Authors
Keywords
Abstract
We collected and analyzed groundwater and other biological samples for the determination of arsenic and other trace elements from an arsenic exposed group and an unexposed group (control group) of people in Bangladesh. The results showed that arsenic in hair, nail, and urine increases with increasing arsenic in drinking water. Along with arsenic in hair and nail samples, I also analyzed Se, Zn, Cu, Hg, Pb, and Sb by ICP-MS after microwave digestion. In both cases (hair and nail), it appears that the mean concentration of Se and Zn are lower in the exposed group than in the control group. Again, the mean concentration of Hg, Pb, and Sb are higher in both hair and nail for the exposed group than the control group.
The regression analyses were carried out between arsenic and other metals and metalloids in hair and nail samples. The linear regression shows negative correlation between As & Se (r = - 0.84, p = 0.00005, n = 16) and As & Zn (r =- 0.78, p =0.0003. n = 16); some positive correlation between As & Pb (r = 0.58, p = 0.008, n = 19), As & Hg (r = 0.745. p 0.0002, n = 19) and As & Sb (r = 0.743, p = 0.0002, n = 19); no significant correlation between As & Cu (r = -0.04, p = 0.87, n = 17) for hair samples. For nail samples, a similar correlation was observed as in hair. The linear regression shows negative correlation between As & Se (r = - 0.53, p = 0.004, n = 27) and As & Zn (r = - 0.55, p = 0.0006, n = 35) and some positive correlation between As & Hg (r = 0.47, p = 0.004, n =35), As & Pb (r = 0.51, p = 0.003, n = 33), and As & Sb (r = 0.57, p = 0.0004, n = 33); no significant correlation between As & Cu (r = 0.15, p = 0.41, n =31) observed.
Also, urine and blood samples were collected from 191 subjects (98 females and 93 males) in the Lagunera area of Mexico. There were five groups that were based on total arsenic concentration (38-116 µg/L) in their drinking water. In urine, statistically significant correlations were not found between the concentrations (µg/g cre) of Se as well as Zn and % inorg As, % MMA, % DMA, as well as the ratios of % DMA to % MMA in urine for females. However, the relative concentrations of Se to As expressed as µg/g cre were positively and significantly correlated with % inorg As (rs= +0.29, p<0.01) & % MMA (rs= +0.25, p<0.05) and negatively correlated with % DMA (rs= -0.34, p<0.001) & the ratios of % DMA to % MMA (rs= -0.31, p<0.01) in urine for females. For males, the ratios of the concentrations (µg/g cre)) of Se or Zn to As were more positively correlated than the concentrations of Se or Zn were with % inorg As levels (rs= +0.26, p<0.05 vs. rs= +0.17 not significant and rs= +0.22, p<0.05 vs. rs= +0.086 not significant, respectively), but it was more negatively correlated with % DMA levels (rs= -0.25, p<0.05 vs. rs= -0.12 not significant and rs= -0.19 not significant vs. rs= -0.008 not significant, respectively) in urines.
In blood, the relative concentrations of Se or Zn to As than the corresponding element concentrations in blood were more significantly and positively correlated with urinary % inorg As (rs= +0.36, p<0.001 vs. rs= +0.11 not significant and rs= +0.24, P<0.05 vs. rs= -0.10 not significant, respectively), and negatively correlated with % DMA (rs= -0.34, p<0.001 vs. rs= -0.12 not significant and rs= -0.24, p<0.05 vs. rs=-0.005 not significant, respectively, and with the ratios of % DMA to % MMA (rs= -0.32, p<0.01 vs. rs=-0.063 not significant and rs= -0.29, p<0.01 vs. vs. rs=-0.049 not significant, respectively) for females. The ratios of the concentrations of Se or Zn to As than the concentrations of Se or Zn in blood were also more positively and significantly correlated with urinary % MMA (rs= +0.27, p<0.01 vs. rs= +0.033 not significant and rs= +0.26, p<0.05 vs. rs= +0.05 not significant, respectively) for females. For males, better and significant correlations found between the ratios of the concentrations of Se or Zn to As in blood and the percentage of urinary arsenic metabolites than the correlations found between the corresponding element concentrations in blood and the percentage of urinary arsenic metabolites. The ratios of the concentrations of Se or Zn to As in blood were more positively correlated with urinary % inorg As (rs= +0.25, p<0.05 vs rs= -0.043 not significant and rs= +0.26, p<0.05 vs. rs= +0.036 not significant, respectively). The ratios of urinary % MMA to % inorg As were negatively correlated with the ratios of the concentrations of Se or Zn to As (rs= -0.20 not significant and rs= -0.23, p<0.05, respectively) in blood. But these correlations were not statistically significant with the concentrations of Se or Zn in blood. Also, the ratios of the concentrations of Se or Zn to As in blood were negatively correlated with the ratios of urinary % DMA to % MMA for males, but these correlations were not statistically significant.
In conclusions, the relative concentration of Se or Zn to As may be an important factor for arsenic methylation process. Also, the doses of Se and/or Zn may not be the same for everyone, and it could be dependent on the concentrations of arsenic in their drinking water.
Background
There are many countries in the world where cases of arsenic contamination of groundwater are known. Out of these, Bangladesh calamity (Figs. 1 and 2) is the largest in the world.
Arsenic in urine, hair, nail, and blood are the biomarkers to measure the absorbed dose of inorganic arsenic. The stability of the various biomarkers to serve as indicators of acute or chronic exposure to inorganic arsenic and the various factors (e.g., dietary intake of arsenic compounds) that can influence the indicators - is important. Although absorbed arsenic is removed from the body mainly via the urine, a small amount of arsenic is removed via other routes (e.g., skin, nails, hair, sweat, and breast milk).
