Association of albumin, fibrinogen, and modified proteins with acute coronary syndrome.

Acute coronary syndrome (ACS) comprises a pathophysiological spectrum of cardiovascular diseases related to atherosclerotic coronary plaque erosion. Oxidative stress and inflammation play pivotal roles in the development and progression of atherosclerosis, which affects circulatory proteins, including albumin and fibrinogen, thereby causing an imbalance in albumin to globulin and fibrinogen to albumin ratios. This study aimed to assess the effect of oxidative stress on circulatory proteins, correlate these parameters, and investigate their significance in patients with ACS. In this case-control study, the major blood proteins in patients with ACS and a control group were evaluated using standard methods. Out of 70 ACS cases, 75.7% had ST-elevation myocardial infarction (STEMI), 18.6% had non-STEMI, and 5.7% had unstable angina. The mean cardiac troponin I level in patients was 12.42 ng/mL. The patients demonstrated a significantly reduced level of human serum albumin (HSA), 3.81 ± 0.99 g/dL, compared to controls, 5.33 ± 0.66 g/dL. The albumin to globulin ratio (AGR) was significantly depressed in patients while their mean fibrinogen level and the fibrinogen to albumin ratio (FAR) were significantly higher. Multivariate logistic regression analysis showed that albumin and fibrinogen were significantly associated with the risk of ACS, showing the potential of these parameters to be used for risk assessment of ACS. The ischemia modified albumin (IMA) and protein carbonyls were significantly higher in patients which showed significant positive correlations with FAR. Albumin, IMA and protein carbonyls were found to have high diagnostic sensitivity and specificity for ACS. Overall, these circulatory and modified proteins in ACS patients, particularly lower HSA, AGR, and higher IMA and protein carbonyls may help assess risk.

Introduction

Cardiovascular diseases (CVD) are the most common causes of mortality, accounting for 31% of all deaths worldwide [1]. Coronary artery disease (CAD) is a form of CVD that contributes to the majority of cardiovascular deaths, and it is caused by the impedance of one or more arteries that deliver blood to the heart, usually due to atherosclerosis. Atherosclerotic plaque rupture or erosion, with varying degrees of superimposed thrombosis, has been demonstrated to result in a partial or complete blockage of the blood flow in the coronary arteries. The most ominous manifestation of CAD is acute coronary syndrome (ACS), which refers to the spectrum of clinical presentations comprising unstable angina (UA), non-ST-elevation myocardial infarction (NSTEMI, partial blockage of the coronary artery), and ST-elevation myocardial infarction (STEMI, full blockage of the coronary artery) [2]. It has been reported that survivors of myocardial infarction (MI) attack are at a significant risk of recurrent MI, as well as other CVD symptoms [3].

The most common risk factor for atherosclerosis is the increased production of reactive oxygen species (ROS). Oxidative stress plays a major role in the pathophysiology of ACS [4]. Oxidative stress profoundly affects circulating proteins, and albumin is the most common among these proteins [5]. ROS produced during ischemia generate highly reactive hydroxyl free radicals in the presence of copper, causing site-specific damage to albumin at the N-terminus [6], which decreases the capacity of albumin to bind transition metals, notably, cobalt [7]. This altered form of albumin is designated ischemia modified albumin (IMA), which is a marker of oxidative stress induced protein modification with diagnostic potential in acute myocardial ischemia [8]. Another indicator of protein damage is protein carbonyls, which are produced on amino acid side chains when they are oxidized. Oxidative modifications of polypeptide chains can also contribute to the alterations of protein functions [9].

Serum albumin has essential antioxidant properties, and its lower concentration has been shown to be associated with an increased risk of cardiovascular mortality [10]. Reduced and impaired biological functions of human serum albumin (HSA) are implicated in the pathogenesis of CVD [11]. Also, low albumin to globulin ratio (AGR) has been associated with vascular adverse events and red blood cell aggregability in both acute and chronic CVD [12], including its risk factors such as old age, diabetes, hypertension, and renal insufficiency [13].

Epidemiological studies and clinical observations have found elevated levels of fibrinogen to be independently related to cardiovascular risks [14]. Fibrinogen can be involved in the early stages of atherosclerotic plaque formation. High levels of plasma fibrinogen increase the speed of platelet aggregation and the reactivity of platelets [14]. An increased level of fibrinogen has been suggested as a coronary risk indicator since it reflects the inflammatory condition of the vascular wall [15]; therefore, further understanding of the association could prevent adverse outcome of ACS. The severity of atherosclerosis can be predicted by the fibrinogen to albumin ratio (FAR). Patients with higher FAR are more likely to develop ischemia, and the FAR could be a helpful early diagnostic test for predicting ischemia [16].

Therefore, this study was conducted to investigate the levels of albumin, fibrinogen, FAR, and AGR in patients with ACS and compare the findings with a non-ACS control group. Furthermore, the serum IMA and protein carbonylation were also compared between ACS patients and controls. It was hoped that correlation among these parameters and their association with ACS would reveal some inexpensive, easy to perform, and reliable biomarkers to be used for risk assessment in ACS.

Materials and methods

Subjects

This case-control study was conducted on 130 adult, male participants, aged 30–70, comprising 70 patients suffering from ACS (cases), admitted to the coronary care unit of Dhaka Medical College Hospital, and 60 healthy subjects enrolled from the local community (controls). The study was approved by the institutional ethics committee that conformed to the Helsinki declaration and had been conducted at the Department of Biochemistry and Molecular Biology, University of Dhaka, Bangladesh. All subjects from both the case and the control groups gave full consent to be included in this investigation.

