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D-Fructose/D-Glucose Assay Kit

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D-Fructose D-Glucose Assay Kit K-FRUGL Scheme
Product code: K-FRUGL

110 assays (manual) / 1100 assays (microplate) / 1100 assays (auto-analyser)

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Content: 110 assays (manual) / 1100 assays (microplate) / 1100 assays (auto-analyser)
Shipping Temperature: Ambient
Storage Temperature: Short term stability: 2-8oC,
Long term stability: See individual component labels
Stability: > 2 years under recommended storage conditions
Analyte: D-Fructose, D-Glucose
Assay Format: Spectrophotometer, Microplate, Auto-analyser
Detection Method: Absorbance
Wavelength (nm): 340
Signal Response: Increase
Linear Range: 4 to 80 µg of D-glucose, D-fructose or sucrose per assay
Limit of Detection: 0.66 mg/L
Reaction Time (min): ~ 13 min
Application examples: Wine, beer, fruit juices, soft drinks, milk, jam, honey, dietetic foods, bread, bakery products, candies, desserts, confectionery, ice-cream, fruit and vegetables, condiments, tobacco, cosmetics, pharmaceuticals, paper and other materials (e.g. biological cultures, samples, etc.).
Method recognition: Methods based on this principle have been accepted by AOAC (Method 985.09), EN, NEN, NF, DIN, GOST, OIV, IFU, AIJN, MEBAK and IOCCC

D-Fructose/D-Glucose test kit, an enzymatic UV-method for the measurement and analysis of D-fructose and/or D-glucose in plant and food products.

Note for Content: The number of manual tests per kit can be doubled if all volumes are halved.  This can be readily accommodated using the MegaQuantTM  Wave Spectrophotometer (D-MQWAVE).

See more of our monosaccharide assay kits.

Scheme-K-FRUGL FRUGL Megazyme

  • Extended cofactors stability. Dissolved cofactors stable for > 1 year at 4oC.
  • PVP incorporated to prevent tannin inhibition 
  • Validated by the University of Wine, Suze la Rousse, France 
  • Very competitive price (cost per test) 
  • All reagents stable for > 2 years after preparation (manual analysis applications) 
  • Rapid reaction at either 25 or 37o
  • Mega-Calc™ software tool is available from our website for hassle-free raw data processing 
  • Standard included
  • Suitable for manual, microplate and auto-analyser formats
Validation of Methods
Megazyme publication

Megazyme “advanced” wine test kits general characteristics and validation.

Charnock, S. J., McCleary, B. V., Daverede, C. & Gallant, P. (2006). Reveue des Oenologues, 120, 1-5.

Many of the enzymatic test kits are official methods of prestigious organisations such as the Association of Official Analytical Chemicals (AOAC) and the American Association of Cereal Chemists (AACC) in response to the interest from oenologists. Megazyme decided to use its long history of enzymatic bio-analysis to make a significant contribution to the wine industry, by the development of a range of advanced enzymatic test kits. This task has now been successfully completed through the strategic and comprehensive process of identifying limitations of existing enzymatic bio-analysis test kits where they occurred, and then using advanced techniques, such as molecular biology (photo 1), to rapidly overcome them. Novel test kits have also been developed for analytes of emerging interest to the oenologist, such as yeast available nitrogen (YAN; see pages 2-3 of issue 117 article), or where previously enzymes were simply either not available, or were too expensive to employ, such as for D-mannitol analysis.

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Megazyme publication

Grape and wine analysis: Oenologists to exploit advanced test kits.

Charnock, S. C. & McCleary, B. V. (2005). Revue des Enology, 117, 1-5.

It is without doubt that testing plays a pivotal role throughout the whole of the vinification process. To produce the best possible quality wine and to minimise process problems such as “stuck” fermentation or troublesome infections, it is now recognised that if possible testing should begin prior to harvesting of the grapes and continue through to bottling. Traditional methods of wine analysis are often expensive, time consuming, require either elaborate equipment or specialist expertise and frequently lack accuracy. However, enzymatic bio-analysis enables the accurate measurement of the vast majority of analytes of interest to the wine maker, using just one piece of apparatus, the spectrophotometer (see previous issue No. 116 for a detailed technical review). Grape juice and wine are amenable to enzymatic testing as being liquids they are homogenous, easy to manipulate, and can generally be analysed without any sample preparation.

