Q J Med 2002; 95: 89-97
© 2002 Association of Physicians
Plasma glutamine and glutamate concentrations in Gabonese children with Plasmodium falciparum infection
1 From the Department of Infectious Diseases, St George's Hospital Medical School, Cranmer Terrace, London, UK, 2 Département de Parasitologie, Mycologie et Médecine Tropicale and 5 Départment de Biochimie, Faculté de Médecine et des Sciences de la Santé, Libreville, Gabon, 3 Research Unit, Albert Schweitzer Hospital, Lambaréné, Gabon, 4 Sektion Humanparasitologie, Institut für Tropenmedizin, Universitaet Tübingen, Tübingen, Germany
Received 18 September 2001 and in revised form 31 October 2001
 |
Summary |
|---|
 Top Summary IntroductionMethodsResultsDiscussionReferences |
|---|
Background: Low plasma glutamine levels in critical illness, neonates and burns patients are associated with poor outcome and increased risk of intercurrent infection.
Aim: To investigate the relationship between plasma glutamine/glutamate levels and severity/outcome of malaria.
Design: Two-hospital prospective study, with both febrile and healthy controls.
Methods: We measured plasma glutamine and glutamate concentrations in 239 Gabonese patients: 145 children with malaria (86 with severe, 36 with moderate and 23 with uncomplicated disease), 42 healthy children, 44 febrile controls and eight healthy adults, and related findings to conventional markers of disease severity such as plasma lactate.
Results: Median (IQR) plasma glutamine was lower in uncomplicated falciparum malaria and in moderate malaria than in healthy controls: 353 (287–474) and 379 (293–448) vs. 485 (428–531) µmol/l, respectively; p<0.01 for both malaria groups vs. controls. In contrast, plasma glutamine was within the normal range in those with severe malaria and in febrile control children: 431 (342–525) and 472 (338–547) µmol/l, respectively. Furthermore, plasma glutamine was significantly higher in the children who died with malaria than in survivors: 514 (374–813) (n=12) vs. 399 (316–475) µmol/l (n=133), respectively; p=0.001. There were no significant differences in plasma glutamate concentrations between any of the study groups.
Discussion: In severe malaria, there was a positive correlation between plasma glutamine and lactate levels (p=0.009, r=0.281). This correlation may reflect impaired gluconeogenesis. Glutamine supplementation is probably not justified in severe P. falciparum infection.
|
Introduction |
|---|
|
TopSummaryIntroductionMethodsResultsDiscussionReferences |
|---|
There are 1–2 million deaths from malaria each year, most of them occurring in children aged <5 years in sub-Saharan Africa. The case fatality rate of children admitted to hospital with cerebral malaria is
20%, and has remained unchanged for the past 40 years.1 A better understanding of the pathophysiology of severe infection may suggest new treatments aimed at reducing this unacceptably high mortality. Glutamine is a conditionally essential amino acid that is required by rapidly dividing cells such as leucocytes and enterocytes, particularly in catabolic states.2,3 Glutamine has a wide spectrum of other biological activities, such as renal ammonia genesis,4 glutathione synthesis5 and carbohydrate metabolism.6,7
Low plasma glutamine levels have been reported in a number of conditions, including burns,8 critically ill adults,9 trauma, sepsis,10 and premature infants.11 Glutamine supplementation reduces the incidence of sepsis and late mortality in critically ill adults12, 13 as well as in infants with very low birth weight.11 Plasma glutamine concentrations fall, and plasma glutamate concentrations rise, in response to extreme physical exertion. An elevation in the ratio of these two amino acids indicates a state of over-exertion.14 Physiological changes in malaria, induced by sequestration of parasitized red blood cells and tissue hypoxia, may be similar to those observed following extreme exertion.
