Analytical Biochemistry 402 (2010) 137–145

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Analytical Biochemistry

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Quantification of folate in fruits and vegetables: A fluorescence-basedhomogeneous assay

Harry Martin *, Daniel Comeskey, Robert M. Simpson, William A. Laing, Tony K. McGhieNew Zealand Institute for Plant and Food Research, Palmerston North 4474, New Zealand

a r t i c l e i n f o a b s t r a c t

Article history:Received 18 November 2009Received in revised form 9 March 2010Accepted 26 March 2010Available online 31 March 2010

Keywords:Fluorescence polarizationFolateAlexa FluorFolic acidAssay

0003-2697/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.ab.2010.03.032

* Corresponding author. Fax: +64 6 354 6731.E-mail address: hmartin@hortresearch.co.nz (H. M

A high-throughput, homogeneous, fluorescence polarization, and fluorescence intensity assay has beendeveloped for the measurement of folate in fruits and vegetables. This assay is based on the competitivedisplacement of the fluorescent folate ligands Alexa Fluor (Alexa) 594–folate and Alexa 660–folate frombovine milk folate-binding protein by folates in fruit and vegetable extracts. These fluorescent ligands areemployed because their excitation and emission maxima are in regions of the spectrum with minimalautofluorescence in many extracts. Folate-binding protein and Alexa–folate were typically used at con-centrations of 0.5 lg/ml and 5 nM, respectively, in 20-ll volumes in 384-well microplates. The assay iscomplete within 100 min. The folate estimate is unaffected by the heterogeneity of polyglutamyl residuesthat complicates the liquid chromatography–mass spectrometry (LC–MS)-based methods of quantifica-tion. In this assay, folic acid had an apparent affinity 2.5-fold greater than 5-methyltetrahydrofolate(5MTHF); therefore, it cannot be used to quantify folate when both natural and synthetic folate are pres-ent. 5MTHF-equivalent values were measured in broccoli (240 lg/100 g), strawberry (113 lg/100 g),white grape (32 lg/100 g), orange (44 lg/100 g), tomato (12 lg/100 g), raspberry (31 lg/100 g), banana(29 lg/g), and kiwifruit (36 lg/100 g). These data are similar to published values. However, the assay willnot detect 5-formyltetrahydrofolate which is a significant constituent of the total folate in lettuce, spin-ach, carrot, and peppers.

� 2010 Elsevier Inc. All rights reserved.

1 Abbreviations used: THF, tetrahydrofolate; 5MTHF, 5-methyltetrahydrofolate;AOAC, Association of Official Analytical Chemists; HPLC, high-performance liquidchromatography; MS, mass spectrometry; FBP, folate-binding protein; ELLSA,enzyme-linked ligand sorbent assay; ELISA, enzyme-linked immunosorbent assay;

Introduction

Folate, or vitamin B9, is required for several fundamental bio-logical processes, including nucleotide biosynthesis and aminoacid metabolism (reviewed in Refs. [1,2]), and is an essential com-ponent of the diet. Dietary deficiency of folate is associated withseveral pathological conditions, including neural tube defects [3],anemia [4], cancer [5], and congenital heart defects [6]. Epidemio-logical evidence suggests that folate deficiency is widespread inpopulations in both developing and developed countries [7–9].Mandatory supplementation of foods with folate has been intro-duced in the Unites States and Canada and is being considered inChina, Australia, Ireland, and the United Kingdom [10]. Becausefolate is essential to human nutrition, it is important that it is accu-rately and rapidly quantified in food and medical samples.

Quantification of folate is not straightforward, and severalmethods are used. Some of the difficulties in folate quantificationin foodstuffs and biomedical samples arise from the fact that folateexists in a variety of chemical structures. Folates are composed of apteridine ring linked to para-aminobenzoate that is attached to one

ll rights reserved.

artin).

or more glutamic acid residues. Folic acid is an inexpensive, stablesynthetic analogue of the naturally occurring form of folate, but itoccurs rarely, if at all, in nature and can be readily convertedin vivo to biologically active forms of folate. Consequently, folicacid is used as a food supplement. In natural foods, the pteridinering is always found in the functionally active reduced form, tetra-hydrofolate (THF)1, which is susceptible to oxidative cleavage [2].The structural variations in the pteroyl and p-aminobenzoate por-tions of THF relate to the one-carbon unit transfer function of themolecule and include 10-formyltetrahydrofolate, 5,10-methylene-tetrahydrofolate, and 5-methyltetrahydrofolate (5MTHF). In fruitsand vegetables, folate is present mainly in the 5MTHF form, with asmall percentage also found as THF [11]. Natural folates also varysubstantially in the length of the polyglutamyl chain; one molecule

SPR, surface plasmon resonance; Alexa, Alexa Fluor; FP, fluorescence polarization; FI,fluorescence intensity; BSA, bovine serum albumin; EDA, ethylenediamine; FITC,fluorescein isothiocyanate; 5-formyl THF, 5-formyltetrahydrofolate; DMSO, dimethylsulfoxide; UV, ultraviolet; mP, millipolarization.

138 Quantification of folate in fruits and vegetables: A fluorescence-based / H. Martin et al. / Anal. Biochem. 402 (2010) 137–145

of folate may contain from 1 to as many as 11 glutamic acid residues[12].

