TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE (1996) 90,654-657

The relationship between glucose production and plasma glucose concentration in children with falciparum malaria

Evelien Dekkerl, Johannes A. Romijnl, Catherine Waruiru2, Mariette T. Ackermansl, Gerrit J. Weverling3, Robert W. Sauerwein? Erik Endertl, Norbert Peshu 2, Kevin Marsh2 and Hans P. Sauerwein’ ‘Metabolism Unit, Department of Endocrinology and Metabolism, Academic Medical Centre, Netherlands; z~~~~ Clinical Research Centre, Kilifi Unit, Kilif, Kenya;

University of Amsterdam, Amsterdam, The 3~~~~~, Academic Medical Centre, University of

Amsterdam, Amsterdam, The Netherlands; 4Department of Medical Microbiology, University Hospital, University of Nijmegen, Nijmegen, The Netherlands

Abstract The pathophysiology of hypoglycaemia in children with acute falciparum malaria, a frequent and serious complication, is unknown due to absence of data on glucose kinetics. We investigated the correlation be- tween basal glucose production and plasma glucose concentration in 20 children (8 girls) with acute, un- complicated falciparum malaria by infusion of [6,6-2H2]glucose. Median plasma glucose concentration was 4.5 (range 2.145) mmol/L and median glucose production 5.0 (range 4.1-8.4) mg/kg/min. There was a positive correlation between basal glucose production and plasma glucose concentration (r=O.53, P=O.O16). There was no correlation between the rate of glucose production and the plasma concentrations of alanine, lactate, counter-regulatory hormones or cytokines. It was concluded that, in children with acute uncompli- cated falciparum malaria, endogenous glucose production is an important determinant of plasma glucose concentration, contrary to previous findings in adults with malaria, in whom peripheral uptake seems to be more important than glucose production in determining plasma glucose concentration.

Keywords: malaria, Plasmodium fulcipurum, glucose production, children, hypoglycaemia

Introduction Hyperglycaemia is a frequent finding in acute infec-

tions. Acute falciparum malaria is a peculiar infectious disease with respect to glucose metabolism as hypogly- caemia instead of hyperglycaemia is a common compli- cation, especially in children and pregnant women (LOOAREESUWAN et al., 1985; WHITE et al., 1987). Its in- cidence in children with acute falciparum malaria is 13.2% in Kilifi, Kenya, and it is associated with a much increased risk of death (MARSH et al., 1995).

Hypoglycaemia is caused by inhibition of glucose pro- duction and/or stimulation of glucose disuosal. Basal glucose production in falciparum-malaria has been meas- ured only in adults, with plasma glucose concentrations above 3 mmol/L. In adults with complicated malaria, and in pregnant patients with uncomplicated malaria, average glucose production was increased during acute falciparum malaria compared to convalescence (DAVIS et al., 1993, 1994). In non-pregnant patients there was an inverse correlation between glucose production and plasma glucose concentration (DAVIS et al., 1993). The authors concluded that facilitated peripheral uptake (by the host and/or the parasite) was the factor most decisive in determining plasma glucose concentration in these adults.

There are no data available on glucose production during falciparum malaria in children, although globally children are by far the largest group at risk for the devel- opment of hypoglycaemia (WHITE et al., 1987; MARSH et al., 1995). The first objective of this study was therefore the measurement of basal glucose production in children with acute falci arum malaria by primed, continuous in-

P fusion of [6,6- Hzlglucose. We studied only those chil- dren with plasma glucose concentrations 22.2 mmol/L, because lower blood glucose levels would have necessi- tated intravenous glucose infusion. Theoretically, low levels of gluconeogenic precursors and counter-regula- tory hormones and high levels of cytokines may be im- plicated in the induction of hypoglycaemia. The second objective was therefore the evaluation of the relation be- tween the rate of basal glucose production and these fac- tors.

Address for correspondence: Dr Evelien Dekker, Metabolism Unit, Department of Internal Medicine (F4-222), Academic Medical Centre, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands; phone +31 20 566 2663, fax f31 20 6919658.

