Blackfoot Disease Patients Research Articles

Anti-endothelial cell IgG from patients with chronic arsenic poisoning induces endothelial proliferation and VEGF-dependent angiogenesis

An endemic peripheral vascular disorder due to chronic arsenic poisoning, named Blackfoot disease (BFD), occurs in Taiwan. BFD causes destruction of vascular endothelial cells, and an anti-endothelial cell IgG antibody was found in the sera of BFD patients. We studied the role of this IgG antibody (BFD-IgG) in modulating proliferation and angiogenesis of human umbilical vein endothelial cells (HUVECs) and found that a low concentration of BFD-IgG (200 μg/mL) stimulated endothelial cell growth and increased expressions of vascular cell adhesion molecule-1 (VCAM-1), nerve growth factor (NGF), and vascular endothelial growth factor (VEGF). The apoptosis events appeared not altered by addition of BFD-IgG. An in vitro neoangiogenesis assay demonstrated that BFD-IgG promoted the formation of tube-like structures, which was completely abrogated by anti-VEGF neutralizing antibody and partially by NOS inhibitor, L-NAME. We conclude that BFD-IgG at 200 μg/mL results in cell proliferation and enhanced VEGF-dependent angiogenesis in vitro. Those results suggested that a low concentration of BFD-IgG plays a protective role in the pathogenesis or the progression of BFD.

Copper and Lead Concentrations at Finger and Foot Bone in Blackfoot Disease Patients

Copper and Lead Concentrations at Finger and Foot Bone in Blackfoot Disease Patients

Blackfoot Disease and Arsenic: A Never-Ending Story

Blackfoot disease (BFD) is an endemic peripheral vascular disease confined to the southwestern coast of Taiwan. This article reviews the epidemiology, clinical manifestations and diagnosis, pathology, etiology and pathogenesis of this disease. Sporadic cases of BFD occurred as early as in the early 20th century, and peak incidence was noted between 1956 and 1960, with prevalence rates ranging from 6.51 to 18.85 per 1,000 population in different villages. Typical clinical symptoms and signs of progressive arterial occlusion mainly found in the lower extremities, but in rare cases, the upper extremities might also be involved. Ulceration, gangrene and spontaneous or surgical amputation were typical fate. An extensive pathological study concluded that 30% of the BFD patients had histologic lesions compatible with thromboangiitis obliterans and 70% showed changes of arteriosclerosis obliterans. Epidemiologic studies carried out since mid-20th century revealed that BFD was associated with the consumption of inorganic arsenic from the artesian wells. Recent studies confirmed the existence of preclinical peripheral vascular disease, subclinical arterial insufficiency and defects in cutaneous microcirculation in the residents of the endemic villages. A more recent study suggested that the methylation capacity of arsenic can interact with arsenic exposure in the development of peripheral vascular disease among residents of BFD-endemic areas. The incidence of BFD decreased dramatically after the implementation of tap water in these villages over the past 2–3 decades. The atherogenicity of arsenic could be associated with its effects of hypercoagulability, endothelial injury, smooth muscle cell proliferation, somatic mutation, oxidative stress, and apoptosis. However, its interaction with some trace elements and its association with hypertension and diabetes mellitus could also explain part of its higher risk of developing atherosclerosis. Although humic substances have also been suggested as a possible cause of BFD, epidemiologic studies are required to confirm its etiologic role.

Decrease of fibrinolytic activity in human endothelial cells by arsenite

Blackfoot disease (BFD) is an endemic peripheral vascular occlusive disease that occurred in the southwest coast of Taiwan. It is believed that arsenic in the drinking water from artesian wells plays an important role in the development of the disease. We have previously shown that BFD patients had significant lower tissue-type plasminogen activator (t-PA) antigen level and higher plasminogen activator inhibitor, Type 1 (PAI-1) antigen level than normal controls. The purpose of this study was to investigate the effects of arsenite on the fibrinolytic and anticoagulant activities of cultured macrovascular and microvascular endothelial cells. Incubation of human microvascular endothelial cells (HMEC-1), but not human umbilical vein endothelial cells (HUVECs), with arsenite caused a decrease of t-PA mRNA level, a rise of both PAI-1 mRNA level and PAI activity. Arsenite could also inhibit the thrombomodulin (TM) mRNA expression and reduce the TM antigen level in HMEC-1. In conclusion, arsenite had a greater effect on HMEC-1 as compared to HUVECs in lowering the fibrinolytic activity and may be responsible for the reduced capacity of fibrinolysis associated with BFD.

