Normal red cells are in line 1; red cells respectively from patient 1 (P1), patient 3 (P3) and patient 7 (P7) are in lanes 2, 4, 6 at baseline and in lanes 3, 5, 7 after treatment. (B) Graph reporting the immuno-blot analysis for the quantification of HSP70 expression as detected by densitometry in normal controls (black bar) and in CF patients (gray bar); data are presented as percentage of baseline values (n = 11). (C) Schematic diagram of the working hypothesis. Cellular membrane lipids and proteins are exposed to chronic oxidative stress in CF patients. The altered metabolism of polyunsaturated fatty acids (PUFAs) contributes to cellular damage, reducing membrane fluidity and increasing pro-oxidant environment. The 5-MTHF and vitamin B12 supplementation increases the levels of PUFAs and reduces both protein oxidative damage and HSP70 association on red cell membranes of CF patients.