Journal of Critical Care (2010) xx, xxx–xxx

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Role of glutamine administration on cellular immunity aftertotal parenteral nutrition enriched with glutamine inpatients with systemic inflammatory response syndromeFerda Cetinbas MDa, Birgul Yelken MDa,⁎, Zafer Gulbas MDb

aDepartment of Anaesthesiology and Intensive Care Unit, Eskisehir Osmangazi University Medical School, Eskişehir,Turkiye, 26480bDepartment of Heamatology, Eskisehir Osmangazi University Medical School, Eskişehir, Turkiye, 26480

0d

Keywords:SIRS;Immunity;Glutamine

Abstract Glutamine is an important substrate for enterocyte and other rapidly proliferating cells. Lowplasma and tissue levels present in glutamine in critically ill patients suggest that demand may exceedendogenous supply. Because commercially available amino acid solutions do not contain glutaminebecause of its instability in aqueous solution, conventional total parenteral nutrition (TPN) does notprevent stress-induced glutamine depletion. In this study, we administered intravenous glutamine-supplemented TPN to patients with systemic inflammatory response syndrome (SIRS) to investigate theeffect of glutamine supplementation on immune states. This study is a prospective, randomized clinicaltrial. All patients received TPN given continuously for 6 days. Thirty patients with SIRS were allocatedto either a glutamine group (L-glutamine 0.4g/[kg d]) (n = 15) or a control group (n = 15). Bloodsamples were collected on day 1 and day 6 after admission for C-reactive protein, immunoglobulin (Ig)M, IgG, IgA, C3, C4, and lymphocyte analysis. The Acute Physiologic and Chronic Health Evaluation IIscore and the Simplified Acute Physiologic II (SAPS II) score were used to evaluate the patients afteradmission. Although there was a tendency for decreased T cytotoxic cells and natural killer cells in thecontrol group, no significant difference was observed between the 2 groups. However, an increase inlymphocyte and lymphocyte subgroups in the glutamine group was observed; but there was nodifference between the groups. A low SAPS II score was observed on the sixth day in the glutaminegroup, whereas no difference in SAPS II and Acute Physiologic and Chronic Health Evaluation II scoreswas observed between the 2 groups. There was no difference in IgM, IgG, IgA, C3, and C4 levels andnumbers of B-lymphocytes between the groups. Glutamine-added TPN significantly decreasesleukocyte and natural killer cell count and therefore suppresses inflammation. Furthermore, totallymphocyte count, B- and T-lymphocytes, and their subgroups (helper T-lymphocytes, cytotoxic T-lymphocytes) are increased; although not statistically significant, these increases might be playing a rolein improving the immune system.© 2010 Published by Elsevier Inc.

⁎ Corresponding author. Tel.: +90 222 2399052; fax: +90 222 2393774.E-mail address: birgulby@yahoo.com (B. Yelken).

883-9441/$ – see front matter © 2010 Published by Elsevier Inc.oi:10.1016/j.jcrc.2010.03.011

1. Introduction

Systemic inflammatory response syndrome (SIRS) is oneof several conditions related to systemic inflammation, organ

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dysfunction, and organ failure. It is a subset of cytokinestorm, in which there is abnormal regulation of variouscytokines. Systemic inflammatory response syndrome is alsoclosely related to sepsis, in which patients satisfy criteria forSIRS and have a suspected or proven infection [1-3]. Feverand leukocytosis are features of the acute-phase reaction,whereas tachycardia is often the initial sign of hemodynamiccompromise. Tachypnea may be related to the increasedmetabolic stress due to infection and inflammation, but mayalso be an ominous sign of inadequate perfusion resulting inthe onset of anaerobic cellular metabolism [2-4].

Protein synthesis and degradation increase in criticalpatients. Human cells use mostly branched-chain aminoacids. In critical patients, glutamine levels normally decrease[5,6]. Glutamine is abundantly found in plasma and tissue.Glutamine is synthesized in the skeletal system and secretedinto the bloodstream so that it may reach tissues. Kidneys,liver, small intestine, and immune system cells are the mostimportant consumers of glutamine [7]. Glutamine providesoptimum immune defense; it is the essential component inthe optimum function of neutrophils and macrophages andalso in lymphocyte proliferation [8].

In our study, we aim to investigate the effects ofglutamine dipeptide–added total parenteral nutrition (TPN)therapy on intensive care unit (ICU) patients who meet atleast 2 of the SIRS criteria.

2. Materials and method

Eskişehir Osmangazi University's Faculty of Medicine'shospital ethics committee approval and patients' informedconsent (or relatives on behalf of patients) were provided.We conducted this prospective clinical study on 30 adultpatients aged 20 to 82 years who met at least 2 SIRS criteria.Patients who had sepsis, multiple organ dysfunction, andimmune deficiency or were enterally fed were excluded fromthe study.

