Association between serum free hemoglobin level and cerebral white matter hyperintensity volume in older adults

The association between serum free hemoglobin (sfHb) level and white matter hyperintensity (WMH) volume is controversial. This study is to examine this association considering nonlinearity, sex dimorphism, and WMH type. We enrolled 704 older adults among the participants of the Korean Longitudinal Study on Cognitive Aging and Dementia and visitors to the Dementia Clinic of Seoul National University Bundang Hospital. We measured sfHb level in the venous blood and WMH volume (VWMH) using fluid-attenuated inversion recovery magnetic resonance images. The association between sfHb level and periventricular VWMH was linear in men (linear regression; β =  − 0.18, p = 0.006) and U-shaped in women (restricted cubic spline; F = 6.82, p < 0.001). sfHb level was not associated with deep VWMH in either sex. These findings were also observed in participants without anemia. To conclude, sfHb level is associated with periventricular VWMH in older adults of both sexes. Maintaining an optimal sfHb level may contribute to the prevention of WMH.


Scientific Reports
| (2022) 12:3296 | https://doi.org/10.1038/s41598-022-07325-x www.nature.com/scientificreports/ sfHb level and prevalence of anemia are different between men and women 6 . Third, two studies examined the linear relationship between sfHb level and V WMH only 15,16 despite that the relationship between them may be non-linear 12 . Fourth, two studies measured the severity of cerebral WMH using semi-quantitative visual ratings instead of V WMH 15,16 . Despite of its clinical usefulness, the visual ratings on the severity of cerebral WMH are less sensitive to subtle differences in V WMH compared to the fully quantitative volumetric measures 18 .
This study investigated whether the association of sfHb with V WMH are different by the type of cerebral WMH and the sex of the participants under following hypotheses: (a) lower sfHb level is associated with higher V WMH ; (b) the association between sfHb level and V WMH may differ between men and women; and (c) the association of sfHb level with the volume of PVWMH (V PVWMH ) may be different from that with the volume of DWMH (V DVWMH ).

Methods
Study design, setting, and participants. This cross-sectional study included 358 participants from the Korean Longitudinal Study on Cognitive Aging and Dementia (KLOSCAD) and 346 visitors to the Dementia Clinic at Seoul National University Bundang Hospital (SNUBH) from 2010 to 2020. The KLOSCAD is an ongoing prospective cohort study that randomly samples 6818 community-dwelling Korean older adults. The baseline assessment of the KLOSCAD was conducted from 2010 to 2012 and four consecutive 2-year follow-up assessments had been completed in 2013-2021 19 . The participants of KLOSCAD took one or more brain scan at the baseline or follow-up assessments. The first brain scan of each participant was collected for the current study when the participant took two or more brain scans. Data were collected from the follow-up assessments as well as the baseline assessment of the KLOSCAD because some participants took their first brain scan in the baseline assessment while others took their brain scan at one of follow-up assessments. All participants were aged 55 years or older. The participants with a history of hematological (e.g., myelodysplastic syndrome, multiple myeloma, and polycythemia vera) and other malignancies, gastrointestinal tract resection, traumatic brain injury, multiple sclerosis, brain tumor, intellectual disability, and any dementing illnesses were excluded. Compared to the participants from the KLOSCAD, those from the SNUBH Dementia Clinic were older and showed higher prevalence of mild cognitive impairment, systolic blood pressure, V WMH and V PVWMH but lower burden of comorbidities and better estimated glomerular filtration rate (Supplementary Table S1).
This study was approved by the Institutional Review Board of Seoul National University Bundang Hospital (B-0912-089-010). All participants were fully informed about the study protocol and provided written informed consent.
Clinical assessments. Geriatric psychiatrists conducted face-to-face standardized diagnostic interviews, physical and neurologic examinations, and routine laboratory tests to all participants using the Korean version of the Consortium to Establish a Registry for Alzheimer's Disease Assessment Packet Clinical Assessment Battery 20 . A consensus panel of geriatric psychiatrists determined the diagnoses of dementia according to the Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV) criteria. Trained research nurses assessed the prior and current history of medical illnesses and the burden of comorbidities using the Cumulative Illness Rating Scale (CIRS) 21 . The trained research nurses also measured the mean systolic and diastolic blood pressure and body mass index (BMI). The estimated glomerular filtration rate was calculated from the serum creatinine level, age, and sex 22 . Assessment of sfHb level and anemia. After overnight fasting, trained research nurses collected venous blood samples from the participants for routine laboratory tests, including those for complete blood cell counts, total and high-density lipoprotein cholesterol levels, chemistry panel, thyroid function, and serum folate and vitamin B12 levels and serological tests for syphilis screening. The sfHb concentration in the venous blood was measured by using an ADVIA 2120i system (Siemens, Germany) for the KLOSCAD samples and an XN-9000 Hematology Analyzer (Sysmex, Japan) for the Clinic samples. The anemia was defined as the sfHb level of < 13 g/ dL in men and < 12 g/dL in women according to the World Health Organization criteria 9 .
To segment the WMH, first, we obtained FLAIR images of all participants and we applied a bias correction on the images to correct non-uniformities caused by the bias field owing to different tissue properties and physics of MRI, using the "Segment" tool from Statistical Parametric Mapping software version 8 (SPM8, Wellcome Trust Centre for Neuroimaging, London, UK). We then segmented WMH from bias-corrected FLAIR images by using a fully automated in-house code run on MATLAB 2014a (MATLAB and statistics Toolbox Release 2014a, MathWorks, Inc., Natick, MA) which has been previously shown to work on different protocols from different scanners without any parameter adjustment 23 . The algorithm started with the brain extraction from FLAIR images and computed the surrogate for the V WMH . The algorithm calculated the optimal threshold intensity for classifying WMH with intensities of normal distribution that corresponded to normal tissues and WMH by applying Bayesian decision rule. It then removed false positive WMH which was located at gray matter or noncerebral region. We calculated the total V WMH . Meanwhile, we segmented lateral ventricle of every participant from T1-weighted MR images and measured intracranial volume using Freesurfer software version 5.3.0 (http://  24 . We classified PVWMH as WMH within 13 mm from lateral ventricle, else was classified as DWMH.