Methods Subjects

All children admitted to Kilifi District Hospital in Kenya with a primary diagnosis of malaria during the study period of 6 weeks were considered for inclusion in the study. Inclusion criteria were acute falciparum ma- laria, age between 2 and 10 years, and a fasting period of at least 4 h. Exclusion criteria were admission plasma glucose concentration ~2.2 mmol/L, complicated ma- laria according to the World Health Organization crite- ria (WHO, 1986) (because clinical practice dictates con- stant glucose infusion in all such patients), treatment with quinine (quinine stimulates insulin secrection by the nancreas: HENOUIN et aZ.. 1975). concomitant infec- tious disease; severemalnutrition, a;d severe chronic di- arrhoea (which may induce hypoglycaemia in child- hood; BENNISH et al., 1990). Witnessed informed consent was obtained from the accompanying parent or guard- ian. The study protocol was approved by the Kenya Na- tional Ethical Committee.

Study design Patients were recruited immediately after laboratory

confirmation of the clinical diagnosis and exclusion of quinine use by a quinine ‘dipstick’ test (SILAMUT et al., 1995). Each patient was weighed and treatment with FansidarB, in some cases combined with chloroquine, was given. An intravenous cannula was introduced in a forearm vein for isotope infusion. A second cannula for blood sampling was introduced into a suitable vein of the contralateral arm. The catheters were kept patent by a slow saline drip.

After obtaining a baseline blood sample for determi- nation of background isotope enrichment, plasma glu- cose, plasma cytokine concentrations and basal haemato- logical and biochemical tests, a primed (5.4 mg/kg), continuous (60 pg/kg/min) infusion of [6,6-2H2]glucose (99%) Isotec Inc., Miamisburg, Ohio, USA), dissolved in sterile isotonic saline and sterilized by passage through a Millipore’” filter (size 0.2 pm; Minisart, Sartorius AG, Giittingen, Germany), was administered by a motor driven, calibrated syringe pump (Perfusor Secura FT’“, Braun AG, Melsungen, Germany). The rate of [6,6- 2H2]glucose infusion was calculated from the measured concentration of glucose in the infusate. The time at the start of the infusion was set at t=O.

After 90 min of [6,6-2H2]glucose infusion for equili- bration, 3 blood samples were collected at intervals of 15

GLUCOSE AND MALARIA 655

rnin for determination of plasma glucose concentration and [6,6-*Hz]glucose enrichment. Blood samples for the measurement of concentrations of insulin, counter-regu- latory hormones, alanine and lactate were collected at the end of the study (t= 120 min).

Blood samples for determination of plasma glucose, [6,6-2Hz]glucose enrichment, insulin, counter-regulatory hormones and cytokines were collected in pre-chilled heparinized tubes and samples for alanine and lactate into fluoride tubes. All samples were kept on ice and centrifuged promptly. Aliquots of separated plasma were stored below -20°C and transported on dry ice before as- say.

Assays All measurements were performed in duplicate and all

samples of each individual subject were anapd in the same run. Glucose concentration and [6,6- Hzlglucose enrichment in plasma were measured by gas chromatog- raphy/mass spectometry using selected ion monitoring. The method was adapted from that of REINAUER et al. (1990), using phenyl-@-D-glucose as internal standard.

Plasma insulin concentration was measured by com- mercial radioimmunoassav (RIA) (Pharmacia Diaenos- tics, Uppsala, Sweden), gldcagon by RIA (Daiichi Ridio- isotope Laboratories, Tokyo, Japan, using glucagon antiserum elicited in guinea-pigs against pancreatic spe- cific glucagon; cross-reactivity with glucagon-like sub- stances of intestinal origin less than l%), catecholamines by high-performance liquid chromatography and elec- trochemical detection. after ourification on Biorex 70’” and concentration by’solven; extraction (SMEDES et al., 1982), and cortisol by fluorescence polarization immu- noassay on TDx’“’ (Abbott Laboratories, Chicago, Illi- nois, USA). Plasma alanine concentration was deter- mined bv amino acid analvser (Chromocon 500’“. Kontron,‘Italy) and plasma <lactate by an enzymatic method (Boehringer Mannheim, Almere, The Nether- lands) on a Cobas Bio-Centrifugal’” analyser.

