![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
* Animal Science Department, University of Illinois, Urbana IL 61801 and Molecular and Human Genetics and Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX 77030
2 To whom correspondence should be addressed. E-mail: jcmarini{at}uiuc.edu.
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
KEY WORDS: • mouse models • spf-ash mouse • ornithine transcarbamylase deficiency • urea cycle disorders • urea kinetics
Ornithine transcarbamylase deficiency is the most prevalent urea cycle disorder in humans, with an estimated incidence of 1:14,000 births (1). The Otcspf-ash mouse model of this disorder, available for the last 30 y (2), has been studied extensively. This spontaneous mutation has been described at the gene (3), transcript (4), and protein (5) levels. The mutation consists of a single base substitution (3) that results in inefficient splicing and 2 mutant proteins (3). Only 1 of these proteins is able to translocate into the mitochondria and assemble into the active trimeric structure (3). The resulting enzyme, however, is kinetically indistinguishable from the wild type (6). More recently the Otcspf-ash mouse has also been used as a model for gene therapy (7,8). Despite the continuous effort to describe and characterize this urea cycle disorder model, no determination of urea kinetics has been conducted in mutant mice, which have only 5% of the enzyme activity of control mice. The purpose of the present research is the study of urea metabolism in conscious Otcspf-ash mice under different nitrogen-load conditions.
|
MATERIAL AND METHODS |
|---|
|
TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Experiment 1: Food-deprivation study. Thirty-five 6-wk-old wild-type mice (weight 22.0 ± 2.0 g, mean ± SD) were used to determine the physiological response to food deprivation and to determine the optimal postabsorptive infusion window. Mice were weighed immediately at the beginning of the light cycle (0600), the feed removed, and the mice placed in wire-bottom cages to minimize coprophagy. Water was available at all times. Five mice were weighed and killed by decapitation per time point starting at 0630 (0, 2, 4, 6, 8, 10, and 26 h). Ten IU of heparin (0.15 IU/L saline solution) were injected intravenously in the lateral tail vein 10 min before decapitation, to facilitate blood collection. Blood was centrifuged at 1500 x g for 15 min and the plasma stored frozen at –20°C.
Blood glucose concentration was determined using a glucose meter (Bayer Corporation), and plasma urea concentration by isotopic dilution (9). The liver was excised, the gall bladder removed, and the liver weight recorded. The stomach was opened, rinsed with double distilled water (ddH2O), and the contents transferred to an aluminum pan. Stomach contents were oven dried at 60°C for 48 h and the apparent dry matter determined.
Experiment 2: Complete mixture of amino acids infusion. Twenty seven pairs of siblings (1 control and 1 mutant mice Otcspf-ash per pair, age 42.1 ± 1.0 d) were randomly allotted to 3 treatments. At 0630 the day of infusion, feed was removed and the mice were transferred to a new cage. Mice were weighed at 0930 and infusions started at 1030. The lateral tail vein catheterization procedure has been described in detail elsewhere (10). In brief, intravascular catheters were made of polytetrafluoroethylene (0.15 mm ID, 0.30 mm OD, SUBL-120 Braintree Scientific) and silastic tubing (0.30 mm ID, 0.64 mm OD, Dow Corning). After warming the tail in warm water, a 27-gauge needle was inserted into the lateral tail vein. The needle was removed and the catheter introduced through the puncture and secured to the tail with cyanoacrylate glue. Mice were restrained by the tail during the experiments. The control group (n = 9) received an infusion of isotonic saline (9 g NaCl/L), while groups AA1 (n = 9) and AA2 (n = 9) were infused with a complete mixture of amino acids (AAn)3 (Travasol 5.5%, Baxter) at a rate of 86 and 172 mg nitrogen · kg–1 · h–1, respectively. The infusion rate was set at 18.6 mL · kg–1 · h–1 for all mice, and the estimated osmolarity of the infusates was 300 (saline), 580 (AA1), and 860 millosmoles (AA2). The infusates also contained 0.025 IU/L of sodium heparin (LEO Pharma, Inc.).
