Table Of ContentPreface
Biothiols participate in numerous cellular functions, such as biosynthetic
pathways, detoxification by conjugation, and cell division. Studies on oxida-
tive stress in recent years have amply documented the key role of thiols--
more specifcally the thiol-disulfide status of the cell--in a wide array of
biochemical and biological responses. Awareness of the great importance
of biothiols in cellular oxidative injury has grown along with the recognition
of free radicals in biological processes. The reactions of thiols with free
radicals are not only of interest in free radical chemistry: the most abundant
nonprotein thiol in the cell, glutathione, is essential for the detoxification
of peroxides as cofactors of various selenium-dependent peroxidases. The
high concentration of glutathione in cells clearly indicates its general impor-
tance in metabolic and oxidative detoxification processes. In many ways,
glutathione may be considered the master antioxidant molecule, a phrase
which the late Alton Meister, one of the pioneers in glutathione research
and a contributor to this volume, used. Bolstering of glutathione by other
thiols, both natural (such as a-lipoic acid) and synthetic (such as Ebselen
and several other drugs), has been investigated as a therapeutic approach
to the oxidative component of various pathologies. Moreover, the redox
changes of several thiol-containing proteins may be involved in key regula-
tory steps of the enzyme as well as in cell proliferation.
The contributions to Volumes 251 and 252 of Methods ni Enzymology
(Biothiols, Parts A and B) provide a comprehensive and detailed account
of the methodology relating to the molecular mechanisms underlying the
multiple functions of biothiols, with emphasis on their interaction at the
biochemical and molecular biological levels in cellular reactions, with oxi-
dants and other biological and clinical implications of thiols. The contribu-
tions in this volume (Part B) include methods on glutathione: distribution,
biosynthesis, metabolism, and transport; signal transduction and gene ex-
pression; thioredoxin and glutaredoxin; and synthetic mimics of biological
thiols and thiols inhibitors. In Part A (Volume 251) methods are included
relating to thiyl radicals; chemical basis of thiol/disulfide measurements:
monothiols: measurement in organs, cells, organelles, and body fluids; dithi-
ols: oMipoic acid; and protein thiols and sulfides. Credit must be given to
the experts in various specialized areas selected to provide state-of-the-art
methodology. The topics and methods included in these volumes were
chosen on the excellent advice of the volume advisors, Bob B. Buchanan,
Enrique Cadenas, Carlos Gitler, Arne Holmgren, Alton Meister, and Hel-
mut Sies, to whom I extend my thanks and most grateful appreciation.
LESTER PACKER
iiix
Contributors to Volume 252
Article numbers are in parentheses following the names of contributors.
Affiliations listed are current,
EUQINOM ADOLPHE (34), Laboratoire ed ELIDO-EIRAM NETSIRHC (32, 33, 34), Labora-
Pharmacologie ,erialulleC Ecole Pratique eriot ed euqitueparOhT ,enredoM SOL VA Y
sed Hautes Etudes, 60057 Paris, France Pharma, 15159 Suresnes Cedex, ecnarF
LEAHCIM T. NOSREDNA (17), Stanford Medi- EGROEG L. DALE (8), Department of -ideM
lac School, Beckman ,retneC Stanford, -ilaC ,enic University of Oklahoma Health Sci-
