Abstract: The aim of this study is to evaluate Protein Z (PTZ) and protein C (PTC) levels in newborns suffering from RDS, healthy preterm and full term newborns and to compare PTZ serum levels in RDS preterm infants with healthy preterm before and after recovery. Sixty newborn infants, recruited from the neonatal unit, were enrolled in the study and divided into 3 groups: Group (I): 20 preterm with RDS, Group (II): 20 healthy preterm control newborns (CPT) and Group (III): 20 healthy full term control newborns (CFT). Protein Z and C were measured using ELISA kits. The results of the study showed lower levels of protein Z were obtained in RDS group compared to preterm controls whose levels were significantly lower than in full-term controls. A significant increase in PTZ levels in RDS' group after recovery, when compared to preterm controls. In RDS, no significant correlations existed between PTZ levels (before and after recovery) and routine investigations except for a significant negative correlation with platelets count. No significant differences were found in PTC levels between the 3 studied groups. To conclude: premature newborns suffering from RDS showed decreased serum protein Z levels than normal preterm control newborns with further increase in its pattern after recovery. Further studies are recommended to evaluate the role of PTZ on outcome in premature newborns with RDS and to evaluate the relationship between protein PTZ and PTC and other coagulation factors incriminated in the development of RDS.
INTRODUCTION
Hyaline membrane disease or respiratory distress syndrome is one of the most common lung disorders in premature newborn. It results from a defect in the pulmonary surfactant metabolism. Abnormalities in the coagulation and fibrinolytic systems may play a role in the pathogenesis of RDS and contributes to the progression of the disease (Brus et al., 1997; Jaarsma et al., 2001; Bastarache et al., 2006). This is evidenced by fibrin deposition in pulmonary microcirculation and small airways (Mautone et al., 1997; Idell, 2001). Histo-pathologic studies proved that fibrin constitutes a major part of the hyaline membranes, which could be regarded as a local origin for clots in RDS (Chan et al., 1998). Fibrin deposition has been demonstrated in the pulmonary microcirculation and small airways in RDS suggesting the activation of the clotting system with a special role of protein C (PTC) and protein S. Protein Z (PTZ) -a vitamin k dependent protein-, was added to these factors. PTZ is a vitamin K-dependent plasma protein whose function remained unclear until many researchers found that its deficiency occurs in newborns affected by severe RDS due to activation of the coagulation process (Schettini et al., 2004). The structure of PTZ is very similar to that of the coagulation-related factors VII, IX and X and PTC (Yin et al., 2000). It forms a calcium-dependent complex with activated coagulation factor X leading to its inhibition and the suppression of thrombus formation (Broze, 2001). Deficiency of human PTZ has been incorporated with a pro-thrombotic tendency (Yin et al., 2000). PTC which a vitamin K protein-dependant is an important natural anticoagulant protein (Esmon, 2003) that also plays an anti-inflammatory role in pathogenesis of RDS (Miller et al., 2000; Liu et al., 2008). Present objectives are: (1) to evaluate PTZ and PTC levels in newborns suffering from RDS, healthy preterm and full term newborns and (2) compare PTZ serum levels in RDS preterm with healthy preterm before and after recovery.
MATERIALS AND METHODS
This is a prospective study that has been conducted on 60 newborn infants from the obstetrics and gynecology hospital and neonatal intensive care unit Ain-Shams University Hospital in the period between 2/2007 and 7/2007. Special assessment of the score of Ballard and ultra ultrasound findings were done to determine the gestational age. Neonatal respiratory distress syndrome was based on clinical and radiological criteria (Kero and Makinen, 1979). They were divided into three groups: Group I: comprised 20 newly born developing respiratory distress syndrome within the first 6 h of life and called RDS patients group, Group II: included 20 healthy preterm newborns as preterm control group and Group III: was formed of 20 healthy full term newborns as the full term control group. Blood sampling: 1 umbilical cord sample was collected initially after delivery and before vitamin K administration for all cases. For those newborns that developed RDS, another sample was collected after their recovery to assess protein Z and C levels. Both samples were collected as follows: 9 mL sec in tubes containing 0.11 M sodium citrate (1 mL) then centrifuged within 20 min at 2500 rpm for 10 min. platelet poor plasma was fractioned and frozen at (-70°C) until estimation of PTZ and PTC. The samples were thawed for 15 min at 37°C just before use. For each newborn enrolled in the study, the following was completed:
| • | General examination: (1) Full history taking including parity, gestational age, antenatal history and mode of delivery, (2) thorough clinical examination, (3) expanded score of Ballard, (4) apgar scoring at 1 and 5 min |
| • | Routine laboratory investigations including (1) Blood gases, using blood gas analyzer BEARS supplied by CIBA corning Diagnostics Corp. (medfield, MA, USA), (2) complete blood picture (Coulter counter T660), (3) liver functions, (4) kidney functions test |
| • | Radiological grading of severity of RDS was done (Kero and Makinen, 1979) |
Protein Z estimation: PTZ Concentrations were measured by a quantitative immuno enzymatic kit supplied by HYPHEN Bio Med 95000 Newville-sur-oise-France. Assay principle: the immunoconjugate, which is a polyclonal antibody specific for PTZ coupled to Horse Radish Peroxides (HRP), is introduced into the microwells coated with a polyclonal antibody specific for PTZ then, the diluted tested sample is immediately introduced and the immunological reaction starts, PTZ binds onto the polyclonal antibody coated solid phase through one epitopes and fixes the polyclonal antibody coupled to HRP through free epitopes following a washing step, the peroxides substrate, 3, 3', 5, 5'-tetramethylbenzidine (TMB), in presence of hydrogen peroxide (H2O2), is introduced and a blue colour develops. When the reaction is stopped with sulfuric acid, a yellow colour is obtained and the amount of color developed is directly proportional to the concentration of human PZ in the tested sample.
