Serum Prolidase and Ischemia-Modified Albumin Levels in Neural Tube Defects: A Comparative Study of Myelomeningocele, Meningocele, and Myeloschisis

Introduction

TYPES AND CLINICAL PRESENTATION OF NTDS:

NTDs are classified based on the severity and extent of neural tissue exposure. The 3 primary forms are:

Myelomeningocele: The most severe and common form, characterized by herniation of the spinal cord, meninges, and cerebrospinal fluid (CSF) through an open vertebral defect [5]. Clinically, it presents with motor and sensory deficits, bladder and bowel dysfunction, hydrocephalus, and Chiari II malformation [6] (Figure 1).

Meningocele: Involves a CSF-filled meningeal sac protruding through the vertebral defect, but unlike myelomeningocele, the spinal cord remains intact [7] (Figure 2). Neurological impairment is usually mild or absent, making the prognosis more favorable (Figure 2).

Myeloschisis: The most severe form, in which the spinal cord remains completely open without any meningeal covering, exposing neural tissue directly to the amniotic fluid [8] (Figure 3). This results in severe neurological impairment, paralysis, and high perinatal mortality (Figure 3).

DIAGNOSIS OF NTDS:

The prenatal diagnosis of NTDs relies on biochemical screening and imaging techniques:

Maternal Serum Alpha-Fetoprotein (AFP) Screening: Elevated AFP levels at 16–18 weeks of gestation indicate an increased risk of open NTDs [9].

Ultrasound Imaging: High-resolution fetal ultrasound detects cranial and spinal abnormalities, confirming spina bifida, anencephaly, or encephalocele [10].

Fetal MRI: Provides detailed visualization of soft tissue involvement, Chiari II malformation, and brainstem abnormalities, aiding in surgical planning [11].

OF NTDS:

The management approach depends on the severity of the defect and whether the diagnosis is prenatal or postnatal:

Fetal Surgery: In utero repair of myelomeningocele improves neurological outcomes, reduces hindbrain herniation, and decreases the need for ventriculoperitoneal (VP) shunting [12]. However, fetal surgery carries risks, including preterm birth and uterine rupture.

Postnatal Surgical Repair: Newborns with open spinal defects undergo neurosurgical closure within 48 hours to prevent infection and protect exposed neural tissue [13].

Hydrocephalus Management: Up to 80% of myelomeningocele cases develop hydrocephalus, often requiring VP shunt placement [14].

Multidisciplinary Long-Term Care: Patients require lifelong follow-up by neurosurgeons, orthopedic specialists, urologists, and rehabilitation experts to manage paralysis, orthopedic deformities, and bladder dysfunction [15].

While genetic, nutritional, and environmental factors are known to influence NTDs, their biochemical mechanisms remain unclear. Prolidase is a key enzyme in collagen turnover and extracellular matrix remodeling, which are crucial for neural tube closure [16]. Previous studies suggest that abnormal prolidase activity may contribute to impaired tissue integrity and developmental defects [17,18].

Similarly, ischemia-modified albumin (IMA) is an established biomarker of oxidative stress and ischemic damage. Elevated IMA levels have been reported in conditions involving chronic hypoxia, including pregnancy complications and neonatal hypoxic-ischemic encephalopathy (HIE) [19]. However, the role of IMA in NTDs remains largely unexplored.

Despite advancements in prenatal screening and surgical interventions, the underlying pathogenesis of NTDs remains poorly understood. Biochemical markers such as prolidase and ischemia-modified albumin (IMA) have not been extensively studied in relation to neural tube closure defects. Given that prolidase plays a crucial role in collagen turnover and extracellular matrix integrity, and IMA reflects oxidative stress-related ischemia, their evaluation in NTD patients could provide valuable insights into NTD pathophysiology.

This study aims to compare serum prolidase and IMA levels in infants with NTDs and a control group, thereby exploring their potential role in NTD pathogenesis. A deeper understanding of these biomarkers may contribute to the identification of novel therapeutic targets and improved prenatal risk assessment.

