Am. J. Trop. Med. Hyg., 77(2), 2007, pp. 256–260 Copyright © 2007 by The American Society of Tropical Medicine and Hygiene

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 accumulation 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. mize this problem are therefore desirable.10,11 Furthermore, the anemia so common in this group of children is often treated by blood transfusion; however, blood for transfusion, even though collected into citrate-phosphate-dextrose solution, tends to have low BPG levels, and may therefore, not have the expected therapeutic effect. The purpose of this study was to determine the causes of the acidosis in African children with severe malaria and to examine the importance of the several factors involved in oxygen release to the tissues.

INTRODUCTION Lactic acidosis is strongly associated with mortality in severe Plasmodium falciparum malaria in African children.1,2 The lactic acidosis of malaria has multiple etiologies; it is likely that poor tissue perfusion related to hypotension, dehydration, occlusion of the microcirculation by parasites, inhibition of gluconeogenesis by circulating tumor necrosis factor, and decrease in hepatic blood flow3–5 play significant roles. Because severe anemia is commonly present,3 the delivery of oxygen to the tissues may be further impaired. A major determinant of oxygen release from hemoglobin is the position on the x-axis of the sigmoid curve of the plot of hemoglobin-oxygen saturation against the partial pressure of oxygen (PO2). Shifts of the curve to the left of the normal position result in greater difficulty in oxygen dissociation, i.e., PO2 has to be decreased to a lower level than the fall needed under normal circumstances before the same amount of oxygen is released, whereas right shifts result in the opposite situation. The position of the curve is usually denoted by P50, i.e., the value of PO2 for half-saturation of hemoglobin; higher values than the mean normal (∼27.2 mm of Hg) imply easier dissociation. P50 is affected by several factors; low pH and high PCO2 lead to an increase. In addition, the unique intra-erythrocytic metabolite 2,3–bisphosphoglycerate (BPG) interacts with the hemoglobin molecule to raise P50 and thus facilitate oxygen release. Acidosis of more than a very few hours duration impairs the synthesis of BPG,6 and in severe diabetic ketoacidosis, BPG may decrease to < 10% of its previous value6–8; furthermore, a decrease in BPG has been described in an animal model of severe malaria.9 The simple therapeutic approach would be to treat the acidosis of malaria with bicarbonate infusion; however, this may result in an undesirable left shift of the dissociation curve in a situation in which oxygen dissociation has already been impaired by a fall in BPG caused by acidosis. Therapeutic approaches that mini-

MATERIALS AND METHODS Subjects. There were 62 cases (5 fatal), between 6 and 112 months of age (mean, 33.9 ± 20.0 [SD] months), and 29 control subjects of similar age range and distribution (range, 6–119 months; mean, 33.5 ± 25.26 months). The sex ratio (male:female) was 47:53 in the cases and 40:60 in controls. Table 1 shows the distribution of clinical states in the patients. Venous blood samples were collected in two batches: the first consisting of 23 cases and 8 controls in 1996–1997 and the second consisting of 39 cases and 21 controls in 2003–2004. The controls were in apparent good health and were attending planned follow-up, which was 4 weeks after hospitalization for non-severe malaria. Nevertheless, because of their fairly recent illness, they cannot strictly be regarded as completely well children. The criteria for inclusion for the cases were one or more of the following: 1) severe acidosis, defined as base deficit > 18 mmol/L; 2) base deficit between 10 and 18 mmol/L, but persisting for > 6 hours after admission; 3) coma, but with base deficit < 10 mmol/L (if postictal or hypoglycemic, 1 hour was allowed to elapse after the fit or correction of the hypoglycaemia before collection of the sample for the study); 4) blood hemoglobin ⱕ 5.0 g/dL. The reported duration of the illness before admission was noted but is of uncertain reliability. Methods. The study was approved by the Kenya Medical Research Institute (KEMRI) Scientific Steering Committee (SSC) and the National Ethics Review Committee (ERC). Venous samples were obtained with informed parental consent on admission and before the start of in-patient treatment from children with severe P. falciparum malaria12 and also

* Address correspondence to Robert D. Cohen, Longmeadow, East, Chichester, West Sussex PO18 0JB, UK. E-mail: rcohen@ doctors.org.uk

