the study was carried out in an elementary school in hunan province about 1000  km south 
 of beijing . children who were aged 611 years , were generally healthy and could eat meals at school 
 were eligible to participate in this study . children with fever ( > 38c ) or 
 c - reactive protein > 10  mg / l at the time of enrolment were excluded from the study . children with serum retinol < 070  mol / l were referred to the hospital for 
 treatment . a total of 228 children met the eligibility criteria and were enrolled in the 
 study . this study was conducted according to the guidelines laid down in the declaration of 
 helsinki and all procedures involving human subjects were approved by the committee on 
 human research , institute of nutrition and food safety , the chinese center for disease 
 control and prevention and the institutional review board of tufts - new england medical 
 center . written informed consent was obtained from the parents before each child 's 
 participation . at 2 weeks before the study , all the subjects were instructed to eat their usual diet at 
 home but to avoid nutritional supplements and foods rich in -carotene or vitamin a , such 
 as spinach , carrots , or liver products . albendazole tablets ( 400  mg per child ) were given to all the 
 children 2 weeks before the study . faecal analysis was done 1 d before the study to make 
 sure there was no parasitic infection in any of the children participating in the study . during the 10-week dietary intervention , 228 children were randomly divided into three 
 groups : 4  g spirulina supplement group ( n 78 ) , 2  g spirulina supplement 
 group ( n 74 ) and the control group ( n 76 ) . all the 
 children ate three meals at school with 4 , 2 , or 0  g spirulina in their breakfast as 
 cakes , sweet rice dumpling or rice noodles . the nutrients in their standard daily diet 
 provided at school contained 7531 ( sd 4184 )  kj with 65 ( sd 5 )  g 
 protein , 60 ( sd 5 )  g fat and 200 ( sd 20 ) retinol activity equivalents 
 retinol , 50  % of the rda of vitamin a for chinese children in this age group . at weekends before and after dietary intervention , the study subjects were given 05  mg of 
 h - labelled vitamin a in an oil capsule with a fat - containing meal at school . [ h4]retinyl acetate was administered at baseline , and 
 [ h8]retinyl acetate was administered 10 weeks later to 
 distinguish serum [ h8]retinol from any residual 
 [ h4]retinol . at 3 and 21 d after each h - labelled vitamin a dose , fasting blood samples 
 ( 3  ml ) were collected from all the subjects for the analysis of serum retinol and 
 carotenoid levels as well as the total - body vitamin a stores since the labelled retinol is 
 supposed to attain an equilibrated state after 21  d. blood samples were covered by 
 aluminium foil to protect them from light , kept at room temperature for 30  min and then 
 centrifuged at 4c and 800  g for 15  min . serum was stored at 20c 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 80c . all frozen samples with a frozen ice brick were sent by air 
 to the carotenoids and health laboratory of the us department of agriculture human 
 nutrition research center on aging at tufts university ( boston , ma ) for analysis . h4-labelled retinyl acetate 
 ( 10,19,19,19-[h4]retinyl acetate ) and 
 h8-labelled retinyl acetate ( 10,14,19,19,19 , 
 20,20,20-[h8]retinyl acetate ) were synthesised by cambridge 
 isotope laboratories . 3 gelatin capsule ) containing 05  mg 
 ( 151  mol [ h4]retinyl acetate or 149  mol 
 [ h8]retinyl acetate ) of these stable isotopes dissolved in 170  mg 
 of maize oil . , 
 wheat flour was chosen as the carrier to mix with spirulina ( 2 or 4  g ) to make noodles or 
 other foods . chlorophyll instead of spirulina was added to the wheat flour used for making 
 the foods given to the control group during the same period , giving the foods the same 
