Phosphorus supplementation mitigated food intake

Downloaded from cdn.nutrition.org on July 28, 2017 - First published online on July 27, 2017 in Current Developments in Nutrition. DOI: 10.3945/cdn.117.000943

1 Phosphorus supplementation mitigated food intake and growth of rats fed a low protein diet 1 Rola U. Hammoud1, Mark N. Jabbour2, Ayman N. Tawil2, Hala Ghattas3, Omar A. Obeid1

Last names of authors are respectively: Hammoud, Jabbour, Tawil, Ghattas, Obeid

1

Department of Nutrition and Food Science, 2Pathology and Laboratory Medicine,

3

Epidemiology and Population Health, American University of Beirut. Lebanon.

Corresponding author: Omar Obeid PhD, Professor in Human Nutrition, Department of Nutrition and Food Sciences, Faculty of Agricultural and Food Sciences, American University of Beirut, Beirut, Lebanon. P.O. Box 11-0236, Lebanon. Fax: 00961-1-744460; Tel: 00961-1-350000 Ext 4440. E-mail: [email protected]

Running title: Phosphorus and low protein diet

Word Count: 5446 Number of figures: 2 Number of tables: 3 Online Supporting Material: 1table and 2 figures

The study was supported by a grant from the University Research Board (URB) at the American University of Beirut. Abbreviations include: ATP: Adenosine Triphosphate; EDTA: Ethylenediaminetetraacetic acid; HDL: High density lipoprotein; LDL: Low density lipoprotein. NAFLD: Nonalcoholic fatty liver disease; P: Phosphorus; The authors declare no conflict of interest.


2 1

Abstract

2

Background: Low protein intake is associated with various negative health outcomes at any

3

stage of life. When diets do not contain sufficient protein, phosphorus (P) availability is

4

compromised since proteins are the major sources of P. However, whether mineral

5

phosphorus supplementation mitigates, the problem is unknown.

6

Objective: Our goal was to determine the impact of dietary P supplementation on food intake,

7

weight gain, energy efficiency, body composition, blood metabolites and liver histology of

8

rats fed a low protein diet for 9 weeks.

9

Methods: Six-week old male Sprague-Dawley rats (n=49) were randomly allocated to 5

10

groups and fed ad-libitum 5 iso-caloric diets that only varied in protein (egg white) and P

11

concentrations for 9 weeks. The control group received 20% protein diet (normal P, 0.3%),

12

whereas the 4 other groups were fed low protein (10%) diets with various P concentrations:

13

0.015%, 0.056%, 0.1% and 0.3%. The rats’ weights, body and liver composition, and plasma

14

biomarkers were then assessed.

15

Results: Food intake, weight gain and energy efficiency increased significantly with the

16

addition of P to the low protein diet and were similar among the 0.3%P groups (LP-0.3P and

17

NP-0.3P) regardless of their dietary protein content. Additionally, P supplementation of the

18

low protein diets was observed to reduce plasma urea nitrogen and to increase total body

19

protein content (defatted). Changes in food intake and efficiency, body weight and

20

composition and plasma urea content were highly pronounced at a dietary P level below

21

0.1%, which may represent a critical threshold.

22

Conclusion: In rats, the addition of P to low protein diets improved growth measures mainly

23

due to an enhancement in energy efficiency. Dietary P level of 0.3% mitigated detrimental

24

effects of low protein diets on growth parameters.


3 25

Keywords: low protein diet, phosphorus, weight gain, food intake, energy efficiency,

26

NAFLD, Sprague-Dawley rats


4 27

Introduction

28

Protein deficiency can lead to various adverse health effects at all stages of life. Protein

29

restriction compromises growth and muscle maintenance of adolescents and leads to weight

30

loss, reduced subcutaneous fat, increased susceptibility to infection, general lethargy and

31

delayed wound healing in adults [1]. Additionally, inadequate protein content of maternal

32

diets during pregnancy, was reported to result in negative health outcomes in offspring [2].

33

Animal experiments on rodents have shown that maternal low protein diet during pregnancy

34

is associated with disproportionate patterns of fetal growth [3], increased adult onset of

35

metabolic disorders, and increased adiposity and glucose intolerance [4]. Furthermore, low

36

dietary protein intake in rodents has been consistently found to be associated with increased

37

body fat content [5-8].

38

The metabolic response of animals to protein restriction is believed to be mediated via

39

alterations in food intake [9, 10], organ size and enzymatic function [11, 12]. Though

40

changes in food intake were reported to be inconsistent, where some studies showed an

41

increase [7, 8, 13], while others reported a decrease that was dependent on the extent of

42

protein restriction [10, 14]. This inconsistency may imply that other factors are involved in

43

the regulation of food intake.

44

Although proteins vary in P content and bioavailability [15], the synergistic relationship

45

between dietary protein and P intake, suggest that dietary P modulates the effects of protein

46

restriction. P is an essential mineral and thus plays an important role in cellular metabolism.

47

It is also integrated within adenosine triphosphate (ATP), which acts as a phosphate donor for

48

many metabolic reactions [16]. Such reactions are strongly dependent on body P availability

49

for its production [17, 18] and that is obviously related to dietary P availability.

50

Populations most at risk of developing diseases associated with protein malnutrition, such as

51

Marasmus and Kwashiorkor, are those whose staple foods are low in protein quantity and


5 52

quality (e.g. cassava, maize, rice) [19]. A consequence of low protein diets is low P diets

53

which tends to compound nutritional problems. These populations also consume minimal

54

amounts of animal and dairy products, which are known to be the most important dietary

55

sources of both high quality proteins and P. In contrast, most stable plant-based foods, such

56

as whole wheat or brown rice; that are also commonly consumed by at-risk populations, they

57

are of low protein quantity and quality which contain low amounts of bioavailable P, as P is

58

mainly bound in the form of phytate that makes it unavailable for absorption [20].

