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Longitudinal analyses of serum neurofilament light and associations with obesity indices and bioelectrical impedance parameters

Neurofilament light is a constituent of the neuronal cytoskeleton and released into the blood following neuro-axonal damage. It has previously been reported that NfL measured in blood serum is inversely related to body mass index. However, no reports exist with regard to body composition assessed using bioelectrical impedance analysis or other indicators of obesity beyond BMI. We analyzed the relationship between sNfL and body composition according to the three compartment model. Additionally, associations between sNfL, body shape index, waist-to-height ratio, and BMI were examined. The sample consisted of 769 participants assessed during the baseline examination and 693 participants examined in the course of the follow-up of the BiDirect Study. Associations between sNfL, BMI, BSI, and WtHR were separately analyzed using linear mixed models. Body compartments operationalized as fat mass, extracellular cell mass, and body cell mass were derived using BIA and the relationship with sNfL was analyzed with a linear mixed model. Lastly, we also analyzed the association between total body water and sNfL. We found significant inverse associations of sNfL with BMI and WtHR. The analysis of the three compartment model yielded significant inverse associations between sNfL, body cell mass and body fat mass, but not extracellular mass. Furthermore, total body water was also inversely related to sNfL. A potential mechanism could involve body cell mass and body fat mass as highly adaptive body constituents that either directly absorb sNfL, or promote the formation of new vasculature and thereby increase blood volume.

Neurofilament light polypeptide (NfL) is a protein found in large-caliber axons and a constituent of the neuronal cytoskeleton 1 . When neuroaxonal damage occurs and white matter disintegrates, NfL is released into the extracellular space from where it reaches the cerebrospinal fluid (CSF) and the blood stream 2 . Previous studies have shown that NfL levels are increased in several neurological disorders, e.g. Alzheimer's disease 3 , traumatic brain injury 4 , or multiple sclerosis 5 . Therefore, NfL is considered a general biomarker for neuroaxonal damage and disease activity 6 , particularly since NfL levels can be measured in the blood serum without exposing an individual to the risks of a lumbar puncture. The minimally invasive assessment of serum NfL (sNfL) makes it feasible for the application in large population-based studies.
Previous studies analyzed associations between sNfL and potential confounders in order to improve the interpretability of sNfL levels for the application in clinical settings. These studies revealed that sNfL increases with age in the general population 5,7 and that sNfL is inversely related to body mass index (BMI) and body blood volume 8 . The inverse association between sNfL, BMI, and blood volume is particularly interesting because it www.nature.com/scientificreports/ is only observed if NfL is measured in the blood but not in the CSF. BMI as a surrogate marker of over-and underweight is a well-established risk factor 9 for several adverse disease outcomes and, thus, might be considered a confounder. The etiology of this association is not fully understood. The aims of this study were to analyze the associations between sNfL and several anthropometric indices operationalized as BMI, body shape index (BSI), and waist-to-height ratio (WHtR). BMI does not discriminate 10 between body fat and fat free mass (e.g. skeletal muscle) whereas BSI and WtHR are better indicators of unhealthy body fat distribution 11 . It was hypothesized that BSI and WHtR exhibit an inverse association with sNfL comparable to the already known relationship with BMI. Additionally, we explored associations between sNfL and body composition parameters estimated by bioelectrical impedance analysis (BIA), particularly body fat mass (BFM), body cell mass (BCM), extracellular mass (ECM), and total body water (TBW).

Results
The exclusion criteria and sample characteristics for both time points are summarized in Fig. 1 and Table 1, respectively. In participants (n = 625) who provided data to both time points, sNfL increased by a mean of 0.95 pg/ ml over an average of 2.7 years. The initial linear model for the cross-sectional baseline data showed a significant inverse association between sNfL and BMI (β = − 0.009, 95% confidence interval = − 0.012 to − 0.007, p < 0.001). The separate longitudinal analyses of associations between sNfL and the three anthropometric indices of obesity revealed that BMI (β = − 0.008, 95% confidence interval = − 0.01 to − 0.006, p < 0.001, semi-partial R 2 = 0.034) and WtHR (β = − 0.43, 95% confidence interval = − 0.56 to − 0.30, p < 0.001, semi-partial R 2 = 0.024) were inversely related to sNfL, whereas the association with BSI was not statistically significant (β = − 1.31, 95% confidence interval = − 3.33 to 0.72, p = 0.205, semi-partial R 2 = 0.001). Partial residual plots showing the adjusted associations with the anthropometric indices are shown in Fig. 2. The analysis of the three-compartment model yielded significant associations with BCM and BFM, as shown in Table 2 and Fig. 3, but not with ECM. Furthermore, TBW (β = − 0.005, 95% confidence interval = − 0.007 to − 0.003, p < 0.001, semi-partial R 2 = 0.015) was significantly and inversely related to sNfL.

