DOCSLIB.ORG

Inhibition of Sterol Biosynthesis by Bacitracin (Mevalonic Acid/CQ-Isopentenyl Pyrophosphate/C15-Farnesyl Pyrophosphate) K

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Inhibition of Sterol Biosynthesis by Bacitracin (Mevalonic Acid/CQ-Isopentenyl Pyrophosphate/C15-Farnesyl Pyrophosphate) K Proc. Nat. Acad. Sci. USA Vol. 69, No. 5, pp. 1287-1289, May 1972 Inhibition of Sterol Biosynthesis by Bacitracin (mevalonic acid/CQ-isopentenyl pyrophosphate/C15-farnesyl pyrophosphate) K. JOHN STONE* AND JACK L. STROMINGERt Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138 Contributed by Jack L. Strominger, March 14 1972 ABSTRACT Bacitracin is an inhibitor of the biosyn- muslin eliminated floating particles of fat from the enzyme thesis of squalene and sterols from mevalonic acid, C5- solution enzyme). In some the isopentenyl pyrophosphate, or C15-farnesyl pyrophosphate (Sio experiments, S10 enzyme catalyzed by preparations from rat liver. The antibiotic was centrifuged at 105,000 X g for 90 min to remove micro- is active at extremely low ratios of antibiotic to substrate. somal particles, and this supernatant is referred to as the Sc1t The mechanism of inhibition appears to be the formation enzyme. of a complex between bacitracin, divalent cation, and C15- Bacitracin was generously provided by the Upjohn Co., farnesyl pyrophosphate and other isoprenyl pyrophos- phates. It is similar to the formation of the complex with Kalamazoo, Mich. [2-14C]3RS-mevalonic acid was obtained C55-isopropenyl pyrophosphate in microbial systems. The from New England Nuclear Co., [SH]farnesyl pyrophosphate toxicity of bacitracin for animal cells could be due in part and [2-'4C]isopentenyl pyrophosphate were generous gifts to the formation of these complexes. from Dr. Konrad Bloch. The Enzyme incubations (about 0.5-ml samples) were termi- antibacterial properties of bacitracin result, in part, from nated by in a water bath for 1 an inhibition of the heating boiling min. They were enzyme, C05-isoprenyl pyrophosphate then extracted three times with an volume of phosphatase (EC 2.5.1.1), which is responsible for the re- equal diethyl generation of ether. The ether extracts were filtered through a bed of about C56-isoprenyl phosphate, an essential carrier in 1 ml of basic alumina (Woelm) and washed with a the biosynthesis of bacterial cell walls (1, 2). In a preceding through the further 5 ml of ether. The alumina filtration ensured that all of paper (3), mechanism of inhibition has been shown to be the substrates were removed from the ether extracts. the formation of a complex between bacitracin, a divalent The cation, and the ether eluate was evaporated to about 0.05 ml and diluted to C55-isoprenyl pyrophosphate. Thus, bacitracin 1 ml with toluene. A portion was then assayed for radioac- acts by complexing an essential substrate rather than by tivity. inactivating an enzyme. The proposed mechanism of complex formation suggested the possibility that other pyrophos- RESULTS phates, structurally related to C05-isoprenyl phosphate, might Inhibition by bacitracin of the synthesis of squalene and also complex with bacitracin and that the antibiotic would sterols from various precursors inhibit enzymatic reactions involving these compounds. The biosynthesis of squalene and sterols from mevalonic acid, The biosynthesis of squalene and sterols from mevalonic isopentenyl pyrophosphate, and dimethylallyl pyrophosphate acid involves a number of intermediates amongst which di- is well established (see for example ref. 5). In this complex methylallyl pyrophosphate (Cr-isoprenyl pyrophosphate), reaction sequence, squalene and later products are soluble in geranyl pyrophosphate (trans-Clo-isoprenyl pyrophosphate), diethyl ether while squalene precursors are water soluble and and farnesyl pyrophosphate (all-trans-C16-isoprenyl pyrophos- are strongly adsorbed by alumina. Utilization of these prop- phate) differ from bacterial C05-isoprenyl pyrophosphate only erties allows a simple evaluation of the squalene plus sterol in the length and stereochemistry of the isoprenoid chain. In synthesizing capacity of an enzyme mixture. the present paper, bacitracin is shown to be an effective The conversion of 3RS-mevalonic acid to squalene was inhibitor of this biosynthetic sequence in vitro. The mechanism found to be