scholarly journals Organic-acid transport in resealed haemoglobin-containing human erythrocyte ‘ghosts’

1980 ◽  
Vol 190 (3) ◽  
pp. 653-658 ◽  
Author(s):  
A R Hubbard ◽  
U Sprandel ◽  
R A Chalmers
Keyword(s):  
Organic Acids ◽  
Metabolic Diseases ◽  
Glyoxylic Acid ◽  
Aliphatic Acid ◽  
Erythrocyte Ghosts ◽  
Cinnamic Acids ◽  
Efflux Transport ◽  
External Concentration ◽  
Enzyme Replacement ◽  
Organic Acid Transport

The transport of organic acids across the membrane of resealed haemoglobin-containing erythrocyte ‘ghosts’ prepared by a dialysis technique has been studied. The present work forms part of studies directed towards the use of erythrocyte cellular carriers in enzyme-replacement therapy of inherited metabolic diseases. Oxalic acid, glycollic acid and glyoxylic acid were taken as representative of aliphatic acids of low molecular mass and benzoic and cinnamic acids as representative of unsubstituted aromatic acids. These selected acids are important in the diseases with which the present work is concerned. Comparison of influx and efflux transport characteristics showed that erythrocyte ‘ghosts’ retain transport properties closely similar to those of normal erythrocytes. Rapid transport was observed with all organic acids studied and there was a linear relationship between initial amount of influx and external concentration of aliphatic acid. Saturation of the transport system was not observed up to 1 mM external concentration, and the presence of plasma in the external medium had no effect on transport characteristics. Transport in intact erythrocytes and prepared erythrocyte ‘ghosts’ from patients with hyperoxaluria was also studied.

Enzyme Replacement Therapy for Genetic Disorders Associated with Enzyme Deficiency

2021 ◽  
Vol 28 ◽  
Author(s):  
Marialaura Marchetti ◽  
Serena Faggiano ◽  
Andrea Mozzarelli
Keyword(s):  
Enzyme Replacement Therapy ◽  
Replacement Therapy ◽  
Rare Diseases ◽  
Mammalian Cells ◽  
Metabolic Diseases ◽  
Genetic Disorders ◽  
Life Quality ◽  
Lysosomal Storage Diseases ◽  
Lysosomal Storage ◽  
Enzyme Replacement

: Mutations in human genes might lead to loss of functional proteins, causing diseases. Among these genetic disorders, a large class is associated with the deficiency in metabolic enzymes, resulting in both an increase in the concentration of substrates and a loss in the metabolites produced by the catalyzed reactions. The identification of therapeutic actions based on small molecules represents a challenge to medicinal chemists because the target is missing. Alternative approaches are biology-based, ranging from gene and stem cell therapy, CRISPR/Cas9 technology, distinct types of RNAs, and enzyme replacement therapy (ERT). This review will focus on the latter approach that since the 1990s has been successfully applied to cure many rare diseases, most of them being lysosomal storage diseases or metabolic diseases. So far, a dozen enzymes have been approved by FDA/EMA for lysosome storage disorders and only a few for metabolic diseases. Enzymes for replacement therapy are mainly produced in mammalian cells and some in plant cells and yeasts and are further processed to obtain active, highly bioavailable, less degradable products. Issues still under investigation for the increase in ERT efficacy are the optimization of enzymes interaction with cell membrane and internalization, the reduction in immunogenicity, and the overcoming of blood-brain barrier limitations when neuronal cells need to be targeted. Overall, ERT has demonstrated its efficacy and safety in the treatment of many genetic rare diseases, both saving newborn lives and improving patients’ life quality, and represents a very successful example of targeted biologics.

scholarly journals Saturated hydrogen improves lipid metabolism disorders and dysbacteriosis induced by a high-fat diet

2020 ◽  
Vol 245 (6) ◽  
pp. 512-521 ◽  
Author(s):  
Xiangjie Qiu ◽  
Qiaona Ye ◽  
Mengxing Sun ◽  
Lili Wang ◽  
Yurong Tan ◽  
...  
Keyword(s):  
Fatty Acid ◽  
High Fat Diet ◽  
Metabolic Disorders ◽  
Metabolic Diseases ◽  
Isocitrate Lyase ◽  
Intestinal Flora ◽  
Glyoxylic Acid ◽  
Density Lipoprotein ◽  
High Fat ◽  
Acid Cycle

