The N-terminal of the prohormone brain natriuretic peptide (NT-proBNP) is a 76 amino acid N-terminal inactive protein that is cleaved from proBNP to release Brain Natriuretic Peptide. It is released in response to fluid overload. Brain natriuretic peptide (BNP) is a 32 amino acid cardiac natriuretic peptide hormone originally isolated from porcine brain tissue.
The human BNP gene is located on chromosome 1 and encodes the prohormone proBNP.
The biologically active BNP and the remaining part of the prohormone, NT-proBNP (76 amino acids) can be measured by immunoassay in human blood. 38
The main stimulus for peptide synthesis and secretion is neuronal hypoxia. Katoh and his co-workers demonstrated that BNP was upregulated and released from the human astrocytoma cell line U373MG under hypoxia through c-Src activation, and inhibition of BNP exacerbated hypoxia-induced apoptosis. The administration of exogenous BNP reduced hypoxia-induced apoptosis. The biological effects include diuresis, vasodilatation, inhibition of renin and aldosterone production and of cardiac and vascular myocyte growth.39
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NT-proBNP is cleared from plasma through binding to the natriuretic peptide clearance receptor type C, but it seems relatively resistant to proteolysis by neutral endopeptidase NEP 24.11. N-terminal pro-brain natriuretic peptide levels are elevated in patients with acute ischemic stroke. Gainnokoulos and his co-workers observed that Pro-BNP were elevated in acute ischemic stroke.147
Natriuretic hormones (NH) include three groups of compounds: the natriuretic peptides (ANP, BNP and CNP), the gastrointestinal peptides (guanine and uroguanylin), and endogenous cardiac steroids. These substances induce the kidney to excrete sodium and therefore participate in the regulation of sodium and water homeostasis, blood volume, and blood pressure (BP).
In addition to their peripheral functions, these hormones act as neurotransmitters or neuromodulators in the brain. The ANP, BNP, and CNP and their receptors are expressed in the brain, which implies a possible role for these peptides in brain function. CNP is the most abundantly present NP in the brain suggesting that it acts as a neurotransmitter or neuromodulator rather than a cardiac hormone. 148
Accordingly, the CNP-specific receptor – NPR-B is widely spread throughout the brain:
NPR-B mRNA was detected in the cerebral cortex, the limbic area, preoptic-hypothalamic regions, motor nuclei, and the brainstem .149
NP and BNP are also present in the brain and have interesting neuro-modulatory functions.
BNP expression was first found in the hypothalamus, which is the main source of NP in the brain .150
2.8.1 NATRIURETIC PEPTIDES IN NEUROPROTECTION
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Natriuretic peptides were shown to exert a neuroprotective effect in cultured cells and in vivo. Cortical spreading depression (CSD) is a wave of depolarization followed by transient suppression of electrical activity in the brain. 151Rats preconditioned with an evoked episode of CSD were protected from neuronal damage following cerebral ischemia. 152
Studies revealed that an acute episode of CSD caused an elevation in ANP mRNA and peptide levels in the rat cortex. The elevation was prolonged, overlapped the time window for CSD-induced neuroprotection and accompanied by ANP-dependent activation of cGMP signalling cascades. 153
Increased cGMP levels were previously implicated in the neuroprotective mechanism of CSD.154This notion is supported by studies showing that ANP and BNP caused an elevation in cGMP levels and inhibited apoptosis of PC12 cells .154
However, there is no direct evidence of this effect in the brain. A neuroprotective effect was also demonstrated in rat retinal neurons, where ANP was shown to ameliorate NMDA-induced neurotoxicity, presumably in a dopamine-dependent manner.155
It was postulated that the ANP neuroprotective effect is mediated via the cerebral blood flow. Indeed, an increased number of ANP-immunoreactive astrocytes and other glial cells were found in the white matter surrounding an infarction area in rats .155
This neuro-protective effect may be modulated by cGMP signalling, since cGMP-phosphodiesterase inhibitor was found to have a protective effect in a focal brain injury model in rats .156
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At pharmacological doses, the peptide significantly suppressed the elevation of the brain’s water and sodium content and reduced the area of edema, as revealed by magnetic resonance imaging (MRI). 157
ANP was beneficial even after delayed administration, and reduced brain edema when injected i.c.v. 4 h after induction of haemorrhagic brain injury in rats. 158BNP too was implicated in neuroprotection following brain injury. James and colleagues demonstrated that i.v. administration of BNP improved cerebral blood flow and reduced inflammation in brain injury models in mice, as manifested by reduced neurodegeneration and improved functional outcome.159
2.8.2 N-TERMINAL PRO-BNP A RELIABLE STROKE PREDICTOR
N-Terminal PRO-BNP has been identified as a reliable predictor of stroke. Cushman et al observed that there was an increased risk of stroke across quartiles of NT-proBNP;
participants with NT-proBNP in the top versus the bottom quartile had a hazard ratio of 2.9 (95% confidence interval, 1.9-4.5). Among pathogenetic stroke subtypes, the association was largest for cardioembolic stroke, with a hazard ratio of 9.1 (95% confidence interval, 2.9-29.2). They concluded that NT-proBNP was a major independent risk marker for stroke.