The concentration of total arsenic in urine has often been used as an indicator of recent exposure because urine is the main route of excretion of most arsenic species1,2. Analyses of blood, hair, and fingernail samples are also good indicators of exposure. Because of the short half-life of As in blood, hematological estimation is useful in particular in the diagnosis of acute intoxication. Inorganic As is incorporated into hair and fingernails due to its affinity to the sulfhydryl groups in keratin.Following exposure to inorganic arsenic, the biological half time is about 4 days. It is slightly shorter following exposure to As(V) than to As(III)3-6.In a study of six humans, subjects who ingested radiolabeled 74As-arsenate, 38% of the dose was excreted in the urine within 48 hours and 58% within 5 days4.The results indicate that the data were best fit to a three-compartment exponential function, with 65.9% excreted with a half time of 2.09 days, 30.4% with a half-time of 9.5 days, and 3.7% with a half-time of 38.4 days5.In three subjects, each of whom ingested 500 µg of arsenic in the form of arsenite in water, about 33% of the dose was excreted in the urine within 48 hours and 45% within 4 days6.The methylated metabolites MMA and DMA are excreted in the urine faster than the inorganic arsenic. In humans, about 78% of MMA and 75% of DMA were excreted in the urine within 4 days of ingestion of the dose6. In another study, two subjects ingested mineral water containing 200 µg As(V) and about 66% of the dose was excreted over 7 days7.Several studies on human subjects exposed to inorganic arsenic occupationally, experimentally, or environmentally have shown that, in general, U-As met consists of 10-30% inorganic arsenic (iAs), 10-20% MMA, and 60-80% DMA8,9.
Arsenic concentrations are normally higher in hair and nail than in other parts of the body because of the high content of keratin (and the corresponding high content of cysteine). The -SH groups of keratins might bind trivalent inorganic arsenic (AsIII)10-12.The concentration of arsenic in the root of the hair is in equilibrium with the concentration in the blood. Hair might be considered an excretory pathway, and once incorporated in the hair, the arsenic is not biologically available. Arsenobetaine, the major organic arsenic compound in seafood, is not accumulated in hair13.That implies that arsenic in hair reflects exposure to inorganic arsenic only. But the speciation of arsenicals in water extracts of fingernails and hair at 90°C was carried out by HPLC-ICP-MS14. The results show that fingernails contained iAsIII (58.6%), iAsV (21.5%), MMAV (7.7%), DMAIII (9.2%), and DMAV (3.0%); and hair contained iAsIII (60.9%), iAsV (33.2%), MMAV (2.2%), and DMAV (3.6%)14. Fingernails contained DMAIII, but hair did not. The higher percentage of iAsIII both in fingernails and hair than that of iAsV and other species suggest more affinity of iAsIII to keratin.
Segmental hair analysis (i.e., determination of the concentration along the length of the hair) might provide valuable information on the time of acute arsenic exposure15-17.Human hair grows at the rate of approximately 0.35 mm per day or 13 mm a month.Nail and hair have similar affinities for arsenic but data on nails is limited. However, the main disadvantage of using hair and nail as indicators of exposure to arsenic is that the arsenic concentrations might be influenced by external contamination via air, water, soaps, shampoos, and soil, and cannot be readily removed by washing. Therefore, external arsenic contamination must be completely excluded if hair and nail arsenic levels are to be used for assessment of poisoning. Thus, arsenic concentration in hair and nail can be used as markers / indicators of exposure rather than markers of absorbed dose18.
Most of the absorbed inorganic and organic arsenic has a short half-time in blood, following a three exponential clearance curve19,20.Most of the arsenic in blood is cleared with a half-time of about l hour. The half-times of the second and third phases are about 30 hr and 200 hr, respectively. Arsenic concentrations in blood are increased only for a very short time following absorption. If exposure is continuous and steady, as is often the case with exposure through drinking water, the blood arsenic concentration might reach a steady -state and then reflect the degree of exposure.
Blood is a difficult matrix for chemical analysis, so in general, only total arsenic concentrations are reported. Partial speciation of arsenic in blood has been reported in a few cases21,22. When using total arsenic in blood as an indicator of exposure to inorganic arsenic, the interference from organic arsenic compounds originating from seafood must be considered. Furthermore, because of the low concentrations, the analytical error might be significant unless the more-sensitive methods are used. In a population in northern Argentina with known exposure to inorganic arsenic and very little fish intake, the average blood arsenic concentration was 0.9 µg/L in children and 1.5 µg/L in adults22-24.
In subjects exposed to arsenic via drinking water, blood arsenic concentrations are clearly increased and might reach several tens of micrograms per liter23,25.In people exposed to arsenic in drinking water (200 µg/L) in northern Argentina, arsenic concentration in blood was about 10 µg/L on average22-24. In studies carried out in California and Nevada, an arsenic concentration of 400 µg/L in the water corresponded to about 13 µg/L in the blood, and 100 µg/L in the water corresponded to 3-4 µg/L in the blood25.Obviously, compared with urine, blood is a much less sensitive biomarker of exposure to arsenic via drinking water.
Interaction of arsenic with selenium and zinc are very well known. Nutrition is the main source of selenium exposure of the general population. The content of this element in drinking water is low. Several studies demonstrated that low Se is an efficacious anticarcinogen whereas high Se can induce carcinogenesis, cytotoxicity and genotoxicity26,27. Some studies reported that As and Se can induce similar toxicity via different pathways28.
