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.     

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.     

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.     

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.     

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.     

Canine serum thermostable alkaline phosphatase isoenzyme from a dog with hepatocellular carcinoma

A dog histopathologically diagnosed with hepatocellular carcinoma (HCC) showed very high serum alkaline phosphatase (ALP) activity. A supernatant of ascitic fluid and tumor tissue extracted from the dog also showed much higher ALP activity than normal. ALP isoenzyme analysis of samples was performed using polyacrylamide gel disk electrophoresis, and a wide, broad abnormal band was observed. By various treatments, the abnormal band showed thermostability, which is a characteristic of tumor-associated ALP that has only been reported in humans. The thermostable ALP isoenzyme was not found in sera from 39 dogs with several types of tumor that originated from the liver, except for HCC, nor was it found in 10 dogs with hepatic diseases that did not include hepatic tumors. The thermostable ALP isoenzyme seemed to be associated with canine HCC.     

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.     

Serum bile acid analysis in dogs with experimentally induced cholestatic jaundice

Serum bile acid (SBA) concentration was determined weekly for 4 weeks in dogs with experimentally induced hyperbilirubinemic liver disease. Obstructive jaundice was created in 6 dogs by surgical ligation of the common bile duct, and hepatocellular jaundice was created in 6 sham-operated dogs by administration of dimethylnitrosamine; 6 other sham-operated dogs served as controls. Serum bile acid concentration increased rapidly after bile duct ligation (from 0.6 +/- 0.1 to 69.2 +/- 15.3 mumol/L at 3 days), peaked at 14 days (247.8 +/- 54.1 mumol/L), and then gradually decreased (179.9 +/- 27.1 mumol/L at 28 days). Serum bile acid concentration in dimethylnitrosamine-treated dogs increased more gradually to 38.9 +/- 10.7 mumol/L at 28 days, at which time the serum bilirubin concentration was comparable with that of bile duct-ligated dogs. Mean total SBA values in bile duct-ligated dogs were significantly (P less than 0.01) higher than those in control and dimethylnitrosamine-treated dogs at days 3 through 28, with no overlap of individual values. Serum bile acid concentration at day 28 correlated positively (P less than 0.01) with cholestasis and bile duct proliferation observed in liver biopsy specimens, but did not correlate with necrosis or inflammation. Serum bile acid concentration also correlated positively (P less than 0.01) with serum bilirubin and cholesterol concentrations and with serum alkaline phosphatase and alanine transaminase activities. Results of the study reported here indicated a relationship between SBA concentration and cholestasis in dogs; extrahepatic bile duct obstruction resulted in the highest SBA values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alkaline phosphatase isoenzymes in feline serum using an agarose gel alkaline phosphatase kit method.

Total serum alkaline phosphatase (ALP) activity is the product of the combined activity of isoenzymes from a number of tissue sources. In this study, a commercially available kit for electrophoretic separation of ALP isoenzymes in an agarose gel was used to separate ALP isoenzymes in feline tissue extracts and serum. Five separate bands of ALP activity were identified. These bands were numbered 1 to 5 with band 1 having the most anodal migration. The tissue of origin corresponding to the migration position of the isoenzymes are as follows: Band 3 was the liver isoenzyme, band 4 was the bone isoenzyme and ALP isoenzymes of both intestine and kidney migrate in the position labelled band 5. Band 1 appears to be related to albumin and does not represent true ALP activity. The tissue source of band 2 (a and b) was not identified. Serum ALP activity of mature, healthy cats is primarily of liver origin. Immature cats (< 1 year of age) have a greater proportion of the bone isoenzyme in the serum.     

Prognostic significance of serum alkaline phosphatase activity in canine appendicular osteosarcoma

Sixty-one dogs with appendicular osteosarcoma were treated with amputation and chemotherapy of cisplatin and doxorubicin. Serum samples were obtained before and after treatment for determination of total alkaline phosphatase (TALP) activity as well as the activities of the constituent bone (BALP), liver (LALP), and corticosteroid-induced (CALP) isoenzymes. The relationship between alkaline phosphatase activities and survival was examined by Cox proportional hazards regression analysis and Kaplan-Meier log rank analysis. Mean activity of TALP, BALP, and LALP decreased significantly after treatment (P < .001). TALP and LALP activities before treatment were significantly correlated with survival (P = .006 and .001, respectively). The correlation between BALP activity before treatment and survival approached significance (P = .054). CALP activity and TALP, BALP, and LALP activities after treatment were not significantly correlated with survival. Dogs with normal pretreatment TALP and BALP activities survived significantly longer than dogs with increased pretreatment activities (P = .001 and .003, respectively). Median survival times for dogs with normal or increased TALP activities before treatment were 12.5 and 5.5 months, respectively; and median survival times for dogs with normal or increased BALP activities before treatment were 16.6 and 9.5 months, respectively. In the design of future clinical trials involving dogs with osteosarcoma, consideration should be given to stratifying the randomization according to alkaline phosphatase activity. In addition, alkaline phosphatase activity should be a factor considered by clinicians attempting to tailor the aggressiveness of adjuvant chemotherapy to the needs of individual patients or owners.     

