Hyperphosphatasemia and concurrent adrenal gland dysfunction in apparently healthy Scottish Terriers.

OBJECTIVE: To determine causes of hyperphosphatasemia (high serum alkaline phosphatase [ALP] activity) in apparently healthy Scottish Terriers. DESIGN: Prospective case-controlled study. ANIMALS: 34 apparently healthy adult Scottish Terriers (17 with and 17 without hyperphosphatasemia). PROCEDURES: Serum activities for 3 isoforms (bone, liver, and corticosteroid) of ALP were measured. Concentrations of cortisol, progesterone, 17-hydroxyprogesterone, androstenedione, estradiol, and aldosterone were measured before and after cosyntropin administration (ie, ACTH; 5 microg/kg [2.27 microg/lb], IM). Liver biopsy specimens from 16 dogs (11 with and 5 without hyperphosphatasemia) were evaluated histologically. RESULTS: In dogs with hyperphosphatasemia, the corticosteroid ALP isoform comprised a significantly higher percentage of total ALP activity, compared with the percentage in dogs without hyperphosphatasemia (mean +/- SE, 69 +/- 5.0% and 17 +/- 3.8%, respectively). In 6 dogs with hyperphosphatasemia, but none without, serum cortisol concentrations exceeded reference intervals after ACTH stimulation. Six dogs with and 15 without hyperphosphatasemia had increased concentrations of >or = 1 noncortisol steroid hormone after ACTH stimulation. Serum ALP activity was correlated with cortisol and androstenedione concentrations (r = 0.337 and 0.496, respectively) measured after ACTH stimulation. All dogs with and most without hyperphosphatasemia had abnormal hepatocellular reticulation typical of vacuolar hepatopathy. Subjectively, hepatocellular reticulation was more severe and widespread in hyperphosphatasemic dogs, compared with that in nonhyperphosphatasemic dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Hyperphosphatasemia in apparently healthy Scottish Terriers was most likely attributable to hyperadrenocorticism on the basis of exaggerated serum biochemical responses to ACTH administration and histologic hepatic changes, but none of the dogs had clinical signs of hyperadrenocorticism.

Measurement of serum carboxyterminal cross-linked telopeptide of type I collagen concentration in dogs with osteosarcoma.

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.

Comparison of results from the semiautomated serum bone alkaline phosphatase isoenzyme assay with the periosteal alkaline phosphatase assay for use in rat models.

BACKGROUND: Assessment of bone formation activity is an important component of pharmacologic efficacy and toxicity evaluations for compounds in development for osteoporosis therapies. Antemortem biomarkers of bone formation and remodeling in rodents are uncommon. While the periosteal alkaline phosphatase (ALP) assay is a postmortem and laborious means of testing bone-building activity, the semiautomated ALP isoenzyme assay is an antemortem assay that is performed on an automated chemistry analyzer after 2 simple dilutions of the initial serum sample and a short incubation. OBJECTIVES: The goal of our investigation was to determine if the serum bone ALP (BALP) data obtained from the semiautomated ALP isoenzyme assay had a similar pattern of response when compared with the periosteal ALP (PALP) assay for use in pharmacologic screening in rats. METHODS: Serum and bone tissue samples were obtained from orchidectomized Wistar rats, a model of clinically induced osteoporosis. Subsequent bone formation was initiated via treatment with one of several compounds. In study 1, orchidectomized male rats were given either vehicle, dihydrotestosterone or a testosterone derivative subcutaneously every 4 days for 28 days. In study 2, orchidectomized male rats were given either vehicle or compounds A, B, or C by oral gavage daily for 15 days. Blood and tibias were collected at necropsy. Serum was analyzed for BALP activity using a semiautomated ALP assay. Tibias from the same rats were analyzed for PALP activity. RESULTS: Serum BALP activity paralleled PALP activity within each group when compared with the controls. CONCLUSION: Our data indicate that the semiautomated serum BALP isoenzyme assay may be used as a biomarker of bone-building potential in rat models of osteoporosis. This assay affords many advantages to investigators of musculoskeletal diseases, including the potential to measure multiple data points in a single study.

