2008-02-05 | 氢应该是我们身体内的重要抗氧化物质
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根据日本学者的发现，我最近经过查找文献，发现人体和动物体内存在一定的氢，这些氢的水平已经达到抗氧化的能力，因此我提出这样的一个观点，目前正在计划用实验证明。
下面是我最近写的一个小文，下月将发表。供大家批评指正。如果是正确的话，将会产生很大影响。
Hydrogen should be an endogenous antioxidant in the body
Recently, Ohsawa et al. provide evidence that inhaled hydrogen gas has antioxidant and antiapoptotic activities that protect the brain and liver against ischemia-reperfusion injury[1,2]. In fact, there is some endogenous hydrogen produced by intestinal bacteria within animal and human. The concentration of hydrogen in some mice tissues is reached the antioxidant effect in their paper demonstrates, so we think that hydrogen should be an endogenous antioxidant in the body.
The most lightweight gas diatomic hydrogen, a major component of interstellar space and the fuel that sustains the stars, is rare on Earth. Hydrogen gas directly and violently reacts with oxidizing elements such as chlorine and fluorine and is highly flammable, a property evident in the 1937 Hindenburg zeppelin fire and its use as propellant fuel for the space shuttle. Hydrogen gas is highly diffusible and reacts with hydroxyl radical to produce water[2].
Ohsawa et al. set out to see if hydrogen gas could be used as a therapeutic mitochondrial antioxidant to neutralize oxidative stress after ischemia- reperfusion injury[2]. To induce the production of reactive oxygen species, the authors treated cultured cells with a mitochondrial respiratory complex I inhibitor or subjected them to oxygen or glucose deprivation. After oxidative damage, cells underwent pathological mitochondrial depolarization, ATP depletion, DNA oxidation, lipid peroxidation, and cellular necrosis and apoptosis. When dissolved in the media, hydrogen gas dose- dependently prevented these events and improved cell viability. In these studies, Ohsawa et al found the smallest concentration of hydrogen which can increase cell survival significantly is 25 M. (Figure 1).
Figure 1 Molecular hydrogen protects cultured PC12 cells by scavenging hydroxyl radicals[2]. (a–d) PC12 cells were maintained with 10 g/ml antimycin A, with (+) or without (–) 0.6 mM hydrogen, for 24 h in a closed flask, and immunostained with antibodies to 8-OH-G or HNE. Fluorescence signals in response to 8-OH-G and HNE were quantified using 100 cells from each independent experiment (n= 4). *P < 0.05, **P< 0.01. 
Phase-contrast pictures of PC12 cells 24 h after the exposure to antimycin A, with (+) or without (–) 0.6 mM hydrogen. Arrows indicate dead cells. 
Cell survival was assessed by manually counting the cells (n= 4). *P < 0.05, **P<0.01 (compared with 0 mM hydrogen). 
PC12 cells were exposed to intracellular hydroxyl radical produced by the Fenton reaction, with or without 0.6 mM hydrogen. Cells were preincubated with 1 mM CuSO4, washed, and exposed for 1 h to 0.1 mM ascorbate in order to reduce intracellular Cu2+ to Cu+. The cells were costained with propidium iodide (for dead cells) and Hoechst 33342 to visualize the nuclei. 
Cell survival was assessed by manually counting the cells as in f (n = 5). *P< 0.05, **P < 0.01. Scale bars: 50 M in a,c,e; 100M in g. Histograms represent mean ± s.d.
These studies also indicated that hydrogen gas could reach subcellular compartments such as the nucleus and mitochondria. This is particularly important, as the latter is the primary site of generation of reactive oxygen species after reperfusion and is notoriously difficult to target. Biochemical experiments using fluorescent probes and electron paramagnetic resonance spectroscopy spin traps indicated that hydrogen gas may selectively scavenge the hydroxyl radical. The authors propose this as a unique cytoprotective pathway that specifically quenches the hydroxyl radical while preserving other reactive oxygen and nitrogen species important in signaling.
To test the efficacy of hydrogen gas therapy during oxidative stress, Ohsawa et al. used a rat model of stroke, with middle cerebral artery ligation and reperfusion. Inhalation of 2% hydrogen gas limited the stroke volume if given before the reperfusion phase of injury. Hydrogen gas treatment also reduced brain tissue lipid peroxidation and DNA oxidation, findings that were also noted in cultured cells challenged with reactive oxygen species. The decrease in reperfusion damage improved long term neurological function, such as thermoregulation and weight maintenance, at one week, implying that hydrogen gas can protect cells in vivo.
