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Basic nutritional investigation

Oral glutamine protects against cyclophosphamide-inducedcardiotoxicity in experimental rats through increase of

cardiac glutathione

Valentina Todorova, Ph.D.a,*, Doug Vanderpool, M.D.a, Sarah Blossom, Ph.D.a,Emmanuel Nwokedi, M.D.a, Leah Hennings, DVM, Ph.D.b, Robert Mrak, M.D., Ph.D.b, and

V. Suzanne Klimberg, M.D.a

a Department of Surgery, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USAb Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA

Manuscript received July 15, 2008; accepted January 9, 2009.

bstract Objective: This study evaluated the effects of supplemental oral glutamine (GLN) on acutecardiotoxicity of cyclophosphamide (CPA) in experimental rats. The dose-related cardiotoxicity ofCPA is associated with a rapid decrease in cardiac glutathione (GSH) and oxidative cardiac injury.GLN is a rate-limiting precursor for GSH synthesis during periods of oxidative and other types ofstress when it becomes a conditionally essential amino acid.Methods: Forty-four male Fischer 344 rats were randomized into two groups to receive 1g · kg�1 · d�1 of GLN or glycine by gavage. After 2 d of prefeeding, each of these groups wasfurther randomized into three subgroups to receive intraperitoneally a lethal dose of CPA (450mg/kg), a sublethal dose of CPA (200 mg/kg), or saline (controls). Twenty-four hours later all sixgroups of rats were sacrificed and blood GLN was measured. Cardiac tissue was examined forhistopathologic alterations: GSH and oxidized GSH concentrations.Results: The results showed that dietary GLN decreased cardiac necrosis and maintained normalcardiac GSH levels. Elevated cardiac GSH levels in the GLN group correlated with increasedarterial GLN levels. GLN protected against the acute cardiotoxic effects of CPA and significantlyimproved the short-term survival after lethal and sublethal doses of CPA.Conclusion: These data suggest that GLN may protect against CPA-related cardiac injury throughmaintenance of cardiac GSH metabolism. © 2009 Published by Elsevier Inc.

Nutrition 25 (2009) 812–817www.nutritionjrnl.com

eywords: Cyclophosphamide; Heart; Cardiotoxicity; Glutamine; Glutathione

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ntroduction

Cyclophosphamide (CPA) is an alkylating agent withotent antineoplastic and immunosuppressive propertiesnd possibly the most widely used anticancer drug [1,2].ardiotoxicity associated with high-dose CPA has beenescribed as a complication of several therapeutic regimens3–6]. The incidence of fatal cardiomyopathy varies from.0% to 17.0%, depending on the different regimens and

This study was supported in part by a grant from the Susan G. Komenreast Cancer Foundation to V.K.T.

* Corresponding author. Tel.: � 501-686-6504; fax: � 501-686-8165.

4E-mail address: todorovavalentinak@uams.edu (V. Todorova).

899-9007/09/$ – see front matter © 2009 Published by Elsevier Inc.oi:10.1016/j.nut.2009.01.004

atient populations [7]. In contrast to cardiomyopathy oc-urring months to years after high cumulative doses of anthra-yclines, CPA-induced cardiomyopathy occurs within the ini-ial 2 or 3 wk after treatment [7,8]. Appelbaum et al. [9]bserved acute heart failure 5 to 9 d after treatment in 4 of5 patients treated with CPA 45 mg · kg�1 · d�1 for 4 d.ostmortem examination of the heart revealed fibrin micro-

hrombi in capillaries and fibrin strands within myocytes.oldberg et al. [10] reported congestive heart failure in 17%f patients treated with CPA at 50 mg · kg�1 · d�1 for 4 d,ith a 43% mortality rate. These investigators observed a

ignificant increase in the incidence of CPA-induced cardiacoxicity in patients receiving a dose of 1.55 g · m�2 · d�1 for

d. Acute heart failure secondary to cardiotoxicity has been

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eported about 1 wk after CPA administration, and thencidence rate is about 20% and mortality about 8% afterone marrow transplantation [3,10]. Gottdiener et al. [3]eported congestive heart failure in 28% of patients treatedith 180 mg/kg of CPA within 3 wk of CPA administration.The pathogenesis of CPA-induced cardiotoxicity is

