3. RESULTADOS Y DISCUSIÓN
3.2. Discusiones
3.2.5. Diferencias entre las formas de reconocimiento actual y deseado
All MRS measurements were performed at room temperature. Proton-decoupled ^^P MR spectra were obtained using a 11.7 Tesla Bruker MR spectroscopy system, from perchloric acid extracts of the fi"eeze-clamped tissue samples, with 16K data points and a 45° pulse angle applied at intervals of Is to 20s.
4.5.4 Data Processing
The fi'ee induction decay (FID) was zero filled to 32K and Fourier transformed after line- broadening of 5 Hz. Peak areas for PE, PC, GPE, GPC, MDP and/or PCr were obtained, using the NMRl® spectral integration program, which fitted the data to Lorentzian functions.
4.5.5 Statistical Analysis
Since the data were not normally distributed, non-parametric statistical analysis was applied. Values for metabolite concentrations in the patient and reference populations were compared using the Mann Whitney U test. A p value of <0.05 was considered significant. All metabolite concentrations are quoted as mean values ±1 standard deviation.
4.6 Results
4.6.1 In vivo MRS
The in vivo hepatic MR spectrum contains at least six resonances (Bates et al., 1989)
which can be assigned to phosphomonoesters (PME), inorganic phosphate (Pi), phosphodiesters (PDE), y ATP, a ATP and BATP (ATP) (Figure 4.1). The a ATP peak contains contributions from a ADP and NADH and the y ATP contains contributions from BADP. A small resonance arising from phosphocreatine (PCr) is present in certain spectra because the MR localisation techniques do not always completely exclude signals from anterior and posterior abdominal wall muscle.
The same resonances were seen in the in vivo hepatic ^^P MR spectra obtained from the patient population (Figure 4.2), but there were significant differences in the metabolite ratios compared to healthy individuals. The mean PME/ATP ratio was significantly elevated in the total patient population (p<0.05) (Table 4.3) and the mean PDE/ATP ratio was significantly reduced (p<0.05) (Table 4.3). The mean derived PME/PDE ratio from the 31 cirrhotic patients was therefore elevated considerably (p<0.0005), when compared to the reference population (Table 4.3).
When patients were classified as having compensated or decompensated cirrhosis, there was an increase in the PME/ATP ratio with the severity liver dysfunction (Table 4.4). The mean PME/ATP ratio in the 17 patients with decompensated cirrhosis was significantly different from both healthy volunteers (p<0.0005) and patients with compensated cirrhosis (p<0.005) (Table 4.4). However, there were no significant differences in mean PME/ATP ratios between healthy volunteers and the 14 patients with compensated cirrhosis (Table 4.4).
The PDE/ATP ratio varied inversely with the functional severity of the liver disease (Table 4.4). The mean PDE/ATP ratio in the 17 patients with decompensated cirrhosis was
therefore significantly reduced when compared to healthy volunteers (p<0.05), but not to patients with compensated cirrhosis (Table 4.4). There was also no significant difference in mean PDE/ATP ratios between healthy volunteers and the 14 patients with compensated cirrhosis (Table 4.4).
Furthermore, the derived ratio of PME to PDE increased with worsening liver function (Table 4.4). In patients with decompensated cirrhosis, the mean PME/PDE ratio was significantly different from both healthy volunteers (p<0.0005) and patients with compensated cirrhosis (p<0.005) (Table 4.4). However there was no statistically significant difference between this metabolite ratio when the 18 healthy volunteers and the patients with compensated cirrhosis were compared (Table 4.4).
There were no significant differences in the mean Pi/ATP ratios between the reference population and the either the total patient population (Table 4.3) or the patient subgroups (Table 4.4).
4.6.2 In vitro MRS
A typical in vitro ^^P MR spectrum fi'om a PCA extract of normal liver contains resonances arising fi"om PME, PDE, NTP, NDP and Pi (Figure 4.3). The PME region of the spectrum consists of over 10 resonances, including signal from PE, PC, AMP, coenzyme A, 2,3-DPG and sugar phosphates such as glucose 6-phosphate, glycerol 3-phosphate, 3- phosphoglycerate and ribose 5-phosphate (Bell et a l, 1993). The PDE region contains two major resonances, GPE and GPC (Cohen, 1983, Bell et a l, 1993).
