Dietary chromium — forms and availabilities

The Science of the Total Environment, 28 (1983) 443--454 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

443

DIETARY CHROMIUM - FORMS AND AVAILABILITIES GALE HANSEN STARICH and CLIFTON BLINCOE Division of Biochemistry, University of Nevada, Reno, Nevada, USA

ABSTRACT The chromium naturally occurring in plants eluted from Sephadex G-25 at approximately 2600 D. Total chromium was quantitated with flameless atomic absorption spectrophotometry. A plant ligand tagged with radioactive chromium both in vivo and in vitro migrated on Sephadex G-25 identically to the naturally occurring chromium compound. The molecular weight of the radioactively tagged chromium compound was 2600 daltons on Sephadex G-25. Similar complexes isolated from plant species were found attached to an organic ligand. The ligand appears to have 2 components, differing in composition by an amine group. This extremely stable (KD = 9 x 10-5) anionic complex does not contain peptide or deoxyribose units. When alfalfa was exposed to either Cr(III) or Cr(VI), only Cr(III) was isolated in this organic chromium compound. The alfalfa bioreduction system can be saturated, as evidenced by Cr(VI) isolation of ionic in those plant extracts incubated with high levels of Cr(VI) in vitro. The gastrointestinal chromium physiology studies show that the radioactively labelled plant chromium compounds remained intact through the gastrointestinal tract up to the large intestine.

Some degradation products were identified in the rat cecum. Approximately 30%

of the plant chromium available to the rat was absorbed across the gastrointestinal tract.

INTRODUCTION Chromium is an environmental pollutant and suspect carcinogen, therefore, NIOSH has established exposure levels for the principal forms of chromium (I). Cr(VI) is defined as the most toxic form of mono- and dichromates. These compounds cannot exceed 25 ug per cubic meter of workplace a i r . Other Cr(VI) compounds are defined as "carcinogenic" forms and cannot exceed 1 ug per cubic meter of workplace air (I). More definitive studies of acute toxicity to Cr(III) and Cr(Vl) compounds have been done in rabbits (2,3). While both compounds are toxic if injected intraperitoneally, Cr(VI) produces the more severe growth depression, and liver and kidney damage (4). Detoxificationof Cr(VI) by reduction to Cr(III) is thought to occur in mammals(5). Biologicalantioxidants such as ascorbic acid and glutathione and its associated enzymes may be involved in the bioreduction (5). Plant systems are also sensitive to Cr(VI) (6). Both Cr(VI) and Cr(III) are transported across root tips (7). Both Cr(III) (8) and Cr(VI) (9) have been isolated from plants. A protective 0048-9697/83/$03.00

© 1983 Elsevier Science Publishers B.V.

444

system that reduces Cr(VI) to Cr(III) in plants has been postulated (5). Plant toxicity occurs with excess chromium and includes inhibition of nitrification (10) and reduced growth (6). How plants detoxify Cr(Vl) and the oxidation state of the chromium within higher plants remains to be defined. Like many essential micronutrients, chromium is toxic at high levels and essential in minute quantities.

The definition of essentiality involves linking chromium deficiencies to

specific pathological conditions (11,12). Chromium supplementation is useful in reversal of the symptoms of malnutrition,

hypercholesterolemia,

opacity of lens, and diabetes (11).

Chromium increases the utilization of glucose during recovery from protein-calorie malnutrition (13,14). Chromium deficiencies are characterized initially by impaired glucose tolerance.

As

the deficiency becomes more severe, glucosuria, fasting hyperglycemia, impaired growth, decreased longevity, and corneal opacities occur (15). The initial observation that impaired glucose tolerance could be reversed with yeast extracts was made by Glaser and Halprin (16). The specific compound which improved the glucose tolerance was later isolated and characterized by Mertz and Schwartz (15). The compound, glucose tolerance factor, contains nicotinic acid, amino acids and chromium, and was the first plant chromium-ligand to be identified. The purpose of this study was to examine the form of chromium naturally occurring in food and feed.

The approximate molecular weight and charge of the plant chromium complex

was ascertained

in a preliminary purification.

chromium was also examined.

Further, the oxidation state of the bound

Finally, the bioavailability and stability of the plant chromium

compounds was estimated using rats as a model mammalian system. EXPERIMENTAL Preparation of Radioactively Labeled Plant Material Radioactively labeled chromium was incorporated in vitro into both mature and seedling alfalfa (Medicago satavia) extracts.

