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Abstract

Objective

To determine the mechanism by which liver alkaline phosphatase (LALP) isoenzyme is converted from a membrane-bound enzyme to the soluble enzyme during cholestasis.

Sample Population

Serum and tissues from 2 dogs.

Procedure

The LALP was purified by use of affinity chromatography in samples of serum from dogs with complete bile duct obstruction. Gas chromatography/mass spectrometry was used to detect myo-inositol residues that would be evident when serum LALP had been membrane-attached and released by phospholipase activity. Exclusion chromatography, gel electrophoresis, and octyl-sepharose phase separation of the serum isolate were used to confirm cleavage of the hydrophobic membrane anchor. Western immunoblot analysis was used to distinguish release by glycosylphosphatidylinositol phospholipase D (GPI-PLD) from that by glycosylphosphatidylinositol phospholipase C (GPI-PLC). Intact hepatocytes were incubated with canine serum GPI-PLD to test sensitivity of LALP to release by GPI-PLD. Hepatocyte membrane fragments were treated with serum GPI-PLD and mixtures of taurocholate and taurodeoxycholate to test effects of bile acids on LALP release.

Results

Amounts of myo-inositol per mole of serum LALP isolate were equal to amounts detected with LALP isolated from hepatic tissue. Evaluation of results of western immunoblot analysis and electrophoretic mobility suggested release by GPI-PLD rather than by GPI-PLC. Membrane-bound LALP was resistant to serum GPI-PLD activity in the absence of bile acids; however, incubation in the presence of bile acids caused release of LALP.

Conclusions

Solubilization of LALP during cholestasis involves cleavage of its membrane anchor by endogenous GPI-PLD activity. Action of GPI-PLD is likely enhanced by increased concentrations of hepatic bile acids during cholestasis. (Am J Vet Res 1999;60:1010-1015)

Free access
in American Journal of Veterinary Research

Abstract

Objective—To determine the effect of glucocorticoids on the induction of alkaline phosphatase (ALP) isoenzymes in the liver, kidneys, and intestinal mucosa, 3 tissues that are principally responsible for ALP synthesis in dogs.

Sample Population—Tissues from the liver, kidneys, and intestinal mucosa of 6 dogs treated with 1 mg of prednisone/kg/d for 32 days and 6 untreated control dogs.

Procedure—Using canine-specific primers for the ALP isoenzymes, a reverse transcription-polymerase chain reaction assay was designed to measure liver ALP (LALP) and intestinal ALP (IALP) mRNA and heterogeneous nuclear RNA (hnRNA) expression in tissues from the liver and kidneys and intestinal mucosa of glucocorticoid-treated and control dogs. Tissue ALP isoenzyme activities were compared between the groups.

Results—The LALP activity and mRNA concentrations increased in tissues of the liver and kidneys in dogs treated with prednisone, whereas LALP hnRNA increased only in liver tissues. The IALP activity and mRNA expression increased in intestinal mucosa and liver tissues in prednisone-treated dogs. We did not detect an increase in IALP hnRNA expression in these tissues.

Conclusions and Clinical Relevance—Synthesis of ALP is increased in the liver, kidneys, and intestinal mucosa of dogs in response to prednisone treatment. This response appears to be regulated at the transcriptional level, but mechanisms may differ between LALP and IALP. (Am J Vet Res 2002;63:1083–1088)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To clone segments of the canine liver alkaline phosphatase (LALP) and corticosteroidinduced alkaline phosphatase (CIALP) genes and use those clones to determine the tissue source of CIALP, the kinetics of LALP and CIALP mRNA expression for glucocorticoid-treated dogs, and the correlation between LALP and CIALP transcript concentrations and isoenzyme activities.

Sample Population—Tissues obtained from 7 dogs treated with prednisone (1 mg/kg, SC, q 24 h) for up to 32 days and 1 untreated (control) dog.

Procedure—Gene segments of LALP and CIALP were obtained by reverse transcription-polymerase chain reaction (RT-PCR) assay. The tissue source of CIALP and IALP mRNA was determined by northern blot analysis of tissues from 1 of the glucocorticoidtreated dogs. Hepatic tissues and serum samples were obtained from the 6 remaining glucocorticoidtreated dogs on days 0, 2, 5, 10, and 32 of prednisone treatment, and relative expression of LALP and CIALP mRNA was correlated with LALP and CIALP activity.

Results—A 2,246-base pair (bp) segment of canine LALP and a 1,338-bp segment of CIALP were cloned. Northern blot analysis revealed CIALP mRNA expression in hepatic tissues only after glucocorticoid treatment. Kinetics of LALP and CIALP mRNA expression in the liver of glucocorticoid-treated dogs paralleled liver and serum activities of LALP and CIALP.

Conclusions and Clinical Relevance—The liver is the most likely source for CIALP in dogs. Analysis of kinetics of serum and hepatic LALP and CIALP mRNA suggests that after glucocorticoid treatment, both are regulated by modification of mRNA transcript concentrations, possibly through differing mechanisms. (Am J Vet Res 2002;63:1089–1095)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine the nucleotide and amino acid sequence of atrial natriuretic peptide (ANP) in cats and its typical regions of cardiac expression.

