Natural Glucose

The relative carbon isotope content (V13C value) in each position of glucose from a C4 plant (maize starch) and a C3 plant (sugar beet sucrose) has been determined by stepwise chemical and biochemical degradation of the molecule and stable isotope ratio measurement of the fragments. The suitability of the degradation methods has been tested through their chemical yield and isotope balance. The results from both methods agreed perfectly, revealing a defined and reproducible 13C distribution in glucose from both origins. Most prominent was a relative 13C enrichment by 5 to 6 a-units in position 4 and a depletion by about 5 s-units in carbon 6. As possible reasons for these nonstatistical isotope distributions, isotope effects of the aldolase, the triose phosphate isomerase, and the transketolase reactions during carbohydrate biosynthesis are discussed. The practical importance of the results in regard to isotope distributions in secondary plant products as a means for food authenticity control is outlined. For many years the average natural abundance of carbon isotopes has been used to assign plant natural products to the origin from C3 and C4 plants. Recently, determinations of intramolecular stable carbon isotope distributions in naturally occurring secondary compounds or in products of biochemical reactions have been used as a sophisticated tool to identify their biosynthetic origin as well as isotope effects in their formation (2-4, 10-13, 15). In this context it has sometimes been assumed that carbohydrates as the primary carbon sources in plants have a homogeneous and statistical intramolecular isotope distribution due to the scrambling of their carbon atoms during their synthesis via the Calvin cycle (4, 9). In contradiction to this assumption are observations on the 14C distribution in sugars synthesized during tracer studies on photosynthetic reactions (5). However, in this case the observed isotopic variation could also have been an effect of the short reaction time and would disappear after a longer experimental period. Recently published results of the partial chemical degradation of glucose from maize starch (6) or of the intramolecular isotopic composition of lactate from the fermentation of beet sucrose (E Melzer, unpublished results) indicate a nonstatistical carbon isotope distribution even in natural glucose. Corresponding nonstatistical distributions have also been reported for the carbon bound hydrogen atoms in this substance (9). Therefore, we have systematically studied the complete carbon isotope distribution in glucose from different origins, 609 namely from a C3 plant (sucrose from beet sugar) and from a C4 plant (maize starch hydrolysate). By the combination of biochemical and chemical methods we have been able to determine the relative and absolute 13C abundance in any of the six positions in the glucose molecule. The reliability of the methods used was controlled through the chemical and isotopic yields of the degradation procedures and by their assessment with standard substances. MATERIALS AND METHODS The assignment of the carbon atoms of glucose to the products of the biological degradations used are shown in Figure 1. The fermentation yields with thoroughly washed resting cells ofLactobacillus bulgaricus and Leuconostoc mesenteroides were in the range of 96 to 98 and 90 to 95%, respectively. The degradation of glucose by Saccharomyces cerevisiae and L. bulgaricus, in combination with the chemical degradation of the fermentation products (ethanol and lactic acid, respectively), led to the mean 63"C values for the carbon atoms I + 6, 2 + 5, 3 + 4 of glucose. In spite of the fact that the two fermentation pathways are different, the results were absolutely identical. By the glucose fermentation with L. mesenteroides and a combined chemical degradation of the ethanol obtained, the isotope abundances in carbon atoms 1, 2, and 3 of the glucose molecule could be measured individually. Finally, from the combination of the results of the homofermentative and heterofermentative fermentations the 63 3C values for any individual carbon atom in the glucose molecule were obtained.