ES2545797B1 - Diagnosis of non-alcoholic liver steatosis - Google Patents

Abstract

Diagnosis of non-alcoholic liver steatosis. # The present invention relates to a method of in vitro diagnosis of non-alcoholic liver steatosis (NASH) comprising determining the concentration of alpha-ketoglutarate in a plasma or serum sample obtained from a subject, where a concentration of alpha-ketoglutarate greater than or equal to 3 {mi} M indicates that the subject suffers from NASH. Additionally, the invention relates to a method of in vitro diagnosis of non-alcoholic steatohepatitis, where a concentration of alpha-ketoglutarate greater than or equal to 20 µM in plasma or serum of a subject indicates that the subject suffers from non-alcoholic steatohepatitis. The invention also provides methods for deciding or recommending the administration of a suitable treatment for NASH or non-alcoholic steatohepatitis, as well as for establishing a subject's response to treatment for NASH or non-alcoholic steatohepatitis.

Description

DESCRIPTION Diagnosis of non-alcoholic liver steatosis.

The present invention is related to the field of medicine and the diagnosis of nonalcoholic liver steatosis.

STATE OF THE PREVIOUS TECHNIQUE

Non-alcoholic hepatic steatosis (NASH) is the accumulation of fat in the cytoplasm of hepatocytes. By definition, it occurs in subjects who do not drink alcohol and the expression in the liver of the metabolic syndrome is considered.

The metabolic syndrome comprises a series of alterations among which the

obesity. Other possible components of the metabolic syndrome are insulin resistance, hypertension, dyslipidemia or type 2 diabetes mellitus.

The prevalence of NASH is not well known and is probably undervalued. A large part of the patients remain asymptomatic or have discrete biological alterations. There are no precise serological markers and liver biopsy is required for the definitive diagnosis. The prevalence of NASH in the West is between 20 and 30% of the population, increases in morbidly obese subjects (75-92%), and is rapidly increasing in the pediatric population (approximately 13-14%).

NASH is no longer considered a benign condition, but is a factor to be taken into account in the comorbidity of morbidly obese patients. In addition, it has been shown that the progressive form of NASH, steatohepatitis, can in turn progress to fibrosis, cirrhosis and even hepatocarcinoma, so an adequate diagnosis of NASH and steatohepatitis is required before this evolution occurs.

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The diagnosis of NASH, however, is not straightforward, since most patients with NASH are asymptomatic, and in them the diagnosis begins when some other manifestation of the metabolic syndrome is detected. In the end, the diagnosis of the certainty of NASH and its progression is made by liver biopsy, which is an invasive, uncomfortable and costly procedure for the healthcare system. 35

Due to the difficulty and lack of consensus when performing a biopsy in patients suspected of suffering from NASH, the application of various non-invasive methods is being investigated. However, so far none has demonstrated a good diagnostic definition. 40

Therefore, there is a need to provide non-invasive and easily applied methods in clinical practice for the diagnosis of NASH.

EXPLANATION OF THE INVENTION 45

The inventors have found that alpha-ketoglutarate constitutes a plasma (or serum) biomarker with high clinical efficiency in the diagnosis of NASH. They have developed an in vitro diagnostic method of NASH that comprises determining the concentration of alpha-ketoglutarate in plasma or serum. The concentration of alpha-ketoglutarate increases significantly in patients compared to healthy controls (who do not suffer from NASH).

In particular, the inventors have found that the concentration of alpha-ketoglutarate in the

Plasma of subjects suffering from NASH is greater than 3 µM. Therefore, a first aspect of the invention provides an in vitro diagnostic method of NASH which comprises determining the concentration of alpha-ketoglutarate in a plasma or serum sample obtained from a subject, where a higher concentration of alpha-ketoglutarate or equal to 3 µM indicates that the subject suffers from NASH. Another aspect of the invention relates to the use of alpha-ketoglutarate 5 as a diagnostic marker of EHNA and the use of a concentration of alpha-ketoglutarate in plasma or serum greater than or equal to 3 µM as an EHNA marker.

Without wishing to be bound by theory, the inventors propose that the accumulation of lipids in hepatocytes decreases the production of energy produced by beta-10 oxidation, reduces liver function and impairs glucose metabolism. Results obtained by the inventors through a plasma metabolomic analysis of subjects with different degrees of NASH indicate that these alterations result in relative differences in the concentration of some metabolites, among which alpha-ketoglutarate stands out (results not shown). The inventors have found that the concentration of this metabolite in the blood of the subjects has a good association with hepatic steatosis and, therefore, constitutes a good biomarker for this disease. Thus, another aspect of the invention provides the use of alpha-ketoglutarate as a marker in the diagnosis of NASH.

The clinical utility of the method developed by the inventors is good. The method provides a high sensitivity and specificity (see FIG 3) and far exceeds the clinical utility of other serum diagnostic methods known in the state of the art, such as for example diagnostic methods of NASH based on the determination of alanine aminotransferase (ALT), gamma glutamyl transpeptidase (GGT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) (see tables 2, 3 and 4). In addition, another advantage of the method of the invention is that it allows to determine the progression of the disease, since it has been found that increasing concentrations of plasma alpha-ketoglutarate are indicative of NASH in more advanced stages (FIG 2).

The high predictive value of the method of the invention makes it possible to diagnose NASH with sufficient precision to dispense with verification by liver biopsy. Since the method of the invention can be carried out in a plasma or serum sample obtained from the patient and does not require a biopsy, the diagnosis by this method results in greater comfort and absence of risks for the patient, less complexity of diagnosis and saving time and costs for the patient and / or the healthcare system. 35

The inventors have also found that very high concentrations, greater than 20 µM, in the subjects' plasma may indicate that the disease of these subjects has evolved to steatohepatitis and, therefore, requires immediate and / or more aggressive treatment. 40

The invention also provides, therefore, the use of alpha-ketoglutarate as a marker for the diagnosis of steatohepatitis, as well as an in vitro diagnostic method of steatohepatitis which comprises determining the concentration of alpha-ketoglutarate in a plasma or serum sample. obtained from a subject, where a concentration of alpha-45 ketoglutarate greater than or equal to 20 µM indicates that the subject suffers from steatohepatitis. The invention also provides the use of a concentration of alpha-ketoglutarate in plasma or serum equal to or greater than 20 µM as a marker for the diagnosis of steatohepatitis.

