Main Research Findings

1) Treatment with magnesium L-threonate has been shown to prevent inflammation through the gut biome and memory impairment in mice fed an alcohol-based diet.

2) Magnesium L-threonate has the potential to elicit a neuroprotective effect against damage elicited by oxidative stress in HT22 cells and an animal-based model of Alzheimer’s disease

Selected Data
1) This research team of Liu et al investigated the effects of magnesium-L-threonate supplementation on alcohol-induced cognitive and physiological impairments in mice, through the use of a well-established National Institute on Alcohol Abuse and Alcoholism (NIAAA) model to evaluate the potential protective effects of MgT against alcohol-induced neuroinflammation and oxidative stress. The study included biochemical, histological, behavioral, and microbiome analyses to comprehensively assess the impact of magnesium L-threonate on alcohol-exposed mice.
For the purpose of this study, 24 healthy male C57BL/6 mice, aged 8 weeks and weighing approximately 20 grams were utilized. The mice were housed in controlled conditions with 60% humidity and a temperature of 23 °C, with ad libitum access to food and water. Following a seven-day adaptation period to liquid feeding, the mice were randomly divided into four groups and subjected to a 31-day NIAAA alcohol exposure model. The groups were defined as follows: a control group receiving a liquid control diet, a model group receiving a liquid alcohol diet, a group receiving an alcohol-based diet with a 100 mg/kg dose of magnesium L-threonate delivered via oral gavage, and a group receiving an alcohol-based diet with a 200 mg/kg dose of magnesium L-threonate delivered via oral gavage [1].
On day 31, the final day of the experiment, all mice were given a single 5 g/kg high-dose alcohol exposure delivered via oral gavage, and were euthanized 9 hours later. Tissue samples were then collected, rapidly frozen in liquid nitrogen, and stored at −80°C for further analysis. Biochemical analyses of blood samples were conducted using a Murray biochemical analyzer 800 to measure calcium, magnesium, white blood cell count, and neutrophils. The levels of inflammatory cytokines TNF-alpha and IL-1-beta in serum were quantified using ELISA kits. Oxidative stress markers, including SOD and GSH-Px, were assessed in both brain and colon tissues.
The tissues were then homogenized in phosphate-buffered saline for further ELISA-based quantification of inflammatory and oxidative stress markers such as TNF-alpha, IL-1-beta, 4-HNE, 5-HT, Claudin-1, and ZO-1. Additionally, fresh brain and colon samples were fixed in 4% paraformaldehyde for 48 hours before being embedded in paraffin, sectioned into 5-μm slices, and stained with hematoxylin and eosin for histopathological evaluation using an inverted microscope [1].
To evaluate the effects of alcohol and magnesium L-threonate supplementation on spatial learning and memory, the Morris Water Maze test was performed during the last five days of the study. The test involved four days of navigation training followed by a probe test on the fifth day. During the navigation training phase, mice were placed in the pool facing the wall and given 60 seconds to locate a hidden platform. The time taken to reach the platform was recorded as escape latency. If a mouse was unable to find the platform within the allotted time, it was guided to the platform, and an escape latency of 60 seconds was recorded. This training was repeated four times daily, with the average escape latency recorded each day. On the final test day, the platform was removed, and the mice were allowed to swim freely for 60 seconds. Their swimming trajectories were recorded, and the frequency of platform crossings was noted as a measure of memory retention [1].
To investigate the effects of alcohol and magnesium L-threonate supplementation on gut microbiota, bacterial DNA was extracted from colonic samples using a Genomic DNA Kit. The 16S rRNA gene through the (V3–V4 regions was amplified and sequenced using the Illumina MiSeq platform. Sequences were processed using QIIME2 software, and operational taxonomic units were classified based on 97% sequence similarity. Gene function prediction was performed using PICRUSt2 software, and results were analyzed using the Kyoto Encyclopedia of Genes and Genomes database.
Next, Western blotting was conducted to assess the expression of tight junction proteins and inflammatory markers in colon tissues. Colonic tissue samples were homogenized in a RIPA buffer containing protease inhibitors and centrifuged to extract proteins. Protein concentration was determined using a BCA kit, and equal amounts of protein were separated on 10% SDS-polyacrylamide gels before being transferred to PVDF membranes. The membranes were blocked with 3% BSA solution and incubated overnight at 4°C with primary antibodies targeting ZO-1, Occludin, β-actin, TNF-alpha, and IL-1-beta. After washing, membranes were incubated with secondary antibodies for 2 hours at room temperature and chemiluminescence signals were detected using a Tanon-5200 chemiluminescence analyzer [1].
2) The research team of Xiong et al examined the neuroprotective effects of magnesium L-threonate in a model of HT22 cells and an animal-based model representative of Alzheimer’s disease. To initiate the experiment, HT22 cells were cultured and differentiated followed by maintenance in standard culture medium before being differentiated in an N2 supplement-containing neurobasal medium for 24 hours prior to drug administration. According to previous studies, exposure to 40 μmol/L Aβ25-35 for 24 hours significantly reduces HT22 cell viability. Therefore, a concentration of 40 umol/L was selected for the study. Aβ25-35 was diluted in sterile saline and pre-aged at 37°C for seven days before use. To evaluate the potential protective effects of magnesium L-threonate against Aβ25-35-induced oxidative stress, HT22 cells were pre-treated with 50 μmol/L of magnesium L-threonate for 12 hours before Aβ25-35 exposure [2].
Following treatment with magnesium L-threonate, cell viability was assessed using the CCK-8 assay. After drug treatments were completed, HT22 cells were incubated with 10 μL of CCK-8 reagent, and absorbance was measured at 450 nm using an absorbance reader. Levels of reactive oxygen species were determined using an oxidation-sensitive fluorogenic dichlorodihydrofluorescein diacetate (DCFH-DA) probe and quantified via flow cytometry. HT22 cells were then washed and incubated with a 10 μmol/L DCFH-DA probe prior to flow cytometric analysis, and the proportion of DCFH-DA-labeled cells counted by the research team indicated the levels of intracellular reactive oxygen species [2].
For the purpose of behavioral experimentation, male APP/PS1 transgenic mice and their wild-type littermates were utilized. The animals were housed under standard laboratory conditions with ad libitum access to food and water. Once the mice were six months old, they were divided into three different experimental groups including: mice treated with magnesium L-threonate, a control of untreated mice, and wild-type control mice. The group of mice treated with magnesium L-threonate received a daily dose of 910 mg/kg that was administered through the drinking water for three months, while the untreated control group and the wild type control group were given regular water. After treatment, cognitive function was evaluated using the Morris Water Maze test, and the mice were euthanized under deep anesthesia to collect hippocampal tissue for biochemical analysis.
The mice underwent the Morris Water Maze after three months of treatment with magnesium L-threonate to assess cognitive functioning. Initially, all mice underwent a one-day pre-training session with a visible platform while hidden platform training was conducted over five days and included four, 90 second trials. Mice were released from different starting quadrants in a randomized order and the time taken to find the hidden platform was recorded as a measurement of escape latency. If a mouse failed to locate the platform within 90 seconds, they were guided to the platform, and an escape latency of 90 seconds was assigned. The escape latencies from four trials were averaged for statistical analysis [2].
24 hours after the last hidden platform training session a probe test was conducted, during which the platform was removed. The animals were allowed to swim freely for 90 seconds, while the latency to reach the previous platform location, the percentage of time spent in the target quadrant, and the number of times they crossed the target position were recorded by the research team. After each trial, the mice were dried and placed on an electric blanket to maintain body temperature [2].
Finally, a fluorescein isothiocyanate-annexin V/propidium iodide assay was used to measure apoptosis in HT22 cells. Following experimental treatment, cells were washed, trypsinized, and incubated with the apoptosis detection reagent before analysis via flow cytometry. In a similar manner, apoptosis in hippocampal neurons was assessed using an allophycocyanin-annexin V/propidium iodide kit. The isolated hippocampal tissue was processed into a single-cell suspension, stained with anti-NeuN and Alexa-Fluor-488-conjugated secondary antibodies followed by analysis using flow cytometry. Protein expression in HT22 cells and hippocampal tissues was examined through Western blotting and all proteins were quantified, probed with specific primary antibodies, and visualized using a digital imaging system [2].

