LABORATORY VALIDATION OF A POINT-OF-CARE CARDIAC TROPONIN I ASSAY FOR USE IN WHITE-TAILED DEER (ODOCOILEUS VIRGINIANUS).

The VetScan® i-STAT® 1 Handheld Analyzer and cardiac troponin I (cTnI) cartridges (i-STAT cTnI assay) measured greater median cTnI concentration [cTnI] in free-ranging white-tailed deer (Odocoileus virginianus) hand-injected with anesthetic drugs after physical restraint in Clover traps than in those ground-darted with the same drugs. This suggested that Clover trapping induces myocardial damage, bringing the use of this capture method under scrutiny. The purpose of this study was to confirm the validity of the i-STAT cTnI assay in deer before recommending changes in capture methods. Median [cTnI] measured by the i-STAT cTnI assay ([cTnI]i) in heparinized whole blood collected from 52 healthy, reproductively mature, female deer physically restrained in a chute was 0.01 ng/ml (10-90% percentiles: 0.00-0.03 ng/ml; minimum, maximum: 0.00, 0.07 ng/ml); [cTnI]i was 0.00 ng/ml in 42% of the deer. There was no association between [cTnI]i and either clotting or hemolytic index. [cTnI]i was 0.00 ng/ml when deer skeletal muscle homogenate was added to deer blood with [cTnI]i of 0.00 ng/ml, confirming the i-STAT cTnI assay does not detect skeletal muscle troponins. When deer cardiac muscle homogenate was serially diluted with 1) deer blood, 2) deer plasma, and 3) cow blood, [cTnI]i was directly proportional (Y intercept=-0.09, 0.7, and -0.08 ng/ml, respectively; r2≥0.97) to the fraction of homogenate in each sample. Deer cardiac muscle homogenate was diluted with deer blood to produce three samples with low, intermediate, and high [cTnI]i; serial measurements (n=10) performed on each sample yielded coefficients of variation (CVs) of 8, 20, and 11%, respectively. Corresponding CVs when plasma was used as diluent were 13, 9, and 7%, respectively. [cTnI]i increased when plasma with a low [cTnI]i was stored at 20-24°C for 9 days. Three freeze-thaw cycles caused no systematic change in plasma [cTnI]i.

Detection by ELISA of C-terminal proBNP in plasma from cats with cardiomyopathy.

The B-type natriuretic peptide prohormone (proBNP) is enzymatically cleaved into an inactive N-terminal peptide and a biologically active C-terminal peptide with many beneficial cardiorenal effects. The purpose of this study was to develop and test in cats with cardiomyopathy an immunoassay to quantify the concentrations of C-terminal proBNP in feline plasma. An anti-canine proBNP monoclonal antibody (UI-1021) was shown to have adequate binding affinity to proBNP 80-106 for use in a solid-phase immunoassay, and by epitope mapping to bind within positions 84-87 of feline proBNP. UI-1021 was paired with an affinity-purified rabbit polyclonal detection antibody to feline proBNP 100-106, in a sandwich ELISA with feline proBNP 80-106 standard. The linearity and analytical range and sensitivity of the assay were confirmed from 1.4 to 85 pmol/L. Spike recovery averaged 106.5% (95% confidence interval 78-135%). Within run and intra-assay coefficients of variation were <12%. A protease inhibitor mixture preserved proBNP 80-106 immunoreactivity for at least 5 days in plasma. Clinical verification of the ELISA was done using plasma from 13 cats with cardiomyopathy, whose C-terminal proBNP concentrations ranged from 1.7 to 78.8 pmol/L vs. <1.4-1.8 pmol/L in plasma from 18 healthy cats. Concentrations were found to be substantially lower than reported N-terminal proBNP concentrations, and similar to those of human heart failure patients where relative C-terminal BNP deficiencies have been proposed as contributory to the progression of the disease.

Detection of left ventricular hypertrophy in rats administered a peroxisome proliferator-activated receptor alpha/gamma dual agonist using natriuretic peptides and imaging.

