Main Research Findings

1) Treatment with alpha-GPC has the potential to reduce anti-inflammatory effects in an animal model of intestinal ischemic reperfusion (IR) injury.

2) Due to the structure of the compound, alpha-GPC has the ability to provide anti-inflammatory effects resulting from ischemia-reperfusion stress.

 

Selected Data

1) Ischemia-reperfusion (IR) is frequently linked to inflammatory responses and the development of various pathologies. The research team of Tökés et al. examined the ability of the alpha-GPC to reduce IR-induced intestinal inflammation. For the purpose of this study, 32 adult male Sprague Dawley rats weighing 250-300 grams each, were utilized. The rats were housed in plastic cages and maintained under standard laboratory conditions with ad libitum access to food and water. Test subjects were randomly assigned to 4 different treatment groups including: a control group, a group that received intestinal IR with no treatment, a group that received intestinal IR with pretreatment with 16.56 mg/kg of alpha-GPC, and a group that received intestinal IR followed by treatment with 16.56 mg/kg of alpha-GPC [1].

Following treatment, the animals were anesthetized with 50 mg/kg of sodium pentobarbital and placed supine on a heating pad in order for a tracheostomy to be performed to facilitate breathing. Additionally, the right jugular vein was cannulated for central venous pressure measurements and infusion with 10 ml/kg of Ringer’s lactate, while the right common carotid artery was cannulated to gather measurements of heart rate and mean arterial pressure. For the groups receiving IR, the superior mesenteric artery was clamped for 45 minutes to induce ischemia. After the clamp was removed the intestine was reperfused for 180 minutes. Following the IR process, the research team collected tissue samples from the liver to further examine ATP content, tissue nitrotyrosine and superoxide (SOX) production, and xanthine oxidoreductase (XOR) activity [1].

In order to measure ATP content of the liver samples, the tissue was weighed and placed in trichloroacetic acid, followed by homogenization and centrifugation. After diluting the samples, 100 uL of ATP assay mix was added and luciferase chemiluminescence was utilized to determine ATP content. This procedure was followed by the assessment of intestinal SOX production in the freshly minced biopsy samples. 25 mgs of the tissue were placed in 1 mL of Dulbecco’s solution with 5 uM of lucigenin, followed by performance of chemiluminescence in the presence or absence of the SOX scavenger nitroblue tetrazolium. Finally, XOR activity was assessed by examining colon and ileum tissue samples homogenized in phosphate buffer that contained 50 mM of Tris-HCl, 0.1 mM of EDTA, 0.5 mM of dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 ug.ml of soybean trypsin inhibitor and 10 ug/ml of leupeptin. The resulting homogenate was examined using a fluorometric kinesthetic assay in the presence or absence of the electron-acceptor methylene blue [1].

2) Previous research has found that treatment with alpha-GPC has been found to improve cognitive function and enhance memory. The research team of Lee et al. examined whether the therapeutic abilities of the compound have the potential to enhance cognition and reduce neuronal cell death when induced by epileptic seizure. For the purpose of this study, 8 week old male Sprague-Dawley rats were utilized. Each animal was caged individually and maintained under standard laboratory conditions with a 12 hour light/dark cycle before induction of seizures took place.

In order to induce a seizure the animals were intraperitoneally injected with 127 mg/kg of lithium chloride, followed by 2 mg/kg of scopolamine and 25 mg/kg of pilocarpine, 19 hours later in order to ensure status epilepticus. Status epilepticus was typically achieved 20-30 minutes after injection of pilocarpine. The animals were then placed in individual observation cages in order for the research team to record seizure symptoms characterized by stereotypical mouse and facial movements, blinking, salivation, forelimb clonus, stiffened hind limbs, rearing and sinking [2].

