Citrulline is a nonessential amino acid that is mainly synthetized in enterocytes and metabolized in the small intestine, liver, and kidneys. Its use as a marker of intestinal functional mass has been investigated primarily in humans.1–5 Citrulline is not incorporated into proteins during ribosomal translation of mRNA because it is not coded by a genetic codon. However, this amino acid might be included in protein during posttranslational maturation.
A large amount of amino acids, particularly glutamine and alanine, is metabolized in the postabsorptive phase. On the contrary, citrulline is the only amino acid to undergo considerable production during that phase.6 In enterocytes, citrulline is produced from arginine, glutamine, and derived amino acids (glutamate and ornithine) through the glutamate-to-ornithine enterocyte pathway.3 In mice, arginine and ornithine are the main precursors for citrulline synthesis.7
Ammonia, which is essential for citrulline synthesis, is produced during glutamine deamination to glutamate and stimulates the formation of carbamoyl sulfate. Citrulline is formed from the transamination reaction of carbamoyl sulfate and ornithine. Pyrroline-5-carboxylate synthase is the key mitochondrial enzyme for formation of ornithine.
In rats, pyrroline-5-carboxylate has high activity mainly in duodeno-jejunal enterocytes but also lower activity in the ileum.8,9 The kidneys metabolize citrulline into arginine.10
Citrulline is produced and used in periportal hepatocytes that have a complete urea cycle in rats.11 Citrulline is converted into arginine, which is recycled into ornithine and urea. Consequently, there is no hepatic release of citrulline in rats.11 In dogs, the liver might contribute to the amount of citrulline in circulation,12 but enterocytes contribute to most of it because citrulline produced in the liver is locally metabolized in the urea cycle.13 Therefore, citrulline has been proposed as a marker of global metabolic activity of enterocytes in patients with healthy renal function and has been investigated in humans with various enteric diseases such as so-called short bowel syndrome or villous atrophy–associated disease.
In humans with short bowel syndrome, plasma citrulline concentration is highly correlated with small bowel length.1 Plasma citrulline concentration is also considered a suitable marker to distinguish between permanent and transient intestinal failure,1 defined as a decrease in functioning intestinal mass to less than the minimal amount necessary for the absorption of nutrients. In humans with villous atrophy–associated small bowel disease, such as celiac disease, small bowel lymphoma, or parasitism, plasma citrulline concentration is decreased relative to that of healthy humans and its concentration is correlated with the severity and extent of villous atrophy.3 However, in humans, plasma citrulline concentration is not considered a marker of intestinal absorptive function. Plasma concentration is within reference limits in humans with Crohn disease14 or severe malnutrition3 unless intestinal functional mass is concurrently reduced. Loss of enterocyte mass is not the only cause of low plasma citrulline concentration. In critically ill humans15 and septic dogs,16 plasma citrulline concentration decreases. However, for septic humans, the lowest plasma citrulline concentrations are reportedly found in those with concurrent intestinal dysfunction.15
In dogs, plasma citrulline concentration decreases with the severe acute intestinal damage caused by parvoviral enteritis,17 suggesting that this analyte could also be used as a marker of functional intestinal mass in that species. Therefore, it would be interesting to assess the usefulness of plasma citrulline concentration as a marker for other diseases involving enterocyte damage, such as primary intestinal disease (eg, parasitism or bacterial enteritis) or nonintestinal diseases (eg, congestive heart failure or chronic kidney disease) that could impair intestinal functional mass. Measurement of plasma citrulline concentration could also be a valuable tool in the diagnosis of chronic inflammatory enteropathies because loss of epithelium functional mass might be used to assess disease severity in association with histologic changes.18
Postprandial measurement of plasma citrulline concentration is not recommended in human medicine13 because values decrease slightly after a meal.10 Prolonged starvation is also associated with decreases in plasma citrulline concentration in humans.19 However, to the authors’ knowledge, the effects of preanalytic conditions, such as feeding or circadian variation, on plasma citrulline concentration have not been investigated in dogs. Such effects must be identified before clinical studies can be conducted to evaluate plasma citrulline concentration in dogs with various clinical conditions. The purpose of the study reported here was to evaluate circadian variation and the influence of a meal on plasma citrulline concentration in healthy dogs so that the most suitable timing for diagnostic measurement could be determined. We hypothesized that plasma citrulline concentration would be unaffected by circadian variation in unfed dogs but would be affected by a meal.
