Grant Application - Bile Acid Transfrmations in Gallstone Therapy

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PRIVACY ACT MATERIAL REMOVED FOR CONTINUATION GRANT REQUESTEO BUOCET PERIOD (Name and Address. Street, City. State. Zip Code1 Argonne National Laboratory The University o € Chicago Argonne National Laboratory -20. OEPLRlMENT. SE RVICE. LABORATORY OR EQUIVALENT 3. ORCANIZATIONAl COMPONLNT TO RECEIVE CREDIT FOR INSTITUTIONAL 6. TITLE AN0 AOORESS Of OFFICIAL IN BUSINESS OffICE Of APPLICANT Assistant Vice President & Director, 7. RESEARCH INVOLVING HUMAN SUBJECTS (See InStruCtiOnr) Division of Biological and Argonne National Laboratory 10. OIRECT COSTS 9EPUESTED TOR OUDGET PERIOD 121. CONGRESSIONAL DISTRICT Of APP ll CAN l OR&NlZATlON SHOWN I N ITEM 4 I l l . Narm of business offieid (Item 6 ) 1IC. Nrma ad title 01 rdminirtratire of li cia l(ltm 1%) 121. COUNIY OF APPLICANT ORGANIZATION SHOWN IN ITEM 4 accept. as l o any grant awardcd. the obligation to comply w i t h Public H e a l t h Service terms and condition3 in effect at the time of the award. -(Signalurer required on original COPY only. Use Ink "Per" fig. 158. OFflClAL SICNINC FOR APPLICANT ORCANIZATION OATL

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FRINGE BENEFITSPERSONNEL TIME OR NAME (Last. First. Initial) TITLE OF POSITION XIHRS. UTERATIONS AND RENOVATIONS Funds to be made available to Argonne National Laboratory 'If this isa special rats (e.r. off dte). atplain.

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PRIVACY ACT MATERIAL REMOVED BUDGET FOR CONTINUATION GRANT E€€ort Requested Benefits TOTAL Hachey, David L. Ass is tant Chemist 25% $ $ See attached page for Argonne costing procedures Total personnel cost includes $11 ,5 13 i n indirect costs €or permanent positions Glassware , gas chromatographic supports , coatings , compressed gases, filaments , electron multipliers, paper for viscicorder and line printers $ 3 , 0 0 0 ; intermediates and isotopes for s t a b l e isotoptcally labeled bile acid Quarterly trips , Chicago/Mayo (4 x $1501, Am. SOC. Mass. Funds to be made available t o ANL TQTAL DIRECT COST

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ARGONNE NATIONAL LABORATORY Argonne's costing procedures a r e based on t h e assumption that all costs incurred will be recovered. The cost of work €or other Federal agencies is charged on t h e same basis as work €or ERDA program activities. The procedure is applied through- -out the Laboratory on a consistent basis and uses standard rates. The standard rates are a n average oC base wages, fringe costs, divisional program direction, divisional general expenses and Laboratory indirect cost charged to the effort incurred within each scientfCic division. A standard rate is developed for each major payroll classification within the Division. Standard rates are reviewed periodically and revised a s necessary. All labor cost is distributed within the Division using the standard rates mentioned above. The fringe costs include payroll related items such as annuities, social security and hospital and medical payments. The Division General, Program Direction, and Laboratory Overhead accounts include costs which cannot be charged to a specific program activity directly. Consistent with t h e procedure for costing work for ERDA, no depreciation of ERDA added cost factor is added t o work €or other Federal agencies. The ANL monthly rates estimated €or the period o l this grant areas follows: Ind irect expense 1599 Total, Staff Regular $3652 Total, Staff Temporary $ 1 2 4 7

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SECTION Ill-FISCAL DA,.. FOR CURRENT BUDGET PERIOD 10/0 1/74 Altentlons and Renovations Total Direct Casts ln dim t Costs (If Included in award) Uses wm below to: EXPENDITURES ADDITIONAL TOTAL ESTIMATED 8. List all items of equipment purchased of expected to be purchased during this budget period which have a U n i t cost of SlDOo of morr C. Explain r n y significant balance or deficit shown in any category of Column 5. D. List all other research support for Principal Inmitigator by souru. project title. and annual amount. See Page 4A For Detail.

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FISCAL DATA FOR GRANT NO. AM - 17862-02 FOR CURRENT BUDGET PERIOD Expenditures Remainder Total Estimated Estimated &rent Thru o€ Current Expend iture s Unobligated Budqet Cateqories Budget 6/30/75 Budget Period And Obligations Balance Total Direct Costs 66,255 22,015 Equ i Dmen t purchases 1. Computer inter€ace €or PDP-12 t o drive Biospect mass spectrometer €or spectra acquisition and multiple ion monitoring: constructed b y Electronics Division, ANL. Cost $2,000. Installation being completed. Modi€ication o€ s t a b l e isotope ratiometer to provide intergrals of ion intensity; construction and installation by Electronics Division, ANL. C o s t $1,900. Pumping system €or liquid chromatograph, to be purchased. Cost $4,000. Research Support €or Principal Investigator The remaining 90% of t h e PI'S support is derived a t present €rom the Energy Research and Funding from a n Ancillary Study t o the National Cooperative Gallstone Study t o cover 10% f?? his time is awaiting contract negotiation and award.

