Sucrose phosphate , detection

Genetic deficiency of fructokinase is benign and often detected incidentally when the urine is checked for glucose with a dipstick. Fructose 1-phosphate aldolase deficiency is a severe disease because of accumulation of fructose 1-phosphate in the liver and renal proximal tubules. Table 1-12-4 compares the two conditions. Symptoms are reversed after removing fructose and sucrose from the diet. 

Figure 14.3 Chromatograms of excipients in film-former class under different mobile phase pH. In both plots, the curves from the bottom are blank, HPMC, acacia, sucrose NF, HPC, povidone and Eudragit EPO, respectively. The sample solvent, mobile phase and column used are (a) 20% ACN-80% pH 2, 25 mM phosphate buffer, a gradient from 30% ACN-70% pH 2, 25 mM phosphate buffer to 80% ACN-20% pH 2, 25 mM phosphate buffer and a Zorbax SB-C8, 4.6 x 150 mm, 3.5 p.m column at 35°C, respectively and (b) 20% ACN-80% pH 7, 25 mM phosphate buffer, a gradient from 30% ACN-70% pH 7, 25 mM phosphate buffer to 80% ACN-20% pH 7, 25 mM phosphate buffer and a Zorbax XDB-Cg, 4.6 x 150 mm, 3.5 p,m column at 35°C, respectively. In both cases, samples were injected at 1800 p,L, the flow rate of mobile phase was 1 mL/min and the detection was at 210 nm. An on-bench examination of the mixture of either 80% ACN-20% pH 2, 25 mM phosphate buffer or 80% ACN-20% pH 7, 25 mM phosphate buffer revealed no precipitation, so they were suitable as the mobile phase.

During photosynthesis, chloroplasts convert CO2, water and Pj to triose phosphates that are exported to the cytosol. Phosphate is therefore a substrate of this process and the continued operation of the RPP cycle is dependent on the utilization of triose phosphate for the synthesis of starch (in the chloroplast) and sucrose (in the cytosol). These synthetic processes release Pj, preventing the level of free Pj in the cell from falling to a concentration where photosynthesis may be limited by its availability. Such a limitation of photosynthesis is observed during O2-insensitive CO2 assimilation [56] and is suggested by the increase in CO2 fixation detected on feeding P via the transpiration stream to a cut leaf [57]. It has long been known that isolated chloroplasts require a continuous supply of P in order to sustain photosynthesis. 

A deficiency of fructose-1-phosphate aldolase produces this rare disorder with hypoglycemia and liver failure. Fructose ingestion inhibits glycogenolysis and giuconeogenesis, producing hypoglycemia. Early detection is important because this condition responds to a diet devoid of sucrose and fructose. 

The existence of two separate mechanisms for the synthesis of sucrose raises the question of their respective roles in vivo. Experiments in which C Mabeled n-glucose was supplied to plants have shown that the d-fructofuranosyl moiety of sucrose becomes highly labeled before any label appears in the free D-fructose pool, suggesting that this monosaccharide is not an intermediate in sucrose synthesis. On the other hand, d-fructose 6-phosphate becomes labeled before sucrose, and small proportions of sucrose 6 -phosphate have also been detected among the labeled pro-ducts. These results are compatible with the hypothesis that sucrose 6 -phosphate is synthesized first and is subsequently hydrolyzed to free sucrose. Such a pathway would be clearly irreversible, since it includes a hydrolytic step, and could account for the large accumulation of sucrose in many plants. 

Figure 5.28 AIDA enzyme assays for sucrose phosphorylase (SPO) and phospho-glucomutase (PGM) with selective detection of the unlabeled products fructose and glucose-6-phosphate by 4,4 -o-BBV and 3,3 -o-BBV,...

Tissue was homogenised with an equal volume of buffer (2 ml Tris-HCl (pH 8.0), 0.1 ml mercaptoethanol, 10 g sucrose, and 25 mg NADP per 100 ml H2O - adjusted to pH 7.2 with HCl). Electrophoresis was performed on cellulose acetate plates (Titan III Helena Laboratories). A preliminary survey of 42 enzymes detected four enzyme loci, coding for four different enzyme systems that were polymorphic and consistently resolvable. These enzymes were used for the analysis and were as follows (abbreviation and enzyme commission number in parentheses) hexokinase (HK, E.C. 2.7.1.1), mannose-6-phosphate isomerase (MPI, E.C. 5.3.1.8), 6-phosphogluconate dehydrogenase (6PGD, E.C. 1.1.1.44), and phosphoglucose isomerase (PGI, E.C. 5.3.1.9). 

Kritsunankul et al. [76] proposed flow injection online dialysis for sample pretreatment prior to the simultaneous determination of some food additives by HPLC and UV detection (FID-HPLC). For this, a liquid sample or mixed standard solution (900 pL) was injected into a donor stream (5%, w/v, sucrose) of a FID system and was pushed further through a dialysis cell, while an acceptor solution (0.025 mol/L phosphate buffer, pH 3.75) was held on the opposite side of the dialysis membrane. The dialysate was then flowed to an injection loop of the HPLC valve, where it was further injected into the HPLC system and analyzed under isocratic reversed-phase HPLC conditions and UV detection (230 nm) (Figure 24.6). The order of elution of five food additives was acesulfame-K, saccharin, caffeine, benzoic acid, and sorbic acid, with an analysis time of 14 min. This system has advantages of high degrees of automation for sample pretreatment, that is, online sample separation and dilution and low consumption of chemicals and materials. 

L. mesenteroides also contains a sucrose phosphorylase. However, since it has been demonstrated that sucrose is converted to dextran by dex-transucrase in the absence of a detectable quantity of inorganic phosphate and that the enzyme does not form polysaccharide from glucose-l-phos-phate, it is clear that phosphorylase is not involved in dextran synthesis. The evidence indicates that the dextransucrase acts by a direct transfer of glucose units. The reaction appears to involve the substitution of a 1,6-glucosidic linkage for a glucose-fructose bond and can be represented by. the following equation ... 

Inorganic phosphate is not required for levan synthesis, and it has no effect on the rate of reaction no esterification of phosphate can be detected. It is therefore considered that levan synthesis is the result of the transfer of fructose units from sucrose to a suitable acceptor through the mediation of a fructose-enzyme complex. 

An external site of synthesis of PEP from photosynthetically formed 3-PGA can be detected by rising the P. concentration up to 5 mM and adding exogenously excessive amounts of rabbit PGM and enolase to intact chloroplasts (10 and 2.5 units/50 ug chlorophyll) similar as in /42/. The activity of the shikimate pathway (measured as nmol Phe and Tyr formed) was considerably enhanced as a result of supply of high amounts of PEP by this enzyme reaction and re-import by the phosphate translocator. Consequently, this way might be considered as an additional site of supply optimized under in vitro conditions by adding enzymes in excess. Under in vivo conditions, the carbon flow from triosephosphates of photosynthetic carbon fixation is preponderantly directed to sucrose synthesis /43/ and less to PEP. 

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