iron bioavailability fromamaranthus species: 1—in vitro dialysable iron for estimation of genetic...

J Sci Food Agric 1998, 78, 267È273

Iron Bioavailability from Amaranthus Species:1—In Vitro Dialysable Iron for Estimation ofGenetic Variation

Anusuya Rangarajan* and John F Kelly

Department of Horticulture, Michigan State University, East Lansing, Michigan 48824, USA

(Received 17 April 1997 ; revised version received 17 December 1997 ; accepted 25 February 1998)

Abstract : Green leafy vegetables have the potential to contribute signiÐcantamounts of iron to the diet, if the bioavailability of iron from these foods isimproved. In order to determine the potential for genetic manipulation of thistrait, 46 lines from 12 species of Amaranthus were evaluated for total and bio-available iron in greenhouse and Ðeld experiments. Bioavailable iron was esti-mated using an in vitro assay for dialysable, low-molecular-weight ironcompounds. SigniÐcant di†erences (P\ 0É01) were detected among lines andspecies for total and bioavailable iron. Total iron ranged from 358 to 880 ppm(dry weight) in Ðeld-grown plants and 55 to 123 ppm (dry weight) in greenhouse-grown plants. Bioavailable iron ranged from 41 to 63 ppm in Ðeld-grown plantsand from 24 to 51 ppm in greenhouse-grown plants. Amaranthus tricolor and Alividus had the highest total and bioavailable iron ; A hypochondriacus had thelowest levels of the species tested. Although Ðeld-grown Amaranthus accumulatedhigher levels of total and bioavailable iron, a greater proportion of the total ironwas sequestered in insoluble, unavailable forms. Generally, species with highertotal iron had higher levels of bioavailable iron. These analyses indicated poten-tial for genetic improvement of iron nutritional quality from Amaranthus, espe-cially within the species A tricolor. 1998 Society of Chemical Industry(

J Sci Food Agric 78, 267È273 (1998)

Key words : amaranth ; total iron ; bioavailable iron ; dialysable iron ; solubleiron ; genetic variation ; green leafy vegetables.

INTRODUCTION

Iron (Fe) deÐciency anemia is one of the most prevalentnutritional deÐciencies a†ecting the worldÏs populationtoday, especially among women and children indeveloping countries (Scrimshaw 1991). Most nutritionprograms aimed at decreasing the incidence of Fe deÐ-ciency utilise fortiÐcation of the diet with various Fecompounds (Monsen 1988). An alternative approach

* To whom correspondence should be addressed at : Depart-ment of Fruit and Vegetable Sciences, Cornell University,Ithaca, New York 14853, USA.

would be to enhance the Fe nutritional quality of thediet through improvement of Fe bioavailability fromcommonly consumed foods. Green leafy vegetables(GLVs), such as amaranth (Amaranthus spp), are onesuch food group. Amaranth is an inexpensive, widelyavailable GLV cultivated in many regions of the worldwhere Fe deÐciency anemia is a problem, such asCentral America, Asia and Africa. This crop accumu-lates a higher level of Fe than many other commonlycultivated greens (Ifon and Bassir 1978 ; Makus 1984 ;Chawla et al 1988). Enhancement of the Fe nutritionalquality of this vegetable through plant breeding andvariety selection may be a very cost-e†ective method to

2671998 Society of Chemical Industry. J Sci Food Agric 0022È5142/98/$17.50. Printed in Great Britain(

268 A Rangarajan, J F Kelly

provide populations with an improved source of Fe intheir diet.

