Richard Taylor

Evidence of oxidative stress in chronic venous ulcers

Reactive oxygen species have been implicated in the impaired healing of chronic leg ulcers but little direct evidence is available. We have observed a significant (pu2003<u20030.01) elevation of the allantoin : uric acid percentage ratio, a marker of oxidative stress, in wound fluid from chronic leg ulcers (median 17, range 8–860) compared to both paired plasma (median 2, range 1–8) and acute surgical wound fluid (median 4, range 3–7). However, the allantoin : uric acid percentage ratio did not differ significantly between chronic wounds that healed and those that failed to heal. Neutrophil elastase was elevated 30‐ to 1300‐fold in chronic wound fluid compared to plasma and there was a correlation (ru200a2u2003=u20030.742) between wound fluid elastase and the allantoin : uric acid percentage ratio. Total antioxidant capacity of wound fluid, as measured with a chemiluminescence assay, did not show a correlation (ru200a2u2003=u20030.03) with the observed oxidative stress. These observations suggest that conditions of localized oxidative stress, possibly related to neutrophil‐associated production of reactive oxygen species, are present in chronic leg ulcers. It is possible that future therapeutic strategies aimed at reducing oxidative stress, in addition to good standard care, could improve healing rates of chronic wounds. (WOUND REP REG 2003;11:172–176)

Effect of smoking, smoking cessation, and nicotine patch on wound dimension, vitamin C, and systemic markers of collagen metabolism

BACKGROUNDnPostoperative wound disruption and tissue-destructive disorders are more frequent in smokers than in nonsmokers. Impaired wound healing and altered connective tissue turnover are suggested mechanisms, but exact details remain unknown.nnnMETHODSnFull-thickness, 5-mm punch biopsy wounds were made lateral to the sacrum in 48 smokers and were randomized double-blinded to continuous smoking, abstinence with transdermal nicotine patch (TNP), or abstinence with placebo patch and 30 never smokers. At 1, 4, 8, and 12 weeks, the wounds were excised and fixed for wound measurement, and blood was collected for measurement of vitamin C, procollagen I N-propeptide (PINP), matrix metalloproteinase 8 (MMP), MMP-9, neutrophils, and eosinophils.nnnRESULTSnOne week after wounding, smokers wounds were 3.1 ± 0.1 mm (mean, standard error of thexa0mean) wide and were 1.3 ± 0.1 mm deep compared with the never smokers wounds, measuring 3.7xa0±xa00.1 mm wide and 1.5 ± 0.1 mm deep (P < .01, respectively). Abstinent smokers wounds were 3.3xa0± 0.1 mm wide (NS) and were 1.4 ± 0.1 mm deep (P = .02 compared with smokers). In smokers, vitamin C and PINP were 50.5 ± 9.0 μmol/L and were 52.7 ± 6.6 ng/mL, respectively, compared with 68.8 ± 14.5 μmolL and 64.7 ± 4.7 ng/mL in never smokers (P < .001 and P = .07). Both increased significantly after smoking cessation. Plasma MMP-8 and MMP-9 were correlated with neutrophil blood count, which significantly was affected by smoking status. No effect of TNP was found.nnnCONCLUSIONnSmokers have smaller, more superficial wounds and lesser blood levels of vitamin C andxa0PINP. Smoking cessation resulted in increased wound depth, vitamin C, and PINP as well as axa0decreased neutrophil blood count. These findings suggest that wound contraction and collagen metabolism are affected by a smoking-induced alteration in vitamin C turnover and by a change in inflammatory cell response.

Simple biochemical markers to assess chronic wounds.

We investigated the potential for the biochemical analysis of chronic wound fluid to predict healing using simple and widely available analytes in an out‐patient clinic setting. Wound fluid was collected from 12 patients attending a leg ulcer clinic and analyzed for a variety of analytes, including lactate, total protein, and albumin. Twelve weeks after collection the wound was assessed for healing (defined as complete healing or greater than 50% reduction in wound size). The median total protein (44.3 ± 8.8 g/l) and albumin (25.0 ± 2.3 g/l) concentrations in exudate collected from four healing wounds were significantly higher (p < 0.05) than in exudate from eight nonhealing wounds (median total protein 29.7 ± 7.6 g/l, median albumin 17.0 ± 4.3 g/l). No significant difference was observed for lactate. A second specimen of wound fluid was collected from four of the patients (three nonhealing and one healing). The protein analysis confirmed the pattern observed for the first collection: nonhealing wounds had total protein and albumin which remained low compared to healing wounds. No wound with an exudate albumin of less than 20 g/l healed. Both total protein and albumin are stable analytes which can be easily measured in any laboratory and may offer a simple biomarker of healing in chronic wounds.

