Synonyms: e-folate, erythrocyte folate, RBC-folate.
Method(s): Microbiological assay, using a chloramphenicol-resistant strain of Lactobacillus casei (1). The assay has been adapted to a microtiter plate format and is carried out by a robotic workstation. Notably, samples containing antibiotic(s) that inhibit(s) the growth of Lactobacillus casei may cause assay interference.
What is measured: Biologically active folate species in blood cells. In the cells, about 50 different folate species exist, with variable polyglutamate chain length.
Methods for measurement of e-folate include hemolysate preparation that is difficult to control. First, the various folate species are deconjugated to monoglutamate forms prior to detection. This reaction is catalyzed by endogenous deconjugase, is pH sensitive and difficult to standardize. The various monoglutamates that are formed support the growth of Lactobacillus casei to a variable extent (and show different affinity to folate binders used in competitive binding assays). Besides, distribution between the different folate species is related to the MTHFR 677C->T genotype.
These are the main reasons that values for e-folate show large variation between laboratories, between different assay formats, and that the CVs are usually high. The calibrators used (usually folic acid in aqueous matrix) do not undergo the various steps in the assay, and do not adjust for precipitation, hemolysis, and binding of folates to oxyhemoglobin etc. Finally, folate instability has made external quality control difficult.
Assessment of folate status.
The view has prevailed that e-folate reflects the average folate status over time, corresponding the lifetime of the erythrocytes (about 120 days), and that e-folate is a better indicator than s-folate of folate status in tissues. However, folate status may vary over a shorter time period, and s-folate may better reflect such fluctuation. Besides, total homocysteine is a responsive indicator of folate status in tissues.
Patient/subject: Some antibiotics may inhibit growth of Lactobacillus casei, and may cause artificial, low folate levels.
Matrix: Whole blood.
Volume: Minimum volume is 100 µL, but 250 µL is optimal and allows sample handling and reanalysis.
Preparation and stability: Folate in hemolysate is degraded at room temperature, but also in samples frozen at -20 °C. In some samples stored frozen for years, folate is not detected (2).
Frozen, on dry ice. (for general instruction on transportation, click here.)
The measurement of e-folate should be abandoned.
1. Molloy, A.M., and Scott, J.M. (1997). Microbiological assay for serum, plasma, and red cell folate using cryopreserved, microtiter plate method. Methods Enzymol 281, 43-53.
2. Bailey, L.B., Stover, P.J., McNulty, H., Fenech, M.F., Gregory, J.F., Mills, J.L., Pfeiffer, C.M., Fazili, Z., Zhang, M., Ueland, P.M., et al. (2015). Biomarkers of nutrition for development-folate review. J Nutr 145, 1636S-680S.
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Amino acids: Alanine, Arginine, Asparagine, Aspartic acid, Glutamic acid, Glutamine, Glycine, Histidine, Isoleucine, Kynurenine, Leucine, Lysine, Methionine, Ornithine, Phenylalanine, Proline, Sarcosine, Serine, Threonine, Total cysteine, Tryptophan, Tyrosine, Valine
Amino acids and catabolites: 2-Aminoadipic acid, 2-Hydroxybutyrate, 3-Hydroxysiobutyrate, α-Hydroxyglutaric acid, β-Alanine, β-Aminoisobutyrate, β-Hydroxy B-methylbutyric acid, Phenylacetylglutamine
Acylcarnities: BB, C0, C2, C3, C3, DC, C4, C4-OH , C4-DC , iC5 , C5-DC, C5:1 , C6 , C8, C10, C12, C14 , C14-OH, C16, C16-OH, C18 , C18-OH, C18:1, C18:2
TCA metabolites: α-Ketoglutarate, Citrate, Fumarate, Isocitrate, Lactate, Malate, Pyruvate, Ketone bodies, 3-Hydroxybutyrate, Acetoacetate
AGEs: Carboxyethyllysine, Carboxymethyllysine
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Kynurenines: Kynurenine, Kynurenic acid, Quinaldic acid, Anthranilic acid, 3-Hydroxykynurenine, Xanthurenic acid, 3-Hydroxyanthranilic acid, Picolinic acid, Quinolinic acid, Nicotinic acid, Nicotinamide, N1-methylnicotinamide
Neopterin
Proteins: C-Reactive protein, Calprotectin, Serum Amyloid A
Ratio-derived: Kynurenine/tryptophan ratio, PAr index (PLP, PL, PA)
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SCFAs: Acetate, Butyrate, Formate, Isobutyrate, Isovalerate, Propionate, Valerate, α-Methylbutyrate
Indoles: 3-Indoxyl sulfate, Imidazole propionate, Indole-3-acetamide, Indole-3-acetate, Indole-3-aldehyde, Indole-3-lactate, Indole-3-propionate
Choline metabolites: Choline ,Betaine, DMG, TMAO
Amino acid derived: Phenylacetylglutamine
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B-vitamins, functional markers, and methyl donors: 4-Pyridoxic acid, Betaine, Choline, Cobalamin, Flavin mononucleotide, Folate, Methylmalonic acid, N1-methylnicotinamide, Nicotinamide, Nicotinic acid, Pyridoxal, Pyridoxal 5-phosphate, Pyridoxine, Riboflavin, Thiamine, Thiamine monophosphate, Total homocysteine
Fat-soluble vitamins: 25-hydroxy vitamin D2, 25-hydroxy vitamin D3, α-Tocopherol (Vit. E), All-trans retinol (Vit. A), Phylloquinone (Vit. K1), y-Tocopherol (Vit. E)
Essential amino acids: Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan, Valine
Meat and fish intake: 1-Methylhistidine, 3-Methylhistidine, B-Alanine, Creatine, Creatinine, TMAO
Tobacco use and coffee intake: Cotinine, Trans-3-hydroxycotinine, Trigonelline
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