Scottish Trace Element & Micronutrient Reference Laboratory

Scotland's specialised laboratory for trace elements and vitamins in health and disease

Vitamin B2

Vitamin B2 is a water soluble vitamin.The main circulating forms of vitamin B2 are riboflavin and its two cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). These two forms are detectable in both plasma and blood cells, however, the predominant form is FAD found in red blood cells. FMN and FAD act as cofactors in oxidation-reduction reactions and in the respiratory chain and are thus involved in energy production (eg. cytochrome, glutathione reductase, glutathione peroxidase, xanthine oxidase and methylene tetrahydrofolatereductase). Flavins also have antioxidant functions, removing free radicals to reduce oxidative damage to lipids and other macromolecules. As an antioxidant, FAD is a coenzyme to glutathione reductase. Flavin coenzymes are also involved in the metabolism of four other vitamins – vitamin B12, folic acid, vitamin B6 (for conversion to the coenzyme pyridoxal 5 phosphate) and niacin (conversion of tryptophan to niacin).

Deficiency and Toxicity
Body stores of vitamin B2 are low, so deficiency can develop fairly quickly when intake is inadequate or losses are excessive.

Specific vitamin B2 deficiency is very rare; it usually occurs in combination with other water soluble vitamin deficiencies as a result of malabsorption states (such as coeliac disease and Crohn’s disease), increased losses (e.g. during renal dialysis) and in the elderly and in alcoholics as a result of inadequate nutrition, and impaired digestion and absorption. Functional deficiency may also be induced by diseases such as hypothyroidism and adrenal insufficiency and by drugs such as chlorpromazine, imipramine and amitryptamine.

Symptoms of deficiency are sore throat, inflammation of mouth, tongue and throat, cracks in lips and corners of mouth, normocytic normochromic anaemia, and dermatitis.

Catabolic patients and patients with systemic infections may have increased requirements due to enhanced excretion, increased utilization, decreased intake, defective absorption and poor utilization.

Vitamin B2 is essentially non-toxic.

Assessment of status
Plasma vitamin B2 (FAD) is extensively bound to albumin and so concentrations fall in the presence of systemic inflammatory response independent of vitamin B2 nutritional status. This is because plasma albumin concentrations fall as part of the systemic inflammatory response and also perhaps due to the redistribution of FAD and increased uptake by tissues.

Work done in this unit has shown that the red cell FAD concentration is unaffected by systemic inflammatory response in patients 48 hours after undergoing elective surgery for knee arthroplasty,76 whereas the plasma FAD concentration decreases significantly (see Table). Red cell FAD also reflects supplementation in patients with critical illness.77 We therefore recommend the measurement of FAD in red cells in patients that have evidence of systemic inflammatory response. Results are expressed as nmoles of FAD per g of haemoglobin.

We routinely only report red cell FAD for assessment of vitamin B2 status. However, if required, plasma or whole blood riboflavin, FAD and FMN can be measured to assess vitamin B2 status in patients who are non-inflamed, for monitoring of patients with the genetic neurodegenerative disorder Brown-Vialetto-Van Laere syndrome, and for research studies.

Recommended Daily Allowance
Adults: 1.3 mg/day

Effect of Systemic Inflammatory Response on Plasma Vitamin B2 Concentrations

Table: Baseline, peak/ trough and day 7 concentrations of CRP, plasma and red cell flavin adenine dinucleotide (FAD) and albumin following elective surgery for knee arthroplasty (n = 20).76 Median (range).


Baseline values

(0 h) 


(48 h)

     Final values      

(168 h)


(0–168 h)

C-reactive protein (mg/L) <6 (<6–<6) 170 (70–242) 31 (<6–168) <0.001
Plasma FAD (nmol/L) 62 (52 – 105) 39 (24 – 76) 66 (36 – 97) <0.001
Red cell FAD
(nmol/g Hb)
2.45 (1.70 – 3.90)    2.7 (1.8-3.9)      2.70 (1.90 – 3.80)     0.115

Sample Requirements and Reference Ranges for Vitamin B2

Sample Type Whole blood or packed red blood cells (please label tube as red cells; fasting sample preferred*).
Container Lithium heparin (non-gel) or EDTA. Lithium heparin gel tubes unsuitable.
Precautions Light-sensitive; wrap in tin foil. Send by first class post within 48 hours.
If delivery to Glasgow is outwith 48 hours of sample collection, prepare red cells (minimum volume 300 µL) by removing plasma and buffy layer (mark clearly on tube that they are red cells) and store frozen until sending and then send by first class post (ice or dry ice not required).
Minimum volume 1 mL whole blood or 300 µL** of red cells***
Reference range Red cell FAD: 1.0 to 3.4 nmol/g Hb 77
Mean turnaround time 3.3 days
Method HPLC with fluorimetric detection78
Traceability Calibrated using commercially available standard manufactured in accordance with EN ISO 17511:2003 “In vitro diagnositic medical devices. Measurement of quantities in biological samples. Metrological traceability of values assigned to calibrators and control materials”.
Intermediate Precision (CV) 4.4% at 1.5 nmol/g Hb, 4.2% at 5.6 nmol/g Hb
Measurement Uncertainty, U 1.5 ± 0.1 nmol/g Hb, 5.6 ± 0.5 nmol/g Hb
Analytical Goals (CV) Acceptable 4.8%, Desirable 3.2%****
EQA Scheme INSTAND (whole blood EQA scheme), Düsseldorf, Germany.

* Ideally a fasting sample should be collected, especially if the patient is receiving oral or parenteral vitamin B2 supplementation. If this is not possible, sample should be taken at least 8 hours post treatment for patients receiving oral supplementation or TPN.
** Absolute minimum volume; this volume is insufficient to carry out repeat analysis if analysis fails.
*** Please contact the Laboratory if vitamin B2 in whole blood or plasma is required.
**** Calculated from biological variation data.71



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