K. P. Maier

Metabolism of phenylalanine in liver diseases

Zusammenfassung1. Ein Verfahren zur Aktivitätsbestimmung der Phenylalaninhydroxylase im Leberpunktat des Menschen wurde entwickelt. Die kinetischen Daten des Enzyms wurden bestimmt: DerKm-Wert für Phenylalanin beträgt 1,32 mM, für den Cofaktor 0,08 mM.2. Die Aktivität der Phenylalaninhydroxylase wurde in Punktaten von normaler Leber, Lebercirrhose, Alkohol-Hepatitis und bei anderen Leberkrankheiten bestimmt. Bei allen untersuchten Leberkrankheiten war die Enzymaktivität bezogen auf Feuchtgewicht oder DNA-Gehalt vermindert. In der cirrhotischen Leber betrug die Enzymaktivität nur 20% von der in normaler Leber.3. Nach oraler Gabe von L-Phenylalanin (100 mg/kg) wurde die Phenylalanin- und Tyrosinkonzentration im Serum über 5 Stunden verfolgt. Bei Kranken mit Lebercirrhose und Hepatitis war im Vergleich zu Gesunden die Phenylalaninkonzentration signifikant erhöht, die Tyrosinkonzentration signifikant vermindert.4. Aus dem Abfall der Phenylalaninkonzentration nach Erreichen des Maximalwertes wurde die Phenylalanin-Eliminationsrate, aus dem Anstieg der Tyrosinkonzentration die Tyrosin-Bildungsrate ermittelt. Zwischen beiden kinetischen Parametern besteht eine enge Korrelation. Bei Kranken mit Lebercirrhose und akuter Hepatitis sind Phenylalanin-Eliminationsrate und Tyrosin-Bildungsrate im Vergleich zu Gesunden signifikant vermindert, zwischen Kranken mit Alkoholhepatitis und Gesunden besteht hingegen kein signifikanter Unterschied hinsichtlich der beiden Parameter.5. Es besteht keine Korrelation zwischen der Aktivität der Phenylalaninhydroxylase im Lebergewebe einerseits, der Phenylalanin-Eliminationsrate bzw. Tyrosin-Bildungsrate andererseits.6. Aus diesen Befunden wird die Folgerung gezogen, daß die Zunahme der Serum-Konzentration von Phenylalanin bei Leberkrankheiten, insbesondere Lebercirrhose, teilweise durch einen verminderten Abbau der Aminosäure in der Leber erklärt werden kann. Jedoch sind zusätzlich weitere Faktoren, z.B. portocavale Shunts, von Bedeutung.Summary1. Amethod for the determination of phenylalaninehydroxylase-activity in needle biopsy material of human liver was developed and tested. The kinetic data of the enzyme were determined. TheKm for the substrate phenylalanine is 1,32 mM, for the cofactor 0,08 mM.2. The activity of phenylalaninehydroxylase was determined in biopsies from normal liver, liver cirrhosis, alcoholic hepatitis and other liver diseases. In all liver diseases the enzyme activity related to wet weight or DNA is reduced. The capacity for the hydroxylation of phenylalanine (of a cirrhotic liver) amounts to about 20% of normal liver.3. After an oral load with L-phenylalanine (100 mg/kg) the phenylalanine- and tyrosine-concentration in blood plasma was followed for 5 hours. Patients with liver cirrhosis or acute hepatitis show significant higher concentrations of phenylalanine, and significant lower concentrations of tyrosine than normal persons.4. From the decline of phenylalanine-concentration after the maximum the phenylalanine-elimination rate and from the increase of tyrosine concentration the tyrosine-production rate were calculated. Between phenylalanine-elimination rate and tyrosine-production rate a strong correlation exists. In patients with cirrhosis or acute hepatitis significantly reduced phenylalanine-elimination rate and tyrosine production rate were found compared with persons without liver disease. Patients with alcoholi hepatitis show no significant difference compared with normal persons.5. There is no correlation between the activity of phenylalaninehydroxylase in liver tissue and phenylalanine-elimination rate and tyrosine-production rate, respectively, calculated from the concentration of this amino acids in serum after a phenylalanine load.6. We conclude from these findings that the increased serum concentration of phenylalanine in some liver diseases, especially liver cirrhosis, can partly be explained by the diminished metabolism of phenylalanine in the liver. Portocaval shunts probably contribute to the elevated level of phenylalanine in serum.