The inventors have also found that the plasma or serum concentration of alpha-50 ketoglutarate differentiates obese subjects from non-obese patients. Thin individuals have a plasma or serum alpha-ketoglutarate concentration below 1.5

µM, while obese individuals have a concentration of alpha-ketoglutarate in plasma or serum above 1.5 µM.

The diagnostic method provided by the invention allows the medical professional to recommend the administration of an appropriate treatment to each patient. 5

Therefore, another aspect of the invention provides a method for deciding or recommending the administration of a suitable treatment for non-alcoholic liver steatosis which comprises determining in vitro the concentration of alpha-ketoglutarate in the blood or serum obtained from a subject, where if the concentration of alpha-ketoglutarate is equal to or greater than 3 10 µM the start of treatment is recommended and if the concentration of alpha-ketoglutarate is less than 3 µM a clinical follow-up is recommended.

Another aspect of the invention provides a method for deciding or recommending the administration of a suitable treatment for non-alcoholic steatohepatitis which comprises determining in vitro the concentration of alpha-ketoglutarate in the blood or serum obtained from a subject, where the concentration Alpha-ketoglutarate is equal to or greater than 20 µM. The initiation of a specific treatment for steatohepatitis is recommended. In a preferred embodiment, the medical professional recommends the start of an immediate specific treatment. twenty

The inventors have also found that carrying out serial measurements of the concentration of alpha-ketoglutarate in plasma or serum of the same subject over time allows monitoring the progression of NASH and / or the response to the treatment applied.

 25

Thus, another aspect of the invention provides a method for establishing the response of a subject to treatment for NASH or for non-alcoholic steatohepatitis which comprises determining the concentration of alpha-ketoglutarate in a plasma or serum sample obtained from the subject being undergoing said treatment and comparing it with the concentration of alpha-ketoglutarate determined in a plasma or serum sample obtained from the same subject 30 before undergoing the treatment or in an earlier phase of the treatment, where a concentration lower than that determined before undergoing the treatment or at an earlier stage of treatment is indicative of a good response.

BRIEF DESCRIPTION OF THE DRAWINGS 35

FIG 1. (A) Steatosis distribution in patients represented as a percentage of patients with a determined value of fat infiltration. The number of patients without steatosis were grouped at one point (≤5). (B) Distribution of fibrosis. (C) Distribution of the degree of lobular inflammation. (D) Distribution of the degree of portal inflammation. In all 40 graphs the Y axis represents the frequency (%).

FIG 2. (A) The mean plasma alpha-ketoglutarate concentration was significantly lower in thin controls and in obese patients without steatosis: a, thin controls; b, obese patients without steatosis; c, obese patients with steatosis. (B) The mean concentration of plasma alpha-ketoglutarate was proportional to the degree of steatosis: a, obese patients without steatosis; b, obese patients with mild steatosis; c, obese patients with moderate steatosis; d, obese patients with severe steatosis. In both graphs the Y axis indicates the plasma concentration of alpha-ketoglutarate (µmol / L).

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FIG 3. ROC curve for alpha-ketoglutarate. (A) Without steatosis vs with steatosis. (B) Without steatosis vs with mild steatosis. (C) Without steatosis vs with moderate steatosis. In all

graphics: the Y axis represents sensitivity; The X axis represents 1-sensitivity. AUC: area under the curve.

DETAILED DESCRIPTION OF THE INVENTION

 5

Hepatic steatosis (also called fatty liver) is understood as a wide range of liver disorders, where the fundamental alteration is the excessive accumulation of fat (fatty acids and triglycerides) in liver cells (hepatocytes). When liver steatosis occurs in subjects who do not drink alcohol or who drink very moderately, this disease is called non-alcoholic liver steatosis (NASH). Depending on the degree of development, this disease is usually classified as mild, moderate and severe (Tiniakos DG, et al, Annu Rev Pathol 2010, vol. 5, p. 145-71)

The accumulation of fat in hepatocytes can lead to liver inflammation, with the possibility of developing fibrosis and eventually ending in chronic liver damage (or liver cirrhosis). The term "non-alcoholic steatohepatitis" is used when NASH is accompanied by an inflammatory phenomenon.

The term "obesity" is understood with the usual meaning in the medical field as a chronic disease of preventable multifactorial origin characterized by excessive accumulation of fat or general hypertrophy of adipose tissue in the body; that is, when the natural energy reserve of humans and other mammals, stored in the form of body fat is increased to a point where it is associated with numerous complications such as certain health conditions or diseases and an increase in mortality.

 25

The term "diagnosis" is well known in the state of the art and refers to the procedure by which a disease, nosological entity, syndrome, or any health-disease condition is identified.

Alpha-ketoglutarate (also called 2-ketoglutarate or oxoglutarate, compound of formula 1) is the anion of alpha-ketoglutaric acid (CAS number 328-50-7) and constitutes a biological compound of importance because it is an intermediate of the cycle of Krebs, produced by the deamination of glutamate.

 35 OOOOO

Formula 1

The present invention relates to a method of diagnosing NASH by determining the concentration of alpha-ketoglutarate in plasma or serum extracted from a subject. According to the method provided by the invention, when the concentration of alpha-40 ketoglutarate in the subject's blood or serum is equal to or greater than 3 µM, this indicates that said subject suffers from NASH. Preferably, the subject is a human subject.

NASH is a disease that is usually associated with metabolic syndrome, which includes a series of alterations, among which obesity stands out. Another possible 45 components of the metabolic syndrome are insulin resistance, hypertension, dyslipidemia or type 2 diabetes mellitus.

