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III'IIT' TI ’- «Lu mumujmwuflmwailing" This is to certify that the thesis entitled INCREASING TOTAL AND BIOLOGICALLY ACTIVE CHROMIUM IN WHEAT GRAIN AND SPINACH BY SPRAYING WITH CHROMIUM SALTS presented by Frank Andrea Vicini has been accepted towards fulfillment of the requirements for M.S. Soil Science degree in V Major professor Date )3 ' é ' // 0-7 639 3*: LIBRARY Micirim 9:212: . gig: 1.35.; .33“, ”f‘l’ s “W' M «v- Trmmt w: 25¢ nerd-w per ita- RETQRNING LIBRARY MATERIALS: P‘laco in book return to remove charge from circulation records INCREASING TOTAL AND BIOLOGICALLY ACTIVE CHROMIUM IN WHEAT GRAIN AND SPINACH BY SPRAYING WITH CHROMIUM SALTS By Frank Andrea Vicini A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Crop and Soil Sciences 1981 ABSTRACT INCREASING TOTAL AND BIOLOGICALLY ACTIVE CHROMIUM IN WHEAT GRAIN AND SPINACH BY SPRAYING WITH CHROMIUM SALTS BY Frank Andrea Vicini The potential for incorporation and translocation of chromium (Cr) in plants by foliar absorption was investigated. A greenhouse experiment was conducted by growing winter wheat (v. Augusta) and Spinach (v. Dark.Green Bloomsdale) and Spraying the plants with treatments of 200, 2000, or 4000 g/ha Cr as Cr2(SO4)3 or as CrEDTA. Controls (0 g/ha Cr) were Sprayed with Na EDTA or Na2804. All Cr solu- 2 tions contained 0.037 mg Cr/ml and all Spraying was completed prior to heading. Variable Cr rates were achieved by the number of sprayings. Spraying with either Cr2(SO4)3 or CrEDTA nearly doubled the concen— tration of total Cr in the wheat grain from a control average of 0.29 ug/g to 0.54 ug/g in high treatment plants. The percent of total Cr ex- tractable with 50% ethanol increased from 40 to 50 percent when sprayed with CrEDTA and slightly more when sprayed with Cr2(SO4)3. Similar trends were detected for ammonium hydroxide extractable Cr which in- creased from about 45 percent in controls to 65 percent in the higher treatment plants. Slightly greater values approaching 75 percent were obtained with Cr2(SO4)3 treated plants. Total Cr in Spinach leaves was increased by as much as ten fold Frank Andrea Vicini with the sulfate source being consistently more effective than the EDTA. The percent of the total Cr that was biologically active (extractable with 50% ethanol or ammonium hydroxide) could not be determined. Foliar application of the Cr solutions also increased total wheat grain yield a maximum of 59 percent in the medium treatment plants as compared to controls. Plants Sprayed with Cr2(SO4)3 generally produced greater yield. In addition, all aspects of wheat growth were enhanced in Cr treated plants. to Alice ii ACKNOWLEDGMENTS I would like to express my sincere gratitude to Dr. B. G. Ellis for his guidance and participation in my academic program and research. His enthusiasm for, and skills in soil science are ones I shall always hold in deep respect and admiration. The support and assistance of other members of my guidance commit— tee are gratefully acknowledged; Dr. B. D. Knezek and Dr. Cress. The assistance of Mr. Derek Rhodes, Mr. Cal Bricker, and Ms. Teresa Hughes was deeply appreciated. I also wish to express my appreciation to other graduate students and especially to Ms. Kathy Foster and Mr. Tom Sims. The financial assistance of the Michigan State University Agricul- tural Experiment Station through the Department of Crop and Soil Science is gratefully acknowledged. iii LIST OF TABLES . . . 0 LIST OF FIGURES . . 0 INTRODUCTION . . . . o CHAPTER I. LITERATURE REVIEW History . . . Chromium in Animals and Man TABLE OF CONTENTS - Biological R018 0 o o o o - Mode of action - Physiological action - Human Cr requirement/Nutritional considerations - Chromium content of the human Evidence for Chromium Deficiency - Indirect evidence 0 - Direct evidence a Chromium and Plants - Physiology of Cr in plants — Increasing Cr in crop plants 0 diet in Man - Growth responses of plants from BIOLOGICALLY ACTIVE CHROMIUM EN SPRAYING WITH CHROMIUM SALTS II. INCREASING TOTAL AND GRAIN AND SPINACH BY AbStraCtoo oo 00. Introduction . . . . . Materials 0 o o o o o MGthOdS ooooooo Results and Discussion Conclusions . . . . . List of References . . III. SUMMARY AND CONCLUSIONS LIST OF REFERENCES . 0 APPENDIX . o o o o o o 0 0 iv applied vii 34 34 36 37 38 44 58 6O 63 65 76 TABLE LIST OF TABLES CHAPTER II 1. Determination of total chromium in selected NBS Standard Reference Materials 0 o o o o o o o o o o o o o 2. Determination of chromium in 50% ethanol extracts of selected NBS Standard Reference Materials . . . . . . . 3. Determination of chromium in 0.1 N_NH4OH extracts of selected NBS Standard Reference Materials . . . . . . . 4. Percent of total chromium extracted with 50% ethanol from selected NBS Standard Reference Materials . . . . . 5. Percent of total chromium extracted with 0.1 N_NH40H from selected NBS Standard Reference Materials . . . . . 6. Determination of optimum drying temperature of graphite oven 1.15ng NBS Orchard Leaves 0 o o o o o o o o o o o o 7. Determination of Optimum drying temperature of graphite oven using NBS Spinach Leaves 0 o o o o o o o o o o o o 8. Determination of optimum ashing temperature of graphite oven using NBS Orchard Leaves 0 o o o o o o o o o o o o 9. Determination of Optimum ashing temperature of graphite oven 11$ng NBS Spinach Leaves a o o o o o o o o o o o o 10. Determination of optimum atomization temperature of graphite oven using NBS Orchard Leaves . . . o . . . . . 11. Determination of optimum atomization temperature of graphite oven using NBS Spinach Leaves . . . . . . . . . APPENDIX 12. Analysis of variance of wheat grain yield . . . . . . . 13. Analysis of variance of wheat growth . . . . . . . . . . 14. Analysis of variance of total chromium in wheat grain . V Page 41 41 41 42 42 42 43 43 43 44 44 76 76 76 TABLE 15. 16. 17. Page Analysis of variance of ethanol soluble chromium inwheatgrain..........o..o....... 76 Analysis of variance of NH4OH soluble chromium in Wheatgrainoooooooooooooooooooooo 77 Analysis of variance of total chromium in Spinachleaves..................... 77 vi LIST OF FIGURES FIGURE Page CHAPTER II 1. Total chromium in wheat grain as affected by rate and source of chromium in foliar spray solution . . . . . . . . 45 2. Total chromium in wheat straw as affected by rate and source of chromium in foliar Spray solution . . . . . . . . 46 3. Total chromium in spinach leaves as affected by rate and - source of chromium in foliar Spray solution . . . . . . . . 49 4. Total chromium in Spinach stems as affected by rate and source of chromium in foliar Spray solution . . . . . . . . 50 5. Ethanol extractable chromium in wheat grain as affected by rate and source of chromium in foliar spray solution . . . 52 6. Ammonium hydroxide extractable chromium in wheat grain as affected by rate and source of chromium in foliar SpraySOlUtj-ODoooooooooooooooooooooo 53 7. Wheat grain yield as affected by rate and source of chromium in foliar spray solution . . . . . . . . . . . . . . . . . 55 8. Total dry matter yield of wheat plants as affected by rate and source of chromium in foliar Spray solution . . . . . . 56 9. Enhancement of maturity of wheat as affected by rate of chromium in foliar Spray solution . . . . . . . . . . . . . 57 vii INTRODUCTI ON The trace element chromium (Cr) has recently been recognized as an essential component of the human diet. Experimental evidence has clear- ly established that Cr deficiency results in impaired glucose metabolism and may be a causitive agent in the etiology of certain forms of diabe- tes. In addition, Cr may be a protective agent against both cardiovas- cular and ischemic coronary heart disease. Recent studies have established that deficiencies of Cr may occur in certain pepulation groups, particularly those from industrialized nations such as the United States (US). As a result, a Cr intake of 200 ug/day has been tentatively recommended for adults by the Committee on Recommended Dietary Allowances of the US National Academy of Sciences/ National Research Council in the 1980 revision of the Recommended Die- tary Allowances (RDA). Plant-derived Cr is considered to provide a biologically valuable form of the element in the human diet. But, few plants contain adequate stores of Cr and food processing diminishes the content further. There- fore, researchers have attempted to derive methods to increase the Cr concentration in plants. To date, few of the methods involving soil application of Cr for plant uptake have been successful. However, several studies on foliar application of the element indicate that Cr can be increased in plants through spraying. This study was designed to determine if biologically active Cr 2 concentrations could be substantially increased in plants through foliar spraying. Additional information was sought regarding the ability of plants to translocate Cr to important plant parts. The knowledge gained will be important in helping design crop management practices to produce food and feed crops high in nutritionally effective forms of Cr. CHAPTER I LITERATURE REVIEW HISTORY The history of Cr as an essential micronutrient can be indirectly traced back to the mid 1800's when brewer's yeast, the richest known source of an organic form of Cr, was used in the treatment of diabetes (Herepath, 1854). Several decades later, Glaser and Halpern (1929) no- ted that yeast extracts significantly increased the hypoglycemic action of the hormone insulin. The authors' work was not followed up, however, since the results were explained as a general effect from the high vita- min content of yeast and since they were overshadowed by the great therapeutic benefits of insulin itself. Approximately 25 years later, Mertz and Schwarz (1954) noted that rats fed a Torula yeast-based diet not only developed an expected dietary necrotic liver degeneration, but also experienced a severe hypoglycemia. DesPite this hypoglycemia, sub- sequent work by Mertz and Schwarz (1955) indicated that the animals also developed an intolerance to intravenous glucose administrations. When preparations of brewer's yeast were used to protect the animals against death from the necrotic liver degeneration, the defect in glucose meta- bolism was also corrected. With continued research on the purification of the brewer's yeast preparations, Schwarz and Mertz (1957) were clear- ly able to separate the agent protecting glucose metabolism from that protecting against the liver disease and termed it glucose tolerance 3 factor (GTF). Subsequent research concentrated on purifying yeast extracts to the point where preparations with extreme GTF activity were obtained. Ana- lytical studies of these preparations indicated the prescence of an inorganic constituent, later determined to be trivalent Cr. However, the prescence of other contaminants could not be excluded and other elements in the periodic system (noble gaseS, elements present in the deficient diet) were also tested for their effects on glucose tolerance. Despite complications involving contamination from Cr in cleaning solu- tions, studies performed by Schwarz and Mertz (1959) finally concluded that the primary active component in the yeast extracts, effective in the treatment of the impaired glucose tolerance, was trivalent Cr. Efforts during the following five years contributed to the develop- ment of a mode of action of Cr in biological systems. Mertz et a1. (1961) noted that the trace element increased the glucose uptake by epididymal fat tissue in Cr deficient rats, but only when insulin was present. Studies by Mertz and Roginski (1963) indicated that Cr's effect was at the first step of sugar utilization, tran510cation into the cell. Similar effects were noted for glucose tran5port into isola~ ted rat lens (Farkas and Roberson, 1965) and in insulin-dependent mitochondrial swelling (Campbell and Mertz, 1963). Finally, with the aid of polargraphic studies, Christian et a1. (1963) proposed the hypo- thesis that Cr functions in biological systems by forming a ternary complex with insulin receptors and insulin, facilitating the initiation of insulin action by acting as a cofactor to the hormone. Human studies on diabetic patients were first initiated in 1963 by Mertz and Glinsmann (1964). Chromium supplementation of 150-200 ug/day as CrCl3 was found to improve the impaired glucose metabolism of 40-50% 5 of the subjects tested (Glinsmann and Mertz, 1966; Levine et al., 1968). Studies performed by other investigators showed similar 40-50% resPonses (H0pkins and Price, 1968; Schroeder, 1968). One study by Sherman et a1. (1968) was completely negative. These results were discouraging and led to the hypothesis that not all inorganic Cr complexes were equally uti— lized and that some humans may require preformed