59

Fermentation is known to breakdown phytate and thus improves the bioavailability of P [21];

60

yeast fermentation for about 60 min was reported to reduce phytate content of bread by about

61

10% [22].

62

Under conditions of protein restriction, the intake of P is compromised and therefore it can be

63

assumed that inadequate dietary P intake compounds the negative effects of protein

64

restriction. Accordingly, the present study was performed to assess the impact of P on food

65

intake, weight gain, food efficiency and body composition of rats maintained on a low protein

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diet with varied concentrations of dietary P.

67

Methods

68

Animal Housing

69

Forty-nine, six-week old male Sprague-Dawley rats (Animal care facility, American

70

University of Beirut, Beirut, Lebanon) were housed individually in a temperature (22±1°C)

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and light (12:12 hours light/dark cycle, light on 0700 h) controlled room. The rats had free

72

access to water and were fed ad libitum a semisynthetic powder control diet (Supplemental

73

Table 1) for one week to familiarize them with the environment and the diet.

74

Experimental Diet


6 75

The semi-synthetic powder experimental diets (Supplemental Table 1) were all prepared

76

using the same ingredients and were iso-caloric. Dried egg white was used as the main source

77

of protein since it supplies all essential amino acids and contains negligible amounts of P

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(1.5±0.013 g/kg) [23]. Phosphorus free mineral mix (AIN-93G MIX without P) and

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potassium phosphate (KH2PO4) from Dyets Inc. (Bethlehem, Pennsylvania, USA) were used

80

to modify P content of the diet. KH2PO4 was used because potassium addition does not affect

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growth [24, 25].

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Experimental Design

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The experimental protocol was approved by the Institutional Animal Care and Use

84

Committee at the American University of Beirut. Following the one-week acclimation period,

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each rat (190-315g) was randomly allocated to one of the five experimental groups. Under

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normal conditions, the recommended P content of the rats’ diet is 0.3% [26] and this was the

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dietary proportion used for the control group. Treatments were as follows (Supplemental

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Table 1):

89

•

Control Group: 20% protein and 0.3% P

90

•

0.015% P Group: 10% protein and 0.015% P

91

•


92

•


93

•


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The rats were offered their corresponding diets ad libitum for 9 weeks. At termination,

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overnight fasted rats were anesthetized by isoflurane (Forane®, Abbott, Berks, UK) and

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blood was collected from the superior vena cava. The rats were then euthanized by removing

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the heart and their livers were extracted and weighed. Immediately afterword, two small liver

98

sections were taken for histological analysis and the remaining section was frozen in liquid


7 99

nitrogen, and stored at -80°C. Blood samples were centrifuged at 2200 g for 15 minutes at

100

3°C and plasma aliquots were collected and stored at -80°C. The rat carcasses were stored at -

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20°C for body composition analysis.

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Food Intake and Body Weight and Composition

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The Food intake and body weight were measured twice per week and then averaged to

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calculate weekly changes. Carcasses were dried until constant weight (about 48 hours) at

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105ºC and homogenized, and the fat was extracted using petroleum Ether (BP 40-60ºC).

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Carcassmoisture and fat contents were calculated from the differences in weight. Hepatic fat

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content was determined as follows: about 2g of liver were freeze-dried (2.5 Liter Bench top

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Freeze-Dry System, LABCONCO) for 48 hours and fat was extracted by petroleum ether

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solvent (BP 40-60ºC) for 40 minutes using an ANKOMXT10 extractor (ANKOM Technology

110

2052 O'Neil Road, Macedon NY 14502).

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Blood Analysis

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Fasting plasma glucose, triglyceride, total cholesterol, and total P were determined using an

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enzymatic colorimetric method on the Vitros 350 Chemistry System (Ortho-Clinical

114

Diagnostics, Johnson & Johnson, New York). Plasma insulin concentration was determined

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by enzyme immunoassay using insulin rat ELISA kit (EZRMI-13K) (EMD Millipore

116

Corporation, Billerica, MA, USA).

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Liver Biopsy

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Small liver sections were stained with hematoxylin and eosin (H&E) and oil red (O) to be

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evaluated for Necro-Inflammatory grading and Fatty Droplets. Histological changes were


8 120

assessed by a modification of the scoring system for grading and staging for non-alcoholic

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fatty liver disease (NAFLD) as described by Kleiner et al. [27].

122

Histopathology Examination

123

Rat liver tissue was processed into 3-4 µm thick formalin-fixed paraffin embedded tissue

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sections and stained with hematoxylin and eosin (H&E). Histopathologic examination

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consisted of assessing steatosis grade and distribution with a score=0 (66%). Location was defined as steatosis

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distribution with a score=0 (zone 3); score=1 (zone 1); score=2 (azonal) or score=3

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(panacinar). Microvesicular steatosis was recorded as either score=0 (not present) or score=1

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(present). Lobular inflammation was semi-quantified according to a score=0 (< 2 foci per

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200x field); score=1 (2-4 foci per 200x field) or score=3 (>4 foci per 200x field).

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Oil Red O (ORO) Examination

132

ORO was performed according to previously described protocol [28]. Briefly, fresh frozen rat

133

liver tissue was embedded into Cryomolds and sectioned into 5 µm sections on a cryostat

134

(Leica). Sections were then stained in ORO and semi-quantified using imagej software

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(http://rsbweb.nih.gov/ij). Tissue sections were imaged at five high-power (400x) fields and

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converted to 8-bit grayscale images. This was followed by an image threshold predefined

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according to a rat liver section negative for steatosis and microvesicular steatosis on H&E

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and ORO staining, and image analysis for ORO surface area staining determined.

