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ANALYTICAL CURRENTS Ptc4
MS rises to yeast challenge The yeast two-hybrid system is the standard technique for creating comprehensive inventories of protein complexes. But the development of extremely sensitive MS methods has given researchers a more direct alternative for this type of proteomic analysis. Yuen Ho, Daniel Figeys, and colleagues at MDS Proteomics in Toronto (Canada) and Odense (Denmark), Mount Sinai Hospital–Toronto, and the University of Toronto demonstrate the power of MS by using it to survey protein interactions in the proteome of the yeast Saccharomyces cerevisiae. The researchers call their technique high-throughput mass spectrometric protein complex identification (HMSPCI). They began by choosing a set of “bait” proteins—notably, proteins involved in cellular signaling and DNA damage repair—to lure binding partners. Of the original set, 600 proteins were expressed at detectable levels. To make it easy to isolate the “caught” complexes, the bait proteins were fused with an epitope tag, which could be recognized easily by particular antibodies. This led to the precipitation of the com-
Ptc2 Swi4 Asf1 Smc3 Mms4
Ydr071c Rad53
Rad59 Ptc3
Rad52 Ai1
Msh2
plexes. More than 1500 Rad9 Mph1 Rad28 of these immunoprecipitaHpr5 Ntg1 Mag1 Rfc2 tions were performed, and Anc1 Dun1 Mgt1 RFC Cdc5 Mus81 the resulting samples were Rad24 Rfc3 Rfc5 Cdc16 pooled, resolved by polyPso2 Rfc1 Yku70 acrylamide gel electroRfc4 Rvb1 Xrs2 phoresis, and analyzed Bcy1 Sml1 Rad18 Rvb2 Met18 Mgm101 using automated nanoElc1 MRX Mms2 Rad26 HPLC electrospray ionRad50 PRR Rad3 Mre11 Rfa1 Ccl1 ization MS/MS. Ubc13 Rfa2 Ufd2 Tfb3 Rpo21 Kin28 After weeding out Rad7 Rsp5 Rad23 Rad17 Rad16 “caught” proteins that apMsi1 Rad1 Lif1 Ddc1 Rad10 Rnr3 peared to bind nonspecifRad4 Rnq1 Dnl4 Rnr2 ically, there were 3617 Ctf4 Rad14 Mec3 NER Lcd1 interactions and 1578 difPCNAL Cce1 Rnr1 ferent proteins, which repSelected parts of the DNA damage response network as deresented 25% of the yeast termined by high-throughput mass spectrometric protein genome. Of these, 531 complex identification. Blue arrows indicate known interacproteins corresponded to those that were postulated tions; red arrows indicate new ones. (Adapted with permisto exist on the basis of the sion. Copyright 2002 Nature Publishing Group.) yeast genome sequence. The HMS-PCI data set obtained by the researchers was compared to 3.4-fold more literature-derived with two other data sets from comprehen- interactions per bait protein than the two-hybrid data sets individually and sive high-throughput yeast two-hybrid 1.9-fold more interactions than those screening, using a set of interactions resets combined. (Nature 2002, 415, ported in the literature as a benchmark. The HMS-PCI data set contained 2.6180–183)
The chiral square grid Noninterpenetrating square-grid poly-
mensions of 25 ⫻ 25 Å. The ligand, L, is
analytical application. Uwe Bunz and
mers are porous self-assembled structures
based on a bipyridine structure and in-
Hans-Conrad zur Loye and their co-authors
with well-defined channels or pores. An im-
cludes a very long side chain that projects
at the University of South Carolina de-
portant challenge is to control the size of the
into the channel, shrinking the open space.
scribe the first synthesis of a chiral nonin-
channels. In this paper, the researchers de-
One of these side chains is a chiral fluorene
terpenetrating square-grid coordination
scribe the synthesis of a copper nitrate coor-
unit that sticks out into the polymer channel,
polymer, which could be used for size-
dination compound polymer, [CuL2(NO3)2],
reducing the grid space to about 8 ⫻ 8 Å.
exclusion chemistries.