GTF (organic Cr) to obtain the necessary effect on glucose metabolism. As a result, a second project of extraction, fractionation, and purification was initi- ated which resulted in the production of preparations with outstanding in vitro activity, that potentiated the action of insulin five to ten fold and that were more efficiently absorbed (Mertz and Roginski, 1971). These preparations also were transported across the placenta into the fetus and interchanged with what is now considered the most important Cr pool in the organism. In 1974, the composition of GTF was hypothesized as a dinicotinato amino acid Cr complex, through the use of mass spectrophotometry (Toep- fer, 1974). Synthetic GTF compounds were then prepared but were not clinically useful since they did not meet the criteria of insulin acti- vation in vivo (Toepfer et al., 1977). During the mid '70's, several studies were again conducted on human subjects with yeast extracts or yeasts of relatively high Cr content since pure GTF could neither be extracted or synthesized (Doisy et al., 1976; Liu and Morris, 1978)° The results obtained demonstrated the ex- pected effect on glucose tolerance, but the most important finding was a normalization of the exaggerated insulin re5ponses to glucose loads. In contrast to the partial reSponses obtained when glucose tolerance was measured, nearly every subject responded with a normalization of insulin levels. This suggested that the high circulating insulin levels often 6 seen in adult-onset diabetes may be due to deficient utilization of in— sulin from a direct lack of Cr. The results were also significant in further understanding the mode of action of Cr as a cofactor to insulin. Efforts to establish precise analytical methods for the detection of Cr began almost simulataneously during the 1960's (Schroeder et al., 1962; Feldman et al., 1967; Levine et al., 1968). These studies indi— cated that past reports of Cr concentrations in blood and urine were most probably in error and exaggerated by a factor of more than 1:100. In addition, during the first decade of atomic absorption spectrophoto- metry, the reported levels of Cr in blood and urine were again found to be much lower and approached a range in urine from 5-10 ug/liter. This value was in agreement with other researchers and during 1974, at a Cr analytical workshop held in Columbia, Missouri, the first preliminary calculation on Cr requirement was made. Based on an accepted absorption efficiency of between 0.5 and 1.0% (Donalson and Barreras, 1966; Doisy et al., 1971) of a given oral Cr dose and on a daily obligatory loss of 10 ug/day, Cr intake had to be in excess of 1000 ug/day in order to maintain balance. Since no reasonable diets in the US furnished more than 100 ug/day, it was assumed that the form of Cr in foods had to be better absorbed than simple Cr compounds. Consequently, researchers tested foods for GTF content (Toepfer et al., 1973) and emphasis was placed on the proportion of GTF active Cr in foods rather than on total Cr. A new analytical development in 1978 once again changed the empha- sis in Cr nutrition. Utilizing CEWM atomic absorption spectrophotometry with an ECHELLE monochromator, researchers found urinary Cr excretion to be less than one-tenth of the values previously reported (Guthrie et al., 1979). Confirmation of these results was made by an independent 7 method Of mass spectrophotometry with stable isotopes and through other methods (Liu et al., 1979; Veillon et al., 1979). These results indi- cated that since urine is the predominant route Of excretion Of Cr in an organism, the estimated minimal requirement Of the element is lower by a factor Of ten or more. As Kumpulainen et a1. (1979) point out, this new Cr requirement would now be capable Of being satisifed by many diets previously thought deficient and once again, total Cr in the diet was felt to be a more useful indicator of the value Of Cr in foods. Overall, during the twenty-five or so years Of intensive research on Cr, the following information has been gathered: (l) The establish- ment Of a requirement Of Cr in the glucose metabolism Of animals and man; (2) the mode of action and function Of Cr in biological systems; (3) the degree Of Cr deficiency in man; (4) the estimated human Cr re- quirement; and (5) the ability to assess Cr status both analytically and physiologically. CHROMIUM IN ANIMALS AND MAN Biological Role Chromium may have several biological functions in animals and man. Mertz (1969) noted a stimulatory effect of Cr on several enzyme systems, in vitro. Chromium was also found to be associated with nucleic acid, lipid, and protein metabolism or structure, or both (Wacker and Vallee, 1959; Wacker et al., 1963; Mertz, 1969). However, the major biological function Of Cr is closely associated with that Of insulin. Nanogram quantities Of Cr are required for the Optimal effect Of the hormone in every insulin-dependent system that has been studied (Hambidge, 1974; Mertz, 1979)o An uncorrected deficiency Of Cr in biological tissues necessitates the administration Of unphysiologically large additions 8 Of insulin to produce a normal biochemical re5ponse. As a result Of this close association with the hormone, Cr is essential for the normal glucose metabolism of man and other animals (Liu et al., 1977; Jeejeeb- hoy et al., 1977; Mertz et al., 1978; Freund, 1979). In effect, defi- ciencies Of Cr in human tissues may be a factor in the etiology Of diabetes and in some cases, supplementation with Cr may prevent or delay the appearance Of certain forms of the disease such as maturity-onset diabetes (Glinsmann and Mertz, 1966; Mertz, 1979). Perhaps the most important role Cr plays in biological systems is that the element may be a protective agent against ischemic coronary heart disease. Supplementation with Cr has been proven to lower ele- vated serum cholesterol levels in humans to a degree similar to that produced by a strict limitation of dietary fats (DOisy et al., 1976; Liu and Morris, 1978). Mertz (1969) noted that an elevated serum cholester- 01 level is one Of several risk factors condusive to cardiovascular disease. A second risk factor consists Of elevated fasting insulin levels as well as elevated insulin resPonses in glucose tolerance tests (Stout, 1977). Two studies, one in Finland (Pyrola, 1979) and one in Australia (Welborn, 1979) found a significantly increased risk Of morbi- dity and mortality from coronary heart disease in subjumws with elev- ted insulin levels and reSponses, independent Of glucose tolerance and other risk factors. Mertz (1979) noted that elevated insulin levels are the first consistent and persistent indication of individuals marginally and/or moderately Cr deficient. These elevated levels can and have been normalized by supplementation with Cr (DOisy et al., 1976; Liu and Morris, 1978). These findings are interpreted by Mertz (1979) to be a possible explanation for the Observed correlations Of three investiga- tors who suggest that Cr may play a protective role against certain 9 cardiovascular diseases in humans (Schroeder et al., 1970; Punsar et al., 1975; Newman et al., 1978). Mode Of Action The mode Of action Of Cr has been reviewed by Hambidge (1974), Mertz et a1. (1974), Mertz (1975), and DOisy et a1. (1976). It is clearly established and uncontradicted that Cr potentiates the action Of insulin in vivo and in several in virto systems (Campbell and Mertz, 1963; Mertz, 1979)° This has been demonstrated in studies with Cr- sufficient and Cr-deficient organisms (Liu and Morris, 1978). The addi- tion of various Cr salts to Cr deficient tissues such as the adipose tissue Of rat epididymis, enhances the action Of insulin on glucose up- take, glucose oxidation tO C0 , or its conversion to fat (Mertz et al., 2 1961; Mertz et al., 1965). In addition, Cr stimulates the uptake Of galactose which is also controlled by insulin (Mertz and Roginski, 1963). Polargraphic studies have indicated that Cr potentiates insulin ac- tion by forming a ternary complex between cell membrane sulfhydryl groups and the intrachain disulfide Of the insulin A chain (Christian et al., 1963). This is believed to be the first step by which the insulin hormone increases the flux Of glucose through cell membranes (Mertz and Roginski, 1971). Results compatible with this hypothesis have been re- corded not only in the rat, but also in the squirrel monkey and in the genetically Obese, diabetic mouse (Davidson and Blackwell, 1968; Mertz, 1969; Tuman and DOisy, 1974). However, no recent studies that would support or contradict the formation of a ternary complex have been con- ducted and the exact biochemical mode of action of Cr must remain hypothetical until further evidence is accumulated. 10 Physiological Action The main physiological action Of Cr is clearly established by vir- tue Of its association with insulin. Chromium functions in all insulin- dependent systems where a deficiency Of the element would result in a diminished reSponsiveness Of the hormone (Mertz, 1979; Saner, 1979; Shapcott, 1979). This has been exemplified in the glucose metabolism Of Cr deficient human subjects (Mertz, 1979). These individuals have ade- quate reserve capacity Of their pancreatic function which responds to a marginal Cr deficiency with an increase in the production Of circulating insulin. Total function of all insulin dependent systems is thereby re- tained by producing more of the hormone. However, these increased levels Of circulating insulin mobilize Cr and drastically accelerate depletion Of the marginal Cr stores (Schroeder, 1970; Mertz, 1971; Jeejeebhoy, 1977). If the extra demands for Cr are not met by increased dietary intake, insulin—dependent functions such as glucose metabolism become impaired even though insulin levels remain elevated (Mertz, 1979). In effect, a vicious cycle is established. If Cr supplementa- tion is now initiated at this second stage Of Cr deficiency, one Of three situations has been found to occur: (1) The increased insulin levels will normalize without a change in glucose tolerance (DOisy et al., 1976); (2) Glucose tolerance will be improved without a change in cir- culating insulin levels; or in the best situation, (3) Insulin levels will be normalized and glucose tolerance im- proved significantly (Mertz, 1979). If Cr supplementation is not initiated at the second stage Of Cr deficiency, the extra capacity of the pancreas to produce insulin may be lost and the impaired glucose tolerance and other insulin-dependent 11 functions may exist concurrently with little or deficient insulin levels (Mertz, 1979). Similar to the first and second stage Of Cr de— ficiency, the primary defect Observed in this third stage is a relative resistance Of insulin-dependent functions to respond to endogenous or exogenous insulin due to a direct lack Of available Cr (Jeejeebhoy, 1977; Freund, 1979). Human Cr Requirement/Nutritional Considerations A Cr intake Of 50-200 ug/day has been tentatively recommended for adults in the 1980 revision Of the Recommended Dietary Allownaces (RDA). This range is based in part on the absence Of Signs Of Cr deficiency in the major part of the US pOpulation found to consume approximately 60 ug Cr/day. The safety of 200 ug Cr/day was established by long-term sup-l plementation trials in humans receiving 150 ug Cr/day in addition to their dietary intake (Glinsmann and Mertz, 1966). In addition, long- term balance studies in humans receiving 200-290 ug Cr/day resulted in or near equilibrium (Tipton and Stewart, 1970). Chromium nutrition and metabolism differs from that Of other trace elements by virtue Of a strict dependence on the chemical form Of the element present in biological systems (Anderson et al., 1978). Organic Cr compounds extracted from brewer's yeast or other natural products differ from simple inorganic Cr compounds in intestinal absorption, access to Special Cr compartments, tissue distribution, and in placental transport (Mertz, 1969; Anderson et al., 1978). Mertz (1979) reviewed Cr nutrition and concluded that Cr in the form Of yeast extracts, supposedly as GTF, is more available than inorganic forms in meeting nutritional needs. In a study by Liu and Morris (1978), as little as 4 ug Of yeast Cr/day was sufficient in replacing deficient Cr stores in a majority Of subjects investigated. However, it should be noted that Cr 12 intakes Of 50-200 ug/day, even as poorly absorbed inorganic compounds, have been found to be more than sufficient to balance normal Obligatory losses. In effect, GTF supplementation of diets is not required if sufficient amounts Of non-GTF Cr are used. Chromium nutrition, then, may be considered very similar tO that Of iron (Fe). Both heme-Fe (organic Fe) and GTF-Cr (organic Cr) are signi- ficantly better absorbed and metabolized