Effect of dietary stable isotopic ratios of carbon and nitrogen on the extent of their incorporation into tissues of rats
© Lv et al.; licensee BioMed Central Ltd. 2012
Received: 26 March 2012
Accepted: 9 May 2012
Published: 31 May 2012
This study was conducted to investigate the effect of different dietary ratios of 13 C to 12 C or 15 N to 14 N on their relative incorporation into tissues. Eighty male rats were used in two 21-day feeding trials in which they were fed diets with either high δ13C levels (δ13C = −13.89‰ and δ15N = 2.37‰ in experiment 1 and δ13C = −19.34‰ and δ15N = 4.73‰ in experiment 2) or low δ13C levels (δ13C = −17.90‰ and δ15N = 3.08‰ in experiment 1 and δ13C = −21.76‰ and δ15N = 0.53‰ in experiment 2), meanwhile, the dietary δ15N levels were designed to two ranks. Blood, liver, adipose and muscle tissues were collected on day 0, 3, 7, 14, and 21 for determination of 13 C, 12 C, 15 N and 14 N isotopes. Rat growth rate, antioxidant capacity and metabolic parameters were also assessed. The results indicate that adipose tissue tend to deplete 13 C before the stable isotopic ratios achieved final equilibrium. Therefore, feeds with different isotopic signatures had different incorporation rates into tissues. Low dietary 13 C levels decreased tissue δ13C values whereas high dietary 13 C levels did not alter tissue δ13C values during the 21-d experiment. Blood δ15N values were a reliable parameter in assessing the relative contribution of dietary nitrogen to tissues. This study revealed a relationship between dietary isotopic signatures and their incorporation rates into rat tissues. However, more studies are needed to illustrate the mechanism through which dietary isotopic ratios influence the extent of isotopic incorporation into the tissues.
KeywordsCarbon Diet Nitrogen Stable isotopes
Use of stable isotopes has attracted a great deal of interest in physiological and metabolic research as many researchers are unwilling to use radioactive isotopes . The amount of carbon stable isotope 13 C varies between the C3 and C4 plants due to their use of different photosynthesis pathways while the abundance of stable nitrogen isotope 15 N in plants resembles that of their growing circumstance [2, 3]. Since every feedstuff is characterized by its natural stable isotopic signature [4, 5], the analysis of stable isotopes in tissues is proposed as a method to evaluate the relative contributions of nutrients from different feed sources to those deposited in tissues [6, 7].
Many models have been established to estimate the fractional contribution of various isotope profiles in the diet to those deposited in tissues including one compartment, two compartment, multi-compartment and mixed models . However, the effect of different dietary ratios of 13 C to 12 C or 15 N to 14 N on their relative incorporation into tissues remains unknown.
Numerous factors such as diet quality, nutritional status, body size, age, dietary ontogeny, tissue and elemental composition have been reported to affect the extent of deposition of 13 C and 15 N into tissues [9–12]. The relationship between discrimination factors and dietary isotopic ratios is consistent and dietary isotopic values have been shown to explain 51 % of the variation in isotopic discrimination  . In controlled experiments, dietary isotopic values explained 60 % to 98 % of the variation in isotopic discrimination in different tissues of rats . The stable isotopic analysis, which has been used as an important tool for so many years, is conducted to investigate the turnover and deposition of nutrients from the macroscopic view. It is aimed at not a certain nutrient but the relationship between various nutrients. However, few studies have determined the effect of different dietary ratios of 13 C to 12 C or 15 N to 14 N on their relative incorporation into tissues. If these discrimination factors are not fully understood, it is impossible to estimate the relative contributions of nutrients by using stable isotopic analysis. Therefore, the present study was carried out to investigate the effect of dietary stable isotopic ratios on their isotopic discrimination in tissues by feeding rats diets providing high or low stable isotopic abundance.
Experimental design and animal model
Sprague–Dawley rats were used as the animal model to do the research. These trials were conducted according to protocols approved by the China Agricultural University Animal Care and Use Committee. Eighty, healthy male, 35 days old rats were used. Every two rats were housed in one cage. The sawdust was laid at the bottom of the cages and replaced with the new and clean sawdust to keep the cages neat every other day. The rats were reared individually on a 12-hour light 12-hour dark cycle and fed commercial feed for a 7 day adaption period before the experiment started. Rats had free access to feed and water. All rats were treated in accordance with the “Guide for Care and Use of Agricultural Animals in Research and Teaching” .
