Evaluation of Stable Isotope Labeling Technique in Measuring the Tissues Protein Fractional Synthesis Rates in Rats

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1 Available online at Annals of Clinical & Laboratory Science, vol. 45, no. 2, Evaluation of Stable Isotope Labeling Technique in Measuring the Tissues Protein Fractional Synthesis Rates in Rats Jihong Zhou 1*, Shengxian Fan 2*, Yacheng Cao 3, Mingfang Zhu 3, Yong Han 3, Xueying Cao 1, Yousheng Li 2, and Jieshou Li 2 1 Department of Burns and Plastic Surgery, Jinling Hospital, Nanjing University School of Medicine, Nanjing, 2 Department of Surgery, Jinling Hospital, Nanjing University School of Medicine, Nanjing, and 3 Institute of Soil Science, Chinese Academy of Sciences, Nanjing, China Abstract. Aims: The objective of this study was to evaluate the technique for measuring tissues protein fractional synthesis rates with [L- 15 N]Leucine. Materials and methods: Sixty male Sprague-Dawley rats were randomly assigned to different groups. After intravenous injection with [L- 15 N]Leucine, the enrichment of 15 N in different time intervals was measured with the same dosage and at the same time by variable dosage. Results: During the period from 0 to 30 minutes, there was nearly a linear increase in enrichment of 15 N in both the free amino acid pool and the bond amino acid pool in rats tissues, and the peak was achieved at 30 minutes. With the increase of dosage, the fractional synthesis rates rose. However, when the dosage was more than 1.0 mmol/kg, there was no significant discrepancy. Conclusions: For measuring the tissues protein fractional synthesis rates with [L- 15 N]Leucine in rats, 30 minutes post-injection is optimal, and 1.0 mmol/kg is the most suitable dosage. Key words: stable isotope, [L- 15 N]Leucine, protein fractional synthesis rates. Introduction To understand the control of protein homeostasis at the level of the intact animal, it is necessary to have methods to assess rates of protein synthesis and degradation in tissues in vivo. However, measurement of protein fractional synthesis rates (FSR) has proved less difficult, and several approaches have been widely used. In particular, two methods employing labeled amino acid administration, known as the constant infusion method and flooding method, have been very extensively used in animal studies [1,2]. Both of these approaches have now been modified for use with stable isotopes in human tissues. By contrast, the flooding method has certain practical advantages measurements can be made over a shorter period of time, and acute changes in protein synthesis are allowed. This method is therefore very well suited to studies in hospital patients, when several hours of infusion *These authors contributed equally to this work. Address correspondence to Dr. Yousheng Li, MD; Department of Surgery, Jinling Hospital, 305 East Zhongshan Road, Nanjing, , China; phone: ; fax: ; e mail: liys@ medmail.com.cn might be impractical and when a metabolic steady state might be difficult to maintain, particularly when studies involve tissues that can only be sampled during surgery [3]. The aim of this prospective study was to evaluate the optimal time interval and the most suitable dosage for measuring the tissues protein FSR, and provide important help to experimental and clinical medicine. Material and Methods Ethics statement. This study was carried out in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication revised 1985). Protocols were approved by Animal Care and Use Committee of Jinling Hospital. All surgery was performed under sodium pentobarbital anesthesia, and every effort was made to minimize suffering. Reagents. [L- 15 N]Leucine ( 15 N, 98%+) (Cambridge Isotope Laboratories, Inc, Andover, USA): the [L- 15 N] Leucine should be mixed with normal saline at 1mol/L before application, and kept under controlled conditions of 4 C, away from light /15/ by the Association of Clinical Scientists, Inc.