than their respective inorganic forms. Like Cr, the Fe requirement can be met by inorganic compounds alone, provided the total intake is high enough (Mertz, 1979). These nutritional considerations do not, however, detract from the dominant physiological roles the organic forms of the elements play in biological systems. Both heme-Fe and.GTThCr are vastly superior in performing their necessary biological functions. Chromium Content Of the Human Diet Food-- The typical Western diet is estimated to provide between 50 and 100 ug Cr/day (Levine et al., 1968; Guthrie, 1973; Walker and Page, 1977; Kumpulainen et al., 1979). Few foodstuffs contain appreciable amounts of the element. In general, highly refined foods contain less Cr since refining processes remove the element (Czerniejewski et al., 1964; Schroeder, 1968; Schroeder et al., 1970; Schroeder, 1971). Determination of the Cr content Of plant and animal materials in the diet provides only limited information relating to dietary adequacy (DOisy et al., 1976). Therefore, Toepfer et a1. (1973) conducted an ex- tensive study to correlate Cr content and the GTF activity Of various foodstuffs. In terms of the relative biological values for GTF activi- ty, the authors Obtained the following data: brewer's yeast 44.88, calf's liver 4.52, wheat germ 4.05, whole wheat bread 3.59, white bread 13 2.99, wheat grain 2.96, chicken muscle 1.89, haddock 1.86, patent flour 1.86, and skim milk 1.59. It is Obvious that extreme variabilities exist in the biological values Of Cr in the diet. Overall, brewer's yeast, meat products, cheeses, whole grains, and condiments are excellent sources Of 'active' Cr; whereas, leafy vegeta- bles may contain large amounts Of the element but in a poorly available form. Polished rice, patent flour, and table sugar are also poor sources of Cr. One additional source Of Cr in the diet may be from stainless steel cookware. If acidic foodstuffs are prepared, the high Cr content Of the stainless steel may leach out during the cooking process into the prepared foods (Schroeder et al., 1962). 4 Water-- Durfor and Becker (1962) analyzed drinking water in the US and found that the nation's drinking water supplies only small amounts Of Cr. They reported the Cr values of 100 selected cities and found a mean Of 0.43 ng/ml with a range from non-detectable to 35 ng/ml. The authors also noted that water treatment and purification methods may add or re- move Cr depending upon the nature Of the purification processes. EVIDENCE FOR CHROMIUM DEFICIENCY IN MAN Evidence for the occurrence of Cr deficiency in man has been re- viewed by Schroeder (1970), DOisy (1976), and Shapcott (1979). TO date, only inconclusive information exists to support the belief that Cr de- ficiency is widespread in the human population. However, isolated Cr deficiencies have been detected and could arise from reduced dietary in- take, by reduced absorption from the gut, by excessive loss Of the ele- ment from the body, or possibly through a combination Of all three 14 factors (Shapcott, 1979). Shapcott (1979) pointed out that there are several approaches in current use for the detection Of deficiencies Of essential micronutri- ents in man. The author outlined the approach most applicable to Cr as follows: Indirect Evidence Firstly-- The demonstration Of a clinical syndrome attributable to the de- ficiency, with disappearance Of the Specific symptoms following admini- stration Of the deficient substance. Secondly-- The demonstration Of a probable deficiency through a calculation Of the dietary intake. Direct Evidence Firstly-- The demonstration of decreased tissue levels Of the substance. Secondly-- The demonstration of increased excretion Of the substance. Thirdly-- The demonstration Of decreased levels Of the substance in the blood and hair of individuals. Igdirect Evidence Cause-effect occurrences-- To date, only two cases Of a cause-effect relationship have been detected for Cr deficiency and impaired glucose metabolism (Jeejeebhoy et al., 1977; Freund, 1979). In both studies, the subjects were main- tained on total parenteral nutrition with an exact record Of Cr intake. The symptoms they developed (i.e. decreased glucose tolerance, insulin 15 dependent diabetes) disappeared upon supplementation with Cr. Less conclusive evidence on Cr deficiency has been gathered in studies on malnourished infants. Majaj and Hopkins (1966) found that four infants with Kwashiokor and impaired glucose tolerance had imp provements in their glucose metabolism following supplementation with inorganic Cr. Carter (1968), however, Obtained less conclusive findings on Egyptian children with similar clinical symptoms. HOpkins (1968) also found a varied resPonse to Cr supplements in malnourished Nigerian and Jordanian infants according to their geographical location. The variable results Obtained with the Egyptian and some of the Jordanian infants were believed by the authors to be due to a previously adequate Cr intake. Shapcott (1979) also attributed some of the variability to the belief that glucose tolerance in malnourished infants does not al- ways result from.Cr deficiency. Gurson and Saner (1973) also investigated the effects Of Cr supple- mentation on the impaired glucose tolerance resulting from protein- calorie malnutrition in infants. Nine Of the infants were found to respond to the supplements, five did not. The effects of Cr supplementation on impaired glucose metabolism in adults has also been investigated by several groups. Levine et a1. (1968) found that four out Of ten elderly subjects with impaired glucose tolerance improved while on prolonged Cr therapy. Glinsmann and Mertz (1966) found that three of six diabetics had improved glucose tolerance after extended supplementation with inorganic Cr. In a separate study, Sherman et al0 (1968) did not find any change in the glucose tolerance of both diabetic and normal subjects following 16 weeks of Cr supple- mentation. In all Of the studies in adults, no prediction could be made by any of the authors as to which patients would benefit, nor could any 16 reason be prOposed to explain why only some Of the subjects had improved (Shapcott, 1979). DOisy et a1. (1976) also studied the effect Of Cr supplementation on impaired glucose tolerance. Using brewer's yeast or yeasts high in extractable GTF, the authors not only found improvements in glucose tolerance, but also noted that post glucose serum insulin levels also fell significantly. The significance in their studies was that nearly all the subjects experienced a normalization Of insulin levels follow- ing Cr supplementation. Recent evidence has established that a majority Of the variability experienced in these earlier supplementation studies can be attributed to one of the following situations: (1) Defects in glucose metabolism can result from more than simple Cr deficiencies; (2) Human subjects vary in their ability to absorb, retain, ex- crete and manufacture various Cr compounds depending upon their physiological condition and inherent biochemistry; (3) Human subjects vary in their relative stores of available Cr. Improvements in glucose tolerance or any other insulin-dependent system will be realized from Cr supplementation only if the defect re- sults from a direct deficiency Of the element (Mertz, 1979). Hence, evidence for a cause-effect relationship between Cr deficiency and im- paired insulin-dependent systems would be expected to be inconsistent. Dietary intake Of Cr-- Calculations Of the actual daily intake Of Cr in man are difficult because of the technical problems encountered in analyses at the nano- gram/gram range. However, recent studies have determined that the daily excretion rate Of Cr is about 0.4 tO 1.8 ug/day (Guthrie et al., 1978). 17 If Obligatory losses Of the element are also taken into account, Veillon et al. (1979) estimate the mean minimal Cr requirement to be approxi- mately 1.0 ug/day. Since the absorption Of CrCl3° 6H20 in humans is roughly between 0.5 and 0.69% Of a given dose (Mertz, 1969), a dietary intake Of about 200 ug/day is believed to provide the average require- ment. Excluding some Older studies with outdated and questionable methodologies, Levine et a1. (1968), Guthrie (1973), Walker and Page (1977), and Kumpulainen et a1. (1979) have estimated the typical Western diet to provide between 50 and 100 ug Cr/day. Certain diets may contain as little as 5 ug/day (Levine et al., 1968). Since a varied diet will furnish Cr with an average availability Of about 1-2% (depending upon the chemical form Of the Cr in the food) it is believed that dietary deficiency Of Cr is possible (Toepfer et al., 1973). In addition, Schroeder (1968) found that a high carbohydrate diet (typical in the US and other Western countries) increases urinary Cr excretion. This in turn is believed tO increase the Cr requirement and may further predis- pose individuals tO dietary Cr deficiency. Several other studies have also indicated that the amount of die« tary Cr is marginal under many conditions in Western nations. Glinsmann et a1. (1966), Levine et a1. (1968), and Schelenz (1977) found this to be the case in their respective studies. In other investigations, food refinement was found to diminish the Cr content Of foods and since the North American diet is high in refined foods, Schroeder (1970) believed that these diets may be more Cr deficient. It is also possible that the preparation Of foods may convert some Cr to unavailable forms or that some Cr may actually be lost through cooking (Schroeder, 1971). On the other hand, the widespread use of stainless steel in food processing may introduce some Cr into meals (Shapcott, 1979). 18. Though many of these studies point to possible Cr deficiencies, it is not possible, according to Shapcott (1979) to predict that dietary intake alone may predispose the general populace to Cr deficiency. Un- til standardized methods Of analysis are employed worldwide for Cr, the evidence for Cr deficiency from dietary intake alone must remain specu- lative. Direct Evidence Tissue Cr concentrations-- Many researchers believe that a decreased tissue concentration Of Cr is evidence for Cr deficiency. Schroeder (1970) and Hambidge and Baum (1972) have reported data on the Cr content Of different tissues Of the body from peOples all over the world. Their results Show a highly significant difference between the US and other countries. Though sub- stantial decreases in Cr concentrations in blood and urine have been re- ported with new methodologies, the differences do suggest that North Americans are relatively Cr deficient. Schroeder (1962) and Hambidge and Baum (1972) also found that tissue Cr levels decrease rapidly with age from birth. The authors also noted a more marked decrease in the US. Anderson et a1. (1975) studied tissues from subjects dying from is- chaemic heart disease or by accident and noted that muscle Cr values were somewhat higher in the accident victims. In other tissues, there were no detectable differences. Research by Morgan (1972) found Cr levels in the livers Of diabe— tics to be lower than in those from normal subjects. On the other hand, Eatough et a1. (1977) in a study Of Pima Indians, found no differences in Cr levels in liver, pancreas, muscle, or kidney from.norma1 and dia- betic subjects. It should be noted that since Pima Indians have a very 19 high incidence Of diabetes, the results Obtained point more towards a hereditary factor for their diabetes than a nutritional deficiency. Blood Cr-- Chromium circulates in blood serum as a protein-bound and as small dialysable molecules assumed to be GTF (Shapcott et al., 1977). The low molecular weight form is believed to be the physiologically active sub- stance whose concentration fluctuates in response to acute metabolic changes (Mertz et al., 1974). Many researchers feel, therefore, that measurements of the changes in serum Cr in response to metabolic stimuli could be a meaningful measure of the Cr nutritional status Of an indi- vidual IShapcOtt, 1979). In 1966, Glinsmann et a1. (1966) found that, in re5ponse to a glu- cose challenge, five normal subjects experienced an increase in serum Cr levels while two diabetics did not. After Cr supplementation (and a subsequent improvement in glucose tolerance) the diabetics also showed increased Serum Cr levels following glucose administration. Levine et a1. (1968) noted Similar results but also found a reduced increase in serum Cr in elderly diabetics who did not re5pond to Cr supplementation. Davidson and Burt (1973) found plasma Cr levels decreased in non-preg- nant women and in normal males after a glucose challenge. Little change was found in pregnant women. Liu and Morris (1978) also investigated the serum Cr re5ponse to glucose challenges. They noted decreases in serum Cr