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l -[1-13C] phenylalanine breath test results reflect the activity of phenylalanine hydroxylase in carbon tetrachloride acute injured rat liver
Life Sciences 78 (2006) 838 – 843 www.elsevier.com/locate/lifescie
l-[1-13C] phenylalanine breath test results reflect the activity of phenylalanine hydroxylase in carbon tetrachloride acute injured rat liver Wei-Li Yan a,b,*, Da-Yu Sun a, Xiang-Tong Lin b, Yi-Bin Jiang a, Xu Sun a a b
Department of Gastroenterology, Huashan Hospital Affiliated to Fudas University, Shanghai 200040, P.R. China Department of Nuclear Medicine, Huashan Hospital Affiliated to Fudan University, Shanghai 200040, P.R. China Received 7 March 2005; accepted 23 May 2005
Abstract l-[1-13C] phenylalanine breath tests (PheBTs) have been used to determine the hepatocyte functional capacity of patients. This study investigated the relationship between the PheBT parameter 13C excretion rate constant (PheBT-k) and activity of the phenylalanine metabolic ratelimiting enzyme phenylalanine hydroxylase (PAH) in rat liver. We noted that the time-course curves of 13C excretion presented as a single peak, which appeared 2 min after administration of l-[1-13C] phenylalanine (13C-Phe). 13C excretion during exhalation can be divided into a slow phase and a rapid phase. The PheBT-k in rats with carbon tetrachloride acute liver injury was.significantly lower than that of control rats. The rapid phase 13 C disposition constants of the acute liver injured rats did not differ from that of the controls. The peak value of 13C abundance in the breath of the acute liver injured rats was markedly higher than that of the control group. Total liver PAH activity in the acute liver injured rats was significantly lower than that in the control group. PheBT-k was highly correlated with the total activity of liver PAH (r = 0.92, P < 0.001). The present findings indicate that PheBT results reflect PAH activity levels. The PheBT-k parameter is a sensitive index that can be used to evaluate PAH function in the liver. In addition we demonstrated that the rodent model used in this study is a valuable tool for basic research studies of the breath test. D 2005 Elsevier Inc. All rights reserved. Keywords: l-[1-13C] phenylalanine breath test; Liver; Rat; Phenylalanine hydroxylase; Activity;
Introduction 13
C breath tests have been employed in nutriology, pharmacology and pharmacokinetics, especially in the diagnosis of digestive system diseases, including dysfunction of the pancreas and liver disease (Romagnuolo et al., 2002). The liver mediates critical physiological functions that involve complex biochemical reactions. Timely and accurate measurement of indices of liver function is important for the diagnosis and monitoring of liver diseases, the evaluation of treatment outcome,.assessment of long-term prognosis, and the evaluation of the risks of surgical intervention and liver transplantation. Several 13C-labelled drugs have been used as substrates in breath tests to study of various metabolic pathways in the liver (hepatocyte microsomes, cytoplasm and mitochondria) and to * Corresponding author. Department of Gastroenterology, Huashan Hospital Affiliated to Fudan University, 12 Central Wurumuqi Road, Shanghai 200040, P.R. China. Tel./fax: +86 2150892400. E-mail address: [email protected] (W.-L. Yan). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.05.073
13
C excretion rate constant
investigate hepatocyte function and liver functional reserve (Armuzzi et al., 2002). Burke et al. (1997) was the first to utilize phenylalanine breath tests (PheBTs) to determine the hepatocyte functional capacity in patients with end-stage liver diseases. They found that 13CO2 excretion decreased by up to 80% after intake of l[1-13C] phenylalanine (13C-Phe). Similar results were subsequently obtained in different populations by other researchers (Tugtekin et al., 1999; Lara Baruque et al., 2000; Kobayashi et al., 2001; Ishii et al., 2003a; Festi et al., 2005). Our clinical research has also supported the clinical value of PheBTs (Yan et al., 2003; Sun et al., 2003). It has been reported that patients with liver disease have a diminished capability to eliminate phenylalanine from plasma (Heberer et al., 1980). It is now an accepted notion that those 13CO2 excretion deficits are due to a reduction of the liver’s capability to oxidize phenylalanine. However the lack of direct experimental evidence for this hypothesis might limit the utilization of the breath test (Rating and Langhans, 1997). Recently, in vivo experiments showed of PheBT results can reflect the activity of phenylalanine
W.-L. Yan et al. / Life Sciences 78 (2006) 838 – 843
hydroxylase (PAH) in galactosamine prostrate livers (Ito et al., 2001) and in two thirds partial hepatectomized rat livers (Ishii et al., 2002). However, such experimental models are not instructive for most clinical cases of slight liver injury. There are two kinds of animal breath tests, the metabolic container method.(Schaad et al., 1995; Schoonjans et al., 2002) and the breath mask method (Ito et al., 2001; Ishii et al., 2002; Suzuki et al., 2002), but both have some deficiencies. In this study, we employed an animal model of the breath test developed in our laboratory to determine the activity of PAH, the rate-limiting enzyme of phenylalanine metabolism in the rat liver. We also investigated the relationship between the parameters of PheBT and PAH activity, and sought valid parameters for the animal breath test. Materials and methods Reagents l-[1-13C] phenylalanine (purity 99%, 13C abundance 99%) powder was purchased from ISOTEC Co. (Miamisburg, Ohio, USA). l-phenylalanine, 6-methyl-tetrahydropterin, dihydropterin reductase (DHPR) and catalase were obtained from Sigma (St. Louis, USA). Sodium pentobarbital and magnesium sulfate were obtained from Shanghai Chemical Reagent Co. (Shanghai, China). Lowry protein assay kits were purchased from Shenneng Bochai Biotech Co. (Shanghai, China). Serum liver function test kits were obtained from the Biotech Center, Shanghai Navy Medical Institution (Shanghai, China). Animals Inbred male Sprague –Dawley (SD) rats were provided by the Experimental Animal Center, Fudan University, housed in the specific pathogen-free (SPF) animal rooms, and given free access to water and standard rat chow. Initially, the rats weighed 220 –230g. After feeding for one week, twenty rats weighing 280 –290 g were selected for the experiment. The rats were divided randomly into two groups of 10 rats each. This study was performed according to our institutional guidelines. Acute liver injury model In the liver injury group, rats were treated orally with 2 ml/ kg of carbon tetrachloride (CCl4) at a 60% volume ratio in olive oil after 8 h of fasting with free access to water. After 4 h, the rats were treated with another dose of CCl4. In the control group, rats received only the same volume of olive oil. All of the rats were fasted for 14 h more and weighed before the breath test. Animal model of breath test After weighing, the SD rats were anesthetized by intraperitoneal injection of 50 mg/kg of 5% sodium pentobarbital. The anesthetized rats were fixed on a small animal dissecting table in a supine position. An incision was made at the center of the
