From the Guidelines
The best method to assess ascorbic acid (Vitamin C) levels is through plasma vitamin C analysis, which is the preferred option for status assessment. This is because intracellular leukocyte vitamin C level analysis, although supposed to be more indicative of tissue vitamin C stores, requires more sample volume and preanalytical steps than plasma and has some caveats related to variability of vitamin C in different cell types 1.
When assessing plasma vitamin C levels, it is crucial to follow strict sample collection and pre-analytical procedures, including immediate separation of plasma samples after blood drawing and storage at ultra-low-temperature (-70 and -80 °C), as well as protection against light exposure 1. The determination of plasma ascorbic acid necessitates considerable logistical and analytical effort due to the high susceptibility of vitamin C to degradation related to temperature, light, pH, dissolved oxygen, and the presence of oxidizing/reducing agents 1.
Some key considerations for plasma vitamin C analysis include:
- Using lithium heparin when total vitamin C (sum of AA and DHAA) is determined 1
- Employing efficient analytical methods such as HPLC with ultraviolet (UV), fluorescence or electrochemical detection 1
- Including a typical sample stabilization preanalytic step before HPLC, such as acidification followed by immediate cold storage at ultra-low temperature 1
- Using a reducing agent such as dithiothreitol (DTT) or dithioerythritol (DTE) to convert DHAA into AA for total vitamin C analysis 1
Alternatively, a recently developed point-of-care device that measures the blood static oxidation-reduction potential (sORP) can be used to estimate plasma ascorbic acid levels, which is strongly related to the plasma vitamin C concentration and can be measured in non-acidified, non-reduced plasma within 20 min, directly after centrifugation 1.
From the Research
Assessment Methods for Ascorbic Acid (Vitamin C) Levels
- Biochemical measurements are considered the most objective approach to determining vitamin C nutritional status 2
- The measurement of serum levels of ascorbic acid is the most commonly used and practical procedure for determining vitamin C nutritional status in individuals or population groups 2
- Leukocyte ascorbate levels provide information concerning the body stores of the vitamin, but the measurement is technically more difficult to perform 2, 3
- Newer analytical techniques, such as high-performance liquid chromatography (HPLC), allow for the determination of reduced (ascorbic acid), oxidized (dehydroascorbic acid), or total amounts of vitamin C in biological specimens or foods 3, 4, 5
Blood Components for Assessing Vitamin C Status
- Platelets, PMN leukocytes, and MN leukocytes concentrate ascorbic acid, suggesting an important role in their physiological functions 4
- The ascorbic acid content of PMNs and platelets correlates positively with plasma concentration and supplementation with vitamin C 4
- MN leukocytes maintain the highest levels of ascorbic acid and play a very important function in immunocompetence 4
Analytical Techniques for Measuring Ascorbic Acid
- Automated or manual colorimetric and fluorometric procedures can be used to measure ascorbic acid in biological samples 2
- HPLC with monolithic column and UV-detection is a high-throughput method for assessing plasma ascorbic acid and total ascorbic acid 5
- Reversed-phase HPLC method for ascorbic acid can be used in conjunction with cellular differential centrifugation technique for the determination of ascorbic acid in relatively pure blood cell fractions 4