fornia 50349 ences Center, Oklahoma City, Oklahoma
KIRDERF ~SLUND (29), Medical Nobel Insti- 09137
etut for Biochemistry, MBB, Karolinska In- PAULETTE DECO'Iq'IGNIES (25), Physiologie
,tetutits S-171 77 Stockholm, Sweden elatOg~V ,erialuc~loM ~tisrevinU ed -siraP
K. D. NNAMUA (5), tfahcslleseG far Biotech- Sud, 91405 ,xedeC-yasrO ecnarF
nologische Forschung (GBF), Braunseh- WULF DROGE (26), Division of Immuno-
,giew Germany ,yrtsimehc Deutsches Krebsforschungszen-
ELAINE S. G. BARDES (16), Department of trum, D-69120 Heidelberg ,1 Germany
Molecular Cancer Biology, Duke -revinU H. JANE NOSYD (30), Department of -uceloM
sity Medical ,retneC Durham, North -oraC ral Biology, ehT Scripps hcraeseR ,etutitsnI
lina 01772 La Jolla, California 73029
TREBOR M. BELL (16), Department of -celoM
L. RIKCAF (33), Laboratoire ed Chimie-Phy-
ular recnaC Biology, Duke ytisrevinU Med-
sique, Universitd R~ne Descartes, 07257
laci ,retneC Durham, North aniloraC 01772
siraP Cedex ,60 ecnarF
TSENRE RELTUEB (8), Department of -uceloM
L. FLOHE (5), Gesellschafi far Biotechnolo-
ral and Experimental ,enicideM Scripps -eR
gische Forschung (GBF), ,giewhcsnuarB
hcraes Institute, La Jolla, California 73029
Germany
LEAKIM TDETSNROJB (21, 22), Department of
Medical Biochemistry and Biophysics, ehT HENRY JAY FORMAN (7), Department of Mo-
ralucel Pharmacology and ,ygolocixoT -inU
Medical Nobel Institute for ,yrtsimehcoiB
ytisrev of Southern ,ainrofilaC Los ,selegnA
Karolinska lnstitutet, S-171 77 Stockholm,
ainrofilaC 33009
nedewS
RoY A. TDRAHCROB (16), Department of Mo- SEMAJ A. SHCUF (9), Department of Biochem-
lecular Cancer Biology, Duke ytisrevinU istry, University of_Minnesota, .tS Paul,
Medical Center, Durham, North aniloraC Minnesota 80155
01772 DRAHCIR .S GLASS (31), Department "fo
R. EHOLF-SUILEGIRB (5), Deutsches Institut ,yrtsimehC University of Arizona, ,noscuT
far Erniihrungsforschung, Postdam-Reh- Arizona 12758
briicke, Germany UDUYAR ANHSIRKALAPOG (14), Department
BoB B. NANAHCUB (24), Department of Plant of Pharmacology and Nutrition, School of
Biology, ytisrevinU of ,ainrofilaC ,yelekreB ,enicideM University of Southern -rofilaC
Berkeley, California 02749 nia, Los Angeles, California 33009
ZHEN-HAI CHEN (14), Department of Phar- AHSU ADEMIDNUG (14), Department of -rahP
macology and Nutrition, School of Medi- macology and Nutrition, School of -ideM
,enic ytisrevinU of Southern ,ainrofilaC Los ,enic ytisrevinU of Southern ,ainrofilaC Los
Angeles, California 33009 Angeles, California 33009
ix
X CONTRIBUTORS TO VOLUME 252
LEONARD A. HERZENBERO (17), Stanford BRIAN KETTERER (6), Department of Bio-
lacideM Beckman School, ,retneC ,drofnatS ,yrtsimehc CRC Re- Molecular Toxicology
ainrofilaC 50349 hcraes Group, University egelloC Mid- and
TOD P. HOLLER (12), Parke-Davis Pharma- dlesex School of Medicine, London W1P
,lacituec Division of Com- Warner-Lambert 6DB, Kingdom United
pany, Ann Arbor, Michigan 50184 KAROLY KOBREHEL (24), Laboratoire ed
Technologie sed ,seladrdC INRA, 34060
ARNE HOLMGREN (21, 22, 29), Department of
Montpellier Cedex ,10 ecnarF
Biochemistry and Biophysics, Medical ehT
Medical Nobel Institute for ,yrtsimehcoiB KONDO TAKAHITO (8), Department of -ohtaP
Karolinska lnstitutet, S-171 77 Stockholm, lacigol Biochemistry, Atomic Insti- Disease
Sweden ,etut Nagasaki School Univeristy of Medi-
,enic Japan Nagasaki,
PAUL B. HOPKINS (12), Department of Chem-
