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Ultrasound-based Screening for Ovarian Cancer

Author: Dror Nir, PhD

Occasionally, I check for news on ovarian cancer screening. I do that for sentimental reasons; I started the HistoScanning project aiming to develop an effective ultrasound-based screening solution for this cancer.

As awareness for ovarian cancer is highest in the USA, I checked for the latest news on the NCI web-site. I found that to-date: “There is no standard or routine screening test for ovarian cancer. Screening for ovarian cancer has not been proven to decrease the death rate from the disease.

Screening for ovarian cancer is under study and there are screening clinical trials taking place in many parts of the country. Information about ongoing clinical trials is available from the NCI Web site.”

I also found that:

Estimated new cases and deaths from ovarian cancer in the United States in 2013:

  • New cases: 22,240
  • Deaths: 14,030

To get an idea on the significance of these numbers, lets compare them to the numbers related to breast cancer:

Estimated new cases and deaths from breast cancer in the United States in 2013:

  • New cases: 232,340 (female); 2,240 (male)
  • Deaths: 39,620 (female); 410 (male)

Death rate of ovarian cancer patients is almost 4 times higher than the rate in breast cancer patients!

Therefore, I decided to raise awareness to the results achieved for ovarian HistoScanning in a double-blind multicenter European study that was published in European Radiology three years ago. The gynecologists who recruited patients to this study used standard ultrasound machines of GE-Medical. I would like as well to disclose that I am one of the authors of this paper:

A new computer-aided diagnostic tool for non-invasive characterisation of malignant ovarian masses: results of a multicentre validation study, Olivier Lucidarme et.al., European Radiology, August 2010, Volume 20, Issue 8, pp 1822-1830

Abstract

Objectives

To prospectively assess an innovative computer-aided diagnostic technology that quantifies characteristic features of backscattered ultrasound and theoretically allows transvaginal sonography (TVS) to discriminate benign from malignant adnexal masses.

Methods

Women (n = 264) scheduled for surgical removal of at least one ovary in five centres were included. Preoperative three-dimensional (3D)-TVS was performed and the voxel data were analysed by the new technology. The findings at 3D-TVS, serum CA125 levels and the TVS-based diagnosis were compared with histology. Cancer was deemed present when invasive or borderline cancerous processes were observed histologically.

Results

Among 375 removed ovaries, 141 cancers (83 adenocarcinomas, 24 borderline, 16 cases of carcinomatosis, nine of metastases and nine others) and 234 non-cancerous ovaries (107 normal, 127 benign tumours) were histologically diagnosed. The new computer-aided technology correctly identified 138/141 malignant lesions and 206/234 non-malignant tissues (98% sensitivity, 88% specificity). There were no false-negative results among the 47 FIGO stage I/II ovarian lesions. Standard TVS and CA125 had sensitivities/specificities of 94%/66% and 89%/75%, respectively. Combining standard TVS and the new technology in parallel significantly improved TVS specificity from 66% to 92% (p < 0.0001).

table 3

table 4

An example of an ovary considered to be normal with TVS.

An example of an ovary considered to be normal with TVS.

The same TVS false-negative ovary with OVHS-detected foci of malignancy. The presence of an adenocarcinoma was confirmed histologically.

The same TVS
false-negative ovary with OVHS-detected foci of malignancy. The presence of an
adenocarcinoma was confirmed histologically.

Conclusions

Computer-aided quantification of backscattered ultrasound is  highly sensitive for the diagnosis of malignant ovarian masses.

 Personal note:

Based on this study a promising offer for ultrasound-based screening method for ovarian cancer was published in:  Int J Gynecol Cancer. 2011 Jan;21(1):35-43. doi: 10.1097/IGC.0b013e3182000528.: Mathematical models to discriminate between benign and malignant adnexal masses: potential diagnostic improvement using ovarian HistoScanning. Vaes EManchanda RNir RNir DBleiberg HAutier PMenon URobert A.

Regrettably, the results of these studies were never transformed into routine clinical products due to financial reasons.

Other research papers related to the management of Prostate cancer were published on this Scientific Web site:

Beta-Blockers help in better survival in ovarian cancer

Ovarian Cancer and fluorescence-guided surgery: A report

Role of Primary Cilia in Ovarian Cancer

Squeezing Ovarian Cancer Cells to Predict Metastatic Potential: Cell Stiffness as Possible Biomarker

BRCA1 a tumour suppressor in breast and ovarian cancer – functions in transcription, ubiquitination and DNA repair

Warning signs may lead to better early detection of ovarian cancer

 

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Minimally invasive image-guided therapy for inoperable hepatocellular carcinoma

Curator & Reporter: Dror Nir, PhD

Large organs like the liver are good candidates for focused treatment. The following paper:

Minimally invasive image-guided therapy for inoperable hepatocellular carcinoma: What is the evidence today?

By Beatrijs A. Seinstra1, et. al. published mid-2010, gives a review of the state-of-the-art of the then available methods for local lesions’ ablation. As far as ablation techniques availability, I have found this review very much relevant to today’s technological reality. It is worthwhile noting that in the last couple of years, new imaging-based navigation and guidance applications were introduced into the market holding a promise to improve the accuracy of administrating such treatment. These are subject to clinical validation in large clinical studies.  From the above mentioned publication I have chosen to highlight the parts discussing the importance of imaging-based guidance to the effective application of localized ablation-type therapies.

The clinical need:

Hepatocellular carcinoma (HCC) is a primary malignant tumor of the liver that accounts for an important health problem worldwide. Primary liver cancer is the sixth most common cancer worldwide with an incidence of 626,000 patients a year, and the third most common cause of cancer-related death [1]. Only 10–15% of HCC patients are suitable candidates for hepatic resection and liver transplantation due to the advanced stage of the disease at time of diagnosis and shortage of donors.

Immerging solution:

In order to provide therapeutic options for patients with inoperable HCC, several minimally invasive image-guided therapies for locoregional treatment have been developed. HCC has a tendency to remain confined to the liver until the disease has advanced, making these treatments particularly attractive.

Minimally invasive image-guided therapies can be divided into the group of the tumor ablative techniques or the group of image-guided catheter-based techniques. Tumor ablative techniques are either based on thermal tumor destruction, as in radiofrequency ablation (RFA), cryoablation, microwave ablation, laser ablation and high-intensity focused ultrasound (HIFU), or chemical tumor destruction, as in percutaneous ethanol injection (PEI). These techniques are mostly used for early stage disease. Image-guided catheter-based techniques rely on intra-arterial delivery of embolic, chemoembolic, or radioembolic agents [22]. These techniques enable treatment of large lesions or whole liver treatment, and are as such used for intermediate stage HCC (Figure 1).

Minimally invasive image-guided ablation techniques and intra-arterial interventions may prolong survival, spare more functioning liver tissue in comparison to surgical resection (which can be very important in cirrhotic patients), allow retreatment if necessary, and may be an effective bridge to transplantation [2327].

During the last 2 decades, minimally invasive image-guided therapies have revolutionized the management of inoperable HCC.

The value of image guidance

Accurate imaging is of great importance during minimally invasive loco-regional therapies to efficiently guide and monitor the treatment. It enables proper placement of instruments, like the probe in case of ablation or the catheter in case of intra-arterial therapy, and accurate monitoring of the progression of the necrotic zone during ablation.

can all be employed. In current clinical practice, placement of the catheter in intra-arterial procedures is usually performed under fluoroscopic guidance, while ablation may be guided by ultrasound, CT or MRI.

  • Ultrasound guidance allows probe insertion from every angle, offers real time visualization and correction for motion artifacts when targeting the tumor, and is low cost. However, the gas created during ablation (or ice in the case of cryoablation) hampers penetration of the ultrasound beams in tissue, causing acoustic shadowing and obscuring image details like the delineation between tumor borders and ablation zone.
  • CT is also frequently used to guide minimally invasive ablation therapy, and is a reliable modality to confirm treatment results. In comparison to US, it provides increased lesion discrimination, a more reliable depiction of ablated/non-ablated interfaces, and a better correlation to pathologic size [28]. However, due to its hypervascularity, small HCCs can only be clearly visualized in the arterial phase for a short period of time. Another disadvantage of CT is the exposure of the patient and physician to ionizing radiation.
  • Combining US imaging for probe placement and CT for ablation monitoring reduces this exposure. At the moment, hybrid systems are being developed, enabling combination of imaging techniques, like ultrasound and CT imaging, thereby improving the registration accuracy during treatment [29]. The interest in MRI-guided ablation is growing, as it produces a high-quality image allowing high-sensitivity tumor detection and accurate identification of the target region with multiplanar imaging.
  • MRI also enables real-time monitoring of the temperature evolution during treatment [3035]. However, MRI is an expensive technique, and MRI-guided ablation is still limited in clinical practice. Currently, the most widely used ablation technique for percutaneous treatment of focal hepatic malignancies is radiofrequency ablation (RFA), which has been shown to be safe and effective for the treatment of early stage HCC [4850]. During RFA, a small electrode is placed within the tumor, and a high-frequency alternating electric current (approximately 400 MHz) is generated, causing ionic agitation within the tissue. ….. Most frequently ultrasound is used for image guidance (Figs. 23), but there are reports of groups who use CT, MRI, or fluoroscopic imaging.
Ultrasound guided RFA. a: HCC lesion in a non-surgical patient pre-treatment (pointed out by arrow). b: Just after start treatment, electrode placed centrally in the tumor. c: Gas formation during ablation causes acoustic shadowing

Ultrasound guided RFA. a: HCC lesion in a non-surgical patient pre-treatment (pointed out by arrow). b: Just after start treatment, electrode placed centrally in the tumor. c: Gas formation during ablation causes acoustic shadowing

Contrast-enhanced CT pre- and post-RFA. Same patient as in Fig. 2. a: Hypervascular lesion (biopsy proven HCC) in right liver lobe (pointed out by arrow) before treatment. b: Ablated lesion directly post ablation, with reactive hyperemia around the RFA lesion

Contrast-enhanced CT pre- and post-RFA. Same patient as in Fig. 2. a: Hypervascular lesion (biopsy proven HCC) in right liver lobe (pointed out by arrow) before treatment. b: Ablated lesion directly post ablation, with reactive hyperemia around the RFA lesion

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Other research papers related to the management of Prostate cancer were published on this Scientific Web site:

HBV and HCV-associated Liver Cancer: Important Insights from the Genome

Issues in Personalized Medicine in Cancer: Intratumor Heterogeneity and Branched Evolution Revealed by Multiregion Sequencing

Harnessing Personalized Medicine for Cancer Management, Prospects of Prevention and Cure: Opinions of Cancer Scientific Leaders @ http://pharmaceuticalintelligence.com

Whole-body imaging as cancer screening tool; answering an unmet clinical need?

Personalized Medicine: Cancer Cell Biology and Minimally Invasive Surgery (MIS)

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Mitochondrial Dysfunction and Cardiac Disorders

Curator: Larry H Bernstein, MD, FACP

This article is the THIRD in a four-article Series covering the topic of the Roles of the Mitochondria in Cardiovascular Diseases. They include the following;

  • Mitochondria and Cardiovascular Disease: A Tribute to Richard Bing, Larry H Bernstein, MD, FACP

http://pharmaceuticalintelligence.com/2013/04/14/chapter-5-mitochondria-and-cardiovascular-disease/

  • Mitochondrial Metabolism and Cardiac Function, Larry H Bernstein, MD, FACP

http://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-metabolism-and-cardiac-function/

  • Mitochondrial Dysfunction and Cardiac Disorders, Larry H Bernstein, MD, FACP

http://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-dysfunction-and-cardiac-disorders/

http://pharmaceuticalintelligence.com/2013/04/14/reversal-of-cardiac-mitochondrial-dysfunction/

Mitochondrial Metabolism in Impaired Cardiac Function

Mitochondrial Dysfunction and the Heart

Chronically elevated plasma free fatty acid levels in heart failure are associated with
  • decreased metabolic efficiency and cellular insulin resistance.
The mitochondrial theory of aging (MTA) and the free-radical theory of aging (FRTA) are closely related.
They were in fact proposed by the same researcher about 20 years apart. MTA adds
  • the mitochondria and its production of free radicals
  • into the concept that free-radicals damage DNA over time.
Tissue hypoxia, resulting from low cardiac output with or independent of endothelial impairment,
This dysfunctional state causes loss of mitochondrial mass. Therapies aimed at protecting mitochondrial function
  • have shown promise in patients and animal models with heart failure that will be the subject of Chapter III.

Myocardial function in hypertension

Genetic variation in vitamin D-dependent signaling
  • is associated with congestive heart failure in human subjects with hypertension.
Functional polymorphisms were selected from five candidate genes:
  1. CYP27B1,
  2. CYP24A1,
  3. VDR,
  4. REN and
  5. ACE.
Using the Marshfield Clinic Personalized Medicine Research Project,
  • 205 subjects with hypertension and congestive heart failure,
  • 206 subjects with hypertension alone and
  • 206 controls (frequency matched by age and gender) were genotyped.
In the context of hypertension, a SNP in CYP27B1 was associated with congestive heart failure
(odds ratio: 2.14 for subjects homozygous for the C allele; 95% CI: 1.05–4.39).
Genetic variation in vitamin D biosynthesis is associated with increased risk of heart failure.
RA Wilke, RU Simpson, BN Mukesh, SV Bhupathi, et al. Genetic variation in CYP27B1 is associated

Heart Failure and Coronary Circulation

There is a decrease in resting and peak stress myocardial function in chronic heart failure patients,
  • with recovery of skeletal muscle phosphocreatine following exercise induced by perhexiline treatment.
This suggested that mitochondrial deficiencies, caused by excessive free fatty acids (FFAs)
  • underlie a common cardiac and skeletal muscle myopathy in heart failure patients.
Tissue hypoxia in chronic heart failure from inadequate circulation in heart failure
  • increases the oxidative stress in lean body mass and in the heart itself.
The heterodimeric transcription factor hypoxia-inducible factor (HIF)-1
  • induces changes in the transcription of genes that encode proteins involved in the adaptation to hypoxia.
HIF-1 activity depends on levels of the HIF-1a subunit, which has a short half-life.
HIF-1a increases in rats with experimentally induced myocardial infarction together with elevated levels of
  • GLUT1 and haemoxygenase-1 in the peri-infarct region of the heart
The cardiac metabolic response to hypoxia is considered to be
  • a return to a pattern of fetal metabolism, in which
  • carbohydrates predominate as substrates for energy metabolism.
The reliance on carbohydrate energy source is thought to be a result of  the downregulation of PPARa with a decreased activity of
The sarcolemmal fatty acid transporter protein (FATP) levels are also decreased with palmitate oxidation,
  • transitioning away from fatty acid metabolism proportional to the degree of cardiac impairment.
The hypoxic changes of heart failure drives a switch toward
  1. glycolysis and glucose oxidation
  2. restriction of myocardial fatty acid uptake.
Nevertheless, late in the progression of heart failure, substrate metabolism is insufficient to support cardiac function, because
  • the hypoxic failing heart is no longer able to oxidize fats and may also be insulin resistant.
The author surmises that mitochondrial dysfunction caused by tissue hypoxia might be mediated by the
  • proapoptotic protein BCL2/adenovirus E1B 19kDa interacting protein (Bnip)3.
It  is strongly upregulated in response to hypoxia. In the isolated, perfused rat heart, Bnip3 expression was
  • induced after 1h of hypoxia, with Bnip3 integrating into the mitochondria of hypoxic ventricular myocytes.
This resulted in mitochondrial defects associated with
  1. opening of the permeability transition pore, leading to
  2. loss of inner membrane integrity and
  3. loss of mitochondrial mass.

Mitochondrial Dysfunction caused by Bnip3 Precedes Cell Death.

Experimentally induced myocardial ischemia had evidence of contractile dysfunction but preserved viability. A progressive
  • decline in circulating levels of endothelial progenitor cells was documented 3 months following instrumentation (P<0.001).
Quantitative polymerase chain reaction analysis revealed that
  • chronic myocardial ischemia produced a biphasic response in both
    • hypoxic-inducible factor 1 and
    • stromal-derived factor 1 mRNA expression.
While initially unregulated, a gradual decline was observed over time (from day 45 to 90), in
  • hypoxic-inducible factor 1 and
  • stromal-derived factor 1 mRNA expression .
On serial assessment, endothelial progenitor cell migration was progressively impaired in response to chemo-attractant gradients of:
  1. vascular endothelial growth factor (10-200 ng/mL)
  2. and stromal cell-derived factor-1 (10-100 ng/mL) .
Decreased circulating levels and migratory dysfunction of bone marrow derived endothelial progenitor cells
  • were documented in a reproducible clinically relevant model of myocardial ischemia.

Nitric Oxide (NO) in Myocardial Ischemia and Infarct

Nitric oxide (NO) is a free radical with an unpaired electron; it is an important physiologic messenger,
  • produced by nitric oxide synthases, which catalyze the reaction l-arginine to citrulline and NO.
The constitutive isoforms exists in neuronal and endothelial cells and is calcium dependent. Calcium binds to calmodulin and
  1. the calcium calmodulin complex activates the constitutive NO synthase that releases NO,
  2. relaxing smooth muscle cells through activation of guanylate cyclase and the production cGMP.
Therefore, the NO produced has a negative inotropic effect on the heart and is instrumental in the autoregulation of the coronary circulation.
The inducible form of NO synthase (iNOS), mostly produced in macrophages, is activated by cytokines and endotoxin. It eliminates intracellular pathogens,
damaging cells by inhibiting
  1. ATP production
  2. oxidative phosphorylation
  3. DNA synthesis.
In infection, lipopolysaccharide released from bacterial walls, stimulates production of iNOS. The large amount of NO produced
  • causes extensive vasodilation and hypotension.
We sought to assess whether oxidation products of
  • nitric oxide (NO), nitrite (NO2−) and nitrate (NO3−), referred to as NOx,
  • are released by the heart of patients after acute myocardial infarction (AMI) and
  • whether NOx can be determined in peripheral blood of these patients.
Previously we reported that in experimental myocardial infarction (rabbits) NOx is released mainly by inflammatory cells
  • (macrophages) in the myocardium 3 days after onset of  ischemia.
NOx is formed in heart muscle from NO; It originates through the activity of the inducible form of nitric oxide synthase (iNOS).
Eight patients with acute anterior MI and an equal number of controls were studied. Coronary venous blood was obtained by
coronary sinus catheterization; NOx concentrations in coronary sinus, in arterial and peripheral venous plasma were measured.
Left ventricular end-diastolic pressure was determined. Measurements were carried out 24, 48 and 72 h after onset of symptoms.
The type and location of coronary arterial lesions were determined by coronary angiography. Plasma NO3− was reduced to NO2−
by nitrate reductase before determination of NO2− concentration by chemiluminescence.
The results provided evidence that in patients with acute anterior MI, the myocardial production of nitrite and nitrate (NOx) was increased,
  • as well as the coronary arterial–venous difference.
Increased NOx production by the infarcted heart accounted for the increase of NOx concentration in arterial and the peripheral venous plasma.
The peak elevation of NOx occurred on days 2 and 3 after onset of the symptoms, suggesting that NOx production was at least in part the result of
  • production of NO by inflammatory cells (macrophages) in the heart.
The appearance of oxidative products of NO (NO2− and NO3−) in peripheral blood of patients with acute MI is
  • the result of their increased release from infarcted heart during the inflammatory phase of myocardial ischemia.
Further studies are needed to define the clinical value of these observations.
K Akiyama,  A Kimura, H Suzuki, Y Takeyama, …. R Bing.  Production of oxidative products of nitric oxide in infarcted human heart.  J Am Coll Cardiol. 1998;32(2):373-379.   http://dx.doi.org/10.1016/S0735-1097(98)00270-8
OPA1 Mutation and Late-Onset Cardiomyopathy
No cardiac disorders have been described in patients with OPA1 or similar mutations
  • involving the fission/fusion genes as seen in inherited maladies like Charcot–Marie–Tooth disease.
Our results indicate that, at least for OPA1, cardiac abnormalities are not completely
  • manifest until the development of blindness.

The OPA1-mutant mice survived more than 1 year and appeared healthy.

In patients with these diseases, reduced cardiac function may go undetected
secondary to reduced physical activity secondary to loss of vision.
It would be expected that patients with such mutations would have impaired cardiac reserve with
  • reduced ability to respond to high-stress disease states such as myocardial infarction and sepsis.
The OPA1-mutant mice have reduced cardiac reserve, as shown by
  • the lack of response to isoproterenol or to ischemia/reperfusion injury,
This suggests that patients with OPA1 and related inherited mitochondrial diseases
  • should be screened for abnormalities of cardiac function.
Le Chen; T Liu; A Tran; Xiyuan Lu; …AA. Knowlton. OPA1 Mutation and Late-Onset Cardiomyopathy:
Mitochondrial Dysfunction and mtDNA Instability.  http://jaha.ahajournals.org/content/1/5/e003012.full

Oxidative Stress and Mitochondria in the Failing Heart

The major problem in tissue hypoxia in the failing heart is oxidative stress. Reactive oxygen species (ROS), including
  • superoxide,
  • hydroxyl radicals and
  • hydrogen peroxide,
are generated by a number of cellular processes, including
  • mitochondrial electron transport,
  • NADPH oxidase and
  • xanthine dehydrogenase/xanthine oxidase.
The low availability of oxygen, the final receptor of mitochondrial electron transport (ET), results in
  • electron accumulation in the ET chain as the complexes become highly reduced.
A number of experimental and clinical studies have suggested that ROS generation is
  • enhanced in heart failure because of electron leak, and complexes I and II
  • are implicated as the primary sites of this loss.
Prolonged oxidative stress in cardiac failure results in damage to mitochondrial DNA.
The continued ROS generation and consequent cellular injury leads to functional decline.
Thus, mitochondria are both the sources and targets of a cycle of ROS-mediated injury in the failing heart.
Mice with a cardiac/skeletal muscle specific deficiency in the scavenger enzyme superoxide dismutase
  • developed progressive congestive heart failure
  • with defects in mitochondrial respiration.
Oxidative stress in these mice also caused specific morphological changes in cardiac mitochondria
  • characterized by decreased ATP levels,
  • impaired contractility,
  • dramatically restricted exercise capacity and
  • decreased survival.
This was in part corrected by treatment with the antioxidant superoxide dismutase mimetic, namely
  • manganese5,10,15,20-tetrakis-(4-benzoic acid)-porphyrin.
EUK-8, a superoxide dismutase and catalase mimetic improved survival and contractile parameters in a mutant mouse model
  • of pressure overload-induced oxidative stress and heart failure and in wild-type mice subjected to pressure overload.
In addition, mitochondria show
  • functional impairment and
  • morphological disorganization
in the left ventricle of Hypertrophic Cardiomyopathy (HCM)  patients without baseline systolic dysfunction.
These mitochondrial changes were associated with impaired myocardial contractile and relaxation reserves.
A strategy to protect the heart against oxidative stress could lie with
  • the modulation of mitochondrial electron transport itself.
Mild mitochondrial uncoupling may offer a potential cardioprotective effect by decreasing ROS production
  • preventing electron accumulation at complex III and
  • the Fe–S centres of complex I, and may therefore

mtDNA, Autophagy, and Heart Failure

Mitochondria are evolutionary endosymbionts derived from bacteria and contain DNA similar to bacterial DNA.
Mitochondria damaged by external haemodynamic stress are degraded by the autophagy/lysosome system in cardiomyocytes.
Mitochondrial DNA (mtDNA) that escapes from autophagy cell-autonomously leads to Toll-like receptor (TLR) 9-mediated
  • inflammatory responses in cardiomyocytes and
  • is capable of inducing myocarditis and dilated cardiomyopathy.
Cardiac-specific deletion of lysosomal deoxyribonuclease (DNase) II showed no cardiac phenotypes under baseline conditions,
but increased mortality and caused severe myocarditis and dilated cardiomyopathy 10 days after treatment with pressure overload.
Early in the pathogenesis, DNase II-deficient hearts showed
  • infiltration of inflammatory cells
  • increased messenger RNA expression of inflammatory cytokines
  • accumulation of mitochondrial DNA deposits in autolysosomes in the myocardium.
Administration of inhibitory oligodeoxynucleotides against TLR9, which is known to be activated by bacterial DNA6, or ablation of Tlr9
  • attenuated the development of cardiomyopathy in DNase II-deficient mice.
Furthermore, Tlr9 ablation
  • improved pressure overload-induced cardiac dysfunction and
  • inflammation even in mice with wild-type Dnase2a alleles.
These data provide new perspectives on the mechanism of genesis of chronic inflammation in failing hearts.
T Oka, S Hikoso, O Yamaguchi, M Taneike, T Takeda, T Tamai, et al.  Mitochondrial DNA that escapes from autophagy causes inflammation and heart failure.