Di†erences in total Fe accumulation by amaranthhave been detected (Elias 1977 ; Deutsch 1978 ; Makus1984), and the bioavailability of the leaf Fe has beentested. In a comparison of 10 tropical GLVs usinganemic rats, Ifon and Bassir (1978) found a range of Febioavailabilities from 7É7 to 36É2%, and amaranth hadthe highest percent bioavailable Fe of all genera tested.Using various in vitro simulations of gastrointestinaldigestion, researchers have found between 2É8 and53É6% of total Fe to be bioavailable in amaranthspecies (Reddy and Kulkarni 1986 ; Chawla et al 1988 ;Latunde Dada 1990 ; Reddy and Malewar 1992).Analysis of four amaranth lines from di†erent speciesfor total and bioavailable Fe indicated that the line withhighest total Fe concentration did not have the highestbioavailable Fe concentrations (Reddy and Kulkarni1986). However, the researchers used a “market basketÏapproach in which samples were collected from localmarkets and analysed for Fe. Although this methodprovided information on consumer nutritional intake,there was no characterisation of the e†ect of horticul-tural practices, production or post-harvest environmentor plant genetic variation on Fe bioavailability of theGLVs.

These studies suggested, however, that genetic varia-tion in Fe bioavailability may exist within the genusAmaranthus. There have been no previous reports ofattempts to elucidate genetic variation for Fe bio-availability in a GLV. To determine if sufficient geneticvariability exists to support breeding e†orts for thistrait, Ðeld and greenhouse evaluations were conducted.An in vitro assay for estimation of bioavailable Fe wasadapted for analysis of multiple samples simultaneously(Kapsokefalou and Miller 1991). After identifyinggenetic di†erences, results were conÐrmed using ananimal bioassay, hemoglobin repletion in anemic rats(Rangarajan et al 1998). Genetic screening of amaranthlines, grown under known environmental conditions,indicated sufficient variability to support breedinge†orts to improve Fe nutritional quality of this GLV.This paper summarises the results from in vitro analysisof lines from 12 species of Amaranthus.

MATERIALS AND METHODS

Field evaluation

Thirty-Ðve lines of Amaranthus, from 12 species, wereselected from the Plant Introduction Station collectionat Ames (IA, USA). The lines were selected from bothcultivated and wild types and grain and vegetable typesto represent the broad geographic and genotypic range

of the genus (Table 1). Local cultivar names have beenprovided, when known, for popular types from certaincountries.

The Ðeld evaluation was conducted at the MichiganState University Horticulture Teaching and ResearchCenter (East Lansing, MI, USA). A site with a sandyloam soil (Marlette Ðne sandy loam, mixed mesic Glass-oboric Hapludalf ) was fertilised with 67 kg N per ha asurea. Lines were direct-seeded into 3 m rows with a15 cm in-row spacing, in a randomised complete blockdesign (four replicates), on 12 July 1990. Irrigation sup-plemented rainfall to provide a minimum of 2.5 cm ofwater per week. No pesticides were applied.

Plots were harvested 35 days after seeding, in themorning, to minimise Ðeld heat. Five to seven wholeplants were selected randomly from each line, cut at thesoil line, bagged and stored at 4¡C for up to 5 h prior toprocessing.

Greenhouse evaluation

Twenty-four lines from four species of amaranth (Alividus, A tricolor, A dubius and A hypochondriacus) weregrown in the greenhouse to determine if species and linedi†erences in Fe nutritional quality could be detected inplants grown in a controlled environment (Table 1).Lines were seeded into plastic cell Ñats, in a 1 : 1 (v/v)loam/peat soil media. After 14 days, uniform seedlingswere selected and transplanted into 20-cm diameterplastic pots, with two seedlings per pot. Three pots perline were placed under supplemental lighting (high-pres-sure sodium lamps providing 420 lmol m~2 s~1) in arandomised complete block design, with three blocks.Diurnal temperature were maintained at 30¡C day and18¡C night (^5¡C). Plants were fertilised three timesper week with 200 ppm N in a formulation of20 : 20 : 20(%) N : P2O5 : K2O.

Pots were harvested 28 days after transplanting. Theplants from each block were cut o† at the soil line andpooled, and the samples were stored at 4¡C for up to2 h prior to processing.

Leaf processing

The upper whorl of leaves, starting with the most fullyexpanded leaf, was removed from each stem and bulkedfor each line of all species except A spinosus. For thelines of A spinosus, all leaves were harvested due to thesmall leaf size and prostrate growth habit. To removesurface soil contamination, leaves were washed asfollows : three rinses with distilled water, 15 s rinse in0É1 M and three more distilled-water rinses.HNO3Samples were freeze-dried and ground in a stainlesssteel Wiley Mill, with a 40-mesh screen.

