The Factors Effect on Natural Lung Surfactant Content for the Treatment of Respiratory Distress Syndrome

Introduction
Lung surfactant is a composite of lipoproteins that are produced, stored, and secreted in type II epithelial cells and cover the lung alveolar epithelial surface in the last weeks of pregnancy (Choi et al., 2020). Natural surfactant components include phospholipids, cholesterol, triglycerides, and four types of proteins (Guzmán & Santini, 2019).
Surfactant comprises ̴80%–85% phospholipids, 5%–10% neutral lipids, and 8%–10% protein, with 5%–6% containing four specific surfactant proteins. Eighty-five percent of the phospholipid fraction contains phosphatidylcholines, the most important component (40%) with the highest compaction properties being dipalmitoylphosphatidylcholine (DPPC); 11% consists of phosphatidylglycerol and phosphatidylinositol, which fluidize the lipid monolayer. The remaining fraction comprises various phospholipids with particular functions (Hentschel et al., 2020). DPPC, the main phospholipid of the lung surfactant, decreases surface tension, promotes compliance, and facilitates the lung to expand without complications and reduce the respiratory process (Bae et al., 2019). 
Hydrophobic surfactant proteins B and C play a crucial role in natural surfactant structure and cause both adsorption and distribution of phospholipids at the air-liquid interface (Olmeda et al., 2017; Hentschel et al., 2020). Hydrophilic surfactant proteins A and D have a similar structure and participate in down-regulating the inflammatory response of the lung and innate immunity (Sardesai et al., 2017). 
Neonatal respiratory distress syndrome (NRDS) is one of the most common problems for preterm infants (Magni et al., 2023). It is common in neonates born before 27 to 32 weeks who need ventilation with the infusion of exogenous lung surfactant from natural sources, helping to restore normal lung function (Han & Mallampalli, 2015; Hockenberry & Wilson, 2018). Destruction of type II alveolar epithelial cells due to adult respiratory distress syndrome (ARDS) increases lung compliance due to dysfunction in surfactant manufacture (Cutts et al., 2017). Common pathogens related to ARDS which can have an effective impact on surfactant quality and its content include Streptococcus pneumonia, Pneumocystis jirovecii, Staphylococcus aureus, and a diversity of respiratory viruses such as H1N1 novel influenza A, novel coronavirus (COVID-19) and respiratory syncytial virus (Al-Abedi et al., 2022; Ashrafi et al., 2020; Mojibi et al., 2022; Rawal et al., 2017; Jamaatia et al., 2020). In addition, different factors affect lung surfactant content and its maturation, such as age, sex, intrinsic cortisol, thyroid hormones, prolactin, epidermal growth factor, diabetic mother, and testosterone levels (Han & Mallampalli, 2015).
Extracting, purifying, and identifying pulmonary surfactants also affects the composition and phospholipid ratio (Christmann et al., 2006). Nielson and Torday (1981) mentioned the biological disparity between fetal sexes in the rabbits as an animal model, which may be the cause for male infants to develop respiratory distress syndrome. 
Moreover, some studies indicate that aging could also modify the composition and function of lung surfactant (Christmann et al., 2006). Meanwhile, John Clements reported the first direct measurements of pulmonary surfactant using his homemade Langmuir–Wilhelmy surface balance half a century ago. Subsequently, many more in vitro tensiometric techniques, such as pulsating bubble surfactometry, captive bubble surfactometry, and the constrained sessile drop, have been developed to assess in vitro surfactant function (Stichtenoth et al., 2014). 
Although some research has been conducted on lung surfactant composition, there is still insufficient data to evaluate the effect of age and gender on lung surfactant content, especially in calves, as an appropriate source of exogenous surfactant consumed in respiratory diseases. This study aimed to determine the effect of age and sex on the quality and quantity of lung surfactants and measure their functionality by new methods, which potentially can support finding the best source to obtain natural surfactants for respiratory distress syndrome (RDS) or other respiratory disorders.

Materials and Methods

Study animals
Twenty-four healthy Holstein calves were collected from the Tehran University of Medical Sciences farm, Tehran City, Iran, and divided into the following three age groups: 0-4 months, 4-8 months, and 8-12 months (n=8 in both sexes in each group). All calves’ characteristics, such as age, weight, and gender, were registered. The calves’ general health was assessed through clinical exams, and blood samples were taken to assess their complete blood count and confirm their health. The clinical criteria for admission to the study included the absence of eye and nasal discharge, normal body temperature, and absence of respiratory sounds or cough. The calves were kept under controlled conditions for approximately 12 hours before the procedure (Fozouni & Tahaei, 2023). All experimental procedures followed the guidelines on ethical standards for experimental processes in animals, according to a protocol approved by the Animal Ethics Committee, University of Tehran, Iran. This study adheres to internationally accepted standards for animal research, following the 4Rs principle. All animal experiments adhere to the ARRIVE guidelines (2.0 version) and are conducted in compliance with the U.K.

