DNA-loaded chitosan oligosaccharide nanoparticles with enhanced permeability across Calu-3 cells.

Chitosan oligosaccharide (oligoCS) is a low molecular weight chitosan and its potential for DNA delivery is described here. DNA-loaded oligoCS nanoparticles were prepared by ionic gelation using thiamine pyrophosphate (TPP) as cross-linker. The nanoparticles with oligoCS:DNA: TPP weight ratio of 50:1:25 were approximately 170 nm in diameter with a zeta potential of +40 mV, and were used in the permeability study. The cytotoxicity of oligoCS solutions and nanoparticles was evaluated by MTT assay. The concentrations that exhibited minimal cytotoxicity were employed to investigate their effect on trans-epithelial electrical resistance (TEER) and cellular uptake across the Calu-3 cell layer which was used as a nasal epithelial model. OligoCS nanoparticles were able to cause a significant and reversible decrease in TEER and promote efficient cellular uptake. In addition, the oligoCS nanoparticles were able to enhance paracellular permeability to a greater extent than oligoCS solutions at an equivalent concentration. However, the oligoCS nanoparticles were too large to cross the cell layers through the paracellular route. The transcellular pathway appeared to be the major mechanism of the transportation of oligoCS nanoparticles across the cell layers. OligoCS nanoparticles also allowed efficient DNA incorporation, thereby providing the possibility of controlled nucleic acids release and absorption across epithelial surface.

Mitochondria from cultured cells derived from normal and thiamine-responsive megaloblastic anemia individuals efficiently import thiamine diphosphate.

BACKGROUND: Thiamine diphosphate (ThDP) is the active form of thiamine, and it serves as a cofactor for several enzymes, both cytosolic and mitochondrial. Isolated mitochondria have been shown to take up thiamine yet thiamine diphosphokinase is cytosolic and not present in mitochondria. Previous reports indicate that ThDP can also be taken up by rat mitochondria, but the kinetic constants associated with such uptake seemed not to be physiologically relevant. RESULTS: Here we examine ThDP uptake by mitochondria from several human cell types, including cells from patients with thiamine-responsive megaloblastic anemia (TRMA) that lack a functional thiamine transporter of the plasma membrane. Although mitochondria from normal lymphoblasts took up thiamine in the low micromolar range, surprisingly mitochondria from TRMA lymphoblasts lacked this uptake component. ThDP was taken up efficiently by mitochondria isolated from either normal or TRMA lymphoblasts. Uptake was saturable and biphasic with a high affinity component characterized by a Km of 0.4 to 0.6 microM. Mitochondria from other cell types possessed a similar high affinity uptake component with variation seen in uptake capacity as revealed by differences in Vmax values. CONCLUSIONS: The results suggest a shared thiamine transporter for mitochondria and the plasma membrane. Additionally, a high affinity component of ThDP uptake by mitochondria was identified with the apparent affinity constant less than the estimates of the cytosolic concentration of free ThDP. This finding indicates that the high affinity uptake is physiologically significant and may represent the main mechanism for supplying phosphorylated thiamine for mitochondrial enzymes.

In vivo study of the kinetics of thiamine and its phosphoesters in the deafferented rat cerebellum.

The effects of chemical (CD) and surgical (SD) deafferentation of the cerebellum on different steps of the metabolism of thiamine (Th), thiamine monophosphate (ThMP) and thiamine pyrophosphate (ThPP) were evaluated in vivo in rats. CD was carried out by i.p. injection of 3-acetylpyridine, followed by harmaline and niacinamide. SD was carried out by complete dissection of the peduncles of the left cerebellar hemisphere. Under steady state condition the radioactivity of Th and its phosphoesters was determined in plasma and whole cerebellum after an i.p. injection of thiazole-[2(14)C]-thiamine (30 micrograms:1.25 micro Ci). Analytical data were processed by using an improved mathematical compartmental model, which allowed the calculation of fractional rate constants (FRC), turnover rates (TR) and turnover times (TT). Both CD and SD caused a significant reduction of TR values for Th phosphorylation to ThPP, dephosphorylation of ThPP to ThMP and Th, and ThMP, but not Th, release. TT for all Th compounds were increased compared to controls, indicating a general slowing of thiamine metabolism in the deafferented cerebellum. These results indicate an imbalance in the thiamine metabolism resulting from the impaired activity of cerebellar neurons. The possible implications of the changes in rate of Th compound turnover with respect to biochemical changes in cerebellar ataxia are discussed.

Effects of phenytoin on the in vivo kinetics of thiamine and its phosphoesters in rat nervous tissues.

The in vivo effects of chronic (30 days) and subchronic (10 days) intragastric treatment with phenytoin (PHT) (500 mg/kg) b.wt., suspended in 10% arabic gum water solution) on the uptake and metabolism of thiamine (T), T monophosphate (TMP) and T pyrophosphate (TPP) were evaluated in rat nervous regions (cerebral cortex, brainstem, cerebellum and sciatic nerve) by determining the radioactivity of T and its phosphoesters in plasma and tissues at fixed time intervals (0.25-240 h) after an i.p. injection of thiazole-[2-14C]thiamine (30 micrograms: 1.25 microCi). A nutritionally adequate diet containing T in excess was given to the animals in order to produce a virtually stable content of T compounds in the tissues. Analytical data were processed by using a compartmental model which allowed the calculation of fractional rate constants (FRC), turnover rates (TR) and turnover times. Compared with vehicle-treated controls, animals treated chronically with PHT exhibited lower levels of radiolabelled T compounds in all nervous regions except for the cerebral cortex. These alterations were not found in animals receiving subchronic treatment. Evaluation of FRC values indicated that PHT-induced effects on T metabolism differed depending on the length of PHT treatment and the nervous region considered. Overall, PHT appeared to interfere mainly with T and TMP uptake, TPP dephosphorylation to TMP and TPP turnover times, these effects being particularly prominent in the cerebellum and in the brainstem of chronically treated animals. Since all changes in T uptake and metabolism were observed in the absence of overt behavioural toxicity, these findings may have potential clinical relevance in highlighting possible mechanisms by which PHT therapy can alter brain metabolism.

