Renal arginine synthesis: studies in vitro and in vivo.

Renal arginine synthesis: studies in vitro and in vivo SRINIVAS N. DHANAKOTI, GENE R. HERZBERG, AND

JOHN T. BROSNAN, MARGARET E. BROSNAN

Department of Biochemistry, Memorial University St. John’s, Newfoundland AlB 3X9, Canada

DHANAKOTI, SRINIVAS N., JOHN T. BROSNAN, GENE R. HERZBERG, AND MARGARET E. BROSNAN. Renal arginine synthesis: studies in vitro and in viva. Am. J. Physiol. 259 (Endo-

crinol. Metab. 22): E437-E442, 1990.-Renal arginine synthesis is a major endogenous source of arginine. Argininosuccinate lyase occurs almost exclusively in kidney cortex. In studies with isolated renal cortical tubules, we observed rapid rates of arginine synthesis from citrulline, provided a source of the N atom of the guanidino group of arginine was supplied. Aspartate, glutamate, or glutamine were effective, whereas glycine, alanine, serine, or NH&l were ineffective as this second substrate. Arginine synthesis as a function of citrulline concentration was determined and was found to be highly sensitive to citrulline concentrations in the physiological range (60 PM), suggesting that renal arginine synthesis in vivo could be regulated by circulating citrulline levels. Therefore, arginine synthesis by the kidney was investigated in vivo by measuring the net flux of citrulline and arginine in saline-infused (control group) and citrulline-infused rats. In normal animals, uptake of citrulline was 60.5 * 20.7 nmol. min-‘. 100 g body wt-‘, and a similar arginine release was observed. Citrulline infusion that increased circulating citrulline levels fourfold resulted in a similar increase in renal citrulline uptake (224 & 33 nmol . min-’ ‘100 g-l) and a similar increase in renal production of arginine. The results suggest that the availability of citrulline is a limiting factor for renal arginine synthesis in rats. citrulline; amino acid metabolism; kidney cortical tubules ALTHOUGH THE ABILITY of the kidney to synthesize arginine has been recognized for many years (1, 3, 16), evidence that the kidney is the major biosynthetic source of circulating arginine in the rat was first reported by Featherston et al. (5). In their studies with rats, injected [14C]citrulline was incorporated into muscle protein as [14C]arginine but only in animals with functional kidneys. The kidney readily synthesizes arginine from citrulline, and this citrulline can arise from the intestinal metabolism of glutamine (21) suggesting the existence of an intestinal-renal axis that converts glutamine to arginine. Arginine is considered a dietary nonessential amino acid in humans and most adult animals based on growth and N-balance studies (17, 18). The endogenous synthesis via the intestinal-renal axis provides a ready explanation for the nonessentiality of arginine. This is supported by the fact that arginine is essential in species (cats, ferrets) with low rates of intestinal citrulline synthesis (4, 11), as well as in rats in which intestinal citrulline synthesis is specifically inhibited (7). 0193-1849/90

of Newfoundland,

In the present study, we investigated arginine synthesis from citrulline in isolated cortical tubules and the renal release of arginine in vivo in normal rats and in animals in which blood citrulline levels are increased by three- to fourfold by infusing citrulline. The results suggest that the kidney cortex is the site of arginine synthesis and that the availability of citrulline is a limiting factor. MATERIALS