We have previously reported that plasma glutamine levels are almost halved in acute (predominantly uncomplicated) P. falciparum infection.15 The magnitude of this fall is similar to that observed in sepsis, and may contribute to some of the complications of malaria such as anaemia or secondary infection. However, our previous study was not designed to examine relationships between plasma glutamine levels, disease severity and mortality. This study now compares severity and mortality of malaria with plasma glutamate and glutamine levels, using plasma lactate concentrations as a marker of disease severity in malaria in African children.16,17
|
Methods |
|---|
|
TopSummaryIntroductionMethodsResultsDiscussionReferences |
|---|
This study was conducted in two hospitals: the Albert Schweitzer Hospital in Lambaréné and the Centre Hospitalier de Libreville between September 1999 and July 2000. The study was approved by the Ethics Committees of the Albert Schweitzer Hospital, Lambaréné, Gabon, the Gabonese Ministry of Health and the University of Tübingen.
Inclusion criteria
Admitting physicians referred children aged 12–71 months (inclusive) with a suspected diagnosis of malaria to the study team. These children were screened, with a history, physical examination, blood film examination and measurement of blood lactate, glucose and haematocrit, within an hour of admission to hospital. Malaria was defined as a history of fever within 24 h of presentation and the presence of asexual P. falciparum in the thick or thin blood film. The severity of malaria and control groups was defined as follows:
Severe malaria was malaria with one or more of the following features: Blantyre coma score (BCS) 
2; repeated witnessed convulsions (three or more observed); lactate in whole blood or capillary 
5 mmol/l, glucose in whole blood or capillary 2.2 mmol/l, and severe anaemia (Hb <5 g/dl or haematocrit <15%).
Moderate malaria was defined as malaria without the features of severe disease with one or more of the following features: hyperparasitaemia 250000 asexual forms of P. falciparum/µl, obtundation (BCS 3–4), inability to tolerate an oral route for treatment and inability to sit or stand unaided (prostration), as appropriate for age.
Uncomplicated malaria was defined as malaria without any of the features of severe or moderate malaria.
Febrile controls were children aged 12–71 months admitted to hospital with a core temperature (tympanic) of 37.5 °C with a negative blood film examination and no history suggestive of treated malaria.
Healthy adults and children were adults or children (aged 12–71 months) who were afebrile, aparasitaemic and reported no ill health.
Patients with sickle anaemia were excluded retrospectively by the results of haemoglobin electrophoresis.
Procedures
Parents or guardians gave informed consent for children; adults gave informed consent themselves. A blood sample (2 ml) was taken into a heparinized tube. Whole blood glucose and lactate measurements were made on site with a YSI 2300 analyser (YSI Instrument Co.) and haematocrit was measured with a microhaematocrit centrifuge (Hawksley). Thick and thin blood films were stained with a Field's stain and counted on thin films per 1000 red cells or by the Lambaréné method on thick films.18 Films were defined as negative if there were no asexual malaria parasites in 100 high-power fields of the thick film
Plasma was separated and stored at -80 °C before being transported within a few weeks on dry ice for assay. Children with severe and moderate malaria were treated with parenteral quinine; those with uncomplicated malaria were treated with oral sulphadoxine/pyrimethamine.
Assays for glutamine and glutamate measurements
Glutamine and glutamate concentrations were measured using a YSI 2700 (YSI Instrument Co.). This analyser uses a platinum electrode to measure the current generated on two enzyme-impregnated membranes. The glutamate membrane generates electrons by the glutamate oxidase reaction, and the current measured is proportional to the glutamate concentration. The glutamine electrode uses the reaction catalysed by glutaminase to convert glutamine to glutamate, and measures the current generated by glutamate oxidase to measure total glutamine and glutamate concentration. Glutamine concentrations are thus calculated as the concentration measured by the glutamate electrode minus that detected by the glutamine electrode. We used 1 mM glutamate and glutamine standards to calibrate the machine every four measurements (sample size 65 µl). All plasma samples were measured in duplicate, and the mean was used in calculations.
