briefly , ten american women participated in a randomised cross - over 
 trial , consuming the following treatments : 100  g biofortified cassava ; 100  g biofortified 
 cassava with 15  ml added oil ; and 100  g typical white cassava with 15  ml oil plus a 
 retinyl palmitate ( rp ) tracer , with 2-week washouts between treatments .
rp and bc 
 concentrations in the tag - rich layer ( trl ) of plasma increased with both biofortified 
 cassava treatments .
changes in rp in the trl plasma fraction were combined with fao 
 consumption data to model the daily bc intake and rp formation of african women that could 
 be expected if all of the cassava in their diet was replaced with the variety of 
 biofortified cassava used in our study .
cyanide data from the second and third soaking 
 procedures were used to simulate the effects of marginal processing , such as occurs during 
 drought or in populations unfamiliar with cassava , and was also combined with fao 
 consumption data to model the daily cyanide exposure of african women from consumption of 
 the current variety of biofortified cassava .
the following 
 procedures were conducted for each woman : height , weight and blood pressure measurements ; 
 pregnancy prescreening ; and administration of health questionnaires .
subject demographics 
 and inclusion and exclusion procedures are described in more detail 
 elsewhere 
 .
as expected , rp formation from biofortified cassava in these women 
 varied greatly . also as usual , this variation did not depend on demographic 
 characteristics of the subjects such as body weight , bmi or age .
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 university of california , davis 
 institutional review board no .
biofortified cassava ( gm 905 - 69 ) bred to contain elevated bc concentrations was provided 
 by the international center for tropical agriculture .
typical white cassava , which 
 contained negligible amounts of bc , was acquired from the las montanas supermarket . 

