Correlation of salivary hs-CRP With conventional cardiovascular risk markers and hba1c in prediabetic subjects

Background

“Prediabetes” is used for individuals whose glucose levels do not meet the criteria for diabetes but are too high to be considered normal.¹

The prevalence of prediabetes is growing worldwide, and it is expected that more than 470 million people will develop prediabetes by 2030, and 5–10% of prediabetics will progress to diabetes.²

According to World Health Organization (WHO), prediabetes is a state of intermediate hyperglycemia characterised by, impaired fasting glucose (IFG) /fasting plasma glucose (FPG) of 6.1-6.9 mmol/L (110 to 125 mg/dL) and impaired glucose tolerance (IGT) /2 hour plasma glucose of 7.8-11.0 mmol/L (140-200 mg/dL) after ingestion of 75 g of oral glucose load.³

On the other hand, according to the American Diabetes Association (ADA), IGT (140-199 mg/dL) has the same cut-off value, but IFG (100-125 mg/dL) has a lower cut-off value and has an additional hemoglobin A1c (HbA1c)-based criteria of a level of 5.7%–6.4% to define prediabetes.^1,3

Prediabetes is of great concern, as it is associated with an increased risk of diabetes and cardiovascular diseases. Prediabetes is linked to obesity (mainly abdominal or visceral obesity), hypertension, and dyslipidemia (high triglycerides and/or low HDL cholesterol).¹

HbA1C has an upper hand over FPG and OGTT due to greater convenience (fasting not required) and preanalytical stability. They are also less affected by day-to-day perturbations during stress and illness.¹

Studies in the past 10 years have proved that HbA1C still continues to be the most valuable glycemic risk marker.⁴

C-reactive protein (CRP) in blood is commonly used as a biomarker for inflammation and infection. However, recent studies have targeted the use of salivary CRP as a noninvasive alternative.

Most serological markers for diabetes are detected using invasive techniques. Thus, the development of noninvasive techniques for monitoring biomarkers is needed in the current scenario.^5,6

One of the advantages of using salivary CRP as a biomarker is its noninvasive nature. Saliva can be easily collected and does not require any special preparation, making it a convenient option for both patients and health care providers. Salivary CRP has been found to be as efficacious as blood CRP.

Human saliva is a rich reservoir of analytes (3,000 proteins and 12,000 peptides) and shares 30% proteins and 10% peptides with the serum proteome and peptidome.^5,7

Inflammation induces hepatic synthesis of acute-phase proteins, such as serum ferritin and high-sensitivity C-reactive protein (hs-CRP), which play a significant role in insulin resistance and atherosclerosis. hs-CRP is a sensitive marker of the inflammatory activity in the arterial wall. hs-CRP is a measure of CRP level with greater accuracy. The lower limit of its measurement is 0.01 mg/L and the measurement is more than100 times as sensitive than CRP measurement (lower limit, 5 mg/L).⁸

The American Heart Association (AHA) defines Metabolic Syndrome based on five factors: fasting blood pressure, blood glucose, triglycerides, HDL-C, and waist circumference. High-sensitivity C-reactive sensitivity CRP (hs-CRP) level is a measure of systemic inflammatory conditions and is considered an important biomarker of diabetes.⁹

Chronic low-grade inflammation has been proposed as a key factor for both metabolic syndrome (MetS) and subsequent clinical outcomes, such as cardiovascular diseases (CVD) and cerebrovascular disease. Chronic low-grade inflammation causes disruption of the vascular endothelial glycocalyx by C-reactive protein (CRP), which leads to its dysfunction and increases susceptibility to proatherogenic factors. Inflammation-induced vascular changes cannot be evaluated using cardiac imaging. Therefore, biomarker detection is of great significance.¹⁰

Studies have shown the relationship between salivary hsCRP and type 2 diabetes and the relationship between plasma hs-CRP and prediabetes; however, our study aimed to determine the relationship between prediabetes and salivary hs-CRP so that timely intervention can help to reverse it. Thus, we can save many prediabetics from converting to overt diabetes and decrease the predisposition to cardiac diseases. This will also help reduce the immense economic burden on society.

Aim

To establish a correlation between salivary hs-CRP levels and conventional cardiovascular risk markers and HbA1C in prediabetic subjects.

Objectives

Study model

PURPOSE: To establish a correlation between salivary high-sensitivity C-reactive protein (hs-CRP) levels and conventional cardiovascular risk markers and HbA1C in prediabetic subjects. Thus, we can save many prediabetics from converting to overt diabetes and decrease the predisposition to cardiac diseases. This will also help reduce the immense economic burden on society.

Inclusion criteria

All consenting patients aged 45 years and above with risk factors, attending medicine OPD, or being admitted to the medicine ward of the DMIHER Wardha.

Exclusion criteria

Study design: Cross sectional, observational study

Study: Department of Medicine, Acharya Vinoba Bhave Rural Hospital (AVBRH), JNMC, DMIHER (Deemed University), Wardha.

Study population: This is a cross-sectional, observational study that will be conducted in patients attending medicine OPD or admitted to the medical ward at Acharya Vinobha Bhave Rural Hospital, DMIHER Wardha, fitting into the exclusion and inclusion criteria of the study.