They subsequently recommended that the clinical use of NT-proBNP measurement in primary prevention settings should be considered.160
Similarly, Doi and his co-workers observed that elevated NT-pro-BNP levels were shown to be a significant risk factor for the development of CVD and its subtypes in a general Japanese population, independent of other cardiovascular risk factors. 161
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2.8.3 N-TERMINAL PRO-BNP AND STROKE SEVERITY
Serum PROBNP levels have been found to be clinically useful in predicting stroke severity.
In comparing the role of serum ferritin, PROBNP and homocysteine in determining stroke subtype, severity and mortality, Ustungdag et al observed ninety-two patients over a six-month period. There was a significant difference in the serum ferritin, pro-BNP and homocysteine levels between patients who died and those who survived (p=0.013, p<0.001 and p=0.003 respectively); Pro-BNP levels were significantly higher in patients with NIHSS score >15 than NIHSS=8-15 and NIHSS=1-7 (p=0.016 and p<0.001 respectively. 162
Another study revealed that stroke patients who had Pro-BNP levels of > 150ng/ml had more severe stroke presentations as measured by the NIHSS (>15) than patients with levels
< 150ng/ml who had less severe presentations on admission, thus having lower NIHSS scores (<15). Tomita et al observed that the BNP level in ischemic stroke, positively correlated with the the NIH Stroke Scale (NIHSS) (r2=0.42, p<0.05) and infarct volume (r2`=0.34, p<0.05). Brain infarct volume and NIHSS were independent contributors to the plasma BNP level in ischemic stroke.163
PREDICTIVE VALUE OF NTPROBNP
Studies by Hajdinjak et al observed that PROBNP had a sensitivity of 94% (OR 5.80 [95%
CI 1.3-22.7] ,a specificity of 84% with OR 5.80 [95% CI 1.3-22.7], as well as a PPV , positive predictive value of 95% and a negative predictive value of 83% ,in assessing the
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prognostic value of a single prehospital measurement of N-terminal pro-brain natriuretic peptide and troponin T after acute ischaemic stroke.164
Relationship of admission ProBNP to Stroke Mortality
The relationship between admission Pro-BNP levels and to the 30-day case fatality of stroke has been observed.A new meta-analysis conducted by a team led by Teresa García-Berrocoso, Universitat Autònoma de Barcelona, Spain, has shown that levels of N-terminal ProBNP are associated with post-stroke mortality independent of stroke severity, age, and sex. 165
Shibazaki et al investigated whether the plasma ProBNP level on admission could serve as a biological marker of in-hospital death in patients with acute ischemic stroke.
He prospectively enrolled 335 consecutive patients (125 females; mean age, 72.3 years) with acute ischemic stroke within 24 hours of onset and measured plasma BNP on admission.
