Determination of polycyclic aromatic hydrocarbons in blood plasma of neurology patients

The etiology of many neurological cases cannot be easily delineated, making the investigation, and treatment to be challenging. This study aims to screen the blood of neurology patients presenting for the first time in the hospital for Polycyclic Aromatic Hydrocarbons (PAHs) using Flame Ionization Detector – Gas Chromatography. Fourteen PAHs were detected in the samples. The results showed that flourene and phenanthrene were common to all the patients in the range (1.37 to 8.08 and 1.66 to 8.34 ng/mL respectively), but were not detected in the control samples. Pyrene, fluoranthene and acenaphthene were present in 80, 75 and 70% in the blood plasma of the patients at the range of 2.96 to 236.86 ng/mL, 1.96 to 11.55 ng/mL and 1.08 to 1.81 ng/mL respectively. These were not found in the control samples. The body burden of these congeners was much higher in neurology patients than controls and literature values of similar study. The concentrations detected were statistically significant, and could be possible causative agents. This can also become one of the investigative tools for these diseases.


INTRODUCTION
Polycyclic Aromatic Hydrocarbons (PAHs) are a large group of ubiquitous and environmentally persistent organic compounds.They are formed by thermal decomposition such as in burning of coal, exhaust engines, garbage, wood, or other organic substances such as tobacco and smoked fish or meat (ATSDR, 1995;Pleil et al., 2010).They are present in air, water, food, dust, soot and so represent a constant flow level of exposure to human via skin contact, inhalation and ingestion (Farhadian et al., 2011).
They are chemically related contaminants that are of various structures and varied toxicity.They exist as volatile, semi-volatile and non-volatile organic compounds.They are ranked number nine in the priority *Corresponding author.E-mail: ioolabanji@yahoo.com.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License list of hazardous substances.Over the years Polycyclic Aromatic Hydrocarbons (PAHs) have received much attention because they are suspected to be carcinogenic and they also induce human inflammatory mediated diseases (CDC, 2009;Sexton et al., 2007;Lewtas, 2007;Schober et al., 2007).They have been implicated in adverse reproductive outcomes, somatic mutations and decrease in children's IQ (Sram et al., 1999;Perera et al., 2002).Inhalation of PAHs from diesel engine exhaust has been linked with pulmonary cytokine expression (Sobus et al., 2008;Swanson et al., 2009).Due to their carcinogenic activity, PAHs have been included in the European Union (EU) and the United States Environmental Protection Agency (USEPA) priority pollutant lists.
The body burden of PAHs had been assessed in urine (Hansen et al. 2008).The analysis for PAHs as potential biomarker and measurement of aliphatic hydrocarbon, single ring aromatic compound as well as analysis of PAHs in blood headspace had been reported (Chambers et al., 2008;Kim et al., 2006;Gyorffy et al., 2008).Pleil et al. (2010) analyzed blood and plasma of healthy people to determine PAHs and found them at varying concentrations.
Neurology disease is a disease that affects the nervous system, structural, biochemical or electrical abnormalities in the brain, spinal cord and nerves.Symptoms may include paralysis, seizures, confusion, loss of sensation and many others.Since PAHs had been implicated of reducing children's IQ (Perera et al., 2002) and neurology diseases affect the brain and the nervous system, it is pertinent to investigate whether there is a link between the pollutant and the disease.
In this study, blood plasma of twenty neurology patients and 12 plasma of healthy subjects were analyzed for PAHs to determine how the concentration levels compared with literature values of healthy subjects.
The concentration levels will give an assessment of the PAHs toxicity levels in the patients compared to literature values of healthy subjects and the healthy subjects used as controls.

MATERIALS AND METHODS
The out patients neurology clinic of the Obafemi Awolowo University Teaching Hospital, Ile-Ife, Nigeria was used for sampling twenty neurology patients experiencing it for the first time

Sampling
Ten milliliters of whole blood was collected via the vein using syringe and needles in heparinized bottle and transferred to the laboratory and centrifuged.The plasma was immediately transferred to 20 mL capacity amber bottles to prevent sample degradation, PAHs being UV lightsensitive.These were stored in the refrigerator at 4°C prior to extraction.

Sample extraction and clean up
Liquid -liquid extraction technique was employed using ratio 4:1 nhexane and Dichloromethane (DCM).The extractant (10 mL) and plasma samples (2 mL) were added in the vials, capped and vortex for 20 s at 300 rpm.The organic layer was sucked out using pipette attached with pipette filler into clean and thermally treated amber bottles.The extracts were cleaned up in a column (1 cm x 15 cm, internal diameter and length) with slurry of silica gel as stationary phase, preconditioned by distilled water and hexane/DCM before the samples were eluted, collected and concentrated in a stream of nitrogen gas.

