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Pattern Recognition Analysis for Hepatotoxicity Induced by Acetaminophen using Plasma and Urinary 1H NMR-Based Metabolomics in Humans Ji Won Kim, Sung Ha Ryu, Siwon Kim, Hae Won Lee, Mi-Sun Lim, Sook Jin Seong, Suhkmann Kim, Young-Ran Yoon, and Kyu-Bong Kim Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac402390q • Publication Date (Web): 15 Oct 2013 Downloaded from http://pubs.acs.org on October 20, 2013

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Analytical Chemistry

Pattern Recognition Analysis for Hepatotoxicity Induced by Acetaminophen using Plasma and Urinary 1H NMR-Based Metabolomics in Humans

Ji Won Kim†,‡, Sung Ha Ryu§, Siwon Kim#, Hae Won Lee▽, Mi-Sun Lim◇, Sook Jin Seong▽, Suhkmann Kim#, Young-Ran Yoon▽, and Kyu-Bong Kim*,§,♡ †

Department of Smart Food and Drug, Inje University, Obang-dong, Gimhae, Gyungnam

621-749, Republic of Korea ‡

Pharmacology Department, CKD Research Institute, Jung-dong, Giheung-gu, Yongin-si,

Gyunggi-do, Republic of Korea §

College of Pharmacy, Dankook University, 119 Dandae-ro, Cheonan, Chungnam 330-714,

Republic of Korea #

Department of Chemistry and Chemistry Institute for Functional Materials, Pusan National

University, Busan 609-735, Republic of Korea ▽

Kyungpook National University Graduate School and Hospital, Department of Biomedical

Science and Clinical Trial Center, Jung-gu, Daegu, Republic of Korea ◇

♡

College of Pharmacy, Yeungnam University, Kyungsan, Kyungpook, Republic of Korea Department of medical laser, graduate school, Dankook university, 119 Dandae-ro,

Cheonan, Chungnam 330-714, Republic of Korea

*

To whom correspondence should be addressed. Phone: +82-41-550-1443; Fax: +82-41-

559-7899; E-mail: [email protected].

1

ACS Paragon Plus Environment


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Abstract

Drug-induced liver injury (DILI) is currently an increasingly relevant health issue. However, available biomarkers do not reliably detect or quantify DILI risk. Therefore, the purpose of this study was to comparatively evaluate plasma and urinary biomarkers obtained from humans treated with acetaminophen (APAP) using a metabolomics approach and a proton nuclear magnetic resonance (NMR) platform. APAP (3 g/day, two 500 mg tablets every 8 h) was administered to 20 healthy Korean males (age, 20–29 years) for 7 days. Urine was collected daily before and during dosing and 6 days after the final dose. NMR spectra of these urine samples were analyzed using principal component analysis (PCA) and partial least square-discrimination analysis. Although the activities of aspartate aminotransferase and lactate dehydrogenase were significantly increased 7 days post-APAP treatment, serum biochemical parameters of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total bilirubin, γ-glutamyl transpeptidase and lactate dehydrogenase were within normal range of hepatic function. However, urine and plasma 1H NMR spectroscopy revealed different clustering between pre-dosing and after APAP treatment for global metabolomic profiling through PCA. Urinary endogenous metabolites of trimethylamine-N-oxide, citrate, 3-chlorotyrosine, phenylalanine, glycine, hippurate, and glutarate as well as plasma endogenous metabolites such as lactate, glucose, 3-hydroxyisovalerate, isoleucine, acetylglycine, acetone, acetate, glutamine, ethanol, and isobutyrate responded significantly to APAP dosing in humans. Urinary and plasma endogenous metabolites were more sensitive than serum biochemical parameters. These results might be applied to predict or screen potential hepatotoxicity caused by other drugs using urinary and plasma 1H NMR analyses. Keywords Drug-Induced Liver Injury, Acetaminophen, Metabolomics, Endogenous metabolite, Clinical 2


