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Kidney clearance of d - and l -tryptophan by rats
Camp.
Eiochem.
Physiol.
Vol. 85A, No. 3, pp. 451453,
0300-9629/86 $3.00 + 0.00 Pergamon Journals Ltd
1986
Printed in Great Britain
KIDNEY
CLEARANCE
SHUE-YUAN Division
of Nutritional
OF D- AND BY RATS WANG
Sciences,
L-TRYPTOPHAN
and M. C. NESHEIM
Cornell
University,
(Receiced 11 February
Ithaca,
NY 14853, USA
1986)
Abstract-l.
Renal clearances of D-tryptophan and of L-tryptophan by rats were compared. 2. Both D- and L-tryptophan were reabsorbed by the rat kidney tubules very efficiently. 3. Compared to the rapid excretion of D-tryptophan by the chick, the good retention of this isomer by the rat kidney might be responsible for its efficient utilization by rats.
exposed. A polyethylene tubing was carefully inserted into the bladder avoiding internal bleeding. The opening of the bladder was sutured securely around the tubing for collecting urine quantitatively. A diuretic solution (5% mannitol and 0.12% inulin in saline) was infused at 0. I ml/min starting 41-60min before the first urine collection. A priming dose of inulin (50 mg/kg body wt) was administered i.v. before starting the infusion. Infusion of D- or L-tryptophan (1.35 pmole/min/kg) was begun immediately following the collection of the first urine sample. Urine was collected at 20-min intervals and a blood sample was taken from the tail at the midpoint of each urine collection period. The infusion was continued for 2-3 hr. All clearance studies were completed within 4 hr of withdrawal of food. The blood collected with heparin was immediately centrifuged. Plasma and urine samples were stored at -70°C until analyzed (within 2 days). Four rats were used for D-tryptophan and another four rats were used for L-tryptophan infusion studies. Inulin and tryptophan levels were measured in both the plasma and urine samples. Inulin levels were determined by a resorcinol method (Schreiner, 1950) which was modified for microassay without affecting the accuracy and sensitivity of the method. Tryptophan levels were determined by a fluorescent method (Denckla and Dewey, 1967). All the determinations were in duplicate. Renal clearances were calculated by methods described by Smith (1956).
INTRODUCTION variations are found in utilization of D-tryptophan by different species. In rats (Berg, 1934) and swine (Thompson et al., 1952), D-tryptophan is used for growth almost as efficiently as its L-isomer. The D-isomer is poorly effective as a source of tryptophan in chicks (Wilkening and Schweigert, 1947) and dogs (Czarnecki and Baker, 1982), and is apparently not used by humans (Rose et al., 1954), rabbits (Schayer, 1950), and mice (Celander and Berg, 1953). In several metabolic studies, a considerable portion of administered D-tryptophan was found in the urine of humans (Langner and Berg, 1955), dogs (Triebwasser et al., 1976), and chicks (Ohara et al., 1980), while only traces of administered D-tryptophan was found in the urine of rats (Ohara et al., 1980). In renal clearance studies with chicks, Nesheim et a/. (1974) found that D-tryptophan was cleared from plasma by the kidney at the same rate as inulin in contrast to efficient reabsorption of L-tryptophan by the kidney. Since D-tryptophan was excreted rapidly in the chick urine and there was very little chance for it to be metabolized, they concluded that the poor utilization of D-tryptophan by chicks was at least partially due to the rapid excretion of D-tryptophan by the kidney. To confirm the results of the study of Ohara and his colleague that D-tryptophan would be reabsorbed by the rat kidney and to determine how the rat kidney processes this isomer, a renal clearance study was carried out and is reported in this paper. Wide
RESULTS
Results of the clearances of tryptophan and inulin are shown in Fig. 1. The clearance rate of D-tryptophan was slightly higher (P < 0.05) than that of L-tryptophan, but there was no significant difference in the inulin clearance between the two groups of rats. The clearance ratio of tryptophan to inulin is shown in Fig. 2. There was no significant difference in the clearance ratio for D- and L-tryptophan in rats. The average ratio was less than 0.01 for both D- and L-tryptophan, indicating efficient reabsorption of both tryptophan isomers. Plasma tryptophan rose during either D- or L-tryptophan infusion (Fig. 3), but there was no significant difference between the levels of the two isomers. As shown in Fig. 4, there were traces of tryptophan excreted in the urine over the collection period with tryptophan excreted when more somewhat D-tryptophan was infused than when the L-isomer
MATERIALS AND METHODS
Male Sprague-Dawley rats, weighing 400 g, were kept in separate cages in a room with the temperature kept at 78-80°F and with a 12-hr lightdark cycle. They were allowed free access to a commercial laboratory diet and water, prior to the renal clearance study. The rat was maintained under light anesthesia with Nembutal (5 mg/lOO g body wt) and was restrained on an animal board on its back. A needle from a butterfly infusion set was connected to a compact infusion pump (Model 975, Harvard Apparatus Co., Inc., Dover, Mass.). A small incision on the lower abdomen was made and the bladder was 451
was infused, but the differences were not statistically significant. The results showed that neither D- nor ~~tr~~~~~~a~
was excreted
in the urine
of rats i*
Fig. 4. Urinary concentration of tryptophan during a 2-hr infusion of D- or L.-tryptophan in rats. The experimental conditions were as described in Fig. I.
s~~i~~nt3~aunts.
