TW201920953A - Method for monitoring serum glutamate levels - Google Patents

Abstract

The present invention relates to methods for monitoring serum glutamate levels in a mammal, particularly in a human. The methods are useful to quantify the ability of the mammal to metabolize dietary glutamate as a diagnostic marker for predicting the onset of or propensity for developing a central nervous system (CNS), psychotic, or related disorder, associated with glutamate toxicity. The methods are also useful for designing regimens for modulating serum glutamate levels in a mammal to treat or prevent such a disorder.

Description

Method for monitoring serum glutamate levels

The invention relates to a method for monitoring serum glutamate levels in mammals, especially human individuals. The method is based on determining a relative serum glutamate level in an individual after a predetermined amount of the glutamate-containing protein composition is administered to the individual under controlled fasting and postprandial conditions. This method can be used to quantify the mammal's ability to metabolize dietary glutamate as a predictor of the onset or propensity of the central nervous system (CNS), mental or neurological disease associated with glutamate toxicity Diagnostic markers. This method also helps to design a regimen to regulate serum glutamate levels in mammals to treat or prevent this disease.

For many diseases, such as cancer, tumors, liver, kidney, blood, or hereditary diseases, there are different biomarkers and blood tests to confirm the diagnosis. However, there are currently no precise biomarkers or a single reliable blood test that can be used to correctly diagnose a wide range of neurological and psychiatric disorders. In the case of diseases such as Amyotrophic Lateral Sclerosis (ALS) or Parkinson's disease, many medical examinations and tests are usually required to diagnose whether a patient has the disease. The diagnostic process may include physical examination, blood examination, and imaging examinations, such as magnetic resonance imaging (MRI). It is important to rule out other conditions and fault diagnosis. From the first observation of symptoms, the diagnosis usually takes 9-12 months. There are currently no blood tests that actively diagnose these conditions, and there is no effective way to monitor the effectiveness of specific treatments. For a rapidly developing deadly disease, such as amyotrophic lateral sclerosis (ALS), the median survival time for the initial diagnosis is only 3 years, and clear biomarkers or blood tests to identify the disease may lead to early intervention To save lives.

Our research on possible biomarkers of the central nervous system (CNS) and related diseases has focused on the human intestine, that is, the portion of the gastrointestinal tract from the pyloric sphincter of the stomach to the anus, and the small and large intestine in humans. More than 100 trillion microorganisms became so-called human microorganisms. These microorganisms contain microorganisms that live on and on the human body and perform important functions: these microorganisms synthesize vitamins, help digestion, and help develop and maintain the immune system. The human intestine is the most bacterial-rich organ in the human body. In the human intestine, the number of genes of resident microorganisms far exceeds the genes in the human genome with a ratio of 100: 1, so it provides attractive candidates for drug interference. . Over the past few years, research and treatments focused on microorganisms have proliferated, including inflammatory bowel disease (IBD), childhood asthma, diabetes, obesity, cardiovascular disease, colorectal cancer, and antibiotics Related diseases such as diarrhea. Most of these disease states involve changes in the composition or function of microorganisms, which are called bacterial phase imbalances. Recent studies have shown interactions between the gut microbiota, the gut, and the central nervous system (CNS) (Fang, 2015).

The gut-brain axis is a two-way communication system controlled by the autonomic and enteric nervous systems (ElAidy et al., 2015). Intestinal homeostasis and microorganisms have been observed to play important roles in neurological diseases, such as amyotrophic lateral sclerosis (ALS) (Fang, 2015), Alzheimer's disease (Lukiw, 2013), autism (Cubells, 2013), schizophrenia (Katlyn Nemani, 2015), Parkinson's disease (Filip Scheperjans, 2014), multiple sclerosis (MS) (Westall, 2006), and mental illness Schizophrenia (Katlyn Nemani, 2015). A study by Wu and colleagues (2015) showed intestinal leakage and changes in the composition of microorganisms in a mouse model of amyotrophic lateral sclerosis (ALS) transgenic SOD1-G93A, indicating that the microorganisms are on the side of the muscular atrophy Potential unknown role in sclerosis (ALS) (Shaoping Wu1, 2015). A similar study has been reported in Parkinson's disease. (Timothy R. Sampson, 2016). However, due to the challenge of dealing with a large number of bacteria, fungi, and microorganisms (possibly more than 2,000 species) in the human intestinal microbiota, the actual mechanism of why and how intestinal microbes affect these neurological diseases remains unknown. These microorganisms interact in a finely balanced ecosystem. Another difficulty in diagnosing and treating these diseases is the property of the blood brain barrier (BBB), which is generally impermeable to most substances, which makes it difficult to use drugs, metabolites, biological materials, or inorganic parts. Doing research on the brain is challenging.

The key to our understanding of the causative factors of many neurological diseases comes from the role of healthy individuals and patients with neurological diseases in the digestion of glutamate protein from food intake, and the ability to metabolize glutamate to glutamine Extensive research and clinical observations on the metabolic effects of acid-resident bacterial species, and the selective absorption rate into the body through the mucosa of the small intestine. Other independent studies detailing the association between elevated glutamate in serum and elevated levels of glutamate in brain extracellular fluid (ECF), as well as a range of neurological conditions, these independent studies Surprisingly confirmed our clinical observations. However, none of these other studies has shown a link between intestinal glutamate digestion and various central nervous system diseases, nor has it been possible to determine the steady state collapse of glutamate metabolism that may result from intestinal flora imbalances The role of a class of bacteria and the ultimate association between glutamate digestion and neurological and mental illness states.

Gluten is the main nerve conduction substance in the human central nervous system, and it is the most abundant free amino acid in this system. The glutamate accounts for about 90% of the activity of total nerve-conducting substances in the brain. The beneficial effects of glutamate depend heavily on strict homeostasis by maintaining glutamate concentrations in the extracellular fluid (ECF) of the brain (below the toxic range of 0.3-2 µM / L) (Akiva Leibowitz, 2012 ). Animal models and human clinical studies have revealed the association of pathologically elevated levels of extracellular fluid (ECF) glutamate with several acute and chronic neurodegenerative diseases, including stroke, traumatic brain injury, TBI), intracerebral hemorrhage, cerebral hypoxia, amyotrophic lateral sclerosis (ALS) (ELISABETH ANDREADOU, 2008), dementia and other diseases. These diseases are characterized by the breakdown of the blood-brain barrier that promotes hundreds of times the concentration of glutamate in the brain's extracellular fluid (ECF), which in turn causes glutamate to travel between plasma and brain extracellular fluid. Its concentration gradient moves freely (Akiva Leibowitz, 2012).

Campos and colleagues' use of functional magnetic resonance imaging in a rat model of stroke has shown that a reduction in plasma glutamate levels by plasma glutamate scavengers is associated with a significant reduction in glutamate in the brain, which is associated with improved neural function Relevant. (Francisco Campos, 2011).

