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Non-invasive Quantification of Triglyceride Content in Steatotic Rat Livers by1 H-MRS

Posted Nov 30, 2011 Updated Apr 10, 2023
By Michal Heger PhD , Hendrik A. Marsman MD PhD , Rick Bezemer PhD , Martijn A. Cloos MS , Rowan F. van Golen MS , Thomas M. van Gulik MD PhD
13 min read

Rationale and Objectives

The aims of this study were to explore the relationship between triglyceride (TG) and water in steatotic rat livers and to accordingly test the validity of the currently used steatosis calculation methods from magnetic resonance spectra. The approximations commonly used to derive steatosis degrees from magnetic resonance spectra include the generic types TG/water and TG/(TG + water).

Materials and Methods

Hepatic fat and water content was quantitated by histology, magnetic resonance spectroscopy (MRS), gas chromatography, and dry/wet weight ratio analysis in increasingly (0%–95%) steatotic rats. Correlation analysis was performed to assess the statistical relationships among the steatosis quantification techniques. Subsequently, data were fitted with linear and nonlinear functions to determine the relationship between hepatic water fraction versus hepatic TG content and TG/water ratio versus macrovesicular steatosis degree to test the validity of commonly used steatosis calculation methods.

Results

Histologic analysis of macrovesicular steatosis correlated very strongly with TG content determined by gas chromatography and MRS. A strong positive correlation was also found between gas chromatography–derived and MRS-derived TG content. Biochemical analysis revealed a linear converse relationship between hepatic fat and water content. This relationship was nonlinear when determined by MRS. The MRS-based TG/(TG + water)–type approximations reflected the linear water-fat relationship better than the TG/water–type approximations, particularly when the calculations were performed with a maximum number of TG resonances.

Conclusions

Hepatic fat approximations of the type TG/water overestimate hepatic steatosis degree because hepatic fat accumulation concurs with hepatic water exudation. Consequently, MRS-based approximations should be of the type TG/(TG + water) and contain a maximum number of TG resonances in the denominator.

Hydrogen-1 magnetic resonance (MR) spectroscopy (MRS) has proven a viable tool for the noninvasive quantitation of experimental and clinical hepatic steatosis, a condition reflective of aberrant hepatic synthesis and elimination of fat that is characterized by the intracytoplasmic accumulation and vacuolization of triglycerides (TGs) in hepatocytes. When the TG-containing vesicles become large, such that they cause displacement of the nucleus, the condition is referred to as macrovesicular steatosis (MaS). Otherwise, the condition is referred to as microvesicular steatosis. Because of the favorable packing order of TGs (ie, a high degree of rotational freedom) and the absence of adipocytes in the liver, the resonances emanating from the acyl chains of TGs can be selectively and accurately acquired with clinical MR imaging scanners and quantified at relatively high signal-to-noise ratios . Hepatic TG quantification by MRS has been shown to correlate well with other quantification techniques, such as histologic analysis, computed tomography, ultrasound, and MR imaging , underscoring the clinical potential of this modality. An added benefit of MRS compared to ultrasonography or other radiologic techniques in the experimental setting is that MRS provides information on hepatic fatty acid (FA) composition with respect to saturated, mono-olefinic, and polyolefinic FAs. This additional feature makes MRS also useful for fat metabolic and lipidomic studies.

A number of peak ratios, summarized in Table 1 , have been used for the quantification of hepatic TG content on the basis of MR spectra derived from experimental steatosis models and patients. In the majority of cases, the quantitation of hepatic fat content is de facto semiquantitative, inasmuch as a TG/water or TG/(TG + water) ratio is calculated and expressed as a percentage. Actual quantitative data have been extrapolated in a select number of studies , whereby the fat mass per tissue volume is derived from the weighed relative proton density of tissue TGs versus water, the water mass/tissue mass ratio, and the spectral TG/water or TG/(TG + water) ratio acquired in a voxel. The chief assumptions in these computations ( Table 1 ) are that the proportion of body water to tissue is relatively invariant (unless extreme changes in hydration state occur) and that consequently, “steatosis increases only the volume fraction of fat in the liver without modifying the volume ratio between the water phase and the MR-invisible phase” (ie, proteins with very short T2 times) .

Table 1

Overview of the Different Calculation Methods Commonly Used for the Determination of TG Content From MR Spectra Derived From Patients and Animal Models