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An approach for estimating the maximum specific growth rate of Gluconobacter japonicus in strawberry purée without cell concentration data.

Cañete-Rodríguez, A. M., Santos-Dueñas, I. M., Jiménez-Hornero, J. E., Torija-Martínez, M. J., Mas, A. & García-García, I. (2016). Biochemical Engineering Journal, 105, 314-320.

The estimation of the maximum specific growth rate (µ max) for non-readily culturable bacteria, growing on complex media containing suspended solids, is a difficult task considering the important problems in obtaining reliable measures of cell concentration. An example of this situation can be a culture of Gluconobacter japonicus growing in strawberry purée for producing gluconic acid. Based on the dependency between energy requirements of the genus Gluconobacter and substrate uptake as well as its constant relationship between gluconic acid production and total substrate uptake, the total substrate concentration profile during the exponential growth phase could be used for estimating µ max without cell concentration measures. In this case, the high selectivity of the strain for glucose in comparison to fructose resulted in no fructose consumption during the batch; so, just using the glucose concentrations data during the exponential phase allow us to obtain an estimation of µ max Additionally, a rough estimation of the apparent and stoichiometric yields of cell on glucose is also possible.

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Oligosaccharide Synthesis in Fruit Juice Concentrates Using a Glucansucrase From Lactobacillus reuteri 180.

Johansson, S., Diehl, B., Christakopoulos, P., Austin, S. & Vafiadi, C. (2016). Food and Bioproducts Processing, 98, 201-209.

The application of the glucansucrase GTF180 from Lactobacillus reuteri 180 in fruit juice concentrates for the synthesis of glucooligosaccharides was investigated. Reaction parameters such as temperature, pH, substrate and enzyme loading, Ca+2 addition and incubation time were investigated in high concentration sucrose solutions. The optimum conditions (50°C, pH 4.5, enzyme loading 14.47 U/gsucrose with 1 mM CaCl2, undiluted fruit juice concentrates) were applied in apple and orange juice concentrates. More than 95% of the intrinsic sucrose was converted to oligosaccharide products after 90 min. The main products were leucrose, isomaltose and isomaltotriose. The enzyme was deactivated during standard fruit juice pasteurization conditions (95°C, 15 s). The oligosaccharides were stable during the pasteurization process, showing a good potential for industrial applications. DOSY NMR analysis of the enzymatically modified fruit juice concentrates showed that α1→6 glycosidic linkages are predominant in apple juice, while products in orange juice possess both α1→6 and α1→3 glycosidic linkages. The presence of α1→2 glycosidic linkages were also observed at a lower extent.

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Chemical and sensory profiles of rosé wines from Australia.

Wang, J., Capone, D. L., Wilkinson, K. L. & Jeffery, D. W. (2016). Food Chemistry, 196, 682-693.

The appeal of rosé wine is attributable to its sensory profiles and underlying chemical composition, which are determined by viticultural and oenological inputs. This study provided the first insight into the sensory attributes and volatile profiles of Australian rosé wines. An HS-SPME-GC-MS method and a recently developed HPLC-MS/MS method were used to quantify 51 volatile compounds, including 4 potent sulfur compounds, in 26 commercial rosé wines. Descriptive analysis on all wines was undertaken and the sensory results were correlated with quantitative chemical data to explore relationships between wine composition and sensory profiles. Based on odour activity values, esters were prominent aroma volatiles, and β-damascenone, 3-methylbutyl acetate, ethyl hexanoate and 3-MHA were deemed to be important, in accord with other studies. Wines were described with terms ranging from developed, spicy and savoury to fresh green, citrus, tropical fruit, floral and confectionery.

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Revalorization of strawberry surpluses by bio-transforming its glucose content into gluconic acid.

Cañete-Rodríguez, A. M., Santos-Dueñas, I. M., Jiménez-Hornero, J. E., Torija-Martínez, M. J., Mas, A. & García-García, I. (2016). Food and Bioproducts Processing, 99, 188-196.

