Food Anal. Methods DOI 10.

1007/s12161-011-9335-9

Glucose Measurement in the Presence of Tea Polyphenols

Hui Xu & Xue Leng & Mingzhu Wang & Genyi Zhang

Received: 1 September 2011 / Accepted: 17 November 2011 # Springer Science+Business Media, LLC 2011

Abstract The accuracy of several commonly used methods for glucose measurement in the presence of tea polyphenols (TPLs) was investigated since TPLs (as a representative of bioactive polyphenols) and glucose often co-exist in experiments exploring TPLs effect on carbohydrate metabolism. The results from a model system containing only glucose and TPLs showed a TPLs amount-dependent variation of measured glucose concentration with a relative error (RE) of 5.0% to 35.5% when a dinitrosalicylic acid (DNS) method was used, and for glucose oxidase/peroxidase assay, the results showed a decreased content of glucose with a RE from 56.7% to 102.7%. When a hexose/kinase (HK) method was employed to quantify the glucose content, the accuracy (RE from 0.57% to 4.7%) was comparable to the result obtained by a high performance liquid chromatography (HPLC) method (RE00.73.0%) that was used as the standard control. Starch digestion experiment further demonstrated the invalidity of DNS method and the accuracy of HK method. Thus, the HK method with its accuracy and convenience is the preferred method for glucose measurement in the presence of TPLs or other bioactive polyphenols. Keywords Glucose . Tea polyphenols . DNS method . Glucose oxidase method . Hexokinase method . HPLC method

Introduction Tea polyphenols (TPLs) are well-known bioactive antioxidants, and they have been shown to have a variety of biological functions in which their antihyperglycemia and antioxidation effects make them one important ingredient in diet-based strategies to prevent or delay the incidence of diabetes that are continuously increasing worldwide. TPLs has been shown to attenuate the postprandial glycemia through inhibiting the activity of carbohydrate digestion enzymes (Matsui et al. 2007) and sodium-dependent glucose transporter (Kobayashi et al. 2000) in which the glucose is the central component in these studies. Thus, the determination of glucose content in the presence of TPLs has become a key step to explore TPLs' biological function in blood glucose control. Since glucose is a polyhydroxy aldehyde and a reducing sugar molecule, its reducing property is often used as the basis for its measurement in commonly used dinitrosalicylic acid (DNS) method and glucose oxidase/peroxidase (GOPOD) assay. TPLs are antioxidants and also chemically reducing compounds, and their presence in the system naturally becomes a confounding factor for an accurate measurement of glucose content using these redox reaction-based methods. Although the background signal caused by TPLs was simply deducted to calculate the content of glucose according to many literature reports (Hara and Honda 1990; Koh et al. 2010; Kwon et al. 2008), we found that there exist substantial variations even we carefully take all the measurement details into consideration, and replication of some literature experiments was rarely successful. Apparently, the accuracy of glucose measurement using these redox methods in a system containing

H. Xu : X. Leng : M. Wang : G. Zhang (*) State Key Lab of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, Peoples Republic of China e-mail: genyiz@163.com

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TPLs or reducing compounds need to be examined to ensure the validity of the results. In the current investigation, the accuracy of several commonly used methods including DNS, GOPOD assay, hexosekinase (HK) assay was compared for glucose measurement when a HPLC method was used as the standard control.

under of curve of the glucose peak, and then sample solution (20 l) containing TPLS was measured accordingly. TPLs Content and Glucose Measurement During Starch Hydrolysis The in vitro digestion of starch samples was carried out according to the modified Englyst method (Englyst et al. 1992). Starch samples (100 mg) and/or TPLs (10% w/w, based on starch) in 8 mL sodium acetate buffer (100 mM, CaCl2 1.0 mM, pH 5.2) were first cooked at 95 C for 20 min and placed in a 37C water bath. The enzyme solution (-amylase and amyloglucosidase mixed in a proportion of 300 U:200 U/mL) were pre-incubated at 37C for 5 min. The reaction was started by adding the enzyme solution (2 mL) into the starch samples, and aliquot of 100 L reaction mixtures were put into a plastic tube containing 0.9 mL ethanol at 120 min. The samples were vortexed and then centrifuged at 6,000 rpm for 10 min at room temperature. The concentration of glucose in the system was measured separately by DNS, GOPOD, HK, and HPLC methods using the same sample. Statistical Analysis All the experiments was replicated for three times, and the experimental data of in vitro starch hydrolysis were expressed as the mean SD (standard deviation), and the difference was analyzed using the paired t test in SPSS 11.5 for Windows software (SPSS) to examine the effect of TPLs on the starch hydrolysis measured with different methods. The statistical significance was accepted at p<0.05.

