WO2019040388A1

WO2019040388A1 - Method for monitoring serum glutamate levels

Info

Publication number: WO2019040388A1
Application number: PCT/US2018/047131
Authority: WO
Inventors: Sam Poon Ang; Siew Hock LAU; Karen GARNAAS
Original assignee: New Biotic, Inc.
Priority date: 2017-08-22
Filing date: 2018-08-21
Publication date: 2019-02-28
Also published as: TW201920953A

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

FIELD OF THE INVENTION

The present invention relates to a method for monitoring serum glutamate levels in a mammal, particularly in a human subject. The method is based on determining the relative serum glutamate levels in the subject under controlled fasting and post prandial conditions after administration of a predetermined quantity of a glutamate containing protein composition. The method is useful for quantifying 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 neurological disorder, associated with glutamate toxicity. The method is also useful for designing regimens for modulating serum glutamate levels in a mammal subject to treat or prevents such a disorder. BACKGROUND OF THE INVENTION

For many disease conditions such as cancer, tumors, liver, kidney, blood or genetic disorders, there are distinct biomarkers and blood tests to confirm a diagnosis. However, there are no precise biomarkers or a single reliable blood test available to properly diagnose a broad class of neurological and psychiatric conditions. In the case of a disease such as Amyotrophic Lateral Sclerosis (ALS) or Parkinson's disease, numerous medical examinations and tests are often required to diagnose whether a patient has the condition. The diagnosis process can include physical examinations, blood tests, and imaging procedures, such as magnetic resonance imagining (MRI). It is important to rule out other conditions and false diagnoses. The diagnostic process can typically take 9-12 months from the time symptoms are first observed. There are no blood tests that can positively diagnose these conditions, nor is there an efficient way to monitor the efficacy of a particular treatment. For rapidly advancing fatal diseases such as ALS, where the median survival time from initial diagnosis is only three years, a definitive biomarker or blood test pinpointing the disease could potentially open possibilities for early intervention, thereby saving lives. Our search for a possible biomarker for CNS and related disorders focused on the human gut, i.e. that portion of the gastrointestinal tract running from the pyloric sphincter of the stomach to the anus, and which in humans comprises the small and large intestines. More than 100 trillion microorganisms make up what is known as the human microbiome. This microbiome comprises microorganisms, living in and on the human body, and performing vital functions: These microorganisms synthesize vitamins, aid digestion, and help develop and maintain the immune system. Within the human gut, which is the most bacteria-rich organ of the human body, the genes of the resident microorganisms outnumber those in the human genome by a factor of 100 to 1 , thus providing attractive candidates for pharmaceutical intervention. In the last few years, there has been a surge in microbiome focused research and therapies ranging for disease states such as inflammatory bowel disease (IBD), childhood-onset asthma, diabetes, obesity, cardiovascular disease, colorectal cancer, and antibiotic-associated diarrhea. Most of these disease states involve changes in the composition of or loss of the function of the microbiome, which is referred to as dysbiosis. Recent studies have shown an interaction between the intestinal microbiota, the gut, and the CNS (Fang, 2015).

The gut-brain axis is a bidirectional communication system controlled by the autonomic and enteric nervous systems (EIAidy, et al., 2015). Intestinal homeostasis and the microbiome are observed to play essential 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 schizophrenia (Katlyn Nemani, 2015). A study by Wu and associates (2015) demonstrated a leaky intestine and a shifted profile of the intestinal microbiome in an ALS transgenic SOD1 -G93A mouse model, suggesting a potential unrecognized role of the microbiome in ALS (Shaoping Wu1 , 2015). A similar study was reported for Parkinson's disease. (Timothy R. Sampson, 2016). However, the actual mechanism as to why and how the intestinal microbiome affects these neurological diseases is still unknown, because of the challenges of dealing with large number of bacteria, fungi and microbes (which can number more than 2000 species) in the human gut microbiota. These microorganisms interact with each other in a finely balanced ecosystem. A further difficulty for diagnosing and treating these conditions is a result of the nature of the blood brain barrier (BBB), which on the whole is impermeable to most substances, making it challenging to undertake studies targeting the brain using drugs, metabolic by products, biological materials, or inorganic moieties.

Our key to understand the causative factors for many neurological conditions came about after extensive research and clinical observations in both healthy subjects and neurological patients on the role of glutamate protein digestion from food intake, its metabolism by resident gut bacteria species capable of metabolizing glutamate to glutamine, and their selective absorption rate into the body through the mucosal layer of the small intestines. Our clinical observation was surprisingly corroborated by other independent studies detailing a link between an elevation of glutamate in the blood serum with elevated glutamate levels in the brain extracellular fluid (ECF), and a host of neurological disease conditions. However, none of these other studies showed a link between glutamate digestion in the gut and various CNS disorders, and failed to pin point the role of a class of bacteria which might be responsible for the breakdown in the homeostasis of glutamate metabolism due to gut dysbiosis, and the ultimate connection between glutamate digestion to neurological and psychotic disease states.

Glutamate is the main neurotransmitter of the human central nervous system and is the most abundant free amino acid in the system. Glutamate accounts for approximately 90 percent of the total neurotransmitter activity in the brain. The beneficial effects of glutamate are greatly dependent on strict homeostasis, by maintaining the concentration of glutamate in the brain extracellular fluid (ECF) (below the toxic range of 0.3-2 μΜ/L) (Akiva Leibowitz, 2012). Animal models and human clinical studies reveal the association of pathologically elevated ECF glutamate levels and several acute and chronic neurodegenerative disorders, including stroke, traumatic brain injury (TBI), intracerebral hemorrhage, brain hypoxia, amyotrophic lateral sclerosis (ALS) (ELISABETH ANDREADOU, 2008), dementia and others. These disorders are characterized by a several hundred-fold elevation of the glutamate concentration in the brain's ECF facilitated by a breakdown of the blood brain barrier, thus permitting free movement of glutamate between the blood plasma and brain extracellular fluid, along its concentration gradient (Akiva Leibowitz, 2012). Using functional magnetic resonance imaging in a rat model of stroke, Campos and colleagues showed that decreasing plasma glutamate levels with blood glutamate scavengers was associated with a significant decrease of glutamate in the brain, which was-correlated with neurological improvement. (Francisco Campos, 201 1 ).