Materials and Methods
Animals
Eight sexually intact Beagles (2 males and 6 females) with a mean ± SD age of 2.9 ± 1.4 years and mean body weight of 10.9 ± 2.3 kg were included in the study. Dogs were considered healthy on the basis of results of physical examination, routine serum biochemical analysis, and CBC. Deworming was performed with fenbendazolea (50 mg/kg) for 5 consecutive days before the study began. All dogs were fed their usual diet (a dry dietb characterized as hypoallergenic by the manufacturer) and had free access to water. Sources of protein in the diet were hydrolyzed soy protein isolate and hydrolyzed poultry liver. Daily dietary requirement was estimated as recommended by the manufacturer (in accordance with dog body weight) and was routinely given in 1 meal at 8:00 am. The meal was totally ingested by all dogs within 5 minutes after distribution. Eating was controlled, with dogs provided access only to their own food bowl. Diet, housing conditions, and environment remained constant during the study and had been stable for months before the study began. Dogs were housed in adjacent cages (each 2 × 2 × 4 m), and each set of 2 adjacent cages allowed the occupants free access to a shared outdoor run. The room in which dogs were housed had natural lighting, and no artificial lighting or temperature control was instituted. Dogs were walked on a leash as usual between 8:00 am and 10:00 am for 15 to 20 minutes each day. The study protocol was approved by the committee for the management of research and teaching dogs of the National Veterinary School at the University of Toulouse.
Study design
Plasma citrulline concentration was measured in 2 phases: when dogs were unfed (phase 1) and after dogs were fed a meal (phase 2). In phase 1, all dogs were evaluated during the same day, beginning 12 hours after food withholding (0 hours; 8:00 am). Blood samples (3 mL) were collected from each dog via jugular venipuncture (20-gauge needle and 10-mL syringe) every 2 hours from 0 hours to 12 hours (8:00 pm) and again at 24 hours (8:00 am the following day). Each blood sample was immediately transferred into a 3-mL tube containing 45 U of lithium heparinc cooled to 4°C and centrifuged for 5 minutes at 4°C within 30 minutes after collection. Plasma was harvested and stored at −80°C until the citrulline assays were performed. Phase 2 was completed a week after phase 1, with the only difference being that dogs were fed their daily requirement of food in 1 meal immediately after blood collection at 0 hours. Again, all dogs were evaluated on the same day.
Citrulline assays
Citrulline concentration in thawed plasma samples was measured by ion exchange liquid chromatography with an automated analyzerd at a commercial laboratorye in accordance with a previously published method.20 The detection limit of the method was 2 μmol/L. To assess reproducibility and repeatability of assay results in our experimental conditions, between-day and within-day CVs, respectively, were measured by use of 0-hour samples from 2 study dogs. Between-day CV was determined through repeated measurements of the same sample on 5 consecutive days. Within-day CV was determined through 5 repeated measurements during the same day.
Statistical analysis
Plasma citrulline concentrations were normally distributed (Kolmogorov-Smirnov test) and are reported as mean ± SD. A general linear model was used with dog and measurement point as explanatory variables to identify differences in concentrations among measurement points in each of the 2 phases of the study. Post hoc comparisons were performed with Dunnett or Bonferroni tests with correction for multiple comparisons. To estimate the dispersion of plasma citrulline values within individual dogs, intradog CV of plasma citrulline concentration was calculated for each dog during each phase. Values of P < 0.05 were considered significant.
Results
Mean ± SD between and within-day CVs for the plasma citrulline assay were 2.8 ± 1.4% and 2.1 ± 1.0%, respectively, at mean citrulline concentrations of 51.1 and 50.1 μmol/L, respectively. For phase 1 of the study, in which concentrations were measured in 8 unfed dogs, overall mean plasma citrulline concentration was 53.2 ± 9.7 μmol/L (n = 64 blood samples [8/dog]). Minimum and maximum plasma citrulline concentrations were 34.2 and 83.2 μmol/L, respectively. Significant (P < 0.001) variation over the 24-hour measurement period was detected in plasma citrulline concentration. Although post hoc comparisons did not reveal any significant variation between 0 hours and any other measurement point, significant differences between plasma citrulline concentrations at other points were identified (Figure 1). Specifically, differences were evident between measurements made at 12 hours (mean ± SD concentration, 56.3 ± 10.7 μmol/L) and those made at 2 hours (50.6 ± 9.0 μmol/L; P = 0.049) and 6 hours (49.9 ± 9.4 μmol/L; P = 0.014). Differences were also detected between measurements made at 24 hours (57.7 ± 12.4 μmol/L) and those made at 2 hours (P = 0.004), 4 hours (51.0 ± 8.7 μmol/L; P = 0.007), and 6 hours (P = 0.001). Mean intradog CV for plasma citrulline concentration over the 24-hour period was 7.85 ± 2.72%, with individual intradog CVs ranging from 4.4% to 12.9%.
