Effect of ultrasound beam angle on equine articular cartilage thickness measurement.

The purpose of this study was to determine if variation in the ultrasound beam angle would affect cartilage thickness measurement performed with B-mode ultrasonography. Transverse sections of six fresh equine middle phalanges were obtained from necropsy. Ultrasonographic images of the proximal articular cartilage were obtained in a water bath, in a plane parallel and adjacent to the section plane using a 5-10 MHz linear transducer. Static images were acquired for all six bone specimens with an ultrasound beam angle of 0 degree, 30 degrees, 45 degrees, and 60 degrees. Proximal articular cartilage thickness was measured on ultrasonographic images and on the bone specimen at the same level. A linear mixed-effects model was used to compare articular cartilage thickness measured on specimen and on ultrasonographic images using different ultrasound beam angle. Mean +/- SD cartilage thickness was 1.82 +/- 0.35 mm on bone specimens, 1.72 +/- 0.29 with a 0 degrees angle, 1.99 +/- 0.34 with 30 degrees, 2.06 +/- 0.34 with 45 degrees, and 2.3 +/- 0.38 with 60 degrees. There was a significant difference between macroscopic measurements and ultrasonographic measurements performed with ultrasound angles at 30 degrees, 45 degrees, and 60 degrees. There was a significant increase in cartilage thickness when the ultrasound beam angle decreased (P = 0.0157; R2 = 0.969). Cartilage thickeness measured on ultrasonographic images varies with the ultrasound beam angle and may not be accurate because ultrasound speed in cartilage may be different than the speed used by the ultrasonographic unit for distance calculation.

Bilirubin measured on a blood gas analyser: a suitable alternative for near-patient assessment of neonatal jaundice?

The reliability of a recently released total bilirubin assay for a blood gas analyser was assessed in two Australian hospital laboratories. The instrument computes total bilirubin concentration from multi-wavelength absorbance measurements of undiluted whole blood or plasma. Performance of the Radiometer ABL 735 blood gas analyser bilirubin method (software version 3.6) was compared with a proven Roche diazo method for Hitachi analysers, calibrated using primary standards prepared from NIST SRM 916a bilirubin. Acceptable bilirubin results were found over a wide concentration range for most neonatal samples of whole blood or plasma. For adult specimens, bilirubin results were approximately 10% lower on the blood gas analyser. Within-run imprecision (whole blood) was < 2.5%, between-day imprecision (synthetic controls) < 1.0%, and the bilirubin assay for both whole blood and plasma was linear to 1,000 micromol/L. Using sampling options from 35 microL to 195 microL, bilirubin results differed by less than 3%, with a 95 microL syringe option producing the highest results. We conclude that the Radiometer ABL 735 bilirubin assay is suitable for near-patient assessment of neonatal jaundice using whole blood, thus eliminating the need for sample centrifugation. Verification using laboratory methods can be used when required. A positive correction of approximately 10% is required for adult specimens to conform with Hitachi results (SRM 916a calibration), possibly due to the optical characteristics of the higher proportion of conjugated bilirubin and other substances present in most adult samples.

Evaluation of amylase and lipase in the diagnosis of acute pancreatitis.

BACKGROUND: The diagnosis of acute pancreatitis relies heavily on a raised amylase. METHODS: In the present study patients were prospectively categorized, without knowledge of pancreatic enzyme levels, into acute pancreatitis (AP; n = 51), disease controls (n = 35), indeterminate as to pancreatitis (n = 189) or exclusions (non-pancreatitis diseases where amylase may be elevated; n = 53). RESULTS: Enzyme levels were analysed by receiver operator characteristics (ROC) curves, with specificity > 80%. Day 1 serum lipase gave the greatest diagnostic accuracy (area under ROC curve = 0.128; P = 0.041 vs serum amylase). At the calculated diagnostic threshold of 208 U/L, lipase gave a sensitivity of 67% and a specificity of 97%. Other diagnostic thresholds (day 1) were: serum total amylase, 176 U/L (ROC 0.104, sensitivity 45%, specificity 97%), urinary total amylase, 550 U/L (ROC 0.108, sensitivity 62%, specificity 97%) and serum pancreatic isoamylase, 41 U/L (ROC 0.107, sensitivity 63%, specificity 85%). At delayed diagnosis (3 days) no enzyme was superior to lipase. The combination of lipase and amylase did not increase diagnostic accuracy. CONCLUSION: Serum lipase is recommended for diagnosis of AP, both early and late in the disease. Although highly specific when elevated, all pancreatic enzymes have low sensitivity for diagnosis.

