PRIVACY ACT MATERIAL REMOVED SECTION I REVIEW CROUP Lype PROCRAM CRANT NUMBER (Insert on A11 Pi DEPARTMENT of HEALTH, EDUCATION

PRIVACY ACT MATERIAL REMOVED SECTION I REVIEW CROUP lYPE PROCRAM CRANT NUMBER (Insert on a11 pi DEPARTMENT OF HEALTH, EDUCATION. AN0 WELFARE GMA 5 R01 AM - 17862-02

APPLICATION from: 10/01/74 nrouh: 9/3 0/7 7 FOR CONTINUATION GRANT REQUESTEO BUOCET PERIOD fmm: 1010 1/74 nmugh: 9/3 0176

2A. PRiNCIPAl INVLSTlGATOR OR PROGRAM OlREClOR 4. APPLICANT ORCMIZITION O(me rnd Address-Street, City. State. Zlp Code) (Name and Address. Street, City. State. Zip Code1 .. Klein Peter- D. The University o€ Chicago Argonne National Laboratory 5801 S. Ellis Avenue 9700 S. Cass Avenue Chicagd, Illinois 60637 Argonne, Illinois 60439

28. DEGREE ZC. SOCIAL SECURITY NOl - 5. Pns ACCOUNT NUMBER -PhD 1362177139Al 20. OEPLRlMENT. SE RVICE. LABORATORY OR EQUIVALENT 6. TITLE AN0 AOORESS Of OFFICIAL IN BUSINESS OffICE Of APPLICANT None ORWNlZATlOW 2E. MAJOR SUBOIVISION Assistant Vice President & Director, Argonne National Laboratory OCfice OE Sponsored Programs 3. ORCANIZATIONAl COMPONLNT TO RECEIVE CREDIT FOR INSTITUTIONAL University OF Chicago GRANT PURPOSES 5801 S. Ellis Avenue 20 Other I Chicago, Illinois 60637

7. RESEARCH INVOLVING HUMAN SUBJECTS (See InStruCtiOnr) 8. tNvLNTJONS (See Instructions) 8/6/74 (Current approva; M No 0 Yes-not previously reportsd 0 No Yes APPROVED: __- Oat* pending) 0 Yer.preriously rrparted 9. PERFORMANCE SlTE(S) TELLPHONE lNfC UTION Division of Biological and 11A. PRINCIPAL INVESTIGATOR Medical Research OR Argonne National Laboratory ROCRAM OlREtTOR (Item 211 9700 S. Cass Avenue Ext. 2811 Argonne, Illinois 60439 Ill. Narm of business offieid (Item 6) Cedric L. Chernick 312 I 753-3080 1IC. Nrma ad title 01 rdminirtratire oflicial (ltm 1%) 10. OIRECT COSTS 9EPUESTED TOR OUDGET PERIOD $63,439 (Includes indirect cost of $11 .SI? See items 6 & 11B 121. CONGRESSIONAL DISTRICT Of APPllCANl OR&NlZATlON SHOWN IN ITEM 4 121. COUNIY OF APPLICANT ORGANIZATION SHOWN IN ITEM 4 1st Cook

I. &#'" -1- ;3 i y& -&-;.I /q7$-

accept. as lo any grant awardcd. the obligation to comply with Public Health Service terms and condition3 in effect at the time of the award.

1'5. SICMATURES (Signalurer required on original - COPY only. Use Ink "Per" fig. 158. OFflClAL SICNINC FOR APPLICANT ORCANIZATION OATL nalures not acceptrDle.) -.___ '-?e:$.;*;c: v .- LI &J FROM THROUGH GRANT NUMBER SECTION il-BUDGET (USUALLY 12 MONTHS) 10/01/74 9/30/76 AM - 17862-02 -

PERSONNEL TIME OR FRINGE BENEFITS . EffORT REQUESTED TOTAL NAME (Last. First. Initial) TITLE OF POSITION XIHRS. (b) (C) (1)

(Indicate cost of each Item listed below) TOTAL (Columns (d) and (e) -I CONSULTANT COSTS (See Instruction$) I

OOMESTiC $ TRAVEL

FOREIGN $ I I PATIENT COSTS (See instructions) . $ UTERATIONS AND RENOVATIONS $

Funds to be made available to Argonne National Laboratory

See Page 2A €or Details

INDIRECT - --X SLW' Date of DHEW Asreant: 0 Not Rquested COST --x TOC' 0 Under nrgotlrtlon with: (See' Instructions) 'If this is a special rats (e.r. offdte). atplain. PRIVACY ACT MATERIAL REMOVED

