Role of Pharmaconutrition in ICU in relation to reducing oxidative stress: The REDOXS study Daren K. Heyland MD Professor of Medicine Queen’s University, Kingston, ON Canada On behalf of the REDOXS Study Investigators

A RANDOMIZED TRIAL OF HIGH-DOSE GLUTAMINE AND ANTIOXIDANTS IN CRITICALLY ILL PATIENTS WITH MULTIORGAN FAILURE The REDOXS study Daren K. Heyland MD Professor of Medicine Queen’s University, Kingston, ON Canada On behalf of the REDOXS Study Investigators

Disclosures The investigator-initiated study was funded by the Canadian Institutes of Health Research. Fresenius Kabi provided intravenous glutamine and enteral supplements, and an unrestricted grant-in-aid. Biosyn provided the intravenous selenium to all participating European sites. None of these agencies had a decisional role in the conception, design, conduct, analysis, interpretation of results or decision to publish.

Mito Function In Search of the Magic Pharmaconutrient Heyland DK, Cook DJ, Guyatt G. Does the Formulation of Enteral Feeding Products Influence Infectious Morbidity and Mortality in the Critically Ill Patient: A Critical Review of the Evidence. Crit Care Med 1994;22:

Glutamine levels drop: - following extreme physical exercice - after major surgery - during critical illness Low glutamine levels are associated with: -immune dysfunction -higer mortality in critically ill patients Novak F, Heyland DK, A Avenell et al., Crit Care Med 2002 Oudemans-van Straaten HM, Bosman RJ, Treskes Met al., Intensive Car Med 2001 Glutamine: A conditionally essential amino acid?

Potential Beneficial Effects of Glutamine Fuel for Enterocytes Lymphocytes NuclotideSynthesis Maintenance of Intestinal Mucosal Barrier Maintenance of LymphocyteFunction Preservation of TCA Function Decreased Free Radical availability (Anti-inflammatory action) GlutathioneSynthesis GLNpool Glutamine Therapy Enhanced Heat Shock Protein Shock Protein Anti-catabolic effect Preservation of Muscle mass ReducedTranslocation Enteric Bacteria or Endotoxins Reduction of Infectious complications Increased Autophagy Increased Autophagy Preserved Cellular Energetics- ATP content GLNPool Critical Illness Enhanced insulin sensitivity

* IV Glutamine Enhances Serum HSP-70 in Critically Ill Patients with Sepsis/SIRS ALA-GLN treatment leads to significant enhancement of serum HSP-70 with 7 days of treatment ALA-GLN mediated enhancement of HSP- 70 correlates with decreased ICU length of stay and time on ventilator Ziegler Intensive Care Medicine, 31: , 2005

Mechanism of Enteral Glutamine 3 RCTs of enteral glutamine Burns patients –Increased plasma glutamine –Improved permeability –Decreased endotoxin levels –Reduced GNB infections –Reduced hospital LOS –Reduced mortality Garrell CCM 2003;31:2444, Zhou JPEN ;241; Peng Burns 2004;30:135

Mortality CCM 2002 Current version posted on

OFR CONSUMPTION OFR PRODUCTION Depletion of Antioxidant Enzymes OFR Scavengers Vitamins/Cofactors Infection Inflammation Ischemia OFR production > OFR consumption = Impaired - organ function - immune function - mucosal barrier function Complications and Death OXIDATIVE STRESS Rationale for Antioxidants

In Critical Illness, Low Levels of Se related to Severity of Illness Manzanares ICM 2009;35:882 Healthy Control s ICU Patients ICU +SIRS ICU +MODS

…and Correlate with GPx activity Manzanares ICM 2009;35:882

Effect on Mortality Current version posted on

The Research Protocol In critically ill patients with a clinical evidence of organ failure … –What is the effect of glutamine supplementation compared to placebo –What is the effect of antioxidant supplementation compared to placebo …on 28 day mortality? The Question(s) N Engl J Med 2013;368:

1200 ICU patients Evidence of Multi-organ failure R glutamine placebo Concealed Stratified by site R R antioxidants placebo Factorial 2x2 design Double blind treatment placebo antioxidants The REDOXS study

