Search

Immunoassays used for the measurement of salivary cortisol are limited by variable interference from cortisone. Salivary cortisone is a consequence of the salivary glands expressing 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) which converts cortisol to cortisone. We report a combined salivary cortisol and cortisone (SalF and SalE respectively) liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to address the cortisone cross-reactivity in cortisol immunoassays and as a tool to study 11beta-HSD2 activity. The method was linear up to 400 nmol/L for SalF and 200 nmol/L for SalE and the lower limits of quantitation were 0.39 nmol/L (SalF) and 0.78 nmol/L (SalE). No evidence of ion suppression was found and precision, accuracy and recovery were within internationally accepted limits. No interference was identified from 13 structurally related steroids. SalF, SalE and SalF/SalE were significantly greater in the morning than at bed-time and following stimulation of the adrenal glands. As serum cortisol increased, an exponential rise was observed in SalF and a linear increase in SalE which reached a plateau at higher SalF concentrations. We have developed a novel, robust LC-MS/MS assay for the combined measurement of SalF and SalE. Our results confirm the 11beta-HSD2 activity of the salivary glands resulting in high SalE concentrations and the enzyme saturation at high substrate concentrations. This method can be used as a simple, non-invasive and highly specific tool to assess the value of salivary cortisol as a surrogate for free serum cortisol and as a potential novel way to assess 11beta-HSD2 activity.

Objectives: To review aetiology, clinical signs and outcome of cats surgically treated for septic peritonitis (2000-2007)., Methods: A retrospective study. Inclusion criteria were the identification of intracellular bacteria and degenerate neutrophils and/or a positive culture from abdominal fluid and exploratory coeliotomy. Aetiology, clinical signs, haematological and biochemical parameters, surgical treatment and outcome were recorded and analysed., Results: Twenty-six cats fulfilled the inclusion criteria. Abdominal pain was reported in 10 (38 per cent) and vomiting was reported in 11 (42 per cent) of the cats. The most common aetiology was trauma (31 per cent). The principal source of contamination was the gastrointestinal tract. Hyperlactataemia, hypoproteinaemia and hyperglycaemia were reported in 9, 13 and 14 of the 26 cases, respectively. Non-survivors had significantly higher blood lactate concentrations than survivors (P=0.02). Nineteen cats were managed with primary closure, two with closed suction drains and three with open peritoneal drainage. Twelve (46 per cent) cats survived to discharge., Clinical Significance: In cats, lethargy, depression and anorexia were more common clinical signs than abdominal pain. Lactate level at the time of diagnosis may be a useful prognostic indicator in cats. The proportion of cats that survived was lower than previously reported and owners should be given a guarded prognosis.

Objective: To investigate the efficacy of adhesive incise drapes in reducing bacterial contamination of clean canine surgical wounds., Study Design: Randomized clinical trial., Animals: Dogs (n=100) having elective ovariohysterectomy or stifle surgery., Methods: Dogs were randomly assigned to 1 of 2 groups: drape or no drape. Swabs obtained from the inner edge of the surgical wound at the beginning (swab 1) and end (swab 2) of surgery were submitted for microbial culture. Number of colony forming units was counted for all positive cultures and change in bacterial counts between swabs 1 and 2 was calculated. Percentage adhesive drape adherence at the end of surgery was calculated from a digital photograph of the surgical site. duration of surgery/anesthesia and the anesthetic induction agent used were recorded., Results: There was a significant increase in bacterial counts between swabs 1 and 2 (P=.001). Wound contamination was 14% (6 drape; 8 no drape; P=0.78) with Staphylococcus spp. most commonly isolated. Median percentage drape adherence at the end of surgery was 89.3% (0-100%). Duration of anesthesia was significantly related to wound contamination (P=.013), but duration of surgery and anesthesia induction agent were not., Conclusions: Adhesive incise drapes did not reduce wound contamination of clean canine surgical wounds., Clinical Relevance: Use of adhesive incise drapes in clean surgical procedures is of questionable benefit in dogs.

Background: The recent interest of clinical laboratories in developing serum testosterone assays based on isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) stems from the lack of accuracy of direct immunoassays. In this study, we assessed the accuracy and state of standardization (traceability) of 4 published ID-LC-MS/MS procedures in a method comparison with an ID-gas chromatography (GC)-MS reference measurement procedure listed in the database of the Joint Committee for Traceability in Laboratory Medicine., Methods: The study used 58 specimens from different patient categories. Each specimen was measured in triplicate (ID-LC-MS/MS) and quadruplicate (ID-GC-MS) in independent runs., Results: The testosterone concentrations by ID-GC-MS were 0.2-4.4 nmol/L (women), 0.2-2.0 nmol/L (hypogonadal man), and 10.1-31.3 nmol/L (normogonadal men). For ID-GC-MS, the CV was nearly constant, with a median of 1.0%; for ID-LC-MS/MS, it was concentration-dependent, with a median of up to 8%. Weighted Deming regression gave mean slopes, intercepts, and correlation coefficients of 0.90-1.11, -0.055-0.013 nmol/L, and 0.993-0.997, respectively. The % difference plot showed between 7% and 26% of the results outside a total error limit of 14%, with median deviations from ID-GC-MS between -9.6 and 0.4%., Conclusions: This study demonstrated fairly good accuracy and standardization of the tested ID-LC-MS/MS procedures. Performance differences between procedures were evident in some instances, due to improper calibration and between-run calibration control. This emphasizes the need for thorough validation, including traceability, of new ID-LC-MS/MS procedures.

