INTRODUCTION:

Unusual CV danger is available from the beginning phases of renal deficiency and increments as the infection advances. Besides, when patients arrive at ESRD, the majority of them present cardiovascular complexities. Almost certainly, the high predominance of CV illness saw in persistent kidney sickness results from expansion to conventional danger variables of non-customary factors like aggravation, oxidative pressure, and expanded unbalanced dimethylarginine levels.

An early identification and revision of these variables ought to have incredible clinical and general wellbeing significance. Sadly, up until now, the majority of the endeavors to further develop endurance by centering of regular CV danger variables or dialysis methods have fizzled. Additionally, the understanding of a portion of the customary danger factors is upset by the obstruction of ailing health, which isn't uncommon in CKD patients¹.

Myocardial infraction is a serious prospective sign of ischemia heart disease and prominent reason of disability as well as death in the developed world. In patients with settled disease, MI might be an initial sign of coronary artery illness (CAD) or which might be further repeated^2,3. After atherosclerotic plaque rupture, the cascade of thrombotic events triggers blockage of the coronary artery, interjecting myocardium oxygen as well as supply of blood, leading in infarction. arrhythmias, myocardial rupture or Heart failure follow myocardial necrosis along with infarction. Based on their symptoms, electrocardiographic defects (ECG) and cardiac enzymes, the WHO has described MI. MI can be diagnosed using several clinical characteristics, which includes ECG results, high biochemical biomarker values of myocardial necrosis, and imaging, or pathology can also be described.

The original stage in the pathogenesis of MI is the imbalance between the supply of oxygen and demand in cardiac tissue owing to myocardial ischemia. Also essential as the clinical characteristic of myocardial ischemia is the history of the patient and ECG. Symptoms of possible myocardial ischemia include different part of chest, uncomfortable with mandibular pain or fatigue or dyspnoea⁴. This acute MI-related distress usually lasts 20 minutes. Often, diaphoresis, nausea, or syncope may accompany the distress. MI can happen with or without symptoms of atypical diseases such as palpitations or cardiac arrest⁵.

In this present investigation, efforts have been made to study cardioprotective effect of alpha keto-analogues in isoproterenol induced myocardial infarction in rats.

MATERIALS AND METHODS:

Materials:

Amlodipine was obtained from Sigma Aldrich Chemical Pvt. Ltd., India and Isoproterenol was procured from Sun pharma Research Laboratories, India. Other reagents used in the investigation were of analytical grade.

Animals:

Male rodents, weight about 150-200 gm, purchases from Animal house, facility in R.V. Northland Institute, Dadri, Gautam Budh Nagar, U.P, India. Then animals kept in polypropene cages used under standard laboratory conditions (12 hours light and 12 hours dark cycles) This animal house temperature was maintained at 25 ± 2ºC. Protocol was approved by Institutional Animal Ethics Committee of R.V. Northland Institute (CPCSEA Registration number -1149/PO/Re/S/07/CPCSEA.

Methods:

Preparation of drug solutions:

Alpha Keto-analogues (KA) solution - Weighed amount of KA has been uniform in 5 ml of vehicle.

Amlodipine solution - Weighed amount of the AML has been uniform in 5 ml of vehicle

Isoproterenol solution - Weighed amount of ISO was dissolved in 3 ml of normal saline solution.

Blood collection:

Blood samples (3 - 5ml) has been composed from the rat’s tail vein in sterile centrifuge tubes, after the last treatment dose and the serum was separated for the estimation of the following biochemical parameters.

Tissue collection:

After blood assortment, every one of the rats were sacrificed by cervical separation under sedation and hearts were taken apart out, washed with super cold typical saline, gauged, and saved in fluid nitrogen at -80⁰C for biochemical, histopathological and myocardial infarct size⁶.

Parameters estimated:

Anthropometric parameter like body weight was estimated, Biochemical estimations in serum included Lactate dehydrogenase and Troponin T, Biochemical estimation in cardiac tissues included protein, Catalase (CAT), Histopathological, and Myocardial infarct size determination.