Tumour necrosis factor a (TNF-a) concentrations were measured by enzyme amplified sensitivity assay (EASIA) (Medgenix, Amersfoort, The Netherlands) with a detection limit of 5 pg/mL. Souble TNF-receptors types I and II (sTNF-RI and sTNF-RII) were measured by EASIA, with detection limits of 0.1 and 05 ng/mL, re- spectively. Plasma concentrations of interleukin (IL) 1 were measured by immunoradiometric assay (Medgenix, Amersfoort, The Netherlands). detection limit 10 pg/mL, and IL-6 was determined by an enzyme-linked immunosorbent assay (ELISA) (CLB, Amsterdam, The Netherlands), detection level 2 pg/mL. Plasma concen- trations of IL-10 were measured by ELISA (kindly pro- vided by Schering-Plough Research Institute, Kenil- worth, New Jersey, USA), detection limit 20 pg/mL.

Calculations and statistics Glucose production rate was calculated from the dilu-

tion of the infused tracer in plasma. Because plasma glu- cose concentrations and tracer/tracee ratios for [6,6- 2H2]glucose remained constant during the study, calculations for steady state kinetics were applied, adapted for the use of stable isotopes (WOLFE, 1992).

Data are reported as medians and ranges. A correla- tion coefficient was calculated for plasma glucose con- centrations and glucose production, using linear regres- sion analysis to investigate the relationship. Statistical significance was set at PcO.05.

Results Clinical data

Twenty children (including 8 girls) with uncompli- cated acute falciparum malaria were studied (Table 1). Their illness had a median duration of 3 d (range 1-6 d) and they had not fed for a median period of 12 h (range 4-24 h) before the study. Median axillary temperature was 39.3”C (range 37.0-40~8”C). Albumin concentrations

Table 1. Physical, biochemical and parasitological characteristics of 20 Kenyan children with uncompli- ated falciparum malaria

Age (years) Height (cm) Weight (kg) Plasma glucose (mmol/L) Glucose production (mg/kg/min) Parasite count (per I*L) Haemoglobin (g/dL) Serum albumin (g/L) Serum ASTa (units/L) Serum ALTb (units/L) Serum bilirubin (umol/L)

aAspartate aminotransferase. bAlanine aminotransferase.

Median

4.5 103 14.2 4.5 5.0

205110 9.4

2 12 13

Range

2.0-10.0 79-132

94-23.0 2.1-6.5 4.1-8.4

77-699200 6.6-12.8

29-47 24-92

6-29 3-28

were normal except in one child, who did not differ from the other subjects in terms of weight-for-height or height-for-age by the US National Center of Health Sta- tistics standards (HAMILL et al., 1979). Using these standards 10 children were below the tenth percentile of weight-for-height and 12 were below the tenth percentile of height-for-age. All patients responded quickly to ther- apy and made uneventful recoveries.

Glucose kinetics Plasma glucose concentrations at times t=90 min,

t=105 min and t=120 min were similar. (Table 1). Al- though one of the inclusion criteria was plasma glucose concentration >2.2 mmol/L, one patient developed a plasma glucose level of 2.1 mmol/L during the study. The individual plasma glucose concentrations, includ- ing the low values, were also constant during the time of the study.

Isotopic steady state was obtained because there was no difference in tracer/tracee ratios between t=90 min, t=105 min and t=120 min (data not shown). There was a positive correlation between plasma glucose concentra- tion and basal glucose production (t=0.53, P=O.O16) (Figure).

No correlation was found between age, parasite count or body temperature, duration of fasting or duration of illness at the start of the study and plasma glucose con- centration or the rate of glucose production.

Hormones, precursors and cytokines Plasma concentrations of insulin and glucose counter-

7

- 1 0

o+---+ / / I / I I I 0 3 4 5 6 7 a 9

hepatic glucose production lmg.kg-‘.min-‘1

Figure. The relationship between the rate of hepatic glucose production and plasma glucose concentration in 20 Kenyan children with uncomplicated falciparum malaria (r=O.53, P=O.O16).

