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SUBJECTS AND METHODS

Subjects

The study was carried out in an elementary school in theHunan province of China in healthy schoolchildren (with normalbiochemical test results; see below) aged 6–8 y either initially

free of parasitic infection or veriﬁed free of infection after treatment with 400 mg albendazole (GlaxoSmithKline). Most area residents were local, middle-income, rural, and working

people. Forty-eight percent of the study subjects who were treated (no side effects) were recruited to participate in the study 1 mo before the start of the study meals. Thus, all of the study

subjects at the time of study were free of parasitic infection by the laboratory test. Local annual health evaluations were performed in all children, and biomedical tests were performed

(white blood cell count, hemoglobin count, platelet count, red blood cell count, and hematocrit).

To estimate the sample size for the study, we used our previous studies in the United States with spinach or b-carotene capsules (26, 27) and studies in China (28) for dark-green vegetables for

the power calculations. We found that to determine the differences for 2 categories—marginal and normal vitamin A intakes—we would need a sample size of 30 per category, and to evaluate the

effect of food matrix among sources (spinach, GR, and b-carotene capsules) we would need 10 per group. Considering the possibility of subject withdrawals from the study, we proposed 36

subjects for the marginal vitamin A group and 36 subjects for the normal vitamin A group, with 12 each per group for the spinach group, rice group, and b-carotene capsule group.

From a total of 112 subjects screened, 72 were enrolled in the study, and sufﬁcient serum samples to facilitate both the HPLC and gas chromatography–mass spectrometry analyses were collected from 68 subjects (this varied from 1 to 5 time points per subject). The anthropometric measurements of the subjects were within the range of the data reported for all of China for

this age group (29).

The study recruitment processes and protocol were approved by the Institutional Review Board–Tufts Medical Center in the United States and by the Ethics Review

Committee of Zhejiang Academy of Medical Sciences in China.

Both parents and pupils consented to participate in the study.

Production of intrinsically labeled plant material and preparation of labeled doses

Intrinsically deuterium-labeled GR grains and spinach were harvested from a hydroponic plant system (housed in the USDA Agriculture Research Service Children’s Nutrition Research Center in Houston, TX) by growing the plants with heavy water (deuterium oxide, D2O), introduced into the growth medium shortly after the start of seed ﬁll (in the case of rice), or at the initiation of planting (spinach), as previously described (26). GR grains were harvested at maturity, air dried for 3 d, and then dehulled, milled, and stored at -70C until being shipped to Boston for cooking, as previously described (25). Similarly, spinach plants were harvested and packed as fresh leaves (withice) and were shipped the next day to Boston for cooking and processing as previously described (26).

Cooked GR was divided into 60-g portions (cooked weight)

in individual food containers and kept at -70C until used. On

the day of feeding, the GR doses were brought to room temperature and then steam heated brieﬂy before being served with

lunch.

Cooked, labeled spinach was homogenized, mixed, and divided into 30-g portions in individual containers and stored at -70C until used. On the day of feeding, the spinach doses were brought to room temperature and then steam heated brieﬂy before being served with lunch.

The pure2H8-b-carotene dose was crystalline all-trans-

[

]b-carotene (11,11#,19,19,19,19#,19#,19#-[

]b-carotene),

which was synthesized by scientists of BASF. The chemical

purity was 91.2% (this percentage was used to calculate the

b-carotene in the dose). The [

]b-carotene in the oil capsule

(gelatin, size 1) was made by weighing 0.50 mg b-carotene by

using a Mettler-Toledo XP-26 microbalance with 0.001-mg

measurement readability and dissolving it with 10 mL absolute

ethanol before mixing it with 170 mg corn oil to form a clear oil

solution. The enrichment proﬁles of b-carotene in these plant

foods together with the pure b-carotene for the study were analyzed by liquid chromatography–atmospheric pressure chemical ionization mass spectrometry and are presented in Figure 1.

As a reference dose, 0.5 mg [

C10

]retinyl acetate (8, 9, 10, 11,

12, 13, 14, 15, 19, 20-[

C10

]retinyl acetate) with a chemical purity of 98% and an isotopic purity of 99% (synthesized by Cambridge Isotope Laboratory) in a 170-mg corn oil gelatin capsule (size 3) was taken by the volunteers during the breakfast meal. The study doses were packed in a cooler together with PolarPack ice bricks, prechilled to -70 C, and shipped to the study site in China, where the doses were kept at -20C until

shortly before use.


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