completely inhibited at a bacitracin concentration of inhibition appears to be identical with that of the bacterial of 1 mol per 2 mol of mevalonic acid added. As only the 3R- system, and formation of a complex between farnesyl pyro- mevalonic acid isomer is used as substrate (6), this is equiva- phosphate, bacitracin, and a divalent cation has been lent to a molar ratio of inhibitor to substrate of 1 to 1. The demonstrated. conversions of isopentenyl pyrophosphate to squalene and MATERIALS AND METHODS farnesyl pyrophosphate to squalene were also inhibited by Enzyme solutions were prepared as described by Popjak (4). bacitracin, although these were less sensitive (Fig. 1). The de- Minced livers from young male Sprague-Dawley rats (100- creased sensitivity of the system to bacitracin from mevalonic 120 g) were gently homogenized with a Potter-Elvehjem acid through isopentenyl pyrophosphate to farnesyl pyro- homogenizer in 2.5 volumes of buffer containing 0.1 M Tris- phosphate suggests that more than one of the intermediate re- HC1, 5 mM MgCl2, 2 mM MnCl2, and 30 mM nicotinamide at actions is sensitive to the antibiotic. pH 7.5. Centrifugation for 30 min at 15,000 X g removed Inhibition by bacitracin of the biosynthesis of nuclei, cell debris, and mitochondria, and filtration through isoprenoid pyrophosphate from mevalonic acid and isopentenyl pyrophosphate * Present address: Roche Research Laboratory, Department of An experiment that shows that other reactions in the bio- Biochemistry, Liverpool University, Liverpool, England. synthetic sequence are also inhibited by bacitracin is illus- t To whom reprint requests should be sent. trated in Fig. 2. In this experiment, the absence of micro- 1287 Downloaded by guest on September 26, 2021 1288 Biochemistry: Stone and Strominger Proc. Nat. Acad. Sci. USA 69 (iI97e) somal particles ddes not allow the formation of squalene, and the immediate precursor, farnesyl pyrophosphate, is the major product (7, 8). Hydrolysis of isoprenyl pyrophosphates with alkaline phosphatase liberates free isoprenol, which is extractable by ether and eluted from alumina. The radioac- tivity eluted from the alumina, thus, gives a measure of the ~40 amount of geranyl pyrophosphate and farnqsyl pyrophosphate formed. The observation that, at high bacitracin concentra- K ~~~~~~LipidReleased by Phosphotose tions and with isopentenyl pyrophosphate as substrate, Jittle radioactivity is eluted from the alumina column shows that little or no isopentetiol is isolated by the above procedure. The 20- small amounts of radioactivity that can be extracted from these incubations before treatment with alkaline phosphatase Squalene + Sterois is undoubtedly due to contamination of the enzyme with small 02 0.4 06 Q8 amounts of residual microsomal fragments. Nevertheless, nmol Bacitracin/nmol Substrate this "background" radioactivity is small compared with that eluted after phosphatase treatment, and a strong inhibitory FIG. 2. Percent products formed from mevalonic acid or isopentenyl pyrophosphate in the presence of Sio5 enzyme and effect of bacitracin on the biosynthesis of isoprenyl pyro- various concentrations of bacitracin. Incubation mixtures con- phosphate is apparent. tained 2.5 mg ATP, bacitracin at the appropriate concentration, Inhibition by bacitracin of the dephosphorylation of 0.5 ml of Soi5 enzyme, and either 320 nmol of [2-14C]mevalonic farnesyl pyrophosphate by Escherichia coli acid, or 74 nmol of [14C]isopentenyl pyrophosphate, each with the phosphomonoesterase specific activities given in Fig. 1. The final incubation volume was 0.6 ml. Incubations were terminated by boiling for 1 min and It has been shown that E. coli phosphomonoesterase catalyzes analyzed as in Methods. The remaining aqueous extract was the dephosphorylation of C06-isoprenyl pyrophosphate and warmed at 60° for 30 min to remove all traces of ether, the pH that this dephosphorylation is inhibited by bacitracin (2). In was adjusted to 8.2 with 0.5 M Tris buffer, and the final mixture the present experiment, farnesyl pyrophosphate has been was hydrolyzed at 370 for 60 min with 7.5 Units of Escherichia used as a substrate for the E. coli phosphomonoesterase. Its coli alkaline phosphatase. This second enzyme reaction was dephosphorylation was similarly inhibited by bacitracin, but terminated and analyzed for