Levels Of Aromatic Amino Acids Research Articles

Increased Plasma Citrulline in Mice Marks Diet-Induced Obesity and May Predict the Development of the Metabolic Syndrome

In humans, plasma amino acid concentrations of branched-chain amino acids (BCAA) and aromatic amino acids (AAA) increase in states of obesity, insulin resistance and diabetes. We here assessed whether these putative biomarkers can also be identified in two different obesity and diabetic mouse models. C57BL/6 mice with diet-induced obesity (DIO) mimic the metabolic impairments of obesity in humans characterized by hyperglycemia, hyperinsulinemia and hepatic triglyceride accumulation. Mice treated with streptozotocin (STZ) to induce insulin deficiency were used as a type 1 diabetes model. Plasma amino acid profiling of two high fat (HF) feeding trials revealed that citrulline and ornithine concentrations are elevated in obese mice, while systemic arginine bioavailability (ratio of plasma arginine to ornithine + citrulline) is reduced. In skeletal muscle, HF feeding induced a reduction of arginine levels while citrulline levels were elevated. However, arginine or citrulline remained unchanged in their key metabolic organs, intestine and kidney. Moreover, the intestinal conversion of labeled arginine to ornithine and citrulline in vitro remained unaffected by HF feeding excluding the intestine as prime site of these alterations. In liver, citrulline is mainly derived from ornithine in the urea cycle and DIO mice displayed reduced hepatic ornithine levels. Since both amino acids share an antiport mechanism for mitochondrial import and export, elevated plasma citrulline may indicate impaired hepatic amino acid handling in DIO mice. In the insulin deficient mice, plasma citrulline and ornithine levels also increased and additionally these animals displayed elevated BCAA and AAA levels like insulin resistant and diabetic patients. Therefore, type 1 diabetic mice but not DIO mice show the “diabetic fingerprint” of plasma amino acid changes observed in humans. Additionally, citrulline may serve as an early indicator of the obesity-dependent metabolic impairments.

News from the dark side!

News from the dark side!

High Levels of Aromatic Amino Acids in Gastric Juice during the Early Stages of Gastric Cancer Progression

BackgroundEarly-stage gastric cancer is mostly asymptomatic and can easily be missed easily by conventional gastroscopy. Currently, there are no useful biomarkers for the early detection of gastric cancer, and their identification of biomarkers is urgently needed.MethodsGastric juice was obtained from 185 subjects that were divided into three groups: non-neoplastic gastric disease (NGD), advanced gastric cancer and early gastric cancer (EGC). The levels of aromatic amino acids in the gastric juice were quantitated using high-performance liquid chromatography.ResultsThe median values (25th to 75th percentile) of tyrosine, phenylalanine and tryptophan in the gastric juice were 3.8 (1.7–7.5) µg/ml, 5.3 (2.3–9.9) µg/ml and 1.0 (0.4–2.8) µg/ml in NGD; 19.4 (5.8–72.4) µg/ml, 24.6 (11.5–73.7) µg/ml and 8.3 (2.1–28.0) µg/ml in EGC. Higher levels of tyrosine, phenylalanine and tryptophan in the gastric juice were observed in individuals of EGC groups compared those of the NGD group (NGD vs. EGC, P<0.0001). For the detection of EGC, the areas under the receiver operating characteristic curves (AUCs) of each biomarker were as follows: tyrosine, 0.790 [95% confidence interval (CI), 0.703–0.877]; phenylalanine, 0.831 (95% CI, 0.750–0.911); and tryptophan, 0.819 (95% CI, 0.739–0.900). The sensitivity and specificity of phenylalanine were 75.5% and 81.4%, respectively, for detection of EGC. A multiple logistic regression analysis showed that high levels of aromatic amino acids in the gastric juice were associated with gastric cancer (adjusted β coefficients ranged from 1.801 to 4.414, P<0.001).ConclusionIncreased levels of tyrosine, phenylalanine and tryptophan in the gastric juice samples were detected in the early phase of gastric carcinogenesis. Thus, tyrosine, phenylalanine and tryptophan in gastric juice could be used as biomarkers for the early detection of gastric cancer. A gastric juice analysis is an efficient, economical and convenient method for screening early gastric cancer development in the general population.

