Evidence-based clinical practice guidelines for nonalcoholic fatty liver disease/nonalcoholic steatohepatitis

Nonalcoholic fatty liver disease (NAFLD) is currently the most common cause of chronic liver disease in industrialized countries worldwide, and has become a serious public health issue not only in Western countries but also in many Asian countries including Japan. Within the wide spectrum of NAFLD, nonalcoholic steatohepatitis (NASH) is a progressive form of disease, which often develops into liver cirrhosis and increases the risk of hepatocellular carcinoma. In turn, a large proportion of NAFLD/NASH is the liver manifestation of metabolic syndrome, suggesting that NAFLD/NASH plays a key role in the pathogenesis of systemic atherosclerotic diseases. Currently, a definite diagnosis of NASH requires liver biopsy, though various noninvasive measures are under development. The mainstays of prevention and treatment of NAFLD/NASH include dietary restriction and exercise; however, pharmacological approaches are often necessary. Currently, vitamin E and thiazolidinedione derivatives are the most evidence-based therapeutic options, although the clinical evidence for long-term efficacy and safety is limited. This practice guideline for NAFLD/NASH, established by the Japanese Society of Gastroenterology in cooperation with The Japan Society of Hepatology, covers lines of clinical evidence reported internationally in the period starting from 1983 to January 2012, and each clinical question was evaluated using the GRADE system. Based on the primary release of the full version in Japanese, this English summary provides the core essentials of this clinical practice guideline comprising the definition, diagnosis, and current therapeutic recommendations for NAFLD/NASH in Japan.(AU)

Hepatocyte destruction with enhanced collagen synthesis: characteristic feature of chronic hepatitis C patients on haemodialysis.

Hepatitis C virus (HCV) infection is frequent among patients with end-stage renal disease on haemodialysis and is considered to be an independent risk factor for mortality in this setting. However, only a few of these patients are treated with anti-hepatitis virus treatment before the development of end-stage renal disease. Recent guidelines recommend identification of patients with good prognoses who are in need of interferon treatment, but we know little of patients who must be treated urgently. Ninety-eight patients on haemodialysis (48 anti-HCV-positive and 50 anti-HCV-negative patients) were enrolled in this study; HCV RNA was detected in 43 anti-HCV-positive patients. Univariate analysis and multivariate regression analysis were applied to identify variables independently associated with persistent HCV infection. Seven variables were proven to be associated with persistent HCV infection. Among them, type IV collagen 7S and N-terminal propeptide of type III procollagen (P-III-P) were defined as independent variables useful in distinguishing HCV RNA-positive patients from HCV RNA-negative patients with 0.91 sensitivity, 0.91 specificity, 0.89 positive predictive value and 0.93 negative predictive value. Our observations suggest that hepatocyte destruction with enhanced liver fibrosis is a characteristic clinical feature of persistent HCV infection. Type IV collagen 7S of ≥ 5 ng/mL and/or P-III-P of ≥ 5 U/mL would be useful markers to identify patients in need of interferon treatment, which supports the idea of the Kidney Disease: Improving Global Outcomes guidelines that a good prognosis in patients with HCV infection on haemodialysis should prompt consideration for IFN treatment when applicable.

Overexpression of GalNAc-transferase GalNAc-T3 promotes pancreatic cancer cell growth.

O-linked glycans of secreted and membrane-bound proteins have an important role in the pathogenesis of pancreatic cancer by modulating immune responses, inflammation and tumorigenesis. A critical aspect of O-glycosylation, the position at which proteins are glycosylated with N-acetyl-galactosamine on serine and threonine residues, is regulated by the substrate specificity of UDP-GalNAc:polypeptide N-acetylgalactosaminyl-transferases (GalNAc-Ts). Thus, GalNAc-Ts regulate the first committed step in O-glycosylated protein biosynthesis, determine sites of O-glycosylation on proteins and are important for understanding normal and carcinoma-associated O-glycosylation. We have found that one of these enzymes, GalNAc-T3, is overexpressed in human pancreatic cancer tissues and suppression of GalNAc-T3 significantly attenuates the growth of pancreatic cancer cells in vitro and in vivo. In addition, suppression of GalNAc-T3 induces apoptosis of pancreatic cancer cells. Our results indicate that GalNAc-T3 is likely involved in pancreatic carcinogenesis. Modification of cellular glycosylation occurs in nearly all types of cancer as a result of alterations in the expression levels of glycosyltransferases. We report guanine the nucleotide-binding protein, α-transducing activity polypeptide-1 (GNAT1) as a possible substrate protein of GalNAc-T3. GalNAc-T3 is associated with O-glycosylation of GNAT1 and affects the subcellular distribution of GNAT1. Knocking down endogenous GNAT1 significantly suppresses the growth/survival of PDAC cells. Our results imply that GalNAc-T3 contributes to the function of O-glycosylated proteins and thereby affects the growth and survival of pancreatic cancer cells. Thus, substrate proteins of GalNAc-T3 should serve as important therapeutic targets for pancreatic cancers.