ALS (ELISABETH ANDREADOU, 2008), Alzheimer's, (DAN E. MIULLI, 1993)

Parkinson's (Yasuo Iwasaki, 1992), and multiple sclerosis (FRED C. WESTALL, 1980) patients for instance, have increased plasma levels of glutamate compared to healthy control patients, suggesting a systemic defect of glutamate metabolism as an underlying cause of these disease (Andreas Plaitakis, 1987). Glutamate-mediated excitotoxicity is considered the foremost cause of motor neuron degeneration in ALS patients. Other neurological Diseases related to high levels of glutamate induced toxicity include; autism (Chie Shimmural , 201 1 ), schizophrenia (S.A. Ivanovaa, 2014), epilepsy (Sirpa Rainesalo, 2004), Alzheimer's (DAN E. MIULLI, 1993), and psychotic diseases (S.A. Ivanovaa, 2014).

Glutamate, which is one of the main constituents of dietary proteins, is the most abundant amino acid in the diet. It is also consumed in many processed or hydrolyzed foods and is used as an additive and flavor-enhancing ingredient in the form of monosodium glutamate (MSG). A considerable fraction of dietary glutamate is metabolized into glutamine during the adsorptive process in the small intestine. Excess glutamate that escapes mucosal metabolism is transported by the portal vein to the liver, which controls the composition of the amino acid mixture released to the peripheral circulation. (Lewis Stegink, 1979) Regulation of glutamate was reported by Nakagawa, where no toxic effects were observed when 12.75 g of free glutamate was fed daily to young boys, showing that orally administered free glutamate was efficiently removed by metabolism in healthy subjects (ITSIRO NAKAGAWA, 1960). Other studies also confirmed that plasma glutamate levels are not affected during diurnal meal intake. Similarly, when high protein meals are given to healthy human subjects, these meals failed to raise plasma glutamate (T Palmer, 1973), although there is an increase in glutamine, i.e. the major metabolite of glutamate, demonstrating that there is more efficient absorption of glutamine compared to glutamate across the gut mucosa. These finding are supported by studies from Reeds in 1996 and 1997 showing that enteral glutamate is almost completely metabolized in the first pass by the gastrointestinal tract of infant pigs, and that enteral glutamate is the preferential source for mucosal glutathione synthesis in fed piglets. (PETER J. REEDS, 1996).

The metabolism of glutamate to glutamine occurs in the human small intestines primarily through glutamine synthetase-producing bacteria. These bacteria are generally Gram-positive bacteria including most species of Lactobacillus, such as L. Plantarum, and Gram-negative bacteria such as E. coil, Bacteriodes fragilis, Pseudomonas and Klebsiella. Glutamine synthetase produced by these bacteria in the intestines, is a vital enzyme for converting dietary glutamate into glutamine in the small intestine. A deficiency or disruption of these resident bacteria due to gut dysbiosis leads to an impaired gut with digestive abnormalities. Dysbiosis is an imbalance in the gut flora caused by too few beneficial bacteria and an overgrowth of undesired bacteria, yeast, and/or parasites. Dysbiosis results in inefficient metabolism of dietary glutamate which causes elevated free glutamate blood serum levels, which are often many times greater than the serum basal level of healthy subjects.

The validity of our working model has been collaborated with studies by Leibowitz and associates (2012), namely, when attempts are made to reduce the level of blood serum glutamate, regardless of the mechanism involved, a decreased blood glutamate concentration is associated with an improved neurological outcome (Akiva Leibowitz, 2012). Blood glutamate scavenging is achieved by several mechanisms including: catalyzation of the enzymatic process involved in glutamate metabolism, the redistribution of blood serum glutamate into tissue, and an acute stress response.

It is seen that elevated serum glutamate levels and the compromised ability to metabolize dietary glutamate to glutamine present potentially serious health issues. It is therefore apparent from the foregoing; there is an urgent need to develop reliable diagnostic methods to quantify the effectiveness of glutamate metabolism from the dietary intake in a human subject, in order to predict the onset or progression of many of the aforementioned neurological disease states. Furthermore, such diagnostic methods would provide a means to design effective treatment regimens and to monitor the efficacy of treatment. SUMMARY OF THE INVENTION

In the present invention, we have developed a method to monitor and modulate serum glutamate levels as a biomarker to quantify the efficiency of glutamate metabolism to predict the onset, or severity of, or to track the progression of various neurological conditions. These conditions include, but are not limited to, ALS, Parkinson's disease, Alzheimer's disease, multiple sclerosis, schizophrenia, depression, dementia, autism, peripheral neuropathy, obsessive compulsive disorder (OCD), dementia, restless legs syndrome, and a whole host of other psychotic and related conditions associated with glutamate toxicity. The present invention is achieved through the monitoring of glutamate levels of a human patient at two or more time points, to obtain both fasting and postprandial serum glutamate levels under a controlled regimen involving fasting followed by ingestion of a predetermined high glutamate liquid meal or suspension.

We surprisingly found that in healthy subjects the difference between the fasting serum glutamate and postprandial serum glutamate levels after consuming 14.5g of dietary glutamate for an average 70kg person, are within the levels from 33±16 μιτιοΙ/L to 63±34 μιτιοΙ/L. This serum glutamate differential in healthy subjects is consistent with previous findings (Lewis Stegink, 1979), showing an approximately 2 fold increase in peak plasma glutamate levels compared to fasting levels after administration of a high protein meal containing from about 80 to about 207 mg/kg of total glutamate. In contrast, the differential observed in human subjects exhibiting a neurological condition associated with glutamate toxicity, is abnormally large, e.g., 271 to 340.4 μιτιοΙ/L (see the Examples section). We have also shown that with patients exhibiting improvement of symptoms after treatment, both the fasting serum glutamate and postprandial serum glutamate levels decline, commensurate with a lessening of the severity of or with a regression of the neurological condition.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for monitoring relative serum glutamate levels in a human patient at two or more selected time points, comprising the steps of: (a) fasting the patient, except for water, for a period of at least about 12 hours;

(b) withdrawing by venipuncture from the patient a first (fasting) blood sample;

