Directed evolution of a biterminal bacterial display scaffold enhances the display of diverse peptides

Bacterial cell-surface display systems coupled with quantitative screening methods offer the potential to expand protein engineering capabilities. To more fully exploit this potential, a unique bacterial surface display scaffold was engineered to display peptides more efficiently from the surface exposed C- and N-termini of a circularly permuted outer membrane protein. Using directed evolution, efficient membrane localization of a circularly permuted OmpX (CPX) display scaffold was rescued, thereby improving the presentation of diverse passenger peptides on the cell surface. Random and targeted mutagenesis directed towards linkers joining the native N- and C-termini of OmpX coupled with screening by FACS yielded an enhanced CPX (eCPX) variant which localized to the outer membrane as efficiently as the non-permuted parent. Interestingly, enhancing substitutions coincided with a C-terminal motif conserved in outer membrane proteins. Surface localization of various passenger peptides and mini-proteins was expedited using eCPX relative to that achieved with the parent scaffold. The new variant also permitted simultaneous display and labeling of distinct peptides on structurally adjacent C- and N-termini, thus enabling display level normalization during library screening and the display of bidentate or dimeric peptides. Consequently, the evolved scaffold, eCPX, expands the range of applications for bacterial display. Finally, this approach provides a route to improve the performance of cell-surface display vectors for protein engineering and design.
IntroductionDisplay methodologies have proven invaluable to the discovery, production and optimization of proteins and peptides in a variety of biotechnological applications. Various approaches including phage display (Smith, 1985), mRNA (Wilson et al., 2001) and DNA display (Yonezawa et al., 2003), ribosome display (Mattheakis et al., 1994; Hanes and Pluckthun, 1997), eukaryotic virus display (Muller et al., 2003; Bupp and Roth, 2002), yeast display (Boder and Wittrup, 1997) and bacterial display (Lu et al., 1995) have been developed to screen diverse molecular repertoires for desired activities. In particular, bacterial display libraries have enabled antibody affinity maturation (Daugherty et al., 2000), the discovery of protein-binding peptides (Bessette et al., 2004), the isolation of cell-specific ligands (Nakajima et al., 2000; Dane et al., 2006) and the identification of optimal protease substrates (Boulware and Daugherty, 2006). One of the key advantages of bacterial surface display is the ability to use flow cytometry for quantitative screening of the libraries, allowing for real-time analysis of binding affinity and specificity to optimize the screening process (Wittrup, 2001). Additionally, ease of genetic manipulation, high transformation efficiency and rapid growth rate make Escherichia coli a well-suited host for display. A broad range of bacterial surface display systems have been developed allowing for insertional or terminally fused peptides and proteins to be displayed on the cell surface. Several outer membrane proteins and cellular appendage proteins have been used to present polypeptides as insertional fusions (Charbit et al., 1986; Taschner et al., 2002; Bessette et al., 2004). Ice nucleation protein (Jung et al., 1998), intimins (Christmann et al., 1999) and LppOmpA (Francisco et al., 1992) have been used to display proteins on the C-terminus of a transmembrane scaffold while N-terminal display has been accomplished using autotransporters IgA1 protease (Maurer et al., 1997) and EstA (Becker et al., 2005).Recently, a unique bacterial display scaffold was developed that allows for both N- and C-terminal display from a circularly permuted variant of outer membrane protein OmpX (CPX) (Rice et al., 2006). This scaffold enabled display of peptides on both termini, but with reduced efficiency when compared to that obtained using insertions into OmpX. Reduced membrane localization of CPX may result from slower folding rates and reduced stability that has been described previously for circularly permuted proteins (Heinemann and Hahn, 1995). Regardless, reduced display efficiency makes it necessary to employ longer induction times to achieve sufficient display for screening via fluorescence-activated cell sorting (FACS). Importantly, inefficient display can also create an undesired selection pressure resulting in growth biases, reduced viability or differing levels of passenger localization on the cell surface. As a result, library screening based on cell fluorescence can favor passengers most efficiently localized to the surface, rather than passengers improved for the properties of interest (e.g. binding affinity).A variety of proteins have been circularly permuted to create novel proteins of desired topologies. For instance, Guntas et al. coupled a circularly permuted β-lactamase with maltose binding protein to create a switch in which β-lactam hydrolysis activity is increased in the presence of maltose (Guntas et al., 2005). Also, fluorescent proteins have been circularly permuted to create molecular sensors that respond to Ca2+ (Baird et al., 1999). In these instances, the point of circular permutation can have important consequences for protein function. Random circular permutations often yield inactive or unfolded variants (Graf and Schachman, 1996), and those variants retaining activity often fold more slowly and are less stable than the non-permuted parent (Lindqvist and Schneider, 1997). With the aim of rescuing the reduced surface localization of CPX relative to the non-permuted OmpX, we used targeted combinatorial mutagenesis and directed evolution to identify CPX variants that would localize passenger peptides to the cell surface with an efficiency equivalent to that of native OmpX. This strategy yielded a more robust display scaffold to present diverse peptides as C- and N-terminal fusions on the surface of bacteria.
ResultsCircularly permuted OmpX librariesWe previously reported a new type of protein scaffold for polypeptide display that allows for both N- and C-termini to be exterior to the cell, using circularly permuted outer membrane protein OmpX (CPX) (Rice et al., 2006). The CPX protein scaffold consists of the native OmpX signal sequence, which is cleaved after translocation; a sequence with an embedded SfiI restriction site (GQSGQ) after which peptides may be inserted; a flexible linking sequence (GGQSGQ); amino acids S54–F148 of the mature OmpX; a GGSG linker joining the native C- and N-termini; and finally, amino acids A1–S53 of the mature OmpX. To assess the extent of display, a disulfide-constrained streptavidin-binding peptide (SApep) with the amino acid sequence AECHPQGPPCIEGRK (Giebel et al., 1995) was fused to the N-terminus of CPX, allowing cell labeling using a fluorescently conjugated streptavidin probe and measurement of display levels using cytometry. CPX yielded a reduced level of peptide display when compared to cells displaying the same peptide presented as an insertion within the corresponding region of OmpX (Fig. 1). The reduced display ability requires longer induction periods to allow for sufficient fluorescence labeling and library screening, thereby causing cell stress that can result in growth biases during library screening (Daugherty et al., 1999).Fig. 1Display of a streptavidin-binding peptide using OmpX, CPX and eCPX. Flow cytometric analysis of E. coli displaying a streptavidin-binding peptide (AECHPQGPPCIEGRK) (Giebel et al., 1995) as an insertion into OmpX, an N-terminal fusion to CPX or an N-terminal fusion to eCPX after varying durations of induction. The cells were induced at room temperature for time increments between 0 and 90 min then labeled with 100 nM SA–PE.The CPX scaffold was constructed using an arbitrarily chosen flexible linker (GGSG) to join the native N- and C-termini. Thus, we reasoned that alternative linkers and point mutations within CPX could enhance the display of peptides on the cell surface. To improve the display characteristics of CPX, various regions of the transmembrane protein were targeted for mutagenesis. An optimized linker region joining the native N- and C-termini was identified by generating and screening four libraries allowing for three (3X), four (4X), five (5X) and six (6X) random residues to be inserted in place of the GGSG linker using the degenerate codon NNK. Each library was screened separately using FACS for clones exhibiting a high level of fluorescence after 50 min of induction, indicating increased display of SApep binding to streptavidin–R-phycoerythrin (SA–PE). Under these conditions, the parent CPX scaffold yielded display levels only slightly greater than background autofluorescence, making the selection of improved mutants efficient. After sorting, the display level of several clones was measured using cytometry, and their sequences were determined by DNA sequencing (Table I). Isolated clones exhibited 3- to 15-fold improved display compared to CPX after only a 30 min induction period. The identified linker sequences exhibited a preference for basic residues, and glycine was present at the first position of the linker in 14 of 16 clones characterized. In addition, four of the clones had unintended mutations preceding the native C-terminus, two with the substitution A165V and two others with G166S. Interestingly, these four clones were among the most improved display scaffolds isolated. The average display level of the selected clones from each library increased with increasing linker length, and was highest for 5- and 6-mer linker clones.Table IMutations improving CPX surface localizationClonePositionsLinkerRelative display level165166CPXAGGGSG2.3a/1.5bThree residue linker library CPX-3X-1AGGRK8.9a CPX-3X-2AGGRK8.3a CPX-3X-3AGGTK7.1a CPX-3X-4AGGKK10aFour residue linker library CPX-4X-1AGGSKR18a CPX-4X-2AGGRQK14a CPX-4X-3AGSWPN15a CPX-4X-4VGPRKS22aFive residue linker library CPX-5X-1AGGRTRK24a CPX-5X-2AGGRKRN22a CPX-5X-3VGGATRR32a CPX-5X-4ASGSQSK36aSix residue linker library CPX-6X-1AGGTKRYH35a CPX-6X-2AGGRRHYK28a CPX-6X-3AGGNRRHR24a CPX-6X-4ASGSKQSK38aSecond generation library CPX-L2-1LSGSKSRR33b CPX-L2-2FSGRKNSH19b CPX-L2-3ISGTRGSQ29b CPX-L2-4LSGHRSHR27b CPX-L2-5ISGDRKRR28b CPX-L2-6VAGARGRH24b CPX-L2-7VSGTHNSQ26b CPX-L2-8VSGPNKSR17b