139 140

Statistical Analysis

141

The required number of rats (n=9) was based on previous weight gain data (6.0g/day SD

142

0.95) assuming a 25% difference in the mean with a statistical power of 90% and 5%


9 143

significance level. Data were expressed as means ± SD of all values. Data analysis was

144

performed using the SPSS 23 (IBM SPSS) software program. Results were analyzed by one-

145

way analysis of variance (ANOVA), and specific comparisons were made between each of

146

the five groups using Fisher’s pairwise comparisons. A probability of less than 0.05 was

147

considered to be significant.

148

Results

149

Weight Gain, Food Intake and Energy Efficiency

150

Initial body weight was similar among the groups, while final body weight was significantly

151

different. Final body weight of the control group (NP-0.3P) was significantly greater than

152

those of the LP-0.015 P and LP-0.056 P groups. Among the low protein groups, body weight

153

increased with increased P level in the diet, and the body weight of LP-0.1 P and LP-0.3 P

154

groups were not significantly different from body weight of the NP-0.3P (Table 1).

155

Addition of P to low protein diets was found to increase weight gain (g/day), in which that of

156

LP-0.1P and LP-0.3P was close to that of NP-0.3P group (Figure 1A). Moreover, food

157

intake was improved by increasing P content of the low protein diets. No difference in food

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intake was observed among the LP-0.1P, LP-0.3P and NP-0.3P groups (Figure 1B,

159

Supplemental Figure 1). The difference in food intake among groups started with the

160

introduction of the various diets and persisted throughout the experimental period

161

(supplementary figure 1). The pattern of variation in energy efficiency [weight gain (g)/100

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kcal] was found to parallel that of weight gain, in which energy efficiency of the low protein

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groups increased with the addition of P to the diet, in which that of the LP-0.015P was lower

164

than that of LP-0.056P that was lower than that of LP-0.1P. However, no differences was

165

observed between energy efficiency of the LP-0.1P and LP-0.3P that was similar to that of

166

NP-0.3P. (Figure 1C).


10

167

Body and Liver Composition

168

Body proximate analysis results suggested that the addition of P to low protein diets was

169

associated with an increase in body moisture. However, the proportion of body moisture was

170

found to decrease with the addition of P to low protein diets. The proportion of moisture in

171

the NP-0.3P group was similar to that of the LP-0.3P group and both were significantly less

172

than moisture proportion of LP-0.015 P and LP-0.056 P fed rats. The addition of P to low

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protein diets was associated with a significant increase in total body fat content (P 0.01),

174

but no significant changes were observed in the percentage of body fat (P = 0.23). Defatted

175

carcass weight was used to provide information on body protein status since mineral content

176

is known to be relatively small. The quantity and percentage of defatted rat carcasses of the

177

LP-0.3P and NP-0.3P treatments were similar to each other regardless of protein content of

178

the diets and both were significantly greater than that of the LP-0.015P and LP-0.056P groups

179

(Table 1).

180

In the low protein groups, liver weight increased with increasing dietary P but liver weights

181

of LP-0.1 P and LP-0.3P rats were similar to that of NP-0.3P. However, when liver weight

182

was expressed per 100 g of body weight, no statistically significant difference were detected

183

among treatment groups (P = 0.55). Additionally, no significant differences were observed

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among groups in terms of their percentages of hepatic dry weight or fat content. (Table1).

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Plasma results

186

Total plasma P concentration was significantly different among the groups (P

187

which that of LP-0.015 P treatment had the lowest value, while that of other treatments

188

maintained similar levels. Plasma glucose concentration of the NP-0.3P group was similar to

189

that of the LP-0.3P group. Among the low protein groups, plasma glucose concentration was

0.01), in


11 190

found to increase with increased P content of the diet. Plasma TG, total cholesterol, albumin

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and CRP levels were not found to be significantly different among treatment groups and

192

were not affected by either protein or P content of the diets (Table 2).

193

In the low protein treatments, plasma urea nitrogen was found to decrease with increased P

194

content of the diet up to 0.1% P. No differences in plasma urea nitrogen were observed

195

among the NP-0.3P, LP-0.1P and LP-0.3P groups (Figure 2).

196

Liver histology

197

Analyses of liver photomicrographs (Supplemental Figure 2) indicate that steatosis grade and

198

location, micro vesicular steatosis and portal inflammation were similar among groups and

199

thus were not affected by protein level or P content of the diet. All groups had approximately

200

similar cases of Microvesicular Steatosis. Around 5 to 6 rats from each group presented with

201

Grade 0 or Grade 1 Steatosis, and only 1 rat from each group developed Grade 3 Steatosis.

202

No signs of Mallory Hyaline Bodies, Fibrosis or GlycogenatedNnuclei were detected in all

203

rats. Only lobular inflammation was found to be significantly greater in the low protein

204

groups as compared to the normal dietary protein group. (Table 3).

