Serum alkaline phosphatase isoenzyme activities in canine malignant mammary neoplasms with and without osseous transformation

BACKGROUND: Increased serum activity of total alkaline phosphatase (TALP) has been found in dogs with mammary neoplasms, especially malignant mixed tumors. We hypothesized that the bone isoenzyme of alkaline phosphatase (BALP), a specific indicator of osteoblastic activity and bone formation, may contribute to increased TALP in dogs with mammary neoplasms with osseous transformation. OBJECTIVE: The purpose of this study was to compare serum TALP, BALP, and other ALP isoenzyme activities in dogs with mammary malignant neoplasms with and without osseous transformation. METHODS: Twenty-one female dogs with malignant mammary neoplasms were compared with 21 clinically healthy, age-matched female control dogs. Physical, clinicopathologic (including preprandial and postprandial serum bile acids, ACTH stimulation, and low-dose dexamethasone suppression tests), radiographic, and ultrasonographic examinations were performed on all dogs with tumors to assess coexisting conditions. On the basis of histologic examination of excised tumors, dogs were further classified as having epithelial (n = 11) or mesenchymal/mixed (epithelial-mesenchymal) (n = 10) neoplasms, the latter of which had histologic and radiologic evidence of bone formation. Serum TALP, BALP, liver alkaline phosphatase (LALP), and corticosteroid-induced alkaline phosphatase (CALP) activities were measured using biochemical methods. RESULTS: Dogs with malignant mammary tumors had significantly higher (P < .05) median serum TALP (170 U/L), BALP (59 U/L), LALP (49 U/L), and CALP (24 U/L) activities, compared with control dogs (81, 32, 37, and 5 U/L, respectively). Significantly higher activities of BALP and LALP were found in dogs with epithelial neoplasms; whereas, only CALP activity was higher in dogs with mesenchymal/mixed neoplasms. There was no significant difference in TALP or isoenzyme activitities between epithelial and mesenchymal/mixed groups. CONCLUSION: BALP activity is increased in some dogs with malignant mammary tumors but does not account for the increase in TALP in dogs with neoplasms that have osseous transformation.     

Canine serum alkaline phosphatase isoenzymes detected by polyacrylamide gel disk electrophoresis

Serum alkaline phosphatase (ALP) isoenzymes were studied in normal dogs using a commercially available polyacrylamide gel disk electrophoresis kit (PAG/disk kit). Serum samples taken from the dogs were incubated with neuraminidase, after which most showed ALP isoenzymes as two characteristic stained bands. To determine the origin of each band, ALP isoenzymes of serum and tissue extracts (liver, intestine and bone) were characterized by heating, wheat germ agglutinin (WGA) and levamisole treatments. The results suggested that the band detected on the anode was liver ALP (LALP) and that the band detected on the cathode represented bone ALP (BALP), and both were corticosteroid-induced ALP (CALP). The percentage of each ALP isoenzyme to total ALP activity was estimated by densitometry. The percentage of BALP was the highest in young dogs (age<1 year, 64.7% ), and this value decreased with age. In contrast, the percentage of LALP in young dogs (22.2%) was much lower than that in middle-aged dogs (ages 1 year to 7 years, 59.3%) and old dogs (ages>7 years, 50.4%). The present results suggested that a commercially available PAG/disk kit is capable of detecting three serum ALP isoenzymes in dogs, and further that it may have clinical applications in the evaluation of ALP isoenzymes in veterinary medicine.     

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.     