CPX-L2-9ISGPHNSR23b CPX-L2-10ISHRGYHAQR33baFold fluorescence above background after 50 min of expression; bfold fluorescence above background after 25 min of expression.In an attempt to further enhance peptide display, the amino acid linker that joined the passenger peptide to the N-terminus of the display scaffold was also targeted for mutagenesis and screening for improved variants. A library was created in place of the original linking sequence GGQSGQ by randomizing these six residues. Screening yielded four clones exhibiting a 10-fold improved display when compared with CPX. Sequencing did not reveal a consensus within the target linker region. Instead, clones with enhanced display possessed a non-targeted substitution of either A165V or G166S. Since the randomly selected library members from the initial pool did not possess mutations outside of the intended region, these advantageous substitutions were rare and likely arose from PCR errors. In parallel, a library was generated with random residues at the surface exposed C-terminus of CPX, since native outer membrane proteins (Omps) possess a conserved C-terminal motif thought to aid in Omp membrane insertion or assembly (Bos and Tommassen, 2004). Four clones were isolated from this library that exhibited improved peptide display. Again, these variants did not share consensus in the randomized region, but each carried the spontaneous mutation G166S. These results suggest that the amino acid compositions of the new termini derived from circular permutation have little effect on the rate of assembly and display of CPX, whereas residues A165 and G166 play a key role in proper translocation and insertion of the protein.A final library was designed to combine the display-enhancing mutations identified within the most improved clones from the first generation libraries. A six residue linker library was chosen to identify optimal linkers to connect the native N- and C-termini, since longer linkers appeared to improve display (Table I). After 50 min of induction, individual library members exhibited higher display levels on average when compared with clones with five amino acid linkers (Table I). The first amino acid of the linker was fixed to glycine since it was highly conserved in the isolated clones. The third and sixth positions were restricted to R/K/S/H/Q/N using the codon MRM, given the increased frequency of these residues at the proposed positions in clones with improved function, and the remaining three positions were fully randomized. Positions A165 and G166, where beneficial substitutions were observed, were also fully randomized. Improved variants were identified using two rounds of MACS followed by two rounds of FACS, sorting clones exhibiting the highest display of the SApep after 30 min of induction. Ten clones were isolated after the final FACS screening (Table I). All variant scaffolds identified possessed a more bulky hydrophobic residue (I/L/V/F) in place of alanine at position 165, a consensus for serine at position 166 and a high frequency of basic residues Arg and Lys within the linking region. The display enhancing substitutions A165V and G166S are located immediately upstream of the native C-terminus of OmpX (Fig. 2).Fig. 2Locations of enhancing mutations within eCPX. Image was created using Molecular Operating Environment using the reported structure of OmpX (Vogt and Schulz, 1999), joining the N- and C-termini with the six residue linker GSKSRR, and creating the new termini within the second extracellular loop. Enhancing substitutions A165L and G166S are shown as space filling residues.Expression characteristics of optimized CPXThe scaffold variant exhibiting the most improved display characteristics, CPX-L2-1, or enhanced CPX (eCPX), was then compared to parental OmpX and CPX scaffolds. The cell-surface display level of SApep was measured at incremental times after induction of expression for these three scaffolds. This peptide was displayed either at the N-terminus (CPX and eCPX) or as an insertional fusion within the second extracellular loop of OmpX. The level of display was measured before and after 30, 60 and 90 min of induction using flow cytometry (Fig. 1). The display rate of eCPX was substantially improved relative to that of CPX, and even slightly higher than that of OmpX. After only 30 min of expression, the level of display of eCPX-nSApep was 50-fold above background autofluorescence. Introducing A165L and G166S into OmpX resulted in nearly identical display of SApep relative to that obtained with OmpX (data not shown).To determine whether the enhanced display using the eCPX scaffold was a general effect or specific to the streptavidin binding peptide SApep, several unrelated passenger peptides were fused to the N-terminus of CPX and eCPX and their display levels were compared (Fig. 3). Surprisingly, a disulfide-constrained peptide binding to C-reactive protein (CRPpep) (EWACNDRGFNCQLQR) identified by bacterial display (Bessette et al., 2004) displayed with eCPX yielded nearly 50-fold higher florescence labeling than that for CPX, after only 30 min of expression. In fact, the fluorescence of cells displaying CRPpep from CPX could not be distinguished from background (Fig. 3). Similarly, the T7•tag epitope (T7pep) (MASMTGGQQMG) was displayed more efficiently by eCPX than by CPX. A disulfide-constrained 19-mer peptide binding to vascular endothelial growth factor (VEGF) identified previously using phage display (Fairbrother et al., 1998) was also displayed over 3-fold more efficiently within eCPX. Additionally, an IgG binding mini-protein [a minimized version of the Z-domain from protein A (Braisted and Wells, 1996)] composed of 33-amino acids that form two antiparallel α-helices exhibited a display level roughly 3-fold higher than that from CPX after a 2 h induction period. Finally, P2, a proline-rich peptide (PAPSIDRSTKPPL) known to bind to the C-terminal SH3 domain of Mona (Harkiolaki et al., 2003), was expressed as a C-terminal fusion using both CPX and eCPX. Similar to the improvements of display at the N-terminus, the display level after only 30 min of expression of P2 using eCPX was improved by 9-fold compared to display with CPX. Thus, for all peptides investigated, the eCPX scaffold increased display levels when compared to the parental CPX.Fig. 3Display of various peptides and mini-proteins using eCPX (shaded) and CPX (white) as measured using flow cytometry. The X-axis indicates the fold fluorescence above background for each protein target in the corresponding fluorescent channel. P2 was labeled with Mona, which is fused to the fluorescent protein YPet. CRPpep and V114 were labeled with biotinylated CRP and VEGF, respectively, then labeled with SA–PE. Mini-Z and T7pep were labeled with Alexa488 -conjugated human IgG and anti-T7•tag monoclonal IgG, respectively. SApep was detected with SA–PE.Since the increased expression of the scaffold could potentially result in a reduction in cell viability, the viability of E. coli populations displaying two different peptides was measured as a function of the duration of induction of expression. The IgG binding mini-Z helix was selected since it exhibited reduced display in the improved eCPX scaffold. Viability was measured at 30, 60 and 90 min after induction using CFU assays and compared to that for parental strain (MC1061/pBAD33) lacking an expression cassette. Under ordinary display conditions (i.e. induction for less than 60 min), a measurable loss in viability could not be observed. After prolonged induction (90 min), cells expressing the IgG binding minihelix (miniZ) exhibited a 15% loss of viability (data not shown), while cells displaying SApep exhibited viability indistinguishable from that of cells that do not express the scaffold (MC1061/pBAD33).Biterminal display using eCPXTwo distinct peptides were simultaneously displayed on the structurally adjacent N- and C-termini of eCPX. SApep was fused to the N-terminus, and the P2 peptide fused to the C-terminus (eCPX-nSApep-cP2). Labeling with fluorescent probes SA–PE (red) and YPet-Mona (Nguyen and Daugherty, 2005) (green) enabled independent detection of each peptide using flow cytometry. To determine the ability of eCPX to simultaneously display these two peptides, cells expressing the biterminal display scaffold were labeled with SA–PE only, YPet-Mona only or both probes concurrently. If the peptides bound to their respective receptors independently (i.e. without any steric clashes), there should be no difference between the extent of single color labeling (fluorescence intensity) of the sample labeled with one probe and that labeled with both fluorescent probes simultaneously. However, simultaneous labeling of cells expressing a fusion protein of the form eCPX-nSApep-cP2 with SA–PE and YPet-Mona, or with each probe separately yielded differing extents of labeling, consistent with steric interference between these two large fluorescent probes (290 and 34 kDa, respectively). Specifically, the fluorescence of the cells when labeled with only one probe was always greater than the fluorescence in the corresponding channel of the cells when labeled with both probes simultaneously. In an attempt to reduce steric interference, a long flexible linker of the form (GGGS)5 was inserted between SApep and eCPX, resulting in a total linker length of 26 amino acids causing SApep to be further from the cell surface and thus increasing the distance between the two peptides. Using this long linker, independent labeling of each displayed peptide was improved (Fig. 4). These results indicate that the use of a long, unstructured linker can increase the accessibility of large proteins to peptides simultaneously displayed at both termini of eCPX, without substantially reducing the level of display.Fig. 4Flow cytometric measurement of simultaneous N- and C-terminal display (bi-terminal display). Overlays of two-parameter histograms resulting from clones displaying SApep and P2 on the N- and C-terminus, respectively, with a 6 (A) or a 26 (B) residue linker between SApep and the N-terminus of eCPX. In both (A) and (B) plots, the four distinct populations consist of non-displaying cells mock-labeled with SA–PE and Ypet-Mona (bottom left population), cells that display SApep and P2 labeled with only SA–PE (top left population), with SA–PE and YPet-Mona (top right population), or with only YPet-Mona (bottom right population).
