Sample preparation method for isolation of single-cell types from mouse liver for proteomic studies

It becomes increasingly clear that separation of pure cell populations provides a uniquely sensitive and accurate approach to protein profiling in biological systems and opens up a new area for proteomic analysis. The method we described could simultaneously isolate population of hepatocytes (HCs), hepatic stellate cells (HSCs), Kupffer cells (KCs) and liver sinusoidal endothelial cells (LSECs) by a combination of collagenase-based density gradient centrifugation and magnetic activated cell sorting with high purity and yield for the first time. More than 98% of the isolated HCs were positive for cytokeratin 18, with a viability of 91%. Approximately 97% of the isolated HSCs expressed glial fibrillary acidic protein with a viability of 95%. Nearly 98% of isolated KCs expressed F4/80 with a viability of 94%. And the purity of LSECs reached up to 91% with a viability of 94%. And yield for HCs, HSCs, LSECs and KCs were 6.3, 1.3, 2.6 and 5.0 million per mouse. This systematic isolation method enables us to study the proteome profiling of different types of liver cells with high purity and yield, which is especially useful for sample preparation of Human Liver Proteome Project.     

小鼠Kupffer细胞分离及细胞培养

目的 采用在体胶原酶灌注、不连续密度梯度离心、选择性贴壁3步法分离Kupffer细胞（Kupffer cells,KCs）,探讨其在分离小鼠KCs的应用及其对KCs生物活性的影响.方法 根据原位灌注和梯度离心方法不同随机分为4组：无胶原酶原位灌注＋3层梯度离心组（A）、无胶原酶原位灌注＋双层梯度离心组（B）、胶原酶原位灌注＋3层梯度离心组（C）和胶原酶原位灌注＋双层梯度离心组（D）.采用F4/80（BM8）免疫染色及吞墨实验判断细胞纯度和功能、台盼蓝拒染实验判断细胞的活力,探讨不同方法KCs分离的效果及细胞活性.结果 刚分离的KCs细胞近似圆形,接种l h后收获细胞纯度较高,但细胞得率相对较低.培养4 h后KCs得率相对较高,培养28 d仍能存活.免疫荧光可显示分离的为KCs,台盼蓝染色显示各组细胞的活力均在90 %左右,在体胶原酶灌注和双层梯度离心可以增加KCs的得率,双层梯度离心法可以增加分离KCs的纯度.结论 在体胶原酶灌注对提高KCs得率较为重要,在体胶原酶灌注、不连续密度梯度离心、选择性贴壁3步法分离小鼠KCs的的方法简便、高效、稳定,培养的KCs具有良好的细胞生物学性状.     

Separation of hepatocytes from suspensions of mouse liver cells using programmed gradient sedimentation in gradients of ficoll in tissue sulture medium

Liver cells were obtained in suspension using a solution of lysozyme in Joklik's modification of minimum essential medium. Hepatocytes were separated in 74.2 ± 12.9% purity from other liver cells having different densities using isopycnic centrifugation, in 97.1 ± 1.9% purity from other liver cells having different diameters using velocity or rate-zonal centrifugation. A previously reported computer integration of the differential sedimentation equation was employed in determining the gradient design and the speed and duration of centrifugation which would permit purification of hepatocytes from other liver cells. More than 98% of the hepatocytes separated by velocity sedimentation excluded trypan blue. Velocity sedimentation is superior to isopycnic centrifugation for the separation of hepatocytes from liver cell suspensions because it gives more highly purified hepatocytes and because it requires lower centrifugal forces for shorter periods of time.     

4 in 1: Antibody‐free protocol for isolating the main hepatic cells from healthy and cirrhotic single rat livers

Liver cells isolated from pre‐clinical models are essential tools for studying liver (patho)physiology, and also for screening new therapeutic options. We aimed at developing a new antibody‐free isolation method able to obtain the four main hepatic cell types (hepatocytes, liver sinusoidal endothelial cells [LSEC], hepatic macrophages [HMΦ] and hepatic stellate cells [HSC]) from a single rat liver. Control and cirrhotic (CCl₄ and TAA) rat livers (n = 6) were perfused, digested with collagenase and mechanically disaggregated obtaining a multicellular suspension. Hepatocytes were purified by low revolution centrifugations while non‐parenchymal cells were subjected to differential centrifugation. Two different fractions were obtained: HSC and mixed LSEC + HMΦ. Further LSEC and HMΦ enrichment was achieved by selective adherence time to collagen‐coated substrates. Isolated cells showed high viability (80%‐95%) and purity (>95%) and were characterized as functional: hepatocytes synthetized albumin and urea, LSEC maintained endocytic capacity and in vivo fenestrae distribution, HMΦ increased expression of inflammatory markers in response to LPS and HSC were activated upon in vitro culture. The 4 in 1 protocol allows the simultaneous isolation of highly pure and functional hepatic cell sub‐populations from control or cirrhotic single livers without antibody selection.     

