RESUMO
OBJECTIVE: Cystic fibrosis (CF) is a multisystemic inherited disease. The aim of this study was to determine free carnitine (FC) and acylcarnitine concentrations in CF newborns with various mutations of the CFTR gene perinatally. STUDY DESIGN: FC/acylcarnitines were determined in dried blood spots via liquid chromatography-tandem mass spectrometry (LC-MS/MS) on the third day of life of full-term normal (n = 50) and CF (n = 28) newborns. For infants with elevated immunoreactive trypsinogen values, FC/acylcarnitines were quantified again 48 hours later, followed by mutational analysis of CFTR gene via Sanger sequencing. RESULTS: Initial FC and sums of acylcarnitine concentrations were statistically significantly lower in CF patients than in controls and even lower 48 hours later. The mutations F508del and 621 + 1G > T were predominantly identified among CF patients. CONCLUSION: Low FC and acylcarnitine concentrations were measured perinatally in CF patients, for all CFTR mutations detected. Carnitine supplementation of breastfeeding mothers could be beneficial.
Assuntos
Carnitina/análogos & derivados , Carnitina/sangue , Fibrose Cística/sangue , Biomarcadores , Carnitina/administração & dosagem , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Análise Mutacional de DNA , Alimentos Fortificados , Humanos , Recém-Nascido , Leite Humano , Mutação , Triagem NeonatalRESUMO
During the last years, the food industry is working on the replacement of high energy methodologies with more sustainable techniques for the encapsulation of natural preservatives, in order to enhance their effectiveness as food additives. In the present study, nisin, an antimicrobial agent, was encapsulated in essential oil-containing microemulsions. More specifically, rosemary, thyme, oregano, and dittany essential oil-containing microemulsions were formulated to encapsulate nisin enhancing the system's overall antimicrobial activity. The systems were investigated for the interfacial properties and size of the surfactants' monolayer using electron paramagnetic resonance spectroscopy and dynamic light scattering. Subsequently, nisin-loaded microemulsions were tested for their antimicrobial activity against Lactococcus lactis, Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus, using the well diffusion assay. Finally, this technique was validated by a killing assay. Overall, this study provides important information on the antibacterial activity of nisin-loaded nano-carriers enhanced by essential oils, in relation to the microemulsions' structure.
Assuntos
Anti-Infecciosos/química , Micelas , Nanoestruturas/química , Nisina/química , Óleos Voláteis/química , Anti-Infecciosos/farmacologia , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Espectroscopia de Ressonância de Spin Eletrônica , Emulsões/química , Microbiologia de Alimentos , Lactococcus lactis/efeitos dos fármacos , Listeria monocytogenes/efeitos dos fármacos , Nisina/farmacologia , Origanum/química , Origanum/metabolismo , Rosmarinus/química , Rosmarinus/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Thymus (Planta)/química , Thymus (Planta)/metabolismo , ViscosidadeRESUMO
Reverse micelles (RMs) as nanocarriers of nisin were optimized for the highest water and bacteriocin content. RMs formulated with either refined olive oil or sunflower oil, distilled monoglycerides, ethanol, and water were effectively designed. Structural characterization of the RMs was assessed using Electron Paramagnetic Resonance Spectroscopy and Small Angle X-ray Scattering in the presence and absence of nisin. No conformational changes occurred in the presence of nisin for the nanocarriers. To assess efficacy of the loaded systems, their antimicrobial activity against Staphylococcus aureus and Listeria monocytogenes was tested in lettuce leaves and minced meat, respectively. Antimicrobial activity was evident in both cases. Interestingly, a synergistic antimicrobial effect was observed in lettuce leaves and to a lesser extent in minced meat between nisin and some of the nanocarriers' constituents (probably ethanol). Our findings suggest complex interactions that take place when RMs are applied in different food matrices.
