Canine plasma cortisol (hydrocortisone) measured by radioimmunoassay: clinical absence of diurnal variation and results of ACTH stimulation and dexamethasone suppression tests.

A radioimmunoassay for plasma cortisol (hydrocortisone) was developed and validated for sensitivity, specificity, accuracy, precision, and parallelism. Steroids were extracted with ethyl ether, and cortisol was purified by gel column chromatography prior to assay. [1,2-3H] cortisol and a commercially available sheep antibody to cortisol-21-hemisuccinate were used. Free steriods were separated from bound steroids by centrifugation after adsorption to dextran-coated charcoal. Plasma cortisol was measured by this technique in 6 normal dogs. Circadian rhythm of cortisol secretion was not detected in samples obtained by venipuncture at 8 different hours on 3 separate days, suggesting that adrenal function tests may be started in clinical patients at any time of day. Resting plasma cortisol concentrations averaged 19.4+/-3.0 (SD) ng/ml and ranged from nondetectable (less than 3 ng/ml) to 77.5 ng/ml. Of 144 canine plasma samples, 95% contained less than 50 ng of cortisol/ml. Intramuscular injection of 2.2 units of adrenocorticotropic hormone/kg of body weight caused detectable increase in plasma cortisol concentrations; maximum response (68.3 to 111.6 ng/ml) occurred 1 to 2 hours after injection. Oral administration of dexamethasone suppressed plasma cortisol to nondetectable concentrations for 32 hours in all 6 dogs.     

Serum hydrocortisone (cortisol) values in normal and adrenopathic dogs as determined by radioimmunoassay

Using a specific radioimmunoassay, serum hydrocortisone values were measured in dogs. Between 9:00 AM and 10:00 AM, the base-line hydrocortisone value for 56 clinically normal dogs ranged from 6.0 to 28.5 ng/ml, with a mean value of 17.8 +/- 1.32 ng/ml (mean +/- SEM). Marked differences due to age, sex, body weight, or breed were not observed in the hydrocortisone values. In 11 dogs with definitive hyperadrenocorticism, serum hydrocortisone values were from 32 to 148 ng/ml. In 2 dogs with hyperadrenocorticism, the values were 4.5 and 3.1 ng/ml. The estimation of serum hydrocortisone values by radioimmunoassay is simple and precise, and can be utilized to aid in the diagnosis of adrenopathy in dogs.     

Effects of storage on concentration of hydrocortisone (cortisol) in canine serum and plasma

A specific radioimmunoassay was used to measure concentrations of hydrocortisone (cortisol) in the serum and plasma of 4 dogs. Differences (P greater than 0.05) in concentrations of cortisol were not found between serum and plasma (from EDTA-treated and heparinized blood samples). Differences (P greater than 0.05) in serum or plasma concentrations of cortisol were not found between samples stored at 4 C for various times (10 minutes, 10 hours, 40 hours) after collection, but before removal of RBC. In a study designed to determine the stability of cortisol in serum samples stored at room temperature, degradation was dependent on the initial serum concentrations of cortisol. Decreases (P greater than 0.05) did not occur in concentrations of cortisol in serum samples stored up to 15 days when initial concentrations of cortisol were less than 15 ng/ml. However, when initial concentrations of cortisol were approximately 55 ng/ml and 80 ng/ml, significant (P greater than 0.05) degradation occurred after 9 and 5 days of storage, respectively. Results of this investigation indicate that either serum or plasma of dogs is suitable for radioimmunoassay of cortisol and that samples (with and without added coagulants) incubated at 4 C may be left uncentrifuged for up to 40 hours without cortisol degradation. However, prolonged storage of serum at room temperature is detrimental, particularly for samples having large concentrations of cortisol.     

Sodium retention and cortisol (hydrocortisone) suppression caused by dexamethasone and triamcinolone in equids

Three ponies and 1 horse were bilaterally adrenalectomized (BADX). The initial hypoadrenal episode after BADX was reversed with 20 mg of dexamethasone (DXM) IM (n = 2) or 20 mg of triamcinolone (TMC) IM (n = 2). Nine hypoadrenal crises were reversed with 20 mg of DXM given IM (n = 4) or 20 mg of TMC given IM (n = 5). Sodium and chloride retention and potassium excretion were documented based on changes in serum electrolytes and urinary excretion. Eight intact adult horses were randomly assigned to 2 groups to study the effects of a single IM injection of DXM (0.044 mg/kg of body weight) or TMC (0.044 mg/kg). Cortisol (hydrocortisone) suppression was found to be maximal (nondetectable amounts of cortisol) by 12 hours in both groups. Cortisol was again detectable in the DXM group at 24 hours after injection and was at pretreatment values at 168 hours. Cortisol was not detectable in the TMC group for 192 hours and did not reach pretreatment values until 336 hours. The duration of the gluconeogenic effect was compared with the duration of cortisol suppression exerted by DXM and TMC in these intact animals. Assuming that the decrease in plasma glucose coincides with the decrease in glucocorticoid activity of the respective steroid, a relative hypoadrenocortical state was found in the animals treated with DXM between the 2nd and 7th day after treatment, whereas this state occurred between the 6th and 14th day after treatment with TMC.     