BUDGET FOR CONTINUATION GRANT NO. AM - 17862-02 FROM 10/01/75 THROUGH 9/30/76

PERSONNEL

Salary Frinqe Name. -Title E€€ort Requested Benefits TOTAL Klein, Peter D. Principal Investigator 10% $ $ Hachey, David L. Ass is tant Chemist 25% $ $ Szczepanik, Patricia A. Scientific Associate 25% $ $ Mede, Karin A. Research Associate* 100% $ $ Tserng , Kou-Yi Research Associate* 100% $ $ *Temporary stafE position See attached page for Argonne costing procedures Total personnel cost includes $11 , 5 13 in indirect costs €or permanent positions

Subtotals $51,569 $4,870

TOTAL $56,439

?-. SUPPLIES

Glassware , gas chromatographic supports , coatings , compressed gases, filaments , electron multipliers, paper for viscicorder and line printers $3,000; intermediates and isotopes for stable isotoptcally labeled bile acid $2,000. $ 5,000

TRAVEL Quarterly trips , Chicago/Mayo (4 x $1501, Am. SOC. Mass. Spectrom. , AASLD $ 1,000

OTHER EXPENSES

x= Photocopying , postage, page charges , reprints Funds to be made available to ANL $ 1,000

a L -c TQTAL DIRECT COST $63,439 2 00 ARGONNE NATIONAL LABORATORY Costing Procedures

Argonne's costing procedures are based on the assumption that all costs incurred will be recovered. The cost of work €or other Federal agencies is charged on the 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 an 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 as 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 the procedure for costing work for ERDA, no depreciation of ERDA added cost factor is added to work €or other Federal agencies. The ANL monthly rates estimated €or the period ol this grant are as follows: 1. Sta€f Regular $1770 Fringe (17.7%) 313 Ind irect expense 1599 Total, Staff Regular $3652

2. Staff TeFporary $1138 Fringe (9.3%) 109 Total, Staff Temporary $1247

PACE 3

R c .I. 8- 7 7 1 i SECTION Ill-FISCAL DA,.. FOR FROM THROUGH GRANT NUMBER CURRENT BUDGET PERIOD 10/0 1/74 9/30/75 AM - 17862-01 (USUALLY 12 MONTHS)

ACTUAL ESTIMATED EXPENDITURES ADDITIONAL TOTAL ESTIMATED ESTIMATED CURRENT UNOBLIGATED BUDGEl BAUNCE A BUDGET CATEGORIES (As approved by REMAINDER (Subtract Col. 4 awarding unit) (Insert Date) OF CURRENT COl. 3) from Cot. 1) BUDGET PERIOD (1) (5)

Personnel (Salaries)

Fringe %enofits

Consultant Costs

Equipment

Supplies

Oomlrtlc TRAVEL

Patient Casb

Altentlons and Renovations

Other $66.255 $22,015 I $44,240 I $66,255 -0- Total Direct Casts $66,255 $22.015 I $44,240 I $66,255 - 3- lndimt Costs (If Included in award) -0- -0- 1 -0- I -0- ~ -0- TOTALS * $66,255 t 22,015 f 44,240 $66,255 i -0-

Use swm below to: 8. List all items of equipment purchased of expected to be purchased during this budget period which have a Unit cost of SlDOo of morr C. Explain rny 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.

- --- NIH 2006-1 (Formerly PHS 2590-1) PACE 4 (Use Conttnunton Pager as necessary) Rev. 8-73 FISCAL DATA FOR GRANT NO. AM - 17862-02 FOR CURRENT BUDGET PERIOD FROM lO/O 1/74 THROUGH 9/30/75

Est imated Additional Expenditures and Actual Obligations €or Expenditures Remainder Total Estimated Estimated &rent Thru o€ Current Expend iture s Unobligated Budqet Cateqories Budget 6/30/75 Budget Period And Obligations Balance

Personnel (Salaries) $50,642 $17,634 $33 ,008 $50,642 $ -0- Fringe Benefits 4,613 1,765 2,848 4,613 -0- Equ i pmen t 4,000 -0- 4,000 4,000 -0- . Supplies 5 ,000 1,872 3,128 5,000 -0- Travel 1, 000 744 256 1,000 -0- Other 1,000 -0- 1,000 1, 000 --0- Total Direct Costs 66,255 22,015 44,240 66,255 -0-

TOTALS $66,255 $22,015 $44,240 $66,255 $ -0-

Equ i Dmen t purchases 1. Computer inter€ace €or PDP-12 to drive Biospect mass spectrometer €or spectra acquisition and multiple ion monitoring: constructed by Electronics Division, ANL. Cost $2,000. Installation being completed. 2. Modi€ication o€stable isotope ratiometer to provide intergrals of ion intensity; construction and installation by Electronics Division, ANL. Cost $1,900. 3. Pumping system €or liquid chromatograph, to be purchased. Cost $4,000.