Mitochondrial Dysfunction Increases Expression of Endothelin-1 and Induces Apoptosis

We developed an in vitro model of mitochondrial dysfunction using rotenone, a mitochondrial respiratory chain complex I inhibitor, and studied
  • preproendothelin-1 gene expression and apoptosis.
Rotenone greatly increased the gene expression of preproendothelin-1 in cardiomyocytes.
This result suggests that the gene expression of preproendothelin-1 is induced by the mitochondrial dysfunction.
Furthermore, treatment of cardiomyocytes with rotenone induced an elevation of caspase-3 activity, and caused a marked
  • increase in DNA laddering, an indication of apoptosis.
In conclusion, it is suggested that mitochondrial impairment in primary cultured cardiomyocytes induced by rotenone in vitro,
  • mimics some of the pathophysiological features of heart failure in vivo, and that ET-1 may have a role in myocardial dysfunction
    • with impairment of mitochondria in the failing heart.

Summary

This review focused on the evidence accumulated to the effect that mitochondria are key players in
  • the progression of congestive heart failure (CHF).
Mitochondria are the primary source of energy in the form of adenosine triphosphate that fuels the contractile apparatus,
  • essential for the mechanical activity and the Starling Effect of the heart.
We evaluate changes in mitochondrial morphology and alterations in the main components of mitochondrial energetics, such as
  • substrate utilization and
  • oxidative phosphorylation,
in the context of their contribution to the chronic energy deficit and mechanical dysfunction in HF.
REFERENCES
Zachman AL, Page JM, Prabhakar G, Guelcher SA, and Sung HJ, “Elucidation of adhesion-dependent spontaneous apoptosis in macrophages using phase separated PEG/polyurethane films.”
Acta Biomater. 2012 Nov 2.    http://dx.doi.org/pii: S1742-7061(12)00530-2. 10.1016/j.actbio.2012.10.038.    http://www.ncbi.nlm.nih.gov/pubmed/23128157

Other Related articles published on this Open Access Scientific Journal, include the following:

Perspectives on Nitric Oxide in Disease Mechanisms: The Nitric Oxide Discovery, Function, and Targeted Therapy  Opportunities, 2013, Aviral Vatsa, PhD and Larry H Bernstein, MD, FACP, Editors, Amazon e-Books (forthcoming). http://pharmaceuticalintelligence.com/biomed-e-books/perspectives-on-nitric-oxide-in-disease-mechanisms-v2/

Mitochondria: More than just the “powerhouse of the cell” Ritu Saxena, Ph.D. Consultants: Aviva Lev-Ari, PhD, RN and Pnina G. Abir-Am, PhD 7/9/2012

http://pharmaceuticalintelligence.com/2012/07/09/mitochondria-more-than-just-the-powerhouse-of-the-cell/

Mitochondrial dynamics and cardiovascular diseases, Ritu Saxena, PhD 11/14/2012
http://pharmaceuticalintelligence.com/2012/11/14/mitochondrial-dynamics-and-cardiovascular-diseases/

Mitochondrial Damage and Repair under Oxidative Stress, Larry H Bernstein, MD, FACP 10/28/2012
http://pharmaceuticalintelligence.com/2012/10/28/mitochondrial-damage-and-repair-under-oxidative-stress/

Mitochondria: Origin from oxygen free environment, role in aerobic glycolysis, metabolic adaptation, Larry H Bernstein, MD, FACP 9/26/2012

http://pharmaceuticalintelligence.com/2012/09/26/mitochondria-origin-from-oxygen-free-environment-role-in-aerobic-glycolysis-metabolic-adaptation/

Ca2+ signaling: transcriptional control, Larry H Bernstein, MD, FACP 3/6/2-13
http://pharmaceuticalintelligence.com/2013/03/06/ca2-signaling-transcriptional-control/

MIT Scientists on Proteomics: All the Proteins in the Mitochondrial Matrix identified, Aviva Lev-Ari, PhD, RN 2/3/2013
http://pharmaceuticalintelligence.com/2013/02/03/mit-scientists-on-proteomics-all-the-proteins-in-the-mitochondrial-matrix-identified/

Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function, Larry H Bernstein, MD, FACP 9/16/2012
http://pharmaceuticalintelligence.com/2012/09/16/nitric-oxide-has-a-ubiquitous-role-in-the-regulation-of-glycolysis-with-a-concomitant-influence-on-mitochondrial-function/

Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis, Larry H Bernstein, MD, FACP 2/14/2013
http://pharmaceuticalintelligence.com/2013/02/14/ubiquinin-proteosome-pathway-autophagy-the-mitochondrion-proteolysis-and-cell-apoptosis-reconsidered/

Low Bioavailability of Nitric Oxide due to Misbalance in Cell Free Hemoglobin in Sickle Cell Disease – A Computational Model   Anamika Sarkar, PhD 11/9/2012
http://pharmaceuticalintelligence.com/2012/11/09/low-bioavailability-of-nitric-oxide-due-to-misbalance-in-cell-free-hemoglobin-in-sickle-cell-disease-a-computational-model/

The rationale and use of inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure, , Larry H Bernstein, MD, FACP 8/20/2012

http://pharmaceuticalintelligence.com/2012/08/20/the-rationale-and-use-of-inhaled-no-in-pulmonary-artery-hypertension-and-right-sided-heart-failure/

Clinical Trials Results for Endothelin System: Pathophysiological role in Chronic Heart Failure, Acute Coronary Syndromes and MI – Marker of Disease Severity or Genetic Determination? Aviva Lev-Ari, PhD, RN 10/19/2012

http://pharmaceuticalintelligence.com/2012/10/19/clinical-trials-results-for-endothelin-system-pathophysiological-role-in-chronic-heart-failure-acute-coronary-syndromes-and-mi-marker-of-disease-severity-or-genetic-determination/

Endothelin Receptors in Cardiovascular Diseases: The Role of eNOS Stimulation, Aviva Lev-Ari, PhD, RN 10/4/2012

http://pharmaceuticalintelligence.com/2012/10/04/endothelin-receptors-in-cardiovascular-diseases-the-role-of-enos-stimulation/

Inhibition of ET-1, ETA and ETA-ETB, Induction of NO production, stimulation of eNOS and Treatment Regime with PPAR-gamma agonists (TZD): cEPCs Endogenous Augmentation for Cardiovascular Risk Reduction – A Bibliography, Aviva Lev-Ari, PhD, RN 10/4/2012

http://pharmaceuticalintelligence.com/2012/10/04/inhibition-of-et-1-eta-and-eta-etb-induction-of-no-production-and-stimulation-of-enos-and-treatment-regime-with-ppar-gamma-agonists-tzd-cepcs-endogenous-augmentation-for-cardiovascular-risk-reduc/

Genomics & Genetics of Cardiovascular Disease Diagnoses: A Literature Survey of AHA’s Circulation Cardiovascular Genetics, 3/2010 – 3/2013, L H Bernstein, MD, FACP and Aviva Lev-Ari,PhD, RN  3/7/2013

http://pharmaceuticalintelligence.com/2013/03/07/genomics-genetics-of-cardiovascular-disease-diagnoses-a-literature-survey-of-ahas-circulation-cardiovascular-genetics-32010-32013/

Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production, Aviva Lev-Ari, PhD, RN 7/19/2012

http://pharmaceuticalintelligence.com/2012/07/19/cardiovascular-disease-cvd-and-the-role-of-agent-alternatives-in-endothelial-nitric-oxide-synthase-enos-activation-and-nitric-oxide-production/

Cardiovascular Risk Inflammatory Marker: Risk Assessment for Coronary Heart Disease and Ischemic Stroke – Atherosclerosis.

Aviva Lev-Ari, PhD, RN 10/30/2012

http://pharmaceuticalintelligence.com/2012/10/30/cardiovascular-risk-inflammatory-marker-risk-assessment-for-coronary-heart-disease-and-ischemic-stroke-atherosclerosis/

Cholesteryl Ester Transfer Protein (CETP) Inhibitor: Potential of Anacetrapib to treat Atherosclerosis and CAD, Aviva Lev-Ari, PhD, RN 4/7/2013

http://pharmaceuticalintelligence.com/2013/04/07/cholesteryl-ester-transfer-protein-cetp-inhibitor-potential-of-anacetrapib-to-treat-atherosclerosis-and-cad/

Hypertriglyceridemia concurrent Hyperlipidemia: Vertical Density Gradient Ultracentrifugation a Better Test to Prevent Undertreatment of High-Risk Cardiac Patients, Aviva Lev-Ari, PhD, RN  4/4/2013

http://pharmaceuticalintelligence.com/2013/04/04/hypertriglyceridemia-concurrent-hyperlipidemia-vertical-density-gradient-ultracentrifugation-a-better-test-to-prevent-undertreatment-of-high-risk-cardiac-patients/

Fight against Atherosclerotic Cardiovascular Disease: A Biologics not a Small Molecule – Recombinant Human lecithin-cholesterol acyltransferase (rhLCAT) attracted AstraZeneca to acquire AlphaCore, Aviva Lev-Ari, PhD, RN 4/3/2013

http://pharmaceuticalintelligence.com/2013/04/03/fight-against-atherosclerotic-cardiovascular-disease-a-biologics-not-a-small-molecule-recombinant-human-lecithin-cholesterol-acyltransferase-rhlcat-attracted-astrazeneca-to-acquire-alphacore/

High-Density Lipoprotein (HDL): An Independent Predictor of Endothelial Function & Atherosclerosis, A Modulator, An Agonist, A Biomarker for Cardiovascular Risk, Aviva Lev-Ari, PhD, RN 3/31/2013

http://pharmaceuticalintelligence.com/2013/03/31/high-density-lipoprotein-hdl-an-independent-predictor-of-endothelial-function-artherosclerosis-a-modulator-an-agonist-a-biomarker-for-cardiovascular-risk/

Peroxisome proliferator-activated receptor (PPAR-gamma) Receptors Activation: PPARγ transrepression for Angiogenesis in Cardiovascular Disease and PPARγ transactivation for Treatment of Diabetes, Aviva Lev-Ari, PhD, RN 11/13/2012

http://pharmaceuticalintelligence.com/2012/11/13/peroxisome-proliferator-activated-receptor-ppar-gamma-receptors-activation-pparγ-transrepression-for-angiogenesis-in-cardiovascular-disease-and-pparγ-transactivation-for-treatment-of-dia/

Sulfur-Deficiciency and Hyperhomocysteinemia, L H Bernstein, MD, FACP

http://pharmaceuticalintelligence.com/2013/04/04/sulfur-deficiency-and-hyperhomocusteinemia/

Mitochondrial metabolism and cardiac function, L H Bernstein, MD, FACP
Cardiotoxicity and Cardiomyopathy Related to Drugs Adverse Effects, L H Bernstein, MD, FACP
Lp(a) Gene Variant Association, L H Bernstein, MD, FACP

Predicting Drug Toxicity for Acute Cardiac Events, L H Bernstein, MD, FACP

Amyloidosis with Cardiomyopathy, L H Bernstein, MD, FACP

Mitochondria and Cardiovascular Disease: A Tribute to Richard Bing, Larry H Bernstein, MD, FACP

http://pharmaceuticalintelligence.com/2013/04/14/chapter-5-mitochondria-and-cardiovascular-disease/

Mitochondrial Metabolism and Cardiac Function, Larry H Bernstein, MD, FACP

http://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-metabolism-and-cardiac-function/

Mitochondrial Dysfunction and Cardiac Disorders, Larry H Bernstein, MD, FACP

http://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-dysfunction-and-cardiac-disorders/

Reversal of Cardiac mitochondrial dysfunction, Larry H Bernstein, MD, FACP

http://pharmaceuticalintelligence.com/2013/04/14/reversal-of-cardiac-mitochondrial-dysfunction/

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Mitochondria and Cardiovascular Disease

Curator: Larry H Bernstein, MD, FACP

This article is the FIRST in a four-article Series covering the topic of the Roles of the Mitochondria in Cardiovascular Diseases. They include the following;

  • Mitochondria and Cardiovascular Disease: A Tribute to Richard Bing, Larry H Bernstein, MD, FACP

http://pharmaceuticalintelligence.com/2013/04/14/chapter-5-mitochondria-and-cardiovascular-disease/

  • Mitochondrial Metabolism and Cardiac Function, Larry H Bernstein, MD, FACP

http://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-metabolism-and-cardiac-function/

  • Mitochondrial Dysfunction and Cardiac Disorders, Larry H Bernstein, MD, FACP

http://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-dysfunction-and-cardiac-disorders/

http://pharmaceuticalintelligence.com/2013/04/14/reversal-of-cardiac-mitochondrial-dysfunction/

Richard Bing was one of the founders of IACS. He was the first president of the International Study Group for Research in Cardiac Metabolism 1969 – 1973.
Then he was elected as Lifetime President of that organization which became the International Society for Heart Research.  In 2001 he was recipient of the first of the Academy’s most prestigious Medals of Merit. On October 12, Richard J. Bing, an APS member since 1942, celebrated his 100th birthday and his scientific colleagues acknowledged the milestone in editorials published in several journals.
A movie documentary was chosen to be aired
Richard Bing published over 500 peer reviewed articles on topics ranging from cardiac metabolism in congestive heart failure to echocardiographic studies of the posterior left ventricular wall in experimental myocardial infarction. He was the first to define the physiology of congenital heart disease (Helen Taussig
and Richard Bing, Blue Baby operation, patent foramen ovale, ductus arteriosus, veno-arterial mixing) by threading a catheter into the heart.
In addition to his scientific contributions, Richard Bing wrote more than 200 compositions of music including
the Missa (Chanted Mass) which was performed in the Cathedral of Saint Stefans in Vienna, Austria on October 30, 1993.
  • Bing also wrote a number of non-medical books including a novel and several short stories.
1. Cheng, T. O. Happy 100th Birthday to Dr. Richard John Bing, Int J Cardiol 137(2): 87-101, 2009.
2. Taegtmeyer, H. Richard Bing at 100: Reflections on a Lion in Winter. J Mol Cell Cardiol 47(5): 562-4, 2009.
3.  Taegtmeyer, H. A Lion at Rest: Richard Bing dies at 101. Circ Res. 2011;108:9-11.
Print ISSN: 0009-7330. Online ISSN: 1524-4571  http://circres.ahajournals.org/content/108/1/9
This discussion is another in a series discussing
  • mitochondrial metabolism,
  • energetics
  • regulatory function, and dysfunction,
  • the process leading to apoptosis
  • a larger effect on disease.
Recall with regard to mitochondrial oxidation-reduction reactions and repair that there are
  • organ specific differences in the rates of organelle mutation errors and in the rates of repair.
Consider also the effect of
  • iron-binding in the function of the cell,
  • Ca2+ binding in the creation of the mechanical work.
The protein content of the cell is determined by
  • the balance between protein synthesis and protein degradation.
At constant intracellular protein concentration, i.e. at steady state,
  • rates of protein synthesis and degradation are equal.
The Ubiquitin-Mediated Proteolytic Pathway is involved in
  • complete destruction of its protein substrates, but
  • in limited proteolysis and posttranslational processing
  • it generates biologically active peptides or fragments.
It is essential for the system that ubiquitin recycles. This function is carried out by
    • ubiquitin C-terminal hydrolases (isopeptidases).
Although turnover of protein results in energy dissipation, regulation
  • at the level of protein degradation effectively controls protein levels.
In the mechanisms controlling macroautophagy,
  • protein phosphorylation plays an important role.
Activation of a signal transduction pathway accompanies inhibition of macroautophagy.
Components of this pathway include
  1. a heterotrimeric Gi3-protein,
  2. phosphatidylinositol 3-kinase
  3. p70S6 kinase.
Autophagy, or mitophagy in mitochondria, is a process
  • central to consider in cardiac dysfunction –
either acute coronary syndrome, or chronic congestive heart failure.

 References

Mitochondrial dynamics and cardiovascular diseases    Ritu Saxena
http://pharmaceuticalintelligence.com/2012/11/14/mitochondrial-dynamics-and-cardiovascular-diseases/

Mitochondrial Damage and Repair under Oxidative Stress   larryhbern
http://pharmaceuticalintelligence.com/2012/10/28/mitochondrial-damage-and-repair-under-oxidative-stress/

Mitochondria: Origin from oxygen free environment, role in aerobic glycolysis, metabolic adaptation   larryhbern
http://pharmaceuticalintelligence.com/2012/09/26/mitochondria-origin-from-oxygen-free-environment-role-in-aerobic-glycolysis-metabolic-adaptation/

Ca2+ signaling: transcriptional control     larryhbern
http://pharmaceuticalintelligence.com/2013/03/06/ca2-signaling-transcriptional-control/

MIT Scientists on Proteomics: All the Proteins in the Mitochondrial Matrix identified  Aviva Lev-Ari
http://pharmaceuticalintelligence.com/2013/02/03/mit-scientists-on-proteomics-all-the-proteins-in-the-mitochondrial-matrix-identified/

Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function    larryhbern
http://pharmaceuticalintelligence.com/2012/09/16/nitric-oxide-has-a-ubiquitous-role-in-the-regulation-of-glycolysis-with-a-concomitant-influence-on-mitochondrial-function/

Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis  larryhbern
http://pharmaceuticalintelligence.com/2013/02/14/ubiquinin-proteosome-pathway-autophagy-the-mitochondrion-proteolysis-and-cell-apoptosis-reconsidered/

Low Bioavailability of Nitric Oxide due to Misbalance in Cell Free Hemoglobin in Sickle Cell Disease – A Computational Model   Anamika Sarkar
http://pharmaceuticalintelligence.com/2012/11/09/low-bioavailability-of-nitric-oxide-due-to-misbalance-in-cell-free-hemoglobin-in-sickle-cell-disease-a-computational-model/

The rationale and use of inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure    larryhbern
http://pharmaceuticalintelligence.com/2012/08/20/the-rationale-and-use-of-inhaled-no-in-pulmonary-artery-hypertension-and-right-sided-heart-failure/

Mitochondria and Cardiovascular Disease: A Tribute to Richard Bing, Larry H Bernstein, MD, FACP

http://pharmaceuticalintelligence.com/2013/04/14/chapter-5-mitochondria-and-cardiovascular-disease/

Mitochondrial Metabolism and Cardiac Function, Larry H Bernstein, MD, FACP

http://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-metabolism-and-cardiac-function/

Mitochondrial Dysfunction and Cardiac Disorders, Larry H Bernstein, MD, FACP

http://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-dysfunction-and-cardiac-disorders/

Reversal of Cardiac mitochondrial dysfunction, Larry H Bernstein, MD, FACP

http://pharmaceuticalintelligence.com/2013/04/14/reversal-of-cardiac-mitochondrial-dysfunction/

 

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Cardiotoxicity and Cardiomyopathy Related to Drugs Adverse Effects

Curator: Larry H Bernstein, MD, FCAP

Introduction
This is the second part of a series on toxicities of therapeutic medications, the first being on the impact on drug development of early phase failure to identify toxicities that are found in late stage trials and result in withdrawal.  This portion will go into details of identifying the effects clinically and give some examples.  In the future, the design of therapies, the identification of high probability successful genomic targets, and more accurate patient selection will transform the approach to development, clinical trials, and clinical use of pharmaceuticals in patients.

Cardiotoxicity and Cardiomyopathy

refer to: What are cardiotoxicity and cardiomyopathy?

The Scott Hamilton Cares Initiative
Cardiotoxicity is a condition of heart muscle functional impairment from toxicity as an
  • adverse secondary effect of taking an essential medication, or
  • as a result of interactions between prescribed medications that result in heart damage,
  • usually dose and time related.
If severe, the adverse effect of chemotherapy may lead to cardiomyopathy.  While cardiomyopathy might be a result of treatments, such as chemotherapeutic medications, it may also caused by a group of diseases or disorders, leading to
  • damaged myocardiocytes, and the injury leads to
  • insufficient cardiac output, referred to as
  • heart failure.
Cardiomyopathy has many causes, singly or in combination:
  • viruses – such as,
    • coxsackie B,
    • human immunodeficiency virus (HIV)
  • systemic inflammatory disorder
    • systemic lupus erythematosis
  • Amyloidosis – amyloid protein deposits in the myocardium alone, and/or other organs
  • Infection –
    • bacterial (tetanus),
    • parasitic (Chaga’s disease)
    • Rheumatic fever
  •  high blood pressure
  • Chronic or long-term alcohol use (B vitamin deficiency)
  • Endocrine disease, such as hyperthyroidism
  • Thiamine and Vitamin B deficiency
  • Radiation therapy
  • Medications – anthracyclines.

Anthracyclines may be used to treat leukemia, lymphoma, multiple myeloma, breast cancer, and sarcoma. A commonly used anthracycline is called doxorubicin (Adriamycin®).

  • cardiomyopathy may also result from genetic defects
  • illegal drugs and toxic substances, cocaine, may also produce serious myocardial damage

With certain drugs, such as doxorubicin, there is a dose at which these cardiotoxic effects on the heart may occur.
An echocardiogram, or a radionuclide ventriculography scan, is performed

  • prior to initiating a cardiotoxic medication
  • to determine baseline cardiac function., and
  • repeated at intervals to monitor heart function while receiving cardiotoxic medications.

The ejection fraction (EF) is a percentage of blood pumped out into the body during each heartbeat. An EF of 50%-75% is considered normal.

  • The lower the ejection fraction, the more severe the heart failure may be.
This may determine if the cardiotoxic drug has caused cardiomyopathy.

Symptoms of cardiomyopathy:

  • fatigue
  •  shortness of breath
  •  fever and aching of the joints,
      • all characteristic of a flu-like illness.
  • Or, sudden heart failure or sudden cardiac death without any prior symptoms.
    • swollen feet and ankles
    • distended neck veins
    • tachycardia
    • dyspnea while reclining

Diagnosis:

  • history & physical examination
  • laboratory tests
  • EKG
  • Chest x-ray
  • Echocardiography
  • Cardiac cath
  • Angiography

Treatment:

  • Dexraoxane HCL –  doxorubicin
  • ACE inhibitors
  • Beta-blockers
  • Diuretics
  • Digoxin

 Biomolecular Screening for Drug Toxicity

Multiparameter In Vitro Assessment of Compound Effects on Cardiomyocyte Physiology Using iPSC Cells

O Sirenko, C Crittenden, N Callamaras, J Hesley, Yen-Wen Chen, et al.
 A sufficient percentage of drugs fail in clinical studies due to cardiac toxicity that the development of new, sensitive in vitro assays that can evaluate potential adverse effects on cardiomyocytes is needed. Cell-based models are more clinically relevant than those used in practice. Human-induced pluripotent stem cell–derived cardiomyocytes are especially attractive because
  • they express ion channels and
  • demonstrate spontaneous mechanical and electrical activity
    • similar to adult cardiomyocytes.
This study introduces techniques for measuring the impact of pharmacologic compounds on the beating rate of cardiomyocytes with ImageXpress Micro and FLIPR Tetra systems. The assays employ
calcium-sensitive dyes to monitor changes in Ca2+ fluxes
  • synchronous with cell beating,
This method allows monitoring of the
  • beat rate
  • amplitude, and
  • other parameters.
The system detects
  • concentration-dependent atypical patterns caused by
  • hERG inhibitors and other ion channel blockers.
In addition,
  • both positive and negative chronotropic effects on cardiac rate can be observed and
  • IC50 values determined.
This methodology is well suited for safety testing and can be used to estimate efficacy and dosing of drug candidates prior to clinical studies.
J Biomol Screen Jan 2013;18(1): 39-53  http://dx.doi.org/10.1177/1087057112457590

Estimating the risk of drug-induced proarrhythmia using human induced pluripotent stem cell-derived cardiomyocytes.