Diurnal variation of peripheral plasma levels of testosterone in bulls measured by a rapid radioimmunoassay procedure

A radioimmunoassay technique, using antibodies to testosterone-3-(O-carboxymethyl) oxime-bovine serum albumin, has been applied for the measurement of peripheral plasma levels of testosterone in bulls. The individual as well as the mean 24-hr. pattern of peripheral plasma levels of testosterone in the four bulls investigated showed three distinct peaks. The mean peripheral plasma level of testosterone peaked around 10 p.m., around 6 a.m. and around noon, the mean levels obtained at these times being 6.2, 6.1 and 4.2 ng/ml, respectively. In one bull an additional peak occurred around 2 a.m. (8.2 ng/ml). Significantly lower peripheral plasma levels of testosterone were observed between the peak values.     

Effects of sample handling on adrenocorticotropin concentration measured in canine plasma, using a commercially available radioimmunoassay kit.

A commercially available radioimmunoassay (RIA) kit for measurement of human adrenocorticotropin (hACTH) was validated for use in dogs. Assay sensitivity was 3 pg/ml. Intra-assay coefficient of variation (x 100; CV) for 3 canine plasma pools was 3.0 (mean +/- SD, 33 +/- 0.99 pg/ml), 4.2 (71 +/- 2.4 pg/ml) and 3.7 (145 +/- 3.7 pg/ml) %. Interassay CV for 2 plasma pools measured in 6 assays was 9.8 (37 +/- 3.6 pg/ml) and 4.4 (76 +/- 3.4 pg/ml) %, respectively. Dilutional parallelism was documented by assaying 2 pools of canine plasma at 3 dilutions and correcting the measured result for dilution. Corrected mean concentrations for the first pool were 33 (+/- 0.99), 36 (+/- 4.3), and 33 (+/- 6.8) pg/ml; corrected mean concentrations for the second pool were 145 (+/- 5.4), 141 (+/- 10.8) and 125 (+/- 3.4) pg/ml. Recovery of 1-39hACTH added to canine plasma (6.25, 12.5, 25.0, 50.0, and 100.0 pg/ml) was linear and quantitative (slope = 0.890, R2 = 0.961). To test whether anticoagulant or the protease inhibitor, aprotinin, influences ACTH concentration in canine plasma, ACTH was measured in canine blood collected in 4 tubes containing anticoagulant: heparin (H), heparin + 500 kallikrein inhibitor units (KIU) of aprotinin/ml (HA), EDTA (E), and EDTA + aprotinin (EA). Plasma ACTH concentration was the same when samples containing H and HA, or HA and E were compared, and was significantly (P less than 0.01) lower in samples containing EA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of chronic hypocortisolaemia on plasma cortisol concentrations during intravenous infusions of hydrocortisone sodium succinate in dogs

Intravenous infusions of hydrocortisone sodium succinate (HSS) were given at 0·625 mg kg⁻¹ hour⁻¹ and 0·312 mg kg⁻¹ hour⁻¹ to six dogs. Plasma cortisol concentrations were measured by radioimmunoassay at 0, 15, 30, 45 and 60 minutes and then every 30 minutes for a further five hours. Chronic hypocortisolaemia was induced and maintained with mitotane and the HSS infusions were repeated after 31 and 50 days. No statistically significant difference was observed in the plasma cortisol concentrations after either period of hypocortisolaemia, but the plasma cortisol concentrations tended to be higher in most of the dogs.     

Feline infectious peritonitis. Proteins of plasma and ascitic fluid.

Electrophoreses of sera, plasma and ascitic fluids of cats with natural or experimental infectious peritonitis show important modifications. Special stainings of electrophoreses and chromatographic and immunoelectrophoretic technics characterized some of the modified proteins. In the experimental disease, fibrinogen, haptoglobin, transferrin, and probably orosomucoid are increased; in the natural disease, in addition to these modifications, the gamma-globulins are strongly increased; the immunoglobulins found in the often abundant ascitic fluid belong to the IgG class. Increased proteins such as fibrinogen, haptoglobin and orosomucoid and decreased albumin are aspecific aspects of inflammatory processes, whereas hypergammaglobulinemia appears in the course of immunological response. The rapid evolution of the experimental disease explains the fact that immunoglobulins do not increase.     

Feline T-Lymphotropic Virus (FTLV) (Feline Immunodeficiency Virus Infection) in cats in New Zealand

AIM: To identify and enumerate colony forming units (cfu) of mastitis pathogens in bulk tank milk (BTM) from pasture-fed New Zealand dairy cows in the Waikato region.

METHODS: BTM samples from seven seasonal-calving dairy herds in the Waikato region were collected monthly from August to December 2004 (cows calved during July-September). Milk samples were cultured on blood aesculin and MacConkey agar plates for 24 h, and the number of mastitis pathogens identified and counted.