DiscussionThe application of bacterial display technology to a broader range of protein engineering applications has been hindered by the absence of robust, validated display scaffolds. We recently reported a circularly permuted outer membrane protein X (CPX) having the unique characteristic that both C- and N-termini of the scaffold become localized on the bacterial cell surface (Rice et al., 2006). This ‘biterminal’ scaffold provides a means for expression normalization to improve affinity screening, analogous to yeast display (Boder and Wittrup, 1997), as well as a means to display dimeric peptides. However, the permuted scaffold exhibited reduced surface localization efficiency, precluding presentation of large peptides or the display of two unique peptides simultaneously from structurally adjacent termini. Here, semi-rational design and directed evolution were used to enhance the surface localization efficiency of CPX to improve the display of diverse peptides. To identify CPX scaffold variants with optimal linker sequences for joining the native C- and N-termini, four separate libraries with three, four, five or six random linker amino acids were screened. Improved scaffold variants revealed a preference for longer linkers of five to six residues, a strict consensus for glycine at the first position of the linker, and a high frequency of basic residues in the remaining positions. Unintended substitutions (A165V, G166S) near the native C-terminus of OmpX greatly increased the display level. These substitutions probably arose from rare errors introduced during PCR which were enriched from the large libraries (109). After directed evolution, the final variant with the most improved display (eCPX) carried substitutions A165L and G166S, with a linker sequence of GSKSRR.Two substitutions (A165L, G166S) identified to improve surface localization occur at the native C-terminus of OmpX and have a high degree of similarity to the C-termini of outer membrane proteins that lack periplasmic domains (Table II), even though these Omps share little identity. These results support previous observations that the C-terminal sequence serves an important role in membrane assembly and/or translocation (Struyve et al., 1991). The beneficial eCPX substitutions discovered could potentially enhance the ability of chaperones to recognize and assemble this topological variant. Interestingly, the extended C-terminal motif resulting from directed evolution (−L/VXYRFG) is conserved in OmpA homologs which utilize this motif in their last transmembrane β-strand that precedes a C-terminal periplasmic domain (Table II). Our results suggest that glycine is required to allow the internal, as opposed to terminal, YRF motif to interact with a putative chaperone. The improved linkers also include an increased frequency of charged residues that could serve to increase solubility and decrease aggregation to allow more protein to insert into the membrane.Table IIComparison of bacterial outer membrane proteins’ C-terminal sequences with eCPXOmp IDC-terminal sequenceaeCPXDVGTWILSVGYRFGSKSRRAT…NP_992891NIGTWVLGIGYRFYP_624929NPIVTFLSVGYRFYP_156535NPRTVSLSVGYRFYP_341424DPRTVSLTVGYRFZP_00792472TPVTFNLNVGYRFSequence of last membrane β-strand of OmpA homologsbEAQ20637LVSVGLIYRFGGKTQAPAEAP54071YWGLELSYRFGTPVAAAAAAN42586LLSLGVSYRFGQGEAAPVNP_992692LLSVGVSYRFGQEDAAAPZP_00826943MLSVGVSYRFGQDDVVAPaIdentities between eCPX (with substitutions A165L, G166S) and other bacterial outer membrane protein C-termini.bOmpA homologues with a periplasmic domain following the transmembrane strand exhibit a highly conserved glycine residue following the C-terminal YRF motif of transmembrane proteins.The evolution of an efficiently localized biterminal display scaffold (eCPX) provides several important new capabilities for peptide engineering. First, eCPX increased the display rate of various unrelated peptide fusions, on either the N- or C-terminus when compared to that achieved using the parental CPX. Increased display improves library screening efficiency using FACS and promotes the recovery of peptides that are otherwise difficult to display. For example, the display of CRPpep using CPX was only marginally detectable after 30 min of expression, but the same peptide displayed using eCPX provided a fluorescent signal 50-fold above background. If a peptide library displayed on CPX was screened for CRP binding peptides, this sequence and others like it may not have been isolated due to the difficulties in presenting this peptide using CPX. Though, it should be noted that excessive display levels, above that required for screening by FACS, should be avoided to minimize avidity interactions with target proteins. In fact, using eCPX, induction periods as short as 10 min can be sufficient for affinity screening of peptide libraries (unpublished observations). Importantly, quantifying the display level during library screening by labeling of a C-terminal peptide such as P2 allows fine affinity discrimination (VanAntwerp and Wittrup, 2000); that is, peptides with a high affinity but low display level can be differentiated from peptides with a high display but moderate affinity. Moreover, efficient biterminal display allows for the potential to generate peptide libraries on each terminus where both peptides can bind to distinct epitopes of a target protein, resulting in increased binding affinity and specificity through avidity. Finally, C-terminal display with eCPX should enable new applications such as screening for preferred PDZ C-terminal binding motifs (Harris and Lim, 2001).Engineering of the bacterial display scaffold eCPX yielded a circularly permuted transmembrane protein that has both termini presented on the exterior of the cell and inserts into the outer membrane as efficiently as the non-permuted parent. The amino acid sequence used to join the termini played a major role in the proper function of CPX, and two substitutions adjacent to the native C-terminus aided in display of peptides using eCPX. This novel protein scaffold substantially improves the robustness of bacterial surface display via CPX and, when combined with modern cell sorting instrumentation, creates new opportunities for peptide engineering. More generally, similar linker mutagenesis strategies coupled with efficient screening methods should prove useful for optimizing other display scaffolds and circularly permuted proteins.