Criteria for SIRS were established in 1992 as part of theAmerican College of Chest Physicians/Society of CriticalCare Medicine Consensus Conference [1]. The conferenceconcluded that the manifestations of SIRS include, but arenot limited to, the following: body temperature less than36°C or greater than 38°C, heart rate greater than 90 beatsper minute, tachypnea (high respiratory rate) with greaterthan 20 breaths per minute, an arterial partial pressure ofcarbon dioxide less than 32 mm Hg, white blood cell countless than 4000 cells per cubic millimeter or greater than12 000 cells per cubic millimeter, or the presence of greaterthan 10% immature neutrophils (band forms). Systemicinflammatory response syndrome can be diagnosed when 2or more of these criteria are present.

After being accepted to the ICU, medical treatmentdirected at primary disease, ventilatory support for respira-tory failure, and antibiotherapy according to cultures were

initiated. Total parenteral nutrition was randomly introducedin both groups: 15 patients with and 15 patients withoutglutamine dipeptide. The daily energy provided by isoni-trogenous TPN solution is standardized as 30 kcal/(kg d) forall patients. This TPN solution is composed of 70%carbohydrates and 30% lipids; proteins (Traumamine 6.9%;Eczacibasi, Baxter; Eskisehir, Turkey) are added as 1.5 g/(kgd). Group I received standard TPN without glutaminedipeptide, whereas group II received TPN with 0.4 g/(kg d)glutamine dipeptide (L-alanyl-L-glutamine dipeptide, Dipep-tiven, Fresenius Kabi; Eskisehir, Turkey). Both groupsreceived TPN for 6 days.

Blood samples were taken from every patient atadmission and on the sixth day. Sedimentation; C-reactiveprotein (CRP); white blood cell count (WBC); and, with theflow cytometer method, lymphocytes, lymphocyte sub-groups, absolute percentage of T-lymphocytes, absolutepercentage of B-lymphocytes, absolute percentage of helperT-lymphocytes (Th), absolute percentage of cytotoxic T-lymphocytes (Ts), and ratio of Th to Ts were measured. Thelevels of products of immune system cells—immunoglob-ulin (Ig) A, IgG, and IgM—and the mediators of immunesystem—C3 and C4—were measured from the bloodsamples taken at admission and on the sixth day. Based othe WBC taken at simultaneous blood samples and theabsolute percentage of lymphocytes, we calculated the totallymphocyte count (WBC × absolute percentage of totallymphocytes = total lymphocyte count per cubic millimeter).Based on the total lymphocyte count, lymphocyte subgroupcount (Th count, Ts count, B-lymphocyte, natural killer [NK]cell count) was calculated (eg, total lymphocyte count percubic millimeter × absolute percentage of B-lymphocytes =B-lymphocytes count per cubic millimeter).

Simplified Acute Physiologic Score (SAPS II) and AcutePhysiologic and Chronic Health Evaluation (APACHE II)scores of all critical patients in ICU are recorded for the sakeof objectivity. To evaluate clinical recovery, progress ofdisease, and mortality risk, we calculated daily SAPS II andAPACHE II scores using the parameters.

In our statistical analysis, we used SPSS (Chicago, IL)10.0 program and the free t test and Mann-Whitney U test fornonparametric data. We accepted P b .05 as significant, anddata are presented as mean ± standard deviation. Statisticalanalyses were conducted both within and intergroup.

3. Results

There was no statistical difference according to age, sex,and body weight in both groups (P b .05) (Table 1).

Both groups had an increased erythrocyte sedimentationrate. The increase in group I was found to be statisticallysignificant (P b .01). The difference between groups was notstatistically significant (P N .05). Both groups had increasedCRP, but it was not statistically significant (P N .05) (Fig. 1).

Table 1 Clinical outcomes in patients receiving control TPN(group I) or glutamine-supplemented TPN (group II)

Glutaminegroup(group I, n = 15)

Control group(group II, n = 15)

P value

Age (y) 54.46 ± 5.32 52.26 ± 5.78 N.05Weight (kg) 67.40 ± 9.37 66.05 ± 9.88 N.05Sex (M/F) 3/12 6/9 N.05

Fig. 2 A, Total lymphocyte counts in patients receiving controlTPN (group I) or glutamine-supplemented TPN (group II). B, Meanabsolute percentage of Th counts in patients receiving control TPN

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The mean leukocyte count of group I increased at the sixthday, and the average leukocyte count of group II decreased.This difference was not statistically significant (P N .05).Both groups showed an increased average absolute percent-age of total lymphocyte count at the end of the sixth day, butthis difference was not statistically significant (P N .05) (Fig.2A). Both groups had increased average total lymphocytecount at the end of the sixth day, but this difference was notstatistically significant (P N .05). Group I showed a decreasedaverage absolute percentage of total lymphocyte count, andgroup II had increased average absolute percentage of totallymphocyte count; but this difference was not statisticallysignificant (P N .05). Group I had decreased average T-lymphocyte count, and group II had increased average T-lymphocyte count; but this difference was not statisticallysignificant (P N .05).

