Serum amino acid profiles in clinically normal Noma horses.

Plasma or serum amino acids are used to evaluate nutritional status and metabolic disorders. In this study, we aimed to set reference values of serum amino acid concentrations in the Noma horse, a Japanese native horse. Thirty-one horses were classified into six age groups: neonatal foal (0-4 days), foal (0.5-1 years), youth (5 years), middle age (10 years), old (15 years), and extra-old (>20 years). Horses >5 years of age were analyzed together as the adult group. In the adult horses, there were no significant differences among the serum amino acid concentrations of each age group. The foal group had higher concentrations of alanine, aspartic acid, glutamic acid, α-aminoadipic acid, and 3-methyl-histidine than the adult group. The neonatal foal group had higher serum concentrations of phenylalanine, lysine, alanine, proline, aspartic acid, glutamic acid, ß-alanine, and ß-amino-iso-butyric acid and lower tryptophan concentrations and Fischer's ratios than the adult group. The neonatal foal group had higher ß-amino-iso-butyric acid concentrations and lower tryptophan and 3-methyl-histidine concentrations than the foal group. Therefore, reference values might be set separately in neonatal foals, foals, and adult horses. The data for the serum amino acid concentrations can be used for health care through physiological and pathological evaluations in Noma horses.

Relationships between the age and blood test results or body sizes in Noma horses.

The objective of this study is to analyze the relationships between the age and blood test results or body sizes in Noma horses by using the results of periodical health examination. Out of 45 hematological or physical items examined, statistically significant, but loose correlations were observed in 14 items. Red blood cell count, activities of aspartate aminotransferase, alkaline phosphatase, and creatinine kinase, concentrations of calcium and inorganic phosphorus decreased with aging. Conversely, mean corpuscular volume, mean corpuscular hemoglobin, lipase activity, γ-globulin and chloride concentrations, body height, chest circumference and cannon bone circumference increased with aging. The changes in a few items seemed unique to Noma horse. However, most age-related changes found in this study might be considered as a common trend in horse breeds rather than distinctive characteristic in Noma horse.

Effect of seasons and sex on the physical, hematological, and blood biochemical parameters of Noma horses.

This study aimed to evaluate the influence of seasons and sex on body size and hematological and biochemistry parameters of Noma horses, a native Japanese breed. Body size was larger in winter than in summer. Laboratory testing variables, including erythrocytic parameters and urea nitrogen, total cholesterol, and creatinine kinase levels, were higher in winter, while the eosinophil count was higher in summer. These seasonal differences may be related to increased energy consumption of horses due to heat stress. The higher eosinophil counts may have been related to the dermatitis observed in summer. Stallions tended to have smaller bodies compared with mares. Future studies are necessary to investigate the effect of stress in seasonal and sex-based groups.

Reference values of hematological and blood biochemical parameters for the Noma horse.

The Noma horse is a Japanese breed from the Noma region of Imabari City, Ehime Prefecture. To obtain reference hematological and biochemical values, we performed examinations in 39 clinically healthy, mature Noma horses managed at the Imabari public ranch. Hematological and biochemical results of Noma horses were close to the normal ranges of horses in the U.S.A. The erythrocyte parameters and hepatobiliary enzyme levels in Noma and Kiso horses were lower than those in Japanese racehorses. Noma horses showed higher erythrocyte parameters and triglyceride concentrations and a lower creatinine concentration compared with those in Kiso horses. These data represent the first report of reference values for Noma horses and may be useful to improve their management.

Effect of 2% meloxicam injection in Holstein dairy cows on acute clinical mastitis without systemic symptoms.

