Cholesteryl 14-methylhexadecanoate Research Articles

Alterations of cholesteryl ester metabolism characteristic for cancer

Earlier as well as recent evidence obtained in our laboratory is reviewed indicating that specific changes in cholesteryl ester metabolism are related to tumor growth. Some properties of cholesteryl 14-methylhexadecanoate, an unknown lipid discovered and identified in our laboratory are described and its fundamental role as an essential cofactor in protein synthesis is emphasized. Increased quantities of this ester are present in tissues of tumor-bearing animals and the elevation of its level in serum is proportional to the tumor growth. Evidence has been presented that it may be a useful and reliable marker of malignant tumors in humans. This compound is synthesized in liver cells by enzymes that were recently purified to homogeneity from rat liver. The activity of these enzymes is significantly enhanced in tumor-bearing rats in a close proportion to the tumor growth. Activity of these preparations from normal rat liver may be greatly enhanced by the addition in vitro of serum from cancer patients. Since the enzymes are of lecithin-cholesterol acyl transferase type, apparently cancer serum contains some peculiar molecular species of these phospholipids. Because these lipids may be specific products of tumor cells, attempts are now in progress in this laboratory to identify them and the possibility is studied for their utilization as specific tumor markers.

Activity of cholesterol-esterifying enzymes from rat liver cytosol is affected by malignant growth

Three cholesterol-esterifying enzymes purified recently to apparent homogeneity from rat liver cytosol (Hradec, et al: J Chromatogr B 681: 55-62, 1996) showed in vitro an absolute requirement for natural mixtures of phosphatidylcholines. These phospholipids may be replaced by the addition of minute quantities of serum from cancer patients but not by that of healthy individuals. Individual enzymes showed different sensitivities in this respect. They utilized only phosphatidylcholines containing saturated fatty acids as substrates but not those containing unsaturated fatty acids. As revealed by high-performance liquid chromatography, products of the esterification were esters with saturated C12-C18 fatty acids (including odd-numbered) in comparable proportions. If cancer serum was added as the only source of substrate, enzymes synthesized predominantly cholesteryl 14-methylhexadecanoate. The enzymatic activity and nature of reaction products thus depended on the availability of particular phosphatidylcholines present in the cancer serum but not (or in lower quantities) in the serum of healthy individuals. These results may be of significance for further studies on metabolic changes accompanying the growth of malignant tumours.

Cholesteryl 14-methylhexadecanoate as a possible marker of malignant tumours

Concentration of cholesteryl 14-methylhexadecanoate was determined by high-performance liquid chromatography in serum of 123 healthy individuals, 83 of patients with other diseases than cancer, and of 394 patients with cancer. A mean level of 22.89 nmol/ml in healthy persons was not significantly different from 23.22 nmol/ml found in other diseases than cancer. With a cut-off value of 40.0 nmol/ml, there were 2 falsely positive results in the former and 4 in the later group. Significantly elevated mean concentrations (60-80 nmol/ml) were demonstrated in most patients with cancer irrespective of the clinical stage. No differences were found in this respect between males and females, and in different age groups. The sensitivity of the test was 81.8% for the whole population tested, if progressive and stable diseases as well as partial remissions were evaluated. Lower sensitivities were found in females and in patients more than 60 years old (down to 70.6%). The test showed a significantly greater sensitivity in less advanced stages of the disease (T1 and T2). Considering the type of cancer, highest sensitivity was observed for testicular cancer (86.2%), lower for prostatic (77.8%), mammary (76.5%), ovarian (75.8%), and colorectal tumors (73.3%) as well as for lymphomas (70%). Follow-up of patients with all these types of cancer revealed that changes in their clinical condition are accompanied with significant alterations in the cholesteryl 14-methylhexadecanoate level. When this ester was determined in 807 sera with a known level of other markers (irrespective of diagnosis), a significant correlation was demonstrated with carcinoembryonic antigen and nearly a significant one with prostatic specific antigen. There was no correlation with CA 15-3, CA 19-9, CA 125, or TPA. Because of its sound theoretical basis, absolutely specific method of determination, high specificity (97.1%), and predictive value (94.7%), the cholesteryl 14-methylhexadecanoate level determination may be useful as an alternate procedure supplementing the spectrum of tumor markers generally used.

