Biomarkers in premature calves with and without respiratory distress syndrome

Abstract Background Approaches to the evaluation of pulmonary arterial hypertension (PAH) in premature calves by using lung‐specific epithelial and endothelial biomarkers are needed. Objective To investigate the evaluation of PAH in premature calves with and without respiratory distress syndrome (RDS) by using lung‐specific epithelial and endothelial biomarkers and determine the prognostic value of these markers in premature calves. Animals Fifty premature calves with RDS, 20 non‐RDS premature calves, and 10 healthy term calves. Methods Hypoxia, hypercapnia, and tachypnea were considered criteria for RDS. Arterial blood gases (PaO2, PaCO2, oxygen saturation [SO2], base excess [BE], and serum lactate concentration) were measured to assess hypoxia. Serum concentrations of lung‐specific growth differentiation factor‐15 (GDF‐15), asymmetric dimethylarginine (ADMA), endothelin‐1 (ET‐1), vascular endothelial growth factor (VEGF), and surfactant protein D (SP‐D) were measured to assess PAH. Results Arterial blood pH, PaO2, SO2, and BE of premature calves with RDS were significantly lower and PaCO2 and lactate concentrations higher compared to non‐RDS premature and healthy calves. The ADMA and SP‐D concentrations of premature calves with RDS were lower and serum ET‐1 concentrations higher than those of non‐RDS premature and healthy calves. No statistical differences for GDF‐15 and VEGF were found among groups. Conclusions and Clinical Importance Significant increases in serum ET‐1 concentrations and decreases in ADMA and SP‐D concentrations highlight the utility of these markers in the diagnosis of PAH in premature calves with RDS. Also, we found that ET‐1 was a reliable diagnostic and prognostic biomarker for PAH and predicting mortality in premature calves.


| INTRODUCTION
In cows and heifers, average perinatal mortality on dairy farms ranges from 2% to 20%, and in developed countries between 5% and 8%.
Premature birth remains an important and common cause of calf mortality in the world. [1][2][3][4] The most important problems in premature infants is respiratory distress syndrome (RDS) that leads to increased respiratory effort and inadequate oxygen exchange. [5][6][7][8] The RDS caused by surfactant insufficiency is associated with pulmonary hypertension (PHT) in lambs. 9 In premature newborns, the lung lobes cannot inflate effectively because of surfactant deficiency, and hypoxia develops because gas exchange does not occur adequately. As a result, alveolar and interstitial edema develop related to interstitial inflammation and endothelial and epithelial damage. Developing hypoxia and alveolar and interstitial edema cause narrowing of the pulmonary arteries and therefore development of pulmonary arterial hypertension (PAH). [10][11][12] Studies in premature infants have shown that the development of PAH substantially increases mortality. 13,14 Recently, in human medicine, a noninvasive method for determining PAH employs biomarkers specific to pulmonary epithelial and endothelial damage. 13,[15][16][17][18][19] Growth differential factor-15 (GDF-15), asymmetric dimethyla rginine (ADMA), endothelial-1 (ET-1), vascular endothelial growth factor (VEGF), and surfactant protein-D (SP-D) concentrations were found to change significantly in infants with PHT. 15,18,[20][21][22][23] The concentration of GDF-15 is used as a biomarker to evaluate the prognosis of PHT. 21,23 These biomarkers are used to determine the proliferation and apoptosis of endothelial cells in PHT cases. 23 Asymmetric dimethylarginine is a naturally occurring amino acid that prevents production of nitric oxide (NO), an end product of oxidative stress.
Increased concentrations of ADMA attenuate NO concentrations leading to an increase in vascular tone. Therefore, it has been suggested that ADMA can be considered a useful biomarker in PHT cases. 18 Endothelial-1 is a peptide abundantly present in the human lung and plays an important role in the development of PHT because of the presence of endothelin receptors on vascular smooth muscle cells. 13 Earlier studies indicated that the concentration of ET-1 in plasma and lung tissue is significantly increased in patients with PHT. 15,22 Vascular endothelial growth factor and its receptor concentrations were found to be increased with damage to pulmonary vessels in patients with PHT. 15 Furthermore, VEGF concentrations also increased significantly during hypoxic conditions. 24 Surfactant protein-D is secreted by type II pneumocytes and plays an important role in ensuring and maintaining the surface integrity of alveoli. In patients with acute RDS (ARDS), SP-D concentrations decreased with destruction of type II pneumocytes in the lungs depending on the severity of damage to the lung. 25 The changes in the SP-D concentration in ARDS patients could provide valuable information about the prognosis of the disease. 16,26 Therefore, we aimed to determine: (a) whether PAH had developed in premature calves with and without RDS by evaluating lung epithelial and endothelial damage biomarkers and (b) the importance of these biomarkers in predicting mortality in affected calves.