radioactive products as in Methods. the hydrolysis of p-nitrophenyl phosphate catalyzed by the A-A, percent phosphatase-labile products from isopentenyl E. coli enzyme was not significantly altered by the antibiotic pyrophosphate; O-O, percent phosphatase-labile products (Fig. 3). This result suggests that the bacitracin interacts from mevalonic acid; A-A, percent squalene and sterol from with the substrate rather than with the enzyme. isopentenyl pyrophosphate; *-*, percent squalene and sterol from mevalonic acid. Direct demonstration of formation of a complex between bacitracin and farnesyl pyrophosphate By the method of Hummel and.Dryer (9), ['H farnesyl pyrophosphate was filtered through a column of Sephadex G-25 in buffer containing Mg++. The addition of bacitracin during the period when the elution of 'adioactivity was con- stant resulted in a peak of radioactivity followed by a trough (Fig. 4). This pattern is indicative of the formation of a complex and is the mirror image of the effect observed with ["2PlC,,- isoprenyl pyrophosphate (3). For farnesyl pyrophosphate, which can be kept in aqueous solution without the addition of a detergent,
Load more

Recommended publications
  • 33 34 35 Lipid Synthesis Laptop
    BI/CH 422/622 Liver cytosol ANABOLISM OUTLINE: Photosynthesis Carbohydrate Biosynthesis in Animals Biosynthesis of Fatty Acids and Lipids Fatty Acids Triacylglycerides contrasts Membrane lipids location & transport Glycerophospholipids Synthesis Sphingolipids acetyl-CoA carboxylase Isoprene lipids: fatty acid synthase Ketone Bodies ACP priming 4 steps Cholesterol Control of fatty acid metabolism isoprene synth. ACC Joining Reciprocal control of b-ox Cholesterol Synth. Diversification of fatty acids Fates Eicosanoids Cholesterol esters Bile acids Prostaglandins,Thromboxanes, Steroid Hormones and Leukotrienes Metabolism & transport Control ANABOLISM II: Biosynthesis of Fatty Acids & Lipids Lipid Fat Biosynthesis Catabolism Fatty Acid Fatty Acid Synthesis Degradation Ketone body Utilization Isoprene Biosynthesis 1 Cholesterol and Steroid Biosynthesis mevalonate kinase Mevalonate to Activated Isoprenes • Two phosphates are transferred stepwise from ATP to mevalonate. • A third phosphate from ATP is added at the hydroxyl, followed by decarboxylation and elimination catalyzed by pyrophospho- mevalonate decarboxylase creates a pyrophosphorylated 5-C product: D3-isopentyl pyrophosphate (IPP) (isoprene). • Isomerization to a second isoprene dimethylallylpyrophosphate (DMAPP) gives two activated isoprene IPP compounds that act as precursors for D3-isopentyl pyrophosphate Isopentyl-D-pyrophosphate all of the other lipids in this class isomerase DMAPP Cholesterol and Steroid Biosynthesis mevalonate kinase Mevalonate to Activated Isoprenes • Two phosphates
    [Show full text]
  • WO 2009/005519 Al
    (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (43) International Publication Date PCT (10) International Publication Number 8 January 2009 (08.01.2009) WO 2009/005519 Al (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every A61K 31/19 (2006.01) A61K 31/185 (2006.01) kind of national protection available): AE, AG, AL, AM, A61K 31/20 (2006.01) A61K 31/225 (2006.01) AT,AU, AZ, BA, BB, BG, BH, BR, BW, BY,BZ, CA, CH, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG, (21) International Application Number: ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, PCT/US2007/072499 IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY,MA, MD, ME, MG, MK, MN, MW, (22) International Filing Date: 29 June 2007 (29.06.2007) MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PG, PH, PL, PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, SV, SY, (25) Filing Language: English TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW (26) Publication Language: English (71) Applicant (for all designated States except US): AC- (84) Designated States (unless otherwise indicated, for every CERA, INC. [US/US]; 380 Interlocken Crescent, Suite kind of regional protection available): ARIPO (BW, GH, 780, Broomfield, CO 80021 (US). GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), (72) Inventor; and European (AT,BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, (75) Inventor/Applicant (for US only): HENDERSON, FR, GB, GR, HU, IE, IS, IT, LT,LU, LV,MC, MT, NL, PL, Samuel, T.
