Chromatographic analysis of methyl hippuric acid metabolite in the urine of petroleum workers and evaluation of cytogenetic changes of epithelial cells in buccal mucosa smears

doi:10.21203/rs.3.rs-3770949/v1

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Chromatographic analysis of methyl hippuric acid metabolite in the urine of petroleum workers and evaluation of cytogenetic changes of epithelial cells in buccal mucosa smears

https://doi.org/10.21203/rs.3.rs-3770949/v1

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Purpose: Millions of workers in various occupational settings face potential exposure to hazardous substances, including a wide array of compounds found in petroleum derivatives. Among these compounds, toluene and xylene are prominent examples known for their genotoxic properties. Toluene and xylene, both belonging to the aromatic compound family, give rise to the main metabolic byproducts of hippuric acid, (o, p, m) methyl hippuric acid.

Methods: HPLC approach is used to analyze the urine of petroleum workers who are exposed to toluene and xylene to assess their concentrations of the metabolites. Hippuric acid and (o, p, m) Methyl hippuric acid. The micronucleus assay of buccal mucosa smears can be used to evaluate the genotoxic effects of these exposures in petroleum workers. Occupational exposure to petroleum and its derivatives may cause DNA damage and chromosomal abnormalities in buccal mucosa cells, indicating a potential increased risk of cancer and other health effects.

Results: The Method was developed for Hippuric acid and (o, p, m) Methyl hippuric acid of urinary samples of petroleum workers. The urinary HA, (o, p, m) MHA levels in cases were found to be 26.042 µg/ml with standard deviation value is 12.38. The study focused to examine the cellular alterations in exfoliated oral epithelial cells, particularly cytomorphometry and micronuclei, which were examined using PAP-stained smear to determine the existence of micronuclei.

Conclusions: The urinary HA, (o, p, m) MHA levels were highly significant and the prevalence of the condition was found to be greater among petroleum workers compared to the control population. It is observed that more micro nuclei were found in buccal mucosa of petroleum workers than control subjects. Hence it is suspected to induce toxicity in petroleum workers.

Hippuric acid

m) Methyl hippuric acid

Heptadecanoic acid

Micronucleus assay

The workplace has a significant impact on an individual’s health, which is highly valuable. Environmental pollution is mostly caused by air, noise, heat, and radiation, particularly in metropolitan regions. Hazardous compounds might potentially be exposed by millions of workers in a range of occupational contexts [1, 2]. Petroleum derivatives are crucial in determining the health risks to employees. BTEX (Benzene, Toluene, Ethylbenzene, Xylene) chemicals, which are frequently present in crude oil and petroleum products, are known to have harmful effects on health, including cancer, neurological diseases, and respiratory ailments. Due to their employment activities including the extraction, transportation, and processing of petroleum products, petroleum workers are more likely to be exposed to BTEX chemicals which is called volatile organic compounds (VOC’S) [3–6]. Petroleum derivatives constitute a complex mix of chemicals and are well known genotoxic agents especially toluene and xylene. Toluene and xylene are both hydrocarbons that belong to the family of aromatic compounds. They are quite similar in terms of both features and uses, but they also differ significantly in certain keyways. Toluene is a colourless liquid that is also highly flammable and has a sweet odour with Chemical formula C₇H₈. Several different chemicals and materials, including paints, coatings, adhesives, rubber, and plastic, are produced using it as a solvent. Besides from being a feedstock for the creation of other compounds like benzene and phenol, toluene is also employed as a gasoline additive [7, 8].

Xylene is a colourless, sweet-smelling, highly flammable liquid with a molecular formula of C₈H₁₀. It belongs to the class of aromatic hydrocarbons and is naturally present in coal tar and petroleum. The manufacturing of insecticides, dyes, and plastics all utilize xylene as a solvent, as do the printing, rubber, and leather industries. Xylene is available in three isomeric forms, ortho-xylene, meta-xylene, and para-xylene, which have slightly different properties and applications [9].