METHODS OF ESTIMATION:

Preparation of samples:

1. Serum:

3-5 ml of blood tests were gathered from the tail vein of the rats in sterile axis tubes and covered with parafilm and left undisturbed at 37 °C for 1 h after which they were exposed to cooling in a cooler for 3 h. The coagulation shaped was then taken out and the serum tests were then emptied out. The supernatants so got after centrifugation were the serum tests utilized for investigation. Just non-hemolyzed serum was utilized.

2. Tissue homogenate:

The rats were scarified and their hearts were eliminated immediately, washed in super cold saline, dried on channel paper and gauged 10% w/v homogenate was set up super cold 0.15 M KCl for MDA and protein assessment, in 0.02 M EDTA for GSH assessment and in phosphate cushion (pH - 7.4) for SOD and CAT assessment by utilizing a Teflon tissue homogenizer⁷.

Biochemical estimation in serum:

I. LDH estimation:

Principle :

LDH catalyzes the oxidation of lactate to pyruvate joined by the concurrent decrease of NAD to NADH. LDH action in serum is proportional to the expansion in absorbance because of the decrease of NAD⁸. Reagents provided in the kit were used directly.

CK-MB estimation:

Principle:

Creatinine kinase- MB assay is based on the principle of individual immune prevention through concoction of monoclonal antibody that prevents the creatinine kinase-MB effectively and 50 % of Creatinine kinase- MB activity. Although it is not stirring on the B subunit property of creatinine kinase- MB and the CK-B activity is evaluated. Creatinine kinase-MB property is attained through augmented the creatinine kinase-B property through two increase the levels of found due to simple exercise and the large manifold Intramuscular Injections. In other symptom and evocative history, serum CK estimation is an important limit of choice for MI.

The reaction is evaluated through analyzing the development in the absorbance at the 340 nm that is precisely proportional to creatinine kinase- B subunit property.

Contents of one bottle of CK-MB-2 are dissolved with 1.1ml of CK-MB-1, and the mixture is used after 15min. The reconstituted reagent is stable for 14 days.

Reagents were mixed and the 1^stabsorbance of the analysis was read precisely at 300 secs and consequently at the different nanometers such as 30, 60, 90 and 120 sec at 340 Nano meters. Regulate the adjustment in the absorbance at every minute.

Biochemical estimation in cardiac tissues:

I. TBARS estimation:

Principle :

LPO is a free radical which interceded reaction. Colorimetric reaction of the compound thiobarbituric acid along MDA is a sensitive method for measuring lipid per oxidation. The assay provides an estimate of the amount of TBARS, e.g., MDA. It is also referred to as TBARS test⁹.

REAGENTS:

1. 0.8% TBA solution: 0.8gm of TBA has been mixed and amount has been made up to 100 millilitres to make 52nM in aqueous medium.

2. 30% TCA solution : 30gm of TCA has been mixed and the total amount was made up to 100 millilitres by using distilled water.

Standard TEP reagent - 0.02gm (± 0.004 gm) of TEP (1, 1, 3, 3 – tetra ethoxy propane) was dissolved in 40% ethanol. The above solution is diluted with 100ml distilled water to 1000ml. A third dilution of 100ml of the above solution to 500 millilitres along distilled water was finally done. This dilution contained 4μg of reagent per ml.

Procedure:

1 millilitre of the pre-arranged suspension medium has been utilized. 10% of the cell is homogenated and centrifuged at the round per minute (rpm) of 10,000. 0.5 milliliter of the 30% TCA sought after through utilizing 0.5 milliliter of the 0.8% TBA has been incorporated.

Preparation of standard MDA curve:

0.1, 0.2, 0.3, 0.4, 0.5 and 0.6ml of standard TEP solution were taken in 6 test tubes. 0.5ml of 0.8% TBA reagent and 0.5ml of 30% TCA have been included to each of the tubes and the same procedure as for the sample was followed. A visual representation has been devised between absorbance of optical density and TEP in Figure 1.

