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Neonatal intensive care has markedly improved survival of extremely preterm infants(1). Due to small energy stores and immature enzyme systems, hypoglycemia is common among these infants and may lead to convulsions and neurologic damage(2,3). An immature regulation of the glucose homeostasis can also result in hyperglycemia, which may lead to osmotic diuresis, dehydration, and risk of cerebral hemorrhage(4). The metabolism of glucose may be influenced by several drugs. One such drug is theophylline, which is administered very commonly as prophylaxis and treatment of apnea in preterm infants(5). The pharmacologic effects of theophylline include relaxation of bronchial smooth muscle(6) and stimulation of the hypoxic ventilatory drive(7). Theophylline also has several metabolic effects, including influence on metabolism of glucose(8) as well as on lipolysis(9).

On a cellular level, theophylline has several different effects, among which inhibition of cAMP phosphodiesterase(10) and adenosine A1 receptors(11,12) are the most well known. Under clinical conditions, the effect of theophylline on apnea seems to be mediated primarily by blockade of adenosine A1 receptors(12). Theophylline in therapeutic doses stimulates the release of norepinephrine and epinephrine(13,14). Toxic doses of theophylline given to dogs resulted in increased concentrations of epinephrine and norepinephrine as well as in several metabolic effects including hyperglycemia. The effects could be prevented or partially reversed by β-blockade with propranolol, indicating the existence of β-adrenergic receptor mediation(15).

The inhibition of cAMP phosphodiesterase increases levels of cAMP(16), which in turn may stimulate glycogenolysis, gluconeogenesis, and lipolysis. To obtain these effects, a serum concentration of theophylline 3 to 10 times higher than that achieved in newborn infants during treatment of apnea is necessary(17,18).

Acute administration of theophylline has been reported to increase lipolysis during short-term fasting in adults(9). No increases in concentrations of catecholamines, plasma glucose, insulin, or growth hormone were noted in that study.

Fjeld et al.(19) studied kinetics and mobilization of fuel in response to chronic treatment with theophylline in preterm infants with postnatal ages of 2-5 wk. They found no effect on lipolysis, glucose production, or gluconeogenesis from glycerol in comparison with a control group. Because metabolic effects related to adaptation and maturation are known to occur postnatally, it is important to evaluate the effect of theophylline on glucose metabolism and lipolysis at start of treatment. For this purpose, a tracer dilution technique using stable isotope-labeled glucose and glycerol has been used in the present study to explore changes in production and utilization of glucose and glycerol after acute administration of theophylline in preterm infants (≤32 wk).

METHODS

Subjects. Ten preterm appropriate-for-gestational-age infants with gestational ages of ≤32 wk and birth weights >900 g were recruited from the neonatal intensive care unit at Uppsala University Children's Hospital (Table 1). Decisions to prescribe theophylline because of symptoms of apnea were made independently of the study by a physician in charge of the ward. Parental consent was obtained after oral and written information was provided. The study was approved by the Human Ethics Committee of the Medical Faculty of the University of Uppsala. Other causes of apnea apart from prematurity had been ruled out. Theophylline was administered as an intravenous solution (23 mg/mL; Aminophylline, Kabi Vitrum, Stockholm, Sweden) in a first loading dose corresponding to 6 mg/kg. The infants had gestational ages (estimated by ultrasound examination during pregnancy wk 16-18 and confirmed by maternal menstrual history) of 27 to 32 wk and postnatal ages between 16 and 84 h. They had body weights between 950 and 1827 g. Eight of 10 infants required treatment with continuous positive airway pressure. They were all normoventilated and normoxemic, 4/10 on air and 6/10 with an oxygen supply of 23, 25, 30, 30, 45, and 50%, respectively. Six of the infants received antibiotics because of premature rupture of membranes but none of the infants showed clinical or laboratory evidence of infection. None of the infants received steroids or diuretics. The infants were kept in isolettes at thermoneutral temperature and had body temperatures between 36.2 and 37.4°C. They were given parenteral nutrition and/or fed enterally with breast milk. The total amount of breast milk given during the last 16-24 h before the study ranged from 7 to 64 mL/kg (median 25 mL/kg), corresponding to 1-17 mL/kg (median 2 mL/kg) every second to third hour. The parenteral formula had a fixed composition of nutrients, vitamins, and electrolytes. None of the infants had been fed orally for a minimum of 2 h before the study (Table 1).