Statistical analysis. We compared the continuous variables and categorical variables between groups using
Student's t-tests and Pearson's chi-square tests, respectively. We examined the association between sfHb level and cerebral V WMH using linear regression and restricted cubic spline analyses. In both analyses, we adjusted for age, hypertension, diabetes, stroke, CIRS score, drinking status, smoking status, systolic blood pressure, BMI, total cholesterol level, high-density lipoprotein cholesterol level, glomerular filtration rate, and cohort as covariates.
In these regression analyses, V WMH was normalized to individuals' intracranial volumes and log-transformed.
As sensitivity analysis, we performed the same linear regression analysis and restricted cubic spline analysis in the following two subgroups of participants as in the total participants: (1) subgroup 1 without anemia or iron supplementation (N = 612) and (2) subgroup 2 without a history of stroke (N = 680). The knots for restricted cubic spline analyses were set at the 5th, 35th, 65th, and 95th percentiles 25 , and the sfHb levels at the knots were provided in Supplementary Table S2. We performed all statistical analyses using STATA 12.1 (StataCorp LP; College Station, Texas).

Ethics approval.
This study was performed in line with the principles of the Declaration of Helsinki.
Approval was granted by the Institutional Review Board of Seoul National University Bundang Hospital (B-0912-089-010).
Consent to participate. All participants were fully informed about the study protocol and provided written informed consent.

Results
As summarized in Table 1, female participants had a lower sfHb level than male participants. However, the frequency of anemia was comparable between sexes. Female participants showed smaller intracranial volume and V WMH than male participants. However, after adjusting for intracranial volume, the V WMH was comparable between them (p = 0.201 for total WMH; p = 0.208 for PVWMH; p = 0.770 for DWMH).
In male participants, the linear regression models demonstrated that sfHb level was negatively associated with the log-transformed V WMH and V PVWMH . However, this association was not observed for the log-transformed V DWMH (Table 2). Similar findings were observed for the restricted cubic spline models adjusted for the same covariates as those used in the linear regression models (Table 3). www.nature.com/scientificreports/ In female participants, the associations between sfHb level and the log-transformed V WMH , V PVWMH , and V DWMH were not significant in the regression models. However, the associations between sfHb level and the log-transformed V WMH and V PVWMH were significant in the restricted cubic spline models adjusted for the same covariates as those used in the linear regression models, indicating that the association may be nonlinear ( Table 3). As demonstrated in Fig. 1, which shows the fitted regression with 95% confidence intervals estimated from the restricted cubic spline models, the associations between sfHb level and the log-transformed V WMH and V PVWMH were linear in male participants and U-shaped in female participants.
These results did not change when we analyzed subgroups 1 and 2 separately (Tables 2, 3).

