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Food and Beverage Chemistry/Biochemistry

Bioavailability and Bioactivity of Selenium from Wheat (Triticum aestivum), Maize (Zea mays), and Pearl Millet (Pennisetum glaucum), in Selenium Deficient Rats Anjum Khanam, and Kalpana Platel J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b02614 • Publication Date (Web): 14 May 2019 Downloaded from http://pubs.acs.org on May 14, 2019

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Journal of Agricultural and Food Chemistry

Bioavailability and Bioactivity of Selenium from Wheat (Triticum aestivum), Maize (Zea mays), and Pearl Millet (Pennisetum glaucum), in Selenium Deficient Rats

Anjum Khanam and Kalpana Platel* Department of Biochemistry CSIR - Central Food Technological Research Institute, Mysore – 570020, India.

* Corresponding author Dr. Kalpana Platel Tel.: +91 821 2598005 Mob. +91 9845567705 E-mail address: [email protected]

1 ACS Paragon Plus Environment


1

ABSTRACT:

2

This study examined the bioavailability and bioactivity of selenium (Se) from staple cereals -

3

wheat, pearl millet and maize, in Se deficient rats (Wistar strain (OUT-Wister, IND-cft (2c)).

4

The bioavailability and bioactivity of Se were determined by measuring the Se contents of the

5

tissue and organs, and activities of Se-dependent enzymes. Se deficient rats were repleted with

6

Se through wheat, pearl millet and maize. Wheat diet exhibited the highest bioavailability of Se,

7

followed by pearl millet and maize. The bioactivity of Se, as indicated by the activity of the Se-

8

dependent enzymes, was found to be significantly (p < 0.001) higher in the organs of rats fed the

9

wheat diet, followed by pearl millet and maize diets. Deficiency of Se resulted in a significant

10

decrease (p < 0.001) in the activity of antioxidant enzymes in circulation and organs. The staples

11

wheat, pearl millet and maize have a high bioavailability of Se.

12 13

KEY WORDS: selenium, cereals, glutathione peroxidase, thioredoxin reductase, bioavailability

14 15 16 17 18 19 20 21


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22

INTRODUCTION

23

Selenium (Se) is an essential trace element which is incorporated into selenoproteins, and is

24

recognized to be beneficial for health, in view of its antioxidant activity1. Deficiency of Se

25

increases the susceptibility of tissues to oxidative stress, with general impairment of the immune

26

system, and also leads to changes in the activity of selenoenzymes - glutathione peroxidase

27

(GPX1, GPX3) and thioredoxin reductase (TR1)2.

28

Increased oxidative stress may be involved in the pathogenesis of many chronic diseases, and

29

there is a link between diet and oxidative stress, since the human body derives its main

30

antioxidant defenses from essential nutrients. Se, being an essential trace element, has to be

31

supplied daily through the diet. Dietary supplementation with Se is associated with many

32

potential health benefits for humans3. It incorporates the amino acid selenocysteine into the

33

active site of selenoproteins by co-translation which has vital enzymatic functions4.The major

34

endogenous antioxidants that counteract reactive oxygen species are superoxide dismutase,

35

glutathione peroxidase, glutathione, thioredoxin peroxidase, thioredoxin and catalase5.

36

GPX1, GPX3 and TR1 are the Se dependent enzymes which play a major role in shuffling

37

electrons from NADPH to glutathione and thioredoxin respectively, thereby maintaining the

38

activity of these important antioxidant enzyme systems. GPX1, GPX3 and TR1 contain an amino

39

acid selenocysteine which is essential for the catalytic activity which serves a number of

40

physiological processes, including spermatogenesis and brain development6, 7.TR1 is a

41

selenoprotein that protects against oxidative injury, regulates the thiol redox status by reducing

42

thioredoxin, which in turn reduces protein disulfides.

43

Cereals contribute a major portion of dietary Se, especially in developing countries. Among the

44

cereals, maize, pearl millet and wheat8 have been reported to have the highest Se content9.



45

The bioaccessibility of Se, as determined by us earlier using an in vitro simulated gastrointestinal

46

digestion procedure, was found to be independent of the total content of this trace mineral9.

