Materials and Methods

Animals—Nineteen sexually intact male adult purebred purpose-bred Beagles were obtained from an approved commercial laboratory animal vendor^a for use in the study. The dogs were allowed to acclimatize for 1 month prior to the onset of the study. All dogs were housed at the laboratory dog facilities of the Faculty of Veterinary Medicine at the University of Helsinki in indoor pens; dogs were allowed in outdoor runs for approximately 4 hours daily. The dogs were exposed to natural and artificial light (from 7:00 am to 4:00 pm), and the indoor environmental temperature was maintained at 15° to 24°C. Throughout the study, dogs were fed a diet that consisted of a commercial canned food formulated for dogs^b (dogs were fed twice daily [1.5 cans/dog/d]). Water was freely available at all times. At the beginning of the study, dogs were between 11 and 40 months of age (mean, 18 months) and had a body weight between 12.4 and 17.0 kg (mean, 14.9 kg). Health status of the dogs was determined via physical examinations, and blood samples were obtained from each dog for routine hematologic and biochemical assessment. Prior to the study, the dogs were treated with fenbendazole^c (50 mg/kg, PO, q 24 h for 3 consecutive days), and fecal samples were subsequently collected for endoparasite examination.

The experimental protocol was approved by the Ethics Committee for Animal Experiments of the University of Helsinki. The dogs were cared for and used in experiments in accordance with the prevailing Finnish legislation and the Council of Europe Convention ETS 123 on the use of vertebrate animals for scientific purposes.

GIPFT—In all dogs, GPIFTs were performed by use of ⁵¹Cr-labeled EDTA, lactulose, rhamnose, d-xylose, 3-O-methyl-d-glucose, and sucrose. On the morning of the experiment, 2 fresh test solutions that consisted of distilled water containing 3.7 MBq of ⁵¹Cr-labeled EDTA^d/50 mL and a mixture of 2 g of lactulose, 2 g of rhamnose, 2 g of d-xylose, 1 g of 3-O-methyl-d-glucose, and 8 g of sucrose in 200 mL were prepared and administered to each dog. For subsequent radioactivity measurements, a 1-mL aliquot of the solution containing ⁵¹Cr-labeled EDTA was retained as a counting standard; the aliquot was further diluted (1:50).

Food was withheld the night before and throughout the study, but water was freely available at all times. After food was withheld overnight, the body weight of each dog was measured and a baseline blood sample was collected from each dog. Each dog was sedated with medetomidine^e (25 μg/kg, IM), and the urinary bladder then was emptied by use of a urinary catheter. All dogs then received the ⁵¹Cr-labeled EDTA solution, which was followed immediately by the sugar solution; both solutions were administered via intragastric gavage. To reverse the sedative effects of medetomidine, atipamezole^f (100 μg/kg, IM) was injected, and the dogs were then placed in metabolic cages for a 6-hour period during which urine was collected. After the urine collection was complete, medetomidine^e (25 μg/kg, IM) was again administered to each dog and the urine in each bladder was collected by use of a urinary catheter. The urine collected from the metabolic cage and urinary bladder of each dog was pooled; total urine volume was determined and recorded for subsequent tests. Two 2-mL aliquots of the pooled urine of each dog were retained, one of which was used for radioactivity measurement and the other of which was stored at −20°C until transported on dry ice to a laboratory^g for analysis.

Analysis of sugar concentrations was performed by use of high-performance liquid chromatography in accordance with a method reported elsewhere.¹⁹ The measurement of ⁵¹Cr-labeled EDTA concentrations was performed at the University of Helsinki Central Laboratory of the Department of Clinical Veterinary Sciences. The gamma-ray emission from the aliquot of the counting standard and each urine sample was concurrently counted for 10 minutes by use of a gamma counter^h during the same evening as the experiment. The amount of ⁵¹Cr-labeled EDTA in urine as a percentage of the orally ingested test solution was calculated by use of the following equation:

View Expanded

where CPM is the counts per minute, the first 50 represents the dilution factor, and the second 50 represents the total volume of the test solution.