Mice were infused for 4 h and, at the end of the infusion, mice were killed by decapitation and blood was collected. Plasma was obtained after centrifugation at 1500 x g for 15 min and frozen at –20°C until analysis.
Experiment 3: Glycine-alanine infusion. An equimolar glycine-alanine (1:1 on a molar basis; Sigma-Aldrich) mixture (GAn) was infused at a rate of 86 (GA1, n = 9) and 172 mg nitrogen · kg–1 · h–1 (GA2, n = 9) to pairs of siblings (control and Otcspf-ash; age 45.4 ± 3.8). Food deprivation, infusion schedule, infusion rate, and heparin content of the infusates were as described for the infusion of the AAn. The GA2 treatment had to be shortened to 2 h in mutant mice because they developed hyperammonemia, lethargy, and convulsions. After decapitation, the liver was excised, gall bladder removed, and liver weight recorded. Plasma was obtained after centrifugation, ammonia was measured immediately in fresh plasma, and the rest of the plasma was frozen at –20°C until analysis.
Tracer infusion.
Primed and continuous infusions of 15N15N urea (Cambridge Isotopes) were conducted for 4 h jointly with the amino acid infusion. The prime (55 µmol 15N15N urea · kg–1), also containing heparin (0.5 IU · kg–1), was followed by a continuous infusion (110 µmol 15N15N urea · kg–1 · h–1) utilizing syringe pumps (Harvard Apparatus, Inc.). With this infusion protocol, plateau enrichment was expected to be achieved by 
45 min in control mice and sooner if mutant mice had impaired ureagenesis (11).
Sample analysis. Both plasma urea concentrations and isotopic enrichments were determined by EI GCMS after the urea was derivatized to the tert-Butyldimethylsilyl derivative. Plasma samples were treated identically, with the exception that 13C18O urea was added as internal standard for determining urea concentration. Plasma proteins were precipitated with 10% sulfosalysilic acid and, after centrifugation, the supernatant was transferred to a column containing AG 50 W-X8-cation-exchange resin (Bio-Rad). The column had been previously rinsed with 5% HCl and washed with ddH2O until the efflux from the column was neutral. Following the addition of the urea sample, the column was rinsed with ddH2O and the sample eluted with 2 mol/L NH4OH. The eluate was then evaporated, utilizing a low vacuum concentrator (Thermo Savant). Samples were redissolved with ddH2O and transferred to a 1-mL V-vial (Wheaton). The water was evaporated under a gentle stream of nitrogen gas at 80°C and the sample derivatized with 25 µL of a 1:1 mixture of MTBSFA (Sigma-Aldrich) and acetonitrile at 80°C for 20 min in the tightly capped V-vials. The analysis was performed in a 5973 Agilent GC MSD in SIM mode, monitoring m/z ions 231, 233, and 234.
Ammonia was determined in fresh plasma samples by reductive amination of 2-oxoglutarate and oxidation of NADPH, using a commercial kit (Sigma Chemical).
Calculating urea entry rate from continuous infusion of tracer.
Urea entry rate (UER) was calculated from the isotopic dilution of the tracer at plateau enrichment, as
 |
where UER is the urea entry rate (mmol · kg–1 · h–1), R is the infusion rate (15N15N-urea mmol · kg–1 · h–1) and E is the 15N15N-urea enrichment at plateau (moles percent excess [mpe]).