Chronic treatment with suprapharmacologic doses of peroxisome proliferator-activated receptor (PPAR) agonists has a known potential for causing left ventricular hypertrophy (LVH). The mechanism by which LVH develops is not well understood nor are biomarkers of it well characterized. Natriuretic peptides are important regulators of cardiac growth, blood volume, and arterial pressure and may be useful biomarkers of LVH and hemodynamic changes that precede it. We measured amino-terminal pro-atrial natriuretic peptide (NTproANP), amino-terminal pro-brain natriuretic peptide (NTproBNP), and cardiac troponin I (cTnI) concentrations in serum and plasma, as well as transcripts in left ventricular heart tissue for atrial natriuretic peptide precursor (Nppa), brain natriuretic peptide precursor (Nppb), and myosin heavy chain-beta (Myh7) as potential biomarkers of LVH induced by a PPARalpha/gamma dual agonist in Sprague-Dawley rats. We used magnetic resonance imaging, echocardiography, and hemodynamics to identify structural and functional cardiovascular changes related to the biomarkers. Heart-to-brain weight ratios (HW:BrW) were correlated with NTproANP, NTproBNP, and cTnI concentrations in serum as well as fold change in expression of Nppa and Nppb. LVH was characterized by increased left ventricular wall thickness and inner diameter, increased cardiac output, decreased arterial blood pressure, and increased heart rate. In these studies, each end point contributed to the early detection of LVH, the ability to monitor its progression, and demonstrated the ability of NTproANP concentration in serum to predict LVH and hemodynamic changes.

Canine heterophilic antibodies as a source of false-positive B-type natriuretic peptide sandwich ELISA results.

BACKGROUND: Interference by heterophilic antibodies is a well-known cause of false-positive sandwich ELISA results in human medicine. They are considered rarely in veterinary species and have not been characterized but could become important as newer, highly sensitive sandwich immunoassay technologies are developed. OBJECTIVES: The goals of this study were to use a B-type natriuretic peptide (BNP-32) sandwich ELISA to determine the effect of heterophilic antibodies on test performance; to characterize canine heterophilic antibodies; and to develop and test a method for heterophilic antibody removal. METHODS: A sandwich ELISA was developed using a mouse IgG(1)K monoclonal and a rabbit polyclonal antibody to two synthetic peptides of canine BNP-32. The effects on false-positive results of heterophilic antibody depletion and blocking by various techniques were compared. The titers of canine heterophilic antibodies were compared with various blood antigens from other species and the relative amount of canine IgG was compared with that of IgM heterophilic antibody. RESULTS: Heterophilic antibodies in dog plasma were shown to be capable of causing false-positive ELISA results. They reacted with blood proteins from a variety of animal species at relatively low titers and consisted of both IgG and IgM. Protein A agarose antibody precipitation, in conjunction with mouse IgG(1)K blocking antibody, was effective in eliminating false-positive sandwich ELISA results while retaining adequate test performance. CONCLUSIONS: Canine heterophilic antibodies can interfere with sandwich ELISA assays and cause false-positive test results. An effective technique for their removal that has a potentially broad application was developed, and allows measurement of canine blood constituents at low picomolar concentrations.

Distinguishing cardiac and noncardiac dyspnea in 48 dogs using plasma atrial natriuretic factor, B-type natriuretic factor, endothelin, and cardiac troponin-I.

BACKGROUND: It is challenging to differentiate congestive heart failure (CHF) from noncardiac cause of dyspnea. HYPOTHESIS: Circulating concentrations of atrial natriuretic peptide (NT-proANP), B-type natriuretic peptide (BNP), endothelin-I (ET-1), and cardiac troponin-I (cTnI) can be used to help distinguish between cardiac and noncardiac causes of dyspnea in dogs. ANIMALS: Forty-eight client-owned dogs admitted to a veterinary teaching hospital for respiratory distress. METHODS: Blood samples from patients were prospectively obtained. The etiology of dyspnea was determined by using physical examination, thoracic radiographs, and echocardiography. RESULTS: CHF was diagnosed in 22 dogs, and dyspnea of noncardiac origin (noHD group) was diagnosed in 26 dogs. Analyses revealed significant difference between groups for NT-proANP (geometric mean, 95% confidence [CI]; no HD: 0.26 nmol/mL, 95% CI 0.17-1.09; CHF: 1.38 nmol/mL, 95% CI 1.09-1.74 nmol/mL; P < .0001), BNP (noHD: 12.18 pg/mL, 95% CI 10.91-16.17 pg/mL; CHF: 34.97 pg/mL, 95% CI 23.51-52.02 pg/mL; P < .0001), and ET-1 (noHD: 0.32 fmol/mL, 95% CI 0.23-0.46 fmol/mL; CHF: 1.26 fmol/mL, 95% CI 0.83-1.91 fmol/mL; P < .0001). Plasma cTnI concentrations were not significantly different between groups (noHD: 0.29 ng/mL, 95% CI 0.12-0.72 ng/mL; CHF: 0.42 ng/mL, 95% CI 0.18-0.97, P = .53). Receiver operating curves indicated areas under the curve for NT-proANP, BNP, and ET-1 of 0.946, 0.886, and 0.849, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: Plasma NT-proANP, BNP, and ET-1, but not cTnI, appear useful for distinguishing between dogs with cardiac and noncardiac causes of dyspnea, with plasma NT-proANP having the highest sensitivity (95.5%) and specificity (84.6%).

