β -Alanine supplementation improves fractional anisotropy scores in the hippocampus and amygdala in 60 – 80-year-old men and women

Recently, β -alanine (BA) supplementation was shown to improve cognitive function in older adults with decreased cognitive function. Mechanisms supporting these improvements have not been well defined. This study examined the effects of 10-weeks of BA supplementation on changes in circulating brain inflammatory markers, brain derived neurotrophic factor (BDNF), and brain morphology. Twenty participants were initially randomized into BA (2.4 g ⋅ d (cid:0) 1 ) or placebo (PL) groups. At each testing session, participants provided a resting blood sample and completed the Montreal cognitive assessment (MoCA) test and magnetic resonance imaging, which included diffusion tensor imaging to assess brain tissue integrity. Only participants that scored at or below normal for the MoCA assessment were analyzed (6 BA and 4 PL). The Mann-Whitney U test was used to examine Δ (POST – PRE) differences between the groups. No differences in Δ scores were noted in any blood marker (BDNF, CRP, TNF-α and GFAP


Introduction
β-alanine (BA) is a non-proteogenic amino acid whose direct physiological role is limited, but when consumed as part of the diet or as a supplement combines with histidine to form the dipeptide carnosine (Harris et al., 2010).BA is the rate limiting amino acid in the formation of carnosine (Harris et al., 2006).Elevations in carnosine have been demonstrated in numerous studies to be an effective intracellular buffer that can delay muscle fatigue and improve athletic performance (Hoffman et al., 2018).However, it has other important physiological roles, such as acting as an antioxidant and anti-chelating agent (Hoffman et al., 2018), which expands the potential physiological benefits of BA supplementation.
In a recent study, 10-weeks of BA supplementation (2.4 g⋅day − 1 ) was demonstrated to improve cognitive function in men and women between 60 and 80 years-of-age (Ostfeld et al., 2023).These results were observed only in participants whose baseline cognitive function scores were considered to be at or below normal and who had not been previously diagnosed with a neurocognitive disorder.When cognitive function changes were examined in all participants, including those whose baseline cognitive scores were above normal, no improvements in cognitive function were noted.This suggested that elevations in carnosine levels did not exhibit an ergogenic on cognitive function, but could compensate or offset increases in oxidative stress or other physiological perturbation in the brain to improve cognitive function in individuals with borderline or below normal cognitive function.Previous research in murine models has demonstrated that elevations in brain carnosine were associated with a reduction in anxiety and an improvement in memory in animals exposed to predator scent stress (Hoffman et al., 2015) and low-pressure blast waves (Hoffman et al., 2017).It has been suggested that the increased resiliency to these stressors is related to carnosine's role as an antioxidant (Boldyrev et al., 2013).
The aging process is associated with increased neuroinflammation and greater oxidative stress, all of which can impact cognitive function (Di Benedetto et al., 2017).Supplementing with BA has been demonstrated to improve cognitive function in older adults, but the mechanism remains speculative and has not been fully elucidated.The focus of this pilot study was to determine the effects of 10-weeks of BA supplementation on changes in circulating inflammatory markers, brain derived neurotrophic factor (BDNF), and brain morphology.The study's hypothesis was that BA supplementation would result in significant increases in fractional anisotropy (FA) and BDNF concentrations and decreases in markers of inflammation.A double-blind randomized control trial was conducted to test this hypothesis.

Methods
The participants of this study were a cohort of a larger double-blind randomized controlled study previously published (Ostfeld et al., 2023).Twenty of those participants, who were between the ages of 60-80 y (10 men and 10 women), volunteered to participate in additional testing.In contrast to the larger study, these participants agreed to provide blood samples and participate in magnetic resonance imaging (MRI) assessments.Interviews with these participants occurred prior to study enrollment and revealed that no participant was supplementing with BA prior to the study period and that no participant had been previously diagnosed with dementia, neurocognitive function disorder, or any other medical condition that would preclude participation in the study.All interviews were conducted by a licensed physician.If participant fulfilled all inclusion requirements, no limitation was made on medication use, as there are no known contraindications to BA supplementation with medication use.The study protocol was approved by the Institutional Review Board at Ariel University (AU-HEA-YO-20201117) and the Helsinki Committee at Tel Hashomer Medical Center (7867-20-SMC).Participants were not permitted to use any additional nutritional supplements for at least 6-weeks before the study.Screening for supplement use and medical history was accomplished during the interview with the study physician.This study was registered as a clinical trial with the Israel Ministry of Health (MOH_2021-09-30_010279).

