Early acetaminophen-protein adducts predict hepatotoxicity following overdose (ATOM-5)

Journal Pre-proof Early Acetaminophen-Protein Adducts Predict Hepatotoxicity Following Overdose Angela L. Chiew, Laura P. James, Geoffrey K. Isbister, John W. Pickering, Kylie McArdle, Betty SH. Chan, Nicholas A. Buckley PII:

S0168-8278(19)30677-4

DOI:

https://doi.org/10.1016/j.jhep.2019.10.030

Reference:

JHEPAT 7541

To appear in:

Journal of Hepatology

Received Date: 21 May 2019 Revised Date:

25 October 2019

Accepted Date: 30 October 2019

Please cite this article as: Chiew AL, James LP, Isbister GK, Pickering JW, McArdle K, Chan BS, Buckley NA, Early Acetaminophen-Protein Adducts Predict Hepatotoxicity Following Overdose, Journal of Hepatology (2019), doi: https://doi.org/10.1016/j.jhep.2019.10.030. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

240 patients

1009 serum samples

100

Immediate-Release Acetaminophen Modified-Release Acetaminophen

NAPQI Binds to cellular proteins

Detoxification

Detoxification

Glucuronide & sulphate conjugates

Glutathione conjugates Acetaminophen-protein adducts released

10

Sensitivity%

Acetaminophen Overdose

1

0.1 0

12

24

36

48

60

72

84

96

108 120 132 144 156 168 180

hours post-ingestion

Acute liver toxicity

Elevated ALT

Time course of acetaminophen-protein adducts in those who developed hepatotoxicity (ALT > 1000 U/L)

ROC of initial acetaminophenprotein adducts and ALT to predict hepatotoxicity (ALT > 1000 U/L)

Early Acetaminophen-Protein Adducts Predict Hepatotoxicity Following Overdose

Angela L Chiew (1,2,3), Laura P James (4), Geoffrey K Isbister (3,5), John W Pickering (6), Kylie McArdle (3,5), Betty SH Chan (2,3) Nicholas A Buckley (1,3)

(1) Department of Pharmacology, School of Medical Sciences, University of Sydney

(2) Department of Clinical Toxicology, Prince of Wales Hospital

(3) NSW Poisons Information Centre, Children’s Hospital at Westmead

(4) Arkansas Children’s Hospital and University of Arkansas for Medical Sciences

(5) Department of Clinical Toxicology and Pharmacology, Calvary Mater Newcastle and School of Medical Practice, University of Newcastle

(6) Department of Medicine, University of Otago Christchurch, and Emergency Department Christchurch Hospital, Christchurch, New Zealand,

Corresponding Author:

Dr Angela Chiew

Department of Clinical Toxicology, Prince of Wales Hospital

Baker Street Randwick 2032 NSW Australia

Phone: +61 412575580

[email protected]

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Keywords: hepatotoxicity, poisoning, biomarker, acute liver injury, paracetamol poisoning

Word Count: 5123

Number of figures and tables: 10

Conflict of interest: Dr. James is part owner of Acetaminophen Toxicity Diagnostics (ATD), LLC, which is developing a lateral flow assay to measure acetaminophen protein adducts in blood. The remaining authors declare: no support from any organization for the submitted work; no financial relationships with any organizations that might have an interest in the submitted work in the previous three years; no other relationships or activities that could appear to have influenced the submitted work.

Financial Support: This research was partially supported by an NHMRC Program Grant 1055176. Laura James is the Principal Investigator of a Small Business Technology Transfer Award (R42DK079387) from the National Institutes of Diabetes, Digestive, and Kidney Diseases of the United States. Geoff Isbister is funded by an NHMRC Senior Research Fellowship ID1061041.

Author’s Contributions: AC, NB, GI: were involved in the design, data collection and analysis and drafting the manuscript. KM, BC: were involved in data collection and drafting the manuscript. LJ: performed the assay and was involved in drafting the manuscript. JP: performed the integrated discrimination improvement and risk assessment plots and was involved in drafting the manuscript.

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Clinical Trial registration: Australian Toxicology Monitoring (ATOM) Study – Australian Paracetamol Project: ACTRN12612001240831 (ANZCTR) Date of registration: 23/11/2012

Abstract:

Background and Aims: Acetaminophen-protein adducts are a specific biomarker of toxic acetaminophen (paracetamol) metabolite exposure. In patients with hepatotoxicity (ALT>1000U/L), a concentration >1.0nmol/mL is sensitive and specific in identifying cases secondary to acetaminophen. Our aim was to characterise acetaminophen-protein adduct concentrations in patients following acetaminophen overdose and determine if they predict toxicity.

Methods: A multi-centre prospective observational study, recruiting patients ≥14 years with acetaminophen overdose regardless of intent or formulation. Three serum samples were obtained within the first 24h of presentation and analysed for acetaminophen-protein adducts. Acetaminophen-protein adduct concentrations were compared to ALT and other indicators of toxicity.

Results: 240 patients participated, and 1009 samples were analysed. 204(85%) were acute ingestions with a median ingested dose of 20g(IQR:10-40). 228(95%) were treated with intravenous acetylcysteine at a median time of 6h(IQR:3.5-10.5) post-ingestion. 36(15%) patients developed hepatotoxicity, of which 22 had an ALT≤1000 U/L at the time of initial acetaminophen-protein adduct measurement. Those who developed hepatotoxicity had a higher initial acetaminophen-protein adduct concentration vs those who didn’t, 1.63nmol/mL (IQR:0.76–2.02, n=22) vs. 0.26nmol/mL (IQR:0.15–0.41, n=204)(p<0.0001), respectively. The ROCAUC for

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acetaminophen-protein adducts and ALT for prediction of hepatotoxicity in these patients was 0.98 (95%CI:0.96-1.00, n = 226, p<0.0001) and 0.89 (95%CI:0.82 – 0.96, n=219, p<0.0001) respectively. An acetaminophen-protein adduct concentration of 0.58nmol/mL was 100% sensitive and 91% specific for identifying patients that would develop hepatotoxicity, who presented with an initial ALT<1000U/L. When utilised in risk prediction models the addition of acetaminophen-protein adducts to initial ALT and time to presentation improved prediction of hepatotoxicity. The improvement was similar to that obtained from more complex prediction models including time adjusted acetaminophen concentrations.

Conclusion: Acetaminophen-protein adduct concentration on presentation predicted which patients with acetaminophen overdose subsequently developed hepatotoxicity, regardless of time of ingestion, or intent. An adduct threshold of 0.58nmol/L was required for optimal prediction.

Lay summary: Acetaminophen poisoning is one of the commonest causes of liver injury. This study examined a new biomarker of acetaminophen toxicity, which measures the amount of toxic metabolite exposure called acetaminophen-protein adduct. We found that those who developed liver injury had a higher initial level of acetaminophen-protein adducts than those who did not.

