Single polyprenol and dolichol isolation by semipreparative high-performance liquid chromatography technique

A new method of separation of single polyprenols (or dolichols) from a mixture of isoprenoid alcohols is described. Application of a high-performance liquid chromatography (HPLC) apparatus equipped with a semipreparative ODS column resulted in preparation of long-chain (dihydro)polyprenols of high purity (>95%).This approach substantially decreases the time scale of the conventional chromatographical preparative procedure. The method can be widely used in chemical and biochemical projects, where single polyprenols or dolichols are required.     

Bioactive Components of Sea Buckthorn <Emphasis Type="Italic">Hippophae rhamnoides</Emphasis> L. Foliage

The composition of lipophilic components of sea buckthorn leafy shoots, a large tonnage waste in the production of sea buckthorn oil and during renewing the cultural plantings of sea buckthorn, was studied. Hexane was used as an extraction solvent for raw materials; it provides a high degree of lipophilic component extraction and is an analogue of extraction gasoline used in the food and perfume industries. The chemical composition of the hexane extract of sea buckthorn leafy shoots was studied by gas chromatography–mass spectrometry and high-performance liquid chromatography. Sixty-seven neutral and twenty-nine acidic components, including polyprenols, dolichols, triterpene alcohols and acids, sterols, were identified. β-Sitosterol was the main component of the sterol fraction. Its content was 6.9% of the extract mass, which is much higher than in the essential extracts of leaves and pulp of sea buckthorn fruit. It is mostly found in the free form in the extract. The acidic fraction contains highly active triterpene acids (up to 5% of the extract mass) along with the major aliphatic acids. Components with the chain length of 11 and 17 isoprene units predominate in the fraction of polyprenols and dolichols (up to 4.2%). The results allow us to consider sea buckthorn leafy shoots as a promising source of biologically active compounds.     

Polyisoprenoid alcohols from the mushroom Lentinus edodes

Lipids extracted from the shiitake mushroom Lentinus edodes contain dolichols composed of 15 up to 19 isoprene units with Dol-17 as the dominating prenologue. Identification of dolichols was achieved by the application of 2D-TLC, HPLC and electrospray ionization mass spectrometry. Additionally a family of polyprenols (-unsaturated counterparts) with the same chain-length was also detected. Dolichols comprised approximately 0.002% of the fresh weight of the mushroom. Dolichols accompanied by traces of polyprenols are for the first time found in the mushroom tissue.     

Electrospray ionization mass spectrometry analysis of polyisoprenoid alcohols via Li+ cationization

Direct analysis of polyisoprenoids by electrospray ionization mass spectrometry (ESI-MS) often produces poor results requiring off-line time and sample-consuming derivatization techniques. We describe a simple ESI-MS approach for the direct analysis of polyisoprenoids using several dolichols and polyprenols with different chain sizes as proof-of-principle cases. Lithium iodide is used to promote cationization by intense formation of [M+Li]+ adducts. Thus, detection of polyisoprenoids with mass determination can be performed with high sensitivity (limit of detection [LOD] approximately 100 rhoM), whereas characteristic collision-induced dissociations observed for both dolichols and polyprenols permit investigation of their structure. Using ESI(Li+)-MS and ESI(Li+)-MS/MS analysis, we screened for polyprenol products of an octaprenyl pyrophosphate synthase of Plasmodium falciparum and dolichols in a complex mixture of compounds produced by Leishmania amazonensis and P. falciparum.     

Dolichols of the fern Matteucia struthiopteris

Dolichols isolated from leaves of the fern Matteucia struthiopteris were present as a mixture of prenologues composed of 14 up to 20 isoprene units with Dol-16 dominating. They comprised approximately 0.004% of the fresh weight of fresh plant tissue and were accompanied by traces of polyprenols (Pren-14 up to Pren-17, Pren-16 dominating). Their structure was confirmed by electropray ionization mass spectrometry (ESI-MS). This is the first time that dolichols have been reported as dominating polyisoprenoid alcohols in plant photosynthetic tissue.     

Uptake and modification of dietary polyprenols and dolichols in rat liver

Short and long dolichols and polyprenols in free form or esterified with fatty acids were incorporated into liposomes and administered to rats through a gastric tube. The free alcohols were taken up by the liver to different extents. While uptake in other organs was less, it also involved the fatty acid esters. The use of systems other than liposomes did not increase the efficiency of uptake. Most of the administered lipids were recovered in the lysosomes. Exogenous dolichols and polyprenols were both partly esterified in the liver and, to some extent, also phosphorylated; a portion of the polyprenols was also alpha-saturated. These results indicate that various polyisoprenes are taken up, to a small extent, from the diet by tissues under normal conditions and in liver these dietary lipids undergo terminal modifications.     