Figure 1: Standard curve for TBARS

Calculation:

The amount of MDA present in a specimen has been determined conferred to the equation moles of

OD at 540nm x Amount of test mixture

MDA =_{-------------------------------------------------------------------------------------}

0.156

II. Protein Estimation:

Principle:

Protein reacts with Folin Ciocalteau reagent to produce a shaded perplexing, absorbance of which is resolved spectrophotometrically at 750nm¹⁰. The response includes two stages. The initial step is the decrease of copper as in the biuret test. The subsequent advance includes the decrease of the Folin reagent by the tyrosine, tryptophan and cysteine present in the protein.

Procedure:

5ml of solvent was added to suspension of 1ml from the supernatant obtained after centrifugation, 10% tissue at 10,000rpm get homogenate and allowed to rest 10min. 0.5Ml of debilitated Folin's reagent was added and chamber was shaken to mix the solution. After 30min, the annihilation was observed at 750nm and recorded.

Preparations of the adjustment bend for protein:

5ml of cow like serum, egg white’s arrangement (2 mg/ml) was ready and different volumes were putted in 6 test tubes. The fixation of protein in the 6 cylinders was evaluated.

Catalase Estimation:

Principle:

In the UV range, H2O2 appeara persistent expansion in ingestion accompanied by diminishing frequency. decay of H2O2 can be trailed by reduction in eradication at 240 nm. The distinction in termination (ΔE240)/unit time is a proportion of the catalase movement.

Procedure:

Phosphate support was ready by blending disodium hydrogen phosphate with potassium dihydrogen phosphate in 40:60 proportions. In 50mM/L potassium phosphate support with a proportion of 1:10w/v heart tissue was homogenized. The homogenate was centrifuged at 10,000rpm for 20min. at 4ºC in a cooling rotator. Supernatant (50µl) was taken in cuvette containing 2.95ml of 19mM/L arrangement of H2O2 arranged in potassium phosphate cushion. The vanishing of H2O2 was checked at 240nm frequency at 1 min. span for 3min.

Calculation:

Activity of catalase was calculated as n moles of H2O2 consumed/min/mg of protein

𝛥 A/m in ute X Vol ume of test so lution

= -------------------------------------------------------

0.181 X Volume of Sample X mg of protein

I.GSH estimation:

Principle:

This spectrophotometric technique depends on the strategy for Ellman for example 5, 5'- dithiobis-(2-nitrobenzoic corrosive), DTNB is diminished by SH gatherings to frame 1mole of 2-nitro-5-mercaptobenzoic corrosive per mole of SH¹¹.

Procedure:

A known amount of heart tissue ranging from (300-600 mg) was homogenized in 0.02 M EDTA (5-8ml) and thereafter cold refined water 4.0ml was added to it. Ensuing its well mixing, 50% TCA 1ml was added and shaken unpredictably for 10min by utilizing a vortex blender. After 10min. mixture was moved to hub tubes (flushed in EDTA) and for 15min centrifuged at 6000 rpm. Following centrifugation, supernatant of 2ml was mixed with 4.0ml of 0.4M Tris support (pH 8.9). The whole solution was mixed well and 0.01M DTNB 0.1ml was added to it.

Calculation:

GSH in tissue was investigated from the accompanying condition and communicated as µmoles/mg protein.

Absorbance at 412 nm

GSH (µmol/mg of protein) = ---------------------------------

E X D X mg of protein

Where,

E = Extinction coefficient of DTNB,

D = Dilution factor

III. SOD ESTIMATION:

Principle:

Pyrogallol auto-oxidizes quickly in watery arrangement; elevated the pH quicker is auto- oxidation and a few middle items are framed. Accordingly, the arrangement initially gets yellow-brown accompanied by a range appear ought to linking 400-425nm¹². After hardly any min, shading starts to become green lastly following not many hours, a yellow tone shows up. So the auto-oxidation is concentrated basically during the underlying advance in 420nm absorbance, which is observed for various min after an enlistment time of approximately 10 sec. Superoxide free extremist catalyzes the auto-oxidation of pyrogallol. A straightforward and fast strategy for measure of SOD is depicted, in view of the capacity of the compound to hinder the auto-oxidation of pyrogallol.

