Nora Berghoff

Development and analytical validation of a radioimmunoassay for the measurement of alpha1-proteinase inhibitor concentrations in feces from healthy puppies and adult dogs.

Canine α1-proteinase inhibitor (cα1-PI), a proteolysis-resistant protein with a molecular weight similar to albumin, has been shown to be clinically useful as a marker for gastrointestinal protein loss in dogs. A competitive, liquid-phase radioimmunoassay was developed and analytically validated. Fecal samples were collected from 101 healthy pet dogs of various breeds and ages, and fecal cα1-PI (Fcα1-PI) concentrations were compared between dogs of different age groups. A reference interval for Fcα1-PI concentration was calculated using the central 95th percentile. Analytical sensitivity of the assay was 2.2 µg Fcα1-PI/g feces. Observed-to-expected ratios for the serial dilution and spiking recovery of 9 and 6 fecal extracts ranged from 90.4 to 152.0% and from 71.3 to 112.3%, respectively. Coefficients of variation for intra- and interassay variability for 6 fecal extracts were ≤10.8% and ≤12.5%, respectively. The reference intervals for the mean and maximum Fcα1-PI from fecal samples collected on 3 consecutive days were 2.2–13.9 µg/g and 2.2–21.0 µg/g, respectively. Fcα1-PI was significantly higher in dogs <1 year of age (P < 0.0001 for both mean and maximum Fcα1-PI for the 3 samples). The radioimmunoassay described is sensitive, linear, precise, reproducible, and accurate for clinical use, thus allowing reliable quantification of Fcα1-PI in clinical patients. Using this assay, a mean or a maximum Fcα1-PI for 3 sampling days of >13.9 µg/g or >21.0 µg/g, respectively, should be considered abnormal in dogs >1 year of age. Fecal cα1-PI concentrations in dogs <1 year of age were significantly higher and should be carefully interpreted in this age group.

Association between serum cobalamin and methylmalonic acid concentrations in dogs.

The aim of this study was to evaluate the association between serum methylmalonic acid (MMA), a proposed marker of cellular cobalamin deficiency, and serum cobalamin concentrations in dogs. Serum samples from 555 dogs were grouped according to their serum cobalamin concentrations (<150 ng/L to 1000 ng/L). Additionally, serum samples were collected from 43 healthy dogs to calculate a reference interval for canine serum MMA. MMA was measured using a GC/MS method. Groups were compared using a Kruskal-Wallis test with Dunns post test. Proportions of dogs above the upper limit of the reference interval were calculated and a χ2-test for trend was performed to evaluate the association between serum cobalamin and MMA concentrations. The reference interval for serum MMA was calculated to be 414.7-1192.5 nmol/L. Dogs with serum cobalamin concentrations <251 ng/L had significantly higher MMA concentrations (P<0.05) and the χ2-test for trend showed a trend for increasing serum MMA concentrations with decreasing serum cobalamin concentrations (P<0.0001). Additionally, a number of dogs with normal serum cobalamin concentrations had increased serum MMA concentrations, suggesting that some of these dogs may have cobalamin deficiency on a cellular level. Further studies are warranted to determine if these dogs should receive cobalamin supplementation.

Partial characterization of cobalamin deficiency in Chinese Shar Peis

A total of 22,462 serum sample results from dogs being evaluated for gastrointestinal disease at the Gastrointestinal Laboratory, College of Veterinary Medicine, Texas A&M University were evaluated retrospectively. The proportion of dogs with serum cobalamin concentrations below the reference interval and median serum concentrations were compared between Shar Peis and other dog breeds. Serum samples were also obtained prospectively from 22 healthy and 32 Shar Peis with chronic gastrointestinal disease and 59 healthy dogs of other breeds, and serum concentrations of cobalamin, folate, and methylmalonic acid were determined and compared. Overall, 64.0% (89/139) of serum samples from Shar Peis showed serum cobalamin concentrations below the limit of the reference interval and 38.1% (53/139) of these were below the detectable limit for the assay. The median serum cobalamin concentration in Shar Peis was significantly lower than in other breeds. Shar Peis with gastrointestinal disease had significantly lower serum cobalamin and higher serum methylmalonic acid concentrations compared to healthy Shar Peis. Healthy Shar Peis had significantly increased serum methylmalonic acid concentrations compared to healthy dogs of other breeds. There were no meaningful differences in folate concentrations between groups. In conclusion, Shar Peis have a high prevalence of cobalamin deficiency compared to other breeds and healthy Shar Peis may have subclinical cobalamin deficiency.

