Authentication of beef versus horse meat using 60 MHz 1H NMR spectroscopy.

This work reports a candidate screening protocol to distinguish beef from horse meat based upon comparison of triglyceride signatures obtained by 60 MHz (1)H NMR spectroscopy. Using a simple chloroform-based extraction, we obtained classic low-field triglyceride spectra from typically a 10 min acquisition time. Peak integration was sufficient to differentiate samples of fresh beef (76 extractions) and horse (62 extractions) using Naïve Bayes classification. Principal component analysis gave a two-dimensional "authentic" beef region (p=0.001) against which further spectra could be compared. This model was challenged using a subset of 23 freeze-thawed training samples. The outcomes indicated that storing samples by freezing does not adversely affect the analysis. Of a further collection of extractions from previously unseen samples, 90/91 beef spectra were classified as authentic, and 16/16 horse spectra as non-authentic. We conclude that 60 MHz (1)H NMR represents a feasible high-throughput approach for screening raw meat.

60 MHz 1H NMR spectroscopy for the analysis of edible oils.

We report the first results from a new 60 MHz 1H nuclear magnetic resonance (NMR) bench-top spectrometer, Pulsar, in a study simulating the adulteration of olive oil with hazelnut oil. There were qualitative differences between spectra from the two oil types. A single internal ratio of two isolated groups of peaks could detect hazelnut oil in olive oil at the level of ∼13%w/w, whereas a whole-spectrum chemometric approach brought the limit of detection down to 11.2%w/w for a set of independent test samples. The Pulsar's performance was compared to that of Fourier transform infrared (FTIR) spectroscopy. The Pulsar delivered comparable sensitivity and improved specificity, making it a superior screening tool. We also mapped NMR onto FTIR spectra using a correlation-matrix approach. Interpretation of this heat-map combined with the established annotations of the NMR spectra suggested a hitherto undocumented feature in the IR spectrum at ∼1130 cm-1, attributable to a double-bond vibration.

Effectiveness of two statin prescribing strategies with respect to adherence and cardiovascular outcomes: observational study.

BACKGROUND: There is considerable evidence that statins can reduce cardiovascular events. Currently high-risk patients are treated to a target cholesterol concentration. An alternative prescribing strategy (the 'fire-and-forget' approach) would instead deploy low-dose statins more widely. It has been suggested that for the same cost this approach might prevent more cardiovascular events. We have compared the treat-to-target and fire-and-forget statin prescribing strategies with respect to adherence and cardiovascular outcomes. METHODS: We used a population-based record-linkage database containing several data sets linked by a unique patient identifier. We identified two cohorts of patients. Patients in the treat-to-target cohort were prescribed a statin, and subsequent measurement of their cholesterol was followed by upward titration of their statin dose if necessary. Patients in the fire-and-forget cohort were prescribed a statin, but no further cholesterol measurement was observed during the follow-up period. FINDINGS: Adherence to statin treatment in patients treated to target was significantly better than in patients treated on a fire-and-forget basis (adjusted odds ratio 2.51, 95%CI 2.26-2.78). We found a lower cardiovascular disease (CVD) event rate in patients treated to target than in fire-and-forget patients (hazard ratio of CVD or cardiovascular death 0.41 (0.35-0.48) even after adjustment was made for adherence and baseline CVD risk). INTERPRETATION: Our findings suggest that adherence to statins is worse in patients treated on a fire-and-forget basis than in patients treated to a target cholesterol concentration, and that this prescribing strategy is associated with worse cardiovascular outcomes.

Inefficacy of selegiline in treatment of canine pituitary-dependent hyperadrenocorticism.

OBJECTIVE: To evaluate selegiline, a monoamine oxidase-B inhibitor, for treating dogs with pituitary-dependent hyperadrenocorticism. DESIGN: Prospective clinical trial using client-owned dogs with pituitary-dependent hyperadrenocorticism treated at The University Veterinary Centre, Sydney, from September 1999 to July 2001. PROCEDURE: Eleven dogs with pituitary-dependent hyperadrenocorticism treated with selegiline were monitored at days 10, 30 and 90 by clinical examination, tetracosactrin stimulation testing, urinary corticoid:creatinine ratio measurement and client questionnaire. Endogenous adrenocorticotropic hormone measurements were also performed on most dogs on days 0 and 90. No dog treated with selegiline had satisfactory control of disease. CONCLUSION: Selegiline administration was safe and free of side-effects at the doses used, but did not satisfactorily control disease in pituitary-dependent hyperadrenocorticism affected dogs.