    [Show full text]
  • Squalene, Olive Oil, and Cancer Risk: a Review and Hypothesis
    Vol. 6, 1101-1103, December 1997 Cancer Epidemiology,Blornarkers & Prevention 1101 Review Squalene, Olive Oil, and Cancer Risk: A Review and Hypothesis Harold L. Newmark’ of unsaturated fatty acids. In Italy, about 80% of edible oil is Strang Cancer Research Laboratory, The Rockefeller University, New York, olive oil,2 suggesting a protective effect of olive oil intake. In New York 10021, and Laboratory for Cancer Research, College of Pharmacy, another Italian case-control study of diet and pancreatic cancer Rutgers University, Piscataway, New Jersey 08855-0789 (8), increased frequency of edible oil consumption was associ- ated with a trend toward decreased risk. Edible oil in Italy consists of about 80% olive oil, thus suggesting a protective Abstract effect of olive oil in pancreatic cancer.2 Epidemioiogical studies of breast and pancreatic cancer Animal studies on fat and cancer have generally shown in several Mediterranean populations have demonstrated that olive oil either has no effect or a protective effect on the that increased dietary intake of olive oil is associated with prevention of a variety of chemically induced tumors. Olive oil a small decreased risk or no increased risk of cancer, did not increase tumor incidence or growth, in contrast to corn despite a higjier proportion of overall lipid intake. and sunflower oil, in some mammary cancer models (9 -1 1) and Experimental animal model studies of high dietary fat also in colon cancer models (12, 13), although in an earlier and cancer also indicate that olive oil has either no effect report, olive oil exhibited mammary tumor-promoting proper- or a protective effect on the prevention of a variety of ties similar to that ofcorn oil (14), in contrast to the later studies chemically induced tumors.
    [Show full text]
  • A Key Mammalian Cholesterol Synthesis Enzyme, Squalene Monooxygenase, Is Allosterically Stabilized by Its Substrate
    A key mammalian cholesterol synthesis enzyme, squalene monooxygenase, is allosterically stabilized by its substrate Hiromasa Yoshiokaa, Hudson W. Coatesb, Ngee Kiat Chuab, Yuichi Hashimotoa, Andrew J. Brownb,1, and Kenji Ohganea,c,1 aInstitute for Quantitative Biosciences, The University of Tokyo, 113-0032 Tokyo, Japan; bSchool of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia; and cSynthetic Organic Chemistry Laboratory, RIKEN, 351-0198 Saitama, Japan Edited by Brenda A. Schulman, Max Planck Institute of Biochemistry, Martinsried, Germany, and approved February 18, 2020 (received for review September 20, 2019) Cholesterol biosynthesis is a high-cost process and, therefore, is the cognate E3 ligase for SM (9, 10), catalyzing its ubiquitination tightly regulated by both transcriptional and posttranslational in the N100 region (11). negative feedback mechanisms in response to the level of cellular Despite its importance in cholesterol-accelerated degradation, cholesterol. Squalene monooxygenase (SM, also known as squalene the structure and function of the SM-N100 region has not been epoxidase or SQLE) is a rate-limiting enzyme in the cholesterol fully resolved. Thus far, biochemical analyses have revealed the biosynthetic pathway and catalyzes epoxidation of squalene. The presence of a reentrant loop anchoring SM to the ER membrane stability of SM is negatively regulated by cholesterol via its and an amphipathic helix required for cholesterol-accelerated N-terminal regulatory domain (SM-N100). In this study, using a degradation (7, 8), while X-ray crystallography has revealed the SM-luciferase fusion reporter cell line, we performed a chemical architecture of the catalytic domain of SM (12). However, the genetics screen that identified inhibitors of SM itself as up-regulators highly hydrophobic and disordered nature of the N100 region of SM.