with particularly large channels—grid di-
(Angew. Chem., Int. Ed. 2002,41, 583–585)
Here is a material that is just waiting for an
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ANALYTICAL CURRENTS Beefing up top-down Whereas many MS strategies for proMS/MS spectrum. ECD breaks more The researchers use this heavy-hitting teomic analysis sort through fragments bonds than CAD and generates some approach to determine proteins in gefrom a protein digest, the “top-down” unique cleavages. Thus, a combination netically modified E. coli. For example, approach begins with the whole protein of CAD- and ECD-generated MS/MS analysis of one cell extract yielded 102 and then uses the molecular ion and spectra provides the higher resolution. molecular weight values. However, none MS/MS to determine the seof the molecular weights quence. That works well, up corresponded to the four kDa 1 2 to a point. Efforts to imenzymes involved in thi7756.11 prove the sequence specificity amin biosynthesis that the 10693.5 (a) (b) ThiS by going beyond MS/MS researchers had targeted. 8+ 46 fragmentation can prove diffiMS/MS of one parent ion 29 970 971 972 973 974 cult because the remaining species identified it with the m/z 20 unbroken bonds have high DNA-predicted sequence ThiS? 15 dissociation energies. So, for the enzyme THiS, which 6 Fred McLafferty and his cohelps synthesize the thiazole 3 workers at Cornell University moiety of thiamin. Further 800 1000 1200 1400 m/z take out the “big gun”— purification produced a mix7756.05 7+ b64 7567.05 (c) electron capture dissociation ture that ECD MS/MS ThiS Gly Gly Gly-OH (ECD)—and demonstrate a showed to be modified forms ∆m = 57.0 Da ∆m = 57.1 Da ∆m = 75.0 Da 8+ top-down analysis with MS/ of THiS and the related en7+ b65 7+ b66 MS for several novel and difzyme THiG. In other stud7624.04 7681.09 ficult-to-find enzymes. ies, ECD helped identify The key to top-down phosphate noncovalent 1080 1085 1090 1095 1100 968 970 972 974 analysis is FTMS, which deadducts and another modim/z termines mass with a very fied protein with an unexSearching for THiS. (a) Gel electrophoresis of a cell lysate with mohigh resolution. Collisionally pected molecular weight. lecular weight markers in lane 1. (b) FTMS spectra of the mixture. activated dissociation (CAD) (J. Am. Chem. Soc. 2002, (c) CAD MS/MS of the isolated 7756.11 ions. is often used to generate the 124, 672–678)
Nanotube problem now soluble Typical carbon nanotubes (NTs) defy disso-
tions of azomethine ylides.
lution. Their characteristic insolubility in common solvents has limited manipulation
Historically, there have been complicated, roundabout attempts to add long alkyl
days. All of the functionalized NTs had approximately the same solubility of 50 mg/mL without sonication, which is known to alter
chains or wrap NTs
NT length. Although only the results for
stricting how their unique
with polymers that
single-wall NTs (SWNTs) were reported,
COURTESY OF MAURIZIO PRATO AND PAOLO BRAIUCA
and processing, thus reproperties have been applied. Maurizio Prato and colleagues at Università di Trieste (Italy), University of Notre Dame, and Universität Erlangen (Germany) overcome this obstacle with a new method to functionalize carbon NTs by 1,3-dipolar cycloaddi-
182 A
Molecular model of a section of an oligoethylene glycol-pyrrolidine-functionalized single-walled carbon nanotube.
A N A LY T I C A L C H E M I S T R Y / A P R I L 1 , 2 0 0 2
functionalized the tips
the researchers say that their method also
and sidewalls, but
worked with multiwall NTs.
