Human Liver Biopsy Samples Research Articles

Phagocytosis of Apoptotic Bodies by Hepatic Stellate Cells Occurs in Vivo and Is an Important Mechanism in Liver Fibrogenesis

Hepatic stellate cells (HSC) are able to phagocytose apoptotic bodies (AB) in vitro, and this results in a fibrogenic response with up-regulation of TGF-β1 and collagen I gene expression. We have previously shown that phagocytosis induces activation of the NADPH oxidase, with generation of superoxide; however, the exact effect of this on fibrogenic signaling is not known. Therefore, our aims were to (1) demonstrate the role of NADPH oxidase in up-regulation of TGF-β1 and procollagen α 1 (I) gene expression, and (2) visualize phagocytic HSC in vivo, in livers undergoing fibrogenesis. Methods: LX-1 cells were exposed to ABs in the presence or absence of NADPH oxidase inhibitor, diphenylene iodonium (DPI, 20 μM). Total RNA was extracted after 48 hours, and quantitative real-time PCR was performed to evaluate TGF-β1 and procollagen α 1 (I) mRNA expression. The copy numbers were normalized to the expression of GAPDH. For the in vivo studies, we used livers from bile duct ligated (BDL) rats, micropigs diets containing 40% of calories as ethanol for 14 weeks, and human liver biopsy samples with recurrent post-transplant hepatitis C. All liver sections were processed for electron microscopy (EM). Results: Our PCR studies demonstrated that expression of procollagen α 1 (I) significantly increased (2.7-fold) in cells phagocytosing ABs, while adding DPI almost entirely prevented this increase. The expression of TGF-β1 also significantly increased (1.94-fold) in cells exposed to ABs, and this was only partially inhibited by DPI. The EM studies in the 3 models of fibrogenesis clearly revealed the presence of myofibroblast-type cells with intracellular ABs, and abundant microfilaments in the same cells. Conclusions: (1) Phagocytosis of ABs by HSC induced up-regulation of procollagen α 1 (I), and this was prevented by inhibition of NADPH oxidase. Phagocytosis also induced up-regulation of TGF-β1, however inhibition of NADPH oxidase did not prevent this effect, suggesting that different signaling pathways linking phagocytosis of ABs to fibrogenesis. (2) Our in vivo, EM studies in fibrogenesis of different etiologies (cholestasis, ethanol, hepatitis C), and in different species, clearly demonstrated that HSC are phagocytosing ABs in vivo. These results strongly suggest that phagocytosis of ABs by HSC may represent an important mechanism contributing to HSC activation during fibrogenesis.

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

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.

A Novel Immunocompetent Rat Model of HCV Infection and Hepatitis

Hepatitis C virus (HCV) infects millions of people worldwide. Therapy is limited, and treatment does not produce a sustained response in the majority of patients. Development of new agents has been hampered by the lack of a convenient animal model. The aim of this study was to determine whether an immunocompetent rat, tolerized and transplanted with a human hepatoma cell line (Huh 7 cells), could be used to sustain an HCV infection. Fetal rats were tolerized in utero with 10(5) Huh 7 cells. One day after birth, rats were transplanted with 5 x 10(6) Huh 7 cells and, a week later, inoculated with HCV, genotype 1. In tolerized, transplanted, and HCV-infected rats, Huh 7 cells were found in the liver, and HCV viral replication was detected by the presence of negative strand HCV RNA. HCV levels in serum were measured at 11,000 copies/mL at week 4, peaked at 22,500 copies/mL by week 12. In tolerized, transplanted, inoculated rats, but not controls, serum alanine aminotransferase (ALT) values increased to 60 IU/L by week 4 and reached a peak of approximately 120 IU/L by week 13. Histology showed foci of mononuclear infiltrates in portal and central regions. HCV-inoculated immunocompetent rats tolerized and transplanted with Huh 7 cells support HCV gene expression, viral replication, and develop biochemical and histologic evidence of hepatitis.

Ultrasonography and element analysis of human liver biopsy samples

Introduction: In a previous study ultrasonography (US) was correlated with the results of element analysis of human liver biopsy samples. Total reflexion X-ray fluorescence spectrometry (TXRF) was previously successfully used for this purpose. Nevertheless, those elements whose concentration was under 1µg/g could only be detected with less reliability, or even were under the detection limit.