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neck, followed by separation of the trachea, making a Tshaped opening at the free end, and inserting a troclear cannula that was fixed by ligation with nylon thread. The operating surface was covered with saline-soaked gauze. The temperature of viscera was kept at 37.5 T 0.5 -C during the experiment. Mechanical ventilation was performed with a small animal breath machine that supplied air containing 21% oxygen and discharged the air directly into air. The tidal volume of mechanical.ventilation was 2.5 ml, ventilation frequency was 52 times/min, and the exhalation/inspiration ratio was 1 : 1 (Bohlen and Nase, 2002; Harf et al., 1983; Torbati et al., 1999). After 15 min of ventilation, a 10 ml breath collection tube (Labco Ltd., Buckinghamshire, UK) was used to collect exhaled air for 20 s, which acted as background. A 1.6% solution of 13C-Phe saline was introduced into the caudal vein with a 13C-Phe dosage of 20 mg/kg of body weight. Exhaled air samples (20 s duration) were collected immediately, and another 28 samples was collected each minute for 20 min and then every 5 min between 20 and 60 min after injection. 13C abundance in the samples was determined with an air isotope ratio mass spectrometer to obtain a y 13C () value at different time points: y 13C () = [(13C atom % / 13C atom %std) 1] 1000. In the formula, 13C atom % equals ( 13C atom / 12 C atom) 100, 13 C atom %std represents the (13C atom / 12C atom of the international standard material Pee dee Belemnite) 100 which equaled 0.0112372. The y 13C value at the 0 timepoint was subtracted from the y 13C value at subsequent time points to get the altered y 13C value. Specifically, Dy13C () = [(13C atom% t min 13C atom% 0 min) / 13C atom%std] 1000. Least squares regression analysis of the logarithm of Dy 13C vs. time was used to calculate the rapid phase 13C disposition constant and slow phase 13C disposition constant (i.e. 13C excretion rate constant, PheBT-k) (Wensing et al., 1990; Krahenbuhl et al., 1989; Reichen et al., 1987). Sample collection At the end of the breath test, we separated a segment of the internal carotid artery.trunk, ligated the distal end, and incised the free segment to kill the rats by intubation and bloodletting. The blood samples were kept at room temperature for 30 min before centrifugation at 1500 rpm for 15 min. Serum was then collected for the detection of ALT (alanine transaminase), AST (aspartate transaminase), TBA (total bile acid), AKP (alkaline phosphatase) and TBIL (total bilirubin). The livers were removed immediately after bloodletting, washed with cold saline, and weighed wet. Part of each liver was fixed with 10% neutral formalin, embedded with paraffin, sectioned, stained with hematoxylin and eosin (H & E), and enveloped by optic resin. The rest of the liver tissue was stored in liquid nitrogen for later use. Preparation of samples for analysis and enzyme extraction SD rat livers were removed from liquid nitrogen. Two gram samples of each liver were broken into pieces, added to 10 ml
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of 3 mmol/l Tris KCl buffer (0.15 mol/L KCl, pH7.2), and homogenized with an homogenate machine. Sediment was removed from the resulting homogenate by centrifugation at 15000 rpm for 10 min in freezing conditions. An ultrasound cell crusher (JY-92 Ultrasound Cell Crusher, Xinyi Scientific Apparatus Institute, Ningbo, China) was used to break up cells in the liquid phase (10 cycles of a 10 s fragmentation time at 10 s intervals). The disrupted cells were centrifuged at 15,000 rpm for 10 min in freezing conditions. Lipids were then removed from the liquid phase with filter paper. After a final spin at 200,000 g for 30 min in a CS150 GX ultracentrifuge (HITACHI, Tokyo, Japan), the liquid phases were preserved in tubes at 20 -C until spectrophotometric analysis.
Statistical analysis
Determination of enzyme activity
The relative weight of livers from the acute liver injury group (total wet weight of liver/body weight %) was 3.28 T 0.26%, which was markedly lower than that in the control group 4.76 T 0.34% ( P < 0.001). Morphological observations of normal rats under the light microscope showed that the the liver lobule structure was complete and clear, and that hepatocytes were arranged along the central vein in a strap-shaped manner (Fig. 1A). In cells from the acute liver injured rats, the liver lobule structure was destroyed. Central degeneration (chaotic swelling, loosened cytoplasm and ballooning degeneration) and necrosis were found in the lobules. The degenerated and necrotic hepatic cells were distributed along the central vein in a path-shaped manner in area III (Fig. 1B) and extended to area II in a band-shaped manner (Fig. 1C), while the dead cells were distributed diffusely in severe cases (Fig. 1D). Serum biochemical results for liver function in rats of control group were as follows: ALT was 46.60 T 13.95 U/L (26 – 67 U/L), AST was 96.20 T 25.38 U/L (68 – 146 U/L), TBA
A WFZ-MV-2000 ultraviolet spectrophotometer (Heli Instrument Company, Shanghai, China) was used to measure absorbance. It was used with a cuvette that had an inner diameter of 1 cm and at a wavelength of 340 nm. Absorbances were read at 0 min and 5 min, followed by a calculation of the decrease in absorbance, A=absorbance at 5 min -absorbance at 0 min. The volume of the sample fluid was 500 Al, including distilled water 421.3 Al, liver homogenate (50 Al), 10 mmol/L magnesium sulfate (1.7 Al), 5 mmol/L l-phenylalanine (5 Al), 50 Ag/Al catalase (10 Al), and dihydropterin reductase (5 Al). After 5 min of shaking and incubation to activate the enzyme, 2.5 mmol/L 6-methyltetrahydropterins (3.5 Al) and 5 mmol/L NADH (3.5 Al) were added to make a total volume of 500 Al. The constituent fluids in the tube used to blank the spectrophotometer were same as in the analysis tube except that the sample fluid was replaced by 50 Al of distilled water.
All of the data were expressed as a mean T standard deviation (x¯ T s). Student’s t test for means comparisons and correlation analysis were performed using SPSS11.0 software. The breath test parameters and the results of the biochemical analyses of blood samples in the normal control group and the acute liver injury model group were compared, and the correlation between breath test data and total PAH activity in rat liver was analyzed. A value of P < 0.05 was considered to be statistically significant. Results
Fig. 1. H and E staining of control and injured liver tissue. (A) Liver lobule structure was intact in control tissue. (B – D) CCl4-injured liver showed central degeneration and necrosis in the lobules. The degenerated and necrotic hepatic cells were distributed along the central vein in a path-shaped manner in area III (B) and extended to area II in a band-shaped manner (C). In severe cases the dead cells were distributed diffusely (D) (200).
