Studies on the dehydrogenative polymerization of monolignol β-glycosides: Part 5. UV spectroscopic monitoring of horseradish peroxidase-catalyzed polymerization of monolignol glycosides

Holzforschung, Vol. 64, pp. 173–181, 2010 • Copyright � by Walter de Gruyter • Berlin • New York. DOI 10.1515/HF.2010.026

2010/113

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Studies on the dehydrogenative polymerization of monolignol

b-glycosides. Part 6: Monitoring of horseradish peroxidase-

catalyzed polymerization of monolignol glycosides

by GPC-PDA

Yuki Tobimatsu1,2, Toshiyuki Takano1,*, HiroshiKamitakahara1 and Fumiaki Nakatsubo3

1 Division of Forest and Biomaterials Science, GraduateSchool of Agriculture, Kyoto University, Kyoto, Japan

2 Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA

3 Research Institute for Sustainable Humanosphere, KyotoUniversity, Kyoto, Japan

*Corresponding author.Division of Forest and Biomaterials Science, Graduate Schoolof Agriculture, Kyoto University, Kitashirakawa-oiwakechoSakyo-ku, Kyoto, 606-8502, JapanPhone: q81-75-753-6255Fax: q81-75-753-6300E-mail: [email protected]

Abstract

Horseradish peroxidase (HRP)-catalyzed dehydrogenativepolymerization of guaiacyl (G) and syringyl (S)-type mono-lignol g-O-glucosides, isoconiferin (iso-G) and isosyringin(iso-S), which contain a hydrophilic glucosyl unit on g-posi-tion of coniferyl alcohol and sinapyl alcohol, respectively,was monitored by gel permeation chromatography coupledwith photodiode array detection (GPC-PDA). Contrary to theconventional dehydrogenative polymerization of monoli-gnols, the polymerization of the glycosides produces water-soluble synthetic lignins (DHPs) in a homogeneous aqueousphase. Taking advantage of this unique reaction system, themethod was developed to follow the changes of molecularweights in the course of DHP formations. Moreover, PDAdetection permits determination of oligomeric S-type qui-none methide intermediates (QMs) formed as stable transientcompounds during polymerization of iso-S. A detailed com-parison of the polymerization profiles revealed entirely dif-ferent behaviors of G- and S-type monomers. The datastrongly support the view that the low reactivity of oligo-meric S-type QMs impedes the formation of DHPs fromS-type monomers. In copolymerization of G- and S-typemonomers, it is conceivable that G-type phenolic hydroxylgroups serve as good nucleophilic reactants to scavenge S-type QMs resulting in efficient production of DHPs. As aconsequence, the present approach can be a powerful tool tostudy the in vitro dehydrogenative polymerization providingfurther mechanistic insights into lignin polymerization invivo.

Keywords: dehydrogenation polymer (DHP); dehydrogena-tive polymerization; gel permeation chromatography (GPC);horseradish peroxidase (HRP); lignin biosynthesis; monoli-gnol b-D-glucoside; photodiode array (PDA) detection; qui-none methide (QM); syringyl lignin.

Introduction

The last stage of lignin biosynthesis in plant tissues is gen-erally defined as a process of the enzymatic dehydrogenativepolymerization of monolignols such as coniferyl alcohol (G-alc), sinapyl alcohol (S-alc), and p-coumaryl alcohol (H-alc),giving rise to guaiacyl (G), syringyl (S), and p-hydroxy-phenyl (H) lignins, respectively. It is well established thatthis polymerization process is easily mimicked in vitro bythe enzymatic oxidative polymerization of monolignols lead-ing to lignin polymer models (dehydrogenation polymers,DHPs) (Freudenberg 1965). As reviewed by several authors(Brunow et al. 1998; Boerjan et al. 2003; Grabber 2005;Ralph et al. 2004, 2008), this versatile model system hasallowed various modifications of the polymerization param-eter to be studied in vitro and provided much information onthe relationship between the reaction environment and theresulting lignin structures in relation to lignin polymerizationin vivo.