The mean absolute percentage of Th count of group Idecreased on the sixth day, but average absolute percentageof Th count of group II increased. This difference was notstatistically significant (P N .05). The average Th count ofgroup I decreased on the sixth day, but average Th count ofgroup II increased. This difference was not statisticallysignificant (P N .05) (Fig. 2B).

Mean absolute percentage of the Ts count in group Idecreased at the sixth day. This difference was statisticallysignificant (P b .05). Average absolute percentage of the Ts

count in group II decreased, but this difference was notstatistically significant (P N .05). The average Ts count ingroup 1 was decreased at the sixth day, but the average Ts

Fig. 1 Median serum concentration of CRP in patients receivingcontrol TPN (group I) or glutamine-supplemented TPN (group II).

(group I) or glutamine-supplemented TPN (group II). C, Meanabsolute percentage of Ts counts in patients receiving control TPN(group I) or glutamine-supplemented TPN (group II).

count in group II increased. This difference was notstatistically significant (P N .05) (Fig. 2C).

The mean Th/Ts ratio of group I decreased at the sixth day,but the mean Th/Ts ratio of group II increased. Thisdifference was not statistically significant (P N .05)(Fig. 3). Both groups had decreased mean in absolutepercentage of NK cell count. The decrease in group I wasfound to be statistically significant (P b .01). The differencebetween the groups was not statistically significant (P N .05).Both groups had decreased average NK cell count. Thedecrease in group I was found to be statistically significant

Fig. 3 The mean Th/Ts ratio in patients receiving control TPN(group I) or glutamine-supplemented TPN (group II).

Fig. 5 A and B, Mean APACHE II and SAPS II scores in patientsreceiving control TPN (group I) or glutamine-supplemented TPN(group II).

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(P b .05). The difference between the groups was notstatistically significant (P N .05) (Fig. 4).

The mean absolute percentage of B-leukocyte count ofgroup II was increased at the sixth day, but this differencewas not statistically significant (P N .05). Both groups hadincreased average B-leukocyte cell count. The differencebetween the groups was not statistically significant (P N .05).

Both groups had increased IgM and IgG cell count, butthis difference was not statistically significant (P N .05).Group I had a decreased average IgA cell count, and group IIhad an increased mean IgA cell count; but these differenceswere not statistically significant (P N .05).

Group I had an increased mean C3 count, and group IIhad a decreased mean C3 count; but these differences werenot statistically significant (P N .05). Group I had adecreased average C4 count, and group II had an increasedaverage C4 count; but these differences were not statisti-cally significant (P N .05).

Mean APACHE II scores were increased in group I, andmean APACHE II scores were decreased in group II; butthese differences were not statistically significant (P N .05)(Fig. 5A). The decrease of SAPS II scores in group I at thesixth day was found to be statistically significant (P b .05).

Fig. 4 Mean absolute percentage of NK cell counts in patientsreceiving control TPN (group I) or glutamine-supplemented TPN(group II).

The difference between the groups was not statisticallysignificant (P N .05) (Fig. 5B).

4. Discussion

Many cells in the immune system proliferate very rapidly.Glutamine facilitates this process, functioning as a biosyn-thetic precursor and as an energy source [9,10]. Glutamineheals the defense system, probably by increasing antioxidantprotection and also by increasing the effectiveness of thebowel-barrier function. Glutamine is very important inoptimizing the immune system, by way of promotinglymphocyte proliferation and optimum functioning ofneutrophils and macrophages [11].

Glutamine has essential properties in lymphocyte prolif-eration and optimum function of neutrophils and macro-phages. In conditions of decreased plasma glutamine levels,T-lymphocytes are suppressed, phagocytic properties ofmacrophages decrease, and IL-1 production also decreases[4]. These very intense uses of glutamine by immune systemcells show that glutamine is very important for protection ofimmune system [3].

In our study, we investigated the effect of glutamine (0.4g/[kg d]) -added TPN on the immune system in SIRS patientsfor 6 days. At admission and at the sixth day, we investigatedimmune cells, specifically lymphocytes and lymphocytesubgroups (T-lymphocytes, Th, Ts, Th/Ts ratio, B-lympho-cytes, NK cells), immune system products (IgM, IgA, IgG),

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serum and membrane proteins required for protectivefunction of immune system (C3, C4), serum CRP, andsedimentation parameters for evaluation of inflammation.