they resulted in limit-
Several analytical methods confirmed
ed solubility. The new
that the NTs had been functionalized and
synthetic strategy
remained intact. Transmission electron mi-
suspends and heats
croscopy showed that although the original
the NTs in dimethyl-
SWNTs aggregated in ~10-nm-diam bun-
formamide with an ex-
dles, functionalized SWNTs formed ~100-nm
cess of glycine and
bundles. (J. Am. Chem. Soc. 2002,124,
aldehyde for five
760–761)
news
New tricks for molecular beacons and the intensity of the fluorescence sigcentration, whereas stem-forming DNA For the oligonucleotide probes known nal changes. In the new assay, the two beacons hybridized to the target more as molecular beacons, the classic targets ds-fragments were brought into close slowly as the salt concentration deare DNA or RNA sequences. But two proximity when they bound to the creased. Stemless DNA beacons had recent papers show that molecular beaDNA-binding protein. poor S/B, though they showed some cons are also useful for other kinds of The assay was tested with CAP, a potential for studying ssDNA flexibility experiments. bacterial transcription factor, and its changes as experimental conditions are Usually, the DNA or RNA targets are single-stranded (ss), but Vadim Demidov, PNA a) Maxim Frank-Kamenetopeners skii, and colleagues at dsDNA Boston University and + Boston Probes, Inc., report that molecular beaStem-forming cons can hybridize to douDNA beacon ble-stranded (ds) DNA. F The researchers use a pair b) Q F Q of short peptide nucleic acid (PNA) “openers” + or to expose the target sequence in a section of F dsDNA. These openers Q bind to homopurine sequences on one strand of Stemless the DNA, displacing the PNA beacon complementary strand and Schematic of the hybridization of molecular beacons to a double-stranded DNA target. (a) PNA “openers” exforming short triplex sections. The displaced strand pose the target sequence in a DNA duplex. (b) Molecular beacons bind to the exposed target and generate a fluorescence signal. is available for binding if a molecular beacon or other oligonucleotide is added at binding site. One fragment of the bindvaried. (J. Am. Chem. Soc. 2002, 124, that point. ing site was labeled with a fluorescent 1097–1103) The researchers tested both a stemdonor, and the other was labeled with a In another paper, Tomasz and Ewa forming DNA beacon and a stemless quencher. When CAP was present, the Heyduk at St. Louis University Medical PNA beacon and found that both output of the fluorescent donor was reSchool describe the use of “molecular would bind to the dsDNA target. In duced. To demonstrate the flexibility of beacons” to detect sequence-specific both cases, the signal-to-background the assay, other types of donor and acDNA-binding proteins. However, unlike ratio (S/B) was close to 10 at the fluoceptor labels and other DNA-binding the classic molecular beacon strategy, rescence emission maximum. This is proteins were used. Further testing indithis one does not involve the hybridizacomparable with the S/B for a stemcated that the assay could work with cell tion of oligonucleotide probes to ssDNA forming DNA beacon hybridizing to extracts, not just purified samples. (Nat. or RNA. Instead, the binding site for ssDNA. Furthermore, the PNA beacon Biotechnol. 2002, 20, 171–176) the DNA-binding protein is cut into two could distinguish mismatched targets ds-fragments, and the pieces are tagged from one that was complementary to with fluorescent labels for a fluorescence the beacon. (This test was not conducted with the stem-forming DNA beacon.) resonance energy transfer (FRET) assay. FRET assays use two fluorescent laIn studies on ssDNA targets, the rebels, a donor and an acceptor, which are searchers compared stem-forming and kept apart until a binding event brings stemless DNA beacons with PNA beacons. The performance of the PNA bea- them together. Then, energy is transferred from the donor to the acceptor, cons was independent of the salt con-
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ANALYTICAL CURRENTS Molecules go with magnetic flow Add magnetism to the list of ways to move analytes through liquids in microscale devices. Henry White and colleagues at the University of Utah explore new forms of magnetohydrodynamic (MHD) flow to transport molecules over macroscopic distances in viscous liquids. The researchers showed that microscopic (~50-µm radius) MHD flow tubes were generated in solution in the gap between two 250-µm-radius Pt microdisk electrodes oriented face-to-face when a magnetic field of 1 T was applied. The flow tubes, which extended ~2.5 cm and were stable indefinitely, transported molecules without significant loss from diffusional broadening or convective mixing. The researchers also transported small “packets” of molecules in pulsed flow and formed large (~3 cm2), microscopically thin (25-µm) rotating sheets of solution.