Effect of otic medications containing glucocorticoids on liver function test results in healthy dogs

Otic medications containing triamcinolone or dexamethasone were administered twice daily for 21 days to 2 groups of 4 healthy dogs each. Serum alkaline phosphatase, gamma-glutamyltransferase, alanine transaminase, and aspartate transaminase activities, and serum bile acid and lipoprotein-X concentrations were assayed for 35 days. Increased serum activities for gamma-glutamyltransferase, alkaline phosphatase, and alanine transaminase were detected by day 7 and peaked at day 21. Increases were greater in dogs given the dexamethasone-containing medication. Enzyme activity returned to baseline by day 35. Serum aspartate transaminase activity and bile acid and lipoprotein-X concentrations did not increase.     

Bile acid concentrations in the diagnosis of hepatobiliary disease in the dog

The clinical usefulness of measuring serum bile acid concentrations as a diagnostic test for hepatobiliary disease, was examined in 150 dogs that were suspected of having hepatic disease. Serum values of total bilirubin (TB), alkaline phosphatase (ALP), alanine transaminase (ALT), and albumin were also measured. Fasting serum bile acid (FSBA) values were determined, using a solid-phase radioimmunoassay for total conjugated bile acids or a direct enzymatic spectrophotometric method. A definitive diagnosis was established by histologic examination of the liver. On the basis of histologic findings, dogs were assigned to groups (1 to 8, respectively) including: extrahepatic bile duct obstruction, cirrhosis, portal systemic vascular anastomosis (PSVA), hepatic necrosis, intrahepatic cholestasis, steroid hepatopathy, neoplasia, and secondary disease. Dogs in group 8 had no morphologic evidence of hepatobiliary disease or had mild hepatic lesions. Test efficacies of FSBA, TB, ALP, ALT, and albumin were expressed using 4 indices: sensitivity, specificity, and positive-predictive and negative-predictive values. The diagnostic efficacy of FSBA was examined alone and in combinations with the other tests. There was wide overlapping of FSBA values among dogs in groups 1 to 7, and there was wide overlapping of ALT and ALP values among dogs in all groups. The specificity of FSBA for the diagnosis of liver disease exceeded 90% at values greater than or equal to 30 mumol/L and reached 100% at greater than or equal to 50 mumol/L. Individual liver tests with the best sensitivity for each group were:FSBA and ALP for extrahepatic bile duct obstruction; FSBA for cirrhosis and PSVA; ALT for hepatic necrosis; and ALP for intrahepatic cholestasis, steroid hepatopathy, and neoplasia. Combinations of tests with the best sensitivity for each group were: FSBA + ALP for extrahepatic bile duct obstruction; FSBA + ALT for cirrhosis and PSVA; FSBA + ALT and TB + ALT for hepatic necrosis; and FSBA + ALP for intrahepatic cholestasis, steroid hepatopathy, and neoplasia. Individual tests had the best sensitivity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diagnostic evaluation of canine serum alkaline phosphatase by immunochemical means and interpretation of results.

Sera of several canine patients contained an isoenzyme of alkaline phosphatase (ALP) that resembled intestinal ALP with respect to heat inactivation, L-phenylalanine inhibition, and sensitivity to anti-canine intestinal ALP antibody, but differed with regard to the electrophoretic migration. The electrophoretic mobility of the isoenzyme was slightly cathodal than that of hepatic ALP, and its migration was reduced, similar to that of hepatic isoenzyme after neuraminidase treatment. This isoenzyme, which could be corticosteroid induced, was in the sera of numerous dogs with hepatobiliary disorders and was different from the hepatic isoenzyme that appeared in the sera of dogs with acute hepatitis, based on anti-canine intestinal ALP antibody interaction, heat inactivation, and electrophoretic migration.     

Plasma bile acid elevation following CCI4 induced liver damage in dogs, sheep, calves and ponies.

Plasma bile acid concentration was determined in normal dogs,sheep, calves and ponies for three days before and six days after liver damage, induced by carbon tetrachloride. In all species, a significant increase in plasma bile acid concentration was associated with a concomitant significant increase in plasma sorbitol dehydrogenase and transferase activity. Plasma bilirubin also significantly increased in all animals except the dogs. Results suggested that plasma bile acid levels could be used to test liver function in domestic animals.     

