Uso intraarticular de opioides y bupivacaína para tratamiento del dolor crónico en la disfunción mecánica temporomandibular / Intraarticular use of opioids and bupivacaine for the management of chronic pain in temporo-maxillary mechanical dysfunction

Objetivo: Valorar la calidad analgésica de la infiltración articular temporomandibular con sulfato de morfina comparado con bupivacaína al 0.5 por ciento y placebo, en pacientes con dolor crónico por disfunción mecánica temporomandibular. Material y métodos: Se estudiaron 30 pacientes con edades comprendidas entre 18 y 60 años de edad, con diagnóstico de disfunción mecánica temporomandibular y dolor crónico mayor de 6 meses sin respuesta a tratamientos previos. Los pacientes se dividieron en tres grupos de 10 cada uno: grupo I: quienes recibieron 2 mg de morfina, grupo II: recibieron 10 mg de bupivacaína al 0,5 por ciento, grupo III: a quienes se les administró placebo. A todos los pacientes se les infiltró un volumen de 2 ml en la articulación temporomandibular. Se valoró la intensidad del dolor por medio de escala visual análoga (EVA), las complicaciones de los procedimientos y los medicamentos analgésicos coadyuvantes previo a la administración de los medicamentos, a los 5 y 60 minutos posterior a la infiltración y cada 7 días durante 4 semanas. Se utilizó para el análisis estadístico la prueba de Mcknemar, prueba exacta de Fisher y prueba independiente de x2 con corrección de Yates, considerando una p < 0,05 para establecer diferencias estadísticas. Resultados: El grupo I mantuvo un promedio de EVA de 1,8, el grupo II con EVA promedio de 3, 5 y grupo III con EVA promedió de 6,4 con' diferencias estadísticas significativas entre los tres grupos. El 30 por ciento de los pacientes del grupo I recibieron medicamentos analgésicos coadyuvantes, mientras que el 60 y 90 por ciento de los pacientes de los grupos II y III respectivamente los requirieron, con diferencias estadísticas significativas entre los tres grupos estudiados. No se registró ninguna complicación asociada al procedimiento en los tres grupos de estudio. Conclusión: La infiltración con morfina es una buena alternativa en el control del dolor crónico en disfunción mecánica temporomandibular, obteniendo una analgesia prolongada y con minimas complicaciones (AU)

(18)O and mass spectrometry in chlorophyll research: Derivation and loss of oxygen atoms at the periphery of the chlorophyll macrocycle during biosynthesis, degradation and adaptation.

Chlorophylls, magnesium-containing tetrapyrrolic pigments of photosynthesis, are widely-distributed in Nature and participate in both light harvesting and in the transduction of light energy to chemical energy for the photosynthetic fixation of carbon dioxide. We briefly discuss the extensive role of various isotopic labelling techniques in elucidating the pathway of tetrapyrrole-pigment biosynthesis and we acknowledge the classic and meticulous research of David Shemin who, approximately 50 years ago, introduced isotopic tracer techniques with (15)N and (14)C isotopes to study the biosynthesis of the carbon/nitrogen macrocycle of haem, an iron tetrapyrrole. The main focus of this review is the application of mass spectrometry and (18)O labelling to the study of the incorporation of oxygen atoms from molecular oxygen or water into the periphery of the chlorophyll macrocycle during biosynthesis and their loss during degradation and light acclimation. In particular, we review the mechanism of formation of the isocyclic ring of chlorophylls, in higher plants, green algae and various photosynthetic bacteria, which concomitantly incurs formation of the 13(1)-oxo group that is present in all photosynthetically-active chlorophylls. In addition we discuss the formation of the ubiquitous 13(3)- and 17(3)-carboxyl groups and also the formation of the 7-formyl group of chlorophyll b and the 3-acetyl group of bacteriochlorophyll a.

Stability of reconstituted solutions of ceftazidime for injections: an HPLC and CE approach.