Efficacy of bezafibrate for chronic GVHD of the liver after allogeneic hematopoietic stem cell transplantation.

Chronic GVHD (cGVHD) of the liver is an important cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-SCT). It is characterized by the destruction of bile duct epithelium followed by progressive cholestasis, which resembles primary biliary cirrhosis (PBC) clinically and histologically. Bezafibrate (BF) is a widely used agent for hyperlipidemia that is also effective in ursodeoxycholic acid (UDCA)-resistant PBC patients. The putative mechanism in cholestasis is that BF upregulates the expression of phosphatidylcholine flippase on bile canaliculi, facilitates phospholipid output into bile and relieves bile duct damage caused by hydrophobic bile salts. Therefore, the effects of BF in patients with cGVHD of the liver were investigated. Of 87 patients with cGVHD who survived more than 100 days after SCT, 8 were given BF to treat liver cGVHD because of a poor therapeutic response to UDCA and immunosuppressants. The serum alkaline phosphatase (ALP) and gamma-glutamyl transpeptidase (gamma-GTP) levels decreased significantly within 1 month after initiation of BF therapy compared with those before BF therapy in all patients (ALP, 964.9.0+/-306.9 to 597.8+/-102.5 IU/l, P=0.012; gamma-GTP, 528.8+/-299.0 to 269.0+/-119.9 IU/l, P=0.012). BF was effective in patients with liver cGVHD, including UDCA-resistant patients. BF could be a novel therapeutic option for liver cGVHD that helps to preserve normal immunity with the antileukemic effect of cGVHD.

Progressive development of insulin resistance phenotype in male mice with complete aromatase (CYP19) deficiency.

Aromatase (CYP19) is a cytochrome P450 enzyme that catalyzes the formation of aromatic C18 estrogens from C19 androgens. It is expressed in various tissues and contributes to sex-specific differences in cellular metabolism. We have generated aromatase-knockout (ArKO) mice in order to study the role of estrogen in the regulation of glucose metabolism. The mean body weights of male ArKO (-/-) mice (n=7) and wild-type littermates (+/+) (n=7) at 10 and 12 weeks of age were 26.7+/-1.9 g vs 26.1+/-0.8 g and 28.8+/-1.4 g vs 26.9+/-1.0 g respectively. The body weights of the ArKO and wild-type mice diverged between 10 and 12 weeks of age with the ArKO males weighing significantly more than their wild-type littermates (P<0.05). The ArKO males showed significantly higher blood glucose levels during an intraperitoneal glucose tolerance test compared with wild-type littermates beginning at 18 weeks of age. By 24 weeks of age, they had higher fasting blood glucose levels compared with wild-type littermates (133.8+/-22.8 mg/dl vs 87.8+/-20.3 mg/dl respectively; P<0.01). An intraperitoneal injection of insulin (0.75 mU insulin/g) caused a continuous decline in blood glucose levels in wild-type mice whereas ArKO males at 18 weeks and older exhibited a rebound increase in glucose levels 30 min after insulin injection. Thus, ArKO male mice appear to develop glucose intolerance and insulin resistance in an age-dependent manner. There was no difference in fasting serum triglyceride and total cholesterol levels between ArKO male mice and wild-type littermates at 13 and 25 weeks of age. However, serum triglyceride and cholesterol levels were significantly elevated following a meal in ArKO mice at 36 weeks of age. Serum testosterone levels in ArKO male mice were continuously higher compared with wild-type littermates. Treatment of ArKO males with 17beta-estradiol improved the glucose response as measured by intraperitoneal glucose and insulin tolerance tests. Treatment with fibrates and thiazolidinediones also led to an improvement in insulin resistance and reduced androgen levels. As complete aromatase deficiency in man is associated with insulin resistance, obesity and hyperlipidemia, the ArKO mouse may be a useful animal model for examining the role of estrogens in the control of glucose and lipid homeostasis.

