Results And Discussion

Recently we described a patient with a defect in the 3-hydroxyacyl-CoA dehydrogenase component of the D-bifunctional protein.10 We have now performed complementation studies and fused fibroblasts from this patient (patient 1) with fibroblasts from the L-bifunctional protein deficient patient known from literature (patient 2)11 followed by measurement of pristanic acid P-oxidation in the fused cells (Table 1). As a control, cells were not fused but only cocultivated after which pristanic acid P-oxidation was measured. Remarkably, pristanic acid P-oxidation was not restored after fusion, indicating that cells from patient 1 and patient 2 did not complement one another. As expected, both cell lines did show complementation when fusions were performed with fibroblasts from a Zellweger patient (Table 1).

The results of the complementation studies suggest that in both patient 1 and patient 2 the defect is in the same gene although patient 1 was described as being D-bifunctional protein deficient and patient 2 was described as being L-bifunctional protein deficient. Importantly, both patients showed elevated levels of C26:0 and accumulation of bile acid intermediates (DHCA, THCA) in plasma.10'11 The accumulation of THCA in plasma and the deficient p-oxidation of pristanic acid in fibroblasts are hard to reconcile with the fact that L-bifunctional protein has been found not to be involved in the p-oxidation of 2-methyl branched-chain fatty acids, like pristanic acid, and bile acid intermediates, like THCA. In fact, recent studies have shown that it is the D-bifunctional protein which is involved in the P-oxidation of these substrates.5'6'9 This led us to measure the activity of D-bifunctional protein in fibroblasts from patient 2.

To this end fibroblasts of patient 2 were incubated with the enoyl-CoA ester of THCA and formation of 24-hydroxy-THC-CoA and 24-keto-THC-CoA was measured (Fig. 1). The results in Fig.1 clearly show that patient 1 is deficient at the level of the 3-hydroxyacyl-CoA dehydrogenase component of D-bifunctional protein because of the increased formation of 24-hydroxy-THC-CoA and lack of 24-keto-THC-CoA formation (Fig. 1B). In contrast, there was only very little formation of 24-hydroxy-THC-CoA and no formation of 24-keto-THC-CoA in patient 2 (Fig. 1C).

These data strongly suggest that it is the D-bifunctional enzyme which is functionally inactive in patient 2. Subsequent studies which include analysis at the molecular level, have revealed distinct mutations in the gene coding for D-bifunctional protein. These results will be described elsewhere (Van Grunsven et al., in preparation).

Taken together, our results resolve the puzzling finding that cells from our patient 1 with a defect in the 3-hydroxyacyl-CoA dehydrogenase component of the D-bifunctional protein failed to show complementation with cells from the patient with presumed L-bifunctional protein deficiency which we have now found to be deficient in the D-specific enzyme. The finding that C26:0 is strongly increased in plasma from both

Table 1. Results of complementation analyses.
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