Inclusion and exclusion criteria

The inclusion criteria of ACS cases were based on characteristic electrocardiogram (ECG) and troponin I value, diagnosed by expert physicians. Patients with acute chest pain and persistent (>20 min) ST-segment elevation were classified as STEMI. Patients with acute chest pain without persistent ST-segment elevation but with elevated troponin I were diagnosed with NSTEMI. Patients who presented with chest pain with no troponin I changes and with normal or undermined ECG were classified as UA. For the control group, apparently healthy volunteers who did not have CVD or any other diseases known to develop oxidative stress were enrolled. Simple random and availability sampling was applied to collect samples. Any patient or control subject suffering from any disease known to develop oxidative stress, including diabetes mellitus, infectious diseases, impaired liver, or renal functions, were excluded from the study. All subjects enrolled in the study had blood glucose levels below 6.5 mmol/L since hyperglycemia also induces oxidative stress, and is common during ACS [17], to avoid false positive results.

Study procedures

This study was conducted from March 2018 to February 2020. In a carefully pre-designed questionnaire, all the general information for each study subject was recorded. The general information included their age, height, weight, blood pressure, family history of CVD, and hypertension. In the case of patients, additional information regarding the duration of chest pain and cardiac troponin levels were also obtained. About 10 mL of venous blood was drawn from each subject, 5 mL was collected into an EDTA containing lavender top tube for plasma collection, and the remainder was taken in a glass tube for serum collection. The serum and plasma were separated, collected in small aliquots, and stored at -20°C until analyzed. The human serum albumin (HSA) level was estimated using bromocresol green method [18]. Briefly, 3 mL of the reagent was added to 20 μL of diluted (1:1) serum sample, mixed, and incubated for 5 minutes. HSA standards were treated in the same way. The concentration of HSA was calculated from the standard graph. The globulin contents of the samples were obtained by direct measurement of total protein (biuret method) and HSA, according to the following equation:

The AGR was estimated by dividing the albumin concentration by the globulin concentration. The plasma fibrinogen level was determined using a thrombin reagent, as described previously [19]. The FAR was calculated from the values obtained by direct measurement of fibrinogen and HSA. The IMA in the serum was determined according to the method of Bar-Or et al. [7] and detailed previously [20]. Protein oxidation was estimated by measuring the protein carbonyl content of the plasma samples by the method described by Levine et al. [21] and detailed previously [22].

Statistical analyses

GraphPad Prism (version 8.0.1, GraphPad Software, USA) was used to conduct independent samples t-test to compare the continuous variables of the two groups and graphical presentation of the analyzed data. For each parameter, the mean ± SD values were computed. The chi-squared/ Fisher’s exact test was used to compare categorical variables. The Spearman correlation analysis, univariate and multivariate logistic regression analyses and the receiver operating characteristic (ROC) curve analysis were done using the Statistical Package for Social Sciences (version 20.0, SPSS Inc., USA). The odds ratio (OR) and 95% confidence interval (CI) were reported. The results were considered significant when the value of p was <0.05.

Results

Baseline features of the study participants

Of the total 70 ACS cases, 53 (75.7%) had STEMI, 13 (18.6%) were ailed with NSTEMI, and 4 (5.7%) had UA. The duration of chest pain, from onset to hospitalization of the patients varied from 0.5 to 120 hours, with a median of 6.0 hours. The cardiac troponin I (cTnI) values, which were measured at the hospital upon diagnosis of ACS, varied from 0.01 to 78 ng/mL with a median of 1.41 ng/mL. The previous history of the patients showed 9 had heart attack or MI, 5 had angina, and 5 suffered cardiac arrest. A comparison of the baseline characteristics of the study participants is shown in Table 1. It was found that the systolic blood pressure (SBP), diastolic blood pressure (DBP), and body mass index (BMI) were not significantly different between the two groups while the mean age of the cases was significantly higher. The family history of CVD and previously diagnosed hypertension were found to be significant risk factors of ACS.

Table 1

Baseline features of the ACS cases and the controls.

Variables	ACS group	Control group	p-value
BMI (kg/m 2 )	26.26 ± 1.41	26.25 ± 3.21	0.98
SBP (mmHg)	127.79 ± 28.32	122.33 ± 10.10	0.21
DBP (mmHg)	83.21 ± 16.83	81.46 ± 7.06	0.50
Age (years)	51.04 ± 9.83	44.35 ± 9.52	<0.01
DCP (hours)	17.81 ± 24.98	N/A	N/A
Troponin I (ng/mL)	12.42 ± 19.03	N/A	N/A
Hypertension (%)	51.43	8.33	<0.0001
Family History of CVD (%)	28.57	11.67	<0.05

DCP = Duration of chest pain, N/A = Not applicable.

Level of human serum albumin

The HSA concentration of the ACS patients varied from 1.70 to 6.27 g/dL with a mean of 3.81 ± 0.99 g/dL, and that in the control subjects varied from 3.81 to 7.24 g/dL with a mean of 5.33 ± 0.66 g/dL (p<0.001). For close comparison, the HSA values were further divided into three categories: lower than 4.0 g/dL, 4.0–6.0 g/dL, and greater than 6.0 g/dL. Of the patients, 57.1% had HSA lower than 4.0 g/dL, 40.0% had values between 4.0 and 6.0 g/dL, and 2.9% had HSA levels greater than 6.0 g/dL; while among the controls, 1.7% had HSA lower than 4.0 g/dL, 82.8% had values between 4.0 and 6.0 g/dL, and 15.5% had HSA levels greater than 6.0 g/dL. A chi-squared test showed significant difference between the cases and controls for the categories mentioned above (Fig 1). Among the ACS group having HSA lower than 4.0 g/dL, 42.9% were STEMI, 12.8% were NSTEMI, and 1.4% were UA patients.

