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The Journal of Nutrition Vol. 128 No. 11 November 1998,
pp. 1995-2000
Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, CA 95616 and * Department of Food Science and Human Nutrition, Colorado State University, Fort Collins, CO 80523
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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Total activity [pyridoxal 5'-phosphate (PLP) added in the assay] of hepatic tyrosine aminotransferase (TAT) measured in cats at 0300, 0900, 1500 and 2100h was 10.3 ±1.1, 14.0 ± 0.7, 9.8 ± 1.3 and 11.0 ± 0.7 nkat/g liver, indicating little diurnal variation. Activity after 18 h of food deprivation was 10.0 ± 0.3 nkat/g liver, also not different from cats that were eating ad libitum. These findings support the idea that cats have only limited changes in the activity of hepatic TAT compared with rats. Total TAT activity was measured in cats fed high protein (550 g/kg) and low protein (180 g/kg) diets for 4 wk. Cats fed a high protein diet had activities significantly higher (about twice) than cats fed the low protein diet. Hepatic TAT activity of vitamin B-6-deficient cats (diet without pyridoxine for 9 wk) was compared with cats given the same diet with 8 mg pyridoxine/kg. Total hepatic TAT activity in deficient cats was significantly (P < 0.05) lower per gram soluble or total protein (but not per gram liver) than control cats; holoenzyme activity and percentage of active enzyme in deficient cats were also significantly lower by 75 and 64%, respectively. The apparent Km of TAT from cats for tyrosine (2.1 mmol/L) was similar to that for rats (1.9 mmol/L), but higher for PLP in cats (0.16 µmol/L) than rats (0.034 µmol/L). Part of the reason for the higher plasma tyrosine in vitamin B-6-deficient cats than rats is the higher Km of TAT for PLP in cats than rats.
KEY WORDS: pyridoxal phosphate · tyrosine aminotransferase · vitamin B-6 deficiency · feline · dietary protein
Tyrosinemia has been reported in vitamin B-6 deficient cats (Bai et al, 1989), but has not been observed in other vitamin B-6-deficient animals, including humans (Park and Linkswiler 1971 Animals.
Specific pathogen-free cats from the Feline Nutrition and Pet Care Center at University of California, Davis were used in all experiments. They were vaccinated against panleukopenia and housed in individual metabolism cages (0.9 × 0.9 m) in a temperature-controlled (24 ± 3°C) room. Cats were kept on a 12-h light:dark schedule (lights on 0900 h). Food and water were available ad libitum. Adult Sprague-Dawley albino rats (Simonsen Laboratory, Gilroy, CA) and Zucker rats (UCD Nutrition Department breeding colony) were used in Experiment 1. Cats and rats were maintained according to a protocol approved by the University of California Animal Use and Care Administrative and Advisory Committee and according to NIH guidelines (NRC 1985).
Diets.
The compositions of the purified diets used in Experiments 2 and 4 are shown in Table 1. The diets for the vitamin B-6-deficient group contained no added pyridoxine (PN), and the diet for the control group contained 8 mg added PN/kg diet. The diet used in Experiment 3 contained 350g/kg vitamin-free casein and proportional adjustment of starch and sucrose as previously reported (Bai et al. 1989
Experimental design.
Experiment 1 was conducted to determine the apparent Michaelis constants of TAT for tyrosine and PLP from cat and rat livers. Livers were obtained from two adult and two juvenile cats and two albino adult Sprague-Dawley rats and two adult Zucker rats. Cats were anesthetized by intravenous injection of sodium pentobarbital; a laparotomy was performed and the liver rapidly removed, blotted and weighed. Each liver was immediately placed on crushed ice for transport to the laboratory. Rats were stunned by a sharp blow on the head, decapitated and the liver was removed, weighed and chilled. All assays were done on fresh liver preparations except the determination of the Km for PLP, which was done on a purified enzyme preparation stored at Plasma tyrosine and cystathionine.
Samples of blood (3 mL) were taken in heparinized syringes from the jugular vein of unanesthetized cats, centrifuged at 1500 × g for 10 min, and the plasma was separated and stored at TAT assay procedure.
