NoteBioscience of Microbiota, Food and Health Vol. 31 (4), 103–108, 2012
Suppressive Effect of High Hydrogen Generating High Amylose Cornstarch on Subacute Hepatic Ischemia-reperfusion Injury in Rats
Hiroki TANABE1, Yumi SASAKI1, Tatsuro YAMAMOTO1, Shuhachi KIRIYAMA2 and Naomichi NISHIMURA1*
1Nayoro City University, Kita 8-1, Nishi 4, Nayoro, Hokkaido 096-8641, Japan2Luminacoid Laboratory, Mure 4-1-4, Mitaka, Tokyo 181-0002, Japan
Received March 1, 2012; Accepted June 12, 2012
We examined whether feeding high hydrogen generating resistant starch could suppress subacute hepatic ischemia-reperfusion injury. Rats were fed a control diet with or without 20% high amylose cornstarch (HAS) supplementationfor 14 days. On day 12, rats were subject to ischemia-reperfusion treatment. Portal hydrogen concentration washigher in the HAS group compared with the control group. Increased plasma alanine and aspartate aminotransferaseactivities due to ischemia-reperfusion treatment tended to decrease, and a significant reduction was observed by HASfeeding when compared with the control group. In conclusion, HAS, which enhances hydrogen generation in thehindgut, alleviated subacute hepatic ischemia-reperfusion injury. 
Key words:  hydrogen; high amylose cornstarch; large intestine; ischemia-reperfusion; antioxidant
Oxidative damage is a cause of various diseases, suchas ischemia-reperfusion (IR) injury, atherosclerosis anddiabetes [1, 2]. Alleviation of oxidative damage istherefore considered an effective measure in preventingthese diseases. Many researchers have been investigatingthe effectiveness of various antioxidants, such asascorbic acid, tocopherols, polyphenols, and carotenoidsagainst such diseases. Recently, the antioxidative effectsof H2 on other organs have been reported in IR-treatedrodents administered H2 via inhalation of H2 gas oradministration of H2 water [3–9]. It is clear from thesestudies that H2 is an effective antioxidant in vivo.Previously, we found that H2 generated from highamylose cornstarch (HAS) and pectin by fermentation inthe large intestine alleviated initial acute hepatic IRinjury, which was induced by 45 min of reperfusionfollowing 30 min of ischemia [10]. Also, the portal H2concentration in rats fed HAS and pectin wascomparable to or more than in those administered H2 viainhalation of H2 gas [3] or administration of H2 water[11]. Therefore, H2 production via large-bowelfermentation is a continuous and available means ofsupplying significant amounts of H2 in vivo. However,the effect of H2 generated in the large intestine on
subacute hepatic IR injury remains unclear.Hepatic IR injury has been demonstrated to occur in abiphasic pattern: first, there is an initial acute phasecharacterized by hepatocellular damage and generationof reactive oxygen species at 1–6 hr, and then there is asubacute phase characterized by massive neutrophilinfiltration and production of inflammatory mediators at18–24 hr [12–14]. Many studies have demonstrated theeffect of H2 molecules on initial acute hepatic IR injury.However, to our knowledge, the effect of H2 molecules,including H2 generated by large intestinal fermentation,on oxidative stress in the subacute phase remains poorlyunderstood. During the subacute phase of injury, theredox burden in damaged tissues is amplified byneutrophils, and then the injury worsens. Therefore, it isimportant to clarify the effect of H2 generated in thelarge intestine on subacute hepatic IR injury. Increasedurinary excretion of 8-hydroxy-2'-deoxyguanosine (8-OHdG), which is an oxidation product of guanine, isobserved due to oxidative damage of DNA [15–17]. Wepresumed that urinary 8-OHdG could be used as amarker of subacute hepatic IR injury. In the presentstudy, to determine the suppressive effect of resistantstarch (RS) on subacute hepatic IR injury, we examinedthe effects of HAS, which results in production of moreH2 in the large intestine than pectin, on liver damage in arat model of subacute hepatic IR.
