Interorgan ammonia metabolism in liver failure: the basis of current and future therapies

Hepatic encephalopathy complicates the course of both acute and chronic liver disease and its treatment remains an unmet clinical need. Ammonia is thought to be central in its pathogenesis and remains an important target of current and future therapeutic approaches. In liver failure, the main detoxification pathway of ammonia metabolism is compromised leading to hyperammonaemia. In this situation, the other ammonia‐regulating pathways in multiple organs assume important significance. The present review focuses upon interorgan ammonia metabolism in health and disease describing the role of the key enzymes, glutamine synthase and glutaminase. Better understanding of these alternative pathways are leading to the development of new therapeutic approaches.     

Leucine metabolism in stable cirrhosis

Alterations in protein and amino acid metabolism have been postulated to explain the frequent observations of muscle wasting and decreased plasma branched-chain amino acid concentrations in cirrhosis. In order to investigate the changes in protein metabolism, we have measured the rates of leucine turnover and oxidation in six stable, biopsy-proven cirrhotics and six age and sex-matched healthy control subjects after an overnight fast, using [1-13C]leucine tracer. Following a primed constant-rate infusion of [1-13C]leucine, the 13C enrichments of plasma leucine and expired CO2 were used to estimate leucine turnover and oxidation, respectively. Fat-free body mass was estimated from the measurements of total body water as quantified by H2[18O] tracer dilution. The rates of CO2 production and oxygen consumption were measured hourly during the study period, using open-circuit respiratory calorimetry. Urinary urea, ammonia and total nitrogen excretion rates were quantified from timed urine samples. Even though the plasma leucine levels were lower in cirrhotics as compared with controls (100.5 +/- 17.1 vs. 138.3 +/- 20.4 mumoles per liter, mean +/- S.D., p less than 0.001), the rates of leucine turnover were not significantly different in the two groups (89.4 +/- 19.0 vs. 87.8 +/- 19.0 mumoles per kg X hr). In contrast, the rates of leucine oxidation were significantly reduced in cirrhosis (8.1 +/- 2.5 vs. 12.7 +/- 3.1 mumoles per kg X hr, p less than 0.01). When all subjects were considered, the leucine oxidation rate was correlated with plasma leucine concentration (r = 0.62, p less than 0.03).(ABSTRACT TRUNCATED AT 250 WORDS)     

Regional differences in cerebral blood flow and cerebral ammonia metabolism in patients with cirrhosis

Clinical and histopathological findings hint at regional differences in the brain's sensitivity to metabolic changes in cirrhosis. The aim of the present study was to examine regional differences in cerebral ammonia metabolism in patients with cirrhosis and grade 0-to-I hepatic encephalopathy (HE). (13)N-ammonia, (15)O-water positron emission tomography (PET) and magnetic resonance imaging (MRI) were performed. Quantitative values of cerebral blood flow (CBF) and the initial cerebral ammonia uptake rate (K1) were derived for several regions of interest from images of the desired parameters after interactive coregistration with the patients' MRI-studies. CBF (mL/mL/min), K1 (mL/mL/min), and the ammonia extraction fraction (K1/CBF) showed marked regional variance with the highest levels in the thalamus, the lenticular nucleus, and the cerebellum. In conclusion, the regional differences in cerebral ammonia uptake correspond to the distribution of histopathological changes in the brain of patients with cirrhosis as well as clinical features of HE, characterized by signs of basal ganglia and cerebellar dysfunction with corresponding signs of functional impairment, especially of the frontal cortex and cingulate gyrus.     