Iron bioavailability from Amaranthus species 269

TABLE 1Amaranthus accessions evaluated for total and bioavailable Fe

under Ðeld and/or greenhouse conditions

Species, cultivar L ocationb PI number Originand (linea)

A caudatus “ChuaÏ (1) f PI 166045 IndiaA caudatus (2) f PI 490609 EquadorA cruentus (1) f Ames 5598 BeninA cruentus (2) f PI 433228 GuatemalaA cruentus (3) f Ames 8269 United StatesA cruentus (4) f PI 511715 GuatemalaA dubius (1) f, g Ames 2098 IndiaA dubius (2) f, g Ames 1967 IndiaA dubius “StubbyÏ (3) f, g Ames 5114 TaiwanA dubius f, g Ames 5674 Zambia“ImbondweÏ (4)

A dubius “NoudomÏ (5) g Ames 1997 GhanaA dubius (6) g Ames 5105 SeychellesA hybridus (1) f PI 482049 ZimbabweA hybridus (2) f PI 494768 ZambiaA hybridus (3) f PI 210995 AfghanistanA hypochondriacus (1) f, g PI 477917 MexicoA hypochondriacus (2) f, g Ames 2171 NepalA hypochondriacus (3) g Ames 5474 MexicoA hypochondriacus g Ames 5691 Nepal“JulmaÏ (4)

A hypochondriacus (5) g Ames 2156 NepalA hypochondriacus (6) g PI 477915 IndiaA lividus (1) f, g PI 288277 IndiaA lividus (2) f, g Ames 2206 Hong KongA lividus (3) f, g Ames 5146 IndiaA lividus (4) g Ames 2035 IndiaA lividus (5) g Ames 5103 Hong KongA lividus (6) g Ames 5387 IndiaA palmerii (1) f Ames 5306 SenegalA rudis (1) f Ames 10827 USAA species (1) f PI 511752 PeruA species (2) f PI 337611 UgandaA spinosus (1) f Ames 2043 IndonesiaA spinosus (2) f PI 482058 ZimbabweA tricolor f, g Ames 5379 Taiwan“Tiger leafÏ (1)

A tricolor “Red leafÏ (2) f, g PI 419057 TaiwanA tricolor “Red leafÏ (3) f Ames 5147 TaiwanA tricolor “Red leafÏ (4) f, g Ames 2154 TaiwanA tricolor f Ames 2205 Hong Kong“White leafÏ (5)

A tricolor f Ames 2209 Hong Kong“White leafÏ (6)

A tricolor “ChulaiÏ (7) f, g PI 173837 IndiaA tricolor (8) f Ames 1980 ZaireA tricolor f Ames 5111 Papua“AupamalipÏ (9) New Guinea

A tricolor f Ames 5113 Taiwan“DuraderaÏ (10)

A tricolor g Ames 2208 Hong Kong“Tiger leafÏ (11)

A tricolor (12) g Ames 5166 IndiaA virdis (1) f PI 540445 Indonesia

a Line numbers correspond to di†erent accessions of that species.b Location of evaluation ; f\ Ðeld, g\ greenhouse.

Fe quantiÐcations

Total and bioavailable Fe were determined in duplicatefor each sample. Total Fe was quantiÐed using atomicabsorption spectrophotometry (Video 12, Instrumen-tation Laboratory AA Spectrophotometer, Andover,MA, USA). Samples were wet-ashed with andHClO4

diluted to 10 ml, and Fe concentration wasH2O2 ,determined by comparison of absorption at 248É3 nm toa standard curve (Alder and Wilcox 1985).