istry, University of Washington, ,elttaeS KUMAR SUSHIL (22), Department of lacideM
Washington 59189 and Biochemistry Biophysics, ehT Medical
Nobel Institute for Biochemistry, Karolin-
TOSHIYUKI HORI (36), Department of -igoloiB
S-171 ska Institutet, 77 Stockholm, Sweden
lac Institute Responses, for Virus ,hcraeseR
Kyoto ,ytisrevinU Kyoto, Japan ECNERWAL H. LASH (2), Department of -rahP
macology, Wayne State School University
STEVAN R. HUBBARD (13), Department of
of ,enicideM Michigan Detroit, 10284
Biochemistry and Molecular Biophysics,
LEMAIRE MARTINE (25), Physiologie elat~g~V
Columbia University, New York, New
,erialuc~loM ~tisrevinU ed ,duS-siraP 50419
York 23001
,xedeC-yasrO ecnarF
RONALD S. HUTCHISON (23), Department of
RuI-MING LIu (7), Departments of raluceloM
Plant Biology, ehT Ohio State ,ytisrevinU
,ygolocamrahP and Pathology, Toxicology,
Columbus, Ohio 01234
,scirtaideP University of Southern -rofilaC
EMMAMUELLE ISSAKIDIS (25), Physiologie
nia, Los Angeles, California 33009
elat~g~V ,erialuc~loM dtisrevinU ed -siraP
TE-CHUNG LIU (20), Department of ,noitirtuN
Sud, 91405 ,xedeC-yasrO ecnarF
Case Western Reserve University, Cleve-
ROLE D. ISSELS (11), Klinik Medizinische lII, land, Ohio 60144
tatisrevinU ,nehcniEM D-81366 Munich,
M. MAIORINO (5), Department of Biological
;ynamreG Institut and far Klinische -am~iH
Chemistry, University of Padua, Padua,
,eigolot GSF ,nehcniEM D-81377 Munich,
Italy
Germany
THOMAS MEIER (11), Institute far Klinische
TAKEO IWAMOTO (7), Department of Bio-
,eigolotamaH GSF ,nehcnitM Mu- D-81377
,yrtsimehc State Kansas ,ytisrevinU Manhat-
nich, Germany
,nat Kansas 60566
ALTON MEISTER* (3), Department of Bio-
IWATA SATOSHI (36), Department of Biologi- ,yrtsimehc Cornell ytisrevinU Col- Medical
lac Institute Responses, for Virus ,hcraeseR
,egel New York, New York 12001
Kyoto ,ytisrevinU Kyoto, Japan
DAVID J. MEYER (6), Department of Bio-
JEAN-PIERRE JACQUOT (25), Physiologie -g~V ,yrtsimehc CRC Re- Molecular Toxicology
elat~ ,erialuc~loM ~tisrevinU ed ,duS-siraP hcraes Group, University egelloC Mid- and
50419 ,xedeC-yasrO ecnarF dlesex School of Medicine, London WIP
DEAN P. JONES (1), Department of Biochemis- 6DB, Kingdom United
try, Emory School University of ,enicideM AWALSORYM MIGINIAC-MASLOW (25), Physi-
Atlanta, Georgia 22303 ologie elat~g~V ,erialuc~loM ~tisrevinU ed
DANIEL JORE (33), Laboratoire ed Chimie- ,duS-siraP 91405 ,xedeC-yasrO ecnarF
Physique, ~tisrevinU R~ne ,setracseD 07257
Cedex Paris ,60 ecnarF *Deceased
CONTRIBUTORS TO VOLUME 252 xi
MASASHI MIZUNO (19), Department of -celoM HEIKE SCHENK (26), Division of Immuno-
ular and Cell Biology, ytisrevinU of -rofilaC ,yrtsimehc Deutsches Krebsforschungszen-
,ain Berkeley, Berkeley, California 02749 trum, D-69120 ,grebledieH Germany
DONALD R. ORT (23), Photosynthesis Re- D. SCHOMBUR6 (5), tfahcslleseG far Biotech-
search Unit, USDA/ARS, Department of nologische Forschung (GBF), Braunsch-
Plant Biology, University of Illinois, -rU ,giew Germany
,anab Illinois 10816 KLAUS SCHULZE-OSTHOFF (26), Division of
LESTER PACKER (18, 19), Department of Mo- Immunochemistry, Deutsches Krebs-
ralucel and Cell Biology, ytisrevinU of -ilaC forschungszentrum, D-69120 ,grebledieH
fornia, Berkeley, Berkeley, ainrofilaC 02749 Germany
MULCHAND V. PATEL (20), Department of PETER NNAMRUHCS (28), Laboratoire ed Bio-
,yrtsimehcoiB State ytisrevinU of New York chimie Vegetale, ~tisrevinU ed ,letdhcueN