Data analysis. Regression analysis was performed on data from Experiment 1 using the proc reg statement of SAS (SAS Inst.). Data from the 3 x 2 and 2 x 2 factorial arrangement of treatments in Experiments 2 and 3 were analyzed utilizing the proc mixed procedure of SAS. Fixed effects were genotype (control or Otcspf-ash), infusion level, and their interaction; litter was the random effect of the model. Least square means are ± SEM and differences were considered significant at P < 0.05. If significant differences were obtained, the post-hoc Tukey procedure for multiple pairwise comparisons was also applied.
|
RESULTS |
|---|
|
TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
25% of this weight loss occurred within the first 2 h. Likewise, the stomach contents decreased linearly and quadratically (P < 0.001), losing most of its contents within 2 h after feed removal; the further loss of material after 8 h suggests that the contents lost after 2 h were poorly digestible material. An increase in the stomach contents of dry matter observed at 26 h was due to the mice eating the cloth that was used for bedding after 10 h of food deprivation. This was easily observed because the cloth was blue and no feed contents were identified. This time point was removed from the statistical analysis. Glucose concentration remained unchanged (P = 0.35) for the first 10 h after feed removal, but dropped (P < 0.001) to 50% at 26 h. Liver weight, as percentage of initial body weight, decreased linearly and quadratically (P < 0.001) with time. After 26 h of food deprivation, liver weight was 60% of its initial weight. Urea concentration decreased quadratically (P < 0.04) with time after feed removal.
|
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
The nitrogen load infused was roughly equivalent to maintenance (AA1 and GA1) or 2 times maintenance nitrogen requirements (AA2 and GA2) (12). However, the total parenteral nutrition solution infused by Sitren et al. (12) also contained an energy source, which was omitted in the present experiments in order to impose a greater nitrogen load to the urea cycle. Despite the nitrogen load imposed, mutant mice were able to synthesize urea at the same rate as control animals when infused with a complete mixture of amino acids. When the GA mixture was infused, however, mutant mice were unable to synthesize urea at the same rate as controls. Furthermore, UER was depressed in Otcspf-ash when the level of GA infusion was increased. As a consequence, plasma ammonia concentration increased and hyperammonemic symptoms developed in the mice that received the GA2 treatment.
A chronic hepatic adaptation by mutant mice to the reduced OTC activity has been reported and consists in an enlarged liver (20% compared with controls), also seen in human patients (13,14), and an increase in mitochondrial protein (15). An acute liver enlargement in Otcspf-ash mice was also evident in the current experiments, shown by the further increase in liver weight of mice on GA2 treatment.
Mutant mice have only 5 and 2% residual enzyme activity in the liver (15,16) and small intestine (16), respectively. The OTCD affects ureagenesis at 2 different levels: 1) by reducing OTC in liver and thus production of urea directly, and 2) by reducing OTC activity in enterocytes and the endogenous synthesis of citrulline. A reduction in citrulline synthesis results in a reduction of arginine synthesis by the kidney, and as a consequence, arginine becomes an essential amino acid (17).
The difference in response to nitrogen load between the complete AA and the GA mixture was probably due to the presence of arginine in the complete mixture. It has been shown that wild-type mice were able to increase the hepatic concentration of urea cycle intermediates after an injection of NH4Cl, whereas Otcspf-ash mice failed to do so (18,19). The ability of urea cycle intermediates to prevent ammonia toxicity after a lethal dose of ammonia has been shown not only in mice (20) but also in other species (21). The fact that an increase in urea cycle intermediates increases the ability to detoxify ammonia has been further supported by observations in isolated hepatocytes from Otcspf-ash mice (22), in which ornithine increased urea synthesis rate and reduced orotic acid production. This effect of ornithine on urea synthesis seems to be mediated by an increase in OTC activity (21), increased carbamoyl phosphate synthetase activity (23,24), or a decrease in carbamoyl phosphate degradation (25).
It seems that the inability to detoxify ammonia by the liver, of mutant mice is mainly due to the decrease of urea cycle intermediates, rather than the reduction in hepatic OTC activity. This is further supported by research conducted in transgenic mice. The correction of the intestinal phenotype with a human OTC transgene in mutant mice, thus regaining the ability to synthesize arginine de novo, reduced orotic acid excretion to levels similar to wild type mice (26). Furthermore, the reduction in orotic aciduria was better correlated with intestinal OTC than with hepatic OTC activity after gene therapy (16).