Compared with healthy controls, such as amyotrophic lateral sclerosis (ALS) (ELISABETH ANDREADOU, 2008), Alzheimer's disease (DAN E. MIULLI, 1993), and Parkinson's disease (Yasuo Iwasaki, 1992 ), And elevated plasma glutamate levels in patients with multiple sclerosis (FRED C. WESTALL, 1980), suggesting that systemic defects in glutamate metabolism are the underlying cause of these diseases (Andreas Plaitakis, 1987) . Glutamate-modulated excitotoxicity is considered the leading cause of motor neuron degeneration in patients with amyotrophic lateral sclerosis (ALS). Other neurological diseases associated with high levels of glutamate-induced toxicity include: autism (Chie Shimmura1, 2011), schizophrenia (SAIvanovaa, 2014), epilepsy (Sirpa Rainesalo, 2004), Azerbaijan Zheimer's disease (DAN E. MIULLI, 1993), and mental illness (SAIvanovaa, 2014).

Gluten is one of the main components of dietary protein and is the most abundant amino acid in the diet. It is also used in many processed or hydrolyzed foods and as an additive and flavor enhancing ingredient in the form of monosodium glutamate (MSG). A significant portion of dietary glutamate is metabolized to glutamate during small intestine adsorption. Excess glutamate that escapes mucosal metabolism is delivered to the liver through the portal vein, and the liver controls the composition of the amino acid mixture released into the peripheral circulation. (Lewis Stegink, 1979) Nakagawa reported the regulation of glutamate. No toxic effects were observed when young boys were fed 12.75 g of free glutamate daily, showing that free glutamate given orally was taken by healthy individuals. The metabolic effects are effectively removed (ITSIRO NAKAGAWA, 1960). Other studies have also confirmed that day and night food intake does not affect plasma glutamate levels. Similarly, when high-protein diets are provided to healthy human individuals, these diets do not increase plasma glutamate, although the main metabolite of glutamate, i.e., glutamic acid, is increased (T Palmer, 1973) Indicates that the absorption of glutamic acid is more effective than that of glutamate in the intestinal mucosa. These findings were supported by Reeds research in 1996 and 1997, which showed that intestinal glutamate was almost completely metabolized during the first passage of piglets through the gastrointestinal tract and that intestinal glutamate was fed Preferred source of mucosal glutathione synthesis in piglets. (PETER J. REEDS, 1996).

The metabolism of glutamate in the human small intestine to glutamate occurs mainly through bacteria producing glutamate synthase. These bacteria are usually Gram-positive bacteria, including most species of Lactobacillus, such as Lactobacillus embryonicus, and Gram-negative bacteria, such as E. coli, Bacteroides fragile, Pseudomonas, and Klebsiella. The glutamate synthase produced by these bacteria in the intestine is an important enzyme for converting dietary glutamate to glutamate in the small intestine. The lack or destruction of these resident bacteria caused by imbalance of the intestinal flora causes intestinal damage, accompanied by digestive abnormalities. Bacterial phase imbalance is an imbalance in the intestinal flora caused by too few beneficial bacteria and excessive growth of bad bacteria, yeasts and / or parasites. Bacterial phase imbalances lead to inefficient metabolism of dietary glutamate, resulting in elevated free glutamate serum levels, which are often many times higher than the serum basal levels of healthy individuals.

The effectiveness of our working model is consistent with the study of Leibowitz and colleagues (2012), that is, when trying to reduce serum glutamate levels, regardless of the mechanisms involved, the decrease in blood glutamate concentration is related to the nerves The results of functional improvement are correlated (Akiva Leibowitz, 2012). Blood glutamate clearance is achieved through several mechanisms, including: catalyzing enzymatic processes involving glutamate metabolism, redistribution of serum glutamate into tissues, and acute stress response.

It can be seen that there are potentially serious health problems with elevated serum glutamate levels and impaired ability to metabolize dietary glutamate to glutamate. Therefore, it can be seen from the above; there is an urgent need to develop reliable diagnostic methods to quantify the effectiveness of glutamate metabolism in the dietary intake of human individuals in order to predict the onset or progression of many of the aforementioned neurological disease states. In addition, this diagnostic method will provide a means to design effective treatment protocols and monitor the efficacy of treatment.

In the present invention, we have developed a method for monitoring and regulating serum glutamate levels as a biomarker to quantify the efficiency of glutamate metabolism to predict the onset, severity or track of various neurological diseases Progression of neurological diseases. These diseases include, but are not limited to, amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, multiple sclerosis, schizophrenia, depression, dementia, autism, peripheral neuropathy, obsessive-compulsive Obessive compulsive disorder (OCD), dementia, restless legs syndrome, and a large group of other mental illnesses and related disorders related to glutamate toxicity. The invention is achieved by monitoring human patients' glutamate levels at two or more time points to obtain fasting and postprandial serum glutamate levels under a controlled regimen that includes fasting and then ingestion A predetermined high glutamate liquid meal or suspension.

We were surprised to find that in healthy individuals an average of 70 kg of people consumed 14.5 grams of dietary glutamate, the difference between fasting serum glutamate and postprandial serum glutamate levels was 33 ± 16 µmol / L to 63 ± 34 µmol / L. This difference in serum glutamates in healthy individuals is consistent with previous findings (Lewis Stegink, 1979), showing that compared to fasting plasma glutamate levels, taking about 80 to about 207 mg / kg bran After a high-protein diet with total glutamate, the peak plasma glutamate level increased approximately two-fold. In contrast, the differences observed in human individuals exhibiting neuropathy associated with glutamate toxicity are unusually large, such as 271 to 340.4 µmol / L (see Examples section). We have also shown that for patients with improved symptoms after treatment, fasting serum glutamate and postprandial serum glutamate levels have fallen, consistent with a reduction or resolution of the severity of neuropathy.

The invention relates to a method of monitoring the relative serum glutamate level of a human patient at two or more selected time points, comprising the steps of: (a) subjecting the patient to fasting for a period of at least about 12 hours, with the exception of water; (b) removing the first (fasted) blood sample from the patient by venipuncture; (c) transferring the first blood sample to a first container, optionally containing between about 0 ° C to about 5 ° C Pre-chilled anticoagulant; (d) orally administered to the patient an aqueous solution or suspension containing about 5 to about 15 grams of glutamic acid (glutamate); (e) during the administration of step (d) About 15 minutes to about 90 minutes after the aqueous solution or suspension, a second (postprandial) blood sample is taken from the patient through a venipuncture; (f) transferring the second blood sample to a second container, optionally containing Pre-chilled anticoagulant between about 0 ° C and about 5 ° C; (g) each of the first and second blood samples is centrifuged to separate serum from platelets in the blood sample to provide a first ( (Fasting) serum samples and second (postprandial) serum samples, (h) by adding deproteinizing agent to each serum sample Deproteinize the first serum sample and the second serum sample separately; (i) centrifuge each serum sample from step (h) to isolate the protein from the serum in the sample to provide a first (prohibited (E) a protein-free serum sample and a second (postprandial) protein-free serum sample; (j) analyzing the first and the second protein-free serum sample to determine the glutamate level in each sample; and (k) The glutamate level in the second sample is compared to the glutamate level in the first sample to determine the relative ratio of post-meal and fasted glutamate levels in the sample.