Animals—5 healthy adult mixed-breed cats.

Procedure—Total RNA was extracted from samples obtained from the left and right atrium, left and right ventricle, and interventricular septum of each cat. The RNA was used to produce cDNA for sequencing and northern blot analysis. Genomic DNA was extracted from feline blood samples. Polymerase chain reaction primers designed from consensus sequences of other species were used to clone and sequence the feline ANP gene.

Results—The feline ANP gene consists of 1,072 nucleotides. It consists of 3 exons (123, 327, and 12 nucleotides) separated by 2 introns (101 and 509 nucleotides). It has several typical features of eukaryotic genes and a putative steroid-response element located within the second intron. Preprohormone ANP consists of 153 amino acids. The amino acid sequence of the active form of feline ANP (ANP-30) is identical to that of equine, bovine, and ovine ANP-30 and differs from that of human, canine, and porcine ANP-28 only by 2 carboxy-terminal arginine residues. The ANP mRNA was detected only in the left and right atria.

Conclusions and Clinical Relevance—The genetic and protein structure and principal regions of cardiac expression of feline ANP are similar to those of other species. Results of this study should be helpful in future studies on the natriuretic response in cats to diseases that affect cardiovascular function. (Am J Vet Res 2002;63:236–240)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To map canine mitochondrial proteins and identify qualitative and quantitative differences in heart mitochondrial protein expression between healthy dogs and dogs with naturally occurring and induced dilated cardiomyopathy (DCM).

Sample Population—Left ventricle samples were obtained from 7 healthy dogs, 7 Doberman Pinschers with naturally occurring DCM, and 7 dogs with induced DCM.

Procedures—Fresh and frozen mitochondrial fractions were isolated from the left ventricular free wall and analyzed by 2-dimensional electrophoresis. Protein spots that increased or decreased in density by ≥ 2-fold between groups were analyzed by matrixassisted laser desorption/ionization time-of-flight mass spectrometry or quadrupole selecting, quadrupole collision cell, time-of-flight mass spectrometry.

Results—Within narrow pH gradients of control canine heart mitochondrial samples, a total of 1,528 protein spots were revealed. Forty subunits of heart mitochondrial proteins that differ significantly from control tissues were altered in tissue specimens from dogs with naturally occurring and induced forms of DCM. The most affected heart mitochondrial proteins in both groups were those of oxidative phosphorylation (55%). Upregulation of manganese superoxide dismutase was suggestive of heart oxidative injury in tissue specimens from dogs with both forms of DCM. Evidence of apoptosis was associated with overexpression of the heart mitochondrial voltage-dependent anion channel-2 protein and endonuclease G in tissue specimens from dogs with induced DCM.

Conclusions and Clinical Relevance—Alterations of heart mitochondrial proteins related to oxidative phosphorylation dysfunction were more prevalent in tissue specimens from dogs with induced or naturally occurring DCM, compared with those of control dogs.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To identify qualitative and quantitative differences in cardiac mitochondrial protein expression in complexes I to V between healthy dogs and dogs with natural or induced dilated cardiomyopathy (DCM).

Sample Population—Left ventricle samples were obtained from 7 healthy dogs, 7 Doberman Pinschers with naturally occurring DCM, and 7 dogs with DCM induced by rapid right ventricular pacing.

Procedures—Fresh and frozen mitochondrial fractions were isolated from the left ventricular free wall and analyzed by 2-dimensional electrophoresis. Protein spots that increased or decreased in density by 2-fold or greater between groups were analyzed by matrixassisted laser desorption/ionization time-of-flight mass spectrometry or quadrupole selecting, quadrupole collision cell, time-of-flight mass spectrometry.

Results—A total of 22 altered mitochondrial proteins were identified in complexes I to V. Ten and 12 were found in complex I and complexes II to V, respectively. Five were mitochondrial encoded, and 17 were nuclear encoded. Most altered mitochondrial proteins in tissue specimens from dogs with naturally occurring DCM were associated with complexes I and V, whereas in tissue specimens from dogs subjected to rapid ventricular pacing, complexes I and IV were more affected. In the experimentally induced form of DCM, only nuclear-encoded subunits were changed in complex I. In both disease groups, the 22-kd subunit was downregulated.

Conclusions and Clinical Relevance—Natural and induced forms of DCM resulted in altered mitochondrial protein expression in complexes I to V. However, subcellular differences between the experimental and naturally occurring forms of DCM may exist.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine reference ranges for results of hematologic analyses of healthy Belgian Tervuren, to compare results of hematologic analyses for healthy Belgian Tervuren with results for healthy dogs of other breeds, and to determine prevalence of physiologic leukopenia in Belgian Tervuren.

Design—Cohort study.

Animals—180 healthy Belgian Tervuren and 63 healthy dogs of other breeds.

Procedure—Blood samples were analyzed by use of an automated device. Reference ranges were calculated for Belgian Tervuren by use of standard methods.