The present results show that despite only vestigial OTC activity, Otcspf-ash mice are able to maintain ureagenesis at the same rate as control animals, depending on the amino acids infused. This implies that Otcspf-ash mice are able to dispose of ammonia, without apparent adverse effects, when a balanced mixture of amino acids is provided, despite reduced enzyme activity.
|
FOOTNOTES |
|---|
3 Abbreviations used: AAn complete amino acid mixture infused at rate n; ddH2O double distilled water; GAn glycine-alanine mixture infused at rate n; mpe, moles percent excess; UER urea entry rate.
Manuscript received 6 December 2005. Initial review completed 31 December 2005. Revision accepted 13 January 2006.
|
LITERATURE CITED |
|---|
|
TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
1. Brusilow SW, Horwich AL. Urea cycle enzymes. In: Scriver CR, Beaudet AL, Sly WS and Valle D, editors, The metabolic and molecular bases of inherited disease. 8th ed. New York: McGraw-Hill; 2001.
2. Doolittle DP, Hulbert LL, Cordy C. A new allele of the sparse fur gene in the mouse. J Hered. 1974;65:194–5.
3. Hodges PE, Rosenberg LE. The spf-ash mouse: A missense mutation in the ornithine transcarbamylase gene also causes aberrant mRNA splicing. Proc Natl Acad Sci USA. 1989;86:4142–6.
4. Rosenberg LE, Kalousek F, Orsulak MD. Biogenesis of ornithine transcarbamylase in spfash mutant mice: two cytoplasmic precursors, one mitochondrial enzyme. Science. 1983;222:426–8.
5. Dubois N, Cavard C, Chasse JF, Kamoun P, Briand P. Compared expression levels of ornithine transcarbamylase and carbamylphosphate Synthetase in liver and small-intestine of normal and mutant mice. Biochim Biophys Acta. 1988;950:321–8.[Medline]
6. Briand P, Francois B, Rabier D, Cathelineau L. Ornithine transcarbamylase deficiencies in human males. kinetic and immunochemical classification. Biochim Biophys Acta. 1982;704:100–6.[Medline]
7. Batshaw ML, Yudkoff M, McLaughlin BA, Gorry E, Anegawa NJ, Smith IAS, Hyman SL, Robinson MB. The sparse fur mouse as a model for gene therapy in ornithine carbamoyltransferase deficiency. Gene Ther. 1995;2:743–9.[Medline]
8. Mian A, McCormack M, Mane V, Kleppe S, Ng P, Finegold M, O'Brien WE, Rodgers JR, Beaudet AL, Lee B. Long-term correction of ornithine transcarbamylase deficiency by WPRE-mediated overexpression using a helper-dependent adenovirus. Mol Ther. 2004;10:492–9.[Medline]
9. Calder AG, Garden KE, Anderson SE, Lobley GE. Quantitation of blood and plasma amino acids using isotope dilution electron impact gas chromatography/mass spectrometry with U-(13)C amino acids as internal standards. Rapid Commun Mass Spectrom. 1999;13:2080–3.[Medline]
10. Marini JC, Lee B, Garlick PJ. Non-surgical alternatives to invasive procedures in mice. Lab Anim. 2006;40:, in press.
11. Marini JC, Lee B, Garlick PJ. In vivo urea kinetic studies in conscious mice. J Nutr. 2006;136:202–6.
12. Sitren HS, Heller PA, Bailey LB, Cerda JJ. Total parenteral nutrition in the mouse: development of a technique. J Parenter Enteral Nutr. 1983;7:582–6.