Discussion
1) The study conducted by the research team of Liu et al assessed the effects of magnesium L-threonate on alcohol-induced damage in mice, focusing on inflammation, gut microbiota, oxidative stress, and cognitive impairment. Compared to control mice, the animals fed a liquid alcohol diet exhibited weight loss, poor hair quality, and increased inflammatory markers. Chronic alcohol consumption activated neutrophils and worsened inflammation, as indicated by elevated white blood cell and neutrophilic granulocyte levels. However, treatment with magnesium L-threonate significantly reduced these markers, suggesting its potential in preventing alcohol-related inflammation. Additionally, inflammatory cytokines such as TNF-alpha and IL-1-beta were lower in the groups treated with magnesium L-threonate, indicating a reduction in alcohol-induced inflammatory responses [1].
To evaluate the effect of treatment with magnesium L-threonate on colon structure, the researchers performed histopathological analysis. Control mice had well-organized colonic epithelial cells and crypts, while alcohol-fed mice displayed significant structural damage, inflammation, and loss of goblet cells. Treatment with magnesium L-threonate alleviated these lesions, preserved epithelial integrity, and reduced overall inflammation. Additionally, alcohol consumption is known to damage the gut by disrupting the intestinal epithelial barrier and increasing permeability. Considering that oxidative stress is a key factor in alcohol-related damage, the study assessed its role in the pathogenesis of alcohol-induced gut damage. Administration of magnesium L-threonate was found to significantly suppress oxidative stress by increasing antioxidant enzyme levels, such as superoxide dismutase and glutathione peroxidase, while also reducing inflammatory cytokines [1].
Furthermore, Intestinal tight junction proteins, including ZO-1 and Claudin-1, play a crucial role in maintaining gut integrity. Alcohol-fed mice exhibited decreased levels of these proteins, leading to increased intestinal permeability and inflammation. However, magnesium L-threonate treatment prevented the alcohol-induced reduction in tight junction proteins, reinforcing the intestinal barrier and reducing inflammation. Chronic alcohol consumption also disrupts gut microbiota, resulting in dysbiosis and reduced bacterial diversity. Sequencing analysis revealed that alcohol-fed mice had lower microbial diversity, which was partially restored by treatment with magnesium L-threonate.