Abbreviations: ALI: acute liver injury, ALT: alanine aminotransferase, APAP-protein adduct: acetaminophen protein adduct, AST: aspartate aminotransferase, ATOM: Australian Toxicology Monitoring, IDI: integrated discrimination improvement, IR: immediate release, IQR: interquartile range, MR: modified release, NAPQI: N-acetylp-benzoquinone imine, PIC: Poisons Information Centre, RSTI: repeated

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supratherapeutic ingestion, ROC: receiver operator characteristic, ROCAUC: ROC area under the curve

Early Acetaminophen-Protein Adducts Predict Hepatotoxicity Following Acetaminophen Overdose (ATOM-5)

Acetaminophen (paracetamol) is one of the most common medications resulting in hospital presentations and admissions following deliberate self-poisoning and accidental overdose worldwide (1). Its major toxic effect is acute liver injury (ALI) and it is the most common cause of acute liver failure in North America, Europe and Australia (2-4). The American Association of Poison Control Centres, which annually provides over 2.1 million tele-consults for the US and associated territories, received over 100 000 calls regarding acetaminophen exposure in 2017 (5).

The mainstay of treatment of acetaminophen poisoning is acetylcysteine. Indications for treatment are determined by reported dose and time of ingestion, acetaminophen concentration and hepatic aminotransferase values (6, 7). This approach has various limitations including following an acute ingestion an accurate time of exposure is required be able to plot a acetaminophen concentration on the Rumack-Matthew nomogram. Acetaminophen concentrations have a low specificity and low positive predictive value in determining who will develop ALI. Furthermore, the aminotransferases, utilised as the traditional biomarkers of ALI, can remain within the normal range for up to 24 h post-exposure and are not specific for acetaminophen induced ALI (4). Acetylcysteine is a highly efficacious treatment and has dramatically decreased the rates of ALI and mortality from acetaminophen. However, the literature has reported cases of ALI that occurred despite early antidote treatment, particularly in large ingestions, or rarely in patients despite 5

acetaminophen concentrations below the Rumack-Matthew nomogram treatment line (8-11).

Hence, there has been increasing interest into novel biomarkers that may be able to stratify patients on presentations for their risk of liver injury and/or biomarkers that are specific for acetaminophen toxicity. Novel biomarkers include those that are liver specific [e.g. microRNA-122 (miR-122)], or reflect mechanisms of toxicity, such as the involvement of activated immune cells [e.g. high mobility group box-1 (HMGB1), keratin-18]; mitochondrial dysfunction [i.e. glutamate dehydrogenase (GLDH)] or oxidative metabolism [e.g. acetaminophen-protein adducts (APAP-protein adducts)] (12-14).

Acetaminophen is primarily metabolised into non-toxic metabolites, but a small percentage is metabolised via cytochrome P450 to produce the reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI) (15). NAPQI is detoxified by irreversible glutathione-dependent conjugation reactions to mercapturic acid and cysteine conjugates (16). In overdose, the increased formation of NAPQI depletes hepatic glutathione and NAPQI covalently binds to critical cellular proteins (17). It has been hypothesised that covalent binding reduces the activity of critical proteins, although additional mechanisms, such as mitochondrial injury and oxygen and nitrogen stress, have been linked to hepatic cell death. APAP-protein adducts represent NAPQI covalently bound to cysteine groups on proteins that are released into blood during hepatocyte lysis (18).

Previous studies evaluating APAP-protein adducts in clinical samples have examined the utility of this biomarker in patients with severe ALI and acute liver failure following acetaminophen ingestion. In these studies, APAP-protein adducts ≥ 6

1.0 nmol/mL distinguished cases of acetaminophen related acute liver failure from those due to other causes (19-22). However, there is little data on APAP-protein adduct concentration profiles within the first 24 h following toxic acetaminophen ingestion. In addition, it is unknown whether APAP-protein adducts may be able to predict acute liver injury earlier than traditional biomarkers.

Methods

Design and setting

This study was nested within the Australian Toxicology Monitoring (ATOM) Collaboration, which consists of prospective observational studies to investigate various drugs and toxins in overdose. ATOM is a multi-centre collaboration comprising five toxicology units in NSW and Queensland Australia and through calls to the NSW and QLD Poisons Information Centre (PIC). The Australian Paracetamol Project collected clinical data and at least three serum samples in the first 24 h of admission at > 4 h post-ingestion or the time of admission, 4 h later, and 1–2 h before completion of the 21 h treatment protocol with acetylcysteine. If available, serum samples collected for patient management were also analysed. The ATOM study received ethical approval from Human Research and Ethics Committees in NSW that covered all involved institutions and patients provided informed consent for participation in the study.

Selection of participants

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Patients were recruited from January 2013 until January 2015; there were two methods to identify and recruit patients. Firstly, two toxicology units (Prince of Wales and Calvary Mater), recruited all admitted patients ≥ 14 years who were assessed for acetaminophen ingestion regardless of intent (acute or repeated supratherapeutic) or preparation (immediate or modified release). These units each manage around 1000 toxicology patients annually. Secondly, higher-risk patients were recruited through telephone calls to the New South Wales (NSW) Poisons Information Centre (PIC). The NSW PIC receives approximately 65 000 calls per year from NSW (population approximately 7.95 million) (23, 24). Hence, patients ≥ 14 years were identified through the NSW PIC for inclusion if they met any of the following criteria: any acute immediate release acetaminophen ingestion of ≥ 35 g or any modified-release ingestion of ≥ 10 g or 200 mg/kg (whichever was less) over a period of less than 8 h or any patients with an alanine aminotransferase (ALT) ≥ 500 U/L (10 X the upper limit of normal) following a acetaminophen ingestion (regardless of intent or preparation). These patients were recruited from over 28 hospitals from around NSW.

Methods and measurements

Clinical data was collected on a preformatted clinical datasheet and from medical records. Data collected included demographic information, overdose exposure (time and dose ingested), co-ingestions including ethanol, laboratory results, treatments, and outcomes. The ingestion was defined as acute if the ingestion occurred over less than an 8 h period (as per the Australian acetaminophen treatment guidelines) (25). All other ingestions were considered to be repeated supratherapeutic ingestion (RSTI). In Australia, acetaminophen is available as immediate release (IR) and

8

modified release (MR) formulations. Each MR acetaminophen tablet contains 665 mg of acetaminophen of which 69% is slow-release and 31% immediate-release acetaminophen in a bilayer tablet. There were some patients for whom an ALT was not recorded at the completion of acetylcysteine treatment or 24 h post-ingestion for those not requiring acetylcysteine. These patients were assumed not to develop hepatotoxicity.