AND

METHODS

Animals. Male Sprague-Dawley rats (Charles River, Montreal, Canada) weighing 300-425 g were used for all experiments. They were allowed water and Purina rat chow ad libitum. In vitro studies. Tubules were prepared from the kidney cortex of rats by the method of Guder et al. (6). The viability of tubules during incubation was determined by measuring the leakage of lactate dehydrogenase (LDH). Briefly, the LDH activity (12) was determined both in the pellet and supernatant fractions after incubations for 0 and 60 min. All incubations were carried out in KrebsHenseleit medium (pH 7.4) at 37’C in a Dubnoff shakingwater bath. Incubations were terminated with 0.2 ml 30% perchloric acid (PCA). After removal of the precipitated protein by centrifugation, an aliquot of the PCA extract was filtered through a 0.45pm filter (Millipore), and arginine was determined by a modification of the high-pressure liquid chromatography (HPLC) procedure developed by Seiler et al. (19). The modification employed involved a gradient elution system instead of an isocratic procedure. Separation of arginine was achieved using a Beckman Ultrasphere I. P. column (4.6 mm ID, 250 mm length) containing 5-pm spherical silica core with chemically bonded C1a groups. The separation column was guarded by a precolumn (2.1 mm ID, 70 mm length) filled with pellicular ODS (Cl8 groups bonded to 37- to 53.pm particles; Whatman, Clifton, NJ). The gradient elution system consisted of 0.05 M sodium acetate (pH 4.5), containing 10 mM octane sulfonic acid (OSA) and 10% methanol (buffer A) and 0.2 M sodium acetate (pH 4.5), containing 10 mM OSA, 10:3 (vol/vol) acetonitrile, and 10% methanol (buffer B). The column was eluted with 100% buffer A for the first 5 min; then from 5-15 min with 50% buffer A + 50% buffer B, and finally, for 5 min with 100% buffer B. Arginine eluted at about 17 min. After the run was completed, the column was washed for 10 min with 100% buffer A before a new

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E438

RENAL

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injection. The flow rate was maintained at 1 ml/min throughout. The identity of the arginine peak was confirmed both by spiking the PCA extract with arginine, as well as by treatment with arginase, which eliminated the peak. Replicate analyses agreed within 3%. The recovery of arginine added to the samples was between 98 and 100%. In one study the extent to which newly synthesized arginine was degraded by arginase in the tubules was determined. In this study [ 14C]ureido-citrulline was used as a substrate after purifying it according to the method of Hurwitz and Kretchmer (8), and [14C]urea was determined in neutralized extracts as 14COZafter urease treatment. Briefly, an aliquot of PCA extract was neutralized with KsP04, and then the neutralized extract (pH 7.4) was incubated with 1 mg Jackbean urease (Sigma, grade 6) at 37’C for 60 min. Incubations were terminated with 0.2 ml 30% PCA and 14COZ collected in center wells containing 0.2 ml NCS (Amersham) and counted in a scintillation counter. In Go studies. Studies were carried out to measure renal citrulline removal and arginine synthesis in animals with elevated blood citrulline levels (citrulline infusion) and in control animals (saline infusion). The methodology used was similar to that previously employed in this laboratory for the measurement of renal blood flow (10). After anesthesia with pentobarbital sodium (60 mg/kg body wt ip), the animal was placed on a heating pad, and the trachea was cannulated with a small piece (2.5 cm long x 2.5 mm ID) of polyethylene tubing. The right jugular vein was then catheterized with PE-50 tubing (Clay-Adams, Parsippany, NJ) for the infusion of inulin and citrulline. For the control animals, a priming dose of 1.75 &i of [c&o&14C]inulin (New England Nuclear, Lachine, Quebec) in 0.8 ml of 10% mannitol0.45% NaCl was then given through the saphenous vein and followed by the continuous infusion of the same solution at a rate of 0.037 ml/min using a Harvard Apparatus model 975 compact infusion pump. The left ureter was then catheterized with a short piece of PE-10 tubing fitted inside a length of PE-50 tubing. Urine was collected between 20 and 40 min. At the end of the urine collection period, l-ml blood samples were drawn from the left renal vein and the abdominal aorta. The blood samples were centrifuged at 12,000 g in a Beckman JA21 centrifuge for 10 min at 4’C. Aliquots of plasma (0.30.5 ml) were treated with an equal volume of 10% sulfosalicylic acid containing 62.5 nmol of 2=aminoethylcysteine (AEC) as an internal amino acid standard. This sample was used for the analysis of amino acids after removal of the precipitated proteins by centrifugation and adjustment of the supernatant to pH 2.2 * 0.1 with lithium hydroxide. An aliquot (25 ~1) of plasma was used for the determination of inulin radioactivity. Samples of the urine were treated in a similar manner. Amino acid analysis was carried out by ion-exchange chromatography using a Beckman model 121 MB amino acid analyzer (2). The experiments involving citrulline infusion were carried out identically to the controls except that citrulline was present at 100 mM in the priming solution and at 30 mM in the infusion solution. In these solutions,