Arterial tissue of arsenic, selenium and iron in Blackfoot disease patients.

In order to identify arsenic as one of the major factors that cause Blackfoot disease on the southwest coast of Taiwan, an atomic absorption spectrophotometric method was used to determine arsenic, selenium and iron concentrations in the tissue of plantar digital arteries. Samples from 31 patients with Blackfoot disease and 30 controls with road traffic accident or occupational injuries were studied. The results indicate that the arterial tissue from Blackfoot disease patients had higher arsenic concentration (3.06+/-1.42 microg/g) than that from healthy controls (0.59+/-0.28 microg/g). The variability was very large 418% at p<0.001. It was also noted that the concentrations of selenium (1.23+/-0.41 microg/g and 1.05+/-0.13 microg/g in patients and controls respectively; with variability 17.1%) and iron (72.7+/-34.9 microg/g and 35.2+/-16.5 microg/g in patients and controls respectively; with variability 106.5%) were both higher than those of controls. However, only the iron concentration was significantly different (p<0.05).

In vitro cytotoxicity of IgG antibodies on vascular endothelial cells from patients with endemic peripheral vascular disease in Taiwan

A unique peripheral vascular disorder called ‘blackfoot disease’ is endemic in a limited area on the south-west coast of Taiwan. Clinically, the signs and symptoms of blackfoot disease (BFD) are similar to those of arteriosclerosis and Buerger’s disease. A destruction of vascular endothelial cells (ECs) takes place at an early stage in the affected limbs. Currently, the cause of BFD is believed to be artesian drinking water containing a high concentration of arsenic and/or humic substances, although the mechanism of EC destruction is not entirely understood. The purpose of the present study was to examine the factors related to EC damage in BFD. Thus, we investigated the effects of purified IgG collected from patients with BFD (BFD-IgG) and from normal controls (N-IgG) on cultured EC. We found that: (1) EC binding activity of BFD-IgG was significantly higher than that of N-IgG; (2) BFD-IgG, at a concentration higher than 100 μg/ml but not N-IgG, induced concentration-dependent EC cytotoxicity; (3) BFD-IgG at a concentration of 100 μg/ml stimulated neither the release of von Willebrand factor nor the expression of intercellular adhesion molecule-1 by EC. Fluorescent video microscopic examination revealed an increase in transcapillary and interstitial diffusion of nailfold capillary loops in clinically normal fingers of BFD patients. These findings strongly suggested that immunological mechanisms played a significant role in the pathogenesis of BFD. We propose that only persons who produce the IgG anti-endothelial cell antibody are potential victims of BFD.

Determination of urinary trace elements (As, Hg, Zn, Pb, Se) in patients with Blackfoot disease

An endemic peripheral vascular disorder resulting in gangrene of the lower extremities, especially of the feet, is called ‘Blackfoot disease (BFD)’. Clinically, the symptoms and signs of Blackfoot disease are similar to those of Buerger's disease. In this study, the objective is to examine the amount of arsenic, mercury, zinc, lead, and selenium in urine samples from BFD patients. After pre-treatment with acids, the samples were digested by means of a microwave oven. The determination of arsenic mercury, zinc, lead and selenium were by hydride atomic absorption spectrometry (HAAS), cold vapor atomic absorption spectrometry (CVAAS), flame atomic absorption spectrometry (FAAS), graphite furnace absorption spectrometry (GFAAS), respectively. The results indicated that urinary arsenic, mercury and lead of the BFD patients were significantly higher than those of the normal controls, while urinary zinc and selenium were significantly lower than those of the normal controls. The possibility that these elements are involved in the etiology of diseases is discussed.

