To investigate reproducibility of proton magnetic resonance spectroscopy (1H-MRS) to measure hepatic triglyceride content (HTGC).
To investigate reproducibility of proton magnetic resonance spectroscopy (1H-MRS) to measure hepatic triglyceride content (HTGC).
In 24 subjects, HTGC was evaluated using 1H-MRS at 3.0 Tesla. We studied “between-weeks” reproducibility and reproducibility of 1H-MRS in subjects with fatty liver. We also studied within liver variability and within day reproducibility. Reproducibility was assessed by coefficient of variation (CV), repeatability coefficient (RC), and intraclass correlation coefficient (ICC).
The CV of between weeks reproducibility was 9.5%, with a RC of 1.3% HTGC (ICC 0.998). The CV in fatty livers was 4.1%, with a RC of 1.3% HTGC (ICC 0.997). Within day CV was 4.5%, with a RC of 0.4% HTGC (ICC 0.999). CV for within liver variability was 14.5%.
Reproducibility of 1H-MRS to measure HTGC for “between-weeks” measurements and in fatty livers is high, which is important for follow-up studies. Within liver variability displays a larger variation, meaning that liver fat is not equally distributed and during consecutive measurements the same voxel position should be used. J. Magn. Reson. Imaging 2009;30:444–448. © 2009 Wiley-Liss, Inc.
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HEPATIC STEATOSIS (FATTY LIVER) is present in approximately one-third (20–33.6%) (1, 2) of the general population in Western countries and is associated with a variety of disorders, including obesity, type 2 diabetes, hepatitis, and drug toxicities (3). Liver biopsy is the reference standard for the assessment of hepatic steatosis. The utility of liver biopsy is limited because of its invasiveness, sampling errors, complications such as bleeding, and inter-observer variability (4–6). Noninvasive methods such as ultrasound, Computed Tomography, Magnetic Resonance Imaging (MRI), and proton magnetic resonance spectroscopy (1H-MRS) have been used to detect hepatic steatosis and attempts have been made to grade hepatic triglyceride content (HTGC) with these methods (1, 2, 7–9). Of these imaging techniques MRI and 1H-MRS are recognized as the most accurate noninvasive techniques for hepatic fat quantification. These techniques permit the breakdown of the MR signal into water and fat signal components and allow quantification of hepatic fat. To date, there are three clinically used MR imaging techniques for the detection and quantification of hepatic fat including chemical shift imaging, frequency-selective imaging, and 1H-MRS. Each technique has important advantages and disadvantages and is increasingly used in diagnosis, treatment, and follow-up of fatty liver disease (10–12).
1H-MRS has proven to be a very sensitive noninvasive method to detect hepatic triglyceride content (13) and has shown to correlate with liver biopsy results (14–16). Noninvasive 1H-MRS is also suitable to determine HTGC and follow up patients in clinical trails (17). Despite the increasing use of 1H-MRS in determining hepatic steatosis, there is sparse literature addressing the reproducibility of this technique (18, 19).
Knowledge of normal variability in measurements is important, especially when consecutive investigations are planned to follow the course of hepatic steatosis during treatment. Literature exists for “within day” and “within liver” reproducibility, indicating coefficients of variation in the range of 3.6–8.5% HTGC (1, 20, 21) (within day) and 11.0–14.5% HTGC (1, 21) (within liver). However, in longitudinal studies knowledge of “between weeks” reproducibility is necessary. To our knowledge only Longo et al (22) studied between weeks reproducibility in two subjects.
In this study, we investigated four aspects of reproducibility of 3.0 Tesla 1H-MRS to measure hepatic fat content. Primarily this concerned (I) “between weeks” reproducibility when measurements are repeated after 4 weeks and (II) reproducibility of 1H-MRS to measure HTGC in subjects with fatty liver. Secondarily, (III) we investigated “within liver” variability when measuring HTGC in two different voxels in different parts of the liver. (IV) We also investigated “within day” reproducibility of 1H-MRS when two acquisitions in the liver are made on the same day.
