Chemistry and Biochemistry
Michael Van Stipdonk
Autism Spectrum Disorder, Analytical Chemistry, Isotope Dilution Mass Spectrometry, Clinical Analysis
Our research team has been focused on quantifying important biological analytes in human blood samples to delineate the biochemical processes underlying certain states of dysfunction and evaluate treatment efficacy. These efforts have been guided by laboratory measurements with the input of medical experts. Speciated isotope dilution mass spectrometry (SIDMS) explained in detail in EPA Method 6800 has been successfully applied in the quantification of an important biomarker analyte for immune function and detoxification processes: glutathione. Past research has proven the molecule a biomarker for autism spectrum disorder (ASD) in the form of the reduced/oxidized glutathione (GSH/GSSG) ratio, and literature has implicated this ratio a potential biomarker for various other disease states.
Previous researchers found the assessment of the GSH/GSSG ratio so challenging that literature disagreed on the ‘healthy’ ratio by over three orders of magnitude. The quantification continued to be a stumbling block for researchers until the Kingston research group published an accurate and precise methodology for whole blood extraction and SIDMS implementation in Analytical Chemistry. This dissertation work has centered around the development and optimization of a new extraction technique for the analysis of glutathione. Protocols have been minimized onto quantitative dried blood spot (Q-DBS) cards, enabling self-sampling and the potential for international assessment of patients. As discussed in Chapter 1, manual and fully automated extractions of DBS samples have been successfully validated according to guidance from the Federal Drug Administration (FDA) detailed in the “Bioanalytical Method Validation: Guidance for Industry” document. These newly developed methods have produced statistically similar data to that of validated methodology within the +/15% acceptance criteria. DBS cards boast various benefits to patient, analyst, and medical professional alike and the reported findings in Chapter 1 demonstrate that DBS cards are a potential replacement for phlebotomic blood draw sampling in a clinical setting.
The reduced/oxidized glutathione (GSH/GSSG) ratio can suffer broader uncertainty in the calculation due to the concentration of GSSG in blood being roughly ten times lower than that of GSH. To mitigate this issue, a novel instrumental signal boosting analytical technique termed ‘Thor’s Hammer’ or ‘Metaspiking’ has been developed to improve upon the IDMS and SIDMS capabilities of quantifying low-level analytes. Thor’s Hammer, discussed in Chapter 2, has enabled a full order of magnitude lower quantification capability beyond what the previous methodology has accomplished. In applying Thor’s Hammer, the accuracy and precision of GSSG quantification has improved greatly and the uncertainty in GSH/GSSG ratio determination has decreased.
Glutathione’s role in detoxification of xenobiotic material from the body is an important aspect of a person’s immunological health, though it can sometimes hinder medical efforts to treat certain diseases, such as cancer. In the field of oncology, several therapeutics are used for patients to rid their bodies of cancerous tissue and cells. One complication with a famous cancer drug, cisplatin, is its rapid expulsion from the body via glutathione Phase II detoxification. To track the elimination of cisplatin from a patient’s system, a new method to evaluate glutathione-conjugated cisplatin in blood and urine has been developed. Two additional platinated drug compounds, carboplatin and oxaliplatin, have been synthesized and approved for clinical use in recent decades. These compounds were synthesized to avoid bodily detection and slow down elimination from the body. Future efforts can strive to further develop this method for quantitative purposes enabling the determination of blood and urine concentration of cisplatin, carboplatin, and oxaliplatin with IDMS and SIDMS mathematics to enable a better biochemical view of the drugs bodily pathways.
Ebert, A. (2022). SPECIATED ISOTOPE DILUTION MASS SPECTROMETRY IN COMBINATION WITH THOR’S HAMMER METASPIKE TECHNOLOGY FOR THE ASSESSMENT OF LOW-LEVEL BIOMARKERS FOR HUMAN HEALTH EVALUATION (Doctoral dissertation, Duquesne University). Retrieved from https://dsc.duq.edu/etd/2210