Dieter Häussinger

Proton magnetic resonance spectroscopy of the brain in symptomatic and asymptomatic patients with liver cirrhosis

BACKGROUND & AIMSnHepatic encephalopathy (HE) is associated with typical changes of cerebral metabolite pattern observed by proton magnetic resonance (MR) spectroscopy consisting of a depletion of myo-inositol and an increase of glutamine. The aim of this study was to determine whether abnormalities in brain metabolism in neurologically asymptomatic patients with liver cirrhosis can be detected by spectroscopy.nnnMETHODSnIn a prospective study, 39 patients with liver cirrhosis were examined clinically according to standardized neuropsychological tests to define whether overt, subclinical, or no hepatic encephalopathy was present. All patients underwent proton MR spectroscopy at short echo times.nnnRESULTSnSpectroscopy allowed for the diagnosis of subclinical HE in 4 of 4 cases and of overt HE in 10 of 11 cases. In 24 cases of mere liver cirrhosis and normal neuropsychological testing, a typical metabolic pattern with a depletion of myo-inositol and an increased glutamine peak were found. A good correlation between the glutamine signal and the clinical grading was found.nnnCONCLUSIONSnProton MR spectroscopy allows for the diagnosis of HE and subclinical HE, also shows the typical pathological metabolite pattern in patients with cirrhotic livers without subclinical HE, and might be more sensitive than neuropsychological testing. Glutamine could serve as a spectroscopic marker for the clinical state of the patients.

Glutamine metabolism in mammalian tissues

Enzymology and Transport.- Enzymology of Glutamine.- Enzymes of Renal Glutamine Metabolism.- Enzymes of Cerebral Glutamine Metabolism.- Glutamine Transport Across Biological Membranes.- Intestine and Liver.- Metabolism of Vascular and Luminal Glutamine by Intestinal Mucosa in Vivo.- Hepatic Glutamine and Ammonia Metabolism Nitrogen and Redox Balance and the Intercellular Glutamine Cycle.- Cellular Distribution and Regulation of Glutamine Synthetase in Liver.- Liver Glutaminase.- Mechanism and Control of Deprivation-Induced Protein Degradation in Liver: Role of Glucogenic Amino Acids.- Kidney.- Renal Glutamine Metabolism and Hydrogen Ion Homeostasis.- Effects of 2-Oxoglutarate and Glutamate on Glutamine Metabolism by Rat Kidney Mitochondria.- Role of Fatty Acids in Simultaneous Regulation of Flux Through Glutaminase and Glutamine Synthetase in Rat Kidney Cortex.- Other Tissues.- Cyclic Nucleotide Regulation of Glutamine Metabolism in Skeletal Muscle.- Cerebral Glutamine/Glutamate Interrelationships and Metabolic Compartmentation.- Glutamine Metabolism in Lymphoid Tissues.- Glutamine Metabolism by Cultured Mammalian Cells.- Clinical Aspects.- Ammonia Detoxication and Glutamine Metabolism in Severe Liver Disease and its Role in the Pathogenesis of Hepatic Encephalopathy.- Molecular Targets of Anti-Glutamine Therapy with Acivicin in Cancer Cells.

Water, K + , H + , lactate and glucose fluxes during cell volume regulation in perfused rat liver

The present study has been performed to test for ion release from isolated perfused rat liver exposed to hypotonic perfusates. Replacement of 40 mmol/l NaCl in perfusate by 80 mmol/l raffinose leads to slight alkalinization and slight decrease of liver weight. Subsequent decrease of perfusate osmolarity by omission of raffinose results in an increase of liver weight and a parallel increase of effluent sodium, chloride and potassium activity pointing to net uptake of solute free water. While effluent chloride and sodium activities approach perfusate activities within less than 2 min, a second, 6 min lasting increase of effluent potassium activity is observed, pointing to potassium release by the liver. This transient increase of effluent potassium activity is paralleled by a decrease of liver weight. Throughout exposure to hypotonic perfusates, lactate, pyruvate and glucose release by the liver is significantly decreased and effluent pH is rendered alkaline. Readdition of 80 mmol/l raffinose leads to rapid decrease of liver weight and a parallel decrease of effluent sodium, chloride and potassium activities followed by a 10–20 min lasting decrease of effluent potassium activity, pointing to net uptake of potassium, which almost matches the net release observed before. The transient decrease of potassium activity is paralleled by an increase of liver weight, an increase of effluent glucose, lactate and pyruvate concentration and an acidification of the effluent. Similar decrease of effluent potassium activity, acidification of effluent and increase of effluent glucose, lactate and pyruvate concentration are observed, if perfusates are made hypertonic by addition of raffinose. In conclusion, both, volume regulatory decrease (VRD) and increase (VRI) can be elicited in liver and are in large part achieved by movements of potassium. Lactate and pyruvate production is decreased throughout exposure to hypotonic perfusates and enhanced following exposure to hypertonic perfusates.