205

Discussion

206

The study was designed to investigate the impact of dietary P supplementation on several

207

growth parameters of rats maintained on low protein diet. In animals, food intake was

208

reported to be regulated in order to serve two objectives. First, the satisfaction of

209

nutritional needs for growth and maintenance, which entails long-term regulation of intake

210

and this is usually accompanied by an increase in food intake. In support, diet selection and

211

protein restriction studies have shown that young animals are capable of adjusting their intake


12 212

in order to support their nutritional protein requirements [29, 30] for growth and

213

maintenance. Second, the maintenance of homeostasis that relates to acute or short-term

214

regulation of food intake [31] and this is usually associated with a decease in food intake in

215

order to avoid the toxicity of amino acid accumulation that is common with the ingestion of

216

low quality proteins.. Thus, reduced food intake of the low protein low P diets (LP-0.015P;

217

LP-0.056P) as compared to the other low protein groups (LP-0.1P; LP-0.3P), despite the

218

similarity in dietary protein contents, is likely to have been the outcome of a dietary

219

adaptation for the maintenance of homeostasis [32-34]. Adequate protein metabolism in

220

terms of synthesis and degradation depends on the availability of essential amino acids as

221

well as energy needed to catalyze these reactions. Protein synthesis is an expensive energy-

222

requiring process, in which 4 ATP equivalents are required for the formation of 1 peptide

223

bond, or the equivalent of 0.67 kcal per one gram of protein synthesized [30]. Several

224

observations indicate that this process is highly dependent on P availability [35-37]. In mice,

225

dietary P restriction was reported to lower gastrocnemius muscle tension, due to a slow rate

226

of ATP synthesis, and this was reversed by P supplementation [35]. Additionally, ATP

227

depletion by 2.4-dinitrophenol [36] or fructose infusion [37] was reported to reduce protein

228

synthesis. Hence, under condition of low P intake, the body loses its capacity to synthesize

229

protein due to reduced ATP availability that is dependent on exogenous P replenishment [35,

230

36] and ultimately amino acids accumulate in circulation. Consequently, food intake is

231

reduced to protect against amino acid toxicity. The ability of P to alter protein metabolism

232

was supported by the fact that dietary P content was inversely related to plasma urea

233

nitrogen. Thus, it can be concluded that the observed increase in food intake with addition of

234

P to low protein diet is likely to have been attributed to an improvement in amino acid

235

homoeostasis due to an enhancement in protein metabolism (e.g. protein synthesis). The

236

facts that proteins are the main sources of dietary P and that bodily protein metabolism is


13 237

dependent on P, makes it reasonable to postulate that reduced food intake under low protein

238

diets [38] is highly related to alteration of amino acid homeostasis in a manner which mimics

239

that of low quality protein diet. Accordingly, the reported changes in food intake following

240

protein (casein) manipulation [10] may have been partially related to P content of the diet,

241

especially since 50% of P (0.15% of diet) in rodents diet is derived from casein [24].

242

On the other hand, measures of body composition seems to be highly dependent on P

243

availability in the diet as indicated by the resemblances in body composition between LP-

244

0.3P and NP-0.3P groups.. This is further supported by the findings where differences in

245

body weight between the normal P containing (LP-0.3P and NP-0.3P) and the P depleted

246

(LP-0.015P and LP-0.056P) groups were associated with changes in both body fat and

247

protein. For example, the 10% reduction in water (%) of the NP-0.3P and LP-0.3P groups

248

was almost replaced by 5% increase in fat (%) and 5% increase in protein (%), while the

249

opposite was true for the P depleted groups (LP-0.015P and LP-0.056P). In the latter, the

250

sharp reduction in weight gain was associated with an increase in water (%). Increased water

251

retention is known to be the main feature of Kwashiorkor, which was not explained by

252

protein restriction alone ([10, 39] and is known to be associated with hypophosphatemia [40]

253

and poor dietary P availability [41]. The inability of P restriction to increase hepatic water

254

retention argues against its involvement in the development of kwashiorkor. It is worth

255

noting that body composition changes following P manipulation were mainly attributed to

256

energy efficiency since the magnitude of changes in food intake was modest.

257

In addition, the high resemblance between LP-0.1P and the control groups (LP-0.3P and NP-

258

0.3P) in weight gain, food intake and body weight was not reflected by similarities in body

259

composition, asthe percentage of body protein (defatted percentage) of the 0.3% P (LP-0.3P

260

and NP-0.3P) groups was higher than that of the LP-0.1P group.. This further confirms the


14 261

importance of P in protein metabolism and indicates that P is capable of altering body

262

composition. In the present study, no significant alterations in liver composition and

263

histology were detected despite the fact that dietary P restriction in mice was reported to

264

increase hepatic lipid accumulation, especially under condition of increased cholesterol

265

content of the diet [42].

266

Moreover, the failure of dietary P restriction to significantly affect plasma P content, except

267

under extreme condition of restriction (LP-0.015P), is in line with human findings [43] in

268

which plasma P is reported not to be a indicator of P intake. Whatever, dietary P

269

manipulation (between 0.056%P and 0.3%P) was able to manipulate food intake, energy

270

efficiency, weight gain and body composition though plasma P was not affected. This further

271

confirms that plasma P level is neither a good marker of P intake nor an indicator of

272

changes in food intake or weight measures.

273

Furthermore, the observed elevated plasma glucose in the control group may have been

274

related to the effect of egg white protein on insulin secretion [44-46]. Human studies have

275

shown that insulin incremental area following 50g of egg white ingestion was much lower

276

than that of cottage cheese in both healthy [45] and diabetic subjects [44]. Additionally, the

277

ingestion of a breakfast with whole egg, egg white or egg yolk was associated with a rise in

278

blood glucose concentration following egg white ingestion, as compared to that of whole egg

279

or egg yolk meals [46]. However, the resemblance in plasma glucose between LP-0.3Pand

280

NP-0.3P implies that plasma glucose is not solely dependent on the quantity of egg white in

281

the diet. In low protein (egg white) groups, the increase in plasma glucose with the addition

282

of P possibly indicates that egg white ingestion was able to blunt the effect of P on insulin

283

sensitivity [47] or the presense of varied glycogen storage between the groups. The latter


15 284

option may prevail, since plasma glucose is affected by hepatic glycogen content that is

285

known to be stimulated by P intake [48].

