Objective
To determine the concentration of amino acids in women receiving the first course of antenatal betamethasone and to evaluate the umbilical venous and arterial amino acid concentrations at the time of elective cesarean section after betamethasone administration.
Study Design
Blood samples were collected from 34 pregnant women at risk of premature delivery before and 24 and 48 hours after the first course of betamethasone. In addition, maternal and cord blood samples were collected in 13 women undergoing an elective cesarean section between 24 and 192 hours after betamethasone.
Results
Maternal amino acid concentrations were significantly increased after the first dose of betamethasone. Overall total amino nitrogen increased 17.5% 24 hours after betamethasone administration and 20.5% after 48 hours. The concentration of most amino acids was increased both in the umbilical vein and artery after maternal betamethasone administration.
Conclusion
The concentration of maternal and fetal amino acids increases significantly after betamethasone administration.
There is no doubt that the administration of antenatal corticosteroids has radically changed the outcome of preterm infants since it was first introduced in 1972. Since then, researchers have studied the effects of this treatment for the reduction of the incidence and severity of respiratory distress syndrome as well as for other derivative effects, that is, beneficial short-term effects have been reported associated with a significant reduction of the risk of cerebroventricular hemorrhage, necrotizing enterocolitis, and intensive care admission. In addition, an association with less developmental delay and probably less cerebral palsy in childhood have been reported as beneficial long-term effects. In the late nineties, however, animal studies began to raise some concerns regarding fetal growth: 1-3 doses administered to the mother, but not to the fetus, in early gestation have been reported to induce growth restriction in premature lambs although this finding has not been confirmed in mice. In human pregnancies, an independent association between repeated antenatal corticosteroids and impairment of fetal growth, as measured by birthweight, head circumference, and length, has been reported. One possible mechanism may be through an influence on the maternal and fetal insulin-insulin-like growth factor-growth hormone (IGF-GH) axis : a single course of antenatal betamethasone has been found to increase maternal glucose, insulin and insulin-like growth factor 1 (IGF-1) but not IGF binding protein levels, whereas, in the fetal compartment, this treatment was associated with a decrease for GH and IGF-II levels. Recently it has been shown that fetuses exposed ≤48 hours to corticosteroids exhibit a significant increase of glucose, insulin, amino acids (AAs) and free fatty acid concentrations accompanied by a significant decrease in IGF-I levels. However, in that study the maternal concentration of amino acids was not measured. Hence any possible influence upon umbilical venous concentrations could not be determined.
The aim of this study was 2-fold: first, to determine the plasma concentration of AAs in women receiving the first course of antenatal betamethasone to enhance fetal lung maturation. Second, to evaluate the umbilical venous and arterial AA concentrations with respect to maternal concentrations in a group of women undergoing an elective caesarean section after betamethasone administration.
Materials and Methods
The protocol for the study was approved by the review board of the Department of Medicine, Surgery, and Dentistry of the San Paolo Hospital. Written informed consent was given by all study participants.
Patients
Studies of maternal concentrations in at-risk pregnancies
Thirty-four pregnant women at risk of premature delivery who received their first course of antenatal betamethasone at 30.2 ± 3.3 weeks (range, 24.6–35.5 weeks) of gestation (Celestone cronodose; Schering-Plough, Summit, NJ; 12 mg + 12 mg; 24 hours apart) were recruited for the study. Thirty-three of 34 women were white, age was 33 ± 4.5 years, prepregnancy body mass index was 22.3 ± 3.3 kg/m 2 . Eight pregnancies were multiple (6 twins and 2 triplets), 14 were complicated by intrauterine growth restriction, 2 by preeclampsia and 1 by preterm premature rupture of membranes: in the remaining 9 pregnancies no other complication but threatened preterm delivery was present.
Three maternal “arterialized” venous samples were collected from an antecubital vein after at least 4 hours of fasting: M0, before the first dose; M1, 24 hours after the first dose; and M2, 48 hours after the first dose. The “arterialization” of the brachial venous samples was obtained by placing the arm between 2 heated thermophores whose temperature was adjusted until the maternal peripheral blood oxygen saturation was >90%: the sampling site of the brachial vein was proximal to the wrist.
Maternal and fetal studies at cesarean section
Thirteen pregnant women underwent an elective cesarean section between 24 and 192 hours (mean, 92 ± 65 hours) after betamethasone administered according to the same protocol (M0, before the first dose; M1, 24 hours after the first dose; and M2, 48 hours after the first dose). Indication for cesarean section was intrauterine growth restriction (7 cases), multiple pregnancy (4 cases), preeclampsia (1 case), and fetal Rh immunization (1 case).
Four patients (2 multiple, 1 intrauterine growth restriction, and 1 with preeclampsia) are also included in the analysis of maternal concentrations. In all cases, cesarean section was performed under general anesthesia: none of the mothers had entered labor. At the time of fetal extraction, umbilical arterial and venous blood samples were obtained from a doubly clamped segment of the cord. A maternal arterial sample was collected simultaneously.
The data obtained in these patients were compared with historical data collected in 2 groups of pregnant patients undergoing elective cesarean section under general anesthesia: a first group of 16 pregnant patients at term who did not receive antenatal betamethasone and a second group of 10 pregnant patients receiving an AA formulation (Freamine 8.5% III; Baxter, Deerfield, IL) before cesarean section. The latter represent a group with high maternal AA concentrations without exposure to betamethasone. Complete data on these patients have been previously published.