Within-day coefficients of variation (CV) for standard solutions containing the following concentrations of glutamine were: 0.4 mmol/l, 12%; 0.6 mmol/l, 10%; 1 mmol/l, 9% (n=6). For glutamate, the within-day CVs were: 0.4 mmol/l, 9%; 0.6 mmol/l, 8%; 1 mmol/l, 14% (n=6). The between-day CVs for the same solutions were (glutamine): 0.4 mmol/l, 13% (n=11); 0.6 mmol/l, 12% (n=9); 1 mmol/l, 9% (n=9); and (glutamate) 0.4 mmol/l, 9% (n=11); 0.6 mmol/l, 9% (n=9); 1 mmol/l, 9% (n=9). Plasma transaminase measurements and creatine phosphokinase (CPK) were analysed on autoanalysers in the Department of Biochemistry, St George's Hospital Medical School.
Statistical analyses
Statistical analyses used Systat (version 5.2) and Stata Statistical Software (Release 6.0). After checking distributions with the Shapiro-Wilks W test, and transforming the data logarithmically (parasitaemia only) or by Box-Cox transformations, as appropriate, we analysed normally distributed data by two tailed Student's T test (paired if appropriate) and non-parametric data with the Mann-Whitney U statistic. Proportions were compared with Fisher's exact test, and correlations were assessed by Pearson linear regression analysis. ANOVA tests were used for multiple comparisons, with Tukey's post hoc test.
|
Results |
|---|
|
TopSummaryIntroductionMethodsResultsDiscussionReferences |
|---|
Demographic and clinical data
Glutamine and glutamate were assayed in 239 patients: 145 children with P. falciparum parasitaemia, 44 febrile controls (one fatality), 42 healthy children and eight healthy adult controls.
In the malaria group, 86 children had severe disease (12 fatalities), 36 children had moderate malaria and 23 children had uncomplicated disease. The admission characteristics of the patients are shown in Table 1. Healthy control children were older than children with malaria: median (IQR) age 54 (35–65) months vs. 25 (18–37) months. The age of the febrile controls was similar to that of the children with malaria. BMI values of children in all study groups were comparable.
|
Laboratory data
Plasma lactate
Plasma lactate levels were higher in children with malaria than in healthy controls: median (IQR) plasma lactate 2.4 (1.6–4.0) vs. 1.26 (0.1–1.7) mmol/l (Figure 1). Elevations in plasma lactate concentrations were proportional to disease severity: median (IQR) plasma lactate was 3.4 (2.3–5.8) mmol/l in severe malaria, 1.8 (1.5–3.3) mmol/l in moderate malaria and 1.5 (1.2–1.9) mmol/l in uncomplicated malaria (Figure 1). A 
2 test for trend confirmed a significant positive correlation between lactate concentrations and severity in these groups (p=0.0001).
|
Plasma glutamine
The median (IQR) plasma glutamine concentrations were lower in children with malaria than in healthy or febrile controls: 413 (323–487) µmol/l, 485 (428–531) µmol/l and 472 (338–547) µmol/l, respectively (p<0.001). Plasma glutamine concentrations in the six diagnostic groups are shown in Figure 2a, and differences between these groups are summarized in Table 2. The plasma concentrations of glutamine of the children with moderate malaria and uncomplicated malaria were lower than those in the healthy or febrile control groups. Children with severe malaria had plasma glutamine concentrations similar to those in the control groups and higher than those with moderate malaria (p=0.026).
|
|
There was a positive correlation between plasma lactate concentrations and plasma glutamine in patients with severe malaria (r=0.281, p=0.009). In contrast, there was a negative correlation between plasma glutamine and plasma lactate concentrations in children with moderate malaria (r=-0.345, p=0.039) and in children with uncomplicated malaria (r=-0.421, p=0.045). There was no relationship between plasma glutamine concentrations and age, sex, coma score, haematocrit, peripheral parasitaemia or any other demographic, clinical or laboratory variable.
Plasma glutamate
Figure 2b shows plasma glutamate concentrations in all study groups. There were no significant differences in plasma glutamate concentrations between any of the groups in this study. There was a weak positive correlation between plasma glutamate and glutamine (r=0.299, p<0.001). There were no differences between plasma glutamate/glutamine ratios in any of the six groups.
