Direct spectrofluorimetric methods as alternatives to compendial ones used for the quality control of biopharmaceuticals: development, validation and application

Abstract A simple, rapid, precise, accurate and sustainable spectrofluorimetric method (SFM) was developed, validated and applied for the determination of 4-aminobenzoic acid and aromatic amino acids (phenylalanine, tryptophan and tyrosine). These compounds are used in biopharmaceutical formulations and therefore must be analyzed by quality control laboratories to meet the criteria established in pharmacopoeias. In general, potentiometric titration (PT) is described in the compendia as the official analytical technique. However, this method showed low sensitivity and selectivity, and moreover was performed with a non-aqueous solvent (acetic acid), which led to higher consumption of reagents and consequently to the formation of residues. Therefore, the SFM was developed in aqueous medium at pH 7.2 using phosphate buffer. It was successfully validated according to the ICH guidelines and showed good linearity range (r>0.999), specificity, accuracy and precision (within and between days) and robustness. The test results were compared between the SFM and PT using raw material samples, while according to the F- and t-tests at 95% confidence level, no statistical difference was found between the methods

A review of experimental models in colorectal carcinogenesis / Modelos experimentais de carcinogênese colorretal

Colorectal cancer is the leading cause of malignancy of the gastrointestinal tract. A better understanding of the molecular and cellular changes that lead to the disease is necessary to develop early diagnosis and optimal treatment modalities. Rodent models are rapid, reproducible and exhibit an adenoma-carcinoma sequence similar to that found in humans. The objective of this manuscript is to review the most common chemical carcinogens used to induce experimental tumors and the usual methods of evaluation.

O câncer colorretal é a principal neoplasia maligna do trato gastrointestinal. Um melhor entendimento dos processos moleculares e celulares é necessário para o desenvolvimento de estratégias que permitam um diagnóstico precoce e um tratamento mais eficaz. Modelos que utilizam roedores são rápidos, reprodutíveis e permitem o estudo da sequencia adenoma-carcinoma de forma similar a encontrada em humanos. O objetivo desse manuscrito é revisar os principais modelos de carcinogênese química e os métodos mais usuais para avaliação dos resultados.

Cloning and Expression Analysis of Phenylalanine Ammonia-Lyase Gene in the Mycelium and Fruit Body of the Edible Mushroom Flammulina velutipes

Phenylalanine ammonia-lyase (PAL) gene is known to be expressed in plants, and is involved in the differentiation, growth and synthesis of secondary metabolites. However, its expression in fungi remains to be explored. To understand its expression in mushroom fungi, the PAL gene of the edible mushroom Flammulina velutipes (Fvpal) was cloned and characterized. The cloned Fvpal consists of 2,175 bp, coding for a polypeptide containing 724 amino acids and having 11 introns. The translated amino acid sequence of Fvpal shares a high identity (66%) with that of ectomycorrhizal fungus Tricholoma matsutake. Distinctively, the Fvpal expression in the mycelium was higher in minimal medium supplemented with L-tyrosine than with other aromatic amino acids. During cultivation of the mushroom on sawdust medium, Fvpal expression in the fruit body correspondingly increased as the mushroom grew. In the fruiting body, Fvpal was expressed more in the stipe than in the pileus. These results suggest that F. velutipes PAL activity differs in the different organs of the mushroom. Overall, this is first report to show that the PAL gene expression is associated with mushroom growth in fungi.

Improving 3-dehydroshikimate production by metabolically engineered Escherichia coli / 生物工程学报

In the aromatic amino acid biosynthetic pathway 3-dehydroshikimate (DHS) is a key intermediate. As a potent antioxidant and important feedstock for producing a variety of important industrial chemicals, such as adipate and vanillin, DHS is of great commercial value. Here, in this study, we investigated the effect of the co-expression of aroFFBR (3-deoxy-D-arabino-heptulosonate 7-phosphate synthase mutant with tyrosine feedback-inhibition resistance) and tktA (Transketolase A) at different copy number on the production of DHS. The increased copy number of aroFFBR and tktA would enhance the production of DHS by the fold of 2.93. In order to further improve the production of DHS, we disrupted the key genes in by-product pathways of the parent strain Escherichia coli AB2834. The triple knockout strain of ldhA, ackA-pta and adhE would further increase the production of DHS. The titer of DHS in shake flask reached 1.83 g/L, 5.7-fold higher than that of the parent strain E. coli AB2834. In 5-L fed-batch fermentation, the metabolically engineered strain produced 25.48 g/L DHS after 62 h. Metabolically engineered E. coli has the potential to further improve the production of DHS.

