Enzymatic assessment of glucose metabolic disorders

Abstract

The present invention relates to methods and means for detecting and / or monitoring of glucose metabolic disorders with enzyme-based methods.

Description

[0001] ENZYMATIC ASSESSMENT OF GLUCOSE METABOLIC DISORDERS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of evaluation and detection of metabolic disorders, in particular to methods and means for detecting and / or monitoring of glucose metabolic disorders with enzyme-based methods.

[0004] BACKGROUND OF THE INVENTION

[0005] Assessment of risk factors for various diseases is crucial for prevention of diseases and early detection of diseases. Thereby, the determination of analytes in samples provided by humans is gaining importance in in vitro diagnostic kits or assays.

[0006] The hydroxy acid 2-hydroxybutyric acid (2-HBA, a-hydroxybutyric acid, aHB, alpha-Hydroxybutanoic acid, 2-Hydroxybutanoic acid, a -Hydroxybutanoic acid) or its respective salt a-hydroxybutyrate has been described in several clinical cohort studies (Alesi et al., 2021 ; Cobb et al., 2016; Gall et al., 2010; Lu et al., 2021 ; Wang et al., 2021) and analyzed in serum / plasma to identify reliable and potent predictors for different types of metabolic disorders such as diabetes (Type II, gestational diabetes). From hundreds of compounds, 2-HBA occurred repeatedly as one of the most suitable predictors.

[0007] 2-HBA is a chiral molecule that occurs in a L- and D-enantiomer and resembles the major metabolite lactate. So far only GC / LC-MS has been successfully used to quantify 2-HBA in human serum and urinary samples in clinical studies.

[0008] US 2019 / 0107530 A1 discloses a method for assessing the risk of developing occult pancreatic beta cell dysfunction in a patient by measuring the level of 2-HBA in a sample of the patient.

[0009] WO 2017 / 210097 A1 discloses the detection and determination of analytes such as 2-HBA in samples by mass spectrometry.

[0010] WO 2015 / 010042 A2 discloses clinical testing of biomarkers such as 2-HBA to predict the likelihood of a subject having impaired glucose tolerance or insulin resistance. Thereby, methods for determination of the biomarkers are e.g. mass spectrometry, NMR, or devices for immunological detection.

[0011] Gestational diabetes mellitus (GDM), defined as glucose intolerance that first occurs or is identified in pregnancy, is estimated to affect 14 % of pregnancies.

[0012] This condition represents a major risk factor for both maternal and infant adverse pregnancy outcomes. Treatment of GDM, as well as weight control following diagnosis, reduces the incidence of some adverse outcomes, strongly underscoring the importance of accurate and minimally burdensome universal screening, as well as early diagnosis.

[0013] Current guidelines recommend GDM screening at 24-28 weeks gestation. Traditional glycaemic markers (e.g. HbA1c, fasting plasma glucose) fail as screening tests for GDM due to their low sensitivity. This explains why glucose load tests (GLTs) are still universally used as the standard of care. A “two-step” approach, consisting of a 1-hour 50 g GLT pre-screening followed by a 3-hour 100 g oral glucose tolerance test (OGTT) for those who fail the GLT, is mostly used in the USA, whereas a “one-step” approach, consisting of a 2-hour 75 g load, is used in many countries around the world (Andrews et al. 2022).

[0014] Both approaches are expensive, time consuming, unpleasant and have poor reproducibility. The failure rate of the OGTT to make a diagnosis of GDM is nearly 10 %, with vomiting during the test being the major reason for this failure. This explains the need for a simpler, easier-to-use, cost-effective, sensitive, and specific biomarker for the screening and diagnosis of GDM (Andrews et al. 2022).

[0015] Currently a number of tests exists that can diagnose patients as diabetic or prediabetic, including pre-diabetic conditions, such as occult pancreatic beta cell dysfunction or post-prandial hyperglycemia. Such tests include glucose, insulin, pro-insulin, c-peptide, HbAlc, fructosamine, glycation gap, 1 ,5-anhydroglucitol (1 ,5-AG), OGTT, Clamp-Like Index (CLIX) scoring, HOMA IR scoring, and IRI scores based on combinations of AHB, L-GPC, and Oleic Acid weighted by insulin or BML Used alone or in combination some of these tests can detect the presence of Type 2 diabetes, prediabetes (metabolic syndrome) and early insulin resistance. Additionally, there are tests that may detect some cases of Type 1 Diabetes (TIDM, sometimes referred to as childhood-onset or early- onset) such as anti-GAD antibody and other auto-antibodies to pancreatic islet cells, as Type 1 diabetes usually involves development of auto-antibodies.

[0016] The best current predictors of fasting normoglycemic patients who are actually at risk of developing diabetes are OGTTs and CLIX scoring of OGTTs. Both of these techniques involve testing multiple analytes at multiple time-points, requiring the patient to have a blood sample drawn at baseline (fasting), drink a beverage containing a known quantity of glucose, and subsequently contacting patient blood samples and measuring the levels of various analytes (e.g. glucose, insulin, pro-insulin, c-peptide, creatinine, etc...) at fasting baseline, and then at various time point intervals after dosing with the glucose load. Most OGTTs and CLIX scoring require a patient to remain in the doctor's office for 2 hours post dose, and most clinicians only test baseline samples and the 2 hour time point, and not the labor-intensive 3-5 additional times blood draws during the 2-hour period necessary for CLIX scoring, due to labor and cost constraints. Additionally, complicated and laborious mathematical calculations need to be performed in order to optimize detection of at-risk individuals with these techniques, and kidney function (approximated by blood creatinine levels / eGFR) needs to be accounted for, causing an additional step. In standard OGTTs, 1 -hour time points are rarely obtained and tested for determination of early insulin resistance and / or beta cell dysfunction, even though it is known from the literature that impaired first-phase insulin secretion response to glucose load at the 1 hour time point is a predictor of risk of development of diabetes and resulting cardio-diabetic complications such as atherosclerosis, coronary artery disease, diabetic retinopathy, etc.

[0017] 2-HBA is a biomarker for insulin resistance and impaired glucose tolerance in prediabetic cohorts (Cobb et al. 2016). 2-HBA was also shown to be a biomarker for postload glucose during an OGTT in pregnant women (Raczkowska et al. 2021). 2-HBA was found to correlate with abnormal glucose tolerance with an OGTT for the diagnosis of GDM (68 normal glucose tolerance, 44 abnormal glucose tolerance). The AUC was 0.75, the sensitivity 70 % and specificity 72 %.

[0018] EP2943790A1 describes a method for detecting the presence of or likelihood of developing occult pancreatic beta cell dysfunction in a patient, comprising: (a) measuring a level of alpha-hydroxybutyrate (AHB) in a single fasting baseline biological sample of the patient; (b) comparing the level of AHB in the single fasting baseline biological sample to a reference AHB level; and (c) determining the presence of or likelihood of developing occult pancreatic beta cell dysfunction in said patient based on the comparison in step (b). An increased AHB level at fasting baseline indicates that a normoglycemic, normo- insulinemic and / or non-dyslipidemic patient has developed or has an increased likelihood of developing occult pancreatic beta cell dysfunction.

[0019] Daff S. et al (1994) discloses mutants of Saccharomyces cerevisiae flavocytochrome b2 having altered substrate specificity.

[0020] US2014127728A1 discloses reagent materials and associated test elements.

[0021] WO2015157407A1 discloses methods for small molecule biochemical profiling of an individual subject for diagnosis of a disease or disorder.

[0022] WO2013072275A1 discloses an analytical apparatus for detecting at least one analyte in a sample, in particular for detecting blood glucose. US2022186277A1 discloses analyte sensors including one or more NAD(P)- dependent enzymes and an internal supply of NAD(P) for the detection of an analyte.

[0023] Sarswat Prashant K. et al. (2016) discloses fabrication and response of alphahydroxybutyrate sensors for rapid assessment of cardiometabolic disease risk.

[0024] So far, 2-HBA can only be measured by expensive laboratory techniques (i.e. LC- MS, GC-MS, or NMR). However, methods such as GC / LC-MS are highly sophisticated and time consuming; hence, fast and accurate measurement of 2-HBA is highly desired for scaled diagnostic purpose.

[0025] Thus, there is a pressing need in the art for methods and means enabling 2-HBA detection and / or quantification based on specific, reliable, fast, simple, accessible, and portable methods.

[0026] SUMMARY OF THE INVENTION

[0027] It is the objective of the present invention to provide methods and means for detecting and / or monitoring glucose metabolic disorders based on specific, reliable, fast, simple, accessible, and portable methods.

[0028] The objective is solved by the subject matter of the present invention.

[0029] The inventors of the present invention surprisingly found that 2-HBA levels can be determined enzymatically in samples obtained from a human. Having a specific, reliable, simple, accessible, and portable method such as an enzymatic determination of 2-HBA allows several different applications and uses such as diagnosing a metabolic disorder in a subject, determining the risk of having or developing a metabolic disorder, monitoring a metabolic disorder, discriminating between normal glucose tolerance and impaired glucose tolerance. The herein provided methods and means are convenient for patients. The herein provided methods and means are characterized by easy standardization and high reproducibility. Another advantage of the herein described methods and means is the application for testing for gestational diabetes, as applying the herein provided methods and means could lead to a substantial reduction (up to 70 %) of burdensome oral glucose tolerance test (OGTT) procedures that have to be performed in pregnant women. Furthermore, the herein described methods and means could be an easy way to screen for gestational diabetes mellitus (GDM) also in early pregnancy e.g., before the 14thgestational week. Another advantage of the present invention is that the methods and means provided herein can be integrated in standard blood analysis. According to the invention there is provided an in vitro method of diagnosing a metabolic disorder in a subject comprising the sequential steps of: i. enzymatically determining the concentration of 2-hydroxybutyric acid (2-HBA) in a sample from said subject; and ii. comparing said concentration of 2-HBA with a predetermined cut-off value of 25 pM 2-HBA or higher, wherein the metabolic disorder is diagnosed if said concentration of 2-HBA is equal or above the predetermined cut-off value.

[0030] According to the invention there is further provided an in vitro method of determining the risk of having or developing a metabolic disorder in a subject comprising the sequential steps of: i. enzymatically determining the concentration of 2-HBA in a sample from said subject; ii. comparing said concentration of 2-HBA with a predetermined cut-off value of 25 pM 2-HBA or higher; and iii. assigning to said subject a high risk of having or developing said metabolic disorder if said concentration of 2-HBA is equal or above the predetermined cut-off value.

[0031] According to the invention there is further provided an in vitro method of monitoring a metabolic disorder in a subject comprising the sequential steps of: i. enzymatically determining the concentration of 2-HBA in a sample from said subject; and ii. comparing said concentration of 2-HBA with a previous concentration of 2-HBA from said subject.

[0032] According to a specific embodiment, the metabolic disorder is a glucose metabolism disorder, or a cardiometabolic disease, preferably the glucose metabolism disorder is selected from the group consisting of gestational diabetes, impaired glucose tolerance, insulin resistance, prediabetes, and diabetes.

[0033] According to the invention there is further provided an in vitro method of discriminating between normal glucose tolerance and impaired glucose tolerance in a subject comprising the sequential steps of: i. enzymatically determining the concentration of 2-HBA in a sample from said subject; ii. comparing said concentration of 2-HBA with a predetermined cut-off value of 25 pM 2-HBA or higher; iii. assigning to said subject impaired glucose tolerance if said concentration of 2-HBA is equal or above the predetermined cut-off value; and assigning to said subject normal glucose tolerance if said concentration of 2-HBA is below the predetermined cut-off value.

[0034] According to a specific embodiment, an enzyme having 2-HBA oxidizing activity is used for enzymatically determining the concentration of 2-HBA, preferably, the enzyme is an enzyme having 2-HBA dehydrogenase activity, more preferably FCb2 or LDH.

[0035] According to a specific embodiment, the sample is a blood sample.

[0036] According to a specific embodiment, the concentration of 2-HBA is determined colorimetrically, photometrically, fluorimetrically, through phosphorescence, through chemiluminescence, or electrochemically.

[0037] According to a specific embodiment, further comprising selectively removing lactate in said sample prior to enzymatically determining the concentration of 2-HBA.

[0038] According to the invention there is further provided a use of an enzyme having 2- HBA oxidizing activity for diagnosing a metabolic disorder in a subject, for determining the risk of having or developing a metabolic disorder, for monitoring a metabolic disorder in a subject, or for discriminating between normal glucose tolerance and impaired glucose tolerance in a subject, wherein the concentration of 2-hydroxybutyric acid (2-HBA) is determined enzymatically in a sample from said subject.

[0039] According to the invention there is further provided a kit for diagnosing a metabolic disorder, for determining the risk of having or developing a metabolic disorder, for monitoring a metabolic disorder in a subject, for discriminating between normal glucose tolerance and impaired glucose tolerance, or for discriminating between gestational diabetes and non-gestational diabetes, said kit comprising an enzyme having 2-HBA oxidizing activity.

[0040] According to a specific embodiment, the enzyme is an enzyme having 2-HBA dehydrogenase activity, preferably FCb2 or LDH.

[0041] According to a specific embodiment, further comprising an enzyme for selectively removing lactate from the sample.

[0042] According to a specific embodiment, further comprising cytochrome, preferably cytochrome C.

[0043] According to a specific embodiment, further comprising an instruction manual, wherein said instruction manual comprises a description of converting a determined 2- HBA concentration to the diagnosis of a metabolic disorder, to the risk of having or developing a metabolic disorder, to the monitoring of a metabolic disorder in a subject, to the discrimination between normal glucose tolerance and impaired glucose tolerance, or to the discrimination between gestational diabetes and non-gestational diabetes.

[0044] BRIEF DESCRIPTION OF THE FIGURES

[0045] Fig. 1 : XpressGT® test kit calibration curve with calibration samples of 0-200 pM 2-HBA. The change of absorbance at 550 nm was measured in 2-HBA standards over 180 seconds and linear least squares regression was fitted to these data yielding the colorimetric slope plotted on the y-axis. Data points represent means ± SD of 3 technical replicates with SD smaller than symbol size if not visible.

[0046] Fig. 2: Comparison between observed and expected 2-HBA concentration measured with the XpressGT® test kit and LC-MS. Commercial plasma pool samples were spiked with different 2-HBA concentrations and measured with both methods. Data points represent means ± SD of 3 technical replicates (XpressGT®; open circles) and means of 2 replicates (LC-MS; triangles) and the equation obtained by linear regression analysis of observed versus expected values and the coefficient of determination R2 is indicated above the data plot.

[0047] Fig. 3: Stability of 2-HBA levels during cool storage and after freeze-thawing in different media. Single donor serum and plasma samples spiked with 100 pM 2-HBA and containing anticoagulants as indicated were stored at 4 °C and 2-HBA concentrations measured with the XpressGT® test kit after 0 to 46 h of storage (a). The same type of samples was exposed to three freeze-thaw cycles and then measured for the remaining 2-HBA contents (b). Data represent means ± SD of 3 technical replicates.

[0048] Fig. 4: Between-assay precision (intermediate precision) of 2-HBA determination using the XpressGT® test kit. 40 independent measurements of sample triplicates of a 100 pM 2-HBA standard were carried out over a period of 10 days by 2 operators. Data show observed 2-HBA concentrations for each independent data point for each operator and the overall average value calculated from these data as a dashed line.

[0049] Fig. 5: Comparison of 2-HBA measurement from standards, spiked plasma and randomized serum samples (n=75) measured with the XpressGT® test kit and LC- MS. XpressGT®-data were plotted against LC-MS-data yielding a linear relation with slope, intercept and R2 as indicated above. Overall, an underestimation bias of 93 % (dotted line) for XpressGT® measurements was derived from these measurements. Data represent means ± SD of 2 (LC-MS) and 3 technical replicates (XpressGT®). Blue lines indicate +20 %, +10 %, -10 %, -20 % (from top to bottom) deviation from the ideal match (blue dashed line) between both methods.

[0050] Fig. 6: Comparison between 2-HBA concentration measured with the XpressGT® test kit on a clinical chemistry analyzer and LC-MS. 79 anonymized Li-Hep plasma samples from patients were analyzed. Data points represent a measurement of XpressGT on an Abbot Alinity clinical chemistry analyzer and LC-MS analysis. The equation obtained by linear regression analysis of observed versus expected values and the coefficient of determination R2 is indicated in the data plot.

[0051] Fig. 7: Top: Display for continuous measurement of the XpressGT 2-HBA [pM] (index test) by binary status of GDM Diagnosis (Composite reference standard (RS)). Bottom: Receiver-operating characteristic (ROC) curves showing the classification performance for the index test XpressGT 2-HBA [pM] related to GDM Diagnosis (Composite reference standard (RS)). AUC, area under the curve.

[0052] Fig. 8: Top: Display for continuous measurement of the XpressGT 2-HBA [pM] (index test) by binary status of fasting plasma glucose (FPG, OGTT Omin.) at the cut-off used for GDM diagnosis (< 92 mg / dL). Bottom: Receiver-operating characteristic (ROC) curves showing the classification performance for the index test XpressGT 2-HBA [pM] related to fasting plasma glucose (FPG, OGTT Omin.) at the cut-off used for GDM diagnosis (< 92 mg / dL). AUC, area under the curve.

[0053] Fig. 9: Top: Display for continuous measurement of the XpressGT 2-HBA [pM] (index test) by binary status of the 1h OGTT value (OGTT 60 min.) at the cut-off used for GDM diagnosis (< 180 mg / dL). Bottom: Receiver-operating characteristic (ROC) curves showing the classification performance for the index test XpressGT 2-HBA [pM] related to 1 h OGTT value (OGTT 60 min.) at the cut-off used for GDM diagnosis (< 180 mg / dL). AUC, area under the curve.

[0054] Fig. 10: Example of an enzymatic assay principle for the determination of 2-HBA in two steps.

[0055] Fig. 11 : Technical triplicate reactions for L-lactate removal and 2-HBA measurement in presence and absence of L-lactate interferant and LOx catalyst.

[0056] DETAILED DESCRIPTION

[0057] Some aspects described herein have been developed with the assistance of artificial intelligence (Al), yet the inventor(s) being natural person(s) have / has contributed to the prior art by the subject matter described herein that is significantly more than any such Al-assisted aspects. This invention is based on a concept and reduction to practice, for which the inventor(s) has / have made a significant contribution, thereby providing a novel and inventive solution to the problem underlying the invention.

[0058] Unless indicated or defined otherwise, all terms used herein have their usual meaning in the art, which will be clear to the skilled person. Reference is for example made to the standard handbooks, such as Sambrook et al, “Molecular Cloning: A Laboratory Manual” (4th Ed.), Vols. 1 -3, Cold Spring Harbor Laboratory Press (2012); Krebs et al., “Lewin's Genes XI”, Jones & Bartlett Learning, (2017); and Berg et al, “Stryer Biochemie” Springer Verlag, 2018.

[0059] The subject matter of the claims specifically refers to artificial products or methods employing or producing such artificial products, which may be variants of native (wildtype) products. Though there can be a certain degree of sequence identity to the native structure, it is well understood that the materials, methods, and uses of the invention, e.g., specifically referring to isolated nucleic acid sequences, amino acid sequences, expression constructs, transformed host cells and modified proteins and enzymes, are “man-made” or synthetic, and are therefore not considered as a result of “laws of nature”.