biofortified cassava was placed on dry ice and air freighted to san francisco , and then 
 transported by road to the western human nutrition research center , davis , ca , usa . the 
 roots were washed , peeled and flash frozen , and then stored in a food - safe freezer at 
 20c in the metabolic kitchen and human feeding laboratory . before preparation , the roots 
 were thawed overnight at 4c , and then rinsed twice with deionised ( di ) water .
tips from 
 the distal and proximal ends were cut ( 12  cm ) and the peel removed .
four volumes of di water were added to the cassava , mixed and 
 changed every 2  h for 8  h. following the last draining , the cassava was rinsed with di 
 water , and simmered ( 95c ) in ten volumes of di water for 30  min , until it was just 
 tender .
boiled cassava was drained , cooled and aliquots were stored in 50  ml polypropylene 
 centrifuge tubes wrapped in aluminium foil under n2 at 20c , in a food - safe 
 freezer . as a precaution to preserve carotenoids , all procedures were conducted under dim 
 light to minimise light exposure .
briefly , 100  g cassava were mixed with quick - cooking oatmeal ( 40  g ) , 
 imitation butter flavouring ( 05  g ) , pears canned in light syrup ( 150  g ) , salt ( 05  g ) , 
 honey ( 21  g ) , raisins ( 189  g ) and unfortified rice milk ( 2455  g ) .
the porridge , total 
 weight 577  g , was microwaved until a temperature of 165f was reached .
the first contained no added oil , but had 6  g of fat 
 in the meal .
this meal had a very low fat content , representing the fat content from a 
 monotonous staple diet .
the second porridge had 15  ml of added oil , for a total of 20  g 
 fat .
this meal had a fat content representing a diversified diet . however , the 20  g fat 
 porridge reflects a less typical fat intake for a single meal ( breakfast ) of 41  % , which 
 is almost half the average daily per capita fat intake for sub - saharan 
 africa 
 .
each step of cassava preparation was analysed for cyanogenic glycosides using a la motte 
 cyanide in water test kit ( lamotte ) .
cassava preparations ( 1  g ) were added to a 15  ml test 
 tube , were mashed with a mortar , and then mixed with 7  ml of di water by vortexing for 
 1  min .
preparations were left at room temperature ( 22c ) for 20  min and then vortexed for 
 20  s before filtering through a 02  m pall gelman acrodisc ( sigma aldrich ) into a lamotte 
 test tube .
the water extracts were tested by following the manufacturer 's specifications . 
 to confirm the results
, the final prepared samples were analysed by applied specialization 
 and consulting llc , bothwell , wa , using alkaline hydrolysis , distillation and ion 
 chromatography with pulsed amperometric detection .
subjects ate a low - fat , low - carotenoid , low - va diet for 7  d prior to each intervention . 
 for the first 4  d , subjects were asked to avoid dark green , red , orange and yellow fruits 
 and vegetables ( which are good sources of carotenoids ) , liver , giblets , herring , 
 va - fortified cereals and multivitamins . for the last 3 
d prior to each cassava treatment , 
 subjects came to the western human nutrition research center and were given a take - home 
 diet that was low fat , low carotenoid and low va 
 .
each cassava treatment consisted of one large bowl of cassava porridge , described 
 earlier .
the subjects ate 
 no other meals that day until dinner time , approximately 105  h later . at dinner time , 
 they were given a low - carotenoid , low - va , low - fat dinner .
treatments were randomly assigned , with 
 researchers blinded to the treatments given . blood collection and analysis
blood samples were collected from the subjects at baseline and five 
 more times postprandially ( at 2 , 35 , 5 , 725 and 95  h ) .
a 1  ml sample of plasma was 
 added to a 22  ml polyallomer ultracentrifuge tube ( beckman coulter ) and overlaid with a 
 prepared nacl salt solution ( density  =  1006 
this was ultracentrifuged in a beckman 
 coulter optima tlx containing a swing - out rotor type beckman tla 100 ultracentrifuge at 130  000  g for 20  min at 4c .
the tube was then placed in a beckman centritube slicer and 
 sliced at a fixed position to isolate the trl plasma fraction 
 .
carotenoids and va were extracted from 1  g homogenised samples of the cassava porridges 
 and the pre - test day diet using a liquid 
liquid extraction was used to isolate carotenoids and va 
 within the trl plasma fraction .
methanol ( 1  ml ) was added to the trl plasma fraction , and 
 then compounds were extracted twice with 1  ml hexane .
plasma and food sample injections ( 20  l ) were analysed by reversed - phase hplc with 
 electrochemical detection 
 .
the system contained an esa model 582 solvent delivery system ( dionex 
 ( esa ) ) , an esa model 542 autosampler and an esa model 5600 coularray electrochemical 
 detector .
carotenoids and retinoids were separated with an esa md-150 column 
 ( 150  mm    32  mm , 3  m pore size ) , using a ch30 eppendorf column heater ( eppendorf ) at 
 37c .
gradient elution was with methanol02  m - aqueous ammonium acetate ( 90:10 , 
 v / v ) and methanol  isopropanol  ammonium acetate ( 78:20:2 , by vol . ) 
 , at a flow rate of 08  ml / min .
cell potentials were set at 400  mv for 
 carotenoids and 700  mv for retinoids .
retention times were 32 , 126 , 148 and 202  min 
 for retinol , echinenone ( internal standard ) , rp and bc , respectively .
inter - assay precision was monitored using a spiked pooled plasma 
 sample purchased from utak .
subjects were aged 29 ( sd 2 )  years , had a bmi of 231 ( sd 
 7 )  kg / m and normal cholesterol , tag , glucose , hb and haematocrit .
rp 
 concentrations were 87 ( sem 135 ) and 749 ( sem 115 ) nmol   
h / l for 
 biofortified cassava with added oil , and biofortified cassava without added oil , 
 respectively 
 .
initial cyanide concentrations varied from 280 to 551  parts per 
 million ( ppm ) , decreasing to 404  ppm after the second wash and 061  ppm after the third 
 wash .
data from the feeding study were combined with fao cassava consumption estimates to model 
 the bc intake , the bc to va conversion and cyanide exposure of african women .
equations 
 ( 1)(3 ) describe the methodology : ( 1 ) rs is the ratio of va formed from ingested bc for the sth subject ; 
 s ranges from one to ten ; it represents each of the ten subjects in the 
 feeding study .
rpbs and rprs are the auc ( nmol    h ) of the newly digested rp in the plasma trl fractions for the 
 biofortified and white cassava with rp tracer , respectively , for the sth 
 subject .
rp nmol / l plasma    00427 litre    kg body weight was used for the 
 sth subject to convert from rp / l to rp / total blood plasma volume prior to 
 calculating auc .
mw is the molecular weight of va 
 ( 2865  g / mol ) .
d is the mean ingested bc dose from the biofortified 
 cassava , which was 2020  g .
: ( 2 ) vaij represents the va formed ( g / d ) for the ith individual , where 
 i ranges from one to 5000 to represent the number of simulated 
 individuals ; and for the jth country , where j ranges 
 from one to six to represent each of the six african countries with the highest cassava 
 consumption . at present , only mean cassava consumption data are available for these countries .
the 
 mean consumption data used were from the following six african countries : angola 
 ( 378 
g / d ) , central african republic ( 371  g / d ) , congo ( 763 
g / d ) , 
 mozambique ( 591  g / d ) and nigeria ( 313  g / d ) 
 .
cj represents the per capita daily cassava consumption ( g ) consumed in the 
 jth country .
y is the ratio of the weight of cooked 
 cassava ( g)/raw cassava ( g ) , which was 071 .
b is the mean bc content of 
 the cooked cassava ( g / g ) .
ri is the amount of va formed from the ingested bc , calculated as a sampled ratio 
 derived from a log - normal distribution , which was developed with the means and standard 
 deviations of the log - transformed vector of rs values .
rs is described in equation ( 1 ) 
 earlier ; for the ith individual , with i representing the 
 number of simulated individual ratios ( which are indexed from one to 5000 ) . the number of individuals simulated was increased progressively to 5000 where a stable 
 pattern developed : a 1  % or less change in the means and standard deviations for replicate 
 runs .
the log - normal distribution was employed to account for the inter - individual 
 variability of the formation of va from bc in larger groups of women .
yjp is the cyanide exposure ( mg / kg per d ) from consumption of the cassava porridge in 
 the jth country , j ranges from one to six ( representing 
 the six african countries with the greatest intakes of cassava ) ; and from the 
 pth soak , where p ranges from one to two .
this 
 represents the data obtained from the second and third cassava soakings in our preparation 
 procedure .
cj represents the daily per capita cassava consumption ( g ) consumed in the 
 jth country .
y is the cooked cassava yield of 071 , 
 explained in equation ( 2 ) earlier . 

