Real‐time ALT and LDH activities determined in viable precision‐cut mouse liver slices using hyperpolarized [1‐¹³C]pyruvate—Implications for studies on biopsied liver tissues

Abstract

Precision‐cut liver slices (PCLS) are widely used in liver research as they provide a liver model with all liver cell types in their natural architecture. The purpose of this study was to demonstrate the use of PCLS for hyperpolarized metabolic investigation in a mouse model, for potential future application in liver biopsy cores. Fresh normal liver was harvested from six mice. 500 μm PCLS were prepared and placed in a 10 mm NMR tube in an NMR spectrometer and perfused continuously. 31P spectra were acquired to evaluate the presence of adenosine triphosphate (ATP) and validate viability in all samples. Hyperpolarized [1‐13C]pyruvate was flushed into the NMR tube in the spectrometer. Consecutive 13C NMR spectra were acquired immediately after the injection using both non‐selective (five injections, two livers) and selective RF excitation (six injections, three livers). The 31P spectra showed the characteristic signals of ATP, confirming the viability of the PCLS for more than 2.5 h in the spectrometer. After each of the [1‐13C]pyruvate injections, both [1‐13C]lactate and [1‐13C]alanine signals were detected. Selective RF excitation aimed at both [1‐13C]lactate and [1‐13C]alanine enabled better visualization and quantification of the metabolic activity. Using this acquisition approach only the newly formed metabolites are observed upon excitation, and their intensities relative to those of hyperpolarized pyruvate enable quantification of metabolite production rates. This rate of lactate and alanine production appeared to be constant throughout the measurement time, with alanine production about 2.3 times higher than lactate. In summary, the viability of PCLS in an NMR spectrometer was demonstrated and hyperpolarized [1‐13C]pyruvate metabolism was recorded. This study opens up the possibility of evaluating alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) activities in human liver biopsies, while preserving the tissue architecture and viability. In healthy, well‐perfused liver slices the ratio of ALT to LDH activity is about 2.3.

Differentiation of Heterogeneous Mouse Liver from HCC by Hyperpolarized 13C Magnetic Resonance

“…The LDH/ALT activities ratio determined here is, in principle, a parameter that is translational to in vivo studies and clinical studies, provided that the product-selective saturating excitation acquisition approach [21,33] can be implemented in vivo. The ability to detect only newly synthesized hyperpolarized metabolites in each repetition time enables this enzyme activity quantification.…”

“…Liver metabolism was studied using hyperpolarized substrates in various pre-clinical models [18][19][20][21][22][23][24]. Previous pre-clinical studies on HCC using hyperpolarized metabolites include a couple of studies on orthotopically implanted McA-RH7777 cells in rats.…”

“…In all of the studies cited above, the absolute LDH/ALT enzyme activities ratio was not determined. Such an absolute determination of enzyme activities ratio was made possible recently with the product-selective saturation excitation approach [21,33] which was applied here. Therefore, due to the complexity of interpreting enzyme activities ratios from hyperpolarized metabolite signals, these prior results cannot be directly compared to the current results except for the presence of these enzyme activities in HCC and liver tissues.…”

“…ALT is considered one of the most liver-specific enzymes in serum laboratory tests [14] as its serum level is one of the most important tests for evaluation of liver injury [15,16]. Both LDH and ALT activities can be monitored by following the metabolism of hyperpolarized [1- 13 C]pyruvate in liver tissues [17][18][19][20][21][22][23][24][25][26][27]. While LDH activity may be correlated to malignant processes, ALT activity may reflect the healthy liver physiology.…”

“…MDR2-KO is a well-established model of chronic inflammatory liver disease in which heterogeneous liver inflammation and fibrosis, and HCC tumors develop spontaneously [28][29][30]. Mouse liver tissues were investigated here ex vivo, in the form of precision cut liver slices (PCLS) [31,32], using a recently developed methodology for studying hyperpolarized substrate metabolism in these isolated liver tissues in well-perfused conditions [21]. In addition, we have used a unique acquisition approach that is based on hyperpolarized product selective saturating excitations [33] to determine the absolute value of the enzyme activities ratio in real-time, in the living cells.…”