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A Future for Plasma Metabolomics in Cardiovascular Disease Assessment

Curator: Larry H Bernstein, MD, FCAP

 

 

Plasma metabolomics reveals a potential panel of biomarkers for early diagnosis
in acute coronary syndrome  

CM. Laborde, L Mourino-Alvarez, M Posada-Ayala,
G Alvarez-Llamas, MG Serranillos-Reus, et al.
Metabolomics – manuscript draft

In this study, analyses of peripheral plasma from Non-ST Segment Elevation
Acute Coronary Syndrome patients and healthy controls by gas chromatography-
mass spectrometry permitted the identification of 15 metabolites with statistical
differences (p<0.05) between experimental groups.
In our study, 6 amino acids were found decreased in NSTEACS patients when
compared with healthy control group suggesting either a decrease in anabolic
activity of these metabolites or an increase in the catabolic pathways. Of both
possibilities, the increased catabolism of the amino acids can be explained
considering simultaneously the capacity of glycogenic and ketogenic amino
acids along with the gradual hypoxic condition to which cardiac muscle cells
have been exposed.

Additionally, validation by gas chromatography-mass spectrometry and liquid
chromatography-mass spectrometry permitted us to identify a potential panel
of biomarkers formed by 5-OH tryptophan, 2-OH-butyric acid and 3-OH-butyric
acid. Oxidative stress conditions dramatically increase the rate of hepatic
synthesis of glutathione. It is synthesized from the amino acids cysteine, glutamic
acid and glycine. Under these conditions of metabolic stress, the supply of cysteine
for glutathione synthesis become limiting and homocysteine is used to form
cystathionine, which is cleaved to cysteine and 2-OH-butyric acid. Thus elevated
plasma levels of 2-OH-butyric acid can be a good biomarker of cellular oxidative
stress for the early diagnosis of ACS.  Another altered metabolite of similar
structure was 3-OH-butyric acid, a ketone body together with the acetoacetate,
and acetone. Elevated levels of ketone bodies in blood and urine mainly occur
in diabetic ketoacidosis. Type 1 diabetes mellitus (DMI) patients have decreased
levels of insulin in the blood that prevent glucose enter cells so these cells use
the catabolism of fats as energy source that produce ketones as final products.
This panel of biomarkers reflects the oxidative stress and the hypoxic state that
disrupts the myocardial cells and consequently constitutes a metabolomic
signature that could be used for early diagnosis of acute coronary syndrome.
We hypothesize that the hypoxia situation comes to “mimic” the physiological
situation that occurs in DMI. In this case, the low energy yield of glucose
metabolism “forces” these cells to use fat as energy source (through catabolism
independent of aerobic/anaerobic conditions) occurring ketones as final
products. In our experiment, the 3-OH-butyric acid was strongly elevated in
NSTEACS patients.

 

Current Methods Used in the Protein Carbonyl Assay
Nicoleta Carmen Purdel, Denisa Margina and Mihaela Ilie.
Ann Res & Rev in Biol 2014; 4(12): 2015-2026.
http://www.sciencedomain.org/download.php?f=Purdel4122013ARRB8763-1

The attack of reactive oxygen species on proteins and theformation of
protein carbonyls were investigated only in the recent years. Taking into
account that protein carbonyls may play an important role in the early
diagnosis of pathologies associated with reactive oxygen species
overproduction, a robust and reliable method to quantify the protein
carbonyls in complex biological samples is also required. Oxidative
stress represents the aggression produced at the molecular level by
the imbalance between pro-oxidant and antioxidant agents, in favor of
pro-oxidants, with severe functional consequences in all organs and
tissues. An overproduction of ROS results in oxidative damages
especially to proteins (the main target of ROS), as well as in lipids,or
DNA. Glycation and oxidative stress are closely linked, and both
phenomena are referred to as ‘‘glycoxidation’’. All steps of glycoxidation
generate oxygen-free radical production, some of them being common
with lipidic peroxidation pathways.
The initial glycation reaction is followed by a cascade of chemical
reactions resulting in the formation of intermediate products (Schiff base,
Amadori and Maillard products) and finally to a variety of derivatives
named advanced glycation end products (AGEs). In hyperglycemic
environments and in natural aging, AGEs are generated in increased
concentrations; their levels can be evaluated in plasma due to the fact
that they are fluorescent compounds. Specific biomarkers of oxidative
stress are currently investigated in order to evaluate the oxidative status
of a biological system and/or its regenerative power. Generaly, malondi-
aldehyde, 4-hydroxy-nonenal (known together as thiobarbituric acid
reactive substances – TBARS), 2-propenal and F2-isoprostanes are
investigated as markers of lipid peroxidation, while the measurement
of protein thiols, as well as S-glutathionylated protein are assessed
as markers of oxidative damage of proteins. In most cases, the
oxidative damage of the DNA has 8-hydroxy-2l-deoxyguanosine
(8-OHdG) as a marker.  The oxidative degradation of proteins plays an
important role in the early diagnosis of pathologies associated with
ROS overproduction. Oxidative modification of the protein structure
may take a variety of forms, including the nitration of tyrosine residues,
carbonylation, oxidation of methionine, or thiol groups, etc.