Ingredient and chemical composition of the experimental diets
Gross energy, MJ/kg
Crude protein, %
Methionine + cysteine, %
In the second trial, 38 male rats with an average weight of 163.8 ± 3.3 g were randomly divided into two groups (n = 19) and fed diets in which the stable carbon isotope ratios were formulated to be lower than that of the tissues of rats (i.e. -19‰ δ13C value) with the exception of adipose tissue. Rats in group 1 were fed a diet in which the carbon and nitrogen isotopic values were −21.76‰ and 0.53‰, respectively. Rats in group 2 were fed a diet with a carbon isotopic value of −19.34‰ and the nitrogen isotopic value of 4.73‰ (Table 1). The rats were fed the experimental diets for 21 days and rat weight and feed intake were determined weekly to calculate average daily gain and average daily feed intake. Additionally, plasma parameters were determined to examine the influence of dietary stable isotopic ratio on rat metabolic physiology.
The experimental diets were formulated to be isocaloric (18.7 ± 0.2 MJ/kg) and isonitrogenous (20.78 ± 0.18 % CP) by using different ratios of fish meal, corn, casein, soybean meal and wheat middlings. The stable isotope ratios of these five ingredients were measured prior to the initiation of the study and the proportion of these ingredients in the diets were set in order to obtain the target carbon and nitrogen isotopic ratios. The chemical composition of the experimental diets and their stable carbon and nitrogen isotopic ratios are shown in Table 1.
Chemical analysis of feed ingredients
The feed ingredients were determined for gross energy by adiabatic oxygen bomb calorimeter (Parr Instruments, Moline, IL), crude protein through Kjeldahl N , calcium (procedure 4.8.03, AOAC, 2000)  and phosphorus (procedure 3.4.11, AOAC, 2000) . Amino acid in the feed of experiment 1 and 2 were analyzed according to the procedures 4.1.11 of AOAC (2000)  and the procedure 998.15 of AOAC (1995) .
Sample collection and preparation
In order to collect tissue samples, 50 mg/kg BW of sodium pentobarbital (Beijing Solarbio Science & Technology Company, Beijing, China) was injected into the abdominal cavity of the rats. The abdominal cavity was opened and 5 mL of blood was obtained from the hepatic artery using 9 mL heparinized tubes (Greiner Vacuette, Monroe, NC). The rats were then killed by cervical dislocation. Samples of liver, muscle and adipose tissue were then obtained.
The blood samples were separated into two parts, one part was stored at −80°C for stable isotope analysis, and the other part was centrifuged at 1,200 × g for 15 min to obtain plasma and was then immediately stored at −20°C until analysis. The liver sample was collected from the left lobe and rinsed briefly in physiological saline (0.9 %, m/v) to diminish blood contamination of the tissue. The adipose tissue on the bilateral paradidymis was collected from the belly. The gastrocnemius on the left leg was removed for use as a muscle sample. One part of the tissues were for the determination of stable isotopes, one was for the analysis of antioxidant and metabolic parameters, and one was for bulk density measurement.
All samples involving feed ingredients, feed and tissues were freeze-dried at −40°C for 48 h (Virtis Genesis-250es; SP Scientific, Stone Ridge, NY). The dried samples were milled into finer particles and then passed through 80 mesh screen. Then, the appropriate quantities were removed to tin capsules and compactly packaged to ensure the samples did not leak from the tin capsules. Finally, the tin capsules were weighed for analysis.
Stable isotope analysis
Bulk density measurement
Antioxidant capacity and metabolic parameters
Liver samples were immediately homogenized in ice-cold phosphate buffered saline (10 mL/g tissue) with a glass homogenizer and the homogenate was centrifuged for 15 min at 1,200 × g. The supernatant obtained and the plasma were used to assay for total antioxidant capacity (T-AOC), total nitric oxide synthase (TNOS), inducible nitric oxide synthase (iNOS), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), glutamic-pyruvate transaminase (GPT), glutamic-oxaloacetic transaminase (GOT), urea nitrogen, maleic dialdehyde (MDA) and creatinine. All these enzymes activities were determined using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing City, China) in accordance with the manufacturer’s instructions.
The influence of the dietary treatments on 13 C and 15 N incorporation, growth, antioxidant capacity as well as metabolic parameters were analyzed using the one-way time-repeated GLM procedures of SAS (8.02; SAS Institute Inc., Cary. NC, USA). The interaction between group and time-repeated was also considered. Differences were considered significant when P < 0.05.
Performance and organ weights
There were no significant differences in growth rate and feed intake of rats which received the different stable isotopic ratio diets in either experiment 1 or 2 (data was not shown). Weights of kidney and heart were not altered by the dietary treatments and neither were the bulk density of liver, kidney and heart (data was not shown).
