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Triglyceride Hydrolysis by Corynebacterium Acnes in Vitro

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Triglyceride Hydrolysis by Corynebacterium Acnes in Vitro CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector THE JOURNAL OF INVESTIGATiVE DERMATOLOGY Vol. 52, Nu. 3 Copyright 1969 by The Williams & Wilkins Co. Printed in U.S.A. TRIGLYCERIDE HYDROLYSIS BY GORYNEBACTERIUM ACNES IN VITRO* ROBERT E. KELLUM, M.D. AND KATHE STRANGFELD The pathogenetic factors in acne vulgaris,and fructose without gas formation and liquefying gelatin. The organisms were isolated from either an inflammatory process involving the piloseba-normal human skin or from comedones, pustules ceous units of human skin, have not been de-or clinically normal skin of patients with acne fined although the free fatty acids have beenvulgaris. implicated by indirect evidence. For example, B. Lipids. Three series of triglycerides were used the intradermal injection of human skin sur-in this study. Tricaprylin (C8 triglyceride), tn— caprin (C10 triglyceride) or trilaurin (C1 triglyc- face lipid produces an inflammatory reactioneride) were chosen as representative of triglycerides and a histologic picture resembling acne vul-of shorter chained fatty acids, tripalmitin (Co garis (1). The free fatty acid fraction oftriglyceride) as representative of triglycerides of human skin surface lipid has been shown to belong chain saturated fatty acids, and triolein (un- saturated C-is triglyceride) as representative of primarily responsible for this reaction (1, 2). triglycerides of long chain unsaturated fatty acids. These fatty acids are incorporated into the Each bacterial strain was incubated with indi- triglycerides synthesized by the human seba-vidual triglycerides in screw-topped culture tubes ceous gland and are liberated in the sebaceouscontaining 8 ml of thioglycolate medium, and 1 ml duct and on the skin surface by the hydrolyzingof glycerol as emulsifier. (Later experiments used action of bacterial or endogenous enzymes. Thea ratio of 10 ml thioglycolate and 3 ml glycerol. No differences were observed in the hydrolyzing microorganism, Corynebacterium acnes (C.activities of three organisms incubated in both acnes, Pro pioni bacterium acnes) has been sug-systems.) Each triglyceride was dissolved in hexane gested as the most likely source of bacterial(10 mg/ml) and 0.5 ml (containing 5 mg of the tri- enzymes (3). Direct evidence is not available,glyceride) was added to each tube of thioglycolate. The hexane was blown off with nitrogen and gentle however, regarding ability of this organism toheating. The tubes were sterilized by autoclave hydrolyze triglycerides of long and short chainfor fifteen minutes at 120° C. (Prior studies demon- fatty acids. To test the hypothesis of fattystrated no breakdown of triglyceride with the acid liberation from triglycerides by the hy-sterilization procedure.) Individual bacterial strains were innoculated (50—100 million organisms) into a drolytic action of C. acnes, in vitro incubationsculture tube containing one of the triglycerides. of fifteen strains of C. acnes were done withAn appropriate series of control tubes, containing three representative triglycerides. (a) medium and glycerol, (b) medium, glycerol ond organism, and (c) medium, glycerol and tri- MATERIALS AND METHODS glyceride, was established for each of the 45 tri- glyceride-microorganism combinations. The tubes A. Bacteriology. Fifteen unselected isolates ofwere sealed and incubated in an oven for seven C. acnes were obtained from those maintained indays at 35° C. The contents of each tube were culture in the microbiology laboratory of the de-agitated daily on a Vortex mixer. Growth of the partment of dermatology. The following criteriaC. acnes organisms was evident in the inoculated were used for identification of the micro-organism: tubes about the 3rd day. On the 7th day, each tube thin pleomorphic rod, Gram positive, catalasewas extracted three times with 4 ml portions of positive, growing as a tiny, pin point, round tochloroform, and the lipids ere studied by thin domed, smooth, white colony at 35360 C. underlayer chromatography (TLC) and gas liquid anaerobic conditions, with minimal or no growthchromatography (GLC). in the presence of air, fermenting glucose, galactose, C. Thin layer chromatography (TLC). Con- Received March 3, 1968; accepted for publi-ventional techniques of thin layer chromatography cation August 21, 1968. were used (4). A slurry of 25 grams of silica gel Supported in part by funds from U.S. ArmyH (No. 7736, according to Stahl) in 70 ml of glass Research and Development Command under con-distilled water was layered (025 mm. in thickness) tract DA-49-193-MD-2184 and by IJSPHS Train-onto 20 X 20 cm glass plates with a Desaga- ing Grant TI-AM-5300 from the National Insti- tute of Arthritis and Metabolic Diseases. Brinkmann adjustable applicator. The plates were Joyce Stewart gave valuable technical assistance. air dried, then heat activated for 30 minutes at * From the Department of Dermatology, Uni-120° C and allowed to cool. The extracted lipids versity of Oregon