Material and Methods

ETHICAL CONSIDERATIONS:

The study was reviewed and approved by the Clinical Research Ethics Committee of Yüzüncü Yıl University Medical Faculty (Approval Date: 02.11.2022; Approval Number: 03). The project was initially titled ‘Investigation of Prolidase Levels and Ischemic Modified Albumin Levels in Meningomyelocele Defect.’ However, the study protocol submitted to the Ethics Committee included patients with all subtypes of neural tube defects (myelomeningocele, meningocele, and myeloschisis), ensuring that ethical approval was granted for the broader scope.

Before participation, detailed information about the study objectives, potential risks, and benefits was provided to all parents/guardians of the participants. The consent process was conducted in accordance with ethical guidelines, ensuring that parents/guardians understood the study before signing the informed consent form. Written informed consent was obtained from all parents or legal guardians of the participating children. The study strictly adhered to the ethical principles outlined in the Declaration of Helsinki.

The study included 45 patients diagnosed with NTD who were being followed up in the neurosurgery unit of the hospital, as well as 45 children known not to have NTD who were being followed up for other reasons in the same unit. The age and sex distributions of the participants included in the study were matched to ensure similarity.

DIAGNOSIS AND CLASSIFICATION OF NEURAL TUBE DEFECTS:

Diagnosis of neural tube defects (NTDs) was established based on a combination of clinical examination, imaging studies, and intraoperative findings, in accordance with current clinical guidelines (Brea & Munakomi, 2023) [12]. The following criteria were used to classify NTD types:

Myelomeningocele: Diagnosed based on a visible neural placode with overlying meningeal protrusion, confirmed by prenatal ultrasound, postnatal MRI, or CT scan. Clinical examination typically revealed a spinal defect with associated neurological deficits.

Meningocele: Identified by the presence of a cerebrospinal fluid-filled sac without neural tissue involvement, confirmed using MRI or CT imaging. Clinical assessment showed a cystic protrusion without significant neurological impairment.

Myeloschisis: Diagnosed in cases where there was a completely open spinal cord without meningeal covering, confirmed intraoperatively and with imaging. These cases frequently exhibited severe neurological deficits and were identified through direct visualization and MRI findings.

SERUM PROLIDASE LEVEL MEASUREMENT: Serum prolidase levels were determined using a colorimetric method based on the technique proposed by Myara et al (1982) [13]. Serum samples were incubated with a Tris-HCl buffer Tris-HCl buffer (50 mmol/L, pH 7.8) containing 1 mmol/L glutathione (GSH; Sigma-Aldrich, USA, Cat. No: G4251) and 50 mmol/L MnCl2 (Merck, Germany, Cat. No: 105926) at 37°C for 30 minutes. The reaction was terminated with glacial acetic acid (Sigma-Aldrich, USA, Cat. No: 537020), and ninhydrin solution (Sigma-Aldrich, USA, Cat. No: N7285) was added before heating at 90°C for 20 minutes. The absorbance of the samples was read at 515 nm using a Shimadzu UV-1800 spectrophotometer (Kyoto, Japan). All samples were centrifuged at 3000 RPM for 10 minutes at 4°C before analysis using a Hettich Universal 320R centrifuge (Hettich, Germany).

The assay was performed in duplicate for each sample, and quality controls with known prolidase levels were included in every batch. The intra-assay and inter-assay coefficients of variation (CV) were <5%, ensuring the reliability of the measurements. The detection limit of the assay was X μmol/L [13]. Reference values for prolidase levels in healthy individuals were taken from Vural et al (2010) [16].

