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Vitamin A equivalence of spirulina β-carotene in Chinese adults as assessed by using a stable-isotope reference method^1,2,3,4

¹ From the National Institute for Nutrition and Food Safety, Beijing, China (JW, LL, and SY); the Institute of Health Food, Zhejiang Academy of Medical Science, Zhejiang, China (YW, WL, and YF); Qingdao University Medical College, Qingdao, China (ZW); USDA Human Nutrition Research Center on Aging at Tufts University, Boston, MA (JQ, RMR, and GT); University Hospital, Zurich, and Nestlé Foundation, Lausanne, Switzerland (PMS); and USDA-ARS Children's Nutrition Research Center, Houston, TX (MAG)

² The contents of this publication do not necessarily reflect the views or policies of the US Department of Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.

³ Supported by the Nestlé Foundation and by grants no. 58-1859-7-707 and 58-6250-6-001 from the US Department of Agriculture–Agricultural Research Service.

⁴ Reprints not available. Address correspondence to S Yin, National Institute for Nutrition and Food Safety, 29 Nan Wei Road,. Xuan Wu District, Beijing, 100050, China. E-mail: shianyin{at}gmail.com.

	ABSTRACT

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Background: Spirulina is a high-protein food supplement that contains carotenoids.

Objective: The objective of the study was to determine the vitamin A equivalence of spirulina β-carotene in humans.

Design: Spirulina was grown in a 23 atom% ²H₂O cultural solution. Spirulina β-carotene showed the greatest enrichment as [²H₁₀]trans β-carotene. Ten healthy Chinese men with a mean (±SD) serum retinol concentration of 1.7 ± 0.3 µmol/L and a body mass index (in kg/m²) of 23 ± 3 consumed 5.8 µmol [¹³C₁₀]retinyl acetate in oil as a reference dose with a breakfast containing 13 g fat. One week later, each subject consumed 7.9 µmol trans β-carotene in spirulina with a breakfast containing 22 g fat. All subjects followed diets low in carotenoid and vitamin A. Forty blood samples were collected from each subject over a span of 56 d. Concentrations and enrichments of retinol and β-carotene in serum samples were determined by using HPLC and a mass spectrometer.

Results: Compared with the serum response to [¹³C₁₀]retinyl acetate dose, the mean conversion factor of spirulina β-carotene to retinol was 4.5 ± 1.6 (range: 2.3–6.9) by weight. It was estimated that 80% of the conversion occurred within the first 24 h after spirulina administration.

Conclusion: In a group of well-nourished, normal-weight Chinese men following low-vitamin A diets, 4.5 mg spirulina β-carotene consumed with 22 g fat has the same vitamin A activity as does 1 mg retinyl acetate.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Vitamin A is essential for life (1), and a severe deficiency of vitamin A causes blindness and mortality (2, 3). However, long-term excessive intake of preformed vitamin A can be harmful to humans by causing liver malfunction and birth defects (4). As observed in many studies, β-carotene is an effective (5) and safe (6, 7) source of vitamin A. High doses (180 mg/d) of β-carotene were given to humans for several months without any observed adverse effects other than changes in skin color (8). Increases in lung cancer risk were observed in long-term smokers who took a supplement of 20 mg β-carotene/d (9) and in asbestos workers who took a supplement of 30 mg β-carotene/d (10). However, a β-carotene supplement 25 mg/d was safe for nonsmokers (11). Dietary intake of β-carotene, usually 10 mg/d, can be a safe source of vitamin A (12-15). In many countries, plant foods rich in β-carotene are major sources of vitamin A. For example, in China, 59% of vitamin A comes from vegetables (16). However, little has been known about the vitamin A activity of β-carotene in plant foods.

