MSc Chemistry
Track Analytical Sciences
Literature Thesis
Detection of human chlorine exposure: bioanalytical
approaches and challenges
By
Elena Michopoulou
UvA#: 12375152, VU#: 2646133
June 2020 12 ECTS-credits
Period:1st of April to October 17th, 2020
Daily supervisor: Second reviewer:
Dr. Isabelle Kohler Prof. Dr. A.C van Asten
Examiner:
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Content
Abstract 2
Abbreviations 3
1. Introduction 4
2. Theoretical background of Chlorine and chlorine compounds 5
2.1 Chlorine toxicity in swimming pools and in the air 7
2.2 Chlorine exposure treatment 8
2.3 Chlorine pathology 8
2.4 Modifications of chlorine and reactions with biological targets 10
2.5 Biomarkers and metabolomics 12
3.1 Biomarker candidates for chlorine exposure 13
3.1.1 3-Chlorotyrosine and 3-5 chlorotyrosine biomarkers 13
3.1.2 3-Nitrotyrosine biomarker 14
3.1.3 Chlorohydrin phospholipids and chlorofatty aldehydes 14
3.1.4 8-isoprostane biomarker 17
3.1.5 Glutathione and glutathione sulfonamide biomarkers 17
3.2 Metabolomics-based analysis of chlorine biomarkers 18
3.2.1 Targeted metabolomics-based approaches 18
3.2.1.1 Sample preparation 23
3.2.1.2 Analysis 24
3.2.2 Targeted lipidomic Approaches 25
3.2.2.1 Sample preparation 27
3.2.2.2 Analysis 27
4. Discussion 29
5. Conclusions and future perspectives 31
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Abstract
Chlorine is a reactive chemical that has been widely used in industry over the last two centuries. Exposure to chlorine has occurred in several situations, resulting in severe problems for human health. The aim of this review is to investigate the different sources of exposure and the exact mechanism behind the toxicology and pathology of chlorine and chloride. When Cl2 is absorbed, it reacts with different compounds in the organism such as amino acids, lipids and low-molecular-weight antioxidants like GSH, causing both acute and chronic respiratory injuries. The exact pathological mechanisms remain unknown; however, few hypotheses have been introduced over the past few years and will be discussed.
This literature review focuses on the exposure of chlorine to humans, via a variety of different factors; from swimming pools, household bleach, to armed conflicts where it was used as chemical warfare. Moreover, emphasis will be given on the mechanism behind chlorine exposure as many compounds are formed during chlorine interaction with organic tissue. Chlorine gas, which is the main source of exposure, produces hypochlorous and hypochloric acid releasing highly reactive oxygen radicals. Furthermore, focus will be given on the mechanism of chlorine exposure especially during inhalation and its interactions with different compounds in the epithelial lining fluid (ELF). The modifications of biological molecules indicate that there can be numerous potential biomarkers for detecting chlorine gas exposure from amino acids to unsaturated phospholipids. This review will offer an insight into the feasible biomarkers using targeted metabolomics-based approaches that provide information on known metabolites such as, 3-chlorotyrosine. However, this biomarker candidate is not unambiguous, thus poorly specific to chlorine exposure. Moreover, more approaches will be discussed using lipidomics, presenting the identification of potential biomarker candidates for chlorine exposure. This review includes detailed information on the analytical techniques that have been used for the identification of the potential biomarkers. The aim of this study is to provide enough data on the feasible biomarker candidates considering their pathology when interacted with Cl2 or HOCl. Finally, the most promising candidates will be referred and a novel approach for the discovery of new metabolite candidates will be introduced as well.
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Abbreviations
AEGL acute exposure guideline levels RADS reactive airway dysfunction syndrome OPCW Organization for the Prohibition of the Chemical Weapon
CFC chlorofluorocarbon ELF epithelial lung lining fluid GSH glutathione
UA uric acid AA ascorbic acid MPO myeloperoxidase
DON dissolved organic nitrogen DPBs disinfected by-products
THMs trihalomethanes
CHCl3 chloroform
GSTT1-1 glutathione-S-transferase T1-1
ALI acute lung injury O- oxygen radicals
ARDS acute respiratory distress syndrome GC gas chromatography MS mass spectrometry UHPLC ultra high pressure liquid chromatography
BALF Bronchoalveolar lavage fluid HRMS high resolution mass spectrometry LC liquid chromatography
SIM selected ion monitoring
SRM selected reaction monitoring TFA trifluoroacetic acid
MTBE methyl tert-butyl ether
MPA mobile phase A MPB mobile phase B
ACN acetonitrile
DART-MS/MS direct analysis in real time tandem mass spectrometry RP reversed phase
DTPA pentetic acid MRM multiple reaction monitoring NO2Tyr 3-nitrotyrosine 2-Cl-PA 2-chloropalmitic acid 2-Cl-SA 2-chlorostearic acid
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1. Introduction
In 1774, the Swedish chemist Carl Wilhelm Scheele discovered chlorine (Cl) when he combined hydrochloric acid with manganese dioxide. Although, at that time, he thought that the contained gas was oxygen. In 1810, the British chemist Humphry Davy insisted that this gas was a new element and identified it as chlorine (Humans, 1991). Chlorine is a naturally greenish-yellow gas and has been used as a reactive gas for more than two centuries. This element can be very useful in food and water industry, but also poisonous and harmful to humans and the environment (Watt, 2002).
Chlorine has many applications in industry, especially in chemical synthesis of, for instance, PVC (polyvinyl chloride), pesticides, plastics, and dyes. Moreover, for many decades it has been used not only as a germicide for drinking water and swimming pools but also as a household disinfectant. However, due to its availability, it has been repeatedly used in armed conflicts worldwide as the toxicity of chlorine gas compared to other chemical warfare agents is low but can be lethal at high exposure levels. According to acute exposure guideline levels for chlorine exposure, exposure to 50 ppm for more than 10 minutes is life threatening especially for individuals with respiratory problems (Hemström et al., 2016). Chlorine exposure can display a variety of symptoms that affect almost entirely the respiratory system, ranging from sensory irritation and cellular changes to pulmonary disease and pneumonitis. In some cases, it can cause reactive airway dysfunction syndrome (Hoyle and Svendsen, 2016).
Although the severity of chlorine exposure in humans is obvious, there is limited information concerning the possible direct or indirect biomarkers than are linked to its toxicity in humans. Moreover, the pathological mechanisms associated to chlorine toxicity are not entirely known yet, rendering the detection of acute and chronic exposure very challenging (Hemström et al., 2016; C. W. White and Martin, 2010). The identification of new potential biomarker candidates is complicated as the myeloperoxidase enzyme in humans produces hypochlorous acid under inflammatory conditions. Indeed, there is an endogenous reaction of hypochlorous production due to oxidative injury in the lungs which that can be a result of a chronic disease. Two biomarker candidates have been proposed for chlorine exposure, i.e., 3-chlorotyrosine and 3,5-chlorotyrosine. However, they are not specific for chlorine exposure as they are present also during inflammatory response when individuals are not exposed to chlorine (Buss et al., 2003; Crow et al., 2016). Other potential biomarker candidates have been reported, such as lipid-based compounds (Ford et al., 2016; Hemström et al., 2016). The epithelial lining fluid in the lung being composed of lipids, the latter are prone to reaction with chlorine and hypochlorous acid, which may lead to the formation of specific biomarker candidates of chlorine exposure (Hemström et al., 2016).
Overall, there is a lack of scientific knowledge on the exact pathological mechanism of chlorine and the possible biomarker candidates. This literature study first describes the different sources of exposure and the underlying mechanisms of the toxicity. Moreover, the different techniques for the discovery of potential markers of chlorine exposure will be discussed, including sample preparation approaches and analytical methods. Special
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attention will be put on the mechanism and pathology of chlorine exposure and more specifically the potential biomarker candidates will be reported, as well the limits of detection/quantitation that were achieved. The aim of this study is to eventually propose a targeted (quantitative) metabolomic approach focusing on a panel of relevant possible biomarker candidates.
