This cross-sectional study with comparison group entitled “Subclinical risk markers for CVD in MHO subjects” was carried out over a period of three years (January 2016 to January 2019) in a tertiary care hospital. Institutional ethical committee (DMIMSU), clearance was taken before starting the study.

All cases were randomly selected from the university students, staff, workers, and various health check-up camps organised by the hospital. Relevant demographic data (information comprised of sex, age, race, occupation and postal address) was collected after taking due consent. History of Diabetes mellitus, systemic hypertension was taken. Detailed drug history was obtained. Subjects with infections, sepsis, coronary artery disease, chronic liver and kidney disease, rheumatologic disorders, alcoholics, women on contraceptive pills, and subjects not giving consent were excluded. Metabolic unhealthy with normal weight phenotypes were also excluded.

Detailed physical examination and anthropometric measurement in form of BMI, WC was calculated. Biochemistry analysis including FBS, TG, HDL-C was estimated.

Obesity in this study was defined as per the Body Mass Index (BMI) categories for Asian Indians that has been revised based on consensus guidelines. The revised guidelines categorise obesity as a BMI ≥25 Kg/m² [12].

MS was defined as per the Modified National cholesterol education programme adult treatment panel III (NCEP ATP III) criteria as proposed by the AHA/NHLB [13].

MHO was defined as subjects with; BMI ≥25 Kg/m² (As per BMI category for Asians) with less than 3 MS criteria as per revised NCEP ATP III guidelines [12,14].

In this study MHNO controls were defined as: BMI ≤25 Kg/m² with less than 3 MS variables [15].

After taking due consent from the participants, Serum hs-CRP, CA-IMT, and USG to detect fatty liver was done.

Anthropometric data including weight, height, BMI, WC was measured by standard methods [16]. Blood Pressures (BP) was measured as per standard protocol [17]. Subjects with BP ≥130/85 mm of Hg or with ongoing treatment for hypertension was included as a parameter for defining MS.

Fasting plasma glucose was estimated by the Glucose Oxidase (GOD)/Peroxidase (POD) method, serum HDL by direct enzymatic method, TG were estimated using a LIQUID STABLE GPO-PAP method by machine Robonic Semi Automatic Chemical Analyser.

Quantitative hs-CRP was estimated by a solid phase ultra-sensitive enzyme immunoassay based on two-site sandwich enzyme immunoassay technique. A serum hs-CRP levels <1, 1-3 and >3 mg/L are taken as as low, intermediate, and high-risk groups for CVD risk as per guidelines. This study considered hs- CRP value ≥3 mg/L as abnormal reflecting sub clinical vascular inflammation [18].

All patients underwent USG of the abdomen to detect fatty changes in the liver, performed by a single experienced radiologist. It was performed using a high resolution B-mode ultrasonography system Prosound Alpha 7 with curved linear arrays mid-frequency probe of 3-5 MHz.

Diagnosis of NAFLD was made by the presence of an increase echogenicity of liver as compared to the kidneys [19].

Cases were examined in the supine position, the neck was oriented in 45 degree, and B-mode ultrasound images obtained as per guidelines by one examiner who was unaware of participant’s clinical characteristics. Common Carotid Artery (CCA) was scanned using Aloka prosound in B-mode at Acharya Vinoba Bhave Rural Hospital (AVBRH). IMT thickness was measured as per protocol. Two measurements were taken from each side and averaged; then, the mean CIMT was calculated as the average of measurements obtained from both CCAs [20]. As per the latest ESH/ESC hypertension guidelines (2013), carotid IMT >0.9 mm has been taken as a marker of asymptomatic organ damage [21,22].

The quantitative data were expressed as mean±SD. Pearson’s correlation coefficient was used to test correlations between variables. Analysis of Variance Tests (ANOVA) and chi-square tests of independence were used for continuous and categorical variables, respectively. Logistic regression analysis was performed, adjusting for age, gender, WC, hypertension, FBS, LDL, HDL, and TG. A p-value <0.05 was considered to be statistically significant.

Total number of participants in group MS were 65, in MHO group were 222 and in control group number of participants were 81.

The MHO group was younger (36.64±14.82) as compared to MS group (45.18±15.88) and control group (37.79±13.63), and prevalence of females were more in MHO group (54.95%). Using One-way ANOVA; All the variables differed significantly across the groups. The male to female ratio in MHO group was 1:1.2. All the variables and anthropometric determinants of metabolic syndrome (WC, SBP, DBP) and the parameters of metabolic syndrome (FBS, TG) differed significantly across the groups [Table/Fig-1].