L Guo, RMC Abrams, JE Babiarz, JD Cohen, S Kameoka, et al.
 Early prediction of drug-induced toxicity is needed in the pharmaceutical and biotechnology industries to decrease late-stage drug attrition.
  • Cardiotoxicity accounts for about one third of safety-based withdrawn pharmaceuticals.
This study reports a high-throughput functional assay,  detailing a model that accurately detects
  • drug-induced cardiac abnormalities.
It employs
  • induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs).
  • Using 96-well plates with interdigitated electrode arrays  detect
    • assess impedance,
    • the rhythmic, synchronous contractions of the iPSC-CMs
Treatment of the iPSC-CMs with 28 different compounds with known cardiac effects resulted in
  • compound-specific changes in the beat rate and/or
  • the amplitude of the impedance measurement.
Changes in impedance for the compounds tested were comparable with the results from a related technology,
  • electric field potential assessment obtained from microelectrode arrays.
Using the results from the set of compounds,
  • an index of drug-induced arrhythmias was calculated,
  • which enabled the determination of a drug’s proarrhythmic potential.
This system of interrogating human cardiac function in vitro opens new opportunities for predicting cardiac toxicity and studying cardiac biology.
Toxicol Sci. Sep 2011; 123 (1):281-9  21693436

Determination of the Human Cardiomyocyte mRNA and miRNA Differentiation Network by Fine-Scale Profiling.

JE Babiarz, M Ravon, S Sridhar, P Ravindran, B Swanson,  et al.
This study is a detailed comparison of the mRNA and miRNA transcriptomes
  • across differentiating human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and
  • biopsies from fetal, adult, and hypertensive human hearts
Gene ontology analysis of the mRNA expression levels of the hiPSCs differentiating into cardiomyocytes
revealed 3 distinct groups of genes:
  • pluripotent specific,
  • transitional cardiac specification, and
  • mature cardiomyocyte specific.
Hierarchical clustering of the mRNA data revealed that the transcriptome of hiPSC cardiomyocytes
  • stabilizes 20 days after initiation of differentiation.
But analysis of cells continuously cultured for 120 days indicated that
  • the cardiomyocytes continued to mature toward a more adult-like gene expression pattern.
Analysis of cardiomyocyte-specific miRNAs (miR-1, miR-133a/b, and miR-208a/b) revealed
  • an miRNA pattern indicative of stem cell to cardiomyocyte specification.
A biostatistitical approach integrated the miRNA and mRNA expression profiles revealing a
  • cardiomyocyte differentiation miRNA network and
  • identified putative mRNAs targeted by multiple miRNAs.
Together, these data reveal the
miRNA network in human heart development and
  • support the notion that overlapping miRNA networks
  • re-enforce transcriptional control during developmental specification.
Stem Cells Dev. 2012 Jul 20;21 (11):1956-65  22050602

Comparative Gene Expression Profiling in Human Induced Pluripotent Stem Cell Derived Cardiocytes and Human and Cynomolgus Heart Tissue.

D Puppala, LP Collis, SZ Sun, V Bonato, X Chen, B Anson, et al.  Compound Safety Prediction.
Cardiotoxicity is one of the leading causes of drug attrition. Current in vitro models insufficiently predict cardiotoxicity.  The authors describe
  • the gene expression profile of human induced pluripotent stem cell derived cardiocytes (iCC)
  • post-thaw over a period of 42 days in culture and
  • compare this profile to human fetal and adult as well as
  • adult cynomolgus nonhuman primate (NHP: Macaca fascicularis) heart tissue.
The results indicate that iCC express relevant cardiac markers such as
  • ion channels (SCN5A, KCNJ2, CACNA1C, KCNQ1 and KCNH2),
  • tissue specific structural markers (MYH6, MYLPF, MYBPC3, DES, TNNT2 and TNNI3),
  • transcription factors (NKX2.5, GATA4 and GATA6), and
  • lack the expression of stem cell markers (FOXD3, GBX2, NANOG, POU5F1, SOX2, and ZFP42).

A functional evaluation of contractility of the iCC showed

  • functional and pharmacological correlations with myocytes isolated from adult NHP hearts.
The results suggest that stem cell derived cardiocytes may represent
  • a novel in vitro model to study human cardiac toxicity with potential ex vivo and in vivo translation.
Toxicol Sci. Sep 14 2012;:   22982684

Characterization of Human Induced Pluripotent Stem Cell Derived Cardiomyocytes:
Bioenergetics and Utilization in Safety Screening.

P Rana, B Anson, S Engle, Y Will.   Compound Safety Prediction. Pfizer Global R&D, Groton CT.
Cardiotoxicity remains the number one reason for drug withdrawal from the market and FDA issued black box warnings; thus
  • demonstrating the need for more predictive preclinical safety screening,
  • especially early in the drug discovery process.
Whereas hERG screening has become routine to mitigate proarrhythmic risk,
  • the development of in vitro assays predicting additional on- and off-target biochemical toxicities
  • will benefit from cellular models exhibiting true cardiomyocyte characteristics
    • such as, native tissue-like mitochondrial activity.
An hypothesis was tested  for using human stem cell derived tissue cells by using a combination of
  • flux analysis,
  • gene and protein expression, and
  • toxicity-profiling techniques
    • to characterize mitochondrial function
    • in induced pluripotent stem cell (iPSC)-derived human cardiomyocytes
      • in the presence of differing carbon sources
      • over extended periods in cell culture.
Functional analyses demonstrate that iPSC-derived cardiomyocytes:
1) are capable of utilizing anaerobic or aerobic respiration depending upon the available carbon substrate,
2) are bioenergetically closest to adult heart tissue cells when cultured in galactose or galactose supplemented with fatty acids, and
3) show a dose dependent toxicity profile to a variety of kinase inhibitors with known clinical cardiac liabilities.
Furthermore, gene and protein expression analyses revealed that in comparison to adult cardiac tissue,
  • iPSCs-derived cardiomyocytes possess a qualitatively similar expression pattern of mitochondrial genes,
  • an up-regulation of apoptotic and antioxidant genes, and
  • a mitochondrial transcription pattern that is similar across different carbon substrates
  • despite showing changes in protein levels and functional bioenergetic adaptation.
Toxicol Sci. 2012 Jul 27;:   22843568

Decreasing cardiac chamber sizes and associated heart dysfunction in COPD – role of hyperinflation.

H Watz, B Waschki, T Meyer, G Kretschmar, A Kirsten, M Claussen, H Magnussen
This study examined the relationship of
  • lung function with heart size and heart dysfunction and
  • associated consequences for 6-minute walk distance (6MWD)
    • in patients with COPD of different severity.
METHODS:   138 patients with COPD (GOLD I-IV)
  • the size of all cardiac chambers,
  • left ventricular diastolic dysfunction (relaxation and filling), and
  • global right ventricular dysfunction (Tei-index)
    • were measured by echocardiography .
  • lung function (spirometry, bodyplethysmography, diffusion capacity) and
  • 6MWD …. were measured.
RESULTS: Size of all cardiac chambers decreased with GOLD stages. Overall,
moderate relationships existed between
  • variables of lung function and cardiac chamber sizes.
  • Static hyperinflation (inspiratory-to-total lung capacity ratio [IC/TLC],
  • functional residual capacity, and residual volume)
showed stronger associations with
  • cardiac chamber sizes than
  • airway obstruction or diffusion capacity.
IC/TLC ratio correlated best with cardiac chamber sizes and was
  • an independent predictor of cardiac chamber sizes
    • after adjustment for body surface area.
Patients with an IC/TLC ratio <!–= 0.25 had a significantly–>
  • impaired left ventricular diastolic filling pattern and
  • a significantly impaired Tei-index
    • compared to patients with an IC/TLC ratio > 0.25.
An impaired left ventricular diastolic filling pattern was independently associated with
  • a reduced 6MWD.
An increasing severity of COPD is associated with a decreasing heart size.
Hyperinflation in patients with COPD might have an important role with respect to
  • heart size and
  • cardiac dysfunction
Chest. Feb 26 2010;:   20190002  Cit:4

Cardiovascular Events After Clarithromycin Use in Lower Respiratory Tract Infections
Analysis of Two Prospective Cohort Studies

S Schembri, PA Williamson, PM Short, A Singanayagam, A Akram, et al. British Medical Journal
Acute exacerbations of chronic obstructive pulmonary disease and community acquired pneumonia are common causes of admission to a hospital.
Antibiotics, including clarithromycin, are commonly prescribed during acute exacerbations of chronic obstructive pulmonary disease, especially
  • in the presence of increased breathlessness,
  • sputum volume, and
  • purulence.
 Use of macrolide antibiotics in community acquired pneumonia has been consistently associated with improved short term mortality in observational studies,
and national and international guidelines therefore recommend their use in combination with β lactams for patients admitted to hospital.

Widespread use of macrolide antibiotics has been accompanied by concerns about adverse effects on cardiovascular morbidity and mortality.
A retrospective study of erythromycin use in 1,249,943 patients identified an increase in deaths from cardiovascular disease.
Azithromycin was shown to have a similar association with increased cardiovascular deaths

  • during the time of administration.

CLARICOR (Effect of Clarithromycin on Mortality and Morbidity in Patients with Ischemic Heart Disease trial) was a double blind, placebo controlled trial
showing that a two week course of clarithromycin administered to patients with coronary heart disease 

  • increased cardiovascular and all cause mortality
The increased mortality rate (clear of pulmonary infection)
  • persisted for three years after discontinuation of the drug.

A recent meta-analysis of 17 trials of antibiotics in coronary heart disease showed

  • increased long term mortality after macrolides, primarily due
  • to increased deaths from cardiovascular disease.

However, no studies have examined the long term effect of clarithromycin on cardiovascular events and mortality in patients

  • after acute exacerbations of chronic obstructive pulmonary disease or community acquired pneumonia.

Therefore, this prospective cohort study was undertaken to examine the association of clarithromycin with cardiovascular events

  • in the setting of acute exacerbations of chronic obstructive pulmonary disease and community acquired pneumonia.

Population.

  • 1343 patients admitted to hospital with acute exacerbations of chronic obstructive pulmonary disease
  • and 1631 patients admitted with community acquired pneumonia.

Main Outcome Measures.

Hazard ratios for cardiovascular events at one year (defined as hospital admissions with

  • acute coronary syndrome, decompensated cardiac failure, serious arrhythmia, or sudden cardiac death) and
  • admissions for acute coronary syndrome (acute ST elevation myocardial infarction, non-ST elevation myocardial infarction, and unstable angina).

Secondary outcomes were all cause and cardiovascular mortality at one year.

Results.

  •  268 cardiovascular events occurred in the acute exacerbations of chronic obstructive pulmonary disease cohort and
  • 171 in the community acquired pneumonia cohort over one year.

After multivariable adjustment, clarithromycin use in acute exacerbations of chronic obstructive pulmonary disease

  • was associated with an increased risk of cardiovascular events and acute coronary syndrome—
  • hazard ratios 1.50 (95% confidence interval 1.13 to 1.97) and 1.67 (1.04 to 2.68).
After multivariable adjustment, clarithromycin use in community acquired pneumonia
  •  was associated with increased risk of cardiovascular events (hazard ratio 1.68, 1.18 to 2.38)
  • but not acute coronary syndrome (1.65, 0.97 to 2.80).
This association was found between clarithromycin use in acute exacerbations of COPD and
  • cardiovascular mortality (adjusted hazard ratio 1.52, 1.02 to 2.26)
  • but not all cause mortality (1.16, 0.90 to 1.51) .

No association was found between clarithromycin use in community acquired pneumonia and all cause mortality or cardiovascular mortality.
Use of β lactam antibiotics or doxycycline was not associated with increased cardiovascular events in patients with

  • acute exacerbations of chronic obstructive pulmonary disease, suggesting an effect specific to clarithromycin.

 Timing of Cardiovascular Events

The study found no significantly increased risk of cardiovascular events while patients were taking clarithromycin in the COPD cohort
(hazard ratio 1.73, 0.71 to 4.25), but
  • an increased risk was present after the clarithromycin course ended (1.41, 1.05 to 1.89).
In the community acquired pneumonia cohort, the hazard ratio for association between clarithromycin use and cardiovascular events
was 1.84 (0.75 to 4.51) during clarithromycin use and 1.66 (1.14 to 2.43) after the antibiotic was stopped.

Association With Duration of Antibiotic Use

Longer courses of clarithromycin were associated with more cardiovascular events. The median duration of treatment was seven days in both cohorts.
Less than three days of clarithromycin treatment was not associated with cardiovascular events in the chronic obstructive pulmonary disease cohort
(hazard ratio 0.89, 0.50 to 1.57) or the community acquired pneumonia cohort (0.63, 0.15-2.65), compared with patients who did not receive clarithromycin.

Effect of Age and Cardiovascular Risk Status

The hazard ratios of the effect of clarithromycin on cardiovascular events in such patients were
  • 1.35 (0.94 to 1.95) in those with a high cardiovascular risk and 0.88 (0.20 to 3.96) in those with a low risk.

The lowest hazard ratios for cardiovascular events were in patients aged 60 or below (1.01, 0.36 to 2.91).
The hazard ratio was 1.47 (1.01 to 2.14) for patients aged 60-79, and a higher risk was associated with

  • clarithromycin use in patients aged over 80 (hazard ratio 1.68, 1.05 to 2.69).

Use of Other Antibiotics

Use of β lactam or doxycycline was not associated with increased cardiovascular events
  • (hazard ratios 1.06 (0.83 to 1.37) and 0.96 (0.61 to 1.51), respectively)
in the chronic obstructive pulmonary disease cohort compared with patients not receiving antibiotics.

Possible Explanations for Findings

There was a strong association between prolonged (more than seven days) courses of clarithromycin and
  • cardiovascular events,
    • which strengthens the case for a true biological cause.

The association between duration of antibiotic treatment and cardiovascular events

  • could also represent residual confounding by severity of illness.

How do the results point to the effect on outcome after cessation of the drug?  The authors support an ischaemic mechanism.
Clarithromycin may activate macrophages, leading to an inflammatory cascade resulting in more vulnerable plaques that

  • over time may lead to acute coronary syndromes or sudden cardiac death by plaque rupture.

 Conclusion

Prolonged courses of clarithromycin (more than seven days) may be associated with

  • increased risk of cardiovascular events,
  • especially in patients with a pre-existing history of coronary heart disease.

This may be of particular importance given recent data supporting long term macrolide use

  • to prevent exacerbations of chronic obstructive pulmonary disease.

Biomarkers Role in Drug Development

Biomarkers: An indispensible addition to the drug development toolkit

Biomarkers are becoming an essential part of clinical development. In this white paper, Thomson Reuters explores
the role of biomarkers as evaluative tools in improving clinical research and the challenges this presents.
The potential of biomarkers to

  • improve decision making,
  • accelerate drug development and
  • reduce development costs

is discussed with insights into a faster alternative to the conventional drug development approach and the promise of

  • safer drugs,
  • in greater numbers,
  • approved more quickly.
The attrition rate for drugs in clinical development is high: the percentage of tested products
  • entering phase I trials that eventually gain regulatory approval has been estimated at a paltry 8%.
Many of these failures happen late in clinical trials, with the consequence that expenditure in clinical drug development is increasing.
One study calculated that the cost of developing a drug increased by over 50% between 2002 and 2007. The related concern is that
very few drugs are making it out of the clinical research pipeline.
In 2007, the FDA approved just 17 new molecular entities and 2 biologic licenses, the lowest number since 1983.
The problem is mainly due to a gap in the industry’s ability to predict a drug candidate’s performance early, and with a large degree of certainty.
The convention in clinical research has been to measure the performance of novel therapies using clinical outcomes. This approach is
laborious, inexact and, as the US Food and Drug Administration (FDA ) puts it, decades old.

Why and what kinds of biomarkers do we determine are ESSENTIAL?

Biomarkers — a measure of
  • a normal biological process in the body,
  • a pathological process, or
  • the response of the body to a therapy —
may offer information about
  • the mechanism of action of the drug,
  • its efficacy, its safety and
  • its metabolic profile.
They feature heavily in the FDA ’s Critical Path Opportunities List for their potential
  • to speed the development and approval of medical products.
  • Moreover, they can predict drug efficacy more quickly than conventional clinical endpoints.
The first three examples are measures of drug efficacy and treatment response, but are not indicators of TOXICITY.

In 1960, researchers discovered that some patients with chronic myelogenous leukemia (CML), a form of adult leukemia

  • in which there is a proliferation of myeloid cells in the bone marrow,
  • have a specific genetic change associated with their cancer, a shortened version of chromosome.

The Philadelphia chromosome is caused by a translocation between chromosomes 9 and 22. The consequence of this genetic swap

  • is the creation of the BCR-ABL ‘oncogene’;
  • this cancer-causing gene produces a protein with elevated tyrosine kinase activity
  • that induces the onset of leukemia.

Researchers were able to use the Philadelphia chromosome as a biomarker

  • to indicate which patients would benefit from drug candidates (tyrosine-kinase inhibitors)
    • specifically targeting the rogue protein.

The drug imatinib (Gleevec) is a Tyr kinase inhibitor and

  • decreases the proliferation of Philadelphia chromosome+ cells,
  • slowing the progression of the disease.

Specific mutations in the BCR–ABL gene were biomarkers that predicted resistance to imatinib,

  • leading to the development of newer tyrosine-kinase inhibitors dasatinib and nilotinib.

In the late 1980’s, scientists discovered that HIV viral load could be used as a marker of disease progression
Viral load was used to show that patients receiving combination therapy had

  • a higher reduction in viral load than those on monotherapy.

Eventually, the viral load biomarker was used in the development and assessment of Highly Active Antiretroviral Therapy (HAART)
treatment regimens taken by many people living with HIV today.

The HER-2 gene and receptor was also discovered in the mid 1980’s. Between 20–30% of breast cancer patients show an

  • over-expression of the HER-2 receptor on their cancer cells (usually postmenopausal).

This biomarker indicates a higher risk of adverse outcomes, but it gave clinicians a new target for novel therapies, and

  • the antibody trastuzumab (Herceptin) was developed
  • to target HER-2 receptors in these ‘overexpressing’ patients.

 Preventing Drug Development Disasters

The need for biomarkers to guide clinical research is perhaps best highlighted in the stories of recent drug development failures.
Between 1995 and 2005, at least 34 drugs were withdrawn from the market, mainly as a result of hepatotoxic or cardiotoxic effects.

Many of us are familiar with the withdrawal in 2004 of the anti-inflammatory drug rofecoxib (Vioxx) due to concerns about its

  • increased risk of heart attack and stroke, and more recently with
  • the extremely serious adverse effects in the phase I clinical trial and subsequent failure of the monoclonal antibody, TGN1412.

TG N1412, a ‘superagonist’, produced by the firm TeGenero, stimulates an immune response. While originally intended to treat B cell
chronic lymphocytic leukemia and rheumatoid arthritis, it had been tested pre-clinically with no toxic or pro-inflammatory effects.
In 2006, six healthy male volunteers took part in a phase I clinical trial to test the safety of the candidate. Within 90 minutes of receiving the drug,

  • all six men were experiencing the beginnings of a ‘cytokine storm’, a term that describes
  • a cascade of proinflammatory cytokine release
  • leading to organ failure due to hypotension.

Although all the men survived, they required weeks of hospitalization. The cost of a failure, such as TGN1412,

  • in terms of patient health and lost resources is huge.

The TGN1412 trial failure highlighted a need for improved preclinical safety testing. It has been suggested that had procedures using safety biomarkers to

  • guide dosing and predict the toxicity of this drug been used, the disaster may not have occurred.

Biomarkers today

Today you “would not even conceive” of developing a new drug without simultaneously looking for biomarkers for
  • efficacy,
  • safety, and
  • to measure the pharmacodynamics of the drug,
says Dr Jeffrey Ross, Head of Pathology at the Albany Medical Center in New York, involved in the original work on HER-2.
The field of oncology is leading the way in the use of biomarkers in drug development. “Clinical trials are designed upon biomarker assays,”
“abstracts of phase II and III cancer trials talk about what biomarkers were selected.
  • In vivo biomarkers,
  • imaging biomarkers,
  • blood and tissue based biomarkers,

One example of a biomarker in use in oncology is circulating tumor cells (CTCs), a biomarker present in the blood of cancer patients.
At the moment, CTCs are used in the development of anti-cancer drugs as

  • an objective and direct measurement of the response of the cancer to a novel agent.

The way that clinical trials had been done previously was to enroll all patients

  • with a given disease independent of gene or phenotypic makers.
  • By selecting a population with the particular gene which is predicted
  • to be important for response to a novel therapeutic, then
    • a smaller clinical trial should be sufficient to see whether it works or not.

The chemotherapy drug irinotecan (Camptosar) is an example of personalized medicine,

  • using a biomarker to guide both clinical practice and subsequent clinical trials.

Irinotecan is used to treat advanced colorectal cancer. Once administered, irinotecan is

  • activated to the metabolite SN-38, and then
  • eventually inactivated in the body by the UGT1A1 enzyme.

In 2005, the US Food and Drug Administration added a warning to the label of the drug, stating that patients

  • homozygous for a particular a version of the UGT1A1 gene — the UGT1A1*28 allele,
  • associated with decreased UGT1A1 enzyme activity —
    • should be given a reduced dose.
Because patients with this allele clear the drug less quickly from their body than the rest of the population,
  • they effectively receive a greater exposure to the drug from the same dose.
As a consequence, they are at higher risk of potentially life-threatening side effects such as neutropenia (a decrease in white blood cells) and diarrhea.
The toxicity of irinotecan has long been a concern, and this biomarker now allows clinicians to better identify those patients who are at high risk of
  • serious side-effects (about 10% of the population are homozygous for UGT1A1*28).
And while this pharmacogenomics information has helped improve the clinical use and efficacy of irinotecan, it has also fed back into
the development of other drugs; this new understanding prompted the use of the UGT1A1 biomarker to guide other studies,
including several new irinotecan and oxaliplatin-based chemotherapy regimens.

Using preclinical biomarkers as evidence of efficacy

  • biomarkers can accelerate research by substituting for clinical symptoms as a measure of efficacy.
  • biomarkers can also replace clinical symptoms when it comes to measuring drug safety
  • an efficacy biomarker plus a safety biomarker will define not just whether a drug will work, but also what kind of dose might be relevant in humans

 Improving efficacy in cardiology

Consider the role of inflammatory marker C-reactive-protein (CRP) in cardiovascular disease. CRP is released by inflamed atherosclerotic plaques in the arteries
of individuals with coronary heart disease, and increased levels of CRP are associated with a greater risk of plaque rupture, but also of a silent heart attack.
CRP is being used as a biomarker to measure drug efficacy, in particular whether rosuvastatin (Crestor)

  • reduces the risk of cardiovascular morbidity and mortality
  • in apparently healthy individuals with low LDL-cholesterol levels but elevated CRP.