2. Theoretical background of Chlorine and chlorine compounds
Chlorine (from the Greek word chloros/χλώρος meaning pale green) is a naturally chemical greenish-yellow gas at room temperature and pressures (Committee, 1980). Chlorine is a halogen compound, a part of the VII group in the periodic table which includes also bromine, fluorine and iodine. Industrially, it is typically produced by the electrolysis of alkali chlorides and obtained from the electrolysis of sodium chloride (NaCl) in water. The reaction also yields sodium hydroxide and hydrogen gas (equation 1) (Hannan, 2007; Watt, 2002):
2𝑁𝑎𝐶𝑙 + 2𝐻2𝑂 → 2𝑁𝑎𝑂𝐻 + 𝐻2 ↑ + 𝐶𝑙2 ↑ (1)
Chlorine also produces a weak solution of hypochlorous acid (HOCl) and hydrochloric acid (HCl) as it is moderately soluble in water (Equation 2). For domestically uses liquid bleach is used which is a dilute solution of sodium hypochlorite.
𝐶𝑙2 + 𝐻2𝑂 → 𝐻𝑂𝐶𝑙 + 𝐻𝐶𝑙 (2)
This element also exists as chloride ion (Cl-) which is then produced differently compared to chlorine. It is found in the form of salts like NaCl and KCl (Chlorine, 2000). The terminology “gaseous chlorine” represents the diatomic molecule as it is present in nature. The chlorine atom has a rearrangement of 17 electrons in total and 7 electrons in its outermost orbit, which allow the two chlorine atoms to bond together achieving better stability in comparison with the single atom. This is also one of the reasons why chlorine reacts with many elements, to fill the space in its outer orbit (Watt, 2002).
As it has been referred above, hypochlorous acid is formed when chlorine gas hydrolyses in water and hypochlorite ions are formed when hypochlorous acid dissociates into H+ and OCl-. Sodium hypochlorite (NaOCl) is the most used irrigant agent in endodontics. It is also a disinfectant and bleaching agent which is used in industry and healthcare. When dissolved in water, it is referred as liquid bleach (Bello et al., 2019; Slaughter et al., 2019). Furthermore, in higher concentrations, it is more dangerous and aggressive whilst in lower concentrations it is biocompatible (Estrela et al., 2002). Sodium hypochlorite presents interesting properties. Indeed, as it can act like an organic or fat solvent, it degrades fatty acids, converting them into fatty acid salts and glycerol. Moreover, NaOCl is able to form salt and water by neutralizing amino acids (Estrela et al., 2002). However, it has some disadvantages like cytotoxicity and chemical instability (Bello et al., 2019).
It needs to be noted that large water systems supply tens of hundreds of millions of gallons of water per day in Canada and in the USA where they use chlorine and sodium hypochlorite for water disinfection (Cheremisinoff, 2018). Calcium hypochlorite [Ca(OCl)2] is a yellow-white alkaline solid powder, relatively stable, and has more
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available chlorine than sodium hypochlorite (Vogt et al., 2010; Yigit et al., 2009). It is known as bleaching powder and is commonly used as a germicide in pool water but can cause serious problems to swimmers. This compound is used in smaller water systems as it is easily stored and transported (Cheremisinoff, 2018; Connell, 2006; Yigit et al., 2009). Moreover, it is used in industrial food processing protecting the food from different germs (Dutta and Saunders, 2012). Another interesting property is that it can produce homemade bombs when combined with oxidized substances like glycerin or weak domiciliary acids like cola vinegar (Yigit et al., 2009).
The main difference between chlorine, sodium hypochlorite and calcium hypochlorite in their use depends on various factors such as availability, cost, equipment maintenance, and simplicity of application. Chlorine is cheaper than its salts, although it is more difficult in its applicability as it is more toxic, and it can form explosive compounds with other chemicals. NaOCl presents difficulty in transportation and storage, while Ca(OCl)2 is stored and transported easily (Yigit et al., 2009).
Furthermore, chlorine has many hazardous effects in the environment. The use of chlorofluorocarbons (CFCs) leads to the so-called “ozone hole”. CFCs have a variety of applications as refrigerants, aerosol propellants, and blowing agents for foams causing increasing ultraviolet light to reach the Earth’s surface (Finlayson-Pitts, 2013). In the organism, chloride (Cl-) is the most abundant negative ion and the most important extracellular anion. It is also referred to, as “the queen of electrolytes” as it assists in many functions such as maintaining the osmotic pressure, muscular activity, and transportation of water in a different fluid area. In this way, the concentration of salts in the body remains relatively constant over time (Berend et al., 2012).
In the United States, chlorine is produced and transported in large amounts, it is considered a chemical threat for its availability and acute toxicity. Chlorine is highly toxic when inhaled, causing respiratory problems. It can be released by accident in household and industrial settings. Moreover, chlorine bleach can be harmful if mixed with other chemical agents that can produce chlorine gas (Agabiti et al., 2001; Hoyle and Svendsen, 2016). In industrial settings, exposure can occur via an accidental release or leak during its transport which has caused many casualties in the past (Evans, 2005). Many incidents have been reported in the US where chlorine gas was released by accident causing many injuries and fatalities (Branscomb et al., 2010). Numerous occupations with potential exposure to chlorine can be accounted but the largest scale exposures can occur during war when chlorine has been used as a chemical warfare agent. The last one remains the biggest concern as it can be used in terrorist attacks (Hoyle and Svendsen, 2016). The use of chlorine gas as a chemical weapon has been widely reported over the past years. It was first introduced in the 1st World War where the German army released more than 150 tons of chlorine gas in April of 1915 in Belgium where this attack killed over 5000 and caused many injuries. Later, in 2007 Iraq and in 2014 in Syria, this toxic gas was again used causing multiple deaths and casualties (Achanta and Jordt, 2019).
7 2.1 Chlorine toxicity in swimming pools and in the air
In most swimming pools, chlorination using sodium or calcium hypochlorite, chlorine gas, chlorinated isocyanate or chlorine dioxide is essential in order to disinfect water. Although, exposure to these chlorine-based disinfectants could increase the risk of asthma among swimmers. Harmful byproducts are produced like chloramines especially monochloramine (NH2Cl) and dichloramine (NHCl2) from hypochlorous acid and nitrogenous compounds which are found in urine and sweat. These compounds are produced from the reaction between free chlorine and dissolved organic nitrogen. The main reaction that is taking place according to equation 3 is:
𝐻𝐶𝑙𝑂 + 𝑅 − 𝑁𝐻2 → 𝐻2𝑂 + 𝑅 − 𝑁𝐻𝐶𝑙 (3)
This reaction is slow, i.e., longer than 10 hours.(Florentin et al., 2011) Furthermore, other disinfected by-products (DPBs) that are present in a high concentration in the air and in pool water are trihalomethanes (THMs) which are present also in tap water. These compounds are generated from the complex reaction between chlorine and organic matter. They are referred by the formula CHX3 where X represents a halogen atom. There are many variables that can affect the formation of THMs, such as chlorine concentration, pH, temperature, organic matter (and its precursors) and bromide ion concentration.(Amy et al., 1987) In swimming pools the most commonly presented THMs are chloroform (CHCl3), bromodichloromethane (CHBrCl2), dibromochloromethane (CHClBr2) and bromoform (CHBr3).(Richardson et al., 2010) The bromination reaction is faster than the chlorination, that is why the formation of brominated THMs are preferred.(Florentin et al., 2011)
Regarding the toxicity of chloramines, there is no specific data. However, chloramines are responsible for the irritation of the eyes and respiratory tract. The irritation in the latter is caused particularly by trichloramine whilst, monochloramine and dichloramine ratio affect the odor and the taste. Concerning the toxicity of trihalomethanes, they are well absorbed and metabolized during inhalation exposure. The liver metabolizes the THMs to carbon dioxide and carbon monoxide. Chloroform is the most known THM and humans absorb approximately 50% by inhalation and a smaller but significant portion of chloroform from water while showering (Florentin et al., 2011). It can follow two possible mechanism routes; oxidation by cytochrome P450 and a reductive route interrelated with theta-class glutathione-S-transferase T1-1 (GSTT1-1) leading to the formation of some toxic metabolites like phosgene (Yang et al. 2018; Florentin, Hautemanière, and Hartemann 2011).
