Abstract
Synchronous non-alcoholic fatty liver disease (NAFLD) and carotid artery plaque formation increase the risk of mortality in patients with cardiovascular disease (CVD). Metabolic status and host gut flora are associated with NAFLD and CVD, but the risk factors require further evaluation.
To evaluate the risk factors associated with NAFLD and CVD, including gut-flora-related examinations.
This cross-sectional study included 235 subjects aged over 40 years who underwent abdominal ultrasound examination and carotid artery ultrasound examination on the same day or within 12 months of abdominal ultrasound between January 2018 and December 2019. All subjects underwent blood tests, including endotoxin and trimethylamine-N-oxide.
The synchronous NAFLD and carotid artery plaque subjects had a higher proportion of men and increased age compared with those without NAFLD and no carotid artery plaque. The synchronous NAFLD and carotid artery plaque group had increased body mass index (BMI), blood pressure, hemoglobin A1C (5.71% vs 5.42%), triglyceride (TG) (164.61 mg/dL vs 102.61 mg/dL), and low-density lipoprotein (135.27 mg/dL vs 121.42 mg/dL). In multiple logistic regression analysis, increased BMI, mean systolic blood pressure, and TG > 110 mg/dL were independent risk factors for synchronous NAFLD and carotid artery plaque formation. Endotoxin and trimethylamine-N-oxide levels were not significantly different between the 2 groups.
Host metabolic status, such as elevated BMI, TG, and systolic blood pressure, are associated with synchronous NAFLD and carotid artery plaque in asymptomatic adults. Aggressive TG control, blood pressure control, and weight reduction are indicated in patients with NAFLD.
Keywords: blood pressure, body mass index, carotid stenosis, non-alcoholic fatty liver disease, triglycerides
1. Introduction
Cardiovascular disease (CVD) and cerebrovascular accidents (CVAs) are leading health problems and important causes of mortality worldwide.[1] The majority of CVD and CVAs are attributable to atherosclerosis, which is characterized by hardening of the arteries and narrowing of the lumen. The pathogenesis of atherosclerosis involves blood vessel inflammation, injury response, degeneration, and thrombosis, and these conditions lead to thickening of the vascular intima and trigger CVD or CVAs.[2,3] The risk factors for atherosclerotic plaque formation include diabetes mellitus (DM), hyperlipidemia, hypertension, sex, habitual smoking, and family history.[4] Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease worldwide and is characterized by an excessive accumulation of fat in the liver parenchyma of patients who consume little to no alcohol.[5] The worldwide prevalence of NAFLD in the general population is 15% to 40% in Western countries, and has been reported to be 11.3% in China, 27% in Hong Kong, and 15% to 45% in South Korea, Japan, and Taiwan.[6–10] Several studies have suggested that metabolites, exercise, and food components play important roles in NAFLD progression.[11]
Our previous study demonstrated that hyperglycemia combined with Helicobacter pylori (H pylori) infection might increase the risk of synchronous colorectal adenoma and carotid artery plaque.[12,13] Additionally, we demonstrated a correlation between CVD and NAFLD, in that when an NAFLD patient had atherosclerosis, the prevalence of CVD events was approximately 2-fold higher than that in the general population. Combined H pylori infection and NAFLD have also been shown to increase carotid artery plaque formation.[14] These findings suggest that the human gut microbiota may play a role in the development of atherosclerosis and affect NAFLD formation. Recent studies have also found changes in the host gut microbiota in patients with colon cancer, ulcerative colitis, obesity, coronary heart disease, delirium, and senile dementia.[15,16] The gut microbiota may influence host immunity and physiology by producing several types of bacterial products and metabolites, such as trimethylamine-N-oxide (TMAO), endotoxins, and short-chain fatty acids. Furthermore, previous studies have demonstrated a connection between the gut microbiota and atherosclerosis through the metabolite TMAO. TMAO enhances foam cell formation and has the potential to promote atherosclerosis, myocardial infarction, and stroke. Endotoxemia has also been shown to be associated with carotid atherosclerosis and CVD.[17–20] However, some previous studies found no connection between a higher level of TMAO and carotid artery disease.[21]
Previous studies have shown controversial results regarding the link between atherosclerosis and gut flora dependent metabolites. Since some studies noted some connection between CVD, NAFLD, carotid atherosclerosis, gut microbiota, and gut-flora dependent metabolites and some presented controversial results, the relationship between these indicators, events, and diagnosis remains unclear. Therefore, in the current study, we aimed to explore the association between host metabolic status, gut-flora dependent metabolites, and synchronous carotid artery disease and NAFLD in asymptomatic adults.