Several methods of folate analysis in foods are currently in use(reviewed in Ref. [13]). The most common method is the microbi-ological assay, which relies on the folate-dependent growth of Lac-tobacillus rhamnosus measured by turbidity [14,15]. This approachis slow and unsuitable for automated analysis. The Association ofOfficial Analytical Chemists (AOAC) has approved the use of themicrobiological method for folate analyses in foods [16,17]. Inthese methods, the time required for analyses is 48–72 h and thelimit of detection is 0.16 lg/100 g.

A second approach is the measurement of folate by chemicalmeans; both high-performance liquid chromatography (HPLC)[11,18,19] and (recently) mass spectrometry (MS) methods havebeen developed [20,21]. Both the physicochemical and microbio-logical methods have the disadvantage that the sample matrixand the variation in the chemical structure of folate forms withinthe sample require extensive sample preparation. Digestion of thesamples with a combination of enzymes to degrade the matrixtypically involves treatment with amylase, proteinase, and conju-gase to release the sample from the matrix and remove the poly-glutamyl chain prior to folate quantification [14,20,22,23]. A thirdapproach to folate analysis is the use natural folate-binding pro-teins (FBPs) in enzyme-linked ligand sorbent assay (ELLSA) [24]or anti-folic acid antibody in enzyme-linked immunosorbent as-say (ELISA) [25,26]. The microfluidic assay of Hoegger andcoworkers is designed for the analysis of folate in foods [26]. Fo-late immobilized in microplates is used to capture an FBP or anti-folate antibody to which a reporter enzyme has been coupled.Competition between free folate in the sample and bound folatefor the enzyme-labeled folate receptor reduces the amount of en-zyme bound in the wells that is subsequently measured colori-metrically [27,28]. Fourth, surface plasmon resonance has alsobeen used effectively as a means of quantifying folate in complexfood samples such as milk and cereals [29,30], again using an FBPto directly quantify bound folate in solution. The AOAC has ap-proved the use surface plasmon resonance (SPR) for the quantifi-cation of folate in foods [17]. This method, known as Qflex, usesan antibody to 5MTHF. The limit of detection for the Qflex meth-od is 0.1 lg/100 ml for liquid samples and more than 4 lg/.100 gfor solid samples. The antibody to 5MTHF cross-reacts by 8% withTHF.

One problem with fluorescence-based assays in plant samplesis that the colored samples can quench the fluorescence from thereporter ligand. Alexa Fluor (Alexa) 594–folate and Alexa 660–fo-late ligands have excitation and emission maxima in regions ofthe spectrum with minimal autofluorescence in many fruit andvegetable extracts. Fluorescence polarization (FP) copes wellwith colored and particulate samples because, in contrast tofluorescence intensity (FI), it is a ratiometric measurement ratherthan an absolute measurement. We use the term ‘‘homogeneousassay” here to mean that all of the assay reagents are present insolution in the same reaction vessel (microplate well) at thesame time.

We previously used FP with Alexa ligands for the analysis ofthe biotin (vitamin B7) in whole extracts of leaves [31]. Herewe report the development of a simple FP assay for folate in fruitsand vegetables using bovine milk folate-binding protein as thereceptor for folate and Alexa 594 coupled to folic acid as the fluo-rescent ligand in a competitive displacement assay. The assay canalso be performed as a straightforward FI assay by exploiting theincrease in fluorescence that occurs when certain folate-coupledfluors bind to FBP [32]. Typically, each assay data point consumesnanogram quantities of FBP and picogram quantities of fluores-cently tagged folate, with data available in 100 min starting fromthe raw sample.

Materials and methods

Materials

Black, shallow, 384-well untreated assay plates were purchasedfrom Nunc (Rochester, NY, USA). Bovine serum albumin (BSA),Hepes buffer, 2-mercaptoethanol, ethylenediamine (EDA),pyridine, sodium-L-ascorbate, fluorescein isothiocyanate (FITC),N,N0-dicyclohexylcarbodiimide, N-hydroxysuccinimide, 5-methyl-tetrahydrofolic acid, a-amylase, and bovine milk folate-bindingprotein were supplied by Sigma–Aldrich (St. Louis, MO, USA). Alexa660 and Alexa 594 carboxylic acid, succinimidyl esters were sup-plied by Invitrogen (Carlsbad, CA, USA). THF, 5-formyltetrahydrofo-late (5-formyl THF), pteroyl tri-, penta-, and hepta-c-L-glutamicacids were purchased from Schircks Laboratories (Jona, Switzerland).

Methods

Coupling of folic acid to a fluorescent tag via EDAThe method used was based on that of Wang and coworkers

[33].

Preparation of EDA folates (alpha and gamma)Folic acid (1 g) was dissolved in 50 ml of dimethyl sulfoxide

(DMSO) at 60 �C, followed by the addition of dicyclohexylcarbodi-imide (0.56 g) and N-hydroxysuccinimide (0.52 g) and stirring for5 h at 60 �C. After cooling to room temperature, EDA (1.5 ml) andpyridine (0.23 ml) were added and stirred for a further 6 h at roomtemperature. Then 50 ml of acetonitrile was added to precipitatethe product, the reaction mixture was centrifuged, and the solventwas removed. The solid was washed with diethyl ether (3 � 50 ml)and dried under a flow of nitrogen to give a pale yellow powder.EDA–folate alpha and EDA–folate gamma were separated fromeach other, and from unreacted starting material, by preparativeHPLC (see below).