Study protocol
Participants met with the study team on two separate occasions.During the first visit (PRE), participants received an explanation about the study, provided their informed consent, and were randomized into either the BA (7 men and 3 women; 65.1 ± 3.3 y; 170.9 ± 9.6 cm and 79.6 ± 13.4 kg) or the placebo group (PL; 3 men and 7 women; 67.3 ± 6.4 y; 166.0 ± 8.4 cm and 66.9 ± 11.3 kg).Before taking their first supplement, participants provided a resting blood sample, completed a cognitive function assessment, and were then walked over to the MRI facility for imaging.All assessments were performed again 10 weeks later, at the conclusion of the study (POST), in the same order.

Supplement protocol
Participants were instructed to consume two tablets (600 mg apiece) twice daily during the 10-week study (total supplement or placebo consumed per day was 2.4 g).Ten weeks of supplementation was chosen based upon the desire to achieve a total BA intake of 168 g.Previous studies have demonstrated that a total of 168 g of BA consumed will increase tissue concentrations of carnosine (Church et al., 2017;Varanoske et al., 2017).Participants were instructed to consume the supplement with their regular meals.The supplement and placebo were identical in appearance.The BA tablet was a sustained-release formulation, and the PL tablet consisted of the same ingredients but contained hydroxypropyl methylcellulose instead of the BA.The BA (SR Carno-syn®) and PL tablets were provided by Natural Alternatives International (Carlsbad, CA, USA).Supplement compliance was tracked by counting the number of tablets remaining in the bottles following 5weeks of supplementation and at POST testing.To be included in the final analysis, participant compliance was set at 80 %.

Blood measurements
During each testing session, a resting blood sample was obtained.All blood samples were obtained from an antecubital arm vein using a 20gauge disposable needle equipped with a Vacutainer tube holder.Blood samples were collected into Vacutainer tubes containing Silica Clot Activator.All blood draws were obtained with the subjects in a seated position.The blood was allowed to clot at room temperature and centrifuged at 1500g for 15 min.The resulting serum was placed into 1.5-ml microcentrifuge tubes and frozen at − 80 • C for later analysis.

Biochemical analysis
Serum concentrations of BDNF, C-reactive protein (CRP) and tumor necrosis factor alpha (TNF-α) were analyzed via a multiplex assay (EMD Millipore, Billerica, MA, USA), Glial fibrillary acidic protein (GFAP) was analyzed with enzyme-linked immunosorbent assay (ELISA) kits from Biotest Inc., (Tel Aviv, Israel) per the manufacturer's instructions.Samples were thawed once and analyzed in duplicate by the same technician using an Absorbance 96 spectrophotometer (Byonoy GmbH, Hamburg, Germany) for the ELISA assay and MagPix (EMD Millipore) for the other measures.Mean intra-assay variability was <10 %.

Cognitive function assessment
Cognitive function was assessed using the Montreal Cognitive Assessment (MoCA) (Nasreddine et al., 2005).The MoCA focuses on several cognitive domains including executive function, memory, attention, language, abstraction, delayed recall, and orientation.It was developed as a screening instrument for the detection of mild cognitive impairment in older adults.Research has indicated that it has excellent test-retest reliability and positive and negative predictive values for mild cognitive impairment (Freitas et al., 2012).There are well-over 1000 publications that have examined MoCA in older adults, with reliability and validity established in >40 languages including Hebrew (Lifshitz et al., 2012).

Magnetic Resonance Imaging (MRI)
Data acquisition was performed at Tel Aviv University's Strauss Center for Neuroimaging.Participants were scanned in a Siemens Prisma 3 T MRI scanner with a 64 channels head coil and a gradient system reaching 80 mT/m.Each scan session included a high-resolution T1w anatomical scan, with magnetization prepared rapid gradient echo (MPRAGE) sequence: TR/TE = 2400/2.78ms, TI = 1000 ms, with a resolution of 0.9mm 3 .T2w anatomical images, were acquired using a SPACE (SPC) sequence, with the following parameters: TR/TE = 3200/ 554, with a resolution of 0.9mm 3 .
Diffusion tensor imaging (DTI) scanning was performed using a single shot diffusion weighted spin-echo echo-planar imaging (ss-DWEPI) sequence with the following parameters: TR/TE = 6900/53 ms; FOV 208 mm; acquisition matrix 122 × 122; slice thickness 1.7 mm without gap; isotropic voxel size 1.7 mm 3 ; B 1000 s/mm 2 (Δ/δ = 23.1/11.4 ms) and 3 images of b0.DTI was applied on 64 non-collinear directions.The outcome measures derived from the DTI scan were fractional anisotropy (FA) and apparent diffusion coefficient (ADC).FA is an index that represents the increase in diffusivity into brain tissue ranging from 0 to 1.