Materials and methodsBacterial strains, reagents and plasmidsAll experiments were performed with E.coli strain MC1061 (F- araD139 Δ(ara-leu)7696 galE15 galK16 Δ(lac)X74 rpsL (StrR) hsdR2 (rK− mK+) mcrA mcrB1) (Casadaban and Cohen, 1980). All plasmid constructs utilize pBAD33 (Cmr) (Guzman et al., 1995), with the promoter araBAD operon and the p15A origin of replication (low-copy number). KOD Hot Start DNA Polymerase (Novagen) was used for PCRs. Primers were from Operon, restriction enzymes from New England BioLabs, SA–PE from Molecular Probes and streptavidin-coated magnetic microbeads (MyOne™ Streptavidin T1) were from Invitrogen. Qiagen mini-preps and gel extraction kits were used for DNA preparation. Ni-NTA agarose for protein purification was from Qiagen, and B-PER II bacterial protein extraction reagent was from Pierce Biotechnology.Vector and library constructionConstruction of circularly permutated OmpX (CPX) was described previously (Rice et al., 2006). To monitor the display level, a streptavidin binding peptide was fused to the N-terminus of CPX as described in earlier work; this plasmid was termed pB33CPX-nSApep. To generate the libraries that joined the original N- and C-termini of OmpX with three to six random residues, primers PD1237–1240 (see Supplementary data available at PEDS Online) were used (respectively) as the reverse primer and PD179 as the forward primer, with pB33CPX-nSApep as the template. Primers are listed in the supplementary material. The random positions were encoded using NNK codons allowing for all amino acids and the amber stop codon. The product of the PCR reaction was gel purified then used as a forward primer for the next reaction, using PD180 as a reverse primer, and again with pB33CPX-nSApep as the template. The product was then gel purified, digested with SfiI and gel purified again. The digested insert was ligated into the similarly digested vector pB33CPX-nSApep. Ligation products were desalted and electroporated into electro-competent MC1061 yielding 7.5 × 107, 7.5 × 107, 1.5 × 108, 5.0 × 108 transformants, respectively. To create the second generation library (CPX-directed), primers PD1282 and PD179 were used to randomize positions A165 and G166 using pB33CPX-nSApep as the template. The product was then used as a template for PCR with PD1281 and PD179, adding the second generation library residues. The primer encoded for G at the first position of the linker and used a restricted codon of MRM to encode for residues R, K, S, H, Q or N at positions 3 and 6 of the linker; the remaining positions used NNK. The product of the previous PCR reaction was then used as a forward primer for the next reaction, using PD180 as a reverse primer and pB33CPX-nSApep as the template. The product was then digested and ligated into the similarly digested vector pB33CPX-nSApep. Ligation products were desalted and electroporated into electro-competent MC1061 yielding 1.0 × 109 transformants.The various binding peptides were fused to the N-terminus of CPX and eCPX using a linker of GGQSGQS. PCR was used with pB33CPX-nSApep as the template, PD180 as the reverse primer and with forward primers PD1192/PD1193 for the CRP binding peptide (EWACNDRGF.NCQLQR) or PD961/PD962 for the VEGF binding peptide (VEPNCDIHVMWEWECFERL). The products were digested with SfiI and ligated into similarly digested vector and electroporated into MC1061. Primers PD1130–1133 were used in an assembly PCR to create the forward primer for the mini-Z-domain (FNMQQQRRFYEALHDPNLNEEQRNAKIKSIRDD). This primer with PD180 and template pB33CPX-nSApep was used in PCR. The product was digested with SfiI and ligated into similarly digested vector and electroporated into MC1061. The CPX-T7pep (MASMTGGQQMG) was created using overlap extension PCR with the products from the PCR reaction with primers PD179/PD705 and PD180/PD706. The products were digested with SfiI and ligated into similarly digested vector and electroporated into MC1061. To transfer these peptides to eCPX, the vectors containing the peptide CPX fusion was digested with PstI and KpnI, the smaller fragment was gel extracted and ligated to the similarly digested vector of eCPX, transferring the displayed peptide to the eCPX plasmid. To insert the P2 peptide (PAPSIDRSTKPPL) at the C-terminus of CPX and eCPX, PCR was used with PD179 as the forward primer and primers PD950/PD951 as the reverse primers with pB33CPX-nSApep at template. The primers also encoded a linker of GGQSGQ preceding the P2 peptide. The products were digested with SfiI and ligated into similarly digested vector and electroporated into MC1061. The gene is eCPX-nSApep-cP2. The streptavidin binding peptide was removed using KpnI and HindIII and ligation with a similarly cut insert that contained no N-terminally fused peptide, creating CPX-cP2.To insert an extended linker of (GGGS)5 between the streptavidin binding peptide and eCPX-cP2, PCR was used with forward primer PD179 and reverse primers PD1429/PD1430/PD31 with pB33eCPX-nSApep-cP2 as template. The product was then gel extracted and used as the forward primer with PD180 as the reverse primer and pB33eCPX-nSApep-cP2 as template. The product was gel extracted and digested with SfiI and ligated to similarly digested vector. The portion after the OmpX signal sequence was now GQSGQ (encoding a SfiI site), AECHPQGPPCIEGRK (the streptavidin binding peptide), (GGGS)5 (the additional linker) and GGQSGQ (original linker) followed by the S54 of eCPX with the P2 peptide on the C-terminus; the gene construct is termed eCPX-nSApep-linker-cP2.Magnetic selection and screening by FACSMagnetic selections were performed for the first round of selection using the libraries CPX-5x, CPX-6x and CPX-directed. An overnight culture of cells corresponding to 5× the library diversity was inoculated to LB medium containing 34 µg/ml chloramphenicol (Cm) for a final cell concentration of 0.05 OD600, or 100 µl of overnight cultures into 5 ml LB Cm, which ever was greater. The cultures were then grown at 37°C to 0.5 OD600 with shaking (250 rpm), at which time the culture was moved to room temperature (22°C) to equilibrate and then induced with l-arabinose to a final concentration of 0.04% (w/v). The cells were induced by shaking (250 rpm) for 50 min, at room temperature. A volume of cells corresponding to 5× the library diversity was concentrated by centrifugation (3000 g, 4°C, 5 min) and resuspended in cold PBS to 10–30 OD600. Dynal MyOne SA beads were added to a ratio of approximately one bead per four cells. Magnetic separation was used to wash the beads four times with a volume of LB equivalent to the volume used in the initial labeling, and the beads plus bound cells were finally resuspended in LB with Cm and 0.2% glucose (w/v) for overnight growth.For flow cytometric sorting, 50 µl of overnight cultures of the libraries were inoculated to 5 ml LB Cm. Cells were induced as described in the previous paragraph, in future rounds of sorting the induction time was decreased to 30 min. Ten microliter of cells were labeled with 100 µl of 100 nM SA–PE in PBS on ice for 45 min, pelleted by centrifugation and the supernatant was removed. Cells were resuspended in ice-cold PBS at approximately 107 cells/ml and immediately analyzed and sorted using a FACSAria cytometer with 488 nm excitation. Between 1 and 5% of the most labeled cells were collected and amplified for further rounds of analysis and/or sorting by growing overnight in LB medium containing glucose and Cm. A subset of the sort was plated directly on agar for isolation of single clones. Typically 4–10 selected clones were assayed for antigen binding by flow cytometry, and the identity of each peptide insert was determined by DNA sequencing.Determination of peptide display levelsTo compare the display level of CPX and eCPX as a function of induction time, CPX and eCPX, cells were subcultured 1:50 from overnight stocks into 5 ml LB Cm and grown for 2 h with shaking (250 rpm) at 37°C. The cells were then moved to room temperature (22°C) to equilibrate and induced with 0.04% (w/v) l-arabinose still shaking at 250 rpm. Samples (5 µl) were taken prior to induction, 30, 60 and 90 min after induction then added to 50 µl of 100 nM SA–PE in PBS and incubated on ice for 45 min. The cells were then centrifuged (3000 g, 5 min), supernatant removed and resuspended in 500 µl ice cold PBS. Cells were immediately analyzed with a FACSAria using 488 nm excitation and fluorescence data collected at 576 nm.To compare the display of various peptides using CPX and eCPX, cultures were started using 50 µl of overnight culture in 5 ml LB Cm. Cultures expressing the mini-Z domain, SApep, P2 and T7pep were grown until an OD600 of 0.4 and moved to room temperature (22°) to equilibrate and then induced with l-arabinose to a final concentration of 0.04% (w/v) for 30 min or 2 h for the mini-Z domain. Cultures expressing the CRPpep and V114 peptides were induced at 37°C at an OD600 of 0.4 for 30 min. Induced cells (5 µl) were added to 50 µl of PBS containing the respective antigens at the following concentrations: 50 nM YPet-Mona, 65 nM biotinylated VEGF, 100 nM biotinylated CRP, 300 nM Alexa labeled human IgG, 100 nM SA–PE and 6.7 nM anti-T7•tag monoclonal IgG. Samples were labeled on ice for 45 min. Biotinylated samples were spun down at 3000 g for 5 min and the supernatant removed. Cells were resuspended in 50 µl of PBS with 10 nM SA–PE and put on ice for 45 min. Before cytometric analysis, samples were spun down at 3000 g for 5 min, supernatants removed and 500 µl of PBS added to resuspend the cells. Samples were excited at 488 nm; fluorescence data were collected at 576 nm for SA–PE labeled samples and 530 nm for Alexa labeled samples and YPet conjugated samples.For the dual labeling experiments, cells were subcultured 1:50 from overnight stocks into 5 ml LB Cm and grown for 2 h shaking (250 rpm) at 37°C. The cells were then induced with 0.04% (w/v) l-arabinose. Cells expressing eCPX-nSApep-cP2 were expressed for 25 min at 37°C, and cells expressing eCPX-nSApep-linker-cP2 were expressed for 45 min at 37°C. Cells (5 µl) were labeled with 50 µl of PBS with 100 nM SA–PE only, 40 nM Ypet-Mona only or with both probes simultaneously. The negative controls were non-displaying cells labeled with SA–PE and Ypet-Mona. The cells were incubated at room temperature for 45 min, centrifuged at 3000 g for 5 min and supernatant removed. The cells were left on ice before resuspension with 500 µl ice cold PBS and analyzed using cytometry with 488 nm excitation and measuring emission at 576 and 530 nm.
Supplementary dataSupplementary data are available at PEDS online.
FundingNational Science Foundation CAREER award (BES-0449399) to P.S.D., U.S. Army Research Office—Institute for Collaborative Biotechnologies (DAAD19-03-D-0004).
Supplementary Material
[Supplementary Data]

directed evolution
bacterial display
ompx
circular permutation
peptide display


Fatty acid-induced mitochondrial uncoupling in adipocytes as a key protective factor against insulin resistance and beta cell dysfunction: do adipocytes consume sufficient amounts of oxygen to oxidise fatty acids?