This excrement dem~nst~ted that D-tryptophan was reabsorbed by the rat kidney almost as efficiently as L-tryptophan. Time
(mini DiSCUSSION
These studies ~cmonstr3te &at. t&z rat kidney has the ability to retain ~tr~~to~~~~ by kidnegf reabsorption quite effieiendy. This ability, along with
four rats.
an active D-amino acid oxidase (Wang, 1985) is probably responsible for the ability of this species to use o-tryptophan noarly as efficiently as the L- form for growth. fn contrast to a&se findings, chickens do not retain D*tryptophan by kidney t&.&u ~~bso~~~~ to any significant extent (Nesheim et al., 1974). Thus, even though some D-amino acid oxidase activity is present, in chicken liver and kidney (Wang, 1985) the trypa tophan excretion occurred in the urine before appreciable conversion to ~-tr~~to~~an coutd take ptace. IS seems IikikeZy that human beings and dcrgs do xx% make &krez3t
use of ~-t~~~~~~a~
ii-xsimiiar feas5ns in
view of the substantial urinary excretion of D-tryptophan observed in these species (Langner and Berg, 1955; Triebwasser et al., 1976).
Rg. 2. The rztioof tryptaphan clearanw to inullin zAm?mx? in rats during a 2&r in&ion of D or Nryptophan. The experimental conditions were as described in Fig. 1.Arrows mark the beginning of infusion of each isamer.
5 8 h^
EE
i$
L
Fig, 3. Plasma concentration of tryptophan during a 2&r in&&an of D- or L-tryptophan in rats. The experimenta’t conditions were as described in Fig. 1.
Berg C. P. (1934) Tryptophan metabolism: TV.The influence for growth and far kynurenic acid production. J. bioi. Chem. 104, 373-384, Cclander D. R. and Berg C. P. (1953) The availability of a-histidine, related &nmidazotes,and u-tryptophan in the rn#Use. J. h&f. C&n%. fB3, 339-350, Czarnecki G. L. and Baker fi. H. [1982> Ut~~~~tjo~ d 0” and ~tryptopban by the growing dog. f. Anim. SG%53, 1405-1410. Denckla W. D. and Dewey H. K. (1967) The determination of tryptophan in plasma, liver and urine. J. Cab. Med. 69, I#-169. Langner B. R. and rterg C. P. (I%%) metabolism of ~tr~~to~b~ in the norm& burn= subjrsc. J+ kif3L C&m. 214, 699-707. Nesheim M. C., Crasby L. 0. and Kuenzel W. 3. j1974) T&
oxidation and kidney clearance of U-tryptophan by chicks. Proc. SW. rxp, Biol. Med. 147, 850-854. Qhara 1., Otsuka Sal,, Yugari Y., and Ariyoshi S. (1980) Inversion of D-tryptophan to t-tryptophan and excretion patterns in the rat and chick. J. Nutr, Zr@,64-648.
D-tryptophan Rose W. C., Lambert G. F. and Coon M. J. (1954) The amino acid requirements of man: VII. General procedures. The tryptophan requirement. J. bid. Chem. 211, 815-827. Schayer R. W. (1950) Studies of the metabolism of tryptophan labeled with iSN in the indole ring. J. biol. Chem. 187, 777-786. Schreiner G. E. (1950) Determination of inulin by means of resorcinol. Proc. Sot. exp. Bid. Med. 74, 117-120. Smith H. W. (1956) Principles of Renal Physiology, Section III, pp. 2530. Oxford University Press, New York.
CkaranCC
453
Thompson C. M., Reber E., Whitehair C. K. and MacVicar R. (1952) Utilization of D-tryptophan by swine. J. Anim. Sci. 11, 712-720. Triebwasser K. C., Swan P. B., Henderson L. M. and Budry J. A. (1976) Metabolism of D- and L-tryptophan in dogs, J. Nutr. 106, 642-652. Wang S-Y. (1985) Studies of species differences in utilization of D- and L-tryptophan. Ph.D. thesis, Cornell Univ., Ithaca, New York. Wilkening M. C. and Schweigert B. S. (1947) Utilization of D-tryptophan by the chick. i. bid. Chem. 171, 209-212.