Modern societies produce massive surpluses of food, by-products and wastes that increase the interest for their revalorization. This work examines the use of a culture of Gluconobacter japonicus CECT 8443, without pH control, to convert selectively the glucose content of industrially pasteurized strawberry purée into gluconic acid for the development of new beverages. However, depending on the initial concentration of glucose, the microorganism could transform the acid formed into other compounds; for this reason, in this work the effect of initial sugar concentration on the preservation of the acid was investigated. The results show that the gluconic acid formed in strawberry purée containing no added sugars started to disappear after glucose depletion, but the acid concentration remained constant if sugar-enriched purée was used. The use of this industrial substrate resulted in the presence of yeasts and hence in some fructose uptake; however, the fructose consumption was negligible until after 20-30 h. The use of food by-products is an excellent opportunity not only to recover valuable compounds but for the development of new chemical and biotechnological approaches for their revalorization. This strategy should improve regional economies and contribute to a sustainable management of these underexploited resources.

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Influence of ursolic acid on glucooligosaccharides synthesized by dextransucrase from Leuconostoc mesenteroides Lm 28.

Vasileva, T., Bivolarski, V., Bozov, P. & Iliev, I. (2015). Journal of BioScience & Biotechnology, 4(2).

A study of modulation of the reactions catalyzed by dextransucrase from Leuconostoc mesenteroides Lm 28 strain in the presence of triterpenoid ursolic acid was carried out. This compound showed concentration dependent inhibition of the studied dextransucrase and Ki = 1.9 mM, which is about 5 times higher than Ki value of the known glucansucrase inhibitor acarbose. Ursolic acid affected significantly the acceptor reactions catalyzed by Lm 28 dextransucrase in the presence of maltose and sucrose to maltose ratio 2. Increasing concentrations of ursolic acid shifted concentration and degree of polymerization (DP) distribution of the synthesized glucooligosaccharides (GOS) to acceptor products with DP ≤ 5. The oligosaccharide synthesis scheme applied in this study is a promising approach for production of GOS with controlled length of the chain.

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Characterization of composition traits related to organoleptic and functional quality for the differentiation, selection and enhancement of local varieties of tomato from different cultivar groups.

Figàs, M. R., Prohens, J., Raigón, M. D., Fita, A., García-Martínez, M. D., Casanova, C., Borràs, D., Plazas, M., Andúja, I. & Soler, S. (2015). Food Chemistry, 187, 517-524.

Tomato (Solanum lycopersicum) local varieties are having an increasing demand. We characterized 69 local tomato accessions from eight cultivar groups for proximate composition traits, major sugars, acids and antioxidants. A large diversity was found, with differences among accessions of almost tenfold for lycopene. Significant differences were found among cultivar group means for most traits. The Cherry and Penjar groups generally presented higher dry matter, soluble solids content, titratable acidity, taste index, β-carotene, ascorbic acid, total phenolics, and antioxidant activity that the other groups. Wide ranges of variation were found within each cultivar group. Positive correlations were found between proximate traits related to taste and antioxidants. The multivariate principal components analysis confirms the distinct profile of the Cherry and Penjar groups and the large variation within groups. The results will be useful for the differentiation, enhancement and selection of local tomato varieties with improved organoleptic properties and functional quality.

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Differential adsorption of Ochratoxin A and anthocyanins by inactivated yeasts and yeast cell walls during simulation of wine aging.

Petruzzi, L., Baiano, A., De Gianni, A., Sinigaglia, M., Corbo, M. R. & Bevilacqua, A. (2015). Toxins, 7(10), 4350-4365.

The adsorption of ochratoxin A (OTA) by yeasts is a promising approach for the decontamination of musts and wines, but some potential competitive or interactive phenomena between mycotoxin, yeast cells, and anthocyanins might modify the intensity of the phenomenon. The aim of this study was to examine OTA adsorption by two strains of Saccharomyces cerevisiae (the wild strain W13, and the commercial isolate BM45), previously inactivated by heat, and a yeast cell wall preparation. Experiments were conducted using Nero di Troia red wine contaminated with 2 μg/L OTA and supplemented with yeast biomass (20 g/L). The samples were analyzed periodically to assess mycotoxin concentration, chromatic characteristics, and total anthocyanins over 84 days of aging. Yeast cell walls revealed the highest OTA-adsorption in comparison to thermally-inactivated cells (50% vs. 43% toxin reduction), whilst no significant differences were found for the amount of adsorbed anthocyanins in OTA-contaminated and control wines. OTA and anthocyanins adsorption were not competitive phenomena. Unfortunately, the addition of yeast cells to wine could cause color loss; therefore, yeast selection should also focus on this trait to select the best strain.

