Tyrosine supplementation in chronic experimental uremia

BIOCHEMICAL

MEDICINE

30,

101-110

(1983)

Tyrosine Supplementation CAROLYN Department

L. ABITBOL,

of Pediatrics, and Department

in Chronic Experimental Uremia’

SUSAN MANDEL, KRYSTYNA RAUL A. WAPNIR

North Shore University Hospital, of Pediatrics, Cornell University New York, New York 10021

MROZINSKA,

Manhas.yet, Nenv Medicai College,

AND York II030

Received August 16. 1982

It has been suggested that tyrosine (Tyr) may be an essential amino acid in uremia (1). Aberrations in the metabolism of proteins have been reproduced in the uremic animal and have consistently resulted in abnormalities of plasma and tissue levels of Tyr and phenylalanine (Phe). The low Tyr and decreased Tyr:Phe ratio have been associated with poor nutritional intakes (2) and decreased conversion of Phe to Tyr (3). Lower plasma Tyr levels in uremia appear not to be due to increased urinary excretion (4), defective intestinal absorption (5), or increased degradation of Tyr (6). The current study was undertaken tc examine the possible relationship between Tyr deficiency and growth failure in chronic experimental uremia and to explore the effects of a substantial dietary supplementation of Tyr on growth-related parameters. We also examined some aspects of the alterations in Phe and Tyr metabolism in response to the supplementation, in an attempt to clarify the origin of uremic hypotyrosinemia. MATERIALS AND METHODS Young male Wistar rats (Charles River Breeding Labs, Wilmington, Mass.) weighing !90-100 g were rendered uremic by a subtotal nephrectomy. It was performed by a modification of the standard 5/6 nephrectomy described by Chantler et al. (7). The operation was carried out in two stages via a flank incision. On Day I, a right total nephrectomy was performed under ether anesthesia. Two days later, a left subtotal nephrectomy was conducted by removing a large portion of cortex by a radical wedge resection. This surgery accomplishes a highly significant 60% Supported in part by PHS NIH Grant SO8 RR 09128-03. 101 0006-2944/83 $3.00 Copyright 0 1983 by Academic Press. Inc. All rights of reproduclion in any form reserved.

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decrease in creatinine clearance. as compared to control animals (8). The controls were sham operated with exteriorization of the kidneys and decapsulation. The study protocol consisted of a recovery period of I week following the second surgery during which all animals were maintained at similar weights by restricting the intake of the controls to 10 g/day of powdered commercial feed (Purina Rat Chow, Ralston Purina Co.. St. Louis, MO. ). They were then divided into two groups according to the diet given. Diet 1 was the standard Purina Lab Chow: diet II was the same Purina Lab Chow with the addition of 3.5% Tyr (Sigma Chemical Co.. St. Louis, MO.). The addition of the Tyr increased the concentration of nitrogen from 3.7 g/l00 g feed to 4.3 g/l00 g feed. Three subgroups of animals were studied for each dietary treatment: uremic rats (U); their pair-fed controls (C,) and controls fed ad libitum CC,). Pair-feeding was performed by feeding control animals the quantity of food consumed by a matched U rat during the preceding day. This continued for 21 days, at which time the animals were sacrificed by exsanguination under ether anesthesia. The right tibia was removed, cleaned, and measured as an indication of linear growth. The gastrocnemius muscle was removed, freed from collagenous material. and weighed as a measure of muscle mass. Heparinized plasma was obtained from the aorta at the time of sacrifice. This was deproteinized with 10% trichloroacetic acid and frozen at -20” C until analysis. Tyr and Phe were determined on plasma by the method of Wong or (11. (9) using a Farrand Mark I spectrofluorometer. Liver and kidney tissue were removed (all that was remaining in the U and a cross-sectional piece in the C,, and C,,). homogenized, and analyzed for phenylalanine hydroxylase (PheH) and y-glutamyl transpeptidase (GGTP). PheH was analyzed according to the method of McGee et ~11.(10) by formation of Tyr from Phe. GGTP was determined by the method of Smith and Heizer (11) by the measurement of liberated I)nitroaniline. Liver and kidney tissue protein was assayed by the Lowry procedure (12). Tissue protein was determined in liver and reported as milligrams protein per gram wet weight of tissue. RESULTS Experirnrntnl

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The rats that underwent the two-stage nephrectomy developed moderate chronic uremia, as expected. Their plasma creatinine was very significantly elevated when compared to either Cr or C,., controls, regardless of whether the rats were supplemented with Tyr or not (Table I). This finding is comparable to other studies in which a similar procedure was followed (7.X).