This study aimed the efficacy of meloxicam (MX) in treating acute clinical mastitis (ACM) without systemic symptoms in Holstein cows by studying improvement in udder pain, changes in prostaglandin E2(PGE2) and bradykinin (BK) levels in the milk, and milk yield (MY) after healing. Forty-two cows with ACM were randomly assigned to the MX treatment group (T group; n=21) and the control group (C group; n=21). At onset of illness (day 0), the T group received a 0.5 mg/kg subcutaneous (SC) injection of MX whereas the C group received 15 mL SC of saline solution as a placebo. Udder tenderness (UT) was measured, and milk samples were collected on days 0-3. There was little change in the MY of the T group before and after healing, whereas MY in the C group was significantly lower than after healing. UT on day 3 in the T group was significantly lower than that in the C group. PGE2 levels significantly decreased from day 0 to day 3 in both groups. A significant negative correlation between PGE2 and linear score was observed on day 1 in the T group, but not in the C group. In ACM without systemic symptoms, the administration MX may be useful for restoring MY and reducing udder pain after healing.

Arsenic speciation analysis of urine samples from individuals living in an arsenic-contaminated area in Bangladesh.

OBJECTIVES: Chronic inorganic arsenic (iAs) exposure currently affects tens of millions of people worldwide. To accurately determine the proportion of urinary arsenic metabolites in residents continuously exposed to iAs, we performed arsenic speciation analysis of the urine of these individuals and determined whether a correlation exists between the concentration of iAs in drinking water and the urinary arsenic species content. METHODS: The subjects were 165 married couples who had lived in the Pabna District in Bangladesh for more than 5 years. Arsenic species were measured using high-performance liquid chromatography and inductively coupled plasma mass spectrometry. RESULTS: The median iAs concentration in drinking water was 55 µgAs/L (range <0.5-332 µgAs/L). Speciation analysis revealed the presence of arsenite, arsenate, monomethylarsonic acid (MMA), and dimethylarsinic acid in urine samples with medians (range) of 16.8 (7.7-32.3), 1.8 (<0.5-3.3), 13.7 (5.6-25.0), and 88.6 µgAs/L (47.9-153.4 µgAs/L), respectively. No arsenobetaine or arsenocholine was detected. The concentrations of the 4 urinary arsenic species were significantly and linearly related to each other. The urinary concentrations of total arsenic and each species were significantly correlated with the iAs concentration of drinking water. CONCLUSIONS: All urinary arsenic species are well correlated with each other and with iAs in drinking water. The most significant linear relationship existed between the iAs concentration in drinking water and urinary iAs + MMA concentration. From these results, combined with the effects of seafood ingestion, the best biomarker of iAs exposure is urinary iAs + MMA concentration.

Surveillance of antimicrobial-resistant Escherichia coli in Sheltered dogs in the Kanto Region of Japan.

There is a lack of an established antimicrobial resistance (AMR) surveillance system in animal welfare centers. Therefore, the AMR prevalence in shelter dogs is rarely known. Herein, we conducted a survey in animal shelters in Chiba and Kanagawa prefectures, in the Kanto Region, Japan, to ascertain the AMR status of Escherichia coli  (E. coli) prevalent in shelter dogs. E. coli was detected in the fecal samples of all 61 and 77 shelter dogs tested in Chiba and Kanagawa, respectively. The AMR was tested against 20 antibiotics. E. coli isolates derived from 16.4% and 26.0% of samples from Chiba and Kanagawa exhibited resistance to at least one antibiotic, respectively. E. coli in samples from Chiba and Kanagawa prefectures were commonly resistant to ampicillin, piperacillin, streptomycin, kanamycin, tetracycline, and nalidixic acid; that from the Kanagawa Prefecture to cefazolin, cefotaxime, aztreonam, ciprofloxacin, and levofloxacin and that from Chiba Prefecture to chloramphenicol and imipenem. Multidrug-resistant bacteria were detected in 18 dogs from both regions; ß-lactamase genes (blaTEM, blaDHA-1, blaCTX-M-9 group CTX-M-14), quinolone-resistance protein genes (qnrB and qnrS), and mutations in quinolone-resistance-determining regions (gyrA and parC) were detected. These results could partially represent the AMR data in shelter dogs in the Kanto Region of Japan.