656 EVELIEN DEKKERETAL.

Table 2. Plasma concentrations of insulin, counter- regulatory hormones, gluconeogenic precursors and cytokines in 20 Kenyan children with uncomplicated falciparum malaria

Normal

Patient? fasting range

Insulin (milliunits/L) 4 (l-25) 5-25 Cortisol (I*mol/L) 0.62 (0.14-2.27) 0.22-0.65 Glucagon (rig/L) 165 (45-498) 40-140 Noradrenaline (nmol/L) 1.18 (0.45-3.90) ~3.25 Adrenaline (nmol/L) 0.60 (0.15-1.98) co.55 Alanine (umol/L) 132 (74-290) 158-314 Lactate (mmol/L) 1.3 (0.8-2.7) 0.6-2.0 TNF-a (pg/mL) 135 (18-468) <20 sTNF-RI (ng/mL) 6.4 (2.1-14.7) 0.3-2.9 sTNF-RI1 (ng/mL) 44.4 (12.6-121.0) 1.9-8.5 IL-1 (na/mL) <lo (<lo) <lo ._ - IL-6 (pg/mLj IL-10 (pg/mL

47 (61342 j <lo 605 (185-3215) <20

aMedian (range in parentheses). Abbreviations: TNF=tumour necrosis factor, sTNF-RI and sTNF-RII=soluble TNF recep- tors, IL=interleukin.

regulatory hormones, alanine and lactate are shown in Table 2. Plasma insulin concentrations were appropri- ately low and plasma concentrations of the counter-regu- latory hormones were appropriately high in all children, with 2 exceptions. One child had a plasma insulin con- centration of 25 milliunits/L; glucose production in this child was 5.4 mg/kg/min. Another child had a low plasma cortisol concentration (0.14 Fmol/L), and a glu- cose production rate of 45 mg/kg/min. These values for glucose production were both intermediate in our study group. The plasma concentrations of the other gluco- regulatory hormones in these 2 children were appropri- ate.

Plasma alanine concentrations were slightly low and plasma lactate concentrations were normal.

There was no correlation between the rate of glucose production and plasma concentrations of insulin or the counter-regulatory hormones, or between glucose pro- duction and plasma concentrations of the gluconeogenic precursors alanine and lactate (either separately or com- bined).

Plasma concentrations of the cytokines TNF-a, sTNF-RI and RII, IL-l, IL-6 and IL-10 are also shown in Table 2. There was no correlation between any of these cytokines and the rate of glucose production.

Discussion This was the first study of glucose production in chil-

dren with falciparum malaria. The children we studied required hospital admission, but had no defining feature of severe malaria (WHO, 1986). They were thus interme- diate between mild out-patient malaria cases and those with the highest risk of hypoglycaemia. In these chil- dren there was a positive correlation between plasma glucose concentration and basal glucose production, in- dicating that children with the lowest plasma glucose concentration had the lowest glucose production rates. Our data suggested that plasma glucose concentrations are primarily determined by glucose production in chil- dren with uncomplicated falciparum malaria.

The clinical data indicated that the Kenyan children evaluated in this study had a weight-for-height and height-for-age around the tenth percentile of the US standards (HAMILL et al., 1979). None the less, physical examination indicated that the children were well pro- portioned and had no other underlying disease; their plasma albumin concentrations supported this finding. Hence, stunted growth in the past and not malnourish- ment at the time of the study was the most probable ex- planation for the low anthropometric values.

The declaration of Helsinki, serving as a guideline for clinical research, does not allow invasive studies in healthy children. We were therefore unable to obtain data from healthy Kenyan children for comparison with our patients. Studies in convalescence were inappropri- ate, for the same reason. There are only 2 published studies on basal glucose production measured by means of a non-recycling isotope in healthy children of the same age grouu elsewhere in the world (BIER et al.. 1977: HAYM~ND et & 1978). Basal glucose production was, on average, approximately 30% lower in the Kenyan chil- dren in our study than in healthy American children with similar body weight (BIER et al., 1977). The present study suggested that, contrary to the findings in adults, glucose production in children with uncomplicated fal- ciparum malaria is not increased. Although the compari- son with healthy American children may not be optimal, this does not invalidate our observation of a positive cor- relation between plasma glucose concentration and glu- cose production in children with malaria.