To determine the apparent Km of TAT for PLP in rat and cat liver, a partial purification of the enzyme through the "heat treatment" step of the purification procedure of Hayashi et al. (1967) B-6 vitamers.
B-6 vitamers in plasma (pyridoxal phosphate [PLP] and pyridoxal [PL]) and liver (PLP and pyridoxamine phosphate [PMP]) were analyzed by reverse-phase ion-pairing HPLC, as described by Sampson and O'Connor (1988) Statistical analysis.
All values were expressed as means ± SEM and were subjected to ANOVA. Student's t test (Mendenhall et al. 1986 Experiment 1.
The apparent Kmvalues of TAT from cats and rats for tyrosine were similar, 2.1 and 1.9 mmol/L, respectively (Table 2). However, the apparent Km of TAT from cats and from rats for PLP was 0.16 and 0.034 µmol/L, respectively.
Experiment 2.
Total TAT activities in nkat/g wet liver during a 24-h period were significantly different for only one time period comparison, 0900 vs. 1500 h (Table 3). Even though these time points were significantly different, they deviated <30% from the mean value. Hepatic TAT activity of cats after 18 h of food deprivation was not significantly different than that of cats that consumed food ad libitum.
Experiment 3.
Vitamin B-6-deficient cats had a significantly lower mean body weight gain than that of control cats (Fig. 1A). The linear component of the growth curves of cats from vitamin B-6-deficient and control groups were 9 and 14 g/d, respectively. During the experimental period, the daily food intake of B-6-deficient cats was 56 ± 2 g/d, which was significantly less than that of control cats (69 ± 2 g/d) (Fig. 1B).
Experiment 4.
Hepatic total TAT activity in cats fed the high protein diet was about twice that of cats fed the low protein diet (Table 6)(P < 0.05).
Tyrosine aminotransferase (TAT) is a dimeric enzyme that contains structural features that support transamination (Hargrove et al. 1989
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
, Swendseid et al. 1964). Tyrosinemia occurs in humans as a result of tyrosine aminotransferase deficiency (Richner-Hanhart Syndrome, tyrosinemia type II) and is the only enzyme defect causing tyrosinemia that requires pyridoxal 5'-phosphate (PLP)4 as a coenzyme. This metabolic disorder is characterized by tyrosinemia, tyrosinuria and p-hydroxyphenyl-pyruvaturia, -lacturia and -aceturia (Nyhan 1984). The concentration of tyrosine in the blood is the balance between dietary intake of tyrosine and phenylalanine, protein synthesis and degradation, and irreversible loss through hepatic catabolic pathways. Tyrosine aminotransferase (TAT) (L-tyrosine: 2-oxoglutarate aminotransferase, EC 2. 6. 1. 5), a PLP-dependent enzyme, initiates the catabolism of tyrosine in the liver and is the rate-limiting enzyme in tyrosine catabolism (Dickson et al. 1981). We have suggested that tyrosinemia induced by vitamin B-6 deficiency in cats could be the result of inadequate TAT activity, and/or due to some peculiarity in the apoenzyme of TAT and/or its affinity for PLP. These studies were conducted to test the hypothesis that the tyrosinemia observed in vitamin B-6-deficient cats may be the result of the enzyme having a lower capability in cats to adapt to low levels of PLP compared with that in rats. To evaluate the adaptive changes in activity of hepatic TAT in cats, four experiments were conducted. The first experiment was designed to compare apparent Michaelis constants of the enzyme for tyrosine and PLP from cat and rat livers. Because the activity of TAT in the rat liver has been reported to be at least four times greater several hours after the onset of darkness than during the daily light period (Wurtman and Axelrod 1967), the second experiment was designed to examine whether hepatic TAT in cats had a daily rhythm, similar to that observed in other animals (Civen et al. 1967, Wurtman and Axelrod 1967). The objectives of the third and fourth experiments were to examine effects of vitamin B-6 deficiency and high dietary protein, respectively, on TAT activity in cats.
MATERIALS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
). The diet for the rats used for the kinetic study was a complete commercial pelleted diet (Rat Diet # 5012, PMI Feed, St Louis, MO).
