Rosemary O'Neill

Determination of hepatic iron concentration in fresh and paraffin-embedded tissue: Diagnostic implications

BACKGROUND/AIMS Determination of hepatic iron concentration (HIC) is essential for the evaluation of hereditary hemochromatosis. Occasionally, only paraffin-embedded liver biopsy specimens are available, or fresh biopsy specimens have been placed in saline for transport. This study aimed to describe a method for extraction of liver tissue from paraffin blocks, determine the accuracy of measurement of HIC in recovered tissue compared with fresh tissue, and determine the effect of immersion in saline on HIC. METHODS HIC was measured in both fresh and deparaffinized liver specimens (n = 41). Accurate measurements were defined as either a normal result in both specimens or a result in the deparaffinized specimen that was within 30% of the fresh measurement. RESULTS Measurements of HIC in fresh and deparaffinized tissue showed an excellent linear relationship (r = 0.95). In deparaffinized samples > or = 0.4 mg, accurate measurements were seen in 24 out of 29 specimens, compared with 6 out of 12 specimens weighing < 0.4 mg (P < 0.01). The hepatic iron index calculated from results in deparaffinized samples > or = 0.4 mg correctly classified all patients. Immersion of fresh biopsy specimens in saline for 1 hour resulted in up to 50% iron loss (P < 0.05). CONCLUSIONS Accurate measurement of HIC in deparaffinized liver biopsy specimens is possible. Calculation of the hepatic iron index from deparaffinized liver tissue can facilitate diagnosis of hemochromatosis when fresh tissue is not available. Samples should not be transported in saline.

PATHOPHYSIOLOGY OF IRON TOXICITY

There are several inherited and acquired disorders that can result in chronic iron overload in humans, and the major clinical consequences are hepatic fibrosis, cirrhosis, hepatocellular cancer, cardiac disease, and diabetes. It is clear that lipid peroxidation occurs in experimental iron overload if sufficiently high levels of iron within hepatocytes are achieved. Lipid peroxidation is associated with hepatic mitochondrial and microsomal dysfunction in experimental iron overload, and lipid peroxidation may underlie the increased lysosomal fragility that has been detected in liver samples from both iron-loaded human subjects and experimental animals. Reduced cellular ATP levels, impaired cellular calcium homeostasis, and damage to DNA may all contribute to hepatocellular injury in iron overload. Long-term dietary iron overload in rats can lead to increased collagen gene expression and hepatic fibrosis, perhaps due to activation of hepatic lipocytes. The mechanisms whereby lipocytes are activated in iron overload remain to be elucidated; possible mediators include aldehydic products of iron-induced lipid peroxidation produced in hepatocytes, tissue ferritin, and/or cytokines released by activated Kupffer cells.

Iron-induced peroxidative injury to isolated rat hepatic mitochondria**

Peroxidative injury to the mitochondrial inner membrane with resultant defects in oxidative metabolism may be partially responsible for hepatocellular injury in iron overload. We examined the effects of iron-induced lipid peroxidation in vitro on hepatic mitochondrial morphology and function and determined if various inhibitors of free-radical-mediated injury could be protective. Normal rat liver mitochondria were prepared by differential centrifugation and were incubated with 1, 2, and 3 microM Fe2+, NADPH, and with and without oxygen radical scavengers, iron chelators, and antioxidants. There was a direct linear relationship between the concentration of added iron and the degree of lipid peroxidation as measured by malondialdehyde (MDA) production (r = .85). With 3 microM Fe2+ there was a decrease in the respiratory control ratio (RCR) for all four substrates tested; this decrease in RCR was due to a decrease in the state 3 respiratory rate for all substrates, with no changes in the state 4 respiratory rate for glutamate, beta-hydroxybutyrate, or succinate. Oxygen radical scavengers failed to prevent iron-induced lipid peroxidation or to protect against associated mitochondrial dysfunction. Iron chelators and antioxidants prevented MDA formation and mitochondrial function was maintained. Iron-induced lipid peroxidation in vitro produces an irreversible inhibitory defect in mitochondrial electron transport that may be specific at complex IV (cytochrome oxidase).