The carbonylation of protein represents the introduction of carbonyl
groups (aldehyde or ketone) in the protein structure, through several
mechanisms: by direct oxidation of the residues of lysine, arginine,
proline and threonine residues from the protein chain, by interaction
with lipid peroxidation products with aldehyde groups (such as 4-
hydroxy-2-nonenal, malondialdehyde, 2-propenal), or by the
interaction with the compounds with the carbonyl groups resulting
from the degradation of the lipid or glycoxidation. All of these
molecular changes occur under oxidative stress conditions.
There is a pattern of carbonylation, meaning that only certain
proteins can undergo this process and protein structure determines
the preferential sites of carbonylation. The most investigated
carbonyl derivates are represented by gamma-glutamic
semialdehyde (GGS) generated from the degradation of arginine
residue and α-aminoadipic semialdehyde (AAS) derived from lysine.

A number of studies have shown that the generation of protein
carbonyl groups is associated with normal cellular phenomena like
apoptosis, and cell differentiation and is dependent on age, species
and habits (eg. smoking) or severe conditions’ exposure (as
starvation or stress). The formation and accumulation of protein
carbonyls is increased in various human diseases, including –
diabetes and cardiovascular disease.

Recently, Nystrom [7] suggested that the carbonylation process
is associated with the physiological and not to the chronological
age of the organism and the carbonylation may be one of the causes
of aging and cell senescence; therefore it can be used as the marker
of these processes. Jha and Rizvi, [15] proposed the quantification of
protein carbonyls in the erythrocyte membrane as a biomarker of aging

PanelomiX: A threshold-based algorithm to create panels of
biomarkers

X Robin, N Turck, A Hainard, N Tiberti, F Lisacek. 
T r a n s l a t i o n a l  P r o t e o m i c s   2 0 1 3; 1: 57–64.
http://dx.doi.org/10.1016/j.trprot.2013.04.003

The computational toolbox we present here – PanelomiX – uses
the iterative combination of biomarkers and thresholds (ICBT) method.
This method combines biomarkers andclinical scores by selecting
thresholds that provide optimal classification performance. Tospeed
up the calculation for a large number of biomarkers, PanelomiX selects
a subset ofthresholds and parameters based on the random forest method.
The panels’ robustness and performance are analysed by cross-validation
(CV) and receiver operating characteristic(ROC) analysis.

Using 8 biomarkers, we compared this method against classic
combination procedures inthe determination of outcome for 113 patients
with an aneurysmal subarachnoid hemorrhage. The panel classified the
patients better than the best single biomarker (< 0.005) and compared
favourably with other off-the-shelf classification methods.

In conclusion, the PanelomiX toolbox combines biomarkers and evaluates
the performance of panels to classify patients better than single markers
or other classifiers. The ICBT algorithm proved to be an efficient classifier,
the results of which can easily be interpreted. 

Multiparametric diagnostics of cardiomyopathies by microRNA
signatures.
CS. Siegismund, M Rohde, U Kühl,  D  Lassner.
Microchim Acta 2014 Mar.
http://dx.doi.org:/10.1007/s00604-014-1249-y

MicroRNAs (miRNAs) represent a new group of stable biomarkers
that are detectable both in tissue and body fluids. Such miRNAs
may serve as cardiological biomarkers to characterize inflammatory
processes and to differentiate various forms of infection. The predictive
power of single miRNAs for diagnosis of complex diseases may be further
increased if several distinctly deregulated candidates are combined to
form a specific miRNA signature. Diagnostic systems that generate
disease related miRNA profiles are based on microarrays, bead-based
oligo sorbent assays, or on assays based on real-time polymerase
chain reactions and placed on microfluidic cards or nanowell plates.
Multiparametric diagnostic systems that can measure differentially
expressed miRNAs may become the diagnostic tool of the future due
to their predictive value with respect to clinical course, therapeutic
decisions, and therapy monitoring.

