Proteomic but Not Enzyme-Linked Immunosorbent Assay Technology Detects Amniotic Fluid Monomeric Calgranulins from Their Complexed Calprotectin Form

Abstract

Four proteomic biomarkers (human neutrophil peptide 1 [HNP1], HNP2 [defensins], calgranulin C [Cal-C], and Cal-A) characterize the fingerprint of intra-amniotic inflammation (IAI). We compared proteomic technology using surfaced-enhanced laser desorption-ionization-time of flight (SELDI-TOF) mass spectrometry to enzyme-linked immunosorbent assay (ELISA) for detection of these biomarkers. Amniocentesis was performed on 48 women enrolled in two groups: those with intact membranes (n ‫؍‬ 27; gestational age [GA], 26.0 ؎ 0.8 weeks) and those with preterm premature rupture of the membranes (PPROM; n ‫؍‬ 21; GA, 28.4 ؎ 0.9 weeks). Paired abdominal amniotic fluids (aAFs)-vaginal AFs (vAFs) were analyzed in PPROM women. Quantitative aspects of HNP1-3, Cal-C, Cal-A, and calprotectin (a complex of Cal-A with Cal-B) were assessed by ELISA. SELDI-TOF mass spectrometry tracings from 16/48 (33.3%) aAFs and 13/17 (88.2%) vAFs were consistent with IAI (three or four biomarkers present). IAI (by SELDI-TOF mass spectrometry) was associated with increased HNP1-3 and Cal-C measured by ELISA. However, immunoassays detected Cal-A in only 4 of the AFs even though its specific SELDI-TOF mass spectrometry peak was identified in 19/48 AFs. Calprotectin immunoreactivity was decreased in AFs retrieved from women with IAI (P ‫؍‬ 0.01). In conclusion, IAI is associated with increased HNP1-3 levels. In the absence of isoform-specific ELISAs, mass spectrometry remains the only way to discriminate the HNP biomarker isoforms. Monomeric Cal-A is not reliably estimated by specific ELISA as it binds to Cal-B to form the calprotectin complex. Cal-C was reliably measured by SELDI-TOF mass spectrometry or specific ELISA.Premature delivery (PTD; birth before 37 weeks in humans) is a significant public health problem. While complicating 7 to 12% of deliveries, prematurity is associated with 75% of infant mortality and 50% of long-term neurological handicaps, including blindness, deafness, developmental delay, cerebral palsy, and chronic lung disease (4,10,18,22). The etiologies of most preterm births remain unknown (2, 5).Clinically, PTD presents as either preterm labor with intact membranes or with preterm premature rupture of the membranes (PPROM). Several distinct pathophysiological pathways are implicated as triggers for PTD (21): excessive myometrial stretching, decidual hemorrhage, maternal and fetal stress, and inflammation (9). Inflammation may be initiated along various pathways of which infection is but one. Microbial invasion of the amniotic cavity, as evidenced by a positive amniotic fluid (AF) culture, occurs in 10% of the patients with preterm labor and intact membranes and in 38% of the patients with PPROM (25). We previously demonstrated that it is not necessarily the intrauterine infection that causes poor outcomes but rather the resulting intrauterine inflammation which can damage the fetus long before triggering labor (7). Thus, specific and sensitive tests for intrauterine inflammation rather than for bacterial infection may be more ...

Impaired lipid transport in gestational diabetes mellitus

“…Establishing an apolipoprotein pattern unique to gestational diabetes could allow for early detection and intervention well before the onset of clinical symptoms [17]. Mass spectrometry affords a more sensitive method than standard biochemical approaches (eg, ELISA assays) to evaluate more subtle changes in protein composition [18]. Our results are concordant with a recent study by Kim and colleagues reporting early changes in apolipoproteins in GDM cases using a comparable mass spectrometry approach [19].…”

Monomeric Calgranulins Measured by SELDI-TOF Mass Spectrometry and Calprotectin Measured by ELISA as Biomarkers in Arthritis

“…The recent identification via SELDI-TOF analysis of several truncated forms of S100A8 and S100A12 in cystic fibrosis patients suggests that C-terminal truncations affect protein function (34 ). The presence of S100A8 and S100A12, but not calprotectin, have been found to be characteristic of intra-amniotic inflammation (22 ), and SAA variants with different properties, such as differential susceptibility to matrix metalloproteinase digestion, have been described (38 ). These findings demonstrate the relevance of developing new proteomic approaches sufficiently powerful for investigating proteins in their modified or monomeric forms.…”

“…In the absence of ELISAs specific for the monomeric forms of S100A8 and S100A9 and of a commercially available test for S100A12, we have hypothesized that mass spectrometry (MS) might be a possible approach for detecting these proteins (22 ). S100A8 has already been identified by 2-dimensional gel electrophoresis to be present in synovium (13 ), but not in serum (12 ).…”

Proteomic Biomarkers of Intra-amniotic Inflammation: Relationship with Funisitis and Early-onset Sepsis in the Premature Neonate

“…Presence of calgranulin A was predictive of early neonatal sepsis. Because the monomeric calgranulin A is not reliably estimated by immunoassays (ELISA) as it binds to calgranulin B to form the calprotectin complex, mass spectrometry remains the only way to discriminate between biomarker isoforms at this time (35). The observation that calgranulin C is strongly associated with funisitis while calgranulin A is linked with neonatal sepsis (a more advanced stage of disease) suggests that there is a specific temporal sequence in the appearance of these biomarkers in women with intra-amniotic infection/inflammation.…”