Procedure:

The supernatant was inspected for activity of SOD by following the pyrogallol auto-oxidation restriction. Cytosolic supernatant 100µl was added to TrisHCl support (pH 8.5). The last 3ml volume was changed accompanied by a comparative pad. Somewhere near pyrogallol 25µl was added and alteration in absorbance at 420nm were observed at 1min stretch for 3min. The augmentation at 420nm absorbance after extension of pyrogallol was ruined by the SOD presence.

Calculation:

SOD 1unit is portrayed as the measure of protein needed to cause 50% hindrance of pyrogallol auto-oxidation per 3ml of test combination and given by the equation:

Unit of SOD per ml of test = (A-B) × 100A × 50

Information was communicated as protein SOD units per mg.

I. HW/BW ratio:

The weight of the heart and the body of the rats have been noted down, HW/BW proportion have been reported.

II. Histopathological analysis:

Following steps were involved in the hematoxylin and eosin (H and E) staining:

Dehydration:

Impact fair and square of thiobarbituric corrosive responsive substance (TBARS) 80%, 95% and 100%.

Clearing:

The reagent used for clearing ought to be miscible with dehydrant and paraffin. When dehydrantwas eliminated, the tissue cleared and got clear, implying consummation of the interaction. CHCL3 has been utilized as the clearing specialist.

Impregnation:

Complete removal of the clearing expert by substitution was finished by paraffin, as it invades the tissues. Impregnation was finished with 3 paraffin showers for 3 h. Paraffin with a dissolving point of 56-58°C was used. Security measure was taken so that warming, more than 5°C was done, which may withdraw and solubilize the tissue. The tissue was then cast in to the squares of paraffin wax.

Sectioning:

Segments of the tissue squares of thickness 3-5 µm were cutted with the help of rotating microtome. The part strips were made to drift on water and afterward situated on glass slides to kill wrinkles fairly warmed and dried.

Hydration:

Segments were hydrated accompanied by xylene for 2 min and 70% alcohol for 2 min. They were flushed accompanied by refined H₂O.

Staining:

Segments were stainedaccompanied by 1 % haematoxylin, washed in refined H₂O The stained segment was then covered with glycerine jam and cover slips were set cautiously on the part, taking consideration that no air pocket ought to enter. The slides were seen under the magnifying instrument and the photos were taken accordingly. After that the translation of photos was done and the perceptions were recorded. The score 0–3 was relegated to 0 - no harm, 1 – little harm, 2 –average harm, and 3 - most extreme harm. Outcomes got were then communicated as aggregate scores from 0 to 9.

Statistical analysis:

All information were communicated as mean ± standard Deviation (SD). Gatherings of results were analyzed by utilizing single direction examination of fluctuation (ANOVA) trailed by the Dunnett's t-test to notice critical contrasts between gatherings. Qualities has been viewed as genuinely huge just when p < 0.05.5.

RESULTS:

Eﬀect of alpha Keto-analogues and amlodipine on HW/BW Ratio (%)

The ISP rationale appear a critical (P<0.001) ascend in the HW/BW proportion when contrast and Normal saline batch. Pre-treatment accompanied by alpha Keto-analogues (20 and 40mg/kilo); amlodipine (5mg/kilo) showed significant reduced in HW/BW proportion (%) as contrast to the ISP rationale. Table 2 summarizes the Changes in body weight and heart weight in experimental groups and table 3 summarizes the Effect of alpha Keto-analogues and amlodipine on Heart to Body weight ratio.

Table 2 - Changes in body weight and heart weight in experimental groups.