Two hours after the onset of status epilepticus the animals were treated with 10 mg/kg of diazepam; 2 mg/kg of diazepam was delivered until recurrent seizure activity stopped. Following seizure induction the test subjects were divided into 4 groups including: sham seizure induction treated with a vehicle or alpha-GPC and seizure-induced animals treated with a vehicle or alpha-GPC. 250 mg/kg of alpha-GPC were administered to the animals once daily, starting immediately for 1 week or 3 weeks. For the late treatment group, the compound was administered once daily for 3 weeks starting 3 weeks after seizure induction. Instead of the active compound, the control group was administered 0.9% normal saline in the same doses as the experimental group [2].

In order to test the ability of alpha-GPC to improve seizure-induced cognitive impairments, the animals underwent the Morris water maze test two weeks after the seizure. The animals were pretested 1 week prior to administration of alpha-GPC, and then retested following treatment to identify improvements in cognitive functioning. The water maze training pool was filled with opaque water and included a circular escape stage submerged under the water’s surface. The testing took place over 5 days and 4 trials were completed each day. The trail began by placing the animal at a random starting point and allowing them to swim for 120 seconds or until they found the escape platform. A camera and tracking system recorded the route and swim trajectories of the animals for further analysis [2].

Following the treatment protocol and Morris water maze testing the animals were euthanized at 1, 3, or 6 weeks post-seizure. The brains were dissected and fixed in a preservative solution for 1 hour followed by fixation overnight with 30% sucrose solution to act as a cryoprotectant. After 2 days the brains were frozen with powered dry ice, cut with cryostats into 30 um thick slices, and maintained in a storage solution for further histological examination to take place. In order to evaluate the neuroprotective effects of alpha-GPC the brain sections were immunohistochemically stained by NeuN, followed by incubation with monoclonal anti mouse-NeuN antibody, biotinylated anti-mouse IgG, and ABC compound diluted in the primary antiserum. Immune responses were visualized with 3,3-diaminobenzidine and samples were mounted on slides to observe immunoreactions [2].

The research team then worked to detect blood brain barrier disruptions, as well as the presence of immature neurons. First, to detect disruptions in the blood brain barrier the ABC immunoperoxidase process was used to detect IgG-like immunoreactivity after staining brain sections with anti-rat IgG. Next, the presence of immature neurons were detected by immunostaining brain sections with guinea pig anti-DCX antibody followed by 24 hours of incubation with the primary DCX antibody. The sections were washed and incubated again in biotinylated goat anti-guinea pig diluted with the primary antiserum in order for the researchers to examine and quantify the intensity of dendritic signaling.

Finally, the research team detected activation of choline acetyltransferase (ChAT) by homogenizing the collected hippocampal specimens in a RIPA buffer compound followed by 20 minutes of centrifugation. The resulting supernatant was stored for further examination while hippocampal protein content was measured and the proteins were diluted, separated, and transferred to a polyvinylidene difluoride membrane. The membranes were then incubated with the primary antibody overnight, washed, and incubated again for an hour with anti-rabbit IgG secondary antibody conjugated with horseradish peroxidase [2].

 

Discussion

1) When looking at the hemodynamic parameters measured in the experiment there were no significant changes noticed by the research team. However, there was a noticeable reduction in mean arterial pressure seen across each experimental group that remained reduced until the end of the study. In regards to heart rate there were no statistically significant differences between the groups throughout the entire experiment. In comparison to the control group, mesenteric vascular resistance was significantly increased in the IR group of test subjects for up to 225 minutes of reperfusion time. When the IR animals were pretreated with alpha-GPC there was a pronounced decrease in mesenteric vascular resistance. That being said, when the IR animals were treated after injury the benefits were shown to diminish over a period of time [1].

Figure 1: Changes in mean arterial blood pressure throughout the process of intestinal ischemia-reperfusion.

Figure 2: Changes in mesenteric vascular resistance throughout the process of intestinal ischemia-reperfusion.

