Environmental Geochemistry and Health 1994 16(2) page 65
Humic substances shorten human plasma prothrombin time Fung-Jou Lu*, Wen-Wei Lin and Tien-Shang Huang 1 Departments of Biochemistry and 1Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China
Abstract The prothrombin time of the normal human pooled plasma was shortened by Aldrich humic acid, well water humic substances and lignin at final concentrations ranging from 5 x 10 3 mg mL -1 to 5 x 10 2 mg mL q, with a maxmum effect at 1 x 10 -2 mg mL -1. At this concentration the shortening was 5.5 s, 4.4 s, and 3.5 s by Aldrich humic acid, well water humic substances and lignin respectively. However, monomeric components of humic acid such as syringic acid, protocatechuic acid, pyrogallol, vanillic acid, gallic acid, resorcinol, ferulic acid, catechol, caffeic acid and p-coumaric acid did not have such ability to shorten prothrombin time at the same final concentration ranges. Certain reducing agents such as t-butanol ( 2.5 x 1 0 3 --2.0 x 1 0 2 mg mL "1 ), glutathione (8.0 x 1 0 2 --1.0 mg mL -1), ascorbic acid (4.2 x 1 0 2 -5.0 x 10- 1 mg mL q) and dithiothreitol (1.0 x 10 -2 -1.7 x 10lmg m L -1) could prevent the shortening effects of humic substances or lignin on prothrombin time. These results suggested that humic substances and lignin with a polymerised structure had an ability to affect activities of some blood coagulating enzymes.
Keywords: Humic
substances, prothrombin time.
Introduction
Humic substances exist abundantly in dead plants, soil, well water and other sources. It is a polymeric compound of high molecular weight and its c o m p o s i t i o n m a y vary in d i f f e r e n t areas (Hartenstein, 1981). Phenolic and phenoliccarboxylic components are monomeric degradative by-products of humic substances (Burges et al., 1964; Keyser et al., 1976; Choudhry, 1981). Humic acid is able to inhibit the activities of certain enzymes such as protease (Ladd and Butler, 1969), indoleacetic acid oxidase (Mato et al., 1972), phosphatase (Malcolm and Vaughan, 1979a), invertase (Malcolm and Vaughan, 1979b), malate dehydrogenase (Pflug and Ziechmann, 1981), ribonuclease (Marty and Bastide, 1982), catalase ( Serban and Nissenbaum, 1986 ) , and peroxidase (Serban and Nissenbaum, 1986). Epidemiological studies showed that well water humic substances might be one of the aetiological factors of K a s h i n - B e c k d i s e a s e , an e n d e m i c c h r o n i c degenerative joint disorder, found in Mainland China (Zhai et al., 1990). Humic substances have also been implicated as a cause of environmental goitrogen (Gaitan et al., 1980; source Gaitan et al., 1983; Cooksey et al., 1985).
*To whom correspondence should be addressed at: Department of Biochemistry, College of Medicine, National Taiwan University, Jen-Ai Road, Sec. 1, Taipei, Taiwan, Republic of China.
We have isolated a blue-green fluorescent humic substances from well water where Blackfoot disease is endemic in an area of the southwest coast of Taiwan (Lu et al., 1975). Blackfoot disease is an endemic peripheral vascular disease. Clinically, patients first experience numbness or coldness of one or more of the extremities resulting in black discolouration, ulceration or gangrenous change in extremities (Tseng et al., 1961). Our previous experimental results showed that this fluorescent humic substances of well water could induce blackening of the tail and feet of mice (Lu, 1990a), inhibit plasmin activity (Lu and Lee, 1992) and shorten prothrombin time of normal human pooled plasma (Lu et al., 1990), and suggested that well water humic substance might be one of the aetiological factors of Blackfoot disease (Lu, 1990b). Here, we report that humic substance (both from well water and Aldrich humic acids) as well as lignin, a polymer of phenolic compounds, have the ability to shorten normal human plasma prothrombin time, whereas, monomeric compounds of humic acid do not have such ability. The ability to shorten the prothrombin time of humic substance can be inhibited by some reducing agents such as t-butanol, glutathione, a s c o r b i c acid and dithiothreitol. Materials and Methods Chemicals
Aldrich humic acid was obtained from Aldrich
66
Human plasma prothrombin COOH
Pr0C0caLechuicacid
-
OH
Pyr ll01
Vanillic acid
3HC 3
OH
C t ho]
2
COCI-t
Gallic aci0
H=:CHCO~:=CI--tCOOH
Syringic acid C H=CH(XXlq
4 i -5
*-
-6
i -
-4
I -3
J -
-1
log (mg m1-1) -ell
Ferulit. OH
p"c0u ic acid
Caffeic acid
acid
OH
R rcinol Figure 1 Monomers of humic acid.