In a particular embodiment of the method of the invention, the subject suffers from some clinical manifestation of the metabolic syndrome. Preferably, the subject on which the method of the invention is carried out is an obese individual and, preferably, suffers from morbid obesity. In other embodiments, the subject suffers from insulin resistance, hypertension and / or diabetes 5 type 2 mellitus.

The discrimination value for the concentration of alpha-ketoglutarate capable of differentiating between subjects who have or do not have this disease has been established at 3 µM because with this value a good predictive capacity is obtained, that is, the majority of subjects who 10 suffer from NASH have alpha-ketoglutarate concentrations equal to or greater than 3 µM in plasma / serum. In any case, the discrimination value from which the diagnosis of NASH is established may depend on the desired objective for the diagnosis.

Ideally, a discrimination value should be established whereby all subjects with NASH 15 are correctly classified as such (100% sensitivity), while all subjects who do not have NASH, “healthy” subjects, are also correctly classified (100% specificity). However, this ideal does not exist, since the usual thing is that a high sensitivity has a certain loss of specificity associated with it (some false positives are obtained), and vice versa, a very high specificity is usually associated with a loss of 20 sensitivity (some are obtained false negatives). As a general rule, a very sensitive test is chosen when you want the number of undetected patients to be minimal. Instead, a very specific test is chosen when you want to ensure that a subject really has a disease.

 25

The ROC curve (acronym for Receiver Operating Characteristic, or Receiver Operating Characteristic) is the representation of the ratio or ratio of true positives (VPR = Reason for Positive True) versus the ratio or ratio of false positives (FPR = Ratio of False Positive) also as the threshold, or value, of discrimination is varied (value from which we decide that a case is a positive). The inventors have determined the ROC curve for the association of plasma alpha-ketoglutarate with EHNA (see FIG 3). In view of the results produced by this analysis, the inventors have determined a value of discrimination for alpha-ketoglutarate that gives the method of diagnosis of NASH adequate sensitivity and specificity.

 35

In a particular embodiment, the discrimination value for plasma / serum alpha-ketoglutarate concentration in the method of the invention is 4 µM. That is, a particular embodiment provides an in vitro diagnostic method of NASH which comprises determining the concentration of alpha-ketoglutarate in a plasma or serum sample obtained from a subject, where a concentration of alpha-ketoglutarate greater than or equal to 4 µM indicates that the subject suffers from NASH. In another particular embodiment of the method of the invention, the concentration of alpha-ketoglutarate indicating that the subject suffers from NASH is greater than or equal to 8 µM.

The inventors have found that both discrimination values provide a good predictive ability; a value of 8 µM provides a high specificity (> 85%) while a value of 4 µM provides a high sensitivity (> 97%).

In another particular embodiment of the method of the invention, increasing concentrations of alpha-ketoglutarate indicate higher degree of EHNA in the subject, that is, the higher the concentration of alpha-ketoglutarate determined in the plasma or serum sample obtained from the major subject. It is the degree of EHNA that this suffers. In one embodiment, a concentration of alpha-ketoglutarate comprised from 3 µM to 9.6 µM is indicative of mild NASH, a

alpha-ketoglutarate concentration from 9.7 µM to 13.9 µM is indicative of moderate NASH, and an alpha-ketoglutarate concentration greater than 14 µM is indicative of severe NASH. In a preferred embodiment, a concentration of alpha-ketoglutarate comprised of 6.6 µM to 9.6 µM is indicative of mild EHNA, a concentration of alpha-ketoglutarate comprised of 10 µM to 13 µM is indicative of moderate EHNA, and a concentration of alpha-5 ketoglutarate greater than 14.5 µM is indicative of severe NASH.

Alpha-ketoglutarate concentrations equal to or greater than 20 µM are also indicative of steatohepatitis. Alpha-ketoglutarate may also indicate more serious lesions in the liver, for example, very high concentrations of this marker could indicate that the subject suffers from hepatocarcinoma. In contrast, a concentration of alpha-ketoglutarate in blood or serum less than 1.5 µM indicates the absence of obesity.

As mentioned above, the method of the invention far exceeds the clinical utility of other serum diagnostic methods known in the state of the art for the diagnosis of NASH. The inventors have constructed multivariable models that combine the values of some of these previously known markers and have found that their yield is always lower than that of the method of the invention based on the determination of alpha-ketoglutarate alone (results not shown). In contrast, the addition of alpha-ketoglutarate to any of these markers significantly improved the prediction (see Table 5 in the "Examples" section). Thus, other embodiments of the method of the invention contemplate determining, in addition to alpha-ketoglutarate, one or more additional markers, for example an additional marker, two, three, four or five additional markers. In a particular embodiment, the additional marker is ALT. ALT (also called alanine transaminase), is an aminotransferase enzyme with a high concentration in the liver and to a lesser extent in the 25 kidneys, heart and muscles. In a particular embodiment, the additional marker is GGT (gamma glutamyl transpeptidase). GGT is a liver enzyme known in the state of the art as a laboratory marker of liver disease. In a particular embodiment, the additional marker is AST. AST (also known as aspartate transaminase) is an aminotransferase enzyme that is found in various tissues of the body and rises in any situation where there is liver damage and lung damage. In a particular embodiment, the additional marker is LDH. LDH is a catalyst enzyme that is found in many tissues of the body, but its presence is greater in the heart, liver, kidneys, muscles, red blood cells, brain and lungs. It is known that the increase in LDH levels may be indicative of liver damage. 35

In a particular embodiment, the alpha-ketoglutarate determination of the method of the invention is carried out in a plasma sample. The determination of alpha-ketoglutarate in plasma or serum can be carried out by any method capable of determining this compound with good precision. The prior art contemplates various methods for the determination of alpha-ketoglutarate, such as, for example, colorimetric, chromatographic methods (gas chromatography, or GC, high efficiency liquid chromatography, or HPLC), or spectrometric (nuclear magnetic resonance) , or NMR, mass spectrometry, or MS), or a combination thereof. In a particular embodiment, the determination of alpha-ketoglutarate in the method of the invention is carried out by chromatographic techniques. In another particular embodiment, the determination is carried out by chromatographic techniques associated with mass spectrometry. The inventors have proven that colorimetric methods do not serve the intended purpose.