SYNTHESIS

citrulline replaced an osmotically identical quantity of NaCl. Preliminary experiments demonstrated that both [ 14C]inulin and citrulline levels in blood were maintained at plateau levels between 20 and 40 min. Glomerular filtration rate (GFR) was calculated from urinary inulin excretion in the 20- to 40-min clearance period. Renal plasma flow (RPF) was calculated using the expression derived by Wolf (22). ChemicaZs. All substrates were obtained from Sigma Chemical (St. Louis, MO) with the exception of [ 14C]ureido-citrulline (42.4/mmol; New England Nuclear). StatikticaZ anaZysis. All results are means & SD. Significant differences between means were determined using paired or unpaired t test as appropriate. Significant differences between individual treatments and the control in Table 1 were determined by Dunnett’s procedure. P values c 0.05 were taken as statistically significant. RESULTS

Arginine synthesis in isolated tubules. In preliminary experiments we found that more than 90% of renal argininosuccinate lyase occurred in the cortex. The measured activities were 40.6 & 7.6, 5.0 & 0.9, and 5.9 k 0.3 prnol. h-l g wet wt-’ of cortex, medulla, and papilla, respectively. We therefore studied arginine synthesis in cortical tubules. Arginine synthesis was strictly dependent on added citrulline. In its absence the rate was minimal (l-2 nmol .30 min-’ . mg dry wt-‘). Arginine synthesis during incubation with citrulline, aspartate, and lactate was linear with time for up to 45 min and with increasing quantity of tubules for up to 2 mg dry wt (Fig. 1, A and B). This linearity indicates the absence of any inhibition by the end products or any other factors generated during the course of the reaction. In the absence of lactate the rates of arginine synthesis were reduced by ~40% (data not shown). This is probably due to lactate serving as an energy source, since ATP is required in the arginino-succinate synthetase reaction. Because kidney is known to contain an arginase (9), it was important to determine the extent to which arginine synthesized in these experiments was further metabolized. Accordingly, experiments were performed in which [14C]citrulline was used as substrate so that [14C]arginine would be produced. Any metabolism of this arginine via arginase would result in the production of [14C]urea which was detected as 14COZafter urease treatment. With such experiments we were able to show that ~6% of arginine was metabolized by this route (data not shown). In the presence of lactate, arginine synthesis from citrulline alone was about 20 nmol.30 minlo mg dry wt-l and this was increased about fourfold by addition of aspartate, glutamate, or glutamine, whereas glycine, alanine, serine, or NH4Cl did not stimulate arginine synthesis (Table 1). The enzyme argininosuccinate synthase uses aspartate as an N donor for the second N atom in the guanidino group of arginine. Thus glutamate and glutamine were effective, presumably because of the high renal activities of glutaminase and aspartate aminotransferase. l


RENAL

ARGININE

E439

SYNTHESIS

0.0

1.0 1.5 2.0 2.5 3.0 Citrulline (mbl) FIG. 2. Dependence of arginine synthesis in rat renal cortical tubules on citrulline concentration. Tubules (~1 mg dry wt) were incubated for 15 min with aspartate and lactate (5 mM each) at 37OC. Values are means k SD, (n = 4).

TB

350

0.5

300 250

2. Arteriovenous differences across the kidney in blood, plasma, and blood cells for citrulline and a&nine in normal and citrulline-infused rats

TABLE

200 150

100

Normal (n = 5)

50

0 0.0

0.5

1.0

1.5

2.0

Dry wdght

2.5

3.0

3.5

4.0

4.5

(mg)

FIG. 1. Arginine synthesis in rat renal cortical tubules as a function of time (A) and tubule quantity (B). Tubules were incubated with 0.5 mM citrulline and 5 mM each of aspartate and lactate. In A, 0.925 mg of tubules were used; in & tubules were incubated for 30 min. A representative experiment is shown.

