Miloski Et Al Pp 7-18

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AMERICAN JOURNAL OF UNDERGRADUATE RESEARCH VOL. 7, NO. 2

Comparison of Biochemical and ChemicalDigestion and Detection Methods for

CarbohydratesKatie Miloski, Kelly Wallace, Ashley Fenger, Ellen Schneider

Kestas Bendinskas*Department of Chemistry

State University of New York—OswegoOswego, New York 13126 USA

Received: August 1, 2008 Accepted: September 8, 2008

ABSTRACT

There is a multitude of chemical and biochemical detection methods for sugars. Which oneswould be most practical in an undergraduate laboratory setting? How to best detect non-reducingdisaccharides? How to make such lab fun for students to perform? After trying severalspectrophotometric methods, it was found that chemical detection by dinitrosalicylic acid andbiochemical detection by hexokinase/glucose-6-phosphate dehydrogenase reagent are mostappropriate. Sucrose, a non-reducing disaccharide was digested chemically with hydrochloricacid and biochemically with invertase. It was concluded that chemical detection and biochemicaldetection compliment each other. Chemical digestion method was preferred over the digestion byinvertase. These methods were applied for testing the validity of sugar ingredients printed ondrink labels as well as the measurement of sugar levels in ripening bananas at two differentconditions. The comprehensive comparison of these methods and the detection of sugar concentrations in interesting samples might serve as a basis for an undergraduate chemistrylaboratory.

I. INTRODUCTION

Carbohydrates are organic

compounds that are either aldehydes or ketones and have many hydroxyl groups.Carbohydrates serve a multitude of biological functions for all known organismson Earth. They are an invaluable energysource and provide structural, storage,informational, recognition, shock absorbingand protective functions. Monosaccharides,the simplest carbohydrate units, arecommonly known as ‘simple sugars.’ Twomonosaccharides can form a glycosidiclinkage by undergoing a condensationreaction, which yields a disaccharide. A

disaccharide can be digested and brokendown into its two original monosaccharideunits. When glycosidic linkages are formedbetween more than two monosaccharideunits, a polysaccharide is produced [1].

Sugars can be detected in naturaland processed foods and drinks by chemical

and biochemical methods. Such detectionscan be useful for not just determiningamount of sugar in food or drink samples,

but also in determining validity of labels. Thefocus of this study is to not only provide bothchemical and biochemical methods of sugar digestion and detection that can be utilizedin an undergraduate biochemistrylaboratory, but to determine if one set of methods works more efficiently than theother.

Sucrose, commonly known as tablesugar, is present in natural fruits andvegetables. Sucrose can be digestedbiochemically using invertase (Reaction 1,Fisher and Kohtes [2]) and chemically using

hydrochloric acid (Reaction 2, Fieser andWilliamson [3]) into glucose and fructosemonosaccharides. The biochemicaldetection method using hexokinase withglucose-6-phosphate dehydrogenase ishighly specific to glucose only (Reaction 3,Avigrad and Englard [4] and Kunst [5]). Thechemical detection method, which utilizesdinitrosalicylic acid (DNS reagent), detectsglucose, fructose, and other mono-

* bendinsk oswe o.edu

7




saccharides (Reaction 4, Bernfeld [6] andMiller [7]). Sucrose cannot be detected byeither detection method directly. Therefore,samples containing sucrose must bedigested so that the glucose and fructoseproducts can be detected. If samplescontain sucrose and some other sugars aswell, monosaccharides have to be measuredinitially before sucrose digestion. Thesucrose concentration can then be

calculated when the total monosaccharidesare measured after hydrolysis of the sample.

Another biochemical methodalternative to hexokinase is detection usingglucose oxidase and peroxidase [8-9].Alternatives to the chemical DNS methodsare the well known Nelson’s method [10]and the ortho-toluidine method of Dubovski[11]. These methods were tried, but notchosen for the reasons discussed in theresults section.

+ HClH

++ Cl

-

O

OHH

HH

OHOH

H OH

H

OH

O O-

H

OH

OH

H

H

OH

OH

O

H

OH

OH

H

H

OH

OH

O

HH

H

OHOH

H OH

H

OH

O

O

H

OH

OH

H

H

OH

OH

O

HH

H

OHOH

H OH

H

OH

O

+ H2OO OH

H

OH

OH

H

H

OH

OH

O

OHH

HH

OHOH

H OH

H

OH

+ H2O Glucose

Reaction 2: Sucrose is protonated by HCl then undergoes a hydrolysis reaction to yield glucoseand fructose as the final products.