Characterization of canine mitochondrial protein expression in natural and induced forms of idiopathic dilated cardiomyopathy.

OBJECTIVE: To map canine mitochondrial proteins and identify qualitative and quantitative differences in heart mitochondrial protein expression between healthy dogs and dogs with naturally occurring and induced dilated cardiomyopathy (DCM). SAMPLE POPULATION: Left ventricle samples were obtained from 7 healthy dogs, 7 Doberman Pinschers with naturally occurring DCM, and 7 dogs with induced DCM. PROCEDURES: Fresh and frozen mitochondrial fractions were isolated from the left ventricular free wall and analyzed by 2-dimensional electrophoresis. Protein spots that increased or decreased in density by >or= 2-fold between groups were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or quadrupole selecting, quadrupole collision cell, time-of-flight mass spectrometry. RESULTS: Within narrow pH gradients of control canine heart mitochondrial samples, a total of 1,528 protein spots were revealed. Forty subunits of heart mitochondrial proteins that differ significantly from control tissues were altered in tissue specimens from dogs with naturally occurring and induced forms of DCM. The most affected heart mitochondrial proteins in both groups were those of oxidative phosphorylation (55%). Upregulation of manganese superoxide dismutase was suggestive of heart oxidative injury in tissue specimens from dogs with both forms of DCM. Evidence of apoptosis was associated with overexpression of the heart mitochondrial voltage-dependent anion channel-2 protein and endonuclease G in tissue specimens from dogs with induced DCM. CONCLUSIONS AND CLINICAL RELEVANCE: Alterations of heart mitochondrial proteins related to oxidative phosphorylation dysfunction were more prevalent in tissue specimens from dogs with induced or naturally occurring DCM, compared with those of control dogs.

Correlation of mitochondrial protein expression in complexes I to V with natural and induced forms of canine idiopathic dilated cardiomyopathy.

OBJECTIVE: To identify qualitative and quantitative differences in cardiac mitochondrial protein expression in complexes I to V between healthy dogs and dogs with natural or induced dilated cardiomyopathy (DCM). SAMPLE POPULATION: Left ventricle samples were obtained from 7 healthy dogs, 7 Doberman Pinschers with naturally occurring DCM, and 7 dogs with DCM induced by rapid right ventricular pacing. PROCEDURES: Fresh and frozen mitochondrial fractions were isolated from the left ventricular free wall and analyzed by 2-dimensional electrophoresis. Protein spots that increased or decreased in density by 2-fold or greater between groups were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or quadrupole selecting, quadrupole collision cell, time-of-flight mass spectrometry. RESULTS: A total of 22 altered mitochondrial proteins were identified in complexes I to V. Ten and 12 were found in complex I and complexes II to V, respectively. Five were mitochondrial encoded, and 17 were nuclear encoded. Most altered mitochondrial proteins in tissue specimens from dogs with naturally occurring DCM were associated with complexes I and V, whereas in tissue specimens from dogs subjected to rapid ventricular pacing, complexes I and IV were more affected. In the experimentally induced form of DCM, only nuclear-encoded subunits were changed in complex I. In both disease groups, the 22-kd subunit was downregulated. CONCLUSIONS AND CLINICAL RELEVANCE: Natural and induced forms of DCM resulted in altered mitochondrial protein expression in complexes I to V. However, subcellular differences between the experimental and naturally occurring forms of DCM may exist.

Physiologic VDD versus nonphysiologic VVI pacing in canine 3rd-degree atrioventricular block.