Many antioxidants or enzymes that scavenge reactive oxygen species limit cytotoxicity after ischemia and reperfusion. In the presence of catalytically active metals, however, detoxification of superoxide to hydrogen peroxide by superoxide dismutase generates the more potent hydroxyl radical. This radical reacts indiscriminately with and damages molecular targets such as nucleic acids, lipids and proteins. Ohsawa et al. have proposed that selective hydroxyl radical scavenging is how hydrogen gas protects cells from oxidative damage after ischemia-reperfusion[2].
It is important to pay attention to the smallest effect concentration of Ohsawa et al’s paper. In vitro experiment shown in Figure 1f in their paper demonstrates that 25 M hydrogen is still effective in cultured cells. In vivo experiment, 2% gas gives 32 M hydrogen in water. Hydrogen is dissolved in lipids more than water, thus may give 60 M in the brain.
In fact, there is production of hydrogen within animals and human. Carbohydrates that are incompletely absorbed by the small intestine within animals reach the colon where they are fermented by intestinal bacteria [3,4]. These floras are primarily anaerobes present in animal faeces or in the colon and perform hydrogen producing reactions associated with short chain volatile fatty acid production. Along with these fatty acids the gases hydrogen and CO2 are produced. These gases associated with fermentations are not utilized by the host, but are primarily either lost in faeces or flatus, or assimilated by methaneproducing bacteria [4,5]. Some studies indicate that a significant proportion of the hydrogen produced by the colonic flora is absorbed into the bloodstream and can even be detected on the breath [6,7]. For example, an estimated 14% [6] or 20% [4] of the total colonic hydrogen production was reported to be carried through the human bloodstream and then released into the lungs.
Some researcher reported that there is plenty of hydrogen in stomach and liver tissues. Olson and Maier determined the average hydrogen content of the mucus layer of the mouse stomach to be 43 M [8] (Table 1). These measurements were taken on different days and at different times during the day and ranged in concentrations from 17 to 93 M[9]. It may be expected that the type of diet of the animal would affect the colonic flora fermentation responses; diet would then affect the hydrogen concentrations in tissues, but was not studied in this paper. Maier et al also found the average hydrogen concentration is over 53 M(Table 2). Molecular hydrogen levels ranged from 118 to 239 µM in the small intestine of live mice (the mean value for 12 determinations was 168 µM), and spleen and liver tissue hydrogen levels were approximately 43 µM[10].
Table 1. Hydrogen concentrations in mouse stomachs[8].
Mouse nohydrogen range(M)Sites measured
125-938
235-888
317-297
419-778
A 50 m size microelectrode probe was used to measure hydrogen in the mucus lining area of the stomach in mice.
Table 2. Microelectrode-determined hydrogen concentrations in mouse livers[9]
Mouse nohydrogen range(M)Mean SDsites measured*
143–6354 910
229–8953 1812
343–6857 1112
* The sites measured included all lobes of the liver in mice.
These studies show that the concentration of hydrogen, at least in liver, stomach, small intestine and spleen, is reached the concentration which need to neutralize oxidative stress after ischemia-reperfusion injury. So we think the hydrogen should be the endogenesis antioxidant in the body.
Although exogenous hydrogen can act as a therapeutic antioxidant as described by Ohsawa et al, there is also a gap between the endogenous and exogenous hydrogen. further studies are required to elucidate whether endogenous hydrogen is an antioxidant. In order to examine our proposal, we can restrain the intestinal bacteria to produce hydrogen by some special antibiotics, and check the oxidative stress index in some tissue.
References
1.Fukuda K, Asoh S, Ishikawa M,et al. Inhalation of hydrogen gas suppresses hepatic injury caused by ischemia/reperfusion through reducing oxidative stress[J]. Biochem Biophys Res Commun. 2007,361:670-674.
2.Ohsawa I, Ishikawa M, Takahashi K, et al. Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals[J]. Nat Med. 2007,13: 688-694 .
3.Maier, R.J. Availability and use of molecular hydrogen as an energy substrate for Helicobacter species[J]. Microbes Infect. 2003,5:1159-1163.
