The clinical usefulness of the molar ratio of branched-chain amino acids to tyrosine (BTR) in discriminating stage of chronic liver diseases]

We determined the molar ratio of branched-chain amino acids to tyrosine (BTR) in plasma and in serum by enzymatic method and compared it with Fischer ratio (the molar ratio of branched-chain amino acids to tyrosine and phenylalanine) in plasma obtained by conventional HPLC method. BTR in plasma and in serum was well correlated with plasma Fischer ratio. The normal range (mean +/- 2SD) of BTR was determined to be 4.41-10.05 in 210 normal subjects. In addition, we investigated the distribution of BTR values in patients with various liver diseases. BTR value decreased according to the severity of liver disease. We evaluated the clinical usefulness of BTR in patients with chronic liver diseases by cumulative distribution analysis (CDA) graph and receiver operating characteristic curve (ROC) analysis. The area under the curve for BTR analyzed by ROC for CH versus LC.HCC group was the highest (86.3%) of any for various concurrently-measured liver function tests, and was significantly higher than AST/ALT, ALT, AST, gamma-GT (each, p less than 0.001) and ALB (p less than 0.05). These diagnostic results showed that BTR is a superior indicator in discriminating between liver cirrhosis and chronic hepatitis.     

Correlation between plasma amino acid profiles and the various stages of hepatitis B infection

The amino acid metabolism in patients with hepatitis B virus (HBV) infection is significantly changed. In this study, we analyzed the relationship between the amino acid profiles and varying clinical stages of HBV infection, and investigated their significance. The plasma amino acid concentrations in 115 patients with HBV infection and 32 healthy donors were detected and analyzed, and the main indicators of liver function were measured. Correlation analysis was performed between the amino acid profiles (Fischer's ratio, branched-chain amino acid to tyrosine ratio [BTR]) and the key indicators of liver function in patients with HBV infection. Fisher's ratio and the BTR of patients with HBV infection was found to differ from that of the healthy controls, and was also found to significantly correlate with the stage of HBV infection. Changes in the BTR were closely related to the level of key indicators of liver function, and a significant relationship was detected between the Fischer's ratio and the BTR (r=0.928, p<0.001). These results suggest that Fischer's ratio and the BTR can indirectly reflect the degree of liver cell injury. Determining and tracking the plasma amino acid profiles could, therefore, be used for the diagnosis, treatment selection, and prognosis of patients with varying stages of HBV infection.     

Transition of branched-chain amino acids and tyrosine ratio (BTR) in the blood of acute hepatitis patients

The molar ratio of branched-chain amino acids to tyrosine (BTR) correlates well with the Fischer ratio, and can be measured in a short period of time. It is regarded as the method of analysis that will eventually replace the Fischer ratio. But clinical significance of BTR in terms of acute liver disorders has not been examined thoroughly as of yet. In this study, we measured BTR of 34 patients with acute hepatitis, and examined the transition of the acute period of acute hepatitis and its recovery process. Thirty-four patients diagnosed with acute viral hepatitis became subjects of examination (16 patients of A type, 15 patients of B type, 1 patient of C type, 2 patients of non-A, non-B, non-C type). Out of the 34 patients, 11 were in serious stages (HPT under 40%), including 3 in fulminant condition. By using preserved serum obtained during the acute period (within 1 week of the highest transaminase value), recovery period (within 4 weeks), and treatment period (3 months and later), measurements were conducted with Diacolor:BTR (enzymatic analysis, ONO Pharmaceutical Co., Ltd.), and the results were compared with those of 50 healthy subjects (25 men, 25 women). BTR correlated well with the Fischer ratio for chronic hepatic patients, and with albumin (Alb), PT, and ICGR15 as well, proving that it is useful as an indicator of hepatic reserve ability. But BTR has not been thoroughly examined as it relates to acute liver disorders. In this study, BTR fell in the acute period, correlating with the serious period, proving that it is a useful indicator. For acute liver damage, BTR supports conventional indicators (Alb, Ch-E, HGF, etc.) for assessing serious damage. Also, it has been suggested that measuring the passage of BTR could be the indicator of true recovery, including amino acid metabolism for liver disorders.     