[0060] The terms “comprise”, “contain”, “have” and “include” as used herein can be used synonymously and shall be understood as an open definition, allowing further members or parts or elements. “Consisting” is considered as a closest definition without further elements of the consisting definition feature. Thus “comprising” is broader and contains the “consisting” definition.

[0061] The term “about” as used herein refers to the same value or a value differing by + / -5 % of the given value.

[0062] As used herein and in the claims, the singular form, for example “a”, “an” and “the” includes the plural, unless the context clearly dictates otherwise.

[0063] As used herein, “amino acids” refer to twenty-two naturally occurring amino acids encoded by sixty-one triplet codons. Those amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, pyrrolysine, selenocysteine, serine, threonine, tryptophan, tyrosine, and valine. These 22 amino acids can be split into those that have neutral charges, positive charges, and negative charges:

[0064] The “neutral” amino acids are shown below along with their respective three-letter and single-letter code and polarity: Alanine (Ala, A; nonpolar, neutral), Asparagine (Asn, N; polar, neutral), Cysteine (Cys, C; nonpolar, neutral), Selenocysteine (Sec, U, nonpolar, neutral), Glutamine (Gin, Q; polar, neutral), Glycine (Gly, G; nonpolar, neutral), Isoleucine (He, I; nonpolar, neutral), Leucine (Leu, L; nonpolar, neutral), Methionine (Met, M; nonpolar, neutral), Phenylalanine (Phe, F; nonpolar, neutral), Proline (Pro, P; nonpolar, neutral), Serine (Ser, S; polar, neutral), Threonine (Thr, T; polar, neutral), Tryptophan (Trp, W; nonpolar, neutral), Tyrosine (Tyr, Y; polar, neutral), Valine (Vai, V; nonpolar, neutral), and Histidine (His, H; polar, positive (10 %) neutral (90 %)).

[0065] The “positively” charged amino acids are: Arginine (Arg, R; polar, positive), Lysine (Lys, K; polar, positive), and Pyrrolysine (Pyl, O, positive).

[0066] The “negatively” charged amino acids are: Aspartic acid (Asp, D; polar, negative), and Glutamic acid (Glu, E; polar, negative).

[0067] The term “metabolic disorder” as used herein refers to a disorder that negatively alters the body's processing and distribution of macronutrients, such as proteins, fats, and carbohydrates.

[0068] According to one embodiment, the metabolic disorder is a glucose metabolism disorder, or a cardiometabolic disease. Preferably, the glucose metabolism disorder is selected from the group consisting of gestational diabetes, impaired glucose tolerance, insulin resistance, prediabetes, and diabetes.

[0069] The term “glucose metabolism disorder” as used herein refers to an abnormal peripheral tissue insulin sensitivity and pancreatic 0-cell dysfunction. Abnormal peripheral tissue insulin sensitivity and pancreatic 0-cell dysfunction may be presented even before glucose level exceeds the optimal thresholds, which is increasingly acknowledged as prediabetes.

[0070] In general, alterations of glucose metabolism are recognized as one of the most important risk factors for the development and complications of cardiometabolic diseases. The relationship between glucose metabolism disorders and cardiometabolic diseases is complex and mediated by multiple dysregulated pathways. Insulin resistance, the main driving force of disturbed glucose homeostasis, is strongly associated with obesity and metabolic syndrome. In addition to metabolic disorders, which include impaired glucose tolerance, abdominal obesity, decreased high-density lipoprotein cholesterol (HDL-C) level and elevated triglycerides (TG), metabolic syndrome is characterized by a procoagulant, proinflammatory and prooxidant state, which further increases the likelihood of developing ischemic cardiovascular (CVD) and cerebrovascular diseases.

[0071] It is known that the control of blood glucose levels is critical. Insulin is the hormone that brings blood glucose into cells. Without sufficient insulin to bring glucose into the cells, blood glucose becomes elevated, and the cells "starve" for glucose and the body must use alternative pathways to produce energy for vital organs, like generating ketone bodies and free fatty acids (FFA's) to fuel the brain and heart, respectively. The pancreatic beta cells normally secrete insulin in response to a meal or a "glucose load" during an oral glucose tolerance test (OGTT), thus bringing down the level of blood glucose by bringing it into the cells of the body. This process of glucose homeostasis can be dysregulated in a number of ways, resulting in poor control of blood glucose levels. When glucose balance is dysregulated such that blood glucose varies to higher than normal for short or long periods of time, this means that the patient has developed or is developing diabetes.

[0072] The term “diabetes” as used herein encompasses all of the multiple forms of diabetes. Decades ago, the predominant type of diabetes was known as "early onset" and it was an acute illness usually occurring in childhood or adolescence in which the patient would suddenly go from healthy to very sick, with high blood sugar due to rapid and catastrophic failure of the pancreas to produce enough insulin. The patient would require injections of insulin in order to maintain normal levels of blood sugar and survive. Today, this is called Type 1 Diabetes Mellitus (TIDM) and it is recognized that the cause is usually a viral infection and / or an autoimmunity, and that this form occurs in adults as well as children. Full-blown TIDM requires that patients be treated with exogenous insulin, because patients do not make enough insulin by themselves to survive. However, there are milder forms of TIDM that progress more slowly to insulin-dependence, or in which a patient may need insulin for a short period of time, and then go off of the insulin and maintain their normal blood glucose regulation. Type 2 Diabetes (T2DM) is physiologically different to TIDM. T2DM is characterized by abnormally high blood glucose and abnormally high insulin levels. Also, T2DM does not have an acute onset of symptoms like TIDM. In contrast, it develops gradually over time, usually years, and therefore T2DM is often also called "adult onset" diabetes. T2DM is related to diet and lifestyle factors such as eating a high-sugar, high-carbohydrate diet, lack of exercise, and development of obesity, in particular abdominal obesity. Because more children are also becoming obese, more cases of T2DM are developing in childhood. The consequences for development of T2DM is a radical increase in the risk of cardiovascular disease, termed cardio-diabetes, such as increased risk of heart attacks, strokes, high blood pressure, atherosclerosis, and coronary artery disease. According to one embodiment, diabetes is Type 1 Diabetes Mellitus (T1 DM), or Type 2 Diabetes (T2DM).

[0073] The term “insulin resistance” as used herein refers to the earliest stage of T2DM, is called "insulin resistance" and most patients exhibit signs of the "metabolic syndrome”. The development of T2DM is preceded by years of abnormal metabolism during which lifestyle and diet intervention, including weight loss, can completely prevent and reverse the development of the disease in most people. The initial clinical presentation associated with insulin resistance is hyperinsulinemia, impaired glucose tolerance, dyslipidemia, and hypertension. Dyslipidemia comprises hypertriglyceridemia and decreased high-density lipoprotein (HDL) cholesterol. It is also known that chronic inflammation can help drive the development of insulin resistance. Insulin resistance is a change in physiologic regulation such that a fixed dose of insulin causes less of an effect on glucose metabolism than occurs in normal individuals (blood glucose does not drop as much or as fast as it should in response to increases in insulin). The normal compensatory response to insulin resistance is an even higher increase in insulin secretion that results in hyperinsulinemia. If the hyperinsulinemia is sufficient to overcome the insulin resistance, glucose regulation remains normal; if not, type 2 diabetes ensues.

[0074] The term "metabolic syndrome" is associated with insulin resistance and is a cluster of metabolic abnormalities involving body fat distribution, lipid metabolism, thrombosis, blood pressure regulation, and endothelial cell function. This cluster of abnormalities is referred to as the insulin resistance syndrome or the metabolic syndrome. Eventually, blood glucose remains elevated even in the fasting state as the insulin resistant patient progresses towards T2DM. The pancreatic beta cells must work very hard to pump out this much insulin, and over time, the pancreatic islets (and the beta cells they contain) are damaged due to what can be thought of as exhaustion. The beta cells begin to secrete more immature insulin (pro-insulin) in an attempt to keep up with the demand, and therefore in the blood of people who are insulin resistant and well on their way to developing T2DM biomarkers of pancreatic beta cell dysfunction such as higher levels of insulin, pro-insulin and c-peptide are seen. The following review describes insulin resistance and all the various tests and indices used to diagnose insulin resistance "Surrogate markers of insulin resistance: A review" by Bhawna Singh and Alpana Saxena, 2010. The term “cardiometabolic disease”, or “cardiometabolic syndrome” as used herein refers to a cluster of conditions including abdominal obesity, insulin-resistant glucose metabolism, dyslipidemia, and increased blood pressure.

[0075] The term “impaired glucose tolerance” (IGT) as used herein refers to 2-h plasma glucose (PG) during 75-g OGTT in the range of 140 mg / dL (7.8 mmol / L) to 199 mg / dL (11 .0 mmol / L) (IGT). The term IGT is also described e.g., in ElSayed, N. A., et al. (2023).

[0076] According to one embodiment, impaired glucose tolerance is a form of prediabetes.

[0077] The term “normal glucose tolerance” (NGT) as used herein refers to 2-h plasma glucose (PG) during 75-g OGTT of < 140 mg / dL (7.8 mmol / L) and fasting plasma glucose (FPG) of < 100 mg / dL (5.6 mmol / L). The term NGT is also described e.g., in ElSayed, N. A., et al. (2023).

[0078] The term “prediabetes” as used herein refers to a condition of individuals whose glucose levels do not meet the criteria for diabetes yet but have abnormal carbohydrate metabolism. The term also refers to a risk factor for progression to diabetes and cardiovascular disease (CVD). People with prediabetes are defined by the presence of impaired fasting glucose (IFG) and / or IGT and / or A1C 5.7-6.4% (39-47 mmol / mol). The term prediabetes is also described e.g., in ElSayed, N. A., et al. (2023).

[0079] Doctors screen patients for prediabetes and diabetes if they have known risk factors, a family history of diabetes, high blood pressure, BMI greater than 25, or if they have abnormal cholesterol levels (defined as HDL-C below 35 mg / dL (0.9 mmol / L) or triglyceride level above 250 mg / dL (2.83 mmol / L). If the patient has pre-diabetes, doctors will usually test fasting blood glucose, HbAIC, total cholesterol, HDL cholesterol, low- density lipoprotein (LDL) cholesterol, and triglycerides at least once a year.

[0080] If full-blown T2DM develops and is left undiagnosed and untreated, patients must be treated with insulin-sensitizing drugs which may help make their cells more responsive to insulin, and the pancreas does not have to work as hard. Blood glucose balance can be maintained with insulin sensitizing drugs or maintained and / or reversed by the addition of diet and lifestyle modifications and weight loss. Unlike full-blown TIDM, T2DM may be reversible in many patients. However, if T2DM progresses far enough, the pancreatic beta cells become unable to secrete enough insulin on their own due to exhaustion and the patient may progress to the last stage of T2DM wherein they cannot make enough insulin, and therefore will become insulin-dependent and must inject exogenous insulin to survive because their pancreatic beta cells no longer function. This is the worst stage of T2DM and can be fatal because while a patient can be administered exogenous insulin, their body may still be resistant to its effects. These patients are at dramatically increased risk for cardio-diabetic morbidity and mortality.

[0081] In general, the development of type 2 diabetes (T2D) or type 2 diabetes mellitus (T2DM) development of diabetes is a continuous process which often occurs in the following stages (see e.g., Tabak, A. G. et al (2012)):

[0082] • The first stage of diabetes development is a long compensatory period when insulin resistance is present and accompanied by increased rates of insulin secretion and an increased P-cell mass.

[0083] • The second stage is the stable adaptation period when P-cells are no longer fully compensating for increased insulin resistance.

[0084] • The third stage of diabetes development is when the P-cells become unable to compensate for a given insulin resistance and consequently glucose levels start to increase rapidly.

[0085] For example, disorders of glucose metabolism on the sliding scale of T2DM are defined per the following laboratory test values:

[0086] - Insulin resistance (IR): is a risk factor for type 2 diabetes and cardiovascular disease (CVD) progression. IR is a state in which higher concentrations of insulin are required to exert normal effects; blood glucose levels may be normal but fasting insulin levels may be high because of compensatory insulin secretion by the pancreas. When coupled with b-cell dysfunction, IR is a major pathophysiological determinant of dysglycemia (impaired fasting glycemia, IFG, and impaired glucose tolerance, IGT) and T2D. The gold standard for assessing insulin resistance in humans is the hyperinsulinemic-euglycemic clamp test. Tam, C. S. et al. (2012) However, this procedure is mostly confined to clinical research settings due to cost and time constraints. Fasting insulin and derived indices (HOMA, QUICKI) have been used as proxy markers for detection of IR.

[0087] - Pre-diabetes: is a risk factor for progression to diabetes and cardiovascular disease (CVD). Prediabetes can be diagnosed by three laboratory tests: o fasting plasma glucose: 100 mg / dL (5.6 mmol / L) to 125 mg / dL (6.9 mmol / L) o oral glucose tolerance test: 2-h PG during 75-g OGTT 140 mg / dL (7.8 mmol / L) to 199 mg / dL (11.0 mmol / L) o HbA1c: 5.7-6.4% (39-47 mmol / mol).

[0088] - Impaired fasting glucose (IFG): can be diagnosed by a fasting plasma glucose test: 100 mg / dL (5.6 mmol / L) to 125 mg / dL (6.9 mmol / L)

[0089] - Impaired glucose tolerance (IGT): can be diagnosed by an oral glucose tolerance test: 2-h PG during 75-g OGTT 140 mg / dL (7.8 mmol / L) to 199 mg / dL (11.0 mmol / L)

[0090] - Diabetes mellitus (DM): any of the following four criteria may be used (results must be confirmed by retesting on a subsequent occasion): o fasting glucose >126 mg / dL o glycosylated hemoglobin (HbAlc) level >6.5% o 2-hour glucose level >200 mg / dL during glucose tolerance testing o random glucose values >200 mg / dL in the presence of symptoms of hyperglycemia.

[0091] The terms “gestational diabetes”, “gestational diabetes mellitus” or “GDM” as used herein refer to a diabetic condition characterized by elevated blood glucose levels, carbohydrate intolerance and / or reduced insulin sensitivity that is brought on by pregnancy. In some cases, GDM diagnosis in each country may rely on different standards set by the professional bodies from such countries that issue recommendations to physicians practicing there. GDM may affect up to 18% of pregnancies with adverse outcomes that affect both the mother and offspring, including both short term and long term effects. Currently, diagnosis and monitoring of GDM in female subjects relies heavily on the measurement of blood glucose levels. Blood glucose is in constant flux and influenced by a number of external factors including meals and level of activity. Glucose levels may change on an hourly basis. This complicates GDM testing by imposing diet requirements and / or restrictions on subjects undergoing testing.

[0092] In general, GDM is one of the most prevalent disorders affecting pregnant women and carries with it a greater risk for complications during pregnancy, at the time of birth and even after birth. Additionally, such complications may affect both mother and offspring. Individuals with GDM lack the ability to adequately break down carbohydrates into energy. In some cases, GDM diagnosis may be carried out through the detection of high blood glucose levels and / or through the observation of a decreased ability to respond to a glucose challenge during pregnancy. Such diagnosis occurs most often in the second trimester and / or third trimester, specifically in weeks 24-28 of gestation. Although mechanisms leading to GDM are still unclear, in some cases, it is believed that hormones that become elevated during pregnancy may interfere with normal insulin signaling, including, but not limited to insulin resistance. This insulin signaling dysfunction leads to decreased cellular glucose uptake and elevated blood glucose levels.

[0093] According to specific embodiments, pregnant subjects may be placed into different subcategories of disease based on certain criteria.

[0094] In general, it is known that GDM diagnosis can be accomplished with either of the following two strategies (ElSayed, N. A., et al. (2023)):

[0095] One step strategy: Perform a 75-g OGTT, with plasma glucose measurement when patient is fasting and at 1 and 2 h, at 24-28 weeks of gestation in individuals not previously diagnosed with diabetes. The OGTT should be performed in the morning after an overnight fast of at least 8 h. The diagnosis of GDM is made when any of the following plasma glucose values are met or exceeded: Fasting: 92 mg / dL (5.1 mmol / L), 1 h: 180 mg / dL (10.0 mmol / L), 2 h: 153 mg / dL (8.5 mmol / L)

[0096] Two-step strategy: Step 1 : Perform a 50-g GLT (nonfasting), with plasma glucose measurement at 1 h, at 24-28 weeks of gestation in individuals not previously diagnosed with diabetes. If the plasma glucose level measured 1 h after the load is 130, 135, or 140 mg / dL (7.2, 7.5, or 7.8 mmol / L, respectively), proceed to a 100-g OGTT. Step 2: The 100-g OGTT should be performed when the patient is fasting. The diagnosis of GDM is made when at least two of the following four plasma glucose levels (measured fasting and at 1 , 2, and 3 h during OGTT) are met or exceeded: Fasting: 95 mg / dL (5.3 mmol / L), 1 h: 180 mg / dL (10.0 mmol / L), 2 h: 155 mg / dL (8.6 mmol / L), 3 h: 140 mg / dL (7.8 mmol / L).

[0097] The “one-step strategy” have been adopted internationally as the preferred approach (ElSayed, N. A., et al. (2023)). However, factors determining the screening strategy may be different for each country and may be controlled by professionals in such countries responsible for providing recommendations to physicians practicing in such countries.

[0098] The term “determining” as used herein refers to detecting and / or quantifying 2- HBA. The term “detecting” the 2-HBA refers to the general determination if 2-HBA is present. Detection does not require the exact quantification of 2-HBA but rather provides the user of the method with the information if e.g., 2-HBA is present with a concentration above a certain threshold. These thresholds are to be adapted to the respective application and sample. The term “quantifying” refers to the determination of the concentration or amount of 2-HBA. Quantification may refer to the determination of an exact amount of an analyte or may alternatively refer to a semi-quantitative determination of an analyte e.g., if the amount of the analyte in a sample is in a certain range. Such a range may be a concentration range suitable for the respective purpose of the determination of the analyte.

[0099] According to one embodiment, the method described herein comprises the quantification or detection of 2-HBA. Depending on the specific type of application of the method, the method may comprise determining the amount of 2-HBA or determining if 2- HBA is present in a certain concentration range. Further, the method described herein may comprise determining if 2-HBA is present above or below a certain threshold.

[0100] According to one embodiment, the result of the enzymatic determination is a 2- HBA concentration value.

[0101] According to one embodiment, the determined 2-HBA value is compared to a cutoff value.

[0102] The term “enzymatically determining” as used herein refers to determining an analyte by using an enzyme.

[0103] According to one embodiment, described herein is a method for enzymatically determining 2-HBA in a sample, said method comprising the sequential steps of: i. incubating the sample with an enzyme having 2-HBA oxidizing activity; and ii. determining the 2-HBA in the sample.

[0104] The term “2-hydroxy butyric acid” (2-HBA) as used herein refers is abbreviated as “2-HBA” herein and refers to 2-hydroxybutyric acid or its respective salt i.e., 2-hydroxybutyrate. Synonyms for the abbreviation 2-HBA are: HBA, a-HB, a-HBA, aHB. Synonyms for 2-hydroxybutyric acid are: alpha-hydroxybutanoic acid, 2-hydroxybutanoic acid, and a-hydroxybutanoic acid.

[0105] According to another specific embodiment, 2-HBA is a chiral molecule having the two enantiomers (R)-2-hydroxybutyric acid and (S)-2-hydroxybutyric acid.