Quantitative Determination of Equine Alkaline Phosphatase lsoenzymes in Foal and Adult Serum

Automated and semiautomated assays were developed and validated for the determination of equine alkaline phosphatase isoenzymes including intestinal (IALP), bone (BALP), and liver (LALP). The addition of levamisole selectively inhibited more than 97%of LALP while inhibiting only 55% of IALP. Because these percentages were highly reproducible in an automated system, the IALP activity could be calculated in a sample. Bone alkaline phosphatase isoenzyme was selectively precipitated by adding an equal volume of wheat germ agglutinin (5 mg/mL), incubating for 30 minutes at 37C, and centrifugating. The LALP activity was determined from the supernatant fluid and BALP activity was calculated by subtraction from total ALP activity. The within-run coefficient of variation for determination of BALP activity was 4.7%.These assays were used to identify and quantify the isoenzymes present in pony foal sera through the first 21 days of life, in horse foal sera before colostrum ingestion and during the first 21 days of life, and in adult horse and pony sera. Intestinal ALP activity was not found in sera of any of the foals or adult ponies or horses. A majority of serum ALP activity of newborn foals is of bone origin (80 to 92%)which decreases markedly over the first 21 days. In adults, only 17.9% (51.2 ± 18.1 U/L) of serum ALP is derived from bone. The absolute LALP activity in foal serum is similar to that in adults.     

Changes of serum alkaline phosphatase activity in dry and lactational cows

Serum alkaline phosphatase (ALP) activities were measured during dry and lactational periods to investigate the influence of lactation on serum ALP activity in cows. Higher levels of serum ALP activity were seen in lactational periods than in dry periods. The serum activities of bone-specific ALP (BALP), liver ALP (LALP), tartrate resistant acid phosphatase (TRAP) and aspartate aminotransferase also increased in lactational periods. ALP activities in the bone extract and in whey were decreased at similar rates by the addition of lectin. Moreover, since the ALP band in whey was observed to have the same migration in polyacrylamide gel (PAG) disk electrophoresis as that of the bone extract, analysis of ALP isoenzymes by lectin affinity or PAG disk electrophoresis could not distinguish ALP originating from the mammary gland from that of bone. In this study, it was clear that the increased level of serum ALP activity was due to increases of BALP and LALP in lactational periods. However, the extent of the influence of ALP originating from the mammary glands on serum ALP activity was unknown. Judging from changes of BALP and TRAP activities in the serum and the correlation between the both, it was guessed that ALP originating from the mammary glands influenced serum ALP activity.     

Subcellular location of corticosteroid-induced alkaline phosphatase in canine hepatocytes

Dogs received either 4 mg/kg of prednisone or sterile saline daily for 32 days. Serum samples were assayed every 4 days for total alkaline phosphatase (ALP) and corticosteroid-induced ALP isoenzyme (CIALP) activity. The initial and major increase of serum ALP was attributed to the liver isoenzyme of ALP (LALP), however, CIALP began to increase by day 8 and was significantly increased by day 24. Prior to treatment and on day 32, sections of liver from control and prednisone-treated dogs were stained for ALP activity after blocking the staining activity of LALP with levamisole. The staining activity of CIALP was compared to the staining activity of LALP in liver sections from control dogs and from dogs in which the bile duct was ligated. It was determined that CIALP was located in that area of the hepatocyte membranes which comprise the bile canaliculi.     

Doxorubicin and BAY 12-9566 for the treatment of osteosarcoma in dogs: a randomized, double-blind, placebo-controlled study

BACKGROUND: This study was designed to assess the efficacy of a matrix metalloproteinase inhibitor in prolonging posttreatment survival for dogs with appendicular osteosarcoma after treatment with amputation and doxorubicin chemotherapy. HYPOTHESIS: Survival will be prolonged in dogs receiving BAY 12-9566. ANIMALS: The study included 303 dogs with appendicular osteosarcoma. METHODS: Dogs were treated with doxorubicin (30 mg/m2) every 2 weeks for 5 treatments starting 2 weeks after amputation. Dogs were randomly allocated to receive a novel nonpeptidic biphenyl inhibitor of matrix metalloproteinases (MMPs, BAY 12-9566; 4-[4-4-(chlorophenyl)phenyl]-4-oxo-2S-(phenylthiomethyl) butanoic acid) or placebo after doxorubicin chemotherapy. RESULTS: Median survival for all 303 dogs was 8 months; and 1-year, 2-year, and 3-year survival rates were 35%, 17%, and 9%, respectively. Treatment with BAY 12-9566 did not influence survival. Multivariate analysis revealed that increasing age (P = .004), increasing weight (P = .006), high serum alkaline phosphatase (ALP) (P = .012) and high bone ALP (P < .001) were independently associated with shorter median survival times. Additional analyses on available data indicated that as the number of mitotic figures in the biopsy increased (P = .013), and as plasma active MMP-2 concentrations increased (P = .027), the risk of dying increased. CONCLUSIONS AND CLINICAL IMPORTANCE: Doxorubicin is an effective adjuvant to amputation in prolonging survival for dogs with appendicular osteosarcoma.     