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TABLE 1 Distribution of clinical states in the older and more recent series Clinical category

Cerebral malaria* Prostrate† Respiratory distress‡ Severe anemia (Hb < 5 g/dL)§ Cerebral malaria and respiratory distress Cerebral malaria and anemia Respiratory distress and anemia Cerebral malaria, respiratory distress, and anemia Totals

Old series

New series

Total

8 0 5 3

15 8 1 12

23 8 6 15

4 0 2

1 1 1

5 1 3

1 23

0 39

1 62

Clinical states: (in all states, P. falciparum asexual parasitemia was present). * Cerebral malaria—coma (inability to localize a painful stimulus, scoring two or less on the Blantyre coma scale23). † 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. ‡ Respiratory distress—deep breathing (Kussmaul acidotic breathing) or chest in-drawing without any finding on auscultation. § Severe anemia—hemoglobin concentration ⱕ 5 g/dL and the patient needing blood transfusion.

from control subjects. All measurements were made at The KEMRI Center for Geographic Medicine Research-Coast, with the exception of those for 3-hydroxybutyrate and BPG. The latter two metabolites were measured on deproteinizates of plasma and spun-down erythrocytes shipped on CO2 snow to the United Kingdom, where they were estimated by magnetic resonance spectroscopy (31P- or 1H-NMR). Laboratory methods. Venous blood pH and PCO2 were determined using an IL 1620 blood gas analyser, and hemoglobin (Hb), mean corpuscular volume (MCV), and mean corpuscular hemoglobin concentration (MCHC) were determined by Coulter counter. Whole blood lactate was measured using an Analox lactate analyser. After deproteinizing packed cells in 3:1 vol/vol 0.6 mol/L perchloric acid, the supernatant after centrifugation was stored at −20°C before shipment to the United Kingdom on solid CO2 and further storage at –20°C before analysis by 31P-nuclear magnetic resonance spectroscopy as previously described.8 BPG was expressed as mol/mol Hb. BPG was also measured on aliquots of four packs of blood for transfusion. For 3-hydroxybutyrate, plasma samples obtained 0–4.5 hours after the initial sample were stored at −20°C and deproteinized with 20% perchloric acid, and the supernatant after centrifugation was neutralized with an equal volume of a mixture of KOH (5 mol/L) and KHCO3 (5 mol/L). 3-Hydroxybutyrate and salicylate were estimated on these samples by 1H-NMR spectroscopy13 (at either 500 or

600 MHz at room temperature in a Bruker AMX spectrometer using the following parameters: pre-saturation on H2O, acquisition with a 30° pulse and 10-second recycling time for full relaxation). In vivo P50 was calculated from the following equation.6 Log10P50 = log10关26.6 + 0.5共MCHC − 33兲 + 0.69共BPG −14.5兲兴 − 0.0013BD + 0.48共7.4 − pH兲 + 0.024(T − 37兲 In this equation, BPG is expressed in ␮mol/g Hb, in contrast to mol/mol Hb used elsewhere in this paper. BE (base excess) in the original equation6 has been replaced by BD (base deficit), together with the consequent sign change. T is temperature (°C). Statistical methods. Because there were no significant differences between the two batches in respect of mean BPG, blood lactate, temperature, base deficit, and pH, and only small significant differences (old series first) for hemoglobin (6.7 versus 5.3 g/dL), MCHC (31.83 versus 30.73 g/dL), and packed cell volume (0.22 versus 0.18), the results from the two batches were pooled for analysis; for the same reasons, the control results were also pooled. Means are given ±SE, except when otherwise stated. Means were compared using one-way analysis of variance (Student t test) or the Kruskal-Wallis test if the variances were non-homogeneous. Correlations were assessed by Pearson test or Spearman rank test if the data were not distributed with bivariate normality. Multiple stepwise regression analysis (to P < 0.05) was used to identify independent determinants of variables of interest; this procedure adjusts for the influence on the variable under consideration of each of the other variables. Two-tailed tests of significance were used. RESULTS In four samples of blood taken into citrate-phosphatedextrose for transfusion, BPG was 0.12, 0.57, 0.64, and 0.94 mol/mol Hb. These values were lower than the expected range in samples taken from normal subjects and analyzed immediately.6 Table 2 shows the mean concentrations of metabolites, venous pH, and in vivo P50 in the cases and control subjects. As expected, blood lactate was substantially higher and venous pH was lower in the cases; multiple stepwise regression showed that the only independent determinants of pH in the cases were blood lactate (mmol/L) and BPG (mmol/mmol Hb)