Vegetables intrinsically labeled with a stable isotope have been produced (17, 18), and a stable-isotope reference method was developed to calculate the vitamin A equivalence of β-carotene in vegetables (19). In the stable-isotope reference method (5), the subjects consumed a single dose or multiple doses of β-carotene and vitamin A labeled with stable isotopes. The labeled retinols from the doses were absorbed into the blood and diluted in the body pool of vitamin A. According to the molecular mass obtained by using a sensitive mass spectrometer, serum retinols derived from β-carotene, vitamin A, or endogenous unlabeled retinol were significantly different. To calculate the vitamin A equivalence of β-carotene, the amount of newly formed retinol from β-carotene was compared with that from vitamin A. One study using the stable-isotope reference method found that 20.9 mg spinach β-carotene or 14.8 mg carrot β-carotene could provide 1 mg retinol (20).

Spirulina is an alga that is rich in protein and carotenoids (mainly β-carotene) (21-23). One supplementation study in humans found that the incidence of a symptom of vitamin A deficiency, Bitot's spot, decreased from 80% to 10% after 1 g spirulina/d was provided for 150 d to 5000 preschool-age children in Madras, India (24). However, no data are available on a direct and quantitative assessment of the vitamin A value of spirulina β-carotene. In the present study, intrinsically deuterium-labeled spirulina was consumed by healthy Chinese men to evaluate the vitamin A equivalence of spirulina β-carotene by using a known amount of [¹³C₁₀]retinyl acetate as a reference dose.

	SUBJECTS AND METHODS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Subjects
Subjects were recruited from 3 villages on the outskirts of the city of Jining in the Shandong Province of China. The diets for this population were composed of cereal, vegetables, and a small amount of foods of animal origin. Study and recruitment information was advertised in the 3 villages, and men (aged 40–60 y, n = 195) voluntarily participated in the prescreening process.

Ten healthy men with normal but relatively low serum retinol concentrations were selected from the 195 volunteers and enrolled in the study. All subjects were nonsmokers or had stopped smoking >2 mo before the study, and they had not taken vitamins or other supplements in the previous month. No smoking, alcohol, supplements, or medicines were permitted during the preparation period and the study. All subjects were negative for Helicobacter pylori infection and hepatitis B surface antigen, had normal liver function, and had no metabolic disease or cardiovascular disease.

Written informed consent was obtained from all volunteers under the guidelines established by the Institutional Review Board (IRB) of Tufts-New England Medical Center, and by the Institute of Nutrition and Food Hygiene Ethical Review Committee of the Chinese Academy of Preventative Medicine.

Study design
The study procedure is shown in Figure 1. Two weeks before the study, the subjects were instructed to eat their usual diet at home but to avoid nutritional supplements and foods rich in β-carotene or vitamin A, such as spinach, carrots, green or red pepper, or liver products. Each subject kept a 3-d food record (2 weekdays and one weekend day) at the beginning of the study. After that 2-wk period, volunteers were housed for 10 d in a metabolic unit established in Jining. Meals low in carotenoids or vitamin A were served.

View larger version (11K): [in this window] [in a new window]   FIGURE 1.. Experiment scheme of dosing and time for blood sample collection. Ten healthy Chinese men consumed 2 mg [13C10]retinyl acetate (RAc) as a reference dose and 5 g spirulina containing 4.3 mg [2H10]β-carotene on day 1 and day 8, respectively. , Blood sample collection; d, day; h, hour of the day.

Blood samples (10 mL) were collected from all subjects at 0 (fasting blood), 5, 6, 7, 8, 9, 11, and 13 h on days 1 and 8. Blood was drawn at 0, 5, and 11 h on days 2 and 9 (see Figure 1) and at 0 and 11 h on days 3 and 10. Fasting blood samples (10 mL) were obtained on days 4, 5, 6, 7, 11, 13, 15, 19, 23, 27, 32, 39, 46, and 56. Blood samples were covered by aluminum foil to protect them from light, kept in a 37 °C water bath for 30 min, and then centrifuged at 4 °C and 800 x g for 15 min. Serum was stored at –20 °C for 10 d and then packed with dry ice and transported to the Institute for Nutrition and Food Safety (Beijing, China), where it was stored at –70 °C. All frozen samples, on dry ice, were sent by air to the Carotenoids and Health Laboratory of the US Department of Agriculture (USDA)–Human Nutrition Research Center on Aging (HNRCA) at Tufts University (Boston, MA) for analysis.