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Fig. 2. PheBT 13C excretion time-course curve. 13C content in breath rose rapidly, peaked 2 min after injection in most rats, and then slowly dropped. This pattern of excretion produced acute, smooth singular peaks. The peak values differed between the control and acute liver injury groups ( P < 0.05). ? normal control group (n = 10); > CCl4 induced acute liver injury group (n = 10).
was 6.60 T 4.67 Amol/L (1 – 15 Amol/L), AKP was 117.50 T 29.76 U/L (72 – 156 U/L), and TBIL was 1.65 T 0.18 Amol/L (1 –2.2Amol/L). In rats with acute liver injury, ALT was 604.30 T 216.00 U/L (363 –1004 U/L), AST was 196.60 T 88.63 U/L (95 – 387 U/L), TBA was 132.50 T 102.09 Amol/L (23 –368 Amol/L), AKP was 216.10 T 57.35 U/L (129 – 318 U/L), and TBIL was 10.66 T 6.90 Amol/L (4.6 –25.4.Amol/L). All indices were significantly higher in rats with acute liver injury than in the control group ( P < 0.01). One half minute after intravenous administration of 13CPhe into the caudal vein of rats in both the normal and acute liver injury groups, 13CO2 was collected from the vent of a rodent ventilator. 13CO2 concentrations increased rapidly, and reached a peak value 2 min after injection in most rats (9 / 10 in normal group and 8 / 10 in liver injury group) (Fig. 2). 13 CO2 concentrations then slowly dropped, presenting an acute and smooth single peak on the 13C excretion timecourse curve within 60 min in all rats. The peak value of 13C excretion was 170.65 T 22.85 in the normal group and 204.33 T 35.80 in the acute liver injury group ( P < 0.05) (Fig. 2). The 13C excretion time-course curve revealed that 13C excretion could be divided into slow and rapid phases in both Table 1 Comparison of different indices between acute liver injury rats induced by CCl4 and controls (x¯ T s) Index Peak value of 13C excretion () 13 C rapid disposition constant (10 2 min 1) 13 C excretion rate constant PheBT-k (10 2 min Total activity of PAH in liver (U/L/100 mg protein)
1
Acute liver injury
Normal control
(n = 10)
(n = 10)
204.33 T 35.80 (133.27¨265.19) 9.46 T 3.27 (2.26¨13.15)
170.65 T 22.85 (135.02¨207.32) 10.17 T 2.10 (7.74¨13.10)
2.45 T 0.25 (2.03¨2.79)
2.98 T 0.19 (2.74¨3.29)
P value
<0.05 >0.05
<0.001
) 1281.59 T 131.22 1523.21 T136.28 <0.001 (1025.49¨1447.75) (1339.00¨1805.50)
P < 0.05 vs. control was considered statistically significant. Data are expressed as means T standard deviation (range).
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Fig. 3. There was a high correlation between the total activity of phenylalanine hydroxylase (PAH) in whole liver and the 13C excretion rate constant PheBT-k (r = 0.92, P < 0.001). Data from the controls (n = 10) and the CCl4-induced acute liver injured rats (n = 10) were included in this analysis.
groups after the intravenous administration of 20 mg/kg l[1-13C] phenylalanine. In normal rats, the rapid disposition constant was (10.17 T 2.10) 10 2 min 1 and PheBT-k was (2.98 T 0.19) 10 2 min 1. In the acute liver injury group, the rapid disposition constant was (9.46 T 3.27) 10 2 min 1 and PheBT-k was (2.45 T 0.25) 10 2 min 1. There was no significant difference in the rapid disposition constant between the two groups ( P > 0.05). However, the difference in the two PheBT-k values was highly significant ( P < 0.001) (Table 1). In rats with acute liver injury, the PheBT parameter PhBT-k was negatively correlated with serum levels of ALT (r = 0.74, P < 0.05), AKP (r = 0.73, P < 0.05), TBA (r = 0.82, P < 0.01) and TBIL (r = 0.67, P < 0.05). No correlation was found between PhBT-k and serum AST levels (r = 0.16, P > 0.05). There was a strong correlation (r = 0.92, P < 0.001) between PheBT-k and total PAH activity in both normal rats and those with acute liver injury (Fig. 3). Total PAH activity in the liver of acute liver injured rats (1281.59 T 131.22 U/L/100 mg protein) was markedly lower than that of the controls (1523.21 T136.28 U/L/100 mg protein) ( P < 0.001) (Table 1). Discussion The liver has very important roles in amino acid metabolism and protein synthesis, and injury to the liver can destroy these essential functions. Previous research (Loda et al., 1984; Becker et al., 1987; Pearl et al., 1987) has shown that the survival rate of patients with hepatic encephalopathy is correlated with energy generation in the liver and elimination of amino acids from plasma. The rate of amino acid elimination can reflect hepatocyte function and the functional reserve of the liver, and could predict the outcome of surgical intervention. However, the amino acid elimination test is not widely applied in clinical practice because this examination is rather complex and time-consuming. Phenylalanine is one of the eight essential amino acids for humans. It cannot be produced by metabolic processes, and must be obtained from dietary sources. This amino acid is mainly decomposed in the liver, but with a low extraction from
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plasma into the liver, E = 0.2 (Matthews et al., 1993), that is even lower in patients with liver diseases (Tessari et al., 1994). Its elimination by the liver is heavily dependent on hepatocyte metabolism, but little on the hemodynamics of the liver. Studies have.found that after intravenous administration of phenylalanine to patients with liver diseases, decreased elimination was consistent with a loss in tyrosine production (Matthews et al., 1993), and that the phenylalaine elimination rate in the central vein was highly correlated with the total amino acid elimination rate (Levine and Conn, 1967). Many researchers (Burke et al., 1997; Tugtekin et al., 1999; Lara Baruque et al., 2000; Kobayashi et al., 2001; Ishii et al., 2003a; Festi et al., 2005; Yan et al., 2003; Sun et al., 2003) have independently evaluated hepatocyte function and liver functional reserve by PheBTs, and found that breath test parameter levels were reduced. The extent of this decrease was somewhat correlated with many blood biochemical indices and the results of Indocyanine Green (ICG) examination. Therefore, it was proposed that the oxidation of phenylalanine was reduced in patients with chronic liver diseases. Recently, Ishii et al. (2003b) found that PheBT results could reflect pathological changes in the livers of patients with liver diseases. This finding is supported by in vivo experiments that PheBT reflects the pathological and morphological changes of liver tissue in rats after hepatectomy (Ishii et al., 2002). 