The stability of aqueous reconstituted ceftazidime injection vials containing ceftazidime pentahydrate blended with anhydrous sodium carbonate was investigated in different storage conditions (4 degrees C and 10 degrees C for 7 days in a refrigerator, 20 and 30 degrees C for 24 h) with validated HPLC and (micellar) CE methods. Stability indicating data were obtained for ceftazidime and two degradation products: pyridine and the delta2-ceftazidime isomer. Other degradation products were also identified (the complementarity of the two used experimental procedures was useful in such exercise) and characterized by their UV spectra and retention times. Stability data (7 days at 4 degrees C in a refrigerator and 18 h at room temperature) resulted in agreements with the manufacturers prescription and point out the need of a strict temperature control of the refrigerator's compartment used to store the reconstituted solution.

LTB4 as marker of 5-LO inhibitory activity of two new N-omega-ethoxycarbonyl-4-quinolones.

The supposed 5-LO inhibitory activity of two N-omega-ethoxycarbonyl-4-quinolones was tested determining leukotriene B4 (LTB4) in RBL-1 cell cultures, pretreated with the two compounds of interest. LTB4, obtained by solid-phase extraction (SPE) from cell cultures supernatants, was determined by micellar electrokinetic chromatography (MEKC). The analysis was performed using an uncoated capillary, filled with borate buffer at pH 8.3, containing 12.5 mM SDS as micelles generator. Therefore, following the decreasing of LTB4 it was possible to verify the 5-LO inhibitory activity of two quinolone derivatives. To asses the suitability of the use of LTB4 as marker of the activity of the new compounds, the analysis was repeated using quercetin, a well known 5-LO inhibitor.

Analysis of ceftazidime and related compounds by micellar electrokinetic chromatography.

A micellar electrokinetic chromatographic method for the separation and quantification of ceftazidime, its delta2-isomer and pyridine (two ceftazidime related impurities) was developed and validated. Optimised conditions were obtained using an electrolyte system consisting of 25 mM sodium tetraborate, at pH 9.2, and 75 mM sodium dodecylsulphate. A limit of detection of 0.2 microg ml(-1) and a limit of quantitation of 0.6 microg ml(-1) were estimated for pyridine and delta2-isomer: this means that levels of < 0.1% of pyridine and delta2-isomer in ceftazidime can be determined. Calibration curves for all analytes were linear over the studied ranges with correlation coefficients >0.999. Good reproducibility for migration times and corrected peak areas were achieved (RSD % 0.3 and 1.0, respectively). The results demonstrate that the method is reproducible, accurate and appropriate for ceftazidime assay in pharmaceutical samples.

Biosynthesis of the 3-acetyl and 13(1)-oxo groups of bacteriochlorophyll a in the facultative aerobic bacterium, Rhodovulum sulfidophilum--the presence of both oxygenase and hydratase pathways for isocyclic ring formation.

Using (18)O-labelling and mass spectrometry, we have examined bacteriochlorophyll a formation in Rhodovulum sulfidophilum, formerly known as Rhodobacter sulfidophilus, which forms large amounts of BCh1 a both aerobically in the dark and anaerobically in the light. R. sulfidophilum, growing under strict anaerobiosis in the light, possesses hydratases which incorporate (18)O label from H2(18)O into both the 13(1)-oxo and 3-acetyl oxygens; in addition, the four carboxyl oxygens at C13(3) and C17(3) were labelled by H2(18)O. Under aerobic conditions in the dark, the labelling of the 13(1)-oxo group by H2(18)O was reduced indicating that (16)O was being incorporated into this group from air. R. sulfidophilum, grown in the dark under an atmosphere initially containing 50% (18)O2 in Ar, possessed an oxygenase which incorporated (18)O label from (18)O2 specifically into the 13(1)-oxo group; under these conditions the acetyl and carboxyl groups remained unlabelled. Thus, both an oxygenase and hydratase operate in R. sulfidophilum to form the 13(1)-oxo group of ring E of BCh1 a; the 3-acetyl group oxygen, however, arises only from water via a hydratase.

Analysis of venlafaxine by capillary zone electrophoresis.