O OH

H

OH

OH

H

H

OH

OH

+ H2OInvertase

Fructose

O

H

OH

OH

H

H

OH

OH

O

HH

H

OHOH

H OH

H

OH

O

O

OHH

HH

OHOH

H OH

H

OH

Glucose

Sucrose

Reaction 1: Sucrose is broken down by invertase into glucose and fructose via a hydrolysisreaction.

8




OH

O

H OH

OH H

H OH

H OH

O

O

H OH

OH H

H OH

H OH

P

O-

O-

O

O

O

H OH

OH H

H OH

H OH

P

O-

O-

O

O

O

H OH

OH H

H OH

H OH

P

O-

O-

O

+ ATP Hexokinase

+ ADP

+ NAD+

Glucose-6-PhosphateDehydrogenase

+ NADH

O

H OH

OH H

H OH

H OH

P

O-

O-

O

O-

O

Glucose

Reaction 3: Glucose is phosphorylated by ATP using hexokinase. This product is then oxidized.The absorbance of NADH is measured.

OH

O

H OH

OH H

H OHH OH

DNS

OH

NO O

N

O

O

O

OH+

OH

O

H OH

OH H

H OH

H OH

OH

OH

NH2

N

O

O

O

OH

+

Glucose

Reaction 4: Glucose (or fructose) and DNS undergo a reduction-oxidation reaction during DNSdetection. The sugar is the reducing agent and DNS is the oxidizing agent.

II. METHODS

a. Preparation of the Standards andStandard Curves

All chemicals were purchased fromSigma-Aldrich. Stock solutions (50 mL each)of 40 mM fructose, 40 mM glucose, and 40mM sucrose were prepared weekly andstored refrigerated until needed. Desired

concentrations of samples for the standardcurve for each sugar were prepared byperforming dilutions of the stock solution.

b. Preparation of the Drink Samples

Table 1 exhibits the drink samplesused in this study along with information oneach sample’s label. It is important tochoose drinks that have contrasting sugars.

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Drink SampleSugar

Ingredient(s)

Grams sugar per 240 mL

servingSugar (mM) expected

(1:20 dilution)

Sierra Mist Free no sugar 0 0

Tea with 3 Domino ®

sugar

packets sucrose 12.6 7.7

Pedialyte ®

glucose 6 6.9

Sprite

High-fructosecorn syrup(mainly glucoseand fructose) 26

Gatorade ®

Rain

fructose,glucose, &sucrose mixture 14

Table 1. Label and calculated information of each drink sample.

Carbonated drinks were degassed byopening the bottles for 30 minutes, andplacing samples under the aspirator-generated vacuum for 10 minutes. Sampleswere diluted 1:20 with water. Expectedconcentrations were determined based onthe grams of the sugar included, its specificmolecular weight, and volume of one serving(240 mL). Actual concentration of eachindividual type of sugar could not bedetermined accurately in the samplescontaining sugar mixtures. The mass of sugar listed on the label is inclusive of allsugars in the entire mixture. Therefore,determining the number of grams of fructoseor glucose in the sample is not possible fromthe label alone. For example, Sprite’singredients contain high fructose corn syrup(HFCS). In the United States, the mainsource of fructose in diets is HFCS [12].HFCS are quite similar to sucrose’scomposition. However, sucrose is composedof 50 % glucose and 50% fructose, whereasHFCS contains approximately 42 % fructoseand the rest glucose (55 %) and other simple sugars [13]. Hence, the calculations

of expected concentration per simple sugar become complicated. This is when detectionmethods come in handy for unknownconcentrations. Using a highly glucosespecific biochemical detection method, theactual grams of glucose in the sample canbe found. Knowing this and the total amountof sugar in the sample, fructose can bedetermined.

c. Biochemical Digestion

Samples containing sucrose(specifically Gatorade Rain, tea with sugar,and the standard) were biochemicallydigested in triplicate via a hydrolysis reactionusing invertase enzyme. The stock invertasewas made by mixing 0.01 g of invertasesolid with 1 mL of 50 mM sodium phosphatebuffer at a pH of 7. A 180 µl aliquot of 0.2 Msodium acetate buffer (pH = 4.5) was addedto 450 µL of sample, followed by an additionof 9 µL invertase stock. Water was thenadded to the samples to bring each to thedesignated volume, 900 µL. Samples wereleft to digest at room temperature for 30minutes.