Alexa 594–EDA–folate gamma, Alexa 660–EDA–folate gamma, FITC–EDA–folate alpha, and FITC–EDA–folate gamma

Alexa 594 succinimidyl ester (150 lg) was taken up in 200 ll ofDMSO and slowly added to a large excess of the EDA–folate gammain 200vll of carbonate buffer and then shaken for 1 h at room tem-perature. The crude reaction mixture was filtered through a 0.45-lm syringe filter, and the Alexa 594–EDA–folate gamma productwas isolated by preparative HPLC. Alexa 660–EDA–folate gamma,FITC–EDA–folate alpha, and FITC–EDA–folate gamma were simi-larly prepared from the corresponding EDA–folate with Alexa660 succinimidyl ester and FITC, respectively. The fluorescentprobes were stored in carbonate buffer at �80 �C.

Analytical HPLC. Reaction mixtures and product isolated by pre-parative HPLC were analyzed using a Waters Alliance 2690 HPLCsystem equipped with a 996 photodiode array detector. The sepa-ration column was a Zorbax SB-C18 HHRT (4.6 � 150 � 4.6 mm i.d.,1.8vlm, Agilent Technologies, Santa Clara, CA, USA) protected witha guard column containing C18 packing. Solvents A (5 mM phos-phate buffer, pH 7.0) and B (acetonitrile) were applied as follows:flow 0.70 ml/min, t 0 min, B 0%; t 5 min, B 0%; t 20 min, B 20%; t23 min, B 20%; and t 24 min, B 0%. The sample injection volumewas 5 ll, and the column was maintained at 40 �C. Compoundswere monitored at 590 and 280 nm.

Preparative HPLC. Reaction mixtures were filtered through a 0.45-lm syringe filter and injected onto a preparative Shimadzu HPLCsystem composed of two LC-8A pumps, an SIL-10AP autosampler,a CTO-20A column oven, an SPD-20A (dual wavelength ultraviolet

Quantification of folate in fruits and vegetables: A fluorescence-based / H. Martin et al. / Anal. Biochem. 402 (2010) 137–145 139

[UV]/visible) detector, an FRC-10A fraction collector, and a CBM-20A controller. The separation column was a Gemini-NX(21.2 � 150 mm i.d., 5 lm, Phenomenex, Torrance, CA, USA) withsolvents A (10 mM carbonate buffer, pH 10.4) and B (acetonitrile)applied as follows: t 0 min, B 4%; t 1 min, B 4%; t 8 min, B 20%; t12 min, B 50%; and t 15 min, B 4%. The column was maintainedat 30 �C. The injection volume was 500–1000 ll, and productswere monitored at 590 nm (folate–EDA–Alexa 594) and 280 nm(folate–EDA, folate–EDA–Alexa 660, and folate–EDA–FITC. Frac-tions were collected separately from multiple chromatographicruns, and like samples were combined, evaporated by rotary evap-orator (45 �C), and dried under vacuum.

FP and FI measurementAll fluorescence readings were performed using the Tecan Sa-

fire2 fluorescence microplate reader (Tecan, Grödig, Austria) at22 �C in a volume of 20 ll. For measurement of FP, excitation/emis-sion wavelengths of 470/525, 590/625, and 635/695 nm were usedfor fluorescein, Alexa 594, and Alexa 660, respectively. In each case,a bandwidth of 10 nm was set. For FI wavelength scans, a fixed gapof 40 nm was set between excitation and emission with a 10-nmbandwidth.

Fluorescent displacement assay procedureThe inhibitor was allowed to equilibrate in the microplate well

with FBP for 15 min prior to the addition of the fluorescent probe.After a further 15 min, when the fluorescence readings had stabi-lized, final fluorescence readings were taken.

Sample preparationHomogenization and assay buffer consisted of 100 mM Hepes,

0.1% 2-mercaptoethanol, 0.2 mg/ml BSA, and 1% sodium ascorbate(pH 7.4). Samples of fruits and vegetables were diced and mixedwith assay buffer in a ratio of 1:4 (w/v) and were manually homog-enized in a Dounce homogenizer. After 5 min of sonication at max-imum power using a Soniclean model 250T sonicator (Soniclean,Thebarton, South Australia), samples were heated to 95 �C for10 min in sealed microcentrifuge tubes. A stepwise description ofsample preparation time and assay time is shown in Table 1.

Size exclusion chromatographySamples (100 ll) containing 10 nM Alexa–folate were chro-

matographed on a Superdex 200 HR10/300 column equilibratedwith assay buffer containing 0.02% Tween 20 using an Äkta Ex-plorer system (Pharmacia). Fractions (200 ll) were collected inblack 96-well microplates. The column was calibrated using BSA

Table 1Stepwise sample preparation and assay time.