286

The ability of P intake to manipulate food intake and body composition requires further

287

investigations to determine the level of P intake that is capable of improving body

288

composition.

289 290

Low body protein content (defatted %) was present under condition of 0.1%P (LP-0.1P)

291

(about 0.25 mg P/kcal) and lower despite having normal protein intake, while higher levels of

292

P intake improved body protein content. The level of P intake (0.25mg/Kcal of highly

293

bioavailable P) resembles that consumed by most people, which was reported to be about 0.5

294

mg P/kcal [49] assuming a 50% bioavailability (due to the significant contribution of plant

295

sources). Such level is not thought to enhance protein status. In support, a recent study on

296

humans reported an improvement in body weight and waist circumference of overweight and

297

obese subjects following 12 weeks of P supplementation [50]. Although body composition

298

was not measured, an increase in lean body mass percent would be expected.

299

Under moderate protein restriction (10%), P addition (0.3%) was able to significantly

300

improve food intake, weight gain and energy efficiency similar to a normal protein diet

301

(20%) containing same level of P (0.3%). Body protein content (defatted %) was high with

302

0.3% P diet, irrespective of protein content (10 or 20%). Looking at the pattern of changes of

303

the different measures, it seems that dietary phosphorus level of 0.1% may represent a critical

304

threshold. However, it is not clear whether P would be able to exert these effects under added

305

protein restriction. Our results may have implications on the management of malnutrition and

306

may have financial value since the cost of protein is much higher than that of P. Additionally,

307

our findings suggest that not only protein quantity but also P content of the diet is of extreme

308

importance for improving food intake, weight gain and energy efficiency.


16

309

Author’s Contributions

310

OO: designed the study, performed the statistical analysis, supervised all the work and had

311

primary responsibility for the final content; RH: collected the data and wrote a draft

312

manuscript; OO and RH analyzed the data; MJ, AT, HG: critically revised the manuscript for

313

important intellectual content. All authors have read and approved the final manuscript.


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18 11. Harper AE. Effect of variations in protein intake on enzymes of amino acid metabolism. Can J Biochem 1965;43(9):1589-603. 12. Muramastu K, Ashida K. Relationsnip Between the Nutritive Value of Dietary Protein and Liver Xanthine Oxidase Activity in Young Rats. Agric Biol Chem 1962;26(1):25-9. 13. Colombo J-P, Cervantes H, Kokorovic M, Pfister U, Perritaz R. Effect of different protein diets on the distribution of amino acids in plasma, liver and brain in the rat. Ann Nutr Metab 1992;36(1):23-33. 14. Beck B, Dollet J-M, Max J-P. Refeeding after various times of ingestion of a low protein diet: effects on food intake and body weight in rats. Physiol Behav 1989;45(4):761-5. 15. Boaz M, Smetana S. Regression equation predicts dietary phosphorus intake from estimate of dietary protein intake. J Am Diet Assoc 1996;96(12):1268-70. 16. Amanzadeh J, Reilly RF. Hypophosphatemia: an evidence-based approach to its clinical consequences and management. Nat Clin Pract Nephrol 2006;2(3):136-48. 17. Solomon S, Kirby D. The refeeding syndrome: a review. JPEN J Parenter Enteral Nutr 1990;14(1):90-7. 18. Morris Jr RC, Nigon K, Reed EB. Evidence that the severity of depletion of inorganic phosphate determines the severity of the disturbance of adenine nucleotide metabolism in the liver and renal cortex of the fructose-loaded rat. J Clin Invest 1978;61(1):209. 19. Waterlow J. Protein-energy malnutrition: the nature and extent of the problem. Clin Nutr 1997;16:3-9. 20. Ravindran V, Ravindran G, Sivalogan S. Total and phytate phosphorus contents of various foods and feedstuffs of plant origin. Food Chem 1994;50(2):133-6. 21. Pozrl T, Kopjar M, Kurent I, Hribar J, Janes A, Marian S. Phytate degradation during breadmaking: the influence of flour type and breadmaking procedures. Czech J Food Sci 2009;27(1):29-38.


19 22. Lopez H, Krespine V, Guy C, Messager A, Demigne C, Remesy C. Prolonged fermentation of whole wheat sourdough reduces phytate level and increases soluble magnesium. J Agric Food Chem 2001;49(5):2657-62. 23. EPA 200- 7/8 (1991) Methods for the determination of metals in environmental samples. 24. Murai I1, Shukuin S, Sugimoto M, Ikeda S, Kume S. Effects of high potassium chloride supplementation on water intake and bodyweight gains in pregnant and lactating mice. Anim Sci J. 2013 Jun;84(6):502-7. doi: 10.1111/asj.12025. Epub 2013 Jan 17. 25. Jodas EM, Voltera AF, Ginoza M, Kohlmann O Jr, dos Santos NB, Cesaretti ML. Effects of physical training and potassium supplementation on blood pressure, glucose metabolism and albuminuria of spontaneously hypertensive rats.J Bras Nefrol. 2014 JulSep;36(3):271-9. 26. . Reeves PG, Nielsen FH, Fahey Jr GC. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J Nutr 1993;123(11):1939-51. 27. Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, Ferrell LD, Liu YC, Torbenson MS, Unalp-Arida A, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatol 2005;41(6):1313-21. 28. Mehlem A, Hagberg CE, Muhl L, Eriksson U, Falkevall A. Imaging of neutral lipids by oil red O for analyzing the metabolic status in health and disease. Nat Protoc 2013;8(6):1149-54. 29. Leathwood PD, Ashley DV. Strategies of protein selection by weanling and adult rats. Appetite 1983;4(2):97-112. 30. Shariatmadari F, Forbes J. Growth and food intake responses to diets of different protein contents and a choice between diets containing two concentrations of protein in broiler and layer strains of chicken. Br Poult Sci 1993;34(5):959-70.