The mean ± SD of the plasma BNP level of the deceased group was significantly higher than that of the survival group (731.5±1,070.9 vs. 213.1±384.5 pg/mL, p=0.001). The optimal cut-off level, sensitivity, and specificity of BNP levels to distinguish the deceased group from the survival group were 240 pg/mL, 75.0% and 73.0%, respectively. 166
Yip et noted that elevated ProBNP levels were strongly and independently correlated with UFCO in patients after ischemic stroke.167
However, Etgen et al recruited 174 consecutive patients with MRI-confirmed ischemic stroke, serial measurements of cTnT, cTnI, and NT-proBNP were performed at 3 different time points in the hyperacute phase (at admission, on days 1 and 2). He concluded that neither NT-proBNP nor cardiac troponins was related to clinical prognosis. 168
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2.8.4 PRO-BNP AND POST-STROKE FUNCTIONAL OUTCOME
Elevated serum Pro-BNP levels have been found to independently influence short term post-stroke functional outcome as determined by the Modified Rankin Scale as well as the Glasgow Outcome Score. A study by Chang and his co-workers observed that plasma levels of Pro-BNP in patients with an unfavorable outcome were significantly higher than those in patients with a favorable outcome [3432 (interquartile range, 1100-54991) vs. 978 (interquartile range, 123-1705) pg/ml; P=0.000].169
Similarly , Yip et al , in assessing the “Time course and prognostic value of plasma levels of N-terminal pro-brain natriuretic peptide in patients after ischemic stroke.”, observed that an increased Pro-BNP level was strongly and independently correlated with unfavorable clinical outcomes in patients after ischemic stroke. 167
Similarly, elevated Pro-BNP level were associated with unfavourable outcomes in haemorrhagic stroke patients. James and his co-workers, in an attempt to determine the predictive value of S100b and brain natriuretic peptide (BNP) in order to determine a discharge prognosis after primary supra-tentorial intracerebral haemorrhage (ICH), concluded that Serum S100b and BNP levels in the first 24 h after injury accurately predict neurological function at discharge after supra-tentorial ICH.170
A study by Goya revealed that a high BNP level upon admission was associated with mortality within 1 month after ICH. A cut-off BNP level of 60.0 pg/ml could predict death within 1 month of ICH. Multivariate logistic regression analysis showed that a plasma BNP of >60.0 pg/ml (OR 4.7; 95% CI 1.43-15.63; p = 0.011) was independently associated with mortality within 1 month after ICH. (OR 4.7; 95% CI 1.43-15.63; p = 0.011) At 6 weeks,
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the dependent patients had higher concentrations of NT-proBNP than independent patients.171
2.8.5 PROBNP AND STROKE SUBTYPE
Ustundag observed that Pro-BNP levels were higher in the cardioembolic stroke patients than in athero-thrombotic stroke patients. High plasma levels of Pro-BNP and MR-pro-ANP are associated with a substantially increased risk of cardioembolic stroke, but not with other subtypes of ischemic stroke.162
In a recent study, Ninety-two patients were included (66 with ischemic stroke) with a mean age of 58·6 years. Twenty-eight (42·4%) ischemic strokes had a cardioembolic cause. Mean N-Terminal Pro-Brain Natriuretic Peptide values for cardioembolic stroke were significantly higher (P<0·001) (491·6; 95% confidence interval 283·7-852·0 pg/ml) than for non-cardio-embolic ischemic stroke (124·7; 86·3-180·2 pg/ml. It was therefore concluded that N-terminal Pro-brain natriuretic peptide was a biomarker with a good accuracy to predict ischemic stroke of cardioembolic cause, namely associated with atrial fibrillation.172
CHAPTER THREE
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3.1.0 METHODOLOGY
This prospective study was carried out at the Lagos University Teaching Hospital, Idi-Araba, Lagos. The study site was well suited for the study as it is a tertiary referral centre in Lagos, South Western, Nigeria. The study was a prospective cohort study.
3.1.1 APPROVAL
Approval of the study protocol was obtained from the LUTH Health research and ethics committee. Strict attention to ethical considerations was a component of the case control study, which ensured confidentiality and the right of potential participants to withhold consent or withdrawal participation at any time without jeopardizing their care.