2. Methods
2.1. Subjects and study design
This was a cross-sectional observation study that involved healthy men and women aged 40 years or older who participated in a comprehensive health-screening examination in the MacKay Memorial Hospital, Taiwan, from January 2018 until December 2019. Asymptomatic individuals who had undergone abdominal ultrasound examination as part of a health check-up were enrolled for analysis. A carotid artery ultrasound survey was arranged on the same day or within 12 months of colonoscopy when the participants accepted annual physical check-ups. As our objective was to evaluate the association between serum endotoxin and TMAO with carotid artery plaque and non-alcoholic fatty liver disease, we excluded patients who met the following criteria: previously proven acute myocardial infarction or stroke; incapacitated or could not undergo carotid artery ultrasound examination; lacked data regarding abdominal ultrasound or carotid artery ultrasound examination, or basic blood tests samples; alcoholic drinking habit or other secondary fat accumulation as a result of steatogenic medication or hereditary disorders; other severe concomitant disease, such as infections, connective tissue disease, malignancies, heart failure, and liver or kidney disease; or positive for hepatitis B surface antigen or hepatitis C antibodies. After excluding 276 subjects, a total of 235 study participants (174 men and 61 women) were enrolled for further study.
2.2. Clinical data collection and questionnaire
Clinical data including fasting plasma glucose AC, hemoglobin A1C (HbA1c), triglyceride (TG), and low-density lipoprotein (LDL) levels were obtained from participants on the same health check-up day as when the abdominal ultrasound examination was performed. These data were reported by the central lab of our hospital which has standardized protocols with routine quality assurance evaluation. Carotid artery ultrasound examination data were collected on the same day, or within 12 months of the endoscopy examination for the participants who accepted an annual healthy examination. Baseline characteristics (age, height, weight, personal medical history, current medicine use, family history of first degree-relatives, and smoking) were obtained from a questionnaire completed at the time of the examination. The study was approved by the MacKay Memorial Hospital Institutional Review Board (18MMHIS185).
2.3. Endotoxin and TMAO
Pierce limulus amebocyte lysate (LAL) chromogenic endotoxin quantitation kit (Thermo Scientific; Waltham, US) was used for the in vitro quantitative detection of human plasma endotoxin concentration. Briefly, purified extract of Escherichia coli (0113:H10) was used as a control standard endotoxin. PM (100 μL) extract was mixed with LAL reagent (100 μL) followed by incubation at 37 °C for 60 minutes. Then substrate solution was added to the incubated fraction and vortexed for 10 minutes to ensure complete mixing. Presence of endotoxins was confirmed by developing yellow color and mixture was measured spectrophotometrically (PerkinElmer EnSpire 2300) at 405 to 410 nm. Concentration of endotoxin was calculated from the standard curve prepared using the series of different endotoxin standard concentration, that is, 1.0, 0.5, 0.25, 0.1 EU/mL.[22]
Early gas chromatography–mass spectrometry studies employed a complicated multistep derivatization protocol that first reduced TMAO to the gas trimethylamine (TMA), which was subsequently derivatized with 2,2,2-trichloroethyl chloroformate and detected as N,N-dimethyl-2,2,2-trichloroethyl carbamate. The coefficient of determination r2 was required to be over 0.98.[23]
2.4. Scanning protocol and definition of carotid artery lesion and NAFLD in ultrasound examination
Ultrasonography of the common carotid artery, carotid bifurcation, and internal carotid artery of the left and right carotid arteries was performed using a 7.5-MHz linear-array transducer (ATL Ultra-Mark IV). On a longitudinal, two-dimensional ultrasound image of the carotid artery, the anterior (near) and posterior (far) walls of the carotid artery were displayed as 2 bright white lines separated by a hypoechogenic space. Frozen images of the arterial wall were saved, and measurements were subsequently performed on the stored digital images. A plaque was defined as a distinct area with an I–M thickness >50% greater than that of neighboring sites.[24] The definition of NAFLD requires that firstly, there is evidence of hepatic steatosis, either by imaging or histology, and secondly, that there are no causes of secondary hepatic fat accumulation, such as significant alcohol consumption, use of steatogenic medication, or hereditary disorders. An experienced gastroenterologist blinded to the study aims performed the abdominal ultrasound using an HD-15 ultrasound system (Philips Medical Systems, Cleveland, OH). Fatty liver diagnosis was made by ultrasound based on standard criteria, including parenchyma brightness, liver-to-kidney contrast, deep beam attenuation, and bright vessel walls.
2.5. Statistical analysis
The following variables were recorded for each subject: age, sex, body mass index (BMI), HbA1c, lipid levels, smoking status, TMAO, and endotoxin level. A t test was applied for continuous variables when the data fit a normal Gaussian distribution, and data for continuous variables were expressed as the mean ± SD. Categorical variables were tested using the chi-square test or Analysis of Variance test and expressed as numbers (percentage). Unadjusted odds ratios (ORs) with 95% confidence intervals (CIs) were computed for potential predictors of colorectal polyps and adenomas. Multiple variable logistic regression analysis was applied to compute the adjusted OR (95% CI) for predictors of carotid artery plaque and NAFLD. Variables with a P-value <.2 on univariate analysis were selected for multivariate logistic regression. The final model was developed using a stepwise backward approach. All variables with P < .05 were considered to be statistically significant and remained in the final model. All analyses were performed using SAS version 9.1 (SAS Institute, Cary, NC).
3. Results
3.1. Demographics of NAFLD and carotid artery plaque statuses
In a total of 235 patients, the NAFLD patients (n = 133) had older mean age and higher levels of BMI, HbA1c, glucose AC, systolic blood pressure (SBP), diastolic blood pressure (DBP), and TG compared with controls (all P < .05) (Table 1), as well as the current oral medicine treatment participants, including diabetes, hypertension, and hyperlipidemia. The proportion of participants who were smokers was also higher in the NAFLD group. The TMAO and endotoxin level was not significantly different between the 2 groups.