Laboratory Tests for the Diagnosis and Management of Chronic Canine and Feline Enteropathies

Chronic enteropathies are commonly encountered in both cats and dogs. Although definitive diagnosis often requires collection of gastrointestinal biopsies for histopathologic evaluation, less invasive laboratory tests can be highly informative and should be performed prior to biopsy collection. Tests for determination of infectious causes comprise those for helminthic, protozoal, bacterial, or fungal organisms. Intestinal function and disease may be assessed by measuring serum concentrations of cobalamin, folate, and C-reactive protein, and fecal concentrations of α(1)-proteinase inhibitor. Ongoing research has led to development of tests for serum perinuclear antineutrophilic cytoplasmic antibodies, and fecal inflammatory markers, including S100-proteins and N-methylhistamine.

Serum cobalamin and methylmalonic acid concentrations in dogs with chronic gastrointestinal disease

OBJECTIVE To determine the prevalence of hypocobalaminemia or methylmalonic acidemia (or both) in dogs with chronic gastrointestinal disease. SAMPLE Serum samples from 56 dogs with chronic gastrointestinal disease and 43 control dogs. PROCEDURES Serum cobalamin and methylmalonic acid (MMA) concentrations were measured in all samples and compared between groups. A correlation between serum cobalamin and MMA concentrations and the canine chronic enteropathy clinical activity index was evaluated via the Spearman rank correlation. RESULTS 20 of 56 (36%) dogs with gastrointestinal disease had hypocobalaminemia. Serum cobalamin concentrations were significantly lower in dogs with gastrointestinal disease than in control dogs. Five of 56 (9%) dogs with chronic gastrointestinal disease and 5 of 20 (25%) hypocobalaminemic dogs had increased MMA concentrations. There was a significant negative correlation (Spearman r = -0.450) between serum cobalamin and MMA concentrations in dogs with gastrointestinal disease. No correlation was found between the canine chronic enteropathy clinical activity index and serum cobalamin or MMA concentrations. CONCLUSIONS AND CLINICAL RELEVANCE These data indicated the prevalence of hypocobalaminemia in dogs with chronic gastrointestinal disease was 20 of 56 (36%). Five of 20 (25%) hypocobalaminemic dogs had increased serum MMA concentrations, which indicated that although hypocobalaminemia was common in these dogs, it did not always appear to be associated with a deficiency of cobalamin on a cellular level. Hypocobalaminemia is a risk factor for negative outcome in dogs with chronic gastrointestinal disease and should be considered in every patient with corresponding clinical signs.

The relationship of serum cobalamin to methylmalonic acid concentrations and clinical variables in cats.

