Pyroglutamate (5-oxoproline) measured with hydrophilic interaction chromatography (HILIC) tandem mass spectrometry in acutely ill patients
Introduction
Pyroglutamic acid (PGA), also commonly referred to as 5-oxoproline, is a highly polar and water soluble (pKa 3.61) endogenous l-glutamate derivative and intermediate in the gamma-glutamyl cycle, that infrequently causes an increased anion gap metabolic acidosis in humans [1]. Markedly increased urinary excretion of PGA is a defining feature of two rare inborn errors of glutathione metabolism: glutathione synthase (MIM #266130) and 5-oxoprolinase (MIM *614243) deficiency. The former is characterized by a persistent increased anion gap metabolic acidosis, encephalopathy and hemolysis, while the latter appears to be clinically more benign [2], [3], [4]. A transient acquired form of PGA mediated metabolic acidosis is also recognised and is typically associated with therapeutic drugs such as acetaminophen, flucloxacillin, netilmicin, vigabatrin and typically occurs in the setting of acute illness and compromised liver or renal function [5], [6], [7], [8], [9].
The hepatic metabolism of acetaminophen produces a highly reactive electrophile intermediate, N-acetyl-p-benzoquinoneimine (NAPQI), which is neutralized by conjugation to glutathione and further metabolized to acetaminophen-cysteine conjugates and excreted by the kidneys. The resulting intracellular cysteine depletion is proposed to result in a futile ATP-depleting cycle that leads to excessive PGA formation [10], [11], [12]. The non-enzymatic cyclization of a high energy phosphorylated glutamate intermediate is schematically depicted in Fig. 1 [10]. The mechanism whereby other therapeutic drugs elicit increased PGA is obscure, but may fit a similar scheme. Acetaminophen and antibiotics are widely used in healthcare settings, but there is little information about the prevalence of increased PGA which may be partly due to the lack of a readily available laboratory assay.
The laboratory measurement of PGA is challenging. Gas chromatography with tedious sample extraction and derivatization steps of urine and plasma is frequently cited in case reports. Liquid chromatography of small polar hydrophilic compounds poses a challenge, and modified ion exchange chromatography with ion paring presents one alternative [13]. Hydrophilic interaction liquid chromatography (HILIC) is an alternative modality suited for the separation of small organic acids with a polar stationary phase combined with an organic polar mobile phase traditionally associated with reverse phase chromatography [14], [15]. An advantage of the high organic content of the mobile phase is that it also promotes rapid evaporation during electron spray ionization with increased mass spectrometer analytical sensitivity. Chromatographic separation of PGA from glutamate and glutamine is required before tandem mass spectrometry as spontaneous cyclization may occur in the ionization source [16].
Our objective was to develop and validate an HILIC tandem mass spectrometry method to measure PGA blood concentration in a clinical laboratory setting. We determined a reference interval in healthy volunteers and compared this to a random sample of acutely ill patients admitted to an intensive care unit (ICU), many of whom had acute hepatic or renal dysfunction and who were receiving medications hypothesized to increase PGA.
Section snippets
Clinical samples
Non-fasting samples from 65 apparently healthy volunteers were collected to establish the range of normal plasma PGA concentrations. None of the volunteers regularly used any medication nor reported any health issues. We used surplus plasma from samples collected for clinical indications from 109 patients admitted to a mixed medical-surgical ICU. The age, sex, laboratory parameters, therapeutic drug administration, length of stay and outcome (ICU mortality) were obtained by retrospective
Method validation
To address spontaneous cyclization of glutamine and glutamate to PGA in the ionization source, the compounds needed to be chromatographically resolved as is demonstrated in Fig. 2. We also investigated the reported spontaneous cyclization caused by sample deproteinization [17] with a recovery experiment. At 40, 400 and 4000 μmol/L, the recoveries were 94.8, 98.7 and 106.9% respectively which demonstrated that this reported phenomenon was not a significant issue with this assay. The analytical
Discussion
We describe an HILIC tandem mass spectrometry method capable of measuring PGA in a healthy population with similar detection capability compared to an ion paring HPLC method, but without the use of ion paring reagents such as heptaflurobutyric acid [15] or the requirement for derivitization as with gas chromatography. The method is suitable for a routine laboratory with a simple sample preparation procedure and a short chromatography time which facilitates the provision of results in a
Acknowledgements
This work was supported by a grant from the Royal Brisbane and Women's Hospital Foundation-(HREC/13QRBW/263). We acknowledge the contribution of Leslie Johnson in assisting CW with the initial method development.
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