Table 1.
Demographics of participants with and without NAFLD.
| NAFLD status | |||
| Variable | Non-NAFLD (n = 102) | NAFLD (n = 133) | P-value |
| Age, mean (SD), yr | 52.09 (8.30) | 53.43 (7.05) | .183 |
| Sex (male %) | 61 (60%) | 113 (85%) | .000 |
| BMI, mean (SD), kg/m2 | 22.15 (2.56) | 25.52 (2.61) | .000 |
| HbA1c, mean (SD), % | 5.44 (0.34) | 5.73 (0.78) | .001 |
| Systolic blood pressure, mean (SD), mmHg | 116.27 (15.97) | 123.50 (13.10) | .000 |
| Diastolic blood pressure, mean (SD) mm Hg | 74.15 (10.14) | 79.06 (10.70) | .000 |
| Glucose AC, mean (SD), mg/dL | 94.03 (9.77) | 103.92 (19.58) | .000 |
| Total cholesterol, mean (SD), mg/dL | 202.94 (34.71) | 205.65 (37.96) | .575 |
| Triglyceride, mean (SD), mg/dL | 99.45 (56.57) | 160.39 (86.70) | .000 |
| LDL, mean (SD), mg/dL | 124.00 (30.78) | 131.65 (33.26) | .073 |
| Plasma hs-CRP, mean (SD), mg/L | 0.15 (0.38) | 0.18 (0.24) | .430 |
| Smoking, no. (%) | 20 (20%) | 37 (28%) | .000 |
| Anti-platelet agent used, no. (%) | 3 (3%) | 11 (8%) | .000 |
| Anti-lipid agent used, no. (%) | 8 (8%) | 18 (14%) | .000 |
| DM control agent used, no. (%) | 2 (2%) | 11 (8%) | .000 |
| Hypertension control agent used, no. (%) | 9 (9%) | 30 (23%) | .000 |
| Hb A1c level ≥6.5%, no. (%) | 0 (0%) | 12 (9%) | .000 |
| TMAO, mean (SD), μM | 2.95 (6.73) | 2.22 (2.94) | .408 |
| Endotoxin, mean (SD), EU/mL | 0.68 (1.18) | 0.95 (1.74) | .230 |
AC = ante cibum; BMI = body mass index; CRP = C-reactive protein; DM = diabetes mellitus; HbA1c = hemoglobin A1C; LDL = low-density lipoprotein; NAFLD = non-alcoholic fatty liver disease; no. = number; SD = standard deviation; TMAO = trimethylamine-N-oxide.
As to the carotid artery plaque status, patients with carotid artery plaque formation (n = 83) had a higher level of the BMI, SBP, DBP, and LDL than in those without (n = 152) (Table 2). In terms of gut-flora dependent metabolites, the endotoxin and TMAO level were not significantly different between the 2 groups.
Table 2.
Demographics of with and without carotid artery plaque.
| Carotid artery plaque status | |||
| Variable | Plaque (–) (n = 152) | Plaque (+) (n = 83) | P-value |
| Age, mean (SD), year | 51.85 (7.78) | 54.67 (7.04) | .006 |
| Sex (male %) | 106 (70%) | 68 (82%) | .042 |
| BMI, mean (SD), kg/m2 | 23.59 (2.83) | 24.91 (3.34) | .002 |
| HbA1c, mean (SD), %) | 5.57 (0.65) | 5.66 (0.64) | .339 |
| Systolic blood pressure, mean (SD), mm Hg | 118.17 (14.24) | 124.39 (15.12) | .002 |
| Diastolic blood pressure, mean (SD), mm Hg | 75.54 (9.65) | 79.47 (12.08) | .007 |
| Glucose AC, mean (SD), mg/dL | 99.29 (18.54) | 100.25 (13.03) | .675 |
| Total cholesterol, mean (SD), mg/dL | 201.36 (33.19) | 210.17 (41.58) | .078 |
| Triglyceride, mean (SD), mg/dL | 130.51 (82.32) | 140.59 (78.35) | .363 |
| LDL, mean (SD), mg/dL | 125.29 (29.14) | 133.90 (37.07) | .050 |
| Plasma hs-CRP, mean (SD), mg/L | 0.17 (0.32) | 0.18 (0.28) | .841 |
| Smoking, no. (%) | 42 (28%) | 15 (18%) | .102 |
| Anti-platelet agent used, no. (%) | 7 (5%) | 7 (8%) | .236 |
| Anti-lipid agent used, no. (%) | 12 (8%) | 14 (17%) | .036 |
| DM control agent used, no. (%) | 7 (5%) | 6 (7%) | .400 |
| Hypertension control agent used, no. (%) | 16 (11%) | 23 (28%) | .001 |
| Hb A1c level ≥6.5%, no. (%) | 7 (5%) | 5 (6%) | .637 |
| TMAO, mean (SD), μM | 2.48 (5.45) | 2.70 (4.30) | .817 |
| Endotoxin, mean (SD), EU/mL | 0.90 (1.74) | 0.69 (0.97) | .367 |
AC = ante cibum; BMI = body mass index; CRP = C-reactive protein; DM = diabetes mellitus; HbA1c = hemoglobin A1C; LDL = low-density lipoprotein; NAFLD = non-alcoholic fatty liver disease; no. = number; SD = standard deviation; TMAO = trimethylamine-N-oxide.