Fig 1

Distribution of human serum albumin (HSA) levels within the ACS patient group and control group.

Albumin to globulin ratio

The AGR in the cases varied from 0.25 to 2.67 with a mean of 1.15 ± 0.58, and the corresponding value in the controls varied from 0.57 to 5.46 with a mean of 2.02 ± 1.18 (p<0.0001). Within the spectrum of ACS cases, the mean AGR in STEMI (1.11 ± 0.55) and NSTEMI (1.15 ± 0.64) cases were significantly lower than the controls, while the UA cases with a mean of 1.69 ± 0.73 were not significantly different (Fig 2a). For further analysis, the AGR values were divided into three categories: lower than 1.5, 1.5–2.0, and greater than 2.0. A significant difference between the proportions of controls and ACS patients in different categories was found in the chi-squared test (Fig 2b). Among the 71.4% of ACS cases with AGR below 1.5, 57.1% were STEMI, 12.9% were NSTEMI, and 1.4% had UA.

Fig 2

(a) Comparison of albumin to globulin ratios (AGR) between the controls and different subgroups of ACS cases. The mean AGR in STEMI and NSTEMI cases were significantly lower than the controls, while the differences in UA cases were not significant (NS). (b) Comparison of the proportions of controls and ACS patients in different categories. Of the ACS patients, 71.4% had AGR lower than 1.5, 22.9% with AGR between 1.5 and 2.0, and 5.7% had values greater than 2.0; among the controls, 37.9% had AGR below 1.5, 34.5% ranged between 1.5 and 2.0, and 27.6% had AGR greater than 2.0 (p<0.001, controls vs. patients, all categories).

Level of fibrinogen and fibrinogen to albumin ratio

The mean fibrinogen level of the controls was found to be 29.2 ± 9.2 mg/dL, ranging from 19.3–73.5 mg/dL. On the other hand, the corresponding level of the patients was 60.5 ± 57.9 mg/dL, with the values varying from 17.6–331.3 mg/dL. The mean fibrinogen levels in the STEMI, NSTEMI, and UA cases were 60.6, 70.8, and 27.9 mg/dL, respectively. In the control group, the mean FAR was 0.0058 ± 0.0022, whereas the value was 0.0186 ± 0.0267 in the ACS group, which was significantly higher (p<0.001). Within the spectrum of ACS cases, the FAR of STEMI and NSTEMI patients varied significantly from the control group, with the mean values 0.0200 ± 0.0294 and 0.0174 ± 0.0170, respectively. The mean FAR in UA cases was 0.0053 ± 0.0007, which did not vary significantly from the controls.

Serum IMA and protein carbonyls levels in the cases and controls

Investigation of serum IMA revealed a significant (p<0.0001) elevation in the ACS group compared to the control group, and the mean values were 2.18 ± 0.61 U/mL and 1.52 ± 0.38 U/mL, respectively. The control group had a mean protein carbonyl level of 1.63 ± 1.06 nmol/mg compared to 3.16 ± 1.29 nmol/mg in the ACS group, which was significantly higher (p<0.0001).

Correlation between different parameters

A significant positive correlation was found between serum IMA and protein carbonyls in ACS patients (Fig 3a) with a Spearman correlation coefficient, ρ, of 0.317 (p<0.05). There were also significant positive correlations between cTnI and fibrinogen, IMA and fibrinogen, IMA and FAR (Fig 3b), protein carbonyls and fibrinogen, and protein carbonyls and FAR (Fig 4a) in patients with ρ values of 0.508 (p<0.05), 0.321 (p<0.05), 0.387 (p<0.01), 0.300 (p<0.05), and 0.362 (p = 0.01), respectively. No such correlations were observed in the controls. Further, a significant, strong negative correlation between HSA and cTnI with a ρ of -0.643 (p<0.01) was also seen in the cases (N = 30) who were brought to the hospital within 7.5 hours (median) of the onset of chest pain (Fig 4b).

Fig 3

(a) A significant positive correlation between IMA and protein carbonyls in ACS patients with a rho value of 0.317 (p<0.05); and (b) between IMA and FAR in ACS cases with a rho value of 0.387 (p<0.01).

Fig 4

(a) A significant positive correlation between protein carbonyls and FAR in ACS patients with a rho value of 0.362 (p = 0.01); and (b) a significant, strong negative correlation (rho = -0.643, p<0.01) between serum albumin and cardiac troponin I levels in ACS cases who were brought to the hospital with acute chest pain.

Diagnostic value of different parameters in ACS

The receiver operating characteristics (ROC) curve was analyzed to assess the clinical sensitivity and specificity of different parameters in ACS patients and controls (Fig 5a). Albumin was identified as the best biomarker in distinguishing ACS from controls, with an area under the curve (AUC) value of 0.906 (Fig 5b), followed by IMA and protein carbonyl with AUC value of 0.865 and 0.840, respectively (Fig 5c). The AUC value of AGR was 0.762, which had a cut-off value of 1.5 and provided 71.4 and 62.1% of sensitivity and specificity, respectively (Fig 5b).

Fig 5

(a) The cutoff value, AUC, sensitivity and specificity of albumin, fibrinogen, AGR, FAR, IMA and protein carbonyl (PC) for distinguishing ACS from controls; (b) Receiver operating characteristic (ROC) curve of albumin (ALB) and AGR for distinguishing ACS from controls; (c) ROC curve of IMA and PC for distinguishing ACS from controls.