Methods
All blood samples were collected into heparinized syringes that were immediately sealed and stored in ice. Plasma for AA analyses was separated by centrifugation at -4°C and frozen at –70°C until the time of analysis.
Maternal glucose concentration and umbilical arterial pH at delivery were measured in duplicate on a Radiometer ABL 330 analyzer (Copenhagen, Denmark).
Plasma was quickly thawed and deproteinized with a solution of 10% sulfosalycilic acid with nor-leucine added as an internal standard and buffered with LiOH to pH 2.2. After centrifugation at 14,000 rpm for 10 minutes, the supernatant fraction was filtered through a millipore filter and loaded into a Dionex high-performance liquid chromatography (Dionix Corp, Sunnyvale, CA) with refrigerated autosampler.
The samples were analyzed by cation exchange column with 3 buffers changed by gradient isothermally. Ninhydrin was used as color reagent and a dual wavelength spectrophotometer with 440 nm and 570 nm wavelengths were used for concentration determinations. Column, buffers, and ninhydrin reagent were purchased from Pickering laboratory. All the instrument operation and data processing were controlled by Dionix’s AI-450 software (Dionix Corp). Samples from each study were analyzed on a single column in the same run, with a variance of ± 2%.
Statistical analysis
Data are presented as mean ± SEM. AA concentration differences between the three maternal samples in the study during pregnancy were tested with analysis of variance for repeated measures and with the Scheffè multiple comparison procedure. The Student t test for paired samples was used to test the difference between the umbilical venous and arterial fetal samples in the group receiving antenatal betamethasone and the historical control group. The relationships between fetal and maternal AA concentrations were analyzed by linear regression analysis determined by the least-squared method using Statistica for Windows (StatSoft, Tulsa, OK) software. Differences between slopes were tested with the t test. P < .05 were considered significant.
Results
Studies of maternal concentrations in at-risk pregnancies
Table 1 presents the mean concentration of individual AA in M0, M1, and M2. The concentration of all AA, except arginine, lysine, histidine, glutamic and aspartic acid, asparagine, and taurine was significantly increased after betamethasone administration. Alanine was the nonessential AA whose concentration increased the most throughout the study (36.6% from M0 to M1; 44.3% from M0 to M2).
| Amino acid | M0 | M1 | M2 | P |
|---|---|---|---|---|
| Arginine | 65.5 ± 5.3 | 77.1 ± 5.2 | 75.9 ± 4.1 | |
| Phenylalanine | 52.2 ± 2.1 | 59.3 ± 2.2 | 58.5 ± 2.1 | .04 |
| Lysine | 150.6 ± 9.5 | 163.9 ± 10 | 178.9 ± 9.3 | |
| Histidine | 88.6 ± 3.1 | 93.4 ± 4.4 | 90.8 ± 3.1 | |
| Methionine | 21.3 ± 1 | 29.3 ± 1.3 a | 30.3 ± 1.8 a | .001 |
| Threonine | 187.2 ± 9.2 | 234.4 ± 14.3 b | 266.6 ± 15.9 a | .001 |
| Valine | 154.5 ± 4.9 | 179.7 ± 6.6 c | 175 ± 5.5 d | .01 |
| Leucine | 77.2 ± 2.8 | 89.9 ± 3.7 e | 88.5 ± 3.6 | .02 |
| Isoleucine | 46.5 ± 1.7 | 59 ± 2.1 a | 60 ± 1.9 a | .001 |
| Serine | 95.3 ± 3.5 | 115.6 ± 4.3 c | 111.3 ± 4.3 f | .002 |
| Glycine | 152.7 ± 9 | 190.9 ± 9.6 f | 186.8 ± 9.7 d | .01 |
| Alanine | 312.7 ± 15.7 | 493.4 ± 27.2 a | 561.2 ± 19.3 a | .001 |
| Glutamine | 429 ± 18.6 | 504.6 ± 23.9 | 517.1 ± 25.7 e | .02 |
| Glutamate | 59.1 ± 5 | 61.3 ± 7 | 58.5 ± 6 | |
| Asparagine | 49.8 ± 3.6 | 63.8 ± 6.2 | 63.6 ± 5 | |
| Aspartate | 8.2 ± 1 | 9.1 ± 1.2 | 8.5 ± 1 | |
| Proline | 157.8 ± 7.6 | 194.2 ± 10.4 f | 216.1 ± 9 a | .001 |
| Taurine | 26.1 ± 1 | 32.3 ± 2.5 c | 27.7 ± 2.1 | |
| Tyrosine | 39.3 ± 1.9 | 49.3 ± 3.2 f | 47.4 ± 2.1 | .01 |
| N essential, mg/L | 19.1 ± 0.7 | 21.8 ± 0.9 b | 22.5 ± 0.8 c | .01 |
| N nonessential, mg/L | 25.2 ± 0.9 | 31.8 ± 1.3 a | 33.2 ± 1.1 a | .001 |
| Total n, mg/L | 44.3 ± 1.3 | 53.7 ± 1.9 a | 55.7 ± 1.7 a | .001 |
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