Budget balances None

Research Support €or Principal Investigator The remaining 90% of the PI'S support is derived at present €rom the Energy Research and Development Administration.

Funding from an Ancillary Study to the National Cooperative Gallstone Study to cover 10% f?? his time is awaiting contract negotiation and award. 0 0 c -J -00

Page 4P. APPLICANT: wtw GRANT NUMBER SHOWN ON PACE 1 ,-) CUTNUMBER SECTION IV-SUMMARY PROGRESS REPORT AM 17862-01

PRINCIPAL INVESTIGATOR OR PROGRAM DIRECTOR (Last. flrst. Inillal) PERIOD WVfRED BY THIS REPOdT Klein, Peter D. fROM THROUGM NAME OF ORGANIZATION Argonne National Laboratory 10/01/75 09/30/76

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 in profersion8i pononnri and any changrs in offort. .. 3. Progross Rapor(. (Sea Instructions)

PAPERS AM) ABSTRACTS SUPPORTED BY AM 17862-01 (October 1, 1974-September 30, 1975)

Klein, P. D., J. R. Haumann and D. L. Hachey 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.

Szczepanik, P. A., D. L. Hachey and P. D. Klein 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.

Hachey, D. L., P. A. Szczepanik, J. B. Watkins, A. M. Tercyak and P. D. Klein Bile salt alkyl ethers: Evidence for a new class of biliary lipids in human bile. Gastroenterology 67, 795 (1974).

CHANGES IN PROFESSIONAL EFFORT

No changes, in Professional Personnel or Distribution of Effort,are planned for year 02. Klein, Peter D. PRIVACY ACT MATERIAL REMOVED

I. A. The overall objectives of AM 17862 are:

1. To use gas chromatography-mass spectrometry to define the biliary bile acid composition and characterize unusual bile acids in gallstone patients at the Mayo Clinic before and after treatment with chenodeoxycholic acid.

2. To use gas chromatography-mass spectrometry to measure plasma and liver concentrations of lithocholic acid in these patients by inverse isotope dilution procedures employing deuterium-labeled lithocholic acid.

3. 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 to be determined from a single sampling of the bile acid pool, after labeling with stable isotopes.

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 in conjunction with gas chromatograph-magnetic sector mass spectrometers or gas quadrupole mass spectrometers.

2. To expand the existing library of bile acid identification techniques to include gas chromatographic mobility, electron impact ionization spectra and chemical ionization spectra for all bile acids likely to occur in human bile.

3. To apply these techniques to the examination of biliary bile acids in samples collected at the Mayo Clinic from gallstone patients before and after treatment with chenodedxycholic acid, cholic acid, or placebo.

11. PROGRESS DURING CURRENT YEAR IN RELATION TO GOALS

1. Development of instrumentation for stable isotope ratio measurements and multiple ion detection.

Abstract We have designed and constructed a stable isotope ratiometer- multiple ion detector (SInfID) unit which can drive existing gas chromatograph-quadrupole or magnetic sector mass spectrometers to Klein, Peter D. PRIVACY ACT MATERIAL REMOVED

modtor up to six ions in turn. Each of the three pairs. of ions 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 to 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 okt at its conclusion. All six ions can be monitored in the analog mode by parallel 'outputs to a multipen recorder. Experience gained in the construction of this prototype indicates that SIRMID units could be commercially available for $1OK or about 1/3 to 1/6th of cost of even an inexpensive computer system.

The manuscript describing this instrument will be published in the August 1975 issue of Clinical Chemistry.

2. Development of bile acid identification techniuues and exoansion of the data base for bileacidslikely to occur in human bile.

Abstract:

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 to ion structures for any given ion mass. Chemical ionization yields highly simplified mass spectra with two or three ions predominating for each compound. When the relative retention times of bile acids as their methyl ester acetates are combined with selective ion monitoring techniques in chemical ionization mass spectrometry, the retention time and ion mass number form a coordinate system which can be a powerfull tool in the charac- kization of bile acid mixtures.

The manuscript describing this work has been submitted to the Journal of Lipid Research.

3. Application of these techniques to the examination of biliary bile acid collected at the Mayo Clinic from gallstone patients before and after treatment. Klein, Peter D. PRIVACY ACT MATERIAL REMOVED

Biliary bile acid samples from three groups of patients: placebo, chenodeoxycholic and cholic acid, before and after treatment' for six months were obtained from Dr. Alan Hofmann. Five patients

in each category, totalling 30 bile samples were studzed by gas , chromatography-chemical ionization mass spectrometry, using the SIRMID system for selective ion monitoring. Specific identification of all major gas chromatographic peaks was made for cholesterol, lithocholic, deoxycholic, chenodeoxycholic, ursodeoxycholic and cholic acid. Additionally, specific searches were made for the presence of any monoketo monohydroxy, mono keto dihydroxy, or di keto monohydroxy bile acids, for cholate epimers, and for the presence of methoxy or ethoxy bile acids (see below).

