Measurement of Serum Acetaminophen–Protein Adducts in Patients With Acute Liver Failure

Article (PDF Available)inGastroenterology 130(3):687-94 · April 2006with140 Reads
DOI: 10.1053/j.gastro.2006.01.033 · Source: PubMed
Acetaminophen toxicity is the most common cause of acute liver failure (ALF) in the United States and Great Britain, but may be underrecognized in certain settings. Acetaminophen-protein adducts are specific biomarkers of drug-related toxicity in animal models and can be measured in tissue or blood samples. Measurement of serum adducts might improve diagnostic accuracy in acute liver failure (ALF) patients. We measured serum acetaminophen-protein adducts using high-pressure liquid chromatography with electrochemical detection in coded sera of 66 patients with ALF collected prospectively at 24 US tertiary referral centers. Samples were included from 20 patients with well-characterized acetaminophen-related acute liver failure, 10 patients with ALF owing to other well-defined causes, 36 patients with ALF of indeterminate etiology, and 15 additional patients without ALF but with known acetaminophen overdose and minimal or no biochemical liver injury. Acetaminophen-protein adducts were detected in serum in 100% of known acetaminophen ALF patients and in none of the ALF patients with other defined causes, yielding a sensitivity and specificity of 100%. In daily serial samples, serum adducts decreased in parallel with aminotransferase levels. Seven of 36 (19%) indeterminate cases demonstrated adducts in serum suggesting that acetaminophen toxicity caused or contributed to ALF in these patients. Low adduct levels were present in 2 of 15 patients with acetaminophen overdose without significant liver injury. Measurement of serum acetaminophen-protein adducts reliably identified acetaminophen toxicity, and may be a useful diagnostic test for cases lacking historical data or other clinical information.
Measurement of Serum Acetaminophen–Protein Adducts in
Patients With Acute Liver Failure
and The Acute Liver Failure Study Group
*University of California at San Francisco, San Francisco, California;
University of Arkansas for Medical Sciences and Arkansas Children’s
Hospital Research Institute, Little Rock, Arkansas;
University of Arkansas for Medical Sciences, Little Rock, Arkansas;
University of
Washington, Seattle, Washington;
University of Texas Southwestern Medical Center, Dallas, Texas;
University of Michigan, Ann Arbor,
Michigan; **University of Texas Southwestern Medical Center, Dallas, Texas;
Albert Einstein Medical Center, Philadelphia, Pennsylvania;
University of Pittsburgh, Pittsburgh, Pennsylvania; and
University of Texas Southwestern Medical Center, Dallas, Texas
Background & Aims: Acetaminophen toxicity is the most
common cause of acute liver failure (ALF) in the United
States and Great Britain, but may be underrecognized in
certain settings. Acetaminophen–protein adducts are
specific biomarkers of drug-related toxicity in animal
models and can be measured in tissue or blood sam-
ples. Measurement of serum adducts might improve
diagnostic accuracy in acute liver failure (ALF) patients.
We measured serum acetaminophen–protein
adducts using high-pressure liquid chromatography with
electrochemical detection in coded sera of 66 patients
with ALF collected prospectively at 24 US tertiary refer-
ral centers. Samples were included from 20 patients
with well-characterized acetaminophen-related acute
liver failure, 10 patients with ALF owing to other well-
defined causes, 36 patients with ALF of indeterminate
etiology, and 15 additional patients without ALF but with
known acetaminophen overdose and minimal or no bio-
chemical liver injury. Results:
adducts were detected in serum in 100% of known
acetaminophen ALF patients and in none of the ALF
patients with other defined causes, yielding a sensitivity
and specificity of 100%. In daily serial samples, serum
adducts decreased in parallel with aminotransferase
levels. Seven of 36 (19%) indeterminate cases demon-
strated adducts in serum suggesting that acetamino-
phen toxicity caused or contributed to ALF in these
patients. Low adduct levels were present in 2 of 15
patients with acetaminophen overdose without signifi-
cant liver injury. Conclusions:
Measurement of serum
acetaminophen–protein adducts reliably identified acet-
aminophen toxicity, and may be a useful diagnostic test
for cases lacking historical data or other clinical infor-
cetaminophen (N-acetyl-P-aminophenol; paraceta-
mol) is the most widely used analgesic-antipyretic
in the United States and Europe, with more than 1
billion tablets sold annually in the United States alone.
Present in more than 600 separate products, acetamino-
phen is generally safe when taken in modest doses (4
g/d, the maximum dose recommended on the package
label), but is also a potentially lethal dose-dependent
toxin. More than 56,000 emergency visits and nearly 500
deaths in the United States each year result from acet-
aminophen toxicity, owing to either intentional or un-
intentional overdoses.
Indeed, acetaminophen toxicity is
currently the most common cause of acute liver failure in
the United States and the United Kingdom.
Intentional acetaminophen overdose is usually easy to
diagnose when a history of a 1-time large-dose ingestion
is given or elevated acetaminophen blood levels are
found. By contrast, in unintentional cases the diagnosis
can be elusive for several reasons: patients unaware of the
risks associated with acetaminophen may not recall tak-
ing the medication; markedly elevated aminotransferase
levels are characteristic of acetaminophen toxicity but are
not diagnostic; plasma acetaminophen concentrations are
often undetectable by the time liver injury has occurred;
and unintentional cases present late when encephalopa-
thy may already be present.