Centrifugal separation of cells in sputum specimens from patients with undifferentiated carcinoma

Ethanol-fixed cells in sputum from patients with undifferentiated carcinoma of the lung were separated in aqueous Ficoll using a discontinuous density gradient centrifugation technique. The selective enrichment of small cell undifferentiated (e.g., oat cell) or large cell undifferentiated carcinoma cells was achieved while removing most of the leukocytes (80-90%) and macrophages (65-75%) from specimen fractions containing the greatest relative frequencies of cancer cells. The maximum purity of small cell carcinoma cells (0.04%) occurs in moderate density (rho = 1.121 g/ml) gradient fractions and results in a 2.4-fold enrichment relative to unprocessed specimens. In contrast, the maximum purity of large cell carcinoma cells (0.22%) is obtained in very high density (rho = 1.172 g/ml) gradient fractions and results in a 1.2-fold enrichment in comparison with unprocessed specimens. Microscopic examination of Papanicolaou-stained specimen fractions reveals that these enrichments were achieved while retaining diagnostically significant cytomorphologic and tinctorial features necessary for cancer screening and diagnosis. Peak purity ranges of undifferentiated cancer cells significantly overlap comparable ranges for material from bronchogenic adenocarcinoma and squamous cell carcinoma.     

Activation, isolation, identification and culture of hepatic stem cells from porcine liver tissues

Objectives: Utility of hepatic stem cells could provide a novel solution to the severe shortage of human donor livers, for treatment of liver‐related diseases, due to their ability to proliferate and differentiate into functional hepatocytes. Porcine liver tissues also offer an alternative source from human donor livers. However, morphology, phenotype, successful isolation and culture of porcine hepatic stem cells still require much investigation. Materials and methods: In the present study, we performed partial hepatectomy to activate hepatic oval cells and developed a procedure utilizing enzymatic digestion and density gradient centrifugation to isolate and purify oval cells derived from porcine livers. We identified ovoid cells by their morphological characteristics and phenotypic properties, thereby providing definitive evidence for the presence of hepatic stem cells in porcine livers. Moreover, we established a culture system, using various growth factors, to provide nourishment for these cells. Results and conclusions: By transmission electron microscopy, oval‐shaped cells with ovoid nuclei, a high nucleus/cytoplasm ratio and few organelles were demonstrated. Flow cytometry and immunocytochemistry showed that freshly isolated oval cells expressed albumin, cytokeratin 19, alpha fetoprotein (AFP) and OV6 at high levels. Immunofluorescence revealed that porcine hepatic oval cells after culture expressed stem‐cell factor, c‐kit, Thy‐1, CK19, OV6, and AFP. Taken together, this study provides a novel insight into morphological and phenotypic characteristics of porcine hepatic stem cells. Our ability for isolation and culturing porcine hepatic stem cells offers an abundant source of cells for transplantation and tissue engineering to help alleviate liver disease.     

建立同时分离培养小鼠肝细胞及肝星状细胞的技术

目的:建立一种简便、经济、高产的同步分离培养肝细胞以及肝星状细胞的方法。方法:在参照国内外方法的基础上加以改良,首先采用肝脏原位胶原酶灌注消化的方法,获得总细胞悬液,经多次低速离心分离肝细胞;再用Nycodenz作为分离介质,通过密度梯度离心法从非实质细胞中得到肝星状细胞。通过台盼蓝染色方法鉴定细胞的活力,用倒置相差显微镜、立体显微镜、CK-18、白蛋白免疫荧光细胞化学染色对培养的肝细胞形态以及功能进行检测。使用Desmin、α-SMA免疫荧光细胞化学对肝星状细胞进行鉴定。结果:成功的在体外同步分离、培养肝细胞及肝星状细胞,肝细胞产率为5-6×107/只小鼠,两只小鼠肝星状细胞产率达1×106个。细胞存活率及纯度均可达90%。肝细胞在培养24h后呈不规则铺路石样形态,此为典型的肝细胞形态,其标志分子CK-18以及白蛋白免疫荧光染色阳性。倒置相差显微镜下可见贴壁后的肝星状细胞呈典型的星形细胞形态,且其标志分子Desmin、α-SMA免疫荧光染色阳性。结论:改良的原位灌注以及分离方法可以同时分离并且培养具有高活性和功能的肝细胞和肝星状细胞。     