Amylase measurement with 2-chloro-4-nitrophenyl maltotrioside as substrate.

The use of 2-chloro-4-nitrophenyl maltotrioside (CNP-G3) as substrate to measure amylase (EC 3.2.1.1) activity in serum directly without the use of auxiliary enzymes was evaluated at two centres. The method was precise (within-run C.V. < 2% and between-run C.V. < 3%), there was no lag phase, background absorbance was low and there were minimal effects of pH changes. When compared with a method which uses 4,6-ethylidene (G7)-p-nitrophenyl (G1)-alpha-D-maltoheptaoside (EPS-G7) as substrate, the CNP-G3 method had greater sensitivity and longer reagent stability (21 days compared with 2 days at 4 degrees C). The activity measured with the CNP-G3 method correlated well with methods using either EPS-G7 and maltotetraose as substrates.

The effect of (L)-cysteine and (L)-2-oxothiazolidine-4-carboxylic acid (OTZ) on urinary oxalate excretion: studies using a hyperoxaluric rat model.

PURPOSE: To determine the efficacy of (L)-cysteine and (L)-2-oxothiazolidine-4-carboxylic acid (OTZ) in reducing urinary oxalate excretion under hyperoxaluric conditions and to determine whether by inclusion of glycolate in a standard diet, cysteine:glyoxylate adduct can be detected in hyperoxaluric rats given either compound. MATERIALS AND METHODS: Hyperoxaluria (200% above basal) was induced 2 days prior to commencement of the studies and maintained throughout. After a 3 days baseline, animals were randomly allocated to a control or treatment group. Standard diet containing either (L)-cysteine or OTZ was then fed to the treatment groups for 5 days while standard diet alone was fed to the control groups. Urinary oxalate excretion was subsequently monitored and average daily rates were then compared with basal values. Plasma and urine were analyzed for adduct. RESULTS: Both (L)-cysteine and OTZ significantly reduced urinary oxalate excretion relative to the basal hyperoxaluric level (28.6 +/- 1.5 micromol./day). While (L)-cysteine reduced oxalate excretion over the 5 day treatment period by only 7.82 +/- 1.39 micromol./day (27%), OTZ reduced it by 12.34 +/- 1.58 micromol./day (43%). Adduct could not be detected in plasma or urine in this study. CONCLUSIONS: This study confirms that both (L)-cysteine and OTZ are effective in reducing urinary oxalate excretion under hyperoxaluric conditions, with OTZ being more effective than (L)-cysteine. These compounds were shown to be 3- to 4-fold more effective in reducing urinary oxalate excretion under hyperoxaluric conditions when compared with the results from previous studies under normooxaluric conditions.

(D)-penicillamine increases hepatic oxalate production resulting in hyperoxaluria.

