SM Women’s Health, Issues and Care

Research Article

Association between Biomass Fuel for Cooking and Serum Nitric Oxide among Women in Karachi, Pakistan

Ambreen Kazi1,2*


Objective: The study determines the association between biomass fuel use and serum nitric oxide (NO) levels among women in villages of Gadap town, Karachi, Pakistan.

Materials and methods: A cross sectional study was conducted in Gadap town, Karachi, Pakistan. A total of 83 women between 15-45 years of age were recruited with 43 biomass users and 40 using natural gas as primary cooking fuel. Particulate matter (PM2.5) and Carbon monoxide (CO) was measured in kitchens using an aerosol monitor Sidepak and Monoxor II, respectively. Serum Nitric Oxide (NO) was measured through Griess reaction. Multivariate regression was conducted to determine the association between serum NO levels and biomass.

Results: CO and PM2.5 levels in kitchens among biomass [CO: 20.22 (±12.2), PM 2.5:4.46 (±3.6)] users were significantly higher compared to natural gas users [CO: 1.22 (± 1.22), PM2.5:0.05 (± 0.02)]. Blood serum NO levels among women were significantly (p<0.001) higher in biomass users (280.8±25 μmol/L) compared to natural gas users (230.4±10 μmol/L). Multivariate regression analysis found unit change in biomass use associated with 0.25 unit (p<0.02) change in NO levels, after adjusting for age and socioeconomic status.

Conclusion: Women who are using biomass as fuel are exposed to high levels of CO and PM2.5 pollutants. High NO levels in biomass users may indicate enhanced in vivo inflammatory responses. Further studies are needed to identify the role of serum NO levels in causing respiratory and cardiovascular diseases among biomass users.


Increase in population and limited access to clean energy resources has compelled almost half of developing nations to rely on solid fuels [1]. Solid fuels are economical and conveniently available; however, their constant use is found to be associated with various adverse health effects [1]. Indoor air pollution (IAP) mainly due to solid fuels, like wood, coal, cow dung etc, contribute approximately 1.6 million deaths annually[1].Smoke released from the burning of biomass fuels include carbon monoxide (CO), nitric oxides (NOx), sulphur oxides (SOx), particulate matter (PM), formaldehyde and polycyclic hydrocarbons. Among these, CO and PM2.5, are the most commonly studied pollutants showing association with chronic obstructive lung diseases (COPD), acute lower respiratory infections, lung cancers, tuberculosis, blindness, cardiac diseases and interstitial lung diseases and adverse pregnancy outcomes [2-3].

Systematic review by Naeher et al [2] found chronic exposure to biomass leads to worsening of localized and systemic inflammatory effects. Experimental animal studies demonstrated various inflammatory changes secondary to biomass exposure: like damage to respiratory epithelial lining, altered pulmonary morphology, increased number of alveolar macrophages and oxidative stress, increased cytokine influx and airway hyper reactivity, altered blood indices level, metaplasia and DNA damage [4,5].Studies have found significant increase in proinflammatory and inflammatory markers in biomass users [6-8]. Ghio et al. exposed volunteer human subjects to prolonged wood smoke (>20 hrs) in a controlled environment. Thebronchoalveolar lavage of volunteers showed neutrophilic influx suggesting pulmonary inflammatory response [9]. Among different inflammatory markers, cytokines and nitric oxide have received special attention by environmental scientists [6-8,10]. It may be because serum NO plays an important role in many biological processes in the body and its levels are affected in various diseases like respiratory, cardiovascular and inflammatory [10,11]. Banerjee et al found serum nitric oxide levels almost twice as higher in rural Indian women using biomass as compared to gas users [8].

Several studies have determined detrimental effects of biomass exposure though experiments involving animals or in vitro/in-vivo methods [2,4]; however few studies are available on human subjects with simultaneous measurements, both in the environment and the body [6-8]. An estimated 87% of rural and 62% of overall households in Pakistan, rely on biomass fuel for their daily household energy requirement hence exposing women to increased health risk [1]. The natural experiment of getting exposed to high levels of biomass in a real life situation, especially during winters is commonly available in the sub-urban and rural areas of a developing country like Pakistan. This study explored the association between biomass and serum NO levels in women residing in villages of sub-urban Gadap town, Karachi.

Materials and Methods

The cross sectional study was conducted during February, 2012 in semi-urban community of Gadap town of Karachi. Gadaptown is located in the north-west of Karachi and according to 2002census has population of 289,732 [ DataSource]. The town is divided into eight union councils, each having several villages. Almost half of Gadap town lacks access to basic facilities like drinking water and gas. The area comprises small villages with mud houses and brick made streets.

