JournalofHepatology, 1992; 14: 26.5-275 @ 1992 Elsevier Science Publishers B.V. All rights reserved. 016%8278/92/!$.05.00

265

HEPAT 00946

Phosphorus-

toni’, David J. Bryant*, Ala ngamin4, Stephen

and Marsha Y.

Gilbey’,

.

Collins*, Glyn A. Coutts*, phrey J. F. Wodgson6

organ’

‘NMR Unit, Hammersmith Hospital, ‘GEC Hirst Research Centre, 3Medical Unit (Cellular Mechanisms Research Group), London Hospital Medical College, 4Department of Surgery, Hammersmith Hospital, ‘Department of Medicine, Hammersmith Hospital, 6Department of Gastroenterology, Hammersmith Hospital and ‘Academic Department of Medicine, Royal Free Hospital, London, United Kingdom (Received

13 December 1990)

Phosphorus-31 magnetic resonance spectroscopy of the human liver was undertaken in 28 healthy adult individuals and in 49 patients with liver disease of varying aetiology. Data localised to the liver were obtained using chemical shift imaging techniques. The mean (+ 1 S.D.) of the peek area ratio phosphomonoesters (PME)/phosphodiesters (PDE) in healthy adult individuals, from spectra obtained with pulse angle 45” and repetition time 1 s, was 0.24 + 0.07. The intra-examination variability of this ratio was 20%, the intra-subject variability 27% and the inter-subject variability 32%. An increase in the PME/PDE was observed in the 31Phepatic MR spectrum from primary or secondary tumours in all 17 patients studied, which invariably represented an increase in PME/.ATP and, in some cases, a reduction in PDE/ATP. The spectra did not show aetiulogical characteristics. A non-specific elevation in PME/PDE was also observed in the 31Phepatic MR spectra of 10 (40%) of 25 patients studied who had diffuse liver diseases, such as cirrhosis and infiltrating malignancies. The spectral pattern did not distinguish between diseases of varying aetiologies, but there was a linear correlation between increasing PME/PDE and a reduction in plasma albumin concentrations @ = 0.03). In three patients with hepatic malignancy and abnormal hepatic 31P-MRS, marked spectral changes were observed after successful treatment to debulk the tumour. Only minor changes were observed in the abnormal spectrum of a fourth patient in whom treatment was unsuccessful. Hepatic 31P-MR spectroscopy may prove useful for monitoring disease processes and treatment effects in well characterised patient populations.

In vivo magnetic resonance spectroscopy (MRS) is a non-invasive and non-destructive method for obtaining localised chemical information. Information is obtained in the form of a spectrum, in which signal intensity is plotted against frequency. The peak position depends on chemical environment and the signal intensity is related to the concentration of species present. Each spectral peak has a characteristic time for recovery after signal excitation, known as the spin-lattice relaxation time, T,, and therefore the appearance of a spectrum can be dependent on acquisition parameters. Correspondence: Dr. 1. Jane Cox, NMR Unit, Hammersmith

Phosphorus-31 magnetic resonance spectroscopy (31PMRS) can be used to monitor metabolic events within the liver. A number of centres have used this technique to obtain hepatic spectra from patients with liver disease of varying aetiology and severity (l-11) but the total clinical experience remains small. Between February 1986 and January 1989 hepatic 31PMRS examinations were undertaken, using a prototype spectroscopy system, in 28 healthy adult individuals to obtain data on the intra-examination and intra/inter-subject variabilities for the individual metabolite ratios and to de-

Hospital, DuCane Road, London W12 OHS. U.K.

I.J. COX

266 lineate the spectral changes observed in relation to parameters. These data were then used to assess spectral changes in 49 patients with focal and diffuse liver disease of varying aetiology and severity.

Patients and Methods

Permission for these studies was obtained from the Research Ethics Committee of the Royal Postgraduate Medical School, London, U.K., and Royal Free Hospital, London, U.K. Informed consent was obtained for each individual. Subjects were screened to exclude pregnancy or the presence of metallic implants, including pacemakers. Spectroscopy

A Picker prototype spectroscopy system was used in these studies (12-14). It is based on a whole body magnet (Oxford Magnet Technology) which operates at I.6 Tesla, 68 MHz resonance frequency for protons and 27.5 MHz for phosphorus. A saddle-shaped transmitter coil was used which enclosed the lower thoracic cage and upper abdomen thus encompassing the liver. The coil was double-tuned to 68 MI& and to 27.5 MHz. A double-tuned surface receiver coil (diameter 60 mm or 150 mm) was placed lateral to the liver in the mid-axillary line. The proton channel was used for shimmir,g and an image was obtained to provide anatomical detail in the region of interest, and to relate the subject’s anatomy to the magnet co-ordinate system. Either partial saturation sequences (PS 500/17) or spin echo (SE 500/22) sequences were used. For phosphorus spectroscopy a trapezoid shaped radiofrequency pulse of width 200~s at half height was used. In general the pulse angle was 45” and the time between successive excitation pulses (the repetition time (TR)) was 1 s, unless otherwise specified. Spatial localisation to the liver was achieved using either a two-dimensional chemical shift imaging (2D CSI) pulse sequence to obtain localised spectra in the form of a series of parasagittal planes (12,15), or a four-dimensional (4D) chemical shift imaging sequence to localise in three spatial dimensions (13,14). The nominal spatial resolution was typically 20 mm or 30 mm in each direction. There was a total delay of 3.1 ms before the start of data acquisition which accommodated the phase encoding gradient pulse and initial decay of the associated eddy currents. Typically, 32 data collects were acquired at each of eight gradient amplitudes to produce a 2D CSI data set, with a resulting data collection time of 4 min for a TR of

et al.