Capillary electrophoresis has been used for the separation of venlafaxine and two of its impurities deriving from the synthesis process. The electrophoretic experiments were performed using background electrolytes at different pHs in the 2.5-9.2 range in order to study the effective mobilities and resolution of the three examined compounds. The optimum experimental conditions for the baseline resolution of the three analytes was found at pH 6.5. Very good repeatability for both migration time and corrected peak areas was achieved. The calibration curve was studied for venlafaxine (concentration range 26-224 micrograms/mL), and the plot of the peak area ratio (sample/internal standard [IS]) versus venlafaxine concentration was linear with a correlation coefficient of 0.9991. The effect of different cyclodextrins (CDs), namely, gamma-cyclodextrin (gamma-CD), hydroxypropyl-beta-CD (HP-beta-CD), and alpha-cyclodextrin (alpha-CD), on effective mobility and enantiomeric resolution (R) of venlafaxine (Wy45030) and its impurities (imp1 and imp2) was studied at different pHs, and the best results were obtained at pH 9.2. Venlafaxine was baseline resolved in its enantiomers using gamma-CD or HP-beta-CD, while imp1 (Wy45494) was baseline resolved using alpha-CD.

Origin of the two carbonyl oxygens of bacteriochlorophyll a. Demonstration of two different pathways for the formation of ring E in Rhodobacter sphaeroides and Roseobacter denitrificans, and a common hydratase mechanism for 3-acetyl group formation.

A respiring culture of Rhodobacter sphaeroides, grown in the dark under defined aerobic conditions, produced cells capable of immediately commencing adaptation to photosynthetic growth on exposure to light and further reduction of oxygen tension. Adaptation was complete after 12 h and the bacteriochlorophyll a content increased 10-20-fold. This adaptation was performed in the presence of either H2(18)O or 18O2. The extracted bacteriochlorophyll a was examined by mass spectrometry to determine the origin of both the 3-acetyl adn 13(1)-oxo oxygen atoms: both were derived from water. The derivation of the 13(1)-oxo group from water in R. sphaeroides indicates that the formation of isocyclic ring E from the 13-propionic acid methylester side chain of Mg(2+)-protoporphyrin IX monomethylester is an anaerobic process involving a hydratase. This is very different to the situation in higher plants and green algae where the formation of isocyclic ring E is an aerobic process in which the 13(1)-oxo group is derived from molecular oxygen via an oxygenase. In contrast to adapting R. sphaeroides cells, the 13(1)-oxo group of bacteriochlorophyll a in growing cells of the obligate aerobic chemotrophic bacterium Roseobacter denitrificans, was labelled by 18O2 and is, therefore, derived from molecular oxygen like in higher plants and green algae; however, the 3-acetyl group was not labelled by 18O2. Thus, while the 13(1)-oxo group has different origins in R. sphaeroides and R. denitrificans, the 3-acetyl group arises in both bacteria by enzymic hydration of the vinyl group of a chlorophyll a derivative.

The derivation of the oxygen atoms of the 13(1)-oxo and 3-acetyl groups of bacteriochlorophyll a from water in Rhodobacter sphaeroides cells adapting from respiratory to photosynthetic conditions: evidence for an anaerobic pathway for the formation of isocyclic ring E.

Using mass spectrometry, we have demonstrated 18O-labelling of both the 13(1)-oxo and 3-acetyl groups of newly-formed bacteriochlorophyll a synthesized by Rhodobacter sphaeroides cells during adaptation from respiratory to photosynthetic conditions in the presence of H218O. This derivation of the 13(1)-oxo group of bacteriochlorophyll a from water provides a stark contrast with that of chlorophylls in higher plants where ring E formation is an aerobic process in which the 13(1)-oxo group arises from molecular oxygen via an oxygenase activity. The formation of the 3-acetyl group of bacteriochlorophyll a, however, is consistent with the enzymic hydration of the 3-vinyl group of a derivative of chlorophyll a.

The derivation of the formyl-group oxygen of chlorophyll b in higher plants from molecular oxygen. Achievement of high enrichment of the 7-formyl-group oxygen from 18O2 in greening maize leaves.

