Human biomarkers for measuring riboflavin intake and status
Riboflavin status can be assessed by different methods, as seen in the table below.
Table 2: Assessment methods of riboflavin status (4, 5, 7)
| Biomarker | Analysis type | Sample | Benefits | Intricacies |
|---|
| EGRAC |
Indirect analysis |
RBC |
Only a marker for B2 deficiency |
Does not reflect status, fresh RBS are needed |
|---|
| FAD |
Direct analysis |
RBC (plasma) |
Reflects status (reflects intake) |
Does not reflect intake (status) |
|---|
| Urinary flavin |
Direct analysis |
Urine |
Reflects recent intake |
Not a marker for low B2 levels |
|---|
| EPPOA |
Indirect analysis |
Plasma |
Suitable for G6PD deficient individuals |
Not readily available |
|---|
EGRAC: erythrocyte glutathione reductase activity coefficient; EPPOA: erythrocyte pyridoxine phosphate oxidase activity FAD: Flavin adenine dinucleotide; RBC: red blood cells
Erythrocyte glutathione reductase activity coefficient (EGRAC)
This assay has been commonly used to determine riboflavin adequacy. The enzyme activity of the erythrocyte glutathione reductase is measured before and after exposure to FAD. The results are expressed as EGRAC; an EGRAC of 1.0 indicates no stimulation by FAD due to more than adequate riboflavin status (4, 7). The IOM suggest the following interpretation of EGRAC: <1.2 is acceptable, 1.2-1.4 is low, >1.4 is deficient. This assay cannot be used for individuals with glucose 6-phosphate dehydrogenase (G6PD) deficiency due to the resulting increased avidity of EGR towards FAD (4).
Method
Erythrocyte Glutathione Reductase Activity Coefficient (EGRAC), see method in reference (9): Becker et al. International Journal for Vitamin and Nutrition Research 1991;61:180-7
FAD analysis
Vitamin B2 vitamers (riboflavin, FMN, FAD) can be simultaneously analysed using chromatographic techniques, such as HPLC with fluorescence detection, or LC-MS. Riboflavin in plasma is accepted as general clinical analysis of status and supplementation monitoring (7).
Method
Riboflavin metabolites in plasma by:
HPLC-FLD, see method in reference (4): IOM DRIs for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic acid, Biotin, and Choline. Washington, DC: National Academy Press
LC-MS, see method in reference (10): Meisser Redeuil et al. Journal of Chromatography A 2015.Nov 27;1422:89-98
Urinary vitamin B2
Status can be assessed by analysis of urinary excretion in a random 24h specimen, expressed as either total riboflavin excreted or in relation to creatinine excretion (5). Since little riboflavin is stored in the body, urine measurements are a good proxy for dietary intake (7).
Method
Riboflavin in urine, see method in reference (11): Chen et al. Journal of Chromatography B 2005;820:147-50.
Erythrocyte pyridoxine phosphate oxidase activity (EPPOA)
This assay has been described for plasma samples and appears to be suitable for population with high prevalence of G6PD deficiency (5, 8). However, isolation and purification of the riboflavin-apoprotein prior to analysis is required (8).
Method
Erythrocyte pyridoxine phosphate oxidase activity (EPPOA), see method in reference (8): Kodentsova et al. Annals of Nutrition and Metabolism 1995;39:355-60.
Quality control and technical assistance
Laboratory accreditation and validation
LabCorp (headquartered in Burlington, North Caroline, USA) is a leading global life science company. Licensure/accreditation documentation may be requested by sending an email to: NOQ@Labcorp.com
This is a paid service, with a wide number of options, e.g. Vitamin B2, whole blood
ARUP Laboratories: A National Reference Laboratory is a US national clinical and anatomic pathology reference laboratory and a worldwide leader in innovative laboratory research and development. A non-profit enterprise of the University of Utah, ARUP offers an extensive menu of highly complex and unique medical tests.
For accreditation documentation see this page.
E.g. Vitamin B2, plasma
For other laboratory accreditation and validation schemes, see the OpeN-Global laboratory accreditation page.
Technical assistance
For questions on riboflavin methods or for technical assistance, please sophie.moore@kcl.ac.uk or write to:
Dr Daniela Hampel, PhD
Project Scientist
USDA/ARS Western Human Nutrition Research Center
Department of Nutrition, University of California Davis
Email: dhampel@ucdavis.edu or daniela.hampel@ars.usda.gov