The present invention also relates to a method for monitoring relative serum glutamate levels from a human individual, comprising the steps of: (i) providing a first (fasting) blood sample from the fasting state at the first time point; Obtained by an individual, wherein the individual preferably fasts other than water for a period of at least about 12 hours; (ii) provides a second (postprandial) blood sample, which is in a fasted state during step (i) The subject obtained from the subject at a second time point of about 15 minutes to about 90 minutes after oral administration of an aqueous solution or suspension containing about 5 to about 15 grams of glutamic acid (glutamate); Transferring the first blood sample to a first container, optionally containing an anti-coagulant precooled between about 0 ° C to about 5 ° C; (iv) transferring the second blood sample to a second container , Optionally containing an anti-coagulant precooled between about 0 ° C and about 5 ° C; (v) each of the first and second blood samples is centrifuged to separate the platelets from the blood sample Serum to provide a first (fasted) serum sample and a second (postprandial) serum sample, (vi) permeate through each serum Deproteinizing agent is added to the sample to deproteinize each of the first serum sample and the second serum sample; (vii) centrifuging each serum sample from step (vi) to separate protein from the serum in the sample To provide a first (fasted) protein-free serum sample and a second (post-prandial) protein-free serum sample; (viii) analyze the first and second protein-free serum samples to determine the glutamate in each sample Levels; and (ix) comparing the glutamate level in the second sample to the glutamate level in the first sample to determine glutamate levels after meals and fasting in the sample Relative ratio.

In another aspect, the invention relates to a method, wherein in step (d) or (ii), the aqueous solution or suspension comprises glutamic acid equivalent to about 70 mg / kg to about 225 mg / kg based on the weight of the individual (Glutamate).

In another aspect, the invention relates to a method, wherein in step (d) or (ii), the aqueous solution or suspension comprises equivalent to about 10 grams of glutamic acid (glutamate).

In another aspect, the invention relates to a method, wherein in step (d) or (ii), the aqueous suspension or solution is an aqueous suspension or solution of a digestible protein.

In another aspect, the invention relates to a method, wherein in step (d) or (ii), the aqueous solution or suspension comprises a glutamic acid (glutamate equivalent to about 150 mg / kg based on the weight of the individual) ).

In another aspect, the invention relates to a method, wherein in step (d) or (ii), the aqueous suspension or solution of the digestible protein is substantially free of glutamate.

In another aspect, the invention relates to a method, wherein in step (d) or (ii), the aqueous suspension or solution is a solution or suspension of whey protein.

In another aspect, the invention relates to a method, wherein in step (d) or (ii), the aqueous suspension or solution of the whey protein is substantially free of glutamate.

In another aspect, the invention relates to a method, wherein in step (d) or (ii), the aqueous suspension or solution comprises about 75 grams of whey protein, suspended or dissolved in about 200 to about 250 ml of water or fruit juice in.

In another aspect, the invention relates to a method, wherein in step (d) or (ii), the fruit juice is apple juice.

In another aspect, the invention relates to a method, wherein the time in step (e) or (ii) is about 60 minutes.

In another aspect, the invention relates to a method, wherein in step (b) or (i), the first (fasting) blood sample has a volume of about 1 to about 10 ml, and wherein in step (e) or In (ii), the volume of the second (postprandial) blood sample is about 1 to about 10 ml.

In another aspect, the invention relates to a method, wherein in step (b) or (i), the first (fasted) blood sample has a volume of about 5 ml, and wherein in step (e) or (ii) The second (postprandial) blood sample has a volume of about 5 ml.

In another aspect, the invention relates to a method, wherein the anticoagulant in step (c) or (iii) and the anticoagulant in step (f) or (iv) are selected from EDTA (ethylenediamine Tetraacetic acid), lithium heparin, sodium citrate, and sodium heparin.

In another aspect, the invention relates to a method, wherein the anticoagulant in step (c) or (iii) and the anticoagulant in step (f) or (iv) is EDTA (ethylenediamine tetra Acetic acid).

In another aspect, the invention relates to a method, wherein in step (g) or (v), the centrifugation is performed at about 0 ° C to about 5 ° C on the first blood sample and the second blood sample, respectively. Performed at about 17,000 × g for about 10 minutes.

In another aspect, the invention relates to a method, wherein in step (h) or (vi), the deproteinizing agent is selected from the group consisting of perchloric acid, trichloroacetic acid, and tungstic acid.

In another aspect, the invention relates to a method, wherein in step (h) or (vi), the deproteinizing agent is perchloric acid.

In another aspect, the invention relates to a method, wherein in step (h) or (vi), the deproteinizing agent is perchloric acid having a concentration of about 0.2 N to about 0.4 N and a volume of about 5 ml.

In another aspect, the invention relates to a method, wherein in step (i) or (vii), the centrifugation is performed at about 0 ° C to about 5 ° C on the first blood sample and the second blood sample, respectively. Performed at about 19,000 × g for about 10 minutes.

In another aspect, the invention relates to a method, wherein the analysis in step (j) or (viii) is performed by an enzyme-linked immunosorbent assay (ELISA).

In another aspect, the invention relates to a method comprising the further step (I): if the ratio of the glutamate level in the second sample to the glutamate level in the first sample is greater than about 2, treating the occurrence or risk or progression of abnormally elevated (excessive) serum glutamate or a disease associated therewith in the human individual.

The invention also relates to a method comprising when the relative ratio of post-meal and fasted glutamate levels in the sample is greater than about 2 (eg, post-meal level: fasted level = 2.5: 1, 3: 1, 3.5: (1: 4: 1, 4.5: 1, 5: 1 or higher), the individual is diagnosed with abnormally elevated (excessive) serum glutamate or with a disease associated with it or at risk or its progression.

In another aspect, the invention relates to a method, wherein in step (I), the method of treating abnormal (excessive) serum glutamate is by increasing the intestinal glutamate synthase activity of the patient.

The present invention also relates to an agent capable of increasing the activity of intestinal glutamate synthetase in the preparation of a medicament for treating abnormally elevated serum glutamate or a disease associated therewith or preventing the progress of the disease in an individual in need thereof. use. In certain embodiments, the agent is a probiotic that is used to modulate the population of bacteria that produce non-pathogenic glutamate synthase in the small intestine of the individual. In certain embodiments, the agent is a probiotic with probiotics to regulate the population of bacteria that produce non-pathogenic glutamate synthase in the individual's small intestine. In certain embodiments, the agent is used for oral administration.

In another aspect, the invention relates to a method, wherein the method of treatment in step (I) comprises administering glutamate synthase to the patient.

The present invention also relates to the use of a glutamate synthetase in the preparation of a medicament for treating an abnormally elevated serum glutamate or a disease related thereto or preventing the progress of the disease in an individual in need.