3.1.2A INCLUSION CRITERIA
1. Consecutive ischemic and hemorrhagic stroke patients confirmed by brain imaging - CT/MRI
2. Presentation within 72hours of onset of acute neurologic symptoms.
3. First -ever stroke.
4. Informed consent from patient or proxy.
3.1.2B EXCLUSION CRITERIA
1. Pre-stroke heart failure as confirmed by the Framingham Criteria or myocardial infarction as confirmed on ECG.
2. Late presentation (>3 days post-stroke).
3. Patients who did not give consent.
4. Cardio-embolic stroke
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3.1.3 RECRUITMENT OF STUDY SUBJECTS (STROKE CASES).
1. Consecutively presenting and consenting stroke patients fulfilling the inclusion criteria were recruited in the study.
3.1.4 PROPOSED METHODOLOGY
Standard Proforma to document age and gender.
PHYSICAL EVALUATION
A detailed general, cardiovascular and neurologic examination was conducted.
Investigations. Patients had a brain CT (if possible), electrocardiogram as well as a random blood sugar was conducted.
SEVERITY ASSESSMENT
Stroke severity was assessed using the NIHSS National Institute of Health Stroke Score for Ischemic strokes and the Glasgow coma scale for the Hemorrhagic strokes. (See appendix1A&C)
FUNCTIONAL OUTCOME ASSESSMENT
This was done after 30 days of the acute stroke using the Modified Rankin Scale. However, patients were followed up every day by means of ward rounds, in order that patients who died or were discharged before 30 days were accounted for. Patients who were discharged earlier than 30 days were followed up in the neurology clinic or by telephone. (see appendix 1B)
3.1.5 DETERMINATION OF BNP
A 5ml venous blood sample was drawn from the study subjects within 72 hours of the stroke. Blood was drawn and placed in a lithium heparin bottle. Samples were centrifuged within 60 minutes of sample collection, and the supernatant was subsequently stored at -80°
at the Central Research Laboratory of LUTH which is equipped with temperature sensors, uninterrupted power supply and backup generators. Samples were depersonalized. A NT-proBNP value less than125ng/ml was considered normal. The NT-NT-proBNP level was
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measured using the Enzyme Linked Immunosorbide Assay method using the Bio-medica BNP Fragment ELISA Kit. CAT .NO.B1-20852 12*8 Tests, rev. No. 080401 Biomedica Gruppe , 2014.
3.1. 6 SAMPLE SIZE CALCULATION
The estimated sample size was between 110 and 130 patients using the Kish formula.
Nf= n
1 + (n/N) n= Z2pq d2
where:
Nf = the final sample size
n = the desired sample size (when population greater than 10,000) z = standard deviation, usually set at 1.96, which corresponds to 95%
confidence level p=proportion of patients with stroke, using prevalence of 50%(0.5)
q = 1-p (1-0.5) =0.5
d = degree of desired accuracy=0.05
N = estimation of population size, that is, new patients with stroke managed annually in LUTH=150
n = (1.96)2 x0.5 x0.5 (0.05)2
= 384
Nf = 384
1+ [(384)/750
= 107.
To allow for an assumed attrition rate of 10% (for possible discharges against medical advice prior to study completion and unavoidable loss to follow-up e.g. from relocation out of town).The sample size of cases was increased to 118 (rounded up to 120 cases and 120
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controls). Controls were included in this study to help determine the normative values for pro BNP in this study population. Age- and gender-adjusted cut points were used.
3.1.7 STATISTICAL ANALYSIS
The collected data was observed to be non-parametric, hence the data had to be log-transformed in order to have a parametric data. Analysis of statistical significance was done using the Student T- test for continuous variables and chi -square for distinct variables p-value =0.005 It was expected that there would be outliers whose NT BNP p-values would fall outside the range of the majority, these would be noted. A correlation was done using the Pearson correlation. Graphic representation (boxplots demonstrating the median, interquartile ranges and outliers) was used in data presentation.
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