Quantitative Determination of Liver Triglyceride Levels with 3T1 H-MR Spectroscopy in Mice with Moderately Elevated Liver Fat Content

Rationale and Objectives

To diagnose hepatic steatosis with noninvasive magnetic resonance (MR)–based measurements, threshold values of liver fat percentages are used. However, these differ between studies. Consequently, the choice of threshold values influences diagnostic accuracy, especially in subjects with borderline hepatic steatosis. In this study, we compared 1 H-MR spectroscopy (MRS) and biochemically determined liver fat content in mice with moderately elevated fat content and studied the diagnostic accuracy of 1 H-MRS using two literature-based threshold values.

Materials and Methods

Fifty mice were divided into three groups: 21 C57Bl/6OlaHSD (B6) mice on a high-fat diet, 20 B6 mice on a control diet, and 9 LDLr−/− mice on a high-fat high-cholesterol diet. 1 H-MRS was performed using multi-echo STEAM at 3T to derive a fat mass fraction ( 1 H-MRS fat content). Biochemical fat content was determined from liver homogenates. Correlation and agreement were assessed with the Pearson correlation coefficient and the Bland–Altman analysis and diagnostic accuracy by calculating sensitivity, specificity, and positive and negative predictive values.

Results

All mice were pooled to form a single cohort. Mean (±standard deviation) biochemical fat content was 32.2 (±13.9) mg/g. Mean 1 H-MRS fat content did not differ at 30.2 (±12.0) mg/g ( P = .13). Correlation r was 0.74 ( P < .0001). Bland–Altman analysis indicated that 1 H-MRS fat content underestimated biochemical fat content by 2.1 mg/g. The diagnostic accuracy of 1 H-MRS depended to a great extent on the chosen reference threshold value.

Conclusions

1 H-MRS measurement of moderately elevated liver fat content in mice correlated substantially with biochemical fat content measurement. Contrary to earlier studies, diagnostic accuracy of 1 H-MRS fat content in borderline liver fat content appears limited.

With the global obesity epidemic reaching epic proportions, the number of people with fatty change of the liver—also referred to as hepatic steatosis or non-alcoholic fatty liver disease (NAFLD)—will rise too . In clinical practice, staging of steatosis on liver biopsy is the reference standard for assessing the amount of liver fat. However, this invasive method is rapidly being overtaken by other noninvasive reproducible measures that include ultrasound, computed tomography (CT), magnetic resonance imaging (MRI), and 1 H-MRS .

Magnetic resonance (MR)–derived measurements of liver fat are usually expressed as ratios between fat and water signal values. However, current threshold values based on MRI and 1 H-MRS for diagnosing “steatosis” differ between studies, field strengths, and populations targeted. For example, the widely used threshold of 5.6% first described by Szczepaniak et al. was found by defining the 95th percentile as threshold value for normal versus elevated in a healthy subgroup ( n = 345) of a large cohort. In that study, no histologic or biochemical reference standard was used to define steatosis, and the authors refer to an earlier publication by Hoyumpa et al. stating that the normal liver contains <5.0% w/w fat. In turn, however, those authors do not refer to other studies to back up this statement. The few studies that have investigated biochemical composition of liver tissue give considerably lower values for normal liver fat content ranging from 0.6% to 1.94% w/w . Even so, the 5.6% threshold value has become a much reported reference value. In a recent study by Tang et al. —which compared the MRI-based proton density fat fraction (PDFF) with biopsy-determined steatosis levels—a 6.4% PDFF threshold was found to optimally distinguish between grade 0 and grade 1 or higher on liver biopsy. The PDFF is the MR signal fat fraction corrected for T 1 and T 2 bias with, at this threshold value, sensitivity and specificity of 96% and 100%. It is important to note that although 1 H-MRS and PDFF measure signal ratios—which correspond nearly one-on-one with volume ratios—these are different metrics than the 5% of hepatocytes-containing fat vacuoles on histology.

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Methods

Ethical Considerations

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Mice

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Magnetic Resonance Imaging

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1 H-MR Spectroscopy

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1 H-MRS Data Analysis

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S(TE)=S(0)×e(−TET2) S

(

)

(

)

(

−

)

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Fatsignalfraction(FSF)=SFATSFAT+SWATER Fat

signal

fraction

(

FSF

)

FAT

WATER

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Fatmassratio(ηm)=SFAT×MWfλf(SFAT×MWfλf+SWATER×MWwλw) Fat

mass

ratio

(

)

FAT

(

FAT

WATER

)

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Biochemical Liver Analysis

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Statistical Analyses

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Results

Biochemical Liver Analysis

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Table 1

Biochemical Fat Content of Diet Groups per Week in Milligrams per Gram

Weeks Control High Fat High Fat High Cholesterol Four 42.2 (11.7); n = 4 29.4 (5.2); n = 4 14.6 (3.7); n = 4 Eight 29.6 (4.9); n = 4 53.9 (15.5); n = 4 30.2 (5.2); n = 5 Twelve 25.5 (10.9); n = 12 36.6 (13.9); n = 13 —

The values are represented as the mean (standard deviation) biochemical fat content divided per diet group (columns) and per diet duration (rows).

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1 H-MR Spectroscopy

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$Figure 2, Example of a 1 H-MR spectrum with estimate fit and residual signal. The lower section of shows the phased unsuppressed echo time = 12 ms 1 H-MR spectrum of a mouse liver with a fat signal fraction of 5.1%, which was converted to a fat mass fraction of 51.1 mg/g. Biochemical determination showed a fat mass fraction of 53.9 mg/g. The middle section of shows the estimate found by fitting peaks in the jMRUI software program, whereas the top section shows the residual signal not accounted for by the signal estimate.$

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Agreement Between Biochemical and 1 H-MRS Fat Content

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Diagnostic Accuracy of 1 H-MRS Fat Content

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Table 2

Diagnostic Accuracy of 1 H-MRS Fat Content at Two Threshold Values

Threshold Value 50 mg/g 19.4 mg/g Steatosis prevalence 7/50 (14%) 41/50 (82%) Sensitivity (%) 14 (0–58) 90 (77–97) Specificity (%) 98 (87–100) 67 (30–93) PPV (%) 50 (3–97) 92.5 (78–98) NPV (%) 87.5 (74–95) 60 (14–73)

The table gives the prevalence of steatosis (defined as biochemical fat content > threshold value). In addition, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 1 H-MRS fat content at two different threshold values of 50 mg/g and 19.4 mg/g are noted. 95% Confidence intervals are given in parentheses for all parameters except prevalence.

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Discussion

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Conclusions and Implications

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Acknowledgments

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Supplementary Data

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Supplementary Material 1

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References

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Quantitative Determination of Liver Triglyceride Levels with 3T1 H-MR Spectroscopy in Mice with Moderately Elevated Liver Fat Content

Rationale and Objectives

Materials and Methods

Results

Conclusions

Methods

Ethical Considerations

Mice

Magnetic Resonance Imaging

1 H-MR Spectroscopy

1 H-MRS Data Analysis

Biochemical Liver Analysis

Statistical Analyses

Results

Biochemical Liver Analysis

1 H-MR Spectroscopy

Agreement Between Biochemical and 1 H-MRS Fat Content

Diagnostic Accuracy of 1 H-MRS Fat Content

Discussion

Conclusions and Implications

Acknowledgments

Supplementary Data

References

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