47

Maize, with the highest total Se content, had the least bioaccessible Se, while the same was

48

highest in wheat, followed by pearl millet. These in vitro findings on the bioaccessibility of Se

49

were validated in the present investigation, using the rat model. The bioavailability of Se in

50

wheat, pearl millet and maize was measured by estimating Se in the tissues and organs, as the

51

absorbed Se reaches the systemic circulation in order to be distributed to organs. The absorbed

52

Se eventually becomes bioactive by the conversion of Se to biologically active selenometablites

53

which is measured in selenoproteins such as GPX1, GPX3 and TR1, etc., and also in antioxidant

54

enzymes (catalase and superoxide dismutase)10. Thus, activity of these selenoproteins and

55

antioxidant enzymes in circulation and various organs and tissues is indicative of the bioactivity

56

of Se. Activities of these enzymes were determined in plasma, erythrocytes, liver, kidney, etc.

57

This study also reports the deposition of organic forms of Se, selenomethionine (SeMet) and

58

selenocysteine (SeCys2) in the two major organs such as liver and kidney of rats. The rat model

59

is often used to study the bioavailability of minerals since the digestive system of rats closely

60

represents human digestive system, and results from such studies are extrapolated to humans to a

61

large extent.

62 63

MATERIALS AND METHODS

64

Materials:

65

Wheat (Triticum aestivum), maize (Zea mays), and pearl millet (Pennisetum glaucum), were

66

procured from the National Seeds Corporation, Mysore, Karnataka. Se standard solution and

67

germanium dioxide were from SRL Chemicals Pvt Ltd; Standard seleno-DL-methionine, 99%


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68

(SeMet) and seleno-DL-cystine, 95% (SeCys2), bovine serum albumin, glutathione reductase,

69

glutathione reduced, glutathione oxidised, tert-butyl hydroxyperoxide, xanthine oxidase,

70

xanthine, cytochrome-C, 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB), Ethylene diamine tetra

71

acetic acid (EDTA), and other fine chemicals were obtained from Sigma–Aldrich Chemical Co.

72

(St. Louis, MO, USA). Nicotinamide adenine dinucleotide phosphate-reduced form (NADPH),

73

Bernhardt–Tommarelli modified salt mixture was procured from SISCO Research Laboratories

74

(Mumbai, India). Nitric acid (69%) and hydrogen peroxide (30%) were procured from Himedia.

75

Methanol (HPLC grade) and formic acid (98–100%) were from Merck (Poole, UK). Casein was

76

purchased from Nimesh Corporation (Mumbai, India). Corn starch and cane sugar powder were

77

purchased from the local market. All other chemicals and solvents were of analytical grade. Milli

78

Q water and acid washed glassware were used throughout the study.

79 80

Animals and diets.

81

Male weanling rats (3 weeks old) of the Wistar strain (OUT-Wister, IND-cft (2c)), obtained

82

from the Institute Experimental Animal Facility were selected for the study which had approval

83

from Institutional Animal Ethics Committee (Approval number-IAEC No.395/15). The

84

Committee is registered under the “Committee for the Purpose of Control and Supervision of

85

Experiments on Animals” (CPCSEA Reg. No: 49/99/CPCSEA). A total of 84 rats were used.

86

They were housed in individual stainless steel cages with stainless steel slotted floor under

87

strictly controlled conditions of temperature (20-28 ºC) and humidity (60-70%); with a 12h dark–

88

light cycle; food and distilled water were provided ad libitum. Initially, these rats were divided

89

into two groups. One group of rats was fed with AIN-76 semi-synthetic diet with a Se content of

90

0.16 mg/kg. The second group of rats was rendered Se-deficient by maintaining them on the



91

AIN-76 semi synthetic diet made deficient in Se by using a mineral mix that did not contain Se.

92

The Se content of the deficient diet was 0.04 mg/kg. Rats were maintained on these diets for a

93

period of six weeks. The microbiological and health status of the animals was regularly

94

monitored by a qualified veterinarian. No adverse events were encountered throughout the period

95

of the study.

96

After a period of 6 weeks, 6 animals from each group were sacrificed by euthanasia by

97

exsanguination from the heart. Blood was collected in heparinized tubes and plasma and

98

erythrocytes were isolated on the same day. Liver, kidney, heart, pancreas, spleen, muscle and

99

brain was quickly excised, washed in 0.9% saline, weighed, and immediately frozen at – 80

100

oC

101

erythrocytes and organs. The activities of enzymes, such as glutathione peroxidase, catalase

102

and superoxide dismutase were estimated in plasma, erythrocyte, liver, kidney, heart, and

103

muscle, where as thioredoxin reductase activity was estimated in liver and kidney. After the