The subjects were free-living from day 11 to day 56. Dietary instructions (a list of vegetables, fruit, and meats that could be eaten) were given to the subjects to avoid large intakes of vitamin A and β-carotene. Dietary record forms with directions were taken home and completed by all subjects for 3 d of each week.

Supplements
[¹³C₁₀]retinyl acetate (8, 9, 10, 11, 12, 13, 14, 15, 19, 20-¹³C₁₀-retinyl acetate) with a chemical purity of 98% and an isotopic purity of >99% was purchased from Cambridge Isotope Laboratory (Andover, MA). Spirulina was grown hydroponically at the USDA-Agricultural Research Service Children's Nutrition Research Center in Houston, TX, by using 23 atom% deuterium oxide in the nutrient media. Algal cells (Spirulina platensis), obtained from the University of Texas Culture Collection of Algae (UTEX No. LB 2340; Austin, TX), were inoculated into 20-L glass carboys and were maintained in a greenhouse with the use of a mixture of sunlight and metal-halide lamps. Cells were grown in a sterilized artificial-seawater nutrient medium (26) enriched with deuterium oxide (23 atom%). Cell cultures were stirred at low speed to keep cells suspended. Individual cultures were grown for 10–12 d before being harvested, at which time cells were allowed to settle, were rinsed with distilled water, and were centrifuged at low speed to pellet the cells. Pelleted cells were freeze-dried and stored at –80 °C until they were shipped on dry ice to the USDA-HNRCA at Tufts University. The pelleted material was powdered, put in gelatin capsules, and weighed. All supplements were subsequently kept at –80 °C until they were given to volunteers or analyzed.

HPLC for retinol and carotenoid concentrations in baseline serum and in spirulina
HPLC (Waters 2695 separation module and 2996 photodiode array detector wavelength 250–600 nm; Waters Corporation, Milford, MA) and a Pronto SIL C₃₀ column (Bischoff Chromatography, Leonberg, Germany) were used to analyze retinol and carotenoid concentrations in baseline serum and spirulina. HPLC mobile phase included solvent A [methanol:tert-butyl methyl ether:water = 85:12:3 (by vol)] and solvent B (methanol:tert-butyl methyl ether:water = 8:90:2 by vol), both of which include 1.5% ammonium acetate in water (27).

To extract retinol and carotenoids from serum, 100 µL serum, 100 µL internal standards (retinyl acetate + echinenone), 500 µL saline (0.85%), and 4 mL of a ratio of chloroform to methanol [(C:M) 2:1, by vol] were added to a glass tube (16 x 100-mm), mixed by using a vortex mixer (Labnet International Inc, Woodbridge, NJ) for 1 min, and then centrifuged at 4 °C and 800 x g for 10 min. The lower layer (chloroform layer) was collected into another glass tube (13 x 100-mm) by using a glass pipette. Hexane (3 mL) was added to the aqueous layer, mixed by vortex, and centrifuged again. The hexane layer and the chloroform layer were combined and dried under N₂ on an N-EVAP nitrogen evaporator (Organomation Associates Inc, South Berlin, MA). The residue was dissolved in 100 µL of a mixture of ethanol and tetrahydrofuran at a ratio of 2:1 (by vol), mixed and sonicated for 20 s, and then transferred to a vial. The dissolved residual solution (20 µL) was injected into the HPLC. A linearly gradient HPLC pump-flow method began with 100% solvent A (A), changed to 45% A and 55% solvent B (B) in 21 min and held for 1 min, changed to 5% A and 95% B in 11 min and held for 4 min, and then changed to 100% A in 2 min and held for 10 min to equilibrate the system before injection of the next sample. The HPLC pump flow was set at 0.4 mL/min.