It is unlikely that the duration of fasting was a deci- sive factor in our study, as the mean duration was only 12 h and literature data suggest that only prolonged fast- ing (> 1 d) is a factor contributing to hypoglycaemia in children with falciparum malaria (TAYLOR et al., 1988; KAWO et al., 1990). Moreover, there was no correlation between the duration of fasting and plasma glucose con- centration or glucose production.

The only antimalarial drug known to influence glu- cose metabolism is quinine, which stimulates insulin re- lease (HENQUIN et al., 1975; WHITE et al., 1983; DAVIS et al., 1990). This was not a confounding variable in our study, as none of the children had detectable plasma qui- nine concentrations and plasma insulin concentrations were appropriately low.

Theoretically, cytokines and/or counter-regulatory hormones could be implicated in the regulation of glu- cose production in our children with malaria as it is well recognized that catecholamines, glucagon and relatively low levels of the cytokines TNF-a and IL-6 can stimu- late glucose production in humans (SHAMOON et al., 1981; GELFAND et al., 1984; VAN DER POLL et al., 1991; STOUTHARD et al., 1995), whereas IL-l induces hypogly- caemia in animal models (FISCHER et al.. 1991). As TNF- a is secreted episodically‘and the concentrations of sol- uble TNF receptors in plasma are considered to be better markers of TNF activity (GODFRIED, 1994), we also included the plasma concentrations of soluble TNF receptors in our analysis. A significant stimulation of counter-regulatory hormone and cytokine production was found in our patients, in accordance with other re- ports in malaria (RERN et al., 1989, 1992; KWIATKOWSKI et al., 1990: PHILLIPS et al., 19%: PEYRON et al.. 1994). However, no correlation was found between the’rate of glucose production and the plasma concentrations of these substances, separately or combined, suggesting that neither of them could be implicated as a major de- terminant for glucose production in these children with uncomplicated malaria.

In untreated malaria, glucose metabolism is deter- mined by factors related to host and parasite, and it has been suggested that a large glucose requirement by the parasite may contribute to the induction of hypoglycae- mia (WHITE et al., 1983). Although some contribution seems plausible, the direct role of the parasite appears to be limited. It can be calculated from data obtained from growth studies in vitro on Plasmodium falciparum para- sites (ZOLG et al., 1984) that, in severe infections, the parasite consumes at the most 10% of total glucose pro- duction, suggesting that changes in the host are the most important precipitating factors for hypoglycaemia. Moreover, if increased peripheral use of glucose were a decisive factor in determining glucose concentration in malaria, a negative correlation would be expected be- tween the rate of glucose production and the plasma glu- cose concentration. In contrast, we found a positive cor-

GLUCOSE AND MALARIA

relation between glucose production and glucose con- centration. Therefore, a major effect on glucose concen- tration by the parasite seems improbable in our patients.

In adults with severe falciparum malaria there is an inverse correlation between plasma glucose concentra- tion and glucose production (DAVIS et al., 1993), suggest- ing that facilitated peripheral glucose uptake rather than decreased production was the most important determi- nant for glucose concentration. In contrast, in children with uncomplicated falciparum malaria there was a posi- tive correlation between glucose production and glucose concentration, suggesting that the rate of production of glucose rather than glucose uptake was the most impor- tant determinant of plasma glucose concentration. These data suggest that glucose metabolism in falciparum ma- laria is differently regulated in children and adults.

Acknowledgements This paper was published with the permission of the Direc-

tor of KEMRI. We are indebted to doctors, nursing and labora- tory staff at the KEMRI unit, Kilifi, and at the Kilifi District Hospital for their pleasant collaboration and helpful advice, to Hazra Moeniralam for her skilful and pleasant assistance dur- ing the studies, to the technicians of the endocrinology labora- tory for their assistance, and to Theunis Eggelte for providing the quinine dipsticks. This work was supported by the Metabo- lic Research Fund AMC, KEMRI, and the Wellcome Trust. J. A. R. is a clinical investigator supported by the Netherlands Or- ganization for Scientific Research and the Dutch Diabetes foundation. K. M. is a Wellcome Trust Senior Fellow in Clini- cal Science.

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Received 30 April 1996; revised 29 July 1996; accepted for publication 31 Ju2y 1996