For phase 2 of the study, in which the same dogs were fed a meal after blood sample collection at 0 hours, mean plasma citrulline concentration was 66.1 ± 12.5 μmol/L (n = 64 samples). Minimum and maximum plasma citrulline concentrations were 38.8 and 92.1 μmol/L, respectively. General linear modeling revealed that feeding had a significant (P < 0.001) effect on plasma citrulline concentration. The mean value at 0 hours (64.4 ± 12.7 μmol/L) was significantly (P = 0.019) lower than the value at 4 hours (72.2 ± 12.7 μmol/L) and significantly (P = 0.011) higher than the value at 24 hours (56.1 ± 12.5 μmol/L; Figure 2). Post hoc comparisons revealed significant differences between several measurement points. Plasma citrulline concentration at 24 hours was significantly lower than the concentration at 2 hours (69.4 ± 14.7 μmol/L; P < 0.001), 4 hours (P < 0.001), 6 hours (69.9 ± 13.0 μmol/L; P < 0.001), 8 hours (68.9 ± 10.9 μmol/L; P < 0.001), and 10 hours (65.1 ± 8.5 μmol/L; P = 0.02). The mean value at 4 hours was significantly (P = 0.015) higher than that at 12 hours (62.8 ± 11.9 μmol/L). Mean intradog CV for plasma citrulline concentration was 10.4 ± 3.6% in phase 2, with individual intradog CVs ranging from 8.4% to 16.5%.
Discussion
The purpose of the present study was to determine the circadian variation in plasma citrulline concentration in healthy fed and unfed dogs and to further define preanalytic conditions for evaluation of plasma citrulline concentration for clinical studies involving dogs. In the present study, plasma citrulline concentration was significantly influenced by a meal. Values increased for 4 hours after the meal, and the concentration at 4 hours was significantly higher than that at 0 hours (before the meal). Plasma citrulline concentration returned to the 0-hour value 6 hours after feeding and did not reach a value significantly lower than the 0-hour value until 24 hours after feeding. We therefore concluded that blood sample collection for plasma citrulline concentration should be performed during the postabsorptive phase, between 8 and 12 hours after a meal. A similar fasting period is also recommended in human medicine.13
The initial increase in plasma citrulline concentration during the postprandial state in the dogs of the present study is not observed in humans. The concentration in humans is usually stable or slightly low (10% to 20%) in the postprandial state and returns to the preprandial value after 2 to 4 hours.10 This postprandial decrease is possibly attributable to the diminution of enzyme activities in intestinal mucosa.21 An increase in plasma citrulline concentration caused by urea-cycle defects, as has been described for Irish Wolfhound puppies,22 was not suspected in the Beagles of the present study.
Postprandial citrulline concentration is influenced by dietary conditions. For example, the concentration was lower than the preprandial concentration when healthy humans who had fasted were fed an arginine-rich diet for 6 days in 1 study.21 Moreover, subjects in that study21 who were fed an arginine-free diet had a significantly higher plasma citrulline concentration than did those fed an arginine-rich diet. However, no difference in plasma citrulline concentration was identified for subjects fed the arginine-free diet between the fasting and fed state. The hypoallergenic commercial diet fed to the dogs of the present study was their usual diet, and they had been fed it for months. Arginine concentration of this diet (1.52 g/100 g of dry matter) exceeded the minimum recommended nutrient concentration for dogs (0.52 g/100 g of dry food in adult dogs).23 One of the main sources of protein of the diet was hydrolyzed soy protein isolate. Genistein, a soy isoflavone, has been found in vitro to increase production of l-citrulline by human endothelial cells after 48 hours of incubation.24 However, an in vivo study25 involving postmenopausal women failed to detect any effect of dietary supplementation with soy isoflavone on citrulline flux. Therefore, the possibility could not be excluded that the specific diet used in the present study could have contributed to the observed postprandial transient increase in plasma citrulline concentration.
Interspecies differences in citrulline metabolism could explain the aforementioned differences between humans and dogs as well. Citrulline is produced exclusively in the intestine in rats,11 pigs,26 and humans.27 However, in dogs, the liver may also play a role.12 Contribution to plasma citrulline concentration by the liver could explain the increase of plasma citrulline concentration in the postprandial state for dogs; however, additional research is needed to support that hypothesis.
In the dogs of the present study, no significant difference was identified between concentrations measured at 0 hours and those measured at any other point in phase 1. The significant difference detected between preprandial and 24-hour postprandial values was therefore unexpected. Pairwise comparisons between measurement points in phase 1 revealed some differences as well. However, in regard to the low intradog CV of plasma citrulline values, these differences were small and probably of marginal relevance, particularly when considering citrulline as a marker of decreases in intestinal mass.
