NMR-Based Metabolic Profiling Identifies ... - ACS Publications

May 16, 2009 - Department of Biomolecular Medicine, Imperial College London, ... rial College London, Sir Alexander Fleming Building, South Kensington...
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NMR-Based Metabolic Profiling Identifies Biomarkers of Liver Regeneration Following Partial Hepatectomy in the Rat Mary E. Bollard,† Nancy R. Contel,‡ Timothy M. D. Ebbels,† Leon Smith,† Olaf Beckonert,† Glenn H. Cantor,§ Lois Lehman-McKeeman,§ Elaine C. Holmes,† John C. Lindon,† Jeremy K. Nicholson,† and Hector C. Keun*,† Department of Biomolecular Medicine, Imperial College London, Division of Surgery, Oncology, Reproductive Biology & Anaesthetics, Faculty of Medicine, Sir Alexander Fleming Building, South Kensington, London, SW7 2AZ, United Kingdom, Incyte Corporation, Wilmington, Delaware 19880-0400, and Discovery Toxicology, Bristol-Myers Squibb Company, Princeton, New Jersey 08543 Received March 2, 2009

Tissue injury and repair are often overlapping consequences of disease or toxic exposure, but are not often considered as distinct processes in molecular studies. To establish the systemic metabolic response to liver regeneration, the partial hepatectomy (PH) model has been studied in the rat by an integrated metabonomics strategy, utilizing 1H NMR spectroscopy of urine, liver and serum. Male Sprague-Dawley rats were subjected to either surgical removal of ∼two-thirds of the liver, sham operated (SO) surgery, or no treatment (n ) 10/group) and samples collected over a 7 day period. A number of urinary metabolic perturbations were observed in PH rats compared with SO and control animals, including elevated levels of taurine, hypotaurine, creatine, guanidinoacetic acid, betaine, dimethylglycine and bile acids. Serum betaine and creatine were also elevated after PH, while levels of triglyceride were reduced. In the liver, triglycerides, cholesterol, alanine and betaine were elevated after PH, while choline and its derivatives were reduced. Upon examining the dynamic pattern of urinary response (the ‘metabolic trajectory’), several metabolites could be categorized into groups likely to reflect perturbations to different processes such as dietary intake or hepatic 1-carbon metabolism. Several of the urinary perturbations observed during the regenerative phase of the PH model have also been observed after exposure to liver toxins, indicating that hepatic regeneration may make a contribution to the systemic alterations in metabolism associated with hepatotoxicity. The observed changes in 1-carbon and lipid metabolism are consistent with the proposed role of these pathways in the activation of a regenerative response and provide further evidence regarding the utility of urinary NMR profiles in the detection of liver-specific pathology. Biofluid 1H NMR-based metabolic profiling provides new insight into the role of metabolism of liver regeneration, and suggests putative biomarkers for the noninvasive monitoring of the regeneration process. Keywords: metabonomics • metabolomics • toxicity • fatty liver • 1-carbon metabolism • liver regeneration

Introduction Regeneration is the ability of an organ to replace tissue mass after partial removal or injury.1 Interest in the process of tissue regeneration and its manipulation as a tool in clinical intervention is currently very high, given recent breakthroughs in the understanding of regeneration of heart muscle2 and brain tissue3 after ischemic damage. Both tissue injury and regeneration occur after a physical insult, so it can be difficult to distinguish between the biochemical effects and effectors of the two responses using a standard injury model. To gain * Corresponding author: Dr Hector Keun, Biomolecular Medicine, Imperial College London, Sir Alexander Fleming Building, South Kensington, London,SW72AZ,U.K.Fax:+442075943226.E-mail:[email protected]. † Imperial College London. ‡ Incyte Corporation. § Bristol-Myers Squibb Company. 10.1021/pr900200v

 2010 American Chemical Society

understanding of NMR-derived metabolic biomarkers associated with the regenerative process, the partial hepatectomy (PH) model was evaluated. Since the liver is unique in its capacity to undergo hyperplasia and to replace lost tissue and cells, there is particular value in this model, in which approximately 2/3 of liver mass is surgically removed, as a means of independently studying hepatic regeneration. The search for the factors responsible for initiating regeneration following PH have resulted in extensive publications detailing the time course of DNA synthesis, gene expression, cellular biochemical and growth regulation signal changes in the partially hepatectomized animal.4-6 Liver regeneration is carried out by all the existing cellular populations found in the mature liver, with hepatocytes being the first cells to proliferate at about 12 h after PH in rodents.6 DNA synthesis begins at 14-16 h postsurgery, peaks at 22-24 h and then again at 36-48 Journal of Proteome Research 2010, 9, 59–69 59 Published on Web 05/16/2009