hought to involve direct endothelial damage, leading inurn to leakage of plasma proteins and erythrocytes [7]. Theistologic findings indicate acute pericarditis and hemor-hagic myocarditis with fibrin platelet microthrombi in cap-llaries and fibrin strands in the interstitium on ultrastruc-ural examination [4]. Wall thickening due to interstitialdema and hemorrhage may decrease left ventricular dia-tolic compliance as left ventricular diastolic dysfunctionnd present as restrictive cardiomyopathy. CPA is an inac-ive prodrug that requires metabolic activation by theytochrome P-450 system. The process of CPA activationroduces hydroxylated active metabolites, e.g., acrolein,hosphoramide mustard, and nitrogen mustard, believed toe toxic, or the inactive compound carboxyphosphamide2]. CPA metabolites can react with carboxyl (-C[O]OH),ercapto (-SH), amino (-NH2), phosphate (-PO3H2), and

ydroxyl (-OH) groups and can form cross-links with DNAnd proteins [8,11,12]. CPA is believed to exert its cardio-oxic effects through damage of the endocardial capillaryndothelium, resulting in increased permeability and micro-hromboses and extravasation of plasma and red blood cellsnto the myocardium [8,13]. CPA administration has beenssociated with increased lipid peroxidation and significantepletion of antioxidant molecules, including glutathioneGSH), catalase, and superoxide dismutase [14–16]. Multi-le clinical studies have suggested that the use of antioxi-ants in combination with chemotherapy and irradiationrolong the survival time of patients compared with ex-ected outcome without antioxidant supplements [17–19].

Glutamine (GLN) is a non-essential amino acid that servesot only as a primary respiratory fuel but also as a necessaryubstrate for nucleotide synthesis in most dividing cells [20].

elbourne [21] demonstrated in kidney tissue under oxidativetress that GLN becomes rate limiting for GSH synthesis. Theafety of enteral and parenteral GLN has been established inumerous studies (reviewed by Sacks [22]).

This study aimed to evaluate the effects of dietary GLNn cardiotoxicity of CPA. We hypothesized that oral GLNould prevent CPA-mediated cardiotoxicity by upregulatingardiac GSH levels and thus would be beneficial to theherapy of patients with cancer.

aterials and methods

xperimental animals and treatment

All studies were approved by the animal care and useommittee at the John L. McClellan Veteran’s Hospital.

orty-four male Fischer 344 (300-g) rats were obtained i

rom SASCO Inc. (Omaha, NE, USA). The rats were main-ained in standard cages in the animal care facility and wereubjected to a 12-h dark/light cycle. During the study pe-iod, the rats were given water ad libitum and were pair-fedpredefined research diet of chow (Harlan Teklad, Madi-

on, WI, USA), which contains 1.84% GLN but no antioxi-ants. After an acclimation period of 1 wk the rats wereandomized to receive 1 g · kg�1 · d�1 of supplementalLN or glycine (GLY) by gavage for a period of 48 hefore and 24 h after chemotherapy with CPA. All rats wereacrificed 24 h after the initiation of CPA. Control animalsere treated identically except that no chemotherapy wasiven.

After 2 d of prefeeding with GLN or GLY by gavage, theats were randomized into one of six groups receiving aethal intraperitoneal dose (LD50 8 h) of CPA (450 mg/kg,),

sublethal intraperitoneal dose of CPA (200 mg/kg), orntraperitoneal saline (control [CON]). Thus the studyroups were as follows: 1) GLN � 450 (n � 10), 2) GLY �50 (n � 10), 3) GLN � 200 (n � 6), 4) GLY � 200 (n �), 5) GLN � CON (n � 6), and 6) GLY � CON (n � 6).he rats were observed for morbidity and mortality. Sur-iving rats were sacrificed at 24 h.

Twenty-four hours after the CPA (or saline) injection theurviving rats were anesthetized intraperitoneally with ket-mine (0.75 mg/100 g of body weight) and acepromazine0.075 mg/100 g of body weight) and heparinized. Arteriallood was withdrawn from the aorta and examined forematocrit, white blood cell count, and GLN content. Ahoracotomy was then performed and the heart removed.entricular muscle was taken for light microscopy and

tudy of GSH metabolism.