Urea‐Cycle Enzymes in Normal Liver and in Patients with Alcoholic Hepatitis

Abstract Urea‐cycle enzymes and omithine‐ketoacid‐transaminase have been measured in biopsy specimens of liver from healthy subjects and from patients suffering from alcoholic hepatitis. Both groups of subjects received a hospital diet of about 100 g of protein daily. Extraction of enzymes from biopsy specimens was performed by a standardized technique.–The DNA content of liver did not vary significantly between the groups, whereas protein content was significantly lower in patients with alcoholic hepatitis than in controls (p < 0.05). Of the enzymes tested, the activities of carbamyl‐phosphate synthetase and arginase were significantly decreased (p < 0.05 and p < 0.005 respectively) in patients with alcoholic hepatitis. Activities of arginosnccinate lyase, ornithine‐ ketoacid‐transaminase and ornithine‐carbamylphosphate transaminase remained unchanged in both groups.–These results demonstrate that alterations in arginase and carbamylphoa‐phate synthetase‐activities in the liver of patients with alcoholic hepatitis precede the histological manifestation of liver cirrhosis, which is associated with a significant decrease in some urea cycle enzymes [3, 15, 16]. Therefore the determination of arginase and carbamylphosphate synthetase in needle‐biopsies of human liver represent sensitive parameters of liver cell necrosis during the course of alcoholic hepatitis.

Activities of urea-cycle enzymes in chronic liver disease

ZusammenfassungDie Aktivitäten der Harnstoffzyklusenzyme wurden in Leberbiopsieproben von Patienten mit chronisch-persistierender Hepatitis (CPH), chronisch-aggressiver Hepatitis (CAH) und mit Lebercirrhose gemessen. Die meisten Aktivitäten der Harnstoffzyklusenzyme unterschieden sich nicht bei Patienten mit CPH im Vergleich zu Lebergesunden. Bei Patienten mit CAH und mit Lebercirrhose konnte ein significanter Abfall (p<0,05) der Enzymaktivitäten im Vergleich mit Kontrollpersonen nachgewiesen werden. Ebenfalls ergaben sich signifikant erniedrigte Aktivitäten der meisten Harnstoffzyklusenzyme bei Patienten mit CAH im Vergleich zu Patienten mit CPH. Keine statistisch signifikanten Differenzen der Enzymaktivitäten ließen sich beim Vergleich von Patienten mit CAH und mit kompletter Lebercirrhose nachweisen.Zusammenfassend konnte gezeigt werden, daß fortschreitende chronische Lebererkrankungen zu zunehmenden Veränderungen von Enzymaktivitäten eines leberspezifischen Stoffwechselabschnittes führen. Die Abnahme der Aktivitäten der Schrittmacherenzyme des Harnstoffzyklus (Carbamylphosphat-Synthetase und Argininosuccinat-Synthetase) ist bereits im Stadium einer CAH nahezu identisch wie bei kompletter Lebercirrhose. Hieraus ergibt sich, daß die ausgeprägten enzymatischen Veränderungen im Stoffwechselweg der sekundären Ammoniakentgiftung den histologischen Veränderungen einer irreversiblen Leberparenchymschädigung vorausgehen.SummaryThe activities of urea-cycle enzymes were measured in liver biopsies of patients suffering from chronic-persistent hepatitis (CPH), chronic-active hepatitis (CAH) and liver cirrhosis. Most of the activities of urea-cycle enzymes did not differ in the case of CPH as compared to controls. Chronic-active hepatitis and liver cirrhosis are associated with a significant (p<0.05) decrease of enzyme activity as compared to normal persons. Most of the urea-cycle enzymes are significantly decreased in patients with CAH in comparison with CPH. No significant differences can be demonstrated in the case of CAH as compared to patients with complete cirrhosis.In conclusion, progression of chronic liver disease is associated with increasing alterations of enzyme activities catalyzing a liver specific metabolic pathway. The decrease of the activities of the key enzymes of the urea cycle (Carbamylphosphate-Synthetase and Arginino-succinate-Synthetase) is nearly identical both in CAH and liver cirrhosis, although CAH may be a reversible disease. Therefore, marked alterations in the metabolic pathway of ammonia detoxification seem to preceed the histological manifestation of irreversible liver damage.