After pressing alfalfa at 2300 kg per cubic centimeter

in a stainless steel cylinder and piston using a Carver Laboratory press, the expressed lic]uid was collected and centrifuged for 30 rain at I0,000 g at 0 to 4°C.

An aliquot of the

supernatant was then incubated with carrier-free 51CRO4-2 or 51CrCI3. The final concentration was 1 uCi chromium-51 per milliliter of plant supernatant.

The incubation mixture was allowed

to stand at room temperature or on ice for 30 rain. The radioactive chromium was incorporated into intact alfalfa by one of two methods depending on the plant material used. In one method, mature alfalfa was exposed to chromium-51 by injecting a millicurie of isotope into the potted plant soils at varying depths (17). Half of the water was applied to the cell surface at a rate that would not allow percolation through the soil and drainage from the pot.

The other half of the water was supplied by capillary

action from a source at the bottom of the pot.

This watering method held the radioactivity

available for plant intake during the 2 to 3 week time period observed.

Liquid expressed

from the foliage was prepared as indicated in the in vitro procedure. The second, and more successful method of in vivo nuclide incorporation was developed using alfalfa seedlings.

One week old hydroponically grown seedlings were immersed in one

liter of distilled water containing a millicurie of 51CRO4-2 or 51CrCl3. The container was

445 sealed and kept at 4°C in the dark for a minimum of three days. The seedlings were then washed three times with triple distilled water and pressed as in the in vitro proeedure.

The

expressed liquid was centrifuged as above before analysis. Saeeromyees earlesbergenis (Brewers yeast) was incubated in Sabourauds broth (DIFCO Co.) eontaining 1 mCi

per liter 51CrCl 3 or 51CrO4 -2 (18). The yeast was harvested by

centrifugation at 5,000 g at 4°C. After resuspension in triple distilled water, the yeast was centrifuged for 30 min at i0,000 g, 0 to 4°C. The yeast pellet was then frozen in liquid nitrogen and ground in a precooled mortar and pestle.

The resulting finely divided powder

was reconstructed in 0.01 M sodium phosphate pH 7.00 buffer and centrifuged as above. The supernatant was collected for further study. Examination of the Chromium Form in Plants Total chromium was determined by flameless atomic absorption spectrophotometry. The oxidation state of the complexed chromium was determined by Faraday magnetic susceptibility and

differential

pulse voltammetry.

Chromium oxidation state

colorimetrically using Feigl's chemical spot tests (19).

was

a l s o examined

Chromium-51 was detected with a

well-type NaI(T1) crystal, single channel pulse height counter and scaler, differential mode, 110 keV window, 320 keV threshold. Gel permeation media used in the plant extract chromatography included Sephadex G-10, G-15, G-25, G-50(R) (Pharmacia Fine Chemicals), Bioge] P-4 and P-6(R) (Biorad Laboratories). The column buffer was 0.01 M sodium phosphate, pH 7.00 unless otherwise noted. Cation exchange media (Dowex 50x8, (NH4+) and CM Sepharose(R) (Pharmacia Fine Chemicals)), and anion exchange media (DEAE Sephacel and QAE Sephadex(R) (Pharmacia Fine Chemicals)) were used to determine the charge of the alfalfa chromium compound. CM Sephadex columns were eluted with 0.01 M sodium acetate pH 7.00 buffer.

The Dowex 50x8 columns were eluted

with 0.01 M sodium phosphate buffer. Samples fractionated on anion exchange resins were eluted with a discontinuous phosphate buffer gradient, 0.01 to 0.4 M, pH 7.00. Bioavailability Studies Endogenous chromium excretion was estimated as described by Comar (20). Twenty microcuries of 51CRO4-2 was injected intraperitoneally into female Wistar rats daily for ten days.

Daily whole-body counting was done with a scintillation counter shielded so that

chromium-51 was not detected.

Excreta were collected and analyzed for chromium-51 daily.

Feeds containing chromium-51 were fed to female Wistar rats.

These animals had

consumed the non-radioactive feed for seven days prior to consuming the radioactively labeled feed. The labeled feed was ingested in less than six hours. Urine and feces were collected and analyzed for chromium-51 as above. RESULTS A variety of gel exclusion media were used to fraetionate plant extracts.

Representative

elution patterns for alfalfa extracts fractionated on Sephadex G-10, G-15, G-25 and G-50 are detailed in Figure 1.