13 C-Phe is phenylalanine with the 12C at the carboxyl outside benzene annulus replaced by 13C. The process of its oxidation and final production of 13CO2 involves the transformation of phenylalanine to tyrosine in the liver by hydroxidation and the further degradation of tyrosine. Phenylalanine hydroxylase, tyrosine transaminase and 4-hydroxyphenylpyruvic oxidase mainly exist in the cytoplasm of hepatocytes (Levine and Conn, 1967; Morgan et al., 1978; O’Keefe et al., 1981) and are involved in the metabolic processing of 13C-Phe and the production of 13CO2 (Burke et al., 1997; Kobayashi et al., 2001). PAH is thought to be the rate-limiting enzyme (Cortiella et al., 1992) in the metabolism of phenylalanine. In a rat model of acute liver injury induced by galactosamine (GalN), Ito et al. (2001) reported that results of the PheBT, Dy13C value at 2 min (r = 0.917), Dy13C at 3 min (r = 0.831) and the area under the curve (AUC) of Dy13C 10 min after administration (r = 0.824), were correlated with total PAH activity. Ishii and his coworkers (Ishii et al., 2002) demonstrated that Dy13C values correlated positively with PAH activity in regenerated rat livers (r = 0.638). However, these liver injures were too serious to resemble most clinical cases of slight liver injury. In our study, the CCl4 induced acute liver injury was much slighter than GalN induced liver injury. Analysis of the parameters of PheBT in control rats vs. those with CCl4-induced acute liver injury suggests that there was a significant difference between the two groups in the peak value of 13C excretion. The parameter was higher in the injured rats, which was inconsistent with the results of Ito et al. (2001). We might attribute the difference to hemodynamic changes in rats after acute liver injury (Tanaka et al., 1999; Makin et al., 1997). It was reported in the literature that total
blood flow increased by 300 –500% in acute chemical liver injury model of rats, which would in turn promote the distribution of 13C-Phe to hepatocytes. According to the pharmacokinetic principle, the rapid disposition constant is determined by the process of distribution and the slow disposition constant (i.e. PheBT-k) is determined by the elimination process (Xu et al., 2001; Xi, 1992). A number of articles have reported on 13C excretion in the breath tests of end-stage hepatopathy patients (Burke et al., 1997; Tugtekin et al., 1999; Lara Baruque et al., 2000; Kobayashi et al., 2001; Ishii et al., 2003a,b; Yan et al., 2003; Sun et al., 2003; Festi et al., 2005) and rat models (Ito et al., 2001; Ishii et al., 2002). Although a decrease in 13C excretion was not observed in our animals, the PheBT-k was significantly lower than that of the control group, which was consistent with data from our clinical research (Yan et al., 2003; Sun et al., 2003). The total activity of PAH in the liver was highly correlated with PheBT-k, the parameter of 13C excretion. This result suggests that the PheBT could be valuable in the diagnosis of liver dysfunction at the enzyme activity level. Most animal breath tests use either the closed metabolic container (Schaad et al., 1995; Schoonjans et al., 2002) or the breath mask method (Ishii et al., 2002; Suzuki et al., 2002). The disadvantage of both is that measurement sensitivity is influenced by a relatively large dead space of ventilation and eddy current caused by airflow. In addition, anesthesia is required for the breath mask method, and inhibition of respiration often occurs due to the great individual differences in tolerance to anesthesia, thus leading to experimental failure. The advantages of our method solved these problems. In conclusion, the results of this study suggest that the decrease in total activity of PAH is the main cause of reduction of the 13C excretion rate in rats with acute liver injury. The 13C excretion rate parameter of PheBT-k in the breath test is a sensitive index that can be used to evaluate the extent of liver injury. Our animal model of the breath test, using basic auxiliary breath machine equipment, is safe, practical and reliable, and will be of widely useful in many areas of basic research that employ breath tests. Acknowledgements This work was supported by a grant from the Science and Technology Foundation of Shanghai Health Board, SHB F01-01406. References Armuzzi, A., Candelli, M., Zocco, M.A., Andreoli, A., De Lorenzo, A., Nista, E.C., Miele, L., Cremonini, F., Cazzato, I.A., Grieco, A., Gasbarrini, G., Gasbarrini, A., 2002. Review article: breath testing for human liver function assessment. Alimentary Pharmacology and Therapeutics 16, 1796 – 1977. Becker, W., Konstantinides, F., Eyer, S., Ward, H., Fath, J., Cerra, F., 1987. Plasma amino acid clearance as an indicator of hepatic function and highenergy phosphate in hepatic ischemia. Surgery 102, 777 – 783. Bohlen, H.G., Nase, G.P., 2002. Dependence of intestinal arteriolar regulation on flow-mediated nitric oxide formation. American Journal of Physiology. Heart and Circulatory Physiology 279, H2249 – H2258.
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O’Keefe, S.J., Abraham, R., El-Zayadi, A., Marshall, W., Davis, M., Williams, R., 1981. Increased plasma tyrosine concentrations in patients with cirrhosis and fulminant hepatic failure associated with increased plasma tyrosine flux and reduced hepatic oxidation capacity. Gastroenterology 81, 1017 – 1024. Pearl, R.H., Clowes Jr., G.H., Bosari, S., McDermott, W.V., Menzoian, J.O., Love, W., Jenkins, R.L., 1987. Amino acid clearance in cirrhosis. A redictor of postoperative morbidity and mortality. Archives of Surgery 122, 468 – 473. Rating, D., Langhans, C.D., 1997. Breath tests: concepts, applications and limitations. European Journal of Pediatrics 156, S18 – S23. Reichen, J., Arts, B., Schafroth, U., Zimmermann, A., Zeltner, B., Zysset, T., 1987. Aminopyrine N-demethylation by rats with liver cirrhosis. Evidence for the intact cell hypothesis. A morphometric-functional study. Gastroenterology 93, 719 – 726. Romagnuolo, J., Schiller, D., Bailey, R.J., 2002. Using breath tests wisely in a gastroenterology practice: an evidence-based review of indications and pitfalls in interpretation. American Journal of Gastroenterology 97, 1113 – 1126. Schaad, H.J., Renner, E.L., Wietholtz, H., Arnaud, M.J., Preisig, R., 1995. Caffeine demethylation measured by breath analysis in experimental liver injury in the rat. Journal of Hepatology 22, 82 – 87. Schoonjans, R., Van Vlem, B., Van Heddeghem, N., Vandamme, W., Vanholder, R., Lameire, N., Lefebvre, R., De Vos, M., 2002. The 13Coctanoic