Arch Toxicol (1991) 65:548-555

Archives of

Toxicology

034057619100077V

9 Springer-Vedag 1991

Taurine, a possible urinary marker of liver damage: a study of taurine excretion in carbon tetrachloride-treated rats Catherine J. Waterfield l, John A. Turton 1, M. David C. Scales 2, and John A. Timbrell I i Toxicology Unit, School of Pharmacy, University of London and 2 Glaxo Group Research, Ware, Herts., UK Received 7 January 1991/Received after revision 9 April 1991/Accepted 15 April 1991

Abstract. Carbon tetrachloride (CC14) caused a dose-dependent increase in urinary taurine which correlated with both the histological and biochemical assessment of liver damage. The peak elevation in urinary taurine occurred within the first 48 h after dosing but there was still significant taurinuria 72 and 96 h after the intermediate dose (1 ml.kg-1) and highest dose (2 ml.kg-l), respectively. Levels of taurine in serum were also elevated over the 24 h period following a hepatotoxic dose (2 ml.kg-l) of CC14. In contrast, although initially elevated, levels of taurine in the liver declined over the 24 h period following dosing and were significantly lower 96 h after a hepatotoxic dose of CC14 (2 ml.kg-1). Male rats showed a different urinary profile for taurine than female rats after dosing with CC14. A reduction in food intake seemed to lower urinary taurine levels although these changes were not statistically significant. There was a significant correlation between the level of urinary taurine and the level of serum AST for individual animals given a hepatotoxic dose of CC14 (2 ml.kg-1). The data presented suggest that: i) taurine is produced by the liver in response to a toxic insult and subsequent leakage from damaged cells leads to increased levels in the urine; ii) the urinary taurine level may be a useful non-invasive marker of liver damage. Key words: Carbon tetrachloride - Taurine - Liver damage - Rat

Introduction Taurine (2-aminoethanesulphonic acid) is a [3-amino acid which is not incorporated into proteins but is found in the cytosol of animal cells, in high concentrations (2-30 mM), mostly unbound (Huxtable and Bressler 1972; Chesney et

Offprint requests to: Dr. J. A. Timbrell, Toxicology Unit, School of Pharmacy, 29/39, Brunswick Square, London WC1N lAX, UK

al. 1978; Wright et al. 1986; Zelikovic and Chesney 1989). In the rat, taurine constitutes the main free amino acid. The diet provides most taurine either directly or by synthesis in the liver and brain from methionine or cysteine via cysteic acid or hypotaurine (Jacobsen and Smith 1968), or via cysteamine in the heart and kidney (Zelikovic and Chesney 1989). The liver and brain are the major organs for the synthesis of taurine which is conjugated with bile acids to form taurocholate in the liver. Taurine is transported from the liver to other organs to maintain tissue levels. The concentration of taurine in the liver is variously reported as between 2 and 10 mM (Garvin 1960; Mikasa et al. 1980; Yoshida and Hara 1985; Hirai et al. 1987). The highest cellular concentrations of taurine are found in cells rich in membranes (e. g. hepatocytes), or where oxidants are generated (e. g. neutrophils and the retina) or in excitable cells (e.g. cardiac muscle) (Thomas et al. 1985; Wright et al. 1986). These findings have led to the suggestion of a central role for taurine as a protective substance. Taurine stabilises membranes, modulates calcium transport and by the formation of the relatively stable taurochloramine molecule is able to dissipate the toxic effects of HOC1, generated by myeloperoxidases from oxygen radicals. The ability of taurine to conjugate with xenobiotics, retinoic acid and bile salts and its role as a major free amino acid in regulating the osmolality of cells, are also examples of protective functions. Plasma levels of taurine are regulated by the kidney where the amino acid is reabsorbed in the proximal tubules by a p-amin 0 acid uptake system for which there is competition with other [3-amino acids, particularly [3-alanine. Excess taurine in the blood results in hypertaurinuria. This has been reported after surgical trauma, X-radiation, muscle necrosis (Kay et al. 1957) and carbon tetrachlorideinduced liver damage (Dent and Walshe 1954; Bowden and Goyer 1962; Cornish and Ryan 1964; Goyer et al. 1964; Angel and Noonan 1974) as part of a general aminoaciduria. Using proton NMR, we have recently shown hypertaurinuria in rats after treatment with several hepatotoxins, including carbon tetrachloride (Sanins et al. 1990). As a