LN measurement

Aliquots of heparinized whole blood were mixed withqual volumes of cold 10% perchloric acid, vortex-mixed,nd centrifuged at 5°C at 3000 � g for 15 min (Sorvallodel RC5). The supernatant was removed and neutralizedith an equal amount of cold 0.48 mol/L of potassiumhosphate (Sigma Chemical Co., St. Louis, MO, USA). Thisas again vortex-mixed and centrifuged at 5°C at 3000 � g for0 min. The supernatant was removed and kept frozen at20°C for later determination of GLN concentration by theicroanalytical method described by Bernt and Bergmeyer

23] and Nahorski [24]. For glutamate (GLU) assay, 100 �Lbove the supernatant was added to 1 mL of reaction mix-ure that contained 0.73 mg of �-nicotinamide adenine dinu-leotide, 10 �L of 0.1 mol/L of ethylenediamine-tetra-aceticcid, 1 �/L of mercaptoethanol in Tris buffer, and 10 �L oflutamate dehydrogenase. The reaction mixture was incu-ated in a 37°C water bath for 30 min, and the fluorescenceas measured using a fluorometer. To assay for total GLN

nd GLU, 50 �L of the supernatant was added to 0.5 mL of.2 mol/L of sodium acetate and 0.025 U of glutaminase,

ncubated in a 37°C shaking water bath for 45 min, and the

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814 V. Todorova et al. / Nutrition 25 (2009) 812–817

eaction was stopped with 0.5 mL of water. This was fol-owed by the procedure described earlier. The GLN andLU concentrations were determined by a standard curve.he net GLN concentration was calculated by subtracting

he GLU concentration from the total GLN plus GLU con-entration. The results are expressed as micromoles periter.

SH measurement

Total GSH and oxidized GSH (GSSG) contents in theearts were measured by a standard enzymatic recyclingethod, as described by Tietze [25] and modified by Ander-

on [26]. Briefly, 0.5 g of tissue was homogenized with 2.5L of 5% 5-sulfosalicylic acid (Sigma Chemical Co.), the

rotein content was measured, and the samples were cen-rifuged at 5000 � g at 4°C for 15 min. Ten microliters ofhe supernatant was added to 1 mL of reaction mix (0.2mol/L of reduced nicotine amide adenine dinucleotide

hosphate, 0.6 mmol/L of 5,5-dithio-bis-2-nitrobenzoiccid, and 1.33 U of GSH reductase) and the absorbance waseasured at 412 nm. To determine GSH disulfide (GSSG)

ontent, 0.5 mL of the supernatant was mixed with 10 �L of-vinyl pyridine and 60 �L of triethanolamine to removeSH by the method of Griffith [27] and then measured

ccording to the procedures described earlier. The data wereormalized by milligram of protein and expressed as nano-oles per milligram of protein.

istopathologic studies

Specimens for light microscopy were immediately fixedn 10% buffered formalin and subsequently embedded inaraffin media. Several 6-�m tissue sections were cut fromach paraffin block and mounted on glass slides. The slidesere stained with hematoxylin and eosin. Histologic eval-ation of the mucosal and tumor specimens was made in alinded fashion. Pathology was graded based on the pres-nce and severity of five parameters, including edema, leu-ocytic infiltration, muscle necrosis, chronic inflammation,nd fibrosis edema, leukocytic infiltration, muscle necrosis,hronic inflammation, and fibrosis. Grading for each com-onent was performed by using a semiquantitative scalehere 0 was normal and 1–4� represented mild through

evere abnormalities. The total cardiac injury score for eacheart was a calculated as an average of all the componentnjury scores.

tatistical analysis

Comparisons across groups were performed using anal-sis of variance and non-paired two-tailed t test (StatView.5 for Windows). All data was expressed as mean �tandard error. Results with P � 0.05 were considered

tatistically significant.

esults

urvival and body weights

One hundred percent survival was established in theLN-supplemented group of rats that received lethal

GLN � 450) and non-lethal (GLN � 200) doses of CPAnd in the control groups of rats (treated with saline). In theLY � 450 group, however, survival was only 20% (P �.05). Body weight was not significantly different in all sixroups.