The Research Protocol Adults (>18) With 2 or more organ failures related to their acute illness : – Requiring mechanically ventilation (P/F<300) – Clinical evidence of hypoperfusion defined by need for vasopressor agents for more than 2 hour –Renal dysfunction : Cr>171 or <500ml/24 hrs –platelet < 50 Inclusion Criteria

The Research Protocol > 24 hrs from admission to ICU Not expected to survive Lack of commitment to full aggressive care Absolute contraindication to EN Severe acquired brain injury –severe head trauma –post cardiac arrest with suspicion of anoxic injury –Grade 4 or 5 SAH –stroke resulting in coma or Intubation Routine cardiac surgery Seizure disorder requiring treatment Burns (>30% BSA) Exclusion Criteria

The Research Protocol Weight 200kg pregnant or lactating prior randomization enrollment in other ICU interventional studies (co-enrollment with PROTECT allowable) End stage cirrhosis Metastatic cancer Exclusion Criteria

Optimizing the Dose of Glutamine Dipeptides and Antioxidants In Critically Ill Patients: A Phase I dose finding study High dose appears safe High dose associated with –no worsening of SOFA Scores –greater resolution of oxidative stress –greater preservation of glutathione –Improved mitochondrial function Heyland JPEN Mar 2007 ParenterallyEnterally Glutamine/day0.35 gms/kg30 gms Antioxidants per day 500 mcg Selenium Vit C 1500 mg Vit E 500 mg B carotene 10 mg Zinc 20 mg Se 300 ug

Glutamine Dipeptides Free L-glutamine has limited solubility and stability Synthetic dipeptides (ala-gln, gly-gln) overcome these difficulties 8.5 gms of dipeptide=6 gms of glutamine Vit C 1500 mg Vit E 500 mg B-carotene 10 mg Zinc 20mg Selenium 300ug Glutamine 30 gms

Enteral Study Supplement EN REDOXS © Formula

Parenteral Study Supplements Dipeptiven and Selenium + Selenium (Biosyn)

Study supplements started within 24 hrs of admission Continued for 28 days or until death or discharge from ICU

Results N Engl J Med 2013;368:

Patient Flow

Patient Characteristics (1) Patient CharacteristicsPLACEBOGLNAOXAOX+GLN P values n Age * 62.8±13.7 ( ) 62.5±15.0 ( ) 63.6±14.3 ( ) 64.3±14.0 ( ) 0.40 Sex 0.45 Female 122 (40.7%)110 (36.5%)130 (42.3%)130 (41.9%) Male 178 (59.3%)191 (63.5%)177 (57.7%)180 (58.1%) APACHEII score * 26.0± 7.4( ) 26.6± 7.6( ) 25.9± 7.1( ) 26.8± 7.4 ( ) 0.39 Charlson Comorbidity Index * 1.7± 1.8( ) 1.5± 1.6( ) 1.8± 1.8( ) 1.8± 1.8( ) 0.24 Functional Comorbidity Index * 1.5± 1.4( ) 1.4± 1.4( ) 1.5± 1.4( ) 1.5± 1.3( ) 0.28 Admission 0.42 Medical 236 (78.7%)238 (79.1%)254 (82.7%)235 (75.8%) Surgical: Elective 26 (8.7%)27 (9.0%)19 (6.2%)35 (11.3%) Surgical: Emergency 38 (12.7%)36 (12.0%)34 (11.1%)40 (12.9%) Primary ICU diagnosis 0.15 Cardiovascular / vascular 70 (23.3%)54 (17.9%)53 (17.3%)60 (19.4%) Respiratory 97 (32.3%)101 (33.6%)94 (30.6%)83 (26.8%) Gastrointestinal 17 (5.7%)21 (7.0%)32 (10.4%)25 (8.1%) Neurologic 2 (0.7%)4 (1.3%)5 (1.6%)2 (0.6%) Sepsis 86 (28.7%)88 (29.2%)98 (31.9%)106 (34.2%) Trauma 10 (3.3%)9 (3.0%)5 (1.6%)7 (2.3%) Metabolic 6 (2.0%)8 (2.7%)9 (2.9%)5 (1.6%) Hematologic 0 (0.0%)2 (0.7%)0 (0.0%)4 (1.3%) Renal 0 (0.0%)2 (0.7%)0 (0.0%)5 (1.6%) Gynecologic 1 (0.3%)0 (0.0%) Orthopedic 0 (0.0%)1 (0.3%)2 (0.7%)3 (1.0%) Other 11 (3.7%) 9 (2.9%)10 (3.2%)