Key aromatic amino acids of anti-hepatoma activity on Parasporin-2 / 生物工程学报

Nine mutants (P2M1-9) were obtained using PCR with 5-BU based on DNA template (P2Y) encoding the active region of Parasporin-2. Mutant proteins were purified after expressing in E. coli BL21 cells, followed by assayed against hepatoma cells and normal liver cells by MTT. They showed diverse anti-hepatoma activities, in which two mutant proteins, P2M1 and P2M8, exhibited high cytotoxicity against hepatoma cell lines SMMC7721 and Be17402, meanwhile leaving normal liver cells Chang-liver unaffected. Structural comparison among P2Y, P2M1 and P2M8 showed that the length of beta-sheet or beta-fold, and the amount of alpha helix greatly affected the anti-hepatoma activity of Parasporin-2. Results based on amino acid alignment, molecular docking between P2Y, P2M1 or P2M8 and receptor, and mimic mutation demonstrated that amino acid residues at the sites of 52, 56, 58 and 208 on P2Y, especially the aromatic amino acids such as Trp, Phe, and Tyr were involved in the interactions.

Asymmetric synthesis of aromatic L-amino acids catalyzed by transaminase / 生物工程学报

Aromatic L-Amino acids are important chiral building blocks for the synthesis of many drugs, pesticides, fine chemicals and food additives. Due to the high activity and steroselectivity, enzymatic synthesis of chiral building blocks has become the main research direction in asymmetric synthesis field. Guided by the phylogenetic analysis of transaminases from different sources, two representative aromatic transaminases TyrB and Aro8 in type I subfamily, from the prokaryote Escherichia coli and eukaryote Saccharomyces cerevisia, respectively, were applied for the comparative study of asymmetric transamination reaction process and catalytic efficiency of reversely converting keto acids to the corresponding aromatic L-amino acid. Both TyrB and Aro8 could efficiently synthesize the natural aromatic amino acids phenylalanine and tyrosine as well as non-natural amino acid phenylglycine. The chiral HPLC analysis showed the produced amino acids were L-configuration and the e.e value was 100%. L-alanine was the optimal amino donor, and the transaminase TyrB and Aro8 could not use D-amino acids as amino donor. The optimal molar ratio of amino donor (L-alanine) and amino acceptor (aromatic alpha-keto acids) was 4:1. Both of the substituted group on the aromatic ring and the length of fatty acid carbon chain part in the molecular structure of aromatic substrate alpha-keto acid have the significant impact on the enzyme-catalyzed transamination efficiency. In the experiments of preparative-scale transamination synthesis of L-phenylglycine, L-phenylalanine and L-tyrosine, the specific production rate catalyzed by TryB were 0.28 g/(g x h), 0.31 g/(g x h) and 0.60 g/(g x h) and the specific production rate catalyzed by Aro8 were 0.61 g/(g x h), 0.48 g/(g x h) and 0.59 g/(g x h). The results obtained here were useful for applying the transaminases to asymmetric synthesis of L-amino acids by reversing the reaction balance in industry.

A Case of Serum Amino Acid Disturbance with Hyperammonemia in Patient with Primary Amyloidosis

There have been reports that hyperammonemia and amino acid disturbance can cause loss of consciousness in patients with multiple myelomas and normal liver function. We experienced a case of a 71-years-old female patient with amyloidosis, who had shown disturbance of consciousness. At that time, the serum ammonia level was elevated; serum amino acids disturbance was also noted. In particular, the decrease in branched-chain amino acids and increase in aromatic amino acids results in a low Fisher ratio. The Fisher ratio, the ratio of branched-chain to aromatic amino acids, has been suggested as an important indicator of consciousness disturbance. We report, for the first time in Korea, a case of amyloidosis, with mental disturbance due to serum amino acid disturbance.