Serum Ischemia-Modified Albumin (IMA) Level Measurement IMA levels were determined using a spectrophotometric cobalt-binding assay based on the method by Bar-Or et al (2000). Serum samples were mixed with 0.1% cobalt chloride (Sigma-Aldrich, USA, Cat. No: C8661, 50 μl) and incubated at room temperature for 10 minutes. Dithiothreitol 1.5 g/L (DTT; Sigma-Aldrich, USA, Cat. No: D9779, 50 μl) was then added, followed by an additional 2-minute incubation. The reaction was stopped using 9 g/L NaCl solution (Sigma-Aldrich, USA, Cat. No: S7653, 1 ml). Sample blanks were prepared without DTT. Absorbance was measured at 470 nm using the Shimadzu UV-1800 spectrophotometer. Reference values for IMA levels in healthy individuals were based on Shevtsova et al (2021) [7].

Each sample was measured in duplicate, and quality controls with known IMA levels were included. The intra-assay and inter-assay coefficients of variation (CV) were <5%. The detection limit for IMA was X ABSU.

QUALITY CONTROL AND VALIDATION: To ensure assay accuracy and reliability, several controls and validation steps were implemented. Positive controls consisted of serum samples spiked with known concentrations of prolidase and ischemia-modified albumin (IMA), while negative controls were prepared by processing serum samples without the addition of substrate or enzyme. A standard curve was generated using serial dilutions of known prolidase and IMA concentrations to enable precise calibration. To assess repeatability and precision, each sample was analyzed in duplicate, and the intra-assay and inter-assay coefficients of variation (CV) were maintained at <5%. Assay linearity and sensitivity were evaluated, with the limit of detection (LOD) determined as X μmol/L for prolidase and X ABSU for IMA. Reference values for prolidase levels and IMA reference values were based on Shevtsova et al (2021) [7]. These measures ensured the robustness and reproducibility of the biochemical analyses.

STATISTICAL ANALYSIS:

All collected data were analyzed using SPSS 20.0 (IBM, USA). To ensure objectivity and minimize bias, all laboratory measurements were performed in duplicate, and the mean value of the 2 readings was used for statistical analysis. Sample processing and measurements were conducted by the same technician, and all sample handling was randomized to prevent systematic errors. Researchers analyzing the data were blinded to case/control status during the evaluation. Data distribution was assessed using the Kolmogorov-Smirnov test, and group comparisons were performed using Student’s t test for normally distributed data and the Mann-Whitney U test for non-normally distributed data. For multiple-group comparisons, the Kruskal-Wallis test was applied. Outliers were identified using boxplot analysis and were excluded if they exceeded ±3 standard deviations from the mean to ensure statistical accuracy. A p value of <0.05 was considered statistically significant.

Results

DEMOGRAPHIC AND CLINICAL CHARACTERISTICS:

Of the patients being followed, 20 (44.4%) were male and 25 (55.6%) were female. The average age of these patients was 21.71±33.53 months, with a median age of 1 month (age range: 1–164 months). When considering age groups, it was found that 25 of the patients (55.6%) were newborns. Among the patients, 34 (75.6%) had meningomyelocele, 5 (11.1%) had meningocele, and 6 (13.3%) had myeloschisis. Considering the lesion level, it was observed that 21 patients (46.7%) had lumbar involvement, and 12 patients (26.7%) had lumbosacral involvement. In terms of clinical findings, 33 patients (73.3%) were plegic, 4 patients (8.6%) had plegia along with pes equinovarus, and 8 patients (17.8%) were paraplegic. Hydrocephalus surgery was performed in 23 patients (51.1%), while 22 patients (48.9%) did not require surgery (Table 1).

IMAGING FINDINGS:

To further clarify the clinical presentation of NTD subtypes, we analyzed imaging studies from the patient cohort. In myelomeningocele cases, MRI revealed a protruding neural placode with an overlying meningeal sac, often associated with ventriculomegaly and Chiari II malformation. Meningocele cases exhibited well-defined cystic formations without direct neural tissue involvement, confirmed using MRI and ultrasound. Myeloschisis cases showed a completely open spinal cord with absent meningeal covering, leading to exposed neural tissue, as observed in postnatal clinical examination and confirmed by MRI. These findings are consistent with established descriptions of NTD [12].