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APPLICANT: wtw GRANT NUMBER SHOWN ON PACE 1 ,-) SECTION IV-SUMMARY PROGRESS REPORT PRINCIPAL INVESTIGATOR OR PROGRAM DIRECTOR (Last. flrst. Inillal) 1. List publlutions: ( 8 ) published and not pnviously reportadd; (b) in press. Provido fiw rrerints It not prOViOUSly submitted. 2. List 811 additions 8nd drlotions i n profersion8i pononnri and any changrs i n offort. PAPERS AM) ABSTRACTS SUPPORTED BY AM 17862-01 (October 1, 1974-September 30, 1975) Argonne National Laboratory NAME OF ORGANIZATION A stable isotope ratiometer-multiple ion detector (SIRMID) unit for quantitative and qualitative stable isotope studies by gas chromatography-mass spectrometry. Clinical Chemistry, in press, August issue. Characterization of bile acid methyl ester acetate derivatives using gas-liquid chromatography, electron impact and chemical ionization mass spectrometry. Manuscript submitted to Journal of Lipid Research. Bile salt alkyl ethers: Evidence for a new class of biliary lipids in human bile. CHANGES IN PROFESSIONAL EFFORT No changes, in Professional Personnel or Distribution of Effort,are planned for year 02.

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PRIVACY ACT MATERIAL REMOVED I. A. The overall objectives of AM 17862 are: 1. To use gas chromatography-mass spectrometry to define the b ilia ry bile acid composition and characterize unusual bile acids i n gallstone patients at the Mayo Clinic before and after treatment with chenodeoxy2. To use gas chromatography-mass spectrometry t o measure plasma and liver concentrations of litho c holi c acid i n these patients by inverse isotope dilution procedures employing deuterium-labeled litho c holi c acid. To develop and validate a dual isotope, single sampling technique based upon deuterium and carbon-13 labeled bile acids which will permit both both bile acid pool size and synthesis rate tobe determined from a single sampling of the bile acid pool, after labeling with stable B. Goals for the current year (10/01/74-09/30/75) 1. To develop instrumentation suitable for stable isotope ratio measurements and multiple ion detection which can be wed i n conjunction with gas chromatograph-magnetic sector mass spectrometers o r gas quadrupole 2. To expand the existing library of bile acid identification techniques t o include gas chromatographic mobility, electron impact ionization spectra and chemical ionization spectra for all bile acids likely to occur i n human bile. 3. To apply these techniques tothe examination of b ilia ry bile acids in samples collected atthe Mayo Clinic from gallstone patients before and after treatment with chenodedxycholic acid, cholic acid, o r placebo. 11. PROGRESS DURING CURRENT YEAR IN RELATION TO GOALS 1. Development of instrumentation for stable isotope ratio measurements and multiple ion detection. W e have designed and constructed as table isotope ratiometermultiple ion detector (SInfID) unit which can drive existing gas chromatograph-quadrupole o r magnetic sector mass spectrometers t o

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PRIVACY ACT MATERIAL REMOVED Klein, Peter D. mod tor up t o six ions in turn. can be selected for quantitation; thus three different or successive, components can be anlaysed in a single gas chromatographic run. A background subtraction option permits the ion intensity in the absence of sample t o be subtracted automatically during sample ' measurement. Displays of accumulated counts and isotope ratio are updated twice per second during the measurement and can be printed oktat its conclusion. mode by parallel 'outputs toa multipen recorder. inthe construction of this prototype indicates that SIRMID units could be commercially available for $1OK o r about 1/3 t o 1/6th of cost of even an inexpensive computer system. Each of the three pairs. of ions All six ions can b e monitored i n the analog The manuscript describing this instrument will be published i n the August 1975 issue of Clinical Chemistry. 2. Development of bile acid identification techniuues and exoansion of the data base for bile acids likely to occur in human bile. The gas chromatographic retention times for forty-eight bile acids as their methyl ester acetate derivatives are given on 0.5% SP-525. Ion tables for electron impact spectra have been compiled which permit direct access t o ion structures for any given ion mass. Chemical ionization yields highly simplified mass spectra with two or three ions predominating for each compound. retention times of bile acids as their methyl ester acetates are combined with selective ion monitoring techniques i n chemical ionization mass spectrometry, the retention time and ion mass number form a coordinate system which can be a powerfull tool in the characki z ation of bile acid mixtures. When the relative The manuscript describing this work has been submitted t o the Journal of Lipid Research. 3. Application of these techniques tothe examination of b ilia ry bile acid collected at the Mayo Clinic from gallstone patients before and