Historically, ventricular demand, nonphysiologic (VVI) pacing has been the most commonly used modality to treat 3rd-degree atrioventricular (AV) block. The goal of this study was to determine the feasibility of using a commercial, single-lead, physiologic (VDD) pacemaker in dogs with 3rd-degree AV block. Furthermore, we hoped to characterize and identify differences in the radiographic, echocardiographic, neurohormonal, and quality of life consequences of physiologic versus nonphysiologic pacing. We evaluated 10 dogs during a 12-week crossover study. Acutely, rate-matched physiologic pacing reduced pulmonary capillary wedge pressure by 19% compared with nonphysiologic pacing. VDD pacing significantly reduced left atrial size normalized to body weight, left atrial-to-aortic root ratio, and left ventricular end-systolic dimension and increased fractional shortening, aortic Doppler velocity, cardiac output, and stroke volume compared with VVI pacing. Variable rate VDD pacing resulted in a significantly slower heart rate (HR) during echocardiography than fixed-rate (100 bpm) VVI pacing. AV synchronous pacing reduced circulating N-terminal proatrial natriuretic peptide (ANP), norepinephrine (NOR), and epinephrine (EPI) concentrations compared with asynchronous pacing. There were no significant differences in systemic blood pressure, thoracic radiographs, or owner-perceived quality of life. The median percentage of AV synchronous pacing during the VDD modality was 99.8% (range, 1.2 to 99.9%). This study confirms the potential to achieve physiologic pacing with a commercial, single-lead system in dogs. VDD pacing improved hemodynamics and neurohormonal profiles over asynchronous pacing although the long-term clinical benefits of these changes remain to be determined.

Clinical pathology approaches to hepatic injury.

Testing the blood for evidence of hepatic damage and dysfunction frequently involves measuring several blood constituents simultaneously to screen for disease. While useful, this approach occasionally leads to apparent disparities between the blood test results, and the results of other diagnostic tests such as histology. In part, these perceived discrepancies may stem from a lack of appreciation for tissue, cellular, and molecular factors that affect the appearance of hepatic disease biomarkers in the blood. Further confusing the matter is that in some instances the mechanisms responsible for the appearance of diagnostic compounds in blood are only partially understood. Many of the known factors that affect hepatic biomarkers are similar to those affecting other tissue markers, while others are unique to the liver, such as those involved with cholestasis. Disease conditions can also cause misleading results by affecting tissue concentrations of test compounds, hepatic mass, and the clearance rate of compounds from the blood. Knowledge of the factors affecting the blood concentrations of biomarkers, as well as investigations into the mechanisms behind changes to hepatic biomarker concentrations, may allow for a better interpretation of blood test results and fewer inconsistencies between diagnostic results.

Validation of an immunoassay for measurement of canine cardiac troponin-I.

Cardiac troponin-I (cTnI) is a sensitive and specific circulating marker of cardiac injury. The amind acid sequence of canine troponin-I suggests that immunoassays designed for humans may be able to quantify canine cTnI. We sought to validate the AccuTnItrade mark system for use in the canine species. Samples of purified canine free cTnI, cardiac troponin I-C (cTnI-C), and cardiac troponin I-T-C (cTnI-T-C) were used to assess the performance characteristics of the assay. Intra-assay precision was 4.2 +/- 3.0% and inter-assay precision was 4.5 +/- 2.7%. The assay demonstrated linearity of serial dilutions from 0.015 to 30 ng/ml for all forms of cTnI (R, 0.998 to 0.999). Mean recovery of cTnI was 92.5 +/- 10.5%, of cTnI-C was 147.2 +/- 19.8%, and of cTnI-T-C was 97.3 +/- 23.5%. Specificity of the assay for the cardiac form of troponin-I was confirmed using samples spiked with canine skeletal muscle troponin-I. The AccuTnItrade mark assay was evaluated in 27 canine patients. Dogs with heart disease (cardiomyopathy or severe mitral valve disease, n = 13) had a higher mean cTnI concentration than controls (disease cTnI = 0.68 ng/ml, range 0.03-5.47 ng/ml vs. control cTnI = 0.03 ng/ml, range 0.01-0.08 ng/ml; P = 0.0003). The AccuTnItrade mark assay possesses sufficient test performance for use with canine plasma and can distinguish a cohort of dogs with heart disease from a cohort of healthy controls. The results of this study suggest that further investigations into the clinical use of the AccuTnItrade mark assay for the diagnosis of canine heart disease are warranted.