d. Chemical Digestion

A separate set of the samplescontaining sucrose was chemically digestedin triplicate by adding 3 µl concentratedhydrochloric acid (HCl) to 450 µL of eachsample. These samples were placed in a90

◦C hot water bath for 15 minutes,

removed, and left to sit at room temperaturefor another 15 minutes. Approximately 40 µlof sodium bicarbonate was added in 10 µLportions to each sample in order toneutralize the acid. The pH was checkedwith pH test strips to ensure a pH of 7.Water was then added to bring each sampleto the designated volume, 900 µL.

10




HK detection of undigested samples

-0.200

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1.600

1.800

0 2 4 6 8 10 12

Concentration of sugars (mM)

A b s o r b a n c e ( a t 3 4 0 n m )

Glucose

Fructose

Sucrose

Figure 1. Hexokinase detection of undigested glucose, fructose, and sucrose.

e. Biochemical Detection

The standards, undigested drinksamples, chemically and biochemicallydigested samples all underwent biochemicaldetection. A 10 µL aliquot of each samplewas pipetted into the wells of a 96-wellmicroplate. A 300 µL aliquot of Hexokinasereagent was added to each well and theplate was left at room temperature for 5minutes. Absorbance of each sample wasread at 340 nm using a 96- well microplatereader (Bio-Tek) and the averageabsorbance for each set of triplicate wascalculated. Using the standard curveconstructed from the absorbance data, theunknown concentrations of the drinksamples were found.

f. Chemical Detection

Another set of chemically andbiochemically digested samples, as well theother standards and drink samplesunderwent chemical detection in triplicate. A450 µL aliquot of each sample was pipettedinto new micro-tubes. A 450 µl aliquot of dinitrosalicylic acid (1% DNS) was added toeach tube. Samples were immersed in a 90

ºC hot water bath for 10-12 minutes. It isimportant that samples and standards aredone at the same time. After removal fromthe bath, 150 µl of sodium potassiumtartrate (40%) was added to each tube. A

300 µL aliquot of each sample was pipettedinto separate wells of the 96-well microplate.Absorbance was read at 575 nm with theplate reader and the average absorbance for each set of triplicate was calculated.

g. Banana Experiment

Two set of bananas were kept for 15days, one at room temperature and onerefrigerated at 4ºC. To determine theamount of starch, the slice was placed ontoa microscope slide, dyed with 4% potassiumiodide, and photographed. Amounts of

sugars were determined after themechanical lysis of 1 g of banana in 10 mLof distilled water and the centrifugation of the sample at 14,000 rpm for 5 minutes.

III. RESULTS AND DISCUSSION

Both hexokinase/ glucose-6-phos-phate dehydrogenase (HK) reagent andglucose oxidase/ peroxidase reagent are

11




HK detection of digested sucrose

0.000

0.100

0.200

0.300

0.400

0.500

0.600

0.7000.800

0.900

1.000

0 2 4 6 8 10 12

Concentration of sucrose (mM)

A b s o r b a n c e ( a t 3 4 0 n m )

Invertase

digestHCl digest

Figure 2. HK detection of sucrose digested by invertase and by hydrochloric acid.

available from Sigma at approximately sameprice for small quantities. The decision wasmade to use HK because it is available inboth large and small quantities, whichmakes it useful in a teaching laboratory.

Also, this reaction takes only 5 minutes, it isglucose specific, and the amount of sampleused is 10 µL. The price, however, was 30¢per measurement. All chemical methodswere an order of the magnitude lessexpensive.

Among chemical methods, Nelson’sassay is often used in laboratoryexperiments. It takes 50 minutes to performthe assay, it detects glucose and fructoseequally well, and has no response for sucrose. However, it uses quite toxic cupper and arsenomolybdate reagents. Ortho-

toluidine method can be used to detectglucose in about 20 minutes. However, itresponds to fructose (7.6 % of glucoseresponse) and sucrose (20.7 % of glucoseresponse), making it completelyinappropriate for complex mixtures. DNStakes 40 minutes to perform. It responds toglucose and fructose but not sucrose. Datais available but not shown for methods wechose not to use.

