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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

Summary

The importance of accurate quantitative blood biochemical analysis for the diagnosis and management of disease is recognized by most veterinarians. In recent years, several biochemical analyzers have become available for the veterinary market. One of these analyzers was evaluated for its suitability in measuring several biochemical variables—alkaline phosphatase, urea nitrogen, creatinine, glucose, alanine transaminase (dog and cat only), and aspartate transaminase (horse only)—in dogs, cats, and horses. Instrument within-day precision ranged from 1.0 to 7.1%, and between-day precision ranged from 1.6 to 7.4%. During the 6-month period of the study, the analyzer required recalibration for only 1 analyte (creatinine).

Concentrations of individual analytes were similar when blood (collected in anticoagulant), plasma, and serum were assayed in parallel. The accuracy of the analyzer, as measured by correlation to a reference method, ranged from 0.861 for creatinine in horses to > 0.950 for each of the other analytes in the 3 species. Mean values for each analyte were similar, except for alkaline phosphatase, which had consistently lower values by use of the analyzer method. A data base was established for reference values in each species.

Free access
in Journal of the American Veterinary Medical Association

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)

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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 establish reference values for a panel of serum markers of bone turnover in dogs of various ages.

Animals

Dogs in 4 age groups (0 to 1 year; 1 to 2 years; 3 to 7 years; > 8 years).

Procedure

Serum concentrations of the carboxyterminal propeptide of type-I procollagen (PICP) and the aminoterminal propeptide of type-I procollagen (PINP), both markers of type-I collagen synthesis (hence, bone formation), were measured by use of commercial human radioimmunoassay kits. Serum concentrations of the carboxyterminal cross-linked telopeptide of type-I collagen (ICTP), a marker for type-I collagen breakdown (hence, bone resorption), also were measured by use of a commercial human radioimmunoassay kit. Serum osteocalcin (OC) concentrations and alkaline phosphatase (ALP) isoenzyme activities were measured by use of techniques developed specifically for dogs.

Results

As expected, the highest values for all of the markers were found in young dogs (< 12 months old). Concentrations of OC and ICTP decreased with age, and were lowest in dogs > 8 years old. Total ALP and bone-specific ALP activities initially decreased with age, then increased in dogs > 8 years old.

Conclusions and Clinical Relevance

Serum markers of bone turnover may be useful diagnostic and prognostic tools for management of dogs with musculoskeletal disorders. (Am J Vet Res 1998;59:250–254)

Free access
in American Journal of Veterinary Research

SUMMARY

Corticosteroid-induced alkaline phosphatase (calp) and intestinal alkaline phosphatase (ialp) from dogs were purified to homogeneity, as determined by polyacrylamide gel electrophoresis. Purification involved an uninterrupted system using deae-cellulose, concanavalin Aagarose, and monoclonal antibody affinity columns. The monoclonal antibody was prepared by use of ialp as the antigen. The 2 isoenzymes were compared, using molecular weight determinations, amino acid analyses, peptide mapping, N-terminal sequencing of the first 10 amino acids, carbohydrate analyses, and recognition by anti-ialp)monoclonal antibody. The data indicated that canine ialp and (calp are identical with regard to recognition by monoclonal antibody and N-terminal amino acid sequence, nearly identical in amino acid content and peptide maps, but different in carbohydrate content. It was concluded that (calp is a product of the same gene as ialp and that differences in glycosyl transferase activities between liver and intestines or the presence of glycosidase activities in or around the intestinal mucosae result in the marked difference in carbohydrate content.

Free access
in American Journal of Veterinary Research

Objective

To investigate intramammary infections in llamas, identify the pathogens responsible, and determine whether effects of intramammary infection could be detected by use of mastitis indicator tests commonly used for cows.

Design

Observational study.

Animals

100 llamas on 10 farms.

Procedure

Milk samples were evaluated by bacterial culturing and by determination of somatic cell count (SCC), using direct microscopic and automated counting methods, California Mastitis Test score, pH, and N-acetyl-β-d-glucosaminidase activity. Correlation coefficients were determined among the various mastitis indicator tests, and test results were determined for milk from infected and uninfected glands.