Plasma lactate and glutamine concentrations and outcome
Plasma lactate concentrations were significantly higher in the children with malaria who died than in those who survived: median (IQR) 7.9 (5.2–11.2) vs. 2.3 (1.6–3.6) mmol/l (p=0.0001). Plasma glutamine concentrations in the 12 children who died with severe malaria were significantly higher than in those who survived their malaria infection: median (IQR) 514 (374–813) vs. 399 (316–475) µmol/l (p=0.001).
Plasma aspartate transaminase (AST), creatinine and creatine phosphokinase (CPK) levels
Overall, plasma creatinine levels were not significantly higher in children with malaria than in healthy children or febrile controls: median (IQR) 41 (31–54) vs. 35 (31–43) µmol/l (p=0.135) or 37 (28–43) µmol/l) (p=0.281), respectively. However, plasma CPK levels were elevated (210 IU/l) in a higher proportion of children with severe malaria (25%, n=21) than healthy children (7%, n=3), febrile controls (7%, n=3), uncomplicated (0%) or moderate malaria (11%, n=4) (p=0.006) (Figure 3). Elevations in CPK were unrelated to plasma glutamine levels.
|
Plasma AST levels were significantly higher in malaria patients than in healthy children or febrile controls: median (IQR) 38 (28–65) IU/l vs. 25 (22–31) IU/l) (p<0.001) or 29 (23–41) IU/l (p=0.003), respectively. There was no significant difference in AST levels between children with severe malaria and those with uncomplicated malaria (p=0.498).
AST levels were correlated with plasma creatinine (r=0.421, p<0.001), glutamine (r=0.392, p<0.001), glutamate (r=0.553, p<0.001) and lactate (r=0.298, p<0.001), in all cases.
|
Discussion |
|---|
|
TopSummaryIntroductionMethodsResultsDiscussionReferences |
|---|
Glutamine is an important substrate for cells with rapid turnover such as gut epithelial cells and white blood cells.3,19 In vitro studies have demonstrated impaired function in white cells cultured in media containing relatively low glutamine concentrations.20 Plasma glutamine plays an important role as a carrier of nitrogen, carbon and energy between organs21 and is used for hepatic urea synthesis, renal ammonia genesis,4 gluconeogenesis22 and nucleotide biosynthesis, and glutamine is a preferential fuel for many cells.3 Glutamine is not classed as an essential amino acid, but becomes conditionally essential in catabolic states. Plasma glutamine levels are low in patients with critical illness,9,23 post trauma,24 in burns patients8 and in pre-term infants.11,25 In these circumstances, glutamine supplementation improves survival, and it decreases the risk of intercurrent infection in pre-term neonates11 and adults with critical illness.12,13 Stable isotope studies have shown that low plasma glutamine levels are a result of higher glutamine utilization in catabolic tissues, which is not compensated by for increased proteolysis.23 Single measurements of plasma glutamine concentrations do not necessarily give an indication of total body glutamine or glutamine metabolic flux. Changes in plasma glutamine may result from changes in volume of distribution, cellular uptake and release of glutamine, as well as changes in utilization and synthesis.
We have previously demonstrated that plasma glutamine concentrations are significantly lowered in children with moderate malaria.15 This study confirms our previous finding that plasma glutamine levels are low in uncomplicated and moderate malaria infection (the magnitude of these falls was comparable). Glutamine concentrations in healthy control children in this study (median (IQR) 485 (428–531) µmol/l) are also in good agreement with reported normal data. There was no relationship between age, plasma glutamate, glutamine or lactate concentrations, suggesting that the higher age of our healthy control children did not influence these values significantly.
In contrast to uncomplicated and moderate malaria, plasma glutamine concentrations were not decreased in children with severe malaria. Moreover, children with severe malaria who subsequently died (n=12) had significantly higher plasma glutamine levels than those who survived (n=74) (p=0.001). There are several possible explanations for this finding: there may be increased glutamine release into plasma in severe malaria; plasma clearance of glutamine may be decreased; the volume of distribution of glutamine may have decreased; or a combination of mechanisms may operate. Previous studies of critically ill patients have described falls in plasma glutamine concentrations,8,10,12 which are probably due to increased tissue utilization.23 A recent study of 80 severely ill adults showed a relationship between low plasma glutamine concentrations on admission and probability of death. Patients with a plasma CPK >150 IU/l had significantly higher plasma glutamine levels than those with plasma CPK 150 IU/l.9 As glutamine levels are within the normal range in children with severe malaria, it is possible that intermediary metabolism is affected in a different way in this condition compared with other critical illnesses.