wp is the cyanide concentration in the raw cassava ( mg / kg ) after the 
 pth soak .
f is a 555  % retention factor for cyanide 
 after the cassava is boiled 
 .
the following benchmarks were used in the evaluation of intake estimates ( g / d ) and 
 exposure estimates ( mg / kg per d ) : the recommended dietary reference intake for va of 
 500  g / d for females 1965 years of age 
 ; the safe upper limit ( for preformed va ) of 3000  g / d 
 ; the rfd for cyanide exposure of 6    10 ( mg / kg per 
 d ) 
 .
the ratio of cyanide exposure to rfd was employed in the comparison of exposure for 
 different countries .
all computational tasks were performed using the r programming 
 language and environment for statistical computing ( r version 2.13.0 , http://cran.r-project.org/ ) .
the control cassava porridge , representing the cassava currently available for use , 
 contained a negligible amount of bc and thus it was estimated that no va was formed from 
 white cassava .
va intake ( g / d ) estimates from consumption of both types of biofortified 
 cassava porridge ( 6  g fat and 20  g fat ) were compared ( fig . 
 1 ) . generally , the presence of added fat increased the absorption of bc and its 
 conversion to va .
however , these differences were non significant , probably due to the small 
 number of subjects tested and the large variations between subjects .
1.distribution of individuals ' daily vitamin a ( va ; g in log base 10 scale ) intake 
 from consumption of both types of porridge : porridge with 6  g fat ( ) and 
 porridge with 20  g fat ( ) .
distribution of individuals ' daily vitamin a ( va ; g in log base 10 scale ) intake 
 from consumption of both types of porridge : porridge with 6  g fat ( ) and 
 porridge with 20  g fat ( ) .
the percentage of each country 's individual daily intake of va ( g / d ) meeting the fao va 
 recommendations of 500  g / d is presented ( table 
 1 ) . biofortified cassava , fed at the average daily intake of people in the respective 
 countries ,
is estimated to meet the nutrient recommendations of 91569982  % ( 6  g fat 
 porridge ) and 84849754  % ( 20  g fat porridge ) of the population of these countries , from 
 the lowest cassava consumption ( nigeria ) to the highest cassava consumption ( congo ) . 