Metabolomic analysis performed by the research team further revealed that treatment with magnesium L-threonate influenced various metabolic pathways associated with gut microbiota. That being said, amino acid metabolism, oxidative phosphorylation, and biosynthesis of cofactors and vitamins were enhanced in magnesium L-threonate-treated mice. Specific pathways, such as alanine, aspartate, and glutamate metabolism, also experienced significant upregulation. This is a crucial finding considering that amino acids, particularly glutamate, play an important role in cognitive functioning related to learning and memory. These findings suggest that administration of magnesium L-threonate may improve cognitive functioning by enhancing amino acid metabolism and improving activity through the gut-brain axis [1].
Considering that alcohol consumption is known to induce neuroinflammation and oxidative stress in the central nervous system, histological analysis of the hippocampus showed severe neuronal damage in alcohol-fed mice, with shrunken cells and disrupted pyramidal cell layers. Treatment with magnesium L-threonate was shown to improve neuronal integrity while reducing damage in the hippocampal regions CA1, CA3, and DG. Additionally, oxidative stress markers such as 4-hydroxy-2-nonenal were significantly increased in alcohol-fed mice but reduced when the animals were administered magnesium L-threonate. Antioxidant enzyme levels, including SOD and GSH-PX, were also restored in mice treated with magnesium L-threonate. These findings suggest an overall improvement in brain oxidative balance [1].
The research team assessed changes in levels of inflammatory cytokines, including IL-1-beta and TNF-alpha, that were shown to be significantly elevated in alcohol-fed mice. However, these levels were reduced following treatment with magnesium L-threonate. Alcohol-fed mice exhibited lower levels of serotonin, while magnesium L-threonate-treated mice showed increased serotonin activity, suggesting improved neurotransmission and cognitive function. Additionally, magnesium ions are found to regulate the activity of NMDA receptors, which are crucial for synaptic plasticity and memory. That being said, the researchers assessed spatial learning and memory using cognitive behavioral experiments. Alcohol-fed mice showed impaired navigation as well as a longer time to find the platform in the Morris water maze test. Comparatively, mice treated with magnesium L-threonate exhibited improved learning and memory, as shown by shorter escape latencies and more frequent crossings over the target area.