20 31. Radcliffe J, Webster A. Regulation of food intake during growth in fatty and lean female Zucker rats given diets of different protein content. Br J Nutr 1976;36(03):457-69. 32. Kabadi UM, Eisenstein AB, Strack I. Decreased plasma insulin but normal glucagon in rats fed low protein diets. J Nutr 1976;106(9):1247-53. 33. Peters JC, Harper AE. Adaptation of rats to diets containing different levels of protein: effects on food intake, plasma and brain amino acid concentrations and brain neurotransmitter metabolism. J Nutr 1985;115(3):382-98. 34. Heard C, Frangi SM, Wright PM, McCartney P. Biochemical characteristics of different forms of protein-energy malnutrition: an experimental model using young rats. Br J Nutr 1977;37(01):1-21 35. Hettleman B, Sabina R, Drezner M, Holmes E, Swain J. Defective adenosine triphosphate synthesis. An explanation for skeletal muscle dysfunction in phosphate-deficient mice. J Clin Invest 1983;72(2):582. 36. Fischbeck KH. Effects of ATP depletion and protein synthesis inhibition on muscle plasma membrane orthogonal arrays. Exp Neurol 1984;83(3):577-88. 37. Bode JC, Zelder O, Rumpelt H, Wittkampy U. Depletion of Liver Adenosine Phosphates and Metabolic Effects of Intravenous Infusion of Fructose or Sorbitol in Man and in the Rat. Eur J Clin Invest 1973;3(5):436-41. 38. Peng Y, Gubin J, Harper A, Vavich M, Kemmerer A. Food intake regulation: amino acid toxicity and changes in rat brain and plasma amino acids. J Nutr 1973;103(4):608-17. 39. Golden MN. Protein deficiency, energy deficiency, and the oedema of malnutrition. Lancet 1982;319(8284):1261-5. 40. Kimutai D, Maleche-Obimbo E, Kamenwa R, Murila F. Hypo-phosphataemia in children under five years with kwashiorkor and marasmic kwashiorkor. East Afr Med J 2009;86(7).


21 41. Manary MJ, Heikens GT, Golden M. Viewpoint: part 3: Kwashiorkor: more hypothesis testing is needed to understand the aetiology of oedema. Malawi Med J 2009;21(3). 42. Tanaka S, Yamamoto H, Nakahashi O, Kagawa T, Ishiguro M, Masuda M, Kozai M, Ikeda S, Taketani Y, Takeda E.. Dietary phosphate restriction induces hepatic lipid accumulation through dysregulation of cholesterol metabolism in mice. Nutr Res 2013;33(7):586-93. 43. de Boer IH, Rue TC, Kestenbaum B. Serum phosphorus concentrations in the third National Health and Nutrition Examination Survey (NHANES III). Am J Kidney Dis 2009;53(3):399-407. 44. Gannon MC, Nuttall FQ, Lane JT, Burmeister LA. Metabolic response to cottage cheese or egg white protein, with or without glucose, in type II diabetic subjects. Metabolism 1992;41(10):1137-45. 45. Nuttall FQ, Gannon MC. Metabolic response to egg white and cottage cheese protein in normal subjects. Metabolism 1990;39(7):749-55. 46. Pelletier X, Thouvenot P, Belbraouet S, Chayvialle J, Hanesse B, Mayeux D, Debry G. Effect of egg consumption in healthy volunteers: influence of yolk, white or whole-egg on gastric emptying and on glycemic and hormonal responses. Ann Nutr Metab 1996;40(2):109-15. 47. Khattab M, Abi-Rashed C, Ghattas H, Hlais S, Obeid O. Phosphorus ingestion improves oral glucose tolerance of healthy male subjects: a crossover experiment. Nutr J 2015;14(1):1. 48. Xie W, Tran TL, Finegood DT, van de Werve G. Dietary Pi deprivation in rats affects liver CAMP, glycogen, key steps of gluconeogenesis and glucose production. Biochem J 2000;352(1):227-32.


22 49. Chang AR, Lazo M, Appel LJ, Gutiérrez OM, Grams ME. High dietary phosphorus intake is associated with all-cause mortality: results from NHANES III. Am J Clin Nutr 2014;99(2):320-7. 50. Ayoub J, Samra M, Hlais S, Bassil M, Obeid O. Effect of phosphorus supplementation on weight gain and waist circumference of overweight/obese adults: a randomized clinical trial. Nutr Diabetes 2015;5(12):e189.

