The levels of cholesterol, ubiquinone and dolichol and the polyprenol composition of dolichol in human hepatocellular carcinomas (hepatomas) with different degrees of differentiation were analyzed and compared with healthy liver tissue. Dolichols were also analyzed in liver metastases. The total level of cholesterol was increased, while the levels of dolichol and ubiquinone were decreased in all hepatomas, but no correlation between these levels and the degree of differentiation of the hepatomas could be observed. The level of dolichol decreased more in the hepatomas than in the liver metastases. The dolichol fraction from hepatomas with a low degree of differentiation contained higher relative amounts of short polyisoprenols (D17) and slightly lower relative amounts of D21 compared with healthy liver tissue, metastatic liver tumors or hepatomas with a high degree of differentiation. The significance of the lipid values found in the different groups is discussed.

Surgical samples of human hepatic tissue were analysed morphologically and biochemically and highly differentiated hepatomas were compared with two control groups: morphologically normal liver tissue surrounding the tumour, and tissue from normal livers. In tumour homogenates cholesterol levels were more than twice, ubiquinone levels about half and the concentration of free dolichol about 10% of the control value. The levels of dolichyl phosphate were basically similar, whereas the phospholipid level was slightly lower in the tumours. In microsomes isolated from hepatomas, the level of cholesterol was about 30% higher than the control value. HMG-CoA reductase activity in microsomes isolated from hepatomas was elevated almost 100% in comparison to control. In hepatomas, no major alterations in the compositions of dolichol or dolichyl phosphate could be observed. The relative amounts of alpha-saturated and alpha-unsaturated polyprenols were also basically unaltered in hepatomas. Liver samples were incubated with 3H-mevalonic acid and radioactivity was monitored in polyprenols. With control tissue, incorporation was considerably higher in alpha-unsaturated polyprenols than in their alpha-saturated counterparts. In the tumours the rates of incorporation into both polyprenol fractions were much lower, although still higher in the alpha-unsaturated fraction. Labelling of polyisoprenols containing 19 isoprene residues was higher than that of 20 residues. The pattern of labelling in the polyisoprenyl-P fraction was similar. In hepatomas the incorporation into cholesterol and ubiquinone-10 was about 100% higher and 50% lower respectively compared with control tissue. The results in this study of hepatomas indicate that the levels of various lipids may be influenced not only by the regulatory enzyme HMG-CoA reductase, but also by other enzymes catalysing reactions subsequent to this regulatory point. It is also suggested that levels of cholesterol, ubiquinone and dolichol may be regulated independently subsequent to the branch point at farnesylpyrophosphate.

A possible role of cell surface glycoconjugates in cell recognition has been envisioned based on recognition of carbohydrates by cell surface proteins such as endogenous lectins, glycosyltransferases, and hydrolases (refs. 18–22 in text). A new possibility that a specific carbohydrate at the cell surface could be recognized by the same or similar carbohydrate on the counterpart cell surface is now suggested by specific interaction between Lex and Lex, but not between Lex and sialylated or non-substituted type 2 chain. A new hypothesis is hereby proposed for carbohydrate-carbohydrate interactions as recognition signals during embryogenesis and organogenesis.

The Lex determinant (Gal beta 1----4[Fuc alpha 1----3]GlcNAc-beta 1----R) has been implicated as having a role in mediating compaction of the mouse embryo at the morula stage (Fenderson, B., Zehavi, U., and Hakomori, S. (1984) J. Exp. Med. 160, 1591-1596). Here, we present evidence suggesting a role for Lex in F9 embryonal carcinoma cell adhesion and a mechanism for Lex recognition based on carbohydrate-carbohydrate interaction. Homotypic aggregation of F9 cells was inhibited by lacto-N-fucopentaose III, and F9 cells showed a preferential interaction with Lex liposomes. The following observations suggest that the structure capable of recognizing Lex per se on F9 cells is Lex: (i) Cell surface-labeled components solubilized in octylglucoside, affinity-bound on an Lex-octyl-Sepharose column, contained glycoproteins reactive with anti-Lex antibody. (ii) Liposomes containing Lex showed significant interaction with Lex glycolipid, but not other glycolipids, coated on a plastic surface. (iii) Liposomes containing Lex glycolipid were found to self-aggregate, whereas liposomes containing paragloboside (nLc4) or sialylparagloboside (IV3NeuAcnLc4) did not. (iv) The diffusibility of 3H-labeled lacto-N-fucopentaitol III (but not I or II), incubated with Lex liposome, from the lower to the upper Boyden chamber through a semipermeable membrane was inhibited. In all these experiments (i-iv), the interaction of Lex to Lex (or Lex to lacto-N-fucopentaose III) was clearly observed only in the presence of Ca2+ and Mg2+ and was enhanced by the presence of Mn2+. These interactions were inhibited by EDTA. The results suggest the novel hypothesis that carbohydrate-carbohydrate interactions may play an important role in controlling cell recognition during F9 cell aggregation and during embryonic development.

