Caprine mucopolysaccharidosis-IIID: clinical, biochemical, morphological and immunohistochemical characteristics.

Several animal models have been developed for the mucopolysaccharidoses (MPSs), a group of lysosomal storage disorders caused by lysosomal hydrolase deficiencies that disrupt the catabolism of glycosaminoglycans (GAG). Among the MPS, the MPS-III (Sanfilippo) syndromes lacked an animal counterpart until recently. In this investigation of caprine MPS-IIID, the clinical, biochemical, morphological, and immunohistochemical studies revealed severe and mild phenotypes like those observed in human MPS III syndromes. Both forms of caprine MPS IIID result from a nonsense mutation and consequent deficiency of lysosomal N-acetylglucosamine 6-sulfatase (G6S) activity and are associated with tissue storage and urinary excretion of heparan sulfate (HS). Using special stains, immunohistochemistry, and electron microscopy, secondary lysosomes filled with GAG were identified in most tissues from affected goats. Primary neuronal accumulation of HS and the secondary storage of gangliosides were observed in the central nervous system (CNS) of these animals. In addition, morphological changes in the CNS such as neuritic expansions and other neuronal alterations that may have functional significance were also seen. The spectrum of lesions was greater in the severe form of caprine MPS IIID and included mild cartilaginous, bony, and corneal lesions. The more pronounced neurological deficits in the severe form were partly related to a greater extent of CNS dysmyelination. These findings demonstrate that caprine MPS IIID is a suitable animal model for the investigation of therapeutic strategies for MPS III syndromes.

Direct evidence of oxidative injury produced by the Alzheimer's beta-amyloid peptide (1-40) in cultured hippocampal neurons.

The beta-Amyloid peptide (A beta) is hypothesized to mediate the neurodegeneration seen in Alzheimer's disease. Recently, we proposed a new hypothesis to explain the toxicity of A beta based on the free-radical generating capacity of A beta. We have recently demonstrated using electron paramagnetic resonance (EPR) spectroscopy that A beta (1-40) generates free radicals in solution. It was therefore suggested that A beta radicals can attack cell membranes, initiate lipoperoxidation, damage membrane proteins, and compromise ion homeostasis resulting in neurodegeneration. To evaluate this hypothesis, the ability of A beta to induce neuronal oxidation, changes in calcium levels, enzyme inactivation, and neuronal death were compared with the ability of A beta to produce free-radicals. Using hippocampal neurons in culture, several methods for detection of oxidation were utilized such as the conversion of 2,7-dichlorofluorescin to 2,7-dichlorofluorescein, and a new fluorescence microscopic method for the detection of carbonyls. The ability of A beta to produce free-radicals was determined using EPR with the spin-trapping compound N-tert-butyl-alpha-phenylnitrone. Consistent with previous studies, we found that preincubation of A beta increased the toxicity of the peptide. There is a strong correlation between the intensity of radical generation by A beta and neurotoxicity. The highest neuronal oxidation and toxicity was seen at a time when A beta was capable of generating the most intense radical signal. Furthermore, little oxidation and toxicity was seen when cultures were treated with freshly dissolved A beta, which did not generate a detectable radical signal. These data are consistent with the hypothesis that free-radical-based oxidative damage induced by A beta contributes to the neurodegeneration of Alzheimer's disease.

Detection of oxidation products in individual neurons by fluorescence microscopy.

In the study of the central nervous system, it is necessary to address mechanisms by which cells are injured. In vitro investigations using cells in culture allow sharply focused mechanistic questions to be addressed; however, these studies have often been limited by the sensitivity constraints of assays. Many assays for oxidative products require large amounts of cells that must be disrupted. The extent of oxidation in individual cells is therefore unknown and the results yield an average among different cell types. This is inconvenient in cultures of the nervous system which often have multiple cell types. Using a newly developed method for visualizing oxidation products in individual cells, we have examined oxidation in neurons in culture. The method uses a hydrazide, biotin-4-amidobenzoic hydrazide, to bind carbonyls generated from oxidation. Biotin is detected by streptavidin conjugated with a fluorescent dye. Neurons in culture were exposed to 0.1 to 100 microM ferrous sulfate and fluorescence was visualized and quantitated using confocal laser microscopy. Low levels of oxidation (0.1 microM ferrous sulfate) were easily detected with this method. Iron concentration and fluorescence intensity correlated highly (r = 0.991). As an indicator of the sensitivity of this new method, carbonyl content in the cultures was also quantitated using the 2,4-dinitrophenylhydrazine assay (DNPH). The DNPH assay failed to detect the low levels of oxidation which were detected by the biotin-4-amidobenzoic hydrazide method. Fluorescence intensity partially paralleled loss of neuronal viability. Low concentrations of iron (0.1 and 1.0 microM) did not produce significant neuronal death; however, higher concentrations (10 and 100 microM) produced 19 and 53% neuronal loss, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Vitamin E is not degraded by ruminal microorganisms: assessment with ruminal contents from a steer fed a high-concentrate diet.