Hippuric acid and methyl hippuric acids are the principal metabolite of toluene and xylene. Ortho-, meta-, and para-xylene are the three isomers of xylene and the chemical characteristics of these compounds are shown in below Table 1 [10, 11]. The two methyl groups on the benzene ring are in different positions in these isomers. An HPLC approach is used to analyze the urine of petroleum workers who are exposed to toluene and xylene to assess their concentrations of the metabolites hippuric acid & (o, p, m) Methyl hippuric acid [12–15].

Table 1

Chemical characteristics of standard compounds
SL NO	VOC’S	Molecular Formula	Molecular weight (g/mol)	Melting point (°C)
1.	HA	C₉H₉NO₃	179.173	187–191 °C
2.	O- MHA	C₁₀H₁₁NO₃	193.2	161–164°C
3.	P- MHA	C₁₀H₁₁NO₃	193.2	160°C
4.	M- MHA	C₁₀H₁₁NO₃	193.2	160°C

The micronucleus assay of buccal mucosa smears can be a useful tool for assessing the genotoxic effects of occupational exposures, such as petroleum work [16]. Petroleum workers may be exposed to various genotoxic agents, including polycyclic aromatic hydrocarbons (PAHs), benzene, xylene and other chemicals [17, 18]. These exposures have been associated with an increased risk of cancer and other health effects. The micronucleus assay of buccal mucosa smears can be used to evaluate the genotoxic effects of these exposures in petroleum workers [19].

Occupational exposure to petroleum and its derivatives may cause DNA damage and chromosomal abnormalities in buccal mucosa cells, indicating a potential increased risk of cancer and other health effects. Exfoliated oral buccal cells have been effectively employed non-invasively to demonstrate the genotoxic consequences of different lifestyle choices, medical procedures, and exposure to possibly cancer-causing and/or mutagenic substances at work [20].

Toluene Metabolism

The primary method of toluene metabolism in the liver is oxidation by cytochrome P450 enzymes. Benzyl alcohol, which is further oxidized to benzoic acid and eliminated in urine as hippuric acid, is the main by-product of the metabolism of toluene as shown in Fig. 1.

Toluene can be metabolized by additional pathways as well, such as conjugation with glutathione and sulphate as well as through the creation of reactive intermediates that can lead to DNA mutations and cellular damage. Thus, there are several routes that can be involved in the metabolism of toluene after inhalation, which can result in the creation of reactive intermediates and the emergence of a variety of adverse health consequences.

Xylene Metabolism

It is a group of three isomeric volatile organic compounds, including ortho-xylene, meta-xylene, and para-xylene. Xylene is largely processed in the liver by cytochrome P450 enzymes by oxidation. Methyl benzoic acids are the primary by-products of xylene metabolism. These acids are then further converted to hippuric acid and eliminated in the urine. Xylene can be subjected to oxidation as well as conjugation with glucuronic acid or sulphate, resulting in the creation of water-soluble metabolites that can be eliminated in urine [21] as shown in Fig. 2. Nevertheless, depending on exposure levels and other variables, the specific metabolites that are produced may vary between the three isomers of xylene they are (o, p, m) MHA as well as the proportional contributions of other metabolic pathways [22]. The metabolism of BTEX compounds can lead to the formation of reactive intermediates, which can cause damage to DNA and other cellular components. This can contribute to the carcinogenic effects and other possible health impacts of BTEX compounds. It is important to minimize exposure to these compounds to reduce the risk of adverse health effect [23–25].

chemicals and reagents

Hippuric acid and (o, p, m) Methyl hippuric acid and Heptadecanoic acid (Internal standard) was procured from sigma Aldrich and Heptadecanoic acid is diluted with acetonitrile. Ethyl Acetate, Isopropyl alcohol and 0.5N Hydrochloric acid 37% was procured from Loba Chemie Pvt Ltd. HPLC grade Acetonitrile, 0.025% Glacial acetic acid and methanol procured from Merck Life Sciences Pvt Ltd. Millipore water was procured from Millipore Direct Q® 3 Water purification system used as a mobile phase and is filtered using membrane filter (0.45µm). Millipore water was used as diluent for solution preparation.