In another aspect, the present invention relates to a method comprising, in step (I), orally administering probiotics to modulate a population of bacteria producing non-pathogenic glutamate synthase in the small intestine of the patient.

In another aspect, the invention relates to a method comprising, in step (I), orally administering probiotics with probiotics to modulate a population of bacteria that produce non-pathogenic glutamate synthase in the small intestine of the patient.

In another aspect, the invention relates to a method for treating a central nervous system or a mental illness.

In another aspect, the present invention relates to a method of treatment, wherein the neurological or mental illness is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, autism, cerebellar atrophy, dementia, epilepsy, severe depression, Multiple sclerosis, obsessive-compulsive disorder, Parkinson's disease, peripheral neuropathy, restless legs syndrome, schizophrenia, stiff people syndrome, and stroke.

In another aspect, the invention relates to a method of diagnosing elevated serum glutamate levels in a human patient, comprising the steps of: (a) subjecting the patient to fasting for a period of at least about 12 hours, with the exception of water; (b) Remove the first (fasted) blood sample from the patient by venipuncture; (c) transfer the first blood sample to a first container, optionally containing pre-cooling between about 0 ° C and about 5 ° C (D) an oral solution or suspension containing about 5 to about 15 grams of glutamic acid (glutamate) in the patient; (e) an aqueous solution or suspension in step (d) About 15 minutes to about 90 minutes after the solution, a second (postprandial) blood sample is taken from the patient through a venipuncture; (f) the second blood sample is transferred to a second container, optionally containing at about 0 ° Pre-chilled anticoagulant between C and about 5 ° C; (g) each of the first and second blood samples is centrifuged to separate serum from platelets in the blood sample to provide a first (fasting) Serum samples and second (postprandial) serum samples, (h) by adding a deproteinizing agent to each serum sample, The first serum sample and the second serum sample are deproteinized; (i) each serum sample from step (h) is centrifuged to isolate protein from the serum in the sample to provide a first (fasted) Protein serum samples and second (postprandial) protein-free serum samples; (j) analyzing the first and second protein-free serum samples to determine the glutamate level in each sample; and (k) when the first When the ratio of the glutamate level in the two samples to the glutamate level in the first sample is greater than about 2, the patient is diagnosed with elevated serum glutamate.

The present invention also provides a method for diagnosing the occurrence or risk or progression of abnormally elevated (excessive) serum glutamate levels or diseases associated therewith in a human individual, comprising the steps of: (i) providing a first (prohibited A) blood sample obtained at a first time point from the individual in a fasting state, wherein the individual preferably fasts other than water for a period of at least about 12 hours; (ii) provides a second (after meal) ) A blood sample from about 15 minutes to about 90 minutes after oral administration of an aqueous solution or suspension containing about 5 to about 15 grams of glutamic acid (glutamate) to an individual in a fasted state. Obtained from the individual at a second time point; (iii) transferring the first blood sample to a first container, optionally containing an anti-coagulant pre-chilled between about 0 ° C and about 5 ° C; (iv) ) Transferring the second blood sample to a second container, optionally containing an anti-coagulant precooled between about 0 ° C and about 5 ° C; (v) each of the first and second blood samples Centrifuging to isolate serum from platelets in the blood sample to provide a first (fasted) serum sample and A second (post-meal) serum sample, (vi) deproteinizing each of the first serum sample and the second serum sample by adding a deproteinizing agent to each serum sample; (vii) centrifugation from step ( vi) each serum sample to isolate protein from the serum in the sample to provide a first (fasted) protein-free serum sample and a second (post-prandial) protein-free serum sample; (viii) analyze the first and A second protein-free serum sample to determine the glutamate level in each sample; and (ix) the ratio of the glutamate level in the second sample to the glutamate level in the first sample At greater than about 2, the individual is diagnosed with an abnormally elevated serum glutamate or with a disease or risk associated with it or its progression.

In another aspect, the present invention relates to a kit for performing the method according to any one of claims 1-22 and 29 of the patent application scope, comprising a reagent capable of specifically detecting glutamate in the sample, and Instructions for implementing the method.

In another aspect, the invention relates to the use of a biomarker in the manufacture of a set, wherein the biomarker is a glutamate in a blood sample from an individual, and the set helps to quantify the individual's metabolism of dietary bran The ability of an amine salt to include obtaining a first (fasted) blood sample from a subject in a fasted state at a first time point; orally administered to the subject in a fasted state contains an amount equivalent to about 5 to about 15 A second (postprandial) blood sample is obtained from the individual at a second time point of about 15 minutes to about 90 minutes after a gram of aqueous solution or suspension of glutamic acid (glutamate); the sample is analyzed for fasting and Postprandial serum glutamate levels; and comparing the levels to obtain a relative ratio of postprandial and fasting serum glutamate levels.

In another aspect, the invention relates to the use of a biomarker in the manufacture of a kit, wherein the relative ratio of post-prandial and fasting serum glutamate levels greater than about 2 indicates poor ability to metabolize dietary glutamate.

In another aspect, the present invention relates to the use of a biomarker in the manufacture of a kit, wherein the relative ratio of post-prandial and fasting serum glutamate levels greater than about 2 indicates that a serum glutamate is abnormally elevated or is associated therewith. The occurrence or risk of a related disease or its progression.

In another aspect, the invention relates to a pharmaceutical composition for treating an abnormally elevated (excessive) serum glutamate or a disease associated therewith or preventing the progression of the disease in an individual in need thereof, comprising the ability to increase the individual An agent for intestinal glutamate synthase activity and a pharmaceutically acceptable carrier.

In another aspect, the present invention relates to a pharmaceutical composition, wherein the agent is a probiotic, and is used to regulate the bacterial population that produces non-pathogenic glutamate synthase in the individual's small intestine.

In another aspect, the present invention relates to a pharmaceutical composition, wherein the agent is a probiotic with a probiotic substance, and is used to regulate the bacterial population that produces non-pathogenic glutamate synthase in the small intestine of the individual.

In another aspect, the present invention relates to a pharmaceutical composition for treating or preventing abnormally elevated (excessive) serum glutamate or a disease associated therewith in an individual in need, comprising glutamate Salt synthase and pharmaceutically acceptable carriers.

definition

As used herein, the following terms have the indicated meanings unless explicitly stated to the contrary.

The term "individual" means a human individual or patient or animal in need of diagnosis or treatment or intervention or prognosis of a disease or condition, such as pain or pruritus, specifically neuropathic pain or pruritus.

The term "therapeutically effective" means the amount of a therapeutic agent required by an individual in need of treatment, such as a human patient or animal, to provide a medical practitioner to understand it as meaningful or demonstrable.

As used herein, the terms "treat (verb)", "treat (verb)" or "treat (noun)" include reducing, weakening, or ameliorating conditions such as elevated serum glutamate levels or related central nervous systems Systemic conditions, or prevent or reduce the risk of infectious symptoms or manifest symptoms, improve or prevent the underlying cause of symptoms, suppress the condition, curb the development of the condition, alleviate the condition, cause the condition to recede, or prevent and / or therapeutically stop the Symptoms of illness.

