Posts Tagged ‘BNP’

More on the Performance of High Sensitivity Troponin T and with Amino Terminal Pro BNP in Diabetes

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


UPDATED on 8/7/2018

Siemens’ high-sensitivity Troponin I (TnIH) assaysgot FDA clearance for use in diagnosing acute myocardial infarction. (Cardiovascular Business) The first high-sensitivity Troponin T test was cleared last year, as MedPage Today reported.



This is the final up to date review of the status of hs troponin T (or I) with or without the combined use of the Brain Type Natriuretic Peptide or its Amino Terminal peptide precursor.  In addition, a new identification of the role of the Atrial Natriuretic Peptide has been reported with respect to arrythmogenic activity.  On the one hand, the diagnostic value of the NT-proBNP has been seen as disappointing, in part because of the question of what information is gained by the test in overt known congestive heart failure, and in part because of uncertainty about following the test during a short hospital stay.  At least, this is the view of this reviewer.  However, in the last several years there has been an emphasis on the value this test adds to prediction of adverse outcomes.   In addition, there has been a hidden nvariable that has much to do with the original reference values that were established for age ranges, without any consideration of pathophysiology that might affect the values within those ranges, leading one to consider values in an aging population as normal, that might well be high.  Why is this?  Aging patients are more likely to have hypertension, and also the onset of type-2 diabetes mellitus, with cardiovascular disease consequences.  Type-2 diabetes mellitus (T2DM), for instance, is associated with insulin resistance and also fat gain with generation of adipokines, but the is also a hyalinization of insulin forming beta-cells of the pancreas, and there is hyalinization of glomeruli (glomerulosclerosis) and afferent arteriolonephrosclerosis with expected decline in glomerular filtrattion rate and hypertension as well.   Of course, this is also associated with hepatosteatosis.   Nevertheless, a reference range is established that takes none of this pathophysiology into account.   While a more reasonable approach has been pointed out, there has been no followup in the literature.

On the other hand, there has been much confusion over the restandardization of a high sensitivity troponin I or T test (hs-Tn(I or T).  The reference range declines precipitously, and there is a good identification of patients who are for the most part disease free, but there is no delineation of patients who are at high risk of acute coronary syndrome with plaque rupture, vs a  host of other cardiovascular conditions.  These have no relationship to plaque rupture, but may be serious and require further evaluation.  The question then becomes whether to admit for a hospital stay, to refer to clinic after an evaluation in the ICU without admission, or to do an extensive evaluation in the emergency department overnight before release for followup.  There is still another dimension of this that has to do with prediction of outcomes using hs-Tn(s) with or without the natriuretic peptides.  Another matter that is not for discussion in this article is the underutilization of hs-CRP.  Originally used for a marker of sepsis in the 1970s, it has come to be tied in with identification of an ongoing inflammatory condition.  Therefore, the existence of a known inflammatory condition in the family of autoimmune diseases, with one exception, might make it unnecessary.

The discussion is broken into three parts:

Part 1.   New findings on the troponins.
Part 2.  The use of combined hs-Tn with a natriuretic peptide (NT-proBNP)
Part 3.  Atrial natriuretic peptide

Part 1.    New findings on the troponins.

Troponin: more lessons to learn

C Liebetrau,HM Nef,andCW.Hamm*
Germany; (GermanCentreforCardiovascularResearch),partnersite
RheinMain,BadNauheim, Germany; and UniversityofGiessen,Medizinische
European Hear tJournal editorial refers to ‘Risk stratification in patients with acute chest pain
using three high-sensitivity cardiac troponin assays’,
by P. Haafetal. troponin entered our diagnostic armamentarium 20 years ago and –
unlike any other biomarker –

  • is going through constant expansion in its application.

Troponin started out as a marker of risk in unstable angina’, then was used

  • as gold standard for risk stratification and therapy guiding in acute coronary syndrome
  •  served further to redefine myocardial infarction, and
  • has also become a risk factor in apparently healthy subjects.

The recently introduced high-sensitivity cardiac troponin (hs-cTn) assays

  • have not only expanded the potential of troponins, but
  • have also resulted in a certain amount of confusion
    • among unprepared users.

After many years troponins were accepted as the gold standard in

  • patients with chest pain by
  • classifying them into troponin-positive and
    • troponin-negative patients.

The new generation of hs-cTn assays has

  • improved the accuracy at the lower limit of detection and
  • provided incremental diagnostic information especially
    • in the early phase of myocardial infarction.

Moreover, low levels of measurable troponins

  • unrelated to ACS have been associated with
    • an adverse long-term outcome.

Several studies demonstrated that

  • these low levels of cardiac troponin measureable 
    • only by hs-Tn assays
  • are able to predict mortality in patients with ACS
  • as well as patients with assumed
    • stable coronary artery disease.

Furthermore, hs-cTn has the potential

  • to play a role in the care of patients
    • undergoing non-cardiac surgery.

The additional determination of hs-cTn

  • improves risk stratification despite
  • established risk scores providing both diagnosis and
  • for prognosis prediction in chest pain patients.

The daily clinical challenge in using the highly sensitive assays is to 

  • interpret the troponin concentrations, especially
  • in patients with concomitant diseases
    • independently from myocardial ischaemia
  • influencing cardiac troponin concentrations
    (e.g. chronic kidney disease, or stroke). 

The troponin test lost its ‘pregnancy test’ quality with the different users.
Different opinions exist on

  • the change of hs-cTn levels compared to simple ‘positive–negative’ interpretation
  • and thereby makes diagnosis finding more complex than before.

This uncertainty probably has the paradigm that

  • serial measurements of troponins are necessary, and also
    • boosted the number of diagnoses of ACS and
    • invasive diagnostic procedures in some locations.

This is more than understandable, with acute chest pain using

  • three high-sensitivity cardiac troponins with their respective baseline value
    • before the diagnosis of acute myocardial infarction (AMI) can be made.

What is a relevant change in concentrations compatible with acute myocardial necrosis and

  • what is only biological variation for the specific biomarker and assay?

Changes in serial measurements between 20% and 200% have been debated, and
the discussion is ongoing. Furthermore, it has been proposed that

  • absolute changes in cardiac troponin concentrations 
    • have a higher diagnostic accuracy for AMI
  • compared with relative changes, and

it might be helpful in distinguishing AMI from other causes of cardiac troponin elevation.

Do we obtain any helpful directives from experts and guidelines for our daily practice?
Foreseeing this dilemma, the 2011 European Society of Cardiology (ESC) Guidelines

  • on non ST-elevation ACS acted.
  • Minor elevations of  troponins were accepted as hs-cTn values in the ‘grey zone’.

This was and still is the rule, but

  • the ESC provided a general algorithm on how to manage patients with limited data.

The ‘Study Group on Biomarkers in Cardiology’ suggested

  • a rise of 50% from the baseline value at low concentrations.

However, this group of experts could also not find a substitute for the missing data

  • needed to validate the proposed recommendation.

The story is just too complex:

  • different troponin assays with
  • different epitope targets,
  • different patient populations,
  • different sampling protocols,
  • different follow-up lengths, and much more.

Therefore, any study that helps us to see better through the fog is welcome here.

Haaf et al. have now presented the results of their study of

  • different hs-cTn assays
    (hs-cTnT, Roche Diagnostics; hs-cTnI, Beckman-Coulter; and  hs-cTnI, Siemens)

    • with respect to the -outcome of patients with acute chest pain.

The authors examine 1117 consecutive patients presenting with acute chest pain.
[340 patients with ACS (30.5%)] from the Advantageous Predictors of Acute Coronary Syndrome
Evaluation (APACE) study. Blood was collected

  • directly on admission and
  • serially thereafter at 2, 3, and 6h.

Eighty-two patients (7.3%) died during the 2-year follow-up. The main finding of the study is that

  1. hs-cTnT predicts mortality more accurately than the hs-cTnI assays, 
  2. -that a single measurement is sufficient
  3. challenges causes of cardiac troponin elevation.

These results of APACE remain in contrast to recent findings from a GUSTO IV cohortof 1335 patients with ACS (Table1).

Table1 Studies investigating high sensitivity troponins for long-term prognosis

Variable                                                       APACE (n 5 1117)              GUSTO IV (n 5 1335)              PEACE (n 5 3567)


Patient cohort                                                   Unstable                            Unstable                               Stable

Blood sampling                                     On admission,1,2,3,6h                    48h after
study randomization           Before randomization

No. of patients with detection limit             883 (79.1%)                                 UKN                                      UKN

No. of patients with hs-cTnT.
99thpercentile                                        401 (35.9%)                              1015 (76%)                          395 (10.9%)

No. of patients with hs-cTnI (Abbott).
detection limit                                           UKN                                             UKN                              3567 (98.5%)

No.of patients with hs-cTnI (Abbott).
99th percentile                                          UKN                                         988(74%)                           105 (2.9%)

No. of patients with NSTEMI                     170 (15.2%)                              100 (100%)                             0 (0%)

Follow-up                                               24 months                                  12 months                            5.2 years

Non-fatal AMI                                           UKN                                              UKN                               209 (5.9%)

Mortality or primary endpoint                    82 (7.3%)                                 119(8.9%)                           203 (5.7%)


Key findings                                    cTnT better than cTnI                      cTnI ¼cTnT                   cTnI better than cTnT

Single cTn sample sufficient

AMI, acute mycordial infaction; cTn, cardiac tropononin; NSTEMI ,non-ST-elevation myocardial infarction; UKN, unknown

NSTEMI defined in the GUSTO IV trial:
  1. one or more episodes of angina lasting ≥ 5min,
  2. within 24h of admission and
  3. either a positive cardiac TnT or I test
    (above the upper limit of a normal for the local assay; during the years 1999 and 2000)
  4. or ≥ 0.5 mm of transient or persistent ST-segment depression.

the prognostic capacity of four different sensitive cardiac troponin assays were compared

  1. hs-cTnT; Roche Diagnostics,
  2. cTnI and hs-cTnI;
  3. Abbott Diagnostics, and
  4. Acc-cTnI; Beckman-Coulter.

In total, 119 patients (8.9%) died during the 1-year follow-up. Looking at their

  • receiver operating characteristic curve (ROC) analyses,
  • there were only negligible diffferences
    • in the area under the curves between the assays.

Contrasting results have also been reported in patients(n 1/4 3.623)

  • with stable coronary artery disease and preserved systolic left ventricular function

from the PEACE trial (Table1).

During a median follow-up period of 5.2 years,

  • there were 203 (5.6%) cardiovascular deaths or
  • first hospitalization for heart failure.

Concentrations of hs-cTnI (Abbott Diagnostics) at or above

  • the limit of detection of the assay were measured in 3567 patients (98.5%), but
  • concentrations of hs-cTnI at or above the gender-specific 99th percentile
    • were found in only 105 patients (2.9%).

This study revealed that

  • there was a strong and graded association
  • between increasing quartiles of hs-cTnI concentrations and
  • the risk for cardiovascular death or heart failure.

Hs-cTnI provided incremental prognostication information

  • over conventional risk markers and
  • other established cardiovascular biomarkers,
  • including hs-cTnT.

In contrast to the APACE results, only hs-cTnI, but

  • no ths-cTnT, was significantly
  • associated with the risk for AMI.

Is there a real difference between cardiac troponin T and cardiac troponin I

  • in predicting long term prognosis?

The question arises of whether there is a true clinically relevant

  • difference between cTnT and cTnI.

Given the biochemical and analytical differences,the two

  • troponins display rather similar serum profiles during AMI.

While minor biological differences between cTnT and cTnI are

  • apparently not relevant for diagnosis
  • and clinical management in the acute setting of ACS.

This is a provocative theory, but appears premature in our opinion.
Above all, the results of the current study appear

  • too inconsistent to allow such conclusions.

In the present study, hs-cTnT (Roche Diagnostics) outperformed

  • hs-cTnI (Siemens and Beckman-Coulter) in terms of
  • very long term prediction of cardiovascular death and
    • heart failure in stable patients.

We don’t know how hs-cTnI from Abbott Diagnostics

  • performs in the APACE consort.

The number of patients and endpoints provided

  • by the APACE registry are rather low.
  • The results could, therefore, be a chance finding.

It is far too early to favour one high sensitivity assay over the other. The findings need confirmation.

Implications for clinical practice

There is no doubt that high-sensitivity assays

  • are the analytical method of choice
    • in terms of risk stratification in patients with ACS.

What is new?
A single measurement of hs-cTn seems to be adequate

  • for long-term risk stratification in patients without AMI.

However, the question of which troponin might be preferable

  • for long-term risk stratification remains unanswered.

Part 2. ability of high-sensitivity cTnT and NT pro-BNP to predict cardiovascular events and death in patients with T2DM

Hillis GS; Welsh P; Chalmers J; Perkovic V; Chow CK; Li Q; Jun M; Neal B; Zoungas S; Poulter N; Mancia G; Williams B; Sattar N; Woodward M
Diabetes Care.  2014; 37(1):295-303 (ISSN: 1935-5548)


Current methods of risk stratification in patients with

  • type 2 diabetes are suboptimal.

The current study assesses the ability of

  • N-terminal pro-B-type natriuretic peptide (NT-proBNP) and
  • high-sensitivity cardiac troponin T (hs-cTnT)

to improve the prediction of cardiovascular events and death in patients with type 2 diabetes.


A nested case-cohort study was performed in 3,862 patients who participated in the Action in Diabetes and Vascular Disease:

Preterax and Diamicron Modified Release Controlled Evaluation (ADVANCE) trial.


Seven hundred nine (18%) patients experienced a

  • major cardiovascular event

(composite of cardiovascular death, nonfatal myocardial infarction, or nonfatal stroke) and

  • 706 (18%) died during a median of 5 years of follow-up.

In Cox regression models, adjusting for all established risk predictors,

  • the hazard ratio for cardiovascular events for NT-proBNP was 1.95 per 1 SD increase (95% CI 1.72, 2.20) and
  • the hazard ratio for hs-cTnT was 1.50 per 1 SD increase (95% CI 1.36, 1.65). The hazard ratios for death were
    • 1.97 (95% CI 1.73, 2.24) and
    • 1.52 (95% CI 1.37, 1.67), respectively.

The addition of either marker improved 5-year risk classification for cardiovascular events
(net reclassification index in continuous model,

  • 39% for NT-proBNP and 46% for hs-cTnT).

Likewise, both markers greatly improved the accuracy with which the 5-year risk of death was predicted.
The combination of both markers provided optimal risk discrimination.


NT-proBNP and hs-cTnT appear to greatly improve the accuracy with which the

  • risk of cardiovascular events or death can be estimated in patients with type 2 diabetes.

PreMedline Identifier: 24089534

Part 3. M-Atrial Natriuretic Peptide

M-Atrial Natriuretic Peptide and Nitroglycerin in a Canine Model of Experimental Acute Hypertensive Heart Failure:
Differential Actions of 2 cGMP Activating Therapeutics.

Paul M McKie, Alessandro Cataliotti, Tomoko Ichiki, S Jeson Sangaralingham, Horng H Chen, John C Burnett
Journal of the American Heart Association 01/2014; 3(1):e000206.
Source: PubMed


Systemic hypertension is a common characteristic in

  • acute heart failure (HF).

This increasingly recognized phenotype

  • is commonly associated with renal dysfunction and
  • there is an unmet need for renal enhancing therapies.

In a canine model of HF and acute vasoconstrictive hypertension

  • we characterized and compared the cardiorenal actions of M-atrial natriuretic peptide (M-ANP),
    a novel particulate guanylyl cyclase (pGC) activator, and
  • nitroglycerin, a soluble guanylyl cyclase (sGC) activator.

HF was induced by rapid RV pacing (180 beats per minute) for 10 days. On day 11, hypertension was induced by continuous angiotensin II
infusion. We characterized the cardiorenal and humoral actions

  • prior to,
  • during, and
  • following intravenous infusions of
  1. M-ANP (n=7),
  2. nitroglycerin (n=7),
  3. and vehicle (n=7) infusion.

Mean arterial pressure (MAP) was reduced by

  1. M-ANP (139±4 to 118±3 mm Hg, P<0.05) and
  2. nitroglycerin (137±3 to 116±4 mm Hg, P<0.05);

similar findings were recorded for

  1. pulmonary wedge pressure (PCWP) with M-ANP (12±2 to 6±2 mm Hg, P<0.05)
  2. and nitroglycerin (12±1 to 6±1 mm Hg, P<0.05).

M-ANP enhanced renal function with significant increases (P<0.05) in

  • glomerular filtration rate (38±4 to 53±5 mL/min),
  • renal blood flow (132±18 to 236±23 mL/min), and
  • natriuresis (11±4 to 689±37 mEq/min) and
  • also inhibited aldosterone activation (32±3 to 23±2 ng/dL, P<0.05), whereas

nitroglycerin had no significant (P>0.05) effects on these renal parameters or aldosterone activation.

Our results advance

the differential cardiorenal actions of

  • pGC (M-ANP) and sGC (nitroglycerin) mediated cGMP activation.

These distinct renal and aldosterone modulating actions make

M-ANP an attractive therapeutic for HF with concomitant hypertension, where

  • renal protection is a key therapeutic goal.

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Erythropoietin (EPO) and Intravenous Iron (Fe) as Therapeutics for Anemia in Severe and Resistant CHF: The Elevated N-terminal proBNP Biomarker


Co-Author of the FIRST Article: Larry H. Bernstein, MD, FCAP

Reviewer and Curator of the SECOND and of the THIRD Articles: Larry H. Bernstein, MD, FCAP


Article Architecture Curator: Aviva Lev-Ari, PhD, RN

This article presents Advances in the Treatment using Subcutaneous Erythropoietin (EPO) and Intravenous Iron (Fe) for IMPROVEMENT of Severe and Resistant Congestive Heart Failure and its resultant Anemia.  The Leading Biomarker for Congestive Heart Failure is an Independent Predictor identified as an Elevated N-terminal proBNP.

NT-proBNP schematic diagram-Copy.pdf_page_1


Anemia as an Independent Predictor of Elevated N-terminal proBNP

Salman A. Haq, MD1, Mohammad E. Alam2, Larry Bernstein, MD, FCAP3,  LB Banko 1, Leonard Y. Lee, MD, FACS4, Barry I. Saul, MD, FACC5, Terrence J. Sacchi, MD, FACC6,  John F. Heitner, MD, FACC7
1Cardiology Fellow,  2  Clinical Chemistry Laboratories, 3 Program Director, Cardiothoracic Surgery, 4 Division of Cardiology,  Department of Medicine, New York Methodist Hospital-Weill Cornell, Brooklyn, NY

(Unpublished manuscript)  Poster Presentation


The effect of correction of mild anemia in severe, resistant congestive heart failure using subcutaneous erythropoietin and intravenous iron: a randomized controlled study

Donald S Silverberg, MDa; Dov Wexler, MDa; David Sheps, MDa; Miriam Blum, MDa; Gad Keren, MDa; Ron Baruch, MDa; Doron Schwartz, MDa; Tatyana Yachnin, MDa; Shoshana Steinbruch, RNa; Itzhak Shapira, MDa; Shlomo Laniado, MDa; Adrian Iaina, MDa

J Am Coll Cardiol. 2001;37(7):1775-1780. doi:10.1016/S0735-1097(01)01248-7


The use of subcutaneous erythropoietin and intravenous iron for the treatment of the anemia of severe, resistant congestive heart failure improves cardiac and renal function and functional cardiac class, and markedly reduces hospitalizations

Donald S Silverberg, MDa; Dov Wexler, MDa; Miriam Blum, MDa; Gad Keren, MDa; David Sheps, MDa; Eyal Leibovitch, MDa; David Brosh, MDa; Shlomo Laniado, MDa; Doron Schwartz, MDa; Tatyana Yachnin, MDa; Itzhak Shapira, MDa; Dov Gavish, MDa; Ron Baruch, MDa; Bella Koifman, MDa; Carl Kaplan, MDa; Shoshana Steinbruch, RNa; Adrian Iaina, MDa

J Am Coll Cardiol. 2000;35(7):1737-1744. doi:10.1016/S0735-1097(00)00613-6


This THREE article sequence is related by investigations occurring by me, a very skilled cardiologist and his resident, and my premedical student at New York Methodist Hospital-Weill Cornell, in Brooklyn, NY, while a study had earlier been done applying the concordant discovery, which the team in Israel had though was knowledge neglected.  There certainly was no interest in the problem of the effect of anemia on the patient with severe congestive heart failure, even though erythropoietin was used widely in patients with end-stage renal disease requiring dialysis, and also for patients with myelofibrosis.  The high cost of EPO was only one factor, the other being a guideline to maintain the Hb concentration at or near 11 g/dl – not higher.  In the first article, the authors sought to determine whether the amino terminal pro– brain type natriuretic peptide (NT-pro BNP) is affected by anemia, and to determine that they excluded all patients who had renal insufficiency and/or CHF, since these were associated with elevated NT-proBNP.  It was already well established that this pro-peptide is secreted by the heart with the action as a urinary sodium retention hormone on the kidney nephron, the result being an increase in blood volume.  Perhaps the adaptation would lead to increased stroke volume from increased venous return, but that is not conjectured.  However, at equilibrium, one would expect there to be increased red cell production to maintain the cell to plasma volume ratio, thereby, resulting in adequate oxygen exchange to the tissues.  Whether that is always possible is uncertain because any reduction in the number of functioning nephrons would make the kidney not fully responsive at the Na+ exchange level, and the NT-pro BNP would rise.  This introduces complexity into the investigation, requiring a removal of confounders to establish the effect of anemia.