Protein C estimation: Protein C% was measured by ELISA test for quantitative determination of protein C antigen in citrated plasma. Assay Principle: The protein C antigen assay is a sandwich ELISA. A capture antibody specific for human protein C is coated to 96-microwell polystyrene plates. Diluted patient plasma is incubated in the wells, allowing any available protein C to bind to the anti-human protein antibody on the microwell surface. The plates are washed to remove unbound proteins or other plasma molecules. Bound protein C is quantified using Horse Radish Peroxidase (HRP) conjugated anti-human protein C detection antibody. Following incubation, unbound is removed by washing. A chromogenic substrate of Tetra Methyl Benzidine (TMB) and hydrogen peroxide (H2O2) are added to develop a colored reaction. The intensity of the color is measured in Optical Density (OD) units with a spectrophotometer at 450 nm. Protein C antigen relative percent concentrations in patient plasma is determined against a curve prepared from the reference plasma provided with the kit.
Statistical methods: Standard computer program SPSS for Windows, release 10.0 (SPSS Inc, USA) was used for data entry and analysis. All numeric variables were expressed as Mean±SD. Comparison of different variables in various groups was done using student’s t test and Mann Whitney test for normal and nonparametric variables respectively. Chi-square (χ2) test was used to compare frequency of qualitative variables among the different groups. Pearson’s and Spearman’s correlation tests were used for correlating normal non-parametric variables respectively. For all tests a probability (p) less than 0.05 was considered significant. Graphic presentation of the results was also done. The p values <0.05 were considered significant and p values <0.01 and <0.001 were considered highly significant.
RESULTS
The results of this study are displayed in Table 1-6. The RDS group were 17 with mean gestational age 32 weeks, mean birth weight 1350 g while the control preterm group were 20 infants with mean gestational age 32 weeks, mean birth weight 2100 g and the control fullterm group were 20 with mean gestational age 39.5 weeks, with mean birth weight 3300 g (Table 1).
| Table 1: | Demographic presentation of the population |
| Table 2: | Laboratory parameters of the studied RDS patients |
| Table 3: | Comparison between protein Z and protein C levels (before and after recovery RDS) with control preterm group and full term |
| S: Significant, NS: Not significant, HS: Highly significant | |
| Table 4: | Correlations between protein Z before and after recovery and gestational age, weight, APGAR 1, APGAR 5, hemoglobin %, total leucocytes counts, platelets counts, pH and PCO2 in patients with RDS, control preterm and control full term |
APG1: APGAR1, APG5: APGAR5, CFT: Control full term, CPT: Control
preterm, GA: gestational age, Hb%: Hemoglobin %, HS: Highly significant,
Negative results: negative correlation, NS: Non significant, P: p-value,
platelets counts PLAT: Positive results:positive correlation., S: Significant,
R: Correlation, TLC: Total leucocytic counts, Wt: Weight |
|
| Table 5: | Correlation between protein C before and after recovery and gestational age, weight, APGAR 1, APGAR 5, hemoglobin %, total leucocytes counts, platelets counts, pH and PCO2 in RDS group, CPT and CFT |
| APG1: APGAR1, APG5: APGAR5, CFT: Control full term, CPT: Control preterm, GA: Gestational age, Hb%: Hemoglobin, Wt: weight, PLAT: Platelets counts, TLC: Total leucocytic counts | |
Laboratory parameter for the RDS group showed mean platelet count 296,000 cm-3, Hb% 12.84 g dL-1 and total leucocytic count 15.42 cells cm-3 to exclude early sepsis with pH 7.24 Co2 51.47 denoting respiratory acidosis (Table 2). In Table 3, RDS group of patients showed a significant decrease in PTZ levels which increased significantly after recovery (p<0.001). Alternatively, protein C% didn’t differ in the same group of patients before and after recovery (p>0.05) PTZ level showed a significant increase in RDS group after recovery which was noticed when compared to preterm controls (p<0.001). A significant difference in PTZ levels existed between preterm and full term controls (p<0.05). With no significant difference in PTC% was noticed either between RDS and preterm control group (p>0.05), or between preterm control and full terms control groups (p>0.05). No correlations were found between PTZ in RDS group (before and after recovery) and gestational age, weight, APGAR 1, APGAR 5, hemoglobin, TLC, Platelets, pH, or PCO2, except for the presence of a significant negative correlation between PTZ before recovery and platelets count in RDS patients (Table 4).
| Table 6: | Correlation between protein Z and protein C before and after recovery in RDS patients group |
No significant correlations existed between PTC% and the laboratory parameters in all the studied groups included in the work (Table 5). There was a positive correlation between PTZ before and PTZ after and a negative correlation in PTC before and after recovery Table 6.