104

confirmation of Se deficiency, the rats were further divided into 4 experimental groups (with

105

12 animals in each group). Three groups were fed with diet prepared based on wheat, pearl

106

millet, and maize, as a Se source respectively, and the fourth group was fed with the normal

107

AIN-76 semi-synthetic diet that contained sodium selenite as the source of Se. The normal

108

rats and one group of Se-deficient rats were continued on their respective Se-sufficient and

109

deficient diets. Se content in wheat based, pearl millet based and maize based experimental

110

diet was 0.16, 0.19, 0.21 mg/kg respectively. The Se content of the normal diet was 0.16

111

mg/kg. Animals were repleted with Se for a period of 4 weeks and were sacrificed by

112

euthanasia by exsanguination from the heart, at an interval of 2 weeks. Blood and organs were

113

harvested, organs were weighed; and body weight of the rats was monitored weekly.

till the analysis. Deficiency of Se was confirmed by analysing Se concentration in plasma,


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114 115

Diet preparation:

116

(i) Normal and Se deficient diet: Normal and deficient diets were prepared based on AIN-76

117

formulations. Se sufficient mineral mix with 0.01g/kg sodium selenite was used for normal

118

diet, whereas for deficient diet sodium selenite was excluded from the mineral mix (Se

119

deficient mineral mix given in Table 1).The diets were stored at – 4 ºC in air tight containers.

120

(ii) Experimental

diet: Experimental diet was prepared by modifying AIN-76 diet, wherein a

121

major part of the corn starch was replaced by the respective cereal flours. The experimental

122

diet contained Cereal flour (wheat, pearl millet, maize) (50%), corn starch (10%), casein

123

(15%), fat (10%), vitamin mix (1%), mineral mix (4%), and sugar (10%). Thus, the source of

124

Se during the repletion period was sodium selenite (present in the mineral mix added to AIN-

125

76 diet), for the control rats, and Se derived from wheat, pearl millet and maize for the

126

experimental rats. The former represents the inorganic form of Se, while the latter three

127

represent the organic form.

128

Analytical procedures:

129

Blood withdrawn by cardiac puncture was collected in heparinized tubes and centrifuged at 2500

130

rpm for 10 min at 4 oC. Plasma and erythrocytes were separated. The frozen tissues were

131

homogenized in 0.1mol/L sodium phosphate buffer (pH 7) and the homogenate was centrifuged

132

at 10,000 rpm for 30 min at 4 oC and the supernatant was collected and stored at – 80 ºC for

133

subsequent use.

134

Preparation of erythrocytes:



135

The erythrocytes obtained were washed thrice sequentially in 5mM phosphate buffer solution

136

(pH 8), 2.5 and 1.25 mM phosphate buffer (pH 8) respectively, and were hemolysed with

137

hypotonic buffer (5mM sodium phosphate buffer pH 8). The hemolyzate was centrifuged at

138

15000 rpm at 4 oC. The supernatant was collected and stored at -20 oC until use11.

139 140

Hemoglobin estimation

141

Hemoglobin content in erythrocytes was determined by using kits from Agape Pvt. Ltd. India.

142

Following the principle according to Dacie and Lewis12, by measuring the cyanomethemoglobin

143

formed by treating heamoglobin with cyanide potassium ferricyanide (Drabkins reagent) at 540

144

nm.

145

Determination of protein:

146

Protein concentrations of the tissue homogenates were determined by the method of Lowry et al.

147

13

using bovine serum albumin as the standard.

148 149

Enzyme assay:

150

Se dependent enzymes such as GPX 1, GPX 3 and TR1 were measured in tissues and organs.

151

GPX 1, GPX 3 was estimated in plasma, erythrocyte, liver, kidney, heart, and muscle.TR1 was

152

determined in liver and kidney. Catalase (CAT) and superoxide dismutase (SOD) the major

153

antioxidant enzymes were also measured in plasma, erythrocyte, liver, kidney, heart and muscle.

154 155

GPX1, GPX3 activity:


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156

Activities of GPX1, GPX3 in plasma, erythrocyte and homogenate of liver, kidney, heart, and

157

muscle were determined using NADPH oxidation in a coupled reduction System of hydrogen

158

peroxide and oxidized glutathione as described by Flohe and Gunzler14.

159 160

TR1 activity:

161

TR1 activity was measured in liver and kidney homogenate15, 16 . The reduction of DTNB was

162

monitored at 412nm in a potassium phosphate buffer (0.1M, pH 7, containing 10 mM EDTA and

163

0.2 mM NADPH). The activity was expressed as nmol substrate reduced/min/mg protein.