Step Description Time (min)

1 Preparation of buffers and reagentsfrom stock solutions

10

2 Sample weighing 23 Homogenizationa 34 Sonication 55 Heating 106 Robotic dilution of sample/5MTHF

standard in microplate10

7 Robotic addition of FBP 108 Equilibration of FBP with sample folate 159 Robotic addition of Alexa fluor 10

10 Binding of Alexa fluor to FBP 1511 Reading microplate fluorescence 5

Total time 100

a For high throughput, Ultra-Turrax homogenization is recommended.

(MW = 66,400 Da), bovine trypsin (MW = 23,300 Da), and chickenegg white lysozyme (MW = 14,300 Da).

Amylase treatmentType II-A a-amylase (product A6380, Sigma–Aldrich) from

Bacillus sp. was used for amylase treatment of extracts (activityP1500 U/mg protein). Freeze-dried kiwifruit homogenate(200 mg) was weighed out and homogenized in 8 ml of 50 mMphosphate buffer (pH 7.0), 1.0% sodium ascorbate, and 0.1%2-mercaptoethanol. Then 4 mg of amylase was added, and thesample was incubated at 37 �C for 30 min before the amylasewas inactivated by heating to 100 �C for 10 min. Control sampleswere treated in an identical manner except that they received noamylase.

Calculations and statistical analysisMillipolarization (mP) values were calculated from the follow-

ing equation:

Millipolarization ðmPÞ ¼ 1000 � ½ðIS� IPÞ=ðISþ IPÞ�;

where IS is the parallel emission intensity and IP is the perpendicu-lar emission intensity. In our assays, the IP value was adjusted by acorrection factor (G-factor) of 1.176 for fluorescein, 1.185 for Alexa594, and 0.802 for Alexa 660 to compensate for inequalities in thetransmission of light through parallel and perpendicular polarizers.

All statistical analyses (sigmoidal curve fitting and IC50 esti-mates) were performed with Origin software (version 7.5, Origin-Lab, Northampton, MA, USA). Of the assortment of equationsavailable for fitting in the Origin graphic package, the logistic equa-tion was empirically chosen because it fitted well to the experi-mental data sets:

y ¼ A2þ ðA1� A2Þ=½1þ ðx=x0Þp�

where A1 = initial (maximum) Y value, A2 = final (minimum) Y va-lue, p = power and x0 = centre (IC50). The Y value at x0 is half waybetween the two limiting values A1 and A2. Each data point was theaverage of at least two determinations. Data presented are repre-sentative of at least two independent experiments.

Results

The development of a fluorescence-based assay of folate re-quires the use of fluors whose emission maxima are distinct fromthe autofluorescence in the sample. Therefore, we scanned severalfruit and vegetable samples from 500 to 800 nm to determine theirautofluorescent profiles. The results revealed two typical profiles,as shown in Fig. 1. Green-colored fruits and vegetables, in this casegreen kiwifruit, tended to have the characteristic chlorophyll pro-file with minimal autofluorescence emission at 625 nm, whereasstrawberries and raspberries had minimal autofluorescence emis-sion above 690 nm. Therefore, we chose Alexa 594 and Alexa 660as fluorescent labels on folic acid to match these different samples.As shown in Fig. 1, both the green (kiwifruit) and red (raspberry)fruit have high fluorescent emission at 525 nm where fluoresceinemits. Thus, fluorescein–folate is unsuitable in these types of sam-ples. Kiwifruit, however, emits very little fluorescent signal be-tween 575 and 640 nm. For samples with this type of profile,Alexa 595–folate was chosen as a fluorescent label for folate be-cause its fluorescent signal at 625 nm is not compromised by sam-ple autofluorescence. Similarly, the fluorescent profile of raspberryis such that at 695 nm very little autofluorescence occurs. There-fore, Alexa 660 was chosen because it emits strongly at 695 nm.

Although 5MTHF is more appropriate as a competitive displace-ment ligand for fruits and vegetables, its insolubility and chemicalinstability led us to use folic acid instead for the preparation of a

OHO

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glutamate

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fluorescein isothiocyanate

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folic acid

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COOH

O

Fig. 2. Structures of fluorescent-folate ligands: (A) EDA–FITC–folate alpha; (B)EDA–FITC–folate gamma; (C) EDA–Alexa 594–folate gamma.

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fluorescein-folate alpha

fluorescein-folate gamma

Alexa-594-folate gamma

Alexa-660-folate gamma

Alexa-594-folate gamma (mP)

Fig. 3. Changes in FI and FP occurring on binding of fluorescent ligands to FBP:fluorescein–folate alpha (h); fluorescein–folate gamma ( ); Alexa 594–folategamma (D); Alexa 660–folate gamma (s); Alexa 594–folate gamma (mP) (d). Thetwo samples analyzed by gel filtration in Fig. 4 are indicated by arrows.

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kiwifruit

fluorescein

Alexa 594

Alexa 660

raspberry

Fig. 1. Kiwifruit ( ) and raspberry (N) extracts were scanned to reveal theirfluorescence emission profiles in comparison with three different fluorescentcompounds coupled to folic acid: fluorescein (h); Alexa 594 (d); Alexa 660 (D).