This study was a nonrandomized pilot study in 24 individuals in total: six healthy subjects, six subjects with familial hypobetalipoproteinemia (FHBL), and 12 obese subjects (body mass index [BMI] over 30 kg/m2. We included these different subjects to cover a broad spectrum of hepatic fat content. We choose subjects with FHBL as this condition is associated with fatty liver due to triglyceride accumulation in the liver. Obese subjects were chosen as a positive correlation has been established between increased waist circumference and hepatic fat content (20). This study was approved by the Medical Ethics Committee. All participants gave written informed consent. The study sponsor had no influence on study design or analysis.
In all 24 subjects, hepatic triglyceride content in the liver was evaluated using 1H-MRS. This cohort comprised 12 males and 12 females. Mean age was 49.1 years (range, 22–65 years). Fifteen of 24 subjects were obese (BMI > 30.0 kg/m2) with mean BMI of 31.2 kg/m2 (range, 21.8–41.0). Eleven of 24 subjects had features of the metabolic syndrome defined as having at least three risk factors according to the National Cholesterol Education Program Adult Treatment Panel III definition (23). One of the risk factors is abdominal obesity. In all 24 subjects, 1H-MRS measurements were performed twice. Both 1H-MRS scans were performed in fasting condition, in the morning between 8:00 and 10:00 AM. The second 1H-MRS scan was scheduled 4 weeks after the first 1H-MRS scan. Other standardization or lifestyle control was not performed.
We investigated four aspects of reproducibility. Primarily, we investigated subjects for the following: (I) “Between weeks” reproducibility; All subjects were scanned twice. The second scan was performed 4 weeks later. In total, we studied “between weeks” reproducibility in 24 subjects. For both scans the same voxel position was used. (II) “Between weeks” reproducibility of 1H-MRS in fatty livers; only subjects with fatty liver were selected. Szczepaniak et al (1) defined hepatic steatosis as more than 5.6% HTGC measured by 1H-MRS. In a subset of 8 subjects with fatty liver (all six subjects with FHBL and two selected obese subjects), we studied “between weeks” reproducibility. In this subgroup, we studied reproducibility of 1H-MRS in subjects with hepatic steatosis.
Secondarily we chose to investigate 12 subjects for (III) “Within liver” variability; during the first visit 1H-MRS was performed in two different positions (voxels) in the liver, repeated in the same two voxels after 4 weeks. This was performed in the six healthy subjects and the six subjects with FHBL. We compared both voxels within the 4 weeks time span to study within liver variability. This was done to anticipate for heterogeneity of fat in the liver. (IV) “Within day” reproducibility; 12 obese subjects were scanned twice on the same day. The second scan was performed approximately 4 hours after the first; both scans were performed in fasting conditions.
All measurements were performed on a 3.0T Philips Intera scanner (Philips Healthcare, Best, the Netherlands) using a cardiac coil. A voxel of 20 × 20 × 20 mm was positioned in the right hepatic lobe, avoiding inclusion of the diaphragm and edges of the liver, but also vascular and biliary structures. When two voxels were used to assess within liver variability, different positions were chosen in the right hepatic lobe. Voxel size and time for acquisition were standardized for all subjects. Spectra were acquired using first order iterative shimming, a PRESS sequence with TE/TR = 35/2000 ms and 64 signal acquisitions during free breathing. We evaluated the liver 1H-MR spectra by using jMRUI software (24). A ratio from the 1H-MR spectra (Fig. 1) was calculated and defined as the methylene peak versus the reference H2O peak. Calculated peak areas of water and fat were corrected for T2 relaxation (T2 water = 34 ms, T2 fat = 68 ms) (25), and percentage hepatic fat content was calculated according to methods described by Szczepaniak et al (1). Room time, including taking subjects in and out of the scanner, positioning of the subjects in the scanner, acquisition of localizers, acquisition of axial–coronal–transversal T2 weighted images for voxel planning and performing 1H-MR spectroscopy, was 45 min.