table 1.summary of individuals meeting vitamin a ( va ) recommendations and exceeding the upper 
 limit ( ul ) for preformed vacountryporridge type ( g fat)meet recommendations * ( % ) exceed ul for preformed va ( % ) angola695041354angola2088823112central african republic694861278central african republic2088383040congo699824848congo2097545692ghana698563038ghana2094624376mozambique698903352mozambique2095364644nigeria691560808nigeria2084842540 * the number of individuals/5000    100 for each country that met the 500  g / d 
 recommended safe level for va 
 . the number of individuals/5000    100 for each country that exceeded the ul of 
 3000  g / d for preformed va 
 .
summary of individuals meeting vitamin a ( va ) recommendations and exceeding the upper 
 limit ( ul ) for preformed va * the number of individuals/5000    100 for each country that met the 500  g / d 
 recommended safe level for va 
 .  the number of individuals/5000    100 for each country that exceeded the ul of 
 3000  g / d for preformed va 
 .
if the cassava was prepared as in our study , with six soaks and rinses , then 
 there would be no detectable cyanide present in the cassava .
the soaking method used in our 
 study was developed so that we could monitor the amounts of cyanide and bc during 
 preparation .
it is not a common processing method in africa , although it resembles the 
 common method of soaking cassava in a stream or other sources of running water for several 
 hours .
it shows that careful preparation of the biofortified cassava results in a non - toxic 
 product that retains most of its bc .
therefore , we estimated the cyanide exposure ( g / kg per d ) from 
 consumption of the biofortified cassava variety used , as if it were soaked two or three 
 times .
although the processing procedures differ , the cyanide residues left after two or 
 three soakings are similar to those left after the common african processing methods of heap 
 fermentation or soaking in still water 
 . after two and three soakings , the cassava retains some residual cyanide 
 ( table 2 ) . the ratio ( exposure to rfd ) values 
 ( table 3 ) demonstrated a marked reduction in 
 cyanide exposure between the second and the third soaking : 13013171 after the second 
 soaking ; and 196479 after the third soaking
; as expected , the lowest exposure would 
 theoretically be in nigeria , the highest in congo .
table 2.cyanide residue according to the number of soakings(mean values and standard deviations)cyanide residue ( mg / kg)*times soakedmean 
 sd 
 15510682404110306100940250435nd6ndnd , not detected .
* mg cyanide / kg raw biofortified cassava . 
 table 3.summary of cyanide exposurecountrytimes soakedexposure ( g / kg per d)*ratio of exposure to rfdangola29431571angola3142237central african republic29251542central african republic3140233congo219033171congo3287479ghana213742290ghana3207346mozambique214742456mozambique3223371nigeria27811301nigeria3118196rfd , reference dose .
* exposure ( g / kg per d )  =  mg cyanide / kg raw biofortified cassava multiplied by a 
 555  % boiling retention factor for cyanide 
  multiplied by the consumed cooked cassava yield ( g ) for each 
 country , all divided by the mean body weight ( kg ) of the ten subjects. exposure ( g / kg per d)/rfd of 6    10 ( mg / kg per d ) 
 .
cyanide residue according to the number of soakings ( mean values and standard deviations ) *
mg cyanide / kg raw biofortified cassava . summary of cyanide exposure * exposure ( g / kg per d )  =  mg cyanide / kg raw biofortified cassava multiplied by a 
 555  % boiling retention factor for cyanide 
  multiplied by the consumed cooked cassava yield ( g ) for each 
 country , all divided by the mean body weight ( kg ) of the ten subjects . 
exposure ( g / kg per d)/rfd of 6    10 ( mg / kg per d ) 
 .
this study modelled the effectiveness of bc - enhanced cassava as a means of increasing bc 
 intake in specific african countries with va deficiency .
if the current biofortified cassava 
 variety replaced 100  % of the cassava currently consumed , then it would provide sufficient 
 bc to meet the recommended dietary intake of va for almost all women .
currently , when the potential impact of a biofortified food on va status is estimated at 
 all , it is by using conversion factors to calculate the mean retinol equivalents or retinol 
 activity equivalents that are formed by the intervention .
the conversion factor used by the 
 united states institute of medicine to calculate retinol activity equivalents is 12  g 
 bc:1  g va 
 .
if this conversion factor is used to estimate va intake , then 
 individuals eating the average amounts of cassava in angola , central african republic , 
 congo , ghana , mozambique and nigeria would consume 451 , 443 , 910 , 657 , 705 and 373  g / d , 
 respectively .
1 ) 
 should be a superior method of analysis since it addresses inter - individual variation .
thus , 
 it estimates the range of va that individuals would attain from this intervention and the 
 number of individuals meeting or failing to meet recommendations . to our knowledge , this is 
 the first distributional analysis of the potential impact of a biofortified food on va 
 status in any population .
mean va intake from the consumption of biofortified cassava met dietary recommendations , on 
 average ( 500  g / d ) , for the six countries for 96  % of the individuals eating the lower - fat 
 cassava ( 6  g fat porridge ) .
despite the fact that added oil increased the mean intake 
 estimates of va in every country , it also increased the variability in the amount ingested . 
 because of this increase in variability ,
the average percentage of people meeting their 
 dietary recommendation actually decreased slightly , to 92  % ( 20  g fat porridge ) .
thus , the 
 present results suggest that the low intakes of fat typical in africa are sufficient to 
 convert the bc in biofortified cassava to va .
our calculations suggest that the amount of va formed from biofortified cassava would 
 greatly exceed recommendations for some women who replaced white cassava with biofortified 
 cassava in their diets ( fig .
however , the upper 
 limit for va is for preformed va , and not for va formed from carotenoids 
 .
although many aspects of carotenoid metabolism remain obscure , it is 
 known that high carotenoid concentrations are poorly absorbed and converted to va . for 
 example ,
high carotenoid intakes do not cause these acute symptoms even when 
 intakes are prolonged .
in fact , carotenoids were originally believed to be essentially 
 non - toxic even at very high dosages , and no upper limit for carotenoids has ever been set . 