Metabolic and toxicologic interactions between arsenic and selenium are multifaceted and complex. These interactions are of practical significance because large populations are simultaneously exposed to inorganic As in drinking water and varying levels of Se in the diet. Weanling female B6C 31-1 mice were maintained for 28 days on Torulayeast-based diets deficient (0.02 ppm. Se), sufficient (0.2 ppm Se) or excess (2.0 ppm Se) in Se; mice then received by oral dose either 0.5 or 5 mg (As)/kg as [73As] sodium arsenate or 0.5 mg (As)/kg as [73As] sodium arsenite29. Selenium deficient mice dosed with 5 mg (As)/kg AsV exhibited slower whole-body clearance from 8 hours-onward29. Total (urine and feces) cumulative excretion of As derived radioactivity was significantly lower in Se deficient AsV exposed mice at both dose levels compared to Se sufficient mice29.Significantly less As-derived radioactivity was also excreted in the feces of Se deficient mice exposed to 5mg (As)/kg AsV compared to Se sufficient mice29.There was also a trend towards lower cumulative excretion of dimethylarsinic acid in urine of Se deficient compared to sufficient mice which was significant for mice exposed to As(III)29.Overall, studies indicate that Se deficiency is associated with altered As metabolism and deposition.
A new glutathione-arsenic selenium compound has been detected recently in rabbit bile30.Gaiter et al. (1999),injected intravenously with sodium selenite followed immediately with intravenous sodium arsenite. Within 25 min a compound containing arsenic, and selenium was excreted in the bile. This newly compound, the seleno-bis (S-glutathionyl) arenium ion, explains a likely mechanism by which selenium prevents arsenic toxicity.
Inorganic arsenic increases the rate of formation of lipid peroxides and free radicals31.These compounds are associated with cancer as well as with cardiovascular diseases. Experiments have shown that arsenic exposure decreases the selenium content, which counteracts lipid peroxidation32.
The clinical relevance of the interaction of arsenic and zinc is more tenuous. Injected parenterally, zinc protects mice against acute arsenic toxicity by way of an unknown mechanism33, not related to the induction of metallothionein. Lin and Yang34measured unusually low zinc concentrations in blood and urine of Blackfoot-disease patients in Taiwan. Engel and Receveur35estimated the nutritional adequacy of the diet of the Taiwanese population in the Blackfoot-disease endemic area and believed that only zinc might be present in inadequate amounts. Therefore, important factors to consider in an evaluation diet to inhibit arsenic toxicity are methionine, cysteine, vitamin B12, and folic acid as well as essential trace elements such as selenium and zinc.
A marginal zinc status could play a role in the severe vascular manifestations of chronic arsenic exposure with its atherosclerotic and thrombotic manifestations36-38.Low zinc could make endothelial cells more vulnerable to arsenic because zinc appears to be important in vascular endothelial barrier function39,40and membrane integrity in general41.Zinc has been reported to inhibit tumor necrosis factor-induced disruption of endothelial cell integrity39and tumor necrosis factor-mediated DNA fragmentation and cytolysis of murine cells42.
Glutathione is involved in arsenic methylation43 and may stimulate prostacyclin (an inhibitor of platelet aggregation and smooth muscle cell proliferation) in arsenic-exposed endothelial cells44.In zinc-deficient rats, blood glutathione levels and glutathione S-transferase activities were decreased45,46and were restored following zinc replacement46.
Oxygen radicals may be involved in arsenite-induced damage for the addition of the radical scavenging enzyme superoxide dismutase that decreased the frequency of arsenite-induced sister chromatid exchanges in human peripheral lymphocytes47. In rat liver cells, the activity of superoxide dismutase increased upon incubation with physiologic zinc levels48.
In this study, I found that arsenic methylation decreased with increasing the relative concentration of Se or Zn to As in urine and blood of arsenic exposed people. There was also negative correlation with As and Se or Zn, but positive correlation with As and Hg, Pb or Sb in both hair and nail of chronic arsenic exposed people.
Methods and Materials
Reagents
All reagents were of analytical reagent grade and Milli Q water was used throughout49-53.
Analytical procedures
Sample collection and preservation
We collected and analyzed the groundwater and other biological samples for determination of arsenic and other trace elements from an arsenic exposed group and a controlled group of people in Bangladesh. The sampling and preservation procedures are given below:
Water samples
Water samples were collected from both shallow and deep tube wells in pre-washed (with 1:1 HN03) polyethylene bottles after pumping off at least for 3-5 minutes. After collection concentrated nitric acid (1.0 ml per liter) was added as preservative54. Samples, which were not analyzed immediately, were kept in a refrigerator at 4°C.
Hair samples
The hair samples were collected from the people with arsenical skin lesions and without skin lesions from the arsenic affected areas of Bangladesh. A few samples were collected from the control zone of Bangladesh and West Bengal-India where arsenic in groundwater is below 3 µg/L. Samples were collected whenever possible close to the scalp by using stainless-steel scissors. At first the hair samples were washed by distilled water, followed by deionized water and finally with acetone (E. Merck, India Limited) as recommended by International Atomic Energy Agency55. After that it was dried in a hot oven at a temperature of 50-60°C and properly stored in polyethylene bottles or white and fresh paper packets with the proper level for the next step.
Nail samples
The nail samples (nails from hand and feet combined) were collected from the people of arsenic affected areas and few samples were also collected from the control zone. After collection the samples were washed by distilled water until free from dust, followed by deionized water and finally with acetone55.It was then dried in a hot oven at a temperature of 50-60°C and then stored in polyethylene bottles or white and fresh paper packets with proper level.
Skin-scale samples
Samples were collected from severely affected people with hyperkeratosis on the palm and sole. The affected skin becomes soft in warm water to make it easy to cut using stainless steel scissors / ceramic cutters. After collection the samples were washed and dried in the same way adopted for the hair/nail samples and properly stored with proper level56,57.
