Identification of metabolites diagnostic for organic acidurias by simultaneous dual-column capillary gas chromatography.

To offer a procedure with increased resolution compared to packed-column gas chromatography, we developed a dual-capillary method of gas chromatography for diagnosing organic acidurias. We derivatized and injected organic acids repeatedly on two different bonded phase columns (DB-1, DB-1701) to establish a table of methylene units. Compounds in urine specimens were identified by their characteristic pair of methylene units. With this method, we are able to identify 120 metabolites in urine. Accordingly, the procedure provides a cost-effective alternative to routine gas chromatography-mass spectrometry.

Effect of prenatal dexamethasone on immunoreactive 6-ketoprostaglandin F1 alpha levels in fetal rat lungs.

The effect of prenatal glucocorticoid treatment on levels of immunoreactive 6-ketoprostaglandin F1 alpha (PGF1 alpha) (the stable metabolite of prostacyclin) was studied in fetal rat lungs. During late gestation (20-22 days), levels of 6-keto-PGF1 alpha peaked at 21 days in offspring of control mothers. At a maternal dose of 0.2 mg/kg dexamethasone, maximal enhancement of fetal 6-keto-PGF1 alpha levels occurred at 20 days gestation. At a treatment dose of 0.4 mg/kg, however, dexamethasone increased fetal lung 6-keto-PGF1 alpha concentrations throughout late gestation. Because maturation of fetal lung is known to be delayed in males relative to females, we also studied the impact of sex of the fetus on levels of 6-keto-PGF1 alpha. Our results showed no statistically significant differences between females and males in any of the treatment groups at any of the gestational ages studied. These results suggest that prenatal dexamethasone enhances endogenous levels of 6-keto-PGF1 alpha in fetal rat lungs. Since prostacyclin may play important roles in fetal lung maturation and neonatal lung function, the effectiveness of prenatal glucocorticoid therapy for accelerating functional maturity of the fetal lung may in part be due to stimulation of prostacyclin synthesis.

Absorbance of amniotic fluid at 650 nm as a fetal lung maturity test: a comparison with the lecithin/sphingomyelin ratio and tests for disaturated phosphatidylcholine and phosphatidylglycerol.

We compared absorbance of amniotic fluid at 650 nm (A650) against the lecithin/sphingomyelin ratio, the quantity of disaturated phosphatidylcholine, and the presence or absence of phosphatidylglycerol in 374 uncontaminated amniotic fluid samples. In addition, we studied these measures of fetal lung maturity in 80 pregnancies where neonatal outcomes were known. Statistical analysis revealed a significant association between increasing absorbance and biochemical indices of lung maturation. Our results indicate that A650 of amniotic fluid is useful as a measure of lung maturity but that the greatest utility may be for samples with either low absorbance (less than 0.10) or high absorbance (greater than 0.20). Because of large false positive and false negative values between these two absorbances, other lung maturity estimates should be used when absorbances fall within this range. Therefore, A650 may be most useful as a screen or as part of a battery of lung maturity estimates. We are exploring these possibilities as a means of providing better fetal lung maturity estimates.

The effect of prenatal dexamethasone on fetal rat lung prostaglandin synthesis.

[14C]Arachidonic acid conversions were studied in homogenates of lungs from 20, 21, and 22 day fetuses with or without prenatal dexamethasone treatment. The major metabolites were in all cases 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), prostaglandin E2 (PGE2), and 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT). Prostaglandin F2 alpha (PGF2 alpha), prostaglandin D2 (PGD2), and thromboxane B2 (TxB2) were present in small amounts. Dexamethasone treatment significantly stimulated the conversion of [14C]-arachidonic acid in fetal lung homogenates to 6-keto-PGF1 alpha, PGE2, 12-HETE, and HHT, at 20 days gestational age. This effect was dependent on the dose of dexamethasone. These results suggest that dexamethasone accelerates the maturation of the enzymes involved in prostaglandin synthesis. Because dexamethasone is also known to inhibit phospholipase A2, further studies are required to determine the overall in vivo effect of prenatal dexamethasone therapy on fetal lung prostaglandin synthesis.

Delayed pulmonary phosphatidylglycerol synthesis and reversal by prenatal dexamethasone in fetal rats of streptozotocin-diabetic mothers.