In Figures 1 through 4, the standardcurves for both the digested and undigestedstandards using biochemical and chemicaldetection are displayed. Each sample wasdone in triplicate, and Y- error bars show a

single standard deviation for each point.The concentration of the glucose

standard is directly proportional to theabsorbance of the sample detected usingthe HK method, while fructose and sucrosehave no absorbance as they can not serveas substrates for at least one enzyme. Thisshows the specificity of this reaction and itssuitability for the purpose of thisinvestigation.

Both invertase and HCl digestionmethods take about 30 minutes to perform.Sucrose standard is digested and yields

glucose and fructose. Glucose only isdetected using hexokinase. The digestyields a two-fold dilution in this procedure.The absorbance of the samples is expected,therefore, to be half of the absorbance of thesame sample of glucose, which it was. Thisindicates a complete digest in both cases.The only difference between the invertaseand HCl digestion are procedural and fiscal,

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DNS detection of undigested samples

-0.200

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1.600

0 5 10 15 20 25

Concentration of sugars (mM)

A b s o r b a n c e ( a t 5 7 5 n m )

Glucose

Fructose

Sucrose

Figure 3. Hexokinase detection of undigested glucose, fructose, and sucrose.

DNS detection of digested sucrose

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1.600

1.800

0 5 10 15 20 25

Concentration of sucrose (mM)

A b s o r b a n c e ( a t 5 7 5 n m )

Invertase

digestHCl

digest

Figure 4. DNS detection of sucrose digested by invertase and by hydrochloric acid.

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Drink Sample Average Absorbance [sugar] mM

Sprite 0.623 ± 0.0195.31± 0.310 mM glucose, 3.278±

0.310 mM fructose

Sierra Mist Free 0.002 ± 0.002 0.050 ± 0.050

Gatorade 0.650 ± 0.007 NA

Pedialyte 0.513 ± 0.078 7.600 ± 0.900

Tea 0.009 ± 0.001 0.158 ± 0.024

Table 2. Average absorbance and concentration for each undigested drink sample using DNSdetection.

Drink Sample Average Absorbance [glucose] mM

Sprite 0.859 ± 0.102 5.00 ± 0.400

Sierra Mist Free 0.073 ± 0.097 0.226 ± 0.371

Gatorade 1.271 ± 0.069 7.500 ± 0.200

Pedialyte 1.204 ± 0.074 7.090 ± 0.230

Tea 0.050 ± 0.093 0.089 ± 0.344

Table 3. Average absorbance and concentration of glucose in each undigested drink sampleusing the hexokinase detection method.

with the cost of the HCl digest being muchless.

The DNS method detects bothglucose and fructose to similar extent(slopes differ by 10.7 % only). This methodcompliments the HK detection method,which only detects glucose. Usingsubtraction of data from DNS and HK

detection, students can determine theconcentration of fructose in a mixed sample.According to this data, invertase

digest seemed to be incomplete, with slope16.2 % below that for HCl digest. Onepossible explanation is that the DNS reagentstops the invertase reaction, while the HKreagent, being quite compatible with theinvertase, forces the reaction to completion.Thus, DNS detection should be used in

conjunction with the HCl digest. Also, sinceDNS detects both fructose and glucose, the2-fold dilution upon digestion has beencompensated by the production of twodetectable molecules. The absorbance atthe same concentration of sucrose after thedigest is about the same as undigestedglucose/fructose of the same concentration,

not 2 times lower as in HK method.

a. Chemical Detection of Undigested DrinkSamples

DNS detection was successfully runon all the drink samples (Table 2). Theexpected concentration of Pedialyte (6.9mM) fell in range with the concentrationsfound (7.6 ± 0.9 mM). This is a less than

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DNS detection

Drink Sample Average Absorbance [glucose and fructose] mM

Tea 0.504 ± 0.019 7.240 ± 0.007


Hexokinase detection


Tea 0.4667 ± 0.022 5.480 ± 0.021

Gatorade 0.4594 ± 0.035 4.800± 0.170

Table 4. DNS and hexokinase detection of tea and Gatorade drink samples after HCl digestion.

DNS detection

Drink Sample Average Absorbance [glucose and fructose] mM

Tea 0.313 ± 0.008 5.640 ± 0.137


Hexokinase detection


Tea 0.174 ± 0.003 2.010 ± 0.020

Gatorade 0.198 ± 0.014 2.296 ± 0.176

Table 5. DNS and hexokinase detection on tea and Gatorade drink samples after invertasedigestion.

