Increased Levels of Granulocyte-Specific Glycosphingolipids in Preterm Labor Amniotic Fluid

Abstract

In a recent study, Hallman et al. reported high levels of lactosyl ceramide in amniotic fluid from preterm labor and postulated that this glycosphingolipid is of leukocytic origin. However, lactosyl ceramide is an ubiquitous glycosphingolipid also present in other cell types. In this study, the concentration of the granulocyte-specific glycosphingolipids, paragloboside and sialyl paragloboside in term and preterm amniotic fluids, was monitored by HPTLC-immunostaining using monoclonal antibodies E5C2 and 534F8, repectively. Amniotic fluid samples were obtained by transabdominal amniocentesis for clinical indications and divided in preterm not in labor (n = 18); preterm labor (n = 20); term not in labor (n = 20); and term labor (n = 16). The gestational age ranged from 25 to 41 weeks and preterm was defined as less than 37 weeks gestation. Diabetic patients were excluded from this study and none of the patients had clinical signs or symptoms of chorioamnionitis. Samples with macroscopic blood contamination were also excluded. The samples from each group were pooled, lyophilized, the residues extracted with chloroform-methanol-water (C-M-W, v/v/v). and the glycosphingolipid fraction isolated by a combination of peracetylation with pyridine-aceuc anhydride, Florisil column chromatography, and deacetylation with sodium mcthoxide. Aliquots of the purified glycosphingolipid fraction were streaked onto either Whatman HP-K or Merck aluminum-backed silica gel plates for high performance thin layer chromatographic analysis (HPTLC), following reaction with the orcinol-ferric chloride reagent; or HPTLC-immunostaining following reaction with anti-paragloboside and anti-sialyl paragloboside monoclonal antibodies E5C2 and 534F8, respectively. Preterm labor amniotic fluid contained significantly higher concentrations of the granulocyte-specific glycosphingolipids lactosyl ceramide, paragloboside and sialyl paragloboside as compared to term labor amniotic fluid. Preterm and term amniotic fluids with no labor had low to undetectable levels of these glycosphingolipids. These results confirm the findings reported by Hallman et al. and provide additional evidence that supports the hypothesis that the onset of preterm labor may be associated with subclinical infection.