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Zeaxanthin is a predominant xanthophyll in human eyes and may reduce the risk of cataracts and age-related macular degeneration. Spirulina is an algal food that contains a high concentration of zeaxanthin. In order to determine the zeaxanthin bioavailability of spirulina for dietary supplementation in humans, spirulina was grown in nutrient solution with ²H₂O for carotenoid labelling. Single servings of ²H-labelled spirulina (4.0-5.0 g) containing 2.6-3.7 mg zeaxanthin were consumed by fourteen healthy male volunteers (four Americans and ten Chinese) with 12 g dietary fat. Blood samples were collected over a 45 d period. The serum concentrations of total zeaxanthin were measured using HPLC, and the enrichment of labelled zeaxanthin was determined using LC-atmospheric pressure chemical ionisation-MS (LC-APCI-MS). The results showed that intrinsically labelled spirulina zeaxanthin in the circulation was detected at levels as low as 10 % of the total zeaxanthin for up to 45 d after intake of the algae. A single dose of spirulina can increase mean serum zeaxanthin concentration in humans from 0.06 to 0.15 μmol/l, as shown in our study involving American and Chinese volunteers. The average 15 d area under the serum zeaxanthin response curve to the single dose of spirulina was 293 nmol × d/μmol (range 254-335) in American subjects, and 197 nmol × d/μmol (range 154-285) in Chinese subjects. It is concluded that the relative bioavailability of spirulina zeaxanthin can be studied with high sensitivity and specificity using ²H labelling and LC-APCI-MS methodology. Spirulina can serve as a rich source of dietary zeaxanthin in humans.