Analytical method for APAP-protein adducts

Serum samples were frozen at −80° for batched analy sis of APAP-protein adducts using a modification of the previously reported HPLC-EC assay for APAP-protein adducts (21, 26). Assay modifications included centrifugal gel filtration, high efficiency proteolytic digestion, and increased sample injection volumes, resulting in improved sensitivity and efficiency of the assay. Calibration curves were prepared over the concentration range of 0.039 to 20 nmol/mL using drug-free serum spiked with authentic APAP cysteine. Standard curves were linear with regression coefficients of 0.99. Samples having concentrations above the highest standard were diluted so their values fell within the range of the standard curve. Intra- and interassay variations were assessed from the quality control samples. Quality control concentrations ranged from 0.031 to 17.5 nmol/mL, and three replicates were analysed with each analysis. Intra-assay variation ranged from 3.24 to 10.0%. Interassay variation ranged from 5.29 to 10.49%. The lower limit of quantitation of the assay was determined by the lowest quality control concentration measurable with a CV of less than 15%. The lower limit of quantitation for the assay was defined as 0.03 nmol/mL. The laboratory technician was blinded to clinical histories and outcomes. The analysis was performed in the laboratory of Dr. Laura James, located

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at Arkansas Children’s Research Institute, Little Rock, Arkansas (Supplementary CTAT Table).

Outcomes

Clinical Outcomes: •

Acute liver injury: defined as a peak ALT > 1000 U/L (traditional main outcome measure following acetaminophen poisoning) (6). We also examined a peak ALT > 100 U/L (the UK criteria for the use of additional acetylcysteine beyond the standard 20 - 21 h course) (14).

•

Complications: coagulopathy (defined as an INR ≥ 2.0), severe acute kidney injury (AKIN classification stage 3) (27), liver transplant and death.

Pharmacokinetic Outcomes: •

To compare acetaminophen concentrations between patients at different time points, the acetaminophen ratio was calculated for acute ingestions who had a acetaminophen concentration obtained between 4 and 16 h post-ingestion. Ratios were not calculated beyond 16 h, consistent with the original publications of the Prescott nomogram (28, 29).

Acetaminophen Ratio =

•

        (!"#$% )     ''  ( ($)*⁄+  )    

Acetaminophen-aminotransferase multiplication products were calculated as per Sivilotti et al (30). The ALT or aspartate aminotransferase (AST) (in U/L; whichever was greater) and the simultaneously measured serum acetaminophen concentration (in mg/L) were multiplied. Acetaminophen

10

concentrations that were reported as lower than the laboratory limit of detection (range of detection between 1 – 10 mg/L) were recorded for the multiplication product as half this limit. If the lower limit of detection was not reported and a acetaminophen concentration was reported as “not detected”, a lower limit of detection of 5 mg/L was used. (30, 31). Pre-specified previously proposed cut-points of 1500 mg/L x U/L (10 000 µmol/L x IU/L) (30) and 10,000 mg/L x U/L (66 000 µmol/L X IU/L) (31) were utilised. •

The APAP-protein adduct concentration, ALT, aspartate aminotransferase (AST) and acetaminophen-aminotransferase multiplication products measured at the same time point to predict hepatotoxicity were compared using receiver operator characteristic (ROC) curve analysis. For acute ingestions, the APAP-protein adduct concentration used was the serum sample collected at the same time as the serum acetaminophen concentration plotted on the nomogram (≥ 4 h post-ingestion). For RSTI we used the initial sample. If these samples were unavailable, the earliest available serum sample was analysed and the ALT and acetaminophen concentration from that same time point utilised. For ROC analysis only those patients with an initial ALT ≤ 1000 U/L at the time of APAP-protein adduct measurement were included. A further analysis to predict an ALT > 100 U/L was performed with those patients with an initial ALT ≤ 100 U/L.

•

For acute ingestions the initial APAP-protein adduct was compared to age, time to initial APAP-protein measurement, dose of acetaminophen ingested and acetaminophen ratio (at the same time point).

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•

The maximum recorded APAP-protein adduct concentration was compared between those patients who did and did not develop hepatotoxicity (defined as an ALT > 1000 U/L).

•

The initial APAP-protein adduct concentration was compared to the maximum recorded ALT in all patients and the maximum recorded INR where an INR was measured. Patient’s on warfarin were excluded from the INR analysis (n=4).

Statistical Analysis

Data are presented as median and range or interquartile range (IQR) (non-normally distributed data). Comparisons between groups were made using the Mann-Whitney U test. Associations among variables were determined using Spearman’s correlations. The ROC area under the curve (ROCAUC) was determined. We examined the sensitivity, specificity and likelihood ratio of these variables to predict hepatotoxicity. A p value of < 0.05 was considered statistically significant. All analysis was performed using GraphPad PRISM® software version 8.0.2.

We determined the added value of APAP-protein adducts and acetaminophen ratio to the current standard predictors of hepatotoxicity and coagulopathy risk by examining logistic regression prediction models. We compared the AUC of the baseline model and the new models using the DeLong method (32). The baseline model comprised ALT and time to initial sampling (also a proxy for time to treatment). These two variables are known to predict hepatotoxicity and are utilised clinically. Only patients with initial acetaminophen-protein adducts collected on arrival were utilised for this analysis. For RSTI the time of initial sampling was arbitrarily set at 24 hours. The acetaminophen ratio has been used in previous 12

observational studies to compare acetaminophen concentrations between patients at different time points. It is not routinely used in clinical practice so was not included in the baseline model, instead tested as a new marker. To calculate the acetaminophen ratio in those presenting > 16 h post ingestion and RSTI ingestions, the acetaminophen nomogram value was set arbitrarily at the 16 h acetaminophen concentration.

The outcomes examined were hepatotoxicity, INR ≥ 2 and ≥ 5. Patients were only included in each model if they did not have the outcome at the time of initial sampling. In patients where an INR was not measured the INR was presumed to be < 2.0, as all patients who developed acute liver injury had an INR recorded. Each logistic regression model enables the calculation of the probability of the outcome (risk) for each individual. The baseline risk prediction model was constructed and then APAP-protein adducts, and acetaminophen ratio were added to the baseline model to form the new models. The integrated discrimination improvement (IDI) and risk assessment plots were used to assess if APAP-protein adducts had any additional benefit over known risk factors for the outcome (33). The IDI is independent of category and considers separately the actual change in calculated risk for each individual for those with and those without events (33). An alternative baseline model was constructed with acetaminophen ratio included with ALT and time to initial sampling. APAP-protein adducts was added to form the new model tested. All analysis was performed using R 3.5.1 (The R Foundation for Statistical Computing).

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Results

A total of 240 patients (176 from the two toxicology units and 64 from New South Wales PIC [Supplementary Table 1]) participated in the study and provided 1009 serum samples for APAP-protein adduct measurement. In this cohort 164 (68%) were female with a median age of 27 y (IQR: 19 - 44 y). The majority (85%) of patients were acute ingestions, distributed as 158 IR acetaminophen, 35 MR acetaminophen and 11 as a combination of both. Of the acute ingestions 99% ingested the tablets over ≤ 2 h. Thirty-six (15%) meet the criteria for RSTI of which 24 ingested for therapeutic purposes, eight were codeine abusers and four had staggered acute ingestions over a period of > 8 h. Of the RSTI, the majority were IR acetaminophen (n = 30). Patient demographic data, co-ingestions, treatments and outcomes are shown in Table 1.