Evaluation of hepatic histology by near-infrared confocal microscopy: A pilot study

Liver biopsy is a necessary procedure in establishing the tissue diagnosis of many liver conditions and often guides therapeutic strategies. Current histopathologic techniques are either time-consuming or tissue-destroying; hence the potential need for a fast and nondestructive imaging technique of unfixed human liver. This pilot study evaluates the use of near-infrared reflectance confocal microscopy (CM) in the study of human liver histopathology. Without cutting or staining the tissue, CM provides images of bulk parenchyma showing cellular and subcellular detail and depicting morphologic features of hepatic parenchyma in both diseased and nondiseased states. This article presents a series of 12 human liver biopsy samples, providing an overview on the potential of this technique in assessing common findings from light microscopy. HUM PATHOL 33:975-982. Copyright 2002, Elsevier Science (USA). All rights reserved.

Human phenol sulfotransferases SULT1A2 and SULT1A1: Genetic polymorphisms, allozyme properties, and human liver genotype–phenotype correlations

Phenol sulfotransferases (PSTs or phenol SULTs) catalyze the sulfate conjugation of phenolic drugs, xenobiotics, and monoamines. Two human PST isoforms have been defined biochemically, a thermostable (TS), or phenol-preferring, and a thermolabile (TL), or monoamine-preferring form. Pharmacogenetic studies showed that levels of both TS PST activity and TS PST thermal stability (an indirect measure of variation in amino acid sequence) in the platelet were regulated by genetic polymorphisms. Subsequent molecular genetic experiments revealed the existence of three human PST genes, two of which, SULT1A1 and SULT1A2, encode proteins with “TS PST-like” activity. We recently reported common nucleotide polymorphisms for SULT1A1 that are associated with variations in platelet TS PST activity and thermal stability. In the present experiments, we set out to determine whether functionally significant DNA polymorphisms also might exist for SULT1A2, to compare the biochemical properties of all common allozymes encoded by SULT1A2 and SULT1A1, and to study phenol SULT genotype–phenotype correlations in the human liver. We phenotyped 61 human liver biopsy samples for TS PST thermal stability and activity. The open reading frames of SULT1A2 and SULT1A1 then were amplified with the polymerase chain reaction and sequenced for each of these hepatic tissue samples. We observed 13 SULT1A2 alleles that encoded 6 allozymes. These alleles were in linkage disequilibrium with alleles for SULT1A1. Biochemical characterization of common allozymes encoded by both genes suggested that SULT1A1 was primarily responsible for “TS PST phenotype” in the human liver. In summary, both SULT1A2 and SULT1A1 have a series of common alleles encoding enzymes that differ functionally and are associated with individual differences in phenol SULT properties in the liver.

Human nicotinamide N-methyltransferase pharmacogenetics

Nicotinamide N-methyltransferase (NNMT) catalyses the N-methylation of nicotinamide and structurally related pyridines. NNMT enzymatic activity in human liver varies over a five-fold range with a bimodal frequency distribution - raising the possibility of regulation by a genetic polymorphism. We set out to characterize molecular genetic mechanisms that might be involved in the regulation of individual variation in human liver NNMT activity. After Northern blot analysis confirmed that NNMT is highly expressed in the liver, eight human hepatic biopsy samples, four each with 'low' or 'high' levels of activity, were used to perform quantitative Western blot analysis. There was a highly significant correlation (r(s) = 0.96, P < 0.0001) between NNMT activity and immunoreactive protein in these samples. We next determined that a potent promoter was located within the initial 700 bp of the 5'-flanking region of the human NNMT gene. That gene consists of 3 exons, with an initial 1240 bp intron and a second intron that is approximately 14 kb in length. We subsequently isolated DNA from 27 human liver biopsy samples with low, intermediate or high levels of NNMT activity. The three exons, all 1240 bp of intron 1 and approximately 700 bp of the 5'-flanking region of the NNMT gene were amplified from each of these samples with the polymerase chain reaction, followed by DNA sequencing to identify genetic polymorphisms that might correlate with 'NNMT phenotype'. No single nucleotide polymorphisms (SNPs) or insertion/deletion events were detected within either the exons or 5'-flanking regions of NNMT for these 27 samples. Although there were eight SNPs within intron 1, none were systematically related to level of NNMT activity. These results indicate that the exons and 5'-flanking region of the NNMT gene display little or no sequence variation. Therefore, polymorphisms within these areas of the gene are unlikely to be related to wide individual variations in the level of this enzyme activity in the human liver.