The other two articles are related studies by the same group in Israel.  They surmised that there was evidence that was being ignored as to the effect of anemia, and the treatment of anemia was essential in addition to other treatments.  They carried out a randomized trial to determine just that, a benefit to treating the anemia.  But they also conjectured that an anemia with a Hb concentration below 12 g/dl has an deleterious effect on the targeted population.  Treatment by intermittent transfusions could potentially provide the added oxygen-carrying capacity, but the fractionation of blood, the potential for transfusion-transmitted disease and transfusion-reactions, combined with the need for the blood for traumatic blood loss made EPO a more favorable alternative to packed RBCs.  The proof-of-concept is told below.  Patients randomized to receive EPO at a lower than standard dose + iron did better.



In this article, Erythropoietin (EPO) and Intravenous Iron (Fe) as Therapeutics for Anemia in Severe and Resistant CHF: The Elevated N-terminal proBNP Biomarker we provides a summary of three articles on the topic and we shading new light on the role that Anemia Hb < 12 g%  plays as a Biomarker for CHF and for

  • prediction of elevated BNP, known as an indicator for the following Clinical Uses:
Clinical Use
  • Rule out congestive heart failure (CHF) in symptomatic individuals
  • Determine prognosis in individuals with CHF or other cardiac disease
  • Maximize therapy in individuals with heart failure by the use of Subcutaneous Erythropoietin (EPO) and Intravenous Iron (Fe)
Evaluation of BNP and NT-proBNP Clinical Performance
Study Sensitivity(%) Specificity(%) PPV(%) NPV(%)
Diagnose impaired LVEF3
BNP 73 77 70 79
NT-proBNP 70 73 61 80
Diagnose LV systolic dysfunction after MI2
BNP 68 69 56 79
NT-proBNP 71 69 56 80
Diagnose LV systolic dysfunction after MI12
BNP 94 40 NG 96
NT-proBNP 94 37 NG 96
Prognosis in newly diagnosed heart failure patients: prediction of mortality/survival1
BNP 98 22 42 94
NT-proBNP 95 37 47 93
Prognosis post myocardial infarction: prediction of mortality2
BNP 86 72 39 96
NT-proBNP 91 72 39 97
Prognosis post myocardial infarction: prediction of heart failure2
BNP 85 73 54 93
NT-proBNP 82 69 50 91
PPV, positive predictive value; NPV, negative predictive value; LVEF, left ventricular ejection fraction; NG, not given.
Reference Range
BNP: < 100 pg/mL13
proBNP, N-terminal: 300 pg/mL
The NT-proBNP reference range is based on EDTA plasma. Other sample types will produce higher values.
Interpretive Information
Symptomatic patients who present with a BNP or NT-proBNP level within the normal reference range are highly unlikely to have CHF. Conversely, an elevated baseline level indicates the need for further cardiac assessment and indicates the patient is at increased risk for future heart failure and mortality.BNP levels increase with age in the general population, with the highest concentrations seen in those greater than 75 years of age.14 Heart failure is unlikely in individuals with a BNP level <100 pg/mL and proBNP level ≤300 pg/mL. Heart failure is very likely in individuals with a BNP level >500 pg/mL and proBNP level ≥450 pg/mL who are <50 years of age, or ≥900 pg/mL for patients ≥50 years of age. Patients in between are either hypertensive or have mild ischemic or valvular disease and should be observed closely.15BNP is increased in CHF, left ventricular hypertrophy, acute myocardial infarction, atrial fibrillation, cardiac amyloidosis, and essential hypertension. Elevations are also observed in right ventricular dysfunction, pulmonary hypertension, acute lung injury, subarachnoid hemorrhage, hypervolemic states, chronic renal failure, and cirrhosis.NT-proBNP levels are increased in CHF, left ventricular dysfunction, myocardial infarction, valvular disease, hypertensive pregnancy, and renal failure, even after hemodialysis.Although levels of BNP and NT-proBNP are similar in normal individuals, NT-proBNP levels are substantially greater than BNP levels in patients with cardiac disease due to increased stability (half-life) of NT-proBNP in circulation. Thus, results from the two tests are not interchangeable.
  1. Cowie MR and Mendez GF. BNP and congestive heart failure. Prog Cardiovasc Dis. 2002;44:293-321.
  2. Richards AM, Nicholls MG, Yandle TG, et al. Plasma N-terminal pro-brain natriuretic peptide and adrenomedullin. New neurohormonal predictors of left ventricular function and prognosis after myocardial infarction. Circulation. 1998:97:1921-1929.
  3. Hammerer-Lercher A, Neubauer E, Muller S, et al. Head-to-head comparison of N-terminal pro-brain natriuretic peptide, brain natriuretic peptide and N-terminal pro-atrial natriuretic peptide in diagnosing left ventricular dysfunction. Clin Chim Acta. 2001;310:193-197.
  4. McDonagh TA, Robb SD, Murdoch DR, et al. Biochemical detection of left-ventricular systolic dysfunction. Lancet. 1998;351:9-13.
  5. Mukoyama Y, Nakao K, Hosoda K, et al. Brain natriuretic peptide as a novel cardiac hormone in humans: Evidence for an exquisite dual natriuretic peptide system, ANP and BNP. J Clin Invest. 1991;87:1402-1412.
  6. Hunt PJ, Richards AM, Nicholls MG, et al. Immunoreactive amino-terminal pro-brain natriuretic peptide (NT-PROBNP): a new marker of cardiac impairment. Clin Endocrinol. 1997;47:287-296.
  7. Davis M, Espiner E, Richards G, et al. Plasma brain natriuretic peptide in assessment of acute dyspnoea. Lancet. 1994;343:440-444.
  8. Kohno M, Horio T, Yokokawa K, et al. Brain natriuretic peptide as a cardiac hormone in essential hypertension. Am J Med. 1992;92:29-34.
  9. Bettencourt P, Ferreira A, Pardal-Oliveira N, et al. Clinical significance of brain natriuretic peptide in patients with postmyocardial infarction. Clin Cardiol. 2000;23:921-927.
  10. Jernberg T, Stridsberg M, Venge P, et al. N-terminal pro brain natriuretic peptide on admission for early risk stratification of patients with chest pain and no ST-segment elevation. J Am Coll Cardiol. 2002;40:437-445.
  11. Richards AM, Troughton RW. Use of natriuretic peptides to guide and monitor heart failure therapy. Clin Chem. 2012;58:62-71.
  12. Pfister R, Scholz M, Wielckens K, et al. The value of natriuretic peptides NT-pro-BNP and BNP for the assessment of left-ventricular volume and function. A prospective study of 150 patients.Dtsch Med Wochenschr. 2002;127:2605-2609.
  13. Siemens ADVIA Centaur® BNP directional insert; 2003.
  14. Redfield MM, Rodeheffer RJ, Jacobsen SJ, et al. Plasma brain natriuretic peptide concentration: impact of age and gender. J Am Coll Cardiol. 2002;40:976-982.
  15. Weber M, Hamm C. Role of B-type natriuretic peptid (BNP) and NT-proBNP in clinical routine.Heart. 2006;92:843-849.


B-type Natriuretic Peptide and proBNP, N-terminal


Anemia as an Independent Predictor of Elevated N-terminal proBNP

Salman A. Haq, MD1, Mohammad E. Alam2, Larry Bernstein, MD, FCAP3,  LB Banko 1, Leonard Y. Lee, MD, FACS4, Barry I. Saul, MD, FACC5, Terrence J. Sacchi, MD, FACC6,  John F. Heitner, MD, FACC7
1Cardiology Fellow,  2  Clinical Chemistry Laboratories, 3 Program Director, Cardiothoracic Surgery, 4 Division of Cardiology,  Department of Medicine, New York Methodist Hospital-Weill Cornell, Brooklyn, NY

(Unpublished manuscript)  Poster Presentation:

Anemia as an Independent Predictor of Elevated N-Terminal proBNP Levels in
Patients without Evidence of Heart Failure and Normal Renal Function.

Haq SA, Alam ME, Bernstein L, Banko LB, Saul BI, Lee LY, Sacchi TJ, Heitner JF.

Table 1.  Patient Characteristics

Variable No Anemia(n=138) Anemia(n=80)
Median Age (years) 63 76
Men (%) 35 33
Creatinine (mg/dl) 0.96 1.04
Hemoglobin (g/dl) 13.7 10.2
LVEF (%) 67 63
Median NT-proBNP (pg/ml) 321.6 1896.0


A series of slide showing the determination of the representation of normal NT-proBNP range
after removal of patient confounders.







N-terminal proBNP (NT-proBNP) has emerged as a primary tool for diagnosing congestive heart failure (CHF). Studies have shown that the level of

  • NT-proBNP is affected by renal insufficiency (RI) and age, independent of the diagnosis of CHF.

There is some suggestion from recent studies that

  • anemia may also independently affect NT-proBNP levels.


To assess the affect of anemia on NT-proBNP independent of CHF, RI, and age.


We evaluated 746 consecutive patients presenting to the Emergency Department (ED) with shortness of breath and underwent evaluation with serum NT-proBNP.

All patients underwent a trans-thoracic echocardiogram (TTE) and clinical evaluation for CHF. Patients were included in this study if they had a normal TTE (normal systolic function, mitral inflow pattern and left ventricular (LV) wall thickness) and no evidence of CHF based on clinical evaluation. Patients were excluded if they had RI (creatinine > 2 mg/dl) or a diagnosis of sepsis. Anemia was defined using the World Health Organization (W.H.O.) definition of

  • hemoglobin (hgb) < 13 g/dl for males and hgb < 12 g/dl for females.


Of the 746 consecutive patients, 218 patients (138 anemia, 80 no anemia) met the inclusion criteria. There was a markedly significant difference between

  • NT- proBNP levels based on the W.H.O. diagnosis of anemia.

Patients with anemia had a

  • mean NT- proBNP of 4,735 pg/ml compared to 1,230 pg/ml in patients without anemia (p=0.0001).

There was a markedly

  • significant difference in patients who had a hgb > 12 (median 295 pg/ml) when compared to
  • both patients with an hgb of 10.0 to 11.9 (median 2,102 pg/ml; p = 0.0001) and
  • those with a hgb < 10 (median 2,131 pg/ml; p = 0.001).

Linear regression analysis comparing hgb with log NT-proBNP was statistically significant (r = 0.395; p = 0.0001). MANOVA demonstrated that

  • elevated NT- proBNP levels in patients with anemia was independent of age.


This study shows that NT-proBNP is associated with anemia independent of CHF, renal insufficiency, sepsis or age.


B-type natriuretic peptide (BNP) is secreted from the myocardium in response to myocyte stretch. 1-2 BNP is released from the myocytes as a 76 aminoacid N-terminal fragment (NT-proBNP) and a 32-amino acid active hormone (BNP). 3 These peptides have emerged as a primary non-invasive modality for the diagnosis of congestive heart failure (CHF). 4- 7 In addition, these peptides have demonstrated prognostic significance in patients with invasive modality for the diagnosis of

  • congestive heart failure (CHF). 4- 7
  • heart failure 8-9,
  • stable coronary artery disease 10, and
  • in patients with acute coronary syndromes. 11-14

Studies have shown that the level of NT- proBNP is affected by

  • age and renal insufficiency (RI) independent of the diagnosis of CHF. 15,16

There is some suggestion from the literature that

  • anemia may also independently affect NT-proBNP levels. 17-20

Willis et al. demonstrated in a cohort of 209 patients without heart failure that anemia was associated with an elevated NT- proBNP. 17 Similarly, in 217 patients undergoing cardiac catheterization, blood samples were drawn from the descending aorta prior to contrast ventriculography for BNP measurements and

  • anemia was found to be an independent predictor of plasma BNP levels. 18

The objective of this study is to assess the effect of anemia on NT-proBNP independent of CHF, sepsis, age or renal insufficiency.


Patient population

The study population consisted of 746 consecutive patients presenting to the emergency room who underwent NT-proBNP evaluation for the evaluation of dyspnea. Transthoracic echocardiogram (TTE) was available on 595 patients. Patients were included in this study if they had a normal TTE, which was defined as normal systolic function (left ventricular ejection fraction [LVEF] > 45%), normal mitral inflow pattern and normal LV wall thickness. CHF was excluded based on thorough clinical evaluation by the emergency department attending and the attending medical physician. Patients with disease states that may affect the NT- proBNP levels were also excluded:

  1. left ventricular systolic dysfunction (LVEF < 45%),
  2. renal insufficiency defined as a creatinine > 2 mg/dl and
  3. sepsis (defined as positive blood cultures with two or more of the following systemic inflammatory response syndrome (SIRS) criteria: heart rate > 90 beats per minute;
  4. body temperature < 36 (96.8 °F) or > 38 °C (100.4 °F);
  5. hyperventilation (high respiratory rate) > 20 breaths per minute or, on blood gas, a PaCO2 less than 32 mm Hg;
  6. white blood cell count < 4000 cells/mm3 or > 12000 cells/mm³ (< 4 x 109 or > 12 x 109 cells/L), or greater than 10% band forms (immature white blood cells). 21

The study population was then divided into two groups, anemic and non- anemic. Anemia was defined using the world health organization (W.H.O.) definition of hemoglobin (hgb) < 13 g/dl for males and < 12 g/dl for females.The data was also analyzed by dividing the patients into three groups based on hgb levels i.e. hgb > 12, hgb 10 to 11.9 and hgb < 10.

Baseline patient data

Patient’s baseline data including age, gender, ethnicity, hemoglobin (hgb), hematocrit (hct), creatinine, NT- proBNP were recorded from the electronic medical record system in our institution. Chemistry results were performed on the Roche Modular System (Indianapolis, IN), with the NT- proBNP done by chemiluminescence assay. The hemogram was performed on the Beckman Coulter GenS. All TTE’s were performed on Sonos 5500 machine. TTE data collected included LVEF, mitral inflow pattern and LV wall thickness assessment.

Statistical analysis

The results are reported in the means with p < 0.05 as the measure of significance for difference between means. Independent Student’s t-tests were done comparing NT proBNP and anemia. Univariate ANOVAs and multivariate ANOVA (MANOVA) with post hoc tests using the Bonferroni method were used to compare NT- proBNP levels with varying ranges of hgb and age using SPSS 13.0 (SPSS, Chicago, IL). A linear regression analysis was performed using SYSTAT. Calculations included Wilks’Lamda, Pillai trace and Hotelling-Lawley trace. A GOLDMineR® plot was constructed to estimate the effects of age and anemia on NT- proBNP levels. The GOLDMineR® effects plot displays the odds-ratios for predicted NT-proBNP elevation versus the predictor values. Unlike the logistic regression, the ordinal regression, which the plot is derived from, can have polychotomous as well as dichotomous values, as is the case for NT-proBNP.


Of the 746 consecutive patients, 218 patients met the inclusion criteria (fig 1). Baseline characteristics of patients are listed in table 1. The median age for anemic patients was 76 years and 63 years for patients without anemia. One third of patients in both groups were men. The mean hemoglobin for

  • anemic patients was 10.2 g/dl as compared to 13.7 g/dl for non-anemic patients.
  • The mean LVEF of patients with anemia was 64% as compared to 67% for non-anemic patients.

Based on the WHO definition of anemia, 138 patients were determined to be anemic while 80 patients were diagnosed as non-anemic. There was a markedly  significant difference between NT-proBNP levels based on the WHO diagnosis of anemia.

Patients with anemia had a

  • mean NT-proBNP of 4,735 pg/ml compared to 1,230 pg/ml in patients without anemia (p = 0.0001).

Of the 218 patients in the study, 55 patients had a hgb of < 10 g/dl. Analysis using

  • hgb < 10 g/dl for anemia demonstrated a statistically significant difference in the NT-proBNP values.

Patients with a hgb < 10 g/dl had a mean NT- proBNP of 5,130 pg/ml

  • compared to 2,882 pg/ml in patients with a hgb of > 10 g/dl (p = 0.01)

The groups were also divided into three separate categories of hgb for subset analysis:

  • hgb > 12 g/dl,
  • hgb 10 to 11.9 g/dl and
  • hgb < 10 g/dl.

There was a markedly significant difference in

  •  the NT- ProBNP levels of patients who had a hgb > 12 g/dl (median 295 pg/ml) when
  • compared to those with a hgb range of 10.0 g/dl to 11.9 g/dl (median 2,102 pg/ml) (p = 0.0001),

and also a significant difference in

  • NT- proBNP levels of patients with a hgb > 12 g/dl (median 295 pg/ml) when
  • compared to a hgb of < 10 g/dl (median 2,131 pg/ml) (p = 0.001).

However, there was no statistically significant difference in NT-proBNP levels of patients with hgb 10 g/dl to 11.9 g/dl

  • when compared to those with a hgb of < 10 g/dl (p = 1.0).

A scatter plot comparing hgb with log NT-proBNP and fitting of a line to the data by ordinary least squares regression was significant (p = 0.0001) and demonstrated

  • a correlation between anemia and NT-proBNP levels (r = 0.395) (fig. 2).

MANOVA demonstrated that elevated NT- proBNP levels in patients with anemia was independent of age (Wilks’ Lambda [p = 0.0001]). In addition, using GOLDMineR® plots (figure 3a and 3b) with a combination of age and hb scaled as predictors of elevated NT-proBNP,

  • both age and hgb were required as independent predictors.

What about the effect of anemia? The GOLDminer analysis of ordinal regression was carried out in a database from which renal insufficiency and CHF were removed. The anemia would appear to have an independent effect on renal insufficiency. Figure 4 is a boxplot comparison of NT – proBNP, the age normalized function NKLog (NT- proBNP)/eGFR formed from taking 1000*Log(NT- proBNP) divided by the MDRD at eGFR exceeding 60 ml/min/m2 and exceeding 30 ml/min/m2. The transformed variable substantially makes the test independent of age and renal function. The boxplot shows the medians, 97.5, 75, 25 and 2.5 percentiles. There appears to be no significance in the NKLog(NT pro-BNP)/MDRD plot. Table II compares the NT-proBNP by WHO criteria at eGFR 45, 60 and 75 ml/mln/m2 using the t-test with unequal variance assumed, and the Kolmogorov-Smirnov test for nonparametric measures of significance. The significance at 60 ml/min/m2 is marginal and nonexistent at 75 ml/min/m2. This suggests that the contribution from renal function at above 60 ml/min2 can be ignored. This is consistent with the findings using the smaller, trimmed database, but there is an interaction between

  •  anemia, and
  •  eGFR at levels below 60 ml/min/m2


The findings in this study indicate that

  • anemia was associated with elevated NT-proBNP levels independent of CHF, renal insufficiency, sepsis or age.

These findings have been demonstrated with NT-proBNP in only one previous study. Wallis et al. demonstrated that after adjusting for age, sex, BMI, GFR, LVH and valvular disease;

  1. only age,
  2. valvular disease and
  3. low hemoglobin

were significantly associated with increased NT-proBNP. 18.