Creatinine in the dog: a review.

Creatinine is the analyte most frequently measured in human and veterinary clinical chemistry laboratories as an indirect measure of glomerular filtration rate (GFR). Although creatinine metabolism and the difficulties of creatinine measurement have been reviewed in human medicine, similar reviews are lacking in veterinary medicine. The aim of this review is to summarize information and data about creatinine metabolism, measurement, and diagnostic significance in the dog. Plasma creatinine originates from the degradation of creatine and creatine phosphate, which are present mainly in muscle and in food. Creatinine is cleared by glomerular filtration with negligible renal secretion and extrarenal metabolism, and its clearance is a good estimate of GFR. Plasma and urine creatinine measurements are based on the nonspecific Jaffé reaction or specific enzymatic reactions; lack of assay accuracy precludes proper interlaboratory comparison of results. Preanalytical factors such as age and breed can have an impact on plasma creatinine (P-creatinine) concentration, while many intraindividual factors of variation have little effect. Dehydration and drugs mainly affect P-creatinine concentration in dogs by decreasing GFR. P-creatinine is increased in renal failure, whatever its cause, and correlates with a decrease in GFR according to a curvilinear relationship, such that P-creatinine is insensitive for detecting moderate decreases of GFR or for monitoring progression of GFR in dogs with severely reduced kidney function. Low sensitivity can be obviated by determining endogenous or exogenous clearance rates of creatinine. A technique for determining plasma clearance following IV bolus injection of exogenous creatinine and subsequent serial measurement of P-creatinine does not require urine collection and with additional studies may become an established technique for creatinine clearance in dogs.

Trilostane treatment in dogs with pituitary-dependent hyperadrenocorticism.

OBJECTIVE: To evaluate the efficacy of trilostane in treating dogs with pituitary-dependent hyperadrenocorticism. DESIGN: Prospective clinical trial using client-owned dogs with pituitary-dependent hyperadrenocorticism treated at University Veterinary Centre, Sydney from September 1999 to July 2001. PROCEDURE: Thirty dogs with pituitary-dependent hyperadrenocorticism treated with trilostane, a competitive inhibitor of 3beta-HSD, were monitored at days 10, 30 and 90 then 3-monthly by clinical examination, tetracosactrin stimulation testing, urinary corticoid:creatinine ratio measurement and by client questionnaire. RESULTS: Twenty-nine of 30 dogs were successfully treated with trilostane (median dose 16.7 mg/kg; range 5.3 to 50 mg/kg, administered once daily); one responded favourably but died of unrelated disease before full control was achieved. CONCLUSION: Trilostane administration controlled pituitary-dependent hyperadrenocorticism in these dogs. It was safe, effective and free of side-effects at the doses used. Most dogs were initially quite sensitive to the drug for 10 to 30 days, then required higher doses until a prolonged phase of stable dose requirements occurred. Urinary corticoid:creatinine ratio was useful in assessing duration of drug effect. Some dogs treated for more than 2 years required reduction or temporary cessation of drug because of iatrogenic hypoadrenocorticism.

Evaluation of d,l-ethionine as a mechanism for pancreatic islet regeneration in dogs.

OBJECTIVE: To determine whether induction of pancreatic necrosis and islet proliferation by d,l-ethionine has potential for treating dogs with beta-cell insufficiency. DESIGN: Eighteen mixed breed dogs of both sexes were given d,l-ethionine at 100 mg/kg three times weekly for 2 weeks; 6 dogs were euthanased at 2, 14 and 28 d after the last dose. METHODS: Clinical signs during administration and recovery were assessed. Routine biochemical analyses were performed before each ethionine dose and then once weekly. Faecal samples were examined weekly for malassimilated nutrients and blood. Blood coagulation screening tests (OSPT and APTT) were determined on four dogs after ethionine administration. Intravenous glucose tolerance tests were conducted before the first and after the last ethionine dose and then fortnightly. All dogs were necropsied and pancreas, liver, kidney and jejunum were examined microscopically. RESULTS: During ethionine administration all animals displayed vomiting, inappetence, diarrhoea (often with blood), weight loss and depression. Three dogs were euthanased prematurely due to severe illness, but those allowed to recover were eating and brighter 7 d after cessation of ethionine administration. Serum concentrations of TLI, amylase and lipase increased initially, then decreased, during administration but retumed to normal during recovery. Concentrations of ALT, ALP, unconjugated and conjugated bilirubin increased during administration then decreased slowly. Histological examination revealed hepatic lipidosis and necrosis, but no renal or jejunal lesions. In most dogs, faecal examination demonstrated increased undigested starch and muscle, as well as increased digested and undigested fat, during ethionine administration or early during the recovery period, suggesting transient malassimilation. APTT was unchanged but OSPT was prolonged in all dogs. There was no impairment of insulin secretion or glucose intolerance and C-peptide concentrations were unaffected. Immediately after ethionine administration there was delayed insulin degradation and by day 43 there was evidence of increased insulin sensitivity. CONCLUSION: d,l-ethionine administration in dogs appeared not to interfere with insulin secretion, but caused clinical signs and laboratory changes indicative of pancreatic exocrine necrosis, severe hepatobiliary disease and transient malassimilation. Pancreatic and hepatic dysfunction was severe but clinical recovery occurred after ethionine administration ceased. The severe side-effects observed with d,l-ethionine should preclude its potential use for treating diabetes mellitus in dogs.