Bioavailable Fe was estimated using the in vitro assaydescribed by Kapsokefalou and Miller (1991). Thisassay estimates bioavailable Fe from the amount oflow-molecular-weight Fe (dialysable) which remains insolution after a simulation of gastrointestinal digestion.The assay was modiÐed to maximise the number ofsamples per run (28) and minimise the amount of driedleaf material required (0É5 g). Included in each run ofthe assay were two replicates of a spinach (Spinaceaoleracea L) sample established as a standard to compareruns. In a 30-ml sample bottle, 0É5 g of ground leafsample was mixed with 10 ml and the solutionddH2Owas adjusted to pH 2 with 0É5 M HCl. The Ðnal weightof this solution was increased to 14 g. One millilitre ofpepsin solution (4 g pepsin in 100 ml 0É1 M HCl, SigmaP-7000, Sigma Chemical Company, St Louis, MO,USA) was added to each sample bottle and then placedin a shaking water bath, at 37¡C, for 2 h. After thisinitial peptic digestion, dialysis tubing (Spectrapore I,6000È8000 MW cuto†, 32 mm Ñat width) containing10 ml of 0É15 M PIPES bu†er, pH 6É87, was added toeach of the bottles. Solutions were allowed to pH-equilibrate for 1 h prior to addition of 5 ml pancreatinand bile enzyme solution (0É2 g pancreatin, SigmaP-1750, 1É2 g bile salts, Sigma B-8631, in 100 ml of0É1 M After the 2 h pancreatin digestion,NaHCO3).dialysis tubes were removed from the bottles, rinsedwith distilled water, and the contents were emptied intoplastic vials. Colorimetric determination of reduced Fein the dialysed samples was conducted using a ferrozinechromogen (Sigma P-9762) as described by Kapsokefa-lou and Miller (1991).

The percent dialysable Fe was calculated from thevalues obtained for total Fe and bioavailable Fe.Analysis of variance was performed for total, dialysable,and percent dialysable Fe for each experiment.

RESULTS

Field evaluation

SigniÐcant di†erences (P\ 0É01) were detected amonglines (Table 2) and species (Table 3) for total, dialysableand percent dialysable Fe. Total Fe content of leaves


TABLE 2Total, dialysable (lg g~1 dry wt) and percent dialysable Fe in

leaves of 35 lines of Amaranthus grown in the Ðeld

Species, cultivar T otal Fe Dialysable Fe % Dialysable Feand (linea)

A caudatus “ChuaÏ (1) 615 55 10A caudatus (2) 492 48 10A cruentus (1) 595 51 9A cruentus (2) 495 48 11A cruentus (3) 484 46 10A cruentus (4) 552 49 10A dubius (1) 527 57 11A dubius (2) 572 58 11A dubius “StubbyÏ (3) 560 54 10A dubius 525 55 11“ImbondweÏ (4)

A hybridus (1) 596 53 9A hybridus (2) 661 51 8A hybridus (3) 450 52 12A hypochondriacus (1) 358 41 12A hypochondriacus (2) 419 45 11A lividus (1) 848 57 8A lividus (2) 713 50 7A lividus (3) 880 63 8A palmerii (1) 453 49 12A rudis (1) 511 52 12A species (1) 459 47 10A species (2) 550 47 9A spinosus (1) 785 60 8A spinosus (2) 768 51 7A tricolor 764 60 8“Tiger leafÏ (1)

A tricolor “Red leafÏ (2) 579 43 9A tricolor “Red leafÏ (3) 641 52 8A tricolor “Red leafÏ (4) 575 49 9A tricolor 702 52 8“White leafÏ (5)

A tricolor 748 57 8“White leafÏ (6)

A tricolor “ChulaiÏ (7) 805 62 8A tricolor (8) 723 44 6A tricolor 754 57 8“AupamalipÏ (9)

A tricolor 756 45 6“DuraderaÏ (10)

A virdis (1) 581 43 8

LSD0Õ01 189 11 3

a Line numbers correspond to di†erent accessions of that species.

ranged from 358 to 880 ppm. Among the species tested,the 10 lines accumulating the highest concentration oftotal Fe were from the species A lividus, A tricolor andA spinosus (Table 2). The two lines with lowest total Fe,both A hypochondriacus lines, contained approximatelyhalf the total Fe concentration of the two highest lines,both from A lividus. SigniÐcant di†erences (P\ 0É01)were detected for total Fe among lines of the species Atricolor.