SERUM PROLIDASE AND IMA LEVELS IN RELATION TO DEMOGRAPHICS:

The Mann-Whitney U test revealed no significant association between serum prolidase levels and serum IMA levels with respect to sex (for serum prolidase level: U=200.0, p=0.251; and for serum IMA: U=226.0, p=0.581). Additionally, the Spearman correlation test results indicated no significant correlation between patient age and serum prolidase levels or serum IMA levels (for serum prolidase level: correlation coefficient: −0.089, p=0.560; and for serum IMA: correlation coefficient: 0.218, p=0.150).

COMPARISON OF SERUM PROLIDASE AND IMA LEVELS AMONG NTD SUBTYPES:

In patients with meningomyelocele, the average serum prolidase level was 2.20±0.06 IU/L, 2.20±0.07 IU/L in those with meningocele, and 2.22±0.08 IU/L in those with myeloschisis. Statistical analysis revealed no significant difference in serum prolidase levels among the NTD subtypes (p=0.743). Additionally, the average serum IMA level was 0.40±0.01 IU/L in patients with meningomyelocele, 0.41±0.01 IU/L in those with meningocele, and 0.40±0.02 IU/L in those with myeloschisis. Statistical analysis indicated no significant difference in serum IMA levels among the NTD subtypes (p=0.631) (Table 2).

COMPARISON BETWEEN NTD PATIENTS AND CONTROL GROUP:

In patients with NTDs, the mean serum prolidase level was measured at 2.21±0.06 IU/L, compared to 1.07±0.04 IU/L in the control group. Additionally, the average serum IMA level was 0.40±0.01 ABSUs in NTD patients, compared to 0.22±0.01 ABSUs in the control group. Statistical analysis showed that the serum IMA levels and prolidase level in the patient group were significantly elevated compared to those in the control group (for both measurements; p=0.001) (Table 3).

Discussion

COMPARISON WITH PREVIOUS STUDIES:

The prolidase enzyme is responsible for catalyzing the rate-limiting step in collagen recycling and is essential for protein metabolism, collagen turnover, and matrix remodeling. Consequently, it participates in various physiological processes, including wound healing, inflammation, angiogenesis, and cell proliferation, and is also significantly involved in carcinogenesis. Altered prolidase level, whether increased or decreased, has been associated with various diseases [14]. Elevated prolidase level has been shown to lead to the accumulation of the end product, proline, in brain tissue, which can result in cognitive deficits and mental retardation, as well as conditions such as schizophrenia, Parkinson’s disease, Alzheimer’s disease, and cerebral ischemia. Conversely, reduced prolidase level has been suggested to be correlated with mental retardation and developmental delays [15].

In the human embryo, the process of neural tube formation begins on the 19^th day, with the anterior neuropore closing on the 25^th day and the posterior neuropore closing on the 28^th day. Therefore, defects related to closure are expected to be associated with the genetic and metabolic processes active between days 19 and 28. However, although prolidase level and its defects have been studied in various tissues, there are few studies on the sources of prolidase in the fetus and its level during this critical period (days 19–28).

In a study conducted by Vural et al (2010), amniotic fluid from 36 fetuses with NTDs at 16–20 weeks of gestation was examined and compared with that of a control group. It was found that the prolidase level and oxidative stress index in the amniotic fluid of fetuses with NTDs were markedly higher compared to those in the control group [16].

In the present study, serum prolidase level in NTD patients was found to be approximately 2 times higher than in the control group. Additionally, no statistically significant difference in prolidase level was observed among patients with meningomyelocele, meningocele, or myeloschisis (p=0.743). When considered alongside the study by Vural et al, the results of the present study suggest that focusing on fetal rather than maternal factors in studies investigating the role of prolidase level in NTD etiology may be more beneficial, as the elevated prolidase level in the fetus can persist into postnatal life.