They used a 25-µm-radius Pt microprobe electrode to measure spatial distributions of the electrochemical reactant and product. Although the flow tubes and pulsed flow have no immediate practical application, they may be useful for the controlled delivery of small quantities of chemicals in microfluidic systems. (J. Am. Chem. Soc. 2002, 124, 462– 467)
The MHD flow in solution results from the Lorentz force acting on ions moving through the magnetic field, similar to the force experienced by an electron or ion moving through a magnetic field in a vacuum. White’s group characterized MHD flow in a solution of CH3CN/0.2 M [n-Bu4N]-PF6 containing nitrobenzene but noted that, in principle, any redox system would work.
a)
Less noise for MALDI spectra 2,5-dihydrobenzoic acid (DHB) showed
Ubiquitous “chemical noise” limits the sensitivity of MS peptide spectra, particu-
the least noise, more intense peptide ion
larly if the solution concentration is low or
signals, and better reproducibility under
peaks have a low relative abundance.
the optimized conditions of the MALDI ion
Current methods in the spectrometrists’
trap mass spectrometer used. Diagnostic MS/MS spectra produced
“bag of tricks” decrease S/N but have associated problems. Recently, Brian Chait
a singular pattern for almost every m/z
and Andrew Krutchinsky of Rockefeller
tested—a series of fragments separated
University selected a MALDI matrix sys-
by ~154 Da and ~136 Da from the parent
tem with relatively low background noise
ion. These masses correspond to frag-
and then identified the main components
mentations of clustered intact DHB mole-
responsible for the noise and devised a
cules and DHB that has lost a water molecule. Finding that many DHB clusters
scheme to eliminate them.
fragment at activation energies lower
The team compared ~20 MALDI targets and found that the polycarbonate
than the threshold for peptide ions, the
surface of CDs produced the lowest back-
team framed several approaches for pre-
ground. Of the MALDI matrixes tested,
activating the clusters and thereby im-
b)
0 500
Flow patterns between two 250-µm-radius Pt microdisk electrodes facing each other. (a) Gravity-driven flow without a magnetic field (B = 0) and (b) MHD flow at B = 1 T. 184 A
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∆135.3
700
900
1100 m/z
1518.5
tion broke up DHB clusters and removed about
1300
half the chemical noise background. The result
1382.5 1400.3
1168.6
1156.7
y8
proving the S/N. Collisional preactiva-
∆136.2
∆136.8
b9
1265.0
y7
1042.8
y5
914.3
y9
1655.1
1673.3
0.1 fmol 1 min
661.3
Relative abundance
∆154.6
∆154.8
1537.1
∆136.0
100
was a clear single-stage MS spectrum and a high1500
1700
MALDI MS/MS spectrum of the species at m/z 1673, showing the fragments corresponding to the neurotensin peptide and to loss of the matrix fragments. (Adapted with permission. Copyright 2002 Elsevier Science.)