Group	Treatment	Body weight		Heart weight
Group	Treatment	Initial	Final	Heart weight
1	Normal Saline	171.5 ± 7.3	191.5 ± 6.9	0.41 ± 0.18
2	Isoproterenol (ISP)	173.1 ± 5.2	186.1 ± 4.7	0.49 ± 0.18
3	α-Keto-analogues (KA) + ISP (20mg/kg/day)	167.5 ± 6.5	174.24 ± 5.1	0.32 ± 0.18
4	KA + ISP (40mg/kg/day)	164.24 ± 4.9	184.4 ± 5.6	0.36 ± 0.18
5	Amlodipine + ISP (5mg/kg/day)	184.24 ± 6.5	194.2 ± 6.3	0.30 ± 0.18

ISP-Isoproterenol; KA- α-Keto-analogues, Values are expressed by mean ± SD of six samples in each group.

Impact fair and square of thiobarbituric acid reactive substance (TBARS):

TBARS level was dictated by assessing content of myocardial malondialdehyde (MDA) (Figure 6). A huge (P<0.001) ascent in the level of serum TBARS was seen in the ISP group when contrasted and typical saline gathering. Pre-treatment with alpha Keto-analogues (20 and 40mg/kilo) and amlodipine (5mg/kilo) showed critical decline in MDA level when contrasted with the ISP group.

Effect on the anti-oxidant enzymes level

Impact of catalase (CAT), superoxide dismutase (SOD), Glutathione (GSH) in the treatment bunches is addressed in Figure 7, 8 and 9. Rodents treated with ISP batch appear a critical (P<0.001) decline degree of serum SOD, CAT and GSH when contrasted with the Normal Saline gathering. Pretreatment with alpha Keto-analogues (20 and 40mg/kilo) and amlodipine 5mg/kilo) showed a critical expansion in their level when contrasted with ISP group.

Table 7: Impact of alpha Keto-analogues and amlodipine on SOD tissues

S. No	Group	SOD (unit/ mg of protein)
1	Normal Saline	5.1 ± 0.2
2	ISP	1.8 ± 0.1***
3	KA (20mg) + ISO	3.4 ± 0.8^##
4	KA (40mg) + ISO	3.7 ± 0.2^###
5	Amlodipine + ISO	4.6 ± 0.3^###

Values are presented as the mean ± standard deviation (SD) for 6 rats in each group. The probability value (p-value; #p < 0.01, ##p < 0.05 ###p < 0.001, *** p < 0.001)

Table 8: Impact of alpha Keto-analogues and amlodipine on SOD tissues of myocardial

S.no	Group	CAT (nmol H2O2/min/mg protein)
1	Normal Saline	7.13 ± 0.32
2	ISP	1.87 ± 0.36***
3	KA (20mg) + ISO	3.44 ± 0.22^##
4	KA (40mg) + ISO	3.89 ± 0.17^###
5	Amlodipine + ISO	5.58 ± 0.13^###

Values are presented as the mean ± standard deviation (SD) for 6 rats in each group. The probability value (p-value; #p < 001, ##p < 0.05, ### p < 0.001, *** p < 0.001)

Figure 7: Impact of alpha Keto-analogues and amlodipine on SOD tissues of myocardial during isoproterenol actuated myocardial localized necrosis in rodents. Information is communicated as mean ± SD (n=6 creatures for every gathering). Importance was dictated by single direction when contrasted with the Normal Saline gathering. #p < 0.05 when contrasted with the ISP group. ## p < 0.01 when contrasted with the ISP gruop. ### p < 0.001 when contrasted with the ISP group.

Table 9: Impact of alpha Keto-analogues and amlodipine on GSH tissues

S. No	Group	GSH (µmol GSH/mg protein)
1	Normal Saline	1.93 ± 0.12
2	ISP	0.53 ± 0.31***
3	KA (20mg) + ISO	1.42 ± 0.19^##
4	KA (40mg) + ISO	1.39 ± 0.08^###
5	Amlodipine + ISO	2.23 ± 0.23^###

Values are presented as the mean ± standard deviation (SD) for 6 rats in each group. The probability value (p-value; #p < 0.01, ##p < 0.05, ### p < 0.001, *** p < 0.001)

Histopathological analysis:

Figure 10 shows the histopathological changes in different gatherings. Typical Saline gathering appear integrity trustworthiness of myocardial tissue accompanied by striations, fanned appear and congruity accompanied by neighboring myofibrils. The ISO-treated appear critical my degeneration, pyknotic core, myocardial rot, invasion of macrophages, edema, and lymphocytes. Pretreatment accompanied by alpha Keto-analogues (20 and40 mg/kilo) and amlodipine (5 mg/kilo) altogether switched the progressions to ordinary.