Logistic regression analyses of risk factors

To determine the potential of albumin, fibrinogen and other baseline characteristics in predicting risk of ACS, univariate and multivariate logistic regression analyses were conducted. The results (Table 2) of univariate regression analyses found albumin, fibrinogen, family history of CVD, age, hypertension all to be significant risk factors for CVD. Upon multivariate analysis, a low value of albumin level was found to be an independent risk factor for ACS (odds ratio [OR]: 0.16 [0.057–0.454], p = 0.001) after adjusting for age, hypertension, family history of CVD and fibrinogen. In addition, a high value of fibrinogen was also a powerful independent risk factor for ACS (OR: 1.042 [1.002–1.083)], p<0.05) after adjusting for age, hypertension, family history of CVD and albumin (Table 2). Further, the multivariate logistic regression analysis found family history of CVD was a significant risk factor for ACS (p<0.05), while age and hypertension were not.

Table 2

Univariate and multivariate logistic regression analyses of risk factors of ACS.

Variable	UnivariateOR (95% CI)	Univariate(p-value)	MultivariateOR (95% CI)	Multivariate(p-value)
Albumin	0.097 (0.043–0.218)	<0.001	0.16 (0.057–0.454)	0.001
Fibrinogen	1.047 (1.016–1.079)	0.003	1.042 (1.002–1.083)	0.038
Family History of CVD	3.029 (1.179–7.780)	0.021	5.238 (1.269–21.626)	0.022
Age	1.094 (1.051–1.139)	<0.001	1.042 (0.990–1.096)	0.119
Hypertension	11.647 (4.164–32.576)	<0.001	2.344 (0.627–8.769)	0.206

OR = Odds Ratio, CI = confidence interval.

Discussion

This study evaluated alterations in circulatory proteins in patients with ACS. Epidemiologic studies have reported on associations of various inflammatory factors, including low albumin with coronary heart disease (CHD) [10,23,24]. Since hyperglycemia decreases regenerative potential of the myocardium [25], we excluded patients and controls with hyperglycemic random blood sugar levels from our investigation, to evaluate the association of albumin, fibrinogen and modified proteins with ACS independently from hyperglycemia.

In the present study, a significantly lower albumin concentration in the ACS patients suggested that lower albumin levels are indeed associated with ACS. In addition, this study revealed that a low value of albumin level was an independent risk factor for ACS and albumin level lower than 4.71 g/dL had 84.3% sensitivity and 84.5% specificity in identifying individuals at higher risk of ACS. This finding in ACS patients is in accordance with a previous observation that HSA lower than 4.5 g/dL is associated with an increased risk of CHD incidence [23]. One of the novel findings of this study is a significant negative correlation between serum albumin and myocardial cell damage marker, cardiac troponin I, which corroborates the association of low albumin with ACS. These findings suggest that regular monitoring and taking preventive measures to increase HSA concentrations may serve to reduce the risk of ACS.

Another novel finding of the present study is significantly lower AGR in STEMI and NSTEMI cases compared to controls, which corroborated its potential in assessing risk of ACS. The present study further found that 71.4% of patients had AGR values lower than 1.5, and STEMI and NSTEMI cases pooled together comprised 70% of the total patients with such low value of AGR. In one study, an AGR of 1.45 or less was prognostic for future vascular events [26]. Supporting this previous finding, the present study proposes an AGR lower than 1.5 to be considered high risk towards ACS occurrence, which has a sensitivity of 71.4% and a specificity of 62.1%. Therefore, this simple and inexpensive parameter may be considered for risk assessment of ACS.

Plasma fibrinogen level is an independent risk factor for CVD [27]. In this study, the fibrinogen levels in ACS cases were significantly higher than in the controls and a high value of fibrinogen was found to be an independent risk factor for ACS, which is in agreement with another study [28]. This finding indicates the potential of keeping the fibrinogen levels controlled in reducing the risk of ACS. However, it should be mentioned that the fibrinogen values in this study were lower than those reported in the study of Shi et al [30], both for the cases and controls. The present study further found a significant positive correlation of fibrinogen with cTnI. A similar correlation was shown in another study demonstrating the coexistence of inflammation and cardiac injury in patients with ACS [29]. One of the critical findings of the present study is significantly higher FAR in the patients. This observation further supports reports of increased FAR in patients with cardiovascular events, as in the literature [16], and indicates the significance of considering FAR as a predictor of future adverse outcomes of ACS.

Apart from inflammation, myocardial ischemia is the most common underlying cause of ACS. Recent research has found IMA to be an ideal biomarker for ischemia [8], which has been described as a sensitive biomarker for identifying ACS in patients presenting with acute chest pain [30] and thus, a possible diagnostic tool. In the present study, a significantly higher IMA was recorded in ACS cases, which was consistent with previous findings [20,31]. The present study found an IMA level greater than 1.74 U/mL has a diagnostic sensitivity and specificity of 83.0 and 71.2%, respectively and may be used as a reference value upon further validation. Furthermore, the positive correlations between IMA and fibrinogen, and IMA and FAR are indicative of the role of increased fibrinogen, decreased albumin, and increased FAR in ischemia.