Recent advances in laboratory tests for liver diseases]

Many tests for hepatitis C virus (HCV) infection have been developed and have proved useful for prevention of post-blood transfusion hepatitis C. However, there are at least 4 genotypes of HCV and the predominant type is different among countries. None of the tests using antigens from one genotype are sensitive in detecting the antibodies against another genotype. More sensitive tests using a more stable part of the HCV RNA sequences such as 5'-noncoding region must be developed for clinical use. Automated PCR methods and DNA sandwich hybridization methods using branched DNA amplification multimers may be candidates. Recently a hepatocyte growth factor test has been developed in Japan. Multicenter trials of this test reveal that it is useful for assessment of acute severe hepatitis. Tests for collagen type IV, fibronectin receptor, and prolyl hydroxylase have been reported useful for assessment of liver fibrosis. However, serum prolyl hydroxylase is prone to increase in response to hepatocellular damage as well as fibrotic processes. Enzymatic methods for determination of branched amino acids and tyrosine have been developed. The molar ratio of branched amino acids to tyrosine seems to have same pathophysiological meaning as the ratio of branched amino acids to aromatic amino acids (Fischer ratio) in assessment of liver cirrhosis. Lidocaine test is reported to be useful for predicting survival of transplanted liver and also assessing the function of the cirrhotic liver. Profiles of alpha-fetoprotein subfractions based on lectin-reactivity and galactosyl transferase II isoenzyme have been reported to be useful for detecting hepatocellular carcinoma but this remains to be proved.     

大鼠肝部分切除后血清氨基酸的代谢变化

本实验研究了大鼠肝部分切除后72小时血清氨基酸的代谢变化。实验分三纰:正常对照组、假手术组和肝部分切除组。用氨基酸自动分析仪测定了主动脉、下腔脉静,门静脉和肝静脉血清20种氨基酸浓度。结果表明大鼠肝部分切除后72小时,上述血管血清氨基酸浓度均显著升高。肝摄取氨基酸总量显著增加,门静脉区内脏释放氨基酸总量占肝摄取量的87%。二者呈正相关。外周血管血清必需与非必需氨基酸比值和支链与芳香族氨基酸比值均显著下降。外周组织对支链氨基酸摄取严重抑制,而肝脏摄取则显著增加,这不仅为肝再生提供充分支链氨基酸的供应,而且促进肝蛋白质的合成。门静脉区内脏释放和肝脏摄取丙氨酸的量均显著增加,从而为肝糖原异生提供原料的供应。     

Metabolism of phenylalanine in liver diseases

Summary 1. 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. TheK _m 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.Mit Unterstützung der Deutschen Forschungsgemeinschaft und der Sandoz-Stiftung für medizinische Forschung     

Imbalanzen neutraler Plasma-Aminosäuren als pathogenetischer Faktor der Anorexie

Summary Studies in anorectic tumor-bearing rats indicate that anorexia is correlated to imbalances of neutral amino acids in blood and CNS. Consequently plasma amino acids of patients with neoplastic and non-neoplastic internal diseases were studied during phases of anorexia; special regard was given to the precursors of dopamine and serotonin. Anorectic patients were compared to non-anorectic patients with neoplasia. During anorexia, plasma levels of valine and leucine and hence the ratio of the molar concentrations of Val+Leu+Ile/Phe+Tyr were significantly decreased in each anorectic patient as compared to non-anorectic patients whose ratios were always within the normal ranges. As aromatic and branched-chain amino acids compete for penetration of the blood brain barrier, the decrease of the amino acid ratio may induce a raised flux of phenylalanine and tyrosine into the CNS which results in an increased activation of dopaminergic neurons — known to cause anorexia.