*Corresponding author. Mailing address: Naomichi Nishimura,Nayoro City University, Kita 8-1, Nishi 4, Nayoro, Hokkaido 096-8641, Japan. FAX: +81-1654-3-3354. E-mail: nishimura@nayoro.ac.jp
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High amylose cornstarch (Hi-maize 1043) was kindlysupplied from Nippon NSC Ltd. (Tokyo, Japan). Thestudy was approved by the Nayoro City UniversityAnimal Use Committee, and animals were maintained inaccordance with the Guidelines for the Care and Use ofLaboratory Animals, Nayoro City University. Seven-week-old male Sprague-Dawley rats were obtained fromJapan SLC (Shizuoka, Japan). They were housed inindividual cages with screen bottoms made of stainlesssteel in a room maintained at 23 ± 1°C with humidityranging from 50% to 70% under lighting conditions with12 hr of light and 12 hr of darkness, daily. The rats wereacclimated by feeding a laboratory chow diet (CE-2,CLEA Japan, Inc., Tokyo) for 5–6 days in order to selecthigh H2-generating (HG) rats before they were subjectedto the experiments. After the acclimation period, net H2excretion from expired air and flatus per 5 minutes wasmeasured using a GC (Biogas analyzer BAS-1000,Mitleben, Osaka, Japan). Forty-nine rats were assignedinto 4 groups (n = 12–13) so as to balance the net H2excretion and body weight. Two groups wereadministered the C diet, and the remaining group wasadministered the 20% HAS diet for 14 days. The C dietwas composed of 250 g/kg casein, 482.5 g/kg cornstarch,100 g/kg sucrose, 70 g/kg soybean oil, 35 g/kg AIN-93Gmineral mix  [18], 10 g/kg AIN-93 vitamin mix [18], 2.5g/kg choline bitartrate and 50 g/kg cellulose. On day 10,net H2 excretion was measured, and urine was collectedfor 2 days. On day 12, one C group and one HAS groupunderwent IR treatment. The remaining C and HASgroups were sham operated. Furthermore, urine wascollected for 2 days after IR treatment and shamoperation.Under pentobarbital sodium anesthesia (70mg/kg ip), the hepatic artery and portal vein to the leftlateral and median lobes were occluded to interruptblood supply to the liver for 30 min, and then reperfusionwas initiated at 37°C. Rats were euthanized at 48 hr afterreperfusion for sampling. Sham-operated rats wereprepared in a similar manner except without vascularocclusion. One-milliliter samples of blood from theportal vein were successively collected into sealedheparin vials and microtubes for H2 analysis and plasmapreparation. A 1 ml sample of the gaseous phase waswithdrawn using a gas-tight syringe, and the H2concentration was determined using GC (Biogasanalyzer BAS-1000). The remaining blood wasseparated by centrifugation (1,200 × g for 20 minutes at4°C), and plasma samples were stored at –80°C. Theliver was perfused immediately after blood withdrawalwith cold saline. Immediately after perfusion, the medianlobe (ischemic area) was removed, and a portion was
rapidly frozen in liquid nitrogen; the samples were thenstored at –80°C until oxidative stress analyses. Activitiesof alanine and aspartate aminotransferase (ALT andAST) in plasma were measured using a commercial kit(Transaminase CII-test Wako, Wako, Tokyo, Japan).Reduced hepatic glutathione (GSH) and oxidizedglutathione (GSSG) levels were determined using themethod of Anderson [19]. The malondialdehyde (MDA)concentration in the liver was measured according to theprocedure of Ohkawa et al. [20]. Hepatic activity ofsuperoxide dismutase (SOD) and myeloperoxidase(MPO) was determined by the method of Sun et al. [21]and Krawisz et al. [22], respectively. Urine 8-OHdGcontent was measured using a commercial ELISA kit(Highly Sensitive 8-OHdG Check ELISA; Nikken SEILCo., Ltd, Fukuroi, Japan). Allantoin and creatinineexcretion in urine were determined by the methods ofYoung and Conway [23] and Bonsnes and Taussky [24],respectively. Values obtained from the experiments wereexpressed as means ± SEM. Data were subjected toBartlett’s test for homogeneity of variances. For sampleswith equal variances, one-way ANOVA was used,followed by the Tukey-Kramer post hoc test for multiplecomparisons between individual group means. If samplevariances were unequal, we used the Steel-Dwass test[plasma ALT and