To analyze carotenoids in spirulina, 100 mg spirulina powder was weighed and put into a 50-mL glass vial, and then 3 mL methanol was added to the vial. Spirulina in the vial was homogenized by using a Polytron (PT1600E; Kinematica AG, Lucerne, Switzerland) at speed 10 for 30 s in an ice bath, and then it was washed with 5 mL methanol. The vial containing the mixture was stored at 4 °C for 12 h. Then, the vial was mixed by vortex for 30 s and centrifuged at 4 °C and 800 x g for 5 min. The methanol layer was transferred to a 50-mL volumetric flask by using a pipette gun. Tetrahydrofuran was used to extract carotenoids 4 times (10 mL tetrahydrofuran for the first 3 times, and 5 mL for the 4th time); vortex mixing and centrifuging were involved each time. The tetrahydrofuran layers were collected into the methanol extract–containing volumetric flask, to which tetrahydrofuran was added to a volume of 50 mL and mixed well. The extract (1 mL) was evaporated under the N₂ flow of the N-EVAP (27). The residue was resuspended in 1 mL ethanol, and 20 µL was injected into the HPLC. Triplicate samples were analyzed.

A linearly gradient procedure started from 60% solvent A and 40% solvent B for 2 min, then changed to 17% A and 83% B in 7 min and held for 7 min, changed to 100% B in 6 min and held for 6 min, and changed back to 60% A and 40% B in 2 min. The HPLC pump flow was set at 0.4 mL/min. Retinol and carotenoid concentrations were calculated according to the standard curves of all-trans retinol, -carotene, trans β-carotene, 9-cis and 13-cis β-carotene (all: Sigma-Aldrich Inc, Saint Louis, MO) and lutein, zeaxanthin, and β-cryptoxanthin (all: Roche Vitamin Inc, Belvidere, NJ).

EMPOWER PRO software (version 2.0; Waters Corporation) was used to control the HPLC system and data analysis. Carotenoids and retinol were detected at 450 and 340 nm, respectively. The standard response curve of retinol was set at 340 nm under the mobile phase solvent composition of the HPLC system. The amount of retinol injected into the HPLC system was measured by using the spectrophotometer at 325 nm with the use of ethanol as solvent.

HPLC for collecting retinol fraction from serum
To collect retinol from serum, 200 µL serum was used; the same procedures as for baseline serum analysis were followed. The serum extract was dissolved in 75 µL of a ratio of ethanol to tetrahydrofuran [(E:T) 2:1 by vol], mixed and sonicated for 20 s, and then transferred to a vial. Sixty µL was injected into the HPLC.

A Waters HPLC (616 pump, 717 Plus Autosampler, and 996 photodiode array detector; Waters Corporation) with a Pecosphere C₁₈ column (PerkinElmer Life and Analytic Sciences, Shelton, CT) and an FC203 collector (Gilson Inc, Middleton, WI) were used to analyze the concentration and to collect retinol fraction from serum samples. The HPLC mobile phase included solvent A [methanol:tetrahydrofuran:water (M:T:W) = 50:20:30 by vol] and solvent B (M:T:W = 50:44:6 by vol) with 1.5% ammonium acetate in water. A linearly gradient solvent procedure started at 100% solvent A for 6 min, changed to 60% A and 40% B in 5 min, changed to 17% A and 83% B in 11 min, and changed to 100% B in 10 min. Finally, the gradient went back to 100% A in 2 min and held for 8 min to stabilize the system before the next sample was injected. The column temperature was 12 °C, and the flow rate was 1 mL/min. Retinol was eluted at 9 min, so the eluant fluid from 8–10 min was collected into a 2-mL flask (Pyrex Labware; Corning Inc, Corning, NY).