The most widely used biomarker for oxidative damage to proteins is protein carbonyls that reflects cellular damage induced by multiple forms of ROS [9]. In the present study, the protein carbonyls of the ACS cases were significantly higher than the controls, which agreed with previous findings [22,32]. In addition, a protein carbonyl level greater than 2.21 nmol/mg with a sensitivity and specificity of 83.0 and 79.7% showed its association with ACS. Further, a significant positive correlation between protein carbonyls and IMA indicated that both these markers of oxidative protein damage are elevated in ACS patients, which may serve as diagnostic tools in the future. Moreover, significant positive correlations of fibrinogen, and FAR with protein carbonyls suggested their association with oxidative stress-mediated protein damage in patients with ACS.

It is well established that hypertension damages the arteries by making them less elastic which decreases the flow of blood and oxygen to the heart; on the other hand, age can cause the development of additional risk factors such as obesity which may also affect the heart. Although there was a significant difference in age and hypertension between cases and controls, multivariate logistic regression analysis did not find age and hypertension to be significant risk factors for ACS, after adjusting for other variables in this study. This could be because in our study population, age and hypertension were weaker risk factors compared to the other biochemical variables.

Limitations and suggestions

There are some limitations to this investigation. The ACS cases were enrolled from only one hospital, and the study population was relatively small. Additionally, all patients admitted to the hospital with ACS during the study period could not be enrolled due to different comorbid conditions and did not take into account differences in hospital treatment procedures. Finally, the mean age of the ACS group was higher by about 6.7 years than the control group, which was due to difficulties in finding age-matched controls with the stringent inclusion criteria. However, multivariate logistic regression analysis did not find age as a significant risk factor for ACS, after adjusting for other variables including albumin, fibrinogen. Albeit these limitations, the significant findings of the study can be used as a reference for future multicenter studies with larger sample size and long-term follow-up, considering the significance of the studied parameters. Findings of this study suggest monitoring lower albumin levels to avoid adverse outcomes.

Conclusion

The present study found significantly lower levels of albumin and AGR, and higher levels of fibrinogen in patients with ACS, which could be promising serum biomarkers for assessing risk and improving risk stratification in ACS. Increased protein carbonyls and IMA in patients suggest the presence of ongoing oxidative stress and ischemia, respectively, and correlations of these parameters with each other and independently with fibrinogen or FAR indicate the role of increased fibrinogen in ischemia and its association with protein alteration in patients with ACS. Finally, albumin, IMA and protein carbonyl levels may be exploited to detect risk of ACS, owing to their high diagnostic sensitivity and specificity.

20 Jan 2022

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Reviewer #1: The study is interesting and quite well written. The conclusions, if confirmed by larger studies, could have clinical relevance. The main limitation, as pointed out by the authors, is the low sample size.

This reviewer raises some issues that need to be addressed by the authors.

1- The authors included diabetes among the exclusion criteria. However, during ACS, hyperglycemia is also frequently found in non-diabetic individuals. In this setting, tight glycemic control favorably influences the CV outcomes of these patients (Journal of Clinical Endocrinology and Metabolism Volume 97, Issue 3, March 2012, 933-942. doi: 10.1210/jc.2011-2037 - Journal of Diabetes Research, 2018, art. no. 3106056. doi: 10.1155/2018/3106056). The text and tables are missing glucose values during hospitalization for ACS, which should have been included in the multivariate analysis. This issue, which is a limitation of the study, and the above references should be discussed in the manuscript.

2- In multivariate logistic regression analyzes, age and hypertension are not independently correlated with ACS. How do the authors justify these unexpected results?

Reviewer #2: The authors conducted “Assessing Circulatory Proteins in Patients with ACS”. While the idea is interesting, I have several concerns.

• The major weakness of this article is that extensive editing of the English language and style is required. Indeed, some parts are absolutely not comprehensible.

• The article’s main title is ambiguous and should be rephrased to be consistent with the precise goal.

• Line 55-58: Please indicate the source of this text.

• Line 59-61: This text does not appear to be in keeping with the preceding text's context, please find sources that narrate the dangerous events of this disease

• Please pay attention to writing the main and sub-headings of the article, and follow the basic approaches to writing scientific research

• Line 100: Please change the title “Subjects and methods” to “Materials and Methods”

• How was the sample size calculated, and is the number of specific subjects sufficient for this study, please explain it in detail

• Why did you specify the ages of the participants from 30 to 70 years? If you mean that the participants were within the ages you specified, then you must transfer this information to the results section and include the acceptable ages in the study.

• The inclusion and exclusion criteria are confusing and unclear.

• Please add a paragraph entitled Study Procedures, and explain all the details of the study to be clear to the reader

• Please delete the "Study period and blood sample collection" paragraph and move the information about it to the study procedures paragraph

• Please specify the end points of the study in a separate paragraph

• Put all the basic tests under the main title "Measurements"

• In table 1: change “ACS Cases” to “ACS group”, “Controls” to “Control group”, and “Statistics” to “p-value. Also, please find the P-values for BMI, SBP, and DBP and add them to the table.

• Line 167: Cheng the title “Level of serum albumin in the study subjects” to “level of human serum albumin”, and please illustrate the results of this test with a graph.

• finally, this study has several limitations that may affect the results of the study.

Reviewer #3: Nabila Nawar Binti, et al. demonstrate that Albumin, ischemia modified albumin (IMA) and protein carbonyls were found to have high diagnostic sensitivity and specificity for ACS. Of interest, these circulatory and modified proteins in ACS patients, particularly lower HSA, AGR, and higher IMA and protein carbonyls showed the potentials to be used for risk assessment of ACS.

The study is of interest nevertheless, I have several concerns that need to be addressed before the study could be re-submitted.

1) Introduction: please, shorten the introduction focusing on the biomarkers.

2) Methods: Please merge the paragraphs from 2.4 to 2.10 in one single session about the Labo test of the biomarkers/circulating proteins.