Kinetics of thiamin and thiamin phosphate esters in human blood, plasma and urine after 50 mg intravenously or orally.

The concentrations of thiamin and thiamin monophosphate and diphosphate in plasma and whole blood samples were assessed in six healthy subjects for 12 h and in urine for 24 h following an IV and PO bolus dose of 50 mg thiamin HCl. Unphosphorylated thiamin increased rapidly in plasma after IV administration and then decreased to its initial value within 12 h in all but one subject; the half-life was 96 min. Thiamin mono and -diphosphate increased moderately (56%), and decreased slowly; the half-life of diphosphate was 664 min. Within 24 h, 53% of the administered dose was recovered in the urine, indicating a restricted distribution. After oral administration, the peak thiamin concentration in plasma was reached after 53 min and the concentration then had increased to 179% of its initial value. The elimination half-life was 154 min, and only 2.5% of the given dose was recovered in the urine. The relative bioavailability of thiamin was 5.3%. A moderate amount of the administered thiamin was stored in blood. Other body tissues must play an important part, therefore, in the distribution of thiamin.

Actividad de la glucosa-6-fosfato deshidrogenasa en pacientes diabéticos tratados con pirofosfato de tiamina o cocarboxilasa / Activity of clucose-6-phosphate dehydrogenase on diabetic patients treated with thiramine pyrophosphate or cocarboxilase

Trabajos realizados con anterioridad tanto a nivel clínico como experimental en el Instituto de Investigaciones Cientificas Hans Selye, permiten proponer que la cocarboxilasa o pirofosfato de tiamina (PPT) estable en solución, al aplicarse a pacientes con hiperglucemia, mejora la incorporación de la glucosa a los tejidos. Con base en tales hechos, un posterior estudio pretende analizar si el PPT ejerce acción alguna sobre el ciclo de las pentosas. Para demostrar esto, se estudió la conducta de la glucosa-6-fosfato deshidrogenasa (G-6-PD) en el eritrocitos humanos, toda vez que su actividad se ha encontrado disminuida en los pacientes afectados por diabetes mellitus. Para comprobarlo se seleccionaron 10 pacientes de uno y otro sexo, de 45 a 70 años de edad y con diagnóstico de diabetes tipo II. De cada uno se tomó una muestra sanguínea antes de iniciar el traqtamiento con PPT y posteriormente a los 15, 30, 60, 90 y 120 días después de obtener la primera muestra, a fin de determinar la actividad de G-6-PD, así como las de glucosa, colesterol total, HDL, LDL, triglicériados y hemoglobina glucosilada. Los resultados obtenidos demuestran que la glucemia tiende a registrar niveles normales, alcanzando una recuperación física plena en 70 por ciento de los pacientes; en 100 por ciento de ellos también se normalizaron los niveles de colesterol y de triglicéridos. La hemoglobina glucosilada disminuye considerablemente en relación con los valores registrados antes de iniciar el tratamiento. Con respecto a la actividad de la G-6-PD, los autores observaron que ésta se incrementa, en promedio, de 3 a 5 U/g de hemoglobina en los pacientes hiperglucémicos: el máximo incremento de actividad se registra entre el segundo y tercer mes de tratamiento, lo cual indica que PPT participa en: 1) la incorporación de glucosa al interior de los eritrocitos, toda vez que dicha sustancia disminuye la glucosa sérica: 2) la utilización de la glucosa en el metabolismo de esta célilas, y 3) el ciclo de las pentosas, dado que la actividad de la G-6-PD aumenta en forma considerable.

Inactivation of the pyruvate dehydrogenase complex of Escherichia coli by fluoropyruvate.

The pyruvate dehydrogenase complex (PDH complex) of Escherichia coli and its pyruvate dehydrogenase component (E1) are rapidly inactivated by low concentrations of fluoropyruvate in a thiamin pyrophosphate (TPP) dependent process. The inactivation rates for the PDH complex and for its E1 component are similar. Pyruvate protects the PDH complex and the E1 component against inactivation by fluoropyruvate. Dihydrolipoamide protects the E1 component from inactivation. TPP is not covalently bound to the PDH complex or to the E1 component by the inactivating reaction. When [14C]fluoropyruvate is used to inactivate the PDH complex, 14C remains bound to the complex after gel filtration. This bound radioactivity is cleaved from the protein by NH2OH, -OH, and NaBH4 but not by dilute acid. When released by -OH, greater than 90% of the 14C cochromatographs with acetate on DEAE-Sephadex. When released by NaBH4, and 14C is recovered as [14C]ethanol. Colorimetric analysis for sulfhydryl groups on the native E1 component and the inactivated E1 component, using 5,5'-dithiobis(2-nitrobenzoate), reveals that complete inactivation results from covalent modification of 1.37 +/- 0.03 sulfhydryl residues. Fluoropyruvate is known to generate acetyl-TPP at the active site of E1. The available evidence indicates that acetylation of a sulfhydryl group by acetyl-TPP at the active site of the E1 component inactivates the enzyme.