levels in normal and hyperglycemic subjects one hour after glucose, with the latter sub- jects experiencing a greater decrease. Shapcott (1979) pointed out that in analyzing serum Cr changes as a measure Of Cr deficiency, caution should be employed. He points out that serum Cr levels may be influenced by hormonal activity and as a 20 result may not directly reflect tissue stores Of the element. Excretion Of Cr-- The predominant route Of excretion of Cr is the urine. Urinary Cr levels have, therefore, been considered as an index Of Cr nutritional status. However, urinary Cr excretion is controlled by hormonal factors and above all by glucose intake (Shapcott, 1979). Therefore, as is found with changes in Serum Cr levels, little agreement exists between researchers as to changes in urinary Cr levels following a glucose challenge. Schroeder (1968) noted a 50% increase in urinary Cr in diabetic subjects after a glucose challenge. After supplementation with Cr, the increase rose to 200%. Wolf et a1. (1974) also reported an acute rise. in urinary Cr levels following a glucose challenge in normal young wo- men. Gurson and Saner (1978) showed an increase in urinary Cr excretion following a glucose load in 80% Of normal adults tested, but found no changes in eight diabetic subjects. DOisy (1976) found that insulin- dependent diabetics had greater excretions of orally administered radio- active Cr, while maturity-onset diabetics did not. Shapcott and Lang- lOis (1979) could find no obvious correlation between glucose tolerance and fasting Cr excretion or in the change in Cr excretion following a glucose challenge in 102 tests on varied subjects. Hair Cr levels-- Hair analysis is believed to be one Of the best methods to detect Cr deficiency in groups Of individuals. As Shapcott (1979) points out, the levels of Cr in hair are not subject to acute fluctuations in re- Sponse to diet or hormonal factors. Hambidge (1974) did extensive work on hair Cr. The author concluded 21 the following: (1) Hair Cr levels are highest at birth, decline substantially in childhood and less so in the adult; (2) Diabetic subjects have lower hair Cr concentrations; (3) Hair Cr levels show little environmental contamination; (4) Pregnant women have lower hair Cr levels than non-pregnant women. Shapcott et a1. (1979) found similar results as Hambidge (1974). Hair Cr levels were lower in pregnant women and could be specifically associated with pregnancy. In addition, the authors found that hair Cr levels were generally higher at one year Of age than later in life. In another study, Rosson et a1. (1979) found lower hair Cr levels in female adult diabetics than in controls. Male diabetics did not differ from controls. Conclusions As is evident, past measurements of Cr in tissues, blood, urine, or hair have not given precise indications of Cr deficiencies. Because Of analytical and physiological problems which were only recently being solved, previous results are questionable and difficult to interpret. However, Mertz (1979) points out that present knowledge and methodology are sufficient tO assess the Cr status Of pOpulation groups. It is an established fact that Cr deficiencies dO occur (particularly in indus-‘ trialized countries) but the extent, cause, and range remain to be de- termined. CHROMIUM AND PLANTS Plant-derived Cr is considered to be an important component Of the human diet even though indigenous levels in edible plant tissues are 22 generally low (Schroeder et al., 1962). Consequently, researchers have tried to derive methods by which the concentration of Cr in plants may be increased. Many studies have, therefore been undertaken to assess the forms of Cr in soils and their availability to plants and to examine the processes Of uptake and accumulation (Shewry and Peterson, 1974; Shewry and Peterson, 1976; Skeffington et al., 1976; Cary et al., 1977; Lahouti and Peterson, 1979). In addition, several comprehensive reviews on plant uptake and essentiality of Cr have been compiled and provide a broad insight into earlier work on the tOpic (Pratt, 1966; Allaway, 1968; Mertz, 1969; Lisk, 1972; National Research Council, 1974). Physiology Of Cr in Plants, Essentiality-- The essentiality of Cr for plants has been reviewed by Pratt (1966), Lisk (1972), and Huffman and Allaway (1973). Despite many earlier find- ings indicating a stimulatory effect Of Cr on plant yield, an absolute requirement has not been established (Huffman and Allaway, 1973). Pratt (1966) believed that the earlier reports of growth increases from Cr were small and erratic responses that were mostly unverified and did not prove essentiality. Huffman and Allaway (1973) felt that none Of the earlier studies provided sufficient data on Cr which meet the criteria Of essentiality prOposed by Arnon and Stout (1939). Warington (1946) interpreted the stimulatory effects of Cr as a limited substitution Of the chromate ion for molybdenum (MO) in soils and not a direct effect Of the element on plant growth. In animals, Cr is required as a cofactor for insulin (Mertz, 1979). However, as Huffman and Allaway (1973) point out, insulin action is not essential to plants and therefore, the essentiality Of Cr to animals does not provide a basis for its requirement to plants. 23 In a series Of experiments designed to test the essentiality Of Cr to plants, Huffman and Allaway (1973) determined that Cr supplementation was not required for the normal growth Of romaine lettuce, tomato, wheat, or bean. However, since they could not reduce the level of Cr in their solutions below 3.8 X 10-4uM and since they encountered atmospher- ic contamination, the question Of essentiality at very low levels could not be resolved. In reviewing this study, Cary et al. (1977a) concluded that if Cr was essential to the species tested, the levels required would be lower than for any known essential nutrient. Brown et a1. (1972) noted vegetational differences clearly marking serpentine soils (high in Cr) implying that Cr could be essential for the growth Of serpentine plants. Shewry and Peterson (1974) also be- lieved that Cr could be essential for the growth of these plants. How— ever, no clear essentiality relationship could be established by either group of researchers. Absorption and uptake-- Culture solutions-- Plants readily take up different chemical forms Of Cr from culture solutions (Cary et al., 1977a). There are con- flicting views both as to the valence state and rate of uptake, however. Bourque et a1. (1967) working with wheat suggested that only hexavalent Cr (chI) as CrO 2- but not trivalent Cr (CrIII) was absorbed by plant 4 roots. Blincoe (1974) in examining work by Eckert and Blincoe (1970) - + claimed that CrO42 was absorbed rapidly but CrIII, as Cr3 was absorbed only to a limited degree. Skeffington et a1. (1976) concluded that there was no evidence to support the claim by Blincoe (1974). Mytten- + aere and Mousny (1974) working with rice plants concluded that Cr3 is 4 . In addition, they also considered taken up more rapidly than CrO .. + . that CrO 2 had to be reduced to Cr3 before entering the plant cell. 4 24 Other findings by Lyon et a1. (1969), Huffman and Allaway (1973), Shewry and Peterson (1974), and Skeffington et al. (1976) indicate sub- stantial differences in specific activity between roots Of plants grown in either CrVI or CrIII solutions, with those grown in CrIII generally containing the largest amounts Of Cr. Work done by Skeffington et a1. (1976) suggests independent uptake mechanisms for the two valence states Of Cr. It is believed that chI enters the plant via the sulfate pathway whereas CrIII enters passively. The authors believe their conclusions are founded by the fact that the metabolic inhibitors sodium azide and dinitrophenol substantially re- - + duce uptake Of CrO 2 but uptake Of Cr3 was not affected. Also, they 4 point out that CrVI but not CrIII has been found in xylem sap by Lyon et a1. (1969). §2il§f' Experiments involving Cr uptake by plants growing on Cr- treated soils indicate that only small increases in plant absorption Of the element can be expected (Allaway, 1968). Cary et al. (1977b) con- cluded that this limited uptake occurs as a result Of two factors. First, the chemistry of Cr appears to be dominated by a trend Of added Cr to form inert mixed oxides Of CrIII and Fe (Plotnikov et al., 1967; Frissel et al., 1975; Cary et al., 1977b; Grove and Ellis, 1980). These forms Of Cr are so inert that they do not constitute an effective source of Cr for plants unless present in the soil in very large amounts. In effect, Cary et al. (1977b) believe that when soluble sources of Cr are added to soils, the reversion to forms that are unavailable to plants is essentially complete within one growing season. It should be noted, however, that work on soil Cr by Bartlett and Jones (1979) contradicts these findings. The authors reported that added CrIII oxidizes readily to the hexavalent form under conditions found in many field soils. 25 The second factor Cary et al. (1977b) present as evidence that li- mited uptake Of Cr Occurs from soils by plants deals with the levels of specific forms Of the element in soil solution. Although Shewry and Peterson (1977) present evidence that chI is taken up by plants, Cary et al. (1977b) believe the concentration in soil solution may be too low to be Of practical Significance, unless the levels are maintained on an ongoing basis. Foliage-- Substantial increases in plant absorption of Cr have been reported when the element was applied as a foliar spray. Perkins et a1. (1960) applied river water containing Cr-Sl as a contaminant to several plants and reported appreciable increases in absorption of the element. Pickrell and Ellis (1980) reported that Cr-Sl as EDTA was ab; sorbed and slowly translocated by soybean leaves. Sedova (1958) irri- gated vegetables with sewage waste waters containing Cr and reported in- creases Of the element by a factor of three to ten over that Of cone‘ trols. Parr and Taylor (1980) also reported plant absorption Of Cr when chromated recirculating water from cooling towers was applied as a foliar spray. Accumulation and translocation-- Roots-- The majority Of Cr taken up by plants remains in the roots. Shewry and Peterson (1974) found that less than one percent Of the CrO 2"-51 absorbed by barley seedlings was transported to the 4 shoots. Only when the Cr concentration in their nutrient solutions was sufficient to injure the barley roots did substantial amounts Of Cr translocate to the plant tOpS. Huffman and Allaway (1973b) reported that greater than 90% of the Cr-Sl absorbed from solutions by beans and wheat was still present in the roots 20 days after the last addition Of Cr—51 to the solutions. Most of this Cr was in soluble fractions and 26 less than 0.1% Of the total Cr-Sl in the plant was in the seeds. This blockage in transport Of Cr from roots to shoots has also been noted by several other workers (Pratt, 1966; Schuneman, 1974; Skeffing- ton et al., 1976; Cary et al., 1977a). Cary et al. (1977a) concluded that this barrier could not be circumvented by addition Of any organic or inorganic Cr complexes studied. Skeffington et a1. (1976) proposed that the barrier was due largely tO the fact that Cr does not penetrate the root to the vascular tissue to any extent and cannot be translocated longitudinally in the cortex. The authors also believed (through addi- tions of Cr3+—51 or CrO42--51 to various segmented portions of barley roots and stems) that Cr is transported largely by xylem. Once in the - + xylem, CrO 2 was noted to move more rapidly to the shoots than Cr3 4 presumably because Cr3+ is held up by ion exchange on vessel walls, as occurs for divalent calcium (Ca2+) (Bell and Biddulph, 1963). This, they believed, may explain why Cr3+ is less toxic and not transported as well as Cr042-, even though it may be taken up more rapidly. Their con- clusions were further supported by short term experiments using Cr-51. The transport index (percentage of Cr absorbed by a plant that is found in the shoots) increased with increasing concentrations Of CrO4 but + remained virtually the same for Cr3 . Myttennaere and Mousny (1977) also noted similar effects in their experiments. They found CrEDTA + moved faster than Cr3 into shoots, presumably because CrEDTA was not retarded by ion exchange. Analagous effects were noted for CaEDTA (Isermann, 1971). 