Studies have shown that metabolic diseases, such as obesity, are significantly associated with intestinal flora imbalance. The amplification of opportunistic pathogens induced by the glyoxylic acid cycle contributes to intestinal flora imbalance. Promising, though, is that saturated hydrogen can effectively improve the occurrence and development of metabolic diseases, such as obesity. However, the specific mechanism of how saturated hydrogen operates is still not very clear. In this study, after a high-fat diet, the level of total cholesterol, total glyceride, and low-density lipoprotein in the peripheral blood of mice increased, and that of high-density lipoprotein decreased. Intestinal fatty acid metabolism-related gene Apolipoprotein E (ApoE), fatty acid synthase (FAS), intestinal fatty acid-binding protein (I-FAPB), acetyl-CoA carboxylase 1 (ACC1), peroxisome proliferator-activated receptor γ (PPARγ), and stearoyl-CoA desaturase 1 (SCD1) increased significantly. Bacteroides, Bifidobacteria, and Lactobacillus counts in feces decreased considerably, while Enterobacter cloacae increased. The activity of isocitrate lyase in feces increased markedly. Treatment of mice with saturated hydrogen led to decreased total cholesterol, total glyceride, and low-density lipoprotein and increased high-density lipoprotein in the peripheral blood. FAS and I-FAPB gene expression in the small intestine decreased. Bacteroides, Bifidobacteria, and Lactobacillus in feces increased significantly, whereas Enterobacter cloacae decreased. The activity of isocitrate lyase also diminished remarkably. These results suggest that saturated hydrogen could improve intestinal structural integrity and lipid metabolism disorders by inhibiting the glyoxylic acid cycle of the intestinal flora. Impact statement Past studies have shown that hydrogen can improve metabolic disorders, but its mechanism of action remains unclear. It is well known that metabolic diseases, such as obesity, are significantly associated with changes in the intestinal flora. The glyoxylic acid cycle is an essential metabolic pathway in prokaryotes, lower eukaryotes, and plants and could be the portal for mechanisms related to metabolic disorders. Many opportunistic pathogenic bacteria can recycle fatty acids to synthesize sugars and other pathogenic substances using the glyoxylic acid cycle. So, the glyoxylic acid cycle may be involved in intestinal dysbacteriosis under high-fat diet. This study, therefore, seeks to provide the mechanism of how hydrogen improves metabolic diseases and a new basis for the use of hydrogen in the treatment of metabolic disorders.

scholarly journals Engineering of the Recombinant Expression and PEGylation Efficiency of the Therapeutic Enzyme Human Thymidine Phosphorylase

2021 ◽  
Vol 9 ◽  
Author(s):  
Christos S. Karamitros ◽  
Catrina M. Somody ◽  
Giulia Agnello ◽  
Scott Rowlinson
Keyword(s):  
Catalytic Activity ◽  
Thymidine Phosphorylase ◽  
Rational Design ◽  
Metabolic Diseases ◽  
Recombinant Expression ◽  
Bacterial Enzymes ◽  
Gastrointestinal Complications ◽  
E Coli ◽  
Enzyme Replacement ◽  
Arginine Residues

Human thymidine phosphorylase (HsTP) is an enzyme with important implications in the field of rare metabolic diseases. Defective mutations of HsTP lead to mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), a disease with a high unmet medical need that is associated with severe neurological and gastrointestinal complications. Current efforts focus on the development of an enzyme replacement therapy (ERT) using the Escherichia coli ortholog (EcTP). However, bacterial enzymes are counter-indicated for human therapeutic applications because they are recognized as foreign by the human immune system, thereby eliciting adverse immune responses and raising significant safety and efficacy risks. Thus, it is critical to utilize the HsTP enzyme as starting scaffold for pre-clinical drug development, thus de-risking the safety concerns associated with the use of bacterial enzymes. However, HsTP expresses very poorly in E. coli, whereas its PEGylation, a crucial chemical modification for achieving long serum persistence of therapeutic enzymes, is highly inefficient and negatively affects its catalytic activity. Here we focused on the engineering of the recombinant expression profile of HsTP in E. coli cells, as well as on the optimization of its PEGylation efficiency aiming at the development of an alternative therapeutic approach for MNGIE. We show that phylogenetic and structural analysis of proteins can provide important insights for the rational design of N’-terminus-truncation constructs which exhibit significantly improved recombinant expression levels. In addition, we developed and implemented a criteria-driven rational surface engineering strategy for the substitution of arginine-to-lysine and lysine-to-arginine residues to achieve more efficient, homogeneous and reproducible PEGylation without negatively affecting the enzymatic catalytic activity upon PEGylation. Collectively, our proposed strategies provide an effective way to optimize enzyme PEGylation and E. coli recombinant expression and are likely applicable for other proteins and enzymes.

scholarly journals Proteinuria as a presenting sign of combined methylmalonic acidemia and homocysteinemia: case report

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Ru-Yue Chen ◽  
Xiao-Zhong Li ◽  
Qiang Lin ◽  
Yun Zhu ◽  
Yun-Yan Shen ◽  
...  
Keyword(s):  
Organic Acids ◽  
Vitamin B12 ◽  
Metabolic Diseases ◽  
Methylmalonic Acid ◽  
Clinical Manifestations ◽  
Methylmalonic Aciduria ◽  
Methylmalonic Acidemia ◽  
Initial Presentation ◽  
Gas Chromatography Mass Spectrometry ◽  
Persistent Proteinuria