As a result, many cases of
acetaminophen toxicity are unrecognized, leading to a
delay in administration of the potentially life-saving
antidote, N-acetylcysteine (NAC).
Mechanisms of acetaminophen toxicity have been ex-
tensively studied (Figure 1).
Excessive formation of a
highly reactive intermediate, N-acetyl-p-benzoquinone-
imine (NAPQI), occurs when large doses are taken. In
Abbreviations used in this paper: ALF, acute liver failure; CV, coef-
ficient of variation; NAC, N-acetylcysteine; NAPQI, N-acetylparabenzo-
© 2006 by the American Gastroenterological Association Institute
GASTROENTEROLOGY 2006;130:687– 694
the absence of glutathione, covalent binding of NAPQI
to cysteine groups on hepatocyte proteins occurs forming
acetaminophen–protein adducts that may be released
into serum.
9 –11
Studies using radiolabeled acetamino-
phen and anti-acetaminophen antibodies have shown
that this covalent binding represents the initial and
irreversible step in the development of hepatic toxicity.
An accurate and reliable assay that could identify pa-
tients with occult acetaminophen-related liver injury
would be of significant value to clinicians. Utilizing a
highly specific assay employing high-performance liquid
chromatography with electrochemical detection (HPLC-
we measured acetaminophen–protein adducts in
serum in patients with acute liver failure (ALF) of dif-
fering etiologies to determine the validity of such an
assay for detecting acetaminophen hepatotoxicity. We
also compared the presence and quantity of the adducts
detected in serum samples from patients with known
acetaminophen-related ALF, patients with other known
causes of ALF, patients with acetaminophen overdoses
without significant injury, and those with acute liver
failure of indeterminate etiology.
Materials and Methods
Study Participants
Serum and data were prospectively collected from Jan-
uary 1998 to the present at 24 sites participating in the U.S.
ALF Study Group on more than 880 adult patients who met
criteria for ALF, defined as the presence of coagulopathy (pro-
thrombin time 15 seconds or international normalized ratio
1.5) and hepatic encephalopathy within 26 weeks of first
symptoms of illness and without evidence of previous liver
Figure 1. Schematic diagram of acetaminophen metabolic pathways. Metabolism of the parent compound is principally via glucuronidation and
sulfation, with only a small fraction normally being metabolized via cytochrome 450 (in particular, CYP2E1) to NAPQI, the reactive intermediate
form. In the presence of adequate glutathione stores, NAPQI is converted into a harmless, water-soluble conjugate; however, if the capacity to
detoxify is exceeded, then NAPQI can covalently bind to cell proteins via cysteine residues. The acetaminophen–protein adducts so formed may
enter the serum with cell death and release of intracellular proteins.
Informed consent is obtained from the patients’ legal
next of kin before enrollment, according to the guidelines of
local institutional review boards.
The etiology of ALF is initially determined at each study center
by the local site investigator using generally accepted diagnostic
criteria based on history, laboratory values, imaging studies, and,
in some cases, histologic examination of the liver.
A careful
history of acetaminophen ingestion is recorded on the case
report forms (CRFs) for each patient, including total dose
ingested, type of acetaminophen product taken, and duration
of use. At the beginning of the study, we established standard
criteria for acetaminophen ingestion to be used by the sites: (1)
a history of ingestion of doses exceeding the package labeling
(4 g/24 hours) within 7 days of presentation; in most cases,
the quantities greatly exceeded this value; (2) detection of
acetaminophen in plasma of a patient meeting other qualifi-
cations for ALF; (3) ALT levels 3500 IU/L, because these
levels are commonly observed in acetaminophen toxicity and
infrequently or rarely with other causes of ALF.
Each case in
the present study was confirmed to have had rapid onset
disease typical of acetaminophen-induced injury. Data pro-
vided on the CRFs were then queried to determine whether
additional data could be obtained. Each case of presumed
acetaminophen ingestion was then subjected to further evalu-
ation at the central site by a systematic review of the CRF. Site
visits by the overall principal investigator of the study
(W.M.L.) provided a further level of scrutiny of cases by
comparing the original medical record with the CRF to be
certain of the diagnosis. In addition to the 20 cases with
definite acetaminophen toxicity, 10 patients with ALF were
chosen at random representing other well-defined etiologies
including 4 with idiosyncratic drug-induced liver injury, 2
with autoimmune hepatitis, 2 acute hepatitis B, 1 acute hep-
atitis A, and 1 with Wilson disease. In this group, there was
no history of concomitant acetaminophen ingestion. ALF was
considered to be of indeterminate etiology when careful his-
torical review and extensive clinical, radiographic, and labora-
tory evaluation (including toxicology screens, serologic mark-
ers of viral hepatitis A, B, and C, and antinuclear and anti–
smooth-muscle antibodies), and pathologic analysis (where
available) failed to indicate a defined cause. Outcome data and
final diagnosis were recorded on a subsequent CRF 3 weeks
after admission, at the time of liver transplantation, or death.
