Relationship between homocysteine levels and post-stroke cognitive impairment in female and male population: from a prospective multicenter study

INTRODUCTION

Minor stroke and transient ischemic attacks (TIAs) are the common manifestations of acute cerebrovascular events.^[1] Post-stroke cognitive impairment (PSCI) is one of the major residual impairments and is associated with functional outcomes and survival in minor stroke and TIA patients.^[2,3] Homocysteine is an important intermediate in methionine, folate, and one-carbon metabolism, and elevated homocysteine increases the risk of stroke and age-associated cognitive impairment.^[4–7] Homocysteine may be a prognostic biomarker or a modifiable target for preventing PSCI.^[8]

Previous studies that demonstrated the relationship between homocysteine and PSCI have yielded inconsistent results.^[8–12] A cohort study (n = 81)^[11] demonstrated that homocysteine was associated with vascular dementia at 3 months after stroke. A case–control study (82 cases vs. 80 controls)^[10] demonstrated that homocysteine levels were correlated with cognitive impairment after stroke at 3-month follow-up.^[10] However, a larger sample size case–control study (169 cases vs. 103 controls)^[12] demonstrated opposite results that there were no associations between homocysteine and PSCI after 3–6 months. A prospective cohort study (n = 251) demonstrated that homocysteine levels at 3 months had no relationship with vascular dementia at 3-month follow-ups after stroke.^[9] In summary, the evidence on the relationship between homocysteine levels and PSCI is limited. Also, (1) previous studies had small sample size and inconsistent conclusions in the relationship between homocysteine and PSCI and (2) cognitive follow-ups in previous studies exploring the relationship between homocysteine levels and PSCI were for short term (3–6 months). Additionally, homocysteine concentration was associated with sex.^[13,14] Females had lower homocysteine levels than males.^[15–18] But females were more susceptible to the damaging effect of homocysteine in vascular diseases, such as the risk of ischemic stroke and poor prognosis (death and major disability) of acute ischemic stroke, than males.^[15,16,18] But the correlation of homocysteine and PSCI has not been elucidated in female and male populations.

Therefore, in this study, we aimed to explore the association between the homocysteine levels and the risk of short- and long-term PSCI in male and female patients with minor acute ischemic stroke or TIA in a prospective multicenter cohort study.

MATERIALS AND METHODS

Study design

The Impairment of Cognition and Sleep after acute ischemic stroke or transient ischemic attack in Chinese patients (ICONS) study is a nationwide multicenter, prospective registry study investigating the occurrence and associated factors of cognitive impairment and sleep disorder after acute ischemic stroke or TIA.^[19] The rationale and design of the ICONS have been described in detail elsewhere.^[19] The ICONS study enrolled 2625 subjects and excluded prior diagnosis of cognitive impairment. The study was performed according to the principles expressed in the Declaration of Helsinki. The protocol of the ICONS study was approved by the ethics committee of a hospital (blinded as requested; Institutional Review Board approval number KY2015-001-01) and all participating centers. All participants obtained written informed consent from patients or legally authorized representatives.

In the current study, patients who had baseline homocysteine levels and cognitive assessment at 3 or 12 months were included. A total of 1070 participants with clinically confirmed acute minor ischemic stroke or TIA and baseline homocysteine information were included in this study, of which 919 patients had 3-month cognitive assessments and 584 participants had 12-month cognitive assessments. Figure 1 shows the flowchart of participant enrollment.

Figure 1.

Flowchart of participant enrollment. Hcy: homocysteine; ICONS: The Impairment of Cognition and Sleep after acute ischemic stroke or transient ischemic attack in Chinese patients; MoCA: Montreal Cognitive Assessment; NIHSS: National Institutes of Health Stroke Scale; TIA: transient ischemic attack.

RESULTS

Relationship between baseline homocysteine level and the incidence of long-term PSCI

Compared to females with unelevated homocysteine, females with elevated homocysteine had a higher 12-month PSCI incidence rate (18.5% vs. 40.0%). There was no significant difference in 12-month PSCI incidence between unelevated and elevated homocysteine in males (Figure 2).

Figure 2.

Incidence rates of post-stroke cognitive impairment at 3 months (A) and 12 months (B) grouped by sex and homocysteine levels. Hcy: homocysteine.

Table 3 shows that female patients with elevated homocysteine had a higher risk of 12-month PSCI (OR 2.93, 95% CI 1.42–6.07; P = 0.004), which was kept (OR 3.28, 95% CI 1.47–7.34, P = 0.004) when age, education, NIHSS, diabetes mellitus, smoking, baseline levels of folate, vitamin B12, eGFR, and MTHFR C667T genotype were adjusted. The findings based on homocysteine tertiles, elevated homocysteine levels monotonically increased the risk of PSCI (P trend = 0.005). There was no relationship between homocysteine and the risk of 12-month PSCI in males. The same pattern of results was observed when homocysteine was categorized as tertiles.

The current findings remained the same when additional linear regression models were used to explore the relationships between baseline homocysteine and MoCA at 12-month follow-up. The linear regression results showed that higher homocysteine levels were associated with lower MoCA scores in females (β = −0.54, 95% CI −0.92 to −0.16, P = 0.006; Table S3), but not in males (β = 0.32, 95% CI −0.08−0.72, P = 0.113). In addition, a non-linear analysis also showed that the risk of developing PSCI increased with the rise of homocysteine concentration in females, but not in males (Figure S1).