PURPOSE: To determine whether (D)-penicillamine is effective in reducing hepatic oxalate production and urinary oxalate excretion. MATERIALS AND METHODS: (D)-Penicillamine was administered orally to rats to determine its effect on urinary oxalate excretion and used in isolated rat hepatocytes to investigate the effect of (D)-penicillamine on oxalate production from glycolate. Studies involving hepatic aminotransferases and hepatocytes isolated from (D)-penicillamine treated rats were used to clarify the discrepancy between the in vitro and in vivo results. RESULTS: In hepatocytes (D)-penicillamine lead to a significant reduction in oxalate production from glycolate. In vivo however. (D)-penicillamine led to a significant increase in urinary oxalate excretion and a decrease in plasma aminotransferase activity. Hepatic aminotransferases are involved in diverting oxalate precursors from oxalate production. In vitro, (D)-penicillamine was shown to inhibit hepatic aminotransferases. Hepatocytes isolated from (D)-penicillamine-treated rats produced significantly more oxalate than controls. CONCLUSIONS: These results indicate that (D)-penicillamine increases hepatic oxalate production and urinary oxalate excretion. (D)-penicillamine therefore has no therapeutic potential for reducing endogenous oxalate production and urinary oxalate excretion. Moreover, in conditions such as Wilson's Disease which is often associated with hypercalcuria, its use may be contraindicated.

The changing face of pathology--an Australian perspective.

Despite Government at various levels in Australia attempting to force restraint by limiting financial resources, modern medicine and community expectations will continue to place demands on health services. The principal illnesses confronting society are unlikely to change, with conditions such as cancer, cardiovascular disease and diabetes remaining at their current prevalence. Early and more sophisticated detection and monitoring of these diseases will be required, with concomitant increases in costs. Overriding this in Australia is the increase in the age of the population, which will also have a major impact on health costs. This article examines the effect, on pathology services, of changes occurring within the health system in Australia, especially the impact on public hospital pathology.

Giant cell arteritis: diagnosis and management.

Giant cell arteritis should not be a diagnosis of exclusion, an afterthought, or a last thought. There is urgency to establishing this diagnosis and initiating therapy. All practitioners who treat adults will be confronted with these patients. Some will have classic presentations, some will have subtle presentations. When patients complain of fever, fatigue, malaise, weight loss, or painless vision loss, GCA should be suspected. An ESR will aid in the diagnosis (although a normal ESR does not rule it out), and sometimes temporal artery biopsy will provide certainty. Giant cell arteritis is usually easy to recognize, easy to treat, and satisfying to manage.

Indirect sandwich enzyme-linked immunosorbent assay (ELISA) for plasma apolipoprotein E.

We have developed an indirect sandwich ELISA for measuring plasma apolipoprotein E (apo-E), using commercially available antibodies. A monoclonal anti-apo-E was used as the capture antibody and the captured apo-E detected with polyclonal anti-apo-E antiserum (goat). The detecting antibody was quantitated using horseradish peroxidase-conjugated rabbit immunoglobulin to goat immunoglobulins. There was no detectable cross-reactivity between the three antisera. Interference with the assay by apolipoprotein A-1, bilirubin and haemoglobin was not significant up to 1.7 g/L, 1250 mumol/L and 13.0 g/dL, respectively. The ELISA method showed high correlation with an established immunonephelometric method (ELISA apo.E = 1.04 Immunonephelometric apo-E + 16; r2 = 0.954, P < 0.0001, n = 39). The assay has a measuring range between 5 and 560 mg/L. The coefficient of duplicates was 2.0%, within-run coefficients of variation (CV) ranged from 3.7 to 6.0% and between-run CV from 6.1 to 15.1%. The reference range determined for 168 normotriglyceridaemic subjects was 20 to 130 mg/L. In an analysis of the lipoprotein subfractions isolated by ultracentrifugation as the fraction of density less than 1.25 g/mL and separated by gel permeation chromatography, apo-E was found to be associated with very low-density lipoprotein and large high-density lipoprotein.

Idiopathic calcium oxalate urolithiasis and endogenous oxalate production.