(c) transferring the first blood sample to a first container, optionally containing an anticoagulant pre-cooled between about 0 °C to about 5 °C;

(d) orally administering to the patient an aqueous solution or suspension comprising the equivalent of about 5 to about 15 grams of glutamic acid (glutamate);

(e) about 15 minutes to about 90 minutes after the administration of the aqueous solution or suspension of step (d), withdrawing by venipuncture from the patient a second (post prandial) blood sample;

(f) transferring the second blood sample to a second container, optionally containing an anticoagulant pre-cooled between about 0 °C to about 5 °C;

(g) centrifuging each of the first and second blood samples to separate the blood serum from the blood platelets in the blood samples, to provide a first (fasting) serum sample and a second (post prandial) serum sample,

(h) deproteinization of each of the first serum sample and the second serum sample by the addition of a deproteinizing agent to each of the serum samples;

(i) centrifuging each of the serum samples from step (h) to separate the protein from the serum in the samples, to provide a first (fasting) protein free serum sample and a second (post prandial) protein free serum sample;

(j) analyzing the first and second protein free serum samples to determine the glutamate level in each sample; and

(k) comparing the glutamate level in the second sample to the glutamate level in the first sample to determine the relative proportion of the postprandial and fasting levels of glutamate in the samples.

The present invention also relates to a method for monitoring relative serum glutamate levels from a human subject, comprising the steps of:

(i) providing a first (fasting) blood sample which is obtained from the subject at a first time point in a fasting state, wherein the subject is preferably fasted, except for water, for a period of at least about 12 hours; (ii) providing a second (post prandial) blood sample which is obtained from the subject at a second time point that is about 15 minutes to about 90 minutes after oral administration of an aqueous solution or suspension comprising the equivalent of about 5 to about 15 grams of glutamic acid (glutamate) to the subject in the fasting state of step (i);

(iii) transferring the first blood sample to a first container, optionally containing an anticoagulant pre-cooled between about 0 °C to about 5 °C;

(iv) transferring the second blood sample to a second container, optionally containing an anticoagulant pre-cooled between about 0 °C to about 5 °C;

(v) centrifuging each of the first and second blood samples to separate the blood serum from the blood platelets in the blood samples, to provide a first (fasting) serum sample and a second (post prandial) serum sample,

(vi) deproteinization of each of the first serum sample and the second serum sample by the addition of a deproteinizing agent to each of the serum samples;

(vii) centrifuging each of the serum samples from step (vi) to separate the protein from the serum in the samples, to provide a first (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 proportion of the postprandial and fasting levels of glutamate in the samples.

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

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

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

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

In another aspect, the present 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 present invention relates to a method wherein in step (d) or

(ii) the aqueous suspension or solution of the whey protein is substantially free of glutamine.

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

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

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

In another aspect, the present 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 (ii) the second (post prandial) blood sample has a volume of about 1 to about 10 ml.

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

In another aspect, the present invention relates to a method wherein the anticoagulant in step (c) or (iii) and the anticoagulant in step (f) or (iv) is selected from EDTA (ethylene diamine tetraacetic acid), lithium heparin, sodium citrate, and sodium heparin.

In another aspect, the present invention relates to a method wherein the anticoagulant in step (c) or (iii) and the anticoagulant in step (f) or (iv) is EDTA (ethylene diamine tetraacetic acid).

In another aspect, the present invention relates to a method wherein in step (g) or

(v) the centrifuging is performed at about 17,000 x g for about 10 minutes at about 0 °C to about 5 °C on each of the first blood sample and the second blood sample.

In another aspect, the present invention relates to a method wherein in step (h) or

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

(vi) the deproteinizing agent is perchloric acid.

In another aspect, the present 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 present invention relates to a method wherein in step (i) or (vii) the centrifuging is performed at about 19,000 x g for about 10 minutes at about 0 °C to about 5 °C on each of the first blood sample and the second blood sample. In another aspect, the present 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 present invention relates to a method comprising the further step (I) of treating the human subject for an abnormal elevated (excess) serum glutamate or occurrence or risk for a disease associated therewith or its progression if the ratio of the glutamate level in the second sample to the glutamate level in the first sample is greater than about 2.

The present invention also relates to a method comprising diagnosing the subject with an abnormal elevated (excess) serum glutamate or having occurrence or at risk for a disease associated therewith or its progression when the relative proportion of the postprandial and fasting levels of glutamate in the samples is greater than about 2 (e.g. the postprandial level: the fasting level = 2.5: 1 , 3: 1 , 3.5:1 , 4:1 , 4.5:1 , 5:1 or higher). In another aspect, the present invention relates to a method wherein in step (I) the method of treating an abnormal (excess) serum glutamate is by increasing the intestinal glutamine synthetase activity in the patient.

The present invention also relates to use of an agent capable of increasing an intestinal glutamine synthetase activity for manufacturing a medicament for treating an abnormal elevated serum glutamate or a disease associated therewith or preventing progression of such disease in a subject in need. In some embodiments, the agent is a probiotic to adjust the population of non-pathogenic glutamine synthetase producing bacteria in the small intestines of the subject. In some embodiments, the agent is a probiotic with a prebiotic to adjust the population of non-pathogenic glutamine synthetase producing bacteria in the small intestines of the subject. In some embodiment, the agent is for oral administration.

In another aspect, the present invention relates to a method wherein the treating method in step (I) comprises administering glutamine synthetase to the patient.

The present invention also relates to use of a glutamine synthetase for manufacturing a medicament for treating an abnormal elevated serum glutamate or a disease associated therewith or preventing progression of such disease in a subject in need.

In another aspect, the present invention relates to a method in step (I) comprising orally administering a probiotic to adjust the population of non-pathogenic glutamine synthetase producing bacteria in the small intestines of the patient.

In another aspect, the present invention relates to a method in step (I) comprising orally administering a probiotic with a prebiotic to adjust the population of non-pathogenic glutamine synthetase producing bacteria in the small intestines of the patient. In another aspect, the present invention relates to a method for treating a central nervous system or psychotic disorder.