Plasma exogenous creatinine clearance test in dogs: comparison with other methods and proposed limited sampling strategy.

Plasma clearance of creatinine was evaluated for assessment of glomerular filtration rate (GFR) in dogs. In 6 healthy dogs (Experiment 1), we determined 24-hour urine clearance of endogenous creatinine, plasma, and urine clearances of exogenous creatinine administered at 40, 80, and 160 mg/kg in a crossover design (linearity study), plasma iothalamate clearance, and plasma and urine clearances of 14C-inulin. In Experiment 2, plasma creatinine and iothalamate clearances were compared, and a linearity study was performed as for Experiment 1 in 6 dogs with surgically induced renal impairment. Experiment 3 compared plasma creatinine clearance with plasma iothalamate clearance before and 3 weeks after induction of moderate renal impairment in 6 dogs. Plasma creatinine clearances were calculated by both noncompartmental and compartmental analyses. In Experiment 1, plasma inulin clearance was higher (P < .001) than other clearance values. Plasma creatinine clearances at the 3 dose rates did not differ from urine inulin clearance and each other. In Experiment 2, plasma creatinine clearances were about 14% lower than plasma iothalamate clearance (P < .05). In Experiment 3, decreases in GFR assessed by plasma clearances of iothalamate and creatinine were similar. Renal failure decreased the daily endogenous input rate of creatinine by 25%. Limiting sampling strategies for optimizing GFR calculation were proposed, allowing an error lower than 6.5% with 4 blood samples. These results suggest that determination of plasma creatinine clearance by a noncompartmental approach offers a reliable, inexpensive, rapid, and convenient means of estimating GFR in routine practice.

Evidence for a role of phospholipid oxidation products in atherogenesis.

There is growing evidence for the accumulation of phospholipid oxidation products (some of which can also be formed enzymatically) in several chronic disease processes including atherosclerosis. There also is considerable evidence that enzymes involved in hydrolysis of these phospholipids (present in both lipoproteins and cells) may be important in regulation of atherogenesis. In vitro studies suggest that these lipids can activate vascular wall cells to states that contribute to the atherosclerotic process. This review focuses on two types of bioactive phospholipids: phosphatidyl cholines in which the sn-2 fatty acid has been modified by oxidation and lysophosphatidic acid in which both the sn-2 and sn-3 positions have been modified. The mechanism by which these phospholipid oxidation products activate cells has revealed the presence of several different receptors and signal transduction pathways.

Small bowel motility and colonic transit are altered in dogs with moderate renal failure.

Although gastrointestinal complications are common in patients with renal disease, the effects of renal dysfunction on bowel motility and gut transit times are not well known. We assessed gastrointestinal electromyographic activity, gastric emptying rate, orocolonic transit time, oroanal transit time, and xylose absorption before and after surgically inducing a 66% decrease in glomerular filtration rate in dogs. Moderate renal failure induced no gross or microscopic gastrointestinal lesions but caused a 16-42% increase in gastrointestinal motility indexes. We found a 24% decrease in the propagation velocity of the myoelectrical migrating complex in the duodenojejunal segment, a 30% decrease in phase I duration in duodenal and jejunal regions, a 20% increase in the total irregular electrical activity of the small intestine, and a 22% increase in duration of the meal response in the duodenum and jejunum. Renal failure did not change xylose absorption, gastric emptying rate, and orocolonic transit time but decreased colonic transit time by 38%. The mean weight of feces was increased. These results indicate that moderate renal failure alters duodenojejunal motility and decreases colonic transit time.