DISCUSSION
Neonatal RDSs are characterized by leakage of plasma proteins of varying sizes into the airspace, which leads to interstitial and intra-alveolar thrombin generation with subsequent fibrin deposition (Stevens et al., 2000; Idell, 2001); systemic activation of clotting, complement and polymorphonuclear lymphocytes (Ware et al., 2007). PTZ which is a vitamin k dependent protein proved to play a role in the prevention of coagulation (Kemkes-Matthes and Matthes, 2001). In the present study, RDS group showed a high significant increase in PTZ level after recovery (by one or two days) compared to its levels before recovery. This was in contrast to Schettini et al. (2004) who noted no increase in PTZ levels after vitamin K administration in RDS patients. Patients of RDS group showed significant lower levels of PTZ when compared to preterm control group, which increased significantly after recovery in comparison to preterm controls. This was in agreement with Yurdakok and Yigit (1999), Yurdakok et al. (2002) and Schettini et al. (2004). Schettini et al. (2004) related the difference in PTZ levels between RDS patients and preterm controls to the coagulation theory, where there is activation of the coagulation mechanism in RDS patients. PTZ acts as an essential cofactor for PTZ-dependent protease inhibitor (ZIP), which in turn is a potent down-regulator for coagulation factor X. Lower levels of PTZ in RDS patients will result in activation of coagulation with intra-alveolar fibrin deposition which would significantly impair the surfactant's function (Heeb et al., 2002). The current study showed no effect of either gestational age, weight, APGAR score, hemoglobin %, TLCs, pH or PCO2 on PTZ levels in all groups included in the study. This was in agreement with Corral et al. (2007), who noted no effect of either the gestational age or weight on PTZ levels. However, a significant negative correlation was found between PTZ before recovery and platelets counts in RDS group of patients. Yurdakok et al. (2002) stated that the hypercoagulable state in RDS patients is responsible for the increase in the platelets levels. They related the abnormalities in fibrinolytic system in these patients to lung damage and local platelet activation which lead to coagulation abnormalities other than DIC.
In the present study, PTC percent was estimated in patients with RDS as a vitamin K dependent and a natural anticoagulant protein. RDS group showed no significant difference in PTC level before and after recovery. This was in contrast to Schettini et al. (2004), who found an increase in PTC activity in newborns with RDS from day 1 to day 3. No significant difference existed between PTC in the RDS patients' group and preterm control, as well as between preterm and full terms control groups. These results were in accordance with Yurdakok et al. (2002) who reported no significant difference in PTC% in patients with RDS and the control newborns. They related this finding to the fact that the activation of clotting is not prominent in the early stages of RDS. This was in disagreement with Kobayashi et al. (1998) and Idell (2001), who found a decrease in Activated Protein C (APC) levels in the broncho alveolar lavage fluid in patients with moderate to severe RDS when compared to normal controls. They attributed this finding to activation of coagulation in their patient's group. In addition, Bartolome et al. (2008) stated that in response to hypoxia, an inflammatory cascade is initiated and microvascular injury ensues. Specifically, within 10 min leukocyte adherence to the endothelium begins and leukocyte emigration and vascular leak soon follow. The discrepancy in the results between the present work and the others may be due to the use of different samples for PTC assessment.
Similar to PTZ, no significant correlations were found between, PTC before or after recovery in RDS group and gestational age, weight, APGAR 1 and APGAR 5. These results were in agreement with Emmerich et al. (2005), who proved no effect of body weight, or age on PTC, as its deficiency is acquired in a number of clinical coagulation conditions such as RDS.
In the present study, in the RDS group, there was a significant negative correlation between PTC before and after recovery. On the other hand, a significant positive correlation existed between PTZ before and after recovery. PTZ acts by forming a complex with factor Xa at the phospholipids surfaces (Han et al., 2000), while PTC exerts its anticoagulant effect by degrading factor VIIIa and factor Va (Abraham, 2000) and also affects the fibrinolytic activity by forming a complex or degrade plasminogen activator inhibitor, thus diminished PTC activation leads to increased pro-coagulant activity and decreased fibrinolysis in RDS patients. Although, both factors deficiency influence coagulation pathways differently, they end with activation of coagulation pathways (Dahlbäck, 2005).
In view of this study, PTZ levels in RDS patients, showed a significant increase in its level after recovery reaching near normal levels. Negative correlation was present between PTZ and platelets count in RDS patients. Serial measures of plasma PTZ levels in premature may be of benefit in RDS to follow up their condition. Further studies are recommended to evaluate the role of PTZ on outcome in premature newborns with RDS and to evaluate the relationship between protein PTZ and PTC and other coagulation factors incriminated in the development of RDS.
ACKNOWLEDGMENT
Authors are great full to the Chief of Ain Shams University Hospital-NICU department and the parents of the babies.