In another aspect, the present invention relates to a method for treating wherein the neurological or psychotic disorder is selected from Alzheimer's disease, Amyotrophic Lateral Sclerosis, Autism, Cerebellum Atrophy, Dementia, Epilepsy, Major Depression Disorders, Multiple Sclerosis, Obsessive Compulsive Disorder, Parkinson's disease, Peripheral Neuropathy, Restless Leg Syndrome, Schizophrenia, Stiff Man Syndrome, and Stroke. In another aspect, the present invention relates to a method of diagnosing elevated serum glutamate levels in a human patient comprising the steps of: (a) fasting the patient, except for water, for a period of at least about 12 hours;

(k) diagnosing the patient with elevated serum glutamate 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 present invention also provides a method of diagnosing an abnormal elevated (excess) serum glutamate levels or occurrence or risk for a disease associated therewith or its progression in a human subject comprising the steps of:

(i) providing a first (fasting) blood sample which is obtained from the subject at a first time point who is in a fasting state, wherein the subject is preferably fasted, except for water, for a period of at least about 12 hours; (ii) providing a second (post prandial) blood sample which is obtained from the subject at a second time point that is about 15 minutes to about 90 minutes after oral administration of an aqueous solution or suspension comprising the equivalent of about 5 to about 15 grams of glutamic acid (glutamate) to the subject in the fasting state;

(vii) centrifuging each of the serum samples from step (h) to separate the protein from the serum in the samples, to provide a first (fasting) protein free serum sample and a second (post prandial) protein free serum sample;

(ix) diagnosing the subject with an abnormal elevated serum glutamate or having or at risk for a disease associated therewith or its progression when the ratio of the glutamate level in the second sample to the glutamate level in the first sample is greater than about 2.

In another aspect, the present invention relates to a kit for performing the method of any of claims 1 -22 and 29 comprising an agent that is capable of specifically detecting glutamate in the samples, and instructions for performing the method.

In another aspect, the present invention relates to use of a biomarker for manufacturing a kit, wherein the biomarker is glutamate in a blood sample from a subject, said kit useful for quantifying the ability of the subject to metabolize dietary glutamate, comprising obtaining a first (fasting) blood sample from the subject at a first time point in a fasting state; obtaining a second (post prandial) blood sample from the subject at a second time point that is about 15 minutes to about 90 minutes after oral administration of an aqueous solution or suspension comprising the equivalent of about 5 to about 15 grams of glutamic acid (glutamate) to the subject in the fasting state; analyzing the samples to obtain fasting and postprandial serum glutamate levels; and comparing the levels to obtain the relative proportion of the postprandial and fasting serum glutamate levels.

In another aspect, the present invention relates to use of a biomarker for manufacturing a kit, wherein the relative proportion of the postprandial and fasting serum glutamate levels greater than about 2 is indicative of poor ability to metabolize dietary glutamate.

In another aspect, the present invention relates to use of a biomarker for manufacturing a kit, wherein the relative proportion of the postprandial and fasting serum glutamate levels greater than about 2 is indicative of an abnormal elevated serum glutamate or occurrence or risk for a disease associated therewith or its progression.

In another aspect, he present invention relates to a pharmaceutical composition for use in treating an abnormal elevated (excess) serum glutamate or a disease associated therewith or preventing progression of such disease in a subject in need, comprising an agent capable of increasing an intestinal glutamine synthetase activity in the subject and a pharmaceutically acceptable carrier. In another aspect, the present invention relates to a pharmaceutical composition, wherein the agent is a probiotic to adjust the population of non-pathogenic glutamine synthetase producing bacteria in the small intestines of the subject.

In another aspect, the present invention relates to a pharmaceutical composition, wherein the agent is a probiotic with a prebiotic to adjust the population of non-pathogenic glutamine synthetase producing bacteria in the small intestines of the subject. In another aspect, the present invention relates to a pharmaceutical composition for use in treating an abnormal elevated (excess) serum glutamate or a disease associated therewith or preventing progression of such disease in a subject in need, comprising a glutamine synthetase and a pharmaceutically acceptable carrier.

Definitions

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

The term "subject" means a human subject or patient or animal in need of diagnosis or treatment or intervention or prognosis for a disease or condition e.g. pain or pruritus, particularly neuropathic pain or pruritus.

The term "therapeutically effective" means an amount of the therapeutic agent needed to provide a meaningful or demonstrable benefit, as understood by medical practitioners, to a subject, such as a human patient or animal, in need of treatment.

The terms "treat," "treating" or "treatment," as used herein, include alleviating, abating or ameliorating the condition, e.g. the elevated serum glutamate level or the associated central nervous system condition, or preventing or reducing the risk of contracting the condition or exhibiting the symptoms of the condition, ameliorating or preventing the underlying causes of the symptoms, inhibiting the condition, arresting the development of the condition, relieving the condition, causing regression of the condition, or stopping the symptoms of the condition, either prophylactically and/or therapeutically.

As used herein, the term "about" or "approximately" refers to a degree of acceptable deviation that will be understood by persons of ordinary skill in the art, which may vary to some extent depending on the context in which it is used. In general, "about" or "approximately" may mean a numeric value having a range of ± 5% around the cited value.

According to the present invention, glutamate, particular a glutamate level in a blood sample can be used as a marker for quantifying/measuring the ability of the subject to metabolize dietary glutamate and/or for diagnosing an abnormal elevated serum glutamate or occurrence or risk for a disease associated therewith or its progression. As used herein, a biological marker (or called biomarker or marker) is a characteristic that is objectively measured and evaluated as an indicator of normal or abnormal biologic processes/conditions, diseases, pathogenic processes, or responses to treatment or therapeutic interventions. Markers can include presence or absence of characteristics or patterns or collections of the characteristics which are indicative of particular biological processes/conditions. A marker is normally used for diagnostic and/or prognostic purposes. However, it may be used for therapeutic, monitoring, drug screening and other purposes described herein, including evaluation the effectiveness of a cancer therapeutic.

"Diagnosis" as used herein generally includes determination as to whether a subject is likely affected by a given disease, disorder or dysfunction. The skilled artisan often makes a diagnosis on the basis of one or more diagnostic indicators, i.e., a marker, the presence, absence, or amount of which is indicative of the presence or absence of the disease, disorder or dysfunction.