Table 1 Patient characteristics
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Isotope tracers. The tracers used were [6,6-2H2] glucose (isotopic purity 98 atom %) and [2-13C]glycerol (isotopic purity 98 atom %), purchased from Cambridge Isotope Laboratories, Woburn, MA. The [6,6-2H2]glucose and [2-13C]glycerol were dissolved in 0.9% saline solution in concentrations of 4.5 and 1.2 mg/mL, respectively. The solutions were sterile in microbiological cultures and pyrogen-free when tested by the limulus-lysate method(20).

Study design. The study was performed in the neonatal intensive care unit, University Childrens Hospital, Uppsala, Sweden. A peripheral vein catheter was inserted for infusion of the tracers and unlabeled glucose, whereas blood samples were obtained from an umbilical artery catheter or a second peripheral vein catheter inserted for clinical purposes. Saline was used throughout the study period to rinse catheters used for blood sampling to avoid contamination with glucose. One blood sample was taken for measurement of natural isotopic abundance, and then the tracers and a 10% glucose infusion were administered simultaneously as constant rate infusion for three and one-half hours. For [6,6-2H2]glucose, the infusion rate was 0.11 mg · kg-1 · min-1, and for [2-13C]glycerol, the infusion rate was 0.03 mg · kg-1 · min-1. The infusion of glucose, 100 mg/mL, corresponded to 3.33 mg · kg-1 · min-1. No episodes of hypoglycemia occurred during the study. The tracers and the unlabeled glucose were infused with calibrated volumetric pumps (IMED 965 micro, IMED, Oxford, England). Blood samples (300-400 µL per sample, approximately 4 mL blood) were obtained before start of the tracer infusion and every 15 min during the last 2 h and 15 min of the study period (a total of 11 samples). First basal measurements were performed and, after 60 min, theophylline was introduced. The samples were collected into ice-cold EDTA tubes.

Chemical procedures. Plasma was immediately separated by centrifugation and the plasma glucose concentration was measured within 5 min by the glucose oxidase-peroxidase method in a glucose analyzer, Ames Minilab 1 (Bayer AG, Leverkusen, Germany), as described earlier(21). The remaining plasma was frozen at -70°C, awaiting further analysis. An internal standard of [1,1,2,3,3-2H5]glycerol (isotopic purity 98%), obtained from Cambridge Isotope Laboratories was added to the plasma samples for quantitation of plasma glycerol. Precipitation of the plasma proteins was performed with acetone, and the pentaactetate derivative of glucose and triacetate derivate of glycerol were prepared by addition of equal amounts of pyridine and acetic anhydride. The isotopic enrichments of [6,6-2H2]glucose, [2-13C]glycerol, and [1,1,2,3,3-2H5]glycerol were determined by gas chromatography/mass spectrometry. A Finnigan SSQ 70 mass spectrometer (Finnigan MAT, San José, CA) equipped with a Varian 3400 gas chromatograph (Varian Associates Inc., Sunnyvale, CA) with a nonpolar (DB 1) capillary column (15 m × 0.25 mm) was used. The temperature of the oven was set to 180 and 130° for glucose and glycerol, respectively. Chemical ionization with methane was used with selective monitoring of ions. For glucose, the ions monitored were m/z 331 and 333, reflecting unlabeled and dideuterated glucose (M + 2). For glycerol, m/z 159, 160, and 164 corresponded to unlabeled glycerol, 13C-labeled glycerol (M + 1), and the 5-deuterated internal standard (M + 5)(21–23). Due to technical reasons, no data on glycerol concentrations in plasma could be obtained in patients 3 and 6.