ffect of GLN on cardiac GSH metabolism

The GSH level in the GLY � 200 group was signifi-antly lower than the GSH level in all other groups, includ-ng GLN � 200 and GLN � 450 (Table 1). Although onlywo animals survived 24 h in the GLY � 450 group, theardiac GSH in this group of animals was not significantlyifferent from the controls, possibly representing biologicalariation. The GSSG/GSH ratio was significantly elevatedn the GLY � 200 group versus that of the GLN � 200roup (159 � 18 in the GLY � 200 group versus 108 � 12n the GLY � 200, P � 0.05). The GSSG/GSH ratio of theLN � 450 group was not significantly different from the

ontrols.

ffect of dietary GLN on arterial GLN levels

Whole blood arterial GLN concentrations were obtainedn the GLN � 200, GLY � 200, GLN � CON, and GLY �ON groups. Arterial GLN levels were significantly de-reased only in the GLY � 200 group (504.5 � 55.6 versus54.7 � 22.2 in GLY � 200 versus GLN � 200, P � 0.05).rterial GLN in the GLN � 200 group was not signifi-

able 1ffect of dietary GLN on cardiac GSH metabolism of rats treated with

ethal (450 mg/kg of body weight) and sublethal (200 mg/kg) doses ofyclophosphamide

roups Reduced GSH(�g/g tissue)

Total GSH(�g/g tissue)

Oxidative injuryGSSG/GSH � 103

LN � 450 2.46 � 0.14 2.83 � 0.17 136 � 14LY � 450 2.48 � 0.24 2.78 � 0.2 123.4 � 25‡

LN � 200 2.32 � 0.07 2.57 � 0.07 108 � 12LY � 200 1.66 � 0.03* 1.89 � 0.06* 159 � 18†

LN � CON 2.34 � 0.16 2.63 � 0.15 131 � 21LY � CON 2.05 � 0.13 2.40 � 0.16 145 � 20

CON, control; GLN � 200, glutamine plus 200 mg/kg of cyclophosph-mide; GLN � 450, glutamine plus 450 mg/kg of cyclophosphamide;LN � CON, glutamine plus saline; GLY � 200, glycine plus 200 mg/kgf cyclophosphamide; GLY � 450, glycine plus 450 mg/kg of cyclophos-hamide; GLY � CON, glycine plus saline; GSH, glutathione; GSSG,xidized glutathione* P � 0.05, GLY � 200 versus all other groups.† P � 0.05, GLY � 200 versus GLN � 200 group.

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antly different from the control group (587.9 � 31.0ersus 597.3 � 27.0, P � NS).

ardiac pathology

Cardiac pathologic injury scores were determined for theLN � 200, GLY � 200, GLN � CON, and GLY � CONroups (Table 2). The pathologic injury score was elevatednly the GLY � 200 group. The cardiac injury scoresorrelated with the presence of muscle necrosis, edema, andnflammation seen in GLY � 200 group (Fig. 1A) com-ared with the normal cardiac muscle tissue observed inhe GLN � 200 group (Fig. 1B).

iscussion

The results of this study showed that the provision of aLN-enriched diet to animals receiving high doses of CPA

an maintain normal cardiac GSH levels. This appears toonfer survival enhancement even in rats receiving lethaloses of CPA. Further, supplemental oral GLN in this studyrevented acute CPA-induced cardiac myonecrosis and de-reased oxidant injury as determined by the GSSG/GSHatio and pathologic injury score. The GSSG/GSH ratio,hich is indicative of acute oxidative injury, was signifi-

antly elevated in the GLY � 200 group versus that of theLN � 200 group, and the pathologic injury score was

levated only the GLY � 200 group. This observation hasmportant clinical implications and suggests that GLN mayave more than just nutritional advantages.

Although cardiac toxicity is a well-recognized compli-ation of chemotherapeutic regimens, no clinically effectivereatment is presently available. The oxidative stress andormation of reactive oxygen species that occur with cyto-oxic drugs administration cause depletion of GSH, peroxi-ation of cell membranes, denature proteins, and result inell death [28].