"Prognosis" as used herein generally refers to a prediction of the probable course and outcome of a clinical condition or disease. A prognosis of a patient is usually made by evaluating factors or symptoms of a disease that are indicative of a favorable or unfavorable course or outcome of the disease. It is understood that the term "prognosis" does not necessarily refer to the ability to predict the course or outcome of a condition with 100% accuracy. Instead, the skilled artisan will understand that the term "prognosis" refers to an increased probability that a certain course or outcome will occur; that is, that a course or outcome is more likely to occur in a patient exhibiting a given condition, when compared to those individuals not exhibiting the condition.

As used herein, an "abnormal elevated" level can refer to a level that is increased compared with a reference or control level. For example, an abnormal elevated level can be higher than a reference or control level by more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, or 2-fold, 3-fold, 4-fold or more. A reference or control level can refer to the level measured in normal individuals that are not diseased.

As used herein, a material that is described as being "substantially free" of a substance includes less than 5% (w/w), less than 4%, less than 3% (w/w), less than 2% (w/w), less than 1 % (w/w) or a non-detectable amount of the substance. Glutamic Acid, Glutamate, Glutamine and Glutamine Synthetase

Glutamic acid is a naturally occurring alpha-amino acid having the chemical formula C5H9O4N and corresponding to the following chemical structure for the L, i.e. the S, stereoisomer of glutamic acid.

L-Glutamic Acid

Glutamate is the main neurotransmitter of the human central nervous system and is the most abundant free amino acid in the system. Glutamate accounts for approximately 90 percent of the total neurotransmitter activity in the brain.

In its solid form and at slightly acidic pH values, glutamic acid exists as the zwitterion, corresponding to the following chemical structure.

L-Glutamic Acid Zwitterionic Form

Glutamic acid is used by most living organisms in the biosynthesis of proteins. In humans, it is considered a non-essential amino acid because it can be synthesized by the human body. Glutamic acid is widely found in a variety of proteins, including many food products such as meats, fish, dairy products, eggs, and soy protein. The sodium salt, monosodium glutamate, is used as a seasoning and flavor enhancer for foods. The glutamate anion can be depicted by the following chemical structure

Glutamate Anion or by the overall, singly negative zwitte ion

In the human body and most mammals, glutamic acid is metabolized to glutamine. The enzyme glutamine synthetase catalyzes the condensation of glutamate and ammonia to form glutamine as depicted by the following reaction.

Glutamate + ATP + NH3→ Glutamine + ADP + Phosphate

Glutamine synthetase enzyme (GS) is found in small quantities in the brain, kidney, liver, skeletal muscles and the heart. But the bulk of the enzymic activity occurs in the small intestines of humans through microbiome capable of producing glutamine synthetase during digestion of proteins. However, for a variety of reasons some subjects are not able to adequately metabolize dietary glutamate to glutamine, resulting in elevated serum glutamate levels. Glutamine-synthetase-producing bacteria

The metabolism of dietary glutamate to glutamine in the human small intestines primarily through glutamine synthetase-producing bacteria such as Gram-positive bacteria including butyrivibro fibrisolvens, many species of Lactobacillus such as Lactobacillus plantarum and Gram-negative bacteria such as E. coli, Bacteriodes fragilis, Pseudomonas and Klebsiella. Glutamine synthetase produced by these bacteria in the intestines, is a vital enzyme for converting most of the glutamate from food sources into glutamine. A deficiency or disruption of these resident bacteria due to gut dysbiosis leads to an impaired gut with digestive abnormalities, notably elevated glutamate in the blood notably after a protein meal.

Role of Glutamine Synthetase Bacteria in Central Nervous System Disorders

Most neurological patients complained about digestion and gut issues. With the displacement of resident Glutamine Synthetase bacteria, it is our hypothesis which is also collaborated with our clinical observation in this invention, that the capacity to metabolize glutamate in food could be severely impaired leading to inefficiency in conversion of dietary glutamate to glutamine, thereby raising serum glutamate levels. The elevated glutamate in the blood may lead to the breaching of the blood brain barrier resulting in the manifestation of neurological conditions. (William G. Mayhan, 1996). It is difficult and impractical to measure Glutamine Synthetase activity in the intestines and unreliable to monitor it in the blood serum. The current embodiment is designed as a simpler way to measure Glutamine Synthetase activity in a human subject as a biomarker for predicting the onset of or propensity for developing a central nervous system (CNS), psychotic, or related disorder, associated with glutamate toxicity. The method is also useful for designing regimens for modulating serum glutamate levels in a subject to treat or prevent such a disorder.

To perform the methods described herein, a blood sample can be obtained from a subject in need and the marker in the biological sample can be measured via methods known in the art, such as an immunoassay, e.g. ELISA (enzyme-linked immunosorbent assay). In some embodiments, two blood samples are obtained from a subject at two different time points e.g. a first fasting time point and after oral administration of an aqueous solution or suspension comprising glutamic acid (glutamate) a second postprandial time point. A subject in a fasting state is preferably fasted, except for water, for a period of at least about 12 hours. A second postprandial time point is about 15 minutes to about 90 minutes after the oral administration of an aqueous solution or suspension comprising glutamic acid (glutamate).

As known in the art, glutamic acid (glutamate) can be present in a variety of protein- rich food source. Therefore, in some embodiments, the aqueous solution or suspension as used herein can be a nutritional composition comprising a diary protein source such as whey protein, casein protein, or soy protein. Commercially available examples of such nutritional composition include for example Osmolite (Abbott). In certain embodiments, the aqueous solution or suspension comprises the equivalent of about 70 mg/kg to about 225 mg/kg based on the weight of the subject of glutamic acid (glutamate). In one example, the aqueous solution or suspension comprises the equivalent of about 150 mg/kg based on the weight of the subject of glutamic acid (glutamate).

In certain embodiments, the aqueous solution or suspension comprises the equivalent of about 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 the whey protein suspended or dissolved in about 200 to about 250 ml of water or fruit juice. Specifically, the subject during the collection of both samples, the first (fasting) blood sample and the second (post prandial) blood sample, is not allowed to urinate, because doing so will lower the serum glutamate right away and artificially distort (lower the level through excretion), resulting in voided tests. The subject is only allowed to urinate right before the collection of the first (fasting) blood sample and right after the collection of the second (post prandial) blood sample.