Disrupted bone metabolism in contaminant-exposed white storks (Ciconia ciconia) in southwestern Spain.

In 1998, the Aznalcóllar mine tailings dyke in southwestern Spain broke, flooding the Agrio-Guadiamar river system with acid tailings up to the borders of one of the largest breeding colonies of white storks in the western Palearctic, Dehesa de Abajo. Over the following years, a high proportion of nestlings developed leg defects not seen before the spill, prompting this study. Nestlings with deformed legs had significantly lower plasma phosphorous (P) and higher Ca:P ratios than non-deformed cohorts in the first two years, but in the third year, when more, younger birds were studied, plasma P ranged from much higher to much lower in the affected colony compared with reference birds. Coefficients of variation for phosphorous were 19% and 60%, in reference and contaminated colonies, respectively. Storks from the contaminated colony were unable to control P levels and Ca:P ratios within the narrow limits necessary for normal bone development.

Evaluation of the influence of S-adenosylmethionine on systemic and hepatic effects of prednisolone in dogs.

OBJECTIVE: To evaluate the influence of a 1,4-butanedisulfonate stable salt of S-adenosylmethionine (SAMe) administered orally on clinicopathologic and hepatic effects induced by long-term administration of prednisolone in dogs. ANIMALS: 12 healthy dogs. PROCEDURE: Following a pilot study (4 dogs), 2 groups of 4 dogs received prednisolone (2.2 mg/kg) orally once daily (84-day trial). One group received SAMe (20 mg/kg/d divided in 2 doses) for 42 days and then a placebo for 42 days; the other group received treatments in the reverse order. Before and during the trial, numerous variables were monitored, including serum total alkaline phosphatase (ALP) and glucocorticoid-induced ALP (G-ALP) activities, serum haptoglobin concentration, and total and oxidized glutathione (TGSH and GSSG) and thiobarbiturate-reacting substances (TBARS) concentrations in erythrocytes and liver tissue (days 0, 42, and 84). Hepatic specimens also were examined microscopically. RESULTS: The stable salt of SAMe was biologically available; plasma concentrations of SAMe or prednisolone were not affected by coadministration. Compared with baseline values, serum ALP and G-ALP activities and haptoglobin concentrations increased and erythrocyte GSSG and TBARS concentrations decreased with both treatments. Erythrocyte TGSH concentration decreased with the prednisolone-placebo treatment. Administration of SAMe appeared to conserve erythrocyte TGSH values and did not inhibit hepatocyte glycogen vacuolation but increased hepatic TGSH concentration and improved the hepatic tissue GSSG:TGSH ratio. CONCLUSIONS AND CLINICAL RELEVANCE: In dogs, administration of 20 mg of SAMe/kg/d may mitigate the apparent pro-oxidant influences of prednisolone but did not block development of classic clinicopathologic or histologic features of vacuolar hepatopathy.

Serum alkaline phosphatase isoenzyme profiles in phenobarbital-treated epileptic dogs.