Statistical analysis—The Shapiro-Wilk test was used to test for normality of the distribution of each variable. Normally distributed variables were expressed as mean ± SD and range, and other variables were expressed as median and range. The Pearson correlation coefficient was used to measure associations between pairs of probe markers that both had a normal distribution, whereas Spearman rank correlation coefficients were calculated if 1 or both of the probe markers were not normally distributed. Values of P < 0.05 were considered significant. Statistical analysis was performed by use of a commercial software program.ⁱ

Results

All dogs were assessed as healthy on the basis of results of physical examination and hematologic and serum biochemical analysis. Analysis of BUN and SUN concentrations suggested physiologically normal renal function in all dogs. Carrier-mediated absorption in the proximal and distal portions of the small intestine was considered to be physiologically normal as determined on the basis of serum concentrations of folate and cobalamin. Exocrine pancreatic function was considered physiologically normal on the basis of serum trypsin-like immunoreactivity concentrations. Results of examination for fecal endoparasitic ova were negative for all dogs.

Values were normally distributed for percentage urinary recovery of ⁵¹Cr-labeled EDTA (P = 0.155), lactulose (P = 0.331), rhamnose (P = 0.388), and 3-O-methyl-d-glucose (P = 0.121) and for recovery ratios of ⁵¹Cr-labeled EDTA to rhamnose (P = 0.077), lactulose to rhamnose (P = 0.062), and d-xylose to 3-O-methyl-d-glucose (P = 0.181). Values were not normally distributed for percentage urinary recovery of d-xylose (P = 0.035) and sucrose (P < 0.001).

The percentage urinary recovery of the 6 markers used to assess gastrointestinal permeability and absorptive function after simultaneous orogastric administration were calculated. Part of the data on ⁵¹Cr-labeled EDTA has been previously reported.²⁰ Mean ± SD percentage urinary recovery of intestinal permeability markers ⁵¹Cr-labeled EDTA, lactulose, and rhamnose was 6.3 ± 1.6% (range, 4.3% to 9.7%), 3.3 ± 1.1% (range, 1.7% to 5.3%), and 25.5 ± 5.0% (range, 16.7% to 36.9%), respectively. The median and range percentage urinary recovery of the intestinal absorptive function marker d-xylose was 40.3% and 31.6% to 62.7%. Mean ± SD percentage urinary recovery of 3-O-methyl-d-glucose was 58.8 ± 11.0% (range, 40.1% to 87.8%). For sucrose (gastric permeability marker), the median and range percentage urinary recovery was 0% and 0% to 0.8%. Moreover, the mean ± SD urinary recovery ratio was 0.25 ± 0.06 (range, 0.17 to 0.37) for ⁵¹Cr-labeled EDTA to rhamnose, 0.13 ± 0.04 (range, 0.08 to 0.23) for lactulose to rhamnose, and 0.73 ± 0.09 (range, 0.60 to 0.90) for d-xylose to 3-O-methyl-d-glucose. Correlation coefficients and significance were determined for all GIPFT variables evaluated in the study (Table 1).

Discussion

In the study reported here, the percentage urinary recovery of ⁵¹Cr-labeled EDTA for urine samples collected during a 6-hour period in healthy adult male Beagles ranged from 4.3% to 9.7%. In other studies²¹ on healthy Beagles, the mean percentage urinary recovery of ⁵¹Cr-labeled EDTA for urine samples collected during a 24-hour period ranged between 17.3% and 37.6%,²¹ and the percentage urinary recovery for urine samples collected during a 6-hour period ranged between 3.8% and 34.2%.⁶ The high variation in test results in those earlier studies may be explained by the inclusion of both pubertal and adult dogs of both sexes that were fed both canned and dry foods. In addition, some of those dogs in earlier investigations were affected by small intestinal dysbiosis (formerly ascribed as small intestinal bacterial overgrowth).^6,21 To our knowledge, the study reported here was the first in which the ⁵¹Cr-labeled EDTA-to-rhamnose recovery ratio has been determined in dogs.

We found that the urinary lactulose-to-rhamnose recovery ratio for samples collected during a 6-hour period in healthy adult male Beagles ranged from 0.08 to 0.23. As expected, our ranges were considerably different from, but in agreement with, results of other investigations that used healthy dogs of a number of breeds, ages, and sizes, including Irish Setters (0.03 to 0.18),⁵ Greyhounds (0.19 to 0.34),¹⁴ Viszlas (0.07 to 0.26),¹⁴ mixed-breed dogs (0.08 to 0.34),¹⁴ adult Miniature Poodles (0.14 to 0.21),¹⁷ adult Standard Schnauzers (0.13 to 0.26),¹⁷ adult Giant Schnauzers (0.17 to 0.32),¹⁷ adult Great Danes (0.26 to 0.42),¹⁷ and Beagles (0.05 to 0.15).^j The findings for the present study, together with those of previous studies, are consistent with the theory that there are effects of breed, age, and body size and possibly diet on intestinal permeability in dogs.