3.2. Demographics of synchronous NAFLD and carotid artery plaque
There were 56 patients with synchronous fatty liver and carotid artery plaque and 75 patients with no fatty liver and no carotid artery plaque. Several factors, included aging, sex, BMI, HbA1c, SBP, DBP, TG, and smoking status, were related to synchronous NAFLD and carotid artery plaque (Table 3). However, the gut-flora dependent metabolites (TMAO and endotoxin) and hs-CRP levels were similar between the 2 groups. Univariate logistic regression and multiple logistic regression for predictors of synchronous fatty liver and carotid artery plaque is shown in Table 4. In univariate logistic regression, increased age, BMI, SBP, DBP, HbA1c >5.6%, TG >110 mg/dL, and men were found to be relative risk factors of synchronous fatty liver and carotid artery plaque. Multiple logistic regression demonstrated that only increased BMI, SBP, and TG >110 mg/dL were independent risk factors for participants with synchronous fatty liver and carotid artery plaque, while TMAO and endotoxin levels were not. The synchronous NAFLD and carotid artery plaque group had increased BMI, blood pressure, HbA1c (5.71% vs 5.42%), TG (164.61 mg/dL vs 102.61 mg/dL), and LDL (135.27 mg/dL vs 121.42 mg/dL) Moreover, we found no interaction effect of increased BMI, SBP, and TG >110 mg/dL in synchronous fatty liver and carotid artery plaque formation. While calculating the power retrospectively under the assumption of alpha error 0.05 with our sample sizes and variances, the power of TMAO was 0.79 and the power of endotoxin was 0.05.
Table 3.
Demographics of synchronous NAFLD and carotid artery plaque status.
| Variable | Synchronous fatty liver and carotid artery plaque (n = 56) | No fatty liver and No carotid artery plaque (n = 75) | P-value |
| Age, mean (SD), year | 54.21 (6.30) | 50.81 (7.94) | .009 |
| Sex (male %) | 50 (89%) | 43 (57%) | .000 |
| BMI, mean (SD), kg/m2 | 26.09 (2.93) | 22.04 (2.49) | .000 |
| HbA1c, mean (SD), % | 5.71 (0.75) | 5.40 (0.34) | .002 |
| Systolic blood pressure, mean (SD), mm Hg | 124.68 (11.64) | 113.57 (12.95) | .000 |
| Diastolic blood pressure, mean (SD), mm Hg | 79.98 (12.65) | 72.61 (9.43) | .000 |
| Glucose AC, mean (SD), mg/dL | 102.70 (14.12) | 93.61 (10.17) | .000 |
| Total cholesterol, mean (SD), mg/dL | 209.80 (42.43) | 200.03 (32.14) | .137 |
| Triglyceride, mean (SD), mg/dL | 164.61 (82.32) | 102.61 (62.57) | .000 |
| LDL, mean (SD), mg/dL | 135.27 (38.23) | 121.42 (28.88) | .020 |
| Plasma hs-CRP, mean (SD), mg/L | 0.21 (0.32) | 0.17 (0.44) | .585 |
| Smoking, no. (%) | 10 (18%) | 15 (20%) | .000 |
| Anti-platelet agent used, no. (%) | 6 (11%) | 2 (3%) | .000 |
| Anti-lipid agent used, no. (%) | 8 (14%) | 2 (3%) | .000 |
| DM control agent used, no. (%) | 4 (7%) | 0 (0%) | .000 |
| Hypertension control agent used, no. (%) | 16 (29%) | 2 (3%) | .000 |
| Hb A1c level ≥6.5%, no. (%) | 5 (9%) | 0 (0% | .000 |
| TMAO, mean (SD), μM | 1.98 (1.22) | 2.58 (6.81) | .640 |
| Endotoxin, mean (SD), EU/mL | 0.64 (0.87) | 0.64 (1.20) | .987 |
AC = ante cibum; BMI = body mass index; CRP = C-reactive protein; DM = diabetes mellitus; HbA1c = hemoglobin A1C; LDL = low-density lipoprotein; NAFLD = non-alcoholic fatty liver disease; no. = number; SD = standard deviation; TMAO = trimethylamine-N-oxide.
Table 4.