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h. Mitosis follows DNA synthesis 6-8 h later. The lobes remaining after PH enlarge, until at 7-10 days, liver mass equals that existing prior to surgery, and further growth is inhibited.1,7,8 Histological architecture is preserved throughout the process of regeneration, and albumin synthesis, drug metabolism and urea cycling are maintained.8 Metabolism plays a key role in the regulation of events that are necessary for liver regeneration. A drop in S-adenosine methionine (SAM) levels is required for the sensitization of liver cells to hepatocyte growth factor (HGF), a key mitogenic signaling molecule in the regeneration process.9,10 The formation of lipid droplets, comprised primarily of triglycerides, is also associated with liver regeneration, and manipulations that interrupt this process have been shown to prevent mitosis.11 Thus, metabolic studies relating to regeneration are of fundamental importance in understanding the processes involved so that they can be more effectively manipulated. NMR spectroscopy of biofluids and tissues, together with pattern recognition, has been utilized previously in defining onset, progression and recovery from a toxic lesion.12-14 However, to date, the process of liver regeneration itself has not been specifically investigated using high resolution 1H NMR spectroscopy. Despite a significant number of reports over two decades detailing reproducible, NMR-detectable changes in biofluids after hepatotoxin exposure, a fundamental question has persisted: how specific are these commonly observed metabolic perturbations to the severity of liver injury? Since metabolic profiles report on the effect on the entire system, they will inevitably capture a combination of injurious and reparative processes, and the temporal profile of a particular metabolite may reflect the kinetics of several of these processes, including liver regeneration. Hence, a metabolic profiling study on the systemic impact of partial hepatectomy, as is presented here, may have major implications for the field of metabonomics, and in particular applications to toxicology and regenerative medicine.

Experimental Procedures Additional information and discussion on the experimental approach used is available in refs 45 and 46 Partial Hepatectomy. This study was carried out in an AAALAC-accredited facility and was subject to appropriate local review. Male Sprague-Dawley Crl:CD(SD)IGS BR rats n ) 30, 6-8 weeks, were obtained from Charles River Laboratories. Study groups included 10 PH, 10 sham operated (SO) and 10 control rats. Rats were housed in conditions optimal for social and health care for 7 days prior to being singly housed in cages on Nalgene refrigerated metabolism racks where they were held for a 24 h acclimatization period prior to surgery (Day -1). The metabolism racks were held in a room maintained at 21 °C and 55% relative humidity and fluorescent lighting between 06.00 and 18.00. Throughout the acclimatization period, all animals had access to food (Purina chow 5002) and water ad libitum. On Day -1, urine was collected from 0-8 and 8-24 h. Surgeries were conducted on Day 1 utilizing aseptic procedures. On the morning of day 1, the rats assigned to the PH group were anesthetized with isoflurane. Each animal’s abdomen was shaved and disinfected. An incision was made, approximately 1.5 cm long, from just above the xiphoid cartilage extending caudally followed by elevation of the rat’s abdomen. The falciform ligament was dissected so that the left lateral and median lobes of the liver could be easily exteriorized. The lobes 60

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were ligated, transected, and weighed. Muscle and skin layers were closed. Animals were provided with an external source of heat during recovery and were returned to the metabolism cages on day 1, after recovery from surgery, at which time the 0-8 h urine collection was commenced. In the case of rats assigned to the SO group, anesthesia and surgical preparation was conducted as with the PH group. A similar abdominal incision was made, and the median and left lateral liver lobes were exteriorized and then returned to the abdominal cavity and the muscle and skin around the incision closed. On the morning of day 3, five animals in each group were euthanized, and the livers and kidneys from each animal weighed and specimens collected for histopathology and MAS NMR spectroscopy. The remaining 5 animals from each group were euthanized on day 8, and tissue samples taken for histopathology. Sample Collection. Urine samples were collected from rats over solid CO2, in tubes containing 1 mL of 1% (w/v) sodium azide over the following time periods: -24 to -8, -8 to 0, 0-8, 8-24, 24-48, 48-72, 72-96, 96-120, 120-144 and 144-168 h relative to hepatectomy at 0 h. Urine volumes were recorded and samples immediately frozen and stored at -40 °C pending NMR spectroscopic analysis. Approximately 1 mL of serum was collected by venipuncture of the tail vein from all animals at 24, 48 h, and at termination and frozen at -40 °C prior to NMR analysis. Serum samples were analyzed by routine methodologies for assessment of serum clinical chemistry parameters. After tissue samples for MAS NMR were collected and snapfrozen in liquid N2, target organs were preserved in 10% buffered formalin, processed through to paraffin wax, sectioned, stained with hematoxylin and eosin, and examined microscopically. Preparation of Urine Samples for 1H NMR Spectroscopic Analysis. Rat urine samples (400 µL) were mixed with phosphate buffer (200 µL) solution (0.2 M Na2HPO4/0.04 M NaH2PO4, pH ) 7.4), containing 1 mM sodium 3-trimethylsilyl1-(2,2,3,3-2H4) propionate (TSP) and sodium azide at 3 mM in H2O/D2O (80:20), in 96 well plates, using a Gilson 215 liquid handling robot. The well plates were centrifuged at 4000 rpm for 5 min to remove insoluble material, prior to positioning via the Bruker Efficient Sample Transfer (BEST) system (Bruker Biospin, Rheinstetten, Germany). Preparation of Serum and Tissue Samples. Aliquots (200 µL) of serum were added to 400 µL of saline (0.9% NaCl in water/D2O, 90:10) in 96-well plates and further treated as above. Individual tissue samples (∼15 mg) were placed in a tissue culture dish and soaked with D2O for ∼10 s at room temperature, containing 0.5% of TSP, immediately prior to NMR spectroscopy. Each sample was inserted into a 4 mm zirconia rotor with a spherical insert and Kel-F cap (Bruker Biospin, Rheinstetten, Germany). Acquisition of 1H NMR Spectra of Urine. NMR data were acquired on a Bruker DRX-600 spectrometer operating at 600.13 MHz 1H observation frequency, using the BEST flow-injection probe for sample delivery and analysis. One-dimensional (1D) spectra of urine samples were acquired using a 90°-3 µs90°-tm-90°-acquire pulse sequence. Water resonances were suppressed with irradiation at the water frequency during both the recycle delay of 2 s and the mixing time (tm) of 100 ms. Typically, 64 free induction decays (FIDs) were collected into 32k data points using a spectral width of 12 019 Hz and acquisition time of 1.36 s. Spectra were acquired at 300 K. The data were zero filled by a factor of 2 and the FIDs were