Serum Analysis
Serum samples collected at admission to study and for
6 additional days were aliquoted at room temperature within
2 hours of collection and stored at 80°C prior to analysis. For
the 20 patients with established acetaminophen-related ALF,
17 (85%) had a history of excessive acetaminophen ingestion
and 15 had ingested 10 g (median ingestion for the group as
a whole: 24 g). In 3 of 4 where doses were unknown, the ALT
values were 8000, 14,570, and 18,079 IU/L. Fifteen (75%) had
detectable acetaminophen levels and 17 (85%) had serum ALT
3500 IU/L. For this known acetaminophen group, the me-
dian total dose ingested was 21 g, and the median ALT value
was 5600 IU/L. In addition to the 10 patients with ALF where
another diagnosis had been established, 36 consecutive pa-
tients with ALF of indeterminate etiology were selected for
study. All but 2 of the 66 patients studied had samples
available from the day of admission to the ALF study. In 6
patients found to have detectable adducts at admission to
study, we studied serial samples to determine the kinetics of
serum adduct elimination. Finally, we analyzed sera from 15
patients hospitalized with intentional acetaminophen overdose
but minimal or no biochemical evidence of liver injury (and
thus no ALF) to determine whether large ingestions with low
levels of liver injury were associated with development of
serum acetaminophen–protein adducts. All patients in this
group (median SD age, 15 4.2 years; 14 women) were
Table 1. Demographic and Clinical Characteristics of Patients With Acetaminophen– or Non-Acetaminophen–Related ALF
Acetaminophen cases
(n 20)
Non-acetaminophen cases
(n 10)
Range Median Range Median
Age (y) 17–63 32 19–55 41
Sex (% female) 70 40
Coma grade I–IV III I–III II
APAP level (mg/L) 0–346 44 0–10
ALT (IU/L) 257–18,079 5640 22–2102 378
AST (IU/L) 59–16,195 4404 122–1609 796
International normalized ratio (INR) 1.3–20.0 3.2 1.2–8.9 2.6
Bilirubin (mg/dl)
0.8–14.1 5.0 7.4–31.8 20.2
Creatinine (mg/dl)
0.5–6.8 1.3 0.7–4.4 1.8
Adduct concentration (nmol
acetaminophen–CYS/mg protein)
0.36–2.83 1.17 0 0
Transplanted (%) 10 50
Outcome (% alive) 85 60
The acetaminophen levels, which were obtained on 4 of the 10 patients, were 0, 10, 10, and 10 mg/dL.
To convert to mol/L multiply by 17.1.
To convert to mol/L multiply by 88.4.
admitted to the hospital following intentional acetaminophen
overdose for treatment with oral NAC and had daily monitor-
ing of ALT values. The median acetaminophen level at pre-
sentation was 108 mg/dL (range, 2–288 mg/dL). Eight of the
15 were judged to be at risk of toxicity by the Rumack
nomogram (equivalent to a serum concentration of APAP of
150 g/mL at 4 hours); 5 presented 24 hours after the
overdose; and 2 patients did not meet criteria for treatment,
but were nonetheless admitted for treatment based on histor-
ical data (dose of acetaminophen ingested). In this patient
subset, the median time to the first dose of NAC was 9 hours
(range 2.5–38.0 hours) and the median peak value for ALT in
these patients was 26 IU/L (range, 2–104 IU/L); all patients in
this group recovered uneventfully.
Measurement of Acetaminophen–Protein
Serum samples were analyzed for acetaminophen–pro-
tein adducts (acetaminophen-cysteine) by a high-performance
liquid chromatography with electrochemical detection
(HPLC-EC) method as previously reported.
Briefly, samples
were dialyzed, treated with protease, and then precipitated
with trichloracetic acid. Following centrifugation, the result-
ing supernatant was injected onto the HPLC machine using a
Model 582 solvent delivery system with 2 Model 5600A
CoulArray detectors (ESA, Chelmsford, MA). The range of
linearity for this method was 1.0 –30.0 mol/L acetamino-
phen-cysteine in serum. The coefficients of variation (CVs) for
the assay were consistently 10% at concentrations of 1.0,
6.0, and 30.0 mol/L acetaminophen-cysteine adducts. Based
on the CVs for the standard curve for the assay, the lower limit
of quantitation for the assay was defined as 1 mol/L acet-
aminophen-cysteine and thus, a sample containing 1.0
mol/L acetaminophen-cysteine was defined as positive, and
those 1.0 mol/L were considered negative. To standardize
each sample for its protein content, concentrations of acet-
aminophen-cysteine determined by HPLC-EC were then cal-
culated as a fraction of the protein content of the sample
(Bradford method),
the final concentrations being expressed
as nmol acetaminophen-cysteine/mg protein. The summary
data for patient subgroups are presented as median and range
Adducts in Patients With Acetaminophen
and Non-Acetaminophen ALF
Acetaminophen protein adducts were present in
all 20 serum samples from patients with acetaminophen-
related toxicity (median, 1.165 nmol acetaminophen-
cysteine/mg protein; range, 0.36 –2.83). In contrast, no
significant adduct levels were detected in the samples of
patients with ALF of other known etiologies (median,
0.005 nmol acetaminophen-cysteine/mg protein; range,
0.0 0.027). Based on these data, the sensitivity and
specificity of the adduct assay were 100%. In sera from
15 patients with acute acetaminophen overdose without
significant liver injury, the concentration of acetamino-
phen-protein adducts was detectable but only at very low
levels (median, 0.036 nmol acetaminophen-cysteine/mg
protein; range, 0 0.138).