Assuntos
Antibacterianos/administração & dosagem , Portadores de Fármacos/química , Microbiologia de Alimentos/métodos , Nanoestruturas/química , Nisina/administração & dosagem , Antibacterianos/química , Antibacterianos/farmacologia , Bacteriocinas , Portadores de Fármacos/administração & dosagem , Espectroscopia de Ressonância de Spin Eletrônica , Emulsões/química , Lactuca/microbiologia , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/patogenicidade , Carne/microbiologia , Micelas , Monoglicerídeos/química , Nanoestruturas/administração & dosagem , Nisina/química , Nisina/farmacologia , Óleos de Plantas/química , Espalhamento a Baixo Ângulo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidadeRESUMO
BACKGROUND: Deficiencies of galactokinase (GALK) and UDP-epimerase (GALE) are implicated with galactose metabolic disorders. The aim of the study was the identification of mutations in GALK and GALE genes and clinical evaluation of patients. METHODS: Five patients with GALK and five with GALE deficiency were picked up via the Neonatal Screening Program. Additionally, two females, 4 years old, were referred with late diagnosed galactosemia, as rare cases. Mutational analysis was conducted via Sanger sequencing, while in silico analysis tools were utilized for the novel mutation. Psychomotor and speech development tests were performed, as well. RESULTS: The mutation p.Pro28Thr was identified in both alleles in GALK-deficient patients of Roma (gypsy) origin, whereas the novel p.Asn39Ser was detected in two non-Roma patients. In GALE-deficient patients benign and/or likely benign mutations were found. Psychomotor and speech delay were determined in the Roma GALK patients. In each of the late diagnosed females, four mutations were identified in all galactosemia-related genes. CONCLUSIONS: The mutational spectrums of GALE- and GALK-deficient patients in Greece are presented for the first time along with a clinical evaluation. Mutational analysis in all galactosemia-related genes of symptomatic patients is highly recommended for future cases.
Assuntos
Galactoquinase/genética , Galactosemias/genética , Transtornos Mentais/epidemiologia , Mutação , Alelos , Pré-Escolar , Análise Mutacional de DNA , Feminino , Galactosemias/complicações , Galactosemias/patologia , Grécia , Humanos , Lactente , Recém-Nascido , Masculino , Transtornos Mentais/genética , PrognósticoRESUMO
Classical galactosaemia is an inborn error of metabolism due to the deficiency of the enzyme galactose-1-phosphate uridylyltransferase (GALT). The aim of the study was to identify the underlying mutations in Greek patients with GALT deficiency and evaluate their psychomotor and speech development. Patients with GALT deficiency (n = 17) were picked up through neonatal screening. Mutational analysis was conducted via Sanger sequencing, while in silico analysis was used in the cases of novel missense mutations. Psychomotor speech development tests were utilized for the clinical evaluation of the patients. Eleven different mutations in the GALT gene were detected in the patient cohort, including two novel ones. The most frequent mutation was p.Q188R (c.563 A > G). As for the novel mutations, p.M298I (c.894 G > A) was identified in four out of 32 independent alleles, while p.P115S (c.343 C > T) was identified once. Psychomotor evaluation revealed that most of the patients were found in the borderline area (Peabody test), while only two had speech delay problems. The WISK test revealed three patients at borderline limits and two were at lower than normal limits. The mutational spectrum of the GALT gene in Greek patients is presented for the first time. The mutation p.Q188R is the most frequent among Greek patients. Two novel mutations were identified and their potential pathogenicity was estimated. Regarding the phenotypic characteristics, psychomotor disturbances and speech delay were mainly observed among GALT-deficient patients.
Assuntos
Galactosemias/enzimologia , Galactosemias/genética , Galactosiltransferases/genética , Análise Mutacional de DNA , Feminino , Grécia , Humanos , Recém-Nascido , MasculinoRESUMO
Water-in-oil microemulsions with biocompatible components were formulated to be used as carriers of natural antioxidants, such as hydroxytyrosol (HT) and gallic acid (GA). The system was composed of a mixture of natural surfactants, lecithin and monoglycerides, medium chain triglycerides, and aqueous phase. A dual approach was undertaken to study the structure and dynamics of these complicated systems. First, experimental data were collected by using adequate techniques, such as dynamic light scattering (DLS) and electron paramagnetic resonance (EPR) spectroscopy. Following this, a coarse-grained molecular dynamics (CGMD) study based on the experimental composition using the MARTINI force field was conducted. The simulations revealed the spontaneous formation of reverse micelles (RMs) starting from completely random initial conformations, underlying their enhanced thermodynamic stability. The location of the bioactive molecules, as well as the structure of the RM, were in accordance with the experimental findings. Furthermore, GA molecules were found to be located inside the water core, in contrast to the HT ones, which seem to lie at the surfactant interfacial layer. The difference in the antioxidants' molecular location was only revealed in detail from the computational analysis and explains the RM's swelling observed by GA in DLS measurements.
