The present study of over 2000 acute AIS patients had demonstrated an association between elevated tHcy and in-hospital outcomes. We found that having elevated tHcy at baseline was independently associated with the occurrence of in-hospital pneumonia, higher risk of in-hospital mortality as well as poor functional outcome at hospital discharge among AIS patients. Moreover, having pneumonia during hospitalization did not modify the effect of elevated tHcy on patient outcome.
Total homocysteine is a sulfur-containing amino acid derived from methionine metabolism, and was an independent risk factor for stroke [5, 6]. Interestingly, several studies suggested elevated tHcy may reflect inflammation as other markers like hs-CRP [14–17]. A study of 977 acute AIS patients showed a combination of increased hs-CRP, tHcy, or white blood cell had a stronger predictive effect on 1-year poor outcome than individual elevated mediators . Another study from China also found a concomitant effect of hs-CRP and tHcy on 1-year post-stroke depression . Moreover, two studies suggested an association between early tHcy increase and poor outcome in patients with sepsis, and having elevated tHcy was a marker of spontaneous bacterial peritonitis in cirrhotic ascites patients [16, 17]. In our study, we found acute AIS patients with elevated tHcy level had a 1.55-fold increased risk of in-hospital pneumonia. Our study is the first to reveal an association between elevated tHcy and the risk of pneumonia, consistent with previous reports of tHcy as a useful inflammatory marker.
Several studies had investigated the prognostic effect of elevated tHcy at admission on the functional outcome among patients with acute AIS. However, the findings from these studies were inconsistent [7–13, 22]. A secondary analysis of the China Antihypertensive Trial in Acute Ischemic Stroke (CATIS) trial of 3309 AIS patients indicated higher tHcy was associated with worse acute AIS prognosis in women at 3-month follow-up . A study of 3799 AIS cases suggested that having elevated tHcy levels could predict mortality, especially in stroke patients with the large-vessel atherosclerosis subtype during 48 months follow-up . Other studies also noted that the higher level of tHcy may predict early neurological deterioration  and poor functional outcome at hospital discharge . However, no significant association between higher tHcy and poor functional outcome was found in other studies [11–13, 22]. In a study which recruited 594 elderly AIS patients, elevated tHcy at admission was not shown to be a predictor of outcome at 3 months and 1 year . A study of 775 AIS patients in Italy demonstrated higher tHcy was not associated with stroke severity nor outcome measured by the Barthel Index at 6 or 12 months . In our study, we found elevated tHcy level was associated with a 3.35-fold and 1.50-fold increased risk of in-hospital mortality and poor functional outcome at hospital discharge respectively, and these associations were significant in different subgroups and in patients with pneumonia. Variations in sample size, population, definition of clinical outcomes and different statistic methods may have led to discrepant findings from previous studies.
In present study, we also found an interaction between high tHcy and gender on in-hospital mortality. The association between high tHcy and increased risk of in-hospital mortality was observed only in females, but not males. Similar interesting phenomenon was seen in other studies [7, 23]. Zhong et al. using CATIS trial database had demonstrated elevated tHcy was associated with poor prognosis of acute AIS only in women  and a Swedish study also reported an association between higher tHcy and increased risk of 5-year mortality only in women . The mechanism of gender-specific relationship between elevated tHcy and in-hospital mortality is unclear, partially due to early endothelial dysfunction caused by tHcy [24, 25] in women.
The exact mechanisms underlying the relationship between elevated tHcy levels and poor functional outcome after AIS is not yet fully understood. Several hypotheses have been proposed. First, previous studies showed a significant association between elevated tHcy and the presence of cerebral small vessel disease [26, 27], which is a known predictor of poor outcome and mortality after stroke [28, 29]. Second, animal studies indicated elevated tHcy significantly increased the blood-brain barrier dysfunction [30, 31], which may lead to poor functional outcome after stroke. Third, elevated tHcy was reported to increase oxidative stress  and exaggerates microglia activation and neuroinflammation after stroke .
Strengths of our study include having a relatively large number of patients from multiple centers and using sensitivity and subgroup analysis, which enhances the generalizability of the results. The present investigation is also the first to have found an independent association between elevated tHcy and pneumonia during hospitalization. However, there are also several potential limitations. First, a significant proportion of patients were excluded due to lack of homocysteine data, this may cause selection bias and contribute to a lower rate of in-hospital mortality compared to our previous studies. Secondly, in our study some patients had time from onset to admission which exceeded 24 hours, therefore, the levels of tHcy at admission might not accurately reflect the levels at stroke onset. However, our sensitivity analysis showed that the significance of the association remained when we restricted to patients with time from onset to admission ≤ 24 hours. Thirdly, data on pre-stroke disability and Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification, which may also influence in-hospital mortality. Moreover, the level of Vitamin B12 and folic acid, which are significant associated with tHcy were not collected. Finally, the follow-up period of our study is relatively short, thus we were unable to evaluate the combined long-term effect of tHcy on AIS outcomes and lacked information on exact cause of death.