Ascorbic acid in lymphocytes: cell preparation and liquid-chromatographic assay

Area of Science:

• Biochemistry
• Clinical Chemistry
• Nutritional Science

Background:

• Ascorbic acid (vitamin C) is vital for immune function and cellular health.
• Accurate assessment of ascorbic acid levels is crucial for managing various patient populations, including the critically ill.
• Existing methods may lack the sensitivity or speed required for timely nutritional assessment.

Purpose of the Study:

• To develop and validate a rapid, reproducible method for quantifying ascorbic acid concentration in leukocytes.
• To establish reference intervals for ascorbic acid in different blood cell types and plasma.
• To assess the prevalence of ascorbic acid deficiency in hospitalized patients.

Main Methods:

• A high-performance liquid chromatography (HPLC) assay was employed for ascorbic acid quantification.
• A simple, reproducible cell-separation technique using minimal whole blood (≤2 mL) was utilized.
• Leukocyte populations were separated with high purity (>95%) and viability (>99%).

Main Results:

• The HPLC method demonstrated high sensitivity, quantifying ascorbic acid down to 0.1 microgram/mL.
• Analysis was rapid, with completion within 15 minutes post-specimen preparation.
• Preliminary evaluation indicated a higher-than-expected risk of ascorbic acid deficiency among hospital patients.

Conclusions:

• HPLC offers a superior method for nutritional assessment through leukocyte ascorbic acid measurement.
• The assay is versatile, applicable to urine, plasma, and other biological samples.
• Hospitalized patients represent a vulnerable group requiring vigilant monitoring for ascorbic acid deficiency.