Análise biomecânica ex vivo de um modelo de haste intramedular de polipropileno para osteossíntese em úmeros de bezerros / Ex vivo biomechanics assay of a model of polypropylene intramedullary nail for calf humeral osteosynthesis

Foram utilizados 12 pares de úmeros obtidos de bezerros machos da raça Holandesa, com idades entre 15 e 30 dias. Os úmeros esquerdos foram mantidos íntegros, e os direitos foram fraturados de forma oblíqua em sua diáfise, na transição entre os terços médio e proximal. A redução da fratura foi feita pela aplicação intramedular de haste de polipropileno, bloqueada por dois parafusos corticais de aço inoxidável, dispostos transversalmente em cada fragmento ósseo. Seis pares de ossos foram submetidos ao teste de compressão, e seis ao teste de flexão, utilizando-se uma máquina universal de ensaios. Nos testes de compressão, as cargas médias de ruptura foram 738,3N e 473,3N, e nos testes de flexão 322,4N e 117,9N, para os ossos íntegros e fraturados, respectivamente. Comparando-se o grupo de ossos fraturados com o grupo de ossos íntegros, verificou-se que o sistema proposto foi capaz de resistir a 66,4 por cento das cargas médias quando submetido à compressão, e a 36,6 por cento quando submetido à flexão. Úmeros fraturados e tratados com haste intramedular de polipropileno apresentaram resistência limitada se comparados aos ossos íntegros.

A total of 12 pairs of humeri from male calves 15 to 30 days old were used. The left humeri remained unchanged and the right ones were fractured in the diaphysis between proximal and middle thirds. The fracture was fixed with a polypropylene intramedullary nail interlocked with two steel bone screws crossed along each segment. Six pairs of bones were subjected to a compression test and the others to a flexural test using a universal testing machine. In the compression tests, the mean rupture loads were 738,3N and 473,3N, and in the flexural tests they were 322,4N and 117,9N for the intact and fractured bones respectively. Fractured bones fixed with the proposed model were able to resist 66.4 percent of the load during compression and 36.6 percent during bending when in comparison to intact bones. Fractured humeri treated with polypropylene intramedullary nail showed limited resistance compared to intact ones.

Observation of high-energy neutrinos using Cerenkov detectors embedded deep in Antarctic ice.

Neutrinos are elementary particles that carry no electric charge and have little mass. As they interact only weakly with other particles, they can penetrate enormous amounts of matter, and therefore have the potential to directly convey astrophysical information from the edge of the Universe and from deep inside the most cataclysmic high-energy regions. The neutrino's great penetrating power, however, also makes this particle difficult to detect. Underground detectors have observed low-energy neutrinos from the Sun and a nearby supernova, as well as neutrinos generated in the Earth's atmosphere. But the very low fluxes of high-energy neutrinos from cosmic sources can be observed only by much larger, expandable detectors in, for example, deep water or ice. Here we report the detection of upwardly propagating atmospheric neutrinos by the ice-based Antarctic muon and neutrino detector array (AMANDA). These results establish a technology with which to build a kilometre-scale neutrino observatory necessary for astrophysical observations.

Quantitative analysis of urinary acylglycines for the diagnosis of beta-oxidation defects using GC-NCI-MS.

The analysis of acylglycines is an important biochemical tool for the diagnosis of inherited disorders of mitochondrial fatty acid beta-oxidation. A stable isotope dilution gas chromatography negative chemical ionisation mass spectrometry method for the quantitative analysis of short- and medium-chain acylglycines as their bis(trifluoromethyl)benzyl (BTFMB) ester derivatives is described. The diagnostic usefulness of the method was demonstrated in nine patients with medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) deficiency, and seven patients with multiple acyl-CoA dehydrogenation defect (MAD). The urinary acylglycine profiles in these patients were compared to those in controls (n = 19), children on a medium-chain triglyceride (MCT) supplemented diet (n = 4), and patients with various other diseases (n = 5).

Quantitative acylcarnitine profiling in fibroblasts using [U-13C] palmitic acid: an improved tool for the diagnosis of fatty acid oxidation defects.

A method was developed for the investigation of mitochondrial fatty acid beta-oxidation in cultured fibroblasts. Monolayer cultures were incubated without foetal calf serum with commercially available [U-13C] palmitic acid and L-carnitine for 96 h. The acylcarnitines produced by the cells were extracted from the cell suspension and analysed either by quantitative stable isotope dilution gas chromatography chemical ionization mass spectrometry, or by fast atom bombardment mass spectrometry. Characteristic acylcarnitine profiles were obtained for all the different enzyme deficiencies investigated, with the exception of carnitine palmitoyltransferase II deficiency and carnitine/acylcarnitine carrier deficiency which showed similar patterns. Comparison between this method and the 3H-myristate and 3H-palmitate tritium release assays revealed that the method described here is superior, allowing unequivocal identification of patients.

The effect of fasting, long-chain triglyceride load and carnitine load on plasma long-chain acylcarnitine levels in mitochondrial very long-chain acyl-CoA dehydrogenase deficiency.