Plasma endothelin-1 immunoreactivity in normal dogs and dogs with acquired heart disease.

We sought to measure plasma endothelin-1 (ET-1) concentrations in normal dogs and to compare them with those measured in dogs with acquired heart disease with or without pulmonary edema. A sandwich enzyme-linked immunosorbent assay kit was validated and used to measure ET-1 immunoreactivity in plasma samples obtained from 32 normal dogs and 46 dogs with either dilated cardiomyopathy (DCM, n = 27) or degenerative valvular disease (CDVD, n = 19) with (n = 30) or without (n = 16) overt congestive heart failure (CHF). Plasma ET-1 concentrations (geometric mean, 95% confidence interval of geometric mean) were 1.17 (1.04-1.32) fmol/mL in the 32 normal control dogs, 1.25 (0.981-1.60) fmol/mL in 16 dogs with DCM (n = 9) or CDVD (n = 7) without CHF, and 2.51 (2.10-3.01) fmol/mL in 30 dogs with DCM (n = 18) and CDVD (n = 12) with CHE Plasma immunoreactivity of ET-1 was significantly higher in dogs with CHF in comparison with normal dogs (P < .001) and dogs with heart disease without CHF (P < .001). No significant difference was found between normal dogs and dogs with heart disease but without CHF (P > .05). Significant correlations were between plasma ET-I concentrations and left atrial:aortic ratio (P < .0001, r2 = .39), left ventricular internal dimension at end-diastole indexed to aortic diameter (P < .0001, r2 = .30) or body surface area (BSA) (P = .0071, r2 = .10), and left ventricular internal dimension at end-systole indexed to aortic diameter (P = .0003, r- = .17) or BSA (P = .0008, r2 = .15).

Comparative sequences of canine and feline endothelin-1.

BACKGROUND: Endothelin-1 (ET-1, "mature ET-1") is a potent vasoconstrictor peptide that is made along with "big ET-1" from its precursor, preproET-1. Increased plasma concentrations of ET-1 and big ET-1 occur with various forms of cardiovascular disease in humans. Our laboratory is investigating plasma endothelins as diagnostic tests of cardiovascular disease in dogs and cats; however, commercial immunoassays designed specifically for use in dogs and cats are limited. OBJECTIVE: Amino acid sequences of feline and canine big ET-1 were obtained and used to predict antibody cross-reactivity with immunoassay test kits from other species. METHODS: Genomic DNA was extracted from peripheral blood and total RNA was extracted from canine and feline left ventricles for reverse transcription polymerase chain reaction (PCR) and PCR amplification of segments of the canine and feline preprohormone containing the big ET-1 sequences. The derived amino acid sequences were compared with known big ET-1 and ET-1 sequences of several other species, including human, mouse, and rat. RESULTS: Feline and canine big ET-1 had 87-97% and 89-100% homology, respectively, with that of other mammalian species. Canine ET-1 was identical to human, mouse, and rat ET-1. In contrast, the amino acid sequence of feline ET-1 was unique owing to a leucine for methionine substitution at position 7. CONCLUSIONS: It is highly likely that anti-human and anti-rodent ET-1 antibodies will cross-react with mature canine ET-1. In contrast, antibodies to mature ET-1 intended for use with feline tissues and antibodies to big ET-1 in either dogs or cats may have partial or no cross-reactivity depending on the peptide sequences used to produce the antibodies.

Inhibition of nuclear protein phosphatase activity in mouse hepatocytes by the cyanobacterial toxin microcystin-LR.

