Metabolic Acidosis and Other Determinants of Hemoglobin-Oxygen Dissociation in Severe Childhood Plasmodium Falciparum Malaria

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Metabolic Acidosis and Other Determinants of Hemoglobin-Oxygen Dissociation in Severe Childhood Plasmodium falciparum Malaria

Philip Sasi, Shamus P. Burns, Catherine Waruiru, Michael English, Claire L. Hobson, Christopher G. King, Moses Mosobo, John S. Beech, Richard A. Iles, Barbara J. Boucher, and Robert D. Cohen* KEMRI/Wellcome Trust Research Programme, Kilifi, Kenya; Child and Newborn Health Group, Centre for Geographic Medicine Research-Coast, Nairobi, Kenya; Centre for Diabetes and Metabolic Medicine, St Bartholomew’s and The London School of Medicine and Dentistry, London, United Kingdom; School of Applied Sciences, (Biological Sciences), University of Huddersfield, Huddersfield, United Kingdom; Department of Immunology, Royal Victoria Infirmary, Newcastle-upon-Tyne, United Kingdom; Department of Anaesthesia, University of Cambridge, Cambridge, United Kingdom; Department of Clinical Pharmacology, Muhimbili University College of Health Sciences, University of Dar es Salaam, Tanzania

Abstract. Metabolic acidosis is a common complication of severe malaria caused by Plasmodium falciparum. The factors contributing to the acidosis were assessed in 62 children with severe falciparum malaria (cases) and in 29 control children who had recently recovered from mild or moderate malaria. The acidosis was largely caused by the accumu- lation of both lactic and 3-hydroxybutyric acids. The determinants of oxygen release to the tissues were also examined; although there was no difference between cases and controls in respect of 2,3-bisphosphoglycerate and mean corpuscular

hemoglobin concentration, there was a marked increase in P50 in the cases, caused by pyrexia, low pH, and base deficit. There was substantial relative or actual hypoglycemia in many cases. The relationship of these observations to therapeutic strategy is discussed.

INTRODUCTION mize this problem are therefore desirable.10,11 Furthermore, the anemia so common in this group of children is often Lactic acidosis is strongly associated with mortality in se- treated by blood transfusion; however, blood for transfusion, 1,2 vere Plasmodium falciparum malaria in African children. even though collected into citrate-phosphate-dextrose solu- The lactic acidosis of malaria has multiple etiologies; it is tion, tends to have low BPG levels, and may therefore, not likely that poor tissue perfusion related to hypotension, de- have the expected therapeutic effect. The purpose of this hydration, occlusion of the microcirculation by parasites, in- study was to determine the causes of the acidosis in African hibition of gluconeogenesis by circulating tumor necrosis fac- children with severe malaria and to examine the importance 3–5 tor, and decrease in hepatic blood flow play significant of the several factors involved in oxygen release to the tissues. roles. Because severe anemia is commonly present,3 the de- livery of oxygen to the tissues may be further impaired. A MATERIALS AND METHODS major determinant of oxygen release from hemoglobin is the position on the x-axis of the sigmoid curve of the plot of Subjects. There were 62 cases (5 fatal), between 6 and 112 hemoglobin-oxygen saturation against the partial pressure of months of age (mean, 33.9 ± 20.0 [SD] months), and 29 con- oxygen (PO2). Shifts of the curve to the left of the normal trol subjects of similar age range and distribution (range, position result in greater difficulty in oxygen dissociation, i.e., 6–119 months; mean, 33.5 ± 25.26 months). The sex ratio