140 Quantification of folate in fruits and vegetables: A fluorescence-based / H. Martin et al. / Anal. Biochem. 402 (2010) 137–145

fluorescent form of folate. We prepared four fluorescent conjugatesusing EDA as linker between folic acid and the fluorescent probes:Alexa 594–EDA–folate gamma, Alexa 660–EDA–folate gamma,FITC–EDA–folate gamma, and FITC–EDA–folate alpha. We first pre-pared FITC–EDA–folate probes because of its low cost in compari-son with Alexa fluors and because the published method ofMcAlinden and coworkers describes the synthesis of FITC–folate[32]. Fig. 2 shows the structures of the Alexa 594- and FITC-labeledfolate ligands. The alpha and gamma linkages of the folate fluorswere confirmed by digestion with glutamate carboxypeptidase IIfollowed by binding to FBP; gamma linkages are susceptible tohydrolysis, but alpha conjugates are not (data not shown).

The fluorescent–folate conjugates were tested for their abilityto bind to bovine milk folate-binding protein. Fluorescent folateconjugate (5 nM) was titrated against increasing concentrationsof FBP in assay buffer at room temperature, and FP and FI readingswere taken every few minutes. These readings stabilized afterapproximately 15 min, indicating that equilibrium had beenreached. FI of fluorescein–folate also increases sharply on binding(Fig. 3), as noted by McAlinden and coworkers [32]. The gamma–folate conjugate shows substantially greater FI increase than thealpha–folate conjugate, presumably indicating that the interactionwith the hydrophobic pocket responsible for FI increase is favoredwhen the FITC is attached by the gamma linkage. FI increases werealso observed with Alexa 594 and Alexa 660, but the increaseswere more moderate using the Alexa–folate conjugates. The rela-tive increase in FI for the various conjugates were 6.0, 3.0, 2.5,and 1.5 for fluorescein–folate gamma, fluorescein–folate alpha,Alexa 594–folate gamma, and Alexa 660–folate gamma, respec-tively. Polarization increases on FBP binding occurred for each li-gand, indicating that the assay appeared to be feasible by both FPand FI.

Two anomalous but reproducible findings were apparent fromthe FP curve of Fig. 3. First, increasing concentrations of FBP didnot lead to the expected plateau of FP values, although FI valueshad ceased to increase. Second, the mP value of 110 for unboundligand was abnormally high in comparison with Alexa 594–biocy-tin, a molecule of very similar mass. The reason for this secondanomaly remains unclear. To explore these anomalies, the samplesfrom various points on the FP binding curve were analyzed by sizeexclusion chromatography on a Superdex 200 column. The result

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Alexa-594 biocytin (mP)

Vo

Vt

aggregated FBP

Fig. 4. FBP aggregation and anomalous mP value of Alexa 594–folate. Alexa 594–folate samples indicated by arrows in Fig. 3, corresponding to 8 and 2 lg/ml FBP, wereanalyzed by size exclusion chromatography on a Superdex 200 column. In addition, free Alexa 594–folate and Alexa 594–biocytin were run. The mP values of selectedfractions are shown above these fractions.

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Fig. 5. Comparison of folic acid, 5MTHF, 10-formyl folic acid, methotrexate, andaminopterin in a competitive binding assay. Alexa 594–folate was used at 5 nM, andFBP was used at 1 lg/ml. Inhibitors were premixed with FBP. After 15 min, Alexa594–folate was added, and readings were taken after a further 15 min. (A) FP. (B) FI.

Quantification of folate in fruits and vegetables: A fluorescence-based / H. Martin et al. / Anal. Biochem. 402 (2010) 137–145 141

(Fig. 4) shows that at the higher concentration of FBP (8 lg/ml),high-molecular-weight aggregates are visible running just behindVo with an elution volume of 8–12 ml, consistent with the observa-tion of Salter and coworkers [34] that concentrated FBP formsaggregate in the presence of folate ligand. The monomeric FBP–li-gand complex peak running at an elution volume of 17 ml has amass of 26.9 kDa. The quantity of Alexa 594–folate in each Alexa594–folate-containing sample was identical, and the greateramount of fluorescence in the FBP-containing samples is due tothe enhanced FI of FBP-bound fluor. A small quantity of unboundfluorescence is visible at Vt in the FBP-containing fractions, reflect-ing an impurity in the fluorescent–folate preparation. The unboundAlexa 594–folate running with an elution volume of 21 ml, slightlyahead of Vt, has a mass of 1193 Da.

The high mP value of the folate–Alexa 594 is not matched by asimilar high molecular weight on gel filtration. In a study of avidinbinding by an Alexa 594–biocytin conjugate, the fluorescence life-time, spectral maximum, and quantum yield of the fluor were af-fected by the linker used for coupling [35]. Conceivably, theproximity of the EDA–linker/folic acid moieties to the Alexa 594might reduce the lifetime of Alexa 594, thereby imparting an in-crease in polarization that is not related to molecular mass.

