Posts Tagged ‘Near-Infrared Spectroscopy’

Advance in hyperresolution spectroscopy

Larry H. Bernstein, MD, FCAP, Curator



New Indium Antimonide-Based IR Detectors Surpass Previous Limitations

Photonics Spectra    Apr 2016

Advances in indium antimonide (InSb) infrared detectors, detector cooling and system design have resulted in a new generation of application-specific cameras where either SWIR or broadband response is critical.


Due to their high thermal sensitivity and favorable atmospheric transmission in the 3- to 5-µm spectral region, cameras based on InSb sensors have been used in traditional applications such as medium- to long-range surveillance, as well as for scientific and research requirements where detection of very small temperature differences or wavelength-specific imaging is beneficial. With recent developments, InSb focal plane arrays (FPAs), when properly passivated, have become a viable option to extend the wavelength response of today’s cameras below 3 µm, into the short-wave infrared (SWIR) spectral region, visible and even ultraviolet wavelength regions. This broadband response provides the ability to solve a variety of challenges with a single instrument, and to build application-specific systems that surpass limitations of other infrared imaging technologies.
FPAs are in leadless chip carriers
Three passivated but otherwise uncoated InSb focal plane arrays with spectral responses from <200 to >500 nm. FPAs are in leadless chip carriers. Courtesy of IRCameras.
Detector advancements

Indium gallium arsenide (InGaAs) sensors are the most common choice for imaging in the SWIR from 900 to 1700 nm due to their high quantum efficiency, ability to operate at or near room temperature, and relatively low production and packaging costs. “Visible InGaAs” sensors, with a response from 400 to 1700 nm, have also proliferated in recent years. InGaAs sensors, however, have been limited to an upper wavelength cutoff at 1700 nm due to difficulties in achieving uniform pixel-to-pixel response in two-dimensional s at longer wavelengths above 1700 nm.

Unfortunately, this leaves these sensors blind in the wavelength region from 1700 to 2500 nm, which contains significant information for both military and civilian applications. Additionally, InGaAs sensors are often subject to image lag or retention when used in very low-light-level applications requiring high-gain states in the sensor.

With advancements in the production of InSb focal planes, infrared cameras today can be equipped with FPAs providing a wide broadband spectral response unparalleled by other production sensors. Advances in the passivation of InSb FPAs eliminate surface states that retain charge when the detector is exposed to higher energy photons, thus removing the possibility of after-images and image lag, which has been an issue for both InSb and InGaAs sensors.

Typical spectral response of InSb FPA with antireflective coating for <1 to >5 µm.
Typical spectral response of InSb FPA with antireflective coating for <1 to >5 μm. Courtesy of IRCameras.  http://www.photonics.com/images/Web/Articles/2016/3/7/Detectors_Spectral.jpg

Typically, the quantum efficiency of InSb focal planes prior to the application of an antireflection (AR) coating is about 60% from UV wavelengths to approximately 5000 nm. Improved AR coating processes increase the quantum efficiency to greater than 90% over the wavelength range from 1 to >5 µm. More specialized coatings are available that extend the lower wavelength cut to 400 nm and below, resulting in a single sensor that can be utilized for a broad range of imaging applications.

Finally, the use of digital readout integrated circuits for InSb FPAs results in performance improvements over more traditional analog-based FPAs. Digital InSb FPAs require lower power, eliminate pixel crosstalk, are resistant to blooming and simplify downstream electronics by digitizing data within the FPA instead of requiring an analog-to-digital converter external to the FPA.

InSb sensors with the capabilities

described above are available today in formats ranging from quarter video graphics array (VGA) (320 × 256) to high definition (1280 × 1024), with even higher-resolution (2520 × 2048 and beyond) FPAs just over the horizon.

Cooling InSb sensors

InSb sensors require cryogenic cooling to become photoconductive. Typically, InSb sensors are cooled to about 77 K, though for some applications, including imagers intended for SWIR applications, further cooling of the sensor to less than 70 K is desirable to reduce noise due to dark current.

To cool InSb sensors to the cryogenic temperatures necessary, the focal planes can be integrated into either liquid nitrogen (LN2)-cooled Dewar assemblies, or closed-cycle integrated Dewar cooler assemblies (IDCAs). There are, of course, advantages and drawbacks to each approach.

Cut-away view of LN2-cooled Dewar configured with filter wheel.

Cut-away view of LN2-cooled Dewar configured with filter wheel. Courtesy of IRCameras.   http://www.photonics.com/images/Web/Articles/2016/3/7/Detectors_Dewar.jpg

InSb cameras based on a LN2-cooled Dewar assembly offer the ultimate flexibility for users who may wish to modify the system as application requirements change, or to demonstrate a proof-of-concept before acquiring a closed-cycle cooled system that cannot easily be reconfigured. With an LN2-cooled system, it is possible for the user to easily change cold filters, apertures (ƒ-number) and even the optical back working distance to accommodate a variety of optics, or to define and test the interface to spectrometers and other instruments into which a camera may be integrated. It is also easy to replace FPAs in an LN2-cooled camera to allow upgrades to different format sensors or even different sensor materials.

LN2-cooled systems are also vibration-free, which can be an important consideration when used as an OEM component in a highly sensitive metrology product such as an interferometer. LN2-cooled cameras may also be equipped with motorized multi-position cold filter wheels, allowing the use of spectral filters cooled to cryogenic temperatures to limit their thermal emission and prevent the filter’s emissions from affecting measurements.

Image of Antares/Cygnus liftoff Sept. 18, 2013
The disadvantage of LN2-cooled InSb cameras is that they require replenishment of the LN2, either by filling the Dewar manually, or through the use of a bulky tank and autofill system. For those applications that require cooling below 77 K, the boiling point of LN2, it is possible to lower the temperature of the LN2 by increasing the vacuum above the LN2 reservoir in the Dewar. When doing so, however, the temperature of the LN2 may fluctuate with atmospheric temperature changes or with fluctuating vacuum pressure changes in the liquid nitrogen reservoir, resulting in measurement inconsistencies. For some applications, including astronomical observations, researchers will run InSb sensors at much lower temperatures, below the 63 K freezing point of nitrogen, obviating its use as a cooling medium.

Closed-cycle Stirling coolers provide a method for cooling InSb and other cryogenic cameras designed for continuous operation. These devices remove heat from the detector through the expansion and contraction of helium gas, and are intended for environments where the use of LN2 is not feasible, or for applications where the sensor temperature must be driven below 63 K. Until recently, most cameras so configured incorporated a rotary Stirling cooler, for which manufacturers would historically claim a limited mean time to failure (MTTF) on the order of 8,000 hours. Actual experience, however, has demonstrated this figure to be optimistic. Stirling cooler lifetime is impacted by operating conditions — continuous use in high ambient temperatures and repeated power cycling of the cooler tend to negatively impact MTTF.

Flexure bearing Stirling cooler
Over the past several years, the introduction and maturation of linear Stirling coolers based on flexure bearing technology has resulted in significant improvements in cooler MTTF. Today, a Stirling cooler with a flexure bearing dual opposed piston compressor routinely provides 25,000 hours of operation. Additionally, linear coolers are significantly less prone to vibration than rotary coolers, making the linear cooler a more desirable choice for precision metrology instruments.

An important consideration for IR cameras based on closed-cycle Stirling coolers, whether rotary or linear, is that once the system is built, it is difficult to change its configuration. The detector, cold shield, cold finger and cold filter are housed in a metal Dewar that is welded closed, and must be cut open in order to make changes or effect repairs. Great care must be taken when cutting open a closed-cycle Dewar to ensure that damage is not inflicted on any of the internal components. Also, with the exception of highly specialized custom built systems, a typical closed-cycle IDCA does not support the use of a cold filter wheel assembly due to the increased cooling capacity required by the cold wheel and increased Dewar volume. This limits the spectral response of the system to that defined by the combination of the sensor and cold filter installed in the Dewar. The result is a purpose-built, continuously operable product that can be used either as a stand-alone product, or as an OEM component for integration into an end-user system.

Today’s infrared imaging systems based on high-performance InSb FPAs offer unmatched sensitivity for a variety of application-specific requirements where either SWIR or broadband spectral response is essential. Understanding available options for sensor resolution and coatings that drive the FPA spectral response; cooling technologies and their associated conveniences; optical filters that define system spectral response; and optical designs that describe how the system interacts with the outside world are critical considerations in the selection of these highly enabled SWIR/MWIR imaging cameras.

Meet the authors

Elliott Rittenberg serves as vice president of sales and marketing at IRCameras LLC in Santa Barbara, Calif.; email: elliott@ircameras.com. Len Kamlet, Ph.D., is product development manager at IRCameras, and has been working exclusively with infrared technology for 14 years; email: len.kamlet@ircameras.com. Arnold Adams, Ph.D., is chief technical officer at IRCameras; email: arn.adams@ircameras.com.

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Dynamic Protein Profiling

Larry H. Bernstein, MD, FCAP, Curator


Dynamic profiling of the protein life cycle in response to pathogens

The protein lifecycle is regulated by mRNA expression, translation,
and degradation. Image courtesy of Broad Communications.

Cellular protein levels are dictated by the net balance of mRNA expression (the type of RNA that provides genetic information for proteins), protein synthesis, and protein degradation. While changes in protein levels are commonly inferred from measuring changes in mRNA levels (due to the difficulties involved in measuring protein levels), it’s not often clear whether determining RNA levels is actually a good proxy for measuring protein levels.

In their recent article in the journal Science, Broad Institute researchers working in core member Aviv Regev’s and institute member Nir Hacohen’s laboratories, along with the Broad’s Proteomics Platform led by Steve Carr describe a quantitative genomic model that lets them explain the abundance of proteins in cells based on mRNA expression, translation, and degradation. They performed their study in mouse dendritic cells stimulated with LPS, a component of bacteria.

While previous studies had looked at global levels of regulation in rapidly-dividing, unstimulated cells, this work focuses on understanding how much of the change in protein levels is due to a change in mRNA expression, translation, and degradation in specific genes and classes of genes in response to a stimulus – in this case, LPS. For example, would the changes in levels of one class of proteins be mostly driven by changes in the levels of the mRNAs that encode them? On the other hand, would changes in the levels of other groups of proteins occur without changes in mRNA, but rather due to faster translation or slower degradation of the protein? These were the type of questions the scientists were interested in.

Explains co-first author Marko Jovanovic, “Can we, in a dynamic system, integrate RNA and protein life cycle data? People rarely do this, and never systematically. Can we really make a global model of gene expression where we know, in the end, how much each type of regulatory layer is contributing to each gene? You can get a global answer too, but straight percentages of global contribution of RNA levels and the protein life cycle to final protein levels was not my goal. My question was really, do we see that certain classes of genes are controlled one way and certain other classes another way and therefore gain new regulatory insight?”

Since changes in protein levels are not as dramatic and fast as changes in RNA levels, one of the greatest challenges they faced in their study was distinguishing actual signal from noise. Co-first author Michael Rooney explains how they tackled this problem: “While the quantitative accuracy of mass spectrometry has grown tremendously, we realized that statistical strategies for handling stochastic and systematic errors in the data would still be critical to getting correct results. As a first step, we developed a generative statistical model for the data. This allowed us to leverage the entire time course in a manner that was robust to missing values and stochastic variation. Second, we saw that the contribution of translation might be over-estimated if we allowed translation rates and protein levels to be calculated from the same experimental system, because in such a case they would both be confounded by the same systematic errors, making them appear more similar than they actually are. This led us to the novel strategy of creating biological replicates prepared by distinct peptide library protocols.”

In this way, the team was able to robustly build a dynamic model in which the mRNA synthesis rate, the translation rate and the protein degradation rate change over time. Based on this model, it was possible to predict how much of each of the three types of regulation are contributing to the change in the level of each protein and from that measure both globally, per gene class, and per gene, the relative contributions of each type of regulation.

Analyzing the LPS-stimulated dendritic cells, the researchers found that overall mRNA expression dominates the regulation strategies, accounting for up to 90% of the fold changes in protein level variation. This is a significant increase from their pre-stimulation measurements showing regulation of mRNA expression contributing 60-70%, translation 15-25%, and degradation also 10-20%.

What appeared to be regulated more substantially by the protein lifecycle (translation, degradation) were highly expressed genes. And, looking at changes in the number of protein molecules rather than just the relative fold changes in pre- versus post-stimulated cells, what emerges is that post-stimulation, regulation at the level of the protein lifecycle begins to dominate.

The findings lead to a model for the LPS-stimulated system in which protein expression associated with functions critical for a dendritic cell-specific functions is taken care of by regulation at the level of RNA expression. However, the readjustment of the pre-existing proteome when the cells enter a new state (for example, in response to pathogen stimulation) is controlled via regulation of the protein life cycle (translation, degradation) rather than RNA expression.

“We termed this the ‘cupcake model’,” says Jovanovic. “You have to forgive me, this is my European view on how I see people buy cupcakes. They go into the store and choose the cupcake based on the icing, so the icing is kind of the identity of the cupcake. So from one cupcake to another you are basically changing the icing. In our model, the identity of cell states is adjusted by mRNA regulation so mRNA regulation is basically contributing to the icing. However, there’s also the cake part. The cake part is often specifically adjusted to the icing on top of the cupcake. The cake part, analogous to “housekeeping genes’, also needs to change and this is mainly through the protein life cycle. I’m very biased because I don’t like the icing on cupcakes, just the cake part, and so in the same vein, I wanted to know more about how the protein lifecycle contributes to gene expression. I think people have focused too much on the icing. “

So, mRNA changes drive new cell state identity. Protein lifecycle regulation drives readjustment of preexisting “housekeeping genes” such as those encoding ribosomes and factors involved in metabolism to adjust the cell to its new state.

This approach is extensible to test the regulation of gene expression in other perturbed systems as well, and allows researchers for the first time to assess the relative contributions of each of the three levels of protein level regulation – mRNA expression, translation, and degradation – in any perturbed system.