 appearance as those with added spirulina . foods such as noodles and cake were made in 
 advance , packed in coded bags and stored at 20c . food with added spirulina was given to 
 the children 5  d / week for 10 weeks , served with soup or porridge at breakfast time . soups 
 or porridge provided for breakfast contained no -carotene or vitamin a. the feeding 
 periods were supervised and daily consumption was recorded for each individual as the 
 relative amount of the food served . an hplc ( waters 2695 separation module and waters 2996 photodiode array detector ; 
 wavelength 250600  nm ; waters corporation ) and a pronto sil c30 column ( bischoff 
 chromatography ) were used to analyse carotenoid concentrations in 
 spirulina 
 . to analyse carotenoids in spirulina , 100  mg spirulina powder were weighed and put into a 
 50  ml glass vial and then 3  ml methanol were added to the vial . spirulina in the vial was 
 homogenised by using a polytron ( pt1600e ; kinematica ag ) at speed 10 for 30  s in an ice 
 bath and then washed with 5  ml methanol . the vial containing the mixture was stored at 4c 
 for 12  h. then , the vial was mixed by vortex for 30  s and centrifuged at 4c and 800  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 four times ( 10  ml of 
 tetrahydrofuran for the first three times and 5  ml for the fourth time ) ; vortex mixing and 
 centrifuging were involved each time . the tetrahydrofuran layers were collected into the 
 methanol extract contained in a 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 an n - evap nitrogen evaporator ( organomation associates inc . ) 
 . the residue was resuspended in 1  ml ethanol , and 20  l were injected 
 into the hplc . a linear gradient procedure started from 100  % solvent a for 21  min , then changed to 45  % 
 a and 55  % b and held for 1  min , changed to 5  % a and 95  % b in 11  min and held for 4  min 
 and changed back to 100  % a in 2  min and held for 20  min . carotenoid concentrations were calculated according to the standard curves of 
 -carotene , trans--carotene , 9-cis- and 
 13-cis--carotene ( all sigma - aldrich inc . ) , lutein , zeaxanthin and  , 
 -cryptoxanthin ( all roche vitamin inc . ) . to collect retinol from serum , 400  l serum , 100  l internal standards ( retinyl 
 acetate  +  echinenone ) , 500  l saline ( 085  % ) and 5  ml of a mixture of chloroform and 
 methanol in the ratio of 2:1 ( v / v ) were added to a glass tube ( 16    100  mm ) , mixed by 
 using a vortex mixer ( labnet international inc . ) for 1  min and then centrifuged at 4c and 800  g for 10  min . the lower layer ( chloroform layer ) was collected into another glass 
 tube ( 13    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 the n - evap nitrogen evaporator . the residue was 
 dissolved in 100  l of a mixture of ethanol and tetrahydrofuran in the ratio of 2:1 ( v / v ) , 
 mixed and sonicated for 20  s and then transferred to a vial . the dissolved residual 
 solution ( 70  l ) 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 ) and an 
 fc203 collector ( gilson inc . ) were used to analyse the concentration and 
 collect the 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 15  % ammonium acetate in water . a linear gradient solvent procedure started 
 at 100  % solvent a for 6  min , changed to 70 % a and 30  % b in 4  min , changed to 50  % a and 
 50  % b in 5  min , changed to 30  % a and 70  % b in 3  min and changed to 15  % a and 85  % b in 
 4  min . finally , the gradient went back to 100  % a in 16  min and held for 8  min to 
 stabilise the system before the next sample was injected . the column temperature was 12c , 
 and the flow rate was 1  ml / min . retinol was eluted at 9  min , so the eluent fluid from 8 to 
 10  min was collected in a 2-ml flask ( pyrex labware ; corning inc . ) . we added 10  l 
 n , o bis(trimethylsilyl ) trifluoroacetamide ( 10  % , 
