Association between homocysteine and white matter hyperintensities in rural‐dwelling Chinese people with asymptomatic intracranial arterial stenosis: A population‐based study

Abstract Purpose Although homocysteine (Hcy) has been proven to be associated with the incidence of white matter hyperintensities (WMH) in patients with stroke, this association remains unclear in participants with asymptomatic intracranial arterial stenosis (aICAS). This study aimed to investigate the association of Hcy with WMH in participants with aICAS. Materials and methods This was a cross‐sectional study based on the Kongcun Town Study. Participants diagnosed with aICAS by magnetic resonance angiography in the Kongcun Town Study were enrolled in this study. Data on demographics, lifestyle, medical histories, and Hcy levels were collected via interviews, clinical examinations, and laboratory tests. The volume of WMH was calculated using the lesion segmentation tool system for the Statistical Parametric Mapping package based on magnetic resonance imaging. The association between Hcy and WMH volume was analyzed using linear and logistic regression analyses. Results A total of 137 aICAS participants were enrolled in the present study. Hcy was associated with the incidence of severe WMH (4th quartile, ≥4.20 ml) after adjustment for certain covariates [Hcy as a continuous variable, odds ratio (95% confidence interval) (OR (95% CI)): 1.09 (1.00, 1.19), p = .047; as a categorical variable (Hcy ≥15 μmol/L), OR (95% CI): 3.74 (1.37, 10.19), p = .010)]. After stratification according to the degree of aICAS, this relationship remained significant only in the moderate‐to‐severe stenosis group (stenosis ≥50%). (Hcy as continuous variable, OR (95% CI): 1.14 (1.02, 1.27), p = .025; as a categorical variable (Hcy ≥15 μmol/L), OR (95% CI): 5.59 (1.40, 15.25), p = .015). Conclusion Serum Hcy concentration may be positively associated with the volume of WMH in rural‐dwelling Chinese people with moderate‐to‐severe (stenosis ≥50%) aICAS.


| INTRODUC TI ON
White matter hyperintensities (WMH) are a type of cerebral small vessel disease characterized by hyperintensities on T2-weighted magnetic resonance imaging (MRI) scans; these hyperintensities are predominantly located in the bilateral and symmetrical white matter of the brain (Garde et al., 2000;Pantoni, 2010).
Pathological changes associated with WMH are mainly axonal degeneration, demyelination, oligodendrocyte apoptosis (Wardlaw et al., 2019). It has been demonstrated that WMH is associated with an increased risk of ischemic stroke (Conijn et al., 2011;Selvarajah et al., 2009), cognitive decline (Silbert et al., 2008), and dementia (Kester et al., 2014). With the emerging trend of social aging, the incidence of WMH-related diseases may increase and these may pose a serious threat to human health. The pathogenesis of WMH remains unclear; however, chronic cerebral ischemia, hyperperfusion injury, endothelial dysfunction, direct damage to the blood-brain barrier, and inflammatory and genetic factors are hypothesized to be potential causes of WMH (Joutel & Chabriat, 2017).
Homocysteine (Hcy) is a sulfur-containing amino acid formed during methionine metabolism. When the metabolic pathway for methionine is impaired, the plasma concentration of Hcy increases, which may induce a series of vascular pathological changes. In recent years, studies have reported that increased Hcy levels were associated with a high WMH volume in both the general population and patients with stroke, with the pathophysiological mechanism for this being related to endothelial dysfunction leading to microvascular disease (Naveed & Bokhari, 2015;Vermeer et al., 2002;Wright et al., 2005). However, data on the relationship between Hcy and WMH are still lacking in patients with asymptomatic intracranial arterial stenosis (aICAS). As an intermediate state, research about the relationship between Hcy and WMH in this period will help us obtain more information that cannot be obtained from studies on the general population and clinical stroke patients. This may be very helpful for a comprehensive understanding of the relationship between Hcy and WMH. It is assumed that in participants with aICAS, high levels of Hcy may be associated with an increase in WMH volume. Moreover, considering that intracranial stenosis may play an important role in the pathophysiology of WMH, and patients with severe ICAS are more likely to have more serious WMH (Park et al., 2015), it is hypothesized that the effects of Hcy on WMH may be more pronounced in patients with severe stenosis.
Therefore, in the present study, we tested this hypothesis by investigating the association between Hcy and WMH in rural-dwelling Chinese people with aICAS.