Figure 10: Myocardial tissues photomicrographs of various gatherings (H and E, 10^×) [Scale bar 50 µm] with semi quantitative investigation. A Group I /Normal saline, B –GroupII/ISP, C–Group III/KA-20+ISP, D –Group IV/KA-40 +ISP, E –Group V/AML+ISP.5.7 Effect of drug treatment on myocardial infarct size.

Figure 11 a and 11b show the progressions in myocardial infarct size in various treatment gatherings. The Normal Saline gathering showed negligible infarcted area while the ISP batch exhibited 65.97 % infarcted district. Alpha Keto-analogues at 20 mg/kg and 40 mg/kg portion showed 22.91% and 16.7 % infarcted region individually while the pre-treatment with AML restricted the dead tissue to 11.28 %.

Figure 11a: Z;A–Normal Saline, B –ISP, C –KA-20 + ISP D –KA-40 + ISP, E –AML + ISP

Figure 11b: Percentage infarct region among various gatherings; Data in chart are communicated as mean ± SD (each gathering for six rodents). Importance was controlled by single direction versus typical saline control; ###p < 0.001, ##p <0.01, #p > 0.05 versus ISP Group.

Table 10: Percentage infarct region among various gatherings

S. No	Group	Percentage Infract area
1	Normal Saline	0.13 ± 0.29
2	ISP	68.63 ± 0.31***
3	KA (20mg) + ISO	26.22 ± 1.19^##
4	KA (40mg) + ISO	19.79 ± 0.38^###
5	Amlodipine + ISO	17.43 ± 0.93^###

Values are presented as the mean ± standard deviation (SD) for 6 rats in each group. The probability value (p-value; #p < 0.01, ##p < 0.05, ### p < 0.001, *** p < 0.001

DISCUSSION:

Cardiovascular infection and kidney sickness are firmly correlated and the illness of one organ causes brokenness of the other, at last prompting disappointment of the two organs. This is frequently alluded as cardio renal condition (CRS). The pathogenesis of cardiomyopathy isn't plainly characterized, yet its assessment by ISP-initiated cardiovascular harm has exhibited the association of oxidative pressure. Our investigation, in light of preclinical assessment in a murine model showed that a blend of amlodipine accompanied by vinpocetine display solid cardio protective clash.

Feline, SOD, and GSH are free revolutionary rummaging cancer prevention agent catalysts and are the primary line cell protection against oxidative pressure. These catalysts wipe out oxygen extremists like (O2-) and (H2O2) and forestall the arrangement of a more responsive hydroxyl revolutionary (% OH). Pretreatment of creatures accompanied by Alpha Keto-analogues portion conditionally expanded the exercises of GSH, CAT and SOD in ISP-oppressed rodents. The aftereffects of our examination showed that free extremists delivered by ISP were essentially extinguished by alpha Keto-analogues. Hence, this cell reinforcement impact represents the cardio protective property of these medications.

Raised serum CK-MB, GOT, LDH levels are notable markers of myocardial localized necrosis. ISP-instigated MI causes cell harm because of oxygen and glucose shortage. These advances burst of cardiovascular film and resulting spillage of these proteins into the circulatory system. Raised troponin levels anticipate the danger of both cardiovascular demise and resulting dead tissue. In our investigation, we additionally saw expanded degrees of cardiovascular troponin-T in the serum of ISP-incited rodents. Pretreatment accompanied by alpha keto-analogs diminished the age of oxygen extremists and aided in the decline of these catalysts appear safeguarding of the primary trustworthiness of myocardium. It has been accounted for that ISP organization could make critical harm the myocardium. The poisonous gathering (batch-II) appears the penetration of macrophages andmy degeneration. Notwithstanding, pre-treatment accompanied by Alpha Keto-analogues effectively forestalled the harm done by ISP.

