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Arsenic as a promoter in the effect of humic substances on plasma prothrombin time in vitro
THROMBOSIS RESEARCH 58; 537-541,199O 0049-3848/90 $3.00 + .OOPrinted in the USA. Copyright (c) 1990 Pergamon Press plc. All rights reserved.
ARSENIC AS A PROMOTER IN THE EFFECT OF HUMIC SUBSTANCES ON PLASMA PROTHROMBIN TIME IN VITRO
F. J. LU Department
of Biochemistry, College of Medicine, National Taiwan University No. 1, Section 1, Jen-Ai Road, Taipei, Taiwan 10018, Republic of China (Received 2.8.1989; accepted in revised form 28.2.1990 by Editor S. Okamoto)
ABSTRACT Protocatechuic acid can be oxidized and polymerized to from humic substances (humic acid and fulvic acid). Should AS,O, occur in the process of oxidative polymerization, humic acid output can increase by 1.5 - 2.3 times and, in the case of fulvic acid, at least 10 times. After protocatechuic acid is oxidized and polymerized, the resultant humic and fulvic acids both exhibit the ability to shorten the prothrombin time of human pool This ability becomes more apparent plasma in vitro. when AS,O, serves as a promoter in the course of oxidative polymerization as described above. However, AS,O, or protocatechuic acid alone is unable to shorten the prothrombin time of human pool plasma.
INTRODUCTION Residents along the southwestern coast of Taiwan tend to suffer from a variety of peripheral vascular obstructive disorder, reknown locally as "Blackfoot disease" (1). Epidemiologic searchers used to attribute the occurrence of this endemic disease to the arsenic content in the region's well water (2). Recent studies have concluded, however, that fluorescent humic substances in this water are also related to Blackfoot disease (3). Nevertheless, the relationships between arsenic and fluorescent humic substances and their relative or combined effects on Blackfoot diseases remains uncertain. Key Words: Arsenic,
Humic substances, PT, Blackfoot disease. 537
538
ARSENIC, HUMIC SUBSTANCES AND PT
Vol. 58, No. 6
This study demonstrates how humic substances were synthesized in the laboratory and attempts to determine the role arsenic plays in the synthesis. Moreover, the effects of synthetic humic substances on the prothrombin time of human plasma is also discussed here. By means of the experimental model as applied in this study, we expect to identify the correlation among arsenic and fluorescent humic substances and the occurrence of Blackfoot disease.
MATERIALS AND METHODS Synthesis of humic substances Added 1.0 gm of protocatechuic acid [3,4-(HO),CSH5 COOH, 3,4_dihydroxybenzoic acid, Sigma Co.] and 100 ml of H,C to one of two conical flasks, and adjusted pH value to 7.0 using the Ca (OH) 2 solution; the other one was filled with 1.0 gm of protocatechuic acid and 100 ml of H,O and 200 mg of As,O, (Mallinckrodt), pH value also adjusted to 7.0 using the Ca(OH), solution. Then, covered the mounths of flasks with cotton stoppers and put them into water bath at 25'C for oxidative polymerization. During the reaction period, slowly vibrated the flasks and adjust pH values to 7.0 by the Ca(OH), solution every day. Reaction stopped on the 17th day, then centrifugate the contents in the flasks to take out the supernatants. Used hydrochloric acid to acidify the supernatant first, and extracted the unreacted protocatechuic acid and other organic compounds using peroxide free diethyl ether. Dissolved the centrifugated deposit in the solution of O.lM Na4P,07, pH 7.0. Acidified to precipitate the mixture once more, and digested the resultant deposit with O.lN HCl-HF. Then centrifugated again, washed with water several times and finally weighed the precipitate after drying in the vacuum drier. This was for the preparation of humic acid. Pass the completely centrifugated supernatant through a filter paper captaining about 100 gm of activated carbon and the Dowex 50 (H ) column respectively. Vacuum dried the collected supernatant (at 45'C) and extracted with methanol. Vacuum dried another time and applied O.lN HCl-HF for the removal of silica. Then centrifugated again, washed with water several times and finally weighed it after a further vacuum drying. This was for the preparation of fulvic acid. Effects of synthetic humic substances on the prothrombin of human pool plasma
time
Upon preparation of these two synthetic humic substances in various concentrations, mixed them with normal human pool plasma (obtained from National Taiwan University Hospital), to which the reagents for measuring the prothrombin time were added. In the test, 0.03 ml of synthetic humic substances in various concentrations were first mixed with human pool plasma