Comparison of Analytical Values after Changing to the International Standardized Method for Lactate Dehydrogenase and Alkaline Phosphatase Measurements in Mouse and Rat.

Since April 2020, the method for lactate dehydrogenase (LD) and alkaline phosphatase (ALP) activity measurements in Japan has been switched from the Japan Society of Clinical Chemistry (JSCC) reference method, which is only used in Japan, to the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) reference method. However, in some species, the relationship between the blood values of both enzymes measured by the two methods remains unclear. Hence, values measured by these two methods cannot be used interchangeably. Therefore, this relationship was examined in ICR mice and Wistar/ST rats. The LD and ALP values obtained by both methods were plotted on scatter graphs, and regression equations were obtained. To compare the JSCC (x) and IFCC (y) methods, regression equations were generated for LD values in non-hemolytic samples as follows: y = 0.954x - 4.008 for ICR mice and y = 0.963x - 6.324 for Wistar/ST rats. The conversion factors from the JSCC to the IFCC methods were 0.954 (mice) and 0.963 (rats). The conversion coefficients from the IFCC to the JSCC methods were 1.048 (mice) and 1.088 (rats). For ALP values in fasted mouse and rat samples, the regression equations were y = 0.336x - 2.247 and y = 0.314x - 17.626, respectively. The conversion factors from the JSCC to the IFCC methods were 0.336 (mice) and 0.314 (rats). The conversion coefficients from the IFCC to the JSCC methods were 2.978 (mice) and 3.188 (rats). These conversion factors can be used for the mutual conversion of both measured values during the transition period from the JSCC to the IFCC method. However, it should be noted that the conversion coefficients for both LD and ALP were affected by isozyme composition.

Changes in the blood lactate dehydrogenase measurements in canines and felines following the international standardization of the assay method.

Lactate dehydrogenase (LDH) in blood is measured using the Japanese Society of Clinical Chemistry (JSCC) method in Japan and the International Federation of Clinical Chemistry (IFCC) method in other countries. In human clinical practice, the IFCC method replaced the JSCC method due to international standardization. Moreover, veterinary LDH measurement will also eventually shift to the IFCC method. However, the relationship between the IFCC and JSCC methods for LDH in various animals and whether they can be equated or not have not yet been investigated. This study aimed to present the changes in measurements in canines and felines after switching to the IFCC method. The plasma LDH activity of canines (N=177) and felines (N=115), who visited a secondary care veterinary clinic, was measured using the JSCC and IFCC methods. The IFCC/JSCC ratio was <1.0 in 85% of canines and 88% of felines, indicating that the IFCC method tended to show lower values than the JSCC method, presumably because LDH5 is dominant among the LDH isozymes in canines and felines. The increase in the systematic errors of both assays was in the high value range, with some samples exceeding the error tolerance from near the upper end of the reference range. When switching to the IFCC method for LDH measurement in canines and felines, each institution should consider whether the reference range and clinical diagnostic values established by the JSCC method are appropriate for continued use.

Development of a canine blood C-reactive protein-measuring device using a flow-type immunosensor.

This study aimed to construct a measurement system with the same performance as a measurement system using an automated analyzer and immunoturbidimetric reagents (comparative method) using a flow-type immunosensor (FIS) based on the fluorescence-linked immunosorbent assay technology. In the FIS constructed in this study, all control samples were within the indicated values. The coefficient of variation of repeatability and intermediate precision were less than 2.4% and less than 4.4%, respectively. The lower limit of quantification in this measurement system was 3.9 mg/L, and linearity was confirmed for quantification values, ranging from 3.9 to 465 mg/L. Canine plasma samples (N = 39) were used to measure C-reactive protein (CRP) levels using the comparative method (x) and FIS (y). The regression equation between the measurements was y = 1.035 × - 0.002, with a correlation coefficient of 0.9809, indicating a significantly high correlation. Although the Brandt-Altman analysis suggested the possibility of a proportional systematic error between the two measurements, 38 of the 39 canine plasma samples measured fell within the acceptable range of error, indicating that the measurements are highly consistent. These results suggest that the analytical accuracy of the FIS constructed in this study and the quantitative value of canine CRP are equivalent to those of measurement systems using automated analyzers and immunoturbidimetric reagents.