Erythrocyte creatine as a marker of excessive erythrocyte destruction due to hypersplenism in patients with liver cirrhosis.

OBJECTIVE: Erythrocyte creatine is a sensitive marker of erythrocyte age, and can be used to detect slight and continuous hemolysis. Excessive blood cell destruction caused by increased spleen function is important evidence of hypersplenism. This study evaluates the usefulness of erythrocyte creatine as a sensitive marker of excessive erythrocyte destruction due to hypersplenism in patients with liver cirrhosis. DESIGN AND METHOD: Erythrocyte creatine was determined by an enzymatic method in 50 patients with postnecrotic liver cirrhosis and 50 healthy controls. The spleen size was measured by ultrasonography and expressed as a spleen index. RESULTS: The patients with splenomegaly showed significantly higher erythrocyte creatine than those without splenomegaly (p < 0.005) and healthy controls (p < 0.001), but there was no significant difference in erythrocyte creatine between healthy controls and those without splenomegaly. Fourteen (93%) of the 15 patients with abnormally high erythrocyte creatine (> 1.8 micromol/g hemoglobin) had splenomegaly. There were no significant differences in reticulocyte count between healthy controls and the patients with and without splenomegaly. Erythrocyte creatine showed good correlations with spleen index (r = 0.67; p < 0.001) and reticulocytes (r = 0.63; p < 0.001). CONCLUSIONS: Erythrocyte creatine can be used for predicting erythropoietic status and estimating hypersplenism in patients with liver cirrhosis.

Targeted disruption of the aromatase P450 gene (Cyp19) in mice and their ovarian and uterine responses to 17beta-oestradiol.

Aromatase P450 (CYP19) is an enzyme catalysing the conversion of androgens into oestrogens. We generated mice lacking aromatase activity (ArKO) by targeted disruption of Cyp19 and report the characteristic features of the ArKO ovaries and uteri as revealed by histological and biochemical analyses. ArKO females were totally infertile but there were as many developing follicles in their ovaries at 8 weeks of age as in wild-type ovaries. Nevertheless, no typical corpus luteum was observed in the ArKO ovaries. Electron microscopy revealed the presence of well-developed smooth endoplasmic reticulum, few lipid droplets and mitochondria with less organized tubular structures in the ArKO luteinized interstitial cells. These ultrastructural features were different from those of the wild-type interstitial cells, where there are many lipid droplets and mitochondria with well-developed tubular structures, characteristic of steroid-producing cells. When ArKO mice were supplemented with 17beta-oestradiol (E(2); 15 microg/mouse) every fourth day from 4 weeks of age for 1 month, increased numbers of follicles were observed in the ovaries as compared with those of untreated ArKO mice, although no typical corpus luteum was detectable. Ultrastructural analysis revealed the disappearance of the accumulated smooth endoplasmic reticulum in the luteinized interstitial cells after E(2 )supplementation. Transcripts of pro-apoptotic genes such as p53 and Bax genes were markedly elevated in the ArKO ovaries as compared with those of wild-type mice. Although E(2) supplementation did not cause suppression of the elevated expression of p53 and Bax mRNAs, it caused marked enhancement of expression levels of lactoferrin and progesterone receptor mRNAs in the uteri as well as increases in uterine wet weight. At 8 months of age, ArKO mice developed haemorrhages in the ovaries, in which follicles were nearly depleted, while age-matched wild-type females still had many ovarian follicles. Furthermore, macrophage-like cells were occasionally observed in the ArKO ovarian follicles. These results suggested that targeted disruption of Cyp19 caused anovulation and precocious depletion of ovarian follicles. Additionally, analysis of mice supplemented with E(2) demonstrated that E(2) apparently supports development of ovarian follicles, although it did not restore the defect in ovulation.

Effects of ethanol on L-arginine transport in rat Ito cells in relation to nitric oxide production.