The mechanism of formation of the formyl group of chlorophyll b has long been obscure but, in this paper, the origin of the 7-formyl-group oxygen of chlorophyll b in higher plants was determined by greening etiolated maize leaves, excised from dark-grown plants, by illumination under white light in the presence of either H2(18)O or 18O2 and examining the newly synthesized chlorophylls by mass spectroscopy. To minimize the possible loss of 18O label from the 7-formyl substituent by reversible formation of chlorophyll b-7(1)-gem-diol (hydrate) with unlabelled water in the cell, the formyl group was reduced to a hydroxymethyl group during extraction with methanol containing NaBH4: chlorophyll a remained unchanged during this rapid reductive extraction process. Mass spectra of chlorophyll a and [7-hydroxymethyl]-chlorophyll b extracted from leaves greened in the presence of either H2(18)O or 18O2 revealed that 18O was incorporated only from molecular oxygen but into both chlorophylls: the mass spectra were consistent with molecular oxygen providing an oxygen atom not only for incorporation into the 7-formyl group of chlorophyll b but also for the well-documented incorporation into the 13(1)-oxo group of both chlorophylls a and b [see Walker, C. J., Mansfield, K. E., Smith, K. M. & Castelfranco, P. A. (1989) Biochem. J. 257, 599-602]. The incorporation of isotope led to as much as 77% enrichment of the 13(1)-oxo group of chlorophyll a: assuming identical incorporation into the 13(1) oxygen of chlorophyll b, then enrichment of the 7-formyl oxygen was as much as 93%. Isotope dilution by re-incorporation of photosynthetically produced oxygen from unlabelled water was negligible as shown by a greening experiment in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. The high enrichment using 18O2, and the absence of labelling by H2(18)O, unequivocally demonstrates that molecular oxygen is the sole precursor of the 7-formyl oxygen of chlorophyll b in higher plants and strongly suggests a single pathway for the formation of the chlorophyll b formyl group involving the participation of an oxygenase-type enzyme.

Derivation of the formyl-group oxygen of chlorophyll b from molecular oxygen in greening leaves of a higher plant (Zea mays).

Using mass spectroscopy, we demonstrate as much as 93% enrichment of the 7-formyl group oxygen of chlorophyll b when dark-grown, etiolated maize leaves are greened under white light in the presence of 18O2. This suggests that a mono-oxygenase is involved in the oxidation of its methyl group precursor. The concomitant enrichment of about 75% of the 13(1)-oxygen confirms the well-documented finding that this oxo group, in both chlorophyll a and b, also arises from O2. High 18O enrichment into the 7-formyl oxygen relative to the substrate 18O2 was achieved by optimization of the greening conditions in combination with a reductive extraction procedure. It indicates not only a single pathway for Chl b formyl group formation, but also unequivocally demonstrates that molecular oxygen is the sole precursor of the 7-formyl oxygen.

The identification of related substances in 9 alpha-fluoroprednisolone-21 acetate by means of high-performance liquid chromatography with diode array detector and mass spectrometry.

A method for the analysis and identification of the principal related substances in 9 alpha-fluoroprednisolone acetate is described. This compound has been chosen as a model for the investigation of related substances which can be originated in the general procedure for introducing a fluorine substituent at position 9 of a corticosteroid molecule. HPLC procedures, both in reversed and in normal phase were used; a rapid scanning UV detector which allows direct spectrophotometric data to be obtained on chromatographic peaks, proved to be a tool of great importance. Thus, after reversed-phase chromatographic separation and observation of the UV spectra and their respective second derivatives, it was possible to characterize some of the principal effective and potential related substances such as 9 alpha-fluorohydrocortisone acetate, 9 alpha-bromoprednisolone acetate, 9 beta, 11 beta-epoxyprednisolone acetate and 9(11)-dehydroprednisolone acetate, emerging as chromatographic peaks. Identification of 9 alpha-bromoprednisolone acetate and of 9 alpha-fluorohydrocortisone acetate which proved to be the most significant impurities, was confirmed by means of an exhaustive study of the mass spectra of these substances conveniently isolated by normal-phase HPLC. The chromatographic, spectrophotometric and mass-spectrometric characteristics of the studied compounds are reported.

