TABLE 2.

Comparison of folate biomarkers¹

Biomarkers	Serum folate	RBC folate	Serum unmetabolized folic acid	Plasma tHcy
How useful is it for assessing exposure (intake)?	++	+	++	++
	Very sensitive to recent changes in intake but confounded by pregnancy, alcohol ingestion. Repetitive measurements over time may be necessary.	Not reflective of current intake but reflects longer term status and can be useful in conjunction with serum folate. Frequently low concentrations due to vitamin B-12 deficiency.	Quite reflective of current intake of fortified food/supplements but not of total folate	Good correlation with folate especially in nonelderly age groups
How useful is it for assessing status (short-, long-term)?	+	++	Not useful	++
	Not always low in folate deficiency, and low concentrations are not necessarily indicative of folate deficiency	Compromised sensitivity (normal concentrations in folate depletion) and specificity (low concentrations in vitamin B-12 deficiency)	Responds to treatment in days but may take longer to return to normal. Note: paradoxical small increase in tHcy shortly after dosing with folic acid reported.
How useful is it for assessing function?	Probably not useful	Probably not useful	Under investigation	+++
	Specific folate form (bioactive form) at the tissue level will affect function	Specific folate form (bioactive form) at the tissue level will affect function	Unclear whether serum unmetabolized folic acid plays a role in cancer development and/or promotion, immune response, etc	A functional indicator in the absence of confounders
Determinants or confounders (eg, age, sex, pregnancy, lactation, infection, polymorphisms)?	Age?	Age?	SNPs in DHFR?	Age
	Pregnancy	Vitamin B-12		Sex
	Alcohol consumption	Pregnancy		Renal function
	Smoking	Alcohol consumption		Polymorphism: MTHFR
	Drug use interactions (antifolates)	Smoking		Other B vitamins: vitamins B-12, B-6, B-2
	Thalassemia	Drug use interactions (antifolates)
	SNPs (MTHFR 677 C→T)	Thalassemia
		SNPs (MTHFR 677C→T)
Accepted cutoffs indicating deficient/normal/excess states?	Negative balance: serum folate <7 nmol/L (3 ng/mL)	Deficiency: RBC folate <305 nmol/L (140 ng/mL)	None	>12 μmol/L B vitamin deficiency/imbalance
	Subclinical deficiency: serum folate <10 nmol/L (increased Hcy)	Subclinical deficiency: RBC folate <340 nmol/L (increased Hcy)		? lower for females, children
	Reduced risk of NTD-affected pregnancy: serum folate >16 nmol/L (7 ng/mL)	Reduced risk of NTD-affected pregnancy: 900 nmol/L (≈400 ng/mL)
Other relevant issues: modifiers of validity (eg, infection)	Assay standardization	Assay standardization	Availability of standard reference materials with certified values for SFA (currently only reference value available)	Assay standardization
	Availability of standard reference materials with certified values for “total folate”	Availability of standard reference materials with certified values for “total folate”
Population vs individual?	Both	Both	Probably both	Both
Applicable in resource-constrained situations (eg, technical, sample storage, cost)?	Very limited	Very limited	No	Plasma preference
	Serum needs to be frozen within days of collection	Whole blood needs to be processed and frozen within days of collection	Requires chromatographic separation from other folate forms	Continued production of tHcy in erythrocytes and export into serum during clotting; keep EDTA whole blood chilled until processed (maximum: 4–6 h)
	For long-term storage, it needs to be frozen < −40°C	Need to generate accurate hemolysate
	Microbiological assay offers accurate results at low cost, but manual handling required and has limited throughput	Need to determine hematocrit (and serum folate)
		Microbiological assay offers accurate results at low cost, but manual handling required and has limited throughput
Multiple biomarkers needed?	Yes (for unequivocal diagnosis of folate deficiency)	No (but serum folate is a plus)	No	Yes (for unequivocal diagnosis of folate deficiency)
Other (clinical) information needed?	Yes	Yes	Yes	Yes
	RBC folate	Serum vitamin B-12	Dietary intake	Renal function (creatinine)
	Serum vitamin B-12	Possibly serum folate
	Hematologic findings	Hematologic findings
Dietary intake	Dietary intake

¹RBC, red blood cell; tHcy, total homocysteine; SFA, serum folic acid; Hcy, homocysteine; NTD, neural tube defect; MTHFR, methylenetetrahydrofolate reductase; SNPs, single nucleotide polymorphisms; DHFR, dihydrofolate reductase; +, useful; ++, very useful; +++, extremely useful.