No abstract

Abstract— Partially purified myelin from the brains of 17‐day‐old rats was separated into 4 subfractions on a three‐step sucrose gradient by virtue of heterogeneity in density and particle size. Precursor‐product relationships between different membrane fractions were investigated by determining the specific radioactivity of individual lipids in each subcellular fraction 15 min after intracranial injection of an appropriate precursor. Rats were injected with [2‐3H]glycerol. myelin subfractions prepared, and individual lipids separated by TLC. For choline and ethanolamine phospholipids, specific radioactivity was highest in the densest fraction (D), intermediate in the next densest fraction (C), and lowest in the lighter fractions (B and A). Similar results were observed for cerebroside and sulphatide when [3H]galactose was the precursor. These data are consistent with (but do not prove) a precursor‐product relationship for individual lipids from the densest to the lightest subfraction. Another experimental design involving time staggered injections of [3H] and [14C] precursors was developed which enables a more definitive result with regard to precursor‐product relationships to be obtained. A precursor‐product relationship between a given lipid in a dense myelin membrane fraction, and the same lipid in a lighter subfraction, would be indicated by a change in isotope ratio. If there is no precursor‐product relationship. Ihe isotope ratio should be constant. Such experiments were done with [3H] and [14C]glycerol. The data indicated that phosphatidyl ethanolamine and its plasmalogen analog were added first to the densest subfraction and then in turn to the lighter subfractions. In contrast, phosphatidyl choline and its plasmalogen analog were added “simultaneously” (i.e. with delays of much less than 15min) to each of the subfractions. Similar experiments with [3H] and [14C]galactose showed that cerebroside, sulphatide and galactosyl diglyceride also entered the subfractions simultaneously rather than in sequential order. Thus the assembly of the myelin sheath involves an obligate order of addition of certain lipids. while other lipids are probably added in a random order.

The IgM monoclonal autoantibodies of patients with demyelinating paraproteinaemic polyneuropathy recognize a carbohydrate structure present on both myelin-associated glycoprotein (MAG) and protein zero (P0). These autoantibodies are sufficient to cause the disease but the mechanism of demyelination remains unclear. We have analysed nerve biopsies from eight patients with polyneuropathy and anti-MAG antibodies by quantitative immunohistochemistry and find a concordant pattern of reduced expression of myelin markers with the loss of myelinated fibres. We report here novel features of this disease, in particular a selective lack of detectable MAG in a large proportion of myelinated-fibres containing P0, myelin basic protein (MBP) and periaxin. There is also an inverse correlation of the distribution of MAG in peripheral nerve myelin with the serum anti-MAG antibody titres but no correlation of these titres with the loss of myelinated fibres. Double immunofluorescence staining of paraproteinaemic polyneuropathy (PPN) nerves shows anti-MAG IgM deposited on the periphery of myelinated fibres associated with or lacking MAG staining. These data suggest that the binding of anti-MAG antibodies to MAG and/or other myelin component(s) results in MAG downregulation and may have an essential role in the molecular mechanisms leading to demyelination and partial regeneration in this disease.

The abundance and developmental regulation of N-acetylaspartate (NAA) in brain suggest that it plays an important role in brain metabolism. Previous studies demonstrated that NAA transports acetate from the mitochondrion to the cytoplasm where it is utilized for lipid synthesis, however, the metabolic fate of NAA-derived aspartate is not established. To investigate NAA metabolism, rats were injected intracranially with N-([2H3]acetyl)-L-[15N]aspartate ([2H3,15N]NAA) and whole brain metabolites were analyzed using gas chromatography and mass spectrometry techniques (GC/MS). The rapid decline of [2H3,15N]NAA was associated with a rapid appearance of [15N]glutamate, indicating rapid transamination of the [15N]aspartate that was derived from the enzymatic hydrolysis of [2H3,15N]NAA. Inability to detect [15N]NAA in brain extracts in several experiments indicates that the 15N moiety is not reutilized for NAA synthesis and suggests one metabolic role of NAA may be the transport of amino nitrogen from the mitochondrion to the cytoplasm.