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PubMed1984

Stability of enzyme inhibitors and lectins in foods and the influence of specific binding interactions.

Proteins with actual or potential antinutrient or toxicant activity found in foodstuffs include (1) enzyme inhibitors, especially those specific for serine proteinases and alpha-amylases, and (2) lectins (hemagglutinins). These inhibitors and lectins must be inactivated during processing or food preparation, usually by heat, to avoid possible undesirable effects. Knowledge of their heat stabilities thus helps determine conditions required for their inactivation or denaturation. Many are heat-stable proteins, and their conformations can be stabilized or destabilized by interactions with other constituents present in the food or the digestive tract. Differential scanning calorimetric (DSC) results show that specific binding interactions can lead to substantial increases in kinetic thermal stability of proteins. Examples of such stabilization include serine proteinase-proteinase inhibitor, alpha-amylase-amylase inhibitor, and metal ion-lectin complexes. The extent of thermal stabilization of proteinases in complexes with inhibitors is correlated with the equilibrium association constant. Presence of more than one denaturing unit revealed by DSC in complexes involving multiheaded inhibitors can be interpreted in relation to domain structures of the inhibitors. Basic information on stability of the enzyme inhibitors and lectins is relevant to food processing, quality, and safety.

Amylases↗
PubMed2006

The effect of some osmolytes on the activity and stability of mushroom tyrosinase.

The thermodynamical stability and remained activity of mushroom tyrosinase (MT) from Agaricus bisporus in 10 mM phosphate buffer, pH 6.8, stored at two temperatures of 4 and 40 degrees C were investigated in the presence of three different amino acids (His, Phe and Asp) and also trehalose as osmolytes, for comparing with the results obtained in the absence of any additive. Kinetics of inactivation obey the first order law. Inactivation rate constant (kinact) value is the best parameter describing effect of osmolytes on kinetic stability of the enzyme. Trehalose and His have the smallest value of kinact (0.7x10(-4) s-1) in comparison with their absence (2.5x10(-4) s-1). Moreover, to obtain effect of these four osmolytes on thermodynamical stability of the enzyme, protein denaturation by dodecyl trimethylammonium bromide (DTAB) and thermal scanning was investigated. Sigmoidal denaturation curves were analysed according to the two states model of Pace theory to find the Gibbs free energy change of denaturation process in aqueous solution at room temperature, as a very good thermodynamic criterion indicating stability of the protein. Although His, Phe and Asp induced constriction of MT tertiary structure, its secondary structure had not any change and the result was a chemical and thermal stabilization of MT. The enzyme shows a proper coincidence of thermodynamic and structural changes with the presence of trehalose. Thus, among the four osmolytes, trehalose is an exceptional protein stabilizer.

Agaricus↗
PubMed2003

Improved kinetic stability of DTPA- dGlu as compared with conventional monofunctional DTPA in chelating indium and yttrium: preclinical and initial clinical evaluation of radiometal labelled minigastrin derivatives.