1. Arginine production in kidney cortical tubules in the presence of various substrates

TABLE

Arginine

Production Rate

85.OklO.9 Citrulline + aspartate (control) 21.4*2.6* Citrulline 92.4k16.5 Citrulline + glutamate Citrulline + glutamine 88.7k17.9 Citrulline + glycine 21&5.8* 16.7&1.1* Citrulline + alanine Citrulline + serine 14.5*3.3* 32.5*3.5* Citrulline + NH&l Values are means k SD in nmol 30 min-’ mg dry wt?; n = 3 for all except control where n = 8. * Significantly different from control by Dunnett’s procedure (P < 0.05). Tubules equivalent to ~1.0 mg dry wt were incubated for 30 min. All substrates were initially present at 5 mM except citrulline and NH&l, which were present at 0.5 and 2 mM, respectively; 5 mM lactate present in all incubations. l

l

The data in Fig. 2 show the rate of arginine synthesis as a function of citrulline concentration. In this experiment incubations were carried out for 15 min to minimize substrate depletion at low substrate concentrations. The synthesis of arginine from citrulline is very sensitive to citrulline concentrations up to 0.5 mM. Most important, arginine synthesis was very sensitive to citrulline concentration in the region of circulating citrulline concentration (0.06 mM), suggesting that renal arginine syn-

Citrulline Infused (n = 3)

Arterial hematocrit 45.3k2.4 47.0k2.6 Renal venous hematocrit 45.6k2.1 47.3k2.1 Arteriovenous differences (a - 9 Blood citrulline, nmol/ml 11.2&6.2* 42.1&7.6*7 Blood arginine, nmol/ml -13.7*7.4* -38.6*4.8*? Plasma citrulline, nmol 13.2&3.2* 45.6*4.9*? in 1 ml blood Plasma arginine, nmol -10.6&3.6* -42.4*7.5*? in 1 ml blood Blood cell citrulline, nmol in -2.0k3.3 -3.5k3.5 1 ml blood Blood cell arginine, nmol -3.1k8.8 3.w4.1 in 1 ml blood Values are means & SD. Values for a - v for plasma, for both citrulline and arginine, were not significantly different from respective blood a - v values (P > 0.05). * P < 0.05, significantly different from zero. T Significantly different from normal rats, P < 0.05.

thesis in vivo could be regulated by circulating citrulline levels. Renal arginine synthesis in vivo. We therefore carried out experiments to quantify renal arginine synthesis in vivo and to. see how this was affected by increased citrulline levels. Our initial experiments examined whether citrulline uptake and arginine release by the kidney occurred from plasma, blood cells, or both. The data in Table 2 shows that arteriovenous (a-v) differences for citrulline and arginine measured in plasma can account for all of the differences seen in whole blood, and there is no contribution from blood cells. It is also evident that in normal and citrulline-infused rats there is a 1:l molar relationship betwen citrulline uptake and arginine release. Such a relationship has also been shown in rats (21) and in humans (20). We next measured total renal fluxes for these two amino acids in normal rats and in rats infused with citrulline to increase circulating concentrations. Because our data (Table 2) showed that


E440

RENAL

ARGININE

interchange from plasma accounted for all of the flux, we quantified the renal flux by multiplying the plasma a-v differences by the plasma flow rate. The basic physiological parameters of renal function in these experiments are presented in Table 3. The basic values are in agreement with those reported earlier in our laboratory (10).There were no significant differences (P > 0.05) for any of the measured parameters between control and citrulline-infused rats. The data from Table 4 clearly show that citrulline infusion increased plasma citrulline fourfold and also caused comparable increases in renal fluxes of citrulline and arginine. There was also an increase in arterial plasma arginine (174.6 k 26.2 vs. 244.1& 35.0 nmol/ml plasma). Furthermore, the renal uptake of citrulline was not significantly different from the arginine output in either the saline- or the citrullineinfused rats. The urinary excretion of citrulline and arginine in saline and citrulline-infused rats was 0 & 0.01 and 0.09 & 0.05 vs. 0.06 & 0.02 and 0.15 & 0.08 nmol min-‘. 100 g body wt-‘, respectively. Thus a urinary excretion of these amino acids was not a significant factor. Figure 3 plots the relationship of citrulline uptake to arginine release. It is clear from Fig. 3 that for every mole of citrulline taken up there is a mole of arginine released ( ? = 0.85) in normal, saline-, and citrullineinfused rats. DISCUSSION

Arginine production in renal cortical tubules. Because argininosuccinate lyase activity was found to be predominant in cortex, we carried out metabolic studies with isolated cortical tubules. Our results clearly show that TABLE 3. Basic physiological data for saline and citrulline-infused rats Saline

Citrulline

425&37 403&38 BOdY wk lz U/P inulin 237&36 216&44 Glomerular filtration rate 0.70&0.06 0.9320.28 Renal plasma flow rate 1.97kO.32 2.42kO.70 Filtration fraction 0.36kO.04 0.39kO.02 Urine flow rate 3.OkO.28 4.4kl.96 Values are means * SD; n = 5. There were no significant differences (P > 0.05) between the saline- and citrulline-infused rats for parameters measured. Glomerular filtration rate and renal plasma flow rate, ml. min-’ -100 g body wt-‘; urine flow rate, ~1. min-’ . 100 g body wt? U/P, urine-to-plasma.