Patient Characteristics (2) Patient CharacteristicsPLACEBOGLNAOXAOX+GLN P values n Etiology of shock 0.79 Cardiogenic 72 (24.0%)54 (17.9%)57 (18.6%)57 (18.4%) Septic 191 (63.7%)206 (68.4%)218 (71.0%)211 (68.1%) Neurogenic 2 (0.7%)3 (1.0%) 2 (0.6%) Anaphylactic 0 (0.0%)1 (0.3%) 0 (0.0%) Hemorrhagic 13 (4.3%)10 (3.3%)9 (2.9%)16 (5.2%) Uncertain Origin 11 (3.7%)15 (5.0%)13 (4.2%)14 (4.5%) Not in shock 9 (3.0%) 4 (1.3%)5 (1.6%) Other 2 (0.7%)3 (1.0%)2 (0.7%)5 (1.6%) Ethnicity 0.15 White 274 (91.3%)262 (87.0%)274 (89.3%)284 (91.6%) Black or African American 7 (2.3%)15 (5.0%)9 (2.9%)3 (1.0%) Hispanic 2 (0.7%)5 (1.7%)6 (2.0%)6 (1.9%) Asian or Pacific Islander 9 (3.0%)11 (3.7%)8 (2.6%)10 (3.2%) Native 2 (0.7%)5 (1.7%)8 (2.6%)6 (1.9%) Other 6 (2.0%)3 (1.0%)2 (0.7%)1 (0.3%) Inclusion criteria A PaO2/FiO2 ratio of ≤ (94.0%) 285 (94.7%) 287 (93.5%) 285 (91.9%) 0.56 Clinical evidence of hypo-perfusion 277 (92.3%) 278 (92.4%) 286 (93.2%) 293 (94.5%) 0.68 Renal dysfunction 104 (34.7%) 117 (38.9%) 99 (32.2%) 122 (39.4%) 0.20 A platelet count of ≤50 mm3 16 (5.3%) 21 (7.0%) 12 (3.9%) 18 (5.8%) 0.42

Patient Characteristics (3) Patient CharacteristicsPLACEBOGLNAOXAOX+GLN P values n Hours in ICU prior to randomization† 17.9[13.4 to 21.5] 17.7[12.7 to 21.1] 18.4[12.3 to 21.5] 18.0[13.3 to 21.6] 0.85 Number of organ failures (0.3%)2 (0.7%)1 (0.3%)0 (0.0%) 2221 (73.7%)206 (68.4%)236 (76.9%)216 (69.7%) 376 (25.3%)85 (28.2%)69 (22.5%)90 (29.0%) 42 (0.7%)8 (2.7%)1 (0.3%)4 (1.3%) First organ dysfunction to initiation of EN supplements(hours)† 23.0[17.0 to 27.3] 21.4[15.9 to 26.4] 21.5[16.8 to 26.0] 21.7[17.0 to 27.0] 0.52 First organ dysfunction to initiation of PN supplements(hours)† 22.3[16.5 to 26.5] 21.0[14.8 to 25.0] 21.1[16.0 to 25.5] 21.5[16.3 to 26.0] 0.21 *mean±SD(min,max) † Median [IQR](min,max) Counts and percentage for all categorical variables