Thirty-six (15%) patients developed hepatotoxicity of which 28 were acute ingestions. A further 28 (12%) had a peak ALT between 100 and < 1000 U/L of which seven were RSTI, none of which developed an INR ≥ 2.0. Of the 36 with hepatotoxicity, eight had an ALT ≤ 100U/L on presentation and 11 had an ALT of > 1000 U/L on presentation. Of those with hepatotoxicity, 25 developed a peak INR ≥ 2.0 of which eight had severe coagulopathy with an INR ≥ 5.0 and five had severe acute kidney injury (AKIN classification stage 3). There were no deaths but one patient who ingested 50 g of acetaminophen required liver transplantation.

There were 57 (24%) patients who did not have an ALT measured more than 24 h post-ingestion; of these, 45 had an initial acetaminophen concentration below the nomogram line (150mg/L at 4 h) and hence were at low risk for ALI (29). A further ten were RSTI and had clinical laboratory tests repeated after 8 -12 h. In these 14

patients, acetylcysteine was discontinued if the repeat ALT was stable or lower than the earlier ALT value (as per the Australian guidelines) (25). The remaining two were acute ingestions treated within 8 h of ingestion who did not have repeat ALT testing.

APAP-protein adducts

There were 1009 samples available for APAP-protein analysis with a median of 3 samples per patient (IQR: 2 - 5, range: 1 - 29). The APAP-protein profiles of the patients are shown in Fig. 1.

Initial APAP-protein adducts

In this cohort, 203 (85%) patients had an initial serum sample available for APAPprotein adduct analysis that was collected at the same time as the acetaminophen concentration utilised to determine need for acetylcysteine (i.e. at least 4 h postingestion for acute ingestions). In the remaining 37 (15%), we utilised the next available serum sample, for the initial APAP-protein adduct analysis, these were collected at a median time of 5 h (IQR: 3 – 14h) post the initial acetaminophen sample. There were 226 (94%) patients with an ALT ≤ 1000 U/L at the time of initial APAP-protein adduct measurement. Median initial APAP-protein adduct concentrations were significantly lower in those who did not develop hepatotoxicity compared to those who did, 0.26 nmol/mL (IQR: 0.15 – 0.41, range = 0.02 – 2.13, n=204) vs 1.63 nmol/mL (IQR: 0.76 – 2.02, range= 0.58 – 4.93, n=22) (p < 0.0001) respectively (Fig. 2). Those with established hepatotoxicity at the time of initial APAP-protein adduct measurement had a median initial APAP-protein adduct concentration of 4.58 nmol/mL (IQR: 2.13 – 18.87, range = 1.43 – 37.2 n=14), which was significantly higher than those patients with an initial ALT ≤ 1000 U/L who did

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and did not develop hepatotoxicity (p <0.0001) (Fig. 2). Results were re-analysed in those with an initial ALT ≤ 1000 U/L including only the 194 patients who had serum sample available for APAP-protein adducts analysis that was collected at the same time as the initial acetaminophen concentration. The results were similar with a median initial APAP-protein adduct of 0.25 nmol/mL (n = 174, IQR: 0.14 - 0.39) vs 1.69 nmol/mL (n = 20, IQR: 0.71 – 2.03) (p < 0.0001).

In those patients with a presentation ALT ≤ 1000 U/L, the initial APAP-protein adduct, ALT, AST (all collected from the same time point) and the acetaminophenmultiplication product were compared for utility to predict hepatotoxicity (Table 2 and Fig. 3). The initial APAP-protein adduct was superior on ROCAUC analysis (Fig. 3), compared to initial ALT or AST and acetaminophen-multiplication product (Table 2 and Fig. 3). Further, an initial APAP-protein adduct concentration of ≥ 0.58 nmol/mL was both more sensitive and specific than an ALT and AST of > 50 U/L and proposed acetaminophen-multiplication product values of ≥ 1500 mg/L x U/L (Table 2 and Fig. 3). The analysis was repeated only including those patients who had an ALT measured at 24 h or more post-ingestion. For this repeated analysis, the ROCAUC for initial APAP-protein adducts to predict hepatotoxicity remained 0.98, (95%CI: 0.96 – 1.00, n=169, p<0.0001).

The negative and positive predictive values of the initial APAP-protein adduct, ALT, AST and the acetaminophen-multiplication product to predict hepatotoxicity were calculated. As the inclusion criteria varied depending on site of enrolment results were stratified according to site of recruitment (i.e. either the two toxicology units or NSW PIC [high risk patients]) (Supplementary Table 1). The two toxicology units recruited all acetaminophen poisoning patients, in this cohort, an initial APAP-protein

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adduct concentration of ≥ 0.58 nmol/mL had a positive predictive value for hepatotoxicity of 0.48 (95%CI: 0.29 – 0.67) and negative predictive value of 1.00 (95% CI: 0.97 – 1.00) (Supplementary Table 2).

Seven patients presented with an ALT ≤ 50 U/L (within the normal reference range) and subsequently went on to develop hepatotoxicity; six of the seven had serum available from presentation for analysis of APAP-protein adducts (Table 3 and Supplementary Fig. 1). The median initial APAP-protein adduct value was 0.66 nmol/mL (IQR: 0.60 – 0.76 nmol/mL, range: 0.58 – 1.02 nmol/mL) for this cohort.

There were 18 patients who had an initial APAP-protein adduct ≥ 0.58 nmol/L who did not develop hepatotoxicity. Among this subset of patients, seven were RSTI and four were acute MR acetaminophen ingestions. Of these 18 patients, seven developed a peak ALT > 100 U/L.

The median initial APAP-protein adduct measurement and ROCAUC were also analysed with an outcome of peak ALT > 100 U/L. There were 198 (83%) patients with an ALT ≤ 100 U/L at the time of initial APAP-protein adduct measurement. Those who developed a peak ALT > 100 U/L had a higher median initial APAPprotein adduct concentration compared to those who did not, 0.57 nmol/mL (n = 20, IQR: 0.44 – 0.67 nmol/mL) vs. 0.25 nmol/mL (n = 178, IQR: 0.14 – 0.36 nmol/ml ) (p < 0.0001) (Supplementary Fig. 2). Those patients who had an ALT of > 100 U/L on presentation had a significant higher median initial APAP-protein adduct concentration [1.70 nmol/mL (IQR: 0.78 – 2.55, n=42) (p=0.0001)] than the preceding two groups (Supplementary Fig. 2). Analysis of the ROCAUC was performed to predict a peak ALT > 100 U/L, in those with an initial ALT less than this (Table 2 and Fig. 3). The initial APAP-protein adduct was superior on ROCAUC 17

analysis, compared to initial ALT or AST and acetaminophen-multiplication product (Table 2 and Fig. 3).

IDI metrics that quantify the added value of APAP-protein adducts to known risk factors (ALT and time to initial sampling) to predict hepatotoxicity, INR ≥ 2 and INR ≥ 5 are shown in Table 4 and Fig. 4. This was done by adding each individual’s initial APAP-protein adduct concentrations to the baseline model. Both the initial APAPprotein adducts and the initial acetaminophen ratio improved prediction of hepatotoxicity and INR ≥ 2 compared with known risk factors of initial ALT and time to presentation/ treatment. APAP-protein adducts substantially improved the baseline model to predict hepatotoxicity and INR ≥ 2 (ROC-AUC 0.95 vs 0.88, p<0.01 and increases in the IDI metrics for both outcomes) (Table 4 and Fig. 4A). Similar improvements to those seen with APAP adducts were seen when acetaminophen ratio was added to the model (Table 4 and Fig. 4B). However, when acetaminophen ratio was included in the baseline model, APAP-adducts did not further improve the prediction of hepatotoxicity or coagulopathy (Table 4 and Fig. 4C). Results were inconclusive for an INR ≥ 5 (n = 7) but small numbers with this outcome limit interpretation.