Paper cited: Jovanovic, M et al. Dynamic profiling of the protein life cycle in response to pathogens.Science. Feb. 12, 2015. http://dx.doi.org:/10.1126/science.1259038

More Dynamic Protein Profiling

To Capture Fleeting Expressions, Go High-Throughput

  • One of the unexpected findings of the Human Genome Project was that human chromosomes contain only 20,000–25,000 protein-encoding genes, fewer than had been anticipated, …

Transitioning from Traditional Assay Formats to HTRF Technology 
Sensitivity of Fluorescence Coupled to Low Background of Time Resolution

Researchers are working on novel adaptations of HTRF-based assays, as well as their combination with other types of assays, to characterize complex disease pathways that may present multiple drug targets for disease therapy. [iStock/ponsulak]

  • At the 6th Cisbio HTRF symposium, “Charting the Course of Drug Discovery” held recently in Brewster, MA, investigators described how homogeneous time-resolved fluorescence (HTRF®) continues to expand and improve upon the repertoire of available bioassay formats for basic research and drug discovery. Participants described applications of these assays as integral components in studies ranging from identification of allosteric modulators as potential drugs to determination of critical components in protein-modifying biochemical pathways as new drug targets.

    A form of time-resolved fluorescence energy transfer (TR-FRET) technology, HTRF brings together the sensitivity of fluorescence with the homogeneous nature of FRET and the low background of time resolution. As in other FRET systems, HTRF uses two fluorophores—a donor and an acceptor that transfer energy when in close proximity to each other. Excitation of the donor molecule by an energy source such as a laser causes the emission of light waves at donor-specific wave lengths.

    If the donor and acceptor are not within proximity to each other, the donor is excited but no energy transfer occurs and no acceptor emission occurs. Dual-wavelength detection reduces buffer and media interference, and the final signal is proportional to the extent of product formation.

    The HTRF assay can be miniaturized into 384- and 1536-well plate formats, which proponents say, can save reagent costs and minimize quantities of limited target and compound material used in the assay. This assay technology has been applied to many antibody-based assays, including GPCR signaling (cAMP and IP-One), kinase, cytokine, biomarker, and bioprocess (antibody and protein production), as well as assays for protein-protein, protein-peptide, and protein-DNA/RNA interactions.

    Unlike traditional TR-FRET systems that employ fluorophores such as fluorescein and rhodamine that are characterized by immediate and transient emissions, HTRF-specific donors such as europium and terbium cryptate emit relatively long-lived fluorescence upon excitation. Conversely, acceptor molecules rapidly emit fluorescence.

    Thus, the nonspecific short-lived background fluorescence that occurs in FRET assays can be reduced by introducing a time delay ranging from 50-150 microseconds between the initial donor excitation and measurement. In HTRF, therefore, if the donor and acceptor molecules are not within proximity, only donor emissions are detected following a time delay.

    Participants at the symposium focused on novel adaptations of HTRF-based assays, as well as their combination with other types of assays, to characterize convoluted disease pathways that may present multiple drug targets for disease therapy, especially neurodegenerative disorders. In particular, several presenters noted its use in addressing what the conference keynote speaker, Terrance Kenakin, Ph.D., of the University of North Carolina, characterized as “The Perfect Storm” of pharmacology, receptor allostery, and biased signaling. Strictly defined, allosteric molecules regulate proteins by binding to the molecule at a site other than the protein’s active site.

    With regard to the seven transmembrane receptors (7TMRs) also known as G protein-coupled receptors, Dr. Kenakin noted that GPCRs comprise the largest class of receptors in the human genome and are common targets for therapeutics. Originally identified as mediators of 7TMR desensitization, β-arrestins (arrestin 2 and arrestin 3), for example, are now recognized as true adaptor proteins that transduce signals to multiple effector pathways. The introduction of molecular dynamics coupled with new assays, including HTRF, he said, opened new vistas for 7TMRs as therapeutic entities. Specifically, probe-dependent allosteric vectors oriented toward the cell cytosol provided fertile ground for new 7TMR drugs in the form of ligand-producing biased signaling.

    Discovering and Characterizing Allosteric Modulators  

    Positive and negative allosteric modulators (PAMs and NAMs) of GPCRs have emerged as a novel and highly desirable class of compounds, particularly in potential treatment for mental disorders, and for metabolic, neurodegenerative, and neuromuscular diseases. Advocates say they offer some distinct advantages over conventional competitive compounds, including the potential for fine-tuning of GPCR signaling and the promise to address formerly intractable targets.

    Introduced to the market in 2010 for the treatment of secondary hyperparathyroidism in adult patients with chronic kidney disease on dialysis, Cinacalcet, a PAM, activates the calcium-sensing receptor that functions as the principal regulator of parathyroid hormone secretion. Cinacalcet is the first clinically administered allosteric modulator acting on a GPCR, and provided a proof-of-concept for future development of allosteric modulators on other GPCR drug targets..

    Hayley Jones and Jeff Jerman, both of Medical Research Council Technology (MRCT) in the U.K., talked about the characterization of novel PAMs for the dopamine 1 receptor. Although preclinical and clinical data have validated this receptor as a target for drugs to improve cognitive impairment in schizophrenia, Jones noted that, to date, attempts to clinically develop agonists have failed.

    She and her colleagues have approached this problem by targeting D1R via PAM saying that in contrast to “direct” orthosteric D1R agonists, PAMS potentially offer advantages, including physiological spatiotemporal control of dopamine function by enhancing the effect of its endogenous ligand and avoiding over stimulation by self-limiting effects.

    The investigators said they had configured a cell-based HTRF assay to screen a subset of an MRCT compound library using CHO cells that transiently express the human receptor. Inclusion of a submaximal concentration of dopamine in the assays facilitated simultaneous detection of both PAMs and agonists, allowing them to identify novel D1R activators.

    Michelle Arkin, Ph.D., of the University of California, San Francisco, focuses her research on developing small molecule modulators of allosterically regulated enzymes and protein complexes as potential drug leads. Neurodegenerative diseases such as Alzheimer’s and other “taopathies” are characterized by formation of intracellular tangles comprised of aggregated tau proteins. Previous studies have shown that the protein actyltransferase p300 acetylates tau at several sites, competing with ubiquitination and thereby inhibiting tau degradation.

    Dr. Arkin and colleagues developed a high-throughput screen using HTRF to identify p300 inhibitors, designing a suite of counter screens and secondary assays to validate hits. Based on previous findings that the protease caspase-6 clips tau at specific sites and that truncated tau forms are associated with disease progression, the investigators developed selective caspase-6 inhibitors.

    HTRF assays demonstrated, she said, that small molecule compounds inhibit caspase-6 mediated cleavage of tau in cell lysates, concluding that the combination of HTRF enzymatic and biophysical assay formats allow characterization of inhibitors of proteins that may be involved in tauopathy progression.

  • Lack of Suitable Assays

    Martha Kimos, biochemist at the Lieber Institute, noted that the discovery of novel catehechol-o methyltransferase (COMT) inhibitors for use in the treatment of Parkinson ’s disease has been limited due to lack of suitable assays for high-throughput screening. COMT inhibitors like entacapone and tolcapone prolong the action of levodopa by preventing its demethylation by COMT.

    Kimos and her colleagues developed an HTRF assay involving an enzymatic step that uses membrane-bound human COMT as an enzyme substrate and an assay step that measures s-adenosyl-L-homcysteine (SAH) as an enzymatic reaction product. To directly measure SAH release, an anti- SAH antibody labeled with terbium cryptate and a SAH-d2 tracer were used. The SAH released by the enzymatic reaction competes with the SAH-d2 labeled leading to a decrease of the HTRF signal. The assay, the researchers said, showed good potency for tolcapone, with a high degree of translation between data in fluorescence ratio and data in terms of SAH produced, and suitable for kinetic studies, including Km determination.

    At Pfizer USA, Richard Frisbee, a scientist in the hit discovery and lead profiling (HDLP) department, and colleagues have focused on the development of HTS whole blood assays using HTRF, particularly to monitor anti-inflammatory drug potency. They noted that traditional whole-blood formats such as ELISAs for detecting cytokines require multiple assay plate manipulations, including wash steps and incubation steps, have limited throughput, and are relatively time consuming.

    They reported that they had developed a sandwich immunoassay protocol that measures cytokine production in human whole blood in a 384-well format, describing key elements of the assay, including nanoliter spotting of test compounds, miniaturized blood/reagent transfer, and optimized assay incubations. Development of a relatively convenient assay to monitor compound potency in whole blood can facilitate they said, the prediction of compound doses required for therapeutic efficacy.

    Inhibiting the enzyme γ-secretase, which converts amyloid precursor protein to β-amyloid , thus preventing its accumulation in the brain, has been a goal of drug developers.

    Most recently, Bristol-Myers Squibb elected to discontinue development of its inhibitor candidate avagacestat into Phase III trials after disappointing Phase II results. BMS remains in the hunt for drugs to treat Alzheimer’s disease. Despite clinical failures of its and other companies’ other gamma secretase inhibitors, researchers continue to search for next-generation compounds they believe may succeed.

    At BMS, Dave Harden, Ph.D., principal scientist and team leader, biochemical screening in the leads discovery and optimization group, has developed novel assays to identify molecules that inhibit secretase by measuring multiple amyloid beta species in cell supernatant. He and his team have capitalized on terbium cryptate’s properties as a donor fluorophore in HTRF, that has different photophysical properties compared to the donor fluor europium. These properties afford the opportunity to measure more than one interaction within a well due to the multiple emission spectra observed upon excitation. It can therefore serve as a donor fluorophore to green-emitting fluors because it has multiple emission peaks including one at 490 nm as well as the typically used 665 nm (red) emission.

    Dr. Harden and colleagues, in order to “enhance” their screening practices by expanding well information content, enabled two color multiplexed HTRF in multiple settings in large (>1 MM well) screening campaigns. This approach, they reported, successfully identified mechanistically distinct gamma secretase inhibitors by measuring multiple amyloid beta peptide species in cell supernatants. This, and several other examples, the presenters said, demonstrated the power of multiplexed HTRF in maximizing screening outcomes.

    Across the board, meeting presenters demonstrated the flexibility of HTRF assays and their adaptability to multiple research settings. The scientists pointed out that the assays yielded values consistent with other assay results using less versatile and convenient assays formats.

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The Colors of Life Function

Writer and Curator: Larry H. Bernstein, MD, FCAP 

2.5.1 Type 1 Copper Proteins

The Cu(II) state of this category has an intense blue color due to a thiolate ligand
to Cu(II) charge transfer, and unusual EPR properties arising from the asymmetrical
Cu site (distorted trigonal-pyramidal). The proteins all have a low molecular
mass and have, so far, rather arbitrarily been divided into sub-groups, such as
azurins, plastocyanins, pseudoazurins, amicyanins and various other blue
proteins. Of these the azurins, amicyanins, pseudo-azurins and plastocyanins
apparently have similar copper coordination by two histidine, one cysteine and
one methionine residue. Where the function of Type I copper proteins is known,
it is invariably electron transfer. As yet the names for these proteins are all trivial
and are often derived from source, function or color. The different classes are
usually discerned on the basis of their primary and tertiary structure.

The first bacterial blue proteins to be described were called azurins. Rusticyanin is
another example of a bacterial protein. It has unusual properties with a reduction
potential of 680 mV, and is functional at pH 2. The azurins have well-defined electron
-transfer functions.

The so-called pseudo-azurins differ from the azurins in the N-terminal amino acid
sequence and the optical spectra, which resemble those of plastocyanins.

The blue proteins known as plastocyanins occur in plants, blue-green and green
algae. Their electron transfer role is well defined, i.e. from the bc1 complex
(EC to the photooxidized P-700.

Amicyanins are electron carriers between methylamine dehydrogenase and
cytochrome c, with a characteristic amino acid sequence.

Of the remaining blue proteins stellacyanin is a well- known example. Umecyanin,
plantacyanin and mavicyanin are also considered to belong to this group.
Although these proteins undergo redox reactions in vitro, their true biological
function remains unknown. Most of these proteins exhibit an unusual EPR signal
in which the copper hyperfine splitting pattern is poorly resolved. There is good
evidence that at least for stellacyanin, methionine does not function as a ligand
for copper.

2.5.2 Type 2 Copper Proteins

The copper centres in these proteins are spectroscopically consistent with square
planar or pyramidal coordination, containing oxygen and/or nitrogen ligation.
The Cu(II) is EPR active, with a ‘normal’ signal. There is no intense blue color.
This group includes the copper/zinc superoxide dismutase (EC,
dopamine b-monooxygenase (EC, galactose oxidase (EC
and the various copper-containing amine oxidases. Some members of this last
group may also contain an organic prosthetic group, such as PQQ
(see section 10), or a modified amino-acid residue.

2.5.3 Type 3 Copper Proteins

In this group a pair of copper atoms comprise a dinuclear centre, with no EPR
activity as for single Cu’s. The best known example of an enzyme containing a
single Type 3 centre is tyrosinase (catechol oxidase, EC This protein
contains a metal center which is a structural analogue of the dinuclear copper
center in hemocyanin (ref 31).

2.5.4 Multi-Copper Oxidases

In addition to the above, there are several proteins with catalytic activity that
contain Types 1, 2 and 3 centres in various stoichiometric ratios. These
include L-ascorbate oxidase (EC, laccase (EC and
ceruloplasmin (ferro-oxidase, EC, the latter two having aromatic diamine
and diphenol oxidase activity. There is growing evidence that in these proteins
the Type 2 and Type 3 copper centres are juxtaposed. Recently it has been
shown that in L-ascorbate oxidase, a trinuclear copper site is present, consisting
of a type 3 copper site, very close (3.9 Å) and possibly bridged to a type 2 copper
site (ref 32). There is a view that ceruloplasmin functions as a ferro-oxidase
and the Fe(III) produced in this reaction can then oxidize the same substrates
as laccase.

2.5.5 Copper Centres in Cytochrome Oxidase

There are two copper centres that appear to be unique. Both are present in
cytochrome-c oxidase (EC The first appears to be an isolated metal ion
and has been referred to as Cud and CuA. The second appears to be part
of a dinuclear centre with cytochrome a3. It has been referred to as Cuu,
Cua3 and CuB. At the moment the ascriptions CuA and CuB are most frequently
used; however, the recent discovery (ref 33) of a cytochrome oxidase in which
cytochrome a has been replaced by cytochrome b, leads to the recommendation
that CuB shall be referred to as Cua3.

There is a striking similarity between two of the Cu centres of N2O reductase
and CuA (ref 34, 35).

2.5.6 Molybdenum enzymes (general)

Molybdenum enzymes contain molybdenum at the catalytic center responsible
for reaction with substrate. They may be divided into those that contain
the iron-molybdenum cofactor and those that contain the pterin-molybdenum

2.5.7 Additional centers

If a molybdenum enzyme contains flavin, it may be called either a molybdenum
flavoprotein or a flavomolybdenum protein, as indicated above. Other centers
should be treated similarly, e.g. an iron-sulfur molybdenum protein.