Chemical Co., Milwaukee,U.S.A., which was further purified by acid precipitation with 0.1M HC1. Lignin was obtained from Tokyo Kasei Kogyo Co. Protocatechuic acid, pyrogallol, vanillic acid, gallic acid, ferulic acid, catechol, caffeic acid, syringic acid, p-coumaric acid, glutathione, ascorbic acid and dithiothreitol were obtained from Wako Pure Chemical Industries, Japan. The prothrombin time reagent kit (thromborel S, which consisted of thromboplastin containing calcium, was a lyophilised extract from human placenta) was obtained from the Behring Co., Germany. Normal human pooled plasma, was prepared by mixing aliquots from 50 normal persons whose plasma prothrombin time was in the range from 11.0 to 14.0 s. This was obtained from the outpatient division of National Taiwan University Hospital, Taiwan. Extraction of well water humic substances Water was taken from a well located in the Blackfoot disease endemic area in Tainan County on the southwest coast of Taiwan. The water was concentrated, acidified with 1M HC1 and extracted with alcohol to obtain the crude extract of fluorescent humic substances. The crude extract of fluorescent humic substances was further purified by following the previously reported methods (Lu et al., 1988). In short, well water was filtered, condensed and evaporated to dry powder. An alcohol extract of the crude powder was evaporated again and then dissolved in distilled water. Then it was loaded on to a Sephadex G-25 column and eluted with distilled water. The first eluted peak of
Figure 2 Effects of polymers and monomers of humic acid on prothrombin time. 1 Monomers of humic acid: protocatechuic acid, pyrogallol, vanillic acid, resorcinol, gallic acid, ferulic acid, catechol, caffeic acid, syringic acid and p-coumaric acid. 2 Lignin. 3 Well water humic substances. 4 Aldrich humic acid. APT." The change of prothrombin time. n-=4, Mean +_SD. fluorescent humic substances was used for study. Previous study had shown it consisted of various polymers and organometallic complexes. It contained various functional groups such as OH-, C=O stretch of ketone or aldehyde or ester, CO0-, ethers, phenols and aromatic ring (Lu et al., 1988). The estimated MW of this elute fraction was around 5,000 Dalton. Effect of humic substances and monomeric components on the prothrombin time of normal human pooled plasma Humic substances and various m o n o m e r i c components were prepared respectively at several different concentrations (1.0 xl0 -4 -1.0 mg mL-I). 0.03 mL of each test solution was added to 0.07 mL of normal human pooled plasma. The mixture was heated at 37 ° C for 1 min. then added to 0.2 mL of Thromborel S solution ,which was preheated for 15 min and then the prothrombin time measured with a fibrintimer (Behring Co.). Effects of reducing agents on the ability of humic substances to shorten the prothrombin time of normal human pooled plasma Reducing agents (t-butanol, glutathione, ascorbic acid and dithiothreitol) were prepared at different concentrations (7.5 x 10-2 -3.0 mg mL -1). A suitable concentration of each reducing agent was added to humic substances to make a final concentration of humic substances of 0.01 mg mL -l at which concentration of humic substances showed the greatest ability to shorten the prothrombin time. To 0.03 mL of the final mixture 0.07 mL of normal human pooled plasma was added and the rest of the procedure was the same as in the previous section.