[0106] According to one embodiment, the subject is a human.

[0107] According to a specific embodiment, the subject is a pregnant woman, a patient having symptoms of a metabolic disorder, or a person undergoing routine testing.

[0108] The term “risk” as used herein in the context of determining the risk of having or developing a metabolic disorder refers to the likelihood of having or developing a metabolic disorder.

[0109] As used herein, the term “diagnose” or “diagnosis” of a status or outcome generally refers to predicting or diagnosing the status or outcome, determining predisposition to a status or outcome, monitoring treatment of a subject, diagnosing a therapeutic response of a subject, and prognosis of status or outcome, progression, and response to particular treatment.

[0110] According to one embodiment, described herein is an in vitro method of diagnosing a metabolic disorder in a subject comprising the sequential steps of: i. enzymatically determining the concentration of 2-hydroxybutyric acid (2-HBA) in a sample from said subject; and ii. comparing said concentration of 2-HBA with a predetermined cut-off value of 25 pM 2-HBA or higher, wherein the metabolic disorder is diagnosed if said concentration of 2-HBA is equal or above the predetermined cut-off value.

[0111] According to one embodiment, described herein is an in vitro method of determining the risk of having or developing a metabolic disorder in a subject comprising the sequential steps of: i. enzymatically determining the concentration of 2-HBA in a sample from said subject; ii. comparing said concentration of 2-HBA with a predetermined cut-off value of 25 pM 2-HBA or higher; and iii. assigning to said subject a high risk of having or developing said metabolic disorder if said concentration of 2-HBA is equal or above the predetermined cut-off value.

[0112] According to one embodiment, described herein is an in vitro method of monitoring a metabolic disorder in a subject comprising the sequential steps of: i. enzymatically determining the concentration of 2-HBA in a sample from said subject; and ii. comparing said concentration of 2-HBA with a previous concentration of 2-HBA from said subject.

[0113] According to one embodiment, a previous concentration is a concentration obtained from a previous determination of 2-HBA in a sample from the same subject. According to a specific embodiment, the previous concentration may be obtained 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, or more previously to the following determination of 2-HBA.

[0114] According to one embodiment, described herein is an in vitro method of discriminating between normal glucose tolerance and impaired glucose tolerance in a subject comprising the sequential steps of: i. enzymatically determining the concentration of 2-HBA in a sample from said subject; ii. comparing said concentration of 2-HBA with a predetermined cut-off value of 25 pM 2-HBA or higher; iii. assigning to said subject impaired glucose tolerance if said concentration of 2-HBA is equal or above the predetermined cut-off value; and assigning to said subject normal glucose tolerance if said concentration of 2-HBA is below the predetermined cut-off value.

[0115] According to one embodiment, described herein is an in vitro method of discriminating between non-insulin resistance and insulin resistance in a subject comprising the sequential steps of: i. enzymatically determining the concentration of 2-HBA in a sample from said subject; ii. comparing said concentration of 2-HBA with a predetermined cut-off value of 25 pM 2-HBA or higher; iii. assigning to said subject insulin resistance if said concentration of 2-HBA is equal or above the predetermined cut-off value; and assigning to said subject non-insulin resistance if said concentration of 2-HBA is below the predetermined cut-off value.

[0116] According to one embodiment, enzymatical and quantitative determination of 2- HBA in human plasma and serum is described herein.

[0117] According to a specific embodiment, the enzymatical and quantitative determination of 2-HBA in human plasma and serum is intended for professional use and is indicated for diagnosis and monitoring of postprandial hyperglycemia in pregnancy.

[0118] According to a specific embodiment, the kit comprising an enzyme having 2-HBA oxidizing activity for enzymatical and quantitative determination of 2-HBA in human plasma and serum is intended for professional use and is indicated for diagnosis and monitoring of postprandial hyperglycemia in pregnancy.

[0119] According to one embodiment, the methods and means described herein are used to screen for or to diagnose GDM in pregnant woman.

[0120] According to a specific embodiment, in the methods and means described herein for diagnosing or screening for GDM, the result of the enzymatic determination is a 2- HBA concentration value. According to a specific embodiment, the determined 2-HBA value is compared to a predetermined cut-off value. According to a specific embodiment, if the determined 2-HBA concentration is below the predetermined cut-off value, a patient is classified as not having GDM. According to a specific embodiment, if the determined 2-HBA concentration is equal or above the predetermined cut-off value, a patient is classified as having a risk of having or developing GDM. According to a specific embodiment, if the determined 2-HBA concentration is equal or above the predetermined cut-off value, a patient is referred to further OGTT testing. Specifically, in the OGTT testing, the patient is diagnosed as no-GDM or GDM.

[0121] According to one embodiment, the method and means described herein provide a prescreening test for gestational diabetes.

[0122] According to one embodiment, an in vitro method is described herein for screening or pre-screening for gestational diabetes, said method comprising the sequential steps of: i. enzymatically determining the concentration of 2-hydroxybutyric acid (2-HBA) in a sample from said subject; and ii. comparing said concentration of 2-HBA with a predetermined cut-off value of 25 pM 2-HBA or higher, wherein a. if the determined 2-HBA concentration is below the predetermined cut-off value, a patient is classified as not having GDM, and b. if the determined 2-HBA concentration is equal or above the predetermined cut-off value, a patient is referred to further gestational diabetes testing.

[0123] According to one embodiment, further gestational diabetes testing may comprise OGTT testing.

[0124] According to one embodiment, an in vitro method is described herein for screening or pre-screening for gestational diabetes, said method comprising the sequential steps of: i. enzymatically determining the concentration of 2-hydroxybutyric acid (2-HBA) in a sample from said subject; and ii. comparing said concentration of 2-HBA with a predetermined cut-off value of 25 pM 2-HBA or higher, wherein a. if the determined 2-HBA concentration is below the predetermined cut-off value, a patient is classified as not having GDM, and b. if the determined 2-HBA concentration is equal or above the predetermined cut-off value, classified as having a risk of having or developing gestational diabetes. According to one embodiment, the predetermined cut-off value is 25, 26, 27, 28,

[0125] 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 ,

[0126] 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74,

[0127] 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97,

[0128] 98, 99, 100 pM 2-HBA, or higher.

[0129] According to one embodiment, the predetermined cut-off value is less than 250 pM 2-HBA. Specifically, it is less than 250, 249, 248, 247, 246, 245, 244, 243, 242, 241 , 240, 239, 238, 237, 236, 235, 234, 233, 232, 231 , 230, 229, 228, 227, 226, 225, 224, 223,

[0130] 222, 221 , 220, 219, 218, 217, 216, 215, 214, 213, 212, 211 , 210, 209, 208, 207, 206,

[0131] 205, 204, 203, 202, 201 , 200, 199, 198, 197, 196, 195, 194, 193, 192, 191 , 190, 189,

[0132] 188, 187, 186, 185, 184, 183, 182, 181 , 180, 179, 178, 177, 176, 175, 174, 173, 172,

[0133] 171 , 170, 169, 168, 167, 166, 165, 164, 163, 162, 161 , 160, 159, 158, 157, 156, 155,

[0134] 154, 153, 152, 151 , 150, 149, 148, 147, 146, 145, 144, 143, 142, 141 , 140, 139, 138,

[0135] 137, 136, 135, 134, 133, 132, 131 , 130, 129, 128, 127, 126, 125, 124, 123, 122, 121 ,

[0136] 120, 119, 118, 117, 116, 115, 114, 113, 112, 111 , 110, 109, 108, 107, 106, 105, 104,

[0137] 103, 102, 101 , 100, 99, 98, 97, 96, 95, 94, 93, 92, 91 , 90, 89, 88, 87, 86, 85, 84, 83, 82,

[0138] 81 , 80, 79, 78, 77, 76, 75, 74, 73, 72, 71 , 70, 69, 68, 67, 66, 65, 64, 63, 62, 61 , 60, 59,

[0139] 58, 57, 56, 55, 54, 53, 52, 51 , 50, 49, 48, 47, 46, 45, 44, 43, 42, 41 , 40, 39, 38, 37, 36,

[0140] 35, 34, 33, 32, 31 , or 30 pM 2-HBA. Preferably, it is less than 100 pM 2-HBA, even more preferably it is less than 70 or 50 pM 2-HBA.

[0141] According to a specific embodiment, the predetermined cut-off value is between 25 and 250 pM 2-HBA, preferably it is between 25 and 150 pM 2-HBA, even more preferably it is at least 25 pM 2-HBA and less than 100 pM 2-HBA. Even more preferably, it is between 25 and 50pM 2-HBA.

[0142] According to one embodiment, the predetermined cut-off a concentration value or a concentration range of 2-HBA.

[0143] According to a specific embodiment, the predetermined cut-off value is a range of 25-30, 25-35, 25-40, 25-45, 25-50, 25-55, 25-60, 25-65, 25-70, 25-75, 25-80, 25-85, 25- 90, 25-95, 25-100, 30-35, 30-40, 30-45, 30-50, 30-55, 30-60, 30-65, 30-70, 30-75, 30-80, 30-85, 30-90, 30-95, 30-100, 35-40, 35-45, 35-50, 35-55, 35-60, 35-65, 35-70, 35-75, 35- 80, 35-85, 35-90, 35-95, 35-100, 40-45, 40-50, 40-55, 40-60, 40-65, 40-70, 40-75, 40-80, 40-85, 40-90, 40-95, 40-100, 45-50, 45-55, 45-60, 45-65, 45-70, 45-75, 45-80, 45-85, 45- 90, 45-95, 45-100, 50-55, 50-60, 50-65, 50-70, 50-75, 50-80, 50-85, 50-90, 50-95, 50- 100, 55-60, 55-65, 55-70, 55-75, 55-80, 55-85, 55-90, 55-95, 55-100, 60-65, 60-70, 60- 75, 60-80, 60-85, 60-90, 60-95, 60-100, 65-70, 65-75, 65-80, 65-85, 65-90, 65-95, 65- 100, 70-75, 70-80, 70-85, 70-90, 70-95, 70-100, 75-80, 75-85, 75-90, 75-95, 75-100, BOSS, 80-90, 80-95, 80-100, 85-90, 85-95, 85-100, 90-95, 90-100, or 95-100 pM 2-HBA.

[0144] The term “pM” refers to pmol per liter.

[0145] According to one embodiment, in the case of a metabolic disorder the predetermined cut-off value is 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39,

[0146] 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62,

[0147] 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85,

[0148] 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100 pM 2-HBA, or higher.

[0149] According to a specific embodiment, in the case of a metabolic disorder the predetermined cut-off value is a range of 25-30, 25-35, 25-40, 25-45, 25-50, 25-55, 25- 60, 25-65, 25-70, 25-75, 25-80, 25-85, 25-90, 25-95, 25-100, 30-35, 30-40, 30-45, 30-50, 30-55, 30-60, 30-65, 30-70, 30-75, 30-80, 30-85, 30-90, 30-95, 30-100, 35-40, 35-45, 35- 50, 35-55, 35-60, 35-65, 35-70, 35-75, 35-80, 35-85, 35-90, 35-95, 35-100, 40-45, 40-50, 40-55, 40-60, 40-65, 40-70, 40-75, 40-80, 40-85, 40-90, 40-95, 40-100, 45-50, 45-55, 45- 60, 45-65, 45-70, 45-75, 45-80, 45-85, 45-90, 45-95, 45-100, 50-55, 50-60, 50-65, 50-70, 50-75, 50-80, 50-85, 50-90, 50-95, 50-100, 55-60, 55-65, 55-70, 55-75, 55-80, 55-85, 55- 90, 55-95, 55-100, 60-65, 60-70, 60-75, 60-80, 60-85, 60-90, 60-95, 60-100, 65-70, 65- 75, 65-80, 65-85, 65-90, 65-95, 65-100, 70-75, 70-80, 70-85, 70-90, 70-95, 70-100, 75- 80, 75-85, 75-90, 75-95, 75-100, 80-85, 80-90, 80-95, 80-100, 85-90, 85-95, 85-100, 90- 95, 90-100, or 95-100 pM 2-HBA.

[0150] According to one embodiment, in the case of a glucose metabolism disorder the predetermined cut-off value is 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39,

[0151] 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62,

[0152] 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85,

[0153] 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100 pM 2-HBA, or higher.

[0154] According to a specific embodiment, in the case of a glucose metabolism disorder the predetermined cut-off value is a range of 25-30, 25-35, 25-40, 25-45, 25-50, 25-55, 25-60, 25-65, 25-70, 25-75, 25-80, 25-85, 25-90, 25-95, 25-100, 30-35, 30-40, 30-45, 30- 50, 30-55, 30-60, 30-65, 30-70, 30-75, 30-80, 30-85, 30-90, 30-95, 30-100, 35-40, 35-45, 35-50, 35-55, 35-60, 35-65, 35-70, 35-75, 35-80, 35-85, 35-90, 35-95, 35-100, 40-45, 40- 50, 40-55, 40-60, 40-65, 40-70, 40-75, 40-80, 40-85, 40-90, 40-95, 40-100, 45-50, 45-55, 45-60, 45-65, 45-70, 45-75, 45-80, 45-85, 45-90, 45-95, 45-100, 50-55, 50-60, 50-65, 50- 70, 50-75, 50-80, 50-85, 50-90, 50-95, 50-100, 55-60, 55-65, 55-70, 55-75, 55-80, 55-85, 55-90, 55-95, 55-100, 60-65, 60-70, 60-75, 60-80, 60-85, 60-90, 60-95, 60-100, 65-70, 65-75, 65-80, 65-85, 65-90, 65-95, 65-100, 70-75, 70-80, 70-85, 70-90, 70-95, 70-100, 75-80, 75-85, 75-90, 75-95, 75-100, 80-85, 80-90, 80-95, 80-100, 85-90, 85-95, 85-100, 90-95, 90-100, or 95-100 pM 2-HBA.

[0155] According to one embodiment, in the case of a cardiometabolic disease the predetermined cut-off value is 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39,

[0156] 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62,

[0157] 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85,

[0158] 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100 pM 2-HBA, or higher.

[0159] According to a specific embodiment, in the case of a cardiometabolic disease the predetermined cut-off value is a range of 25-30, 25-35, 25-40, 25-45, 25-50, 25-55, 25- 60, 25-65, 25-70, 25-75, 25-80, 25-85, 25-90, 25-95, 25-100, 30-35, 30-40, 30-45, 30-50, 30-55, 30-60, 30-65, 30-70, 30-75, 30-80, 30-85, 30-90, 30-95, 30-100, 35-40, 35-45, 35- 50, 35-55, 35-60, 35-65, 35-70, 35-75, 35-80, 35-85, 35-90, 35-95, 35-100, 40-45, 40-50, 40-55, 40-60, 40-65, 40-70, 40-75, 40-80, 40-85, 40-90, 40-95, 40-100, 45-50, 45-55, 45- 60, 45-65, 45-70, 45-75, 45-80, 45-85, 45-90, 45-95, 45-100, 50-55, 50-60, 50-65, 50-70, 50-75, 50-80, 50-85, 50-90, 50-95, 50-100, 55-60, 55-65, 55-70, 55-75, 55-80, 55-85, 55- 90, 55-95, 55-100, 60-65, 60-70, 60-75, 60-80, 60-85, 60-90, 60-95, 60-100, 65-70, 65- 75, 65-80, 65-85, 65-90, 65-95, 65-100, 70-75, 70-80, 70-85, 70-90, 70-95, 70-100, 75- 80, 75-85, 75-90, 75-95, 75-100, 80-85, 80-90, 80-95, 80-100, 85-90, 85-95, 85-100, 90- 95, 90-100, or 95-100 pM 2-HBA.

[0160] According to one embodiment, in the case of gestational diabetes the predetermined cut-off value is 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39,

[0161] 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62,

[0162] 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85,

[0163] 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100 pM 2-HBA, or higher.

[0164] According to a specific embodiment, in the case of gestational diabetes the predetermined cut-off value is a range of 25-30, 25-35, 25-40, 25-45, 25-50, 25-55, 25- 60, 25-65, 25-70, 25-75, 25-80, 25-85, 25-90, 25-95, 25-100, 30-35, 30-40, 30-45, 30-50, 30-55, 30-60, 30-65, 30-70, 30-75, 30-80, 30-85, 30-90, 30-95, 30-100, 35-40, 35-45, 35- 50, 35-55, 35-60, 35-65, 35-70, 35-75, 35-80, 35-85, 35-90, 35-95, 35-100, 40-45, 40-50, 40-55, 40-60, 40-65, 40-70, 40-75, 40-80, 40-85, 40-90, 40-95, 40-100, 45-50, 45-55, 45- 60, 45-65, 45-70, 45-75, 45-80, 45-85, 45-90, 45-95, 45-100, 50-55, 50-60, 50-65, 50-70, 50-75, 50-80, 50-85, 50-90, 50-95, 50-100, 55-60, 55-65, 55-70, 55-75, 55-80, 55-85, 55- 90, 55-95, 55-100, 60-65, 60-70, 60-75, 60-80, 60-85, 60-90, 60-95, 60-100, 65-70, 65- 75, 65-80, 65-85, 65-90, 65-95, 65-100, 70-75, 70-80, 70-85, 70-90, 70-95, 70-100, 75- 80, 75-85, 75-90, 75-95, 75-100, 80-85, 80-90, 80-95, 80-100, 85-90, 85-95, 85-100, 90- 95, 90-100, or 95-100 pM 2-HBA. Preferably, in the case of gestational diabetes the predetermined cut-off value is between 25 and 35 pM 2-HBA. Preferably, it is about 30pM 2-HBA.

[0165] According to one embodiment, in the case of impaired glucose tolerance the predetermined cut-off value is 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39,

[0166] 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62,

[0167] 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85,

[0168] 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100 pM 2-HBA, or higher.

[0169] According to a specific embodiment, in the case of impaired glucose tolerance the predetermined cut-off value is a range of 25-30, 25-35, 25-40, 25-45, 25-50, 25-55, 25- 60, 25-65, 25-70, 25-75, 25-80, 25-85, 25-90, 25-95, 25-100, 30-35, 30-40, 30-45, 30-50, 30-55, 30-60, 30-65, 30-70, 30-75, 30-80, 30-85, 30-90, 30-95, 30-100, 35-40, 35-45, 35- 50, 35-55, 35-60, 35-65, 35-70, 35-75, 35-80, 35-85, 35-90, 35-95, 35-100, 40-45, 40-50, 40-55, 40-60, 40-65, 40-70, 40-75, 40-80, 40-85, 40-90, 40-95, 40-100, 45-50, 45-55, 45- 60, 45-65, 45-70, 45-75, 45-80, 45-85, 45-90, 45-95, 45-100, 50-55, 50-60, 50-65, 50-70, 50-75, 50-80, 50-85, 50-90, 50-95, 50-100, 55-60, 55-65, 55-70, 55-75, 55-80, 55-85, 55- 90, 55-95, 55-100, 60-65, 60-70, 60-75, 60-80, 60-85, 60-90, 60-95, 60-100, 65-70, 65- 75, 65-80, 65-85, 65-90, 65-95, 65-100, 70-75, 70-80, 70-85, 70-90, 70-95, 70-100, 75- 80, 75-85, 75-90, 75-95, 75-100, 80-85, 80-90, 80-95, 80-100, 85-90, 85-95, 85-100, 90- 95, 90-100, or 95-100 pM 2-HBA.