Interleukin-8 and hIRH (SDF1-alpha/PBSF) mRNA expression and histological activity index in patients with chronic hepatitis C.

Recombinant human intercrine reduced in hepatomas (hIRH)/stromal cell-derived factor 1 (SDF1-alpha)/pre-B-cell growth-stimulating factor (PBSF), a new chemokine, exhibits an in vitro chemotaxis to neutrophils and a mixed in vivo chemotactic activity to neutrophils, lymphocytes, and monocytes in a rat intradermal injection model. We have investigated the messenger RNA (mRNA) expression of interleukin-8 (IL-8) and hIRH, in chronic hepatitis C of differing severity. Levels of expression of IL-8 and hIRH mRNA obtained from 37 human liver biopsy samples were measured by reverse-transcription and semiquantitative polymerase chain reaction (RT-PCR) amplification. We examined the correlation between mRNA expression and components of the histological activity index (HAI). Patients with HAI > or = 8 had a significantly higher corrected IL-8 mRNA expression ratio (0.24 +/- 0.13 [mean +/- SD]; n = 20) than those with HAI < or = 7 (0.05 < or = 0.03; n = 17; P < .0001). Additionally, IL-8 mRNA expression was strongly associated with the severity of portal inflammation (PI) (high PI vs. low PI, 0.22 +/- 0.14 vs. 0.05 +/- 0.04; P < .0001) and with the presence of bile duct lesions (0.29 +/- 0.15 vs. 0.11 +/- 0.1; P < .01). In contrast, hIRH mRNA expression was not associated with the total HAI, any components of the HAI, or bile duct inflammation or injury. These results suggest that hIRH, although having the -CXC-, alpha chemokine motif, and exhibiting in vivo and in vitro inflammatory activity as does IL-8, plays a different role from IL-8 in hepatic inflammation and injury. IL-8 expression is directly associated with inflammation in patients with chronic hepatitis C, while hIRH expression does not correlate with histopathological severity of inflammation.

Immunohistochemical Determination of Hepatic Cytochrome P-4502E1 in Formalin-Fixed, Paraffin-Embedded Sections

Cytochrome P-4502E1 (2E1) is inducible by chronic ethanol consumption that results in enhanced activation of anesthetics and commonly used drugs (such as acetaminophen) to hepatotoxins. Therefore, assessment of hepatic 2E1 is needed in prescribing these drugs for the management of alcoholic patients. Currently, measurement of 2E1 requires either immunohistochemistry on frozen sections or Western blot (WB) analysis of homogenized tissue in excess of that needed for pathology. To obtain a more widely applicable method, we developed a procedure to detect 2E1 by immunohistochemistry in formalin-fixed, paraffin-embedded liver biopsies obtained routinely for diagnosis. Data were collected from rats fed ethanol-containing or control liquid diets for 3 weeks. lmmunostaining was performed using anti-human rabbit 2E1 antibody as the primary antibody, and the immunoreaction was detected by the avidin-biotin immunoperoxidase method after treating sections with target unmasking fluid, an antigen retrieval buffer that enhanced the staining of 2E1. In control rats, 2E1 staining was weak and perivenular. After ethanol feeding, it showed a lobular gradient, strongest perivenular and weakest periportal, similar to that seen in frozen sections. The staining intensity was scored as: 0 (no staining) to 3 (strong staining). The zonal staining was scored as follows: 1 = perivenular zonal staining, 2 = midzonal, and 3 = panlobular. With the product of the two scores, a significant difference was found between alcohol-fed and control rats (5.1 ± 0.3 vs. 0.8 ± 0.2, p < 0.001). 2E1 assessments by WB were also significantly different for these rat pairs (68.5 ± 2.1 vs. 7.9 ± 0.8 arbitrary units/mg protein, p < 0.001), with a parallel increase of immunostaining scores and WB measurement of 2E1 content. This immunohistochemical method was then validated in 14 paraffin-embedded percutaneous human liver biopsy samples. In livers of nonalcoholics, 2E1 staining was seen in the perivenular zone only, whereas in samples of alcoholics, the staining was perivenular to midzonal and sometimes periportal. A significant correlation between the zonal staining scores (rs= 0.67, p < 0.005) or intensity ± zonal staining scores (rs= 0.79, p < 0.001) and WB analysis was found. The immunohistochemical assessments of 2E1 expression in formalin-fixed, paraffin-embedded sections from livers of alcoholics was found to correlate with WB analysis, and lobular distribution was consistent with that seen in frozen sections. The proposed method should therefore be useful for the assessment of 2E1 content in paraffin-embedded liver samples, thereby aiding in the management of heavy drinkers.