Abstract Background Disorders of the metabolism and absorption of vitamin B12 can lead to decrease in activity of methionine synthetase and methylmalonate coenzyme A mutase (MMUT), which results in increased levels of methylmalonic acid and homocysteine in blood and urine. Often, combined methylmalonic acidemia (MMA) and homocysteinemia is misdiagnosed due to a lack of specific symptoms. The clinical manifestations are diverse, but proteinuria as the initial presentation is rare. Case presentation Two cases of MMA with homocysteinemia in children are reported. Proteinuria were a primary presenting symptom, followed by anemia and neurologic symptoms (frequent convulsions and unstable walking, respectively). Screening of amino acids and acyl carnitine in serum showed that the propionyl carnitine:acetylcarnitine ratio increased. Profiling of urinary organic acids by gas chromatography–mass spectrometry revealed high levels of methylmalonic acid. Homocysteine content in blood was increased. Comprehensive genetic analyses of peripheral blood-derived DNA demonstrated heterozygous variants of methylmalonic aciduria type C and homocystinuria (MMACHC) and amnionless (AMN) genes in our two patients, respectively. After active treatment, the clinical manifestations in Case 1 were relieved and urinary protein ceased to be observed; Case 2 had persistent proteinuria and was lost to follow-up. Conclusions Analyses of the organic acids in blood and urine suggested MMA combined with homocysteinemia. In such diseases, reports of renal damage are uncommon and proteinuria as the initial presentation is rare. Molecular analysis indicated two different genetic causes. Although the pathologic mechanisms were related to vitamin B12, the severity and prognosis of renal lesions were different. Therefore, gene detection provides new insights into inherited metabolic diseases.

Organic acids in the study of metabolic diseases

1982 ◽  
pp. 211-238 ◽  
Author(s):  
R. A. Chalmers ◽  
A. M. Lawson
Keyword(s):  
Organic Acids ◽  
Metabolic Diseases

Gas-chromatographic method of analysis for urinary organic acids. I. Retention indices of 155 metabolically important compounds.

1980 ◽  
Vol 26 (13) ◽  
pp. 1839-1846 ◽  
Author(s):  
K Tanaka ◽  
D G Hine ◽  
A West-Dull ◽  
T B Lynn
Keyword(s):  
Organic Acids ◽  
Metabolic Diseases ◽  
Retention Indices ◽  
Chromatographic Retention ◽  
Organic Acidurias ◽  
Gas Chromatographic Retention Indices ◽  
Chromatographic Retention Indices ◽  
Methylene Unit ◽  
Trimethylsilyl Derivatives ◽  
Gas Chromatographic Method

Abstract Gas-chromatographic retention indices are given, in terms of methylene units, for 155 metabolically important compounds (mostly organic acids) as trimethylsilyl derivatives on 10% OV-1 and 10% OV-17 columns. Comprehensive references on metabolic diseases that can be diagnosed by detection of these metabolites are cross-indexed to facilitate the use of the methylene-unit list. With the data presented here, it is now possible to diagnose more than 25 well-defined organic acidurias by use of gas chromatography alone.

Preparation of Methyl Derivatives of Some Organic Acids for Analysis by Gas–Liquid Chromatography

1971 ◽  
Vol 49 (8) ◽  
pp. 949-952 ◽  
Author(s):  
Craig A. Atkins ◽  
David T. Canvin
Keyword(s):  
Liquid Chromatography ◽  
Organic Acids ◽  
Thionyl Chloride ◽  
Glyoxylic Acid ◽  
Gas Liquid Chromatography ◽  
Methyl Derivative ◽  
Oxaloacetic Acid ◽  
Ethereal Diazomethane ◽  
Methyl Derivatives ◽  
Derivatives Of

One microgram to 1 mg of organic acid was completely converted to its methyl derivative when it was dissolved in methanol and treated with an excess of ethereal diazomethane. Single products were obtained from α-ketoglutaric acid, oxaloacetic acid, pyruvic acid, glyoxylic acid, or hydroxypyruvic acid on reaction with thionyl chloride – methanol. The separation of 24 organic acids by gas–liquid chromatography on a Reoplex 400 column is presented.

STUDIES OF THE KETO ACIDS OF WHEAT: II. GLYOXYLIC ACID AND ITS RELATION TO ALLANTOIN

1958 ◽  
Vol 36 (1) ◽  
pp. 179-186 ◽  
Author(s):  
R. M. Krupka ◽  
G. H. N. Towers
Keyword(s):  
Amino Acids ◽  
Organic Acids ◽  
Hydrolysis Product ◽  
Glyoxylic Acid ◽  
Nitrogen Atmosphere ◽  
Carbon 14 ◽  
Keto Acids ◽  
Wheat Tissue

In order to determine the metabolic origin of glyoxylate 12 different carbon-14 labelled compounds, including sugars, organic acids, and amino acids, were fed to wheat tissue. With the exception of glycine none gave rise to glyoxylate. The labelling from glycine was slight and could not have accounted for the large amounts of glyoxylate in the tissue. Of a number of inactive compounds fed to degrained seedlings only allantoin induced an increase in the concentration of glyoxylate. A nitrogen atmosphere was found to increase the content of glyoxylate while decreasing that of allantoin. Allantoin-C14 was synthesized much more readily from glycine-C14 than from glyoxylate-C14. When seedlings previously fed glycine-C14 in air were transferred to nitrogen, considerable radioactivity appeared in glyoxylate. These results suggest that glycine is a precursor and glyoxylic acid a hydrolysis product of allantoin.

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