Antioxidant capacity and critical metabolic parameters
Plasma parameters and hepatic indexes were determined to investigate the influence of different stable isotopic ratios on the antioxidant and metabolic status of the body. In trial 1, the activities of plasma GPT, GOT, T-AOC, SOD, GSH-Px, creatinine, TNOS, iNOS, plasma urea nitrogen and MDA did not differ between rats fed the two dietary treatments (data was not shown). Similarly, no differences were observed in the activities of T-AOC, SOD, MDA, iNOS, TNOS, GSH-Px in the liver along with the period of dietary treatment extending. However, in trial 2, the activity of plasma GOT (6.1 IU/L vs. 15.8 IU/L, P = 0.05) and plasma urea nitrogen (18.3 mmol/L vs. 31.0 mmol/L, P = 0.06) tended to decrease in the rats that ingested the diet with δ13C of −21.76‰ and δ15N of 0.53‰, whereas plasma MDA tended to increase (13.3 nmol/mL vs. 10.9 nmol/mL, P = 0.06) compared with those of rats that ingested δ13C of −19.34‰ and δ15N of 4.73‰.
Stable isotope abundance in tissues
Stable carbon isotopic values showed significant tissue-specificity between adipose tissue and liver, muscle, blood; while each tissue, involving liver, blood, and muscle, had their own nitrogen isotope signature. In particular, both in trial 1 and 2, the adipose tissue 13 C value was about 3.0‰ lower than that of the other tissues, while 13 C values of the liver, blood and muscle were similar. There were observed relationships among 15 N values of the different tissues with liver 15 N > blood 15 N > muscle 15 N.
Influence of diet δ13C values (‰) on δ13C incorporation into tissues in Trial 1
δ13C = −17.90‰
δ13C = −13.89‰
Group × Time
δ15N = 3.08‰
δ15N = 2.37‰
Influence of diet δ15N values (‰) on δ15N incorporation into tissues in Trial 1
δ13C = −17.90‰
δ13C = −13.89‰
Group × Time
δ15N = 3.08‰
δ15N = 2.37‰
Influence of diet δ13C values (‰) on δ13C incorporation into tissues in Trial 2
δ13C = −21.76‰
δ13C = −19.34‰
Group × Time
δ15N = 0.53‰
δ15N = 4.73‰
Influence of diet δ15N values (‰) on δ15N incorporation into tissues in Trial 2
δ13C = −21.76‰
δ13C = −19.34‰
Group × Time
δ15N = 0.53‰
δ15N = 4.73‰
In the present study, we investigated whether different dietary stable isotope ratios influenced rat growth rate, critical parameters relating to body antioxidant capacity or metabolism. Our results showed that dietary isotopic signature had no or little effect on rat body antioxidant capacity, metabolism or growth rates.
Isotopic ratios in foods consumed are reflected in the tissues, proportionate to the amount assimilated for each ingredient source, after accounting for discrimination against heavier isotopes in the digestion and assimilation process [20, 21]. Thus, stable isotope analysis is frequently used to quantify contributions of different food sources to an animal’s diet and nutrient routing [14, 22], which requires a priori estimates of discrimination factors. However, the discrimination factors, particularly, the diet-dependent discrimination factors that influence isotopic incorporation into the tissues remain unknown. It has been emphasized that potential sources of variation for discrimination factors should not be overlooked, which underpins the isotope model used in studies of ecology and animal nutritional physiology .
Many studies have attempted to determine the abundance of 13 C and 15 N in animal tissues and these studies have shown that stable isotopic signature varies among tissues [9, 23–25]. The reasons for tissues differing in δ13C and δ15N values were summarized by Martínez del Rio . For δ13C values, the adipose tissue content and amino acid composition of tissues are two important candidates. It has been proved that adipose tissue synthesis is accompanied by depletion of 13 C . Meanwhile, the difference in δ15N value among tissues is due to differences in their amino acid content and the isotopic composition of individual amino acids. However, effect of different levels of dietary stable isotopic ratios relative to the animal tissues on their incorporation into tissues remains unrevealed.