The relationships between environmental parameters in livestock pen and physiological parameters of Holstein dairy cows.

There has been an increase in temperature and the incidence of extreme weather events, such as heat wave, due to global warming, which has promoted the incidence of livestock diseases. Therefore, it is important to examine the effect of changes in environmental parameters on livestock performance. The aim of this study was to examine the relationship between ambient environmental conditions in livestock pen and the physiological parameters of Holstein dairy cows. The results showed that there was a decrease in the red blood cell counts, hemoglobin concentrations, and mean corpuscular hemoglobin concentration of the cows with increasing pen temperature, wet bulb globe temperature (WBGT), and temperature humidity index (THI). Additionally, high daily variation in temperature caused a decrease in the serum albumin levels of the cows. Moreover, the lowest serum calcium, inorganic phosphorus, and magnesium concentrations were observed in November, and were negatively correlated with the 24-hr temperature, WBGT, and THI range of the pen prior to sampling. Multiple regression analysis showed a positive correlation between serum cortisol concentration and 24-hr WBGT range of the pen prior to samplings and packed cell volume. However, serum cortisol and total protein concentrations were negatively correlated. Overall, the findings of the study suggest that large variation in temperature induced stress in the cows, which could be overcome by increased water consumption and improved protein digestion and absorption by the animals, and the addition of minerals, such as calcium to the diet.

Distal renal tubular acidosis and lethargy associated with zonisamide treatment in a dog with idiopathic epilepsy.

A 3-year-old neutered male golden retriever administered zonisamide for the treatment of seizures showed lethargy and had normal anion gap metabolic acidosis with hypokalaemia, hyperchloremia, and alkaline urine. The serum zonisamide concentration was close to the upper limit, which raised a suspicion of adverse effects of zonisamide. This is the first report showing that the fractional excretion of bicarbonate after compensation for the plasma bicarbonate concentration by a sodium bicarbonate infusion was approximately 5%, indicating distal renal tubular acidosis (RTA). The serum zonisamide concentration decreased, and adverse effects were abated by reducing the zonisamide dosage. Diagnostic therapy with bicarbonate served as a means of compensating for bicarbonate deficiency and contributed to the clinical diagnosis of the condition in zonisamide-associated RTA in dogs.

Canine mast cell tumour cells regulate tryptophan catabolism via the expression of indoleamine 2,3-dioxygenase.

Indoleamine 2,3-deoxygenase (IDO) produced by cancer cells catabolizes tryptophan (TRP) to kynurenine (KYN) in the environment, resulting induction of cancer immune escape through induction of T cell anergy and enhancement of regulatory T cells. Recently, inhibition of IDO has been recognized as one of therapeutic strategies for human neoplastic diseases. However, there have been few reports about IDO-expressing cancers in dogs. In this study, we attempted to examine whether canine mast cell tumour (MCT) cells express IDO and modulate the concentration of TRP and KYN in the environment. BR, MPT-1.2, and MPT-3 cells were used as canine MCT cells. Expression of IDO was examined with RT-PCR and western blotting. Concentrations of TRP and KYN in the culture medium after incubation with canine MCT cells were detected with liquid chromatography-tandem mass spectrometry. The expression of mRNA and protein of IDO were confirmed in all samples extracted from canine MCT cells. TRP concentration in the culture medium was decreased and that of KYN was increased on incubation with canine MCT cells. The ratio of KYN/TRP, widely considered to represent IDO activity, was also significantly elevated. Moreover, treatment with an IDO inhibitor L-1-methyl-tryptophan (L-1MT) clearly diminished the elevation of KYN/TRP ratio induced by the incubation with canine MCT cells. Our results indicate that canine MCT cells could directly regulate the concentrations of TRP and KYN through expressing IDO, suggesting that canine MCT have an immune escape ability. Therefore, inhibition of IDO might be a novel strategy for the treatment of dogs with MCT.