Univariate logistic regression and multiple logistic regression for predictors of synchronous NAFLD and carotid artery plaque.
| Univariate logistic regression | Multiple logistic regression | |||||
| OR | S.E. | P-value | OR | S.E. | P-value | |
| Age | 1.06 | 0.03 | .009 | 1.037 | 0.081 | .639 |
| Male, sex | 6.20 | 3.05 | .000 | 6.761 | 8.330 | .121 |
| BMI | 1.88 | 0.22 | .000 | 1.658 | 0.409 | .040 |
| HbA1c ≥5.6 | 3.05 | 0.17 | .002 | 2.183 | 2.015 | .398 |
| Systolic blood pressure, mean | 1.08 | 0.02 | .000 | 1.156 | 0.085 | .047 |
| Diastolic blood pressure, mean | 1.08 | 0.02 | .000 | 0.954 | 0.086 | .597 |
| Smoking | 0.87 | 0.39 | .758 | 0.290 | 0.339 | .289 |
| LDL >100 mg/dL | 1.52 | 0.65 | .330 | 6.076 | 7.479 | .143 |
| Triglyceride >110 mg/dL | 5.79 | 2.25 | .000 | 14.080 | 15.438 | .016 |
| TMAO, mean | 1.00 | 0.19 | .987 | 1.835 | 0.763 | .144 |
| Endotoxin, mean | 0.97 | 0.06 | .649 | 0.757 | 0.436 | .244 |
BMI = body mass index; HbA1c = hemoglobin A1C; LDL = low-density lipoprotein; NAFLD = non-alcoholic fatty liver disease; OR = odds ratio; S.E. = standard error; TMAO = trimethylamine-N-oxide.
4. Discussion
Several previous studies have demonstrated that the relationship between NAFLD and metabolic syndrome is bidirectional. The presence of NAFLD is a strong predictor of metabolic syndrome, such as abdominal obesity, hypertension, atherogenic dyslipidemia, and dysglycemia, and these features are established risk factors for type 2 DM and CVD. Furthermore, liver fat content has also been shown to be significantly increased in participants with dyslipidemia or metabolic syndrome.[25,26] Since there is a clinically significant association between NAFLD and the development of CVD, type 2 DM, and other systemic diseases, it is necessary to identify those subjects at risk of developing NAFLD. A study by Gastaldelli et al[27] demonstrated that NAFLD subjects are more prone to early carotid atherosclerosis, even in the absence of metabolic syndrome. Furthermore, a study by Wong et al[28] demonstrated that the prevalence of NAFLD was 58%, and more severe CAD was predicted in a coronary angiogram. The cohort study described by Moon et al[29] also showed a strong association between NAFLD and carotid artery plaque. Moreover, Musso et al[30] conducted a meta-analysis study and found that patients with NAFLD have a higher risk of mortality and morbidity as a result of CVD (OR, 2.05; 95% CI, 1.8–2.3; P < .0001) than the matched general population. These growing bodies of evidence support the association of NAFLD with cardiovascular events, independent of traditional risk factors. Furthermore, they have provided evidence that future research should not only focus on the hepatic situation but also the need to survey the possibility of CVD in patients with NAFLD. These studies highlight the importance of synchronous fatty liver and carotid artery plaque, as well as the importance of establishing risk factors or biomarkers for this group of patients.
Our previous study revealed the connection between NAFLD and CVD, as well as several noninvasive diagnostic methods for NAFLD.[12]H pylori infection combined with NAFLD has been demonstrated to increase the risk of carotid artery plaque formation.[14] Indeed, previous studies have highlighted the association between gut microbiota and human diseases, including enteric infections and malignancy, inflammatory bowel disease, metabolic disease, CVD, and mood disorders.[31] Our previous study also demonstrated that the colorectal adenoma ratio is likely to decrease following H pylori eradication.[32] It has been suggested that the gut microbiota contributes to nearly every aspect of the host's growth and development, such that host disease and organ dysfunction may result when an imbalance exists in either the composition, number, or habitat of the gut microbiota.[31] A further possible mechanism of this condition might relate to the role of microbiota in gut inflammation, as it has been shown that when toll-like receptors are stimulated by bacterial products, such as endotoxin, IL-23 increases, and acts on downstream cells, including lymphocytes. Endotoxin is a complex that contains lipopolysaccharide, a cell wall component of gram-negative bacteria that elicits strong immune responses in humans and may function as a copromoter that triggers the formation of carotid artery plaque.[33] However, in the current study, the endotoxin level was not significantly different between participants with and without carotid artery plaque and NAFLD. There are several possible explanations for this observation. First, endotoxin levels might be affected by a high-fat diet.[34] Second, the endotoxin level measured by the LAL assay may be inaccurate due to technical difficulties with the assay and the need to collect samples under lipopolysaccharide-free conditions, as well as its lack of sensitivity.[35]
Recent studies have shown that the direct connection between the gut microbiota and atherosclerosis, at least in part through the metabolite TMAO and elevated TMAO levels, might predict the incident risk of thrombotic events in human subjects.[18,36] However, a study by Skagen et al[21] found that serum γ-butyrobetain and carnitine, but not TMAO or trimethyllysine, were increased in patients with carotid atherosclerosis. TMAO has been proposed to be produced in several steps. The dietary intake of carnitine or phosphatidylcholine leads to its conversion to trimethylamine (TMA) by the intestinal microbiota, which is subsequently oxidized to TMAO by flavins containing monooxygenase in the liver.[37] Since γ-butyrobetain is produced endogenously from trimethyllysine and partly converted to TMA in the colon, it has been suggested that γ-butyrobetain could mediate some of its effects through TMA, and subsequently TMAO.[37] Both microbiota-dependent and endogenous pathways could potentially affect the formation of carotid atherosclerosis. The recent study conducted by Koay et al[38] showed that both “healthy” and “unhealthy” diets could cause an increase in the plasma level of TMAO and no direct association of plasma TMAO and the extent of atherosclerosis, both in mice and humans. According to the author's opinion, this result might be related to the fact that different diets induce changes in the gut microbiome, and the gut itself is a site of significant oxidative production of TMAO. Thus, the TMAO level might not be a straight marker of carotid atherosclerosis.
