In a search for the pathophysiologic mechanisms, we estimated isoprenoid synthesis and concentration, cellular growth, and the activity of the LDL receptor pathway in fibroblasts from patients with mevalonate kinase deficiency (MKD), a severe multisystemic disorder of cholesterol and non-sterol isoprenoid biosynthesis. In response to different concentrations of LDL and non-lipoprotein-bound cholesterol, MKD cells partially counteracted their enzyme defect by increased activities of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase (results from earlier studies) and the LDL receptor pathway, responses similar to the pharmacologic effects seen upon administration of HMG-CoA reductase inhibitors. Rates of N-linked protein glycosylation, estimated as the amount of [14C]galactose-labeled macromolecules secreted into cell culture medium, were significantly decreased in MKD fibroblasts in comparison with control cells which may indicate alterations in the dolichol or dolichol phosphate pool. In response to exogenous cholesterol, the major feedback inhibitor of isoprenoid biosynthesis, growth velocities of MKD fibroblasts declined in comparison with control cells, further suggesting an impairment of non-sterol isoprenoid biosynthesis in MKD. Our results suggest an imbalance in the multilevel regulation of the biosynthesis of cholesterol and non-sterol isoprenoids in MKD, representing an additional causative factor responsible for the pre- and postnatal pathology of MKD.
A sensitive assay is described for quantitating dolichyl phosphate (Dol-P), the polyprenyl phospholipid which participates in N-linked glycosylation. The assay is based on a novel reaction of alkyl phosphates with phenyl chloroformate in which a monosubstituted mixed anhydride of phosphoric and carbonic acid is formed. Evidence in support of the proposed structure for the derivative includes a phosphate to phenyl ratio of 1, infrared spectra, elemental analysis, behavior during ion-exchange chromatography, and reactivity with primary and secondary amines. A simple, rapid procedure is described for the preparation of [14C]phenyl chloroformate from [14C]phenol; use of the radiolabeled reagent allows assay of Dol-P in the subnanomolar range. The assay was applied to the quantitation of total Dol-P levels in rat liver. Dol-P and Dol-PP and their glycosylated derivatives were extracted with organic solvents and degraded to free Dol-P by acid hydrolysis. Following saponification to hydrolyze contaminating phospholipids, Dol-P was purified by using diethylaminoethyl-cellulose chromatography and derivatized with [14C]phenyl chloroformate. Tracer quantities of [3H]Dol-P were added to the tissue before extraction in order to monitor purification and yield. Double-label counting of the isolated derivative allowed calculation of the level of total Dol-P in the original tissue sample. This procedure yielded values of 2.9 +/- 0.9 nmol of Dol-P/g of rat liver.
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