Materials and Methods Normal corn starch was from National Starch and Chemical Company (Shanghai, China). Tea polyphenols (TPLs) were obtained from Lideshi Chemical Industry Co. Ltd. (Rizhao, China) with a total tea catechins of ~99%, and its composition was analyzed using a common HPLC method (Wang et al. 2003). -Amylase (EC 3.2.1.1, type VI-B from porcine pancreas, 19.6 U/mg), amyloglucosidase (EC 3.2.1.3, from Rhizopus mold, 21,100 U/g) were purchased from Sigma Chemical Co. (St. Louis, MO). Glucose oxidase/peroxidase assay (GOPOD) kit and Hexokinase (HK) kit for D-glucose assay were from Megazyme International Ireland Ltd. (Wicklow, Ireland). Glucose Measurement A model system containing pure glucose solution was used to compare the accuracy of glucose measurement by DNS, GOPOD, HK, and HPLC methods. The effect of TPLs on glucose measurement was studied by changing either the glucose or the TPLs content while keeping the other at a constant concentration. Specifically, pure glucose solution with a concentration of 0.0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL with TPLs (0.4 mg/mL) was first prepared, and 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL of tea polyphenol solution with glucose (0.4 mg/mL) were also prepared to examine the effect of TPLs on the glucose measurement by GOPOD and HK assay according to their manuals as well as the DNS method (Miller 1959). HPLC method as the control was also carried out based on the following procedure. Glucose Measurement by HPLC Method An HPLC system comprised of a Waters 600 pump, a Waters 600 controller, and a Waters 2414 refractive index detector operated at 85 C with a mobile phase of distilled water at a flow rate of 0.4 mL/min was used to measure the glucose concentration. The column used was Sugar-Pak-1 (6.5300 mm, Waters, USA) with a guard column of Bond Elute C18 (3 mL, Varian Inc. USA) to absorb TPLs. Pure glucose solution (20 l) with different concentrations (0.0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL) was injected into the column using a Micro injector, and a standard curve was first obtained between the glucose concentration and the area

Results and Discussion Glucose Measurement in the Model System TPLs' antioxidation effect is due to its reducing hydroxyl group in the phenol ring that can delocalize and stabilize the free electrons when the hydrogen atom is transferred to an oxidant. Thus, the reducing property of TPLs determines that they can react with DNS reagent to produce a linear doseresponse relationship (A540 00.109TPLs' concentration (milligrams per milliliter), R2 00.99) between TPLs content and the absorbance at 540 nm, which is the peak wavelength of 3-amino-5-nitrosalicylic acid resulted from a reduction of 3,5-dinitrosalicylic acid. Glucose, as a reducing sugar, also reacts with DNS reagent in a linear manner. Clearly, the fact that both TPLs and glucose react with the DNS reagent to produce the blue colorant of 3-amino-5nitrosalicylic acid demonstrates the confounding effect of

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TPLs on the glucose measurement. After the background signal from TPLs was subtracted, which is the common way used in literature report to calculate reducing sugar content in the presence of TPLs (Hara and Honda 1990), the experimental results (Table 1) from the DNS assay still showed that the presence of TPLs (0.4 mg/mL) decreased the accuracy of glucose measurement with a high value of relative error. When different amount of TPLs was used in the system with a certain concentration of glucose, a TPLs' dose-independent increase of glucose content was observed (Fig. 1a) suggesting the complexity of DNS assay in the presence of TPLs. Thus, the commonly used DNS method is not suitable for an accurate measurement of glucose in the presence of TPLs. GOPOD assay (Trinder 1969) is another method designed specifically for glucose measurement without disturbance from other reducing sugars with the glucose specificity of glucose oxidase. The mechanism of this assay is based on the amount of hydrogen peroxide (H2O2) produced through the action of glucose oxidation by glucose oxidase. The produced H2O2 was then quantified by reacting with phydroxybenzoic acid and 4-aminoantipyrine in the presence of peroxidase to produce a quinoneimine dye with a peak absorbance at 510 nm. Although the enzyme used in the assay is only specific to glucose, the H2O2 produced is a strong oxidant that can react with the reducing agent of TPLs leading to a decreased value of glucose content. Another possibility may be that TPLs act as an alternate substrate (instead of p-hydroxybenzoic acid) for the hydrogen peroxide that is catalyzed by peroxidase. Furthermore, the amount of H2O2 produced is not likely in proportion to the glucose content due to the inhibitory effect of TPLs on the glucose oxidase used in the assay (Ruch et al. 1989). In the presence of TPLs (0.4 mg/mL), the measured value of glucose showed a huge variation (Table 1), and when the glucose content was constant with different content of TPLs, the glucose content also showed a huge variation depending on the TPLs' concentration (Fig. 1b). Since there is no way to eliminate the background signal from TPLs, this method