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study

INTRODUCTION

Acetaminophen (APAP), a mild non-narcotic analgesic and antipyretic agent, is widely used as a pain reliever and fever reducer. It is available in hundreds of singleingredient or combinations of over-the-counter products, and also in numerous prescription products. Its use has achieved great popularity, with more than 1 billion tablets sold annually in the US.1,2 APAP is metabolized by cytochrome P450 (2E1), largely via sulfation and glucuronidation (94%) and produces the toxic intermediate N-acetyl-p-benzoquinoneimine (NAPQI), which leads to hepatotoxicity. NAPQI is predominantly detoxified by glutathione conjugation in adults. The annual percentage of APAP-related acute liver failure (ALF) cases has risen from 28% in 1998 to 51% in 2003.1 In the US, APAP overdoses account for > 56,000 emergency room visits, 26,000 hospitalizations, and 450 deaths annually.3 This druginduced liver injury (DILI) is currently an increasingly relevant health issue. This type of adverse event leads most frequently to a regulatory action on drugs, including failure to approve and withdrawal from the market.4,5 It is estimated that > 1,100 drugs, herbal therapies and illicit drugs are associated with DILI, and drug-induced hepatotoxicity remains the leading cause of ALF from both APAP and non-APAP drugs.6 During the development phase of pharmaceutical drugs, the drop-off rates in clinical trial phases are estimated at > 40% of all candidates due to hepatotoxicity.7 Therefore, many incidents of hepatotoxicity to various drugs have been reported.8-11 The recent US Food and Drug Administration draft guidelines document on “Drug-Induced Liver Injury; Premarketing Clinical Evaluation” represents a formal recommendation regarding consistent approaches to 3



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detecting and investigating liver safety in clinical trials.4 Therefore, it has been an important emerging issue to assess and screen hepatotoxicity of new molecular entities in the early stages of new drug development. In this study, we show a new approach to identify biomarkers for hepatotoxicity induced by APAP using metabolomics in humans. Metabolomics is used to achieve a comprehensive measurement of the metabolome and how it changes in response to stressors, with the biological payoff being illumination of the relationship between the perturbation and affected biochemical pathways.12 Metabolomics studies have focused on the discovery of biomarkers, as well as in the preclinical arena, particularly with regard to toxicity, safety, and efficacy of a particular compound.13-15 Analytical techniques are used in metabolomic studies such as different types of a nuclear magnetic resonance (NMR) or mass spectrometry spectral data to identify new biomarkers or spectral patterns of biomarkers that can be related to toxicity or health status.16 High-resolution NMR (1H-NMR) spectroscopy has been used as a promising analytical tool for generating metabolomics data due to increased sensitivity and better spectral resolution offered by the availability of high field strength instruments along with a cryoprobe to reduce noise levels.16 Data analysis using spectral integration, principal components analysis (PCA), and partial least squares (PLS) makes NMR data meaningful for metabolomics.17, 18 It is a pattern recognition technique that is capable of classifying sample groups based on inherent similarities or dissimilarities in their corresponding biochemical compositions.16 Several studies have used metabolomics data for hepatotoxicants such as APAP, α-naphthylisothiocyante, orotic acid, methapyilene, and bromobenzene.19-26 These studies have shown that changes in the patterns of endogenous metabolites can be linked to the hepatotoxicity induced by the compounds. 19 The purpose of this study was to comparatively evaluate plasma and urinary 4


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biomarkers identified from humans treated with APAP using a metabolomics approach and a proton NMR platform. Unlike prior studies, an additional aspect of this investigation was to identify and quantify the endogenous metabolites.

MATERIALS AND METHODS

Chemicals. APAP (Tylenol® 500 mg tablet) was purchased from Janssen Korea Ltd. (Seoul, Korea). 3-(Trimethylsily)-1-propanesulfonic acid sodium salt (DSS), 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid sodium salt (TSP), and imidazole were obtained from Sigma Aldrich (St. Louis, MO, USA). Sodium azide was purchased from Bio Basic Inc. (Ontario, Canada). All other chemicals used were of the highest grade commercially available. Clinical trials. Healthy Korean male volunteers aged 20-55 years with a body weight of ≥ 55 kg and within ± 20% of ideal weight were eligible to participate in this study, if they had no clinically significant abnormalities judged by clinical history and detailed physical examination, including vital signs and laboratory analyses (blood hematology, biochemistry, and urinalysis). Subjects were excluded, who had a history of hypersensitivity to acetaminophen; had a history or evidence of cardiovascular, pulmonary, gastrointestinal, hepatic, renal, hematologic, endocrine, central nervous system, psychiatric, or malignant disease; had a history of excessive alcohol use (> 21 units of alcohol per week); had a history of drug abuse; had received any prescription drug or herbal remedy within 14 days or overthe-counter remedies within 7 days that may affect this hepatic enzyme prior to dosing; had participated in another investigational drug study within 3 months prior to dosing; had donated whole blood within 2 months or any blood product within 1 month prior to dosing; engaged in excessive smoking (> 10 cigarettes per day); had a history of abnormal diet that 5