PO2 has to be decreased to a lower level than the fall needed (male:female) was 47:53 in the cases and 40:60 in controls. under normal circumstances before the same amount of oxy- Table 1 shows the distribution of clinical states in the patients. gen is released, whereas right shifts result in the opposite Venous blood samples were collected in two batches: the first situation. The position of the curve is usually denoted by P50, consisting of 23 cases and 8 controls in 1996–1997 and the i.e., the value of PO2 for half-saturation of hemoglobin; second consisting of 39 cases and 21 controls in 2003–2004. higher values than the mean normal (∼27.2 mm of Hg) imply The controls were in apparent good health and were attend- easier dissociation. P50 is affected by several factors; low pH ing planned follow-up, which was 4 weeks after hospitaliza- and high PCO2 lead to an increase. In addition, the unique tion for non-severe malaria. Nevertheless, because of their intra-erythrocytic metabolite 2,3–bisphosphoglycerate (BPG) fairly recent illness, they cannot strictly be regarded as com- interacts with the hemoglobin molecule to raise P50 and thus pletely well children. The criteria for inclusion for the cases facilitate oxygen release. Acidosis of more than a very few were one or more of the following: 1) severe acidosis, defined hours duration impairs the synthesis of BPG,6 and in severe as base deficit > 18 mmol/L; 2) base deficit between 10 and 18 diabetic ketoacidosis, BPG may decrease to < 10% of its mmol/L, but persisting for > 6 hours after admission; 3) coma, previous value6–8; furthermore, a decrease in BPG has been but with base deficit < 10 mmol/L (if postictal or hypoglyce- described in an animal model of severe malaria.9 The simple mic, 1 hour was allowed to elapse after the fit or correction of therapeutic approach would be to treat the acidosis of malaria the hypoglycaemia before collection of the sample for the with bicarbonate infusion; however, this may result in an un- study); 4) blood hemoglobin Յ 5.0 g/dL. The reported dura- desirable left shift of the dissociation curve in a situation in tion of the illness before admission was noted but is of un- which oxygen dissociation has already been impaired by a fall certain reliability. in BPG caused by acidosis. Therapeutic approaches that mini- Methods. The study was approved by the Kenya Medical Research Institute (KEMRI) Scientific Steering Committee (SSC) and the National Ethics Review Committee (ERC). * Address correspondence to Robert D. Cohen, Longmeadow, Venous samples were obtained with informed parental con- East, Chichester, West Sussex PO18 0JB, UK. E-mail: rcohen@ sent on admission and before the start of in-patient treatment doctors.org.uk from children with severe P. falciparum malaria12 and also 256 P50 IN SEVERE MALARIA 257

TABLE 1 600 MHz at room temperature in a Bruker AMX spectrom-

Distribution of clinical states in the older and more recent series eter using the following parameters: pre-saturation on H2O, acquisition with a 30° pulse and 10-second recycling time for Clinical category Old series New series Total full relaxation). Cerebral malaria* 8 15 23 6 In vivo P50 was calculated from the following equation. Prostrate† 0 8 8 Respiratory distress‡ 5 1 6 = ͓ + ͑ − ͒ + ͑ − ͔͒ Log10P50 log10 26.6 0.5 MCHC 33 0.69 BPG 14.5 Severe anemia (Hb < 5 g/dL)§ 3 12 15 − 0.0013BD + 0.48͑7.4 − pH͒ + 0.024(T − 37͒ Cerebral malaria and respiratory distress 4 1 5 In this equation, BPG is expressed in ␮mol/g Hb, in contrast Cerebral malaria and anemia 0 1 1 Respiratory distress and anemia 2 1 3 to mol/mol Hb used elsewhere in this paper. BE (base excess) 6 Cerebral malaria, respiratory in the original equation has been replaced by BD (base defi- distress, and anemia 1 0 1 cit), together with the consequent sign change. T is tempera- Totals 23 39 62 ture (°C). Clinical states: (in all states, P. falciparum asexual parasitemia was present). Statistical methods. Because there were no significant dif- * Cerebral malaria—coma (inability to localize a painful stimulus, scoring two or less on the Blantyre coma scale23). ferences between the two batches in respect of mean BPG, † Prostrate—inability to sit upright in a child normally able to do so or inability to drink in the case of children too young to sit. blood lactate, temperature, base deficit, and pH, and only ‡ Respiratory distress—deep breathing (Kussmaul acidotic breathing) or chest in-drawing small significant differences (old series first) for hemoglobin without any finding on auscultation. § Severe anemia—hemoglobin concentration Յ 5 g/dL and the patient needing blood (6.7 versus 5.3 g/dL), MCHC (31.83 versus 30.73 g/dL), and transfusion. packed cell volume (0.22 versus 0.18), the results from the two batches were pooled for analysis; for the same reasons, the from control subjects. All measurements were made at The control results were also pooled. Means are given ±SE, except KEMRI Center for Geographic Medicine Research-Coast, when otherwise stated. Means were compared using one-way with the exception of those for 3-hydroxybutyrate and BPG. analysis of variance (Student t test) or the Kruskal-Wallis test The latter two metabolites were measured on deproteinizates if the variances were non-homogeneous. Correlations were assessed by Pearson test or Spearman rank test if the data of plasma and spun-down erythrocytes shipped on CO2 snow to the United Kingdom, where they were estimated by mag- were not distributed with bivariate normality. Multiple step- netic resonance spectroscopy (31P- or 1H-NMR). wise regression analysis (to P < 0.05) was used to identify independent determinants of variables of interest; this proce- Laboratory methods. Venous blood pH and PCO2 were determined using an IL 1620 blood gas analyser, and hemo- dure adjusts for the influence on the variable under consid- globin (Hb), mean corpuscular volume (MCV), and mean eration of each of the other variables. Two-tailed tests of corpuscular hemoglobin concentration (MCHC) were deter- significance were used. mined by Coulter counter. Whole blood lactate was measured using an Analox lactate analyser. After deproteinizing packed RESULTS cells in 3:1 vol/vol 0.6 mol/L perchloric acid, the supernatant after centrifugation was stored at −20°C before shipment to In four samples of blood taken into citrate-phosphate- the United Kingdom on solid CO2 and further storage at dextrose for transfusion, BPG was 0.12, 0.57, 0.64, and 0.94 –20°C before analysis by 31P-nuclear magnetic resonance mol/mol Hb. These values were lower than the expected spectroscopy as previously described.8 BPG was expressed as range in samples taken from normal subjects and analyzed mol/mol Hb. BPG was also measured on aliquots of four immediately.6 packs of blood for transfusion. For 3-hydroxybutyrate, plasma Table 2 shows the mean concentrations of metabolites, samples obtained 0–4.5 hours after the initial sample were venous pH, and in vivo P50 in the cases and control subjects. stored at −20°C and deproteinized with 20% perchloric acid, As expected, blood lactate was substantially higher and and the supernatant after centrifugation was neutralized with venous pH was lower in the cases; multiple stepwise regres- an equal volume of a mixture of KOH (5 mol/L) and KHCO3 sion showed that the only independent determinants of pH in (5 mol/L). 3-Hydroxybutyrate and salicylate were estimated the cases were blood lactate (mmol/L) and BPG (mmol/mmol on these samples by 1H-NMR spectroscopy13 (at either 500 or Hb)