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Aroma compounds in Ontario Vidal and Riesling icewines. I. Effects of harvest date.

Bowen, A. J. & Reynolds, A. G. (2015). Food Research International, 76, 540-549.

Icewine is a sweet dessert wine made from pressing grapes naturally frozen on the vines. It is likely that freeze/thaw cycles endured by icewine grapes change their chemical and sensory profiles due to climatic events. Our objective was to determine the influence of harvest date on icewine must and wine basic chemical variables and aroma compounds. Riesling and Vidal icewines were made from grapes picked between December 2004 and February 2005; Harvest 1 (H1): 19 December; Harvest 2: 29 December; Harvest 3 (H3): 18 January; and Harvest 4 (H4): 11 February (Vidal only). Icewine musts differed in titratable acidity and pH (Vidal only). All basic wine chemical analytes differed across harvest dates. All aroma compounds differed in Vidal and Riesling wines. Highest concentrations for most aroma compounds were in the last harvest date; 16 of 24 for Vidal and 17 of 23 for Riesling. The latest harvest date had highest ethyl isobutyrate, ethyl 3-methylbutyrate, 1-hexanol, 1-octen-3-ol, 1-octanol, cis-rose oxide, nerol oxide, ethyl benzoate, ethyl phenylacetate, γ-nonalactone and β-damascenone. H1 had highest ethyl butyrate, ethyl hexanoate, linalool, 4-vinylguaiacol and ethyl octanoate. Based on odor activity values, the most odor-potent compounds were β-damascenone, cis-rose oxide, 1-octen-3-ol, ethyl octanoate, ethyl hexanoate, and 4-vinylguaiacol across harvest dates. PCA found most aroma compounds associated with the last harvest date, 4-vinylguaicol excepted, which was associated with H1. Harvest date was considered a discriminating dimension using canonical variant analysis for volatile compounds.

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Enhancement of acetyl xylan esterase activity on cellulose acetate through fusion to a family 3 cellulose binding module.

Mai-Gisondi, G., Turunen, O., Pastinen, O., Pahimanolis, N. & Master, E. R. (2015). Enzyme and Microbial Technology, 79, 27-33.

The current study investigates the potential to increase the activity of a family 1 carbohydrate esterase on cellulose acetate through fusion to a family 3 carbohydrate binding module (CBM). Specifically, CtCBM3 from Clostridium thermocellum was fused to the carboxyl terminus of the acetyl xylan esterase (AnAXE) from Aspergillus nidulans, and active forms of both AnAXE and AnAXE-CtCBM3 were produced in Pichia pastorisCtCBM3 fusion had negligible impact on the thermostability or regioselectivity of AnAXE; activities towards acetylated corncob xylan, 4-methylumbelliferyl acetate, p-nitrophenyl acetate, and cellobiose octaacetate were also unchanged. By contrast, the activity of AnAXE-CtCBM3 on cellulose acetate increased by two to four times over 24 h, with greater differences observed at earlier time points. Binding studies using microcrystalline cellulose (Avicel) and a commercial source of cellulose acetate confirmed functional production of the CtCBM3 domain; affinity gel electrophoresis using acetylated xylan also verified the selectivity of CtCBM3 binding to cellulose. Notably, gains in enzyme activity on cellulose acetate appeared to exceed gains in substrate binding, suggesting that fusion to CtCBM3 increases functional associations between the enzyme and insoluble, high molecular weight cellulosic substrates.

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Lactose fermentation by Kombucha - a process to obtain new milk–based beverages.

Iličić, M., Kanurić, K., Milanović, S., Lončar, E., Djurić, M. & Malbaša, R. (2012). Romanian Biotechnological Letters, 17(1), 7013-7021.