BACKGROUND: Serum total alkaline phosphatase (AP) activity commonly is high in dogs receiving phenobarbital. Specific isoenzymes responsible for this increase are not well documented. OBJECTIVES: The purposes of this study were 1) to qualitatively and quantitatively describe serum AP isoenzymes in phenobarbital-treated dogs and 2) to monitor changes in serum AP isoenzyme activities associated with phenobarbital treatment over time. METHODS: Serum AP isoenzyme activities were determined in a cross-sectional study of 29 dogs receiving phenobarbital (duration of treatment 2 months to 6.5 years). Additionally, in a prospective study of 23 dogs, serum AP isoenzyme activities were determined before and 3 weeks, 6 months, and 12 months after the start of phenobarbital treatment. Isoenzyme activities were quantitatively determined using wheat germ lectin precipitation and levamisole inhibition, and qualitatively (ie, present or absent) evaluated using cellulose acetate affinity electrophoresis. RESULTS: In phenobarbital-treated dogs with high serum total AP activity in the cross-sectional study, the increase was due predominantly to increased activities of the corticosteroid-induced (C-AP) and liver (L-AP) isoenzymes. Prospectively, serum total AP and L-AP activities were significantly higher at 3 weeks, 6 months, and 12 months after the start of phenobarbital treatment compared with pretreatment values. Serum C-AP and bone isoenzyme (B-AP) activities were significantly higher after 6 and 12 months of treatment. B-AP accounted for only a small amount of the total AP activity. No unusual or previously unidentified AP isoenzymes were identified. CONCLUSIONS: Phenobarbital treatment was associated with increased C-AP and L-AP isoenzyme activities and with a minor increase in B-AP activity. No unique "phenobarbital-induced" isoenzyme was identified. Isoenzyme analysis does not appear to be useful for differentiating between high serum total AP due to phenobarbital therapy and other causes.

Alkaline phosphatase expression in tissues from glucocorticoid-treated dogs.

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. PROCEDURES: 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.

Kinetics of mRNA expression of alkaline phosphatase isoenzymes in hepatic tissues from glucocorticoid-treated dogs.

OBJECTIVE: To clone segments of the canine liver alkaline phosphatase (LALP) and corticosteroid-induced 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 glucocorticoid-treated dogs. Hepatic tissues and serum samples were obtained from the 6 remaining glucocorticoid-treated 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.

Semiautomated analysis of alkaline phosphatase isoenzymes in serum of normal cynomolgus monkeys (Macaca fascicularis).

Alkaline phosphatase (ALP) isoenzyme analysis, using a combination of wheat germ lectin (WGL) precipitation, levamisole inhibition and an automated p-nitrophenylphosphate assay was validated for use with serum from monkeys (Macaca fascicularis) and used to determine the activities of liver ALP (LALP), bone ALP (BALP) and intestinal ALP (IALP). Based on serial dilution studies and within-run and between-run coefficients of variation, each assay had excellent linearity and acceptable precision. In addition, liver and intestinal mucosa extracts for tissue specific alkaline phosphatases were used to confirm assay validations. Gender-specific differences for total ALP, LALP, and BALP activities were present in sera from normal monkeys between 2 and 4 years of age. Males had 1.3-fold higher total ALP and LALP activities, and 1.5-fold higher BALP activity compared with females. The majority of ALP activity in normal monkey serum was LALP isoenzyme activity, which ranged from 56.7% to 94.7% of total activity. Serum BALP activity ranged from 5.3% to 42.8%. There was negligible IALP activity in all serum samples.

Quantification of bone alkaline phosphatase in canine serum.

An assay was developed and proven accurate and precise for the quantification of canine serum alkaline phosphatase of bone origin (BAP). The assay uses wheat germ lectin (WGL) which selectively precipitates SAP but not liver alkaline phosphatase (LAP) in serum preincubated for 1 hour at 37 degrees C before conducting the assay. Although a large percentage of corticosteroid-induced alkaline phos- phatase (CAP) is also precipitated by WGL, the activity of this isoenzyme can be determined by utilizing the automated levamisole inhibition assay and BAP determined by subtraction except in those cases in which CAP is very markedly increased. Use of these two assay techniques in combination allows the quantification of LAP, BAP, and CAP activity in canine serum. In sera from adult dogs of various ages, BAP activity represents a mean of 21.27 -/+ 11.4 U/L; however, there was a statistical decrease in BAP activity with age. This allowed the determination of 95% confidence interval for a reference range dependent on age of the dog. Bone AP activity in puppies drops dramatically within the first 3 months, reaching a magnitude of activity consistent with that of the adult dog by approximately 15 months. BAP was increased over adult reference range in five of five dogs tested with osteosarcoma. This assay will now allow conducting a clinical study of the diagnostic significance of bone AP activity in neoplastic and metabolic diseases of bone.