The development of monofunctional DTPA derivatives has been a major breakthrough in the labelling of proteins or peptides with a variety of radiometals. Although this methodology is simple and useful for indium-111 labelling, the stability of these conjugates is too low for most therapeutic nuclides. Cyclic chelators, such as DOTA, have shown excellent kinetic stability with a variety of radiometals, but the labelling procedure is more difficult, requiring ultra-pure reagents and a heating step that sometimes endangers the biomolecule's integrity. The aim of this work was twofold: (a) to develop a novel, open chain chelator which can be easily labelled with various radiometals, displaying higher kinetic stability than monofunctional DTPA, and (b) to evaluate this chelator in vitro and in vivo when conjugated to a CCK-B receptor ligand as a detection modality for receptor-(over-)expressing tumours. DTPA derivatives of Leu(1)- and dGlu(1)-minigastrin were synthesised. All conjugates could be labelled with (111)In or (88/90)Y at high specific activities (8.5-44.4 GBq/micro mol) and with high radiochemical purity. Serum stability testing was performed, and the labelled conjugates were compared concerning their stability against DTPA challenge. The biodistribution of the radiolabelled Leu(1)- and dGlu(1)-minigastrin derivatives was studied in tumour-bearing nude mice, in one healthy human volunteer and in three patients with metastatic medullary thyroid carcinoma. The transchelation of all tested radiometals to serum proteins was significantly slower with the DTPA-Glu conjugates as compared with their Leu analogues (e.g. transchelation t(1/2) of DTPA- dGlu(1)-minigastrin vs its Leu(1) analogue at 37 degrees C in human serum for (111)In: 239 h vs 91 h; for (90)Y: 130 h vs 53 h). In animals, all labelled CCK-B receptor ligands showed fast and specific uptake in CCK-B-receptor-positive tissues, such as the stomach and tumour, as well as a fast renal clearance pattern. However, DTPA-Leu(1)-minigastrin showed higher background activity in the whole body and those organs known to accumulate the respective free radiometal (e.g. (88)Y-DTPA-Leu(1)-minigastrin had bone uptake of 22%ID/g as compared to only 1.2%ID/g with its dGlu(1) analogue). In humans, fast tumour and stomach uptake was observed for both (111)In-labelled compounds, but DTPA- dGlu(1)-minigastrin lacked the liver, spleen and bone marrow uptake observed with its Leu(1) analogue. In conclusion, anionic amino acid derivatives of DTPA may display improved metabolic stability as compared with monofunctional DTPA conjugates. DTPA- dGlu(1)-minigastrin is preferred to "monofunctional" DTPA-Leu(1)-minigastrin for diagnostic application with (111)In for the in vivo detection of CCK-B receptor-expressing tissues.

Animals↗
PubMed2006

cry1Aa lacks stability elements at its 5'-UTR but integrity of its transcription terminator is critical to prevent decay of its transcript.

We analyzed the influence of the 5' and 3' untranslated regions of the Bacillus thuringiensis cry1Aa on its mRNA stability. Although the cry1Aa gene has a stable transcript (8 min), its 5' UTR did not provide stability to the reporter gene uidA. Stability of cry1Aa could be increased to 40 min by addition of an SP82 stability element at the 5' UTR, suggesting that once the 5' and 3' ends were protected initiation of decay could be effectively blocked. We generated mutations in the transcription terminator and found that changes that reduced the stability of the stem, a larger loop, or elimination of the U-trail sharply decreased the half-life of the transcript. Therefore, unlike some stable bacterial transcripts, cry1Aa lacks special features at the end 5' to prevent decay, but its terminator is the main determinant of its stability.

5' Untranslated Regions↗
PubMed2005

Effect of glycosylation on the catalytic and conformational stability of homologous alpha-amylases.

A thermostable alpha-amylase from B. licheniformis (BLA) and a mesophilic amylase from B. amyloliquefaciens (BAA) were covalently coupled to oxidized synthetic sucrose polymers (OSP400 and OSP70) and polyglutaraldehyde (PGA) by reductive alkylation to study the effect of neoglycosylation on the activity, kinetic and thermodynamic stability. The catalytic efficiency of the modified enzymes was comparable to that of the native enzyme. Covalent coupling decreased the rate of inactivation at all the temperatures studied, both in the presence and absence of added Ca2+. The stability of the native enzyme was found to increase upon modification as observed from the increase in tl/2 in the absence of Ca2+ ions by about 1.5 - 13.7 times (at 85 degrees C) in the case of BLA and 5.7 - 8.4 times (at 50 degrees C) for BAA. The highest stability was observed for OSP400 modified enzyme with Delta Cm and Delta Tm values of 0.63 M and 7.92 degrees C for BLA and 0.85 M and 5.3 degrees C for BAA, respectively. The order of stability was OSP400 > OSP70 > PGA > Native for both BLA and BAA. The stability of the modified amylases obtained from the present study were superior compared to most of the single and double mutants obtained by site-directed mutagenesis that were constructed so as to enhance the intrinsic stability of these enzymes.

Bacillus↗
PubMed1992

Thermal stabilities of mutant Escherichia coli tryptophan synthase alpha subunits.