| Study population
The current participants originated from the Kongcun Town Study, an ongoing longitudinal study mainly aimed to investigate: (1) the occurrence and distribution of aICAS; (2) major cardiovascular risk factors or biomarkers related to the development and prognosis of aICAS. The detailed study design of Kongcun Town Study has been described previously Xue et al., 2020). In brief, from October 2017 to May 2018, all participants aged ≥40 years who were free of a history of clinical stroke or transient ischemic attack (TIA) were enrolled. Participants with severe cardiopulmonary insufficiency, vision, or hearing impairments were excluded. The baseline investigation of the Kongcun study included two phases: The phase 1 assessment aimed to collect data (demographic features, health history, lifestyle factors, etc.) and identify people at high risk of aICAS using transcranial Doppler (TCD) examination. In phase 2, those who screened positive for aICAS further underwent structural brain MRI and magnetic resonance angiography (MRA). After a baseline line investigation, 204 participants were positive for aICAS by TCD; of them, 169 participants completed MRI/MRA. Among these 169 participants, 15 subjects were excluded because of previous silent cerebral infarction detected by MRI, and finally, 154 were diagnosed with aICAS. All the 154 participants were included in the present study. Of the 154 participants, 10 participants were excluded because of missing Hcy data, and another seven participants were excluded due to unqualified MR images. Consequently, all analyses were based on 137 participants with complete baseline data on Hcy and MR images.
Conclusion: Serum Hcy concentration may be positively associated with the volume of WMH in rural-dwelling Chinese people with moderate-to-severe (stenosis ≥50%) aICAS.

K E Y W O R D S
arteriosclerosis, asymptomatic intracranial arterial stenosis, homocysteine, population-based study, white matter hyperintensities

| Ethics approval and consent to participate
The study protocol was reviewed and approved by the ethical standards committees on human experimentation at Shandong Provincial Hospital Affiliated to Shandong First Medical University. Written informed consent was obtained from all the participants.

| Assessments of aICAS
Asymptomatic intracranial arterial stenosis was defined as ICAS without a history of stroke or TIA and previous silent cerebral infarction detected by MRI in the present study. A self-report of a physician diagnosis and/or clinical examinations showing typical symptoms were considered as a history of stroke or TIA, which was determined during the interviews and clinical examination. Participants were examined using a portable TCD machine (VIASYS Companion III, Nicolet, Washington, United States) by two experienced physicians.
Those who screened positive according to the criteria for intracranial stenosis by TCD further underwent MRA (Feldmann et al., 2007).
The extent of stenosis in the five evaluated arteries (bilateral middle cerebral arteries, bilateral intracranial segment of internal carotid arteries, and basilar artery) was classified into five grades based on the signal strength of MRA detection: normal, mild (signal reduction <50%), moderate (signal reduction ≥50% and <70%), severe (signal reduction ≥70%), or occlusion (focal signal loss with the presence of distal signal). Detailed information on the assessments of aICAS can be found in previous studies .

| WMH volume measurement
Participants who screened positive by TCD received a brain MRI assessment using a 3-T magnetic resonance scanner (Philips, Achieva, Netherlands), including T1-weighted images, T2-weighted images, proton density images, T2 fluid-attenuated inversion recovery images, and diffusion-weighted imaging, as described previously . WMH was calculated by two trained investigators and a neuroradiologist blinded to clinical information using the lesion segmentation tool (LST) for the Statistical Parametric Mapping package (SPM) (Schmidt et al., 2019). WMH was defined as hyperintensities on proton density and T2-weighted images, with no prominent hypointensity on T1-weighted images. The volume of WMH was divided into four categories; patients with WMH volumes in the highest (4th) quartile were classified into the severe WMH group (≥4.20 ml). Figure 1 shows a flowchart of the WMH volume using LST auto-calculation.