Arsenic, Selenium and Zinc in Finger and Foot Bone from Blackfoot Disease Patients.

Atomic absorption spectrophotometric methods, developed for determination of arsenic, selenium and zinc concentration in finger and foot bone from blackfoot disease patients(BFDPs), by the amputation were developed. Thirty-four cases of BFDPs at the most strict clinical examination and thirty cases of Non-BFDPs(NBFDP) from the traffic accidents nearly at the same condition were compared as control. Arsenic has been claimed to be a major causative agent of blackfoot disease(BFD) in the southwestern coast of Taiwan. Previously we published some reports also supported this conception, as with increasing of zero, first second, third and fourth clinical stages. The increasing of the arsenic concentration in the blood, hair and urine were happened during the BFD progressed. Although at the third and fourth stages, arsenic decreased may be affected by the antagonistic effect of selenium and zinc. For the purpose to this facts, this experiment was carried to check whether at the fourth stages, the concentration of arsenic in BFDPs finger and foot bone by amputation are higher than NBFDPs and even in the blood of BFDPs at the fourth stage or not. The result showed arsenic concentration in finger and foot bone of BFDPs were a little higher than NBFDPs and with the rate of 0.09±0.05 μg/g and 0.08±0.04 μg/g. Zinc concentration in BFDPs finger and foot bone by amputation were lower than the NBFDPs and with the rate of 52.9±20.2μg/g >35.0±13.8 μg/g and had a significantly difference with p 3.59±1.36 μg/g. Selenium concentration of BFDPs in finger and foot bone by amputation were also had a lower selenium concentration than the NBFDPs and with the rate of 0.07±0.04 μg/g 0.04±0.02 μg/ml and had significantly difference with p<0.05. Selenium and zinc may have some antagonistic effect with arsenic and from this stand point of the role, selenium and zinc as either inhibitory agents of BFD. Therefore selenium and zinc may assess in the blood as diagnostic or prognostic aids in BFD.

Arsenic and Iron in the Finger and Foot Muscle from Blackfoot Disease Patients.

Atomic absorption spectrophotometric method, for determination of arsenic and iron concentration in finger and foot muscles from Blackfoot Dim Patients(BFDPs), by amputation were developed. Thirty-four cases of BFDPs, at the most strict clinical examination and thirty cases of Non-BFDPs(NBFDPs) from the traffic accidents. NBFDPs from the same district were compared as the controls. Arsenic has been claimed to be major causative agents of Blackfoot Disease(BFD) in the south-western coast of Taiwan. We published several papers on this and presented a conception that, as with the increase in linical stages from the zero, first, second, third and fourth, the increase in the arsenic concentration in blood, hair and urine accompanies as the BFD progressed, although at the third and fourth stages, arsenic decrease might be induced by the antagonistic effect of selenium, zinc and iron. For the purpose to prove this, the study was carried out to check whether at the fourth stage, the concentration of arsenic in BFDPs finger and foot muscles by amputation are higher than that of NBFDPs and whether it is also the case with the blood of BFDPs . The arsenic in muscles of finger and foot of BFDPs by amputation was more than 10times higher than the NBFDPs at the raze of 0.80±0.57μg/g>0.07±0.05μg/g and they were also about 8times higher than the blood of BFDPs at the fourth clinical stages at the rate of 0.80±0.57μg/g>0.091±0.052μg/ml. Both of them had significantly difference with P<0.01. These evidences directly indicated that arsenic might be a major causative agent of BFD.supplementThe iron in muscles of finger and foot of BFDPs by amputation were also about 2times higher than that of NBFDPs at the rate of 39.63±23.54μg/g>19.82±7.61μg/g and had a significance difference with P<0.05. These results were on the contraiy to the blood, when compared with the blood iron concentration in BFDPs at fourth stage and NBFDPs. The NBFDPs were higher than the BFDPs at the rate of 553.28±92.96μg/ml> 510.71±163.52μg/ml. Therefore iron may have some antagonistic effect to arsenic toxicity for the BFDPs and this fact is worthy of more academic study of the BFD in clinical application.