Cholesteryl 14-methylhexadecanoate modulates protein synthesis and protein phosphorylation in Zajdela rat hepatoma

Cholesteryl 14-methylhexadecanoate modulates protein synthesis and protein phosphorylation in Zajdela rat hepatoma

The role of cholesteryl 14-methylhexadecanoate in the function of eukaryotic peptide elongation factor 1.

The binding of [3H]cholesteryl 14-methylhexadecanoate by a highly purified peptide elongation factor 1 from rabbit reticulocytes is significantly enhanced by GTP and CTP, much less by guanosine 5'-[beta, gamma-methylene]-triphosphate and not at all by ATP or UTP. Removal of endogenous cholesteryl 14-methylhexadecanoate present in the molecule of the factor [Hradec, J. et al. (1971) Biochem. J. 123, 959-966] by digestion with immobilized cholesterol esterase resulted in an almost complete loss of GTPase activity and this could be restored to nearly normal values by the addition of the ester. The same holds true for the GTP-dependent autophosphorylation of the protein-synthesis factor. Cholesteryl 14-methylhexadecanoate was bound only by the beta subunit of the factor. Addition of the alpha subunit, which was inactive on its own, stimulated the binding of the ester to the beta subunit in a sigmoid dependence. The binding of the ester was significantly stimulated by aminoacyl-tRNA but this effect was fully abolished by sodium fluoride, indicating a relation of cholesteryl 14-methylhexadecanoate to the dephosphorylation of the peptide elongation factor. Treatment of the factor with cholesterol esterase decreased its activity in the poly(U)-dependent binding of phenylalanyl-tRNA to ribosome and this activity was again restored by the addition of cholesteryl 14-methylhexadecanoate. The ester thus interacts with the GTP-dependent autophosphorylation of peptide elongation factor 1 and in this way modulates the activity of the factor. A putative scheme is presented explaining the mode of action of cholesteryl 14-methylhexadecanoate.

CTP can replace GTP in reactions catalyzed by eukaryotic peptide elongation factor 1.

In several reactions catalyzed by highly purified peptide elongation factor 1 from rabbit reticulocytes, GTP may be fully replaced by CTP but not by ATP or UTP. This holds true for the factor-dependent binding of aminoacyl-tRNA to ribosomes, GTPase activity, GTP-dependent autophosphorylation of the factor protein and binding of cholesteryl 14-methylhexadecanoate by the factor.

Cholesteryl esters are bound by a peptide-initiation and a peptide-elongation factor.

Significantly higher quantities of cholesteryl 14-methylhexadecanoate than of cholesteryl laurate and cholesteryl palmitate are bound by a homogeneous peptide-initiation factor and purified peptide-elongation factor 1. Cholesteryl 14-methylhexadecanoate may function as a specific allosteric modifier changing the conformation of protein synthesis factors and thus modulating the activity of their binding sites.

Decreased activity of peptide-elongation factors after treatment with cholesterol esterase

1. Peptide-elongation factors were purified from rat liver and treated with cholesterol esterase and phospholipase A2 immobilized on Sepharose 4B. 2. Binding of L-[3H]-phenylalanyl-tRNA to 40S ribosomal subunits was decreased by approx. 70% and to polyribosomes by 30% in the presence of the binding factor incubated with cholesterol esterase. Treatment of this factor with immobilized phospholipase A2 decreased the binding to smaller ribosomal subunits by only about 15%. 3. Poly(U)-dependent phenylalanine polymerization by ribosomal subunits was decreased to approx. 30% of its original value by treatment of both elongation factors with cholesterol esterase. 4. The normal activity of esterase-treated elongation factor in both the binding reaction and peptide-elongation assay was fully recovered by the addition of cholesteryl 14-methyl-hexadecanoate. 5. Different classes of lipids present in peptide-elongation factor 1 have apparently different functions. Whereas phospholipids are required to maintain the strcture of heavy aggregates of this factor, the presence of cholesteryl 14-methylhexadecanoate is obviously necessary for the normal function of peptide-elongation factors.