Fig. 5 shows the time post ingestion relationship between ALT and APAP-protein adducts in those that developed hepatotoxicity following an acute ingestion. The APAP-protein adducts appear to rise earlier and fall faster and the ratio isn’t constant over time.

For the 204 acute ingestions, initial APAP-protein adduct value was compared with age, time to initial adduct concentration, dose ingested and acetaminophen ratio (Fig. 6: A-D). There was a moderate correlation with initial APAP-protein adduct 18

concentration and time to initial measurement [Spearman r = 0.57 (95% CI: 0.46 – 0.66, p<0.0001)]. Dose and acetaminophen ratio had a weak correlation [Spearman r = 0.35 (95%CI: 0.22 – 0.47) and 0.39 (95%CI: 0.25 – 0.51) (p < 0.0001)]. No relationship was noted between age and initial APAP-protein adduct concentration Spearman r = 0.04 (95%CI: -0.10 – 0.18) (p=0.578).

For all patients the initial APAP-protein adduct value was compared with the maximum recorded ALT and INR (Fig. 6E and F); both moderately correlated with initial APAP-protein adduct value, Spearman r = 0.54 (95% CI: 0.44 – 0.63, n=240, p<0.0001) and Spearman r = 0.49 (95% CI: 0.37 – 0.59, n = 205, p<0.0001) respectively.

Cmax APAP-protein adducts

The median maximum recorded APAP-protein adduct concentration was significantly higher in patients who developed hepatotoxicity vs those who did not [9.20 nmol/mL (IQR: 2.67 – 17.75, range: 1.22 – 43.23) (n=36) vs 0.35 nmol/mL (IQR: 0.22 – 0.57, range: 0.02 – 2.38) (n= 204) (p<0.0001) respectively]. In a subset of patients (n = 131), the peak APAP-protein adduct concentration and time to peak concentration could be determined. These are shown in Appendix 1 (supplementary data) and confirm the findings of previous studies, that those with hepatotoxicity had significantly higher peak APAP-protein adduct concentration compared to those that did not, and that the peak APAP-protein adduct concentration moderately correlated with peak ALT (20, 21). We also observed a moderate correlation between peak INR and peak APAP-protein adducts (Supplementary Fig. 3), which has not been

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observed in previous studies. This likely is explained by differences in liver injury severity in the patients in these studies; our patients presented earlier in the course of toxicity, and generally had less severe liver injury and lower INR values (21, 22).

Discussion:

Elevation of hepatic transaminases following acetaminophen overdose is a latent event that may not occur until 36 to 48 hours after the overdose. There is a need for biomarkers that can detect acetaminophen ALI earlier than the delayed increase in aminotransferases. There is limited published data on early APAP-protein adduct concentrations; our study of 240 patients with acetaminophen overdose found that the initial APAP-protein adduct concentration could predict which patients presenting with a normal or moderately elevated ALT would later develop hepatotoxicity. APAPprotein adducts were superior to traditional biomarkers of ALT and AST and acetaminophen-multiplication products.

Several previous studies reported APAP-protein adduct concentrations in patients following acute ingestions of acetaminophen (19, 20, 34, 35). These studies found that APAP-protein adducts were detectable following acetaminophen overdose not resulting in ALI, but in significantly lower concentrations than those who developed ALI (20, 34). Adduct concentrations have also been shown to strongly correlate with ALT concentrations (35). However, these studies did not examine the utility of early (on presentation) measurement of APAP-protein adducts to identify patients at high risk of subsequently developing hepatotoxicity.

20

The need for treatment in those presenting within 8 h of ingestion is generally determined by a timed acetaminophen concentration plotted on the nomogram. The acetaminophen concentration alone does not reflect the contribution of the hepatic metabolism to toxicity. Although the Prescott treatment algorithm is safe to use in most patients, the literature suggests that a very small percentage of patients develop hepatotoxicity despite an acetaminophen concentration below the nomogram (8, 9). Furthermore, some patients with a high initial acetaminophen concentration are at increased risk of ALI despite acetylcysteine treatment within 8 h of ingestion (9-11). However, it is difficult to predict which patients will develop ALI. APAP-protein adducts reflect both hepatic glutathione depletion and the metabolic activation of acetaminophen through cytochrome P450 oxidation (16, 36). Six patients in the present study developed hepatotoxicity despite an ALT ≤ 50 U/L on presentation. Of interest, Patient B required a liver transplant despite antidote treatment at 2.5 h and Patient E developed acute liver failure after ingesting 10 g of acetaminophen with an acetaminophen concentration below the nomogram line (Table 3 and Supplementary Fig. 1). Patient E was not initially treated with acetylcysteine (as per local guidelines) and her time of ingestion was thought to be accurate. Both patients were otherwise well and on no medications. They both had an initial APAP-protein adduct concentration ≥ 0.58 nmol/L, higher than most other patients without hepatotoxicity on presentation (Figure 1). This finding may indicate that these patients had hepatic glutathione depletion or increased NAPQI production. We found only a weak correlation between the initial APAP-protein adduct concentration and acetaminophen dose ingested or the acetaminophen ratio in patients with acute overdose. This finding suggests that APAP-protein adducts may be a measure of early hepatic glutathione depletion.

21

Previous studies have shown that APAP-protein adducts are sensitive and specific for identifying acetaminophen hepatotoxicity and acute liver failure in patients arriving at tertiary care medical centres late in the course of the toxicity. Serum acetaminophen concentrations are often low or undetectable in the later stages of toxicity. A previous study characterizing APAP-protein adduct levels in acetaminophen-related acute liver failure reported a median Tmax value for adducts of approximately 72 hours after the toxic ingestion (20, 21). In addition, an APAPprotein adduct concentration ≥ 1.1 nmol/mL had a sensitivity of 97% and a specificity of 95% in patients with acetaminophen related acute liver failure. Additional studies used an adduct “cut-point” of 1.0 nmol/mL for adducts associated with APAP liver injury, defined as an ALT value > 1000 U/L (22, 37). The current study demonstrated that those with hepatotoxicity on presentation all had APAP-protein adduct concentration ≥ 1.0 nmol/L with many far exceeding this value. Although the APAP-protein adduct assay is not widely available, the current study suggests a role for this biomarker that is distinct from earlier reports. The initial APAP-protein adduct concentration improved prediction of hepatotoxicity and coagulopathy even when used in addition to the initial ALT and time (Fig. 4 and Table 4). The acetaminophen ratio also similarly increased prediction of hepatotoxicity and coagulopathy and is a simple measurement that could be easily utilised in clinical practice (Fig. 4 and Table 4). However, acetaminophen ratio can only accurately be determined in acute ingestions with a recorded time of ingestion. Hence, an initial APAP-protein adduct concentration could be utilised to risk stratify patients, even if the time, dose ingested, or intent was unknown. In particular, the adduct biomarker may be useful in patients where the nomogram or acetaminophen ratio cannot be utilised such as RSTI. An initial APAP-protein adduct concentration ≥ 22

0.58 nmol/L was sensitive in predicting which patients were at risk of hepatotoxicity, prior to a rise in the traditional aminotransferases. APAP-protein adducts may in the future help guide management, by 1) identifying which patients may benefit from treatment despite an acetaminophen concentration below the nomogram line, 2) guiding acetylcysteine dose and/or duration in patients with large overdoses, or 3) identifying the need for treatment in cases of RSTI.