Sample analysis
Samples were analyzed after reconstitution with hexane/DCM at Nigerian Institute of Oceanography and Marine Research (NIOMR) Lagos.The standards of the congeners were supplied and analyzed and used to identify and quantify the PAHs in the samples.The corresponding relative retention time with respect to each congener was used to identify each PAH by Gas Chromatography Flame Ionization Detector (GC-FID) using Agilent Model 7890A (Agilent Technologies, Delaware, MD.USA).Separation was carried out in a GC column HP-5 (Agilent; 30 m × 320 µm × 0.25 µm film thickness) using a temperature programmed GC for elution of analytes.The column temperature was set initially at 60°C and held for 1 min.This was ramped to 210°C at 12°C/min and increased to 320°C at 8°C/min held for 5 min.The total run time was 32.25 min and the detector temperature was 325°C.The flow rate was 1.2 ml/min with injection volume of 1 µL.The inlet mode was splitless with temperature of 270°C.

PAHs concentrations
All twenty patients sampled were found positive to PAHs and exhibited wide variations in concentrations.The United States Environmental Protection Agency (USEPA) classified 16 PAHs as priority pollutants because they are common in environmental media, toxic and they can easily be absorbed by humans (EPA, 2010).Fourteen out of these sixteen on EPA priority list were detected in the neurology patients' plasma and only six PAHs were detected in the plasma of control subjects.Out of the twelve healthy subjects, six were positive to pyrene, two to chrysene, two to Benzo[b]fluoranthrene, six to Benzo[k] fluoranthrene and three were positive to Benzo(g,h,i)perylene which was not detected in the patients.Table 1 shows the mean concentrations of various congeners in the samples.The result shows that, 18 of the patients (80%) had pyrene at varying concentrations in the plasma, 75% had fluoranthrene and 70% had acenaphthene.Fluorene and phenanthrene were present in all the patients screened.With the exception of acenaphthene, the above four analytes had Those that are more than four rings are heavier, because they are particulates and tend to adsorb on the environmental media, while the PAHs that are less than four rings tend to remain in the gaseous state until removed via precipitation.The majority of PAHs analyzed in this study are less than or equal to four rings (about ten), hence, inhalation may likely be one major route of absorption by the patients due to PAHs emission while drinking of shallow well water might be a minor source because of their low solubility in water.Leaching of bitumen used in road construction to surface water could also be an additional source.
The mean concentrations of the PAHs in all the patients are summarized in Figure 1.Pyrene concentration constitutes 43% of all the PAHs concentrations in all the patients, being found in 18 patients.The geometric mean of plasma concentrations of pyrene in this study was 11.33 ng/mL with maximum concentration of 236.86 ng/mL.Its urinary metabolite, 1-hydroxypyrene, had been used as an indicator of exposure to PAHs chemicals (Popp, 1997;CDC, 2005).This metabolite was recommended for measurement as end-of-work-week urine samples by The American Conference of Governmental Industrial Hygienists (ACGIH) as biological exposure index (BEI) for assessment of exposure to mixtures containing PAHs (Heikkila et al., 1995;ACGIH, 2005).
Since the relative amounts of PAH congeners in mixtures vary from place-to place, this does not provide direct information on the relationship between exposure to pyrene and urinary 1-hydroxypyrene concentrations (Alghamdi et al., 2015).Like most PAHs, pyrene is used to make dyes, plastics and pesticides.It has also been used to make another PAH called Benzo(a)pyrene (ATSDR, 1990;Faust, 1993).Pyrene as a common chemical in materials that are daily been used could make people to be more exposed, lifestyle of the patients and occupation could also play a major role in high percentage constitution.
As shown in this study, the statistical concentrations of PAH's found in the patients, and an earlier study of regular subjects by Pleila et al. (2010) and Seracare (the reference blood sample)', it is evident that the body burden of PAH's in the blood of the neurology patients is higher.The individual value in this study measured at ng/mL as compared with the literature values in (Table 1) measured in pg/mL showed the high concentration at which the PAHs were present in the neurology patients.
Figure 2 shows the levels of the difference, what was found in the neurology patients (Blue colour) were higher than the literature values.The bio accumulative effect of the volatile and particulate forms of these PAHs may be capable of neurological damage.Rappaport et al. (2005), in their study, proposed Naphthalene as an alternate biomarker for the determination of PAHs in the body and the metabolites 1and 2-hydroxynapthalene in urine as an alternative to 1hydroxypyrene.Since naphthalene (two ring PAHs) sublimes into gaseous state, it will be a suitable biomarker for industrial exposure to airborne PAHs mixtures.AIOH (2016) suggested that where elevated levels of lower ring number PAHs may be present, the monitoring and assessment could be done using Naphthalene.A proximate matrix of the mixture of PAH was done using the mean concentration of the PAHs, naphthalene correlated with Phenanthrene (0.647), bitumen used in road construction to surface water could also be an additional source.Pyrene (0.595) and Benz(a)Pyrene(0.466)(Table 2).This may be a pointer that the source of absorption is not predominantly through inhalation, but may be dermal, food and air borne.According to Rappaport et al. (2004) where there is a mixture of dermal and airborne exposure, an alternative marker correlating better with the higher number ring compounds could be more suitable as biomarker.Thus, any of the following three could be used as biomarker; Phenanthrene, Pyrene and Benz (a)Pyrene as corroborated by Sobus et al. (2008).Sobus et al. (2009) in their study concluded that levels of naphthalene and phenanthrene in urine reflect airborne exposures to these compounds and are promising surrogates for occupational exposures to PAH mixtures.The control samples used in this study were blood plasma of undergraduate students (majorly, clinical students, >20 ≥30 years); all the three proposed biomarkers were absent in the control samples.This suggests that the occupation of subjects may play major role in the exposure and concentrations of these congeners in the body.In this study, blood plasma was used similar to urine in the sense that, they are both body fluid that represent the current state or on-going activities in the body.Seidel et al. (2008) determined metabolites of phenanthrene in post shift urine of occupational exposed worker (and concluded that phenanthrene was also a reliable biomarker for PAH exposure).The higher member ring, especially, B[a]pyrene; a potential carcinogen correlates with 5 other PAHs (Naphthalene, Pyrene, Benzo (a) Anthracene, Chrysene and benzo[k]fluoranthrene), of which Benzo (a) Anthracene, Chrysene and benzo[k]fluoranthrene were classified as possible carcinogens (IARC, 2010).This suggests that they have common possible source.The high values of these PAHs give course for concern as they have been implicated as being carcinogenic.