Glycated haemoglobin : an assessment of high capacity liquid chromatographic and immunoassay methods

We have assessed five high-throughput systems for the measurement of glycated haemoglobin and have reviewed published evaluations of individual analysers. All systems offered better precision than a widely used electroendosmosis method. The low pressure chromatography and immunoassay systems demonstrated greater between-batch imprecision than the high performance liquid chromatography analysers, the latter achieving the proposed analytical goal of between-batch coefficients of variation less than 5%. Agreement between all systems measuring HbA1 was good but there was variability amongst observed HbA1c values. The systems were also assessed for their quality of chromatographic separation, simplicity of operation, flexibility, cost and potential for interference by other haemoglobins.

Utility of cardiac biomarkers for the diagnosis of type V myocardial infarction after coronary artery bypass grafting: insights from serial cardiac MRI

Objectives To examine, using cardiac magnetic resonance (CMR), the utility of cardiac biomarkers for the determination of myocyte necrosis and function after coronary artery bypass grafting (CABG), and to test the recently updated guidelines for the diagnosis of postoperative myocardial infarction (type V MI). Methods and results Forty patients included in a single-centre randomised trial of two surgical techniques for performing CABG underwent serial assessment with CMR biochemical markers. Cine and delayed enhancement CMR (DE-CMR) for assessment of left ventricular (LV) function and irreversible myocyte necrosis was performed and levels of troponin I (TnI) and creatine kinase-MB isoform (CK-MB) were determined. The area under the curve for TnI strongly correlated with the mass of new myocyte necrosis as assessed by DE-CMR (r=0.83, p<0.001), compared with CK-MB (r=0.39, p=0.06). Furthermore, routine assessment of TnI alone at 24u2005h (>6.6u2005μg/l) predicted type V MI on DE-CMR with a sensitivity of 88% and specificity of 97%, whereas CK-MB predicted type V MI with a sensitivity of 75% and specificity of 87%. Conclusions Biomarkers alone (TnI), at an appropriate threshold appear robust for the detection of type V MI, independently of supplementary evidence, as suggested by the ESC/ACCF/AHA/WHF criteria. Clinical trial registration information The study is listed on the Current Controlled Trials Registry: ISRCTN41388968. URL: http://www.controlled-trials.com.

Optimisation of an enzymatic method for β-galactosidase

Abstract Background : The enzyme β-galactosidase present in the Kupffer cells of the liver has potential as a marker of liver dysfunction prior to transplantation. Spectrophotometric methods have insufficient sensitivity. Methods : Fluorimetric methods have the required sensitivity and we have optimised such a method in a microtitre plate format to improve its utility. β-galactosidase acts on the substrate 4-methylumbelliferyl-galactoside (MUG) to produce 4-methylumbelliferone (4-MU), detected fluorimetrically with excitation wavelength 355 nm and emission wavelength 460 nm. Results : Reaction conditions in a citrate–phosphate buffer were optimised to give maximal enzyme activity: pH was optimal at 4.4 (range investigated 3.6–5.0) and substrate concentration at 3.33 mmol/l. A small specimen volume (10 μl) in 80 μl of substrate solution produced adequate fluorescent yield after an incubation period of 30 to 60 min at 37 °C. Reaction was terminated by addition of 200 μl of glycine–NaOH, pH 12.8. The assay is linear to 3000 U/ml. The intra-assay coefficient of variation (CV%) at 50, 502, and 2012 U/ml was 4.7, 3.1, and 3.4, respectively ( n =10). Inter-assay CV% at 51, 496, and 1986 U/ml was 7.0, 4.0, and 3.9, respectively ( n =10). Conclusions : The assay has greater practical utility and demonstrated significant differences in the perfusate β-galactosidase between cold-stored and warm-perfused livers in a porcine model of transplantation.

The effects of precision, haematocrit, pH and oxygen tension on point-of-care glucose measurement in critically ill patients: a prospective study.

Background Critical care glycaemic control protocols commonly have treatment adjustment (target) ranges spanning ≤2 mmol/L. These require precise point-of-care glucose measurement, unaffected by other variables, to avoid measurement errors increasing glycaemic variability and hypoglycaemic episodes (both strongly associated with mortality in critically ill patients). Methods A prospective 206 intensive care patient study was carried out. Arterial glucose concentrations were measured in duplicate using three point-of-care instruments (MediSense Precision PCχ, HemoCue DM and Radiometer 700), a central laboratory instrument (Siemens ADVIA), and in whole blood and plasma using the Yellow Springs Instruments 2300 instrument. Results Coefficients of variation for the MediSense, HemoCue, Radiometer and Siemens instruments were 5.1%, 2.5%, 2.1% and 2.3%, respectively. Compared with the Siemens instrument, the bias (95% limits of agreement) for the MediSense, HemoCue and Radiometer instruments were 0.0 (−1.4 to 1.4), 0.0 (−1.2 to 1.1) and −0.2 (−0.9 to 0.6) mmol/L, respectively. The whole blood–plasma glucose concentration difference was significantly affected by the haematocrit. MediSense and HemoCue instrument performances were substantially affected by haematocrit. MediSense instrument performance was also affected by pH and PaO2. Radiometer instrument performance was not affected by haematocrit, pH or PaO2. Conclusions The MediSense instrument was too imprecise for use in critically ill patients. The haematocrit range seen is too great to allow fixed-factor conversion between whole blood and plasma values, substantially affecting the accuracy of both glucose meters. However, the Radiometer instrument was unaffected by the haematocrit, pH or pO2, resulting in a performance equivalent to the laboratory method. Instrument performance differences may therefore partially explain the differing results of tight glycaemic control therapy trials.