Overall, the study demonstrated that supplementation with magnesium L-threonate effectively prevents alcohol-induced damage in mice by reducing inflammation, restoring gut microbiota balance, enhancing antioxidant defenses, and improving cognitive function. Magnesium L-threonate improved gut integrity by upregulating tight junction proteins and modulating microbiota composition. The compound also enhances amino acid metabolism, which plays a crucial role in neurotransmission and cognitive health. Magnesium L-threonate was also found to reduce neuroinflammation, oxidative stress, and neuronal damage while improving memory and learning abilities. These findings suggest that the compound has the therapeutic potential to prevent and alleviate alcohol-induced cognitive and systemic impairments, supporting its role as a neuroprotective supplement [1].
2) The initial results of the study conducted by Xiong et al revealed that intracellular levels of reactive oxygen species were significantly increased in Aβ25-35-treated HT22 cells compared to control cells, as measured by the DCFH-DA assay. However, administration of magnesium L-threonate effectively reduced reactive oxygen species levels in Aβ25-35-treated cells, suggesting the antioxidant potential of the compound. In a similar manner, the expression of hypoxia-inducible factor-1-alpha was elevated in Aβ25-35-treated cells, and was significantly downregulated by treatment with magnesium L-threonate. These results indicate that magnesium L-threonate prevents oxidative stress and hypoxic responses in HT22 cells exposed to Aβ25-35 [2].
The PI3K/Akt signaling pathway, known for its role in cell survival and apoptosis regulation, was examined in Aβ25-35-treated HT22 cells. In comparison to cells collected from the control groups, Aβ25-35 exposure resulted in significantly lower ratios of phosphorylated PI3K/PI3K and p-Akt/Akt, indicating pathway inhibition. However, treatment with magnesium L-threonate substantially restored these ratios, suggesting that the compound protects HT22 cells by activating the PI3K/Akt pathway and counteracting Aβ25-35-induced apoptotic signaling.
The activation of the PI3K/Akt pathway was also examined in APP/PS1 mice. Similar to in vitro findings, the p-PI3K/PI3K and p-Akt/Akt ratios were significantly reduced in the transgenic control group compared to the wild type group. However, administration of magnesium L-threonate increased these ratios in the transgenic group treated with the compound, indicating that magnesium L-threonate promotes cell survival and neuroprotection through activation of the PI3K/Akt signaling pathway. The neuroprotective effects of magnesium L-threonate were further evaluated by analyzing neuronal apoptosis in the hippocampus of APP/PS1 mice. The apoptosis rate was significantly elevated in the transgenic control group compared to the wild type group. However, magnesium L-threonate treatment significantly reduced apoptosis in the transgenic group of mice treated with the compound in comparison to the transgenic control group of mice, indicating that MgT mitigates neuronal death [2].

Next, the Morris Water Maze test was performed to assess the effects of magnesium L-threonate on cognitive function in APP/PS1 mice. The transgenic group exhibited significantly prolonged escape latency compared to the wild-type group, indicating impaired spatial memory. However, the transgenic group treated with magnesium L-threonate exhibited a shortened escape latency compared to the transgenic control group, suggesting cognitive improvement. Additionally, the transgenic control group demonstrated a significant decrease in platform crossings and the percentage of time spent in the target quadrant compared to the wild type group. Treatment with magnesium L-threonate increased these cognitive performance markers, with a significant improvement in platform crossings as well as a moderate increase in target quadrant exploration time [2].
During the probe trial of the Morris Water Maze, the transgenic control group took significantly longer to locate the removed platform than the wild type group, while the transgenic group treated with magnesium L-threonate exhibited a shorter latency compared to the transgenic control group, indicating a beneficial effect of magnesium L-threonate on memory retention. However, there were no significant differences in swimming speed or body weight among the groups, confirming that the cognitive improvements were not due to changes in general motor activity or health [2].
Overall, the findings of this study revealed that magnesium L-threonate prevents Aβ25-35-induced oxidative stress and apoptosis in HT22 cells by reducing levels of reactive oxygen species, downregulating HIF-1α expression, and activating the PI3K/Akt pathway. In APP/PS1 mice, administration of magnesium L-threonate significantly improved cognitive function, reduced oxidative stress markers, and protected hippocampal neurons from apoptosis. Additionally, magnesium L-threonate activated the PI3K/Akt pathway in vivo, further supporting the crucial role played in neuroprotection. These results suggest that magnesium L-threonate has potential therapeutic value in reducing neurodegeneration and cognitive deficits associated with Alzheimer’s disease [2].

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

Citations

[1] Liu C, Cheng Y, Guo Y, Qian H. Magnesium-L-threonate alleviate colonic inflammation and memory impairment in chronic-plus-binge alcohol feeding mice. Brain Res Bull. 2021 Sep;174:184-193. doi: 10.1016/j.brainresbull.2021.06.009. Epub 2021 Jun 16. PMID: 34144203.

[2] Xiong Y, Ruan YT, Zhao J, Yang YW, Chen LP, Mai YR, Yu Q, Cao ZY, Liu FF, Liao W, Liu J. Magnesium-L-threonate exhibited a neuroprotective effect against oxidative stress damage in HT22 cells and Alzheimer’s disease mouse model. World J Psychiatry. 2022 Mar 19;12(3):410-424. doi: 10.5498/wjp.v12.i3.410. PMID: 35433327; PMCID: PMC8968501.

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