Significantly higher atherosclerosis risks in patients with obstructive sleep apnea and non-alcoholic fatty liver disease.

INTRODUCTION: There is limited data on the relationship between Obstructive Sleep Apnea (OSA) and Non-Alcoholic Fatty Liver Disease (NAFLD), each associated with increased cardiovascular risk. This study aimed to determine the relationships between severity of OSA, degree of steatosis in NAFLD and cardiovascular risk via CIMT and atherosclerosis markers ie intracellular adhesion molecule-1 (ICAM-1) an Lipoprotein-a (Lp(a)) in a group of patients with OSA.
MATERIALS AND METHODS: This was a cross-sectional, single center study. A total of 110 subjects between 18 to 65 years of age and diagnosed with OSA following sleep study examinations were recruited. Exclusion criteria included seropositive Hepatitis B or Hepatitis C, and significant alcohol intake. RESULT: The prevalence of NAFLD was 81.8%. The mean CIMT (0.08±0.03 vs 0.06±0.01 cm, p = 0.001), ICAM-1 (334.53±72.86 vs 265.46±102.92 ng/mL, p = 0.001) and Lp(a) (85.41±52.56 vs 23.55±23.66 nmol/L, p<0.001) were significantly higher in the NAFLD group compared to the non-NAFLD group. Comparisons between the different groups showed significantly increasing levels of CIMT, ICAM-1 and Lp(a), lowest within the non-NAFLD, followed by the NAFLD 1 and NAFLD 2+3 groups. There was a significant positive correlation between degree of steatosis and the severity of OSA (r = 0.453, p<0.001). Logistic regression analysis revealed that patients with apnea/hypopnea index (AHI) of >30 were 52.77 (CI 6.34, 439.14) times more likely to have NAFLD compared to those with mild AHI (p<0.001).
CONCLUSION: The prevalence of NAFLD is alarmingly high in this group of OSA patients. The degree of steatosis in patients with NAFLD was significantly correlated with severity of OSA, CIMT measurements, ICAM-1 and Lp(a). Our findings underscore screening for NAFLD in patients with OSA to ensure prompt risk stratification and management.

Introduction

The prevalence of obesity has gradually risen globally, with a doubling from 6.4% in 1980 to 12.0% in 2008 and currently poses significant public health problems [1,2]. The prevalence of obesity among Malaysians was estimated at 30.2% in 2015 [3], rising exponentially from what was reported by the Malaysian National Health Morbidity Survey in 1996, which then stood at 5.8% [4]. Obesity has been shown to be strongly associated with various non-communicable diseases (NCD), such as hypertension, type 2 diabetes, coronary heart disease, stroke, obstructive sleep apnea and non-alcoholic fatty liver disease (NAFLD), to list a few.

Obstructive sleep apnea (OSA) is a disorder that is characterized by recurrent upper airway collapse during sleep, leading to sleep fragmentation and daytime sleepiness with chronic intermittent hypoxia (CIH) [5]. It has been associated with a profound number of metabolic disorders including insulin resistance and dysglycemia, dyslipidemia, hypertension and subclinical atherosclerosis [6]. In recent years, despite the plethora of evidence to support the association between OSA and cardiovascular diseases, the void for more knowledge on the complexity of this anatomical and yet systemic illness remain apparent.

Non-alcoholic fatty liver disease (NAFLD) has been increasingly recognized as a liver disease that develops in the absence of alcohol abuse and imposes a major health burden. It is a progressive condition, whereby sustained liver injury could lead to further hepatocytes damage, and thus includes a spectrum of the disease severity, ranging from steatosis without inflammation to non-alcoholic steatohepatitis (NASH) and ultimately liver cirrhosis [7]. The pathophysiology is complex and related to oxidative stress and endothelial inflammation with the production of many pro-inflammatory cytokines including tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), C-reactive protein (CRP) and interleukin-8 (IL-8) [8]. The interest in NAFLD has been increasing of late, with some reports on the clinical and histological features of NAFLD within the country [9,10]. Liver biopsy remains the gold standard to diagnose and categorize the severity of NAFLD. However, although it represents the best diagnostic tool for fatty liver, this procedure is invasive and could not be widely performed on apparently healthy subjects, mainly for ethical as well as practical reasons. Therefore, non-invasive tests and biomarkers of NAFLD are desirable alternatives. Liver ultrasonographic scanning has been demonstrated to have good correlations with histological findings of fatty infiltration, and has been universally accepted as a method for evaluation of severity of fatty liver [11].

The carotid artery intima-media thickness (CIMT) is a recognized tool to identify subclinical atherosclerotic disease [12]. Various cross-sectional studies have reported significantly greater carotid artery wall thickness in patients with NAFLD compared to those without NAFLD [13]. A large population-based study also suggests premature atherosclerosis in patients with NAFLD [14]. In regards to OSA, there is limited data on the measurements of CIMT to represent subclinical atherosclerosis in this group of patients.

Various surrogate markers are currently used in clinical studies to determine subclinical atherosclerosis, which include the Framingham Risk Score (accounting for age, gender, hypertension, smoking, and hyperlipidemia status), carotid artery intima-media thickness (CIMT), high sensitivity C-reactive protein (hsCRP), atheroma formation, mediastinal fat pad, endothelial dysfunction and coronary calcium scores [15]. In addition, plasma levels of intercellular adhesion molecule-1 (ICAM-1) and E-selectin have been demonstrated as molecular markers for atherosclerosis and the development of coronary heart disease [16]. More recently, another marker that has been shown to predict subclinical atherosclerosis is lipoprotein a, Lp(a), which is a low density lipoprotein (LDL)-like particle synthesized within the liver hepatocytes and then released into plasma [17]. A few studies have reported its role as an independent prospective risk factor for coronary heart disease attributed by its action on promoting atherogenesis and inhibiting fibrinolysis [18].

The relationship between OSA and NAFLD has been recognized in more recent years [19]. Benotti et al studied a group of subjects who were undergoing bariatric surgery and demonstrated an association between severity of OSA and histologically examined hepatocytic inflammation in patients with NAFLD, but was unable to reach statistical significance [20]. A few more studies have since provided more convincing evidence on the associations between the 2 conditions but these studies were limited by small sample sizes and mainly involving a specific group of patients undergoing bariatric surgery [21]. In view of the overlapping metabolic abnormalities, it may seem intuitive to assume that the co-existence of NAFLD and OSA would potentially increase cardiovascular risk further. In addition to further understanding that causal relationship between OSA and NAFLD, this study aimed to determine the downstream effect of this double inflammatory cascades on atherosclerosis risk. To the best of our knowledge, to date, there has been no study on the direct associations between CIMT and the relevant atherosclerosis markers particularly ICAM-1 and Lp(a) in patients with OSA and ultrasound- proven NAFLD.