1 s. The total data collection time for a 4D data set was 34 min. Peak areas for phosphomonoesters (PME), inorganic phosphate (Pi), phosphodiesters (PDE) and/3ATP (ATP) were measured using a computerised integration programme. Areas were obtained with two different baselines, one of which (Fig. la) probably underestimated the peak area whereas the other (Fig. lb) may have overestimated it (16). Relative ratios for PMEIATP, PDE/ATP, PMWPDE and Pi/ATP are given as an average of two ratio measurements, calculated with the two different baselines, in the spectra obtained with pulse angle 45”and TR 1 s. pH was derived from the chemical shift of Pi measured, relative to aATP (referenced at -7.50 ppm) (17). Healthy individuals The control population comprised 28 healthy adult indi-

viduals (21 male: seven female; median (range) age 32 (22-59) years), who had no history of liver injury or alcohol abuse and no evidence of liver disease on physical examination. Standard liver function tests were normal in the 13 subjects in whom they werti obtained. None of these individuals was on regular medication and none consumed alcohol in excess of 20 g daily. Phosphorus-31 hepatic MR spectra were obtained in these 28 individuals on 41 occasions, after an overnight fast, but with no other specific preparation. Data were collected from parasagittal planes using 2D CSI with standard acquisition parameters of pulse angle 45”, TR 1 s. The total examination time was 60-90 min. Ten spectra were obtained from one subject, on each of two examinations, in order to assess intra-examination spectral variability. Intra-subject spectral variability was assessed in two subjects by comparing data from single spectra, selected at random, from at least five separate data sets obtained at different examinations. Inter-subject spectral variability was assessed by comparing data from single spectra, selected at random, from onC’.txamination of each of the 28 individuals. Phosphorus-31 hepatic MR spectra were obtained from one subject with TR values ranging from 0.5 s to 20 s, using a 90”pulse angle, in order to illustrate spectral variations due to changes in acquisition parameters. Patients

Forty-nine patients (31 men: 18 women; median (range) age 51(18-72) years) with liver disease of varying aetiology and severity were studied. Three patients (6%) could not tolerate the procedure, and the examination was technically unsatisfactory in a further four (8%). Of the 42 patients in whom the examination was satisfactory, 17 had focal liver lesions, four (two men: two women;

31P-MRS OF THE HUMAN LIVER

mean age 56 (52-65) years) due to priinary malignancies (one cholangiocarcinoma, three hepaxocellular carcinoma) and 13 (nine men: four women; m’ean age 57 (18-72) years) due to secondary malignancies (seven adenocarcinoma, six carcinoid tumours). The presence and location of focal liver lesions were determined by X-ray computed tomography (X-ray CT). The nature of the focal lesion was determined by histological examination of material obtained by needle or wedge biopsy, except in patients with carcinoid metastases in whom the diagnosis was made from the history, physical examination and the finding of increased urinary 5-hydroxyindoleacetic acid (SHIAA) excretion. The remaining 25 patients (16 men: nine women; mean age (range) 48 (27-71) years) had diffuse liver disease (five infiltrating malignancies, ten alcoholic liver injury, five autoimmune liver disease, five miscellaneous liver disorders). A diagnosis of diffuse liver disease was based on the clinical history, physical examination, laboratory test results, radiological investigations and, in 22 (88%) of the 25 patients, histological examination of liver tissue obtained by needle biopsy. On the basis of clinical history and laboratory investigations seven of the ten patients with alcoholic liver disease were judged to have been abstinent from alcohol for at least two weeks before the MRS examination. Three patients were known to have been drinking in the week prior to examination. Whenever possible patients were examined after an overnight fast. In the patients with focal liver disease, the surface receiver coil was placed lateral to the liver in the mid-axillary line over the bu”lk of the tumour mass, as determined by a combination of X-ray CT, magnetic resonance imaging am8 surface markings. Data were obtained from a series of volume elements, each 8-27 cm3, using 4D CSI in 14 of the 17 cases (82%). In three subjects, data were obtained from parasagittal planes of nominal width 20 or 30 mm using 2D CSI. One spectrum was selected for analysis from a volume within the tumour, or alternatively from a volume containing the greatest proportion of tumour. A typical examination took 60-90 min. For patients with diffuse liver disease the centre of the surface receiver coil was placed lateral to the liver in the mid-axillary line. Data were obtained from parasagittal planes only, using 2D CSI, of nominal width 20 or 30 mm. One hepatic spectrum from a plane containing little or no contribution from overlying muscle was selected for analysis. A typical examination took 60 min. In 32 patients, data were obtained with standard acquisition parameters of TR i s, pulse angle 45”. These data have been analysed quantitatively. In the remaining ten subjects, who were studied at the start of our clinical pro-