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may affect absorption, distribution, metabolism, and excretion of drugs; had a positive serologic test result for hepatitis (B or C) and/or human immunodeficiency virus (HIV) infection. The protocol was approved by the Institutional Review Board of Kyungpook National University Hospital (KNUH 2011-02-004-001). Informed consent was signed and presented by each subject, and the study protocol followed the ethical guidelines of the Declaration of Helsinki 1975. On days 1-6, all 20 subjects aged 20-29 years were selfadministered 3 g/day APAP (2ⅹ 500 mg tablets every 8 h). Subjects were admitted to the study center at 8 pm on day 6. After an overnight fast of 10 h, subjects received the last maintenance dose with 240 mL of water in the morning of day 7. During the study period, the subjects were advised to maintain their standard diet. To determine the plasma concentrations of acetaminophen, serial blood samples (8 mL) were collected into heparinized tubes on the day before the first dose (-1 day); and immediately before and at 0.25, 0.5, 1, 1.25, 1.5, 2, 4, 6, 8, and 12 h after the last dose (7 day). Blood samples for metabolic profiling were collected on -1 day, 3 day, 7 day (at 0, 1, and 12 h after the last dose) and the follow-up visit (7±1 day after the last dose) (Fig. 1). Plasma was obtained by centrifuging the blood samples at 3,000 rpm for 10 min at 4℃ (Allegra 6, Beckman Coulter, Fullerton, CA, USA). Urine was collected daily before (-1 day) and during dosing (1–6 days, 7 day-0 h and 7 day-12 h) and 6 days (13 day) after final dosing. Urine and plasma samples were stored at -70℃ until analysis. Blood samples for clinical chemistry were collected at screening (pre-dose), 3 day, 7 day, and the follow-up visit. Clinical Chemistry. The plasma analysis was carried out on a Roche Modular Evo (D2400/P800) (Roche Diagnostics Ltd., Rotkreuz, Switzerland), using appropriate kits to determine the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), 6


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alkaline phosphatase (ALP), total bilirubin (T. bilirubin), γ-glutamyl transpeptidase (γ-GTP), and lactate dehydrogenase (LDH) at Kyungpook National University (Daegu, Korea). Pharmacokinetic analysis. APAP concentrations were analyzed using a Waters ACQUITY™ UPLC, Quattro Premier XE™ Micromass triple quadrupole mass spectrometer at Kyungpook National University (Daegu, Korea) and an ACQUITY™ UPLC Shield RP18 column (2.1 mm × 100 mm, 1.7 µm). Plasma samples were pretreated with acetonitrile using a protein free filter, and 5 µl was used for analysis. The mobile phase consisted of 0.1% formic acid in distilled water (solution A) and 0.1% formic acid in acetonitrile (solution B). APAP was analyzed with a gradient of 10–90% B from 0 to 7 min. AUCinf, AUC0-tz, Cmax, Tmax, T1/2, and Cltotal parameters were calculated using the time-concentration results. The pharmacokinetic parameters for APAP in plasma were determined by a non-compartment model using WinNonlin Pro 5.3 (Pharsight Corp., Mountain View, CA, USA). 1

H NMR urine spectroscopic analysis. After thawing the urine samples at 4°C, they

were centrifuged to remove solids. A 600 µl aliquot of the supernatant was added to a micro centrifuge tube containing 70 µl D2O solution with 5 mM DSS and 10 mM imidazole. DSS was used as the qualitative standard for the chemical shift scale. In addition, 30 µl of 0.42% sodium azide was added. After vortexing, this solution was adjusted to pH 6.8, and the urine sample was analyzed with an NMR spectrometer within 48 h. All spectra were determined using a Varian Unity Inova 600 MHz spectrometer at Pusan National University (Busan, Korea) operating at 26°C. One-dimensional NMR spectra were acquired with the following acquisition parameters: spectral width 24,038.5 Hz, 12.53 min acquisition time, and 128 nt. Additional conditions of a relaxation delay time of 1 s and saturation power of 4 were set to suppress massive water peaks. NMR spectra were reduced to data using Chenomx NMR Suit program (ver. 4.6, Chenomx Inc., Edmonton, Alberta, Canada). The δ0.0-10.0 spectral region 7