Random chemical mutagenesis, in vitro, of the 5' portion of the Escherichia coli trpA gene has yielded 66 mutant alpha subunits containing single amino acid substitutions at 49 different residue sites within the first 121 residues of the protein; this portion of the alpha subunit contains four of the eight alpha helices and three of the eight beta strands in the protein. Sixty-two of the subunits were examined for their heat stabilities by sensitivity to enzymatic inactivation (52 degrees C for 20 min) in crude extracts and by differential scanning calorimetry (DSC) with 29 purified proteins. The enzymatic activities of mutant alpha subunits that contained amino acid substitutions within the alpha and beta secondary structures were more heat labile than the wild-type alpha subunit. Alterations only in three regions, at or immediately C-terminal to the first three beta strands, were stability neutral or stability enhancing with respect to enzymatic inactivation. Enzymatic thermal inactivation appears to be correlated with the relative accessibility of the substituted residues; stability-neutral mutations are found at accessible residual sites, stability-enhancing mutations at buried sites. DSC analyses showed a similar pattern of stabilization/destabilization as indicated by inactivation studies. Tm differences from the wild-type alpha subunit varied +/- 7.6 degrees C. Eighteen mutant proteins containing alterations in helical and sheet structures had Tm's significantly lower (-1.6 to -7.5 degrees C) than the wild-type Tm (59.5 degrees C). In contrast, 6 mutant alpha subunits with alterations in the regions following beta strands 1 and 3 had increased Tm's (+1.4 to +7.6 degrees C). Because of incomplete thermal reversibilities for many of the mutant alpha subunits, most likely due to identifiable aggregated forms in the unfolded state, reliable differences in thermodynamic stability parameters are not possible. The availability of this group of mutant alpha subunits which clearly contain structural alterations should prove useful in defining the roles of certain residues or sequences in the unfolding/folding pathway for this protein when examined by urea/guaninidine denaturation kinetic analysis.

Calorimetry, Differential Scanning↗
PubMed1994

Cationic liposomes improve stability and intracellular delivery of antisense oligonucleotides into CaSki cells.

Antisense oligonucleotides (ODNs) are promising novel therapeutic agents against viral infections and cancer. However, problems with their inefficient delivery and inadequate stability have to be solved before they can be used in therapy. To circumvent these obstacles, a wide variety of improvements, including phosphorothioate ODNs and liposomes as a carrier system, have been developed. This study was designed to compare the effects of two cationic liposomes on the intracellular delivery and stability of ODNs in CaSki cell cultures. Also the stability of 3'-end phosphorothioate ODNs were investigated. The 3'-modification neither had any effect on the delivery, nor protected the ODNs against degradation. The cellular delivery and stability of ODNs was improved with both cationic liposomes, but a cationic liposomal preparations containing dimethyldioctadecylammonium bromide and dioleoylphosphatidylethanolamine (DDAB/DOPE) was more efficient than commercially available N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammoniummethylsulf ate (DOTAP). The improved cellular delivery was largely due to the stabilization of ODNs by cationic liposomes. The improved stability in the culture medium indicates that the cationic liposomes per se protect the ODNs from enzymatic degradation. Indeed, intact ODNs were found in the cytoplasm and nucleus only when delivered by cationic liposomes.

Base Sequence↗
PubMed1977

Comparison of intrinsic stabilities of free and bound enzymes by graphical removal of diffusional effects.

The enhanced stability usually exhibited by enzymes after immobilization may be attributed either to a stabilization effect of the solid matrix on the bound enzyme molecule or to the influences of diffusional limitations on the observed activity. To allow the comparison of the intrinsic statilities of free and bound enzymes a simple graphical procedure for the removal of external diffusional effects of stability curves is described. It is based on the determination of substrate concentration differences between the enzyme micro- and macroenvironment. Application of the method to aspartate aminotransferase bound to collagen membranes indicates that diffusional limitations for oxaloacetate are partly responsible for the observed stability enhancement. Comparison of the graphically obtained intrinsic profile with the stability curve of the soluble enzyme further demonstrate that the binding itself greatly increases the stability of aspartate aminotransferase.

Aspartate Aminotransferases↗
PubMed1982

Stabilization of lipoprotein lipase by endothelial cells.

Lipoprotein lipase, purified from bovine milk, lost 90% of its activity when incubated in Hanks' balanced salt solution for 5 min at 37 degrees C. Bovine pulmonary artery endothelial cells, maintained in culture, markedly stabilized this enzyme. The stabilizing factor of endothelial cells was non-dialyzable, resistant to heating at 100 degrees C and to changes in pH, and unaffected by treatments of cells with proteolytic enzymes or with heparinase (Flavobacterium heparinum enzyme). However, the stabilizing effect on lipoprotein lipase was reduced by 60-70% by the extraction of cells with chloroform/methanol (2:1). The lipid extract of the cells stabilized the enzyme, suggesting that lipid component(s) of the endothelial cells account for their stabilizing effect. Since the endothelial cell is thought to be the site of action of lipoprotein lipase, stabilization of the enzyme by this cell may play a role in its preservation and function in vivo.