Possible sites of interaction of acute renal failure with amino acid utilization for gluconeogenesis in isolated perfused rat liver

Abstract. Experiments were performed to elucidate the mechanisms involved in the enhanced conversion of amino acids to glucose in acute uraemic rats. Increased gluconeogenesis from a mixture of serine, threonine, lysine, glutamate, ornithine and citrulline was confirmed using a non‐recirculating perfusion system. Stimulation was concentration dependent, being most pronounced at physiological amino acid concentrations. Stimulation of glucose and urea formation could be mimicked by using serine alone whereas with lactate and pyruvate inhibition of gluconeogenesis was observed. Serine dehydratase activity was significantly elevated following nephrectomy. Further, the uptake of the non‐metabolizable amino acid α‐aminoisobutyrate was considerably increased. It is concluded that serine may play a special role as substrate for the additional glucose formation in acute uraemic rats, probably mediated by an activation of serine dehydratase. Acceleration of amino acid transport seems to represent an additional component of stimulation of amino acid utilization in acute uraemia.

Studies on Urea Cycle Enzymes in Rat Liver during Acute Uraemia

Abstract. Activities of urea cycle enzymes were measured in the liver of starved rats 12 and 48 h after bilateral nephrectomy. Control experiments (sham‐operated, starved rats) revealed that the activities of only two enzymes of the cycle are altered in the uraemic state: argininosuccinic acid synthetase (EC 6. 3. 4. 5.), which is considered to be rate limiting for urea production and carbamyl phosphate synthetase (EC 2. 7. 2. 5.). Alterations in ornithine concentration of the liver, a possible cause of an increased urea production rate, could not be detected previously (21). Our present results do not support the concept that a decrease of the activity of ornithine‐6‐amino transferase (EC 2. 6. 1. 13), leading to an increase in the ornithine content of the liver is responsible for the accelerated urea production rate in the liver of acute uraemic rats.