3) Methods: please describe how family history of CVD was assessed considering that is one of the independent factor of the multivariate logistic regression.

4) Results: “The duration of chest pain, from onset to hospitalization of the patients varied 158 from 0.5 to 120 hours, with a median of 6.0 hours”. Please, split the symptoms to balloon time for STEMI and NSTEMI/UA.

5) Results: dyslipidemia, previous history of CAD and admission medical therapy should be added to table 1 in order to better describe the 2 study populations.

6) Figure Legends: I am not sure that the figure legend should be in the text. Please, verify.

7) Results: Please merge the paragraphs 3.4 - 3.5 and 3.6 – 3.7 in two different paragraphs.

8) Results: Please add some information regarding the angiographic data (vessels affected by the lesions, PCI performed, numbers of stent).

9) Results: were there any patients without significant coronary artery disease (MINOCA)? Please clarify this information.

10) Results: Please, can the authors provide some data about the standard inflammatory agents such as WBC (white blood cells), neutrophils and lymphocytes counts and CRP.

11) Results: Please, can the authors provide some data about the admission blood glucose level and possible correlations with these circulating inflammatory proteins (albumin, ischemia modified albumin (IMA) and protein carbonyls)?

12) Discussion: Please shorten the discussion, focusing on the main findings.

13) Discussion: Please integrate the discussion with the following ref PMID: 33530978 regarding the inflammatory burden in patients with acute myocardial infarction.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

3 Mar 2022

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Response: We ensure that our manuscript meets PLOS ONE’s style requirements, including those for file naming.

2. Thank you for stating the following financial disclosure:

“This study was funded by a research grant awarded to Dr. LNI by the Ministry of Science and Technology, Government of the People’s Republic of Bangladesh.”

Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

Response: In the cover letter, the following statement has been included "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

Additional Editor Comments:

Please edit the manuscript carefully as suggested by the reviewers.

Check reference style, word count by consulting the instructions for authors.

[Note: HTML markup is below. Please do not edit.]

Response: We have edited the manuscript carefully as suggested by the reviewers. We have checked the reference style, preparation of figures, figure legends and their placement in the text, and word count by following the instructions for authors. The total number of words in our revised manuscript is 4879.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Partly

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The study is interesting and quite well written. The conclusions, if confirmed by larger studies, could have clinical relevance. The main limitation, as pointed out by the authors, is the low sample size.

This reviewer raises some issues that need to be addressed by the authors.

1- The authors included diabetes among the exclusion criteria. However, during ACS, hyperglycemia is also frequently found in non-diabetic individuals. In this setting, tight glycemic control favorably influences the CV outcomes of these patients (Journal of Clinical Endocrinology and Metabolism Volume 97, Issue 3, March 2012, 933-942. doi: 10.1210/jc.2011-2037 - Journal of Diabetes Research, 2018, art. no. 3106056. doi: 10.1155/2018/3106056). The text and tables are missing glucose values during hospitalization for ACS, which should have been included in the multivariate analysis. This issue, which is a limitation of the study, and the above references should be discussed in the manuscript.

Reply to the reviewer: As mentioned by the learned reviewer, the stated references observed that tight glycemic control may increase regenerative potential of the ischemic myocardium (J Clin Endocrinol Metab. 2012; 97:933-942, doi: 10.1210/jc.2011-2037), and hyperglycemia is common during ACS which is a significant and independent mortality predictor among diabetic patients with recent ACS (J Diabetes Res. 2018, art. no. 3106056. doi: 10.1155/2018/3106056). Considering these, we excluded patients and controls with hyperglycemic random blood sugar levels from our study, so that the association of albumin, fibrinogen and modified proteins with ACS could be assessed independently from hyperglycemia. It should be mentioned here that all patients enrolled in the study had blood glucose levels below 6.5 mmol/L on admission.

2- In multivariate logistic regression analyzes, age and hypertension are not independently correlated with ACS. How do the authors justify these unexpected results?

Reply to the reviewer: To determine the potential of albumin, fibrinogen and other baseline characteristics in predicting risk of ACS, both univariate (not shown in the manuscript) and multivariate logistic regression analyses were conducted. In univariate logistic regression, age, hypertension, family history of CVD, albumin and fibrinogen all were independent risk factors for ACS. Upon multivariate logistic regression analysis, age and hypertension were found to be not significant with respect to all other parameters.

Reviewer #2: The authors conducted “Assessing Circulatory Proteins in Patients with ACS”. While the idea is interesting, I have several concerns.

• The major weakness of this article is that extensive editing of the English language and style is required. Indeed, some parts are absolutely not comprehensible.

Reply to the reviewer: We have edited the Introduction, Materials and Methods, and Discussion sections to make them more comprehensible. We believe, the learned reviewer would find an overall improvement of language in the Revised Manuscript.

• The article’s main title is ambiguous and should be rephrased to be consistent with the precise goal.

Reply to the reviewer: The title of the article has been rephrased to be consistent with the precise goal of the study:

Association of albumin, fibrinogen, and modified proteins with acute coronary syndrome

• Line 55-58: Please indicate the source of this text.

Reply to the reviewer: A reference has been added in the manuscript to indicate the source of the text.

• Line 59-61: This text does not appear to be in keeping with the preceding text's context, please find sources that narrate the dangerous events of this disease

Reply to the reviewer: This has been addressed in the manuscript.

• Please pay attention to writing the main and sub-headings of the article, and follow the basic approaches to writing scientific research

Reply to the reviewer: The main and sub-headings of the article have been changed and improved.