Materials and methods

This was a cross-sectional study, conducted at the facilities of the Faculty of Medicine, Universiti Teknologi MARA, Malaysia. We screened 110 subjects consecutively, between the ages of 18 to 65, who were diagnosed with OSA as confirmed by sleep study examinations ie polysomnograpy (PSG). They were subsequently categorized by the severity of their Apnea/ Hypopnea Index (AHI) ie mild AHI 5–15, moderate AHI 15–30, and severe AHI > 30/hr based on the polysomnography report [22]. We excluded patients with seropositive Hepatitis B or Hepatitis C, and had significant alcohol intake of more than 21 units per week in men and 14 units for women. The sampling method used was convenience sampling. Patients who fulfilled the inclusion criteria were approached, explained and informed regarding the main objectives of the study and blood samples were subsequently taken, prior to the initiation of positive airway pressure, if required. Confidentiality was assured to the respondents. A written consent was signed by respondents before proceeding with the study. Patients were allowed to opt out of the study at any time should they wish to. The study was conducted in accordance to the Declaration of Helsinki. This study was approved by the Research Ethics Committee (REC) of Universiti Teknologi MARA (UiTM) REF: 600-IRMI (5/1/6).

All patients had an additional 5 mls of venous blood drawn following an overnight fast, prior to routine clinic appointments. Serum samples were separated from blood following centrifugation and stored at -20οC until further analysis. Biochemical measurements were performed on an automated analytical platform (c501, Roche Diagnostics, Germany) according to standard procedures at an ISO 15189 accredited laboratory. ICAM-1 was performed using the ELISA technique and read with microplate reader, whilst Lp(a) was read using the immunoassay analyzer. We refer to a previous study which has determined a clinically acceptable reference range for ICAM-1 was 128.9–347.48 ng/ml, thus taking values above this to be of clinical significance [23]. In regards to Lp(a), we refer to the report by Nordestgaard et al, which recommended that a value of approximately 50 mg/dL should be the desired level for reduction of cardiovascular risk [24].

Abdominal ultrasound examinations were performed by 2 independent radiologists. Subjects were scanned in the supine position by two independent radiologists using a high frequency 7.5 MHz linear array transducer using Philips iU22 imaging system. All measurements were made at the time of the scan on frozen images of longitudinal scans by using the machine’s electronic caliper. Evidence of NAFLD was confirmed with radiological technique of liver-kidney contrast on degree of steatosis and further divided into four grades, severe (NAFLD-3), moderate (NAFLD-2), mild (NAFLD-1) and normal (non-NAFLD).

For the CIMT measurements subjects were also scanned in the supine position by two independent radiologists utilizing the same equipment. The distance between the 2 lines gave a reliable index of the thickness of the carotid intimal-medial complex. All measurements were made at the time of the scan on frozen images of longitudinal scans by using the machine’s electronic caliper. Carotid segments for far (posterior) walls of each common carotid artery at a distance of 1cm from the bulb will be examined. The average of right and left CIMT were calculated and were recorded into millimeters (mm). The abnormal CIMT was defined as measurement of above 0.80 mm [25].

Statistical analysis

Statistical Package for the Social Sciences (SPSS for Windows Version 22.0, SPSS Inc., Chicago, IL, USA) was used for all statistical analysis. Data on patients’ sociodemographic information and characteristics are presented as mean and standard deviations for parametric data. For non-parametric data, the results are presented with median and interquartile range. Categorical data are presented as numbers of patients and percentages.

Comparisons between NAFLD and non-NAFLD was analyzed using Chi-Squared tests for categorical variables and independent- t test for continuous variables. Comparison for different degrees of steatosis in NAFLD, namely grades 0 (no steatosis), 1 (mild), 2 (moderate) and 3 (severe) groups were analyzed using chi-square test for categorical variables and ANOVA test for continuous variables. Correlation between 2 continuous variables was analyzed using Pearson rho test for normally distributed data and Spearman’s rho test for non-parametric data. Simple linear regression was done for variables which are significantly correlated. Significance level was set at p<0.05. Univariate analysis was performed using linear regression and analysis of variance (ANOVA) to identify potential associated factors. Correlation between two continuous variables was analyzed with Spearman’s rho test for non-normally distributed data. Cohen’s (1988) cut-off points for interpretation of correlation strength are used [26]. A p value <0.05 is considered significant.

Results

A total of 110 patients with OSA were recruited. The baseline characteristics are presented in (. The mean age of the study population was 50.11±13.91 years. The majority of them were males (65.5%) and of Malay ethnicity (78.2%). Most of the participants were non-smokers (61.8%) and obese (72.7%). The mean waist circumference was 106.17±17.53 cm and the mean for hip circumference was 110.78 ±15.01 cm. Hypertension and dyslipidemia were diagnosed in 63.6% (n = 70) and 56.4% (n = 62) of the study population, respectively. However, most of the patients had reasonably good blood pressure control with mean systolic blood pressure (SBP) of 140.72 ±17.78 mmHg and mean diastolic blood pressure (DBP) of 81.03±12.26 mmHg. Type 2 diabetes mellitus (T2DM) was present in 50 subjects (45.5%), with good glycemic control, mean HbA1c of 7.75 ±1.69%, and almost all of them (90%) were on metformin. In regards to the OSA status, almost half (45.5%) of the study population had severe AHI of more than 30. The liver enzymes were within normal range. The mean values for both intercellular adhesion molecule -1 (ICAM-1) and lipoprotein (a) [Lp(a)] were within the low risk ranges, ie 321.97±83.05 ng/ml and 74.16±54.11 nmol/L, respectively. However, high levels were detected in at least half of study population, with ICAM-1 level of ≥347.48 ng/ml observed in 63.6% and high level of Lp(a) of ≥75 nmol/L seen in 50.9%. Finally, mean CIMT in this study population was 0.80±0.20 mm. Refer .

The prevalence of NAFLD within the study population was notably high at 81.8% (95% CI: 74.5, 89.1) (n = 90). Mean weight in the NAFLD group was significantly higher compared to the non-NAFLD group (94.77±21.85 kg vs 74.67±16.80 kg, p < 0.001), with 82.2% of the NAFLD group being obese (p<0.001). The NAFLD group also had significantly higher mean SBP and liver transaminases. Both the atherosclerosis markers ie mean ICAM-1 and Lp(a) were significantly higher in the NAFLD group compared to the non-NAFLD group (334.53±72.86 vs 265.46 ±102.92 ng/mL, p = 0.001, 85.41±52.56 vs 23.55±23.66 nmol/L, p <0.001, respectively). Subsequent categorical analyses showed higher percentage of patients with high ICAM level of >347.48 ng/ml and Lp(a) >75 nmol/L within the NAFLD group compared to the non-NAFLD group, (56.7% vs 5%, p <0.001 and 58.9% vs 1.9%, p <0.001, respectively). Mean CIMT was higher in the NAFLD group compared to the non-NAFLD group (0.80±0.30 vs 0.60±0.10 mm, p = 0.001). The clinically significant abnormal CIMT of >0.8 mm was detected in 64.4% of patients with NAFLD compared to only 5% within the non-NAFLD group, p <0.002. Refer