10% difference between the actual andexpected concentrations of glucose in

Pedialyte. Such a percent difference isacceptable in an undergraduatebiochemistry lab. Sprite is a mixture of simple sugars due to HFCS and has thelarge overall amount of sugars in themixture. The expected absorbance andderived sugar concentrations should behigher than the Pedialyte. This wasobserved. The concentrations of glucose

and fructose in Sprite were estimated by acalculation using the known percentages of

each sugar in HFCS (55% glucose, 42%fructose). Tea had virtually no absorbancesince the sucrose was not digested andundetectable. The same result occurred for the Sierra Mist Free since there are nonatural sugars in it. As for the Gatorade mix,a high absorbance was expected. However because the actual percentages of glucose,fructose, and sucrose components of the

15




total sugar amount are unknown, theexpected concentrations of these sugarscannot be estimated. Because this methodis not specific to any one specific sugar, thisis a major drawback in using this detectionprocedure.

b. Biochemical Detection of UndigestedDrink Samples

Hexokinase successfully detectedglucose in the undigested samples (Table3). The expected concentration of glucose inPedialyte (6.9 mM) was in range of theconcentration measured (7.090 ± 0.230mM). The percent difference between theactual and expected was only 2.68%.Because of the glucose specificity, theabsorbance of glucose alone in Sprite couldbe determined directly and not by

calculation. Note that the concentration of glucose using this method (5.00 ± 0.400mM) is similar to the concentration of glucose estimate found using the DNSmethod (5.31± 0.310 mM). As found in thechemical detection method, tea had noglucose detected. Sierra Mist Free had veryslight glucose detection (0.226 ± 0.371 mM)but the relatively high standard deviationnegates this data point. Gatorade had a highabsorbance of glucose in it and thus a highconcentration of glucose (7.500 ± 0.200mM). Even though the DNS method does

not differentiate between reducing sugarsand their individual concentrations, thehexokinase does allow specific detection of glucose in any drink mixture.

c. Chemical Digestion of Drink SamplesContaining Sucrose

HCl digestion of the drink samplescontaining sucrose (tea and Gatorade; seeTable 4) was successful. After running DNSdetection, the expected concentration of glucose and fructose combined (7.7 mM)

was close to the concentration expectedbased on the label (7.240 ± 0.007) with adifference of 5.97%. The concentrationcould not be determined in Gatoradebecause, as stated earlier, the percentagesof each sugar of the total mixture areunknown. This detection method detectsother simple sugars in the Gatorade as well.The concentrations of glucose determinedby the HK method after chemical digestion

of sucrose are shown in Table 4. Thereason for lower readings in tea is unclear.

d. Biochemical Digestion of Drink SamplesContaining Sucrose

The sucrose in tea and Gatoradedrink samples was digested using theinvertase method. The determinedconcentration of glucose and fructose in tea(5.640 ± 0.137 mM) was lower thanexpected (7.7 mM) with a 36% difference.This is probably due to the invertasereaction, which is a reversible reaction and itdoes not fully go to completion. Hence, theconcentrations of sugar products are lower than expected. Glucose was also detectedin the digested drink samples and theconcentrations are shown in Table 5. Thosereadings were significantly lower thanexpected. The reason for lower readings isunclear.

e. Findings of the banana experiment

It was expected that bananas storedat room temperature for 15 days will ripenmore quickly than those stored in arefrigerator. Bananas would be expected tohave an increase in concentration of sugarswith a concurrent decrease in starch. Thestarch and glucose results were consistentwith the predicted hypothesis. Starch levels

decreased over the fifteen day period. Thedecrease in starch in samples stored atroom temperature was greater than that of samples that were refrigerated. Glucoselevels, measured by HK method, increasedover the fifteen day period, more quickly inroom temperature samples. Data from theDNS method and digests was inconsistent.This data is available, but not provided.

IV. CONCLUSIONS

The advantage of the biochemicaldetection and digestion method is arelatively small amount of sample andenzyme required, compared to the larger amount of sample and DNS reagentrequired by chemical methods. Hexokinaseis also time efficient and highly specific toglucose. Determining glucoseconcentrations of food or drink samples

16




would be highly efficient in anundergraduate biochemistry laboratory usingthe HK detection method. This was found tobe effective both in analyzing drink samplesand bananas. The drawback to biochemicalmethods of digestion and detection used inthis investigation is that the enzymes aremore expensive. Also, invertase seems tofalter in achieving a complete digest under certain conditions.