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was segmented into regions of 0.04 ppm width providing 250 integrated regions in each NMR spectrum. This binning process endowed each segment with an integral value providing an intensity distribution of the whole spectrum with 250 variables prior to the pattern recognition analysis. The spectrum region of water (δ4.5-5.0) was removed from the analysis to prevent variation in water suppression efficiency. We also identified and quantified the spectra using the Chenomx NMR Suit Professional software package ver. 4.6 (Chenomx Inc.). DSS was used as the concentration reference at a concentration of 0.5 mM. 2D NMR analysis was also performed to validate identification of endogenous metabolites. Metabolite concentrations were expressed as relative ratio values normalized to creatinine concentration, assuming a constant rate of creatinine excretion in every urine sample. 1

H NMR plasma spectroscopic analysis. After the plasma samples were thawed at

4°C, a 350 µl aliquot of the supernatant was added to a micro centrifuge tube containing 350 µl D2O solution with 4 mM TSP. TSP was used as a qualitative standard for the chemical shift scale. After vortexing, the plasma samples were analyzed with a NMR spectrometer within 48 h. Analytical condition for plasma samples are identical to urine analysis such as instruments, NMR spectra acquisition parameters, spectral binning, etc. Indetification and quantification of the spectra were also determined by same process of Chenomx NMR library. PCA and partial least square-discrimination analysis (PLS-DA). All data were converted from the NMR suite professional software format into a Microsoft Excel format. One-dimensional NMR spectra data were imported into SIMCA-P (version 12.0, Umetrics Inc., Kinnelon, NJ, USA) for a multivariate statistical analysis performed to examine intrinsic variations in the data set. These data were scaled using centered scaling prior to PCA and PLS-DA. For the scaling process, the average value of each variable was calculated, and then 8


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subtracted from the data. PCA and PLS-DA score plots were used to interpret intrinsic variation in the data. Variable importance plots (VIP) were also utilized to select putative biomarkers for hepatotoxicity by APAP. Statistical analyses. Means and standard deviations of the metabolites were calculated using Microsoft Excel. The statistical significance (P 1.0): lactate, glucose, 3hydroxyisovalerate, isoleucine, acetylglycine, acetone, acetate, glutamine, ethanol, and isobutyrate (Fig. 4 e). Ten endogenous plasma metabolites such as lactate, glucose, 3-hydroxyisovalerate, isoleucine, acetylglycine, acetone, acetate, glutamine, ethanol, and isobutyrate were significantly increased after APAP treatment (Fig. 6). Lactate increased in accordance with 13



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other studies that analyzed human serum,38 rat serum,29 mouse plasma,30 or pig plasma.39 Bernal et al. reported that as lactate is metabolized mainly by the liver, increased lactate concentration might reflect decreases in clearance and impaired hepatic function, and lactate is significantly higher in non-surviving patients than that in survivors from APAP-induced acute liver failure.40 That study concluded that arterial blood lactate measurements in cases of APAP-induced liver failure are more rapid and accurate than that of the King’s College Hospital criteria. Enhanced levels of lactate and glucose are consistent with mitochondrial impairment, which can lead to an inability to use pyruvate in the citric acid cycle.40 Plasma isoleucine levels also increased in our study and other animal studies.27,28,37 Isoleucine prevents a rise in plasma glucose concentration.42 Therefore, isoleucine increased to reduce an increase in glucose concentration. Glutamine level also increased. However, other animal studies reported that a hepatotoxic dose of APAP inhibits glutamine synthetase.43,44 Therefore, additional research is needed to explain this result. Fig. 6 shows expected plasma metabolic pathway change by APAP treatment.

CONCLUDING REMARKS We provided NMR metabolomics-based analyses of urine and plasma samples from subjects dosed with APAP. The metabolomics studies showed pattern recognition using PCA and PLS-DA by distinctly separating clusters in the APAP treatment. We proposed 14 and 10 endogenous metabolites in the urine and plasma, respectively, related to APAP treatment and possible changes in hepatic function. Clinical chemistry did not provide evident hepatic damage or changes due to APAP treatment at a dose of 3 g/day for 7 consecutive days. However, the metabolomics data showed evident changes in urinary and plasma metabolites. 14


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Therefore, NMR metabolomics analyses using biofluid spectra may lead to an effective approach for suggesting APAP-induced acute hepatic changes in clinical study.