We further categorized the NAFLD group according to the severity of the steatosis, with 52% within grade 1 steatosis (n = 47), 46% within grade 2 steatosis (n = 42) and 2% had grade 3 steatosis (n = 1). Grades 2 and 3 were subsequently combined for analyses. There were significant differences in SBP, DBP and liver transaminases between non-NAFLD, NAFLD 1 and NAFLD 2+3 groups. CIMT measurements were significantly highest in the NAFLD 2+3 group, followed by NAFLD 1, compared to the non-NAFLD groups (0.90±0.20, 0.70±0.30, 0.60±010 mm, respectively, p<0.001). Levels of ICAM-1 and Lp(a) were also seen to be significantly highest in the NAFLD 2+3 group, followed by the NAFLD 1 group as compared to the non-NAFLD. Refer

From the overall study population, 24.5% (n = 27) had mild AHI, 30% (n = 33) had moderate AHI and 45.5% (n = 50) had severe AHI. It was interesting to note that there was a significantly higher proportion of patients with NAFLD 2+ 3 within the severe AHI group compared to the mild AHI group (61.9% vs 14.3%, p<0.001). There was indeed a significant correlation between the severity of OSA and NAFLD; r = 0.384, p<0.001. Refer

Logistic regression analysis was performed to ascertain the effects of blood pressure, BMI, glycemia, severity of OSA, waist circumference, hip circumference, ICAM-1 and Lp(a) on the likelihood of OSA patients having NAFLD. Patients with OSA and elevated AHI of >30 were 52.77 times more likely to have NAFLD compared to those with mild AHI (5–15) (p < 0.001). Patients with OSA were also more likely to have NAFLD with co-existing hypertension (OR 4.33, p = 0.005; diabetes mellitus (OR 3.0, p = 0.049; and dyslipidemia (OR 2.92, p = 0.038). In addition, elevated surrogate markers of ICAM-1 and Lp(a) level were more likely to be present in patients with NAFLD with OR 14.53 and 27.22, respectively (p = 0.011 and p = 0.001). Refer .

Multiple logistic regression analysis was performed to adjust for the predictors for NAFLD in OSA patients. The interaction and multicollinearity were also checked within the overall study cohort. All the significant factors in Table 5 were included in this analysis. Notably, the small number in the non-NAFLD group limit the statistical power. Nevertheless, two factors were subsequently identified to be significant predictors for the detection of NAFLD in OSA patients, which were ICAM-1 level (p = 0.002) and Lp(a) (p = 0.003). Elevated ICAM-1 level of ≥347ng/ml demonstrated an OR of 41.69; CI, 3.76–461.97; p = 0.002, whilst high Lp(a) level of ≥75 nmol/L showed an OR of 35.12; CI, 3.47–355.40; p = 0.003) for NAFLD. The Receiver Operative Characteristic (ROC) curve of these factors associated with NAFLD showed an area under the curve of 95.6% (95%CI: 91.9, 99.3). It could, therefore, be inferred that OSA patients who had elevated levels of ICAM-1 and Lp(a) are highly likely to have NAFLD (

Table 5

Analysis of associated factors for NAFLD in OSA patients.

	OR (95%CI)	p-value
Age	1.03 (0.99, 1.07)	0.071
Gender:
Male	1.72 (0.64, 4.81)	0.280
Female	1	Ref
Race:
Malay	1.29 (0.32, 5.16)	0.723
Chinese	0.50 (0.08, 3.27)	0.469
Indian & Others	1	Ref
Hypertension:
Yes	4.33 (1.56, 12.06)	0.005
No	1	Ref
DM:
Yes	3.00 (1.01, 8.95)	0.049
No	1	Ref
Dyslipidemia:
Yes	2.92 (1.06, 8.03)	0.038
No	1	Ref
Ischemic Heart Disease:
Yes	1.62 (0.43, 6.09)	0.476
No	1	Ref
IHD family:
Yes	2.29 (0.77, 6.86)	0.137
No	1	Ref
Smoking:
Never	1	Ref
Current	0.92 (0.26, 3.24)	0.896
Former	6.79 (0.85, 54.42)	0.071
BMI:
Obese	17.27 (4.18, 71.25)	<0.001
Overweight	2.20 (0.50, 9.75)	0.229
Normal	1	ref
Severity of OSA:
Mild	2	Ref
Moderate	6.03 (1.79, 20.32)	0.004
Severe	52.77 (6.34, 439.14)	<0.001
Waist	1.08 (1.04, 1.12)	<0.001
Hip	1.07 (1.03, 1.12)	0.001
SBP	1.05 (1.02, 1.09)	0.003
DBP	1.04 (0.99, 1.09)	0.060
ICAM-1
≥ 347 ng/ml	14.53 (1.86, 113.28)	0.011
< 347 ng/ml	1	ref
Lp(a)
≥ 75 nmol/L	27.22 (3.49, 212.31)	0.002
< 75 nmol/L	1	ref
HbA1c (n = 50)	1.21 (0.95, 5.15)	0.067

ROC curve of the two significant factors associated with having NAFLD.

The area under the curve is 95.6% (95%CI: 91.9, 99.3).

Discussion

The prevalence of NAFLD in this group of OSA patients was notably high (81.8%, n = 90/110). We observed that this number is similar to a study by Turkay et al who reported a prevalence of 71.2% [27]. The most probable reason for this similarity is the high number of obese subjects within both study populations. Over the last decade, there has been increasing interest in understanding the associations between OSA and NAFLD. A recent review on OSA and NAFLD summarized that OSA is indeed associated with increased liver fibrosis and raises a question on the need for screening of these conditions, followed by aggressive therapy to reduce future cardiovascular risks [21]. The association between the high prevalence of NAFLD in OSA patients has been suggested to be attributed to the pathogenesis of fatty liver disease caused by continuous intermittent hypoxia (CIH) that lead to sympathetic activation, endothelial dysfunction, oxidative stress and systemic inflammation [28]. However, data from a cross-sectional study by Norman et al have further recommended that CIH is in fact an independent causal factor for the development of NAFLD [29]. We therefore concur that perhaps there is a direct association between AHI and NAFLD, suggesting some effect of the severity of apnea on hepatic steatosis, albeit not as a significant independent predictor for the latter based on the multivariate analysis.