RESULTS: Colonies identified in BTM included aesculinpositive streptococci, Staphylococcus aureus, coagulase-negative staphylococci (CNS), and coliforms; counts ranged from zero to >1,000 cfu/ml. Counts >1,000 cfu/ml for total aesculin-positive streptococci, CNS and coliforms were present in 48%, 51% and 11% of BTM samples, respectively. Counts of Staph. aureus ranged from zero to 1,000 cfu/ml, but first appeared in BTM samples only in October. Staphylococcus aureus was repeatedly isolated in BTM from 4/7 farms during the testing period.

CONCLUSIONS: Counts of mastitis pathogens in this study appeared high relative to interpretive criteria set by other workers, which may indicate a high prevalence of mastitis risk factors on these farms. Interpretation of results is difficult as aesculinpositive streptococci, CNS and coliforms can be isolated from the environment as well as from cows with clinical or subclinical mastitis. Furthermore, Staph. aureus is inconsistently excreted from infected bovine mammary glands. More extensive study of this method is required in New Zealand to attempt to further validate the interpretation of results of bacterial culture of BTM.

CLINICAL RELEVANCE: This method may be used to monitor challenge from mastitis pathogens over time as part of milk quality control programmes. The technique may be of use as a screening test to provide information to veterinarians, affording them the opportunity to have an input into mastitis control on dairy farms in New Zealand.     

Increased free cortisol in plasma of dogs with portosystemic encephalopathy (PSE)

Dogs with portosystemic encephalopathy (PSE) are known to develop pituitary-dependent hyperadrenocorticism, but there have been no reports on the plasma protein binding of cortisol in these dogs. Since the liver is involved in the synthesis of corticosteroid-binding globulin (CBG) and other transport proteins for cortisol, the binding characteristics of these proteins and thus the biologically-active free fraction of cortisol might be altered in dogs with PSE. We investigated the total concentration of cortisol and the free fraction and the free cortisol concentration in plasma of thirty-two dogs with PSE due to inherited portosystemic shunts or chronic active hepatitis with cirrhosis. We found a significantly higher free fraction (14.7 ± 5.8%, P<0.0001) and free cortisol concentration (26.3 ± 23.1 nM, P<0.001) in these dogs than in healthy controls (8.2 ± 2.3% and 9.2 ± 7.2 nM, respectively). Moreover, basal concentrations of total cortisol in the dogs with PSE were higher than in the healthy controls (190 ± 146 nM v. 107 ± 65, P<0.01). The per cent free cortisol in plasma was not significantly correlated with the concentration of albumin or the total cortisol in plasma. We conclude that there is decreased binding of cortisol in plasma of dogs with PSE due to decreased hepatic synthesis of cortisol binding proteins. The presence of increased concentrations of free cortisol in these dogs indicates that their basal pituitary-adrenocortical activity was increased, probably due to aberrant neurotransmission in brain centers associated with pituitary function, as a result of hepatic encephalopathy.     

Rapid radioimmunoassay of progesterone in unextracted bovine plasma.

A rapid radioimmunoassay for determining progesterone levels in unextracted bovine plasma has been developed. Two antisera have been tested, anti-deoxycorticosterone-21-hemisuccinate human serum albumin and antiprogesterone-11-hemisuccinate bovine serum albumin. The usefulness of an antiserum for rapid assays has been based on its agreement with assays of organic extracts or chromatographically purified extracts of selected bovine samples. To avoid large blank effects and non-specific interference, a plasma volume of 20 microliter that provided adequate sensitivity was not exceeded. All correlations between rapid assays, assays using organic extraction or assays after chromatographic purification were greater than 0.9 and showed regression slopes approximating unity. It was concluded that individual lots of antisera should be evaluated thoroughly before use in a rapid assay.     

Feline Hemoperitoneum 16 Cases (1986-1993)

Sixteen cases of feline, non-traumatic hemoperitoneum were evaluated retrospectively. The median age was 5.75 years (range 1.5 - 16 years). There were eight male and eight female cats. Common presenting complaints (n=13) were anorexia (37%), lethargy (31%), and recumbency (31%). Physical examination findings (n=11) included depressed mentation (100%), hypothermia (89%), pale mucous membranes (82%), and poor quality pulses (80%). The median initial peripheral packed cell volume (n=11) was 24% (range 17-55%). In four out of six cases where abdominocentesis was performed, the packed cell volume of the abdominal fluid ranged from 18% to 24%, and matched the peripheral packed cell volume (range 15 - 26%). Some common abnormalities in the serum chemistry screens 9n=6) were elevated alanine aminotransferase in 83% (5/6) of the cats (range 55-5828 U/l) and elevated alkaline phosphatase in 50% (3/6) of the cats (range 18-402 U/l). Ten cats (63%) were euthanized, three (19%) were presented dead on arrival, two (12%) are still alive, and one (6%) were euthanized, three (19%) were presented dead on arrival, two 912%) are still alive, and one (6%) died. The causes of hemoperitoneum were hepatic neoplasia (31%), hepatic necrosis (19%), hepatic amyloidosis (13%), non hepatic neoplasia (13%), hepatopathy (6%), hepatic rupture (6%), necrotic/hemorrhagic cystitis (6%), and ruptured bladder (6%).