23

LP-0.015P n=9

LP-0.056P n=10

LP-0.1P n=10

LP-0.3P n=10

NP-0.3P n=10

ANOVA P-value

266.1 ± 35.7

265.4 ± 32.8

266.5 ± 27.6

267.8 ± 24.2

266.9 ± 31.9

1.000

383 ± 42.6 a

426 ± 39.5 a

513 ± 50.6 b

536 ± 76.7 b

563 ± 60.6 b

< 0.001

224.4 ± 25.6 a 65.4 ± 2.1 a

237.1 ± 21.4 ac 62.4 ± 3.7 ab

261.1 ± 22.4 ab 57.2 ± 6.3 bc

253.5 ± 17.8 bc 53.9 ± 9.7 c

277.1 ± 19.5 d 55.1 ± 5.6 c

< 0.001 < 0.001

24.9 ± 9.4 a 20.6 ± 5.0

33.8 ± 15.8 ab 23.1 ± 6.1

56.6 ± 32.1 bc 27.3 ± 8.6

64.9 ± 37.7 c 26.7 ± 8.5

56.4 ± 21.2 ab 24.2 ± 5.3

0.006 0.230

94.5 ± 9.9 a 14.0 ± 3.7 a

110.1 ± 14.9 a 14.5 ± 3.9 a

142.7 ± 22.5 b 15.5 ± 5.6 ab

163.2 ± 46.7 bc 19.5 ±5.6 bc

174.0 ± 33.7 c 20.8 ±5.4 c

< 0.001 0.010

12.0 ± 2.1 a

12.5 ± 1.9 a

15.3 ± 2.4 b

16.4 ± 3.3 b

16.3 ± 2.3 b

< 0.001

Weight (g/100g)

3.3 ± 0.4

3.1 ± 0.3

3.2 ± 0.4

3.2 ± 0.2

3.09 ± 0.2

0.545

Water (%)

72.2 ±1.5

71.1 ± 2.2

70.5 ± 1.6

70.7 ± 1.3

70.7 ±1.0

0.187

Fat (%)

8.0 ± 2.0

13.1 ± 5.9

11.0 ± 3.6

11.9 ± 6.5

11.0 ± 2.7

0.186

Variables Initial Body Weight, g Final Body measures Weight (g) Water (g) (%) Fat (g) (%) Defatted (g) (%) Liver Weight (g)

LP-0.015P: 10% protein and 0.015% P; LP-0.056P: 10% protein and 0.056% P; LP-0.1P: 10% protein and 0.1% P; LP-0.3P: 10% protein and 0.3% P; NP-0.3P (Control Group): 20% protein and 0.3% P. Data are expressed as means ± SD of all values. One-way ANOVA analysis is used to detect significant differences between the groups. Significance is set at P-value < 0.05. Categories in the same row not sharing the same subscripts are significantly different.


Table 1: Body Weight (g) and Body Composition of rats fed a control diet or 1 of 4 low protein diets with different P concentrations for 9 weeks.

24

314 Variables

LP-0.015P n=9

LP-0.056P n=10

LP-0.1P n=10

LP-0.3P n=10

NP-0.3P n=10

Phosphorus, mg/dL

5.4 ± 0.9 a

6.5 ± 1.2 ab

7.4 ± 0.9 b

6.4 ± 0.7 ab

6.4 ± 0.9 ab

0.002

148.3 ± 91.7 a

205.0 ± 57.5 ab

245.4 ± 83.3 ab

263.9 ± 68.6 b

277.0 ± 76.5 b

0.004

Insulin, ng/mL

0.4 ± 0.2

0.5 ± 0.4

0.8 ± 0.8

0.8 ± 0.9

0.7 ± 0.9

0.487

TG, mg/dL

35.7 ± 6.7

41.2 ± 17.2

45.0 ± 22.7

40.6 ± 15.8

41.2 ± 13.1

0.807

Cholesterol, mg/dL

87.0 ± 22.1

85.3 ±13.3

84.8 ± 14.7

85.5 ±15.9

105.4 ±25.3

0.081

Albumin, mg/dL

3.2 ± 0.2

3.2 ± 0.3

3.1 ± 0.2

3.1 ± 0.2

3.2 ± 0.3

0.921

CRP, mg/dL

3.2 ± 0.4

2.8 ± 0.4

2.7 ± 0.7

2.7 ± 0.8

3.1 ± 0.3

0.162

Glucose, mg/dL

ANOVA 315 P-value

316 317 318 319 320 321 322 323 324 325 326 327 328 329

LP-0.015P: 10% protein and 0.015% P; LP-0.056P: 10% protein and 0.056% P; LP-0.1P: 10% protein and 0.1% P; LP-0.3P: 10% protein and 0.3% P; NP-0.3P (Control Group): 20% protein and 0.3% P. Data are expressed as means ± SD of all values. Significance is set at P-value < 0.05. Categories in the same row not sharing the same subscripts are significantly different.


Table 2: Plasma Metabolites of rats fed a control diet or 1 of 4 low protein diets with different P concentrations for 9 weeks.

25

LP-0.015P n=9

LP-0.056P n=10

LP-0.1P n=10

LP-0.3P n=10

NP-0.3P n=10

ANOVA P-value

Oil-R-O Image Analysis (µm2)

280 ± 727

1510 ± 2837

1234 ± 1636

2075 ± 3088

664 ±1457

0.407

Steatosis Grade

0.4 ± 1.0

0.5 ± 1.0

0.9 ± 1.1

0.6 ± 1.0

0.3 ± 0.7

0.701

Location

0.7 ± 1.0

0.7 ± 1.3

1.8 ± 1.6

0.9 ± 1.5

0.3 ± 0.9

0.118

Micro vesicular steatosis

0.6 ± 0.5

0.6 ± 0.5

0.9 ± 0.3

0.7 ± 0.5

0.5 ± 0.5

0.385

Variables

Lobular inflammation Portal inflammation

0.7 ± 0.9

ab

0.2 ± 0.4

0.7 ± 1.0

ab

0.2 ± 0.4

1.4 ± 1.1

a

0.1 ± 0.3

0.5 ± 0.7

b

0.1 ± 0.3

0.1 ± 0.3

b

0.1 ± 0.3

0.020 0.895

LP-0.015P: 10% protein and 0.015% P; LP-0.056P: 10% protein and 0.056% P; LP-0.1P: 10% protein and 0.1% P; LP-0.3P: 10% protein and 0.3% P; NP-0.3P (Control Group): 20% protein and 0.3% P. Data are expressed as means ± SD of all values. One-way ANOVA analysis is used to detect significant differences between the groups. Significance is set at P-value < 0.05. Categories not sharing in the same row the same subscripts are significantly different.