Effects of branched-chain amino acids on nitrogen metabolism in patients with cirrhosis

This study was conducted to determine whether an amino acid solution enriched with branched-chain amino acids altered protein catabolic rates and plasma ammonia in patients with cirrhosis. Nine stable subjects were given two peripheral intravenous infusions: a standard amino acid solution (solution A) and a branched-chain-enriched solution containing 97% more leucine (solution B). Each solution was given for separate 9-day (group 1, n = 6) or 3-day (group 2, n = 3) periods. Amino acid solutions delivered 0.7 gm protein.kg-1.day-1. Diets provided an additional 0.3 gm protein plus maintenance calories. Protein turnover was assessed by a primed continuous infusion of [1-14C] leucine in six patients (three patients in group 1 and three patients in group 2). Nitrogen balance and urinary 3-methyl histidine excretion were determined in group 1 patients. Compared with solution A, solution B increased leucine flux and leucine oxidation but had no significant effect on protein synthesis or catabolism based on the plasma specific activity of either leucine or alpha-ketoisocaproic acid. The additional leucine infused with solution B was quantitatively oxidized. Nitrogen balance did not differ with the two solutions and there was also no difference in the urinary excretion of 3-methyl histidine, suggesting that muscle protein catabolism was unchanged. Plasma ammonia concentration decreased significantly during the infusion of solution B and was associated with a slight fall in plasma glucagon concentration. The results indicated that a branched-chain-enriched amino acid solution did not alter protein synthesis or catabolism although it did lower the plasma ammonia when compared with a standard amino acid formula in stable cirrhotic patients.     

Apolipoprotein B metabolism in humans: studies with stable isotope-labeled amino acid precursors

This article reviews the literature from 1986 to early 2001 relating to apoB100 and apoB48 kinetics in humans using amino acid precursors labeled with stable isotopes. The following subjects are reviewed: (1) methodology; (2) normal individuals and the effects of aging; (3) diet; (4) hereditary dyslipidemias: familial hypercholesterolemia, familial combined hyperlipidemia, cholesteryl ester storage disease, cholesteryl ester transfer protein deficiency, lipoprotein lipase deficiency, familial hypobetalipoproteinemia, and truncated forms of apoB; (5) hormonal perturbations: estrogen, insulin, diabetes, obesity, and growth hormone; (6) the nephrotic syndrome; and (7) the effects of the statin class of drugs. Because of the advances which have been made in mass spectrometry techniques, the advantages of using non-radioactive tracers in humans have made stable isotope kinetic studies the present day standard in this area of research.     

Cerebral metabolism of ammonia and amino acids in patients with fulminant hepatic failure.

BACKGROUND & AIMS: High circulating levels of ammonia have been suggested to be involved in the development of cerebral edema and herniation in fulminant hepatic failure (FHF). The aim of this study was to measure cerebral metabolism of ammonia and amino acids, with special emphasis on glutamine metabolism. METHODS: The study consisted of patients with FHF (n = 16) or cirrhosis (n = 5), and healthy subjects (n = 8). Cerebral blood flow was measured by the 133Xe washout technique. Blood samples for determination of ammonia and amino acids were drawn simultaneously from the radial artery and the internal jugular bulb. RESULTS: A net cerebral ammonia uptake was only found in patients with FHF (1.62 +/- 0.79 micromol x 100 g(-1) x min(-1)). The cerebral glutamine efflux was higher in patients with FHF than in the healthy subjects and cirrhotics, -6.11 +/- 5.19 vs. -1.93 +/- 1.17 and -1.50 +/- 0.29 micromol x 100 g(-1) x min(-1), respectively (P < 0.05). Patients with FHF who subsequently died of cerebral herniation (n = 6) had higher arterial ammonia concentrations, higher cerebral ammonia uptake, and higher cerebral glutamine efflux than survivors. Intervention with short-term mechanical hyperventilation in FHF reduced the net cerebral glutamine efflux, despite an unchanged net cerebral ammonia uptake. CONCLUSIONS: Patients with FHF have an increased cerebral glutamine efflux, and short-term hyperventilation reduces this efflux. A high cerebral ammonia uptake and cerebral glutamine efflux in patients with FHF were associated with an increased risk of subsequent fatal intracranial hypertension.     