549 result o f t h e s e initial f i n d i n g s , w e h a v e s u g g e s t e d that taurinuria c o u l d b e u s e d as a q u a n t i t a t i v e , n o n - i n v a s i v e marker o f v a r i o u s t y p e s o f l i v e r d a m a g e . S u c h a u r i n a r y marker w o u l d p r o v i d e , u n l i k e the c o m m o n l y u s e d assays for s e r u m / p l a s m a t r a n s a m i n a s e s o r r o u t i n e h i s t o p a t h o l o g i cal e x a m i n a t i o n , a n o n - i n v a s i v e and r e a d i l y a c c e s s i b l e m e t h o d for the c o n t i n u o u s m o n i t o r i n g o f a n i m a l s or h u m a n s w h e r e l i v e r d a m a g e is s u s p e c t e d . T h e w o r k p r e s e n t e d h e r e is a d e t a i l e d study o f taurine levels in the urine, s e r u m and l i v e r o f rats t r e a t e d w i t h the m o d e l h e p a t o t o x i n , c a r b o n t e t r a c h l o r i d e . C a r b o n tetr a c h l o r i d e was c h o s e n b e c a u s e it has b e e n u s e d e x t e n s i v e l y as a h e p a t o t o x i n to s t u d y t h e p a t h o g e n e s i s and c h a r a c t e r o f hepatic n e c r o s i s and the e f f e c t s o f h e p a t o c y t e injury o n liver f u n c t i o n ( Z i m m e r m a n 1978) and it p r o d u c e s p r e d i c t able e f f e c t s . T h e o b j e c t i v e w a s to e q u a t e u r i n a r y l e v e l s o f taurine w i t h the s e v e r i t y and t y p e o f h e p a t i c d a m a g e p r o duced and to s p e c u l a t e o n the o r i g i n o f the i n c r e a s e d urinary taurine. T a u r i n e w a s q u a n t i t a t e d u s i n g a s i m p l e H P L C method.

Mobile phase buffer for HPLC Sodium dihydrogen phosphate buffer (0.05 M, pH 5.3) in water and methanol as described by Larsen et al. (1980) was prepared. The buffer was filtered and degassed using a Millipore vacuum filter (0.22/am).

Post mortem procedure Animals were anaesthetised with diethylether and exsanguinated from the abdominal aorta. Blood samples were put into Microtainers (Beckton Dickinson and Co., Rutherford, NJ USA) for the separation of serum. Serum samples to be analysed for various blood parameters and taurine were stored at -20 ~C. The liver was removed, weighed, and approximately 1 g taken from the right lobe and stored at -20 ~C for subsequent taurine analysis. The median lobe was placed in 10.5% (v/v) phosphate buffered formalin (pH 7.2) for histological processing. The kidneys were also weighed and placed in fixative for histopathology. Tissues were stained with haematoxylin and eosin or Periodic acid-Schiff (PAS) reaction for glycogen using diastase treatment as a control. Frozen sections (10 lam) were stained for lipid with Oil red 0 in triethyl phosphate and Mayer's haematoxylin as counter stain.

Biochemical measurements Materials and methods

Animals and treatment Male and female rats of the Sprague-Dawley strain (Glaxo Group Research Ltd.) weighing 220-320 g were allowed to acclimatise for 6-10 days after delivery. They were housed in communal cages, fed Rat and Mouse maintenance 691 cube diet (Quest Nutrition Ltd., Wingham, Kent, UK) and water ad libitum. During the experiments animals were housed for up to 10 days either in individual metabolism cages designed to separate and collect faeces and urine (Techmate Ltd, Milton Keynes, UK), or in communal cages when urine was not being collected. Animals were provided with water ad libitum and ground diet when in metabolism cages. Lighting was controlled to give a regular 12 h light-dark cycle (light on at 8 a. m. off at 8 p. m.) and room temperature was maintained at 21 + 2 ~C. All dosing was by the oral route. Body weight, food and water intake and the general condition of animals were monitored daily. Urine samples (4 h or 24 h) were collected over ice throughout the study periods.

Reagents o-Phthalaldehyde (OPA; HPLC grade), tanrine (cell culture tested), homoserine, sodium dihydrogen phosphate and Dowex resins were all supplied by Sigma Chemical Company Ltd (Poole, Dorset, UK). Mercaptoethanol, sodium hydroxide (AfistaO, sulphosalicylic acid and boric acid were obtained from BDH Ltd, (Poole, Dorset, UK), methanol (HPLC grade) from Rathburn (Wakeburn, Scotland, UK) and Coomassie Blue Reagent from Pierce and Warriner (Chester, UK). Water was of UHQ standard, prepared using an Elgastat water purifier.

Preparation of derivatising solution for taurine measurement The tbllowing reagents were used: OPA 40 mg (in 0.8 ml absolute ethanol), 40 Ill mercaptoethanol; 10 ml Na borate buffer (3.1 g boric acid /90 ml UHQ water adjusted to pH 10.3 using 5 M NaOH, then made up to 100 ml (Durldn et al. 1988). The derivatizing solution was kept in a dark bottle at room temperature and used on the day of preparation.