In our study, hs-CRP, endotoxin, and TMAO were ineffective in the detection of NAFLD or carotid artery plaque in asymptomatic adults, and we found that elevated BMI, TG > 110 mg/dL, and elevated SBP were related to synchronous NAFLD and carotid artery plaque after univariate logistic regression and multiple logistic regression analysis (Table 4). Indeed, elevated BMI and TG levels were previously thought to be highly related to NAFLD.[39] In Table 2, we show that both SBP and DBP were significantly higher, and LDL was borderline higher in the carotid artery plaque group. Our study showed that both NAFLD and carotid artery plaque subjects had higher SBP (approximately 125 mmHg) than subjects without NAFLD or carotid artery plaque. A similar result was found in synchronous NAFLD and carotid artery plaque than in non-NAFLD and non-carotid artery plaque participants (Table 3). Although the systolic blood pressure was only borderline elevated (approximately 125 mmHg), it was also demonstrated to be linked to NAFLD and carotid artery plaque formation. Indeed, the study by Lemne et al[40] also reported the presence of carotid intima-media thickness and plaque in borderline hypertension. Our study results demonstrated that the risk of synchronous NAFLD and carotid artery plaque was not linked to TMAO and endotoxin increase in relatively healthy adults with a high BMI and TG if the systolic pressure was borderline elevated (approximately 125 mmHg).
The causal factors and mechanisms of NAFLD are not yet completely understood, and the debate on the role of NAFLD as a primary or secondary illness continues. Although our study provides some factors associated with NAFLD according to a population-based dataset, there are still many hypotheses proposed in the past that we were not able to address in this study, such as the overproduction of reactive oxygen species, the expression of polymorphisms of specific genes such as patatin-like phospholipase domain-containing protein 3 (PNPLA3), the adipose tissue expandability hypothesis, environmental factors contributing to ectopic lipid accumulation, behavioral factors affecting the gut microbiota, and the effects of gut microbiota on host metabolism, nutrient absorption, and immune function.[41]
This study has some limitations. First, although blood samples were taken before meals, and fasting samples were used, we still lacked information on vegetarians versus omnivores in our analyses. Second, this study included subjects with relatively high incomes, who may have been healthier and may not be representative of the general population. Third, as a cross-sectional study, we can only show an association between metabolic status and the risk of synchronous NAFLD and carotid artery plaque; thus, a larger population and long-term follow-up are required to make definite conclusions on the impact of metabolic status or gut-flora dependent metabolites on cardiovascular mortality. Further studies are needed to determine the relationships among metabolic status, gut microbiota and their metabolites, and NAFLD or carotid artery plaque. Other gut microbiota-related metabolites, such as serotonin or short-chain fatty acids, might be included in studies on NAFLD or carotid artery plaque formation.
5. Conclusion
Our study demonstrated that elevated BMI, TG over 110 mg/dL, and elevated SBP are associated with synchronous NAFLD and carotid artery plaque in asymptomatic adults. According to this disease connection, we should not only focus on the hepatic situation, but also survey the possibility of CVD in patients with NAFLD. Given the increasing proportion of obesity in general population, as well as the high mortality and morbidity of CVD when carotid artery plaque is found in combination with NAFLD, more aggressive blood pressure and weight reduction, as well as TG control should be considered in clinical practice.
Acknowledgments
The authors acknowledge the funding of the Department of Medical Research, Mackay Memorial Hospital, grant number MMH-110-34, MMH-109-27 who had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
Author contributions
Conceptualization: Kuang-Chun Hu, Ying-Chun Lin, Hung-I Yeh.
Data curation: Horng-Yuan Wang.
Formal analysis: Lo-Yip Yu, Hung-I Yeh.
Funding acquisition: Kuang-Chun Hu.
Investigation: Ming-Shiang Wu, Chun-Jen Liu, Shou-Chuan Shih, Hung-Ju Ko.
Methodology: Yang-Che Kuo, Ming-Shiang Wu.
Project administration: Ming-Jen Chen.
Resources: Ming-Jen Chen, Chun-Jen Liu, Horng Woei Yang, Lo-Yip Yu, Hung-Ju Ko.
Software: Yang-Che Kuo, Horng Woei Yang.
Validation: Chun-Jen Liu, Shou-Chuan Shih.
Visualization: Shou-Chuan Shih.
Writing – original draft: Ying-Chun Lin.
Writing – review & editing: Kuang-Chun Hu.
Footnotes
Abbreviations: BMI = body mass index, CIs = confidence intervals, CVAs = cerebrovascular accidents, CVD = cardiovascular disease, DM = diabetes mellitus, H pylori = Helicobacter pylori, HbA1c = hemoglobin A1C, LAL = limulus amebocyte lysate, LDL = low-density lipoprotein, LPS = lipopolysaccharide, NAFLD = non-alcoholic fatty liver disease, ORs = odds ratios, SBP = systolic blood pressure, TG = triglyceride, TMA = trimethylamine, TMAO = trimethylamine-N-oxide, TML = trimethyllysine.