267

Fig. 1. A representative “P hepatic MR spectrum obtained from 5 healthy 27-year-old woman using 2D chemical shift imaging (CSI) technique, TR 1 s, pulse angle 45”. The two definitions of the baseline are illustrated and the area under the curve is defined for inorganic phosphate in each case.

gram, data were obtained with a pulse angle of W-90” and TR 1 s. These data have been analysed qualitatively. In four patients with malignancies 31P hepatic spectra v+‘ere obtained before and after varying foms of treatment designed to reduce the tumour mass. The surface receiver coil was positioned in the same location for examinations before and after treatment. Statistical analysis

Data from healthy adult control subjects are presented as mean and standard deviation. Intra-examination and intrafinter-subject spectral variability within the entire healthy adult volunteer data set of each metabolite ratio were assessed by calculation of the coefficients of variation (CV) from a set of selected spectra. Spectral parameters from patient data sets which were two or more standard deviations from the mean control values were classified as abnormal. Linear regression models were used to assess the relationship between the PME/PDE ratio and serum bilirubin, serum alkaline phosphatase, serum aspartate aminotransferase, plasma albumin and blood prothrombin time in 15 patients with diffuse liver disease from whom quantitative spectral data were available.

esuIts Healthy individuals

A representative 31P hepatic MR spectrum from a healthy adult, obtained using 2D CSI, pulse angle 45”, TR 1 s, is shown in Fig. 2. Resonances are assigned to phosphomonoesters (PME), inorganic phosphate (Pi), phosphodiesters (PDE) and adenosine triphosphate (ATP). The PME and PDE peaks are multi-component, the aATP peak contains contributions from aADP and NADH, and the yATi’ peak contains contributions from /?ADP. Spectral data from the 28 healthy adult voluntt summarised in Table 1. The CV of the relative metabolite

I.J. COX et al.

268

60

I

’

I

+lO

0

’

I

’

-1c

I

’

-23

PPm Fig. 2. A 31Phepatic MR spectrum obtained from a healthy 22-yearold woman using 2D CSI, TR 1 s, pulse angle 45”. Resonances are assigned to phosphomonoesters (PME), inorganic phosphate (Pi), phosphodiesters (PDE) and adenosine triphosphate (ATP).

ratics of the entire data set ranged from 22-30%. The intra-examination variability of metabolite ratios, assessed by comparing ten spectra from one subject during one examination, ranged from 15-24%. The in&a-subject variability of metabolite ratios, assessed by comparing spectra from one subject on five different occasions and from another subject on six occasions, ranged from 16-28%. The inter-subject variability, assessed from one spectrum selected at random from each of the 28 individuals, ranged from 22-35%. Phosphorus-31 hepatic MR spectra, obtained with TR 0.5 s alld TR 20 s and 2D CSI, pulse angle 90”, are illustrated in Fig. 3. The spectral pattern varied with TR value. A large increase in the intensity of the PDE resonance was observed in the spectrum obtained at the longer TR value. The increase in Pi and BATP was less marked. This indicates that at least one component of the PDE peak has a much longer T1 than that Of Pi and BATP. Patients Focal liver lesions. Phosphorus-31 hepatic MR spectra

--rc+ +10

0

-10

-20

ppm Fig. 3. Phosphorus-31 hepatic MR spectra from a healthy 56-yearold man from a parasagittal plane acquired using 2D CSI, pulse angle 90”, total number of data collections M-512. (a) TR 0.5 s, (b) TR 20 s. The ‘T,’ of PDE is longer than those of Pi and ATP.

PPm Fii. 4 A 31Phepatic MR spectrum from a 65-year-old man with hepatocellular carcinoma obtained from a 30 mm x 30 mm x 30 mm voxel within the tumour volume, acquired using 4D CSI, pulse angle 45”, TR 1 s, total number of data collections 2048. The PME/PDE is elevated.

TABLE 1 Parameters from hepatic 3’P magnetic resonance spectra of healthy adult subjects3 Study group (n,N,S)b

Entire data set (28,41,121) Intra-examination variability (l,l,lO) Intra-subject variability (L5.5) Inter-subject variability (28,28,28)

Mean f S.D. (coefficient of variation) PME/AW

PDE/ATPE

PhrE/PDEC

P,/ATP’

PH

0.27 f (30%) 0.22 * (15%) 0.25 + (16%) 0.27 + (35%)

1.16 f 0.30 (26%) 1.7 + 0.28 (24%) 1.19 + 0.25 (21%) 1.13 + 0.28 (25%)

Grto.07 (2G%) 0.20 + 0.04 (20%: 0.22 L 0.06 (27% 0.24 i 3.08 (32%)

0.36 + (22%) 0.36 f (14%) 0.35 + (17%) 0.35 + (22%)

0.08

7.23 + 0.14

0.05

7.19 f 0.07

0.06

7.20 + 0.09

0.08

7.31 + 0.15

0.08 0.03 0.04 0.09

6 Relative peak area ratio measurementsare given from spectra obtained with pulse angle 45”, TR 1 s. n = number ofsubjects;N = number of examinations; S = number of spectra. ’ PM& phosphomonoesters; ATP, adenosine triphosphate; PDE, phosphodiesters; Pi, inorganic phosphate.