    [Show full text]
  • Livers of Sharks, Especially Deepwater Shark Species Which Are Especially Vulnerable Theto Overfishing and Often Endangered
    AN EXCLUSIVE STUDY BY BLOOM March 2015 Shark in our beauty BEAST creams and the Beauty Shark in our beauty BEAST creams Some cosmetic brands still use a substance derived from the liver of sharks in the blends of moisturizing creams. In 2012,and BLOOM carried out a worldwide study on the use of shark squalane, a substance extracted from the livers of sharks, especially deepwater shark species which are especially vulnerable theto overfishing and often endangered. The study revealed that the cosmetics sector was the main consumer of animal squalane, even though plant substitutes derived from sugar cane or olive were available. The report pointed to the urgency for corporations to take their environmental responsibility seriously and to modify their supply chain in order to exclusively use plant squalane. Two years later, in 2014, BLOOM proceeded to carry out the largest test ever made on commercial creams to identify the presence of shark squalane in them. In total, BLOOM tested 72 moisturizing creams whose list of ingredients mentioned “squalane”. Labels did not specify whether the squalane was animal-based (shark) or plant-based (olive or sugarcane). Ten creams out of 72 did not contain a sufficient volume of squalane altogether to identify its origin, but results were robust for 62 of the products tested. As a conclusion, one moisturizing cream out of five contains shark squalane. It appearsBeauty that a high proportion of Asian brands still use shark squalane while Western brands, although still massively concerned by this issue in 2012, have globally shifted away from using shark substances.
    [Show full text]
  • Steroidal Triterpenes of Cholesterol Synthesis
    Molecules 2013, 18, 4002-4017; doi:10.3390/molecules18044002 OPEN ACCESS molecules ISSN 1420-3049 www.mdpi.com/journal/molecules Review Steroidal Triterpenes of Cholesterol Synthesis Jure Ačimovič and Damjana Rozman * Centre for Functional Genomics and Bio-Chips, Faculty of Medicine, Institute of Biochemistry, University of Ljubljana, Zaloška 4, Ljubljana SI-1000, Slovenia; E-Mail: [email protected] * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +386-1-543-7591; Fax: +386-1-543-7588. Received: 18 February 2013; in revised form: 19 March 2013 / Accepted: 27 March 2013 / Published: 4 April 2013 Abstract: Cholesterol synthesis is a ubiquitous and housekeeping metabolic pathway that leads to cholesterol, an essential structural component of mammalian cell membranes, required for proper membrane permeability and fluidity. The last part of the pathway involves steroidal triterpenes with cholestane ring structures. It starts by conversion of acyclic squalene into lanosterol, the first sterol intermediate of the pathway, followed by production of 20 structurally very similar steroidal triterpene molecules in over 11 complex enzyme reactions. Due to the structural similarities of sterol intermediates and the broad substrate specificity of the enzymes involved (especially sterol-Δ24-reductase; DHCR24) the exact sequence of the reactions between lanosterol and cholesterol remains undefined. This article reviews all hitherto known structures of post-squalene steroidal triterpenes of cholesterol synthesis, their biological roles and the enzymes responsible for their synthesis. Furthermore, it summarises kinetic parameters of enzymes (Vmax and Km) and sterol intermediate concentrations from various tissues. Due to the complexity of the post-squalene cholesterol synthesis pathway, future studies will require a comprehensive meta-analysis of the pathway to elucidate the exact reaction sequence in different tissues, physiological or disease conditions.
