Membrane and intracellular modes of sugar-dependent increments in red cell stability.

Sugar-dependent increments in red cell stability under osmotic stress can be ascribed to changes either in the membrane or in the intracellular matrix. These two possible modes of action have been tested and characterized. Rheological investigation of membrane-free haemoglobin solutions has shown that D-glucose, but not D-fructose, promotes the formation of a visco-plastic gel structure. Gel strength is a function of glucose concentration, haemoglobin concentration and temperature. The ability of various sugars to promote gel formation correlates with their solution properties. The existence of gel structure reduces K+ and haemoglobin leak from red cells whose membranes were partially destroyed by gamma-radiation. Reduced osmotic swelling in the presence of glucose is also due to gel formation since the glucose effect is lost in resealed red cell ghosts. D-Fructose does not protect red cells against radiation damage; its mode of action in increasing red cell stability under osmotic stress is a membrane effect. Cell sizing using the Coulter Counter has shown that fructose, but not glucose, can increase the maximal volume at lysis. At 50 mM, D-fructose expands the red cell ghost volume by 11.2%; this represents a 7.2% increase in membrane area. Ghost expansion by fructose is fructose concentration dependent (0-100 mM) and is insensitive to temperature variation (0-37 degrees C).     

Addition of oligosaccharide decreases the freezing lesions on human red blood cell membrane in the presence of dextran and glucose

Although incubation with glucose before freezing can increase the recovery of human red blood cells frozen with polymer, this method can also result in membrane lesions. This study will evaluate whether addition of oligosaccharide (trehalose, sucrose, maltose, or raffinose) can improve the quality of red blood cell membrane after freezing in the presence of glucose and dextran. Following incubation with glucose or the combinations of glucose and oligosaccharides for 3 h in a 37 °C water bath, red blood cells were frozen in liquid nitrogen for 24 h using 40% dextran (W/V) as the extracellular protective solution. The postthaw quality was assessed by percent hemolysis, osmotic fragility, mean corpuscle volume (MCV), distribution of phosphatidylserine, the postthaw 4 °C stability, and the integrity of membrane. The results indicated the loading efficiency of glucose or oligosaccharide was dependent on their concentrations. Moreover, addition of trehalose or sucrose could efficiently decrease osmotic fragility of red blood cells caused by incubation with glucose before freezing. The percentage of damaged cell following incubation with glucose was 38.04 ± 21.68% and significantly more than that of the unfrozen cells (0.95 ± 0.28%, P < 0.01). However, with the increase of the concentrations of trehalose, the percentages of damaged cells were decreased steadily. When the concentration of trehalose was 400 mM, the percentage of damaged cells was 1.97 ± 0.73% and similar to that of the unfrozen cells (P > 0.05). Moreover, similar to trehalose, raffinose can also efficiently prevent the osmotic injury caused by incubation with glucose. The microscopy results also indicated addition of trehalose could efficiently decrease the formation of ghosts caused by incubation with glucose. In addition, the gradient hemolysis study showed addition of oligosaccharide could significantly decrease the osmotic fragility of red blood cells caused by incubation with glucose. After freezing and thawing, when both glucose and trehalose, sucrose, or maltose were on the both sides of membrane, with increase of the concentrations of sugar, the percent hemolysis of frozen red blood cells was firstly decreased and then increased. When the total concentration of sugars was 400 mM, the percent hemolysis was significantly less than that of cells frozen in the presence of dextran and in the absence of glucose and various oligosaccharides (P < 0.01). However, when both glucose and trehalose were only on the outer side of membrane, with increase of the concentrations of sugars, the percent hemolysis was increased steadily. Furthermore, addition of oligosaccharides can efficiently decrease the osmotic fragility and exposure of phosphatidylserine of red blood cells frozen with glucose and dextran. In addition, trehalose or raffinose can also efficiently mitigate the malignant effect of glucose on the postthaw 4 °C stability of red blood cells frozen in the presence of dextran. Finally, addition of trehalose can efficiently protect the integrity of red blood cell membrane following freezing with dextran and glucose. In conclusion, addition of oligosaccharide can efficiently reduce lesions of freezing on red blood cell membrane in the presence of glucose and dextran.     

