Diagnostic evaluation of urea nitrogen/creatinine ratio in dogs with gastrointestinal bleeding

Abstract Background Urea nitrogen/creatinine ratio (UCR) is a marker for upper gastrointestinal bleeding (GIB) in people. Objectives To assess the usefulness of UCR to predict occult GIB and distinguish upper from lower GIB in dogs. Animals Eighty‐nine dogs with GIB and 65 clinically healthy dogs. Dogs were grouped according to 65 overt GIB and 24 occult GIB, and based on lesion localization (37 upper, 13 lower, and 8 both). Methods Seventy‐four dogs were included retrospectively and 15 dogs prospectively. Serum urea nitrogen and creatinine concentrations, UCR, hemoglobin concentration, hematocrit, mean corpuscular volume, and mean corpuscular hemoglobin concentration were compared between groups. Logistic regression models were fitted to assess if variables could distinguish occult GIB from being healthy and upper from lower GIB. Results The UCR was significantly higher in dogs with overt GIB compared to control dogs (P = .02) and dogs with occult GIB (P = .05). The UCR was not significantly associated with occult GIB vs being healthy, or upper vs lower GIB (P > .05 each). Dogs with higher hemoglobin concentration and hematocrit had significantly lower odds of having occult GIB than being healthy (P < .0001 each). Conclusions and Clinical Importance The UCR does not seem to be a clinically useful marker of occult GIB and appears to have poor discriminatory ability between upper and lower GIB. An increased UCR in a dog without signs of overt GIB, especially if its hematocrit is within the middle or upper reference interval, does not appear to warrant prompt prescription of gastrointestinal protectants.


| INTRODUCTION
Gastrointestinal bleeding (GIB) is a frequent cause of hospitalization in dogs. [1][2][3] Clinical signs vary from subclinical disease without visible bleeding (occult GIB) to visible hemorrhage (overt GIB) including hematemesis, hematochezia, and melena. 3,4 Gastrointestinal bleeding can further be distinguished into upper and lower gastrointestinal (GI) hemorrhage based on the location of bleeding either orad or aborad to the ligament of Treitz (duodenojejunal junction). 5 To properly manage and treat GIB, accurate identification of the source of GI hemorrhage is required.
The ratio of serum urea nitrogen to creatinine concentration has been used as a simple index to discriminate upper (higher ratio) from lower GIB (lower ratio) sources in human medicine. [5][6][7] Different urea nitrogen/creatinine ratio (UCR) cutoffs to differentiate upper from lower GIB ranging from 30 to 36 have been reported in people. [7][8][9][10][11] Dogs with melena, hematemesis, or both had significantly higher UCR than did control dogs. 1 It is postulated that an increase in serum urea concentration in upper GIB is caused by increased hepatic ureagenesis after metabolism of blood proteins in the GI tract. 6,12,13 Experimental studies in people and dogs showed an association between ingestion of blood and increased serum urea concentration. [12][13][14] An alternative hypothesis is that early prerenal azotemia associated with blood loss and subsequent hypovolemia causes increased serum urea concentration without an increase in serum creatinine concentration. 14,15 To our knowledge, the diagnostic value of the index to predict occult GIB and localize GIB has not been investigated in dogs.
Our primary objective was to evaluate the usefulness of the UCR as a diagnostic marker for occult GIB and to distinguish upper from lower GIB in dogs. We hypothesized that UCR would be higher in dogs with occult GIB compared to healthy dogs but lower compared to dogs with overt GIB, and that UCR would predict occult GIB. Moreover, we anticipated that UCR would be higher in dogs with upper vs lower GIB, and that the ratio could predict upper GIB. As a secondary objective, we compared the diagnostic value of UCR to routine hematological variables in dogs with GIB.