Sample collection

The urine samples were collected from the petroleum workers at 9AM before exposure to the petroleum vapours and again collected at 5PM after the inhalation of petroleum gas in the petroleum stations shown in Table 2. The urinary hippuric acid and (o, p, m) methyl hippuric acid were analysed by HPLC technique. Buccal mucosa smears were collected randomly between working hours of petroleum workers. It can be collected using a cytobrush, which is a small, soft-bristled brush that is designed to gently scrape cells and the workers are asked to rinse their oral cavity with water to remove any debris or food particles that may interfere with the collection of the buccal cells. The cytobrush is then gently inserted into the mouth, with the bristles facing the inside of the cheek. The brush is rotated gently against the cheek for several seconds, to collect a sufficient number of cells and transferred to glass slide, later it should be placed in coplin jar which containing isopropyl alcohol help for fixation of cells for micronucleus assay. Urine and oral buccal mucosa smears were obtained from both groups of participants consisting of 30 petroleum workers and 30 healthy individuals.

Table 2

Petroleum bunks visited to collected urine and buccal mucosa smear sample of petroleum workers
Petrol bunk name	Address line	City	State
Kodagalli Service station	KRS road, Metagalli	Mysuru	Karnataka
Simhadri HP petrol bunk	KR mill colony	Mysuru	Karnataka
Indian oil	Siddhartha layout	Mysuru	Karnataka
Shell fuel station	Gayathripuram	Mysuru	Karnataka
J.N. Khan Fuel Dome	Udayagiri	Mysuru	Karnataka
Essar petrol bunk	Mahadevipura	Mysuru	Karnataka
Sri Vara Ganapathi Fuel Station	Belvathagrama	Mysuru	Karnataka
Indian oil pump	Rajendranagar	Mysuru	Karnataka
HP petrol pump	Bannimantap	Mysuru	Karnataka

PREPARATION FOR HPLC

Selection and preparation of mobile phase

Hippuric acid and (o, p, m) Methyl hippuric acid are polar compounds. The polar nature of these compounds makes them soluble in polar solvents such as water, methanol, acetonitrile. The number of mobile phases in mixture of ratios were attempted for the mobile phase selection. The Hippuric acid and (o, p, m) Methyl hippuric acid APIs were injected with different ratios of MP in combination and flow rate for the optimization of peak. The procedure was continued until sharp peak appearance. The sharp peak appeared at 84:0.025:16 (v/v) of HPLC grade Acetonitrile, Glacial acetic acid and Millipore water.

Standard stock solution preparation

A stock solution with a standard concentration of 100µl/ml was prepared by dissolving 10mg of both hippuric acid and (o, p, m) methyl hippuric acid in 10ml of Millipore water, which was diluted with additional Millipore water. Heptadecanoic acid (Internal standard) was prepared by weighing 10mg and dilute in 100ml acetonitrile to become (100µl/ml). The prepared solution was protected using aluminium foil and kept in refrigerator till analysis at 4ºC.

Sample preparation

A 1ml of urine sample and 1ml Millipore water were pipetted into 50ml beaker, 200µl of heptadecanoic acid (Internal standard) was added. To acidify the mixture, 200µl of 0.5N H₂SO₄ was added and 3 ml of ethyl acetate was added in the mixture and kept in horizontal shaker for 10 min at room temperature. Then the mixture was transferred to centrifuge tube and kept for centrifugation at 4000rpm for 4 minutes. Further the organic phase was transferred to RBF (round bottom flask) and evaporated by rotary evaporator at 50°C until the residue sticks to the bottom surface of the RBF. Then 1 ml Millipore water is added to the residue and transferred to HPLC vials through 0.02µ syringe filter and solution is injected into the HPLC system.