Lung slices from fetal rats of streptozotocin-diabetic mothers incorporated [3H]glycerol and [3H]choline into phosphatidylglycerol and disaturated phosphatidylcholine, respectively. When compared to age-matched fetuses from nondiabetic mothers, lung phosphatidylglycerol synthesis of 21-day fetuses of diabetic mothers was significantly diminished, although [3H]glycerol incorporation into other phospholipids was not impaired. Synthesis of disaturated phosphatidylcholine was not diminished in lungs of 20-, 21-, or 22-day fetuses of diabetic mothers. Prenatal dexamethasone partially reversed the diminished phosphatidylglycerol synthesis at 21 days of gestation; the degree of stimulation was the same as that seen in 21-day fetuses of normal mothers but the maximal rate of [3H]glycerol incorporation was about 60% of that in 21-day fetuses of normal mothers. Fetal lung disaturated phosphatidylcholine synthesis was not stimulated by dexamethasone in diabetic pregnancies, in contrast to that seen in nondiabetic pregnancies. These data suggest that maternal diabetes interferes with the ability of fetal lungs to synthesize phosphatidylglycerol, a finding consistent with the delayed appearance of phosphatidylglycerol in the amniotic fluid of human diabetic pregnancies. In addition, maternal diabetes impairs the responsiveness of disaturated phosphatidylcholine synthesis to dexamethasone. Since phosphatidylglycerol synthesis is enhanced by prenatal dexamethasone, this therapy may still be effective for reducing the adverse impact of maternal diabetes on fetal lung development.

Disturbance of pulmonary prostaglandin metabolism in fetuses of alloxan-diabetic rabbits.

[14C]Arachidonic acid conversion in lung homogenates of 28-day fetuses from control and alloxan-diabetic rabbits was studied. The major metabolites were 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid and prostaglandin E2. Small amounts of 6-ketoprostaglandin F1 alpha, prostaglandin F2 alpha, and thromboxane B2 were also observed. Lung homogenates from fetuses of alloxan-diabetic rabbits convert significantly less [14C]arachidonic acid to prostaglandin E2, whereas all other metabolites were present in similar quantities compared to fetuses of non-diabetic rabbits. These studies suggest that the decreased arachidonic acid conversion to prostaglandin E2 could be partially responsible for the functional delay of lung maturation in offspring of alloxan-diabetic rabbits.

Fetal rat lung phosphatidylcholine synthesis in diabetic and normal pregnancies: a comparison of prenatal dexamethasone treatments.

The effects of maternal diabetes upon fetal lung surfactant phospholipid metabolism were studied using 19-day gestational age fetal rats from mothers with streptozotocin-induced diabetes mellitus. In this experimental animal model, maternal glucose intolerance significantly impaired fetal body and lung development. However, incorporation of [14C]palmitate and [3H]choline into lung total and disaturated phosphatidylcholine was unimpaired in offspring of diabetic mothers. Dexamethasone, which is known to promote fetal lung maturation in normal pregnancies, was administered to diabetic and control mothers during late gestation. Prenatal dexamethasone inhibited lung growth in both diabetic and control pregnancies. While this agent slightly stimulated [14C]palmitate incorporation into total phosphatidylcholine and markedly enhanced [3H]choline incorporation into both disaturated and total phosphatidylcholine in control pregnancies, it failed to stimulate incorporation of either precursor into fetal lung from diabetic pregnancies.

Improved thin-layer chromatography of disaturated phosphatidylcholine in amniotic fluid.

We describe an indirect test of fetal lung maturity: the quantitation of disaturated phosphatidylcholine in amniotic fluid. The lipids in samples of amniotic fluid from 172 patients were reacted with osmium tetroxide, and disaturated phosphatidylcholine was then isolated by thin-layer chromatography. Interfering substances were retained by a pre-adsorbent layer. The charred disaturated phosphatidylcholine, quantitated by densitometry, was compared to standard dipalmitoyl phosphatidylcholine. Both within-run and between-run coefficients of variation were about 10%. Blood and meconium do not interfere. Six infants developed respiratory distress when disaturated phosphatidylcholine concentrations of amniotic fluid drawn within 72 h of delivery were less than 5.5 mg/L. A concurrently determined lecithin/sphingomyelin ratio falsely predicted lung maturity in one of these. In seven other samples for which lecithin/sphingomyelin ratios suggested lung immaturity but disaturated phosphatidyl-choline predicted maturity, none of the infants developed respiratory distress. In normal pregnancies, measurement of disaturated phosphatidylcholine in amniotic fluid appears to be a better predictor of fetal lung maturity than is measurement of the lecithin/sphingomyelin ratio. Further studies are needed to determine if this analysis is a better predictor in diabetic pregnancies.

Free amino acid analysis of untimed and 24-h urine samples compared.

We measured 11 amino acids in untimed urine samples, to determine whether such samples are suited for use in diagnosis of aminoacidurias. Results for untimed samples varied by as much as 25% more than for 24-h collections when amino acid excretions were expressed in terms of urinary creatinine. Values decreased with increasing age for either type of specimen. Urinary amino acid excretions were also determined with untimed or 24-h samples from patients with cystinuria. Lowe's syndrome, nonketotic hyperglycinemia, or phenylketonuria. In all cases studied, the amino acids diagnostic of the diseases significantly exceeded the reference interval obtained for 260 control subjects in six age categories. We conclude that untimed urine samples can be used for diagnosis of these inborn errors of amino acid metabolism, but further studies are needed to evaluate their usefulness for other metabolic disorders.