This paper focuses on fermentation of lactose from a model system (black tea) and from two types of milk (0.9% w/w and 2.2% w/w of fat) by application of Kombucha. Quantities of the applied Kombucha starter were 10% v/v and 15% v/v. All fermentations were performed at 42°C. The process to achieve a desirable pH=4.5 was slower in the model system (16 h) than in milks (9 - 10 h). Regarding starter quantity, 10% v/v proved the optimal. Regarding types of milk, higher fat content guarantees shorter fermentation and higher yield of metabolites. Utilization of lactose was found at a level of ≈20% and ≈30% in milks with 0.9% w/w and 2.2% w/w of fat, respectively. This was correlated with an appearance of intermediates and/or products. Glucose underwent further transformations almost entirely, while galactose showed much lower reactivity. Seven to twelve times higher contents of lactic acid were found compared to acetic acid. Milk-based beverage from the reduced fat sample, inoculated with 10% v/v of Kombucha starter, has the best physical characteristics (syneresis and water holding capacity). It also developed a good texture (especially cohesiveness and index of viscosity). Milk lactose fermentation was a process that could have been used for obtaining new milk-based products.

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Physiological studies of Leuconostoc mesenteroides strain NRRL B-1149 during cultivation on glucose and fructose media.

Bivolarski, V., Vasileva, T., Shukla, R., Goyal, A. & Iliev, I. (2012). Journal of BioScience & Biotechnology, 1(3), 235-240.

Glycosyltransferases are extracellular and cell-associated sucrase enzymes produced mainly by lactic acid bacteria Leuconostoc mesenteroides, oral Streptococcus species and also Lactobacillus species. According to the synthesized polymer (glucan or fructan) in the presence of sucrose, these enzymes are divided into two groups: glucosyltransferases (GTFs) and fructosyltransferases (FTFs). Only Streptococcus, Lactobacillus and Leuconostoc strains are known as producers of both GTFs and FTFs. The enzymes from Lactobacillus and Leuconostoc spp. are implicated in the synthesis of polymers and oligosaccharides (OS) important for human health because of their prebiotic properties and immunomodulating activity. In the present work, we studied the production of extracellular and cell-associated glycosyltransferases by Leuconostoc mesenteroides strain NRRL B-1149 during its growth on media containing glucose or fructose as a main carbon source. The enzyme activities, pH and biomass formation were measured and compared during the cultivation. We have shown that glucose and fructose have not an equal role for enzyme production. The highest extracellular activity was detected at the 4th hour during the cultivation of the strain in medium with fructose - 5.45 U/mg. When the strain was cultivated in medium with glucose, the maximum of extracellular enzyme activity was detected at the 5th hour of the cultivation but the measured activity was about 9 times lower compared to these, obtained after cultivation in fructose medium. The studied strain produced mainly extracellular glycosyltransferases in glucose or fructose medium, which were 92.4% and 97.1% of the total enzyme activity, respectively. In order to characterize the produced enzymes, cell-associated and extracellular enzymes were determined using SDS-PAGE and in situ Periodic Acid Schiff's staining after incubation with 10% sucrose. When the investigated strain was grown in media with sucrose, glucose or fructose, several types of glycosyltransferases were detected - dextransucrase with molecular weight 180 kDa and two fructosyltransferases, corresponding to 120 kDa and 86 kDa molecular weights.

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β-Fructofuranosidase and sucrose phosphorylase of rumen bacterium Pseudobutyrivibrio ruminis strain 3.

Kasperowicz, A., Stan-Glasek, K., Guczynska, W., Pristas, P., Javorsky, P., Vandzurova, A. & Michalowski, T. (2012). World Journal of Microbiology and Biotechnology, 28(3), 1271-1279.

