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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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 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.     

Application of a dot enzyme-linked immunosorbent assay for evaluation of the immune status to canine parvovirus and distemper virus in adult dogs before revaccination.

A growing body of literature has been published indicating that the current practice of annual vaccination of dogs may not be beneficial and in some cases may even be harmful. A number of publications have proposed assessing the immune status of dogs before annual revaccination. In this study the usefulness of a commercially available dot-ELISA kit was evaluated to determine the duration IgG antibody titers to canine parvovirus (CPV) and canine distemper virus (CDV) in 158 dogs vaccinated at least one year ago. Overall, the percentage of dogs with protective antibody titers to both CPV and CDV was 84%. The percentage of dogs with borderline antibody titers was 11% for CPV and 10% for CDV. Four percent of the dogs had no detectable antibody to CPV and 6% had no antibody to CDV. The results reported here are in good agreement with other studies measuring IgG antibody levels. It is concluded that the kit offers veterinarians the opportunity of determining antibody titers and revaccinating only those pets whose antibody titers to specific diseases have waned.     

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.     

Markers of bone metabolism in dog breeds of different size

Serum and urinary markers of bone turnover may be of value in animals as noninvasive tools for determining the response of the skeleton to disease and injury. Although normal values for bone markers have been reported for the Beagle, concerns remain that breed to breed differences will complicate the interpretation of bone marker data in dogs. To explore this, we examined serum bone markers in two breeds of vastly different size, Pomeranians and Irish Wolfhounds. Our hypothesis was that serum concentrations of bone markers are similar in toy and giant dog breeds and fall within the same range as those reported for Beagles. Bone alkaline phosphatase (BALP) and carboxy-terminal telopeptide of type I collagen (ICTP), respectively markers of bone formation and bone resorption, were measured in age matched Pomeranians (n=14) and Irish Wolfhounds (n=14). No statistically significant differences between the mean BALP and mean ICTP serum concentrations from Pomeranians and Irish Wolfhounds were found. All BALP and ICTP concentrations were within the reference range reported for Beagles. The results of this study suggest that serum BALP and ICTP concentrations in giant and toy breeds are the same as in Beagles and that these assays may be used for dogs of all sizes.     

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.     

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.