Gas chromatography–electron capture negative chemical ionization–mass spectrometer for the enrichment of labeled retinol in serum
The retinol fraction from HPLC was dried under N₂. We added 50 µL N,O-bis(trimethylsilyl) trifluoroacetamide (1%, BSTFA-TMCS; Pierce, Rockford, IL) to the residue, capped the flask with a stopper to avoid moisture, and it put into an oven at 100 °C for 60 min. Then the flask was cooled to room temperature. The derivatized retinol (2–4 µL) was injected into a gas chromatography–electron capture negative chemical ionization–mass spectrometer (GC-ECNCI-MS, Agilent 6890 Series GC System and 5973 Network Mass Selective Detector; Agilent Technologies, Andover, MA). A Zebron ZB-1ms Capillary GC column (Phenomenex Inc, Torrance, CA) was used to separate compounds. Helium and methane were used as the carrier gas and the reaction gas, respectively. The GC oven temperature was increased from 50 °C to 220 °C at a speed of 15 °C/min, to 230 °C at 5 °C/min, and to 310 °C at 20 °C/min; it remained at 310 °C for 5 min. This method produced a trans retinol peak at 14 min. The mass spectrometer was set to scan at a mass-to-charge ratio (m/z) of 260 to 280. Retinol molecular mass was equal to 286, and lost one group of H₂O during the negative chemical ionization process, which made the major fragment of retinol at m/z 268 (19).

The enrichment of labeled retinol was calculated by using the following 2 equations:

(1)

and

(2)

Total body stores of vitamin A (in mmol retinol) were calculated as 0.00468 x 10^{37(isotope dose in mmol)}/[¹³C]retinol: [²H]retinol in serum 3 d after the isotope dose (28). [¹³C]retinol:[²H]retinol instead of [²H]retinol:unlabeled retinol was used in the present study, assuming that retinol labeled by ¹³C followed the same kinetics as did deuterated retinol.

The area under the curve (AUC) of labeled retinol was calculated by using INTEGRAL-CURVE of KALEIDAGRAPH software (version 3.5; Synergy Software, Reading, PA) using total serum response (nmol, as y axis) versus time (d, as x axis). The calculation is shown in the following equation:

(3)

where body wt x 0.0435 = total-body serum volume.

Vitamin A equivalence was calculated by using the following equations:

(4)

where 5790 is the reference dose (in nmol); and

(5)

Postabsorptive conversion of β-carotene to retinol
[²H₅]retinol from spirulina [²H₁₀]β-carotene was assumed to follow the same serum kinetics as does [¹³C₁₀]retinol from the [¹³C₁₀]retinyl acetate dose, on the assumption that all [²H₅]retinol was formed in the intestine only. The time span of 24 h was chosen as the cutoff for the intestinal conversion and postabsorptive conversion. If no further conversion occurred, the ratio of [²H₅]retinol to [¹³C₁₀]retinol during the study should be as consistent as the ratio on day 1. The conversion calculations were obtained by using the following equations:

(6)

and

(7)

in both of which equations n d = 3, 7, 15, 24, 31, 38, or 48 d.

Liquid chromatography–atmospheric pressure chemical ionization–mass spectrometer for β-carotene in spirulina and serum
To determine the percentage enrichment of labeled β-carotene, the β-carotene fraction was extracted and collected from the separation on the HPLC system (the same procedure as that used for collecting the retinol fraction, described above). After extraction of carotenoids from 1–2 mL serum by using hexane and a ratio of chloroform to ethanol (C:E) as described above, the carotenoid residue was resuspended in 200 µL chloroform and put on a Strata NH₂–propyl open column (500 mg/3 mL; Phenomenex, Torrance, CA) conditioned with 4 mL hexane. Hexane (4 mL) was used as the eluent. The β-carotene in hexane was evaporated under N₂. The residue was resuspended in 75 µL E:T (1:2 by vol), and 60 µL was injected into the HPLC with a C₁₈ column. A linearly gradient solvent procedure started from 60% solvent A and 40% B, changed to 17% A and 83% B in 4 min and held for 4 min, changed to 100% B in 5 min and held for 25 min, then changed back to 60% A and 40% B in 2 min and held for 8 min to stabilize the system before injecting the next sample. β-Carotene was washed out at 17 min, and the eluant of 16–18 min was collected, dried down, resuspended in 60 µL ethanol, and injected into the liquid chromatograph–mass spectrometer [LC-MS (20, 29)].