BACKGROUND: Nitric oxide (NO) is a potent mediator of hepatic sinusoidal hemodynamics that is synthesized in the hepatic stellate cells (Ito cells, fat-storing cells) and affects these cells. NO production may depend on the induction of inducible nitric oxide synthase and on transport of extracellular L-arginine. The precise mechanism that controls NO production in stellate cells was characterized recently. METHODS: Kinetic analysis of L-arginine transport and reverse transcription-polymerase chain reaction for cationic amino acid transporter (CAT) were carried out by using stellate cells prepared from the male Wistar rat. The effect of ethanol on L-arginine transport and NO production of stellate cells was assessed in the presence of tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma. RESULTS: The L-arginine transport system functioning in the hepatic stellate cells was system y+, possibly mediated by CAT-1 and CAT-2B (Km approximately 50 microM). IFN-gamma in combination with TNF-alpha induced NO production with an enhancement in CAT-2B mRNA expression and L-arginine transport, whereas L-arginine transport and NO production were suppressed by coincubated ethanol. CONCLUSIONS: In hepatic stellate cells, ethanol has suppressive effects on NO production and extracellular L-arginine transport in the presence of TNF-alpha and IFN-gamma. The estimated Km of L-arginine transporter in hepatic stellate cells is very similar to the physiological L-arginine concentration in portal vein. Our findings may support the merit of further studies on the modulation of NO production via access to portal blood L-arginine concentration to control disturbed hepatic sinusoidal blood flow in patients with alcoholic liver disease.

Radiation-induced apoptosis of human peripheral T cells: analyses with cDNA expression arrays and mitochondrial membrane potential assay.

We examined sequential changes in post-irradiated peripheral blood T cells taken from normal volunteers, by using targeted Atlas cDNA Expression Arrays and mitochondrial membrane potential assay. At 1 and 3 hours after 5 Gy irradiation, changes of gene expression were examined by targeted Atlas cDNA Expression Arrays using Apoptosis Array. The Atlas Human Apoptosis Array includes 205 key genes that are known to control apoptosis, including extracellular and cytoplasmic effectors. Concerning Fas, no significant changes of spot intensities were identified between irradiated T cells and non-irradiated ones at both 1 h and 3 h after 5 Gy irradiation. Caspase families, including caspases 9 and 3 also showed no changes between these two groups. An apoptosis regulator bclw showed a remarkable decrease in irradiated T cells. These results suggested that irradiation induced direct apoptosis of T cells by changing the membrane potential of mitochondria. Using a CCD camera-equipped fluorescence microscope and MitoCapture, a mitochondrial membrane potential indicator, we demonstrated 5 Gy radiation induced loss of membrane potential, i.e., an early stage of apoptosis, in human peripheral blood T cells at 10 hours after irradiation.

Eosinophil-induced liver injury: an experimental model using IL-5 transgenic mice.

BACKGROUND/AIMS: In certain liver diseases, activated eosinophils are considered to be important effector cells in addition to T-cell-mediated cytotoxicity. No experimental model, however, has been developed for in vivo analysis of the cytotoxic mechanisms. METHODS: Interleukin-5 (IL-5) transgenic mice (C3H/HeN-TgN(IL-5)Imeg), which exhibit marked eosinophilia without liver injury, were injected once with 25 microg of lipopolysaccharide (LPS) intraperitoneally. The mice were sacrificed weekly and eosinophilic injuries were assessed microscopically. To clarify the role of Kupffer cells and tumor necrosis factor-alpha (TNF-alpha) in the liver injury, gadolinium chloride (GdCl3) and anti-TNF-alpha neutralizing antibody were administrated before the LPS injection. RESULTS: Two weeks after injection, transgenic mice exhibited marked infiltration of eosinophils and extensive lobular necrosis. Transmigration of eosinophils through vascular endothelium and degranulation of eosinophil cytotoxic granules in inflamed areas were observed. These eosinophilic injuries were transient, but liver-specific. Pre-administration of GdCl3 and anti-TNF-alpha markedly reduced the hepatic inflammation, suggesting that LPS-activated Kupffer cells play a key role in producing the cytotoxicity of eosinophils by releasing TNF-alpha. CONCLUSIONS: We have established an experimental model of eosinophil-induced liver injury using IL-5 transgenic mice. Since this model is simple and highly reproducible, it will be useful for analysis of in vivo cytotoxic mechanisms of eosinophils.

Oestrogen at the neonatal stage is critical for the reproductive ability of male mice as revealed by supplementation with 17beta-oestradiol to aromatase gene (Cyp19) knockout mice.

