[Blood products stored in plastic bags: release of plasticizers from the bag material]. / Vérkészítmények müanyag zsákban: lágyító kioldódás a zsákfalból.

The plasticizer di-(2-ethyl-hexyl)phthalate (DEHP) is slowly leached from the wall of the PVC blood bags into the blood products. It will be partly incorporated into the membranes of the blood cells, and finally will be transfused together with the blood products into the recipient, where DEHP might exert toxic effects. DEHP content was determined by HPLC in different blood products stored in plastic bags manufactured by different firms (Medicor--Hungary, MacoPharma--France, Terumo--Japan, Baxter--USA). In whole blood DEHP is present mainly in the plasma and blood bags of different firms show similar results. 1 unit of whole blood contains 20-50 mg DEHP on day 14 of storage, and 30-55 mg on day 28, while red blood cells resuspended in plasma contained only 2-15 mg on day 14. 15-20 mg DEHP was found in 1 unit of plasma stored for 4 days at +4 degrees C. Only 1-4 mg DEHP was detected in 1 unit of erythrocytes (cca. 200 ml) even on day 14 of storage. The supernatant of platelet concentrates contained the relatively highest amount of DEHP (even 50-65 mg was found in 1 unit of platelet concentrates on day 5 of storage). When platelet concentrates were studied, essential differences were recorded between the bags manufactured by different firms. Platelet reactivity is better maintained in storage bags manufactured without DEHP.

Enhanced association of mutant triosephosphate isomerase to red cell membranes and to brain microtubules.

In a Hungarian family with triosephosphate isomerase (TPI; D-glyceraldehyde-3-phosphate keto-isomerase, EC 5.3.1.1) deficiency, two germ-line identical, but phenotypically differing compound heterozygote brothers (one of them with neurological disorder) have been identified with the same very low (<5%) TPI activity and 20- or 40-fold higher erythrocyte dihydroxyacetone phosphate levels as compared with normal controls. Our present studies with purified TPI and hemolysates revealed the binding of TPI, and the binding of human wild-type and mutant TPIs in hemolysate, to the red cell membrane, and the interference of binding with other hemolysate proteins. The binding of the mutant TPI is enhanced as compared with the wild-type enzyme. The increased binding is influenced by both the altered structure of the mutant and the changes in the red cell membrane. Compared with binding of glyceraldehyde-3-phosphate dehydrogenase, the isomerase binding is much less sensitive to ionic strength or blocking of the N-terminal tail of the band-3 transmembrane protein. The binding of TPIs to the membrane decreases the isomerase activity, resulting in extremely high dihydroxyacetone phosphate levels in deficient cells. In cell-free brain extract, tubulin copolymerizes with TPI and with other cytosolic proteins forming highly decorated microtubules as shown by immunoblot analysis with anti-TPI antibody and by electron microscopic images. The efficacy order of TPI binding to microtubules is propositus > brother without neurological disorder > normal control. This distinct microcompartmentation of mutant proteins may be relevant in the development of the neurodegenerative process in TPI deficiency and in other, more common neurological diseases.

Distinct behavior of mutant triosephosphate isomerase in hemolysate and in isolated form: molecular basis of enzyme deficiency.

In a Hungarian family with severe decrease in triosephosphate isomerase (TPI) activity, 2 germ line-identical but phenotypically differing compound heterozygote brothers inherited 2 independent (Phe240Leu and Glu145stop codon) mutations. The kinetic, thermodynamic, and associative properties of the recombinant human wild-type and Phe240Leu mutant enzymes were compared with those of TPIs in normal and deficient erythrocyte hemolysates. The specific activity of the recombinant mutant enzyme relative to the wild type was much higher (30%) than expected from the activity (3%) measured in hemolysates. Enhanced attachment of mutant TPI to erythrocyte inside-out vesicles and to microtubules of brain cells was found when the binding was measured with TPIs in hemolysate. In contrast, there was no difference between the binding of the recombinant wild-type and Phe240Leu mutant enzymes. These findings suggest that the missense mutation by itself is not enough to explain the low catalytic activity and "stickiness" of mutant TPI observed in hemolysate. The activity of the mutant TPI is further reduced by its attachment to inside-out vesicles or microtubules. Comparative studies of the hemolysate from a British patient with Glu104Asp homozygosity and with the platelet lysates from the Hungarian family suggest that the microcompartmentation of TPI is not unique for the hemolysates from the Hungarian TPI-deficient brothers. The possible role of cellular components, other than the mutant enzymes, in the distinct behavior of TPI in isolated form versus in hemolysates from the compound heterozygotes and the simple heterozygote family members is discussed.