is even worse that the DNS assay for glucose measurement in the presence of TPLs. HK assay is another enzymatic method used to measure glucose concentration based on the absorbance of reduced nicotinamide adenine dinucleotide phosphate (NADPH) at 340 nm when nicotinamide adenine dinucleotide phosphate (NADP), which is reacted with glucose-6-phosphate (G-6-P) produced by glucose phosphorylation catalyzed by hexokinase (HK), is reduced by G-6-P dehydrogenase (Finch et al. 1969). Theoretically, since the generation of both G-6-P and NADPH are catalyzed by substrate-specific enzymes, which eliminate the interference from other chemicals, this method should be suitable for the measurement of glucose in the presence of TPLs. The experimental results proved that this method gives a result (Table 1 and Fig. 1c) that is comparable to the result obtained by HPLC, which is the golden standard method used in this investigation (Table 1 and Fig. 1d). Apparently, TPLs, which are noted for its inhibitory effect on many enzymes (Babu and Liu 2008; Hanhineva et al. 2010), do not likely affect the enzymes used in the HK assay. With the consideration of convenience and easy operation without employing expensive equipment such as HPLC system, HK assay was chosen as the method for accurate measurement of glucose in the presence of TPLs or other reducing compounds that are commonly used in food products for various purposes. Glucose Measurement in the Englyst Test In order to verify if the method could be used under a real reaction condition, enzymatic hydrolysis of gelatinized normal corn starch affected by the addition of TPLs was studied. The Englyst method (Englyst et al. 1992) is often used for starch fractionation employing two different enzymes (-amylase and amyloglucosidase) for a complete conversion of starch to glucose that is measured by GOPOD method. However, as what has been found above, the method of GOPOD is not only substantially affected by TPLs, there is also no way to eliminate the background signal

Table 1 The relative errors of glucose measurement methods in the presence of TPLs (0.4 mg/mL) Method DNS GOPOD HK HPLC Glucose concentration (mg/mL) DV RE DV RE DV RE DV RE 0 0.141 0.000 0.027 0.000 0.2 0.271 35.50% 0.000 102.73% 0.190 4.79% 0.206 3.00% 0.4 0.441 10.25% 0.016 96.08% 0.405 1.20% 0.409 2.18% 0.6 0.562 6.33% 0.102 83.01% 0.611 1.84% 0.604 0.72% 0.8 0.760 5.00% 0.285 64.38% 0.797 0.31% 0.817 2.16% 1.0 0.936 6.40% 0.433 56.72% 0.994 0.57% 0.990 1.04%

DV detected value, RE relative error

Food Anal. Methods Fig. 1 The effect of TPLs on the glucose content measured by method of DNS (a), GOPOD (b), HK (c), and HPLC (d). The Original represents the added glucose content in the system, and the Measured represents the glucose content calculated based on the measured results

caused by TPLs. Thus, methods of DNS, HK, and HPLC for glucose measurement were used to study the enzyme hydrolysis of starch in the presence of TPLs. Although it has been shown above that DNS is not suitable for glucose measurement in a simple model system, it is worth reevaluating it due to its simplicity, low-cost, and its ability to measure any reducing sugar molecules, which sometimes is also important for the research. The experiment of starch hydrolysis (Fig. 2) showed a measurement method-dependent manner regarding the

TPLs' effect on starch hydrolysis. In the absence of TPLs, DNS method showed the highest degree of starch hydrolysis compared with the results measured by HK and HPLC method indicating there are other non-glucose reducing sugars (~9.6% of glucose) in the system, and these reducing sugars are likely oligosaccharides resulted from incomplete enzyme digestion. In the presence of TPLs, the lowest degree of starch hydrolysis was shown for DNS method compared with the data obtained by HK and HPLC measurement. In another word, TPLs significantly decreased the