Effect of peptide size on antioxidant properties of African yam bean seed (Sphenostylis stenocarpa) protein hydrolysate fractions.

Enzymatic hydrolysate of African yam bean seed protein isolate was prepared by treatment with alcalase. The hydrolysate was further fractionated into peptide sizes of <1, 1–3, 3–5 and 5–10 kDa using membrane ultrafiltration. The protein hydrolysate (APH) and its membrane ultrafiltration fractions were assayed for in vitro antioxidant activities. The <1 kDa peptides exhibited significantly better (p < 0.05) ferric reducing power, diphenyl-1-picryhydradzyl (DPPH) and hydroxyl radical scavenging activities when compared to peptide fractions of higher molecular weights. The high activity of <1 kDa peptides in these antioxidant assay systems may be related to the high levels of total hydrophobic and aromatic amino acids. In comparison to glutathione (GSH), the APH and its membrane fractions had significantly higher (p < 0.05) ability to chelate metal ions. In contrast, GSH had significantly greater (p < 0.05) ferric reducing power and free radical scavenging activities than APH and its membrane fractions. The APH and its membrane fractions effectively inhibited lipid peroxidation, results that were concentration dependent. The activity of APH and its membrane fractions against linoleic acid oxidation was higher when compared to that of GSH but lower than that of butylated hydroxyl toluene (BHT). The results show potential use of APH and its membrane fractions as antioxidants in the management of oxidative stress-related metabolic disorders and in the prevention of lipid oxidation in food products.

HPLC–FLD determination of serum aromatic amino acids: Application in chronic kidney disease patients

BackgroundThe aromatic amino acids (AAA) are very important amino acids in the body and have been suggested to be involved in many diseases. We describe the development and full validation of a high performance liquid chromatography with fluorescence detection (HPLC–FLD) method for simultaneously quantitative determination of serum AAA. Furthermore, we aimed to explore the clinical significances of AAA for chronic kidney disease (CKD). MethodsSerum samples were deproteinized by perchloric acid. Separation was carried out on a Megres C18 column (4.6mm×250mm i.d., 5μm). The mobile phase consisted of 10% acetonitrile (v/v) in water and was run at a flow rate of 1.0ml/min. The eluted AAA was monitored by measuring the fluorescence intensity using the programmed wavelength detection setting. ResultsAAA determination in a serum sample was achieved in <10min. The linear ranges of the method were 0.55–275μmol/l, 3.05–1220.0μmol/l and 0.049–49μmol/l for Tyr, Phe and Trp, respectively. The limit of detection (LOD) was 0.014μmol/l for Tyr, 0.5μmol/l for Phe, and 0.0049μmol/l for Trp. Recovery and reproducibility were satisfactory. Compared with healthy control subjects, the CKD patients showed lower serum levels of AAA. It should probably available to apply for screening and monitoring of CKD. ConclusionsThe method is simple, fast, accurate, and suitable for routine analysis.

EFFECT OF AMINO ACIDS COMPOSITIONS ON THE LEVEL OF FREE AMINO ACIDS OF THE LIVER UNDER CHRONIC ALCOHOL INTOXICATION

In present study we investigated effect of amino acids composition of BCAA, taurine and BCAA, taurine and tryptophan on the liver amino acids pool of rats under chronic alcohol intoxication. Chronic ethanol intoxication led to increase levels of glutamate, asparagines, glycine, b-alanine, thirozine, and decrease level of alanine in the liver of rats. Administration of composition of BCAA, taurine and tryptophan was found to normalize the levels of aromatic amino acids, glutamate and alanine.