The effect of training stress on enzyme activity in working dogs

The activities were studied in five kinds of enzymes (aspartate aminotransferase - AST, alanine aminotransferase - ALT, lactate dehydrogenase - LD, the thermally stable fraction of lactate dehydrogenase - LD-1, and alkaline phosphatase - ALP) of 30 male dogs. The dogs, divided into two age categories, were studied during a long-continued training (130 days). Both transaminases exhibit characteristic changes in the activity, with a depression at the beginning between the 30th and 40th days of training, followed by a slow increase in AST and by a rapid increase in ALT, continuing until the end of the training period. A statistically significant activity pattern was recorded in LD: the activity declined continuously in both age groups of dogs. LD-1 exhibited an activity depression continuing until the 70th day of training, followed by an increase which reached statistical significance towards the end of the training. ALP activity varied regularly, but always remained significantly below the starting values. The enzymatic activities can be used as partial tests during the scientific management of the training of dogs in relation to the physiological and pathophysiological processes in the bodies of the dogs subjected to the training stress.     

Effects of long-term phenobarbital treatment on the liver in dogs

Long-term administration of phenobarbital has been reported to cause hepatic injury in dogs. Phenobarbital induces hepatic enzymes, and it may be difficult to distinguish the effect of enzyme induction on serum liver enzyme activities from actual hepatic damage. The hepatotoxicity of phenobarbital and the impact of enzyme induction on serum liver enzyme activity were investigated prospectively in 12 normal dogs. Phenobarbital was administered for 29 weeks at 5 mg per kilogram of body weight (range, 4.8— 6.6 mg/kg) PO q12h, resulting in therapeutic serum phenobarbital concentrations (20–40 μg/mL). Serum alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate transaminase (AST), γ-glutamyltransferase (GGT), fasted bile acids (fBA), total bilirubin, and albumin were determined before and during treatment. Lateral abdominal radiographs, abdominal ultrasounds, and histopathologic examinations of liver tissue obtained by ultrasound-guided biopsy were performed before and during treatment. Radiographs revealed a moderate increase in liver size in most dogs. Ultrasonographic examination revealed no change in liver echogenicity or architecture. No evidence of morphologic liver damage was observed histopathologically. ALP and ALT increased significantly ( P < .05), GGT increased transiently, and albumin decreased transiently during the study. There were no significant changes in AST, bilirubin, and fBA. These results suggest that increases in serum ALP, ALT, and GGT may reflect enzyme induction rather than hepatic injury during phenobarbital treatment in dogs. Serum AST, fBA, and bilirubin, and ultrasonographic evaluation of the liver are not affected by the enzyme-inducing effect of phenobarbital and can therefore be helpful to assess liver disease in dogs treated with the drug.     

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.     

Effects of long-term primidone and phenytoin administration on canine hepatic function and morphology

Primidone, phenytoin, or phenytoin and primidone in combination were given to healthy Beagle dogs for 6 months. Serum biochemical changes in dogs given primidone alone or phenytoin and primidone in combination for the entire 6-month test period included increased activities of alanine aminotransferase, alkaline phosphatase (AP), and gamma-glutamyltransferase, and decreased concentrations of albumin and cholesterol. Changes in dogs given phenytoin alone were limited to increased AP activity and decreased albumin concentration. Sulfobromophthalein excretion and conjugated bile acid concentration were within normal limits. All dogs given primidone alone or phenytoin alone remained clinically healthy throughout the treatment period. Three of 8 dogs given both drugs in combination became clinically ill after 9, 14, and 15 weeks of treatment, and were euthanatized. Two of the dogs developed clinical jaundice. In addition to the serum biochemical abnormalities observed in clinically healthy dogs, these dogs developed hyperbilirubinemia, delayed sulfobromophthalein excretion, and increased conjugated bile acid concentrations. Histologic examination of the liver showed intracanalicular casts of bile pigment typical of intrahepatic cholestasis in all 3 dogs. Histologic findings characteristic of treated dogs included hepatocellular hypertrophy attributable to hyperplasia of the smooth endoplasmic reticulum. Single-cell necrosis and multifocal lipidosis were observed in individuals of all treatment groups. Electron microscopy of the liver showed dilated bile canaliculi and damaged sinusoidal epithelium in dogs given both drugs. The elevated serum AP activity, associated with anticonvulsant drug therapy, was found to be exclusively the liver isoenzyme by cellulose acetate electrophoresis. The hepatic AP was localized to primarily the canalicular membranes by enzyme histochemistry. There was a statistically significant positive correlation between the AP activities of liver and serum. The results of this study indicate that long-term administration of anticonvulsant drugs to dogs is associated with clinical, serum biochemical, and histologic evidence of hepatic dysfunction. High drug dosage contributed most to abnormal serum biochemical test results, and combining phenytoin with primidone was responsible for more severe electron microscopic lesions of the liver of surviving dogs and for the death of 3 dogs.