However, the chemical structures of DHPs are generallydifferent from those of native lignins (Terashima et al. 1995,1996; Saake et al. 1996; Landucci et al. 1998). For example,DHPs are well reported to contain relatively small amountsof the b-O-4 interunit linkage, whereas that linkage is themost predominant and accounts for more than half amongthe various interunit linkages in native lignins. It is alsoreported that typical DHPs have lower molecular weightsthan those of isolated lignins such as milled wood lignins(MWL) (Higuchi et al. 1971; Faix et al. 1981; Tanahashi etal. 1982; Cathala et al. 2003). Although many factors affect-ing the in vitro dehydrogenative polymerization have beenraised, a satisfactory synthesis of DHPs resembling nativelignins is still challenging. This emphasizes the need for abetter understanding of the dehydrogenative polymerizationprocess.

Several authors investigated the time-course of the oxi-dative polymerization of monolignols or their analogs usingHPLC (Terashima and Atalla 1995; Aoyama et al. 2002;Sasaki et al. 2004), GC (Kobayashi et al. 2005), capillaryzone electrophoresis (Fournand and Lapierre 2001; Fournand

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Figure 1 Chemical structures of monolignols and monlignol glycosides.

Figure 2 Syringyl quinone methide intermediates (S-type QMs)formed in the dehydrogenative polymerization of the g-O-glycosideof sinapyl alcohol (iso-S) or sinapyl alcohol (S-alc).

et al. 2003), UV spectroscopy (Sterjiades et al. 1993; Taka-hama et al. 1996; Kobayashi et al. 2005; Hatfield et al. 2008)or matrix-assisted laser desorption/ionization time-of-flightmass spectrometry (De Angelis et al. 1996). However, cur-rent analytical data are generally on the chemistry of theproducts obtained in the initial stage of polymerization suchas monomer conversions or oligomerization reactions. Thereare few reports in which the whole process of the dehydro-genative polymerization of monolignols was monitored.

Recently, we investigated the enzymatic dehydrogenativepolymerization of monolignol g-O-glucosides wtriandrin (iso-H); isoconiferin (iso-G); isosyringin (iso-S) (Figure 1)x,which contain D-glucosyl units on the g-positions of mono-lignols (Tobimatsu et al. 2006, 2008a,b). Owing to the pres-ence of highly hydrophilic sugar units attached tomonolignols, the polymerization of the glycosides produceswater-soluble DHPs in a homogeneous aqueous phase,whereas the conventional polymerization of monolignolsyields water-insoluble DHPs in a heterogeneous reactionsystem.

Data in our previous studies reveal that this unique modelsystem based on the glycosides would be a powerful tool tomonitor DHP formation. Moreover, information about thewhole chemistry in the course of the homogeneous poly-merization mixtures can be obtained by techniques of solu-tion-state spectroscopy or liquid chromatography. In ourprevious report, UV spectroscopy was applied for the real-time monitoring of the HRP-catalyzed polymerization of theglycosides (Tobimatsu et al. 2008c). Importantly, ourapproach established the unexpectedly stable presence of S-type quinone methide intermediates (QMs, Figure 2) duringthe polymerization of S-type glycoside, iso-S, whereas nosuch stable intermediates were detectable during the polym-erization of G-type glycoside, iso-G. It has been recognizedthat the preparations of high molecular weight DHPs fromS-type monolignol and analogs are difficult (Freudenbergand Hubner 1952; Yamasaki et al. 1976; Higuchi et al. 1977;Faix and Besold 1978; Sterjiades et al. 1993; Weymouth etal. 1993; Yoshida et al. 1994, 1998; Aoyama et al. 2002;Kobayashi et al. 2005; Tobimatsu et al. 2008a). Data in ourprevious study demonstrate that the low reactivity of S-typeQMs possibly impedes the formation of DHP from iso-S aswell as from S-alc (Tobimatsu et al. 2008c).

In the present study, gel permeation chromatography cou-pled with photodiode array detection (GPC-PDA) is appliedto monitor the HRP-catalyzed polymerization and copoly-merization of G- and S-type glycosides, i.e., iso-G and iso-

S. GPC techniques are suitable to reveal the progressivechanges of molecular masses of oligomer and polymer typeintermediates during polymerization. Simultaneously, thePDA detection offers the UV absorption patterns of thepolymerization products, providing additional informationabout the structural changes during polymerization. Thepolymerization behaviors of iso-G and iso-S will be dis-cussed in terms of the polymerization efficiency and thereactivity of QMs as key intermediates.