164 165

Activities of CAT and SOD:

166

The supernatant of the homogenate was used for enzyme assay. CAT activity was measured by

167

the rate of decrease in hydrogen peroxide at an absorbance at 240 nm for 3 min17. The enzyme

168

activity was expressed as the amount of enzyme that decomposes 1 μM hydrogen peroxide per

169

mg of protein. SOD activity was assayed by quantitating the inhibition of cytochrome-C

170

reduction in the xanthine–xanthine oxidase system as described by Flohe and Otting18.

171 172

Se analysis:

173

Plasma and erythrocyte (100 µL) and sections of various organs such as liver, kidney, heart,

174

spleen, pancreas, and brain, were weighed (500 mg) and subjected to acid digestion in the

175

presence of hydrogen peroxide and nitric acid9. Se was determined by Inductively Coupled

176

Plasma - Atomic Emission Spectroscopy (ICP-AES), as described by us earlier9.

177 178

Selenomethionine (SeMet) and selenocysteine (SeCys2 ) determination by HPLC-MS:



179

SeMet and SeCys2 were determined in liver and kidney by chromatographic separations9. Liver

180

and kidney homogenates (100 µL) were freeze dried and the dried homogenate sample was

181

dissolved in a mixture of 1.0% (v/v) formic acid solution and 10% (v/v) methanol, and the

182

volume was made up to 100 µL19. 20 µL of the sample was injected.

183 184

Statistical analysis:

185

The values are expressed as means with their standard errors of six rats. Statistical analysis of the

186

results was done using Prism 6.0; Graph-Pad Software (San Diego, CA, USA). Results were

187

analysed and the significance between the groups was determined by performing one-way

188

ANOVA with Tukey–Kramer multiple comparison test. Differences between the control and the

189

experimental group for all the parameter were analysed by using student’s t test and the

190

difference was considered statistically significant when P ≤ 0·05.

191 192

RESULTS

193

Organ weights:

194

The body weight and weight of the organs determined is given in the Table 2. The experimental

195

rats did not show any significant differences among the rats fed with the normal and Se deficient

196

diets, during the initial 6-week dietary regimen. All the groups of rats gained weight by the end

197

of the 2-week and 4-week regimen of repletion with Se, while there was no significant change in

198

organ weights. The final body weight of rats supplemented with Se for 2 and 4 weeks ranged

199

from 239 ± 4.0g to 247 ± 6.0g and 276 ± 8.0g to 300 ± 5.0g, n=6 respectively, for the deficient

200

control and experimental rats. Thus, growth was not affected by dietary Se level.

201

Se content in plasma, erythrocytes and various organs:


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202

Se concentration in tissues and organs of the rats studied is given in the Table 3. Brain contained

203

the highest amount of Se, while the lowest amount was present in pancreas. As expected, there

204

was a highly significant decrease (p < 0.001) in the Se concentration in all the organs of the rats

205

rendered Se-deficient. The highest percent decrease in Se was observed in plasma (~90%) and

206

erythrocyte (86%), followed by heart, brain, kidney, spleen, liver, muscle and pancreas.

207

Repletion of the Se in rats with all the four sources, viz., wheat, maize, pearl millet, and

208

sodium selenite, significantly improved the Se concentration of all the organs studied (p
0.05 – ns (not significant), aP < 0.001, bP < 0.01, cP < 0.05, significantly different from

normal group



Table 4.Retention of SeMet and SeCys2 in Liver and Kidney SeMet(ng/g) Liver Kidney Depletion period Normal 3.84 ± 0.09 Deficient 2.98 ± 0.1ns ED(Wheat) ED(PM) ED(Maize) ED(CN) Repletion of Se for 2 week Normal 7.26 ± 0.4 Deficient 1.05 ± 0.1x ED(Wheat) 7.58 ± 0.5a(ns) ED(PM) 6.64 ± 0.29a ns ED(Maize) 6.42 ± 0.5a ns ED(CN) 4.58 ± 0.3c ns Repletion of Se for 4 week Normal 8.53 ± 0.5 Deficient 1.54 ± 0.1x ED(Wheat) 9.6 ± 0.5a ns ED(PM) 7.45 ± 0.6b ns ED(Maize) 6.35 ± 0.19b ns ED(CN) 4.55 ± 0.2cz