Conditions for the isolation and quantitation of dolichyl phosphate, dolichol, cholesterol, and ubiquinone by reversed phase high performance liquid chromatography were investigated. A simple and fast sample preparation procedure using prepacked mini columns was employed. The UV spectra of the fractions obtained were examined and, in the case of dolichol compounds, the maximum absorbance around 205 nm was shown to be linearly dependent on the number of double bonds present in the isoprenolog. The analytical procedure described shows a very broad range of linearity (five orders of magnitude) and detects single dolichyl phosphate isoprenologs in amounts as small as 0.1 ng. The lowest overall recovery, that for dolichyl phosphate, is 77%. Use of isoprenolog 23 and ergosterol as internal standards reduced the variation in the method to 2.5, 4.0 and 5.5% for cholesterol, dolichyl phosphate and dolichol, respectively. The method described was employed to study the lipid composition of rat organs and biological variations in these compositions.

The lipid composition of human hepatocellular carcinomas was examined. The level of dolichol in the tumours was decreased compared to control tissue, whereas the concentration of dolichyl phosphate did not exhibit any major change. A decrease in the amount of dolichyl ester was also observed. The pattern of individual polyisoprenoids in the free dolichol pool was changed in several carcinomas with a relative increase in the shorter dolichols. The isoprenol composition in the dolichyl ester and phosphate fractions of tumours were basically similar to those of controls. alpha-Unsaturated polyisoprenols are present in control livers at a level of 3% of the total free polyisoprenoid fraction, while this value was increased in the tumours. Similar to dolichol, the amount of ubiquinone was also decreased. The content of cholesterol was increased, while the fatty acid pattern of the dolichyl esters showed minor alterations. These modifications in lipid content indicate different mechanisms for the regulation of dolichol and dolichyl phosphate concentrations. The high levels of sterols in contrast to the low polyisoprenol content suggests interference with the regulation of the mevalonate pathway, which is the common biosynthetic route for cholesterol, ubiquinone and dolichol.

The lipid compositions of homogenates and microsomal fractions derived from surgical samples of highly differentiated human hepatoma, morphologically normal regions outside the tumours and from normal livers were analysed. A few enzyme activities were also assayed. Hepatoma microsomes demonstrated considerably lowered levels of cytochromes P-450 and b5. Hepatoma homogenates exhibited increased levels of cholesterol, normal amounts of dolichyl-P and slightly lowered levels of total phospholipid. The levels of dolichol, dolichol ester and ubiquinone in hepatoma homogenates were prominently decreased. In tumour microsomes the levels of cholesterol and dolichyl phosphate were increased considerably while the levels of phospholipid and dolichol were lowered. The phospholipid composition of tumour homogenates was roughly similar to that of control tissue. In tumour microsomes the relative amounts of phosphatidylserine and phosphatidylinositol were about 30% decreased, whereas the major phospholipids showed minor increases in amount. The rate and pattern of incorporation of [3H]glycerol into individual phospholipids in liver slices from control and hepatoma tissue did not differ to any larger extent. The fatty acid composition of tumour homogenates exhibited minor differences in comparison to the control with the greatest changes in the sphingomyelin fraction. In hepatoma microsomes the fatty acid compositions of the major phospholipids were altered moderately, with evident decreases in the relative amounts of the long-chain polyunsaturated fatty acids. In hepatoma homogenates the fatty acid composition of dolichol esters differed only slightly from the control pattern. These results indicate that the major disturbance in the lipid metabolism of highly differentiated hepatomas is localized to the mevalonate pathway, thus affecting mainly the levels of cholesterol, dolichol and ubiquinone.

Homogenates and microsomal fractions prepared from biopsies of highly differentiated human hepatocellular carcinomas were found to contain low levels of dolichol in comparison with control tissue. In contrast, the amount of dolichyl phosphate in tumor homogenates was unchanged and actually increased in the microsomal fraction. The pattern of individual polyisoprenoids, both in the free and the phosphorylated dolichol fractions of hepatomas, did not exhibit any major alterations compared to the control. The rates of incorporation of [3H]mevalonic acid into dolichol and dolichyl phosphate in hepatomas were low. The dolichol monophosphatase activities in microsomal fractions from hepatomas and controls did not show any major differences, whereas the activity of the CTP-dependent dolichol kinase was increased in tumor microsomes. Glycosylation of endogenous dolichyl phosphate and of total protein using certain nucleotide-activated sugars was found to be slightly elevated in microsomal fractions from the tumor itself when compared to the control. The reasons for the differences in the levels of polyisoprenoids in hepatomas and control tissue are discussed.