Immunohistochemical Determination of Hepatic Cytochrome P‐4502E1 in Formalin‐Fixed, Paraffin‐Embedded Sections

Cytochrome P-4502E1 (2E1) is inducible by chronic ethanol consumption that results in enhanced activation of anesthetics and commonly used drugs (such as acetaminophen) to hepatotoxins. Therefore, assessment of hepatic 2E1 is needed in prescribing these drugs for the management of alcoholic patients. Currently, measurement of 2E1 requires either immunohistochemistry on frozen sections or Western blot (WB) analysis of homogenized tissue in excess of that needed for pathology. To obtain a more widely applicable method, we developed a procedure to detect 2E1 by immunohistochemistry in formalin-fixed, paraffin-embedded liver biopsies obtained routinely for diagnosis. Data were collected from rats fed ethanol-containing or control liquid diets for 3 weeks. Immunostaining was performed using anti-human rabbit 2E1 antibody as the primary antibody, and the immunoreaction was detected by the avidin-biotin immunoperoxidase method after treating sections with target unmasking fluid, an antigen retrieval buffer that enhanced the staining of 2E1. In control rats, 2E1 staining was weak and perivenular. After ethanol feeding, it showed a lobular gradient, strongest perivenular and weakest periportal, similar to that seen in frozen sections. The staining intensity was scored as: 0 (no staining) to 3 (strong staining). The zonal staining was scored as follows: 1 = perivenular zonal staining, 2 = midzonal, and 3 = panlobular. With the product of the two scores, a significant difference was found between alcohol-fed and control rats (5.1 +/- 0.3 vs. 0.8 +/- 0.2, p < 0.001). 2E1 assessments by WB were also significantly different for these rat pairs (68.5 +/- 2.1 vs. 7.9 +/- 0.8 arbitrary units/mg protein, p < 0.001), with a parallel increase of immunostaining scores and WB measurement of 2E1 content. This immunohistochemical method was then validated in 14 paraffin-embedded percutaneous human liver biopsy samples. In livers of nonalcoholics, 2E1 staining was seen in the perivenular zone only, whereas in samples of alcoholics, the staining was perivenular to midzonal and sometimes periportal. A significant correlation between the zonal staining scores (rs = 0.67, p < 0.005) or intensity x zonal staining scores (rs = 0.79, p < 0.001) and WB analysis was found. The immunohistochemical assessments of 2E1 expression in formalin-fixed, paraffin-embadded sections from livers of alcoholics was found to correlate with WB analysis, and lobular distribution was consistent with that seen in frozen sections. The proposed method should therefore be useful for the assessment of 2E1 content in paraffin-embedded liver samples, thereby aiding in the management of heavy drinkers.

Simultaneous analysis of cyclin and oncogene expression using multiple monoclonal antibody immunoblots.