How to cite this article: Lin YC, Wang HY, Kuo YC, Chen MJ, Wu MS, Liu CJ, Yang HW, Shih SC, Yu LY, Ko HJ, Yeh HI, Hu KC. Gut-flora metabolites is not associated with synchronous carotid artery plaque and non-alcoholic fatty liver disease in asymptomatic adults: a STROBE-compliant article. Medicine. 2021;100:34(e27048).
The authors have no conflicts of interest to disclose.
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
References
- [1].Benjamin EJ, Blaha MJ, Chiuve SE, et al. American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Heart disease and stroke statistics-2017 update: a report from the American Heart Association. Circulation 2017;135:e146–603. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [2].Sawayama Y, Ariyama I, Hamada M, et al. Association between chronic Helicobacter pylori infection and acute ischemic stroke: Fukuoka Harasanshin Atherosclerosis Trial (FHAT). Atherosclerosis 2005;178:303–9. [DOI] [PubMed] [Google Scholar]
- [3].Gaudio E, Carpino G, Grassi M, Musca A. Morphological aspects of atherosclerosis lesion: past and present. Clin Ter 2006;157:135–42. [PubMed] [Google Scholar]
- [4].Wilson PW, D’Agostino RB, Levy D, Belanger AM, Silbershatz H, Kannel WB. Prediction of coronary heart disease using risk factor categories. Circulation 1998;97:1837–47. [DOI] [PubMed] [Google Scholar]
- [5].Farrell GC, Larter CZ. Nonalcoholic fatty liver disease: from steatosis to cirrhosis. Hepatology 2006;43: (2 suppl 1): S99–112. [DOI] [PubMed] [Google Scholar]
- [6].Chen CH, Huang MH, Yang JC, et al. Prevalence and risk factors of nonalcoholic fatty liver disease in an adult population of Taiwan: metabolic significance of nonalcoholic fatty liver disease in nonobese adults. J Clin Gastroenterol 2006;40:745–52. [DOI] [PubMed] [Google Scholar]
- [7].Fan JG. Epidemiology of alcoholic and nonalcoholic fatty liver disease in China. J Gastroenterol Hepatol 2013;28: (suppl): 11–7. [DOI] [PubMed] [Google Scholar]
- [8].Fan JG, Saibara T, Chitturi S, Kim BI, Sung JJ, Chutaputti A. What are the risk factors and settings for non-alcoholic fatty liver disease in Asia-Pacific? J Gastroenterol Hepatol 2007;22:794–800. [DOI] [PubMed] [Google Scholar]
- [9].Farrell GC, Wong VW, Chitturi S. NAFLD in Asia--as common and important as in the West. Nat Rev Gastroenterol Hepatol 2013;10:307–18. [DOI] [PubMed] [Google Scholar]
- [10].Park SH, Jeon WK, Kim SH, et al. Prevalence and risk factors of non-alcoholic fatty liver disease among Korean adults. J Gastroenterol Hepatol 2006;21(1 pt 1):138–43. [DOI] [PubMed] [Google Scholar]
- [11].Fan JG, Cao HX. Role of diet and nutritional management in non-alcoholic fatty liver disease. J Gastroenterol Hepatol 2013;28: (suppl): 81–7. [DOI] [PubMed] [Google Scholar]
- [12].Hu KC, Wang HY, Liu SC, et al. Nonalcoholic fatty liver disease: updates in noninvasive diagnosis and correlation with cardiovascular disease. World J Gastroenterol 2014;20:7718–29. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [13].Hu KC, Wu MS, Chu CH, et al. Hyperglycemia combined Helicobacter pylori infection increases risk of synchronous colorectal adenoma and carotid artery plaque. Oncotarget 2017;8:108655–64. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [14].Yu LY, Hu KC, Liu CJ, et al. Helicobacter pylori infection combined with non-alcoholic fatty liver disease increase the risk of atherosclerosis: focus in carotid artery plaque. Medicine (Baltimore) 2019;98:e14672–14680. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [15].Nicholson JK, Holmes E, Kinross J, et al. Host-gut microbiota metabolic interactions. Science 2012;336:1262–7. [DOI] [PubMed] [Google Scholar]
- [16].Ley RE, Turnbaugh PJ, Klein S, Gordon JI. Microbial ecology: human gut microbes associated with obesity. Nature 2006;444:1022–3. [DOI] [PubMed] [Google Scholar]
- [17].Li J, Lin S, Vanhoutte PM, Woo CW, Xu A. Akkermansia muciniphila protects against atherosclerosis by preventing metabolic endotoxemia-induced inflammation in Apoe−/− mice. Circulation 2016;133:2434–46. [DOI] [PubMed] [Google Scholar]
- [18].Koeth RA, Wang Z, Levison BS, et al. Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis. Nat Med 2013;19:576–85. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [19].Tang WH, Wang Z, Levison BS, et al. Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk. N Engl J Med 2013;368:1575–84. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [20].Wiedermann CJ, Kiechl S, Dunzendorfer S, et al. Association of endotoxemia with carotid atherosclerosis and cardiovascular disease: prospective results from the Bruneck Study. J Am Coll Cardiol 1999;34:1975–81. [DOI] [PubMed] [Google Scholar]