2 188 Annals of Clinical & Laboratory Science, vol. 45, no. 2, 2015 Animals. Sixty male Sprague-Dawley (SD) rats, weighing 180 to 220 g (Shanghai Experimental Animal Center, The Chinese Academy of Sciences, Nanjing, China), were used. Briefly, the rats were anesthetized with an intraperitoneal injection of ketamine (100 mg/ kg BW), and kept a left external jugular vein cannulation. Meanwhile, we used an anti-rotary transfusion device to connect the intravenous catheter to a micro-injection pump (Zhejiang Medical University, Zhejiang, China). 5% GS was infused at an initial rate of 10 ml/ kg body weight per hour. Two days later, we infused [L- 15 N]Leucine. All the rats were maintained in individual metabolic cages and allowed free access to water. Experimental design. Forty-two rats were randomly assigned into seven groups (n=6 for each group). [L- 15 N] Leucine was injected through intravenous catheter in a single dose of 0.5 mmol/kg and the samples were collected at 15 minutes and 0.5, 1, 2, 4, 8, 12 hours after the injection. The other eighteen rats were randomly assigned into three groups (n=6 for each group); rats in these three groups were respectively injected with [L- 15 N]Leucine in a single dose of 2.0, 1.0, and 0.25 mmol/kg. The samples were collected at 0.5 hour after the injection. Sample analysis. Blood (5ml) and tissues (liver, skeletal muscle, small intestine, g each) were collected while the rats were anesthetized with ketamine. Blood was collected in lithium heparin tubes and centrifuged at 1500g for 5 minutes, and then stored at -20 C until analysis. Tissues were flash-frozen in liquid nitrogen, and subsequently ground into a homogenized powder. The powder was then precipitated in 10 ml of 0.2 mol/l perchloric acid. 5 ml supernatant was taken into a centrifuge tube and centrifuged at 3000g for 15 minutes. Then, a suitable amount of KOH (1 mol/l) was added to neutralize HCLO4. After the reaction was completed, the pellets of KCLO4 were removed by centrifugation at 3000g for 15 minutes. The protein pellets were washed three times in 10ml of 0.2mol/L perchloric acid followed by digestion in 10ml of 0.2mol/L NaOH at 37 C for 1 hour. After re-precipitation with 10ml of 0.2mol/L perchloric acid, the pellets were washed twice with 5 ml of 0.2 mol/l perchloric acid and hydrolyzed in 5mL of 6mol/L HCL for 24 hours at 110 C. The samples (1ml of liquid samples, 1g of solid samples) were put into a dry kjeldahl flask together with 10ml of 3mol/L H 2 SO 4, and then boiled for 6 hours. The boiled liquid was put into a distiller and washed the kjeldahl flask with water for 4 to 5 times; however, the total water consumed should be no more than 35ml. All the liquid was distilled in 20 ml of 10 mol/l NaOH. The distillate was collected in a conical flask in which there were 5 ml of 0.01 mol/l H 2 SO 4. The conical flask was put in a boiling water bath while the distillate was 40ml. When the distillate was dried up, it could be applied to the measurement of enrichment of 15 N. Measurement of enrichment of 15 N. The protein FSR, defined as the percentage of tissue protein renewed by synthesis each day, was calculated from the following equation. EB and EF are the enrichment of 15 N of protein-bound and tissue-free leucine, and t is the period from injection of isotope to immersion of tissue into iced water. The enrichment of 15 N of protein-bound and tissue-free amino acid was detected by mass spectrometry (Finnigan MAT, San Jose, CA). Statistical analysis. FSR E E B( t) B(0 ) = t EF ( t) dt 0 All data were presented as mean ± SD. One-way analysis of variance was used to assess the means among groups, while the Dunnett s T3 test was used to assess pairwise multiple comparisons. Statistical Package for Social Sciences (SPSS Inc, Chicago, Ill) version 11.0 was used for data analysis. Statistical significance was accepted at the p<0.01 level. Results The enrichment of 15 N of free amino acid pool in rats tissues at different time intervals. From 0 to 30 minutes, there is an almost linear increase in enrichment of 15 N of free amino acid pool in rats tissues, with the peak time at 30 minutes (liver atoms%, skeletal muscle 0.48 atoms%, small intestine atoms%). Then, the enrichment of 15 N decreases mildly between 1 to 4 hours and maintains the plateau before 12 hours. Even at 12 hours, the enrichment of 15 N is higher than the natural one (0.364 atoms%) (Figure 1). The enrichment of 15 N of bond amino acid pool in rats tissues at different time intervals. The enrichment of 15 N of bond amino acid pool in rats tissues also rises linearly from 0 to 30 minutes, and attains the peak at 30 minutes (liver atoms%, skeletal muscle atoms%, small intestine atoms%), and then declines gradually. However, the decline is not obvious from 30 minutes to 12 hours; even at 12 hours, the enrichment of 15 N is higher than the natural one (0.364 atoms%) (Figure 2).