1H-MRS analysis: The percentage hepatic fat content was the main endpoint of this study. The reproducibility of the measurements was assessed by means of the Bland-Altman method (26) and repeatability coefficient (RC), which allows calculating the 95% limits of agreement. We also used the intraclass correlation coefficient (ICC), as well as the coefficient of variation (CV). We chose to use the Bland-Altman method, because this method is more suited to study reproducibility between different measurements than correlation coefficients. The repeatability coefficient was defined as 1.96 times the standard deviation of the mean difference between two measurements (26). The CV was investigated because most other literature used this method to study reproducibility of 1H-MRS, so we were able to compare results. The CV was calculated by dividing the standard deviation of the mean difference between two measurements divided by the mean HTGC of all measurements. To study differences between groups and scanning visits we used nonparametric tests for related samples (Wilcoxon signed rank test). A P value < 0.05 was considered significant. For statistical analysis SPSS (SPSS Inc, Chicago, IL) was used.
When performing this 1H-MRS study, we encountered no technical failures, and all 1H-MRS measurements were of sufficient quality for analysis.
Mean HTGC in the first 1H-MRS measurement was 6.8% and did not differ from the second 1H-MRS measurement, 7.0% (P = 0.391). The CV between both scanning sessions was 9.5%. The RC was 1.3% HTGC (Table 1). In Figures 2 and 3 these results are represented in a scatter plot and Bland-Altman plot to show the limits of agreement between both measurements. The ICC between both scanning sessions was 0.998 (P < 0.001), indicating that these measurements are reproducible.
|1) Between weeks (n=24)||9.5%||1.3%||0.998|
|2) Fatty liver (n=8)||4.1%||1.3%||0.997|
|3) Within day (n=12)||4.5%||0.4%||0.999|
Hepatic steatosis is defined as more than 5.6% HTGC measured by 1H-MRS. In this study we identified eight of in total 24 subjects having more than 5.6% hepatic fat (two obese healthy subjects and six subjects with FHBL). Mean HTGC in the first 1H-MRS measurement did not differ (16.7%) from the second 1H-MRS measurement, 16.7% (p = 0.889). CV was 4.1% and RC 1.3% HTGC. The ICC between both scanning session was 0.997 (P < 0.001).
To measure “within liver” reproducibility of 1H-MRS HTGC was assessed in 12 subjects by comparing two voxels positioned in different parts of the right liver lobe (right liver lobe defined as segments IV to VIII by Couinaud), also repeated within 4 weeks. Mean HTGC in the first voxel (9.7%) did not differ from the second voxel, 9.5% (P = 0.831). The CV in this group was 14.0%. In Figures 4 and 5 HTGC measured by 1H-MRS is represented in a scatter plot and Bland-Altman plot to assess agreement between both measurements in two separate voxels in the liver. The ICC between both scanning sessions was 0.996 (P < 0.001).
In total 12 healthy obese subjects underwent 1H-MRS measurements of HTGC twice on the same day. Mean HTGC in the first 1H-MRS measurement was 4.0% and did not differ from the second 1H-MRS measurement, 4.0% (p = 0.583). The CV was 4.5% and the RC was 0.4% HTGC (see Table 1). The ICC between both scanning sessions was 0.999 (P < 0.001).
The reproducibility of 1H-MRS for measuring hepatic fat content was very acceptable for “between weeks” and “fatty liver” measurements. In this study “within liver” variability and “within day” reproducibility was acceptable.
Assessment of “between weeks” reproducibility is essential to detect abnormal variation in longitudinal studies. There is sparse literature addressing “between weeks” reproducibility of 1H-MRS to measure HTGC. Longo et al (22) studied reproducibility in two subjects on 3 consecutive days, with variability of 11% and 7%. From our data, it can be concluded that normal variation in a single subject is lower than 1.3% HTGC. Of interest, the same variability is found for the subpopulation with fatty liver in this study, indicating the robustness of this technique. In addition, our data can be used to calculate sample sizes in intervention trials for detecting drug efficacy, but also for monitoring hepatic steatosis as a side effect of drug treatments.