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The mean ? SEM initial weight (grams) at the beginning of the 3week experimental period was lower in U rats (99.7 -t 4.6, N = 7) than in Cz and C,, animals (121.2 t 3.4. N = 31. P 0.01). This reflected the trauma of the nephrectomy. which was still more profound than the one produced by the sham operation. After 1. 2, and 3 weeks, C., animals gained significantly more weight than their corresponding U or C,, groups fed the same diet (Table 1). However, the C,, animals fed identical diets as U rats grew at a similar pace. Supplementation with Tyr failed to improve weight gain in either U or C,, animals. but increased the weight advantage of the rats fed ad libitum. In addition to total weight gain over the 3-week experimental period. tibia1 length and muscle mass were greater in the C, group fed diet I than in any of the other five groups (Table I). Significantly more tissue protein was found in control animals pair-fed to U rats (C,,) not supplemented with Tyr. There was no difference. however. between U and C,, rats when fed extra Tyr in the diet. Muscle mass, tibia1 length. and weight gain were significantly greater in corresponding C., groups. The difference in muscle mass between the two ad libitum-fed control groups was not significant. Supplementation with Tyr had no effect on linear growth or muscle mass in either control or U animals.

Plasma levels of Tyr and Phe were considered separately and as a ratio (Table 2). U rats fed standard Purina Rat Chow showed a significant decrease in plasma Tyr as compared to C,, and C., animals. Mean plasma Tyr was not significantly different between the C,, and U rats. Plasma Phe levels were unaltered in C, animals in relation to C,, but were significantly greater in U as compared to C,, and C, rats when the commercial feed only- was provided. In Tyr supplemented rats. the Phe levels were lower in Cr and U animals in comparison with the C:, group. In U rats. the Tyr:Phe ratio was lower than in C,, and C,, rats. when there was no extra amino acid to the diet. In all groups supplemented with Tyr, both plasma Tyr and Phe concentrations were elevated above normal values. However. a normal Tyr : Phe ratio, comparable to that shown by C,,, animals fed a standard ration, was reestablished in the U and C, rats provided with Tyr supplementation. Liver PheH activity was similar in all groups with or without Tyr supplementation, whether the data were expressed per gram of tissue or as specific activity (Table 3). Kidney PheH, however. was significantly

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lower in the remaining renal tissue of the U rats when expressed by unit of organ mass, as well as in terms of specific activity in the nonsupplemented animals. Among the Tyr supplemented rats, no differences between the groups for kidney PheH were observed in terms of specific activity, but they were lower for U animals when expressed by tissue weight. Cp rats in the ration-fed series had the highest specific activity of all groups. In the U rats, kidney GGTP was consistently lower, irrespective of Tyr supplementation or whether the results were expressed per gram of tissue or per milligram of protein. Pair-feeding introduced no alteration in the levels of this enzyme. The specimens of the CA group, fed diet II, were not analyzed for GGTP. DISCUSSION

Abnormalities of amino acid metabolism have been a prominent focus of interest in trying to determine the causes of growth failure and inanition frequent in chronic uremia. Determinations of amino acid patterns in the plasma of uremics and comparisons made with normal controls have pointed to suspected deficiencies of a number of specific amino acids. The decline in plasma levels have, in some cases, led to the conclusion that uremics may have increased requirements for certain amino acids (15-17). An example of these findings is the case of histidine, which is universally low in the plasma of uremic patients. This amino acid has been proposed to be essential in renal failure (16). To support this view, it has been shown that uremic patients. when supplemented with histidine, improved their anemia and nitrogen balance (16,17). Tyr has also been consistently recognized as deficient in renal failure. Kopple et af. (6) and Wang et al. (18) examined the various possible etiologies of the Tyr deficiency by studying the role of diet, renal function, Phe hydroxylation, and Tyr degradation in patients with moderate to severe chronic uremia. A discordance between low ratios of Tyr:Phe in uremic versus pair-fed control rats suggested that dietary inadequacies alone could not account for the more profound deficit in uremia (19). The current study also confirms the low ratio in nutritionally deprived U rats and Cp animals, indicating its association with a suboptimal nutritional status. Supplementation with Tyr sufficed to normalize the Tyr: Phe ratio (Table 2). A decrease in the activity of PheH in kidney tissue of U animals is thought to contribute to the low Tyr:Phe ratio (19). Speculation as to whether the decreased enzymatic activity is due to uremic toxins or to absolute loss of functioning renal mass has been prominent among researchers. Young and Parsons (3) demonstrated a 15% in vitro inhibition of PheH in rat liver by uremic plasma, but were unable to document a specific toxin. Previous investigators in this field have reported the results