| Animals
The experimental groups consisted of 50

| Clinical evaluation
Upon admission of premature calves to the clinic, history was taken, and live weight, age, and breed were recorded. Routine clinical examinations of all calves were performed. According to clinical observations and blood gas analysis, premature calves that met the criteria for RDS were enrolled in the trial group, premature calves without RDS were enrolled in the positive control group, and healthy normal-term calves were enrolled in the negative control group. The criteria for prematurity were decreased gestation period (<255 days), low body weight (BW), a short silky hair coat, incomplete eruption of incisors, soft hooves, and weak or no suckling reflex. 7,8,27 2.3 | Criteria for definition of RDS Hypoxia (PaO 2 < 60 mm Hg), hypercapnia (PaCO 2 > 45 mm Hg), tachypnea (breaths per minute >45/min), and abdominal respiration with wheezing were the criteria for distinguishing between calves with or without RDS. 7,8,27,28 A PaO 2 <60 mm Hg and at least 2 other criteria described above were required for a case to be diagnosed as RDS.

| Collection of blood samples
Blood samples were collected from the calves for arterial blood gas analysis and lung-specific biomarker measurements at the time of admission. For uniformity among the groups, all blood samples were taken within the first 12 hours after birth. Blood samples for serum were taken from jugular vein and for blood gas measurement from auricular arteries. Nonanticoagulant tubes were used for serum and sodium heparin-containing plastic syringes were used for blood gas measurement. Blood samples taken for biochemical analyses were kept at room temperature for 15 minutes, then centrifuged at 2000g for 10 minutes. Sera were removed and stored at À80 C. Blood gas measurements were performed within 5 to 10 minutes of collection.  inter-assay coefficients of variation, and detectable ranges were ≤8%, ≤10%, and 7-1500 ng/L for GDF-15; ≤8%, ≤10%, and 0.05-10 nmol/ mL for ADMA; ≤8%, ≤10%, and 2-600 ng/L for ET-1; ≤8%, ≤10%, and 15-3000 ng/L for VEGF; and ≤8%, ≤10%, and 1-400 ng/L for SP-D, respectively.

| Treatment protocol
In premature calves without RDS, only the standard treatment protocol was applied whereas premature calves with RDS were treated using a standard treatment protocol along with oxygen application and nebulizer treatment.

| Oxygen application
Oxygen was administered to premature calves with RDS. Oxygen was passed through water to humidify it and given to the calves using a T A B L E 1 Arterial blood gas parameters of premature and healthy calves

| Blood gas analysis
Arterial blood gas parameters of premature and healthy calves are presented in