TABLE 4. Arterial plasma levels and net renal flux

of citrulline and arginine in salineand citrulline-infused

rats

SYNTHESIS

0

r

* I

I I

I I

I I

I

0

30

60

90

120

150

(A-V)

Plasma

Citrulllne

(nmoles/ml) FIG. 3. Relationship between the a - v difference for citrulline for arginine in saline and citrulline-infused rats.

and

high rates of arginine synthesis from citrulline depend on the presence of a source of the second N atom in the guanidino group of arginine. In that regard, aspartate, glutamate, or glutamine served as an excellent source, whereas glycine, alanine, or serine did not. Higher rates of arginine production were observed in the presence of an oxidizable substrate (lactate). The arginine that was newly synthesized from citrulline was not appreciably metabolized to urea and ornithine, suggesting either that it was not readily available to renal arginase or that the effective renal arginase activity is low. The presence of arginase in different cells than the arginine synthesizing enzymes could explain these results. Clearly it is important to determine the cellular localization of enzymes of arginine synthesis and degradation in kidney to understand these processes fully. Such studies are currently in progress. In renal cortical tubules, arginine synthesis was very sensitive to citrulline concentration up to 0.5 mM and did not appear to be saturated even at 2.5 mM. Because plasma citrulline concentration in vivo is only ~0.06 mM (Table 4), it follows that arginine synthesis could be physiologically regulated by circulating citrulline levels. Arginine synthesis in vivo. There was a good stoichiometry between citrulline removal and arginine production in vivo, indicating that renal arginine synthesis is for export to extrarenal tissues rather than for utilization within the kidney by arginase. A similar conclusion may be drawn from the experiments of Tizianello in humans cw A’comparison of the rates of arginine synthesis in vivo and in vitro is of interest. Rates of 867 and 1,450 nmol. min-’ g wet wt of kidney-’ were found at 0.1 and 0.3 mM citrulline, respectively, in vitro (Fig. 1), whereas in vivo rates of 98 and 331 nmol min-’ . g wet wt of kidney-’ were found at 0.06 and 0.24 mM citrulline (Table 4), respectively. Thus the rates in vitro were about four- to fivefold higher at comparable citrulline concentrations than in vivo. A partial explanation for this discrepancy is that the rates in vivo refer to the whole kidney, whereas the studies in vitro were carried out with a cortex preparation in which arginine svnthesis is enriched. However. l

Arterial Plasma Concentration Citrulline

Arginine

Renal Flux Citrulline

l

Arginine

Saline 62.lk7.8 174.6k26.2 60.5k20.7 -78.9k24.5 Gitrulline 241,9&3&O* 244.1&35.0* 223.6*33.2* -264.6&82.7* Values are means & SD in nmol/ml plasma (arterial plasma concentration) and nmolmin-’ 100 g-l (renal flux); n = 5. * p < 0.05 for comparison with saline infusion. Positive value for flux indicates uptake and negative value indicates output. l


