Corticosteroid-induced Alkaline Phosphatase Research Articles

Use of a combination of routine hematologic and biochemical test results in a logistic regression model as a diagnostic aid for the diagnosis of hypoadrenocorticism in dogs.

OBJECTIVE To assess the discriminatory value for corticosteroid-induced alkaline phosphatase (CiALP) activity and other variables that can be measured routinely on a CBC and biochemical analysis for the diagnosis of hypoadrenocorticism in dogs. SAMPLE Medical records of 57 dogs with confirmed hypoadrenocorticism and 57 control dogs in which hypoadrenocorticism was suspected but ruled out. PROCEDURES A retrospective case-control study was conducted. Dogs were included if a CBC and complete biochemical analysis had been performed. Dogs with iatrogenic hypoadrenocorticism and dogs treated previously with glucocorticoids were excluded. Cortisol concentration for dogs with hypoadrenocorticism was ≤ 2 μg/dL both before and after ACTH administration. Cortisol concentration for control dogs was > 4 μg/dL before or after ACTH administration. RESULTS Area under the receiver operating characteristic (ROC) curve for CiALP activity was low (0.646; 95% confidence interval, 0.494 to 0.798). Area under the ROC curve for a model that combined the CiALP activity, Na-to-K ratio, eosinophil count, activity of creatine kinase, and concentrations of SUN and albumin was high (0.994; 95% confidence interval, 0.982 to 1.000). Results for this model could be used to correctly classify all dogs, except for 1 dog with hypoadrenocorticism and no electrolyte abnormalities. CONCLUSIONS AND CLINICAL RELEVANCE CiALP activity alone cannot be used as a reliable diagnostic test for hypoadrenocorticism in dogs. Combined results for CiALP activity, Na-to-K ratio, eosinophil count, creatine kinase activity, and concentrations of SUN and albumin provided an excellent means to discriminate between hypoadrenocorticism and diseases that mimic hypoadrenocorticism.

Analysis of serum corticosteroid-induced alkaline phosphatase isoenzyme in dogs with hepatobiliary diseases.

To reveal the relationship between canine corticosteroid-induced alkaline phosphatase isoenzyme activity and hepatobiliary diseases. Retrospective analysis of the relationship between serum corticosteroid-induced alkaline phosphatase activity and diagnosis, serum cortisol concentration and alanine transferase activity in dogs with hepatobiliary diseases. Dogs with a history of glucocorticoid administration were excluded. Seventy-two dogs with hepatobiliary diseases were analysed. The serum corticosteroid-induced alkaline phosphatase concentration was increased in dogs with hepatobiliary diseases. There was no correlation between serum cortisol concentration and serum corticosteroid-induced alkaline phosphatase percentage or activity. Dogs with hepatobiliary disease can exhibit high serum alkaline phosphatase activity even if the dogs have not been administrated glucocorticoids and the serum cortisol concentration is normal.

Serum alkaline phosphatase isoenzymes in laboratory beagle dogs detected by polyacrylamide-gel disk electrophoresis

Serum alkaline phosphatase (ALP) activity is frequently measured in toxicity studies. Itoh et al. (2002) reported that a commercially available polyacrylamide-gel (PAG) disk electrophoresis kit used in humans (AlkPhor System, Jokoh Co., Ltd., Tokyo, Japan) for identifying serum ALP isoenzymes was useful for veterinary clinicopathological diagnosis in mongrel dogs. In the present study, based on the report of Itoh et al. (2002), we tried to expand the application range of this kit to laboratory beagle dogs which are commonly used in toxicity studies. In order to identify the origin of each ALP isoenzyme, tissue ALP extracts from the liver, bone and small intestine and serum samples were treated with neuraminidase, anti-small intestinal ALP antibody, ALP inhibitor levamisole and/or wheat germ agglutinin (WGA). The main serum ALP isoenzymes in 5-month-old intact beagle dogs were bone-derived (bone and atypical ALP: corresponding to human variant bone ALP) and they tended to decrease with age. However, liver-derived ALP isoenzyme greatly increased in the serum of cholestasis model dogs. The cholestasis model dogs also had a large molecular ALP detected in the resolving gel. This ALP could be originated from intestinal ALP or corticosteroid-induced ALP (CALP), because the activity remained even after levamisole inhibition. CALP was observed in intact laboratory beagle dogs with individual differences. These results suggest that the present method is a useful tool for detecting serum ALP isoenzymes in laboratory beagle dogs and concomitant levamisole inhibition with another gel is applicable for the evaluation of organ toxicity.

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

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. 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. 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. 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. 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.