Human mesenchymal stem cells towards non-alcoholic steatohepatitis in an immunodeficient mouse model

Non-alcoholic steatohepatitis (NASH) is a frequent clinical picture characterised by hepatic inflammation, lipid accumulation and fibrosis. When untreated, NASH bears a high risk of developing liver cirrhosis and consecutive hepatocellular carcinoma requiring liver transplantation in its end-stage. However, donor organ scarcity has prompted the search for alternatives, of which hepatocyte or stem cell-derived hepatocyte transplantation are regarded auspicious options of treatment. Mesenchymal stem cells (MSC) are able to differentiate into hepatocyte-like cells and thus may represent an alternative cell source to primary hepatocytes. In addition these cells feature anti-inflammatory and pro-regenerative characteristics, which might favour liver recovery from NASH. The aim of this study was to investigate the potential benefit of hepatocyte-like cells derived from human bone marrow MSC in a mouse model of diet-induced NASH. Seven days post-transplant, human hepatocyte-like cells were found in the mouse liver parenchyma. Triglyceride depositions were lowered in the liver but restored to normal in the blood. Hepatic inflammation was attenuated as verified by decreased expression of the acute phase protein serum amyloid A, inflammation-associated markers (e.g. lipocalin 2), as well as the pro-inflammatory cytokine TNFα. Moreover, the proliferation of host hepatocytes that indicate the regenerative capacity in livers receiving cell transplants was enhanced. Transplantation of MSC-derived human hepatocyte-like cells corrects NASH in mice by restoring triglyceride depositions, reducing inflammation and augmenting the regenerative capacity of the liver.     

A MODIFIED PROCEDURE FOR ISOLATION OF ASTROCYTE- AND NEURON-ENRICHED FRACTIONS FROM RAT BRAIN

Our previously published method for isolation of neurons with extensive processes (Farooq et al., 1977) has been modified to permit the isolation of both astrocyte- and neuron-enriched fractions. Rat cerebral tissue is incubated with acetylated trypsin and disrupted. The cell suspension is separated first by differential centrifugation and then by gradient centrifugation on discontinuous Ficoll gradients. The method is reproducible and is applicable equally well to immature and adult animals. The yield of astrocytes of 57% particle purity, and higher weight purity, is 4–7 × 10⁶ cells/brain, amounting to 1.5–2.0 mg of protein. The astrocytes appear to be a mixture of fibrous and protoplasmic types. The yield of neurons of 90% particle purity is 10–14 × 10⁶ cells/brain, amounting to 2.4–3.0 mg of protein. A total yield of neurons of 28–37 × 10⁶ cells/brain can be obtained at 70% purity. These preparations have been characterized by light microscopy and protein, RNA and DNA content.     

Highly purified plasma membranes from rat hepatocytes following rate-isopycnic zonal centrifugation

Plasma membranes from liver parenchymal cells were isolated by rate-isopycnic zonal centrifugation. A method is described for the Beckman size 15 zonal rotor. It involved preparation from a perfused liver of a parenchymal cell-enriched homogenate in isoosmotic sucrose. The nuclear fraction containing membranes was recovered by centrifugation. The resuspended pellet was applied on the gradient of the zonal rotor. The isolated membranes had the same isopycnic banding density as 37% sucrose (w/w). The specific activity of 5′-nucleotidase, a widely used plasma membrane marker, was 105 μmoles·(mg protein)^?1·h^?1 being enriched by a factor of 50 as compared with parenchymal cell homogenate. The plasma membrane fraction was free of the mitochondrial and lysosomal enzymes, succinate dehydrogenase and acid phosphatase. No DNA and 10 μg RNA per mg plasma membrane protein were found. The purity of the membranes and their morphological appearance were controlled by electron microscopy. The preparation consisting of large membrane sheets showed a considerable purification away from other cellular components. A comparison with similar methods indicates that plasma membranes of a higher degree of purity can be obtained from parenchymal cells.     