    [Show full text]
  • Oligodendroglial Energy Metabolism and (Re)Myelination
    life Review Oligodendroglial Energy Metabolism and (re)Myelination Vanja Tepavˇcevi´c Achucarro Basque Center for Neuroscience, University of the Basque Country, Parque Cientifico de la UPV/EHU, Barrio Sarriena s/n, Edificio Sede, Planta 3, 48940 Leioa, Spain; [email protected] Abstract: Central nervous system (CNS) myelin has a crucial role in accelerating the propagation of action potentials and providing trophic support to the axons. Defective myelination and lack of myelin regeneration following demyelination can both lead to axonal pathology and neurode- generation. Energy deficit has been evoked as an important contributor to various CNS disorders, including multiple sclerosis (MS). Thus, dysregulation of energy homeostasis in oligodendroglia may be an important contributor to myelin dysfunction and lack of repair observed in the disease. This article will focus on energy metabolism pathways in oligodendroglial cells and highlight differences dependent on the maturation stage of the cell. In addition, it will emphasize that the use of alternative energy sources by oligodendroglia may be required to save glucose for functions that cannot be fulfilled by other metabolites, thus ensuring sufficient energy input for both myelin synthesis and trophic support to the axons. Finally, it will point out that neuropathological findings in a subtype of MS lesions likely reflect defective oligodendroglial energy homeostasis in the disease. Keywords: energy metabolism; oligodendrocyte; oligodendrocyte progenitor cell; myelin; remyeli- nation; multiple sclerosis; glucose; ketone bodies; lactate; N-acetyl aspartate Citation: Tepavˇcevi´c,V. Oligodendroglial Energy Metabolism 1. Introduction and (re)Myelination. Life 2021, 11, Myelination is the key evolutionary event in the development of higher vertebrates. 238.
    [Show full text]
  • Squalene – Antioxidant of the Future?
    This is a summary of an article which appeared in The South African Journal of Natural Medicine 2007, 33, p106-112. Squalene – antioxidant of the future? HEIDI DU PREEZ is a Sourced primarily from the liver of the deep-sea professional natural scientist, shark, squalene has been referred to as the with a Masters degree in 1 ‘antioxidant of the future’. Heidi du Preez food science. She is currently discusses this unique molecule. studying for a degree in nutritional medicine. Heidi qualene is an isoprenoid hydrocarbon with six isoprene units. It is consults for both the food produced in our bodies and also found in nature. Many and health industries. She S antioxidants are either isoprenoids or have an isoprenoid tail. Vitamin E, vitamin A, beta-carotene and flavonoids are all uses a holistic, naturopathic isoprenoids. Isoprenoids are found abundantly in nature, but biologists approach, incorporating are mainly interested in studying the few, including squalene and diet, supplementation, lycopene, that have extraordinary antioxidant properties. detoxification and spiritual wellbeing in her treatment Since squalene is a pure isoprenoid, containing only isoprene units, it has an effective and stable antioxidant configuration. Squalene is regimen. Her focus is on the considered by many to be the most powerful and stable of the prevention and cure of isoprenoids.2 chronic, metabolic and degenerative diseases. Heidi In its purified form squalene is a colourless, almost tasteless, serves on the council of the transparent liquid without a significant odour. It is the major hydrocarbon in fish oils. Since squalene is a polyunsaturated ‘lipid’, South African Association for derived from fish oil, it should not be confused with being an essential Nutritional Therapy, and is fatty acid.