Materials and methods

The monolignol glycosides, iso-G and iso-S were synthesized aspreviously reported (Takano et al. 2006). HRP (100 U mg-1) waspurchased from Wako Pure Chemical Co. (Osaka, Japan) and usedwithout further purification. Other chemicals were purchased fromNacalai Tesque Inc. (Kyoto, Japan) or Wako Pure Chemical Co. andused as received.

GPC-PDA monitoring of HRP-catalyzed

dehydrogenative polymerization

The HRP-catalyzed polymerization of iso-G or iso-S was carriedout as follows. Three solutions were prepared. Solution A: 2 mg ofHRP in 500 ml of 0.05 M phosphate buffer (pH 6.5); solution B:20 mmol of monomer in 2500 ml of the buffer; solution C: 2500 mlof hydrogen peroxide (24 mmol) aqueous solution. Solutions B and

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Figure 3 Remaining amounts of S-type QMs over a storage timeafter sampling from the HRP-catalyzed polymerization mixturesfrom iso-S determined by GPC-PDA. (For abbreviations see Figures1 and 2.)

C were simultaneously pumped to solution A at a constant rate of1.25, 2.5, or 15 ml h-1 (monomer addition time: 10, 60 or 120 min).Reaction mixtures (100 ml) were sequentially sampled, immediatelymixed with 900 ml of 0.1 M LiCl in N,N-dimethylformamide(DMF), cooled at 08C and subjected to the GPC-PDA analyses with-in 15 min after withdrawing from the reaction mixtures.

The HRP-catalyzed copolymerization of iso-G and iso-S was car-ried out in the same manner as described above, except the com-positions in solutions B and C. The mixture of iso-G and iso-S(iso-G/iso-Ss2/20, 5/20, 10/20, 15/20 mmol) in 2500 ml of 0.05 Mphosphate buffer (pH 6.5) was used for solution B. For solution C,a hydrogen peroxide (1.2 eq. mol for total monomers in solution B)in 2500 ml water was prepared. The addition rate of solutions Band C to solution A was set to 2.5 ml h-1 (monomer addition time:60 min).

The GPC-PDA analyses were performed on a Shimadzu LC-20ALC system (Shimadzu, Japan) equipped with a photodiode arraydetector (SPD-M20A; Shimadzu). Elution conditions: column TSKgel a-M (Tosoh, Japan); eluent 0.1 M LiCl in DMF; flow rate 0.5 mlmin-1; column oven temperature 408C; injection volume 20 ml. Con-ditions for PDA detection: cell temperature 408C; scan region260–400 nm; band width 4 nm; response 1280 ms. Molecularweight calibration is based on polystyrene standards (Shodex,Japan). The data acquisition and computation was done with LC-solution version 1.22 SP1 software (Shimadzu). Before injection,sample solutions were filtered through a 0.45 mm PTFE disposablemembrane (Advantec, Japan).

Stability of S-type QMs from iso-S

in the GPC-PDA monitoring

For quantitative determination of S-type QMs formed in the HRP-catalyzed polymerization of iso-S, the stability of S-type QMs inthe GPC-PDA monitoring was investigated. The solution (3 ml)consisting of 20 mM iso-S and 2 mg of HRP in 50 mM sodiumphosphate buffer (pH 6.5) was kept at 258C under stirring. Thepolymerization was initiated by adding 30 ml of a 0.816% hydrogenperoxide aqueous solution (24 mM) to the mixture. After 10 minreaction time, 100 ml of the reaction mixture containing S-type QMswas sampled, mixed with 900 ml of 0.1 M LiCl in DMF and storedat 08C under identical conditions as described above for polymeri-zation monitoring. At a given storage time after sampling (5, 15,30, 45, 60 min), 20 ml of the solution was injected into the GPC-PDA system. Three runs for each storage time were carried out. Aloss of S-type QMs during the storage was evaluated by the decreasein the GPC peak area detected at 344 nm. A quasi-linear decreasein the peak from S-type QMs with the storage time was observed.The peak area at storage time of 0 was obtained from extrapolationand the proportions of remaining S-type QMs are plotted againstthe storage time in Figure 3. As a result, the loss of S-type QMs at15 min storage time was estimated to be 0.4% (the standard devi-ations were around 2%). This confirms that S-type QMs were semi-quantitatively determined by the presented monitoring experimentsbecause the sample storage time in the monitoring experiments waswithin 15 min, as described above.