Serum trace metals in Blackfoot disease patients.

The purpose of this study was to investigate ultratrace levels of metals in serum of patients with Blackfoot disease (BFD). BFD is an endemic peripheral vascular disorder confined to a limited area along the southwest coast of Taiwan. In this study, graphite furnace atomic absorption spectrophotometry with stabilized temperature platform furnace conditions was used for the determination of selenium, manganese, cobalt, chromium and zinc. This technique includes a dilution of serum with 12 mM ultrapure nitric acid and 1% Triton X-100. The results showed that total manganese, cobalt, chromium and zinc levels in the BFD patients were significantly different from those in normal controls (P < 0.05). The total selenium level in the BFD patients was not different from the normal controls (P > 0.05). The possible connection of these elements with the etiology of the disease is discussed.

Dose-Response Relationship Between Ischemic Heart Disease Mortality and Long-term Arsenic Exposure

The cardiovascular effects of inorganic arsenic have been documented, but the dose-response relationship between ischemic heart disease (ISHD) and long-term arsenic exposure remains to be elucidated. Mortality rates from ISHD among residents in 60 villages of the area in Taiwan with endemic arseniasis from 1973 through 1986 were analyzed to examine their association with arsenic concentration in drinking water. Based on 1 355 915 person-years and 217 ISHD deaths, the cumulative ISHD mortalities from birth to age 79 years were 3.4%, 3.5%, 4.7%, and 6.6%, respectively, for residents who lived in villages in which the median arsenic concentrations in drinking water were <0.1, 0.1 to 0.34, 0.35 to 0.59, and > or = 0.6 mg/L. A cohort of 263 patients affected with blackfoot disease (BFD), a unique arsenic-related peripheral vascular disease, and 2293 non-BFD residents in the endemic area of arseniasis were recruited and followed up for an average period of 5.0 years. There was a monotonous biological gradient relationship between cumulative arsenic exposure through drinking artesian well water and ISHD mortality. The relative risks were 2.5, 4.0 and 6.5, respectively, for those who had a cumulative arsenic exposure of 0.1 to 9.9, 10.0 to 19.9, and > or = 20.0 mg/L-years compared with those without the arsenic exposure after adjustment for age, sex, cigarette smoking, body mass index, serum cholesterol and triglyceride levels, and disease status for hypertension and diabetes through proportional-hazards regression analysis. BFD patients were found to have a significantly higher ISHD mortality that non-BFD residents, showing a multivariate-adjusted relative risk of 2.5 (95% CI, 1.1 to 5.4).

Determination of Se, Mn, Co, Cr and Zn in Urine Specimens of Patients with Blackfoot Disease.

The purpose of this study was to use various atomic absorption spectroscopic techniques to investigate the amount of biological trace metals, including selenium, manganese, cobalt, chromium and zinc, in the urine of Blackfoot disease (BFD) patients. Urine specimens from BFD patients were collected, acidified with a mixed acid solution, purified and preconcentrated with a solid phase extraction cartridge before being digested in a microwave oven at controlled conditions prior to analysis. The optimum composition of the mixed acid was established according to the analysis of standard known sample. The level of urinary selenium and zinc was determined by hydride atomic absorption spectrophotometry (HAAS) and flame atomic absorption spectrophotometry (FAAS), respectively, whereas manganese, cobalt and chromium level were determined using graphite furnace atomic absorption spectrophotometry (GFAAS) with stabilized temperature platform furnace conditions. The validity of the analytical methods was confirmed by the analysis of standard known samples. The result showed that total urinary Mn, Co, Cr and Zn levels in BFD patients were significantly different than those in normal subjects (Mann-Whitney test, p 0.05). The possible connection of these elements with the etiology of the disease is discussed.

Studies on the concentrations of arsenic, selenium, copper, zinc and iron in the hair of blackfoot disease patients in different clinical stages.