Solubilization of liver alkaline phosphatase isoenzyme during cholestasis in dogs.

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.     

Purification and comparison of corticosteroid-induced and intestinal isoenzymes of alkaline phosphatase in dogs

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 un-interrupted system using DEAE-cellulose, concanavalin A-agarose, 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.     

A comparison of two techniques for the determination of serum bone-specific alkaline phosphatase activity in dogs

Bone-specific alkaline phosphatase (BALP) shows potential as a marker of bone formation in the dog. Recent studies have indicated that serum BALP may provide a useful, non-invasive indicator of skeletal health in dogs, and as a diagnostic and prognostic marker in the management of dogs with musculoskeletal or metabolic disorders. Two assay techniques (one based on wheatgerm lectin precipitation followed by a simple enzymatic reaction, the second on a specific enzyme-linked immunoassay) were used to measure serum levels of BALP in 35 dogs of different ages.As expected, BALP concentrations decreased with age. For the enzymatic assay, mean (+/-SD) serum concentrations of BALP activities were 100.3 (+/-11.6) U/liter in dogs under 1 year of age, 25.3 (+/-6.8) U/L in dogs 1 to 2 years of age, 16.5 (+/-7.3) U/L in dogs 2 to 3 years of age, 14.3 (+/-5.6) U/L in dogs 3 to 7 years of age, and 12.3 (+/-4.8) U/L in dogs aged 8 years and older. Corresponding results from the immunoassay were 56.3 (+/-9.8) U/L, 10.7 (+/-4.5) U/L, 7.0 (+/-2.5) U/L, 6.7 (+/-3.6) U/L and 7.0 (+/-2.9) U/L. There was excellent correlation between the results from the two assay techniques (r = 0. 96; P < 0.0001). The correlation between BALP and total ALP activities was poor (r = 0.20 for enzymatic BALP, r = 0.31 for immunoreactive BALP), indicating that total ALP should be considered unreliable as an indicator of BALP activity in canine serum. The immunoassay demonstrated acceptable (13 per cent) cross-reactivity with the liver isoform of ALP.The commercial immunoassay kit is simple and provides fast results. Although the wheatgerm lectin/enzymatic technique is preferred in situations where the activities of all three isoforms of ALP are required, the immunoassay should be considered whenever the activity of BALP is the focus of interest.     

Incidence and prognostic importance of lymph node metastases in dogs with appendicular osteosarcoma: 228 cases (1986-2003)

OBJECTIVE: To determine the incidence of regional lymph node metastasis in dogs with appendicular osteosarcoma and determine whether regional lymph node metastasis was associated with shortened disease-free interval or survival time. DESIGN: Retrospective study. ANIMALS: 228 dogs with appendicular osteosarcoma in which regional lymph nodes were examined histologically at the time of limb amputation. PROCEDURE: Information collected from the medical records included signalment; affected site; initial serum alkaline phosphatase activity; whether treatment involved adjuvant chemotherapy and, if so, chemotherapeutic agents administered and number of treatments; disease-free interval; and survival time. RESULTS: 10 (4.4%) dogs had histologic evidence of regional lymph node metastasis at the time of amputation. Median disease-free interval for dogs without regional lymph node metastasis (238 days; range, 0 to 1,067 days) was significantly longer than median disease-free interval for dogs with regional lymph node metastasis (48 days; range, 2 to 269 days). Median survival time for dogs without lymph node metastasis (318 days; range, 20 to 1,711 days) was significantly longer than median survival time for dogs with lymph node metastasis (59 days; range, 19 to 365 days). CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that regional lymph node metastasis is rare in dogs with appendicular osteosarcoma but that dogs with lymph node metastasis have a poorer prognosis than do dogs without.     