HDL and the inflammatory response induced by LDL-derived oxidized phospholipids.

Oxidation of low density lipoprotein (LDL) phospholipids containing arachidonic acid at the sn-2 position occurs when a critical concentration of "seeding molecules" derived from the lipoxygenase pathway is reached in LDL. When this critical concentration is reached, the nonenzymatic oxidation of LDL phospholipids produces a series of biologically active, oxidized phospholipids that mediate the cellular events seen in the developing fatty streak. Normal high density lipoprotein (HDL) contains at least 4 enzymes as well as apolipoproteins that can prevent the formation of the LDL-derived oxidized phospholipids or inactivate them after they are formed. In the sense that normal HDL can prevent the formation of or inactivate these inflammatory LDL-derived oxidized phospholipids, normal HDL is anti-inflammatory. HDL from mice that are genetically predisposed to diet-induced atherosclerosis became proinflammatory when the mice are fed an atherogenic diet, injected with LDL-derived oxidized phospholipids, or infected with influenza A virus. Mice that were genetically engineered to be hyperlipidemic on a chow diet and patients with coronary atherosclerosis, despite normal lipid levels, also had proinflammatory HDL. It is proposed that LDL-derived oxidized phospholipids and HDL may be part of a system of nonspecific innate immunity and that the detection of proinflammatory HDL may be a useful marker of susceptibility to atherosclerosis.

Dimeric W3SO3 cluster complexes: synthesis, characterization, and potential applications as X-ray contrast agents.

Our continued research on the use of heavy metal cluster complexes as a new class of X-ray contrast agents in medical diagnostic imaging is described. A series of 2:3 cluster-ligand complexes, [(W(IV)3SO3)2L3]4- (L = linear polyaminopolycarboxylate ligands), were isolated from the reaction of aqua ion [W(IV)3SO3(H2O)9]4- (prepared in large quantities through an improved literature process) with respective ligands in refluxing DMF. The salts of [(W(IV)3SO3)2L3]4- complex anions were fully characterized using routine techniques such as elemental analysis, MS, HPLC, UV-vis, IR, and NMR. The solid structures of two complex anions, [(W(IV)3SO3)2(PDTA)3]4- and [(W(IV)3SO3)2(HO-PDTA)3]4-, were determined by X-ray crystallography. They are the first examples wherein two W(IV)3SO3 clusters are complexed and linked by three ligands that contain two terminal iminodiacetate (bis-IDA) groups. Complexation of the unstable aqua ion [W(IV)3SO3(H2O)9]4- with ligands has imparted desired biological compatibility to the tungsten metal cluster. These complexes are stable and highly soluble in H2O. The potential utility of such tungsten cluster complexes as X-ray contrast agents was evaluated in both in vitro and in vivo animal studies. In addition, the syntheses of several new linear polyaminopolycarboxylate ligands used in this study are reported.

Systemic antibacterial drug use in dogs in Australia.

A survey of veterinarians' use of antibacterial drugs was conducted by distributing a questionnaire to Australian practitioners. Respondents were asked to indicate their general patterns of use of various systemic antibacterial drugs and drug combinations in dogs and their approach to certain specified clinical disorders. Overall, antibacterials of the beta-lactam type (penicillins and cephalosporins) were most commonly used. Other antibacterials with substantial use were doxycycline, sulphonamide-trimethoprim, metronidazole, fluoroquinolones and clindamycin. Drug selection for different disorders was generally appropriate when compared with recommendations in recent texts, although inappropriate use was evident in some circumstances.

Ssn6-Tup1 interacts with class I histone deacetylases required for repression.

Ssn6-Tup1 regulates multiple genes in yeast, providing a paradigm for corepressor functions. Tup1 interacts directly with histones H3 and H4, and mutation of these histones synergistically compromises Ssn6-Tup1-mediated repression. In vitro, Tup1 interacts preferentially with underacetylated isoforms of H3 and H4, suggesting that histone acetylation may modulate Tup1 functions in vivo. Here we report that histone hyperacetylation caused by combined mutations in genes encoding the histone deacetylases (HDACs) Rpd3, Hos1, and Hos2 abolishes Ssn6-Tup1 repression. Unlike HDAC mutations that do not affect repression, this combination of mutations causes concomitant hyperacetylation of both H3 and H4. Strikingly, two of these class I HDACs interact physically with Ssn6-Tup1. These findings suggest that Ssn6-Tup1 actively recruits deacetylase activities to deacetylate adjacent nucleosomes and promote Tup1-histone interactions.

