Metabolic Assessment of Human Liver Transplants from Biopsy Samples at the Donor and Recipient Stages Using High-Resolution Magic Angle Spinning ¹H NMR Spectroscopy

Abstract

This work presents the first application of high-resolution magic angle spinning (HR-MAS) 1H NMR spectroscopy to human liver biopsy samples, allowing a determination of their metabolic profiles before removal from donors, during cold perfusion, and after implantation into recipients. The assignment of peaks observed in the 1H HR-MAS NMR spectra was aided by the use of two-dimensional J-resolved, TOCSY and 1H-13C HMQC spectra. The spectra were dominated by resonances from triglycerides, phospholipids, and glycogen and from a variety of small molecules including glycerophosphocholine (GPC), glucose, lactate, creatine, acetate, amino acids, and nucleoside-related compounds such as uridine and adenosine. In agreement with histological data obtained on the same biopsies, two of the six livers were found to contain high amounts of triglycerides by NMR spectroscopy, which also indicated that these tissues contained a higher degree of unsaturated lipids and a lower proportion of phospholipids and low molecular weight compounds. Additionally, proton T2 relaxation times indicated two populations of lipids, a higher mobility triglyceride fraction and a lower mobility phospholipid fraction, the proportions of which changed according to the degree of fat content. GPC was found to decrease from the pretransplant to the posttransplant biopsy of all livers except for one with a histologically confirmed high lipid content, and this might represent a biomarker of liver function posttransplantation. NMR signals produced by the liver preservation solution were clearly detected in the cold perfusion stage biopsies of all livers but remained in the posttransplant spectra of only the two livers with a high lipid content and were prominent mainly in the graft that later developed primary graft dysfunction. This study has shown biochemical differences between livers used for transplants that can be related to the degree and type of lipid composition. This technology might therefore provide a novel screening approach for donor organ quality and a means to assess function in the recipient after transplantation.

Current and future applications ofin vitromagnetic resonance spectroscopy in hepatobiliary disease

“…Further MRI studies in vivo have demonstrated a close relation between hepatic steatosis and body adiposity, and a close correlation between MRI estimation of adiposity and histological assessment in two of these patients [11] . Although studies have shown a close association between in vivo estimates and biopsies, in vitro MRS assessment of lipid content in liver biopsies by MAS MRS would allow direct comparison with histology, reducing the effect of sampling error [33] .…”

“…In a preliminary study, Duarte and colleagues used in vitro 1 H MAS MRS to assess biopsies taken at three time-points from six livers, before removal from donors, during cold perfusion and following implantation into the recipient [33] . The biopsies with the highest concentration of peaks reflecting fatty acyl chain (triglyceride) resonances were also identified as those also estimated to have the highest fat content on histological analysis.…”

“…The biopsies with the highest concentration of peaks reflecting fatty acyl chain (triglyceride) resonances were also identified as those also estimated to have the highest fat content on histological analysis. Other metabolites were identified, including glycerophosphocholine (GPC), which were reported to decrease from pre-to post-transplant [33] . However, in further studies such spectral changes need to be correlated with a range of clinical endpoints, including the premorbid clinical history of the liver donor, the pretransplantation clinical history and nutritional status of the recipient, the subsequent post-transplantation liver function tests, pre-and post-transplantation indices of nutrition in the recipient and ultimately, the final clinical outcome.…”

Metabonomic Study of Lung Cancer and the Effects of Radiotherapy on Lung Cancer Patients: Analysis of Highly Polar Metabolites by Ultraperformance HILIC Coupled with Q-TOF MS

“…Within the past few years, metabonomics has shown enormous potential for use in disease diagnosis, toxicological mechanism research and the identification of biomarkers [3][4][5][6][7][8]. Nowadays, many techniques have been employed in metabonomic studies, such as nuclear magnetic resonance (NMR) [9][10][11][12][13][14][15], gas chromatography/mass spectrometry (GC/MS) [16][17][18] and liquid chromatography/mass spectrometry (LC/MS) [19][20][21]. Most recently, a new LC/MS technology known as ultraperformance liquid chromatography (UPLC) coupled with Q-TOF MS detection has been used widely in metabonomic studies [22][23][24].…”

Analysis of human urine metabolites using SPE and NMR spectroscopy

“…With some spectral editing techniques [27] , one can also separate signals of metabolites according to their physical properties without separating samples [15,27,28] . With high resolution magic-angle spinning NMR and in vivo magnetic resonance techniques, the metabolites in biological tissues can also be analyzed which are exemplified in a number of studies of biological tissues such as the liver [9,19,[28][29][30] and intestine [31,32] .…”