“P-MRS

OF THE HUMAN

TABLE

2

LIVER

269

MR spectral parameters from I? patients with focal hepatic malignancies”

Phosphorus-31

hepatic

No.

I)iagnosis

PMEIATPb (0. I I-0.43) -___

PQEIATPb (0.S6-1.76)c

PME/PDEb ([email protected])c

Primary malignancies 1 Chotangiocarcinoma 2 Hepatocellular carcinoma 3 Hepatocellular carcinoma 4 HepatocelluIar carcinoma

0.37 0.67 0.43 PMEPDE

Secondary malignancies 5 Adenocarcinoma 6 Adenocarcinoma 7 Adenocarcinoma 8 Adenocarcinoma 9 Adenocarcinoma 10 Adenocarcinoma 11 Adenocarcinoma 12 Carcinoid 13 Carcinoid 14 Carcinoid 15 Carcinoid 16 Carcinoid 17 Carcinoid

1.23 0.69 0.65 0.74 0.99 0.57 0.62 1.65 1.20 0.65 0.49 0.69 0.40

1.03 a.72 0.84 0.97 1.29 0.85 1.13 0.72 1.03 0.74 0.63 1.19 0.93

1.2@ 0.92 0.78 0.7x 0.72 0.67 0.54 2.38 1.17 0.87 0.79 0.58 0.44

0.75 0.37-l 65

0.84 0.44-1.29

0.93 0.44-2.38

1-17

Mean Range

P,/ATPb (0.20-0.52)

0.65 0.56 1.47 0.44 0.93 0.44 elevated in region of tumour

p6Ng5-7.51)C

0.27 0.21 0.19

7.28 7.12 6.79 7.25

0.66 0.50 0.22 0.24 0.25 0.27 0.38 0.26 0.21 0.26 0.23 0.2; 0.16

6.98 7.38 7.62 6.85 7.50 7.20 7.28 6.98 ‘7.12 6.98 7.12 7.38 6.98

7.17 0.29 6.79-7.62 0.16-0.66 _ Data obtained with puke angles M-90”, TR Es were analysed qualita-

a Data obtained with pulse an;gle 4Y,TR 1 s, were :nalyssJ ;aantitatively. tively. b PME, phosphomonoesters; ATP, adenosine triphosphate; PDE, phosphodiesters; Pi, inorganic phosphate, c Reference range (?2 S.D. of the mean) of the entire data set obtained from the healthy adult individuals.

from

areas

of primary

turnour

showed

a~

elevated

PME/PDE in all four subjects studied (Table 2) repreIATP and a decrease in senting au increase in P PDE/ATP in two of the three subjects in whom the spectra were quantified. A representative spectrum from one patient is illustrated in Fig. 4. The spectra obtained from non-tumourous areas in two subjects, both of whom were known to have cirrhosis, were abnormal, with PME/PDE elevated in one (No. 2) but not in the other (No. 4). Spectra from areas of secondary tumour displayed an increased PME/PDE in all i3 patients studied (Table 2). Spectra were obtained from non-tumourous areas in 11 subjects and were abnormal in two, showing an elevated PMEIPDE. The Pi/ATP and pH values were generally within the reference ranges for both the primary and secondary tumours, although the pH was outside this range in three individuals (Nos. 3,7 and 8). There was no characteristic spectral pattern for any of the tumour types studied. DifFre liver disease. Hepatic 3’P-MR spectral parameters were within the reference range in 14 (44%) of the 25 patients studied (Table 3). In the remaining 14 patients (16%), spectral abnormalities were observed, predominantly elevation of the PME/PDE (Fig. 5).

Hepatic “P-MR spectra from three of the five patients with diffuse malignancy showed an elevated PME/PDE. AbnormaI hepatic 3’P-MR spectra were observed in three of the ten patients with alcohol-related liver disease (Nos. 24, 25 and 30); these subjects were judged to be abstinent at the time of the MRS examination.. Two of these patients had alcoholic hepatitis, either alone (No. 24) or superimposed on cirrhosis (No. 30). The third patient (No. 25) showed spectral changes at depth but not in

I

+lO

’

I

0

’

I

-10

’

1

I-

-20

PPm :Fig. 5. A 3’P hepatic MR spectrum from a Jl-year-old man with nonA, non-B hepatitis, acquired using 2D CSI. pulse angle 45”, TR 1 s, total number of data collections 256. The PMEiPDE is elevated.

I.J. COX et al.