Furthermore, chlorine gas inhalation can result in both acute and chronic respiratory injuries. The symptoms in acute respiratory injuries commence from minutes after the exposure to hours and it affects exposed tissues mainly. These symptoms include pulmonary edema, tracheobronchitis, temporary airflow dysfunction.(Jonasson et al., 2013) In higher concentrations, chlorine can cause adult respiratory distress syndrome, pulmonary inflammation, respiratory failure and death.(Das and Blanc, 1993) The long-term consequences are referred to as reactive airways dysfunction syndrome is characterized by asthma symptoms including coughing, chest tightness and breathlessness
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which is caused by the inhalation of a highly irritating substance like chlorine gas and results in airflow obstruction. Its symptoms start within 24 hours after exposure and insist on for at least three months (Arif et al., 2013; Carl W. White and Martin, 2010).
2.2 Chlorine exposure treatment
Nowadays, the treatment of chlorine poisoning is nonspecific, but it focuses mostly on reducing the symptoms. In 2005 on a train accident of chlorine poisoning, inhaled β-agonists were used as treatment which is the most common prescribed medication (Van Sickle et al., 2009). In most cases of exposure the usage of humidified oxygen is preferred. However, in case of any airway obstruction the administration of beta-adrenergic agents is suggested. However, some studies in rats showed that administration of oxygen did not provide long-term survival and led to respiratory failure (Okponyia et al., 2018; C. W. White and Martin, 2010). All patients should therefore remain under observation and in extreme cases hospitals need to provide mechanical support, In most cases patients, present mild symptoms with reduction of their symptoms 3 to 5 days after exposure, although, their pulmonary is able to function properly within few months. However, in some cases when the injury has proceeded in the airways, the symptoms may persist for a longer time (Gupta, 2020). Moreover, according to (Honavar et al., 2017) after the administration of nitrite in rabbits 30min post chorine exposure, more than 50% had no injury in the lungs. In reducing the inflammatory injury, many compounds have been used, as corticosteroids and a nitric oxide donor.the development of an antidote for chlorine exposure is in great need but more research needs to be done (Gupta, 2020; Honavar et al., 2017).
2.3 Chlorine pathology
Chlorine gas is moderately soluble in water and releases hypochlorous acid and hydrochloric acid as it dissolves at the airway surface. Thus, the chlorine toxicity involves also other forms such as chloramine, chlorine dioxide and hypochlorous dioxide. The precise mechanism of how Cl2 reacts with oxygen species and other fluids forming reactive oxidants is unknown (Figure 1). What is known, though, is that exposure to chlorine gas can cause direct injury to the lung epithelium and further damage can take place if the inflammatory cells like neutrophils activate and migrate to the airway epithelium with the following production of oxidants and proteolytic enzymes (Tuck et al., 2008; Carl W. White and Martin, 2010). In this way, Cl2 leads to damage to the lower airways as well as the eyes, skin and upper airway. The airway is affected by the nose and any injury from chlorine on the structure and the function of the airway cannot fully be repaired. However, there is not enough available information regarding the injury and compensation of the epithelium airway after chlorine gas exposure (Carl W. White and Martin, 2010).
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Figure 1: Mechanism of the hydration of chlorine gas (Cl2) into the airway leading to the production of HCl and HOCl, both chlorine and hypochlorous acid react with airway constituents. It is indicated that reactive oxygen species are formed by a type of white cells called neutrophils and during an injury at the epithelial mitochondrial (Carl W. White and Martin, 2010).
Oxygen radicals (O-) are released when chlorine gas produces hydrochloric and hypochlorous acid. This occurs when the above interact with tissue and water producing HCl and free O˙, causing major consequences on the tissue (National Research Council (US) Subcommittee on Acute Exposure Guideline Levels, 2004). Oxygen radicals are byproducts of oxygen. They are defined as a molecular fragment that has one or more unpaired electrons in its outermost orbit. These byproducts are highly reactive. In moderate levels they are beneficial but lead to oxidative stress in high concentrations, resulting in tissue damages. Oxygen-free radicals are formed within the mitochondrial membranes, from enzymatic and non-enzymatic reactions. Primarily, they target the unsaturated bonds in lipids, and damage enzymes that contain sulphur, which results in peroxidation and in membrane loss. The main consequences of oxygen radicals are potentially tissue injury, disease and DNA damage (Sailaja Rao et al., 2011). The reactions of oxygen radicals that are taking place due to chlorine inhalation are presented in the equations below:
𝐶𝑙2 + 𝐻2𝑂 ↔ 𝐻𝐶𝑙 + 𝐻𝑂𝐶𝑙 (4)
2𝐻𝑂𝐶𝑙 ↔ 2𝐻𝐶𝑙 + 𝑂2 (5)
𝐻𝑂𝐶𝑙 + 𝑁𝑂2− → 𝑟𝑒𝑎𝑐𝑡𝑖𝑣𝑒 𝑛𝑖𝑡𝑟𝑜𝑔𝑒𝑛 𝑠𝑝𝑒𝑐𝑖𝑒𝑠 → 𝑡𝑦𝑟𝑜𝑠𝑖𝑛𝑒 → 3𝑁𝑇 (6) (𝐶𝑙 ∙ 𝑂𝑁𝑂, 𝐶𝑙 ∙ 𝑁𝑂2) (𝑛𝑖𝑡𝑟𝑎𝑡𝑖𝑜𝑛)
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𝐻𝑂𝐶𝑙 + 𝑂2− ˙ → 𝑠𝑜𝑢𝑟𝑐𝑒 𝑜𝑓 ℎ𝑦𝑑𝑟𝑜𝑥𝑦𝑙 𝑟𝑎𝑑𝑖𝑐𝑎𝑙𝑠 (𝑂𝐻) (7) 𝑂2 + 2 𝑁𝑂˙ → 2𝑁𝑂2− (8)
𝑂2− + 𝑁𝑂˙ → 𝑂𝑁𝑂𝑂 (9)
Equations: Representation of the reactions that most likely take place during the chlorine inhalation. Equation 4: Hydration of chlorine gas leading to the formation of HCl and HOCl. Equation 5: HOCl in the
epithelial lung fluid can form HCl and O2. Equation 6: HOCl reacts with NO2 compounds that exist on the organic tissue, forming reactive nitrogen species that interact with tyrosine. A nitration reaction is taking place between tyrosine and nitrogen species. Equation 7: HOCl reacts with reactive oxygen species such as superoxide O2-˙, forming potentially hydroxyl radical that can be formed both via recruited neutrophils and
via secondary mitochondrial dysfunction. Equation 8: Induction of nitric oxide synthase (iNOS) can lead to formation of nitric oxide (·NO). Equation 9: Induction of nitric oxide synthase (iNOS) can lead to formation of peroxynitrite (ONOO−) (Carl W. White and Martin, 2010).
