Dirk Reijngoud

Determination of low isotopic enrichment of L-[1-C-13]valine by gas chromatography combustion isotope ratio mass spectrometry: a robust method for measuring protein fractional synthetic rates in vivo

A method was developed for measuring protein fractional synthetic rates using the N-methoxycarbonylmethyl ester (MCM) derivative of L-[1-13C]valine and on-line gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). The derivatization procedure can be performed rapidly and GC separation of valine from the other branched-chain amino acids, leucine and isoleucine, is easily obtained. A good linear relationship was observed between the increment of the 13C/12C isotope ratio in CO2 gas derived from the combustion of derivatized valine and the tracer mole ratio of L-[1-13C]valine to unlabelled valine. The limit of quantitation was at an L-[1-13C]valine tracer mole ratio of 0.0002. The method was used to measure the isotopic enrichment of L-[1-13C]valine in standard mixtures and in skeletal muscle of six growing piglets infused with L-[1-13C]valine (2 mg kg-1 h-1 for 6 h). After infusion of L-[1-13C]valine the mean tracer mole ratio in plasma of L-[1-13C]valine at the isotopic steady state was 0.0740 +/- 0.0056 (GC/MS, mean +/- SEM) and the mean tracer mole ratio of valine in muscle protein fraction at 6 h was 0.000236 +/- 0.000038 (GC/C/IRMS). The resulting mean protein fractional synthetic rate in piglet skeletal muscle was 0.052 +/- 0.007% h-1, which is in good agreement with literature data obtained with alternative, more elaborate techniques. By this method protein fractional synthetic rates can be measured at low isotopic enrichment levels using L-[1-13C]valine, the MCM derivative and on-line GC/C/IRMS.

Effects of artificial ventilation on surfactant phospholipid metabolism in rabbits

Surfactant phospholipid metabolism and lung stability were studied in mechanically ventilated and in spontaneously breathing rabbits (control group). During ventilation the dynamic lung-thorax compliance decreased to 79% after 6 h. Static compliance and amount and composition of surfactant phospholipids remained unaltered. These data indicate inactivation of alveolar surfactant during ventilation, which is reversible by a single large inflation. Incorporation of injected radioactively labeled palmitate into saturated phosphatidylcholine (SPC) of the lamellar body fraction increased significantly in the ventilated group; maximal specific activity increased from 20 dpm/nmol SPC at 6 h in the control group to 30 dpm/nmol SPC at 2 h in the ventilated group. The clearance of radioactivity from the lamellar bodies into the alveolar lumen was greatly enhanced in the ventilated group. The results are explained by assuming that as a result of the inactivation of alveolar surfactant, endocytosed surfactant is degraded in the type II cells instead of being recycled and that the degradation products are subsequently reutilized in surfactant synthesis. This interpretation is supported by computer simulations.

Surfactant replacement therapy in surfactant-deficient rabbits: Early effects on lung function and biochemical aspects

Lung-surfactant-deficient rabbits (n = 6) requiring artificial ventilation were subjected to a weaning-off regimen following surfactant replacement therapy. Surfactant-deficient rabbits (n = 6) that did not receive surfactant but underwent the same procedure served as controls. All surfactant-treated rabbits survived (i.e., reestablished spontaneous air breathing) whereas all the control animals died. In the surfactant-treated animals lung function improved in such a way that during the weaning period PaCO2 did not increase and the level of PaO2 remained significantly higher than in the control animals. The static lung compliance and the stability and expansion indices in vitro were significantly higher in the surfactant-treated rabbits. The lamellar body fraction of the lungs of surfactant-treated animals contained a significantly higher amount of surfactant phospholipids than those of the control animals.It is concluded that the animal model used in this study is an excellent tool for testing early effects of different surfactant preparations.