View this table:
Table 1.
Composition of the experimental diets
80°C.
80°C until analyzed for amino acids. Plasma was deproteinized with an equal volume of 60g/L sulfosalicylic acid and centrifuged at 10,000 × g for 12 min at 4°C. The deproteinized supernatant was analyzed for free amino acid concentrations using a Beckman amino acid analyzer (system 7300, Palo Alto, CA).
was used in Experiment 1. The fixed-time assay as described by Mavrides (1987) was used for assaying the enzyme activity. The reaction mixture contained 5.6 µmol tyrosine, 9.0 µmol 
-ketoglutarate, 1 µmol EDTA, 1 µmol dithiothreitol and 80 µmol triethanolamine at pH 7.6. Various pyridoxal 5'-phosphate concentrations were used in a final volume of 1 mL. The mixture minus -ketoglutarate was preincubated at 37°C for 10 min. The reaction was started by the addition of 50 µL of freshly prepared and neutralized solution of -ketoglutarate. After 10 min, the reaction was stopped by the addition of 0.07 mL of 10 mol/L NaOH. After 30 min at room temperature, the absorbance at 330 nm was measured using a spectrophotometer (Gilford model 2000 multiple sample absorbance recorder, Oberlin, OH).
as previously described by Szepesi and Freedland (1967). Liver samples were homogenized in 4 volumes of 0.14 mol/L KCl, centrifuged at 30,000 × g for 30 min at 0-4°C, and the resulting clear portion of the supernatant fraction was used as the enzyme source within 2 h. The supernatants were kept in an ice bath before the assays. Aliquots of the homogenate and the supernatant were assayed for total and soluble liver protein by a modified biuret procedure (Mokrasch and McGilvery 1956). TAT activity was measured at a final dilution 1:100 by determining the amount of p-hydroxyphenylpyruvate (PHPP) formed from tyrosine at 310 nm. One katal of TAT activity is defined as 1 mol of PHPP formed per second. The results are reported per gram wet liver, per gram total protein, per gram soluble protein and/or per 100 g body weight. Apparent Km were calculated by the double reciprocal plot method of Lineweaver and Burk (1934).
. Vitamers other than PLP and PL in plasma and PLP and PMP in liver contributed only a small percentage of the total vitamin B-6; they were not affected by dietary treatment and therefore are not reported.
) or Fisher's Least Significant Difference (LSD) test (Steel and Torrie 1980) was used to evaluate statistical differences with P < 0.05 considered significant.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
View this table:
Table 2.
Apparent Michaelis constants of tyrosine aminotransferase for tyrosine and pyridoxal phosphate from livers of
cats and rats (Experiment 1)
View this table:
Table 3.
Hepatic total tyrosine aminotransferase activities of
cats at four 6-h intervals over 24 h and after
food deprivation (Experiment 2)1,2
View larger version (28K):
[in a new window]
Fig 1.
Effects of a vitamin B-6 deficient diet in cats on: (A) cumulative body weight gain, (B) weekly average daily food intake, (C) concentrations of plasma free tyrosine (Tyr) (Experiment 3) and (D) concentrations of plasma free cystathionine (Experiment 3). Values are means ± SEM ( B6, vitamin B-6-deficient cats, n = 4; +B6, control cats, n = 5). Differences between means are indicated: *P < 0.05.
PLP activity) expressed as a percentage of active enzyme in vitamin B-6-deficient cats was significantly lower than that of control cats whether expressed per gram fresh liver, per 100 g body weight, per gram total protein or per gram soluble protein.
View this table:
Table 4.
Effects of vitamin B-6 deficiency on hepatic tyrosine aminotransferase (TAT) activities in cats (Experiment 3)1
View this table:
Table 5.
Effect of vitamin B-6 deficiency on the pyridoxal phosphate (PLP) and pyridoxal (PL) concentrations in plasma and liver of cats (Experiment 3)1
View this table:
Table 6.