In our study, CHF was excluded based on both a normal TTE and a thorough clinical evaluation. In the only other study directly looking at NT- proBNP levels in anemic patients without heart failure

  • only 25% of patients had TTEs, with one patient having an LVEF of 40%. 17

BNP, the active molecule released after cleavage along with NT- proBNP, has also been studied in relation to blood hemoglobin levels. 18 In 263 patients undergoing cardiac catheterization  blood samples were drawn from the descending aorta prior to contrast ventriculography to determine the value of BNP. Anemia was present in 217 patients. Multivariate linear regression model adjusting for

  1.  age,
  2.  gender,
  3.  body mass index,
  4.  history of myocardial infarction,
  5.  estimated creatinine clearance, and
  6.  LVEF
  • found hgb to be an independent predictor of BNP levels.

In our study, patients with anemia were slightly older than those without anemia. However, both MANOVA and GOLDMineR® plot demonstrated that

  • elevated NT-proBNP levels in patients with anemia was independent of age.

Other studies have found that BNP is dependent on renal insufficiency and age. Raymond et al. randomly selected patients to complete questionnaires regarding CHF and

  1. then underwent pulse and blood pressure measurements,
  2.  electrocardiogram (ECG),
  3.  echocardiography and
  4.  blood sampling. 15

A total of 672 subjects were screened and 130 were determined to be normal, defined as

  • no CHF or ischemic heart disease,
  • normal LVEF,
  • no hypertension,
  • diabetes mellitus,
  • lung disease, and
  • not on any cardiovascular drugs.

They found

  1. older age,
  2. increasing dyspnea,
  3. high plasma creatinine and a
  4. LVEF < 45%

to be independently associated with an elevated NT-proBNP plasma level by multiple linear regression analysis. In another study, McCullough et al. evaluated the patients from the Breathing Not Properly Multinational Study

  • looking at the relationship between BNP and renal function in CHF. 16

Patients were excluded if they were on hemodialysis or had a estimated glomerular filteration rate (eGFR) of < 15 ml/min. They found that the BNP levels correlated significantly with the eGFR, especially in patients without CHF, suggesting

  1. chronic increased blood volume and
  2. increased left ventricular wall tension as a possible cause. 16

Our study was designed to exclude patients with known diseases such as CHF and renal insufficiency in order to demonstrate

  • the independent effect of anemia on elevated NT-proBNP levels.

The mechanism for elevated NT-proBNP levels in patients with anemia is unknown. Some possible mechanisms that have been reported in the literature include

  • hemodilution secondary to fluid retention in patients with CHF 18,
  • decreased oxygen carrying capacity with accompanying tissue hypoxia which
  • stimulates the cardio-renal compensatory mechanism leading to increased release of NT-proBNP. 17

The findings from our study suggest that

  •  NT-proBNP values should not be interpreted in isolation of hemoglobin levels and
  • should be integrated with other important clinical findings for the diagnosis of CHF.

Further studies are warranted

  1.  to assess the relationship between anemia and plasma natriuretic peptides, and
  2. possibly modify the NT-proBNP cutoff points for diagnosing acutely decompensated CHF in patients with anemia.


This study shows that elevated NT-proBNP levels are associated with anemia independent of

  •   CHF,
  •  renal insufficiency,
  •  sepsis and
  •  age.

NT-proBNP levels should be interpreted with caution in patients who have anemia.


1. Brunea BG, Piazza LA, de Bold AJ. BNP gene expression is specifically modulated by stretch and ET-1 in a new model of isolated rat atria.Am J Physiol  1997; 273:H2678-86.

2. Wiese S, Breyer T, Dragu A, et al. Gene expression of brain natriuretic peptide  in isolated atrial and ventricular human myocardium: influence of angiotensin II and diastolic fiber length. Circ 2000; 102:3074-79.

3. de Lemos JA, McGuire DK, Drazner MH. B-type natriuretic peptide in cardiovascular disease. Lancet 2003; 362:316-22.

4.   Dao Q, Krishnaswamy P, Kazanegra R, et al. Utility of B-type natriuretic  peptide in the diagnosis of congestive heart failure in an urgent care setting. J Am  Coll Cardiol 2001; 37:379-85.

5. Morrison LK, Harrison A, Krishnaswamy P, Kazanegra R, Clopton P, Maisel A. Utility of rapid natriuretic peptide assay in differentiating congestive heart failure from lung  disease in patients presenting with dyspnea.
J Am Coll Cardiol  2003; 39:202-09.

6.  Maisel AS, Krishnaswamy P, Nowak RM, et al.  Rapid measurement of B-type natriuretic peptide in the emergency diagnosis of heart failure. N Engl J Med 2002; 347:161-67.

7. Januzzi JL, Camargo CA, Anwaruddin S, et al. The N-terminal Pro-BNP investigation of dyspnea in the emergency department (PRIDE) study. Am J  Cardiol 2005; 95:948-954.

8.  Tsutamoto T, Wada A, Meada K, et al.   Attenuation of compensation of  endogenous cardiac natriuretic peptide system  in chronic heart failure: prognostic role  of plasma  brain natriuretic peptide concentration in patients with chronic  symptomatic  left ventricular dysfunction.
Circulation 1997; 96(2): 509-16.

9.  Anand IS, Fisher LD, Chiang YT, et al. Changes in brain natriuretic peptide and norepinephrine over time and mortality and morbidity in the Valsartan Heart Failure Trial (Val-HEFT). Circulation 2003; 107:1278-1283.

10. Omland T, Richards AM, Wergeland R and Vik-Mo H. B-type natriuretic peptide and long term survival in patients with stable coronary artery disease.
Am J Cardiol 2005; 95:24-28.

11. Omland T, Aakvaag A, Bonarjee VV. et al. Plasma brain natriuretic peptide as an indicator of left ventricular systolic dysfunction and long term prognosis after acute myocardial infarction. Comparison with plasma atrial natriuretic peptide and N-terminal proatrial natriuretic peptide.
Circulation 1996; 93:1963-1969.

12. de Lemos JA, Morrow DA, Bently JH, et al. The prognostic value of B-type natriuretic peptide in patients with acute coronary syndromes. N Engl J Med 2001; 345:1014-1021.

13. Richards AM, Nicholls MG, Espiner EA, et al. B-type natriuretic peptides and  ejection fraction for prognosis after myocardial infarction. Circulation 2003; 107:2786-2792.

14. Sabatine MS, Morrow DA, de Lemos JA, et al.  Multimarker approach to risk  stratification in non-ST elevation acute coronary syndromes: simultaneous  assessment of troponin I, C-reactive protein and B-type natriuretic peptide.
Circulation 2002; 105:1760-1763.

15. Raymond I, Groenning BA, Hildebrandt PR, Nilsson JC, Baumann M, Trawinski   J, Pedersen F.  The influence of age, sex andother variables on the plasma level of N-terminal pro brain natriureticpeptide in a large sample of the general  population. Heart 2003; 89:745-751.

16. McCollough PA, Duc P, Omland T, McCord J, Nowak RM, Hollander JE, et al. B-type natriuretic peptide and renal function in the diagnosis of heartfailure:  an analysis from the  Breathing Not Properly Multinational Study.
Am J Kidney Dis 2003; 41:571-579.

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19. Tsuji H, Nishino N, Kimura Y, Yamada K, Nukui M, et al. Haemoglobin level influences plasma brain natriuretic peptide concentration. Acta Cardiol 2004;59:527-31.

20. Wu AH, Omland T, Wold KC, McCord J, Nowak RM, et al. Relationship  of B-type natriuretic peptide and anemia  in patients withand without heart failure:  A substudy from the Breathing Not Properly(BNP) Multinational Study.
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22. Bone RC, Balk RA, Cerra FB, Dellinger RP, Fein AM, et al.  Definitions for sepsis and organ failure and guidelines for theuse of innovative therapies in sepsis.  The ACCP/SCCM Consensus Conference Committee. Chest. 1992;101(6):1644-55.

Table Legends

Table I. Clinical characteristics of the study population

Table II. Comparison of NT- proBNP means under WHO criteria at different GFR

Table I
Variable No Anemia(n=80) Anemia(n=138)
Median age (years) 63 76
    Men (%) 27 (34) 47 (34)
    Women (%) 53 (66) 91 (66)
Weight (kg) 82.9 80.1
Chest Pain 21 (26) 3 (2)
Hemoglobin (g/dl) 13.7 10.2
Hematocrit (%) 40.5 30.5
Mean Corpuscular Volume 97 87
Creatinine (mg/dl) 0.99 1.07
Median NT-proBNP (pg/ml) 321 1896
Medical History
    HTN (%) 12 (15) 51 (37)
    Prior MI (%) 11 (14) 5 (4)
    ACS (%) 16 (20) 3 (2)
    CAD (%) 2 (1) 3 (2)
     DM (%) 18 (22) 11 (8)
   Clopidogrel 58 (72) 15 (11)
   Beta Blockers 68 (85) 27 (20)
   Ace Inhibitors 45 (56) 18 (13)
   Statins 57 (71) 17 (12)
   Calcium Channel Blocker 17 (21) 8 (6)
LVEF (%) 67 64

HTN: Hypertension CAD: Coronary Artery Disease
MI: Myocardial Infarction DM: Diabetes Mellitus
ACS: Acute Coronary Syndrome LVEF: Left Ventricular Ejection Fraction

Table II
GFR WHO Mean P (F) N NPar
> 45 0 3267 0.022 (4.33) 661
1 4681
> 60* 0 2593 0.031 (5.11) 456 0.018
1 4145
> 60r 0 786 0.203 (3.63) 303 0.08
1 3880
> 75 0 2773 > 0.80 320 0.043
1 3048

*AF, valve disease and elevated troponin T included
r AF, valve disease and elevated troponin T removed


FIGURE 1. Study population flow chart. (see poster)
FIGURE 2. Relationship between proBNP and hemoglobin. (see above)
FIGURE 3. NT-proBNP levels in relation to anemia (see above)

Supplementary Material

Table based on LatentGOLD Statistical Innovations, Inc., Belmont, MA., 2000: Jeroen Vermunt & Jay Magidson)

4-Cluster Model

Number of cases                                   408
Number of parameters (Npar)             24

Chi-squared Statistics
Degrees of freedom (df)                          71                     p-value
L-squared (L²)                                    80.2033                    0.21
X-squared                                            80.8313                     0.20
Cressie-Read                                        76.6761                     0.30
BIC (based on L²)                          -346.5966
AIC3 (based on L²)                        -132.7967
CAIC (based on L²)                       -417.5966

Model for Clusters
 Intercept                Cluster1      Cluster2     Cluster3     Cluster4     Wald     p-value
————–           0.1544           0.1434        0.0115        -0.3093     1.1981     0.75
Cluster Size           0.2870          0.2838       0.2487          0.1805

< 1.5                       0.0843           0.2457       0.0006          0.0084
1.6-2.5                   0.6179            0.6458       0.0709          0.2809
2.5-3.5                  0.2848           0.1067         0.5319          0.5883
> 3.5                      0.0130           0.0018         0.3966         0.1224
> 90                     0.1341             0.7919         0.0063         0.6106
61-90                  0.6019            0.2040          0.1633         0.3713
41-60                  0.2099            0.0041          0.3317         0.0175
< 41                     0.0542            0.0001         0.4987        0.0006
under 51           0.0668           0.5646          0.0568        0.0954
51-70                 0.3462            0.3602          0.3271         0.3880
over 70             0.5870            0.0752          0.6161         0.5166
No anemia      0.7518             0.6556          0.2041         0.0998
Anemia            0.2482             0.3444          0.7959         0.9002

———          Cluster1          Cluster2      Cluster3      Cluster4
Overall           0.2870            0.2838         0.2487        0.1805

< 1.5                0.2492              0.7379           0.0013         0.0116
1.6-2.5            0.4163               0.4243           0.0427        0.1167
2.6-3.5           0.2296               0.0887          0.3723        0.3095
> 3.5              0.0328                0.0023          0.7982        0.1666
> 90              0.1001                0.5998           0.0043        0.2958
61-90           0.5198                 0.1716           0.1136         0.1950
41-60           0.3860                 0.0055          0.5847         0.0238
< 41             0.1205                  0.0002          0.8785         0.0008
< 51            0.0720                 0.7458           0.0910          0.0912
51-70         0.3036                 0.3084           0.2013          0.1867
over 70     0.3773                  0.0409          0.3633           0.2186
No anemia 0.4589              0.3957           0.1076           0.0378
Anemia     0.1342                 0.1844            0.3742           0.3073

Hemoglobin on NT proBNP 3


The effect of correction of mild anemia in severe, resistant congestive heart failure using subcutaneous erythropoietin and intravenous iron: a randomized controlled study

Donald S Silverberg, MDa; Dov Wexler, MDa; David Sheps, MDa; Miriam Blum, MDa; Gad Keren, MDa; Ron Baruch, MDa; Doron Schwartz, MDa; Tatyana Yachnin, MDa; Shoshana Steinbruch, RNa; Itzhak Shapira, MDa; Shlomo Laniado, MDa; Adrian Iaina, MDa

J Am Coll Cardiol. 2001;37(7):1775-1780. doi:10.1016/S0735-1097(01)01248-7


This is a randomized controlled study of anemic patients with severe congestive heart failure (CHF) to assess the effect of correction of the anemia on cardiac and renal function and hospitalization.


Although mild anemia occurs frequently in patients with CHF, there is very little information about the effect of correcting it with erythropoietin (EPO) and intravenous iron.


Thirty-two patients with moderate to severe CHF (New York Heart Association [NYHA] class III to IV)
who had a left ventricular ejection fraction (LVEF) of 40% despite maximally tolerated doses of CHF medications and
  • whose hemoglobin (Hb) levels were persistently between 10.0 and 11.5 g% were randomized into two groups.
Group A (16 patients) received subcutaneous EPO and IV iron to increase the level of Hb to at least 12.5 g%. In Group B (16 patients) the anemia was not treated. The doses of all the CHF medications were maintained at the maximally tolerated levels except for oral and intravenous (IV) furosemide, whose doses were increased or decreased according to the clinical need.


Over a mean of 8.2 +/- 2.6 months,
  • four patients in Group B and none in Group A died of CHF-related illnesses.
  • The mean NYHA class improved by 42.1% in A and worsened by 11.4% in B.
  • The LVEF increased by 5.5% in A and decreased by 5.4% in B.
  • The serum creatinine did not change in A and increased by 28.6% in B.
  • The need for oral and IV furosemide decreased by 51.3% and 91.3% respectively in A and increased by 28.5% and 28.0% respectively in B.
  • The number of days spent in hospital compared with the same period of time before entering the study decreased by 79.0% in A and increased by 57.6% in B.


When anemia in CHF is treated with EPO and IV iron, a marked improvement in cardiac and patient function is seen,
  • associated with less hospitalization and renal impairment and less need for diuretics. (J Am Coll Cardiol 2001;37:1775– 80)

Anemia of any cause is known to be capable of causing congestive heart failure (CHF) (1). In patients hospitalized with CHF the 

  • mean hemoglobin (Hb) is about 12 g% (2,3),

which is considered the lower limit of normal in adults (4). Thus, anemia appears to be

common in CHF. Recently, in 142 patients in our special CHF outpatient clinic, we found that

  • as the CHF worsened, the mean Hb concentration decreased, from 13.7 g% in mild CHF (New York Heart Association [NYHA] class I) to 10.9 g% in severe CHF (NYHA 4), and
  • the prevalence of a Hb 12 g% increased from 9.1% in patients with NYHA 1 to 79.1% in those with NYHA 4 (5).
The Framingham Study has shown that anemia is an
  • independent risk factor for the production of CHF (6).
Despite this association of CHF with anemia,
  • its role is not mentioned in the 1999 U.S. guidelines for the diagnosis and treatment of CHF (7), and
  • many studies consider anemia to be only a rare contributing cause of hospitalization for CHF (8,9).
Recently, we performed a study in which the anemia of severe CHF that was resistant to maximally tolerated doses of standard medications
  • was corrected with a combination of subcutaneous (sc) erythropoietin (EPO) and intravenous iron (IV Fe) (5).
We have found this combination to be safe, effective and additive
  • in the correction of the anemia of chronic renal failure (CRF) in both
  • the predialysis period (10) and the dialysis period (11).
The IV Fe appears to be more effective than oral iron (12,13). In our previous study of CHF patients (5), the treatment resulted in
  • improved cardiac function,
  • improved NYHA functional class,
  • increased glomerular filtration rate,
  • a marked reduction in the need for diuretics and
  • a 92% reduction in the hospitalization rate
compared with a similar time period before the intervention. In the light of these positive results, a prospective randomized study was undertaken
  • to determine the effects of the correction of anemia in severe symptomatic CHF resistant to maximally tolerated CHF medication.

Abbreviations and Acronyms

CABG coronary artery bypass graft
CHF congestive heart failure
CRF chronic renal failure
EPO erythropoietin
%Fe Sat percent iron saturation
GFR glomerular filtration rate
Hb hemoglobin
Hct hematocrit
IU international units
IV intravenous
LVEF left ventricular ejection fraction
NYHA New York Heart Association
PA pulmonary artery
sc subcutaneous
SOLVD Studies Of Left Ventricular Dysfunction


Patients.Thirty-two patients with CHF were studied. Before the study, the patients were treated for least six months in the CHF clinic with

  • maximally tolerated doses of angiotensin-converting enzyme inhibitors, the beta-blockers bisoprolol or carvedilol, aldospirone, long-acting nitrates, digoxin and oral and intravenous (IV) furosemide.

In some patients these agents could not be given because of contraindications and in others they had to be stopped because of side effects. Despite this maximal treatment

  • the patients still had severe CHF  (NYHA classIII), with  fatigue and/or shortness of breath  on even mild exertion or at rest.  All had levels of
  • Hb in the range of 10 to 11.5 g%  on at least three consecutive visits over a three-week period.
  • All had a LVEF of 40%.

Secondary causes of anemia including hypothyroidism, and folic acid and vitamin B12 deficiency were ruled out and

  • there was no clinical evidence of gastrointestinal bleeding.

The patients were randomized consecutively into two groups:

  • Group A, 16 patients, was treated with sc EPO and IV Fe to achieve a target Hb of at least 12.5 g%.
  • Group B, 16 patients, did not receive the EPO and IV Fe.

Treatment protocol for correction of anemia.

All patients in Group A received the combination of sc EPO and IV Fe. The EPO was given once a week at a starting dose of 4,000 international units (IU) per week  and
the dose was increased  to two  or  three  times a week or decreased to once every few weeks as  necessary

  • to achieve and maintain a target Hb of 12.5 g%.

The IV Fe (Venofer-Vifor International, Switzerland), a ferric sucrose product, was given in a dose of 200 mg IV in 150 ml saline over 60 min every two weeks

  • until the serum ferritin reached 400 g/l or
  • the %Fe saturation (%Fe Sat is serum iron/total iron binding capacity 100) reached 40% or
  • the Hb reached 12.5g%. 

The IV Fe was then given at longer intervals as needed to maintain these levels.


Visits to the clinic were at two- to three week intervals depending on the patient’s status. This was the same frequency of visits to the CHF clinic as before then,

  • potassium and ferritin and %Fe Sat were performed on every visit.
  • blood pressure was measured by an electronic device on every visit.
  • LVEF was measured initially and at four- to six-month intervals by MUGA radioisotope ventriculography.

This technique measures

  • the amount of blood in the ventricle at the end of systole and at the end of diastole, thus giving
  • a very accurate assessment of the ejection fraction.

It has been shown to be an accurate and reproducible method of measuring the ejection fraction (14).  Hospital records were reviewed at the end of the intervention period to compare

  • the number of days hospitalized during the study with 
  • the number of days hospitalized during a similar period 
    • when the patients were treated in the CHF clinic before the initial randomization and entry into the study.

Clinic records were reviewed to evaluate the types and doses of CHF medications used before and during the study. The mean follow-up for patients was 8.2 +/-  2.7 months (range 5 to 12 months).  The study was done with the approval of the local ethics committee.Statistical analysis.

An analysis of variance with repeated measures (over time) was performed to compare the two study groups (control vs. treatment) and

  • to assess time trend and the interactions between the two factors.
  • A separate analysis was carried out for each of the outcome parameters.
  • The Mann-Whitney test was used to compare the change in NYHA class between two groups.

All the statistical analysis was performed by SPSS (version 10).