The subject of this study was the fructan and sucrose degrading enzymes of bacterium Pseudobutyrivibrio ruminis strain 3. It was stated that cell extract from bacteria growing on inulin contained β-fructofuranosidase (EC and/or EC and sucrose phosphorylase (EC, while the bacteria maintained on sucrose showed only phosphorylase. Partially purified β-fructofuranosidase digested inulooligosaccharides and sucrose to fructose or fructose and glucose, respectively, but was unable to degrade the long chain polymers of commercial inulin and Timothy grass fructan. Digestion rate of inulooligosaccharides fit Michaelis–Menten kinetics with Vmax 5.64 µM/mg/min and Km 1.274%, respectively, while that of sucrose was linear. Partially purified sucrose phosphorylase digested only sucrose. The digestion products were fructose, glucose-1P and free glucose. The reaction was in agreement with Michaelis–Menten kinetics. The Vmax were 0.599 and 0.584 µM/mg/min, while Km were 0.190 and 0.202% for fructose release and glucose-1P formation, respectively, when bacteria grew on inulin. The Vmax were, however, 1.37 and 1.023 µM/mg/min, while Km were 0.264 and 0.156%, if bacteria were grown on sucrose. The free glucose was hardly detectable for the enzyme originated from inulin grown bacteria, but glucose levels ranged from 0.05 to 0.25 µM/mg/min, when cell extract from bacteria grown on sucrose was used. Release of free glucose was observed when no inorganic phosphate was present in reaction mixture.

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Immobilised yeast grape must deacidification in a recycle fixed bed reactor.

Portugal, I., Ribeiro, S. C., Xavier, A. M. R. B., Centeno, F. & Strehaiano, P. (2011). International Journal of Food Science & Technology, 46(2), 284-289.

Maloalcoholic fermentation (MAF) of grape must by Schizosaccharomyces pombe immobilised in calcium-alginate double-layer beads (ProMalic®) was studied in Erlenmeyer flasks and in a total recycle fixed-bed reactor operating in batch mode. The reaction is pseudo-first order with respect to L-malic acid and under similar conditions deacidification is faster in the recycle reactor. This was attributed to mass transfer limitations which were confirmed in the recycle reactor by studying the influence of yeast load on the rate of MAF. Mass transfer limitations are also responsible for the lower activation energy of fermentation with the immobilised yeast (67 ± 9 kJ mol-1) in comparison with the free cells (126 ± 19 kJ mol-1). Alcoholic fermentation and MAF were performed simultaneously, both in the recycle reactor and in the industrial trials, confirming the efficacy of immobilised S. pombe to reduce grape must acidity without interfering with the main fermentation. Altogether, the present results are useful for the scale-up of a recycle reactor to process large volumes of grape must.

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Comparative effects of fructose and glucose on lipogenic gene expression and intermediary metabolism in HepG2 liver cells.

Hirahatake, K. M., Meissen, J. K., Fiehn, O. & Adams, S. H. (2011). PloS one, 6(11), e26583.

Consumption of large amounts of fructose or sucrose increases lipogenesis and circulating triglycerides in humans. Although the underlying molecular mechanisms responsible for this effect are not completely understood, it is possible that as reported for rodents, high fructose exposure increases expression of the lipogenic enzymes fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC-1) in human liver. Since activation of the hexosamine biosynthesis pathway (HBP) is associated with increases in the expression of FAS and ACC-1, it raises the possibility that HBP-related metabolites would contribute to any increase in hepatic expression of these enzymes following fructose exposure. Thus, we compared lipogenic gene expression in human-derived HepG2 cells after incubation in culture medium containing glucose alone or glucose plus 5 mM fructose, using the HBP precursor 10 mM glucosamine (GlcN) as a positive control. Cellular metabolite profiling was conducted to analyze differences between glucose and fructose metabolism. Despite evidence for the active uptake and metabolism of fructose by HepG2 cells, expression of FAS or ACC-1 did not increase in these cells compared with those incubated with glucose alone. Levels of UDP-N-acetylglucosamine (UDP-GlcNAc), the end-product of the HBP, did not differ significantly between the glucose and fructose conditions. Exposure to 10 mM GlcN for 10 minutes to 24 hours resulted in 8-fold elevated levels of intracellular UDP-GlcNAc (P<0.001), as well as a 74–126% increase in FAS (P<0.05) and 49–95% increase in ACC-1 (P<0.01) expression above controls. It is concluded that in HepG2 liver cells cultured under standard conditions, sustained exposure to fructose does not result in an activation of the HBP or increased lipogenic gene expression. Should this scenario manifest in human liver in vivo, it would suggest that high fructose consumption promotes triglyceride synthesis primarily through its action to provide lipid precursor carbon and not by activating lipogenic gene expression.