Preferably the determination of alpha-ketoglutarate is carried out by gas chromatography and mass spectrometry in combination with flight time and quadrupole (GC-Q-TOF-MS). This technique is particularly suitable for carrying out the diagnostic method of the invention, since it provides a high sensitivity in the determination.

of alpha-ketoglutarate, which in turn allows a better diagnosis. In this technique, the compound of interest, in this case alpha-ketoglutarate, is separated from other compounds in the sample by gas chromatography and then detected by mass spectrometry in flight and quadrupole time. This technique is known in the state of the art, although the conditions for a sensitive detection of alpha-ketoglutarate have been optimized by the inventors. Thus, a particular embodiment of the invention provides an in vitro diagnostic method of NASH as defined above, where the concetration of alpha-ketoglutarate is determined by the GC-Q-TOF-MS technique, said determination comprising the following steps:

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(i) add a standard to the plasma or serum sample obtained from the patient,

(ii) remove the protein,

(iii) derivatize,

(iv) incubate with N-methyl-N-methyl-N (trimethylsilyl) trifluoroacetamide under stirring and in the dark, and

(v) inject the sample into a gas chromatograph coupled in quadrupole at a time of 15 flight and to a mass spectrometer.

The alpha-ketoglutarate concentration is obtained thanks to an alpha-ketoglutarate calibration curve prepared above.

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One embodiment, the standard is deuterated succinic acid or C-13 isotope. Preferably, the standard is deuterated succinic acid. In another embodiment, the protein is removed by precipitation with methanol and centrifugation. In another embodiment, derivatization is carried out with methoxyamine hydrochloride in pyridine. In another embodiment, the gas chromatograph coupled in quadrupole at a time of flight and a mass spectrometer is equipped with a J&W Scientific HP5-MS column. In another embodiment, the conditions of the analysis in the gas chromatograph coupled in quadrupole to a flight time and a mass spectrometer are: helium at a flow rate of 1.5 mL / min, initial temperature of 70 ° C that increases up to 190 ° C at a rate of 12 ° C / min and then at 325 ° C at 20 ° C / min where it is held for 3.25 min. 30

The method provided by the invention allows the medical professional to decide on the administration of a suitable treatment for the subject for whom NASH or steatohepatitis is diagnosed. According to a particular embodiment, the medical professional may recommend the administration of metformin. According to another particular embodiment, the medical professional may recommend weight loss. According to another particular embodiment, the medical professional may recommend bariatric surgery. Bariatric surgery is the set of surgical procedures used to achieve decreased body weight as an alternative or complement to treatment with other non-surgical means. The most commonly used procedure is gastric bypass. 40

Often the most appropriate strategy combines more than one of the previous treatments. Thus, one embodiment provides a method for deciding or recommending the administration of a suitable treatment for NASH which comprises determining in vitro the concentration of alpha-ketoglutarate in plasma or serum obtained from a subject, where if the concentration of alpha-ketoglutarate is equal to or greater than 3 µM, the initiation of at least one treatment selected from the group consisting of metformin administration, weight loss and bariatric surgery is recommended. In other embodiments the value of discrimination to be taken into account to recommend the treatment is 4 µM or 8 µM.

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In addition, since this method allows to determine the progression of the disease, the doctor may recommend a treatment appropriate to the degree of NASH presented by the subject, which is very advantageous. For example, if the subject has mild NASH, the doctor may

recommend only one diet to lose weight, while if the subject has severe NASH, the doctor may recommend, in addition to a diet, additional treatments such as the administration of metmorphine or bariatric surgery. Similarly, if the diagnosis made according to the method of the invention indicates that the subject suffers from steatohepatitis (alpha-ketoglutarate concentrations greater than 20 µM), the doctor may recommend a specific treatment for steatohepatitis. In the latter case, the doctor may recommend alternative or complementary treatments to those mentioned.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. In addition, the word "understand" includes the case "consists of". For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention. The numerical signs relating to the drawings and placed in parentheses in a claim are only to attempt to increase the understanding of the claim, and should not be construed as limiting the scope of protection of the claim. In addition, the present invention covers all possible combinations of particular and preferred embodiments indicated herein.

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EXAMPLES

Materials and methods

1. PATIENTS 25

The ethics committee of the Univertitari Sant Joan de Reus Hospital approved the study protocol, and informed written consent was obtained from the participants (EPINOLS / 12-03-29 / 3proj6). Between 2006 and 2012, patients with morbid obesity (BMI ≥40) were recruited and therefore bariatric surgery was recommended after numerous failed attempts to lose 30 weight using non-surgical means. First of all, we performed a non-directed metabolomic analysis in a limited group of patients with or without steatosis (n = 15 in each group). The presence of steatohepatitis, fibrosis and other hepatocyte lesions were histologically ruled out. These samples were sent to Metabolon, Inc. (Durham, NC). No differences in quality of life were observed between the groups, and the selected patients did not consume alcohol or any medication that could alter liver function, including vitamin supplements. The results obtained led to qualify and verify plasma α-ketoglutarate as a possible biomarker to identify obese patients with NASH. For this, 230 patients were recruited. The exclusion criteria were only those that apply for surgery. Blood was obtained before the operation, and the 40 tissue samples were obtained during the perioperative period (Terra X, et al. Gastrectomy Obes Surg 2013; vol. 23, p. 1790-8). Samples from a group of healthy individuals (BMI <24) matched by sex and age provided by the Biobank (Jove J, et al, J Clin Virol 2011; vol. 51, p. 199-201) were used as control. used to assess differences in plasma α-ketoglutarate levels between thin and obese patients. The plasma aliquots were anticoagulated with EDTA and frozen at -80 ° C 2h after collection.