AST activities, net H2 excretion andportal H2 concentration]. Furthermore, to examine thepossible role of cecal H2 in alleviating oxidative stress,we used the Mann-Whitney U test using only IR rat datafor plasma ALT and AST activity analyses. The Tukey-Kramer test and Mann-Whitney U test were performedusing the SAS JMP software (version 8.0.1; Tokyo,Japan), and the Steel-Dwass test was performed usingKyPlot (version 5.0; KyensLab Inc, Tokyo, Japan).Significance was defined as a p < 0.05.In the first 12 days of the experimental period, bodyweight gain and food intake did not differ among thegroups (Table 1). However, body weight gain and foodintake from days 13–14 of the experimental period werelower in the IR rats compared with the sham-operatedrats. Net H2 excretion on day 10 and portal H2concentration on day 14 were about 10-fold and 5-foldhigher in rats from the HAS group compared with thosein the C group, respectively. Plasma ALT and ASTactivities were significantly higher in the IR-C groupcompared with the sham group. Plasma ALT and ASTactivities in the IR-HAS group tended to decrease, and asignificant reduction was observed in the IR-HAS groupcompared with the IR-C group (p = 0.0725 and 0.0167),respectively. Urinary excretion of 8-OHdG correctedwith creatinine on days 11–12 and days 13–14 did not
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differ among the groups (Table 2). Urinary excretion ofallantoin, which is a metabolite of purine bodies,corrected with creatinine also did not change among thegroups. No significant differences in SOD activity,glutathione concentration and MDA concentration wereobserved. Significant differences were observed inhepatic MPO activity between the groups (Table 3).These parameters are related to oxidative stress andinflammation. Hepatic MPO activity was higher in IRrats compared with sham-operated rats, but nosignificant effect was observed by HAS feeding.Plasma ALT and AST activities are used clinically asa marker of hepatic injury. In the present study, lowplasma ALT and AST activities in IR rats fed the HASdiet were observed compared with those fed the control
diet. Therefore, it is possible that hepatic injury wasalleviated by H2 molecules generated in the largeintestine. H2 generated in vivo should relieve hepatic IRinjury in the subacute phase as well as the initial acutephase. We previously found that pectin and HASincrease cecal H2 production and relieve initial acutehepatic IR injury in rats [10]. Recently, many studieshave reported that H2 molecules exert an antioxidanteffect in vivo. Ohsawa and his collaborator demonstratedthe alleviation effect of H2 gas on IR injury of the brain[3], liver [5] and heart [4]. Furthermore, a similar effectwas shown for the myocardium and intestine [25, 26] viaadministration of H2-rich saline. In the liver, IR injuryhas been demonstrated to occur in a biphasic pattern: aninitial acute phase characterized by hepatocellular
Table 1. Effect of high amylose cornstarch on body weight gain, food intake and net H2 excretion in IR-treated and sham-operated rats
Sham IRTwo-way ANOVA
CHASCHASDietTreatment Interaction
Body weight gain   Start-day 12 (g/12 days)71 ± 372 ± 371 ± 370 ± 2 0.9606 0.6134 0.7514   Days 13–14 (g/2 days)3.7 ± 1.1 3.9 ± 0.5 –3.3 ± 1.4 –3.1 ± 1.80.8598<0.00010.9961Food intake   Start-day 12 (g/12 days)272 ± 4270 ± 5275 ± 8274 ± 60.79420.60650.9485   Days 13–14 (g/2 days)32 ± 133 ± 127 ± 128 ± 10.2106<0.00010.7163Net H2 excretion (mol/5 min)   Start0.327 ± 0.1530.469  ±  0.1800.450  ±  0.1740.345  ±  0.1520.91210.99360.4594
   After 10 days0.130a(0.065–0.420) 0.823b(0.142–0.548) 0.127a(0.055–0.548) 0.603b(0.107–9.88)
C, control; HAS, high amylose cornstarch; IR, ischemia-reperfusion; H2, hydrogen. High H2 generating rats, which we selected, were used. After 30min of ischemia, 45 min of reperfusion was performed in IR rats. Values are expressed as means ± SEM or medians (minimum value-maximum value),n=12–13. Data were analyzed with two-way ANOVA and Tukey-Kramer’s post hoc test or nonparametric multiple test (Steel-Dwass test). Mean valueswithin a row with different superscript letters are significantly different (p < 0.05).