Preparation for micronucleus assay

The collected samples were then placed on the slides, fixed in isopropyl alcohol, and coded for identification of micronuclei. Papanicolaou (PAP) staining was used to stain these smeared slides. Bright field microscopes at 40X magnifications were used to examine the coded slides, to identify the cytomorphometric alterations of exfoliated epithelial cells. Exfoliated oral epithelial cells were analyzed using technique called micronuclei assessment to investigate cellular modifications.". Bright field microscopy was used to study 100 cells in a zigzag pattern on each slide. Precautions must be made to prevent repetitive or overlapping cells.

Method was development for Hippuric acid and (o, p, m) Methyl hippuric acid of urinary samples of petroleum workers.

Liquid chromatographic conditions

Table 3

Optimized Chromatographic conditions of Hippuric and (o, m, p) Methyl hippuric acid
CHROMATOGRAPHIC CONDITIONS
Instrument	Shimadzu Prominence-i LC-2030C Plus
Column	Phenomenex Luna C18 column (5 µm particle size, L ×I.D. 25cm×4.6mm)
Flow rate	0.7 ml/min
Run time	14 min
Injection Volume	10 µL
Wavelength	254nm
Diluent	Acetonitrile
Mobile Phase A	Acetonitrile
Mobile Phase B	0.025% Glacial acetic acid in Milli pore water
Mobile Phase ratio	Acetonitrile: Glacial acetic acid: milliQ water (84:0.025:16)
Column oven Temperature	40°C
Detector	UV

The order of elution observed were as follows the methyl esters of HA, followed by o-, m-, p-MHA, and heptadecanoic acid (used as an internal standard).

The above Figs. 3 and 4 shows the standard and sample chromatograms of Hippuric acid and (o, p, m) Methyl hippuric acid peak elution at different retention time HA is at 4.739, o- MHA at 6.485, p- MHA at 7.536, Heptadecanoic acid (Internal standard) at 11.308 and m-MHA is at 13.441.

In the present study, the urinary HA and (o, p, m) MHA levels in cases were found to be 27.28 µg/ml with standard deviation value is 12.38. Whereas in controls were found to be 6.53 µg/ml with SD value of 5.09 as shown in Table 4 and Fig. 5. The urinary HA & (o, p, m) MHA levels were highly significant and the prevalence of the condition was found to be greater among petroleum workers compared to the control population. The urinary HA & (o, p, m) MHA levels were higher in case of after shift (4 PM) of sample collection than the before shift at (9 AM).

Table 4

Urinary HA and (o, m, p) MHA levels in cases and control after and before shift of petroleum work.
Urinary HA & MHA	Subject	Mean	SD	Highly Significant
	Cases	27.28	12.38
	Control	6.53	5.09

Cytomorphometric analysis were conducted on exfoliated epithelial cells obtained from buccal mucosa smears.

Micronuclei were observed in exfoliated oral epithelial cells obtained from PAP-stained smears. Exfoliated oral epithelial cells were analyzed for cellular changes, including micronuclei and cytomorphometry, by examining 100 cells per slide in a zig-zag pattern under bright field microscopy. The study continued, the number of micro nuclei per 100 cells were analysed in bright field microscope and found to be 47.45 in cases of petroleum workers with standard deviation of 12.83. Whereas in control were found to be 7.24 with SD value of 3.03. The micro nucleus of epithelial cells of buccal mucosa were highly significant and high in cases than control population shown in Fig. 6.