An important aspect of this study is the evidence of subclinical atherosclerosis in this particular cohort of patients with OSA. A study by Targher et al had shown a markedly greater CIMT than control subjects (1.14 ± 0.20 vs. 0.82 ± 0.12 mm; P < 0.001) [30]. Another study by Silvestrini et al similarly showed that CIMT in OSA patients was significantly higher than that of control subjects (1.429 ± 0.34 vs 0.976 ± 0.17 mm, p <0.0001) [31]. However, there is paucity of data on the cumulative atherosclerotic risk of patients with co-existing OSA and NAFLD. Petta et al, showed that in a group of patients with NAFLD who subsequently underwent cardiorespiratory polygraphy to detect OSA, the prevalence of significant thickening of CIMT was higher in patients at high risk compared to those at low risk for OSA (51.2% vs 24.3%, p = 0.006) [32]. However, the study only employed the use of the STOP-BANG questionnaire to identify risk for OSA whilst our study confirmed its diagnosis. Moreover, we demonstrated that the mean CIMT values were highest in those within the severe NAFLD grades 2 and 3 compared to NAFLD 1 and non-NAFLD. There were statistically significant positive correlations between CIMT and SBP (r = 0.233, p = 0.014) among OSA patients as well as in NAFLD (r = 0.217, p = 0.040). This suggests the importance of good SBP control in patients with OSA and NAFLD.

ICAM-1 is proven to be a molecular marker for atherosclerosis and the development of coronary heart disease [33]. Our data is in agreement with previous studies including a meta-analysis which included 51 studies and concluded that ICAM-1 was indeed higher in patients with OSA compared to control group [34]. In this present study we demonstrated that more than half of the study population (63.6%) had high ICAM-1 level (> 347.48 ng/ml). Deneva-Koyvheva et al recommended the clinically acceptable reference interval for ICAM-1 was 128.9–347.48 ng/ml [22]. The mean ICAM-1 level was significantly higher within the NAFLD compared to the non-NAFLD group, which was consistent with previous studies [35,36]. Earlier studies reported a relationship between ICAM-1 and CIMT as subclinical atherosclerosis risk, which concluded that ICAM-1is independent of other known CHD risk factors and is strongly associated with carotid plaques [37-39]. Consequently, we also demonstrated that higher levels of ICAM-1 corresponded to worsening severity of steatosis, which underscores the escalating cardiovascular risk in these patients at the end of the spectrum of the disease.

Lipoproprotien (a) [Lp(a)] is a low density lipoprotein (LDL)-like particle and has been recognized as another inflammatory marker predicting subclinical atherosclerosis [23]. More recently, the Dallas Heart Study showed Lp(a) as a positive predictor for major cardiovascular event (MACE) with hazard ratio of 2.35 [40]. Interestingly our study demonstrated that half of the patients with OSA (50.9%) had significantly high levels of Lp(a) of above 75 nmol/L, of which this Lp(a) cut-off level is regarded as having high atherosclerosis risk based on the evaluation of the Framingham data [23]. High serum Lp(a) levels correlate with premature atherosclerosis and stroke with an an approximate doubling of coronary risk when the Lp(a) level rises above 75 nmol/L, with the risk further escalating to approximately 6‑fold in the addition of a high LDL‑cholesterol [41,42]. To the best of our knowledge, there has been no study to determine the association between the Lp(a) with the severity of OSA as well as the NAFLD populations. There were significant differences in Lp(a), being highest in NAFLD grade 2 group as compared to NAFLD-1 and non-NAFLD groups (92.21 ± 48.80 vs 79.30 ± 56.07 vs 23.55 ± 23.70 nmol/L, p < 0.001). Thus, our data further supports the recommendation by the European Atherosclerosis Society that Lp(a) measurement should be recommended in patients with high cardiovascular risk including family history of premature cardiovascular disease [42] and subsequently those with OSA and NAFLD.

The multivariate analyses demonstrated a few novel points worth noting. Patients with severe OSA as defined by elevated AHI of >30 were 52 times more likely to have NAFLD compared to those with mild AHI, which were further worsened by other comorbidities including hypertension, diabetes mellitus and dyslipidemia. In addition, elevated inflammatory markers of ICAM-1 and Lp(a) are demonstrated to be significant predictors for the detection of NAFLD in OSA patients. These findings point to the understanding that patients with OSA and co-existing NAFLD are at an alarmingly high risk of cardiovascular events or disease. It also suggests that, in addition to other conventional risk factors assessment, these patients should have further tests to stratify their cardiovascular risks among which is the use of these 2 surrogate markers, ICAM-1 and Lp(a).

To date, there is scarcity in the number of studies evaluating the relationship between the OSA severity via AHI with NAFLD. Nonetheless, this study reported a similar finding to Turkay et al, who demonstrated that as NAFLD severity increased from mild to severe form, mean AHI and oxygen desaturation index values were also significantly increased [26]. Conversely, our study showed that increasing severity of OSA, based on AHI values, correlated with severity of NAFLD based on the grades 0–3, r = 0.453, p<0.001. The causal or effect relationship remains vague but suffice to note the synergistic consequence of the co-existing insults.

Comparisons between NAFLD and non-NAFLD groups demonstrated statistically significant higher weight, BMI, systolic blood pressure, waist circumference, hip circumference and waist to hip ratio within the former group. Obesity is a co-existential factor between the 2 conditions and plays an important causative factor for both. Ong et al observed that the prevalence of NAFLD could be as high as 93% among morbidly obese patients and 9% of them had advanced fibrosis (i.e., bridging fibrosis or cirrhosis [43]. Interestingly, our study demonstrated similar finding as 92.5% of our NAFLD and OSA patients were obese. Other co-morbidities including dyslipidemia, hypertension and diabetes mellitus are also well recognized associated factors for NAFLD. However, we emphasize the importance of these co-morbidities on the likelihood of developing NAFLD. OSA patients with high SBP, expanded waist and hip circumferences were more likely to have NAFLD compared to leaner patients. This could add a very important impact on screening for NAFLD in a subgroup of patients who have been diagnosed with severe OSA, and found to be obese, with uncontrolled blood pressure, to prevent major adverse cardiac events and death.

Our data seems to be quite consistent with similar studies within the Asian region, whereby, the prevalence of ultrasound-diagnosed NAFLD among OSA patients has been reported to range between 60% and 90% [44-46]. However, we acknowledge that this is probably an overestimation in comparison to a histopathological diagnosis of NAFLD, which could be much lower in patients with histology-proven NAFLD [47]. Nonetheless, the risk remains high as demonstrated by a cross-sectional study by Musso et al (n = 2,183) which showed that patients with OSA had 2.01 times (95% CI: 1.36–2.97) higher risk to have NAFLD confirmed by histopathological evidence [48]. Other imaging modalities have emerged as diagnostic tools for NAFLD in recent times. From a meta-analysis involving 46 studies, which compared the sensitivity and specificity of various imaging modalities including ultrasonography, computed tomography (CT) scan, magnetic resonance imaging (MRI) and proton magnetic resonance spectroscopy (1H-MRS), the authors concluded that the latter 2 newer techniques were acceptable in the evaluation of hepatic steatosis [49]. In due course, MRI has become increasingly popular in establishing the diagnosis of NAFLD mainly attributed to the availability of quantification of hepatic steatosis by measuring proton density fat fraction (PDFF) [50]. Its use, however, is limited by cost and accessibility. Therefore, liver ultrasonography scanning remains the radiological imaging of choice by virtue of its non-invasiveness and convenience. Furthermore, it has been shown to have a good correlation with histological findings of fatty infiltration, and has been accepted worldwide as a method of evaluation for the different degrees of fatty liver [7,51], with an accuracy of 88% in direct comparison with histopathological findings [52].