Transport of glucose and fructose in rat hepatocytes at 37 degrees C

The kinetic parameters for transport of the nonmetabolizable glucose analogue 3-O-methyl-D-glucose and the relationship between transport and metabolism of D-glucose and D-fructose were determined in isolated rat hepatocytes at 37 degrees C and pH 7.4. 3-O-Methylglucose at a very low concentration (0.1 mM) equilibrated with the intracellular water with a rate constant of 0.41 s-1. Km for equilibrium exchange entry was 5.5 mM and Vmax was 2.2 mM X s-1 and similar results were obtained when using the zero-trans entry protocol. The rate constant for entry of tracer D-glucose was 0.15 s-1 and Km for glucose was about 20 mM. The phosphorylation rate for D-glucose was much slower than the transport rate. The rate constant for D-fructose entry was about 0.04 s-1, the apparent Km was about 100 mM and Vmax about 5 mM X s-1. The concentration dependence of 3-O-methylglucose inhibition of labelled fructose transport revealed biphasic kinetics indicating that fructose was transferred by both the glucose transporter and a fructose transporter. At concentrations lower than 1 mM, fructose metabolism appeared to be limited by the transport step.     

A yeast biosensor for glucose determination

Summary A yeast potentiometric biosensor for glucose determination is described. After induction of glycolytic enzyme synthesis a cell suspension of the yeast Hansenula anomala is retained in calcium alginate gel on the surface of a glass electrode. This biosensor gives a Nernstian response in glucose concentration of 5·10^–4–5·10^–3 mol/l with a response time of 5 min and a life-time of at least 2 months. Mannose and fructose are the only significantly interfering substances. The biosensor was used for measurement of glucose concentration in urine with results comparable to those obtained by a photometric enzymatic method.     

The effect of glucose and glutamine on the intracellular nucleotide pool and oxygen uptake rate of a murine hybridoma

The effects of media concentrations of glucose andglutamine on the intracellular nucleotide pools andoxygen uptake rates of a murine antibody-secretinghybridoma cell line were investigated. Cells takenfrom mid-exponential phase of growth were incubated inmedium containing varying concentrations of glucose(0–25 mM) and glutamine (0–9 mM). The intracellularconcentrations of ATP, GTP, UTP and CTP, and theadenylate energy charge increased concomitantly withthe medium glucose concentration. The total adenylatenucleotide concentration did not change over a glucose concentration range of 1–25 mM but therelative levels of AMP, ADP and ATP changed as theenergy charge increased from 0.36 to 0.96. Themaximum oxygen uptake rate (OUR) was obtained in thepresence of 0.1–1 mM glucose. However at glucoseconcentrations >1 mM the OUR decreased suggestinga lower level of aerobic metabolism as a result of theCrabtree effect.A low concentration of glutamine (0.5 mM) caused asignificant increase (45–128%) in the ATP, GTP,CTP, UTP, UDP-GNac, and NAD pools and a doubling ofthe OUR compared to glutamine-free cultures. Theminimal concentration of glutamine also caused anincrease in the total adenylate pool indicating thatthe amino acid may stimulate thede novosynthesis of nucleotides. However, all nucleotidepools and the OUR remained unchanged within the rangeof 0.5–9 mM glutamine.Glucose was shown to be the major substrate forenergy metabolism. It was estimated that in thepresence of high concentrations of glucose (10–25 mM),glutamine provided the energy for the maintenance ofup to 28% of the intracellular ATP pool, whereas theremainder was provided by glucose metabolism.(Author for correspondence; E-mail:     