Adducts in Serial Samples From Patients
With ALF
In patients for whom serial measurements of ad-
duct levels were made, 2 (Figure 2A and B) had mea-
surable acetaminophen concentrations prior to the study,
whereas another patient (Figure 2C) had a history of
excessive acetaminophen ingestion 3 days prior to pre-
senting with liver failure, but no detectable acetamino-
phen in serum on admission. In this patient the acet-
aminophen-cysteine value from the admission blood
sample was 2.78 nmol acetaminophen-cysteine/mg pro-
tein. The highest levels of adducts in these patients were
associated with the highest serum aminotransferase levels
observed, although peak levels might have occurred prior
to admission to study. The decline of acetaminophen-
cysteine paralleled the resolution of biochemical abnor-
malities. Assuming that the disappearance of adducts
follows first-order kinetics, the half-life of adducts in sera
ranged from 17.1 to 30.7 hours in these patients.
Adducts in Patients With Indeterminate
Analysis of acetaminophen protein adducts in sera
disclosed that 7 of 36 (19.4%) indeterminate patients
had detectable acetaminophen-cysteine adducts (median
value 1.06 nmol acetaminophen-cysteine/mg protein;
range 0.23–3.14). The values and range measured in this
group did not differ significantly from those observed in
well-characterized acetaminophen cases (Figure 3). In
addition, the rate of decline in serum adduct concentra-
tion in indeterminate patients (eg, Figure 2D) was sim-
ilar to that observed in the patients with ALF from
acetaminophen and appeared to parallel the decline in
serum aminotransferases.
In retrospect, despite lacking a history of excessive
acetaminophen ingestion, the adduct-positive indetermi-
nate cases studied (Table 2), displayed clinical features
that were similar to those with known acetaminophen
toxicity: they were younger, more likely to be female,
and had higher aminotransferase and serum creatinine
levels, but lower serum bilirubin values than the adduct-
negative indeterminate ALF group. Although the per-
centage of patients reporting any exposure to acetamin-
ophen (albeit in the therapeutic range) was higher in the
adduct-positive than the adduct-negative group, the per-
centage with detectable serum acetaminophen levels was
identical in both groups (17%). Empiric treatment with
NAC was uncommon in both adduct-positive (14%) and
adduct-negative (21%) cases, suggesting that the physi-
cians caring for these patients considered acetaminophen
toxicity unlikely. In contrast, 92% of suspected acet-
aminophen cases were treated with this well-established
antidote for acetaminophen toxicity. The overall sponta-
neous (without transplant) survival rate was substantially
lower in the indeterminate group (13%) than in the
acetaminophen control group (80%), but was higher for
the adduct-positive (29%) compared with the adduct-
negative (10%) group.
In this study, we evaluated the role of an assay to
measure acetaminophen adducts in patients with known
acetaminophen toxicity, other causes of acute liver fail-
ure, a large number with indeterminate ALF, and a
group of patients who sustained an overdose but dem-
onstrated little evidence of liver damage. The HPLC-EC
assay identified all 30 test cases of ALF with known
diagnoses as acetaminophen related (or not). The tempo-
ral profile of serum adducts paralleled that of aminotrans-
ferase levels, remaining detectable for up to 7 days after
admission to study. More importantly, acetaminophen
was also implicated in cases not previously recognized as
affected, by the detection of adducts in similar quantities
to the known acetaminophen cases in nearly 20% of the
indeterminate group.
The diagnosis of ALF due to acetaminophen is clear
cut when a history of ingestion of an excessive amount of
acetaminophen coupled with an elevated serum acet-
aminophen level is obtained. However, those overdose
patients with the most severe injury and the poorest
prognoses (typically unintentional cases) present late for
medical care when acetaminophen levels are often unde-
tectable and a reliable history may not be obtained owing
to altered mentation.
Moreover, false-positive acet-
aminophen levels may be observed in the presence of
Figure 2. Acetaminophen–CYS adduct levels and ALT levels measured serially from day 1 to 7 in 4 patients with either known acetaminophen
toxicity (A–C) or indeterminate ALF (D). In each case, the decline in serum adduct concentration approximates that of ALT. On presentation,
patients A and B had histories of excessive acetaminophen dosing and detectable serum acetaminophen levels, whereas patient C had a history
of excessive dosing but an undetectable acetaminophen level, and patient D had neither history nor an elevated serum acetaminophen level.
serum bilirubin levels 10 mg/dL, further impeding an
accurate diagnosis.
NAC is a highly effective anti-
dote for acetaminophen poisoning when given early fol-
lowing ingestion, making an accurate diagnosis of signal
It remains uncertain how much benefit
may accrue if NAC is given after onset of injury, but it
may be effective as late as 72 hours following ingestion.