Results—Total WBC counts of Belgian Tervuren ranged from 2.61 to 16.90 X 103/µl. Mean WBC count of Belgian Tervuren (mean ± SEM, 7.04 ± 0.16 X 103/µl) was significantly lower than mean count for control dogs. Significantly more Belgian Tervuren (65/180; 36%) than control dogs (2/63; 3%) had WBC counts < 6.00 X 103/µl. Percentage of Belgian Tervuren with WBC count < 6.00 X 103/µl was low for dogs ≤ 2 years old, increased sharply for dogs between 2 and 4 years old, and was approximately 65% for dogs > 4 years old. Neutrophil, lymphocyte, and monocyte counts were significantly lower, and RBC count, hematocrit, and eosinophil fraction were significantly higher in Belgian Tervuren than in control dogs.

Conclusions and Clinical Relevance—Results suggest that physiologic leukopenia, resulting from low numbers of neutrophils, lymphocytes, and monocytes, may be a typical finding in a large percentage of healthy Belgian Tervuren and is not of clinical importance in otherwise healthy dogs. Healthy Belgian Tervuren may also have RBC counts and hematocrits higher than expected for healthy dogs.( J Am Vet Med Assoc 2000;216:866–871)

Full access
in Journal of the American Veterinary Medical Association

Abstract

Quantitative determination of the corticosteroid-induced isoenzyme of alkaline phosphatase (cap) was evaluated as a screening test for hyperadrenocorticism (hac) in dogs. A series of 40 dogs with hac (cap range, 96 to 14,872 U/L), 30 clinically normal dogs (cap range, 0 to 38 U/L), and 80 dogs with various diseases (non-hac) and without history of exogenous glucocorticoid exposure for a minimum of 60 days (cap range, 0 to 1163 U/L) were used to evaluate the test. Sensitivity and specificity of cap was calculated at various cutoff points for absolute cap activity and for cap activity expressed as a percentage of total alkaline phosphatase activity. A cutoff point of 90 U/L was selected as optimal for use of this assay as a screening test for hac. A prevalence survey then was done of all canine serum samples submitted to our diagnostic laboratory over a 3-month period, to calculate the predictive values of a positive and a negative test result in a clinical population and to determine the relative frequency and magnitude of cap activity in dogs that had received glucocorticoids. The predictive values of a positive and a negative test result at the 90 U/L cutoff value were 21.43% (95% confidence limits, 8.3 to 40.95%) and 100% (95% confidence limit > 96%), respectively. It was concluded that cap isoenzyme activity, determined by routine biochemical analysis by an automated levamisole-inhibition assay, could function as a screening test for hac, however, (the predictive value of a positive test result was too low to recommend the assay as a diagnostic test. The cap activity was > 90 U/L in 52% of dogs receiving glucocorticoids, emphasizing the importance of treatment history in the use of this test. To help eliminate possible workup bias, an additional set of serum samples from 30 dogs diagnosed as having hac at a second institution also were analyzed. Sensitivity of the assay in this sample group, using the 90 U/L cutoff value, was 83.3% (95% confidence limits, 65.3 to 94.4%), suggesting that test sensitivity may vary, depending on the institution in which the assay is used and the selection of cases for diagnostic workup.

Free access
in Journal of the American Veterinary Medical Association
in Journal of the American Veterinary Medical Association

SUMMARY

Assay procedures for determining serum haptoglobin concentration and ceruloplasmin oxidase activity in dogs were validated, and reference values were established. Serum haptoglobin concentration is reported as milligrams per deciliter of cyanmethemoglobin binding capacity, whereas serum ceruloplasmin oxidase activity was determined by use of p-phenylenediamine as substrate. Both assays were used to analyze serum samples from 288 dogs. In each dog’s case record, clinical history and final diagnosis were evaluated to determine whether the dog had an inflammatory condition. Complete blood cell counts were performed in 265 dogs, using simultaneously collected blood samples. Plasma fibrinogen concentration was determined for 161 dogs. A positive correlation (P < 0.01) was found for serum haptoglobin concentration and for ceruloplasmin oxidase activity, compared with wbc counts, segmented neutrophil and band neutrophil counts, and plasma fibrinogen concentration. Ceruloplasmin oxidase activity and haptoglobin concentration were up to 6 times more sensitive than fibrinogen concentration or leukocyte counts in detecting inflammation. Specificity of ceruloplasmin oxidase activity was comparable to fibrinogen concentration and leukocyte counts, whereas haptoglobin concentration was found to be slightly less specific. Specificity of haptoglobin concentration improved slightly (from 0.82 to 0.88) when dogs with a history of glucocorticoid administration were excluded from analysis. Predictive value of a negative test result (haptoglobin concentration < 125 mg/dl; ceruloplasmin oxidase activity < 20 IU/L) and predictive value of a positive test result for haptoglobin concentration and ceruloplasmin activity were comparable to or better than fibrinogen concentration or various oxidase leukocyte counts in detection of inflammation in a variety of disease conditions. We concluded that serum haptoglobin and ceruloplasmin oxidase assays could be used as adjuncts for diagnosis of the inflammation in dogs.

Free access
in American Journal of Veterinary Research