Results and discussion

GPC-PDA monitoring of the polymerization

of iso-G

Dehydrogenative polymerization of iso-G initiated by HRPwas carried out under similar conditions as described pre-

viously for the syntheses of DHPs (Tobimatsu et al. 2006).Aqueous solutions of the monomer and hydrogen peroxidewere added simultaneously to a buffer (pH 6.5, 258C) con-taining HRP. At a given reaction time, an aliquot of thehomogeneous reaction mixtures was sampled and subjectedto GPC-PDA. It was preliminary confirmed that isolatedDHPs from the glycosides are completely soluble in theDMF eluent in the range of concentrations used in the pres-ent work. Therefore, it can be stated that whole polymeri-zation mixtures were accessible in the GPC-PDA analyses.

Figure 4a shows GPC-PDA profiles of iso-G polymeri-zation (monomer addition rate: 2.5 ml h-1). In the profile atthe reaction time of 0 min, the strong absorptions from iso-G at 272 and 300 nm are visible, whereas the absorptionmaximums were observed at 274 nm in the profiles at thereaction time of 30 and 180 min (Figure 4a2). As shown inthe GPC profiles, the elution peaks shifted to higher molarmass regions as reaction time progressed, indicating thegrowth of DHP chains (Figure 4a3). In Figure 5, the weight-and number-average molecular weights (Mw and Mn) – cal-culated based on the elution chromatograms at 274 nmdetection – are plotted against the polymerization time forDHPs with different monomer addition rates. In all the reac-tions, the values of Mw and Mn increased rapidly at the initialstage of the polymerization and further increased graduallyat later stages. The slow addition of the monomer solutionincreases the molecular masses. These results support thewell-known and generally accepted facts that molecularmasses of DHPs prepared by the ‘‘end-wise’’ polymerizationmethods (slow addition of monomers) are higher than thoseof DHPs prepared by the ‘‘bulk’’ polymerization method(simultaneous reaction of all reactants) (Sarkanen 1971;Cathala et al. 1998; Tobimatsu et al. 2006). The results ofGPC-PDA monitoring of iso-G polymerization confirm thatpolymerization proceeds without formation of stable (lowmolecular) intermediates.

GPC-PDA monitoring of the polymerization

of iso-S

HRP-catalyzed polymerization of iso-S was conducted underidentical reaction conditions to those described for iso-Gmonomers. However, the polymerization profiles of iso-S

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Figure 4 GPC-PDA profiles of HRP-catalyzed polymerization of iso-G (a), iso-S (b), and copolymerization of iso-G and iso-S (c).Monomer addition rate: 2.5 ml h-1. The reaction time 0 is defined as the time at which the monomer addition was started.

Figure 5 Product molecular weights calculated based on GPCchromatograms detected at 274 nm during the course of HRP-cat-alyzed polymerization of iso-G. Mw, weight average molecularweight. Mn, number average molecular weight.

(Figure 4b) are different from those of iso-G, a finding whichconfirms the observation made in our previous studies. Asshown by the UV spectra at the elution maximums (Figure4b2), the absorptions from iso-S at 279 nm disappear afterinitiating the polymerization, indicating a complete conver-sion of iso-S into other compounds. The formation of phe-nolic intermediates can be followed by absorptions at 274 nmand characteristic temporary absorptions at 344 nm from rel-atively stable S-type QMs are observable, as evidenced inour previous study (Tobimatsu et al. 2008c). The GPC meas-urements permit the determination of the phenolic interme-diates and S-type QMs separately, based on the maxima at274 nm and 344 nm, respectively (Figure 4b3). The calcu-lated molecular weights of the products formed areplotted against the reaction time in Figure 6. As reaction timeprogressed, the molecular weights of phenolic intermediates(274 nm detection) increased, but these data are much lowerthan those observed in iso-G polymerization experiments(Figure 6a). The monomer addition rate, at least in the rangeinvestigated, shows little effect on the growth of polyphe-nolic chains in the polymerization of iso-S. The molecularweights calculated based on 344 nm detection were even