Glutamine is not traditionally thought of as a rate-limitingubstrate for the synthesis of GSH, although in the pres-

able 2chematic representation of cardiac injury score determined by scoringf 10 random fields on slides of hearts stained with hematoxylin andosin at 24 h

xperimental groups Cardiac injury score*

LN � 200 0.4 � 0.2LY � 200 1.83 � 0.5†

LN � CON 0.4 � 0.2LY � CON 0.7 � 0.5

CON, control; GLN � 200, glutamine plus 200 mg/kg of cyclophosph-mide; GLN � CON, glutamine plus saline; GLY � 200, glycine plus 200g/kg of cyclophosphamide; GLY � CON, glycine plus saline* The injury was score for five parameters: edema, leukocytic infiltra-

ion, muscle necrosis, chronic inflammation, and fibrosis.† Only the GLY � 200 group showed a significant elevation above the

sontrol groups or the GLN � 200 group (P � 0.05).

nce of oxidative stress GLN might become rate limitingn GSH synthesis [21]. GSH is a key regulator of the cellularedox state and the redox environment within the tumorells determines the response of tumors (and protection ofhe normal cells) to chemotherapy and radiation [28–31].everal reports have suggested that the GSH constituentmino acids, including GLN, inhibit tumor promotion, ateast in part, by their interference with the GSH metabolism32–34]. The safety of enteral and parenteral GLN and itsenefits in improving amino acid metabolism [35], immuneunction [36], and outcome in normal volunteers and pa-ients with catabolic diseases [37] has been established inumerous studies, including at least 18 clinical trials [22]. Ahase II study published by our group demonstrated the

ig. 1. (A) Sections of heart ventricle from the group that received glu-amine plus 200 mg/kg of cyclophosphamide reveal individual cardiacuscle cells arranged in diffuse bundles in a connective tissue framework.

ndividual myocytes are seen in cross section to be well stained andreserved. They have centrally located nuclei with abundant cytoplasmutlined by distinct and intact cell walls. The myocytes essentially appearormal. Magnification 200�. (B) Sections of heart ventricle from theroup that received glutamine plus 200 mg/kg of cyclophosphamide revealnjured myocytes with scattered coagulative changes and thin bands ofontraction necrosis. Magnification 400�.

afety and efficacy of oral GLN in escalating doses of

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ethotrexate [38]. We previously showed also that GLNupplementation stimulated host GSH production and de-reased intratumoral GSH levels in breast cancer modelsreviewed by Klimberg [20]).

The present study showed that supplemental oral GLNot only protects against acute cardiotoxic death secondaryo CPA but also modulates GSH metabolism within theentricular muscle in sublethal doses of CPA.

Clinical studies have shown that patients who receiventioxidants with the standard chemotherapy tolerate thereatment better and have prolonged survival time comparedith expected outcome without the antioxidant supplements

19,39–42]. Except for three specific interactions (fla-onoids with tamoxifen, N-acetyl-cysteine with doxorubi-in, and �-carotene with 5-fluorouracil), there is no evi-ence to date that antioxidants interfere with conventionalancer therapeutics in vivo (reviewed by Lamson et al.43]); moreover, data showed that antioxidants and chemo-herapy may enhance the effectiveness of the treatment20,44].

The data presented in this report and the previous resultsrom our group and other investigators suggest an importantole for dietary GLN in intensifying chemotherapeutic reg-mens containing CPA while decreasing their toxicities. Inddition, because of the possible synergistic effect of CPAnd doxorubicin, GLN might be an important adjuvant inoxorubicin/cyclophosphamide breast cancer protocols.

eferences

[1] Gershwin ME, Goetzl EJ, Steinberg AD. Cyclophosphamide: use inpractice. Ann Intern Med 1974;80:531.

[2] Tew K, Colvin OM, Chabner BA. Alkylating agents. In: Chabner BA, Longo DL, editors. Cancer chemotherapy and biotherapy: princi-ples and practice. 2nd ed. Philadelphia: Lippincott-Raven; 1996.

[3] Gottdiener JS, Applebaum ER, Ferrans VJ, Deisseroth A, Ziegler J.Cardiotoxicity associated with high-dose cyclophosphamide therapy.Arch Intern Med 1981;141:758–63.

[4] Birchall IW, Lalani Z, Venner P, Hugh J. Fatal haemorrhagic myo-carditis secondary to cyclophosphamide therapy. Br J Radiol 2000;73:1112–4.