Quantitative determination of cholesteryl 14-methyl-hexadecanoate by radio-gas chromatography

A method was developed for the quantitative determination of radioactive cholesteryl 14-methylhexadecanoate at picomole levels. Intact cholesteryl esters are separated by gas-liquid chromatography on OV-1 silicone, one portion of the effluent being led to the flame ionization detector and the other portion combusted to 14CO 2. For qualitative analyses the radioactivity is assayed by continuous gas flow counting. For quantitative purposes the radioactive gas is trapped in ethanolamine, fractions are collected according to the mass record and the radioactivity is determined by liquid scintillation counting. The latter technique is able to detect 1–2 pmoles of radioactive cholesteryl esters with a reproducibility within ± 10% and is suitable for the quantitative determination of these compounds enzymatically synthesized in vitro.

Influence of cholesteryl 14-methylhexadecanoate on some ribosomal functions required for peptide elongation.

1. Polyribosomes and ribosomal subunits from rat liver were adsorbed on a cellulosic ion-exchange adsorbent, freeze-dried and extracted with organic solvents. The activity of extracted particles in peptide elongation was tested in the presence of purified peptideelongation factors. 2. Chloroform-methanol mixture (2:1, v/v) extracted 1.87+/-0.15 pmol of cholesteryl 14-methylhexadecanoate/pmol of the smaller ribosomal subunit and 0.92+/-0.11 pmol/pmol of the larger subunit. 3. In the presence of transferase I, extracted polyribosomes and 40S subunits bound more phenylalanyl-tRNA than did control non-extracted particles. The same binding as in control mixtures was obtained with extracted particles supplemented with cholesteryl 14-methylhexadecanoate in quantities corresponding to those extracted. 4. The polymerization of phenylalanine was greatly decreased with extracted polyribosomes and subunits and addition of the cholesteryl ester could not fully restore the original activity. 5. Extraction significantly decreased the activity of the P site of peptidyl transferase and normal activity was recovered after the addition of the ester. The A site of peptidyl transferase in extracted polyribosomes showed an increased activity when compared with non-extracted polyribosomes. 6. Cholesteryl 14-methylhexadecanoate apparently affects the function of the ribosomal A site and peptidyl transferase site and probably also that of the guanosine triphosphatase site and P site. The presence of different amounts of the ester in polyribosomes may be one of the mechanisms modulating peptide elongation at the ribosomal level.

Protein synthesis in the liver of rats injected with cholesteryl 14-methylhexadecanoate.

1. Rats were injected intraperitoneally with cholesteryl 14-methylhexadecanoate and killed after various intervals of time up to 3 days; ribosomes and cell sap were isolated from their liver tissue. These fractions were tested for their ability to participate in protein synthesis. 2. Protein synthesis in complete systems containing ribosomes, cell sap and all necessary cofactors was significantly enhanced at 12 and 72h after the injection and significantly inhibited at 24h. At early times after injection isolated ribosomes had a slightly enhanced ability to bind nRNA. Peptide-elongation processes (i.e. binding of aminoacyl-tRNA to ribosomes, peptidyl transfer and polyphenylalanine synthesis) showed significant stimulation or inhibition depending on the time after injection of the ester. 3. A correlation was found between the ability of cell sap to stimulate polyphenylalanine synthesis and the relative cholesteryl 14-methylhexadecanoate content in the postmicrosomal supernatant at different time-intervals after administration of the ester. No significant changes were found in its content in the whole liver tissue. 4. Since the injected ester has previously been shown to accumulate in some enzymic fractions, the changes in its relative content may represent a regulatory mechanism modulating the rate of protein synthesis.

Intermediate reactions in the binding of aminoacyl-transfer ribonucleic acid to rat liver ribosomes. the interaction of cholesteryl 14-methylhexadecanoate.

1. Transferase I from rat liver extracted with iso-octane binds significantly less aminoacyl-tRNA than the non-extracted enzyme. The original activity can be fully restored by the addition of cholesteryl 14-methylhexadecanoate. The binding capacity for GTP is not affected by the extraction. 2. In the presence of extracted transferase I the binding of aminoacyl-tRNA to ribosomes is decreased to 11-26% and the simultaneous binding of GTP to 32-43%. Cholesteryl 14-methylhexadecanoate induces a full reactivation of the extracted enzyme in both respects. 3. Extracted complexes A (aminoacyl-tRNA-GTP-transferase I) become bound to ribosomes to the same extent as the corresponding non-extracted preparations. 4. It is concluded that cholesteryl 14-methylhexadecanoate interacts with the binding site of transferase I for aminoacyl-tRNA and secondarily with that for GTP. It does not affect the binding site for ribosomes.