Ischemia-modified albumin (IMA) has been noted for its potential usefulness in the early evaluation of ischemia, particularly in cardiac diseases, where it can help prevent the progression to infarction and avoid possible complications. IMA has also been frequently studied as a biomarker in various psychiatric disorders, cancers, ischemic stroke, intrauterine growth restriction, and the evaluation of pregnancy and childbirth complications [7]. Although IMA levels are mainly studied in the context of acute ischemic events, a study by Erol et al (2022) demonstrated that elevated IMA levels could also be a good indicator of an underlying chronic ischemic condition [17].

Papageorghiou et al (2008) showed that abnormal intrauterine hypoxia during early pregnancy can lead to oxidative damage to trophoblasts during reperfusion, and it has been suggested that IMA could be used alongside the classic symptoms of preeclampsia (proteinuria, hypertension) after 20 weeks of gestation [19]. In another study, Maternal serum IMA levels showed a 1.5-fold increase in samples collected at the end of the second trimester and just after birth in pregnancies affected by fetal growth restriction due to placental insufficiency, compared to normal pregnancies [20]. Reddy et al (2018) conducted a systematic review and meta-analysis, concluding that IMA levels in maternal serum and fetal cord blood could serve as biomarkers for both normal pregnancy and preeclampsia [21].

There are very few studies investigating IMA levels and their clinical significance in neural tube defects. A study by Güzelmansur et al (2018) reported that serum IMA levels were significantly elevated in pregnant women carrying a fetus with NTDs compared to those in normal pregnancies [22]. Similarly, another study by Özyer et al (2019), which evaluated a total of 61 pregnant women in the second trimester, found similar results [23]. The findings of a more recent study published in 2023 also support these findings [24].

In this study, serum IMA levels in NTD patients were significantly higher than those in the control group. Moreover, there was no significant correlation between serum IMA levels and either sex or age within the patient group. Additionally, no significant differences in serum IMA levels were detected among the different NTD subtypes. A literature review showed that no prior studies have examined serum IMA levels in children with NTDs.

STUDY LIMITATIONS:

Despite the significant findings, this study has some limitations. Firstly, the sample size was relatively small, which may impact the generalizability of the results. A larger, multicenter study would provide more robust data. Secondly, this study only included postnatal serum samples, limiting insight into how prolidase and IMA levels fluctuate throughout fetal development and early postnatal life. Longitudinal studies that track these biomarkers from fetal stages to infancy would be beneficial.

Additionally, while the prolidase and IMA assays used were standardized, potential variations in laboratory conditions and measurement techniques could introduce minor inconsistencies. Further, the study did not assess the influence of confounding factors such as nutritional status, comorbid conditions, or genetic predispositions that might contribute to biomarker variations.

To the best of our knowledge, this study is the first to explore both serum prolidase level and serum IMA levels in NTD patients. We believe this research could set a foundation for future studies in this field. The findings indicate that chronic ischemia caused by oxidative stress can be present in patients with meningomyelocele, meningocele, and myeloschisis. More extensive studies are required to better understand the relationship between NTDs and chronic ischemia.

Conclusions

The pathogenesis of neural tube defects (NTDs), which are associated with high mortality and morbidity, remains incompletely understood. This study aimed to evaluate serum ischemia-modified albumin (IMA) levels, a reliable indicator of oxidative stress, and serum prolidase levels, which play a crucial role in collagen metabolism, in NTD patients compared to a control group. Our findings revealed that both biomarkers were significantly higher in NTD patients, suggesting a potential role for oxidative stress and altered collagen metabolism in NTD pathogenesis.

These results highlight the importance of further investigating the underlying mechanisms leading to persistently elevated serum prolidase and IMA levels in NTD patients. Understanding these mechanisms may provide new insights into the etiology of NTDs and contribute to the development of potential diagnostic or therapeutic strategies. Future research with larger, multicenter cohorts and longitudinal designs is necessary to validate these findings and explore their clinical implications.