Study of the polyol pathway in the porcine epididymis

Concentrations of D-glucose, D-fructose and D-sorbitol were quantified in porcine epididymal fluid by spectrofluorimetric assays and aldose reductase (AR) and sorbitol dehydrogenase (SDH) were located immunohistochemically in the epididymal epithelium. Glucose and fructose concentrations were low (<1 mM) and decreased in the cauda whereas sorbitol concentration (4-7 mM) was rather uniform along the duct. AR was luminally located on microvilli in the caput and corpus with less presence distally and was present in the lumen. SDH was present apically and basally in epithelial cells throughout the epididymis and in the lumen. The observations are consistent with diffusion of circulating glucose into the lumen, its conversion via AR to sorbitol which accumulates in the lumen and the action of SDH on sorbitol to produce fructose. Sperm metabolism of glucose and fructose may explain their lower concentrations in the cauda and sorbitol could be a metabolic substrate or osmolyte required for volume regulation.     

Determination of subcellular concentrations of soluble carbohydrates in rose petals during opening by nonaqueous fractionation method combined with infiltration–centrifugation method

Petal growth associated with flower opening depends on cell expansion. To understand the role of soluble carbohydrates in petal cell expansion during flower opening, changes in soluble carbohydrate concentrations in vacuole, cytoplasm and apoplast of petal cells during flower opening in rose (Rosa hybrida L.) were investigated. We determined the subcellular distribution of soluble carbohydrates by combining nonaqueous fractionation method and infiltration–centrifugation method. During petal growth, fructose and glucose rapidly accumulated in the vacuole, reaching a maximum when petals almost reflected. Transmission electron microscopy showed that the volume of vacuole and air space drastically increased with petal growth. Carbohydrate concentration was calculated for each compartment of the petal cells and in petals that almost reflected, glucose and fructose concentrations increased to higher than 100 mM in the vacuole. Osmotic pressure increased in apoplast and symplast during flower opening, and this increase was mainly attributed to increases in fructose and glucose concentrations. No large difference in osmotic pressure due to soluble carbohydrates was observed between the apoplast and symplast before flower opening, but total osmotic pressure was much higher in the symplast than in the apoplast, a difference that was partially attributed to inorganic ions. An increase in osmotic pressure due to the continued accumulation of glucose and fructose in the symplast may facilitate water influx into cells, contributing to cell expansion associated with flower opening under conditions where osmotic pressure is higher in the symplast than in the apoplast.     

Effects of pre-freeze incubation of human red blood cells with various sugars on postthaw recovery when using a dextran-rapid cooling protocol

Polymer has been used as substitute to replace glycerol for cryopreservation of red blood cells (RBCs). But polymer can not penetrate cell membrane, it can not efficiently protect the inner membrane. In this study, RBCs were incubated with glucose, fructose, galactose or trehalose and frozen in liquid nitrogen for 24 h using dextran as the extracellular protectant. The postthaw quality was assessed by RBC hemolysis, RBC morphology, PS distribution, osmotic fragility, and the 4 °C stability. The results indicated the loading efficiency of monosaccharide was significantly higher than that of trehalose. Adding trehalose and 40% dextran caused more serious hemolysis before freezing. The percent hemolysis of RBCs loaded with high concentration of trehalose was approximately 16% and significantly more than that of RBCs loaded with glucose (approximately 5%, P < 0.05). Intracellular trehalose can not increase the postthaw recovery of RBCs compared with cells frozen without sugar. However, low concentration of intracellular glucose or galactose can reduce the percent hemolysis to less than 5% and significantly less than that of RBCs frozen without sugar (P < 0.05). Finally, the ability of galactose or fructose to maintain the 4 °C stability was significantly more than that of glucose. In conclusion, the injuries caused by trehalose loading may directly lead to postthaw hemolysis and poor quality of RBCs. However, monosaccharide can enhance the recovery of frozen RBCs. The cryoprotective effect of galactose may be better than that of glucose or fructose. In the future, we will continue to look for a safe and efficient trehalose loading process and try to decrease the osmotic fragility of RBCs frozen with polymers and sugars.     

