Journal of the Science of Food and Agriculture J Sci Food Agric 79 :232–236 (1999)

Cost-effective colorimetric microtitre plateenzymatic assays for sucrose, glucose andfructose in sugarcane tissue extractsJames A Campbell, Ros s W Hans en* and John R Wils onCSIRO Tropical Agriculture, 306 Carmody Road, St Lucia,Qld 4067, Aus tralia

Abstract : Quantiücation of sucrose, glucose and fructose is essential for developing a physiological

understanding of growth and development of plant species. This is usually achieved by either HPLC or

enzymatic assays for speciüc sugars. The need for reliable and cost-eþ ective assays of large numbers

of samples led us to compare microtitre plate-adapted enzymatic assays for sucrose, glucose and

fructose with current HPLC protocols. The enzymatic assay techniques were modiüed without loss of

precision or accuracy when compared to the original protocols. Application of HPLC and the modi-

üed enzymatic assay to a range of sugarcane stem extracts found no diþ erence in assay sensitivity.

The enzymatic assay had considerable advantages in terms of sample throughput and cost-

eþ ectiveness.

1999 Society of Chemical Industry(

Keywords: sucrose; glucose; fructose; sugarcane; microtitre plate; enzymatic assay

INTRODUCTION

Quantiücation of non-structural carbohydrates inplant tissues is central to the understanding of wholeplant carbon allocation and general physiology.Assays which determine total sugar content withoutdiscriminating between the constituent sugars havethe advantage of comparative simplicity,1 howevertheir utility is limited by their non-speciücity. Whenhigh levels of discrimination and precision arerequired, the two most commonly employed proto-cols for quantiücation are separation techniques suchas high-performance liquid chromatography (HPLC)and colorimetrically-quantiüed enzymatic analysis.

The HPLC technique separates constituent sugarsof a sample, then allows quantiücation based on thepreviously deüned elution characteristics of sugarstandards.2,3 Modern HPLC protocols have highlevels of sensitivity and speciücity, however through-put is limited by substantial sample preparation(dilution followed by one or more ültration steps andsometimes removal of interfering ions) and run time(from 5–30min per sample).

Enzymatic analysis, through the use of speciücenzymes which catalyse reactions with deünedsugars, oþers greater levels of sensitivity thanHPLC. However, such assays, particularly as com-mercial kits, can be expensive. The unit expense peranalysis can be greatly reduced for enzymatic assaysby modifying original methodologies to enable deter-mination in microtitre plates. Successful modiü-cation reduces the amounts of sample, biochemicals

and enzymes needed, and increases the number ofsamples which can be analysed per day. The use ofpure enzymes in modiüed determination protocolsalso overcomes enzymatic impurities associated withcommercial kits.4 Previous studies have reportedgood agreement between determinations achieved bystandard enzymatic assays and HPLC in the absenceof interfering compounds.5h7

Several enzymatic assays for speciüc sugars havesuccessfully been modiüed for microtitre plate use,although these measure the formation of formazandyes rather than the direct catalytic product,NADH.8,9 We have not noted microtitre plate-adapted NADH detection protocols for the enzy-matic assays of glucose10 and fructose11 used in ourlaboratory. Similarly, there has been no report of amicrotitre plate-adapted protocol for the Birnbergand Brenner12 assay for sucrose, which employssucrose phosphorylase rather than invertase to cleavesucrose into its constituent reducing sugars.

Ongoing experiments into sugarcane physiology inour laboratory yield large numbers of samples forsugar analysis.13 Needing a quantiücation protocolwhich oþered sugar discrimination, high sensitivityand the capacity to process large numbers ofsamples, we compared HPLC and microtitre plate-adapted enzymatic assay techniques. This paperdescribes the modiücation of existing enzymatictechniques for sucrose, glucose and fructose analysisfor use with a microtitre plate reader, compares theprecision and sample throughput of these procedures

* Corres pondence to : Ros s W Hans en, CSIRO Tropical Agricul-ture, 306 Carmody Road, St Lucia, Qld 4067, Aus tralia.

Contract/grant s pons or : Sugar Res earch and Development

Corporation.

(Received 7 Augus t 1997 ; revis ed vers ion received 17 April 1998 ;

accepted 6 May 1998)

( 1999 Society of Chemical Industry. J Sci Food Agric 0022–5142/99/$17.50 232

Microtitre plate-adapted enzymatic assay for sugars

with HPLC analyses, and compares determinationdata for a range of sugarcane tissue extracts mea-sured by the two protocols.