The physicochemical properties of the hydrochloride salt explain its mechanism of toxicity. NaOCl forms mainly hypochlorous acid rather than hypochlorite ion, when it dissolves in water, a reaction that depends on the pH. This oxidizing agent can have toxic effects which are closely related to the pH of the solution and the oxidizing capacity. Sodium hypochlorite can be toxic upon contact with skin and mucous membranes. The production of hypochlorous acid can also occur when it is present in the mouth, the stomach and the respiratory tract. In addition to this, it can produce also free radicals which can lead to cytotoxic injury by denaturing proteins (Slaughter et al., 2019). Moreover, one of its properties is to form chloramine gas by reacting with ammonia-based products like cleaners or nitrogenous products which are present in swimming pools (Tanen et al., 1999). Sodium hypochlorite forms water and salt by neutralizing amino acids. HOCl, which is presented in sodium hypochlorite solution, can react with the protein’s amino group. This happens when HOCl interacts with amino acids in organic tissue and forms chloramines (mainly monochloramine) that interfere with cell metabolism (Estrela et al., 2002; Racioppi et al., 1994). Furthermore, chloramine can react with moisture in the membranes when inhaled and it can produce ammonia, hydrochloric acid and free radicals. These three agents can cause severe injuries like pneumonitis and edema as they irritate the respiratory tract (Slaughter et al., 2019). Moreover, hypochlorite releases chlorine gas when mixed with acid as this reaction is closely correlated with the pH of the solution. When the solution is at acidic pH below 2.4 there is an extended release of chlorine which releases radicals, hypochloric and hypochlorous acid. These products can cause cytotoxic injury in the respiratory tract and damages in the cells and membranes (Slaughter et al., 2019). Ca(OCl)2 has a similar mechanism to NaOCl as it produces Cl2 as it reacts with water and produces hypochlorous acid and radicals that can cause tissue injuries (Yigit et al., 2009).
2.4 Modifications of chlorine and reactions with biological targets
During inhalation, chlorine dissolves in the epithelial lining fluid (ELF) due to its high solubility in water. The ELF is composed of a thin layer which covers the surfaces of the airways and the cells in the epithelial cells of the lung (Squadrito et al., 2010). Cl2 reacts in the ELF with different biological targets, mostly with low-molecular-weight antioxidants like glutathione (GSH). Amino acid residues in proteins and phospholipids are also targets of Cl2, or it reacts with water and hydrolyzes to produce hypochlorous and
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hypochloric acid. This route of reaction depends on the concentration of the antioxidants: the higher concentration, the higher the possibility to react with biological molecules. Moreover, according to Haber’s law, the biological response of chlorine depends also on the concentration and the duration of exposure of chlorine (Yadav et al., 2010). As the concentration of chlorine rises, the injury will progressively occur to more distal lung regions damaging the components of the alveolar epithelial cells and the pulmonary surfactant system (Squadrito et al., 2010).
There are many potential targets that may react with chlorine in the ELF. Their reaction depends mostly on their reactivity which is a function of pH, pKa and their concentration. The ELF has approximately a pH of 7. The amines with smaller pKa values show the larger population of unprotonated amine for the pH value of 7 and they also show the higher reactivity. Due to their low pKa, the amino terminal function in peptides and proteins is highly reactive towards Cl2. The reaction between Cl2 with amine groups produces tyrosine and more specific 3-chlorotyrosine and 3-5chlorotyrosine (Squadrito et al., 2010). Moreover, the ELF has high concentrations of low molecular weight antioxidants like glutathione (GSH), ascorbic acid (AA) and uric acid (UA). These antioxidants have a high reactivity towards HOCl. In the lung ELF, the most abundant antioxidant is GSH but, its concentration is much lower in the nose ELF (van der Vliet et al., 1999).
Unsaturated lipids are considered to be also likely targets for reaction with HOCl, especially plasmalogens, a class of vinyl ether containing phospholipids (Schröter and Schiller, 2016). The latter produce halohydrins during reaction with alkenes which is a typical electrophilic reaction. It must be noted that some lipids carry polar and more reactive functional groups in their head groups than the alkenyl groups in their non-polar regions. Some of these lipids are phosphatidylethanolamine, phosphatidylserine, sphingosine and sphinganine that have a primary amine as a functional group. The most abundant lipids in the lung ELF are phosphatidylethanolamine and phosphatidylserine and they have a high probability to react with Cl2 or HOCl (Squadrito et al., 2010). In biological membranes, there is a high number of phospholipids that are capable of oxidation and can be chlorinated by different enzymatic factors. This is one of the reasons that chlorinated phospholipids manifest effects in acute and chronic inflammation and thus related to inflammatory diseases (Fruhwirth et al., 2007; Mauerhofer et al., 2016).
Furthermore, a monolayer of phospholipids covers the alveolar ELF and any of the amino phospholipids that may exist in this monolayer can likely react with chlorine (Squadrito et al., 2010). The hydrophobic fatty acid chains are exposed to the air whilst the phospholipid polar head groups are headed to the aqueous film where a monolayer is formed by the pulmonary surfactant. The tension on the surface is decreased by the monolayer that has been created. The phospholipid non-polar tails that form a lipophilic phase, dissolve the oxygen that exists in the air and the proteins that exist on the surfactant lead to the oxygen dispersal. In this way the chlorine is able to dissolve in the non-polar tails as it is more lipophilic than oxygen and it can react with the phospholipids to compose chlorohydrins as seen in Figure 2 (Hemström et al., 2016).
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Figure 2: Probable mechanism of the chlorination of the phospholipid bonds (Hemström et al., 2016).
2.5 Biomarkers and metabolomics
The toxicity behind chlorine exposure is obvious, however, there is no evidence of successful treatment. The reason behind this, lies to the fact that there is no known marker that could undeniably prove the obvious poisoning by chlorine. A biomarker is a substance that is identified as indicator/marker of an interaction between a xenobiotic exposure and defined as any feasible biochemical compounds or any other alteration that is measurable in the organism after exposure (Epidemiology, 1991). They are closely correlated with characteristics of a specific disease and currently, they are used increasingly as clinical and diagnostic tools in order to improve the therapeutic response. Proteomics, metabolomics and genomics are all techniques that are used to discover new biomarkers. Proteomics is a technique of identifying the function and structure of proteins from biological samples. This technique holds the key for an early prognosis and diagnosis of a disease. Genomics is a technique that studies the structure, content and the sequence of genome. metabolomics focuses on the identification and quantification of small molecules, with a mass lower than 1kDa, including endogenous and exogenous molecules that are derived from the cellular metabolism.
Metabolomics reflect complex and multiple reactions with the intention to provide insight into cellular physiology (Liu and Locasale, 2017; Nalbantoglu, 2019). This technique rely on two different analytical strategies, i.e., untargeted and targeted approaches. In
untargeted approaches, thousands of metabolites can be measured offering the opportunity
to observe unexpected biochemical changes. One great advantage that this approach offers is the huge collection of data leading to the generation of a certain hypothesis that will be additionally analyzed with targeted approaches (Schrimpe-Rutledge et al., 2016). The chemical identity of every metabolite is not known at the beginning of the study which is the main limitation as it may not be possible to identify all the metabolites of interest. On the other hand, targeted approaches are considered to be a quantitative analysis is performed with a small and specific number of metabolites. For this reason, a targeted analysis provides greater selectivity and sensitivity than untargeted methods. The main differences of these approaches are the number of metabolites that can be measured, the sample preparation required, the analytical method for the analysis and the level of quantification of metabolites (Cajka and Fiehn, 2016; Liu and Locasale, 2017;
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Nalbantoglu, 2019). Taking all this into account these two approaches are interdependent as the first one offers a global analysis whilst the later offers a more subset analysis. (Schrimpe-Rutledge et al., 2016) Many studies in the past few years have used metabolomics as it plays an essential role in the discovery of biomarker candidates for chlorine exposure.
3.1 Biomarker candidates for chlorine exposure
The mechanisms on the modifications of biological molecules discussed in section 2.4 indicate that there can be numerous potential biomarkers for detecting chlorine gas exposure from amino acids to unsaturated phospholipids. The identification of new biomarkers is complicated as they must unambiguously prove the Cl2 exposure. This means that the biomarker must not be produced by any endogenous reactions, but the substance may be a result of more than one source (Hemström et al., 2016).