acid breath test: validation of a new noninvasive method of measuring gastric emptying in rats. Neurogastroenterology and Motility 14, 287 – 293. Sun, D.Y., Yan, W.L., Lin, X.T., Sun, X., 2003. Research on parameters of l-[1-13C] phenylalanine breath test for quantitative assessment of liver function in humans. Chinese Journal of Digestion 23, 309 – 402. Suzuki, S., Ishii, Y., Asai, S., Kohno, T., Mazaki, T., Takahashi, Y., Kohno, T., Ishikawa, K., 2002. l-[1-(13)C] Alanine is a useful substance for the evaluation of liver function. The Journal of Surgical Research 103, 13 – 18. Tanaka, N., Tanaka, K., Nagashima, Y., Kondo, M., Sekihara, H., 1999. Nitric oxide increases hepatic arterial blood flow in rats with carbon tetrachlorideinduced acute hepatic injury. Gastroenterology 117, 173 – 180. Tessari, P., Inchiostro, S., Barazzoni, R., Zanetti, M., Orlando, R., Biolo, G., Sergi, G., Pino, A., Tiengo, A., 1994. Fasting and postprandial phenylalanine and leucine kinetics in liver cirrhosis. American Journal of Physiology 267 (1 Pt 1), E140 – E149. Torbati, D., Ramirez, J., Hon, E., Camacho, M.T., Sussmane, J.B., Raszynski, A., Wolfsdorf, J., 1999. Experimental critical care in rats: gender differences in anesthesia, ventilation, and gas exchange. Critical Care Medicine 27, 1878 – 1884. Tugtekin, I., Radermacher, P., Wachter, U., Barth, E., Weidenbach, H., Adler, G., Georgieff, M., Vogt, J., 1999. Comparison between the oral and intravenous l-[1-13C]phenylalanine breath test for the assessment of liver function. Isotopes in Environmental and Health Studies 35, 147 – 156. Wensing, G., Sabra, R., Branch, R.A., 1990. Renal and systemic hemodynamics in experimental cirrhosis in rats: relation to hepatic function. Hepatology 12, 13 – 19. Xi, L.Z., 1992. Pharmacokinetics. Shanghai Medical University Publishers, Shanghai. Xu, S.Y., Brian, R.L., Chen, X., 2001. Experimental Methods of Pharmacology. People’s Medical Publishing House, Beijing. Yan, W.L., Sun, D.Y., Lin, X.T., Jiang, Y.B., Sun, X., 2003. A quantitative assessment of liver function using l-[1-13C] phenylalanine breath test. Nuclear Techniques 26, 363 – 365.
l-[1-13C] phenylalanine breath test results reflect the activity of phenylalanine hydroxylase in carbon tetrachloride acute injured rat liver Wei-Li Yan a,b,*, Da-Yu Sun a, Xiang-Tong Lin b, Yi-Bin Jiang a, Xu Sun a a b
Department of Gastroenterology, Huashan Hospital Affiliated to Fudas University, Shanghai 200040, P.R. China Department of Nuclear Medicine, Huashan Hospital Affiliated to Fudan University, Shanghai 200040, P.R. China Received 7 March 2005; accepted 23 May 2005
Abstract l-[1-13C] phenylalanine breath tests (PheBTs) have been used to determine the hepatocyte functional capacity of patients. This study investigated the relationship between the PheBT parameter 13C excretion rate constant (PheBT-k) and activity of the phenylalanine metabolic ratelimiting enzyme phenylalanine hydroxylase (PAH) in rat liver. We noted that the time-course curves of 13C excretion presented as a single peak, which appeared 2 min after administration of l-[1-13C] phenylalanine (13C-Phe). 13C excretion during exhalation can be divided into a slow phase and a rapid phase. The PheBT-k in rats with carbon tetrachloride acute liver injury was.significantly lower than that of control rats. The rapid phase 13 C disposition constants of the acute liver injured rats did not differ from that of the controls. The peak value of 13C abundance in the breath of the acute liver injured rats was markedly higher than that of the control group. Total liver PAH activity in the acute liver injured rats was significantly lower than that in the control group. PheBT-k was highly correlated with the total activity of liver PAH (r = 0.92, P < 0.001). The present findings indicate that PheBT results reflect PAH activity levels. The PheBT-k parameter is a sensitive index that can be used to evaluate PAH function in the liver. In addition we demonstrated that the rodent model used in this study is a valuable tool for basic research studies of the breath test. D 2005 Elsevier Inc. All rights reserved. Keywords: l-[1-13C] phenylalanine breath test; Liver; Rat; Phenylalanine hydroxylase; Activity;
Introduction 13
C breath tests have been employed in nutriology, pharmacology and pharmacokinetics, especially in the diagnosis of digestive system diseases, including dysfunction of the pancreas and liver disease (Romagnuolo et al., 2002). The liver mediates critical physiological functions that involve complex biochemical reactions. Timely and accurate measurement of indices of liver function is important for the diagnosis and monitoring of liver diseases, the evaluation of treatment outcome,.assessment of long-term prognosis, and the evaluation of the risks of surgical intervention and liver transplantation. Several 13C-labelled drugs have been used as substrates in breath tests to study of various metabolic pathways in the liver (hepatocyte microsomes, cytoplasm and mitochondria) and to * Corresponding author. Department of Gastroenterology, Huashan Hospital Affiliated to Fudan University, 12 Central Wurumuqi Road, Shanghai 200040, P.R. China. Tel./fax: +86 2150892400. E-mail address: [email protected] (W.-L. Yan). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.05.073
13
C excretion rate constant
investigate hepatocyte function and liver functional reserve (Armuzzi et al., 2002). Burke et al. (1997) was the first to utilize phenylalanine breath tests (PheBTs) to determine the hepatocyte functional capacity in patients with end-stage liver diseases. They found that 13CO2 excretion decreased by up to 80% after intake of l[1-13C] phenylalanine (13C-Phe). Similar results were subsequently obtained in different populations by other researchers (Tugtekin et al., 1999; Lara Baruque et al., 2000; Kobayashi et al., 2001; Ishii et al., 2003a; Festi et al., 2005). Our clinical research has also supported the clinical value of PheBTs (Yan et al., 2003; Sun et al., 2003). It has been reported that patients with liver disease have a diminished capability to eliminate phenylalanine from plasma (Heberer et al., 1980). It is now an accepted notion that those 13CO2 excretion deficits are due to a reduction of the liver’s capability to oxidize phenylalanine. However the lack of direct experimental evidence for this hypothesis might limit the utilization of the breath test (Rating and Langhans, 1997). Recently, in vivo experiments showed of PheBT results can reflect the activity of phenylalanine