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multiplied by an exponential weighting function equivalent to a line broadening of 1 Hz prior to Fourier transformation (FT). The assignments of 1H NMR urine spectra were made with reference to published literature data.15,16 Acquisition of 1H NMR Spectra of Serum. 1-D spectra of serum samples were acquired using the pulse sequence with parameters as described above. In addition, spin-echo spectra were measured using the water-suppressed Carr-PurcellMeiboom-Gill (CPMG) pulse sequence.41 A spin-spin relaxation delay of 64 ms was used for all samples and water suppression irradiation was applied during the relaxation delay (2 s). Spectra were processed as above. The assignments of 1H NMR spectra of serum were made with reference to published literature data.15,16 Acquisition of 1H MAS NMR Spectra of Liver Samples. Spectra were acquired on a Bruker AV-600 spectrometer operating at 600.13 MHz 1H observation frequency, using a standard Bruker high resolution MAS probe with magic-angle gradient at a temperature of 300 K and sample spinning speed of 4 kHz. 1-D spectra of tissue samples were acquired using the pulse sequence described above. Typically, 256 FIDs were collected into 16k data points using a spectral width of 12 kHz, an acquisition time of 2.04 s, recycle delay of 2 s and a mixing time of 150 ms. Additional spectra were acquired for each sample using the CPMG spin-echo pulse sequence. A spin-spin relaxation delay of 360 ms was used for all samples. Spectra were processed as above. The assignment of MAS NMR liver spectra were made with reference to published literature data.17 Quantification of Metabolites and Statistical Analysis. Significant changes in all measured parameters were determined using a Student’s t test. For selected metabolites, an NMR peak-picking algorithm was used to determine statistical significance of perturbations at each time point and time point of maximum effect. With the use of a template fitting procedure for characteristic resonances,18 the excretion profiles over time for these perturbed metabolites were generated, and are presented normalized to the CH2 resonance of creatinine (δ 4.05), to account for variation in urinary concentration and change in body mass associated with normal growth. Creatinine is derived primarily from a nonenzymatic reaction in muscle and its excretion therefore largely reflects total muscle mass. In the specific case of bile acids, time points of significant difference were calculated using integrated spectral intensities and not a spectral template. Only time-points which showed a stastistically significant (p < 0.05) difference in the PH group compared to both sham and control groups were considered to exhibit clearly biologically significant effects in the quantitative analysis presented. Assuming ∼40 metabolites would typically be available for visual inspection and analysis, we would expect no more than 1 metabolite to exhibit such effects by chance, giving acceptable rates of false discovery. The range of the lowest p-value resulting from between group comparisons for each metabolite was 6 × 10-3 to 1 × 10-6.

Results Pathology and Serum Clinical Chemistry Parameters. The livers from PH rats had regenerated to approximately 2/3 the weight of control livers by 48 h and 3/4 the weight of control livers by 168 h. Liver weights of SO and PH rats as a percentage of controls are given in Figure 1 and were consistent with previous reports.1 The body weights of PH rats were significantly less than those of SO or controls at 48 h, even when

Figure 1. Mean liver weights of sham-operated (SO) and hepatectomized (PH) rats as a percentage of controls. Error bars indicate standard errors.