Following induction of ischemic there was a significant reduction in blood flow through the superior mesenteric artery that was not seen in the sham-operated or control group. However, when the animals were treated with alpha-GPC after ischemia was induced, blood flow through the superior mesenteric artery remained unchanged. In the IR animals that were pretreated with alpha-GPC blood flow through the superior mesenteric artery had a tendency to increase [1].

Figure 3: Changes in superior mesenteric artery flow throughout the process of intestinal ischemia-reperfusion.

In terms of microcirculation parameters the red blood cell velocity through the serum was used to measure ileal microcirculatory conditions in the test subjects. Red blood cell velocity was shown to decrease in the IR group of subjects, however when treated with alpha-GPC following ischemia-reperfusion there was a significant increase and a normalizing reduction in red blood cell velocity within 15 minutes of reperfusion time. Additionally, it was noted that in the group of test subjects treated with alpha-GPC prior to ischemia-reperfusion, there was a positive trend observed by the research team.

Figure 4: Changes in red blood cell velocity throughout the process of intestinal ischemia-reperfusion.

When looking at biopsy samples collected from the small intestines of the test subjects, there were no changes in the reactive oxygen species producing capacity in the animals that underwent sham-operations. In the IR group of test subjects there was a significant increase in the reactive oxygen species producing capacity after 15 minutes of reperfusion, in comparison to baseline values and values collected from the sham-operated group of test subjects. The animals that were pretreated with alpha-GPC prior to ischemia-reperfusion injury experienced a significant reduction in the reactive oxygen species producing capacity. Similar results were seen in the group of test subjects that were treated with alpha-GPC following induction of ischemic-reperfusion injury [1].

Figure 5: Changes in reactive oxygen species producing capacity throughout the process of intestinal ischemia-reperfusion.

Furthermore, the research team examined the activation of xanthine oxidoreductase in the small intestine during the IR process, ultimately increasing production of superoxides. Xanthine oxidoreductase activation was found to be significantly higher in the IR animals, compared to the sham-operated test subjects. When the test subjects were treated with alpha-GPC prior to induction of ischemia-reperfusion injury there was a noticeable elevation in xanthine oxidoreductase activity. However, when alpha-GPC was administered to the test subject following induction of ischemia-reperfusion injury, xanthine oxidoreductase activity was significantly reduced in comparison to the IR group, the pretreatment group, and the sham-operated group. That being said, the researchers were able to conclude that treatment with alpha-GPC was more effective at decreasing xanthine oxidoreductase activity and superoxide production when administered after the induction of ischemia-reperfusion injury [1].

Figure 6: Changes in xanthine oxidoreductase activation across all experimental treatment groups.

The final biomarkers assessed by the research team included tissue nitrotyrosine levels and ATP levels in liver samples. Nitrotyrosine formation is indicative of nitrosative stress and is associated with the production of peroxynitrite. Induction of IR injury led to an increase in the levels of nitrotyrosine in the tissues in comparison to the control and sham-operated groups. When the test subjects were treated with alpha-GPC both before and after induction of IR injury there were no noticeable increases in nitrotyrosine, and levels remained similar to the control group of animals. As for ATP levels, due to the production of reactive oxygen species following IR injury, there was a significantly reduced level of ATP in the liver samples in comparison to the sham-operated group. When the animals were treated with alpha-GPC prior to induction of ischemia-reperfusion injury there was an elevation in ATP levels in the liver. However, this increase in ATP was more pronounced when the test subjects were treated with alpha-GPC after induction of ischemia-reperfusion injury, and levels remained similar to the sham-operated group of animals [1].

Figure 7: Changes in nitrotyrosine levels in the small intestine across all experimental treatment groups.

FIgure 8: Changes in ATP levels present in liver samples across all experimental treatment groups.

2) The research team of Lee et al examined the ability of alpha-GPC to reduce cognitive deficits and neuron death following seizure induction. When administered for only 1 week, the nootropic was shown to not elicit any significant effects on neuron death or disruptions in the blood brain barrier. Immunohistochemistry performed using anti-NeuN revealed that seizure induction led to neuronal death in the hippocampus, CA3, CA21, and subiculum. When comparing the experimental groups to the control group, 1 week of early treatment with alpha-GPC did not result in any significant difference in the number of live hippocampal neurons, the disruption of the blood brain barrier, or neuroprotective effects [2].

