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A longitudinal assessment of chronic wound fluid to detect biochemical indicators of healing

Published on Jan 1, 2010
Melissa Laura Fernandez4
Estimated H-index: 4
Abstract
Chronic venous leg ulcers are a detrimental health issue plaguing our society, resulting in long term pain, immobility and decreased quality of life for a large proportion of sufferers. The frequency of these chronic wounds has led current research to focus on the wound environment to provide important information regarding the prolonged, fluctuated or static healing patterns of these wounds. Disruption to the normal wound healing process results in release of multiple factors in the wound environment that could correlate to wound chronicity. These biochemical factors can often be detected through non-invasively sampling chronic wound fluid (CWF) from the site of injury. Of note, whilst there are numerous studies comparing acute and chronic wound fluids, there have not been any reports in the literature employing a longitudinal study in order to track biochemical changes in wound fluid as patients transition from a non-healing to healed state. Initially the objective of this study was to identify biochemical changes in CWF associated with wound healing using a proteomic approach. The proteomic approach incorporated a multi-dimensional liquid chromatography fractionation technique coupled with mass spectrometry (MS) to enable identification of proteins present in lower concentrations in CWF. Not surprisingly, many of the proteins identified in wound fluid were acute phase proteins normally expressed during the inflammatory phase of healing. However, the number of proteins positively identified by MS was quite low. This was attributed to the diverse range in concentration of protein species in CWF making it challenging to detect the diagnostically relevant low molecular weight proteins. In view of this, SELDI-TOF MS was also explored as a means to target low molecular weight proteins in sequential patient CWF samples during the course of healing. Unfortunately, the results generated did not yield any peaks of interest that were altered as wounds transitioned to a healed state. During the course of proteomic assessment of CWF, it became evident that a fraction of non-proteinaceous compounds strongly absorbed at 280 nm. Subsequent analyses confirmed that most of these compounds were in fact part of the purine catabolic pathway, possessing distinctive aromatic rings and which results in high absorbance at 254 nm. The accumulation of these purinogenic compounds in CWF suggests that the wound bed is poorly oxygenated resulting in a switch to anaerobic metabolism and consequently ATP breakdown. In addition, the presence of the terminal purine catabolite, uric acid (UA), indicates that the enzyme xanthine oxidoreductase (XOR) catalyses the reaction of hypoxanthine to xanthine and finally to UA. More importantly, the studies provide evidence for the first time of the exogenous presence of XOR in CWF. XOR is the only enzyme in humans capable of catalysing the production of UA in conjunction with a burst of the highly reactive superoxide radical and other oxidants like H2O2. Excessive release of these free radicals in the wound environment can cause cellular damage disrupting the normal wound healing process. In view of this, a sensitive and specific assay was established for monitoring low concentrations of these catabolites in CWF. This procedure involved combining high performance liquid chromatography (HPLC) with tandem mass spectrometry and multiple reaction monitoring (MRM). This application was selective, using specific MRM transitions and HPLC separations for each analyte, making it ideal for the detection and quantitation of purine catabolites in CWF. The results demonstrated that elevated levels of UA were detected in wound fluid obtained from patients with clinically worse ulcers. This suggests that XOR is active in the wound site generating significant amounts of reactive oxygen species (ROS). In addition, analysis of the amount of purine precursors in wound fluid revealed elevated levels of purine precursors in wound fluid from patients with less severe ulcers. Taken together, the results generated in this thesis suggest that monitoring changes of purine catabolites in CWF is likely to provide valuable information regarding the healing patterns of chronic venous leg ulcers. XOR catalysis of purine precursors not only provides a method for monitoring the onset, prognosis and progress of chronic venous leg ulcers, but also provides a potential therapeutic target by inhibiting XOR, thus blocking UA and ROS production. Targeting a combination of these purinogenic compounds and XOR could lead to the development of novel point of care diagnostic tests. Therefore, further investigation of these processes during wound healing will be worthwhile and may assist in elucidating the pathogenesis of this disease state, which in turn may lead to the development of new diagnostics and therapies that target these processes.
  • References (272)
  • Citations (1)
References272
Newest
Published on Sep 9, 2014in Cochrane Database of Systematic Reviews 7.75
E Andrea Nelson22
Estimated H-index: 22
(University of Leeds),
Sally E. M. Bell-Syer16
Estimated H-index: 16
(Ebor: University of York)
+ 1 AuthorsJoan Webster Rn Ba33
Estimated H-index: 33
(Royal Brisbane and Women's Hospital)
Background Up to 1% of adults will have a leg ulcer at some time. The majority of leg ulcers are venous in origin and are caused by high pressure in the veins due to blockage or weakness of the valves in the veins of the leg. Prevention and treatment of venous ulcers is aimed at reducing the pressure either by removing/repairing the veins, or by applying compression bandages/stockings to reduce the pressure in the veins. The majority of venous ulcers heal with compression bandages, however ulcer...
185 Citations Source Cite
Published on Nov 14, 2012in Cochrane Database of Systematic Reviews 7.75
S. O'Meara23
Estimated H-index: 23
(Ebor: University of York),
Nicky Cullum48
Estimated H-index: 48
(University of Manchester)
+ 1 AuthorsJo C Dumville36
Estimated H-index: 36
(Ebor: University of York)
Venous leg ulcers can occur when blood returning from veins in the legs to the heart is slow or obstructed. These ulcers can take a long time to heal (weeks or months) and can cause distress to patients, as well as being very costly to the health service. Compression bandages help blood to return to the heart from the legs, and there are a variety of types of bandage systems available; some are just a single bandage, while others require the application of several different types of bandages to ...
573 Citations Source Cite
Published on Aug 1, 2009in Annual Review of Biomedical Engineering 12.26
John R. Yates151
Estimated H-index: 151
(Scripps Research Institute),
Cristian I. Ruse15
Estimated H-index: 15
,
Aleksey Nakorchevsky6
Estimated H-index: 6
Mass spectrometry (MS) is the most comprehensive and versatile tool in large-scale proteomics. In this review, we dissect the overall framework of the MS experiment into its key components. We discuss the fundamentals of proteomic analyses as well as recent developments in the areas of separation methods, instrumentation, and overall experimental design. We highlight both the inherent strengths and limitations of protein MS and offer a rough guide for selecting an experimental design based on th...
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Published on Jul 1, 2009in Clinical Biochemistry 2.43
Angela Maria Amorini25
Estimated H-index: 25
(Sapienza University of Rome),
Axel Petzold50
Estimated H-index: 50
+ 10 AuthorsSerafina D'Urso2
Estimated H-index: 2
(University of Catania)
Abstract Objectives In this study, the concentrations of uric acid, purine profile and creatinine in samples of cerebrospinal fluid and serum of multiple sclerosis (MS) patients were measured by HPLC and compared with corresponding values recorded in patients without MS (cerebrospinal fluid) and healthy subjects (serum). Design and methods All samples were deproteinized with ultrafiltration (which ensures minimal sample manipulation and efficient protein removal) and then assayed for the synchro...
69 Citations Source Cite
Published on Jun 1, 2009in Nature 43.07
Philipp Niethammer5
Estimated H-index: 5
(Harvard University),
Clemens Grabher21
Estimated H-index: 21
(KIT: Karlsruhe Institute of Technology)
+ 1 AuthorsTimothy J. Mitchison117
Estimated H-index: 117
(Harvard University)
In animals, within minutes of wounding, leukocytes are recruited to the site of injury across distances of hundreds of micrometres. Early leukocyte recruitment after injury is now shown to be driven by the establishment of an H2O2 gradient from the epithelium to the vasculature in zebrafish.
824 Citations Source Cite
Published on May 1, 2009in Journal of Chromatography B 2.81
Neil R. Kitteringham53
Estimated H-index: 53
(University of Liverpool),
Rosalind E. Jenkins34
Estimated H-index: 34
(University of Liverpool)
+ 2 AuthorsB.K. Park81
Estimated H-index: 81
(University of Liverpool)
The conventional pipeline for biomarker development involves a discovery phase, typically conducted by mass spectrometry (MS), followed by validation and clinical application, usually on an alternative platform, such as immunoassay. Whilst this approach is suitable for the development of single biomarkers, with the current drive towards larger panels of multiplexed biomarkers, the process becomes inefficient and costly. Consequently, the emphasis is now shifting towards performing full biomarker...
254 Citations Source Cite
Anastasia K. Yocum15
Estimated H-index: 15
(UM: University of Michigan),
Arul M. Chinnaiyan125
Estimated H-index: 125
Quantitative targeted proteomics has recently taken front stage in the proteomics community. Centered on multiple reaction monitoring ^ mass spectrometry (MRM^MS) methodologies, quantitative targeted proteomics is being used in the verification of global proteomics data, the discovery of lower abundance proteins, protein post-translational modifications, discrimination of select highly homologous protein isoforms and as the final step in biomarker discovery. An older methodology utilized with sm...
83 Citations Source Cite
Published on Nov 1, 2008in Journal of Pharmaceutical and Biomedical Analysis 2.98
Aidong Wen20
Estimated H-index: 20
(Fourth Military Medical University),
Taijun Hang14
Estimated H-index: 14
(CPU: China Pharmaceutical University)
+ 5 AuthorsXinxin Xu2
Estimated H-index: 2
(CPU: China Pharmaceutical University)
Abstract A rapid, simple and sensitive LC–MS/MS method was developed for simultaneous determination of amoxicillin and ambroxol in human plasma using clenbuterol as internal standard (IS). The plasma samples were subjected to a simple protein precipitation with methanol. Separation was achieved on a Lichrospher C 18 column (150 mm × 4.6 mm ID, dp 5 μm) using methanol (containing 0.2% of formic acid) and water (containing 0.2% of formic acid) as a mobile phase by gradient elution at a flow rate o...
40 Citations Source Cite
Published on Nov 1, 2008in Biochimica et Biophysica Acta 3.79
Chandan K. Sen75
Estimated H-index: 75
(OSU: Ohio State University),
Sashwati Roy64
Estimated H-index: 64
(OSU: Ohio State University)
Physical trauma represents one of the most primitive challenges that threatened survival. Healing a problem wound requires a multi-faceted comprehensive approach. First and foremost, the wound environment will have to be made receptive to therapies. Second, the appropriate therapeutic regimen needs to be identified and provided while managing systemic limitations that could secondarily limit the healing response. Unfortunately, most current solutions seem to aim at designing therapeutic regimen ...
158 Citations Source Cite
Published on Nov 1, 2008in Cell 36.22
Jeannette Winter4
Estimated H-index: 4
(UM: University of Michigan),
Marianne Ilbert18
Estimated H-index: 18
(UM: University of Michigan)
+ 2 AuthorsUrsula Jakob50
Estimated H-index: 50
(UM: University of Michigan)
SUMMARY Hypochlorous acid (HOCl), the active ingredient in household bleach, is an effective antimicrobial produced by the mammalian host defense to kill invading microorganisms. Despite the widespread use of HOCl, surprisingly little is known about its mode of action. In this study, we demonstrate that low molar ratios of HOCl to protein cause oxidative protein unfolding in vitro and target thermolabile proteins for irreversible aggregation in vivo. As a defense mechanism, bacteria use the redo...
204 Citations Source Cite
Cited By1
Newest
Published on Jan 1, 2013in arXiv: Other Quantitative Biology
Zoltán Noszticzius23
Estimated H-index: 23
,
Maria Wittmann13
Estimated H-index: 13
(BME: Budapest University of Technology and Economics)
+ 4 AuthorsJános Szegedi6
Estimated H-index: 6
Background / Aims ClO2, the so-called "ideal biocide", could also be applied as an antiseptic if it was understood why the solution's rapid killing of microbes does not cause any harm to humans or to animals. Our aim was to study both theoretically and experimentally its reaction-diffusion mechanism to find the source of that selectivity. Methods ClO2 permeation measurements through protein membranes were performed and the time delay of ClO2 transport due to reaction and diffusion was determined...