Analytical and regulatory considerations for ferritin-containing pharmaceutical products.

Ferritin, an iron-containing protein widely diffused in nature, has the important biochemical function of being the principal reserve and regulator for Fe3+ levels in blood and tissue. Due to its natural, effectively non-toxic iron content, ferritin has been the object of strong interest in the development of pharmaceutical products for use in iron deficiency treatment. Therefore, the need has arisen for the analytical characterization of this industrial product, be it in the hydroglyceric solution or dry powder form. The main considerations for the characterization of industrial ferritin are the following: a) iron content in both the unmodified product and the precipitated protein; b) protein content and protein/iron ratio; c) protein identification by means of polyacrylamide gel electrophoresis (PAGE); d) confirmation of protein identity and evaluation of molecular weight by means of size exclusion chromatography; e) identification and evaluation of other components of bulk ferritin preparations such as preservatives, excipients for lyophilization, water and concomitant proteins. Eight different samples were examined using the above considerations. Among the qualitative and quantitative results reported, of particular interest are those obtained by means of size-exclusion high performance liquid chromatography (SEC-HPLC). With a UV "diode array" detector it is possible to discern the peaks for ferritin and its molecular aggregates from those of concomitant proteins and preservatives; furthermore, it is possible to evaluate their molecular dimensions. Using this method, the ferritin monomer and other protein fractions can be quantitatively analyzed either by calculating area percent distribution of the chromatographic peaks or by comparing the sample with a highly pure external standard of ferritin.(ABSTRACT TRUNCATED AT 250 WORDS)

Carotenoids, retinoids and alpha-tocopherol in human serum: Identification and determination by reversed-phase HPLC.

A rapid, simple and specific high performance liquid chromatographic procedure for assaying alpha- and beta-carotene is described. The method also enables the simultaneous determination of retinol and dl-alpha-tocopherol in human serum. The same chromatographic procedure can be used to assay the major carotenoids in human serum, provided analyses are replicated and the effluent is monitored at 450 nm. The conditions described also enable determination of licopene, cryptoxanthine and lutein with zeaxanthine. An aliquot of 0.5 ml serum is deproteinized with ethanol (0.5 ml) and extracted with petroleum ether (0.75 ml). The petroleum ether extract is evaporated until dry and then redissolved immediately with 0.5 ml of an eluent mixture consisting of methanol-hexane (85:15, v/v). Aliquots of 50 microl are then injected onto a 250 x 4.6 mm column packed with Spherisorb ODS-2. Owing to its good reproducibility, the procedure can be used for assays with external standards. Clinical applications are described for cases of hypercarotinemia associated with endocrine dysfunctions such as hypothyroidism and diabetes.

Labelling of chlorophylls and precursors by [2-14C]glycine and 2-[1-14C]oxoglutarate in Rhodopseudomonas spheroides and Zea mays. Resolution of the C5 and Shemin pathways of 5-aminolaevulinate biosynthesis by thin-layer radiochromatography.