Many of these metabolites, such as the intermediates of carbohydrate metabolism, vary markedly with ischaemia and it was therefore only sensible to quantify the more stable
region of the spectrum (Dawson, 1955; Perry et a l, 1971; Perry et a l, 1981).
The signal intensity of the PE and PC resonances was increased and the GPE and GPC resonances reduced in spectra from all 31 patients with cirrhosis (Figure 4.4), when compared to spectra from the six histologically normal hvers. The metabolite concentrations (pmol/g wet weight of liver tissue) are summarised in Table 4.5.
There were significantly higher PE (p<0.0005) and PC concentrations (p<0.005) and significantly lower GPE (p<0.005) and GPC concentrations (p<0.0005) in the PCA extracts from all 31 patients than from the six reference livers (Table 4.5).
There was no significant difference between PE, PC, GPE and GPC concentrations from the 14 livers with fiinctionally compensated cirrhosis and those from the 17 Hvers with functionally decompensated cirrhosis (Table 4.5). Both groups had significantly different metabolite concentrations from the reference levels (Table 4.5).
4.7 Discussion
The in vivo hepatic ^^P MR spectra from patients with cirrhosis showed significant differences in metaboHte ratios with respect to the severity of liver injury, the PME/ATP and PME/PDE ratios rising and the PDE/ATP ratio falling with worsening hepatic function.
Previous human in vivo ^^P MR studies of the liver have also shown abnormalities in PME, PME/ATP, PME/PDE and PDE/ATP in patients with cirrhosis (Meyerhoff et al., 1989a; Cox
et ai, 1992b, Rajayanayagam e ta l, 1992; Munakatae^a/., 1993; Menon a/., 1995). Two
of these studies correlated the functional severity of liver injury in cirrhosis with an elevation in PME/ATP and a reduction in PDE/ATP (Munakata et a l, 1993; Menon et a l, 1995). Munakata and colleagues (Munakata et a l, 1993) studied 14 patients with cirrhosis of differing aetiology and found that there was an elevation in PME according to functional
grade, based on the Pugh's modification of the Childs' classification (Pugh et al., 1973) and the aminopyrine breath test. Menon and colleagues studied a much larger series of 85 patients and 16 healthy volunteers (Menon et a i, 1995). The elevation in PME/ATP they observed, in relation to the functional severity of liver injury, was thought to represent increased phospholipid synthesis in the rapidly regenerating liver, while the reduction in PDE/ATP was thought to represent a combination of altered phospholipid metabolism and reduced signal from hepatic endoplasmic reticulum.
This present study used in vitro MRS to correlate the underlying changes in membrane metabolism responsible for the in vivo spectral appearances in this cohort of patients with cirrhosis. Only quantification of PE and PC, intermediates on the pathway of phospholipid biosynthesis, and the phospholipid breakdown products, GPE and GPC was performed, because the relative amounts of other metabohtes contributing to the PME and PDE resonances are known to change even during the short periods of ischaemia in this study (Bell
et a l, 1993). However, post-mortem studies of human brain and animal liver indicate that
the levels of PE, PC, GPE and GPC are not significantly affected by periods of ischaemia of up to one hour (Dawson, 1955; Perry e ta l, 1971; Perry e ta l, 1981).
