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.     

Alkaline phosphatase and alkaline phosphatase isoenzymes in the cat

Feline alkaline phosphatase and alkaline phosphatase isoenzymes have been studied in tissue and serum. Alkaline phosphatase from various organs was quantitated and then subjected to cellulose acetate electrophoresis. The effects of bile duct ligation, prednisolone treatment and phenobarbital treatment on serum alkaline phosphatase was measured. The diagnostic importance of feline serum alkaline phosphatase levels is discussed in light of the results of this and other studies.     

Alkaline phosphatase and its isoenzymes in the tissues and sera of normal dogs

The total alkaline phosphatase (AP) activity and the pattern of its isoenzymes were studied in the tissues and sera of normal adult dogs. Small intestine mucosa showed the greatest total AP activity followed by kidney, bone, pancreas, liver, lung, skeletal muscle and heart muscle. After separation by agarose gel electrophoresis, each tissue showed only one isoenzyme except lung which showed two. The tissue isoenzymes, in decreasing order of migration distance towards the anode, were as follows: fast lung isoenzyme, liver or slow lung isoenzyme, the group consisting of skeletal muscle, bone, small intestine and pancreas isoenzymes and, finally, the kidney isoenzyme. Two isoenzymes occurred in serum. The major band corresponded to liver and the slow lung isoenzyme, while the minor band was considered to be the corticosteroid-induced isoenzyme, previously thought to be absent from normal serum.The AP isoenzyme patterns in lung and skeletal muscle and the presence of an isoenzyme migrating an identical distance to the corticosteroid-induced isoenzyme do not appear to have been reported before in normal dogs.     

Alkaline phosphatase and its isoenzymes in the tissues and sera of normal dogs

The total alkaline phosphatase (AP) activity and the pattern of its isoenzymes were studied in the tissues and sera of normal adult dogs. Small intestine mucosa showed the greatest total AP activity followed by kidney, bone, pancreas, liver, lung, skeletal muscle and heart muscle. After separation by agarose gel electrophoresis, each tissue showed only one isoenzyme except lung which showed two. The tissue isoenzymes, in decreasing order of migration distance towards the anode, were as follows: fast lung isoenzyme, liver or slow lung isoenzyme, the group consisting of skeletal muscle, bone, small intestine and pancreas isoenzymes and, finally, the kidney isoenzyme. Two isoenzymes occurred in serum. The major band corresponded to liver and the slow lung isoenzyme, while the minor band was considered to be the corticosteroid-induced isoenzyme, previously thought to be absent from normal serum. The AP isoenzyme patterns in lung and skeletal muscle and the presence of an isoenzyme migrating an identical distance to the corticosteroid-induced isoenzyme do not appear to have been reported before in normal dogs.     

Comparison of techniques for quantifying alkaline phosphatase isoenzymes in canine serum

Three methods for quantifying the steroid-induced and liver isoenzymes of alkaline phosphatase in canine serum were compared on a group of 29 canine serum samples with increased total alkaline phosphatase activity. Levamisole inhibition, heat inactivation, and affinity electrophoresis with densitometry yielded results that correlated strongly. The relationship between levamisole inhibition and heat inactivation test values was a simple linear one, whereas the relationship between their values and those of electrophoresis was better fitted to an exponential model. The levamisole inhibition and heat inactivation tests provided essentially the same information regarding the relative proportions of steroid-induced and hepatic isoenzymes in canine serum; either test was judged practical for routine clinical application.     

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.     

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.     

Agarose gel electrophoresis of alkaline phosphatase isoenzymes in the serum of hyperthyroid cats

Cats with hyperthyroidism [(increased serum thyroxine (T(4))] commonly have increased serum alkaline phosphatase (ALP) activity in addition to other serum biochemical abnormalities. Serum biochemical profiles were obtained from 10 hyperthyroid cats which had increased serum ALP. Agarose gel electrophoresis of serum from these cats was performed and stained for alkaline phosphatase activity. Alkaline phosphatase activity was calculated for each of the separate bands obtained, and the results were compared to those of tissue extracts, serum from normal cats, and serum from normothyroid cats with increased serum ALP activity. The hyperthyroid cats had increased ALP activity in bands corresponding to isoenzymes originating in the liver, bone, and an unidentified tissue source.     

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.     

Osteosarcoma tissues and cell lines from patients with differing serum alkaline phosphatase concentrations display minimal differences in gene expression patterns

Serum alkaline phosphatase (ALP) concentration is a prognostic factor for osteosarcoma in multiple studies, although its biological significance remains incompletely understood. To determine whether gene expression patterns differed in osteosarcoma from patients with differing serum ALP concentrations, microarray analysis was performed on 18 primary osteosarcoma samples and six osteosarcoma cell lines from dogs with normal and increased serum ALP concentration. No differences in gene expression patterns were noted between tumours or cell lines with differing serum ALP concentration using a gene‐specific two‐sample t‐test. Using a more sensitive empirical Bayes procedure, defective in cullin neddylation 1 domain containing 1 (DCUN1D1) was increased in both the tissue and cell lines of the normal ALP group. Using quantitative PCR (qPCR), differences in DCUN1D1 expression between the two groups failed to reach significance. The homogeneity of gene expression patterns of osteosarcoma associated differing serum ALP concentrations are consistent with previous studies suggesting serum ALP concentration is not associated with intrinsic differences of osteosarcoma cells.     