ACKNOWLEDGMENTS This research was supported by a grant (11182MFDS605) from Ministry of Food and Drug Safety in 2011 and National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (NRF-2011-0013659)

SUPPORTING INFORMATION AVAILABLE Representative urinary or plasma NMR spectra shown in Fig. S-1 and -2 at -1 day, 1 day, 3 day, 7 day, and 13 day post-dosing of acetaminophen (2 × 500 mg tablets every 8 h, daily for 7 days). This material is available free of charge via the Internet at http://pubs.acs.org.

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Figure Legends

Fig. 1 Schematic diagram of scheduled collection of blood and urine in the clinical study. Two tablets of acetaminophen (APAP) were orally administered 8 h daily for 7 days.

Fig. 2 Urinalysis pattern recognition. The score plots are completely separated in the principal components analysis (PCA) (a) (R2X: 0.44, Q2: 0.278) and the partial least squarediscrimination analysis (PLS-DA) (b) (R2X: 0.617, R2Y: 0.508, Q2: 0.363). In targeted profiling, PCA (c) (R2X: 0.331, Q2: 0.0573) and PLS-DA (d) (R2X: 0.425, R2Y: 0.404, Q2: 0.204) are slightly separated between pre-dosing and post-dosing. Variable importance plot (VIP) (e) shows the major metabolites that contributed to separate the clusters.

Fig. 3 A comparison with an animal study (Kim et al., 2008) using PCA. Score plots of predosing (a) (R2X: 0.842, Q2: 0.655), 1 day (b) (R2X: 0.826, Q2: 0.544) and 13 day (e) (R2X: 0.812, Q2: 0.605) were positioned closely with the control group in the animal study, whereas 3 day (c) (R2X: 0.809, Q2: 0.577) and 7 day-12 h (d) (R2X: 0.708, Q2: 0.584) were close to the 1 day and 2 day score plots.

Fig. 4 Pattern recognition of the plasma analysis. The score plots were completely separated in the principal components analysis (PCA) (a) (R2X: 0.742, Q2: 0.703); however, the partial least square-discrimination analysis (PLS-DA) (b) (R2X: 0.804, R2Y: 0.31, Q2: 0.207) results were not distinctly separated except 7 day -12 h. In targeted profiling, PCA (a) (R2X: 0.923, Q2: 0.829) and PLS-DA (b) (R2X: 0.951, R2Y: 0.32, Q2: 0.157) showed similar pattern recognition of global profiling. The variable importance plot (VIP) (e) shows the major 20


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metabolites that contributed to separating the clusters.

Fig. 5 Expected urinary metabolic pathway changes due to acetaminophen (APAP) treatment. Citrate decreased on 1 day and was sustained to 7 day and then recovered on 13 day. This change in citrate level suggests that he TCA cycle might be down-regulated by APAP.

Fig. 6 Expected plasma metabolic pathway change due to acetaminophen (APAP) treatment. Although TCA cycle components were not measured, increased plasma levels of lactate, acetone, and glutamine suggested that the TCA cycle might be down-regulated by APAP.

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Table 1. The serum clinical chemistry of 20 volunteers treated with two-tablets of APAP (500 mg) for 7 consecutive days

Clinical Chemistry

1

2

Scr

3 day

7 day

psv

AST (U/L)

18.1 ± 3.3

18.1 ± 4.9

26.9 ± 8.9*

19.8 ± 4.7

ALT (U/L)

16.6 ± 6.4

15.3 ± 7.2

21.4 ± 10.8

23.7 ± 12.1

ALP (U/L)

63.0 ± 14.0

62.8 ± 14.3

54.1 ± 13.7

62.7 ± 13.9

T.bilirubin (mg/dl)

0.8 ± 0.3

0.7 ± 0.3

0.7 ± 0.3

0.8 ± 0.3

γ-GTP (U/L)

15.9 ± 4.1

15.1 ± 6.1

15.2 ± 8.1

17.3 ± 8.8

LDH (U/L)

293.7 ± 42.3

307.7 ± 46.7

466.5 ± 101.2*

298.7 ± 45.7

*

, p

Pattern Recognition Analysis for Hepatotoxicity ... - ACS Publications

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