Table 2. Effect of high amylose cornstarch on H2 concentration and aminotransferase activities in the portal vein, and urinary 8-OHdG and
allantoin concentrations in IR-treated and sham-operated rats
Sham IRTwo-way ANOVA
CHASCHASDietTreatmentInteraction
Portal   H2 (mol/L)0.780ab(0.197–5.70)3.16b(0.374–17.7)0.353a(0.087–2.35)  2.57b(0.345–16.8)   Plasma ALT (kat/L) 0.159a(0.082–0.352)0.175a(0.090–0.243) 0.384b(0.096–2.68) 0.229ab(0.046–0.992)   Plasma AST (kat/L)1.46ab(1.14–2.43)1.50a(1.32–1.82)1.84b(1.45–9.10)1.39ab,*(0.95–4.96)Urine 8-OHdG (mol/mol creatinine)   Days 10–128.02 ± 1.1810.4 ± 2.48.71 ± 1.3512.5 ± 2.60.12180.47750.7190   Days 12–1411.2 ± 1.99.70 ± 2.3011.6 ± 2.613.8 ± 4.40.90750.45100.5390 Allantoin (mol/mol creatinine)   Days 10–123.56 ± 0.173.60 ± 0.173.54 ± 0.093.60 ± 0.110.72230.96160.9416
   Days 12–144.47 ± 0.124.83 ± 0.244.66 ± 0.114.85 ± 0.130.09430.51750.5901
ALT, alanine aminotransferase; AST, aspartate aminotransferase; C, control; HAS, high amylose cornstarch; IR, ischemia-reperfusion; H2, hydrogen;8-OHdG, 8-hydroxy-2'-deoxyguanosine. High H2 generating rats, which we selected, were used. After 30 min of ischemia, 45 min of reperfusion wasperformed in IR rats. Values are expressed as means ± SEM or medians (minimum value-maximum value), n=12–13. Data were analyzed with two-wayANOVA and Tukey-Kramer’s post hoc test or nonparametric multiple test (Steel-Dwass test). Mean values within a row with different superscriptletters are significantly different (p < 0.05).* p < 0.05 compared with the IR-C group. Values of plasma ALT and AST activities in both IR groups were analyzed by Mann-Whitney’s U test in caseof the variances that were too different between the sham-operated and IR treated groups.
