Mevalonic acid chromatography

Jemal, M., Schuster, A., and Whigan, D. B. (2003). Liquid chromatography/tandem mass spectrometry methods for quantitation of mevalonic acid in human plasma and urine method validation, demonstration of using a surrogate analyte, and demonstration of unacceptable matrix effect in spite of use of a stable isotope analog internal standard. Rapid Commun. Mass Spectrom. 17, 1723-1734. 

In the past decade, eight inherited disorders have been linked to specific enzyme defects in the isoprenoid/cholesterol biosynthetic pathway after the finding of abnormally increased levels of intermediate metabolites in tissues and/or body fluids of patients (Table 5.1.1) [7, 9, 10]. Two of these disorders are due to a defect of the enzyme mevalonate kinase, and in principle affect the synthesis of all isoprenoids (Fig. 5.1.1) [5]. The hallmark of these two disorders is the accumulation of mevalonic acid in body fluids and tissues, which can be detected by organic acid analysis, or preferably, by stable-isotope dilution gas chromatography (GC)-mass spectrometry (GC-MS) [2]. Confirmative diagnostic possibilities include direct measurement of mevalonate kinase activities in white blood cells or primary skin fibroblasts [3] from patients, and/or molecular analysis of the MVK gene [8]. 

Mevalonic acid 5-pyrophosphate [1492-08-6] M 258,1, Purified by ion-exchange chromatography on Dowex-1 formate [Bloch et al. JBC 234 2595 7959], DEAE-cellulose [Skilletar and Kekwick, AB 20 171 7967], on by paper chromatography [Rogers et al. BJ99 381 7966]. Likely impurities are ATP and mevalonic acid phosphate. Stored as a dry powder or as a slightly alkaline (pH 7-9) soln at -20°. 

The main problem in this approach is the very low permeability of mevalonic acid to membranes, resulting in very low incorporation. Positive results have been obtained by the use of cell-free systems incubated with [14C]-mevalonic acid,26,27 [14C]isopentenyl diphosphate,28 or [32P]orthophos-phate.29 Incubation of these radioactive lipids with glycosyl nucleotides labelled in the glycosyl group with a different isotope, followed by extraction and cochromatography in different solvent systems, may indicate that both compounds are present in the same molecule. When the lipid moiety becomes labelled from mevalonic acid or isopentenyl diphosphate, chromatography on DEAE-cellulose columns should be performed, in order to avoid confusion with steryl glycosides. 

Jemal M, Schuster A, Whigan DB (2003) Liquid chromatography/tandem mass spectrometry methods for quantitation of mevalonic acid in human plasma and urine method validation,... 

The first report of the incorporation of radioactively labelled mevalonic acid into dendrobine (82) was by Yamazaki et al. (224). Sodium [2— CJmevalonate [( )-463 ] was administered to stems of D. nobile by the cotton-wick method. After 12 days, the plants were extracted and radioactive dendrobine (82) was isolated by column chromatography. The total incorporation was 0.012%. Subsequent Kuhn-Roth oxidation led to acetic acid that showed the expected activity assuming the biosynthesis pathway a leading to the picrotoxanes via a cadalene as precursor (Scheme 55, pathway c). 

The most adequate method to eliminate matrix effects is the use of an ILIS. An ILIS shows (almost) identical behaviour to the target analyte in both sample pretreatment, chromatography, and analyte ionization. The data reported in the bioanalysis of mevalonic acid [23] indicate that this issue needs proper attention during method development and validation (Ch. 11.3.4). 

Mevalonate kinase (EC 2.7.1.36) phosphorylates mevalonic acid, the NADPH-reduced form of HMG-CoA. The reaction is ATP and Mg dependent. The enzyme was purified from a C. roseus cell suspension culture, in which, after induction a specific activity of 1.5 nkat/mg protein is found [12]. The purification protocol comprised ion-exchange chromatography, hydrophobic interaction chromatography and gel filtration. By gel filtration an M,. of 105,000 was found for mevalonate kinase. 

Fig. 1A,B. pH]-Mevalonic acid incorporation into cytokinins by tobacco crown gall tissue. A Sephadex LH-20 column chromatography of the nucleotide-derived. BuOH-soluble fraction from compactin (5 jLiM)-treated tissue B HPLC of fraction 1 obtained from the analysis shown above (column juBondapak C,. gradient elution with 10-50% MeOH (containing 1% HOAc) in 30 min. 3 ml/min)...

The formation, presence and activity of the non-volatile plant growth regulators in soil have received far less attention than that focussed on ethylene. Indeed the studies that have been undertaken have been somewhat less rigorous than those on ethylene. One of the major and fundamental differences is the ease of chemical analysis. Ethylene can be determined rapidly and unequivocally by gas chromatography. Determination of the non-volatile hormones usually involves a complex extraction procedure and more sophisticated chemical instrumentation. Even if only one of the gibberellins, gibberellic acid (GA ), were to be determined, it v/ould be desirable to label the material by incorporating -mevalonate, a precursor, into the compound bio-... 

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