Quantitative determination of equine alkaline phosphatase isoenzymes 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 37 degrees C, 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.

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.

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.

Evaluation of ivermectin for larvicidal effect in experimentally induced Parascaris equorum infections in pony foals.

A controlled test was carried out on 15 pony foals inoculated with 1,500 +/- 108.8 infective Parascaris equorum eggs. The foals were assigned to 3 treatment groups. Treatments given on postinoculation day 11 included 0.2 mg of ivermectin/kg of body weight, formulated as paste (n = 5), or liquid (n = 5), or no treatment (controls; n = 5). The foals were euthanatized on postinoculation day 25, and examined for larvae in the small intestine, lungs, and liver. Larvae were not found in foals treated with ivermectin liquid or paste, whereas significantly (P less than 0.05) higher mean numbers (960.9; range, 379 to 1,736) of 4th-stage larvae were found in the controls. Histologic and gross examination of lungs and liver revealed pathologic changes attributable to P equorum migration that were similar in all foals. Adverse reactions to treatment were not observed.

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.

Extracutaneous histologic changes accompanying zinc deficiency in pups.

The extracutaneous tissues in pups fed a Zn-deficient diet were examined. Four pups were fed a Zn-deficient diet and 3 littermates were fed a Zn-adequate diet. After 5 weeks, the pups were euthanatized. Lesions found only in pups fed a Zn-deficient diet were located in the buccal mucosa, lymph nodes, spleen, and thymus. In the buccal mucosa, lesions consisted of irregular epithelial hyperplasia and disruption of epithelial cell layers. There was a marked absence of lymphocytes in the thymus and in T-cell areas of the lymph nodes and spleen.

Tissue changes in dogs fed a zinc-deficient ration.

Six-week-old puppies fed a corn-soy based zinc-deficient diet developed lesions of parakeratosis, mild hyperkeratosis, alterations in germinal epithelium, erosions, ulcerations, vesiculation, alopecia, and inflammation of the skin. These changes were prominent in the skin of dependent regions, in areas of stretch and friction, and external contact. The epithelial lesions were reversible by adding zinc to the diet, with complete remission of external lesions by 6 weeks.