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Abkürzungen AS Aminosäuren - CML Chronisch-myeloische Leukämie - Ile Isoleucin - Leu Leucin - m männlich - M Median - Phe Phenylalanin - Tyr Tyrosin - Val Valin - VZK Verzweigtkettige Aminosäuren - w weiblich Herrn Prof. Dr. W. Kaufmann zur Vollendung des 60. Lebensjahres gewidmet     

Metabolic Effects of Mixture Containing Branched-Chain Amino Acids and Taurine during Subchronic Poisoning with Barbiturates

We studied the effect of a mixture containing branched-chain amino acids and taurine on the pool of free amino acids and their derivatives during chronic phenobarbital poisoning. Subchronic barbiturate poisoning produced by daily intraperitoneal injection of phenobarbital caused imbalance in the content of some amino acids in blood plasma and liver of rats. Treatment with the mixture of branched-chain amino acids and taurine normalized the content of amino acids in the liver and blood plasma of animals with subchronic phenobarbital poisoning. The mixture of branched-chain amino acids and taurine corrects metabolic processes and normalized the peripheral pool of amino acids. Our findings extend the range for application of amino acids in clinical practice.     

A vegetable protein-rich diet for the treatment of liver cirrhosis

Twelve patients were administered a vegetable protein-rich diet, which was low in methionine and high in the branched-chain amino acid (BCAA) to aromatic amino acid (AAA) molar ratio, and an animal protein-rich diet, high in methionine and low in the BCAA/AAA molar ratio. These diets were administered successively for one week each. Actually ingested amounts of tyrosine and methionine were significantly lower during the feeding of the vegetable protein-rich diet than the animal protein-rich diet. Serum methionine concentrations increased while on the animal protein-rich diet and decreased following the switch to the vegetable protein-rich diet. No other amino acid concentrations were affected. Significant differences were not observed in nitrogen balance or serum protein concentrations.     

Tryptophan and tyrosine ratios to neutral amino acids in endogenous depression. Relation to antidepressant response to amitriptyline and lithium + L-tryptophan

The plasma ratios of tryptophan and tyrosine to those amino acids that compete with them during transport across the blood-brain barrier have been determined in depressed patients before and after treatment for four weeks with amitriptyline or lithium + L-tryptophan. There was no relation between the absolute plasma concentrations of free or total tryptophan or tyrosine and the clinical response to amitriptyline. There was also no relation between pre-treatment ratio of plasma tyrosine to competing amino acids and response to amitriptyline, but depressives with subnormal tryptophan ratio improved significantly more than patients with supernormal tryptophan ratio with comparable serum drug levels. The therapeutic response to lithium + L-tryptophan was predicted neither by the absolute plasma concentrations of free or total tryptophan or tyrosine nor by the tyrosine ratio, but there was also a trend towards greater improvement in patients with subnormal compared with supernormal tryptophan ratio. The results suggest that the pre-treatment plasma ratio of tryptophan to competing amino acids is a useful predictor of clinical response to amitriptyline. The possible mode of action of amitriptyline and lithium + L-tryptophan is briefly discussed.     

Neutral amino acids monitoring in phenylketonuric plasma microdialysates using micellar electrokinetic chromatography and laser-induced fluorescence detection

Neutral and non-polar amino acids such as phenylalanine (Phe), valine (Val), tyrosine (Tyr), threonine (Thre) and GABA are hard to resolve by capillary zone electrophoresis (CZE). Their separation is possible by adding a surfactant to the mobile phase. This method is called micellar electrokinetic chromatography (MEKC). We used MEKC with laser-induced fluorescence detection (LIFD) to separate and quantitate these amino acids in plasma microdialysates of patients with phenylketonuria (PKU). This disease is an inborn enzymatic defect with decreased conversion of Phe to Tyr that causes severe neurological damage and mental deterioration, which is diagnosed by measuring plasma Phe and Phe/Tyr ratio. The amino acids tested had linear concentration-signal relation. PKU patients had significantly higher Phe, lower Tyr, 21 times higher Phe/Tyr ratio and decreased values of Val and Thre than controls. These results show that microdialysis of biological fluids coupled with MEKC-LIFD is a convenient technique to measure neutral amino acids in clinical disorders such as PKU.     