Plasma lactate concentrations in this study agree well with previous observations that correlate elevations in blood lactate with clinical assessment of disease severity, as well as with fatal outcome.16,17,26 These data confirm that lactate measurements are a valuable and useful objective method for defining severity of malaria for uncomplicated, moderate and severe malaria.
We also measured CPK as an indicator of muscle damage and potential source of glutamine into the plasma, and AST and creatinine to assess liver and kidney impairment. There were no relationships between plasma CPK, creatinine and plasma glutamine concentrations, suggesting that the raised plasma glutamine concentrations in severe malaria are unrelated to either muscle damage or renal dysfunction as assessed by these crude plasma indicators. The relationship between plasma glutamine and disease severity in malaria is complex, and further investigation of intermediary metabolism in severe childhood P. falciparum infection is needed before the issue of glutamine supplementation in malaria can be resolved.
|
Acknowledgments |
|---|
We would like to thank the medical, nursing, and laboratory staff of the Albert Schweitzer Hospital, and of the Centre Hospitalier de Libreville, especially Dr Josseaume, Dr Tchoua, Professor Ngaka and Professor R. Tchoua for advice and aid in conducting this study. We would also like to thank M. Obiang Nestor, Batchelili Batchelili, Ekoumembia Michel, Mozogho Emmanuel and Mbandinga Frankie for their assistance. We thank Martin Hurl of YSI for the loan of the YSI2700 analyser. This work is a part of a program for research in tropical medicine based at St George's Hospital Medical School, London and funded by the Wellcome Trust. TP was funded by the Special Trustees of St Georges Hospital Medical School.
|
Notes |
|---|
Address correspondence to Professor S. Krishna, Department of Infectious Diseases, St George's Hospital Medical School, Cranmer Terrace, London SW17 ORE. e-mail: s.krishna{at}sghms.ac.uk
|
References |
|---|
|
TopSummaryIntroductionMethodsResultsDiscussionReferences |
|---|
1. Newton CR, Krishna S. Severe falciparum malaria in children: current understanding of pathophysiology and supportive treatment. Pharmacol Ther1998; 79:1–53.[ISI][Medline]
2. Wilmore DW, Shabert JK. Role of glutamine in immunologic responses. Nutrition1998; 14:618–26.[ISI][Medline]
3. Castell LM, Bevan SJ, Calder P, Newsholme EA. The role of glutamine in the immune system and in intestinal function in catabolic states. Amino Acids1994; 7:231–43.
4.
Welbourne TC. Interorgan glutamine flow in metabolic acidosis. Am J Physiol1987; 253:F1069–76.
5. Hong RW, Rounds JD, Helton WS, Robinson MK, Wilmore DW. Glutamine preserves liver glutathione after lethal hepatic injury. Ann Surg1992; 215:114–19.[ISI][Medline]
6. Rennie MJ, Ahmed A, Khogali SE, Low SY, Hundal HS, Taylor PM. Glutamine metabolism and transport in skeletal muscle and heart and their clinical relevance. J Nutr1996; 126(Suppl.):1142–9S.