The LC–atmospheric pressure chemical ionization–MS (LC-APCI-MS) system included an Agilent 1100 LC (Agilent Technologies) equipped with a C₁₈ Prizm column (Keystone Scientific, Bellefonte, PA) and an Esquire mass spectrometer (Bruker Daltonic Inc, Billerica, MA). The LC mobile phase contained 75% methanol and 25% E:M:T (75:20:5 by vol). The mass spectrometer was set to scan at m/z 530–560 in 0.1-Da steps with a scan speed of 5.25 scans/s.

Statistical analysis
We used SPSS software (version 13.0; SPSS Institute, Chicago, IL) for statistical analyses. Bivariate correlation was used to find the correlations between variables. The paired t test was used to compare the concentrations of serum retinol and carotenoids before and after retinyl acetate and spirulina were ingested.

	RESULTS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Subject characteristics and fasting serum concentrations of retinol and carotenoids at the beginning of the study are listed in Table 1. The mean (±SD) BMI and serum retinol concentration of the 10 subjects was 23.4 ± 3.1 and 1.71 ± 0.33 µmol/L, respectively. Mean serum concentrations of -carotene, β-carotene, and β-cryptoxanthin were lower than the mean values in American men aged 20 y, but the mean serum concentration of lutein + zeaxanthin (0.44 µmol/L) was similar to the mean value in American men aged 20 y—ie, 0.40 µmol/L (30).

Table 2

Figure 2

View larger version (12K): [in this window] [in a new window]   FIGURE 2.. Enrichment profile of β-carotene (β-C) in deuterated spirulina determined by using a liquid chromatography–atmospheric pressure chemical ionization–mass spectrometer.

The mean (±SD) highest enrichment of [²H₅]retinol in the circulation was 3.7 ± 1.3% (range: 2.4–6.6%), and the mean highest enrichment of [¹³C₁₀]retinol in the circulation was 7.8 ± 1.4% (range: 5.2–9.8%). The greatest concentrations of [¹³C₁₀]retinol and [²H₅]retinol both appeared at 11–24 h after the corresponding doses. Representative serum response curves are shown in Figure 3.

View larger version (10K): [in this window] [in a new window]   FIGURE 3.. Serum response curves of [13C10]retinol from retinyl acetate () and [2H5]retinol from spirulina β-carotene (•) in 3 subjects. A, subject 1; B, subject 6; C, subject 7.

Table 3

Table 4

Table 5

	DISCUSSION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
In the stable-isotope reference method, the labeled β-carotene can be administered as a single dose or in multiple doses. Although multiple doses were more related to usual dietary practice, the single-dose method can provide an accurate metabolic curve for isotope tracers because there are more blood samples and less influence from diets than in the multiple-dose method (29). In the current study, a single dose of spirulina β-carotene was given to the subjects 7 d after a single dose of labeled retinyl acetate to avoid the potential absorption competition. Diets low in vitamin A and β-carotene (174 µg retinol activity equivalents/d, 19% of the US recommended dietary allowance or 37% of the intake recommended in China) were followed by the subjects to minimize the influence of subsequent dietary vitamin A or β-carotene on the labeled retinol metabolic curve.

We found that the mean conversion factor of spirulina β-carotene to vitamin A was 4.5 by weight, which was in the range of mean conversion factors (1.3–9.1 by weight) of commercial β-carotene (dose 20 mg) for normal converters (5, 29, 31). Several factors could contribute to the high vitamin A equivalence of spirulina β-carotene. As observed in previous studies, fat provided with the β-carotene dose was a major influencing factor for conversion efficiency. In the single-dose method, β-carotene was usually dissolved in more oil (6 mg in 6 g oil) than was retinyl acetate (3 mg in 170 mg oil) to improve the absorption and conversion (20, 29, 31). Edwards found that an increased retinol concentration in plasma triacylglycerol–rich lipoprotein could be detected when carrot or spinach was consumed with 20 g fat instead of 1 g fat, but the fat amount did not affect the AUC of serum [²H₄]retinol from [²H₄]retinyl acetate (32). In our study, the labeled spirulina and the vitamin A doses were both administered with a liquid drink containing 10 g fat. An additional 9 g fat in the breakfast meal was provided 1 h after the spirulina dose to benefit the conversion. However, 71 and 70 g fat were consumed by the subjects on day 1 and day 8, respectively. Therefore, we adjusted the proportion of fat in the 3 meals, but the total fat and energy for the day did not change. We felt that this was an acceptable dietary practice when taking a fat-soluble supplement. The low vitamin A diets during the study may contribute to the high vitamin A equivalence. No control groups (no spirulina or vitamin A dose) were included to determine whether the vitamin A nutriture would change during the study period. Therefore, our results on the conversion factors were obtained under optimal conditions.