Urine samples
Spot urine samples were collected from the people of arsenic affected areas in pre-washed polyethylene bottles during sampling when first void samples were not available, and few samples were collected from the control zone. The samples were not subjected to any chemical treatment. Immediately after collection, the samples were stored in a cool box with ice and later, after being brought back to the laboratory, kept at- 20°C until analyses were carried out.
Sampletreatmentforanalysis
Decomposition of many biological substances by HNO3, alone under pressure in Teflon vessels at temperature up to l60°C has been found to be incomplete. But for decomposition of hair, nail, and organs less violet, decomposition procedures e.g., mixture of nitric and with sulfuric acid58 or nitric acid with hydrogen peroxide59 or nitric acid alone60 are adequate. In this study, HNO3-H2O2 were used for the decomposition of hair, nail, and skin-scale for some specific samples. In our earlier publication61, Teflon bomb acid digestion had been discussed for the determination of arsenic in hair, nail, and skin-scale samples. But for routine analysis of large numbers of samples, this procedure is not suitable. Hot plate acid digestion procedure is very simple, and a large number of samples can be digested at a time by this technique which I had adopted throughout this study. To check the validity of this method, a known amount of arsenic was spiked in hair samples during digestion. Percentage recovery (98± 8%) was evaluated by spiking fifteen hair samples with known amounts of arsenic(V). Standard hair sample (NCSDC 73347) was analyzed by this procedure and found in good agreement with certified value.
Acid digestion of hair, nail, and skin-scales samples
Hair samples
0.02 to 0.07 g of hair sample was taken in a 25 ml Borosil glass beaker and added 5 ml concentrated nitric acid (E. Merck. India). Then closed the lid and heated on a hot plate with a temperature of 90- 100°C for a few minutes. Heating was discontinued and kept overnight. Next morning the lid was opened and l ml concentrated HNO3 added again, and the sampleevaporated about l00°C in an exhaust chamber. Nitric acid was added, if necessary, till the color of the solution turned into pale yellow. On reaching a final volume of about l ml, heating was discontinued. The pale-yellow liquid was diluted and filtered through a Millipore membrane (0.45µm) filtering apparatus, then adjusted to a fixed volume61.
Nail samples
0.02 to 0.07 gm of nail sample was taken in a 25 ml Borosil glass beaker and added 5 ml concentrated nitric acid. Then closed the lid and heated on a hot plate with a temperature of 90-100°C for a few minutes. Heating was discontinued and kept overnight. Next morning the lid was opened and 1 ml concentrated HNO3 added again, and the sample evaporated at about 100°C in an exhaust chamber. Heating was continued with time-to-time addition of a known volume of concentrated HNO3 until the color of the solution turned to almost colorless. Next steps taken were like those of hair samples61.
Skin-scale samples
0.05-0.2 gm of sample was taken in a Borosil glass beaker and next steps taken were like those of nail digestion61.
Microwave digestion of hair and nail samples.
Samples for digestion were weighed in the Teflon vessel (advanced composite vessel, HP 500) and added 2: l (V/V)of nitric acid and hydrogen peroxide (Waka Pure Chemicals Ind. Ltd., Japan). The vessels were closed using the lid provided. For safety of the vessel, rupture membrane was inserted in the lid. Vessels were set in the turntable of the microwave digestion machine (Model: MDS 2100, USA) and the below settings were programmed (Table 1).
| Table 1: Optimum parameters for sample digestion by microwave system | |||||
| Stages | 1 | 2 | 3 | 4 | 5 |
| Power (watt) | 80 | 80 | 80 | 0 | 0 |
| PSI | 70 | 120 | 170 | 20 | 20 |
| Time (min) | 20 | 20 | 20 | 20 | 20 |
| TAP (min) | 5 | 5 | 5 | 5 | 0000 |
Hair, nail, and skin-scale samples were analyzed by FI-HG-AAS
Digested samples were analyzed by Fl-HG-AAS method against arsenate as the standard
| Table 2: Optimum-Parameters for arsenic determination by flow infection (Fl) system | ||
| Parameters | PerkinElmer (Model 3100) | Varian (Model Spectra AA-20) |
| Lamp Current | 400mA (EDL power supply) | 10mA (hollow cathode) |
| Wavelength | 193.7 nm | 193.7 nm |
| Slit | 0.7 nm | 0.5 nm |
| HCl flow rate | 1.25 ml/min | 1 ml/min |
| HCl concentration | 5M | 5M |
| NaBH4 flow rate | 2 ml/min | 1.5 ml/min |
| NaBH4concentration | 1.25% (W/V) in 0.5% (W/V)NaOH solution | 1.25% (W/V) in 0.5% (W/V)NaOH solution |
| Carrier gas | Nitrogen | Nitrogen |
| Carrier gas flow rate | 130 ml/min | 50 ml/min |
| Flame | Air-acetylene | Air-acetylene |
| Table 3: Analytical performance of FI-HG-AAS for the determination of arsenic | ||
| Parameter | Perkin-Elmer | Varian |
| Sensitivity (Au/ng) | 3.8 X 10-2 | l.9x10-2 |
| Detection limit | 0.1 ng/ml | 0.16 ng/ml |
| Quantitation limit | 0.3 ng/ml | 0.47 ng/ml |
| Precision (CV%) | 1.97 | 3.0 |
| Sample frequency | 100/h | 70/h |
| Table 4: Analysis of Standard Reference Materials (SRM) for arsenic by FI-HG-AAS | ||
| Samples | Certified Value | Found Value |
| CRM (BND 301) NPL waterSRM (QCS) Metals in Water GBW 07601(Hair)NIST, SRM 2670 (Urine) Elevated level Normal levelNIST, SRM 1577b (Bovine liver) * NIST, SRM 1572 (Citrus Leaves) * CRM 278 (Muscle Tissue) *NIES-2 (Pond Sediment)Chinese River Sediment 81-101 (of 1981) | 990 ±200 (µg/L)17.6 ± 2.21 (µg/L)0.28 (µg/gm)480±100 (µg/L)60a (µg/L)0.047 ±0.006 (µg/gm)3. 1 ± 0.3 (µg/gm)5.9 ± 0.2 (µg/gm)12.2 ± 2 (µg/gm)56.0 ± 10.0 (µg/gm) | 960 ± 40 (µg/L)16 ± 3.5 (µg/L)0.278 (µg/gm)477 ± 30 (µg/L)42.4 ± 2.4 (µg/L)0.043 ± 0.003 (µg/gm)± 0.5 (µg/gm)6.1 ± 0.3 (µg/gm)9.85 ± 0.5 (µg/gm)53.79 ± 2.0 (µg/gm) |
aNot certified value, for information only
* Samples were analyzed after washing followed by FI-HG-AAS.
