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The recently reported phenomenon that red blood cells (RBC) from Alzheimer disease (AD) patients and normal individuals, which have identical electrophoretic mobilities (EPM) in phosphate-buffered saline (PBS), have different EPM in appropriately selected polymer solutions, has been further explored. Of a number of in vitro treatments to which AD and normal RBC were subjected prior to EPM measurements in bottom phase (from a dextran-poly(ethylene glycol) (PEG) aqueous phase system) only trypsin eliminated the difference. Thus, the differential polymer interaction between AD and normal RBC, thought to be the basis for their dissimilar EPM, can be abolished by appropriate proteolytic modification of the cell surfaces and suggests protein as a source of difference. Because young and old RBC from normal individuals, which have the same EPM in PBS, have different EPM in certain polymer solutions, and the RBC from AD patients have been reported to age abnormally, we also compared the young and old RBC subpopulations from these two sources. By the criterion of cell electrophoresis in polymer solutions the differences between AD and normal RBC and between young and old RBC are distinct. The EPM of AD and normal RBC differ in bottom phase or PEG but not in dextran solution; while the EPM of young and old RBC differ predominantly in dextran. We speculate that since the observed difference in EPM of RBC from AD patients and normals depends on protein(s) yet is anticoagulant-related (being obtained only when blood is collected in citrate or oxalate) it might be the result of an interaction (Ca(2+)-mediated?) between the surfaces of these cells and protein component(s) of their respective, compositionally differing sera.

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It has recently been found that electrophoresis in solutions of appropriately selected polymers in phosphate-buffered saline (PBS) can differentiate between some closely related cell populations which have identical electrophoretic mobilities (EPM) in PBS (e.g., human young and old red blood cells (RBC); RBC from Alzheimer patients and normal individuals). The EPM differences detected in polymer solutions are most likely a consequence of cell- and polymer-specific interactions. Aspects of the relation between the electrophoresis in aqueous polymer solutions of native and in vitro treated young and old RBC (from human and rat) and their partitioning in a charge-sensitive dextran-poly(ethylene glycol) (PEG) aqueous phase system (i.e., a system with a Donnan potential between the phases, top phase positive) have been examined further and are discussed. Unlike the behavior of RBC from Alzheimer patients and normal individuals in which an EPM difference can be detected in PEG solutions but not in dextran, differences in EPM between human young and old RBC are detectable in solutions of either polymer. Selected enzyme treatments of human young and old RBC or their fixation with aldehyde eliminates the EPM differences in dextran; while neuraminidase treatment or formaldehyde fixation of rat young and old RBC retains EPM differences in dextran between these cells. In these latter cases partitioning differences are also in evidence and are in the same direction as the cells' relative EPM (i.e., old RBC < young RBC). The earlier hypothesis that cell partitioning is 'more sensitive' than cell electrophoresis in detecting differences in surface charge between cells bears reexamination because human young and old RBC, which cannot be differentiated by single-tube partitioning in a charge-sensitive phase system, have different EPM in polymer solutions. The difference between these cells can be detected by partitioning but only by use of a multiple-extraction procedure. It is then found to be in a direction similar to the cells' relative EPM in dextran (i.e., human old RBC > young RBC). Rat young and old RBC have different partitions (rat old RBC < young RBC) and different EPM (also rat old RBC < young RBC). Thus, while cell partitioning in a charge-sensitive dextran-PEG aqueous phase system and cell electrophoresis in polymer solution seem to reflect, at least with these cell subpopulations, qualitatively analogous differences in surface properties (in that increasing partitions and EPM are concomitant), there are instances in which either of these physical measurements discerns surface differences which escape detection by the other.

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A correlation, with some exceptions, between the partitioning behavior of red blood cells (RBCs) from different species in charge-sensitive dextran-poly(ethylene glycol) (PEG) aqueous phase systems and their relative electrophoretic mobilities (EPMs) in phosphate-buffered saline (PBS) has previously been reported. This relationship has now been further probed by carrying out RBC electrophoresis in media (i.e., dextran-rich bottom or PEG-rich top phases) more closely approximating the environment in which RBC partitioning takes place to see whether a better correlation would ensue. The ratios of viscosity-corrected EPMs of different species' RBCs in (diluted) dextran-rich or PEG-rich phases/EPMs of the respective species' RBCs in PBS differ for a number of species, and from each other, reflecting thereby differences in kind (i.e., dextran or PEG) and nature of polymer interaction with these RBCs. There is a general tendency for EPMs in any of the tested media to correlate with both the cells' relative partition ratios as well as with their relative EPMs in one of the other media. However, examination of the behavior of different species' RBCs taken two species at a time indicates that their relative EPMs in any two suspending media or in one suspending medium and partitioning often differ. Thus, both the cell partition ratio and the cell EPMs obtained in polymer media must, at least in some cases, reflect surface properties other than or in addition to the charge reflected by EPM measurements in PBS or saline. Cell electrophoresis in polymer solutions thereby provides an additional parameter for discriminating between surface properties of certain closely related cell populations.

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Blood from patients diagnosed as having Alzheimer disease and from subjects without memory impairment or dementia was collected in citrate. The erythrocytes were washed and electrophoresed in phosphate-buffered saline as well as in a number of polymer solutions in phosphate-buffered saline. The electrophoretic mobilities of red cells from Alzheimer patients and from normals were indistinguishable when measured in phosphate-buffered saline. In the dextran-rich bottom phase obtained from a dextran-poly(ethylene glycol) aqueous phase system, but not in a dextran solution alone, the electrophoretic mobilities differ (P < 0.001). In a poly(ethylene glycol) solution the mobilities also differ although, on a percentage basis, less so than in the bottom phase. It would appear that a differential adsorption of appropriately selected polymer(s) on the Alzheimer and normal red blood cells renders surface differences electrophoretically detectable.

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N-[7-Nitrobenz-2-oxa-1,3-diazol-4-yl]psychosine sulfate (NBD-PS), a fluorescent analog of cerebroside sulfate (CS), was synthesized and tested as an alternative to the radiolabeled forms of CS used for assaying arylsulfatase A (ASA) in its physiological role as a cerebroside sulfate sulfohydrolase. NBD-PS simulates the natural substrate for ASA. Protocols have been developed for its use in differentiating low enzyme activities in diagnostic samples. Hydrolysis of NBD-PS is specific for ASA and optimal assay parameters were identical to those determined for CS. Differentiations between each of the major phenotypes for ASA activity were possible in the set of samples tested. One particular advantage was the ability to discriminate between individuals exhibiting arylsulfatase A pseudodeficiency and the truly deficient individuals with metachromatic leukodystrophy. Differential diagnosis was possible with fibroblast extracts by an assay that is more sensitive than procedures employing radioisotopes. Reaction products may be analyzed quantitatively by HPLC, or semiquantitatively with TLC. NBD-PS provides a simpler, safer, and more cost-effective means of performing natural substrate enzyme assays for ASA. Phenotyping with the fluorescence assay is an effective alternative to the laborious radioactive CS preparations and tissue culture loading studies that have previously been necessary.

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