As used herein, the words "about" or "approximately" refer to the degree of acceptable deviation that will be understood by one of ordinary skill in the art, which may vary to some extent, depending on the context in which it is used. Generally, "about" or "approximately" can mean a value with a range of ± 5% around the quoted value.

According to the present invention, glutamate levels, in particular blood glutamate levels, can be used as markers for quantifying / measuring an individual's ability to metabolize dietary glutamate and / or for diagnosing abnormal elevations The occurrence or risk of high serum glutamate or a disease associated with it or its progression. As used herein, biological markers (or biomarkers or markers) have characteristics that are objectively measured and evaluated as normal or abnormal biological processes / conditions, diseases, pathogenic processes, or responses to treatment or therapeutic intervention Instructions. A marker may include the presence or absence of a feature or pattern or set of features indicative of a particular biological process / condition. Markers are often used for diagnostic and / or prediction purposes. However, it can be used for treatment, monitoring, drug screening, and other purposes described herein, including assessing the effectiveness of cancer therapeutics.

As used herein, "diagnosis" generally includes determining whether an individual may be affected by a given disease, disorder, or dysfunction. The skilled person usually makes a diagnosis based on one or more diagnostic indicators, which are markers whose presence, absence, or amount is indicative of the presence or absence of a disease, disorder, or dysfunction.

"Prognosis" as used herein generally refers to the prediction of possible processes and outcomes of a clinical disorder or disease. A patient's prognosis is usually performed by assessing factors or symptoms of the disease that indicate a favorable or unfavorable process or outcome for the disease. It should be understood that the term "prognosis" does not necessarily refer to the ability to predict the course or outcome of a disease with 100% accuracy. In contrast, the skilled person will understand that the term "prognosis" refers to an increased probability that a process or result will occur; that is, when compared to an individual who does not exhibit the disorder, in patients who exhibit the given disorder Processes or results are more likely to occur.

As used herein, an "abnormally elevated" level may refer to an increased level compared to a reference or control level. For example, the abnormally elevated level may be more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or 2 times, 3 times higher than the reference or control level. Times, 4 times or more. Reference or control levels can refer to levels measured in normal individuals who are not diseased.

As used herein, materials described as "substantially free of" a substance include less than 5% (w / w), less than 4%, less than 3% (w / w), less than 2% (w / w), less than 1% (w / w) or undetectable amount of substance.

bran Amino acid glutamic acid ), Glutamate ( glutamate ), Glutamic acid ( glutamine ) And glutamate synthase ( glutamine synthetase )

Glutamic acid is a naturally occurring a-amino acid, which has the chemical formula C ₅ H ₉ O ₄ N and corresponds to the following chemical structure of L, which is the S, stereoisomer of glutamic acid. L-glutamic acid

Gluten is the main nerve conduction substance in the central nervous system of the human body and is the most abundant free amino acid in the system. The glutamate accounts for about 90% of the activity of total nerve-conducting substances in the brain.

In its solid form and slightly acidic pH, glutamic acid exists as a zwitterion, corresponding to the following chemical structure. L-glutamic acid zwitterionic form

Most organisms use glutamic acid in the biosynthesis of proteins. In humans, it is considered a non-essential amino acid because it can be synthesized by the human body. Gluten is widely found in many proteins, including many foods such as meat, fish, dairy products, eggs, and soy protein. Sodium salt, sodium glutamate, is used as a seasoning and flavor enhancer for foods.

The glutamate anion can be described by the following chemical structure Glutamate anion or through monolithic, negative zwitterions .

In humans and most mammals, glutamate is metabolized to glutamate. The glutamate synthetase catalyzes the condensation of glutamate and ammonia to form glutamate, as shown in the following reaction. Glutamate + ATP + NH ₃ → Glutamic acid amide phosphate + ADP +

Glutamate synthetase (GS) is found in small amounts in the brain, kidney, liver, skeletal muscle and heart. However, most of the enzyme activity occurs in the human small intestine through microorganisms capable of producing glutamate synthase during protein digestion. However, for various reasons, some individuals cannot adequately metabolize dietary glutamate to glutamate, resulting in elevated serum glutamate levels.

Bacterium synthase producing bacteria

The metabolism of glutamate by the dietary glutamate in the human small intestine is mainly caused by bacteria producing glutamate synthase, such as Gram-positive bacteria, including Vibrio fibrinolyticus, various lactic acid bacteria such as lactobacillus germ and Gram-negative bacteria such as E. coli, V. fragile, Pseudomonas and Klebsiella. The glutamate synthase produced by these bacteria in the intestine is an important enzyme that converts most of the glutamate from food sources to glutamate. The lack or destruction of these resident bacteria caused by imbalance of the intestinal flora causes intestinal damage and digestive abnormalities, especially elevated glutamate in the blood after a protein meal.

bran Role of Transcriptase Bacteria in Central Nervous System Diseases

Most patients with the nervous system complain of digestive and intestinal problems. With the replacement of resident glutamate synthase bacteria, our hypothesis is also consistent with our clinical observations in the present invention, that is, the ability of glutamate metabolism in food may be severely impaired, leading to dietary glutamate Conversion to glutamate is inefficient, thereby increasing serum glutamate levels. Elevated glutamate in the blood can cause damage to the blood-brain barrier, leading to the manifestation of neurological diseases. (William G. Mayhan, 1996). It is difficult and impractical to measure glutamine synthetase activity in the intestine, and monitoring glutamine synthetase activity in serum is not reliable. The current embodiment is designed as a simpler way to measure the activity of glutamate synthase in a human individual as a predictor of the onset of central nervous system (CNS), mental illness, or related conditions associated with glutamate toxicity or Tendency to biomarkers. This method also helps to design a regimen for regulating serum glutamate levels in an individual to treat or prevent this condition.

To perform the methods described herein, blood samples can be obtained from individuals in need, and markers in biological samples can be measured by methods known in the art, such as immunoassays such as ELISA (enzyme-linked immunosorbent assay). In some embodiments, two blood samples are obtained from an individual at two different time points, such as the first fasting time point and the first time after oral administration of an aqueous solution or suspension containing glutamic acid (glutamate). After meals. With the exception of water, individuals in the fasted state preferably fast for a period of at least about 12 hours. The second postprandial time point is about 15 minutes to about 90 minutes after oral administration of an aqueous solution or suspension containing glutamic acid (glutamate).

As is known in the art, glutamic acid (glutamate) can be found in a variety of protein-rich food sources. Thus, in some embodiments, the aqueous solution or suspension as used herein may be a nutritional composition comprising a dimeric protein source such as whey protein, casein or soy protein. Commercially available examples of such nutritional compositions include, for example, infiltration stones (Abbott Corporation).

In certain embodiments, the aqueous solution or suspension comprises glutamic acid (glutamate) equivalent to about 70 mg / kg to about 225 mg / kg based on the weight of the individual. In one example, the aqueous solution or suspension comprises glutamate (glutamate) equivalent to about 150 mg / kg based on the weight of the individual.