Comparison of regression for blood ALP levels using methods of the Japan Society of Clinical Chemistry and the International Federation of Clinical Chemistry and Laboratory Medicine in bovine, canine, feline, and human testing.

Livestock and companion animal health have a direct impact on human health. Research on clinical laboratory technology for veterinary medicine is as important as that on human laboratory technology. Reagents and analysis equipment for human medical laboratory tests are often used in veterinary medicine. Medical laboratories in Japan utilize the Japan Society of Clinical Chemistry (JSCC) method for blood alkaline phosphatase (ALP) analysis. The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) method is used worldwide for ALP catalytic concentration measurement. When the IFCC method is used, human blood ALP activity is approximately one-third of the JSCC method's activity. The JSCC method for ALP measurement was switched to the IFCC method in medical laboratories in Japan in April 2020 for global standardization purpose. It is uncertain whether conventional JSCC method reagents will continue to be supplied. In veterinary medicine, the relationship between the JSCC and IFCC methods in terms of ALP measurement is almost unclear. This study investigated the regression between JSCC and IFCC methods measuring ALP in bovine, canine, feline, and human. The regression formulas for bovine, canine, feline, and human ALP values using the conventional JSCC (x) and IFCC (y) methods are y = 0.379x + 0.124, y = 0.289x + 8.291, y = 0.358x + 0.432, and y = 0.337x + 2.959, respectively. These results suggested that the IFCC method measurement could be estimated by approximately one-third of the JSCC method measurement in animal species such as bovine, canine, and feline. By applying the conversion factors proposed in this study, a very good correlation could be obtained between the two methods for each animal.

Distribution of urinary gamma-glutamyltransferase activity in 40- to 74-year-old Japanese women.

Urinary gamma-glutamyltransferase (u-γGT) concentration (U/L) and excretion (urinary creatinine-corrected u-γGT; u-γGT/u-Cre, U/g creatinine) are useful markers for kidney disease. However, there is limited information available on u-γGT and u-γGT/u-Cre distribution in the elderly Japanese population. In this study, we investigated the distribution of u-γGT and u-γGT/u-Cre in 113 Japanese women aged 40-74 years. The u-γGT was assessed from spot urine samples (collected from 09:00 to 14:00) spectrophotometrically according to the Japan Society of Clinical Chemistry reference measurement procedure using l-γ-glutamyl-3-carboxy-4-nitroanilide as the substrate. The u-Cre was measured enzymatically using creatininase, creatinase, sarcosine oxidase, and peroxidase. None of the participants was diagnosed with any kidney disease. Median u-γGT and u-γGT/u-Cre values (central 95% interval values) were 29.7 (5.3-144.0) U/L and 57.9 (32.9-122.7) U/g creatinine, respectively. The distribution of u-γGT tended to decline with age. There was a statistically significant difference in the u-γGT value between the 40-59- and 60-74-year-old groups. In contrast, there was no significant difference in the u-γGT/u-Cre between each age group. The u-Cre level also declined with age. It is suggested that the decline of u-γGT with aging would be masked by the u-Cre correction.

Regression formula to predict the International Federation of Clinical Chemistry and Laboratory Medicine measure of alkaline phosphatase activity in canine blood based on the Japan Society of Clinical Chemistry reference method.