The JUPITER study (Justification for the Use of Statins in Primary Prevention: an Intervention Trial Evaluating Rosuvastatin) was halted in March 2008

  • due to firm evidence that the drug is indeed more beneficial than placebo and
  • improves the prognosis of individuals with high CRP levels.
A related biomarker of cardiovascular risk called neopterin. Just as CRP is produced by inflamed atherosclerotic plaques at risk of rupture,
neopterin is produced by activated macrophages in this inflammatory process. Circulating neopterin levels are higher in patients with ACS and may be
a marker of coronary disease activity. In addition, “Neopterin could also potentially be a marker of drug efficacy because
if you reduce the number of active macrophages in the plaque or the circulation, the levels of neopterin also decrease,” says Dr Juan Carlos Kaski,
Professor of Cardiovascular Science and Director of the Cardiovascular Biology Research Centre at St George’s University of London.

Other uses of biomarkers

These types of biomarkers can be used to drive critical ‘go/no go’ decision in drug development
Mechanistic or ‘target’ biomarkers can be used in pre-clinical or phase I trials to measure the pharmacological effect of the drug, i.e.
  • whether the drug interacts with its receptor, enzyme, or protein target,
  • whether it is distributed to the site where it needs to act,whether there is some
    • form of downstream pharmacology, and
  • the dose ranges in which the drug is pharmacologically active.
Drugs such as 5-HT4 receptor agonists (e.g. cisapride, mosapride), used in gastro-esophageal reflux disease (GERD), stimulate
  • the secretion of aldosterone as a side-effect.
Although aldosterone is not linked to GERD (and can’t be used as a biomarker of the disease), the hormone can be used
  • as a mechanistic biomarker in drug development to assess whether
    • novel 5-HT4 agonists in development have a pharmacological effect.

Discovering new biomarkers

The fundamental issue we have to deal with, both with target selection and developing better biomarkers,
  • is a better understanding of pathophysiology.
The clinical need is huge, not least in diseases like chronic obstructive pulmonary disease (COPD), an illness about which we know very little.

“COPD has very few markers to indicate severity and disease progression,” says Dr Trevor Hansel, Medical Director of the National Heart &
Lung Institute Clinical Studies Unit in London. Many pharmaceutical companies have begun to invest in ‘omics’ —

  • genomics,
  • proteomics,
  • metabonomics —

to begin to sort through this mountain of molecules and characterize biomarkers based on a molecular understanding of disease.

The ‘omics’ approach enables
  • the detection of small changes in tissue composition through protein profiling technologies such as
  • mass spectrometry and gel electrophoresis.

Essentially, it is about capturing a molecular profile from a clinical sample and converting this into

  • information about a clinical condition — for example the stage of disease or
  • what players are involved in the disease pathways.
“We will be able to look at diseases and catagorize them based on
  • biochemical or physiological findings, rather than just on symptoms” …  David Roblin, Pfizer

Companion Diagnostics and the Drug–Diagnostic Codevelopment Model

 Jan Trøst Jørgensen   Drug Development Research Nov 2012; 73(7):390-397.   http://dx.doi.org/10.1002/ddr.21029

The concept of using a predictive or selective diagnostic assay in relation to drug development goes back to the 1970s when

  • the selective estrogen receptor modulator, tamoxifen (AstraZeneca) was developed for metastatic breast cancer.
Clinical data showed that the estrogen receptor status correlated well with the clinical outcome when the patients were treated with tamoxifen.
It is only within the last decade that this model has gained widespread acceptance. The drug and the diagnostic are interdependent, and
if the development project proves successful, the companion diagnostic assay (CoDx) will end up determining the conditions for the use of the drug.
This gatekeeper role obviously requires that the CoDx assays adhere to the same strict rules and regulations that are known from the development of drugs.
For any CoDx assay, it must be documented that it is robust and reliable and that it possesses clinical utility. The article focus on some of the most important
aspects of the CoDx development process with emphasis on the clinical validation and clinical utility but also other critical issues, such as,
  • the biomarker selection process,
  • determination of the cut-off value, and
  • the analytical validation.

 Detecting Potential Toxicity in Mitochondria

 Brad Larson, Principal Scientist; Peter Banks, Scientific Director; BioTek Instruments, Winooski, Vt.
Mitochondrial dysfunction may be
  • inherited,
  • arise spontaneously, or
  • develop as a result of drug toxicity.
Mitochondrial toxicity as a result of pharmaceutical use may damage key organs, such as the liver and heart. For example,
  • nefazodone—a depression treatment—was withdrawn from the U.S. market after it was shown to
significantly inhibit mitochondrial respiration in liver cells, leading to liver failure. Troglitazone, an anti-diabetic and anti-inflammatory,
was withdrawn from all markets after research concluded that it caused acute mitochondrial membrane depolarization, also leading to liver failure.
Drug recalls are costly to a manufacturer’s bottom line and reputation, and more importantly, can be harmful or even fatal to users. As drug
discovery continues to evolve, much lead compound research now includes careful review of its interaction and potential toxicity with mitochondria.
Cytotoxicity and ATP production are measured in cancerous and normal hepatocytes using a known inducer of cellular necrosis. (All figures: BioTek Instruments)
Cell-based mitochondrial assays in microplate format may include
  • mitochondrial membrane potential,
  • total energy metabolism,
  • oxygen consumption, and
  • metabolic activity;
and offer a truer environment for mitochondrial function in the presence of drug compounds compared to isolated mitochondria-based tests.
Combining more than one assay in a multiplex format increases the amount of data per well while decreasing data variability arising from running the assays separately.
The aggregated data also provides a more encompassing analysis of the drug’s effect on mitochondria than a single test.

Human cardiac muscle

Human cardiac muscle (Photo credit: Carolina Biological Supply Company)

English: Non-sustained run of ventricular tach...

English: Non-sustained run of ventricular tachycardia on telemonitoring from a patient with chemotherapy-induced cardiomyopathy. (Photo credit: Wikipedia)

English: Doxorubicin 3D model Русский: Трёхмер...

English: Doxorubicin 3D model Русский: Трёхмерная модель молекулы доксорубицина (Photo credit: Wikipedia)

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2013 – YEAR OF THE ULTRASOUND

Author – Writer: Dror Nir, PhD

To those of you who did not know, 2013 is the year of the ultrasound: http://www.ultrasound2013.org/. This initiative was launched by AIUM and its objectives:

  • Raise awareness of the value and benefits of ultrasound among patients, health care providers, and insurers
  • Provide ultrasound education and evidence-based guidelines for health care providers
  • Educate insurers about the cost savings and patient benefits associated with performing an ultrasound study when scientific evidence supports its potential effectiveness compared to other imaging modalities
  • Educate patients about the benefits of ultrasound as the appropriate imaging modality for their care
  • Encourage the incorporation of ultrasound into medical education

 Quoting from the ultrasound first web-site:

The initiative is designed to call attention to the safe, effective, and affordable advantages of ultrasound as an alternative to other imaging modalities that are more costly and/or emit radiation. For a growing number of clinical conditions, ultrasound has been shown to be equally effective in its diagnostic capability, with a distinct advantage in safety and cost over computed tomography and magnetic resonance imaging. Despite this advantage, evidence suggests that ultrasound is vastly underutilized. Ultrasound First focuses on educating health care workers, medical educators, insurers, and patients of the benefits of ultrasound in medical care. “There is growing support and public awareness for the need to reduce and carefully monitor patients’ exposure to radiation during medical imaging. The use of ultrasound as an alternative imaging modality will help achieve that goal while reducing cost,” states AIUM President Alfred Abuhamad, MD. “Many health care workers and insurers are unacquainted with the range of conditions for which ultrasound has been shown to have superior diagnostic capabilities. Disseminating this knowledge to health care workers and incorporating ultrasound in medical protocols where scientific evidence has shown its diagnostic efficacy will undoubtedly improve patient safety and reduce cost. The time to act is now.”

 A primary component of Ultrasound First is providing clinical evidence for the use of ultrasound. To that aim, the Journal of Ultrasound in Medicine has launched a special feature, the Sound Judgment Series, consisting of invited articles highlighting the clinical value of using ultrasound first in specific clinical diagnoses where ultrasound has shown comparative or superior value. Clinical conditions that will be addressed in the series include postmenopausal bleeding, right lower quadrant pain, pelvic pain, right upper quadrant pain, and shoulder pain, among others. This series will serve as an important educational resource for health care workers and educators.  On the clinical evidence page one can find reasoning for why ultrasound first. Not much related to cancer diagnosis and management. The only interesting claim is:Ultrasound-guided surgery: Its use to remove tumors from women who have palpable breast cancer is much more successful than standard surgery in excising all the cancerous tissue while sparing as much healthy tissue as possible.”

In support of this initiative The Journal of Ultrasound in Medicine has launched a special series, Sound Judgment, comprised of invited articles highlighting the clinical value of using ultrasound first in specific clinical diagnoses where ultrasound has shown comparative or superior value. So far it includes only two items related to management of cancer: Sonography of Facial Cutaneous Basal Cell Carcinoma, A First-line Imaging Technique; by Ximena Wortsman, MD, and Quantitative Assessment of Tumor Blood Flow Changes in a Murine Breast Cancer Model After Adriamycin Chemotherapy Using Contrast-Enhanced Destruction-Replenishment Sonography; by Jian-Wei Wang, MD et. al. The devoted readers of our Open Access Scientific Journal might find the article by Dr. Wortsman, MD bringing complementary information to a previous post of mine: Virtual Biopsy – is it possible?. Qouting from this article: “Cutaneous basal cell carcinoma is the most common cancer in human beings, and the face is its most frequent location. Basal cell carcinoma is rarely lethal but can generate a high degree of disfigurement. Of all imaging techniques, sonography has proven to support the diagnosis and provide detailed anatomic data on extension in all axes, the exact location, vascularity, and deeper involvement. This information can be used for improving management and the cosmetic results of patients.”

 The article gives clear presentation of the problem and includes demonstrative pictures:

f1

Figure: Basal cell carcinoma with dermal involvement (transverse view, nasal tip). Grayscale sonography (A) and 3-dimensional reconstruction (B, 5- to 8-second sweep) show a 10.1-mm (wide) × 1.4-mm (deep) well-defined hypoechoic oval lesion (between markers in A and outlined in B) that affects the dermis (d) of the left nasal wing. Notice the hyperechoic spots (arrowheads) within the lesion. The nasal cartilage (c) is unremarkable; asterisk indicates basal cell carcinoma.

Basal cell carcinoma with dermal and subcutaneous involvement (transverse view, frontal region). A, Grayscale sonography shows a 11.4-mm (wide) × 6.6-mm (deep) well-defined oval hypoechoic lesion that involves the dermis (d) and subcutaneous tissue (st). There are hyperechoic spots (arrowheads) within the tumor. B, Color Doppler sonography shows increased vascularity within the tumor (asterisk). C, Three-dimensional sonographic reconstruction (5- to 8-second sweep) highlights the lesion (asterisk, outlined); b indicates bony margin of the skull.

Basal cell carcinoma with dermal and subcutaneous involvement (transverse view, frontal region). A, Grayscale sonography shows a 11.4-mm (wide) × 6.6-mm (deep) well-defined oval hypoechoic lesion that involves the dermis (d) and subcutaneous tissue (st). There are hyperechoic spots (arrowheads) within the tumor. B, Color Doppler sonography shows increased vascularity within the tumor (asterisk). C, Three-dimensional sonographic reconstruction (5- to 8-second sweep) highlights the lesion (asterisk, outlined); b indicates bony margin of the skull.

f3

Figure: Pleomorphic presentations of basal cell carcinoma lesions on grayscale sonography (transverse views). Notice the variable shapes of the tumors.

f4

Figure: Frequently, blood flow can be detected within the tumor and its periphery, with slow-flow arteries or veins. The latter vascular data can orient the clinician about the distribution and amount of blood flow that he or she will face during surgery. Despite the fact that basal cell carcinomas usually do not present high vascularity, it should be kept in mind that many of basal cell carcinoma operations are performed in the offices of clinicians and not in the main operating rooms of large hospitals. Nevertheless, the finding of high vascularity within a clinically diagnosed basal cell carcinoma may suggest another type of skin cancer that could occasionally mimic basal cell carcinoma, such as squamous cell carcinoma, Merkel cell carcinoma, or a metastatic tumor. The above figure presents variable degrees of vascularity in basal cell carcinoma lesions going from hypovascular to hypervascular on color and power Doppler sonography (transverse views)

f5

Figure: The depth correlation between sonography (variable frequency) and histologic analysis in facial basal cell carcinoma has been reported to be excellent. Thus, the intraclass correlation coefficient for comparing thickness for the two methods (sonography and histologic analysis) that has been described in literature is 0.9 (intraclass correlation coefficient values ≥0.9 are very good; 0.70–0.89 are good; 0.50–0.69 are moderate; 0.30–049 are mediocre; and ≤0.29 are bad). Two rare sonographic artifacts have been described in basal cell carcinoma. One is the “angled border” that is produced by an inflammatory giant cell reaction underlying the tumor, which may falsely increase the apparent size of the tumor. The other is the “blurry border,” which is produced by large hypertrophy of the sebaceous glands surrounding the lesion. According to the literature, both artifacts can be recognized by a well-trained operator. The figure above presents the sonographic involvement of deeper layers such as the nasal cartilage and orbicularis muscles in the face is of critical importance and may change the decision about the type of surgery. Basal cell carcinoma with nasal cartilage involvement (3-dimensional reconstruction, 5- to 8-second sweep, transverse view, left nasal wing). Notice the extension of the tumor (asterisk, outlined) to the nasal cartilage region (c); d indicates dermis.

Basal cell carcinoma with involvement of the orbicularis muscle of the eyelid (m). Grayscale sonography (transverse view, right lower eyelid) shows that the tumor (asterisk) affects the muscle layer (arrows).

Basal cell carcinoma with involvement of the orbicularis muscle of the eyelid (m). Grayscale sonography (transverse view, right lower eyelid) shows that the tumor (asterisk) affects the muscle layer (arrows).

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How Methionine Imbalance with Sulfur-Insufficiency Leads to Hyperhomocysteinemia

Curator: Larry H Bernstein, MD, FACP

The Open Clinical Chemistry Journal, 2011; 4: 34-44

http://occj.com/1874-2416/11 2011/
http://dx.doi.org/11.2011/occl/1874-2416/
Bentham Open   Open Access

Introduction:  The following document is a seminal article concerning the relationship between hyoerhomocysteinemia and cardiovascular and other diseases. It provides a new insight based on the metabolism of S8 and geographic factors affecting the distribution, the differences of plant and animal sources of dietary intake,
and the great impact on methylation reactions.  The result is the finding that hyperhomocysteine is a “signal”, just as CRP is a measure of IL-6, IL-1, TNFa -mediated inflammatory response.  A deficiency of S8 due to the unavailability of S8, leads to CVD, and is seen in sulfur deficient regions with inadequate soil content and with veganism.  Hyperhomocysteinemia is also an indicator of CVD risk in the well fed populations, and that gives us a good reason to ASK WHY?

I have trimmed the content to make the necessary points that would be sufficient for this content.  The article can be viewed at OCCJ online.

The Oxidative Stress of Hyperhomocysteinemia Results from Reduced Bioavailability of Sulfur-Containing Reductants

Yves Ingenbleek*
Laboratory of Nutrition, Faculty of Pharmacy, University Louis Pasteur Strasbourg, France

Abstract

A combination of subclinical malnutrition and S8-deficiency

  • maximizes the defective production of Cys, GSH and H2S reductants,
  • explaining persistence of unabated oxidative burden.

The clinical entity

  • increases the risk of developing cardiovascular diseases (CVD) and stroke
    • in underprivileged plant-eating populations
    • regardless of Framingham criteria and vitamin-B status.

Although unrecognized up to now,

  • the nutritional disorder is one of the commonest worldwide,
  • reaching top prevalence in populated regions of Southeastern Asia.

Increased risk of hyperhomocysteinemia and oxidative stress may also affect

  • individuals suffering from intestinal malabsorption or
  • westernized communities having adopted vegan dietary lifestyles.

Vegetarian subjects

  • consuming subnormal amounts of methionine (Met) are characterized by
  • subclinical protein malnutrition causing reduction in size of their lean body mass (LBM) best
  • identified by the serial measurement of plasma transthyretin (TTR).

As a result, the transsulfuration pathway is depressed at cystathionine-beta-synthase (CbS) level

  • triggering the upstream sequestration of homocysteine (Hcy) in biological fluids and
  • promoting its conversion to Met.

Maintenance of beneficial Met homeostasis is

  • counterpoised by the drop of cysteine (Cys) and glutathione (GSH) values downstream to
  • CbS causing in turn declining generation of hydrogen sulfide (H2S) from enzymatic sources.

The biogenesis of H2S via non-enzymatic reduction is further inhibited in areas where

  • earth’s crust is depleted in elemental sulfur (S8) and sulfate oxyanions.

Keywords: Vegetarianism, malnutrition, sulfur-deficiency, hyperhomocysteinemia, oxidative stress, hydrogen sulfide, cardiovascular diseases, developing countries, Asia.

Homocysteine (Hcy) Generated by Transmethylation Pathway and Degraded via Transsulfuration Pathway

Homocysteine (Hcy) is a nonproteogenic sulfur containing amino acid (SAA)

  • generated by the intrahepatic transmethylation (TM) of dietary Met.
  • It may either be recycled to Met following remethylation (RM) pathways or
  • catabolized along the transsulfuration (TS) cascade.

Under normal circumstances, the Met-Hcy cycle stands under the regulatory control of three water soluble B-vitamins:

  • folates (5-methyl-tetrahydrofolates, B9) are regarded as the main factor working as donor of the CH3 group involved in the remethylation process,
  • pyridoxine (pyridoxal-5’-phosphate, PLP, B6) plays the role of co-factor of both
  • cystathionase enzymes belonging to the TS pathway and cobalamins (B12) ensure that of methionine-synthase.

Met-Hcy-Met Cycle

The main steps of the Met _ Hcy _Met cycle are summarized in Fig. (1).

FIGURE 1 NR H2S

Fig. (1). Schematic representation of the methionine cycle and homocysteine degradation pathways.

Compounds: ATP, adenosyltriphosphate; THF, tetrahydrofolate; SAM, S- adenosylmethionine; SAH, adenosylhomocysteine; Cysta, cystathionine; Cys, cysteine;
GSH, glutathione; H2S, hydrogen sulfide; Tau, taurine; SO4-2 , sulfate oxyanions.
Enzymes: (1) Met-adenosyltransferase; (2) SAM-methyltransferases; (3) adenosyl-homocysteinase; (4) methylene-THF reductase; (5) Metsynthase; (6) cystathionine
-b-synthase
, CbS;  (7) cystathionine-b-lyase, CbL; (8) g-glutamyl-synthase; (9) g-glutamyl-transpeptidase; (10)oxidase; (11) reductase; (12) cysteine-dioxygenase, CDO.

Metabolic pathways

Met molecules supplied by dietary proteins are

  • submitted to TM processes
  • releasing Hcy which may in turn either
    • undergo Hcy_Met RM pathways or be
    • irreversibly committed into TS decay.

Impairment of CbS activity in protein malnutrition, entails

  • supranormal accumulation of Hcy in body fluids,
  • stimulation of (5) activity and maintenance of Met homeostasis.

This last beneficial effect is counteracted by

  • decreased concentration of most components generated downstream to CbS,
  • explaining the depressed CbS- and CbL-mediated enzymatic production of *H2S along the TS cascade.

The restricted dietary intake of elemental S is a limiting factor for

  • its non-enzymatic reduction to **H2S which contributes to
  • downsizing a common body pool (dotted circle).(Fig 1)

Combined protein- and S-deficiencies work in concert

  • to deplete Cys, GSH and H2S from their body reserves,
  • impeding these reducing molecules from countering
  • the oxidative stress imposed by hyperhomocysteinemia.

Hyperhomocysteinemia

Hyperhomocysteinemia (HHcy) is an acquired metabolic anomaly first identified by McCully [1]

The current consensus is that dietary deficiency in any of
three water soluble vitamins may operate as causal factor of HHcy.

  • PLP–deficiency may trigger the upstream accumulation of Hcy in biological fluids [2] whereas
  • the shortage of vitamins B9 or B12 is held responsible for its downstream sequestration [3,4].

HHcy is regarded as a major causal determinant of CVD

  1. working as an independent and graded risk factor
  2. unrelated to the classical Framingham criteria such as
  • hypercholesterolemia,
  • dyslipidemia,
  • sedentary lifestyle,
  • diabetes and
  • smoking.

Hcy may invade the intracellular space of many tissues and locally generate [5]

  • endothelial dysfunction working as early harbinger of blood vessel injuries and atherosclerosis.

Most investigators contend

  • that production of harmful reactive oxygen and nitrogen species (ROS, NOS), notably
    • hydrogen peroxide (H2O2), superoxide anion (O2 .-) and peroxinitrite (ONOO.-),
    • constitutes a major culprit in the development of HHcy-induced vascular damages [7-10].

Accumulation of ROS
associated with increased risk for

  • cardiovascular diseases [11]
  • stroke [12],
  • arterial hypertension [6],
  • kidney dysfunction [13],
  • Alzheimer’s disease [14],
  • cognitive deterioration [15],
  • inflammatory bowel disease [16] and
  • bone remodeling [17].

These effects overlook the protective roles played by

  • extra- and intracellular reductants such as cysteine (Cys) and glutathione (GSH)
    • in the sequence of events leading from HHcy to tissue damage.

Hydrogen Sulfide (H2S)

After the discovery of nitric oxide (NO) and carbon oxide (CO), hydrogen sulfide (H2S) is the

  • third gaseous signaling messenger found in mammalian tissues [18].

H2S is a reducing molecule displaying strong scavenging properties

  • as the gasotransmitter significantly attenuates [19, 20] or
  • even abolishes [21,22] the oxidative injury imposed by HHcy burden.

The endogenous production of the naturally occurring H2S reductant depends on

  • Cys bioavailability through
  • the mediation of TS enzymes [23,24].

H2S may also be produced in human tissues starting from elemental sulfur,

  • by a non-enzymatic reaction requiring the presence of Cys, GSH, and glucose [25,26].

It would be worth disentangling the respective roles played by

  1. Cys,
  2. GSH
  3. H2S
  • for the prevention and restoration of HHcy-induced oxidative lesions.
  •  but the plasma concentration of Cys and GSH is severely depressed in
  • subclinically malnourished HHcy patients [27],
    •  impeding appropriate biogenesis of H2S molecules.

The present paper reviews the biological consequences

  • resulting from the complex interplay existing between the 3 reducing molecules,
  • to gain insight into the pathophysiologic mechanisms associated with HHcy states.

CLINICAL BACKGROUND

Numerous surveys have conclusively shown that the water soluble vitamin deficiency concept,

  • provides only partial causal account of the HHcy metabolic anomaly.

The components of body composition, mainly

  • the size of lean body mass (LBM),
  • constitutes a critical determinant of HHcy status [28,29].

Because nitrogen (N) and sulfur (S) concentrations

  • maintain tightly correlated ratios in tissues, we hypothesize 
  • defective N intake and accretion rate would cause concomitant and
  • proportionate depletion of total body N (TBN) and total body S (TBS) stores [30].

Our clinical investigation undertaken in Central Africa in apparently healthy but

  • nevertheless subclinically malnourished vegetarian subjects has
  • documented that reduced size of LBM could lead to HHcy states [27].

The field study conducted in the Republic of Chad, populated by the Sara ethnic group [27], is a  semi-arid region and

  • the staple food consists mainly of cassava, sweet potatoes, beans, millets and groundnuts.

Participants were invited to fill in a detailed dietary questionnaire whose results were compared with values reported in food composition tables [32-34] [27].
The dietary inquiry indicates that participants

  • consumed a significantly lower mean SAA intake (10.4 mg.kg-1.d-1)[27]
  • than the Recommended Dietary Allowances (RDAs) (13 mg.kg-1.d-1)[33,34].