Image processing
Image processing was carried out using the pipelines provided by the Human Connectome Project (HCP-pipelines) as previously described in detail (Glasser MF, Sotiropoulos SN, Wilson JA, Coalson TS, Fischl B, Andersson JL.WU-Minn HCP Consortium, 2013).Briefly, the N-back task data underwent motion and distortion corrections and nonlinear alignment to the MNI template space.Furthermore, the data were denoised using FMRIB's ICA-based Xnoiseifier (FIX) (Griffanti et al., 2014;Salimi-Khorshidi et al., 2014), and then resampled and "projected" onto a surface representation of 91,282 vertices ("grayordinates") in standard space.Data were aligned and registered using Multimodal Surface Matching (MSMAll) (Robinson et al., 2018;Robinson et al., 2014).

N-back task analysis
Activity estimates were computed from pre-processed functional scans using FSL's FILM (FMRIB's Improved Linear Model with autocorrelation correction) (Woolrich et al., 2001).The task was designed in a blocked design manner, and the different blocks were convolved with a double gamma canonical hemodynamic response function (Glover, 1999) to generate the main model regressors.Z-score task-activation maps for the 2back > 0back contrast were used for the analysis, for both pre-and post-intervention.The task fmri was acquired using multiband gradient echo planar imaging pulse sequence with tr/te = 2000/30 ms, isotropic resolution of 2 mm3, mb = 2, Ipat = 2, with full brain coverage (no gaps).

Statistical analysis
Recent findings reported that 10-weeks of BA supplementation resulted in significant improvements in cognitive performance in participants whose baseline MoCA score were at or below normal (i.e., a score of 26) (Ostfeld et al., 2023).Thus, in the present study, only those participants that scored a 26 or below on the PRE MoCA were analyzed.Of the 20 participants who volunteered to participate, 10 participants had MoCA scores at that level (six participants from the BA group, and four participants from the PL group).Of the BA group, three of the participants were men and three were women, while the PL group consisted of three women and one man.The cognitive function, blood, and MRI measures were examined as a delta (Δ) score (POST -PRE).Due to the small sample size the non-parametric Mann-Whitney U test was used to examine differences between BA and PL.All statistical analyses were performed using SPSS v27 software (SPSS Inc., Chicago, IL), and an alpha level of p ≤ 0.05 was used to determine statistical significance.All data are reported as mean ± SD.

Results
Supplement compliance was met by all participants.The mean compliance rate was 90.3 ± 7.5 %, and no adverse events were reported.MoCA scores at PRE were 24.0 ± 0.6 for BA and 23.8 ± 3.2 for PL.Following the 10-week supplement intervention MoCA scores for BA and PL increased to 26.7 ± 1.6 and 24.5 ± 1.4, respectively.However, the Mann-Whitney analysis revealed no significant difference (p = 0.331) in the Δ score between BA (2.7 ± 2.1) and PL (0.8 ± 2.6).In addition, comparisons of the n-back assessment performed in the MRI device revealed no significant differences in the Δ scores of accuracy or reaction time in the 0-back assessment (p = 0.184 and p = 0.286, respectively), 1-back assessment (p = 1.00 and p = 0.522, respectively) and 2-back assessments (p = 0.668 and p = 0.831, respectively) (see Table 1).
Changes in resting BDNF, CRP, TNF-α, and GFAP concentrations can be examined in Fig. 1a-d, respectively.No significant changes were noted in circulating BDNF (p = 0.393), CRP (p = 0.190), TNF-α (p = 0.111) or GFAP (p = 0.190) concentrations subsequent to β-alanine supplementation.Results from the MPRAGE and DTI analysis focused on the hippocampus and amygdala, brain regions that are primarily involved with cognitive function and emotion.Analysis included total volume and volume of both white and gray matter within these brain regions.No significant difference (p = 1.00) in the total volume of the left hippocampus between BA (− 0.001 ± 0.398 ml) and PL (0.011 ± 0.026 ml) was noted.However, changes in total volume of the right hippocampus (0.026 ± 0.257 ml and − 0.493 ± 0.044 ml), and both right (− 0.005 ± 0.034 ml and − 0.056 ± 0.018 ml) and left amygdala (0.045 ± 0.067 ml and − 0.043 ± 0.043 ml) trended towards significance (p = 0.063 for all regions) between BA and PL, respectively.These results indicated that the change of total brain volume for BA tended to be greater than PL.
Table 2 provides absolute volume changes in both gray and white matter in the hippocampus and amygdala.No changes in the volume of gray matter was noted between BA and PL in either the left (p 0.7620 or right hippocampus (p = 0.171) or the left (p = 0.257) or right amygdala (p = 0.352).Similarly, no changes were observed in the volume of white matter in the left (p = 0.171) or right hippocampus (p = 0.762), nor in the left (p = 0.352) or right amygdala (p = 0.352).
Changes in FA in the hippocampus and amygdala are depicted in Fig. 2. Analysis of the DTI data resulted in changes in FA scores that were significantly greater for BA than PL in the right hippocampus (p = 0.033) and the left amygdala (p = 0.05).No between group differences were noted in the change in FA in the left hippocampus (p = 0.522) or right amygdala (p = 0.670).No difference between the groups were noted in the change score of ADC in either the left or right hippocampus (p = 0.831 and p = 0.394, respectively) or in the left or right amygdala (p = 0.831 and p = 0.522).