The Japan Society of Clinical Chemistry reference method (JSCC method) is used to measure alkaline phosphatase (ALP) activity only in Japan. Other countries use the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) reference method to measure ALP activity. Since April 2020, human medical institutions in Japan have been gradually switching to the IFCC method. However, it is unclear whether the supply of reagents required for the JSCC method will be steady in the future. Additionally, the comparison of the performances and accuracies of these two methods for measuring ALP values remains uncertain in several animal species. In this investigation, we measured canine ALP activity using both methods and developed a formula to interconvert the two resulting values. The regression formula for ALP values measured using the modified JSCC (x) and IFCC (y) methods was determined as log10 y=0.960 log10 x-0.395 (r=0.997). However, the correlation between values based on JSCC and IFCC methods can change depending on the composition of ALP isozymes. Therefore, the developed formula can currently serve as a provisional strategy in calculating ALP levels. Nevertheless, this formula might avoid confusion in the clinical field during the transition from the JSCC to the IFCC method when both measurement values co-exist.

Metabolism of 3-[5'-deoxy-5'-(dimethylarsinoyl)-ß-ribofuranosyloxy]-2-hydroxypropylene glycol in an artificial digestive system.

Seaweeds contain large amounts of organoarsenic compounds, mostly arsenosugars (AsSug) and arsenolipids (AsLipid). AsSug is mainly metabolized into dimethylarsinic acid (DMA V ) in humans. However, this metabolic process is not well understood. We investigated the metabolism of an AsSug, 3-[5'-deoxy-5'-(dimethylarsinoyl)-ß-ribofuranosyloxy]-2-hydroxypropylene glycol (AsSug328), in the gastrointestinal tract using an in vitro artificial gastrointestinal digestion system. AsSug328 was incubated with gastric juice for 4 h, with bile-pancreatic juice for 0.5 h, and finally with enteric bacteria solution for 24 h. The conversion of arsenic compounds after artificial digestion was analyzed by HPLC-ICP-MS and HPLC-ESI-Q-TOF-MS. Our results show that artificial gastrointestinal digestion converted AsSug328 into thio-AsSug328. However, no formation of DMA V was detected. Under the artificial digestion system, the 5-deoxyribofuranose structure of AsSug was maintained. Therefore, AsSug should be absorbed in the intestinal tract after its sugar moiety is partially decomposed. They are then possibly metabolized to DMA V in the liver and subsequently excreted through urine.

Differences in apoptotic signaling and toxicity between dimethylmonothioarsinic acid (DMMTAV) and its active metabolite, dimethylarsinous acid (DMAIII), in HepaRG cells: Possibility of apoptosis cascade based on diversity of active metabolites of DMMTAV.

Dimethylmonothioarsinical acid (DMMTAV), a metabolite of arsenosugars (AsSug) and arsenolipids (AsLP), which are major organoarsenicals contained in seafoods, has been a focus of our attention due to its toxicity. It has been reported that the toxicity of DMMTAV differs according to the host cell type and that dimethylarsinous acid (DMAIII), which is a higher active metabolite of inorganic and organo arsenic compounds, may be the ultimate substance. To further elucidate the details of the mechanisms of DMMTAV, we carried out toxicological characterization by comparing DMMTAV and DMAIII using HepaRG cells, which are terminally differentiated hepatic cells derived from a human hepatic progenitor cell line that retains many characteristics, e.g, primary human hepatocytes including the morphology and expression of key metabolic enzymes (P450 s and GSTs, etc.) and complete expression of all nuclear receptors. HepaRG cells were induced to undergo differentiation by DMSO, which result red in increased levels of metabolic enzymes such as P450 and GST, in non-differentiated cells the cellular toxicities of DMMTAV and DMAIII were reduced and the induction of toxicity by DMMTAV was increased by GSH but not by DMAIII. Both DMAIII and DMMTAV induce apoptosis and increase caspase 3/7 activity. DMAIII exposure increased the activity of caspase-9. On the contrary, DMMTAV exposure resulted in markedly elevated activity of caspase-8 as well as caspase-9. These results suggest there are differences between the signaling pathways of apoptosis in DMAIII and DMMTAV and that between their active metabolites. Consequently, the ultimate metabolic substance of toxicity induction of DMMTAV may not only be DMAIII, but may also be partly due to other metabolic substances produced through the activation mechanism by GSH.