Blood sample can be obtained by different ways known in the art e.g. peripheral vein puncture (venipuncture). The blood samples can be subjected to processing with an anti-coagulate, centrifugation and/or deproteinization, to obtain protein free serum samples. The serum samples as obtained can be analyzed for the glutamate level in each sample by methods known in the art such as an immunoassay, e.g. ELISA.

According to the present invention, if an increase in the level of the marker(s) is observed, particularly, the level of the marker in a second postprandial obtained sample being 2-fold or more to that in an earlier fasting obtained sample, the subject is deemed as having an abnormal elevated serum glutamate or occurrence or risk for a disease associated therewith or its progression.

After a subject has been determined as having an abnormal elevated serum glutamate or occurrence or at risk for a disease associated therewith or its progression, the subject can be subjected to a further test (such as a conventional physical examination, including imaging tests, e.g., X-ray mammograms, magnetic resonance imaging (MRI) or ultrasound to conform the disease occurrence and/or determine the stage/phase of progression. In some embodiments, the methods described herein can further comprise treating the subject to at least lower the abnormal elevated serum glutamate level or alleviate a symptom associated with the disease. The present invention also provides a composition as a pharmaceutical composition for treatment.

In particular embodiments, a glutamine synthetase or an agent capable of increasing an intestinal glutamine synthetase activity can be used as an active ingredient to manufacture a medicament for treating an abnormal elevated (excess) serum glutamate or a disease associated therewith or preventing progression of such disease in a subject in need. Such agent can be a probiotic, optional with a prebiotic to adjust the population of non-pathogenic glutamine synthetase producing bacteria in the small intestines of the subject.

As used herein, "pharmaceutically acceptable" means that the carrier is compatible with the active ingredient in the composition, and preferably can stabilize said active ingredient and is safe to the individual receiving the treatment. Said carrier may be a diluent, vehicle, excipient, or matrix to the active ingredient. Some examples of appropriate excipients include lactose, dextrose, sucrose, sorbose, mannose, starch, Arabic gum, calcium phosphate, alginates, tragacanth gum, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, sterilized water, syrup, and methylcellulose. The composition may additionally comprise lubricants, such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preservatives, such as methyl and propyl hydroxybenzoates; sweeteners; and flavoring agents. The composition of the present invention can provide the effect of rapid, continued, or delayed release of the active ingredient after administration to the patient.

According to the present invention, the form of said composition may be tablets, pills, powder, lozenges, packets, troches, elixers, suspensions, lotions, solutions, syrups, soft and hard gelatin capsules, suppositories, sterilized injection fluid, and packaged powder.

The composition of the present invention may be delivered via any physiologically acceptable route, such as oral, parenteral (such as intramuscular, intravenous, subcutaneous, and intraperitoneal), transdermal, suppository, and intranasal methods. Regarding parenteral administration, it is preferably used in the form of a sterile water solution, which may comprise other substances, such as salts or glucose sufficient to make the solution isotonic to blood. The water solution may be appropriately buffered (preferably with a pH value of 3 to 9) as needed. Preparation of an appropriate parenteral composition under sterile conditions may be accomplished with standard pharmacological techniques well known to persons skilled in the art, and no extra creative labor is required. Also described herein is a kit for performing the method of the invention, which comprises an agent that is capable of specifically detecting glutamate in the samples. Such agent can be, for example, an antibody, to perform an immunoassay. An antibody as used herein can refer to an immunoglobulin molecule having the ability to specifically bind to a particular target antigen. An antibody as used herein includes not only intact (i.e. full-length) antibody molecules but also antigen-binding fragments thereof retaining antigen binding ability e.g. Fab, Fab', F(ab')2 and Fv. An antibody as used herein can include humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, or multispecific antibodies (e.g., bispecific antibodies). Antibodies as described herein are commercially available or can be made by methods known in the art e.g. by a hybridoma method.

In some embodiments, the immunoassay can be in a sandwich format. Particularly, the kit comprises a capture antibody paired with a detection antibody that comprises a detectable label such as an enzymatic label, a fluorescent label, a metal label and a radio label. In certain examples, the kit is an ELISA sandwich kit, comprising a microtiter plate with wells to which a capture antibody has been immobilized, a solution containing a detection antibody and a color developing reagent. Particularly, the kit may further comprise additional reagents or buffers, a medical device for collecting a biological sample form a subject, and/or a container for holding and/or storing the sample. In some examples, the kit may comprise a detection device configured to detect the results of the immunoassay and produce a signal proportional to the glutamate level in each well; and a reader configured to read the signal and preferably further to indicate a positive result, when the relative proportion of the postprandial and fasting levels of glutamate in the samples as detected is greater than about 2; the reader can be further configured to indicate poor ability to metabolize dietary glutamate, an abnormal elevated serum glutamate or occurrence or risk for a disease associated therewith or its progression. In some embodiments, the reader can indicate a negative result, when the relative proportion of the postprandial and fasting levels of glutamate in the samples as detected is less than about 2; and the reader can be further configured to indicate normal ability to metabolize dietary glutamate, a normal level of serum glutamate or less likelihood of occurrence or risk for a disease associated with an abnormal elevated serum glutamate or its progression.

The kit can further comprise instructions for using the kit to detect glutamate levels in the samples and calculate to obtain the relative proportion of the postprandial and fasting levels of glutamate in the samples.

EXAMPLES

The following examples further describe and demonstrate embodiments within the scope of the present invention. The Examples are given solely for purpose of illustration and are not to be construed as limitations of the present invention, as many variations thereof are possible without departing from the spirit and scope of the invention. Example 1 : Method for Monitoring Serum Glutamate Levels - Normal Subject

