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Uncovering the mechanisms responsible for intravenous paracetamol-induced hypotension

ABSTRACT: Intravenous (IV) administration of paracetamol (APAP) is well-documented to cause severe hypotension, particularly in critically ill patients. We have previously shown that a metabolite of APAP – N-acetyl-p-benzoquinone imine (NAPQI) – is a potent vasodilator, but whether APAP can be metabolised to NAPQI in the vasculature is unknown. Here, we show that NAPQI can be generated by the human coronary endothelial cells (HCAECs) treated with APAP, by staining for APAP-adducts and showing a drop in cell viability and glutathione levels, similar to direct NAPQI treatment. We show that glutathione depletion following APAP treatment is caused the cytochrome p450 enzymes (CYPs) expressed in HCAECs by utilizing a blocker of the CYPs, ketoconazole. Furthermore, mass spectrometry analysis of HCAECs treated with 10 mM APAP for 24h shows upregulation of proteins involved in redox biology and the pentose phosphate pathway, as well as CYP20A1 and POR. Lastly, we show that myeloperoxidase can also metabolize APAP to NAPQI at physiologically relevant concentrations. Taken together, this study shows that APAP can be metabolized within the vasculature to the potent vasodilator NAPQI. We suggest that this is the mechanism by which critically ill patients can present with IV APAP-induced hypotension.

INSTRUMENT(S): Bruker Daltonics timsTOF series

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cardiac Blood Vessel Endothelial Cell, Cell Culture

SUBMITTER: Joakim Bastrup

LAB HEAD: Thomas Jepps

PROVIDER: PXD047218 | Pride | 2025-05-20

REPOSITORIES: Pride

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	AllRawFiles.zip	Other
	Sample_overview.xlsx	Xlsx
	UP000005640_9606.fasta	Fasta
	UP000005640_9606_additional.fasta	Fasta
	checksum.txt	Txt

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Paracetamol metabolism by endothelial cells – potential mechanism underlying intravenous paracetamol-induced hypotension

Project description:We have shown previously that a metabolite of acetaminophen (APAP), N-acetyl-p-benzoquinone imine (NAPQI), is a potent vasodilator, which could underlie the hypotension observed when APAP is administered intravenously. However, it is unknown whether APAP metabolism to NAPQI is possible in the vasculature. In this study, we examine the hypothesis that APAP is metabolized by cytochrome P450 enzymes within the endothelium, which may be accelerated in critically ill patients by the presence of elevated myeloperoxidase (MPO). Exposure of human coronary artery endothelial cells (HCAECs) to APAP resulted in the formation of protein-bound APAP adducts, together with a loss of metabolic activity. Proteomic analysis of HCAECs exposed to APAP showed upregulation of CYP20A1 and cytochrome P450 reductase (POR), together with proteins involved in the pentose phosphate pathway and maintaining redox homeostasis. Furthermore, proteomic analysis of mesenteric arteries and livers from rats administered intravenous APAP showed that APAP metabolism likely took place in the vascular wall and not in the liver. Moreover, similar proteomic pathway changes were identified in HCAECs and mesenteric arteries. Intracellular thiols were depleted in HCAECs upon APAP treatment, which was partially attenuated by ketoconazole, consistent with the involvement of cytochrome P450 enzymes (CYP) in the metabolism of APAP to a thiol-reactive metabolite such as NAPQI. Evidence was also obtained for the metabolism of APAP to a thiol-reactive intermediate by MPO, in accordance with NAPQI formation. Taken together, these data provide a putative mechanism to explain the presentation of hypotension in critically ill patients following IV APAP administration.

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