The mean age in Group A (EPO and Fe) was 75.3 +/-  14.6 years and in group B was 72.2 +/-  9.9 years. There were 11 and 12 men in Groups A and B, respectively.
Before the study the two groups were similar in
  1. cardiac function,
  2. comorbidities,
  3. laboratory investigations and
  4. medications
  • (Tables 1, 2 and 3), except for IV furosemide (Table 3),
which was higher in the treatment group. The mean NYHA class of Group A before the study was 3.8  0.4 and was 3.5  0.5 in Group B. The contributing factors to CHF in Groups A and B, respectively, are seen in Table 1 and were similar.
Table 1. Medical Conditions and Contributing Factors to Congestive Heart Failure in the 16 Patients Treated for the Anemia and in the 16 Controls

Table 1 medical conditions heart failure anemia

Table 2. The Effect of Correction of Anemia by Intravenous Iron and Erythropoietin Therapy on Various Parameters in 16 Patients in the Treatment (A) and 16 in the Control (B) Group

Table 2 medications to treat heart failure anemia

p values are given for analysis of variance with repeated measures and for independent t tests for comparison of baseline levels between the two groups.
BP  blood pressure; Fe Sat  iron saturation; Hb  hemoglobin; IV  intravenous; NS  not stated; Std Dev.  standard deviation.

The main contributing factors to CHF were considered to be

  • ischemic heart disease (IHD) in 11 and 10 patients respectively,
  • hypertension in two and two patients,
  • valvular heart disease in twoand two patients, and
  • idiopathic cardiomyopathy in one and two patients, respectively.

A significant change after treatment was observed in the two groups in the following parameters:

  • IV furosemide,
  • days in hospital,
  • Hb,
  • ejection fraction,
  • serum creatinine and
  • serum ferritin.
In addition, the interaction between the study group and time trend was significant in all measurements except for blood pressure and %Fe Sat. This interaction indicates that
  • the change over time was significantly different in the two groups.
Table 3. The Effect of Correction of Anemia by Intravenous Iron and Erythropoietin Therapy on Various Parameters in 16 Patients in the Treatment (A) and 16 in the Control (B) Group

Table 3  CHF aneia EPO

p values are given for analysis of variance with repeated measures and for independent t tests for comparison of baseline levels between the two groups.
BP  blood pressure; Fe Sat  iron saturation; Hb  hemoglobin; IV  intravenous; NS  not stated; Std Dev.  standard deviation.

We find in the comparisons of Tables 2 and 3:

  1. before treatment the level of oral furosemide was higher in the control group (136.2 mg/day) compared with the treatment group (132.2 mg/day).
  2. after treatment, while the dose of oral furosemide of the treated patients was reduced  to 64.4 mg/day
  • the dose of the nontreated patients was increased to 175 mg/day.

The same results of improvement in the treated group and deterioration in the control group are expressed in the following parameters:

  1. IV furosemide, days in hospital,
  2. Hb,
  3. ejection fraction and
  4. serum creatinine.

The NYHA class was

  • 3.8 +/- 0.4 before treatment and 2.2 +/- 0.7 after treatment in Group A  (delta mean = – 1.6) and
  • 3.5 +/-  0.7 before treatment and 3.9 +/- 0.3 after treatment in Group B. (delta mean = 0.4)

The improvement in NYHA class was significantly higher (p < 0.0001) in the treatment group compared with the control group (Table 4).

Table 4. Changes from Baseline to Final New York Heart Association (NYHA) Class
Initial minus final

Table 4  changes from NYHA baseline  CHF anemia

The improvement in NYHA class was statistically higher (p <  0.0001) in the treatment group compared with control.

There were no deaths in Group A and four deaths in Group B.

Case 1: A 71-year-old woman with severe mitral insufficiency and severe pulmonary hypertension  (a pulmonary artery [PA] pressure of 75 mm Hg) had persistent NYHA 4 CHF  and died during mitral valve surgery  seven months after onset of the study. She was hospitalized for 21 days  in the seven months before randomization and for 28 days  during the seven months after randomization.

Case 2:

A 62-year-old man with a longstanding history of hypertension complicated by IHD, coronary artery bypass graft (CABG) and atrial fibrillation had persistent NYHA 4 CHF  and a PA pressure of 35 mm Hg,  and died from pneumonia and septic shock eight months after onset of the study. He was hospitalized for seven days in the eight months before randomization and for 21 days during the eight months  after

Case 3:
A 74-year old man with IHD, CABG, chronic obstructive pulmonary disease, a history of heavy smoking and diabetes had persistent NYHA 4 CHF and a PA pressure of  28 mm Hg, and died of pulmonary  edema and cardiogenic shock nine months after onset of the study. He was hospitalized for 14 days in the nine months before  randomization and for 41 days during the nine months after randomization.

Case 4:
A 74-year-old man with a history of IHD, CABG, diabetes, dyslipidemia, hypertension and atrial fibrillation, had persistent NYHA 4 CHF and a PA pressure of 30 mm Hg,  and died of pneumonia and septic shock   six months after onset of the study. He was hospitalized for five days in the six months before randomization and for 16 days during the nine months after randomization.


 Main findings.

The main finding of the present study is that the correction of

  • even mild anemia in patients with symptoms of very severe CHF despite being on maximally tolerated drug therapy
  • resulted in a significant improvement in their cardiac function and NYHA functional class.

There  was also a large

  • reduction in the number of days of  hospitalization compared with a similar period before the  intervention.
  • all this was achieved despite a marked reduction in the dose of oral and IV furosemide.

In the group in whom the anemia was not treated, four  patients died during the study. In all four cases

  • the CHF was unremitting and contributed to the deaths. 

In addition,  for the group as a whole, 

  • the LVEF, the NYHA class and  the renal function worsened.

There was also need for

  • increased oral and IV furosemide as well as increased  hospitalization.

Study limitations.

The major limitations of this study are

  1. the smallness of the sample size and
  2. the fact that randomization and treatment were not done in a blinded fashion.

Nevertheless, the two groups were almost identical in

  1. cardiac, renal and anemia status;
  2. in the types and doses of medication they were taking before and during the intervention and
  3. in the number of hospitalization days before the intervention.

Although the results of this study, like those of  our previous uncontrolled study (5), suggest that

  • anemia may play an important role in the mortality and morbidity of  CHF,
  • a far larger double-blinded controlled study should be carried out to verify our findings.

Anemia as a risk factor for hospitalization and death in CHF.

Our results are consistent with a recent analysis of 91,316 patients hospitalized with CHF (15). Anemia was found to be a stronger predictor of

  • the need for early rehospitalization than  was hypertension,  IHD or the presence of a previous CABG.  

A recent analysis of the Studies Of Left Ventricular Dysfunction (SOLVD) (16) showed that

  • the level of hematocrit (Hct) was an independent risk factor for mortality.

During a mean follow-up of 33 months the mortality was

  • 22%, 27% and 34% for those with a Hct of 40, 35 to 40 and 35 respectively.

The striking response of our patients to

  • correction of mild anemia suggests that the failing heart may be very susceptible to anemia.

It has, in fact, been found in both animal (17) and human studies (17–19) that

  • the damaged heart is more vulnerable to anemia and/or ischemia than is the normal heart.

These stimuli may result in a more marked reduction in cardiac function than occurs in the normal heart and may explain why,  in our study,

  • the patients were so resistant to high doses of CHF medications and
  • responded so well when the anemia was treated.

Our findings about the importance of anemia in CHF are not surprising when one considers that, in dialysis patients,

  • anemia has been shown to be associated with an increased prevalence and incidence of CHF (20) and that
  • correction of anemia in these patients is associated with improved
    • cardiac function (21,22),
    • less mortality (23,24) and
    • fewer hospitalizations (23,25).

Effect of improvement of CHF on CRF.

Congestive heart failure can cause progressive renal failure (26,27). Renal ischemia is found very early on

  • in patients with cardiac dysfunction (28,29), and
  • chronic ischemia may cause progression of renal failure (30). Indeed, the development of
  • CHF in patients with essential hypertension has been found to be one of the most powerful predictors of
  • the eventual development of end-stage renal disease (31).

Patients who develop CHF after a myocardial infarction experience a

  • fall in the glomerular filtration rate (GFR) of about 1 ml/min/month if the CHF is not treated (32).

In another recent analysis of the SOLVD study, treating the CHF with

  • both angiotensin-converting enzyme inhibitors and beta-blockers resulted in better preservation of the renal function than did
  • angiotensin-converting enzyme inhibitors alone (26),
suggesting that the more aggressive the treatment of the CHF, the better the renal function is preserved. In the present study, as in our previous one (5), we found that the deterioration of GFR was prevented with
  • successful treatment of the CHF, including correction of the anemia, whereas
  • the renal function worsened when the CHF remained severe

All these findings suggest that early detection and treatment of CHF and systolic and/or diastolic dysfunction from whatever cause could prevent

  • the deterioration not only of the cardiac function
  • but of the renal function as well.

This finding has very broad implications in the prevention of CRFbecause most patients with advanced CRF have

  • either clinical evidence of CHF or at least some degree of systolic dysfunction (33).

Systolic and/or diastolic dysfunction can occur early on in many  conditions, such as

  • essential hypertension (34),
  • renal disease of any cause (35,36) or
  • IHD, especially after a myocardial infarction (37).

The early detection and adequate treatment of this cardiac dysfunction, including correction of the anemia, could prevent this cardiorenal insufficiency. To detect cardiac dysfunction early on, one would need  at least an echocardiogram and MUGA radio-nucleotide ventriculography. These tests should probably be done not only in patients with signs and symptoms of CHF,   but in all patients where CHF or systolic  and/or diastolic dysfunction are suspected, such as those with a history of heart disease or suggestive changes of ischemia or hypertrophy on the electrocardiogram, or in patients with hypertension or renal disease.

Other positive cardiovascular effects of EPO treatment.

Another possible explanation for the improved cardiac function in this study may be the direct effect that EPO itself has on improving cardiac muscle function (38,39) and myocardial cell growth (39,40) unrelated to its  effect of the anemia. In fact EPO may be  crucial in the formation of the heart muscle in utero (40). It may also improve  endothelial function (41).  Erythropoietin may be superior to blood transfusions  not only  because adverse reactions to EPO are infrequent, but also because

  • EPO causes the production and release of young cells from the bone marrow into the blood.

These cells have an oxygen dissociation curve that is shifted to the right of the normal curve, causing the release of

  • much greater amounts of oxygen into the tissues than occurs normally (42).

On the other hand, transfused blood consists of older red cells with an oxygen dissociation curve that is

  • shifted to the left, causing the release of much less oxygen into the tissues than occurs normally (42).

The combination of IV Fe and EPO. The use of IV Fe along with EPO has been found to have an additive effect, 

  • increasing the Hb even more than would occur with EPO alone while at the same time
  • allowing the dose of EPO to be reduced (10 –13).
  • The lower dose of EPO will be cost-saving and also reduce the chances of hypertension developing (43).
 We used iron sucrose (Venofer) as our IV Fe medication because, in our experience, it is extremely well tolerated (10,11) and  
  • has not been  associated  with any serious side effects in more than 1,200 patients over six years.

Implications of treatment of anemia in CHF. The correction of anemia is not a substitute for the well-documented effective therapy of CHF but seems to be  an important, if not vital,  addition to the therapy. It is surprising, therefore,  that judging from  the  literature  on CHF,

  • such an obvious treatment for improving CHF is so rarely considered.

We believe that correction of the anemia will have an important role to play in

  • the amelioration of cardiorenal insufficiency, and that this improvement will have
  • significant economic  implications as well.


The authors thank Rina Issaky, Miriam Epstein, Hava Ehrenfeld and Hava Rapaport for their secretarial assistance.
Reprint requests and correspondence: Dr. D. S. Silverberg, Department of Nephrology, Tel Aviv Medical Center, Weizman 6, Tel Aviv, 64239, Israel.


The use of subcutaneous erythropoietin and intravenous iron for the treatment of the anemia of severe, resistant congestive heart failure improves cardiac and renal function and functional cardiac class, and markedly reduces hospitalizations

Donald S Silverberg, MDa; Dov Wexler, MDa; Miriam Blum, MDa; Gad Keren, MDa; David Sheps, MDa; Eyal Leibovitch, MDa; David Brosh, MDa; Shlomo Laniado, MDa; Doron Schwartz, MDa; Tatyana Yachnin, MDa; Itzhak Shapira, MDa; Dov Gavish, MDa; Ron Baruch, MDa; Bella Koifman, MDa; Carl Kaplan, MDa; Shoshana Steinbruch, RNa; Adrian Iaina, MDa

J Am Coll Cardiol. 2000;35(7):1737-1744. doi:10.1016/S0735-1097(00)00613-6


This study evaluated the prevalence and severity of anemia in patients with congestive heart failure (CHF) and

  • the effect of its correction on cardiac and renal function and hospitalization.


The prevalence and significance of mild anemia in patients with CHF is uncertain, and the role of erythropoietin with intravenous iron supplementation in treating this anemia is unknown.


In a retrospective study, the records of the 142 patients in our CHF clinic were reviewed to find
  • the prevalence and severity of anemia (hemoglobin [Hb]12 g).
In an intervention study, 26 of these patients, despite maximally tolerated therapy of CHF for at least six months, still had had severe CHF and were also anemic. They were treated with
  • subcutaneous erythropoietin and intravenous iron sufficient to increase the Hb to 12 g%.
The doses of the CHF medications, except for diuretics, were not changed during the intervention period.


The prevalence of anemia in the 142 patients increased with the severity of CHF,
  • reaching 79.1% in those with New York Heart Association class IV.
In the intervention study, the anemia of the 26 patients was treated for a mean of 7.2 5.5 months.
  • The mean Hb level and mean left ventricular ejection fraction increased significantly.
  • The mean number of hospitalizations fell by 91.9% compared with a similar period before the study.
  • The New York Heart Association class fell significantly,
  • as did the doses of oral and intravenous furosemide.
  • The rate of fall of the glomerular filtration rate slowed with the treatment.


Anemia is very common in CHF and its successful treatment is associated with a significant improvement in
  • cardiac function,
  • functional class,
  • renal function and
  • in a marked fall in the need for diuretics and hospitalization.
Abbreviations and Acronyms
ACE Angiotensin-converting enzyme
CHF congestive heart failure
COPD chronic obstructive pulmonary disease
CRF chronic renal failure
CVA cerebrovascular accident
EPO erythropoietin
Fe iron
g% grams Hb /100 ml blood
GFR glomerular filtration rate
Hb hemoglobin
Hct hematocrit
IV intravenous
LVEF left ventricular ejection fraction
LVH left ventriculr hypertrophy
NYHA New York Heart Association
%Fe Sat percent iron saturation
sc subcutaneous
TNF tumor becrosis factor
ACE Angiotensin-converting enzyme
CHF congestive heart failure
COPD chronic obstructive pulmonary disease
CRF chronic renal failure
CVA cerebrovascular accident
EPO erythropoietin
Fe iron
g% grams Hb /100 ml blood
GFR glomerular filtration rate
Hb hemoglobin
Hct hematocrit
IV intravenous
LVEF left ventricular ejection fraction
LVH left ventriculr hypertrophy
NYHA New York Heart Association
%Fe Sat percent iron saturation
sc subcutaneous
TNF tumor becrosis factor

The mean hemoglobin (Hb) in patients with congestive heart failure (CHF) is about 12 g Hb per 100 ml blood (g%) (1–3), which is considered to be the lower limit of normal in adult men and postmenopausal women (4). Thus, many patients with CHF are anemic, and

  • this anemia has been shown to worsen as the severity of the CHF progresses (5,6).
Severe anemia of any cause can produce CHF, and treatment of the anemia can improve it (7). In patients with chronic renal failure (CRF) who are anemic,
  • treatment of the anemia with erythropoietin (EPO) has improved many of the abnormalities seen in CHF,
  • reducing left ventricular hypertrophy (LVH) (8 –10),
  • preventing left ventricular dilation (11) and,
    • in those with reduced cardiac function, increasing the left ventricular ejection fraction (LVEF)(8 –10),
    • the stroke volume (12) and
    • the cardiac output (12).
In view of the high prevalence of anemia in CHF, it is surprising that we could find no studies in which EPO was used in the treatment of the anemia of CHF, and the use of EPO is not included in U.S. Public Health Service guide-lines of treatment of the anemia of CHF (13). In fact, anemia has been considered
  • only a rare contributing factor to the worsening of CHF, estimated as contributing to
  • no more than 0% to 1.5% of all cases (14 –16).
Perhaps for this reason, recent guidelines for the prevention and treatment of CHF do not mention treatment of anemia at all (17). If successful treatment of anemia could improve cardiac function and patient function in CHF,
  • this would have profound implications, because,
  • despite all the advances made in the treatment of CHF, it is still a major and steadily increasing cause of hospitalizations, morbidity and mortality (18 –20).
The purpose of this study is to examine
  • the prevalence of anemia (Hb 12 g%) in patients with different levels of severity of CHF and
  • to assess the effect of correction of this anemia in severe CHF patients
  • resistant to maximally tolerated doses of CHF medication.
A combination of subcutaneous (SC) EPO and intravenous (IV) iron (Fe) was used. We have found this combination to be additive in improving the anemia of CRF (21,22).



The medical records of the 142 CHF patients being treated in our special outpatient clinic devoted to CHF were reviewed to determine the prevalence and severity of anemia and CRF in these patients. These patients were referred to the clinic either from general practice or from the various wards in the hospital.

Intervention study.

Despite at least six months of treatment in the CHF clinic,
  • 26 of the above patients had persistent, severe CHF (New York Heart Association [NYHA] class III),
  • had a Hb level of 12 g% and were on
    • angiotensin-converting enzyme [ACE] inhibitors, the 
    • alpha-beta-blocker carvedilol,
    • long-acting nitrates,
    • digoxin, 
    • aldactone and
    • oral and IV furosemide.

These 26 patients participated in an intervention study. The mean age was 71.76  8.12 years. There were 21 men and 5 women. They  all had a

  • LVEF below 35%,
  • persistent fatigue and
  • shortness

    of breath on mild to moderate exertion and often at rest, and had

  • required hospitalizations at least once during their follow-up in the CHF clinic for pulmonary edema.
In 18 of the 26 patients, the CHF was associated with ischemic heart disease either
  • alone in four patients, or
  • with hypertension in six,
  • diabetes in four,
  • the two together in three, or with
  • valvular heart disease in one.
Of the remaining eight patients,
  • four had valvular heart disease alone and
  • four had essential hypertension alone.
Secondary causes of anemia including
  • gastrointestinal blood loss (as assessed by history and by three negative stool occult blood examinations),
  • folic acid and vitamin B12 deficiency and
  • hypothyroidism were ruled out.
Routine gastrointestinal endoscopy was not carried out. The study passed an ethics committee.
Table 1. Initial Characteristics of the 142 Patients With CHF Seen in the CHF Clinic
Age, yearsMale/female,  %Associated conditionsDiabetesHypertensionDyslipidemiaSmoking

Main cardiac diagnosis
Ischemic heart disease

Dilated CMP

Valvular heart disease


LVEF,  %

Left atrial area (n 15 cm2)

Pulmonary artery pressure  (15 mm Hg)

Previous hospitalizations/year

Serum Na, mEq/liter

Serum creatinine, mg%

Hemoglobin, g%

70.1 +/- 11.1










32.5 +/- 12.2

31.3  +/- 10.3

43.1  +/-14.9

3.2  +/- 1.5

139.8  +/- 4.0

1.6   +/-  1.1

11.9   +/- 1.5

CMP  cardiomyopathy; LVEF  left ventricular ejection fraction; NYHA  New York Heart Association class.

Correction of the anemia.

All patients received the combination of SC EPO and IV Fe. The EPO was given once a week at a starting dose of 2,000 IU per week subcutaneously, and the dose was increased or decreased as necessary to achieve and maintain a target Hb of 12 g%. The IV Fe (Venofer-Vifor International, St. Gallen, Switzerland), a ferric sucrose product, was given in a dose of 200 mg IV in 150 ml saline over 60 min every week until the serum ferritin reached 400  g/liter or the percent Fe saturation (%Fe Sat: serum iron/total iron binding capacity   100) reached 40% or until the Hb reached 12 g%. The IV Fe was then given at longer intervals as needed to maintain these levels.

Medication dose.

Except for oral and IV furosemide therapy, the doses of all the other CHF medications, which were used in the maximum tolerated doses before the intervention, were kept unchanged during the intervention period.

Duration of the study.

The study lasted for a mean of 7.2  5.5 months (range four to 15 months).