Comparison of Bayer Advia Centaur immunoassay results obtained on samples collected in four different Becton Dickinson Vacutainer tubes.

Background: Medicines and Healthcare products Regulatory Agencys (MHRAs) Medical Device Alert MDA/2004/048 described bias in some endocrine test results obtained on a few immunoassay platforms, particularly the Bayer Advia Centaur® instrument, when using blood specimens collected into Becton Dickinson (BD) VacutainerTM SSTTMII Advance tubes. As users of BD tubes and the Advia Centaur® instrument, we addressed our concerns about the quality of the results that we had previously reported by undertaking an independent study. Method: We compared the results of 15 immunoassays performed on Bayer Advia Centaur® using blood specimens collected into four different BD Vacutainer® tubes (plain, old and newly released BD SSTTMII Advance, and BD PSTTMII). Results: Compared with plain tubes, old SSTTMII Advance tube results showed no bias for testosterone, CA15-3, follicle-stimulating hormone and folate assays, but gave a positive bias for cortisol and a negative bias for vitamin-B12. Compared with plain tubes, BD PSTTMII tubes gave no significant bias for thyroid function tests, prolactin, parathyroid hormone, and CA125, but gave a negative bias for steroid assays, and a positive bias for gonadotrophins. The results obtained using new BD SSTTMII Advance tubes were generally comparable with those on plain tubes. Conclusions: Only for cortisol did our findings support the bias described by MHRA. Based on our results, apart from vitamin-B12 and possibly cortisol, there may have been no significant influence on clinical decisions as a result of using the old BD SSTTMII Advance specimen tubes. New BD SSTTMII Advance tubes and plain tubes give generally comparable results. BD PSTTMII tubes should not be used for steroid hormone measurements on the Bayer Advia Centaur® instrument.

Improved immunoturbidimetric assay for cystatin C

An immunoturbidimetric assay for cystatin C was optimized with respect to assay imprecision. After investigating the optimum pH, polyethylene glycol concentration and specimen volume, two modifications were introduced: an increase in specimen volume to 25µL; and an extension of the pre-incubation period to 240s. These modifications produced an assay with between-batch imprecision (coefficient of variation, n=10 or 11) ranging from 3·9% at 0·72mg/L to 1·3% at 5·29mg/L. The assay was susceptible to interference from lipaemia and haemolysis but not bilirubinaemia in both the original and modified protocol. Extending the preincubation to 240s improved tolerance to common interferences and retained assay applicability in the routine clinical setting.

Phenylalanine: application of a simple HPLC technique to its measurement in dried blood spots

Recent recommendations regarding the management of phenylketonuria suggest that to attain maximum achievable intelligence quotients plasma phenylalanine levels need to be more strictly controlled. Twice weekly monitoring and a target range of 120-300 1-LII10l/L have been advocated. To achieve the necessary turnround time and meet the analytical requirements of such a policy laboratories need simple, rapid methods for phenylalanine estimation which are both accurate and precise particularly at the lower end of the suggested range. High performance liquid chromatography (HPLC) can be accurate and precise but most methods require derivatization, gradient separation or fluorimetric detection.s- A method based on isocratic separation and ultraviolet (UV) detection of the aromatic amino acids by HPLC without derivatization has been used for the measurement of phenylalanine in plasma deproteinized with perchloric acid (PCA).s This report describes the extension of the method to the estimation of phenylalanine in extracts of dried blood spots on paper which are considered to be a safe and convenient method of collecting blood at home and are also used for neonatal screening. HPLC was performed at 30°C on Waters equipment (Millipore (UK) Limited, Watford WD1 8YW, UK) using a 501 pump, 712 automated injector and 455 UV detector at 206 nm. Data acquisition and calculation of results based on peak height was performed by Baseline 810 software on a NEC Advanced Personal Computer. The octadecylsilane analytical column was as used by Atherton and Green.s Stock phenylalanine standard (5 mmol/L), stock internal standard (c-methylphenylalanine, 5 mmol/L) and working internal standard (500 ILmol/L) were