RENAL

ARGININE

because cortex comprises 63% of the kidney in the rat (13) such enrichment cannot account for a four- to fivefold discrepancy. It is not likely that the second N source is limiting in vivo as there was a linear response in arginine output to increased citrulline concentration. A more likely explanation relates to the different modes of delivery of citrulline to the tubules in vivo and in vitro. The tubules (in vitro) are open ended and thus have a continuous supply of citrulline available to them. The total delivery of citrulline in vivo depends on external factors such as GFR and renal plasma flow and offers a limit to the extent to which citrulline can be converted to arginine. The increased rate of arginine synthesis on citrulline infusion could be due either to the increased citrulline concentration per se or to the increased delivery in the plasma or the filtrate. The increased citrulline uptake on infusion of citrulline closely paralleled the increase in the filtered load and, indeed, was not significantly different from it, suggesting that this may be the route of the increased citrulline uptake. This suggestion requires further experimentation before firm conclusions can be drawn. A most striking observation in the present study was the marked sensitivity of renal arginine synthesis to citrulline concentrations in the physiological range. A similar dependence is evident from the data obtained by Tizianello in humans (20). These findings imply that the intestinal production of citrulline may be the principal site of regulation of endogenous arginine synthesis. This is consistent with the data from Windmueller and Spaeth that most (83%) of the citrulline produced in the intestine is converted to arginine in kidney. Citrulline uptake by the kidney during citrulline infusion was 223 & 33 nmol min-’ . 100 g-l, so that the extra citrulline metabolized was 163 & 39 nmol. mine1 . 100 g-l after deducting the uptake in saline-infused rats (60.5 & 20.7 nmol . min-‘0 100 g-l). This accounted for 58% of the infused citrulline (281 k 29 nmol . mine1 100 g-l) and thus indicates that the kidney remains the principal organ for citrulline metabolism even when citrulline levels are elevated. It is known that weanling rats (18) or rats in which intestinal citrulline synthesis is specifically inhibited (7) require exogenous arginine. However, in these studies it was demonstrated that citrulline can effectively substitute for arginine. This implies that the body contains a system for the conversion of citrulline to arginine that is not saturated (i.e., can accommodate an increased delivery of citrulline). The kinetic properties of the renal system for synthesizing arginine uncovered here strongly suggest that it could serve as this system. It could also serve as the system responsible for the metabolism of any dietary citrulline. This is made use of in patients suffering from lysinuric protein intolerance (14, 15). In this condition, there is defective intestinal absorption and renal reabsorption of dibasic amino acids such as lysine, arginine, and ornithine. Oral arginine supplementation cannot therefore be useful in correcting hyperammonemia arising from deficiency of urea cycle intermediates arginine and ornithine. In this regard, oral citrulline administration (2-3 g) prevented hvperammol

l

E441

SYNTHESIS

nemia as indicated by a marked decrease in erotic acid excretion when compared with arginine supplementation. However, there was a great increase in urinary arginine excretion after citrulline supplementation, indicating both a conversion of citrulline to arginine and a defect in the reabsorption of arginine. Although citrulline is not found in protein (with very minor exceptions), considerable quantities of free citrulline occur in some foods. For example, the watermelon, CitruZZus udgaris, contains about 100 mg/lOO g. We postulate that the kidney can initiate the metabolism of such exogenous citrulline by converting it to arginine, which can be subsequently metabolized in the liver or extrahepatic tissues. We thank D. Hall for the amino acid analyses. This work was supported by grants from the Medical Council and the Kidney Foundation of Canada. Address reprint requests to M. E. Brosnan. Received 4 December 1989; accepted in final form 18 May

Research

1990.

REFERENCES H., AND J. W. DUBNOFF. The conversion of citrulline to arginine in kidney. J. BioZ. Chem. 141: 717-738,194l. 2. BROSNAN, J. T., K. MAN, D. E. HALL, S. A. COLBOURNE, AND M. E. BROSNAN. Interorgan metabolism of amino acids in streptozotocin-diabetic ketoacidotic rat. Am. J. PhysioZ. 244 (Endocrinol. 1. BORSOOK,

IKetub. 7): El!%El58, 1983. 3. COHEN, P. P., AND M. HAYANO.

arginine

(transamination)

The conversion of citrulline to by tissue slices and homogenates. J.

BioZ. Chem. 4. DESHMUKH,

166: 239-250,1946. D. R., AND T. C. SHOPE. Arginine requirement and ammonia toxicity in ferrets. J. Nutr. 113: 1664-1667, 1983. 5. FEATHERSTON, W. R., Q. R. ROGERS, AND R. A. FREEDLAND.