7. Stumvoll M, Perriello G, Meyer C, Gerich J. Role of glutamine in human carbohydrate metabolism in kidney and other tissues. Kidney Int1999; 55:778–92.[ISI][Medline]
8. Parry-Billings M, Evans J, Calder PC, Newsholme EA. Does glutamine contribute to immunosuppression after major burns? Lancet1990; 336:523–5.[ISI][Medline]
9. Oudemans-van Straaten HM, Bosman RJ, Treskes M, van der Spoel HJ, Zandstra DF. Plasma glutamine depletion and patient outcome in acute ICU admissions. Intensive Care Med2001; 27:84–90.[ISI][Medline]
10. Vente JP, von Meyenfeldt MF, van Eijk HM, van Berlo CL, Gouma DJ, van der Linden CJ, et al. Plasma-amino acid profiles in sepsis and stress. Ann Surg1989; 209:57–62.[ISI][Medline]
11. Neu J, Roig JC, Meetze WH, Veerman M, Carter C, Millsaps M, et al. Enteral glutamine supplementation for very low birth weight infants decreases morbidity. J Pediatr1997; 131:691–9.[ISI][Medline]
12. Griffiths RD. Outcome of critically ill patients after supplementation with glutamine. Nutrition1997; 13:752–4.[ISI][Medline]
13. Griffiths RD, Jones C, Palmer TE. Six-month outcome of critically ill patients given glutamine-supplemented parenteral nutrition. Nutrition1997; 13:295–302.[ISI][Medline]
14. Smith DJ, Norris SR. Changes in glutamine and glutamate concentrations for tracking training tolerance. Med Sci Sports Exerc2000; 32:684–9.[Medline]
15. Cowan G, Planche T, Agbenyega T, Bedu-Addo G, Owusu-Ofori A, Adebe-Appiah J, et al. Plasma glutamine levels and falciparum malaria. Trans Roy Soc Trop Med Hyg1999; 93:616–18.[Medline]
16. Krishna S, Waller DW, ter Kuile F, Kwiatkowski D, Crawley J, Craddock CF, et al. Lactic acidosis and hypoglycaemia in children with severe malaria: pathophysiological and prognostic significance. Trans Roy Soc Trop Med Hyg1994; 88:67–73.[ISI][Medline]
17. Agbenyega T, Angus B, Bedu-Addo G, Baffoe-Bonnie B, Griffin G, Vallance P, et al. Plasma nitrogen oxides and blood lactate concentrations in Ghanaian children with malaria. Trans Roy Soc Trop Med Hyg1997; 91:298–302.[Medline]
18. Planche T, Krishna S, Kombila M, Engel K, Faucher JF, Ngou-Milama E, et al. A comparison of methods for the rapid laboratory assessment of children with malaria. Am J Trop Med Hyg2001; 65:599–602.[Abstract]
19. Newsholme EA. The possible role of glutamine in some cells of the immune system and the possible consequence for the whole animal. Experientia1996; 52:455–9.[ISI][Medline]
20.
Spittler A, Winkler S, Gotzinger P, Oehler R, Willheim M, Tempfer C, et al. Influence of glutamine on the phenotype and function of human monocytes. Blood1995; 86:1564–9.
21. Nurjhan N, Bucci A, Perriello G, Stumvoll M, Dailey G, Bier DM, et al. Glutamine: a major gluconeogenic precursor and vehicle for interorgan carbon transport in man. J Clin Invest1995; 95:272–7.
22. Hankard RG, Haymond MW, Darmaun D. Role of glutamine as a glucose precursor in fasting humans. Diabetes1997; 46:1535–41.[Abstract]
23.
Jackson NC, Carroll PV, Russell-Jones DL, Sonksen PH, Treacher DF, Umpleby AM. The metabolic consequences of critical illness: acute effects on glutamine and protein metabolism. Am J Physiol1999; 276:E163–70.
24. Houdijk AP, Rijnsburger ER, Jansen J, Wesdorp RI, Weiss JK, McCamish MA, et al. Randomised trial of glutamine-enriched enteral nutrition on infectious morbidity in patients with multiple trauma. Lancet1998; 352:772–6.[ISI][Medline]
25. Roig JC, Meetze WH, Auestad N, Jasionowski T, Veerman M, McMurray CA, et al. Enteral glutamine supplementation for the very low birthweight infant: plasma amino acid concentrations. J Nutr1996; 126(Suppl.):1115–20S.
26. Allen SJ, O'Donnell A, Alexander ND, Clegg JB. Severe malaria in children in Papua New Guinea. Q J Med1996; 89:779–88.[Abstract]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||