We acknowledge the limitations of this study such as the single center data collection, the cross-sectional design and the relatively small number of subjects. Furthermore, a comparison with a group of healthy subjects without OSA would have been ideal. The number of subjects with high degree of steatosis ie grade 3 was very small and made sub-group analysis difficult. The use of fibroscan to assess the degree of steatosis would also be interesting to be incorporated as a research tool. The exclusion of viral or autoimmune hepatitis were obtained based on medical records and patients’ disclosures only and not from actual serological testing. Future studies to address these limitations would help strengthen the findings from this study.

Conclusion

In conclusion, this study revealed a high prevalence of NAFLD within a group of OSA patients. We were able to demonstrate that severity of OSA, categorized by the AHI, was significantly associated with severity of NAFLD. Subclinical atherosclerosis, represented by abnormal levels of CIMT, ICAM-1 and Lp(a), were significantly predominant among NAFLD subjects. ICAM-1 and Lp(a) were strong predictors for detection of NAFLD in the study cohort. This study highlighted the elevated cardiovascular risk of patients with severe OSA and NAFLD, the importance of early screening and detection of this chronic inflammatory condition. It also stresses the importance of optimizing cardiovascular risk factors in this group of high-risk patients as part of a preventive management strategy to avoid the consequential debilitating and fatal cardiovascular events.

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8 Apr 2021

PONE-D-21-03573

Significantly Higher Atherosclerosis Risks in Patients with Obstructive Sleep Apnoea and Non Alcoholic Fatty Liver Disease

PLOS ONE

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We will update your Data Availability statement on your behalf to reflect the information you provide.

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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: Partly

Reviewer #2: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: No

**********

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: No

Reviewer #2: No

**********

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: 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: Degree of steatosis is not the same as stage/grade of NAFLD. Grade is determined by degree of inflammation (NAS score) and stage by fibrosis on biopsy (gold standard). If biopsy is not available severity can be assessed by specialized non invasive markers and scores like Fibroscan, NALFD fibrosis score etc. Hence in this paper the term grade/stage of NAFLD should be replaced by degree of steatosis.

It would be useful if the authors had any such surrogate markers of NAFLD severity and study the relationship of NAFLD severity with atherosclerosis markers.

What is the significance of table 5? It would seem that the authors are saying that in patients who do not have NAFLD, CIMT is not related to any metabolic or cardiovascular factor which does not seem to be true clinically. I think there are too few patients in Non NAFLD group to such correlation analysis in this subgroup.

Table 6 and table 7 are almost duplicate. One of them can be removed.

How many parameters were entered into the multivariate analysis model? With only 20 patients without NAFLD in the study, doing a multivariate analysis with many dependent variables will severely limit the power of the calculations.

Which parameters were studied in the multivariate analysis? It is surprising to note that BMI/obesity did not come out as independent predictors of NAFLD in this study. were these parameters included in the multivariate analysis?

"Inversely it could, therefore, be inferred that these OSA patients who had NAFLD had a 95.6% possibility of having elevated atherosclerosis risk as suggested by elevated levels of ICAM-1 and Lp(a)." I dont think this is a correct conclusion. AUROC of 95.6% for ICAM and LPa simply suggests that a predictive score using these 2 parameters together has 95.6% accuracy in predicting presence of NAFLD. To assess the probability of atherosclerotic risk in NAFLD one would have to calculate how many of NAFLD patients had elevated levels of one or more markers of atherosclerosis using acceptable cut-offs for each such marker and then calculate the atherosclerotic risk associated with each of those markers.

"However, there is paucity of data on the cumulative atherosclerotic risk of patients with co-existing OSA21

and NAFLD; and to the best of our knowledge this would be the first study utilizing CIMT and the inflammatory markers to demonstrate this." This is a misleading conclusion. Please see the point above.

"We concur with the findings by Petta et al, which showed that OSA was independently associated with subclinical carotid atherosclerosis as evaluated by CIMT in patients with NAFLD". The authors have not presented any such data showing association of CIMT with OSA, since there was no non OSA control group in this study to compare CIMT values with.

"Furthermore, our data demonstrated a significant rise in ICAM-1 levels with severity of OSA as categorized by

increasing AHI, from mild 259.16 ± 57.40 ng/mL, moderate 299.65 ± 90.41 ng/mL and severe 370.63 ± 57.71 ng/mL, p < 0.001". This data has not been presented anywhere in the results section.

The discussion is too long with many repetitions and can be condensed.

Reviewer #2: This was an interesting study that examined potential links between OSA, liver disease and CV risks. The authors studied 110 patients with suspected OSA. Authors found that OSA severity was significantly associated with liver disease. CV risk (CIMT) was elevated in liver patients. I had the following questions/comments:

1. In the discussion, other limitations that should be mentioned include: relatively small numbers, single centre, cross sectional nature

2. I wasnt sure if these were consecutive patients? if not, how were they selected?

3. When was the blood taken in relation to PSG (i.e. time frame)?

4. I assume none of these patients were treated?

5. Was there an analysis of desaturation indices (e.g. 4% ODI, mean saturation, % time below 90% etc). I would suspect these might be highly related to liver disease?

6. I wasnt sure why ICAM specifically was the inflammatory marker selected?

7. Mean BMI, AHI, saturation, etc should be added to Table 1. Did any patients have obesity hypoventialtion syndrome? This might have increased right sided pressures and lead to more liver swelling?

8. There should be more description of the ultrasound methods to detect liver disease. How validated are they in the context of substantial obesity +/- high right sided pressures?

9. In the last paragraph of page 8, do you mean carotid ultrasound or abdominal?

10. Was there an analysis of BMI, AHI as continuous variables? It was difficult to understand if Tables 7 and 8 reflected multivariate models? This should be made clearer.

11. The CI around the OR for severe osa is very wide-- these should be added to the abstract so readers understand the instability of these measures.

12. Was there a multivariate analysis looking at determinants of CIMT? I think you need this to determine whether liver disease is an independent risk factor.

**********

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: Yes: Swastik Agrawal

Reviewer #2: 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.

16 Apr 2021

We would like to thank both Reviewers for their constructive comments and suggestions. We hereby provide the responses to the questions and comments.

Reviewer #1:

Degree of steatosis is not the same as stage/grade of NAFLD. Grade is determined by degree of inflammation (NAS score) and stage by fibrosis on biopsy (gold standard). If biopsy is not available severity can be assessed by specialized non invasive markers and scores like Fibroscan, NALFD fibrosis score etc. Hence in this paper the term grade/stage of NAFLD should be replaced by degree of steatosis.