    [Show full text]
  • Simultaneous Determination of Pigments, Tocopherols, and Squalene in Greek Olive Oils: a Study of the Influence of Cultivation and Oil-Production Parameters
    foods Article Simultaneous Determination of Pigments, Tocopherols, and Squalene in Greek Olive Oils: A Study of the Influence of Cultivation and Oil-Production Parameters Ioannis Martakos, Marios Kostakis, Marilena Dasenaki * , Michalis Pentogennis and Nikolaos Thomaidis Laboratory of Analytical Chemistry, Department of Chemistry, National and Kapodistrian, University of Athens, Panepistimiopolis Zographou, Athens 15771, Greece; [email protected] (I.M.); [email protected] (M.K.); [email protected] (M.P.); [email protected] (N.T.) * Correspondence: [email protected]; Tel.: +30-210-7274430; Fax: +30-210-727475 Received: 4 November 2019; Accepted: 17 December 2019; Published: 29 December 2019 Abstract: A new facile and fast method was developed in this study for the determination of pigments (chlorophylls and carotenoids), tocopherols (α-, sum of (β + γ), and δ), and squalene in olive oil. This method consisted of a dilution of olive oil in 2-propanol, followed by reversed phase-high-pressure liquid chromatography equipped with a diode array detector (RP-HPLC-DAD). Chromatographic separation was performed using a C18 column, while the mobile phase consisted of acetonitrile and methanol using a gradient elution program. The methodology was optimized, validated, and applied to the analysis of 452 samples of Extra Virgin Olive Oil (EVOOs) and Virgin Olive Oil (VOOs) originated from five islands of the Northeastern Aegean Region, in Greece. From the obtained results, it was indicated that the carotenoid, tocopherol, and squalene content was relatively high, while the chlorophyll content was low. Furthermore, the acquired results were studied and compared in order to obtain useful information about the correlation of the concentration levels of these compounds in olive oil to different cultivation and olive oil production parameters.
    [Show full text]
  • Chromatographic Analysis and Viosynthesis of Peppermint Oil
    CHROMATOGRAPHIC ANALYSIS AND BIOSYNTHESIS OF PEPPERlflWI' OIL 'lEBl'ENES by ROBERT LEWIS Dtm'NDJG ' A THESIS submitted to OREGON STATE COLLEGE in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE June 1956 *PIHTIDT Redacted for Privacy : ii.. Intrtru* lr'*'s of Drfttd of Slrdltqr e Stfitp of Drqc Redacted for Privacy 6bIEn of Drr# tf ghdrtr;f Redacted for Privacy 6htil& of 8cheo:l' {trt*En Ccltt . Redacted for Privacy DrE oE erktr'8*boo1' Art th.ilIt tr lur:rtrted ,{r, th IPS ., *?e.{ tB drffrtr {rxtrtn I wish to thank Dr. w. D. Loomis for his help and understanding during the progress of these investi-­ gations. I al.ao 1Fish to thank Dr. w. H. Slabaugh and Dr. c. 1!. Wa.ng tor their help on vartoua aapeets ot this work. TABLE OF CONTENTS Page INTRODUCTION • • • • • ~ . ~ .. • • • • • • • • • • • 1 CHROMATOGRAPHY OF PEPPERMINT OIL • • • • • . •· • • • • 8 Introduction • • • • • • • • • • • • • • • • • • 8 Experimental • • • • . • • • • • • • • • • • • • • 10 Preparation and Use of Chromatograms • • • • 11 Detection or Colorless Compounds • • • • • • 13 Experimental Results • • • • • • • • • • • • • • 14 Dependence of Rf on the Composition of the Solvent • • • • • • • • • • • • • • 16 Dependence of Rr on Temperature • • • • • • 17 Discussion • • • • • • • • • • • • • • • • • • • 19 Theoretical Treatment • • • • • • • • • • • 20 METABOLISM OF l-cl4-ACETATE BY EXCISED PEPPERMIIT LEAVES • • • • • • • • • • • • • • • • • 29 Tracer Study I • • • • • • • • • • • • • • • • • 29 Time study • • • • • • • • • • • • • •