Despite the great effort that has gone into investigating urolithiasis, this condition still persists as one of the major ailments of the urinary tract. Calcium oxalate urolithiasis is the most common form, accounting for some 60 to 80% of total stones. This review examines the elements (i.e., urine volume and pH and urinary excretion of calcium, oxalate, citrate, urate, magnesium, pyrophosphate, and glycosaminoglycans) that give rise to idiopathic calcium oxalate urolithiasis. Treatment strategies for idiopathic calcium oxalate urolithiasis, including lithotripsy, also are discussed. Urinary oxalate excretion is a major risk factor for calcium oxalate urolithiasis, with 85 to 95% of the urinary load derived endogenously. The factors controlling endogenous oxalate production are reviewed, including pathways for the diversion of glyoxylate from oxalate production. The use of beta-aminothiols and other substances to reduce endogenous oxalate production in subjects with idiopathic calcium oxalate urolithiasis is also discussed. A review of current methodologies for the determination of urinary oxalate is also included.

The efficacy of (L)-2-oxothiazolidine-4-carboxylate (OTC) and (L)-cysteine in reducing urinary oxalate excretion.

The effects of orally administered (L)-cysteine and (L)-2-oxothiazolidine-4-carboxylate (OTC) on urinary oxalate excretion were investigated in male Porton rats, as (L)-cysteine has been shown to form an adduct with glyoxylate in vitro. Feeding of OTC (204 +/- 1 mg. per day) for 5 days increased urinary cyst(e)ine, p < 0.001; sulphate, p < 0.001; phosphate, p < 0.05; and calcium, p < 0.05; and decreased urinary pH, p < 0.001. In addition, OTC feeding significantly decreased urinary oxalate excretion when compared with controls, p < 0.05 (delta OTC-delta Control -4.26 +/- 1.55 nmol./day/gm.). In the 5-day period after cessation of OTC feeding, all urinary parameters returned to control levels. At the completion of this recovery period there were no significant differences in any of the clinically significant plasma parameters. When fed for 22 days (191 +/- 3 mg. per day) OTC decreased urinary oxalate compared with controls, p < 0.05 (delta OTC-delta Control -9.47 +/- 4.24 nmol./day/gm.). Other urinary parameters (uric acid, magnesium, calcium, phosphate, creatinine, pH and volume) were not significantly altered by OTC feeding. Again, at the completion of this feeding period there were no significant differences in any of the clinically significant plasma parameters. (L)-cysteine feeding for 5 days (184 +/- 10 mg. per day) increased urinary sulphate, p < 0.001; and magnesium, p < 0.05, and decreased urinary pH, p < 0.001. In addition, (L)-cysteine feeding did not significantly change urinary oxalate excretion when compared with the controls (delta(L)-Cysteine-delta Control -2.94 +/- 2.14 nmol./day/gm.). However, at the completion of this feeding period, plasma urate, p < 0.02; and glucose, p < 0.05, were decreased, and plasma potassium, p < 0.01, was increased. these results indicate that orally administered OTC is effective in reducing urinary oxalate excretion without altering plasma biochemistry. It is suggested that (L)-cysteine-glyoxylate adduct formation is the mechanism by which OTC reduces urinary oxalate excretion through a reduction in endogenous oxalate production.

Formation of the L-cysteine-glyoxylate adduct is the mechanism by which L-cysteine decreases oxalate production from glycollate in rat hepatocytes.