quality MS/MS spectrum for 100-amol peptide samples. (J. Am. Soc. Mass Spectrom. 2002,13, 129– 134)
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“Magic” NMR locates protein The mechanisms of how peptides and proteins insert themselves into bacterial membranes composed of lipids are virtually impossible to work out without at least some structural information. Using standard solid-state magic-angle spinning NMR equipment, Mei Hong and colleagues at Iowa State University show that a two-dimensional (2-D) technique can help elucidate the structures of membrane-bound proteins. Currently, there are several ways to use NMR for analyzing the orientation topology of proteins and peptides bound to bacteria. However, these methods need complex labeling and micelles that may not appropriately mimic lipid bilayers, are difficult to perform on large membrane proteins, or require low temperatures. Instead, the researchers perform 2-D 1 H spin-diffusion experiments that track the magnetization transfer of methyl protons at the end of the lipid chains to
the protein. Using this NMR data and numerical simulations called spindiffusion curves, they can (b) (a) determine the protein’s depth inside or position CH3 on the membrane. They tested their technique on a 13C-labeled colicin 1a— an E. coli protein that kills competing bacterial Proteins can either (a) be embedded in or (b) lay across a strains—which has a lipid bilayer. However, the transfer of NMR magnetization membrane-embedded differs. (a) Thick arrows show the efficient pathway for domain and a DNA/ magnetization transfer from the lipid chain termini to a procationic lipid complex that tein in close proximity. (b) Thin arrows show a slow, ineffihas surface-bound DNA cient magnetization transfer over a long distance. rods. They find that more colicin residues are found on the membrane surface nique for small peptides, they do recthan inside the bilayer. ommend 31P NMR of lipids for collectExperiments are designed for 13C or 15 31 N detection in the protein or P deing data quickly without 13C enrichment tection in the DNA. Although the reor similar isotope labeling. (J. Am. Chem. searchers don’t recommend the techSoc. 2002, 124, 874–883)
Mass spectrometer is a space “Dustbuster” J. L. Beauchamp and colleagues at the California Institute of Technology have designed an impact-ionization time-of-
flight mass spectrometer dubbed the “Dustbuster”, which analyzes cosmic dust for deep space missions. It weighs only 0.5 kg. The cylindrically symmetric Dustbuster is only 10 cm in diameter and 20 cm in length and has a 65-cm2 copper target plate area with a reflectron. Beauchamp’s group reports that the instrument is considerably smaller than previous in situ dust analyzers. The instrument simultaneously optimizes mass resolution, particle detection, and ion collection. The researchers see the new mass spectrometer as an answer to the National Aeronautics and Space Administration’s emThe Dustbuster mass spectrometer. (Adapted with permisphasis on developing smaller, sion. Copyright 2002 American Institute of Physics.) lighter, and lower-powered
instruments for space missions. Dust particles enter through the front grid of the Dustbuster, hit the target plate, and are partially ionized by the impact. The ions, which have broad energy and angular distributions, are then accelerated through a modified reflectron, which focuses ions of specific m/z. Then the ions leave the reflectron, pass through the drift tube, and are detected by a 5-cm2 microchannel plate detector. The researchers tested their Dustbuster using laser desorption ionization to simulate dust impacts. From these experiments, they reported mass resolutions (m/⌬m) ranging from 60 to 180. The mass spectrometer, they add, can be combined with other instrument components to determine dust particle trajectories and sizes. A tantalum plate for dust impact studies is planned. (Rev. Sci. Instrum. 2002, 73, 185–189)
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ANALYTICAL CURRENTS Fluorescence polarization tracks ligands The binding of lipophilic ligands with nuclear receptors affects many aspects of human physiology, including activating or repressing gene expression and treat-
ing diseases. Identifying specific ligand– receptor pairs can lead to a better understanding of physiological processes. Siqi Lin, Cindy Bock, and colleagues at DuPont Pharmaceuticals in Wilmington, Del., and 250 Matrical, Inc., in Chadds Ford, Penn., have devel200 oped a reproducible fluorescent polarization (FP) 150 screening method for ligand–receptor binding. 100 The new technique handles high-throughput 50 assays of crude cellular 0 extracts. 1 2 3 4 The researchers moniMillipolarization value of dexamethasone fluorescein probe tor the binding of probe (1) in buffer solution; (2) in glucocorticoid receptor lysate; ligands with receptor mol(3) in glucocorticoid receptor lysate with unlabeled dexamecules by measuring a ethasone; and (4) in crude non-glucocorticoid receptor lysate. change in polarization. (Adapted with permission. Copyright 2002 Elsevier Science.) When low-molecularMillipolarization units
weight probe fluorophores are excited by plane-polarized light, their rapid rotation depolarizes the emitted light, and a low polarization value is recorded. However, when a probe binds with an appropriate nuclear receptor, the highmolecular-weight pair tumbles more slowly and causes the polarization to increase. In a model system of a glucocorticoid receptor with a dexamethasone fluorescein (Dex-fl) ligand, the lowest concentration with a reasonable fluorescent intensity was 5 nM for Dex-fl. The sensitivity of FP analysis was demonstrated by adding competing ligands to the system. Dex-fl binding decreased with the increasing concentration of competitors and in an order of affinity that is consistent with a previously established radiolabeled filter binding assay. (Anal. Biochem. 2002, 300, 15–21)
CE tackles DNA adducts Covalent attachment of an agent, such as
migration time reproducibility, the re-
step, but the high detection limit is already
a benzo[a]pyrene, to DNA is often an early
searchers use the deoxyadenosine phos-
sufficient to determine many endogenous
step in carcinogenesis; thus, analysis of
phate dAMP as a standard and show that
adducts. (Angew. Chem., Int. Ed. 2002,41,
these difficult-to-detect complexes is im-
times can be determined with a standard
445–448)
portant. Oliver Johannes Schmitz and
deviation of < 3%. Peak
colleagues at the Deutsches Krebsfor-
heights are measured
schungszentrum Heidelberg (Germany)
using fluorescein as an
introduce a new method for determining
internal standard.