Figure 2. Mean body weights of control, sham-operated and hepatectomized rats. Error bars indicate standard errors.

liver weights were taken into account (p < 0.01). Body weights over time are given in Figure 2. The histopathological changes observed in the liver of rats after SO and PH are shown in Table 1 and Figure 3. The major findings in all of the PH animals at 48 h were moderate hepatocellular hypertrophy, mildly increased mitotic figures, and mild lipid accumulation, consistent with the expected outcome of liver regeneration in the PH model.7,19 A summary of the serum clinical chemistry parameters for PH, SO and control rats is given in Table 2. PH rat serum had significantly elevated levels of AST, alkaline phosphatase (ALP), and ALT at 24 h compared with controls (p < 0.01). AST and ALP levels remained elevated at 48 h in PH rats (p < 0.01). Creatinine, glucose, calcium, albumin and total protein were depleted at 24 h in PH rats compared with controls (p < 0.01). As expected, the small ligated stub of the resected lobes in the PH animals was necrotic, and in some, thrombi were observed. It is feasible that these small stubs of necrotic tissue may release enzymes such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and hence may play a minor role in the metabolic response observed after PH. It is acknowledged that, although the PH procedure is widely accepted as the classical model of liver regeneration, it may not be a ‘pure’ regenerative model.1,20 However, the effect of the presence of the ligated stubs is likely to be minimal and of Journal of Proteome Research • Vol. 9, No. 1, 2010 61

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Table 1. Summary of Histopathology Data observation in the liver

incidence

mean severity (range)a

Peritonitis, fibrinous inflammation, serosal, diffuse Hepatocellular necrosis, subserosal, focal Peritonitis, fibrinous inflammation, serosal, diffuse Regenerative hypertrophy, panlobular, diffuse. Increased mitotic figures, regenerative, pan-lobular, diffuse. Lipid accumulation and vacuolar degeneration, pan-lobular, diffuse. Peritonitis, fibrinous inflammation, serosal, diffuse. Hepatocellular necrosis, infarct, panlobular, diffuse (surgical stub). Vascular thrombosis, focal (surgical stub) Peritonitis, granulomatous, with mineralization, serosal, focal. Hepatocellular necrosis, infarct, panlobular, focal (surgical stub).

3/5 2/5 1/5 5/5 5/5 5/5 3/5 3/5 1/5 4/5 2/5

1.7 (1-2) 2.0 2.0 2.8 (2-3) 1.8 (1-2) 1.4 (1-2) 2.3 (2-3) 4.7 (4-5) 5.0 3.0 (2-4) 3.0

intervention (time point)

SO (48 h) SO (168 h) PH (48 h)

PH (168 h) a

Mean severity was derived from a scale of 1-5, in which 1 ) minimal, 2 ) mild, 3 ) moderate, 4 ) marked, and 5 ) severe.

significance only to the earliest sample time points since levels of ALT (with a low half-life) are normalized by 48 h post PH. There were no treatment-related renal findings. 1 H NMR Spectroscopic Analysis of Urine, Serum, and Liver. Visual inspection of the urinary NMR spectra indicated that several metabolites exhibited altered excretion patterns relative to control, in particular after the PH procedure (Figure 4). Perturbations observed in urine samples from PH rats included elevated levels of hypotaurine, taurine and guanidinoacetic acid (GAA), with maximum excretion in the 0-8 h urine samples. In addition, 2-oxoglutarate (2-OG) and citrate were depleted in the 0-48 h urine and then elevated in the 72-120 h urine samples. Hippurate excretion in the urine followed a similar metabolic profile as 2-OG and citrate. In addition, bile acids were observed in the 24-48 h urine samples from all rats after PH and in the 8 h urine samples from two animals only. Creatine, betaine and dimethylgycine (DMG) showed elevated excretion, reaching maximum concentrations at 8-24 h, 48-72 h and 72-96 h post PH, respectively. Mean time profiles of selected urinary metabolites are shown in Figure 5 to illustrate the change in concentration of urinary metabolites for the 3 groups. All effects of PH seen in metabolic profiles are summarized in Table 3. Metabolic profiles of MAS NMR data from liver samples were examined by visual inspection and exploratory pattern recognition analysis (Supplementary Figure 1). In 48 h liver samples from PH rats, lipid and cholesterol resonances were higher in concentration in the MAS NMR spectra than those in the SO and control samples. In addition, alanine and betaine were elevated in 48 h liver samples, while choline, phosphocholine (PC), glycerophosphocholine (GPC), glucose and glycogen were depleted in concentration. Typical MAS NMR spectra of 48 h liver samples from PH, SO and control rats are given in Figure 6 for comparison. A few perturbations were observed in the serum from PH rats compared with serum from SO animals. These included elevated levels of betaine and creatine in 48 h samples and reduced triglyceride resonances in 168 h serum samples (spectra not shown).