Branched-chain amino acids and muscle ammonia detoxification in cirrhosis

Branched-chain amino acids (BCAA) are used as a therapeutic nutritional supplement in patients with cirrhosis and hepatic encephalopathy (HE). During liver disease, the decreased capacity for urea synthesis and porto-systemic shunting reduce the hepatic clearance of ammonia and skeletal muscle may become the main alternative organ for ammonia detoxification. We here summarize current knowledge of muscle BCAA and ammonia metabolism with a focus on liver cirrhosis and HE. Plasma levels of BCAA are lower and muscle uptake of BCAA seems to be higher in patients with cirrhosis and hyperammonemia. BCAA metabolism may improve muscle net ammonia removal by supplying carbon skeletons for formation of alfa-ketoglutarate that combines with two ammonia molecules to become glutamine. An oral dose of BCAA enhances muscle ammonia metabolism but also transiently increases the arterial ammonia concentration, likely due to extramuscular metabolism of glutamine. We, therefore, speculate that the beneficial effect of long term intake of BCAA on HE demonstrated in clinical studies may be related to an improved muscle mass and nutritional status rather than to an ammonia lowering effect of BCAA themselves.     

Effect of insulin and plasma amino acid concentration on leucine metabolism in cirrhosis

Clinically stable patients with cirrhosis demonstrate insulin resistance with regard to glucose metabolism. However, much less is known about the two major factors, insulin and plasma amino acid concentration, that regulate protein metabolism in cirrhotic patients. To examine this question, we performed paired euglycemic insulin clamp studies in combination with 14C-leucine and indirect calorimetry. In the first study insulin alone was infused, and the plasma amino acid concentration was allowed to decline. During the second study a balanced amino acid solution was infused with insulin to increase the total plasma amino acid concentration approximately twofold. Insulin-mediated glucose disposal (4.68 vs. 6.45 mg/kg-min, p less than 0.01) was significantly impaired by 30% in cirrhotic patients during both insulin clamp studies. In the postabsorptive state, cirrhotic patients manifested low plasma leucine (76 vs. 102 mumol/L) and alpha-ketoisocaproate (19 vs. 30 mumol/L) concentrations, but all parameters of leucine turnover were normal. When insulin alone was infused, the endogenous leucine flux (an index of protein degradation) declined similarly in cirrhotic patients (30.8 mumol/m2-min) and control (26.9) subjects, and this was accompanied by a similar decrease in plasma leucine concentration (31% vs. 33%). The decline in circulating leucine concentration was accompanied by a parallel decline in leucine oxidation (5.1 vs. 4.6 mumol/m2-min) and nonoxidative (28.9 vs. 26.0 mumol/m2-min) leucine disposal, which were of similar magnitude in cirrhotic patients and control subjects, respectively. In both cirrhotic patients and control subjects, combined hyperinsulinemia/hyperaminoacidemia elicited a similar stimulation of nonoxidative leucine disposal (an index of protein synthesis) and leucine oxidation while causing a greater suppression of endogenous leucine flux than observed with insulin alone. Thus the suppressive effect of insulin on protein degradation and the stimulatory effect of insulin/amino acid infusion on protein synthesis are not impaired in cirrhotic patients, demonstrating a clear-cut dissociation between the effects of insulin on protein and glucose metabolism.     

Plasma amino acid response to protein ingestion in patients with liver cirrhosis

The metabolic effects of a protein-rich meal were studied for 3 h in 10 controls and in 20 cirrhotic patients. After protein ingestion, blood glucose did not vary significantly. Insulin and glucagon levels rose in controls and, more markedly, in cirrhotics. Aromatic amino acids and tryptophan increased more in cirrhotics as a result of their decreased liver function. Similarly, branched-chain amino acids increased by 153 +/- 14 nmol/ml X min (mean +/- SE) in controls and by 259 +/- 27 nmol/ml X min in cirrhotics (p less than 0.02), in the presence of a markedly increased insulin response. Branched-chain amino acid metabolism mainly occurs in skeletal muscle under insulin control; in cirrhosis, it might be reduced as a consequence of insulin resistance. To support this hypothesis, the effects of the protein meal were compared with those of an oral glucose load in 15 cirrhotic patients. Branched-chain amino acid response to protein ingestion significantly correlated with blood glucose response to oral glucose (r = 0.714), and with insulin resistance during the glucose tolerance test, when assessed by the insulinogenic index (r = 0.628). Similarly, in 8 patients, increased branched-chain amino acid response also correlated with the index of tissue sensitivity to insulin obtained by means of the glucose clamp technique during continuous insulin infusion (r = -0.809). We conclude that liver cirrhosis is characterized by an abnormal branched-chain amino acid response to protein ingestion, which matches the well-known intolerance to oral glucose. Both alterations are possibly due to decreased peripheral insulin activity on substrates.     