Study MFS [T] TG Peak Frequency [ppm] TG Quantification 0.8–1.0 1.2–1.4 2.0–2.1 2.2–2.4 5.3 -CH 3 -CH 3 - -CH 2 -CH=CH- -COO-CH 2 - -CH=CH- Clinical studies Szczepaniak et al 1.5 × AUC(-CH 2 -)/AUC(water) Szczepaniak et al 1.5 × × AUC(-CH 2 -, -CH 3 )/[AUC(-CH 2 -, -CH 3 ) + AUC(water)] Rijkelijkhuizen et al 1.5 × × × AUC(-CH 2 -, -CH 3 , -CH 2 -CH=CH-)/AUC(water) Machann et al 1.5 × AUC(-CH 2 -)/(AUC(-CH 2 -) + AUC(water)] d’Assignies et al 1.5 × × × AUC(-CH 2 -, -CH 3 , -CH 2 -CH=CH-)/[AUC(-CH 2 -,-CH 3 ,-CH 2 -CH=CH-) + AUC(water)] Rigazio et al 1.5 × × × × AUC(-CH 2 -, -CH 3 , -CH 2 -CH=CH-, COO-CH 2 -)/[AUC(-CH 2 -, -CH 3 , -CH 2 -CH=CH-, COO-CH 2 -) + AUC(water)] Hwang et al 1.5 × AUC(-CH 2 -)/[AUC(-CH 2 ,-) + {AUC(water) × 0.72}] Borra et al 1.5 × × AUC(-CH 2 -, -CH 3 )/[AUC(-CH 2 -, -CH 3 ) + {AUC(water) × 0.7}] Animal studies: mice Calderan et al 4.7 × × Not specified Garbow et al 4.7 × × × × × AUC(-CH 3 -,-CH 2 -, -CH 2 -CH=CH-, COO-CH 2 -, -CH=CH-)/AUC(water) Animal studies: rats Ling et al 2.0 × × I(-CH 2 -)/I(water) Hockings et al 2.0 × × × AUC(-CH 3 -, -CH 2 -, -CH 2 -CH=CH-)/AUC(water) Lim et al 3.0 × × AUC(-CH 3 -, -CH 2 -)/AUC(water) Herling et al 4.7 × × AUC(-CH 3 -, -CH 2 -)/AUC(water) Animal studies: rabbits Szczepaniak et al 1.5 × AUC(-CH 2 -)/AUC(water) Animal studies: dogs Szczepaniak et al 1.5 × AUC(-CH 2 -)/AUC(water)

AUC, area under the curve; I, intensity; MFS, magnetic field strength; MR, magnetic resonance; TG, triglyceride.

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Materials and methods

Experimental Setup and Steatosis Induction

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1 H-MRS

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Histology

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Lipid Extraction and GC

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Determination of Water Content

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Data Normalization

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Curve Fitting

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∑95x=1{[f(x)=ax+b]−[f(x)=A+Bx+Cx2]}ω, ∑

x

=

1

95

{

[

f

(

x

)

=

a

x

+

b

]

−

[

f

(

x

)

=

A

+

B

x

+

C

x

2

]

}

ω

,

where x represents the histologic MaS degree; f ( x ) = ax + b and f ( x ) = A + Bx + Cx 2 are generic linear and polynomial equations, respectively; and ω indicates that {[ f ( x ) = ax + b ] − [ f ( x ) = A + Bx + Cx 2 ]} comprises natural numbers. The function sums up the difference between y values of the linear and polynomial equations for each steatosis degree ( x ) and expresses the cumulative difference as a number between zero (ie, no difference between the linear fit and the polynomial fits at each steatosis degree) and ∞. Thus, the higher Σ is, the greater the difference between the linear and polynomial fit. Σ was solved per approximation (specified later) for the respective fits.

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Statistical Analysis

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Results

Steatosis Induction and TG Profiles in MCD Diet–fed Animals

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Figure 1, Fat content determination by histological analysis (a) , gas chromatography (GC) (b) , and 1H-magnetic resonance spectroscopy (MRS) (c) plotted as a function of MCD diet time. Data are plotted as mean ± SD. #, # #, and # # # designate a P-value of ≤ .05, ≤ .01, and ≤ .001 respectively, vs. t = 0 wk; ‡ and ‡‡ designate a P-value of ≤ .05 and ≤ .01, respectively, vs. t = 1 wk. Scatter plots of histology vs. GC (d) , histology vs. MRS (e) , and GC vs. MRS (f) , curve fitted with a linear fit and supplemented by 95% confidence interval bands (dotted lines). Spearman's correlation coefficient (ρ) and the goodness of fit are indicated in the upper left corner. TG content was calculated from MR spectra by AUC(-CH 2 -) [AUC(-CH 2 -) + AUC(water)]. MaS, macrovesicular steatosis.

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TG Accumulation and Water Exudation Is a Nonlinear Process in Increasingly Steatotic Rat Livers

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Figure 2, Color-transformed histologic images (hematoxylin and eosin) of rat livers exhibiting a progressive accumulation of fat in macrovesicular compartments (yellow) with MCD diet time, indicated in weeks in the upper left corner . The liver parenchyma appears red to brown. The subimposed unsuppressed magnetic resonance (MR) spectra were acquired from the livers from which the respective histologic sections were biopsied. Note the gradual decline of the water peak (4.7 ppm) amplitude with an increasing histopathologic macrovesicular steatosis degree (upper left corner) and the corollary increase in the methylene peak (1.3 ppm) amplitude, as indicated by the direction of the black arrows in the very left and very right MR spectra. The methylene (-CH 2 -) and water (H 2 O) peaks are characterized in the very left spectrum (0% steatosis).