Results

Evidence of intramammary infection was evident in 76 of 369 (21%) milk samples, with 54 of 94 (57%) llamas having at least 1 infected gland. Staphylococcus sp other than Staphylococcus aureus were the predominant pathogens. None of the llamas had clinical signs of mastitis, and significant differences were not detected in SCC, California Mastitis Test score, pH, or N-acetyl-β-d-glucosaminidase activity between infected and uninfected samples. California Mastitis Test scores were negative or trace for 307 of 313 (98%) samples, and SCC were low. In contrast, pH and N-acetyl-β-d-glucosaminidase activity of milk from uninfected glands were higher than values reported for milk from uninfected cows, and neither variable was significantly correlated with the number of somatic cells in samples of llama milk.

Clinical Implications

Although intramammary infections develop in llamas, inflammation (mastitis) appears to be rare. Values for mastitis indicator tests used for cows cannot be directly extrapolated to llamas. Subclinical mastitis is apparently not an important problem in llamas in the United States. (J Am Vet Med Assoc 1996;209:1457–1463).

Free access
in Journal of the American Veterinary Medical Association

Abstract

Objective—To evaluate the usefulness of carboxyterminal cross-linked telopeptide of type I collagen (ICTP) concentrations for screening dogs for the presence of osteosarcoma.

Sample Population—32 client-owned dogs with osteosarcoma (27 dogs with osteosarcoma of the appendicular skeleton and 5 dogs with osteosarcoma of the axial skeleton) and 44 non–tumor-bearing control dogs.

Procedures—Serum was obtained from blood samples collected from dogs with osteosarcoma and from clinically normal dogs. The serum ICTP concentration was determined by use of a commercially available radioimmunoassay for ICTP.

Results—Mean ± SD serum ICTP concentration in the tumor-bearing dogs was 7.32 ± 2.88 ng/mL, and in clinically normal dogs, it was 6.77 ± 2.31 ng/mL; values did not differ significantly. Mean serum ICTP concentration in dogs with appendicular osteosarcoma, compared with that of clinically normal dogs, was not significantly different. Mean serum ICTP concentration in dogs with axial skeletal tumor location was 10.82 ± 2.31 ng/mL, compared with a value of 6.73 ± 2.28 ng/mL in dogs with appendicular osteosarcoma.

Conclusions and Clinical Relevance—On the basis of the results of this study, serum ICTP concentrations are not a clinically useful screening tool for the detection of appendicular osteosarcoma in dogs. Despite the observation that serum ICTP concentration was higher in dogs with axial osteosarcoma than in clinically normal dogs, serum ICTP concentration determination is not a suitable screening test for osteosarcoma.

Full access
in American Journal of Veterinary Research

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

Objective—

To determine whether alkaline phosphatase activity in dogs with appendicular osteosarcoma can be used as a prognostic indicator.

Design—

Retrospective study.

Animals—

75 dogs with appendicular osteosarcoma.

Procedure—

Serum total alkaline phosphatase (TALP) and bone-specific alkaline phosphatase (BALP) activities were determined from archival serum samples obtained at various times during treatment of appendicular osteosarcoma and follow-up evaluations. Associations among activities of TALP and BALP and survival and disease-free intervals, percentage of bone length involved with tumor, histologic subtype, and method of surgical treatment were evaluated.

Results—

High activities of TALP and BALP before surgery were significantly associated with shorter survival and disease-free intervals in dogs undergoing surgery (amputation or limb-sparing procedure) and adjuvant chemotherapy. Activity of BALP significantly decreased in 29 dogs for which postoperative samples were available. Failure of BALP activity to decrease after surgery was correlated with shorter survival and disease-free intervals.

Clinical Implications—

Activities of TALP and BALP in serum are important prognostic factors for appendicular osteosarcoma in dogs. Prognostic factors may help clinicians initiate more aggressive treatment for dogs that are at higher risk of death or relapse. (J Am Vet Med Assoc 1998:213:1002-1006)

Free access
in Journal of the American Veterinary Medical Association