Enzyme stabilization by glass-derived silicates in glass-exposed aqueous solutions.

OBJECTIVES: To analyze the solutes leaching from glass containers into aqueous solutions, and to show that these solutes have enzyme activity stabilizing effects in very dilute solutions. METHODS: Enzyme assays with acetylcholine esterase were used to analyze serially succussed and diluted (SSD) solutions prepared in glass and plastic containers. Aqueous SSD preparations starting with various solutes, or water alone, were prepared under several conditions, and tested for their solute content and their ability to affect enzyme stability in dilute solution. RESULTS: We confirm that water acts to dissolve constituents from glass vials, and show that the solutes derived from the glass have effects on enzymes in the resultant solutions. Enzyme assays demonstrated that enzyme stability in purified and deionized water was enhanced in SSD solutions that were prepared in glass containers, but not those prepared in plastic. The increased enzyme stability could be mimicked in a dose-dependent manner by the addition of silicates to the purified, deionized water that enzymes were dissolved in. Elemental analyses of SSD water preparations made in glass vials showed that boron, silicon, and sodium were present at micromolar concentrations. CONCLUSIONS: These results show that silicates and other solutes are present at micromolar levels in all glass-exposed solutions, whether pharmaceutical or homeopathic in nature. Even though silicates are known to have biological activity at higher concentrations, the silicate concentrations we measured in homeopathic preparations were too low to account for any purported in vivo efficacy, but could potentially influence in vitro biological assays reporting homeopathic effects.

Laboratory research in homeopathy: con.

Alternative medical approaches to human diseases such as cancer are becoming increasingly popular, but reports on their success rates have been highly variable. Homeopathy is an alternative medical practice often applied to less critical human diseases but one that has also been applied sporadically to the treatment of cancer. Animal studies on the use of homeopathy to treat experimental cancer are few and the evidence provided to date is far from conclusive. The debate presented here concerns the utility of animal studies on cancer treatment with homeopathic preparations. As part of a Point-Counterpoint feature, this review and its companion piece in this issue by Khuda-Bukhsh (Integr Cancer Ther. 2006;5:320-332) are composed of a thesis section, a response section in reaction to the companion thesis, and a rebuttal section to address issues raised in the companion response.

Characterization of the N-acetylaspartate biosynthetic enzyme from rat brain.

Aspartate N-acetyltransferase (Asp-NAT; EC 2.3.1.17) activity was found in highly purified intact mitochondria prepared by Percoll gradient centrifugation as well as in the three subfractions obtained after the sucrose density gradient centrifugation of Percoll purified mitochondria; citrate synthase was used as a marker enzyme for mitochondria. The proportion of recoverable activities of Asp-NAT and citrate synthase were comparable in mitochondrial and synaptosomal fractions but not in the fraction containing myelin. Asp-NAT was solubilized from the pellet of the rat brain homogenate (26 000 g for 1 h) for the recovery of maximum activity and partially purified using three protein separation methods: DEAE anion exchange chromatography, continuous elution native gel electrophoresis and size-exclusion high performance liquid chromatography. Asp-NAT activity and the optical density pattern of the eluted protein from size-exclusion column indicated a single large protein (approximately 670 kDa), which on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed at least 10 bands indicative of an enzyme complex. This seemingly multi-subunit complex Asp-NAT was stable towards ionic perturbations but vulnerable to hydrophobic perturbation; almost 95% of activity was lost after 10 mm 3-[(3-cholamidopropyl)dimethylammonia]-1-propanesulfonate (CHAPS) treatment followed by size-exclusion chromatography. Asp-NAT showed an order of magnitude difference in Km between l-aspartate (l-Asp, approximately 0.5 mm) and acetyl CoA (approximately 0.05 mm). Asp-NAT showed high specificity towards l-Asp with 3% or less activity towards l-Glu, l-Asn, l-Gln and Asp-Glu. A model on the integral involvement of NAA synthesis in the energetics of neuronal mitochondria is proposed.

Developmental increase of aspartoacylase in oligodendrocytes parallels CNS myelination.

Canavan disease, an autosomal-recessive neurogenetic disorder, is caused by mutations in aspartoacylase, an enzyme that deacetylates N-acetylaspartate to generate free acetate in the brain. Earlier studies have shown that aspartoacylase is primarily restricted to myelin synthesizing cells (oligodendroglia) in the CNS. These findings have led us to investigate the developmental expression of aspartoacylase gene in the rat brain in an attempt to shed more light on the role of this enzyme in myelination. In situ hybridization using a 35S riboprobe based on murine aspartoacylase cDNA was used in this study. The probe hybridized mostly to the white matter tracts with different densities depending on the age of the animal and region of the brain examined. Little or no hybridization signals were detected in the 1-day-old rats, whereas the signal was clearly detectable in most of the white matter regions of the CNS in the 11-day-old rats. The signal density markedly increased at postnatal day 17, the peak of myelination. Thereafter, the hybridization signals decreased somewhat but still could be observed in the adult animals. Thus, the developmental expression pattern of aspartoacylase gene in the postnatal brain closely parallels myelination in the CNS. In the CNS, the hybridization signal of ASPA appeared to be restricted primarily to oligodendrocytes, the primary myelin synthesizing cell type in the CNS. However, the signal was not detectable in rat sciatic nerve (Schwann cells) of the peripheral nervous system. These findings indicate that the role of N-acetylaspartate in myelin synthesis is restricted to the CNS. Furthermore, they provide additional support for the acetate deficiency hypothesis of Canavan disease and also make a stronger case for acetate supplementation as an immediate and inexpensive therapy for Canavan disease.

Aspartoacylase is restricted primarily to myelin synthesizing cells in the CNS: therapeutic implications for Canavan disease.

Canavan disease is a devastating neurodegenerative childhood disease caused by mutations in aspartoacylase, an enzyme that deacetylates N-acetylaspartate to generate free acetate in the brain. Localization of aspartoacylase in different cell types in the rat brain was examined in an attempt to understand the pathogenesis of Canavan disease. In situ hybridization histochemistry with a riboprobe based on murine aspartoacylase cDNA was used in this study. The hybridization signal was detectable primarily in the myelin-synthesizing cells, namely oligodendroglia. These findings provide strong additional support for insufficient myelin synthesis as the pathogenic basis of Canavan disease and make a compelling case for acetate supplementation as a simple and noninvasive therapy for this fatal disease with no treatment.

A radiometric assay for aspartoacylase activity in cultured oligodendrocytes.

Recent studies have shown that aspartoacylase (ASPA), the defective enzyme in Canavan disease, is detectable in the brain only in the oligodendrocytes. Studying the regulation of ASPA is central to the understanding the pathogenesis of Canavan disease and to the development of therapeutic strategies. Toward this goal, we have developed a sensitive method for the assay of ASPA in cultured oligodendrocytes. The method involves: (a) chemical synthesis of [14C]N-acetylaspartate (NAA) from L-[14C]Asp; (b) use of [14C]NAA as substrate in the assay; and (c) separation and quantitation of the product L-[14C]Asp using a TLC system. This method can detect as low as 10pmol of product and has been optimized for cultured oligodendrocytes. Thus, this method promises to be a valuable tool for understanding the biochemical mechanisms involved in the cell-specific expression and regulation of ASPA in oligodendrocytes.