Visits were at weekly intervals initially and then at two- to three-week intervals depending on the patient’s status. This was the same frequency of visits to the CHF clinic as before the intervention study.
  • A complete blood count, serum creatinine, serum ferritin and % Fe Sat were performed on every visit.
  •  An electronic device measured the blood pressure on every visit.
  • The LVEF was measured by a multiple gated ventricular angiography heart scan initially and at four- to six-month intervals.
Hospital records were reviewed to compare the number of hospitalizations during the time the patients were treated for the anemia with the number of hospitalizations
  • during a similar period of time that they were treated in the CHF clinic 
    before the anemia was treated.
Clinic records were reviewed to evaluate the types and doses of CHF medications used 
before and during the study.

Period of time that they were treated in the CHF clinic before the anemia was treated.

Clinic records were reviewed to evaluate the types and doses of CHF medications used before and during the study.  The glomerular filtration rate (GFR) was calculated from the serum creatinine by the formula: 1/serum creatinine in mg% x 100 GFR in ml/min. The rate of change of the GFR before and during the intervention period was calculated by comparing the change in GFR per month in the year before the intervention with that during the intervention.

Statistical analysis.

Mean standard deviation was calculated. One-way analysis of variance (ANOVA) was performed to compare parameter levels between the four NYHA groups. Hochberg’s method of multiple comparisons (23) was used for pair-wise comparison between two groups. A p value of less than 0.05 was considered statistically significant. In the intervention study, the significance of the difference between the initial values and those at the end of the study for the individual parameters in the 26 treated patients was assessed by paired student’s t test; p < 0.05 was considered statistically significant. All the statistical analysis was performed by the SPSS program (Version 9, Chicago, Illinois).


CHF: the whole study group.

The clinical, biochemical and hematological characteristics of the 142 patients seen in the clinic are shown in Tables 1 and 2.

  • Sixty-seven patients (47%) had severe CHF as judged by a NYHA class of IV (Table 2).
  • Seventy- nine of the 142 patients (55.6%) were anemic (Hb  12 g%).

The mean Hb level fell progressively from 13.73 +/- 0.83 g% in class I NYHA to 10.90 +/- 1.70 g% in class IV NYHA (p  0.01). The percentage of patients with Hb  12 g% increased from 9.1% in class I to 79.1% in class IV.
Fifty eight patients (40.8%) had CRF as defined as a serum creatinine  1.5 mg%. The mean serum creatinine increased from 1.18 +/_  0.38 mg% in class I NYHA, to 2.0 +/-    1.89 mg% in class IV NYHA, p  0.001. The percentage of patients with an elevated serum creatinine ( 1.5 mg%)      increased from 18.2% in class I to 58.2% in class IV.

The mean ejection fraction fell from 37.67 +/-  15.74% in class I to 27.72 +/-  9.68% (p  0.005) in class IV.

Table 2. LVEF and Biochemical and Hematological Parameters by NYHA Class in 142 Patients With CHF 
NYHA Class I II III IV  Significantly Different Pairs*

 *p  0.05 at least between the two groups by pair-wise comparison between groups.

†p  0.05 at least between the groups by ANOVA.

No. of patients





(total 142) (%)

    (7.7)    (18.3)    (26.8)    (47.2)

Hb, g%†

13.73 (0.83)

13.38 (1.26)

11.95 (1.48)

10.90 (1.70) 

1–3, 1–4, 2–3, 2–4

Serum creatinine,





1–2, 1–3, 1–4


    (0.38)     (0.29)      (0.38)     (1.89)

LVEF, %†

37.67 (15.74)

32.88 (12.41)

32.02 (10.99)

27.72 (9.68)

1–4, 2–4

Hb 12 g%,  (%)


5 (19.2) 

20 (52.6) 

53 (79.1)

Serum creatinine

    2      5     12     39

1.5 mg%,  (%) 





The intervention study: medications.

The percentage of patients receiving each CHF medication before and after the intervention period and the reasons for not receiving  them are seen in Table 3.

Table 3. Number (%) of the 26 Patients Taking Each Type of Medication Before and During the Intervention Period and the Reason Why the Medication Was Not Used

Medication    No. of Patients  (%)         Reason for Not Receiving the Medications (No. of Patients)
BP  blood pressure; CRF  chronic renal failure; IV  intravenous.

The main reason for not receiving:

  • 1) ACE inhibitors was the presence of reduced renal function;
  • 2) carvedilol was the presence of chronic obstructive pulmonary disease (COPD);
  • 3) nitrates was low blood pressure and aortic stenosis and
  • 4) aldactone was hyperkalemia.
Table 4. Mean Dose of Each Medication Initially and at the End of the Intervention Period in the 26 Patients

                                       No. of Patients                                 Initial Dose ^                 Final Dose^
Carvedilol (mg/day)                      20                                                        26.9 15.5                                   28.8 14.5
Captopril (mg/day)                          7                                                        69.6 40.0                                 70.7 40.4
Enalapril (mg/day)                        13                                                        25.7 12.5                                   26.9 12.6
Digoxin (mg/day)                          25                                                       0.10 0.07                                    0.10 0.07
Aldactone (mg/day)                       19                                                        61.2 49.2                                   59.9 47.1
Long-acting nitrates                      23                                                        53.2 13.2                                   54.1 14.4
Oral furosemide (mg/day)           26                                                      200.9 120.4                                78.3 41.3*
IV furosemide (mg/month)         26                                                      164.7 178.9                                  19.8 47.0*
*p  0.05 at least vs. before by paired Student’s t test.
^  +/-

The mean doses of the medications are shown in Table 4. 

The mean dose of oral furosemide was 200.9 +/-  120.4 mg/day before and 78.3 +/-  41.3 mg/day after the intervention (p   0.05). The dose of IV furosemide was 164.7 +/-  19.8,  178.9 mg/month before and  7.0 mg/month after the intervention (p  0.05).  

The doses of the other CHF medications were almost identical in the two periods.

Clinical results.

There were three deaths during the intervention period. An 83-year-old man died after eight months of respiratory failure after many years of COPD, a 65-year-old man at eight months of a CVA with subsequent pneumonia and septic shock and a 70-year-old man at four months of septicemia related to an empyema that developed after aortic valve replacement.
Three patients, a 76-year-old man, an 85-year-old woman and a 72-year-old man, required chronic hemodialysis after six, 16 and 18 months, respectively. The serum creatinines of these three patients at onset of the anemia treatment were 4.2, 3.5 and 3.6 mg%, respectively. All three had improvement in their NYHA status but
  • their uremia worsened as the renal function deteriorated, demanding the initiation of dialysis.

In no cases, however, was pulmonary congestion an indication for starting dialysis.

Functional results (Table 5).

During the treatment period, the NYHA class fell from a mean of 3.66 +/- 0.47 to 2.66 +/- 0.70 (p 0.05), and
  • 24 had some improvement in their functional class.
The mean LVEF increased from 27.7 +/- 4.8 to 35.4  +/- 7.6% (p 0.001), an increase of 27.8%.
Compared with a similar period of time before the onset of the anemia treatment, the mean number of hospitalizations fell from 2.72 +/-  1.21 to 0.22 +/-  0.65 per patient (p   0.05)a decrease of 91.9%.
No significant changes were found in the mean systolic/diastolic blood pressure.

Hematological results (Table 5).

  • The mean hematocrit (Hct) increased from 30.14 +/- 3.12%) to 35.9  +/- 4.22% (p < 0.001).
  • The mean Hb increased from 10.16 +/- 0.95 g%) to 12.10 +/-  1.21 g% (p <  0.001).
  • The mean serum ferritin increased from 177.07 +/-  113.80  g/liter to 346.73 +/- 207.40 g/liter (p  0.005).
  • The mean serum Fe increased from 60.4 +/- 19.0 g% to 74 +/- .80  20.7 g% (p  0.005). 
  • The mean %Fe Sat increased from 20.05   6.04% to 26.14 =/- 5.23% (p  0.005).
  • The mean dose of EPO used throughout the treatment period was 5,227  +/- 455 IU per week, and
  • the mean dose of IV Fe used was 185.1 +/- 57.1 mg per month.
In four of the patients, the target Hb of 12 g% was maintained despite stopping the EPO for at least four months.

Renal results (Table 5).

The changes in serum creatinine were not significant. The estimated creatinine clearance fell at a rate of 0.95 + 1.31 ml/min/month before the onset of treatment of the anemia and increased at a rate of 0.85 + 2.77 ml/min/month during the treatment period.
Table 5. The Hematological and Clinical Data of the 26 CHF Patients at Onset and at the End of the Intervention Period

————–                                         Initial ^                                    Final^
Hematocrit, vol%                              30.14 3.12                            35.90 4.22*
Hemoglobin, g%                                10.16 0.95                              2.10 1.21*
Serum ferritin, g/liter                    177.07 113.80                       346.73  207.40*
Serum iron, g%                                  60.4 19.0                               74.8  20.7*
% iron saturation                              20.5 6.04                               26.14 5.23*
Serum creatinine, mg%                   2.59 0.77                                 2.73 1.55
LVEF, %                                              27.7 4.8                                   35.4  7.6*
No. hospitalizations/patient          2.72 1.21                                 0.22   0.65*
Systolic BP, mm Hg                       127.1 19.4                                128.9  26.4
Diastolic BP, mm Hg                       73.9 9.9                                   74.0   12.7
NYHA (0–4)                                     3.66 0.47                                2.66 0.70*
*p  0.05 at least vs before by paired Student’s t test.     ^ +/-
BP  blood pressure; LVEF  left ventricular ejection fraction; NYHA  New York Heart Association.


The main findings in the present study are that anemia is common in CHF patients and becomes progressively more prevalent and severe as CHF progresses. In addition, for patients with resistant CHF, the treatment of the associated anemia causes a marked improvement in their

  1. functional status,
  2. ejection fraction and
  3. GFR.
        • All these changes were associated with a markedly
            • reduced need for hospitalization and
            • for oral and IV furosemide.

The effect of anemia on the ischemic myocardium.

We used the IV Fe together with EPO to avoid the Fe deficiency caused by the use of EPO alone (38,39).
The Fe deficiency will cause

  • a resistance to EPO therapy and
  • increase the need for higher and higher doses to maintain the Hb level (39,40).

These high doses will not only be expensive but may increase the blood pressure excessively (41). The IV Fe reduces the dose of EPO needed to correct the anemia, because

  • the combination of SC EPO and IV Fe has been shown to have an additive effect on correction of the anemia of CRF (21,22,39,42).

Oral Fe, however, has no such additive effect (39,42). The relatively low dose of EPO needed to control the anemia in our study may explain why

  • the blood pressure did not increase significantly in any patient.

We used Venofer, an Fe sucrose product, as our IV Fe supplement because, in our experience (21,22,43), it has very few side effects and, indeed, no side effects with its use were encountered in this study.

The Effect of Anemia Correction on Renal Function.

Congestive heart failure is often associated with some degree of CRF (1–3,27–29), and

  • this is most likely due to renal vasoconstriction and ischemia (28,29).

When the anemia is treated and the cardiac function improves,

  • an increase in renal blood flow and glomerular filtration is seen (7,28).

In the present study, renal function decreased as the CHF functional class worsened (Table 2). The rate of deterioration of renal function was slower during the intervention period. Treatment of anemia in CRF has been associated with

  • a rate of progression of the CRF that is either unchanged (30) or is slowed (31–33).

It is possible, therefore, that adequate treatment of the anemia in CHF may, in the long term, help slow down the progression of CRF.

Possible Adverse Effects of Correction of the Anemia.

There has been concern, in view of the recent Amgen study (34), that correction of the Hct to a mean 42% in hemodialysis patients might increase cardiovascular events in those receiving EPO compared with those maintained at a Hct of 30%. Although there is much uncertainty about how to interpret this study (35), there is a substantial body of evidence that shows

  • correction of the anemia up to a Hb of 12 g% (Hct 36%) in CRF on dialysis is safe and desirable (35–38), and
  • results in a reduction in mortality, morbidity and in the number and length of hospitalizations.

The same likely holds true for the anemia of CHF with or without associated CRF. Certainly, our patients’ symptoms were strikingly improved, as was their cardiac function (LVEF) and need for hospitalization and diuretics. It remains to be established

  • if correction of the anemia up to a normal Hb level of 14 g% might be necessary in order to further improve the patient’s clinical state.

The Role of Fe Deficiency and its Treatment in the Anemia of CHF.

We used the IV Fe together with EPO to avoid the Fe deficiency caused by the use of EPO alone (38,39). The Fe deficiency will cause

  • a resistance to EPO therapy and increase the need for higher and higher doses to maintain the Hb level (39,40).

These high doses will not only be expensive but may

  • increase the blood pressure excessively (41).

The IV Fe reduces the dose of EPO needed to correct the anemia, because the combination of SC EPO and IV Fe has been shown to have an additive effect on correction of the anemia of CRF (21,22,39,42). Oral Fe,  however, has no such additive effect (39,42). The relatively low dose of EPO needed to control the anemia in our study may explain

  • why the blood pressure did not increase significantly in any patient.

We used Venofer, an Fe sucrose product, as our IV Fe supplement because, in our experience (21,22,43), it has very few side effects and, indeed, no side effects with its use were encountered in this study.

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English: Amino acid sequence of the molecule o...

English: Amino acid sequence of the molecule of the brain natriuretic peptide (BNP) 32 (functional). Português: Sequência de aminoácidos da molécula de BNP 32 (funcional). (Photo credit: Wikipedia)

Assessing Cardiovascular Disease with Biomarker

Author and Curator: Larry H Bernstein, MD, FCAP


A Changing expectation from cardiac biomarkers.

This article on Assessing Cardiovascular Disease with Biomarkers will demonstrate the unique role in the discipline evolution that each of the following biomarkers has played in our understanding of CVD risk:

The article is introduced with an entire section on the evolution of our knowledge of cardiac biomarkers and how concepts from thermodynamics have transformed
the way we investigate biochemical mechanisms, and how we have gone from a macro- to a micro- landscape of high complexity.  The same concepts from physics
have also transformed the mathematical stage upon which we model data.  BIG Data is not just about business!  We have entered a new domain of knowledge enabling.

(1)  Enzymes and Isoenzymes

  • AST, ALT, LD, alkaline phosphatase
  • Isoenzymes evolution and genomic loci for polypeptides
  • Emergence of pathway divergence and regulation from gene-loci peptide changes
  • A reflection to implications for biomarkers and therapeutic development based on critical links

(2)  Natriuretic Peptides

  •       Cause of Death: silent cardiac target organ damage (cTOD) (no so sign of cardiac disease)
  •       B Type natriuretic peptide in evolution of CHF
  •       2D and Doppler echocardiography and BNP serum level
  •       Amino terminal pro B-type Natriuretic Peptide
  •       Renal Effect on NT-proBNP
  •       pro-atrial natriuretic peptide

(3)     CRP as Biomarker, theory that lowering the C-reactive protein (CRP) level with statin therapy is predictive of cardiovascular outcomes independent of lowering the low-density lipoprotein (LDL) cholesterol level

(4)     CRP as an Inflammatory Agent

Acute phase reaction is a systemic response: physiological condition  in the beginning of an inflammatory process.

(5)     troponins and hs-troponins (I, T)

(6)     New Candidate  Biomarkers for NSTEMI

(7)    Guidelines for Cardiovascular Risk Assessment

(8)     Statistical Issues to be Resolved

Historical perspective

The use of cardiac markers emerged in the late 1950s, when the physician was faced with the problem of a patient with recent onset of squeezing, crushing, or heaviness in the chest, with or without a Q-wave or definitive ST elevation (acute injury), and perhaps a non specific elevation of the neutrophil count.   A medical student at Albert Einstein Medical school at the time, Arthur Karmen identified the first enzymatic test for acute myocardial infarct (MI), serum glutamic oxaloacetic acid transaminase (SGOT), which is renamed Aspartate Aminotransferase (AST) in a seminal study with Wroblewski and LaDue[1].  The enzyme is ubiquitous, and the authors published another observation that the SGPT, now referred to as Alanine Aminotransferase, has a greater specific activity in liver and myocardial infarct can be distinguished from necrotizing liver disease by using AST and ALT.  These two enzymes were among the three enzymes,with lactate dehydrogenase (LD) and alkaline phosphatase that appeared on the original Technicon (later Siemens) SMA-12 profile, prior to the designated panels used today.  At that time it was common for the pathologist to stain the heart lesion at autopsy in identifying the “ischemic necrosis” postmortem.

In 1957 Hunter and Markert described the five isoenzymes of lactate dehydrogenase, the most anodal migrating pattern was associated with heart and the most cathodal isoenzymes with liver, the five bands being combinations of two subunits.  These were described as different variants of the same enzyme having identical functions, but different tissue specific patterns, such that,  enzyme variants have altered gene loci that results in an amino acid change but catalyze the same reaction.  When mutation modifies the enzymatic catalysis, or its pattern of gene expression, then any of two (or more) variants may be favoured by natural selection and become specialized to different cell environments.  His group suggested that a single gene might somehow encode an array of isozymes differing in “structural variations,” a concept that seems to presage our current understanding of alternative mRNA splicing and post-translational protein modification. A former student of George beadle, he transformed the “concept of one gene one enzyme”  to “one gene one polypeptide”. By treating the enzyme with denaturing agents it was learned that LDH is a tetramer of two types of polypeptide chains (Appella and Markert, 1961). Thus the multiple-gene hypothesis was partially correct: Two different LDH subunits, each encoded by a distinct gene, re-sort themselves in various tetrameric combinations to give rise to five different isozymes (Markert, 1963). During the succeeding years Markert and his students and postdocs elucidated how the study of isozymes could contribute to our understanding of the biochemical variation that underlies cell differentiation and evolution, culminating in the new perspective presented in a Science paper (Markert et al., 1975) entitled “Evolution of a Gene.”

In the early 1960’s Nathan Kaplan postulated that the major LD-isoenzyme types were associated with fundamental differences in the metabolism of the tissue of origin, either catabolic (heart) or anabolic (liver), and skeletal muscle would appear to be in the same class as liver (ignore the ratio of fast and slow twitch), which was elaborated on further by studies of the flight wing patterns of birds.   These isoenzymes not only had different migration in an electrophoretic field and could be separated chromatographically, but they also had different kinetic properties. They all have the same Km, but the purified heart LD is inhibited by a ternary complex of the enzyme, the NAD, and pyruvate that forms, slowing the reaction in the forward direction (pyruvate to lactate).

At about the same time, Masahiro Chiga discovered that adenylate kinase, the enzyme that converts ATP to ADP, from skeletal muscle can be inhibited by inorganic S (myokinase), which led Bernstein and Russell to publish on the identification of adenylate kinase from heart in myocardial infarction using sulfhydryl inhibition in J Molec Cellular Cardiology.  Burton Sobel in the early 1970s showed that CK and the MB isoenzyme of CK, which has a more rapid increase and disappearance than the AST or LD ,  could be used to estimate the amount of cardiac damage in MI.   This meant that a test could be done at any time of day or night with a result in less than an hour.  He applied this to determining whether the extent of infarction was an important determinant of prognosis after myocardial infarction and furthermore, whether the extent of infarction could be modified by interventions that reduce myocardial oxygen requirements or increase myocardial oxygen supply. This work has had a major impact on how patients with acute myocardial infarction are treated and led to a reduction of mortality secondary to treatments, such as thrombolysis, that were validated initially with the methods developed. This led to an immunoassay for CK isoenzyme MB that was offered by Roche on the Cobas analyzer, and by Dupont on the ‘aca’. What emerged is a new imperative to reduce infarct size under the rubrick – “Time is Muscle”.


  1. Karmen Arthur, Wróblewski Felix, LaDue John S. TRANSAMINASE ACTIVITY IN HUMAN BLOOD. J Clin Invest. 1955; 34(1):126–133.
  2. LADUE JS, WROBLEWSKI F, KARMEN A. Serum glutamic oxaloacetic transaminase activity in human acute transmural myocardial infarction. Science 1956; 75(11).
  3. Hunter, R. L. and C.L. Merkert. (1957) Histochemical demonstration of enzymes separated by zone electrophoresis in starch gels. Science 125: 1294-1295.
  4. Bernstein L, Kerrigan M, Maisel H. Lactic dehydrogenase isoenzymes in lens and cornea. Exp Eye Res 1965; 5(3):999-1005. ICID: 844979
  5. Nathan O. Kaplan Papers. MSS 0099. UC San Diego::Mandeville Special Collections Library.