氨在肝硬化患者中的预后作用

【据《Hepatology》2019 年9 月报道】题:氨在肝硬化患者中的预后作用(作者Shalimar 等)氨被认为是肝性脑病(HE)发病机制的核心,但其在肝硬化和急性失代偿期患者中的预后作用尚不清楚。本研究的目的是确定氨水平与HE 严重程度之间的关系,以及其与器官功能障碍和短期死亡率的关系。     

Plasma amino acid levels in patients with colorectal cancers and liver cirrhosis with hepatocellular carcinoma

BACKGROUND/AIMS: A variety of cancer-bearing patients have been shown to have disturbances in carbohydrate, lipid and protein metabolism. The complex of metabolic derangements of protein in cancer patients may be reflected by alteration in the plasma free amino acid profile. In this study, we try to investigate the plasma free amino acid profile in patients with colorectal cancer and liver cirrhosis with hepatocellular carcinoma, which are the most common cancers in Taiwan. METHODOLOGY: Fasting venous blood samples were drawn from sixteen control volunteers and 42 cancer-bearing patients including 14 early stage colorectal cancer patients (Duke A and B), 18 late stage ones (Duke C and D) and 10 liver cirrhotic patients with hepatocellular carcinoma. Seventeen amino acid levels were measured using a Beckman amino acid analyzer. RESULTS: About one third of early or late colorectal cancer patients had body weight loss more than 10% in half a year and were defined as malnourished. For individual amino acids, in early colorectal cancer patients, the plasma level of most essential amino acids and non-essential amino acids decreased (significantly in Tyr, Ala, Met, Phe and Thr). In late stage colorectal cancer patients and patients with liver cirrhosis with hepatocellular carcinoma, plasma levels of most essential amino acids and non-essential amino acids decreased more obviously. For group amino acids, the plasma levels of essential amino acids, non-essential amino acids, gluconeogenic amino acids and branched-chain amino acids were also lower in the cancer patients than those in control volunteers. The difference was also noticeably significant in patients with late stage colorectal cancer and liver cirrhosis with hepatocellular carcinoma. The plasma free amino acid patterns in colorectal cancer patients are quite different from those in patients with non-gastrointestinal cancer and weight loss. The plasma level of essential amino acids and branched-chain amino acids was not kept within normal range in colorectal cancer patients. Elevation of plasma aromatic amino acids and methionine levels usually observed in liver cirrhotic patients without hepatocellular carcinoma was not apparent in our cirrhotic patients with hepatocellular carcinoma. CONCLUSIONS: The plasma free amino acid patterns in our colorectal cancer patients and cirrhotic patients with hepatocellular carcinoma were rather characteristic. The results will offer useful tools for improving diagnosis and therapy.     

Myocardial amino acid metabolism in patients with chronic ischemic heart disease

Summary In nine patients with ischemic heart disease the authors investigated the arterio-coronary venous difference of free amino acids in serum at rest and during pacing. At rest aspartate was the only amino acid with a marked positive arterio-coronary venous difference. At the peak of pacing, in addition to aspartate, there is a significant positive arterio-coronary venous difference in glutamate, leucine and isoleucine and a significantly negative difference in cystine-cysteine and glutamine with asparagine. When expressed in per cent of the arterial level, the negative difference in alanine is also significant. Among the mutual correlations of arterio-coronary venous differences the negative correlation between alanine and lactate is most significant, which suggests that under normal conditions pyruvate is transformed rather to alanine, while in ischemia lactate is formed from pyruvate, and released from the heart muscle. There is also a positive correlation between alanine and glutamine and between leucine, isoleucine and glutamate.On the other hand, cystine-cysteine correlates very significantly but inversely with leucine, isoleucine and glutamate.The arterio-coronary venous difference of aspartate, though significantly positive, does not correlate with any other amino acid. The arterio-coronary vernous differences of ammonia and uric acid correlate inversely, whereby uric acid, contrary to ammonia, is practically not released from the heart muscle.