Blood Analytes

The blood lipid profiles of rural subjects were confined within normal ranges

  • ruling out this class of parameters as causal risk factors for CVD disorders.

The normal levels measured for pyridoxine, folates, and cobalamins

  •  precluded these vitamins from playing any significant role in the rise of Hcy

plasma concentrations [27]. Analysis of plasma SAAs revealed

  • unmodified methioninemia, significantly 
  • elevated Hcy values (18.6 umol/L)
  • contrasting with significantly decreased plasma Cys and GSH values [27].

The significant lowering of classical

  • anthropometric parameters
    •  (body weight, BW;
    • body mass index, BMI)
  • together with that of the main plasma and urinary biomarkers of
    • metabolic (visceral) and
    • structural (muscular) compartments point to

an estimated 10 % shrinking of LBM [27].

Transthyretin (TTR)  and Lean Body Mass (LBM)

We have attached peculiar importance to the measurement of plasma transthyretin (TTR)

  1. this indicator integrates the evolutionary trends outlined by body protein reserves [35],
  2. providing from birth until death an overall and balanced estimate of LBM fluctuations [29].
  • In the absence of any superimposed inflammatory condition,
    • LBM and TTR profiles indeed reveal striking similarities [29].

Scientists belonging to the Foundation for Blood Research (Scarborough, Maine, 04074, USA) have recently published a large number of TTR results recorded
in 68,720 healthy US citizens aged 0-100 yr which constitute a comprehensive reference material to follow the shape of LBM fluctuations in relation with sex and age [29].

  • TTR concentrations plotted against Hcy values reveal a strongly negative correlation (r = –0.71)  [29,30], confirming that
      • the depletion of TBN and TBS stores plays a predominant role in the development of HHcy states.

The body of a reference man weighing 70 kg contains 64 M of N (1,800 g) and 4,400 mM of S (140 g) [36]. Our vegetarian subjects consume diets providing
low fat and high fiber content conferring a large spectrum of well described health benefits notably for the prevention of several chronic disorders such as
cancer and diabetes, together with an effective protection against the risk of hypercholesterolemia-induced CVD [37,38].
Plant-based regimens, however, do not supply appropriate amounts of

  • nitrogenous substrates of good biological value which are required to adequately fulfill mammalian tissue needs [30].
  • vegetable items contain suboptimal concentrations of both SAAs [33,34,39] below the customary RDA guidelines.

This dietary handicap may be further deteriorated by

  • unsuitable food processing [40] and by
  • the presence in plant products of naturally occurring anti-nutritional factors
    • such as tannins in cereal grains and
    • anti-trypsin or anti-chymotrypsin inhibitors in soybeans and kidney beans [41].

LBM loss

LBM shrinking may be the result of either

  • dysmaturation of body protein tissues as an effect of protracted dietary SAA deprivation
  • or of cytokine-induced depletion of body stores.

Although causally unrelated and evolving along dissimilar adaptive processes,

  • both physiopathologic entities lead to comparable LBM downsizing best
    • identified by declining plasma TTR ( measured alone or within combined formulas )
    • and subsequently rising Hcy values.

All parameters are downregulated with the sole exception of RM flux rates, indicating that

  • maintenance of Met homeostasis remains a high metabolic priority in protein-depleted states.

Stressful disorders are characterized by

  • overstimulation of all
  1. TM
  2. RM
  3. TS flux rates.

The severity and duration of initial impact determine the magnitude of protein tissue breakdown,

  • rendering an account of N : S urinary losses,
  • fluctuations of albuminuria and of
  • insulin resistance striving to contain LBM integrity.

Both physiopathologic entities are compromized in reducing the oxidative burden imposed by HHcy states owing to

  • defective synthesis and/or
  • enhanced overconsumption of Cys-GSH-H2S reducing molecules,
  • a condition still worsened by its co-existence with elemental S-deficiency.

IMPAIRMENT OF THE TRANSSULFURATION PATHWAY

The hypothesis that subclinical protein malnutrition might be involved in the occurrence of HHcy states via inhibition of cystathionine-b-synthase (CbS) activity
first arose in Senegal in 1986 [42] and was later corroborated in Central Africa [43]. The concept was clearly counterintuitive in that it was unexpected that

  • high Hcy plasma values might result from low intake of its precursor Met molecule.

Despite the low SAA intake of our vegetarian patients [27], plasma Met concentrations disclosed noticeable stability permitting

  • maintenance of the synthesis and functioning of myriads of Met dependent molecular, structural and metabolic compounds

These clinical investigations have received strong support from recent mouse [45] and rat [46] experiments submitted to Met-restricted regimens.
At the end of the Met-deprivation period, both animal species did manifest meaningful HHcy states (p<0.001) contrasting with

  • significantly lower BW (p<0.001) reduced by 33 % [45] and 44 % [46] of control, respectively.
  • the uniqueness of Met behavior stands in accordance with balance studies performed on large mammalian species showing
  • that the complete withdrawal of Met from otherwise normal diets causes the greatest rate of body loss,
    • nearly equal to that generated by protein-free regimens [47,48].

This efficient Met homeostatic mechanism is classically ascribed to a PLP-like inhibition of CbS activity exerted through

  • allosteric binding of S-adenosylmethionine (SAM) to the C-terminal regulatory domain of the enzyme [49,50].

The loss of CbS activity may develop via a (post)translational defect

  • independently from intrahepatic SAM concentrations [45].

We have postulated the existence of an independent sensor mechanism set in motion by TBS pool shrinkage and

  • reduced bioavailability of Met – its main building block – working as an inhibitory feedback loop of CbS activity [30].

Such Met-bodystat, likely to be centrally mediated, is to maintain unaltered Met disposal in conditions of

  • decreased dietary provision implies the fulfillment
  • of high metabolic priorities of survival value [30,44].

Whereas HHcy may be regarded as the dark side of a beneficial adaptive machinery [43],

  • impairment of the TS pathway also depresses the production of compounds situated downstream to the CbS blockade level,
  • notably Cys and GSH, keeping in mind that Cys may undergo reversible GSH conversion (Fig. 1).

The plasma concentration of both Cys and GSH reductants is indeed significantly decreased in our vegetarian subjects

  • by 33 % and 67 % of control, displaying negative correlations (r = –0.67 and –0.37, respectively) with HHcy values [27].

Reduced dietary intake of the preformed Cys molecule [27] and diminished Cys release from protein breakdown in malnourished states [51]

  • may contribute to the lowering effect.

The significantly decreased GSH blood levels may similarly be attributed to dietary composition since the tripeptide is mainly found in meat products

  • but is virtually absent from cereals, roots, milk and dairy items [52] and
  • because regimens lacking SAAs may lessen the production of blood GSH and its intrahepatic sequestration [53].

BIOGENESIS OF HYDROGEN SULFIDE

The TS degradation pathway schematically proceeds along two main PLP-dependent enzymatic reactions working in succession (Fig. 1).

  • The first is catalyzed by CbS (EC 4.2.1.22) governing the replacement of the hydroxyl group of serine with Hcy to generate Cysta plus H2O.
    • Cys may however substitute for serine and the replacement of its sulfhydryl group with Hcy releases Cysta and H2S instead of water [54].
  • The second is regulated by cystathionine-g-lyase (CgL, EC 4.4.1.1.) hydrolyzing Cysta to release Cys and alpha-ketobutyrate plus ammonia as side-products [55].
    •  Cys may also undergo nonoxidative desulfuration pathways leading to H2S or sulfanesulfur production [56] under the control of CbS or CgL enzymes.
    •  Cys may otherwise undergo oxidative conversion regulated by cysteine-dioxygenase (CDO, EC 1.13.11.20) which
      • catalyzes the replacement of the SH- group of Cys by SO3 – to yield cysteine-sulfinate [56].

This last compound may be further decarboxylated to hypotaurine that is finally oxidized to Tau (67 %) and SO4 2- oxyanions (33 %) [56]. CbS and CgL,  both cytosolic enzymes,

  • their relative contribution to the generation of H2S may vary according to
    • animal strains,
    • tissue specificities and
    • nutritional or physiopathological circumstances [23,24].

CbS and CgL are expressed in most organs such as liver, kidneys, brain, heart, large vessels, ileum and pancreas [57,58] potentially

  • subjected to HHcy-induced ROS injury while keeping the capacity to desulfurate Cys and to
  • locally produce H2S as cytoprotectant signaling agent.

CbS is the principal TS enzyme found in

  • cerebral glial cells and astrocytes [59].

CgL predominates in the

  • vascular system [60] whereas
      • both enzymes are present in the renal proximal tubules [61].

H2S is the third gaseous substrate found in the biosphere [18] after NO and CO. All three gases are characterized by

  • severe toxicity when inhaled at high concentrations.

In particular, H2S produced by anaerobic fermentation is

  • capable of causing respiratory death by
  • inhibition of mitochondrial cytochrome C oxidase [62].

NO, CO and H2S are synthesized from arginine, glycine and Cys, respectively, exerting at low concentrations major biological functions in living organisms.
Most of our knowledge on these atypical signal messengers [63] are derived from animal experiments and tissue cultures. These transmitter molecules may

  • share some properties in common such as penetration of cellular membranes independently from specific receptors [64].

They are also manifesting dissimilar activities: whereas NO and CO activate guanylyl cyclase to generate biological responses via cGMP-dependent kinases,

  • H2S induces Ca2+-dependent effects through ATP-sensitive K+ channels [65].

Some of these potentialities may work in concert while others operate antagonistically. For instance,

  • NO and H2S express vasorelaxant tone on endogenous smooth muscle [66]
  • but reveal different effects on large artery vessels [67].

These gaseous substances maintain whole body homeostasis through complex interactions and multifaceted crosstalks between signaling pathways.
Elemental S (32.064 as atomic mass) is a primordial constituent of lava flows in areas of volcanic or sedimental origin usually presenting as crown-shaped
stable octamolecules – hence its S8 symbolic denomination – which may conglomerate to form brimstone rocks. The vegetable kingdom is

  • unable to assimilate S8 and requires as prior step its natural or bacterial oxidation to SO4 2- derivatives before launching
  • the synthesis of SAA molecules along narrowly regulated metabolic pathways [30,44].

Distinct anabolic processes are identified in mammalian tissues which lack the enzymatic equipment required to organize sulfate oxyanions

  • but possess the capacity of direct S8 conversion into H2S.

S8 is poorly soluble in tap waters [68] may be taken up and transported to mammalian tissues loosely fastened to serum albumin (SA) [69].
S may also be covalently bound to intracellular S-atoms taking the form of sulfane-sulfur compounds [70] either

  • firmly attached to cytosolic organelles or in
  • untied form to mitochondria [57,58,71,72] to undergo
  • later release in response to specific endogenous requirements [71].

Sulfane-sulfur compounds are somewhat unstable and may decompose in the presence of reducing agents allowing the restitution of S [70,71].
S may either endorse the role of stimulatory factor of several mammalian apoenzyme activities as shown for

    • succinic dehydrogenase [73] and NADH dehydrogenase [74] or
  • operate as inhibitory agent of other mammalian apoenzymes such as
    • adenylate kinase [75] and liver tyrosine aminotransferase [76].

Elemental S resulting from dietary supply or from sulfane-sulfur decay may be subjected to

non-enzymatic reduction in the presence of Cys and GSH [25,26] and/or reducing equivalents obtained from

  • glucose oxidation [25], hence yielding at physiological pH additional provision of H2S.

The gaseous mediator is a weakly acidic molecule endowed with strong lipophilic affinities. In experimental models, the blockade of the TS cascade

  • at CbS or CgL levels significantly depresses or even
  • abolishes the vitally required production of Cys
  • operating at the crossroad of multiple converting processes (Fig. 1).

Addition of Cys to the incubation milieu

  • resumes the generation of H2S [19] in a Cys concentration-dependent manner [77].

The compounds situated downstream both cystathionases in the context of SAA deprivation

  • keep their functional potentialities
  • but are unable to express their converting Cys – H2S capacities
    • in the absence of precursor substrate.

Summing up

inhibition of CbS activity contributes to

  • promote efficient RM processes and
  • maintenance of Met homeostasis

but entails as side-effects

  1. upstream sequestration of Hcy molecules in biological fluids
  2. while decreasing the bioavailability of Cys and GSH
    • working as limiting factors for H2S production.

These last adverse effects thus constitute the Achilles heel of a remarkable adaptive machinery.

ROLES PLAYED BY HYDROGEN SULFIDE

The first demonstration that human tissues may reduce S to H2S was incidentally provided in 1924 when a man given colloid sulfur

  • for the treatment of polyarthritis did rapidly exhale the typical rotten egg malodor [78].
  • H2S may be produced by the intestinal flora [79] and serves as a metabolic fuel for colonocytes [80].
  • Prevention of endogenous poisoning by excessive enteral production is insured by the detoxifying activities of mucosal cells [81],
    • hindering any systemic effect of the gaseous substrate.

The normal H2S concentration measured in mammalian plasmas usually ranges from 10 to 100 μM with a mean average turning around 40-50 μM [19,21,82,83].
This H2S plasma level, appearing as the net product of organs possessing CbS and CgL enzymes and supplemented by the non-enzymatic conversion of S,

  • flows transiently into the vasculature and freely penetrates into all body cells.
  • Supposing that the gaseous reductant is evenly distributed in total body water (45 L in a 70 kg reference man) allows an estimate of
    • bioavailable H2S pool turning around 2 mM which represents, in terms of S participation, largely less than 1 / 1,000 of TBS.

The peculiar adaptive physiology of vegetarian subjects renders very unlikely that their TBS pool might be solicited to release

  • S-substrates prone to undergo conversion to nascent H2S molecules since
  •  they adapt to declining energy and nutrient intakes
  • by switching overall body economy toward downregulated steady state activities.

The release from TBS of substantial amounts of S-compounds occurs

  • only during the onset of hypercatabolic states as documented in trauma patients [31]
  • and in infectious diseases [84], exacting as preliminary step
  • cytokine-induced breakdown of tissue proteins, a selective hallmark of stressful disorders [85].

H2S in fulfilling ROS Scavenger Tasks

The limited disposal of H2S endogenously produced might be readily exhausted in fulfilling ROS scavenging tasks at the site of oxidative lesions.
All body organs generating H2S from TS enzymes are

  • simultaneously producers and consumers of the gaseous substrate whose actual concentration
  • reflects the balance between synthetic and catabolic rates [86].

Clinical investigations show that H2S concentrations found in cerebral homogenates from Alzheimer’s disease (AD) patients are

  • very much lower than expected from values measured in healthy brains [87], suggesting that
  • the gaseous messenger is locally submitted to enhanced consumption rates reflecting disease severity.

The concept is strongly supported by studies pointing to the

  • negative correlation linking the severity of AD to H2S plasma values [88].
  • in pediatric [89] and elderly [90] hypertensive patients as well
  • more severe HHcy-dependent oxidative burden is
    • associated with more intense H2S uptake rates.
  • These H2S cleansing properties are mainly exerted by mitochondrial organelles
    • known to be centrally involved in oxidative disorders [20,91].

Malnourished subjects deprived of Cys and GSH disposal thus incur the risk of H2S-deficiency

  • rendering them unable to properly overcome HHcy-imposed oxidative lesions.

The rapid exhaustion of H2S stores have detrimental consequences as shown disclosing

  • the beneficial effects of exogenous administration of commonly used sulfide salt donors (Na2S and NaHS)
  • generating H2S gas once in solution.

Such supply significantly augments

  • H2S plasma concentrations allowing to counteract ROS damages. 

H2S was primarily recognized as a physiological substrate working as

  • neuromodulator [92] and soon later as
  • vasorelaxant factor [65].

H2S is now regarded as endowed with a broader spectrum of biological properties [18],

  • operating as a general protective mediator
    • against most degenerative organ injuries,
  • being capable of neutralizing or
  • abolishing most ROS harmful effects.

Table 1 collects findings displaying that H2S may promote the synthesis and activity of several

  • anti-oxidative enzymes (catalases, Cu- and Mn-superoxide dismutases, GSH-peroxidases) and
  • stimulate the production of anti-inflammatory reactants (interleukin-10) or
  • conversely downregulate
    • pro-oxidative enzymes (collagenases, elastases),
    • pro-inflammatory cytokines (interleukine-1b, tumor-necrosis factor a) and
    • immune reactions (hyperleukocytosis, diapedesis, phagocytosis).

It has been calculated that 81.5% of H2S undergoes catabolic disintegration in the form of hydrosulfide anion (HS-) or sulfide anion (S2-) [117].
Since S is the main element in the diprotonated H2S molecule (34.08 as molecular mass), it may be considered that

  • partial or complete repair of HHcy-induced lesions constitutes the therapeutic proof that
  • S-deficiency is causally involved in the development of ROS damages.

The concept is sustained by the observation that all synthetic drugs (diclofenac, indomethacine, sildenafil) utilized as surrogate providers of H2S [64,118] are

  • characterized by a large diversity of molecular conformations but
  • share in common the presence of Satom(s) mimicking, once released,
  • H2S-like pharmacological properties.

It remains to be clarified whether the beneficial effects of S-fortification to S-deficient subjects are mediated, among other possible mechanisms, via

  • stimulation [73,74] of anti-oxidative enzymes or inhibition [75,76] of pro-oxidative enzymes.

It is only very recently that the essentiality of S has been recognized, causing Hcy elevation in deficient individuals [119]. It is worth reminding that the

  • gaseous NO substrate may work in concert or antagonistically [66,83] to fine-tuning the helpful properties exerted by H2S on body tissues.

Preliminary studies suggest for instance that NO operates, in combination with H2S, as a potential modulator of endothelial remodeling since

  •  NO-synthase isoforms contribute to the activation of  metalloproteinases involved in the regulation of the collagen/elastin balance defining vascular elastance [83,120].

SUBCLINICAL MALNUTRITION AS WORLDWIDE  SCOURGE

A growing body of data collected along the last decades indicates that

large proportions of mankind still suffer varying degrees of protein and energy deficiency that is associated with

  • increased morbidity and mortality rates.

The determinants of malnutrition are complex and interrelated, comprising

  • socioeconomic and political conditions,
  • insufficient dietary intakes,
  • inadequate caring practices and
  • superimposed inflammatory burden.

Children living in developing countries are paying a heavy toll to chronic malnutrition [121,122] whereas adult populations are handicapped by

  • feeble physical and working capacities,
  • increased vulnerability to infectious complications and
  • reduced life expectancy [123,124].

Cross-sectional studies collected in the eighties indicate that chronic malnutrition remains a worldwide scourge with

  • top prevalence recorded in Asia, whereas
  • sub-Saharan Africa endures medium nutritional distress and
  • Latin America appears as the least affected [125,126].

Along the last decades, significant progresses have been achieved in some countries such as Vietnam [127] and Bangladesh [128]

  • owing to appropriate education programs and improved economic development.

Inequalities however persist between middle class population groups mainly located in affluent urban areas and

  • underprivileged rural communities remaining stagnant on the sidelines of household income growth.

Representative models of these socio-economic disparities in global nutrition and health are illustrated in the two most populated countries in the world, China and India.
Large surveys undertaken in 105 counties of China and recently published have concluded that the rural communities haven’t yet reached the stage of overall welfare [129].
In India, similar investigations have documented that extreme poverty still prevails in the northern mountainous states of the subcontinent [130]. Taken together, southern
Asian countries fail to overcome malnutrition burden [131]. In some African countries, there exists even upward trends suggesting nutritional

deterioration over the years [132] still aggravated by a severe drought. The assessment of malnutrition in children usually rely on anthropometric criteria such as height-for-age, weight for-height, mid upper arm circumference and skinfold thickness allowing to draw the degree of stunting and wasting from these estimates. In adult subjects, BW and BMI are currently selected parameters to which some biochemical measurements are frequently added, notably SA, classical marker of protein nutritional status, and creatininuria (u-Cr), held as indicator of sarcopenia. The former biometric approaches are very useful in that they correctly provide a static picture of the declared stages of malnutrition but fail to recognize the dynamic mechanisms occurring during the preceding months and the adaptive alterations running behind.

Table 1. Reversal of HHcy-Induced Oxidative Damages by Administration of Exogenous H2S

BRAIN EFFECTS

H2S is overproduced in response to neuronal excitation [93], and

  1. increases the sensitivity of N-methyl-D-aspartate (NMDA) reactions to glutamate in hippocampal neurons [23,94].
  2.  improves long-term potentiation, a synaptic model of memory [92,93]
  3. stimulates the inhibitory effects of catalase and superoxide dismutase (SOD) in oxidative stress of endothelial cells [95].
  4.  regulates Ca 2+ homeostasis in microglial cells [96]and it inhibits TNFa expression in microglial cultures [97].
  5.  protects brain cells from neurotoxicity by preventing the rise of ROS in mitochondria [98].

CARDIOVASCULAR EFFECTS

  1. H2S releases vascular smooth muscle,
  2. inhibits platelet aggregation and
  3. reduces the force output of the left ventricule of the heart [18].
  4. maintains vascular smooth muscle tone [66] and
  5. insures protection against arterial hypertension [99].
  6. modifies leucocyte-vascular epithelium interactions in vivo  by
    1. modulating leucocyte adhesion and
    2. diapedesis at the site of inflammation [100].
  7. attenuates myocardial ischemia-reperfusion injury by
    1. depressing IL-1b and mitochondrial function [20].
  8. upregulates the expression of depressed anti-oxidative enzymes in heart infarction and
    1. inhibits myocardial injury [21].
  9. alleviates smooth muscle pain by
    1. stimulating K+ ATP channels [101].
  10. prevents apoptosis of human neutrophil cells
    1. by inhibiting p38 MAP kinase and caspase 3 [102].
  11. potentiates angiogenesis and wound healing [103].

RENAL EFFECTS

  1. H2S downregulates the increased activity of metalloproteinases 2 and 9 involved in extracellular matrix degradation (elastases, collagenases) [19].
  2. Prevents apoptotic cell death in renal cortical tissues [19].
  3. Improves the expression of desmin (marker of podocyte injury) and
  4. restores the drop of nephrin (component of normal slit diaphragm) in the cortical tissues
    1. resulting in reduced proteinuria [19].
  5. Induces hypometabolism revealing protective effects on renal function and survival [104].
  6. Normalizes GSH status and production of ROS in renal diseases [19].
  7. Controls renal ischemia-reperfusion injury and dysfunction [105].
  8. Depresses the expression of inflammatory molecules involved in glomerulosclerosis [106].
  9. Increases renal blood flow, glomerular filtration and urinary Na+ excretion [77].

OTHER ORGAN EFFECTS
Gastrointestinal

  1. H2S insures protection against ROS stress in gastric mucosal epithelia [22].
  2. Accelerates gastric ulcer healing [107].
  3. Reduces gastric injury caused by nonsteroidal anti-inflammatory drugs [108].
  4. Relaxes ileal smooth muscle tone and increases colonic secretions [79].
  5. Attenuates intestinal ischemia-reperfusion injury by increasing SOD and GSH peroxidase status [109].
  6. Stimulates insulin secretion [110] and controls inflammatory events associated with acute pancreatitis [111].
  7. Alleviates hepatic ischemia-reperfusion injury [112].

Pulmonary

  1. Prevents lung oxidative stress in hypoxic pulmonary hypertension caused by low GSH content [113].
  2. Promotes SOD and catalase activities and reduces the production of malondialdehyde in oxidative lung injury [114].
  3. Reduces lung inflammation and remodeling in asthmatic animals [115] and in pulmonary hypertension [116].  ..(see OCCJ 2011;4:34-44)

Assessing Protein-Depleted States

  1.  SA is an insensitive marker of protein-depleted states compared to TTR [134]
  2. SA is an indicator of population than of individual protein status in subclinical PEM.
  3. u-Cr is likewise a meagerly informative tool as 10 % loss of muscle mass is required before it reaches significantly decreased urinary concentrations [135].