Bronchoalveolar lavage method (BAL) 
BAL was performed in the anesthetized calves with propofol (Fresenius Kabi, USA) (Diprivan® 5 mg/kg) using a sterilized and flexible catheter with a 3-5 mL balloon cuff (Supa Co, Iran). The head and neck of the calf were extended to facilitate the passage of the sterile BAL catheter. The BAL catheter was introduced into the trachea via a tracheal tube, and its positioning was confirmed by repeated coughing. The balloon cuff was then inflated with 3 mL of air, and subsequently, 5 aliquots of 200-300 mL pre-warmed sterile saline solution (37 °C) were infused. Immediately after infusion, the lavage fluid was aspirated by applying negative pressure (Danlois et al., 2000). The lavaged fluid was mixed and pooled in a sterile tube maintained on ice and immediately transferred to the biochemistry laboratory. The calves were under critical care support after the BAL procedure to prevent bronchial complications. To obtain a cell-free supernatant, BALF was centrifuged at 400×g for 15 min.

Cytology
For cytological evaluation of BALF, the cells were precipitated by centrifuging and stained using Wright-Giemsa staining and direct smear preparation (Allen et al., 1992).

Crude surfactant extraction
Cell-free BALF supernatants were centrifuged at 20000×g for 60 min at 4 ºC, allowing separation of crude surfactant pellets (CSP). Subsequently, the supernatant was discarded, and CSP was separated into several aliquots and stored at -80 ºC for further analysis.

Calf lung surfactants extract analysis 
The obtained pellets were re-suspended in CaCl2-NaCl (Merck, Whitehouse Station, NJ) solution, and the lipid part of the calf lung surfactant was extracted by modified Bligh and Dyer method (Bligh & Dyer, 1959). The lower phase-separated and concentrated with a rotary evaporator (IKA Co, rv10 digital) and stored at -20 ºC. For the triglyceride assay, the dried extract was redissolved in isopropanol and vortexed, and then the saponification reagent was added. After mixing, the periodate solution was added and kept at room temperature for 5 min. 
Subsequently, acetylacetone reagent was added, and samples were heated at 65 ºC for 15 min in a water bath. Finally, the optical density (OD) was read at 410 nm vs blank (Neri & Fring, 1973). To evaluate total lipid content, sulfuric acid was added to a test tube containing the sample, mixed well, and heated in a boiling water bath for 10 minutes. After cooling the samples, the phosphovanillin reagent was added and mixed. Finally, tubes were incubated at 37 °C for 15 minutes, cooled at room temperature, and OD was read at 540 nm.
The analysis of total cholesterol was carried out using the total cholesterol assay mentioned before by Loeffler and McDougald (Loeffler & McDougald, 1963). Briefly, isopropanol was added to the conical test tube containing the sample, mixed well, and then put at room temperature for 5 min, later centrifuged for 5 min.
Subsequently, clear supernatant was transferred to a clean test tube, and FeCl3.6H2O was added to each tube; after mixing, sulfuric acid was added, and OD read at 550 nm using blank.
The aqueous phase was collected and stored at -20 ºC to analyze the hydrophilic proteins. The phospholipids classes distribution was determined by thin-layer chromatography (TLC) on silica gel plates (Merck, Whitehouse Station, NJ; 60 F 254) using a mobile phase containing chloroform/methanol/2-propanol/triethylamine/H2O (Touchstone et al., 1983). The samples and standards include L-α-phosphatidylcholine from Soy (CAS No.=840054C, Avanti polar lipids Inc) and 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (CAS No.=850355C, Avanti polar lipids Inc) and 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (CAS No.=850468C, Avanti polar lipids Inc) were placed on plates and after drying, the TLC plate was put in the mobile phase containing tank. Then, the samples were stained with 10% H2SO4 and 8% H3PO4 and incubated at 150 °C (oven) for 15 minutes. Finally, the phospholipids were determined using Power Scan 2017 software (Mokra et al., 2016).