4.Wolin, M.J. and Miller, T.L. Acetogenesis from CO2 in the human colonic ecosystem, in Acetogenesis [M]//Drake, H.L., Chapman and Hall, New York.,1994: 365-385.
5.Miller, T.L. and Wolin, M.J. Pathways of acetate, propionate, and butyrate formation by the human fecal microbial flora[J]. Appl Environ. Microbiol. 1996, 62:1589-1592.
6.Bond, Jr, J.H., Levitt, M.D. and Prentiss, R. Investigation of small bowel transit time in man utilizing pulmonary hydrogen measurements[J]. J Lab Clin Med. 1975,85:546-555.
7.Tharanathan, R.N. Food derived carbohydrates structural complexity and functional diversity[J]. Crit Rev Biotechnol.2002, 22: 65-84.
8.Olson, J. W., and R. J. Maier. Molecular hydrogen as an energy source for Helicobacter pylori[J]. Science. 2002, 298:1788-1790.
9.R.J. Maier, J. Olson and A. Olczak..Hydrogen-oxidizing capabilities of Helicobacter hepaticus and in vivo availability of the substrate[J]. J. Bacteriol.2003,185:2680-2682.
10.Maier RJ.Use of molecular hydrogen as an energy substrate by human pathogenic bacteria[J]. Biochem Soc Trans.2005,33:83-85.
Phase-contrast pictures of PC12 cells 24 h after the exposure to antimycin A, with (+) or without (–) 0.6 mM hydrogen. Arrows indicate dead cells. Cell survival was assessed by manually counting the cells (n= 4). *P < 0.05, **P<0.01 (compared with 0 mM hydrogen). PC12 cells were exposed to intracellular hydroxyl radical produced by the Fenton reaction, with or without 0.6 mM hydrogen. Cells were preincubated with 1 mM CuSO4, washed, and exposed for 1 h to 0.1 mM ascorbate in order to reduce intracellular Cu2+ to Cu+. The cells were costained with propidium iodide (for dead cells) and Hoechst 33342 to visualize the nuclei. Cell survival was assessed by manually counting the cells as in f (n = 5). *P< 0.05, **P < 0.01. Scale bars: 50 M in a,c,e; 100M in g. Histograms represent mean ± s.d.
These studies also indicated that hydrogen gas could reach subcellular compartments such as the nucleus and mitochondria. This is particularly important, as the latter is the primary site of generation of reactive oxygen species after reperfusion and is notoriously difficult to target. Biochemical experiments using fluorescent probes and electron paramagnetic resonance spectroscopy spin traps indicated that hydrogen gas may selectively scavenge the hydroxyl radical. The authors propose this as a unique cytoprotective pathway that specifically quenches the hydroxyl radical while preserving other reactive oxygen and nitrogen species important in signaling.
To test the efficacy of hydrogen gas therapy during oxidative stress, Ohsawa et al. used a rat model of stroke, with middle cerebral artery ligation and reperfusion. Inhalation of 2% hydrogen gas limited the stroke volume if given before the reperfusion phase of injury. Hydrogen gas treatment also reduced brain tissue lipid peroxidation and DNA oxidation, findings that were also noted in cultured cells challenged with reactive oxygen species. The decrease in reperfusion damage improved long term neurological function, such as thermoregulation and weight maintenance, at one week, implying that hydrogen gas can protect cells in vivo.
Many antioxidants or enzymes that scavenge reactive oxygen species limit cytotoxicity after ischemia and reperfusion. In the presence of catalytically active metals, however, detoxification of superoxide to hydrogen peroxide by superoxide dismutase generates the more potent hydroxyl radical. This radical reacts indiscriminately with and damages molecular targets such as nucleic acids, lipids and proteins. Ohsawa et al. have proposed that selective hydroxyl radical scavenging is how hydrogen gas protects cells from oxidative damage after ischemia-reperfusion[2].
It is important to pay attention to the smallest effect concentration of Ohsawa et al’s paper. In vitro experiment shown in Figure 1f in their paper demonstrates that 25 M hydrogen is still effective in cultured cells. In vivo experiment, 2% gas gives 32 M hydrogen in water. Hydrogen is dissolved in lipids more than water, thus may give 60 M in the brain.