Sugar availability modulates polyisoprenoid and phytosterol profiles in Arabidopsis thaliana hairy root culture

Sugars are recognized as signaling molecules regulating the biosynthesis of secondary metabolites in plants. Here, a modulatory effect of sugars on dolichol and phytosterol profiles was noted in the hairy roots of Arabidopsis thaliana. Arabidopsis roots contain a complex dolichol mixture comprising three groups (‘families’) of dolichols differing in the chain-length. These dolichols, especially the longest ones are accompanied by considerable amounts of polyprenols of the same length. The spectrum of polyisoprenoid alcohols, i.e. dolichols and polyprenols, was dependent on sugar type (glucose or sucrose) and its concentration in the medium. Among the long-chain dolichols Dol/Pren-20 (dolichol or prenol molecule composed of 20 isoprene residues) and Dol/Pren-23 were the main components at 0.5% and 2% glucose, respectively. Moreover, the ratio of polyprenols versus respective dolichols was also modulated by sugar in this group of polyisoprenoids, with polyprenols dominating at 3% sucrose and dolichols at 2% glucose. Glucose concentration affected the expression level of genes encoding cis-prenyltransferases, enzymes responsible for elongation of the polyisoprenoid chain. The most abundant phytosterols of the A. thaliana roots, β-sitosterol, stigmasterol and campesterol, were accompanied by corresponding stanols and traces of brassicasterol, stigmast-4,22-dien-3-one and stigmast-4-en-3-one. Similar to the polyisoprenoids, sterol profile responded to the sugar present in the medium, β-sitosterol dominating in roots grown on 3% or lower glucose concentrations and stigmasterol in 3% sucrose. These results indicate an involvement of sugar signaling in the regulation of cis-prenyltransferases and phytosterol pathway enzymes.     

On the specificity of dolichol kinase and DolPMan synthase towards isoprenoid alcohols of different chain length in rat liver microsomal membrane.

1. A wide range of dolichols differing in the length of hydrocarbon chain (from 11 to 32 isoprene residues) were found to be phosphorylated in the presence of CTP in rat liver microsomes. 2. Fully unsaturated polyprenols of the same chain length as dolichols were poor substrates for dolichol kinase at low detergent (Nonidet P-40) concentration. At higher concentration of detergent, both dolichols and polyprenols were equally effective. 3. In the transfer of mannosyl residues from GDPMan, the dolichyl phosphates generated in rat liver microsomes were all good lipid acceptors, while fully unsaturated polyprenyl phosphates were not.     

Separation, quantitation and distribution of dolichol and dolichyl phosphate in rat and human tissues

Two procedures for quantitative determination of dolichol were studied and these were applied to analyze tissue and subcellular distribution. In the first procedure the dolichols were oxidized with Cr2O3 and reduced with NaB3H4. The radioactivity in the individual dolichols was measured using reversed-phase thin-layer chromatography. In the second procedure, dolichols were analyzed by high-pressure liquid chromatography. For determination of dolichyl phosphates the lipid extract was subjected to acid and alkaline hydrolysis, and after hydrolysis with acid phosphatase the distribution was determined by high-pressure liquid chromatography. Recovery was monitored by the addition of dolichol D15 and D23 phosphate to the homogenate. Rat spleen had the highest dolichol content (114 micrograms/g) followed by lower content in rat liver and brain. The distribution pattern was similar in all organs, with 18 and 19 isoprene residues as dominating components. Human organs contain considerably higher concentrations of dolichol, with the 19 and 20 isoprene residues as the main components. In rat liver, outer mitochondrial and Golgi membranes, lysosomes and plasma membranes contain considerable amounts of dolichol. A drastic increase in dolichol content was observed in rat liver hyperplastic nodules while human liver cirrhosis and hepatocarcinoma showed a marked decrease in dolichol. In the latter case, the distribution pattern was also changed. Of the total amount of dolichol present in the tissues, 2% was phosphorylated in human liver, 10% in human testis and 18% in rat liver. In rat liver mitochondria and in microsomes 4 and 31%, respectively, of the polyprenols were in activated form. The results demonstrated that dolichyl phosphate and dolichol concentrations were regulated by different mechanisms and that the two forms possessed an independent distribution.     

In vitro plant tissue cultures accumulate polyisoprenoid alcohols

In vitro cultivated plant cells and tissues were found to synthesize polyisoprenoids. Taxus baccata suspension cell cultures accumulated polyisoprenoids of the same pattern as the parental tissue; methyl jasmonate or chitosan treatment almost doubled their content. All the root cultures studied accumulated dolichols as predominant polyisoprenoids. Roots of Ocimum sanctum grown in vitro accumulated approx. 2.5-fold higher amount of dolichols than the roots of soil-grown plants. Dolichols dominated over polyprenols in all Triticum sp. tissues studied.     

Synthesis of dolicyl- and polyprenyl sulfates]

Dolichyl and polyprenyl sulfates were synthesized as analogues of dolichyl and polyprenyl phosphates by the interaction of dolichols and polyprenols with the pyridine-sulfuric anhydride complex.     

Preparative separation of naturally occurring mixtures of polyprenols on hydroxyalkoxypropyl-Sephadex.