ARSENIC, HUMIC SUBSTANCES AND PT
Vol. 58, No. 6
539
Preheated the mixture for 1 minute in the water respectively. bath at 37OC. Then, Thromborel S solution which had been preheated for 15 minutes (37'C) was added. Finally, used Fibrintimer (Behring) to measure the prothrombin time. RESULTS AND DISCUSSION After oxidative polymerization for 17 days, _1.0 _ gm of protocatechuic acid can produce 2.3 mg (+As,O,) or I.0 mg (-As,O,) of humic acid, and 149.3 mg (+As,O,) or 13.7 mg (-As,O,) of fulvic acid. Should As,O, be added in the course of oxidative polymerzation, output of humic acid can increase by 1.5 - 2.3 times (n = 81, and for fulvic acid, output will increase by at least 10 times (n = 8). In conclusion, As,O, was found to act as a catalyst for the synthesis of humic substances. Synthetic humic substances, whether humic acid or fluvic acid, are all able to shorten the prothrombin time of human pool plasma within a certain concentration range (0.01 - 0.25 mg/ml). That is to say, they all have the ability to promote this effect on blood coagulation (Fig.). With As,O, as a catalyst, such ability would become more obvious. However, it is interesting that As,O, or protocatechuic acid or Ca(OH), has no way to promote the effect of blood coagulation when acting alone. Fig.
XI.5_
1
,
-1.0'A-A \
A
-1.5A.
prothrorrbin time by two scffple means
\,,
z -2.0
A
:I
\
Effect of synthetichumic substanceson humanplasma
0 hunic acid without A~203 '0 hunic acid with A~203
=A.
fulvic acid without A~203 : fulvic acid with As2O-j
.\/ /
-3.5, 0*-*/a
12
-4.0
-0.6
-0.4
-0.2
0
log [mg/mll Early on, researcher
for the etiology of Blackfoot disease
540
ARSENIC, HUMIC SUBSTANCES AND PT
Vol. 58, No. 6
found that the concentration of arsenic in drinking well water was high at the endemic areas (1). This had a positive correlation with the epidemical degree of the disease (2). Therefore, they presumed that arsenic is closely related to Blackfoot disease. However, they did not know what role arsenic was playing in the Blackfoot disease. Subsequently, the author also found there was strong greenish-blue fluorescence present in the well water of Blackfoot disease endemic areas (4). In laboratory, the greenish-blue fluorescent substances extracted from the well water could cause symptoms in mice to result similar to those of human Blackfoot disease (5). Later, the author further proved that such fluorescent substances are humic substances (6). which contain cancer-causing phthalate esters and more than 30 metallic elements. With humic substances as the core, those metallic elements and other organic substances combined together to form an organo-metallic complex, which the author assumed would also be the pathologic agent of many endemic diseases(7). In addition, the author proved the fluorescent humic substances existing in the well water can shorten the prothrombin time of human plasma in vitro, that is, to accelerate the coagulation of blood (8). Hence, it was concluded that such fluorescent humic substances are closely related with vascular obstructive diseases. In this experiment, synthetic humic substances (humic acid or fulvic acid) all were proved to accelerate the coagulation of human plasma. Should As,O, be added as catalyst, such ability would become more apparent. AszG3r however, has no way to do so alone itself. This experiment revealed that humic acid, acting as the core in the complex would accelerate the coagulation of plasma, while arsenic which combines with humic acid together is an auxiliary agent only REFERENCES 1. TSENG, W.P. CHEN, W.Y. SUNG, J.L. and CHEN, J.S.: A clinical study of Blackfoot disease in Taiwan: An endemic peripheral Memoirs Co11 Med N.T.U.; 7, l-8, 1961. vascular disease. 2. CHEN, K.P. and WU, H.Y.: Epidemiologic studies on Blackfoot disease: 2. A study of source of drinking water in relation to the disease. J. Formosan Med Assoc; 61, 611-618, 1962. 3. LU. F.J. SYE, H.B. WU, H.U. KUO, T.L. LEE, C.J. and HU, H.T.: Study on the fluorescent humic substances in well water at BlackCorrelation between foot disease epidemic areas in Taiwan: fluorescent intensity, arsenic content, pH value and total dissolved solids in well water and Blackfoot diseases epidemic index. J Formosan Med Assoc; 87S, 105-106, 1988 4. LU, F.J. YANG, C.K. and LIN, K-H.: Physico-chemical characteristics of drinkinq water in Blackfoot disease endemic areas in Chia-I and Tainan-Hsiens. J. Formosan Med Assoc; 74, 596-605, 1975.