CONCLUSION:

The administration of intraperitoneal injections of isoproterenol induced MI in rodents as evidenced by abnormal alterations in biochemical and histopathological markers. Alpha keto- analogues showed cardio protection when given alone in a limitation of MI. administration of alpha keto-analogues showed significant cardio protection and the effect at higher dose was more pronounced than their individual doses. In this way, thinking about every one of the outcomes, we can close those alpha keto-analogues can be used to treat myocardial Infarction. The present research is a genuine illustration of medication repurposing and offers remedial benefit for the patients who foster MI.

ABBREVIATIONS:

MI: Myocardial infarction; KA: Alpha-Keto-analogues; HR: Heart Rate; SAP: Systolic Arterial Blood Pressure; MAP: Mean Arterial Blood Pressure; DAP: Diastolic Arterial Blood Pressure; ISO: Isoproterenol; GSH: Glutathione; CK-MB: Creatine Kinase-MB; SOD: Superoxide dismutase; LDH: Lactate Dehydrogenase; TBARS: Thiobarbituric Acid Reactive Substance.

ETHICS APPROVAL:

Protocol was approved by Institutional Animal Ethics Committee of R.V. Northland Institute (CPCSEA Registration number -1149/PO/Re/S/07/CPCSEA.

ACKNOWLEDGEMENTS:

The authors are highly thankful to the management of R.V. Northland Institute for their continuous support and encouragement.

CONFLICT OF INTEREST:

None to declare.

REFERENCES :

1. Anderson JL, Morrow DA. Acute myocardial infarction. New England Journal of Medicine. 2017 May 25; 376(21): 2053-64.

2. Park KC, Gaze DC, Collinson PO, Marber MS. Cardiac troponins: from myocardial infarction to chronic disease. Cardiovascular Research. 2017 Dec 1; 113(14): 1708-18.

3. Nabel EG, Braunwald E. A tale of coronary artery disease and myocardial infarction. New England Journal of Medicine. 2012 Jan 5; 366(1): 54-63.

4. Anavekar NS, McMurray JJ, Velazquez EJ, Solomon SD, Kober L, Rouleau JL, White HD, Nordlander R, Maggioni A, Dickstein K, Zelenkofske S. Relation between renal dysfunction and cardiovascular outcomes after myocardial infarction. New England Journal of Medicine. 2004 Sep 23; 351(13): 1285-95.

5. Fathima SN. An Update on Myocardial Infarction. Current Research and Trends in Medical Science and Technology. 2021;1.

6. Lobo Filho HG, Ferreira NL, Sousa RB, Carvalho ER, Lobo PL, Lobo Filho JG. Experimental model of myocardial infarction induced by isoproterenol in rats. Brazilian Journal of Cardiovascular Surgery. 2011; 26: 469-76.

7. Bruce AJ, Baudry M. Oxygen free radicals in rat limbic structures after kainate-induced seizures. Free Radical Biology and Medicine. 1995 Jun 1; 18(6): 993-1002.

8. Lum G, Gambino SR. A comparison of serum versus heparinized plasma for routine chemistry tests. American Journal of Clinical Pathology. 1974 Jan 1; 61(1): 108-13.

9. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry. 1979 Jun 1; 95(2): 351-8.

10. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov 1; 193(1): 265-75.

11. Sedlak J, Lindsay RH. Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent. Analytical Biochemistry. 1968 Jan 1; 25: 192-205.

12. Marklund S, Marklund G. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. European Journal of Biochemistry. 1974 Sep; 47(3): 469-74.

Received on 19.12.2024 Revised on 26.02.2025

Accepted on 28.04.2025 Published on 22.07.2025

Available online from July 26, 2025

Res.J. Pharmacology and Pharmacodynamics.2025;17(3):165-174.