Table 2 shows the optimum parameters for arsenic determination with both Perkin-Elmer and Varian instruments. Low acid concentrations showed lower sensitivity probably due to incomplete reaction. The optimum HCl concentration was 5M and 1.25% NaBH4produced the maximum sensitivity in 5M HCI.
Several transition metals can interfere with the determination of arsenic during hydride generation62 for the batch system. However, the flow injection hydride generation (FI-HG) system showed better tolerance toward hydride forming elements than the batch system63,65.This might be due to shorter reaction time and smaller sample volume. In such a short reaction period most of the interfering transition metal ions could not be reduced to metal and thus, could not absorb or decompose the hydride64. Sensitivity, detection limit and precision were determined for the proposed method. The results are summarized in Table 3. The analytical characteristics were evaluated in accordance with IUPAC recommendation66. To check the accuracy of the techniques I analyzed various types of SRM samples. The results are given in Table 4 andthe results show good agreement with the certified values.
ICP-MS Analysis
ICP-MS is an element selective detector. Twenty microliters (20 µL) of the microwave acid digested sample were injected into a carrier stream of Milli Qwater with a sample loop. The Chromatographic areas were measured, and the concentrations of elements were calculated against the individual element standard curve. The experimental conditions of ICP-MS are given in
Table 5.
To check the accuracy of the techniques I analyzed various types of NIST, SRM samples. The results are given in Table 6 and 7, and itshowsgood agreement with the certified values.
| Table 5: Instrumental conditions for ICP-MS | |
| Mobile phase | Milli Q water |
| Flow rate | 1 ml/min |
| Infection volume | 20 µL |
| Radio frequency (RF) | 1300 W |
| RF refracting power | Below 5 W |
| Flow of plasma gas | 15 L/ min |
| Flow of Carrier gas | 1.2 L/min |
| Measuring time | 2 min |
| Table 6: Analytical results of Standard Reference Material (SRM) by FI-HG-AAS | ||
| Sample | Certified value (As,µg/gm) | Found value (As,µg/gm) |
| NIST, SRM 1572 (Citrus Leaves) | 3.1 ± 0.3 | 3.5 ± 0.5 |
| NIES -2 (Pond Sediment) | 12.2± 2 | 9.85 ±0.5 |
| Chines River Sediment 81-101 (of 1981) | 56.0± 10.0 | 53.79 ± 2.0 |
| NIST, SRM 2709 (San Joaquin Soil) | 17.7 ± 0.8 | 16.87 ± 0.34 |
| NIST, SRM® 1568a (Rice Flour) | 0.29 ± 0.03 | 0.28 ± 0.04 |
| NIST, SRM 15708 (Spinach Leaves) | 0.068 ± 0,012 | 0.062± 0.014 |
| NIST, SRM 1573a (Tomato Leaves) | 0.112 ± 0.004 | 0.100 |
| Table 7: Analytical results of NIST Standard Reference Material (SRM) by ICP-MS | ||||||||||||||||
| Samples | Certified Value (µg/gm) | Found Value (µg/gm) | ||||||||||||||
| As | Se | Mn | Cu | Hg | Pb | Ni | Zn | As | Se | Mn | Cu | Hg | Pb | Ni | Zn | |
| NIST2709(San Joaquin Soil) | 17.7 ± 0.8 | 1.57±0.08 | 538±17 | 34.6±0.7 | 1.4±0.08 | 18.9±0.5 | 88±5 | 106±3 | 16.58±1.79 | 1.87 ± 0.07 | 495±11.32 | 29.35±1.39 | 1.28±0.06 | 21.8±1.9 | 72.17±3.53 | 102.35±4.26 |
| NIST 1568a(Rice Flour) | 0.29±0.03 | 0.38±0.04 | 20.0±1.6 | 2.4 ± 0.3 | 0.0058±0.0005 | <0.01 | - | 19.4 ± 0.5 | 0.29±0.06 | 0.41 ± 0.08 | 17.8±1.8 | 1.9±0.7 | <0.005 | <0.003 | <0.01 | 18.07±0.26 |
| NIST 1515(Apple leaves) | 0.038±0.007 | 0.05±0.009 | 54±3 | 5.64±0.24 | 0.044±0.004 | 0.47±0.024 | 0.91 ± 0.12 | 12.50±0.30 | 0.039±0.007 | 0.052±0.01 | 55.16±0.22 | 5.28±0.26 | 0.43±0.001 | 0.47±0.028 | 1.16±0.04 | 12.98±0.05 |
| NIST 1570a(Spinach Leaves) | 0.068±0.012 | 0.117±0.009 | 75.9±1.9 | 12.20±0.60 | 0.03±0.003 | 0.20 | 2.14±0.10 | 82±3 | 0.062±0.005 | 0.127 ± 0.019 | 67.96±3.14 | 10.29±0.22 | 0.034±0.006 | 0.16±O.01 | 2.27±0.09 | 74.85±4 |
| NIST 1573a(Toma to leaves) | 0.112±0.004 | 0.054±0.003 | 246±8 | 4.70±0.14 | 0.034±0.004 | - | 1.59±0.07 | 30.9±0.70 | 0.100±0.01 | 0.058±0.006 | 182±9 | 4.273±0.26 | 0.034±0.008 | <0.003 | 1.47 ± 0.11 | 26.86±2.86 |
Urine and Blood Collection
Urine and blood samples were collected from 191 subjects (98 females and 93 males), aged 18-77years in the Lagunera area of Mexico. There werefive groups, based on total arsenic concentration (38-116 µg/L) in their drinking water. The collection, processing, and analysis procedures of those samples were previously described67.