[5] Kamezaki K, Fukuda T, Makino S, Harada M. Cyclophosphamideinduced cardiomyopathy in a patient with seminoma and a history ofmediastinal irradiation. Intern Med 2005;44:120–3.

[6] Brockstein BE, Smiley C, Al-Sadir J, Williams SF. Cardiac andpulmonary toxicity in patients undergoing high-dose chemotherapyfor lymphoma and breast cancer: prognostic factors. Bone MarrowTransplant 2000;25:885–94.

[7] Taniguchi I. Clinical significance of cyclophosphamide-induced car-diotoxicity. Intern Med 2005;44:123–35.

[8] Nieto Y, Cagnoni PJ, Bearman SI, et al. Cardiac toxicity followinghigh-dose cyclophosphamide, cisplatin, and BCNU (STAMP-I) forbreast cancer. Biol Blood Marrow Transplant 2000;6:198–3.

[9] Appelbaum FR, Strauchen JA, Graw RG, et al. Acute lethal carditiscaused by high-dose combination chemotherapy: a unique clinicaland pathological entity. Lancet 1976;1:58–62.

10] Goldberg MA, Antin JH, Guinan EC, Rappeport JM. Cyclophosph-amide cardiotoxicity: an analysis of dosing as a risk factor. Blood

1986;68:1114–8.

11] Fleming RA. An overview of cyclophosphamide and isosfamidepharmacology. Pharmacotherapy 1997;17:146S–54.

12] Huang Z, Waxman DJ. Modulation of cyclophosphamide-based cy-tochrome P450 gene therapy using liver P450 inhibitors. Cancer GeneTher 2001;8:450–8.

13] Slavin RE, Millan JC, Mullins GM. Pathology of high dose intermit-tent cyclophosphamide therapy. Hum Pathol 1975;693–709.

14] Patel JM, Block ER. Cyclophosphamide-induced depression of theantioxidant defense mechanisms of the lung. Exp Lung Res 1985;8:153–65.

15] Patel JM. Stimulation of cyclophosphamide-induced pulmonary mi-crosomal lipid peroxidation by oxygen. Toxicology 1987;45:79–91.

16] Dorr RT, Lagel K. Effect of sulfhydryl compounds and gluta-thione depletion on rat heart myocyte toxicity induced by 4-hydroperoxycyclophosphamide and acrolein in vitro. Chem Biol In-teract 1994;93:117–28.

17] Sudharsan PT, Mythili Y, Selvakumar E, Varalakshmi P. Lupeol andits ester meliorate the cyclophosphamide provoked cardiac lysosomaldamage studied in rat. Mol Cell Biochem 2006;282:39–44.

18] Manda Kl, Bhatia AL. Prophylactic action of melatonin againstcyclophosphamide-induced oxidative stress in mice. Cell Biol Toxi-col 2003;19:367–72.

19] Singh DK, Lippman SM. Cancer chemoprevention part 1: retinoidsand carotenoids and other classic antioxidants. Oncology 1998;12:1643–60.

20] Klimberg VS. Glutamine, cancer and its therapy. Am J Surg 1996;172:418–24.

21] Welbourne T. Ammonia production and glutamine incorporation intoglutathione in the functioning rat kidney. Can J Biochem 1979;57:233–7.

22] Sacks GS. Glutamine supplementation in catabolic patients. AnnPharmacother 1999;33:348–54.

23] Bernt E, Bergmeyer HU. L-Glutamate UV-assay with glutamatedehydrogenase and NAD. In: Bergmeyer HU, editor. Methods ofenzymatic analysis. Volume 4. 2nd English ed. Deerfield Beach, FL:Verlag-Chemie International; 1974, p. 1704–8.

24] Nahorski SR. Fluorometric measurement of glutamine and asparagineusing enzymic methods. Anal Biochem 1971;42:136–42.

25] Tietze F. Enzymatic method for quantitative determination of nano-gram amounts of total and oxidized glutathione: applications to mam-malian blood and other tissues. Ann Biochem 1969;27:502–22.

26] Anderson ME. Enzymatic and chemical methods for the determina-tion of glutathione. In: Dolphin D, editor. Glutathione. Volume 1.New York: John Wiley & Sons; 1983, p. 340–65.