Formation of thiazolidine-2,4-dicarboxylic acid, the L-cysteine-glyoxylate adduct, is the putative mechanism by which L-cysteine reduces hepatic oxalate production from glycollate [Bais, Rofe and Conyers (1991) J. Urol. 145, 1302-1305]. This was investigated in isolated rat hepatocytes by the simultaneous measurement of both adduct and oxalate formation. Different diastereoisomeric ratios of cis- and trans-adduct were prepared and characterized to provide both standard material for the enzymic analysis of adduct in hepatocyte supernatants and to investigate the stability and configuration of the adduct under physiological conditions. In the absence of L-cysteine, hepatocytes produced oxalate from 2 mM glycollate at a rate of 822 +/- 42 nmol/30 min per 10(7) cells. The addition of L-cysteine to the incubation medium at 1.0, 2.5 and 5.0 mM lowered oxalate production by 14 +/- 2, 25 +/- 3 (P < 0.05) and 38 +/- 3% (P < 0.01) respectively. These reductions were accompanied by almost stoichiometric increases in the levels of the adduct: 162 +/- 6, 264 +/- 27 and 363 +/- 30 nmol/30 min per 10(7) cells. Adduct formation is therefore confirmed as the primary mechanism by which L-cysteine decreases oxalate production from glycollate. As urinary oxalate excretion is a prime risk factor in the formation of calcium oxalate stones, any reduction in endogenous oxalate production is of clinical significance in the prevention of this formation.

IFCC methods for the measurement of catalytic concentration of enzymes. Part 8. IFCC method for lactate dehydrogenase (L-lactate: NAD oxidoreductase, EC 1.1.1.27).

Human lactate dehydrogenase is a tetramer made up of two types of subunits, either H (heart) or M (muscle). Combination of these subunits gives rise to the five isoenzymes of lactate dehydrogenase which are found in mammalian tissues. The relative proportions of the individual isoenzymes found in serum of patients is related to the severity of the lesion in the organ or tissue from which they originate and the half-life of the individual tissue-specific enzymes. Thus, one cannot predict the relative proportions of the different isoenzymes in any one patient sample.Lactate dehydrogenase catalyses the reversible oxidation of lactate to pyruvate and either reaction can be measured readily. However, in this method, the lactate to pyruvate reaction has been selected because of the following reasons; the time-course of the reaction is more linear, the reaction results in an increase in absorbance and optimization of substrates is possible (see appendix A).The principles applied in the selection of the conditions of measurement are those stated in previous publications by the IFCC's Committee on Enzymes [1]. Human serum and tissue extracts have been used as the sources of enzymes. The final concentration of substrates and the pH have been selected on the basis of experiments and empirical optimization techniques and have been confirmed by calculation from rate equations. The catalytic and physical properties of the isoenzymes differ, but because of the importance of the heart specific isoenzyme (LD1) in the assessment of coronary heart disease and as a tumour marker, this method has been optimized for this isoenzyme. However, the method is also suitable, although less optimally, for the determination of the other isoenzymes of lactate dehydrogenase which may be present in serum.

The effects of hypercholesterolaemia, simvastatin and dietary fat on the low density lipoprotein receptor of unstimulated mononuclear cells.

The in vivo expression and regulation of the LDL receptor of circulating mononuclear cells was studied using a sensitive spectrophotometric assay with low density lipoproteins conjugated to colloidal gold (LDL-gold). The high plasma cholesterol of familial hypercholesterolemic subjects was shown to be related to a low in vivo LDL receptor activity; cells from a homozygote had virtually no activity and those from 24 heterozygotes expressed 45% of the activity of cells from 35 normals. The average receptor activity of cells from 18 polygenic hypercholesterolemic (PH) subjects was not significantly different from normal but a low expression may have been a factor in six of these subjects. Simvastatin increased the LDL receptor activity of cells from the PH subjects by 70% while lowering their plasma cholesterol by 26%, but reducing the fat intake from 38% to 20% of energy and cholesterol from 239 to 96 mg/day had no effect on the receptor despite a 10% reduction in plasma cholesterol. Upregulation of the LDL receptor may therefore have been involved in the lowering of plasma cholesterol by simvastatin but not by the reduction in dietary fat and cholesterol.

Influence of season, age, and sex on renal stone formation in South Australia.