F. Lu, W. Lin and T. Huang
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Humic substances shorten human plasma prothrombin time Fung-Jou Lu*, Wen-Wei Lin and Tien-Shang Huang 1 Departments of Biochemistry and 1Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China
Abstract The prothrombin time of the normal human pooled plasma was shortened by Aldrich humic acid, well water humic substances and lignin at final concentrations ranging from 5 x 10 3 mg mL -1 to 5 x 10 2 mg mL q, with a maxmum effect at 1 x 10 -2 mg mL -1. At this concentration the shortening was 5.5 s, 4.4 s, and 3.5 s by Aldrich humic acid, well water humic substances and lignin respectively. However, monomeric components of humic acid such as syringic acid, protocatechuic acid, pyrogallol, vanillic acid, gallic acid, resorcinol, ferulic acid, catechol, caffeic acid and p-coumaric acid did not have such ability to shorten prothrombin time at the same final concentration ranges. Certain reducing agents such as t-butanol ( 2.5 x 1 0 3 --2.0 x 1 0 2 mg mL "1 ), glutathione (8.0 x 1 0 2 --1.0 mg mL -1), ascorbic acid (4.2 x 1 0 2 -5.0 x 10- 1 mg mL q) and dithiothreitol (1.0 x 10 -2 -1.7 x 10lmg m L -1) could prevent the shortening effects of humic substances or lignin on prothrombin time. These results suggested that humic substances and lignin with a polymerised structure had an ability to affect activities of some blood coagulating enzymes.
Keywords: Humic
substances, prothrombin time.
Introduction
Humic substances exist abundantly in dead plants, soil, well water and other sources. It is a polymeric compound of high molecular weight and its c o m p o s i t i o n m a y vary in d i f f e r e n t areas (Hartenstein, 1981). Phenolic and phenoliccarboxylic components are monomeric degradative by-products of humic substances (Burges et al., 1964; Keyser et al., 1976; Choudhry, 1981). Humic acid is able to inhibit the activities of certain enzymes such as protease (Ladd and Butler, 1969), indoleacetic acid oxidase (Mato et al., 1972), phosphatase (Malcolm and Vaughan, 1979a), invertase (Malcolm and Vaughan, 1979b), malate dehydrogenase (Pflug and Ziechmann, 1981), ribonuclease (Marty and Bastide, 1982), catalase ( Serban and Nissenbaum, 1986 ) , and peroxidase (Serban and Nissenbaum, 1986). Epidemiological studies showed that well water humic substances might be one of the aetiological factors of K a s h i n - B e c k d i s e a s e , an e n d e m i c c h r o n i c degenerative joint disorder, found in Mainland China (Zhai et al., 1990). Humic substances have also been implicated as a cause of environmental goitrogen (Gaitan et al., 1980; source Gaitan et al., 1983; Cooksey et al., 1985).
*To whom correspondence should be addressed at: Department of Biochemistry, College of Medicine, National Taiwan University, Jen-Ai Road, Sec. 1, Taipei, Taiwan, Republic of China.
We have isolated a blue-green fluorescent humic substances from well water where Blackfoot disease is endemic in an area of the southwest coast of Taiwan (Lu et al., 1975). Blackfoot disease is an endemic peripheral vascular disease. Clinically, patients first experience numbness or coldness of one or more of the extremities resulting in black discolouration, ulceration or gangrenous change in extremities (Tseng et al., 1961). Our previous experimental results showed that this fluorescent humic substances of well water could induce blackening of the tail and feet of mice (Lu, 1990a), inhibit plasmin activity (Lu and Lee, 1992) and shorten prothrombin time of normal human pooled plasma (Lu et al., 1990), and suggested that well water humic substance might be one of the aetiological factors of Blackfoot disease (Lu, 1990b). Here, we report that humic substance (both from well water and Aldrich humic acids) as well as lignin, a polymer of phenolic compounds, have the ability to shorten normal human plasma prothrombin time, whereas, monomeric compounds of humic acid do not have such ability. The ability to shorten the prothrombin time of humic substance can be inhibited by some reducing agents such as t-butanol, glutathione, a s c o r b i c acid and dithiothreitol. Materials and Methods Chemicals
Aldrich humic acid was obtained from Aldrich
66
Human plasma prothrombin COOH
Pr0C0caLechuicacid
-
OH
Pyr ll01
Vanillic acid
3HC 3
OH
C t ho]
2
COCI-t
Gallic aci0
H=:CHCO~:=CI--tCOOH
Syringic acid C H=CH(XXlq
4 i -5
*-
-6
i -
-4
I -3
J -
-1
log (mg m1-1) -ell
Ferulit. OH
p"c0u ic acid
Caffeic acid
acid
OH
R rcinol Figure 1 Monomers of humic acid.