[0170] According to one embodiment, in the case of insulin resistance the predetermined cut-off value is 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43,

[0171] 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66,

[0172] 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89,

[0173] 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100 pM 2-HBA, or higher.

[0174] According to a specific embodiment, in the case of insulin resistance the predetermined cut-off value is a range of 25-30, 25-35, 25-40, 25-45, 25-50, 25-55, 25- 60, 25-65, 25-70, 25-75, 25-80, 25-85, 25-90, 25-95, 25-100, 30-35, 30-40, 30-45, 30-50, 30-55, 30-60, 30-65, 30-70, 30-75, 30-80, 30-85, 30-90, 30-95, 30-100, 35-40, 35-45, 35- 50, 35-55, 35-60, 35-65, 35-70, 35-75, 35-80, 35-85, 35-90, 35-95, 35-100, 40-45, 40-50, 40-55, 40-60, 40-65, 40-70, 40-75, 40-80, 40-85, 40-90, 40-95, 40-100, 45-50, 45-55, 45- 60, 45-65, 45-70, 45-75, 45-80, 45-85, 45-90, 45-95, 45-100, 50-55, 50-60, 50-65, 50-70, 50-75, 50-80, 50-85, 50-90, 50-95, 50-100, 55-60, 55-65, 55-70, 55-75, 55-80, 55-85, 55- 90, 55-95, 55-100, 60-65, 60-70, 60-75, 60-80, 60-85, 60-90, 60-95, 60-100, 65-70, 65- 75, 65-80, 65-85, 65-90, 65-95, 65-100, 70-75, 70-80, 70-85, 70-90, 70-95, 70-100, 75- 80, 75-85, 75-90, 75-95, 75-100, 80-85, 80-90, 80-95, 80-100, 85-90, 85-95, 85-100, 90- 95, 90-100, or 95-100 pM 2-HBA.

[0175] According to one embodiment, in the case of prediabetes the predetermined cut-off value is 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45,

[0176] 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68,

[0177] 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 ,

[0178] 92, 93, 94, 95, 96, 97, 98, 99, 100 pM 2-HBA, or higher.

[0179] According to a specific embodiment, in the case of prediabetes the predetermined cut-off value is a range of 25-30, 25-35, 25-40, 25-45, 25-50, 25-55, 25-60, 25-65, 25-70, 25-75, 25-80, 25-85, 25-90, 25-95, 25-100, 30-35, 30-40, 30-45, 30-50, 30-55, 30-60, SOOS, 30-70, 30-75, 30-80, 30-85, 30-90, 30-95, 30-100, 35-40, 35-45, 35-50, 35-55, 35-60, 35-65, 35-70, 35-75, 35-80, 35-85, 35-90, 35-95, 35-100, 40-45, 40-50, 40-55, 40-60, 40- 65, 40-70, 40-75, 40-80, 40-85, 40-90, 40-95, 40-100, 45-50, 45-55, 45-60, 45-65, 45-70, 45-75, 45-80, 45-85, 45-90, 45-95, 45-100, 50-55, 50-60, 50-65, 50-70, 50-75, 50-80, SO- 85, 50-90, 50-95, 50-100, 55-60, 55-65, 55-70, 55-75, 55-80, 55-85, 55-90, 55-95, 55- 100, 60-65, 60-70, 60-75, 60-80, 60-85, 60-90, 60-95, 60-100, 65-70, 65-75, 65-80, 65- 85, 65-90, 65-95, 65-100, 70-75, 70-80, 70-85, 70-90, 70-95, 70-100, 75-80, 75-85, 75- 90, 75-95, 75-100, 80-85, 80-90, 80-95, 80-100, 85-90, 85-95, 85-100, 90-95, 90-100, or 95-100 pM 2-HBA.

[0180] According to one embodiment, in the case of diabetes the predetermined cut-off value is 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45,

[0181] 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68,

[0182] 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 ,

[0183] 92, 93, 94, 95, 96, 97, 98, 99, 100 pM 2-HBA, or higher.

[0184] According to a specific embodiment, in the case of diabetes the predetermined cut-off value is a range of 25-30, 25-35, 25-40, 25-45, 25-50, 25-55, 25-60, 25-65, 25-70, 25-75, 25-80, 25-85, 25-90, 25-95, 25-100, 30-35, 30-40, 30-45, 30-50, 30-55, 30-60, SO- 65, 30-70, 30-75, 30-80, 30-85, 30-90, 30-95, 30-100, 35-40, 35-45, 35-50, 35-55, 35-60, 35-65, 35-70, 35-75, 35-80, 35-85, 35-90, 35-95, 35-100, 40-45, 40-50, 40-55, 40-60, 40- 65, 40-70, 40-75, 40-80, 40-85, 40-90, 40-95, 40-100, 45-50, 45-55, 45-60, 45-65, 45-70, 45-75, 45-80, 45-85, 45-90, 45-95, 45-100, 50-55, 50-60, 50-65, 50-70, 50-75, 50-80, SO- 85, 50-90, 50-95, 50-100, 55-60, 55-65, 55-70, 55-75, 55-80, 55-85, 55-90, 55-95, 55- 100, 60-65, 60-70, 60-75, 60-80, 60-85, 60-90, 60-95, 60-100, 65-70, 65-75, 65-80, 65- 85, 65-90, 65-95, 65-100, 70-75, 70-80, 70-85, 70-90, 70-95, 70-100, 75-80, 75-85, 75- 90, 75-95, 75-100, 80-85, 80-90, 80-95, 80-100, 85-90, 85-95, 85-100, 90-95, 90-100, or 95-100 pM 2-HBA.

[0185] According to one embodiment, an enzyme having 2-HBA oxidizing activity is used for enzymatically determining the concentration of 2-HBA, preferably, the enzyme is an enzyme having 2-HBA dehydrogenase activity, more preferably FCb2 or LDH.

[0186] According to one embodiment, the sample is a blood sample.

[0187] According to one embodiment, the concentration of 2-HBA is determined colorimetrically, photometrically, fluorimetrically, through phosphorescence, through chemiluminescence, or electrochemically.

[0188] According to one embodiment, methods described herein may further comprise selectively removing lactate in the sample prior to enzymatically determining the concentration of 2-HBA.

[0189] According to one embodiment, described herein is the use of an enzyme having 2-HBA oxidizing activity for diagnosing a metabolic disorder in a subject, for determining the risk of having or developing a metabolic disorder, for monitoring a metabolic disorder in a subject, or for discriminating between normal glucose tolerance and impaired glucose tolerance in a subject.

[0190] Specifically, the concentration of 2-hydroxybutyric acid (2-HBA) is determined enzymatically in a sample from said subject.

[0191] According to one embodiment, described herein is a kit for diagnosing a metabolic disorder, for determining the risk of having or developing a metabolic disorder, for monitoring a metabolic disorder in a subject, for discriminating between normal glucose tolerance and impaired glucose tolerance, or for discriminating between gestational diabetes and non-gestational diabetes, said kit comprising an enzyme having 2-HBA oxidizing activity.

[0192] According to one embodiment, the enzyme having 2-HBA oxidizing activity is an enzyme having 2-HBA dehydrogenase activity, preferably FCb2 or LDH.

[0193] According to one embodiment, the kit described herein further comprises an enzyme for selectively removing lactate from the sample. The term “lactate” refers to lactic acid or the salt thereof. Specifically, in the context of selectively removing lactate from the sample, and in the context of 2-HBA determining, the term “lactate” refers to L-lactic acid or the salt thereof, i.e. , L-lactate.

[0194] According to one embodiment, the sample may be any material for which determining the presence of 2-HBA is relevant or of interest. In particular, the sample is a human or animal sample, specifically any one of body fluid, interstitial fluid, blood, blood plasma, blood serum, dermal fluid, urine, tears, sweat, saliva, skin, flesh, tissue, eyeballs, cornea, and gastric fluid.

[0195] According to a specific embodiment, human blood is used as a sample in the methods and means described herein. Specifically, blood contains 2-HBA and contains also lactate.

[0196] According to one embodiment, the blood sample is serum, or plasma.

[0197] According to one embodiment, the blood sample is a fasting, or non-fasting sample.

[0198] According to one embodiment, the sample is a fasting or non-fasting human serum or plasma sample.

[0199] According to one embodiment, the sample is obtained from a pregnant woman during pregnancy.

[0200] According to one embodiment, the method described herein has a sensitivity of 70%, or more.

[0201] According to one embodiment, the method described herein has a specificity of 65%, or more.

[0202] According to one embodiment, the method described herein has a negative predictive value (NPV) of 93%, or more.

[0203] In general, negative predictive value is the proportion of the cases giving negative test results who are already healthy. It is the ratio of subjects truly diagnosed as negative to all those who had negative test results (including patients who were incorrectly diagnosed as healthy). This characteristic can predict how likely it is for someone to truly be healthy, in case of a negative test result.

[0204] According to one embodiment, the method described herein has a positive predictive value (PPV) of 25, or more.

[0205] In general, positive predictive value is the proportion of cases giving positive test results who are already patients. It is the ratio of patients truly diagnosed as positive to all those who had positive test results (including healthy subjects who were incorrectly diagnosed as patient). This characteristic can predict how likely it is for someone to truly be patient, in case of a positive test result.

[0206] According to one embodiment, the sample is obtained from a pregnant woman during the 5-28, 6-24, 7-28, 8-28, 9-28, 10-28, 11-28, 12-28, 13-28, 14-28, 15-28, 16-28, 17-28, 18-28, 19-28, 20-28, 21-28, 22-28, 23-28, 24-28, 5-14, 6-14, 7-14, 8-14, 9-14, IQ- 14, 11-14, 12-14, or 13-14 gestational week.

[0207] According to a specific embodiment, the sample is obtained from a pregnant woman during the 24-28 gestational week.

[0208] According to one embodiment, the method described herein is performed using a clinical chemistry analyzer.

[0209] According to one embodiment, 2-HBA is determined on a clinical analyzer with the kit described herein.

[0210] According to one embodiment, the method described herein is performed using a clinical chemistry assay.

[0211] According to one embodiment, the method described herein is implemented on a point of care device.

[0212] According to one embodiment, the kit described herein comprises a point of care device, wherein said point of care device comprises an enzyme having 2-hydroxybutyric acid oxidizing activity.

[0213] According to one embodiment, the method can be performed using a high throughput clinical chemistry analyzer.

[0214] According to one embodiment, the method described herein further comprises providing treatment to the subject, preferably selected from the group consisting of diet modification, exercise, insulin therapy, and oral medication.

[0215] According to one embodiment, a therapy guidance may be effectuated based on the result obtained from the methods described herein. Suitable therapy guidance includes one or more of the following: performing a confirmatory OGTT and / or additional diagnostic testing, prescribing a drug therapy, increasing monitoring frequency of patient condition, and recommending appropriate risk-reduction therapy such as making or maintaining diet and lifestyle choices. The therapy guidance may involves administration of antioxidants, administration of anti-coagulants, administration of anti-dyslipidemic drugs, avoidance of drugs or agents known to damage pancreatic cells; discontinued administration of current drug therapy, administration of agents specific for post-prandial hyperglycemia (e.g. cycloset), administration of drugs that enhance, and / or augment, and / or spare pancreatic beta cell function, administration of an anti-viral agent, an immunosuppressant or insulin or an insulin analog or combinations thereof. The therapy guidance may also include one or more of the following: increased frequency of physician's follow-up, referral for oral glucose tolerance test (OGTT) and / or CLIX test, repetition of tests for monitoring disease progression, patient referral for comprehensive testing for type I diabetes; testing for auto-antibodies to pancreatic cell antigens, other biomarkers for autoimmune diseases, viral DNA / RNA and / or antibodies to viral capsid proteins for Enterovirus family members or combinations thereof. Lifestyle choices involve changes in diet and nutrition, changes in exercise, smoking elimination or a combination thereof.

[0216] According to one embodiment of the invention, the sample contains or is suspected to contain 2-HBA and lactate.

[0217] According to a specific embodiment, the sample contains or is suspected to contain lactate at a concentration in the range of 1 to 2 mM.

[0218] According to a specific embodiment, the sample contains or is suspected to contain at least a 10-fold higher concentration of lactate than 2-HBA.

[0219] According to the invention, the methods and means described herein enable the determination of 2-HBA in a sample suspected to contain also lactate e.g., in a blood sample which contains also lactate, by selectively removing lactate prior to the determination of the 2-HBA.

[0220] According to one embodiment, described herein is a method for determining a 2- HBA in a sample comprising 2-HBA and lactate, said method comprising the sequential steps of: i. selectively removing lactate from the sample; ii. incubating the sample with an enzyme having 2-HBA oxidizing activity; and iii. determining the 2-HBA in the sample.

[0221] According to one embodiment, described herein is a method for determining 2- HBA in a sample comprising the 2-HBA and lactate, said method comprising the steps of: i. selectively removing lactate from the sample prior to step ii. and iii.; ii. incubating the sample with an enzyme having 2-HBA oxidizing activity; and iii. determining the 2-HBA in the sample. According to a specific embodiment, described herein is a method for determining 2-hydroxybutyric acid in a sample comprising 2-HBA and lactate, said method comprising the sequential steps of: a. selectively removing lactate from the sample by an enzyme having lactate oxidase activity; b. incubating the sample with an enzyme having 2-HBA dehydrogenase activity; and c. determining 2-HBA in the sample.

[0222] According to a specific embodiment, described herein is a method for determining 2-HBA in a sample comprising 2-HBA and lactate, said method comprising the steps of: a. selectively removing lactate from the sample by an enzyme having lactate oxidase activity prior to step b. and c.; b. incubating the sample with an enzyme having 2-HBA dehydrogenase activity; and c. determining 2-HBA in the sample.

[0223] According to one embodiment, described herein is a method for determining a 2- HBA in a sample comprising the 2-HBA and optionally lactate, said method comprising the sequential steps of: i. optionally selectively removing lactate from the sample; ii. incubating the sample with an enzyme having 2-HBA oxidizing activity; and iii. determining the 2-HBA in the sample.

[0224] According to one embodiment, described herein is a method for determining a 2- HBA in a sample comprising the 2-HBA and optionally lactate, said method comprising the steps of: i. optionally selectively removing lactate from the sample prior to step ii. and iii.; ii. incubating the sample with an enzyme having 2-HBA oxidizing activity; and iii. determining the 2-HBA in the sample.

[0225] According to one embodiment, selectively removing lactate from the sample is optional.

[0226] The term “selectively removing” as used herein refers to the removal of lactate while not substantially changing the amount or concentration of the 2-HBA in the sample. Specifically, the term “removing” as used herein refers to the modification of lactate e.g., to the oxidation of lactate to pyruvate. Non-limiting examples of methods for selectively removing lactate are enzyme reaction, precipitation, emulsion liquid membrane separation techniques, adsorption, extraction, polymerization, and esterification.

[0227] Non-limiting examples of extracting lactate are extraction with niosomes, microfiltration, and reactive extraction (see Roque L., et aL, 2020).

[0228] A non-limiting example of polymerization is polymerization using catalysts and heat (see Lunt, James, 1998, and Chafran, Liana S., et al., 2019).

[0229] A non-limiting example of esterification is vapor permeation-assisted esterification (see Khunnonkwao, Panwana, et aL, 2012).

[0230] According to a specific embodiment, modification of lactate is accompanied by the accumulation of modified lactate in the sample. Thereby, the modified lactate is not necessarily removed from the sample, but may remain in the sample also during the determination of the 2-HBA. For example, if oxidation of lactate is accompanied by the accumulation of its reaction product pyruvate, the reaction product pyruvate is not necessarily removed from the reaction mixture and may remain in the sample.

[0231] According to an alternative embodiment, the modified lactate may be removed from the sample.

[0232] According to a specific embodiment, pyruvate can remain in the sample, e.g., if a lactate oxidase characterized by a low product inhibition is used. Alternatively, the reaction product pyruvate may be removed from the reaction mixture.

[0233] The term “enzyme” as used herein refers to any substance composed wholly or largely of protein or polypeptides that catalyzes or promotes, more or less specifically, one or more chemical or biochemical reaction(s).

[0234] The term “activity” as used herein e.g., in the context of an enzyme activity, shall refer to the catalyzed reaction of the enzyme. Thereby, an enzyme having an activity is a functionally active molecule such as a functional enzyme. A functional enzyme is specifically characterized by a catalytic center recognizing the enzyme substrate and catalyzing the conversion of the substrate to a conversion product. Enzyme variants are considered functional or functionally active upon determining their enzymatic activity in a standard test system, e.g., wherein the enzymatic activity is at least 50 % of the activity of the parent (not modified or wild-type enzyme), or at least any of 60 %, 70 %, 80 %, 90 %, 100 %, or even more than 100 %.

[0235] Enzyme activity is generally given in units. Thereby, one unit of enzymatic activity is defined as the amount of enzyme that catalyzes the reaction of 1 pmol of substrate per minute under the respective conditions of the determination method. For example, one unit of enzymatic activity is defined as the amount of enzyme that oxidizes 1 pmol of substrate such as e.g., lactate or 2-HBA, per min under the respective conditions of the determination method. The specific activity is given in “U / mg”, “U mg-1” or “U per mg”. Volumetric activity is given in units per volume such as in “U / mL”, “U / ml”, “U per mL”, “U per ml”, “U mL1”, or “U ml1”.

[0236] As used herein and if an enzyme is used in step i. for selectively removing lactate, such as enzyme is referred to as herein also as enzyme A.

[0237] According to one embodiment of the invention, enzyme A is an enzyme having lactate oxidizing activity. In step i, enzyme A does not substantially change the concentration of 2-HBA to be determined in the sample.

[0238] According to a specific embodiment, enzyme A is selected from the group consisting of lactate oxidases, lactate monooxygenases, and lactate dehydrogenases.

[0239] According to one embodiment of the invention, in step ii. an enzyme having 2-HBA oxidizing activity is used. Thereby, the enzyme having 2-HBA oxidizing activity is herein also referred to as enzyme B.

[0240] According to a specific embodiment, enzyme B has 2-HBA oxidizing activity but may also be able to oxidize lactate. Lactate oxidizing activity of enzyme B does not disturb the determination of the 2-HBA as lactate is removed in step i.

[0241] According to a specific embodiment, enzyme B is selected from the group consisting of 2-HBA dehydrogenases, 2-HBA oxidases, and 2-HBA monooxygenases. Thereby, enzymes such as LDH, FCb2, LOx, or hydroxy acid oxidase may be used as enzyme B.

[0242] In general, the use of e.g., LDH, FCb2, and LOx as enzyme A or as enzyme B depends on the specific substrate specificity of the specific enzyme and the specific 2- HBA to be determined in the sample.

[0243] According to one embodiment of the invention, enzyme A is an enzyme capable of modifying lactate to such an extent that the enzyme B cannot catalyze a reaction with the modified lactate.

[0244] According to a specific embodiment of the invention, enzyme A is an enzyme capable of selectively oxidizing lactate but does not substantially oxidize the 2-HBA.

