Ovarian Cyst Fluid of Serous Ovarian Tumors Contains Large Quantities of the Brain Amino Acid N-acetylaspartate

BACKGROUND: In humans, N-acetyl L-aspartate (NAA) has not been detected in other tissues than the brain. The physiological function of NAA is yet undefined. Recently, it has been suggested that NAA may function as a molecular water pump, responsible for the removal of large amounts of water from the human brain. Ovarian tumors typically present as large cystic masses with considerable fluid accumulation. METHODOLOGY AND PRINCIPAL FINDINGS: Using Gas Chromatography-Mass Spectrometry, we demonstrated that NAA was present in a high micromolar concentration in oCF of epithelial ovarian tumors (EOTs) of serous histology, sometimes in the same range as found in the extracellular space of the human brain. In contrast, oCF of EOTs with a mucinous, endometrioid and clear cell histological subtype contained a low micromolar concentration of NAA. Serous EOTs have a cellular differentiation pattern which resembles the lining of the fallopian tube and differs from the other histological subtypes. The NAA concentration in two samples of fluid accumulation in the fallopian tube (hydrosalpinx) was in the same ranges as NAA found in oCF of serous EOTs. The NAA concentration in oCF of patients with serous EOTs was mostly 10 to 50 fold higher than their normal serum NAA concentration, whereas in patients with other EOT subtypes, serum and cyst fluid NAA concentration was comparable. CONCLUSIONS AND SIGNIFICANCE: The high concentration of NAA in cyst fluid of serous EOTs and low serum concentrations of NAA in these patients, suggest a local production of NAA in serous EOTs. Our findings provide the first identification of NAA concentrations high enough to suggest local production outside the human brain. Our findings contribute to the ongoing research understanding the physiological function of NAA in the human body

Glutathione and antioxidant enzymes serve complementary roles in protecting activated hepatic stellate cells against hydrogen peroxide-induced cell death

Background: In chronic liver disease, hepatic stellate cells (HSCs) are activated, highly proliferative and produce excessive amounts of extracellular matrix, leading to liver fibrosis. Elevated levels of toxic reactive oxygen species (ROS) produced during chronic liver injury have been implicated in this activation process. Therefore, activated hepatic stellate cells need to harbor highly effective anti-oxidants to protect against the toxic effects of ROS.Aim: To investigate the protective mechanisms of activated HSCs against ROS-induced toxicity.Methods: Culture-activated rat HSCs were exposed to hydrogen peroxide. Necrosis and apoptosis were determined by Sytox Green or acridine orange staining, respectively. The hydrogen peroxide detoxifying enzymes catalase and glutathione-pefoxidase (GPx) were inhibited using 3-amino-1,2,4-triazole and mercaptosuccinic acid, respectively. The anti-oxidant glutathione was depleted by L-buthionine-sulfoximine and repleted with the GSH-analogue GSH-monoethylester (GSH-MEE).Results: Upon activation, HSCs increase their cellular glutathione content and GPx expression, while MnSOD (both at mRNA and protein level) and catalase (at the protein level, but not at the mRNA level) decreased. Hydrogen peroxide did not induce cell death in activated HSCs. Glutathione depletion increased the sensitivity of HSCs to hydrogen peroxide, resulting in 35% and 75% necrotic cells at 0.2 and 1 mmol/L hydrogen peroxide, respectively. The sensitizing effect was abolished by GSH-MEE. Inhibition of catalase or GPx significantly increased hydrogen peroxide-induced apoptosis, which was not reversed by GSH-MEE.Conclusion: Activated HSCs have increased ROS-detoxifying capacity compared to quiescent HSCs. Glutathione levels increase during HSC activation and protect against ROS-induced necrosis, whereas hydrogen peroxide-detoxifying enzymes protect against apoptotic cell death. (C) 2013 Elsevier B.V. All rights reserved.</p