Although NAC may be given empirically when acet-
aminophen toxicity is a possibility, a rapid and accurate
biomarker for acetaminophen injury would allow its use
only when appropriate.
Extensive time course and dose–response studies in
animal models have characterized acetaminophen–pro-
tein adduct formation in acetaminophen toxicity.
duct formation appears in the liver following depletion of
glutathione and the appearance of unconjugated NAPQI,
the reactive metabolite of acetaminophen (Figure 1).
Serum adducts are presumed to be of hepatic origin based
on the time course for their appearance relative to their
formation in the liver.
Thus, acetaminophen–protein
adducts in serum represent liver injury specific to acet-
The presence of adducts in the indeter-
minate cases does not clarify whether acetaminophen
toxicity was the only cause or whether another etiology
plus acetaminophen together caused the liver injury;
however, the levels of adducts were similar to those
found in our known ingestions, and the clinical similar-
ities of the cases (low bilirubin, high ALT levels) indicate
that acetaminophen may well have been the sole agent,
unrecognized because of encephalopathy or specific de-
nial of the ingestion.
Our study has limitations. Foremost, we focused
mainly on patients with ALF and have not yet studied
sera from patients with therapeutic exposure to acet-
aminophen or with acute acetaminophen overdose and
moderate to severe liver injury without encephalopathy.
In patients with acute overdose and early NAC admin-
istration, injury was prevented and adducts were found
in low concentrations in only 2 of the 15 patients. The
Figure 3. Serum levels of acetaminophen–CYS adducts in patient
groups. (A) Patients with ALF secondary to known acetaminophen
overdose. (B) Patients with ALF owing to nonacetaminophen causes.
(C) Patients with acetaminophen overdose but no ALF. (D) Patients
with ALF of indeterminate etiology and detectable serum adducts. (E)
Patients ALF of indeterminate etiology and negative adducts. The
boxes represent the 25th–75th IQR and the horizontal line represents
the median. The extremes of the population are represented by the
Table 2. Demographic and Clinical Characteristics of Patients With Indeterminate ALF
Indeterminate Group With Adducts (N 7)
Group Without
Adducts (N 29)
Range Median Range Median1 2 3 4567
Age (y) 33 42 23 32 17 35 29 17–42 32 17–76 39
ALT (IU/L) 5760 4773 8960 849 4288 6430 2639 849–8960 4773 29–3724 881
AST (IU/L) 14,580 11,029 18,928 1569 6465 8850 1629 1549–18,928 8850 42–7310 778
normalized ratio
8.2 8.1 3.8 2.2 1.7 6.2 3.0 1.7–8.2 3.8 1.3–24.8 2.7
Bilirubin (mg/dL)
2.2 6.7 7.1 10.9 8.5 2.5 5.3 2.2–10.9 6.7 1.8–52.2 23.7
Creatinine (mg/
3.8 2.9 3.4 3.9 0.5 2.2 3.6 0.5–3.9 3.4 0.7–6.3 1.4
level (mg/L)
16 10 2 10 0 ND 10 0–16 0–15
2.71 3.14 1.77 0.94 1.06 0.48 0.23 0.23–3.14 1.06 0.0–0.138 0.000
ND, not determined.
To convert to mol/L multiply by 17.1.
To convert to mol/L multiply by 88.4.
finding of minimal amounts of adduct formation in
patients without overt liver injury is similar to the
results observed in experimental animal models; early
treatment with NAC in mice can abort toxicity but small
amounts of adducts are still observed in the liver in this
The HPLC-EC assay used in this study is labor
intensive and not yet available in hospital clinical
laboratories. However, refinement of the assay has
reduced its turnaround time to 2–3 hours. By con-
firming the diagnosis of acetaminophen toxicity, the
practicing physician may be able to consider and use
NAC earlier and may prevent future episodes of in-
tentional or unintentional toxicity, for example, by
confronting the patient and arranging psychiatric
evaluation if appropriate. Recent data suggest that
acetaminophen overdoses are often repeated, making
an accurate diagnosis of acetaminophen toxicity essen-
Identification of a surreptitious acetamino-
phen overdose may also have implications with regard
to liver transplant candidacy in certain situations, and
also has important forensic and medicolegal implica-
tions. Confirming the diagnosis of acetaminophen
hepatotoxicity may preclude further etiologic evalua-
tion, thereby decreasing health care costs; refuting the
diagnosis may lead to additional testing. Measurement
of adducts might more accurately define the epidemi-
ology of acetaminophen toxicity, leading to public
health measures to limit cases. Finally, treating all
ALF patients with NAC when a specific diagnosis
cannot be determined may be appropriate in light of
our finding that unrecognized cases of acetaminophen
toxicity regularly occur.