Table 5

Comparing the mean of micro nuclei per 100 cells in oral epithelial cells of buccal mucosa smears
Micro Nucleus Assay	Groups	Mean	SD	Highly Significant
	Cases	47.45	12.83
	Control	7.24	3.03

As the study conducted above Fig. 8 shows that the 20 cases (petroleum workers) were included in the study to monitor their inhalation of toluene and xylene by workers at different shifts (before and after) to know the concentration of HA & (o, p, m) MHA inhaled by the workers which implies increase in the concentration of HA & (o, p, m) MHA analysed by HPLC method as shown in the Graph. Whereas the after-shift cases were found to be highly significant than control population and also increases in the micro nucleus of epithelial cells in buccal mucosa smears were found to be highly significant than control population and the cells were visualized in bright field microscope for micro nucleus assay shown in Fig. 7.

System suitability

System suitability parameters were determined by considering various factors like tailing factor, retention time and theoretical plates of HA & (o, p, m) MHA. To analyse the system suitability parameters a concentration of 100µg/ml of HA & (o, p, m) MHA were injected into RP-HPLC. As summarized in Table 6 the tailing factor, retention time and theoretical plates was found to be considered within the acceptable criteria. Hence, it was proficient that the recommended validated analytical procedure fulfilled the system suitability requirements.

Table 6

System suitability results
Standard Drugs	Parameters	Acceptance criteria	Results
HA o-MHA p-MHA m-MHA	Theoretical plates	NLT 2000	3547 2460 4210 2040
HA o-MHA p-MHA m-MHA	Tailing factor	NMT 2.0	1.589 1.609 1.220 1.284

Data interpretation

From Table no 6, It is concluded that the system suitability test parameters are being satisfied.

LINEARITY

For, HA and (o, p, m) MHA of standard calibration curve over the concentration ranges from 10µg/ml to 160µg/ml for Hippuric acid further 20µg/ml to 320µg/ml for 0- MHA, 20µg/ml to 320µg/ml for p- MHA and 10µg/ml to 160µg/ml for m- -MHA was checked and Calibration curve graph for Hippuric and (o, p, m) Methyl hippuric acid was shown in Fig. 16.

Table 7

Correlation coefficient results
Standard drugs	Correlation coefficient
Hippuric acid	0.999
o - Methyl hippuric acid	0.998
p - Methyl hippuric acid	0.998
m - Methyl hippuric acid	0.999

Acceptable limit:

R ² value should be NLT 0.95.

Data interpretation:

The linearity data shown in Fig. 9, concludes that HA & (o, p, m) MHA is linear in the range of 10 to 320µg/ml concentration and Table 14 shows peak area and concentration of the standard compounds.

The correlation was 0.9987 which is more that acceptable limit, therefore linearity parameters was approved.

LOD and LOQ

The sensitivity of proposed RP-HPLC procedure was calculated by using the method calibration standards

𝐿𝑂𝐷 = 3.3 × 𝑁/ 𝑆 𝐿𝑂𝑄 = 10 × 𝑁/ 𝑆

Where, N – Standard deviation and S – slope.

Data Interpretation: It may be determined that the peaks were identifiable at LOD level concentration. The LOD and LOQ of the standard compounds are shown in Table 8

Table 8

Results of LOD and LOQ
Standard Drugs	LOD (µg/ml)	LOQ (µg/ml)
Hippuric acid	0.77	2.24
o - Methyl hippuric acid	0.30	0.87
p - Methyl hippuric acid	1.42	4.23
m - Methyl hippuric acid	0.45	1.37

ACCURACY

Accuracy is determined by the closeness b/w the values obtained and true values of the developed method which is indicated as percentage recovery. The percentage recovery of samples were in range of 98–102%. The values of percentage recovery of HA and (o, p, m) MHA found to be considerably within the acceptance criteria. Hence, the suggested method is accurate for quantification of HA and (o, p, m) MHA.

Acceptance limit

The mean %recovery at each level should be between 102.0% and 98.0%.

SUMMARY

The sensitive RP-HPLC method for quantification of Hippuric and (o, m, p) Methyl hippuric acid was developed and validated and the study investigated cellular changes, such as cytomorphometry and micronuclei, in exfoliated oral epithelial cells and 100 cells were observed in zig-zag pattern on each slide using bright field microscopy.