Effects of exogenous hexoses on bovine in vitro fertilized and cloned embryo development: Improved blastocyst formation after glucose replacement with fructose in a serum-free culture medium

To evaluate the embryotrophic role of three hexoses (glucose, fructose, and galactose), bovine embryos derived from somatic cell nuclear transfer (SCNT) or in vitro-fertilization (IVF) were cultured in a modified synthetic oviductal fluid (mSOF), which contained either glucose (1.5 or 5.6 mM), fructose (1.5 or 5.6 mM), or galactose (1.5 or 5.6 mM). Compared to 1.5 mM glucose, use of 1.5 mM fructose significantly enhanced blastocyst formation in both SCNT (23 vs. 33%) and IVF embryos (26 vs. 34%), while 5.6 mM fructose did not improve blastocyst formation. Using 1.5 mM galactose did not improve blastocyst formation in SCNT embryos (22 vs. 23%), whereas it significantly inhibited blastocyst formation in IVF embryos (26 vs. 0%). In both SCNT and IVF embryos, 5.6 mM glucose or galactose significantly inhibited embryo development. In a second experiment, in glucose-free mSOF, fructose at concentrations of 0.75, 1.5, 3.0, or 5.6 mM was able to support to morula (32-42 vs. 12%) and blastocyst formation (30-38 vs. 12%) compared to 0 mM fructose. In Experiment 3, addition of fructose (1.5, 3.0, or 5.6 mM) to mSOF containing 1.5 mM glucose did not further promote blastocyst formation in SCNT embryos compared with replacement with 1.5 mM fructose only. Replacement of glucose with 1.5 mM fructose significantly increased total blastomeres (143 vs. 123 cells) and trophectodermal (TE) cells (116 vs. 94 cells) and decreased inner cell mass (ICM) to TE cell ratio (0.24 vs. 0.31) in blastocysts, compared to 1.5 mM glucose. The combined addition of 1.5 mM fructose and glucose significantly increased ICM cell number (36.7 cells) and ICM/TE ratio (0.46). In conclusion, fructose might be a more efficient energy substrate than glucose for producing large number of transferable blastocysts derived from SCNT.     

Glucose-induced positive cooperativity of fructose phosphorylation by human B-cell glucokinase

Human B-cell glucokinase displays sigmoidal kinetics towards D-glucose or D-mannose, but fails to do so towards D-fructose. Yet, D-glucose, D-mannose and 2-deoxy-D-glucose confer to the enzyme positive cooperativity towards D-fructose. For instance, in the presence of 5 mM D-[U-¹⁴C]fructose, its rate of phosphorylation is increased to 214.3 ± 11.0%, 134.0 ± 4.3% and 116.5 ± 3.0% of paired control value by D-glucose, D-mannose and 2-deoxy-D-glucose (each 6 mM), respectively. D-glucose and, to a lesser extent, D-mannose also display reciprocal kinetic cooperativity. D-fructose, however, fails to affect D-glucose or D-mannose phosphorylation under conditions in which positive cooperativity is otherwise observed. These findings are relevant to the reciprocal effects of distinct hexoses upon their phosphorylation by B-cell glucokinase and, as such, to the metabolic and functional response evoked in pancreatic islet B-cells by these sugars, when tested either separately or in combination. (Mol Cell Biochem 175: 263–269, 1997)     

Membrane depolarization induced by transcellular osmosis in internodal cells ofNitella flexilis

Summary Membrane depolarization induced by transcellular osmosis was studied using internodal cells ofNitella flexilis. Transcellular osmosis was induced by using sorbitol or methanol as the osmotic agent. In the endosmotic cell half, the membrane often generated an action potential and depolarized further with a concomitant decrease in membrane resistance. This osmosis-induced depolarization was a graded response dependent on the external osmotic gradients. However, in the exosmotic cell half, both membrane potential and membrane resistance changed insignificantly. Membrane depolarization occurred also in cells made inexcitable by bathing in 0.1–1 mM KCl solution.Effects of temperature and internal osmotic pressure on osmosis-induced depolarization were investigated. The magnitude of depolarization at low temperature (2 or 4°C) was larger than that at room temperature (around 20°C). Membrane depolarization was accelerated by lowering the internal osmotic pressure and inhibited by raising it.Not only the plasmalemma but the tonoplast also responded significantly to endosmosis.     