Assessment of corticosteroid‐induced alkaline phosphatase as a prognostic indicator in canine lymphoma

To examine the incidence of elevated corticosteroid-induced alkaline phosphatase (sALP) in dogs with lymphoma and to determine if sALP is a reliable prognostic indicator in canine lymphoma. The medical records of 62 canine lymphoma patients treated with a combination chemotherapy protocol from 1994 to 2003 at the University of Illinois Veterinary Teaching Hospital were examined. Variables assessed with respect to response rate and remission duration included age, bodyweight, sex, breed, World Health Organization stage (I to V), substage (a or b), pretreatment administration of corticosteroid, and serum levels of alkaline phosphatase, sALP and alanine aminotransferase. sALP was not statistically significant with respect to response rate or duration of remission, nor was preinduction glucocorticoid administration. Stage was significant with respect to achieving remission. It was found that sALP is not a useful prognostic indicator for response rate and remission duration in dogs with lymphoma.

Characterization and localization of alkaline phosphatase in canine seminal plasma and gonadal tissues

Alkaline phosphatase (AP) is a useful indicator of the presence of the sperm-rich (2nd) fraction in the canine ejaculate. Two AP isoenzymes originating from separate genes have been identified in the dog: tissue nonspecific (TNS) and intestinal. Bone, liver, and corticosteroid-induced AP are different isoforms of the TNS and intestinal isoenzymes. Using gel electrophoresis and levamisole inhibition assays, it was determined that seminal plasma AP (SAP) is a unique isoform of canine TNS AP whose glycosylation is distinct from either of the TNS AP isoforms commonly found in canine serum. Using immunocytochemistry, SAP activity was localized to the epididymal and seminiferous tubular epithelium. The ability to distinguish SAP from bone AP, liver AP and corticosteroid-induced AP could be beneficial to the practitioner in determining the quality of a semen sample.

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

To clone segments of the canine liver alkaline phosphatase (LALP) and corticosteroid-induced alkaline phosphatase (CIALP) genes and use those clones to determine the tissue source of CIALP, the kinetics of LALP and CIALP mRNA expression for glucocorticoid-treated dogs, and the correlation between LALP and CIALP transcript concentrations and isoenzyme activities. Tissues obtained from 7 dogs treated with prednisone (1 mg/kg, SC, q 24 h) for up to 32 days and 1 untreated (control) dog. Gene segments of LALP and CIALP were obtained by reverse transcription-polymerase chain reaction (RT-PCR) assay. The tissue source of CIALP and IALP mRNA was determined by northern blot analysis of tissues from 1 of the glucocorticoid-treated dogs. Hepatic tissues and serum samples were obtained from the 6 remaining glucocorticoid-treated dogs on days 0, 2, 5, 10, and 32 of prednisone treatment, and relative expression of LALP and CIALP mRNA was correlated with LALP and CIALP activity. A 2,246-base pair (bp) segment of canine LALP and a 1,338-bp segment of CIALP were cloned. Northern blot analysis revealed CIALP mRNA expression in hepatic tissues only after glucocorticoid treatment. Kinetics of LALP and CIALP mRNA expression in the liver of glucocorticoid-treated dogs paralleled liver and serum activities of LALP and CIALP. The liver is the most likely source for CIALP in dogs. Analysis of kinetics of serum and hepatic LALP and CIALP mRNA suggests that after glucocorticoid treatment, both are regulated by modification of mRNA transcript concentrations, possibly through differing mechanisms.

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.

Separation and quantification of corticosteroid-induced, bone and liver alkaline phosphatase isoenzymes in canine serum.

Quantifying alkaline phosphatase (ALP) isoenzymes in canine serum would provide a useful index in a clinical laboratory. To achieve this goal, we tested a semi-automatic assay combining wheat germ lectin (WGL) precipitation and chemical inhibition of isoenzymes of the TNS gene with levamisole to quantify bone ALP (BALP) and corticosteroid-induced ALP (CALP), respectively. The liver ALP (LALP) isoenzyme was then calculated from the equation: TALP = BALP + LALP + CALP BALP, LALP and CALP standards from serum of puppies, bile-duct ligated dogs and dogs on 4.4 mg/kg/day prednisolone for 30 days, respectively, were used. The suitability of standard sera was tested by affinity electrophoresis. Levamisole (4.2 mM) inhibits 98% of BALP and LALP but only 42% CALP. Multiplying measured CALP by 1.8 gives the total CALP value in serum. WGL precipitated 92.3% BALP, 23.3% LALP and 26.8% heated CALP standards. These values were used to adjust precipitated ALP to obtain the exact levels of BALP. WGL was then tested on pooled serum standards in which the relative proportions of all the ALPs were known and controlled. BALP was adequately quantified except when LALP and CALP levels were extremely high. The assay was also applicable under conditions resulting in high ALP. Therefore, combining WGL and levamisole inhibition provides an adequate separation and quantification of canine ALP isoenzymes. The method has great potential for diagnostic use and should be tested further for routine implementation.