[0245] According to one embodiment of the invention, enzyme A is an enzyme having lactate oxidizing activity. In general, the term “oxidizing” in the context of an oxidizing agent such as an enzyme having oxidizing activity, refers to an agent that oxidizes a substance and gains or “accepts” an electron from said substance. Thereby, the enzyme has “substance oxidizing activity”. Such a substance may also be referred to as substrate. Therefore, an enzyme having lactate oxidizing activity catalyzes the oxidation of lactate. An enzyme having 2-HBA oxidizing activity catalyzes the oxidation of 2-HBA. Herein, the term “capable of oxidizing” may be alternatively used for the term “oxidizing” in the context of an enzyme having oxidizing activity.

[0246] In general, an enzyme having a substance oxidizing activity gains or accepts one or more electrons from the substance. Thereby, the enzyme itself or a cofactor of the enzyme, gets reduced. In the reduced state, an enzyme cannot catalyze another oxidation reaction of a substance. Therefore, the enzyme or the cofactor of the enzyme needs to be re-oxidized by transferring the gained electrons to an electron acceptor before another oxidation reaction of a substance can be catalyzed.

[0247] According to one embodiment, the enzyme or the cofactor may be re-oxidized by transferring the gained electrons to oxygen, to a molecule with electrochemical activity such as a redox mediator, or to an electrode.

[0248] In general, the preference or degree of electron transfer of an enzyme to an electron acceptor differs depending on the specific enzyme and on the specific electron acceptor used.

[0249] In general, an enzyme having substance oxidizing activity is known as “oxidase” if the enzyme uses dioxygen as preferred electron acceptor for the reoxidation of the enzyme. If an enzyme having a substance oxidizing activity uses an electron acceptor different than dioxygen as preferred electron acceptor for the reoxidation of the enzyme, then such an enzyme is generally known as a “dehydrogenase”.

[0250] According to a specific embodiment of the invention, enzyme A may be an enzyme having lactate oxidase activity or alternatively an enzyme having lactate dehydrogenase activity.

[0251] The term “lactate oxidase activity” refers to the activity of an enzyme catalyzing the oxidation of lactate with dioxygen as electron acceptor forming pyruvate and hydrogen peroxide as products. Thereby, two electrons are transferred from lactate to the cofactor of the enzyme e.g., to FAD, and the so gained electrons are subsequently transferred towards dioxygen producing hydrogen peroxide. According to a specific embodiment of the invention, if an enzyme having lactate oxidase activity is used as enzyme A, dioxygen may be used as electron acceptor for the re-oxidation of the enzyme.

[0252] According to one embodiment, enzyme A is a lactate oxidase (LOx). Lactate oxidases belong to the enzyme family of E.C 1.1.3.2. Specifically, various lactate oxidases may be used in the method described herein e.g., lactate oxidase from Aerococcus viridans, Nostoc sp. (PCC7120), Lactobacillus jensenii, Lysinibacillus sphaericus, Chlamydomonas reinhardtii, Alicycliphilus denitrificans, Lacticaseibacillus rhamnosus, Lentilactobacillus hilgardii, Roseobacter sp. (strain GAI101), Streptococcus iniae, and Pediococcus acidilactici. Specifically, the lactate oxidase may be a functional variant of any one of the foregoing lactate oxidases having at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99 % sequence identity with the respective amino acid sequence of the foregoing lactate oxidases.

[0253] According to another embodiment, enzyme A may be a lactate monooxygenase. Therefore, various lactate monooxygenases may be used in the method described herein e.g., lactate 2-monooxygenase from Mycolicibacterium smegmatis.

[0254] According to a specific embodiment, the lactate oxidase is characterized by its enantiomer selectivity and specificity towards the natural lactate substrate L-lactate.

[0255] According to a specific embodiment of the invention, enzyme A may be an engineered variant of an enzyme. For example, an engineered variant may be an oxidase engineered towards using lactate as substrate while not using e.g., 2-HBA as substrate by decreasing the enzyme activity with 2-HBA.

[0256] According to an alternative embodiment of the invention, enzyme A may be an engineered variant of an enzyme which is naturally not capable of oxidizing lactate. For example, an oxidase naturally oxidizing a different molecule than lactate may have been engineered towards using lactate as substrate.

[0257] In a specific embodiment, the LOx described herein is a functionally active variant of a LOx peptide sequence and comprises one or more point mutations in the nucleotide sequence encoding the LOx sequence, compared to the respective parent LOx sequence. Specifically, it comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, point mutations, specifically resulting in one or more amino acid substitutions, additions, or deletions, or the like. Specifically, the functional variant of the LOx peptide sequence is a full-length LOx peptide sequence comprising point mutations, or it is a fragment of the full-length LOx peptide sequence with retained enzymatic activity. Specifically, depending on the use of the LOx as described elsewhere herein as enzyme A or as enzyme B, a variant of a LOx sequence is functionally active if it is capable of converting L-lactate to pyruvate or capable of converting a 2-HBA to the respective oxidized 2-HBA. Specifically, a functionally active variant of a LOx sequence has at least 10, 20, 30, 40, 50, 60, 70, or even more % of the enzymatic activity of the corresponding parent LOx sequence with lactate or 2-HBA. Specifically, a functionally active variant of a LOx sequence has at least 10, 20, 30, 40, 50, 60, 70, or even more % of the enzymatic activity of the corresponding parent LOx sequence, wherein said enzymatic activity is determined with the following assays.

[0258] The lactate oxidase activity of an enzyme can be determined for example by the Amplex Red assay. Thereby, oxidase activity is measured using a peroxidase-coupled reaction containing 7.1 U / mL horseradish peroxidase (181 U / mg; Sigma) and 0.05 mM AmplexRed (resorufin: E560 nm = 54.0 mM-1 cm-1). Oxygen is present at ambient concentrations of ~250 pM (Kadowaki, M. A. S. et al. (2020)). The oxidase activity of an enzyme with 2-HBA as substrate can be determined using the method for determining lactate oxidase activity and using the 2-HBA instead of lactate.

[0259] The term “lactate dehydrogenase activity” as used herein refers to the activity of an enzyme catalyzing the oxidation of lactate with an electron acceptor different from dioxygen forming an oxidized lactate molecule e.g., pyruvate, and the respective reduced electron acceptor as products. Such a reaction is e.g., performed by a lactate dehydrogenase (LDH) or by a flavocytochrome b2 (FCb2).

[0260] According to a specific embodiment of the invention, if an enzyme having lactate dehydrogenase activity is used as enzyme A, a molecule different from dioxygen is used as electron acceptor for the re-oxidation of the enzyme. In even more specific embodiments, a system for regeneration of the used electron acceptor may be implemented in the method described herein.

[0261] According to a specific embodiment, a system for regeneration of an electron acceptor may comprise an enzymatic, chemical, electrochemical, homogeneous catalytic, photocatalytic, or heterogeneous catalytic regeneration system.

[0262] The term “lactate dehydrogenase” is abbreviated herein as LDH. In general, a LDH is an enzyme catalyzing the oxidation of lactate to pyruvate whereby two electrons are transferred from lactate to the cofactor of the LDH e.g., FMN or FAD, and the so gained electrons are subsequently transported towards a suitable electron acceptor like DCIP. Thereby, the LDH is commonly irresponsive or almost irresponsive to accepting dioxygen as an electron acceptor.

[0263] According to one embodiment, a LDH is used in the method described herein as enzyme A. Specifically, various LDHs may be used in the method described herein such as but not limited to NAD+ dependent LDH e.g., from Sus scrofa, Homo sapiens, Mus musculus, Rattus norvegicus, Lactobacillus easel, Geobacillus stearothermophilus, Lactiplantibacillus pentosus, Deinococcus radiodurans, Thermus caldophilus, Thermotoga maritima, Bacillus subtilis, and Thermus thermophilus; or FAD- or FM Independent LDH e.g., from Pediococcus acidilactici. Specifically, the LDH may be a functional variant of any one of the foregoing LDHs having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99 % sequence identity with the respective amino acid sequence of the foregoing LDHs.

[0264] According to a specific embodiment of the invention, enzyme A may be an engineered variant of an enzyme. For example, an engineered variant of a dehydrogenase e.g., a lactate dehydrogenase, engineered towards using lactate as substrate while not using e.g., 2-HBA as substrate by decreasing the enzyme activity with 2-HBA.

[0265] According to an alternative embodiment of the invention, enzyme A may be an engineered variant of an enzyme which is naturally not capable of oxidizing lactate. For example, a dehydrogenase naturally oxidizing a different molecule than lactate may be engineered towards using lactate as substrate and thus, an engineered variant may also be an enzyme having lactate dehydrogenase activity.

[0266] According to a specific embodiment, the LDH used as enzyme A is characterized by its enantiomer selectivity and specificity towards the natural lactate substrate L-lactate.

[0267] In a specific embodiment, the LDH described herein is a functionally active variant of a LDH peptide sequence and comprises one or more point mutations in the nucleotide sequence encoding the LDH sequence, compared to the respective parent LDH sequence. Specifically, it comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20, point mutations, specifically resulting in one or more amino acid substitutions, additions or deletions, or the like. Specifically, the functional variant of the LDH peptide sequence is a full-length LDH peptide sequence comprising point mutations, or it is a fragment of the full-length LDH peptide sequence with retained enzymatic activity. Specifically, depending on the use of the LDH as described elsewhere herein as enzyme A or as enzyme B, a variant of a LDH sequence is functionally active, if it is capable of converting L-lactate to pyruvate or capable of converting a 2-HBA to the respective oxidized 2-HBA. Specifically, a functionally active variant of a LDH sequence has at least 10, 20, 30, 40, 50, 60, 70, or even more % of the enzymatic activity of the corresponding parent LDH sequence with lactate or 2-HBA. Specifically, a functionally active variant of a LDH sequence has at least 10, 20, 30, 40, 50, 60, 70, or even more % of the enzymatic activity of the corresponding parent LDH sequence, wherein said enzymatic activity is determined with the following assays.

[0268] The lactate dehydrogenase activity of an enzyme or the enzymatic activity of a LDH or a variant thereof can be determined by a DCIP assay assessing the enzymatic activity from the colorimetric reduction of 2,6-dichlorophenol-indophenol sodium salt hydrate (DCIP) at 30 °C and 520 nm or alternatively at 600 nm (molar extinction coefficient £520 nm= 6.8 mM-1 cm-1 ; molar extinction coefficient £6oo nm= 8.98 mM-1 cm-1), e.g. as previously described (W.J. Bao, S.N. et al. (1993), Krondorfer, I., et al. (2014), Harreither, W. et al. (2011)). The assay mixture is buffered at pH 7.4 with 11 mM potassium phosphate, 137 mM NaCI, 3 mM KCI and contains 10 mM lactate and 120 pM DCIP, which acts as an electron acceptor. One unit of enzymatic activity is defined as the amount of enzyme that oxidizes 1 pmol of lactate per min under the assay conditions. The reaction stoichiometry of lactate: DCIP is 1 : 1 , since two electrons are gained per lactate molecule and transferred to a single DCIP molecule. For the detection of activity with other substrates, lactate can be exchanged for other compounds. For the detection of the 2-HBA dehydrogenase activity, the lactate is exchanged for this specific 2-HBA. For example, if the 2-HBA dehydrogenase activity of an enzyme is measured, the lactate is exchanged for 2-HBA.

[0269] The enzymatic activity of LDH variants can also be determined by assessing the colorimetric reduction of 500 pM 1 ,4-benzoquinone (1-4-BQ) (molar extinction coefficient £290 nm = 2.24 mM‘1cm"1) or 160 pM ferrocenium hexafluorophosphate (FcPFe) (molar extinction coefficient £300 nm = 4.3 mM-1cm-1). The assay mixture is formulated as is described for the DCIP assay but contains 500 pM 1 ,4-benzoquinone or 160 pM ferrocenium hexafluorophosphate instead of DCIP. The reaction stoichiometry of lactate: 1 ,4-benzoquinone is 1 : 1 , since two electrons are gained per lactate molecule and transferred to a single molecule of 1 ,4-benzoquinone. The reaction stoichiometry of lactate : ferrocenium hexafluorophosphate is 1 : 2, since two electrons are gained per lactate molecule and transferred individually to two molecules ferrocenium hexafluorophosphate. For the detection of activity with other substrates, lactate can be exchanged for other compounds (Brugger, D, et al. (2014), Sygmund, C. et al. (2011)).

[0270] The determination of the enzymatic activity of a LDH or a variant thereof can be also determined for oxygen as electron acceptor. Thereby, specifically the oxidase activity of a LDH is measured. The oxidase activity might not be detectable if the capability of the LDH to transfer electrons to oxygen is very low. As an example of a suitable method, the Amplex Red assay can be used. Thereby, oxidase activity is measured using a peroxidase-coupled reaction containing 7.1 U / mL horseradish peroxidase (181 U / mg; Sigma) and 0.05 mM AmplexRed (resorufin: £560 nm = 54.0 mM"1cm-1). Oxygen is present at ambient concentrations of ~250 pM (Kadowaki, M. A. S. et al. (2020)).

[0271] According to a specific embodiment, the enzyme having lactate oxidizing activity such as e.g., lactate oxidase activity or lactate dehydrogenase activity, used as enzyme A in the method described herein does not substantially alter the concentration or amount of the 2-HBA. For example, the enzyme having lactate oxidase or dehydrogenase activity used as enzyme A does not have or does substantially not have 2-HBA oxidase or 2-HBA dehydrogenase activity.

[0272] According to a specific embodiment, enzyme A has a specific activity with the 2- HBA of less than 10, 5, 4, 3, 2, 1 % relative to the specific activity of said enzyme with lactate. Specifically, enzyme A has a specific activity with the 2-HBA of 0.0, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 % relative to the specific activity of said enzyme with lactate.

[0273] According to a specific embodiment, the sample is treated for at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 minutes or even longer with the enzyme A.

[0274] According to a specific embodiment, the sample is treated at room temperature with the enzyme A.

[0275] According to a specific embodiment, the sample is treated at 10, 15, 20, 25, 30, 35, or 40 °C with the enzyme A.

[0276] According to a specific embodiment, the enzyme A is added to a final concentration in the sample of at least 0.01 mg / mL. Specifically, the enzyme A is added to a final concentration in the sample of 0.01 to 0.1 mg mL-1or even higher.

[0277] According to a specific embodiment, the duration of selectively removing lactate (pretreatment step, step i.) depends on the specific activity of the enzyme with lactate and on the final concentration of the enzyme in the sample. Thus, the duration of the pretreatment step may be adapted depending on the specific enzyme and on the specific concentration of enzyme in the sample.

[0278] According to a specific embodiment, the enzyme A and optionally the catalase may be inactivated after selectively removing lactate and prior to incubation with the enzyme B. For example, the sample may be heated up to 100 °C for 5, 10, 15, or 20 minutes for inactivating the enzyme(s).

[0279] According to a specific embodiment, the determination of the 2-HBA as described herein may be corrected by a pre-determined factor. Such a pre-determined factor may be derived from the side-reactivity of enzyme A with the specific 2-HBA.

[0280] According to a specific embodiment, enzyme A does not substantially change the concentration of 2-HBAJn the sample. Thereby, the degree of this substantially change of the concentration of 2-HBA may be predetermined and a correction factor for the determination of 2-HBA may be determined.

[0281] According to one embodiment of the invention, hydrogen peroxide may be produced as a by-product in the step of selectively removing lactate from the sample. Therefore, an agent capable of removing hydrogen peroxide may be added in the method described herein. Such an agent may be added in step i. of the method described herein i.e. , in the step of selectively removing lactate from the sample. Alternatively, such an agent may be added after step i. but before step ii., or simultaneously to step ii.

[0282] According to a specific embodiment, the agent capable of removing hydrogen peroxide is an enzyme having catalase activity. Catalase activity refers to the activity of an enzyme catalyzing the reaction of decomposing hydrogen peroxide to water and molecular oxygen. Non-limiting examples of such enzymes are catalases, e.g., catalase from Aspergillus niger, bovine liver, human erythrocytes, and any other known catalase.

[0283] The catalase activity of an enzyme or the enzymatic activity of a catalase or a variant thereof can be determined spectrophotometrically by continuous spectrophotometric rate reduction determination at 240 nm. Thereby, the rate of disappearance of H2O2 is followed by observing the rate of decrease in the absorbance at 240 nm. One unit of catalase will decompose 1.0 pmole of H2O2 per minute at pH 7.0 at 25 °C, while the H2O2 concentration falls from 10.3 mM to 9.2 mM. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase is described by Beers, R. F. Jr, & Sizer, I. W. (1952). According to a specific embodiment, catalase is added to the sample in step i.. Thereby, catalase decomposes the hydrogen peroxide produced by the lactate oxidase to water and molecular oxygen. The produced molecular oxygen may again serve as electron acceptor for the lactate oxidase.

[0284] According to one embodiment, catalase is used to boost treatment and / or detection reactions with lactate oxidase.

[0285] According to one embodiment of the invention, enzyme B is an enzyme having 2- HBA oxidizing activity. Enzyme B may be an enzyme having 2-HBA dehydrogenase activity or an enzyme having 2-HBA oxidase activity.

[0286] The term “2-HBA oxidizing activity” as used herein refers to the activity of an enzyme catalyzing the oxidation of a 2-HBA. Alternatively, the term “hydroxy acid oxidizing activity” may be used as the enzyme may also catalyze the oxidation of lactate as described elsewhere herein.

[0287] The term “2-HBA dehydrogenase activity” as used herein refers to the activity of an enzyme catalyzing the oxidation of a 2-HBA with an electron acceptor different from dioxygen and thereby forming an oxidized hydroxy acid and the respective reduced electron acceptor as products. Such enzymes having dehydrogenase activity are commonly being irresponsive or almost irresponsive to accepting dioxygen as an electron acceptor. Alternatively, the term “hydroxy acid dehydrogenase activity” may be used as the enzyme may also catalyze the oxidation of lactate as described elsewhere herein.

[0288] In general, the enzyme having hydroxy acid dehydrogenase activity or hydroxy acid oxidase activity may also have the respective lactate dehydrogenase or lactate oxidase activity. Since the lactate present in the sample is selectively removed from the sample in step i., there is no lactate present anymore in step ii. and thus, the enzymatic reaction of step ii. is not disturbed by lactate irrespective if the used enzyme has the theoretical capability of oxidizing lactate.

[0289] According to one embodiment of the invention, the enzyme having 2-HBA dehydrogenase activity is an enzyme having 2-HBA dehydrogenase activity. For example, flavocytochrome b2 (FCb2) and lactate dehydrogenase (LDH) are enzyme having 2-HBA dehydrogenase activity.

[0290] According to one embodiment, the enzyme having 2-HBA dehydrogenase activity is flavocytochrome b2 (FCb2) or lactate dehydrogenase (LDH). According to another embodiment of the invention, such an enzyme having hydroxy acid dehydrogenase activity may also be a functionally active variant of e.g., FCb2 or LDH.

[0291] The term “lactate dehydrogenase” (LDH) is described elsewhere herein as an enzyme catalyzing the oxidation of lactate to pyruvate whereby two electrons are transferred from lactate to the cofactor of the LDH e.g., FMN and the subsequent transport of the so gained electrons towards a suitable electron acceptor like DCIP whilst commonly being irresponsive or almost irresponsive to accepting dioxygen as an electron acceptor. However, in the herein described invention, if the term “LDH” or “lactate dehydrogenase” is used in the context of enzyme B, it is referred to an enzyme catalyzing the oxidation of the 2-HBA, to its corresponding oxidized form whereby two electrons are transferred from the 2-HBA to the cofactor of the LDH e.g., FMN and the subsequent transport of the so gained electrons towards a suitable electron acceptor like DCIP whilst commonly being irresponsive or almost irresponsive to accepting dioxygen as an electron acceptor.