Fig. 2 The effect of TPLs on starch digestion measured by different methods. Different labels represent significant difference at p<0.05

Fig. 3 The HPLC chromatogram of TPLs

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starch hydrolysis by ~15.5% whereas no inhibitory effect on starch hydrolysis was shown when the hydrolysis was measured by either HK or HPLC method. Although TPLs' inhibitory effect on -amylase leading to a decreased starch hydrolysis has been documented in many literature reports (Hara and Honda 1990; Kwon et al. 2008) using the DNS method, which is consistent with our current data using the same method, the result obtained by HK or HPLC method is obviously contradictory to those literature studies. This measurement method-dependent inconsistency concerning the function of TPLs on starch digestion indicates the validity of each measurement method needs to be reconsidered. Theoretically, DNS method could measure any reducing compounds based on its reaction mechanism, and both the reducing sugars and TPLs in the system could react with DNS reagents. After the background signal of TPLs is deduced, there are glucose and other non-glucose reducing oligosaccharides in the reaction system, and so the measured reducing sugar content should be higher than the concentration of glucose alone that is obtained by HK and the HPLC measurement. However, this reasoning is not consistent with the experimental results, and there are only two possibilities to explain this result: either we accept that TPLs affect enzyme activity or DNS method itself is incompatible with TPLs. However, considering the high accuracy of glucose measurement using both HK and HPLC method in the model system, a lowest value of starch hydrolysis (lowest reducing sugar) measured by DNS method is not likely due to the TPLs' inhibitory effect on starch hydrolysis under the current dosage of TPLs, but the DNS method itself is incompatible with TPLs. The presence of TPLs in the DNS procedure might directly or indirectly reduce its sensitivity to reducing sugars leading to a lower value. The incompatibility between TPLs and DNS assay might be due to the special chemical properties of TPLs. TPLs are antioxidant and chemically reducing compounds comprising of ()-epicatechin (15.7%), ()-epicatechin gallate (10.62%), ()-epigallocatechin (5.88%), and ()-epigallocatechin-3gallate (EGCG; 66.90%; Fig. 3). Although there have been many studies on TPLs' antioxidation and other biological functions (Higdon and Frei 2003), the actual chemical structure of TPLs under various conditions is essential to their biological functions as TPLs are sensitive to environmental conditions including pH, temperature, oxygen concentration, metal ions, and their concentrations. The study of Sang et al. (2005) showed that the major component of EGCG can undergo auto-oxidation to form dimmers and cause epimerization of gallocatechin gallate (GCG) at normal cell culture conditions. The remaining TPLs decreased quickly along the increase of pH from 4.0 to 7.0, and highest GCG was formed at pH 5.0 (Chen et al. 2000). An expanded profile of catechins dimmers including catechin homodimer and heterodimers under simulated digestive tract conditions

was also formed through auto-oxidation (Neilson et al. 2007). Thus the stability of TPLs in the current study of enzyme digestion of starch is a changeable parameter, and a simple deduction of the background signal from TPLs, which is commonly found in literatures (Hara and Honda 1990; Kwon et al. 2008), is not a scientific way to eliminate the TPLs' confounding effect in DNS assay. As the DNS procedure required, high temperature (boiling water bath) and alkaline conditions would dramatically changes the structure of TPLs and so their antioxidant property. Furthermore, the interaction between starch and TPLs (Liu et al. 2011) in the starch digestion might be another factor that affects the accuracy of glucose measurement using DNS. All these would contribute to the incompatibility of DNS method with TPLs for an accurate measurement of reducing sugars in a real starch digestion system and model system. Glucose measurement is one of the most basic and simple tasks in the field of carbohydrate research, and there have been many methods available for its measurement based on different mechanisms. DNS is not only one of the earliest methods developed for reducing sugar measurement, it is also widely used with its simplicity and inexpensive reagents. Other enzyme-based methods increase the specificity for glucose but the price of kit is often more expensive than DNS reagents. Comparatively, HPLC method is the most accurate procedure, but a high quality HPLC system is not likely available in many laboratories, especially in developing countries. When all this factors are taken into consideration, the HK method with its simple operation and specificity to glucose should be used to measure glucose in a reducing environment containing TPLs or other polyphenols.
Funding Sources The current investigation was supported by National Natural Science Foundation of China (project no. 21076095).

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