In vitro digestion provided estimates of bioavailableFe (dialysable) from 41 to 63 ppm, and signiÐcant di†er-


leaves of 12 Amaranthus species grown in the Ðeld

Species T otal Dialysable %DialysableFe Fe Fe

A caudatus 554 51 10A cruentus 531 49 10A dubius 546 56 10A hybridus 569 52 10A hypochondriacus 388 43 12A lividus 814 57 7A palmerii 453 49 12A rudis 511 54 12A species 504 47 9A spinosus 776 55 7A tricolor 705 52 8A virdis 581 43 8

LSD0Õ01 174 7 3

ences (P\ 0É01) were detected among lines (Table 2)and species (Table 3). Within-species di†erences in dia-lysable Fe were signiÐcant (P\ 0É01) for A tricolor andA lividus. A signiÐcant correlation was not detectedbetween total and dialysable Fe in the Ðeld-grownmaterial.

SigniÐcant di†erences also were measured for thepercent dialysable Fe in Ðeld-grown plants, rangingfrom 6 to 12% (Table 2). Lines from A tricolor and Alividus had the lowest percentages of dialysable Fe,whereas A hypochondriacus had the highest (Table 2).

Greenhouse evaluation

SigniÐcant di†erences (P\ 0É01) also were detected forboth total and dialysable Fe among lines (Table 4) andspecies (Table 5) grown in the greenhouse. Total Feranged from 55 to 123 ppm, and dialysable Fe rangedfrom 24 to 51 ppm. Total Fe was correlated with dialys-able Fe (r \ 0É84). The species A lividus accumulated thehighest levels of total and dialysable Fe and A hypo-chondriacus accumulated the lowest levels (Table 5),similar to results from the Ðeld evaluation (Table 3).Amaranthus tricolor was the only species with signiÐcantdi†erences in total Fe among individual lines (LSD0Õ01for total Fe\ 14). No signiÐcant di†erences weredetected for percent bioavailable Fe in greenhouse-grown material. An average of 43% of the total Fe wasmeasured as bioavailable.

For the 13 lines grown in both environments, thetotal Fe levels in the greenhouse-grown plants weremuch lower than Ðeld-grown plants. However, the rela-tive di†erences in total Fe levels and ranking among thefour species tested in the two environments (A dubius, A



leaves of 24 lines of Amaranthus grown in the greenhouse

Species, cultivar T otal Dialysable %Dialysableand (linea) Fe Fe Fe

A dubius (1) 75 31 42A dubius (2) 82 35 43A dubius “StubbyÏ (3) 93 35 39A dubius 84 34 41“ImbondweÏ (4)

A dubius “NoudomÏ (5) 89 37 43A dubius (6) 81 33 41A hypochondriacus (1) 60 27 45A hypochondriacus (2) 73 33 45A hypochondriacus (3) 61 28 46A hypochondriacus 61 28 46“JulmaÏ (4)

A hypochondriacus (5) 64 28 45A hypochondriacus (6) 55 24 44A lividus (1) 109 45 42A lividus (2) 113 51 45A lividus (3) 107 48 45A lividus (4) 118 48 41A lividus (5) 123 51 42A lividus (6) 110 45 41A tricolor 82 38 47“Tiger leafÏ (1)

A tricolor “Red leafÏ (2) 89 41 46A tricolor “Red leafÏ (4) 101 39 38A tricolor “ChulaiÏ (7) 75 38 49A tricolor 96 41 43“Tiger leafÏ (11)

A tricolor (12) 79 38 48

LSD0Õ01 28 10 NS

a Line numbers correspond to di†erent accessions of that species.

hypochondriacus, A lividus and A tricolor) were signiÐ-cant among both lines and species (P\ 0É05, Spear-manÏs rank correlation.). Relative di†erences in dialysedFe were not maintained between environments.