Assuntos
Simulação de Dinâmica Molecular , Emulsões , Micelas , Tensoativos , ÁguaRESUMO
A fully automated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of omeprazole in human plasma. Utilization of 96-well plates and robotic liquid handling workstations, rendered the whole procedure very fast, compared to the manual respective procedure of Liquid-Liquid Extraction (LLE). Sample analysis was performed by reversed phase LC-MS/MS, with positive electrospray ionization, using multiple reaction monitoring (MRM). The method required low plasma volumes and analysis of samples was completed in short run times. It was fully validated and applied to a pharmacokinetic study after per os administration of 20mg tablet formulations of omeprazole. The obtained concentrations were used for the calculation of the basic omeprazole pharmacokinetic parameters. Some variations observed in pharmacokinetic parameters among subjects were attributed to differences of CYP2C19 genotype. Therefore, a novel molecular method was developed in which DNA analysis was conducted by using Real Time-Polymerase Chain Reaction (Real Time-PCR). As source of biological material, Dried Blood Spots (DBS) were utilized, offering an alternative and advantageous strategy for such kind of studies.
Assuntos
Antiulcerosos/sangue , Citocromo P-450 CYP2C19/genética , Omeprazol/sangue , Cromatografia Líquida de Alta Pressão/métodos , Genótipo , Humanos , Extração Líquido-Líquido/métodos , Polimorfismo Genético , Reação em Cadeia da Polimerase em Tempo Real/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
Water-in-oil (W/O) microemulsions based on either refined olive oil (ROO) or sunflower oil (SO), distilled monoglycerides (DMG), and ethanol were used as nisin carriers in order to ensure its effectiveness as a biopreservative. This work presents experimental evidence on the effects of ethanol concentration, hydration, the nature of oil, and the addition of nisin on the nanostructure of the proposed inverse microemulsions as revealed by electrical conductivity measurements, dynamic light scattering (DLS), small angle X-ray scattering (SAXS), and electron paramagnetic resonance (EPR) spectroscopy. Modeling of representative SAXS profiles was applied to gain further insight into the effects of ethanol and solubilized water content on the inverse swollen micelles' size and morphology. With increasing ethanol content, the overall size of the inverse micelles decreased, whereas hydration resulted in an increase in the micellar size due to the penetration of water into the hydrophilic core of the inverse swollen micelles (hydration-induced swelling behavior). The dynamic properties of the surfactant monolayer were also affected by the nature of the used vegetable oil, the ethanol content, and the presence of the bioactive molecule, as evidenced by EPR spin probing experiments. According to simulation on the experimental spectra, two populations of spin probes at different polarities were revealed. The antimicrobial effect of the encapsulated nisin was evaluated using the well diffusion assay (WDA) technique against Lactococccus lactis. It was found that this encapsulated bacteriocin induced an inhibition of the microorganism growth. The effect was more pronounced at higher ethanol concentrations, but no significant difference was observed between the two used vegetable oils (ROO and SO).