Discussion
The results of this pilot study suggest that 10-weeks of BA supplementation did not alter any of the inflammatory markers or BDNF.However, significant improvements in FA values were noted in both the hippocampus of the right hemisphere and the amygdala of the left hemisphere, and a trend towards a greater total volume in the BA group.DTI measures provide an indication of tissue integrity by assessing the diffusion of water molecules (Lerner et al., 2014).An increase in FA is indicative of an increase in tissue integrity among neural fibers (e.g., axons, neurons) indicating less diffusion, while a decrease in FA indicates diffusivity in all directions and characterizes non-ordered regions such as gray matter and white matter where multiple fibers cross (Assaf, 2019;Assaf and Pasternak, 2008).In contrast, ADC represents the degree of diffusion in each direction.An increase in ADC indicates greater diffusion, hence greater tissue disruption (Lerner et al., 2014).The significant increase in FA observed in BA is suggestive of greater tissue integrity of the hippocampus and amygdala, brain regions that are primarily responsible for emotion and cognitive function.These results were supportive of previous research that indicated that differences in FA values were predictive of cognitive function (Li et al., 2023).Total volume tended to increase in BA, while no changes were noted in the volume of gray matter or white matter in these specific brain regions.
Both gray matter and white matter have been reported to have important roles in cognitive function (Paulsen et al., 2020;Lampe et al., 2019).
Additional study with larger sample size may be warranted to further examine the effect of BA on changes in volume of both gray and white matter.
The changes in FA may provide some insight into the potential benefit of BA supplementation on improvements in cognitive performance.Although no significant change in MoCA score was seen in this pilot study, the small sample size was likely a confounding factor.Our previous study examining 100 participants was appropriately powered.This was a pilot examination to examine the potential mechanism that may be responsible for the changes previously reported.Interestingly, the change in MoCA scores in this pilot study was nearly 3.4-fold greater in BA than PL.In addition, the lack of change in the circulating inflammatory markers and BDNF may not reflect what is occurring within the brain regions.Changes in circulating BDNF concentrations have been previously demonstrated to be related to improved cognitive function (Orlando et al., 2023), but the present study was not powered for this specific question.Previous studies, using similar total dosages, have reported significant elevations in BDNF expression and reductions in GFAP expression in the hippocampus following BA supplementations in animals that were exposed to stress (Hoffman et al., 2015;Hoffman et al., 2017).
In summary, the results of this partially support our hypothesis that BA supplementation can improve FA scores but did not support any change in circulating BDNF or inflammatory marker responses.This pilot study provides a potential mechanism of how BA supplementation may improve cognitive function as reflected by improved tissue integrity within the hippocampus and amygdala.Additional research appears warranted to further explore this potential mechanism.

Table 2
Changes in absolute volume in gray and white matter of the hippocampus and amygdala in older men and women supplementing with BA or PL.

Declaration of competing interest
Authors declare that they have no conflict of interest to report.This research was funded by CarnoSyn® Brands of Natural Alternatives International, Inc. (Carlsbad, CA, USA).The funding source had no involvement in collection, interpretation, or analysis of data or of writing of the manuscript.

Fig. 2 .
Fig. 2. Δ Changes in Fractional Anisotropy (FA) in the Hippocampus and Amygdala.Fig. 2a contains ΔFA in Left hippocampus; 2b depicts ΔFA in the right hippocampus; 2c depicts ΔFA for the left amygdala; 2d contains ΔFA for the right amygdala.Significantly greater increases in FA were noted for BA in the right hippocampus and left amygdala.All data are reported as mean ± SD.BA = β-alanine; PL = Placebo.

Table 1 N
-back performance measures in older men and women supplementing with BA or PL.