EXPERIMENTAL

Plant growth and sugar extraction

Sugarcane (Saccharum spp) plants of the Australiancommercial variety Q117 were grown in a controlledenvironment according to established protocols13 aspart of an experiment investigating the eþects oftemperature on growth, development and sugaraccumulation of sugarcane. At maturity, plants weredestructively harvested, and fresh samples of leaf(80–100mg) and internode (120–150mg) wereexcised from mid-leaf or the mid-point of each inter-node for sugar extraction. The tissue samples wereincubated twice for 6h in 1ml of 80% ethanol inwater (v/v) at 75¡C, and the pooled extracts werevacuum-dried then reconstituted in water. This pro-tocol extracts 99% of total sugars in sugarcane stemsamples of this type. A total of 75 diþerent reconsti-tuted extracts were analysed by both microtitreplate-adapted enzymatic assay and HPLC, followingappropriate dilution with water (sugar concentrationsof between 0.1 and 1.0mg ml~1 for enzymatic assay,and between 1 and 5mg ml~1 for HPLC).

Sugar determination by microtitre plate-adapted

enzymatic assay

All sugar determination assays were modiüed foruse with a Molecular Devices ‘ThermoMax’ platereader ütted with a 340nm, 10nm bandwidth inter-ference ülter. The plate-reader was linked to a per-sonal computer, and data were calibrated andtabulated by the Softmax software package. Assayswere performed in polystyrene ‘U’ or ‘Flat’ wellassay plates, of standard 96 well conüguration. End-point determination for all three assays was based onthe reduction of NAD to NADH, which wasdetected colorimetrically at 340nm. To compensatefor any optical imperfections in the microtitre plates,absorbances were corrected against a second wave-length (562nm). All assays were against standardseries of analytical grade sugar : 0.1, 0.2, 0.35, 0.5and 0.75mg ml~1 sucrose, and 0.1, 0.25, 0.35, 0.5,0.75 and 1.5mg ml~1 glucose and fructose. Twoduplicated water blanks were included on eachmicroplate, one as a zero point for the calibrationcurve and the other as a reagent blank, the absorb-ance of which was subtracted from all samples andstandards. All samples and standards were deter-mined as the mean value of two replicate assay wells.

Sucrose determination was modiüed from theoriginal protocol of Birnberg and Brenner.12 Thisprotocol is based on the cleavage of sucrose intoglucose-1-phosphate (G1P) and fructose by sucrosephosphorylase (SucP, EC 2.4.1.7) (eqn 1), isomer-isation of G1P to glucose-6-phosphate (G6P) by

phosphoglucomutase (PGluM, EC 5.4.2.2) (eqn 2),and ünal conversion of G6P and NAD to gluconate6-phosphate (6PGlcU) and NADH by glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49)(eqn 3). NADH is stoichiometrically balanced withthe initial concentration of sucrose.

SucPSucrose ] P

i——— Õ G1P ] fructose (1)

PGluMGlP ——— Õ G6P (2)

G6PDHG6P ] NAD ——— Õ 6PGlcU ] NADH (3)

For the microtitre plate-adapted enzymatic assay, abuþer is prepared by mixing buþer solutions A andB to achieve pH 7.0. Solution A contains 10mM

and 1mM while Solution B con-K2HPO4 MgSO4tains 10mM and 1mM TheKH2PO4 MgSO4 .reagent mixture, sufficient for one full plate of 96wells, is prepared by dissolving 8.3mg nicotinamideadenine dinucleotide (NAD) (Sigma N-7004) in25ml of assay buþer. Enzymes are added to thismixture as 80ll (32 U) PGluM (Boehringer Mann-heim 108 375), 73 ll (75 U) G6PDH (BoehringerMannheim 165 875) and 53 ll (5 U) SucP (SigmaS-7660). The reaction is achieved by adding 250 ll ofthe reagent mixture to 13.5 ll of standard or appropri-ately diluted sample, incubating at room temperaturefor 70 min, then assaying for NADH production.