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PRIVACY ACT MATERIAL REMOVED Klein, Peter D. B ilia ry bile acid samples from three groups of patients : placebo, chenodeoxycholic and c holi c acid, before and after treatment' for six months were obtained from Dr. Alan Hofmann. i n each category, totalling 30 bile samples were studzed by gas , chromatography-chemical ionization mass spectrometry, using the SIRMID system for selective ion monitoring. of a l l major gas chromatographic peaks was made for cholesterol, litho c holi c, deoxycholic, chenodeoxycholic, ursodeoxycholic and c holi c acid. Additionally, specific searches were made for the presence of any monoketo monohydroxy, mono keto dihydroxy, or d i keto monohydroxy bile acids,for cholate epimers, and for the presence of methoxy o r ethoxy bile acids (see below). number of patients studies, regardless of treatment protocol. Lithocholate and ursodeoxycholate were consistently present inall patients in concentrations up t o 10%. acid resulted in bile which was 80-96% chenodeoxycholic. treatment reduced chenodeoxycholate concentrations but increased deoxycholate. Placebo patients showed levels of litho cho late as high as those inpatients receiving chenodeoxycholic acid. following bile acids were detected i n concentrations between 0.01 epimers were not detected i n t h e concentration ranges covered, but were being monitored on a less abundant ion. tu ents turned out t o be incompletely acetylated di- or tri-hydroxy bdle samples studied from these patients. No abnormal bile acids were fouud in any of the limited Treatment with chenodeoxycholic No methoxy o r ethoxy bile acids were found i n any A collateral study with Dr. John Watkins a t Children's Hos pic d Medical Center employed these techniques to identify a new bil4acid believed t o be 3-ethoxy, 7a, 12aOH cholanic acid in children with been found i n premature infants and i n one patient with cholelfth.sl8. It appears t o be present in fairly high concentrations (1% of total bile acids in children with CF). This bile acid, as well as the methoxy analoqua has Electron impact and chemical

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PRIVACY ACT MATERIAL REMOVED ionization spectra are identical to those obtained from authentic campounds synthesized i n our laboratory; the thin-layer chromatographic behaviour is identical, but the GC behaviour of the methyl ester acetates differ in retention time, indicating a positional or epimeric distinction between standard and patient sample. Although this compound was not found i n any of the gallstone patients studied in the current series, further exploration and identification of this bile acid appears t o be important. been noted i n human fecal bile acids by S js vd let al. (J. Lipid groups, particularly ethyl ether s, is highly unusual i n nature and their effect on bile acid metabolism, enterohepatic circulation and excretion should be further investigated. time their distribution and occurrence i n other disease states should be determined. t i v e procedures of chemical ionization mass spectrometry and selective ion monitoring mean that it is now possible to detect these and other constituents of bile acid mixtures at levels as which cannot be resolved gas chromatographically. The existence of ether The extremely selective and highly sensiM, even inthe presence of components present a t 0.9 Pi I11 SIGNIFICANCE OF THESE STUDIES TO HEALTH PROBLEMS The ability to screen b ilia ry bile acid mixtures for the presence or absence of more than 40 known bile acids and bile acid derivatives, and t o do so rapidly means that a variety of liver diseases, including chronic active liver disease, cirrhosis, and pediatric c hole stasis can be examined for their relation ship to bile acid metabolism. chenodeoxycholic acid, or with other agents can be monitored to establish the metabolic and bacterial consequences on b ilia ry composition. Moreover, treatment of gallstones with IV RESEARC'f€ GOALS FOR "HE COMING YEAR A. Characterization of b ilia ry bile acids by gas chromatography-mass spectrometry Extend present series of gallstone patients as supplied by Mayo. Examine four patients in whom deoxycholic acid was fed. Examine b ilia ry bile acids from baboons maintained on high levels

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PRIVACY ACT MATERIAL REMOVED 4. As practical study patients in the following categories, as bile b. Chronic active liver disease d. Pediatric disorders of hepatic origin - B. Development of inverse isotope dilution techniques for quant it ation of bile acids i n plasma and tissue 1. Synthesis of 38 *H labeled b i l e acid standards’ for: 2. Determination of detection limits for unlabeled bile acids in plasma a. As free bile acids c. As sulfated conjugates As glycine or taurine conjugates 3. Validation of inverse isotope dilution techniques Against conventional gas chromatographic techniques Against radioimmune assays where available C. Development of bile acid kinetic measurement techniques suitable for plasma 1. Synthesis of ~ 4 - l ~ ~labeled b i l e acids 2. Individual and simultaneous measurement of bile acid kinetics for deoxycholic, chenodeoxycholic and cholic acid In bile samples following gallbladder contraction I n post prandial plasma samples from the same patients

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PRIVACY ACT MATERIAL REMOVED The undersigned agrees to accept responsibility for the scientific and technical conduct of the project and for provision of required progress reports if a grant 1 i s awarded as the result of this application. u