Eiochem.
Physiol.
Vol. 85A, No. 3, pp. 451453,
0300-9629/86 $3.00 + 0.00 Pergamon Journals Ltd
1986
Printed in Great Britain
KIDNEY
CLEARANCE
SHUE-YUAN Division
of Nutritional
OF D- AND BY RATS WANG
Sciences,
L-TRYPTOPHAN
and M. C. NESHEIM
Cornell
University,
(Receiced 11 February
Ithaca,
NY 14853, USA
1986)
Abstract-l.
Renal clearances of D-tryptophan and of L-tryptophan by rats were compared. 2. Both D- and L-tryptophan were reabsorbed by the rat kidney tubules very efficiently. 3. Compared to the rapid excretion of D-tryptophan by the chick, the good retention of this isomer by the rat kidney might be responsible for its efficient utilization by rats.
exposed. A polyethylene tubing was carefully inserted into the bladder avoiding internal bleeding. The opening of the bladder was sutured securely around the tubing for collecting urine quantitatively. A diuretic solution (5% mannitol and 0.12% inulin in saline) was infused at 0. I ml/min starting 41-60min before the first urine collection. A priming dose of inulin (50 mg/kg body wt) was administered i.v. before starting the infusion. Infusion of D- or L-tryptophan (1.35 pmole/min/kg) was begun immediately following the collection of the first urine sample. Urine was collected at 20-min intervals and a blood sample was taken from the tail at the midpoint of each urine collection period. The infusion was continued for 2-3 hr. All clearance studies were completed within 4 hr of withdrawal of food. The blood collected with heparin was immediately centrifuged. Plasma and urine samples were stored at -70°C until analyzed (within 2 days). Four rats were used for D-tryptophan and another four rats were used for L-tryptophan infusion studies. Inulin and tryptophan levels were measured in both the plasma and urine samples. Inulin levels were determined by a resorcinol method (Schreiner, 1950) which was modified for microassay without affecting the accuracy and sensitivity of the method. Tryptophan levels were determined by a fluorescent method (Denckla and Dewey, 1967). All the determinations were in duplicate. Renal clearances were calculated by methods described by Smith (1956).
INTRODUCTION variations are found in utilization of D-tryptophan by different species. In rats (Berg, 1934) and swine (Thompson et al., 1952), D-tryptophan is used for growth almost as efficiently as its L-isomer. The D-isomer is poorly effective as a source of tryptophan in chicks (Wilkening and Schweigert, 1947) and dogs (Czarnecki and Baker, 1982), and is apparently not used by humans (Rose et al., 1954), rabbits (Schayer, 1950), and mice (Celander and Berg, 1953). In several metabolic studies, a considerable portion of administered D-tryptophan was found in the urine of humans (Langner and Berg, 1955), dogs (Triebwasser et al., 1976), and chicks (Ohara et al., 1980), while only traces of administered D-tryptophan was found in the urine of rats (Ohara et al., 1980). In renal clearance studies with chicks, Nesheim et a/. (1974) found that D-tryptophan was cleared from plasma by the kidney at the same rate as inulin in contrast to efficient reabsorption of L-tryptophan by the kidney. Since D-tryptophan was excreted rapidly in the chick urine and there was very little chance for it to be metabolized, they concluded that the poor utilization of D-tryptophan by chicks was at least partially due to the rapid excretion of D-tryptophan by the kidney. To confirm the results of the study of Ohara and his colleague that D-tryptophan would be reabsorbed by the rat kidney and to determine how the rat kidney processes this isomer, a renal clearance study was carried out and is reported in this paper. Wide
RESULTS
Results of the clearances of tryptophan and inulin are shown in Fig. 1. The clearance rate of D-tryptophan was slightly higher (P < 0.05) than that of L-tryptophan, but there was no significant difference in the inulin clearance between the two groups of rats. The clearance ratio of tryptophan to inulin is shown in Fig. 2. There was no significant difference in the clearance ratio for D- and L-tryptophan in rats. The average ratio was less than 0.01 for both D- and L-tryptophan, indicating efficient reabsorption of both tryptophan isomers. Plasma tryptophan rose during either D- or L-tryptophan infusion (Fig. 3), but there was no significant difference between the levels of the two isomers. As shown in Fig. 4, there were traces of tryptophan excreted in the urine over the collection period with tryptophan excreted when more somewhat D-tryptophan was infused than when the L-isomer
MATERIALS AND METHODS
Male Sprague-Dawley rats, weighing 400 g, were kept in separate cages in a room with the temperature kept at 78-80°F and with a 12-hr lightdark cycle. They were allowed free access to a commercial laboratory diet and water, prior to the renal clearance study. The rat was maintained under light anesthesia with Nembutal (5 mg/lOO g body wt) and was restrained on an animal board on its back. A needle from a butterfly infusion set was connected to a compact infusion pump (Model 975, Harvard Apparatus Co., Inc., Dover, Mass.). A small incision on the lower abdomen was made and the bladder was 451
was infused, but the differences were not statistically significant. The results showed that neither D- nor ~~tr~~~~~~a~
was excreted
in the urine
of rats i*
Fig. 4. Urinary concentration of tryptophan during a 2-hr infusion of D- or L.-tryptophan in rats. The experimental conditions were as described in Fig. I.
s~~i~~nt3~aunts.