3+ 2- It should be noted, however, that even though Cr and CrO4 enter the vascular tissue of a plant root with difficulty, once there they are tranSported readily. Skeffington et a1. (1976) fed both ions to the + base of cut stems Of barley seedlings and noted that 24% Of the Cr3 and 27 53% Of the CrO42- absorbed were transported beyond the basal 0.5 cm Of the roots. Foliage-- Foliar applied Cr is absorbed readily but translocation of the element appears to be slow. Pickrell and Ellis (1980) reported Cr-Sl as EDTA to be translocated throughout the soybean plant when applied to the leaves. Parr and Taylor (1980) concluded that chromate applied to the leaves Of plants was not translocated basipetally even though it was absorbed readily. Uptake and translocation differences among plant Species-- Although the tendency to retain Cr by plant roots is a common physiological occurrence in almost all species tested, there are both qualitative and quantitative differences. Of the food crops tested, Cary et al. (1977a) found that the leafy vegetables that tend to accu— mulate Fe appear to be the most efficient in translocating Cr to the edible tops Of plants. Under conditions where a supply Of Cr was main- tained in the nutrient solution, Cary et al. (1977a) were able to in- crease Cr in the leaves Of Spinach and lettuce almost an order of magni- tude higher than in concentrations reported in a survey Of Cr in foods by Thomas et a1. (1974). In a study of several plant species, Lahouti and Peterson (1979) found that the specific activity Of the tops Of nine crop plants re- vealed a 10 fold difference between Species supplied with CrVI and an eight fold difference between species supplied with CrIII. Of the nine crOps examined, cauliflower plants accumulated the most Cr in both shoots and roots and mung beans the least. Species differences of ap- proximately five fold were recorded for the roots. In terms of specific activity or total activity, the authors found the concentration Of Cr in shoots was greatest in cauliflower followed by beetroot, radish and 28 carrot when supplied with CrVI. In plant roots, the greatest con- centration was found in radish followed by cauliflower, carrot, and beetroot. With CrIII, the greatest concentration in both shoots and roots occurred in cauliflower followed by radish, beetroot, and carrot. The least accumulation was recorded in mung bean and barley. Chemical forms in plants-- The chemical form Of Cr in plants is Of considerable importance since not all Cr compounds are nutritionally useful. Toepfer et a1. (1973) reported that the Cr in leafy vegetables was not active in the potentiation of insulin unless the vegetables were subjected to acid hy- drolysis. Chen et a1. (1973) found that oxalate increased the intesti— nal absorption of Cr compared with CrCl3, an indication that Cr oxalate complexes, a probable form Of Cr in plants (Lyon et al., 1969), may be a nutritionally available source of Cr. Toepfer et a1. (1973) also re- ported that the unknown Cr compounds in wheat grain and other cereals, though present at very low concentrations, are biologically active. Bean leaves, though they contain higher amounts of Cr, are not as nu- tritionally valuable since they contain poorly absorbable Cr (Huffman and Allaway, 1973). Various studies have shown that the Cr compounds in the bean leaves are of low molecular weight, but do not coincide with complexes of oxalic or citric acid. Several other studies have reported the occurrence Of unknown low molecular weight complexes from barley (Shewry and Peterson, 1974; Skeffington et al., 1976) and lucerne (Blin- coe, 1974) while trioxalate chromate was identified by Lyon et a1. (1969) in the Cr accumulating plant Leptospermum scoparium. Lahouti and Peterson (1979) also reported the occurrence Of a Cr containing compound in cauliflower with properties identical to trioxalate Cr. The exact structure Of the compound could not be determined, however. 29 Increasing Cr in Crop Plants-Overview Most work clearly indicates that plants may restrict the movement of nutritionally effective forms Of Cr from the soil into human and animal diets. Of the Cr taken up from the soil, most accumulates in plant roots and translocation is minimal. Chromium translocated to plant tOps is mostly present in the leaves and this Cr cannot be readily absorbed or utilized by animals. Foliar application Of Cr to crOps can substantially increase the levels of Cr in plant tissues but further studies are required to evaluate the nutritional significance Of this Cr. Chromium might only be expected to be present in nutritionally effective forms in the seeds of plants and only very little of the Cr taken up from soils (or possibly leaves) by plants is translocated to seeds. Recently, Mertz (1979) pointed out that Cr requirements can be met without nutritionally effective forms Of the element, as long as a total Cr intake is met. This would imply that the crop species which accumu- late Cr readily (i.e. Spinach, cauliflower, etc.) might be adequate in meeting nutritional needs. However, refining processes tend to diminish the total Cr content Of plants prior to consumption, placing greater im- portance on the form of the element remaining and to its biological value. In light Of this, Mertz (1979) points out that the important contribution Of nutritionally effective forms of Cr is still substantial and continued research with plants to provide high concentrations Of nu- tritionally effective forms of Cr is required. Growth Responses Of Plants fromquplied Cr Plant growth stimulation and phytotoxicity by Cr have been reported and reviewed by Pratt (1966), Mertz (1969), and Huffman and Allaway (1973). While low rates Of application may prove stimulatory (Arnon, 30 1937; Warrington, 1946; Haas et al., 1961; Farrar, 1968) higher rates generally tend to be toxic (Koenig, 1910; Robinson and Edington, 1935; Hunter and Vergnano, 1953; Soane and Saunder, 1959; Haas et al., 1961). In addition, several studies have shown Cr to have no significant effect on plant yield (Hewitt, 1953; Kusaka et al., 1971; Turner and Rust, 1971). Yield increases-- Soil applied Cr-- Reports of increased yields due to Cr applica— tion to soils have been cited in reviews by Allaway (1968), Mertz (1969) and the National Research Council (1974). Crop yields have been report- ed tO improve by the application Of Cr to soils in Germany (Koenig, 1911; Scharrer and Schropp, 1935; Gericke, 1943), France (Bertrand and De Wolf, 1965; Bertrand, 1967; Bertrand and De Wolf, 1968), Poland (Ku- rylowicz and Gasiorowski, 1959), and Russia (Dobrolyubskii, 1955; DO- brolyubskii et al., 1963). Leep (1974) found that corn plants grown on a muck soil treated with CrIII had a higher yield than control plants. In a study in Poland (Kurylowicz and Gasiorowski, 1959), the application Of a fertilizer containing 0.43% Cr increased the growth Of flax as com- pared to controls. The effect was Observed only when the flax was grown on sand, indicating, according to the authors, that the results from the Cr were apparent on the basis Of a previous deficiency. Addition of 0.05% and less Of chromous acetate to soils in Germany (Koenig, 1911) had stimulatory effects on the growth Of carrots, lupines, and cucum- bers, as well as on certain weeds. The results were confirmed and ex- tended by Scharrer and Schropp (1935) in sand and water cultures where CrIII, as Opposed to CrVI increased the yields of rye, oats, wheat, corn and peas and significantly stimulated root growth. Haas and Brusca (1961) reported stimulatory effects Of Cr on citrus 31 and avocado. They did not, however, report Cr analyses Of their nutri— ent solutions or plant tissues. Bertrand and De Wolf (1968) studied the effect Of Cr additions to a 'Cr-deficient' soil in France on the growth Of potatoes. Applications Of 40 g Of Cr per hectare increased yields 42%, from 32.7 in controls to 46.5 tons per hectare. In spite Of these reported yield increases, no change in leaf Cr was found. The authors Obtained similar results on the yields Of beets, peas, and carrots. Other reports on soil applied Cr have shown no significant effects Of the element on plant growth. Kusaka (1971) found that turnips grown on soils treated with 400 ppm Cr experienced no differences in yields from controls. Hewitt (1953) grew plants on purified nutrient solutions containing low levels Of Cr and found no growth reSponse in tomato and) other species tested. Turner and Rust (1971) found no significant re- sponse in soybean growth when Cr was added to unpurified nutrient solu- tions. However, the Cr concentration in their plant tissue was below the detection limit Of their analytical method. Foliar applied Cr-- Studies on foliar application of Cr solutions seem to be more consistent with respect to growth effects. CrIII as a foliar Spray was found to increase the yield and sugar content, lower the acidity and enhance the maturation Of grapes (Shcheglov and Baev, 1973; Dobrolyubskii and Viktrova, 1974). When chromic sulfate was ap- plied tO the soil (600 g/ha) or directly to the vine (200 mg/bush) the weight Of grapes improved 21%, the size and sugar content by 18% and 23% respectively, and the yield increased from 205 to 245 kg/ha (Dobrolyub— skii and Slavvo, 1958). Optimal results were Obtained by giving 5 mg/ vine. The authors concluded that the more rapid ripening under the in- fluence of Cr could be explained by the active participation Of Cr3+ in different oxidation—reduction processes occurring in the plants. At the 32 same time, they noted that the activity Of a number Of grape enzymes was increased (i.e. catalase, ascorbic oxidase, polyphenol oxidase, inver- tase) and the chlorophyll content, organic acids, glucose, and fructose were elevated as compared to controls. The authors did not report the nature Of their control solutions, however. In other studies, Bykun et a1. (1980) treated corn seeds with a solution Of 0.01% Cr(NO3)2 and increased the yield, enhanced plant growth and photosynthesis and positively affected the development of the stem conducting bundles over control plants. Growth reductions—- Reports on growth reductions due to Cr have been cited by Pratt (1966), Mertz (1969), and Huffman and Allaway (1973) in their reviews. Toxicity symptoms in soybeans were produced by 0.5 ppm CrVI in nutrient culture or 5 ppm chI in soil culture (Turner and Rust, 1971). Schuene- man (1974) found that 50 ppm CrIII applied on a sandy soil inhibited growth Of 70% Of the crops tested and that 100 ppm CrIII applications adversely affected 100% of the plantS. Hexavalent Cr has generally been found to be more toxic to plants than CrIII. Mortvedt and Giordano (1975) found that as little as 20 ppm soil Cr, in the hexavalent form, lowered the yield of corn plants. They also found chI to be generally more toxic than CrIII at an application rate Of 80 ppm. Luzzati and Siragusa (1979) added 25-250 ppm CrVI to soils and found growth impairments to plants treated with concentrations greater than 50 ppm. The authors also noted differences in plant resis- tance to Cr. The toxicity Of chI was found tO be greatest on neutral as Opposed to acidic soils. Ghini and Vercellino (1966) found that both CrIII and CrVI adversely affected the growth of wheat as low as 1 ppm solution Cr. Hewitt (1953) also concluded from long term growth Of 33 . 2- several plant SpeCleS that CrO 4 inhibits the growth Of roots and shoots to a greater extent than Cr3+. The general symptoms of Cr toxicity appear to vary among plants. Turner and Rust (1971) and Schueneman (1974) found severe wilting to be the predominant symptom Of toxicity in their studies. Ghini and Ver- cellino (1966) also noted the wilting effect on their wheat plants but also Observed that both CrIII and CrVI, at 0.1 ppm, inhibited water up- take by wheat seeds after planting. Hewitt (1953) and Anderson et a1. (1973) found Fe chlorosis to be the predominant symptom Of toxicity in their studies. Inhibitory effects Of Cr on plant growth are thought to be the re- sult of specific interactions between Cr and phosphorous (P) (Robinson) et al., 1935; Soane and Saunder, 1959; Vegnano, 1959; Spence and Millar, 1963) or Fe (Hewitt, 1948; Hewitt, 1953; De KOck, 1956; Walker and Gro- ver, 1957; Cannon, 1960). Hewitt (1953) interpreted the Cr toxicity in his studies to be an Fe—Cr interaction during plant uptake or transloca- tion since he was able to bring about plant recovery by foliar applica- tion Of Fe. Sedova (1958) concluded that part of the decreases in plant growth he witnessed were do to inhibition of nitrification by CrIII or CrVI additions to soils. Cary et al. (1977a) point out that even though plants Show visual symptoms of toxicity, the levels of Cr in plant tops generally are the same as for normal plants. CHAPTER II INCREASING TOTAL AND BIOLOGICALLY ACTIVE CHROMIUM.IN WHEAT GRAJN AND SPINACH BY SPRAYING WITH CHROMIUM SALTS ABSTRACT Chromium (Cr) has recently been recognized as an essential compo- nent in animal and human nutrition. As a result, a Recommended Dietary Allowance (RDA) has been tentatively established for the element. Plants provide a major biologically valuable source Of Cr in the human diet; yet, indigenous levels in edible plant tissues are generally too low to meet daily intake needS. Therefore, research has been directed towards increasing Cr in certain crop plants. But, most plant Species greatly restrict the uptake of Cr from soils. The objective of this study was to determine if total and biologically active Cr (as measured by ethanol and ammonium hydroxide extraction) could be increased in wheat grain and Spinach by Spraying plant leaves with either Cr2(SO4)3 or CrEDTA. A greenhouse experiment was conducted by growing winter wheat (v. Augusta) and spinach (v. Dark Green Bloomsdale) and Spraying the plants with treatments of 200, 2000, and 4000 g/ha Cr as Cr2(SO4)3 or as CrEDTA. Controls (0 g/ha Cr) were Sprayed with Na2EDTA or NaZSO4. All Cr solu— tions contained 0.037 mg Cr/ml and all Spraying was completed prior to heading. Variable Cr rates were achieved by the number Of Sprayings. 