Arsenics species analysis in urine
An HPLC-ICP-MS (High Performance Liquid Chromatography- Inductively Coupled Plasma-MassSpectrometry)speciation method was used for themeasurement of arsenic species (AsV, AsIII, MMAV, and DMAV)including arsenobetaine (AsB)67.
Trace elements analysis in urine
After acid digestion, we analyzed trace elements (As, Se, Zn, Co, Cu, Mn, Ni, Cd, Pb, and Hg) in urine samples collectedfrom the subjects67.
Trace elements analysis in whole blood
Whole blood samples were analyzed for total As, Se, Zn, Co, Cu, Mn, Ni, Cd, Pb, and Hg concentrations using Perkin Elmer ElanDRCe ICP-MS67.
Results and Discussion
Total arsenic in biological samples (hair, nail, skin-scales, and urine) of the villagers in Bangladesh where we have identified arsenic patients
Literature survey shows that arsenic concentration in the body tissue and fluids are increasing with increase of arsenic concentration in the drinking water68. Since urine, hair and nail are available, these are used as the universal biomarker. Urinary arsenic has been considered as the most reliable indicator of recent exposure to inorganic arsenic and is used as the main biomarker of exposure68-70. In case of ingestion of inorganic arsenic, experimental studies show that around 60-75% of the dose is excreted in the urine within a few days71-74. In my study, I measured totalarsenic (Urinaryinorg+metabolites), and inorganic arsenic and its metabolites in urine. Arsenic in hair and nails play an important role in evaluating arsenic poisoning from oral ingestion of arsenic. Although, determined arsenic concentration in normal hair and nail is not usually considered very reliable for biologic monitoring due to external contamination75,76. But the concentration of arsenic in hair and nail is usually quite high in arsenic affected villages in Bangladesh and external contamination is not a major problem. Also, arsenic contamination from dust is not a major problem too because villagers have higher arsenic body burden due to arsenic coming from highly arsenic contaminated drinking water.
Statistical presentation of arsenic in hair, nail, urine (inorganic+methylated arsenic), and skin scale samples from the villagers of the arsenic affected villages of Bangladesh where we have found arsenic patients and are presented in Table 8. About 40-50% of these samples were from peoplehaving arsenical skin lesions and rest of the samples from non-patients but they were living in the arsenic affected villages. The analytical report shows95.11%, 83.15%, and 93.77% of the samples we had analyzed have arsenic in urine, hair, and nail above normal/ toxic level (hair), respectively.During our dermatological survey in the affected villages, we have observed that out of 5 people drinking the same arsenic contaminated water, 2 may not show arsenical skin lesions, but their hair, nail, and urine contain high levels of arsenic like other members. Thus, many of the villagers may not have arsenical skin lesions, but they are sub-clinically affected. However, we do not expect such elevated level of arsenic in biological samples from all villagers. The probable reason for such elevated levels of arsenic in hair, nail, and urine is that we have collected these samples from those villagers where arsenic patients exist and many tube wells are highly contaminated. The picture may be different in areas where groundwater is not much contaminated. The overall results from 50 districts show that 37% of the tube wells are safe to drink, according to the WHO recommended value (10 µg /L). Therefore, about 37% of the people should not show an elevated level of arsenic in the biological samples. A study was carried out by our group in 199877to understand why body burden is higher among those using safe water for drinking and cooking, while living in arsenic affected villages78. In this study, safe water from a source having less than 3 µg/Larsenic was supplied for 2 years to 5 affected families to study the loss of arsenic through urine, hair, and nail. The study finally showed that despite having safe water for drinking and cooking, the study group could not avoid an intake of arsenic from contaminated food, food materials contaminated by washing, and the occasional drinking of arsenic contaminated water78. Arsenic in groundwater and in hair, nail, urine of the controlled population is presented in Table 9.