In certain embodiments, the aqueous solution or suspension contains approximately 10 grams of glutamic acid (glutamate).

In certain embodiments, the aqueous solution or suspension comprises a digestible protein. For example, the aqueous solution or suspension is a solution or suspension of whey protein. Preferably, the aqueous solution or suspension is substantially free of glutamine.

In certain embodiments, the aqueous suspension or solution comprises about 75 grams of whey protein suspended or dissolved in about 200 to about 250 ml of water or fruit juice.

In particular, when collecting two samples, the individuals of the first (fasting) blood sample and the second (postprandial) blood sample are not allowed to urinate because doing so would immediately lower serum glutamate and artificially distort (Reduced by excretion), rendering the test ineffective. The subject urinates only before the first (fasting) blood sample is collected and immediately after the second (post-prandial) blood sample is collected.

Blood samples can be obtained by different methods known in the art, such as peripheral venipuncture (venipuncture). Blood samples can be anticoagulated, centrifuged, and / or deproteinized to obtain protein-free serum samples. The glutamate levels in each of the obtained serum samples can be analyzed by methods known in the art, such as immunoassays such as ELISA.

According to the present invention, if an increase in the level of the marker is observed, specifically, the level of the marker in the sample obtained after the second meal is twice or more than that obtained in the fasting sample obtained earlier, the individual is considered to have The occurrence or risk of abnormally elevated serum glutamate or a disease associated therewith or its progression.

After it is determined that the individual has abnormally elevated serum glutamate or is at risk of developing or associated disease or its progression, the individual may be subjected to further testing (e.g., routine physical examinations, including imaging tests, such as X-ray breasts) Photography, magnetic resonance imaging (MRI) or ultrasound to match the stage / period of disease onset and / or determination of progression.

In some specific embodiments, the methods described herein may further comprise treating the individual to at least reduce abnormally elevated serum glutamate levels or reduce disease-related symptoms.

The present invention also provides a composition as a pharmaceutical composition for treatment.

In a specific embodiment, glutamate synthase or an agent capable of increasing intestinal glutamate synthase activity can be used as an active ingredient to prepare an abnormally elevated (excess) amount for the treatment of an individual in need. Serum glutamate or a disease associated with it or a drug that prevents the progression of this disease. Such an agent may be a probiotic, optionally containing a probiotic to regulate the population of bacteria that produce non-pathogenic glutamate synthase in the small intestine of an individual.

As used herein, "pharmaceutically acceptable" means that the carrier is compatible with the active ingredient in the composition and preferably stabilizes the active ingredient and is safe for the individual being treated. The carrier can be a diluent, carrier, excipient, or base of the active ingredient. Some examples of suitable excipients include lactose, dextrose, sucrose, sorbose, mannose, starch, acacia, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, Polyvinylpyrrolidone, cellulose, sterile water, syrup and methyl cellulose. The composition may additionally contain lubricants such as talc, magnesium stearate and mineral oil; wetting agents; emulsifiers and suspending agents; preservatives such as methyl and propyl hydroxybenzoate; sweeteners; and flavoring agents . The composition of the present invention can provide a rapid, sustained or delayed release effect of the active ingredient after administration to a patient.

According to the invention, the composition may be in the form of tablets, pills, powders, dragees, sachets, troches, elixirs, suspensions, lotions, solutions, syrups, soft and hard gelatin capsules, suppositories, sterile injections And packaging powder.

The compositions of the invention can be delivered by any physiologically acceptable route, such as oral, parenteral (e.g., intramuscular, intravenous, subcutaneous, and intraperitoneal), transdermal, suppository, and intranasal methods. For parenteral administration, it is preferably used in the form of a sterile aqueous solution, which may contain other substances sufficient to make the solution isotonic with blood, such as salt or glucose. The aqueous solution may be appropriately buffered as needed (preferably pH is 3 to 9). The preparation of suitable parenteral compositions under sterile conditions can be accomplished using standard pharmacological techniques well known to those skilled in the art, and does not require additional creative labor.

Also described herein is a kit for carrying out the method of the invention comprising a reagent capable of specifically detecting glutamate in a sample. Such a reagent may be, for example, an antibody to perform an immunoassay. An antibody as used herein may refer to an immunoglobulin molecule that has the ability to specifically bind a specific target antigen. As used herein, antibodies include not only complete (ie, full-length) antibody molecules, but also antigen-binding fragments, such as Fab, Fab ', F (ab') 2, and Fv, which retain antigen-binding capacity. An antibody as used herein may include a humanized antibody, a chimeric antibody, a diabody, a linear antibody, a single chain antibody, or a multispecific antibody (eg, a bispecific antibody). Antibodies as described herein are commercially available or can be prepared by methods known in the art, for example, by fusion tumor methods.

In some embodiments, the immunoassay can be in a sandwich format. Specifically, the set comprises a capture antibody paired with a detection antibody that includes a detectable label, such as an enzyme label, a fluorescent label, a metal label, and a radioactive label. In some examples, the kit is an ELISA sandwich kit, comprising a microtiter plate having a well to which a capture antibody is fixed, and a solution containing a detection antibody and a color development reagent. In particular, the kit may further contain additional reagents or buffers, medical devices for collecting biological samples from individuals, and / or containers for holding and / or storing samples.

In some examples, the kit may include a detection device configured to detect the results of the immunoassay and generate a signal proportional to the level of glutamate in each well; when the glutamate is detected in the sample When the relative ratio of postprandial and fasting levels of salt is greater than about 2, the reader is configured to read the signal and preferably further indicate a positive result; the reader may be further configured to indicate the metabolism of dietary glutamate Poor ability, abnormally elevated serum glutamate, or the occurrence or risk of disease associated with it or its progression. In some embodiments, the reader may indicate a negative result when the relative ratio of postprandial and fasting levels of glutamate in the detected sample is less than about 2, and the reader may be further configured to indicate normal The ability to metabolize dietary glutamate, normal levels of serum glutamate or lower likelihood of occurrence or risk of diseases associated with abnormally elevated serum glutamate or its progression.

The set may further include instructions for using the set to detect glutamate levels in the sample and calculate the relative ratio of postprandial and fasting levels of glutamate in the sample.

Examples

The following examples further describe and illustrate specific embodiments within the scope of the invention. The examples are given for the purpose of illustration only and should not be construed as a limitation on the present invention, as many changes can be made thereto without departing from the spirit and scope of the invention.