Flame atomic absorption spectrophotometric methods were developed for, arsenic, selenium, copper, zinc and iron in hair samples. Data from blackfoot disease patients at five clinical stages were compared with those from healthy controls. The copper and zinc concentrations showed only slight differences in all clinical stages, which indicated the less relation to blackfoot disease. The decrease of selenium and iron in all stages was attributed to the antagonistic effect of arsenic; arsenic increased in the first and second stages, but decreased in the later stages. The decrease of selenium and iron during the progression of the disease is thought to be due to persistence of the antagonistic effect of arsenic in the initial stages, so that very low concentrations of selenium are found in the advanced stages, despite the later decrease of arsenic. There was also a progressive decrease of iron with advance of the disease, and the later stages also showed a decrease in haemoglobin. It was shown that arsenic is a major cause of blackfoot disease, and that it antagonises selenium and iron, which decreased in the advanced clinical stages of the disease.

Impaired fibrinolysis in patients with blackfoot disease

Blackfoot disease is a unique endemic and chronic progressive arteriosclerotic vascular disease in southwest area of Taiwan. In this study, we determined the plasma levels of tissue plasminogen activator (tPA), plasminogen activator inhibitor (PAI), urokinase plasminogen activator (uPA), and von Willebrand factor (vWF) antigen in Blackfoot disease patients, in comparison with normal controls from non-endemic areas and the endemic area, Putai. Blackfoot disease patients had mean tPA antigen level of 7.9 ng/ml (n = 27) which was significantly lower (p < 0.05) than both the normal controls with 11.0 ng/ml (n = 20) and the Putai normal controls with 9.7 ng/ml (n = 39). However, the mean PAI-1 antigen level in the patient group was 41.2 ng/ml (n = 28) which was significantly higher (p = 0.0001) than both the normal controls with 19.7 ng/ml (n = 23) and the Putai normal controls with 21.3 ng/ml (n = 40). Furthermore, in the patient group, a significantly lower (p < 0.005) mean uPA antigen level (2.3 ng/ml, n = 18) was noted as compared with that in the normal controls (3.2 ng/ml, n = 14). No significant difference was observed in vWF antigen level between patients and normal controls. This study suggests that a reduced capacity for fibrinolysis is associated with Blackfoot disease.

Simultaneous determination of Zn, Cd, Pb, and Cu in urine of patients with blackfoot disease using anodic stripping voltammetry.

Blackfoot disease (BFD) is an endemic peripheral vascular disorder resulting in gangrene of the lower extremities, especially the feet, among residents in a limited area on the southwest coast of Taiwan. In the present study, the concentrations of zinc, cadmium, lead, and copper in urine of BFD patients with matched normal controls are investigated by differential pulse anodic stripping voltammetry (DPASV) on a hanging mercury drop electrode (HMDE). The analytical results indicate that urinary copper, cadmium, and lead of the BFD patients are significantly higher than those of the controls. In addition, the patients showed a significantly lower concentration of zinc in the urine than the normal controls. The possible connection of these elements with the etiology of the disease is discussed.

Studies on the concentration of arsenic, selenium, copper, zinc and iron in the blood of blackfoot disease patients in different clinical stages.

Flame atomic absorption spectrophotometric methods were developed for the determination of zinc, copper, arsenic, iron and selenium in blood samples. Data from blackfoot disease patients in five clinical stages were compared with those from healthy controls. Copper concentrations were the same for all clinical stages. Arsenic increased in the initial three stages but decreased thereafter, although arsenic was previously considered to be the major causative agent of the disease. The decrease of arsenic in the later stages was attributed to the antagonistic effect of selenium, and the decrease of iron during the progress of the disease is thought to be due to the antagonistic effect of arsenic in the initial stages and the loose of haemoglobin in the later stages.

Determination of Arsenic and Mercury Concentrations in Urine of Patients with Blackfoot Disease.