Two separate groups of women were invited to participate, the first group; Biomass users (n=42, who were using wood, cow dung, crop residues or coal (locally purchased or collected) as cooking fuel for atleast the last five years) and the second group Current gas users (n=41, who were using gas as cooking fuel for at least the last 5 years). The biomass users were selected consecutively from six villages of Gadap town and natural gas users were selected from the nearest adjacent village with availability of gas as cooking fuel. A team of two local residents was formed to identify the households in the village according to the inclusion criteria. Only those households that had a properly constructed (with four walls and roof) external kitchen (not linked internally with rooms), were selected for this study. One woman, doing the major cooking (the primary cook) for the family, was selected from each house. Females who were suffering from high grade fever, physician diagnosed infectious disease (hepatitis, typhoid, tuberculosis) or chronic disease (diabetes and hypertension), or were pregnant, were current smokers, were not included in the study. The study was approved by Aga Khan University Ethics Review Committee (Human Subject Assurance Number: FWA00001177). Due to cultural reasons, signed informed consent was taken from the woman and her husband (elder of the house in case husband was away) before starting the interviews/measurements (thumb impression was taken in case of illiterate woman). The data collectors first explained to the women about the procedure of the study which included structured interviews, anthropometric measurements, and blood sample collection and kitchen air measurements. Participation was on voluntarily basis and there were no refusals. All participants were assigned a unique identifier number (questionnaire and the blood samples) in order to maintain confidentiality.

A team of two health persons, one male environmental scientist and one female phlebotomist approached each selected house. Male data collector was responsible for recording PM2.5 and CO measurements, and female phlebotomist conducted the interview, took anthropometric measurements and blood samples. All interviews were conducted in Sindhi language, which is locally understood and spoken. Detailed interviews comprised of socio-demographic variables (age, education, occupation, number of family members, income, source of water supply); kitchen information(duration of kitchen use, type of kitchen, type of stove, presence of chimney, number of windows and doors and time spent in cooking); smoking history (current smoker, passive smoker, other burning sources such as burning of garbage or incense inside the house); medical history related to allergic symptoms (coughing, sneezing, wheezing, redness of eyes, watery eyes or sticky eyes). Anthropometric measurements included weight and height. Weight was measured by using the electronic weighing scale whereas height was measured using measuring tape according to the standard procedure.

Trained phlebotomist collected and stored 3ml blood sample from each woman using aseptic venipuncture technique. Whole blood samples were transported to laboratory in cold boxes within 3 hours of collection. Blood was centrifuged at 800g for 10 minutes to separate serum, which was subsequently collected, aliquoted and stored at -700C until used for NO level analysis.

Exposure to indoor air pollution was determined by measuring fine particulate matter (PM2.5) and carbon monoxide (CO). Women were asked to do cooking in a manner similar to their daily routine. All measurements were taken exactly five minutes after the fire was lit to its maximum (mentioned according to participant) and by standing at a distance of 1 meter from the stove. Real-time measurement of particulate matter of <2.5micron aerodynamic diameter (PM2.5) was made using a handheld laser operated monitor (Sidepak-Model AM510, TSI Inc., MN, USA), a laser guided photometer with an inbuilt 2.5 micro impactor. Prior to each measurement, the device was zero-calibrated with a HEPA filter according to manufacturer’s specifications and air flow rate was set to 1.7 L/min. CO level was measured using portable CO Analyzer (Bacharach, Monoxor-II USA). CO monitor had the ability to measure between 0 and 2,000 parts per million (ppm) with an accuracy of ±2 ppm in low CO concentrations. Spot monitoring for PM2.5 and CO measurement were done.

Nitric Oxide Assay Kit Thermo (R&D Systems, UK) was used and total nitrate (Nitrite and Nitrate) was estimated using Griess Reagent I and Griess Reagent II followed by measurement at dual wavelength (540 nm/ 690 nm). For the Nitrate Reduction assay, NADH and Nitrate Reductase were added to all wells prior to addition of Griess reagents and subsequent reading at dual wavelength was done. Range of detection for Nitrite was 3.125μM -200μM and for Nitrate 3.125μM -100μM (Sensitivity of assay: 0.222μM).

Data were analyzed using SPSS computer statistical software package (IBMSPSS statistics version 21). The continuous variables were calculated as mean (±SD), whereas, categorical were measured as percentages. Serum NO levels, and air pollutants PM2.5 and CO were analyzed as continuous variable. Comparison between two groups was done using student’s t-test for independent samples with the significance level at<0.05. Correlation coefficient was estimated for PM2.5 and CO levels in the kitchen during cooking and serum NO levels. Multivariate linear regression was performed to determine the association between serum NO and type of fuel used, after adjusting for potential confounders. Model fit was used assessed using the R square value.