Wischmeyer et al [12] investigated infection morbidityand nitrogen control by administering glutamine-added TPNto severe burn patients for 14 days. They used CRP as aparameter of all inflammations. Their results show thatglutamine-added TPN statistically decreases total inflamma-tion measured by CRP. Our study shows that a glutamine-added TPN group has lower CRP levels than the controlgroup, but this difference is not statistically significant. Webelieve that the differences between these studies may beassociated with different patient groups and the duration ofglutamine administration.

In trauma patients, the effect of administering glutamine,arginine, and omega-3 fatty acid solutions added to theenteral nutrition on the duration of hospital stay and healingrate of immune system was investigated [13]. C-reactiveprotein is used as a parameter for inflammation. Totallymphocytes, lymphocyte subgroups (T-lymphocytes, Th,Ts, B-lymphocytes), immune system products (IgM, IgA,IgG), and serum and membrane proteins required forprotective function of immune system (C3, C4) are measuredas parameters of the immune system. They showed adecrease in respect to the control group, but this decrease wasnot statistically significant. In our study, we showed that bothgroups had increased total lymphocyte count and that groupII had higher levels of T-lymphocytes, Th, Th/Ts, and B-lymphocytes but that this difference was not statisticallysignificant. Immunoglobulin M levels are increased in bothgroups, but IgA levels are increased only in group II.Average C3 levels are increased in group I but decreased ingroup II, but none of the parameters were significant.Although, in the Wischmeyer et al study, they also addedomega-3 and used different means of nutrition, the results aresimilar to our study.

The effect of glutamine-added vs glutamine-free multi-fiber nutrition solutions on the immune system of 16 acutepancreatitis patients [14] was investigated. They evaluatedIgG, IgM, C3, and C4 parameters for immune system andCRP as acute phase reactant. There was no significantdifference in CRP, but IgG levels were clinically improved inthe glutamine-added group; also, IgM levels were markedlyimproved. There was no significant difference in C3 and C4

parameters. Similar to our study, CRP, IgG, C3, and C4 levelswere correlated in their study.

T-lymphocyte response according to glutamine level wasinvestigated in 20 postoperative colorectal resection patientsreceiving glutamine-added TPN for 6 days [15]. They foundthat T-lymphocyte count elevation was statistically signifi-cant. Similar to our study, the T-lymphocyte levels of groupII increased; but this increase was not statistically significant.We think that the difference in these studies might beassociated with different patient groups and age intervals.

Cellular immunity changes due to perioperative gluta-mine infusion after cardiopulmonary bypass were investi-

gated [6]. They found that T-cell–derived inflammatoryresponse, indicating a sufficient supply of glutamine, showsno significant change. We think that the difference betweenthese studies might be associated with different durations ofglutamine administration and patient numbers. Crawford andCohen [16] investigated glutamine effect on lymphocytecount and immunoglobulin production. They showed thatimmunoglobulin production improved, but that there was nochange in B-lymphocytes, T-lymphocytes, Th, and Ts.Therefore, it is suggested that glutamine support mightimprove lymphoblastic transformation and differentiation ofplasma cells. Boelens et al [17] investigated glutamine-addedenteral nutrition on lymphocyte subgroup count andimmunoglobulin production in 38 trauma patients. Theyfound out that both groups had increased levels of serumIgM, IgA, and IgG, but that these increases were notstatistically significant. These results are similar to our study.

L-Glutamine and L-alanine effects on immune systemcells were investigated in vitro [18]. It was shown that L-glutamine– and L-alanine–added nutrition leads to increasedT-lymphocyte count. This increase in T-lymphocyte countdepends on glutamine dose. In the same study, they could notfind any change in NK cells and Ts cells. In our study, wefound a statistically significant decrease in both groups;group I showed a more marked significance.

Jones et al [19] investigated glutamine-added enteralnutrition effect on survival. They used APACHE II scores forthis purpose and found no difference between groups. In ourstudy, we observed decreased APACHE II scores in group II,but found no significant difference between groups.Nevertheless, we found a statistically significant decreasein SAPS II scores.

In conclusion, glutamine dipeptide–added TPN signif-icantly decreases SAPS II scores, leukocytes, and NK cellcount, which might be associated with suppressinginflammation and improving clinical recovery. Further-more, total lymphocyte count, B- and T-lymphocytes, andtheir subgroups (Th, Ts) are increased. Although notstatistically significant, these increases may play a role inimproving the immune system. Larger and controlledclinical trials are needed to determine the potential efficacyof glutamine-supplemented nutrition as an adjunctivetherapy in critically illness.

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