 bstfa tmcs ; pierce ) to the residue , capped the flask with a stopper to avoid moisture and 
 put it into an oven at 70c for 30  min . the derivatised retinol ( 3  l ) was injected into a gc - electron capture negative chemical 
 ionisation  mass spectrometer ( agilent 6890 series gc system and 5973 network mass 
 selective detector ; agilent technologies ) . a zebron zb-1  ms capillary gc column 
 ( phenomenex inc . ) was used to separate compounds . he and ch4 were used as the 
 carrier gas and the reaction gas , respectively . the gc oven temperature was increased from 
 53 to 220c at a speed of 40c / min , to 230c at 15c / min and to 320c at 40c / min ; it 
 remained at 320c for 5  min . the mass spectrometer was set to scan at a mass - to - charge ratio 
 ( m / z ) of 260280 . retinol molecular mass was equal to 
 286 , and lost one group of h2o during the negative chemical ionisation process , 
 which made the major fragment of retinol at m / z 
 268 
 . the ratio of non - h - labelled to h - labelled retinol ( h : d ) in serum 
 was determined by gc - ms , and total - body stores of vitamin a were calculated as previously 
 reported 
 . briefly , a modified version of the formula of bausch and 
 rietz 
  for the assessment of vitamin a stores in rats was used as follows : 
 total - body vitamin a stores ( in mmol 
 retinol )  =  f    dose    ( s    a    ( h : d  
 1 ) ) , 
  where f  =  05 ( fraction of the dose to be stored in 
 liver ) 
 ,  dose is the labelled vitamin a in mmol , s  =  065 
 ( correction for h : d in serum ) 
 , and a  =  e , a 
 correction factor to the h : d that is due to the continued daily intake of dietary vitamin 
 a ( k  =  ln2/t1/2 ; 
 t1/2 is the half - life of vitamin a turnover in the liver and 
 is estimated to be about 32  d for the children 

 , and t is the time ( in d ) since the isotope dose was 
 administered ) , and h : d ( h : h ) is the enrichment of retinol after a 
 labelled dose of vitamin a. means , standard deviations , medians and ranges are reported . anova was used to determine 
 the differences in serum concentration of the major carotenoids , vitamin e and retinol 
 between the groups . the student 's paired t test was used to compare the 
 mean concentrations of total - body vitamin a stores before and after a 10-week 
 intervention . a probability of < 005 was considered to be statistically 
 significant . all the statistical analyses were performed with spss software ( version 13.0 ; 
 spss inc . ) . the characteristics of the three groups of children at baseline are listed in table 1 . these three groups were well balanced with 
 respect to age , height , weight and bmi . 1.serum retinol concentrations of children by group at baseline ( ) and 
 3 d after the end of the intervention ( ) . group 1 , control group 
 ( n 76 ) ; group 2 , group given food with 2 g added spirulina , 5 
 d / week for 10 weeks ( n 74 ) ; group 3 , group given food with 4 g added 
 spirulina , 5 d / week for 10 weeks ( n 78 ) . 2.serum -carotene concentrations of children by group at baseline ( ) , 3 
 d after the end of the intervention ( ) and 21 d after the end 
 of the intervention ( ) . group 1 , control group 
 ( n 76 ) ; group 2 , group given food with 2 g added spirulina , 5 d / week 
 for 10 weeks ( n 74 ) ; group 3 , group given food with 4 g added 
 spirulina , 5 d / week for 10 weeks ( n 78 ) . anova was used to 
 compare differences between the groups ( p  <  005 ) . table 1.characteristics of study subjects by group ( mean values , standard deviations and 
 ranges)group 1 ( n 76)group 2 ( n 74)group 3 ( n 78)mean 
 sd 
 rangemean 
 sd 
 rangemean 
 sd 
 rangeage ( years)770960100791360110741060100weight ( kg)211391513602143814032221143144328height ( cm)1222661124142012231251084141212187210741378bmi ( kg / m)140151142151401411820014117113186hb ( g / l)12741169401550125496980156012311279301540group 1 , control group ; group 2 , group given food with 2 g added spirulina , 5 