2. CLINICAL PARTICULARS AND BIOCHEMICAL ANALYSIS

The most relevant data were collected from the medical records or by standard methods of the routine laboratory (Simó JM, et al, Clin Chem 1998, vol. 10, p. 1233–41). The BMI was calculated as the weight in kilograms divided by the height in square meters. To evaluate possible histological alterations, sections of hematoxylin and eosin were stained with

2-μm thick liver. The degree of steatosis was assessed using image analysis software and expressed as a percentage (AnalySIS image software system, Soft Imaging System, Munster, Germany). Patients were considered not to have steatosis when the presence of fat in the liver was ≤5%. Patients with steatosis were arbitrarily classified as mild: 6-30%, moderate: 31-60%, and severe:> 61% (Tiniakos DG, et al, 5 Annu Rev Pathol 2010, vol. 5, p.145-71 ; Young J, et al, Atherosclerosis 2007).

Livers from patients with or without steatosis (n = 6 in each group) were also used to measure the concentration of α-ketoglutarate in tissue, and to determine the shape and size of mitochondria by transmission electron microscopy (n = 3 in each group).

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3. METABOLOMIC PLATFORM NOT DIRECTED

Epiciments Samples were extracted in the external reference laboratory (Evans AM, et al, Anal Chem 2009, vol. 81, p. 6656-67). The variability of the instrument and the general process was 4% and 9% respectively. fifteen

Chromatography Chromatographic conditions have been previously described (Evans AM, et al, Anal Chem 2009, vol. 81, p. 6656-67). Simply put, the liquid chromatography and mass spectrometry platform (LC-MS, LC-MS2) is based on a Waters ACQUITY UPLC and a Thermo-Finnigan LTQ mass spectrometer, which consists of an ionization source 20 by electrospray and a linear ion trap. Samples for gas chromatography / mass spectrometry (GC / MS) were derivatized and analyzed on a Thermo-Finnigan Trace DSQ fast-scanning singlequadrupole spectrometer by electron impact ionization (Evans AM, et al, Anal Chem 2009, vol. 81, p. 6656–67). The metabolites were identified using a reference library of approximately 25 2,800 chemical standards that included retention times, mass (m / z), and MS or MS2 spectrum.

4. MEASUREMENT OF THE CONCENTRATION OF α-CETOGLUTARATE IN PLASMA THROUGH GAS CHROMATOGRAPHY AND MASS SPECTROMETRY (FLIGHT TIME-30 QUADRUPOL)

Pretreatment of the sample. A solution of internal standards (25 μL, 2 mg / L) (50 μL) was added to the previously plasma aliquots thawed on ice at 4 ° C and deprotected with methanol, mixed using a vortex (2 min), and centrifuged (15,000xg, 15 min, 4 ° C). The supernatant was dried and stored at -80 ° C. Samples were derivatized using 30 μL of methoxyamine hydrochloride in pyridine (30 mg / ml) and incubated 1.5 hours at 37 ° C with stirring. Subsequently, 30 µL of N-methyl-N- (trimethylsilyl) trifluoroacetamide (Sigma-Aldrich, Steinheim, Germany) was added with stirring and incubated in the dark for 1h before analysis. A calibration curve of α-ketoglutaric acid (Fluka, St. Gallen, Switzerland) (0-82.5 M) was prepared immediately before each test. Deuterated succinic acid (Isotec Stable Isotopes, Miamisburg, OH, USA) was used as the reference standard.

Chromatographic analysis The samples (1 μL) were automatically injected into a 7890A gas chromatograph coupled to a 7200 quadrupole-flight time mass spectrometer (GC-MS-QTOF) (Agilent Technologies, Santa Clara, USA) equipped with a J&W Scientific HP5-MS column (19091S-433). Helium was used as a carrier gas at a flow rate of 1.5 ml / min with constant flow. The oven program was set at an initial temperature of 70 ° C which was increased to 190 ° C at a speed of 12 ° C / min, followed by an increase to 50 325 ° C at a speed of 20 ° C / min and a retention final at 325 ° C for 3.25 min. The ionization was carried out by electronic impact with an electronic energy of 70 eV and an emission intensity of 35μA. Primary data was processed using MassHunter

B.05.00 (gilent). The α-ketoglutaric acid in plasma was quantified using a certain ion and was identified using qualifying ions and retention time.

5. STATISTICAL ANALYSIS

 5

Significantly altered metabolites, which were corrected for multiple tests, were defined from a value of p <0.05 (17). Welch t-tests and / or Wilcoxon rank sum tests were performed for pairwise comparisons. ANOVA was used in some cases. Multivariate statistics were used to refine the raw data and for pattern recognition. 10

Random Forests is a supervised classification technique based on a set of decision trees (Goldstein BA, et al, BMC Genet 2010, 11:49. Doi: 10.1186 / 1471-2156-11-49). This method provides an unbiased estimate of how well the sample classes can be predicted in a new data set (prediction accuracy) and a selection of the variables that make the greatest contributions to the classification. Linear Discriminant Analysis (LDA) was also used as a classical method of classification and Principal Component Analysis (PCA) as an unsupervised data analysis, which measures innate variation in data sets. Ingenuity Pathway Analysis (IPA), a functional analysis, was used to explore biomolecular interaction networks and thus identify the signaling and metabolic pathways and compare the affected pathways. Fisher's test was used to calculate a p-value and determine the probability of the association between metabolites and the canonical pathway. The network score is based on the hypergeometric distribution and was calculated using the Fisher test. Logistic regression analysis and ROC (Receiver Operating Characteristic) curves describe and evaluate the binary classification (Xia J, et al, Metabolomics 2013; vol. 9, p. 280-99). The statistical software used includes the "R" program

and the SPSS 18.0 package.

Results 30

1. METABOLITES IN PLASMA AND HEPATIC ESTEATOSIS

The basic characteristics of the selected patients are shown in Table 1.