Preparation of ion exchange columns. Before determination of taurine in samples, interfering substances were removed using ion exchange resins. Ion exchange columns were prepared in a way similar to that described by Anzano et al. (1978), using two types of ion exchange resin: (i) a strongly cationic resin Dowex-50W-X8 (H§ form, 200-400 or 100-200 mesh); (ii) a strongly anionic resin Dowex-l-X4 (CI- form, 100-200 mesh). Dowex-50W removes almost all cations (except taurine and cysteic acid) and urea. Dowex-1 removes anions and the cysteic acid which remains (Sorbo 1961; Datta and Naryanaswami 1983). Previously washed Dowex-50W (H § and Dowex- 1 (CI-) resins were packed into columns (0.6• 8.0 cm and 0.6 x 6.0 cm, respectively), stacked one above the other and washed with HCI (1 M, 10 ml) and UHQ water (10 ml), immediately prior to use. Serum and tissue samples were purified using smaller columns with 1.5 ml apparent volume of Dowex-I (CI-) layered directly on top of a similar volume of Dowex-50W (H+).

Preparation of urine samples. Urine samples C0-24 h) were diluted to 25 ml, centrifuged at 4000 rpm for 10 rain to remove hair and food debris, and frozen (-20~ in aliquots (5 ml) until analysis. Aliquots (1 ml) were put onto prepared, stacked, ion exchange columns for purification and the first 1.0 ml of eluate discarded. The elation of taurine was completed by the addition of 8 x0.5 ml aliquots of UHQ water. Homoserine as internal standard, (0.5-1.0 ml; 4 raM) was added to the eluted sample (Durkin et al. 1988), giving a final concentration (0.40.8 lttmol/ml) similar to the expected taurine concentration in the eluate.

Preparation of serum samples. Sulphosalicylic acid (200/.tl; 0.2 M) was added to serum (200 lal), mixed, allowed to stand (5 min) to precipitate protein, and centrifuged (11 000 g; 3 rain). The supernatant was placed onto a dual bed ion exchange column and the first 200 /al of eluate discarded. The protein pellet was resuspended twice in water (0.5 ml), centrifuged, the supernatant added to the column and the eluate collected. The pellet was washed with a further 0.5 ml water and the washing added to the column. Elation was completed with 3 • 0.5 ml water. Homoserine (0.4 mM; 100 p.1) was added to the eluate as internal standard.

Preparation of tissue samples. Liver (0.1-0.2 g) was homogenised in sulphosalicylic acid ( 1.0 ml; 0.2 M; 4 ~C). The homogenate and washings (1 ml) were centrifuged (4000 rpm; 10 rain; 4" C) and the supernatant added to a dual bed ion exchange column. All the eluate was collected. The pellet was resuspended twice in water (0.5 ml), centrifuged and the supernatant added to the column. Elation was completed with 5 x0.2 ml water. Homoserine (100 ILl;4 mM) was added to the eluate as an internal standard.

550 Table 1. Preparation of fluorescent taurine adduct

e-

200

,,r Eluate (pl)

Derivatizing solution (pl)

Mobile phase buffer (~tl)

Urine Serum Tissue

50 50 100

450 0 400

50 200 125

Measurement oftaurine. Taurine was measured in the eluates from the ion exchange columns using HPLC with fhiorimetric detection (modified from Larsen et al. 1980). The fluorescent taurine adduct was formed in the different ehiates as shown in Table 1. The ratio of eluates: derivatizing solution was dependent on the pH of the eluate and the expected taurine concentration. For example, the serum eluates were too dilute to be further diluted with buffer. The eluate and derivatizing solution were mixed (30 s), mobile phase buffer added (pH 5.3) and the derivatised sample (10 p_l) injected onto the HPLC column (Waters p.Bondapak C~s) exactly 1.5 rain after addition of the derivatising solution, lsocratic elution was carried out at a flow rate of 2 ml/min at ambient temperature. Homoserine was eluted first, well separated from taurine which had a retention time between 3 and 4 min (dependent on the characteristics of the column). Adducts were measured using a fluorimetric detector (Tridet, Perkin, Elmer) and integrator. After calibration, taurine concentrations were determined by integration of the peak areas of the eluted homoserine and taurine aducts.

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Fig. 1. Effect of various doses of CCI4 on the levels of taudne in 4-hourly urine samples collected from male rats over the 24-h period after dosing Control ; 0.5 ml.kg- l - - ; 1.0 ml.kg -I. . . . ; 2.0 ml.kg-~ . . . . . Values are means _+ SEM from four animals. * p

Taurine, a possible urinary marker of liver damage: a study of taurine excretion in carbon tetrachloride-treated rats.

Carbon tetrachloride (CCl4) caused a dose-dependent increase in urinary taurine which correlated with both the histological and biochemical assessment...
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