The mean age of participants was 25.4 (±7.5) years, and no significant difference in age was observed between the two groups. Similarly, no statistically significant difference was found for education, marital status, number of children and income between the two groups. Question on the type of biomass used found that almost all used dry wood as primary cooking fuel except for 2 participants who were using cow dung as primary and wood as secondary fuel. Around 40% of biomass users had no window in the kitchen as compared to 33% gas users (p=0.29). Among those who had windows, majority of them (93% gas users and 75% biomass users) kept their windows and doors open while cooking (p=0.09). Around 50% of biomass users and 32% of gas users mentioned burning incense inside their homes. More women (28%) in biomass group were passive smokers as compared to natural gas users (12%). Allergic symptoms like watery eyes were more frequent among natural gas users whereas upper respiratory symptoms although not statistically significant were more frequent among biomass users (Table I).

Table 1: Comparison of socio-demographic characteristics of women using biomass or gas as cooking fuel in Gadap town, Karachi.


Biomass users (n=42) (%)

Gas users (n=41) (%)

p value

Age (years)

(Mean+/- SD)

25.4 (±8.5)

23.7 (±6.9)


Educational status


1 (2.4)

1 (2.4)



12 (28.6)

12 (29.3)


14 (33.3)

12 (29.3)


15 (35.7)

16 (39.0)

Marital status


20 (47.6)

20 (48.8)



22 (52.4)

21 (51.2)


(mean +/- SD)

21.1 (±3.5)

20.4 (±3.7)


Passive smoker


30 (71.4)

36 (87.8)


12 (28.6)

5 (12.2)


Drinking water supply

Tap water

30 (71.4)

13 (31.7)

Well water

12 (28.6)

28 (68.3)


Animals in the household


5 (11.9)

7 (17.1)


37 (88.1)

34 (82.9)


Monthly income (PKR)

>10 000

17 (40.5)

18 (43.9)


6000-10 000

16 (38.1)

14 (34.1)


9 (21.4)

9 (22.0)

Eye & and respiratory symptoms

Itching of eyes during cooking



27 (63.4)

27 (65.8)



15 (36.6)

14 (34.1)

Redness of eyes during cooking









Tears while cooking



23 (54.7)




19 (45.2)



29 (69)

31 (75.6)



13 (31)

10 (24.4)

Sore throat


24 (57.1)

27 (65.9)



18 (42.9)

14 (34.1)




32 (78)



9 (22)

9 (22)



30 (71.4)

32 (78.0)



12 (28.6)

9 (22.0)

The mean CO levels inside the biomass and natural gas users kitchen was 24.9 (±15.0)mg/m3 and5.50(±4.4) mg/m3, respectively. The mean PM2.5 levels inside the biomass and natural gas userskitchen was [1.22 ppm (±0.8) and 0.05 ppm (±0.02)] respectively. There was significant correlation between CO and PM2.5 levels (correlation coefficient r=0.73, p<0.01). Mean serum NO levels were higher among biomass users (280.8 ±10 μmol/L) as compared to natural gas users (230±10μmol/L).

Univariate analysis found biomass use (0.32, 95%CI 16.9, 69.3), literacy level (0.42, 95%CI 20, 53), age in years (0.29, 95%CI 1.1, 5.8) and income level (-0.35, 95% CI -0.004, -0.00) were significantly associated with serum NO levels (Table II). Multivariate linear regression analysis found significant association between serum NO levels and biomass use after adjusting for income level and age; biomass use led to 0.25 times increase in serum NO levels compared to natural gas users (0.25, 95%CI 3.4, 66.7 μmol/L) (Table III). The adjusted R² for the model was 0.28, explaining 28% variance in the serum NO levels due to biomass use.

Table 2: Univariate linear regression analysis between NO levels (μgm/dl) and sociodemographic, allergic and type of fuel used by women in Gadap town, Karachi, Sindh (n=83).




95% CI

Biomass use



(16.9, 69.3)

Literacy level



(20.0, 53.0)

Body Mass Index (BMI)



(-0.10, 43.3)

Passive smoking



(57.2, -24.4)

Age of respondent (in years)



(1.1, 5.8)

Household income level




Table 3: Multivariate linear regression analysis between NO levels and type of fuel used by women in Gadap town, Karachi, Sindh (n=83).