    [Show full text]
  • Squalene: a Multi-Task Link in the Crossroads of Cancer and Aging
    Functional Foods in Health and Disease 2013; 3(12):462-476 Page 462 of 476 Review Open Access Squalene: a multi-task link in the crossroads of cancer and aging Alvaro L. Ronco1,2 and Eduardo De Stéfani3 1Department of Epidemiology and Scientific Methods, IUCLAEH School of Medicine, Prado and Salt Lake p.16, Maldonado 20100, Uruguay; 2Unit of Oncology and Radiotherapy, Pereira Rossell Women’s Hospital, Bvar. Artigas 1550, Montevideo 11300, Uruguay; 3Epidemiology Group, Department of Pathology, Clinicas Hospital, Av. Italia s/n, Montevideo 11300, Uruguay Correspondent author: Alvaro Ronco, MD, 1Department of Epidemiology and Scientific Methods, IUCLAEH School of Medicine, Prado and Salt Lake p.16, Maldonado 20100, Uruguay Submission date: September 5, 2013; Acceptance date: December 14, 2013; Publication date: December 20, 2013 ABSTRACT Since its discovery in the beginning of the XXth century, squalene has been recognized as an important link in metabolic pathways. More recently, it has been further recognized as an intermediate step in the biosynthesis of cholesterol. Its well known antioxidant capability, together with its ability to protect skin, improve the immune system, and modulate the lipid profile, confer a high potential to this natural substance, which is spread all across the body structure, though mainly in the epithelial tissues, and in particular the skin sebum. This review will focus mainly on its major properties, which are related to anticancer properties, the maintenance of the oxidation/antioxidation balance, and its antiaging capabilities. Although the substance was originally obtained from shark liver oil, it is currently possible to obtain useful amounts from vegetable sources like extra virgin olive oil, therefore avoiding the dependence on capturing the aforementioned animal species.
    [Show full text]
  • Improved Method of Administering Beta-Hydroxy
    (19) TZZ _ ¥_T (11) EP 2 512 236 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: A01N 37/00 (2006.01) A61K 31/19 (2006.01) 19.10.2016 Bulletin 2016/42 (86) International application number: (21) Application number: 10838355.5 PCT/US2010/061367 (22) Date of filing: 20.12.2010 (87) International publication number: WO 2011/075741 (23.06.2011 Gazette 2011/25) (54) IMPROVED METHOD OF ADMINISTERING BETA-HYDROXY-BETA-METHYLBUTYRATE (HMB) VERBESSERTES VERFAHREN ZUR VERABREICHUNG VON BETA-HYDROXY-BETA-METHYLBUTYRAT (HMB) PROCÉDÉ AMÉLIORÉ PERMETTANT D’ADMINISTRER DU BÊTA-HYDROXY-BÊTA-MÉTHYLBUTYRATE (HMB) (84) Designated Contracting States: • NISSEN, Steve AL AT BE BG CH CY CZ DE DK EE ES FI FR GB Ames GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO Iowa 50014 (US) PL PT RO RS SE SI SK SM TR • ABUMRAD, Naji Nashville (30) Priority: 18.12.2009 US 287857 P Tennessee 37220 (US) (43) Date of publication of application: (74) Representative: Evans, Jacqueline Gail Victoria 24.10.2012 Bulletin 2012/43 Marches Intellectual Property Limited Wyastone Business Park (73) Proprietor: Metabolic Technologies, Inc. Wyastone Leys Ames, IA 50010 (US) Ganarew Monmouth NP25 3SR (GB) (72) Inventors: • RATHMACHER, John (56) References cited: Story City US-A- 4 992 470 US-A- 5 028 440 Iowa 50248 (US) US-A- 5 087 472 US-A- 6 031 000 • FULLER, John US-A1- 2004 220 266 US-A1- 2005 215 640 Zearing US-A1- 2005 215 640 Iowa 50278 (US) • BAIER, Shawn Remarks: Polk City Thefile contains technical information submitted after Iowa 50226 (US) the application was filed and not included in this specification Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations.
    [Show full text]