Furthermore, the high negative predictive value and sensitivity of APAP-protein adducts mean they could be used to identify very low risk patients who still currently receive treatment with acetylcysteine. For example, in the United Kingdom the threshold for acute acetaminophen overdose is lower than in most other countries [dose > 75mg/kg/day or level above nomogram line set at 100 mg/L at 4 h (not 150 mg/L)]. This greatly increases the number of patients treated with acetylcysteine. An estimated 31 000 additional patients are treated per death from hepatotoxicity prevented (38). Among these patients who are already low risk, a low initial APAPprotein adducts may be able to identify very low risk patients who do not require treatment. However, future prospective studies utilising a treatment algorithm that incorporates currently available tests and APAP-protein adducts are needed to confirm our findings and to test these potential applications.

Two prospective cohort studies of acetaminophen overdose have shown that biomarkers such as miR-122, HMGB1, and full-length K18 at hospital presentation can predict acute liver injury (ALT > 100 U/L) better than ALT alone (14). In particular, miR-122 had higher hepatic specificity over current biomarkers (14, 39) and HMGB1 predicted a subsequent INR of more than 1.5 (14). In contrast APAPprotein adducts are a specific biomarker of acetaminophen toxicity and measures the

23

toxic metabolite bound to cellular protein. Hence it may give an indication that NAPQI production exceeds hepatic glutathione detoxification capacity.

Limitations of this study include its reliance of patient report of APAP timing and dose. However, generally these aspects of clinical history are carefully recorded as they drive treatment decisions. Secondly, the number of patients with hepatotoxicity was low, particularly those with an initial ALT < 50 U/L who went on to develop ALI. More patients are required to validate our findings and confirm the best early cut-off to predict hepatotoxicity. Similarly, larger datasets of RSTI and MR ingestions are required to confirm our results in these patient groups. Many patients in this study with an initial non-toxic acetaminophen concentration did not have a repeat ALT at 24 h. Although uncommon, some patients may have developed ALI.

Conclusion

This study demonstrates that APAP-protein adducts could be utilised as an early biomarker to predict hepatotoxicity following an acetaminophen overdose. The initial APAP-protein adduct concentration can be used regardless of the dose, preparation, intent or time of ingestion. APAP-protein adducts may be useful in the future to determine which patients require alteration to the usual treatment algorithms.

24

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Acknowledgments

The authors wish to thank the staff of the New South Wales Poisons Information Centre for assisting with identifying patients and Lynda Letzig for the analytical analysis of paracetamol protein adducts.

30

Figure Legend:

Fig. 1. APAP-protein adduct profiles divided according to ingestion intent.

A-C: Acute ingestions (ingestion over ≤ 8 h duration): A. Peak ALT < 100 U/L B. Peak ALT between 100 and 1000 U/L C. Peak ALT > 1000 U/L (hepatotoxicity)

Open circle (

): immediate release acetaminophen ingestion, Solid triangles (

):

modified release acetaminophen ingestion

D. Repeated supratherapeutic ingestions.

Closed circle (

): Peak ALT < 100 U/L or unchanged from baseline, Cross (X):

Peak ALT between 100 and 1000 U/L, Closed square (

): hepatotoxicity.

Fig. 2. Box and whiskers plot of initial APAP-protein adduct concentration stratified according to presentation ALT (i.e. ALT ≤ or > 1000 U/L) and subsequent outcome ALT > 1000 U/L. The individual points show APAP-protein adduct concentrations stratified according to type of ingestion acute immediate release ingestion ( ), modified release acute ingestion ( ), RSTI ( X ). Box and whiskers plot bar are the median value, box represents the 1st and 3rd quartile, bars the 10th and 90th centiles.

31

Fig. 3. Receiver operator curve analysis for initial APAP-protein adduct (A), ALT (B), AST (C) and acetaminophen-multiplication product (D) to predict hepatotoxicity (ALT > 1000 U/L) (solid line) and ALT > 100 U/L (dotted line).

Fig. 4. Risk assessment of baseline model and new model to predict hepatotoxicity (i), INR ≥ 2 (ii) and INR ≥ 5 (iii). A. Baseline model (ALT and time to initial sampling) and new model with the addition of APAP-protein adducts. B. Baseline model (ALT and time to initial sampling) and new model with the addition of acetaminophen ratio. C. Baseline model (ALT, time to initial sampling and acetaminophen ratio) and new model addition of APAP-protein adducts Risk assessment plots depict baseline model (dotted line) and new model (bolded line) after adding biomarker concentration to baseline model. Sensitivity versus the calculated risk and 1-Specificity versus the calculated risk for subjects who developed the outcome (event, black line) and did not develop (non-event, red line) are shown respectively. The movement of black line towards the upper right corner illustrates the addition of the biomarker to increase the predicted values for those developing the event. The movement of red line towards the lower left corner illustrates the addition of the biomarker decreased the predicted values for those who did not develop the outcome (non-event).

Fig. 5. ALT vs APAP-protein adduct concentration in acute ingestions that developed hepatotoxicity (ALT > 1000 U/L). Stratified according to time the concentration was measured post-ingestion.

32

Fig. 6. Correlation in acute ingestions of initial APAP-protein adducts and age (n= 203) (A) time to initial APAP-protein adduct concentration (n = 203) (B), acetaminophen dose ingested (n= 203) (C) and acetaminophen ratio (n=173) (D).

Correlation in all patients (acute and RSTI) of initial APAP-protein adducts and peak ALT (n=240) (E) and peak INR (n=205) (F).

Open circle ( ) represents those individuals with a peak ALT < 1000 U/L and red squares ( ) those with hepatotoxicity.