Discussion Liver Regeneration May Contribute to the Metabolic Perturbations Previously Associated with Hepatotoxicity. We have characterized the metabolic effects of PH in the rat for the first time using NMR-based metabolic profiling. A majority of the urinary metabolites observed to change after PH has previously been observed to vary after exposure to hepatotoxins, including such endogenous biomarkers as creatine, taurine, 62

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Figure 3. Hepatocellular hypertrophy 48 h after partial hepatectomy. (A) Liver from rat 14, 48 h after sham surgery; (B) liver from rat 22, 48 h after partial hepatectomy; (C) liver from rat 27, 168 h after partial hepatectomy. In panel B, note the hepatocellular cytoplasmic enlargement, reduced density of hepatocytes, increased cytoplasmic eosinophilia, and mitotic figure (*).

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Table 2. Summary of Serum Clinical Chemistry Parameters group

time

CRE

ALT

AST

ALP

GLU

Ca

ALB

TP

Control SO PH Control SO PH Control SO PH

24 24 24 48 48 48 168 168 168

31 ( 1 27 ( 1 25 ( 1* 34 ( 1 21 ( 1 20 ( 1 29 ( 1 31 ( 2 25 ( 1

66 ( 2 61 ( 4 486 ( 73* 60 ( 2 45 ( 4 117 ( 15 65 ( 6 60 ( 3 69 ( 6

123 ( 5 180 ( 12* 974 ( 180* 121 ( 19 117 ( 19 298 ( 33* 88 ( 4 102 ( 8 120 ( 8

399 ( 15 349 ( 19 746 ( 39* 356 ( 24 316 ( 20 724 ( 38* 410 ( 32 377 ( 33 426 ( 14

128 ( 2 136 ( 7 104 ( 3* 139 ( 7 161 ( 9 132 ( 9 146 ( 6 152 ( 9 143 ( 3

2.9 ( 0 2.9 ( 0 2.7 ( 0* 3.0 ( 0 2.9 ( 0 2.9 ( 0 3.0 ( 0 3.0 ( 0 2.9 ( 0

44 ( 0 40 ( 0* 39 ( 1* 43 ( 1 37 ( 1* 35 ( 1* 44 ( 1 40 ( 1 36 ( 1*

63 ( 1 61 ( 1 55 ( 0* 61 ( 1 58 ( 1 51 ( 1* 65 ( 1 63 ( 1 58 ( 1*

a Values are presented as means and standard deviations. Key: *p < 0.01 PH or SO compared with controls Student’s t tests. N ) 5 per group. CRE, creatinine (µM); ALT, alanine aminotransferase (IU/L); AST, aspartate aminotransferase (IU/L); ALP, alkaline phosphatase (IU/L); GLU, glucose (mg/dL); Ca, calcium (mM); ALB (g/L), albumin; TP, total protein (g/L). Urea nitrogen, sodium, potassium and phosphorus were unchanged. BIL, bilirubin (µM) was not measurable for the majority of samples.

Figure 4. 1H NMR spectra δ 0.5-4.5 at 600 MHz of urine from a rat 0, 8, 24, 48, and 72 h post partial hepatectomy.

citrate, 2-OG, hippurate, GAA, betaine and bile acids.13,14,21-27 While there is some evidence for liver injury at early time points, metabolic events occur that are particular to the regenerative period. Thus, our current data indicate that cellular regeneration, in addition to cell death or loss of normal function, could be a major contributing factor to the systemic

perturbations seen in previous metabonomic studies of hepatotoxicity. The Importance of One-Carbon Metabolism to Liver-Specificity of Changes in Systemic Metabolic Profiles Post PH. The four metabolites GAA, creatine, betaine and DMG are intimately associated with the cycling between SAM and Journal of Proteome Research • Vol. 9, No. 1, 2010 63

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Figure 5. Metabolic mean time profiles for major metabolites. Error bars indicate standard errors of selected urinary metabolites and are shown for contol and hepatectomy groups only. (A) citrate, (B) 2-OG, (C) hippurate, (D) taurine, (E) bile acids, (F) GAA, (G) creatine, (H) betaine, (I) DMG, (J) creatinine excretion. All concentrations (A-I) are expressed relative to creatinine. Black circles, control; shaded squares, sham operated; red triangles, partial hepatectomy. 64

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Table 3. Summary of Urinary Metabolite Perturbations Observed after Partial Hepatectomy Relative to Sham-Operated Ratsa Chemical shift δ and multiplicity