Vol. 58, No. 6
ARSENIC, HUMIC SUBSTANCES AND PT
541
Fluorescent compounds in drinking 5. LU, F.J. and LIU, T.M.: water of Blackfoot disease endemic areas: Animal experimental model. J. Formosan Med Assoc; 85, 352-358, 1986. 6. LU, F.J. YAMAMURA, Y. YAMAUCHI, H.: Studies on fluorescent compounds in water of a well in Blackfoot disease endemic areas in Taiwan: Humic substances. J. Formosan Med Assoc; 87, 66-75, 1988. 7. LU, F.J.: Contributions of fluorescent humic substances existing in the drinking well water of Blackfoot disease endemic area in Taiwan to environmental toxicology. J. Formosan Med Assoc; 88S, 76-83, 1989. 8. LU, F.J. SHIH, S.R. LIU, T.M. and SHOWN, S.H.: The effect of fluorescent humic substances existing in the well water of Blackfoot disease endemic areas in Taiwan on prothrombin time and activated partial thromboplastin time in vitro. Thrombosis Res. in printing.
ARSENIC AS A PROMOTER IN THE EFFECT OF HUMIC SUBSTANCES ON PLASMA PROTHROMBIN TIME IN VITRO
F. J. LU Department
of Biochemistry, College of Medicine, National Taiwan University No. 1, Section 1, Jen-Ai Road, Taipei, Taiwan 10018, Republic of China (Received 2.8.1989; accepted in revised form 28.2.1990 by Editor S. Okamoto)
ABSTRACT Protocatechuic acid can be oxidized and polymerized to from humic substances (humic acid and fulvic acid). Should AS,O, occur in the process of oxidative polymerization, humic acid output can increase by 1.5 - 2.3 times and, in the case of fulvic acid, at least 10 times. After protocatechuic acid is oxidized and polymerized, the resultant humic and fulvic acids both exhibit the ability to shorten the prothrombin time of human pool This ability becomes more apparent plasma in vitro. when AS,O, serves as a promoter in the course of oxidative polymerization as described above. However, AS,O, or protocatechuic acid alone is unable to shorten the prothrombin time of human pool plasma.
INTRODUCTION Residents along the southwestern coast of Taiwan tend to suffer from a variety of peripheral vascular obstructive disorder, reknown locally as "Blackfoot disease" (1). Epidemiologic searchers used to attribute the occurrence of this endemic disease to the arsenic content in the region's well water (2). Recent studies have concluded, however, that fluorescent humic substances in this water are also related to Blackfoot disease (3). Nevertheless, the relationships between arsenic and fluorescent humic substances and their relative or combined effects on Blackfoot diseases remains uncertain. Key Words: Arsenic,
Humic substances, PT, Blackfoot disease. 537
538
ARSENIC, HUMIC SUBSTANCES AND PT
Vol. 58, No. 6
This study demonstrates how humic substances were synthesized in the laboratory and attempts to determine the role arsenic plays in the synthesis. Moreover, the effects of synthetic humic substances on the prothrombin time of human plasma is also discussed here. By means of the experimental model as applied in this study, we expect to identify the correlation among arsenic and fluorescent humic substances and the occurrence of Blackfoot disease.