1. Optimized method conditions for RP-HPLC

Through various optimization trials the chromatographic conditions were optimized in Shimadzu, HPLC instrument using UV detector at 254nm. Separation was achieved by using SHIMADZU C18 column, Acetonitrile and 0.025% glacial acetic acid in Millipore water (84:0.025:16), flow rate 0.7 ml/min at 40°C temperature. The retention time of HA & (o, p, m) MHA were found to be 4.739, 6.485, 7.563, 13.441) minute as shown in Fig. 3. The lesser retention time is the major merits of this method.

2. Cytomorphometric analysis of Micronucleus assay of oral epithelial cells in buccal mucosa smears

The micronucleus assay of buccal mucosa smears can be a useful tool for assessing the genotoxic effects of occupational exposures like petroleum workers. Cytomorphometric analysis is performed to assure the difference between case (petroleum workers) and control population. the number of micro nuclei per 100 cells were analysed in bright field microscope and found to be 47.45 in cases of petroleum workers with standard deviation of 12.83. Whereas in control were found to be 7.24 with SD value of 3.03 Fig. 6. The micro nucleus of epithelial cells of buccal mucosa were highly significant and high in cases than control population.

Finally, as the concentration of HA and (o, p, m) MHA increases in urine sample so as the cytomorphometric changes indicates the increase in micronuclei exfoliated epithelial cells in buccal mucosa smears by the amount of toluene and xylene inhalation by the petroleum workers.

The HPLC method was developed for the quantification of Hippuric acid and (o, m, p) Methyl hippuric acid. Validated the method as per USFDA guidelines. The technique is established to be accurate, precise and linear which proves the reliability of proposed technique. Therefore, the projected RP-HPLC technique can be employed for regular detection of Hippuric acid and (o, p, m) Methyl hippuric acid. Cytomorphometric changes is assessed for the epithelial cells in buccal mucosa smears for the evaluation of micronuclei in smears. Papanicolaou stain is the best staining procedure to identify the micronuclei of exfoliated oral epithelial cells. This stain is reliable, accurate, less time with low cost. To ensure the safety and well-being of gas station employees, it is vital to minimize their exposure to petroleum products and to educate them about the potential risks involved. Using appropriate personal protective equipment is recommended for petrol pump workers to prevent exposure to the harmful effects of toluene and xylene.

Funding: This work was supported by the JSS Academy of Higher Education & Research, Mysore [Grant number JSSAHER/REG/RES/URG/54/2011-12/14009 dated 06/03/2023].

Availability of data and materials: The datasets generated and analyzed during the current sudy are available from the correponding author on reasonable request.

Conflict of interest: The authors declare that they have no confict of interest.

Ethical approval: The study was discussed and reviewed and same has been cleared and aproved by the Institutional Ethical Committee (JSSMC/IEC/240323/20 NCT/2022-23 dated 27/03/2023).

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No competing interests reported.

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Chromatographic analysis of methyl hippuric acid metabolite in the urine of petroleum workers and evaluation of cytogenetic changes of epithelial cells in buccal mucosa smears

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Abstract

Figures

INTRODUCTION

Toluene Metabolism

Xylene Metabolism

METHODS

chemicals and reagents

Sample collection

Selection and preparation of mobile phase

Standard stock solution preparation

Sample preparation

Preparation for micronucleus assay

RESULTS AND DISCUSSION

Liquid chromatographic conditions

System suitability

Data interpretation

LINEARITY

Acceptable limit:

Data interpretation:

LOD and LOQ

ACCURACY

Acceptance limit

SUMMARY

1. Optimized method conditions for RP-HPLC

2. Cytomorphometric analysis of Micronucleus assay of oral epithelial cells in buccal mucosa smears

CONCLUSION

Declarations

References

Additional Declarations

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