The data imply that the magnitude of subclinical malnutrition is largely

  • underscored when classical biometric and laboratory investigations are performed.

Moreover, ruling out the protein component involved in HHcy epidemiology and confining solely attention to the B-vitamin triad led to unachieved conclusions.

  • surveys undertaken in Taiwan [136] and in India [137] established HHcy variance turning around 30 %, indicating that
  • a sizeable percentage of subjects do not come within the vitamin shortage concept.
  • only one recent review recommending the use of TTR in vegetarian subjects [138].

The main reason for making the choice of TTR is grounded on the striking similar plasma profile disclosed by this marker with both LBM and Hcy [29].
Under healthy conditions, the 3 parameters –

  • TTR,
  • LBM,
  • Hcy –
    • indeed show low  concentrations at birth,
    • linear increase without sexual difference in preadolescent children,
    • gender dimorphism in teenagers with higher values recorded in adolescent male subjects
    • thereafter maintenance of distinct plateau levels during adulthood [29,139,140].

Under morbid circumstances, the plasma concentrations of

  • Hcy manifest gradual elevation
  • negatively correlated with LBM downsizing and
  • TTR decline.

In vegetarian subjects and subclinically malnourished patients,

  • rising Hcy and
  • diminished TTR plasma concentrations look as mirror image of each other,
    • revealing divergent distortion from normal and
    • allowing early detection of preclinical steps
    • at the very same time both SA and u-Cr markers still remain silent.

Any disease process characterized by quantitative or qualitative dietary protein restriction or intestinal malabsorption

  • may cause LBM shrinking,
  • downregulation of TTR concentrations and
  • subsequent HHcy upsurge.

These conditions are documented in frank kwashiorkor [141], subclinical protein restriction [27,43] and anorexia nervosa [142].
In patients submitted to weight-reducing programs,

  • LBM was found the sole independent variable
  • negatively correlated with rising Hcy values [143].

Morbid obesity may be alleviated by medical treatment [143] or surgical gastroplasty [144,145],

  • conditions frequently associated with secondary malabsorptive syndromes and malnutrition [146],

How does this account play out in the typical patient with excessive body fat, lipoprotein disoreder, and perhaps diabetes and disordered sleep – an account of acquired HHcy?
Have the studies been done?  Would you expect to see a clear benefit from reduced HHcy_emia  based on a 30 min daily walk, and

  • eating of well fat trimmed meats, fruits and vegetables, and fish, flax seed, or krill oil?

In westernized countries, subclinical protein-depleted states are illustrated in immigrants originating from

  • developing regions but keeping alive their traditional feeding practices [147] or
  • by communities having adopted, for socio-cultural reasons, strict vegan dietary lifestyles [148].

THE ADDITIONAL BURDEN OF S-DEFICIENCY

After N, K and P, elemental S is recognized as the fourth most important macronutrient required for plant development. The essentiality of S in the vegetable kingdom
arose from observations made many decades ago by pedologists and agronomists [149,150] revealing that the withdrawal of sulfate salts from nutrient sources produces
rapid growth retardation,

  • depressed chlorophyllous synthesis,
  • yellowing of leaves and
  • reduction in fertility and crop yields.

A large number of field studies, mainly initiated for economical reasons, has provided continuing gain in fundamental and applied knowledge and led to the overall consensus

  • that SO4 2- -deficiency is a major wordwide problem [151,152].

Field investigations have shown that the concentration of SO4 2- oxyanions in soils and drinking waters

  • may reveal considerable variations ranging from less than 2 mg/L to more than 1 g/L,
  • meaning a ratio exceeding 1 / 500 under extreme circumstances [30].

The main causal factors responsible for unequal distribution of SO4 2- oxyanions are geographical distance from eruptive sites and

  • intensity of soil weathering in rainy countries.

SO4 2- -dependent nutritional deficiencies entail detrimental effects to most African and Latin American crops [151]

  • reaching nevertheless top incidence in southeastern Asia [151,153].
  • and the Indo-Gangetic plain extending from Pakistan to Bangladesh and covering the North of India and Nepal [154].
  1. Intensive agricultural production,
  2. lack of animal manure and
  3. use of fertilizers providing N, K and P substrates
  4. but devoid of sulfate salts may further aggravate that imbalanced situation.

As global population increases steadily and the production of staple plants predicted to escalate considerably,

  • SO4 2- deficient disorders are expected to become more pregnant along the coming years [155] with significant harmful impact for mankind.

Nevertheless, effective preventive efforts are developed in some countries aiming at fortification of soils mainly

  • by ammonium sulfate or calcium sulfate (gypsum) salts,
  • resulting in meaningful improvements in crop yield,
  • SAAs content and biological value and
  • opening more optimistic perspectives for livestock and human consumption [152,155-158].

Contrasting with the tremendously high amounts of data accumulated over decades by pedologists and agronomists on sulfate requirements and metabolism,
the available knowledge on elemental sulfur in human nutrition looks like a black hole. Despite the fact that S8 follows H, C, O, N, Ca and P as the seven most
abundant element in mammalian tissues, it appears as a forgotten item. Not the slightest attention is dedicated to S8 in the authoritative “Present Knowledge
in Nutrition” series of monographs even though they go over most oligo- and trace-elements in minute detail.

The geographical distribution of S8 throughout the earth’s crust is not well-known

  • as extreme paucity of measurements in soils and tap waters prevents reaching a comprehensive overview.

Nevertheless, and because S8 is the obligatory precursor substrate for the oxidative production of sulfate salts,

a decremental dispersion pattern paralleling those of SO4 2- oxyanions is likely to occur with

  • highest values recorded in the vicinity of volcano sources
  •  and lowest values found in remote and washed-out areas.

Obviously, a great deal of research on elemental S remains to be completed by clinical biochemists before rejoining the status of plant agronomy.
Taken together, these data imply that subclinically malnourished subjects living in areas recognized as

  • SO4 2- -deficient for the vegetable kingdom also
  • incur increased risks to become S8-depleted.

This clinical entity most probably prevails in all regions, notably Northern India, where protein malnutrition [130] and sulfur-deficiency [154] coexist.
Combination of both nutritional deprivations explains why the bulk of local dwellers, including young subjects [159,160], may develop HHcy states and CVD disorders

  • characterized by strong refractoriness to vitamin-B supplementation [160] or
  • high incidence of stroke [161] unrelated to the classical Framingham criteria.

The current consensus is that “the problem of CVD in South Asia is different in etiology and magnitude from other parts of the world” [162]. These disquieting findings are
confirmed in several Asian countries [163] and have prompted local cardiologists to exhort their governments to focus more attention on CVD epidemiology [164].

CONCLUDING REMARKS

  1.  vegetarian subjects are not protected against the risk of CVD and stroke which should no longer be regarded as solely affecting populations living in westernized societies
  • whose morbidity and mortality risks are stratified by classical Framingham criteria.
  • Likewise hypercholesterolemia, hyperhomocysteinemia should be incriminated as
    • emblematic risk factor for a panoply of CVD and related disorders.
  • Whereas the causality of cholesterol and lipid fractions largely prevails in affluent societies consuming high amounts of animal-based items,
    • that of homocysteine predominates in population groups whose dietary lifestyle gives more importance to plant products.

 MAIN PHYSICO-CHEMICAL AND METABOLIC CHARACTERISTICS* OF 3 CARRIER-PROTEINS INVOLVED IN THE STRESS RESPONSE

CBG

TTR

RBP

Molecular mass (Da.)

42,650

54,980

21,200

Conformation

monomeric

tetrameric

monomeric

Amino acid sequence

383

4 x 127

182

Carbohydrate load

18 % glycosylated

unglycosylated

unglycosylated

Hormonal binding sites

one for cortisol

two for TH

one for retinol

Association constant (M-1)

3 x 107

7 x 107 (T4)

1.9 x 107

Normal plasma concentration

30 mg/L.

300 mg/L.

50 mg/L.

Biological half-life

5 days

2 days

14 hrs

Bound ligand  concentration

120 µg/L.

80 µg TT4/L.

500 µg/L.

Free ligand concentration

5 µg/L.

20 ng FT4/L.

1 µg/L.

Ratio free : bound ligands

4 %

0.034 %

0.14 %

Distribution volume of free moieties

18 L.

12 L.

18 L.

STIMULATORY AND INHIBITORY EFFECTS MODULATED

BY GLUCOCORTICOIDS

TARGET SYSTEMS

 

INDUCED EFFECTS

REF.

Thymidine kinase

_

transcription of induced DNA into RNA

112

Alkaline phosphodiesterase I

_

cleavage of phosphodiester bonds

113

Tyrosine transaminase

_

transfer of tyrosine amino group

114

Tryptophane oxygenase

_

formylkynurenine and Trp catabolites

115

Alkaline phosphatase

_

release of P from phosphoric esters

116

Phosphoenolpyruvate carboxykinase (liver)

_

glycolysis from pyruvate and ATP production

117

Mannolsyltransferases

_

dolichol-linked glycosylation of APRs

118

Haptoglobin

_

APR combining with hemoglobin

119

α1-Anti (chymo) trypsin (α1 AT, α1 ACT)

_

serpin molecules allowing N-sparing effects

120

α1-Acid glycoprotein (AGP)

_

glycosylated APR with antibody-like actions

121

Serum amyloid protein (SAA)

_

defense systems against oxidative burst

122

γ-Fibrinogen

_

clotting processes and tissue repair

123

C-Reactive Protein (CRP)

_

complement processes and opsonization

124

Corticosteroid-binding globulin (CBG)

_

CBG levels, favoring free hypercortisolemia

100

Phosphoenolpyruvate carboxykinase (adipocytes)

_

ATP turnover and glycolysis

113

THE DUAL MORBID ENTITIES CAUSING LBM DOWNSIZING AND SUBSEQUENT Hcy UPSURGE 

Primary causal factor

  1. Reduced dietary intake of methionine (39,151,152)
  2. Cytokine-induced tissue breakdown (164,165)

Main clinical conditions

  1. Protein malnutrition,
  2. veganism,
  3. intestinal malabsorption (139,155,156,158-160,281)
  4. Trauma,
  5. sepsis,
  6. burns,
  7. Inflammatory & neoplastic disorders (163,166,170,176,179,180)

Physiopathologic mechanisms

  1. Unachieved LBM replenishment (30,33)
  2. Excessive LBM losses (33,167,179)

Overall protein metabolic status

  1. Downregulated
  2. Upregulated

Plasma biomarker(s) of protein status

  1. Transthyretin (TTR) (144,145)
  2. TTR coupled with CRP or other inflammatory indices (31,177,178,284,285)

Insulin resistance status

  1. Normal or low (286)
  2. Increased in proportion of tissue breakdown (177,178,181-183)

status of Cys-GSH-H2S reducing molecules

  1. Decreased enzymatic and non-enzymatic production (39,161,162,287)
  2. Increased production cancelled out by tissue overconsumption (78,171)

Urinary SO42- and S-compounds

  1. Decreased kidney output (76,78,79)
  2. Variable depending on exogenous SAA supply and
  • extent of tissue breakdown (78,163,168,173)

Transmethylation pathway

  1. Depressed (48,93)
  2. Overstimulated (169)

Remethylation pathway

  1. Stimulated (76,83,153)
  2. Overstimulated (169)

Transsulfuration pathway

  1. Inhibited (49,76,83)
  2. Overstimulated (170,173)


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Received: September 30, 2011 Revised: October 12, 2011 Accepted: October 12, 2011
© Yves Ingenbleek; Licensee Bentham Open.
This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.

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Protein Malnutrition

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

A large part of  the world’s population is undernourished by the standards of Western Europe and North America. Scientists and nonscientists alike recognize as one of the major challenges of our time the problem of how to ensure that the production and distribution of food keep pace with the increasing number of mouths to be fed. In the world as a whole the most widespread and serious dietary deficiency is that of protein. This fact emerges clearly from the reports of the expert committees of WHO and FAO (World Health Organization, 1951, 1953). Nevertheless, many protein chemists, even those associated with medical research, may not realize the extent and severity of protein malnutrition, because it occurs chiefly in the technically underdeveloped countries far from where they work.

Dietary histories and response to treatment point to deficiency of total protein as the primary cause of the clinical syndrome kwashiorkor. The level of calorie intake has an important influence on the pattern of the disease. Deficiency of one or more specific amino acids, or amino acid imbalances in the diet, may perhaps be responsible for some of the symptoms and signs, particularly those whose incidence varies from one part of the world to another. All these variations on a theme are covered by the general term protein malnutrition. The onset is often precipitated by the added burden of diarrhea, infection, and parasitic infestation. The nutritional state influences the resistance to infection, and conversely the presence of an infection affects the state of nutrition. A further contributory factor may be the psychological upheaval in the child when the next baby in the family is born. At the root of all these causes lie poverty, ignorance, and disruption of the family life.

The planning of preventive measures cannot be effective unless it is based on some knowledge of the magnitude of the problem to be tackled. At a very rough estimate, in some countries perhaps 10% of the children suffer from severe protein malnutrition at some age between birth and 4 years. The marginal deficiency states must be much more common, Clinical signs and biochemical changes are of little value in diagnosing the early case; a deficit in body weight still seems to be the best criterion. Prevention ideally would be by greater production and consumption of animal protein, and by the increased use of skim milk and of surplus fish at present often wasted. However, animal protein is likely to remain scarce and expensive. Plant sources are being investigated with a view to encouraging not only domestic production, but also the production on an industrial scale of cheap foodstuffs rich in protein. A preventive program that is nutritionally sound may fail if account is not taken of local food habits, traditions, and customs. Protein requirements are affected by the quality of protein, the intake of calories, and by the state of the body (growth, the presence of disease, etc.). The maintenance requirement and the amount required for growth in children can be estimated, but the requirement for health is still unknown. For the time being, the allowances of protein recommended for people in the world as a whole are based empirically on the known physiological requirement with an arbitrarily added wide margin of safety.

The absorption of nitrogen is remarkably efficient even in severely malnourished infants. In general the nitrogen of plant proteins is less well absorbed than that of milk. When a baby receives a diet in which the protein is derived entirely from vegetabIe sources, incomplete absorption of nitrogen may play a significant part in the production of protein malnutrition. The malnourished baby who responds to treatment is able to retain and utilize nitrogen very efficiently; there is no evidence of any impairment in the mechanisms of protein synthesis. It is possible, however, that these mechanisms may be irreversibly damaged in babies who die, and that this may be the cause of death. The level of calorie intake has an important influence on the efficiency of utilization of nitrogen. An adequate calorie intake promotes conservation of nitrogen in the body as a whole when supplies of protein are short, but this protective effect may not be exerted equally in all organs. In this way the level of calorie intake may modify the pattern of protein depletion. A greater than normal calorie intake is needed for the restoration of depleted protein stores.

The discussion of protein metabolism in protein malnutrition has been purposely limited to a narrow field-to studies made on man, and to the few animal experiments that have a direct bearing on those studies. For technical reasons most of the work discussed relates to plasma proteins. There is a conflict of evidence between results obtained in man and animals about the effect of protein depletion or a low protein diet on the rate of catabolism of plasma albumin. It is of great importance to settle this point. A priori there seems no reason why the rate of protein catabolism should be affected by nutritional state. Preliminary studies with radioactive methionine in infants suggest, as working hypotheses, that in protein malnutrition there may be an increase in the reutilization of amino acids liberated by tissue catabolism, and an apparent concentration of protein synthesis in the more essential organs at the expense of the less essential. There is some experimental support for both these ideas, but further work is badly needed. The concept of protein stores or reserve protein is based entirely on dynamic and not on chemical considerations. It is suggested that the essential difference between a “labile” and a “fixed” protein is a difference in turnover rate. An attempt is made to show that the changes produced by protein depletion in the protein content of organs such as liver and muscle are a necessary consequence of the metabolic characteristics of proteins in those organs. There may be no need to invoke the help of homeostatic or compensatory regulations to explain the changes found in protein depletion.

Aging and growth are processes during which some metabolic adjustments must take place. It is believed that it may be better to regard the changes which are found in protein malnutrition in a similar light: as evidence of an alteration in functional pattern, rather than of damage or disease. Protein malnutrition in man has two aspects-a practical and a theoretical one. From the practical point of view it is an extremely common disease with a high mortality, and there is every reason to believe that it will become more common unless urgent preventive measures are taken. Theoretically it raises many questions that are of interest in relation to other branches of medicine and biochemistry. It is believed that the two aspects are linked, and that progress towards prevention is still impeded by our lack of basic knowledge as well as by our failure to apply what is already known. In protein malnutrition there is no sharp line between health and disease. The simple concept of specific deficiency diseases that grew from the discovery of vitamins is not applicable. We have to go back instead to the ideas of an earlier era, when nutrition was regarded as a branch of physiology, concerned with the functions, fate, and metabolic interrelationships of the major nutrients.

It is a characteristic of protein metabolism that nitrogen balance can be maintained at many different levels of protein intake. These different steady states are achieved by adjustments of the amount and distribution of proteins in the body as a whole, in organs, and in cells. It is believed that these changes in amount and distribution of proteins must result in alterations of metabolic pattern, with a gradation of change from an optimum, which cannot be defined, to a state of irreversible breakdown incompatible with life. In the intermediate stages function is modified and efficiency perhaps impaired. It seems possible that variations in diet, and particularly in the amount and quality of the protein, may underlie many of the differences in incidence and symptomatology of disease which are gradually being uncovered in different parts of the world.

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Amyloidosis with Cardiomyopathy

Author: Larry H Bernstein, MD, FACP
Introduction
Amyloidosis describes the various clinical syndromes that occur as a result of damage by amyloid deposits in tissues and organs throughout the body.  Systemic amyloidosis is a relatively rare multisystem disease caused by the deposition of misfolded protein in various tissues and organs. The term amyloid describes the deposition in the extracellular space of certain proteins in a highly characteristic, insoluble fibrillar form.  The disease entity is a disorder of misfolded or misassembled proteins.  There is extracellular amyloid fiber laid down as cross β-sheets disrupting organ function, which may affect the pancreas, kidney, autonomic nervous system, the heart, and in one form causes carpal tunnel syndrome.
It may present to almost any specialty, and diagnosis is frequently delayed. Cardiac involvement is a leading cause of morbidity and mortality, especially in primary light chain (AL) amyloidosis and in both wild-type and hereditary transthyretin amyloidosis. The heart is also occasionally involved in acquired serum amyloid A type (AA) amyloidosis and other rare hereditary types. Clinical phenotype varies greatly between different types of amyloidosis, and even the cardiac presentation has a great spectrum. The incidence of amyloidosis is uncertain, but it is thought that the most frequently diagnosed AL amyloidosis has an annual incidence of 6 to 10 cases per million population in the United Kingdom and United States.
The molecular basis for this particular phenomenon came with the extensive work done on multiple myeloma, antibody structure, and light chains.  In 1950, the discovery of a familial amyloid polyneuropathy was described in Portugal, and there were similar diseases in Sweden and Japan.  There were 72 known variants of transthyretin (TTR) in 1995, and now there are 100.  In addition, the occurance of different TTR associated variants with and without (amyloid) is found is Brazil, UK, US, Israel, Spain, France, Germany, Denmark, and Africa.  The table of variants, organ damage, and geographic location is too large to place on this document. If we refer to amyloid cardiomyopathy, it is exclusively a primary amyloidopathy, not secondary to light chain disorders or an inflammatory disease.  If we consider amyloidosis, we also have to consider family history, organ dysfunction, and we have to make a distinction between primary cardiac involvement, autonomic nervous system instability, and the two coexisting.  Familial amyloid polyneuropathy (FAP) is an extremely debilitating and progressive disease that is only treatable by liver transplantation.  Primary amyloid cardiomyopathy has been treated by heart transplant.  The qualifying statement here is, it depends.

Primary and Secondary Amyloidoses

Amyloid was originally described by pathologists based on microscopy. Amyloidoses are a systemic primary or secondary disease. There are distinctions to be made based on location and type.  The clinical significance of amyloid disease varies enormously, ranging from incidental asymptomatic deposits to localized disease through to rapidly fatal systemic forms that can affect multiple vital organs.
Common causes of secondary amyloidosis are – light chain production (AL) as in plasma cell dyscrasia, amyloid A (AA), senile systemic amyloidosis (diagnosed rarely in life).  The systemic amyloidoses are designated by a capital A (for amyloid) followed by the abbreviation for the chemical identity of the fibril protein. Thus, TTR amyloidosis is abbreviated ATTR, and immunoglobulin light chain type amyloidosis is abbreviated AL. Both normal-sequence TTR and variant-sequence TTR form amyloidosis. Normal-sequence TTR forms cardiac amyloidosis in elderly people, termed senile cardiac amyloidosis (SCA). When it was recognized that SCA is often accompanied by microscopic deposits in many other organs, the alternative name senile systemic amyloidosis (SSA) was proposed. Both terms are now used.
Currently available therapy is focused on reducing the supply of the respective amyloid fibril precursor protein and supportive medical care, which together have greatly improved survival. Chemotherapy and anti-inflammatory treatment for the disorders that underlie AL and AA amyloidosis are guided by serial measurements of the respective circulating amyloid precursor proteins, i.e. serial serum free light chains in AL and serum amyloid A protein in AA type.
Quality of life and prognosis of some forms of hereditary systemic amyloidosis can be improved by liver and other organ transplants. Various new therapies, ranging from silencing RNA, protein stabilizers to monoclonal antibodies, aimed at inhibiting fibril precursor supply, fibril formation or the persistence of amyloid deposits, are in development; some are already in clinical phase.
Ann Clin Biochem May 2012; 49(3 ): 229-241   http://acb.2011.011225v1 49/3/229

What is transthyretin (TTR)?

TTR is a  tetramer of 4 127 amino acid subunits synthesized by the liver that circulates as a transporter of thyroxin, and with retinol-binding protein, transports vitamin A.  It was originally defined by the migration in electrophoresis more anodal to albumin, hence, prealbumin.  It is present in cerebrospinal fluid, secreted by the choroid plexus.  The TTR monomer contains 8 antiparallel beta pleated sheet domains. TTR can be found in plasma and in cerebrospinal fluid and is synthesized by the choroid plexus of the brain and, to a lesser degree, by the retina. Its gene is located on the long arm of chromosome 18 and contains 4 exons and 3 introns.
The concentration in serum can be expected to be above 20 mg/dL in a health adult, but the protein decreases by 1 mg/dL/day postoperatively, and it decreases with acute or chronic renal failure, pneumonia or sepsis, rising again with the onset of anabolism.  Patients in the pulmonary intensive care unit have TTR levels that remain low for 7-10 days, but followup data for the remainder of the hospital stay or in relationship to readmission in the six months after release from hospital care was not part of the study.
A decrease in TTR is associated with the systemic inflammatory response, whereby, the liver reprioritizes the synthesis of proteins with an increase in acute phase reactants (APRs), namely, C-reactive protein (CRP) and a-1 acid glycoprotein, and decreased albumin and TTR.  The inflammatory condition maintains a euthyroid status with decreased TTR because of the availability of free thyroxine in equilibrium with the lower binding protein.  This has been referred to sick euthyroid status. The role in thyroxine transport is not insignificant, as chronic protein malnutrition is associated with hypothyroidism, as originally described by Prof. Yves Ingenbleek, Univ. Louis Pateur, Starsbourg, Fr. in Senegalese children with Kwashiorkor.  However, the importance of TTR as a unique biomarker is not to be downgraded because of what is often refered to as “an inverted APR”.
Transthyretin was discovered to be a good reflection of the “lean body mass”, by Vernon Young, MIT, and Ingenbleek, as a result of 3 decades of study. The ratio of S:N being 1:20 in plant proteins and 1:12.5 in animal sources, is closely related to methylation reactions and sustained deficiency of S intake results in elevated homocysteine level.

What is FAP?