270 TABLE 3 Phosphorus-31 hepatic MR spectral parameters from 25 patients with diffise hepatic disordersa No.

Diagnosis

PMEIATPb (0.11-0.43)

PDE/ATPb (0.56-1.76)

PME/PDEb (0.10-0.38)

Pi/ATPb (0.20-0.52)~

PH (6.95-7.51)

Malignancies 18 19 20 21 22

Carcinomatosis Non-Hod&ins lymphoma Hodgkins lymphoma Multiple endocrine adenomatosis II Infiltrative cholangiocarcinoma

0.74 0.41 0.23 0.31 1.14

1.08 0.80 0.76 1.79 0.93

0.68 0.51 0.28 0.17 1.23

0.31 0.29 0.30 0.25 0.35

7.28 7.38 7.38 7.38 7.28

1.04 0.23 0.68 0.72 0.94 spectra from superficial planes normal, those at depth showed elevated PME/PDE no obvious abnormality no obvious abnormality no obvious abnormality no obvious abnormality 0.70 0.40 1.74 0.29 1.03 0.28 no obvious abnormality

0.28 0.30

7.12 6.98 6.94

0.39 0.81 0.40 1.05 PMElPDE elevated 0.28 0.72 no obvious abnormality

0.49 0.36

0.20 0.43

0.38

0.21

7.38 7.05 7.48

0.60 0.88 1.84 1.31 1.51

0.95 0.88 0.40 0.35 0.62

0.26 0.22 0.54 0.39 0.55

7.28 7.28 6.98 7.38 7.20

Alcoholic liver disease 23 Fatty liver 24 Hepatitis 25 Cirrhosis 26 27 28 29 30 31 32 Autoimmune 33 34 35 36 37

Cirrhosis Cirrhosis Cirrhosis Cirrhosis Cirrhosis + hepatitis Cirrhosis + hepatitis Cirrhosis + hepatitis disease Chronic Chronic Chronic Primary Primary

active hepatitis active hepatitis active hepatitis/cirrhosis biliary cirrhosis biliary cirrhosis

Miscellaneous 38 Non-A, non-B hepatitis Fatty liver of pregnancy z Caroli syndrome 41 Amyloidosis 42 Sclerosing cholangitis

0.24

0.53 0.77 0.73 0.46 1.02

0.33 0.29

7.28 7.28 7.28 7.48 7.20 7.20 7.38

7.63

a Data obtained with pulse angle 45”, TR 1 s, were analysed quantitatively. Data obtained with pulse angles 60-90”, TR 1 s were analysed qualitatively. b PME, phosphomonoesters; ATP, adenosine triphosphate; PDE, phosphodiesters; Pi, inorganic phosphate. c Reference range (+2 SD. of the mean) of the entire data set obtained from the healthy adult individuals.

superficial planes. This patient had profound hyperlipidaemia and the observed spatial variation in spectral parameters might reflect patchy fat deposition within the liver parenchyma. The spectral parameters from the patients with well-compensated cirrhosis, who were abstinent at the time of the MRS examination (Nos. 26-29), showed no obvious abnormality. The spectral parameters were within the reference range for the three patients who had been drinking in the week prior to the MRS examination, one of whom had fatty change (No. 23), and two of whom had alcoholic cirrhosis,with superimposed alcoholic hepatitis (Nos. 31 and 32). Phosphorus-31 hepatic MR spectra were abnormal in two of the five patients with autoimmune liver disease (NOS. 33 and 35) and in all five patients with miscellaneous liver disorders (Nos. 38-42). In the 15 patients with diffuse liver injury in whom

quantitative spectral data were available there was no significant relationship between the PME/PDE and values of serum bilirubin, aspartate transaminase, alkaline phosphatase or prothrombin time. However, there was a significant correlation between the PME/PDE and plasma albumin levels (p = 0.03). Response to treatment. The PMElPDE ratio was elevated in tumour regions before treatment in the four monitored patients (Table 4). In the three subjects in whom there was a clinical and radiological response to treatment the PME/PDE reverted towards reference values in each case (Figs. 6 and 7). In the subject who underwent embolisation of the tumour circulation, Pi/ATP increased after treatment (Fig. 7). In the fourth patient, in whom tumour vessel embolisation was unsuccessful, there was some reduction in the PME/PDE but no change in the PJATP,

0.29

reduction in mediastinal Non-Hodgkins ’ combination chemotherapy and tumour bulk asessed by X-ray lymphoma CT, accompanied by a radiotherapy reduction in serum 0.51 alkaline phosphatase

19

0.23

0.79

tumour blood supply not effectively interrupted

Carcinoid

15

7.38

7.12

7.20

a PME, phosphomonoesters; ATP, adenosine triphosphate; PDE, phosphodiesters; Pi, inorganic phosphate. b Reference range (2 2 SD. of the mean) of the entire data set obtained from healthy adult individuals.

arterial embohsation

0.20

2.29

arterial embolisation

successful timbolisation of tumour arterial blood supply

Carcinoid

12

0.38 7.28

(6.95-7.51)b

(0.10-0.38)b

(0.20-0.52)b

pH

PMEJPDE* Pi/ATPa

Pre-treatment

0.54

oral 5-tluorouracil

Adenocarcinoma

11

Effect of treatment at time of repeat MRS study

marked reduction in tumour mass assessed by MRI

Treatment

Diagnosis

No.