| Patient population
This project was a multicenter, observational, retrospective, and prospective study. Seventy-four dogs were included retrospectively and

| Data collection
For all eligible dogs, medical records were reviewed for the following data: signalment, history, clinical signs, medications at the time of presentation, results of blood tests, results of diagnostic tests to identify bleeding lesions, final diagnosis, and survival to discharge. For each dog enrolled prospectively, muscle condition and systemic blood pressure at the time of blood collection were recorded. Only temporally relevant blood test results were included.
All laboratory tests were performed by reference laboratories (Animal Health Laboratory, University of Guelph, Guelph, Canada; IDEXX Reference Laboratories Ltd., Antech Diagnostics Reference Laboratories) or using standard in-house analyzers. Information on biochemistry and hematology analyzers used at the reference laboratories is given in Table S1 and Table S2. The biochemistry analyzers all measured the entire molecule urea using the kinetic test method. [17][18][19] Because urea concentration and its ratio to creatinine concentration traditionally are reported using serum urea nitrogen concentration in mg/dL, we converted urea concentrations

| Tests to diagnose GIB
The diagnostic evaluation performed to identify bleeding GI lesions varied and included at least 1 of the following: conventional GI endoscopy, VCE, exploratory laparotomy, or necropsy. For all dogs, conventional endoscopy (upper or lower GI endoscopy or both) was performed and assessed by a board-certified internist or a resident in internal medicine.
All VCE examinations were performed after a 12-to 24-hour fast, and capsules were administered PO or endoscopically.  Descriptive statistics were reported for all variables. Categorical variables were presented as frequencies or percentages. Numerical data were tested for normality using the Shapiro-Wilk test and inspection of QQ plots. Normally distributed data were expressed as mean ± SD. Non-normally distributed data were expressed as median and range. Chi-squared or Fisher's exact tests were used for comparisons of proportions of sex and neuter status between overt and occult GI bleeders and clinically healthy dogs.

| Statistical analyses
To compare serum urea nitrogen and creatinine concentrations, and UCR between GI bleeders with and without corticosteroid administration, independent t-tests with logarithmically transformed data to meet test assumptions were performed. Dogs that had received corticosteroids were excluded from the remaining statistical analyses if a statistically significant difference was identified for serum urea nitrogen or creatinine concentration or UCR between them and dogs not treated with corticosteroids.
To assess if the variables serum urea nitrogen concentration,

| RESULTS
One-hundred seventeen dogs with GIB were identified for inclusion in the study, 28 were receiving corticosteroids, and 89 were not.  (1), and marked muscle mass loss (1) corticosteroids were excluded from the study. No dogs were diagnosed with hyperadrenocorticism.

| Study population
In total, 89 dogs with GIB were included in our analyses. There were 65 dogs with overt and 24 dogs with occult GIB (Figure 1).
Sixty-five clinically healthy dogs were included. Of these, 37 dogs were blood donors, 19 dogs were part of an unrelated research study, and 9 dogs were presented for elective castration or spay.
Signalment and clinical signs of overt GI bleeders, occult GI bleeders, and control dogs are presented in Tables 1 and 2, respectively. Control dogs were significantly younger compared to overt GI bleeders (Table 1)

| UCR and hematological variables in dogs with overt and occult GIB and clinically healthy dogs
Descriptive statistics and comparison testing for serum urea nitrogen and creatinine concentrations and UCR in dogs with overt and occult GIB and clinically healthy dogs are presented in Table 5. Serum urea nitrogen concentration was significantly lower in dogs with occult GIB compared to control dogs and dogs with overt GIB. Serum creatinine concentration in dogs with overt and occult GIB was significantly lower compared to healthy control dogs. In dogs with overt GIB compared to control dogs and dogs with occult GIB, UCR was significantly higher.
Serum urea nitrogen and creatinine concentrations were significantly associated with the odds of having occult GIB compared to being clinically healthy, but the UCR was not significantly associated with this outcome (Table 6). Dogs with serum urea nitrogen concentration of ≤12 mg/dL (first quartile) were significantly more likely to have occult GIB than were dogs with serum urea nitrogen concentration ranging from >12 to ≤21.5 mg/dL (second and third quartiles, Table 6). The odds of occult GIB were lower in dogs with higher serum creatinine concentrations (Table 6). Urea nitrogen/creatinine ratio was not a statistically significant variable, even after controlling for the confounding effects of weight loss and anorexia. For each of the logistic regression models used, no evidence of lack of model fit was found and no outliers were identified.
Hemoglobin concentration and Hct were significantly lower in dogs with overt and occult GIB compared to healthy dogs (Table 5).
Mean corpuscular volume was not significantly different between groups (Table 5). Overt GI bleeders had significantly lower MCHC compared to healthy dogs (Table 5).
Using logistic regression models, dogs with higher Hb concentration and Hct had significantly lower odds of occult GIB (Table 6).
No significant association was found between MCV or MCHC and the odds of having occult GIB after adjusting for age (Table 6).