EFFECT OF AMINO ACIDS COMPOSITIONS ON THE LEVEL OF FREE AMINO ACIDS OF THE LIVER UNDER CHRONIC ALCOHOL INTOXICATION

In present study we investigated effect of amino acids composition of BCAA, taurine and BCAA, taurine and tryptophan on the liver amino acids pool of rats under chronic alcohol intoxication. Chronic ethanol intoxication led to increase levels of glutamate, asparagines, glycine, b-alanine, thirozine, and decrease level of alanine in the liver of rats. Administration of composition of BCAA, taurine and tryptophan was found to normalize the levels of aromatic amino acids, glutamate and alanine.

Antisense inhibition of enolase strongly limits the metabolism of aromatic amino acids, but has only minor effects on respiration in leaves of transgenic tobacco plants

Enolase catalyses the reversible conversion of 2-phosphoglycerate and phosphoenolpyruvate in glycolysis. Phosphoenolpyruvate constitutes an important branch point in plant metabolism. It is converted to pyruvate by pyruvate kinase and organic acids by phosphoenolpyruvate carboxylase. Phosphoenolpyruvate also acts as a precursor for the synthesis of aromatic amino acids in plastids. Tobacco (Nicotiana tabacum) enolase antisense plants were analysed for changes in metabolite composition, respiration and photosynthetic parameters. Antisense repression resulted in up to a 95% reduction in total enolase activity. It also resulted in fundamental changes in foliar metabolism. Although 2-phosphoglycerate remained largely unaltered, there was a substantial decrease in phosphoenolpyruvate. The levels of aromatic amino acids and secondary phenylpropanoid metabolites that are derived from these compounds decreased strongly, as did branched chain amino acids. The level of pyruvate was unaltered, as was the rate of respiration. There were substantial increases in tricarboxylic acid cycle intermediates, including a 16-fold increase in isocitrate, an increase in the total free amino acid content, including a 14-fold increase in asparagine and glutamine, and a 50% decrease in free sugars. We conclude that a decrease in enolase activity affects secondary pathways, such as the shikimate branch of amino acid biosynthesis, but does not inhibit the rate of respiration.

Albumin dialysis improves hepatic encephalopathy and decreases circulating phenolic aromatic amino acids in patients with alcoholic hepatitis and severe liver failure

IntroductionThe aim of this study was to assess the effects of albumin dialysis on hepatic encephalopathy and circulating levels of amino acids in severe alcoholic hepatitis.MethodsThe study was carried out in nine patients with severe alcoholic hepatitis and four with primary biliary cirrhosis treated with the molecular adsorbent recirculating system. Besides standard liver function tests, circulating levels of ammonia, total, branched chain and aromatic amino acids, the presence and severity of hepatic encephalopathy, and number connection test were measured before and after each treatment.ResultsThere were eight episodes of encephalopathy in patients with alcoholic hepatitis. Albumin dialysis was associated with significant improvement in encephalopathy (p = 0.02), and a decrease in total amino acid levels (2490 ± 152 μM to 2229 ± 114 μM, p < 0.001). Moreover, the Fischer's ratio, which was significantly lower in patients with alcoholic hepatitis (1.32 ± 0.08) than in controls (3.20 ± 0.16), increased by 17% after albumin dialysis (p < 0.02) because of a significant decrease in phenolic aromatic amino acids (193 ± 17 μM to 165 ± 9 μM, p = 0.04). No differences were observed in circulating ammonia. Changes in phenolic aromatic amino acids and the Fischer's ratio were more prominent in patients with encephalopathy and higher bilirubin removal. Albumin dialysis did not significantly affect the amino acid profile in the controls.ConclusionsAlbumin dialysis results in a significant decrease in circulating phenolic aromatic amino acids and improvement of hepatic encephalopathy in patients with severe liver failure.