MATERIALS AND METHODS Synthesis of humic substances Added 1.0 gm of protocatechuic acid [3,4-(HO),CSH5 COOH, 3,4_dihydroxybenzoic acid, Sigma Co.] and 100 ml of H,C to one of two conical flasks, and adjusted pH value to 7.0 using the Ca (OH) 2 solution; the other one was filled with 1.0 gm of protocatechuic acid and 100 ml of H,O and 200 mg of As,O, (Mallinckrodt), pH value also adjusted to 7.0 using the Ca(OH), solution. Then, covered the mounths of flasks with cotton stoppers and put them into water bath at 25'C for oxidative polymerization. During the reaction period, slowly vibrated the flasks and adjust pH values to 7.0 by the Ca(OH), solution every day. Reaction stopped on the 17th day, then centrifugate the contents in the flasks to take out the supernatants. Used hydrochloric acid to acidify the supernatant first, and extracted the unreacted protocatechuic acid and other organic compounds using peroxide free diethyl ether. Dissolved the centrifugated deposit in the solution of O.lM Na4P,07, pH 7.0. Acidified to precipitate the mixture once more, and digested the resultant deposit with O.lN HCl-HF. Then centrifugated again, washed with water several times and finally weighed the precipitate after drying in the vacuum drier. This was for the preparation of humic acid. Pass the completely centrifugated supernatant through a filter paper captaining about 100 gm of activated carbon and the Dowex 50 (H ) column respectively. Vacuum dried the collected supernatant (at 45'C) and extracted with methanol. Vacuum dried another time and applied O.lN HCl-HF for the removal of silica. Then centrifugated again, washed with water several times and finally weighed it after a further vacuum drying. This was for the preparation of fulvic acid. Effects of synthetic humic substances on the prothrombin of human pool plasma
time
Upon preparation of these two synthetic humic substances in various concentrations, mixed them with normal human pool plasma (obtained from National Taiwan University Hospital), to which the reagents for measuring the prothrombin time were added. In the test, 0.03 ml of synthetic humic substances in various concentrations were first mixed with human pool plasma
ARSENIC, HUMIC SUBSTANCES AND PT
Vol. 58, No. 6
539
Preheated the mixture for 1 minute in the water respectively. bath at 37OC. Then, Thromborel S solution which had been preheated for 15 minutes (37'C) was added. Finally, used Fibrintimer (Behring) to measure the prothrombin time. RESULTS AND DISCUSSION After oxidative polymerization for 17 days, _1.0 _ gm of protocatechuic acid can produce 2.3 mg (+As,O,) or I.0 mg (-As,O,) of humic acid, and 149.3 mg (+As,O,) or 13.7 mg (-As,O,) of fulvic acid. Should As,O, be added in the course of oxidative polymerzation, output of humic acid can increase by 1.5 - 2.3 times (n = 81, and for fulvic acid, output will increase by at least 10 times (n = 8). In conclusion, As,O, was found to act as a catalyst for the synthesis of humic substances. Synthetic humic substances, whether humic acid or fluvic acid, are all able to shorten the prothrombin time of human pool plasma within a certain concentration range (0.01 - 0.25 mg/ml). That is to say, they all have the ability to promote this effect on blood coagulation (Fig.). With As,O, as a catalyst, such ability would become more obvious. However, it is interesting that As,O, or protocatechuic acid or Ca(OH), has no way to promote the effect of blood coagulation when acting alone. Fig.