Sephadex G-25 produces the best separation of the chromium complex

446 G-25

G-IO G-15

G-50

100

f

80

\

'° l IO B

0

0.0

0.5

.

1.0

1.5

2.0

2.5

I

I

I

3.0

3.5

4.0

Ve/Vo

Fig. I. Mature alfalfa e x t r a c t labelled in vitro with 51CRO4-2. Fractionation with the sephadex gels noted was w i t h 0.01 M phosphate buffer, pH 7.00, at #oc in 1.5 x 50 crn c o h . ~ ns.

from high molecular weight compounds and 51CRO4-2 salts. Two 51CRO4-2 labeled peaks distinct from inorganic chromium were frequently found after Biogel P-6 chromatography (Fig 2). An unlabeled alfalfa extract was then ehromatographed on Sephadex G-25 to examine the distribution of naturally occurring chromium in the plant. Flameless atomic absoprtion spectrophotometry (FAAS) was used to qualitate total chromium in the resultant fractions. The total chromium peak eluted in the same position on all gels examined as the in vitro labelled 51CRO4-2 peak. From this, it was concluded that the 51CRO4-2 was acting as a valid label for the chromium normally present in alfalfa. In examining the elution patterns of the alfalfa extract on Sephadex G-25 and Biogel P-6 (Fig 1 and 2), the major difference noted was the separation of two distinct components on Biogel P-6. Frequently, gel permeation media have an affinity for the metal or ligand causing the metal-ligand bond to dissociate (21). Because Sephadex gels are highly charged, it seemed possible that chromium complex affinity for the gel was producing a poor separation. Since Biogels have fewer charged sites (22), the double peak was thought to represent improved resolution of the two component systems. To reduce these interactions, gels were saturated with chromium to cover any sites that might interact with the metal and therefore reduce labile metal-ligand bond dissocation (21). There was no difference in the elution pattern produced by the potassium chromate saturation of the gels. Both anion and cation exchange media were used to determine the charge on the chromium complex and to futher purify it.

Anion exchange ge]s such as DEAE Sephace] and

447

100

80

~

60

E ~

40

20

0

10

20

30

40

50

60

70

80

Fraction Number

Fig. 2. Mature a l f a l f a e x t r a c t labelled in v i t r o with 51CRO4-2 and subsequently f r a c t i o n a t e d w i t h Biogel P-6 and P-4. Buffers and conditions were as noted in Figure I.

QAE Sephadex bind the partially purified chromium complex tightly. The chromium complex elutes from these gels with 0.4 M sodium phosphate buffer, pH 7.00. The chromium complex is shown to be displaced from the ion exchange site in a minimum number of fractions in Figure 3.

The lack of peak complexity may be indicative of a like net negative charge on

these complexes. The cation exchange media CM Sephadex and Dowex 50x8 (NH4+) did not retard the radioactively labeled chromium complex. Chromium saturation of the above ion exchange gels prior to chromium complex separation did not alter the elution pattern routinely seen. The ionic affinity was apparently for the organic moiety of the complex rather than the chromium itself.

These data indicate that chromium exists in alfalfa as a reasonably

homeogenous anion. The molecular weight assignment from gel permeation chromatography of the chromium compounds prepared from a variety of higher plants is given in Table I.

Columns were

standardized with insulin B chain, streptomycin sulfate, oxytocin and NADH. It indicates that the same complex is formed in most higher plants investigated. The exceptions are the in vitro preparations of Brewers yeast and apples. The alfalfa seedlings have a limited capacity to complex chromium resulting in some inorganic chromium appearing in the chromotograms. Table 1 also shows that the alfalfa complex formation is independent of the oxidation state of the chromium incubated with the plant. The exception was in that litt]e chromic ion was taken up from soil. This was probably due to reduced solubility of the chromic ion in alkaline soils (23). The oxidation state of the chromium bound to the plant-chromium complex was determined both directly and indirectly. Figure 4 is the differential pulse voltammogram that

448 1900 1800 1700 1000 1500 1400 1300 1200 1100 1000 90O 80O 7O0 600 5OO 400 3OO 200 100 0

DEAE Sephacel

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Fraction Number

Fig. 3. Discontinuous gradient elution of alfalfa chromium complex on anion exchange resins. In vitro 51CRO4-2 labelled alfalfa extract was partially purified on Sephadex G25 before application to the 1.5(d) x 25 cm column in 0.0IM phosphate buffer, pH 7.00, at 4oc.

includes the determination of the oxidation state of the chromium within the plant complex and the redox potential of the chromium in a NH4+/NH4C1 buffer system.