Phosphorus-31 hepatic MR spectral data from four patients studied before and after treatment for hepatic malignancies

TABLE 4

13

1

1

10

Time between start of treatment and reneat MRS (weeks) ’ ’ ’

0.23

0.59

1.75

0.15

(0.10-0.38)b

PME/PDE’

0.36

0.20

0.55

0.25

(0.20-0.52)b

Pi/AW

Post-treatment

7.55

7.35

7.28

7.50

(6.95-7.51)b

pH

I.J. COX et al.

272

(W

(W

-+1Q

0

wm Fig. 6. Phosphorus-31hepatic MR spectra from a 56.year-old man with hepatic metastasesfrom a carcinoid tumour, obtained from a parasagittalplane using standard data collection parameters. (a) Be-

fore and (b) 1 week after cmbolisation of the tumour arterial blood supply. PME/PDE was elevated before treatment. After embolisation the PMEIPDE decreased towards reference values but is still abnormal and P,/ATp is increased. There is a small contribution from phosphocreatine (PCr), arising from overlying superficial muscle.

Discussion The hepatic 31P-MR spectrum consists of several resonances, including PME, Pi, PDE and ATP (18). The PME peak contains contributions from sugar phosphates, other glycolytic intermediates and precursors of phospholipid synthesis including phosphorylcholine and phosphorylethanolamine. The PDE peak contains contributions from phospholipid membrane breakdown products, including glycerophosphorylcholine (GPC) and glycerophosphorylethanolamine (GPE), and from phospholipid headgroups (19,20), possibly from endoplasmic reticulum

(ER) (21). The signal from phospholipid headgroups appears to contribute to over 50% of the PDE signal (20), at least at low magnetic field strengths (~2 Tesla). ATP and Pi play a key role in energy metabolism and pathological changes affecting the hepatic energy state might therefore be reflected in a lower ATPlp, ratio. At the present time there are two major problems associated with hepatic 31P-MRstudies. First, patients are required to lie flat and still for approx. 1 h. The patients in the present study were all relatively well yet 6% could not tolerate the procedure. It is unlikely that the examination time could be reduced appreciably given the time needed

Fig. 7. Hepatic MR images and slP-MR spectra from a 43.year-old man with hepatic secondaries from a primary adenocarcinoma be-

for spectral localisation. Second, difficulties arise in spectral analysis because the signal-to-noise ratio (SNR) of

fore and after treatment with oral 5fluorouracil. (a) Pre-treatment “P spectrum from a single voxel within the tumour from a 4D data set of nominal dimensions30 x 30 x 30 mm. The PME/PDE is ele-

the spectra is generally low so that many of the signals overlap, and the lineshapes probably cannot be described by simple Lorentzian or Gaussian functions. These prob-

vated. (b) Post-treatment 31P spectrum showing a return of the PME/PDE to reference values. (c) Pre-treatment MR image obtained with a short T, inversion recovery sequence (IR 1500~44NK9. (d) Post-treatment MR image.

31P-MRSOF THE HUMAN LIVER lems are compounded when the data are incompletely collected, for example using chemical shift imaging (CSI) techniques where there is a delay before start of data acquisition, and when the data are collected in the presence of motion artefact from breathing. However, if the technique for collecting data and the method for spectral analysis are standardised then valid comparison can be made between population groups. In the present study the variability of the spectral area ratio measurements for healthy subjeits was high. For the various metabolite ratios the intra-examination variability ranged from 14 to 24%, the &a-subject variability from 16 to 28%, and the inter-subject variability from 22 to 35%. Angus and colleagues (3) quoted a comparable intra-subject variability of lo-20%. The intra-examination variation is intrinsically high; additional error is introduced because subject and coil positioning cannot be exactly replicated in every case. This explains the increased magnitude of the intrtimter-subject variability. Because the inter-subject variability of the PME/PDE was 32% for healthy individuals, differences in this ratio in excess of 60% are needed to be considered abnormal at the 5% probability level. In healthy individuals the hepatic 31P-MB spectral pattern varies according to spectral acquisition parameters. Thus, it is important when comparing data from different centres to take account of the methodology used. In disease states Tt values may vary (10,16) so that it is probably even more important to take account of methodological variation when comparing the results of patient studies from different centres if T1 values are not measured. The differences which result from changes in acquisition parameters can be reduced if fully relaxed spectra are acquired, but this may prolong the examination time, which is often impractical. In the majority of patients with focal liver lesions data were collected using 4D CSI methods which allowed three-dimensional localisation of the liver lesion and simultaneous acquisition of spectra from both the tumour area and the surrounding liver tissue. This locahsation technique has not been routinely used previously to study focal liver lesions (5-8). The increase we observed in the PME/ATP in the spectra from both primary and secondary tumours is consistent with data previously reported (15-8). Our finding of a reduction in the PDE/ATP in the spectra obtained from some primary tumours has not been previously reported in adults but further studies are required before comment on the significance of this finding can be made. In 40% of the patients with diffuse liver disorders the PME/PDE was elevated, generally as a result of an increase in the PME/ATP. The spectra did not show aetio-