Nutritional Outcomes Glutamine No Glutamine p values AntioxidantsNo Antioxidants P values * Adequacy of calories from total nutrition Mean±SD(range) 47.8±29.0 ( ) 48.4±29.5 ( ) ±29.7 ( ) 47.9±28.8 ( ) 0.74 Adequacy of protein from total nutrition Mean±SD(range) 43.4±27.7 ( ) 43.2±27.9 ( ) ±28.3 ( ) 42.8±27.3 ( ) 0.60 Adequacy of calories from EN Mean±SD(range) 45.0±29.4 ( ) 46.1±30.1 ( ) ±29.9 ( ) 45.7±29.5 ( ) 0.84 Adequacy of protein from EN Mean±SD(range) 40.8±27.8 ( ) 41.1±28.2 ( ) ±28.3 ( ) 40.8±27.7 ( ) 1.00 Type of Nutrition EN Only 459 (75.1%)466 (76.8%)462 (74.9%)463 (77.0%) PN Only 8 (1.3%)13 (2.1%)14 (2.3%)7 (1.2%) EN+PN 37 (6.1%)30 (4.9%)35 (5.7%)32 (5.3%) None 107 (17.5%)98 (16.1%)106 (17.2%)99 (16.5%) Time of initiation of EN(hours) Mean±SD(range) 28.8±40.6 ( ) 26.2±25.7 ( ) ±38.9 ( ) 26.2±28.3 ( ) 0.94

Compliance with Study Procedures Glutamine No Glutamine p values Antioxidants No Antioxidants P values * % EN supplements received Mean±SD (range) 71.0±26.4 ( ) 70.9±27.2 ( ) ±26.7 ( ) 71.7±26.9 ( ) 0.18 % PN supplements received Mean±SD (range) 88.8±17.6 ( ) 89.5±17.8 ( ) ±18.0 ( ) 89.2±17.3 ( ) 0.74 Duration of EN study supplement Median (Q1,Q3) 6.8 [3.2 to 13.6] 6.8 [3.5 to 13.2] [3.3 to 13.0] 6.9 [3.2 to 13.8] 0.61 Duration of PN study supplement Median (Q1,Q3) 7.9 [4.8 to 14.8] 7.9 [5.0 to 14.0] [4.9 to 14.7] 7.9 [5.0 to 14.5] 0.63

Primary Outcome

Primary outcome of 28 day mortality using all 1218 evaluable patients (ITT) Glutamine (glut) Antioxidants (AOX) YesNo AOX OR conditioned on Glut Overall adjusted OR of AOX Yes 101/310 (32.6%)97/301 (32.2%) 1.02 (0.72, 1.43) 1.09 ( ; p=0.48*) No 89/307 (29.0%)76/309 (25.3%) 1.20 (0.84, 1.72) Glut OR conditioned on AOX 1.18 ( )1.40 ( ) Overall adjusted OR for glut 1.28 ( ; p=0.049*) AOX by glut interaction p=0.49 OR=odds ratio. ORs are presented with 95% confidence intervals in parentheses. An OR>1 indicates increased mortality with treatment. All odds ratios adjust for presence of shock at baseline. Overall ORs also adjust for other treatment factor. *To account for the two interim analyses, we pre-specified statistical significance of the final analysis at a two-sided p<0.044 in our protocol. Thus, our primary outcome did not reach statistical significance for either intervention.

Primary outcome of 28 day mortality using 1025 patients who received ≥5 days of supplements Glutamine (glut) Antioxidants (AOX) YesNo AOX OR conditioned on Glut Overall adjusted OR of AOX Yes55/255 (21.6%)60/246 (24.4%)0.85 ( ) 0.98 ( ; p=0.90) No55/263(20.9%)49/261 (18.8%)1.14 ( ) Glut OR conditioned on AOX1.03 ( )1.39 ( ) Overall adjusted OR for glut 1.20 ( ; p=0.23) AOX by glut interaction p=0.33 OR=odds ratio. ORs are presented with 95% confidence intervals in parentheses. An OR>1 indicates increased mortality with treatment. All odds ratios adjust for presence of shock at baseline. Overall ORs also adjust for other treatment factor.