TABLE 2 Mean values for the variables assessed on admission

Cases (N) 95% confidence limits Controls (N) 95% confidence limits Blood glucose (mmol/L) 5.13 (60) 4.40–5.90 4.24 (8) 3.75–4.72 Blood lactate (mmol/L) 4.83 (61)* 3.32–4.84 1.68 (8) 1.17–2.18 Plasma 3-OHB (mmol/L) 10.47 (54)† 6.88–14.07 1.20 (17) −0.08–2.47 Venous pH 7.26 (58)† 7.23–7.30 7.42 (8) 7.38–7.46 Hb (g/dL) 5.85 (62)† 5.29–6.41 9.86 (29) 9.46–10.25 MCHC (g/dL) 31.14 (61)† 30.69–31.60 31.11 (28) 30.09–32.14 Base excess (mmol/L) −11.60 (58)† −13.60 to −9.60 −2.03 (8) −3.50 to −0.55 2,3-BPG (mmol/mmol Hb) 0.78 (51) 0.65–0.90 0.88 (26) 0.72–1.03

In vivo P50 (mm of Hg) 30.15 (41)* 28.08–32.23 22.84 (8) 21.34–24.34 Salicylate was not detected in any sample. * P < 0.01. † P < 0.001. 258 SASI AND OTHERS