An ALS patient (male, age 51 ) with his Comprehensive Stool Analysis Report (Table 1 ) showed no growth of E Coli bacteria, one of the main glutamine synthetase bacteria in the small intestine, which in normal situation should be at 4+ (range is from No Growth, 1 +, 2+, 3+, 4+, arbitrary units). The patient's serum fasting glutamate level was at 141 μιηοΙ/L, whereas a normal serum fasting glutamate level should be around 30 μιηοΙ/L. His serum glutamate level increased to 271 μιηοΙ/L 90 minutes after feeding with 1000 mL isotonic nutrition from Abbott (#00668 Osmolite 1 Cal Ready-to Hang Institutional), while a normal post prandial serum glutamate should be around 60 μιηοΙ/L (patient #1 in Table 2). Normal plasma free glutamate of healthy people per the Peters study in 1969 should be 29.90 to 30.85 μη-iol/L (4.4-4.5ppm) (J H Peters, 1969). We thus show with this example show that the ALS patient's serum glutamate to be about 9 times higher than normal. Another ALS patient (male, age 69) exhibited a post prandial serum glutamate level of 340.4 μιτιοΙ/L, more than 1 1 times higher than that for a healthy individual (patient #2 in Table 2). These elevated levels can lead to a cascading effect where the high concentration of free glutamate in the blood can breach the blood brain barrier leading to toxic conditions in the brain and the death of neurons. This observation is consistent with several studies suggesting that loss of gut microbiota affects the integrity of the blood brain barrier (Viorica Braniste, 2014). It is our working model that gut dysbiosis can lead to the inability of the body to efficiently metabolize glutamate, which in turn causes an elevation of serum glutamate levels. Furthermore, chronically elevated serum glutamate levels have been shown to compromise the blood brain barrier, leading to glutamate toxicity in the brain (William G. Mayhan, 1996) . This toxicity is the result of a domino effect involving dysfunctional glutamate transporters and over-reacting glutamate receptors leading to astrocytosis and eventual death of astrocytes, calcification of neuron cells from an impaired Ca channel; ultimately neuron necrosis and a myriad of effects manifesting as a wide spectrum of neurological and psychotic disorders (Maria Ankarcrona, 1995). Example 2: Method for Monitoring Serum Glutamate Levels - Normal Subject Baseline Levels

The present method was applied to subjects known to be in good health and having a serum glutamate concentration of about 29.90 - 30.6 μιηοΙ/L (4.4 - 4.5 ppm). See, (J H Peters, 1969), which describes normal levels for healthy individuals.

Venous blood was obtained after an overnight fast from 77 healthy persons. Of which there were 37 male and 40 female, aged from 14 to 56 years old, mean is 32 years old, fasting serum glutamate level is found to be between 1 1 to 33 μιτιοΙ/L. See, (T. L. PERRY, 1975), which also describes this normal range.

The present method demonstrates that the ratio of postprandial to fasting glutamate levels is in the normal range of about 2:1 (See (Lewis Stegink, 1979). The diagnostic method shows that normal subjects are normally metabolizing dietary glutamate to glutamine.

Example 3: Method for Monitoring Serum Glutamate Levels - Subject Exhibiting Amyotrophic Lateral Sclerosis

The present method was applied to a subject (male, age 77) diagnosed with Amyotrophic Lateral Sclerosis and having a fasting serum glutamate concentration of about 33 μιτιοΙ/L. The Post Prandial serum glutamate level measured 60 minutes after taking 10 gram of dietary glutamate is 184 μιτιοΙ/L, which is 5.6 times higher than fasting serum glutamate (patient #3 in Table 2).

The present method demonstrates that the ratio of postprandial to fasting glutamate levels is above the normal range of about 2. The diagnostic method shows that the subject is not normally metabolizing dietary glutamate to glutamine.

Table 1 : Comprehensive Stool Analysis: Beneficial Flora for ALS patient # 1

Note: Range is 1 + to 4+, where 4+ is normal and No growth being highly

abnormal (arbitary units)

Table 2: Fasting and Post Prandial Serum Glutamate (Glu) Levels for 3 ALS

Patients

NA: Data not available

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The entire disclosure of each of the patent documents, including certificates of correction, patent application documents, scientific articles, governmental reports, websites, and other references referred to herein is incorporated by reference herein in its entirety for all purposes. In case of a conflict in terminology, the present specification controls.

Equivalents

The invention can be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are to be considered in all respects illustrative rather than limiting on the invention described herein. In the various embodiments of the methods and systems of the present invention, where the term comprises is used with respect to the recited steps or components, it is also contemplated that the methods and systems consist essentially of, or consist of, the recited steps or components. Furthermore, the order of steps or order for performing certain actions is immaterial as long as the invention remains operable. Moreover, two or more steps or actions can be conducted simultaneously.

In the specification, the singular forms also include the plural forms, unless the context clearly dictates 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 the case of conflict, the present specification will control.

Furthermore, it should be recognized that in certain instances a composition can be described as composed of the components prior to mixing, because upon mixing certain components can further react or be transformed into additional materials.

All percentages and ratios used herein, unless otherwise indicated, are by weight.

Claims

WHAT IS CLAIMED IS:

1 . A method for monitoring relative serum glutamate levels from a human subject, comprising the steps of:

(i) providing a first (fasting) blood sample which is obtained from the subject at a first time point in a fasting state, wherein the subject is preferably fasted, except for water, for a period of at least about 12 hours;

(ii) providing a second (post prandial) blood sample which is obtained from the subject at a second time point that is about 15 minutes to about 90 minutes after oral administration of an aqueous solution or suspension comprising the equivalent of about 5 to about 15 grams of glutamic acid (glutamate) to the subject in the fasting state of step

(i);

2. The method according to claim 1 , wherein in step (ii) the aqueous solution or suspension comprises the equivalent of about 70 mg/kg to about 225 mg/kg based on the weight of the subject of glutamic acid (glutamate).

3. The method according to claim 1 , wherein in step (ii) the aqueous solution or suspension comprises the equivalent of about 10 grams of glutamic acid (glutamate).

4. The method according to claim 1 , wherein in step (ii) the aqueous solution or suspension comprises the equivalent of about 150 mg/kg based on the weight of the subject of glutamic acid (glutamate).

5. The method according to claim 3 wherein in step (ii) the aqueous suspension or solution is of a digestible protein.

6. The method according to claim 5 wherein in step (ii) the aqueous suspension or solution of the digestible protein is substantially free of glutamine.

7. The method according to claim 5 wherein in step (ii) the aqueous suspension or solution is a solution or suspension of whey protein.

8. The method according to claim 7 wherein in step (ii) the aqueous suspension or solution of the whey protein is substantially free of glutamine.

9. The method according to claim 8 wherein in step (ii) the aqueous suspension or solution comprises about 75 grams of the whey protein suspended or dissolved in about 200 to about 250 ml of water or fruit juice.