Using an in vitro incubation system containing undiluted ruminal contents from a steer fed a high-concentrate, corn-based diet, we examined microbial degradation of DL-alpha-tocopherol acetate (TA). Gas production, pH, and fermentation acid profiles were done in an initial experiment to ensure conditions for reproducible, viable cultures over 24 h. The pH decreased from 5.7 to 4.9, gas production averaged 3.4 mL/mL of ruminal contents, and > 300 mM fermentation acids were produced. We then monitored the fate of TA added to bottles containing ruminal contents. Three methods of TA extraction were tried, of which two were used in experiments. The two methods used were 1) hot ethanol in a Soxhlet apparatus and 2) chloroform/methanol. Each of these was used to extract added TA from a set of three in vitro experiments. Concentrations of TA were determined at 0 h and after 4, 8, and 24 h at 39 degrees C. In the three hot ethanol extracted experiments, TA recoveries were 85% at 0 h. With time of incubation, TA levels either 1) remained constant, 2) decreased then returned to the initial value, or 3) decreased by approximately 50%. These inconsistent results indicated that this extraction method was unacceptable. In the latter three experiments we used a chloroform/methanol extraction method. Recoveries of added TA averaged 96% overall. Thus, the level of TA remained constant during the 24-h period, suggesting that microbial destruction of TA does not occur. Rather, the previously reported losses of vitamin E may be attributable to incomplete extraction of tocopherol from high-concentrate ruminal contents.

The effect of glutathione on the vitamin E requirement for inhibition of liver microsomal lipid peroxidation.

Vitamin E dependent inhibition of rat liver microsomal lipid peroxidation in an NADPH and ADP-Fe+3 containing system occurred at lower vitamin E concentrations in the presence of glutathione (GSH). Using microsomes from rats fed a vitamin E deficient diet, vitamin E was shown to be required for inhibition. Inhibition also required the presence of a storage labile microsomal component, since no inhibition was observed when using microsomes that had been stored for one month. This observation provides evidence that direct reduction of reversibly oxidized vitamin E by GSH does not appear to contribute significantly to inhibition of peroxidation. During GSH and vitamin E dependent inhibition of lipid peroxidation, vitamin E (reduced form) concentrations remained constant, indicating that GSH maintained vitamin E concentrations. Without GSH, vitamin E concentrations dropped rapidly. By adding vitamin E to microsomes, it was found that inhibition of lipid peroxidation in the presence of GSH occurred at about five-fold less vitamin E than in the absence of GSH. Inhibition at these lower levels of vitamin E was 85-90% complete. Results indicate that GSH can be used to maintain vitamin E (reduced form) concentrations, thereby lowering the concentration of vitamin E necessary to inhibit microsomal lipid peroxidation.

Importance of the polyunsaturated fatty acid to vitamin E ratio in the resistance of rat lung microsomes to lipid peroxidation.

Rat lung microsomes and liposomes made from isolated lung microsomal lipids were found to be much more resistant to lipid peroxidation than those from liver in both enzymatic and nonenzymatic systems. The polyunsaturated fatty acid (PUFA) content of isolated lung microsomal lipids was 28% of total fatty acids, while liver was 54%. The vitamin E (alpha-tocopherol) content of isolated lung microsomal lipids was 2.13 nmol/mumol lipid phosphate and that of liver was 0.43. Individually, neither the lower PUFA content nor higher vitamin E levels could account for the resistance of lung microsomal lipids to peroxidation. Distearoyl-L-alpha-phosphatidylcholine and/or alpha-tocopherol were added to liver microsomal lipids to achieve different PUFA to vitamin E ratios at PUFA contents of 28% or 54%, and the resulting liposomes were subjected to an NADPH-dependent lipid peroxidation system utilizing cytochrome P450 reductase, EDTA-Fe+3, and ADP-Fe+3. Liposomes having PUFA to vitamin E ratios less than approximately 250 nmol PUFA/nmol vitamin E were resistant to peroxidation, whereas lipid peroxidation, as evidenced by malondialdehyde production, occurred in liposomes having higher ratios. When lipid peroxidation occurred, 40%-60% of the liposomal vitamin E was irreversibly oxidized. Irreversible oxidation did not occur in the absence of lipid peroxidation. These studies indicated that the low PUFA to vitamin E ratio in lung microsomes and isolated microsomal lipids was sufficient to account for the observed resistance to lipid peroxidation.