Mortality Outcomes P=0.07 P=0.049 P=0.02 Note: all P values pertain to GLN vs No GLN; no significant differences between AOX vs. No AOX

Overall 6 Month Survival All evaluable patients (ITT) Glutamine vs. no-Glutamine (P=0.02)

Overall 6 Month Survival All evaluable patients (ITT) Antioxidant vs. no- Antioxidant (P=0.87)

Pre-specified Sub-group Analysis Glutamine vs. No Glutamine 28 day mortality, OR with 95% CI)

Pre-Specified Sub-group Analysis Antioxidant vs. No Antioxidants 28 day mortality, OR with 95% CI

Other Clinical Outcomes No differences between groups –SOFA –Need for dialysis –Duration of mechanical ventilation –PODS –infections –ICU and Hospital LOS

Effect of Glutamine on HRQOL Short Form-36 at 3 months

Effect of Glutamine on HRQOL Short Form-36 at 6 months

Plasma Levels of Glutamine in Subset of Patients P <0.001

Plasma Levels of Selenium in Subset of Patients P <0.001

Adverse Events ARM PLACEBOGLNAOXAOX+GLN n Number of patients ever had an UREA>50 12(4.0%)43(14.3%)12(3.9%)39(12.6%) Number of patients ever had diarrhea >750 ml or >5/day 112 (37.2%)105 (34.9%)107 (34.9%)120 (38.8%) Proportion of study days with diarrhea >750 or >5/day 368/4032=9.1%358/3866=9.3%336/3933=8.5%448/4109=10.9% Signs of phlebitis or extravasations 7(2.2%)10(3.3%) 18(5.8%)

Post-hoc Secondary Analyses

Kaplan-Meier Survival Curve by Treatment Arm

Selected Subgroup Analyses OR (95% CI) compared to placeboP-values* SubgroupDeaths/n (%)GLN aloneAOX aloneGLN+AOX Overall 363/1218 (30%)1.40 ( )1.20 ( )1.42 ( ) Study Setting Region 0.37 Canada303/1044 (29%)1.41 ( )1.14 ( )1.29 ( ) USA44/131 (34%)1.56 ( )1.43 ( )3.43 ( ) Europe16/43 (37%)0.86 ( )2.40 ( )0.89 ( ) Baseline Patient Characteristics Admission category 0.52 Surgical 59/255 (23%) 2.16 ( )1.94 ( )1.58 ( ) Medical 304/963 (32%) 1.28 ( )1.08 ( )1.43 ( ) Cancer patients 0.74 No 297/1048 (28%) 1.48 ( )1.15 ( )1.42 ( ) Yes 66/170 (39%) 1.05 ( )1.43 ( )1.38 ( ) Etiology of Shock 0.71 Cardiogenic 74/240 (31%) 1.24 ( )1.62 ( )2.19 ( ) Septic 256/826 (31%) 1.43 ( )1.06 ( )1.21 ( ) Other/Unkown/None 33/152 (22%) 1.45 ( )1.45 ( )1.83 ( ) Vasopressors 0.37 <15 mcg/min162/595 (27%)1.58 ( )1.66 ( )1.50 ( ) >=15 mcg/min201/623 (32%)1.32 ( )0.92 ( )1.39 ( ) Renal dysfunction No216/776 (28%)0.93 ( )0.90 ( )1.14 ( ) Yes147/442 (33%)2.75 ( )2.16 ( )2.15 ( ) OR-odds ratio; CI-confidence interval; GLN-Glutamine; AOX-antioxidants

Examination of Treatment Effect by Baseline Renal Dysfunction and Post-Baseline Dialysis Multivariable SubgroupOR (95% CI) Compared To Placebo Arm Renal Dysfunction Ever On Dialysisdeaths/n (%)GLN aloneAOX aloneGLN+AOX No 158/634 (25%) 1.1 ( ) 1.3 ( ) NoYes58/142 (41%) 0.4 ( )0.5 ( )0.6 ( ) YesNo76/240 (32%) 3.9 ( )3.3 ( )1.6 ( ) Yes 71/202 (35%)1.8 ( ) 1.4 ( )3.1 ( ) OR-odds ratio; CI-confidence interval; GLN-glutamine; AOX-antioxidants Cells in bold indicate treatment arm had significantly higher 28 day mortality than placebo at p<0.05.