Isolation and characterization of biliary epithelial cells from rainbow trout liver

Summary Lectin binding and density gradient centrifugation were explored for isolating epithelial cells from trout liver. Hepatocytes exhibited preferential attachment to coverslips coated withPhaseolus vulgaris erythroagglutinin. Biliary epithelial cells attached with glycine max agglutinin; however, significant attachment of cellular debris limited the use of glycine max agglutinin. Percoll-density gradient centrifugation separated liver cells into two distinct populations with biliary cells and hepatocytes banding at densities of 1.04 and 1.09, respectively. A discontinuous gradient composed of 13% Ficoll (wt/wt) separated biliary cells from hepatocytes. The recovery of highly enriched biliary epithelial cells from trout liver using Ficoll gradients yielded approximately 8 million cells (0.1 ml packed cells) from 10 g liver. Western blot analysis demonstrated that the cytokeratin profile for extracts from biliary epithelial cell-enriched populations differ significantly from those seen with whole liver extracts or with extracts from hepatocyte-enriched populations. Ficoll-gradient purified biliary cells and hepatocytes attached to culture plates coated with trout skin extract and carried out linear incorporation of leucine into protein and thymidine into DNA for 24 h. A mixture of growth hormones (insulin, epidermal growth factor, and dexamethasone) stimulated thymidine incorporation into DNA; however, long-term culture of dividing biliary epithelial cells was not achieved. Chemical analysis of neutral and acidic glycolipids indicated that hepatocytes and biliary cells have similar glycolipid profiles with an exception in the region of GM3 mobility, which is attributable to differences in the ceramide moiety. These studies provide a starting point for further characterization of unique cell types of the trout liver that may be important in their response to toxic and carcinogenic agents.     

Simple separation of chicken gonadal primordial germ cells with and without foreign genes

Simplicity is the key element of an inexpensive technique described that is superior in performance to previous methods. It can make it the rapid method of choice to obtain reasonable yields of purified primordial germ cells (PGCs) for immediate production of germline chimeric chickens with integrated foreign genes. After Ficoll centrifugation, the purity of PGCs from gonads was 80.9+/-0.08% (mechanical) compared with 86.1+/-0.19% (enzymatic). GFP gene and lacZ-transduced chicken gonadal primordial germ cells (gPGCs) examined 72h after transduction had a transfection efficiency of approximately 61% and approximately 64%, respectively. After 10 days of G418 selection, approximately 90 and 92% of pure gPGCs did not contain other cells following this Ficoll gradient centrifugation method of preparation.     

Separation and subtyping of planarian neoblasts by density-gradient centrifugation and staining

A method has been devised to isolate neoblasts from planarians in high purity and high yield. Specimens of Dugesia polychroa and Dugesia tahitiensis were disintegrated mechanically. After several prepurification steps consisting in sequential filtering through increasingly fine meshes and differential centrifugation, the resulting cell suspension was separated by centrifugation in discontinuous density gradients formed from Percoll solutions. Isosmotic conditions were applied. Two gradients are recommended, one isopycnic four-step gradient (densities 1.03, 1.05, 1.07, 1.09) to obtain one single fraction with a high yield of neoblasts in high purity and one six-step gradient for preparing subfractions of neoblasts somewhat less pure, but in still greater amounts. This latter gradient (densities 1.04, 1.05, 1.06, 1.065, 1.07, 1.09) was run under conditions of rate zonal centrifugation and used for further subtyping of neoblasts by specific staining with azure A – eosin B. A first survey of tentative types based on morphological criteria is given. The viability of neoblasts isolated in the way described was tested in primary cell cultures. In current experiments, 6-week-old cultures had a viability of roughly 50%, with mitoses up to 1 week after the isolation of neoblasts. This revised version was published online in July 2006 with corrections to the Cover Date.     

All-In-One: Advanced preparation of Human Parenchymal and Non-Parenchymal Liver Cells

Background & Aims

Liver cells are key players in innate immunity. Thus, studying primary isolated liver cells is necessary for determining their role in liver physiology and pathophysiology. In particular, the quantity and quality of isolated cells are crucial to their function. Our aim was to isolate a large quantity of high-quality human parenchymal and non-parenchymal cells from a single liver specimen.