To the Editor: The phenotype of patients with various forms of congenital lipodystrophy has indicated that proper storage of triacylglycerol in peripheral adipocytes is essential to prevent formation of ectopic fat deposits and development of type 2 diabetes [1]. A similar situation with induced peripheral lipodystrophy and elevated risk for type 2 diabetes is seen in patients undergoing highly active anti-retroviral therapy (HAART). In these patients, there is a 30–50% decline in mitochondrial copy number as a result of HAART [2].Based on these and other observations, we have recently put forward the hypothesis that one of the functions of mitochondria in adipose tissue is to prevent leakage of fatty acids into the circulation [3]. This mitochondrial activity attenuates the development of ectopic triacylglycerol deposits in the liver and other tissues that would otherwise contribute to the development of whole body insulin resistance and pancreatic beta cell damage. Mitochondria contribute to efficient confinement of fatty acids within adipocytes by oxidative removal of fatty acids, liberated from the triacylglycerol pool, by uncoupled beta oxidation. In addition, mitochondria may provide glyceroneogenic substrates, which contribute to re-esterification of fatty acids. As a net result, there are less fatty acids available to the circulation for redistribution to other compartments of the body [3]. A recent study provided supporting evidence for this concept by showing that the mitochondrial DNA copy number in human adipocytes is positively associated with lipogenesis in adipocytes [4].Recently, Frayn et al. have argued against this biochemical mechanism [5]. They point out that fatty acid oxidation is not a major pathway in white adipocytes and that oxygen consumption by adipose tissue is insufficient to oxidise substantial amounts of fatty acids [6].We fully agree with the point made by the authors that fatty acid oxidation by adipocytes is not a major pathway compared with muscle. However, we hold a different view on the quantitative interpretation of these data: in our model there is no need for a high rate of fatty acid oxidation in adipose tissue per time unit, as fatty acid redistribution from peripheral tissue to the liver is a slow process, taking several years. For instance, in individuals in whom mitochondrial copy number is acutely reduced as result of starting HAART, it takes 18–24 months before redistribution of body fat becomes clinically manifest [7].Therefore, the amount of fatty acids that undergoes redistribution per time unit is very small. Thus, mitochondria in adipocytes only need to remove a small amount of fatty acids per time unit to protect against redistribution of body fat. By using the oxygen consumption data of Frayn et al. and others [5, 6] or using similar data from other studies [8, 9], one can calculate that oxygen consumption by adipose tissue is sufficient to prevent redistribution of >1.5 kg of fat out of 10 kg of peripheral adipose tissue to the liver within a 3 year period, a realistic clinical situation.Therefore, in our opinion, adipose tissue is able to oxidise substantial amounts of fatty acids over a long period by mitochondrial beta oxidation. This process may over time attenuate ectopic deposition of those fatty acids. Since mitochondria also contribute to formation of glycerol 3-phosphate, needed for re-esterification of fatty acids within adipocytes, mitochondria in adipocytes have the ability to protect the organism to a certain extent against leakage of fatty acids into the circulation and thus, redistribution of body fat. As Frayn et al. [5] have pointed out, brown adipose tissue is indeed more active in uncoupled oxidative disposal of fatty acids compared with white tissue. Recent evidence also suggests that brown adipose tissue is present in variable amounts and at multiple sites in humans, contrary to earlier reports [10]. This recently recognised situation further enhances the capacity of the adipose compartment to remove fatty acids through uncoupled beta oxidation.In order to understand the mechanism by which mitochondria in adipocytes contribute to the control of body fat distribution during the development of the metabolic syndrome, additional studies are needed, comprising quantification of fatty acid fluxes in relation to re-esterification and oxidation in adipose tissue. These studies should include study of adipose tissue from individuals with normal glucose tolerance and from those with developing glucose intolerance.

fatty acids
adipocytes
haart
mitochondria
type 2 diabetes mellitus


Safe addition of vitamins and minerals to foods: setting maximum levels for fortification in the Netherlands

Background In 2004, the European Court of Justice decided that the prohibition of fortification with vitamin A, vitamin D, folic acid, selenium, copper, and zinc in the Netherlands conflicts with the principle of free movement of goods in the European Union. This decision led to a change in the Dutch policy, resulting in a more flexible handling of requests for exemption from this prohibition to fortify. Therefore, an investigation was proposed in which it would be determined whether a general exemption could be granted for food fortification with a certain maximum safe amount per micronutrient.
Introduction
Foods are fortified with micronutrients and other bioactive compounds to prevent deficiencies or to provide additional health effects. Furthermore, the use of health claims on products after fortification is thought to result in better marketing. As fortification with micronutrients should be safe for the whole population, unacceptably large intakes of micronutrients from all sources should be prevented. For this purpose, tolerable upper intake levels (ULs) are established [28].
Because of the small range between recommended daily intakes and ULs and the lack of proof of a nutritional need, fortification with vitamin A, vitamin D, folic acid, selenium, copper, and zinc is prohibited in the Netherlands [1]. Addition of these six micronutrients was only allowed in the case of substitution (e.g., vitamins A and D in margarine as a substitute for butter) or restoration (for instance, because of losses during processing) [1]. In December 2004, the Court of Justice of the European Communities decided that the prohibition of fortification with these six micronutrients contradicted the free movement of goods, a fundamental principle in the Treaty Establishing the European Community [5]. This decision means that market introduction of fortified foods can only be prohibited if it can be proved that the degree of fortification (e.g., level of fortification or quantity of foods fortified) may harm public health. This decision led to a change of Dutch policy, resulting in a more flexible handling of requests for exemption from the Dutch Commodity Act that prohibits fortification with these six micronutrients [6]. Exemption will only be granted if there is no harm to public health, based on the results of the most recent international scientific research.
Because of this court decision, each request for individual exemption had to be evaluated for the risk of exceeding the UL. At first, requests for individual exemption were evaluated in order of receiving. To evaluate the risk of exceeding the UL, consumption of the new fortified product by the Dutch population was simulated by replacing the unfortified food products with the new fortified product within the Dutch National Food Consumption Survey (DNFCS) (e.g., the consumption of all kinds of breakfast cereals as monitored within the DNFCS was replaced by the new fortified breakfast cereals). The resulting habitual daily intake (P95) of the micronutrient was compared with the UL. If the UL was not exceeded, the exemption was granted. This approach resulted in the exemption of very few products and turned out to be an unrealistic worst-case approach. Furthermore, the procedure resulted in an administrative overload and long waiting periods for the industry. Therefore, and in anticipation of upcoming European legislation [11], an investigation was proposed in which it would be determined whether a general exemption could be granted for food fortification with a certain maximum safe amount per micronutrient. A model was needed to calculate the national maximum level of a specific micronutrient that can safely be added to foods. Several such models have recently been developed [11–14, 25]. First, an International Life Science Institute (ILSI) Europe model (2002) [14], which was followed by models by Domke et al. [12, 13] and Rasmussen et al. [25]. The latter is an adapted version of the ILSI Europe model [14]. These models were developed for the purpose of calculating the maximum amount of micronutrients that can be added to each 100 kcal of a given food [14, 25] or each food portion [12]. The intake of micronutrients from nonfortified foods and dietary supplements must be taken into account for this calculation. Domke et al. also estimate the maximum amount of micronutrients in dietary supplements by dividing the space between the UL and the level of intake from nonfortified foods. Most models are theoretical approaches, and data regarding their practical use in decision- or policy-making have not yet been published, although the published acceptable levels of addition per 100 kcal by Rasmussen et al. have been implemented as the Danish maximum levels for fortification (Rasmussen, personal communication, 2006). Besides these models that calculate a maximum fortification level, models that calculate the optimal intake for the population (taking into account both too low and too high intakes) are also being developed [15, 26]. In view of the present application, the derivation of maximum safe fortification levels (MSFLs), these models on optimal intake were not taken into account. The question how to create an optimal micronutrient intake is outside the scope of this paper.
On the basis of the existing models to calculate a maximum fortification level, a model adapted to the Dutch practical situation was developed for the Dutch authorities to derive for the fortification with micronutrients. This article first describes the model for the derivation of maximum amounts of micronutrient fortification in the Netherlands, and then it illustrates the use of the micronutrient specific data and assumptions in the calculation of the MSFLs for folic acid, vitamin A, and vitamin D. The article concludes with the considerations and the final allowed fortification level (AFL) of the risk management.
Methods
Models to calculate safe maximum levels of addition of micronutrients are published, among others, by ILSI, Rasmussen and Domke [12–14, 25]. We developed a model for the Dutch situation, based on (elements of) the models of ILSI, Rasmussen and Domke. Below, we will amplify on the main differences between the models and our filling-in for the Dutch model.