Adjusted Beta

95% CI

Biomass use



(3.4, 66.7)

Total household income



(-.003, 0.001)

Age of respondent (years)



(-1.9, 2.6)


Similar to previous studies, high mean levels of CO and PM2.5 levels were found in kitchens of biomass users during peak cooking hours. These values were consistent with studies conducted by Siddiqui et al (8 hr mean CO: 29.4 ppm, 8 hr mean PM2.5: 2.74 mg/ m3) [12-14] (24 hr mean PM2.5 1169 μg/m3). Immediate exposure to these pollutants may cause watery and red eyes and upper respiratory symptoms; however, no significant difference was noted in the number of women belonging to both groups.

Our study demonstrated high levels of Nitric oxide among the biomass users. Nitric oxide was first discovered as a biologically active molecule in the late 1980s. It is produced from L-arginine in the body at numerous sites such as the vascular endothelial cells, and plays an important role in neurotransmission and in relaxation of vascular smooth cells [15,16]. However, high levels of NO, referred as inducible or iNOS is expressed only under the influence of certain substances such as cytokines, and/or endotoxins that are released during inflammatory and infectious processes resulting in a sustained production (hours to days) of NO [8]. This inducible NO acts on the non specific host defense mechanism and its raised levels have been observed in a number of conditions including sepsis, asthma, rheumatoid arthritis, tuberculosis, inflammatory bowel disease, Alzheimer disease, multiple sclerosis, etc. [1-2,17,18]. The inorganic free radical (NO) act as an important mediator in a number of physiological as well as pathological processes. In our study, initiation of inflammation secondary to biomass use is one of the major proposed mechanisms leading to adverse health outcomes [6-8,17].

It is proposed that increased serum NO levels affects the activation of macrophages cytokine production by host cells via release of P-select in expressing platelets or release of reactive oxygen species (ROS) [18-20]. Review on air pollution from India and Pakistan found association between indoor air pollutants and cardiovascular diseases [21]. Study from rural Pakistan found women using biomass for cooking purpose are 4.8 times (95% CI 1.5 to 14.8) at risk of developing acute coronary syndrome [22]. The constant and regular exposure to biomass resulted in very high serum NO levels, which were significantly higher as compared to Iranian population (mean value Iranian population 22.7 ± 0.02 μmol/L) [23]. These levels even exceeded those suffering from chronic inflammatory disease like diabetes mellitus (166.8 ± 3.2 μmol/L) [24], suggesting that biomass exposure may lead to severely poor health status, that can differ based on the duration of exposure. Trials focusing on improved stoves with less exposure to biomass smoke have shown beneficial health effects [25].

A natural experiment in which women are exposed to high biomass levels in a real life situation is the main strength of our study. Both exposure to biomass and outcome as serum NO levels were measured using validated methods. CO and PM2.5 were measured using equipment which has been tested and used by prior studies conducted within Pakistan and in South Asian region [26,27]. Similarly, serum NO levels were measured via Griess method that gives estimates close to accurate levels.

This study had some limitations. It was a cross sectional study hence difficult to establish causality between biomass use and serum NO levels. Women who were exposed to biomass for last five years were included; hence it is difficult to comment if any other type of fuel was used prior to five years or if there was any contribution due to neighborhood burning of biomass. More than one time measurements of indoor pollutants, NO and other cytokines (Interleukins-6, Tumour necrosis factor) would have strengthened the findings. The results demonstrated high levels of NO, which may be due to the difference in the detection kit method (Banarjee, utilized the ELISA method, whereas we used Griess reaction method).

High indoor air pollutants secondary to biomass is a wellestablished finding, however, detection of high serum NO levels among biomass users invite large scale follow up studies to explore underlying pathophysiological mechanism responsible for causing adverse health effects in humans.


Women who are using biomass as fuel are exposed to high levels of CO and PM2.5 pollutants. Immune markers, like high serum NO levels in biomass users may indicate enhanced in vivo inflammatory responses. Further studies are needed to identify the role of serum NO levels in causing respiratory and cardiovascular diseases among biomass users.

Funding Agency

I shall like to thank the Aga Khan University Research Council for granting us the seed money to conduct this preliminary study. The funding agency had no role in the conduction, analysis, write up and publication of the study.


I shall like to acknowledge Prof Zafar Fatmi for his guidance and support in conducting this study. I shall like to thank Prof. Zahra Hasan for facilitating the laboratory measurements and interpretations. I shall also like to acknowledge Miss Mussarat Ashraf for her technical support in the laboratory and to Mr. Mohammad Ubaidullah Hassan Qureshi for his voluntary participation in data collection and data entry process.


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Citation: Kazi A. Association between Biomass Fuel for Cooking and Serum Nitric Oxide among Women in Karachi, Pakistan. SM Womens health issues care. 2018; 1(1): 1001.

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