Methods

Hepatocytes, Kupffer cells, liver sinusoidal endothelial cells, and stellate cells were isolated from liver tissues by collagenase perfusion in combination with low-speed centrifugation, density gradient centrifugation, and magnetic-activated cell sorting. The purity and functionality of cultured cell populations were controlled by determining their morphology, discriminative cell marker expression, and functional activity.

Results

Cell preparation yielded the following cell counts per gram of liver tissue: 2.0±0.4×10⁷ hepatocytes, 1.8±0.5×10⁶ Kupffer cells, 4.3±1.9×10⁵ liver sinusoidal endothelial cells, and 3.2±0.5×10⁵ stellate cells. Hepatocytes were identified by albumin (95.5±1.7%) and exhibited time-dependent activity of cytochrome P450 enzymes. Kupffer cells expressed CD68 (94.5±1.2%) and exhibited phagocytic activity, as determined with 1μm latex beads. Endothelial cells were CD146⁺ (97.8±1.1%) and exhibited efficient uptake of acetylated low-density lipoprotein. Hepatic stellate cells were identified by the expression of α-smooth muscle actin (97.1±1.5%). These cells further exhibited retinol (vitamin A)-mediated autofluorescence.

Conclusions

Our isolation procedure for primary parenchymal and non-parenchymal liver cells resulted in cell populations of high purity and quality, with retained physiological functionality in vitro. Thus, this system may provide a valuable tool for determining liver function and disease.     

The Annexin A2-Notch regulatory loop in hepatocytes promotes liver fibrosis in NAFLD by increasing osteopontin expression

BackgroundThe mechanisms underlying the progression of liver disease from simple hepatic steatosis to advanced nonalcoholic steatohepatitis (NASH) and liver fibrosis warrant further investigation. Increased mRNA levels of Annexin A2 protein (Anxa2) have been observed in patients with NASH. However, the role of Anxa2 in NASH remains unclear.MethodsThe protein levels of Anxa2 were analyzed in the livers of mice and patients with NASH. Anxa2-knockout and -knockdown mice were generated, and NASH was induced through a high fructose, palmitate, and cholesterol (FPC) diet or methionine- and choline-deficient (MCD) diet.FindingsWe found elevated expression of Anxa2 in the livers of patients and mice with NASH. Anxa2 knockdown but not knockout ameliorated liver fibrosis in both FPC and MCD diet–fed mice. Liver-specific Anxa2 overexpression increased collagen deposition in mice fed a normal diet. Mechanistically, Anxa2 overexpression in hepatocytes promoted hepatic stellate cell activation in a paracrine manner by increasing osteopontin expression. Notch inhibition suppressed the exogenous overexpression of Anxa2-induced osteopontin and endogenous Anxa2 expression. Additionally, Anxa2 overexpression accelerated the progression of nonalcoholic fatty liver disease (NAFLD) in mice fed a high-fat diet. Moreover, Anxa2 levels were higher in NAFLD patients with advanced liver fibrosis than in those with mild liver fibrosis, as determined using the Gene Expression Omnibus database.InterpretationIn conclusion, we found increased Anxa2 expression in hepatocytes promoted liver fibrosis in NASH mice by increasing osteopontin expression. The Anxa2-Notch positive regulatory loop contributes to this process and represents a novel target for the treatment of NASH-related liver fibrosis.     

Isolation and culture of hepatic lipocytes, Kupffer cells, and sinusoidal endothelial cells by density gradient centrifugation with Stractan