The ILSI Europe and Rasmussen et al. models differ in several aspects from Domke et al.’s model. Instead of the calculation per portion size as applied by Domke et al., both ILSI Europe and Rasmussen et al. calculate the maximum fortification level per 100 kcal. For our Dutch model, we also calculate with 100 kcal portions because the variation in mean daily energy intake is low compared to for instance the variation in micronutrient or carbohydrate intake [29]. Moreover, there is no univocal definition of portion size in the Netherlands. Another important difference is that Domke et al. not only establish maximum fortification levels, but they also establish maximum levels for dietary supplements. Because maximum safe addition levels of vitamins and minerals in dietary supplements will be regulated via European Union legislation, we did not determine them in our model [11]. Similarly to ILSI Europe and Rasmussen et al., we estimated the current intake of micronutrients from dietary supplements and took them into account. Our Dutch model is principally based on the ILSI Europe model [14] and Rasmussen et al.’s extended model [25], with adaptations to the Dutch practical situation. The formula is equal to the one used by Rasmussen et al., however some of the factors are differently derived (see below).
Our model calculates the maximum safe level for fortification with a micronutrient per 100 kcal of the food (MSFL) for various age groups with the mathematical formula presented in Box 1. The lowest MSFL, i.e., the MSFL for the most sensitive group, is then advised to be the overall maximum acceptable fortification, as is common practice in toxicological risk assessment.
Tolerable upper intake level (UL)
The UL is the maximum level of total chronic daily intake of a nutrient that is unlikely to pose a risk of adverse health effects to humans. In general, the UL is related to total daily intake from all sources including nonfortified foods as well as fortified foods and dietary supplements. In Europe, the Scientific Committee on Food (SCF) and the Scientific Panel on Dietetic Products, Nutrition and Allergies (NDA) set age-specific ULs [28]. Due to lack of data, the ULs for children are usually extrapolated from the ULs for adults based on bodyweight. In anticipation of future European policy [11], these age-specific European ULs were used as model input rather than the national ULs, which is similar to the model of Rasmussen et al. [25]. ILSI Europe has also used European ULs, although only the values for men [14].
Box 1. Formula for the calculation of the maximum safe fortification level with micronutrients per 100 kcal food (MSFL) [25].
Current intake of energy and micronutrients from nonfortified foods (EI95 & CI95)
Micronutrients are naturally present in the diet (further referred to as ‘background diet’). Because it is important to consider the intake from all possible sources to assess the risk, the intake from the background diet has to be included in the calculations. To estimate the intake of energy and micronutrients from the background diet, it is important to use the most up-to-date country- and age-specific data available. In view of the precautionary principle [3, 10], we calculated the age-specific habitual intake (also referred to as usual intake) at the 95th percentile of the distribution of energy and the specific micronutrients from the background diet on the basis of data from the most recent Dutch National Food Consumption Surveys (DNFCS): DNFCS-3 and DNFCS-2003. The DNFCS-3 was carried out in 1997–1998 for the whole population (1–97 years) and the DNFCS-2003 was carried out in 2003 for young adults (19–30 years) [16, 23]. In the DNFCS-3, a 2-day dietary record method was used to collect data on two consecutive days. In the DNFCS-2003, data were collected with a 2-day, 24-hour recall method using EPIC-SOFT, a computer-assisted interview method. We estimated the habitual intake of both micronutrients and energy with statistical correction for the within-person variation using the Iowa State University method (C-SIDE software) [18, 20].
Rasmussen et al.’s model uses results from the Danish dietary survey in which a 7-day prospective food recall method was used to calculate the daily intake of micronutrients from nonfortified foods (95th percentile) and energy intake (95th percentile) [25]. ILSI Europe used pan-European estimates of the 95th percentile of intake of micronutrients based on seven national surveys. Some of these surveys already include voluntary fortification of foods with micronutrients and the intake of dietary supplements [14]. Furthermore, the pan-European 95th percentile of energy intake was estimated from five national surveys.
Current intake of micronutrients from dietary supplements (SI)
In addition to the intake from the background diet, the micronutrient intake from dietary supplements (SI) has to be taken into account in order to get a good estimate of the total intake from all sources. In the Netherlands, similar to other Western countries, the use of dietary supplements has increased and takes a more important place in the total micronutrient intake [22]. Preferably, the habitual micronutrient intakes of nonfortified foods and dietary supplements are calculated together. This method takes into account the facts that not everybody uses dietary supplements, not all supplement users use the supplements daily, and supplement users and nonusers may have different eating habits [19]. However, due to lack of information about supplement use, a calculation of combined habitual intake is not possible at this time. Therefore, we used two realistic high-intake scenarios of dietary supplements based on the limited Dutch dietary supplement data available: one for adults (≥18 years old) and one for children (1–17 years old). The scenario for adults was based on the P90-P95 of both the intake of micronutrients from dietary supplements in young adults (19–30 years) reported by the DNFCS-2003 [22] and the levels of micronutrients present in dietary supplements (for adults) available in the Netherlands [4]. The scenario for children was based on P90–P95 of the infants’ intake of dietary supplements [9] and the levels of micronutrients present in dietary supplements for children, that are available in the Netherlands [4].
We determined the SI differently than Rasmussen et al. and ILSI Europe did. Rasmussen et al. chose to set the SI at 100% of the reference values for the recommended daily intake of vitamins and minerals because this is the content of the most used multi-vitamin–mineral supplement in Denmark [25]. The ILSI Europe model does not include the intake from dietary supplements because only a minority of the population is expected to use them [11, 14].
Fraction of energy intake that can and will be fortified (PFFn)
Not all foods on the market can or will be fortified. The fraction of total energy intake that will finally be fortified consists of two parts: (1) the proportion of total energy intake that can be fortified; for example, it is unlikely that fresh products such as vegetables, fruits and meat will be fortified; and (2) the proportion of the energy intake of foods available for fortification that actually will be fortified. Due to practical constraints such as costs and processing, not all foods available for fortification will be fortified with a particular micronutrient.
The first part of the fraction was set at 30%, which is lower than the 50% set by both ILSI Europe and Rasmussen et al. Main argument for the lower percentage was that the large part of the energy intake in the Dutch population is delivered by the traditional dinner, which consists in general of fresh products like meat, vegetables, and potatoes. The second part of the fraction was equal to the one ILSI Europe and Rasmussen et al. set at 50% (worst case). This resulted in a fraction of the energy intake available for fortification (PFFn) of 15% (i.e., 30% * 50%).
Results
Risk assessment: deriving MSFLs
We illustrate the applicability of the Dutch model by deriving MSFLs of folic acid, vitamin A, and vitamin D. Tables 1, 2, 3 present the factors used for the calculation of the MSFLs for folic acid, vitamin A, and vitamin D. The classification into age groups is based on the age categories used by NDA/SCF to define the ULs [28] and is different for folic acid, vitamin A, and vitamin D. We calculated the 95th percentile of habitual energy intake for each age group. The habitual energy intake of children increases with age (Tables 1–3). The 95th percentile of the habitual energy intake for men is greater than that for women; there is a small decline of habitual energy intake with age for both genders.