Enzyme-coenzyme-substrate complex. of pyridine nucleotide depend. dehydrogenases 1958.  box 39, folder 5.
Enzymatic studies with analogues of diphosphopyridine nucleotide 1959. box 39, folder 12.
Heterogeneity of the lactic dehydrogenases of new-born and adult rat heart as determined with enzyme analogs 1961. box 39, folder 37.
Regulatory effects of enzyme action 1961. box 39, folder 38.
Inhibition of dehydrogenase reactions by a substance formed from reduced dpn 1961. box 39, folder 40.
Lactic dehydrogenases: functions of the two types 1964. box 39, folder 67.
Lactate dehydrogenase – structure and function. 1964. box 40, folder 4.
Role of the two types of lactic dehydrogenases 1964. Box 40, folder 9.
Structural and functional properties of h and m subunits of lactic dehydrogenase 1965. Box 40, folder 12.

  • Bernstein LH, Everse J, Shioura N, Russell PJ. Detection of cardiac damage using a steady state assay for lactate dehydrogenase isoenzymes in serum. J Mol Cell Cardiol 1974; 6(4):297-315. ICID: 825597
  • Bernstein LH, Everse J.  Determination of the isoenzyme levels of lactate dehydrogenase. Methods Enzymol 1975; 41 47-52.
  • Bernstein LH. Automated kinetic determination of lactate dehydrogenase isoenzymes in serum. Clin Chem 1977; 23(10):1928-1930. ICID: 825616
  • Bernstein LH, Scinto P. Two methods compared for measuring LD-1/total LD activity in serum. Clin Chem 1986; 32(5):792-796. ICID: 825581
  1. Shell WE, Kjekshus JK, Sobel BE: Quantitative assessment of the extent of myocardial infarction in the conscious dog by means of analysis of serial changes in serum creatine phosphokinase activity. J Clin Invest 50:2614-2626, 1971.
  2. Bergmann SR, Fox KAA, Ter-Pogossian MM, Sobel BE (Washington University), Collen D (University of Leuven): Clot-selective coronary thrombolysis with tissue-type plasminogen activator. Science 220:1181-1183, 1983.
  3. Van de Werf F, Ludbrook PA, Bergmann SR, Tiefenbrunn AJ, Fox KAA, de Geest H, Verstraete M, Collen D, Sobel BE: Coronary thrombolysis with tissue-type plasminogen activator in patients with evolving myocardial infarction. N Engl J Med 310:609-613, 1984.
  • Adan J, Bernstein LH, Babb J. Can peak CK-MB segregate patients with acute myocardial infarction into different outcome classes?  Clin Chem 1985; 31(2):996-997. ICID: 844986
  • Bernstein LH, Reynoso G.  Creatine kinase B-subunit activity in serum in cases of suspected myocardial infarction: a prediction model based on the slope of MB increase and percentage CK-MB activity. Clin Chem 1983; 29(3):590-592. ICID: 825549
  • Bernstein LH, Horenstein JM, Sybers HB, Russell PJ.  Adenylate kinase in human tissue. II. Serum adenylate kinase and myocardial infarction. J Mol Cell Cardiol 1973; 5(1):71-85. ICID: 825590

A Metabolic Functional Meaning of Existence of Isoenzymes

There are many examples of  isozymes, such as glucokinase, a variant of hexokinase which is not inhibited by glucose 6-phosphate. It has different regulatory features and lower affinity for glucose (compared to other hexokinases). Alkaline and acid phosphatase isoenzymes were used briefly for a time in clinical diagnostics.  These isoenzymes are oligomeric proteins that have distinct subunits that affect their binding with substrate.  A distinctive type of protein that can form two or more different homo-oligomers, comes apart and changes shape to convert between forms is called a morpheein. The alternate shape may reassemble to a different oligomer, and the shape of the subunit dictates which oligomer is formed. Morpheeins can interconvert between forms under physiological conditions and can exist as an equilibrium of different oligomers. Features of morpheeins can be exploited for drug discovery. A small molecule compound can shift the equilibrium either by blocking or favoring formation of one of the oligomers. The equilibrium can be shifted using a small molecule that has a preferential binding affinity for only one of the alternate morpheein forms. This introduces the concept of allostericity.  Most allosteric effects can be explained by a model put forth by Monod, Wyman, and Changeux, and also by a model described by Koshland, Nemethy, and Filmer. Both postulate that enzyme subunits exist in one of two conformations, tensed (T) or relaxed (R), and that relaxed subunits bind substrate more readily than those in the tense state.  This concept provides a foundation for another generation of biomarkers than was the focus of the 20th century, and only has been investigated since the 1980’s, and takes another dimension after the completion of the Human Genome Project, opening a “Pandora’s box”. This moved biomedical science forward into an emerging field of ‘OMICs’, which tied small molecules into regulatory processes, transcription, and the possibility of identifying new biomarkers and developing new biomolecules that could modify disease progression.


  1. Bu Z, Callaway DJ. “Proteins MOVE! Protein dynamics and long-range allostery in cell signaling”. Adv in Protein Chemistry and Structural Biology 2011; 83: 163–221. doi:10.1016/B978-0-12-381262-9.00005-7. PMID 21570668.
  2.  Monod J, Wyman J, Changeux JP. On the nature of allosteric transitions:A plausible model. J Mol Biol, May 1965; 12:88-118.
  3.   Koshland DE, Némethy G, Filmer D. Comparison of experimental binding data and theoretical models in proteins containing subunits. Biochemistry. Jan 1966; 5(1):365-8
  4.  Jaffe EK. “Morpheeins – a new structural paradigm for allosteric regulation”. Trends Biochem Sci 2005; 30(9): 490–497. doi:10.1016/j.tibs.2005.07.003. PMID 16023348.
  5.  Huang Z, Zhu L, Cao Y, Wu G, Liu X, et al.  ASD: a comprehensive database of allosteric proteins and modulators. Nucleic Acids Res 2011; 39: D663-669

Fundamental Transformative Concepts Carried Over from Physics to Biomolecular Processes.

A colleague once noted that we are learning more and more about less and less.  This is the remarkable evolution of our thinking from macrostates to microstates and segmentation of processes, further leading us to exploration of interactions between states.  This has required a breakdown and a repeated remodeling or resynthesis of ideas based on new findings in science.  It has gradually driven medicial science to a greater dependence on chemistry and physics in underlying principle.  We can better envision the mechanism of evolution from the concepts put forth.

In 1824 Sadi Carnot published the concept that heat is lost in the conversion into work, using the term “caloric”, equivalent to entropy in the second law of thermodynamics.  Clausius then develops the concepts of interior work in 1854, i.e. that “which the atoms of the body exert upon each other”, and exterior work, i.e. that “which arise from foreign influences [to] which the body may be exposed”, anticipating the concept of entropy. He enunciated the passage of the quantity of heat Q from the temperature T1 to the temperature T2 has the equivalence-value entropy, symbolized by S :  dS = Q (1/T2 – 1/T1), which led to his 1865 statement on irreversible heat loss: I propose to name the quantity S the entropy of the system, after the Greek word [τροπη trope], the transformation. I have deliberately chosen the word entropy to be as similar as possible to the word energy.”  In 1876, physicist J. Willard Gibbs, building on the work of Clausius, Hermann von Helmholtz and others, proposed that the measurement of “available energy” ΔG in a thermodynamic system could be mathematically accounted for by subtracting the “energy loss” TΔS from total energy change of the system ΔH, and in 1877, Ludwig Boltzmann formulated the alternative definition of entropy S defined as:

S = kBlnΩ


kB is Boltzmann’s constant and

Ω is the number of microstates consistent with the given macrostate.

An analog to thermodynamic entropy is information entropy. Claude Shannon set out to mathematically quantify the statistical nature of “lost information” in phone-line signals  and developed  a concept of information entropy, a fundamental cornerstone of information theory. The close similarity between his new quantity and earlier work in thermodynamics is attributed to a visit and discussion with Jon von Neumann in 1949. Shannon then called the “measure of uncertainty” or attenuation in phone-line signals with reference to his new information theory.  This led to the elucidation of a signal (as opposed to noise, by Solomon Kullback, which became the basis for the measure of an optimum diagnostic decision point of a laboratory test by Bernstein and Rudolph, related to Eugene Rypka’s “Syndromic Clustering”.  The loop was closed by the Japanese mathematician Akaike, who brought Fisher’s statistical formulations and Kullback-Liebler distance into alignment.   This is not a digression because it has been central to underlying principles in resolution in spectroscopy, and to classification of biochemical molecular features.

Although Boltzmann first linked entropy and probability in 1877, it seems the relation was never expressed with a specific constant until Max Planck first introduced k, and gave an accurate value for it (1.346×10−23 J/K, about 2.5% lower than today’s figure), in his derivation of the law of black body radiation in 1900–1901. Before 1900, equations involving Boltzmann factors were not written using the energies per molecule and the Boltzmann constant, but rather using a form of the gas constant R, and macroscopic energies for macroscopic quantities of the substance. The iconic terse form of the equation S = k log W on Boltzmann’s tombstone is in fact due to Planck, not Boltzmann. Planck actually introduced it in the same work as his h. Planck noted in his 1920 Nobel Prize acceptance : “:This constant is often referred to as Boltzmann’s constant, although, to my knowledge, Boltzmann himself never introduced it — a peculiar state of affairs.”  The Kullback–Leibler divergence (also information divergence, information gain, relative entropy, or KLIC) is a non-symmetric measure of the difference between two probability distributions P and Q, was  introduced by Solomon Kullback and Richard Leibler in 1951. KL-divergence of a model from reality may be estimated, to within a constant additive term, by a function (like the squares summed) of the deviations observed between data and the model’s predictions. When trying to fit parametrized models to data there are various estimators which attempt to minimize Kullback–Leibler divergence, such as, the familiar maximum likelihood  estimator.


  1. Planck, Max (2 June 1920), The Genesis and Present State of Development of the Quantum Theory (Nobel Lecture)
  2. Kalinin M, Kononogov S. “Boltzmann’s Constant, the Energy Meaning of Temperature, and Thermodynamic Irreversibility”, Measurement Techniques 2005; 48 (7): 632–36, doi:10.1007/s11018-005-0195-9
  3. Kullback S, Leibler RA “On Information and Sufficiency”. Annals of Mathematical Statistics 1951; 22 (1): 79–86. doi:10.1214/aoms/1177729694. MR 39968.
  4. Kullback S (1959) Information theory and statistics (John Wiley and Sons, NY).
  5. Jaynes ET(1957) Information theory and statistical mechanics, Physical Review 106:620
  6. Jaynes ET(1957) Information theory and statistical mechanics II, Physical Review 108:171
  7. Burnham KP and Anderson DR. (2002) Model Selection and Multimodel Inference: A Practical Information-Theoretic Approach, Second Edition (Springer Science, New York) ISBN 978-0-387-95364-9.
  8. Rudolph RA, Bernstein LH, Babb J.  Information induction for predicting acute myocardial infarction. Clin Chem 1988; 34(10):2031-2038. ICID: 825568

A New Imperative

Cardiovascular Biomarkers


[A] Aids in the Prevention of Cardiac Events by Detecting Silent Ischemic Lesions and Selecting Patients for Imaging

12/17/12 · Emily Humphreys

Physicians use risk factors, such as history, exercise level, diabetes, blood pressure, lipid profiles, and other laboratory measurements to ascertain risk for cardiac events, which are not foolproof in predicting all cardiac events. Nonetheless, 40% to 50% of sudden cardiac deaths (SCD) occur before risk factors are able to predict cardiac events.2,3 Those who die suddenly with no so sign of cardiac disease often have silent cardiac target organ damage (cTOD).  While patients with silent ischemia have a 21-fold increase in risk of a coronary event.4 It has also been shown that cTODs such as left ventricular hypertrophy (LVH), left ventricular systolic dysfunction (LVSD), left ventricular diastolic dysfunction (LVDD), and left atrial enlargement (LAE) each independently predict cardiovascular events5,6,7,8 Nadir et al. hypothesized that identification of silent cTOD would aid in the prevention of cardiovascular events, including SCDs.9 To identify cTOD present, The Nadir group evaluated several known cardiac biomarkers including: B-type natriuretic peptide (BNP), high-sensitivity cardiac troponin T (hs-cTnT), microalbuminuria, the estimated glomerular filtration rate, and uric acid.  The lab results of 300 asymptomatic individuals recruited for the study were compared with primary screening using transthoracic echocardiography, stress echocardiography, and/or myocardial perfusion imaging.
  • 34% of study volunteers had evidence of a cTOD. Out of all biomarkers analyzed, BNP levels were significantly higher in those with cTOD compared with those without. BNP levels were also higher in those who had more than one form of cTOD compared with those who had a single form of cTOD.
  • Hs-cTnT also performed well, but BNP levels had the highest correlation to imaging data. The gold standard diagnostic tool for cardiovascular disease is imaging studies, such as echocardiography.
  • It is not standard practice to investigate healthy individuals for possible cTOD and would be costly and time consuming to perform imaging on these individuals.
  • Biomarkers like BNP could be used as a primary screening tool with follow-up image studies performed, if necessary.

The eventual hope is to reduce the mortality of cardiovascular diseases and prevent silent cTOD from leading to more serious and potentially life-threatening cardiac events.


1. Roger, V.L. (2012) ‘AHA statistical update: Heart disease and stroke statistics-2012 update. A report from the american heart association‘, Circulation, 125 (2012), (pp. e2-e220)

2. Chiuve, S.E., et al., (2006) ‘Healthy lifestyle factors in the primary prevention of coronary heart disease among men: Benefits among users and nonusers of lipid-lowering and antihypertensive medications‘ Circulation, 114 (2006), (pp. 160-167)

3.De Vreede-Swagemakers, J.J., et al. (1997) ‘Out-of-hospital cardiac arrest in the 1990s: A population-based study in the Maastricht area on incidence, characteristics and survival‘, Journal of the American College of Cardiology, 30 (1997), (pp. 1500-1505)

4. Rutter, M.K., et al. (2002) ‘Significance of silent ischemia and microalbuminuria in predicting coronary events in asymptomatic patients with type 2 diabetes‘, Journal of the American College of Cardiology, 40 (2002), (pp. 56-61)

5. Tsang, T.S., et al. (2003) Prediction of risk for first age-related cardiovascular events in an elderly population: The incremental value of echocardiography‘, Journal of the American College of Cardiology, 42 (2003), (pp. 1199-1205)

6. Gosse, P., (2005) ‘Left ventricular hypertrophy—the problem and possible solutions‘,The Journal of International Medical Research, 33 (Suppl 1) (2005), (pp. 3A-11A)

7. Benjamin, E.J., et al. (1995) ‘Left atrial size and the risk of stroke and death‘ The Framingham Heart Study Circulation, 92 (4), (pp. 835-41)

8. Redfield, M.M., et al. (2003) ‘Burden of systolic and diastolic ventricular dysfunction in the community: Appreciating the scope of the heart failure epidemic‘, JAMA, 289 (2003), (pp. 194-202)

9. Nadir, M.A., et al., (2012) ‘Improving the primary prevention of cardiovascular events by using biomarkers to identify individuals with silent heart disease‘, Journal of American College of Cardiology, 60 (11), (pp. 960-968) Tags: 

[B] Evaluating CHF patients in the emergency department

The role of B-type natriuretic peptide in the evaluation of congestive heart failure patients in emergency department

Congestive heart failure (CHF) is a severe cardiovascular disorder seen in the Emergency Department (ED). B-type Natriuretic Peptide (BNP) is usually ordered to evaluate the CHF severity.

However, it is difficult to interpret serum BNP level when different clinical entities existed.

The aim of this study is to illustrate the correlation between serum BNP level and

  • relevant clinical variables and
  • further determine the role of serum BNP in different CHF patients.

High variability of serum BNP levels exists in CHF patients with weak-to-moderate correlation effects particularly on obesity and diastolic/systolic HF.

Physicians should be cautious on interpreting BNP in different CHF populations.

[C]   NT-proBNP compared with ECHO

Comparison of N-Terminal Pro B-Natriuretic Peptide and Echocardiographic Indices in Patients with Mitral Regurgitation.  Shokoufeh Hajsadeghi1, Niloufar Samiei2, Masoud Moradi3, Maleki Majid2, et al. Corresponding author email:


Introduction: Echocardiographic indices can form the basis of the diagnosis of systolic and diastolic left ventricular (LV) dysfunction in patients with Mitral regurgitation (MR). However, using echocardiography alone may bring us to a diagnostic dead-end. The aim of this study was to compare N-Terminal pro B-natriuretic peptide (BNP) and echocardiographic indices in patients with mitral regurgitation.

Methods: 2D and Doppler echocardiography and BNP serum level were obtained from 54 patients with organic mild, moderate and severe MR.

Results: BNP levels were increased with symptoms in patients with mitral regurgitation (NYHAI: 5.7 ± 1.1, NYHAII: 6.9 ± 1.5, NYHAIII: 8.3 ± 2 pg/ml, P , 0.001). BNP plasma level were significantly correlated with MPI (myocardial performance index)(r = 0.399, P = 0.004), and following echocardiographic indices: LVEDV (r = 0.45, P , 0.001), LVESV (r = 0.54, P , 0.001), LVEDD (r = 0.48, P , 0.001), LVESD (r = 0.54, P , 0.001), dp/dt (r = −0.32, P = 0.019) and SPAP (r = 0.4, P = 0.006).

Conclusion: The present study showed that BNP may be useful in patients with MR and may confirm echocardiographic indices.

Keywords: mitral regurgitation, N-Terminal pro-B natriuretic peptide, echocardiographic indices.

The hypothesis assumes that there is a linear sequence of most effective screening that comes out of this study, from a b-type natriuretic peptide to the imaging.  It’s not clear that that is the case, and moreover, silent myocardial infarct is taken and lumped with other serious conditions affecting the myocardium, presumably through compromise of the end-artery circulation to the heart (R, L, and circumflex coronaries).  There is no mention of whether the patients were screened out for peripheral, carotid, or other associated artery disease (superior mesenteric).

I’ll assume that that is the case.  I see a problem with a linear, monothetic, “gold standard” approach, when the disease and the diagnosis of it is multivariate and requires a method that uses a classificatory approach.  We’ll return to that.

English: A Wiggers diagram, showing the cardia...

English: A Wiggers diagram, showing the cardiac cycle events occuring in the left ventricle. (Photo credit: Wikipedia)

[D]  reference normal for NT-proBNP


Background: The natriuretic peptides, B-type natriuretic peptide (BNP) and NT-proBNP that have emerged as tools for diagnosing congestive heart failure (CHF) are affected by age and renal insufficiency (RI).This study evaluates the reference value for interpreting NT-proBNP concentrations. Increasing concentrations of NT-proBNP are associated with co-morbidities, not merely CHF, resulting in altered volume status or myocardial filling pressures.

Methods: NT-proBNP was measured in a population with normal trans-thoracic echocardiograms (TTE) and free of anemia or renal impairment.

Selection of Patients: Study participants were seen in acute care for symptoms of shortness of breath suspicious for CHF.

Results: The median NT-proBNP for patients under 50 years is 27.6 pg/ml with an upper limit of 445 pg/ml, and for patients over 50 years the median was 142.3 pg/ml with an upper limit of 475.3 pg/ml. We introduce a transformed NT-proBNP that normalizes for decrease in glomerular filtration rate and eliminates the age factor.

Conclusion: We suggest that NT-proBNP levels can be more accurately interpreted only after removal of the major co-morbidities that affect an increase in this peptide in serum. The PRIDE study guidelines should be applied until presence or absence of comorbidities is diagnosed. With no comorbidities, the reference range for normal over 50 years of age can be reduced below 800 pg/ml. The effect shown in previous papers likely is due to increasing concurrent comorbidity with age.

Key Words: Congestive Heart Failure, Natriuretic peptides, Anemia, Chronic renal insufficiency

Statistical treatment:

The combined acute and blood donor study sets were kept separate and each analyzed for central tendency, distribution and variability. The two were combined after the comorbidities described above were extracted from the acute care study group. This resulted in a population that should be representative of an unaffected study population that could be used to establish a most representative reference range. The database was replicated several times and then patient rows were randomly deleted until there was an expanded combined and mixed data set with 6,700 entries. All of the database sets were used for analyses.