High plasma branched-chain amino acids:aromatic amino acids ratio in children on the ketogenic diet: a mechanism in controlling epilepsy.

BACKGROUND: The authors proposed that ketogenic diets will produce an increase in the ratio of branched chain amino acids (BCAAs) and aromatic amino acids (BCAAs) in plasma of children who are on the diets. SUBJECTS AND METHOD: A sample of plasma amino acids sample before initiation of fasting and on day 10 of the dietary treatment was obtained in patients with refractory epilepsy who were newly admitted for initiation of ketogenic diet. Plasma amino acids were determined by high performance liquid chromatography equipment. RESULTS: There are 20 patients with refractory epilepsy participating in this study. Outcomes of ketogenic diet therapy were satisfactory. Nineteen cases out of 20 cases had a significantly higher ratio of plasma BCAAs:ARAAs during ketogenic diets than before the diet (P < 0.001). CONCLUSION: The ketogenic diets produced an increased ratio of plasma BCAAs:ARAAs. Whether the increased ratio of plasma BCAAs:ARAAs plays an important role in controlling epilepsy is yet to be elucidated.

Myeloperoxidase-generated phenothiazine cation radicals inactivate Trypanosoma cruzi dihydrolipoamide dehydrogenase

Peroxidase/H2O2/phenothiazine systems irreversibly inhibit Trypanosoma cruzi dihydrolipoamide dehydrogenase (LADH). Inactivation of the parasite enzyme depended on (a) phenothiazine structure; (b) peroxidase nature; (c) incubation time and (d) the presence of a cation radical scavenger. With the myeloperoxidase/H2O2/system, promazine, trimeprazine, thioridazine, promethiazine, prochlorperazine, chlorpromazine and perphenazine were the most effective derivatives out of twelve phenothiazines studied. An electronegative substituent at position 2 of the phenothiazine ring such as Cl, or trifluoromethyl, propionyl and nitrile groups decreased or nullified phenothiazine activity. Myeloperoxidase/H2O2/, horseradish peroxidase/H2O2/, and myoglobin/H2O2/systems activated phenothiazines producing the corresponding cation radicals, myeloperoxidase being the most selective one with respect to phenothiazine structure. The myoglobin/H2O2/system activated phenothiazines that were scarcely active or inactivate with the MPO/H2O2/system, such as the trifluoromethyl derivatives. Production of phenothiazine cation radicals was demonstrated by optical spectroscopy. Phenothiazine cation radical stability depended on their structure as illustrated by promazine and thioridazine. Thiol compounds (GSH, N-acetyl-cysteine and penicillamine), aromatic aminoacids (L-tyrosine, L-tryptophan, and the corresponding peptides) and ascorbate scavenged phenothiazine cation radicals, thus preventing LADH inactivation. Comparison of the summarized phenothiazine effects with those of phenothiazines on T. cruzi suggest the role of cation radicals in phenothiazines chemotherapeutic actions.

Study on the Protective Effects of 6R-Tetrahydrobiopterin on the Oxidative Neuronal Injury in Mouse Cortical Cultures

OBJECTIVE: 6R-Tetrahydrobiopterin(BH4) is a cofactor for the aromatic amino acid hydroxylases which is essential for the biosynthesis of catecholamines and serotonin. It also acts as a cofactor for nitric oxide synthase, and stimulates the release of some neurotransmitters such as dopamine, serotonin, acetylcholine and glutamate. Recently, it has been reported that BH4 could induce cellular proliferation and enhance neuronal survival. This study was performed to investigate the antioxidative effect of BH4 on the various oxidative insults in mouse cerebral cortical cell cultures. METHODS: Iron ion(FeCl2), zinc ion(ZnCl2), sodium nitroprusside(SNP) and buthionine sulfoximine(BSO, a glutathione depletor) were used as oxidants. Cell death was assessed by measurement of lactate dehydrogenase efflux to bathing media at the end of exposure. RESULT: All 4 oxidants induced neuronal cell death associated with cell body swelling, which was markedly inhibited by trolox(100nM), a vitamin E analog. BH4(10-100nM) markedly inhibited the neuronal cell death induced by all 4 oxidants(20nM Cu2+, 20nM Zn2+, 1nM SNP or 1mM BSO). However, BH4 failed to inhibit the neuronal cell death induced by 24hr exposure to 20nM NMDA. CONCLUSION: These results suggest that BH4 has antioxidative action independently of any actions of enzyme cofactor.