0002-HC ,let~hcueN Switzerland
ta Buffalo, Buffalo, New York 41241
MICHAEL MING SHI (7), Departments of Mo-
ANDREW F. G. QUEST (16), Department of
ralucel Pharmacology, Toxicology, Pathol-
Molecular Cancer Biology, Duke -revinU
,ygo and ,scirtaideP ytisrevinU of Southern
sity Medical ,retneC Durham, North -oraC
,ainrofilaC Los Angeles, California 33009
lina 01772
HELMUT SIES (35), -isrevinU-enieH-hcirnieH
EC1RTAP RAT (34), Laboratoire de Pharma-
,tlit Instimt far Physiologische Chemie .1
cologie Cellulaire, Ecole Pratique sed
10004-D Diisseldorf Germany
Hautes Etudes, 60057 Paris, France
FRANK J. T. STAAL (17), Department of Im-
DONALD J. REED (10), Department of Bio-
munology, Netherlands Cancer ,etutitsnI
chemistry and Biophysics, Environmental
6601 ,XC Amsterdam, 'sdnalrehteN
Health Sciences ,retneC Oregon State -inU
THRESSA C. STADTMAN (31), Laboratory of
,ytisrev ,sillavroC Oregon 13379
Biochemistry, National Heart, Lung, and
YHTOM1T W. ROBINSON (7), Departments of Blood Institute, National Institutes of
Molecular Pharmacology, Toxicology, -aP ,htlaeH Bethesda, Maryland 29802
,ygoloht and ,scirtaideP ytisrevinU of South-
YUICHIRO J. SUZUKI (18), Department of Mo-
nre California, Los Angeles, California
ralucel and Cell Biology, ytisrevinU of -ilaC
33009
fornia, Berkeley, Berkeley, California 02749
JOAQUfN ROMA (15), Department of Physiol- MARC THEVENIN (34), Laboratoire ed Toxi-
ogy, Experimental Toxicology and Neuro-
,eigoloc Facult( ed ,eicamrahP 07257 siraP
toxicology Unit, School of Medicine, Uni-
Cedex ,60 ecnarF
ytisrev of ,aicnelaV 46010 ,aicnelaV Spain
F. URSINI (5), Department of ,yrtsimehC -inU
FRANCISCO J. ROMERO (15), Department of ytisrev of ,enidU 00133-1 ,enidU Italy
Physiology, Experimental Toxicology and
NATARAJ N. VAKNAKAMUROKKATTEV (20),
Neurotoxicity Unit, School of ,enicideM
Department of Biochemistry, State -revinU
ytisrevinU of Valencia, 46010 ,aicnelaV
sity of New York ta Buffalo, Buffalo, New
niapS
York 41241
A. ROVERI (5), Department of Biological JEAN-MICHEL WARNET (34), Laboratoire ed
Chemistry, University of Padua, Padua,
,eigolocixoT Facult~ ed Pharmacie, 07257
ylatI
Paris Cedex ,60 ecnarF
MARJORIE RUSSEL (27), Laboratory of -teneG MICHAEL P. WASHBURN (4), Department of
,sci ehT Rockefeller ,ytisrevinU New York, Biochemistry, Michigan State ,ytisrevinU
New York 12001 East Lansing, Michigan 42884
NORIHITO SATO (36), Department of Biologi- WILLIAM W. WELLS (4), Department of Bio-
lac Responses, Institute for Virus ,hcraeseR ,yrtsimehc Michigan State ,ytisrevinU East
Kyoto ,ytisrevinU Kyoto, Japan Lansing, Michigan 42884
xii CONTRIBUTORS TO VOLUME 252
HTEBAZILE TTOLLIW (16), Department of Mo- DIAN GNEP XU (4), Department of Biochemis-
lecular Cancer Biology, Duke University try, Michigan State University, East Lan-
Medical Center, Durham, North Carolina sing, Michigan 48824
01772
AKIRA IHCUAMAY (36), Department of Bio-
AUHSOJ H. (24), WONG Department of Plant
logical Responses, Institute for Virus Re-
Biology, University of California, Berkeley,
search, Kyoto University, Kyoto, Japan
Berkeley, California 94720
NEW QIN XIE (16), Department of Molecular (36), YODOI JUNJI Department of Biological
Cancer Biology, Duke University Medical Responses, Institute for Research, Virus Ky-
Center, Durham, North Carolina 27710 oto University, Kyoto ,10-606 Japan
1 GLUTATHIONE DISTRIBUTION IN NATURAL PRODUCTS 3
I Glutathione Distribution in Natural Products:
Absorption and Tissue Distribution
yB DEAN .P SENOJ
Introduction
Glutathione (GSH) is present in most plant and animal tissues from
which the human diet is derived. Much of this GSH is bioavailable because
cells of the gastrointestinal tract have Na+-dependent and Na+-independent
uptake mechanisms, 3-1 and net absorption of GSH from the lumen into
the vascular circulation occurs in the small intestine. 4 In addition, GSH can
function as an antioxidant in the ingesta, can maintain ascorbate in a re-
duced and functional form, 5 can directly react with and inactivate toxic
electrophiles in the diet, 6 and can be broken down to yield cysteine. 7 Studies
have shown that dietary GSH enhances metabolic clearance of dietary
peroxidized lipids and decreases their net absorption 9'8 and that consump-
tion of foods high in GSH content is associated with about a 50% reduc-
tion in risk of oral and pharyngeal cancer, w Therefore, an improved under-
standing of the distribution of GSH in foods and the factors affecting
GSH absorption and distribution can be expected to provide the basis
for improved food selection and preparation to reduce risk of chronic dis-
eases.
1 M. K. Hunjan and D. F. Evered, Biochim. Biophys. Acta 815, 481 (1985).
2 L. J. Dahm, P. .S Samiec, J. W. Eley, E. W. Flagg, R. J. Coates, and D. P. Jones, in "Free
Radicals: From Basic Science to Medicine" (G. Poli, E. Albano, and .M U. Dianzani, eds.),
p. 506. Birkhaeuser, Basel, .3991
3 .M T. Vincenzini, F. Favilli, and T. Iantomasi, Biochim Biophys, Acta 1113, 31 (1992).
4 T. M. Hagen and D. P. Jones, Am. J. Physiol. 252, G607 (1987).
5 B. .S Winkler, Biochim. Biophys. Acta 925, 852 (1987).
P. S. Samiec, L. J. Dahm, E. W. Flagg, R. J. Coates, J. W. Eley, and D. P. Jones, in "Free
Radicals and Antioxidants in Nutrition" (F. Corongiu, S. Banni, .M A. Dessi, and C. Rice-
Evans, eds.), p. 269. Richelieu Press, London, .3991
7 N. Tateishi and Y. Sakamoto, in "Glutathione: Transport and Turnover in Mammals"
(Y. Sakamoto, T. Higashi, and N. Tateishi, eds.), p. .31 Japan Scientific Societies Press,
Tokyo, .3891
8 D. P. Kowalski, R. M. Feeley, and D. P. Jones, J. Nutr. 120, 1115 (1990).
9 T. Y. Aw and .M W. Williams, Am. J. Physiol. 263, G665 (1992).
m E. W. Flagg, R. J. Coates, D. P. Jones, T. E. Byers, R. .S Greenberg, G. Gridley, J. K.
McLaughlin, W. J. Blot, M. Haber, S. Preston-Martin, J. B. Schoenberg, D. F. Austin, and
J. F. Fraumeni, Am. J. Epidemiol. 139, 354 (1994).
Copyright © 1995 by Academic Press, Inc.
METHODS IN ENZYMOLOGY, VOL. 252 llA rights of reproduction in any form reserved.
4 ENOIHTATULG 11
Glutathione Contents of Foods
General Considerations
Glutathione is readily oxidized by food processing and preparation, and
many foods contain reactive electrophilic compounds that can remove GSH
even after food is ingested. 6 Thus, analytically precise measures of GSH
in foods only provide an index of GSH delivered to gastrointestinal epithe-
lia. In addition, oxidized forms of GSH, such as oxidized glutathione
(GSSG), are also present in foods and are at least partially available as
GSH because the intestine contains a GSSG reductase mechanism, tl Conse-
quently, direct measures of GSH probably underestimate GSH availability;
whereas, measures of total GSH (GSHt) following reduction of food ex-
tracts with dithiothreitol probably overestimates GSH that is bioavailable.