Metabolite

alanine betaine

1.48 d, 3.79 q 3.27 s, 3.9 s

cholesterol choline citrate

0.66 m, 0.7 m, 0.84 m 3.20 s, 3.51 m, 4.07 m 2.56 d, 2.7 d

creatine

3.04 s, 3.92 s

dimethylglycine

2.92 s, 3.57 s

R-and β-glucoseb

guanidinoacetate

3.40-3.9, 4.5 d 5.24 d 3.63-3.98, 5.4 d 3.24 s, 3.67 d,d, 3.78 m, 4.36 (m) 3.8 s

hippurate

7.55 d, 7.64 t, 7.84 t, 3.97 s

hypotaurine triglycerides

2-oxoglutarate

2.66 t, 3.37 t 0.87 t, 1.29 m, 1.30 m, 1.59 m 2.03 m, 2.23 m 2.80 m, 5.30 m 2.45 t, 3.02 t

taurine

3.27 t, 3.45 t

trimethylamine-N-oxide

3.27 s

phosphocholine Bile acids

3.22 s, 3.60 m, 4.18 m 0.66-0.76 m (C-18 methyl) 0.90-1.00 m (C-19, C-21 methyls)c

glycogen GPC

Urinary perturbations in PH rats

v 8-120 h (24-96 h*) Max effect 48-72 h

Serum perturbations in PH rats

Liver perturbations in PH rats

v 48, 168 h

v 48 h v 48, 168 h v 48 h V 48 h

V 0-48 h (0-48 h*) v 72-120 h v 8-96 h (8-48 h*) Max effect 8-24 h v 48-96 h (72-96 h*) Max effect 72-96 h

v 48 h

V 48 h V 48 h V 48 h v 0-48 h (0-72 h*) Max effect 0-8 h V 0-72 h (8-48 h*) v 96-144 h* v 0-8 h V 168 h

v 48 h

V 0-48 h (0-48 h*) v72-120 h (72-120 h*) v 0-96 h (0-24 h*) Max effect 0-8 h v 0-96 h Max effect 48-72 h V 48 h v0- 8 h 2 rats only v 24-48 h (24-48 h*)

a Key: s ) singlet, d ) doublet, t ) triplet, q ) quartet, m ) multiplet. *Time points where PH value was significantly different from both sham and controls (p < 0.05). Calculated using an automated peak fitting and integration algorithm (Crockford et al.)18 with the exception of the bile acids, for which significant time points were calculated using the signal integrated between 0.66 and 0.76 ppm). b Refer to literature for detailed assignments (Nicholson et al.;15 Fan).16 c Beckwith-Hall et al.13

homocysteine in 1-carbon metabolism and are all perturbed by PH (Figure 7). GAA is directly converted to creatine, via a reaction that consumes molar equivalents of SAM, while betaine is converted to DMG via betaine-homocysteine methyltransferase (BHMT). Analysis of the dynamics of metabolic response show that the increased urinary excretion of GAA, creatine, betaine and DMG occurs in sequence, with differing onset, maximum effect time point and duration of each temporal profile (Figure 5F-I). For instance, GAA and creatine exhibit similar metabolic trajectories. However, GAA reaches a maximum concentration at 0-8 h, prior to creatine (8-24 h) (Figure 4 and Figure 5F). This pattern of change, together with the fact that GAA is the immediate precursor of creatine (Figure 7), suggests that both metabolites could be coregulated by a process that temporarily inhibits GAA conversion to creatine, as may occur with a transient drop in SAM levels. While acute dietary restriction has been associated with elevation of creatine, this has not been reported together with elevation of urinary GAA. The elevated levels of betaine (maximum effect at 48-72 h) can also be explained by a drop in SAM if this was to precipitate a fall in homocysteine required for conversion of betaine to DMG. A transient fall in hepatic SAM levels is known to precede regeneration after PH and is considered necessary for this to occur.9,10,28 The particular role of the liver in the synthesis of creatine, together with the liver-specificity

of BHMT transferase expression, means that the altered excretion of compounds involved in 1-carbon metabolism could be a useful probe for liver-specific processes, such as regeneration. Metabolic Consequences of Ischemia-Reperfusion Injury and the Partial Hepatectomy Model. Several reports describe similar metabolic consequences to those observed here that have been attributed to ischemia-reperfusion (I/R) injury. Niemann et al.42 and Behrends et al.43 both reported markedly elevated serum levels of betaine within 24 h postischemiareperfusion injury in Zucker and Wistar rat models. A later study from Park et al.44 that used Lewis rats also reports elevation of betaine from baseline after I/R, but levels did not reflect the lesser degree of injury in young versus old animals. Behrends et al. also report increases in serum creatine during the same period, which was not reported by Niemann et al. but was seen in the Park study. The surgical procedure used in these studies differs from the model here in several important respects: the left and median lobes are ligated and the ischemic tissue is reperfused in the I/R model. In the hepatectomy model, the left lateral and median lobes are ligated and surgically removed, leaving only a small remnant of ischemic tissue, which is not reperfused. The bulk of the residual tissue (the remaining lobes) is not directly manipulated by the procedure. Although there is necrosis of the small stubs, the Journal of Proteome Research • Vol. 9, No. 1, 2010 65