The C-5 of 5-aminolaevulinate, a tetrapyrrole precursor which accumulates when inhibitory laevulinate is present, is derived from either the C-2 of glycine by the 5-aminolaevulinate-synthase-mediated Shemin pathway or the C-1 of 2-oxoglutarate by the C5 pathway. Thin-layer-radiochromatographic procedures are described for determining whether [2-14C]glycine or 2-[1-14C]oxoglutarate labelled the macrocycle of bacteriochlorophyll a, in addition to or rather than the methyl ester or phytyl ester moieties of the side-chains. The method was also used for detecting whether the same substrates label the formaldehyde (C-5) or the succinate (C-1 to C-4) fragments, obtained by periodate cleavage of 5-aminolaevulinate. These methods therefore can readily distinguish between the Shemin and C5 pathways as was demonstrated by using Rhodopseudomonas spheroides and Zea mays (maize), respectively, as examples of each pathway. Both [2-14C]glycine and, to a lesser extent 2-[1-14C]oxoglutarate labelled the macrocycle of bacteriochlorophyll a formed during adaptation of respiring R. spheroides cells to photosynthetic (anaerobic, illuminated) conditions. This and earlier evidence suggested augmentation of the Shemin pathway by a minor C5 pathway contribution. The present studies revealed only Shemin pathway activity: with laevulinate present, [2-14C]glycine formed 5-[5-14C]aminolaevulinate as proved by H14CHO production during periodate cleavage. These methods were sufficiently sensitive also to detect the incorporation of 14CO2, from degradation of either substrate, into 5-aminolaevulinate via the Shemin pathway thus labelling the succinate fragment produced with periodate: this explains bacteriochlorophyll a labelling by 2-[1-14C]oxoglutarate and proves double labelling of 5-aminolaevulinate by [2-14C]glycine. The same techniques were applied to etiolated maize leaves exposed to aerobic illuminated conditions with laevulinate and either 2-[1-14C]oxoglutarate or [2-14C]glycine as substrates. Only the C5 pathway was detected: 2-[1-14C]oxoglutarate was converted to 5-[5-14C]aminolaevulinate, which yielded H14CHO on periodate cleavage. This is not inconsistent with our earlier 13C-NMR studies [Porra, R.J., Klein, O. and Wright, P. E. (1983) Eur. J. Biochem. 130, 509-516] showing that the C5 pathway formed all the 5-aminolaevulinate for chlorophyll biosynthesis.(ABSTRACT TRUNCATED AT 400 WORDS)

[Oral load test with beta-carotene in normal and dysthyroid subjects]. / La prova da carico per os con beta-carotene nei soggetti normali e distiroidei.

An oral load of beta-carotene (500 mg) was administered to four normal, four hypo and four hyperthyroid subjects. Plasma beta-carotene content was determined at the 2nd, 4th, 6th, 8th, 10th, 12th and 24th hour after administration and at every 24th hour thereafter for 5 consecutive days. Plasma assays were performed by HPLC. No significant differences were revealed by Student's T test for one group to the other. The authors sustain that, as there is no impairment in intestinal uptake of beta-carotene in disthyroid subjects, the elsewhere described increase in carotinemia in hypothyroids is due to other mechanisms.

[Effect of renal and liver failure on blood levels of vitamin A, its precursor (beta-carotene) and its carrier proteins (prealbumin and retinol binding protein)]. / Influenza dell'insufficienza renale ed epatica sul tasso ematico della vitamina A, dei suoi precursori (beta-carotene) e dei suoi vettori proteici (PA e RBP).

Plasma beta-carotene and retinol assay was performed by high pressure liquid chromatography (HPLC) in subjects with chronic renal failure or liver cirrhosis. In the same subjects blood prealbumin (PA) and retinol binding protein (RBP) were determined by immunological technique. A considerable increase of retinol and in a lesser extent of beta-carotene was noted in the blood of patients with renal insufficiency. In cirrhotic patients it was shown a marked decrease both of beta-carotene and retinol plasma concentrations. PA and RBP there were greatly increased in renal failure and decreased in liver cirrhosis. This results suggest that kidney and liver chronic failure interfere with vitamin A metabolism throughout their action on metabolic processes of synthesis and elimination of PA and RBP.

[Behavior of vitamin A, beta-carotene, retinol binding protein and prealbumin in the plasma of hypo- and hyperthyroid subjects]. / Comportamento della vitamina a, dela beta-carotene, della proteina legante il retinolo e della prealbumina nel plasma di soggetti ipo ed ipertiroidei.

Plasma concentrations of beta-carotene and retinol, determined by HPLC, and of transport proteins, ascertained by immunodiffusion technique, in hypo and hyperthyroid subjects are reported. In hypothyroid subject a considerable increase in carotene was noted. This was not the case for retinol. In hyperthyroids both beta-carotene and retinol levels were found to be normal. Transport protein (PA and RBP) levels were found to be lower only in cases of hyperthyroidism but unchanged for hypothyroids. According to the Authors the results show that the alteration in plasma carotene levels to be found in hypothyroid subjects is not the direct consequence of a lack of thyroid hormone in the metabolism of vitamin A but the indirect effect of thyroid disease.