The fluorescent–folate conjugates were tested in a displace-ment assay using a selection of folate biologically active analoguesas competitive inhibitors of binding of the labeled folate. The re-sults, shown in Fig. 5 using Alexa 594–folate as the probe, con-firmed that the fluorescent probe is displaced effectively by folicacid, 10-formyl folic acid, and 5MTHF. Both of the dihydrofolatereductase inhibitors, methotrexate and aminopterin, had roughly1000-fold higher IC50 values for FBP than folic acid, indicating thatthe assay cannot be applied to measurement of these drugs in bio-logical samples. In a separate assay (inhibition curves not shown),folinic acid (5-formyl THF) and THF were assayed, and the IC50 dataare combined and summarized in Table 2. The published affinitiesof some natural folates and folic acid, determined by SPR [36], arealso shown in this table.

A major problem in the established folate assays is the variabil-ity in the length of the polyglutamyl side chain. We compared aseries of folic acid forms of increasing polyglutamyl chain length.Mono-, tri-, penta-, and heptaglutamyl folic acid were assayed fortheir ability to inhibit binding of the fluorescent Alexa 594 probe.

Table 2Comparison of IC50 values for folate analogues tested in Fig. 5.

FP (nMIC50 ± SD)

FI (nMIC50 ± SD)

Biacore (SPR)affinity (pM) [36]

Folic acid 6.2 ± 0.07 5.2 ± 0.21 205MTHF 15.0 ± 0.18 9.9 ± 0.33 20005-Formyl THF 804 ± 33.5 302 ± 18.1 12,00010-Formyl folic acid 7.3 ± 0.16 5.5 ± 0.21 –THF 12.6 ± 0.58 9.5 ± 0.20 250Methotrexate 7063 ± 287 3358 ± 276 –Aminopterin 12,523 ± 764 4153 ± 889 –

Note. The IC50 values for THF and folinic acid are also shown. The IC50 data arecompared with the published affinities of these compounds determined by SPR. SD,standard deviation.

Table 3Influence of folic acid glutamyl chain length on inhibition of binding of Alexa 594–folate to folate-binding protein.

Folic acid, glutamyl chain length

1 3 5 7

FP (nM IC50 ± SD) 5.3 ± 0.3 5.0 ± 0.2 5.1 ± 0.2 5.1 ± 0.4FI (nM IC50 ± SD) 5.3 ± 0.4 4.9 ± 1.0 4.6 ± 1.0 5.2 ± 1.0

Note. SD, standard deviation.

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µg folate/100 g (USDA)

Fig. 6. (A) Comparison of selected fruit and vegetable extracts in an FP assay. FBPwas present at 1 lg/ml, Alexa 594–folate was used at 5 nM. The raw data are shownuncorrected for background FP. (B) Estimates of folate content of a selection of fruitsand vegetables. The FP and FI estimates are compared with U.S. Department ofAgriculture (USDA) published estimates [37].

142 Quantification of folate in fruits and vegetables: A fluorescence-based / H. Martin et al. / Anal. Biochem. 402 (2010) 137–145

The data (Table 3) show that no significant difference in IC50 valuescould be discerned in the polyglutamyl series.

a-Amylase is routinely used to prepare sample of fruits andvegetables for folate analysis because it digests the carbohydratematrix in which the folate is enmeshed. We compared the appar-ent folate content of a sample of kiwifruit that had been subjectedto the standard a-amylase treatment with a control sample receiv-ing no a-amylase treatment. The data show that amylase treat-ment of kiwifruit did not cause any detectable alteration in thefolate available to FBP (Table 4).

The applicability of the assay to a selection of fruits and vegeta-bles was then tested. Fig. 6A shows the dose–response curves forbroccoli, carrot, feijoa, orange, and tomato. (Feijoa (Feijoa sellowi-ana), also known as the pineapple guava or the guavasteen, is afruit of the myrtle family originating in South America and nowalso commonly cultivated in New Zealand.) A standard curve wasprepared using 5MTHF for FP and FI. The IC50 values derived formeach fruit were then compared with the standard curve, allowingthe folate content of each fruit to be estimated. A comparison ofthe fluorescence scans of the fruit showed that raspberry andstrawberry are unsuited to the use of Alexa 594 (Fig. 1), and theywere instead analyzed using Alexa 660. The results are comparedby both FP and FI with data published by the U.S. Department ofAgriculture [37] (Fig. 6B). The variability in folate levels that mayoccur due to climate, storage, cultivar, and the like means that thiscomparison can be only loosely approximate. For example, thecontent of folates in raw tomatoes ranged from 4.1 to 35.3 lg/100 g fresh weight [38], whereas a study of folate in nine straw-berry cultivars revealed a 7.5-fold variation in folate content from12.8 to 96.0 lg/100 g fruit [39].

Interference from fluorescence in the sample may affect the fo-late estimate. For folate-rich samples, the background fluorescence

Table 4Effect of amylase treatment on folate estimation in Actinidia deliciosa (green kiwifruit)based on FP data.

A. deliciosa sample dilution to achieve IC50 SD

No treatment 579 29.1Amylase treated 606 17.4

Note. SD, standard deviation.

can be diluted out until autofluorescence is negligible while the fo-late in the sample is still sufficiently high to cause maximal inhibi-tion of binding. However, in some samples, the backgroundfluorescence impinges on the signal from the fluorescent ligandat the higher extract concentrations of the dose–response curve.So long as the majority of the fluorescent signal comes from thefluorescent ligand, the sample (background) fluorescence in paral-lel and perpendicular planes can be subtracted from the readingstaken in the presence of the fluorescent ligand. Fig. 7 shows theuse of Alexa 660–folate to generate an IC50 from an extract ofstrawberry. Background correction of the readings adjusts the po-sition of maximal inhibition to the same value as the unboundAlexa 660.