Effects of sorbent suspension dialysis on plasma amino acid levels in cirrhotic patients with refractory hepatic encephalopathy

In cirrhotic patients, plasma amino acid levels are severely deranged. A decreased ratio of branched-chain to aromatic amino acids (Fischer ratio) has been implicated in the pathogenesis of hepatic encephalopathy. In this prospective study, we investigated the effects of extracorporeal detoxification on amino acid levels using a sorbent suspension dialysis system. Twenty patients with documented cirrhosis and hepatic encephalopathy grade II-III not responding to standard treatment were randomized to receive either six hours of sorbent dialysis and standardized conventional medical treatment or ongoing medical treatment alone. In contrast to previous uncontrolled studies, no significant effect on amino acid levels, Fischer ratio or clinical grade of hepatic encephalopathy was detected in either treatment group. In conclusion, a 6-hour treatment with sorbent dialysis did not significantly influence plasma levels of amino acids and did not ameliorate the clinical grade of hepatic encephalopathy.     

Effects of insulin and glucagon on serum amino acid concentrations in liver disease

The effects of insulin and glucagon administration on serum amino acid levels were investigated in patients with severe liver disease, since simultaneous injection of pancreatic hormones has been recently introduced as a therapeutic approach. The changes in serum amino acid concentrations, as observed 3 h after ceasing a 3 h infusion of insulin and glucagon in 500 ml glucose solution, were an elevation of serum branched chain amino acid (BACA) levels and of the molar ratio of BCAA/aromatic amino acid (AAA) levels in patients with liver cirrhosis. Similar increases of serum BCAA levels during the infusion were also observed in patients with fulminant hepatitis. The results suggest that insulin-glucagon therapy for severe liver disease has no harmful side effects at least with respect to alterations in the serum aminogram.     

内毒素对实验性肝硬化大鼠血浆氨基酸系列的影响

实验性肝硬化大鼠血浆氨基酸系列改变表现为支／芳比值降低，但无肝性脑病表现，腹腔注射对正常大鼠无明显影响的微量内毒素，肝硬化大鼠即出现嗜睡与错迷症状，肝细胞损伤加重，氨基酸分析表明血浆谷氨酰胺、牛磺酸水平显著高于肝硬化组，酪氨酸水平与肝硬化组比较显著降低，提示内毒素能加剧肝硬化大鼠血浆氨基酸失衡，为肝性脑病重要的诱发因素。     

A simple and rapid method for determining blood ammonia levels

Blood ammonia levels in patients with various liver diseases were determined quantitatively by a simple and rapid method using the Amitest Meter System. The results were compared to those obtained by an enzymatic method and were well correlated. This simple Amitest is also useful in animal experiments, particularly when there is a need to determine blood ammonia levels serially. This paper test was evaluated as being accurate and reliable for clinical and experimental use.     

Diagnosis of MSUD by newborn screening allows early intervention without extraneous detoxification

Maple syrup urine disease (MSUD) is a genetic metabolic disorder resulting from the defective activity of branched-chain 2-ketoacid dehydrogenase complex. Due to the metabolic block, high concentrations of the branched-chain amino acids (BCAA) leucine, valine, isoleucine, and allo-isoleucine as well as their corresponding branched-chain 2-keto acids accumulate in patients on a BCAA-unrestricted diet or during episodes with increased protein catabolism. Early diagnosis and management are essential to prevent permanent brain damage. Newborn screening by tandem MS allows for detection of elevated BCAA concentrations in blood in patients with classical MSUD before they show severe encephalopathic symptoms. Here, we report that newborn screening by expanded tandem MS enables for reversing the intoxication in newborns with MSUD within 24-48 h without any need for extraneous detoxification and thus decreasing the risk of brain damage during a particularly vulnerable period.     