As a greater dilution is required for sucrose determi-nation compared to glucose and fructose determi-nations in sugarcane tissue extracts, the latter areachieved in di†erent microplates to those used forsucrose determination. Glucose determination wasmodiÐed from the protocol of Kunst et al.10 The basisof this method is the phosphorylation of glucose toform G6P by hexokinase (HK, EC 2.7.1.1) (eqn 4), fol-lowed by the conversion of G6P and NAD to 6PGlcUand NADH by G6PDH (eqn 3) and quantiÐcation ofNADH as for the Ðnal step of the sucrose determi-nation reaction sequence. The adapted protocol forfructose determination was modiÐed from that ofBeutler.11 This involves the phosphorylation of fruc-tose to fructose-6-phosphate (F6P) by HK (eqn 5), theconversion of F6P to G6P by phosphoglucose isom-erase (PGluI, EC 5.3.1.9) (eqn 6), and the previouslydescribed conversion of G6P and NAD to 6PGlcUand NADH (eqn 3), the latter being quantiÐedspectrophotometrically. As the fructose assay isaccomplished in the same well as the glucose assay,they are temporally separated, and fructose (which isconverted to F6P then G6P during the assay) is deter-mined by subtraction of glucose prior to isomerisationfrom total glucose at the end of the assay.

HKGlucose] ATP ÈÈÈÕ G6P] ADP (4)

HKFructose ] ATP ÈÈÈÕ F6P] ADP (5)

PGluIF6P ÈÈÈÕ G6P (6)

J Sci Food Agric 79 :232–236 (1999) 233

JA Campbell, RW Hansen, JR Wilson

Figure 1. Standard curves for s ucros e glucos e and(L), (K)fructos e obtained us ing the microtitre plate-adapted(|)enzymatic as s ays . Points are mean values from 10 independent

s tandard s eries ^SEM, which is generally too s mall to beres olved in this repres entation.

For glucose and fructose assay, a bu†er is prepared tocontain 50 mM triethanolamine-HCl, 5 mM MgSO4 ,0.02% bovine serum albumin (BSA) and 0.5 mM

dithiothreitol (ClelandÏs Reagent). The pH is adjustedto 7.6 using 0.5 M NaOH. A solution of adenosine-5-triphosphate (ATP) as the disodium salt is prepared bydissolving 300 mg ATP (Boehringer Mannheim 127523) and 300 mg in 6 ml water. A reagentNa2CO3mixture, sufficient for one full microtitre plate, is pre-pared by dissolving 19.6 mg NAD in 9 ml of tri-ethanolamine bu†er, adding 18 ml of water, and 900 llof the ATP solution. Enzymes are added to thismixture as 36.9 ll (52 U) HK (Boehringer Mannheim1426 362) and 23.4 ll (26 U) G6PDH (BoehringerMannheim 165 875).

For glucose assay, 5 ll of appropriately dilutedsample or standard was added to each assay well, fol-lowed by 250 ll of reagent mixture. The combinedsolutions were mixed well and incubated for 15 minbefore determining the production of NADH at340 nm. After completion of the glucose assay, thefructose assay is initiated by addition of 2 ll (1.4 U) ofPGluI (diluted 1 : 5 with water) (Boehringer Mann-

Figure 2. Standard curves for s ucros e glucos e and(…), (=)fructos e obtained us ing HPLC. ‘Res pons e’ is a quantification(>)of integrated peak height. Points are mean values from 10

independent s tandard s eries . SEM is too s mall to be res olved in

this repres entation.

heim 128 139) to each microplate well. After mixingand 15 min incubation the total NADH is determinedand fructose is calculated by di†erence.

Sugar determination by HPLC

Analyses were performed using a Waters HPLCsystem comprising a 600E Multi-Solvent DeliverySystem, a 717plus Autosampler with refrigeratedsample compartment and a 410 Diþerential Refrac-tometer. System control and data handling wereachieved using the Waters Millennium Chromatog-raphy Manager software.

Aliquots of sample (100ll) were transferred to lowvolume inserts in 4 ml HPLC vials for analysis.Separation of sugars was achieved using a4.6 mm] 250 mm steel cartridge Waters carbohydratecolumn packed with 4lm Nova-Pak spherical silicabonded with trifunctional amino propylsilane (PartNo WAT044355). The mobile phase comprised ace-tonitrile :water (75 : 25) at a Ñow rate of 1.4 mlmin~1.Separated sugars were detected using a di†erential ref-ractometer.

Standards of analytical grade sugars were run atfour levels to construct calibration curves from whichunknowns were read. The standards contained fruc-tose and glucose at levels between 0.5 and 4 mg ml~1and sucrose at levels between 2.5 and 12 mg ml~1.Multi-level calibration curves through zero were usedwith bracketing (standards run before and afterbatches of samples then averaged).

Comparison of determination techniques

Sucrose, glucose and fructose determinationsachieved by both enzymatic assay and HPLC weretested against known sugar concentrations (Figs 1and 2) and each-other (Fig 3) by regression analysis.