To confirm that 5MTHF present in an extract is not masked byendogenous factors and is available to FBP, whole unprocessedhomogenates of broccoli and strawberry were spiked with 5MTHFto bring the final concentrations of spiked 5MTHF to 100 and50 nM, respectively. After the addition of 5MTHF, the spiked sam-ple and unspiked control samples were mixed and left for 2 h at4 �C in sealed Eppendorf tubes. These samples were then analyzedby the FP method using Alexa 594–folate for broccoli and Alexa

0

100

200

300

400

0.001 0.01 0.1 1 10µl Strawberry extract / well

mP

Val

ue

0

20000

40000

60000

80000

Flu

ores

cenc

e in

tens

ity

background corrected mP (strawberry)

uncorrected mP (strawberry)

unbound Alexa-660 folate mP

fluorescence intensity (strawberry extract)

fluorescence intensity (strawberry extract + Alexa-660 folate + FBP)

Fig. 7. Use of Alexa 660 for analysis of strawberry extract. Parallel and perpendic-ular fluorescence readings of the sample prior to the addition of the Alexa 660 folatecan be subtracted from the FP readings to correct for sample interference at highsample concentrations. h: strawberry extract, FBP, and Alexa 660–folate FP valueswithout background correction; d: strawberry extract, FBP, and Alexa 660–folateafter subtraction of the fruit extract fluorescence readings taken before Alexa 594addition; : FI of the fruit extract; D: fluorescence intensity of the fruit extract plusAlexa 594–folate. The mP value of the free Alexa 660–folate is designated by thegray line.

Table 5Effect of spiking with known quantities of 5MTHF on the IC50 of whole homogenatesof broccoli and strawberry based on FP data

nM 5MTHF

Spikedbuffer

Unspikedsample

Expected nMin spiked sample

Spikedsample

Broccoli 100.0 ± 4.65 183.6 ± 9.91 283.6 275.4 ± 14.58Strawberry 50.0 ± 6.22 64.2 ± 3.84 114.2 116.2 ± 5.96

90000

120000

150000

hyperbolic fit of fluorescein-folate-gammaEquation: y = A1*exp(x/t1) + y0R2 = 0.9958

inte

nsity

Quantification of folate in fruits and vegetables: A fluorescence-based / H. Martin et al. / Anal. Biochem. 402 (2010) 137–145 143

660–folate for strawberry. The data (Table 5) show that the esti-mated 5MTHF concentration in each extract is increased by the ex-pected amount (from 183.6 ± 9.91 to 275.4 ± 14.60 nM for broccoliand from 64.2 ± 3.84 to 116.2 ± 5.96 nM for strawberry).

We have not attempted to provide a limit of detection for thisassay because of the wide variation in folate and background fluo-rescence in the samples of fruits and vegetables. However, thecoefficient of variation for 5MTHF in assay buffer is 5.9% assayedby FP. The same samples of carrot and green kiwifruit were ana-lyzed by the FP assay and by the Australian National MeasurementInstitute (Melbourne, Australia) using the L. rhamnosus method[14,15]. The FP results for these samples were 17.6 lg folate/100 g carrot and 35.8 lg folate/100 g kiwifruit, whereas the Na-tional Measurement Institute values were 15.8 lg/100 g and47.0 lg/100 g, respectively.

0.01 0.1 10

30000

60000

Flu

ores

cenc

e

FBP concentration [µg/ml]

Fig. 8. Hyperbolic fit of fluorescein–folate gamma data (minimum to maximumintensity) from Fig. 3.

Discussion

The advantages of this solution-based fluorescent assay for fo-late in fruits and vegetables are its speed, simplicity, and applica-bility to a wide range of colored plant extracts. Laboratoriesequipped with fluorescence measurement capability will be ableto perform this assay without recourse to microbiological facilitiesor HPLC/MS equipment. However, because this assay cannot dis-tinguish between various forms of folic acid such as 5-MTHF, 10-formyl folic acid, and the polyglutamyl forms, the use of analyticalchemistry approaches will be required to differentiate these folate

analogues. Because the form of folate in plants is mainly 5MTHF[11], this degree of resolution might be unnecessary for manyapplications. It is also likely that other folate-binding receptorssuch as antibodies could also be used to distinguish folic acid from5MTHF. However, this assay cannot be applied to the quantifica-tion of folate in vegetables that have a significant proportion oftheir folate in the form of 5-formyl THF because the FBP used asa receptor will not bind this ligand appreciably. 5-Formyl THF rep-resents up to 43% of folate in spinach [40,41], 0–67% of folate in let-tuce varieties [11,40], up to 27% of folate in carrot [11,42], and from6% to 54% in peppers [11,43]. Thus, for these vegetables, this FP andFI method may greatly underestimate the folate content. Conceiv-ably, an antibody specific for 5-formyl THF might allow the quan-tification of this folate form by FP.