| Table 8: Status of biological samples collected from the people of arsenic affected villages in Bangladesh where we had identified arsenic patients | ||||
| Parameters | Ar Arsenic in hair (µg/kg) | A Arsenic in nail (µg/L) | Ars Arsenic in urine (µg/L) | Arse Arsenic in skin scales (µg/kg) |
| No. of observation | 4386 | 4321 | 1084 | 705 |
| Mean | 3390 | 8570 | 280 | 5730 |
| Median | 2340 | 6400 | 116 | 4800 |
| Minimum | 280 | 260 | 24 | 600 |
| Maximum | 28060 | 79490 | 3086 | 53390 |
| Standard deviation | 3330 | 7630 | 410 | 9790 |
| % of samples having arsenic above normal/toxic(hair) level | 83.15 | 93.77 | 95.11 | - |
Normal level of arsenic in hair ranges from 80 - 250 µg/kg; 1000 µg/kg is the indication of toxicity79
Normal level of arsenic in nail ranges from 430 - 1080 µg/kg80
Normal excretion of arsenic in urine ranges from 5 - 40 µg/day81
There is no normal value for skin scale in literature
| Table 9: Parametric presentation of arsenic in hair, nail, and urine of control population of Patia police station of Chittagong district, Bangladesh where arsenic in groundwater was below 3 µg/L. | |||
| Parameters | A Arsenic in hair (µg/kg) | A Arsenic in nail (µg/kg) | Ar Arsenic in urine (µg/L) |
| No. of observation | 62 | 62 | 62 |
| Mean | 410 | 830 | 31 |
| Minimum | 210 | 90 | 6 |
| Maximum | 850 | 1580 | 94 |
| Standard deviation | 180 | 680 | 20 |
Normal level of arsenic in hair ranges from 80 - 250 µg/kg; 1000 µg/Kg is the indication of toxicity79
Normal level of arsenic in nail ranges from 430 - 1080 µg/kg80
Normal excretion of arsenic in urine ranges from 5 - 40 µg/day81
From our overall study in Bangladesh, we have observed that arsenic in hair, nail, and urine increases with increasing arsenic in drinking water. Figures 3-5 show our findings. It appears that correlations are not strongly positive (for hair samples r = 0.251, p = 0.01, n = 739; for nail samples r = 0.220, p = 0.01, n = 691; and for urine r = 0.547, p = 0.01, n = 910). Probable reason is that people are not drinking from the same source all the time. Our field data indicates that most of the villagers’ drink water from more than one tube well.
Total arsenic, and other metals and metalloids in some biological samples [hair and nail] measured by using ICP-MS after microwave digestion
Along with arsenic in hair and nail samples, I also analyzed Se, Zn, Cu, Hg, Pb, and Sb by ICP-MS after microwave digestion. Our analysis of Standard human hair (NCS, CRM DC 73347) sample by the same procedure is in well agreement (Table 10). Statistical presentation of As, Se, Zn, Cu, Hg, Pb, and Sb in hair and nail samples of exposed and control population are shown in Tables 11 and 12, respectively. It appears in both cases (hair and nail) the mean concentration of Se and Zn are lower in exposed group than control group [for Se 167 vs. 295 µg/kg (hair) and 88 vs. 194 µg/kg (nail); and for Zn 92,013 vs. 123,255 µg/kg (hair) and 72,465 vs. 91,089 µg/kg (nail), where arsenic concentration (mean value) in hair 2,749 vs. 187 µg/kg and in nail 6,864 vs. 462 µg/kg for exposed and control group, respectively]. Again the mean concentration of Hg, Pb, and Sb are higher in both hair and nail for exposed group than control group [for Hg 789 vs. 476 µg/kg and 646 vs. 506 µg/kg; for Pb 5,531 vs. 4,587 µg/kg and 5,010 vs. 4,272; and for Sb 283 vs. 150 and 1049 vs. 225 µg/kg for exposed and control group, respectively, where arsenic concentration (mean value) in hair 2,749 vs. 187 µg /kg and in nail 6,864 vs. 462 µg/kg for exposed and control group, respectively]. But the mean concentration of Cu in both hair and nail is higher [6,778 vs. 5,677 µg/kg (hair) and 5,420 vs. 4,730 µg/kg (nail)] in control group than exposed group, respectively.
Table 10: Analysis of Standard Human Hair sample (NCS, CRM DC 73347) for arsenic by ICP-MS after microwave digestion
| Sample | Elements | Certified value (µg/gm) | Found value (µg/gm) |
| Human hair (CRM73347) | As | 0.28 ± 0.04 | 0.28 ± 0.01 |
| Se | 0.60 ± 0.03 | 0.709 ± 0.13 | |
| Zn | 190±5.00 | 182.84 ± 5.6 | |
| Cu | 10.60 ± 0.70 | 12.10 ± 0.57 | |
| Hg | 0.36 ± 0.05 | 0.35 ± 0.01 | |
| Pb | 8.8 ± 0.90 | 10.37±0.13 | |
| Sb | 0.095 ± 0.012 | 0.109 ± 0.005 |
Table 11: Statistical presentation of As, Se, Zn, Cu, Hg, Pb, and Sb in hair of chronic exposed and control population of MadaripurSadar police station of Madaripur district, Bangladesh and Bhupatinagar police station of Medinipur district, West Bengal-India, respectively
| Parameters | Exposed group (from Madaripur, Bangladesh)where arsenic in drinking water > 50 µg/L | Control group (from Medinipur, West Bengal-India) where arsenic in drinking water < 3 µg/L | |||||||||||||
| As | Se | Zn | Cu | Hg | Pb | Sb | As | Se | Zn | Cu | Hg | Pb | Sb | ||
| [µg/kg] | [µg/kg] | ||||||||||||||
| No. of observation | 19 | 16 | 16 | 17 | 19 | 19 | 18 | 30 | 13 | 28 | 17 | 19 | 17 | 18 | |
| Mean | 2749 | 167 | 92013 | 5677 | 789 | 5531 | 283 | 187 | 295 | 123255 | 6778 | 476 | 4587 | 150 | |
| Median | 2666 | 149 | 90306 | 5812 | 711 | 3444 | 224 | 178 | 216 | 122274 | 5794 | 440 | 3739 | 123 | |
| Minimum | 1009 | 50 | 62270 | 3448 | 139 | 1148 | 69 | 53 | 139 | 60195 | 3467 | 242 | 1375 | 50 | |
| Maximum | 6675 | 285 | 139088 | 10235 | 1766 | 16945 | 665 | 520 | 890 | 210650 | 12413 | 755 | 10787 | 322 | |
| Standard deviation | 1650 | 71 | 22996 | 1886 | 397 | 5085 | 170 | 109 | 217 | 39058 | 2642 | 144 | 3004 | 76 | |