unfortunately , results from the alpha - tocopherol , beta - carotene cancer prevention ( atbc ) 
 study and the beta - carotene and retinol efficacy trial ( caret ) lung cancer trials have 
 demonstrated that smokers who ingested pharmacological doses of bc increased their risk for 
 cancer , suggesting that it is useful to take extra caution when consuming higher than 
 recommended carotenoid intakes 


 .
because food - based interventions are intended to reach all populations 
 including young children and pregnant women , we believe it is appropriate to take a 
 conservative approach . furthermore , these equations can be used for other nutrients and 
 toxicants , where estimating the upper safe limit of intake is more useful .
the present results suggest that food - based interventions in which biofortified cassava is 
 substituted for typical cassava should be monitored for signs of carotenoid overconsumption . 

fortunately , this can be monitored easily since consuming large amounts of carotenoids can 
 lead to carotenodermia 

 , which appears as a yellowing of the skin .
for all the ten subjects , the conversion ratio of bc to va varied 3400  % ( 03106:1 ) for 
 the 20  g porridge compared with 900  % ( 14121:1 ) for the 6  g fat porridge ; this greater 
 variation is also depicted by a greater interquartile range for the 20  g fat porridge as 
 compared with the 6  g fat porridge ( fig .
1 ) . the 
 wide range of conversion ratios of bc to va seen in this study are as expected ; similar 
 ranges are seen in other studies , even those that provide bc as purified supplements instead 
 of food 