Figure 1: Changes in the amount of live NeuN (+) cells located in CA1, CA3, the hilus, and the hippocampus after 1 week of treatment with alpha-GPC administered immediately following seizure induction.

Comparatively, when alpha-GPC was administered for 3 weeks there was a significant reduction in hippocampal neuronal death and disruption of the blood brain barrier. The changes were detected using immunohistochemistry that revealed neuronal death and IfG leakage in the hippocampus following seizure induction. 3 weeks of immediate administration of alpha-GPC led to decreased neuron death in the CA3 and hilus regions when compared to the group treated with a vehicle. Similar results were reported in terms of IgG leakage in the experimental group compared to the control group. These results reported by the research team indicated that 3 weeks of treatment with alpha-GPC immediately following induction protected the hippocampus of the test subjects from neuronal death and blood brain barrier disruption [2].

Figure 2: Changes in the amount of live NeuN (+) cells located in CA1, CA3, the hilus, and the hippocampus after 3 weeks of treatment with alpha-GPC administered immediately following seizure induction.

In addition to immediate treatment with the nootropic, the research team also assessed how treatment administered 3 weeks after induction of injury affects hippocampal neuron death and disruption of the blood brain barrier. 6 weeks after the induction of seizure the brain sections collected from each group of test subjects were stained with NeuN and IgG. In the seizure induced group treated with a vehicle, the amount of live neurons in the hippocampus decreased by 62%. In the seizure induced group treated with alpha-GPC, there was only a 25% decrease in the number of live neurons in the hippocampus. Overall, these findings suggested that neuronal death and blood brain barrier disruption were significantly decreased when alpha-GPC was administered for 3 weeks, beginning 3 weeks after a seizure was induced [2].

Figure 3: Changes in the amount of live NeuN (+) cells located in CA1, CA3, the hilus, and the hippocampus after 3 weeks of treatment with alpha-GPC administered 3 weeks after seizure induction occurred.

Treatment with alpha-GPC was also examined to determine the ability of the compound to improve cognitive functioning measured through Morris water maze testing. The test subjects completed the water maze at 3 weeks post-seizure, before injection of alpha-GPC, and at 5 weeks post-seizure, after injection of alpha-GPC. Prior to treatment the animals were not able to reach the escape platform within the 120 second time limit. However, after treatment the animals were able to successfully reach the escape platform, indicating improved performance and cognitive functioning [2].

Figure 4: Changes in performance on the Morris water maze test recorded at 3 weeks post-seizure, prior to treatment with alpha-GPC, and 5 weeks post-seizure, after treatment with alpha-GPC.

 

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] Tőkés T, Tuboly E, Varga G, et al. Protective effects of L-alpha-glycerylphosphorylcholine on ischaemia-reperfusion-induced inflammatory reactions. Eur J Nutr. 2015;54(1):109-118. doi:10.1007/s00394-014-0691-2

[2] Lee SH, Choi BY, Kim JH, et al. Late treatment with choline alfoscerate (l-alpha glycerylphosphorylcholine, α-GPC) increases hippocampal neurogenesis and provides protection against seizure-induced neuronal death and cognitive impairment. Brain Res. 2017;1654(Pt A):66-76. doi:10.1016/j.brainres.2016.10.011

 

 

Mechanisms and Effects of Alpha-GPC

Alpha-glycerophosphocholine (Alpha-GPC) is a choline compound that contains a phospholipid. The main benefits of the compound are improvement in cognition, primarily memory and attention, and the ability to help combat Alzheimer’s disease. The compound breaks down into choline and glycerol-1-phosphate. Choline is a precursor for acetylcholine, a neurotransmitter that plays a large role in both the central and peripheral nervous system. Glycerol-1-phosphate is shown to support and protect cellular membranes.