DISCUSSION

In this study, we demonstrated that females had lower baseline homocysteine levels than males. Elevated homocysteine levels at baseline had no relationship with PSCI at short term (3 months). Elevated homocysteine levels were significantly associated with the risk of long-term (12-month) PSCI in females with minor acute ischemic stroke or TIA, but not in males. Additional adjustment for sociodemographics and vascular risk factors still supported the relationship between homocysteine and PSCI. Linear regression analysis and restricted cubic spline (non-linear) analysis further confirmed the sex-specific relationship between homocysteine and 12-month follow-up PSCI.

In the current study, we conducted cognitive follow-ups at two time points (3 and 12 months), which allowed us to explore both short- and long-term effects of homocysteine on the PSCI. Different factors were associated with PSCI in the short term and long term.^[23,24] Previous observational studies using a small sample size (<200 subjects)^[10,11] demonstrated that homocysteine was associated with vascular dementia or PSCI in the short term. However, a prospective cohort study^[9] or a case–control study^[12] based on large sample sizes (200–300 cases) demonstrated that there was no association between homocysteine and PSCI at 3–6 months, which is consistent with the findings in our study using data from a large national prospective stroke cohort (943 cases).

We found that elevated homocysteine was associated with long-term (12-month) PSCI, but not with short-term (3-month) PSCI in females. There are two consensuses that put forward the concept of classifying PSCI according to the time of appearance of cognitive impairment after stroke.^[25,26] It is divided into early-onset (3–6 months after stroke) and delayed-onset (6 months or more after stroke) PSCI, which have different mechanisms. Early-onset PSCI depends on a complex interplay between features of the stroke lesion, such as large size or strategic site, and brain resilience, such as cognitive reserve and brain reserve. Delayed-onset PSCI is driven mostly by severe small vessel disease or concurrent Alzheimer disease pathology. Neuroimaging studies have demonstrated that homocysteine contributes to small vessel disease, including white matter lesions, silent infarcts, and brain atrophy.^[27,28] Also, homocysteine showed significant associations with Alzheimer’s disease and homocysteine-lowering treatments are recommended to prevent Alzheimer disease in randomized controlled trials and meta-analysis.^[5,29] So we speculated that elevated homocysteine might promote PSCI through small vessel disease or Alzheimer disease pathology, which may explain our finding that homocysteine levels are associated with long-term incidence of PSCI.

In the current study, elevated homocysteine was associated with PSCI in females, but not in males. Accumulating evidence suggests that homocysteine promoted white matter lesions and PSCI pathogenesis by endothelial dysfunction.^[30–32] Estrogen is thought to protect vascular endothelial cells.^[33] In postmenopausal females, the estrogen levels decrease^[13] and homocysteine levels increase,^[33] which may aggravate endothelial dysfunction and speed up the progress of PSCI. In the present study, average age of females was above 60 years, which is a postmenopausal state. The decreasing estrogen levels in females, which leads to elevated homocysteine levels and weakened endothelial protective effect, may be one explanation for the sex difference observed in the association of homocysteine and PSCI. Elevated homocysteine caused by low folic acid was reported to have a higher risk of the 5-year composite vascular outcome, including vascular dementia, vascular cognitive impairment, or fatal stroke among females, but not in males,^[34] which could support the findings in our study. Other evidences showed that females were more susceptible to the damaging effect of homocysteine in PSCI than males, which could also be supported by previous clinical findings that elevated homocysteine was a stronger risk factor in females than in males in vascular disease,^[33] including ischemic stroke incidence,^[15] poor prognosis (death and major disability) after stroke onset,^[16] and neurodegenerative cognitive impairment.^[17]

Our research had several limitations. Firstly, although folate, vitamin B12, renal function, and MTHFR genotype were taken into consideration, homocysteine levels are also affected by diet, vitamin supplements, gastrointestinal factors that lead to B vitamins’ malabsorption, and other genetic mutations (e.g., the cystathionine β-synthase genotype), of which data were not collected during the follow-up. Secondly, magnetic resonance imaging is an efficient instrument to evaluate brain lesions.^[35] However, brain magnetic resonance imaging data of 12-month follow-up were not available. Therefore, we cannot evaluate brain structure changes, especially white matter lesions and brain atrophy, which might be associated with high homocysteine levels and cognitive impairments. Further studies are warranted to evaluate the potential intermediary role of brain structural changes in the relationship between homocysteine and long-term PSCI. Thirdly, one of the inclusion criteria was to be enrolled within 7 days after the onset of stroke or TIA in this prospective registry ICONS study. The mean (interquartile range) hours from event onset to hospitalization was 13.6 (3.2–46.7) in patients with 3-month cognitive follow-up and 16.4 (3.3–47.0) with 12-month cognitive follow-up. The blood samples used to test homocysteine were obtained on the second day of hospitalization. Hence, there was time delay from stroke or TIA onset to admission homocysteine levels. Although due to the practical limitation of the time interval between hospitalizations of stroke patients, homocysteine in similar studies also had a certain time delay.^[7,22,36] This may be a factor to accurately reflect the levels of homocysteine at stroke onset. Collecting blood samples within 24 hours after the stroke onset in further studies may remedy this limitation. Fourthly, 45.9% (491/1070) of patients were lost to follow-up in MoCA and could not be included in the long-term PSCI analysis. Compared with the included population, the excluded population has milder stroke symptoms, lower homocysteine levels, and higher folic acid and vitamin B12 levels, which demonstrated that there is a selection bias due to loss to follow-up. The median homocysteine of the included group with 12-month MoCA was 17.2 μmol/L. The research conclusions based on the included group are restrictive to the population with high homocysteine and cannot be extended to other populations.

In conclusion, elevated homocysteine levels at baseline were independently associated with 12-month PSCI in female patients with minor acute ischemic stroke or TIA, which should be further validated in future clinical studies.