Aromatase P450 (CYP19) is an enzyme responsible for the conversion of androgens to oestrogens. We generated CYP19 knockout (ArKO) mice by targeted disruption of Cyp19 and studied the role of oestrogens in male reproductive ability. Approximately 85% of ArKO males were unable to sire offspring. However, no obvious difference was found in testicular and epididymal weights, numbers of sperm in the epididymis or the ability of sperm to fertilize eggs in vitro between wild-type and ArKO males. An examination of mating behaviour demonstrated that ArKO males showed an impairment in mounting behaviour against sexually mature females. The inability of more than 90% of ArKO males to sire offspring was reversed by repeated subcutaneous injections of 17beta-oestradiol when initiated on the day of birth. The effects of 17beta-oestradiol on reproduction were concentration dependent and evident when supplementation was initiated on day 7, but not on day 15 after birth. These findings suggest that oestrogens acting during neonatal life are required for normal mating behaviour in adulthood.

A loss of aggressive behaviour and its reinstatement by oestrogen in mice lacking the aromatase gene (Cyp19).

Aromatase P450 (CYP19) is an enzyme responsible for conversion of androgens to oestrogens. We generated CYP19 knockout (ArKO) mice by targeting disruption of the CYP19 gene and observed that the ArKO males exhibited a complete loss of aggressive behaviour against intruder mice when examined using a resident-intruder paradigm. The defect in the behaviour of ArKO males was reinstated when the mice received supplements of 17beta-oestradiol soon after birth. Nevertheless, the cumulative duration of the behaviour displayed by the treated mice during the test period of 15 min was 19+/-10 s, which was much shorter than that displayed by wild-type males, 90+/-17 s. When the supplementation was started at 7 days after birth, the defect was not restored. These findings illustrate an absolute requirement for oestrogen during the neonatal stage of a male's life for the development of the potential for aggression observed in adulthood. Furthermore, the present study demonstrates that ArKO males are a useful model in which to investigate the neural mechanisms by which aggressive behaviour is controlled.

Alternations in hepatic expression of fatty-acid metabolizing enzymes in ArKO mice and their reversal by the treatment with 17beta-estradiol or a peroxisome proliferator.

We generated aromatase gene knockout mice (ArKO mice) by targeting disruption of Cyp19, which encodes an enzyme responsible for conversion of androgens to estrogens. We found that ArKO males developed hepatic steatosis spontaneously with aging, indicating that the function of Cyp19 is required to maintain constitutive lipid metabolism in male mice. Plasma lipoprotein analysis using a gel permeation chromatography revealed that high density lipoprotein (HDL)-cholesterol levels were slightly higher in ArKO males than in wild-type males, whereas no other obvious alternations in the profiles were detected. Nevertheless, analysis of lipoprotein compositions by SDS-polyacrylamide gel electrophoresis demonstrated apparent reduction in the amounts of apolipoprotein E, functioning in receptor-mediated clearance of lipoproteins in the liver, in the IDL/LDL fraction of ArKO males as compared with that of wild-type males. Biochemical analysis on the ArKO livers revealed suppression of mRNA expression and activity of enzymes involved in fatty acid beta-oxidation. The impairment was reversed to the wild-type levels by treatment with 17beta-estradiol or bezafibrate, the latter is a synthetic peroxisome proliferator. These findings indicated a pivotal role of estrogen in supporting constitutive hepatic expression of genes involved in fatty acid beta-oxidation and in maintaining lipid homeostasis.

Toremifene-induced fatty liver and NASH in breast cancer patients with breast-conservation treatment.

We have described fatty liver, diagnosed by computed tomography scanning (CT) in more than 30% of patients with breast cancer who received tamoxifen. Therefore, it is urgent to elucidate the frequency and the degree of fatty liver induced by toremifene, an analogue of tamoxifen, which is also used in breast cancer. We enrolled 52 breast cancer patients who were treated with breast-conservation treatment and administered oral toremifene for 3-5 years as adjuvant endocrine therapy. We evaluated the degree of fatty liver by abdominal CT performed annually. CT demonstrated toremifene-induced fatty liver in four (7.7%) of 52 breast cancer patients. Toremifene-induced fatty liver did not correlate with abnormal levels of AST, ALT, GGT or total cholesterol. One patient who demonstrated moderate fatty liver by CT was histologically diagnosed as non-alcoholic steatohepatitis (NASH) by liver biopsy. The incidence of toremifene-induced fatty liver was significantly lower than that induced by tamoxifen. Accordingly, in terms of fatty liver and NASH, toremifene is considered to be more appropriate agent than tamoxifen. Though toremifene is less likely to induce fatty liver, the possibility remains that toremifene-induced steatohepatitis occurs. Because the diagnosis of fatty liver or NASH can be easily missed if only a blood test is performed, it is necessary to screen fatty liver by annual CT examination for patients who receive an antiestrogen agent.