2.5.8 Molybdenum enzymes containing the iron-molybdenum cofactor

The only enzymes at present known to belong to this group are the nitrogenases
(EC; and EC see pp 89-116 in (ref 36) and pp 91-100 in (ref 37).

2.5.9 Molybdenum enzymes containing the pterin-molybdenum cofactor

These enzymes [see pp 411-415 in (ref 36) and (ref 38)] may be divided
into those in which the molybdenum bears a cyanide-labile sulfido (or thio
– see Note 1) ligand i.e. containing the S2- ligand as Mo=S) and those
lacking this ligand. The former group includes xanthine oxidase (EC,
xanthine dehydrogenase (EC, aldehyde oxidase (EC and
purine hydroxylase (EC: see Note 2 and 3). These may be called ‘molybdenum-
containing hydroxylase’ as is widely done. Molybdenum enzymes lacking the
sulfide (thio) ligand include sulfite oxidase (EC, NAD(P)+-independent
aldehyde dehydrogenase and nitrate reductases (assimilatory and dissimilatory)

2.5.10 Molybdenum enzymes containing the pterin-molybdenum cofactor

These enzymes [see pp 411-415 in (ref 36) and (ref 38)] may be divided into those
in which the molybdenum bears a cyanide-labile sulfido (or thio – see Note 1)
ligand i.e. containing the S2- ligand as Mo=S) and those lacking this ligand. The
former group includes xanthine oxidase (EC, xanthine dehydrogenase
(EC, aldehyde oxidase (EC and purine hydroxylase. These
may be called ‘molybdenum-containing hydroxylase’ as is widely done.
Molybdenum enzymes lacking the sulfide (thio) ligand include sulfite oxidase
(EC, NAD(P)+-independent aldehyde dehydrogenase and nitrate
reductases (assimilatory and dissimilatory) (EC

2.5.11 Metal-Substituted Metalloproteins

Scientists from several areas, dealing with spectroscopy and electron-transfer
mechanisms, often use metalloproteins in which a metal at the active site has
been substituted by another metal ion, like Co, Zn, Hg, Cd. Examples are zinc-
substituted cytochromes and cobalt-substituted ferredoxins.

The names for such modified proteins are easily given by using indications
like: ‘zinc-substituted ….’. In case of multi-metal proteins, where ambiguity might
arise about which metal has been substituted, one could easily add in parentheses
the name of the metal that has been replaced, such as: cobalt- substituted [Fe]

In formulae fragments or short names one could use the following notation:
[3Fe1Co-4S]2+, cytochrome c'[Fe[arrow right]CoFe], plastocyanin[Cu
[arrow right]Hg].

Ambler, R.P. (1980) in From Cyclotrons to Cytochromes (Kaplan, N.O. &
Robinson, A., eds) Academic Press, New York

Moore, G. & Pettigrew, F.(1987) Cytochromes c, Springer-Verlag, Berlin

Bartsch, R.G. (1963) in Bacterial Photosynthesis (Gest, H., San Pietro, A. &
Vernon, L.P., ed.) p. 315, Antioch Press, Yellow Springs, Ohio.

Stiefel, E.I. & Cramer, S.P. (1985) in Molybdenum Enzymes (Spiro, T.G., ed.),
Wiley-Interscience, New York, 89-116.

Smith B.E. et al. (1988), in Nitrogen Fixation Hundred Years After (Bothe,
H., de Bruijn, F.J. & Newton, W.E., ed.), Gustav Fischer, Stuttgart, New York,

Type-2 copper-containing enzymes.
MacPherson IS1, Murphy ME.
Cell Mol Life Sci. 2007 Nov;64(22):2887-99.

Type-2  Cu sites are found in all the major branches of life and are often
involved in the catalysis of oxygen species. Four type-2 Cu protein
families are selected as model systems for review: amine oxidases,
Cu monooxygenases, nitrite reductase/multicopper oxidase, and
CuZn superoxide dismutase. For each model protein, the availability
of multiple crystal structures and detailed enzymological studies provides
a detailed molecular view of the type-2 Cu site and delineation of the
mechanistic role of the Cu in biological function. Comparison of these
model proteins leads to the identification of common properties of the
Cu sites and insight into the evolution of the trinuclear active site found
in multicopper oxidases.

Copper proteins and copper enzymes.
Cass AE, Hill HA.
Ciba Found Symp. 1980;79:71-91.

The copper proteins that function in homeostasis, electron transport, dioxygen
transport and oxidation are discussed. Particular emphasis is placed on the
role of the ligands, their type and disposition which, in conjunction with other
residues in the active site, determine the role of the copper ion. It is proposed that
copper proteins can be considered in four groups. Those in Group I contain a
single copper ion in an approximately tetrahedral environment with nitrogen and
sulphur-containing ligands. Group II proteins have a single copper ion in a square-
planar-like arrangement. Group III proteins have two copper ions in close
proximity. Group IV consists of multi-opper proteins, composed of sites
representative of the other three groups.

Such centers owe their name to the intense blue coloration of the corresponding
Cu(II) proteins. The color is particularly distinctive since the metal centers are
so optically diluted in these metalloenzymes that only intense absorption in the
visible region, resulting from symmetry allowed electronic transitions, can give
rise to conspicuous colors. In contrast, the comparatively pale blue color of normal
Cu(II)) is the result of forbidden electronic transitions between d-orbitals of
different symmetry; in Cu2+(aq) this gives a molar extinction coefficient of
10 M-1cm-1 from a broad absorption between 10,000 cm-1 and 15,000 cm-1
compared to about 3000 M-1cm-1 observed for blue Cu(II) centers.  For the
T1 centers the intense absorption is attributed to a ligand-to-metal charge
transfer between the Cu2+ and a bonded cysteinate ligand. Typically, as in
azurin or plastocyanin this occurs around 16,000 cm-1. Ceruloplasmin has
three T1 centers, and the blue absorption is at 16,400 cm-1 (610nm).

Plastocyanine geometry

around the copper Crystal structures show a very irregular ‘tetrahedral’ coordination
with two sulphurs from methionine and cysteinate, and two histidine nitrogens.
However a comparison of azurin with plastocyanin shows that the geometry
is in some ways closer to a trigonal bipyramid, with and without one extra apical
ligand, so that azurin has a weakly bound glutamine oxygen, and plastocyanine
does not. The T1 coppers in caruloplasmin are in plastocyanine-type domains.
Each of these are coordinated to two histidines and a cysteine, in two of the T1
domains there is also a methionine residue, the third T1 domain has a leucine
residue which may only have a van der Waals type contact with the copper.

T1 copper centers are functional in the reversible electron transfer:

Cu2+ + e-   =   Cu+

The strongly distorted geometry represents a compromise (entactic-state
situation) between d10 Cu(I), with its preferred tetrahedral or trigonal
coordination through soft sulfur ligands, and d9 Cu(II) with preferential
square planar or square pyramidal geometry and nitrogen ligand
coordination.   This irregular, high energy arrangement at the metal
center resembles the transition-state geometry between the tetrahedral
and square planar equilibrium configurations of the two oxidation states
involved and permits enhanced rates of electron transfer. The potential
range for proteins with T1 copper centers runs from 180 mV in
stellacyanin to 680 mV in rusticyanin.

Zinc proteins: enzymes, storage proteins, transcription factors, and replication
Coleman JE.
Annu Rev Biochem. 1992;61:897-946.

In the past five years there has been a great expansion in our knowledge of
the role of zinc in the structure and function of proteins. Not only is zinc
required for essential catalytic functions in enzymes (more than 300 are known
at present), but also it stabilizes and even induces the folding of protein
subdomains. The latter functions have been most dramatically illustrated
by the discovery of the essential role of zinc in the folding of the DNA-binding
domains of eukaryotic transcription factors, including the zinc
finger transcription factors, the large family of hormone receptor proteins,
and the zinc cluster transcription factors from yeasts. Similar functions are
highly probable for the zinc found in the RNA polymerases and the zinc-
containing accessory proteins involved in nucleic acid replication. The rapid
increase in the number and nature of the proteins in which zinc functions
is not unexpected since zinc is the second most abundant trace metal found in
eukaryotic organisms, second only to iron. If one subtracts the amount of iron
found in hemoglobin, zinc becomes the most abundant trace metal found
in the human body.

Zinc Coordination Spheres in Protein Structures
ACS ChemWorx
Mikko Laitaoja , Jarkko Valjakka , and Janne Jänis
Inorg. Chem., 2013, 52 (19), pp 10983–10991
Sept 23, 2013

A statistical analysis in terms of zinc coordinating amino acids, metal-to-ligand
bond lengths, coordination number, and structural classification was performed,
revealing coordination spheres from classical tetrahedral cysteine/histidine binding
sites to more complex binuclear sites with carboxylated lysine residues. According
to the results, coordination spheres of hundreds of crystal structures in the PDB
could be misinterpreted due to symmetry-related molecules or missing electron
densities for ligands.

Protein-folding location can regulate manganese-binding versus copper- or
Tottey S, Waldron KJ, Firbank SJ, Reale B, Bessant C, Sato K, Cheek TR, et al.
Nature. 2008 Oct 23;455(7216):1138-42. http://dx.doi.org:/10.1038/nature07340

Metals are needed by at least one-quarter of all proteins. Although metallo-
chaperones insert the correct metal into some proteins, they have not been
found for the vast majority, and the view is that most metalloproteins acquire
their metals directly from cellular pools. However, some metals form more
stable complexes with proteins than do others. For instance, as described
in the Irving-Williams series, Cu(2+) and Zn(2+) typically form more stable
complexes than Mn(2+). Thus it is unclear what cellular mechanisms manage
metal acquisition by most nascent proteins. To investigate this question, we
identified the most abundant Cu(2+)-protein, CucA (Cu(2+)-cupin A), and the
most abundant Mn(2+)-protein, MncA (Mn(2+)-cupin A), in the periplasm of
the cyanobacterium Synechocystis PCC 6803. Each of these newly identified
proteins binds its respective metal via identical  ligands within a cupin fold.
Consistent with the Irving-Williams series, MncA only binds Mn(2+) after
folding in solutions containing at least a 10(4) times molar excess of Mn(2+)
over Cu(2+) or Zn(2+). However once MncA has bound Mn(2+), the metal
does not exchange with Cu(2+). MncA and CucA have signal peptides for
different export pathways into the periplasm, Tat and Sec respectively. Export
by the Tat pathway allows MncA to fold in the cytoplasm, which contains only
tightly bound copper or Zn(2+) (refs 10-12) but micromolar Mn(2+) (ref. 13). In
contrast, CucA folds in the periplasm to acquire Cu(2+). These results reveal
a mechanism whereby the compartment in which a protein folds overrides its
binding preference to control its metal content. They explain why the cytoplasm
must contain only tightly bound and buffered copper and Zn(2+).

Predicting copper-, iron-, and zinc-binding proteins in pathogenic species of the
Paracoccidioides genus
GB Tristão, L do Prado Assunção, LPA dos Santos, CL Borges, MG Silva-Bailão,
CM de Almeida Soares, G Cavallaro and AM Bailão*
Front. Microbiol., 9 Jan 2015 http://dx.doi.org:/10.3389/fmicb.2014.00761

Approximately one-third of all proteins have been estimated to contain at least
one metal cofactor, and these proteins are referred to as metalloproteins. These
represent one of the most diverse classes of proteins, containing metal ions that
bind to specific sites to perform catalytic, regulatory and structural functions.
Bioinformatic tools have been developed to predict metalloproteins encoded by
an organism based only on its genome sequence. Its function and the type of
metal binder can also be predicted via a bioinformatics approach.  Paracoccidioides
complex includes termodimorphic pathogenic fungi that are found as saprobic
mycelia in the environment and as yeast, the parasitic form, in host tissues. They
are the etiologic agents of Paracoccidioidomycosis, a prevalent systemic mycosis
in Latin America. Many metalloproteins are important for the virulence of several
pathogenic microorganisms. Accordingly, the present work aimed to predict the
copper, iron and zinc proteins encoded by the genomes of three phylogenetic species
of Paracoccidioides (Pb01, Pb03, andPb18). The metalloproteins were identified
using bioinformatics approaches based on structure, annotation and domains. Cu-,
Fe-, and Zn-binding proteins represent 7% of the total proteins encoded by
Paracoccidioides spp. genomes. Zinc proteins were the most abundant metallo-
proteins, representing 5.7% of the fungus proteome, whereas copper and iron
proteins represent 0.3 and 1.2%, respectively. Functional classification revealed that
metalloproteins are related to many cellular processes. Furthermore, it was observed
that many of these metalloproteins serve as virulence factors in the biology of the
fungus. Thus, it is concluded that the Cu, Fe, and Zn metalloproteomes of the
Paracoccidioides spp. are of the utmost importance for the biology and virulence
of these particular human pathogens.

Zinc finger proteins: new insights into structural and functional diversity
John H Laity, Brian M Lee, Peter E Wright
Current Opinion in Structural Biology Feb 2001; 11(1): 39–46

Zinc finger proteins are among the most abundant proteins in eukaryotic genomes.
Their functions are extraordinarily diverse and include DNA recognition, RNA
packaging, transcriptional activation, regulation of apoptosis, protein folding
and assembly, and lipid binding. Zinc finger structures are as diverse as their
functions. Structures have recently been reported for many new zinc finger
domains with novel topologies, providing important insights into structure/function
relationships. In addition, new structural studies of proteins containing the
classical Cys2His2 zinc finger motif have led to novel insights into mechanisms
of DNA binding and to a better understanding of their broader functions in
transcriptional regulation.

Zinc Finger Proteins

Zinc finger (ZnF) proteins are a massive, diverse family of proteins that serve a
wide variety of biological functions. Due to their diversity, it is difficult to come up
with a simple definition of what unites all ZnF proteins; however, the most common
approach is to define them as all small, functional domains that require coordination
by at least one zinc ion (Laity et al., 2001). The zinc ion serves to stabilize the
integration of the protein itself, and is generally not involved in binding targets.
The “finger” refers to the secondary structures (α-helix and β-sheet) that are
held together by the Zn ion. Zinc finger containing domains typically serve
as interactors, binding DNA, RNA, proteins or small molecules (Laity et al., 2001).