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Measurement of glucose and fructose in clinical samples using gas chromatography/mass spectrometry.

Wahjudi, P. N., Patterson, M. E., Lim, S., Yee, J. K., Mao, C. S. & Lee, W. N. P. (2010). Clinical Biochemistry, 43(1-2), 198-207.

Objective: The impact of increased fructose consumption on carbohydrate metabolism is a topic of current interest, but determination of serum level has been hindered due to low concentration and interference from serum glucose. We are reporting a method for the quantification of glucose and fructose in clinical samples using gas chromatography/mass spectrometry (GC/MS). The accuracy and precision of GC/MS and an enzymatic assay were compared. Design and methods: Mass spectrometry fragmentation patterns of methyloxime peracetate derivatized aldose and ketose were determined. Unique fragments for glucose and fructose were used for quantitative analysis using isotope labeled recovery standards. Results: Methyloxime peracetate derivatives of glucose and fructose showed characteristic loss of acetate (M-60) or ketene (M-42) under chemical ionization (CI). Under electron impact (EI) ionization, a unique C1–C2 fragment of glucose was formed, while a C1–C3 fragment was formed from keto-hexoses. These unique fragments were used in the quantitative assay of glucose and fructose in clinical samples. In clinical samples, the GC/MS assay has a lower limit of detection than that of the enzymatic assay. In plasma samples from patients evaluated for diabetes the average serum glucose and fructose were 6.19 ± 2.72 mM and 46 ± 25.22 μM. Fructose concentrations in many of these samples were below the limit of detection of the enzymatic method. Conclusion: Derivatization of aldose and ketose monosaccharides to their respective O-methyloxime acetates for GC/MS analysis is a facile method for determination of serum/plasma glucose and fructose samples.

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Endocrine and metabolic effects of consuming fructose- and glucose-sweetened beverages with meals in obese men and women: Influence of insulin resistance on plasma triglyceride responses.

Teff, K. L., Grudziak, J., Townsend, R. R., Dunn, T. N., Grant, R. W., Adams, S. H., Keim, N. L., Cummings, B. P., Stanhope, K. L. & Havel, P. J. (2009). Journal of Clinical Endocrinology & Metabolism, 94(5), 1562-1569.

Context: Compared with glucose-sweetened beverages, consumption of fructose-sweetened beverages with meals elevates postprandial plasma triglycerides and lowers 24-h insulin and leptin profiles in normal-weight women. The effects of fructose, compared with glucose, ingestion on metabolic profiles in obese subjects has not been studied. Objective: The objective of the study was to compare the effects of fructose- and glucose-sweetened beverages consumed with meals on hormones and metabolic substrates in obese subjects. Design and Setting: The study had a within-subject design conducted in the clinical and translational research center. Participants: Participants included 17 obese men (n = 9) and women (n = 8), with a body mass index greater than 30 kg/m2. Interventions: Subjects were studied under two conditions involving ingestion of mixed nutrient meals with either glucose-sweetened beverages or fructose-sweetened beverages. The beverages provided 30% of total kilocalories. Blood samples were collected over 24 h. Main Outcome Measures: Area under the curve (24 h AUC) for glucose, lactate, insulin, leptin, ghrelin, uric acid, triglycerides (TGs), and free fatty acids was measured. Results: Compared with glucose-sweetened beverages, fructose consumption was associated with lower AUCs for insulin (1052.6 ± 135.1 vs. 549.2 ± 79.7 µU/ml per 23 h, P < 0.001) and leptin (151.9 ± 22.7 vs. 107.0 ± 15.0 ng/ml per 24 h, P < 0.03) and increased AUC for TG (242.3 ± 96.8 vs. 704.3 ± 124.4 mg/dl per 24 h, P < 0.0001). Insulin-resistant subjects exhibited larger 24-h TG profiles (P < 0.03). Conclusions: In obese subjects, consumption of fructose-sweetened beverages with meals was associated with less insulin secretion, blunted diurnal leptin profiles, and increased postprandial TG concentrations compared with glucose consumption. Increases of TGs were augmented in obese subjects with insulin resistance, suggesting that fructose consumption may exacerbate an already adverse metabolic profile present in many obese subjects. Compared to glucose-sweetened beverages, consumption of fructose-sweetened beverages increases postprandial triglycerides in obese subjects, potentially exacerbating the known adverse metabolic profile associated with obesity.