The in vitro ^^P MR spectra fi'om PCA extracts of liver tissue obtained at the time of transplant hepatectomy in this cohort of 31 patients revealed an elevation in PE and PC and a reduction in GPE and GPC, which mirrored the elevation in PME/ATP and the reduction in PDE/ATP seen in vivo in the preoperative period. The increase in PE and PC in the liver extracts fi'om patients in this study may be due to increased cell turnover as the cirrhotic liver attempts to regenerate (Murphy et a l, 1992), while the reduction in GPE and GPC levels is expected in common with previous studies on rapidly proliferating cells, because these metabolites are recirculated back into the synthetic pathways (Cox et a l, 1992a; Morikawa
There were no clear distinctions between the measured metabolite levels in liver tissue from patients classified as having compensated cirrhosis and those classified as having decompensated cirrhosis. This may reflect the inadequacies of the clinical grading system (Pugh et al., 1973), which is subject to a number of extrahepatic influences. However, cirrhosis of the liver is not a uniform process and therefore the in vitro liver extracts may have been subject to a degree of sampling error, since the in vivo results are composite information from a large area of liver, but the in vitro data are from l-2g representative samples of tissue, taken immediately after recipient hepatectomy. The results from the previous chapter, showing a regional variation in metabolite concentrations from the same liver (Table 3.3), imply strongly that this latter supposition is correct.
The contributions of other metabolites such as glycolytic intermediates to the PME signal are not assessable in human in vitro studies, owing to instability with minimal ischaemic insults (Hems and Brosnan; 1970). Similarly, endoplasmic reticulum (ER) is also an important component of the PDE resonance in vivo (Bates et al., 1989; Murphy et a l, 1989). In order to fuUy resolve the question of the reduction in the PDE/ATP ratio observed in vivo
in these patients, electron microscopy studies are required to quantify the amount of ER in end-stage liver disease.
However the results of this study, correlating in vivo and in vitro hepatic MR spectroscopy in a cohort of patients undergoing liver transplantation, suggest that the changes in PE, PC, GPE and GPC are responsible, to a large extent, for the PME/ATP and PDE/ATP abnormalities seen in patients with cirrhosis of the liver. The variation of metabolite ratios with in vivo hepatic spectra with respect to liver injury, suggests that ^^P MR spectroscopy may be of use in the functional grading of liver disease and because it is not blood flow dependent or subject to extrahepatic influences, unlike other grading systems or dynamic liver function tests, it may provide useful information for the timing of liver transplantation in individual patients.
4.8 References
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(1985). Beneficial effects of azathioprine and prediction of prognosis in primary biliary cirrhosis: Final results of an international trial. Gastroenterology 89: 1084-1091.
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Cox, I.J., Bell, J.D., Peden, C.J., Iles, R.A., Foster, G.S., Watanapa, P. and Williamson, R.C.N. (1992a). In vivo and in vitro ^^P magnetic resonance spectroscopy of focal hepatic malignancies. NMR Biomed. 5:114-120.
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Hems, D.A., and Brosnan, J.T. (1970). Effects of ischemia on content of metabolites in rat liver and kidney in vivo. Biochem. J. 120: 105-111.
Jalan, R., and Hayes, P C.(1995). Quantitative tests of liver function. Aliment. Pharmacol Therapeut. 9: 263-270.
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Table 4.1 Liver function tests, prothrombin time and Pugh's score o f study patients on the day o f in vivo hepatic MRS examination, mean (range) values
Patient Serum Serum Serum Plasma Prothrombin Pugh's
group aspartate alkaline bilirubin albumin time score"
transaminase phosphatase
(n) (u/L) (u/L) (p.mol/L) (g/L) (s)
(5-40)* (35-130)* (5-17)* (35-50)* (12-14)* (5-15) Compensated*’ 161 935 177 40 14 6-7 cirrhosis (82-236) (110-3063) (35-460) (31-48) (12-15) (n=14) Decompensated'’ 172 324 143 31 16 8-14 cirrhosis (40-626) (90-1133) (25-388) (22-40) (13-25) (n=17)
^degree of functional impairment (Pugh et ai, 1973). ^compensated cirrhosis = Pugh's score < 7
decompensated cirrhosis = Pugh's score > 8
Table 4.2 Liver function tests, prothrombin time and Pugh's score o f the study patients on the day o f transplantation mean (range) values
Patient group (n) Serum aspartate transaminase (u/L) (5-40)* Serum alkaline phosphatase (u/L) (35-130)* Serum bilirubin (pmol/L) (5-17)* Plasma albumin (g/L) (35-50)* Prothrombin time (s) (12-14)* Pugh’s score* (5-15) Compensated 157 820 138 39 14 6-7 cirrhosis^ (n=14) (92-263) (160-2150) (23-293) (31-49) (12-15) Decompensated 150 317 155 33 17 8-12 cirrhosis" (n=17) (42-461) (93-1111) (34-610) (27-40) (13-25)
Megree of functional impairment (Pugh et a l, 1973). ^compensated cirrhosis = Pugh's score < 7
"decompensated cirrhosis = Pugh's score > 8
Table 4.3 In vivo hepatic MRS metabolite ratios in the patient and reference populations. mean (+ 1 SD) values Study group (n)
PME/ATP Pi/ATP PDE/ATP PME/PDE
Healthy volunteers (n=18) 0.85±0.25 0.86+0.31 3.86±0.71 0.22±0.06 All patients (n=31) 1.44±0.75* 1.00±0.54 3.24±1.06* 0.52+0.40**
T able 4.4 In vivo hepatic MRS metabolite ratios in the patient subgroups and the reference population.