lower than those of phenolic intermediates based on 274 nmdetection and remained almost constant around Mns1200,which is corresponding to degree of polymerization(DP)sca. 3 (Figure 6b). This clearly indicates that oligo-meric S-type QMs with less reactivity accumulate signifi-cantly during iso-S polymerization. The accumulation ofthese products can be determined by the quantitative evalu-ations of GPC chromatograms detected at 344 nm (Figure7). Preliminary experiments demonstrated that the loss of S-type QMs after sampling is negligibly small (0.4%) underthe present analytical conditions (Figure 3). Accordingly, theS-type QMs reached a maximum shortly after the reactionstarted and decreased very slowly as reaction time pro-gressed. Although slow addition of the monomer solutionsuppressed accumulation of S-type QMs at the initial stageof polymerization, still high levels of S-type QMs accumu-lations were observed in all polymerization experiments withiso-S.

These findings support the results in our previous studies(Tobimatsu et al. 2008c). The low reactivity of S-type QMscan be explained by reduced positive charge density at thea-positions owing to the presence of two electron-donatingmethoxyl groups, and can also be related to the steric hin-drance for the formation of syringyl a-O-4 structures. It isreported that the analogous quinone methide, 2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone reacts very slowlyin aqueous media. Bolton et al. (1990, 1992) pointed out thatthis low reactivity is as a result of the lack of hydrogenbonding between the shielded oxo group and water mole-cules, suppressing charge separation of the quinone methide.The same is true for the low reactivity of S-type QMs duringthe dehydrogenative polymerization. Recently, a productionof DHP with high yield was observed in HRP-catalyzedpolymerization of sinapyl alcohol in the presence of stronglynucleophilic azide ion, which serves as a QM scavenger(Tobimatsu et al. 2008d, 2010). The findings of the presentpaper can be readily rationalized by the low reactivity of theS-type QMs, which considerably impedes the subsequentpolymerizations to DHP.

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Figure 6 Product molecular weights calculated based on GPC chromatograms detected at 274 nm (a) and at 344 nm (b) during the courseof HRP-catalyzed polymerization of iso-S. Mw, weight average molecular weight. Mn, number average molecular weight.

Figure 7 Plots of GPC peak area detected at 344 nm in HRP-catalyzed polymerization of iso-S.

GPC-PDA monitoring of copolymerization

of iso-G and iso-S

It has been recognized for many years that S-type monomerscan be converted to high molecular weight DHPs in the pres-ence of G- or H-type monomers (Freudenberg and Hubner1952; Schweers and Faix 1973; Tobimatsu et al. 2008b). Itis obvious that the presence of G- or H-type monomers great-ly enhances the enzymatic oxidation rates of S-type mono-mers, which is generally interpreted that the former act asradical mediators from enzyme to S-type monomers (Taka-hama et al. 1996; Aoyama et al. 2002; Fournand et al. 2003;Sasaki et al. 2004; Hatfield et al. 2008; Tobimatsu et al.2008b). In copolymerization experiments with iso-G and iso-S, the accumulation of S-type QMs was suppressed, andTobimatsu et al. (2008c) suggested that G-type monomersmight also serve as nucleophiles for S-type QMs in theircopolymerization. In other words, the oligomeric S-typeQMs rearomatize more rapidly in the presence of iso-G andthus a subsequent polymerization of the resulting phenolicintermediates becomes possible and DHPs with highermolecular weights arise.

To confirm this concept, the time dependent copolymeri-zation of iso-G and iso-S was monitored by GPC-PDA. Theconcentration of iso-S was set to constant and the concen-tration of iso-G was systematically varied. Therefore, thepotential amount of S-type QMs was the same in all mon-omer feed ratios.