3.1.1 3-Chlorotyrosine and 3-5 chlorotyrosine biomarkers
Two feasible biomarkers chlorotyrosine and dichlorotyrosine are produced through the electrophilic addition of chlorine to the meta positions of the aromatic ring. However, they can be formed through the peroxidase enzyme, myeloperoxidase which is highly expressed in neutrophil granulocytes, a subtype of white blood, and it is triggered during an inflammatory response (Sochaski et al., 2008). When stimulated, neutrophils produce oxidants like superoxide radicals and hydrogen peroxide, and release myeloperoxidase (MPO). The later one generates hypochlorous acid by the oxidation of chlorine. HOCl is a strong oxidant and participates in several reactions with cells and tissue components. In addition, a minor reaction is taking place with the hypochlorous acid and the tyrosine residues in proteins forming chlorotyrosine products (Buss et al., 2003). Thus, in the case of these two biomarkers they can be present also in individuals that are not being exposed to chlorine but in individuals that present chronic lung disease. For this reason, chlorotyrosine has been proposed as a biomarker for neutrophil oxidant activity due to its high concentration on individuals with respiratory problems (Buss et al., 2003; Sochaski et al., 2008). Despite these two chloramines not being unambiguous biomarkers, they are known to be chemically stable to the heated acidic conditions needed for their analysis. Moreover, they have been used extensively
as a biomarker for cases of chronic inflammatory disease, but they have also been detected in the past in rat nasal tissue after exposure to chlorine gas. The main problem with these two biomarkers is that it needs to separate the chronic inflammatory response from the acute chlorine exposure (Crow et al., 2016; Sochaski et al., 2008).
Figure 3: (A): structure of chlorotyrosine, (B):
14 3.1.2 3-Nitrotyrosine biomarker
As it has been referred already above the enzyme myeloperoxidase which generates reactive oxygen species like hypochlorous acid is able to lead to the formation of 3-nitrotyrosine (NO2Tyr). This happens by the nitration of the tyrosine phenol ring by reactive nitrogen species. Both 3-chlorotyrosine and 3-nitrotyrosine are byproducts of myeloperoxidase’s activity which can produce reactive nitrating agents such as NO2˙ that
is generated by the oxidation of nitrite NO2- and nitryl chloride (NO2Cl2) (Curtis, n.d.; Eiserich et al., 1998; Hazen Stanley L. et al., 1999). These reactive agents react with tyrosine and form 3-nitrotyrosine damaging biological molecules like free or protein bound tyrosine. Thus, the potential pathways of tyrosine chlorination and nitration by MPO can lead to both 3-nitrotyrosine and 3 chlorotyrosine (Curtis, n.d.).
Figure 4: Nitration of tyrosine to 3-nitrotyrosine by the nitrating agents (Ahsan, 2013).
3.1.3 Chlorohydrin phospholipids and chlorofatty aldehydes
The next group of potential biomarkers according to the mechanisms that have been referred in chapter 2.4 are the chlorinated lipids that are been formed through the chlorination of the vinyl ether bond of plasmalogens. According to (Spickett, 2007) the main products from the reaction of HOCl and unsaturated phosphatidylcholines are chlorohydrins. However, in complex biological samples, the outcome can be even more complicated leading to lipid peroxidation. A chlorine atom has numerous effects on the function and properties of the lipid that can cause problems to cells or tissues. The chlorine atom attacks lipids that contain reactive headgroups or the vinyl ether bond of plasmalogens leading to a variety of complex and possible products (Spickett and Pitt, 2015). These lipids are formed due to plasmalogen oxidation and are produced during chlorine exposure as the lung and surfactant are full of plasmalogens. These chlorinated lipid levels remain high even 24 hours after exposure, long after other biomarkers have returned to normal levels (Ford et al., 2016).
Moreover, the reaction of HOCl with unsaturated lipids is relatively slow in contrast with amino acids. Thus, most products generated by HOCl would be in most cases other biomolecules, but this is closely dependent on the local concentration of biomolecules and HOCl. So, in locations where the concentration of unsaturated phospholipid levels exceeds those of other molecules such as proteins, there will be a higher possibility of formation of chlorinated phospholipids (Spickett, 2007). This happens in BALF where the concentration of phospholipids is relatively high in contrast with blood. (Hemström et al., 2016) showed that after chlorine exposure, in the first 48 hours, there is a lung dysfunction which is related
15
to the decrease in pulmonary phospholipid content. This is one of the reasons that there is an overall disappearance of the biomarkers discovered in BALF after a certain amount of time after exposure. This disappearance is attributed also to other factors such as metabolism due to the severe lung damage, Moreover, the phospholipids are recognized by macrophage receptors which can lead to the removal of alveolar surfactants.
Figure 5: Representative products of phosphatidylethanolamine and phosphatidylserine due to their reactions
with Cl2 and HOCl (Squadrito et al., 2010).
Figure 6: Structures of the potential chlorohydrin biomarkers: (a)chlorohydrin of palmitoyl-oleyl
16 palmitoyl- linoleyl phosphatidylglycerol, (d) chlorohydrin of oleic acid, (e) 9-chloro fatty acid and (f) α-chloro fatty acid (Hemström et al., 2016).
(Ford et al., 2016) detected 2-chloropalmitaldehyde, 2-chlorostearaldehyde and their oxidized products, 2-chloropalmitic acid and 2-chlorostearic acid, it should be noted that the last two were also detected by (Hemström et al., 2016). The levels of these chlorinated lipids were elevated and at their highest after chlorine exposure but then declined after 72 hours. Glutathione adducts were detected at high also at 4 hours in plasma, this happens because glutathione (GSH) leads to a nucleophilic substitution of the above chlorinated species producing glutathionylated adducts.
Figure 7: HOCl oxidation and plasmalogen-derived products
It was shown that free and esterified 2-Cl-PA and 2-Cl-SA levels increased in plasma like in the lung after chlorine exposure, however, their levels decreased over 6-12 hours. The 2 chlorofatty aldehydes that reacted with glutathione formed a lot faster in the lung that in the plasma. Increased levels of inflammatory cells in the BAL were also observed because of the chlorinated fatty acids. Furthermore, for all the chlorinated lipids that have been detected their levels were high immediately after exposure which suggests their direct formation. It should be noted that the esterified 2-chlorofatty acids in plasma have a longer half-life in plasma compare to free 2-chlorofatty acids meaning probably that the esterified species are more stable. Collectively, all the above data indicate that plasmalogens are significant targets for chlorine gas and Cl-lipids should be considered as selective biomarkers for chlorine exposure (Ford et al., 2016).
HOCl in many cases reacts with plasmalogens leading to the formation of α-chlorofatty aldehydes and lysophospholipids that can be further metabolized to α-chlorofatty acid or alcohol. Reactions with the vinyl ether bond have a relatively high constant which means that the reaction between plasmalogens and HOCl is more feasible. However, many inflammatory reactions are attributed to myeloperoxidase (MPO) which is the main reason behind the complexity of finding an unambiguous biomarker (Hemström et al., 2016; Wacker et al., 2013).
17 3.1.4 8-isoprostane biomarker
8-isoprostane, according to (Elfsmark et al., 2018) is linked with oxidative stress in airways and in blood circulation post chlorine exposure. After exposing mice to Cl2, high levels of 8-isoprostane were detected. This compound is closely associated with an early inflammatory cytokine response. Cytokines such as IL-1β, IL-6 are produced and activated by macrophages and are produced by inflammatory reactions (Elfsmark et al., 2018; Zhang and An, 2007). Isoprostanes are linked also with lipid peroxidation, due to their formation on phospholipids at sites of free radical generation. Hydroperoxidases are generated by the lipid oxidation and go through a fragmentation producing a broad range of isoprostane derivatives. Lipids are the main targets of peroxidation and for this reason are used for the estimation of oxidative stress status (Ito et al., 2019).
According to (Elfsmark et al., 2018), 8-isoprostane is highly expressed in BALF and in serum after chlorine exposure indicating a process of ongoing oxidative stress. Concerning the link of chlorine exposure to isoprostane there are two hypotheses, as the exact mechanism is not entirely known. The first one is that the chlorine or hypochlorous acid is some kind of mediator for the peroxidation of arachidonic acid and the other hypothesis is that Cl2 is responsible for the dysfunction of the enzymes that break the isoprostanes (Elfsmark et al., 2018; Yadav et al., 2011). Arachidonic acid is a fatty acid and an integral constituent of cells providing fluidity and flexibility. Isoprostane is a product of free radicals with arachidonic acid that forms on cell membrane disrupting the biological cell membrane.