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of enzyme activity only in terms of organ mass rather than specific activity. Hence, a differentiation between the inhibitory effects of uremic toxins on specific activity as opposed to an absolute effect due to a decreased functional renal mass was not possible. In the current study. decreased PheH was again noted relative to the renal mass. The deficiency. however, was less marked in terms of specific activitv. and only in the rats not supplemented with Tyr, suggesting that the synthesis of the enzyme is lower in the stump of kidney remaining in the U rats. Supplementation with Tyr appeared to have a protective effect which may be linked to more residual tissue remaining intact in the kidney. GGTP was also assayed as an index of the enzyme integrity of the kidney, as compared to PheH. Both enzymes are considered to be presenr. primarily. in the renal cortex. In these experiments. we found that the activity of GGTP in terms of renal mass. as well as its specific activity. was decreased in U animals, regardless of their supplementation with TYJ or not. These data suggest that loss of physiologically active renal mass is responsible for the decreased hydroxylation of Phe to Tyr. to a far greater extent than the effect of uremic toxins 01‘ other endogenous factors. This information also indicates that renal PheH may have a significant role in the overall balance of the Phe metabolic pathway. since there were no alterations caused by uremia or nutritional restrictions on hepatic PheH. In addition, nutritional supplementation of Tyr appeared not to have an inhibitory effect on PheH either in liver or in kidney. The experimental model used mimics human chronic uremia in terms of decreased appetite and growth. The present study also examined the effect of substantial dietary supplementation of Tyr on the growth of the moderately uremic rat. The negative results obtained in terms of linear growth rate and muscle mass must be considered only tentative. since the small number of uremic rats imposed a JatheJ large type II error. In addition. the design. involving only the one level of TYJ supplement. did not allow discrimination as to appropriate or excessive dose. It is known, however, that TYJ, when added to a protein-sufficient diet, causes no toxicity or impairment of growth in rats even with a 7ci; Tyr supplement (19.20). Our use of a 3.5% Tyr supplement added to a baianced commercial diet was well within the nutritional tolerance of rodents. Moreover, no ocular or dermal lesions were noted in our experimental animals which were described by Harper of rrl. (2 I ) when Tyr was used to supplement a protein-deficient diet. The elevated plasma TYJ levels in all animals receiving the supplement suggested over-compensation of the initial deficiency. Also, this effect of such dietary manipulation on the absorption and/or utilization of other amino acids is not well established. Competition for intestinal absorption sites between amino acids transported by common carrier\, i.c.. those

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specific for neutral aromatic amino acids, has been demonstrated previously in vivo (19,22). Moreover, the use of a free amino acid as a compensatory treatment can also be open to criticism, since it is now generally accepted that the absorption of protein occurs mostly at the di- and tripeptide level, with ultimate hydrolysis to single amino acids taking place in the enterocyte (23). Therefore, a dietary supplementation with a Tyr-containing peptide may have produced more significant results. Under our experimental conditions, the correction of the low plasma Tyr levels present in chronic experimental uremia by substantial dietary Tyr supplementation had no effect in terms of linear growth and weight gain or accumulation of muscle mass and tissue protein. Therefore, a small functional renal mass causing a loss of endogenous Tyr production by the kidney does not appear to make Tyr a rate-limiting, or essential, amino acid, under these experimental conditions. SUMMARY

The occurrence of low tyrosine tissue levels in uremic subjects, possibly due to impaired phenylalanine hydroxylation, suggests that tyrosine may be an essential amino acid in uremia. Additional dietary tyrosine may thus re-dress the deficiency. This study examined growth and tyrosine/ phenylalanine metabolism in uremic rats during tyrosine supplementation. Rats made uremic (U) by 7/8 nephrectomy were compared to pair-fed (C,) and ad Iibitum-fed (C,), sham-operated controls. Two sets of each group of rats were studied after 21 days on the resepctive diets: I = Purina Lab Chow; 11 = same + 3.5% tyrosine. Plasma tyrosine was below normal in U and &-fed diet 1. With diet II, the tyrosine:phenylalanine ratio in U was lower than both C, and C,. In rats fed diet II, the tyrosine : phenlalanine ratio became indistinguishable among the three groups. Growth parameters in U and C, were similar, regardless of the diet. Body weight gain, tibia1 length, muscle mass, and tissue protein did not improve in uremic animals supplemented wilh tyrosine. The specific activity of liver phenylalanine hydroxylase in U was not different from CA or Cr. However, loss of cortical renal mass appeared to be the major determinant of decreased kidney phenylalanine hydroxylation in experimental uremia. This alteration is likely to be the greatest contributory factor to the alteration of plasma levels of tyrosine and phenylalanine. The data presented do not support a proposed essentiality of tyrosine in uremia. ACKNOWLEDGMENTS We thank Mr. Robert S. Brody for statistical consultation. The unfortunate death of Ms. Krystyna Mrozinska imparts added significance to this manuscript for which she worked so diligently.

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