Nitrogen Metabolism and Flower Symmetry of Petunia Corollas Treated with Glyphosate

A change of flower shape was observed in petunia corollas treated with 0.5 mM glyphosate. Glyphosate changed the flower symmetry from the actinomorphic type to the zygomorphic type. Corollas treated with glyphosate showed an increased free amino acid content. Free amino acid profiles in petunia corollas revealed that glyphosate had no significant effect on aromatic amino acid levels but increased the level of proline. Soluble protein content in glyphosate-treated corollas did not cause any significant changes. The contents of soluble phenolics, lignin, and IAA in the corollas were not significantly affected by the glyphosate treatment. In contrast, glyphosate reduced the nitrate content and the RNA content of petunia corollas by 45% and 63% of the control, respectively. However, the DNA content in glyphosate-treated corollas was similar to that of the control. Low concentrations of glyphosate did not show any phytotoxic effects on the whole plants and any remarkable changes on aromatic amino acid metabolism and protein synthesis. However, glyphosate reduced the RNA content of petunia corollas and changed the flower symmetry from the actinomorphic type to the zygomorphic type. The results of nonprotein nitrogen metabolism in glyphosate-treated petunia corollas suggested that glyphosate application at low concentration may influence the regulation of flower symmetry through the change of RNA biosynthesis.

The Two Chorismate Mutases from both Mycobacterium tuberculosis and Mycobacterium smegmatis : Biochemical Analysis and Limited Regulation of Promoter Activity by Aromatic Amino Acids

Chorismate mutase (CM) catalyzes the rearrangement of chorismate to prephenate in the biosynthetic pathway that forms phenylalanine and tyrosine in bacteria, fungi, plants, and apicomplexan parasites. Since this enzyme is absent from mammals, it represents a promising target for the development of new antimycobacterial drugs, which are needed to combat Mycobacterium tuberculosis, the causative agent of tuberculosis. Until recently, two putative open reading frames (ORFs), Rv0948c and Rv1885c, showing low sequence similarity to CMs have been described as "conserved hypothetical proteins" in the M. tuberculosis genome. However, we and others demonstrated that these ORFs are in fact monofunctional CMs of the AroQ structural class and that they are differentially localized in the mycobacterial cell. Since homologues to the M. tuberculosis enzymes are also present in Mycobacterium smegmatis, we cloned the coding sequences corresponding to ORFs MSMEG5513 and MSMEG2114 from the latter. The CM activities of both ORFs was determined, as well as their translational start sites. In addition, we analyzed the promoter activities of three M. tuberculosis loci related to phenylalanine and tyrosine biosynthesis under a variety of conditions using M. smegmatis as a surrogate host. Our results indicate that the aroQ (Rv0948c), *aroQ (Rv1885c), and fbpB (Rv1886c) genes from M. tuberculosis are constitutively expressed or subjected to minor regulation by aromatic amino acids levels, especially tryptophan.

Physicochemical and bitterness properties of enzymatic pea protein hydrolysates.

The effects of different proteolytic treatments on the physiochemical and bitterness properties of pea protein hydrolysates were investigated. A commercial pea protein isolate was digested using each of 5 different proteases to produce protein hydrolysates with varying properties. After 4 h of enzyme digestion, samples were clarified by centrifugation followed by desalting of the supernatant with a 1000 Da membrane; the retentates were then freeze-dried. Alcalase and Flavourzymetrade mark produced protein hydrolysates with significantly higher (P < 0.05) degree of hydrolysis when compared to the other proteases. Flavourzyme, papain, and alcalase produced hydrolysates that contained the highest levels of aromatic amino acids, while trypsin hydrolysate had the highest levels of lysine and arginine. Papain hydrolysate contained high molecular weight peptides (10 to 178 kDa) while hydrolysates from the other 4 proteases contained predominantly low molecular weight peptides (</= 23 kDa). DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging activity of the Flavourzyme hydrolysate was significantly (P < 0.05) the highest while alcalase and trypsin hydrolysates were the lowest. Inhibition of angiotensin converting enzyme (ACE) activity was significantly higher (P < 0.05) for papain hydrolysate while Flavourzyme hydrolysate had the least inhibitory activity. Sensory analysis showed that the alcalase hydrolysate was the most bitter while papain and alpha-chymotrypsin hydrolysates were the least. Among the 5 enzymes used in this study, papain and alpha-chymotrypsin appear to be the most desirable for producing high quality pea protein hydrolysates because of the low bitterness scores combined with a high level of angiotensin converting enzyme inhibition and moderate free radical scavenging activity.