3 Measurement of protein FSR using stable isotope 189 increasing dosage of [L- 15 N] Leucine injected intravenously, but the increase in enrichment of 15 N of the bond amino acid pool in rats tissues is not so significant as that of the free amino acid pool (Figure 4). Figure 1. The enrichment of 15 N of free amino acid pool in rats tissues at different time intervals. The enrichment of 15 N of free amino acid pool increases from 0 to 30 minutes, and the peak time is at 30 minutes. All data are presented as mean ± SEM. *P<0.01. Figure 2. The enrichment of 15 N of bond amino acid pool in rats tissues at different time intervals. The enrichment of 15 N of bond amino acid pool increases from 0 to 30 minutes, and the peak time is at 30 minutes. All data are presented as mean ± SEM. *P<0.01. The protein FSR in tissues with variable dosage at same time intervals. The protein FSR in tissues rises with increasing dosage of intravenously injected [L- 15 N]Leucine. Among the three groups with dosages of [L- 15 N]Leucine 1.0 mmol/kg, 0.5 mmol/kg, and 0.25 mmol/kg, the discrepancy is significant. There is no significant variation between the two groups with [L- 15 N] Leucine dosages of 1.0 mmol/ kg and 2.0 mmol/kg. The only differences among the tissues are the values (Table 1). The enrichment of 15 N of free amino acid pool in plasma and tissue with variable dosage. The enrichment of 15 N in the free amino acid pool in tissues is significant higher than that in plasma when injected with dosages of 1.0mmol/kg and 2.0mmol/kg (Table 2). Discussion The enrichment of 15 N of free amino acid pool in rats tissues with variable dosage at same time intervals. With the increase in the dosage of [L- 15 N] Leucine injected intravenously, the enrichment of 15 N of free amino acid pool in rats tissues increased significantly, and the only difference among the tissues are the values (Figure 3). The enrichment of 15 N of bond amino acid pool in rats tissues with variable dosage at same time intervals. The enrichment of 15 N of the bond amino acid pool in rats tissues rises along with the Advances in the understanding of the physiological control of protein synthesis in animal and human tissues in vivo have owed much to the use of two isotopic techniques the constant infusion method and the flooding method [1,3]. While these two approaches in general provide reasonable agreement, particularly in animals, there are instances when they disagree, requiring evaluation of the assumptions on which each method is based. For the constant infusion method, the important question is how to measure the enrichment of the direct precursor of protein synthesis and whether a

4 190 Annals of Clinical & Laboratory Science, vol. 45, no. 2, 2015 Figure 3. The enrichment of 15 N of free amino acid pool in rats tissues with variable dosage at same time intervals. With the increasing of the dosage of [L- 15 N]Leucine injected intravenously, the enrichment of 15 N of free amino acid pool in rats tissues increased significantly. All data are presented as mean ± SEM. P<0.01. is fully resolved, but our preference has been to use the flooding method, most recently with [L- 15 N]Leucine, because there is little indication that the food does indeed alter protein synthesis. Moreover, the flooding method has certain practical advantages, such as that measurements can be made over a shorter period of time. It also allows acute changes in protein synthesis [4,5]. The method is therefore very well suited to studies in hospital patients, when several hours of infusion might be impractical and a metabolic steady state difficult to maintain, particularly when studies involve tissues that can only be sampled during surgery. Therefore, our study used the flooding method to evaluate the optimal time interval and the most suitable dosage for measuring the tissues protein FSR. Figure 4. The enrichment of 15 N of bond amino acid pool in rats tissues with variable dosage at same time intervals. With the increasing of the dosage of [L- 15 N]Leucine injected intravenously, the enrichment of 15 N of bond amino acid pool in rats tissues increased significantly. All data are presented as mean ± SEM. P<0.01. measurement of, for example, ketoisocaproate (KIC) will adequately serve under all metabolic and nutritional conditions. For the flooding method, the relevant question is whether the increased concentration of the labeled amino acid will alter the rate of protein synthesis. Neither of these questions Theoretically, the rate of protein synthesis can be measured by detecting the enrichment of some tracer amino acid, and should follow these steps: choosing the tracer amino acid; changing the dosage of tracer amino acid; choosing the precursors to measure; setting the time to measure [4]. Our study was based on the above theories. First, we simplified the application of tracer amino acid, that is applying flooding method. However, in order to avoid affecting the protein metabolism by injecting a large amount of amino acid, we initially used a smaller dosage of [L- 15 N]Leucine, and then measured the enrichment of 15 N in both free and bond amino acids in different tissues (liver, small intestine, skeletal muscle) at different times. The present