Examples 1 : Method for monitoring serum glutamate levels - Normal individuals

A comprehensive stool analysis report (Table 1) from an ALS patient (male, 51 years old) showed no E. coli growth, which is one of the major glutamate synthase bacteria in the small intestine and should normally be 4+ (Range from no growth, 1+, 2+, 3+, 4+, arbitrary units). In this patient, the serum fasting glutamate level was 141 µmol / L, while the normal serum fasting glutamate level should be around 30 µmol / L. 90 minutes after feeding with 1000 mL isotonic nutrients (model # 00668 Osmolite 1 Cal Ready-to Hang Institutional) from Abbott, the patient's serum glutamate level rose to 271 µmol / L, while normal postprandial serum bran The amine salt should be about 60 µmol / L (patient # 1 in Table 2). According to a study by Peters in 1969, the normal plasma free glutamate in healthy humans should be 29.90 to 30.85 µmol / L (4.4-4.5 ppm) (J H Peters, 1969). Therefore, we use this example to show that serum glutamate in patients with ALS is about 9 times higher than in normal people. Another ALS patient (male, 69 years old) showed a postprandial serum glutamate level of 340.4 µmol / L, which was more than 11 times higher than that of a healthy individual (patient # 2 in Table 2). These elevated levels can lead to cascade effects in which high concentrations of free glutamate in the blood can disrupt the blood-brain barrier, leading to a toxic state in the brain and the death of neurons. This observation is consistent with several studies that the loss of the gut microbiota can affect the integrity of the blood-brain barrier (Viorica Braniste, 2014). Our mode of operation is that intestinal disorders cause the body to fail to metabolize glutamate effectively, which leads to elevated serum glutamate levels. In addition, chronically elevated serum glutamate levels have been shown to impair the blood-brain barrier, leading to glutamate toxicity in the brain (William G. Mayhan, 1996). This toxicity is a consequence of the domino effect, which involves dysfunctional glutamate transporters and overreacted glutamate receptors, leading to astrocytic proliferation and eventual death of astrocytes from impaired calcium channels Calcification of nerve cells; eventual neuronal necrosis and myriad effects manifested in a wide range of neurological and psychiatric disorders (Maria Ankarcrona, 1995).

Examples 2 : Method for monitoring serum glutamate levels - Normal Individual Baseline Level

This method is suitable for individuals who are known to be in good health and whose serum glutamate concentration is approximately 29.90-30.6 µmol / L (4.4-4.5 ppm). See (J H Peters, 1969), which describes normal levels in healthy individuals.

Venous blood was obtained after fasting overnight from 77 healthy humans. There were 37 males and 40 females, aged 14 to 56 years, with an average age of 32 years. Fasting serum glutamate levels were between 11 and 33 µmol / L. See (T.L.PERRY, 1975), which also describes this normal range.

This method demonstrates that the ratio of postprandial to fasting glutamate levels is within the normal range of about 2: 1 (see (Lewis Stegink, 1979)). Diagnostic methods show that normal individuals usually metabolize dietary glutamate to glutamate.

Examples 3 : Method for monitoring serum glutamate levels - Individuals exhibit amyotrophic lateral sclerosis

This method is suitable for individuals diagnosed with amyotrophic lateral sclerosis and fasting at a serum glutamate concentration of about 33 µmol / L (male, 77 years old). The postprandial serum glutamate level measured 60 minutes after taking 10 grams of dietary glutamate was 184 µmol / L, which is 5.6 times higher than the fasting serum glutamate (patient # 3 in Table 2).

This method demonstrates that the ratio of postprandial to fasting glutamate levels is above the normal range of about 2. Diagnostic methods show that individuals generally do not metabolize dietary glutamate to glutamate.

Table 1 : Comprehensive Fecal Analysis: Beneficial Flora in Patients with ALS # 1 Note: The range is 1+ to 4+, where 4+ is normal and no growth is highly abnormal (arbitrary units)

TABLE 2: 3 ALS patients fasting and postprandial serum glutamate (Glu) level NA: No data

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Include by reference

The entire disclosure of each patent document, including correction certificates, patent application documents, scientific articles, government reports, websites, and other references mentioned herein, is incorporated herein by reference in its entirety for all purposes. In case of conflict of terms, this specification shall prevail.

Equivalent

The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing specific embodiments are to be considered in all respects illustrative and not restrictive of the invention described herein. In various specific embodiments of the methods and systems of the present invention, wherein the term "comprising" is used for the steps or components, it is also contemplated that the methods and systems consist essentially of or consist of the steps or components Or component composition. Furthermore, as long as the invention is still operational, the order of the steps or the order in which certain actions are performed is not important. In addition, two or more steps or actions may be performed simultaneously.

In the description, the singular form also includes the plural form unless the context clearly indicates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the manual shall prevail.

In addition, it should be recognized that in some cases, a composition may be described as consisting of components before mixing, as certain components may be further reacted or converted into additional materials while mixing.

Unless otherwise stated, all percentages and ratios used herein are by weight.

Claims (37)