DNA adducts using micellar electrokinetic cence detection, and fluorescent labeling
ing a detection limit of
dCMP
1 dGMP
850 pM. That translates
AP site
8-HO-dGMP
to two DNA adducts
apurinic sites and various endogenous and
per 106 unmodified nu-
0
exogenous DNA adducts in several types
cleotides. This number
12
of samples, such as oligonucleotides and
could be improved, say
human DNA. The same conditions for sep-
the authors, by using
aration and derivatization are used in all
electrostacking with re-
analyses. Moreover, to overcome CE’s poor
versed field as a focusing
186 A
dTMP
2
Frel
lacks is sensitivity, yield-
The method is selective, detecting
dAMP
What the method
chromatography, laser-induced fluoresof nucleotides.
3
A N A LY T I C A L C H E M I S T R Y / A P R I L 1 , 2 0 0 2
14
16 t /min
18
20
Analysis of apurinic sites and the difficult-to-detect hydroxy deoxyguanosine phosphate 8-HO-dGMP, which is a marker of oxidative stress. (Adapted with permission. Copyright 2002 Wiley-VCH Verlag.)
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RESEARCH PROFILES The unexpected gas-phase Raman spectrometer Chen. The technique also requires relaChemistry (pp 1618–1623), Chen and In 1999, Peter Chen, Candace Joyner, his co-workers describe their new method tively long collection periods, which and students Sheena Patrick and Kanika poses problems in analyzing samples as ideal for spectroscopic imaging, comBenton ran into “accidents” in Chen’s that change over time. Chen knew that bustion diagnostics, pump–probe speclaboratory at Spelman College in Atthe collection time of CARS, which may lanta, Ga. The mishaps became blessings troscopy, and other applications that be used to increase the intensity of a may benefit from high spectral, spatial, in disguise. Raman signal and overcome spectral inand temporal resolution. One accident happened when the reterference, could be dramatically reThe spectrometer uses a degenerate searchers tried to fix a loose hydrogen OPO as the broadband source that cov- duced using multiplex methods. But gas valve on a Raman shifter as they finding an adequate broadband source ers a continuous range of >3000 cm–1. worked to increase its output and build of light for multiplex CARS had been The OPO is pumped at 683 nm by the a new gas-phase Raman spectrometer. backward SRS from the hydrogen Raman an obstacle. Chen began thinking that “We went to tighten this little valve a shifter. When the beam from the broad- perhaps using the degenerate OPO little bit, and as we had the wrench and could solve the problem. band source is added to the 532-nm were tightening this valve, the power Finding out about the measurements were flucbackward SR S that pumps tuating,” recalls Joyner. OPO the OPO at 683 nm was For Chen, the moment Sample Raman cell D D also a “lucky” accident. was distressing. “At first, One day, when he walked you know, when someL L F L L into the lab, Chen noticed body does something in W Monochromator the bright beam going an experiment, and it with CCD backwards toward the starts making the readings original pump laser. “We go kind of crazy, your first hadn’t planned on actually tendency is to say, ‘Stop Nd:YAG laser M M using that,” he says. “The doing that!’” reason it was a really nice Layout of the multiplex OPO CARS: D, dichroic mirror; F, filter; L, lens; But then Chen and his accident is because it turns M, mirror; W, wedged window. team began asking why out that backward stimuthe Raman shifter output lated Raman scattering was so sensitive to adjustprovides a really high quality beam, and narrow-band beam from an Nd:YAG ments on the valve. They fiddled with for OPOs you need a very high quality laser, a new beam that contains the the instrument and discovered that the Raman signal is created. The signal’s in- pump beam.” cause of the fluctuations wasn’t the The applications for his new method tensity is amplified by the nonlinear opvalve but the Raman shifter. As they look promising to Chen. The National tical effect, explains Chen’s group. The moved the Raman cell with respect to the original pumped laser beam, the en- 0.5 g per serving would have the asterisked footnote, “*Includes __ g trans fat.” If a product contained < 0.5 g trans fat and < 0.5 g saturated fat per serving, the label could claim the product is “trans fat-free.” The proposed FDA rule on trans fat content also would