pH = 7.394 − 0.028 ͑blood lactate) partly related to the inanition caused by the illness. However, + 0.158 ͑BPG͒ ͑adjusted R2 = 0.715͒ pre-admission administration of quinine cannot be excluded as a cause of hypoglycemia. Age, PCO2, hemoglobin, temperature, fever duration, The only independent determinants of blood pH identified blood glucose, and plasma 3-hydroxybutyrate were excluded were blood lactate and BPG. The fact that 3-hydroxybutyrate as independent determinants of venous pH by this analysis. was excluded as an independent determinant of pH could be Plasma 3-hydroxybutyrate rose with decreasing blood glucose a reflection of its interaction with other variables, such as (Figure 1). Mean temperature in the cases was 38.64 ± 0.16 body temperature, fever duration, and again, the period of ס (N 59) and 36.81 ± 0.10 in the eight controls in whom inanition consequent on the illness. temperature was measured. The cases were markedly more The cases were markedly more anemic than the controls, anemic than controls, and the mean calculated in vivo P50 and the mean calculated in vivo P50 much higher in cases than much higher in the cases at the time of admission. It should be in controls at the time of admission. There was marked ke- noted that the full data set required for the calculation of in tosis in many of the cases, as reflected by elevated 3-hydroxy- 2 vivo P50 was available in only eight of the control children. butyrate, a phenomenon previously described. Salicylate in- There was marked ketosis in many cases. Mean blood glucose toxication from pre-admission parental medication with aspi- did not differ significantly between cases and controls, al- rin is another theoretical cause for ketosis,14 but no salicylate though 19 cases had a blood glucose concentration < 4 was detectable. mmol/L on admission. For salicylate, the lower limit of de- Perhaps surprisingly, BPG did not differ significantly be- 1 ␮ ∼ tectability in the H-NMR spectra was 50 mol/L ( 0.7 mg/ tween cases and controls, possibly because of opposing effects dL), and there were no resonances in any of the spectra at the of acidosis and anemia on its synthesis. The higher in vivo P50 chemical shift of salicylate that exceeded that value. in the cases was not therefore caused by a compensatory in- Mean BPG was not significantly different between cases crease in BPG or to differences in MCHC. It was principally and controls. The higher in vivo P50 in the cases was not related to changes in venous pH, base deficit, and tempera- caused by a compensatory increase in BPG or to differences in ture. BPG is produced in a side-reaction of the glycolytic MCHC. It was principally related to changes in lactate, pathway unique to erythrocytes. Acidosis inhibits glycolysis at venous pH, base deficit, and temperature; lactate was an in- the step catalyzed by phosphofructokinase-1, the activity of 15–17 dependent determinant of in vivo P50 in the cases [in vivo which is decreased by low pH. The source of the elevated ס P50 0.567(lactate) + 26.2]. blood lactate may therefore not be immediately apparent. However, hepatic gluconeogenesis from lactate is inhibited by 18 DISCUSSION an effect of low pH on pyruvate carboxylase, one of the enzymes specific to gluconeogenesis from lactate or pyruvate. This study characterizes the determinants of acidosis and This enzyme has an obligatory requirement for activation by hemoglobin-oxygen dissociation in Kenyan children with se- acetyl coenzyme A, and activation is inhibited by low pH.18–20 vere malaria caused by P. falciparum. Although, overall, It is possible that this effect is an important reason in these mean blood glucose was not significantly different between cases for raised blood lactate, when not accounted for by cases and controls, individual instances of hypoglycemia in circulatory insufficiency, and could be a target for pharmaco- the cases were a common phenomenon, and, because the logic intervention. In addition, lactate production by the para- samples were taken before known administration of poten- site itself could be a factor.21 tially hypoglycemic agents such as quinine, may have been As pointed out above, the controls cannot be regarded as completely well children, and this may partly account for the

discrepancy between the mean in vivo P50 in the controls (22.5 mm of Hg) and the range found in healthy adults (26–28 mm of Hg). This may be partially because of the marked correction of the anemia22 that had taken place in the controls, but it should be pointed out that the equation of Bell- ingham and others6 was established in European adults as opposed to African children, and the possibility of a system- atic error arising because of this cannot be excluded. Hemo-

globinopathies, which may affect P50, are uncommon in these children; there was only one instance (sickle cell trait) among the subjects studied. It should also be noted that, whereas much of Kenya is at moderate altitude (∼1,600 m), the children in this study came from more coastal regions (0–400 m), and the BPG content of erythrocytes is therefore unlikely to have been significantly influenced by the hypoxia of higher altitudes. The observations in this series may have implications for therapeutic strategy. Obviously, apart from specific anti- malarial therapy, attention should be given to counteracting hypotension, dehydration, hypoglycemia, and anemia. Such FIGURE 1. Relationship of blood glucose (GLU) to plasma 3-hy- -general measures might also permit the cautious administra ס droxybutyrate (OHB) in the cases. The fitted exponential is OHB 21.237e−0.5114GLU (P < 0.01). tion of alkali, which if given without such support, might have P50 IN SEVERE MALARIA 259

[email protected]. Catherine M. Waruiru, Department of Immu- nology, Royal Victoria Infirmary, Queen Victoria Road. Newcastle- upon-Tyne NE1 4LP, UK, E-mail: [email protected]. Michael English, Child and Newborn Health Group, Centre for Geo- graphic Medicine Research-Coast, PO Box 43640, 00100 GPO, Nairobi, Telephone: 254-2027-15160; E-mail: Menglish@nairobi .kemri-wellcome.org. John S. Beech, Department of Anaesthesia, University of Cambridge, Cambridge, UK, E-mail: [email protected]. Richard A. Iles, Department of Radiology and Physics, Institute of Child Health, 30 Guilford Street, London WC1N 3JH, UK, E-mail: [email protected]. Barbara J. Boucher and Robert D. Cohen, Longmeadow, East Dean, Chichester, West Sussex PO18 0JB, UK, Telephone: 440-1243-811230, Fax: 440-1243-811924, E-mails: [email protected] and [email protected]. Reprints requests: R. D. Cohen, Longmeadow, East, Chichester, West Sussex PO18 0JB, UK. E-mail: [email protected].

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