Secys2 (ng/g) Liver Kidney

4.79 ± 0.3 1.17 ± 0.04y -

3.02 ± 0.2 2.08 ± 0.15y -

3.08 ± 0.2 2.17 ± 0.18y -

11.39 ± 0.8 1.59 ± 0.2 10.51 ± 0.5a ns 9.9 ± 0.6az 4.2 ± 0.2b z 8.24 ± 0.4ax

3.4 ± 0.3 2.44 ± 0.1 ns 5.53 ± 0.23 b ns 3.05 ± 0.06 ns 3.26 ± 0.2 ns 4.04 ± 0.3 ns

7.36 ± 0.4 2.23 ± 0.18x 8.19 ± 0.7a ns 5.7 ± 0.35a ns 4.1 ± 0.3ns x 7.08 ± 0.4a ns

12.2 ± 0.6 2.03 ± 0.18a 13.01 ± 0.8a ns 11.42 ± 0.49ay 7.3 ± 0.7az 10.08 ± 0.9ax

9.86 ± 0.5 5.2 ± 0.6 ns 8.8 ± 0.5 ns 6.5 ± 0.1bx 6.3 ± 0.17ax 9.04 ± 0.4cx

12.42 ± 0.87 6.8 ± 0.3x 10.9 ± 0.7b ns 4.6 ± 0.3ns x 5.6 ± 0.4ns x 11.9 ± 0.56ns x

Values are mean ± SEM, n=6, P > 0.05 – ns (not significant), aP < 0.001, bP < 0.01, cP < 0.05, significantly different from deficient group. P > 0.05 – ns (not significant), xP < 0.001, yP < 0.01, zP < 0.05, significantly different from normal group.


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Table 5. Glutathione Peroxide Activity of Organs and Tissues of Se Deficient and Se Repleted Rats Groups

Plasma (mm/min/dl)

Erythrocyte (mm/min/mg Hb)

Liver (µm/min/mg pro)

Kidney (µm/min/mg pro)

Heart (µm/min/mg pro)

Muscle (µm/min/mg pro)

Se depletion period (6weeks)

Normal Deficient

48.30 ± 3.4 28.48 ± 3.0a

Repletion of Se for 2 week Normal 46.2 ± 3.4 Deficient 15.54 ±2.1 ED(wheat) 39.99 ± 2.8a ED(pearl millet) 40.8 ± 0.8a ED(Maize) 25.79 ± 1.0ya Control normal 35.10 ± 3.9a Repletion of Se for 4 week Normal 61.89 ± 5.1 Deficient 14.7 ± 3.4 73.77± 3.8a ED(wheat) ED(pearl millet) 68.18 ± 4.7 a ED(Maize) 69.15 ± 3.9 a Control normal 56.25 ± 4.1a

204 ± 2.4 150.1 ± 2.4b

520.8 ± 2.5 131.2 ± 1.7a

332.6 ± 2.6 70.1 ± 1.6a

290.2 ± 99.7 99.7 ± 3.5a

19.5 ± 5.1 12.5 ± 2.1c

157.5 ± 11.0 68.25 ± 2.9 107.6 ± 4.6c 76.3 ± 6.1 ns 60.3 ± 4.7 ns 71.73 ± 1.2 ns

317.2 ± 4.9 95.9 ± 1.5 237.2 ± 9.5a 232.2 ± 6.3a 180.6 ± 3.8za 195.3 ± 5.8za

306.1 ± 6.8 41.3 ± 3.4 247.6 ± 6.7a 217.23 ± 9.1a 168.2 ± 6.8b 192.5 ± 10.2a

294.0 ± 5.2 85.25 ± 4.5 213.6 ± 8.2a 190.1 ± 6.1a 181.2 ± 2.4ya 229.8 ± 1.7a

15.98± 0.9 9.61 ± 1.0 11.68 ± 0.9 ns 11.32 ± 3.1 ns 10.65 ± 2.3 ns 13.4 ± 4.7 ns

212.7 ± 8.44 130.0 ± 7.6 205.1 ± 11.4 c 177.0 ± 10.1 ns 162.9 ± 7.4 ns 172.2 ± 9.3 ns

333.09 ± 4.7 66.6 ± 1.6 278.26 ± 14.1a 235.23 ± 8.3xa 203.53 ± 11.1za 196.3 ± 11.3za