Prospective Study on Efficacy of Oral Supplement of Branched-Chain Amino Acid Granules on the Nutritional Status of the Cirrhotics / 대한간학회지

BACKGROUND/AIMS: A prospective comparative study was conducted to investigate the efficacy of orally administered branched-chain amino acids (BCAA) in cirrhotic patients. METHODS: Forty-seven patients with liver cirrhosis of viral etiologies, whose hypoalbuminemia could not be corrected with adequate protein intake, were randomly assigned to either the BCAA group (n=31) or the control group (n=16). The selection criteria were ages between 16 and 70 years, patients whose Child-Pugh scores were less than 13 points and who were willing to participate in the study. Most patients (87.1%) belonged to Child-Pugh class B. Patients in the BCAA group received oral supplementation with branched-chain amino acid granules (12 g/day, each packet containing total 4 g of BCAA, i.e. leucine, 1904 mg; valine, 1144 mg; isoleucine, 952 mg) for 12 weeks. Patients had complete blood counts and chemistry at entry and once every month. Serum ferritin and amino acid concentrations in plasma were determined. Anthropometric parameters including body weight, body fat contents and body mass index were assessed at the beginning and at the end of the 3-month period. RESULTS: In the BCAA group, there was a significant increment in plasma levels of isoleucine, valine (p < 0.001) whereas levels of aromatic amino acids did not show substantial change. Total BCAA concentration and BCAA/AAA (aromatic amino acid) molar ratio (Fischer's ratio) also increased significantly after the administration of oral BCAA (p < 0.001). In contrast, patients in the control group showed no significant change in assessed parameters. CONCLUSION: Oral supplementation of BCAA to cirrhotic patients improved several parameters reflecting nutritional status without causing encephalopathy or other serious adverse effects.

Amino Acid Derangements in Plasma and Erythrocytes of Patients with Sepsis / 대한임상병리학회지

BACKGROUND: Sepsis is a major insult leading to increased muscle breakdown and oxidation of amino acids. Disturbed plasma and brain amino acid levels may be important in the altered mental status observed in patients with infections and alteration in mental status due to sepsis has been associated with an increased mortality rate. Erythrocytes have been suggested as transport cells for amino acids and may be better than plasma for utilization and storage of amino acids in the body. Several reports suggested that amino acid alterations in plasma and erythrocytes have had an important role in sepsis. So we investigated the association between severity and prognosis of sepsis and amino acid levels in plasma and erythrocytes. METHODS: Heparinized plasma and erythrocytes were taken from 25 healthy controls, 10 infected patients and 15 septic patients. The amino acid levels were determined using high performance liquid chromatography after deproteinization with sulfosalicylic acid. RESULTS: Plasma amino acid pattern showed elevated levels of the aromatic amino acids (tyrosine and phenylalanine) and sulfur-containing amino acid (cystine) but decreased level of branched chain amino acid (isoleucine) in septic patients compared with healthy controls or infected patients. Patients with septic encephalopathy had higher levels of tyrosine, phenylalanine and sulfur-containing amino acid (methionine) than patients without septic encephalopathy. The ratios of erythrocytes to plasma showed no significant decrease in septic patients compared with healthy controls or infected patients. However, in patients with septic encephalopathy, methionine, leucine and phenylalanine levels were lower than in patients without septic encephalopathy. CONCLUSIONS: The results showed a characteristic pattern of amino acid derangements in plasma and erythrocytes of septic patients. This pattern was more prominent in severe sepsis. It was suggested that the severity and prognosis of septic patients could be predicted by measuring the amino acid levels of plasma and erythrocytes.