34 35 Spraying with either Cr2(SO4)3 or CrEDTA nearly doubled the concen- tration of Cr in wheat grain from a control average of 0.29 ug/g to 0.54 ug/g in high treatment plants. The percent Of Cr extractable with 50% ethanol increased from about 40 to 50 percent when sprayed with CrEDTA and slightly more when sprayed with Cr2(SO4)3. Similar treands were no— ted for ammonium hydroxide extractable Cr which increased from about 45 percent in controls to 65 percent in the higher treatment plants. Gen- erally greater values approaching 75 percent were Obtained with plants treated with Cr2(SO4)3. Total Cr in Spinach leaves was increased by as much as ten fold with the sulfate source being consistently more effective than the EDTA. The percent Of the total Cr that was biologically active could not be determined. Foliar application of the Cr solutions also increased total wheat grain yield a maximum Of 59 percent in the medium treatment plants as compared to controls. Plants Sprayed with Cr2(SO4)3 generally produced greater yield. 36 INTRODUCTION The trace element chromium (Cr) has recently been recognized as an essential component of the human diet. Experimental evidence has clear- ly established that Cr deficiency results in impaired glucose metabolism and may be a causitive agent in the etiology of certain forms of diabe— tes (Mertz, 1979). In addition, Cr may be a protective agent against both cardiovascular and ischemic coronary heart disease (Schroeder et al., 1970; Punsar et al., 1975; Newman et al., 1978). Deficiencies Of Cr may occur in certain pOpulation groups, partic- ularly those from industrialized nations such as the United States (US) (DOisy et al., 1976; Shapcott, 1979). As a result, a Cr intake of 200 ug/day has been tentatively recommended for adults by the Committee on‘ Recommended Dietary Allowances of the US National Academy Of Sciences/ National Research Council in the 1980 revision of the Recommended Die- tary Allowances (RDA). Plant-derived Cr is considered to provide a biologically valuable form Of the element in the human diet (Schroeder et al., 1962). How- ever, indigenous levels of Cr in edible plant tissues are generally tOO low to meet daily intake requirements and food processing diminishes the content further (Czerniejewski et al., 1964; Schroeder, 1968, 1971; Schroeder et al., 1970). Several studies have, therefore, been under- taken tO derive methods to increase the concentration of Cr in plants (Cary et al., 1977a, 1977b; Huffman and Allaway, 1973). TO date, few Of the methods involving soil application of Cr for plant uptake have been successful. However, research on foliar application Of the element in- dicates that Cr can be increased in certain plants through Spraying (Perkins, 1960; Parr and Taylor, 1980). The primary Objective Of this experiment was to determine if 37 biologically effective forms Of Cr (collectively termed 'glucose toler- ance factor' or GTF) could be increased in plant tissues through foliar application Of the element. Additional information was sought regarding the ability of plants to translocate Cr to important plant organs. The knowledge gained will be important in helping design crOp management practices to produce food and feed crops high in nutritionally effective forms Of Cr. MATERIALS Plant Culture Winter wheat (v. Augusta) and Spinach (v. Dark Green Bloomsdale) were grown in sterilized polyethylene pots containing 6.8 kilograms of Hillsdale sandy loam (Typic Hapludalfs, coarse-loamy, mixed, mesic) and 3.7 kilograms of Houghton muck soil (euic, mesic, typic, medisaprist), respectively. In the greenhouse, pots were arranged in a randomized complete-block design with 24 pots per cr0p type. Following soil ferti- lization and watering, 22 wheat seeds and 24 Spinach seeds were planted per pot to a soil depth of 1.25 cm and 0.95 cm, respectively. Seeds were allowed to germinate in darkness for approximately 72 hours. Three days after emergence, wheat seedlings were thinned to 19 plants per pot and Spinach seedlings 4 plants per pot. Soils were watered to field capacity with distilled/deionized water and fertilized with 100 ppm ni- trogen every 2-3 weeks, as required. The experiment was conducted in a climate controlled greenhouse with a 14 hour light, and a 10 hour dark period. Air temperature was set for 27C during the light period and 16C during the dark. Water was administered when soils dried to 70% of their field capacity weight. After three weeks growth (15 cm height), wheat plants were trans- ferred to a vernalization chamber maintained at 4C. Lighting was 38 provided 24 hours per day and watering conducted every 10-14 days to maintain field capacity. Following eight weeks in the chamber, the wheat plants were returned to the greenhouse. Composition Of Foliar Spray Solutions The experiment was conducted using a 2X4 source by rate factorial with three replications. Treatments imposed were 0, 200, 2000, and 4000 grams of Cr/ha as CrEDTA or Cr2(SO4)3. Each Cr solution was formulated to contain 0.037 mg Cr/ml. Variable Cr rates were achieved by the num~ ber Of sprayings in order to prevent toxicity as a result Of Spraying single concentrated doses. Control solutions (0 grams Cr/ha) consisted Of either Na EDTA or Na 80 prepared to contain equimolar concentrations 2 2 4 Of either the EDTA or SO 2- present in the two Cr solutions. All solu- 4 tions were prepared with a surfactant to aid in foliar absorption of Cr. METHODS Foliar Application Of Cr Solutions Foliar treatments were initiated after 85 days growth for the wheat plants and 28 days for the Spinach. Individual plants were separately sprayed every third day with 10 ml Of their respective Spray solutions until their total Cr allotment was reached. All Spraying was completed prior to heading in the wheat plants. NO visible symptoms Of Cr toxici- ty were apparent at any rate Of Cr application. Uniform leaf coverage was achieved using a plastic atomizer at— tached to a portable air-compressor. Individual solutions were sprayed on each plant in an enclosed Spray chanber, one pot at a time. Soils were covered during each treatment and no detectable Spray drift was produced. Plant Harvesting Wheat plants were harvested at maturity and separated into heads, 39 stems, and leaves. All plant materials were then oven dried at 70C for 72 hours, weighed to determine yield, and ground to pass a 40 mesh sieve.in an aluminum blade grinder. The ground wheat was then stored in air-tight containers in darkness at 25C until analyzed. Spinach plants were harvested 55 days after planting and separated into leaves, stems, and roots. All spinach materials were then weighed to determine yield, washed in a solution Of sodium laurly sulfate fol- lowed by destilled/deionized water, and oven dried at 70C for 72 hours. The dried Spinach tissues were then ground to pass a 20 mesh sieve and stored in air-tight containers in darkness at 25C until analyzed. Cr Analyses Total Cr-- Total Cr was determined by a modification Of the method Of Kump- ulainen et al., 1979b. Samples of dried plant material were oven dried at 70C for two hours. Plant samples Of 300 mg dry weight were weighed into 10 m1 Coors porcelain crucibles and dry ashed overnight at 500C in a muffle furnace. After cooling, 20 ul Of concentrated sulfuric acid (ultrapure) and 40 ul of 50% H202 (Fisher Scientific) were added and the samples carefully evaporated to dryness on a hot plate. The crucibles were heated two additional hours at 500C. Only one acid treatment was necessary for the completion Of the digestion of both plant materials. The ash was dissolved in 1.0 m1 Of 7.5 NHNO3 (redistilled) and analyzed for Cr using a Varian-AA6 atomic absorption SpectrOphotometer equipped with a CR-90 graphite furnace and background corrector. Chromium was read at 357.9 nm and sample size for analysis was 5 ul using a program cycle consisting of: drying, 40 s at 110C; ashing, 20 s at 1400C; and atomization, 1 s at 2400C. Nitrogen was used as the car- rier gas. Working standards in the range of 10-500 ppb were prepared 40 twice a week in 7.5 N_HNO from 1 mg/ml Cr stock solution. 3 Ethanol soluble Cr-- Ethanol soluble Cr was determined by a modification of the method of Toepfer et al., 1973. Two grams Of plant material were suspended in 30-40 ml Of 50% ethanol in covered plastic containers. The mixture was shaken for 10 minutes in a water bath set at 80C. The extracted sample (after cooling) was centrifuged at 1500 X g for 15 mdnutes. The super- natant was decanted and set aside and the residue washed with 50% etha- nol and centrifuged again. The supernatants were combined and 2.0 ml placed into a 10 ml Coors porcelain crucible and slowly evaporated to dryness on a hot plate. The residue was dry ashed in the same manner as for total Cr. The ash was dissolved in 1.0 m1 Of 7.5 N HNO3 and ana- ‘ lyzed for Cr. Ammonium hydroxide soluble Cr-- The ammonium hydroxide extractable Cr was determined by a modifi- cation Of the method of Kumpulainen et al., 1979a. Two grams Of plant material were suspended in 15 ml of 0.1 N_NH4OH and shaken for 60 man- utes at 30C. The extracted sample was then centrifuged at 1500 X g for 15 minutes and the clear extract stored in plastic bottles at 4C. For Cr analysis Of the extract, 2.0 ml were placed into a 10 ml Coors porcelain crucible and carefully evaporated to dryness on a hot plate. The residue was dry ashed and digested in the same manner as for total Cr and then dissolved in 1.0 ml Of 7.5 N_HNO for Cr analysiS. 3 The validity Of the digestion and extraction procedures was tested by checking for Cr Obtained from analyses Of NBS Orchard Leaves (SRM 1571), NBS Brewer's Yeast (SRM 1569) and NBS Spinach Leaves (SRM 1570). The results Of these determinations are presented in Tables 1-5. The Optimum temperature Settings for the graphite furnace were 41 determined by analyzing for Cr recovered from.NBS Orchard Leaves. The results Of these determinations are presented in Tables 6-11. Table 1. Determination Of total chromium in selected NBS Standard Reference Materials. Sample ' Replicates Total Cr* Ref. Value** ....... ug/g dry wt! NBS Orchard Leaves 10 2.6 - 0.50 2.6 - 0.30 NBS Spinach Leaves 15 4.5 - 0.50 4.6 - 0.30 NBS Brewer's Yeast 8 2.2 - 0.10 2.1 - 0.05 * Values as analyzed. **Certified for\Cr by the National Bureau Of Standards, Washington,-D.C. e Mean * s.d. Table 2. Determination Of chromium in 50% ethanol extracts Of selected NBS Standard Reference Materials. Sample Replicates Extracted Cr* Ref. Value** ng/ml t NBS Orchard Leaves 10 7.5 - 0.90 7.1 - 0.20 NBS Brewer's Yeast 8 3.3 - 0.70 * Values as analyzed. **Knmpulainen et a1. (1979). * Mean 1 Sod. Table 3. Determination Of chromium in 0.1 N_NH OH extracts of selected NBS Standard Reference Materials. 4 Sample Replicates Extracted Cr* Ref. Value** ng/ml ¢ NBS Orchard Leaves 10 14.5 - 1.50 13.9 - 1.20 NBS Brewer's Yeast 8 6.8 - 0.90 6.2 - 0.60 * Values as analyzed. **Kumpulainen et alo (1979). t Mean 1 Sod. 42 Table 4. Percent of total chromium extracted with 50% ethanol from selected NBS Standard Reference Materials. Sample Replicates Extracted Cr* Ref. Value** % NBS Orchard Leaves 10 5.8 5.5 NBS Brewer's Yeast 8 ~-- 3.1 * Values as analyzed. **Kumpulainen et a1. (1979). Table 5. Percent of total chromium extracted with 0.1 N_NH OH from selected NBS Standard Reference Materials. 