Cell dysfunction or dysregulation in cancer generally results from complex gene interactions, numerous cellular events and environmental influences which modify gene expression or post-translational protein modifications. Genetic analysis in itself cannot always predict or diagnose multigenic diseases. The major technical difficulty is thus to detect, identify and measure simultaneously the expression of several genes and the post-translational modifications of their products. In order to progress to this direction, this paper describes a simple immunoblot method using several monoclonal anti-bodies to simultaneously analyze oncogene expression and cell cycle specific checkpoints in patient solid biopsies and transformed cell lines. One mg of normal human liver biopsy and HEPG2 (hepatoblastoma-derived cell line) protein samples have been separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were stained with amido black, scanned and tested separately with the nine monoclonal antibodies p53, c-myc, PCNA, MEK1, pan-ras, Cip1, Cdc2, Kip1, and TCTP. The nine antibodies of interest were then combined to form a mixture, and simultaneously used as the primary antibodies. This antibody mixture simultaneously detected the nine proteins of interest on both samples and it demonstrated the extensive expression changes and the presence of various isoforms most likely due to post-translational modifications of gene products.

Human dehydroepiandrosterone sulfotransferase pharmacogenetics: quantitative Western analysis and gene sequence polymorphisms.

Dehydroepiandrosterone sulfotransferase (DHEA ST) catalyzes the sulfation of DHEA and other hydroxysteroids. DHEA ST enzymatic activity in individual human liver biopsy samples has been shown to vary over a five-fold range, and frequency distribution histograms are bimodal, with approximately 25% of subjects included in a high activity subgroup. We set out to characterize the molecular basis for variation in human liver DHEA ST activity. The first step involved performing quantitative Western analysis of cytosol preparations from 92 human liver samples that had been phenotyped with regard to level of DHEA ST enzymatic activity. There was a highly significant correlation (r(s) = 0.635, P < 0.0001) between levels of DHEA ST activity and immunoreactive protein. We next attempted to determine whether the expression of DHEA ST might be controlled, in part, by a genetic polymorphism. DNA was isolated from three "low" and three "high" DHEA ST activity liver samples. Exons and the 5'-flanking region of the DHEA ST gene (STD) were amplified for each of these samples with the polymerase chain reaction (PCR). When compared with "wild type" STD sequence, some of the samples contained a T --> C transition at DHEA ST cDNA nucleotide 170, located within exon 2, resulting in a Met 57 --> Thr change in amino acid. Other samples contained an A --> T transversion at nucleotide 557 within STD exon 4 that resulted in a Glu 186 --> Val change. STD exons 2 and 4 were then sequenced for DNA isolated from an additional 87 liver samples that had been phenotyped with regard to level of DHEA ST enzymatic activity. The allele frequency for the exon 2 polymorphism in these samples was 0.027, whereas that for the exon 4 polymorphism was 0.038, but neither polymorphism was systematically related to the level of enzyme activity in these samples. Transient expression in COS-1 cells of cDNA that contained the nucleotide 170 and 557 polymorphisms, either separately or together, resulted in decreased expression of both DHEA ST enzymatic activity and level of immunoreactive protein, but only when the nucleotide 557 variant was present. Identification of common genetic polymorphisms within STD will now make it possible to test the hypothesis that those polymorphisms might alter in vivo expression and/or function of this important human steroid-metabolizing enzyme.

Morphometric characteristics of human hepatocellular peroxisomes in alcoholic liver disease.

Hepatocellular peroxisomes harbor one of the metabolic pathways for ethanol metabolism (i.e., catalase in the presence of H2O2-generating enzymes). We studied the morphometric characteristics of these organelles in 26 biopsy samples of patients with different alcohol-induced lesions (12 with steatosis, 5 with hepatitis, and 9 with cirrhosis) and compared the findings with those obtained in seven control livers. All 33 human liver biopsy samples were stained for catalase activity to facilitate peroxisomal identification. Morphometric analysis of the peroxisomes was performed on calibrated electron micrographs. The numerical density of the peroxisomes was significantly increased to 183%, whereas the mean peroxisomal diameter (dcircle) revealed a significant decrease to 89%. This resulted in a normal volume density of the peroxisomal compartment, whereas the surface density was significantly induced. Peroxisomal shape was not different between alcoholic and control livers. When alcoholic livers were divided into three subgroups according to histopathological findings, similar morphometric results were obtained when compared with control livers, although significantly was sometimes lost. No differences in peroxisomal characteristics were found among alcoholic subgroups. The mean peroxisomal diameter per human liver (alcoholic and control) was inversely correlated to the numerical density. It is concluded that the peroxisomal adaptation in human alcoholic liver is such as to create an efficient environment for a presumably increased peroxisomal metabolism.