 35

Table 1. Clinical characteristics and selected biochemical variables in morbidly obese patients divided according to the degree of statosis.

  Without steatosis (n = 76) Mild steatosis (n = 86) Moderate steatosis (n = 52) Severe steatosis (n = 16) Clinical characteristics Men, n (%) 8 (10.5) 12 (13.9) 10 (19.2) 4 ( 25.0) BMI, kg / m2 47.0 (44.1-51.0) 45.9 (43.2-49.5) 48.1 (44.1-51.1) 44.5 (42.9-47.8) Biochemical variable * Total cholesterol, mmol / L 4.0 (3.4-4.9) 4.5 (3.9- 5.4) 4.9 (4.2-5.5) b 4.2 (3.5-5.6) HDL-cholesterol, mmol / L 0.9 (0.7-1.1) 0.9 (0.7-1.0) 0.9 (0.8-3.4) 0.8 (0.5-1.1) LDL-cholesterol, mmol / L 2.4 (1.9-3.0) 2.7 (2.6-3.3) 3.0 (2.3-3.4) 2.6 (1.9-3.0) Triglycerides, mmol / L 1.6 (1.2-1.9) 2.2 (1.6-2.7) to 2.0 (1.7-3.0 ) b 2.5 (1.6-3.0) AGNEs, mEq / L 1.1 (1.0-1.4) 1.2 (1.0-1.5) 1.1 (0.8-1.6) 1.0 (0.9-1.7) Glucose, mmol / L 7.1 (5.9-8.4) 7.4 ( 6.2-10.0) 7.9 (7.0-9.8) b 7.6 (6.7-13.4) Insulin, pmol / L 74.6 (41.9-117.6) 79.3 (42.5-122.3) 82.7 (56.8-137.3) 91.5 (40.8-172.2) HOMA-IR 3.3 (1.9-6.1) 3.9 (2.6-6.1) 3.9 (2.7-8.0) 3.61 (2.02-11.6) AST, µKat / L 0.5 (0.3-0.8) 0.5 (0.4-0.7) 0.9 (0.6-1.1) b, d 1.1 (0.6-2.0) c, e ALT, µKat / L 0.5 (0.4-0.6) 0.4 (0.4-0.7) 0.9 (0.5-1.2) b, d 0.6 (0.4-1.9) GGT, µKat / L 0.2 (0.2-0.3) 0.3 (0.2-0.8) 0.4 (0.3-0.6) b 0.4 (0.3-0.8) c LDH, µKat / L 2.5 (2.2-2.9) 2.7 (2.3-3.3) 3.2 (2.6-4.0) b, d 4.0 (2.8-6.7) c, e Total bilirubin, mmol / L 7.0 ( 5.0-10.0) 8.0 (5.3-11.8) 8.5 (4.5-11.0) 9.5 (5.8-20.8) Leptin, ng / mL 68.5 (55.4-97.5) 73.7 (51.9-97.5) 83.2 (59.5-114.1) 87.8 (75.2-185.4 ) Adiponectin, µg / mL 3.6 (2.4-4.3) 2.6 (1.8-4.2) 2.4 (1.8-3.9) 2.2 (1.6-3.4)

* Values are represented as median (interquartile range). ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, Body Mass Index; CRP, C-reactive protein; GGT, Gamma-glutamyl transpeptidase; LDH, lactate dehydrogenase; AGNEs, non-esterified fatty acids. Significant difference (p <0.05) in comparisons: if steatosis vs mild steatosis; b without steatosis vs moderate steatosis; c without steatosis vs 5 severe steatosis; d mild steatosis vs moderate steatosis; e mild steatosis vs severe steatosis.

In the metabolomic study, 316 metabolites were identified, of which 38 were significantly different between groups. Finally, under more demanding conditions, 19 metabolites were selected for further consideration (data not shown). The relative abundance of disturbances in amino acids and lipid metabolism showed a series of alterations (results not shown). The following alterations support the hypothesis of a defective liver function: 1) the levels of the three branched-chain amino acids (leucine, isoleucine and valine) were increased in patients with 15 NASH, and 2) chain keto acid levels Branched (3-methyl-2-ocobutytate, 3-methyl-2-oxovalerate and 4-methyl-2-ocopentanoate) were found slightly elevated in the plasma of patients with NASH. Steatosis can also sequester beta-oxidation fatty acids in liver cells using an alternative source of energy. This hypothesis was reinforced by the slight reduction in the levels of 3-hydroxybutyrate, which is a ketone body that is formed during the beta-oxidation of fatty acids, and by a significant increase in the concentration of alpha-ketoglutarate and succinylcarnitine in the serum of patients with steatosis.

A Random Forest analysis resulted in a predictive capacity of> 80% to differentiate 25 patients with or without NASH and revealed that plasma alpha-ketoglutarate as a primary differentiator in a list of metabolites sorted according to their importance in the classification scheme (results not shown ). The application of LDA and PCA yielded similar results for grouping and pattern recognition. In addition, logistic regression analysis and ROC analysis provided a short list of possible biomarkers. Among the possible 30 markers, alpha-ketoglutarate stood out as a prediction value.