We also studied reproducibility of two different voxel positions in the liver to investigate possible effect of heterogeneity of hepatic fat content in the liver on 1H-MRS measurements. Of interest, in our study the largest variation in HTGC % of 1H-MRS measurements was “within liver” variation. Our data are comparable to the existing literature. Johnson et al (21) also found a CV of 14.5%, studied in five subjects. Szczepaniak et al (1) found a lower CV of 11% studied 10 subjects. Thomas et al (20) found a substantial 2 voxel inter individual variation (1–50%) in 12 volunteers. These results indicate a difference in hepatic fat content in different parts of the liver. In repeating 1H-MRS measurements of HTGC one should be aware of this and must perform measurements in the same voxel positions in the liver.
Furthermore, we found fairly good “within day” reproducibility, fitting within the range of CV's reported in the literature. Johnson et al (21) found a CV of 3.6% in five subjects, and Thomas et al (20) a CV of 7% in 34 subjects. A higher CV was found by Szczepaniak et al (1) in 10 subjects (CV of 8.5%). Machann et al (27) studied reproducibility in five healthy subjects and found variation between 0.3% and 1.7%. Another study by Machann et al (28) showed variations up to 10% in five healthy volunteers.
In our data, “within day” reproducibility is better than “between weeks” reproducibility. This finding suggests that variations are partly related to the MR scanner itself and partly to physiologic variation of liver fat. “Within day” reproducibility will be mainly related to measurement variability, whereas “between weeks” reproducibility contains both measurement variability and physiologic variations.
This study has some limitations. Although the number of patients was larger than in previous studies, still the number of patients is limited. As this was an exploratory pilot study no formal sample size calculation was carried out. We chose a sample size that was considered appropriate to address the study aims.
We did not compare our 1H-MRS results with reference standard liver biopsy due to its invasiveness. This would have been unethical in the majority of individuals in this study. Furthermore, we studied different aspects of reproducibility of 1H-MRS in different groups. Not all groups underwent all reproducibility investigations. However, our primary study aim—reproducibility of 1H-MRS for “between weeks” measurements—was performed in all subjects.
Standardization and lifestyle control was not implemented in this study. We think this is not strictly necessary because standardization and lifestyle control can be implemented in many different ways. In daily clinical practice patients are not standardized as well, and no lifestyle control is performed. Moreover, in clinical practice patients do not always obey rules on standardization and lifestyle control.
In this study, 1H-MRS is performed during free breathing. This is a potential limitation because the volume interrogated by 1H-MRS is blurred in the longitudinal direction by 2 to 3 cm respiratory excursions of the liver. In this study, we did not encounter 1H-MRS acquisition problems caused by respiratory excursions. Voxels were carefully positioned in the right liver, avoiding the diaphragm by at least 4 cm. This way 1H-MRS was always performed in liver tissue. 1H-MRS during free breathing was not a disadvantage in this study.
Finally, it should be noted that, in this study, a 3.0T magnet of one vendor is used. It is unclear if these results can be extrapolated to 1.5T scanners and other vendors. No direct benefit of the increased spectral resolution at 3.0T is to be expected in HTGC measurements. It might even be that differences in, for example, shimming, B1-homogeneity, amount of eddy currents at 3.0T result in a less reproducible measurement (29). The same effects might play a role when comparing different scanners in different institutes and of different vendors. However, we do not expect these differences to be large, because the “within day” and “within liver” variability found in this study compares well with results obtained at 1.5T.
In conclusion, 3.0T 1H-MRS for the measurement of hepatic fat content is highly reproducible in a spectrum varying from low to high hepatic fat content. “Between weeks” reproducibility is clinically most relevant and displays a variation of 9.5%. We also showed that 1H-MRS is highly reproducible in subjects with fatty liver. Because the variation in HTGC measurements between two voxel positions in the liver exceeds the “between weeks” variability, the same voxel position should be used in consecutive 1H-MRS measurements. Furthermore, “within liver” and “within day” reproducibility of 3.0T 1H-MRS to measure HTGC is comparable with reproducibility of 1H-MRS reported in the literature for 1.5T.
We thank Nikki Bodegom for her contribution in performing the 1H-MRS scans. This study was supported in part by a research Grant from Johnson & Johnson Pharmaceutical Research & Development, a Division of Janssen Pharmaceutica NV, Beerse, Belgium.