Figure 3, Histology micrograph (hematoxylin and eosin) of two adjacent and equally sized hepatocytes in a steatotic liver taken from a rat that had been fed the methionine/choline-deficient diet for 3 weeks. The left hepatocyte (green) contains a triglyceride (TG)–incorporating macrovesicle that has pushed the contents of the cell toward the cell membrane, as evidenced by the perimembranous localization of the nucleus. The right hepatocyte (red) does not contain any TG-filled vesicles. Inasmuch as TGs are lipophilic, they are encapsulated into vesicles that physically separate lipophilic from hydrophilic constituents (ie, that separate TGs from the aqueous cytoplasm, respectively). Any environmental effects on relaxation times, if at all present, are therefore only expected to occur at the surface of the microvesicles (arrowheads) . Because volume is an order of magnitude greater than surface, any possible effects on relaxation times by the formation of macrovesicles will be negligible when relatively large, intrahepatically positioned voxels are scanned. This, in combination with the experimentally determined reduction in intrahepatic water content with increasing steatosis degree (Fig 4B), provides strong evidence that the reduction in water peak amplitudes with increasing steatosis is the result of water exudation rather than of shifts in the relaxation times.

Figure 4, (a) Integrated peak values as determined by magnetic resonance spectroscopy (MRS) (area under the curve [AUC]; arbitrary units) for the water resonance (4.7 ppm) (gray) and the cumulative methylene (-CH 2 -, 1.3 ppm) and methyl (-CH 3 , 0.9 ppm) resonances (black) plotted as a function of histologic macrovesicular steatosis (MaS) degree. The linear regression functions are y = −0.0009 x + 0.2356 ( R 2 = 0.39) for water and y = 0.0021 x − 0.0015 ( R 2 = 0.85) for triglycerides (TGs). AUC, area under the curve. (b) TG content as determined by gas chromatography (GC) plotted against the respective water fraction deduced from the dry/wet weight ratio. Spearman's correlation coefficient (ρ), goodness of fit ( R 2 ), and the slope are provided in the upper right corner . The 95% confidence intervals are included for the linear fit (dotted lines) . (c) A ratio of the data in (A) was calculated as AUC(-CH 2 -)/AUC(water) and plotted pairwise against histopathologic MaS degree. The R 2 value of the exponential fit is indicated in the upper left corner , and 95% confidence intervals are included for the nonlinear fit (dotted lines) . (d) To validate the TG/(TG + water) ratios as determined in (C) by MRS, nonparametric correlation and linear regression analysis was performed on the fat/water ratio as determined by MRS versus GC and the dry/wet weight ratio. Spearman's ρ is indicated in the upper left corner , as well as the R 2 value of the linear fit and its slope. Ninety-five percent confidence intervals are included for the linear fit (dotted lines) . Ratios were normalized (Norm.) to the maximum value of the data set.

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Accuracy of Approximations for TG Content Determination from MR Spectra

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Figure 5, Scatterplots of histologic macrovesicular steatosis (MaS) degree plotted against magnetic resonance spectroscopy (MRS)–derived triglyceride (TG)/water ratios calculated by AUC(-CH 2 -)/AUC(water) (a) , AUC(-CH 2 - + -CH 3 )/AUC(water) (b) , and AUC(-CH 2 -, -CH 3 , -CH 2 -CH=CH-)/AUC(water) (c) and TG/(TG + water) ratios calculated by AUC(-CH 2 -)/[AUC(-CH 2 -) + AUC(water)] (d) , AUC(-CH 2 -, -CH 3 )/[AUC(-CH 2 -, -CH 3 ) + AUC(water)] (e) , and AUC(-CH 2 -, -CH 3 , -CH 2 -CH=CH-, =CH-CH 2 -CH=, -CH=CH-)/[AUC(-CH 2 -, -CH 3 , -CH 2 -CH=CH-, =CH-CH 2 -CH=, -CH=CH-) + AUC(water)] (f) . The resonances (in ppm) used in the formulas are indicated above each respective panel. The goodness-of-fit values of the linear fit (black) and the second-order polynomial fit (gray) are provided in the upper left corner. The extent of convergence of both fits (Σ; see text) is given in the lower right corner and represents the sum of all light gray-shaded areas. AUC, area under the curve.

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Figure 6, Fatty acid (FA) profile maps categorized according to FA subgroup. Individual FAs are indicated in vertical text. Low grayscale values (black) represent low mean FA concentrations, whereas high grayscale values (white) represent high mean FA concentrations. The time indication (in weeks) on the left-hand side designates methionine/choline-deficient diet duration. The values in red indicate the minimum and maximum concentrations in milligrams FA per gram of liver tissue.

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Discussion

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Conclusions

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Academic Radiology, Volume 18, Issue 12
Journals General Radiology
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