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Figure 6. 1H NMR spectra δ 0.5-4.5 at 600 MHz of 48 h liver from control (CTL), sham-operated (SO) and hepatectomized (PH) rats.

predominant change is hepatocellular hyperplasia and hypertrophy in the remaining lobes.

confirm 1-carbon intermediates such as betaine as useful markers of liver physiology.

There are also specific differences between the observations made in our study and reports of the metabolic effects of I/R injury. The levels of hepatic injury were much more severe in the I/R model than following hepatectomy (e.g., mean ALT 10-60 times higher in I/R). Furthermore, the excretion of betaine peaks at 48-72 h posthepatectomy after ALT levels are normalized and after the expected zenith of I/R injury.42-44 Therefore, while we clearly cannot rule out a contribution to the metabolic response from ischemia during surgery, the characteristics of the partial hepatectomy make I/R injury unlikely to dominate the effect of regeneration and/or loss of normal hepatic function in our study. It is also possible that loss of function and pro-regenerative processes contribute to the metabolic perturbations previously observed in I/R injury. Regardless, both our data and metabonomic studies of I/R

The Link between Dietary Intake and Organic Acid Excretion. The geometries of the metabolic profiles can reveal similarities between specific metabolites in common metabolic pathways (Figure 7), and these relationships presumably reflect a common process underlying the physiological change.26,29 The urinary metabolic time courses for citrate, 2-OG and hippurate in PH rats exhibit similar biphasic profiles (Figure 5A-C), whereby, at early time points (0-48 h), their levels are decreased compared with SO and control urine samples, and at later time points (96-120 h), the levels are raised. These organic acids are frequently observed to share a common excretory time profile in toxicity studies12-14,25 and hence appear to be ‘coregulated’ in response to a wide range of stressors. Perturbations in urinary tricarboxylic acid (TCA) cycle intermediates and hippurate are most likely nonspecific markers of physiological stress related to elevations in energy demand and are altered by diet, food intake and weight

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Figure 7. Metabolic pathways involved in liver regeneration observed by NMR measurements. Changes in the concentration of individual metabolites and the time posthepatectomy that a response was detected are highlighted. Parentheses indicate urinary effects. SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; GAA, guanidinoacetic acid; DMG, dimethylglycine; Ptd-Etn, phosphatidylethanolamine; PtdC, phosphatidylcholine; CDP-choline, cytidine 5-diphosphocholine; P-choline, phosphocholine; Ser, serine.

loss.26,30,31 Food consumption was not measured in this study; however, changes in body weight, which reflect nutrient availability, correlate well with alterations in TCA cycle intermediates. Body weights of PH rats fall over the first 48 h, compared with SO and control animals, and show a higher average increase between 48 and 168 h (24.9%), than SO (18.5%) and control animals (17.3%). Taurinuria as a Marker of Hepatotoxicity and Liver Regeneration. In the present study, elevated levels of hypotaurine were observed in the 0-8 h urine samples from PH rats and taurine excretion was significantly increased in the 0-24 h urine samples, peaking at 8 h (Figure 5D,E). Hypertaurinuria has previously been described as a marker of liver dysfunction, including necrosis and steatosis, when in combination with other metabolic events such as hypercreatinuria. It has been observed after treatment of rats with several wellknown hepatotoxins including carbon tetrachloride, ethionine, galactosamine and hydrazine.13,14,22,32 As an end point for sulfur metabolism, taurine excretion has often been associated with an upregulation of cysteine production and hence protective mechanisms such as protein or glutathione synthesis.33-35 Alternatively, it has been postulated that elevated taurinuria observed after damage of liver cells by hepatotoxins may be a result of liver regeneration rather than necrosis.36 Given the early time point of taurinuria, it is difficult to distinguish between pathological and regenerative contributions to the observed increases in taurine excretion. Bile Aciduria. A mixture of bile acids was observed in the 24-48 h urine samples from all rats in the PH treatment group. Alteration of bile acids during liver cell proliferation has previously been observed in rats after PH.19 Tsuda et al. suggest that during DNA synthesis, predominantly in the first 24 h following PH, liver cells are not capable of conjugating all free bile acids with taurine, and therefore, there is an increase in urinary excretion of these compounds.19 Bile aciduria has previously been observed by NMR spectroscopy after treatment of rats with the model hepatotoxins galactosamine and R-naphthyl isothiocyanate.13 As with taurinuria, the early time point