XI.5_
1
,
-1.0'A-A \
A
-1.5A.
prothrorrbin time by two scffple means
\,,
z -2.0
A
:I
\
Effect of synthetichumic substanceson humanplasma
0 hunic acid without A~203 '0 hunic acid with A~203
=A.
fulvic acid without A~203 : fulvic acid with As2O-j
.\/ /
-3.5, 0*-*/a
12
-4.0
-0.6
-0.4
-0.2
0
log [mg/mll Early on, researcher
for the etiology of Blackfoot disease
540
ARSENIC, HUMIC SUBSTANCES AND PT
Vol. 58, No. 6
found that the concentration of arsenic in drinking well water was high at the endemic areas (1). This had a positive correlation with the epidemical degree of the disease (2). Therefore, they presumed that arsenic is closely related to Blackfoot disease. However, they did not know what role arsenic was playing in the Blackfoot disease. Subsequently, the author also found there was strong greenish-blue fluorescence present in the well water of Blackfoot disease endemic areas (4). In laboratory, the greenish-blue fluorescent substances extracted from the well water could cause symptoms in mice to result similar to those of human Blackfoot disease (5). Later, the author further proved that such fluorescent substances are humic substances (6). which contain cancer-causing phthalate esters and more than 30 metallic elements. With humic substances as the core, those metallic elements and other organic substances combined together to form an organo-metallic complex, which the author assumed would also be the pathologic agent of many endemic diseases(7). In addition, the author proved the fluorescent humic substances existing in the well water can shorten the prothrombin time of human plasma in vitro, that is, to accelerate the coagulation of blood (8). Hence, it was concluded that such fluorescent humic substances are closely related with vascular obstructive diseases. In this experiment, synthetic humic substances (humic acid or fulvic acid) all were proved to accelerate the coagulation of human plasma. Should As,O, be added as catalyst, such ability would become more apparent. AszG3r however, has no way to do so alone itself. This experiment revealed that humic acid, acting as the core in the complex would accelerate the coagulation of plasma, while arsenic which combines with humic acid together is an auxiliary agent only REFERENCES 1. TSENG, W.P. CHEN, W.Y. SUNG, J.L. and CHEN, J.S.: A clinical study of Blackfoot disease in Taiwan: An endemic peripheral Memoirs Co11 Med N.T.U.; 7, l-8, 1961. vascular disease. 2. CHEN, K.P. and WU, H.Y.: Epidemiologic studies on Blackfoot disease: 2. A study of source of drinking water in relation to the disease. J. Formosan Med Assoc; 61, 611-618, 1962. 3. LU. F.J. SYE, H.B. WU, H.U. KUO, T.L. LEE, C.J. and HU, H.T.: Study on the fluorescent humic substances in well water at BlackCorrelation between foot disease epidemic areas in Taiwan: fluorescent intensity, arsenic content, pH value and total dissolved solids in well water and Blackfoot diseases epidemic index. J Formosan Med Assoc; 87S, 105-106, 1988 4. LU, F.J. YANG, C.K. and LIN, K-H.: Physico-chemical characteristics of drinkinq water in Blackfoot disease endemic areas in Chia-I and Tainan-Hsiens. J. Formosan Med Assoc; 74, 596-605, 1975.
Vol. 58, No. 6
ARSENIC, HUMIC SUBSTANCES AND PT
541
Fluorescent compounds in drinking 5. LU, F.J. and LIU, T.M.: water of Blackfoot disease endemic areas: Animal experimental model. J. Formosan Med Assoc; 85, 352-358, 1986. 6. LU, F.J. YAMAMURA, Y. YAMAUCHI, H.: Studies on fluorescent compounds in water of a well in Blackfoot disease endemic areas in Taiwan: Humic substances. J. Formosan Med Assoc; 87, 66-75, 1988. 7. LU, F.J.: Contributions of fluorescent humic substances existing in the drinking well water of Blackfoot disease endemic area in Taiwan to environmental toxicology. J. Formosan Med Assoc; 88S, 76-83, 1989. 8. LU, F.J. SHIH, S.R. LIU, T.M. and SHOWN, S.H.: The effect of fluorescent humic substances existing in the well water of Blackfoot disease endemic areas in Taiwan on prothrombin time and activated partial thromboplastin time in vitro. Thrombosis Res. in printing.