Thank you for the clarification. This has been amended accordingly throughout the manuscript. Stages of NAFLD has been replaced as grades of steatosis Grade 0-4.

It would be useful if the authors had any such surrogate markers of NAFLD severity and study the relationship of NAFLD severity with atherosclerosis markers.

Thank you for the suggestion. Unfortunately we do not have access to Fibroscan. This is a very good suggestion for us to continue with the study at a later opportunity. We have included this in the limitation as well.

What is the significance of table 5? It would seem that the authors are saying that in patients who do not have NAFLD, CIMT is not related to any metabolic or cardiovascular factor which does not seem to be true clinically. I think there are too few patients in Non NAFLD group to such correlation analysis in this subgroup.

The reviewer has highlighted a valid point. Table 5 has been removed as suggested. Table 6 has been adjusted to Table 5 accordingly.

Table 6 and table 7 are almost duplicate. One of them can be removed.

Thank you for highlighting that. We have removed Table 7. Table 8 has been adjusted to table 6 accordingly.

How many parameters were entered into the multivariate analysis model? With only 20 patients without NAFLD in the study, doing a multivariate analysis with many dependent variables will severely limit the power of the calculations.

Thank you for the comments. 10 parameters were included in the multivariate analysis as described in table 7 previously. We have acknowledged the small number of n=20 in the Non-NAFLD group in the result section.

This sentence has been added : “All the variables in Table 5 were included in this analysis. Notably, the small number in the non-NAFLD group limit the statistical power. Nevertheless, two factors….”

Which parameters were studied in the multivariate analysis? It is surprising to note that BMI/obesity did not come out as independent predictors of NAFLD in this study. were these parameters included in the multivariate analysis?

Only the significant parameters were included in the multivariate analysis ie SBP, DBP, BMI, WC, ALP, ALT, AST, GGT, ICAM-1, Lp(a).

Yes, BMI was included in the analysis, however it was not significant.

"Inversely it could, therefore, be inferred that these OSA patients who had NAFLD had a 95.6% possibility of having elevated atherosclerosis risk as suggested by elevated levels of ICAM-1 and Lp(a)."

I don’t think this is a correct conclusion. AUROC of 95.6% for ICAM and LPa simply suggests that a predictive score using these 2 parameters together has 95.6% accuracy in predicting presence of NAFLD. To assess the probability of atherosclerotic risk in NAFLD one would have to calculate how many of NAFLD patients had elevated levels of one or more markers of atherosclerosis using acceptable cut-offs for each such marker and then calculate the atherosclerotic risk associated with each of those markers.

Thank you for the comment. The statement has been amended to include your comment.

"It could, therefore, be inferred that OSA patients who had elevated levels of ICAM-1 and Lp(a) are highly likely to have NAFLD.”

"However, there is paucity of data on the cumulative atherosclerotic risk of patients with co-existing OSA

and NAFLD; and to the best of our knowledge this would be the first study utilizing CIMT and the inflammatory markers to demonstrate this."

This is a misleading conclusion. Please see the point above.

Thank you for the comment. We have removed the statement of “……and to the best of our knowledge this would be the first study utilizing CIMT and the inflammatory markers to demonstrate this."

"We concur with the findings by Petta et al, which showed that OSA was independently associated with subclinical carotid atherosclerosis as evaluated by CIMT in patients with NAFLD".

The authors have not presented any such data showing association of CIMT with OSA, since there was no non OSA control group in this study to compare CIMT values with.

Thank you for the comment. We have revised the statement to state the following:

Petta et al, showed that in a group of patients with NAFLD who subsequently underwent cardiorespiratory polygraphy to detect OSA, the prevalence of significant thickening of CIMT was higher in patients at high risk compared to those at low risk for OSA (51.2% vs 24.3%, p = 0.006).

"Furthermore, our data demonstrated a significant rise in ICAM-1 levels with severity of OSA as categorized by

increasing AHI, from mild 259.16 ± 57.40 ng/mL, moderate 299.65 ± 90.41 ng/mL and severe 370.63 ± 57.71 ng/mL, p < 0.001".

This data has not been presented anywhere in the results section.

Thank you for the comment. You are absolutely right, we have not presented this data in the result section. We have removed this statement in the discussion section.

Reference no 35 has been removed. Subsequent references have been adjusted accordingly.

The discussion is too long with many repetitions and can be condensed.

Thank you for the comment. The discussion has been refined to avoid repetitions without losing the relevant findings of the study.

Reviewer #2: This was an interesting study that examined potential links between OSA, liver disease and CV risks. The authors studied 110 patients with suspected OSA. Authors found that OSA severity was significantly associated with liver disease. CV risk (CIMT) was elevated in liver patients. I had the following questions/comments:

1. In the discussion, other limitations that should be mentioned include: relatively small numbers, single centre, cross sectional nature

Thank you for the comment. This has been added in the limitation. . “The single center data collection, the cross-sectional design and the relatively small number of subjects are areas of improvement for future studies.”

2. I wasn’t sure if these were consecutive patients? if not, how were they selected?

Thank you for the question. Patients were recruited based on convenient sampling. This has been added in the method section.” We screened 110 subjects consecutively, between the ages of 18 to 65, who were diagnosed with OSA….

3. When was the blood taken in relation to PSG (i.e. time frame)?

Thank you for the question. PSG was done at a much earlier date just to establish the diagnosis of OSA.

4. I assume none of these patients were treated?

Thank you for the question. Upon recruitment, the patients have not initiated treatment yet.

5. Was there an analysis of desaturation indices (e.g. 4% ODI, mean saturation, % time below 90% etc). I would suspect these might be highly related to liver disease?

Thank you for the question. We understand the relevance. Unfortunately, these were not available in our analysis.

6. I wasn’t sure why ICAM specifically was the inflammatory marker selected?

Thank you for the question. We acknowledge the many surrogate markers for cardiovascular disease including ICAM-1, which is an intercellular adhesion molecule that are expressed on the surface of vascular endothelial cells. It is a valid and recognised research tool, serving as one of the many molecular markers for atherosclerosis and the development of coronary heart disease.

7. Mean BMI, AHI, saturation, etc should be added to Table 1. Did any patients have obesity hypoventilation syndrome? This might have increased right sided pressures and lead to more liver swelling?

Thank you for the suggestion. Mean BMI and AHI has been added in Table 1. But we were unable to include the saturation indices. The study also did not look into obesity hypoventilation syndrome and its associated complications. This would however be an interesting variable for future studies in the area.

8. There should be more description of the ultrasound methods to detect liver disease. How validated are they in the context of substantial obesity +/- high right sided pressures?