 d / week for 10 weeks ; group 3 , group given food with 4 g added spirulina , 5 d / week 
 for 10 weeks . serum retinol concentrations of children by group at baseline ( ) and 
 3 d after the end of the intervention ( ) . group 1 , control group 
 ( n 76 ) ; group 2 , group given food with 2 g added spirulina , 5 
 d / week for 10 weeks ( n 74 ) ; group 3 , group given food with 4 g added 
 spirulina , 5 d / week for 10 weeks ( n 78 ) . serum -carotene concentrations of children by group at baseline ( ) , 3 
 d after the end of the intervention ( ) and 21 d after the end 
 of the intervention ( ) . group 1 , control group 
 ( n 76 ) ; group 2 , group given food with 2 g added spirulina , 5 d / week 
 for 10 weeks ( n 74 ) ; group 3 , group given food with 4 g added 
 spirulina , 5 d / week for 10 weeks ( n 78 ) . anova was used to 
 compare differences between the groups ( p  <  005 ) . characteristics of study subjects by group ( mean values , standard deviations and 
 ranges ) group 1 , control group ; group 2 , group given food with 2 g added spirulina , 5 
 d / week for 10 weeks ; group 3 , group given food with 4 g added spirulina , 5 d / week 
 for 10 weeks . the provitamin a carotenoid content in spirulina powder consisted mainly of zeaxanthin , 
 all - trans and 9-cis--carotene . the powder contained 
 about 091  mg zeaxanthin , 107  mg -carotene and 034  mg 9-cis--carotene 
 per g ( table 2 ) . table 2.carotenoid content of spirulina powder ( mean values and standard deviations)content ( mg / g dry weight ) 
 n 
 mean 
 sd 
 zeaxanthin3091006trans--carotene31070049-cis--carotene3034001 carotenoid content of spirulina powder ( mean values and standard deviations ) in the intervention study , wheat flour , as a food vehicle to mix with spirulina ( 2 or 4  g ) , 
 was used to make noodles , cakes or rice dumplings . after it was cooked , a serving size of 
 cake ( 70  g ) , with added 2 or 4  g spirulina , was found to contain 273 and 367  mg 
 -carotene , respectively . an amount of 70 g ( with added 2  g spirulina ) or 140  g ( with added 
 4  g spirulina ) of rice noodles given to children in different groups could provide 234 and 
 469  mg -carotene , respectively ( table 3 ) . table 3.carotenoid content in cooked food with added spirulina ( g per serving , 
 n 3 ) ( mean values and standard deviations)zeaxanthintrans--carotene9-cis--carotenemean 
 sd 
 mean 
 sd 
 mean 
 sd 
 cake with 2  g spirulina164018273032068006cake with 4  g spirulina193013367024101010rice noodles with 2  g spirulina139023234034043005rice noodles with 4  g spirulina278046469068087009 carotenoid content in cooked food with added spirulina ( g per serving , 
 n 3 ) ( mean values and standard deviations ) there were no significant differences in baseline serum concentrations of retinol , 
 -carotene , cryptoxanthin , lutein / zeaxanthin and vitamin e of children in the three groups 
 ( table 4 ) . serum retinol concentrations did not 
 change significantly over time in the three groups , even in the children supplemented with 
 2  g ( p  =  0621 ) or 4  g ( p  =  0358 ) spirulina for 10 weeks 
 ( fig . serum -carotene level increased 
 significantly for children supplemented with 2  g ( p  =  0031 ) and 4  g 
 ( p  =  0012 ) spirulina after 10 weeks as shown in fig . children supplemented with 4  g spirulina had a serum -carotene 
 concentration almost twice as high as those supplemented with 2  g spirulina 
 ( p  =  0026 ) . an obvious reduction in serum -carotene concentrations was 
 observed 3 weeks after the spirulina supplementation finished ; however , it was still 
 significantly higher than that of the control group . table 4.baseline serum concentrations of retinol , carotenoids and vitamin e of subjects by 
 group ( mol / l ) ( mean values and standard deviations)group 1group 2group 3mean 
 sd 
 mean 
 sd 
 mean 
 sd 