RESULTS AND DISCUSSION

Figure 1 shows the linear increase in absorbance at340nm with concentration of ; sucrose (n \ 60,R2\ 0.994, P \ 0.001), glucose (n \ 70, R2\ 0.997,P \ 0.001) and fructose (n \ 70, R2\ 0.999,P \ 0.001). Holmes14 recently reported a protocolfor sucrose determination in medical samples by amicrotitre plate-adapted, invertase-mediated assay(linear range 100nM–1mM). The linear ranges foraccurate determination in this study are greater thanthose reported by Holmes ;14 sucrose (300nM–2.2mM), glucose (560nM–8.3mM) and fructose(560nM–8.3mM), although minimum detectablelevels are higher in this study. Modiücation of theoriginal sucrose assay of Birnberg and Brenner12reduced sample and reagent volumes (and reagentcost) by 67%. Modiücation of the existing glucoseand fructose assays eþected volume and costreductions of more than 90% over commercial kits.Using the adapted enzymatic protocol, up to 80samples per day could be analysed for sucrose,glucose and fructose.

234 J Sci Food Agric 79 :232–236 (1999)

Microtitre plate-adapted enzymatic assay for sugars

Figure 3. Plots of s ucros e glucos e and fructos e(L), (K) (|)determinations obtained us ing the microtitre plate-adapted

enzymatic as s ays agains t determinations achieved by HPLC.

Figure 2 shows repeated analysis of 10 diþerentseries of standard solutions by HPLC. Sugar concen-tration was linearly related to response for all threesugars ; sucrose (n \ 50, R2\ 0.999, P \ 0.001,linear range 7.3mM–36.2mM), glucose (n \ 50,R2\ 0.995, P \ 0.001, linear range 3.1mM–24.0mM) and fructose (n \ 50, R2\ 0.995,P \ 0.001, linear range 3.2mM–23.5mM). Reýectingadvances in HPLC technologies, these data indicate

linear responses at sugar concentrations approx-imately twenty times lower than previously reportedresults with this protocol.15 Using this HPLC proto-col, up to 40 samples could be analysed per day.

Figure 3 shows the relationships between the con-centrations of the three sugars of interest in the 75sugarcane tissue extracts as determined by HPLCand the microtitre plate-modiüed enzymatic assays.Sugarcane stem is a convenient source of plantmaterial for such protocol comparisons. The range ofdevelopmental stages existing along an individualstem has been reported,13,16 and this range yieldstissue extracts with diþerent concentrations and rela-tive contributions of sucrose, glucose and fructose.There was good agreement between HPLC- andenzymatic assay-derived sugar determinations for therange of samples examined; sucrose (n \ 75,R2\ 0.984, P \ 0.001), glucose (n \ 69, R2\ 0.977,P \ 0.001) and fructose (n \ 65, R2\ 0.962,P \ 0.001). There was no bias of either protocol ateither low or high concentrations, and we concludethat in terms of precision neither protocol can beconsidered substantially superior to the other. Giventhe faster analysis time and lower cost per determi-nation of the microplate-adapted enzymatic determi-nation of sugars, we have chosen this method as thestandard for our studies of sugarcane physiology.There is considerable scope for adaptation to micro-titre plate scale of a wide range of enzymatic assays.We believe that such adaptations should provideequally reliable and resource-efficient quantiücationprotocols for other compounds of interest.

CONCLUSION

Existing enzymatic assay methods for the quantiüca-tion of sucrose, glucose and fructose have been modi-üed for application with microplate techniqueswithout loss of assay sensitivity. Precision and accu-racy of the modiüed protocols compare favourablywith current HPLC determination techniques, andallow faster and less expensive analysis per samplethan HPLC. A comparison of sucrose, glucose andfructose determinations by both techniques onextracts from sugarcane internodes indicated goodagreement for results achieved by either protocol.

Given the precision and cost-eþectiveness of themodiüed enzymatic techniques, we suggest thatother laboratories interested in quantifying sugars inlarge numbers of samples consider their developmentand use.

ACKNOWLEDGEMENTS

The authors thank Dr Graham Bonnett for his criti-cal review of this manuscript, Mr Peter Tuckett forhis expert technical assistance and Dr Peter Jones ofthe CSIRO Division of Mathematical and Informa-tion Sciences for his advice on statistical analysis.RWH thanks Dr John Patrick and his staþ at the

J Sci Food Agric 79 :232–236 (1999) 235

JA Campbell, RW Hansen, JR Wilson

University of Newcastle for introduction to some ofthese techniques. This work was partially funded bythe Sugar Research and Development Corporation.

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