DISCUSSION

These studies represented the Ðrst attempt to determineif genetic variation exists for Fe bioavailability from a


leaves of four Amaranthus species grown in the greenhouse

Species T otal Dialysable %DialysableFe Fe Fe

A tricolor 87 39 45A dubius 84 34 41A lividus 113 48 43A hypochondriacus 62 28 45

LSD0Õ01 9 2 NS

GLV. The signiÐcant di†erences observed for total andbioavailable (dialysable) Fe among the species ofAmaranthus indicated potential genetic variability thatmay be utilised to improve the Fe nutritional quality ofthis GLV. Although di†erent species are preferred forconsumption in various regions of the world, this genusis known for its ability to form interspeciÐc hybrids(Kulakow and Jain 1990), allowing for such variabilityto be utilised to enhance Fe bioavailability. Amaranthustricolor, A lividus and A spinosus, which accumulatedhigh levels of total Fe, are used predominately as GLVs,whereas A hypochondriacus, which accumulated thelowest levels of total Fe, is predominately cultivated forgrain. Although A spinosus is consumed as a GLV insome regions of Asia, this species has a prostrate growthhabit and axillary spines and has been referred to as anoxious weed (Martin and Ruberte 1997).

Generally, di†erences among amaranth species fortotal and dialysable Fe were greater than within indi-vidual species, for the two production environmentstested. Within the species A tricolor, signiÐcant di†er-ences (P\ 0É05) were observed for total and dialysableFe among lines tested, in both production environ-ments. Amaranthus tricolor has been reported pre-viously to accumulate higher levels of total Fe thanother Amaranthus species (Elias 1977 ; Deutsch 1978 ;Makus 1984). The high Fe concentrations observedamong lines of A lividus and A tricolor, the variability intotal and available Fe observed among lines withinthese species, and their popularity as cultivated veget-able types suggests these species may be good candi-dates for breeding e†orts to improve the Fe nutritionalquality of this GLV. Other species, such as A cruentus(popular GLV types in Central America and WestAfrica) would also be good candidates for crossing tohigher Fe accumulating lines to determine heritabilityof this trait and potential enhancement of Fe concentra-tion.

Although the in vitro dialysable Fe assay has beenreported to be highly correlated with in vivo bio-availability measurements (Schricker et al 1981), it isuseful only to make relative comparisons among testedmaterials. Determination of actual bioavailability wouldrequire animal or human feeding studies and an exami-nation of meal interactions (Forbes et al 1989 ; Rangara-jan et al 1998). Using a conversion of dry weight equalto 10% fresh weight (FW) for Ðeld-grown plants(average value from data not reported), the lines con-taining the lowest and highest amounts of dialysable Fecontributed 4É1 and 6É3 mg bioavailable Fe kg~1 FW.Chawla et al (1988) detected a similar value for Fe bio-availability from amaranth, 4É5 mg kg~1 FW. Based onthese estimates of bioavailable Fe, a 100 g portion (atypical serving) of amaranth could provide 40È60% ofthe daily Fe needs for men and children (approximately1 mg per day) and 27È41% for women (1É5 mg per day)(National Research Council 1989).


The narrow range of values observed for dialysableFe across both environments, 41È63 ppm in the Ðeld-grown amaranths and 23È51 ppm in the greenhouse-grown plants, suggested a fairly constant amount ofbioavailable Fe for this leaf material (Tables 2 and 4).However, the actual chemical nature of this dialysableFe in GLVs remains unknown. This dialysable Fe frac-tion may consist of speciÐc Fe compounds within plantsthat are physiologically active, may not vary greatly inconcentrations among di†erent production environ-ments (metabolically essential enzymes) and can besolubilised readily under the conditions of the in vitroassay. Such “activeÏ Fe compounds have been extractedfrom leaf material using weak acids or Fe chelators(Abadia et al 1984). The relationship between the“activeÏ Fe fraction and the bioavailable Fe fraction ofleaves has not been investigated.