Many factors affect food contents of nutrients, and established food
analysis protocols, such as those of the Association of Official Analytical
Chemists, 21 should be used whenever possible. As water content is highly
variable in stored foods, dry weight or total nitrogen contents should be
determined for all samples. Conditions of growth, seasonal and geographic
conditions, plant variety, duration and conditions of storage and processing,
and preparation are all expected to affect GSH content of foods, but no
systematic studies of the effects of these factors on GSH content are
available.
The methods that we use to assay GSH contents of foods were developed
to provide a database for epidemiological studies of dietary intake of GSH
and risk of diseases in humans. 31 We based our food items on the National
Cancer Institute's Health Habits and History Questionnaire (HHHQ), 51'41
which includes 89 food items and groups that account for over %09 of
calories, fiber, and 81 major nutrients in diets in the United States. 61,41 For
each individual food item or group, a specific food and method of prepara-
tion is selected based on those most commonly reported in the second
National Health and Nutrition Survey, a population-based survey con-
ducted in the United States between 1976 and 1980.
11 T. M. Hagen, G. T. Wierzbicka, B. B. Bowman, T. Y. Aw, and D. P. Jones, Am. Z PhysioL
259, G530 (1990).
21 S. Williams (ed.), "Official Methods of Analysis." (1141 pages). Association of Official
Analytical Chemists, Arlington, Virginia, 1984.
31 D. P. Jones, R. J. Coates, E. W. Flagg, J. W. Eley, G. Block, R. .S Greenberg, E. W. Gunter,
and B. Jackson, Nutr. Cancer 17, 57 (1992).
41 G. Block, C. M. Dresser, A. M. Hartman, and M. D. Carroll, Am. J. Epidemiol. 122,13 (1985).
51 G. Block, A. M. Hartman, C. M. Dresser, M. D. Carroll, J. Gannon, and L. Gardner, Am.
J. Epidemiol. 124, 453 (1986).
61 G. Block, C. M. Dresser, A. M. Hartman, and M. D. Carroll, Am. J. EpidemioL 122, 27 (1985).
1 GLUTATHIONE DISTRIBUTION IN NATURAL PRODUCTS 5
Standard Protocol
For each food item, at least three different brand name products or
three different samples (different sources) are purchased from major chain
supermarkets, cafeterias, or restaurants. When samples are stored prior to
assay, this is done under usual storage conditions, that is, in a refrigerator
for perishable foods and in a freezer for frozen foods. Prior to sampling,
foods are prepared according to common habits in the United States. Non-
edible portions are removed, and foods that are normally cooked are pre-
pared accordingly. In general, steamed or boiled foods are cooked in a
small amount of water until tender and then drained. Pan-fried foods, such
as pork chops, are fried at medium heat for 5 min or until done (loss of
pink or red color in meats).
Because GSH can be rapidly lost on homogenization, we developed
a standard protocol that provides a correction for GSH loss based on
simultaneously assayed samples with known amounts of GSH added. Five
g-1 portions are taken from one sample of each food item and treated as
follows. One portion is placed in an aluminum tray and dried for at least
24 hr at 501 ° to obtain a dry weight. The other four portions are added to
beakers containing 01 ml of ice-cold homogenization solution A, B, C, or
D. Solution A is 0.9% (w/v) NaC1 containing 5 mM EDTA. Solutions B,
C, and D are made from solution A by adding 01 ~M GSH, 20/zM GSH,
or 5 mM dithiothreitol, respectively.
Foods that are solutions or homogenates are mixed on a vortex mixer
prior to removal of 1-ml aliquots for addition to test tubes containing 5.0
ml of ice-cold %03 (w/v) trichloroacetic acid. Emulsions (e.g., milk) are
extracted 71 to remove lipids before acidification. For other foods, portions
are homogenized using a Polytron for a minimum duration to obtain a
homogeneous suspension, and 1-ml of aliquots are immediately transferred
to ice-cold 30% trichloroacetic acid.
Following centrifugation to remove insoluble material, 0.5-ml aliquots
are transferred to microcentrifuge tubes containing 1.0 ml of 40 mM io-
doacetic acid. 81 Each solution is adjusted to pH 8 by addition of i M KHCO3
in I M KOH and incubated for 1 hr at room temperature. One-half milliliter
of 1-fluoro-2,4-dinitrobenzene (1.5% in ethanol; v/v) is then added, and
samples are incubated overnight at room temperatures in the dark. 8a Sam-
ples are then stored at 0o-4 ° for up to 2 weeks prior to assay by high-
performance liquid chromatography (HPLC).