33

Table 1 Acute Ingestion All Patients (n= 240)

Females (%) Median Age (years) (IQR) Median weight (kg) (IQR)

Median Dose ingested (g) (IQR)

(n=204)

RSTI (n=36)

IR

MR$

(n=158)

(n= 46)

164 (68%)

114 (72%)

29 (63%)

21 (58%)

27.0

24

27.5

44

(19 – 44)

(19 – 38)

(17.0 – 50.5)

(28 - 50)

66

65

74

70

(57 – 80)

(55 – 80)

(59 – 82)

(50 – 90)

(n=239)

(n=157)

(n=46)

(n=36)

20¥

20

28.9

(10 -40)

(10 – 36)

12g/24h (IQR: 6.5 – 16.5)

(17 – 44.3)

For a median of 2 d (IQR: 1-5) (n=34)

0.30¥

0.28

0.44

(0.17 -0.55)

(0.16 - 0.55)

(0.25 – 0.69)

Co-ingested agents that slow gut emptying (i.e., opioids or anticholinergic) (%)

85 (35%)

60 (38%)

10 (22%)

15 (42%)

Co-ingested ethanol (%)

43 (18%)

29 (18%)

9 (20%)

5 (14%)

3¥

3.3

2.6

(1.8-8.5)

(1.8 -8.5)

(1.9 – 8.1)

(n=203)

(n=157)

(n=46)

21 (9%)

15 (9%)

6 (13%)

1.8¥

1.8

Median Dose ingested (g/kg) (IQR)

Median time to presentation (h) (IQR)

Received Activated Charcoal (%) Median time to

0

2.0

34

Activated Charcoal ALT at presentation not elevated (< 50 U/L or at their baseline)

(1 -3.3)

(1 - 3.3)

(1.2 – 3.8)

179 (75%)

127 (80%)

37 (80%)

15 (42%)

91 (58%)

33# (72%)

28* (78%)

228 (95%)

151 (96%)

43 (93%)

34 (94%)

6¥

6

4.5

(3.5 – 10.5)

(3.9 – 10.8)

(3 – 9.4)

76 (32%)

42 (27%)

26 (57%)

8 (22%)

Acetaminophen concentration above the nomogram and/or ALT > 50 U/L in those presenting > 8h post ingestion Commenced on acetylcysteine Median time to acetylcysteine (h)(IQR) Required prolonged treatment with acetylcysteine (i.e. > 20 – 21h)

Outcomes: Hepatotoxicity (ALT > 1000 U/L)

36 (15%)

22 (14%)

6 (13%)

8 (22%)

Peak ALT b/w > 100 U/L and 1000 U/L

26 (11%)

15 (11%)

4 (9%)

7 (19%)

Table 1: Demographic data, treatment and outcome of patients

$ Includes patients who ingested MR acetaminophen (n=35) or a combination of MR and IR (n=11) ¥ Acute ingestions only # either initial or repeat acetaminophen concentration above nomogram, 4 patients first acetaminophen concentration below the nomogram, repeat concentration above. *Meet criteria for acetylcysteine as per the Australian guidelines either acetaminophen concentration > 20mg/L or ALT > 50 U/L. Table 2 35

ROC AUC

Best Cut-off

(CI 95%)

Sensitivity

Specificity

Likelihood

(95% CI)

(95% CI)

Ratio

100 %

91.2 %

11.33

(85.1 – 100%)

(86.5 – 94.4%)

72.7%

81.7%

(51.2 – 86.9%)

(75.7 – 86.5%)

81%

85.1%

(60.0 - 92.3%)

(79.3 – 89.5%)

95.5%

55.7%

(78.2 – 99.8%)

(48.8 – 62.3%)

54.6%

94.1

(34.7 – 73.1%)

(90 – 96.6%)

Outcome: Hepatotoxicity (ALT > 1000 U/L) Initial APAPprotein adduct concentration

0.98

≥ 0.58 nmol/mL

(0.96 – 100) (P<0.0001)

(n= 226) ALT (n= 219)

AST (n=207)

Acetaminophen – multiplication product

0.89

> 50 U/L

(0.82 – 0.96) (p<0.0001) 0.89

> 50 U/L

(0.80 – 0.97) (p< 0.0001) 0.88 (0.81 – 0.95) (p<0.0001)

≥ 1500 mg/L x U/L ≥ 10 000 mg/L x U/L

(n = 225)

3.98

5.43

2.15

9.23

Outcome: ALT > 100 U/L Initial APAPprotein adduct (n=198)

ALT (n= 196)

AST (n=187)

Acetaminophen – multiplication

0.86

≥ 0.58 nmol/mL

(0.76 – 0.95) (p<0.0001)

0.77

> 50 U/L

(0.67 – 0.88) (P<0.0001) 0.71

> 50 U/L

(0.61 – 0.88) (p=0.0004) 0.71

≥ 1500 mg/L x U/L

50 %

94.4%

(30.0 – 70.1%)

(90.0 – 96.9%)

30.0%

89.2%

(14.6 – 51.9%)

(83.8 – 93.0%)

47.6%

94.0%

(28.3– 67.6%)

(89.3 – 96.7%)

75.0%

47.5%

8.90

2.78

7.91

1.43

36

Patient Age

product

(0.58 – 0.84)

(n = 197)

(p=0.002)

Amount (g)/ formulation of

(53.1 – 88.8%)

(40.2 – 54.8%)

Initial Pathology Results Acetylcysteine

≥ 10 000 mg/L x U/L

20.0%

97.2

(8.07 – 41.6%)

(93.5 – 98.8%)

Outcome

7.08

Table 2: Comparison of the initial APAP-protein adducts, ALT, AST and acetaminophen-multiplication product (from the same time point) ability to predict hepatotoxicity in those with an initial ALT ≤ 1000 U/L and ALT > 100 U/L in those with an initial ALT ≤ 100 U/L. Comparison of various proposed cut-offs to predict hepatotoxicity are shown.

37

Time postingestion

Acetaminophen concentration (mg/L)

ALT (U/L)

(hours)

A: 20M (120kg)

7

50g IR and codeine

242

APAPprotein adduct

Acetaminophen multiplication product

(nmol/mL)

(mg/L*U/L)

0.63

7744

27

10.5 h Standard acetylcysteine

ALT: 1519 U/L Peak INR: 2 Recovered

B: 15F (55kg)

50g IR

C: 16F

30g IR + ibuprofen

(60kg)

D: 29M (109kg)

E: 27F

7

517

25

0.59

15510

2.5 h Standard acetylcysteine

10.3

167

18

0.58

3006

12 h Increased acetylcysteine dose

17.5*

96g IR + codeine

10g IR

4

199

100

39

47

1.02

0.78

7773

5980

(65kg)

Liver transplant

Peak ALT: 1113 U/L Peak INR: 1.5 Recovered

9h

Peak INR: 3.9

Standard acetylcysteine for 20 h then increased dose

Peak ALT: 7606 U/L

54.6 h

Peak ALT: 20761 U/L

Standard acetylcysteine

Recovered

INR: 7.3 Intubated, acute renal failure requiring dialysis. Recovered

F: 20F (50kg)

RSTI 10g over 24 h for flu symptoms

on arrival

99

47

0.69

6141

5 h post presentation

Peak ALT: 4072 U/L

Standard acetylcysteine

INR: 2.1 Recovered

Table 3 *Initial acetaminophen level = 204mg/L at 8.5h Table 3: Clinical details of the six patients who developed hepatotoxicity whose initial ALT ≤ 50 U/L at the time of initial APAP-protein adduct measurement. 38

Table 4

Model

Additional variable

Total

Event

(n)

(n)