Familial amyloid polyneuropathy (FAP), also called transthyretin-related hereditary amyloidosis, transthyretin amyloidosis or Corino de Andrade’s disease, is an autosomal dominant neurodegenerative disease. It is a form of amyloidosis, and was first identified and described by Portuguese neurologist Mário Corino da Costa Andrade, in the 1950s.FAP is distinct from senile systemic amyloidosis (SAS), which is not inherited, and which was determined to be the primary cause of death for 70% of supercentenarians who have been autopsied.
Familial amyloid polyneuropathy (FAP) is an extremely debilitating and progressive disease that is only treatable by liver transplantation.  Primary amyloid cardiomyopathy has been treated by heart transplant.  The qualifying statement here is, it depends.  Those patients with TTR-amyloidopathy have a specific gene substitution in the TTR gene. Consequently, there is circulation TTR, but it is not effectively involved in thyroxine transport.

Characteristics.

Usually manifesting itself between 20 and 40 years of age, it is characterized by pain, paresthesia, muscular weakness and autonomic dysfunction. In its terminal state, the kidneys and the heart are affected. FAP is characterized by the systemic deposition of amyloidogenic variants of the transthyretin protein, especially in the peripheral nervous system, causing a progressive sensory and motor polyneuropathy. The age at symptom onset, pattern of organ involvement, and disease course vary, but most mutations are associated with cardiac and/or nerve involvement. The gastrointestinal tract, vitreous, lungs, and carpal ligament are also frequently affected. When the peripheral nerves are prominently affected, the disease is termed familial amyloidotic polyneuropathy (FAP). When the heart is involved heavily but the nerves are not, the disease is called familial amyloid cardiomyopathy (FAC). Regardless of which organ is primarily targeted, the general term is simply amyloidosis-transthyretin type, abbreviated ATTR.

Genetics.

  1. TTR mutations accelerate the process of TTR amyloid formation and are the most important risk factor for the development of clinically significant ATTR. More than 85 amyloidogenic TTR variants cause systemic familial amyloidosis. The variant TTR is mostly produced by the liver. Amyloidogenic TTR mutations destabilize TTR monomers or tetramers, allowing the molecule to more easily attain an amyloidogenic intermediate conformation. The tetramer has to dissociate into misfolded monomers to aggregate into a variety of structures including amyloid fibrils. Because most patients are heterozygotes, they deposit both mutant and wild type TTR subnits.
  2. Familial amyloid polyneuropathy has an autosomal dominant pattern of inheritance. FAP is caused by a mutation of the TTR gene, located on human chromosome 18q12.1-11.2. A replacement of valine by methionine at position 30 (TTR V30M) is the mutation most commonly found in FAP.
  3. The disease in the TTR V30M kindreds was termed FAP because early symptoms arose from peripheral neuropathy, but these patients actually have systemic amyloidosis, with widespread deposits often involving the heart, gastrointestinal tract, eye, and other organs.
  4. TTR V122I: This variant, carried by 3.9% of African Americans and over 5% of the population in some areas of West Africa, increases the risk of late-onset (after age 60 years) cardiac amyloidosis. It appears to be the most common amyloid-associated TTR variant worldwide. Affected patients usually do not have peripheral neuropathy.
  5. TTR T60A: This variant causes late-onset systemic amyloidosis with cardiac, and sometimes neuropathic, involvement. This variant originated in northwest Ireland and is found in Irish and Irish American patients.
  6. TTR L58H: Typically affecting the carpal ligament and nerves of the upper extremities, this variant originated in Germany. It has spread throughout the United States but is most common in the mid-Atlantic region.
  7. TTR G6S: This is the most common TTR variant, but it appears to be a neutral polymorphism not associated with amyloidosis. It is carried by about 10% of people of white European descent.

Cardiac transthyretin (TTR) amyloidosis

Cardiac amyloidosis of transthyretin fibril protein (ATTR) type is an infiltrative cardiomyopathy characterised by ventricular wall thickening and diastolic heart failure. More than 27 different precursor proteins have the propensity to form amyloid fibrils. The particular precursor protein that misfolds to form amyloid fibrils defines the amyloid type and predicts the patient’s clinical course. Several types of amyloid can infiltrate the heart, resulting in progressive diastolic and systolic dysfunction, congestive heart failure, and death.  Increased access to cardiovascular magnetic resonance imaging has led to a marked increase in referrals to St George’s University of London, London (Dr. Jason Dungu) of Caucasian patients with wild-type ATTR (senile systemic) amyloidosis and Afro-Caribbean patients with the hereditary ATTR V122I type. Both subtypes present predominantly as isolated cardiomyopathy. The differential diagnosis includes cardiac amyloid light-chain (AL) amyloidosis, which has a poorer prognosis and can be amenable to chemotherapy.

Clinical Presentation

Cardiac amyloidosis, irrespective of type, presents as a restrictive cardiomyopathy characterized by progressive diastolic and subsequently systolic biventricular dysfunction and arrhythmia.1 Key “red flags” to possible systemic amyloidosis include nephrotic syndrome, autonomic neuropathy (eg, postural hypotension, diarrhea), soft-tissue infiltrations (eg, macroglossia, carpal tunnel syndrome, respiratory disease), bleeding (eg, cutaneous, such as periorbital, gastrointestinal), malnutrition/cachexia and genetic predisposition (eg, family history, ethnicity). Initial presentations may be cardiac, with progressive exercise intolerance and heart failure. Other organ involvement, particularly in AL amyloidosis, may cloud the cardiac presentation (eg, nephrotic syndrome, autonomic neuropathy, pulmonary or bronchial involvement). Pulmonary edema is not common early in the disease process, but pleural and pericardial effusions and atrial arrhythmias are often seen. Syncope is common and a poor prognostic sign. It is typically exertional or postprandial as part of restrictive cardiomyopathy, sensitivity to intravascular fluid depletion from loop diuretics combined with autonomic neuropathy, or conduction tissue involvement (atrioventricular or sinoatrial nodes) or ventricular arrhythmia. The latter may rarely cause recurrent syncope. Disproportionate septal amyloid accumulation mimicking hypertrophic cardiomyopathy with dynamic left ventricular (LV) outflow tract obstruction is rare but well documented. Myocardial ischemia can result from amyloid deposits within the microvasculature. Atrial thrombus is common, particularly in AL amyloidosis

Diagnosis and Treatment

imaging – Cardiovascular Magnetic Resonance in Cardiac Amyloidosis*.

Cardiac amyloidosis can be diagnostically challenging. Cardiovascular magnetic resonance (CMR) can assess abnormal myocardial interstitium. In cardiac amyloidosis, CMR shows a characteristic pattern of global subendocardial late enhancement coupled with abnormal myocardial and blood-pool gadolinium kinetics. The findings agree with the transmural histological distribution of amyloid protein and the cardiac amyloid load.
 *AM Maceira; J Joshi; SK Prasad; J Charles Moon, et al. Royal Brompton Hospital, London;
The diagnosis of amyloidosis requires histological identification of amyloid deposits. Congo Red staining renders amyloid deposits salmon pink by light microscopy, with a characteristic apple green birefringence under polarized light conditions. Additional immunohistochemical staining for precursor proteins identifies the type of amyloidosis.  Ultimately, immunogold electron microscopy and mass spectrometry confer the greatest sensitivity and specificity for amyloid typing.
Treatment of cardiac amyloidosis is dictated by the amyloid type and degree of involvement. Consequently, early recognition and accurate classification are essential.
Novel diagnostic and surveillance approaches using imaging (echocardiography, cardiovascular magnetic resonance), biomarkers (brain natriuretic peptide [BNP], high-sensitivity troponin), new histological typing techniques, and current and future treatments, including approaches directly targeting the amyloid deposits.

Etiology

Amyloidosis is caused by the extracellular deposition of autologous protein in an abnormal insoluble β-pleated sheet fibrillary conformation—that is, as amyloid fibrils. More than 30 proteins are known to be able to form amyloid fibrils in vivo, which cause disease by progressively damaging the structure and function of affected tissues. Amyloid deposits also contain minor nonfibrillary constituents, including serum amyloid P component (SAP), apolipoprotein E, connective tissue components (glycosaminoglycans, collagen), and basement membrane components (fibronectin, laminin). Amyloid deposits can be massive, and cardiac or other tissues may become substantially replaced. Amyloid fibrils bind Congo red stain, yielding the pathognomonic apple-green birefringence under cross-polarized light microscopy that remains the gold standard for identifying amyloid deposits.

AL Amyloidosis

AL amyloidosis is caused by deposition of fibrils composed of monoclonal immunoglobulin light chains and is associated with clonal plasma cell or other B-cell dyscrasias. The spectrum and pattern of organ involvement is very wide, but cardiac involvement occurs in half of cases and is sometimes the only presenting feature. Cardiac AL amyloidosis may be rapidly progressive. Low QRS voltages, particularly in the limb leads, are common. Thickening of the LV wall is typically mild to moderate and is rarely >18 mm even in advanced disease. Cardiac AL amyloid deposition is accompanied by marked elevation of the biomarkers BNP and cardiac troponin, even at an early stage. Involvement of the heart is the commonest cause of death in AL amyloidosis and is a major determinant of prognosis; without cardiac involvement, patients with AL amyloidosis have a median survival of around 4 years, but the prognosis among affected patients with markedly elevated BNP and cardiac troponin (Mayo stage III disease) is on the order of 8 months.

Hereditary Amyloidoses

Mutations in several genes, such as transthyretin, fibrinogen, apolipoprotein A1, and apolipoprotein A2 can be responsible for hereditary amyloidosis, but by far the most common cause is variant ATTR amyloidosis (variant ATTR) caused by mutations in the transthyretin gene causing neuropathy and, often, cardiac involvement.

TTR gene mutation

 The most common is the Val122Ile mutation. In a large autopsy study that included individuals with cardiac amyloidosis, the TTR Val122Ile allele was present in 3.9% of all African Americans and 23% of African Americans with cardiac amyloidosis. Penetrance of the mutation is not truly known and is associated with a late-onset cardiomyopathy that is indistinguishable from senile cardiac amyloidosis.

Pathology, Presentation, and Management of Amyloidoses

More than 100 genetic variants of TTR are associated with amyloidosis. Most present as the clinical syndrome of progressive peripheral and autonomic neuropathy. Unlike wild-type ATTR or variant ATTR Val122Ile, the features of other variant ATTR include vitreous amyloid deposits or, rarely, deposits in other organs. Cardiac involvement in variant ATTR varies by mutations and can be the presenting or indeed the only clinical feature. For example, cardiac involvement is rare in variant ATTR associated with Val30Met (a common variant in Portugal or Sweden), but it is almost universal and develops early in individuals with variant ATTR due to Thr60Ala mutation (a mutation common in Ireland).

Senile Systemic Amyloidosis (Wild-Type ATTR)

Wild-type TTR amyloid deposits are found at autopsy in about 25% of individuals >80 years of age.  The prevalence of wild-type TTR deposits leading to the clinical syndrome of wild-type ATTR cardiac amyloidosis is unknown. Wild-type ATTR is a predominantly cardiac disease, and the only other significant extracardiac feature is a history of carpal tunnel syndrome, often preceding heart failure by 3 to 5 years. Extracardiac involvement is most unusual.
Both wild-type ATTR and ATTR due to Val122Ile are diseases of the >60-year age group and are often misdiagnosed as hypertensive heart disease. Wild-type ATTR has a strong male predominance, and the natural history remains poorly understood, but studies suggest a median survival of about 7 years from presentation. Recent developments in cardiac magnetic resonance (CMR), which have greatly improved detection of cardiac amyloid during life, suggest that wild-type ATTR is more common than previously thought: It accounted for 0.5% of all patients seen at the UK amyloidosis center until 2001 but now accounts for 7% of 1100 cases with amyloidosis seen since the end of 2009. There appears to be an association between wild-type ATTR and history of myocardial infarctions, G/G (Val/Val) exon 24 polymorphism in the alpha2-macroglobulin (alpha2M), and the H2 haplotype of the tau gene36; the association of tau with Alzheimer’s disease raises interesting questions as both are amyloid-associated diseases of aging.
ECG of a patient with cardiac AL amyloidosis showing small QRS voltages (defined as ≤6 mm height), predominantly in the limb leads and pseudoinfarction pattern in the anterior leads.
Echocardiography is characteristic. Typical findings include concentric ventricular thickening with right ventricular involvement, poor biventricular long-axis function with normal/near-normal ejection fraction and valvular thickening (particularly in wild-type or variant ATTR). Diastolic dysfunction is the earliest echocardiographic abnormality and may occur before cardiac symptoms develop. Biatrial dilatation in presence of biventricular, valvular, and interatrial septal thickening 53 is a useful clue to the diagnosis.
Transthoracic echocardiogram with speckle tracking. The red and yellow lines represent longitudinal motion in the basal segments, whereas the purple and green lines represent apical motion. This shows loss of longitudinal ventricular contraction at the base compared to apex.

Biomarkers.

High-sensitivity troponin is abnormal in >90% of cardiac AL patients, and the combination of BNP/NT-proBNP plus troponin measurements is used to stage and risk-stratify patients with AL amyloidosis at diagnosis. Very interestingly, the concentration of BNP/NT-proBNP in AL amyloidosis may fall dramatically within weeks after chemotherapy that substantially reduces the production of amyloidogenic light chains. The basis for this very rapid phenomenon, which is not mirrored by changes on echocardiography or CMR, remains uncertain, but a substantial fall is associated with improved outcomes.

Cardiac Magnetic Resonance.

CMR provides functional and morphological information on cardiac amyloid in a similar way to echocardiography, though the latter is superior for evaluating and quantifying diastolic abnormalities. An advantage of CMR is in myocardial tissue characterization. Amyloidotic myocardium reveals subtle precontrast abnormalities (T1, T2), but extravascular contrast agents based on chelated gadolinium provide the key information.

CMR with the classic amyloid global, subendocardial late gadolinium enhancement pattern in the left ventricle with blood and mid-/epimyocardium nulling together.
Recently, the technique of equilibrium contrast CMR has demonstrated much higher extracellular myocardial volume in cardiac amyloid than any other measured disease. It is anticipated that accurate measurements of the expanded interstitium in amyloidosis will prove useful in serial quantification of cardiac amyloid burden.
Sequential static images from a CMR TI scout sequence. As the inversion time (TI) increases, myocardium nulls first (arrow in image 3), followed by blood afterwards (arrow in image 6), implying that there is more gadolinium contrast in the myocardium than blood—a degree of interstitial expansion such that the “myocrit” is smaller than the hematocrit.

Tissue biopsy.

To confirm amyloidosis, including familial TTR amyloidosis, the demonstration of amyloid deposition on biopsied tissues is essential. With Congo red staining, amyloid deposits show a characteristic yellow-green birefringence under polarized light. Tissues suitable for biopsy include: subcutaneous fatty tissue of the abdominal wall, skin, gastric or rectal mucosa, sural nerve, and peritendinous fat from specimens obtained at carpal tunnel surgery. Sensitivity of endoscopic biopsy of gastrointestinal mucosa is around 85%; biopsy of the sural nerve is less sensitive. It is ideal to show that these amyloid deposits are specifically immunolabeled by anti-TTR antibodies.

Serum variant TTR protein.

TTR protein normally circulates in serum or plasma as a soluble protein having a tetrameric structure [Kelly 1998, Rochet & Lansbury 2000]. Normal plasma TTR concentration is 20-40 mg/dL (0.20-0.40 mg/mL).  Pathogenic mutations in TTR cause conformational change in the TTR protein molecule, disrupting the stability of the TTR tetramer, which is then more easily dissociated into pro-amyloidogenic monomers.

After immunoprecipitation with anti-TTR antibody, serum variant TTR protein can be detected by mass spectrometry. Approximately 90% of TTR variants so far identified are confirmed by this method. Mass shift associated with each variant TTR protein is indicated.

Molecular genetic testing.

  • TTR is the only gene in which mutations are known to cause familial TTR amyloidosis.
  • Identified in many individuals of different ethnic backgrounds; found in large clusters in Portugal, Sweden, and Japan.
  • The gene has four exons; and all the hitherto-identified mutations are in exons 2, 3, or 4.
GeneReviews designates a molecular genetic test as clinically available only if the test is listed in the GeneTests Laboratory Directory by either a US CLIA-licensed laboratory or a non-US clinical laboratory.
  • Molecular genetic testing of TTR by sequence analysis (may be preceded by targeted mutation analysis)
  • Although deletion/duplication testing is available clinically, no exonic or whole-gene deletions or duplications involving TTR have been reported to cause familial transthyretin amyloidosis.
  • However, with newly available deletion/duplication testing methods, it is theoretically possible that such mutations may be identified in affected individuals in whom prior testing by sequence analysis of the entire coding region was negative.
  • Predictive testing for at-risk asymptomatic adult family members requires prior identification of the disease-causing mutation in the family.
  • Prenatal diagnosis and preimplantation genetic diagnosis (PGD) for at-risk pregnancies require prior identification of the disease-causing mutation in the family.

Genetically Related (Allelic) Disorders

Familial euthyroid hyperthyroxinemia is caused by normal allelic variants in TTR, including Gly6Ser, Ala109Thr, Ala109Val, and Thr119Met (see Table 5) [Nakazato 1998, Benson 2001, Saraiva 2001]. The TTR protein binds approximately 15% of serum thyroxine. These mutations increase total serum thyroxine concentration because of their increased affinity for thyroxine, however, they increase neither free thyroxine nor free triiodothyronine. Therefore, individuals with these sequence variants develop no clinical symptoms (i.e., they are euthyroid).
Senile systemic amyloidosis (SSA; previously called senile cardiac amyloidosis) results from the pathologic deposition of wild-type TTR, predominantly in the heart. Pathologic deposits are also seen in the lungs, blood vessels, and the renal medulla of the kidneys [Westermark et al 2003]. SSA affects mainly the elderly but is rarely diagnosed during life.
Sensorimotor neuropathy and autonomic neuropathy progress over ten to 20 years. Various types of cardiac conduction block frequently appear. Cachexia is a common feature at the late stage of the disease. Affected individuals usually die of cardiac failure, renal failure, or infection.

Cardiac amyloidosis.

Cardiac amyloidosis, mainly characterized by progressive cardiomyopathy, has been reported with more than two thirds of TTR mutations. In some families with specific TTR mutations, such as Asp18Asn, Val20Ile, Pro24Ser, Ala45Thr, Ala45Ser, His56Arg, Gly57Arg, Ile68Leu, Ala81Thr, Ala81Val, His88Arg, Glu92Lys, Arg103Ser, Leu111Met, or Val122Ile, cardiomyopathy without peripheral neuropathy is a main feature of the disease.

Cardiac amyloidosis is usually late onset. Most individuals develop cardiac symptoms after age 50 years; cardiac amyloidosis generally presents with restrictive cardiomyopathy. The typical electrocardiogram shows a pseudoinfarction pattern with prominent Q wave in leads II, III, aVF, and V1-V3, presumably resulting from dense amyloid deposition in the anterobasal or anteroseptal wall of the left ventricle. The echocardiogram reveals left ventricular hypertrophy with preserved systolic function. The thickened walls present “a granular sparkling appearance.”
Among the mutations responsible for cardiac amyloidosis, Val122Ile is notable for its prevalence in African Americans. Approximately 3.0%-3.9% of African Americans are heterozygous for Val122Ile . The high frequency of Val122Ile partly explains the observation that in individuals in the US older than age 60 years, cardiac amyloidosis is four times more common among blacks than whites.

Leptomeningeal (oculoleptomeningeal) amyloidosis.

Amyloid deposition is seen in the pial and arachnoid membrane, as well as in the walls of vessels in the subarachnoid space associated with TTR mutations including Leu12Pro, Asp18Gly, Ala25Thr, Val30Gly, Ala36Pro, Gly53Glu, Gly53Ala, Phe64Ser, Tyr69His, or Tyr114Cys.  Individuals with leptomeningeal amyloidosis show CNS signs and symptoms including: dementia, psychosis, visual impairment, headache, seizures, motor paresis, ataxia, myelopathy, hydrocephalus, or intracranial hemorrhage. When associated with vitreous amyloid deposits, leptomeningeal amyloidosis is known as familial oculolepto-meningeal amyloidosis (FOLMA). In leptomeningeal amyloidosis protein concentration in the cerebrospinal fluid is usually high, and gadolinium-enhanced MRI typically shows extensive enhancement of the surface of the brain, ventricles, and spinal cord.

Genotype-Phenotype Correlations.

In subsets of families with the Val30Met mutation, considerable variation in phenotypic manifestations and age of onset is observed. It is hypothesized that genetic modifiers and non-genetic factors contribute to the pathogenesis and progression of familial TTR amyloidosis. The vast majority of individuals with familial TTR amyloidosis are heterozygous for a TTR mutation. It has been clinically and experimentally demonstrated that the normal allelic variant c.416C>T (Thr119Met) has a protective effect on amyloidogenesis in individuals who have the Val30Met mutation.

Cardiac amyloidosis is caused by Asp18Asn, Val20Ile, Pro24Ser, Ala45Thr, Ala45Ser, His56Arg, Gly57Arg, Ile68Leu, Ala81Thr, Ala81Val, His88Arg, Glu92Lys, Arg103Ser, Leu111Met, or Val122Ile. Peripheral and autonomic neuropathy are absent or less evident in persons with these mutations.
Leptomeningeal amyloidosis is associated with Leu12Pro, Asp18Gly, Ala25Thr, Val30Gly, Ala36Pro, Gly53Glu, Gly53Ala, Phe64Ser, Tyr69His, or Tyr114Cys.

Penetrance.

It is generally accepted that the penetrance is much higher in individuals in endemic foci than outside of endemic foci. In Portugal, cumulative disease risk in individuals with the Val30Met mutation is estimated at 80% by age 50 and 91% by age 70 years, whereas the risk in French heterozygotes is 14% by age 50 and 50% by age 70 years. In Sweden, the penetrance is much lower: 1.7% by age 30, 5% by age 40, 11% by age 50, 22% by age 60, 36% by age 70, 52% by age 80, and 69% by age 90, respectively.

Nomenclature

The neuropathy associated with TTR mutations, now called familial TTR amyloidosis, was formerly referred to as one of the following:
  • Familial amyloid polyneuropathy type I (or the Portuguese-Swedish-Japanese type)
  • Familial amyloid polyneuropathy type II (or the Indiana/Swiss or Maryland/German type)

Prevalence

The Val30Met mutation, found worldwide, is the most widely studied TTR variant and is responsible for the well-known large foci of individuals with TTR amyloid polyneuropathy in Portugal, Sweden, and Japan. Numerous families with various non-Val30Met mutations have also been identified worldwide.

 Small transthyretin (TTR) ligands as possible therapeutic agents in TTR amyloidoses.

Almeida MR, Gales L, Damas AM, Cardoso I, Saraiva MJ. Porto, Portugal.
Curr Drug Targets CNS Neurol Disord. 2005 Oct;4(5):587-96.
In transthyretin (TTR) amyloidosis TTR variants deposit as amyloid fibrils giving origin, in most cases, to peripheral polyneuropathy, cardiomyopathy, carpal tunnel syndrome and/or amyloid deposition in the eye. The amino acid substitutions in the TTR variants destabilize the tetramer, which may dissociate into non native monomeric intermediates that aggregate and polymerize in amyloid fibrils that further elongate. Since this is a multi-step process there is the possibility to impair TTR amyloid fibril formation at different stages of the process namely by tetramer stabilization, inhibition of fibril formation or fibril disruption. Based on the proposed mechanism for TTR amyloid fibril formation we discuss the action of some of the proposed TTR stabilizers such as derivatives of some NSAIDs (diflunisal, diclofenac, flufenamic acid, and derivatives) and the action of amyloid disrupters such as 4′-iodo-4′-deoxydoxorubicin (I-DOX) and tetracyclines. Among all these compounds, TTR stabilizers seem to be the most interesting since they would impair very early the process of amyloid formation and could also have a prophylactic effect.