Medical School, Portland, Ore-from each series of four tubes were applied to a gon 97201. single activated chromatographic plate in 300 g 255 256 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY amounts bracketed with appropriate referencein the central portion of the plate was scraped standards. The following reference standards (infrom the plate into a beaker, extracted three times 20 g amounts) were used as appropriate: Tn-with 2 ml portions of chloroform, filtered through caprylin, tricaprin, tnilaurin, tripalmitin, triolein,fat free filter paper and blown to dryness with stearic acid, palmitic acid, dipalmitin, and mono-nitrogen, The methyl esters of the free fatty acids palmitin. The plates were developed for 20 minuteswere prepared with BF, in methanol (8) and in a glass developing tank (27 X 27 X 7 cm inter-studied by gas liquid chromatography to confirm nal dimensions) lined with solvent-saturated What-the presence of the specific fatty acid. GLC condi- man 3 MM paper. tions were: 20 ft. seamless, stainless steel column, All solvents were redistilled prior to use. The2.5 mm internal diameter, packed with 8.8% development system used for TLC was hexane-SE-30 on 6.5 grams of silanized, acid-washed diethyl ether-acetic acid (70:30:1). After develop-Chromosorb W, 80—100 mesh, flow rate —20 cc per ment, the plates were air dried, sprayed withminute of helium carrier gas, at 150° and 205° C. dichiorofluorescein or by the method of JonesAppropriate reference compounds were used as et al. (5) for visualization, viewed and photo-external standards, and relative carbon numbers graphed under ultraviolet light. The plates werewere calculated by the method of Woodford and then exposed to iodine vapors and photographed.van Gent (9). Identical thin layer plates, spotted and developed in parallel, were stained with bromocresol green RESULTS (6) or by the method of Kwapniewski and Sliwiok, as previously modified (7), for the detection of free This study reports striking differences in the carboxylic acids. The free fatty acids were separated from eachability of 15 isolates of C. acnes to hydrolyze sample by TLC. Plates were layered with silica gel triglycerides in vitro. Figure 1 summarizes the II (0.25 mm in thickness), predeveloped in chloro-results of the study of triglyceride hydrolysis form:methanol (9:1, V:V) to remove possibleby thin layer chromatography with confirma- contaminating lipids, air dried, then heat activatedtion of the presence of the specific fatty acid for 30 minutes at 120° C and allowed to cool. The extracted lipids were applied to the activatedby gas chromatography. Although four strains chromatographic plates in 5 to 10 mg samples,of C. acnes possessed substantial hydrolyzing flanked by reference standards on the two outsideactivity against all three triglycerides, the re- lanes of each plate. The plates were developed asmaining eleven isolates apparently lacked such described above, air dried, and the reference stand- ard lanes were sprayed with dichlorofluoresceindiverse ability and split only certain triglyc- with the center of the plate screened by plasticerides. Repeated incubations with several film. The silica gel containing the free fatty acidsisolates confirmed a consistent pattern of hy- Organism Short chain triglyceride* Tripalmitin Triolein C. acnes(C.D.C.) none evident ?trace trace C. aenes(AA'287) trace trace trace C. acnes(AA8 271) yes yes yes C. acnes(AA457) trace none evident yes C. acne.s(AA 1459) trace none evident yes C. acnes(AA*451) yes yes yes C. acnes(AA452) none evident trace yes C. acnes(AA455) yes ?trace yes C. acne.s(AA456) trace trace yes C. aenes(AA545) none evident none evident trace C. acnes(AA 542) none evident none evident ?trace C. acnes(AA544) yes yes yes C. acnes(AA,t'546) yes trace yes C. aenes(AA54l) yes none evident yes C. Genes(AA543) yes yes yes * Thefirst three organisms were incubated with both C8 and C10 triglycerides. The remaining twelve were cultured with Cu triglyceride. FIG. 1. Degree of hydrolysis of three representative triglycerides by 15 isolates of Coryne bacterium acnes in vitro, as determined by thin-layer chromatography, special TLC staining with bromocresol green and by cupric acetate-hematoxylin, and confirmed by gas chromatography. triglyceride free fatty acid 13-diglyceride 1,2-diglyceride monoglyceride 1) Lane No. Fm. 2. Thin layer chromatography plate showing hydrolysis of tripalmitin by C.acnes *'271; (Lane 1), tripalmitin standard 20 g; (2) media + organism control, 300 tg; (3) tripalmitin, 1, 3-dipalmitin, 1, 2-dipalmitin, monopalmitin standards, 20 tg each; (4) palmitic acid standard, 20 cg; (5) tripalmitin + organism, 300 pg (note presence of free fatty acid, 1,3- and 1,2—diglyccride from hydrolysis of triglyceride); (6) tripalmitin, palmitic acid, diglyceridcs and monoglyceride standards, 20 pg each; (7) media control, 300 cg; (8) media + tripalmitin control, 300 pg. triglyceride free fatty acid 1,3-diglyceride 1,2—diglyceride monoglyceride Lane No. Fio. 3. Thin layer chromatography plate showing failure of C. acnei 287 to hydrolyze significant amounts of triglyceride.
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