Figure 4c displays GPC-PDA profiles of the copolymeri-zation products at 60 min reaction time. Formations of thephenolic intermediates and S-type QMs could be detectedseparately by their absorptions at 274 nm and 344 nm,respectively. As readily visible, the copolymerization profileswere much affected by the feed ratios. The molecularweights calculated based on PDA detection at 274 nm and344 nm are plotted against reaction time in Figure 8. Theincreasing iso-G content in the monomer mixtures contrib-uted to the increment of molecular weights of phenolic inter-mediates (Figure 8a). It is obvious that the directparticipation of iso-G plays an important role for the poly-merization of iso-S and accelerates the formation of DHPswith high molecular masses. The molecular weights calcu-lated based on 344 nm detection also increased systemati-cally with increasing iso-G content (Figure 8b). In addition,it was clearly observed that the GPC peak areas detected at344 nm were reduced with increasing iso-G content as shownin Figure 9. This indicates that accumulations of S-type QMsintermediates are suppressed in the presence of iso-G.

The data presented here fully support the concept sug-gested in our previous studies. It is obvious that QMs rea-romatize more rapidly in the copolymerization of G-type andS-type monomers than in the homopolymerization of S-typemonomers and leads to DHPs with higher molecular masses.Two mechanisms are conceivable for interpretation: (1) high-er reactivity of G/S-type QMs resulting from cross-couplingsbetween G- and S-type substrates and (2) high nucleophili-city of G-type phenolic hydroxyl groups towards S-typeQMs. Our previous study showed significantly high reacti-vity of a G-type phenolic compound toward S-type QMs andsupports the second explanation (Tobimatsu et al. 2008c).

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Figure 8 Product molecular weights calculated based on GPC chromatograms detected at 274 nm (a) and at 344 nm (b) during the courseof HRP-catalyzed copolymerization of iso-G and iso-S. Mw, weight average molecular weight. Mn, number average molecular weight.

Figure 9 Plots of GPC peak area detected at 344 nm in HRP-catalyzed copolymerization of iso-G and iso-S.

Obviously, the results of a copolymerization between G- andS-type monomers should be a high abundance of non-cyclica-aryl ether bonds in DHPs. Indeed, many authors demon-strated the predominant occurrence of non-cyclic a-arylethers in DHPs (Saake et al. 1996; Landucci et al. 1998;Ammalahti and Brunow 2000). Nevertheless, it is still ques-tionable to conclude that S-type QMs intermediates arescavenged via nucleophilic additions of G-type phenoliccompounds during lignin formation in plant cell (lignifica-

tion in vivo), because native lignins contain much less a-etherified b-O-4 substructures than non-etherified ones (Edeand Brunow 1992; Saake et al. 1996; Crestini and Argyro-poulos 1997).

Conclusions

GPC-PDA techniques have been applied to monitor theHRP-catalyzed dehydrogenative polymerization of mono-lignol glycosides. Working with water-soluble monolignolglycosides has the advantage of dehydrogenative polymeri-zation in a homogeneous phase. The GPC-PDA method issuited to follow the changes of the molecular weights in thecourse of DHP formations. In addition, a PDA detector per-mits a semi-quantitative determination of the oligomeric S-type quinonemethide intermediates (S-type QMs), which arerelatively stable and impede further polymerization.

Based on the results presented in this study and in previouspublications (Tobimatsu et al. 2008c,d, 2009), we proposethat the formation of unreactive oligomeric S-type QMs isresponsible for the low yield and low molecular mass ofDHP in typical dehydrogenative polymerization of S-typemonomers. It seems probable that G-type monomers quenchstoichiometrically the S-type QMs in copolymerization

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experiments and the molecular mass of the DHP becomeshigher. So far, the contributions of stable S-type QMs inDHP formations have not been the focus of much attention.Subsequent studies should focus on the reactivity of S-typeQMs under various polymerization conditions. Such studiesare expected to also provide new clues for understanding thefactors controlling lignin polymerization in vivo.

Acknowledgements

This research was supported by a Grant-in-Aid for Young Scientists(�20-2841) from the Japan Society for the Promotion of Science(JSPS).

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Received August 20, 2009. Accepted September 9, 2009.Previously published online January 14, 2010.