Calculations. The glycerol concentration in plasma was calculated from the ion current ratio 159/164 during periods of approximate steady state (mean CV 11%) using a standard curve (cf.21,22). The standard solutions were prepared by adding an amount of the internal standard, equal to that added to the plasma samples, to increasing amounts of unlabeled glycerol. The corresponding CV for plasma glucose concentration during approximate steady state averaged 6%. Ra of glucose and glycerol were calculated from isotopic enrichments of [6,6-2H2]glucose and [2-13C]glycerol obtained during the periods of approximate steady state (mean CV of m/z 333/331 and of m/z 160/159 were both 4%) using standard curves obtained by gradually increasing amounts of labeled glucose and glycerol in relation to the corresponding unlabeled compounds(22). GPR and endogenous rate of glycerol Ra were calculated as follows: GPR = (i × 100/IE) - glucose infusion rate; endogenous glycerol Ra = i × 100/IE, where i is the infusion rate of the tracer and IE is the isotopic enrichment of the tracer in plasma [given as labeled (tracer)/unlabeled substrate in %] and glucose infusion rate of unlabeled glucose(22). Glucose Ra = GPR + exogenous unlabeled and labeled glucose. It was not possible to calculate enteral contributions to the Ra of glucose and glycerol. Since the given amounts of breast milk were small and the time without oral feeding before study was at least 2 h, any enteral contribution of glucose or glycerol must have been small.

Statistical analysis. The results are presented as mean ± SD or, when abnormal distribution, as median and range. Paired t test was used to test statistical significance of plasma Ra of glucose and glycerol as well as GPR and glycerol concentration in plasma before and after initiation of theophylline treatment.

RESULTS

Calculations with regard to plasma glucose and plasma glycerol as well as isotope enrichment of glucose and glycerol were performed at approximate steady state obtained 60-90 min after start of the tracer infusion as well as 45-90 min after administration of theophylline. The Ra of glycerol and GPR were calculated during these two periods. The plasma glucose concentration averaged 4.0 ± 1.9 mmol/L before and 4.7 ± 2.1 mmol/L (p = 0.0006) after theophylline administration (Fig. 1a and Table 2a). The mean GPR was 6.0 ± 2.5 mg · kg-1 · min-1 before and 4.2 ± 1.9 mg · kg-1 · min-1 (p = 0.002) after theophylline administration (Fig. 1b and Table 2a). GPR was positively correlated (p < 0.001) with the plasma glucose before (r = 0.90) and after (r = 0.84) theophylline administration (Fig. 2). The median plasma glycerol concentration (n = 8, cf. "Methods") was 67.1 (7.5-784.9) µmol/L before and 121.5 (44.0-802.7) µmol/L after administration of theophylline (Fig. 3a and Table 2b). The mean glycerol production rate was 5.9 ± 2.6 µmol · kg-1 · min-1 before and 6.7 ± 3.0 µmol · kg-1 · min-1 after theophylline administration (Fig. 3b and Table 2b). In 8/10 infants, it was possible to quantitate gluconeogenesis from glycerol (Fig. 4a and Table 2c), whereas, in two of the infants, the conversion was under the limit of detection. The median fraction of glycerol transformed into glucose was 21.2% (6.2-61.6%) before and 37.9% (10.6-92.1%) after theophylline administration (Fig. 4b and Table 2c). The median percentage of glucose derived from glycerol corresponded to 1.8% (0.3-7.8%) before and 5.8% (1.1-28.9%) (p = 0.04) after theophylline administration.

Figure 1
figure 1

Plasma glucose concentrations (a) and GPR (b) before and after administration of theophylline (unfilled indicates before; filled, after).

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Table 2a P-glucose, glucose Ra, and GPR before and after theophylline
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Figure 2
figure 2

Correlation between GPR and plasma glucose before (––––) and after (–) theophylline administration.

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Figure 3
figure 3

Plasma glycerol concentrations (a) and glycerol production rate (b) before and after administration of theophylline (unfilled indicates before; filled, after). *No data available; cf. study design.

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Table 2b P-glycerol, glycerol Ra before and after theophylline
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Figure 4
figure 4

Fraction of glycerol turned into glucose before and after theophylline administration (a) and fraction of glucose derived from glycerol before and after theophylline administration (b) (unfilled indicates before; filled, after). *No conversion detectable.

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Table 2c Fraction of glycerol converted to glucose and fraction of GPR obtained from glycerol before and after theophylline
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DISCUSSION

The present study shows that initiation of theophylline therapy was associated with a rise in plasma glucose concentration in a group of preterm infants (≤32 wk). Despite an increased fraction of glucose obtained from glycerol after the start of theophylline administration, the treatment was associated with a decrease in the GPR, indicating a parallel fall in glucose utilization. The rate of lipolysis was not influenced by the medication.

