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The area at risk (risk mass/left ventricular mass) in the sham-operated group was 48.3 �� 3.7% for the 1 min ischaemia protocol, and no significant differences were observed among all the groups (Fig. 2B). The infarct size (infarct mass/risk mass) in the sham-operated group was 30.0 �� 2.2% for the 1 min ischaemia protocol and 30.0 �� 2.4% for the 3 min ischaemia protocol. Preconditioning with 1 min ischaemia and 10 min reperfusion did not reduce the infarct size (29.4 �� 2.3%), whereas when preconditioning with 3 min ischaemia and 10 min reperfusion was performed, the infarct [http://en.wikipedia.org/wiki/INPP5D INPP5D] size was significantly reduced to 17.5 �� 4.2% (Fig. 2C). Changes in BH4 contents and the expression of GTPCH mRNA in hearts were determined 24 h after the IPC protocol. Consistent with the infarct size-limiting effect, BH4 contents in hearts were increased by preconditioning with a 3 min ischaemia protocol, but not with a 1 min ischaemia protocol (Fig. 3A). Oxidized biopterin (BH2 + biopterin) levels in the sham group for the 3 min ischaemia protocol and in the IPC group for the 3 min ischaemia protocol were 0.019 �� 0.001 and 0.035 �� 0.004 pmol (mg wet weight)?1, respectively, and total biopterin levels were 0.225 �� 0.016 and 0.350 �� 0.025 pmol (mg wet weight)?1, respectively. Thus, [http://www.selleckchem.com/products/RO4929097.html RO4929097 in vivo] the ratio of BH4 levels to total biopterin levels was slightly decreased by IPC (sham group, 0.957 �� 0.001; IPC group, 0.897 �� 0.014; P [http://www.selleckchem.com/products/Nutlin-3.html Selleckchem Nutlin 3] in the acquisition of resistance to I�CR injury, the effects of DAHP, an inhibitor of BH4 synthesis, were examined. Hamadate et al. (2008) reported that BH4 content in rat aorta was diminished to 65% of the control value by administration of DAHP (100 mg kg?1, i.p.) for 5 h. Although administration of DAHP (100 mg kg?1, i.p.) to sham-operated rats 2 h before determination of BH4 slightly decreased the cardiac BH4 levels (Fig. 4A), its administration eliminated both the elevation of BH4 content by IPC (Fig. 4A) and the infarct size-limiting effect by IPC (Fig. 4C).