Adjusted Analysis The 28-day mortality rates in the placebo, glutamine, antioxidant and combination groups were 25%, 32%, 29% and 33% respectively. Compared to placebo, the unadjusted OR (95% CI) of mortality was 1.4 ( , P =0.063), 1.2 ( , P =0.31) and 1.4 ( , P=0.049) in the glutamine, antioxidant and combined groups respectively. After adjusting for all statistically significant baseline characteristics, the corresponding adjusted ORs remained virtually unchanged at 1.4 ( , P =0.054) 1.2( , P =0.34) and 1.4 ( , P =0.10)

Conclusions Glutamine and antioxidants at doses studied in this study do not improve clinical outcomes in critically ill patients with multi-organ failure Glutamine may be harmful For both glutamine and antioxidants, the greatest signal of harm was in patients with multi-organ failure that included renal dysfunction upon study enrollment. Patients with multi-organ failure not uniformly associated with low plasma glutamine levels May have provided insufficient selenium

Where did we go wrong with Glutamine? Moved from RCTs of IV glutamine in PN patients (meta-analysis) to patient with multi-organ failure thinking they would be more glutamine deficient Dosing study was inadequate –No plasma glutamine levels –No kinetics/dynamics in renal failure Others?

e Experimental Diet enriched with Glutamine, AOX, and Omega 3 FFAs A van Zanten, unpublished data

Future Trials Require Bedside Testing?

Where does that leave Glutamine?

Updated Meta-analysis of IV Glutamine (n=28 RCTs) Overall Mortality Note: Does not include EN GLN studies nor REDOXS study RR=0.87 (0.75,1.02) P=0.08

Updated Meta-analysis of IV Glutamine (n=28 RCTs) Hospital Mortality Note: Does not include EN GLN studies nor REDOXS study RR=0.68 (0.51,0.89) P= 0.005

Updated Meta-analysis of IV Glutamine (n=28 RCTs) Hospital Mortality Influence of the methodological quality of the study

Updated Meta-analysis of IV Glutamine (n=28 RCTs) Hospital Mortality Influence of the number of study sites involved in the trial

Updated Meta-analysis of IV Glutamine (n=28 RCTs) Infection Note: Does not include EN GLN studies nor REDOXS study RR=0.86 (0.73,1.03) P=0.10

Updated Meta-analysis of IV Glutamine (n=28 RCTs) ICU Length of Stay Note: Does not include EN GLN studies nor REDOXS study WMD=-2.46 (-4.74, -0.18) P=0.03

Updated Meta-analysis of IV Glutamine (n=28 RCTs) Hospital Length of Stay Note: Does not include EN GLN studies nor REDOXS study WMD=-2.42 (-4.60, -0.24) P=0.03

Canadian Nutrition CPGs: IV Glutamine Recommendation: When parenteral nutrition is prescribed to critically ill patients, parenteral supplementation with glutamine should be considered*. However, we strongly recommend that glutamine NOT be used in critically ill patients with multi-organ failure. here are insufficient data to generate recommendations for intravenous glutamine in critically ill patients receiving enteral nutrition. *downgraded from ‘strongly recommend’

Canadian Nutrition CPGs: EN Glutamine No new studies since 2009 Conclusions are: –1) Glutamine supplemented enteral nutrition may be associated with a reduction in mortality in burn patients, but inconclusive in other critically ill patients. –2) Glutamine supplemented enteral nutrition may be associated with a reduction in infectious complications in burn and trauma patients. –3) Glutamine supplemented enteral nutrition is associated with a significant reduction in hospital length of stay in burn and trauma patients. Recommendation: Enteral glutamine should be considered in burn and trauma patients. There are insufficient data to support the routine use of enteral glutamine in other critically ill patients.* *warning against use in multi-organ failure

Canadian Nutrition CPGs: Combined IV+ EN Glutamine Recommendation: Based on one level 1 study (REDOXS), we strongly recommend that high dose combined parenteral and enteral glutamine supplementation NOT be used in critically ill patients with multi-organ failure.

Written by Dr. Heyland and R. Dhaliwal. For more information, please contact Margot Viola at or visit Thanks for nibbling on our NIBBLE. Is the patient in shock or have multi-organ failure, particularly renal failure? Patient is PN dependent Is the patient: Burns? Trauma? Is EN possible? Give EN Glutamine gm/kg/day as long as they are on EN Do not give any glutamine, neither EN or PN Give IV Glutamine 0.35 gm/kg/day as long as they are on PN Yes No Yes Do not give glutamine No

Questions Interpretive Comments?