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hydroxylase (PAH) in galactosamine prostrate livers (Ito et al., 2001) and in two thirds partial hepatectomized rat livers (Ishii et al., 2002). However, such experimental models are not instructive for most clinical cases of slight liver injury. There are two kinds of animal breath tests, the metabolic container method.(Schaad et al., 1995; Schoonjans et al., 2002) and the breath mask method (Ito et al., 2001; Ishii et al., 2002; Suzuki et al., 2002), but both have some deficiencies. In this study, we employed an animal model of the breath test developed in our laboratory to determine the activity of PAH, the rate-limiting enzyme of phenylalanine metabolism in the rat liver. We also investigated the relationship between the parameters of PheBT and PAH activity, and sought valid parameters for the animal breath test. Materials and methods Reagents l-[1-13C] phenylalanine (purity 99%, 13C abundance 99%) powder was purchased from ISOTEC Co. (Miamisburg, Ohio, USA). l-phenylalanine, 6-methyl-tetrahydropterin, dihydropterin reductase (DHPR) and catalase were obtained from Sigma (St. Louis, USA). Sodium pentobarbital and magnesium sulfate were obtained from Shanghai Chemical Reagent Co. (Shanghai, China). Lowry protein assay kits were purchased from Shenneng Bochai Biotech Co. (Shanghai, China). Serum liver function test kits were obtained from the Biotech Center, Shanghai Navy Medical Institution (Shanghai, China). Animals Inbred male Sprague –Dawley (SD) rats were provided by the Experimental Animal Center, Fudan University, housed in the specific pathogen-free (SPF) animal rooms, and given free access to water and standard rat chow. Initially, the rats weighed 220 –230g. After feeding for one week, twenty rats weighing 280 –290 g were selected for the experiment. The rats were divided randomly into two groups of 10 rats each. This study was performed according to our institutional guidelines. Acute liver injury model In the liver injury group, rats were treated orally with 2 ml/ kg of carbon tetrachloride (CCl4) at a 60% volume ratio in olive oil after 8 h of fasting with free access to water. After 4 h, the rats were treated with another dose of CCl4. In the control group, rats received only the same volume of olive oil. All of the rats were fasted for 14 h more and weighed before the breath test. Animal model of breath test After weighing, the SD rats were anesthetized by intraperitoneal injection of 50 mg/kg of 5% sodium pentobarbital. The anesthetized rats were fixed on a small animal dissecting table in a supine position. An incision was made at the center of the
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neck, followed by separation of the trachea, making a Tshaped opening at the free end, and inserting a troclear cannula that was fixed by ligation with nylon thread. The operating surface was covered with saline-soaked gauze. The temperature of viscera was kept at 37.5 T 0.5 -C during the experiment. Mechanical ventilation was performed with a small animal breath machine that supplied air containing 21% oxygen and discharged the air directly into air. The tidal volume of mechanical.ventilation was 2.5 ml, ventilation frequency was 52 times/min, and the exhalation/inspiration ratio was 1 : 1 (Bohlen and Nase, 2002; Harf et al., 1983; Torbati et al., 1999). After 15 min of ventilation, a 10 ml breath collection tube (Labco Ltd., Buckinghamshire, UK) was used to collect exhaled air for 20 s, which acted as background. A 1.6% solution of 13C-Phe saline was introduced into the caudal vein with a 13C-Phe dosage of 20 mg/kg of body weight. Exhaled air samples (20 s duration) were collected immediately, and another 28 samples was collected each minute for 20 min and then every 5 min between 20 and 60 min after injection. 13C abundance in the samples was determined with an air isotope ratio mass spectrometer to obtain a y 13C () value at different time points: y 13C () = [(13C atom % / 13C atom %std) 1] 1000. In the formula, 13C atom % equals ( 13C atom / 12 C atom) 100, 13 C atom %std represents the (13C atom / 12C atom of the international standard material Pee dee Belemnite) 100 which equaled 0.0112372. The y 13C value at the 0 timepoint was subtracted from the y 13C value at subsequent time points to get the altered y 13C value. Specifically, Dy13C () = [(13C atom% t min 13C atom% 0 min) / 13C atom%std] 1000. Least squares regression analysis of the logarithm of Dy 13C vs. time was used to calculate the rapid phase 13C disposition constant and slow phase 13C disposition constant (i.e. 13C excretion rate constant, PheBT-k) (Wensing et al., 1990; Krahenbuhl et al., 1989; Reichen et al., 1987). Sample collection At the end of the breath test, we separated a segment of the internal carotid artery.trunk, ligated the distal end, and incised the free segment to kill the rats by intubation and bloodletting. The blood samples were kept at room temperature for 30 min before centrifugation at 1500 rpm for 15 min. Serum was then collected for the detection of ALT (alanine transaminase), AST (aspartate transaminase), TBA (total bile acid), AKP (alkaline phosphatase) and TBIL (total bilirubin). The livers were removed immediately after bloodletting, washed with cold saline, and weighed wet. Part of each liver was fixed with 10% neutral formalin, embedded with paraffin, sectioned, stained with hematoxylin and eosin (H & E), and enveloped by optic resin. The rest of the liver tissue was stored in liquid nitrogen for later use. Preparation of samples for analysis and enzyme extraction SD rat livers were removed from liquid nitrogen. Two gram samples of each liver were broken into pieces, added to 10 ml
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of 3 mmol/l Tris KCl buffer (0.15 mol/L KCl, pH7.2), and homogenized with an homogenate machine. Sediment was removed from the resulting homogenate by centrifugation at 15000 rpm for 10 min in freezing conditions. An ultrasound cell crusher (JY-92 Ultrasound Cell Crusher, Xinyi Scientific Apparatus Institute, Ningbo, China) was used to break up cells in the liquid phase (10 cycles of a 10 s fragmentation time at 10 s intervals). The disrupted cells were centrifuged at 15,000 rpm for 10 min in freezing conditions. Lipids were then removed from the liquid phase with filter paper. After a final spin at 200,000 g for 30 min in a CS150 GX ultracentrifuge (HITACHI, Tokyo, Japan), the liquid phases were preserved in tubes at 20 -C until spectrophotometric analysis.