庚型肝炎病毒致病性的初步研究

目的 探讨庚型肝炎病毒（ＨＧＶ）的致病性。方法 应用ＲＴ－ｎＰＣＲ检测３６８例肝炎患者血清ＨＧＶ ＲＮＡ和血清酶的变化,并对其中１例单独庚型肝炎肝硬化病例进行肝脏活组织是检测。结果 在７１例急性黄疸型肝炎中检出单纯性ＨＧＶ ＲＮＡ阳性７例,１５５例慢性肝炎中检出单纯性ＨＧＶ ＲＮＡ阳性２２例,５１例肝硬化中检出单纯ＨＧＶ ＲＮＡ阳性３例。其中１例肝穿组织免疫组化证实为ＨＧＶ ＮＳ ５Ａｇ阳性。结论 争性黄疸型肝炎及乙、丙型肝炎病毒携带者,其慢性肝炎、肝硬化和肝癌中均可检出ＨＧＶ ＲＮＡ,ＨＧＶ感染可为单独感中与乙／丙型肝炎病毒混合或重叠感染,肝脏病理和免疫组化检查证实庚型肝炎病毒是一种嗜肝病毒,其定位主要存在于细胞浆内,可引起慢性病毒性肝炎甚至肝硬化。庚型肝炎病毒很可能具有致病性。     

肝病患者庚型肝炎病毒感染的研究

目的 研究肝病患者庚型肝炎病毒感染状况。方法 用酶链免疫吸附试验（ＥＬＩＳＡ）检测１５４例门诊和住院肝病患者（均无输血史）的抗－ＨＧＶ,１５４例中有５４例同时采用逆转录聚合酶链反应（ＲＴ－ＰＣＲ）检测ＨＧＶ ＲＮＡ,分别计算抗－ＨＧＶ和ＨＧＶ ＲＮＡ的阳性率。以ＨＧＶ ＲＮＡ阳性为ＨＧＶ感染的判断标准,来分析各类肝病患者ＨＧＶ感染状况。结果 １５４例肝病患者抗－ＨＧＶ阳性者３１例,阳性率２０．１３     

Characterization of Full-Length Genomes of Hepatitis B Virus Quasispecies in Sera of Patients at Different Phases of Infection

Hepatitis B virus (HBV) infection results in different clinical presentation due to different levels of immune response. Our study aimed to characterize HBV full-length genome quasispecies (QS) in patients with different phases of infection to better understand its pathogenesis. Forty treatment-naive HBV-infected patients were enrolled, including 10 cases of acute hepatitis B (AHB), 9 cases of immunotolerant (IT) HBV carriers, 11 cases of chronic hepatitis B (CHB), and 10 cases of acute-on-chronic liver failure (ACLF). The present study was conducted by clone-based sequencing. QS heterogeneity within each open reading frame was calculated. The mutation frequency index (MFI) and amino acid variations within the large HBsAg, HBcAg, and HBxAg regions were analyzed based on the different infection phases. In total, 606 HBV full-length sequences were obtained. HBV QS had higher heterogeneity in ACLF and CHB than that in IT among chronically infected individuals. AHB patients had the lower QS heterogeneity at onset than those with chronic infection. ACLF patients had the highest frequency of mutations in the core promoter and precore region. A triple mutation (A1762T/G1764A/G1896A) was observed more frequently in genotype C than in genotype B. The MFI indicated that specific peptides of the studied regions had more frequent mutations in ACLF. Furthermore, several amino acid variations, known as T- and B-cell epitopes, were potentially associated with the immunoactive phase of infection. More HBV genome mutations and deletions were observed in patients with more severe diseases, particularly in specific regions of the core and preS regions, the clinical significance and mechanism of which need to be further investigated.