The shapes of the dose–response curves for FP and FI assays dif-fer markedly. FBP is known to bind its ligand in a multistep fashioninvolving a conformational change in FBP [44]. Initial binding islow affinity, but the final step leads to high-affinity binding thatis associated with high-intensity fluorescent intensity signal. McA-linden and coworkers accounted for the FI increase on the basisthat fluorescein falls into a hydrophobic pocket on FBP and so thisincreases the fluorescent signal [32]. Therefore, the FI dose–re-sponse curve is distorted and has a hyperbolic appearance ratherthan a sigmoidal appearance (Fig. 8). Because the mP value is cal-culated from the FI readings in each plane, it follows that the FPdose–response curve will itself be affected by the fact that thebound fluor disproportionately contributes to the unrotated (Is)signal and so raises the FP ratio to more than would be expectedfrom the actual ratio of bound-to-unbound fluor. However, boththe FP and FI estimates are based on reference to a standard curvethat takes this effect into account. The empirical nature of the esti-mate means that although the IC50 values of the standard curveand samples might not reflect the true IC50 in terms of the fractionof receptor occupied, they do function to quantify folate in practice.The logistic equation employed for curve fitting gives high R2 val-ues, typically 0.98 and above, when fitting mP and FI values forAlexa 594 and 660 fluors. However, the distorted nature of thefluorescein–folate gamma dose–response curve (Fig. 3) does notfit the chosen logistic equation. The steeper increase in FI aroundthe IC50 compared with the FP slope at IC50 (Fig. 3) means thatthe error associated with FI estimates tends to be somewhat higherthan the FP IC50 error. In addition, the ratiometric nature of FP con-fers an advantage over FI in terms of precision (Tables 2 and 3).

A simpler assay design would be to premix the FBP with the fo-late–fluor to minimize the number of pipetting operations. How-

144 Quantification of folate in fruits and vegetables: A fluorescence-based / H. Martin et al. / Anal. Biochem. 402 (2010) 137–145

ever, in practice, a significant portion of the binding does not ap-pear to be reversible. In addition, preincubation of sample withFBP improves sensitivity. Therefore, we recommend preincubationof sample with FBP for 15 min followed by the addition of the fluo-rescent probe. Fluorescence readings should be taken approxi-mately 15 min after probe addition when fluorescence hasstabilized.

Although the affinities of 5MTHF, THF, and folic acid for FBP dif-fer markedly (Table 1), the assay methodology tends to minimizethe differences in affinity. Preincubating the sample with FBP be-fore the addition of the fluorescent ligand maximizes detection ofunlabeled forms of folate, and the IC50 values do not reflect the trueaffinities of the unlabeled folate analogues because the assay mightnot allow sufficient time for full equilibration of all natural folateforms with the fluorescent ligand. Although the on-rates of 5MTHFand THF are similar, the off-rate of 5MTHF is roughly 5-fold fasterthan that of THF [36] The empirical nature of the assay tends tounderestimate this difference by relating the sample to a standardcurve prepared under the same conditions as the sample. In addi-tion, the reagents are used at concentrations substantially higherthan their affinities. Thus, the assay is more of a titration of theamount of folate than a measure of the affinity of the particularform of folate. However, as can be seen from Table 1 and 5-formylfolate is greatly underestimated by the FP and FI method.

Amylase treatment of kiwifruit did not affect the folate estimate(Table 3). This may be due in part to the fact that the fluorescencetechnique allows the use of very dilute suspensions of homogenatethat contain insoluble material. It is possible that the fine suspen-sion of fruit homogenate allows diffusion of folate and FBP withoutinterfering with the fluorescent signal. Thus, the fluorescence-based assay may be more permissive of the presence of insolublematerial than an HPLC or microbiological approach. We found thata substantial pellet formed when the kiwifruit homogenate wassubjected to high-speed centrifugation and that the supernatantcontained roughly 20% less folate than whole homogenate (datanot shown). This may be due to a weak binding of folate to the pel-leted material. Although amylase treatment of kiwifruit extract didnot increase the folate estimate, amylase treatment might be re-quired for other samples with a more robust carbohydrate matrixor higher carbohydrate content such as rice and peas.

The sample sizes required for this assay allow us to easily ex-plore different regions of fruits and vegetables for folate content.For example, we separated 10 kiwifruit seeds from kiwifruit pulpand compared the folate levels in each sample. The kiwifruit seedscontained 15% (w/w) more folate than the pulp, but seeds com-prised only 3% of the original sample weight (data not shown). Thistype of analysis may be worth undertaking in circumstances wherefolate may be present in a fruit but not bioavailable. However, inour seed extraction procedure, amylase was not used and, there-fore, our data may underestimate the true level of folate in seeds.

We have described an assay of folate that, due to its speed, lowcost, and simplicity, permits the high-throughput screening oflarge numbers of fruit and vegetable extracts to aid in selectionof high-folate cultivars.

Acknowledgments

We thank Ali Chancel Mimbi (Massey University, New Zealand)for his excellent technical assistance with FP and HPLC, and wethank Denis Lauren (New Zealand Institute for Plant and Food Re-search) for his kind gift of folic acid analogues.

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