Statistical analysis
Determination of enzyme activity
The relative weight of livers from the acute liver injury group (total wet weight of liver/body weight %) was 3.28 T 0.26%, which was markedly lower than that in the control group 4.76 T 0.34% ( P < 0.001). Morphological observations of normal rats under the light microscope showed that the the liver lobule structure was complete and clear, and that hepatocytes were arranged along the central vein in a strap-shaped manner (Fig. 1A). In cells from the acute liver injured rats, the liver lobule structure was destroyed. Central degeneration (chaotic swelling, loosened cytoplasm and ballooning degeneration) and necrosis were found in the lobules. The degenerated and necrotic hepatic cells were distributed along the central vein in a path-shaped manner in area III (Fig. 1B) and extended to area II in a band-shaped manner (Fig. 1C), while the dead cells were distributed diffusely in severe cases (Fig. 1D). Serum biochemical results for liver function in rats of control group were as follows: ALT was 46.60 T 13.95 U/L (26 – 67 U/L), AST was 96.20 T 25.38 U/L (68 – 146 U/L), TBA
A WFZ-MV-2000 ultraviolet spectrophotometer (Heli Instrument Company, Shanghai, China) was used to measure absorbance. It was used with a cuvette that had an inner diameter of 1 cm and at a wavelength of 340 nm. Absorbances were read at 0 min and 5 min, followed by a calculation of the decrease in absorbance, A=absorbance at 5 min -absorbance at 0 min. The volume of the sample fluid was 500 Al, including distilled water 421.3 Al, liver homogenate (50 Al), 10 mmol/L magnesium sulfate (1.7 Al), 5 mmol/L l-phenylalanine (5 Al), 50 Ag/Al catalase (10 Al), and dihydropterin reductase (5 Al). After 5 min of shaking and incubation to activate the enzyme, 2.5 mmol/L 6-methyltetrahydropterins (3.5 Al) and 5 mmol/L NADH (3.5 Al) were added to make a total volume of 500 Al. The constituent fluids in the tube used to blank the spectrophotometer were same as in the analysis tube except that the sample fluid was replaced by 50 Al of distilled water.
All of the data were expressed as a mean T standard deviation (x¯ T s). Student’s t test for means comparisons and correlation analysis were performed using SPSS11.0 software. The breath test parameters and the results of the biochemical analyses of blood samples in the normal control group and the acute liver injury model group were compared, and the correlation between breath test data and total PAH activity in rat liver was analyzed. A value of P < 0.05 was considered to be statistically significant. Results
Fig. 1. H and E staining of control and injured liver tissue. (A) Liver lobule structure was intact in control tissue. (B – D) CCl4-injured liver showed central degeneration and necrosis in the lobules. The degenerated and necrotic hepatic cells were distributed along the central vein in a path-shaped manner in area III (B) and extended to area II in a band-shaped manner (C). In severe cases the dead cells were distributed diffusely (D) (200).
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Fig. 2. PheBT 13C excretion time-course curve. 13C content in breath rose rapidly, peaked 2 min after injection in most rats, and then slowly dropped. This pattern of excretion produced acute, smooth singular peaks. The peak values differed between the control and acute liver injury groups ( P < 0.05). ? normal control group (n = 10); > CCl4 induced acute liver injury group (n = 10).
was 6.60 T 4.67 Amol/L (1 – 15 Amol/L), AKP was 117.50 T 29.76 U/L (72 – 156 U/L), and TBIL was 1.65 T 0.18 Amol/L (1 –2.2Amol/L). In rats with acute liver injury, ALT was 604.30 T 216.00 U/L (363 –1004 U/L), AST was 196.60 T 88.63 U/L (95 – 387 U/L), TBA was 132.50 T 102.09 Amol/L (23 –368 Amol/L), AKP was 216.10 T 57.35 U/L (129 – 318 U/L), and TBIL was 10.66 T 6.90 Amol/L (4.6 –25.4.Amol/L). All indices were significantly higher in rats with acute liver injury than in the control group ( P < 0.01). One half minute after intravenous administration of 13CPhe into the caudal vein of rats in both the normal and acute liver injury groups, 13CO2 was collected from the vent of a rodent ventilator. 13CO2 concentrations increased rapidly, and reached a peak value 2 min after injection in most rats (9 / 10 in normal group and 8 / 10 in liver injury group) (Fig. 2). 13 CO2 concentrations then slowly dropped, presenting an acute and smooth single peak on the 13C excretion timecourse curve within 60 min in all rats. The peak value of 13C excretion was 170.65 T 22.85 in the normal group and 204.33 T 35.80 in the acute liver injury group ( P < 0.05) (Fig. 2). The 13C excretion time-course curve revealed that 13C excretion could be divided into slow and rapid phases in both Table 1 Comparison of different indices between acute liver injury rats induced by CCl4 and controls (x¯ T s) Index Peak value of 13C excretion () 13 C rapid disposition constant (10 2 min 1) 13 C excretion rate constant PheBT-k (10 2 min Total activity of PAH in liver (U/L/100 mg protein)
1
Acute liver injury
Normal control
(n = 10)
(n = 10)
204.33 T 35.80 (133.27¨265.19) 9.46 T 3.27 (2.26¨13.15)
170.65 T 22.85 (135.02¨207.32) 10.17 T 2.10 (7.74¨13.10)
2.45 T 0.25 (2.03¨2.79)
2.98 T 0.19 (2.74¨3.29)
P value
<0.05 >0.05
<0.001
) 1281.59 T 131.22 1523.21 T136.28 <0.001 (1025.49¨1447.75) (1339.00¨1805.50)
P < 0.05 vs. control was considered statistically significant. Data are expressed as means T standard deviation (range).
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Fig. 3. There was a high correlation between the total activity of phenylalanine hydroxylase (PAH) in whole liver and the 13C excretion rate constant PheBT-k (r = 0.92, P < 0.001). Data from the controls (n = 10) and the CCl4-induced acute liver injured rats (n = 10) were included in this analysis.