A procedure is described for the efficient preparation of individual polyprenols from naturally occurring mixtures of dolichols, ficaprenols, and betulaprenols.     

S--vinyl homocysteine, an analog of ethionine that is highly mutagenic for S. typhimurium TA100.

Two types of bovine pituitary gland polyprenols were resolved by silica gel chromatography; i. e., the high molecular weight dolichols of 17 to 23 isoprene units with the OH-terminal isoprene residue saturated, and a fully unsaturated decaprenol. The latter compound was found to be a mixture of molecules differing in the proportion of $\text{cis}$- and $\text{trans}$- isoprene units.     

High-performance liquid chromatographic method for the determination of dolichols in tissues and plasma

High-performance liquid chromatographic methods for the determination of dolichols in tissues and plasma have been developed. The tissue concentration of dolichols was measured by high-performance liquid chromatography with uv detection and plasma levels of dolichols were determined fluorometrically after derivatization with anthracene-9-carboxylic acid. In both methods, 2,2-didecaprenylethanol was used as an internal standard. The method with fluorescence detection was sufficiently sensitive to measure the concentration of dolichols in human plasma.     

Synthesis of Dolichyl and Polyprenyl Sulfates

Dolichyl and polyprenyl sulfates were synthesized as analogues of dolichyl and polyprenyl phosphates by the interaction of dolichols and polyprenols with the pyridine–sulfuric anhydride complex.     

2D-LC/MS techniques for the identification of proteins in highly complex mixtures

Today, 2D online or offline liquid chromatography/mass spectrometry is state of the art for the identification of proteins from complex proteome samples in many laboratories. Both 2D liquid chromatography methods use two orthogonal liquid chromatography separation techniques. The most commonly used techniques are strong cation exchange chromatography for the first dimension and reversed phase separation for the second dimension. In order to improve sensitivity the reversed phase separation is usually performed in the nanoflow scale and mass spectrometry is used as the final detection method. The high-performance liquid chromatography techniques complement the 2D-gel techniques supporting their weaknesses. This is especially true for the gel separation of hydrophobic membrane proteins, which play an important role in living cells as well as being important targets for future pharmaceutical drugs.     

Metabolism of exogenous polyisoprenoids by animal cells cultured in vitro

The content and type of dolichols in chicken embryo fibroblasts was estimated and the presence of enzymic activities of various dolichyl phosphate sugar transferases was documented. Chicken embryo fibroblasts effectively take up various polyprenoids from the culture medium and catalyse both formation of polyprenyl fatty acid esters and their hydrolysis. With fully unsatured polyprenols the hydrogenation of the terminal alpha-isoprene residue was observed. In baby hamster kidney cells the formation of mannolipids was enhanced by exogenous polyprenols.     

2D-LC/MS techniques for the identification of proteins in highly complex mixtures

Today, 2D online or offline liquid chromatography/mass spectrometry is state of the art for the identification of proteins from complex proteome samples in many laboratories. Both 2D liquid chromatography methods use two orthogonal liquid chromatography separation techniques. The most commonly used techniques are strong cation exchange chromatography for the first dimension and reversed phase separation for the second dimension. In order to improve sensitivity the reversed phase separation is usually performed in the nanoflow scale and mass spectrometry is used as the final detection method. The high-performance liquid chromatography techniques complement the 2D-gel techniques supporting their weaknesses. This is especially true for the gel separation of hydrophobic membrane proteins, which play an important role in living cells as well as being important targets for future pharmaceutical drugs.     

The half-lives of dolichol and dolichyl phosphate in rat liver

Rat liver dolichol and dolichyl-P were labeled by injection of [³H]mevalonate into the portal vein and their rates of synthesis and breakdown determined. In the initial phase the radioactivity appeared in -unsaturated polyprenols. Subsequent saturation required 90 min. The half-lives of dolichols in microsomes were between 80 and 118 h, and shorter dolichols had shorter values of T_1/2. The half-lives of dolichols in lysosomes were between 115 and 137 h, while microsomal dolichyl-P exhibited a T_1/2 of 32 h. Injected dolichol was recovered in the lysomes of hepatocytes and exhibited a rate of breakdown which was slower than that of the endogenous compound. These results indicate differences in the catabolism of dolichol at different subcellular locations, as well as differences between the catabolism of dolichol and dolichyl-P.     

The characterization and stereochemistry of biosynthesis of dolichols in rat liver

It was shown that rat liver contains a series of dolichols with chain lengths of from 17 (or possibly 16) to 21 isoprene residues, the main constituent of the mixture being dolichol-18. By using double-labelled radioactive mevalonates it was demonstrated that each of these dolichols possesses three biogenetically trans-isoprene residues and that the remaining residues are biogenetically cis, suggesting that these polyprenols are biosynthesized from all-trans-farnesyl pyrophosphate by the cis additions of isoprene residues, followed by saturation of the alpha-isoprene residue. The results obtained with these radioactive mevalonates also indicated that the activity of isopentenylpyrophosphate isomerase is low relative to the activity of prenyltransferase in rat liver.