Clusterin regulates transthyretin amyloidosis.

Lee KW, Lee DH, Son H, Kim YS, Park JY, et al.  Gyeongnam National University, South Korea
Biochem Biophys Res Commun 2009;388(2):256-60.   http://dx.doi.org/10.1016/j.bbrc.2009.07.166.
Clusterin has recently been proposed to play a role as an extracellular molecular chaperone, affecting the fibril formation of amyloidogenic proteins. The ability of clusterin to influence amyloid fibril formation prompted us to investigate whether clusterin is capable of inhibiting TTR amyloidosis. Here, we report that clusterin strongly interacts with wild-type TTR and TTR variants V30M and L55P under acidic conditions, and blocks the amyloid fibril formation of TTR variants. In particular, the amyloid fibril formation of V30M TTR in the presence of clusterin is reduced to level similar to wild-type TTR. We also demonstrated that clusterin is an effective inhibitor of L55P TTR amyloidosis, the most aggressive form of TTR diseases. The mechanism by which clusterin inhibits TTR amyloidosis appears to be through stabilization of TTR tetrameric structure.

Prognosis.

Cardiac amyloidosis in general has a poor prognosis, but this differs according to amyloid type and availability and response to therapy. Treatment may be classified as follows: supportive therapy (ie, modified heart-failure treatment including device therapy); therapies that suppress production of the respective amyloid fibril precursor protein (eg, chemotherapy in AL amyloidosis); and novel strategies to inhibit amyloid fibril formation or to directly target the amyloid deposits or stabilize the precursor protein (especially in ATTR with drugs such as tafamidis or diflunisal). Cardiac transplantation, although rarely feasible, can be very successful in carefully selected patients.

Reducing Amyloid Fibril Precursor Protein Production

Treatment of amyloidosis is currently based on the concept of reducing the supply of the respective amyloid fibril precursor protein. In AL amyloidosis, therapy is directed toward the clonal plasma cells using either cyclical combination chemotherapy or high-dose therapy with autologous stem cell transplantation.
The newer treatment options include bortezomib (a proteosome inhibitor)105 and the newer immunomodulatory drugs lenalidomide and pomalidomide. Bortezomib combinations appear to be especially efficient in amyloidosis with high rates of near-complete clonal responses, which appear to translate into early cardiac responses.106–108 Phase II (bortezomib in combination with cyclophosphamide or doxorubicin) and phase III (bortezomib, melphalan, and dexamethasone compared to melphalan and dexamethasone as front-line treatment) trials are underway.
AA amyloidosis is the only other type of amyloidosis in which production of the fibril precursor protein can be effectively suppressed by currently available therapies. Anti-inflammatory therapies, such as anti-tumor necrosis factor agents in rheumatoid arthritis, can substantially suppress serum amyloid A protein production, but very little experience has been obtained regarding cardiac involvement, which is very rare in this particular type of amyloidosis.
TTR is produced almost exclusively in the liver, and TTR amyloidosis has lately become a focus for novel drug developments aimed at reducing production of TTR through silencing RNA and antisense oligonucleotide therapies. ALN-TTR01, a systemically delivered silencing RNA therapeutic, is already in phase I clinical trial. Liver transplantation has been used as a treatment for variant ATTR for 20 years, to remove genetically variant TTR from the plasma. Although this is a successful approach in ATTR Val30Met, it has had disappointing results in patients with other ATTR variants, which often involve the heart. The procedure commonly results in progressive cardiac amyloidosis through ongoing accumulation of wild-type TTR on the existing template of variant TTR amyloid. The role of liver transplantation in non-Val30Met–associated hereditary TTR amyloidosis thus remains very uncertain.

Inhibition of Amyloid Formation

Amyloid fibril formation involves massive conformational transformation of the respective precursor protein into a completely different form with predominant β-sheet structure. The hypothesis that this conversion might be inhibited by stabilizing the fibril precursor protein through specific binding to a pharmaceutical has lately been explored in TTR amyloidosis. A key step in TTR amyloid fibril formation is the dissociation of the normal TTR tetramer into monomeric species that can autoaggregate in a misfolded form. In vitro studies identified that diflunisal, a now little used nonsteroidal anti-inflammatory analgesic, is bound by TTR in plasma, and that this enhances the stability of the normal soluble structure of the protein. Studies of diflunisal in ATTR are in progress. Tafamidis is a new compound without anti-inflammatory analgesic properties that has a similar mechanism of action. Tafamidis has just been licensed for neuropathic ATTR, but its role in cardiac amyloidosis remains uncertain, and clinical trial results are eagerly awaited. Higher-affinity “superstabilizers” are also in development.

Conclusion

Cardiac amyloidosis remains challenging to diagnose and to treat. Key “red flags” that should raise suspicion include clinical features indicating multisystem disease and concentric LV thickening on echocardiography in the absence of increased voltage on ECG; the pattern of gadolinium enhancement on CMR appears to be very characteristic. Confirmation of amyloid type is now possible in most cases through a combination of immunohistochemistry, DNA analysis, and proteomics. A variety of novel specific therapies are on the near horizon, with potential to both inhibit new amyloid formation and enhance clearance of existing deposits.

Future Prospects

Jeffery W. Kelly, the former Dean of Graduate Studies (2000-2008) and Vice President of Academic Affairs (2000-2006), currently is the Chairman of Molecular and Experimental Medicine and the Lita Annenberg Hazen Professor of Chemistry within the Skaggs Institute of Chemical Biology at The Scripps Research Institute in La Jolla, California.
The work on folding proteins by the Kelly Group focuses on
[1] understanding protein misfolding and aggregation and on developing both chemical
[2] and biological strategies
[3] to ameliorate diseases caused by protein misfolding and/or aggregation.
Besides studying the structural and energetic basis behind protein folding, his laboratory also studies the etiology of neurodegenerative diseases linked to protein aggregation, including Alzheimer’s disease, Parkinson’s Disease, and the familial gelsolin and transthyretin-based amyloidoses–publishing over 250 peer-reviewed papers in this area to date. He has also provided insight into genetic diseases associated with loss of protein function, such as lysosomal storage diseases.
Kelly has cofounded three biotechnology companies, FoldRx Pharmaceuticals (with Susan Lindquist), now owned by Pfizer, Proteostasis Therapeutics, Inc. (with Andrew Dillin and Richard Morimoto) (a private corporation) and Misfolding Diagnostics (with Xin Jiang and Justin Chapman; a private corporation). The Kelly laboratory discovered the first regulatory agency-approved drug that slows the progression of a human amyloid disease using a structure-based design approach. This drug, now called Tafamidis or Vyndaqel, slowed the progression of familial amyloid polyneuropathy in an 18 month placebo controlled trial and in an 18 month extension study sponsored by FoldRx Pharmaceuticals (acquired by Pfizer in 2010). Vyndaqel or Tafamidis  was approved for the treatment of Familial amyloid Polyneuropathy by the European Medicines Agency in late 2011. Kelly also discovered that diflunisal kinetically stabilizes transthyretin, enabling a placebo controlled clinical trial with it to ameliorate familial amyloid polyneuropathy–the results of which will be announced in 2013. Proteostasis Therapeutics, Inc. is developing first-in-class drugs that adapt the proteostasis network to ameliorate both loss-of-function misfolding diseases and gain-of-toxic function diseases linked to protein aggregation.
In addition to discovering the first drug that slows the progression of a human amyloid disease, the Kelly Laboratory is credited with demonstrating that transthyretin conformational changes alone are sufficient for amyloidogenesis, discovering the first example of functional amyloid in mammals, making major contributions toward understanding β-sheet folding, discovering the “enhanced aromatic sequon”–sequences that are more efficiently glycosylated by cells and sequences which stabilize the proteins that they are incorporated into as a consequence of N-glycosylation and was corresponding author on and contributed some of the key experimental data demonstrating that altering cellular proteostasis capacity has the potential to alleviate protein misfolding and aggregation diseases.
Native state kinetic stabilization as a strategy to ameliorate protein misfolding diseases: a focus on the transthyretin amyloidoses. Johnson SM, Wiseman RL, Sekijima Y, Green NS, Adamski-Werner SL, Kelly JW.  http://www.ncbi.nlm.nih.gov/pubmed/16359163
Small molecule-mediated protein stabilization inside or outside of the cell is a promising strategy to treat protein misfolding/misassembly diseases. Herein we focus on the transthyretin (TTR) amyloidoses and demonstrate that preferential ligand binding to and stabilization of the native state over the dissociative transition state raises the kinetic barrier of dissociation (rate-limiting for amyloidogenesis), slowing and in many cases preventing TTR amyloid fibril formation. Since T119M-TTR subunit incorporation into tetramers otherwise composed of disease-associated subunits also imparts kinetic stability on the tetramer and ameliorates amyloidosis in humans, it is likely that small molecule-mediated native state kinetic stabilization will also alleviate TTR amyloidoses.
Energetic characteristics of the new transthyretin variant A25T may explain its atypical central nervous system pathology.
Sekijima Y, Hammarström P, Matsumura M, Shimizu Y, Iwata M, Tokuda T, Ikeda S, Kelly JW.
Lab Invest. 2003 Mar;83(3):409-17.   http://www.ncbi.nlm.nih.gov/pubmed/12649341
Transthyretin (TTR) is a tetrameric protein that must misfold to form amyloid fibrils. Misfolding includes rate-limiting tetramer dissociation, followed by fast tertiary structural changes that enable aggregation. Amyloidogenesis of wild-type (WT) TTR causes a late-onset cardiac disease called senile systemic amyloidosis. The aggregation of one of > 80 TTR variants leads to familial amyloidosis encompassing a collection of disorders characterized by peripheral neuropathy and/or cardiomyopathy. Prominent central nervous system (CNS) impairment is rare in TTR amyloidosis. Herein, we identify a new A25T TTR variant in a Japanese patient who presented with CNS amyloidosis at age 42 and peripheral neuropathy at age 44. The A25T variant is the most destabilized and fastest dissociating TTR tetramer published to date, yet, surprising, disease onset is in the fifth decade. Quantification of A25T TTR in the serum of this heterozygote reveals low levels relative to WT, suggesting that protein concentration influences disease phenotype. Another recently characterized TTR CNS variant (D18G TTR) exhibits strictly analogous characteristics, suggesting that instability coupled with low serum concentrations is the signature of CNS pathology and protects against early-onset systemic amyloidosis. The low A25T serum concentration may be explained either by impaired secretion from the liver or by increased clearance, both scenarios consistent with A25T’s low kinetic and thermodynamic stability. Liver transplantation is the only known treatment for familial amyloid polyneuropathy. This is a form of gene therapy that removes the variant protein from serum preventing systemic amyloidosis. Unfortunately, the choroid plexus would have to be resected to remove A25T from the CSF-the source of the CNS TTR amyloid. Herein we demonstrate that small-molecule tetramer stabilizers represent an attractive therapeutic strategy to inhibit A25T misfolding and CNS amyloidosis. Specifically, 2-[(3,5-dichlorophenyl)amino]benzoic acid is an excellent inhibitor of A25T TTR amyloidosis in vitro.
Prevention of Transthyretin Amyloid Disease by Changing Protein Misfolding Energetics
Per Hammarström*, R. Luke Wiseman*, Evan T. Powers, Jeffery W. Kelly†
Science 31 Jan 2003; 299(5607):713-716    http://dx.doi.org/10.1126/science.1079589
Genetic evidence suggests that inhibition of amyloid fibril formation by small molecules should be effective against amyloid diseases. Known amyloid inhibitors appear to function by shifting the aggregation equilibrium away from the amyloid state. Here, we describe a series of transthyretin amyloidosis inhibitors that functioned by increasing the kinetic barrier associated with misfolding, preventing amyloidogenesis by stabilizing the native state. The trans-suppressor mutation, threonine 119 → methionine 119, which is known to ameliorate familial amyloid disease, also functioned through kinetic stabilization, implying that this small-molecule strategy should be effective in treating amyloid diseases.
R104H may suppress transthyretin amyloidogenesis by thermodynamic stabilization, but not by the kinetic mechanism characterizing T119 interallelic trans-suppression.
Sekijima Y, Dendle MT, Wiseman RL, White JT, D’Haeze W, Kelly JW.
Amyloid. Jun 2006;13(2):57-66.    http://www.ncbi.nlm.nih.gov/pubmed/16911959
The tetrameric protein transthyretin (TTR) forms amyloid fibrils upon dissociation and subsequent monomer misfolding, enabling misassembly. Remarkably, the aggregation of one of over 100 destabilized TTR variants leads to familial amyloid disease. It is known that trans-suppression mediated by the incorporation of T119M subunits into tetramers otherwise composed of the most common familial variant V30M, ameliorates disease by substantially slowing the rate of tetramer dissociation, a mechanism referred to as kinetic stabilization of the native state. R104H TTR has been reported to be non-pathogenic, and recently, this variant has been invoked as a trans-suppressor of amyloid fibril formation. Here, we demonstrate that the trans-suppression mechanism of R104H does not involve kinetic stabilization of the tetrameric structure, instead its modest trans-suppression most likely results from the thermodynamic stabilization of the tetrameric TTR structure. Thermodynamic stabilization increases the fraction of tetramer at the expense of the misfolding competent monomer decreasing the ability of TTR to aggregate into amyloid fibrils. As a consequence of this stabilization mechanism, R104H may be capable of protecting patients with modestly destabilizing mutations against amyloidosis by slightly lowering the overall population of monomeric protein that can misfold and form amyloid.

Amyloidosis, Node, Congo Red. The amyloid depo...

Amyloidosis, Node, Congo Red. The amyloid deposits are strongly congophilic when viewed before white light. (Photo credit: Wikipedia)

Amyloidosis

Amyloidosis (Photo credit: Boonyarit Cheunsuchon)

English: Intermed. mag. (H&E). Image:Cardiac a...

English: Intermed. mag. (H&E). Image:Cardiac amyloidosis high mag he.jpg (Photo credit: Wikipedia)

English: Intermed. mag. (H&E). Image:Cardiac a...

English: Intermed. mag. (H&E). Image:Cardiac amyloidosis high mag he.jpg (Photo credit: Wikipedia)

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Treatment for Infective Endocarditis

Curator: Larry H Bernstein, MD, FACP

UPDATED on 3/4/2019

WATCH VIDEO

https://consultqd.clevelandclinic.org/tricuspid-valve-reconstruction-for-infective-endocarditis-operative-highlights-video/amp/?__twitter_impression=true

Tricuspid Valve Reconstruction for Infective Endocarditis: Operative Highlights (Video)

There are no easy solutions for acute infective tricuspid valve endocarditis in IV drug users, as the risk of prosthetic endocarditis in this population is high. Complete valve resection without replacement is feasible but leads to progressive right-sided heart failure. Reconstruction of the tricuspid valve with autologous pericardium is an alternative option, as demonstrated in the video case study below.

A 29-year-old female drug abuser with fever, hemoptysis and MRSA bacteremia was started on IV antibiotics. She looked frail and had prominent jugular venous pressure as well as 95 percent saturation on 2 liters of nasal cannula oxygen. She was not on inotropes and had a pulmonary artery pressure of 40/20 mmHg with a good cardiac index. Chest CT showed a large left pleural effusion with associated atelectasis of the left lung. The right lung had manifestations of septic emboli and a smaller pleural effusion.

A Cleveland Clinic surgical team led by cardiothoracic surgeon Faisal Bakaeen, MD, proceeded to excise the patient’s extensive infected and devitalized tissue around the tricuspid valve, leaving only a portion of the anterior leaflet to serve as a reference for reconstruction using autologous pericardium. Dr. Bakaeen walks us through the essential surgical steps — and their underlying rationale — in the narrated operative video below.

SOURCE

https://consultqd.clevelandclinic.org/tricuspid-valve-reconstruction-for-infective-endocarditis-operative-highlights-video/amp/?__twitter_impression=true

 

An article that appeared in NEJM compares early surgery versus conventional treatment for infective endocarditis.
Early Surgery versus Conventional Treatment for Infective Endocarditis
Duk-Hyun Kang, Yong-Jin Kim, Sung-Han Kim, Byung Joo Sun, et al.

N Engl J Med June 28, 2012; 366:2466-2473. http://doi.org/10.1056/NEJMoa1112843

Background and Purpose: While current guidelines advocate surgical management for complicated left-sided infective endocarditis and early surgery for patients with infective endocarditis and congestive heart failure, the indications for surgical intervention to prevent systemic embolism remain unclear. Surgery is favored by experience with complete excision of infected tissue and valve repair, and low operative mortality, but it does not remove concerns about residual active infection, which results in two sets of guidelines, the 2006 ACC-AHA for class IIa indication only for recurrent emboli and persistent vegetation, and the 2009 ESC guidelines for class IIb indication for very large, isolated vegetations. The Early Surgery versus Conventional Treatment in Infective Endocarditis (EASE) trial was conducted to determine whether early surgical intervention woulddecrease rate of death or embolic events.

Patient Enrollment: The study enrolled 76 consecutive patients, 18 years of age or older, with left-sided, native-valve infective endocarditis and a high risk of embolism. For all patients with suspected infective endocarditis, blood cultures were obtained and transthoracic echocardiography was performed within 24 hours after hospitalization. Patients were only eligible for enrollment if they had received a diagnosis of definite infective endocarditis and had severe mitral valve or aortic valve disease and vegetation with a diameter greater than 10 mm. Patients were excluded if they had moderate-to-severe congestive heart failure, infective endocarditis complicated by heart block, annular or aortic abscess, destructive penetrating lesions requiring urgent surgery, or fungal endocarditis, or were over 80 years age, or coexisting major embolic stroke with a risk of hemorrhagic transformation at the time of diagnosis, and a serious coexisting condition. Patients were also excluded if they had infective endocarditis involving a prosthetic valve, right-sided vegetations, or small vegetations (diameter, ≤10 mm) or had been referred from another hospital more than 7 days after the diagnosis of infective endocarditis.
The protocol specified that patients who were assigned to the early-surgery group should undergo surgery within 48 hours after randomization. Patients assigned to the conventional-treatment group were treated according to the AHA guidelines, and surgery was performed only if complications requiring urgent surgery developed during medical treatment or if symptoms persisted after the completion of antibiotic therapy. Details of the study procedures are provided in the Supplementary Appendix, available at NEJM.org.

Study End Points: The primary end point was a composite of in-hospital death or clinical embolic events that occurred within 6 weeks after randomization. An embolic event was defined as a systemic embolism fulfilling both prespecified criteria: the acute onset of clinical symptoms or signs of embolism and the occurrence of new lesions, as confirmed by follow-up imaging studies. Prespecified secondary end points, at 6 months of follow-up, included death from any cause, embolic events, recurrence of infective endocarditis, and repeat hospitalization due to the development of congestive heart failure.

Clinical and Echocardiographic Characteristics of the Patients at Baseline, According to Treatment Group:

The mean age of the patients was 47 years, and 67% were men. The mitral valve was involved in 45 patients, the aortic valve in 22, and both valves in 9. Severe mitral regurgitation was observed in 45 patients, severe aortic regurgitation in 23, severe aortic stenosis in 3, severe mitral regurgitation and stenosis in 1, and both severe mitral regurgitation and aortic regurgitation in 4. The median diameter of vegetation was 12 mm (interquartile range, 11 to 17). All patients met the Duke criteria for definite endocarditis; the most common pathogens in both groups were viridans streptococci (in 30% of all patients), other streptococci (in 30%), and Staphylococcus aureus (in 11%). Characteristics of Antibiotic Therapy, According to Treatment Group: There were no significant between-group differences in terms of control of the underlying infection, the antibiotic regimen used, or the duration of antibiotic therapy.

Surgical Procedures: All patients in the early-surgery group underwent valve surgery within 48 hours after randomization; the median time between randomization and surgery was 24 hours (interquartile range, 7 to 45). Of the 22 patients with involvement of the mitral valve, 8 patients underwent mitral-valve repair and 14 underwent mitral-valve replacement with a mechanical valve. Of the 15 patients with involvement of the aortic valve or both the mitral and aortic valves, 14 underwent mechanical-valve replacement and 1 underwent valve replacement with a biologic prosthesis. Concomitant coronary-artery bypass grafting at the time of valve surgery was performed in 2 patients (5%).

Conventional Therapy: Of the 39 patients assigned to the conventional-treatment group, 30 (77%) underwent surgery during the initial hospitalization (27 patients) or during follow-up (3). The surgical procedures included 11 mitral-valve repairs, 6 mitral-valve replacements (with 5 patients receiving a mechanical valve and 1 a biologic prosthesis), 11 aortic-valve replacements (with 9 patients receiving a mechanical valve and 2 a biologic prosthesis), and 2 combined aortic-valve replacements (with 1 patient receiving a mechanical valve and 1 a biologic prosthesis) and mitral-valve repairs. In 8 patients (21%), indications for urgent surgery developed during hospitalization (median time to surgery after randomization, 6.5 days [interquartile range, 6 to 10]). Elective surgery was performed in an additional 22 patients owing to symptoms or left ventricular dysfunction more than 2 weeks after randomization. Surgical results are shown in the Supplementary Appendix.

Primary End Point: The primary end point of in-hospital death or embolic events within the first 6 weeks after randomization occurred in one patient (3%) in the early-surgery group, as compared with nine (23%) in the conventional-treatment group (hazard ratio, 0.10; 95% confidence interval [CI], 0.01 to 0.82; P=0.03). In the early-surgery group, one patient died in the hospital and no patients had embolic events; in the conventional-treatment group, one patient died in the hospital and eight patients had embolic events (Table 3TABLE 3).
http://www.nejm.org/na101/home/literatum/publisher/mms/journals/content/nejm/2012/nejm_2012.366.issue-26/nejmoa1112843/production/images/small/nejmoa1112843_t3.gif

At 6 weeks after randomization, the rate of embolism was 0% in the early-surgery group, as compared with 21% in the conventional-treatment group (P=0.005). No patient in either group had an embolic event or was hospitalized for congestive heart failure during follow-up. Recurrence of infective endocarditis within 6 months after discharge was not observed in any patient in the early-surgery group but was reported in 1 patient in the conventional-treatment group. Among the 11 patients (28%) in the conventional-treatment group who were treated medically and discharged without undergoing surgery, 1 (3%) died suddenly, 7 (18%) had symptoms related to severe valve disease or recurrence of infective endocarditis (3 of whom underwent surgery during follow-up), and 3 (8%) had no symptoms or embolic events (Table S3 in the Supplementary Appendix).
There was no significant difference between the early-surgery and conventional-treatment groups in all-cause mortality at 6 months (3% and 5%, respectively; hazard ratio, 0.51; 95% CI, 0.05 to 5.66; P=0.59) (Figure 2AFIGURE 2).
http://www.nejm.org/na101/home/literatum/publisher/mms/journals/content/nejm/2012/nejm_2012.366.issue-26/nejmoa1112843/production/images/small/nejmoa1112843_f2.gif
Kaplan–Meier Curves for the Cumulative Probabilities of Death and of the Composite End Point at 6 Months, According to Treatment Group.

At 6 months, the rate of the composite of death from any cause, embolic events, recurrence of infective endocarditis, or repeat hospitalization due to the development of congestive heart failure was 3% in the early-surgery group, as compared with 28% in the conventional-treatment group (hazard ratio, 0.08; 95% CI, 0.01 to 0.65; P=0.02). The estimated actuarial rate of end points was significantly lower in the early-surgery group than in the conventional-treatment group (P=0.009 by the log-rank test) (Figure 2B).

Conclusion: Early surgery performed within 48 hours after diagnosis reduced the composite primary end point of death from any cause or embolic events by effectively reducing the risk of systemic embolism. Moreover, these improvements in clinical outcomes were achieved without an increase in operative mortality or recurrence of infective endocarditis.

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