The phospholipid contents of the extracted samples were confirmed using high-performance liquid chromatography with C8 reversed-phase column 150×4.6 mm 5 µm (waters) along with refractive index detection. The mobile phase was prepared by combining 500 mL acetonitrile, 450 mL methanol, and 100 mL 50 mM acetic acid to a final ratio of 50:45:10. The mobile phase was degassed for 10 minutes. The extracted samples were injected into the column containing a mobile phase of acetonitrile, methanol, and acetic acid (50:45:10) at a 1 mL/min flow rate.
The total protein of the surfactant was measured by the Micro-Bradford method (Bradford, 1976). Besides, surfactant protein (SP)-A, -B, -C, and -D contents were analyzed using enzyme-linked immune sorbent assay (ELISA) technique by commercial kits (CAS No.: E0890b, U1622b and U1623b and E1039b).
A profile analysis tensiometer method was used (PAT1, Sin-interface Technology, Germany) to evaluate extracted lung surfactant in situ. Measurements of dynamic interfacial tension and dilatational viscoelasticity at the water-lipid interface were performed by the drop profile analysis tensiometer (Vatanparast et al., 2017). Briefly, the measurement process is based on image achievement of drop profile calculated by the Gauss-Laplace equation in which all the experiments are conducted at 25 °C and atmospheric pressure.

Data analysis
Statistical analysis was conducted using SPSS software, version 21. Significance levels were set at the P<0.05 using a one-way analysis of variance (ANOVA) and independent t-test. All experimental procedures involving animals were approved by the Ethics Committee of the Faculty of Veterinary Medicine of the  University of Tehran, Tehran, Iran.

Results

Clinical parameters and BALF cytology
Table 1 presents that general hematological parameters were recorded in the normal range in all groups. 

The data showed no significant difference between the cytological content of BALF at different ages and sex (Tables 1 and 2) (P>0.05).

Phospholipids and protein contents
The results showed that sex and age did not affect SP-A, SP-B, SP-D, and the total protein contents of the extracted surfactant (P>0.05). However, BALF in female groups was significantly enriched by SP-C compared to male groups at the same age (P=0.003). On the other hand, the amount of SP-C increased considerably in group A in females compared to those of similar sex in other groups. Furthermore, SP-C decreased in the males of group C compared with the males in group A (P=0.04). 

In addition, a decreasing trend by aging was observed in the amount of SP-C and SP-D in 
both female and male groups. As Figure 1 shows, the phospholipids composition of the extracted surfactant was confirmed by TLC (Figure 1). No significant differences were found in age and sex on the number of total cholesterols and glycerides (P˃0.05). 

Total phospholipids in the female groups were significantly higher than the male groups in the same age group (P=0.002). Additionally, total lipids in group A were significantly higher than the other groups for males and females (P=0.003) (Table 3). It can be inferred from the present data that all measured factors except total glycerides have a descending trend by aging in both sexes. 

Surface tension measurement results
Based on the obtained results, the surface tension of male samples was recorded at 44.76±0.38 mN/m in 0-4 months, 45.97±1.25 mN/m in 4-8 months, and 55.06±0.45 mN/m in 8-12 months old animals (Figure 2).

It was construed from the results that the surface tension of female samples was recorded 24. 85±1.30 mN/m in 0-4 months, 25.02±1.05 mN/m in 4-8 months and 50.36±0.8 mN/m in 8-12 months old animals (Figures 3, 4, and 5). 