Alternating carboplatin and doxorubicin as adjunctive chemotherapy to amputation or limb-sparing surgery in the treatment of appendicular osteosarcoma in dogs

Thirty-two dogs with appendicular osteosarcoma treated by amputation or limb sparing had adjuvant chemotherapy of alternating doses of carboplatin (300 mg/m2 IV) and doxorubicin (30 mg/m2 IV) every 21 days for a total of 3 cycles. Efficacy, toxicity, and previously identified prognostic factors for osteosarcoma were evaluated. The median progression free survival was 227 days (range 180-274), and the median overall survival was 320 days (range 153-487). The 1-year survival rate was 48%, and the 2-year survival rate was 18%. Age, sex, surgical procedure, and alkaline phosphatase activity above the reference ranges were not prognostic for survival. There was minimal toxicity associated with the chemotherapy. This protocol could be useful for the adjuvant treatment of appendicular osteosarcoma of dogs.     

Association of Blood Monocyte and Lymphocyte Count and Disease‐Free Interval in Dogs with Osteosarcoma

Background: Identification of biomarkers that predict outcomes in dogs with osteosarcoma (OSA) would be valuable to veterinarians and owners. Leukocyte numbers in peripheral blood are associated with outcomes in some types of cancer in humans. Hypothesis/Objectives: We hypothesized that increased numbers of monocytes would be associated with reduced disease‐free interval (DFI) in dogs with OSA. Animals: Medical data from 69 dogs with appendicular OSA treated with amputation and chemotherapy were selected for study. Methods: Retrospective study. Statistical associations were assessed by univariate and multivariate analysis. Information about DFI and leukogram values, tumor location, and serum alkaline phosphatase was abstracted from the medical record. Results: Higher numbers of circulating monocytes (>0.4 × 10³ cells/μL) and lymphocytes (>1.0 × 10³ cells/μL) before treatment were found to be significantly (P < .05) associated with shorter DFI in dogs with OSA. Other parameters associated with poor outcomes were increased alkaline phosphatase, primary tumor location, and age. Conclusion and Clinical Importance: These results indicated that pretreatment evaluation of monocyte and lymphocyte counts provided prognostic information for dogs with appendicular OSA. Notably, most animals in this study had monocyte counts within the normal reference range, indicating that variations within the reference range of leukocyte values might also have prognostic significance.     

The course of some bone remodelling plasma metabolites in healthy horses and in horses offered a calcium-deficient diet

An inquiry was carried out to assess the concentrations of plasma metabolites related to bone remodelling in 21 saddle horses of Warmblood breed aged 4-26 years, five draught horses of Ardennes breed aged 4-10 years, and 10 Ardennes foals aged 9-11 months. They were fed according to normal feeding practice in Belgium. The changes in some bone remodelling plasma metabolite concentrations were studied when an unbalanced diet was offered and later corrected for four Warmblood horses. Bone formation was evaluated by bone alkaline phosphatase (BALP), total alkaline phosphatase (TALP) and osteocalcin (bone gla-protein, OC). Bone resorption was assessed by hydroxyproline (HYP). Total calcium, ionized calcium, phosphorus (P) and 25-hydroxyvitamin D3 [25-(OH)D] concentrations were more or less constant. The comparison of four bone remodelling factors between the Ardennes and Warmblood horses showed higher concentrations in the Ardennes breed. Bone marker concentrations decreased according to age. The correction of the unbalanced Ca : P diet induced inconsistent effects at plasma level. The interpretation of the different bone parameters appeared to be difficult if not associated with other parameters such as a complete anamnesis and clinical examination of the animal in addition to dietary evaluation.     