ZnF Protein Families

Cys2His2 was the first domain discovered (also known as Krüppel-type). It was
initially discovered as a repeating domain in the IIIA transcription factor in
Xenopus laevis (Brown et al., 1985; Miller et al., 1985). IIIA has nine repeats
of the 30 amino acids that make up the Cys2His2 domain. Each domain forms
a left-handed ββα secondary structure, and coordinates a Zn ion between
two cysteines on the β-sheet hairpin and two histidines in the α-helix, hence
the name Cys2His2 (Lee et al., 1989). These resides are highly conserved,
as well as a general hydrophobic core that allows the helix to form. The other
residues can show great sequence diversity (Michael et al., 1992). Cys2His2
zinc fingers that bind DNA tend to have 2-4 tandem domains as part of a
larger protein. The residues of the alpha helices form specific contacts with a
specific DNA sequence motif by “reading” the nucleotides in major groove
of DNA (Elrod-Erickson et al., 1996; Pavletich and Pabo, 1991). Cys2His2
proteins are the biggest group of transcription factors in most species. Non-
DNA binding proteins can have much more flexible tertiary structure.
Examples of Cys2His2 proteins include the Inhibitor of Apoptosis (IAP) family
of proteins and the CTFC transcription factor.

Treble clef fingers are a very diverse group of ZnF protiens both in terms of
structure and function. What makes them a family is a shared fold at their core
that looks a little like a musical treble clef, especially if you squint (Grishin,
2001). Most treble clef finger motifs have a β hairpin, a variable loop region,
a β hairpin, and an α helix. The “knuckle” of the β hairpin and the α helix contain
the Cys-x-x-Cys sequence necessary to coordinate the Zn ion. Treble clef
fingers often form the core of protein structures, for example the L24E and
S14 ribosomal proteins and the RING finger family.

Zinc ribbons are a little less structurally complex than the other two major groups.
Zinc ribbons contain two zinc knuckles, often β hairpins, coordinating a zinc ion via
a two Cys residures separated by 2-4 other residues on one knuckle, and a Cys-x-x-
Cys on the other (Hahn and Roberts, 2000). Examples of zinc ribbon-containing
proteins include the basal transcription factors TFIIS and TFIIB that for a complex
with RNAPII to bind DNA, and the Npl4 nuclear core protein that uses a zinc ribbon
to bind ubiquitin (Alam et al., 2004). Cys2His2, treble clef fingers, and zinc ribbons
form the majority of zinc fingers, but there are several other smaller groups that
don’t fit neatly into these three. Green fluorescent protein as a marker for gene

Metallothionein proteins expression, copper and zinc concentrations, and lipid
peroxidation level in a rodent model for amyotrophic lateral sclerosis
E Tokuda, Shin-Ichi Ono,  K Ishige, A Naganuma, Y Ito, T Suzuki
Toxicology Jan 2007; 229(1–2): 33–41

It has been hypothesized that copper-mediated oxidative stress contributes to the
pathogenesis of familial amyotrophic lateral sclerosis (ALS), a fatal motor neuron
disease in humans. To verify this hypothesis, we examined the copper and zinc
concentrations and the amounts of lipid peroxides, together with that of the
expression of metallothionein (MT) isoforms in a mouse model [superoxide
dismutase1 transgenic (SOD1 Tg) mouse] of ALS. The expression of MT-I and
MT-II (MT-I/II) isoforms were measured together with Western blotting, copper
level, and lipid peroxides amounts increased in an age-dependent manner in the
spinal cord, the region responsible for motor paralysis. A significant increase was
already seen as early as 8-week-old SOD1 Tg mice, at which time the mice had not
yet exhibited motor paralysis, and showed a further increase at 16 weeks of age,
when paralysis was evident. Inversely, the spinal zinc level had significantly
decreased at both 8 and 16 weeks of age. The third isoform, the MT-III level,
remained at the same level as an 8-week-old wild-type mouse, finally increasing
to a significant level at 16 weeks of age. It has been believed that a mutant SOD1
protein, encoded by a mutant SOD1, gains a novel cytotoxic function while
maintaining its original enzymatic activity, and causes motor neuron death
(gain-of-toxic function). Copper-mediated oxidative stress seems to be a probable
underlying pathogenesis of gain-of-toxic function. Taking the above current
concepts and the classic functions of MT into account, MTs could have a disease
modifying property: the MT-I/II isoform for attenuating the gain-of-toxic function
at the early stage of the disease, and the MT-III isoform at an advanced stage.

Prion protein expression level alters regional copper, iron and zinc content in
the mouse brain
MJ Pushie,  IJ Pickering, GR Martin, S Tsutsui, FR Jirik and GN George
Metallomics, 2011,3, 206-214 http://dx.doi.org:/10.1039/C0MT00037J

The central role of the prion protein (PrP) in a family of fatal neurodegenerate
diseases has garnered considerable research interest over the past two decades.
Moreover, the role of PrP in neuronal development, as well as its apparent role
in metal homeostasis, is increasingly of interest. The host-encoded form of the
prion protein (PrPC) binds multiple copper atoms via its N-terminal domain
and can influence brain copper and iron levels. The importance of PrPC to the
regulation of brain metal homeostasis and metal distribution, however, is not
fully understood. We therefore employed synchrotron-based X-ray fluorescence
imaging to map the level and distributions of several key metals in the brains of
mice that express different levels of PrPC. Brain sections from wild-type, prion
gene knockout (Prnp−/−) and PrPC over-expressing mice revealed striking
variation in the levels of iron, copper, and even zinc in specific brain regions as
a function of PrPC expression. Our results indicate that one important function
of PrPC may be to regulate the amount and distribution of specific metals within
the central nervous system. This raises the possibility that PrPC levels, or its
activity, might regulate the progression of diseases in which altered metal
homeostasis is thought to play a pathogenic role such as Alzheimer’s,
Parkinson’s and Wilson’s diseases and disorders such as hemochromatosis.

Zinc & Copper Imbalances: Immense Biochemical Implications
Mar 27, 2013 by Michael McEvoy

The status of zinc and copper levels may have profound implications for
many people. Much has been written about the significance of these two
trace elements for many, many years. Many health conditions may be
directly caused by abnormal zinc and copper levels.

With all of the recent attention given to methylation status, gene mutations,
MTHFR, and the associated neurological and mental/behavioral disorders
that may ensue, zinc and copper status remains a pivotal ratio in these regards.

While zinc toxicity and copper deficiency are possible, the subject of this
article is on the more common imbalance: copper toxicity and zinc deficiency.

The Physiological Roles Of Zinc & Copper

Zinc and copper are antagonists. The balance between these two trace
elements is an example of the effects of biological dualism. While zinc
toxicity is possible, far more common is zinc deficiency and copper toxicity.
Both zinc and copper play essential roles in the body, and there can be a
number of causes for why imbalances ensue.

It may be easier to identify the roles that zinc doesn’t play in the body,
than the roles it does play. Zinc is an essential trace element that activates
several hundred enzymatic reactions. These reactions are fundamental
to life and biological activity. Some of the activities that zinc are involved in:

  • DNA & RNA synthesis
  • Gene expression
  • Nervous system function
  • Immune function & immune signaling such as cell
  • Neuronal transmission
  • Brain function
  • Zinc possesses powerful anabolic activities in the cells
  • Formation of zinc proteins known as “zinc fingers”
  • Zinc is essential for blood clotting and platelet formation
  • Zinc is involved in Vitamin A synthesis
  • Folate is made available through zinc enzyme reactions
  • Along with copper, Zinc makes up the antioxidant
    system, ZnCu superoxide dismutase
  • Steroidal hormone synthesis
  • Growth & development of children
  • Testosterone and semen formation
  • The highest concentration of zinc is found in the
    male prostate gland

Copper is an essential trace element serving many important functions
as well. However, copper is well documented to induce several toxic effects
in the body, when elevated. Because copper is a pro-oxidant when free and
unbound, it can quickly generate free radicals.

The major sources for copper toxicity are: exposure to industrial forms
of copper such as copper pipes, copper cookware, birth control, exposure
to copper-based fungicides. Diets high in copper and low in zinc may play
a role in copper toxicity. Pyrrole disorder, which causes depletion of zinc,
may result in elevated levels of copper.

Some of the essential roles copper plays in the body:

  • Connective tissue formation
  • ATP synthesis
  • Iron metabolism
  • Brain health via neurotransmitter synthesis
  • Gene transcription
  • Synthesis of the antioxidant superoxide dismutase
  • Skin pigmentation
  • Nerve tissue: myelin sheath formation
  • Copper tends to rise when estrogen is dominant

Perhaps one of the first reports that zinc and copper imbalances play
a role in human health and disease was their detection in mental
disorders made by Carl Pfeiffer, MD, PhD. Dr. Pfeiffer identified a
condition known as pyrrole disorder, sometimes referred to as
pyrroluria or “mauve factor”.

As it turns out, pyrrole disorder is a major biochemical imbalance
in many people with chronic illnesses such as chronic Lyme disease,
autism, schizophrenia, depression, bi-polar, and chronic fatigue
syndrome. Pyrroles are a byproduct of hemoglobin synthesis.
Apparently, some individuals are more predisposed towards producing
higher amounts of pyrroles. When pyrroles are excessive, they irreversibly
bind to zinc and vitamin B6, causing their excretion. Consequently,
it is common that once zinc levels become depleted, copper levels tend to rise.

Copper Toxicity

Problems associated with copper toxicity include: pyrrole disorder,
estrogen dominance, schizophrenia, depression, anxiety disorder,
chronic fatigue, migraines, liver toxicity, thyroid conditions, chronic
candida yeast infections, PMS, to name a few. Some research has
even implicated copper toxicity with Alzheimer’s Disease and with
cardiovascular disease. Perhaps one of the primary mechanisms
through which copper toxicity can damage tissues is through its
initiation of oxidative stress and free radical formation. Free copper
ions that are not bound to copper proteins such as ceruloplasmin,
are pro-oxidants, and are highly reactive.

Empirical research from clinicians, indicates that there are different
types of copper imbalances. For example, if there is a lot of free,
unbound copper present, this may cause a situation of nutritive
copper deficiency. Another copper imbalance is when high pyrroles
depress zinc levels, and copper levels concomintantly rise. If high
pyrroles are present, B6 will also be lost in high amounts. In a general
but very real sense, all forms of copper excess will affect zinc status,
due to the dualistic nature of zinc and copper.

Copper & Estrogen

It has been known for many years that copper can cause a rise in
estrogen, and conversely estrogen may raise copper. Estrogen
dominance has been extensively studied in its role in breast
cancer development. One possible, critical role that can cause
estrogen to become carcinogenic, is through its oxidation induced by
Once oxidized, estrogen forms volatile hydroxyl radicals and
the associated DNA damage and “mutagenesis”.

Zinc Deficiency

As mentioned previously, pyrrole disorder will directly depress
zinc status, causing high levels of its excretion. When zinc is
lost, copper rises. Because of their essential roles in neuro-
transmitter synthesis, low zinc and high copper levels can
directly effect cognition, behavior and thought processes.
Zinc has been studied in biochemical reactions involving
calcium-driven, synaptic neurotransmission, as well as in
glutamate/GABA balance and with limbic brain function.

Zinc & Reproduction

Zinc is essential for steroidal hormone synthesis, and is a
well known catalyst for testosterone synthesis, as well as
leutinizing hormone. Zinc has demonstrated its ability to
prevent miscarriage and toxicity during pregnancy. The male
prostate gland reportedly contains the highest concentration
of zinc in the body.

Zinc & Brain Function

Much attention has been given to excitotoxicity, such as the
effects induced by MSG (monosodium glutamtate). Excess
stimulation of the excitatory neurotransmitter glutamate,
may cause severe physical and psychological reactions in
certain individuals. Zinc has been studied for its ability to
enhance GABA 
(glutamate’s antagonistic neurotransmitter)
activity and to suppress excess glutamate.

Studies on mice demonstrated that when depleted of zinc
for two weeks, the mice developed seizures, most likely due
to GABA deficiencies and glutamate excess.

There is an emerging body of evidence that demonstrates
that Alzheimer’s disease may involve copper toxicity and
zinc deficiency. Not only can excess copper cause zinc
depletion, but so can excess lead.

The hippocampus, a major part of the limbic brain, records
memories and is responsible for processing meaningful
experiences. Numerous studies site that if hippocampal
cells are deprived of zinc, the hippocampal cells die. In
addition to hippocampus cell death induced by zinc
deprivation, the amygdala, the other major limbic gland
experiences cell death as well, when deprived of zinc.

Green Fluorescent Protein

Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC.
Science. 1994 Feb 11;263(5148):802-5.

A complementary DNA for the Aequorea victoria green fluorescent protein (GFP)
produces a fluorescent product when expressed in prokaryotic (Escherichia coli)
or eukaryotic (Caenorhabditis elegans) cells. Because exogenous substrates and
cofactors are not required for this fluorescence, GFP expression can be used
to monitor gene expression and protein localization in living organisms.


The green fluorescent protein (GFP) is a protein composed of 238 amino acid
residues (26.9 kDa) that exhibits bright green fluorescence when exposed
to light in the blue to ultraviolet range. Although many other marine organisms
have similar green fluorescent proteins, GFP traditionally refers to the protein
first isolated from the jellyfish Aequorea victoria. The GFP from A. victoria
has a major excitation peak at a wavelength of 395 nm and a minor one at
475 nm. Its emission peak is at 509 nm, which is in the lower green portion
of the visible spectrum. The fluorescence quantum yield (QY) of GFP is 0.79.
The GFP from the sea pansy (Renilla reniformis) has a single major excitation
peak at 498 nm.

In cell and molecular biology, the GFP gene is frequently used as a reporter of
expression. In modified forms it has been used to make biosensors, and many
animals have been created that express GFP as a proof-of-concept that a gene
can be expressed throughout a given organism. The GFP gene can be introduced
into organisms and maintained in their genome through breeding, injection with a
viral vector, or cell transformation. To date, the GFP gene has been introduced
and expressed in many Bacteria, Yeast and other Fungi, fish (such as zebrafish),
plant, fly, and mammalian cells, including human. Martin Chalfie, Osamu Shimomura,
and Roger Y. Tsien were awarded the 2008 Nobel Prize in Chemistry on 10 October
2008 for their discovery and development of the green fluorescent protein.


In Aequorea victoria a protein called aequorin releases blue light upon binding
with calcium. This blue light is then totally absorbed by the GFP, which in turn
gives off the green light as in the animation below.

In 1994 GFP was cloned. Now GFP is found in laboratories all over the world where
it is used in every conceivable plant and animal. Flatworms, algae, E. coli and pigs
have all been made to fluoresce with GFP.