Risk assessment
The Toxicity Equivalency Factor (TEF) methodology was developed by the U.S. Environmental Protection Agency (EPA) to evaluate the toxicity and assess the risks of a mixture of structurally related chemicals with a common mechanism of action.A TEF is an estimate of the relative toxicity of a chemical compared to a reference chemical   (Ramírez et al., 2011;WHO, 2000)  Chrysene, Dibenzo(a,h)anthracene, fluoranthene, indeno(1,2,3-cd)pyrene, phenanthrene and pyrene whose BaP eq should not be more than 0.1 µg/L.Only Benzo (g,h,i)perylene and indeno(1,2,3-cd) pyrene were not detected in this study.Although the PAHs mixture were more than 10 congeners in this study, only 8 of the congeners were used in the calculation and the concentrations totaled 14.459 ng/mL or 14.459 µg/L which is well above the expected value of 0.1 µg/L.The mixture also reflected all classes of classification of PAHs ranging from carcinogenicprobable carcinogennot classifiable.Napthalene has a maximum Concentration limit of 70 µg/L in water (since there is no limit set for PAHs in the blood by any standard organization), but the mean value in this study was 6.17 µg/L.Maximum Contamination Level(MCL) of some PAHs in water given by USEPA (1984) were 0.2 ng/mL for Benzo(a)pyrene, Benzo(b)fluoranthene, Benzo(k)fluoranthene and chrysene while Benz[a]anthracene and Dibenzo(a,h) anthracene were given MCL of 0.1 ng/mL and 0.3 ng/mL respectively.The mean values for these PAHs were found to be higher in the plasma of the neurology patients.The non-classifiable member of PAHs is capable of causing problem(s) at a high concentration which may not be known for now.

Conclusion
The study shows that the concentration levels of the PAHs detected in the plasma samples of the neurology patients are significantly higher at 95 % level than the control samples.The MCL values provided for water and the literature values of the similar research were lower in concentration than what was found in the neurology patients.Volatile PAHs are most prevalent in the samples and there was high correlation in the concentrations of the volatile congeners, thus, they could be used as biomarkers in the blood/plasma analysis.This result represents a veritable baseline study of PAHs in Neurology Patients in the country.Further work could be done in assessing the level of these PAHs in the same group of patients as they continue their treatment, and also to further establish the actual roles of these congeners in the causation of these neurological diseases.This can further open up a new vista in therapeutic approaches to treatment.

Figure 1 .
Figure 1.Percentage composition of PAHS in blood of neurology patients.
was used to estimate the Excess Lifetime Neurology Risk due to PAH exposures.Benzo (a) pyrene equivalents (BaP eq ) were determined by multiplying individual PAH concentrations with the corresponding TEF developed by US EPA, 2010 and Nekhavhambe (2014) based on the toxicity equivalent factor of individual PAH.Thus: BaP eq = (Cn x TEFn) Total Toxicity Equivalent factor = Where, Cn = individual PAH concentration in the complex mixture, TEFn = toxic equivalent factor of individual PAH, and k = total number of PAH compounds The total toxicity of the congeners was calculated on the basis of the method for calculating the concentration of 10 group of PAH to reflect a toxicity equivalent factor (TEF) based on benzo(a)pyrene in order to normalize the toxicity.Adding the concentrations of the 10 congeners together directly will lead to over estimation of the toxicity (WDNR, 2015), thus the use of toxicity equivalent factor.The 10 PAHs include Benzo(a)anthracene, Benzo(b) fluoranthene, Benzo(g,h,i) perylene, Benzo(k) fluoranthene,

Table 1 .
Statistical comparison of the concentrations of PAHs in plasma of neurology patients with the normal subjects in the literature.
Skupinska et al. (2004)stated that size influences the fate of the gaseous PAHs mixtures.

Table 2 .
Proximity Matrix of Mixture of PAHs in Neurology Patients.

Table 3 .
Risk Assessment Table of PAHs in ng/mL.