AUC

AUC (baseline model)

(alternative model)

(95% CI)

(95% CI)

IDI

IDI

Events

non-events

Difference in AUC

(95% CI)

(95% CI)

P value

0.95

0.111

0.012

(0.91 - 0.99)

(0.059 - 0.167)

(0.004 - 0.020)

0.95

0.205

0.022

(0.90 - 0.99)

(0.079 - 0.331)

(0.009 - 0.0354)

Outcome: Hepatotoxicity Baseline model (ALT + time to initial sampling)

APAP-adducts

0.009 0.88 (0.80 - 0.97)

Baseline model (ALT + time to initial sampling)

Acetaminophen ratio

0.07 194

19

Baseline model (ALT, time to initial sampling & acetaminophen ratio)

0.95

0.94

APAP-adducts

-0.007

-0.001 0.14

(-0.011 -0.002)

(-0.002 - 0.001)

0.95

0.199

0.015

(0.91 - 0.99)

(0.076 - 0.321)

(0.005 - 0.026)

0.90

0.212

0.016

(0.82 - 0.99)

(0.067 - 0.357)

(0.005 - 0.028)

0.90

0.95

0.054

0.004

(0.82 - 0.99)

(0.92 - 0.99)

(-0.001 - 0.108)

(-0.002 - 0.011)

0.84

-0.001

0

(0.71 - 0.96)

(-0.014 - 0.013)

(-0.003 - 0.003)

0.87

0.097

0.004

(0.74 to 1)

(-0.019 - 0.213)

(-0.004 - 0.011)

0.87

0.87

0.0007

0

(0.74 - 1)

(0.74 to 1)

(-0.002 - 0.004)

(-0.001 - 0.001)

(0.90 – 0.99)

(0.89 – 0.99)

Outcome: INR ≥ 2 Baseline model (ALT + time to initial sampling)

APAP-adducts

0.006 0.88 (0.81 - 0.94)

Baseline model (ALT + time to initial sampling)

Acetaminophen ratio

0.07 195

14

Baseline model (ALT, time to initial sampling & acetaminophen ratio)

APAP-adducts

0.07

INR ≥ 5 Baseline model (ALT + time to initial sampling)

0.009

APAP-adducts 0.80 (0.66 - 0.94)

Baseline model (ALT + time to initial sampling)

Acetaminophen ratio

0.26 200

7

Baseline model (ALT, time to initial sampling & acetaminophen ratio)

APAP-adducts

1

39

Table 4: Integrated discrimination improvement (IDI) for APAP-adducts and acetaminophen ratio on arrival to predict hepatotoxicity, INR ≥ 2 and INR ≥ 5. IDI-event represents the mean increased change in calculated risk for those who developed the outcome (event). IDI-non-event represents the mean increased change in calculated risk for those not developing an event after adding each biomarker. AUC: area under the receiver operator characteristic curve; IDI: integrated discriminatory improvement; CI: 95% confidence interval.

40

A. 2.5

Immediate-Release Acetaminophen

2.0

Modified-Release Acetaminophen

1.5

1.0

0.5

APAP-Protein Adducts (nmol/mL)

APAP-Protein Adducts (nmol/mL)

B.

2.5

Immediate-Release Acetaminophen

2.0

Modified-Release Acetaminophen

1.5

1.0

0.5

0.0

0.0 0

12

24

36

48

60

72

0

12

24

hours post-ingestion

36

48

60

72

84

96

108 120

hours post-ingestion

C.

D.

100

Immediate-Release Acetaminophen

100

Hepatotoxicity APAP-Protein Adducts (nmol/mL)

APAP-Protein Adducts (nmol/mL)

Modified-Release Acetaminophen 10

1

10

Peak ALT > 100 and £ 1000 U/L Peak ALT < 100 U/L

1

0.1

0.1 0

12

24

36

48

60

72

84

96

108 120 132 144 156 168 180

hours post-ingestion

0.01 0

12

24

36

48

60

72

hours post-admission

84

96

Initial APAP-Protein Adducts (nmol/mL)

100

100

10

10

1

1

0.58

0.1

0.1

0.01

0.01

Peak ALT £1000 U/L (n=204)

Acute Immediate-Release Acetaminophen Ingestion

Peak ALT >1000 U/L (ALT on presentation < 1000 U/L (n = 22)

Peak ALT >1000 U/L (ALT on presentation > 1000 U/L) (n=14)

Acute Modified-Release Acetaminophen Ingestion

RSTI

B.

A.

75

75

Sensitivity%

100

Sensitivity%

100

50

25

50

25

ALT = 50 U/L

APAP-Protein Adduct = 0.58 nmol/mL 0

0 0

25

50

75

100

0

100% - Specificity%

50

75

100

100% - Specificity%

C.

D.

100

100

75

75

Sensitivity%

Sensitivity%

25

50

50

multiplication product = 1500 mg/L x U/L

25

25

multiplication product = 10 000 mg/L x U/L

AST = 50 U/L 0

0

0

25

50

75

100

0

25

50

75

100% - Specificity%

100% - Specificity% ALT > 100 U/L

Hepatotoxicity (ALT >1000IU/L)

100

INR ≥ 2

Hepatotoxicity

INR ≥ 5

A(i).

A(ii).

A(iii).

B(i).

B(ii).

B(iii).

C(i).

C(ii).

C(iii).

10000

Measurement hours post-ingestion of acetaminophen

ALT (U/L)

1000

< 8h 8-16 h 16-24h

100

24-48 h 48-72 h >3 days 10

1 0.01

0.1

0.58 1

10

APAP-Protein adduct (nmol/mL)

100

A.

B.

128

Time to APAP-protein adduct (hours)

128

Age (years)

64

32

16

64

32

16

8

R=0.04

0.01

0.1

1

10

R=0.57

4 100

0.01

0.1

Initial APAP-protein adducts (nmol/mL)

1

10

100

Initial APAP-protein adducts (nmol/mL)

C.

10

256

D.

Acetaminophen ratio

Acetaminophen dose ingested (grams)

128 64 32 16 8 4

1

0.1

R=0.37

R=0.35

2 0.01

0.1

1

10

0.01 0.01

100

0.1

E. 16

10000

8

Maximum INR

100000

Maximum ALT (U/L)

1

10

100

Initial APAP-protein adducts (nmol/mL)

Initial APAP-protein adducts (nmol/mL)

1000

100

F.

4

2

R= 0.54 1

10

R= 0.53 1 0.01

0.1 1 10 Initial APAP-protein adduct (nmol/mL)

100

Peak ALT £ 1000 U/L

0.5 0.01

0.1 1 10 Intial APAP-protein adduct (nmol/mL)

Hepatotoxicity (ALT >1000IU/L)

100

Highlights Early Acetaminophen-Protein Adducts Predict Hepatotoxicity Following Acetaminophen Overdose (ATOM-5) • •

•

APAP-protein adducts represent NAPQI (toxic metabolite) bound to cellular proteins They are higher on arrival in those who develop liver injury even when ALT is normal They may be an early biomarker to predict hepatotoxicity after acetaminophen overdose