The Biological Significance of Cell Volume

To survive, cells have to avoid excessive alterations of their volume. To this end, cells have developed a complex machinery of cell volume regulatory mechanisms comprising transport across the cell membrane and metabolism. Upon cell swelling, they loose electrolytes mainly via selective K+ channels and unselective ion channels and/or KCl symport, upon cell shrinkage they accumulate ions by Na+,K+,2Cl- cotransport and parallel operation of Na+/H+ exchange and Cl-/HCO3- exchange. In addition, cell shrinkage stimulates glycogenolysis, proteolysis and formation of organic osmolytes such as amino acids, methylamines and polyols. Cell swelling stimulates formation of glycogen and proteins and cellular release of organic osmolytes. Alterations of cell volume do play a crucial role in the regulation of cell function, as illustrated by four examples: 1. Epithelial transport may lead to cell swelling, which then triggers volume regulatory mechanisms modifying transcellular transport. 2. Insulin swells hepatocytes by activation of Na+,K+,2Cl- cotransport and Na+/H+ exchange, glucagon shrinks those cells by activation of ion channels. The respective volume changes participate in the regulation of cellular protein and glycogen metabolism by these hormones. 3. Growth factors and expression of ras oncogene activate Na+,K+,2Cl- cotransport and Na+/H+ exchange, leading to the respective cell swelling. 4. Hepatocyte swelling triggers a hepatorenal reflex decreasing renal blood flow.

Altered cell volume regulation in ras oncogene expressing NIH fibroblasts

Expression of the Ha-ras oncogene has been reported to stimulate the dimethylamiloride sensitive Na+/H+ exchanger and Na+, K+, 2Cl− cotransport, both transport systems which are involved in cell volume regulation. The present study has been performed to test for an influence of ras oncogene expression on cell volume regulation in NIH 3T3 fibroblasts expressing the Ha-ras oncogene (+ ras). As controls served NIH 3T3 fibroblasts not expressing the ras oncogene (− ras). In isotonic extracellular fluid, the cell volume of + ras cells (2.70±0.08 pl) is significantly greater than the cell volume of −ras cells (2.04±0.10 pl). Both, + ras and − ras cells exhibit a regulatory cell volume increase in hypertonic extracellular fluid and a regulatory cell volume decrease in hypotonic extracellular fluid. The regulatory cell volume decrease is inhibited by 1 mmol/l quinidine and barium, the regulatory cell volume increase is inhibited in − ras and + ras cells by dimethyl-amiloride (100 μmol/l) and, only in + ras cells, by furosemide (100 μmol/l) and bumetanide (10 μmol/l). In conclusion, expression of the ras oncogene leads to a shift of the set point for cell volume regulation to greater cell volumes, which may contribute to the activation of the Na+/H+ exchanger and Na+, K+, 2Cl− cotransport.

Metabolism of Amino Acids and Ammonia

Amino acids are not only essential building blocks for the synthesis of peptides, proteins, amino sugars, purines, and pyrimidines, but also a major source of energy in different organs. Apart from this, several amino acids or their derivatives are important for organ-specific functions, such as neurotransmission in the brain or stimulation of hormone secretion by endocrine glands.

Alkalinization of acidic cellular compartments following cell swelling

Osmotic swelling of rat hepatocytes increases fluorescence of Acridine orange and of fluorescein isothiocyanate (FITC)‐dextran, both indicative of alkalinization of acidic intracellular vesicles. Similar to osmotic cell swelling, insulin and glutamine lead to an increase in Acridine orange fluorescence, an effect virtually abolished upon osmotic reversal of glutamine‐induced cell swelling. Barium, which blocks K+ channels in the plasma membrane, similarly leads to cell swelling and increase of Acridine orange fluorescence. Since proteolysis is governed by lysosomal pH, these observations indicate that the anti‐proteolytic action of osmotic cell swelling is mediated by lysosomal alkalinization. Thereby, insulin, glutamine and barium probably exert their anti‐proteolytic action by cell swelling and subsequent lysosomal alkalinization.