10. The method according to claim 9 wherein in step (ii) the fruit juice is apple juice.

1 1 . The method according to claim 5 wherein in step (ii) the second time point is about 60 minutes after oral administration of the aqueous solution or suspension to the subject in the fasting state.

12. The method according to 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 (post prandial) blood sample has a volume of about 1 to about 10 ml.

13. The method according to 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 (post prandial) blood sample has a volume of about 5 ml.

14. The method according to claim 1 wherein the anticoagulant in step (iii) and the anticoagulant in step (iv) is selected from EDTA (ethylene diamine tetraacetic acid), lithium heparin, sodium citrate, and sodium heparin.

15. The method according to claim 14 wherein the anticoagulant in step (iii) and the anticoagulant in step (iv) is EDTA (ethylene diamine tetraacetic acid).

16. The method according to claim 1 wherein in step (vii) the centrifuging is performed at about 17,000 x g for about 10 minutes at about 0 °C to about 5 °C on each of the first blood sample and the second blood sample.

17. The method according to claim 1 wherein in step (vi) the deproteinizing agent is selected from perchloric acid, trichloroacetic acid, and tungstic acid.

18. The method according to claim 17 wherein in step (vi) the deproteinizing agent is perchloric acid.

19. The method according to claim 18 wherein in step (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.

20. The method according to claim 1 wherein in step (vii) the centrifuging is performed at about 19,000 x g for about 10 minutes at about 0 °C to about 5 °C on each of the first blood sample and the second blood sample.

21 . The method according to claim 1 wherein the analysis in step (viii) is performed by an enzyme-linked immunosorbent assay (ELISA).

22. The method according to claim 1 comprising diagnosing the subject with an abnormal elevated (excess) serum glutamate or having or at risk for a disease associated therewith or its progression when the relative proportion of the postprandial and fasting levels of glutamate in the samples is greater than about 2.

23. Use of an agent capable of increasing an intestinal glutamine synthetase activity for manufacturing a medicament for treating an abnormal elevated (excess) serum glutamate or a disease associated therewith or preventing progression of such disease in a subject in need.

24. Use of a glutamine synthetase for manufacturing a medicament for treating an abnormal elevated (excess) serum glutamate or a disease associated therewith or preventing progression of such disease in a subject in need.

25. Use of claim 23, wherein the agent is a probiotic to adjust the population of nonpathogenic glutamine synthetase producing bacteria in the small intestines of the subject, which is particularly for oral administration.

26. Use of claim 25, wherein the agent is a probiotic with a prebiotic to adjust the population of non-pathogenic glutamine synthetase producing bacteria in the small intestines of the subject.

27. Use of claim any of claims 23 to 26, wherein the disease is a central nervous system or psychotic disorder.

28. Use of claim 27, wherein the neurological or psychotic disorder is selected from Alzheimer's disease, Amyotrophic Lateral Sclerosis, Autism, Cerebellum Atrophy, Dementia, Epilepsy, Major Depression Disorders, Multiple Sclerosis, Obsessive Compulsive Disorder, Parkinson's disease, Peripheral Neuropathy, Restless Leg Syndrome, Schizophrenia, Stiff Man Syndrome, and Stroke.

29. A method of diagnosing an abnormal elevated (excess) serum glutamate levels or occurrence or risk for a disease associated therewith or its progression in a human subject comprising the steps of:

(i) providing a first (fasting) blood sample which is obtained from the subject at a first time point who is in a fasting state, wherein the subject is preferably fasted, except for water, for a period of at least about 12 hours;

(ii) providing a second (post prandial) blood sample which is obtained from the subject at a second time point that is about 15 minutes to about 90 minutes after oral administration of an aqueous solution or suspension comprising the equivalent of about 5 to about 15 grams of glutamic acid (glutamate) to the subject in the fasting state;

(v) centrifuging each of the first and second blood samples to separate the blood serum from the blood platelets in the blood samples, to provide a first (fasting) serum sample and a second (post prandial) serum sample, (vi) deproteinization of each of the first serum sample and the second serum sample by the addition of a deproteinizing agent to each of the serum samples;

30. A kit for performing the method of any of claims 1 -22 and 29 comprising an agent that is capable of specifically detecting glutamate in the samples, and instructions for performing the method.

31 . Use of a biomarker for manufacturing a kit, wherein the biomarker is glutamate in a blood sample from a subject, said kit useful for quantifying the ability of the subject to metabolize dietary glutamate, comprising obtaining a first (fasting) blood sample from the subject at a first time point in a fasting state; obtaining a second (post prandial) blood sample from the subject at a second time point that is about 15 minutes to about 90 minutes after oral administration of an aqueous solution or suspension comprising the equivalent of about 5 to about 15 grams of glutamic acid (glutamate) to the subject in the fasting state; analyzing the samples to obtain fasting and postprandial serum glutamate levels; and comparing the levels to obtain the relative proportion of the postprandial and fasting serum glutamate levels.

32. Use of claim 31 , wherein the relative proportion of the postprandial and fasting serum glutamate levels greater than about 2 is indicative of poor ability to metabolize dietary glutamate.

33. Use of claim 31 , wherein the relative proportion of the postprandial and fasting serum glutamate levels greater than about 2 is indicative of an abnormal elevated serum glutamate or occurrence or risk for a disease associated therewith or its progression.

34. A pharmaceutical composition for use in treating an abnormal elevated (excess) serum glutamate or a disease associated therewith or preventing progression of such disease in a subject in need, comprising an agent capable of increasing an intestinal glutamine synthetase activity in the subject and a pharmaceutically acceptable carrier.

35. The pharmaceutical composition of claim 34, wherein the agent is a probiotic to adjust the population of non-pathogenic glutamine synthetase producing bacteria in the small intestines of the subject.

36. The pharmaceutical composition of claim 34, wherein the agent is a probiotic with a prebiotic to adjust the population of non-pathogenic glutamine synthetase producing bacteria in the small intestines of the subject.

37. A pharmaceutical composition for use in treating an abnormal elevated (excess) serum glutamate or a disease associated therewith or preventing progression of such disease in a subject in need, comprising a glutamine synthetase and a pharmaceutically acceptable carrier.