Researchers have theorized that Alpha-GPC works by increasing the synthesization and the expression of acetylcholine in the brain. This is what allows it to evoke such drastic effects on memory, learning, and attention. Acetylcholine is often closely related to Alzheimer’s as the most common treatment for the disease is acetylcholinesterase inhibitors that promote increased levels of acetylcholine in the brain (https://examine.com/supplements/alpha-gpc/).

Evidence has shown that Alpha-GPC also promotes the secretion of Growth Hormone (GH) due to higher circulating levels of choline. In addition to the increased levels of GH, acetylcholine is responsible for skeletal muscle contraction, which indicates involvement in the improvement of physical performance. Various studies have administered Alpha-GPC to animals and found that administration of the nootropic led to a drastic improvement in various physical performance tests. These studies also emphasized that even a short supplementation period led to increased muscle mass and improved performance.

 

Alpha-GPC as a Byproduct of Wheat Fermentation

As it was mentioned above, Alpha-GPC has shown promise in treating Alzheimer’s disease and other forms of dementia due to the correlation between the compound and the neurotransmitter, acetylcholine. Since the 1990s animal-based studies have been conducted in order to determine how effective Alpha-GPC is against memory loss. The animals being tested were given either a placebo or active Alpha-GPC for 10 days. After the 10 days memory loss was induced through use of the drug, scopolamine. Overall the study concluded that administration of Alpha-GPC was capable of significantly reducing memory impairments and promoting general improvement to cognitive functions associated with Alzheimer’s and dementia (https://www.onnit.com/academy/alpha-gpc-benefits/).

Due to the potential Alpha-GPC has shown in treating Alzheimer’s and memory loss, researchers have begun to find a sustainable way of synthesizing the compound. Researchers Oyeneye et. Al examined how the fermentation of wheat could possibly produce sufficient levels of Alpha-GPC. Current methods of production include the hydrolysis of phosphocholine or the condensation of glycerol derivatives with the use of catalysts. The enzymatic method of phosphocholine hydrolysis is effective as it reduces the amount of excess chemical reagents being produced. Further purification of Alpha-GPC comes from the use of chromatography. While this sufficiently produces Alpha-GPC it is not a sustainable method of synthesization and incredibly costly to the laboratories and researchers looking to further their experiments.

The researchers found that AC Andrew was the type of wheat cultivar best suited to produce large amounts of Alpha-GPC due to its high starch content. Typically when fermentation occurs ethanol is produced until it reaches its peak at approximately 72 hours, however this study highlighted the fact that after ethanol production had stopped, Alpha-GPC continued to form without altering the fermentation process at all. That being said, when the goal of fermentation is specifically to produce Alpha-GPC, there are no excess levels of ethanol produced. These findings support the goal of synthesizing Alpha-GPC in an energy-efficient and low expense manner (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7271372/).

 

GPC Promotion of Longevity and Health in Roundworms

In addition to its benefits on cognition, researchers Liu et. Al examined how GPC could combat symptoms of aging in Caenorhabditis elegans, more commonly known as the roundworm. Since GPC levels tend to decrease while aging, the roundworms were given an initial dosage of 10 mM of GPC to see how the compound would affect whole lifespan, mean lifespan, and maximum lifespan. The dosage capped at 50 mM and led to a drastic increase in whole, mean, and maximum lifespan.

Furthermore, it was noted that motor ability declines from aging so the researchers measured the roundworms’ activity levels by examining body bending rate and pharyngeal pumping rate at different points throughout their lifespan. Following a 50 mM dose of GPC there was a drastic improvement in both of these rates. Specifically, GPC increased the rate of body bending in the mid-late life stage and increased the rate of pharyngeal pumping in the early-mid life stage. These results suggest that treatment with GPC helps to alleviate the symptoms of age-induced decline (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8875989/).

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