The ethanol metabolite, linolenic acid ethyl ester, stimulates mitogen-activated protein kinase and cyclin signaling in hepatic stellate cells

Chronic ethanol consumption can result in hepatic fibrosis and cirrhosis. In addition to oxidative metabolism, ethanol can be metabolized by esterification with fatty acids to form fatty acid ethyl esters (FAEE) such as linolenic acid ethyl ester (LAEE). We have previously demonstrated that LAEE has promitogeinc and activating effects on hepatic stellate cells (HSC), but the mechanisms of these actions are not known. Intracellular signaling through MAP kinase pathways, including extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) can influence the activity of the transcription factor AP-1, while cell-cycle regulatory proteins such as cyclin E and cyclin-dependent kinase (CDK), play an important role in cell proliferation. In this study, we demonstrate that treatment of HSC with LAEE increases cyclin E expression and cyclin E/CDK2 activity, which may underlie the promitogenic effects of this compound. In addition, LAEE increases ERK and JNK activity, and these pathways play an important role in the activation of AP-1-dependent gene expression by LAEE. The stimulation of intracellular signaling pathways in HSC by this well-characterized ethanol metabolite may contribute to ethanol-induced hepatic fibrogenesis.

Aldehydic products of lipid peroxidation do not directly activate rat hepatic stellate cells

Abstract Background and Aim : Activation of hepatic stellate cells (HSC) results in the transdifferentiation of the resting (quiescent) phenotype to one characterized by loss of vitamin A droplets, increased α‐smooth muscle actin (SMA) expression and increased collagen production. Aldehydic products of lipid peroxidation have been shown to increase collagen production by cultured fibroblasts and by passaged HSC, but it is unclear whether these products of lipid peroxidation can initiate the activation of HSC. In the present study the effects were examined of two aldehydic products of lipid peroxidation, malondialdehyde (MDA) and 4‐hydroxynonenal (HNE), on activation of rat HSC in early culture as measured by SMA and desmin expression, and collagen production.

Effect of phorbol ester and platelet-derived growth factor on protein kinase C in rat hepatic stellate cells

Background/Aims: Hepatic stellate cells (HSC) play a key role in hepatic fibrogenesis and thus, it is important to understand the intracellular signalling pathways that influence their behaviour. This study investigated the expression and regulation of protein kinase C (PKC) in HSC.

Effect of protein kinase C activation and inhibition on rat hepatic stellate cell activation.

Protein kinase C (PKC) may play a role in the intracellular signaling pathways responsible for transforming hepatic stellate cells into myofibroblasts. This study examined the effects of inhibitors and activators of PKC on hepatic stellate cell activation. Stellate cells isolated from normal rats were incubated with either 10−5 M chelerythrine, 10−7 M bisindolylmaleimide I hydrochloride (BIM), or 10−6 M staurosporine (PKC inhibitors), or 10−7 M phorbol myristate acetate (PMA) or 10−6 M thymeleatoxin (PKC activators). Chelerythrine suppressed α-smooth muscle actin expression and proliferation by 49% and 33%, respectively. BIM inhibited α-smooth muscle actin expression by 60%, but had no significant effect on proliferation. Staurosporine decreased proliferation by 86% and completely prevented α-smooth muscle actin expression. PKC activators had divergent effects on proliferation and α-smooth muscle actin expression. PMA and thymeleatoxin caused a 2.8- to 3.2-fold increase in proliferation, while suppressing α-smooth muscle actin expression by 50–70%. The demonstration that hepatic stellate cell activation can be suppressed by PKC inhibitors suggests a role for PKC in the regulation of hepatic stellate cell activation.

Determination of α-tocopherol in rat liver by high-performance liquid chromatography utilizing ultraviolet detection

A rapid, sensitive analytical procedure was developed to quantitate alpha-tocopherol in rat liver. Liver is homogenized in acetone, fractionated by reversed-phase high-performance liquid chromatography and alpha-tocopherol quantitated by monitoring the column effluent at 280 nm. A single analysis requires 18 min. The method is linear from 1.0 to greater than 30.0 micrograms alpha-tocopherol per g of liver (wet weight). The average relative standard deviation of the slope of the standard curve is 2.3% and the single-day coefficient of variation is 6.3%. The use of an acetone extraction, reversed-phase column chromatography and quantitation of alpha-tocopherol at 280 nm results in a sensitive and reproducible system for the determination of alpha-tocopherol concentrations in rat liver. Preliminary studies have shown the assay to be useful for investigation of the effects of age and diet on hepatic alpha-tocopherol concentration.