The importance of GFP was recognized in 2008 when the Nobel Committee awarded
Osamu Shimomura, Marty Chalfie and Roger Tsien the Chemistry Nobel Prize ”
for the discovery and development of the green fluorescent protein, GFP.”

Why is it so popular? Well, I like to think of GFP as the microscope of the twenty-
first century. Using GFP we can see when proteins are made, and where they can go.
This is done by joining the GFP gene to the gene of the protein of interest so that
when the protein is made it will have GFP hanging off it. Since GFP fluoresces, one
can shine light at the cell and wait for the distinctive green fluorescence associated
with GFP to appear.

A variant of yellow fluorescent protein with fast and efficient maturation for
cell-biological applications
T Nagai, K Ibata, E Sun Park, M Kubota, K Mikoshiba & A Miyawaki
Nature Biotechnology 20, 87 – 90 (2002)  http://dx.doi.org:/10.1038/nbt0102-87

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria
has provided a myriad of applications for biological systems. Over the last
several years, mutagenesis studies have improved folding properties of GFP.
However, slow maturation is still a big obstacle to the use of GFP variants for
visualization. These problems are exacerbated when GFP variants are expressed
at 37°C and/or targeted to certain organelles. Thus, obtaining GFP variants that
mature more efficiently is crucial for the development of expanded research
applications. Among Aequorea GFP variants, yellow fluorescent proteins (YFPs)
are relatively acid-sensitive,and uniquely quenched by chloride ion (Cl−)3. For
YFP to be fully and stably fluorescent, mutations that decrease the sensitivity
to both pH and Cl− are desired. Here we describe the development of an
improved version of YFP named “Venus”. Venus contains a novel mutation,
F46L, which at 37°C greatly accelerates oxidation of the chromophore, the rate-
limiting step of maturation. As a result of other mutations, F64L/M153T/
V163A/S175G, Venus folds well and is relatively tolerant of exposure
to acidosis and Cl−. We succeeded in efficiently targeting a neuropeptide
Y-Venus fusion protein to the dense-core granules of PC12 cells. Its secretion
was readily monitored by measuring release of fluorescence into the medium.
The use of Venus as an acceptor allowed early detection of reliable signals of
fluorescence resonance energy transfer (FRET) for Ca2+ measurements in brain
slices. With the improved speed and efficiency of maturation and the increased
resistance to environment, Venus will enable fluorescent labelings that were not
possible before.

Rhodopsin-like Protein from the Purple Membrane of Halobacterium halobium
Nature New Biology 29 Sep 1971; 233, 149-152  | http://dx.doi.org:/10.1038/

HALOPHILIC bacteria require high concentrations of sodium chloride and lower
concentrations of KCl and MgCl2 for growth. The cell membrane dissociates into
fragments of varying size when the salt is removed1. One characteristic fragment—
termed the “purple membrane” because of its characteristic deep purple colour—
has been isolated in relatively pure form from Halobacterium halobium. We can
now show that the purple colour is due to retinal bound to an opsin-like protein,
the only protein present in this membrane fragment.


Stoeckenius, W. , and Rowen, R. , J. Cell Biol., 34, 365 (1967).

Stoeckenius, W. , and Kunau, W. H. , J. Cell Biol., 38, 337 (1968).

Blaurock, A. E. , and Stoeckenius, W. , Nature New Biology, 233, 152 (1971).

Sehgal, S. N. , and Gibbons, N. E. , Canad. J. Microbiol., 6, 165 (1960).

Kelly, M. , Norgård, S. , and Liaach-Jensen, S. , Acta Chem. Scand., 2A, 2169 (1970).

Shapiro, A. L. , Vinnela, E. , and Maizel, jun., J. V. , Biochem. Biophys. Res.
Commun., 28, 815 (1967).

The monomerization of the Purple protein, a member of the GFP-family
Corning, Brooke

Green fluorescent protein (GFP) has been used extensively since its discovery
in the 1960s to report and visualize gene expression. For years it has been the only
known naturally occurring fluorescent pigment that is encoded by a single gene,
making it extremely useful in various fields of biology, because the expression of
this gene directly leads to the appearance of the fluorescent green color. Recently,
however, many more proteins with similar properties to GFP, and available in a
variety of colors, have been isolated from the class of marine organisms called
Anthozoa, which includes the corals. This increase in the availability of colored
proteins in GFP family in turn has expanded the number of available biotech-
nology applications. However, some of these newly discovered GFP-like
proteins do not have wild-type forms that readily allow for the creation of
fusion proteins, particularly because of oligomerization. It is widely accepted
that almost all members of the GFP-family form dimers or tetramers in their
functional forms.

This study investigates a GFP-ike protein, Purple, isolated from two species,
Galaxea fascicularis and Montipora efflorescens. Purple protein forms oligomers
when expressed, which would then interfere with the normal expression of a  protein
to be tagged in gene fusion experiments. We selectively mutated 3 amino acids,
which we believed were responsible for oligomerization in Purple. These 3
residues were chosen based on sequence similarities to a very similar protein,
a mutant form of the Rtms5 chromoprotein from Montipora efflorescens. While
we had hoped that the resulting triple-mutant Purple protein would form
monomers in vivo while retaining its purple coloration, this turned out to
be incorrect. The resulting mutants had lost their ability to turn purple. However,
we also determined that we had successfully changed the oligomerization
state of Purple by examining the relative molecular mass of one our
mutant proteins, which turned out to be half the size of the original
purple protein. It is possible that by adding additional mutations in
the future, the original spectral properties could be recovered. If
successful, this would further expand the utility of the GFP family.

Rhodopsin, also known as visual purple, from Ancient Greek ῥόδον
(rhódon, “rose”), due to its pinkish color, and ὄψις (ópsis, “sight”), is
a light-sensitive receptor protein. It is a biological pigment in photo-
receptor cells of the retina. Rhodopsin is the primary pigment found
in rod photoreceptors. Rhodopsins belong to the G-protein-coupled
receptor (GPCR) family. They are extremely sensitive to light, enabling
vision in low-light conditions. Exposed to light, the pigment
immediately photobleaches, and it takes about 45 minutes to regenerate
fully in humans. Its discovery was reported by German physiologist
Franz Christian Boll in 1876.

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Coronary Circulation Combined Assessment: Optical Coherence Tomography (OCT), Near-Infrared Spectroscopy (NIRS) and Intravascular Ultrasound (IVUS) – Detection of Lipid-Rich Plaque and Prevention of Acute Coronary Syndrome (ACS)

Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC


Article Curator: Aviva Lev-Ari, PhD, RN

The clinical motivations for coronary artery imaging include identifying and characterizing obstructive lesions, analyzing suitability for various feasible interventions, and assessing comparative risk with and without interventions. With improvements in non-invasive detection of fixed obstructions in the coronary arteries, it should not be surprising that half of the lesions that cause heart attacks (myocardial infarction) among those who had recent imaging consisted of unstable plaques that were less than 50% obstructive. Therefore there is growing interest not only in more reliable detection of lesions that exceed 50% obstruction, but also improved characterization of lesions that are not obstructive but may be unstable.

By way of analogy, think of impaired blood supply to the heart as a traffic jam in a roadway. The best time to check for a traffic jam is during rush hour. The corresponding clinical scenario is stress testing. There are three major roadways in the heart: left anterior, left circumflex, and right, each with branches (forks). The two left major vessels stem from a short but treacherous left main (“widow maker”). A temporary traffic jam results in symptoms of impaired delivery (angina, from hunger due to late delivery of food). Alternatively, a prolonged traffic disruption can result in suicidal tissue destruction (starvation). A fixed obstruction consists of potholes and landslides resulting in a persisting shutdown of half or more of the lanes in the highway. An unstable plaque consists of a less severe abnormality that can cause accidents (plaque rupture, local hemorrhage, sudden occlusion). A road may shutdown not only from progressive road damage, but also a truck can flip over and shutdown a relatively clean roadway.

Among patients who had recent coronary imaging prior to the onset of a heart attack, half do not have occlusive lesions. Instead of slow progressive reduction in vessel diameter leading to a critically severe flow reduction, the mechanism in the cases of no severe narrowing is attributed to unstable plaque, meaning plaque with thin fibrous caps that rupture, causing sudden thrombosis. Stress tests focus on detection of fixed obstructions and do not warn who has unstable plaque. Thus the next great frontier for coronary imaging is not just to identify flow reducing lesions, but also to identify unstable plaque even if it is not currently flow limiting. This article presents candidate imaging methods and their current capabilities.

Coronary imaging methods include:

  • intra-coronary ultrasound (IVUS)
  • optical coherence imaging (fiberoptic)
  • computed tomographic xray angiography (CTA)
  • magnetic resonance angiography (MRA)
  • near infra-red spectroscopic imaging (NIRS)

    NIRS-IVUS Imaging To Characterize the Composition and Structure of Coronary Plaques 

    David Rizik, MD1 and James, A. Goldstein, MD2

    1. Scottsdale Healthcare Hospital, Scottsdale, AZ

    2. Department Cardiovascular Medicine, William Beaumont Hospital, Royal Oak, MI

    This supplement,


    authored by highly experienced interventional cardiologists expert in the field of coronary plaque characterization, contains a detailed description of the new NIRS-IVUS combination catheter, and the clinical information obtained during its use in over 90 hospitals in over 10 countries. Case vignettes, cohort outcomes, reviews, and plans for future studies are also presented. It is our hope that this information will be useful in the near term to those seeking to improve PCI. For the longer term, we believe that the NIRS-IVUS system is an excellent candidate for evaluation as a detector of vulnerable plaque. Success in the prospective studies that are planned will make it possible to detect vulnerable plaques and thereby enhance efforts to prevent coronary events.

    Imaging Methods for Detection of Intravascular Plaque – Direct, Robust and/or Validated

    Cap Thickness – OCT

    Expansive Remodeling – IVUS & NIRS-IVUS [Combination TVC System & TVC Insight Catheter]

    Plaque Volume – IVUSNIRS-IVUS

    Calcification – Angiography, IVUS & NIRS-IVUS

    Thrombus – Angioscopy & OCT

    Inflammation Macrophages – Indirect by OCT

    Lipid Core – IVUS & NIRS-IVUS

    Requires Blood-Free FOV – Angioscopy & OCT

    based on Table 1 p.5


    Comparative Intravascular Imaging for Lipid Core Plaque: VH-IVUS vs OCT vs NIRS

    Eric Fuh, MD and Emmanouil S. Brilakis, MD, PhD

    VA North Texas Healthcare System, Dallas, TX and Division of Cardiology, Dept of Medicine, UT Southwestern Medical Center, Dallas, TX


    VH-IVUS, OCT, and NIRS can assist in the detection and evaluation of lipid core plaque. Comparative studies have shown important differences between modalities, but are all limited from lack of comparison with the gold standard of histology. Given the different strengths and weaknesses of each modality, combination imaging will likely provide the best results.41 Further refinement of the clinical implications of LCP detection and its impact on optimizing treatment strategy selection will stimulate advances in LCP detection imaging.

    OCT and NIRS can image through calcified lesions, whereas IVUS cannot. LCPs are often accompanied by neovascularization, which can only be visualized by OCT. VH-IVUS may classify stents, which usually appear white (misclassified as “calcium”) surrounded by red (misclassified as “necrotic core”), although this does not appear to be a limitation for NIRS and OCT.54

    Reference 41:

    Bourantas CV, Gracia-Gracia HM, Naka KK, et al. Hybrid intravascular imaging: current applications and prospective potential in the study of coronary atherosclerosis, JACC 2013;61:1369-1378


    The miniaturization of medical devices and the progress in image processing have allowed the development of a multitude of intravascular imaging modalities that permit more meticulous examination of coronary pathology. However, these techniques have significant inherent limitations that do not allow a complete and thorough assessment of coronary anatomy. To overcome these drawbacks, fusion of different invasive and noninvasive imaging modalities has been proposed. This integration has provided models that give a more detailed understanding of coronary artery pathology and have proved useful in the study of the atherosclerotic process. In this review, the authors describe the currently available hybrid imaging approaches, discuss the technological innovations and efficient algorithms that have been developed to integrate information provided by different invasive techniques, and stress the advantages of the obtained models and their potential in the study of coronary atherosclerosis.


    Reference 54

    Kim SW, Mintz GS, Hong YJ, et al. The virtual histology intravascular ultrasound appearance of newly placed drug-eluting stents. Am J Cardiol. 2008;102:1182-1186.

    American Journal of Cardiology
    Volume 102, Issue 9 , Pages 1182-1186, 1 November 2008

    The Virtual Histology Intravascular Ultrasound Appearance of Newly Placed Drug-Eluting Stents

    Received 17 January 2008; received in revised form 17 March 2008; accepted 17 March 2008. published online 13 June 2008.

    Intravascular ultrasound (IVUS) is used before and after intervention and at follow-up to assess the quality of the acute result as well as the long-term effects of stent implantation. Virtual histology (VH) IVUS classifies tissue into fibrous and fibrofatty plaque, dense calcium, and necrotic core. Although most interventional procedures include stent implantation, VH IVUS classification of stent metal has not been validated. In this study, the VH IVUS appearance of acutely implanted stents was assessed in 27 patients (30 lesions). Most stent struts (80%) appeared white (misclassified as “calcium”) surrounded by red (misclassified as “necrotic core”); 2% appeared just white, and 17% were not detectable (compared with grayscale IVUS because of the software-imposed gray medial stripe). The rate of “white surrounded by red” was similar over the lengths of the stents; however, undetectable struts were mostly at the distal edges (31%). Quantitatively, including the struts within the regions of interest increased the amount of “calcium” from 0.23 ± 0.35 to 1.07 ± 0.66 mm2 (p <0.0001) and the amount of “necrotic core” from 0.59 ± 0.65 to 1.31 ± 0.87 mm2 (p <0.0001). Most important, because this appearance occurs acutely, it is an artifact, and the red appearance should not be interpreted as peristrut inflammation or necrotic core when it is seen at follow-up. In conclusion, acutely implanted stents have an appearance that can be misclassified by VH IVUS as “calcium with or without necrotic core.” It is important not to overinterpret VH IVUS studies of chronically implanted stents when this appearance is observed at follow-up. A separate classification for stent struts is necessary to avoid these misconceptions and misclassifications.

    Table 2. Comparison of three intravascular imaging modalities for the detection of coronary lipid core plaque.