2. THE ALFA-CETOGLUTARATE AS A BIOMARCATOR

The results obtained identify and qualify alpha-ketoglutarate as a biomarker for diagnosis (Biomarkers Definitions Working Group. Clin Pharmacol Ther 2001, vol. 69, p. 89-95). With a 95% confidence interval, the area under the ROC curve obtained with these data ranges between 0.90 and 0.96 with a specificity of 0.93 and a sensitivity of 0.8. However, these results were calculated with a limited number of carefully selected patients. To reduce procedural insecurity, the analysis was extended to a wide range of morbidly obese patients (Table 1) in which the degree of steatosis was widely distributed. The degree of steatosis was predominantly mild, while the other liver abnormalities were in principle benign (FIG 1). Approximately 25% of the patients were sensitive to insulin but this condition was not related to the presence of NASH. Plasma alpha-ketoglutarate was mild but significantly associated with HOMA values (ρ = 0.25, p = 0.01). It was estimated that at least 115 cases would be necessary to determine whether plasma alpha-ketoglutarate improves the performance of other commonly used biomarkers using a non-differential approach. These minimum requirements were increased to 230 cases to ensure validity in a target group in which the positive and negative results are not distributed uniformly and in which the addition of fibrosis and / or inflammation could complicate the interpretation. In order to achieve the objectives sought, the colorimetric test commonly used for the determination of alpha-ketoglutarate is not adequate, so the chromatography technique of

Gas-mass spectrometry in quadrupole flight time to determine the concentration of this metabolite with good sensitivity. A significant association was observed between circulating triglycerides and steatosis in these patients (ρ = 0.42, p <0.001; Table 1), which is similar to that observed with alpha-ketoglutarate levels (ρ = 0.49, p <0.001) . Thin controls exhibited significantly lower levels of plasma alpha-ketoglutarate 5 than obese patients [1.1 μM (0.82-1.37) versus 7.5 μM (5.5-10.8)], without any overlap. The alpha-ketoglutarate values were also significantly higher in obese patients with NASH than in those without NASH, which clearly indicates the potential of this metabolite to discriminate the different phases in the progression of these conditions (FIG 2). These differences were also observed in liver tissue; in samples without steatosis the concentration of alpha-ketoglutarate was significantly lower [22.1 μM / 100 mg dry matter (11.3-31.3)] compared to those with steatosis [57.8 μM / 100 mg dry matter (32.8-63.1)]. Among the variables determined, only plasma alpha-ketoglutarate agreed significantly with the degree of steatosis, which can be differentiated between mild, moderate or severe. Curves 15 AUC and ROC demonstrated that alpha-ketoglutarate has good clinical utility (FIG 3). In contrast, as shown in Tables 2, 3 and 4, other liver function tests common in the laboratory and plasma triglycerides did not demonstrate good clinical utility. Surprisingly, the one considered as the "gold standard" for the diagnosis of steatosis, ALT, showed the worst performance. twenty

Table 2. Values obtained from the ROC curve in the differentiation of patients without steatosis vs patients with steatosis for the AST, ALT, GGT and LDH markers.

 Variable

 Area Error P-value 95% CI Sensitivity (%) Specificity (%)

 lower

 Higher

 AST

 0.675 0.060 0.006 0.556 0.793 80.0 48.6

 ALT

 0.618 0.061 0.064 0.498 0.738 80.0 33.4

 GGT

 0.685 0.058 0.003 0.571 0.799 80.0 47.5

 LDH

 0.632 0.062 0.038 0.511 0.753 80.0 35.2

Table 3. Values obtained from the ROC curve in the differentiation of patients without steatosis vs 25 patients with mild steatosis for the AST, ALT, GGT and LDH markers.

 Variable

 Area Error P-value 95% CI Sensitivity (%) Specificity (%)

 lower

 Higher

 AST

 0.589 0.072 0.226 0.447 0.731 80.0 41.8

 ALT

 0.518 0.073 0.804 0.375 0.662 80.0 23.0

 GGT

 0.608 0.072 0.138 0.467 0.750 80.0 22.4

 LDH

 0.546 0.075 0.534 0.400 0.692 80.0 16.5

Table 4. Values obtained from the ROC curve in the differentiation of patients without steatosis vs patients with moderate steatosis for the AST, ALT, GGT and LDH markers.

 Variable

 Area Error P-value 95% CI Sensitivity (%) Specificity (%)

 lower

 Higher

 AST

 0.758 0.066 0.002 0.629 0.887 80.0 59.5

 ALT

 0.756 0.068 0.002 0.623 0.889 80.0 49.7

 GGT

 0.756 0.065 0.002 0.629 0.883 80.0 61.6

 LDH

 0.716 0.075 0.010 0.568 0.864 80.0 57.0

 30

Multivariable models were also constructed by combining the values of the available markers, but the calculated yields were lower than when alpha-ketoglutarate was used alone (results not shown). On the contrary, the addition of alpha-ketoglutarate

any of these known markers improved their performance, as illustrated in Table 5 for GGT.

Table 5. Values obtained from the ROC curve in the differentiation of patients without steatosis vs patients with steatosis for alpha-ketoglutarate, GGT markers and the combination of both.

 Variable

 Area Error P-value 95% CI Sensitivity (%) Specificity (%)

 lower

 Higher

 α-KGa

 0.743 0.050 <0.001 0.645 0.840 80.0 62.5

 GGT

 0.685 0.058 0.003 0.571 0.799 80.0 47.5

 GGT + α-KG

 0.727 0.055 <0.001 0.619 0.834 80.0 57.0

to alpha-ketoglutarate

The optimal value of discrimination with clinical utility (plasma alpha-ketoglutarate concentration value) for any marker depends on the use. Ideally, all 10 patients with NASH should be classified correctly, but this ideal is only obtained at the cost of some patients being incorrectly classified as steatosis free. For example, 8 μM or 4 μM discrimination values (representing two mathematically possible candidates) for alpha-ketoglutarate provide a similar positive predictive capacity (> 80%), but while 8 μM provides high specificity (> 85% ), 15 4 μM provides high sensitivity (> 97%).

Ultimately, the results show that plasma alpha-ketoglutarate levels can distinguish thin controls from obese patients with 100% prediction accuracy, as well as predict obese patients with or without NASH better than the 20 biomarkers known in the state. of technique Thus, these results support the clinical utility and performance of plasma alpha-ketoglutarate for the diagnosis of NASH and obesity.

LIST OF REFERENCES CITED IN THE APPLICATION. 25

Tinakos DG, Vos MB, Brunt EM. "Nonalcoholic fatty liver disease: pathology and pathogenesis." Annu Rev Pathol 2010, vol. 5 p. 145-71.