13. Staudt M, Wermuth B, Freisinger P, Hassler A, Pontz BF. Symptomatic ornithine carbamoyltransferase deficiency (point mutation H202P) with normal in vitro activity. J Inherit Metab Dis. 1998;21:71–2.[Medline]
14. Vella S, Steiner F, Schlumbom V, Zurbrugg R, Wiesmann UN, Schaffner T, Wermuth B. Mutation of ornithine transcarbamylase (H136R) in a girl with severe intermittent orotic aciduria but normal enzyme activity. J Inherit Metab Dis. 1997;20:517–24.[Medline]
15. Cohen NS, Cheung CW, Raijman L. Altered enzyme activities and citrulline synthesis in liver mitochondria from ornithine carbamoyltransferase-deficient sparse-fur ash mice. Biochem J. 1989;257:251–7.[Medline]
16. Saheki T, Mori K, Kobayashi K, Horiuchi M, Shige T, Obara T, Suzuki S, Mori M, Yamamura K. Importance of ornithine transcarbamylase (OTC) deficiency in small intestine for urinary orotic acid excretion: analysis of OTC-deficient spf-ash mice with OTC transgene. Biochim Biophys Acta. 1995;1270:87–93.[Medline]
17. Brosnan ME, Brosnan JT. Renal arginine metabolism. J Nutr. 2004;134:2791S–5S.
18. Li MX, Nakajima T, Fukushige T, Kobayashi K, Seiler N, Saheki T. Aberrations of ammonia metabolism in ornithine carbamoyltransferase-deficient spf-ash mice and their prevention by treatment with urea cycle intermediate amino acids and an ornithine aminotransferase inactivator. Biochim Biophys Acta. 1999;1455:1–11.[Medline]
19. Saheki T, Fukushige T, Kakinoki H, Kobayashi K. No increase of ornithine concentration in the liver of ornithine transcarbamylase-deficient spf-ash mice following intraperitoneal injection of ammonium chloride. Contrib Nephrol. 1997;121:109–16.[Medline]
20. Sarhan S, Knoedgen B, Seiler N. Protection against lethal ammonia intoxication - synergism between endogenous ornithine and L–carnitine. Metab Brain Dis. 1994;9:67–79.[Medline]
21. Goodman MW, Zieve L, Konstantinides FN, Cerra FB. Mechanism of arginine protection against ammonia intoxication in the rat. Am J Physiol. 1984;247:G290–5.
22. Rabier D, Cathelineau L. Synthesis of the urea by means of isolated hepatocytes from Spf-Ash mice (mutation for ornithine transcarbamylase). Diabetes Metab. 1984;10:348–348.
23. Banko G, Zollner H. Does ornithine stimulate carbamoylphosphate synthetase? Int J Biochem. 1985;17:503–7.[Medline]
24. Cohen NS, Cheung CW, Raijman L. The effects of ornithine on mitochondrial carbamyl-phosphate synthesis. J Biol Chem. 1980;255:10248–55.
25. Lof C, Wanders RJA, Meijer AJ. Activity of carbamoyl-phosphate synthetase (ammonia) in isolated rat-liver mitochondria - cycling of carbamoyl phosphate in the absence of ornithine. Eur J Biochem. 1982;124:89–94.[Medline]
26. Jones SN, Grompe M, Munir MI, Veres G, Craigen WJ, Caskey CT. Ectopic correction of ornithine transcarbamylase deficiency in sparse fur mice. J Biol Chem. 1990;265:14684–90.
This article has been cited by other articles:
|
|
 |
|
  Y. C. Luiking, M. M. Hallemeesch, M. C. van de Poll, C. H. C. Dejong, W. J. de Jonge, W. H. Lamers, and N. E. P. Deutz Reduced citrulline availability by OTC deficiency in mice is related to reduced nitric oxide production Am J Physiol Endocrinol Metab, December 1, 2008; 295(6): E1315 - E1322. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Marini, A. Erez, L. Castillo, and B. Lee Interaction between murine spf-ash mutation and genetic background yields different metabolic phenotypes Am J Physiol Endocrinol Metab, December 1, 2007; 293(6): E1764 - E1771. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. Marini, B. Lee, and P. J. Garlick Ornithine Restores Ureagenesis Capacity and Mitigates Hyperammonemia in Otcspf-ash Mice J. Nutr., July 1, 2006; 136(7): 1834 - 1838. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
