Advancing the total testing process of vitamins B1 and C for critical care
Creators: name: COLLIE, Jake
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 320202
subject: Clinical chemistry (incl. diagnostics)

description: Vitamin B1 and vitamin C play a crucial role in energy production and immune defence in the human body. Their use in critical care has increased over the last decade as they can be administered without adverse reactions and are inexpensive making them potential simple, economical and efficient treatment options. However, there is conflicting evidence on the effectiveness of these vitamins in critical care. Much of the current information on vitamin B1 and C administration in critical care is based on randomised control trials (RCTs) in which the levels of the analyte, before and during the intervention, are absent. Without insight into the status of the concentrations of the analytes, within the participants before commencing and during the intervention, any conclusions on the outcomes are made without key evidence. Trials which have included analyte quantification often do so without including detailed information on the sample handling and processing protocols or with inappropriate methodologies.
 
The aims of this thesis were to improve the total testing process of vitamin B1 in whole blood and C in plasma, focusing on their application in RCTs. These aims were achieved by collaborating with several intensive care units to construct and implement RCTs with vitamin B1 and C interventions. This included the successful development of an appropriate method for the quantification of whole blood thiamine pyrophosphate (TPP), the most abundant and biologically active form of vitamin B1, and studies to support the overall integrity of the vitamin C analytical methods. Together, these methods underpin the quality of the data generated for the RCT, as demonstrated in this thesis, through the validation of relevant pre-analytical, analytical and post-analytical components related these vitamins.

Timely analysis of plasma ascorbate samples is required to reduce any pre-analytical loss due to the rapid oxidation of vitamin C after sample collection. For RCTs this becomes prohibitively expensive as multiple analyses are required due to the slow nature of the recruitment process. The work performed in this thesis overcame this difficulty through the construction and validation of an innovative sample handling and processing protocol of plasma vitamin C samples. The primary outcome of this was a significant improvement to sample stability by reducing vitamin C oxidation and allowing for long-term sample storage and batch analyses, a necessity for RCTs. This was accomplished though the addition of a stabilising agent, during sample handling and processing, that was validated using a popular commercial kit. Secondary outcomes from this work included improved accuracy and reliability of plasma vitamin C results to provide strong analytical evidence of the prevalence of vitamin C deficiency in critical care. Additionally, this has made vitamin C analysis cost effective and more attractive for inclusion into future RCTs.
 
The development of this vitamin C sample handling and processing protocol minimised pre-analytical errors and allowed for this thesis to conduct a robust sub-study, within the RCT, on the pharmacokinetics of high dose vitamin C in patients with septic shock. This work validated that 6-hourly doses of 1.5g of vitamin C maintains supranormal levels. This confirmed that the bioavailability of vitamin C is sustained, within the study population, throughout the duration of the RCT strengthening the correlation between intervention and outcome. These plasma vitamin C results supported the RCT conclusion that despite the analytical evidence, which validated the administration protocol, there was no significant difference in mortality or length of stay between the high dose vitamin C intervention or control groups.
 
This thesis developed and validated an analytical method, using liquid chromatography tandem mass spectrometry (LC-MS/MS), for the quantification of whole blood TPP. This was established for a RCT investigating patients in critical care with hypophosphatemia. The primary outcome of the work, performed in this thesis, was to validate the administration protocol by providing analytical evidence that sufficient levels of TPP were maintained during the treatment period. A secondary outcome was to identify the prevalence of TPP deficiency in critical care. This work validated that the administration protocol rapidly improved TPP concentrations and maintained supranormal levels for the duration of the RCT. Furthermore, it provided evidence of a large percentage of TPP deficient patients within the study population. These TPP measurements supported the RCT conclusion that despite the validation of the administration protocol, and the improvement in TPP concentrations in the deficient population, there was no significant difference in mortality or length of stay between the intervention or control groups.
 
This thesis has highlighted the importance of including appropriate analytical methodologies and discussed the pre-analytical, analytical and post-analytical concepts behind them. This has led to the development and validation of the LC-MS/MS method for whole blood TPP and a sample handling and processing protocol for plasma vitamin C. Through these outcomes this work can be utilised as a basis for the standardisation for TPP methodologies allowing for traceability and improving overall analytical performance. Furthermore, this work supports future high dose vitamin C or TPP RCTs, of various study populations, in validating their administration protocols by verifying cellular uptake or metabolic use through analyte quantification. Above all, this thesis has provided evidence based analytical procedures to ensure accurate analyses to supporting clinicians in making informed decisions for the ongoing improvement of patient care.