High-frequency oscillation affects surfactant phospholipid metabolism in rabbits

Surfactant phospholipid metabolism was studied in anesthetized rabbits ventilated with high-frequency oscillation at a frequency of 5 Hz and a mean airway pressure of 5 cm H2O. Blood gases were normal although some atelectasis was evident after 1 h. The static compliance of the lungs and amount and composition of surfactant phospholipids of the lamellar body and alveolar lavage fraction were comparable to values found for spontaneously breathing rabbits. The data obtained for the incorporation of radioactively labeled palmitate into phospholipids are compatible with intracellular degradation of newly synthesized surfactant phospholipids. This hypothesis is supported by two observations. First, the rapid initial increase in specific activity of SPC and PC of the lamellar body fraction is not accompanied by a similar rapid increase in specific activity of SPC and PC of the lamellar body fraction is not accompanied by a similar rapid increase in specific activity of the alveolar lavage fraction. Second, a dissociation occurs between the metabolism of PC and SPC for the lamellar body fraction but not for the alveolar lavage fraction. The change in metabolism might be caused by the absence of large pressure swings during this pattern of ventilation.

Postnatal Glucose Kinetics in Newborns of Tightly Controlled Insulin-Dependent Diabetic Mothers

ABSTRACT: Infants of diabetic mothers are at risk of developing hypoglycemia postnatally. Strict control of blood glucose during pregnancy might result in adequate glucose homeostasis in the neonate. We followed 15 mother-infant pairs from the beginning of pregnancy until birth. Glucose kinetics in the infants were measured on the first day of life, using a stable isotope dilution technique. Furthermore, levels of alternative substrates, FFA, and ketone bodies were measured. All infants received i. v. glucose from birth onward at a rate of 3.4 ± 0.7 mg/kg/min (mean ± SD). There was no relationship between the parameters of control of the insulin-dependent diabetes mellitus in the mothers and glucose kinetics in their infants. Glucose turnover was 5.2 ± 1.1 mg/kg/min, glucose production rate (GPR) was 1.8 ± 1.1 mg/kg/min. GPR was significantly lower in the infants studied at the end of the first day of life (p < 0.01), irrespective of the glucose infusion rate. Furthermore, the lower GPR was associated with an increased concentration of ketone bodies, suggesting an increased production of ketone bodies in these infants. The relatively high GPR measured in the infants who were studied in the first hours postnatally may be the result of postnatal hormonal stimulation of glycogenosis and/or gluconeogenesis. From this study, we conclude that glucose kinetics in infants of tightly controlled diabetic mothers appear to be normal. Interestingly, despite the near-optimal insulin therapy in the mothers, there is a relationship between the SD scores of birth weight and the mean 3rd-trimester blood glucose values.

GLUCOSE-METABOLISM IN PRETERM AND TERM INFANTS OF DIABETIC MOTHERS IN THE 1ST HOURS OF LIFE

There are almost no data on endogenous glucose production rate (EGPR) and glucose appearance rate (Ra gluc) in preterm infants of diabetic mothers (idm).Therefore we studied 5 preterm and 8 term idms within the first 24 h after birth (mean 8 h). Ra gluc and EGPR were measured, prior to oral feeding, with the prime-dose constant-rate infusion technique, using 6,6-dideuteroglucose. To prevent hypoglycaemia all infants received a low dose glucose i.v. (mean 3.4 mg/kg/min) Results are shown in the table (mean, range).There are no significant differences in EGPR and Ra gluc between preterm and term idms. In preterm idms EGPR is lower than reported for premature infers born to non-diabetic mothers.

COMPARISON OF 2 SURFACTANTS - INVITRO SURFACE-PROPERTIES AND EARLY EFFECTS IN SURFACTANT DEFICIENT RABBITS

In search of the most effective surfactant to use for surfactant replacement therapy, we compared an artificial porcine derived (APS) and neutral sheep surfactant preparation (NSS) with respect to in vitro characteristics and in vivo early effects. In 6 animals APS and 6 others NSS was administered endotracheally. Results: APS had the lowest in vitro minimal surface tension, 7 vs. 21 mN/m (Wilhelmy balance) and nearly zero vs. 22 mN/m (oscillating bublle) and a larger hysteresis compared to NSS. Within 1 min. following endotracheal surfactant instillation, however, PaO2 increased to significantly higher levels in the NSS group animals: 48.9 ± 16.2 vs. 25.1 ± 12.0 kPa (mean±SD, p<0.05). Similarly, static lung compliance was significantly higher in the NSS group: 1.04 ± 0.33 vs. 0.06±0.10 ml/cmH2O/kg body weight (p<0.05). We conclude that early in vivo effects of surfactant preparations do not seem to relate to in vitro properties.