The measurement of 24 h urinary free cortisol is used in the investigation of patients with symptoms of hypercortisolism. Many different methods have been published for the measurement of cortisol, but most of these methods involve cumbersome pre-extraction of the cortisol prior to analysis. We have developed a method using in-well protein precipitation which serves to clean up the sample without requiring lengthy sample preparation. A Shimadzu SIL-HT autosampler was used to inject 50 microL of extract onto a Phenomemex Gemini C18 guard column attached to a Waters Xbridge C18 column. The eluant was introduced directly into a Waters Quattro Micro tandem mass spectrometer. The method was found to be linear up to 3448 nmol/L with a lower limit of detection of 5.3 nmol/L. Precision and accuracy were acceptable, and no interference was noted from compounds such as prednisolone or fenofibrate. This assay was compared to a previously published method, which uses solid phase extraction prior to LC-MS/MS analysis. We have developed a simplified, robust assay for the quantitation of urinary free cortisol that will increase the throughput of the assay and avoid the use of neurotoxic solvents such as dichloromethane.

Background: We aimed to develop a sensitive assay to quantitate serum concentrations of both androstenedione and testosterone within the female range simultaneously, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for use in the routine clinical laboratory and to compare this method with immunoassay., Method: Samples (200 microL) were prepared by liquid-liquid extraction using (1 mL) methyl-tert-butyl-ether. Deuterated androstenedione and testosterone were used as internal standards., Results: The standard curve was linear to 50 nmol/L, the lower limit of quantitation was 0.25 nmol/L, and intra- and inter-assay coefficients of variation were < 10% for both androgens over the range 0.3-35 nmol/L. There was a poor relationship between the LC-MS/MS and the radioimmunoassay methods for androstenedione with the LC-MS/MS generally giving lower results. For testosterone, the LC-MS/MS and immunoassay methods compared well at all concentrations. However, when female samples only were examined, the agreement deteriorated., Conclusions: We have developed a sensitive and precise LC-MS/MS method, which gives more accurate results for all androstenedione measurements and low testosterone concentrations than immunoassay.

The goal of this study was to evaluate the relationships between self-reported and measured height, weight, and body mass index (BMI) in a sample of eighth-grade students. The study population consisted of eighth-grade students in eastern North Carolina who completed a cross-sectional survey, self-reported their height and weight, and had their height and weight measured (N = 416). Fifty-nine percent of the sample was male; 42% African American, 46% white, and 12% other races. Mean self-reported weight (62.9 kg) was significantly lower than mean measured weight (64.4 kg). Mean self-reported BMI (22.8 kg/m2) was significantly lower than mean measured BMI (23.3 kg/m2). Race and BMI category were significantly associated with reporting errors. Specifically, African American and white students were significantly less likely to under-report their height compared to other race students. African American students were more likely to underestimate their weight compared to other race students. BMI was more likely to be underestimated in African American and white students compared to other race students. Students who were at risk for overweight and those that were overweight were more likely to underestimate their weight and BMI than students who were normal weight. Approximately 17% of students were misclassified in BMI categories when self-reported data were used. The results indicate that eighth-grade students significantly underestimate their weight, but on average provide valid estimates of their height. Race and measured BMI category influence this discrepancy. School-based research that addresses the prevalence of obesity in adolescents should utilize measured height and weight when feasible.

An eight-year-old, male neutered, crossbred dog was presented for investigation of a lingual mass of four months duration. Oral examination revealed a 7 cm × 5 cm soft, fluctuant mass at the caudal aspect of the tongue. Ultrasound examination of the mass demonstrated mixed echogenicity, with cavitations containing hypoechoic and anechoic regions. Lingual haemangiosarcoma was diagnosed on histopathological examination of multiple biopsy samples, with confirmation of the vascular endothelial origin of tumour cells by positive immunolabelling for factor VIII-related antigen.

Background: Glomerular filtration rate (GFR) can be estimated using creatinine clearance or calculated formulae, methods which rely on creatinine quantification in serum. Creatinine is most frequently measured using the Jaffe method, but this is prone to interference by, for example, bilirubin, protein and ketones. Recent amendments to this assay have been made by manufacturers in an attempt to compensate for the protein interference., Methods: We developed a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the analysis of creatinine in serum, using a strong cation exchange column to give a short run time of 1.5 min and minimal ion suppression. The method had a lower limit of quantification of 5 micromol/L and the intra- and inter-assay imprecision was <5% and <7%, respectively. Serum samples (n = 182) were subsequently analysed by enzymatic, compensated Jaffe and LC-MS/MS methods and results compared., Results: The compensated Jaffe and enzymatic methods gave similar results to the LC-MS/MS method at concentrations below 150 micromol/L. At concentrations above this value, creatinine was underestimated by both the compensated Jaffe and enzymatic methods compared with the LC-MS/MS method., Conclusions: We have developed a novel, simple, sensitive LC-MS/MS assay for use in comparative studies or when the results of either Jaffe or enzymatic assays are in question.

The bovine herpesvirus-1 (BHV-1) genome was analysed by Southern blot hybridization using the herpes simplex virus type 1 (HSV-1) DNA polymerase gene as a probe. A 2.5 kilobase region which hybridized specifically to the HSV-1 DNA polymerase gene was identified within the Hind III G fragment at approximate map units 0.334-0.352. In order to provide further evidence that this is the location of the BHV-1 DNA polymerase gene, the 2.5 kilobase region was cloned and part of it sequenced. An uninterrupted stretch of over 800 nucleotides was obtained and an open reading frame spanning the entire sequence was identified. The amino acid sequence that it encodes shows striking homology to a region within the C-terminal half of other herpesvirus DNA polymerases.