Prior to the initiation of this experiment, when the rats were fed commercial diets, the ranking of tissue isotopic abundance for 13 C was liver, muscle and blood > adipose tissue. The same distribution order was maintained when rats ingested different 13 C diets for the 21-day experiment. These results were somewhat different from some other reported studies. For example, tissue δ13C values were shown to vary among the tissues with the order of abundance being hair > brain > muscle > liver > adipose tissue in the gerbil . There are limited data on the tissue 13 C turnover of rats, although the half-life of blood carbon was reported as 24.8 days . We designed the duration of the present trials based on the mouse, using a half-life of liver carbon of 6.4 days  and that of muscle of 16.5 days . Since rats have a slower metabolic rate per unit mass than mice , the half-life for rat tissue carbon might be longer than that of mice. No difference was observed in the present study for muscle, liver and blood 13 C which might be partly due to the fact that these tissues had not equilibrated with dietary 13 C during the 21 day experiment. Before isotopic incorporation achieves equilibrium, the metabolic rate that is tissue protein turnover, including synthesis and catabolism, shapes the stable isotopic composition of the bodies as well as the tissues, although the growth, metabolic rate and nutritional status of the animal providing the tissues would also influence it to certain extent .
Regardless of diet, adipose tissue δ13C value was about 3.0‰ lower than that of other tissues, which was in accordance with previous studies . It has been suggested that adipose tissue depletes 13 C faster than other tissues, whereas liver, muscle, blood and hair enrich 13 C [8, 14, 24]. Increases in the mass of tissues through growth have an additional dilution affect which results in faster equilibration to the new diet than would occur by metabolic turnover alone .
In the current study, the abundance of tissue 15 N values were ranked as liver > blood > muscle. The discrimination factor (δ15N tissue - δ15N diet) varied from 1.14‰ to 5.81‰, which is similar to previous studies [14, 30].
Most animal tissues undergo continual incorporation of stable isotopes including 13 C and 15 N, and so the stable isotope values change over time depending on the diet fed and tissue-specific metabolic rates . In the present study, an interesting phenomenon was observed in that different dietary δ13C values, compared with that of the tissues, differentially modulated the stable carbon isotope incorporation into tissues. When rats were fed low stable carbon isotopic ratio diets, δ13C values in liver, muscle and blood decreased significantly which is consistent with previous research . However, we found that when rats were fed high isotopic ratio diets, the δ13C values of the tissues were not altered during the 21-day experiments. It seems that the tissues tend to assimilate 12 C rather than 13 C and remain at a certain δ13C value. The finding that adipose tissue δ13C values were unaltered by dietary stable carbon isotope values in the short term might be due to the fact that adipose tissue has the highest percentage of carbon element among the tissues analyzed.
Isotopic composition turnover rates vary among tissues, with high rate in tissues such as blood and liver, somewhat lower rates in muscle, and low rates in long-lived tissue such as bone . In the present study, it was surprising to find that the liver δ15N value was rapidly decreased at day 3 and then remained constant over the remaining 21 days of the dietary treatments. However, we cannot explain why the liver δ15N value was decreased at day 3 and then remained the δ15N level. Since liver is a central organ of metabolism in the body, perhaps the liver isotopic composition change was balanced by the bodies’ metabolism. Blood δ15N value also decreased significantly at day 3 or 14, and further decreased to day 21. The time divergence existing in the two trials might be due to the protein quality of the diets. Because a 21-day dietary treatment is essentially a short-term study, we did not observe a decrease in muscle δ15N value during the experiments.
Combining the results of two feed trials, it could be concluded that the liver is the most sensitive organ reflecting the difference of dietary δ15N values, the muscle followed second among three tested organs. However, the muscle was resistant to further decrease in δ15N once it obtained a new equilibrium of δ15N values. Blood δ15N value was similar between dietary treatments but it was decreased along with the increased duration of rat exposure to the dietary treatment, which could be accounted for that the difference of δ15N between dietary treatments is far smaller than that between the diets and the initial blood δ15N. In fact, blood δ15N reduced linearly together with the increased length of rat exposure to the lower δ15N diets, which suggested that blood δ15N values be suitable for assessing the extent of dietary nitrogen contribution to the rat’s tissues. Furthermore, the relationship between dietary isotopic ratios and their incorporation rate in tissues warrant more studies. Particularly, in estimating discrimination factors of stable isotopes, the half-life of the stable isotopes in given tissues should be further researched.
Collectively, dietary stable isotopic ratios of carbon or nitrogen may play a vital role in estimating their incorporation into the tissues in different animals. Dietary 13 C incorporation had been shown to be more complex than 15 N. In the short term, before the stable isotopes achieved equilibrium, the tissues tend to deplete 13 C during the turnover process. Therefore, different levels of dietary δ13C values have been shown to have different incorporation rates into the tissue. Lower dietary δ13C values decrease the tissue δ13C values, whereas the higher dietary δ13C values did not alter the tissue δ13C values as quickly.
Total antioxidant capacity
Total nitric oxide synthase
Inducible nitric oxide synthase
This study was supported by the Program for New Century Excellent Talents in University (NCET-06-0115).
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