3.1.5 Glutathione and glutathione sulfonamide biomarkers
(Harwood et al., 2009) examined the possibility of glutathione (GSH) and some of its adducts as a new feasible biomarker for chlorine exposure. HOCl reacts with different factional groups but mostly with thiols as this reaction is faster with a rate constant of 107 M−1·s−1, thus more favorable (Harwood et al., 2006). Glutathione sulfonamide (GSA) is formed when neutrophils and endothelial cells react with HOCl but in lower yields than glutathione disulfide. In the ELF concentrations of glutathione are relatively high making it, a likely target for HOCl. Moreover, GSA is produced in the airways of children with respiratory infections such as cystic fibrosis, thus it has the potential to detect lung infections (Kettle et al., 2014). GSA is a chemically stable product that is formed from the following reaction of GSH and HOCl as seen in figure 8 and it is considered to be a selective biomarker of HOCl
Figure 8: The formation of glutathione sulfonamide (GSA) product, through the reaction of glutathione
18
3.2 Metabolomics-based analysis of chlorine biomarkers
Metabolomics have already been used for the assessment of chlorine exposure providing insight on known metabolites such as chlorotyrosine, but also for the discovery of new biomarkers candidates, like chlorohydrin phospholipids. Moreover, in the upcoming paragraphs, the advances in analytical methods with the most suitable biomarkers are presented and compared. Also, targeted and untargeted metabolomics will be reviewed in detail, for the analysis of potential biomarker candidates of chlorine exposure.
3.2.1 Targeted metabolomics-based approaches
Table 1 presents the studies using targeted metabolomics-based approaches for quantitation
of known metabolites of chlorine exposure:
Table 1: Review of targeted metabolomic studies for the analysis of biomarker candidates of chlorine
exposure. (TFA): trifluoroacetic acid, (ΑCN): acetonitrile, (SPE): solid phase extraction, (LLE): liquid liquid extraction, (LOD): limit of detection, (LLOQ): lower limit of quantification, (ELISA): enzyme linked immunosorbent assay, (DART): direct analysis in real time, (MPA): mobile phase A, (MPB): mobile phase B, (EC-NCI): electron capture – negative chemical ionization
Biomarker candidates
Matrix Sample preparation Analytical
method/Detection
Gradient conditions LOD Volume
injected Reference 3-chlorotyrosine & 3,5-chlorotyrosine Rat nasal tissue
For the chlorotyrosine preparation:
SPE
Cation exchange column (Waters MCX Oasis)
Solvents:
Methanol & TFA Washed twice (TFA) Elution with methanol
For the tissue sample preparation: LLE: 7x10-4 L chloroform & diethyl ether 7x10-4 L Derivatization GC/MS SIM mode capillary column (RTX-5MS, 30 m, mm i.d., 0.25-pm film thickness *0.5min: 100oC
*At 0.5min increased
rate 20oC/min.
*Held for 2 min at 300
oC..
*Total run time 13.75min For chlorotyrosine: 500fg/mol & For 3.5 chlorotyrosine: 1100fg/mol Samples of 1x10-6 L injected (Sochaski et al., 2008) 3-chlorotyrosine, 3,5-chlorotyrosine Whole blood, serum & plasma
SPE HLB 96-well plate
Samples adjusted to pH=1 with TFA Solvents: Conditioned with 1x10-3 L methanol and 1x10-3 L water. Washed with 2% methanol in water (1x10-3 L)
& eluted with 1x10-3 L
methanol UHPLC-MS/MS Column for HPLC : Hypercarb 3 μm, 2.1 × 30 mm SRM mode Mobile phase : (A): MPA: 0,1 formic acid
+ HPLC water (B): MPB: 0,1 formic acid in ACN *Initial: 98% A + 2%B * *1min hold *linear 3min 2%MPA
and 98%MPB *5.01min 98%A + 2%B Cl-Tyr: 0.43ng/ml Cl2-Tyr: 0.396ng/ml Samples of 50 x10-6 L injected (Crow et al., 2016)
20
Flow rate: 250μL/min
MS: Agilent 6490 triple
quadrupole mass analyzer with a Jet Stream ESI
source 3-chlorotyrosine Human tissue specimens SPE Solvents: Wash: 2x10-3 L 0.1% TFA Eluted: 2x10-3 L H 2O: methanol (1:1 vol/vol) Derivatization GC-MS SIM mode 30m DB-17 capillary column Temperature gradient of 20oC/min: 150oC to 250oC Detection limit ≤ 1x 10-16 mole Samples of 1x10-6 L injected (Hazen et al., 1997) 3-chlorotyrosine & 3- nitrotyrosine Human plasma SPE Supelco C18 column (500
mg of sorbent) & Chrom P column (Supelco; 250 mg of sorbent Solvents: 2x10-3 L of 100% methanol Washed with 8x10-3 L 0.1% TFA(pH 5) + sample, washed with
water Derivatization LC-MS/MS analysis (A):(4/95/1, methanol/ water/acetic acid pH=3 (B)(95/4/1, methanol/ water/acetic acid pH=3.2 EC-NCI GC/MS SIM mode Derivatization HPLC column: a C18 column (Zorbax; 5-mm resin, 1x150 5-mm MS: Finnigan LCQ ion trapping instrument GC column: 15-m DB-5ms column MS: HP 5973 mass detector For the LC: For 2 min: 100% A Over 6min: 5-50% B For 4min 50% B For 2 min 50-100% B Over 2 min: 100-0% B Equilibration 100% A for 22 min Flow rate: 50μL/min
For the GC:
180 oC to 300 oC at 40
oC/min
ESI/MS
For 3-chlorotyrosine: Full scan: 54 fmol
SRM: 7,4 fmol For 3-nitrotyrosine:
Full scan: 39 fmol SRM: 3,2 fmol
GC/MS
For 3-chlorotyrosine Full scan: 2,0 fmol
SIM: 0,05 fmol For 3-nitrotyrosine:
Full scan: 6,5 fmol SIM: 0,07 fmol For the LC: 2x10-6 L of sample For the GC: 1x10-6 L of sample (Gaut et al., 2002)
21 3-chlorotyrosine & 3-nitrotyrosine Human plasma Minimal sample preparation: diluted with
50%ACN
DART-MS/MS
MRM mode MS : 5500 triple quadrupole MS/MS
Flow rate : 2,8 L/min Greatest response at 350 oC For chlorotyrosine LOD: 0,1μg/ml LLOQ: 0,3μg/ml For nitro-tyrosine LOD: 0,2μg/ml LLOQ: 0,6μg/ml -(Song et al., 2015)
8 isoprostane BALF &
Serum from mice
BALF & serum:
SPE : affinity column
eluted with ethanol
ELISA Absorbance at 450nm - -Samples 50x10-6 L (Elfsmark et al., 2018)
3-chlorotyrosine Plasma &
white blood cell SPE: C18 column Solvents: Methanol/water/water-washed ether at 4oC Derivatization GC-MS SIM mode -1 fmol 1x10-6 L of sample (Himmelfarb et al., 2001)
3-chlorotyrosine Urine samples SPE
C18 column Solvents: Pre-washed with 2x10-3 L methanol and 5x10-3 L of 0.1%(v/v) TFA/water (pH 5.0)
Washed with water and eluted with 4x10-3 L of
30% (v/v) methanol in water.
