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.
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
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.
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
3 25
Keywords: low protein diet, phosphorus, weight gain, food intake, energy efficiency,
26
NAFLD, Sprague-Dawley rats
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
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
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
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
66
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)
71
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
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
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
78
(1.5±0.013 g/kg) [23]. Phosphorus free mineral mix (AIN-93G MIX without P) and
79
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
81
growth [24, 25].
82
Experimental Design
83
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,
85
each rat (190-315g) was randomly allocated to one of the five experimental groups. Under
86
normal conditions, the recommended P content of the rats’ diet is 0.3% [26] and this was the
87
dietary proportion used for the control group. Treatments were as follows (Supplemental
88
Table 1):
89
•
Control Group: 20% protein and 0.3% P
90
•
0.015% P Group: 10% protein and 0.015% P
91
•
0.056% P Group: 10% protein and 0.056% P
92
•
0.100% P Group: 10% protein and 0.1% P
93
•
0.300% P Group: 10% protein and 0.3% P
94
The rats were offered their corresponding diets ad libitum for 9 weeks. At termination,
95
overnight fasted rats were anesthetized by isoflurane (Forane®, Abbott, Berks, UK) and
96
blood was collected from the superior vena cava. The rats were then euthanized by removing
97
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
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
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 -
101
20°C for body composition analysis.
102
Food Intake and Body Weight and Composition
103
The Food intake and body weight were measured twice per week and then averaged to
104
calculate weekly changes. Carcasses were dried until constant weight (about 48 hours) at
105
105ºC and homogenized, and the fat was extracted using petroleum Ether (BP 40-60ºC).
106
Carcassmoisture and fat contents were calculated from the differences in weight. Hepatic fat
107
content was determined as follows: about 2g of liver were freeze-dried (2.5 Liter Bench top
108
Freeze-Dry System, LABCONCO) for 48 hours and fat was extracted by petroleum ether
109
solvent (BP 40-60ºC) for 40 minutes using an ANKOMXT10 extractor (ANKOM Technology
110
2052 O'Neil Road, Macedon NY 14502).
111
Blood Analysis
112
Fasting plasma glucose, triglyceride, total cholesterol, and total P were determined using an
113
enzymatic colorimetric method on the Vitros 350 Chemistry System (Ortho-Clinical
114
Diagnostics, Johnson & Johnson, New York). Plasma insulin concentration was determined
115
by enzyme immunoassay using insulin rat ELISA kit (EZRMI-13K) (EMD Millipore
116
Corporation, Billerica, MA, USA).
117
Liver Biopsy
118
Small liver sections were stained with hematoxylin and eosin (H&E) and oil red (O) to be
119
evaluated for Necro-Inflammatory grading and Fatty Droplets. Histological changes were
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
8 120
assessed by a modification of the scoring system for grading and staging for non-alcoholic
121
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
124
sections and stained with hematoxylin and eosin (H&E). Histopathologic examination
125
consisted of assessing steatosis grade and distribution with a score=0 (66%). Location was defined as steatosis
127
distribution with a score=0 (zone 3); score=1 (zone 1); score=2 (azonal) or score=3
128
(panacinar). Microvesicular steatosis was recorded as either score=0 (not present) or score=1
129
(present). Lobular inflammation was semi-quantified according to a score=0 (< 2 foci per
130
200x field); score=1 (2-4 foci per 200x field) or score=3 (>4 foci per 200x field).
131
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
135
(http://rsbweb.nih.gov/ij). Tissue sections were imaged at five high-power (400x) fields and
136
converted to 8-bit grayscale images. This was followed by an image threshold predefined
137
according to a rat liver section negative for steatosis and microvesicular steatosis on H&E
138
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%
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
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
158
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
162
kcal] was found to parallel that of weight gain, in which energy efficiency of the low protein
163
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).
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
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
173
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
184
among groups in terms of their percentages of hepatic dry weight or fat content. (Table1).
185
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
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
11 190
found to increase with increased P content of the diet. Plasma TG, total cholesterol, albumin
191
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
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
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
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
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
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
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
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
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.
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
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.
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
17 References
1. Wu G. Dietary protein intake and human health. Food Funct 2016;7(3):1251-65. 2. Geraghty AA, Lindsay KL, Alberdi G, McAuliffe FM, Gibney ER. Nutrition During Pregnancy Impacts Offspring’s Epigenetic Status—Evidence from Human and Animal Studies. Nutr Metab Insights 2015;8(Suppl 1):41. 3. Langley-Evans S, Sherman R, Welham S, Jackson A. Fetal Exposure to Maternal Low Protein Diets during Discrete Periods of Pregnancy Induces Hypertension in the Rat. Clin Sci 1997;92(s36):13P-P. 4. Bhasin KKS, van Nas A, Martin LJ, Davis RC, Devaskar SU, Lusis AJ. Maternal lowprotein diet or hypercholesterolemia reduces circulating essential amino acids and leads to intrauterine growth restriction. Diabetes 2009;58(3):559-66. 5. Meyer J. Interactions of dietary fiber and protein on food intake and body composition of growing rats. Am J Physiol 1958;193(3):488-94. 6. Noblet J, Henry Y, Dubois S. Effect of protein and lysine levels in the diet on body gain composition and energy utilization in growing pigs. J Anim Sci 1987;65(3):717-26. 7. White BD, He B, Dean RG, MARTIN R. Low Protein Diets Increase Neuropeptide Y Gene Expression in the Basomedial Hypothalamus of rats. J Nutr 1994;124:1152-60. 8. White B, Dean R, Martin R. An association between low levels of dietary protein, elevated NPY gene expression in the basomedial hypothalamus and increased food intake. Nutr Neurosci 1998;1(3):173-82. 9. Meyer J, Hargus W. Factors influencing food intake of rats fed low-protein rations. Am J Physiol 1959;197(6):1350-2. 10. Du F, Higginbotham DA, White BD. Food intake, energy balance and serum leptin concentrations in rats fed low-protein diets. J Nutr 2000;130(3):514-21.
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
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.
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
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.
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
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).
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
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.
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
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.
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
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.
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
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.
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
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.
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
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). .
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
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
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
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
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
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
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
Online Supporting Material
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
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
Online Supporting Material
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).
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
Online Supporting Material
30