4 Sample Replicates Extracted Cr* Ref. Value** % NBS Orchard Leaves 10 4.2 4.0 NBS Brewer's Yeast 8 2.3 2.2 * Values as analyzed. **Kumpulainen et a1. (1979). Table 6. Determination Of Optimum drying temperature Of graphite oven using NBS Orchard Leaves. Drying Temperature Total Cr* Certified Value** Recovery C ug/g dry wt. % 90 2.43 2.60 93.4 100 2.46 2.60 94.6 110 2.63 2.60 101.1 120 2.48 2.60 95.4 130 1.51 2.60 58.2 * Values as analyzed. **Prepared and certified for Cr by the National Bureau Of Standards, Washington , D .C. 43 Table 7. Determination Of Optimum drying temperature Of graphite oven using NBS Spinach Leaves. Drying Temperature Total Cr* Certified Value** Recovery C ug/g dry wt. % 100 4.48 4.60 97.4 110 4.59 4.60 99.8 120 4.39 4.60 95.4 130 3.23 4.60 70.2 * Values as analyzed. **Prepared and certified for Cr by the National Bureau Of Standards, Washington, D.C. Table 8. Determination Of Optimum using NBS Orchard Leaves. ashing temperature Of graphite oven Ashing Temperature ' Total Cr* Certified Value** Recovery C ug/g dry wt. % 1200 2.51 2.60 96.5 1300 2.53 2.60 97.3 1400 2.61 2.60 100.4 1500 2.52 2.60 96.9 * Values as analyzed. **Prepared and certified for Cr by the National Bureau of Standards, Washington, D.C. Table 9. Determination of optimum using NBS Spinach Leaves. ashing temperature Of graphite oven Ashing Temperature Total Cr* Certified Value** Recovery C ug/g dry wt. % 1200 4.50 4.60 97.8 1300 4.55 4.60 98.9 1400 4.63 4.60 100.6 1500 4.51 4.60 98.0 * Values as analyzed. **Prepared and certified for Cr by the National Bureau Of Standards, Washington, D.C . 44 Table 10. Determination Of Optimum atomization temperature Of graphite oven using NBS Orchard Leaves. Atomization Temp. Total Cr* Certified Value** ' Recovery C ug/g dry wt. % 2100 2.54 2.60 97.7 2200 2.55 2.60 98.1 2600 2.61 2.60 100.4 2800 2.50 ... . 2.60 96.2 * Values as analyzed. **Prepared and certified for Cr by the National Bureau Of Standards, waShirlgton , D. C 0 Table 11. Determination Of Optimum atomization temperature Of graphite oven using NBS Spinach Leaves. Atomization Temp. Total Cr* Certified Value** Recovery C ug/g dry wt. % 2100 4.53 4.60 98.5 2200 4.52 4.60 98.3 2600 4.61 4.60 100.2 2800 4.51 4.60 98.0 * Values as analyzed. **Prepared and certified for Cr by the National Bureau Of Standards, Washington, D.C. RESULTS AND DISCUSSION Uptake and Translocation Of Cr Wheat-- Figures 1 and 2 demonstrate the existence Of significant differ- ences in the levels of total Cr in various wheat tissues after harvest- ing. Similar to findings reported by Toepfer et al. (1973) and Jones and Buckley (1977), the indigenous levels of Cr in the grain of control plants averaged 0.29 ug/g. Low, medium, and high treatment plants had substantially increased Cr concentrations Of 0.49, 0.52, and 0.55 ug/g, Total Chromium (ppm) 0.7 0.5 0.4 0.2 45 D Cr(EDTA) Source '7‘ Cr2(SO4)3 Source - r __ / / / .. r‘é J. / $0.05 9 ¢ .. ¢ ¢ 2 2 / a r % ¢ ¢ / / / a a - 2 / / % ¢ ¢ ' a % % a Chromium Application Rate (g/ha) Fig. 1--Total chromium in wheat grain as affected by rate and source of chromium in foliar spray solution. Total Chromium (ppm) 3.0 2.0 1.0 46 D Cr ( EDTA) Source Cr2 (804)3 Source L. 4L LSD \\\\\\\\\\\\\\\\\\\\\ MW\\\\\\‘\V \\\\\\\\\\J O 20 O 2000 4000 Chromium Application Rate (g/ha) Fig. 2--Tota1 chromium in wheat straw as affected by rate and source of chromium in foliar spray solution. 47 respectively. These concentrations are almost twice as great as the largest increases achieved by Cary et al. (1977a) in plant uptake stud— ies Of Cr conducted on buckwheat. Total Cr levels also increased in wheat straw from an average of 1.05 ug/g in controls to 2.02, 2.10, and 2.23 ug/g in low, medium, and high treatment plants, respectively. As found with the wheat grain, no significant differences in either the pattern or extent Of uptake and translocation of the element were experienced as a result Of the chemi- cal form Of Cr in the foliar spray solutions. This is contrary to re- sults Obtained by Skeffington et a1. (1976) and Cary et al. (1977a) who indicated that CrEDTA is generally more mobile in plants. But, in both studies Cr was applied to plant roots, not leaves. Since all Spraying was completed prior to heading and since both aerial and soil contamination were prevented, the increased levels Of Cr in both wheat grain and straw can only be attributed to significant translocation Of the element from that absorbed by the leaves. However, most workers have not been able to produce significant translocation Of Cr to the seeds Of plants when working with root uptake of the element (Skeffington et al., 1976; Cary et al., 1977a, 1977b; Lahouti and Peter- son, 1979). Skeffington et a1. (1976) proposed that Cr could not pene- trate plant roots to the vascular tissue to any great extent and could not be translocated longitudinally in the cortex. The authors also point out, however, that although the element enters the vascular system of a plant with difficulty once there it can be transported readily. This finding is exemplified in foliar absorption studies Of Cr by Per- kins et a1. (1960) and Parr and Taylor (1980) who also reported substan- tial absorption and translocation of Cr when applied to plant leaves. If it is assumed that the leaves of plants provide a more accessible 48 route for Cr to enter the vascular system Of plants, then the signifi- cant translocation of the element to other plant tissues can be under- stood. Though no toxicity symptoms were apparent at any level Of Cr appli- cation, absorption and/or translocation Of Cr appears to have leveled Off at the medium rate Of spraying. High treatment plants Sprayed with twice as much Cr had concentrations nearly equal to those sprayed at the medium rate. Since the variable application rates were achieved by the number Of sprayings, this effect can be attributed tO either (1) a de- creased ability tO absorb and/or translocate Cr by the leaves at latter stages Of maturity or (2) other small differences in the physiological status Of the plant with age (Lyon et al., 1969). Spinach-- Figures 3 and 4 indicate the levels Of total Cr in various spinach tissues after harvesting and washing. Both the pattern and degree of uptake of the element in the leaves were affected by the chemical form Of Cr in the foliar Spray solutions. Total Cr levels in spinach leaves were approximately two times greater in Cr2(SO4)3 treated plants than in those treated with CrEDTA. In Cr2(SO4)3 treated plants, total Cr in- creased from a control average Of 0.69 ug/g to 6.9, 9.1, and 11.5 ug/g in low, medium, and high treatment plants, respectively. Chromium le- vels in plants treated with CrEDTA increased from 0.72 ug/g in controls to 3.6, 4.7, and 6.9 ug/g in low, medium, and high treatment plants, re- spectively. However, total Cr levels in spinach stems Of low, medium, and high treatment plants, though significantly greater than in controls did not differ with respect to the form Of Cr in the foliar spray solu- tions. Although the levels of Cr in Spinach leaves are slightly greater Total Chromium (ppm) . 3.0!- 49 [j Cr (EDTA) Source Cr2(SO4)3 Source 12.0 .- 11.0 - 10.0 - I 05 2.0 r.- 1.0 '- 1 \\\\\\\\\\\\\\\\ l \X\\\\\\\\\\\\\\\\\\\\ m 0 200 4000 N O O O Chromium Application Rate (g/ha) Fig. 3--Total chromium in spinach leaves as affected by rate and source Of chromium in foliar spray solution. Total Chromium (ppm) 0.30 0.25 0.20 0.15 0.10 0.05 50 D Cr(EDTA) Source a Cr2 ( 804 ) 3 Source 7 b\\\\\\\\\\\\\X\ I .- LSD \\\X\\\\\\“\\\\\ 8 \\\\\\\\\\\\\\\\\\ 2000 4000 O N Chromium Application Rate (g/ha) Fig. 4-- Total chromium in spinach stems as affected by rate and source of chromium in foliar Spray solution. 51 greater than those Obtained by Cary et al. (1977a) in root uptake stud— ies, these results must be viewed with caution. NO tests were conducted tO determine the location Of the Cr in the spinach tissues after washing and it is suspected that a significant portion Of this Cr may be attri- buted to remnants Of surface contamination from foliar spraying. Extraction Of Cr in Wheat Toepfer et a1. (1973) reported that some Of the Cr in wheat grain may be present as GTF and the level Of this Cr soluble in 50% ethanol correlates positively with GTF activity. Also, Anderson et a1. (1978) studied methods for the extraction Of labelled biologically active Cr (GTF) in brewer's yeast and determined that the release Of labelled Cr (GTF) from the yeast cells was pH dependent and more than 85% Of this Cr could be extracted with 0.1 N NH4OH. Using both Of these extract- ants, researchers have been able to assess the GTF value Of various foods by expressing the Cr extracted by either ethanol or NH4OH as a percentage Of the total Cr. Figures 5 and 6 show the percentages Of total Cr extracted by either 0.1 N_NH OH or 50% ethanol from wheat grain. The percent Of to- 4 tal Cr extractable with NH OH was consistently greater than with ethanol 4 at all treatment levels. In general terms, the values Of total or etha- nol extractable Cr in wheat grain controls were Of a similar order to those quoted by Cary and Allaway (1971), Toepfer et a1. (1973), Wolf et a1. (1974), Cary and Olson (1975) and Welch and Cary (1975).. Ethanol extractable Cr increased from an average of 38.2% in controls to 55.7, 53.6, and 48.9% in low, medium, and high treatment plants, reSpectively. Ammonium hydroxide extractable Cr increased from a control average Of 43.7% tO 65.9, 63.6, and 57.1% in low, medium, and high treatment plants, respectively. Though nO significant differences in either the pattern Percent of Total Chromium 52 [:1 Cr(EDTA) Source Cr2 ($04!)3 Source 5. '05 _/ _, _. / 2 4. .. a 2 Z 3. .. / / / / / ¢ / / .0 - 4 / /- / % f 2 4 10 — //t l / / Chromium Application Rate (g/ha) Fig. 5--Ethanol extractable chromium in wheat grain as affected by rate and source Of chromium in foliar Spray solution. Percent of Total Chromium 100 90 8O 70 60 50' 4O 3O 20 10 53 D Cr (EDTA) Source a Cr2 ($04) 3 Source 7 .. / a / L. 1 1,313.05 _¢ F“; ”'7’ - / ¢ p / _ 7 / / é / / - / / / / / / - / / ¢ / § / ' Q /, 4 Chromium Application Rate (g/ha) Fig. 6--Ammonium hydroxide extractable chromium in wheat grain as affected by rate and source Of chromium in foliar spray. 54 or degree Of extractability Of the element occurred as a result Of the form of Cr in the foliar Spray solutions, both ethanol and NH4OH ex- tractable Cr levels were slightly higher for plants Sprayed with the sulfate source, for most concentrations. These apparent increases in GTF-Cr cannot be explained from any previous research. However, it has been postulated that GTF-Cr may be produced by rapidly dividing cells. In recognition Of the substantial increases in plant growth attributable to Cr, the increases in GTF-Cr may have resulted from increased metabolic activity. Affects on Plant Growth Figures 7 and 8 demonstrate the effects Of foliar application Of Cr solutions on wheat growth. Total wheat grain yield increased a maximum of 59% in the medium treatment plants, as compared to controls. Over- all, wheat grain yield rose from an average Of 16.1 g/pot in controls to 19.8, 25.7, and 21.5 g/pot in the low, medium, and high treatment plants, respectively. In a similar fashion, total dry matter yield Of wheat in- creased substantially from an average Of 100 g/pot in controls to 116.2, 136.3, and 133.0 g/pot in low, medium, and high treatment plants, re- spectively. In both wheat grain and total dry matter, no significant differences in yield were experienced as a result Of the form Of Cr in the spray solutions even though plants sprayed with Cr2(SO4)3 generally produced greater yield. Figure 9 demonstrates the enhancement of maturity in wheat growth produced by the foliar application Of either Cr solution. Virtually all phases Of wheat growth were significantly accelerated in Cr treated plants. Since control plants were Sprayed with solutions Of either NaEDTA or Na 804, these increases can only be attributed tO the Cr ap- 2 plications. Dry Weight Of Grain (g/pot) 30 25 20 15 10 55 E] Cr(EDTA) Source a Cr2 (804 ) 3 Source / ., é - / / 4 .. » LSD.05 / / 7 —/ / / / / / r- / / / )- / fl / W / / / / _ / / / / / / / / / / / / / / / ~ / / / a a a Chromium Application Rate (g/ha) Fig. 7--Wheat grain yield as affected by rate and source of chromium in foliar Spray solution. Total Dry Matter Yield (g/pot) 150 100 50 56 D Cr(EDTA) Source P Eza Cr2(SO4)3 Source ~b LSD 05 \\\\\\\\\\\\\\\\\\\\\\\\\\\ K\\\\\\\\\\\\\\\\\\\\\\\\H \\Y\\\\\\\\\\\\\\\\ 8 \\\\\\\\\\\\\\\\\\\\\\\\ O N 2000 :35 O O O Chromium.Application Rate (g/ha) Fig. 8--Total dry matter yield of wheat plants as affected by rate and source of chromium in foliar spray solution. Percent Of total heads harvested reaching maturity 57 Control Low Rate Medium Rate High Rate .9310 I I I O 4 8 12 16 20 24 28 32 36 40 Days Fig. 9--Maturity Of wheat as affected by rate Of chromium in foliar Spray solution. 