The HPLC procedure is essentially that described by Reed et al. 8~ using
71 j. Folch, M. Lees, and G. H. Sloane Stanley, J. BioL Chem. 226, 497 (1957).
81 D. J. Reed, J. R. Babson, P. W. Beatty, A. E. Brodie, W. W. Ellis, and D. W. Potter, Anal.
Bioehem. 106, 55 (1980).
6 ENOIHTATULG 11
001
80
06
$
02
I i I I I I i I i I
0 01 02 03 04 05
Time (rain)
FIG. .1 Analysis of GSH in potato by HPLC. The sample 1( g) was homogenized in 01
ml of solution A and derivatized as described. Twenty-five microliters were injected on the
HPLC system. The peak at 40.0 min is the S-carboxymethyl-N-dinitrophenyl derivative of
GSH. The GSSG derivative is the peak at 44.3 min.
an Ultrasil NH2 column from Beckman (Norcross, GA) or an aminopropyl
column from Custom LC (Houston, TX). Initial conditions are 1 ml/min
flow with 80% solvent A (80% methanol) plus o%02 solvent B 5( M sodium
acetate, pH 4.6, in %08 methanol). Initial conditions are maintained for 01
min followed by a linear gradient to 95% B over 40 min and reequilibration
with initial conditions for 01 min prior to analysis of the next sample.
Derivatives are detected at 563 nm and quantified relative to standards by
integration (Fig. 1).
Integral values for portions taken in solutions A, B, and C are plotted
as a function of added GSH content (0, ,01 and 20/zM), and the negative
of the x axis intercept is taken as the GSH concentration (Fig. 2). Values are
multiplied by 11 to convert back to sample concentrations and appropriately
recalculated to be expressed in terms of millimolar or as mg/g dry weight.
Values are converted to milligrams per usual portion size by use of the
HHHQ medium portion sizes from the HHHQ database. Total GSH values
are estimated from the same graphical representation as the negative of
the x axis intercept of a line through the point (0,D) with the same slope
as the line through the data from portions A-C (see Fig. 2).
1 GLUTATHIONE DISTRIBUTION IN NATURAL PRODUCTS 7
006
• .~ ~ 400 _/ IO,BJ J
~2oo
.=
-10 0 10 20
GSH Added (~tNO
.~1F .2 Graphical analysis of the GSH content of bell pepper from multiple samples with
a method of additions. Integral values from replicate samples homogenized in solutions A,
B, and C were graphed sa a function of the respective amounts of GSH in each solution,
namely, ,0 ,01 and 02 ~M. These are identified sa points (0,A), (10,B), and (20,C) (filled
circles). Extrapolation of the linear least-squares best-fit line back to the x axis provides an
estimate of the negative of the GSH content. For most foods, the slope of this line approximated
that of the GSH contents of solutions A-C without added food sample (open circles). For
some foods, GSH-reactive materials result in less than complete recovery of added GSH
(filled squares). Extrapolation of the linear least-squares best-fit line for these points thus
swolla a correction for incomplete recovery. For estimation of GSHt, the integral value for
the sample taken in solution D is plotted sa (0,D), and a line si drawn through this point
with a slope equivalent to the best tif for the corresponding solutions A-C. The negative of
the x intercept is taken sa the GSHt concentration. All values are corrected for dilution prior
to expression per wet weight, dry weight, or total nitrogen.
Glutathione Content in Food Groups
Although knowledge of GSH contents of foods remains incomplete,
studies have provided insight into the range of values and provide some
useful generalizations. Of particular importance, considerable variation in
GSH content occurs in different foods, and estimated daily intakes of GSH
vary considerably between individuals.
The GSH contents of representative foods are given in Table I. Dairy
products, eggs, fats, and oils, most beverages, and most cereal grain products
have very little GSH. In contrast, fresh fruits and vegetables and freshly
cooked meats are high in GSH. Frozen foods generally have GSH contents
comparable to fresh foods, but other forms of preservation usually result