Novel aspects of peroxisome composition and function in the liver

Peroxisomes are organelles that are involved in important liver functions, including bile salt biosynthesis and lipid metabolism. In this thesis we report several novel aspects of peroxisome composition and function in different liver cell types. We analyzed the function of liver peroxisomes with respect to bile salt homeostasis and liver fibrosis. We obtained proof that unconjugated bile acids transit through hepatocyte peroxisomes to become (re-)conjugated. This implies the presence of putative bile salt transporters in the peroxisomal membrane. Moreover, we show that typical peroxisomal proteins in hepatocytes might be present at different subcellular locations in other liver cell types. In activated hepatic stellate cells (aHSCs), we detected the peroxisomal membrane protein PMP70 in cellular fibers and showed that PMP70 is required for the development of the α -smooth muscle actin network. Therefore, PMP70 might be required for aHSC plasticity and contractility during liver fibrosis. In addition, we performed a detailed analysis on peroxisomal membrane proteins and their putative association with cholesterol-enriched membrane microdomains (lipid rafts). We observed that caveolin-1, which resides in the plasma membrane in liver endothelial cells and hepatic stellate cells, localizes to the peroxisomal membrane of hepatocytes. This may be important for peroxisomal processes involved in liver regeneration. Finally, we found that hepatocyte peroxisomes contain different subtypes of lipid rafts. At least one of these subtypes is crucial for peroxisome biogenesis. Taken together, this thesis demonstrates novel functions of peroxisomes in the liver, that may have implications for several liver diseases, in particular liver fibrosis and cholestatic disorders.

Caveolin-1 Is Enriched in the Peroxisomal Membrane of Rat Hepatocytes

Caveolae are a subtype of cholesterol-enriched lipid microdomains/rafts that are routinely detected as vesicles pinching off from the plasma membrane. Caveolin-1 is an essential component of caveolae. Hepatic caveolin-1 plays an important role in liver regeneration and lipid metabolism. Expression of caveolin-1 in hepatocytes is relatively low, and it has been suggested to also reside at other subcellular locations than the plasma membrane. Recently, we found that the peroxisomal membrane contains lipid microdomains. Like caveolin-1, hepatic peroxisomes are involved in lipid metabolism. Here, we analyzed the subcellular location of caveolin-1 in rat hepatocytes. The subcellular location of rat hepatocyte caveolin-1 was analyzed by cell fractionation procedures, immunofluorescence, and immuno-electron microscopy. Green fluorescent protein (GFP)-tagged caveolin-1 was expressed in rat hepatocytes. Lipid rafts were characterized after Triton X-100 or Lubrol WX extraction of purified peroxisomes. Fenofibric acid-dependent regulation of caveolin-1 was analyzed. Peroxisome biogenesis was studied in rat hepatocytes after RNA interference-mediated silencing of caveolin-1 and caveolin-1 knockout mice. Cell fractionation and microscopic analyses reveal that caveolin-1 colocalizes with peroxisomal marker proteins (catalase, the 70 kDa peroxisomal membrane protein PMP70, the adrenoleukodystrophy protein ALDP, Pex14p, and the bile acid-coenzyme A:amino acid N-acyltransferase BAAT) in rat hepatocytes. Artificially expressed GFP-caveolin-1 accumulated in catalase-positive organelles. Peroxisomal caveolin-1 is associated with detergent-resistant microdomains. Caveolin-1 expression is strongly repressed by the peroxisome proliferator-activated receptor-alpha agonist fenofibric acid. Targeting of peroxisomal matrix proteins and peroxisome number and shape were not altered in rat hepatocytes with 70%-80% reduced caveolin-1 levels and in livers of caveolin-1 knockout mice. CONCLUSION: Caveolin-1 is enriched in peroxisomes of hepatocytes. Caveolin-1 is not required for peroxisome biogenesis, but this unique subcellular location may determine its important role in hepatocyte proliferation and lipid metabolism

NAA (µmol/L) in ovarian cyst fluid and ascites.

<p>NAA (µmol/L) in ovarian cyst fluid and ascites.</p

NAA (µmol/L) concentration of individual patients with serous (n = 36) and mucinous (n = 23) tumors.

<p>Values are presented by dots on a logarithmic scale. A horizontal line represents the cut-off value of 1.1 µmol/L NAA.</p

Overview of biological fluid samples grouped by origin.

<p>Overview of biological fluid samples grouped by origin.</p

NAA (µmol/L) in oCF of patients with serous (n = 9), mucinous (n = 6), endometrioid (n = 8) and clear cell (n = 2) ovarian cancer.

<p>NAA (µmol/L) in oCF of patients with serous (n = 9), mucinous (n = 6), endometrioid (n = 8) and clear cell (n = 2) ovarian cancer.</p