Discussion
The present study analyzed associations between sNfL, anthropometric indices, bioelectrical impedance parameters according to the three-compartment model, and TBW. The results showed significant inverse associations between sNfL levels and BMI, WtHR, BCM, BFM, and TBW. However, no significant estimates were obtained for BSI and ECM.
Our results replicated earlier findings regarding the inverse relationship between sNfL and BMI 5,8 , and extended these findings to anthropometric indices such as the WtHR. The latter is a better indicator of unhealthy body fat distribution 11 . However, the BSI is also an indicator of unhealthy body fat distribution but was not significantly associated with sNfL. These findings were complemented by the BIA, showing an inverse association between sNfL, BFM, and BCM. This relationship could be explained by the role of adipose tissue and BCM in angiogenesis and blood volume. BCM, which includes highly adaptive and metabolically active tissue 12 (e.g. skeletal muscle), is related to the formation of new blood vessels depending on the level of exercise and can increase the circulating blood volume 13,14 . This contrasts with ECM, which is metabolically inactive and less adaptive compared to BCM 15 . It has also been observed that expanding adipose tissue is paralleled by increased blood vessel formation 16 , which consequently manifests in larger blood volume 17 . Thus, it is suggested that BFM and metabolically active BCM 12 may be related to increased blood volume thereby decreasing the concentration of proteins of neuronal origin, or a direct distribution into tissue. This perspective is also supported by the inverse relationship between sNfL and TBW, and the previously observed inverse association between sNfL and blood volume 8 , calculated based on weight and height 18 . The current results from the BIA support and extend these previous observations. Apart from sNfL, previous studies that employed blood-based analyses using ultrasensitive assays have observed similar inverse associations between BMI and proteins originating from the brain. In particular, an inverse association between standardized BMI and glial fibrillary acidic protein (GFAP) levels has been reported recently 19 . An inverse relationship between BMI and phosphorylated tau protein 20 has also been observed, although there is conflicting evidence 21 .
Together with previous findings, the present results suggest attenuated sNfL concentrations related to body composition, captured by ultrasensitive assays measuring proteins of neuronal origin in the blood. Future studies analyzing sNfL should therefore consider to include BMI (or other indicators of body composition) in their statistical models as an easy to measure confounder, e.g. in the field of dementia where it has been observed that BMI declines over time prior to the clinical onset of Alzheimer's disease 22 , or apply age and BMI adjusted percentile scores 5 for individual application. Further studies analyzing the relationship between body composition and attenuated concentrations of proteins of neuronal origin measured in the blood are required.
The BMI does not allow a distinction between body fat and other body constituents 10 and does not capture sarcopenia 23 , a type of muscle loss associated with aging. However, a fine-grained assessment of body composition using BIA can overcome these limitations by distinguishing between BCM, BFC, and ECM. Although body composition can be even more precisely assessed using other methods, body composition estimates derived with BIA are in good agreement with those derived by computer tomography or hydrostatic weighting 23,24 , which are unfeasible to conduct in larger samples. A major strength of our study is the large, population-based sample with repeated assessments of BIA parameters and anthropometric indicators of obesity over the course of 2.7 years. However, the age range of the current sample was limited between 35 and 65 years during recruitment and a steeper association between sNfL and age has been observed in older samples 5,7 . We were not able to provide results for other body indices such as the waist-to-hip ratio because hip circumference was not assessed in the course of the study. In conclusion, we observed an inverse association between sNfL and body composition, particularly BCM, BFM, and TBW, suggesting a compartment-specific attenuation of neuronal biomarkers captured by ultrasensitive assays. ), and WHtR ( waist circumference height ) were calculated based on the respective measurements. Body composition was evaluated using a BIA 2000-S device (Data Input GmbH, Pöcking, Germany) and categorized into BCM, BFM, and ECM according to the threecompartment model [26][27][28] . The three-compartment model has proved beneficial over other models of body composition like the two-compartment model, which separates the body into BFM and fat free mass but does not capture BCM 27,29 . Additionally considering BCM and ECM with the three-compartment model allows a reliable estimation of body composition using BIA 23 .
Serum neurofilament light. Non-fasting blood samples were taken, centrifuged and stored at − 80 °C within two hours. Later, sNfL was quantified by blinded, board-certified technicians using a commercially available kit (NF-Light, Quanterix) and a single molecule array (SIMOA) HD-X analyzer (Quanterix, Lexington, MA, USA). The coefficients of variation were below 15% for all samples. Due to the right-skewed distribution of sNfL, the variable was log 10 -transformed prior to the analyses.
Statistical analysis. In a first step, we aimed to replicate previously reported cross-sectional associations between sNfL as a dependent variable and BMI with a linear regression model for the baseline data, adjusted for age and sex. Next, longitudinal associations between sNfL as dependent variable and BMI, BSI and WtHR as respective independent variables were analyzed with three separate linear mixed models using the lmerTest package 30 for R. Additional linear mixed models were run to investigate associations between sNfL and BCM, ECM and BFM according to the three-compartment model of body composition, and to examine associations between sNfL and TBW. All models were adjusted for age and sex. A random intercept was included in all longitudinal models to account for the correlated structure of repeated measurements over time. P values below 0.05 (two-tailed) were considered significant. All analyses were conducted with R version 4.1.2.

Data availability
The dataset used and analyzed during the current study is available from the corresponding author on reasonable request.   Figure 3. Partial residual plots showing the sex-and age-adjusted inverse associations between sNfL and body fat mass, and extracellular mass, and body cell mass according to the three-compartment model. The bottom right panel shows the sex-and age-adjusted inverse association between sNfL and total body water. The 95% confidence intervals are shown as grayish shaded areas.