In addition, this efficient conversion of spirulina β-carotene may be due to the simple cell structure of spirulina, which is composed of protein and peptidoglycans that are easily digested (21). It has been reported that the absorption of iron in spirulina was significantly lower than that of the iron in ferrous sulfate and whole egg but significantly greater than that of the iron in whole wheat (33). In addition, the high natural free fatty acid (1–10%) in spirulina (21) may help the absorption of β-carotene (34).

It is well known that β-carotene could either be cleaved into retinol in the intestine by carotenoid monooxygenase I and II (35, 36) or be absorbed intact into the circulation (37). β-Carotene that is absorbed intact into the circulation is stored in human tissue or cleaved into retinol thereafter (37, 38). The part of the conversion that occurred after absorption of the β-carotene was called postabsorptive conversion of β-carotene. However, it was nearly impossible to directly differentiate between the retinol from preabsorptive conversion and that from postabsorptive conversion, because the same retinol was formed. A mathematical method using 24 h as a cutoff for preabsorption and postabsorptive conversin was developed to calculate the postabsorptive conversion (29). Haskell et al (39), using the deuterated retinol–dilution technique, found that plasma-labeled retinol peaked within the first 24 h. Dueker et al (40) reported that labeled retinol appeared at 5.5 h, and there was a linear rise for 28 h before the decline began. The metabolic curve of labeled retinol in the present study showed that the greatest concentration of labeled retinol in serum from the vitamin A dose or from the spirulina β-carotene dose appeared at 11–24 h. Because the intestine is the most important organ for the conversion of β-carotene to retinol, and because all of the metabolic curves reached their peaks in 24 h, it is reasonable to use 24 h as the cutoff for intestinal and postabsorptive conversion. According to the calculations, only 1 in 10 subjects in the present study did not show postabsorptive conversion (Table 5).

Since the late 1970s, the nutritive value and health benefits of spirulina have been reported, and those reports triggered the commercial production of spirulina. Millions of people in the world have used it as a health food supplement, taking 3–20 g/d. Rarely have there been any reports of allergies or sensitivities (41). The present study, using the stable-isotope reference method, found that, when consumed with 22 g fat, 4.5 mg spirulina β-carotene provided 1 mg vitamin A for 10 well-nourished, normal-weight Chinese men. Therefore, 4.1 g spirulina powder could meet the US recommended dietary allowance of 900 µg retinol activity equivalents for men.

	ACKNOWLEDGMENTS

 
We thank David A Dworak and Chee-Ming Li for assistance with producing the labeled spirulina.

The authors' responsibilities were as follows—JW: conducted the human study, collected and analyzed samples and data, and wrote the manuscript; SY, JW, and ZW: supervised the human study and revised the manuscript; WL and YF, nurses: collected blood samples; GT and RR: designed the study and revised the manuscript; GT and JQ supervised the liquid chromatographic, gas chromatographic, and mass spectrometric analyses; PS: proposed the project and revised the manuscript; and MG-C: produced the labeled spirulina, analyzed the carotenoid content in spirulina, and transported the spirulina and liquid diets from United States to China. PS is affiliated with the University Hospital (Zurich, Switzerland) and Nestlé Foundation (Lausanne, Switzerland). None of the other authors had a personal or financial conflict of interest, and no financial benefit was obtained from this research study.

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TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 

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