Influence of steroids on urea-cycle enzymes in chronic human liver disease

ZusammenfassungDie Aktivitäten der Enzyme des Harnstoffzyklus wurden in Leberbiopsien von Normalpersonen, von Patienten mit alkoholischer Fettleber-Hepatitis und chronisch-aggressiver Hepatitis bestimmt.Prednison wurde 5 Tage vor der Biopsie in fallender Dosierung (1,5 mg-0,5 mg/kg/Körpergewicht) verabreicht an Patienten mit alkoholischer Fettleber-Hepatitis und an Lebergesunde. Patienten mit chronisch-aggressiver Hepatitis standen unter einer Steroidtherapie (15–20 mg Prednison/tgl.) für mehrere Monate.Die Aktivitäten der Harnstoffzyklus-Enzyme wurden bei lebergesunden Personen durch Steroide in der genannten Dosis nicht verändert. Bei Patienten mit alkoholischer Hepatitis waren die meisten Harnstoffzyklus-Enzyme signifikant erniedrigt (P<0,05) im Vergleich zu lebergesunden Kontrollpersonen. Die Gabe von Glucocorticoiden veränderte die Enzymaktivitäten nicht.Die Aktivitäten der meisten Harnstoffzyklus-Enzyme sind signifikant (p<0,05) erniedrigt bei (unbehandelten) Patienten mit chronisch-aggressiver Hepatitis. Bei einigen dieser Patienten konnte nach Glucocorticoidgabe eine Remission der Erkrankung aufgrund klinischer, biochemischer und histologischer Parameter nachgewiesen werden.Bei diesen Patienten kam es zu einem signifikanten Anstieg der geschwindigkeitslimitierenden Enzyme des Harnstoffzyklus (ASAS und CPS). Dagegen blieb der Anstieg der Enzymaktivitäten aus bei solchen Patienten, welche trotz Steroidgaben nicht in eine — auch histologisch nachweisbare — Remission kamen.SummaryActivities of Krebs-Henseleit enzymes were determined in liver biopsies of normal persons and in patients suffering from alcoholic hepatitis and chronic active hepatitis. Prednisone was administered for five days in falling dosis (1.5 mg–0.5 mg/kg/body weight) to patients with alcoholic hepatitis and to controls. Patients with chronic active hepatitis received 15–20 mg prednisone daily for more than three months.In healthy persons prednisone did not influence the activities of Krebs-Henseleit enzymes.In patients with alcoholic hepatitis most of the urea-cycle enzymes are significantly decreased (p<0.05) when compared to controls. After glucocorticoid administration enzyme activities remained unchanged.Activities of most of the urea-cycle enzymes are significantly (p<0.05) decreased in untreated patients with chronic active hepatitis.In some of these patients, glucocorticoid administration was associated with a remission as proved by clinical, biochemical and histological data.Activities of the rate-limiting enzymes of the ureacycle (ASAS, CPS) increased significantly in these patients.By contrast, alterations of enzyme activities could not be observed in patients who failed to respond favourably to steroid treatment.

Intrazelluläre Lokalisation von Enzymen des Krebs-Henseleit-Cyclus in der Leber

Enzymes of the Krebs-Henseleit urea-cycle were localized by means of differential centrifugation and fractional tissue extraction in rat liver and in human liver. Argininosuccinatlyase (ASAL) and Argininosuccinatsynthetase (ASAS) represent enzymes of the soluble cytoplasmic fraction. Ornithine-ketoacid-transaminase(OKT), carbamyl-phosphate-synthetase (CPS) and ornithine-carbamyl-transferase (OCT) are localized in the mitochondrial and nuclei fractions of the liver cell. Most of the arginase activity is bound to subcellular structures (probably to nuclei). A small portion of arginase-activity was found in the soluble cytoplasmatic fraction. The enzymes of the Krebs-Henseleit urea-cycle are equally distributed in rat liver and in human liver. Differences in the subcellular localisation of (mitochondrial) enzymes in human liver could be attributed to mitochondrial breakage during tissue preparation and do not represent in-vivo conditions.SummaryEnzymes of the Krebs-Henseleit urea-cycle were localized by means of differential centrifugation and fractional tissue extraction in rat liver and in human liver.Argininosuccinatlyase (ASAL) and Argininosuccinatsynthetase (ASAS) represent enzymes of the soluble cytoplasmic fraction. Ornithine-ketoacid-transaminase(OKT), carbamyl-phosphate-synthetase (CPS) and ornithine-carbamyl-transferase (OCT) are localized in the mitochondrial and nuclei fractions of the liver cell. Most of the arginase activity is bound to subcellular structures (probably to nuclei). A small portion of arginase-activity was found in the soluble cytoplasmatic fraction. The enzymes of the Krebs-Henseleit urea-cycle are equally distributed in rat liver and in human liver. Differences in the subcellular localisation of (mitochondrial) enzymes in human liver could be attributed to mitochondrial breakage during tissue preparation and do not represent in-vivo conditions.ZusammenfassungEnzyme des Krebs-Henseleit-Harnstoffcyclus wurden durch Differentialzentrifugation und fraktionierte Extraktion in Rattenleber und in menschlicher Leber lokalisiert.Argininosuccinatlyase (ASAL) und Argininosuccinatsynthetase (ASAS) sind Enzyme der löslichen Cytoplasmafraktion. Ornithin-Ketosäure-Transaminase (OKT), Carbamyl-Phosphat-Synthetase (CPS) und Ornithin-Carbamyl-Transferase (OKT) sind in der Kern- und Mitochondrienfraktion der Leberzelle lokalisiert. Der größte Teil der Arginase-Aktivität ist an subcelluläre Strukturen (möglicherweise an Kerne) gebunden. Ein kleiner Teil der Arginase-Aktivität wurde in der löslichen Cytoplasmafraktion gefunden. Die Enzyme des Krebs-Henseleit-Harnstoffcyclus sind in der Rattenleber und in der menschlichen Leber identisch verteilt. Unterschiedliche Resultate hinsichtlich der subzellulären Lokalisation mitochondrialer Enzyme des Krebs-Henseleit-Cyclus in der menschlichen Leber beruhen auf der Schädigung von Mitochondrienmembranen während der Gewebspräparation und nicht auf unterschiedlichen in-vivo Bedingungen.

