The objective of this prospective study was to determine if a correlation could be established between the ground-level concentrations of sulfur dioxide and methemoglobin concentrations in pregnant women when a coal-powered thermoelectric power plant was in operation ("dirty" period) and when it was closed ("clean" period). The location of the power plant, Plomin 1, in Labin, Croatia, was taken into consideration. Blood and urine samples of each pregnant woman in the study were tested three times in the clean period (n = 138) and three times in the dirty period (n = 122), with 1 month between each test. I observed a correlation between the increase in mean values of methemoglobin and the ground-level concentration of S[O.sub.2] on corresponding dates during the dirty period (r = 0.72, p < 0.01). In the clean period, the negative mean value of methemoglobin was significant (r = -0.60, p [less than or equal to] < 0.05), whereas in the dirty period, the positive mean value of methemoglobin was significant (r = 0.73, p [less than or equal to] < 0.01). The increase of maternal methemoglobin could be a useful biomarker to determine when the health of pregnant women is threatened by toxic substances in the environment. Key words." biomarker, environmental toxicants, maternal methemoglobinemia, precursor, pregnancy, toxic substances. Environ Health Perspect 111:1902-1905 (2003). doi:10.1289/ehp.6055 available via http://dx.doi.org/[Online 13 November 2003]
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In recent years, researchers have focused on explaining the role of oxygen, free radicals, and oxidative stress during embryogenesis and placental stages, and development of pathologic pregnancy, especially preeclampsia and fetal intrauterine growth restriction (IUGR). Because I found no evidence that methemoglobin levels had been tested during human pregnancy in an air-polluted environment, I reevaluated research performed in this laboratory in the past. My objective was to identify a biomarker for methemoglobin to be used as a precursor and proof of the presence of oxidants before clinically manifested symptoms occur, even in early pregnancy. The statistical analysis was incomplete, thus requiring additional research, which ,was delayed because of the Croatian War for independence that lasted from 1991 to 1995.
Methemoglobinemia is a condition in which hemoglobin is oxidized to the ferric form and is unable to transport oxygen to tissues, therefore causing hypoxia. The physiologic level of methemoglobin is 1% in peripheral blood, and it may increase because of a variety of genetic, dietary, idiopathic, toxic, and other Factors. Methemoglobinemia primarily occurs when erythrocytes are affected by xenobiotics and pharmaceutical compounds with toxicologic properties, such as volatile organic compounds, oxidants, nitrogen oxides, peroxynitrites, phenacetin, and sulfonamides.
Materials and Methods
To determine the toxic substances in the environment and the level of air pollution, I chose to study the population living near Plomin 1, a coal-powered thermoelectric power plant in Labin, Croatia. The plant, with a 110-m-tall chimney, is the single major air polluter within a 40-km radius of the target population. Every hour of operation, the plant emits about 8.5 tons (18,080 mg/[m.sup.3], or 6,900.8 ppm) of sulfur dioxide in addition to nitrogen oxides (N[O.sub.x]), carbon dioxide, carbon monoxide, total suspended particles, iron, titanium, vanadium, chromium, nickel, copper, zinc, selenium, lead, and other products of coal combustion. The coal from this area has a high sulfur content (9-11%) and a high level of radioactivity (the activity of [sup.238]U is 300 Bq/kg, which is 10-15 times higher than the average for other types of coal in the world). In the approximately 700,000 tons of crude waste from coal combustion surrounding the plant, the concentration of radionuclides was 5-10 times higher than that in the unburned coal (Saric 1996). Because the plant was closed from 19 February 1989 to 6 September 1989, I was able to carry out research during two separate periods: the "clean" period from April to July 1989 and the "dirty" period from December 1989 to March 1990. In the dirty period, the daily ground-level concentrations of S[O.sub.2] were monitored at three different locations.
Air quality data and samples. Daily minimum, maximum, and average air temperatures; quantity and type of precipitation; wind direction and strength; and relevant data on weather conditions were provided by the Labin meteorologic station. Air quality (S[O.sub.2], fumes, and particulates) was analyzed by an acidimetric method based on the British standards recommended by the World Health Organization (WHO 1976). Briefly, air samples were collected in a weak solution of hydrogen peroxide; after particulates were removed by filtration, the quantity of the absorbed S[O.sub.2] in the [H.sub.2][O.sub.2] solution was determined by titration. The Regional Institute of Health Care (Pula, Croatia) performed the air quality, measurements.
Subjects. Pregnant women were selected for the research target group from patients of the Primary Health Center, which is responsible for the health of about 25,500 residents, of which about 6,180 women of reproductive age bad a permanently assigned obstetrician or gynecologist. Subjects were informed about the purpose of the research and gave written consent. Out of 273 women who were pregnant at the time of the study, 260 women were considered representative based on the criteria that they were pregnant and came to the center for regular monthly checkups during the clean and dirty periods. Patients received care from the Primary Health Center, the Obstetric-Gynecological Clinic in Rijeka, or the regional Obstetric-Gynecological Hospital in Pula.
Blood and urine samples from women in the study were tested three times in the clean period (n = 138) and three times in the dirty period (n = 122), with 1 month between each test. All 260 of the pregnant women in the study lived in Labin and the surrounding area.
The pregnant women were divided into six groups on the basis of the location of their places of residence within zones defined by concentric circles around the Plomin 1 plant. Most of the pregnant women lived in the zones 3.5-7.5 km (71.63%) and 7.5-12.5 km from the plant (21.63%). The town of Labin, with 12,000 inhabitants, is also in these zones.
The Obstetric-Gynecological Clinic and the Obstetric-Gynecological Hospital, where women from the area surrounding Plomin 1 were treated, provided data on reproductive loss for the clean and dirty periods.
Blood samples. According to the planned prospective study, 2 cm3 of each blood sample (heparinized) was tested. Hemoglobin and methemoglobin were measured by spectrophotometric method in the toxicology laboratory, at the Department of Occupational Medicine (Rijeka, Croatia). Briefly, the erythrocytes were lysed with Triton X-100 and spectrophotometry was performed using the cyanohematin method. The maximal absorption of methemoglobin is 630-633 mm.
Samples (10 mL buffer, 0.1 mL blood from exposed or nonexposed pregnant women, or one drop of Triton X-100) were placed into test tubes, mixed well by turning the tubes five to eight times, and then left for 5 min. The mixture was then poured into another test tube, and the photometry was performed toward water at 633 mm (measurement A1). Afterward, one drop of neutral sodium cyanide was added to the mixture, which was stirred, left for 5 rain, and measured again at 633 mm (measurement A2).
Methemoglobin (g/L) = (A1 - A2) x F
F is determined by adding 0.1 mL potassium ferricyanide solution to 10.0 mL buffer and 0.1 mL normal blood. The mixture was then stirred and left for 2-3 min so that all of the hemoglobin could oxidize into methemoglobin. The absorption was measured at 633 mm (measurement A3). A drop of neutral sodium cyanide was then added to the mixture, and after 5 min, the absorption was measured again at 633 mm (measurement A4). The difference (A3 - A4) corresponds to the absorption of total hemoglobin in the form of methemoglobin. I also used the cyanohematin method to determine the difference in absorption between hemoglobin and methemoglobin.
F = g/L hemoglobin / A3 - A4
Percentage methemoglobin in total hemoglobin = (g/L methemoglobin / g/L hemoglobin) x 100 The mean of two blood samples was used to determine F.
The standards used to relate absorbance values to hemoglobin and methemoglobin concentrations were 120-160 g/L and 0.0-2.5 g/L, respectively.
Results