273 logical characteristics but there was a significant relationship between increasing PME/? E and a reduction in rhaps suggesting a replasma albumin concentrations, lationship between the presence of spectral abnormalities and the severity of the liver injury. E/ATP has been reported by other workers in similar groups of patients, including Dixon and colleagues (9) who reported data from patients with diffuse lymphomatous infiltration of the liver, and Obcrhaensli and co-workers (1) and Meyerhoff 2nd colleagues (4) who reported spectra from patients with viral hepatitis. There is no clear concensus regarding the spectral findings in patients with alcohol-related liver disease. In our study thiee of ten individuals with alcoholic liver injury had abnormal spectra. Of these one had alcoholic hepatitis, one cirrhosis with superimposed alcoholic hepatitis and one cirrhosis and profound hyperlipidaemia. All three showed an elevation in the PME/PDE and one a reduced pH. None of the data from the three patients who were actively drinking in the week prior to the MRS examination showed any spectral abnormalities, and we are therefore unable, on the basis of these studies, to attribute any changes to alcohol abuse per se. However, it is possible that any changes induced by chronic alcohol abuse might have reversed in this time period. Angus and colleagues (3) reported an elevated PME/ATP in 16 patients with alcoholic hepatitis, but observed no significant spectral changes in 13 patients with alcoholic steatosis or cirrhosis; pH was within the control range in all subjects studied. Meyerhoff and colleagues (4) on the other hand, reported no significant change in relative metabolite ratios in nine patients with alcoholic cirrhosis and ten patients with alcoholic hepatitis. However, they reported a change in absolute metabolite concentrations in both these patient groups, although these were inappropriately calculated using T1 values from healthy subjects. They also reported a reduction in pH in patients with alcoholic cirrhosis and an increased pH in patients with alcoholic hepatitis. Further studies are obviously required in this important patient group. However, great care must be taken to characterise the patient population both in terms of the degree of liver damage and the current drinking behaviour. It is likely that alcoholic abuse may of itself result in spectral abnormalities distinct from those associated with the presence of liver disease per se. Spectral changes were observed in patients with hepatic malignancy, studied before and after treatment, which reflected :he efficacy of the treatment procedures used. These finlings confirm those of previous studies in which 31p-MRS has been used to monitor the efficacy Of chemotherapy (5) and hepatic tumour embolisation (2,6).

I.J. COX et al.

274 The biochemical interpretation of the non-specific changes in the PME/PDE is difficult since both these resonances are multicomponent. In vitro magnetic resonance studies of aqueous extracts (22) suggest that phosphorylethanolamine and phosphorylcholine are the major components of the elevated PME resonance in diseased liver, and GPC and GPE are reduced in the PDE resonances. No information is available regarding the level of phospholipid headgroups from these aqueous extract studies. Both phosphorylethanolamine and phosphorylcholine are intermediates in the biosynthesis of phospholipids whereas GPE and GPC are thought to reflect phospholipid degradation. An increase in phosphorylethanolamine and/ or phosphorylcholine may therefore indicate the cells’ adaptation to the increased demand for phospholipid biosynthesis by increasing the supply of the precursor. When rapid cell growth and/or proliferation take place cell membrane synthesis is very active and in turn demand on the synthesis of membrane components, for example phospholipids, is high. Enzymes which are responsible for reactions that supply phospholipid precursors may therefore be activated, for example ethanolamine kinase (23) or choline kinase (24). It is interesting that the spectral parameters reported in liver tumours and in diseased liver

Oberhaensli RD, Rajagopalan B, Galloway GJ, Taylor DJ, Radda GK. Study of human liver disease with P-31 magnetic resonance spectroscopy. Gut 1990; 31: 463-7. Meyerhoff DJ, Karczmar GS, Weiner MW. Abnormalities of the liver evaluated by 31PMRS. Invest Radio1 1989; 24: 980-4. Angus PW, Dixon RM, RajagopalanB, et al. A study of patients with alcoholic liver disease by 31P NMR spectroscopy. Clin Sci 1990; 78: 39-47. MeyerhoffDJ, BoskaMD, Thomas AM, Weiner MW. Alcoholic liver disease: quantitative image guided P-31 MR spectroscopy. Radiology 1989; 173: 393-400. Maris JM, Evans AG, McLaughlinA, et al. ‘lP nuclearmagnetic resonance spectroscopicinvestigationof human necroblastoma in situ. N Engl J Med 1985; 312: 1500-S.