Chemical Co., Milwaukee,U.S.A., which was further purified by acid precipitation with 0.1M HC1. Lignin was obtained from Tokyo Kasei Kogyo Co. Protocatechuic acid, pyrogallol, vanillic acid, gallic acid, ferulic acid, catechol, caffeic acid, syringic acid, p-coumaric acid, glutathione, ascorbic acid and dithiothreitol were obtained from Wako Pure Chemical Industries, Japan. The prothrombin time reagent kit (thromborel S, which consisted of thromboplastin containing calcium, was a lyophilised extract from human placenta) was obtained from the Behring Co., Germany. Normal human pooled plasma, was prepared by mixing aliquots from 50 normal persons whose plasma prothrombin time was in the range from 11.0 to 14.0 s. This was obtained from the outpatient division of National Taiwan University Hospital, Taiwan. Extraction of well water humic substances Water was taken from a well located in the Blackfoot disease endemic area in Tainan County on the southwest coast of Taiwan. The water was concentrated, acidified with 1M HC1 and extracted with alcohol to obtain the crude extract of fluorescent humic substances. The crude extract of fluorescent humic substances was further purified by following the previously reported methods (Lu et al., 1988). In short, well water was filtered, condensed and evaporated to dry powder. An alcohol extract of the crude powder was evaporated again and then dissolved in distilled water. Then it was loaded on to a Sephadex G-25 column and eluted with distilled water. The first eluted peak of
Figure 2 Effects of polymers and monomers of humic acid on prothrombin time. 1 Monomers of humic acid: protocatechuic acid, pyrogallol, vanillic acid, resorcinol, gallic acid, ferulic acid, catechol, caffeic acid, syringic acid and p-coumaric acid. 2 Lignin. 3 Well water humic substances. 4 Aldrich humic acid. APT." The change of prothrombin time. n-=4, Mean +_SD. fluorescent humic substances was used for study. Previous study had shown it consisted of various polymers and organometallic complexes. It contained various functional groups such as OH-, C=O stretch of ketone or aldehyde or ester, CO0-, ethers, phenols and aromatic ring (Lu et al., 1988). The estimated MW of this elute fraction was around 5,000 Dalton. Effect of humic substances and monomeric components on the prothrombin time of normal human pooled plasma Humic substances and various m o n o m e r i c components were prepared respectively at several different concentrations (1.0 xl0 -4 -1.0 mg mL-I). 0.03 mL of each test solution was added to 0.07 mL of normal human pooled plasma. The mixture was heated at 37 ° C for 1 min. then added to 0.2 mL of Thromborel S solution ,which was preheated for 15 min and then the prothrombin time measured with a fibrintimer (Behring Co.). Effects of reducing agents on the ability of humic substances to shorten the prothrombin time of normal human pooled plasma Reducing agents (t-butanol, glutathione, ascorbic acid and dithiothreitol) were prepared at different concentrations (7.5 x 10-2 -3.0 mg mL -1). A suitable concentration of each reducing agent was added to humic substances to make a final concentration of humic substances of 0.01 mg mL -l at which concentration of humic substances showed the greatest ability to shorten the prothrombin time. To 0.03 mL of the final mixture 0.07 mL of normal human pooled plasma was added and the rest of the procedure was the same as in the previous section.
F. Lu, W. Lin and T. Huang
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1
2
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3
67 I
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-2 o -3 03
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