Assuntos
Portadores de Fármacos , Etanol/química , Lactococcus lactis/efeitos dos fármacos , Nisina/farmacologia , Água/química , Condutividade Elétrica , Emulsões , Lactococcus lactis/crescimento & desenvolvimento , Micelas , Monoglicerídeos/química , Nisina/química , Azeite de Oliva/química , Marcadores de Spin , Óleo de Girassol/químicaRESUMO
Water-in-oil (W/O) microemulsions and emulsions based on medium chain triglycerides (MCT) were successfully formulated with the addition of emulsifiers and used as encapsulation matrices for hydroxytyrosol (HT), an antioxidant naturally found in extra virgin olive oil. The digestibility of these edible W/O dispersions by recombinant dog gastric lipase (rDGL) and porcine pancreatic lipase (PPL) was then tested at different pH values using a pHstat device. rDGL and PPL displayed a much lower activity on the W/O microemulsion than that on the W/O emulsion and MCT alone. This was explained by the presence of higher amounts of emulsifiers (4.9% w/w lecithin and monoglycerides) in the composition of W/O microemulsions compared to W/O emulsions (1.3% w/w emulsifiers). These surfactants also induced a shift of maximum lipase activity towards lower pH values, which usually reflects the competition between surfactants and lipases for binding at the lipid-water interface. rDGL and PPL were then used consecutively in a two-step digestion model mimicking the conditions found in the human gastrointestinal tract. Direct titration and back-titration of free fatty acids allowed the continuous estimation of lipolysis rates under both gastric and duodenal conditions. Gastric lipolysis of W/O microemulsions was reduced 6 to 9-fold compared to W/O emulsions. This inhibition had a major impact on the overall lipolysis, although duodenal lipolysis was less affected by the dispersion type. The presence of HT had also some minor effects on lipolysis rates.
Assuntos
Química Farmacêutica/métodos , Emulsões/química , Lipase/metabolismo , Lipólise , Preparações Farmacêuticas/química , Álcool Feniletílico/análogos & derivados , Estômago/enzimologia , Água/química , Animais , Digestão , Cães , Emulsificantes/química , Ensaios Enzimáticos , Ácidos Graxos/metabolismo , Ácidos Graxos não Esterificados , Concentração de Íons de Hidrogênio , Lecitinas/química , Lipase/química , Monoglicerídeos/química , Azeite de Oliva/metabolismo , Álcool Feniletílico/química , Proteínas Recombinantes , Tensoativos/química , TriglicerídeosRESUMO
Two edible Water-in-Oil (W/O) dispersions, an emulsion that remains kinetically stable and a microemulsion which is spontaneously formed, transparent and thermodynamically stable, were developed for potential use as functional foods, due to their ability to be considered as matrices to encapsulate biologically active hydrophilic molecules. Both systems contained Medium Chain Triglycerides (MCT) as the continuous phase and were used as carriers of Hydroxytyrosol (HT), a hydrophilic antioxidant of olive oil. A low energy input fabrication process of the emulsion was implemented. The obtained emulsion contained 1.3% (w/w) of surfactants and 5% (w/w) aqueous phase. The spontaneously formed microemulsion contained 4.9% (w/w) of surfactants and 2% (w/w) aqueous phase. A comparative study in terms of structural characterization of the systems in the absence and presence of HT was carried out. Particle size distribution obtained by Dynamic Light Scattering (DLS) technique and interfacial properties of the surfactants' layer, examined by Electron Paramagnetic Resonance (EPR) spectroscopy indicated the involvement of HT in the surfactant membrane. Finally, the proposed systems were studied for the scavenging activity of the encapsulated antioxidant toward galvinoxyl stable free radical showing a high scavenging activity of HT in both systems.
Assuntos
Portadores de Fármacos , Emulsões , Álcool Feniletílico/análogos & derivados , Espectroscopia de Ressonância de Spin Eletrônica , Álcool Feniletílico/administração & dosagem , Tensoativos/químicaRESUMO
BACKGROUND: Hawkinsinuria is a rare inborn error of tyrosine metabolism. OBJECTIVES: To study novel hawkinsinuria cases by monitoring their biochemical profile and conducting a mutation analysis. SUBJECTS AND METHODS: Among 92,519 newborns that underwent expanded newborn screening, two unrelated cases with high tyrosine blood levels were further investigated by chromatographic techniques and via genetic testing for 4-hydroxyphenylpyruvate dioxygenase (HPD) gene. RESULTS: Elevated levels were monitored for blood/plasma tyrosine and for the specific diagnostic markers in urine. The two newborns were put on a special low tyrosine diet. Till completion of the 1st year of their life, liver function tests and brain MRI were normal. The mutation A33T was identified in both cases, while one neonate carried an additional novel mutation of HPD gene (V212M). CONCLUSIONS: Two mutations of HPD gene, A33T, which are associated with hawkinsinuria and a novel one (V212M) were detected for the 1st time in Greek newborns.