The age dependent levels of serum ALP isoenzymes and the diagnostic significance of corticosteroid-induced ALP during long-term glucocorticoid treatment.

Three isoenzymes of total alkaline phosphatase (TALP) are known in canine serum: Bone alkaline phosphatase (BALP), liver alkaline phosphatase (LALP) and corticosteroid-induced alkaline phosphatase (CALP). Using an assay developed by combining selective precipitation of BALP by wheat germ lectin (WGA) and an automated levamisole inhibition method for quantifying CALP, age-related reference ranges of the isoenzymes in 75 canine serum samples were investigated. BALP comprised 96, 38 and 26% of TALP in young, middle aged and old dogs, respectively, and CALP was respectively 12, 11 and 27% of TALP. LALP was less than 10% in the young but represented more than 50% of TALP in middle aged and old dogs. Furthermore, the significance of monitoring LALP and CALP and their relationship to hepatopathy in dogs receiving long term prednisolone therapy was assessed. In this study, TALP increased in all dogs receiving prednisolone. But only LALP was responsible in dogs with minor vacuolization of the liver, while in severely degenerated cases both LALP and CALP increased. It is concluded that a high TALP due solely to LALP, rather than LALP and CALP represents lesser liver pathologic involvement. Monitoring the 2 isoenzymes has greater significancy in assessing in the level of liver damage than relying on an increased TALP value alone. Quantifying the individual isoenzymes may further be useful in assessing the clinical significance of these isoenzymes in various conditions that result in elevated TALP values.

Canine corticosteroid-induced alkaline phosphatase in serum was solubilized by phospholipase activity in vivo

In this study, gas chromatography/mass spectrometry revealed the presence of stoichiometric amounts of myo-inositol in association with serum corticosteroid-induced isozyme of alkaline phosphatase (CALP) in canine serum. Such remnants are consistent with prior membrane attachment of serum CALP and its release into serum by endogenous phospholipase activity. Serum CALP was further shown to behave similarly to CALP released from hepatocyte membranes by glycosyl phosphatidylinositol phospholipase D (GPI-PLD) and differently from CALP solubilized by GPI-phospholipase C (PLC) on both native polyacrylamide gel electrophoresis and Western blot analysis using anti-cross-reacting determinant antibody. In addition to bile canalicular surfaces, CALP activity was found over hepatocyte sinusoidal surfaces by histochemical staining of canine liver sections. A significantly higher ratio of CALP to total alkaline phosphatase activity was observed in serum as opposed to bile in 10 of 11 paired serum and bile samples from dogs. This suggested that bile is not likely to be the source of serum CALP and is consistent with the release of CALP from hepatocyte basolateral surfaces directly into serum. It was concluded that serum CALP was once membrane bound and was released by phospholipase activity into serum. Our findings are consistent with release of CALP from the sinusoidal surfaces of hepatocytes into serum either by endogenous GPI-PLD activity or release by GPI-PLC followed by modification of the phosphatidylinositol remnant in vivo.

Assessment of corticosteroid-induced alkaline phosphatase isoenzyme as a screening test for hyperadrenocorticism in dogs

Abstract Quantitative determination of the corticosteroid-induced isoenzyme of alkaline phosphatase (cap) was evaluated as a screening test for hyperadrenocorticism (hac) in dogs. A series of 40 dogs with hac (cap range, 96 to 14,872 U/L), 30 clinically normal dogs (cap range, 0 to 38 U/L), and 80 dogs with various diseases (non-hac) and without history of exogenous glucocorticoid exposure for a minimum of 60 days (cap range, 0 to 1163 U/L) were used to evaluate the test. Sensitivity and specificity of cap was calculated at various cutoff points for absolute cap activity and for cap activity expressed as a percentage of total alkaline phosphatase activity. A cutoff point of 90 U/L was selected as optimal for use of this assay as a screening test for hac. A prevalence survey then was done of all canine serum samples submitted to our diagnostic laboratory over a 3-month period, to calculate the predictive values of a positive and a negative test result in a clinical population and to determine the relative frequency and magnitude of cap activity in dogs that had received glucocorticoids. The predictive values of a positive and a negative test result at the 90 U/L cutoff value were 21.43% (95% confidence limits, 8.3 to 40.95%) and 100% (95% confidence limit &gt; 96%), respectively. It was concluded that cap isoenzyme activity, determined by routine biochemical analysis by an automated levamisole-inhibition assay, could function as a screening test for hac, however, (the predictive value of a positive test result was too low to recommend the assay as a diagnostic test. The cap activity was &gt; 90 U/L in 52% of dogs receiving glucocorticoids, emphasizing the importance of treatment history in the use of this test. To help eliminate possible workup bias, an additional set of serum samples from 30 dogs diagnosed as having hac at a second institution also were analyzed. Sensitivity of the assay in this sample group, using the 90 U/L cutoff value, was 83.3% (95% confidence limits, 65.3 to 94.4%), suggesting that test sensitivity may vary, depending on the institution in which the assay is used and the selection of cases for diagnostic workup.