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.     

Serum alkaline phosphatase activity in Scottish Terriers versus dogs of other breeds

OBJECTIVE: To determine whether Scottish Terriers have higher serum alkaline phosphatase (ALP) activities and a higher prevalence of diseases commonly associated with high serum ALP activity than do dogs of other breeds. DESIGN: Retrospective case-control study. ANIMALS: 85 Scottish Terriers and 340 age-matched control dogs that were not Scottish Terriers. PROCEDURE: Medical records were reviewed, and data for year of evaluation, age, sex, breed, serum ALP activity, and final diagnosis were recorded. RESULTS: Scottish Terriers had a significantly higher mean serum ALP activity than did control dogs (1,520 U/L vs 306 U/L). Regardless of breed, dogs that had a disease commonly associated with high serum ALP activity had a significantly higher mean serum ALP activity than did dogs without such diseases (1,304 U/L vs 427 U/L). Scottish Terriers were 2.4 times as likely to have a disease commonly associated with high serum ALP activity than were control dogs, but Scottish Terriers with diseases commonly associated with high serum ALP activity had a significantly higher mean ALP activity than did control dogs with such diseases (2,073 U/L vs 909 U/L), and Scottish Terriers without such diseases had a significantly higher mean serum ALP activity than did control dogs without such diseases (1,349 U/L vs 228 U/L). CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that Scottish Terriers have higher serum ALP activities than do dogs of other breeds. Although Scottish Terriers also have a higher prevalence of diseases associated with high serum ALP activity, this alone did not explain the higher mean serum ALP activity in the breed.     

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.     

Lactate dehydrogenase and its isoenzymes in the tissues and sera of clinically normal dogs

The total activity of lactate dehydrogenase (LDH) and the percentage distribution of its isoenzymes in the tissues and sera of clinically normal adult dogs are presented. Total LDH activity was greatest in skeletal muscle followed by heart muscle, kidney, small intestinal mucosa, liver, lung, pancreas and bone. Each tissue had a unique isoenzyme pattern and the proportions of the isoenzymes in serum suggested that liver is the source of normal serum LDH. The tissue isoenzyme patterns were similar to those obtained by other authors in human beings, horses, cattle, sheep and cats although in liver, differences between ruminants and monogastric animals including dogs were evident. The data presented provide a basis for the interpretation of serum LDH isoenzyme patterns in canine disease.     

The expression of histamine H4 receptor mRNA in the skin and other tissues of normal dogs

The histamine 4 (H₄) receptor was first cloned and characterized in 2000 using the human H₃ receptor DNA sequence. The H₄ receptor has been shown to participate in various aspects of inflammation, such as chemotaxis, upregulation of adhesion molecule expression and modulation of cytokine secretion. The primary goal of this study was to determine whether H₄ receptor mRNA is expressed in normal canine skin by performing an RT‐PCR. An additional goal was to determine the expression of this receptor in the colon, liver, spleen and kidney. Tissues were collected from five healthy, young‐adult pit bull dogs. Samples were immediately placed in RNAlater^® solution and stored at ?20°C until processed. The amplified products in all skin samples in addition to the colon, liver, spleen and kidney (variable expression) had the expected size of 400–500 bp. The sequenced amplicons matched the National Center for Biotechnology Information published sequence for the canine H₄ receptor. The study results showed that canine normal skin expresses the H₄ receptor mRNA. Further studies using immunohistochemistry should be conducted to demonstrate the expression of the H₄ receptor at the protein level and to localize the expression of this receptor in the skin.     

Duration of increased serum alkaline phosphatase activity in dogs receiving different glucocorticoid doses

Exposure to exogenous glucocorticoids can variably increase the serum alkaline phosphatase (alp) activity, however, the duration of this effect in dogs has not been determined. In this study, three groups of ten clinically normal adult dogs were administered different types of glucocorticoids at therapeutic doses. Group 1 received prednisone 1 mg kg day(-1)p.o. for 3 weeks; Group 2 received a single dose of methylprednisone acetate 1.1 mg kg day(-1)s.c.; Group 3 received dexamethasone 0.25 mg kg day(-1)p.o. for 1 week. In Group 1 elevations were statistically significant on days 7, 14 and 21 (P<0.01, P<0.001, P<0.001, respectively). After discontinuing therapy serum alp returned to baseline levels in 7 days. In Group 2, serum alp activity remained significantly elevated for 3 weeks after therapy (P<0.05, P<0.001, P<0.01 on days 7, 14 and 21 respectively). In Group 3, serum alp levels were significantly increased after 1 week of therapy (P<0.001) returning to basal levels 2 weeks after discontinuing glucocorticoid administration. In conclusion, duration of increased serum alp activity was variable and with the protocols assessed a 3-week period for short-acting glucocorticoids and more than 4 weeks for long-acting methylprednisone may be required to return to baseline levels in all dogs.