• Line 100: Please change the title “Subjects and methods” to “Materials and Methods”

Reply to the reviewer: The title has been changed in the manuscript.

• How was the sample size calculated, and is the number of specific subjects sufficient for this study, please explain it in detail.

Reply to the reviewer: Acute coronary syndrome (ACS, formerly called ischemic heart disease) is caused by a sudden onset of cardiac tissue ischemia secondary to impaired blood flow. Bangladeshi people have high susceptibility to ischemic heart disease (IHD) although no population-based data is available. One study found the prevalence of IHD in Bangladeshi men to be 4.6% (Zaman et al, 2007; PMID: 19124932). It may be mentioned here that all our study participants were men, which had been inadvertently omitted from the manuscript. It has now been mentioned under the heading “Subjects” in the manuscript. Therefore considering the available prevalence rate, and owing to the difficulties in collecting blood samples from patients admitted to the CCU by following the stringent inclusion criteria of this study, we had to consider a single parameter (namely, human serum albumin) to calculate the sample size. The formula used is:

n = Z2p(1-p)/d2, where n = sample size; Z = confidence level at 95%; p = expected prevalence (here, 4.6%); and d = precision (±5%).

By substituting all values in the formula, we get:

n = 1.962×0.046(1-0.046)/0.052 = 67.4

Thus, we collected 70 samples from ACS patients.

Please note that this number is not sufficient as there are other parameters in this study. We have mentioned this in the limitations of our study.

• Why did you specify the ages of the participants from 30 to 70 years? If you mean that the participants were within the ages you specified, then you must transfer this information to the results section and include the acceptable ages in the study.

Reply to the reviewer: In a recent study carried out in Bangladesh on 800 hospitalized patients with ACS, it was found that approximately 5% of patients were less than 30 years of age while about 10% were aged 70 years or more (Ahmed et al., 2018; doi.org/:10.3329/bhj.v33i1.37018). We found a similar scenario in our preliminary study; hence we selected this age range to enroll study participants.

• The inclusion and exclusion criteria are confusing and unclear.

Reply to the reviewer: We have revised the relevant section and clarified the inclusion and exclusion criteria of the study subjects in the manuscript.

• Please add a paragraph entitled Study Procedures, and explain all the details of the study to be clear to the reader

Reply to the reviewer: We have added a paragraph entitled Study Procedures, and explained all the details of the study to be clear to the reader.

• Please delete the "Study period and blood sample collection" paragraph and move the information about it to the study procedures paragraph

Reply to the reviewer: We have provided the information on study period and blood sample collection to the study procedures paragraph.

• Please specify the end points of the study in a separate paragraph

Reply to the reviewer: Since our study was a case-control study, we did not specify any end points. In future similar observational cohort studies or clinical trials, the study end points could include all cause mortality, recurrent MI, cardiovascular deaths, stroke, etc.

• Put all the basic tests under the main title "Measurements"

Reply to the reviewer: All the basic tests have been put under the heading “Study procedures”, as suggested by Reviewer #3 (point no. 2, below).

• In table 1: change “ACS Cases” to “ACS group”, “Controls” to “Control group”, and “Statistics” to “p-value. Also, please find the P-values for BMI, SBP, and DBP and add them to the table.

Reply to the reviewer: All suggestions of the learned reviewer have been incorporated in Table 1 of the revised manuscript.

• Line 167: Cheng the title “Level of serum albumin in the study subjects” to “level of human serum albumin”, and please illustrate the results of this test with a graph.

Reply to the reviewer: The change has been made in the manuscript. The results of human serum albumin in the ACS and Control groups have been illustrated with a figure (Fig 1).

• finally, this study has several limitations that may affect the results of the study.

Reply to the reviewer: The limitations of this study have been discussed in the manuscript. However, the conclusions, if confirmed by larger studies, could have clinical relevance.

Reviewer #3: Nabila Nawar Binti, et al. demonstrate that Albumin, ischemia modified albumin (IMA) and protein carbonyls were found to have high diagnostic sensitivity and specificity for ACS. Of interest, these circulatory and modified proteins in ACS patients, particularly lower HSA, AGR, and higher IMA and protein carbonyls showed the potentials to be used for risk assessment of ACS.

The study is of interest nevertheless, I have several concerns that need to be addressed before the study could be re-submitted.

1) Introduction: please, shorten the introduction focusing on the biomarkers.

Reply to the reviewer: The introduction has been shortened and focused on the biomarkers.

2) Methods: Please merge the paragraphs from 2.4 to 2.10 in one single session about the Labo test of the biomarkers/circulating proteins.

Reply to the reviewer: The paragraphs 2.4 to 2.10 have been merged in one single session under the heading “Study procedures”.

3) Methods: please describe how family history of CVD was assessed considering that is one of the independent factors of the multivariate logistic regression.

Reply to the reviewer: Details have been added in the manuscript under the heading “Study Procedures”. Briefly - in a carefully pre-designed questionnaire, all the general information for each study subject was recorded which included their age, height, weight, blood pressure, any family history of CVD, and hypertension.

4) Results: “The duration of chest pain, from onset to hospitalization of the patients varied 158 from 0.5 to 120 hours, with a median of 6.0 hours”. Please, split the symptoms to balloon time for STEMI and NSTEMI/UA.

Reply to the reviewer: Unfortunately, this information was not available from the hospital.

5) Results: dyslipidemia, previous history of CAD and admission medical therapy should be added to table 1 in order to better describe the 2 study populations.