DOI: 10.52711/2321-5836.2025.00027

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Group (N)		Drug Treatment	Dosage mg/kg of Body weight and Route of administration
I	Normal Control	1ml (0.9%NS)	1ml/kg 0.9% normal saline (p.o) for 14^thdays.
II	Toxic control	Isoproterenol (ISP)	85mg/kg ISP (i.p) on 13^thand 14^thday.
III	Alpha Keto-analogues	KA + ISP	20mg/kg/day Alpha Keto-analogues (p.o) for 14 days + 85mg/kilo ISP (i.p) on 13^thand 14^thday.
IV	Alpha Keto-analogues (KA)+(ISP)	KA + ISP	40mg/kg/day Alpha Keto-analogues (p.o) for 14 days + 85mg/kilo ISP (i.p) on 13^thand 14^thday.
V	Amlodipine +ISP	AML + ISO	5mg/kg/day amlodipine (p.o) for 14 days + 85mg/kilo ISP (intraperitoneal) on 13^thand 14^thday.

S.No	Group	LDH (IU/L)	CK-MB (IU/L)	SGOT (IU/L)
1	Normal Saline	215.9 ± 6.2	201.1± 12.6	111.4 ±1.7
2	ISP	567.7 ± 5.7***	534.4± 31.4***	251.1 ± 1.5***
3	KA (20mg) + ISO	498.9 ± 12.2^#	413.2± 10.6^#	191.8 ± 4.2^#
4	KA (40mg) + ISO	485.8 ± 10.8^##	422.7± 20.7^#	198.3 ± 3.6^#
5	Amlodipine + ISO	405.5 ± 8.1^###	201.1± 10.9^###	174.1 ± 1.8^###

Tissue collection:

Parameters estimated:

Table 1: Experimental protocol Treatment

METHODS OF ESTIMATION:

Preparation of samples:

2. Tissue homogenate:

Biochemical estimation in serum:

Biochemical estimation in cardiac tissues:

REAGENTS:

Procedure:

Preparation of standard MDA curve:

Calculation:

II. Protein Estimation:

Procedure:

Preparations of the adjustment bend for protein:

5ml of cow like serum, egg white’s arrangement (2 mg/ml) was ready and different volumes were putted in 6 test tubes. The fixation of protein in the 6 cylinders was evaluated.

Catalase Estimation:

Principle:

Procedure:

Calculation:

I.GSH estimation:

Principle:

Procedure:

Calculation:

III. SOD ESTIMATION:

Principle:

Procedure:

Calculation:

I. HW/BW ratio:

II. Histopathological analysis:

Following steps were involved in the hematoxylin and eosin (H and E) staining:

Statistical analysis:

Effect of alpha Keto-analogues and amlodipine on the cardiac injury marker enzymes (LDH, CK-MB and SGOT) levels:

Impact fair and square of thiobarbituric acid reactive substance (TBARS):

Effect on the anti-oxidant enzymes level

Histopathological analysis:

S. No	Group	Heart to body weight ratio (%)
1	Normal Saline	0.278 ± 0.01^##
2	ISP	0.467 ± 0.01
3	KA (20mg)	0.379 ± 0.02
4	KA (40mg)	0.309 ± 0.02^###
5	Amlodipine + ISP	0.302 ± 0.01^###

S. No	Group	Trop-T level in serum (ng/ml)
1	Normal Saline	0.19 ± 0.02
2	ISP	3.67 ± 0.01***
3	KA (20mg) + ISO	2.97 ± 0.02^##
4	KA (40mg) + ISO	2.88 ± 0.02
5	Amlodipine + ISO	1.57 ± 0.01^###

S. No	Group	TBARS (nmol MDA/ mg protein)
1	Normal Saline	0.13 ± 0.02
2	ISP	0.57 ± 0.01***
3	KA (20mg) + ISO	0.44 ± 0.02^##
4	KA (40mg) + ISO	0.38 ± 0.07
5	Amlodipine + ISO	0.28 ± 0.03^###