A stable isotope method for in vivo assessment of human insulin synthesis and secretion.

AIMS: In vitro, beta cells immediately secrete stored but readily releasable insulin in response to a rise of glucose. During a prolonged insulin response, this is followed by newly synthesized insulin. Our aim was to develop an in vivo test to determine the ratio between readily available and newly synthesized insulin after a stimulus in humans by labelling newly synthesized insulin. METHODS: A stable isotope tracer of 1.0 g 13C leucine with C-peptide as target peptide was administered 45 min prior to 75 g glucose load of a frequently blood sampled 210-min oral glucose tolerance test (OGTT). Our OGTT also encompassed collection of urine, which has a high content of C-peptide. Prior, the optimal conditions under which the tracer 13C leucine was administered for enrichment of (pre) proinsulin were established. Also, techniques to obtain urinary C-peptide under highly purified circumstances were set up. Our main outcome measure was the stable isotope enrichment of de novo C-peptide, which we related to early plasma insulin and glucose AUC. Twelve healthy Caucasian individuals (M4F8, age 41.8 ± 2.3, BMI 28.3 ± 1.7) with normal glucose tolerance underwent our OGTT. RESULTS: We found that during a 75-g OGTT, newly synthesized insulin contributed approximately 20 % of total insulin secretion. The pattern of isotope enrichment obtained by collecting multiple urine voids was suggestive that the newly synthesized insulin contributes to the late phase of insulin secretion. De novo C-peptide correlated negatively with both early plasma insulin AUC (r = -0.629, P = 0.028) and early plasma glucose AUC (r = -0.605, P = 0.037). CONCLUSIONS: With stable isotope technique added to OGTT, we were able to measure newly synthesized insulin in healthy individuals. This new technique holds the promise that it is feasible to develop a direct in vivo beta cell function test.

Surfactant phosphatidylcholine pool size in human neonates with congenital diaphragmatic hernia requiring ECMO.

OBJECTIVE: We measured surfactant phosphatidylcholine (PC) pool size and half-life in human congenital diaphragmatic hernia (CDH) patients who required extracorporeal membrane oxygenation (ECMO). Study design Surfactant PC pool size and half-life were measured by endotracheal administration of deuterium-labeled dipalmitoylphosphatidylcholine in 8 neonates with CDH on ECMO (CDH-ECMO), in 7 neonates with meconium aspiration syndrome on ECMO (MAS-ECMO), and in 6 ventilated infants (NON-ECMO). RESULTS: Lung PC pool size in the CDH-ECMO group was 73 +/- 17 mg/kg (mean +/- SEM), which was not significantly different from the MAS-ECMO (50 +/- 18 mg/kg) and the NON-ECMO group (69 +/- 38 mg/kg). Surfactant PC concentration in tracheal aspirates was not different between groups (~6 mg/mL). However, the percentage of palmitic acid in surfactant PC was significantly lower in the MAS-ECMO (56.3%) and the NON-ECMO (55.8%) group compared with the CDH-ECMO (67.6%) group. Surfactant PC half-life (~24 hours) was not different between the groups. A correlation was found between the surfactant PC half-life and the duration of ECMO. CONCLUSIONS: These data show no decreased surfactant PC pool size in high risk CDH patients who require ECMO. A shorter half-life of surfactant PC, indicating a faster turnover, may result in a faster improvement of the pulmonary condition during ECMO.

The effect in premature infants of prenatal corticosteroids on endogenous surfactant synthesis as measured with stable isotopes.

Most in vitro studies show that prenatal administration of corticosteroids stimulates the synthesis of surfactant phosphatidylcholine (PC), but studies in animals are controversial. Whether prenatal corticosteroids stimulate surfactant PC synthesis in humans has not been studied. We studied endogenous surfactant PC synthesis in relation to prenatal corticosteroid treatment in 27 preterm infants with respiratory distress syndrome. Infants received a 24-h infusion of the stable isotope [U-(13)C]glucose, starting approximately 5 h after birth. We measured (13)C-incorporation into palmitic acid in surfactant PC from serial tracheal aspirates and in plasma triglycerides and phospholipids by isotope-ratio mass spectrometry. Premature infants had received either zero (n = 11), one (n = 4), or two doses (n = 12) of prenatal betamethasone (12 mg intramuscularly). The fractional synthesis rate (FSR) of surfactant PC from glucose was 1.7 +/- 0.3%/d without corticosteroid treatment, 2.9 +/- 1.4%/d with one dose of prenatal corticosteroid, and 5.8 +/- 1.3%/d after two doses of prenatal corticosteroid. Using multiple regression analysis, we found that the FSR of surfactant PC increased by 40% (confidence interval: 7 to 82%/d, p < 0.02) per dose of corticosteroid and doubled after two doses of corticosteroid. The (13)C-enrichment of plasma triglycerides and phospholipids was not increased by corticosteroid. These data show for the first time that prenatal corticosteroid treatment stimulates surfactant synthesis in the preterm infant.

Metabolism of endogenous surfactant in premature baboons and effect of prenatal corticosteroids.

We studied the synthesis of surfactant and the effect of prenatal betamethasone treatment in vivo in very preterm baboons. Ten pregnant baboons were randomized to receive either betamethasone (beta) or saline (control) 48 and 24 h before preterm delivery. The newborn baboons were intubated, treated with surfactant, and ventilated for 6 d. They received a 24-h infusion with the stable isotope [U-(13)C]glucose as precursor for the synthesis of palmitic acid in surfactant phosphatidylcholine (PC). Palmitic acid in surfactant PC became enriched 27 +/- 2 h after the start of the isotope infusion and was maximally enriched at 100 +/- 4 h. The fractional synthesis rate of PC palmitate in the beta group (1.5 +/- 0.2%/d) was increased by 129% above control (0.7 +/- 0.1%/d) (p < 0.02, Mann- Whitney U test). The absolute synthesis rate of PC in the beta group [1.6 +/- 0.3 micromol/kg/d] was increased by 128% above controls [0.7 +/- 0.2 micromol/kg/d] (p < 0.02). These data show that the synthesis of endogenous surfactant from plasma glucose as precursor is a slow process. It is shown, for the first time in vivo, that prenatal glucocorticosteroids stimulate the synthesis of surfactant PC in the very premature baboon.