    Intravascular Imaging Modalities for Detecting LCP

    Vol. 25, Supplement A, 2013


     VH-IVUS (20 MHz)                        OCT                          NIRS-IVUS (40 MHz)

    Hybrid intravascular imaging  No No Yes

    Axial resolution, μm 200 10 100

    Imaging through blood ++ – ++

    Need for blood column clearance during image acquisition No Yes No

    Imaging through stents No Yes Yes

    Imaging through calcium No Yes Yes for NIRS – No for IVUS

    Imaging neovascularization No Yes No

    Detection of non-superficial LCPs Yes No No

    Evaluation of LCP cap thickness + ++ *

    Detection of thrombus – + *

    Expansive remodeling ++ – ++

    Need for manual image processing for LCP detection Yes Yes No

    ++ = excellent; + = good; ± = possible; – = impossible; * = potential under investigation

    VH-IVUS = virtual histology intravascular ultrasound; OCT = optical coherence tomogra-phy; NIRS = near-infrared spectroscopy; LCP = lipid core plaque 

    The Search for Vulnerable Plaque — The Pace Quickens


    Ryan D. Madder, MD1, Gregg W. Stone, MD2, David Erlinge, MD3, James E. Muller, MD4


    1Frederik Meijer Heart & Vascular Institute, Spectrum Health, Grand Rapids, Michigan;

    2New York Presbyterian Hospital, Columbia University and Car-diovascular Research Foundation, New York, New York;

    3Department of Cardiology, Lund University, Lund, Sweden;

    4Infraredx, Inc., Burlington, Massachusetts

    Disclosure: Drs. Madder and Erlinge report no financial relationships or conflicts of interest regarding the content herein.

    Dr. Stone is a consultant for Infraredx, Inc., Volcano Corp., Medtronic, and Boston Scientific, and is a member of the scientific advisory boards for Boston Scientific and Abbott Vascular.

    Dr. Muller is a full-time employee of Infraredx, Inc from which he receives salary and equity.

    Address for Correspondence: Email: ryan.madder@spectrumhealth.org

    The search for the vulnerable plaque has been a lengthy endeavor requiring the work of multiple individuals and institutions over many years. It is disappointing that in more than 2 decades since the “vulnerable plaque” concept was formulated, over 40 million coronary events have occurred. However, it is encouraging that positive answers are now available for most of the questions related to a vulnerable plaque detection and treatment strategy. As shown in Table 1, most of the essential preconditions of a successful vulnerable plaque strategy are present. This positive information has accelerated the pace of work in this area. The pathophysiology of coronary events is well-understood; powerful imaging methods are available; and therapies, both existing and novel, may well be effective (although appropriately powered randomized trials are required to demonstrate their safety and effectiveness). The time is approaching for the conduct of prospective outcome trials to determine the value of a vulnerable plaque strategy for more effective prevention of coronary events.

    Table 1. Essential Components of a Strategy to Prevent Coronary Events by the Detection and Treatment of Vulnerable Plaques

    Essential Components Evidencefrom  Published Research
    Pathophysiology of Coronary Events
    Are the causes of coronary events known? Yes Constantinides and others have shown that most coronary events are caused by rupture of a thin-capped LRP with subsequent formation of an occlusive thrombus.1-5
    Are LRPs focal? Yes Cheruvu et al demonstrated that ruptures and TCFA occupy less than 4% of the length of arteries studied at autopsy.8
    Are LRPs stable over time? Yes Kubo et al demonstrated that most fibroatheromas by radiofrequency IVUS remain fibroatheromas over time.39
    Detection of Suspected Vulnerable Plaque by Invasive Imaging (For Secondary Prevention)
    Can invasive imaging safely detect LRP? Yes Waxman et al, Ino et al, and many others have demonstrated the safety of detecting LRP in patients.40
    Do cross-sectional studies show increased LRP concentrated at culprit sites? Yes Madder et al, Erlinge et al, Ino et al have shown LRP concentrated at the culprit site across the spectrum of ACS.14,16,41
    Do prospective studies show that suspected vulnerable plaque can be detected in advance? Yes PROSPECT, VIVA, PREDICTION established the principle by proving that increased plaque burden predicted events but prediction lacked specificity.23-25
    Is more specific detection of vulnerable plaque possible? ? NIRS-IVUS and OCT may provide more specific detection of VP, but have not yet been tested in a prospective study.
    Can Vulnerable Plaques be Treated?
    Is systemic treatment of LRPs possible with current agents? Yes YELLOW study showed a reduction in LRP with rosuvastatin.33
    Is focal treatment of LRPs possible with current methods? Yes Ruptured LRPs are routinely stented in ACS in clinical practice with good outcomes.
    Can systemic treatment be enhanced with new agents? ? PCSK9 inhibitors, Apo A1 Milano, other agents in development may be more effective than statins, but more costly.35,36
    Can focal treatments be enhanced with new methods? ? Bioresorbable vascular scaffolds and/or drug-coated balloons may be useful for VP.
    Primary Prevention
    Can demographic and serum biomarkers be used as a first step in a screening strategy? Yes Framingham Risk Score, improved serum biomarkers, and genetic markers can identify individuals at increased risk.
    Can non-invasive imaging with CTA detect LRP and increased risk? Yes Motoyama et al have identified CTA markers associated with future events.26
    Will a strategy of detection and treatment of vulnerable plaque, if proven to be successful, be cost-effective for secondary prevention? Probably Bosch et al demonstrated that for patients already undergoing invasive imaging, the added costs of detection and treatment of VP are likely to be less than the cost of second events, leading to a cost-saving approach that also improves health.38
    Will a strategy of detection and treatment of vulnerable plaque, if proven to be successful, be cost-effective for primary prevention? ? Bosch et al: For primary prevention the cost of screening would be greater than for secondary prevention. Cost-effectiveness would depend upon cost, the accuracy of detection, and effectiveness of therapy.38
    ACS = acute coronary syndrome; CTA = coronary computed tomographic angiography; LRP = lipid-rich plaque; TCFA = thin-capped fibroatheroma; 


    1. Constantinides P. Plaque fissures in human coronary thrombosis. J Atheroscler Res. 1966;6:1-17.

    2. Friedman M, Van den Bovenkamp GJ. The pathogenesis of a coronary thrombus. Am J Pathol. 1966;48:19-44.

    3. Burke AP, Farb A, Malcom GT, et al. Coronary risk factors and plaque morphology in men with coronary disease who died suddenly. N Engl J Med. 1997;336:1276-1282.

    4. Farb A, Tang AL, Burke AP, et al. Sudden coronary death. Frequency of active coronary lesions, inactive coronary lesions, and myocardial infarction. Circulation. 1995;92:1701-1709.

    5. Virmani R, Kolodgie FD, Burke AP, Farb A, Schwartz SM. Lessons from sudden coronary death: a comprehensive morphological classification scheme for atherosclerotic lesions. Arterioscler Thromb Vasc Biol. 2000;20:1262-1275.

    6. Waksman R, Serruys PW. Handbook of the Vulnerable Plaque. Martin Dunitz: London, England, 2004.

    7. Libby P, Ridker PM, Hansson GK. Progress and challenges in translating the biology of atherosclerosis. Nature. 2011;473:317-325.

    8. Cheruvu P, Finn A, Gardner C, et al. Frequency and distribution of thin-cap fibroatheroma and ruptured plaques in human coronary arteries – a pathologic study. J Am Coll Cardiol. 2007;50:940-949.

    9. Hong M, Mintz GS, Lee CW, et al. Comparison of coronary plaque rupture between stable angina and acute myocardial infarction: a three-vessel intravascular ultrasound study in 235 patients. Circulation. 2004;110:928-933.

    10. Fujii K, Kobayashi Y, Mintz GS, et al. Intravascular ultrasound assessment of ulcerated ruptured plaques. A comparison of culprit and non-culprit lesions of patients with acute coronary syndromes and lesions in patients without acute coronary syndromes. Circulation. 2003;108:2473-2478.

    11. Ehara S, Kobayashi Y, Yoshiyama M, et al. Spotty calcification typifies the culprit plaque in patients with acute myocardial infarction. An intravascular ultrasound study. Circulation. 2004;110:3424-3429.

    12. Lee SY, Mintz GS, Kim SY, et al. Attenuated plaque detected by intravascular ultrasound: clinical, angiographic, and morphologic features and post-percutaneous coronary intervention complications in patients with acute coronary syndromes. J Am Coll Cardiol Intv. 2009;2:65-72.

    13. Asakura M, Ueda Y, Yamaguchi O, et al. Extensive development of vulnerable plaques as a pan-coronary process in patients with myocardial infarction: an angioscopic study. J Am Coll Cardiol. 2001;37:1284-1288.

    14. Ino Y, Kubo T, Tanaka A, et al. Difference of culprit lesion morphologies between ST-segment elevation myocardial infarction and non-ST-segment elevation acute coronary syndrome. J Am Coll Cardiol Intv. 2011;4:76-82.

    15. Madder RD, Smith JL, Dixon SR, Goldstein JA. Composition of target lesions by near-infrared spectroscopy in patients with acute coronary syndrome versus stable angina. Circ Cardiovasc Interv. 2012;5:55-61.

    16. Madder RD, Goldstein JA, Madden SP, et al. Detection by near-infrared spectroscopy of large lipid core plaques at culprit sites in patients with acute ST-segment elevation myocardial infarction. J Am Coll Cardiol Intv. In press, 2013.

    17. Hoffmann U, Moselewski F, Nieman K, et al. Noninvasive assessment of plaque morphology and composition in culprit and stable lesions in acute coronary syndrome and stable lesions in stable angina by mulitdetector computed tomography. J Am Coll Cardiol. 2006;47:1655-1662.

    18. Motoyama S, Kondo T, Sarai M, et al. Multislice computed tomographic characteristics of coronary lesions in acute coronary syndromes. J Am Coll Cardiol. 2007;50:319-326.

    19. Madder RD, Chinnaiyan KM, Marandici AM, Goldstein JA. Features of disrupted plaques by coronary computed tomographic angiography: correlates with invasively proven complex lesions. Circ Cardiovasc Imaging. 2011;4:105-113.

    20. Muller JE, Tofler GH, Stone PH. Circadian variation and triggers of onset of acute cardiovascular disease. Circulation. 1989;79;733-743.

    21. Kolodgie FD, Burke AP, Farb A, et al. The thin-cap fibroatheroma: a type of vulnerable plaque: the major precursor lesion to acute coronary syndromes. Curr Opin Cardiol. 2001;16:285-292.

    22. Yamagishi M, Terashima M, Awano K, et al. Morphology of vulnerable coronary plaque: insights from follow-up of patients examined by intravascular ultrasound before an acute coronary syndrome. J Am Coll Cardiol. 2000;35:106-111.

    23. Stone GW, Maehara A, Lansky A, et al. A prospective natural-history study of coronary atherosclerosis. N Engl J Med. 2011;364:226-235.

    24. Calvert PA, Obaid DR, O’Sullivan M, et al. Association between IVUS findings and adverse outcomes in patients with coronary artery disease: the VIVA (VH-IVUS in Vulnerable Atherosclerosis) study. J Am Coll Cardiol Imaging. 2011;4:894-901.

    25. Stone PH, Saito S, Takahashi S, et al. Prediction of progression of coronary artery disease and clinical outcomes using vascular profiling of endothelial shear stress and arterial plaque characteristics: the PREDICTION study. Circulation. 2012;126:172-181.

    26. Motoyama S, Sarai M, Harigaya H, et al. Computed tomographic angiography characteristics of atherosclerotic plaques subsequently resulting in acute coronary syndrome. J Am Coll Cardiol. 2009;54:49-57.

    27. Stone GW, Maehara A, Mintz GS. The reality of vulnerable plaque detection. J Am Coll Cardiol Imaging. 2011;4:902-904.

    28. Madder RD, Steinberg DH, Anderson RD. Multimodality direct coronary imaging with combined near-infrared spectroscopy and intravascular ultrasound: Initial US experience. Catheter Cardiovasc Interv. 2013;81:551-7.

    29. Kume T, Akasaka T, Kawamoto T, et al. Measurement of the thickness of the fibrous cap by optical coherence tomography. Am Heart J. 2006;152:755.e1-4.

    30. Nissen SE, Tuzcu EM, Schoenhagen P, et al. Effect of intensive compared with moderate lipid-lowering therapy on progression of coronary atherosclerosis: a randomized controlled trial. JAMA. 2004;291:1071-1080.

    31. Nissen SE, Nicholls SJ, Sipahi I, et al. Effect of very high-intensity statin therapy on regression of coronary atherosclerosis: the ASTEROID trial. JAMA. 2006;295:1556-1565.

    32. Nicholls SJ, Ballantyne CM, Barter PJ, et al. Effect of two intensive statin regimens on progression of coronary disease. N Engl J Med. 2011;365:2078-2087.

    33. Kini AS, Baber U, Kovacic JC, et al. Changes in plaque lipid content after short-term, intensive versus standard statin therapy: the YELLOW trial. J Am Coll Cardiol. 2013 (In press).

    34. Takarada S, Imanishi T, Kubo T, et al. Effect of statin therapy on coronary fibrous-cap thickness in patients with acute coronary syndrome: assessment by optical coherence tomography study. Atherosclerosis. 2009;202:491-497.

    35. Stein EA, Gipe D, Bergeron J, et al. Effect of a monoclonal antibody to PCSK9, REGN727/SAR236553, to reduce low-density lipoprotein cholesterol in patients with heterozygous familial hypercholesterolaemia on stable statin dose with or without ezetimibe therapy: a phase 2 randomised controlled trial. Lancet. 2012;380:29-36.

    36. Nissen SE, Tsunoda T, Tuzcu EM, et al. Effect of recombinant ApoA-I Milano on coronary atherosclerosis in patients with acute coronary syndromes: a randomized controlled trial. JAMA. 2003;290:2292-2300.

    37. Braunwald, E. Epilogue: What do clinicians expect from imagers? J Am Coll Cardiol. 2006;47:C101-C103.

    38. Bosch JL, Beinfeld MT, Muller JE, Brady T, Gazelle GS. A cost-effectiveness analysis of a hypothetical catheter-based strategy for the detection and treatment of vulnerable coronary plaques with drug-eluting stents. J Interv Cardiol. 2005;18:339-349.

    39. Kubo T, Maehara A, Mintz GS, et al. The dynamic nature of coronary artery lesion morphology assessed by serial virtual histology intravascular ultrasound tissue characterization. J Am Coll Cardiol. 2010;55:1590-1597.

    40. Waxman S, Dixon SR, L’Allier P, et al. In vivo validation of a catheter-based near-infrared spectroscopy system for detection of lipid core coronary plaques: initial results and exploratory analysis of the SPECTroscopic Assessment of Coronary Lipid (SPECTACL) multicenter study. J Am Coll Cardiol Imaging. 2009;2:858-868.