---

Myokardialer Aminosäuren-Stoffwechsel bei Patienten mit chronisch-ischämischen Herzleiden

Zusammenfassung Die Autoren untersuchten bei 9 Patienten mit ischämischen Herzleiden die arterio-koronarvenöse Differenz von freien Fettsäuren im Serum unter Ruhebedingungen und während künstlichem Schrittmacherantrieb. Bei körperlicher Ruhe war Aspartat die einzige Aminosäure mit einer ausgesprochenen positiven arterio-koronarvenösen Differenz. Auf dem Höhepunkt des Schrittmacherantriebs war zusätzlich zum Aspartat eine positive arteriovenöse Differenz für Glutamat, Leucin und Isoleucin und eine signifikant negative Differenz für Cystin-Cystein und Glutamin zusammen mit Asparagin zu verzeichnen. Ausgedrückt als Prozentsatz des arteriellen Spiegels war die negative Differenz auch bei Alanin signifikant. Bei wechselseitiger Korrelation der arteriovenösen Differenzen war die negative Beziehung zwischen Alanin und Lactat am eindeutigsten, was dafür spricht, daß unter normalen Bedingungen Pyruvat eher zu Alanin umgeformt wird, während im Zustand der Ischämie Lactat aus Pyruvat gebildet und vom Herzmuskel freigesetzt wird. Auch ergibt sich eine positive Korrelation zwischen Alanin und Glutamat und zwischen Leucin, Isoleucin und Glutamat. Andererseits korreliert Cystin-Cystein hoch signifikant, aber invers mit Leucin, Isoleucin und Glutamat.Die arterio-koronarvenöse Differenz von Aspartat, obwohl signifikant positiv, korreliert nicht mit irgendeiner anderen Aminosäure. Die arteriovenösen Differenzen von Ammoniak und Harnsäure korrelieren invers, wobei Harnsäure im Gegensatz zu Ammoniak praktisch nicht aus dem Herzmuskel freigesetzt wird.

---

---

With 3 figures and 1 table     

肝硬化与肝癌患者血浆氨基酸分析

目的探讨肝硬化与肝癌患者血浆氨基酸变化及其临床意义。方法用日立835氨基酸分析仪对46例肝硬化患者和44例肝癌患者血浆氨基酸进行测定。方法用日立835氨基酸分析仪对46例肝硬化患者和44例肝癌患者血浆氨基酸进行测定。结果肝硬化时血浆谷氨酸、胱氨酸、蛋氨酸、酪氨酸显著升高（P〈0.01）,苏氨酸、苯丙氨酸明显升高（P〈0.05）;肝癌时血浆苏氨酸、酪氨酸、苯丙氨酸显著升高（P〈0.01）,丝氨酸明显升高（P〈0.05）;两组中BCAA/AAA（支链氨基酸/芳香族氨基酸）比值均较正常对照组显著降低（P〈0.01）,缬氨酸、亮氨酸较正常对照组明显降低（P〈0.05）。说明以上血浆氨基酸分析与对照组之间的差异有统计学意义。结论血浆氨基酸分析对肝硬化与肝癌诊断和治疗有一定指导意义。     

Features of myocardial metabolism of some amino acids and ammonia in patients with coronary artery disease

Fractional myocardial extraction/release of glutamate, glutamine, alanine, ammonia, asparagine, glucose and lactate was studied in 12 subjects with normal coronary anatomy (controls) and 28 patients with coronary artery disease (CAD) during rest and atrial pacing. At rest patients with CAD showed an increased myocardial extraction of glutamate, glucose and lactate and an augmented glutamine and alanine release compared with controls. In all CAD patients myocardial ammonia and asparagine release was found at rest, while all controls showed myocardial extraction of these compounds. Myocardial glutamate extraction correlated positively with glucose and lactate extraction, glutamine and alanine release and inversely with ammonia release in CAD patients at rest. In patients with two- and three-vessel disease pacing-induced ischaemia resulted in a pronounced decrease in myocardial glutamate extraction and glutamine release, augmented myocardial production of ammonia and asparagine and a conversion of lactate extraction into lactate release. During pacing myocardial glutamate extraction was related to alanine and glutamine release and correlated inversely with ammonia and lactate release in these patients. The results indicate that glutamate extraction is closely connected with glucose and lactate extraction and ammonia binding via glutamine formation in the hearts of CAD patients and, thus, with the energy supply of ischaemic myocardium. An assessment of myocardial exchange of the nitrogenous compounds we have studied, complimentary to lactate, is a promising biochemical test for the identification of ischaemic heart disease in man.     