This excrement dem~nst~ted that D-tryptophan was reabsorbed by the rat kidney almost as efficiently as L-tryptophan. Time
(mini DiSCUSSION
These studies ~cmonstr3te &at. t&z rat kidney has the ability to retain ~tr~~to~~~~ by kidnegf reabsorption quite effieiendy. This ability, along with
four rats.
an active D-amino acid oxidase (Wang, 1985) is probably responsible for the ability of this species to use o-tryptophan noarly as efficiently as the L- form for growth. fn contrast to a&se findings, chickens do not retain D*tryptophan by kidney t&.&u ~~bso~~~~ to any significant extent (Nesheim et al., 1974). Thus, even though some D-amino acid oxidase activity is present, in chicken liver and kidney (Wang, 1985) the trypa tophan excretion occurred in the urine before appreciable conversion to ~-tr~~to~~an coutd take ptace. IS seems IikikeZy that human beings and dcrgs do xx% make &krez3t
use of ~-t~~~~~~a~
ii-xsimiiar feas5ns in
view of the substantial urinary excretion of D-tryptophan observed in these species (Langner and Berg, 1955; Triebwasser et al., 1976).
Rg. 2. The rztioof tryptaphan clearanw to inullin zAm?mx? in rats during a 2&r in&ion of D or Nryptophan. The experimental conditions were as described in Fig. 1.Arrows mark the beginning of infusion of each isamer.
5 8 h^
EE
i$
L
Fig, 3. Plasma concentration of tryptophan during a 2&r in&&an of D- or L-tryptophan in rats. The experimenta’t conditions were as described in Fig. 1.
Berg C. P. (1934) Tryptophan metabolism: TV.The influence for growth and far kynurenic acid production. J. bioi. Chem. 104, 373-384, Cclander D. R. and Berg C. P. (1953) The availability of a-histidine, related &nmidazotes,and u-tryptophan in the rn#Use. J. h&f. C&n%. fB3, 339-350, Czarnecki G. L. and Baker fi. H. [1982> Ut~~~~tjo~ d 0” and ~tryptopban by the growing dog. f. Anim. SG%53, 1405-1410. Denckla W. D. and Dewey H. K. (1967) The determination of tryptophan in plasma, liver and urine. J. Cab. Med. 69, I#-169. Langner B. R. and rterg C. P. (I%%) metabolism of ~tr~~to~b~ in the norm& burn= subjrsc. J+ kif3L C&m. 214, 699-707. Nesheim M. C., Crasby L. 0. and Kuenzel W. 3. j1974) T&
oxidation and kidney clearance of U-tryptophan by chicks. Proc. SW. rxp, Biol. Med. 147, 850-854. Qhara 1., Otsuka Sal,, Yugari Y., and Ariyoshi S. (1980) Inversion of D-tryptophan to t-tryptophan and excretion patterns in the rat and chick. J. Nutr, Zr@,64-648.
D-tryptophan Rose W. C., Lambert G. F. and Coon M. J. (1954) The amino acid requirements of man: VII. General procedures. The tryptophan requirement. J. bid. Chem. 211, 815-827. Schayer R. W. (1950) Studies of the metabolism of tryptophan labeled with iSN in the indole ring. J. biol. Chem. 187, 777-786. Schreiner G. E. (1950) Determination of inulin by means of resorcinol. Proc. Sot. exp. Bid. Med. 74, 117-120. Smith H. W. (1956) Principles of Renal Physiology, Section III, pp. 2530. Oxford University Press, New York.
CkaranCC
453
Thompson C. M., Reber E., Whitehair C. K. and MacVicar R. (1952) Utilization of D-tryptophan by swine. J. Anim. Sci. 11, 712-720. Triebwasser K. C., Swan P. B., Henderson L. M. and Budry J. A. (1976) Metabolism of D- and L-tryptophan in dogs, J. Nutr. 106, 642-652. Wang S-Y. (1985) Studies of species differences in utilization of D- and L-tryptophan. Ph.D. thesis, Cornell Univ., Ithaca, New York. Wilkening M. C. and Schweigert B. S. (1947) Utilization of D-tryptophan by the chick. i. bid. Chem. 171, 209-212.