Fraktionierte Zellextraktion zur Differenzierung cytoplastischer, mitochondrialer und lysosomaler Enzyme der Leber

SummaryEnzymes of extramitochondrial, mitochondrial and lysosomal origin were differentiated by the method of fractional tissue extraction from rat liver. It could be confirmed that soluble enzymes of the extramitochondrial (cytoplasmatic) compartment (glycerine aldehydphosphate-dehydrogenase, argininosuccinate lyase) and soluble enzymes of the mitochondrial matrix can be differentiated easily according to their different intracellular localisation. Additionally, our results emphasize that soluble enzymes of both compartements and marker-enzymes of the lysosomal cell space which are known to be structurally-bound, can be differentiated too. However, the method failed to establish significant differences between enzymes located in the submitochondrial compartment (adenylatekinase, monaminooxidase and succinic-dehydrogenase) and lysosomal enzymes (cathepsin-D and N-acetyl-D-glucosaminidase).ZusammenfassungAm Modell der Rattenleber wurde die fraktionierte Zellextraktion zur Differenzierung von Enzymen extramitochondrialen, mitochondrialen und lysosomalen Ursprungs angewandt.Mit dieser Methode ist eine Trennung löslicher Enzyme des extramitochondrialen Zellabschnittes (Glycerinaldehydphosphatdehydrogenase, Argininosuccinatlyase) von solchen der mitochondrialen Matrix (Glutamatdehydrogenase, Ornithin-Ketosäure-Transaminase) möglich. Darüber hinaus machen die Ergebnisse deutlich, daß auch die Marker-Enzyme des lysosomalen Zellabschnittes differenziert extrahiert und gemessen werden können.Die gleichzeitige Bestimmung von strukturgebundenen Enzymen des submitochondrialen Zellabschnittes (Monaminooxidase, Adenylatkinase, Succinatdehydrogenase) und von strukturgebundenen lysosomalen Enzymen (Kathepsin D und N-Acetyl-D-Glucosaminidase) ergab, daß derartige Enzyme mit dem Verfahren der fraktionierten Zellextraktion nicht unterschieden werden können.Enzymes of extramitochondrial, mitochondrial and lysosomal origin were differentiated by the method of fractional tissue extraction from rat liver. It could be confirmed that soluble enzymes of the extramitochondrial (cytoplasmatic) compartment (glycerine aldehydphosphate-dehydrogenase, argininosuccinate lyase) and soluble enzymes of the mitochondrial matrix can be differentiated easily according to their different intracellular localisation. Additionally, our results emphasize that soluble enzymes of both compartements and marker-enzymes of the lysosomal cell space which are known to be structurally-bound, can be differentiated too. However, the method failed to establish significant differences between enzymes located in the submitochondrial compartment (adenylatekinase, monaminooxidase and succinic-dehydrogenase) and lysosomal enzymes (cathepsin-D and N-acetyl-D-glucosaminidase).

Isozymes of Aspartate Aminotransferase in Rat Liver during Acute Uraemia

Abstract. Total activity of aspartate‐aminotransferase (GOT; EC 2.6.1.1) and activities of the cytoplasmic (c‐GOT) and mitochondrial (m‐GOT) isozymes were measured in rat liver 24 and 48 h after bilateral nephrectomy.