27] Griffith OW. Determination of glutathione and glutathione disulfideusing glutathione reductase and 2-vinylpyridine. Anal Biochem 1980;106:207–12.

28] Mitchell JB, Russo A. The role of glutathione in radiation and druginduced cytotoxicity. Br J Cancer 1987;8(suppl):96S–104.

29] Schafer FQ, Buettner GR. Redox environment of the cell as viewedthrough the redox state of the glutathione disulfide/glutathione cou-ple. Free Radic Biol Med 2001;30:1191–212.

30] Friezen C, Kiess Y, Debatin KM. A critical role of glutathione indetermining apoptosis sensitivity and resistance in leukemia cells.Cell Death Differ 2004;11(suppl 1):S73–85.

31] Tormos C, Javier Chaves F, Garcia MJ, Garrido F, Jover R, O’ConnorJE, et al. Role of glutathione in the induction of apoptosis and c-fosand c-jun mRNAs by oxidative stress in tumor cells. Cancer Lett2004;208:103–13.

32] Perchellet JP, Owen MD, Posey TD, Orten DK, Schneider BA.Inhibitory effects of glutathione level-raising agents and D-alpha-tocopherol on ornithine decarboxylase induction and mouse skintumor promotion by 12-O-tetradecanoylphorbol-13-acetate. Carcino-genesis 1985;6:567–57.

33] Baruchel S, Wang T, Farah R, Jamali M, Batist G. In vivo selectivemodulation of tissue glutathione in a rat mammary carcinoma model.

Biochem Pharmacol 1995;50:1505–8.

[

[

[

[

[

[

[

[

[

[

[

817V. Todorova et al. / Nutrition 25 (2009) 812–817

34] Wang T, Chen X, Schecter RL, Baruchel S, Alaoui-Lamali M, Mel-nychuk D, Batist G. Modulation of glutathione by a cysteine pro-drugenhances in vivo tumor response. J Pharamacol Exp Ther 1996;276:1169–73.

35] Chen K, Nezu R, Sando K, Haque SM, Iiboshi Y, Masunari A, et al.Influence of glutamine-supplemented parenteral nutrition on intesti-nal amino acid metabolism in rats after small bowel resection. SurgToday 1996;26:618–23.

36] O’Riordain MG, De Beaux A, Fearon KC. Effect of glutamine onimmune function in the surgical patient. Nutrition 1996;12(suppl):S82–4.

37] Luo M, Fernandez-Estivariz C, Jones DP, Accardi CR, Alteheld B,Bazargan N, et al. Depletion of plasma antioxidants in surgicalintensive care unit patients requiring parenteral feeding: effects ofparenteral nutrition with or without alanyl-glutamine dipeptide sup-plementation. Nutrition 2008;24:37–44.

38] Rubio IT, Cao Y, Hutchins L, Westbrook KC, Klimberg VS. Effectof glutamine on methotrexate efficacy and toxicity. Ann Surg 1998;

227:772–80.

39] Jaakkola K, Lahteenmaki P, Laakso J, et al. Treatment with antiox-idant and other nutrients in combination with chemotherapy andirradiation in patients with small-cell lung cancer. Anticancer Res1992;12:599–606.

40] Whelan RL, Horvath KD, Gleason NR, et al. Vitamin and calciumsupplement use is associated with decrease adenoma recurrence inpatients with a previous history of neoplasia. Dis Colon Rectum1999;42:212–7.

41] Lamm DL, Riggs DR, Shriver JS, et al. Megadose vitamins in bladdercancer: a double-blind clinical trial. J Urol 1994;151:21–6.

42] Wagdi P, Fluri M, Aeschbacher B, et al. Cardioprotection in patientsundergoing chemo- and/or radiotherapy for neoplastic disease. JpnHeart J 1996;37:353–9.

43] Lamson DW, Matthew S, Brignall MS. Antioxidants in cancer ther-apy; their actions and interactions with oncologic therapies, AlternMed Rev 1999;4:304–29.

44] Conklin KA. Chemotherapy-associated oxidative stress: impact onchemotherapeutic effectiveness. Integr Cancer Ther 2004;3:294 –

300.