Nutritional lipidomics: Molecular metabolism, analytics, and
diagnostics
JT. Smilowitz, AM. Zivkovic, Yu-Jui Y Wan, SM. Watkins, et al.
Mol. Nutr. Food Res2013, 00, 1–17.
http://dx.doi.org:/10.1002/mnfr.201200808

The term lipidomics is quite new, first appearing in 2001. Its definition
is still being debated, from “the comprehensive analysis of all lipid
components in a biological sample” to “the full characterization of
lipid molecular species and their biological roles with respect to the
genes that encode proteins that regulate lipid metabolism”. In principle,
lipidomics is a field taking advantage of the innovations in the separation
sciences and MS together with bioinformatics to characterize the lipid
compositions of biological samples (biofluids, cells, tissues, organisms)
compositionally and quantitatively.

Biochemical pathways of lipid metabolism remain incomplete and the
tools to map lipid compositional data to pathways are still being assembled.
Biology itself is dauntingly complex and simply separating biological
structures remains a key challenge to lipidomics. Nonetheless, the
strategy of combining tandem analytical methods to perform the sensitive,
high-throughput, quantitative, and comprehensive analysis of lipid
metabolites of very large numbers of molecules is poised to drive
the field forward rapidly. Among the next steps for nutrition to understand
the changes in structures, compositions, and function of lipid biomolecules
in response to diet is to describe their distribution within discrete functional
compartments lipoproteins. Additionally, lipidomics must tackle the task
of assigning the functions of lipids as signaling molecules, nutrient sensors,
and intermediates of metabolic pathways.

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Summary of Genomics and Medicine: Role in Cardiovascular Diseases


Summary of Genomics and Medicine: Role in Cardiovascular Diseases

Author: Larry H. Bernstein, MD, FCAP

The articles within Chapters and Subchapters you have just read have been organized into four interconnected parts.
  1. Genomics and Medicine
  2. Epigenetics – Modifyable Factors Causing CVD
  3. Determinants of CVD – Genetics, Heredity and Genomics Discoveries
  4. Individualized Medicine Guided by Genetics and Genomics Discoveries
The first part established the
  • rapidly evolving science of genomics
  • aided by analytical and computational tools for the identification of nucleotide substitutions, or combinations of them
that have a significant association with the development of
  • cardiovascular diseases,
  • hypercoagulable state,
  • atherosclerosis,
  • microvascular disease,
  • endothelial disruption, and
  • type-2DM, to name a few.
These may well be associated with increased risk for stroke and/or peripheral vascular disease in some cases,
  • essentially because the involvement of the circulation is systemic in nature.

Part 1

establishes an important connection between RNA and disease expression.  This development has led to
  • the necessity of a patient-centric approach to patient-care.
When I entered medical school, it was eight years after Watson and Crick proposed the double helix.  It was also
  • the height of a series of discoveries elucidating key metabolic pathways.
In the period since then there have been treatments for some of the important well established metabolic diseases of
  • carbohydrate,
  • protein, and
  • lipid metabolism,
such as –  glycogen storage disease, and some are immense challenges, such as
  • Tay Sachs, or
  • Transthyretin-Associated amyloidosis.
But we have crossed a line delineating classical Mendelian genetics to
  • multifactorial non-linear traits of great complexity and
involving combinatorial program analyses to resolve.
The Human Genome Project was completed in 2001, and it has opened the floodgates of genomic discovery.  This resulted in the identification of
genomic alterations in
  • cardiovascular disease,
  • cancer,
  • microbial,
  • plant,
  • prion, and
  • metabolic diseases.
This has also led to
  • the identification of genomic targets
  • that are either involved in transcription or
  • are involved with cellular control mechanisms for targeted pharmaceutical development.
In addition, there is great pressure on the science of laboratory analytics to
  • codevelop with new drugs,
  • biomarkers that are indicators of toxicity or
  • of drug effectiveness.
I have not mentioned the dark matter of the genome. It has been substantially reduced, and has been termed dark because
  • this portion of the genome is not identified in transcription of proteins.
However, it has become a lightning rod to ongoing genomic investigation because of
  • an essential role in the regulation of nuclear and cytoplasmic activities.
Changes in the three dimensional structure of these genes due to
  • changes in Van der Waal forces and internucleotide distances lead to
  • conformational changes that could have an effect on cell activity.

Part 2

is an exploration of epigenetics in cardiovascular diseases.  Epigenetics is
  • the post-genomic modification of genetic expression
  • by the substitution of nucleotides or by the attachment of carbohydrate residues, or
  • by alterations in the hydrophobic forces between sequences that weaken or strengthen their expression.
This could operate in a manner similar to the conformational changes just described.  These changes
  • may be modifiable, and they
  • may be highly influenced by environmental factors, such as
    1. smoking and environmental toxins,
    2. diet,
    3. physical activity, and
    4. neutraceuticals.
While neutraceuticals is a black box industry that evolved from
  • the extraction of ancient herbal remedies of agricultural derivation
    (which could be extended to digitalis and Foxglove; or to coumadin; and to penecillin, and to other drugs that are not neutraceuticals).