Aromatic Amino Acid Metabolism during Liver Failure

Liver failure is associated with hepatic encephalopathy (HE). An imbalance in plasma levels of aromatic amino acids (AAA) phenylalanine, tyrosine, and tryptophan and branched chain amino acids (BCAA) and their BCAA/AAA ratio has been suggested to play a causal role in HE by enhanced brain AAA uptake and subsequently disturbed neurotransmission. Until recently, data on this subject and the role of the liver and splanchnic bed were scarce, particularly in humans, due to inaccessibility of portal and hepatic veins. Here, we discuss, against a background of relevant literature, data obtained in patients undergoing liver resection or with a transjugular intrahepatic portasystemic stent shunt (TIPSS), where these veins are accessible. The BCAA/AAA ratio remained unchanged after major liver resection, but plasma AAA levels were inversely correlated (P < 0.001) with residual liver volume, in keeping with the observed hepatic AAA uptake. In patients with stable cirrhosis and a TIPSS, the plasma BCAA/AAA ratio was lower than in controls (1.19 ± 0.09 vs. controls: 3.63 ± 0.34). Gastrointestinal bleeding in cirrhotics with a TIPSS induced disturbances in BCAA levels and the BCAA/AAA ratio and induced catabolism, which could partly be corrected by isoleucine administration. AAA may be important in the pathogenesis of HE, but it is unlikely that they are the sole factors. HE most likely is a syndrome with multifactorial pathogenesis, where hyperammonemia, AAA/BCAA imbalances, inflammation, brain edema, and neurotransmitter changes interact. Novel therapies to normalize AAA levels in patients with liver failure (such as the molecular adsorbent recirculating system dialysis device) should probably be combined with supplementation of e.g. isoleucine and enhancing ammonia excretion by the kidneys.

Preparation and scaling up of a low phenylalanine enzymatic hydrolysate of bovine whey proteins

We describe the preparation of pancreatic enzymes hydrolysate of milk whey proteins containing low levels of aromatic amino acids. Pancreatin and trypsin/chymotrypsin (6.3% w/w protein) when used to hydrolyze whey proteins for 27 h at 37±2 ºC, released 74% of the Phe, 100% of the Tyr and 100% of the Trp as free amino acids. Most of the free aromatic amino acids present in 2 kg hydrolysate were separated from the remaining peptides and other amino acids by gel filtration on a 15 liter Sephadex G-25 column eluted with 5% acetic acid at 60 liters h-1 at 25ºC. The product, recovered in 37% yield, contained 0.70 mmol Phe, 0.41 mmol Tyr, and <0.01 mmol Trp/100 mmol recovered amino acids. The hydrolysate had a general amino acid composition similar to the whey proteins from which it was prepared and could be used as a nitrogen source for patients with phenylketonuria or tyrosinemia after the addition of appropriate aromatic amino acids.

Amino acid ratios in plasma and tissues in a rat model of liver cirrhosis before and after exercise

In human liver cirrhosis (LC), the Fischer ratio (branched-chain amino acid (BCAA)/aromatic amino acid (AAA)) is used as a marker of hepatic encephalopathy. Similarly, the plasma BCAA levels decrease while AAA levels increase in LC rats. However the BCAA and AAA ratio and the influence of exercise on this ratio remains to be clarified in various tissues of the LC rat model. Normal and LC rats received 2-weekly injections of olive oil or CCl 4 for 10 weeks, respectively, and half of the rats from each group were subjected to one time exercise on a treadmill. BCAA, AAA, glutamate (GLU) and glutamine (GLN) concentrations were measured in plasma, liver, brain, heart and skeletal muscles. Decreased BCAA and increased AAA concentrations in brain and heart were also observed in LC rats and these changes were significantly amplified by exercise. The Fischer ratio in the LC group with and without exercise was decreased in skeletal muscles, while it remained unchanged in the liver. Decreased GLU and increased GLN concentrations were shown in most LC samples following exercise. In conclusion, specific alterations of the amino acid patterns were observed in various tissues in the LC group. These alterations were further emphasized by exercise.