Advantages regarding chemicaldigestion and detection are that reagentsare inexpensive and readily available for students in a laboratory setting. In terms of efficiency, the chemical methods are just asefficient as the biochemical methods basedthe results of this research. The DNSmethod is nonspecific and detects allreducing sugars. This detection method ismore time consuming and requires a larger

sample size than the HK method. Hence,neither biochemical nor chemical detectionmethods are exclusively superior to oneanother. Rather, they compliment eachother.

On a side note, if a drink samplecontains HFCS or a mixture of sugars, likeSprite or Gatorade respectively, thedetermination of sugar concentrationsbecomes too complicated and discouragingfor an undergraduate lab when usingnonspecific detection methods, such asDNS. However, if a class was to use drink

samples containing sugar mixtures, theycould determine the glucose concentrationin the samples quite easily using thehexokinase method. An example of possiblestudent explorations is comparing glucoselevels in multiple brands of cola. In addition,the validity of diet and regular soda labelscould be investigated. Students could alsobe provided unknown samples anddetermine if the samples were of regular or diet soda.

Taking into account the time andfinancial constraints of a typical collegiate

laboratory setting, it is recommended to useboth HK and DNS methods to detectglucose and all reducing sugars prior todigestion. A chemical digestion, which usesreagents that are cheaper and moreavailable in a typical lab, as well as yieldinga more complete digestion of sucrose,should be followed by the DNS detection inorder to calculate the concentration of sucrose present in the sample. The

proposed lab for undergraduate curriculumcan be requested from the correspondingauthor of this publication.

ACKNOWLEDGEMENTS

The authors wish to thank Dr. JimMackenzie and Dr. James Seago for their help and insight.

REFERENCES

1. Voet, D., Voet, J.G., Pratt C.W.Carbohydrates, Funamentals of Biochemistry (3

rdEdn.) 2008, Wiley,

219- 244.2. Fisher and Kohtes, 1951. Helv Chim

Acta, 34, 1123.

3. Fieser, L.F. and Williamson K.L., 1979Hydrolysis of a Disaccharide. Organic Experiments , D.C Heath and Company,196-197.

4. Avigrad, G. and Englard, S., 1968.“Phosporylation of Yeast Hexokinase.”Journal of Biological Chemistry 243:1511.

5. Kunst, A.; Draeger, B.; Zeigenhorn, J. InUV-Methods with Hexokinase andGlucose-6-Phosphate Dehydrogenase;Bergmeyer, H. U., Ed.; Methods of.Enzymatic Analysis (3

rd. Edn) 1984; pp

163.172.6. Bernfeld, P. In Amylases, α and β;

Colowick, S. P., Kaplan, N. D., Eds.;Methods Enzymol. 1, 1955; pp 149.158.

7. Miller, G., 1959. “Use of dinitrosalicylicacid reagent for determination of reducing sugar.” Analytical Chemistry 31: 426-428.

8. Eliot F. Beach and James J. Turner, “AnEnzymatic Method for GlucoseDetermination in Body Fluids,” Clinical Chemistry , Vol. 4, No. 6, 1958, 462-475.

9. Bergmeyer, H. U.; Bernt, E. In

Determination with Glucose Oxidaseand Peroxidase; Bergmeyer, H. U., Ed.;Methods of. Enzymatic Analysis (2nd Edn) 1974; pp 1205.1212.

10. Nelson, N., J. Biol. Chem. 153, 375(1944).

11. Dubovski, K.M., “An o-toluidine methodfor body-fluid glucose determination 1”Clin. Chem. 1962 May-Jun; 8:215-35.

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12. K. J. Melanson, L. Zukley, J. Lowndes,V. Nguyen, T. J. Angelopoulos and J. M.Rippe. “Effects of high-fructose cornsyrup and sucrose consumption oncirculating glucose, insulin, leptin, andghrelin and on appetite in normal-weight

women.” Nutrition , 23, 103 (2007).13. CRA, Corn Refiners Association. 2007.

‘HFCS Facts.” Web URLwww.hfcsfacts.com Accessed August 12008.

UNI Physics provides students with the personal mentoring typical of smaller colleges but adds theresources of a larger research university. Students and faculty work together on research projects, andstudents have the opportunity to continue research during summers as paid Undergraduate ResearchFellows in Physics. www .uni.edu/physics

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A federally-funded initiative to educate a high-tech professionalworkforce in nanoscience and nanotechnology

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