groups after the intravenous administration of 20 mg/kg l[1-13C] phenylalanine. In normal rats, the rapid disposition constant was (10.17 T 2.10) 10 2 min 1 and PheBT-k was (2.98 T 0.19) 10 2 min 1. In the acute liver injury group, the rapid disposition constant was (9.46 T 3.27) 10 2 min 1 and PheBT-k was (2.45 T 0.25) 10 2 min 1. There was no significant difference in the rapid disposition constant between the two groups ( P > 0.05). However, the difference in the two PheBT-k values was highly significant ( P < 0.001) (Table 1). In rats with acute liver injury, the PheBT parameter PhBT-k was negatively correlated with serum levels of ALT (r = 0.74, P < 0.05), AKP (r = 0.73, P < 0.05), TBA (r = 0.82, P < 0.01) and TBIL (r = 0.67, P < 0.05). No correlation was found between PhBT-k and serum AST levels (r = 0.16, P > 0.05). There was a strong correlation (r = 0.92, P < 0.001) between PheBT-k and total PAH activity in both normal rats and those with acute liver injury (Fig. 3). Total PAH activity in the liver of acute liver injured rats (1281.59 T 131.22 U/L/100 mg protein) was markedly lower than that of the controls (1523.21 T136.28 U/L/100 mg protein) ( P < 0.001) (Table 1). Discussion The liver has very important roles in amino acid metabolism and protein synthesis, and injury to the liver can destroy these essential functions. Previous research (Loda et al., 1984; Becker et al., 1987; Pearl et al., 1987) has shown that the survival rate of patients with hepatic encephalopathy is correlated with energy generation in the liver and elimination of amino acids from plasma. The rate of amino acid elimination can reflect hepatocyte function and the functional reserve of the liver, and could predict the outcome of surgical intervention. However, the amino acid elimination test is not widely applied in clinical practice because this examination is rather complex and time-consuming. Phenylalanine is one of the eight essential amino acids for humans. It cannot be produced by metabolic processes, and must be obtained from dietary sources. This amino acid is mainly decomposed in the liver, but with a low extraction from
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plasma into the liver, E = 0.2 (Matthews et al., 1993), that is even lower in patients with liver diseases (Tessari et al., 1994). Its elimination by the liver is heavily dependent on hepatocyte metabolism, but little on the hemodynamics of the liver. Studies have.found that after intravenous administration of phenylalanine to patients with liver diseases, decreased elimination was consistent with a loss in tyrosine production (Matthews et al., 1993), and that the phenylalaine elimination rate in the central vein was highly correlated with the total amino acid elimination rate (Levine and Conn, 1967). Many researchers (Burke et al., 1997; Tugtekin et al., 1999; Lara Baruque et al., 2000; Kobayashi et al., 2001; Ishii et al., 2003a; Festi et al., 2005; Yan et al., 2003; Sun et al., 2003) have independently evaluated hepatocyte function and liver functional reserve by PheBTs, and found that breath test parameter levels were reduced. The extent of this decrease was somewhat correlated with many blood biochemical indices and the results of Indocyanine Green (ICG) examination. Therefore, it was proposed that the oxidation of phenylalanine was reduced in patients with chronic liver diseases. Recently, Ishii et al. (2003b) found that PheBT results could reflect pathological changes in the livers of patients with liver diseases. This finding is supported by in vivo experiments that PheBT reflects the pathological and morphological changes of liver tissue in rats after hepatectomy (Ishii et al., 2002). 13 C-Phe is phenylalanine with the 12C at the carboxyl outside benzene annulus replaced by 13C. The process of its oxidation and final production of 13CO2 involves the transformation of phenylalanine to tyrosine in the liver by hydroxidation and the further degradation of tyrosine. Phenylalanine hydroxylase, tyrosine transaminase and 4-hydroxyphenylpyruvic oxidase mainly exist in the cytoplasm of hepatocytes (Levine and Conn, 1967; Morgan et al., 1978; O’Keefe et al., 1981) and are involved in the metabolic processing of 13C-Phe and the production of 13CO2 (Burke et al., 1997; Kobayashi et al., 2001). PAH is thought to be the rate-limiting enzyme (Cortiella et al., 1992) in the metabolism of phenylalanine. In a rat model of acute liver injury induced by galactosamine (GalN), Ito et al. (2001) reported that results of the PheBT, Dy13C value at 2 min (r = 0.917), Dy13C at 3 min (r = 0.831) and the area under the curve (AUC) of Dy13C 10 min after administration (r = 0.824), were correlated with total PAH activity. Ishii and his coworkers (Ishii et al., 2002) demonstrated that Dy13C values correlated positively with PAH activity in regenerated rat livers (r = 0.638). However, these liver injures were too serious to resemble most clinical cases of slight liver injury. In our study, the CCl4 induced acute liver injury was much slighter than GalN induced liver injury. Analysis of the parameters of PheBT in control rats vs. those with CCl4-induced acute liver injury suggests that there was a significant difference between the two groups in the peak value of 13C excretion. The parameter was higher in the injured rats, which was inconsistent with the results of Ito et al. (2001). We might attribute the difference to hemodynamic changes in rats after acute liver injury (Tanaka et al., 1999; Makin et al., 1997). It was reported in the literature that total
blood flow increased by 300 –500% in acute chemical liver injury model of rats, which would in turn promote the distribution of 13C-Phe to hepatocytes. According to the pharmacokinetic principle, the rapid disposition constant is determined by the process of distribution and the slow disposition constant (i.e. PheBT-k) is determined by the elimination process (Xu et al., 2001; Xi, 1992). A number of articles have reported on 13C excretion in the breath tests of end-stage hepatopathy patients (Burke et al., 1997; Tugtekin et al., 1999; Lara Baruque et al., 2000; Kobayashi et al., 2001; Ishii et al., 2003a,b; Yan et al., 2003; Sun et al., 2003; Festi et al., 2005) and rat models (Ito et al., 2001; Ishii et al., 2002). Although a decrease in 13C excretion was not observed in our animals, the PheBT-k was significantly lower than that of the control group, which was consistent with data from our clinical research (Yan et al., 2003; Sun et al., 2003). The total activity of PAH in the liver was highly correlated with PheBT-k, the parameter of 13C excretion. This result suggests that the PheBT could be valuable in the diagnosis of liver dysfunction at the enzyme activity level. Most animal breath tests use either the closed metabolic container (Schaad et al., 1995; Schoonjans et al., 2002) or the breath mask method (Ishii et al., 2002; Suzuki et al., 2002). The disadvantage of both is that measurement sensitivity is influenced by a relatively large dead space of ventilation and eddy current caused by airflow. In addition, anesthesia is required for the breath mask method, and inhibition of respiration often occurs due to the great individual differences in tolerance to anesthesia, thus leading to experimental failure. The advantages of our method solved these problems. In conclusion, the results of this study suggest that the decrease in total activity of PAH is the main cause of reduction of the 13C excretion rate in rats with acute liver injury. The 13C excretion rate parameter of PheBT-k in the breath test is a sensitive index that can be used to evaluate the extent of liver injury. Our animal model of the breath test, using basic auxiliary breath machine equipment, is safe, practical and reliable, and will be of widely useful in many areas of basic research that employ breath tests. Acknowledgements This work was supported by a grant from the Science and Technology Foundation of Shanghai Health Board, SHB F01-01406. References Armuzzi, A., Candelli, M., Zocco, M.A., Andreoli, A., De Lorenzo, A., Nista, E.C., Miele, L., Cremonini, F., Cazzato, I.A., Grieco, A., Gasbarrini, G., Gasbarrini, A., 2002. Review article: breath testing for human liver function assessment. Alimentary Pharmacology and Therapeutics 16, 1796 – 1977. Becker, W., Konstantinides, F., Eyer, S., Ward, H., Fath, J., Cerra, F., 1987. Plasma amino acid clearance as an indicator of hepatic function and highenergy phosphate in hepatic ischemia. Surgery 102, 777 – 783. Bohlen, H.G., Nase, G.P., 2002. Dependence of intestinal arteriolar regulation on flow-mediated nitric oxide formation. American Journal of Physiology. Heart and Circulatory Physiology 279, H2249 – H2258.
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