[0292] According to a specific embodiment, several different lactate dehydrogenases are known and described elsewhere herein. Thereby, the lactate dehydrogenase may be FAD-dependent or NAD+-dependent. For example, a FAD-dependent lactate dehydrogenase is the lactate dehydrogenase from Pediococcus acidilactici which was previously described as lactate oxidase (see Ashok, Y., et al., 2020).

[0293] According to one embodiment of the invention, a LDH is used in the method described herein as enzyme B.

[0294] According to a specific embodiment, a LDH is used in the method described herein as enzyme B as an enzyme capable of oxidizing 2-HBA.

[0295] According to an alternative embodiment of the invention, a FCb2 is used in the method described herein as enzyme B.

[0296] According to a specific embodiment, a FCb2 is used in the method described herein as enzyme B as an enzyme capable of oxidizing 2-HBA.

[0297] The term “FCb2” refers to an L-lactate-cytochrome c oxidoreductase (EC 1.1.2.3; flavocytochrome b2, FCb2, L-lactate cytochrome c oxidoreductase). In general, FCb2 catalyzes the electron transfer from L-lactate to cytochrome c in yeast mitochondria. In yeast, L-lactate is converted to pyruvate by L-lactate cytochrome c- oxidoreductase (EC 1.1.2.3), which is herein referred to as “Flavocytochrome b2” or “FCb2”. Native yeast flavocytochrome b2 (FCb2) has two functional domains that are connected via a “hinge” linker (57 kDa monomer). The FCb2 from S. cerevisiae is the best studied representative and has been crystallized (PDB 1 FCB). However, in the herein described invention, if the term “FCb2” or a synonym thereof is used in the context of an enzyme having the capability of oxidizing the 2-HBA_to be determined with the method described herein, these terms refer to an enzyme catalyzing the oxidation of the 2-HBA to its corresponding oxidized form whereby the FCb2 catalyzes the electron transfer from 2-HBA to cytochrome c.

[0298] According to a specific embodiment, the FCb2 described herein may comprise a sequence based on the mature form of FCb2 naturally found in the yeast mitochondrial intermembrane space, which comprises a cytochrome b2 domain, a flavin domain, a hinge region connecting the cytochrome b2 domain and the flavin domain and a tail region at its C-terminus. A mature FCb2 peptide sequence is the sequence of an FCb2 peptide as it is naturally found in the yeast mitochondrion, specifically in the mitochondrial intermembrane space.

[0299] According to a specific embodiment, the FCb2 described herein comprises a FCb2 peptide sequence comprising at least a yeast heme domain and a yeast flavin domain.

[0300] In a specific embodiment, the FCb2 described herein is a functionally active variant of a FCb2 peptide sequence found in the yeast mitochondrial intermembrane space and comprises one or more point mutations in the nucleotide sequence encoding the FCb2 sequence, compared to the respective native mature FCb2 sequence. Specifically, it comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 point mutations, specifically resulting in one or more amino acid substitutions, additions or deletions, or the like. Specifically, the functional variant of the FCb2 peptide sequence is a full-length mature FCb2 peptide sequence comprising point mutations, or it is a fragment of the full-length mature FCb2 peptide sequence with retained enzymatic activity. Specifically, a variant of a FCb2 sequence is functionally active if it is capable of converting the 2-HBA_to be determined with the method to the corresponding oxidized form. Specifically, a functionally active variant of a FCb2 sequence has at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or even more % of the enzymatic activity of the corresponding wild type FCb2 sequence with the 2-HBA as substrate. Specifically, a functionally active variant of a FCb2 sequence has at least 10, 20, 30, 40, 50, 60, 70, or even more % of the enzymatic activity of the corresponding wild type FCb2 sequence, wherein said enzymatic activity is determined with the CytC assay and the respective 2- HBA as substrate. The enzymatic activity of a flavocytochrome b2 variant can be readily determined by assays known in the art, such as assays determining the colorimetric reduction of cytochrome c, ferricyanide or 2,6-dichloroindophenol (DCIP). Specifically, the FCb2 described herein is determined by the CytC assay described by Diep Le et al. (2009) using the respective 2-HBA or alternatively lactate as substrate.

[0301] According to a specific embodiment, the FCb2 may comprise the amino acid sequence of a FCb2 from S. cerevisiae, W. anomalus, K. marxianus, O. parapolymorpha, Candida glabrata, Kluyveromyces lactis, Lachancea thermotolerans, Saccharomycodes ludwigii, Naumovozyma castelli, Zygosaccharomyces bailii, Zygosaccharomyces parabalii, Lachancea mirantina, Tetrapisispora phaffii, Saccharomyces eubayanus, Saccharomyces kudriavzevii, Saccharomyces paradoxus, Vanderwaltozyma polyspora, Lachancea dasiensis, Wickerhamomyces ciferri, Kluyveromyces dobzhanskii, Kazachstania naganishii, Zygosaccharomyces mellis, Kazachstania saulgeensis, Candida boidinii, Lachancea fermentati, Zygosaccharomyces rouxii, Cyberlindnera fabianii, Cyberlindnera jadinii, Kazachstania africana, Lachancea quebecensis, Kuraishia capsulata, Torulaspora delbrueckii, Komogatella pastoris, Komagatella phaffii, Lachancea nothofagi, or Naumovomyces dairenensis. Specifically, the FCb2 may be a functional variant of any one of the foregoing and comprise an amino acid sequence having 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99 % with the amino acid sequence of an FCb2 of any one of the foregoing.

[0302] Specifically, the recombinant FCb2 described herein comprises a peptide sequence derived from the FCb2 of Saccharomyces cerevisiae, Kluyveromyces marxianus, Wickerhamomyces anomalus, Naumovozyma castelli or Cyberlindera fabianii.

[0303] Amino acid sequences of polypeptides derived from organisms may be readily derived from publicly available databases such as e.g., from databases provided by the National Center for Biotechnology Information (NCBI).

[0304] According to another alternative embodiment, enzyme B may also be an enzyme having 2-HBA_oxidase activity.

[0305] The term “2-HBA oxidase activity” as used herein refers to the activity of an enzyme catalyzing the oxidation of a 2-HBA with dioxygen as electron acceptor forming the respective oxidized 2-HBA and hydrogen peroxide as products. Thereby, two electrons are transferred from the 2-HBA to the cofactor of the enzyme e.g., to a FAD cofactor, and the so gained electrons are subsequently transferred towards dioxygen producing hydrogen peroxide. Alternatively, the term “hydroxy acid oxidase activity” may be used as the enzyme may also catalyze the oxidation of lactate as described elsewhere herein.

[0306] According to a specific embodiment of the invention, an enzyme having 2-HBA oxidase activity may be an enzyme selected from the enzyme class EC: 1.1.3.15.

[0307] According to a specific embodiment, an enzyme having 2-HBA oxidase activity may be hydroxy acid oxidase from Mus musculus, Rattus norvegicus, Homo sapiens, Arabidopsis thaliana, or any other known enzyme having hydroxy acid oxidase activity.

[0308] According to a specific embodiment, the enzymes used in the method described herein may be active at acidic, neutral, or alkaline pH ranges. Specifically, the enzymes of the invention may be used at a pH of 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or at any pH of a body fluid e.g., of sweat or blood. Usually, blood has a pH between 7.35 and 7.45.

[0309] According to a specific embodiment, in the method described herein a lactate dehydrogenase-based detection system (flavocytochrome b2, Fcb2) is applied in combination with a lactate oxidase (LOx) dependent lactate removal. Specifically, the enzymes are characterized either by high turnover rates with the analyte of interest (2- HBA) or high specificity to the otherwise interfering substances such as lactate.

[0310] According to one embodiment, the combination of an oxygen-dependent enzyme and an oxygen-independent enzyme for unaffected detection or unaffected colorimetric reaction is described herein. For example, the colorimetric reaction with FCb2 is unaffected by lactate removal with LOx.

[0311] According to another embodiment, the method described herein may comprise the combination of an engineered lactate oxidase variant with specificity for L-lactate and an engineered flavocytochrome b2 variant with improved reactivity towards 2-HBA. Thereby, the lactate oxidase variant is used in a pre-treatment step to specifically oxidize L-lactate. The improved FCb2 variant is used to oxidize 2-HBA and colorize a reagent concomitantly to translate 2-HBA concentration into a quantifiable signal (e.g., absorbance, fluorescence).

[0312] The term “incubating” as used herein refers to contacting the sample with an enzyme to allow the enzyme to react with the 2-HBA. Thereby, incubating may be performed directly in the sample e.g., after adding the enzyme. Alternatively, an aliquot of the sample may be taken from the sample and mixed with a suitable liquid e.g., a buffer, prior to adding the enzyme. Depending on the specific determination method, such a suitable liquid may also comprise other compounds such as e.g., a compound necessary for the colorimetric detection or an electron acceptor.

[0313] According to one embodiment of the invention, the step of incubating the sample with an enzyme having 2-HBA oxidizing activity may be sequentially or simultaneously performed with the step of determining the 2-HBA in the sample.

[0314] According to one embodiment of the invention, the enzyme having 2-HBA oxidizing activity may be any enzyme capable of oxidizing the 2-HBA and transferring the so gained electrons to a suitable electron acceptor. Such an electron acceptor may be any suitable molecule which can be used for the colorimetric, photometric, fluorimetric, through phosphorescence, through chemiluminescence, or electrochemical detection of the electron transfer reaction. Thereby, the signal obtained from detecting the electron transfer reaction is used for the determination of the 2-HBAJn the sample.

[0315] According to an alternative embodiment, if an enzyme having hydroxy acid oxidase activity is used, such an electron acceptor may also be dioxygen. In this case, hydrogen peroxide is produced through the electron transfer from 2-HBA to dioxygen. Hydrogen peroxide can also be detected by suitable methods such as e.g., by colorimetric, photometric, fluorimetric, through phosphorescence, through chemiluminescence, bioelectrochemical, electrochemical methods, possibly using enzyme-based assays. Thereby, the presence or amount of 2-HBA can be calculated from the detection of hydrogen peroxide.

[0316] According to yet another alternative embodiment of the invention, the electron acceptor used in the oxidation reaction of the 2-HBA may also be a polypeptide. Such a polypeptide may be naturally attached to the enzyme capable of oxidizing the 2-HBA or synthetically attached to said enzyme. For example, the enzyme FCb2 comprises a heme domain which accepts electrons from the cofactor of the catalytically active domain of the FCb2 i.e. , flavin domain, capable of oxidizing the 2-HBA. Furthermore, a polypeptide used as electron acceptor may transfer the so gained electrons to a terminal electron acceptor. Examples of such a terminal electron acceptor are molecules such as cytochrome c, ferricyanide or 2,6-dichloroindophenol (DCIP) which can be used in colorimetric and photometric detection methods. Another example of such a terminal electron acceptor is an electrode surface. The presence or amount of 2-HBA in the sample can be derived from a signal obtained by the specific detection method. According to one embodiment of the invention, determining the 2-HBA is performed colorimetrically, photometrically, fluorimetrically, through phosphorescence, through chemiluminescence, or electrochemically.

[0317] The term “colorimetrically” as used herein refers to the application of a colorimetric analysis for the determination of the presence or the amount of 2-HBA in the sample. In a colorimetric analysis the concentration of a chemical element or chemical compound in a solution is determined with the aid of a color reagent. The color reagent may be visually detected or detected with suitable equipment e.g., a colorimeter. In enzymatic analysis as described herein, the color reaction is preceded by a reaction catalyzed by an enzyme such as the enzyme capable of oxidizing a 2-HBA. A general example of such a colorimetric determination is the detection of a colored complex formed by a peroxidase from hydrogen peroxide and ABTS.

[0318] The term “photometrically” as used herein refers to the usage of a photometer or a spectrophotometer for enzymatically determining a substance in a sample, or alternatively also for determining the enzyme activity, by following the course of an enzyme reaction by measuring the changes in the intensity of the light absorbed or scattered by the reaction solution. A colorimetric detection may be coupled or performed by a photometer.

[0319] The term “fluorimetrically” as used herein refers to the detection of fluorescence by determining the difference in the fluorescence spectra of substrate from product to measure the enzyme reaction or determine the substrate or product concentration. Thereby, the substrate and product may be different from the direct substrates and products of enzymes described herein e.g., different from 2-HBA and oxidized 2-HBA. The Amplex red assay which is elsewhere described herein is an example of an assay based on a fluorimetric detection method.

[0320] The term “electrochemically” as used herein refers to the usage of an electrochemical biosensor based on the measurement of biological binding eventdependent changes in conductance, resistance, or capacitance of the biosensor surface. In such an electrochemical biosensor, one of the electrodes is immobilized with a biological recognition molecule. The binding of the analyte to the biological recognition molecule triggers a change in the electrical properties due to oxidation and reduction reactions taking place as a result of biological interaction activity, thus providing the sensor signal. Electrochemical biosensors rely mostly on enzyme-catalyzed reactions to produce current / potential difference which is then detected. According to one embodiment of the invention, the electron acceptor used in the oxidation reaction of the 2-HBA_for the determination of the amount of 2-HBA in the sample may also be an electrode equipped with an enzyme capable of oxidizing the 2- HBA. Such an electrode may be part of a biosensor.

[0321] According to one embodiment of the invention, detecting the oxidation of 2-HBA by the enzyme capable of oxidizing the 2-HBA may be carried out by a sensor, specifically a bio-electrochemical sensor, configured to detect and / or quantify 2-HBAJn a sample via (bio)electrochemical redox reactions. These reactions typically can be transduced to an electrical signal that can be correlated to an amount or concentration of the analyte 2- HBA.

[0322] “Electrochemical biosensors” can be impedimetric, potentiometric, or amperometric. In an amperometric biosensor, a biochemical signal is transduced into a quantifiable amperometric signal.

[0323] As described in Rocchitta G. et al. (2016) amperometric biosensors are commonly divided into three main generations depending on the electron transfer method used for the measurement of the biochemical reaction or the degree of separation of the biosensor components (transducer, enzyme, mediators, and cofactors). First-generation biosensors measure the concentration of analytes and / or products of enzymatic reactions that diffuse to the transducer surface and generate an electrical response. They are also called mediatorless amperometric biosensors. Commonly, oxidases are used in first-generation biosensors. Oxidases need molecular oxygen as a second substrate so the oxidase- based biosensors are oxygen dependent. Second-generation biosensors require an electron mediator for the transfer of electrons obtained from enzymatic reactions to the transducer surface and thereby generate an electrical response. In third-generation biosensors, direct electron transfer is enabled between the redox-active biomolecule i.e. , the enzyme, and the electrode surface.

[0324] According to one embodiment of the invention, for the determination of 2-HBA by electrochemical means, an electrode comprising an enzyme capable of oxidizing the 2- HBAJs contacted with the sample. This contact between electrode and sample can be performed by any approach which brings the electrode and the sample in contact in order that the enzyme is allowed to react with the 2-HBA or with the sample suspected to contain 2-HBA.

[0325] The term “electrode” refers to any suitable surface for accepting electrons from the enzyme via mediatorless, mediated, or direct electron transfer. Thereby the electrode is of a material capable of accepting electrons. Furthermore, the electrode may be of any material suitable or modified with any material to adsorb or immobilize the enzyme capable of oxidizing the 2-HBA. Non-limiting examples of such a material are platinum, gold, boron doped diamond and carbons such as graphite, pyrolytic graphite and glassy carbon where all of them can additionally be modified with carbon nanotubes (single or multi-walled), carbon fibers, nanoparticles e.g. gold nanoparticles or promoters as e.g., thiols. The electrode may be also of any material to increase the specific surface are of the electrode.

[0326] According to one embodiment of the invention, the electrode may be used as single electrode or as a stack of electrodes of e.g., 2, 3, 4, 5, or more electrodes.

[0327] According to one embodiment of the invention, the electrode provided herein is a working electrode.

[0328] According to one embodiment, the electrode comprising the enzyme capable of oxidizing the 2-HBA of the invention enables the detection and / or quantification of 2-HBA based on mediatorless, mediated, or direct electron transfer.

[0329] Mediatorless electron transfer typically employs the transfer of electrons from hydrogen peroxide, produced through the enzymatic reaction, to the electrode.

[0330] Mediated electron transfer in biosensors typically employs a two-step procedure in which the enzyme takes part in a first redox reaction with the substrate and is in turn reoxidized by a redox mediator. Finally, the redox mediator is oxidized by the electrode.

[0331] Redox mediators are artificial electron transferring agents that can readily participate in the redox reaction with the biological component and thus help in rapid electron transfer to the electrode. A “redox mediator” is an electron-transfer agent for carrying electrons between an analyte, an analyte-reduced or analyte-oxidized enzyme, and an electrode, either directly, or via one or more additional electron-transfer agents. A redox mediator that includes a polymeric backbone may also be referred to as a redox polymer.

[0332] Specifically, said redox polymer may comprise a transition metal complex, preferably an osmium-containing complex.

[0333] According to one embodiment of the invention, the oxidation of 2-HBA is performed in the presence of a redox mediator. The method of the invention can also be performed in the presence of more than one redox mediator e.g., in the presence of two or more different redox mediators. Thereby, the redox mediator may be present on the electrode, in an enzyme composition comprising the enzyme and the redox mediator, or may be present in the sample.

[0334] According to one embodiment, the redox mediator may be any molecule or material able to carry electrons between the enzyme capable of oxidizing the 2-HBA and electrode. Specifically said redox mediator is selected from the group consisting of any one of organic redox mediators, soluble redox mediators, insoluble redox mediators, redox polymers, transition metal complexes, polymeric transition metal complexes, wired redox mediators, sandwich compounds, and derivatives of these redox mediators.

[0335] Polymeric transition metal complexes comprise a polymeric backbone, spacers, and transition metal complexes.

[0336] Specifically, redox polymers are polymers comprising redox species. Non-limiting examples of such redox species used in redox polymers are osmium (Os), ruthenium (Ru), iron (Fe), cobalt (Co), or any transition metal. Non-limiting examples of polymers used for redox polymers are poly(vinylpyridine), poly(thiophene), poly(aniline), poly(pyrrole), or poly(acetylene). An example of a redox polymer is Os-containing poly(vinylpyridine).