| Hcy and covariates
Trained investigators conducted face-to-face questionnaires, physi- Step two: L-cystathionine was further transformed into Hcy, pyruvic acid, and NH 3 under the catalysis of cystathione β-catabolase; Step three: pyruvate produced by the F I G U R E 1 Flowchart of the measurement of white matter hyperintensities volume using lesion segmentation tool auto-calculation system above cyclic reaction was detected by lactic dehydrogenase and oxidized nicotinamide adenine dinucleotide (NADH), and the rate of conversion of NADH to nicotinamide adenine dinucleotide was directly proportional to the Hcy content. High levels of Hcy (hHcy) were defined as serum Hcy ≥15 μmol/L, which is a commonly used standard (Sachdev, 2004;Wright et al., 2005). The height and weight of participants were measured without shoes, and body mass index (BMI) was calculated (kg/m 2 ). Blood pressure (mmHg) was measured twice with a sphygmomanometer after a 5-min rest each time.
Hypertension was defined as blood pressure ≥140/90 mmHg or the use of antihypertensive drugs. Diabetes mellitus was defined as a history of diabetes, a glucose level of ≥7.0 mmol/L, or self-reported use of oral antidiabetic drugs or insulin. Hypercholesterolemia was defined as total cholesterol ≥6.20 mmol/l or an ongoing prescription of hypolipidemic drugs. Smoking was assessed by enquiring about the amount of packs smoked per year, and alcohol intake was categorized as never, former, or current. Carotid ultrasound examination was performed by two experienced physicians in a local clinic to measure extracranial arterial stenosis (ECAS) with a 7 MHz linear transducer (Siemens ACUSON P500) according to the peak systolic blood flow velocity (Grant et al., 2003). A diagnosis of ECAS was established when one or more of the following arteries were observed to be stenotic to any degree by ultrasound: the common carotid artery, ICA, and external carotid artery.  Compared to patients in the 1st -3rd quartile group, patients in the 4th quartile group were older (p < .001), had a higher systolic pressure (p = .003) and were more likely to smoke (p = .003) and drink (p = .017). The mean serum Hcy level was also significantly higher in the 4th quartile group than in the 1st -3rd quartile group (p <.001). The proportion of participants with hHcy was also significantly higher in the 4th quartile than in the 1st -3rd quartile group (p < .001). There were no significant differences in other variables between the two groups ( Table 1).

| RE SULTS
The multiple regression analysis showed that when WMH was analyzed as a continuous variable, Hcy was not significantly associated with WMH after adjusting for covariates. However, as a categorical variable (for 4th and 1st-3rd quartile groups), the serum Hcy level was independently associated with a high volume of WMH

| D ISCUSS I ON
The results of this study revealed that serum Hcy levels may be independently associated with the severity of WMH in rural Chinese people with moderate-to-severe ICAS (MRA signal reduction ≥50%).
Previous studies on the impact of Hcy on WMH have been carried out in the general population, with inconsistent results. The Northern Manhattan Study found that higher levels of Hcy were associated with WMH after adjusting for sociodemographics and vascular risk factors in a community-based sample (Wright et al., 2005).

Several other studies involving community-based participants
also reported a similar association between Hcy and WMH (Raz et al., 2012;Sachdev et al., 2004;Vermeer et al., 2002). However, two studies failed to find any association between WMH and Hcy in general participants, one study aimed to evaluate the relationship between Hcy levels and findings on brain MRI and found that higher plasma Hcy levels are associated with a smaller brain volume but not with WMH (Seshadri et al., 2008). In another study, after adjusting for age and sex, Hcy levels were not found to be associated with WMH (Longstreth et al., 2004). Different study populations, age ranges, sample sizes, and ethnicities may have led to the differing results in these studies.