Thank you for the suggestion. We have added the this statement. “Evidence of NAFLD was confirmed with radiological technique of liver-kidney contrast on degree of stetatosis and further divided into four grades, severe (NAFLD-3), moderate (NAFLD-2), mild (NAFLD-1) and normal (non-NAFLD)…..” on Page 9. The examinations were performed by 2 independent, trained radiologists based according to universally accepted criteria (Joy, D., Thava, V. R., & Scott, B. B. (2003). Diagnosis of fatty liver disease: is biopsy necessary? Eur J Gastroenterol Hepatol, 15(5), 539-543. doi:10.1097/01.meg.0000059112.41030.2e)

9. In the last paragraph of page 8, do you mean carotid ultrasound or abdominal?

Thank you for highlighting this. We have added – “For the CIMT measurements subjects were also scanned in the supine position by two independent radiologists utilizing the same equipment. The distance between the 2 lines gave a reliable index of the thickness of the carotid intimal-medial complex. All measurements were made at the time of the scan on frozen images of longitudinal scans by using the machine’s electronic caliper. Carotid segments for far (posterior) walls of each common carotid artery at a distance of 1cm from the bulb will be examined. The average of right and left CIMT were calculated and were recorded into millimeters (mm)….”

10. Was there an analysis of BMI, AHI as continuous variables? It was difficult to understand if Tables 7 and 8 reflected multivariate models? This should be made clearer.

Thank you for the comment. There were analysis of BMI and AHI as continuous variables and we have added the mean±SD in Table 1. We have removed Tables 5 and 7 as suggested by Reviewer 1. Table 8 has been adjusted to Table 6. It is a multivariate analysis.

11. The CI around the OR for severe OSA is very wide-- these should be added to the abstract so readers understand the instability of these measures.

Thank you for the suggestion. This has been added in the abstract.

12. Was there a multivariate analysis looking at determinants of CIMT? I think you need this to determine whether liver disease is an independent risk factor.

Thank you for the suggestion. This was not assessed in this paper but worthwhile addressing in future studies.

Thank you again for your suggestions. We look forward to your positive review.

Submitted filename: Response Letter for Reviewers Final.docx

Click here for additional data file.

3 May 2021

PONE-D-21-03573R1

Significantly Higher Atherosclerosis Risks in Patients with Obstructive Sleep Apnoea and Non Alcoholic Fatty Liver Disease

PLOS ONE

Dear Dr. Abdul Ghani,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please note that Reviewer #2 has two additional comments to be addressed. I concur with both.

Please submit your revised manuscript by Jun 17 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

James Andrew Rowley

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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

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

Reviewer #2: (No Response)

**********

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

Reviewer #2: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

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

Reviewer #2: No

**********

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

Reviewer #2: No

**********

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: (No Response)

Reviewer #2: The authors have addressed the majority of my comments and the paper improved. I just had afew more:

1. I wasnt sure why desaturation parameters were not available? Isnt this part of the PSG report (event indices such as mean saturation, % below 90% would have been useful).

2. It seems like the blood samples were taken after PSG. To clarify, were any of the patients on treatment at the time of the blood samples (it was unclear to me when recruitment was)?

**********

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: Yes: Swastik Agrawal

Reviewer #2: 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.

4 May 2021

Dear Sir,

Thank you again for your kind responses and effort towards this manuscript. I hereby provide the details of the amendments as required by the Editors and Reviewer 2.

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

Reviewer #2: No

Author response: The raw data has been uploaded as supporting document.

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

Reviewer #2: No

Author response: The manuscript has been checked, corrected and revised thoroughly.

Reviewer 2:

The authors have addressed the majority of my comments and the paper improved. I just had a few more:

1. I wasn’t sure why desaturation parameters were not available? Isn’t this part of the PSG report (event indices such as mean saturation, % below 90% would have been useful).

Thank you for the question. I apologize for the ambiguous response previously.

The data is available. However, in relation to the current data presented in this manuscript, the PSG served only as a diagnostic tool to identify patients with OSA and subsequent categorization on severity based on the AHI. Further discussion on OSA would make the manuscript too lengthy and subsequently exceed the stipulated maximum word count.

However, we do acknowledge the relevance of the event indices that you have mentioned above and we enthuse over a future report to determine the associations between those indices and the studied atherosclerosis risks.

2. It seems like the blood samples were taken after PSG. To clarify, were any of the patients on treatment at the time of the blood samples (it was unclear to me when recruitment was)?

Thank you for the question. All patients were recruited prior to initiation of treatment (if deemed necessary by the treating physician).

This statement has been added to the methodology section. “Patients who fulfilled the inclusion criteria were approached, explained and informed regarding the main objectives of the study prior to the initiation of mechanical ventilation, if required.”

Thank you again for your suggestions. We look forward to your positive review.

Submitted filename: Response Letter for Reviewers 2.docx

Click here for additional data file.

13 May 2021

PONE-D-21-03573R2

Significantly Higher Atherosclerosis Risks in Patients with Obstructive Sleep Apnoea and Non Alcoholic Fatty Liver Disease

PLOS ONE

Dear Dr. Abdul Ghani,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please note that while Reviewer #2 is most satisfied with your response, they still have two points to clarify.  Please do so in your response and in the manuscript.

Please submit your revised manuscript by Jun 27 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

James Andrew Rowley

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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

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 #2: (No Response)

**********

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 #2: Yes

**********

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

Reviewer #2: Yes

**********

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Reviewer #2: (No Response)

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Reviewer #2: Yes

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Reviewer #2: Thank you for the responses- a minor point. "Patients who fulfilled

the inclusion criteria were approached, explained and informed regarding the main objectives

of the study prior to the initiation of mechanical ventilation, if required." Can you change mechanical ventilation to "positive airway pressure". Can you also explicitly state that samples were taken prior to initiation of therapy (this only says they were recruited prior to treatment).

**********

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Reviewer #2: No

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17 May 2021

Dear Sir,

Thank you again for your kind responses and effort towards this manuscript. I hereby provide the details of the amendments as required by Reviewer 2.

Reviewer #2: Thank you for the responses- a minor point. "Patients who fulfilled

the inclusion criteria were approached, explained and informed regarding the main objectives

of the study prior to the initiation of mechanical ventilation, if required." Can you change mechanical ventilation to "positive airway pressure". Can you also explicitly state that samples were taken prior to initiation of therapy (this only says they were recruited prior to treatment).

This sentence has been further amended to: “Patients who fulfilled the inclusion criteria were approached, explained and informed regarding the main objectives of the study and blood samples were subsequently taken, prior to the initiation of positive airway pressure, if required.”

Thank you again for your suggestions. We look forward to your positive review.

Submitted filename: Response Letter for Reviewers 3.docx

Click here for additional data file.

2 Jun 2021

Significantly Higher Atherosclerosis Risks in Patients with Obstructive Sleep Apnea and Non Alcoholic Fatty Liver Disease

PONE-D-21-03573R3

Dear Dr. Abdul Ghani,

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Additional Editor Comments (optional):

Reviewers' comments:

22 Jun 2021

PONE-D-21-03573R3

Significantly Higher Atherosclerosis Risks in Patients with Obstructive Sleep Apnea and Non-Alcoholic Fatty Liver Disease

Dear Dr. Abdul Ghani:

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.