The Ðeld-grown amaranth lines accumulated betweensix and 11 times the amount of total Fe when comparedto greenhouse-grown plants. As a result, the percentbioavailable (dialysable) Fe was low (6È12%) comparedto greenhouse-grown plants (38È47%) despite the higherabsolute quantity of bioavailable Fe detected in Ðeld-grown plants (Tables 2 and 4). To examine further thedi†erences in the total Fe fractions of plants grown inthe greenhouse and Ðeld, four species were analysed forsoluble Fe, by measurement of Fe in solution after invitro digestion (Rangarajan 1995). In Ðeld-grownmaterial, an average of only 23% of the total Fe couldbe solubilised during the in vitro digestion, compared to82% of the total Fe that was soluble in greenhouse-grown plants. A fractionation of the Fe in Ðeld-grownspinach revealed only 7% of the leaf Fe was soluble(Lee and Clydesdale 1981). The remaining insoluble Fefraction included Fe precipitates and those Fe com-pounds resistant to the digestion conditions of theassay. Soil Fe (highly insoluble) embedded in leafmaterial may have contributed to the total Fe concen-tration of Ðeld-grown plants (Cary et al 1994) but totalFe concentration were maintained across Ðeld replicateswith each season tested (Rangarajan 1995). Alternative-ly, Ðeld-grown plants may accumulate higher levels ofother plant compounds (ascorbic acid, tannins,phosphorous) which may change the Fe bioavailability(Gillooly et al 1983 ; Hazell and Johnson 1987 ; LatundeDada 1990 ; Reddy and Malewar 1992). Bioavailable Fein spinach (greenhouse-grown) was positively correlatedwith ascorbate and oxalate and negatively with phos-phorus concentrations (Reddy and Malewar 1992 ;Reddy et al 1993). These relationships have not beenexamined in Ðeld-grown GLVs. Increases in the low-molecular-weight, soluble forms of Fe or changes inlevels of secondary compounds may be alternativeapproaches for genetic improvement of Fe nutritionalquality of GLVs.

Researchers have concluded that the Fe from GLVsis highly bioavailable, based on calculations of percent

bioavailable Fe after a greenhouse evaluation (Wien etal 1975 ; Van Campen and Welch 1980). The resultsfrom this study indicated that conclusions regarding Febioavailability based solely on greenhouse evaluationsmay be incorrect. The relative di†erences in total Feamong the amaranth species tested were maintainedbetween the two production environments, but thee†ects of secondary compounds or the di†erences in theFe forms in Ðeld-grown material had a great e†ect onthe percent of bioavailable Fe. Attempts to increase thetotal Fe concentrations in greenhouse-grown plantsgenerally have been unsuccessful. The di†erences in thesoil pH, the light intensity and light quality of con-trolled environments may be more important than soilFe concentrations in explaining the di†erences observedin total Fe accumulation from plants grown in Ðeldenvironments (Abadia 1992). Although di†erences weredetected among species in the greenhouse evaluation,replicated Ðeld analyses must be utilised for Ðnal deter-minations of Fe nutritional value.

Any assay of Fe bioavailability only provides a rela-tive measure of nutritional quality among tested foods.Actual bioavailability will vary based upon the diverseinteractions of diet components and the health andcharacteristics of the animal population considered. Forplant breeders attempting to improve the Fe nutritionalquality of GLVs, inexpensive, rapid screening tools areneeded to assess relative di†erences among plantspecies. The modiÐcation of the in vitro method of esti-mating Fe bioavailability used in this study was a reli-able method for evaluation of plant material. The smallamount of plant material needed, the low cost(especially compared to animal studies) and the highernumber of samples analysed per run favoured its appli-cability to the evaluation of plant material. This methodalso may be adaptable for the study of nutritional con-tribution of other plant minerals (Reykdal and Lee1991) or materials (Latunde Dada 1991). The use oftotal Fe analysis for broad screening of total Fecontent, combined with in vitro dialysable and solubleFe analyses for more speciÐc screening of select highand low Fe lines, is a very efficient method for evalu-ation of genetic variability of Fe nutritional quality andidentiÐcation of interesting species and lines for furtherstudy.

ACKNOWLEDGEMENTS

The authors would like to acknowledge the NationalPlant Introduction Station, Ames, Iowa, for provisionof seed for the screening.

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