Unrecognized hepatic steatosis and non-alcoholic steatohepatitis in adjuvant tamoxifen for breast cancer patients.

Adjuvant tamoxifen has become the treatment of choice against estrogen receptor-positive breast cancer. Adverse effects are rarely observed and since symptoms of hepatic steatosis, non-alcoholic steatohepatitis and cirrhosis are usually negligible, such effects are not well characterized despite large cohort studies of adjuvant tamoxifen. This issue remains to be systematically studied. The present study consisted of 136 breast cancer patients treated with or without tamoxifen. Patients had laboratory tests once each month and underwent abdominal computed tomography (CT) annually for 5 years. The extent of hepatic steatosis was assessed by CT as the liver/spleen ratio. While receiving adjuvant tamoxifen, 40 of 105 patients developed hepatic steatosis (liver/spleen ratio <0.9) without obvious changes in body mass index. Twenty-one had a liver spleen ratio of <0.5, whereas none of the 31 patients treated without tamoxifen had a ratio <0.9 or <0.5 (p<0.0001 and p<0.0001, respectively). Hepatic steatosis was recognized in 35 of the 40 patients within the first 2 years of receiving adjuvant tamoxifen and 21 of the 40 had increased transaminase levels. Liver biopsy revealed NASH in 6 of 7 patients among the 21 with a liver/spleen ratio of <0.5. A subset of individuals given adjuvant tamoxifen developed progressive hepatic steatosis without significant changes in the body mass index. We suggest a liver/spleen ratio of <0.5 as a criterion upon which liver biopsy should be recommended since NASH frequently occurred in such patients.

Altered expression of fatty acid-metabolizing enzymes in aromatase-deficient mice.

Hepatic steatosis is a frequent complication in nonobese patients with breast cancer treated with tamoxifen, a potent antagonist of estrogen. In addition, hepatic steatosis became evident spontaneously in the aromatase-deficient (ArKO) mouse, which lacks intrinsic estrogen production. These clinical and laboratory observations suggest that estrogen helps to maintain constitutive lipid metabolism. To clarify this hypothesis, we characterized the expression and activity in ArKO mouse liver of enzymes involved in peroxisomal and mitochondrial fatty acid beta-oxidation. Northern analysis showed reduced expression of mRNAs for very long fatty acyl-CoA synthetase, peroxisomal fatty acyl-CoA oxidase, and medium-chain acyl-CoA dehydrogenase, enzymes required in fatty acid beta-oxidation. In vitro assays of fatty acid beta-oxidation activity using very long (C24:0), long (C16:0), or medium (C12:0) chain fatty acids as the substrates confirmed that the corresponding activities are also diminished. Impaired gene expression and enzyme activities of fatty acid beta-oxidation were restored to the wild-type levels, and hepatic steatosis was substantially diminished in animals treated with 17beta-estradiol. Wild-type and ArKO mice showed no difference in the binding activities of the hepatic nuclear extracts to a peroxisome proliferator response element. These findings demonstrate the pivotal role of estrogen in supporting constitutive hepatic expression of genes involved in lipid beta-oxidation and in maintaining hepatic lipid homeostasis.

Multifrequency method for dielectric monitoring of cold-preserved organs.

To answer a growing need for non-invasive monitoring of biological organs, we have developed an automated system capable of repeated dielectric measurements over the frequency range 10 kHz-100 MHz. Further, we propose a novel method of data analysis that may convert the acquired, individual dispersion curves into a diagram of the time course of specific phenomenological parameters, such as the characteristic frequency. By using this new procedure, unattended, long-term monitoring of temporal changes in the dielectric behaviour of excised liver lobes stored at 4 degrees C was successfully realized. The 'multifrequency' method presented here was definitely superior to the conventional 'fixed-frequency' method in providing reliable results.