The results are reported in means with p < 0.05 as the measure of significance for difference between means. Independent Student’s t-tests were applied comparing NT-proBNP and anemia. Univariate ANOVAs were used to compare NT-proBNP levels with varying ranges of hemoglobin and age using SPSS 15.0 (SPSS, Chicago, IL). A linear regression analysis with linear fitting and confidence interval was performed using SYSTAT 12 (SYSTAT, San Jose, CA). The results are reported in means with p < 0.05 as the measure of significance for difference between means. Linear regression analysis, Independent Student’s t and Mann-Whitney tests were applied comparing NT-proBNP for age intervals. Reference range was determined using MedCalc (Mariakerke, Belgium).

We observe the following changes with respect to NT-proBNP and age:
  • Sharp increase in NT-proBNP at over age 50
  • Increase in NT-proBNP at 7 percent per decade over 50
  • Decrease in eGFR at 4 percent per decade over 50
  • Slope of NT-proBNP increase with age is related to proportion of patients with eGFR less than 90
  •  NT-proBNP increase can be delayed or accelerated based on disease comorbidities
Adjustment of the NT-proBNP for eGFR and for age over 50 difference

We have carried out a normalization to adjust for both eGFR and for age over 50:

  • Take Log of NT-proBNP and multiply by 1000
  • Divide the result by eGFR (using MDRD[9] or Cockroft Gault[10])
  • Compare results for age under 50, 50-70, and over 70 years
  • Adjust to age under 50 years by multiplying by 0.66 and 0.56.

GFR (mL/min/1.73 m2) = 186 x (Scr)-1.154 x (Age)-0.203 x (0.742 if female) x (1.210 if African-American) (conventional units)

The equation does not require weight because the results are reported normalized to 1.73 m2 body surface area, which is an accepted average adult surface area.

Comparison of the mean + standard deviation of 607 blood donors and NYMH inpatients for the MDRD and Cockroft Gault (eCG), respectively gave 114.3, 43.7(MDRD); 105.0, 40.1 (eCG). The eCG is predicted by the regression: eCG = 0.059 + 0.918*MDRD. The mean + standard deviation for the age under 50 years and 50 or older is 106.5 + 14.7, 100.9 + 14.5 (MDRD); and 102.5 + 18.5, 98.4 + 20.8 (eCG). These differences are significant at < 0.0001, and 0.010, respectively.

The means comparison of the normalized NT-proBNP (NKLog[NT-proBNP]/eGFR) results in 23.4 and 18.7 for 307 non-donors and 300 donors, significant at p < 0.0001, assuming unequal variance). The difference is not significant for the MDRD normalized NT-proBNP (16.5, 6.6). The normalized by eCG result for 324 under age under 50 years and 283 age 50 years and older is 17.7 versus 18.2, significant at p = 0.0001. The MDRD calculated adjustment is 16.8 vs 16.9, which is not significant. The relationship between these is NKLog(NT-proBNP)/eCG = 4.47 + 0.948*NKLog(NT-proBNP)/MDRD. Figure 4 is a plot of the regression of NKLog(NTproBNP)/MDRD vs NKLog(NTproBNP)/eCG predicted over the full study population.

The reference range for the normalized Klog(NT-proBNP)/MDRD is described by a mean 13.99, median 13.12, and standard deviation 6.14 with a nonparametric upper limit of 24.7. A ROC curve is constructed comparing the NT-proBNP, the NKLog(NTproBNP)/MDRD and the ratio NTproBNP to NKLog(NTproBNP)/MDRD in the expanded full database. The area under the curve is 0.944 (0.938-0.950) for NKLog(NTproBNP)/MDRD with a base of 571 patients with early CHF and 6115 patients without. The reference range for NKLog(proBNP)/MDRD can be referenced to the percentiles as follows: 20, 8.78; 40, 11.92; 60, 14.85; 90, 21.10; 95, 24.73; 97.5, 29.54.

Conclusion: We suggest that NT-proBNP levels can be accurately assessed only after removal of the major confounding co-morbidities that increase this peptide in serum. We established our new range after establishing absence of co-morbidities. The value of this approach for screening purposes is an allowance for a considerably lower reference normal with a higher specificity based on the donor studies and the mixture model. This study finds that the reference range for NT-proBNP is age-dependent past age 50 years, mainly as the change relates to eGFR, and we introduce an age adjusted alternative measure, the normalized NT-proBNP using the MDRD transformation described.

NT-pro BNP reference range with blood donors and patients

Measure                                            NT-proBNP (pg/ml)                         After trimming extremes

Highest                                                    1110                                                                   599.4

Arithmetic mean                                   179.6                                                                   118.2

Geometric mean                                        68.7                                                                      54.4

Median                                                          52.6                                                                     42.6

Standard deviation                                250.5                                                                  150.6

D’Agostino-Pearson                          P = 0.0026                                                    P = 0.0091


< 50 years                                                  526.9                                                                 445.0

> 50 years                                                1000                                                                    565.0

Upper Limit of Normal                           772.9                                                                475.3

95% confidence interval                   637.1 – 873.73                                      442.7 – 531.0

Bernstein LH, Zions MY, Alam ME, Haq SA, Heitner JF, et al.  What is the best approximation of reference normal for NT-proBNP? Clinical Levels for Enhanced Assessment of NT-proBNP (CLEAN)

Renal Effect on NT-proBNP

NT-proBNP is excreted by the kidney.  Therefore, GFR has to be taken into account in adjusting the reference range.  BNP, unlike the propeptide, is eliminated 80% by vascular endothelium.  What would be the effect of vascular endothelium erosion?  We don’t know.

The Cockroft Gault equation is widely used in hospitals for adjusting medication doses in hospital patients. The MDRD equation was developed for patients with renal insufficiency and has been shown to be comparable to CG for the population the MDRD is applied. However, the MDRD is only reported to a CLCR of only 60 ml/min and is not validated for age over 65 years. On the other hand, the body weight and BMI, necessary for calculating the CG formula are not routinely done for all patients or in all hospitals. We used 307 inpatients and calculated the MDRD up to 100 ml/min/m2, then used the results to predict the CG. The regression for MDRD versus the CG resulted in an r = 0.884, and a regression equation: CG = -21.1 + 1.172*(MDRD). The initial prediction of CG from MDRDe from 198 of the patients is defined by the regression: CGe = -64.6 + 1.866*MDRDe. (Deming)(95% CI: Intercept -84.5 to -42.8; slope 1.40 to 2.33).  The means, medians, standard deviations, and 97.5th percentiles, respectively, of the age, MDRDe and CGe (calculated from weight data) for the 307 patients are: age- 61.2, 62.0, 17.4, 91.3; MDRD – 121.5, 107.5, 55.9, 212.3; CG – 111.7, 98.7, 51.4, 195.0.

The NT-proBNP was adjusted using a log transform and the estimated GFR (glomerular filtration rate by the original method of Levey et al.  The result for reference corrected Nt proBNP is shown in Table 2.

Table 2.

Kruskal-Wallis test

Factor codes MDRD60
Sample size




Average Rank







Test statistic


Corrected for ties  Ht


Degrees of Freedom (DF)


Significance level

P < 0.0001

[E]   Mid-region proANP in Emergency Room

Mid-region pro-hormone markers for diagnosis and prognosis in acute dyspnea: results from the BACH (Biomarkers in Acute Heart Failure) trial.
J Am Coll Cardiol 2010 May 11;55(19):2062-76 (ISSN: 1558-3597)
Maisel A; Mueller C; Nowak R; Peacock WF; Landsberg JW; Ponikowski P; Mockel M; Hogan C; Wu AH; Richards M; Clopton P; Filippatos GS; Di Somma S; Anand I; Ng L; Daniels LB; Neath SX; Christenson R; Potocki M; McCord J; Terracciano G; Kremastinos D; Hartmann O; von Haehling S; Bergmann A; Morgenthaler NG; Anker SD
VA San Diego Healthcare System, San Diego, California 92161, USA.
OBJECTIVES: Our purpose was to assess the diagnostic utility of midregional pro-atrial natriuretic peptide (MR-proANP) for the diagnosis of acute heart failure (AHF) and the prognostic value of midregional pro-adrenomedullin (MR-proADM) in patients with AHF. BACKGROUND: There are some caveats and limitations to natriuretic peptide testing in the acute dyspneic patient. METHODS: The BACH (Biomarkers in Acute Heart Failure) trial was a prospective, 15-center, international study of 1,641 patients presenting to the emergency department with dyspnea. A noninferiority test of MR-proANP versus B-type natriuretic peptide (BNP) for diagnosis of AHF and a superiority test of MR-proADM versus BNP for 90-day survival were conducted. Other end points were exploratory. RESULTS: MR-proANP (> or =120 pmol/l) proved noninferior to BNP (> or =100 pg/ml) for the diagnosis of AHF (accuracy difference 0.9%). In tests of secondary diagnostic objectives, MR-proANP levels added to the utility of BNP levels in patients with intermediate BNP values and with obesity but not in renal insufficiency, the elderly, or patients with edema. Using cut-off values from receiver-operating characteristic analysis, the accuracy to predict 90-day survival of heart failure patients was 73% (95% confidence interval: 70% to 77%) for MR-proADM and 62% (95% confidence interval: 58% to 66%) for BNP (difference p < 0.001). In adjusted multivariable Cox regression, MR-proADM, but not BNP, carried independent prognostic value (p < 0.001). Results were consistent using NT-proBNP instead of BNP (p < 0.001). None of the biomarkers was able to predict rehospitalization or visits to the emergency department with clinical relevance. CONCLUSIONS: MR-proANP is as useful as BNP for AHF diagnosis in dyspneic patients and may provide additional clinical utility when BNP is difficult to interpret. MR-proADM identifies patients with high 90-day mortality risk and adds prognostic value to BNP. (Biomarkers in Acute Heart Failure [BACH]; NCT00537628). [Copyright 2010 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.].
Comment In: RefSource:J Am Coll Cardiol. 2010 May 11; 55(19):2077-9/PMID:20447529


[A] Predictor of benefit of lowering CRP with statin

Sever PS, Poulter NR, Chang CL, et al; ASCOT Investigators. Evaluation of C-reactive protein prior to and on-treatment as a predictor of benefit from atorvastatin: observations from the Anglo-Scandinavian Cardiac Outcomes Trial. Eur Heart J. 2012;33:486-494

The theory that lowering the C-reactive protein (CRP) level with statin therapy is predictive of cardiovascular outcomes independent of lowering the low-density lipoprotein (LDL) cholesterol level was first advanced by the JUPITER investigators.[1]

  • This study further fueled the debate on whether CRP measurement should be routine for monitoring cardiovascular disease risk. The ASCOT investigators entered this debate when they analyzed data from their trial to determine whether on-statin C-reactive protein level was associated with cardiovascular outcomes.[4]
  • The data in the nested case-control study were from persons recruited to ASCOT in the United Kingdom and Ireland, 9098 of whom were randomly assigned in the blood pressure-lowering arm to receive either amlodipine with or without perindopril or atenolol with or without bendroflumethiazide.[5] Within the whole blood-pressure-lowering group, 4853 persons with a total cholesterol level of 6.5 mmol/L or less (≤ 250 mg/dL) were further randomized to receive atorvastatin or placebo as part of the lipid-lowering arm.[6]
  • For the case-control study, 485 cardiovascular cases were age- and sex-matched with 1367 controls. As expected, the investigators found that baseline LDL cholesterol and CRP levels both predicted cardiovascular events.
  • However, neither the baseline nor the on-treatment CRP level was related to the magnitude of statin efficacy in the prevention of cardiovascular events, whereas the on-treatment LDL cholesterol level was strongly predictive. Including CRP in the Framingham model resulted in a “modest” (2.1%) improvement in risk prediction overall.

The investigators noted that this finding was in line with other prospective studies that showed statistically significant, but modest, absolute improvements with the use of CRP in clinical risk prediction.[7,8] They concluded that “in this hypertensive population selected on the basis of traditional, common coexisting risk factors, CRP did not usefully improve the prediction of cardiovascular events and, critically, reduction in CRP associated with statin therapy was not a predictor of cardiovascular outcome alone or in combination with LDL-cholesterol.”

Eugene Braunwald downplayed the ASCOT investigators’ conclusions in observing “the ASCOT results, albeit quite limited in size, are in fact remarkably similar to those of the [CARE, AFCAPS/TexCAPS, REVERSAL, A to Z], and JUPITER trials, especially in light of the fact that the dose of atorvastatin [in ASCOT] was only 10 mg, while some of the other trials used considerably larger equivalent doses of statins.”

My own take on this is that for at least two decades, there was a belief that the LDL lowering was the main effect of statins, until the (deep frozen) specimens were reexamined from the Framingham study using a hs CRP assay.  The investigation was to determine whether there is a predictor of CVD that is present when the traditional features are not present (which would have to include diabetes and hypertension).  The basis for the use of hsCRP became to identify patients who had sufficient risk to be treated with a statin.  The essential focus seemed to turn on whether statin treatment has efficacy in view of the differential between the LDL or the CRP on the magnitude of the effect.  The muscular effect of a statin then comes into view with the size dose and length of treatment.  However, the CRP measurement identified a relationship between development of the vascular disease and the inflammatory process independently of the STATIN treatment benefit.

Prof. Sever (Medscape): The key result that we found in the initial paper was that CRP, although an independent predictor of cardiovascular events in the hypertensive population, was really only a weak predictor, which is confirmed by the meta-analyses. Furthermore, when you incorporate CRP into a Framingham-style model, it really does not add any benefit or give any more information than if it had not been included in the model. LDL cholesterol is a much more important biomarker. Our second important conclusion addressed the question of whether, after starting a patient on statin therapy, the magnitude of lowering CRP by the statin and the level to which CRP has been reduced predicts cardiovascular outcome. The simple answer from our analyses was that it did not and that the benefits were all related to lowering LDL cholesterol. Our population comprised patients with stable hypertension and no history of coronary disease; likewise, the diabetes population in CARDS had no history of coronary heart disease. Oddly, PROVE IT-TIMI 22 involved persons who were selected from a high-risk coronary heart disease population simply because they had a high level of an inflammatory marker. So, to a certain extent, this is like comparing apples and oranges, and to find some unifying hypothesis on the basis of widely heterogeneous patient populations seems to be a little naive.

[B] Predictor of cardiovascular disease

Acute Phase Reactants as Novel Predictors of Cardiovascular Disease  M. S. AhmedA. B. JadhavA. Hassan, and Qing H. Meng SRN Inflammation 2012; Article ID 953461, 18 pages. doi:10.5402/2012/953461

  • Acute phase reaction is a systemic response which usually follows a physiological condition that takes place in the beginning of an inflammatory process.
  • This physiological change usually lasts 1-2 days. However, the systemic acute phase response usually lasts longer.
  • The aim of this systemic response is to restore homeostasis.
  1. These events are accompanied by upregulation of some proteins (positive acute phase reactants) and
  2. downregulation of others (negative acute phase reactants) during inflammatory reactions.

Cardiovascular diseases are accompanied by the elevation of several positive acute phase reactants such as

  • C-reactive protein (CRP),
  • serum amyloid A (SAA),
  • fibrinogen,
  • white blood cell count,
  • secretory nonpancreatic phospholipase 2-II (sPLA2-II),
  • ferritin, and
  • ceruloplasmin.

Cardiovascular disease is also accompanied by the reduction of important transport proteins such as

  • albumin, transferrin,
  • transthyretin,
  • retinol-binding protein,
  • antithrombin, and
  • transcortin.

In this paper, we will be discussing the biological activity and diagnostic and prognostic values of acute phase reactants with cardiovascular importance.

The potential therapeutic targets of these reactants will be also discussed.


[C] CRP as an Inflammatory Biomarker

CRP is an acute phase protein [78] produced in the liver in response to interleukin- (IL-) 6 which is stimulated, in turn, by tumour necrosis factor-α (TNF-α) and IL-1 [89].

Recent studies suggest that CRP plays a pivotal role in many aspects of atherogenesis including

  • LDL uptake by macrophage,
  • release of proinflammatory cytokines,
  • expression of monocyte chemotactic protein-1,
  • intercellular adhesion molecule-1, and vascular cellular adhesion molecule-1 [1012].

Activation of inflammation and the acute phase reaction appear to play an important role, not only in the pathogenesis of atherosclerosis, but also in the initiation of the acute coronary syndrome (ACS) [13,14]. Cesari et al. suggested that the inflammatory markers CRP, IL-6, and TNF-α are independent predictors of cardiovascular events in older persons [14].

Diagnostic Value

CRP is also an early ischemic marker and elevated CRP is predictive of future adverse events [2223]. High-sensitivity CRP (hs-CRP) rises acutely after tissue injury, including myocardial infarction (MI). Intense cytokine production and inflammatory cell infiltration occur in the area of ischemia and necrosis. This increase of hs-CRP levels, in part, correlates with infarct size [2425]

CRP can be also used for patients screening in the primary prevention population [36]. Ockene et al. indicated that CRP is generally expressed at low levels (<1 mg/L) in healthy adults and levels remain relatively stable in the absence of an acute inflammatory stimulus [37].

Patients with unstable angina and CRP >3 mg/L at discharge are more likely to be readmitted for recurrent cardiovascular instability or MI within 1 year [38].

Pietilä et al. indicated that hs-CRP measurement is the strongest correlative factor for future clinical events due to arterial inflammation, myocardial infarction, unstable angina, stroke, and peripheral vascular disease in both diseased and apparently healthy asymptomatic patients [40].

The CRP plasma level also is the best risk assessment in patients with

  • either stable or unstable angina,
  • long term after myocardial infarction, and
  • in patients undergoing revascularization therapies [41].
  • One study showed the only independent cardiovascular risk indicators using multivariate, age adjusted and traditional risk analysis were CRP and Total/HDL cholesterol ratio.
  • If CRP, IL-6, and ICAM-1 levels are added to lipid levels, risk assessment can be improved over lipids alone.
  • Moreover, serum CRP may indicate the vulnerability of the plaque [40].

Prognostic Value

  • elevation of hs-CRP levels predicts a poor cardiovascular prognosis [42].
  • The extent of the inflammatory response to injury appears to have prognostic significance, which is independent of the extent of myocardial injury.
  • hs-CRP response after MI has been shown to predict future CHD morbidity and mortality independent of infarct size [43].
  • CRP is also a predictor of incident type 2 diabetes. As well, it adds a prognostic information on vascular risk at all levels of the metabolic syndrome [44].


III.  Troponin(s) and hs-TnI/cTnT

Comparison of diagnostic accuracy between three different rules of interpreting high sensitivity troponin T results. Francesco Buccelletti; Leonarda Galiuto; Davide Marsiliani; Paolo Iacomini; et al. Intern Emerg Med 2012; 7, 365


With the introduction of high sensitivity troponin-T (hs-TnT) assay, clinicians face more patients with ‘positive’ results but without myocardial infarction. Repeated hs-TnT determinations are warranted to improve specificity. The aim of this study was to compare diagnostic accuracy of three different interpretation rules for two hs-TnT results taken 6 h apart. After adjusting for clinical differences, hs-TnT results were recoded according to the three rules.

  • Rule1: hs-TnT >13 ng/L in at least one determination.
  • Rule2: change of >20 % between the two measures.
  • Rule3: change >50 % if baseline hs-TnT 14-53 ng/L and >20 % if baseline >54 ng/L.

The sensitivity, specificity and ROC curves were compared. The sensitivity analysis was used to generate post-test probability for any test result. Primary outcome was the evidence of coronary critical stenosis (CCS) on coronary angiography in patients with high-risk chest pain.

183 patients were analyzed (38.3 %) among all patients presenting with chest pain during the study period. CCS was found in 80 (43.7 %) cases.

The specificity was 0.62 (0.52-0.71), 0.76 (0.66-0.84) and 0.83 (0.74-0.89) for rules 1, 2 and 3, respectively (P < 0.01). Sensitivity decreased with increasing specificity (P < 0.01).

Overall diagnostic accuracy did not differ among the three rules (AUC curves difference P = 0.12). Sensitivity analysis showed a 25 % relative gain in predicting CCS using rule 3 compared to rule 1. Changes between two determinations of hs-TnT 6 h apart effectively improved specificity for CCS presence in high-risk chest pain patients.

There was a parallel loss in sensitivity that discouraged any use of such changes as a unique way to interpret the new hs-TnT results.

Advances in identifying NSTEMI biomarkers [Published 31 August 2012 | Article by Excerpta Medica | Tags: elderly, ami, biomarkers, diagnosis]

In the run-up to the ESC conference at the end of August, we review some recently published research on the hot topic of biomarkers for NSTEMI.