Measurement of mRNA by quantitative PCR with a nonradioactive label.

This report describes the development of a method to measure mRNA in small samples of human tissue by the polymerase chain reaction with a nonradioactive label. In this method RNA is reverse-transcribed in the presence of a control RNA, and subsequently amplified by the polymerase chain reaction during which a nonradioactive label (digoxigenin-11-dUTP) is incorporated. Gel blotting and immunological detection of digoxigenin followed by a chemiluminescent reaction provide an intense signal on film. This allows the detection and quantitation of 3-hydroxy-3-methylglutaryl (HMG) CoA reductase mRNA in 12 ng of RNA. We demonstrate that this is a sensitive and reproducible method, and that quantitation is linear with respect to the amount of mRNA present. The application of this method to the measurement of low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA levels in circulating peripheral blood mononuclear cells and human liver biopsy samples is discussed. The use of chemiluminescent reagents instead of radioactive labels allows this procedure to be performed safely in laboratories not equipped for radioactivity.

Glucuronidation of 7‐Hydroxy‐4‐methylcoumarin by Microsomes from Normal and Diseased Human Livers

Glucuronidation of 7-hydroxy-4-methylcoumarin (7-H-4-MC) by microsomal UDP-glucuronosyltransferases from 12 human liver biopsy samples was studied. Glucuronidation rates were highest for livers with mild cholestasis while those for livers with mild fatty changes were similar to normal livers. The lowest rates were exhibited by livers showing primary or metastatic carcinoma on histological examination. Thus, glucuronidation of not only bile acids but also of other substrates such as 7-H-4-MC may be elevated in cholestasis. These results are also consistent with other studies stating that glucuronidation might be spared in certain mild hepatic diseases.

The ontogeny of human hepatic microsomal glucose-6-phosphatase proteins

We have studied 250 human liver biopsy samples to determine the ontogeny of the microsomal glucose-6-phosphatase (EC 3.1.3.9) system. Human hepatic glucose-6-phosphatase enzyme activity develops at 11 weeks' gestation and slowly increases to approximately 10% of adult activity at term. In the first week after birth, activity rises to adult values. Increases in enzyme activity coincide with increasing concentrations of the glucose-6-phosphatase enzyme protein. The phosphate/pyrophosphate transport protein (T2) of the human hepatic glucose-6-phosphatase complex develops at a different rate from that of the enzyme. Our study shows that the development of rat and human glucose-6-phosphatase activities are completely different. We conclude that deficiencies of the proteins in the microsomal glucose-6-phosphatase complex can be diagnosed with much more certainty perinatally than prenatally.

Human liver nicotinamide N-methyltransferase: Ion-pairing radiochemical assay, biochemical properties and individual variation

Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide, pyridine, and structurally related compounds. The products of this reaction are positively charged pyridinium ions that cannot be removed from aqueous solution by simple organic solvent extraction. We developed an assay for human liver NNMT based on the extraction of the positively charged reaction product into 60% isoamyl alcohol in toluene in the presence of the ion-pairing reagent 1-heptanesulfonic acid. Nicotinamide was the methyl acceptor for the reaction, and [ 14C-methyl]- S-adenosyl- l-methionine (Ado-Met) served as the methyl donor. Apparent K m values of human liver NNMT for nicotinamide and Ado-Met were 347 and 1.76 μmol/l, respectively. The product of the reaction was identified as N 1-methylnicotinamide (NMN) by high performance liquid chromatography. NNMT activity was inhibited by the reaction products, NMN and S-adenosyl- l-homocysteine. NNMT activity was not affected by inhibitors of other methyltransferases including Ca 2+, SKF 525A and 3,4-dimethoxy-5-hydroxybenzoic acid. Individual variation in NNMT activity was studied by measuring hepatic enzyme activities in 163 human liver biopsy samples obtained during clinically-indicated surgery. The average NNMT activity in these samples was 51.5 ± 32.5 U per mg protein (mean ± SD), and there was not a significant correlation of enzyme activity with patient age or with the time of storage of the biopsy samples at −80°C. The distribution of activities was bimodal, and approximately 26% of the samples were included in a subgroup with high NNMT activity. It will now be possible to test the hypothesis that individual differences in hepatic NNMT activity might be related to variation in the N-methylation of pyridine compounds and to individual differences in either toxicity or the therapeutic efficacy of such compounds.