Invertase and phosphatase of yeast in a phosphate-limited continuous culture

Summary Deficiency of inorganic phosphate caused the hyper production of invertase and the derepression of acid phosphatase in a continuous culture ofSaccharomyces carlsbergensis. The specific invertase activity was 40,000 enzyme units per g dry cell weight at a dilution rate lower than 0.05 h^–1 with a synthetic glucose medium of which the molecular ratio of KH₂PO₄ to glucose was less than 0.006. This activity is eight fold higher than in a batch growth and 1.5 fold as much as the highest enzyme activity observed so far in a glucose-limited continuous culture.For the hyper production of invertase, it is necessary to culture the yeast continuously by keeping the Nyholm's conservative inorganic phosphate concentration at less than 0.2 m mole per g dry weight cell. The derepression of acid phosphatase brought about by phosphate deficiency, was similar in both batch and continuous cultures.Nomenclature D dilution rate of continuous culture (h^–1) - E_i invertase concentration in culture (enzyme unit l^–1) - E_p acid phosphatase concentration in culture (enzyme unit l^–1) - P inorganic phosphate concentration in culture (mM) - S glucose concentration in culture (mM) - X cell concentration in culture (g dry weight cell l^–1) Greek Letter specific rate of growth (h^–1) Suffix f feed - 0 initial value     

Taste Responses to Fructose and Tannic Acid Among Gorillas (Gorilla gorilla gorilla)

We investigated the taste responses to fructose and tannic acid compounds among 6 western gorillas (Gorilla gorilla gorilla) at the San Francisco Zoo. We presented the subjects with 12 reference concentrations of 3–300 mM fructose, paired with tap water in paired-choice experimental trials. We subsequently presented them with tap water paired with 8 dilute concentrations of tannic acid (0.25–6 mM) dissolved in 100 mM fructose solutions and 8 concentrations (0.25–6 mM) dissolved in 300 mM fructose solutions. The gorillas exhibited a broadly similar preference threshold for fructose (50 mM) to those of other nonhuman primate and human samples. The gorillas tolerated moderate levels of tanninc acid, especially when presented in a very sweet package. The depressing effect of tannic acid on the ingestion of fructose solutions appeared to increase progressively with tannin concentration and was lower as fructose concentration increased. The inhibition threshold for tannic acid solutions was reached at 4 mM tannic acid in 100 mM fructose solution, and at 6 mM tannic acid in 300 mM fructose solutions. These results suggest that gorillas use sweetness as a criterion for food selection and regard those with moderate levels of tannins as palatable. Our findings corroberate food preference studies and nutritional analyses of wild gorilla foods indicating that they prefer sugary foods and readily consume ones containing moderate levels of tannins. Taste responses may facilitate a flexible frugivorous/folivorous diet among gorillas.     

Interaction of D-fructose and fructose 1-phosphate with yeast phosphofructokinase and its influence on glycolytic oscillations.

Fermentation of D-fructose- and D-glucose induced glycolytic oscillations of different period lengths in Saccharomyces carlsbergensis. Recent studies suggested, that D-fructose or one of its metabolites interacted with phosphofructokinase (ATP:D-fructo-6-phosphate 1-phosphofructokinase, EC 2.7.1.11), the core of the glycolytic 'oscillator'. In order to explore the kinetics of interaction, the influence of D-fructose and fructose 1-phosphate on purified yeast phosphofructokinase was studied. D-fructose concentrations up to 0.3 mM stimulated the enzyme, while a further increase led to competitive inhibition. The Hill coefficient for fructose 6-phosphate decreased from 2.8 to 1.0. Fructose 1-phosphate acted in a similar way, up to 1 mM activation and inhibition competitive to fructose 6-phosphate at higher concentration (2.0--3.5 mM) with the same effect on the Hill coefficient. The inhibition patterns obtained with D-fructose or fructose 1-phosphate suggest a sequential random reaction mechanism of yeast phosphofructokinase with fructose 6-phosphate and MgATP2-. The mode of interaction of phosphofructokinase with D-fructose and fructose 1-phosphate is discussed. The influence of both effectors resulted in altered enzyme kinetics, which may cause the different period lengths of glycolytic oscillations.     