Plasma citrulline concentrations in dogs with conditions requiring critical care or parvoviral enteritis have been reported.16,17,28 Mean ± SD or median (range) concentrations in healthy dogs (39.2 ± 0.2 μmol/L,28 57.6 μmol/L [33.7 to 143.2 μmol/L],16 and 38.6 μmol/L [11.4 to 96.1 μmol/L]17) were similar to the mean concentration for dogs in phase 1 of the present study (53.2 ± 9.7 μmol/L). The decrease in plasma citrulline concentration reported for dogs with parvoviral enteritis17 is higher than that identified in the present study. In that other study,17 mean plasma citrulline concentration in parvovirus-infected dogs was 94% lower than in healthy dogs, whereas the maximum intradog CV in the present study was 16.5%.
Variations in plasma citrulline concentration for dogs in the present study were also less important than those identified in dogs with enteric diseases other than those associated with villous atrophy. For example, compared with in control dogs, mean plasma citrulline concentration was reportedly 68% lower in dogs with superficial necrolytic dermatitis and 54% lower in dogs with chronic hepatitis,28 and median plasma citrulline concentration was 60% lower in critically ill dogs.16 Therefore, circadian variation in citrulline concentration for dogs in the present study was significant from a statistical perspective but considered of minimal relevance from a clinical perspective.
Although reference intervals are not available for plasma citrulline concentration in dogs, value ranges for healthy control dogs used in various studies (33.7 to 143.2 μmol/L16 or 11.4 to 96.1 μmol/L17) suggest wide interindividual variation. Interindividual variation was also evident for dogs in the present study; however, that variation was narrower than in the other studies,16,17 with extreme values varying from 49.9 to 72.2 μmol/L. Moreover, interindividual variation did not prevent detection of significant differences in plasma citrulline concentration between diseased dogs and healthy dogs in those other studies.16,17
Reproducibility and repeatability of the ion exchange liquid chromatography assay used in the present study were good. Therefore, this method appeared to be appropriate for use in other studies to evaluate the usefulness of plasma citrulline concentration as a marker of gastrointestinal conditions in dogs. Chronic idiopathic enteropathy can lead to conditions ranging from focal epithelium loss to widespread ulceration, depending on the histologic severity of the lesions.18 We therefore believe that plasma citrulline concentration could be a used as a marker of disease severity in dogs with chronic idiopathic enteropathy.
The authors acknowledge that exercise could contribute to variations in plasma citrulline concentration, and the exercise that dogs in the present study received might have influenced the results. Dogs were walked on a leash as usual for a maximum of 20 minutes between 8:00 am and 10:00 am or between the 0-hour and 2-hour measurement points. Exercise leads to a significant decrease in plasma citrulline concentration in horses,29 probably because of exercise-induced protein breakdown. Because leash walking is a far less strenuous exercise than the training protocol used for horses in that study,29 we believe that any influence of exercise on the results of the present study was probably minimal. However, leash walking could have contributed to the apparent, albeit nonsignificant, decrease in plasma citrulline concentration observed between 0 hours and 2 hours (when dogs were walked) in phase 1 (Figure 1). Indeed, exercise might have partially reduced the magnitude of the increase in plasma citrulline concentration observed after feeding during phase 2. Exercise did not appear to have been involved in the increase in plasma citrulline concentration observed after feeding in phase 2 because no similar increase was observed during phase 1, which was conducted in identical conditions. Another limitation of the present study was the low number of dogs used to determine circadian variation in plasma citrulline concentration. A larger sample size would be needed to provide reference intervals for plasma citrulline concentration in healthy dogs.
Overall, no clinically relevant variations in plasma citrulline concentration were identified in unfed dogs in the study reported here. Plasma citrulline concentrations during a 24-hour period were not significantly different from those measured after 12 hours of food withholding. The diet that dogs were fed influenced plasma citrulline concentration, which was significantly higher 4 hours after feeding than before feeding but returned to the preprandial value 6 hours after feeding. Therefore, we conclude that, for proper evaluation of plasma citrulline concentration in dogs, blood samples should be collected 8 to 12 hours after a meal.
Acknowledgments
Presented as a poster at the 23rd Congress of the European College of Veterinary Internal Medicine, Liverpool, England, September 2013.
The authors thank Isabelle Cuvelier and Didier Olichon for performance of the assays.
ABBREVIATIONS
CV | Coefficient of variation |
Footnotes
Panacur, Intervet, Beaucouze, France.
Hypoallergenic DR 21 canine dry diet, Royal Canin, Aimargues, France.
Lithium heparin tube (3 mL), Venosafe, Terumo Europe, Leuven, Belgium.
AminoTac, Jeol, Tokyo, Japan.
Cerba Laboratory, Saint Ouen l'Aumone, France.
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