 retinol101021099023103034lutein / zeaxanthin041017034017033018-crypaxanthin010004010006010006-crypaxanthin053028051038051042-carotene002001002001002002trans--carotene012008011008011010vitamin e566104553116573105group 1 , control group ; group 2 , group given food with 2 g added spirulina , 5 
 d / week for 10 weeks ; group 3 , group given food with 4 g added spirulina , 5 d / week 
 for 10 weeks . baseline serum concentrations of retinol , carotenoids and vitamin e of subjects by 
 group ( mol / l ) ( mean values and standard deviations ) group 1 , control group ; group 2 , group given food with 2 g added spirulina , 5 
 d / week for 10 weeks ; group 3 , group given food with 4 g added spirulina , 5 d / week 
 for 10 weeks . total - body vitamin a stores of the children in the three groups are presented in table 5 . there was no significant difference in 
 total - body vitamin a stores of children in the three groups at baseline . a significant 
 increase was found in children supplemented with 2 or 4  g spirulina 
 ( p  <  0001 ) , with a median increase of 0160 and 0269  mmol , 
 respectively . a small but not significant increase was observed in the control group 
 ( p  =  0539 ) . table 5.total-body stores of vitamin a in subjects before and after intervention ( median 
 values , mean values and standard deviations)before intervention ( mmol)after intervention ( mmol ) 
 n 
 medianmean 
 sd 
 medianmean 
 sd 
 group 152027703160232034404150321group 25302770345023904370632**0471group 35902870340022205660807**0681group 1 , control group ; group 2 , group given food with 2 g added spirulina , 5 
 d / week for 10 weeks ; group 3 , group given food with 4 g added spirulina , 5 d / week 
 for 10 weeks . * * mean value was significantly different from that at baseline ( p 
 < 001 ; student 's paired t test ) . total - body stores of vitamin a in subjects before and after intervention ( median 
 values , mean values and standard deviations ) group 1 , control group ; group 2 , group given food with 2 g added spirulina , 5 
 d / week for 10 weeks ; group 3 , group given food with 4 g added spirulina , 5 d / week 
 for 10 weeks . * * mean value was significantly different from that at baseline ( p 
 < 001 ; student 's paired t test ) . spirulina ( spirulina platensis ) is a photoautotroph alga and has a long 
 history of use in the diet 
 . there are reports that spirulina was used traditionally by mexicans and 
 natives in the lake chad area 
 . a certain amount of spirulina powder is mixed with traditional foods 
 and cooked using methods such as steaming , baking , etc . about 90  % of -carotene in 
 spirulina can be preserved . as shown in table 3 , a 
 serving of cake ( 70  g ) with 2  g added spirulina can provide 273  mg -carotene . a serving 
 with 4  g added spirulina contained 367  mg -carotene , which indicated that wheat flour is 
 an appropriate food vehicle to deliver spirulina -carotene to the study subjects . therefore , it is not surprising that the serum -carotene concentrations of the subjects 
 increased significantly after consumption of the foods with a certain amount of added 
 spirulina . an obvious decrease in serum -carotene levels was observed 3 weeks after the 
 intervention study finished . however , the children 's serum -carotene concentration still 
 significantly higher than the baseline level . the greater improvement in serum -carotene concentration compared with the changes in 
 serum retinol is not surprising because it is recognised that circulating retinol is 
 homeostatically controlled over the physiological range of liver vitamin a 
 concentrations 
 . serum retinol values tend to fall precipitously when liver vitamin a 
 concentrations are < 007  mol / g ( 20  g / g ) and tend to increase steeply when liver 
 concentrations are > 105  mol / g ( 300  g / g ) 
 . within the cited range , however , circulating concentrations respond 
 with a very shallow slope to increments in liver vitamin a across the aforementioned range . 