Xia J, Broadhurst DI, Wilson M, Wishart DS. "Translational biomarker Discovery in clinical 30 metabolomics: an introductory tutorial". Metabolomics 2013, vol. 9, p. 280-99.

Young J, Espinel E, Rull A, Beltrán-Debón R, Aragonès G, Rodríguez-Gallego E, et al. "Serum fatty acid synthase concentration is increased in patients with hepatitis viral infection and may assist in the prediction of liver steatosis." J Clin Virol 2011, vol. 51, p. 199-201. 35

Simó JM, Castellano I, Ferré N, Joven J, Camps J. “Evaluation of a homogeneous assay for high-density lipoprotein cholesterol: limitations in patients with cardiovascular, renal, and hepatic disorders”. Clin Chem 1998, vol. 10, p. 1233–41.

 40

Young J, Rull A, Ferré N, Escolà-Gil JC, Marsillach J, Coll B, et al. "The results in rodent models of atherosclerosis are not interchangeable: the influence of diet and strain." Atherosclerosis 2007, vol. 195, p. 85-92.

Evans AM, DeHaven CD, Barrett T, Mitchell M, Milgram E. “Integrated, nontargeted ultrahigh 45 performance liquid chromatography / electrospray ionization tandem mass spectrometry platform for the identification and relative quantification of the small-molecule complement of biological systems”. Anal Chem 2009, vol. 81, p. 6656–67.

Goldstein BA, Hubbard AE, Cutler A, Barcellos LF. "An application of Random Forests to a genome-wide association dataset: methodological considerations and new findings." BMC Genet 2010, vol. 11:49. doi: 10.1186 / 1471-2156-11-49.

 5

Terra X, Auguet T, Guiu-Jurado E, Berlanga A, Orellana-Gavaldà JM, Hernández M, et al. “Long-term Changes in Leptin, Chemerin and Ghrelin Levels Following Different Bariatric Surgery Procedures: Roux-en-Y Gastric Bypass and Sleeve” Gastrectomy. Obes Surg 2013, vol. 23, p. 1790-8.

 10

Biomarkers Definitions Working Group. "Biomarkers and surrogate endpoints: preferred definitions and conceptual framework". Clin Pharmacol Ther 2001, vol. 69, p. 89-95.

Claims (17)

1. In vitro diagnostic method of non-alcoholic hepatic steatosis (NASH) comprising determining the concentration of alpha-ketoglutarate in a plasma or serum sample 5 obtained from a subject, where a concentration of alpha-ketoglutarate greater than or equal to 3 µM indicates that the subject suffers from NASH. 2. A method according to claim 1, wherein the concentration of alpha-ketoglutarate indicating that the subject suffers from NASH is greater than or equal to 4 µM. 10 3. The method according to claim 2, wherein the concentration of alpha-ketoglutarate indicating that the subject suffers from NASH is greater than or equal to 8 µM. 4. Method according to any of claims 1-3, wherein increasing concentrations of alpha-ketoglutarate indicate a greater degree of NASH in the subject. 5. A method according to claim 4, wherein a concentration of alpha-ketoglutarate of 3 µM to 9.6 µM is indicative of mild EHNA, a concentration of alpha-ketoglutarate of 9.7 µM to 13.9 µM is indicative of Moderate NASH, and a concentration of alpha-ketoglutarate greater than 14 µM is indicative of severe NASH. 6. In vitro diagnostic method of non-alcoholic steatohepatitis which comprises determining the concentration of alpha-ketoglutarate in a plasma or serum sample obtained from a subject, where a concentration of alpha-ketoglutarate greater than or equal to 20 µM indicates that subject suffers from non-alcoholic steatohepatitis. 7. Method according to any of claims 1-6, wherein an additional diagnostic marker of non-alcoholic liver steatosis is determined.  30 Method according to claim 7, wherein at least one additional marker selected from the group comprising alanine aminotransferase (ALT), gamma glutamyl transpeptidase (GGT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) is determined. 9. Method according to any of claims 1-8, wherein the concentration of alpha-35 ketoglutarate is determined by chromatography associated with mass spectrometry. 10. Method according to claim 9, wherein the concentration of alpha-ketoglutarate is determined by gas chromatography and mass spectrometry in combination with flight time and quadrupole. 40 11. Method for deciding or recommending the administration of a suitable treatment for NASH which comprises determining in vitro the concentration of alpha-ketoglutarate in the plasma or serum obtained from a subject, where if the concentration of alpha-ketoglutarate is equal or higher at 3 µM the initiation of treatment is recommended and if the concentration of alpha-45 ketoglutarate is less than 3 µM a clinical follow-up is recommended. 12. Method according to claim 11, wherein if the concentration of alpha-ketoglutarate is equal to or greater than 3 µM, the initiation of at least one treatment selected from the group consisting of metformin administration, weight loss and surgery is recommended. bariatric fifty 13. Method for deciding or recommending the administration of a suitable treatment for non-alcoholic steatohepatitis which comprises determining in vitro the concentration of alpha- ketoglutarate in plasma or serum obtained from a subject, where if the concentration of alpha-ketoglutarate is equal to or greater than 20 µM, the initiation of a specific treatment for steatohepatitis is recommended. 14. In vitro method for establishing a subject's response to treatment for NASH or nonalcoholic steatohepatitis which comprises determining the concentration of alpha-ketoglutarate in a plasma or serum sample obtained from the subject being subjected to said treatment and compare it with the concentration of alpha-ketoglutarate determined in a plasma or serum sample obtained from the same subject before undergoing the treatment or in an earlier stage of the treatment, where a concentration lower than that determined before 10 undergoing the treatment or in a Previous phase of treatment is indicative of a good response. 15. Method according to any of claims 1-14, wherein the subject suffers from metabolic syndrome. fifteen 16. Method according to any of claims 1-14, wherein the subject suffers from morbid obesity. 17. Use of alpha-ketoglutarate as a marker in the diagnosis of alcoholic NASH or non-alcoholic steatohepatitis.