Paul Pirozzola of Leco Corp. Magdi Mossoba of the Center for Food Safety and Applied Nutrition and colleagues have developed an attenuated total reflection IR method that quantitatively measures trans fat content. The American Oil Chemists’ Society (AOCS) and AOAC International have adopted the test as an official method. “The new FTIR methods are a big advance, especially in terms of speed of analysis and sample preparation,” says Frederick van de Voort of McGill University. The alternative is GC, but Ratnayake says that this technique is more sensitive than necessary and takes more time. However, GC is necessary to separate and identify specific trans fatty acids. Scientists also seem to be particularly interested in supercritical fluid extraction (SFE) methods for optimizing total fat analysis and possibly as a sample preparation technique. Phillip Liescheski of Isco, Inc., and colleagues have investigated online SFE coupled to FTIR using shortening and margarine as test samples. Pirozzola adds that Leco is in the process of validating an SFE method for trans fat analysis per AOCS and AOAC standards. Jocelyn Alfieri of Silliker (Canada) says that low-volume or solvent-free extractions such as SFE could be used for trans fatty acids and other nutritional components. However, the equipment is expensive, and the procedures depend on the particular food. Finally, Dane Bicanic of Wageningen University and Research Centre (The Netherlands) and colleagues are investigating thermal lens spectrometry in combination with HPLC as a method to detect trans fatty acids. a –Laura Ruth PHOTODISC
What will a consumer be reading on a processed food label about the trans fat content of a food item? Both the U.S. and Canadian governments have been revising nutrition labels to include information about trans fatty acids, which are made through the hydrogenation process that solidifies liquid oils. According to the U.S. Food and Drug Administration (FDA), the food policy initiative of adding trans fat content to the label of processed foods, such as crackers, cookies, and snack foods, is currently on FDA’s “B” list of priorities and is to be finalized as a rule in 2002 or 2003. FDA has delayed finalizing the trans fatty acid labeling initiative, originally proposed in 1999, to wait for the results of an upcoming study on cholesterol that is being jointly drafted by the National Institutes of Health (NIH) and the U.S. Department of Agriculture, says Ann Wilkes of the Snack Food Association. Scientific studies have indicated that consuming trans fats contributes to increased blood levels of low-density lipoprotein, or “bad” cholesterol, which can increase the risk of coronary heart disease. FDA is under pressure to recognize the problem, and the proposed rule is in response to a petition submitted by the Center for Science in the Public Interest in 1994. According to William Ratnayake of the Canadian Bureau of Nutritional Sciences, both the FDA and Health Canada have proposed food-labeling rules that require the amount of trans fat per serving to be added to the amount of saturated fat per serving. Thus, the amount and percent daily value per serving on the nutrition facts panel
change cholesterol claims, and products that are labeled “low saturated fat”, “reduced saturated fat”, “lean”, or “extra lean” would have to account for the amount of trans fat. Anticipating a need for easy methods to quantify trans fatty acids in food, researchers have been developing new laboratory methods. “We believe that the renewed interest was spurred by the federal regulations for nutritional labeling approximately two years ago,” says
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