Septic encephalopathy. Diagnosis und therapy

23% of all septic patients develop septic encephalopathy which is associated with an increased mortality rate. Symptoms such as agitation, confusion and disorientation ranging from stupor to coma often develop in early sepsis. Severe hypotension is significantly associated with the development of septic encephalopathy. Several other factors which may play a role are also discussed: effects of inflammatory mediators on the brain, inadequate cerebral perfusion pressure, blood-brain barrier derangements, disturbances of the cerebral microcirculation, cerebral ischemia e.g. due to hypocapnia,metabolic changes, altered amino acid levels, transmitter imbalances, liver insufficiency, multiple organ failure and infections of the CNS, respectively. Compared to patients with an isolated infection,patients in septic shock have increased levels of aromatic amino acids such as phenylalanine and tryptophan in the plasma and brain as well as decreased levels of branched chain amino acids. Patients who died had higher levels of aromatic amino acids than the survivors. The correlation between aromatic amino acids and the APACHE II score was significant. The tryptophan metabolite quinolinic acid which can be synthesized in activated macrophages could act as an excitatory transmitter on the N-methyl-D-aspartate (NMDA) -receptor. Observations from experimental models indicate that activated NMDA receptors activate the neuronal isoform of the NO-synthase and other calcium dependent enzymes. This releases free radicals which may damage the DNA and activate the nuclear enzyme Poly-ADP-ribose-synthetase (PARS), resulting in energy depletion and cell death. Sepsis is the main cause of metabolic encephalopathies in critically ill patients. The differential diagnoses include hepatic, renal,hypoxic-ischemic or cardiovascular encephalopathies as well as encephalopathies,metabolic disorders and organ dysfunctions of other origin. Therapeutic interventions are numerous,however, so far only investigated in few controlled studies. The primary therapeutic goal is to maintain an adequate perfusion pressure and to prevent hypoxia and hypocapnia. Although the infusion of branched chain amino acids is controversial, experimental investigations demonstrated improvements improvements in an animal model with septic encephalopathy. Further investigations with respect to glutamate receptor antagonists, new radical scavengers, NO- and PARS-inhibitors may show whether these substances are suitable for the prophylaxis or early therapy of septic encephalopathy.

Purification and characterization of a copper-binding protein from Asian periwinkle Littorina brevicula

The Asian periwinkle, Littorina brevicula, is highly resistant to a wide range of heavy metal concentrations and its metal-binding protein(s) are induced in the presence of cadmium (Cd) and zinc (Zn). In this study, we isolated and characterized a novel copper-binding protein (Cu-BP). Following purification by Sephacryl S-100 chromatography, Cu-BP contained an equal amount of Zn in non-exposed physiological conditions. However, Zn is replaced by Cu at the binding site upon addition of excess Cu (100 μM CuCl 2) to the cytosol or after a long period (60 days) of exposure of the periwinkles to the metal ion (150 μg/l CuCl 2). The ligand was further purified by DEAE-Sepharose anion-exchange chromatography and C 18 reverse-phase HPLC. The molecular weight of the purified protein was determined as 11.38 kDa by MALDI-TOF MS analyses. This Cu-BP is distinct from common mollusk metallothionein (MT) in that it contains significantly lower number of Cys (8 residues) and high levels of aromatic amino acids, Tyr and Phe. The protein additionally contains His and Met, which are absent in the MT-like Cd-BP of L. brevicula. The finding that Cu-BP in the Asian periwinkle is distinct from MT-like Cd-BP suggests that the timely expression of specific metal-binding proteins allows added protection against each heavy metal in severely polluted conditions.