TABLE 2 Mean values for the variables assessed on admission

Blood glucose (mmol/L) Blood lactate (mmol/L) Plasma 3-OHB (mmol/L) Venous pH Hb (g/dL) MCHC (g/dL) Base excess (mmol/L) 2,3-BPG (mmol/mmol Hb) In vivo P50 (mm of Hg) Salicylate was not detected in any sample. * P < 0.01. † P < 0.001.

Cases (N)

95% confidence limits

Controls (N)

95% confidence limits

5.13 (60) 4.83 (61)* 10.47 (54)† 7.26 (58)† 5.85 (62)† 31.14 (61)† −11.60 (58)† 0.78 (51) 30.15 (41)*

4.40–5.90 3.32–4.84 6.88–14.07 7.23–7.30 5.29–6.41 30.69–31.60 −13.60 to −9.60 0.65–0.90 28.08–32.23

4.24 (8) 1.68 (8) 1.20 (17) 7.42 (8) 9.86 (29) 31.11 (28) −2.03 (8) 0.88 (26) 22.84 (8)

3.75–4.72 1.17–2.18 −0.08–2.47 7.38–7.46 9.46–10.25 30.09–32.14 −3.50 to −0.55 0.72–1.03 21.34–24.34

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pH = 7.394 − 0.028 共blood lactate) + 0.158 共BPG兲 共adjusted R2 = 0.715兲 Age, PCO2, hemoglobin, temperature, fever duration, blood glucose, and plasma 3-hydroxybutyrate were excluded as independent determinants of venous pH by this analysis. Plasma 3-hydroxybutyrate rose with decreasing blood glucose (Figure 1). Mean temperature in the cases was 38.64 ± 0.16 (N ⳱ 59) and 36.81 ± 0.10 in the eight controls in whom temperature was measured. The cases were markedly more anemic than controls, and the mean calculated in vivo P50 much higher in the cases at the time of admission. It should be noted that the full data set required for the calculation of in vivo P50 was available in only eight of the control children. There was marked ketosis in many cases. Mean blood glucose did not differ significantly between cases and controls, although 19 cases had a blood glucose concentration < 4 mmol/L on admission. For salicylate, the lower limit of detectability in the 1H-NMR spectra was 50 ␮mol/L (∼0.7 mg/ dL), and there were no resonances in any of the spectra at the chemical shift of salicylate that exceeded that value. Mean BPG was not significantly different between cases and controls. The higher in vivo P50 in the cases was not caused by a compensatory increase in BPG or to differences in MCHC. It was principally related to changes in lactate, venous pH, base deficit, and temperature; lactate was an independent determinant of in vivo P50 in the cases [in vivo P50 ⳱ 0.567(lactate) + 26.2]. DISCUSSION This study characterizes the determinants of acidosis and hemoglobin-oxygen dissociation in Kenyan children with severe malaria caused by P. falciparum. Although, overall, mean blood glucose was not significantly different between cases and controls, individual instances of hypoglycemia in the cases were a common phenomenon, and, because the samples were taken before known administration of potentially hypoglycemic agents such as quinine, may have been

FIGURE 1. Relationship of blood glucose (GLU) to plasma 3-hydroxybutyrate (OHB) in the cases. The fitted exponential is OHB ⳱ 21.237e−0.5114GLU (P < 0.01).