In fact, there is production of hydrogen within animals and human. Carbohydrates that are incompletely absorbed by the small intestine within animals reach the colon where they are fermented by intestinal bacteria [3,4]. These floras are primarily anaerobes present in animal faeces or in the colon and perform hydrogen producing reactions associated with short chain volatile fatty acid production. Along with these fatty acids the gases hydrogen and CO2 are produced. These gases associated with fermentations are not utilized by the host, but are primarily either lost in faeces or flatus, or assimilated by methaneproducing bacteria [4,5]. Some studies indicate that a significant proportion of the hydrogen produced by the colonic flora is absorbed into the bloodstream and can even be detected on the breath [6,7]. For example, an estimated 14% [6] or 20% [4] of the total colonic hydrogen production was reported to be carried through the human bloodstream and then released into the lungs.
Some researcher reported that there is plenty of hydrogen in stomach and liver tissues. Olson and Maier determined the average hydrogen content of the mucus layer of the mouse stomach to be 43 M [8] (Table 1). These measurements were taken on different days and at different times during the day and ranged in concentrations from 17 to 93 M[9]. It may be expected that the type of diet of the animal would affect the colonic flora fermentation responses; diet would then affect the hydrogen concentrations in tissues, but was not studied in this paper. Maier et al also found the average hydrogen concentration is over 53 M(Table 2). Molecular hydrogen levels ranged from 118 to 239 µM in the small intestine of live mice (the mean value for 12 determinations was 168 µM), and spleen and liver tissue hydrogen levels were approximately 43 µM[10].
Table 1. Hydrogen concentrations in mouse stomachs[8].
Mouse nohydrogen range(M)Sites measured
125-938
235-888
317-297
419-778
A 50 m size microelectrode probe was used to measure hydrogen in the mucus lining area of the stomach in mice.
Table 2. Microelectrode-determined hydrogen concentrations in mouse livers[9]
Mouse nohydrogen range(M)Mean SDsites measured*
143–6354 910
229–8953 1812
343–6857 1112
* The sites measured included all lobes of the liver in mice.
These studies show that the concentration of hydrogen, at least in liver, stomach, small intestine and spleen, is reached the concentration which need to neutralize oxidative stress after ischemia-reperfusion injury. So we think the hydrogen should be the endogenesis antioxidant in the body.
Although exogenous hydrogen can act as a therapeutic antioxidant as described by Ohsawa et al, there is also a gap between the endogenous and exogenous hydrogen. further studies are required to elucidate whether endogenous hydrogen is an antioxidant. In order to examine our proposal, we can restrain the intestinal bacteria to produce hydrogen by some special antibiotics, and check the oxidative stress index in some tissue.
References
1.Fukuda K, Asoh S, Ishikawa M,et al. Inhalation of hydrogen gas suppresses hepatic injury caused by ischemia/reperfusion through reducing oxidative stress[J]. Biochem Biophys Res Commun. 2007,361:670-674.
2.Ohsawa I, Ishikawa M, Takahashi K, et al. Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals[J]. Nat Med. 2007,13: 688-694 .
3.Maier, R.J. Availability and use of molecular hydrogen as an energy substrate for Helicobacter species[J]. Microbes Infect. 2003,5:1159-1163.
4.Wolin, M.J. and Miller, T.L. Acetogenesis from CO2 in the human colonic ecosystem, in Acetogenesis [M]//Drake, H.L., Chapman and Hall, New York.,1994: 365-385.
5.Miller, T.L. and Wolin, M.J. Pathways of acetate, propionate, and butyrate formation by the human fecal microbial flora[J]. Appl Environ. Microbiol. 1996, 62:1589-1592.
6.Bond, Jr, J.H., Levitt, M.D. and Prentiss, R. Investigation of small bowel transit time in man utilizing pulmonary hydrogen measurements[J]. J Lab Clin Med. 1975,85:546-555.
7.Tharanathan, R.N. Food derived carbohydrates structural complexity and functional diversity[J]. Crit Rev Biotechnol.2002, 22: 65-84.
8.Olson, J. W., and R. J. Maier. Molecular hydrogen as an energy source for Helicobacter pylori[J]. Science. 2002, 298:1788-1790.
9.R.J. Maier, J. Olson and A. Olczak..Hydrogen-oxidizing capabilities of Helicobacter hepaticus and in vivo availability of the substrate[J]. J. Bacteriol.2003,185:2680-2682.
10.Maier RJ.Use of molecular hydrogen as an energy substrate by human pathogenic bacteria[J]. Biochem Soc Trans.2005,33:83-85.
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