[0337] According to a specific embodiment, in the case the redox mediator comprises osmium the redox mediator may be an osmium transition metal complex with one or more ligands, each ligand having a nitrogen-containing heterocycle such as 2,2'-bipyridine, 1 ,10-phenanthroline, 1 -methyl, 2-pyridyl biimidazole, or derivatives thereof. The redox mediator may also have one or more ligands covalently bound in a polymer, each ligand having at least one nitrogen-containing heterocycle, such as pyridine, imidazole, or derivatives thereof. One example of an electron transfer agent includes (a) a polymer or copolymer having pyridine or imidazole functional groups and (b) osmium cations complexed with two ligands, each ligand containing 2,2' -bipyridine, 1 ,10-phenanthroline, or derivatives thereof, the two ligands not necessarily being the same. Some derivatives of 2,2' -bipyridine for complexation with the osmium cation include but are not limited to 4,4'-dimethyl-2,2'- bipyridine and mono-, di-, and polyalkoxy-2, 2'-bipyri dines, including 4,4'-dimethoxy-2,2'- bipyridine. Derivatives of 1 , 10-phenanthroline for complexation with the osmium cation include but are not limited to 4,7-dimethyl- 1 , 10-phenanthroline and mono, di-, and polyalkoxy-1 , 10- phenanthrolines, such as 4,7-dimethoxy- 1 ,10- phenanthroline. Polymers for complexation with the osmium cation include but are not limited to polymers and copolymers of poly(1 -vinyl imidazole) and poly(4-vinyl pyridine). Suitable copolymer substituents of poly(1 -vinyl imidazole) include acrylonitrile, acrylamide, and substituted or quaternized N-vinyl imidazole, e.g., electron transfer agents with osmium complexed to a polymer or copolymer of poly(1 -vinyl imidazole). An example of a redox polymer is also derived from poly(1 -vinylimidazole) or a copolymer of (1 -vinyl imidazole) bound to a metal ion selected from the group consisting of Os.sup.3+ / 2+, Ru.sup.3+ / 2+, and Fe.sup.3+ / 2+.

[0338] The term “transition metal” refers to an element whose atom has a partially filled d sub-shell, or which can give rise to cations with an incomplete d sub-shell. Thereby, transition metals are elements in the d-block of the periodic table and also lanthanides and actinides.

[0339] Non-limiting examples of transition metal complexes include complexes comprising titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, hafnium, tantalum, tungsten, rhenium, osmium, iridium, and platinum. Specific examples of transition metal complexes are ferricyanide, ruthenium hexamine, metalloporphyrins such as heme b or heme c. In these complexes, the transition metal is coordinatively bound to one or more ligands, which are typically mono-, di-, tri-, or tetradentate.

[0340] Non-limiting examples of transition metal complexes include complexes comprising lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, and lutetium.

[0341] Non-limiting examples of transition metal complexes include complexes comprising actinium, thorium, protactinium, uranium, neptunium, plutonium, americium, curium, berkelium, californium, einsteinium, fermium, mendelevium, nobelium, and lawrencium.

[0342] A sandwich compound is a chemical compound featuring a metal bound by haptic covalent bonds to two arene ligands. The arenes have the formula CnHn, substituted derivatives (for example Cn(CH3)n) and heterocyclic derivatives (for example BCnHn+i). A special class of sandwich complexes are metallocenes. A metallocene contains a transition metal and two cyclopentadienyl ligands coordinated in a sandwich structure, i.e. , the two cyclopentadienyl anions are on parallel planes with equal bond lengths and strengths. Non-limiting examples of sandwich compounds and metallocenes are ferrocene, 1 ,1'-dimethyl ferrocene [DMF], and ferrocene monocarboxylic acid.

[0343] Organic redox compounds are organic molecules capable to act as a redox mediator. Non-limiting examples of organic redox mediators are organic molecules such as quinones, compounds having a quinoid structure such as benzoquinones or phenanthroline quinones, phenazine such as 1 -methoxyphenazine methosulfate, tetracyanoquinodimethane (TCNQ), N,N,N', N'-tetramethyl-p-phenylenediamine (TMPD), DCIP, tetrathiafulvalene (TTF), and derivative of these molecules.

[0344] According to one embodiment, a direct electron transfer biosensor may be used for the determination of 2-HBA, wherein the biosensor comprises an electrode comprising an enzyme capable of oxidizing the 2-HBA and said enzyme is able to transfer the electrons gained from the oxidation of the 2-HBA directly to the electrode surface. An example of such an enzyme capable of oxidizing the 2-HBA_enabling direct electron transfer is FCb2.

[0345] According to one embodiment of the invention, the enzyme capable of oxidizing the 2-HBA is immobilized on the electrode by adsorption, physical entrapment in a polymer, complex formation, preferably via an additional complexing linker, covalent binding, in particular cross-linking, or ionic binding and / or the immobilized enzyme can be cross-linked, in particular by bifunctional agents, to increase stability or activity. Crosslinking agents are e.g., dialdehydes such as glutaraldehyde.

[0346] According to one embodiment of the invention, the electrode of the invention is part of a biosensor. Thereby, a specific use of the electrodes of the invention is in the provision of a biosensor, more specifically a first, second, or third-generation 2-HBA biosensor using mediatorless, mediated, or direct electron transfer properties to detect 2- HBA and / or to measure the 2-HBA concentration. The biosensor may be suitable for use at acidic, neutral, or alkaline pH. The biosensor may be suitable for use at room temperature or at body temperature. Specifically, the biosensor may be suitable for the detection and / or quantification at 4 °C, 10 °C, 15 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C,

[0347] 38 °C, 39 °C, 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, 45 °C or higher.

[0348] According to another embodiment, the biosensor may have one or more electrodes comprising the enzyme capable of oxidizing the 2-HBA. In further embodiments, the 2- HBA.biosensor includes: a working electrode comprising a conductive material, wherein the enzyme capable of oxidizing the 2-HBAJs in proximity to the conductive material. One or more other electrodes may be included such as one or more counter electrodes, one or more reference electrodes and / or one or more counter / reference electrodes.

[0349] The particular configuration of the biosensor may depend on the use for which the biosensor is intended and the conditions under which it will operate. In a specific embodiment of the present invention, the biosensor may be a single use biosensor for the detection of 2-HBA. Thereby, the biosensor may be a biosensor strip.

[0350] According to another embodiment of the invention, a kit for determining, e.g., detecting and / or quantifying. 2-HBA in a sample is provided comprising an enzyme capable of selectively removing lactate from the sample and an enzyme capable of oxidizing the 2-HBA. These enzymes are described elsewhere herein.

[0351] According to one embodiment, a kit is provided for determining 2-HBA in a sample.

[0352] According to one embodiment, the kit may further comprise an agent for selectively removing hydrogen peroxide, specifically an enzyme, specifically a catalase.

[0353] According to one embodiment, in the kit described herein, the enzyme capable of oxidizing the 2-HBA may be part of an electrode. Specifically, the electrode may be part of a biosensor.

[0354] According to one embodiment, the kit described herein further comprises an instruction manual.

[0355] According to one embodiment, the kit may also comprise auxiliary substances, like buffers, molecules necessary for detection e.g., electron acceptors, and containers such as a sample holding means, and / or 2-HBA standards. 2-HBA standards may be used to calibrate the assay. The kit may also comprise a reader for a signal, especially an electrochemical signal such as a potentiostat, a computer readable memory device with software for calibration and / or measurement calculations.

[0356] According to one embodiment of the present invention, the enzymes described herein may be recombinantly expressed by methods commonly known in the art. For example, the enzymes described herein may be expressed using standard methods for cloning, transformation, and recombinant production in suitable host organisms e.g., in Escherichia coli or in Pichia pastoris.

[0357] The terms “increase in activity”, “increased activity”, or the like used herein may refer to a detectable increase in activity of an enzyme. The terms “increase in activity”, or “increased activity” used herein may mean that a modified enzyme (variant) shows higher activity than a comparable enzyme of the same type, like an enzyme that does not have the particular modification. As another example, the modified enzyme may comprise sequence alterations in the polypeptide or the nucleotide sequence encoding the enzyme. For example, activity of a modified or engineered enzyme may be higher than activity of a non-engineered enzyme of the same type, for example, a wild-type enzyme by about 5 % or more, about 10 % or more, about 15 % or more, about 20 % or more, about 30 % or more, about 50 % or more, about 60 % or more, about 70 % or more, or about 100 % or more. The activity of a particular protein or enzyme in a recombinant or engineered cell may be higher than the activity of a protein or enzyme of the same type in a parent cell, for example, a non-engineered cell by about 5 % or more, about 10 % or more, about 15 % or more, about 20 % or more, about 30 % or more, about 50 % or more, about 60 % or more, about 70 % or more, or about 100 % or more. Increased activity of an enzyme or protein in a cell may be verified by any methods known in the art. Similarly, the term “decrease in activity”, “decreased activity”, or the like used herein may refer to a detectable decrease in activity of an enzyme.

[0358] An increase or decrease in activity may also target only a specific substrate of interest. For example, an enzyme used herein may have a decreased activity with 2-HBA as a substrate but a substantially non-altered or even increased activity with lactate.

[0359] The term “functional variant” or “functionally active variant” also includes naturally occurring allelic variants, as well as mutants or any other non-naturally occurring variants. As is known in the art, an allelic variant, or also referred to as homologue, is an alternate form of a nucleic acid or peptide that is characterized as having a substitution, deletion, or addition of one or more nucleotides or amino acids that does essentially not alter the biological function of the nucleic acid or polypeptide. Specifically, a functional variant may comprise a substitution, deletion and / or addition of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues, or a combination thereof. Specifically, substitutions, deletions and / or additions may be conservative modifications. Specifically, substitutions, deletions and / or additions do not decrease the enzyme’s specific activity. Specifically, a functionally active variant of the enzyme capable of oxidizing 2-HBA_as described herein comprises specific enzymatic activity towards a 2- HBA of at least 1 U / mg, as determined by the respective assay as described herein.

[0360] Specifically, a functional variant as described herein comprises no more than or up to 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25,26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acid substitutions, deletions and / or additions. Specifically, these modifications may be conservative modifications. Specifically, these modifications do not decrease the enzyme’s specific activity. Specifically, a functionally active variant as described herein comprises up to 15, preferably up to 10 or 5, amino acid substitutions, deletions and / or additions. Specifically, these modifications may be conservative modifications. Specifically, these modifications do not decrease the enzyme’s specific activity.

[0361] Specifically, a functionally active variant described herein comprises at least 40, 50, 60, 70, 80 or 90 % or even more of the enzymatic activity of the respective wild type enzyme.

[0362] Functional variants may be obtained by sequence alterations in the polypeptide or the nucleotide sequence e.g., by one or more point mutations, wherein the sequence alterations retain or improve a feature of the enzyme, such as its stability or activity for example. Such sequence alterations can include, but are not limited to, (conservative) substitutions, additions, deletions, mutations and insertions. Conservative substitutions are those that take place within a family of amino acids that are related in their side chains and chemical properties. Examples of such families are amino acids with basic side chains, with acidic side chains, with non-polar aliphatic side chains, with non-polar aromatic side chains, with uncharged polar side chains, with small side chains, with large side chains etc.

[0363] A point mutation is particularly understood as the engineering of a polynucleotide that results in the expression of an amino acid sequence that differs from the nonengineered amino acid sequence in the substitution, or exchange, deletion, or insertion of one or more single (non-consecutive) or doublets of amino acids for different amino acids.

[0364] According to a specific embodiment, the enzymes described herein may comprise one or more tag sequences, specifically N-terminal tag sequences. Specifically, such tag sequence is C-terminal of the N-terminal methionine of the enzymes described herein. Such tag sequence may comprise any number of amino acids of more than 2, 4, 5, 6 or 10 amino acids and up to 20 or 50 or more amino acids. Specifically, tag sequences used herein may be any tag sequence known to the person skilled in the art. Specifically, tag sequences used herein are selected from affinity tags, solubility enhancement tags or monitoring tags.

[0365] Affinity tags are amino acid sequences that can be used for example for the purification of proteins where they are attached to. These affinity tags have high affinity to appropriate ligands of a solid support, like chromatography resins or directly to the resins. By selectively binding of the protein having the affinity tag to the particular resin the protein can be purified highly effective by only one chromatography step. According to a specific embodiment, affinity tag sequences used herein are selected from histidine (His) tag, specifically a poly-histidine tag, poly-arginine tag, FLAG tag, Strep tag, streptavidin-binding peptide (SBP) tag, calmodulin-binding peptide (CBP) tag, S-tag, HA tag, c-Myc tag, and SUMO tag, or any other tag known to be useful for the efficient purification of a protein it is fused to. Preferably, the tag is a His tag comprising one or more H, specifically a hexahistidine tag. Specifically, proteins comprising a poly-, or hexahistidine tag (His-tag) can be captured and purified using chromatography, e.g. by Immobilized Metal Affinity Chromatography (IMAC).

[0366] Solubility enhancement tags can be fused N-terminal to the enzymes described herein. Solubility enhancement tags can increase the titer of the soluble protein when expressed in a host cell, e.g. in the cytosol of P. pastoris, compared to expression of the proteins without the tag. According to a further specific embodiment, solubility enhancement tag sequences used herein are selected from calmodulin-binding peptide (CBP), poly Arg, poly Lys, protein D tag (dTAG), Z domain of Staphylococcal protein A, and thioredoxin or any other tag known to improve the solubility of the protein it is fused to e.g. during expression in a host cell. Specifically, the solubility enhancement tag is a T7 tag, preferably selected from the group consisting of T7A, T7A1 , T7A2, T7A3, T7A4, T7A5, T7B, T7B1 , T7B2, T7B3, T7B3, T7B4, T7B5, T7B6, T7B6, T7B7, T7B8, T7B9, T7B10, T7B11 , T7B12, T7B13, and T7C.

[0367] According to a further specific embodiment, the monitoring tag sequence used herein is m-Cherry, GFP or f-Actin or any other tag useful for detection or quantification of the recombinant enzyme during production steps including fermentation, isolation and purification by simple in situ, inline, online or atline detectors, like UV, IR, Raman, fluorescence and the like.

[0368] According to one embodiment, described herein is a lactate oxidase (LOx) comprising SEQ ID NO:4, or a LOx comprising an amino acid sequence having at least 50, 55, 60, 65, 70, 75, 85, 90, 95, 96, 97, 98, or 99 % sequence identity with SEQ ID NO:4.

[0369] According to one embodiment, described herein is a flavocytochrome b2 (FCb2) comprising SEQ ID NO:5, or a FCb2 comprising an amino acid sequence having at least 50, 55, 60, 65, 70, 75, 85, 90, 95, 96, 97, 98, or 99 % sequence identity with SEQ ID NO:5.

[0370] According to one embodiment, described herein is a lactate oxidase (LOx) comprising SEQ ID NO:1 , or a LOx comprising an amino acid sequence having at least 50, 55, 60, 65, 70, 75, 85, 90, 95, 96, 97, 98, or 99 % sequence identity with SEQ ID NO:1.

[0371] According to one embodiment, described herein is a flavocytochrome b2 (FCb2) comprising SEQ ID NO:2, or a FCb2 comprising an amino acid sequence having at least 50, 55, 60, 65, 70, 75, 85, 90, 95, 96, 97, 98, or 99 % sequence identity with SEQ ID NO:2.

[0372] According to one embodiment, described herein is a flavocytochrome b2 (FCb2) comprising SEQ ID NO:3, or a FCb2 comprising an amino acid sequence having at least 50, 55, 60, 65, 70, 75, 85, 90, 95, 96, 97, 98, or 99 % sequence identity with SEQ ID NO:3.

[0373] According to one embodiment, enzyme A is a lactate oxidase (LOx) comprising SEQ ID NO:1 , SEQ ID NO:4, or a LOx comprising an amino acid sequence having at least 50, 55, 60, 65, 70, 75, 85, 90, 95, 96, 97, 98, or 99 % sequence identity with SEQ ID NO:1 or SEQ ID NO:4.

[0374] According to one embodiment, enzyme A is a lactate oxidase (LOx) comprising SEQ ID NO:4, or a LOx comprising an amino acid sequence having at least 50, 55, 60, 65, 70, 75, 85, 90, 95, 96, 97, 98, or 99 % sequence identity with SEQ ID NO:4.

[0375] According to one embodiment, enzyme B is a flavocytochrome b2 (FCb2) comprising SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, or a FCb2 comprising an amino acid sequence having at least 50, 55, 60, 65, 70, 75, 85, 90, 95, 96, 97, 98, or 99 % sequence identity with SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:5.

[0376] According to one embodiment, enzyme B is a flavocytochrome b2 (FCb2) comprising SEQ ID NO:5.

[0377] According to one embodiment, described herein is an in vitro method of diagnosing a metabolic disorder in a subject comprising the sequential steps of: i. enzymatically determining the concentration of 2-hydroxybutyric acid (2-HBA) in a sample from said subject by using a flavocytochrome b2 (FCb2) comprising SEQ ID NO:5, or a or a FCb2 comprising an amino acid sequence having at least 50, 55, 60, 65, 70, 75, 85, 90, 95, 96, 97, 98, or 99 % sequence identity with SEQ ID NO:5; and ii. comparing said concentration of 2-HBA with a predetermined cut-off value of 25 pM 2-HBA or higher, wherein the metabolic disorder is diagnosed if said concentration of 2-HBA is equal or above the predetermined cut-off value.

[0378] According to one embodiment, described herein is an in vitro method of diagnosing gestational diabetes in a subject comprising the sequential steps of: i. enzymatically determining the concentration of 2-hydroxybutyric acid (2-HBA) in a sample from said subject by using a flavocytochrome b2 (FCb2) comprising SEQ ID NO:5, or a FCb2 comprising an amino acid sequence having at least 50, 55, 60, 65, 70, 75, 85, 90, 95, 96, 97, 98, or 99 % sequence identity with SEQ ID NO:5; and ii. comparing said concentration of 2-HBA with a predetermined cut-off value of 25 pM 2-HBA or higher, wherein the gestational diabetes is diagnosed if said concentration of 2-HBA is equal or above the predetermined cut-off value.

[0379] According to one embodiment, described herein is an in vitro method of determining the risk of having or developing a metabolic disorder in a subject comprising the sequential steps of: i. enzymatically determining the concentration of 2-HBA in a sample from said subject by using a flavocytochrome b2 (FCb2) comprising SEQ ID NO:5, or a FCb2 comprising an amino acid sequence having at least 50, 55, 60, 65, 70, 75, 85, 90, 95, 96, 97, 98, or 99 % sequence identity with SEQ ID NO:5; ii. comparing said concentration of 2-HBA with a predetermined cut-off value of 25 pM 2-HBA or higher; and iii. assigning to said subject a high risk of having or developing said metabolic disorder if said concentration of 2-HBA is equal or above the predetermined cut-off value.

[0380] According to one embodiment, described herein is an in vitro method of determining the risk of having or developing a gestational diabetes in a subject comprising the sequential steps of: i. enzymatically determining the concentration of 2-HBA in a sample from said subject by using a flavocytochrome b2 (FCb2) comprising SEQ ID NO:5, or a FCb2 comprising an amino acid sequence having at least 50, 55, 60, 65, 70, 75, 85, 90, 95, 96, 97, 98, or 99 % sequence identity with SEQ ID NO:5; ii. comparing said concentration of 2-HBA with a predetermined cut-off value of 25 pM 2-HBA or higher; and iii. assigning to said subject a high risk of having or developing said gestational diabetes if said concentration of 2-HBA is equal or above the predetermined cut-off value.

[0381] According to one embodiment, described herein is an in vitro method of monitoring a metabolic disorder in a subject comprising the sequential steps of: i. enzymatically determining the concentration of 2-HBA in a sample from said subject by using a flavocytochrome b2 (FCb2) comprising SEQ ID NO:5, or a FCb2 comprising an amino acid sequence having at least 50, 55, 60, 65, 70, 75, 85, 90, 95, 96, 97, 98, or 99 % sequence identity with SEQ ID NO:5; and ii. comparing said concentration of 2-HBA with a previous concentration of 2-HBA from said subject.