 in this study , no significant changes in serum retinol were observed after a 10-week 
 intervention even though the children 's total - body vitamin a stores increased significantly . this also implies that serum retinol concentration is not a sensitive indicator of vitamin a 
 status over a wide range of vitamin a nutriture , which may be the reason why some 
 intervention studies are not as efficacious as hoped 

  with serum retinol concentration as a biomarker to evaluate vitamin a 
 status . the paired isotope dilution technique is one of the most advanced and accurate methods 
 currently available for assessing vitamin a status 

 . this procedure has been used to assess the total - body stores of vitamin 
 a in children , adults , or both in the usa 

 , bangladesh 

 , philippines 

 , peru 
  and china 

 . the present study employed the paired isotope dilution technique to 
 evaluate the changes of total - body vitamin a stores of children supplemented with spirulina . the estimated mean total - body vitamin a stores in chinese school - age children at baseline 
 were lower than the value of 102  mmol reported for one 6-year - old child in the 
 usa 
  and higher than the mean value of chinese children reported by tang 
 et al . after children were supplemented with 2 or 4  g spirulina for 10 weeks , 
 their total - body vitamin a stores increased by 833 and 1373 % , respectively , which 
 approached the value reported for us children . for subjects in the control group without 
 spirulina supplementation , their total - body vitamin a stores remained stable during the 
 10-week period . therefore , we could conclude that spirulina supplementation contributes to 
 the increase in total - body vitamin a stores in the intervention group . it is well known that -carotene can be cleaved into retinol in 
 the intestine by carotenoid mono - oxygenases i and ii after it is absorbed from 
 food 

 . our previous study showed that the conversion factor of spirulina 
 -carotene to vitamin a is as high as 45 to 1 by weight in chinese adults 
 . by this conversion factor , 2 or 4 g spirulina can provide about 05 or 
 1  mg vitamin a. therefore , it is not difficult to explain that total - body vitamin a stores 
 of children supplemented with spirulina can be increased significantly . green - yellow 
 vegetables and sweet potatoes are also good dietary sources of -carotene . it has been 
 reported that green - yellow vegetables can sustain the total - body vitamin a stores of 
 kindergarten children , but no obvious increase was observed 
 . found that consumption of 
 orange - fleshed sweet potatoes could improve vitamin a liver stores 
 . recently , studies have shown that golden rice also has a vitamin a 
 equivalence as high as 38 ( sd 17 ) to 1 by weight 
 ; however , no data on its effect on total - body vitamin a stores have been 
 reported . from this study , it is safe to conclude that spirulina can significantly increase subjects 
 serum -carotene and total - body vitamin a stores monitored by using a paired stable - isotope 
 dilution technique , which suggests that spirulina is a good dietary source of -carotene and 
 vitamin a.
spirulina is an alga rich in high - quality protein and carotenoids . 
 it is unclear whether 
 spirulina can improve the total - body vitamin a stores of school - age children in china with 
 a high prevalence of vitamin a malnutrition . 
 we aimed to evaluate the efficacy of 
 spirulina in improving the total - body vitamin a stores of school - age children in rural 
 areas of china when they consumed spirulina in their daily meals . 
 a total of 228 children 
 ( 611 years ) were recruited and randomly divided into three groups supplemented with 4  g 
 ( containing 418  g -carotene ) , 2  g ( containing 254  g -carotene ) or 0  g spirulina 
 5  d / week for 10 weeks , respectively . before and after the intervention period , each child 
 was given 05  mg [ 2h4]retinyl acetate and 
 [ 2h8]retinyl acetate , respectively . to assess vitamin a stores , 
 blood samples ( 3  ml ) 
 were collected on the third and the twenty - first day after each 
 labelled retinyl acetate dose for a retinol enrichment analysis using a gc mass 
 spectrometer . 
 the concentrations of retinol and -carotene in serum samples were also 
 determined by using hplc . after the 10-week intervention , serum -carotene concentrations 
 of children with 2 or 4  g spirulina supplement increased by 0160 and 0389  mmol / l , 
 respectively . 
 total - body vitamin a stores increased significantly , with a median increase 
 of 0160  mmol in children taking 2  g spirulina and of 0279  mmol in children taking 4  g 
 spirulina . 
 spirulina is a good dietary source of -carotene , which may effectively 
 increase the total - body vitamin a stores of chinese school - age children .