TA B L E 2 Association between Hcy and WMH by generalized regression analysis
Unlike the results drawn for general participants, in crosssectional studies involving patients with stroke (Tseng et al., 2009), Parkinson's disease (Shen et al., 2019), or rheumatoid arthritis (Anan et al., 2009), researchers have reached a relatively consistent conclusion that Hcy is a risk factor for cerebral white matter lesions.
Prospective studies have also obtained similar results. For example, in a longitudinal population-based study involving participants aged 60 years or older who were free of dementia, the total Hcy concentration was confirmed to be related to the progression of WMH (Hooshmand et al., 2016). Our study is consistent with these studies, and to the best of our knowledge, this is the first study to focus on the association between Hcy and WMH in participants with aICAS.
A noteworthy finding of our study was that the relationship between Hcy and WMH was only significant in the moderate-to-severe stenosis (≥50%) aICAS group after adjusting for confounders. This finding suggests that, to a certain extent, stenotic intracranial arteries may be involved in the pathophysiology of WMH caused by Hcy, even in the absence of clinical stroke. Compared with general participants or those with mild stenotic aICAS, participants with greater than 50% stenosis of an intracranial artery may have more serious vascular pathological changes, including large conduit vessels and microcirculation (Ritz et al., 2014), which may lead to the brain tissue being more vulnerable to abnormal Hcy levels. The mechanisms by which Hcy causes the incidence of WMH are: 1) inhibition of endothelial nitric oxide via the production of reactive oxygen species or the accumulation of asymmetric dimethylarginine, leading to functional suppression of the blood-brain barrier (BBB) (Kamath et al., 2006;Woo et al., 1997), infiltration of toxic materials into neural tissues, and blockage of interstitial fluid clearance (Doubal et al., 2010;Hainsworth & Fisher, 2017;Hassan et al., 2003); 2) activation of the coagulating pathway by increasing the expression of prothrombotic factors, such as β-thromboglobulin, tissue plasminogen, and factor VIIc (Nishinaga et al., 1993;Schreiner et al., 2002); and 3) promoting the growth of vascular smooth muscle cells (Chen et al., 2000).
This study has the following strengths: First, to the best of our knowledge, this is the first study to explore the association between Hcy and WMH in rural-dwelling Chinese people with aICAS.
As a high-risk group for stroke, research on these participants helps us provide more data on the relationship between Hcy and WMH, and further explore the possible mechanisms for Hcy causing WMH.
Second, the use of automated, quantitative measurements of WMH avoided the subjective factors of previously used semiquantitative methods and may have produced more reliable results. For example, in another study using automatic quantitative segmentation methods to measure the volume of WMH, the upper quartile range of WMH volume was greater than or equal to 5.93 ml (4th Q ≥ 5.93 ml), which is basically consistent with the ranges of the present study (Wong et al., 2006). Indeed, the lesion prediction algorithm implemented in SPM's LST toolbox has been shown to produce consistently good performance and in many cases is considered a robust gold standard for lesion segmentation (Guerrero et al., 2018). However, there are several limitations to be addressed. First, when WMH was used as however, data on serum folate and vitamin B12 levels are lacking in the present study, and the effect of these vitamins on the variation of Hcy level could not be examined. Third, it has also been reported that the association between Hcy and WMH may differ depending on the location of the brain being examined (Gao et al., 2015). Our study did not compare the incidence of WMH in different locations (periventricular versus. deep WMH); therefore, the association between WMH and Hcy in different parts of the brain could not be ascertained.

| CON CLUS ION
In conclusion, the present study showed that Hcy is a risk factor for white matter lesions in patients with moderate-to-severe aICAS (≥50%) in rural Chinese dwellings. Future prospective studies with a larger sample size are warranted to confirm our findings. At the same time, further studies including healthy individuals without ICAS will help to clarify whether aICAS is involved in the pathophysiological pathway of WMH caused by Hcy.

ACK N OWLED G M ENTS
We would like to thank all the study participants, staff of the participating hospitals, and the Steering Committee Members of this study.

CO N FLI C T O F I NTE R E S T S
The authors declare that they have no competing interests.

E TH I C A L A PPROVA L
This study was approved by the ethical standards committee on human experimentation at Shandong Provincial Hospital Affiliated to Shandong First Medical University.

CO N S E NT TO PU B LI S H
Not applicable.

PE E R R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1002/brb3.2205.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.