Table 12: Statistical presentation of As, Se, Zn, Cu, Hg, Pb, and Sb in nail of chronic exposed and control population of MadaripurSadar police station of Madaripur district, Bangladesh and Bhupatinagar police station of Medinipur district, West Bengal-India, respectively
| Parameters | Exposed group (from Madaripur, Bangladesh) where arsenic in drinking water > 50 µg/L | Control group (from Medinipur, West Bengal-India) where arsenic in drinking water < 3 µg/L | ||||||||||||
| As | Se | Zn | Cu | Hg | Pb | Sb | As | Se | Zn | Cu | Hg | Pb | Sb | |
| [µg/kg] | [µg/kg] | |||||||||||||
| No. of observation | 40 | 27 | 35 | 31 | 35 | 31 | 39 | 29 | 11 | 26 | 17 | 18 | 18 | 19 |
| Mean | 6864 | 88 | 72465 | 4730 | 646 | 5010 | 1049 | 462 | 194 | 91089 | 5420 | 506 | 4272 | 225 |
| Median | 536 | 50 | 64878 | 3914 | 494 | 3864 | 850 | 420 | 150 | 80000 | 4941 | 452 | 2791 | 198 |
| Minimum | 1011 | 50 | 38057 | 1506 | 137 | 1239 | 97 | 189 | 103 | 63081 | 1575 | 233 | 455 | 77 |
| Maximum | 21174 | 202 | 124300 | 8971 | 1984 | 18190 | 4104 | 960 | 430 | 162000 | 12905 | 1062 | 11852 | 449 |
| Standard deviation | 4615 | 56 | 24170 | 2093 | 436 | 3491 | 834 | 198 | 93 | 25990 | 2885 | 247 | 3680 | 99 |
The regression analyses were carried out between arsenic and other metals and metalloids in hair and nail samples. The linear regression shows negative correlation between As & Se (r = - 0.84, p = 0.00005, n = 16) and As & Zn (r =- 0.78, p =0.0003. n = 16); somewhat positive correlation between As &Pb (r = 0.58, p = 0.008, n = 19), As & Hg (r = 0.745. p 0.0002,n = 19) and As & Sb (r = 0.743, p = 0.0002, n = 19); no significant correlation between As & Cu (r = -0.04, p = 0.87, n = 17) for hair samples. For nail samples a similar correlation observed as in hair. The linear regression shows negative correlate between As & Se (r = - 0.53, p = 0.004, n = 27) and As & Zn (r = - 0.55, p = 0.0006, n = 35) and somewhat positive correlation between As & Hg (r = 0.47, p = 0.004, n =35), As &Pb (r = 0.51, p = 0.003, n = 33), and As & Sb (r = 0.57, p = 0.0004, n = 33); nosignificant correlation between As & Cu (r = 0.15, p = 0.41, n =31) observed. Filon, J., et al (2020)82 found statistically significant mean positive correlations between Pb and As in the hair of children with autism spectrum disorders (ASD). Epidemiologic studies suggest synergistic effects from binary combinations of Pb-As83.
Toxicological and metabolic interactions of selenium (Se) with arsenic (As) have been reported in many experimental studies. However, for human populations, possible interactions between As and Se, and their toxicological significance have not been established. Miyazaki, K., et al (2003)84have examined the relationship between Se and As in spot urine samples collected from the inhabitants of two rural communities of northeast Bangladesh and negative correlation between UAs and USe was found in both females ((r = –0.25, p < 0.01) and males (r = –0.16, p < 0.05)84. They explain this inverse correlation might be due to inverse intakes of these two elements, i.e., high As containing tube well water may contain low Se and vice versa84.
Segmental hair analysis report
Arsenic concentration is normally higher in hair and nail than in other parts of the body because of the high content of keratin,the -SH group of which might bind trivalent inorganic arsenic85-87. Therefore. Hair and nail might be considered an excretorypathway of arsenic from the body. Arsenobetaine, the major organic arsenic compound in seafood, is not accumulatedin hair88.
That implies that arsenic in hair and nail reflects exposure to inorganic arsenic only. Experimental studies in which radiolabeled DMA was administered to mice and rats showed very low incorporation of DMA in skin and hair compared withthat of inorganic arsenic 89. However, others reported presence of DMA in hair and nail90, 91.
Segmented hair analysis (i.e., determination of the concentration along the length of the hair) might provide valuable information on the time of acute arsenic exposure92-94. For example, Smith, H. (1964)92reported a case in which a single fatal ingestion of 800 mg of arsenic trioxide gave rise to a concentration of only 860 µg/kg for the whole length of hair 30 cm long. Further, analysis showed that the concentration of arsenic in the first millimeter, including the root, was 90,000 µg/kg. In another fatal case95of arsenic poisoning, centimeter segmental analysis revealed that the concentrations of arsenic varied between 28,000 and 226,000 µg /kg.
For my experiment I had collected long hair samples from four chronic female patients having arsenical skin lesions. Before washing I cut the samples in different segments from root to top (each segment about 4 cm length) and analyzed them by ICP-MS after microwave digestion. The analytical results are shown in Table 13. Table 13 shows the distribution of arsenicin different segments of hair sample (from root to top). It appears that arsenic concentration in hair is decreasing trend with increased length from root to top. However, the decrease is not sharp as observed for acute toxicity92.
Trace elements concentrations in urine and whole blood of arsenic exposed people from subjects in the Lagunera areaof Mexico 67
The concentrations of As, Se, and Zn in urine and blood are reported in Table 14. The elements concentrations in urine expressed as ug/g cre were higher for females (F) compared to males (M). But the concentrations of As and Zn were lower, but Se was almostsame in blood for females compared to males.
Influence of the relative concentrations of Se or Zn to arsenic in urine on the percentage of urinary arsenic metabolites 67
The