Relative importance of kidney and liver in synthesis of arginine by the rat. Am. J. Physiol. 224: 127-129, 1973. 6. GUDER, W. G., W. WIESNER, AND B. STUKOWSKI. Metabolism of isolated kidney tubules: oxygen consumption, gluconeogenesis, and the effect of cyclic nucleotides in tubules from starved rats. HoppeSeyZer’s 2. Physiol. Chem. 352: 1319-1328, 1971. 7. HOOGENRAAD, N., N. TOTINO, H. ELMER, C. WRAIGHT, P. ALEWOOD, AND R. B. JOHNS. Inhibition of intestinal citrulline synthesis causes severe growth retardation in rats. Am. J. PhysioZ. 249 (Gastrointest. Liver Physiol. 12): G792-G799, 1985. 8. HURWITZ, R., AND N. KRETCHMER. Development of argininesynthesizing enzymes in mouse intestine. Am. J. PhysioZ. 251 (Gastrointest. Liver Physiol. 14): GlO3-GllO, 1986. 9. KAYSEN, G. A., AND H. J. STRECKER. Purification and properties of arginase of rat kidney. Biochem. J. 133: 779-788, 1973. 10. LOWRY, M., D. E. HALL, M. S. HALL, AND J. T. BROSNAN. Renal

metabolism of amino acids in vivo: studies on serine and glycine fluxes. Am. J. Physiol. 252 (Renal Fluid Electrolyte Physiol. 21): F304-F309,1987. 11. MORRIS, J.

G., AND Q. R. ROGERS. Arginine: an essential amino acid for the cat. J. Nutr. 108: 1944-1953, 1978. 12. MORRISON, G. R., F. E. BROCK, D. T. SOBRAL, AND R. E SHANK. Cold-acclimatization and intermediary metabolism of carbohydrates. Arch. Biochem. Biophys. 114: 494-501, 1966. 13. PFALLER, W., AND M. RI~INGER. Quantitative morphology of the rat kidney. Int. J. Biochem. 12: 17-22, 1980. 14. RAJANTIE, J., SIMELL, AND J. PERHEENTUPA. Oral administration of urea cycle intermediates in lysinuric protein intolerance: effect on plasma and urinary arginine and ornithine. MetuboZism 32: 4951, 1983. 15. RAJANTIE,

J., 0. SIMELL, J. RAPOLA, AND J, PERHEENTUPA. Lysinuric protein intolerance: a two-year trial of dietary supplementation therapy with citrulline and lysine. J. Pediutr. 97: 927-932,

1980. 16. RATNER,

S., AND B. PETRACK. The mechanism of arginine synthesis from citrulline in kidney. J. Biol. Chem. 200: 175-185, 1953.


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of man. Nutr. Abstr. 17. ROSE, W. C. The amino acid requirements Rev. 27: 631-647,1957. 18. SCULL, C. W., AND W. C. ROSE. Arginine metabolism 1. The relation of the arginine content of the diet to the increments in tissue arginine during growth. J. I3iol. Chem. 89: 109-123, 1930. 19. SEILER, N., S. SARHAN, AND B. KNODGEN. Developmental changes of nutreanine in vertebrate brains. Int. J. Dev. Neurosci. 3: 31732i, 1985. 20. TIZIANELLO, A. G., DEFERRARI, G. GARIBOTTO, G. GURRERI, AND

SYNTHESIS C. ROBAUDO. Renal metabolism of amino acids and ammonia in subjects with normal renal function and in patients with chronic renal insufficiency. J. Clin. Invest. 65: 1162-l 173, 1980. 21. WINDMUELLER, H. G., AND A. E. SPAETH. Source and fate of circulating citrulline. Am. J. Physiol. 241 (Endocrinol. Metab. 4): E473-E480,1981. 22. WOLF, A. V. Total renal blood flow at any urine flow or extraction fraction. Am. J. Physiol. 133: 496-497, 1941.


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DNA synthesis in human lymphocyts: intermediates in DNA synthesis, in vitro and in vivo.

Transplantation of human renal cell carcinoma to the nude mice: as an intermediate of in vivo and in vitro studies.

PEG-Ursolic Acid Conjugate: Synthesis and In Vitro Release Studies.

Experimental studies of bone mechanoadaptation: bridging in vitro and in vivo studies with multiscale systems.

The effect of kynurenic acid on the synthesis of selected cytokines by murine splenocytes - in vitro and ex vivo studies.

Microenvironmental arginine depletion by macrophages in vivo.

Intravenous glutamine supplementation enhances renal de novo arginine synthesis in humans: a stable isotope study.

Novel 2,4-diaminopyrimidines bearing tetrahydronaphthalenyl moiety against anaplastic lymphoma kinase (ALK): Synthesis, in vitro, ex vivo, and in vivo efficacy studies.