BACKGROUND Serum cobalamin concentration [CBL] suggests CBL deficiency in cats but serum methylmalonic acid concentration [MMA] more accurately indicates CBL deficiency. OBJECTIVE To examine the ability of [CBL] to predict CBL deficiency defined by increased [MMA], and relationships of [CBL] and [MMA] with select clinical and clinicopathological variables. ANIMALS One hundred sixty-three client-owned cats with [CBL] measurements, 114 cats with simultaneous [MMA] measurements; 88 cats with medical information. METHODS Prospectively collected [CBL] and [MMA] were compared using scatter plots, receiver operating characteristic and correlative analyses with historical [CBL] thresholds and those identified in the study. [CBL] and [MMA] were compared retrospectively to specific clinical and clinicopathological variables. RESULTS [CBL] correlated negatively with [MMA] (τ = -0.334, P < .0001). [MMA] ≥ 1,343 nmol/L identified CBL deficiency. [CBL] = 209 pg/mL optimized sensitivity (0.51), specificity (0.96), PPV (0.89), and NPV (0.74) for detecting [MMA] ≥ 1,343 nmol/L. Prevalence of CBL deficiency was 42% (48/114) when defined by [MMA] ≥ 1,343 nmol/L versus 23% (27/114) by [CBL] ≤ 209 pg/mL. Unexpectedly, 23 and 45% of 48 cats with [MMA] ≥ 1,343 nmol/L had [CBL] > 900 pg/mL and 290 pg/mL (historical thresholds). [CBL] correlated with mean corpuscular volume (τ = -0.199, P = .013) and [MMA] with hematocrit (τ = -0.28, P = .006). CONCLUSIONS AND CLINICAL IMPORTANCE Cobalamin deficiency ([MMA] ≥ 1,343 nmol/L) occurred in 42% of cats and is predicted with high specificity by [CBL] ≤ 209 pg/mL. CBL status correlates with microcytosis and anemia. Discordance between [CBL] and [MMA] cautions against relying on any single marker for determining CBL status.

Fecal and urinary N-methylhistamine concentrations in dogs with chronic gastrointestinal disease.

Due to their ability to release inflammatory mediators, such as histamine, mast cells are potentially important in gastrointestinal disease. The purpose of this study was to measure N-methylhistamine (NMH), a histamine metabolite, in fecal and urine samples from dogs with chronic gastrointestinal disease. Fecal and urinary NMH concentrations were compared between dogs with chronic gastrointestinal disease and control dogs, and/or to control ranges. Correlation between fecal and urinary NMH concentrations, serum C-reactive protein (CRP) concentration, the clinical disease activity index (CCECAI), and gastrointestinal mucosal mast cell numbers (where available) in dogs with gastrointestinal disease was evaluated. Seven of 16 dogs with gastrointestinal disease had increased urinary or fecal NMH concentrations, but there was no correlation between NMH concentrations and the CCECAI or mucosal mast cells numbers. Urinary NMH concentrations were positively associated with histological grading and serum CRP concentrations. The lack of correlation between NMH concentrations and the CCECAI suggests that NMH may not be a good marker for clinical disease activity in dogs as determined by the CCECAI. Based on their association with severity of intestinal mucosal inflammation, urinary NMH concentrations may potentially have clinical utility as a marker of intestinal inflammation in certain groups of dogs with chronic gastrointestinal disease, but future studies in a larger number of dogs are necessary to further characterize the role of mast cell-mediated inflammation in dogs with chronic gastrointestinal disease.

Development and analytic validation of a gas chromatography–mass spectrometry method for the measurement of sugar probes in canine serum

OBJECTIVE To develop and analytically validate a gas chromatography-mass spectrometry (GC-MS) method for the quantification of lactulose, rhamnose, xylose, 3-O-methylglucose, and sucrose in canine serum. SAMPLE POPULATION Pooled serum samples from 200 dogs. Procedures-Serum samples spiked with various sugars were analyzed by use of GC-MS. The method was analytically validated by determination of dilutional parallelism, spiking recovery, intra-assay variability, and interassay variability. RESULTS Standard curves ranging from 0.5 to 500 mg/L for each sugar revealed a mean r(2) of 0.997. The lower detection limit was 0.03 mg/L for lactulose, rhamnose, xylose, and methylglucose and 0.12 mg/L for sucrose. The observed-to-expected ratios for dilutional parallelism had a mean +/- SD of 105.6 +/- 25.4% at dilutions of 1:2, 1:4, and 1:8. Analytic recoveries for the GC-MS assays of sugars ranged from 92.1% to 124.7% (mean +/- SD, 106.2 +/- 13.0%). Intra-assay coefficients of variation ranged from 6.8% to 12.9% for lactulose, 7.1% to 12.8% for rhamnose, 7.2% to 11.2% for xylose, 8.9% to 11.5% for methylglucose, and 8.9% to 12.0% for sucrose. Interassay coefficients of variation ranged from 7.0% to 11.5% for lactulose, 6.4% to 9.4% for rhamnose, 6.8% to 13.2% for xylose, 7.0% to 15.9% for methylglucose, and 5.5% to 9.4% for sucrose. CONCLUSIONS AND CLINICAL RELEVANCE The GC-MS method described here was accurate, precise, and reproducible for the simultaneous measurement of sugar probes in canine serum.

