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Materials and Methods:
Tissue extraction and NMR spectroscopy. Water-soluble metabolites were extracted with
perchloric acid.12 The lyophilized water-soluble samples were dissolved in D2O,
centrifuged, and adjusted to pH 7.2. For 31P-NMR measurements, the samples were
treated with 100 mM EDTA and adjusted to pH 7.2. NMR spectra were recorded on
DRX-600 or WB-360 Bruker spectrometers. 1H-NMR spectra were recorded with a 5mm H,C,N-inverse-triple-resonance probe, flip angle 40, repetition time 15 s, spectral
width 7,183 Hz. 13C-NMR spectra were recorded with a 5-mm 1H/13C dual probe,
repetition time 2.5 s, flip angle 27, composite pulse decoupling with WALTZ-16,
spectral width 47,619 Hz. 31P-NMR spectra were recorded with a 5-mm QNP probe, flip
angle 80, repetition time 3.5 s, spectral width 5,155 Hz, composite pulse decoupling
with WALTZ-16.
Quantification of metabolite concentrations and fractional 13C-enrichments. Total
metabolite concentrations, used for determinations of the fractional 13C enrichments of
metabolites detected in 13C-NMR spectra, were analyzed from 1H-NMR spectra of PCA
extracts using (trimethylsilyl)propionic-2,2,3,3d4-acid as external standard. ATP
concentrations were quantified from 31P-NMR spectra of perchloric acid extracts, using
hexamethylphosphoric acid triamide as external standard (31.5 ppm). The amounts of 13C
in specific carbon positions of metabolites (glutamate, succinate) were derived from 13C-
NMR spectra as described previously.12 The integrals of the signals were corrected for
natural abundant 13C, nuclear Overhauser enhancement and saturation effects.
Labeling of glutamate with 13C. [U-13C]glucose allows determination of metabolic
pathways used to synthesize downstream compounds. Briefly, via glycolysis, 1 molecule
[U-13C]glucose is transformed into 2 molecules [U-13C]pyruvate. [U-13C]pyruvate is
transformed via pyruvate dehydrogenase (PDH) to [1,2-13C]acetyl-CoA, or is
carboxylated via pyruvate carboxylase (PC) to [1,2,3-13C]oxaloacetate, which condense
with acetyl-CoA into citrate. Glucose flux through these 2 pathways can be quantified
from 13C-NMR spectra from the 13C-labelling pattern in glutamate synthesized from the
Krebs cycle intermediate -ketoglutarate. Glutamate is subsequently incorporated into
the (-glutamyl)-residue of GSH. The NMR analysis was performed on tissue extracts
obtained 45 min after injection of the [U-13C]glucose tracer, i.e. at 2.25 h (tracer injection
at 1.5 h after APAP) and at 6.75 h (tracer injection at 6 h).
Rationale for treatment regimen: The injection of GSH/NAC at 90 min after APAP is
based on the assumption that under the conditions of these experiments, the majority of
NAPQI formation is over at that time and that most newly synthesized GSH is not used to
scavenge NAPQI but to scavenge peroxynitrite. In one of the previous studies,15 we
injected GSH at different times after APAP and showed that when GSH is being resynthesized at the time when NAPQI is still formed, there is not only complete protection
but also no increase of GSSG formation (indicator for the mitochondrial oxidant stress).
In striking contrast, GSH injected at 90 min or later still protected but showed elevated
GSSG levels similar to or even higher than APAP alone. This indicated that the delayed
GSH treatment did not prevent the mitochondrial oxidant stress, which is presumably
initiated by binding of NAPQI to mitochondrial proteins.15 These data provided strong
evidence that the delayed treatment with GSH at 90 min has only a limited impact on
protein binding of NAPQI. In addition to this more indirect evidence, there are at least 4
additional publications which directly support our conclusion. Roberts et al. demonstrated
by immunohistochemistry maximum of APAP protein adducts at 1 h after APAP. S1
Pumford et al. measured protein adducts by an ELISA method and showed maximal
binding between 1 and 2 h after APAP. S2 Muldrew et al. measured liver APAP protein
adducts by HPLC with electrochemical detection and showed maximal adduct formation
at 1 h after APAP.S3 In addition, James et al. showed by immunohistochemistry that NAC
protected against APAP in the presence of protein adduct formation when given at 1 h
after APAP.18 However, there appeared to be some effect on protein binding at this early
time point.18 In contrast, Salminen et al. did not detect any effect of NAC on APAP
protein adducts when administrated at 1 or at 3 h after APAP.S4
All experiments were performed with doses of APAP of 300-400 mg/kg resulting in a
similar injury as we report here. Thus, based on this extensive evidence it is justified to
conclude that delayed treatment (1.5 h) with GSH or NAC has a limited effect on protein
binding. In addition, GSH and NAC act through the same mechanism, i.e. promotion of
hepatic GSH synthesis to the same degree (Figure 3). Thus, if there is any effect on
protein binding by GSH and NAC, the effect would be exactly the same and would not
explain or impact the differences between GSH and NAC investigated in this manuscript.
In other words, a potential effect of GSH and NAC on protein binding has no impact on
the conclusions in this manuscript.
Supplemental References:
S1.
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 adduct in acetaminophen hepatotoxicity. Am J Pathol
1991;138:359-371.
S2.
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 acetaminophen-treated mice. J Pharmacol Exp Ther 1989;248:190-196.
S3.
Muldrew KL, James LP, Coop L, McCullough SS, Hendrickson HP, Hinson JA,
Mayeux PR. Determination of acetaminophen-protein adducts in mouse liver and serum
and human serum after hepatotoxic doses of acetaminophen using high-performance
liquid chromatography with electrochemical detection. Drug Metab Dispos 2002;30:446451.
S4.
Salminen WF Jr, Voellmy R, Roberts SM. Effect of N-acetylcysteine on heat
shock protein induction by acetaminophen in mouse liver. J Pharmacol Exp Ther
1998;286:519-524.
Figure Legends:
Supplemental Figure 1:
Histological assessment of liver injury (hematoxilin & eosin, H&E) in control animals or
mice treated with 300 mg/kg acetaminophen (APAP) for 6 h. Some of the animals
received additionally 10 ml/kg saline, 0.65 mmol/kg GSH or 0.65 mmol/kg Nacetylcysteine (NAC) iv 1.5 h after APAP. The representative pictures show extensive
centrilobular necrosis as indicated by the loss of basophilic staining, vacuolization, cell
swelling and karyolysis in APAP-treated animals. Both, GSH and NAC treatment
improved the area of necrosis with GSH being more effective than NAC. (x200 for all
panels)
Supplemental Figure 2:
Concentrations of 13C-labelled [4,5-13C]glutamate and the Krebs cycle intermediate [2,313
C]succinate (nmol/g wet weight), as calculated from their resonances in 1H- and 13C-
NMR spectra of liver extracts. The mice were treated with 300 mg/kg APAP and some
subsequently received additionally 10 ml/kg saline, 0.65 mmol/kg N-acetylcysteine (lNAC), 1.95 mmol/kg NAC (h-NAC), a mixture of 3 amino acids (0.65 mmol/kg of
glycine, glutamic acid and NAC) (3AS) or a mixture of 2 amino acids (0.98 mmol/kg
glycine and glutamic acid) (2AS) iv 1.5 h after APAP. Data represent means ± SE of n =
5 animals per group. *P<0.05 (compared to controls)