Microcystin-LR (MCLR) is a cyanobacterial hepatotoxin and protein phosphatase inhibitor that contaminates water reservoirs worldwide. MCLR localizes to the cytosol of hepatocytes, however, immunohistochemical studies indicate that it accumulates in the nucleus. MCLR toxicosis is associated with decreased hepatic protein phosphatase activity, but effects in nuclear protein phosphatase activity have not been investigated. Balb/c mice were given lethal (100 microg/kg) or sublethal (12, 23 and 45 microg/kg) i.p. doses of MCLR and hepatic nuclear extracts were analyzed for protein phosphatase 1 and 2A activity. There was profound inhibition of nuclear protein phosphatase activity within 50 min of lethal dosing, however an inhibition was not detected with sublethal doses. MCLR immunohistochemistry revealed widespread lobular staining in the lethal group and centrilobular staining in the sublethal groups. At the cellular level there was nuclear and cytoplasmic staining of equal intensity. As an indicator of nuclear protein phosphatase activity, the phosphorylation of p53, a nuclear phosphoprotein and known substrate for protein phosphatases 1 and 2A, was evaluated. Balb/c mice were treated with sublethal doses of MCLR or saline vehicle after induction of hepatic p53 by the DNA damaging agent diethylnitrosamine (DEN). P53 was immunoprecipitated and probed with phosphoserine specific antibodies by Western blotting. There was greater phosphoserine reactivity of p53 protein in animals treated with MCLR relative to saline treated controls, consistent with increased phosphorylation of serine sites. It is concluded that an interaction of this toxin with nuclear protein phosphatases occurs within 50 min of lethal dosing, which leads to a profound inhibition of enzymatic activity. Even sublethal doses of MCLR that do not result in significant inhibition of activity in bulk nuclei, result in detectable changes in phosphorylation of p53.

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.

Evaluation of the effect of pH on in vitro growth of Malassezia pachydermatis.

The purpose of this study was to evaluate the effects of pH on the growth of canine Malassezia pachydermatis isolates in vitro. Yeast growth was monitored by measuring the optical density with a spectrophotometer. The growth of American Type Culture Collection and field strains of M. pachydermatis was optimal between the pH values of 4.0 and 8.0, and inhibited at the ranges of 1.0 to 3.0 and 9.0 to 10.0. An analysis of covariance showed no significant differences among the growth curves at pH levels 5.0 to 8.0. Although specific contrast tests showed that the growth slope at pH 4.0 was significantly different from that at pH 5.0 to 8.0, only small, random differences were found when the growth slope at pH 4.0 was compared to the individual slopes at pH 5.0, 6.0, 7.0, and 8.0. The findings of this study suggest that topical acidifying products could be beneficial therapeutic options for cutaneous yeast infections in dogs.

Cloning and characterization of feline brain natriuretic peptide.

Brain (B-type) natriuretic peptide (BNP) is a cardiac hormone involved in regulation of fluid balance and blood pressure homeostasis of mammalian species. BNP sequence is species-specific and considered to be a significant prognostic and diagnostic marker for cardiac dysfunction. Using conventional polymerase chain reaction and amplification of cDNA 3'- and 5'-ends, a total of 1500 nucleotides encompassing the entire feline BNP gene were characterized. The feline BNP gene is organized in three exons separated by two introns. The complete transcript of 736 nucleotides was characterized, including 396 nucleotides encoding feline preproBNP. The preproBNP consisted of a signal peptide of 26 amino acids and a proBNP of 106 residues. The predicted mature BNP comprised 35 amino acids with likely 26- and 29-aa isomers, including a histidine residue at the C-terminus. Based on the similarity of BNP prepropeptide sequences, a phylogenetic relationship is presented for mammalian species including human, cat, cattle, dog, mouse, rat, sheep and swine.

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.

Validation of human haptoglobin immunoturbidimetric assay for detection of haptoglobin in equine and canine serum and plasma.

The Incstar(R) SPQ II human haptoglobin (Hpt) (Incstar Corporation, Stillwater, MN) immunoturbidimetric assay was validated for the determination of serum and plasma Hpt concentrations in dogs and horses. The anti-human Hpt antiserum supplied with the assay, displayed monospecificity to both dog and horse serum Hpt by immunoelectrophoresis and Western blotting techniques. The automated immunoturbidimetric assay results correlated well with the cyanmethemoglobin binding assay (r=0.953 for canine serum and r=0.941 for equine serum), and had excellent precision at both high and low serum Hpt concentrations (within run and between run coefficients of variation near or less than 5%). The assay was linear in both species by serial dilution of pooled-high serum with pooled-low serum, saline and with Hpt-free serum. Interference from hemolysis (> 25 mg/dl hemoglobin) and lipemia greater than 100 mg/dl caused a false decrease and false increase respectively in Hpt yield with the immunoturbidimetric assay. The anti-Hpt antibody supplied with the assay kit, once diluted with polymer diluent and stored at 4 degrees C, was stable for up to 6 days and gave consistent results.