The urinary d-xylose–to–3-O-methyl-d-glucose recovery ratio ranged from 0.60 to 0.90 in urine samples collected during a 6-hour period in healthy adult male Beagles in the present study, which was consistent with results of similar studies on a group of Beagles (0.40 to 0.59)^19,j and in other breeds, including adult Miniature Poodles (0.52 to 0.65),²¹ adult Standard Schnauzers (0.51 to 0.68),¹⁷ adult Giant Schnauzers (0.54 to 0.62),¹⁷ and adult Great Danes (0.56 to 0.62).¹⁷ Findings from the present study are in agreement with those of another study,¹⁷ which suggested that in contrast to the apparent morphological differences, the carrier-mediated mechanisms that allow intestinal absorption are similar among adult dogs of different breeds.

Studies in humans (r = 0.98; P = 0.001) and cats (r = 0.85; P = 0.03) have revealed a correlation between ⁵¹Cr-labeled EDTA and lactulose concentrations when compared as single markers^8,9 or as ratios to rhamnose concentrations (r = 0.97; P = 0.002).⁹ However, analysis of the data from the present study suggested that the correlation between the percentage urinary recovery of ⁵¹Cr-labeled EDTA and lactulose after their concurrent oral administration in Beagles when compared as single markers (r = 0.64; P = 0.003) or as ratios to rhamnose (r = 0.50; P = 0.030) was not as prominent as previously determined in humans and cats. The lower correlation between these 2 markers in the dogs of the present study may be explained by the possible partial metabolization or breakdown of the sugar probes (eg, lactulose metabolism by resident intestinal bacteria).

In general, ⁵¹Cr-labeled EDTA is considered to be the ideal permeability marker because of its biological inertness. This is especially useful in individuals with an overgrowth of bacteria because the inertness will prevent bacterial degradation of the molecule. It is known that apparently healthy Beagles may have evidence of small intestinal dysbiosis,²¹ a condition that is consistent with bacterial degradation of saccharides such as lactulose and that may be exacerbated if bacteria that typically colonize the colon are able to colonize the small intestine.²²

No significant positive correlation was detected in the present study between the percentage urinary recovery of ⁵¹Cr-labeled EDTA and that of the other sugar markers (rhamnose, d-xylose, 3-O-methyl-d-glucose, and sucrose), which is consistent with current knowledge of the GIPFT markers.² Interestingly, the correlation (r = 0.90; P < 0.001) between rhamnose, a marker reflecting nonmediated transcellular diffusion throughout the small intestine, and 3-O-methyl-d-glucose, a molecule absorbed across the small intestine via a specific carrier-mediated transport system, may indicate that both markers may be absorbed in parallel by the intact mucosa of the small intestines of healthy Beagles. Moreover, the correlation (r = 0.63; P = 0.004) between rhamnose and d-xylose, the intestinal absorption mechanism of which is similar to that of 3-O-methyl-d-glucose but limited to the jejunum, adds support to this suggestion.

It has generally been assumed that the ratio of 2 molecules provides a more reliable index of intestinal damage and dysfunction than does evaluation of single markers alone.² Nevertheless, the findings for the present study, in which we compared the percentage urinary recovery of ⁵¹Cr-labeled EDTA and lactulose with the ⁵¹Cr-labeled EDTA-to-rhamnose recovery ratio (r = 0.73; P < 0.001) and lactulose-to-rhamnose recovery ratio (r = 0.80; P < 0.001), suggested that equivalent results were obtained in healthy dogs when a single marker was evaluated or a combination of 2 markers was used as a ratio. However, evaluation of a single marker for the assessment of intestinal absorptive function is not recommended on the basis of the findings of the present study in healthy Beagles.

In the study reported here, we provided data on the percentage urinary recovery of ⁵¹Cr-labeled EDTA, lactulose, rhamnose, d-xylose, 3-O-methyl-d-glucose, and sucrose used for GIPFTs in healthy adult male Beagles in a controlled experimental setting. These data add to current information about integrity of the physiologically normal gastrointestinal mucosa and absorptive capacity in this breed. These data could be used in future studies with Beagles. Although there was a significant correlation between the percentage urinary recovery of the intestinal permeability markers ⁵¹Cr-labeled EDTA and lactulose, there was a slight discrepancy between results for the present study in dogs and published results for other species. Further studies are needed to clarify this disagreement. Meanwhile, caution is warranted when GIPFT results are obtained via the use of lactulose and other sugar probes, particularly in dogs suspected of having small intestinal dysbiosis.