Serum and fecal canine α1-proteinase inhibitor concentrations reflect the severity of intestinal crypt abscesses and/or lacteal dilation in dogs.

Gastrointestinal (GI) protein loss, due to lymphangiectasia or chronic inflammation, can be challenging to diagnose. This study evaluated the diagnostic accuracy of serum and fecal canine α1-proteinase inhibitor (cα1PI) concentrations to detect crypt abscesses and/or lacteal dilation in dogs. Serum and fecal cα1PI concentrations were measured in 120 dogs undergoing GI tissue biopsies, and were compared between dogs with and without crypt abscesses/lacteal dilation. Sensitivity and specificity were calculated for dichotomous outcomes. Serial serum cα1PI concentrations were also evaluated in 12 healthy corticosteroid-treated dogs. Serum cα1PI and albumin concentrations were significantly lower in dogs with crypt abscesses and/or lacteal dilation than in those without (both P <0.001), and more severe lesions were associated with lower serum cα1PI concentrations, higher 3 days-mean fecal cα1PI concentrations, and lower serum/fecal cα1PI ratios. Serum and fecal cα1PI, and their ratios, distinguished dogs with moderate or severe GI crypt abscesses/lacteal dilation from dogs with only mild or none such lesions with moderate sensitivity (56-92%) and specificity (67-81%). Serum cα1PI concentrations increased during corticosteroid administration. We conclude that serum and fecal α1PI concentrations reflect the severity of intestinal crypt abscesses/lacteal dilation in dogs. Due to its specificity for the GI tract, measurement of fecal cα1PI appears to be superior to serum cα1PI for diagnosing GI protein loss in dogs. In addition, the serum/fecal cα1PI ratio has an improved accuracy in hypoalbuminemic dogs, but serum cα1PI concentrations should be carefully interpreted in corticosteroid-treated dogs.

Urinary and faecal N-methylhistamine concentrations do not serve as markers for mast cell activation or clinical disease activity in dogs with chronic enteropathies.

BackgroundThis study sought to correlate faecal and urinary N-methylhistamine (NMH) concentrations with resting versus degranulated duodenal mast cell numbers in dogs with chronic enteropathies (CE), and investigate correlations between intestinal mast cell activation and clinical severity of disease as assessed by canine chronic enteropathy clinical activity index (CCECAI), and between urinary and faecal NMH concentrations, mast cell numbers, and histopathological scores. Twenty-eight dogs with CE were included. Duodenal biopsies were stained with haematoxylin and eosin (H&E), toluidine blue, and by immunohistochemical labelling for tryptase. Duodenal biopsies were assigned a histopathological severity score, and duodenal mast cell numbers were counted in five high-power fields after metachromatic and immunohistochemical staining. Faecal and urinary NMH concentrations were measured by gas chromatography–mass spectrometry.ResultsThere was no correlation between the CCECAI and faecal or urinary NMH concentrations, mast cell numbers, or histopathological score – or between faecal or urinary NMH concentration and mast cell numbers. Post hoc analysis revealed a statistically significant difference in toluidine blue positive mast cells between two treatment groups (exclusion diet with/without metronidazole versus immunosuppression (IS)), with higher numbers among dogs not requiring IS.ConclusionFaecal and urinary NMH concentrations and duodenal mast cell numbers were not useful indicators of severity of disease as assessed by the CCECAI or histological evaluation. The number of duodenal mast cells was higher in dogs that did not need IS, i.e. in dogs responding to an exclusion diet (with/without metronidazole), than in dogs requiring IS. Further studies comparing the role of mast cells in dogs with different forms of CE are needed.