makes it difficult to distinguish between pathological and regenerative contributions to the observed bile aciduria. However, the combination of the two events could be associated with a general loss of normal liver function expected post hepatectomy and during regeneration. Perturbations in Serum and Liver Tissue. In the liver tissue of PH rats, an increase in mobile lipids, particularly triglycerides, and cholesterol was clearly seen at 48 h by MAS NMR spectroscopy, which agrees with previous studies which have repeatedly observed fatty liver and elevated cholesterol up to 48 h post PH.7,11,37,38 The precise mechanistic basis of the steatosis (confirmed in our study by histopathology) is not known, but it is presumably the result of an increase in liver triglyceride synthesis or/and alteration to the balance of triglyceride import and export. The hypolipidaemia observed in the 168 h serum samples from PH rats supports the idea that transport processes are affected, although no clear differences were seen at 48 h. Triglycerides are normally exported from the liver in the form of very low density lipoprotein (VLDL) particles, which also contain relatively high concentrations of free cholesterol. Since VLDL synthesis requires the availability of phospholipids, particularly phosphatidylcholine (PtdC), an insufficiency of PtdC or its precursors can lead to decreased secretion of triglycerides from the liver and hence fatty liver.37 From MAS NMR spectroscopy of the liver from PH rats, depleted levels of PC and GPC were evident at 48 h after surgery. In the PH model, there are several factors that could combine to effect such a deficit: regenerating liver requires phospholipids for cell membrane synthesis;39 choline is entirely dietary in origin and reduced food intake is a possible consequence of PH; the conversion of phosphatidylethanolamine (PtdEtn) to PtdC requires SAM, which is depleted during PH (Figure 7). In our study, we also observed a dramatic increase in liver concentration and urinary excretion of betaine, which might imply an increased conversion of choline to betaine. Interestingly, it has been suggested that fat in the liver may provide a nutritional source for replicating hepatocytes and hence is necessary for Journal of Proteome Research • Vol. 9, No. 1, 2010 67

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Bollard et al. 7,11

normal regeneration rather than a passive consequence of it. In our study, depleted levels of glucose and glycogen observed at 48 h in the liver are suggestive of augmented glycolysis and glycogenolysis, and may also indicate increased energy demands.40

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Conclusion Tissue injury and repair are distinct consequences of disease and toxicity, and hence, it is important to differentiate these processes. In the present work, we have applied metabolic profiling to characterize the effect of partial hepatectomy, a primarily regenerative model, on the systemic metabolism of the rat. Several of the metabolic changes that occur during the regenerative phase in this model have been previously observed with hepatotoxicity, and hence, such effects in toxicity studies may represent liver regeneration and not just reflect hepatocellular necrosis or liver dysfunction. NMR spectroscopic based analysis provides a holistic approach to the study of tissue regeneration, as changes in metabolic pathways can be studied simultaneously in urine, serum and tissue samples. The approach of NMR profiling has provided new insight into the interactions between lipid metabolism, 1-carbon metabolism and SAM recycling in hepatic regeneration, supporting the notion that metabolic changes have an active mechanistic role to play in regeneration and are not just a reactive consequence of proliferation. These results provide further confidence in the ability of urinary metabonomics to report the presence of liver specific pathology and will directly help in providing biomarkers for identifying toxic events in preclinical studies. Biomarkers indicated in the current work may prove useful in future attempts to apply metabonomics to noninvasive monitoring of the regeneration process in the clinic. Abbreviations: PH, partial hepatectomy; NMR, nuclear magnetic resonance; SO, sham-operated; MAS, magic angle spinning; DNA, deoxyribonucleic acid; SAM, S-adenosine methionine; HGF, hepatocyte growth factor; TSP, sodium 3-trimethylsilyl-1-(2,2,3,3-2H4) propionate; BEST, Bruker efficient sample transfer; 1-D, 1-dimensional; FID, free induction decay; FT, Fourier transformation; CPMG, Carr-Purcell-Meiboom-Gill; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; GAA, guanidinoacetic acid; 2-OG, 2-oxoglutarate; DMG, dimethylglycine; PC, phosphocholine; GPC, glycerophosphocholine; BHMT, betaine-homocysteine methyltransferase; TCA, tricarboxylic acid cycle; VLDL, very low density lipopoproteins; PtdC, phosphatidylcholine; Ptd-Etn, phosphatidylethanolamine.

Acknowledgment. Many thanks to Letitia Cheatham and Philip Czerniak for the design and undertaking of surgical procedures at Dupont Pharamceuticals, and to all the members of the COMET consortium for financial support and access to data. Supporting Information Available: Principal Components Analysis of 1H MAS NMR spectra of liver tissue. This material is available free of charge via the Internet at http:// pubs.acs.org.

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