The adaptive response of hepatic total tyrosine aminotransferase (TAT) activities in cats fed high and low protein diets (Experiment 4)1
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
). This enzyme has been isolated from rats and frogs (Donner et al. 1978, Ohisalo et al. 1977), and the characteristics of TAT from rat livers have been reported (Hayashi et al. 1967). Aminotransferase reactions are complicated, multistep processes, and their complete kinetic analyses require that velocity-substrate relationships for a reactant be studied at several concentrations of the other substrates. We examined the velocity of the TAT reaction by using liver cell extracts and a purified enzyme preparation as the enzyme source and saturating levels of the other reactants. The substrate concentrations at which we found half-maximal velocities are referred to as the "apparent" Michaelis constants. The apparent Km values of TAT from cat and rat livers for tyrosine were similar (Table 2). The rat values agreed well with those reported by Hayashi et al. (1967) of 1.4 and 1.7 mmol/L from two different assay methods. The apparent Km that we found for TAT from cat livers for PLP was approximately five times that found for rats, leading us to suggest that the decline in PLP in the liver of vitamin B-6-deficient cats decreases TAT activity in vivo, thus explaining why plasma tyrosine increases in vitamin B-6-deficient cats and not in vitamin B-6-deficient rats (Bai et al. 1989 and 1991).
). Wurtman and Axelrod (1967) observed that TAT activity in rat liver was at least four times greater several hours after the onset of darkness than during the light period. Yamamoto et al. (1979) suggested that the daily rhythm of TAT in rats is a result of the daily rhythm of food intake. Rats do not usually eat in the first half of the light period but consume 8-10 meals during the dark period. In this study of cats, the activity of hepatic TAT had minimal diurnal variation (Table 3). The reason cats show so little diurnal rhythm in TAT is probably related to their normal eating pattern, which is to take many small meals evenly distributed during both the light and dark periods (Kane et al. 1981). Although some cats eat more during the light period than the dark period, other cats have the reverse pattern (Kane et al. 1981, Thorne 1982).
found that the TAT enzyme activity without added PLP in rats fed a vitamin B-6-deficient diet for 5 wk was decreased 21%, even though total TAT activity was not affected. In contrast, Lee et al. (1977) reported that vitamin B-6 deficiency caused a 40% decrease in total TAT activity and a 65% decrease in enzyme activity assayed without added PLP in rats fed the deficient diet for 12 wk. In an earlier report, Lin et al. (1958) indicated that total TAT activity and TAT enzyme activity without added PLP decreased by a similar amount (40 and 45%) in rats fed a vitamin B-6-deficient diet for 13-14 wk. There is general agreement that in vitamin-B-6 deficient rats, TAT enzyme activity decreases without added PLP, but there is some disagreement concerning whether total TAT activity decreases. Lee et al. (1977) found that the rate of synthesis of TAT was reduced to 46% of control in the vitamin B-6-deficient rats. They also showed that the rate of TAT degradation did not differ between control and vitamin B-6-deficient rats. They concluded that coenzyme deficiency resulted in a significant reduction in TAT levels and that much of this enzyme was free of coenzyme in the vitamin B-6-deficient state, but its degradation proceeded at the normal rate. In contrast, Reynolds and co-workers (Reynolds 1978, Sloger et al. 1978) concluded that vitamin B-6 deficiency blocked TAT "inactivation" (i.e., degradation) in rat liver. However, they used in vitro techniques based on die-away of TAT enzyme activity in liver homogenates, whereas the conclusions of Lee et al. (1977) were based on more direct measures of TAT protein degradation using specific antisera with immunoprecipitation. In our study, vitamin B-6 deficiency caused a 20-30 % decrease in total TAT activity, whereas enzyme activity (measured at 1:100 dilution without PLP added) decreased by 75%. This reduction of enzyme activity without added PLP in vitamin B-6-deficient cats was more severe than that reported in B-6-deficient rats (21, 45 or 65%) and is consistent with the effect of the fourfold higher Km of TAT for PLP in cats vs. rats. This greater reduction of enzyme activity without added PLP in vitamin B-6-deficient cats than rats thus appears to explain why vitamin B-6 deficiency causes tyrosinemia in cats, but not in rats.