| Biomarker analysis
Serum biomarker concentrations in the premature and healthy calves are presented in Table 2. Serum ADMA and SP-D concentrations of the RDS group were found to be significantly lower than those of the control group (P < .05). Serum ET-1 concentration of the RDS group were significantly higher than those of the non-RDS and control groups (P < .05). No statistical differences (P > .05) were found among all groups for GDF-15 and VEGF concentrations ( Table 2). Correlations between arterial blood gas parameters and ADMA, ET-1, and SP-D concentrations in the premature and healthy calves are presented in Table 3. Positive correlations between blood pH and PaO 2 , SO 2 , and BE were found whereas a negative correlation was found among PaCO 2 , lactate, and ET-1 concentrations (P < .01). A positive correlation between blood PaCO 2 and lactate concentrations (P < .01) and ET-1 (P < .05) and negative correlations among PaO 2 , SO 2 , and BE (P < .01) were identified. Positive correlations between blood PaO 2 and SO 2 (P < .01), BE (P < .05), and SP-D (P < .05), and negative correlation between lactate and ET-1 concentrations (P < .01) were found. A negative correlation between lactate concentration and BE and a positive correlation with ET-1 concentration (P < .01) were found. A negative correlation was identified between blood BE and ET-1 concentrations (P < .01).
Correlation results between some arterial blood gas parameters and ET-1 concentrations in the premature and healthy calves are presented in Table 3 and Figure 1. A negative correlation between ADMA and ET-1 concentrations (P < .01; Table 3; Figure 1) and a positive correlation with SP-D concentrations (P < .01) were identified (Table 3; Figure 2). A negative correlation (P < .01) was found between ET-1 and SP-D concentrations ( Figure 2).
The results of the premature calves also showed that the concentrations of ET-1 from the nonsurvivor calves were significantly higher  The causes of high mortality in premature calves with RDS have been reported to be severe hypercapnia and hypoxemia. 7,8,27,[30][31][32]  Earlier studies on premature calves with RDS have found variable severity of changes in blood gases and acid-base balance. 7,8,31 In addition to hypercapnia and hypoxia in premature calves with RDS, mixed acidosis (respiratory-metabolic acidosis) is common. 3,6,8 In our study, a positive correlation between pH and PaO 2 , SO 2 , and BE and a negative correlation between PaCO 2 and lactate concentrations were observed.
Blood pH and BE are commonly accepted as valuable parameters for detecting mixed (respiratory-metabolic acidosis) acidosis in calves after birth. 33,34 The decrease in blood pH occurs as a result of insufficient removal of CO 2 from the lung and accumulation in the blood. 35 In addition to PCO 2 , a high L-lactate concentration also contributes to the development of acidosis. 7,8,36 Lactate is produced under hypoxic conditions and poor tissue perfusion, and is used as an indirect marker of tissue hypoxia. 5,7,8,37,38 A study in premature infants reported a strong correlation between lactate concentrations and BE and found that the effect of lactate on blood pH increased mortality rates. 39 Base deficit often is used as an indirect indicator of lactic acidosis. 40 Lactate plays an important role in the development of acidosis in newborns with asphyxia and is responsible for metabolic acidosis. It remains in the blood at high concentrations much longer than Endothelin-1 is a peptide abundantly found in the human lung and has been reported to play an important role in the development of PHT because of the presence of endothelin receptors (ET-A and ET-B) on vascular smooth muscle cells. 13 Endothelin-1 concentration was found to increase significantly in cases of pulmonary hypertension. 15,22 A previous study reported higher concentrations of ET-1 in cases of PHT compared to normal healthy individuals. 42 On the other hand, it was determined that the mortality rate was higher in patients with PHT having high concentrations of ET-1. 13 In our study, serum F I G U R E 3 Receiver operator characteristic curve analysis graph based on serum ET-1 concentration for surviving and dying premature calves. Performed ROC analysis to determine the relationship of ET-1 concentration with mortality in premature calves determined that the ET-1 concentration has prognostic importance to determining mortality. ET-1, endothelin-1; ROC, receiver operating characteristic curve ET-1 concentrations of premature calves with RDS were significantly increased compared to those of the non-RDS premature and control group calves. Although ET-1 can be expressed in numerous tissues and organs, concentrations of ET-1 mRNA were at least 5-fold higher in lung than in any other organ. 43 These findings suggest the development of PHT in premature calves with RDS as has been confirmed by previous studies in humans. 15,22,42 Also, serum ET-1 concentrations of nonsurvivor premature calves were found to be higher than those of surviving premature calves (Table 4). Receiver operating characteristic curve analysis to determine the relationship of ET-1 concentrations with mortality in premature calves determined that the ET-1 had prognostic importance in determining mortality with a cutoff of 34 ng/L ( decreased in response to RDS. 8 However, in infants with RDS, blood ET-1 concentrations increased, the mechanism for which has not been fully elucidated. However, it was found in rats that increases in plasma and pulmonary ET-1 concentrations are positively correlated with the severity of hypoxia and that ET-1 plays a role in hypoxia-related pulmonary arterial narrowing or PHT. 44 In our study, correlations related to ET-1 concentrations are consistent with those of earlier studies, 9,44 and we conclude that hypoxia and acidosis during RDS may cause ET-1 synthesis and release into the bloodstream. The increase in serum ET-1 concentration observed in premature calves with RDS may be related to the pulmonary vasoconstriction caused by hypoxia. Nitric oxide (NO) is produced from L-arginine by NO synthase and plays a central role in maintaining low pulmonary vascular resistance. [45][46][47] Nitric oxide production is dependent on oxygen, and lack of NO synthesis under hypoxic conditions contributes to chronic hypoxic pulmonary vasoconstriction. 48,49 A significant increase in the concentration of ADMA occurred with endothelial damage. 17 Increased ADMA concentrations cause a decrease in NO, which then increases vascular tone.
Therefore, ADMA may be a useful biomarker in identifying PHT. 18 A previous study determined that the secretion of ADMA decreased the activity of connexin 43, causing pulmonary endothelial dysfunction. 50 An increase in the concentration of ADMA is considered to be an indicator of poor prognosis in patients with PHT, congestive heart failure, and portopulmonary hypertension and is negatively correlated with right atrial pressure and positively with venous oxygen saturation. 18 Plasma ADMA concentrations were increased in infants with RDS. 51 In a study conducted in premature infants with bronchopulmonary dysplasia, plasma ADMA concentrations were found to be higher in preterm infants with PAH than in infants without PAH. 14  Research examining genetic predisposition to RDS focused on genes for surfactant protein and found that the lack of SP-D gene alleles in premature infants was associated with RDS development. 53 In rabbits in which surfactant was experimentally removed from the lung, plasma and pulmonary ET-1 concentrations were increased. 54 Mice that lack SP-D have increased NO production and inducible NO synthase (iNOS) expression in the bronchoalveolar (BAL) fluid. 55 In Although our results were similar to those found in human medicine, some limitations still exist. The most important limiting factor in our study is that the presence PAH was not confirmed by invasive or noninvasive methods. Therefore, the biomarker findings of our study should be interpreted carefully until they are evaluated together with direct measurements of PAH.

| CONCLUSION
Significant changes occurred in blood gases and acid-base balance in premature calves with RDS. Serum ET-1 concentrations were found to be high and ADMA and SP-D concentrations were low in premature calves with RDS. Thus, serum ET-1 may be useful in the diagnosis of PAH in premature calves with RDS, and a cutoff of 34 ng/mL corresponds to a sensitivity of 87% and a specificity of 82% for prediction of mortality.

This research was supported by Selcuk University Scientific Research
Project Office with project number 20401007.