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on behalf of

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PLOS ONE

Table 1

Sociodemographic and baseline characteristics of overall study population.

Variables	N (%)	Mean±SD
Age		50.11±13.91
Gender
Male	72 (65.5%)
Female	38 (34.5%)
Ethnicity
Malay	86 (78.2%)
Chinese	9 (8.2%)
Indian & *Others	15 (13.6%)
Comorbidities
Hypertension:	70 (63.6%)
Diabetes mellitus	50 (45.5%)
Dyslipidemia	62 (56.4%)
Ischemic Heart Disease	7 (6.4%)
**Others	7 (6.4%)
Smoking Status
Never	68 (61.8%)
Current	17 (15.5%)
Former	25 (22.7%)
Weight mean±SD, kg		91.12±22.36
Height mean±SD, cm		162.76±10.40
BMI mean±SD, kg/m2		34.67±9.44
BMI
Obese (≥27.5)	80 (72.7%)
Overweight (23–27.4)	18 (16.4%)
Normal (≤22.9)	12 (10.9%)
Waist Circumference mean±SD cm		106.17±17.53
Hip Circumference mean±SD, cm		110.78±15.01
Waist hip ratio		0.95±0.08
Systolic blood pressure, mean±SD, mmHg		140.72±17.78
Diastolic blood pressure mean±SD, mmHg		81.03±12.26
Mean Apnea Hypopnea Index (AHI)
Severity of Obstructive Sleep Apnea (AHI),
Mild (5–15)
Moderate (16–30)	27 (24.5%)
Severe (>30)	33 (30.0%)
	50 (45.5%)
NAFLD status
Yes	90 (81.8%)
No	20 (18.2%)
Degree of steatosis
Normal	20 (18.2%)
Grade 1	47 (42.7%)
Grade 2	42 (38.2%)
Grade 3	1 (0.9%)
Carotid Intima Media thickness mean (SD) mm		0.80±0.20
≥ 0.80 mm	54 (49.1%)
< 0.80 mm	56 (50.9%)
ALT mean±SD, U/L		32.14±23.67
ALP mean±SD, U/L		78.71±23.31
AST mean±SD, U/L		21.87±16.16
GGT mean±SD, U/L		53.39±57.02
Intracellular Adhesion Molecule-1, mean±SD, ng/ml		321.97±83.05
≥ 347.48 ng/ml	70 (63.6%)
< 347.48 ng.ml	40 (36.4%)
Lipoprotein (a), mean±SD, nmol/L		74.16±54.11
≥ 75 nmol/L	56 (50.9%)
< 75 nmol/L	54 (49.1%)
HbA1c (n = 50), mean±SD, %		7.75±1.69

Table 2

Comparison between non-NAFLD and NAFLD group.

Variables	NAFLD (N = 90)	Non-NAFLD (N = 20)	p-value
Systolic blood pressure mean±SD, mmHg	143.23±16.33	129.35±19.96	0.001
Diastolic blood pressure mean±SD, mmHg	82.08±11.95	76.30±12.81	0.056
ALT mean±SD, U/L	34.93±25.05	19.57±8.47	0.008
ALP mean±SD, U/L	79.68±23.74	74.35±21.30	0.358
AST mean±SD, U/L	34.27±16.72	21.05±6.11	0.001
GGT mean±SD, U/L	59.10±61.26	27.70±15.12	0.025
Severity of OSA (AHI), n (%)
Mild (5–15)	13 (14.4%)	14 (70.0%)	<0.001
Moderate (16–30)	28 (31.1%)	5 (25.0%)
Severe (>30)	49 (54.4%)	1 (5.0%)
Intracellular Adhesion Molecule-1, mean±SD, ng/ml	334.53 ±72.86	265.46 ± 102.92	0.001
≥ 347.48 ng/ml	39 (56.7%)	1 (5%)
< 347.48 ng.ml	51 (43.3%)	19 (95%)	0.001
Lipoprotein (a), mean±SD, nmol/L	85.41±52.56	23.55±23.66	<0.001
≥ 75 nmol/L	53 (58.9%)	5 (1.9%)	<0.001
< 75 nmol/L	37 (41.1%)	95 (33.9%)
Carotid Intima Media thickness mean±SD, mm	0.80±0.30	0.60±0.10	<0.001
≥ 0.80 mm	58 (64.4%)	1 (5%)	0.002
< 0.80 mm	32 (35.6%)	19 (95%)

Table 3

Comparison of clinical parameters between different groups of NO NAFLD, NAFLD 1 and NAFLD 2+3.

Variables	Non-NAFLD (N = 20)	NAFLD Grade 1 (N = 47)	NAFLD Grades 2 +3 (N = 43)	p-value
Systolic Blood Pressure, mean±SD, mmHg	129.35±19.96	138.55±16.43	148.26±14.95	<0.001
Diastolic Blood Pressure, mean±SD, mmHg	76.30±12.81	79.77±10.45	84.67±12.21	0.027
ALP mean±SD, U/L	74.35±21.30	79.57±23.58	79.59±23.44	0.669
ALT mean±SD, U/L	19.56±8.47	30.70±22.00	39.00±27.72	0.008
AST mean±SD, U/L)	21.06±6.12	31.18±12.37	37.00±19.85	0.001
GGT mean±SD, U/L)	27.70±15.12	59.30±73.79	58.93±45.12	0.085
Intracellular Adhesion Molecule-1, mean±SD, ng/ml	265.46±102.92	333.74±78.00	334.16±68.04	0.003
Lipoprotien (a), mean±SD, nmol/L	23.55±23.70	79.30±56.07	92.21±48.80	<0.001
CIMT mean±SD, mm	0.60±0.10	0.70±0.30	0.90±0.20	<0.001

Table 4

The correlation between severity of NAFLD and OSA (AHI).

OSA Classification	NAFLD Classification			p-value for associationa	r	p-value for correlationb
	Normal	NAFLD Grade 1	NAFLD Grade 2
Mild	14 (70.0)	7 (14.9)	6 (14.3)	<0.001*	0.384	<0.001*
Moderate	5 (25.0)	18 (38.3)	10 (23.8)
Severe	1 (5.0)	22 (46.8)	26 (61.9)

Notes

* Statistically significant at α = 0.05.

Statistical test

a chi-square test

b Kendal tau’b.

Table 6

Multivariate analysis in overall cohort.

	Adj. OR (95%CI)	p-value
ICAM-1
≥ 347ng/ml	41.69 (3.76, 461.97)	0.002
> 347ng/ml	1
Lp(a)
≥ 75 nmol/L	35.12 (3.47, 355.40)	0.003
> 75 nmol/L	1

Notes

Sensitivity: 94.4; Specificity: 75.0.

Model fit (p>0.05), Hosmer and Lemeshow: 0.967.

Cox & Snell: 41.3.

Forward Method.

No multicollinearity and interaction problem.