mean (± 1 SD) values
Study group
(n )
PME/ATP Pi/ATP PDE/ATP PME/PDE
Healthy volunteers (n=18) 0.85±0.25 0.86±0.31 3.86±0.71 0.22±0.06 Compensated cirrhosis^ (n=14) 0.95±0.29 0.79±0.32 3.74±1.03 0.28±0.13 Decompensated cirrhosis^ (n=17) 1.84±0.78<» T 1.18±0.63 2.84±0.93* 0.73±0.11 ❖ V
''compensated cirrhosis = Pugh's score <7 (Pugh et a l, 1973). ^decompensated cirrhosis = Pugh's score >8 (Pugh et a i, 1973). significant difference from healthy volunteers: *p<0.05; ❖p<0.0005 significant difference from patients with compensated cirrhosis: T p<0.005
Table 4.5 Metabolite concentrations obtained from in vitro MRS o f liver tissue, mean (± 1 SD) values Study group (n) PE PC GPE GPC Normal liver (n=6) 0.16±0.03 0.16±0.04 2.35±0.46 2.46±0.37 All cirrhosis 1.37±0.S6 ❖ 0.45±0.35* 0.31± 0.47^ 0.19±0.43^ Compensated cirrhosis^ (n=14) 1.41±0.76 ❖ 0.35±0.16* 0.22± 0.28^ 0.09±0.18^ Decompensated cirrhosis"" (n=17) 1.34±0.96 ❖ 0.53±0.43t 0.38±0.58<* 0.26±0.55<-
All values expressed as pmol/g wet weight o f liver tissue ^compensated cirrhosis = Pugh's score < 7 (Pugh et a i, 1973). ""decompensated cirrhosis = Pugh's score > 8 (Pugh et a i, 1973).
Figure 4.1
In vivo hepatic M R spectra acquired with TR 5000ms from:
a) a healthy volunteer
b) a patient with compensated cirrhosis and c) a patient with decompensated cirrhosis
PM E = phosphomonoester; Pi = inorganic phosphate, PDE = phosphodiester;
p ATP
+20
+10
0
ppm
Figure 4.2
a) In vivo hepatic MR spectrum from a patient with compensated cirrhosis.
b) In vitro hepatic MR spectrum from a perchloric acid extract of liver tissue
from the same patient.
MDP = methylene diphosphonate (external reference standard used for quantification of metabolites), NDP = nucleotide diphosphates, NTP = nucleotide triphosphates.
(a)
PDE PCrp ATP
PM E+20
+10
-10
-20
ppm
MDP PME PDE NTT/NDP — I - - 1 0 4- O -2 0 10 20 PPMFigure 4.3
In vitro hepatic MR spectrum from a perchloric acid extract of liver tissue from a patient
with decompensated cirrhosis. The spectrum has been expanded to show the PME and PDE regions.
PE = phosphoethanolamine, PC = phosphocholine, GPE = glycerophosphorylethanolamine, GPC = glycerophosphorylcholine.
MDP PME n NTP/NDP PDE NTP •10 PPM PE PC GPE GPC PPM
Chapter 5