58 Results both consistent and contrary to these findings have been reported by several researchers. However, only few studies have in- vestigated the effects Of foliar sprays on plant growth. Trivalent Cr as a foliar Spray was found by Dobrolyubskii (1959), Shcheglov and Baev (1973), and Dobrolyubskii and Viktorova (1974) to increase the yield and enhance the maturation Of grapes. In explaining these results, the au- thors concluded that the growth effects were due to an active participa- tion Of Cr3+ in different oxidation-reduction processes which occur in plants. This in turn was speculated to cause favorable changes in the activity Of certain enzyme systems (i.e. the photosynthetic process). Also, the element was believed to cause an increase in the production and retention of chlorophyll in plant leaves. Other studies have spec? ulated on the reasons for Cr's affects on plant growth, but without additional research on the biochemical changes accompanying yield in- creases from foliar applied Cr, the conclusions presented by Dobrolyub- skii and Viktorova (1974) remain the only reasonable explanation for Cr's Observed effects. CONCLUSIONS The primary Objective Of this study was tO provide a basis for de- signing crOp management practices that might increase the Cr concentra- tion in food and feed crops. Results Obtained with both plant species clearly indicate that both inorganic and GTF-Cr can be increased in cer- tain plant tissues to levels that may represent significant increases in dietary intake if foliar application methods are employed. The nutritional implications of these increases are several in num- ber. First, Mertz (1979) indicated that Cr needs can be satisfied Sims ply by meeting a total Cr intake. These findings point out that foliar application Of Cr to certain plants can significantly increase the 59 concentration Of the element in the human diet without impaired plant growth. Second, ethanol and NH4OH extractions indicate that GTF-related Cr substantially increased in the wheat grain. From a biological stand- point, GTF-Cr is superior to inorganic Cr and the human requirement for Cr is significantly less if GTF—Cr is used (Liu and Morris, 1978; Mertz, 1979). The benefits Of consuming grains with a higher concentration Of biologically effective Cr are Obvious but also, since food refinement leads to a loss Of the element in the human diet, the form of Cr which remains becomes more important. Since the percentage Of total Cr re- lated to GTF activity increased in the wheat grain, the amount remaining after processing would be vastly superior to inorganic Cr. Thirdly, since both types Of extractions indicated increases in GTF related Cr,' it can be assumed that the GTF content of foods, or at least certain plants, can be manipulated anthropogenically. This would imply that other methods Of crop management, less involved than foliar spraying, might be employed tO alter Cr quantity and quality in plants. 60 LIST OF REFERENCES Anderson, R.A., M.M. Polansky, E.E. Roginski, and W. Mertz. 1978. Fac- tors affecting the retention and extraction Of yeast chromium. J. Agric. Food Chem. 26:858—861. Cary, E.E., and W.H. Allaway. 1971. Determination Of chromium in plants and other biological materials. J. Agric. Food Chem. 19:1159-1161. Cary, E.E., W.H. Allaway, and O.E. Olson. 1977a. Control Of chromium concentrations in food plants: I. Absorption and translocation Of chromium by plants. J. Agric..FOOd Chem. 25:300-304. Cary, E.E., W.H. Allaway, and O.E. Olson. 1977b. Control Of chromium concentrations in food plants: II. Chemistry Of chromium in soils and its availability to plants. J. Agric. Food Chem. 25:305-309. Cary, E.E., and O.E. Olson. 1975. Atomic absorption spectrophotometric determination Of chromium in plantS. J. Assoc. Off. Anal. Chem. 58:433—435. Czerniejewski, C.P., C.W. Shank, W.G. Bechtel, and W.B. Bradley. 1964. The minerals Of wheat flour, and bread. Cereal Chem. 41:65—72. Dobrolyubskii, O.K. 1959. The effect Of chromium trace fertilizer on the biochemical processes Of the grape. Biochemistry (USSR) (Eng— lish Transl.) 24:577-582. Dobrolyubskii, O.K., and G.M. Viktorova. 1974. Effect of chromium on the chemical composition Of grapevines. Agrokhimiya 10:135-140. (cf. Chem. Abstr. 82:42325h). DOisy, R.J., D.H.P. Streeten, J.M. Freiberg, and A.J. Schneider. 1976. Chromium metabolism and biochemical effects. pp. 79-104. IQ_A.S. Prasad and D. Oberleas (eds.). Trace elements in human health and disease: Vol. II. Essential and toxic elements. Academic Press, New York. Huffman Jr., E.W.D., and W.H. Allaway. 1973. Chromium in plants: Dis- tribution in tissues, organelles, and extracts and availability Of bean leaf Cr to animals. J. Agric. Food Chem. 21:982-986. Jones, G.B., and R.A. Buckley. 1977. Levels Of chromium in wheats and some other animal feedstuffs in Australia. J. Sci. Food Agric. 28 3265-2 68 o Kumpulainen, J.T., R.A. Anderson, M.M. Polansky, and W.R. Wolf. 1979a. Chromium content Of biologically active extracts Of standard refer- ence materials. pp. 79-84. In_D. Shapcott and J. Hubert (eds.). Developments in nutrition and metabolism. Elsevier/North Holland Biomedical Press, Amsterdam. 61 Kumpulainen, J.T., W.R. Wolf, C. Veillon, and W. Mertz. 1979b. Deter- mination Of chromium in selected United States diets. J. Agric. Food Chem. 27:490-494. Lahouti, M., and P.J. Peterson. 1979. Chromium accumulation and dis- tribution in crop plants. J. Sci. Food Agric. 30:136-142. Liu, V.J.K., and J.S. Morris. 1978. Relative chromium response as an indicator Of chromium status. Amer. J. Clin. Nutr. 31:972-976. Lyon, G.L., P.J. Peterson, and R.R. Brooks. 1969. Chromium-51 distri- bution in tissues and extracts of Leptospermum scoparium. Planta 88:282-287. Mertz, W. 1979. ChromiumpAn overview. pp. 1-14. lp_D. Shapcott and J. Hubert (eds.). DevelOpments in nutrition and metabolism: Vol. II. Chromium in nutrition and metabolism. Elsevier/North Holland Biomedical Press, Amsterdam. Newman, H.A., R.F. Leighton, R.R. Lanese, and N.A. Freeland. 1978. Se- rum Cr and angiographic determined coronary artery disease. Clin. Chem. 24(4):541-544. Parr, P.D., and F.G. Taylor, Jr. 1980. Incorporation of chromium in vegetation through root uptake and foliar absorption pathways. Environ. Exp. Bot. 20:157-160. Perkins, R.W., J.M. Nielsen, W.C. Roesch, and R.C. McCall. 1960. zinc- 65 and chromiumPSl in foods and people. Science 132:1895-1897. Punsar, S., O. Eramtsa, M.J. Karvonen, A. Rytranen, P. Hilska, and H. Vornamo. 1975. Coronary heart disease and dringking water. J. Chronic Dis. 28:259-287. Schroeder, H.A. 1968. The role Of chromium in mammalian nutrition. Amer. J. Clin. Nutr. 21:230-244. Schroeder, H.A. 1971. Losses Of vitamins and trace minerals resulting from processing and preservation Of foods. Amer. J. Clin. Nutr. 24:562-573. Schroeder, H.A., J.J. Balassa, and I.H. Tipton. 1962. Abnormal trace metals in man: Chromium. J. Chronic Dis. 15:941-964. Schroeder, H.A., A.P. Nason, and I.H. Tipton. 1970. Chromium deficien- cy as a factor in atherosclerosis. J. Chronic Dis. 23:123-142. Shapcott, 1979. The detection of chromium deficiency. pp. 113-127. In D. Shapcott and J. Hubert (eds.). DevelOpments in nutrition and metabolism: Vol. II. Chromium in nutrition and metabolism. El- sevier/North-Holland Biomedical Press, Amsterdam. 62 Shcheglov, A.T., and A.I. Baev. 1973. Effect Of leaf feeding Of grapes with chromium preparations on the yield and quality Of the berries. Tr. Stovrop. Skh. Inst. 3:99-100. (cf. Chem. Abstr. 82:3244m). Skeffington, R.A., P.R. Shewry, and P.J. Peterson. 1976. Chromium up- take and tranSport in barley seedlings. Planta .132:209-214. Toepfer, E.W., W. Mertz, E.E. Roginski, and M.M. Polansky. 1973. Chromium in foods in relation to biological activity. J. Agric. Food Chem. 21:69-73. Welch, R.M., and E.E. Cary. 1975. Concentrations Of chromium, nickel, and vanadium in plant materials. J. Agric. Food Chem. 23:479-482. Wolf, W., W. Mertz, and R. Masironi. 1974. Determination Of chromium in refined and unrefined sugars by oxygen plasma ashing flameless atomic absorption. J. Agric. Food Chem. 22:1037-1042. CHAPTER III SUMMARY AND CONCLUSIONS In a brief review Of the results of this study, the following gen- eral conclusions concerning the effects of Cr applied to plants by foliar spraying were drawn: (1) The levels of total and biologically active Cr can be signi- ficantly increased in wheat seeds through foliar Spraying of the ele- ment. Since wheat products are extensively used as food for animal and human consumption, these increases could represent important increases in dietary intake of the element. Though foliar spraying of Cr may be prohibitively expensive on a large scale, crop management practices could be designed to produce smaller quantities Of high Cr wheat for use as valuable components Of blended foods and animal feeds. This could indirectly increase the levels of Cr in the food chain and ultimately in man. Considering the morbidity of diseases caused by Cr deficien- cies, the economics and viability of foliar Spraying needs to be further investigated. Dietary supplements Of both inorganic and organic Cr are currently available, but do to cost and lack of appeal cannot be as sig- nificant in reducing the incidence of Cr deficiencies as an increase in food Cr. (2) Foliar applied Cr translocates to the seeds of wheat plantS. This translocation is substantial but cannot be explained from previous findings. 63 64 (3) Both types of Cr compounds (Cr2(SO4)3 and CrEDTA) behave simi- larly in wheat plants when sprayed on leaveS. (4) Biologically active Cr concentrations (as measured by ethanol and NH4OH extraction) can be anthropogenically manipulated in wheat grain. This finding represents the most important result of this study since it implies (for the first time) that the levels of GTF related Cr are not an unalterable characteristic of plants. As a result, other methods of crop management, perhaps not as involved or expensive as fo- liar spraying, might be undertaken to increase the levels of GTF-Cr in plants. In addition, this finding Opens up other important areas of research (i.e. studies of the differences between plant species and va- rieties to accumulate or produce GTF-Cr) not previously considered worthwhile. (5) Wheat growth appears to be increased and maturity enhanced by the foliar application of low levels of Cr salt solutions. Due to the inconsistency of the reported effects of Cr on plants, however, the following steps should be employed to test the validity of the reported yield increases Of this study: (a) Further studies should be conducted (using the medium rate of Cr application) to test if Cr's effects can be repeated in field conditions; (b) various physiological processes in plants (and their respective enzyme systems) should be investigated if further growth changes are Observed; and (0) tests should be performed to determine if Cr's effects can be substituted with other elements or compounds of similar nature. The benefits of even substantially smaller increases in crop yields from Cr necessitate further investigation of the element on crop growth. LIST OF REFERENCES LIST OF REFERENCES Allaway, W.H. 1968. Agronomic controls over the environmental cycling Of trace elements. Adv. Agron. 20:235-274. Anderson, A.J., D.R. Meyer, and F.K. Mayer. 1973. 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Determination of chromium in refined and unrefined sugars by oxygen plasma ashing flameless atomic absorption. J. Agric. Food Chem. 22:1037-1042. APPENDIX 76 APPENDIX Table 12. Analysis of variance of wheat grain yield. Source gf SS MS F(calc.) F(tabular) Replicates 2 197.60 98.80 7.06* 4.86* Cr-Source 1 12.80 12.80 0.91 6.30 Cr-Trmt. 3 259.94 86.65 6.19* 4.24 Cr-Source X Treatment 3 68.41 22.80 1.95 4.24 Error 14 163.65 11.69 Table 13. Analysis of variance of wheat growth(total dry matter yield). Source g; 53 MS F(calc.) F(tabular) Replicates 2 3786.63 1893.32 6.62* 4.86 Cr-Source 1 277.92 277.92 0.97 6.30 Cr-Trmt. 3 5032.78 1677.59 5.87* 4.24 Cr-Source X Treatment 3 872.28 290.76 1.02 4.24 Error 14 4001.38 285.81 Table 14. Analysis of variance of total chromium in wheat grain. Source g; 53 MS 45(ca1c.) F(tabular) Replicates 2 0.012 0.006 0.50 4.86 Cr-Source 1 0.010 0.010 0.83 6.30 Cr-Trmt. 3 0.229 0.076 6.33* 4.24 Cr-Source X Treatment 3 0.097 0.032 2.66 4.24 Error 14 0.161 0.012 Table 15. Analysis of variance of ethanol soluble chromium in grain. Source g; 88 MS , F(calc.) F(tabular) Replicates 2 224.53 112.27 2.39 4.86 Cr-Source 1 36.75 36.75 0.79 6.30 Cr-Trmt. 3 1091.57 363.85 7.78* 4.24 Cr-Source X Treatment 3 316.82 105.61 2.26 4.24 Error 14 655.04 46.79 77 Table 16. Analysis of variance of NH OH soluble chromium in wheat grain. 4 Source gg 88 MS F(calc.) F(tabular) Replicates 2 44.31 22.10 0.28 4.86 Cr-Source 1 225.09 225.09 2.86 6.30 Cr-Trmt. 3 1799.08 599.70 7.63* 4.24 Cr-Source X Treatment 3 568.03 189.31 2.40 4.24 Error 14 1100.42 78.60 Table 17. Analysis of variance of total chromium in spinach leaveS. Source g; SS MS F(calc.) F(tabular) Replicates 2 0.623 0.312 0.99 4.86 Cr—Source 1 56.108 56.108 177.56* 6.30 Cr-Trmt. 3 233.683 77.894 246.50* 4.24 Cr-Source X Treatment 3 20.642 6.881 21.78* 4.24 Error 14 4.426 0.316