Determinants of bioactivity of oxidized phospholipids. Specific oxidized fatty acyl groups at the sn-2 position.

We previously described 3 bioactive oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC) containing oxovaleroyl (POVPC), glutaroyl (PGPC), and epoxyisoprostane (PEIPC) groups at the sn-2 position that were increased in minimally modified/oxidized low density lipoprotein (MM-LDL) and rabbit atherosclerotic lesions. We demonstrated specific and contrasting effects of POVPC and PGPC on leukocyte-endothelial interactions and described an effect of PEIPC on monocyte binding. The major purpose of the present study was to determine the effects of structural changes on the bioactivities of these 3 lipids. We demonstrate herein that the group at the sn-2 position determines the specific bioactivity and that the substitution of stearoyl for palmitoyl at the sn-1 position or ethanolamine for choline at the sn-3 position of the phospholipid did not alter bioactivity. Oxidized PAPC, oxidized 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine, and oxidized 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphorylethanolamine stimulated monocyte binding and inhibited lipopolysaccharide-induced expression of the neutrophil-binding molecule E-selectin. Furthermore, all oxovaleroyl phospholipids but not the glutaroyl phospholipids induced monocyte binding without an increase in vascular cell adhesion molecule-1 (VCAM-1) expression and inhibited lipopolysaccharide-induced E-selectin expression. In contrast, glutaroyl phospholipids but not oxovaleroyl phospholipids stimulated E-selectin and VCAM-1 expression. We further demonstrate that all parts of the phospholipid molecules are required for these bioactivities. Hydrolysis with phospholipase (PL) A(1), PLA(2), and PLC strongly reduced the bioactivities of POVPC, PGPC, and mixed isomers of PEIPC. PLD had a smaller but still significant effect. The effects of POVPC and PEIPC could be abolished by sodium borohydride treatment, indicating the importance of the reducible groups (carbonyl and epoxide) in these molecules. In summary, these studies identify 6 new bioactive, oxidized phospholipids that are increased in MM-LDL and, where measured, in atherosclerotic lesions. They thus suggest that a family of phospholipid oxidation products containing oxovaleroyl, glutaroyl, and epoxyisoprostane at the sn-2 position play an important role in the regulation of leukocyte-endothelial interactions, bioactivity being in part controlled by several types of phospholipid hydrolases.

Normal high density lipoprotein inhibits three steps in the formation of mildly oxidized low density lipoprotein: steps 2 and 3.

Treatment of human artery wall cells with apolipoprotein A-I (apoA-I), but not apoA-II, with an apoA-I peptide mimetic, or with high density lipoprotein (HDL), or paraoxonase, rendered the cells unable to oxidize low density lipoprotein (LDL). Human aortic wall cells were found to contain 12-lipoxygenase (12-LO) protein. Transfection of the cells with antisense to 12-LO (but not sense) eliminated the 12-LO protein and prevented LDL-induced monocyte chemotactic activity. Addition of 13(S)-hydroperoxyoctadecadienoic acid [13(S)-HPODE] and 15(S)-hydroperoxyeicosatetraenoic acid [15(S)-HPETE] dramatically enhanced the nonenzymatic oxidation of both 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) and cholesteryl linoleate. On a molar basis 13(S)-HPODE and 15(S)-HPETE were approximately two orders of magnitude greater in potency than hydrogen peroxide in causing the formation of biologically active oxidized phospholipids (m/z 594, 610, and 828) from PAPC. Purified paraoxonase inhibited the biologic activity of these oxidized phospholipids. HDL from 10 of 10 normolipidemic patients with coronary artery disease, who were neither diabetic nor receiving hypolipidemic medications, failed to inhibit LDL oxidation by artery wall cells and failed to inhibit the biologic activity of oxidized PAPC, whereas HDL from 10 of 10 age- and sex-matched control subjects did. We conclude that a) mildly oxidized LDL is formed in three steps, one of which involves 12-LO and each of which can be inhibited by normal HDL, and b) HDL from at least some coronary artery disease patients with normal blood lipid levels is defective both in its ability to prevent LDL oxidation by artery wall cells and in its ability to inhibit the biologic activity of oxidized PAPC.