Normal Biotin Levels and Testing Methods
Normal biotinidase activity in serum is 7.57 ± 1.41 nmol/min/mL, and the diagnosis of biotin-related disorders is made through enzymatic biotinidase activity testing in serum or plasma, not by measuring biotin levels directly. 1
Standard Reference Ranges for Biotinidase Activity
The American College of Medical Genetics and Genomics has established the following reference ranges for biotinidase enzyme activity 1:
- Normal individuals: 7.57 ± 1.41 nmol/min/mL serum
- Heterozygotes (carriers): 3.49 ± 0.72 nmol/min/mL serum
- Partial biotinidase deficiency: 1.47 ± 0.41 nmol/min/mL (10-30% of mean normal activity)
- Profound biotinidase deficiency: 0.12-0.19 nmol/min/mL (<10% of mean normal activity)
Important caveat: Full-term newborns have only 50-70% of mean normal adult biotinidase activity, so age-specific reference ranges must be used when testing infants. 1
How to Test for Biotin Status
Primary Testing Method: Enzymatic Biotinidase Assay
The gold standard for diagnosing biotin-related disorders is measuring biotinidase enzyme activity in serum or plasma using the colorimetric biotin-4-amidobenzoic acid substrate method. 1
Sample collection requirements 1:
- Collect 3-5 mL whole blood in red-top or green-top (heparin) tube
- Separate serum/plasma by centrifugation within 1 hour
- Freeze immediately at -80°C (critical: -20°C storage causes enzyme degradation and falsely low results)
- Ship on dry ice to laboratory
Critical pitfall: Sulfa drugs interfere with the assay and cause falsely elevated results, so a substrate-free blank must be run for each patient sample to detect interfering substances. 1
Alternative Testing Methods
Fluorescent assay using biotinyl-6-aminoquinoline can be used but is more expensive than the colorimetric method, though it works well with dried blood spots for newborn screening. 1
What NOT to Use as Sole Testing
Urine organic acids (3-hydroxyisovalerate, 3-methylcrotonylglycine) and plasma acylcarnitines (3-hydroxyisovalerylcarnitine) should never be used alone to diagnose biotinidase deficiency because many deficient patients have normal metabolite levels and will be missed, while these abnormalities can occur in other conditions leading to misdiagnosis. 1
Confirmatory Molecular Testing
When enzymatic testing shows abnormal results, DNA analysis should be performed to distinguish between profound deficiency, partial deficiency, and heterozygous carrier status, particularly since almost all partial deficiency patients carry the D444H mutation. 1 This requires 1-2 mL whole blood in EDTA (lavender-top) tube shipped at room temperature. 1
Critical Clinical Context: Biotin Supplementation Interference
A major pitfall in modern practice is that high-dose biotin supplements (often taken for hair/nail health) interfere with biotin-streptavidin immunoassays, causing falsely low troponin levels that can lead to missed myocardial infarctions—a life-threatening diagnostic error. 2 This represents the most dangerous clinical scenario related to biotin testing. 2
When ordering laboratory tests that use biotin-streptavidin technology (thyroid hormones, troponin, b-HCG, vitamin D, hepatitis/HIV serology), specifically ask patients about biotin supplement use and discontinue supplementation before testing. 2, 3
Quality Control Requirements
Each laboratory must establish its own reference ranges using 20-50 normal individuals and multiple affected individuals across all deficiency categories, with ongoing proficiency testing documented. 1 Positive and normal controls must be included with each patient sample batch. 1