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Adult Upis ceramboides do not survive freezing in the summer but tolerate freezing to -60 degrees C in midwinter. The accumulation of two cryoprotective polyols, sorbitol and threitol, is integral to the extraordinary cold-hardiness of this beetle. U. ceramboides are the only animals known to accumulate high concentrations of threitol; however, the biosynthetic pathway has not been studied. A series of (13)C-labeled compounds was employed to investigate this biosynthetic pathway using (13)C{(1)H} NMR spectroscopy. In vivo metabolism of (13)C-labeled glucose isotopomers demonstrates that C-3-C-6 of glucose become C-1-C-4 of threitol. This labeling pattern is expected for 4-carbon saccharides arising from the pentose phosphate pathway. In vitro experiments show that threitol is synthesized from erythrose 4-phosphate, a C(4) intermediate in the PPP. Erythrose 4-phosphate is epimerized and/or isomerized to threose 4-phosphate, which is subsequently reduced by a NADPH-dependent polyol dehydrogenase and dephosphorylated by a sugar phosphatase to form threitol. Threitol 4-phosphate appears to be the preferred substrate of the sugar phosphatase(s), promoting threitol synthesis over that of erythritol. In contrast, the NADPH-dependent polyol dehydrogenase exhibits broad substrate specificity. Efficient erythritol catabolism under conditions that promote threitol synthesis, coupled with preferential threitol biosynthesis, appear to be responsible for the accumulation of high concentrations of threitol (250 mm) without concomitant accumulation of erythritol.