335.98 ± 4.5 32.9 ± 6.7a 271.1 ± 5.9a 202.8 ± 12.1ya 178.3 ± 6.8za 200.9 ± 18.1za

316.3 ± 8.9 60.26 ± 3.6 201.1 ± 11.5a 153.3 ± 9.2xa 149.7 ± 5.8xa 175.77 ± 4.1xa

20.92 ± 8.9 12.40 ± 3.6 15.6 ± 9.1 ns 15.0 ± 5.1 ns 14.12 ± 3.4 ns 15.34 ± 4.2 ns

Values are mean ± SEM, n=6, P > 0.05 – ns (not significant), aP < 0.001, bP < 0.01, cP < 0.05, significantly different from deficient group. P > 0.05 – ns (not significant), xP < 0.001, yP < 0.01, zP < 0.05, significantly different from normal group. The enzyme unit is defined as the amount of enzyme that transforms 1 mmol NADPH per minute (for GPX) or 1 nmol DTNB/min (for TR).



Liver

unit/mg protein

250 200

*

**

150

***

**

***

100 50

***

0

***

DP

normal ED(PM) Kidney

RP (2nd wk)

RP (4th wk)

ED(wheat)

deficient

ED(CN)

ED(maize)

unit/mg protein

250 ***

**

150 100 50 0

Figure 1

ns

ns

200

***

***

***

DP

RP (2nd wk)

normal

deficient

ED(PM)

ED(maize)

RP (4th wk)

ED(wheat) ED(CN)


Page 36 of 39

Page 37 of 39


B 5000

unit/dL

4000

Erythrocyte 150

plasma

***

ns

* ***

3000

*** **

* ns

ns

*

2000

unit/g Hb

A

***

100 ***

* *

50

ns c a

1000

0

0

DP

RP (2nd wk)

ED(PM)

ED(maize)

RP (2nd wk)

ED(wheat)

deficient

normal

ED(CN)

RP (4th wk)

ED(CN)

ED(maize)

ED(PM)

Liver

C 1500

D

***

ns

ns

unit/mg protein

* a

500

0

DP

RP (2nd wk)

normal E

ED(PM) Heart 400

**

***

unit/mg protein

100

normal

ED(PM) Figure 2

deficient ED(maize)

normal

deficient

RP (4th wk)

ED(CN)

ED(CN)

*** **

ns

*

5

DP normal

ED(wheat)

ED(wheat)

ED(maize) ***

***

RP (4th wk)

10

0 RP (2nd wk)

RP (2nd wk)

*

200

**

DP

*

300

DP

*

ns

ED(PM) Muscle 15

**

**

500

F

***

0

* ***

0

ED(CN)

***

*** *

**

ED(wheat)

ED(maize) ***

***

1000

RP (4th wk)

deficient

***

Kidney 1500

**

ns

1000

unit/mg protein

unit/mg protein

ED(wheat)

deficient

normal

DP

RP (4th wk)

ED(PM)


RP (2nd wk) deficient ED(maize)

RP (4th wk) ED(wheat) ED(CN)


B

A

Erythrocyte

plasma

8000

Page 38 of 39

200

***

**

4000 2000

C 80

DP

RP (2nd wk)

Liver normal

deficient

***

ED(PM)

ED(maize)

ns

*

DP

normal E

Heart ED(PM) 100

RP (2nd wk)

deficient ED(maize)

80

***

ED(wheat) ED(CN)

***

40

RP (2nd wk)

RP (4th wk)

ED(wheat)

deficient

ED(CN)

ED(maize)

***

60 ***

40

***

20

DP

RP (2nd wk)

normal

deficient

2000

Muscle ED(PM)

ED(maize)

1500

***

F

***

***

ED(PM)

0

RP (4th wk)

80 60

normal

ED(CN)

20 0

DP

Kidney

unit/mg protein

40

unit/mg protein

50

D

60

**

100

RP (4th wk)

ED(wheat)

*

**

0

0

unit/mg protein

unit/mg protein

***

150

unit/mg protein

unit/mg protein

***

6000

***

1000

RP (4th wk)

ED(wheat) ED(CN)

***

500

20 0

0 DP

normal ED(PM)

RP (2nd wk)

deficient ED(maize)

RP (4th wk)

ED(wheat)

DP normal

ED(CN)

ED(PM)

Figure 3


RP (2nd wk) deficient ED(maize)

RP (4th wk) ED(wheat) ED(CN)

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Table of Contents (TOC) Graphic.


and Pearl Millet

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