Blackfoot disease (BFD) is a peripheral vascular disease resulting in gangrene of the lower extremities. In the present work, the objective was to examine the amount of arsenic and mercury in urine of BFD patients. The urine specimens were acidified with nitric acid and digested in a microwave oven. A solid phase extraction (SPE) cartridge was used for sample purification and preconcentration. The analytical technique for the determination of arsenic was by hydride atomic absorption spectrophotometry (HAAS) and for mercury by cold vapor atomic absorption spectrophotometry (CVAAS). The sensitivity and accuracy of the analytical techniques were checked with Lanornorm control urine. Arsenic and mercury concentrations in the urine of normal controls and BFD patients were found to be 11.3±4.7 μg/1 and 33.6±23.1 μg/1 for As ; 5.0±1.8 μg/1 and 11.6±5.9 μg/1 for Hg, respectively. The life background of the BFD patients is also briefly mentioned.

Baseline and sodium arsenite-induced sister chromatid exchanges in cultured lymphocytes from patients with Blackfoot disease and healthy persons.

A significantly higher frequency of baseline sister chromatid exchange (SCE) was found in the cultured lymphocytes of 13 Blackfoot disease patients (BFP) in comparison with that of healthy persons (HP). Twelve of these BFP consumed well water containing a high concentration of arsenic for 15 years or longer and had switched to drinking tap water 12 years before the time of this study. Sodium arsenite was found to be effective in increasing the SCE frequency and delaying the cell growth of the lymphocytes from both BFP and HP. However, the SCE increment induced by sodium arsenite as well as the progression of the cell divisions in the cultured lymphocytes showed no significant difference between BFP and HP.

黒脚病と癩病の鑑別診断

By the end of World War II, a specific disease, which might have existed in the past, yet was not known to the public, was found in the down-stream basin of the Pa-Chang River on the south west coast of Taiwan. The disease was found to cause gangrene of the extremities, especially the foot, and mutila-tion, and was called the "black-foot" disease by the local people, though it could probably be arsenical gangrene by nature. This disease has been feared by the people for, once infected, patients suffer as much as the lepers. In fact, several clinical symptoms of this specific disease, such as red swelling of toes and fingers, the initial stage of paresthesia, breaking of fingers and toes, the later stage of mutilation, resemble some symptoms of leprosy. Therefore, the dif-ferential diagnosis of this "black-foot" disease in the course of the diagnosis of leprosy has become quite necessary in this country. Since few health workers in the field of leprosy control in this country have encountered this disease, it is helpful to describe the similarity and differences of these two diseases for reference.1. Paralysis and gangrene found in black-foot disease patients are caused by long-term intake of arsene with a result in changes primarily in the blood system, such as intimal arteritis, particularly capillar sclerosis, obliterans, and even thrombosis. Peripheral nerves are injured to cause peripheral multiple neuritis, and therefore paralysis. The trophic nerves are then injured, and the deep tissue therefore becomes necrotic, resulting in gangrene and mutilation2. Leprosy is the only bacillary infection which attacks the peripheral nerves. Paresthesia occurs naturally. The Mycobacterium leprae parasitizes and multiplies in the small arteria, the round cells are then infiltrated, and the con-nective tissue becomes thicker. As a result, the arterial lumen becomes nar-rower, and macula circumscripta and paralysis occur. The severity and the frequency of the red-swelling of fingers and toes in leprosy are less than seen in Black foot disease.3. Although these two diseases are caused by different agents, their local symptoms, such as the red-swelling of fingers and toes, numbness, cold feeling, contraction of fingers and toes, dwindle and remnant of nails, are similar.4. While anhidrosis is found in the leprosy patients, hyperidrosis is found in the black-foot disease patients, 5. Other symptomatological, and bacteriological findings mentioned in this article can be of reference to the differential diagnosis of these two diseases. In one word, there is neither superficial nerve hypertrophy, nor leprotic erup-tion in the black-foot disease. At the ulcerous lesions, hyperesthesia instead of anesthesia occurs. Since the black-foot disease is a nonbacillary disease, no acid-fast bacilli can be found.6. It was found in this study that the increase of arsenical content in water in these areas might probably have something to do with earthquakes.Acknowledgement: This work was aided by grants from the Natioual Council on Science Development of the Republic of China.