Reply to the reviewer: The lipid profile of the study participants was measured in the study but not included in the manuscript. There were no significant differences in serum levels of cholesterol, triglycerides, and LDL cholesterol among the two study populations (it should be mentioned here that the patients were already hospitalized and under cholesterol-lowering drugs). However, the HDL cholesterol was found significantly diminished in the patient group in contrast to that of the control group. The previous history of the patients showed 9 had MI, 5 had angina, and 5 suffered cardiac arrest (this information has been added in the manuscript under the heading “Baseline features of the study participants”). The admission medical therapy data of the patients was unavailable.

6) Figure Legends: I am not sure that the figure legend should be in the text. Please, verify.

Reply to the reviewer: The PLOS ONE style template has been checked regarding the placement of the figure legend.

7) Results: Please merge the paragraphs 3.4 - 3.5 and 3.6 – 3.7 in two different paragraphs.

Reply to the reviewer: This has been addressed in the manuscript.

8) Results: Please add some information regarding the angiographic data (vessels affected by the lesions, PCI performed, numbers of stent).

Reply to the reviewer: Unfortunately, the suggested information was not available from the hospital.

9) Results: were there any patients without significant coronary artery disease (MINOCA)? Please clarify this information.

Reply to the reviewer: Clinicians did not report any such observation.

10) Results: Please, can the authors provide some data about the standard inflammatory agents such as WBC (white blood cells), neutrophils and lymphocytes counts and CRP.

Reply to the reviewer: We have done the blood platelet count (data not shown) and measured fibrinogen levels in plasma of the study participants and found both the inflammatory parameters to be significantly higher in the ACS group. However, in a recently published paper from our lab, the WBC count of ACS patients was found to be 11.76 ± 2.49 × million cells/mL compared to 7.37 ± 1.77 × million cells/mL in the control subjects (p<0.001); the neutrophil count was significantly higher and lymphocyte count was significantly lower in patients (Afr.J.Bio.Sc. 4(1) (2022) 37-47). In the present study, some of the patients had CRP level between 6.0 and 20.0 mg/L.

11) Results: Please, can the authors provide some data about the admission blood glucose level and possible correlations with these circulating inflammatory proteins (albumin, ischemia modified albumin (IMA) and protein carbonyls)?

Reply to the reviewer: All patients enrolled in the study had blood glucose levels below 6.5 mmol/L on admission. Individual values were not recorded for further analysis.

12) Discussion: Please shorten the discussion, focusing on the main findings.

Reply to the reviewer: We have shortened the discussion, focusing on the main findings.

13) Discussion: Please integrate the discussion with the following ref PMID: 33530978 regarding the inflammatory burden in patients with acute myocardial infarction.

Reply to the reviewer: Unfortunately, we couldn’t integrate the discussion with the suggested reference (PMID 33530978) since the study focused on the interplay between hyperglycemia, inflammation and infarct size in a cohort of patients admitted with acute myocardial infarction, including cases of MINOCA, which did not exactly relate to our study.

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Response: We uploaded our figure files (a total of 5 figures) to the PACE digital diagnostic tool to check whether the figures met PLOS requirements; all figures were converted to the accepted formats of PLOS ONE.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

20 Apr 2022

1 Jun 2022

In response to the first question raised by Reviewer #1, the following lines have been inserted in the manuscript:

Line # 109: “All subjects enrolled in the study had blood glucose levels below 6.5 mmol/L since hyperglycemia also induces oxidative stress, and is common during ACS [17], to avoid false positive results.”

Line # 245: “Since hyperglycemia decreases regenerative potential of the myocardium [25], we excluded patients and controls with hyperglycemic random blood sugar levels from our investigation, to evaluate the association of albumin, fibrinogen and modified proteins with ACS independently from hyperglycemia.”

As for the second question, we are apologetic for failing to clarify the second issue both in our manuscript and ‘Response to Reviewers’. To address this important issue, we have included an extra column in Table 2 in the accompanying revised version [PONE-D-21-36646R1], to present the statistical findings of the univariate logistic regression analyses along with those of the multivariate regression data. Also, the following lines have been inserted in the manuscript:

Line # 297: “It is well established that hypertension damages the arteries by making them less elastic which decreases the flow of blood and oxygen to the heart; on the other hand, age can cause the development of additional risk factors such as obesity which may also affect the heart. Although there was a significant difference in age and hypertension between cases and controls, multivariate logistic regression analysis did not find age and hypertension to be significant risk factors for ACS after adjusting for other variables in this study. This could be because in our study population, age and hypertension were weaker risk factors compared to the other biochemical variables.”

To our understanding, since multivariate regression analysis considers more than one factor of independent variables that influence the variability of dependent variables, the conclusion drawn is more accurate, more realistic, and nearer to the real-life situation for our study population.

In response to the Editor comments, we have reviewed our reference list to ensure that it is complete and correct. Reference numbers 17 and 25 have been newly inserted in the Manuscript, as suggested by Reviewer #1. We have addressed the reviewers' comments in detail, and revised our manuscript accordingly.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

11 Jul 2022

Association of albumin, fibrinogen, and modified proteins with acute coronary syndrome

PONE-D-21-36646R2

Dear Dr. Islam,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Arturo Cesaro, MD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The paper appears to be improved after changes were made based on the reviewers' comments.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: All issues raised by this reviewer were addressed by authors. The manuscript is methodologically correct. The conclusions were supported by results. In this revised version the authors improved the original manuscript.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

**********

18 Jul 2022

PONE-D-21-36646R2

Association of albumin, fibrinogen, and modified proteins with acute coronary syndrome

Dear Dr. Islam:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

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on behalf of

Dr. Arturo Cesaro

Academic Editor

PLOS ONE