Oberhaensli RD, Hilton Jones D, Bore PJ, et al. Biochemical investigation of human tumours in vivo with phosphorus-31 magnetic spectroscopy. Lancet 1986; ii: 8-11. - --resonance _ 7 cox l.r, Sargentoni J, Calam J, Bryant DJ, Iles RA. Four dimensional phosphorus-31 chemical shift imaging of carcinoid metastases in the liver. NMR Biomed 1988; 1: 56-60. 8 Glazer GM, Smith SR, Chenevert TL, et al. Image localised 31P magnetic resonance spectroscopy of the human liver. NMR Biomed 1989; 1: 184-9. 9 Dixon RM, Angus PW, Rajagopalan B, Radda GK. “P magnetic resonance studies of patients with lymphoma. In: Proceedings of the Eighth Annual Meeting of the Society of Magnetic Resonance in Medicine, Amsterdam 1989; 69 (Abstract). 10 Horikawa Y, Naruse S, Tanaka C, et al. Semi-quantitative analysis of “P MRS in human liver cirrhosis and tumours. In: Proceedings of the Eighth Annual Meeting of the Society of Magnetic Resonance in Medicine, Amsterdam 1989,568 (Abstract). 11 Oberhaensli RD, Rajagopalan B, Taylor DJ, et al. Study of liver

tissue are similar to those observed in infant liver (25). This is consistent with studies of the pattern of several enzyme activities in animals which suggest that both in the regenerating and pre-cancerous liver the dividing cells exhibit retroversion, that is appear to become foetal in their behaviour (26). It may become possible in time to resolve the PME and PDE peaks in vivo, either by improving the spectral resolution, by use of proton decoupling (27) or by more detailed analysis of the relaxation parameters (16). This would provide greater insight into the nature of the spectral abnormalities described. In conclusion, although phosphorus-31 magnetic resonance spectroscopy may have limited application in differentiating the aetiology of liver injury it may prove valuable in monitoring disease processes and treatment effects.

Acknowledgements We thank Neal Alexander of the Department of .Medical Physics, Royal Postgraduate Medical School, for statistical advice ahd the Department of Health and the Medical Research Council for their financial support.

metabolism in glucose-6-phosphatase deficiency (glycopn storage disease Type IA) by 31P magnetic resonance spectroscopy. Paediatr Res 1988; 23: 375-80. 12 Bailes DR, Bryant DJ, Bydder GM, et al. Localised phosphorus31 spectroscopy of normal and pathological human organs in vivo using phase encoding techniques. J Magn Reson 1987; 74: 158-70. 13 Bailes DR, Bryant DJ, Case HA, et al. In vivo implementation of 3D phase encoded spectroscopy with a correction for field inhomogeneity. J Magn Reson 1988; 77: 460-70. 14 Cox IJ, Bryant DJ, Collins AG, et al. Four dimensional chemical shift imaging of phosphorus metabolites of normal and diseased human liver. J Comput Assist Tomogr 1988; 12: 369-76. 15 Brown TR, Kincaid BM, Ugurbil K. NMR chemical shift imaging in three dimensions. Proc Nat1 Acad Sci USA 1982; 79: 3523-6. 16 Cox IJ, Coutts GA, Gadian DG, et al. Saturation effects in phosphorus-31 magnetic resonance spectroscopy of the human liver. Magn Reson Med 1991; 17: 53-61. 17 Taylor DJ, Bore PJ, Styles P, Gadian DG, Radda GK. Bioenergetics of intact human muscle; a 3’P nuclear magnetic resonance study. Mol Bid Med 1983; 1: 77-94. 18 Cohen SM. Application of nuclear magnetic resonance to the study of liver physiology and disease. Hepatology 1983; 3: 738-49. 19 Bates TE, Williams SR, Gadian DG. Phosphodiesters in the liver. The effect of field strength on the 31Psignal. Magn Reson Med 1989; 12: 145-50. 20 Murphy EJ, Rajagopalan B, Brindle KM, Radda GK. Phospholipid bilayer contribution to 31PNMR spectra in vivo. MagnRe-

son Med 1989;12:282-9. 21 WilliamsSR, Murphy EJ, Bates TE, et al. Endoplasmicreticulum contributes to the phosphodiestersignal in 31PMRS. In: Proceedings of the Ninth Annual Meeting of the Society of Magnetic

3’P-MRS OF -i-HE HUMAN LIVER Resonance in Medicine, New York, August 1990; 284 (A& stract). 22 Bell JD, Cox IJ, Sargentoni J, et al. In-vivo and in-vitro IEIRS study of the PME and PDE peaks in human !iver. In: Proceedings of the Eighth Annual Meeting of the Society of Magnetic Resonance in Medicine, Amsterdam 1989; 1053(Abstract). 23 Weinhold PA, Rethy VB. Ethanolamine phosphokinase: activities and properties during liver development. Biochii Biophys Acta 1972; 276: 143-54. 24 Weinhold PA, Scott Skinner R, Sanders RD. Activity and some properties of choline kinase, choline-phosphate cytidyltransfe-

275 rase and choline pbosphotransferase during liver development in the rat. Biochim Biophys Acta 1973; 329: 43-51. 25 Iles RA, Cox IJ, Bell JD, et al. 3’P MRS of ihe human paediatric liver. NMR Biomed 1990; 3: 90-4. 26 Curtin NJ, SneU K. Enzymic retrodifferentiation during hepatocarcinogenesis and liver regeneration in rats in vivo. Br J Cancer 1983; 48: 495-505. 27 Luyten PR, Brutnik 6, Sloff FM, et al. Broadband proton decoupling in human 31PNMR spectroscopy. NMR Biomed 1989; 1: 177-83.