Hyperplastic nodules and hepatocarcinomas were produced in rat liver by 2-acetylaminofluorene-containing diet. The homogenates and isolated microsomes were analyzed for the content of lipid intermediates and glycosylation reactions. The dolichol content of hyperplastic nodules increases four times in the homogenate and six times in the microsomes. In developed hepatocarcinoma, the amount of dolichol was doubled. Concerning the distribution pattern of the polyprenols, there is a change in the relative amounts of dolichols with 18 and 19 residues. In contrast to the free alcohol, dolichyl phosphate was greatly decreased in nodules, a finding which might be explained by a decreased dolichol kinase and an increased dolichol monophosphatase activity. The percentage of total phosphorylated dolichol was related to the glycosylating capacity. In microsomes, mitochondria, and homogenate from normal liver and in homogenate from hyperplastic liver nodules, the percentages of dolichyl phosphate were 23, 2, 16, and 4, respectively. At maximal glycosylation in vitro, only part of the total dolichyl phosphate was glycosylated. Dolichol-mediated protein glycosylation exhibited a general decrease in the microsomes from nodules and cancer tissue; it is suggested that the main cause of the decrease is a shortage of the available dolichyl phosphate which is rate limiting and which also contributes to the synthesis of the modified oligosaccharide chain.

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 Chromatograph v. 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 μg/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.

The distribution of mannosyl-, glucosaminyl- and glucosyltransferases in rough and smooth microsomes isolated from rat liver homogenate has been investigated. Amphomycin and tunicamycin were used as inhibitors of dolichol-mediated glycosylation, and diazobenzene sulfonate and proteolytic enzymes were used as nonpenetrating surface probes. Under in vitro conditions only 20–30% of the proteins glycosylated are of the secretory type. Nonpenetrating surface probes, which interact with components on the outer surface of rough microsomal vesicles, decrease glycosylation of both secretory and membrane proteins to a great extent. Inhibitor sensitive glycosylation is present in both the outer and inner compartments of the microsomal membranes. In contrast, the surface probes and the inhibitors of dolichol-mediated glycosylation do not significantly affect protein glycosylation in smooth microsomes. When dolichol phosphate sugars were used as substrates, instead of nucleotide sugars, the probes used inhibited protein glycosylation in both subfractions. Glycosylation of externally added Lipidex-bound dolichol monophosphate and of ovalbumin were in agreement with the above results. It appears that both rough and smooth microsomes may possess several types of glycosylating pathways. The most prominent of these in rough microsomes under the conditions used is the dolichol mono- and pyrophosphate-mediated glycosylation of endogenous proteins, where the enzymes involved in the initial steps are distributed at the outer surfaces of the microsomal vesicles. The dominating pathway in smooth microsomes appears to function in completion of the oligosaccharide chain of the protein and this process does not involve lipid intermediates and cannot be influenced by nonpenetrating surface probes.

The biosynthesis of dolichol and dolichylmonophosphate in rat liver was studied using [3H]mevalonate as precursor. The radioactive precursor was either injected into the portal vein of the rat or added to the incubation medium containing isolated hepatocytes, followed by the isolation of microsomes and mitochondria from the liver or the hepatocytes. In both systems dolichol in microsomes was highly labeled after a short labeling period followed by a rapid decrease. During this period the labeling of mitochondrial dolichol was low. The specific radioactivity of dolichyl-P in microsomes of both systems was higher in the initial phase than in dolichol and increased further with time. The mitochondrial labeling was also increased but was at a much lower level.

Conference Abstract| April 01 1981 DOLICHOL AND DOLICHOL-MEDIATED GLYCOSYLTRANSFERASES IN HEPATOCYTES I. Eggens; I. Eggens 1Dept. Biochem., Univ. Stockholm, Dept. Pathol., Karolinska Inst., and Dept. Biochem., Polish acad. Sci., Warszaw, Poland Search for other works by this author on: This Site PubMed Google Scholar T. Chojnacki; T. Chojnacki 1Dept. Biochem., Univ. Stockholm, Dept. Pathol., Karolinska Inst., and Dept. Biochem., Polish acad. Sci., Warszaw, Poland Search for other works by this author on: This Site PubMed Google Scholar O. Abrahamson; O. Abrahamson 1Dept. Biochem., Univ. Stockholm, Dept. Pathol., Karolinska Inst., and Dept. Biochem., Polish acad. Sci., Warszaw, Poland Search for other works by this author on: This Site PubMed Google Scholar G. Dallner G. Dallner 1Dept. Biochem., Univ. Stockholm, Dept. Pathol., Karolinska Inst., and Dept. Biochem., Polish acad. Sci., Warszaw, Poland Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1981) 9 (2): 148P. https://doi.org/10.1042/bst009148pb Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation I. Eggens, T. Chojnacki, O. Abrahamson, G. Dallner; DOLICHOL AND DOLICHOL-MEDIATED GLYCOSYLTRANSFERASES IN HEPATOCYTES. Biochem Soc Trans 1 April 1981; 9 (2): 148P. doi: https://doi.org/10.1042/bst009148pb Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1981 Biochemical Society1981 Article PDF first page preview Close Modal You do not currently have access to this content.