In conclusion, we demonstrated that measurement of
serum acetaminophen–protein adducts represents a sen-
sitive and specific method to confirm or exclude acet-
aminophen as the cause of severe liver injury in patients
with ALF. As such, the assay should help to identify
acetaminophen-induced liver injury in situations where
historical or laboratory data are lacking. Because occult
acetaminophen toxicity appears responsible for a signif-
icant proportion of cases of indeterminate ALF, a rapid
test for adducts is a worthwhile goal. Until such a test is
readily available, use of NAC for patients with ALF
where the diagnosis is uncertain, particularly where char-
acteristic low bilirubin and high aminotransferase levels
are observed, seems indicated.
1. Nourjah P, Willey M. Epidemiology of acetaminophen-related over-
dose. Department of Health and Human Services, Center for Drug
Evaluation and Research, Food and Drug Administration, 2002.
2. Ostapowicz G, Fontana RJ, Schiodt FV, Larson A, Davern TJ, Han
SH, McCashland TM, Shakil AO, Hay JE, Hynan L, Crippin JS, Blei
AT, Samueal G, Reisch H, Lee WM. Results of a prospective study
of acute liver failure at 17 tertiary care centers in the United
States. Ann Intern Med 2002;137:947–954.
3. Bernal W. Changing patterns of causation and the use of trans-
plantation in the United Kingdom. Semin Liver Dis 2003;23:227–
4. Schiodt FV, Rochling FA, Casey DL, Lee WM. Acetaminophen
toxicity in an urban county hospital. N Engl J Med 1997;337:
5. Lee WM. Drug-induced hepatotoxicity. N Engl J Med 2003;349:
474 485.
6. Jollow DJ, Mitchell JR, Potter WZ, Davis DC, Gillette JR, Brodie
BB. Acetaminophen-induced hepatic necrosis. II. Role of covalent
binding in vivo. J Pharmacol Exp Ther 1973;187:195–202.
7. Mitchell JR, Thorgeirsson SS, Potter WZ, Jollow DJ, Keiser H.
Acetaminophen-induced hepatic injury: protective role of glutathi-
one in man and rationale for therapy. Clin Pharmacol Ther 1974;
16:676 684.
8. Bessems JG, Vermeulen NP. Paracetamol (acetaminophen)-in-
duced toxicity: molecular and biochemical mechanisms, ana-
logues and protective approaches. Crit Rev Toxicol 2001;31:55–
9. James LP, Mayeux PR, Hinson JA. Acetaminophen-induced hepa-
totoxicity. Drug Metab Dispos 2003;31:1499 –1506.
10. Roberts DW, Pumford NR, Potter DW, Benson RW, Hinson JA. A
sensitive immunochemical assay for acetaminophen-protein ad-
ducts. J Pharmacol Exp Ther 1987;241:527–533.
11. Pumford NR, Hinson JA, Potter DW, Rowland KL, Benson RW,
Roberts DW. Immunochemical quantitation of 3-(cystein-S-yl)
acetaminophen adducts in serum and liver proteins of acetamin-
ophen-treated mice. J Pharmacol Exp Ther 1989;248:190 –196.
12. Pumford NR, Hinson JA, Benson RW, Roberts DW. Immunoblot
analysis of protein containing 3-(cystein-S-yl) acetaminophen ad-
ducts in serum and subcellular liver fractions from acetamino-
phen-treated mice. Toxicol Appl Pharmacol 1990;104:521–532.
13. Muldrew KL, James LP, Coop L, McCullough SS, Hendrickson HP,
Hinson HA, Mayeux PR. Determination of acetaminophen-protein
adducts in mouse liver and serum and human serum after hep-
atotoxic doses of acetaminophen using high- performance liquid
chromatography with electrochemical detection. Drug Metab Dis-
pos 2002;30:446 451.
14. Trey C, Davidson CS. The management of fulminant hepatic
failure. Prog Liver Dis 1970;3:282–298.
15. Zimmerman HJ, Maddrey WC. Acetaminophen (paracetamol) hep-
atotoxicity with regular intake of alcohol: analysis of instances of
therapeutic misadventure. Hepatology 1995;22:767–773.
16. Bradford MM. A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principle of pro-
tein-dye binding. Anal Biochem 1976;72:248 –254.
17. Bertholf RL, Johannsen LM, Bazooband A, Mansouri V. False
positive acetaminophen results in a hyperbilirubinemic patient.
Clin Chem 2003;49:695– 698.
18. Polson J, Orsulak P, Wians F, Murray N, Balko J, Fuller D, Rossaro
L, McGuire B, Lee WM. Elevated bilirubin may cause false posi-
tive acetaminophen levels in hepatitis patients. Hepatology
19. Smilkstein MJ, Knapp GL, Kulig KW, Rumack BH. Efficacy of oral
N-acetylcysteine in the treatment of acetaminophen overdose.
Analysis of the national multicenter study (1976 to 1985). N Engl
J Med 1988;319:1557–1562.
20. Jones AL. Mechanism of action and value of N-acetylcysteine in
the treatment of early and late acetaminophen poisoning: a
critical review. J Toxicol Clin Toxicol 1998;36:277–285.
21. Roberts DW, Bucci TJ, Benson RW, Warbritton AR, McRae TA,
Pumford NR, Hinson JA. Immunohistochemical localization and
quantification of the 3-(cystein-S-yl)-acetaminophen protein ad-
duct in acetaminophen hepatotoxicity. Am J Pathol 1991;138:
359 –371.