From this work

it was ascertained that there were no interfering trace metals in the plant-chromium preparation. Only Cr(III) was identified in this buffer system.

The redox potential for Cr(III) Cr ° was -.26

volts. Data supporting the existence of some Cr(VI) in the plant-chromium complex has also been observed. Faraday magnetic susceptibility was determined on a variety of plant-chromium complex preparations.

In all cases, the chromium complex was repelled from the field,

indicating a diamagnetic configuration (24). Cr(VI) is diamagnetic.

Supporting data used the

independent method of Feigl's spot tests (19) for Cr(III) and Cr(VI) (20). The colorimetric assay indicated the presence of Cr(VI). Peptides were not associated with this chromium-containing complex as indicated by a variety of colorimetric tests and ultraviolet absorption at 285 nm.

A primay amine (detected

449

TABLE 1 Molecular weight est~nates of plant chromium complexes. 10,000g supernatants of noted plant extracts were fractionated on Sephadex G-25 with 0.01 M phosphate buffer, pH 7.00. Columns were saturated with either Cr(Iil) or Cr(Vl) before complex fractionation. Column standardization was done with insulin B chain, streptomycin sulfate, oxytocin and NADH. Plant Source

Molecular Weight in daltons

Mature Alfalfa In vivoa~ In vitrou Hay Alfalfa Seedlings In vivo In vitro

Cr(IIl) ~ 2600

Cr(VI) - ~ 2600 2900

2600 2800

2400 2300

Apple In vitro

1700

Crested Wheatgrass In vivo In vitro

3000 3000

Brewer's Yeast In vivo In vitro

2300

2300 1500

a Chromium in noted oxidation state placed in the plant growth media. b Chromium in noted oxidation state incubated with plant extracts.

Chromiumredox

(z

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potential

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AppliedPotential(volts) Fig. #. Polarographic record of purified alfalfa chromium complex in NH0+/NHoCI buffer. Redox potential determined against a standard calom el electrode.

450

with fluorescamine (25)) was associated with the high molecular weight peak resolved on Biogel P-6. Treatment of the high molecular weight fractions with cation exchangers removed the fluorescamine reactive material.

All of the bound chromium-51 then appeared in the lower

molecular weight peak of the Biogel P-6 chromatograph. In order for any alfalfa chromium complex to be useful as a dietary source of chromium, it had to be stable under conditionsnormallyfound in the gut. The chromium complex integrity was maintained after most of the treatments described in Table 2. This chromium complex is extremely stable once formed (Kd = 9 x 10-5). This was determined by dialysis of the chromium-51 complex against increasing concentrations of Cr(VI) salts. Onlyprolonged heating, acidic digestion, or extended dialysis destroyed the chromium complex. An alfalfa chromium complex was found throughout the gastrointestinal tract.

After intraperitoneal injection of

radioisotope, the gut contents were collected and analysed (Table 3).

A 1400 dalton complex

(on Biogel P-6) was isolated from the liquid portion of the gut contents in the distal small intestine.

This molecule migrated at 2800 D on Sephadex 6-25. A lower molecular weight

chromium containing componentwas identified in the cecum. The chromium complex contained in the digestive tract was an anion, as assigned by those ionic exchange media noted in Table 3. An alfalfa chromium complex appears to form in the digestive tract.

Inorganicchromium

isolated in the cecum may be a breakdown product of the alfalfa compound described here.

TABLE 2 Study of the stability of the alfalfa chromi~'n cornplex. Molecular weight of chromium compound estimated with Sephadex G-25. Separation conditions in Figure I, Alfalfa Chromium Complex Found After These Treatments Dialysis (24 hrs.) -amylase, trypsin, pepsin digestion (1 to 10 ng m1-1) Drying (90° C) Storage at 4°C for 30 days Boiling (30 rain) pH shifts 1.00 to II.00 Ionic strength shifts from 0.01 to 1.0 M HCI digestion (30 rain) Treatments That Destroyed Alfalfa Chromium Complex(a) NHO3 digestion (30 min) High pH (2 hrs) Dialysis (48 hrs) Boiling (2 hrs) la) Only inorganic chromium identified

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TABLE 3 Molecular weight and charge assignment of radioactive chromit~n complex isolated from selected areas of the digestive tract in rats, Animals were injected intraperitoneally for ten consecutive days with 51CrO4 -2. Samples were prepared from the liquid phase of the gut contents. Molecular Weight Estimate Area of Digestive Tract

Gel

M.W. (daltons)

Lower Small Intestine

Biogel P-6

1368 + 171

Ceacum

Biogel P-6

1338 + 89 504 + 122

Charge Determinationa Media

Charge

CM Sephadex

(-)

DEAE Sephacel

(-)

QAE Sephadex

(-)

achromium-51 alfalfa complex prepurified on Sephadex G-25.