For protein precipitation: Chloroform and methanol
Derivatization GC-MS 15m DB-1701 capillary column (0,25-mm internal diameter, 0,25-mm film thickness) Temperature maintained for 1min at
150oC and increasing to 300oC at 20oC/min - 1x10-6 L of sample (Mani et al., 2007)
22 Glutathione (GSH), Glutathione disulphide (GSSG) & Glutathione sulphonamide (GSA) Biological samples neutrophils and endothelial cell & BALF BALF: 2x10-4 μL + N-ethylmaleimide (final concentration 10Mm) Rest for 20 min + labeled
internal standards & 80% cold ethanol + centrifuge for protein
precipitation +
Dry + reconstituted with H2O
LC-MS/MS
Thermo Hypercarb column (100×2.1 mm)
MPA: H2O & 0,5% formic
acid
MPB:
acetonitrile/propan-2-ol (50/50) & 0,5% formic acid
Flow rate: 0,2 mL/min
MS: Thermo Finnigan LCQ
Deca XP Plus ion trap mass spectrometer
100% MPA to 30% MPB over 15min. 5min wash with 100%
MPB Returned to initial conditions LOQ: 0,1 pmol 50 μL of samples (Harwood et al., 2009)
23 3.2.1.1 Sample preparation
The main purpose of the sample pretreatment is to ensure the removal of all the compounds that may interfere with the analytical system and to release the metabolites from the sample matrix. The sample preparation depends mainly on the matrix, the analytical method and detection that is going to be used as well, and the analyte. In recent years numerous sample pretreatment techniques are used like solid phase extraction (SPE), liquid liquid extraction (LLE) and protein removal (Raterink et al., 2014).
In the targeted metabolomics approaches reported in the literature (Table 1), SPE is the most frequently used technique as it has several advantages over LLE. In SPE the sample passes through the stationary phase and the analytes are separated according to the degree they absorbed in the stationary phase. SPE is easier than LLE as it is simpler to separate the liquid from a solid rather than two liquids. Moreover, SPE offers a higher extraction recovery and ensures better clean-up. Other advantages that make SPE more appealing are the low solvent consumption, the increased extraction efficiency, the higher selectivity, the better reproducibility and the removal of interfering substances (Ahadi et al., 2011). SPE consists of mainly 4 steps: equilibration of the column, conditioning, washing and elution. For the conditioning of the C18 column, methanol is used as a solvent in most studies according to Table 1. For the second step of the SPE a small percentage of TFA is used or methanol and for the elution methanol as polar compounds will elute best with polar solvents, like methanol. It should be noted that the most used sorbent for SPE was the C18 column (Crow et al., 2016; Gaut et al., 2002; Hazen et al., 1997; Mani et al., 2007). Furthermore, it needs to be noted that for the analysis of 3-chlorotyrosine with DART-MS/MS that (Song et al., 2015) used, the sample pre-treatment is minimal. Dilution of the samples was achieved with 50% of ACN because this compound is volatile and has low viscosity. The 50% of ACN displayed a higher response of chlorotyrosine providing an efficient reproducibility. Even though this method is fast and offers good reproducibility, it is not sensitive enough.
Moreover, during sample preparation (Crow et al., 2016) in the step of SPE used a hydrophilic lipophilic balance (HLB). In this research different sorbent types of SPE were tested like hydrophilic interaction chromatography (HILIC) on silica and HLB sorbent. Both had similar recoveries, however, the first one required a further step, making it time consuming in contrast with the latter one. Thus, the HLB sorbent offered similar recoveries with HILIC by lowering the pH to 1 with TFA. The main advantage of HLB sorbent over a traditional C18 column, is the extraction and recovery of a broad range of analytes from polar to non-polar and from acidic to basic using a single sorbent. The recoveries that have been achieved were 60% and 77% for 3-chlorotyrosine and 3,5-chlorotyrosine, respectively.
Another study of (Gaut et al., 2002) used also SPE for the analysis of human plasma with LC-MS/MS and electron capture - negative chemical ionization GC/MS. For the sample preparation, a C18 column was used like in the study of (Hazen et al., 1997). The column was conditioned with 0.002L of 100% methanol and washed with 0,008L of 0.1% TFA pH 5. After these two steps, the loading of the sample was followed and washed with 2ml
24
water. The amino acids (3-nitrotyrosine and 3-chlorotyrosine were eluted with 25% of methanol. It needs to be noted that for the GC/MS analysis, a second step of SPE was used to remove any impurities, because human plasma contains contaminants that could interfere with the GC column The recoveries for 3-chlorotyrosine in this procedure was higher than 90%.
Moreover, in the studies where GC/MS was used for the analysis of the amino acids, a step of derivatization was essential. Derivatization is essential in GC when there is a need to detect non-volatile compounds. During this procedure the properties of the analyte change in order to have better separation and sensitivity (Moldoveanu and David, 2018). (Sochaski et al., 2008) used for the derivatization of chlorotyrosine, N-propyl/heptafluorobutyryl whilst, (Gaut et al., 2002) used N-methyl-N-(t-butyldi-methylsilyl)trifluoroacetamide and trimethylchloro-silane (MtBSTFA). Other studies (Mani et al., 2007) and (Hazen et al., 1997; Himmelfarb et al., 2001) for the step of derivatization used ethyl heptafluorobutyrate and heptafluorobutyryl to analyze the chlorinated amino acids with GC-MS, respectively.
One of the major problems of analyzing, GSH and GSSG according to (Harwood et al., 2009) is their oxidation during the process. Thus, a step of blocking is introduced to the sample preparation in order to prevent their oxidation. Therefore, the addition of N-ethylmaleimide (NEM) to BALF samples after collection is essential. NEM blocks the thiol groups and prevents the oxidation of the products during processing. Moreover, the GSH-NEM adduct presents better chromatographic properties than unalkylated GSH.
3.2.1.2 Analysis
The two most frequently used techniques for the analysis of the chlorinated amino acids are GC-MS and LC-MS/MS. However, LC-MS/MS is more sensitive when biological samples are analyzed. Furthermore, it needs to be noted that the common feature in all the studies listed in table 1 is the use of the internal standard 3-[13C6]chlorotyrosine. For a quantitative bioanalytical assay, the most suitable internal standard is a stable isotopically labeled analogue. This analogue has almost identical physical and chemical properties to the unlabeled analyte. Thus, the ratio of the peak area of the analyte to the labeled analogue remains constant despite any alterations in sample processing (Wang et al., 2007). The internal standard is essential to compensate for the errors during sample preparation and to reduce the matrix effects.(Zhou et al., 2017) To confirm detection for each analyte, calibration curves were constructed by plotting the response ratio versus the calibrator concentration.
Sensitivity is one of the most critical issues in a bioanalytical assay because biomarkers candidates typically exist in small amounts in biological samples. For this reason, detection limit is a crucial factor to determine whether a molecule can be identified and quantified. According to (Gaut et al., 2002) EC-NCI GC/MS using HP 5973 mass detector offered a much higher sensitivity of about 100-fold in contrast with LC-MS/MS, using Finnigan LCQ ion trapping. Thus, for an equivalent amount of analyte, the GC-MS should be more sensitive especially for trace amounts of chlorotyrosine. LC-MS/MS required lengthy equilibration time as the time of analysis was 1.5 hour per sample while in GC/MS only 5 minutes per sample were required. Moreover, in this study, they were unable to detect
3-25
chlorotyrosine with LC-MS/MS in human plasma while in GC-MS they were able to detect and quantify trace amounts of this amino acid. This problem could be overcome by analyzing larger amounts of analyte; however, limited amount of biological sample can be introduced to the mass spectrometer.
One high-throughput method was developed by (Crow et al., 2016) using HPLC-MS/MS for the detection and quantitation of two chlorine adducts, 3-chlorotyrosine and 3,5-chlorotyrosine. It needs to be noted that this was the first simultaneous measurement of chlorotyrosine and 3,5-chlorotyrosine in whole blood, serum or plasma with a run time of only 5 min. This method was validated to assess detection limits, precision, accuracy and linearity. Another novel method for the simultaneous determination of 3-chlorotyrosine and 3-nitrotyrosine in human plasma was introduced by (Song et al., 2015). The analysis that was developed used direct analysis in real time-tandem mass spectrometry (DART MS/MS). The main advantage of this method is that limited, or no sample preparation is needed. This method was also validated however the sensitivity was relatively low with limits of detection 0,2μg/ml and 2μg/ml for 3-chlorotyrosine and 3-nitrotyrosine respectively. It needs to be noted, that the sample matrix affected the sensitivity, as the signal did not increase much when the concentration of chlorotyrosine was doubled. intra- and inter-day precisions (RSD) for chlorotyrosine were 4.0%-8.4% and 2.5-5.4% respectively and the accuracies were approximately from -1.7% to7.5%. The RSD precisions for 3-nitrotyrosine were 2.3%–7.9% and 1.1%–5.5% and the accuracies were 3.7% to 0.2%. in almost all targeted metabolomic studies the most attractive detection method is MS as it offers quantitative analyses with high selectivity, sensitivity and the possibility of identification (Dettmer et al., 2007).