Effect of Fructose Supplementation on Sorbitol Accumulation and myolnositol Metabolism in Cultured Neuroblastoma Cells Exposed to Increased Glucose Concentrations

Aldose reductase activity is increased in neuroblastoma cells grown in media containing 30 mM fructose and/or 30 mM glucose. Neuroblastoma cells cultured in media supplemented with increased concentrations of glucose and fructose amass greater amounts of sorbitol than do cells exposed to media containing only high glucose concentrations. The increase in sorbitol content is dependent on the fructose and glucose concentration in the media. The increase in sorbitol content caused by exposing neuroblastoma cells to media containing 30 mM glucose/30 mM fructose is due to a protein synthesis sensitive mechanism and not to an alteration in the redox state. The addition of sorbinil to media containing 30 mM glucose blocks the increase in sorbitol content. In contrast, sorbinil treatment of media containing 30 mM glucose/30 mM fructose does not totally block the increase in sorbitol levels. myo-Inositol accumulation and incorporation into inositol phospholipids and intracellular myo-inositol content are decreased in cells chronically exposed to media containing 30 mM glucose or 30 mM glucose/30 mM fructose compared to cells cultured in unsupplemented media or media containing 30 mM fructose. However, maximal depletion of myo-inositol accumulation and intracellular content occurs earlier in cells exposed to media containing 30 mM glucose/30 mM fructose than in cells exposed to media supplemented with 30 mM glucose. Sorbinil treatment of media containing 30 mM glucose/30 mM fructose maintains cellular myo-inositol accumulation and incorporation into phospholipids at near normal levels. myo-Inositol content in neuroblastoma cells chronically exposed to media containing 30 mM glucose or 30 mM glucose/30 mM fructose recovers within 72 h when the cells are transferred to unsupplemented media or media containing 30 mM fructose. In contrast, the sorbitol content of cells previously exposed to media containing 30 mM glucose or 30 mM glucose/30 mM fructose then transferred into media containing 30 mM fructose remains elevated compared to the sorbitol content of cells transferred into unsupplemented media. These data suggest that fructose may be activating or increasing sorbinil-resistant aldose reductase activity as well as partially blocking sorbitol dehydrogenase activity. The presence of increased concentrations of fructose in combination with increased glucose levels may enhance alterations in cell metabolism and properties due to increased sorbitol levels.     

Effect of Irradiance, Sugars and Nitrogen on Leaf Size of In Vitro Grown Ceratonia Siliqua L.

Carob (Ceratonia siliqua L.) has compound pinnate leaves consisting of 4 – 6 pairs of leaflets. However, in conditions of in vitro culture only one pair of leaflets develops. With increasing irradiance from 9.3 to 74.1 µmol m^–2 s^–1, leaf area increased 5-fold. Sucrose also significantly increased leaf area and the maxima were at concentration 147 mM at high irradiance and 233.6 mM at low irradiance. Sucrose was superior to fructose, glucose and combination of both in increasing leaf area. Decreasing concentration of KNO₃ and NH₄NO₃ caused a 3-fold decline of leaf area.     

The effect of increasing glucose in CPD on the quality of stored blood

Changes in quality of blood units containing one and a half or double amounts of glucose, stored at +4 degrees C for three weeks were analysed. An experimental preservative containing glucose and fructose (1 : 1) was also used. No other additives (purine or purine-nucleoside) were applied. A standard CPD preservative of the National Inst. of Haematology and Blood Transfusion was used as control. The pH, plasma free haemoglobin, K+ content, red blood cell (RBC) ATP and 2,3-DPG content, and RBC fragility index were determined in each sample. Increase of glucose concentration, the addition of fructose had a beneficial effect on blood pH, and on plasma free haemoglobin and K+ concentration. 150% glucose improved the 2,3-DPG maintenance in stored blood.     