partly related to the inanition caused by the illness. However, pre-admission administration of quinine cannot be excluded as a cause of hypoglycemia. The only independent determinants of blood pH identified were blood lactate and BPG. The fact that 3-hydroxybutyrate was excluded as an independent determinant of pH could be a reflection of its interaction with other variables, such as body temperature, fever duration, and again, the period of inanition consequent on the illness. The cases were markedly more anemic than the controls, and the mean calculated in vivo P50 much higher in cases than in controls at the time of admission. There was marked ketosis in many of the cases, as reflected by elevated 3-hydroxybutyrate, a phenomenon previously described.2 Salicylate intoxication from pre-admission parental medication with aspirin is another theoretical cause for ketosis,14 but no salicylate was detectable. Perhaps surprisingly, BPG did not differ significantly between cases and controls, possibly because of opposing effects of acidosis and anemia on its synthesis. The higher in vivo P50 in the cases was not therefore caused by a compensatory increase in BPG or to differences in MCHC. It was principally related to changes in venous pH, base deficit, and temperature. BPG is produced in a side-reaction of the glycolytic pathway unique to erythrocytes. Acidosis inhibits glycolysis at the step catalyzed by phosphofructokinase-1, the activity of which is decreased by low pH.15–17 The source of the elevated blood lactate may therefore not be immediately apparent. However, hepatic gluconeogenesis from lactate is inhibited by an effect of low pH18 on pyruvate carboxylase, one of the enzymes specific to gluconeogenesis from lactate or pyruvate. This enzyme has an obligatory requirement for activation by acetyl coenzyme A, and activation is inhibited by low pH.18–20 It is possible that this effect is an important reason in these cases for raised blood lactate, when not accounted for by circulatory insufficiency, and could be a target for pharmacologic intervention. In addition, lactate production by the parasite itself could be a factor.21 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 Bellingham and others6 was established in European adults as opposed to African children, and the possibility of a systematic error arising because of this cannot be excluded. Hemoglobinopathies, 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 antimalarial therapy, attention should be given to counteracting hypotension, dehydration, hypoglycemia, and anemia. Such general measures might also permit the cautious administration of alkali, which if given without such support, might have

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[email protected]. Catherine M. Waruiru, Department of Immunology, Royal Victoria Infirmary, Queen Victoria Road. Newcastleupon-Tyne NE1 4LP, UK, E-mail: [email protected]. Michael English, Child and Newborn Health Group, Centre for Geographic 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].

REFERENCES

FIGURE 2. Relationship of blood lactate to blood glucose in the cases. The fitted quadratic function is lactate ⳱ 0.112(glucose)2 − 1.22(glucose) + 6.31 (P < 0.001). A linear fit was not significant.

clinically important adverse effects on oxygen dissociation.10,11 Some comment is needed on the plot of blood lactate against blood glucose (Figure 2). The curvilinear regression through the points is a quadratic function, of significance P < 0.02, and with a minimum blood lactate at plasma glucose 5.5 mmol/L. The biphasic nature of this curve may result from the inhibition of gluconeogenesis seen in malaria accounting for the negative relationship between blood lactate and blood glucose < 5.5 mmol/L, and with the positive relationship at blood glucose above that level resulting from a dominant effect of increased substrate supply to the glycolytic pathway. One possibility arising from this data is that a strategy of a modest but deliberate sustained degree of elevation of blood glucose to > 5.5 mmol/L might lead to an increase in erythrocyte BPG concentration sufficient to improve oxygen release to the tissues within a clinically relevant time frame. There is no current evidence to support such a strategy, but its possible value could be assessed in a suitably designed clinical trial. Received February 26, 2007. Accepted for publication April 27, 2007. Acknowledgments: This study was published with the permission of the Director of the Kenya Medical Research Institute (KEMRI). The authors thank the parents/guardians of children enrolled in the study for accepting the invitation to participate; the staff of Kilifi District Hospital and the KEMRI Centre for Geographic Medicine Research–Coast for valuable support and assistance during data collection; and Jacktone Obeiro for collaboration in the early stages of the project. The American Society of Tropical Medicine and Hygiene (ASTMH) assisted with publication expenses. Financial support: This study was funded through the KEMRI/ Wellcome Trust Research Programme. P. Sasi was supported by a Research Capacity Strengthening Grant from WHO (TDR/MIM Grant 980074) to Professor Gilbert Kokwaro. Authors’ addresses: Philip Sasi and Moses Mosobo, KEMRI/ Wellcome Trust Research Programme, Centre for Geographic Medicine Research-Coast, PO Box 230-80108, Kilifi, Kenya, E-mail: [email protected]. Shamus P. Burns, Claire L. Hobson, and Christopher G. King, School of Applied Sciences (Biological Sciences), University of Huddersfield, Huddersfield, UK, E-mail:

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