Hepatic amino-acid metabolism in liver cirrhosis and in the long-term course after liver transplantation

Abstract The aim of this study was to investigate the impact of orthotopic liver transplantation (OLT) on plasma levels and splanchnic turnover of key amino acids for muscular (branched-chain amino acids: BCAAs) and hepatic metabolism (aromatic amino acids (AAAs) and methionine) in 48 patients with cirrhosis, 14 patients after OLT, and 46 controls. Also, hepatic amino-acid supply and resting energy expenditure were measured. BCAA levels (no hepatic uptake) decreased in cirrhosis (P<0.001) and were improved, although not normalized, after OLT (P<0.001). AAA and methionine levels were raised in cirrhosis (P<0.001) and normalized after OLT (P<0.001). Hepatic supply of these amino acids increased in patients graded Child B and C and decreased significnatly after OLT. Splanchnic uptake of AAAs and methionine increased significantly in Child-B and decreased in Child-C patients. After OLT, splanchnic extraction of AAAs and methionine was as in Child A. Circulating AAAs and methionine correlated with indocyanine-green half-life (r= 0.71, P <0.001) and resting energy expenditure (r= 0.50, P <0.001), indicating that levels of circulating AAAs and methionine in cirrhosis are determined by hepatic and extrahepatic metabolic factors. This study demonstrates persistent changes in muscular metabolism of BCAAs after OLT, while the hepatic amino-acid metabolism in normalized due to (1) a significant reduction in the rate of peripheral proteolysis, and (2) improved liver function compared with that in patients with cirrhosis.

Plasma amino acids in cirrhosis and after liver transplantation: influence of liver function, hepatic hemodynamics and circulating hormones

Liver cirrhosis is characterized by substantial changes in amino acid (AA) metabolism, resulting in a deranged plasma AA profile. To investigate the effect of liver transplantation (OLT), we studied arterial AA profiles in 52 patients with advanced cirrhosis, 16 stable patients over 6 months after OLT and 48 controls. Changes in AA levels were correlated with portal pressure (hepatic venous pressure gradient), functional hepatic blood flow (indocyanine green extraction) and circulating hormone levels (catecholamines, insulin, C-peptide). Fourteen of 18 measured AA were significantly altered in cirrhosis and 11 of 18 remained abnormal after OLT compared with controls. Aromatic AA (AAA) and methionine were elevated in cirrhosis (p < 0.001 each), increasing with disease stage, and normalized after OLT. Branched chain AA (BCAA) levels were decreased in cirrhosis (p < 0.001) and were unrelated to disease stage. After OLT, BCAA levels remained subnormal (p < 0.01), although higher than in cirrhosis (p < 0.001). AAA levels increased with decreasing functional hepatic blood flow (r = -0.67; p < 0.001) and increasing portal pressure (r = 0.59; p < 0.001). BCAA levels decreased with increasing catecholamine (r = - 0.54, p < 0.001) and insulin levels (r = - 0.40, p = 0.001). We conclude that despite normal liver function, AA metabolism is only partially normalized after OLT. AAA levels mainly determined by hepatic metabolic function and functional liver blood flow return to normal, while BCAA levels remain subnormal, indicating persistent changes in muscular AA metabolism after OLT.

Systemic Induction of Salicylates in Salix myrsinifolia(Salisb.)

We studied the induction of salicylates in mature leaves of Salix myrsinifolia Salisb. (Salicaceae) following severe wounding by a specialist leaf-beetle Phratora vitellinae L. (Chrysomelidae). Levels of individual salicylates and aromatic amino acids and their total levels were determined in leaves at different developmental stages. Induction of salicylates depended on: (1) the individual compound; (2) the developmental stage of the plant organ; and (3) the genotype of the plant. Induction of salicylates was systemic: levels of salicylates rose in unwounded young immature and mature leaves, but no local response was detected in wounded leaves. In addition, there were clear clonal variations in both the constant and induced levels of salicylates: clones with the highest levels of salicylates were also most capable of increasing this level in response to herbivore attack i.e. no trade-off between constant and induced levels was detected. Furthermore, the levels of three aromatic amino acids, Phe, Tyr and Trp, increased in immature leaves of herbivore-affected plants, which may indicate induction of enzymes of the shikimate pathway by wounding. The increase in salicylates was suggested to be a consequence of an increased rate of synthesis rather than that of translocation. The induced levels of salicylates did not affect the subsequent feeding of highly specialized P. vitellinae. However, the ability to increase levels of salicylates may reduce grazing by generalist herbivores which are not able to tolerate high levels of salicylates.