Evidence for expression in human liver of halothane-induced neoantigens recognized by antibodies in sera from patients with halothane hepatitis.

Previous investigations have shown that antibodies in sera from patients with halothane hepatitis recognize neoantigens, expressed in livers of halothane-exposed rabbits and rats, which consist of a halothane metabolite bound covalently to specific microsomal proteins. These studies have suggested that the patients' antibodies may play a role in the pathogenesis of the hepatitis. In the present investigation, human liver biopsy samples were analyzed using an immunoblotting method to seek evidence for expression of halothane-induced neoantigens in humans. Sera from four patients with halothane hepatitis, which recognized halothane-induced rabbit liver neoantigens of 100, 76 and 57 kD, reacted strongly with antigens of very similar molecular weights that were expressed in livers from two patients who had died of cardiac failure following recent anesthesia with halothane. The antigens were not expressed in normal human liver or in livers from three patients who died of cardiac failure following anesthesia with agents other than halothane. The human antigens were not recognized by antibodies present in various control sera. Recognition of the 100- and 76-kD human antigens by the patients' antibodies was greatly reduced by absorption of sera with liver microsomes from halothane-exposed rabbits, but not by absorption of sera with control rabbit microsomes. These results indicate that humans exposed to halothane express liver neoantigens which are analogous to the halothane metabolite-protein neoantigens characterized previously in halothane-exposed animals.

Relationship between unusual hepatic acyl coenzyme A profiles and the pathogenesis of Reye syndrome.

This study examines the relationship between impaired fatty acid oxidation and the pathogenesis of Reye syndrome. We present a hypothesis proposing that many clinical signs of this childhood disease are caused by accumulation of unusual acyl CoA esters, precursors to deacylated metabolites found in the patients' blood and urine. A new method was developed to measure acyl CoA compounds in small human liver biopsy samples, offering several advantages over previous techniques. A major finding was an accumulation in Reye syndrome patients of short- and medium-chain acyl CoA intermediates of fatty acid and branched-chain amino acid oxidation. These metabolites included octanoyl, isovaleryl, butyryl, isobutyryl, propionyl, and methylmalonyl CoA esters. The findings were explained in a model of hepatic fatty acid oxidation involving three interrelated pathways: mitochondrial beta-oxidation, peroxisomal beta-oxidation, and omega-oxidation in the endoplasmic reticulum. The results suggest that pathogenesis in Reye syndrome stems from generalized mitochondrial damage resulting in accumulation of acyl CoA esters. High levels of these compounds lead to inhibition of mitochondrial pathways for ureogenesis, gluconeogenesis, and fatty acid oxidation. The inhibited pathways, in turn, could cause the hyperammonemia, hypoglycemia, and hypoketonemia observed in patients. The model also explains underlying biochemical differences between patients with Reye syndrome and medium-chain acyl CoA dehydrogenase deficiency, another disorder of fatty acid metabolism. Acetyl CoA levels, in the latter disease, were dramatically decreased, compared with both human controls and Reye syndrome patients.

Multiple NMR T2 relaxation values in human liver tissue.

Twenty-one human hepatic liver biopsy samples were evaluated by nuclear magnetic resonance spectroscopy. This spectroscopy was performed by obtaining multiple and not single T2 values (T2-long and T2-short). Neoplastic tissue had greater T2-long and T2-short values than diffuse liver disease. The T2-short correlated, r = 0.947 (p less than 0.01), with the percentage of cancer cellularity within the tissue specimens. There was no correlation between the multiple T2 values and the degree of fibrosis and inflammation. Minimal correlation was noted between the T2-long and percentage of fat within the diffuse disease (cirrhotic) specimens, r = 0.636 (p less than 0.05). The possible reasons for the above findings are discussed.