Discussion
The results of this study in older adults showed that the association between sfHb level and V WMH was sexually dimorphic and confined to PVWMH. The sfHb level showed a negative linear association with V PVWMH in men and a U-shaped association in women. Furthermore, these associations remained significant when participants without anemia were analyzed separately. To our knowledge, this is the first study to identify that the association between sfHb level and V WMH differed by the type of WMH. Our findings demonstrated that a lower sfHb level was associated with increasing V PVWMH only in both sexes. This result is in line with a previous study on stroke patients. In stroke patients, sfHb level was associated with V PVWMH , but not with V DWMH 26 . When the sfHb level decreases, cerebral blood flow increases to compensate for cerebral deoxygenation 7,27 . As the microvasculature in white matter is scarcer and its' connection to the pial capillary plexus is longer, more indirect and tortuous than those in gray matter 28,29 , the compensatory increase of cerebral blood flow against deoxygenation associated with low sfHb level in white matter is far lower than that in cerebral gray matter 30 . Within cerebral white matter, periventricular white matter is more vulnerable to focal or systemic cerebral hypoperfusion than deep white matter because periventricular white matter has less collateral blood supplies for ventriculofugal vessels than deep white matter 31 . PVWMH was associated with decreased cortical blood flow but DWMH was not 17 . The current study suggests that dynamic autoregulation of cerebral blood flow against altered sfHb levels may be more decompensated in periventricular white matter than in deep white matter. The impaired dynamic autoregulation of cerebral blood flow is closely associated with the cerebrovascular risk factors known to increase dementia risk 32 , and that could partially explain why the PVWMH is more strongly associated with the risk of dementia than DWMH 5 . Maintaining optimal sfHb level may be beneficial to reduce the risk of PVWMH and cognitive decline associated with PVWMH in older adults. Table 2. Linear association between serum free hemoglobin level and white matter hyperintensity volume. Hb hemoglobin, V WMH volume of total white matter hyperintensity, V PVWMH volume of periventricular white matter hyperintensity, V DWMH volume of deep white matter hyperintensity. *Adjusted for age, hypertension, diabetes, stroke, cumulative illness rating sore, drinking status, smoking status, systolic blood pressure, body mass index, total cholesterol level, high-density lipoprotein cholesterol level, glomerular filtration rate, and cohort; the stroke was not adjusted as a covariate for the analysis of subgroup 2. a Participants without anemia or iron supplementation. b Participants without a history of stroke. Significance values are given in bold. www.nature.com/scientificreports/ This study also found the sexually dimorphic association between sfHb level and V PVWMH . Our finding is in line with a previous study reported a sexually dimorphic association between the risk of stroke and sfHb level. In that study, a U-shaped association between the risk of stroke and sfHb level was found in women only 33 . Regardless of sex, the increase in sfHb level may reduce cerebral blood flow by elevating blood viscosity and activating platelets 34 . In women, reduced blood estrogen levels by increasing age may weaken the ability to compensate for the reduced cerebral microcirculation associated with a high sfHb level, because estrogen is known to facilitate cerebral microvasculature dilatation and regulates the coagulation pathway 35,36 . In men, however, serum androgen which constricts brain microvasculature decreases with increasing age. Therefore, older men may have better ability to compensate for the reduced cerebral blood flow induced by high sfHb level than older women 36 .
The inconsistent results on the sfHb-WMH association in previous studies [12][13][14][15][16] may be, at least in part, attributable to the dimorphisms in the sfHb-WMH association by sex and the type of WMH demonstrated in the current study. The sfHb-WMH association with increasing age might have been prominent specifically in this study because of the participants with higher age and lower sfHb level compared to the most previous studies [12][13][14][15] . In men with the sfHb level below the normal range and women with the sfHb level below or above the normal range, the risk of PVWMH and associated cognitive impairments needs to be evaluated and their abnormal sfHb should be quickly managed. In addition, optimal sfHb levels for men and women should be investigated in future research because the dimorphic sfHb-WMH association was observed in the older adults without anemia.
This study has several limitations. First, this is a cross-sectional design, which limits the interpretation of the causal relationship between sfHb level and V WMH . Second, this study analyzed data from two separate cohorts with different participants and measured sfHb levels using different automated systems. However, our analytic models included cohort as a covariate and still showed significant associations between hemoglobin with WMH. Third, the chronicity and the etiology of low or high sfHb levels were not identified. Finally, this study was limited to the Asian population. As the normal range of hemoglobin and the prevalence of red cell disorders are differentially distributed by ethnicities, our findings should be replicated in various populations.

Summary
Our study provides evidence that sfHb level differentially affects V WMH according to sex and lesion type. Older adults with abnormal sfHb levels may be at high risk of periventricular white matter lesions; thus, modifiable factors should be screened to prevent adverse outcomes related to WMH. Table 3. Restricted cubic spline model for association between serum free hemoglobin level and white matter hyperintensity volume. Hb hemoglobin, V WMH volume of total white matter hyperintensity, V PVWMH volume of periventricular white matter hyperintensity, V DWMH volume of deep white matter hyperintensity. *Adjusted for age, hypertension, diabetes, stroke, cumulative illness rating sore, drinking status, smoking status, systolic blood pressure, body mass index, total cholesterol level, high-density lipoprotein cholesterol level, glomerular filtration rate, and cohort; the stroke was not adjusted as a covariate for the analysis of subgroup 2. a Participants without anemia or iron supplementation. b Participants without a history of stroke. Significance values are given in bold.

Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.