HPLC-ICP-MS speciation analysis of arsenic in urine of Japanese subjects without occupational exposure.

The toxicity and carcinogenicity of arsenic depend on its species. Individuals living in Japan consume much seafood that contains high levels of organoarsenics. Speciation analysis of urinary arsenic is required to clarify the health risks of arsenic intake. There has been no report of urinary arsenic analysis in Japan using high performance liquid chromatography with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). We performed speciation analysis of urinary arsenic for 210 Japanese male subjects without occupational exposure using HPLC-ICP-MS. The median values of urinary arsenics were as follows: sodium arsenite (AsIII), 3.5; sodium arsenate (AsV), 0.1; monomethylarsonic acid (MMA), 3.1; dimethylarsinic acid (DMA), 42.6; arsenobetaine (AsBe), 61.3; arsenocholine, trimethylarsine oxide, and unidentified arsenics (others), 5.2; and total arsenic (total As), 141.3 microgAs/l. The median creatinine-adjusted values were as follows: AsIII, 3.0; AsV, 0.1; MMA, 2.6; DMA, 35.9; AsBe, 52.1; others 3.5; and total As, 114.9 microgAs/g creatinine. Our findings indicate that DMA and AsBe levels in Japan are much higher than those found in Italian and American studies. It appears that the high levels of DMA and AsBe observed in Japan may be due in part to seafood intake. ACGIH and DFG set the BEI and BAT values for occupational arsenic exposure as 35 microgAs/l and 50 microgAs/l, respectively, using the sum of inorganic arsenic (iAs), MMA, and DMA. In the general Japanese population, the sums of these were above 50 microgAs/l in 115 (55%) samples. We therefore recommend excluding DMA concentration in monitoring of iAs exposure.

Effectiveness of arsenic mitigation program in Bangladesh--relationship between arsenic concentrations in well water and urine.

BACKGROUND: Arsenic in drinking water remains a major public problem in Bangladesh, although arsenic mitigation programs began there a decade ago. The purpose of this study was to examine the effectiveness of this program by determining the relationship between current arsenic levels in well water and the high level of urinary arsenic excretion. METHODS: A community-based cross-sectional study was conducted in the Pabna district of Bangladesh between May and July 2005. We included 174 married couples and collected their drinking water from 138 wells. The allowable limit for arsenic in drinking water is 50 microg/L in Bangladesh, while the normal level of urinary arsenic is < or =40 microg x 1.5 L(-1) x day(-1) by Dhaka Community Hospital. RESULTS: Of 348 subjects, 304 exceeded the urinary arsenic level of 40 microg x 1.5 L(-1) x day(-1). Of all wells, 44.2% had arsenic levels >50 microg/L. Multiple-adjusted odds ratios of urinary arsenic level >40 microg x 1.5 L(-1) x day(-1) were 8.90 (95% CI: 3.31-23.93) for the arsenic level in well water of 11-50 microg/L, and 53.07 (11.91-236.46) for that of 51-332 microg/L, compared with < or =10 microg/L. When the Bangladeshi standard arsenic level in drinking water of 50 microg/L was used, the sensitivity in detecting subjects with a urinary arsenic level >40 microg x 1.5 L(-1) x day(-1) was 50%, although when the World Health Organization (WHO) guideline value of 10 microg/L was used, it was 76.3%. CONCLUSIONS: Green marked wells, which the Bangladesh government regards as safe, are not always safe. The mitigation programs should use the WHO guideline arsenic level to determine the safety of well water for drinking.