    41. Erlinge D, Muller JE, Puri R, et al. Validation of a near-infrared spectroscopic signature of lipid located at culprit lesions in ST-segment elevation myocardial infarction. European Atherosclerosis Society. June 2013 (abstract).


    Proposed Algorithm for Vulnerable Plaque Screening and Treatment 


    Page 31A in


    Long-term Consequences of a Lipid Core Plaque

    Christos V. Bourantas, MD, PhD1, Hector M. Garcia, MD, PhD1, Roberto Diletti, MD1, Carlos A.M. Campos, MD1, Yaojun Zhang, MD, PhD1, Scot Garg, MRCP, PhD2, Patrick W. Serruys, MD, PhD1

    1Department of Interventional Cardiology, Erasmus University Medical Centre, Thoraxcenter, Rotterdam, The Netherlands and 2Department of Cardiology, East Lancashire NHS Trust, Haslingden Road, Blackburn, Lancashire, United Kingdom.

    Disclosures: The authors report no financial relationships or conflicts of interest regarding the content herein.

    Address for correspondence:  Email: p.w.j.c.serruys@erasmusmc.nl

    The advent of intravascular imaging in the 1980s allowed us to study in vivo plaque morphology and its prognostic implications.

    • Angioscopy and intravascular ultrasound (IVUS) were the first imaging techniques that provided information about the composition of plaque and allowed detection of its lipid component.7,8

    However, the first applications of these modalities in the clinical setting not only underscored their potential value in the study of atherosclerosis but also highlighted their limitations in characterizing atheroma.9-11 Therefore an effort was made over the last few years to develop advanced techniques that would allow more reliable assessment of a plaque’s composition. Today several modalities are available for this purpose including:

    • the radiofrequency analysis of the IVUS backscatter signal (RF-IVUS),
    • near-infrared spectroscopy (NIRS),
    • optical coherence tomography (OCT),
    • magnetic resonance spectroscopy,
    • intravascular magnetic resonance imaging,
    • Raman spectroscopy,
    • photoacoustic imaging, and
    • time resolved spectroscopic imaging (Figure 1).

    Some of these modalities are still in their infancy, while others have already been used in the clinical setting providing robust evidence about the prognostic implications of the differing compositions of the plaque. The aim of this review article is to present the most recent evidence about the long-term consequences of the atheroma’s phenotype. 

    Current Evidence from NIRS-based Clinical Studies

    NIRS relies on the principle that different organic molecules absorb and scatter NIRS light to different degrees and wavelengths. Recent advances in device technology enabled the development of a catheter suitable for assessing the plaque in human coronaries that is able to emit NIR light and acquire the scattered signal. Spectral analysis of the obtained signal provides a color-coded display, called a chemogram (Figure 1C), which provides the probability that lipid core is present in the superficial plaque (studied depth approximately: 1 mm). Several studies have examined the reliability of this technique using histology as the gold standard and demonstrated a high overall accuracy in detecting lipid-rich plaques while others demonstrated its feasibility in the clinical setting.19-20

    The European Collaborative Project on Inflammation and Vascular Wall Remodeling in Atherosclerosis (NCT01789411) – NIRS sub-study was the first prospective trial designed to evaluate the prognostic implications of an increased lipid component, as detected by NIRS, in coronary plaques. Two hundred three patients that underwent X-ray angiography, and PCI if it was indicated, had NIRS in a non-culprit coronary segment and were followed-up for 1 year. Twenty-eight patients sustained a MACE during the follow-up period; 21 of these events were non-culprit lesion related. Lipid plaque burden index appeared to be an independent predictor of MACE (hazard ratio: 4.04, 95% confidence interval: 1.33-12.29; P=0.01). 

    Currently, the Chemometric Observation of Lipid Rich Plaque of Interest in Native Coronary Arteries (COLOR, NCT00831116) registry is recruiting patients. This study is planning to recruit 2000 patients that will be investigated with NIRS imaging, and aims to examine the association between the presence of a necrotic core in the atheroma and subsequent coronary events. Preliminary results indicate that the absence of lipid-rich plaques is related with better outcomes (www.infraredx.com/the-color-registry). 

    Current Evidence From OCT-based Clinical Studies

    OCT imaging with its high resolution appears able to provide detailed assessment of the superficial plaque and visualize structures that are unseen by other techniques such as the presence of micro calculations of thin-capped fibroatheroma (TCFA). However, a significant limitation of this technique is its poor penetration (1-2 mm), which does not permit through visualization of plaque burden, as well as its low capacity in differentiating lipid from calcific tissue when these are deeply embedded in the vessel wall.21

    In this analysis, 53 patients who underwent PCI had OCT imaging in non-obstructive lesion sat baseline and repeat angiography at 7 months follow-up. They found that plaques with a TCFA phenotype, exhibiting vessel walldiscontinuities, macrophages, neo-vessels, and thrombi were morelikely to progress and cause significant angiographic obstructions.22

    Future Perspective in Plaque Imaging – Conclusions

    Cumulative data derived from intravascular imaging studies have provided robust evidence about the prognostic implications of plaque’s composition and burden, and demonstrated a strong association between the presence of lipid-rich plaques and future cardiovascular events. Plaque pathology and quantification of lipid components is done by hybrid catheters able to acquire different intravascular imaging data.23

    References on page 26A in


    1.Kragel AH, Reddy SG, Wittes JT, Roberts WC. Morphometric analysis of the composition ofatherosclerotic plaques in the four major epicardial coronary arteries in acute myocardial infarctionand in sudden coronary death. Circulation. 1989;80:1747-1756.

    2.ᆳacteristics of coronary atherosclerotic plaques underlying fatal occlusive thrombi. Br Heart J.1983;50:127-134.

    3.Clark E, Graef I, Chasis H. Thrombosis of the aorta and coronary arteries. Archives of Pathology.1936;22:183-212.

    4.Virmani R, Kolodgie FD, Burke AP, Farb A, Schwartz SM. Lessons from sudden coronary death:a comprehensive morphological classification scheme for atherosclerotic lesions. ArteriosclerThromb Vasc Biol. 2000;20:1262-1275.

    5.Stary HC, Chandler AB, Glagov S, et al. A definition of initial, fatty streak, and intermediatelesions of atherosclerosis. A report from the Committee on Vascular Lesions of the Council onArteriosclerosis, American Heart Association. Circulation. 1994;89:2462-2478.

    6.ᆳrotic lesions and a histological classification of atherosclerosis. A report from the Committee onVascular Lesions of the Council on Arteriosclerosis, American Heart Association. Circulation.1995;92:1355-1374.

    7.Di Mario C, The SH, Madretsma S, et al. Detection and characterization of vascular lesionsby intravascular ultrasound: an in vitro study correlated with histology. J Am Soc Echocardiogr. 1992;5:135-146.

    8.ᆳdation by histomorphologic analysis and association with stable and unstable coronary syndromes.J Am Coll Cardiol. 1996;28:1-6.

    9.Hiro T, Leung CY, Russo RJ, et al. Variability in tissue characterization of atherosclerotic plaqueby intravascular ultrasound: a comparison of four intravascular ultrasound systems. Am J CardImaging. 1996;10:209-218.

    10.ᆳdial infarction: ability of optical coherence tomography compared with intravascular ultrasoundand coronary angioscopy. J Am Coll Cardiol. 2007;50:933-939.

    11.ᆳated with future risk of acute coronary syndrome: detection of vulnerable patients by angioscopy.J Am Coll Cardiol. 2006;47:2194-2200.

    12.ᆳnary syndrome using integrated backscatter intravascular ultrasound. J Am Coll Cardiol.2006;47:734-741.

    13.Amano T, Matsubara T, Uetani T, et al. Lipid-rich plaques predict non-target-lesion ischemicevents in patients undergoing percutaneous coronary intervention. Circ J. 2011;75:157-166.

    14.ᆳsclerosis. N Engl J Med. 2011;364:226-235.

    15.Calvert PA, Obaid DR, O’Sullivan M, et al. Association between IVUS findings and adverseᆳsclerosis) Study. JACC Cardiovasc Imaging. 2011;4:894-901.

    16.Granada JF, Wallace-Bradley D, Win HK, et al. In vivo plaque characterization using intravascularultrasound-virtual histology in a porcine model of complex coronary lesions. Arterioscler ThrombVasc Biol. 2007;27:387-393.

    17.Sales FJ, Falcao BA, Falcao JL, et al. Evaluation of plaque composition by intravascular ultrasound“virtual histology”: the impact of dense calcium on the measurement of necrotic tissue. ᆳvention. 2010;6:394-399.

    18.ᆳtual histology intravascular ultrasound in porcine coronary artery disease. Circ Cardiovasc Imaging. 2010;3:384-391.

    19.ᆳmens with a novel catheter-based near-infrared spectroscopy system. JACC Cardiovasc Imaging. 2008;1:638-648.

    20.Waxman S, Dixon SR, L’Allier P, et al. In vivo validation of a catheter-based near-infrared spectrosᆳcopy system for detection of lipid core coronary plaques: initial results of the SPECTACL study.JACC Cardiovasc Imaging. 2009;2:858-868.

    21.Manfrini O, Mont E, Leone O, et al. Sources of error and interpretation of plaque morphology byoptical coherence tomography. Am J Cardiol. 2006;98:156-159.

    22.Uemura S, Ishigami K, Soeda T, et al. Thin-cap fibroatheroma and microchannel findings inoptical coherence tomography correlate with subsequent progression of coronary atheromatousplaques. Eur Heart J. 2012;33:78-85.

    23.ᆳplications and prospective potential in the study of coronary atherosclerosis. J Am Coll Cardiol.2013;61:1369-378.

    24.ᆳtroscopy and intra-vascular ultrasound catheter to identify composition and structure of coronaryplaque. EuroIntervention. 2010;5:755-756.

    25.ᆳᆳgrated Biomarker and Imaging Study-3 (IBIS-3). EuroIntervention. 2012;8:235-241.


    NIRS-IVUS Imaging Identifies Lesions at High Risk of Peri-Procedural Myocardial Infarction

    James A. Goldstein, MD, Simon R. Dixon, MBChB*, Gregg W. Stone, MD

    From the Department of Cardiovascular Medicine, William Beaumont Hospital, Royal Oak, MI.

    Address for correspondence: James A. Goldstein, MD, FACC, Department of Cardiovascular Medicine, William Beaumont Hospital, 3601 West 13 Mile Road, Royal Oak, Michigan 48073. Email: jgoldstein@beaumont.edu

    Disclosures: Dr. Goldstein is a consultant for and owns equity in Infraredx, Inc. Dr. Stone is a consultant for Infraredx, Inc., Volcano Corp., Medtronic, and Boston Scientific, and is a member of the scientific advisory boards for Boston Scientific and Abbott Vascular. Dr. Dixon reports no financial relationships or conflicts


    Percutaneous coronary intervention (PCI) is associated with distal embolization complications, including peri-procedural myocardial infarction (PPMI), including no-reflow, in 3%-15% of cases. These complications are predominantly related to distal embolization of lipid core plaque (LCP) components. Catheter-based near-infrared spectroscopy (NIRS) provides rapid, automated detection of LCPs, the magnitude of which appears associated with a high-risk of PPMI. Employing this technique may facilitate development of preventive measures such as embolic protection devices (EPDs).

    J INVASIVE CARDIOL 2013;25 (Suppl A):14A-16A

    Key words: Distal embolization, lipid core plaque, near-infrared spectroscopy, peri-procedural myocardial infarction

    Figures 1. A 62-year-old man with stable angina underwent coronary angiography, which demonstrated a complex hazy ulcerated culprit lesion in the mid-right coronary artery (Figure 1A, solid arrow). Neither the angiogram nor an intravascular ultrasound image indicated the presence of thrombus. NIRS demonstrated a large yellow signal spanning the circumference of the culprit site (Figure 1B, white rectangle), indicating the presence of a napkin-ring LCP; a smaller LCP was evident distally (Figure 1, open arrow).

    Figure 2. Balloon angioplasty was performed (Figure 2A, arrow), which led to prompt no-reflow (Figure 2B, arrow) associated with severe bradyarrhythmia and profound hypotension (Figure 2C). After brief cardiopulmonary resuscitation and pharmacological support with atropine and dopamine, physiologic rhythm and blood pressure were restored and stenting resulted in excellent angiographic outcome. However, the patient developed a peri-stenting non-transmural infarction (peak creatine kinase of 512 ng/mL) and required an additional day of hospital care in an intensive care unit. (Goldstein JA, et al. JACC Cardiovasc Imaging. 2009;2(12):1420-1424. Reproduced with permission.)

    On Page 14A in


    Pharmacological Therapy of Lipid Core Plaque

    Jason C. Kovacic, MD, PhD, Annpoorna Kini, MD, MRCP

    From The Zena and Michael A. Wiener Cardiovascular Institute, Mount Sinai School of Medicine, New York, New York.

    Address for correspondence: Dr. Annapoorna Kini, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1030, New York, NY, 10029. Email: annapoorna.kini@mountsinai.org

    Disclosures: Dr. Kovacic is supported by National Institutes of Health Grant K08HL111330 and has received research support from AstraZeneca. Dr. Kini acknowledges honoraria from Medscape and has received research grant support from InfraReDx.

    A new group of terms is slowly creeping in to the atherosclerotic disease lexicon: “Lipid Arc,” “Lipid Core Plaque,” “Lipid-Rich Plaque,” “Lipid Core Burden Index” and other similar phrases. While clinicians and researchers have long been aware of the central importance of lipid in the biology of atherosclerosis, the growing use of these terms is driven by the recent widespread use of novel imaging modalities that provide accurate detection, and even quantification, of the extent of lipid that is contained within the core of an atherosclerotic plaque. Our ability to detect and quantify lipid in plaques is opening up new therapeutic opportunities for modifying the atherosclerotic disease process, which may ultimately be of benefit to patients.

    At the present time there are 3 methods that are commonly used to measure the extent of lipid in atherosclerotic plaques. Perhaps most familiar of these is coronary computer tomographic (CT) scanning. While more commonly used to quantitate calcification or luminal stenosis, CT scanning is readily able to quantitate the extent of lipid associated with an atherosclerotic lesion. However, while several studies have reported various Hounsfield Unit (HU)-based criteria to distinguish lipid-rich from fibrous plaques, the HU cut-off points have so far been inconsistent. The use of CT for detecting lipid-rich plaque is further limited by its relatively low spatial resolution and the fact that the HU values for distinguishing between fibrous and lipid-rich plaques are overlapping.1 In contrast, optical coherence tomography (OCT) offers perhaps the greatest spatial resolution of all clinically available coronary imaging devices. OCT can offer exquisite detail of abluminal coronary artery anatomy, including detection of lipid core plaque. However, while automated systems are being developed, at the present time the quantitation of lipid by OCT is a somewhat specialized process that typically involves detailed off-line analysis.