Quantification of bone alkaline phosphatase in canine serum.

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

Preliminary experience with the diagnostic value of the canine corticosteroid-induced alkaline phosphatase isoenzyme in hypercorticism and diabetes mellitus.

The present study communicates the interim experience with the diagnostic value of the canine corticosteroid-induced alkaline phosphatase isoenzyme (CIAP), qualitatively assessed by agarose gel electrophoresis, for the diagnosis of Hypercorticism (prolonged exposure to increased concentrations of exogenous or endogenous corticosteroids) and Diabetes mellitus. A total of 106 canine serum samples with a total alkaline phosphatase activity greater than 2.90 mu kat/l were included. CIAP was identified in 28 out of 29 cases of Hypercorticism, in 7 out of 9 cases of Diabetes mellitus, and in 33 out of 68 cases of various other diseases. Sensitivity and specificity with respect to Hypercorticism were 0.97 and 0.48, respectively, while for Diabetes mellitus sensitivity and specificity were 0.78 and 0.37. From this, the positive and the negative predictive values were calculated using standard formulas. The main conclusion may be summarized as follows: In the dog, only the negative finding of CIAP seems to be of diagnostic value in ruling out Hypercorticism as a cause for an increased total alkaline phosphatase activity.

Effect of Glucocorticoids on Alkaline Phosphatase, Alanine Aminotransferase, and Gamma-Glutamyltransferase in Cultured Dog Hepatocytes

Glucocorticoid(GC)-induced hepatopathy in the dog is characterized by abnormal liver morphology and increases in serum alanine aminotransferase (ALT), gamma-glutamyltransferase (GGT), and the liver alkaline phosphatase isoenzyme (LALP) and by the appearance of an unusual isoenzyme of alkaline phosphatase known as the corticosteroid-induced alkaline phosphatase isoenzyme (CALP). It has not been shown whether the increases in serum ALT, GGT, and LALP are as a result of an increase in production of these enzymes or as a result of the GC-induced hepatocellular swelling and possible membrane alterations. Also, it has been assumed that the mechanism of production of CALP is via GC-induced gene derepression and de novo protein synthesis; however, this hypothesis has not been directly tested. Using isolated dog hepatocytes maintained in a confluent monolayer culture in the presence and absence of GC or cyclic AMP, no statistical increase in serum ALT, GGT, or LALP was observed. A combination of GC and cyclic AMP also caused no statistical increase in ALT and GGT; however, we demonstrate that these conditions clearly stimulated the de novo synthesis of LALP. These conditions do not induce the synthesis of CALP as determined by a sensitive immunoassay. The data obtained using this in vitro model suggest that the primary mechanism(s) of the in vivo increase of serum ALT and GGT in GC treated dogs may be other than that of de novo protein synthesis. Likewise, in vitro production of CALP may be a mechanism more complex than the conditions tested in this study.

Corticosteroid-induced alkaline phosphatase isoenzyme in the diagnosis of canine hypercorticism.

Corticosteroid-induced isoenzyme of alkaline phosphatase (AP) can easily be demonstrated in canine plasma as a routine procedure because of its greater heat stability at 65 degrees C in comparison with that of other AP-isoenzymes. In this study the accuracy of this test for the diagnosis of hypercorticism was investigated. The AP-65 degrees C test had its highest efficiency when applied to plasma AP levels exceeding 150 units/litre. In a group of 146 dogs, clinically suspected of having hyperadrenocorticism, the test had a sensitivity of 0.92 and a positive predictive value for a positive test result of 0.89. Its lack of specificity (0.44) makes it unsuitable as a diagnostic test. The main application of AP-65 degrees C is in detecting hypercorticism in dogs by routine laboratory measurements, as was demonstrated in 711 dogs, in which a positive predictive value for the presence of hypercorticism of 0.89 was found.

A Technique for Automated Quantification of Canine Glucocorticoid-induced Isoenzyme of Alkaline Phosphatase.

A sensitive assay for the corticosteroid-induced alkaline phosphatase isoenzyme (CAP), adaptable to most clinical chemistry autoanalyzers, is described and validated. This assay is based on the greater than 98% inhibition of liver alkaline phosphatase isoenzyme (LAP) activity with 4.2 mM levamisole, as compared to the 42% inhibition of CAP activity. Analysis of serum with total alkaline phosphatase (AP) activity within the reference range, resulted in a reference range of 0 to 19 U/L for CAP activity. Analysis of serum from 160 clinical patients with AP activity above the reference range, revealed 73% with increased CAP activity ranging from 20 to 7,000 U/L. The diagnostic significance of this increased CAP activity is discussed.

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.