Results. No abnormal bile acids were fouud in any of the limited number of patients studies, regardless of treatment protocol. Lithocholate and ursodeoxycholate were consistently present in all patients in concentrations up to 10%. Treatment with chenodeoxycholic acid resulted in bile which was 80-96% chenodeoxycholic. Cholate treatment reduced chenodeoxycholate concentrations but increased deoxycholate. Placebo patients showed levels of lithocholate as high as those in patients receiving chenodeoxycholic acid. The following bile acids were detected in concentrations between 0.01 and 0.5%; 3808 7aOH, 3aOH 7=0, 3a0H 12-0, 3aOH 7-0 12aOH. Cholat. epimers were not detected in the concentration ranges covered, but were being monitored on a less abundant ion. A few small constituents turned out to be incompletely acetylated di- or tri-hydroxy bile acids. No methoxy or ethoxy bile acids were found in any bdle samples studied from these patients.

A collateral study with Dr. John Watkins at Children's Hospicd Medical Center employed these techniques to identify a new bil4 acid believed to be 3-ethoxy, 7a, 12aOH cholanic acid in children with cystic fibrosis. This bile acid, as well as the methoxy analoqua has been found in premature infants and in one patient with cholelfth.sl8. It appears to be present in fairly high concentrations (1% of total bile acids in children with CF). Electron impact and chemical

. Klein, Peter D. PRIVACY ACT MATERIAL REMOVED

ionization spectra are identical to those obtained from authentic campounds synthesized in 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 in any of the gallstone patients studied in the current series, further exploration and identification of this bile acid appears to be important. It has been noted in human fecal bile acids by Sjsvdl et al. (J. Lipid Res. -7, 524 (1966)) but not identified. The existence of ether groups, particularly ethyl ethers, is highly unusual in nature and their effect on bile acid metabolism, enterohepatic circula- tion and excretion should be further investigated. At the same time their distribution and occurrence in other disease states should be determined. The extremely selective and highly sensi- tive 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 low as M, even in the presence of components present at 0.9 Pi which cannot be resolved gas chromatographically.

I11 SIGNIFICANCE OF THESE STUDIES TO HEALTH PROBLEMS The ability to screen biliary bile acid mixtures for the presence or absence of more than 40 known bile acids and bile acid derivatives, and to do so rapidly means that a variety of liver diseases, including chronic active liver disease, cirrhosis, and pediatric cholestasis can be examined for their rela- tionship to bile acid metabolism. Moreover, treatment of gallstones with chenodeoxycholic acid, or with other agents can be monitored to establish the metabolic and bacterial consequences on biliary composition. IV RESEARC'f€ GOALS FOR "HE COMING YEAR A. Characterization of biliary bile acids by gas chromatography-mass spectrometry c 1. Extend present series of gallstone patients as supplied by Mayo. c) 2. Examine four patients in whom deoxycholic acid was fed. c=) E 3. Examine biliary bile acids from baboons maintained on high levels of cheno.

A Klein, Peter D. PRIVACY ACT MATERIAL REMOVED

4. As practical study patients in the following categories, as bile is supplied: a. Biliary cirrhosis b. Chronic active liver disease c. Alcoholic cirrhosis d. Pediatric disorders of hepatic origin

- B. Development of inverse isotope dilution techniques for quantitation of bile acids in plasma and tissue

1. Synthesis of 38 *H labeled bile acid standards’ for: a. Lithocholic b. Deoxycholic c. Chenodeoxycholic d. Ursodeoxycholic e. cholic

2. Determination of detection limits for unlabeled bile acids in plasma a. As free bile acids b. As glycine or taurine conjugates c. As sulfated conjugates

3. Validation of inverse isotope dilution techniques a. Against conventional gas chromatographic techniques b. Against radioimmune assays where available

C. Development of bile acid kinetic measurement techniques suitable for plasma samples

1. Synthesis of ~4-l~~labeled bile acids a. Deoxycholic b. Chenodeoxycholic .c. Cholic

2. Individual and simultaneous measurement of bile acid kinetics for deoxycholic, chenodeoxycholic and cholic acid a. In bile samples following gallbladder contraction 5 0 b. In post prandial plasma samples from the same patients 0 s=r J cx) 4

1 ! Klein. Peter D. PRIVACY ACT MATER IAL 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

is awarded as the result of this application. u

JuNe 2a. 1t75 Rsb.kP.3 Date Principal Investigator