Effect of cholesteryl 14-methylhexadecanoate on the RNA polymerase activity of rat liver nuclei in vivo and in vitro

Effect of cholesteryl 14-methylhexadecanoate on the RNA polymerase activity of rat liver nuclei in vivo and in vitro

The role of cholesteryl 14-methylhexadecanoate in peptide elongation reactions.

1. Peptide-elongation factors were purified from rat liver and human tonsils and the contents of cholesteryl 14-methylhexadecanoate were determined in fractions obtained during enzyme purification. The relative contents of this compound in purified enzyme preparations was several times higher than that in the crude starting material. Elongation factors from human tonsils contained a significantly larger quantity of the cholesteryl ester than enzyme from rat liver. 2. Transfer enzymes extracted with various organic solvents showed variable decreased activities in both binding and peptidization assay. The decrease of enzymic activity was proportional to the amount of cholesteryl 14-methylhexadecanoate extracted from a given enzymic preparation. In systems containing both extracted elongation factors the polyphenylalanine synthesis was limited by the residual activity of the less active transfer factor. 3. The original enzymic activity of extracted transferases was fully recovered by the addition of pure cholesteryl 14-methylhexadecanoate in quantities corresponding to those extracted. 4. Increase of the relative contents of this cholesteryl ester during enzyme purification, decrease of the enzymic activity after the extraction and its recovery by the addition of this compound indicates that the presence of this ester in elongation factors is essential for the normal function of these enzymes.

Changes of aminoacyl-tRNA synthesis in the liver of rats administered cholesteryl 14-methylhexadecanoate

Changes of aminoacyl-tRNA synthesis in the liver of rats administered cholesteryl 14-methylhexadecanoate

Effect of cholesteryl 14-methylhexadecanoate on the activity of some amino acid-transfer ribonucleic acid ligases from mammalian tissues.

1. l-Tyrosine-, l-alanine-, l-tryptophan- and l-threonine-tRNA ligases (where tRNA is transfer RNA) were purified from mammalian tissues and the relative contents of cholesteryl 14-methylhexadecanoate were determined in fractions obtained during the isolation. Purified enzymes were extracted with various organic solvents. 2. Cholesteryl 14-methylhexadecanoate contents in purified ligases were up to 210-fold that in the starting material. Different enzymes showed different contents of this cholesteryl ester. 3. Extracted enzymes lost in most cases their ability to catalyse formation of the aminoacylhydroxamate and aminoacyl-tRNA complexes. Enzymes extracted with various solvents showed a variable decreased activity. 4. The original activity could be restored to 70-100% by the addition of cholesteryl 14-methylhexadecanoate. Cholesteryl palmitate, cholesteryl margarate and cholesteryl stearate were inactive in this respect. 5. Incubation mixtures of extracted enzymes with cholesteryl 14-methylhexadecanoate added showed an initial delay in the time-course of both reactions assayed. 6. It is concluded that the effect of cholesteryl 14-methylhexadecanoate on the activity of amino acid-tRNA ligases seems to be specific and that this compound may play some role in the function of these enzymes.

Effect of lipids, in particular cholesteryl 14-methylhexadecanoate, on the incorporation of labelled amino acids into transfer ribonucleic acid in vitro.

1. Rat liver pH5 enzymes and cell sap extracted with various organic solvents showed a variable decreased incorporation of labelled amino acids into s-RNA (;soluble' or transfer RNA) in vitro. 2. The original enzymic activity could be fully restored, though at different rates, by the addition of lipid extracts in quantities corresponding to those originally present. 3. Of the main lipid groups separated from the extract, only free cholesterol and cholesteryl esters were able to reactivate the extracted pH5 enzymes in the same way as the whole lipid extract. 4. Addition of pure cholesteryl 14-methylhexadecanoate also fully restored the enzymic activity. 5. There was no energy-dependent incorporation of labelled amino acids into ribosomal protein in the presence of extracted cell sap. Addition of cholesteryl 14-methylhexadecanoate fully restored the activity of the cell sap to incorporate labelled leucine and lysine into ribosomal protein and enhanced the incorporation of labelled protein hydrolysate and phenylalanine over the level found with the corresponding non-extracted preparations. 6. It is concluded that lipids play an important role in the synthesis of aminoacyl-s-RNA complexes and that cholesteryl 14-methylhexadecanoate may be the active lipid in this respect.