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Metabolic and endocrine profiles in response to systemic infusion of fructose and glucose in rhesus macaques.

Adams, S. H., Stanhope, K. L., Grant, R. W., Cummings, B. P. & Havel, P. J. (2008). Endocrinology, 149(6), 3002-3008.

Diurnal patterns of circulating leptin concentrations are attenuated after consumption of fructose-sweetened beverages compared with glucose-sweetened beverages, likely a result of limited postprandial glucose and insulin excursions after fructose. Differences in postprandial exposure of adipose tissue to peripheral circulating fructose and glucose or in adipocyte metabolism of the two sugars may also be involved. Thus, we compared plasma leptin concentrations after 6-h iv infusions of saline, glucose, or fructose (15 mg/kg•min) in overnight-fasted adult rhesus monkeys (n = 9). Despite increases of plasma fructose from undetectable levels to about 2 mm during fructose infusion, plasma leptin concentrations did not increase, and the change of insulin was only about 10% of that seen during glucose infusion. During glucose infusion, plasma leptin was significantly increased above baseline concentrations by 240 min and increased steadily until the final 480-min time point (change in leptin = +2.5 ± 0.9 ng/ml, P < 0.001 vs. saline; percent change in leptin = +55 ± 16%; P < 0.005 vs. saline). Substantial anaerobic metabolism of fructose was suggested by a large increase of steady-state plasma lactate (change in lactate = 1.64 ± 0.15 mm from baseline), which was significantly greater than that during glucose (+0.53 ± 0.14 mm) or saline (−0.51 ± 0.14 mm) infusions (P < 0.001). Therefore, increased adipose exposure to fructose and an active whole-body anaerobic fructose metabolism are not sufficient to increase circulating leptin levels in rhesus monkeys. Thus, additional factors (i.e. limited post-fructose insulin excursions and/or hexose-specific differences in adipocyte metabolism) are likely to underlie disparate effects of fructose and glucose to increase circulating leptin concentrations.

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Sourdough-leavened bread improves postprandial glucose and insulin plasma levels in subjects with impaired glucose tolerance.

Maioli, M., Pes, G. M., Sanna, M., Cherchi, S., Dettori, M., Manca, E. & Farris, G. A. (2008). Acta Diabetologica, 45(2), 91-96.

Sourdough bread has been reported to improve glucose metabolism in healthy subjects. In this study postprandial glycaemic and insulinaemic responses were evaluated in subjects with impaired glucose tolerance (IGT) who had a meal containing sourdough bread leavened with lactobacilli, in comparison to a reference meal containing bread leavened with baker yeast. Sixteen IGT subjects (age range 52–75, average BMI 29.9 ± 4.2 kg/m²) were randomly given a meal containing sourdough bread (A) and a meal containing the reference bread (B) in two separate occasions at the beginning of the study and after 7 days. Sourdough bread was leavened for 8 h using a starter containing autochthonous Saccharomyces cerevisiae and several bacilli able to produce a significant amount of D-and L-lactic acid, whereas the reference bread was leavened for 2 h with commercial baker yeast containing Saccharomyces cerevisiae. Plasma glucose and insulin levels were measured at time 0, 30, 60, 120, and 180 min. In IGT subjects sourdough bread induced a significantly lower plasma glucose response at 30 minutes (p = 0.048) and a smaller incremental area under curve (AUC) Δ 0–30 and Δ 0–60 min (p = 0.020 and 0.018 respectively) in comparison to the bread leavened with baker yeast. Plasma insulin response to this type of bread showed lower values at 30 min (p = 0.045) and a smaller AUC Δ 0–30 min (p = 0.018). This study shows that in subjects with IGT glycaemic and insulinaemic responses after the consumption of sourdough bread are lower than after the bread leavened with baker yeast. This effect is likely due to the lactic acid produced during dough leavening as well as the reduced availability of simple carbohydrates. Thus, sourdough bread may potentially be of benefit in subjects with impaired glucose metabolism.

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