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.     

Serial evaluation of protein C and antithrombin in dogs with sepsis

BACKGROUND: Protein C (PC) and antithrombin (AT) activities are decreased in humans with severe sepsis, and persistent changes are associated with decreased survival. In dogs with sepsis, PC and AT have been shown to be decreased at the time of diagnosis. HYPOTHESIS: PC and AT activities change significantly over time in dogs with sepsis and may be related to outcome. ANIMALS: Twelve dogs with naturally occurring sepsis. METHODS: Blood was collected from 12 dogs with sepsis, defined as histopathologic or microbiologic confirmation of infection and two of the following: hypo- or hyperthermia, tachycardia, tachypnea, leukopenia, leukocytosis, or >3% bands. The time of 1st sampling was considered day 1 and sampling was repeated every 24 hours for 5 days or until discharge or death. Changes over time were analyzed by ANOVA with repeated measures, and the association between PC and AT and outcome was determined by a 2-equation treatment effects model. RESULTS: Nine dogs and 11 dogs had decreased PC and AT activity on day 1, respectively (mean PC, 66.0 +/- 25.8%; mean AT, 48.1 +/- 16.5%). PC activity significantly decreased from day 1 to day 2 (P= .001), then increased over time. Changes in PC (P < .001) and AT (P < .001) over time were likely associated with outcome with nonsurvivors having lower PC and AT activities than survivors. CONCLUSION: Results of this preliminary study show that PC and AT activities change significantly over time in dogs with sepsis and both are likely related to survival.     

TP53 gene mutations in canine osteosarcoma

Objective— To investigate mutations of the TP53 gene in canine osteosarcoma (OS).
Study Design— Clinical historic cohort study.
Animals— Client-owned dogs.
Methods— OS (n=59) were screened for mutations of the complete TP53 gene using polymerase chain reaction and the mutation was analyzed by single-strand conformational polymorphism. Clinical outcome of dogs with TP53-mutated OS were compared with dogs with OS without a mutation after complete surgical excision of the primary tumor.
Results— TP53 gene mutations were observed in 24 of 59 (40.7%) OS; 3 mutated OS had 2 mutations. The alterations consisted mainly of point mutations (74%). Dogs with mutated OS had a significantly shorter survival time (ST) after surgery than dogs with normal tumor TP53 gene expression ( P =.03). Other significant prognosticators for ST and disease-free interval included elevated serum alkaline phosphatase ( P <.01) and tumor grade ( P =.01).
Conclusion— TP53 genetic mutations are common in canine OS and may have a prognostic value.
Clinical Relevance— Mutations of the TP53 gene may influence survival and should be considered when evaluating canine OS.     

Tissue sources of serum alkaline phosphatase in 34 hyperthyroid cats: a qualitative and quantitative study

The concentration of serum alkaline phosphatase (SALP) is commonly elevated in hyperthyroid cats. Agarose gel electrophoresis, in tris -barbital-sodium barbital buffer, with and without the separation enhancer neuraminidase, was used to investigate the sources of the constituent isoenzymes of SALP in serum samples from 34 hyperthyroid cats, comparing them to sera from five healthy cats and to tissue homogenates from liver, kidney, bone and duodenum. Contrary to previous reports, treatment of serum with neuraminidase made differentiation of the various isoenzymes more difficult to achieve. A single band corresponding to the liver isoenzyme (LALP) was found in 100 per cent of healthy cats. Eighty-eight per cent of the hyperthyroid cats showed two bands, corresponding to the liver and bone (BALP) isoenzymes while 12 per cent showed a LALP band alone. In hyperthyroid cats, there was a significant correlation between the serum L-thyroxine concentrations and the SALP concentrations. These findings suggest pathological changes in both bone and liver in most cases of feline thyrotoxicosis.