Table 1Maximum safe fortification levels and allowed fortification level of folic acid based on Dutch consumption data and European tolerable upper levels of intake for specific age groupsAge groupnUL (µg/day)CI95a (µg/day)SI (µg/day)EI95 (SE) (kcal/day)Max safe fortification level (MSFL) (µg/100 Kcal)Allowed fortification level (AFL) (µg/100 kcal)PFFn = 0.15PFFn = 0.10bChildren1–3c25420003001,890 (67)0Children4–6c27630003001,995 (56)0Children7–10c31240003002,456 (63)27Children11–14c33660003003,154 (117)63Adolescents15–17c28080003003,518 (150)95Men18–30c5251,00006004,010 (112)67100100Women18–30c5791,00006002,867 (62)93140Men19–30d3521,00006003,751 (130)71106100Women19–30d3981,00006002,581 (76)103155Men31–50c9501,00006003,529 (64)76113Women31–50c1,1001,00006002,721 (48)98147Men51–65c4201,00006003,427 (109)78117Women51–65c4791,00006002,647 (74)101151Men>65c2601,00006003,106 (119)86129Women>65c4101,00006002,432 (65)110164aDietary intake of folic acid only, folate is not taken into account because the UL is based on folic acid onlybAdditional calculation with exactly the same results as for PFFn = 0.15 in combination with a scenario of low dietary supplement intake (i.e., 400 µg), only calculated for adultscBased on consumption data from the DNFCS-3 (1997–1998) [2]dBased on consumption data from the DNFCS 2003 [17, 21]The most sensitive age groups are bolded; CI95, 95th percentile of habitual dietary folic acid intake; EI95, 95th percentile of habitual energy intake; SI, supplement intake; PFFn, proportion of energy intake that can and will be fortified; UL, tolerable upper intake levelTable 2Maximum safe fortification levels of vitamin Aa based on Dutch consumption data and the European upper levels of intake for specific age groupsAge groupNUL (µg /day)CI95 (SE) (µg /day)SI (µg /day)EI95 (SE) (kcal/day)Max safe fortification level (MSFL) (µg /100 kcal) PFFnb = 0.15Children1–3c2548001,057 (167)8001890 (67)0Children4–6c2761,1001,553 (262)8001995 (56)0Children7–10c3121,5001,055 (116)8002456 (63)0Children11–14c3362,0001,153 (131)8003154 (117)10Adolescents15–17c2802,6001,429 (205)8003518 (150)70Men19–30b3523,0001,886 (261)1,2003751 (130)0Women19–30b3983,0001,203 (162)1,2002581 (76)154Men≥18c2,1553,0002,363 (132)1,2003642 (50)0Women18–50c1,6793,0001,350 (83)1,2002785 (39)108Women>50c8891,5001,441 (108)1,2002555 (53)0aNot including provitamin AbBased on consumption data the DNFCS 2003 [17, 21]cBased on consumption data from the DNFCS-3 (1997–1998) [2]The most sensitive age groups are bolded; CI95, 95th percentile of habitual dietary vitamin A intake; EI95, 95th percentile of habitual energy intake; SI, supplement intake; SE, standard error; PFFn, proportion of energy intake that can and will be fortified; UL, tolerable upper intake levelTable 3Maximum fortification levels and allowed fortification level of vitamin D based on Dutch consumption data and European tolerable upper levels of intake for specific age groupsAge groupnUL (µg/day)CI95 (SE) (µg/day)SI (µg/day)EI95 (SE) (kcal/day) Max safe fortification level (MSFL) (µg/100 Kcal)Allowed fortification level (AFL) (µg/100 kcal)PFFn = 0.15PFFn = 0.10aChildren1–3b254254.8 (0.6)101,890 (67)3.65.4Children4–10b276254.4 (0.2)102,334 (50)3.04.54.5Children11–13b312506.5 (0.6)102,935 (100)7.611.4Adolescents14–18b336507.6 (0.5)103,509 (116)6.29.2Men19–30c352506.5 (0.4)103,751 (130)6.08.9Women19–30c398505.2 (0.4)102,581 (76)9.013.5Men31–50b950508.9 (0.4)103,529 (64)5.98.8Women31–50b1,100506.4 (0.3)102,721 (48)8.212.3Men51–60b3145011.3 (1.2)103,410 (128)5.68.4Women51–60b332506.7 (0.6)102,623 (90)8.512.7Men61–70b197509.8 (1.0)103,315 (124)6.19.1Women61–70b270506.6 (0.6)102,528 (87)8.813.2Men>70b1695012.2 (1.8)103,067 (141)6.09.1Women>70b287508.6 (0.8)102,487 (86)8.412.6aAdditional calculation with exactly the same results as for PFFn = 0.15 in combination with low scenario of dietary supplement intake (i.e., 5 µg)bBased on consumption data from the DNFCS-3 (1997–1998) [2]cBased on consumption data from the DNFCS 2003 [21]The most sensitive age groups are bolded; CI95, 95th percentile of habitual dietary vitamin D intake; EI95, 95th percentile of habitual energy intake; SI, supplement intake; SE, standard error; PFFn, proportion of energy intake that can and will be fortified; UL, tolerable upper intake level
Folic acid
Folic acid is the synthetic form of folate, a natural component in the diet. An UL has been established only for folic acid and not for folate. For this reason, the calculation does not consider the intake of folate from the background diet. At the time the food consumption surveys were conducted, fortification with folic acid was not permitted in the Netherlands, which results in an intake from the background diet (CI95) of 0 µg/day. The SI of folic acid was estimated to be 300 µg/day for children and adolescents and 600 µg/day for adults. Filling in these factors in the formula of Box 1 resulted in a maximum fortification level for folic acid of 0 µg/100 kcal for children 1–6 years old to 110 µg/100 kcal for women older than 65 years (Table 1). For children and adolescents, the MSFL for folic acid fortification increased with age because of an increase in UL over the age groups. For adults, men generally had lower MSFLs than women due to a higher energy intake. Based on the most sensitive age group (i.e., children 1–6 years) the MSFL is 0 µg/100 kcal. For the most sensitive adult group (i.e., men 18–30 years old) the MSFL is 67–71 µg/100 kcal. Our calculated MSFL for the most sensitive group is lower for folic acid than the acceptable level of addition of Rasmussen et al., 23 µg/100 kcal for the most sensitive group (1–3 years old), while we calculated 0 µg/100 kcal for children 1–6 years old. This difference is mainly caused by a lower estimation of the SI by Rasmussen et al., which was based on Danish legislation. In our calculations, the current intakes from dietary supplements were estimated based on the amount of folic acid available in dietary supplements on the Dutch market.
Vitamin A
Part of the vitamin A is consumed as provitamin A (for instance, carotenoids). However, the UL is defined for the intake of vitamin A as pure retinol and retinyl esters only because it is thought that the intake of provitamin A will not significantly contribute to the toxicity of high intakes of vitamin A [28]. When we use the term ‘vitamin A’, we refer to pure retinol and retinyl esters. Due to the risk of osteoporosis, the UL for postmenopausal women is much lower than that for other women [28]. In our study population, the distinction between premenopausal and postmenopausal women could not be made exactly; therefore, it was assumed that women older than 50 years are representative for the group of postmenopausal women.
When the food consumption surveys were carried out, vitamin A addition was only allowed for margarine and low-fat margarine as a substitute for butter. This substitution will continue in the Netherlands, and therefore the fortification of (low-fat) margarine with vitamin A was included in the calculation of the CI95. The SI was estimated to be 800 µg vitamin A/day for children and adolescents and 1,200 µg vitamin A/day for adults. The habitual dietary vitamin A intake varied from 1,055 µg/day to 2,363 µg/day. For children, the age group 4–6 years had the greatest vitamin A intake (1,553 µg/day). Men had a larger intake than women. The vitamin A intake of premenopausal and postmenopausal women (i.e., older than 50 years) was comparable (Table 2). Filling in these factors in the formula (Box 1) resulted in a MSFL for vitamin A of 0 µg vitamin A/100 kcal for children 1–10 years old, men and post-menopausal women, 154 µg vitamin A/100 kcal for women 19–30 years old and for older children and adolescents, the MSFL is 10–70 µg vitamin A/day. In conclusion, the MSFL for vitamin A will be 0 µg vitamin A/100 kcal on the basis of the most sensitive groups (i.e., children 1–10 years, men 18 years old or more, and postmenopausal women). Our results for vitamin A were the same as Rasmussen et al.’s, i.e., 0 µg vitamin A/100 kcal.
Vitamin D
At the time when the DNFCS-3 and the DNFCS-2003 were carried out, addition of vitamin D was only allowed for margarine and low-fat margarine as a substitute for butter. Because this substitution is expected to continue in the Netherlands, this fortification practice was included in the calculation of the CI95. The 95th percentile of habitual dietary vitamin D intake varied from 4.4 µg/day to 12.2 µg/day (CI95). Vitamin D intake increased with age for children, although children 1–3 years old had a slightly greater intake than children 4–10 years old. For men, the 95th percentile of habitual vitamin D intake lies between 6.5 µg/day and 12.2 µg/day and for women, between 5.2 µg/day and 8.6 µg /day (Table 3). The SI was estimated to be 10 µg vitamin D/day for children, adolescents, and adults.
Entering these factors into the formula (Box 1) resulted in a MSFL for vitamin D of 3.0 µg/100 kcal for children 4–10 years old to 9.0 µg/100 kcal for women 19–30 years old. The MSFL increased with age for children, although children 4–10 years old had a slightly lower MSFL than children 1–3 years old. Due to a smaller habitual energy and vitamin D intake, the MSFL for women is greater than that for men. In conclusion, the MSFL of vitamin D is 3.0 µg/100 kcal based on the most sensitive group (i.e., children 4–10 years). For the most sensitive adult group (men 51–60 years old), the MSFL is 5.6 µg/100 kcal. Our MSFL for vitamin D (3.0 µg/100 kcal for children 4–10 years old) is greater than Rasmussen et al.’s acceptable level of addition: 1 µg/100 kcal for children 7–10 years old. This is mainly due to the difference in PFFn. Rasmussen et al. used a more strict PFFn of 25% whereas we used a PFFn of 15%.
Risk-management: setting AFLs for general exemption
After the risk was assessed, a risk manager considered all the important issues and decided what maximum level of fortification will be allowed and included in a general exemption. In the Netherlands, proposed changes and additions to the Dutch Commodity Act are discussed with stakeholders (e.g., organizations of trade and industry, consumers organizations, authorities) in a Regular Consultation on the Commodity Act. Representatives of individual companies cannot participate in the Regular Consultation on the Commodity Act.