The best examples are the importance of

  • n-3 fatty acids, and
  • fiber
  • dietary sulfur (in the source of methionine), folic acid, vitamin B12
  • arginine combined with citrulline to drive eNOS
  • and of iodine, which can’t be understated.
In addition, meat consumption involves the intake of fat that contains

  • the proinflammatory n-6 fatty acid.

The importance of the ratio of n-3/n-6 fatty acids in diet is not seriously discussed when

  • we look at the association of fat intake and disease etiology.
Part 2 then leads into signaling pathways and proteomics. The signaling pathways are
  • critical to understanding the inflammatory process, just as
  • dietary factors tie in with a balance that is maintained by dietary intake,
    • possibly gut bacteria utilization of delivered substrate, and
    • proinflammatory factors in disaease.
These are being explored by microfluidic proteomic and metabolomic technologies that were inconceivable a half century ago.
This portion extended into the diagnosis of cardiovascular disease, and
  • elucidated the relationship between platelet-endothelial interaction in the formation of vascular plaque.
It explored protein, proteomic, and genomic markers
  1. for identifying and classifying types of disease pathobiology, and
  2. for following treatment measures.

Part 3

connected with genetics and genomic discoveries in cardiovascular diseases.

Part 4

is the tie between life style habits and disease etiology, going forward with
  • the pursuit of cardiovascular disease prevention.
The presentation of walking and running, and of bariatric surgery (type 2DM) are fine examples.
It further discussed gene therapy and congenital heart disease.  But the most interesting presentations are
  • in the pharmacogenomics for cardiovascular diseases, with
    1. volyage-gated calcium-channels, and
    2. ApoE in the statin response.

This volume is a splendid example representative of the entire collection on cardiovascular diseases.

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Genomics and Medicine: The Physician’s View


Genomics and Medicine: The Physician’s View

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

 

Genomics has had a rapid growth of research into variability of human genetics in both healthy populations in the study of population migration, and in the study of genetic sequence alterations that may increase the risk of expressed human disease.  This is the case for cardiology, cancer, inflammtory conditions, and gastrointestinal diseases. For the most part, genomics research in the last decade has shed light on potential therapeutic targets, but the identification of drug toxicities in late phase trials has been associated with a 70 percent failure rate in bringing new drugs to the market.   Despite good technologies for investigative studies, initial work is carried out on animals and then the transferrability of the work from a “model” to man has to be assured.  That is the first issue of concern.

Secondly, there is a well considered reluctance on the part of experienced and well prepared physicians to be “early” adopters to newly introduced drugs, with the apprehension that unidentified clinical problems can be expected to be unmasked.  It is, however, easier to consider when a new drug belongs to an established class of medications, and it has removed known adverse effects.  In this case, the adverse effects are known side effects, but not necessarily serious drug reactions that would preclude use.

A third consideration is the cost of drug development, and the cost of development is passed on to the healthcare organization in the purchasing cost. We can rest assured that the Pharmacy and Therapeutics Review Committee will not cease meeting on a regular schedule anytime soon.  Further, how do the drug failures become embedded in the cost of the pharmaceutical budget passed on to the recipient.  Historically, insurance is an actuarial discipline.  But in the lifetime of an individual, they are bound to see a physician for acute or chronic medical attention.  Only the timing cannot be predicted.  As a result, dealing with the valid introduction of new medications is a big concern for both the public and the private insurer.

How does this compute for the physician provider.  The practice of medicine is not quickly adaptive, as the physician’s primary concern is to do no harm.   Genomics testing is not widely available, and it is for the most part not definitive for diagnostic purposes as things stand today.  It may provide assessment of risk, or of survival expectation.  The physician uses a step by step assessment, using the patient and family history, a focused physical exam, laboratory and radiology, proceeding to other more specialized exams.  Much of the laboratory testing is based on the appearance in the circulation of changes in blood chemistry of the nature of electrolytes, circulating cells in the blood and of the blood forming organ, proteins, urea and uric acid.  They are not exquisitely sensitive, but they might be sufficient for their abnormal concentrations appearing at the time the patient presents with a complaint. What tests are ordered is determioned by a need for relevant information to make a medical decision.

The relevant questions are:

1. acuity of symptoms and signs.
2. actions to be taken.
3. tests that are needed to clarify the examination findings.

once a provisional diagnosis is obtained, referrals, additional testing, and medication orders are provided based on the assessment.

Where does genetic testing fit into this? At this point, it will only be used

  1. to confirm a restricted list of diagnoses that have a high association with the condition, and
  2. only with the participation of a medical geneticist, when
  3. profiling the patient and other members of the family is required.

10d0de1 Vitruvian Man by Leonardo da Vinci

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