OBJECTIVE: To investigate trends in renal stone formation in the South Australian population, between 1977 and 1991 (3634 stones), with respect to age, sex and seasonal variation. RESULTS: The frequency of the different stone types was: calcium oxalate (with or without phosphate), 68%; uric acid, 17%; infection stones (magnesium ammonium phosphate), 12%; and pure calcium phosphate, 3%. No significant seasonal variation was observed with calcium oxalate or calcium phosphate stones. The incidence of uric acid stones increased significantly during summer and autumn (P < 0.001 and P < 0.01 respectively), and that of infection stones decreased significantly during spring and summer (P < 0.05 and P < 0.01 respectively). Calcium oxalate, uric acid and calcium phosphate stones were more frequent in male subjects; male to female ratio 2.8:1, 3.7:1 and 1.4:1 respectively. However, there was an increased frequency of calcium oxalate stones in women 20 to 25 years of age; male to female ratio 0.7:1. Infection stones were more common in female subjects; male to female ratio 0.7:1. CONCLUSIONS: This study demonstrates significant seasonal variation in uric acid and infection stones. Men are at a higher risk of forming stones than women, with the exception of infection stones. Additionally, with calcium oxalate stones, women may have distinct periods of higher risk. This study confirms that calcium oxalate stones are the most common stone type, which is in accordance with studies from other industrialised countries.

Effect of morphine or phenobarbital on teicoplanin elimination pharmacokinetics.

Elimination kinetics following a single dose of teicoplanin in rats pre-treated with morphine sulphate (MS), phenobarbital sodium (Pb), and normal saline (NS) were determined. A microbioassay was used to measure teicoplanin levels. A significant increase in the total clearance of teicoplanin was found in rats pre-treated with MS as compared to controls (P < 0.048). Wide variability was observed in the renal and non-renal clearances of teicoplanin. The mean renal clearance for rats pre-treated with MS, Pb and NS was 0.61 +/- 0.07.mL/min/kg, 0.60 +/- 0.13 mL/min/kg, and 0.46 +/- 0.02 mL/min/kg, respectively; the mean non-renal clearance was 0.33 +/- 0.18 mL/min/kg, 0.17 +/- 0.15 mL/min/kg, and 0.08 +/- 0.03 mL/min/kg, respectively. The differences among the groups for renal and non-renal clearance were not statistically significant. The mean apparent volume of distribution of teicoplanin at steady state was significantly lower in the Pb-pre-treated rats as compared to controls (P < 0.043). The mean half-life for MS-, Pb-, and NS pre-treated groups was 8.1 +/- 3.1 h, 5.9 +/- 3.3 h, and 34.6 +/- 20.7 h, respectively. The differences in mean half-life among the groups achieved statistical significance (P < 0.016). The increase in the total clearance of teicoplanin can best be explained by an increase in both renal elimination and hepatic metabolic pathways.

Lipid parameters and apolipoprotein B RFLP studies: comparison of normal and coronary heart disease groups as defined by angiography.

We have compared the lipid and apolipoprotein values and the frequency of DNA polymorphisms of the apolipoprotein B gene detected with the restriction enzymes, Xba I and Eco RI in 122 patients with coronary heart disease (CHD) and 80 control subjects. The patients with coronary heart disease (CHD) were defined by > 70% stenosis in at least one major coronary artery whereas the controls showed no signs of coronary artery narrowing at angiography. When males and females were considered separately, differences in triglyceride, total cholesterol and high density lipoprotein-cholesterol (HDL-cholesterol) between CHD and control subjects were significant only for females. The polymorphism studies showed no significant differences between the control and CHD subjects except for a difference in the frequency in the females of the Xba I polymorphism (p < 0.05). The X1 allele (absence of the restriction enzyme cutting site) occurred significantly more often in the patient group than in the controls. Individuals with the X1X2 genotype had the highest serum total cholesterol whereas those with the X1X1 genotype had the lowest HDL-cholesterol value. Generally, the associations between the Xba I and Eco RI alleles and serum lipid levels were weak and inconsistent. Furthermore, even after careful selection of disease and control groups, a useful role for restriction fragment length polymorphism studies in assessing CHD risk in individual patients was not demonstrated.