Animals↗
PubMed1981

Stability of lactate dehydrogenases. I. Chemical modification of lysines.

The lysine residues of lactate dehydrogenase (L-lactate: NAD+ oxidoreductase, EC 1.1.1.27) can be amidinated by methyl-4-hydroxy-3-nitrobenzimidate to introduce nitrophenolate anions. This modification results in lowered thermal stability, as does acetylation. The conversion of these groups into uncharged aminophenol groups without further modification of the enzyme itself stabilizes the enzymes from pig heart and muscle and from chicken muscle, as does acetamidination, but the unusually stable enzyme from chicken heart reverts only to the stability of the native form. The results allow for the following conclusions. Destabilization is brought about at many points at the surface of lactate dehydrogenases by neutralization of positive charges. Stabilization, in contrast, is concluded to be due to modification of one lysine at position 241 of the sequence. This lysine must have been changed to arginine during the evolution of heart-type lactate dehydrogenases in going from lower to higher reptiles. This exchange has been conserved in the enzymes from the hearts of birds and therefore the enzyme from chicken heart is very stable and cannot further be stabilized by modification of lysines. From X-ray structure analysis, the stabilization by exchange of Arg for Lys at position 241 or by amidination is explained by the formation of additional ion pairs with aspartic acid57 of the Q-related subunits.

Animals↗
PubMed1990

Effects of reduced malto-oligosaccharides on the thermal stability of pullulanase from Bacillus acidopullulyticus.

We investigated the effects of the reduced malto-oligosaccharides, D-glucitol (G1-OH), maltitol (G2-OH), maltotriitol (G3-OH), maltotetraitol (G4-OH), and maltopentaitol (G5-OH) on the thermal stability of Bacillus acidopullulyticus pullulanase (EC 3.2.1.41). The thermal stability depended on the concentration of D-glucitol; after heat treatment for 90 min at 60 degrees in the presence of 0.56, 0.28, 0.14, or 0M G1-OH, the residual activity was 100, 80, 32, and 10% of the control, respectively. Stability increased with the number of glucosyl residues in the alditols added; the effects of G3-OH, G4-OH, and G5-OH on stability were remarkable. Addition of 30% G2-OH, G3-OH, and G4-OH also contributed to the thermal stability of the pullulanase immobilized onto chitosan beads treated with glutaraldehyde. A high concentration of G2-OH stabilized other debranching amylases, Klebsiella pneumoniae pullulanse, Bacillus sectorramus pullulanase, and Pseudomonas amyloderamosa isoamylase (EC 3.2.1.68) under heat treatment for 48 h at 60 degrees, as well as the pullullanase of B. acidopullulyticus.

Amylases↗
PubMed1991

Cumulative site-directed charge-change replacements in bacteriophage T4 lysozyme suggest that long-range electrostatic interactions contribute little to protein stability.

Bacteriophage T4 lysozyme is a basic molecule with an isoelectric point above 9.0, and an excess of nine positive charges at neutral pH. It might be expected that it would be energetically costly to bring these out-of-balance charges from the extended, unfolded, form of the protein into the compact folded state. To determine the contribution of such long-range electrostatic interactions to the stability of the protein, five positively charged surface residues, Lys16, Arg119, Lys135, Lys147 and Arg154, were individually replaced with glutamic acid. Eight selected double, triple and quadruple mutants were also constructed so as to sequentially reduce the out-of-balance formal charge on the molecule from +9 to +1 units. Each of the five single variant proteins was crystallized and high-resolution X-ray analysis confirmed that each mutant structure was, in general, very similar to the wild-type. In the case of R154E, however, the Arg154 to Glu replacement caused a rearrangement in which Asp127 replaced Glu128 as the capping residue of a nearby alpha-helix. The thermal stabilities of all 13 variant proteins were found to be fairly similar, ranging from 0.5 kcal/mol more stable than wild-type to 1.7 kcal/mol less stable than wild-type. In the case of the five single charge-change variants, for which the structures were determined, the changes in stability can be rationalized in terms of changes in local interactions at the site of the replacement. There is no evidence that the reduction in the out-of-balance charge on the molecule increases the stability of the folded relative to the unfolded form, either at pH 2.8 or at pH 5.3. This indicates that long-range electrostatic interactions between the substituted amino acid residues and other charged groups on the surface of the molecule are weak or non-existent. Furthermore, the relative stabilities of the multiple charge replacement mutant proteins were found to be almost exactly equal to the sums of the relative stabilities of the constituent single mutant proteins. This also clearly indicates that the electrostatic interactions between the replaced charges are negligibly small. The activities of the charge-change mutant lysozymes, as measured by the rate of hydrolysis of cell wall suspensions, are essentially equal to that of the wild-type lysozyme, but on a lysoplate assay the mutant enzymes appear to have higher activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Arginine↗
PubMed1995