New clues to the pathophysiology of hepatorenal failure

SummaryIn patients with advanced liver disease, decreases in renal blood flow, glomerular filtration rate, and urinary output are frequently observed. The deterioration in renal function is usually not due to a unique cause but is the result of the concerted action of several mechanisms operating in parallel; decreased plasma protein formation and increased intrahepatic vascular resistance lead to sequestration of blood volume, favoring hypovolemia and reduction in cardiac output. At the same time enhanced formation of nitroxide leads to peripheral vasodilation; bacterial endotoxin escaping clearance by the diseased liver stimulates the expression of a long-acting nitroxide synthase. Furthermore, vasodilating intestinal mediators such as substance P escape inactivation by the liver. In the face of peripheral vasodilation the maintenance of blood pressure requires an increase in cardiac output, which is achieved by activation of sympathetic nervous tone, renal vasoconstriction, enhanced release of renin, angiotensin, aldosterone, and antidiuretic hormone, leading to renal retention of sodium and water. Renal vasoconstriction is opposed by vasodilatatory prostaglandins, and renal failure may be triggered by inhibition of prostaglandin formation. On the other hand, vasoconstrictive eicosanoids, such as thromboxane B2 and leukotriene E2, which escape hepatic inactivation, may contribute to renal vasoconstriction. Beyond these mechanisms disturbed hepatic regulation of renal function may participate in the generation of hepatorenal syndrome. The liver regulates renal function via both a hepatorenal reflex decreasing renal blood flow and a hypothetical liver-borne diuretic factor increasing renal blood flow. Both enhanced hepatorenal reflex activity and decreased formation of the liver-borne diuretic factor could participate in the pathogenesis of hepatorenal syndrome.

Net prostaglandin release by perfused rat liver after stimulation with phorbol 12-myristate 13-acetate

Phorbol myristate acetate, which was shown previously to elicit eicosanoid synthesis in primary cultures of Kupffer cells, led to a net release of prostaglandins (PG) D2 and E2 from the perfused rat liver. While a substantial amount of PGD2 (the major prostaglandin of Kupffer cells) left the liver, very little PGE2 was found in the effluent. Considerable amounts of immunologically reactive PGD2 and E2 were secreted with the bile. PGE2 rather than PGD2 was able to stimulate glycogenolysis and to increase perfusion pressure. These effects were, however, strongly dependent on the direction of the flow. If the liver was perfused in a retrograde fashion, i.e., from the vena cava to the portal vein, phorbol myristate acetate or PGE2 exerted only minor effects. These observations suggest a topological heterogeneity of producer and responder cells, respectively, in the liver sinusoid.

The role of calcium in cell shrinkage and intracellular alkalinization by bradykinin in Ha-ras oncogene expressing cells

In ras oncogene expressing cells, bradykinin leads to intracellular alkalinization by activation of the Na+/H+ exchanger. This effect is paralleled by oscillatory increase of intracellular calcium activity and cell shrinkage. Staurosporine (1 μmol/l) is not sufficient to prevent bradykinin induced intracellular alkalinization, thus pointing to a protein kinase C independent pathway for the activation of Na+/H+ exchange. The present study has been performed to elucidate, whether the increase of intracellular calcium contributes to cell shrinkage and activation of the Na+/H+ exchanger. To this end, the effects of the calcium ionophore ionomycin have been tested. Ionomycin leads to a dose dependent increase of intracellular calcium activity. At 100 nmol/l ionomycin intracellular calcium is increased from 114 ± 17 nmol/l to 342 ± 24 nmol/l (n = 9), a value within the range of intracellular calcium concentrations following application of bradykinin. The calcium increase is paralleled by a decrease of cell volume by 12 ± 2% (n = 5) and an increase of intracellular pH from 6.78 ± 0.02 to 6.90 ± 0.03 (n = 11), values similar to those following application of bradykinin. The alkalinizing effect of ionomycin is completely abolished in the presence of the novel Na+/H+ exchange inhibitor HOE 694 (10 μmol/l), but is not inhibited by 1 μmol/l staurosporine. Inhibition of K+ and Cl− channels by barium (5 mmol/l) and ochratoxin‐A (5 μmol/l) prevents both ionomycin induced cell shrinkage and protein kinase C independent intracellular alkalinization. It is concluded that bradykinin leads to intracellular alkalinization mainly by increasing intracellular calcium concentration. Calcium triggers calcium sensitive K+ channels, and presumably Cl− channels, the subsequent loss of cellular KCl leads to cell shrinkage which, in turn, activates Na+/H+ exchange.