Discussion
Lung surfactant is a composite of lipoproteins that are produced, stored, and secreted in type II epithelial cells and cover the lung alveolar epithelial surface in the last weeks of pregnancy. It reduces the surface tension at the air-water interface, improves alveolar ventilation exchanges of respiratory gases, prevents pulmonary edema formation, and finally prevents the alveoli from collapsing (Khawar & Marwaha, 2023; Singh et al., 2021). The qualitative and quantitative lung surfactant change due to infectious and noninfectious diseases. One of the more significant findings from this study is that the quality of isolated lung surfactant content in female calves under 4 months of age is higher than in other groups, which can be potentially used as the best source to obtain natural surfactant to prescribe for respiratory diseases. On the other hand, it probably rationalizes the high incidence of infectious diseases such as RDS due to viral and bacterial pneumonia in the elderly because of the reduction in quantity and functionality of natural lung surfactants.
The analysis of BALF undertaken in this study demonstrated that SP-C, SP-D, total phospholipid, total lipid content, and functionality in BALF depended on gender, and in female groups, it was significantly higher than in the male groups. Additionally, it would be interesting that the only compositions changed by aging are SP-C and SP-D, and the other surfactant contents were not dependent on it. It can be inferred from the PAT data that both age and sex could affect the amount of surface tension as increased by aging and decreased in the female group in comparison to the male group. It possibly reflects the high incidence and case fatality rate of infectious diseases such as COVID-19 among males in comparison to females (Jamaatia et al., 2020).
SP-B is an important component of surfactant substitute mixtures that can alter PL membrane association, enhancing the surfactant-like properties and the uptake of phospholipids (PLs) vesicles by type II cells in vitro while resisting surface tension by increasing the lateral stability of the phospholipid’s monolayer (Hockenberry & Wilson, 2018).
Exogenous surfactant prepared from natural sources or synthetic form is mainly used to treat NRDS and meconium aspiration. In addition to respiratory distress syndrome, surfactant deficiency is observed in many other clinical situations in term and preterm infants and adults. So, scientists should be encouraged to develop different methods of extraction and recognize the best sources for exogenous surfactants (Han & Mallampalli, 2015; Sardesai et al., 2017).
Total lipids and phospholipids in the lung surfactant are responsible for the surface active function of pulmonary surfactant by substituting interfacial water molecules, and its ultimate objective is to reduce surface tension at the water-air interface (Han & Mallampalli, 2015; Cañadas et al., 2020). Pulmonary surfactant proteins, particularly SP-B and SP-C, show a strong affinity for interfaces and ensuing surface-active properties indicated by many researchers as key constituents in achieving the optimal dynamic and mechanical properties of surfactant membranes (Sardesai et al., 2017).
The study results by Torday et al. (1981) revealed that male infants had a higher risk for RDS in comparison to females, and the reason for this phenomenon is a delay in surfactant production in response to the inhibitory effect by testes-derived hormones or diminishing the response of the male to corticosteroids stimulating surfactant synthesis. Another study showed differences in the two genders by proteomic analysis, which may be due to varying expression of specific proteins in which the level of SP-C and SP-D in the female was higher than the male; this can be the cause of more respiratory abnormalities in males (Sardesai et al., 2017; Rahmanian et al., 2014). Seaborn et al. (2010) found that by blocking lung maturation, testicular hormones can contribute to higher morbidity and mortality rates in male infants.
Another important factor that can change the surfactant content is aging due to reduced alveolar, alveolar-capillary, and lung parenchyma surface area. An age-related decrease in surfactant in monkeys could be explained by a lower number of type II alveolar cells per unit lung volume with aging and decreased alveolar surface tension produced by surfactants (Shimura et al., 1986; Dezfouli et al., 2022; Vanstapel et al., 2021). Furthermore, the decreases occur with the alveolar septal area and the total surface area of the lung parenchyma, which can cause a decrease in surfactant composition (Pruthi & Multani, 2012).
Christmann et al., (2006) showed that surface tension and phospholipid composition of surfactants in neonatal foals were significantly different compared to adult horses, which may influence the biophysical and immunologic functions of surfactants. However, studies on the whole-lung tissue extracted surfactant from humans, rats and rhesus monkeys revealed no significant alterations in the content of disaturated phosphatidylcholine with aging (Egberts et al., 1987; Ghidoni et al., 2015).

Conclusion
Finally, owing to the importance of surfactant replacement therapy in preterm foals, lambs, calves, and babies, obtaining the best source with new method identifications to acquire the natural surfactant to produce exogenous surfactant is necessary. Based on the obtained results, female BALF was more enriched in SP-C and total PL contents than differences in surfactant content; besides, PAT analysis between neonate males and females may be attributed to surfactant phosphatidylcholine synthesis in late pregnancy, but further investigation is necessary by longer age intervals. 

Ethical Considerations

Compliance with ethical guidelines
All experimental procedures followed the guidelines on ethical standards for experimental processes in animals, according to a protocol approved by the Animal Ethics Committee, University of Tehran, Tehran, Iran. This study adheres to internationally accepted standards for animal research, following the 4Rs principle. All animal experiments adhere to the ARRIVE guidelines (2.0 version) and are conducted in compliance with the UK.

Funding
This research was financially supported by the Institute of Biomedical Research of Veterinary Medicine, the University of Tehran, Tehran, Iran and Persian Darou Alborz Research and Technology, Tehran, Iran.

Authors' contributions
Supervision and project administration: Zohre Eftekhari and Sirous Sadeghian Chaleshtori; Conceptualization, methodology, formal analysis, data curation, resources, review and editing: All authors; Investigation and writing the original draft: Marzieh Mokhber Dezfouli; Validation: Marzieh Mokhber Dezfouli and Sirous Sadeghian Chaleshtori. 

Conflict of interest
The authors declared no conflict of interest.

Acknowledgments
The authors gratefully acknowledge the financial support from the Institute of Biomedical Research of Veterinary Medicine, the University of Tehran, Tehran, Iran and Persian Darou Alborz Research and Technology fund.

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