Duration of study: 2 years

Tools:

Outcome measures

Sample size: 95 patients

Formula:

Zα is 95% confidence level = 1.96

Z1-β is 80% power of study = 0.8413

P is prevalence of prediabetes = 14%¹¹

E is the absolute error = 10%

Study reference: Jose J et al.¹¹

Statistical analysis

The data were entered in MS EXCEL and analyzed using R Studio software version 4.3.1.

Quantitative data was analyzed using Student’s T-test and ANOVA analysis.

Methods

After explaining the study in detail to the subjects included in the study, informed consent written and oral was obtained.

In the medicine OPD/ward, the subjects will undergo an assessment of vitals and measurement of anthropometric indices.

The values of the same will be recorded in designated proforma.

As per Performa, relevant history will be obtained from the subjects after fulfilling the inclusion and exclusion criteria.

Further, the subjects will be instructed regarding the need for overnight fasting for 8 hours after dinner and to report to the medicine OPD the next morning for collection of blood samples for lipid profile estimation.

Salivary samples will also be collected.

Anthropometric measurements

The following parameters will be included

Obesity is defined as a BMI of 25 or higher.

WHO criteria for BMI for Asia-pacific region were used in this study:

Waist circumference

Lipid profile

Fasting Lipid Profile—The fasting lipid profiles of all patients will be examined, including Triglycerides, Total cholesterol HDL and LDL. Standard laboratory procedures were used to measure all the biochemical parameters. After a 10- to 12-hour overnight fast, blood samples were collected and centrifuged for five minutes at 3000 rpm to determine the serum lipid levels. This will be performed with a Virtos integrated 5600 ortho clinical diagnostics analyzer.

Estimation of serum triglyceride

Estimation of serum HDL

By Using the Direct Enzymatic Method, the Virtos Integrated System - Ortho Diagnostics Chemicals Analyzer, serum HDL was evaluated.

Estimation of serum cholestrol

Using the Enzymatic Method, the Virtos Integrated System - Ortho Diagnostics Chemicals Analyzer serum cholesterol will be assessed.

Showing machine for FLP: Fully automated analyzer–Virtos interated system-5600- Ortho clinical diagnostics

Samples will be processed in the central clinical lab (CCL) of AVB Rural Hospital, Sawangi (Meghe), Wardha.

Total Cholesterol

Normal—Less than equal to 200 mg/dl

Borderline—Between 200-239 mg/dl

High—240 mg/dl or more

LDL Cholesterol

Normal—Less than equal to 100 mg/dl

Borderline—Between 130-159 mg/dl

High—160 mg/dl or more

HDL Cholesterol

Normal—In Males 40 mg/dl or more

In Females 50 mg/dl or more

Abnormal—Less than 40mg/dl in Males

Less than 50 mg/dl in Females

Triglyceride levels

Normal—Less than equal 149 mg/dl

Borderline—Between 150-199 mg/dl

High—200 mg/dl or more

Fasting Blood Glucose, 2 Hour Postmeal Glucose, HbA1C

ADA Criteria 2019—

Prediabetes—

FBG-100-125 mg/dl

2h post glucose—140-199 mg/dl

HbA1C—5.7-6.4%

Fasting plasma glucose

It is estimated by the glucose oxidase-peroxidase (GOD-POD) method using a Robonic Semi Automatic Chemical Analyzer.

PRINCIPLE: Glucose oxidase oxidizes (GOD) glucose to gluconic acid and hydrogen peroxide. Hydrogen peroxide reacts with phenol and 4-amino-antipyrin (4AAP) in the presence of peroxidase (POD) to form quinone-imine dyes. The intensity of the color was directly proportional to the amount of glucose in the sample and was measured at 505 nm (500 – 520 nm or with a green filter).

Specimen collection

After 8 h of overnight fasting, the subject’s venous sample was taken from the anterior cubital vein and collected in a sugar bulb containing sodium fluoride (NaF) as an anticoagulant. This will be done to prevent glycolysis and improve separation of serum or plasma as soon as possible.

Reagent storage and stability

The liquid glucose reagent (Anamol Laboratories Pvt. catalogue number of the reagent: 2939149) is stable until the expiry date when stored at 2-8°C.

Procedure

1000 μL The reagent will be added to 10 μL of plasma (dissolved in an anticoagulant). Incubation was performed for 15 min at room temperature and the results were read using a Robonic Semi Automatic Chemical Analyzer.

Calculation

Glucose (mg/dl) = Optical Density of Test × 100/Optical Density of Standard

HbA1C

Using the Enzymatic Method, the Virtos Integrated System 5600 - Ortho Diagnostics Chemicals Analyzer serum HbA1C was assessed.

Salivary sample collection for hs–CRP

Two milliliters of unstimulated saliva were collected by draining in a sterile container (Eppendorf tube). All samples were maintained at a stable room temperature. Patients will be instructed not to consume food or any other substance for two hours before saliva collection. All samples were taken in the morning between 9am-11am.

Laboratory method

Saliva was centrifuged at 3000 rpm for 10 min and CRP levels were estimated.¹²

Dissemination

Articles arising from this study will be published in the index journals.

Study status

Not yet started, is expected to begin from October 2023.

Ethical considerations

The Institutional Ethics Committee of Datta Meghe Institute of Higher Education and Research (DU) has granted approval to the study protocol (Reference number: REF/2023/07/070519, Date: 17/07/2023).

After explaining the study in detail to the subjects included in the study, informed consent written and oral was obtained.