A method for isolating purified populations of hepatic lipocytes, Kupffer cells, and sinusoidal endothelial cells suitable for culture, using density gradient centrifugation on the polysaccharide material Stractan is described. A nonparenchymal cell digest of liver from either normal rats or rats treated with modest doses of vitamin A is layered on a discontinuous gradient of 6, 8, 12, ind 20% Stractan; lipocytes are separated efficiently from other nonparenchymal cells and are removed from the top of the gradient. Kupffer cells and sinusoidal endothelial cells, which migrate to denser interfaces in the gradient, are further purified by differential plating and selective trypsinization, respectively. Isolated highly viable lipocytes free of contaminants adhere and spread progressively over several days in primary culture and display both intrinsic vitamin A fluorescence and positive immunostaining for desmin. Lipocytes survive for prolonged periods on plain plastic, and collagen synthesis by these cells remains relatively constant for at least 28 days. Based on serial assay of DNA content, lipocytes in primary culture proliferate, beginning 7 days after plating. Kupffer cells and sinusoidal endothelial cells isolated by Stractan density centrifugation likewise retain their typical morphologic and functional characteristics in culture; the purity of these cell isolates has been confirmed by using specific fluorescent markers. This investigation demonstrates that Stractan density gradient centrifugation is an efficient, sensitive, and reproducible method for isolating pure populations of hepatic nonparenchymal cells.     

Separation of leucocytes in the dogfish (Scyliorhinus canicula) using density gradient centrifugation and differential adhesion to glass coverslips

Summary The blood of the dogfish, S. canicula, contains several types of leucocytes, namely thrombocytes, monocytes, lymphocytes and four populations of granulocytes. Three of these granulocyte types, G1, G3 and G4, are eosinophilic while G2 is heterophilic/neutrophilic. All of the leucocyte types, with the exception of G2 granulocytes and monocytes, can be separated by means of their differential adherent properties to glass and by density gradient centrifugation. Thrombocytes, G3 and G4 granulocytes can be separated in good purity by single-step methods while G1 granulocytes and lymphocytes require a combination of density gradient centrifugation followed by adherence to glass to remove contaminating thrombocytes. Depending on the cell type, between 11–45% of cells with consistently high viability can be recovered after separation. Separated populations of the thrombocytes and granulocytes will be especially useful for studies on the role of such cell types in inflammation.     

Enrichment and characterization of clonogenic epithelial cells from adult rat liver and initiation of epithelial cell strains

Summary A highly efficient method is described for obtaining prolifertive epithelial cells from adult rat livers for the reproducible establishment of liver epithelial cell strains. When cells were isolated from livers of 10-to 15-wk-old male Fischer 344 rats by a collagenase-perfusion method, collected by centrifugation at 50×g for 5 min, and cultured in Williams' medium E containing fetal bovine serum and dexamethasone, colonies of epithelial cells different in size and morphology from hepatocytes were obtained. Sequential perfusion with collagenase and dispase yielded numerous epithelial cell colonies. When isolated cells were fractionated by differential centrifugation, the great majority of hepatocytes were sedimented at 50 ×g for 1 min, whereas many non-hepatocytic cells remiined in the supernatant and could be sedimented by a second centrifugation at 50×g for 5 min. Culture of the two fractions revealed that almost all the epithelial cell colonies were derived from cells in the non-hepatocytic cell fraction. The epithelial cells were cytochemically negative for γ-glutamyl transpeptidase activity, whereas an increase in the activity was detected in hepatocytes with duration in culture. Ultrastructural characteristics of hepatocytes were not found in the cells of newly established cell strains. These results suggest that adult rat liver epithelial cells propagable in culture were derived from a cell type other than the hepatocyte.     

Identification of metallothionein in parenchymal and non-parenchymal liver cells of the adult rat.

Parenchymal and non-parenchymal cells were isolated from the livers of control, starved, Zn2+-injected and Cd2+-injected rats. Parenchymal cells were prepared by differential centrifugation after perfusion of the liver with collagenase. Non-parenchymal cells were separated from parenchymal cells by unit-gravity sedimentation and differential centrifugation. Yields of 2 x 10(8) non-parenchymal cells with greater than 95% viability and less than 0.2% contamination with parenchymal cells were obtained without exposing cells to Pronase. Metallothioneins-I and -II were identified in parenchymal cells and non-parenchymal cells from Zn2+-treated rats. The metallothionein contents of parenchymal cells, non-parenchymal cells and intact liver were quantified by a competitive 203Hg-binding assay. Administration of heavy-metal salts significantly increased the metallothionein content of both cell populations, although the concentration of the protein was approx. 2.5-fold greater in parenchymal cells than in non-parenchymal cells. Overnight starvation increased the metallothionein content of parenchymal cells without altering that of non-parenchymal cells. The potential significance of this differential response by different liver cell types with regard to the influence of Zn2+ on stress-mediated alterations in hepatic metabolism is discussed.