, Reynolds 1978). The disparity between liver PLP concentration and the percentage of total TAT present as the holoenzyme suggests that PLP concentration available to TAT must be much lower than the overall mean liver PLP concentration. Information about localized PLP concentration, and even about PLP distribution among PLP-dependent enzymes within a tissue is limited (Bosron et al. 1978). Allgood et al. (1993) speculated that variation in PLP concentration among tissues or cells within tissues may modulate steroid-responsive gene expression [which could include TAT, because it contains a glucocorticoid response element (Lee et al. 1977)]. If these authors are correct, i.e., that local alterations of PLP concentrations occur within cell types, or, by extension, within cell compartments, then our TAT data may be the result of localized vitamin B-6 deficiency near TAT, possibly exacerbated by the low affinity of TAT for PLP (Lee et al. 1977). To evaluate this possibility, knowledge of both the subcellular PLP concentrations within hepatocytes and the molar concentration of all PLP-dependent enzymes would be required. Such information is lacking in the literature. The results in rats of Lee et al. (1977) discussed above, suggest that the decreased total TAT caused by vitamin B-6 deficiency found in this study may be due to decreased synthesis of TAT, rather than to increased degradation. There was a small decrease in food intake that would have reduced the quantity of tyrosine available; on the other hand, there was a decrease in weight gain. The mechanism by which vitamin B-6 deficiency decreases the activity of TAT deserves further investigation.
). A similar effect is seen in rats (Stabler, et al. 1997). It is tempting to speculate that, as for plasma tyrosine and TAT, vitamin B-6 deficiency depressed flux though the PLP-dependent enzyme, cystathionase. Such an effect has been reported in liver of vitamin B-6-deficient rats (Takeuchi et al. 1991). Direct confirmation of such an effect in cats awaits future study.
). The ratios of PLP to PL in plasma of cats and rats are not the same [vitamin B-6-replete cats ~5:1; vitamin B-6-replete rats ~1:1 (Sampson and O'Connor 1989)]; therefore, changes occurring with vitamin B-6 deficiency are species dependent. In vitamin B-6 deficiency, PLP falls to nondetectable levels in cats, whereas PL is reduced to about one fifth normal. In rats, both vitamers are equally depressed (Sampson and O'Connor 1989). However, although the magnitude of the reduction in hepatic PLP in vitamin B-6-deficient cats was similar to that of vitamin B-6-deficient rats, PMP concentration in rats was not altered by deficiency, whereas it was reduced to about one third normal in vitamin B-6-deficient cats.
; Szepesi and Freedland 1967). Rogers et al. (1977) reported that the activities of most of the hepatic aminotransferases and urea cycle enzymes in cats were not influenced by protein intake, but TAT was an exception. They reported that hepatic TAT activities from adult cats fed a high (700 g soybean protein/kg diet) and a low (17 5g soybean protein/kg diet) protein diet for 5 wk were 3.7 ± 0.7 and 1.2 ± 0.5 nkat/g liver, respectively (mean ± SEM, n = 5). In addition, they found that hepatic TAT activity in adult cats consuming a diet containing 350 g protein/kg diet was not significantly different from that of cats that were fed or food deprived (6.8 ± 1.8 vs. 5.3 ± 0.5 nkat/g liver). Silva and Mercer (1985) later reported that flux rate of TAT in hepatocytes derived from cats fed a high protein (700 g soybean protein/kg) diet was two to three times higher than that of hepatocytes from cats fed a low protein (175 g soybean protein/kg) diet.
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FOOTNOTES |
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Manuscript received 15 December 1997. Initial reviews completed 22 January 1998. Revision accepted 16 June 1998.
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ACKNOWLEDGMENTS |
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The authors wish to thank R. A. Freedland and E. Avery for valuable discussions and assistance with enzyme and protein assays.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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recent advances.
J. Small Anim. Pract.
1982;
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K. Russell, P. R. Murgatroyd, and R. M. Batt Net Protein Oxidation Is Adapted to Dietary Protein Intake in Domestic Cats (Felis silvestris catus) J. Nutr., March 1, 2002; 132(3): 456 - 460. [Abstract] [Full Text] [PDF]
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