Several consultations regarding the results (Tables 1–3) of the risk assessment for fortification with folic acid, vitamin A, and vitamin D resulted in a general exemption for folic acid of 100 µg/100 kcal and for vitamin D of 4.5 µg/100 kcal. No general exemption was given for vitamin A. A main general consideration is that this national policy will be temporary, as European legislation is expected to come into effect within a few years [11].
Folic acid
The UL of folic acid for adults is based on the risk of masking vitamin B12 deficiency, for children an UL is extrapolated from this adult value based on bodyweight. Because of an ongoing discussion about the value of the UL for children extrapolated from the UL for adults, the risk manager decided to focus on the results of the risk assessment for adults only. It was thought that such an extrapolation would be conservative because the risk of vitamin B12 deficiency for children was thought to be low. Furthermore, at that time, exemptions had been requested for only a few product types and some specific brands. Therefore, the risk manager expected that the chance of children consuming all available folic-acid-fortified products was small. In discussions with the stakeholders, which considered the temporary character of this national policy and the number of requests for exemption, the PFFn of 15% was judged to be rather conservative. Using the dietary intake at the 95th percentile of the distribution (CI95) and the 90–95th percentile of intake from dietary supplements (SI) was also judged to be quite conservative. Therefore, two additional calculations were done, one with a PFFn of 10% and the other with a low intake scenario of dietary supplements (i.e., 400 µg for adults). Both scenarios resulted in a maximum level for fortification in the most sensitive adult group (men aged 18–30 years) of 100–106 µg/100 kcal (Table 1). For practical reasons, the risk manager rounded this value off to an AFL of 100 µg folic acid/100 kcal. Because of this relatively liberal AFL and the fact that the value was based on the MLF of adults, it was decided to monitor the intake of folic acid from fortified foods. If the results of the monitoring show that the long-term intake exceeds the UL in specific population groups, appropriate measures can be taken.
Vitamin D
For vitamin D the derivation of the UL for children is based on critical effects observed in children, whereas for folic acid UL-values for children were established by extrapolation only [28]. Therefore, the maximum fortification levels for children from the risk assessment were considered. As for folic acid, this will be a temporary policy for only a couple of years and it was thought that the PFFn of 15% and the high-intake scenario from dietary supplements are rather conservative estimates. In addition, only one request for exemption was received at that time for vitamin D fortification, and the risk manager did not expect more exemption requests. Thus, similar additional calculations with a PFFn of 10% or a scenario of smaller intakes from dietary supplements (i.e., 5 µg) were done for vitamin D. The calculations resulted in a MSFL of 4.5 µg vitamin D/100 kcal for the most sensitive group (children aged 4–10 years) (Table 3). The risk manager adopted this MSFL and set the AFL at 4.5 µg/100 kcal. Again, the risk manager desires monitoring of vitamin D and energy intake for insight into current intakes and proportions of energy intake fortified. If long-term intakes exceed the ULs, appropriate measures can be taken.
Vitamin A
In contrast to the liberalization for folic acid and vitamin D, the risk manager did not liberalize the results from the risk assessments for vitamin A. To the opinion of the risk manager, exceeding the UL of vitamin A has more serious consequences for public health than exceeding the ULs for folic acid and vitamin D. In addition, the 95th percentile of habitual dietary vitamin A intake of several age groups already exceeded the UL, even without considering the vitamin A intake from dietary supplements. Furthermore, at this time no request for exemption has been received, although one product for which an exemption for folic acid was requested also contained vitamin A. So the AFL was set at 0 µg/100 kcal. The stakeholders in the consultation agreed with this decision.
Exception for low-energy products
An exception was made for light variants of products (low-energy products). According to the model, products low in energy may be fortified with smaller amounts of the micronutrient than the more energy-dense counterparts. In view of the obesity trend and the healthy image of light products, this was considered undesirable. Therefore, light products may be fortified with amounts similar to the amounts approved for their more energy-dense counterparts.
Discussion
We presented a model for the derivation of maximum amounts of micronutrient fortification in the Netherlands. A novelty of this publication is that besides the theoretical model, the practical applicability for the Netherlands is shown including both the risk-assessment and risk-management aspects. The model has already contributed to new Dutch policy-making, as the allowed level for fortification for folic acid and vitamin D are applied as a general exemption in 2007 [8].
Applicability of the model
Although the model can already be applied with the available data in the Netherlands, it is recommended to improve the monitoring of consumption of fortified foods and dietary supplements within the Netherlands. Such data are very useful to check the model parameters and to keep them up-to date. If necessary, AFLs can be adjusted. It is recommended to monitor the intake of fortified foods and dietary supplements as part of the national food consumption survey [7, 27]. Food frequency questionnaires specifically for fortified functional foods and dietary supplements in a representative sample of the population would be a useful tool. In addition composition databases of fortified foods and dietary supplements are recommended [27].
Risk management
The discussions about the risk-assessment results with the different stakeholders worked well for the risk manager. This resulted in broad insight into the various points of view of the stakeholders. Ultimately the risk manager took the final decision, and in all these cases the stakeholders agreed with the final outcome. The risk assessment was based on the precautionary principle. However, with a view to the fact that this will be a temporary policy, the outcome of the risk assessment was judged to be rather conservative for folic acid and vitamin D. In contrast, the market may change, the date the European legislation will come into effect can be later than expected, and people may consume more fortified foods than estimated. Therefore, monitoring the intake of micronutrients from fortified foods is a very important tool for warranting the safety of the population. If monitoring shows that some groups within the population have habitual intakes that exceed the ULs, adaptations should be made. Especially children may be vulnerable. A food consumption survey is currently being conducted among children in the Netherlands. These data will provide insight into the micronutrient intake from natural foods, fortified foods and dietary supplements during 2005/2006.
Additional considerations
Some factors that were not taken into account in MSFL risk assessment are worth considering, and they can be implemented in the model or taken into account in the risk management if necessary. First, we made an exception for the fortification level of ‘light’ products in our risk management. This exception was not considered in the calculation and can therefore theoretically lead to micronutrient intakes that are too large even if the energy intakes are not exceptionally high. Monitoring the use of fortified ‘light’ products will be necessary to avoid frequent users exceeding the UL. Second, some products are intended for a specific population or target group only. If there is little risk that the product will be used by nontarget groups, the risk manager can make an exception to the model. Instead of using the calculated MSFL for the most vulnerable group, the MSFL for the specific target group can be used as a maximum for this specific product. Again, monitoring of the use of these products is important, and mentioning the target groups, as well as nontarget groups, on the product package might be considered.
The vitamin levels in products may decrease over time. Therefore, it is possible that the amount of vitamin added by manufacturers is greater than the amount declared on the packages [24]. To be able to protect all consumers and avoid underestimation of the intake, this so-called overage should be integrated into the MSFL in the model. Rasmussen et al. also noted this problem. In Denmark the currently accepted practice is to add up to 150% of the declared amount. Our calculated MSFL includes overage.
International applicability of the risk-assessment model
Currently, the European Commission is preparing to set maximum and minimum amounts for vitamins and minerals in both foods and dietary supplements [11]. The simultaneously setting of both the maximum levels for dietary supplements and for foodstuffs can result in a well based division of micronutrients over dietary supplements and fortified foods. Once the choice has been made which micronutrient is allowed to be added to dietary supplements and/or fortified foods, models like the one presented can be helpful to set the MSFL and AFL for micronutrients in food products.
The applicability of the model for this purpose depends on the availability of the input data. When calculating MSFLs valid for Europe as a whole, the availability of European data is necessary. However, data from a food consumption survey that cover all Member States of the European Union representatively are not available. Such data are not even available for all Member States individually. Furthermore, consumption data available in various Member States are often collected with different methods, in a different time frame, and for different specific population groups and the fraction of energy intake that can be fortified may differ greatly from country to country because of the various traditional diets. This makes it difficult to estimate the total habitual intake for each micronutrient and energy type at a European level.
To overcome these problems, MSFLs in Europe can be set by using the available country-specific calculations and to select the most sensitive country (i.e., the country with the lowest MSFL). The maximum safe level for fortification for this country can then be applied in all Member States.
Conclusion
As this paper illustrates, our model for risk assessment can be used in the Netherlands to help risk managers to set maximum levels for safe addition of vitamins to foods. This has resulted in two general exemptions, one for folic acid at 100 µg/100 kcal and one for vitamin D at 4.5 µg/100 kcal. The model is flexible and can be adapted to new insights. For example, aspects concerning overage and ‘light’ products can be included with minor changes in the formula. Monitoring of the total micronutrient intake after applying a general exemption, as well as monitoring of the fortified fraction of the energy intake and the effect of the exception for light products, is recommended for evaluating the model parameters and AFLs and for making adjustments when needed.

safe addition
vitamins
minerals
food
fortification
micronutrients
tolerable upper intake level
maximum fortification levels