| UCR and hematological variables in dogs with upper and lower GIB
Descriptive statistics and results of comparison testing for serum urea nitrogen and creatinine concentrations, and UCR in dogs with upper GIB, lower GIB, and hemorrhage at both sites are presented in Table 7. No significant difference was found between these groups for serum urea nitrogen and creatinine concentrations and UCR.
Serum urea nitrogen and creatinine concentrations and UCR were not significant predictors of upper GIB (  When assessing serum urea nitrogen and creatinine concentrations separately, serum urea nitrogen concentration was significantly lower in occult GI bleeders compared to healthy dogs and compared to overt GI bleeders. Unexpectedly, dogs with a serum urea nitrogen concentration of ≤12 mg/dL were significantly more likely to have occult GIB than were dogs with serum urea nitrogen concentration ranging from >12 to ≤21.5 mg/dL. Anorexia could have blunted a possible increase in serum urea nitrogen concentration and therefore also an increase in UCR secondary to occult GIB. Decreased or absent food intake and therefore decreased protein digestion could lead to lower serum urea nitrogen concentrations, potentially masking increases associated with GIB. 26 In fact, based on previous study results, serum urea nitrogen concentrations may be more influenced by dietary protein intake than GIB. 14  The sensitivity of UCR to diagnose GIB of any form also may be decreased in our study because the maximum change in serum urea nitrogen concentration could have been missed depending on the timing of the analysis vs timing of hemorrhage. In dogs, the peak serum urea nitrogen concentration occurs approximately 4.5 to 10 hours after blood digestion and serum urea nitrogen concentration decrease to baseline by 24 hours. 12 The timing of GIB to blood collection may preclude useful findings, particularly in dogs with suspected GIB receiving GI protectants. In the dogs in our study, 42.7% were receiving GI protectants at the time of blood sampling. Including dogs receiving GI protectants may be a limitation in our study. Successful treatment of gastroduodenal ulceration using protein-pump inhibitors or other GI protectants could have blunted a possible increase in UCR. Ideally, dogs receiving GI protectants would have been excluded from the study. However, most dogs enrolled retrospectively had been prescribed these medications before referral, and excluding these dogs would have decreased patient enrollment.
Additionally, withholding GI protectants in dogs enrolled prospectively despite suspected or confirmed GIB would not have been ethical.
Excluding these patients would have resulted in very small sample size, likely precluding sufficient statistical power. We calculated descriptive statistics after excluding dogs that received GI protectants, which produced very similar results compared to the original data (Tables S3 and S4). We therefore believe that the overall potential influence of GI protectants on our results was small.
Another limitation is that hematological and biochemical analyses were performed at different laboratories and using different in-house analyzers. Therefore, results could have been influenced by interassay and interlaboratory variation especially given the inclusion of samples run on in-house analyzers, which could have been a source of bias.
Only 9 of 154 (5.8%) samples however had in-house blood analysis performed and, even after exclusion of these, UCR was not associated with occult GIB and could not distinguish between upper and lower GIB (Tables S5-S8)

| CONCLUSION
Our data suggest that the UCR is not useful in predicting occult GIB and does not have strong discriminatory ability to distinguish upper from lower GIB in dog. Consequently, increased UCR in a dog without signs of overt GIB, especially if Hct is within the middle or upper reference interval, does not support prompt prescription of GI protectants. Other factors, such as anorexia, high protein diet, weight loss, muscle condition, and corticosteroid administration should be considered when interpreting UCR.
either GI disease (metronidazole, sulfasalazine, tylosin) or extra-GI disease (ampicillin, cefazolin, and doxycycline). These drugs are not licensed for canine use in Canada and were chosen as they are considered safe and their use in these patients was deemed indicated.

INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) OR OTHER APPROVAL DECLARATION
For data gathered retrospectively, the authors declare no IACUC or other approval was needed. For data gathered prospectively, the study was approved by the University of Guelph Animal Care Committee, and owner consent was obtained before blood collection and administration of video capsule endoscopy.

HUMAN ETHICS APPROVAL DECLARATION
Authors declare human ethics approval was not needed for this study.

SUPPORTING INFORMATION
Additional supporting information may be found online in the Supporting Information section at the end of this article.