The bioavailability of different plant chromium complexes to the rat is detailed in Table 4. The three plant forms were absorbed similarly by the rat.

The alfalfa chromium complexes

studied were excreted in the same proportion in the feces and urine. The Brewers yeast radiochromium was only found in the feces.

This could be indicative of a Iow percentage of

free ionic chromium within the yeast cells, or simply that the level of chromium in the urine was too low to detect.

All of the plant forms studied were more available to the rat than

inorganic chromium salts (26). DISCUSSION The alfalfa chromium complex is both similar and dissimilar to other chromium compounds previously described. As Huffman and Allaway found in the bean leaf system (7), the alfalfa root transport system showed little chromium oxidation state preference (Table 1).

The

inavailability of Cr(HI) to aIfalfa root uptake is probably produced by olation at alkaline pH valves as noted in Schroeder (23). However, Bourque (27) and Skeffington et al (9) have shown preferential uptake of Cr(VI) by plant roots. The alfalfa chromium complex is anionic like the chromium compound isolated from Leptospermum scoparium (8) (Figure 3).

Alfalfa may

be able to reduce the Cr(VI) to Cr(III), as was described by MeKee and Wolf (28) in organic compost. The complex may contain a mixture of the two oxidation states, as Sehroeder et al (29) described in oak leaves and corn oil. The mixture of chromium oxidation states probably represents a saturation of the plant bioreduction systems. Baugh (5) has indicated that biological systems can reduce Cr(VI) to Cr(III). As such, this process may serve to reduce the toxicity of chromium in the biosphere. Very little radioactive label was attached to protein with in vivo labelling techniques, in contrast to that of Bourque et al (27). We found little association of chromium-51 with plant orffanelles, similar to that described by Huffman and Allaway (7). It is important to separate the chemical characteristics of the alfalfa chromium complex

452

TABLE 4 Bioavailability of various plant chromium forms to t h e r a t . A l f a l f a and brewers y e a s t c o n t a i n i n g chromium-51 complex were c o n s u m e d by t h e rats within 6 hours. E x c r e t a c o l l e c t e d and analyzed as noted in experim e n t a l s e c t i o n , d Available Cr Absorbed

% Total Excreted in Feces

% Total Excreted in Urine

Seedlings a

35.2 + 14.8%

63.6 + 15.1%

1.3 + 0.5%

Matureb

37.1 + 12.0%

59.6 + 6.8%

3.3 + 2.8%

29.8 + 11.1%

70.2 + 11.1%

None Detectedc

Alfalfa

Brewers Yeast

In vivo label of seedlings. b In vitro labeled alfalfa extract applied to rat chow. e Very low radioactivity incorporated into yeast probably rendered urine counts too low to detect. d Inorganic chromium bioavailability currently under study.

from the Brewers Yeast GTF. Glucose tolerance factor is reported to have a molecular weight of 400 daltons and contain Cr(III).

It is extracted from Brewers yeasts with hot ethanol.

A

primary purification step is elution from cation exchangers with ammonium buffers (31). Possible molecular components of GTF include nicotinic acid, glycine, cysteine and glutamic acid.

The intact alfalfa chromium complex is about 2600 daltons and contains both Cr(III)

and Cr(VI). The complex requires no extraction from alfalfa and is practically insoluble in non-polar solvents. The alfalfa chromium complex does not bind to cation exchangers, although an amine compound associated with it does.

The alfalfa chromium complex is a polydispersed

collection of anions (Fig. I). It may be a two component system, as evidenced by the improved resolution produced by Biogel P-6 chromatography (Fig. 2). This was not ~roduced by chromium affinity to the gel.

The elution pattern was very similar to the plant saceharides described

by John et al (32) on Biogel P-2.

In short, then, the chromium form isolated from alfalfa is

distinct from the Brewers yeast GTF. Separation of unlabelled alfalfa extract shows that the chromium naturally occurring in alfalfa has the same molecular weight as the radioactively labeled organic form,

and as such represents an alternate source of dietary chromium.