Understanding and increasing protein stability.

This review surveys the processes leading to loss of protein function and the types of molecular interaction that help stabilize proteins. It considers the effects of organic solvents on stability and the special features of thermophilic proteins. The deliberate manipulation of stability by protein engineering is discussed using the enzyme subtilisin as example. Both random and rational mutations of this protein have led to variants with greatly improved tolerances of high temperatures and organic solvents. One can also use chemical modification to modify protein stability and some of the main approaches are reviewed. The chemical and genetic strategies are complementary and have been combined to stabilize cytochrome c by metal-mediated cross-linking following site-specific mutagenesis. The article concludes by summarizing the beneficial effects of certain additives on protein stability.

Enzyme Stability↗
PubMed1987

Stabilization of phosphofructokinase with sugars during freeze-drying: characterization of enhanced protection in the presence of divalent cations.

Phosphofructokinase purified from rabbit skeletal muscle is fully inactivated after freeze-drying and dissolution. The addition of trehalose or maltose to the enzyme solution prior to freeze-drying results in a recovery of up to 80% of the original activity. Slightly less stabilization is imparted by sucrose, whereas glucose and galactose at concentrations up to 500 mM are relatively ineffective at protecting phosphofructokinase. Addition of ionic zinc to enzyme-sugar mixtures prior to freeze-drying greatly enhances the stabilization imparted by the above sugars. This effect is not simply due to the summation of the individual protective capacities of zinc and the sugar. Zinc alone affords no protection, but a high degree of stabilization is achieved when zinc is added to a sugar solution, even when the sugar is at a concentration at which, by itself, it is totally ineffective. In the presence of a constant sugar concentration (100 mM), freeze-dry stabilization of phosphofructokinase is increased as the concentration of zinc is increased. When the zinc concentration is held constant (0.9 mM) and the sugar concentration varied, the maximum stabilization is noted with less than 200 mM sugar. At higher solute concentrations the degree of enhancement decreases such that with 500 mM sugar the addition of zinc results in only a slight increase in protection. Several other organic solutes (proline, 4-hydroxyproline, glycine, trimethylamine N-oxide, glycerol and myo-inositol) that afford cryoprotection to phosphofructokinase, an effect enhanced by the addition of zinc, do not stabilize the enzyme during freeze-drying, even if zinc is present. The addition of ionic copper, cadmium, nickel, cobalt, calcium and manganese to trehalose-phosphofructokinase solutions prior to freeze-drying also increases the percentage of activity recovered after dissolution. Magnesium is ineffective in this respect.

Animals↗
PubMed1995

The use of capillary electrophoresis to monitor the stability of a dual-action cephalosporin in solution.

Ro 23-9424 is a broad-spectrum antibacterial agent consisting of a cephalosporin and a quinolone moiety which are held together by an ester linkage. This compound has limited solubility in water but is more soluble at alkaline pH. Such high pH values, however, lead to stability problems due to the lability of the ester linkage, and result in the formation of the free quinolone and cephalosporin moieties. The balance between solubility and stability presents a challenge in formulation development for this compound. Thus, it is important to effectively monitor the stability of Ro 23-9424 after it has been reconstituted in different solvent systems. In this manner, a diluent which does not lead to degradation of the drug can be identified. A capillary electrophoresis method was developed and validated to monitor the stability of Ro 23-9424. The method was found to meet acceptable criteria for method precision, system precision, linearity and limits of detection and quantitation. The method was used to monitor the stability and measure the half-life of Ro 23-9424 in water and in an L-arginine-sodium benzoate-saline diluent designed to mimic the drug delivery system. This work shows that capillary electrophoresis can be used to compare the stability of a drug in different solutions as an aid in formulation development.