We studied a 10-year-old patient with very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency who was originally (mis)diagnosed as having systemic carnitine deficiency. He was subjected to a fasting test, a long-chain triglyceride (LCT) loading test (1.5 g/kg) and an intravenous carnitine clearance test (0.25 mumol/kg per min). Plasma acylcarnitines were analysed using a quantitative GC-CI-MS method. During fasting, all long-chain acylcarnitines with a chain length of C14 and higher (especially C14:1) increased dramatically. Total plasma long-chain acylcarnitine reached a concentration of 28.6 mumol/L. LCT loading resulted in a moderate increase, mainly of the C18 esters. The carnitine infusion, which led to a supranormal plasma free carnitine concentration, gave only a slight but generalized rise of long-chain acylcarnitines. Although only one patient could be tested, the results suggest that the accumulation of potentially toxic long-chain acylcarnitines in VLCAD deficiency is provoked by fasting, LCT loading and carnitine supplementation. Therapy should be adjusted accordingly.

Carnitine palmitoyltransferase II specificity towards beta-oxidation intermediates--evidence for a reverse carnitine cycle in mitochondria.

Using isolated rat liver mitochondria, in the absence or presence of malonyl-CoA (an inhibitor of carnitine palmitoyltransferase I), we have found that carnitine palmitoyltransferase II (CPT II) is active with palmitoyl-CoA as well as with its beta-oxidation intermediates. A partially purified CPT II fraction from rat liver mitochondria was shown to be able to convert 3-hydroxypalmitoyl-CoA to 3-hydroxypalmitoylcarnitine, which could be identified by fast-atom-bombardment mass spectrometry. This apparent broad specificity of CPT II was further evaluated by kinetic studies using purified CPT II. It was found that CPT II readily accepts 3-oxopalmitoyl-CoA, palmitoyl-CoA, 3-hydroxypalmitoyl-CoA and 2,3-unsaturated palmitoyl-CoA as substrates with decreasing order of affinity. The apparent Vmax values found for the first three compounds were of the same order of magnitude; the 2,3-unsaturated acyl-CoA was the poorest substrate. Kinetic studies with purified CPT II showed 3-hydroxypalmitoyl-CoA to have the lowest K0.5 value (20 +/- 6 microM) of all the CoA esters studied; the highest K0.5 value (65 +/- 17 microM) was found for the 3-oxo intermediate. These findings support the hypothesis that CPT II is involved in the export of toxic long-chain acyl-CoA esters from the mitochondria by first converting them into the corresponding carnitine esters, followed by transport out of the mitochondria and subsequently out of the cell.

Simultaneous analysis of plasma free fatty acids and their 3-hydroxy analogs in fatty acid beta-oxidation disorders.

We present a new derivatization procedure for the simultaneous gas chromatographic-mass spectrometric analysis of free fatty acids and 3-hydroxyfatty acids in plasma. Derivatization of target compounds involved trifluoroacetylation of hydroxyl groups and tert-butyldimethylsilylation of the carboxyl groups. This new derivatization procedure had the advantage of allowing the complete baseline separation of free fatty acids and 3-hydroxyfatty acids while the superior gas chromatographic and mass spectrometric properties of tert-butyldimethylsilyl derivatives remained unchanged, permitting a sensitive analysis of the target compounds. Thirty-nine plasma samples from control subjects and patients with known defects of mitochondrial fatty acid beta-oxidation were analyzed. A characteristic increase of long-chain 3-hydroxyfatty acids was observed for all of the long-chain 3-hydroxyacyl-CoA dehydrogenase-deficient and mitochondrial trifunctional protein-deficient plasma samples. For medium-chain acyl-CoA dehydrogenase deficiency and very-long-chain acyl-CoA dehydrogenase deficiency, decenoic and tetradecenoic acids, respectively, were the main abnormal fatty acids, whereas the multiple acyl-CoA dehydrogenase-deficient patients showed variable increases of these unusual intermediates. The results showed that this selective and sensitive method is a powerful tool in the diagnosis and monitoring of mitochondrial fatty acid beta-oxidation disorders.

Quantitative analysis of plasma acylcarnitines using gas chromatography chemical ionization mass fragmentography.

A stable isotope dilution gas chromatography chemical ionization mass spectrometry (GC-CI-MS) method was developed for the quantitative profiling of plasma acylcarnitines. The clean-up procedure was comprised of a solid-phase cation exchange extraction using PRS-columns from which the acylcarnitines were eluted with a barium chloride solution. Isolated acylcarnitines were transformed into acyloxylactones and analyzed by positive GC-CI-MS using isobutane as reactant gas. The selected monitoring of a common ion at m/z [85]+ and the protonated molecular ion enabled a selective and sensitive detection of all C2-C18 acylcarnitines. An accurate quantitation was achieved by the use of stable isotope-labeled internal standards (C2-C18) and acylcarnitines could be analyzed in the sub-nanomolar range. Control values for C2-C18 acylcarnitines in plasma were established. Concentrations ranged from 0.02 micromol/L for C14-acylcarnitine to 4.90 micromol/L for C2-acylcarnitine. The diagnostic suitability of the method was demonstrated for patients with medium-chain acyl-CoA dehydrogenase deficiency and very long-chain acyl-CoA dehydrogenase deficiency.