Technique
Thin-Layer Chromatography as an Analytical Tool


Qualitative Analysis


Quantitative Analysis





Thin-Layer Chromatography as a Preparative Tool

Thin-layer chromatography of lipids

Complete separation of lipid classes on a single thin-layer plate.

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In autopsy tissues of 12 cases of Tay-Sachs disease the N-acetyl-β-hexosamini-dase A and B activities were investigated using chromogenic and physiological substrates.

In three cases of Tay-Sachs disease, classified as the variant O, the enzyme activities A and B were missing; in eight cases, classified as the variant B, the enzyme activity A was missing. In another case, both enzyme activities wcre shown to be enhanced in brain tissue (‘variant AB’), using a chromogenic substrate.

The three enzymic variants showed different glycolipid storage patterns of Tay-Sachs-ganglioside (TSG) and its asialo residue, the trihexosylceramide (THC) in the nervous tissues. Additional storage of kidney globosidc was found in the visceral tissues of the O variant. A decrease of the non-accumulated lipids, especially of those characteristic for myelin, was observed. The quantitative lipid determinations were performed by means of a thin-layer densitometric micromethod (standard deviation 2–5 per cent).

Evidence is presented that the different storage patterns result from the corresponding enzyme alterations in the three variants. An essential condition for this statement was the isolation of the storage compounds from Tay-Sachs tissues and their radioactive labelling by the addition of tritium to the double bond in their sphingosine moiety. In a previous investigation it was shown that enzyme A degrades the storage compounds TSG, THC and kidney globoside while enzyme B acts on THC and kidney globoside only. In agreement with this finding, a highly concentrated mixture of both enzymes from normal tissues hydrolyses the main storage compound, the Tay-Sachs-ganglioside. This hydrolysis was reduced when corresponding enzyme preparations from tissues of variants of Tay-Sachs disease (including variant AB) acted on TaySachs ganglioside.

Some properties of the N-acetyl-β-D-hexosaminidases from normal and from pathological tissues were determined with chromogenic and physiological substrates. The relationship between the enzymes A and B is discussed.

Nelson Stephens

Papers

Quantitative carbon-14 and tritium assay of thin-layer chromatography plates