Anti-Infective Agents↗
PubMed1995

Issues in evaluation of bioanalytical method selectivity and drug stability.

Evaluation of selectivity is one of the most controversial aspects of method validation and application of methods to studies. The focus of selectivity testing should address the question: Above what level will interference significantly impact on study conclusions? Four key issues will be addressed: the statistical relevance of any selectivity test performed; a criterion for significant interference; experimental methods to establish selectivity; and criteria for acceptance. To ensure that compound integrity is maintained throughout the work-up process, statistically meaningful methods of stability evaluation which are associated with specific acceptance criteria are required. Suitable methods for evaluating stability of analyte and/or solutions of analyte, in process stability, processed sample stability, long term stability and freeze-thaw stability, as well as meaningful acceptance criteria, are presented.

Animals↗
PubMed1995

N,N'-ethylene-di-L-cysteine (EC) complexes of Ga(III) and In(III): molecular modeling, thermodynamic stability and in vivo studies.

There are several reports in the literature on the evaluation of chelates containing two nitrogens and two sulfurs (N2S2) as four coordinate ligands (in solution) for gallium and indium radiopharmaceuticals. No thermodynamic stability constants of these ligands were reported, and the stability of these complexes in vivo has been questioned. L,L-Ethylenedicysteine (EC) is a N2S2 ligand that also contains two carboxylic acid moieties for complexation of Ga(III) and In(III) in a hexacoordinate environment. The stability constants of Ga- and In-EC have been determined by potentiometric methods. The stability of In-EC was found to be greater than that of Ga-EC, with stability constants (log K's) of 33.0 and 31.5, respectively. A molecular mechanics evaluation of the Ga- and In-EC complexes support the thermodynamic results. 67Ga- and 111In-labeled complexes of EC were prepared and analyzed by thin layer chromatography and electrophoresis. Both complexes were evaluated in biodistribution studies in normal Sprague-Dawley rats. 111In-EC cleared rapidly through the hepatobiliary system, whereas 67Ga-EC remained in the liver at 1 h post-injection. Although 67Ga-EC was retained in the liver, suggesting instability of the complex in vivo. 67Ga-EC was stable in rat plasma in vivo at 2 h post-injection. Because of the high thermodynamic and in vivo stability of In-EC, derivatives of EC may have applications as bifunctional chelates for 111In-labeled proteins and peptides. More lipophilic analogues of 68Ga-EC may also have potential as myocardial PET imaging agents.

Animals↗
PubMed2004

Thermal unfolding of medium-chain acyl-CoA dehydrogenase and iso(3)valeryl-CoA dehydrogenase: study of the effect of genetic defects on enzyme stability.

Genetic defects affecting acyl-CoA dehydrogenases (ACAD)-key enzymes in the degradation of fatty acids and branched chain amino acids-are increasingly recognized as being more widespread than originally thought. For the medium-chain acyl-CoA dehydrogenase (MCAD), the K304E mutation is the most common genetic defect among Caucasian populations. The effect of substrate or substrate analog binding on the stability of wild-type MCAD and isovaleryl-CoA dehydrogenase (i3VD) and their genetic mutants (K304E- and T168A-MCAD and A282V-i3VD) is examined. Binding to the mutant ACADs is generally approximately 10-fold weaker compared to wild-type proteins. Thermal stability of wt-MCAD (melting point approximately 53.6 degrees C) is significantly higher compared to wt-i3VD ( approximately 49.3 degrees C). With the exception of the A282V-i3VD mutant, a high degree of stabilization (5-11 degrees C) is induced by conversion into the reduced enzyme form complexed with product. The results are discussed based on the 3D-structures of the enzymes, and it is concluded that in the case of K304E-MCAD thermal stability as such is not a major contribution to the clinical phenotype. With the T168A-MCAD and A282V-i3VD mutants, however, the diminished thermal stability and minor stabilization by ligands must be regarded as an important factor contributing to the manifestation of the disease.

Acyl-CoA Dehydrogenase↗