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damage at 3–6 hr and a subacute phase characterized bymassive neutrophil infiltration at 18–24 hr, [12, 13],which is similar to the conditions used in our presentexperiment. However, the effect of H2 molecules,including H2 generated by large intestinal fermentationon oxidative stress in a subacute phase, remains poorlyunderstood. Our present study is the first to demonstratethe suppressive effect of H2 on hepatic IR injury in thesubacute phase.Plasma ALT and AST activities after 48 hr ofreperfusion in the present study were lower than after 24h of reperfusion in our previous study [10]. This mayindicate that the liver had recovered from injury due torelieved oxidative stress during the 24–48 hr period ofreperfusion. The production of reactive oxygen speciesby neutrophils exacerbates inflammation [27]. HepaticMPO activity, which indicates the presence ofneutrophils, was higher after 48 hr of reperfusion due tothe IR treatment. This result was consistent with thereport of Kawaguchi et al., in which the hepatic MPOcontent after 48 hr of reperfusion was shown to be highdue to IR treatment [28]. However, in the present study,HAS feeding had no significant effect on hepatic MPOactivity. Because H2 exerts an antioxidant effect in theinitial acute phase, the effect of H2 generated from HASin the subacute phase may be dependent on lowinflammation in the initial acute phase. That is, themigration and infiltration of neutrophils may occur lessfrequently during low inflammation in the initial acutephase. A few studies have reported the relationshipbetween large intestinal fermentation and liver MPOactivity; however, further study, including histologicalevaluation, is required to evaluate the effect of H2
derived from dietary fiber and HAS in the subacutephase on recruitment of neutrophils. Also, oxidativestress markers (hepatic glutathione, MDA concentrationand activities of SOD) were not affected by HASfeeding. When rats were subjected to IR treatment(reperfusion time, 15 min–24 hr), the oxidative stressmarkers were altered, as an oxidative status was caused.This discrepancy is likely to be caused by the differentreperfusion times. HAS might have no effect on thesemarkers in this study because the rats had enough time torecover from oxidative stress. In this study, we alsoexamined whether HAS feeding inhibited urinaryexcretion of 8-OHdG, which is an oxidation product ofguanine, in IR rats. Increased urinary excretion of 8-OHdG is observed due to oxidative damage of DNA,caused by various factors such as brain IR [15], diabetes[16, 17] and atherosclerosis [17]. Unfortunately, nochange in the urinary oxidation product could bedetected as a result of IR and HAS treatment in thepresent study. We used urine collected during the 0–48hr period of reperfusion, and it included urine samplescollected during the 0–24 hr period of reperfusion. Urinecollected between 0–24 h may exhibit a change in 8-OHdG excretion due to IR treatment and HAS. In conclusion, we found that high H2 generating HASalleviated subacute hepatic IR injury as well as initialacute IR injury. However, various substances such asshort-chain fatty acids as well as H2 are produced byfermentation in the large intestine. Therefore, furtherstudy is required to evaluate the effect of H2 derivedfrom dietary fiber and RS in the large intestine on thesubacute phase.This research was supported in part by a Grant-in-Aid
Table 3. Effect of high amylose cornstarch on hepatic glutathione and MDA levels and SOD and MPO activities in IR-treated and sham-
operated rats
Sham IRTwo-way ANOVA
CHASCHASDietTreatment Interaction
Glutathione   Total (mol/g tissue)9.78 ± 0.1910.0 ± 0.39.96 ± 0.3010.5 ± 0.30.16640.24410.6182   GSH (mol/g tissue) 9.45 ± 0.209.62 ± 0.259.64 ± 0.3110.1 ± 0.30.22170.18930.5356   GSSG (mol/g tissue)0.326 ± 0.0270.405 ± 0.0470.318 ± 0.0260.336 ± 0.0360.17010.27490.3756   GSH/GSSG30.9 ± 2.426.7 ± 2.432.2 ± 2.332.6 ± 2.30.41740.13060.3396SOD activity   Total (U/g tissue)8660 ± 3407160 ± 8208400 ± 8407350 ± 6700.07520.95750.7488   Mn type (U/g tissue)417 ± 66262 ± 38370 ± 120279 ± 380.84350.11240.6759   Mn type/Total 0.0488 ± 0.00800.0378 ± 0.00460.0464 ± 0.01530.0384 ± 0.00470.32810.92130.8775MDA (nmol/g tissue)135 ± 11152 ± 9148 ± 8151 ± 100.32190.55750.4786
MPO (U/g tissue)0.0247 ± 0.00420.0326 ± 0.00690.112 ± 0.0640.153 ± 0.0480.56250.01670.6954
C, control; HAS, high amylose cornstarch; IR, ischemia-reperfusion; GSH, reduced glutathione; GSSG, oxidized glutathione; SOD, superoxidedismutase; MDA, malondialdehyde; MPO, myeloperoxidase. High H2 generating rats, which we selected, were used. After 30 min of ischemia, 45 minof reperfusion was performed in IR rats. Values are expressed as means ± SEM, n=12–13. Data were analyzed with two-way ANOVA. Totalglutathione and GSSG concentrations were expressed as GSH equivalent.
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for Scientific Research (C) (21500781) from the JapanSociety for the Promotion of Science, Japan. 
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