Prompt and accurate diagnosis of acute non-ST elevation myocardial infarction (NSTEMI) can be particularly challenging in elderly patients, as they often present with

  • atypical symptoms and/or have an inconclusive ECG.
  • the diagnostic value of cardiac troponin T (cTnT), an established marker of cardiac injury, is often limited as there is often non-coronary troponin elevation caused by concomitant conditions such as acute congestive heart failure.
  • Identifying new sensitive and specific biomarkers of NSTEMI in elderly patients is therefore important, and circulating microRNAs (miRs) are emerging as good candidates.

researchers evaluated the diagnostic potential of plasma levels of various miRs in elderly patients enrolled at presentation:

  • 92 acute NSTEMI patients (complicated by congestive heart failure in three-quarters of cases),
  • 81 patients with acute congestive heart failure without acute MI, and
  • 99 age-matched healthy people (the control group).

The researchers, from centers in Italy, found that levels of miR-1, miR-21, miR-133a, and miR-423-5p were 3-10 times higher in the patients with NSTEMI, compared with controls. Levels of miR-499-5p, meanwhile, were more than 80 times higher in the NSTEMI patients compared with the patients in the control group.

  • Levels of miR-499-5p and miR-21 were also significantly higher in the NSTEMI group compared with the group of patients with acute congestive heart failure without acute MI.
  • The researchers also found that miR-499-5p was similar to cTnT in being able to distinguish NSTEMI patients from the other two groups.
  • Also, a subgroup analysis of patients with a modest elevation in cTnT level at presentation (0.03-0.10 ng/mL) revealed that miR-499-5p had a diagnostic accuracy superior both to cTnT and to high sensitivity cTnT in differentiating NSTEMI from acute congestive heart failure.

International Journal of Cardiology


IV. Predicting cardiovascular mortality in NSTEMI patients

[A]  Japanese researchers studied 258 consecutive patients hospitalized for unstable angina and NSTEMI within 24 hours of the onset of chest symptoms, and followed them up for a median period of 49 months after admission. During this period there were 38 cardiovascular deaths (14.7%).

They reported that high-mobility group- box 1 (HMGB1), a nuclear protein and signaller of tissue damage, was “a potential and independent predictor of cardiovascular mortality in patients hospitalized for unstable angina and NSTEMI.

  • HMGB1,
  • cardiac troponin I,
  • Killip class greater than 1, and
  • age

were each independently and significantly associated with cardiovascular mortality.


[B]  William LaFramboise et al.       BMC Med. 2012 Dec 5;10(1):157)
see Report by Aviva Lev-Ari (  Coronary artery disease in symptomatic patients referred for coronary angiography: Predicted by Serum Protein Profiles

Significant differences were detected in circulating proteins from patients requiring revascularization including increased apolipoprotein B100 (APO-B100), C-reactive protein (CRP), fibrinogen, vascular cell adhesion molecule 1 (VCAM-1), myeloperoxidase (MPO), resistin, osteopontin, interleukin (IL)-1beta, IL-6, IL-10 and N-terminal fragment protein precursor brain natriuretic peptide (NT-pBNP) and decreased apolipoprotein A1 (APO-A1). Biomarker classification signatures comprising up to 5 analytes were identified using a tunable scoring function trained against 239 samples and validated with 120 additional samples. A total of 14 overlapping signatures classified patients without significant coronary disease (38% to 59% specificity) while maintaining 95% sensitivity for patients requiring revascularization. Osteopontin (14 times) and resistin (10 times) were most frequently represented among these diagnostic signatures. The most efficacious protein signature in validation studies comprised osteopontin (OPN), resistin, matrix metalloproteinase 7 (MMP7) and interferon gamma (IFNgamma) as a four-marker panel while the addition of either CRP or adiponectin (ACRP-30) yielded comparable results in five protein signatures.


V.  Assessing Cardiovascular Risk

Agency for Healthcare Research and Quality (AHRQ)

Assessing Cardiovascular Risk: Guideline Synthesis

Agency for Healthcare Research and Quality (AHRQ) Authors and Disclosures Posted: 03/01/2012


The Third MI Definition: An Expert Interview With Joseph Alpert In the new definition, the diagnosis of acute MI remains unchanged: That is, it applies where there is evidence of myocardial necrosis in a clinical setting consistent with acute myocardial ischemia. However, the criteria for diagnosis have been updated, with an emphasis on the biomarker cardiac troponin.

  1. The first essential criterion for diagnosis of MI is detection of a rise or fall in cardiac troponin, or CKMB if troponin is not available, with at least 1 value above the 99th percentile upper reference limit, plus at least 1 the following criteria:
  2. Symptoms of ischemia;
  3. ECG changes of new or presumed new ischemia (significant ST-segment T-wave changes or new left bundle branch block);
  4. Development of pathologic Q waves on ECG; or
  5. Imaging evidence of new loss of viable myocardium or new regional wall-motion abnormality.Other criteria include those for MI in sudden unexpected cardiac death and for MI during percutaneous coronary intervention (PCI) and coronary artery bypass graft surgery (CABG).

The guidance document supports the use of high-sensitivity cardiac troponin assays, especially for distinguishing myocardial injury not related to myocardial ischemia, such as that associated with heart failure or renal failure. These assays are available in Europe, and not in the United States. MI is designated as ST-segment elevation MI or non- ST-segment elevation MI, and as in the 2007 version, it is classified into 5 types on the basis of pathologic, clinical, and prognostic differences. These types have been updated in the latest version.

  1. Type 1 MI (spontaneous MI) is related to atherosclerotic plaque rupture or other event leading to thrombus formation in ≥ 1 of the coronary arteries, leading to decreased myocardial blood flow with ensuing necrosis;
  2.  Type 2 MI arises from a condition other than CAD;
  3.  Type 3 MI is deemed to have occurred when cardiac death occurs with symptoms suggestive of myocardial ischemia, but without biomarker values having been obtained; and
  4. Type 4 and 5 MIs are related to PCI and CABG, respectively, and have been redefined since 2007.

The new document also describes situations in which troponin levels are elevated in conditions where myocardial injury with necrosis is associated with predominantly nonischemic myocardial injury, such as heart failure, renal failure, myocarditis, arrhythmias, or pulmonary embolism.


VI Statistical Problems

The normal or “bell shaped” curve is a plot of numerical values along the x-axis and the frequency of the occurrence on the y-axis.  If the set of measurements occurs as a random and independent event, we refer to this as parametric, and the distribution of the values is a bell shaped curve with all but 2.5% of the values included within both ends, with the mean or arithmetic average at the center, and with 67% of the sample contained within 1 standard deviation of the mean.  We view a reference range in terms of a homogeneous population.  This means that while all values might not be the same, the values are scattered within a distance from the mean that becomes less frequent as the distance is larger so that we can describe a mean and a 95% confidence interval around the mean.  In the problem we are discussing, the classic reference value could be determined with outliers removed, and it would most likely fit to a Receiver Operator Characteristic curve.  This became blurred when the high sensitivity assay was introduced, which included NOISE, which is really not noise but heterogeneous elements related to [a] vascular events that are not caused by plaque rupture, or [b] ischemia related to “piecemeal necrosis” which continued might predict a serious future event.  Hidden variables include – age, diurnal variation, racial factors, and disease (hypertension, CHF, type 2 diabetes, renal failure).

A majority have no ST elevation on EKG, considered definitive for AMI.  This makes the finding of elevated and increasing cardiac specific enzyme or protein in serum of paramount importance for specifying damaged cardiac muscle in a context of insufficient circulation.   We examine the classification of AMI using a combination of features of chest pain, EKG, and a sensitive cardiac marker, derived from the cardiac muscle filament.   An optimum value for a test cutoff is, such as cTnT, can be derived without using a prior determination of disease status. This is an alternative to first carrying out a study with a training set, and then repeating it with a validation set, provided there is sufficient information for classifying the data..  We have to construct a self-classifying table of ordered classes that have assigned measurable risks.   Haberman (4) discusses the underlying assumptions used by Magidson for association models of cross-classified data in calculating the maximum likelihood estimates (MLE) by using the log-likelihood ratio and a sum of squares representing deviations of parameters from their constraints. The Latent Class Analysis (LCA) developed by Magidson and Vermunt allows use of a traditional LCA or a regression model (Statistical Innovation. Belmont, MA).  .  The LCA model uses the variables – chest pain, EKG, and troponin T – to classify the data and to test the underlying structure using powerful fit measures, such as L2.  Chest pain has the value of “typical” or “other”.   EKG has the value ST depression or any other (for a non Q-wave study).  “cTnT” has the value  assigned by rank in the scaling intervals.   The results of such an analysis are displayed in Table 1.

Table 1. This LCA classification was constructed using the troponin T before hsTnT was available.

CTnT (mg/L) MI (6%)  Not MI (94%)
0-0.03 0.0008 0.8485
0.031-0.055 0.0009 0.0791
0.056-0.080 0.0009 0.0369
0.081-0.099 0.0010 0.0106
0.10-0.199 0.2026 0.0238
> 0.20 0.7939 0.0012

Eugene Rypka. Syndromic classification: A process for amplifying information using S-Clustering.  Nutrition 1996; 14(12: 827-829.

Stuart W Zarich, Keith Bradley, Inder Dip Mayall, Larry H Bernstein. Minor elevations in troponin T values enhance risk assessment in emergency department patients with suspected myocardial ischemia: analysis of novel troponin T cut-off values. Clin Chim Acta 2004; 343(1-2):223-229

Haberman SJ. Computation of maximum likelihood estimates in association models. J Am Stat Assoc 1995;90:1438-1446

Rudolph RA, Bernstein LH, Babb J.  Information Induction for the Diagnosis of  Myocardial Infarction. Clin Chem 1988; 34: 2031-8.

Vermoent JK, and Magidson J. Latent class cluster analysis. JA Hagenaars and AL McCutcheon (eds.), Advanced Latent Class Analysis. Cambridge, Cambridge University Press, 2000.

Bernstein LH, Qamar A, McPherson C, Zarich S. Evaluating a new graphical ordinal logit method (GOLDminer) in the diagnosis of myocardial infarction utilizing clinical features and laboratory data.   Yale J Biol Med 1999; 72: 259-268.


VII. Conclusions I have made a number of comments to follow up on.  The diagnosis of myocardial infarct has been extended as a result of the emergence of the high sensitivity troponins, but the laboratory methods have not caught up with the technology as the identification of myocardial ischemia is broken down with fine granularity in order to

  • identify the high risk patients early
  • and manage them effectively at the earliest stage of disease evolution

We no longer ponder over

  1. ECG findings of new Q-qave, not previously seen
  2. ST elevation
  3. T-wave inversion

These are an indication of plaque rupture. There are strong associations between CRP, hyperhomocysteinemia, and then add the troponins and b-type natriuretic  and the pro b-type peptides.  These associations have to be analyzed by “syndromic classification”, described by Eugene Rypka. The study first described found great value in the BNP and proBNP.  Despite having the creatinine clearance, the NT-proBNP can’t be adequately interpreted without adjusting for the estimated glomerular filtration rate, and using a log transform for the high fold-increase with age.

There is much more to be done with capturing the information from the data we are generating.  The problem of classification requires accurate data measurement, but it also requires that features in scaled data are combined in meaningful ways.  That job is far from completed.


Below I paste all discussions on this post that are taking place on LinkedIn Group: Innovations in Cardiology:

Kathy Dowd, AuD • I am an audiologist representing the Academy of Doctors of Audiology for an initiative of early identification of hearing loss in adults with chronic diseases, including cardiovascular disease, thyroid disease, diabetes, etc. I am working on a new product that will automatically screen hearing of patients with these conditions and route them to audiologists for evaluation, treatment and counseling. Hearing loss is unidentified for most adults for 7-10. The psychological impact of hearing loss includes depression, isolation, confusion and poor job performance. We are focused on educating healthcare professionals on the need to identify this ‘silent epidemic’ as a co morbidity with most major illnesses.

Aviva Lev-Ari, PhD, RN • Dr. William LaFramboise

Thank you for your comment above and the reference to your most recent publication. It is very helpful. We are on the same page on this topic.

May I bring to the attention of the readers three sources which are shading additional light on that matter.

To Stent or Not? A Critical Decision

Obstructive coronary artery disease diagnosed by RNA levels of 23 genes – CardioDx heart disease test wins Medicare coverage

Follow William
William LaFramboise • Thank you Aviva. The CardioDx approach is promising with heavy commercial support for use in a primary care practitioner’s office. However, RNA acquisition, purification and qRT-PCR expression analysis takes 2-3 days, is performed off-site, derives from a small subset of circulating inflammatory cells and is not yet directly correlated with functional proteomics. So its value in the Emergency Room and Chest Clinic is currently limited. The proteomics test we published revealed systemic serum changes in a small number of proteins known to be involved in atherosclerosis. It has a faster turnaround time (minutes to hours) that could be implemented in an emergency room or chest clinic. We are predominantly interested in using our test to “rule out” symptomatic patients who currently undergo coronary angiography but do NOT have clinically significant CAD. Our goal is to eliminate unecessary catheterizations while catching all patients that should undergo coronary angiography with a high probability of percutaneous intervention. Currently, the patients we are targeting all undergo catheterization; our test will hopefully allow us to identify at least some of these patients who do not have CAD and eliminate this expensive and risky procedure. However, we are in the pioneering stages of developing our test so there are miles to go before we establish and validate clinical efficacy.


Larry Bernstein • What you have indicated is practical proteomics. There is more to be done in line with what Dr Lev-Ari has indicated based on additional voluminous literature. What you have done with a not so large data set, and probably underpowered looks very interesting.

I f you were willing to share the data, now that it is publihed, I think that we can sharpen the results using a method of “identifying anomalies”, and even come up with estimated probabilities for meaningful classes. I think that the best you can get is defined by Kullback-Liebler distance.

Larry H Bernstein, MD

Biomarker classification signatures comprising up to 5 analytes were identified using a tunable scoring function trained against 239 samples and validated with 120 additional samples. A total of 14 overlapping signatures classified patients without significant coronary disease (38% to 59% specificity) while maintaining 95% sensitivity for patients requiring revascularization. Osteopontin (14 times) and resistin (10 times) were most frequently represented among these diagnostic signatures. The most efficacious protein signature in validation studies comprised osteopontin (OPN), resistin, matrix metalloproteinase 7 (MMP7) and interferon gamma (IFNgamma) as a four-marker panel while the addition of either CRP or adiponectin (ACRP-30) yielded comparable results in five protein signatures.

Proteins in the serum of CAD patients predominantly reflected (1) a positive acute phase, inflammatory response and (2) alterations in lipid metabolism, transport, peroxidation and accumulation.

Follow William
William LaFramboise • Our study ( comprised discovery research using targeted immunochemical screening of retrospective patient samples using both Luminex and Aushon platforms as opposed to shotgun proteomics. Hence the costs constrained sample numbers. Nevertheless, our ability to predict outcome substantially exceeded available methods:

The Framingham CHD scores were statistically different between groups (P <0.001, unpaired Student’s t test) but they classified only 16% of the subjects without significant CAD (10 of 63) at a 95% sensitivity for patients with CAD. In contrast, our algorithm incorporating serum values for OPN, RES, CRP, MMP7 and IFNγ identified 63% of the subjects without significant CAD (40 of 63) at 95% sensitivity for patients with CAD. Thus, our multiplex serum protein classifier correctly identified four times as many patients as the Framingham index.

The addition of clinical variables to our scoring system should improve the acuity of our test as we move into the next phase. I appreciate your input and will contact you directly for further insights

Larry Bernstein • Bill La Fram..

our algorithm incorporating serum values for OPN, RES, CRP, MMP7 and IFNγ identified 63% of the subjects without significant CAD (40 of 63) at 95% sensitivity for patients with CAD

I think you can improve the algorithm with strong clinical features. The Goldman algorithm is stronger than the Framingham Index. Maybe its because the FI is epidemiological and is a measure of long term risk being present and does not indicate significant features at the time of presenting. The best features of the Goldman algorithm (without lab work) are – ECG (which may be arguable with NSEMI), but the presence of Afib or tachyarrhythmia could be added to that in weighting, and radiation actually should include symptoms of gall bladder (vagal nerve branch), and onset, characteristic and duration of pain, and SOB.

In your algorithm there isn’t any assessment of the hypercoagulable state, blood flow or Viscosity. There is a strong relationship between hyperhomocysteinemia and CVD, and the HHCys has ties to impaired methyl group transfers that maybe proinflammatory through more than one interaction: countering eNOS, related to Lp(a), un unknown relationship to iNOS (which becomes a counterpoise to eNOS), an effect on blood flow and viscosity, and a relationship to platelet aggregation.

Lp(a) was originally considered of less weight, except that it occurred in thin people from Asian Indian descent. The relationship to apo(B) and to dense LDL particles is now a factor. Sam Filligane uses Lp(a) in his ambulatory practice, and he also uses the PLAC test that Aviva has posted on.

The biggest problem we have is the amount of variability in the data physicians use. It makes metaanalysis a poor solution to the problem. The standardization of laboratory “panels” set up after CLIA 88 puts a real burden on the physician for unsubstantiated “cost benefits” in the light of today’s knowledge.


English: Four chamber view on cardiovascular m...

English: Four chamber view on cardiovascular magnetic resonance imaging. (Photo credit: Wikipedia)

Other related articles on Assessing Cardiovascular Disease with Biomarkers published on this Open Access Online Scientific Journal, include the following:


Dr. Lev-Ari’s research on Assessing Cardiovascular Disease with Biomarkers includes

  • Biomarkers in vascular biology,
  • Biomarkers in molecular cardiology and
  • circulating Endothelial Progenitor Cells (cEPCs) as a Biomarker for cardiovascular marcovascular disease risk


Lev-Ari, A., (2012U). Cardiovascular Outcomes: Function of circulating Endothelial Progenitor Cells (cEPCs): Exploring Pharmaco-therapy targeted at Endogenous Augmentation of cEPCs

Lev-Ari, A., (2012T). Endothelial Dysfunction, Diminished Availability of cEPCs, Increasing CVD Risk for Macrovascular Disease – Therapeutic Potential of cEPCs

Lev-Ari, A., (2012S). Vascular Medicine and Biology: CLASSIFICATION OF FAST ACTING THERAPY FOR PATIENTS AT HIGH RISK FOR MACROVASCULAR EVENTS Macrovascular Disease – Therapeutic Potential of cEPCs

Lev-Ari, A. (2012a). Resident-cell-based Therapy in Human Ischaemic Heart Disease: Evolution in the PROMISE of Thymosin beta4 for Cardiac Repair

Lev-Ari, A. (2012b). Triple Antihypertensive Combination Therapy Significantly Lowers Blood Pressure in Hard-to-Treat Patients with Hypertension and Diabetes

Lev-Ari, A. (2012h). Macrovascular Disease – Therapeutic Potential of cEPCs: Reduction Methods for CV Risk

Lev-Ari, A. (2012xx). Coronary artery disease in symptomatic patients referred for coronary angiography: Predicted by Serum Protein Profiles

Lev-Ari, A. (2013a) forthcoming, based on:

Part III: (2006c) Biomarker for Therapeutic Targets of Cardiovascular Risk Reduction by cEPCs Endogenous Augmentation using New Combination Drug Therapy of Three Drug Classes and Several Drug Indications. Northeastern University, Boston, MA 02115

Special Considerations in Blood Lipoproteins, Viscosity, Assessment and Treatment                                    Larry Bernstein

New Insights on Nitric Oxide donors – Part IV                Larry Bernstein

The Essential Role of Nitric Oxide and Therapeutic NO Donor Targets in Renal Pharmacotherapy             Larry Bernstein

A second look at the transthyretin nutrition inflammatory conundrum                                                                  Larry Bernstein

What is the role of plasma viscosity in hemostasis and vascular disease risk?                                                        Larry Bernstein

Biochemistry of the Coagulation Cascade and Platelet Aggregation – Part I                                                            Larry Bernstein

Laboratory, Innovative Technology, Therapeutics                                                                                                            Larry Bernstein

Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis                 Larry Bernstein

Overview of new strategy for treatment of T2DM: SGLT2 inhibiting oral antidiabetic agents                             aviralvatsa

Mitochondrial dynamics and cardiovascular diseases          ritusaxena

Nitric Oxide and it’s impact on Cardiothoracic Surgery        tildabarliya

Telling NO to Cardiac Risk                                                                  sjwilliamspa

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