A method of monitoring the relative serum glutamate level of a human individual, comprising the steps of: (i) providing a first (fasting) blood sample obtained at a first time point from the individual in a fasting state, wherein the The subject preferably fasts other than water for a period of at least about 12 hours; (ii) provides a second (postprandial) blood sample, which is administered orally to the subject in the fasted state in step (i) A second time point from about 15 minutes to about 90 minutes after the aqueous solution or suspension containing about 5 to about 15 grams of glutamic acid (glutamate) is obtained from the individual; (iii) the first blood The sample is transferred to a first container, optionally containing an anti-coagulant precooled between about 0 ° C to about 5 ° C; (iv) the second blood sample is transferred to a second container, optionally containing Anti-coagulant pre-cooled between about 0 ° C and about 5 ° C; (v) each of the first and second blood samples is centrifuged to separate serum from platelets in the blood sample to provide a first One (fasting) serum sample and a second (postprandial) serum sample, (vi) by adding a deproteinizing agent to each serum sample To deproteinize each of the first serum sample and the second serum sample; (vii) centrifuge each serum sample from step (vi) to isolate the protein from the serum in the sample to provide a first One (fasting) protein-free serum sample and a second (post-prandial) protein-free serum sample; (viii) analyzing the first and second protein-free serum samples to determine the glutamate level in each sample; and (ix) comparing the glutamate level in the second sample to the glutamate level in the first sample to determine the relative ratio of glutamate levels after meals and fasting in the sample . The method of claim 1, wherein in step (ii), the aqueous solution or suspension comprises glutamic acid (glutamate) equivalent to about 70 mg / kg to about 225 mg / kg based on the weight of the individual. The method of claim 1, wherein in step (ii), the aqueous solution or suspension contains equivalent to about 10 grams of glutamic acid (glutamate). The method of claim 1, wherein in step (ii), the aqueous solution or suspension comprises glutamic acid (glutamate) equivalent to about 150 mg / kg based on the weight of the individual. The method of claim 3, wherein in step (ii), the aqueous suspension or solution is an aqueous suspension or solution of digestible protein. The method of claim 5, wherein in step (ii), the aqueous suspension or solution of the digestible protein is substantially free of glutamine. The method of claim 5, wherein in step (ii), the aqueous suspension or solution is a solution or suspension of whey protein. The method of claim 7, wherein in step (ii), the aqueous suspension or solution of the whey protein is substantially free of glutamine. The method of claim 8, wherein in step (ii), the aqueous suspension or solution comprises about 75 grams of whey protein, suspended or dissolved in about 200 to about 250 ml of water or fruit juice. The method of claim 9, wherein in step (ii), the fruit juice is apple juice. The method of claim 5, wherein in step (ii), the second time point is about 60 minutes after orally administering the aqueous solution or suspension to the individual in a fasted state. The method of claim 1, wherein in step (i), the first (fasting) blood sample has a volume of about 1 to about 10 ml, and wherein in step (ii), the second (after meal) The blood sample has a volume of about 1 to about 10 ml. The method of claim 12, wherein in step (i), the first (fasting) blood sample has a volume of about 5 ml, and wherein in step (ii), the second (postprandial) blood sample has About 5 ml volume. The method of claim 1, wherein the anticoagulant in step (iii) and the anticoagulant in step (iv) are selected from EDTA (ethylenediaminetetraacetic acid), lithium heparin, sodium citrate, And heparin sodium. The method of claim 14, wherein the anticoagulant in step (iii) and the anticoagulant in step (iv) are EDTA (ethylenediaminetetraacetic acid). The method as claimed in claim 1, wherein in step (vii), the centrifugation is performed for each of the first blood sample and the second blood sample at about 17,000 × g at about 0 ° C to about 5 ° C for about 10 minute. The method of claim 1, wherein in step (vi), the deproteinizing agent is selected from the group consisting of perchloric acid, trichloroacetic acid, and tungstic acid. The method of claim 17, wherein in step (vi), the deproteinizing agent is perchloric acid. The method of claim 18, wherein in step (vi), the deproteinizing agent perchloric acid has a concentration of about 0.2 N to about 0.4 N and a volume of about 5 ml. The method of claim 1, wherein in step (vii), the centrifugation is performed on the first blood sample and the second blood sample at about 19,000 × g at about 0 ° C to about 5 ° C for about 10 times, respectively. minute. The method of claim 1, wherein the analysis in step (viii) is performed by an enzyme-linked immunosorbent assay (ELISA). The method of claim 1, comprising diagnosing that the individual has an abnormally elevated (excessive) serum glutamate or has an associated glutamate when the relative ratio of postprandial and fasted glutamate in the sample is greater than about 2. Disease or its risk or its progression. Use of an agent capable of increasing intestinal glutamate synthetase activity in the preparation of a drug for treating abnormally elevated (excessive) serum glutamate or a disease associated therewith or preventing the progress of the disease in an individual in need . Use of a glutamate synthetase in the preparation of a medicament for treating abnormally elevated (excessive) serum glutamate or a disease associated therewith or preventing the progress of the disease in an individual in need thereof. The use according to claim 23, wherein the agent is a probiotic, and is used for regulating the bacterial population that produces non-pathogenic glutamate synthase in the small intestine of the individual, especially for oral administration. The use as claimed in claim 25, wherein the reagent is a probiotic with probiotics to regulate the bacterial population that produces non-pathogenic glutamate synthase in the small intestine of the individual. The use according to any one of claims 23 to 26, wherein the disease is a central nervous system or a mental illness. The use according to claim 27, wherein the neurological or mental illness is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, autism, cerebellar atrophy, dementia, epilepsy, severe depression, multiple sclerosis, obsessive-compulsive Disease, Parkinson's disease, peripheral neuropathy, restless legs syndrome, schizophrenia, stiff people syndrome, and stroke. A method for diagnosing the abnormally elevated (excessive) serum glutamate levels or the occurrence or risk of a disease associated therewith or the progression thereof in a human individual, comprising the steps of: (i) providing a first (fasting) blood sample, It is obtained from the individual in a fasted state at a first time point, wherein the individual preferably fasts for a period of at least about 12 hours other than water; (ii) provides a second (postprandial) blood sample, which At a second time point from about 15 minutes to about 90 minutes after oral administration of an aqueous solution or suspension containing about 5 to about 15 grams of glutamic acid (glutamate) to the subject in the fasted state The individual obtains; (iii) transferring the first blood sample to a first container, optionally containing an anticoagulant precooled between about 0 ° C and about 5 ° C; (iv) transferring the second The blood sample is transferred to a second container, optionally containing pre-chilled anticoagulant between about 0 ° C and about 5 ° C; (v) the first and second blood samples are each centrifuged to remove from the Serum is separated from platelets in a blood sample to provide a first (fasted) serum sample and a second (postprandial) serum (Vi) deproteinizing each serum sample by adding a deproteinizing agent to each serum sample; and (vii) centrifuging each serum sample from step (vi) To separate proteins from the serum in the sample to provide a first (fasted) protein-free serum sample and a second (post-prandial) protein-free serum sample; (viii) analyze the first and second protein-free serum samples to Determining the glutamate level in each sample; and (ix) diagnosing the glutamate level in the second sample when the ratio of the glutamate level in the second sample to the glutamate level in the first sample is greater than about 2. The individual has abnormally elevated serum glutamate or has a disease or risk associated with it or its progression. A kit for carrying out the method according to any one of claims 1 to 22 and 29, comprising a reagent capable of specifically detecting glutamate in the sample, and instructions for carrying out the method. Use of a biomarker in the manufacture of a kit, wherein the biomarker is a glutamate in a blood sample from an individual, the kit is useful for quantifying the individual's ability to metabolize dietary glutamate, and is included in the A first (fasted) blood sample is obtained from the individual in a fasted state at a point in time; the individual in a fasted state is orally administered with a content of about 5 to about 15 grams of glutamic acid (glutamic acid A second (postprandial) blood sample from the individual at a second time point from about 15 minutes to about 90 minutes after the aqueous solution or suspension of salt); analyzing the sample to obtain fasting and postprandial serum glutamate levels; And compare the levels to get the relative ratio of the post-prandial and fasting serum glutamate levels. The use of claim 31, wherein the relative ratio of post-prandial and fasting serum glutamate levels greater than about 2 indicates a poor ability to metabolize dietary glutamate. The use of claim 31, wherein the relative ratio of the post-prandial and fasting serum glutamate levels is greater than about 2 indicating the abnormally elevated serum glutamate or the occurrence or risk of a disease associated therewith or the progression thereof. A pharmaceutical composition for treating abnormally elevated (excessive) serum glutamate or a disease associated therewith in an individual in need or preventing the progression of the disease, comprising an enzyme capable of increasing intestinal glutamate synthase in the individual Active agents and pharmaceutically acceptable carriers. The pharmaceutical composition of claim 34, wherein the agent is a probiotic, and is used to regulate the bacterial population that produces non-pathogenic glutamate synthase in the small intestine of the individual. The pharmaceutical composition of claim 34, wherein the agent is a probiotic with a probiotic substance, and is used to regulate the bacterial population that produces non-pathogenic glutamate synthase in the small intestine of the individual. A pharmaceutical composition for treating abnormally elevated (excessive) serum glutamate or a disease associated therewith in an individual in need or preventing the progress of the disease, comprising a glutamate synthetase and a pharmaceutically acceptable Carrier.