For the analysis and detection of GSH,GSSG and GSA in biological samples, (Harwood et al., 2009) used LC-MS/MS. The method that (Harwood et al., 2009) developed for the detection of GSH and its adducts, within a single run was validated and it provided satisfactory results. The accuracy for all three targets was between 83%-113%, the relative standard deviations for intra-day precision<10% and for inter-day precision <20%. It should be noted that the recoveries from BALF were greater than 90%. The extraction recovery was satisfactory, although for greater sensitivity solid phase extraction seems necessary especially for biological samples. Furthermore, for the validation and quantitation of the assay, isotopically labeled internal standards of 10 pmol GSH-NEM, 5 pmol GSSG and 5 pmol GSA were used and the corresponding calibration curves were generated.
3.2.2 Targeted lipidomic Approaches
Table 2 presents the studies using targeted lipidomic-based approaches for the analysis of
26
Table 2: Review of targeted lipidomic studies for the analysis of biomarker candidates of chlorine exposure
Biomarker candidates
Matrix Type of Sample preparation Analytical method/Detection Method LOD Volume
injected Reference Phosphatidylglycer ol chlorohydrins BALF from mice exposed to chlorine LLE
Matyash method MTBE lipid extraction
Solvents:
Methanol, MTBE, MQ water
LC-HRMS MS and MS/MS
(A):80%MQ/20%ACN + 10 mM ammonium acetate (B):90%isopropanol/ 10%ACN/1%1M
ammonium acetate Flow rate: 300nL/min
Column:C18 column (150 × 0.075 mm, 2 μm particles) MS: Bruker Impact HD Qq-TOF 0-3min: 90%A /10%B 3-15min: 10-95%B 15-25min: 95%B 25-26min: 10%B <500pM 5μL (Hemström et al., 2016) 2-chlorostearaldehyd e, 2-chloropalmitic acid, and 2-chlorostearic acid Lungs & plasma
LLE: for plasma and lung
Bligh and Dyer method Solvents: methanol: water (1:1 v:v)
LC-MS/MS
C18 column (50 × 2,0 mm)
Flow rate: 200μL/min MPA: 0,15% formic acid + MQ water MPB: acetonitrile-isopropanol (3:2, v/v) + 0,15% formic acid MS: triple quadrupole 0-2min: 65% MPA 35% MPB 2-5min: 100%MPB 25μL (Ford et al., 2016)
27 3.2.2.1 Sample preparation
The biological samples in targeted lipidomic studies as seen in Table 2 require LLE for the extraction of lipids. in contrast with targeted metabolomic approaches, where SPE was the tool of extraction in most cases. The sample pre-treatment includes also, separation of low molecular weight compounds from lipids and proteins using delipidation or deproteinization techniques (Vuckovic, 2012).
In (Hemström et al., 2016) study for the sample preparation of lipids, liquid liquid extraction (LLE) was used and more specifically Matyash method. The upper layer in this method is the lipid organic phase and the main objective of this extraction method is the increased number of extracted lipids that can be detected (Gil et al., 2018). Moreover, MTBE extraction provides faster and cleaner lipid recovery minimizing any dripping losses. This extraction method has also similar or even better recoveries compared with other standard protocol extractions like Folch or Bligh and Dyer. These techniques use chloroform as solvent which is toxic and carcinogenic whilst MTBE is not. One of the main advantages using MTBE, is the higher polarity in contrast with chloroform and the higher capacity for solubilizing water. This improves the extraction efficiency of acidic lipids. MTBE has a lower density than water, thus it exists in the upper of the two-phase extraction whilst, chloroform has higher density than water, so it appears at the down layer. For this reason (Hemström et al., 2016), for the lipid extraction used MTBE, as it has less chances of contaminating the sample during extraction, Another advantage of MTBE, is that it is stable and there is no danger of degrading lipids (Eggers and Schwudke, 2016; Matyash et al., 2008). The phase separation was achieved after the addition of water followed by vortexing and centrifuging. The next step was the collection of the supernatants which were pooled, and the samples were extracted again.
In contrast with (Hemström et al., 2016), (Ford et al., 2016) for the LLE of the lipids used the Bligh and Dyer method in the same way as (Wacker et al., 2013). This method was first introduced by Bligh and Dyer in 1959 to isolate lipid fractions using a solvent consisting of chloroform/methanol/water (2:2:1.8, v/v/v) (Sündermann et al., 2016). According to (Wacker et al., 2013) the first step for the lung tissue was the homogenization with the addition of internal standard followed by vortex. The next step was the addition of methanol and chloroform while in the standards were added methanol. After that (Wacker et al., 2013) continued with the addition of chloroform and saline in order to extract and re-extract the upper phase until the lower chloroform layer would become clearer. Moreover, the aqueous layer was saved because the GSH adducts remain to this layer and for the organic layer, it was washed with methanol and water. At the end, dilution of the combined aqueous layer was followed by water and extracted. Finally, the sample preparation of internal standard of 3-[13C9]chlorotyrosine was achieved using solid phase extraction with a C18. The equilibration of the column was achieved with 0.1% TFA in water and for the elution 0.1%TFA in methanol (Ford et al., 2016).
3.2.2.2 Analysis
(Hemström et al., 2016)used for the analysis, liquid chromatography-high resolution mass (LC-HRMS) with nano ESI spray ion ionization. In this study, they exposed mice to
28
chlorine gas and detect chlorinated biomolecules that are present in the BALF. For the identification of L-α-phosphatidyl-DL-glycerol and L-α-phosphatidylcholine, a mix of phosphatidylglycerol chlorohydrins was used as a reference standard. (Hemström et al., 2016) found two plausible candidates, chloro-stearic and chloro-palmitic acids that were also identified by (Ford et al., 2016). Moreover, the identification of two other unknown compounds was achieved, which were palmitoyl-oleyl phosphatidylglycerol and palmitoyl-linoleyl phosphatidylglycerol as seen in the figure 6.
The identities of the two biomarkers were confirmed by the reference standards as they had identical MS/MS spectra and retention times. However, due to the ion suppression, the sensitivity was low, and the limit of detection was estimated below 500pM. A positive ion mode was also tried; however, the absence of a mobile proton did not offer a satisfactory fragmentation and it was not favored. Furthermore, one of the most challenging issues was the quantification of the phosphatidylglycerol chlorohydrin biomarker as there was no isotopically labeled internal standard. The only feasible way to achieve this was by using synthetized chlorohydrin of palmitoyl-oleyl phosphatidylglycerol (Hemström et al., 2016).
(Ford et al., 2016) suggested two other possible biomarkers which were glutathione adducts of 2-chloropalmitaldehyde (2-Cl-Pald) and 2-chloropalmitic acid (2Cl-Sald). Increased levels of these two chlorinated lipids were detected in lungs and plasma of mouse and rat models. For all free chlorinated lipids high levels were increased immediately after exposure but were decreased and undetectable after a few hours. It should be noted, that in this study the detection of 3-chlorotyrosine was achieved but the levels of this amino acid were declined 12-24 hours post exposure. However, the chlorofatty acids provided higher sensitivity as they remained at relatively high levels after 24 hours post exposure.
Figure 9: Exposure of400ppm for 30 min of Cl2 in rats. (A,B): Levels of free and esterified chlorofatty acids in the lungs, (C,D): Levels of free and esterified chlorofatty acids in plasma. Data are mean ± SEM (n = 3). *P < 0.05 relative to air by t-test (Ford et al. 2016).