Correlations between zinc, amino acids and ammonia in liver cirrhosis

In view of the universal metabolic importance of zinc in the organism, it was the purpose of the present work to determine the concentrations of zinc in serum, of amino acids and ammonia in plasma of patients with liver cirrhosis, and investigate that correlations might exist between these substances. The study involved 18 patients with decompensated liver cirrhosis without coma and eleven with coma. The subjects with normal livers were used as controls. While confirming known data (reduced zinc levels, imbalance of plasma amino acids, hyperammonaemia in chronic liver diseases) the findings also revealed correlations between the above substances. A negative correlations existed between zinc and ammonia. Decreases in zinc serum levels were accompanied by increases in plasma ammonia concentrations in hepatic coma (p less than 0.05). Plasma levels of amino acids did not correlate with serum zinc concentrations.     

Vitamin B6 repletion in cirrhosis with oral pyridoxine: failure to improve amino acid metabolism

This study evaluated the effect of daily oral pyridoxine supplementation in patients with cirrhosis. Eight subjects were treated with 25 mg of pyridoxine for 28 days. Before and after the supplementation period, B6 status was assessed by measuring fasting plasma vitamer levels and response to a 25 mg oral pyridoxine load. In addition, a 24-hr urine collection was analyzed during each load study for B6 metabolites. The data indicated that supplementation achieved repletion of peripheral B6 stores, as evidenced by: (i) a significant (p less than 0.005) rise in fasting plasma pyridoxal phosphate after supplementation (mean +/- S.D. = 56.8 +/- 30.5 nmoles per liter) as compared to initial levels (17.0 +/- 17.8 nmoles per liter); (ii) a higher (p less than 0.05) percentage excretion of the pyridoxine load as urinary 4-pyridoxic acid (31.0 +/- 9.3%) compared to the initial load (19.6 +/- 5.8%), and (iii) a postsupplementation area under the plasma concentration vs. time curve for pyridoxal phosphate (377 +/- 529 nmoles.hr per liter), which was decreased (p less than 0.005) from the presupplementation value (934 +/- 756 nmoles.hr per liter). The postsupplementation fasting plasma pyridoxal phosphate concentrations were within the normal range. The consequences of B6 repletion on amino acid metabolism were measured by oral protein loads (n = 4) or oral methionine loads (n = 4). No significant changes were observed for methionine or any other amino acid in regard to plasma fasting concentration, peak concentration or AUC. Although the vitamin B6 deficiency of cirrhosis was corrected by daily oral pyridoxine supplementation, there was apparently no improvement in the deranged amino acid metabolism.     

Cholesterol and bile acid metabolism in moderately advanced, stable cirrhosis of the liver.

We have selected for this study a well-defined group of patients with moderately advanced but compensated alcoholic cirrhosis. They were well-nourished and had no ascites, varices, azotemia, or encephalopathy. Liver biopsy showed little or no necrosis and inflammation despite wide-spread fibrosis. Serum bilirubin, transaminase, alkaline phosphatase, albumin and globulins were essentially normal. Biochemical evidence for liver disease was restricted to modest elevation of BSP retention, gamma GTP, serum bile acid concentrations, and urinary bile acid excretion. Except for changes in the interrelationships among the three biliary lipids, they were generally spared the abnormalities of sterol metabolism described in other patients with more advanced, more active liver disease. Thus, striking abnormalities in the metabolism of cholesterol and bile acids probably require severe reductions in functioning hepatocellular mass, major portal-systemic shunting, high disease activity, or all three to become manifest.