By using Pearson’s Correlation Coefficient significant positive correlation was found between CIMT and BMI, WC, FBS, SBP, TG, NAFLD and hs-CRP [Table/Fig-2]. By using Multiple regression analysis NAFLD and hs-CRP were significantly associated with CIMT [Table/Fig-3]. By using Pearson’s Correlation Coefficient significant positive correlation was found between hs-CRP level and BMI, WC, FBS, SBP, DBP, TG, NAFLD and CIMT [Table/Fig-4]. By using Multiple regression analysis BMI, WC, NAFLD and CIMT were significantly associated with high hs-CRP levels [Table/Fig-5].

By using Pearson’s Correlation Coefficient significant positive correlation was found between NAFLD and BMI, WC, FBS, SBP DBP, TG, CIMT and hs-CRP [Table/Fig-6]. By using Multiple regression analysis WC, CIMT and hs-CRP were significantly associated with NAFLD [Table/Fig-7].

Prevalence of high hs-CRP in metabolic syndrome group was 76.92%. The adjusted odd’s ratio (AOR: adjusted with WC, HDL, FBS, TG) was (2.41; CI 1.92-3.51) compared to MHNO population. Prevalence of high hs-CRP in MHO group was 65.32%. AOR was (1.98; CI 1.29-3.12) compared to MHNW population. In MHO females the AOR was 1.65 as compared to MHNO. The prevalence of NAFLD in MHO group was 59.01%. In metabolic syndrome group the overall AOR was 2.21(1.21-2.51). In MHO group the overall AOR for NAFLD as compared to MHNO group was 1.81 (CI: 1.11-2.13). Prevalence of abnormal CIMT in MHO group was 24.32% in MHO group as compared to control 3.70%. The adjusted odd’s ratio (AOR: adjusted with WC, HDL, FBS, TG) of abnormal CIMT in overall MHO subjects was around 1.6 (95% CI 1.12-1.91), in males it was 1.06 (95% CI 0.82-1.51), and in females it was 1.76 (95% CI 0.41-2.12) as compared to normal control [Table/Fig-8].

MS had the higher risk values followed by MHO and lastly the control group. The subclinical risk parameters like hs-CRP, CIMT and NAFLD showed similar trends across the groups which were statistically significant (p<0.05). As these findings suggest, though MHO individuals had less than three metabolic risk parameters and decreased levels of all these risk markers for developing cardiovascular disease when compared with MS, still they had significantly higher values of these variable than controls. This also suggests that MHO may have an increased risk of progressing to MS in future. Studies have confirmed that MHO usually progresses to MS in future being a snap shot of an abnormal pre-metabolic state [23,24]. It is needless to deny MHO as a pre-metabolic state and it would progress to overt MS with its consequent CVD risks in future.

By using multiple regression analysis, NAFLD and hs-CRP were significantly associated with CIMT values in MHO group [Table/Fig-3]. As hs-CRP is also a risk marker of vascular inflammation and NAFLD suggests the altered milieu of adipose tissue metabolism, both contribute to elevated CIMT irrespective of the presence or absence of other abnormal metabolic parameters.

Jung CH et al., studied the degree of subclinical coronary atherosclerosis in asymptomatic Korean population categorising them into different phenotypes [25]. The study concluded that, MHO subjects have substantial subclinical coronary atherosclerotic burden. Zhao M et al., in their study assessed CIMT in MHO children and adolescent population [26]. In comparison with metabolically healthy normal weight, Odds Ratios (ORs) for high CIMT were 3.91 (2.46-6.21) for MHO, concluding that CIMT was higher for both MHO and metabolically healthy overweight compared with metabolically healthy normal weight. Jae SY et al., accessed cardiorespiratory fitness association between MHO phenotype and CIMT [27]. Compared with metabolically healthy normal weight, MHO was associated with a higher prevalence of subclinical carotid atherosclerosis (odds ratio, 1.39; 95% CI, 1.12-1.72). Metabolically healthy obesity was associated with a higher prevalence of subclinical carotid atherosclerosis.

Roberson LL et al., examined four studies reporting a mean difference in Common Carotid Artery Intima Media Thickness (CCA-IMT) between MHO and MHNW individuals, of which two studies reported significantly higher levels in the MHO [7].

In this study, the mean hs-CRP in MHO was 4.01±1.68 and in the control group it was 2.16±0.56 (p=0.0001), suggesting that MHO portray trends of abnormal subclinical inflammation.

By using Multiple regression analysis BMI, WC, NAFLD and CIMT were significantly associated with elevated hs-CRP [Table/Fig-5]. An increased WC denotes visceral obesity which contributes to inflammation, insulin resistance, and fat deposition in liver. MHO population is vulnerable for all these abnormalities compared to MHNO individuals.