- [21].Skagen K, Trøseid M, Ueland T, et al. The Carnitine-butyrobetaine-trimethylamine-N-oxide pathway and its association with cardiovascular mortality in patients with carotid atherosclerosis. Atherosclerosis 2016;247:64–9. [DOI] [PubMed] [Google Scholar]
- [22].Roy R, Jan R, Joshi U, Bhor R, Pai K, Satsangi PG. Characterization, pro-inflammatory response and cytotoxic profile of bioaerosols from urban and rural residential settings in Pune, India. Environ Pollut 2020;264:114698. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [23].daCosta KA, Vrbanac JJ, Zeisel SH. The measurement of dimethylamine, trimethylamine, and trimethylamine N-oxide using capillary gas chromatography-mass spectrometry. Anal Biochem 1990;187:234–9. [DOI] [PubMed] [Google Scholar]
- [24].Tahmasebpour HR, Buckley AR, Cooperberg PL, Fix CH. Sonographic examination of the carotid arteries. Radiographics 2005;25:1561–75. [DOI] [PubMed] [Google Scholar]
- [25].Bugianesi E, McCullough AJ, Marchesini G. Insulin resistance: a metabolic pathway to chronic liver disease. Hepatology 2005;42:987–1000. [DOI] [PubMed] [Google Scholar]
- [26].Perseghin G. Viewpoints on the way to a consensus session: where does insulin resistance start? The liver. Diabetes Care 2009;32: (suppl): S164–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [27].Gastaldelli A, Kozakova M, Højlund K, et al. Fatty liver is associated with insulin resistance, risk of coronary heart disease, and early atherosclerosis in a large European population. Hepatology 2009;49:1537–44. [DOI] [PubMed] [Google Scholar]
- [28].Wong VW, Wong GL, Yip GW, et al. Coronary artery disease and cardiovascular outcomes in patients with non-alcoholic fatty liver disease. Gut 2011;60:1721–7. [DOI] [PubMed] [Google Scholar]
- [29].Moon SH, Noh TS, Cho YS, et al. Association between nonalcoholic fatty liver disease and carotid artery inflammation evaluated by 18F-fluorodeoxyglucose positron emission tomography. Angiology 2015;66:472–80. [DOI] [PubMed] [Google Scholar]
- [30].Musso G, Gambino R, Cassader M, Pagano G. Meta-analysis: natural history of non-alcoholic fatty liver disease (NAFLD) and diagnostic accuracy of non-invasive tests for liver disease severity. Ann Med 2011;43:617–49. [DOI] [PubMed] [Google Scholar]
- [31].Sekirov I, Russell SL, Antunes LC, Finlay BB. Gut microbiota in health and disease. Physiol Rev 2010;90:859–904. [DOI] [PubMed] [Google Scholar]
- [32].Hu KC, Wu MS, Chu CH, et al. Decreased colorectal adenoma risk after helicobacter pylori eradication: a retrospective cohort study. Clin Infect Dis 2019;68:2105–13. [DOI] [PubMed] [Google Scholar]
- [33].Grivennikov SI, Wang K, Mucida D, et al. Adenoma-linked barrier defects and microbial products drive IL-23/IL-17-mediated tumour growth. Nature 2012;491:254–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [34].Pendyala S, Walker JM, Holt PR. A high-fat diet is associated with endotoxemia that originates from the gut. Gastroenterology 2012;142: 1100.e2-1101.e2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [35].Munford RS. Detoxifying endotoxin: time, place and person. J Endotoxin Res 2005;11:69–84. [DOI] [PubMed] [Google Scholar]
- [36].Zhu W, Gregory JC, Org E, et al. Gut microbial metabolite TMAO enhances platelet hyperreactivity and thrombosis risk. Cell 2016;165:111–24. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [37].Koeth RA, Levison BS, Culley MK, et al. γ-Butyrobetaine is a proatherogenic intermediate in gut microbial metabolism of L-carnitine to TMAO. Cell Metab 2014;20:799–812. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [38].Koay YC, Chen YC, Wali JA, Luk AW, Li M, Doma H. Plasma levels of trimethylamine-N-oxide can be increased with ‘healthy’ and ‘unhealthy’ diets and do not correlate with the extent of atherosclerosis but with plaque instability. Cardiovasc Res 2021;117:435–49. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [39].Vanni E, Marengo A, Mezzabotta L, Bugianesi E. Systemic complications of nonalcoholic fatty liver disease: when the liver is not an innocent bystander. Semi Liver Dis 2015;35:236–49. [DOI] [PubMed] [Google Scholar]
- [40].Lemne C, Jogestrand T, de Faire U. Carotid intima-media thickness and plaque in borderline hypertension. Stroke 1995;26:34–9. [DOI] [PubMed] [Google Scholar]
- [41].Tarantino G, Citro V, Capone D. Nonalcoholic fatty liver disease: a challenge from mechanisms to therapy. J Clin Med 2020;9:15. [DOI] [PMC free article] [PubMed] [Google Scholar]