22. Webster PA, Roberts DW, Benson RW, Kearns GL. Acetamino-
phen toxicity in children: diagnostic confirmation using a specific
antigenic biomarker. J Clin Pharmacol 1996;36:397– 402.
23. James LP, Farrar HC, Sullivan JE, Givens TE, Kearns GL, Wasser-
man GS, Walson PD, Hinson JA, Pumford NR. Measurement of
acetaminophen-protein adducts in children and adolescents with
acetaminophen overdoses. J Clin Pharmacol 2001;41:846 851.
24. James LP, McCullough SS, Lamps LW, Hinson JA. Effect of
N-acetylcysteine on acetaminophen toxicity in mice: relationship
to reactive nitrogen and cytokine formation. Toxicol Sci 2003;75:
458 467.
25. Rumack BH, Peterson RC, Koch GG, Amara IA. Acetaminophen
overdose. 662 cases with evaluation of oral acetylcysteine treat-
ment. Arch Intern Med 1981;23:380 –385.
26. Hawton K, Harris L, Hall S, Simkin S, Bale E, Bond A. Deliberate
self-harm in Oxford 1990 –2000: a time of change in patient
characteristics. Psychol Med 2003;33:987–995.
Received June 30, 2005. Accepted November 9, 2005.
Address reprint requests to: Timothy J. Davern II, MD, Division of
Gastroenterology and Liver Transplant Program, University of Califor-
nia, San Francisco, 513 Parnassus Avenue, Room S-357, San Fran-
cisco, California 94143-0538. e-mail:; fax:
Supported by NIDDK: DK R-01 58369 and DK067999 (to LJ), the
Jean Roberts and the Rollin and Mary Ella King Funds at the South-
western Medical Foundation, Dallas, and the Stephen B. Tips Fund of
Northwestern Medical Foundation. We are grateful to all the coordi-
nators, patients, and families who make this study possible.
Thanks to Sadie McFarlane for expert artwork in Figure 1.
The U.S. Acute Liver Failure Study Group who participated in this
study includes William M. Lee (Principal Investigator), University of
Texas Southwestern Medical Center, Dallas, Texas; Anne Larson,
University of Washington, Seattle, Washington; Jeffery S. Crippin,
Washington University, St. Louis, Missouri; Timothy J. Davern and
Nathan Bass, University of California at San Francisco, San Fran-
cisco, California; Sukru Emre, Mt. Sinai Medical Center, New York,
New York; Timothy M. McCashland, University of Nebraska, Omaha,
Nebraska; J. Eileen Hay, Mayo Clinic, Rochester, Minnesota; Natalie
Murray, Baylor University Medical Center, Dallas, Texas; A. Obaid
Shakil, University of Pittsburgh, Pittsburgh, Pennsylvania; Andres T.
Blei, Northwestern University, Chicago, Illinois; Atif Zaman, Oregon
Health Sciences University, Portland, Oregon; Steven H.B. Han,
University of California, Los Angeles, Los Angeles, California; Robert
J. Fontana, University of Michigan, Ann Arbor, Michigan; Brendan
McGuire, University of Alabama at Birmingham, Birmingham, Ala-
bama; Raymond Chung, Massachusetts General Hospital, Boston,
Massachusetts; Steven Lobritto, Robert Brown and Michael Schil-
sky, Columbia-Presbyterian Medical Center, New York, New York; M.
Edwyn Harrison, Mayo Clinic Scottsdale, Phoenix, Arizona; Adrian
Reuben, Medical University of South Carolina, Charleston, South
Carolina; Santiago Munoz, Albert Einstein Medical Center, Philadel-
phia, Pennsylvania; Rajender Reddy, University of Pennsylvania,
Philadelphia, Pennsylvania; R. Todd Stravitz, Virginia Common-
wealth University, Richmond, Virginia; Lorenzo Rossaro, University
of California, Davis Medical Center, Sacramento, California; and Raj
Santayanarana, Mayo Clinic, Jacksonville, Florida.
    • "Circulating levels of APAP protein adducts have been established as a biomarker of APAP toxicity in experimental models and clinical samples (Davern et al., 2006; Muldrew et al., 2002 ). Serum samples were analyzed for APAP protein adducts by highperformance liquid chromatography with electrochemical detection (HPLC-EC) as previously reported (Davern et al., 2006; Muldrew et al., 2002). Measurement of serum ALT was performed in the clinical chemistry laboratories at the participating institutions using standardized methods. "
    [Show abstract] [Hide abstract] ABSTRACT: Phospholipids are an important class of lipids that act as building blocks of biological cell membranes and participate in a variety of vital cellular functions including cell signaling. Previous studies have reported alterations in phosphatidylcholine (PC) and lysophosphatidylcholine (lysoPC) metabolism in acetaminophen (APAP)-treated animals or cell cultures. However, little is known about phospholipid perturbations in humans with APAP toxicity. In the current study, targeted metabolomic analysis of 180 different metabolites including 14 lysoPCs and 73 PCs was performed in serum samples from children and adolescents hospitalized for APAP overdose. Metabolite profiles in the overdose group were compared to those of healthy controls and hospitalized children receiving low dose APAP for treatment of pain or fever (therapeutic group). PCs and lysoPCs with very long chain fatty acids (VLCFAs) were significantly decreased in the overdose group, while those with comparatively shorter chain lengths were increased in the overdose group compared to the therapeutic and control groups. All ether linked PCs were decreased in the overdose group compared to the controls. LysoPC-C26:1 was highly reduced in the overdose group and could discriminate between the overdose and control groups with 100% sensitivity and specificity. The PCs and lysoPCs with VLCFAs showed significant associations with changes in clinical indicators of drug metabolism (APAP protein adducts) and liver injury (alanine aminotransferase, or ALT). Thus, a structure-dependent reduction in PCs and lysoPCs was observed in the APAP-overdose group, which may suggest a structure-activity relationship in inhibition of enzymes involved in phospholipid metabolism in APAP toxicity.