In a study by Shaharyar S et al., the prevalence of abnormal hs-CRP values in MHO was 22% [28]. The overall AOR of high hs-CRP was 2.45 (males 2.51, females 3.59). The conclusion of this study was similar to the current study suggesting MHO as a non healthy entity. In van Wijk DF et al., study there was a higher multivariable-adjusted hazard ratio for CHD in MHO subjects with CRP levels >2 mg/L [29]. The higher CRP levels were associated with a CHD risk. Iglesias Molli AE; in their study found that hs-CRP was significantly high in MHO than normal controls similar to the current study [30]. Bennett NR et al., analysed 342 men and 404 women with MHO [31]. Approximately 15% of the participants had high risk hsCRP (>3 mg/L), in logistic regression models high WC was associated with significantly higher odds of high hs-CRP (OR 7.8, 95% CI 4.8-12.9, p<0.001) in MHO subjects.

In this study, out of 222 MHO subjects 84 (37.8%) had grade 1 NAFLD, 45 (20.3%) had grade 2 NAFLD, and 2 (0.9%) had grade 3 NAFLD overall prevalence was 59.01%. In males the prevalence was 28.83% and in females it was 30.18%.

In Shaharyar S et al., study the prevalence of NAFLD in MHO was 40% [28]. The overall AOR of high NAFLD in MHO was 5.08. Heianza Y et al., in their study found that the prevalence of NAFLD in MHO was around 47.8% [32]. This may explain the increased risk of insulin resistance contributed by hepatic fat, there by risk of development of diabetes among the MHO phenotypes.

Chang Y et al., in another study found that the incident rate of NAFLD was 29.7 per 1,000 person-years in MHO [33]. The multivariable adjusted hazard ratio for the development of NAFLD in overweight and obese with normal-weight participants were 2.15 (95% CI; 2.06-2.26) and 3.55 (95% CI; 3.37-3.74), respectively. Overweight and obesity were strongly and progressively associated with an increased incidence of NAFLD.

Jung CH et al., investigated the effect of Fatty Liver Disease (FLD) as a risk factor for development of incident type 2 diabetes in Metabolically Healthy Obesity MHO [34]. MHO subjects with high fatty liver index had an elevated risk of incident type 2 diabetes (multivariate-adjusted HR, 1.99 (95% CI 1.36-2.92). Haskins IN et al., in their study determined the prevalence and characteristics of NAFLD in individuals with MHO [35]. In the MHO group, 23% had lobular inflammation, 8.5% had hepatocyte ballooning, 8.2% had steatohepatitis, and 4.4% had liver fibrosis. They concluded that NAFLD though asymptomatic is highly prevalent in MHO. Zhang H et al., in their study examined 485 obese adults with magnetic resonance spectroscopy [36]. MHO individuals were 1.9 times more likely to have metabolic syndrome per 1 SD increase in IHTG content. The risk for high CIMT increased 29% per 1 SD change in IHTG content in MHO group.

Gutierrez Y et al., evaluated liver fibrosis in patients with NAFLD, in MHO and Metabolically Unhealthy Obese (MUHO) [37]. MHO patients showed a significantly lower prevalence of advanced liver fibrosis than MUHO on transient elastography, but they still had NAFLD as a risk factor.

The present study showed that MHO groups have a higher prevalence of elevated hs-CRP levels, abnormal CIMT and NAFLD as compared to MHNO group. The inference of the study directs towards the subclinical vascular inflammatory triggers of obesity per se. Obesity in absence of metabolic risk factors is not entirely benign and is associated with subclinical vascular inflammation that can predispose to future development of MS and incident CVD.

Adipose tissue is highly metabolic and produces inflammatory cytokines. The pro-inflammatory markers present in MHO individuals are Complement C3, Alpha necrosis factor, CRP, Interleukin 6, leptin, resistin, TNF-a, IL-6, IL-18 [38]. These adipokines contribute to the metabolic disturbances and CVD morbitities associated with obesity. Obesity results in a pro-inflammatory metabolic state and may explain the higher prevalence of elevated inflammatory markers like hs-CRP, higher CIMT and NAFLD in our MHO subjects [39].

The present study is agreement, in that MHO subjects have increased levels of the abnormal anthropometric and metabolic variables as compared to MHNO individuals. Subclinical vascular inflammatory markers are elevated in MHO making them vulnerable for development of MS, CVD and type II diabetes in future.

This study is cross-sectional in design so it only throws light on associations of high risk subclinical pro-inflammatory markers with MHO phenotype and wouldn’t prove the causation. This study defined MHO using a single criteria (NCEP ATP III).

There is no paucity of data that emphasises that MHO is actually not a “healthy” phenotype. This study indicates that both MHO phenotype and MS are associated with higher burden of inflammation when compared to MHNO subjects. It is high time; we question the benignity of MHO. Rather as a warning awareness, MHO should be replaced by the term “Premetabolic syndrome” in the era of evidence based medicine.