    A specific intra-coronary imaging catheter for the quantitation of coronary artery lipid content is now available and FDA approved: diffuse reflectance near-infrared spectroscopy (NIRS). The application of NIRS to identify lipid deposition within coronary arteries has been validated ex vivo2-5 and in vivo.6,7 Although NIRS itself is essentially only able to detect and quantitate lipid, design changes and technological advances to this catheter have now made it possible to combine intravascular ultrasound (IVUS) and NIRS technology on a single instrument. In one of the few clinical studies published to date using this device, NIRS has already shown that a high lipid burden in a target lesion undergoing percutaneous coronary intervention (PCI) is associated with an increased likelihood of peri-procedural myocardial infarction.7

    It is well known that the reduction of cholesterol levels by statin therapy is associated with significant decreases in plaque burden. REVERSAL,8 ASTEROID,9 and more recently the SATURN II10 trial showed that in patients with coronary artery disease (CAD), lipid lowering with high-dose statin therapy reduced progression of plaque atheroma burden, even causing plaque regression of some lesions. However, while reduction in atheroma burden and plaque size are important anatomical endpoints, a major unresolved question had been the mechanism of action of statins and the unanswered question of whether they reduce plaque lipid content. Indeed, a high burden of plaque lipid is one of the cardinal features of a rupture-prone vulnerable lesion.11 Therefore, the ability to reduce plaque lipid content may have important effects on lesion stability and therefore, might impact clinical endpoints.

    The advent of sensitive imaging tools for the evaluation of plaque lipid content has paved the way for the investigation of potential pharmacological therapies for lipid core plaque. In particular, the ability of NIRS to provide an automated quantitation of plaque lipid provides a ready-made platform for this task. We recently completed the YELLOW study of high-dose statin therapy for the potential reduction of coronary artery lipid content as assessed by NIRS. We randomized 87 patients with multivessel CAD undergoing elective PCI to rosuvastatin 40 mg daily vs conventional statin therapy. Following PCI of the culprit lesion, non-culprit lesions with a fractional flow reserve (FFR) <0.8 were interrogated using IVUS and NIRS. Changes in plaque composition were assessed after 6-12 weeks during follow-up angiography. The core finding of this study was that high-dose statin therapy was associated with significant reductions in the lipid content of coronary atherosclerotic plaques. Interestingly, despite reduced plaque lipid content, in this relatively short time period concordant changes in gross lesion characteristics such as total atheroma volume or % plaque burden were not observed.12 In short, the YELLOW study identified that even before gross atheroma regression occurs, lipid removal from plaques is an early event upon initiation of high-dose statin therapy. Furthermore, the results of the YELLOW study are concordant with the known acute benefits of statin therapy in patients presenting with acute coronary syndromes, where the early introduction of these agents is known to be of clinical benefit.13 While the YELLOW study was the first of this nature and the results remain to be replicated in a larger trial, these findings have revived interest in the concept of the “vulnerable plaque” because it appears possible that by causing lipid core reduction over a just few weeks, high-dose statin therapy may have rapid plaque stabilizing effects. We are now embarking on the YELLOW II study, where we will further explore the utility of high-dose rosuvastatin for the early reduction of plaque lipid content and potential mechanistic pathways.

    What other agents might have therapeutic efficacy for lipid core reduction? This question is perhaps more complex than it might first appear, because at the present time we do not know the specific mechanism whereby high-dose rosuvastatin causes lipid reduction in plaques. Theoretically it may be due to reduced LDL, increased HDL, other mechanisms or a combination of these effects. Potentially, other agents that are already available such as bile acid sequestrants, ezetimibe, and fibrates may have a weak lipid core reducing effect. However, we would underscore the fact that at the present time the utility of these agents is speculative, and no other agent (apart from high-dose rosuvastatin in the YELLOW study) has been shown to reduce lipid content in vivo in human plaques. Furthermore, given the fact that these other agents are far less potent in their overall effect than rosuvastatin 40 mg/day, it may be clinically challenging to determine if they have efficacy for lipid core reduction beyond that of statins.

    In addition to pharmacotherapy, it must be remembered that we have several non-pharmacological treatments in our armamentarium that may impact lipid core reduction. For example, exercise is known to be associated with reduced plaque lipid content,14 and proper adherence to current guidelines with respect to lifestyle and diet are of paramount importance in any patient in whom it is considered desirable to reduce plaque lipid content.

    Looking ahead, there are several emerging and investigational agents that may hold promise for lipid core reduction. Microsomal triglyceride transfer protein (MTP) is expressed in the liver, intestine, and the heart and is required for the proper assembly of VLDL and chylomicrons. In animals, treatment with an MTP inhibitor leads to a rapid reduction in plasma lipid levels, with a significant decrease in lipid content and monocyte-derived (CD68+) cells in atherosclerotic plaques.15 On December 21, 2012, the first of the MTP inhibitors was approved for clinical use. Lomitapide (marketed as Juxtapid) was approved by the FDA as an adjunct to a low fat diet and other lipid-lowering treatments for patients with homozygous familial hypercholesterolemia. However, concerns have been raised due to hepatic side effects and liver toxicity. As a result, lomitapide will carry a boxed warning and will only be available through a restricted program.16 Another new drug that was recently given restricted approval in the US for homozygous familial hypercholesterolemia is mipomersen. This agent is an antisense therapeutic that targets messenger RNA for apolipoprotein B, leading to reduced apoB protein and LDL levels. While showing efficacy for lowering LDL,17 safety concerns have thus far prohibited this agent from gaining approval for use in Europe. PCSK9 inhibitors are yet another novel class of agents that may hold promise for reducing lipid core plaque. PCSK9 is involved in the degradation of the LDL receptor (LDLR), and by inhibiting PCSK9 it is believed that this permits more LDL receptors to remain active and participate in LDL removal from the blood, thereby reducing plasma LDL and cholesterol levels. Denis et al18 recently demonstrated that gene inactivation of PCSK9 in mice reduced aortic cholesterol accumulation and atherosclerotic lesion development in atherosclerosis-prone mice. Based on their powerful LDL lowering effect, intense efforts are currently underway to develop clinically efficacious PCSK9 inhibitors with several agents already moving to phase II/III human studies.19 While all of these new and emerging therapies are cause for optimism, the recent experience with CETP-inhibitors and the overall failure of this class so far to stand up to rigorous testing as HDL raising agents in phase III studies20,21 serves to remind us that not all “promising future therapies” survive through the arduous clinical testing pipeline.

    In conclusion, there is renewed interest in the concept of “plaque regression” and pharmacological therapy for “lipid core reduction.” This has been driven by our increasing ability to image and quantify these phenomena, and more recently by the provocative findings that high-dose statin therapy may achieve both of these clinical endpoints. Further studies are now required to evaluate novel agents, define mechanisms of action and, most importantly, to confirm that atherosclerotic lipid core reduction is associated with plaque stabilization and fewer clinical endpoints.

    References, pp. 27A-28A in the Supplement

    1. Kristanto W, van Ooijen PM, Greuter MJ, et al. Non-calcified coronary atherosclerotic plaque visualization on CT: effects of contrast-enhancement and lipid-content fractions. Int J Cardiovasc Imaging. 2013; online ahead of print.

    2. Cassis LA, Lodder RA. Near-IR imaging of atheromas in living arterial tissue. Anal Chem. 1993;65:1247-1256.

    3. Jaross W, Neumeister V, Lattke P, et al. Determination of cholesterol in atherosclerotic plaques using near infrared diffuse reflection spectroscopy. Atherosclerosis. 1999;147:327-337.

    4. Moreno PR, Lodder RA, Purushothaman KR, et al. Detection of lipid pool, thin fibrous cap, and inflammatory cells in human aortic atherosclerotic plaques by near-infrared spectroscopy. Circulation. 2002;105:923-927.

    5. Wang J, Geng YJ, Guo B, et al. Near-infrared spectroscopic characterization of human advanced atherosclerotic plaques. J Am Coll Cardiol. 2002;39:1305-1313.

    6. Waxman S, Dixon SR, L’Allier P, et al. In vivo validation of a catheter-based near-infrared spectroscopy system for detection of lipid core coronary plaques: initial results of the SPECTACL study. JACC Cardiovasc Imaging. 2009;2:858-868.

    7. Goldstein JA, Maini B, Dixon SR, et al. Detection of lipid-core plaques by intracoronary near-infrared spectroscopy identifies high risk of periprocedural myocardial infarction. Circ Cardiovasc Interv. 2011;4:429-437.

    8. Nissen SE, Tuzcu EM, Schoenhagen P, et al. Statin therapy, LDL cholesterol, C-reactive protein, and coronary artery disease. N Engl J Med. 2005;352:29-38.

    9. Nissen SE, Nicholls SJ, Sipahi I, et al. Effect of very high-intensity statin therapy on regression of coronary atherosclerosis: the ASTEROID trial. JAMA. 2006;295:1556-1565.

    10. Nicholls SJ, Ballantyne CM, Barter PJ, et al. Effect of two intensive statin regimens on progression of coronary disease. N Engl J Med. 2011;365:2078-2087.

    11. Varnava AM, Mills PG, Davies MJ. Relationship between coronary artery remodeling and plaque vulnerability. Circulation. 2002;105:939-943.

    12. Kini AS, Baber U, Kovacic JC, et al. Changes in plaque lipid content after short-term, intensive versus standard statin therapy: The YELLOW Trial. J Am Coll Cardiol. 2013;62:21-29.

    13. Hulten E, Jackson JL, Douglas K, et al. The effect of early, intensive statin therapy on acute coronary syndrome: a meta-analysis of randomized controlled trials. Arch Intern Med. 2006;166:1814-1821.

    14. Yoshikawa D, Ishii H, Kurebayashi N, et al. Association of cardiorespiratory fitness with characteristics of coronary plaque: assessment using integrated backscatter intravascular ultrasound and optical coherence tomography. Int J Cardiol. 2013;162:123-128.

    15. Hewing B, Parathath S, Mai CK, et al. Rapid regression of atherosclerosis with MTP inhibitor treatment. Atherosclerosis. 2013;227:125-129.

    16. Cuchel M, Bloedon LT, Szapary PO, et al. Inhibition of microsomal triglyceride transfer protein in familial hypercholesterolemia. N Engl J Med. 2007;356:148-156.

    17. Raal FJ, Santos RD, Blom DJ, et al. Mipomersen, an apolipoprotein B synthesis inhibitor, for lowering of LDL cholesterol concentrations in patients with homozygous familial hypercholesterolaemia: a randomised, double-blind, placebo-controlled trial. Lancet. 2010;375:998-1006.

    18. Denis M, Marcinkiewicz J, Zaid A, et al. Gene inactivation of proprotein convertase subtilisin/kexin type 9 reduces atherosclerosis in mice. Circulation. 2012;125:894-901.

    19. Roth EM, McKenney JM, Hanotin C, et al. Atorvastatin with or without an antibody to PCSK9 in primary hypercholesterolemia. N Engl J Med. 2012;367:1891-1900.

    20. Schwartz GG, Olsson AG, Abt M, et al. Effects of dalcetrapib in patients with a recent acute coronary syndrome. N Engl J Med. 2012;367:2089-2099.

    21. Kovacic JC, Fuster V. From Treating Complex Coronary Artery Disease to Promoting Cardiovascular Health: Therapeutic Transitions and Challenges, 2010-2020. Clin Pharmacol Ther. 2011;90:509-518.



    The Journal of Invasive Cardiology®

    KEY SOURCE for this Article

    Journal of Invasive Cardiology, August 2013, Vol 25/Supplement A

    Print ISSN 1042-3931 / Electronic ISSN 1557-2501


    NIRS-IVUS Imaging To Characterize the Composition and Structure of Coronary Plaques

    D. RIZIK AND J.A. GOLDSTEIN……………………………………..2A


    Imaging of Plaque Composition and Structure with the TVC Imaging System™ and TVC Insight™ Catheter

    B. SHYDO, ET AL…………………………………………………………5A

    Comparative Intravascular Imaging for Lipid Core Plaque: NIRS vs VH-IVUS vs OCT

    E. FUH AND E.S. BRILAKIS……………………………………………9A

    Plaque Characterization and PCI Procedural Outcomes

    NIRS-IVUS Imaging Identifies Lesions at High Risk of

    Peri-Procedural Myocardial Infarction

    J.A. GOLDSTEIN, ET AL……………………………………………..14A

    Case Vignettes:

    Multiple Plaque Ruptures in a Patient with ST-Segment Elevation Myocardial Infarction: Does Infrared Spectroscopy Evidence Explain a Significant Change in the Angiogram?

    M.J. LIM AND J.M. STOLKER……………………………………….16A

    Missing the Culprit Yellow Plaque

    D. ERLINGE…………………………………………………………….18A

    The Use of Near-Infrared Spectroscopy to Optimize Stent Length

    G.A. STOUFFER ………………………………………………………19A

    Employing NIRS-IVUS to Guide Optimal Lesion Coverage—Avoidance of Geographic Miss

    I. HANSON, ET AL……………………………………………………..20A

    Peri-Procedural Myocardial Injury Unraveled: Combined

    Assessment by Optical Coherence Tomography, Near-Infrared

    Spectroscopy, and IVUS

    A. KARANASOS, ET AL………………………………………………..22A

    Plaque Characterization and Long-Term 

    Clinical Outcomes

    Long-term Consequences of a Lipid Core Plaque

    C.V. BOURANTAS, ET AL…………………………………………….24A

    Pharmacological Therapy of Lipid Core Plaque

    J.C. KOVACIC AND A. KINI………………………………………….27A

    The Search for Vulnerable Plaque — The Pace Quickens

    R.D. MADDER, ET AL…………………………………………………29A

    Case Vignettes:

    Observations from Intracoronary Near-Infrared Spectroscopy in Patients with ST-Segment Elevation Myocardial Infarction

    R.D. MADDER…………………………………………………………34A

    NIRS Imaging of Cardiac Allograft Vasculopathy

    G. WEISZ ……………………………………………………………….35A

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