5 Measurement of protein FSR using stable isotope 191 Table 1. Protein FSR (% d -1 ) in different tissues with different dosage of [L- 15 N]Leucine. group n liver skeletal muscle small intestine ±0.020 # 0.354±0.052 # 2.286±0.052 # ±0.014 # * ±0.057 # * 5.717±0.057 # * ±0.014* 1.752±0.063* 6.788±0.054* ±0.015* 1.757±0.033* 6.822±0.027* The protein FSR in tissues is elevated along with the increasing dosage of [L- 15 N]Leucine injected intravenously. Among the three groups with the dosage of [L- 15 N]Leucine injected intravenously 1.0mmol/kg, 0.5mmol/kg and 0.25mmol/kg, the discrepancy is significant. There is no significant variation between the two groups with the dosage of [L- 15 N]Leucine 1.0mmol/kg and 2.0mmol/kg. All data are presented as mean±sem. P<0.01. # P<0.01 compared group 0.25 and group 0.5, as determined by analysis of variance followed by Student-Newman-Keuls test. *P<0.01 compared group 0.5 and group 1.0, as determined by analysis of variance followed by Student-Newman- Keuls test. P>0.05 compared group 1.0 and group 2.0, as determined by analysis of variance followed by Student- Newman-Keuls test. Table 2. The enrichment of 15 N of free amino acid pool of plasma and different tissues. group n plasma liver skeletal muscle small intestine ± ±0.021 # 0.622±0.013* 0.515± ± ±0.020 # 0.856±0.019* 0.666±0.013 The enrichment of 15 N of free amino acid pool in tissues is significantly higher than that in plasma while injected with the dosage of 1.0mmol/kg and 2.0mmol/kg. All data are presented as mean ±SEM. P<0.01. # P<0.01 compared to plasma, as determined by analysis of variance followed by Student-Newman-Keuls test. *P<0.01 compared to plasma, as determined by analysis of variance followed by Student-Newman-Keuls test. P<0.01 compared to plasma, as determined by analysis of variance followed by Student-Newman-Keuls test. study demonstrated that the peak time was 30 minutes; whether free or bond amino acids, the only difference was the value. Therefore, the recommended measurement time is recommended 30 minutes. As for the selection of dosage, by measuring the enrichment of 15 N in different time intervals with the same dosage of [L- 15 N]Leucine, we found that as the the dosage of [L- 15 N]Leucine injected intravenously increased, the enrichment of 15 N in the free amino acid pool in rats tissues increased significantly. In contrast, the enrichment of 15 N in the bond amino acid pool in rats tissues rose evidently but not as significantly as in the free amino acid pool. Similarly, the protein FSR in tissues rose with the increasing dosage of [L- 15 N]Leucine injected intravenously. Among the three groups with intravenously injected dosages of [L- 15 N]Leucine of 1.0 mmol/kg, 0.5 mmol/kg and 0.25 mmol/kg, the discrepancy is significant. There is no significant variation between the two groups whose dosages of [L- 15 N]Leucine were 1.0 mmol/kg and 2.0 mmol/ kg. The only differences among the tissues are the values. These results show that when the dosage exceeds 1.0mmol/kg, the protein FSR is not elevated significantly as the dosage of [L- 15 N]Leucine increases. Therefore, when measuring the protein FSR in tissues, the most suitable dosage is 1.0 mmol/kg. The L-type amino acid is the most common tracer amino acid, among which the most commonly used is leucine ( 13 C or 15 N labeled), followed by lysine, glycine, valine, and phenylalanine [5-7]. Leucine is one of the essential amino acids and is necessary for the process of tissue protein synthesis [8]. The technology of labeling leucine with 15 N is comparatively mature. The above advantages are the reasons why we selected [L- 15 N]leucine as the tracer amino acid.

6 192 Annals of Clinical & Laboratory Science, vol. 45, no. 2, 2015 In conclusion, data from our study show that when measuring tissue protein fractional synthesis rates (FSR) with [L- 15 N]Leucine in rats, 30 minutes post-injection is the optimal time, and 1.0 mmol/ kg is the most suitable dosage. This method does not require a prolonged steady state or much time, and is much simpler and more accurate and effective for clinicians to implement. Acknowledgment This work was supported by a grant from the National Natural Science Foundation of China (no and no ). References 1. Lengqvist J, Sandberg A. Stable isotope labeling methods in protein profiling. Methods Mol Biol 2013; 1023: Wilkinson DJ, Franchi MV, Brook MS, et al. A validation of the application of D(2)O stable isotope tracer techniques for monitoring day-to-day changes in muscle protein subfraction synthesis in humans. Am J Physiol Endocrinol Metab 2014; 306: E Garlick PJ, McNurlan MA, Essen P, Wernerman J. Measurement of tissue protein synthesis rates in vivo: a critical analysis of contrasting methods. Am J Physiol 1994; 266: E Wolfe RR. Skeletal muscle protein metabolism and resistance exercise. J Nutr 2006; 136: 525S-528S. 5. Parks EJ, Matthews DE. A.S.P.E.N Research Workshop on using tracers to measure carbohydrate, fat, and amino acid metabolism in humans. JPEN J Parenter Enteral Nutr 2004; 28: Smith K, Barua JM, Watt PW, Scrimgeour CM, Rennie MJ. Flooding with L-[1-13C]leucine stimulates human muscle protein incorporation of continuously infused L-[1-13C]valine. Am J Physiol 1992; 262: E Zeisel SH, Waterland RA, Ordovás JM, Muoio DM, Jia W, Fodor A. Highlights of the 2012 Research Workshop: Using nutrigenomics and metabolomics in clinical nutrition research. JPEN J Parenter Enteral Nutr 2013; 37: Connell A, Calder AG, Anderson SE, Lobley GE. Hepatic protein synthesis in the sheep: effect of intake as monitored by use of stable-isotope-labelled glycine, leucine and phenylalanine. Br J Nutr 1997; 77: