Method Validation for New Nitrogen/Sulfur Species Analysis Techniques

Study Overview
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Brief Summary
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Plasma redox status is well known to alter with age but previously differences have only been reported for two specific redox couples; reduced and oxidized cysteine and reduced and oxidized glutathione. (Jones DP, Rejuvenation Res. 2006) The overall aim of this project is to develop new methods to determine how other Reactive Nitrogen and Sulfur species, such as free and protein-bound thiols (organic compounds that contain a Sulfhydryl group, -SH) and Hydrogen Sulfide (H2S) change with age and gender in a medium-sized cohort of healthy individuals (n=100 of either sex; age 18-70). Our group has recently developed a novel analytical platform to measure thiol-containing compounds in biological samples that is based on the reaction with the thiol-alkylating agent N-ethylmaleimide (NEM). We have used this chemical for years as stabilisation agent in other analyses to quantify Nitric Oxide metabolites.(Feelisch et al, FASEB 2002 and Levett et al, Sci Rep 2011) We now wish to develop a similar method to measure thiol containing compounds using a novel rapid LC-MS/MS based technology for screening large numbers of individuals. The project will therefore have 2 stages: Initial method development will require occasional use of whole blood, plasma and red blood cells to establishing optimal conditions. E.g. to compare heparin and EDTA as anticoagulants to serum; investigate effects of temperature and pH; identify optimal concentration of derivatisation/reduction agents and optimal reaction times. We then aim to establish the normal distribution of these novel compounds across healthy individuals of different gender and varying ages.

Official Title
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Method Validation for New Nitrogen/Sulfur Species Analysis Techniques

Conditions
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Redox Status

Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Aged 18 years or above Currently healthy (i.e. no inter-current illness/infection) Capacity and ability to give written informed consent Exclusion Criteria: Current or previous smoker Other medical co-morbidity (e.g. hypertension, diabetes, asthma, COPD, cardiovascular disease, current or previous malignant disease etc.) Surgery within previous 28 days Excessive alcohol intake (i.e above UK's recommended intake limit of 14 units per week). Pregnant or currently breast-feeding On current medication Taken vitamins / minerals / herbal remedies within previous 28 days Difficult venous access Needle phobia Participating in another similar/conflicting research study already

Ages Eligible for Study
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Minimum Age: 18 Years

Sexes Eligible for Study
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All

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Stage 1<br>Samples from up to 20 participants will be used to finalise the analytical methods | |
| Stage 2<br>Samples from up to 200 participants will be used to then validate the normal ranges of these markers across different age ranges in both genders | |

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Concentration of reactive (nitrogen or sulfur containing) species |  | Baseline |