    Full-text · Article · Aug 2016
    • "Acetaminophen (n-acetyl-p-aminophenol, APAP, also known as Tylenol, Paracetamol) is a very safe and effective analgesic and antipyretic drug at therapeutic dosage. However, APAP overdose can cause severe liver toxicity characterized by depletion of GSH, protein adduct formation [3, 4], generation of highly active free radicals, mitochondrial damage, and nuclear DNA fragmentation [5] that leads to cell death and hence necrosis. While some species like rat are relatively resistant to APAP toxicity, the mouse is the preferred model as several studies have demonstrated dose dependent response to either oral or intraperitoneal APAP challenge [6, 7]. "
    [Show abstract] [Hide abstract] ABSTRACT: Some botanicals have been reported to possess antioxidative activities acting as scavengers of free radicals rendering their usage in herbal medicine. Here we describe the potential use of “SAL,” a standardized blend comprised of three extracts from Schisandra chinensis , Artemisia capillaris , and Aloe barbadensis , in mitigating chemically induced acute liver toxicities. Acetaminophen and carbon tetrachloride induced acute liver toxicity models in mice were utilized. Hepatic functional tests from serum collected at T24 and hepatic glutathione and superoxide dismutases from liver homogenates were evaluated. Histopathology analysis and merit of blending 3 standardized extracts were also confirmed. Statistically significant and dose-correlated inhibitions in serum ALT ranging from 52.5% ( p = 0.004 ) to 34.6% ( p = 0.05 ) in the APAP and 46.3% ( p < 0.001 ) to 29.9% ( p = 0.02 ) in the CCl 4 models were observed for SAL administered at doses of 400–250 mg/kg. Moreover, SAL resulted in up to 60.6% and 80.2% reductions in serums AST and bile acid, respectively. The composition replenished depleted hepatic glutathione in association with an increase of hepatic superoxide dismutase. Unexpected synergistic protection from liver damage was also observed. Therefore, the composition SAL could be potentially utilized as an effective hepatic-detoxification agent for the protection from liver damage.
    Full-text · Article · Mar 2016
    • "Thus, it was postulated that bile acids may represent very sensitive markers of drug induced liver injury and be tested as potential candidate biomarkers for future application in liver screening panels. APAP protein adducts reflect the contribution of oxidative metabolism to APAP toxicity [42] and are specific to APAP exposure [5]. In addition, the sensitivity of the analytical assay for quantitation of APAP protein adducts is such that very low levels of adducts can be quantified in subjects receiving APAP in the clinical setting [43]. "
    [Show abstract] [Hide abstract] ABSTRACT: Metabolomics approaches have enabled the study of new mechanisms of liver injury in experimental models of drug toxicity. Disruption of bile acid homeostasis is a known mechanism of drug induced liver injury. The relationship of individual bile acids to indicators of oxidative drug metabolism (acetaminophen protein adducts) and liver injury was examined in children with acetaminophen overdose, hospitalized children with low dose exposure to acetaminophen, and children with no recent exposure to acetaminophen. Nine bile acids were quantified through targeted metabolomic analysis in the serum samples of the three groups. Bile acids were compared to serum levels of acetaminophen protein adducts and alanine aminotransferase. Glycodeoxycholic acid, taurodeoxycholic acid, and glycochenodeoxycholic acid were significantly increased in children with acetaminophen overdose compared to healthy controls. Among patients with acetaminophen overdose, bile acids were higher in subjects with acetaminophen protein adduct values > 1.0 nmol/mL and modest correlations were noted for three bile acids and acetaminophen protein adducts as follows: taurodeoxycholic acid (R=0.604; p<0.001), glycodeoxycholic acid (R=0.581; p<0.001), and glycochenodeoxycholic acid (R=0.571; p<0.001). Variability in bile acids was greater among hospitalized children receiving low doses of acetaminophen than in healthy children with no recent acetaminophen exposure. Compared to bile acids, acetaminophen protein adducts more accurately discriminated among children with acetaminophen overdose, children with low dose exposure to acetaminophen, and healthy control subjects. In children with acetaminophen overdose, elevations of conjugated bile acids were associated with specific indicators of acetaminophen metabolism and non-specific indicators of liver injury.
    Full-text · Article · Jul 2015
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