Table 3: Liver histology results of rats fed a control diet or 1 of 4 low protein diets with different P concentrations for 9 weeks.


26 Figures Figure 1. Mean daily food intake, weight gain, and energy efficiency of rats fed a control diet or 1 of 4 low protein diets with different P concentrations for 9 weeks.

▲

: Control Group: 20% protein and 0.3% of P; :Low protein groups (10% protein) with 0.015%P, 0.056%P, 0.1%P or 0.3%P. Food intake, weight gain, and energy efficiency are expressed as Mean ± SD as a function of the level of phosphorus in the diet, n = 9 or 10. Energy efficiency was calculated as the average weight gained per day per 100 kcal of the corresponding diet consumed. One-way analysis of variance (ANOVA), and specific comparisons were made between each of the five groups using Fisher’s pairwise comparisons. Values that do not share the same subscript are significantly different (P-value < 0.05).

●

Figure 2. Plasma urea nitrogen concentrations (mM) of rats fed a control diet or 1 of 4 low protein diets with different P concentrations for 9 weeks.

▲

●

: Control Group: 20% protein and 0.3% of P; :Low protein groups (10% protein) with 0.015%P, 0.056%P, 0.1%P or 0.3%P. The plasma urea nitrogen concentrations are expressed as Mean ± SD as a function of level of phosphorus in the diet, n = 9 or 10. One-way analysis of variance (ANOVA), and specific comparisons were made between each of the five groups using Fisher’s pairwise comparisons. Values that do not share the same subscript are significantly different (P-value < 0.05). .


7

A

cd

Weight Gain (g/day)

6 5 4

2 1

d

c

3

Low Protein

b a

Control

0 0 0.015

B

0.05

0.1

0.15

0.2

0.25

0.3

Food Intake (g/day)

28 b

26 b 24 22

a

a

20

b

18

Low Protein

16

Control

14

C

Energy Efficiency (g/100kcal)

0 0.015

0.05

0.1

0.15

0.2

0.25

0.3

d

6 5 4

c cd

3 b

2 1

Low Protein

a

Control

0 0 0.015

0.05

0.1

0.15

0.2

Dietary Phosphorus (%)

0.25

0.3


Plasma Urea Nitrogen (mM)

7

Low Protein Control

6 bc 5 a ab

4

bc c

3 0 0.015

0.05

0.1

0.15

0.2

Dietary Phosphorus (%)

0.25

0.3

Online Supporting Material

Composition of diet

LP-0.015P

LP-0.056P

LP-0.1P

LP-0.3P

NP-0.3P

Egg white*, g/100g (% energy)

10 (10)

10 (10)

10 (10)

10 (10)

20 (20)

Corn Oil, g/100g (% energy)

10 (22)

10 (22)

10 (22)

10 (22)

10 (22)

Corn Starch, g/100g (% energy)

35 (34)

35 (34)

35 (34)

35 (34)

30 (29)

Sucrose, g/100g (% energy)

35 (34)

35 (34)

35 (34)

35 (34)

30 (29)

Cellulose*, g/100g

5.5

5.5

5.4

5.2

5.2

Mineral mix (AIN-93G)* 2, g/100g

3.5

3.5

3.5

3.5

3.5

Vitamin mix (AIN-93VX)*, g/100g

1

1

1

1

1

410

410

410

410

410

-

0.041

0.091

0.291

0.282

0.015

0.056

0.106

0.306

0.312

Total Energy, kcal/100g KH2PO4*, g/100g Total Phosphorus, g/100g a

As formulated. P: phosphorus. LP-0.015P: 10% protein and 0.015% P; LP-0.056P: 10% protein and 0.056% P; LP-0.1P: 10% protein and 0.1% P; LP-0.3P: 10% protein and 0.3% P; NP-0.3P (Control diet): 20% protein and 0.3% P. 1 1.5mg P/g Egg White 2 Phosphorous Free Mineral Mix (AIN-93G MIX without phosphorus) * From Dyets Inc., Bethlehem, Pennsylvania, USA


Supplemental Table 1: Compositiona (macro and micronutrients) of the experimental diets (control and 4 low protein diets with different P concentrations).

28

Average Weekly Food Intake (g/day)

35

LP-0.015P

LP-0.1P

LP-0.056P

LP-0.3P

NP-0.3P

30

25

20

15

10 0

1

2

3

4

5

6

7

8

Weeks

Supplemental Figure 1. Mean weekly food intake of rats fed a control diet or 1 of 4 low protein diets with different P concentrations.

9



LP-0.015P: 10% protein and 0.015% P; LP-0.056P: 10% protein and 0.056% P; LP-0.1P: 10% protein and 0.1% P; LP-0.3P: 10% protein and 0.3% P; NP-0.3P (Control diet): 20% protein and 0.3% P.

Statistical analysis using one way analysis of variance (ANOVA) with Fisher’s pair wise comparison shows that weekly food intake of the low protein low P groups (LP-0.015P; LP-0.056P) was constantly lower than that of the other groups



29

Supplemental Figure 2: Hepatic histopathological assessment of rats fed a control diet or 1 of 4 low protein diets with diffe ifferent P concentrations for 9 weeks. Staining and original magnification: Haematoxylin and eosin (H&E) (x 100) and Oil-Red (x 400).



30