 .
in fact , subjects living under controlled conditions , with very similar 
 demographic characteristics and health histories , have widely divergent values for 
 carotenoid absorption and conversion 

 .
the reasons for this variation are not completely understood , although 
 several genetic polymorphisms appear to be important 

 .
since demographic characteristics are not predictive of bc absorption 
 and metabolism , they are not included in our model .
this method allowed us to control the 
 detoxification process and ensure that the volunteers in our study consumed no cyanide . 

popular detoxification 
 methods in africa include heap drying , soaking in running or still water and 
 fermentation 


 .
some of these methods , especially garification , remove most of the 
 cyanide from cassava ; although the most common preparation methods leave cyanide residues , 
 which can exceed the who guideline of 10  mg hcn equivalents / kg 

 .
for example , 77  % of the cassava chips tested in australia exceeded the 
 guideline , with a mean of 642  mg hcn equivalents / kg 

 .
this mean concentration is greater than our cyanide residues after 
 soaking once ( table 2 ) .
even so , individuals 
 living in congo who prepare cassava by methods that leave cyanide residues similar to our 
 data from soaking cassava twice could be exceeding the rfd by thirty - two times ( table 3 ) .
these results suggest that cyanide levels in 
 biofortified cassava and its preparations could be a health concern .
certain limitations on the bc intake and cyanide exposure modelling must be addressed . per 
 capita data
were used in this study and represent the average intake for an entire country , 
 which does not account for inter- and intra - individual variation in eating habits . 

currently , data on inter- and intra - individual variation of cassava intake for these 
 countries nationwide are unavailable , and there is little information on the range of 
 cassava consumption even at the local level .
however , studies show a range of consumption 
 from 0 to 75  % of energy , with means ranging from 15 to > 50  % energy 

 .
more studies are required in order to gather detailed food consumption 
 data for african countries which will improve the accuracy of both the va and the cyanide 
 estimates .
a second limitation is that this study used ratios of va conversion from ingested 
 biofortified cassava for ten american women .
these ratios will most probably differ from 
 those of african women because of differences in the frequency of genetic 
 polymorphisms 

 , microflora , health history , va status and diet 
 . the techniques provided herein can be applied to studies of larger 
 groups of people in africa to increase the accuracy of these ratios .
furthermore , the approach outlined in this study is not only suitable for the estimation of 
 the risk  benefit from consumption of biofortified cassava , but can also be used to calculate 
 the risk  benefit of other dietary interventions .
cassava crossbreeding and specific 
 processing have been employed in this study to demonstrate a means of increasing nutrient 
 intake , while minimising exposure to a food component of toxicological concern .
the approach 
 used in this study can be used for other foods with similar nutrient toxicological 
 concerns .
 vitamin a ( va ) deficiency causes disability and mortality . 
 cassava can be crossbred to 
 improve its -carotene ( bc ) content ; typical white cassava contains negligible amounts of 
 bc . 
 however , cassava contains cyanide and its continued consumption may lead to chronic 
 disability . 
 our objective was to estimate the risk  benefit of consuming bc - enhanced 
 cassava to increase va intake . 
 a total of ten american women were fed white and 
 bc - enhanced cassava . 
 bc and cyanide data from the feeding study were combined with african 
 cassava consumption data to model the potential daily bc , va and cyanide intakes of 
 african women . if bc - enhanced cassava replaced white cassava in the diets , it could 
 theoretically meet recommended va intakes for the following percentages of individuals 
 from six african countries that consume cassava as a staple crop : angola ( 95  % ) , central 
 african republic ( 95  % ) , congo ( about 100  % ) , ghana ( 99  % ) , mozambique ( 99  % ) and nigeria 
 ( 92  % ) . 
 cyanide intake after minimal preparation of cassava could be thirteen to 
 thirty - two times the reference dose ( rfd ) , a toxicological exposure reference , but could 
 be completely removed by extensive soaking . 
 this study demonstrates that consumption of 
 bc - enhanced cassava , processed to maintain bc and remove cyanide , theoretically increases 
 va intakes for african populations and other areas of the world where cassava is a staple 
 crop . 
Experimental methods
Results
Discussion