A chromium compound of any dietary significance would have to be stable under conditions encountered in the alimentary canal.

Sayato et al (33) found the intestine to be

the most active area of endogenous chromium excretion into the alimentary canel.

The liquid

contents of the small intestine and cecum were analyzed for chromium-51 labeled compounds after intraperitoneal injections of the nuclide.

A medium molecular weight species identified

on Biogel P-6 was similar in size to that previously found in alfalfa. an anion.

This molecule was also

A low molecular weight component, probably inorganic chromium, was identified in

the cecum contents. This may represent a breakdown product of the alfalfa chromium complex. A breakdown product of bean leaf chromium was associated with poor intestinal absorption in the rat ( 7 ) .

Apparently, the alfalfa chromium complex is stable in the alimentary canal.

Cr(III) and Cr(VI) are generally not well absorbed in the digestive tract (34). The 3%

453

absorption of sodium chromate-51 described by Mackenzie et al (26) is one of the highest published. Less than 1% absorption was the average literature value (26). Chromic oxide is often used as an unabsorbed gut content marker (35). Rollison (36) felt that complexing agents that would stabilize the chromium near neutrality would markedly improve intestinal absorption. The absorption of the rats consuming the two different forms of chromium complex from alfalfa was examined. B o t h mature and seedling alfalfa labeled with chromium-51 were approximately 30% bioavailable. Of the amount of chromium-51 absorbed, less than 3% of it was excreted with the urine. The urinary chromium is usually considered to be the percentage of ionic chromium existing in the diet (37). This level of absorption is comparable to that described in Robles et al (38). In their work, the total chromium recovered in the feces after feeding various alfalfa preparations was 24.7% for both leaf and stem preparations. Because Mertz (11) has described GTF as being more absorbable than inorganic chromium, we used an in vivo labeled Brewers yeast in similar feeding experiments. The intestinal absorption of Brewers yeast chromium was comparable to the alfalfa data. The lack of urinary excretion of chromium-51 contained in the Brewer's yeast may be due to the low incorporation of nuclide into the Brewer's yeast. It may also suggest that the Brewer's veast contains very little inorganic or uncomplexed chromium. This level of absorption of Brewer's yeast chromium is within the 10 to 25% availability reported by Casey and Hambridge (37). In conclusion, we have shown that higher plants reduce the more toxic Cr(VI) to Cr(III) and assemble it into a medium molecular weight complex. The same complex was found in a variety of higher plants. This complex is highlystable to conditions that would be encountered in food/feed preparation. This complex is considerably more absorbable than inorganic chromium. In a contaminated environment, animals may be exposed to a variety of chemical forms of chromium, each of which have their own specific bioavailabilities. To assess toxicity, it is thus necessary to consider not only the total amount of chromium in the biosphere but also its chemical form(s). REFERENCES 1 NIOSH, Occupational Exposure to Cr(VI): U.S. Department of HEW, Publication No. 76-129, 1975, p.2. 2 S. Tandon, D. Saxena, J. Gaur and S. Chandra, Comparative Toxicity of Trivalent and Hexavalent Chromium Alterations in Blood and Liver. Environ. Res., 15(1978)90. 3 K. Nakamuro, K. Yoshikawa, Y. Sayato and H. Kurata,Comparative Studies of Chromosomal Abberation and Mutagenticity of Trivalent and Hexavalent Chromium. Mutat. Res., 58(1978)175. 4 E. Underwood (Ed.), Trace Elements in Human and Animal Nutrition, 3rd Edition, Academic Press, New York, 1971, p. 253. 5 M. Baugh, Toxic Effects of Chromium. Chem. and Eng. News. 27(1981)55. 6 A.R.C. Haas and J.N. Brusca, The Effects of Chromium on Citrus and Avocado Grown in Nutrient Solutions. Calif. Agric., 15(1961)10. 7 E. Huffman and W. Allaway, Chromium in Plants: Distribution in Tissues, Organelles and Extracts and Availability of Bean Leaf Cr to Animals. Agric. and Food Chem. 21(1973)982. 8 G. Lyon, P. Peterson and R. Borrks, Chromium-51 Distribution in Tissues and Extracts of Leptospermum scoparium. Planta, 88(1969)282. 9 R.A. Skeffington, P. Shewry and P. Peterson, Chromium Uptake and Transport in Barley Seedlings (Hordeum vulgare L.). Planta 132(1976)209.

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