Even though plasma glucose increased, hyperglycemia was not induced during the study period. This is in line with the clinical experience that theophylline treatment is not associated with adverse metabolic effects in newborns. In fact, Srinivasan et al.(16) have reported on a similar increase of plasma glucose after a loading dose of theophylline in preterm infants. Although studies on the effect of theophylline in adult humans(9) have resulted in unchanged or increased glucose production, the mean rate in our infants declined. It was, however, still well within the range reported for term and preterm infants. In fact, Corssmit et al.(24) have shown that pentoxyfylline (a xanthine derivative) can inhibit hepatic glucose production in adults without any associated changes in concentrations of glucoregulatory hormones. A relationship between glucose utilization and glucose concentration has been proposed earlier(25–27) and is supported by the positive correlation between GPR and plasma glucose levels found both before and after theophylline administration. The shift of the regression line after theophylline administration indicates the occurrence of a new regulatory set point between GPR and plasma glucose levels.

The level of plasma glycerol varied considerably between the infants. This is not surprising in view of earlier data on preterm infants(22). In fact, the endogenous plasma glycerol Ra and the gluconeogenic contributions from glycerol were similar to what has been reported earlier in extreme prematurity but lower than what has been found in more mature infants(19,28–30). Gluconeogenesis from glycerol contributed little to glucose production before the start of theophylline, whereas a greater part of glucose derived from glycerol after treatment. Even if small, such a contribution may support the hepatic glucose output and prevent a further decrease of the GPR.

The present findings are in some contrast to the concept that theophylline has stimulatory effects on glucose turnover and lipolysis. This concept has been based on studies in vitro(11,18), in animals(10,15), and in adults(9), but there are very limited data on the effect of theophylline on glucose turnover and lipolysis in newborn infants. The only study focusing on infants is that by Fjeld et al.(19) who recently concluded that in preterm infants with postnatal ages of 2-5 wk, chronic administration of theophylline had no effect on lipolysis, GPR, or gluconeogenesis from glycerol. Our data and those of Fjeld et al.(19) indicate that preterm infants have different regulatory mechanisms for glucose and glycerol homeostasis in comparison with adults(31,32). It should be pointed out that in contrast to studies in vitro(11,18) and in adults(9), the study of Fjeld et al.(19) and the present study were performed using theophylline in clinical routine dosage. The fact that there were some discrepancies between the results of our patients and those of Fjeld et al.(19) may be due to differences in postnatal age and to the occurrence of adaptive mechanisms. The latter group of infants had been medicated for several weeks in contrast to our patients who were studied at the very first bolus dose of theophylline given.

Because the stimulatory effect of theophylline on lipolysis has been ascribed to a possible inhibition of adenosine receptor binding, an increase of adenosine receptors after chronic exposure to theophylline has been proposed(11). Such a mechanism could explain the variation in results between the chronic exposure in preterm infants as reported by Fjeld et al.(19) in comparison with the data reported for adults acutely exposed to theophylline(9). Our infants, studied on average on the second day of postnatal life, i.e. at the time when theophylline commonly is instituted against apneic spells, probably still are subject to postnatal stimulation of lipolysis by TSH. Such stimulation is induced immediately after birth by the marked rise of TSH(33). In vitro studies have shown that adipocytes from newborn infants respond to TSh much more efficiently than those of older infants and children(33). At the time of study in the present investigation, there may well have been a maximal stimulation of lipolysis by the combined effect of TSH and catecholamines. This could explain the lack of additional stimulatory effect of theophylline on lipolysis. Except for the possible effects of theophylline in the infants, differences in gestational and postnatal ages as well as time without oral feeding may have influenced the individual results.

In conclusion, we compared rates of glucose production and lipolysis in preterm newborn infants before and after start of theophylline treatment. No effects on lipolysis were obtained, whereas the plasma level and production rate of glucose as well as the fraction of glucose obtained from glycerol were changed. The figures obtained, however, were within the ranges reported for newborn infants of varying degrees of maturity. The results are in line with the lack of adverse metabolic effects of theophylline on production and metabolism of energy substrates in the newborn infant.