Sugar utilization by yeast during fermentation

Summary When glucose and fructose are fermented separately, the uptake profiles indicate that both sugars are utilized at similar rates. However, when fermentations are conducted in media containing an equal concentration of glucose and fructose, glucose is utilized at approximately twice the rate of fructose. The preferential uptake of glucose also occurred when sucrose, which was first rapidly hydrolyzed into glucose and fructose by the action of the enzyme invertase, was employed as a substrate. Similar results were observed in the fermentation of brewer's wort and wort containing 30% sucrose and 30% glucose as adjuncts. In addition, the high levels of glucose in the wort exerted severe catabolite repression on maltose utilization in theSaccharmyces uvarum (carlsbergensis) brewing strain. Kinetic analysis of glucose and fructose uptake inSaccharomyces cerevisiae revealed aK _m of 1.6 mM for glucose and 20 mM for fructose. Thus, the yeast strain has a higher affinity for glucose than fructose. Growth on glucose or fructose had no repressible effect on the uptake of either sugar. In addition, glucose inhibited fructose uptake by 60% and likewise fructose inhibited, glucose uptake by 40%. These results indicate that glucose and fructose share the same membrane transport components.     

The effects of d- and l-glyceraldehyde on glucose oxidation, insulin secretion and insulin biosynthesis by pancreatic islets of the rat

d-glyceraldehyde stimulated insulin secretion from isolated rat pancreatic islets in static incubation and perifusion systems. At low concentrations (2–4 mM) d-glyceraldehyde was a more potent secretagogue than glucose. The insulinotropic action of 15 mM d-glyceraldehyde was not affected by d-mannoheptulose, was potentiated by cytochalasin B (5 μg/ml) and theophylline (4 mM), and was inhibited by both adrenalin (2 μM) and somatostatin (10 μg/ml). D-glyceraldehyde at a concentration of 1.5 mM produced a 10-fold increase of l-[4,5-³ H]leucine incorporation into proinsulin and insulin without a significant increase into other islet proteins. Glucose at 1.5 mM did not stimulate proinsulin biosynthesis. d-Glyceraldehyde at concentrations higher than 1.5 mM, in marked contrast to glucose, progressively inhibited incorporation of labelled leucine into proinsulin + insulin and other islet proteins. d-glyceraldehyde also inhibited the oxidation of glucose. l-Glyceraldehyde did not stimulate proinsulin biosynthesis and had less effect than the d-isomer on insulin release and glucose oxidation. The results strongly suggest that metabolites below d-glyceraldehyde-3-P are signals for insulin biosynthesisand release. Interaction of d-glyceraldehyde with a “membrane receptor” cannot, however, be excluded with certainty.     

Amperometric mediated carbon paste biosensor based on D-fructose dehydrogenase for the determination of fructose in food analysis

A new mediated amperometric biosensor for fructose is described. The sensor is based on a commercially available D-fructose dehydrogenase. The enzyme is incorporated in a carbon paste matrix containing Os(bpy)₂Cl₂ as redox mediator that achieves electron transfer at 0·1 V (versus Ag/AgCl) with maximum apparent current densities of 1·2 mA/cm². The dependence of the steady-state current on the loading of the mediator and the enzyme, other electrode construction parameters, the operating potential, the pH and the temperature was studied. In the steady-state mode the response current was directly proportional to D-fructose concentration from 0·2 to 20mM with a detection limit of 35 μM (signal-to-noise ratio, S/N, 3). In the flow injection analysis mode the response current was directly proportional to D-fructose concentration from 0·5 to 15 M with a detection limit of 115 μM (S/N 3). The sensor was used for the determination of fructose in food samples in a flow injection system and validated with a commercial enzyme kit.