[0382] According to one embodiment, described herein is an in vitro method of monitoring a gestational diabetes in a subject comprising the sequential steps of: i. enzymatically determining the concentration of 2-HBA in a sample from said subject by using a flavocytochrome b2 (FCb2) comprising SEQ ID NO:5, or a FCb2 comprising an amino acid sequence having at least 50, 55, 60, 65, 70, 75, 85, 90, 95, 96, 97, 98, or 99 % sequence identity with SEQ ID NO:5; and ii. comparing said concentration of 2-HBA with a previous concentration of 2-HBA from said subject.

[0383] According to a specific embodiment, the metabolic disorder is a glucose metabolism disorder, or a cardiometabolic disease, preferably the glucose metabolism disorder is selected from the group consisting of gestational diabetes, impaired glucose tolerance, insulin resistance, prediabetes, and diabetes.

[0384] According to one embodiment, described herein is an in vitro method of discriminating between normal glucose tolerance and impaired glucose tolerance in a subject comprising the sequential steps of: i. enzymatically determining the concentration of 2-HBA in a sample from said subject by using a flavocytochrome b2 (FCb2) comprising SEQ ID NO:5, or a FCb2 comprising an amino acid sequence having at least 50, 55, 60, 65, 70, 75, 85, 90, 95, 96, 97, 98, or 99 % sequence identity with SEQ ID NO:5; ii. comparing said concentration of 2-HBA with a predetermined cut-off value of 25 pM 2-HBA or higher; iii. assigning to said subject impaired glucose tolerance if said concentration of 2-HBA is equal or above the predetermined cut-off value; and assigning to said subject normal glucose tolerance if said concentration of 2-HBA is below the predetermined cut-off value.

[0385] According to a specific embodiment, in the methods described herein the sample is a blood sample.

[0386] According to a specific embodiment, in the methods described herein the concentration of 2-HBA is determined colorimetrically, photometrically, fluorimetrically, through phosphorescence, through chemiluminescence, or electrochemically. According to a specific embodiment, the methods described herein further comprise selectively removing lactate in said sample prior to enzymatically determining the concentration of 2-HBA by a using a lactate oxidase (LOx) comprising SEQ ID NO:4, or a LOx comprising an amino acid sequence having at least 50, 55, 60, 65, 70, 75, 85, 90, 95, 96, 97, 98, or 99 % sequence identity with SEQ ID NO:4.

[0387] According to one embodiment, described herein is a use of a flavocytochrome b2 (FCb2) comprising SEQ ID NO:5, or a FCb2 comprising an amino acid sequence having at least 50, 55, 60, 65, 70, 75, 85, 90, 95, 96, 97, 98, or 99 % sequence identity with SEQ ID NO:5, for diagnosing a metabolic disorder in a subject, for determining the risk of having or developing a metabolic disorder, for monitoring a metabolic disorder in a subject, or for discriminating between normal glucose tolerance and impaired glucose tolerance in a subject.

[0388] According to one embodiment, described herein is a kit for diagnosing a metabolic disorder, for determining the risk of having or developing a metabolic disorder, for monitoring a metabolic disorder in a subject, for discriminating between normal glucose tolerance and impaired glucose tolerance, or for discriminating between gestational diabetes and non-gestational diabetes, said kit comprising a flavocytochrome b2 (FCb2) comprising SEQ ID NO:5, or a FCb2 comprising an amino acid sequence having at least 50, 55, 60, 65, 70, 75, 85, 90, 95, 96, 97, 98, or 99 % sequence identity with SEQ ID NO:5.

[0389] According to one embodiment, described herein is a kit for diagnosing a metabolic disorder, for determining the risk of having or developing a metabolic disorder, for monitoring a metabolic disorder in a subject, for discriminating between normal glucose tolerance and impaired glucose tolerance, or for discriminating between gestational diabetes and non-gestational diabetes, said kit comprising a flavocytochrome b2 (FCb2) comprising SEQ ID NO:5, or a FCb2 comprising an amino acid sequence having at least 50, 55, 60, 65, 70, 75, 85, 90, 95, 96, 97, 98, or 99 % sequence identity with SEQ ID NO:5, for determining the concentration of 2-HBA, and a lactate oxidase (LOx) comprising SEQ ID NO:4, or a LOx comprising an amino acid sequence having at least 50, 55, 60, 65, 70, 75, 85, 90, 95, 96, 97, 98, or 99 % sequence identity with SEQ ID NO:4, for selectively removing lactate from the sample.

[0390] According to one embodiment, described herein is a kit for diagnosing a gestational diabetes, and / or for discriminating between gestational diabetes and non-gestational diabetes, said kit comprising a flavocytochrome b2 (FCb2) comprising SEQ ID NO:5, or a FCb2 comprising an amino acid sequence having at least 50, 55, 60, 65, 70, 75, 85, 90, 95, 96, 97, 98, or 99 % sequence identity with SEQ ID NO:5; and a lactate oxidase (LOx) comprising SEQ ID NO:4, or a LOx comprising an amino acid sequence having at least 50, 55, 60, 65, 70, 75, 85, 90, 95, 96, 97, 98, or 99 % sequence identity with SEQ ID NO:4. Specifically, flavocytochrome b2 (FCb2) is used for determining the concentration of 2-HBA. Specifically, lactate oxidase (LOx) is used for selectively removing lactate from the sample.

[0391] According to one embodiment, an enzyme having ascorbate oxidizing activity may be used before, together with, or after enzyme A for reducing an interference with ascorbate.

[0392] According to a specific embodiment, an interference with ascorbate may mean that the 2-HBA determination described herein is disturbed by the presence of ascorbate in the sample.

[0393] According to one embodiment, a catalase may be used together with enzyme A.

[0394] According to one embodiment, the kit described herein may further comprise an enzyme having ascorbate oxidizing activity.

[0395] According to one embodiment, the kit described herein may further comprise catalase.

[0396] According to a specific embodiment, the kit described herein may comprise a reagent 1 and a reagent 2, wherein reagent 1 comprises a lactate oxidase (LOx) comprising SEQ ID NO:4, or a LOx comprising an amino acid sequence having at least 50, 55, 60, 65, 70, 75, 85, 90, 95, 96, 97, 98, or 99 % sequence identity with SEQ ID NO:4; and wherein reagent 2 comprises a flavocytochrome b2 (FCb2) comprising SEQ ID NO:5, or a FCb2 comprising an amino acid sequence having at least 50, 55, 60, 65, 70, 75, 85, 90, 95, 96, 97, 98, or 99 % sequence identity with SEQ ID NO:5.

[0397] According to a specific embodiment, reagent 1 further comprises catalase and / or ascorbate oxidase.

[0398] According to a specific embodiment, reagent 2 further comprises cytochrome C.

[0399] According to a specific embodiment, reagent 2 further comprises bovine serum albumin.

[0400] According to a specific embodiment, reagent 1 and 2 are buffered solutions, preferably having pH 7.4.

[0401] According to a specific embodiment, the buffered solution is a phosphate buffered saline. According to a specific embodiment, reagent 1 comprises lactate oxidase (LOx), and catalase in a phosphate buffered saline solution. Preferably, said LOx comprises SEQ ID NO:4, or a LOx comprising an amino acid sequence having at least 50, 55, 60, 65, 70, 75, 85, 90, 95, 96, 97, 98, or 99 % sequence identity with SEQ ID NO:4.

[0402] According to a specific embodiment, the concentration of lactate oxidase in reagent 1 is in the range of 0.01 to 20, 0.01 to 15, 0.01 to 10, 0.01 to 5, 0.01 to 1 , 0.05 to 20, 0.05 to 15, 0.05 to 10, 0.05 to 5, 0.05 to 1 , 0.1 to 20, 0.1 to 15, 0.1 to 10, 0.1 to 5, 0.1 to 1 , or 0.1 to 0.5 mg / mL. Preferably, at 0.1 mg / mL.

[0403] According to a specific embodiment, the concentration of catalase in reagent 1 is in the range of 0.01 to 20, 0.01 to 15, 0.01 to 10, 0.01 to 5, 0.01 to 1 , 0.05 to 20, 0.05 to 15, 0.05 to 10, 0.05 to 5, 0.05 to 1 , 0.1 to 20, 0.1 to 15, 0.1 to 10, 0.1 to 5, 0.1 to 1 , or 0.1 to 0.5 mg / mL. Preferably, at 0.1 mg / mL.

[0404] According to a specific embodiment, reagent 2 comprises a flavocytochrome b2 (FCb2), cytochrome C, and bovine serum albumin in a phosphate buffered saline solution. Preferably, said FCb2 comprises SEQ ID NO:5, or a FCb2 comprising an amino acid sequence having at least 50, 55, 60, 65, 70, 75, 85, 90, 95, 96, 97, 98, or 99 % sequence identity with SEQ ID NO:5.

[0405] According to a specific embodiment, the concentration of FCb2 in reagent 2 is in the range of 0.01 to 20, 0.01 to 15, 0.01 to 10, 0.01 to 5, 0.01 to 0.1 , 0.01 to 1 , 0.05 to 20, 0.05 to 15, 0.05 to 10, 0.05 to 5, 0.05 to 1 , 0.1 to 20, 0.1 to 15, 0.1 to 10, 0.1 to 5, 0.1 to 1 , or 0.1 to 0.5 mg / mL. Preferably, at 0.01 mg / mL.

[0406] According to a specific embodiment, the concentration of cytochrome C in reagent 2 is in the range of 10 to 150, 10 to 140, 10 to 130, 10 to 120, 10 to 110, 10 to 100, 10 to 90, 20 to 150, 20 to 140, 20 to 130, 20 to 120, 20 to 110, 20 to 100, 20 to 90, 30 to 150, 30 to 140, 30 to 130, 30 to 120, 30 to 110, 30 to 100, 30 to 90, 40 to 150, 40 to 140, 40 to 130, 40 to 120, 40 to 110, 40 to 100, 40 to 90, 50 to 150, 50 to 140, 50 to 130, 50 to 120, 50 to 110, 50 to 100, 50 to 90, 60 to 150, 60 to 140, 60 to 130, 60 to 120, 60 to 110, 60 to 100, 60 to 90, 70 to 150, 70 to 140, 70 to 130, 70 to 120, 70 to 110, 70 to 100, or 70 to 90 pM.

[0407] According to a specific embodiment, the concentration of bovine serum albumin is in the range of 0.1 to 1 , 0.1 to 0.5, 0.1 to 0.4, 0.1 to 0.3, 0.2 to 1 , 0.2 to 0.5, 0.2 to 0.4, or 0.2 to 0.3 % (w / v). According to a specific embodiment, in the methods, uses, and means described herein, reagent 2 is used for enzymatically determining the concentration of 2-hydroxybutyric acid (2-HBA) in a sample.

[0408] According to a specific embodiment, in the methods, uses, and means described herein, reagent 1 is used for selectively removing lactate from the sample.

[0409] According to a specific embodiment, the kit described herein further comprises an instruction manual.

[0410] According to a specific embodiment, the instruction manual comprises a description of converting a determined 2-HBA concentration to the diagnosis of a metabolic disorder, to the risk of having or developing a metabolic disorder, to the monitoring of a metabolic disorder in a subject, to the discrimination between normal glucose tolerance and impaired glucose tolerance, or to the discrimination between gestational diabetes and non-gestational diabetes. Specifically, the instruction manual provides a description on how to obtain the diagnosis of a metabolic disorder, the risk of having or developing a metabolic disorder, the monitoring of a metabolic disorder in a subject, the discrimination between normal glucose tolerance and impaired glucose tolerance, or the discrimination between gestational diabetes and non-gestational diabetes from the determined concentration of 2-HBA.

[0411] According to one embodiment, in the methods described herein an enzyme having ascorbate oxidizing activity may be used for reducing an interference with ascorbate.

[0412] According to one embodiment, reagent 1 may further comprise herein an enzyme having ascorbate oxidizing activity, preferably ascorbate oxidase.

[0413] According to a specific embodiment, the concentration of ascorbate oxidase in reagent 1 is in the range of 0.0001 to 20, 0.0001 to 15, 0.0001 to 10, 0.0001 to 5, 0.0001 to 1 , 0.0005 to 20, 0.0005 to 15, 0.0005 to 10, 0.0005 to 5, 0.0005 to 1 , 0.001 to 20, 0.001 to 15, 0.001 to 10, 0.001 to 5, 0.001 to 1 , 0.001 to 0.01 , 0.001 to 0.005, 0.05 to 20, 0.05 to 15, 0.05 to 10, 0.05 to 5, 0.05 to 1 , 0.1 to 20, 0.1 to 15, 0.1 to 10, 0.1 to 5, 0.1 to 1 , or 0.1 to 0.5 mg / mL. Preferably, at 0.003 mg / mL.

[0414] According to one embodiment, an enzyme having ascorbate oxidizing activity is ascorbate oxidase.

[0415] According to a specific embodiment, ascorbate oxidase is ascorbate oxidase from Cucurbita spp.. Specifically, ascorbate oxidase from Cucurbita spp. is commercially available and is known under CAS Number: 9029-44-1 and under EC Number: 232-852- 6. According to one embodiment, in the methods described herein a catalase may be used for removal of hydrogen peroxide.

[0416] The term “sequence identity” as used herein is understood as the relatedness between two amino acid sequences or between two nucleotide sequences and described by the degree of sequence identity or sequence complementarity. The sequence identity of a variant, homologue or orthologue as compared to a parent nucleotide or amino acid sequence indicates the degree of identity of two or more sequences. Two or more amino acid sequences may have the same or conserved amino acid residues at a corresponding position, to a certain degree, up to 100 %. Two or more nucleotide sequences may have the same or conserved base pairs at a corresponding position, to a certain degree, up to 100 %.

[0417] Sequence similarity searching is an effective and reliable strategy for identifying homologs with excess (e.g., at least 50 %) sequence identity. Sequence similarity search tools frequently used are e.g., BLAST, FASTA, and HMMER.

[0418] Sequence similarity searches can identify such homologous proteins or polynucleotides by detecting excess similarity, and statistically significant similarity that reflects common ancestry. Homologues may encompass orthologues, which are herein understood as the same protein in different organisms, e.g., variants of such protein in different organisms or species.

[0419] To determine the % complementarity of two complementary sequences, one of the two sequences needs to be converted to its complementary sequence before the % complementarity can then be calculated as the % identity between the first sequence and the second converted sequences using the above-mentioned algorithm.

[0420] “Percent (%) identity” with respect to an amino acid sequence, homologs and orthologues described herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific polypeptide sequence, after aligning the sequence and introducing gaps, if necessary, and not considering any conservative substitutions as part of the sequence identity. Those skilled in the art can determine appropriate parameters for the alignment, including any algorithms needed to achieve the highest scoring alignment over the full length of the sequences being compared. In case of percentages determined for sequence identities, it is possible that arithmetical decimal places may result which are not possible with regard to full nucleotides or amino acids. In this case, the percentages shall be rounded up to whole nucleotides or amino acids. For purposes described herein, the sequence identity between two amino acid sequences is determined using standard methods, e.g. using the NCBI BLAST program version 2.2.29 (Jan-06-2014) or online using the multiple sequence alignment tool EMBL- EBI Clustal Omega (Sievers, F. et al. (2011)).

[0421] “Percent (%) identity” with respect to a nucleotide sequence e.g., of a nucleic acid molecule or a part thereof, in particular a coding DNA sequence, is defined as the percentage of nucleotides in a candidate DNA sequence that is identical with the nucleotides in the DNA sequence, after aligning the sequence and introducing gaps, if necessary, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent nucleotide sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software. Those skilled in the art can determine appropriate parameters for the alignment, including any algorithms needed to achieve the highest scoring alignment over the full length of the sequences being compared.

[0422] Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, MAFFT based algorithms: multiple alignment using fast fourier transform, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomies.org.cn), and Maq (available at maq.sourceforge.net).

[0423] According to a specific embodiment, sequence identity is determined using Clustal W.

[0424] In a structure alignment the maximal set of corresponding pairs of amino acid residues that gives a good structural match when the structures are overlaid, i.e., superposed, is identified. Thereby, the positions of the protein’s backbone C-alpha atoms and / or location of secondary structural elements are considered in this alignment. Tools for performing a structure alignment are available, e.g., the protein data bank provides a tool for pairwise structure alignment. Specifically, structure superposition is also a tool for determining corresponding amino acid positions in different enzymes. Structure superposition can be performed using the Molecular Graphics System PyMOL, (Schrodinger) using the command “align”. The examples described herein are illustrative of the present invention and are not intended to be limitations thereon. Many modifications and variations may be made to the techniques described and illustrated herein without departing from scope of the invention.

[0425] EXAMPLES

[0426] Example 1 : Enzymatically determining 2-HBA by XpressGT® on spectrophotometric plate readers

[0427] Chemicals

[0428] (S)-2-hydroxybutyric acid (>97 %, CAS 3347-90-8), sodium (S)-lactate (> 98 %, CAS 867-56-1), human plasma (pool, lyophilized, citrate anticoagulant), human serum (male, sterile filtered), and phosphate buffered saline tablets were purchased from Sigma. The latter were dissolved in 200 mL reverse osmosis-generated water with the pH adjusted to 7.40 with NaOH / phosphoric acid. (RS)-2-hydroxybutyric acid sodium salt (>97 %, racemic, CAS 5094- 24-6) was from Thermo Fisher Scientific, 2-hydroxybutanoic-d3 acid sodium salt (isotope labeled, racemic, CAS 1219798-97-6) was from Toronto Research chemicals. Sterile human serum and human plasma in sodium citrate, sodium EDTA, and in lithium heparin were obtained from Biotrend CliniSciences Group and were from healthy donors, sterile pooled and gender balanced.

[0429] Materials used for liquid chromatography-MS / MS were Elo-Mel isotonic infusion solution from Fresenius Kabi, Graz, Austria, and human serum albumin (200 g / L infusion solution) from Kedrion Biopharma, Austria. Formic acid 99-100%, AnalaR NORMAPUR®, methanol >99,8%, and HiPerSolv CHROMANORM® were from VWR (Pennsylvania, USA). Purified water was prepared using the Milli Q® Academic water purification System from Millipore GmbH, Vienna, Austria.

[0430] The XpressGT® test kit

[0431] The XpressGT® test kit is an enzymatic test kit for the fast and accurate determination of 2-HBA levels in serum or plasma samples. The kit can be used on spectrophotometric plate readers as well as on automated laboratory analyzers and consists of two reagents that are employed to adjust reaction conditions and neutralize interferences (Reagent 1) and colorimetric determination of the 2-HBA analyte using an engineered hydroxy butyrate dehydrogenase in conjunction with a tailored colorimetric redox mediator (Reagent 2).

[0432] Reagent 1 : LOx (0.1 mg / mL) and catalase (0.1 mg / mL) in PBS (11 mM, pH 7.4) Reagent 2: FCb2 (0.01 mg / mL), cytochrome C (80 pM) and bovine serum albumin (0.25%) in PBS (11 mM, pH 7.4)

[0433] LOx having the following amino acid sequence is used in reagent 1 (LOx L19; WP_1 95852629.1 _LOx_L19_A.sanguinicola; SEQ ID NO:4): MSAEYKAPSEVKDLTIYNTIELEDLAKEVMPKGGFDYLAGGSGEEFTLKRNVQCFKSQ GILPRVLADVEFPETETEIFGQKLKVPFIMAPIAAHETKEAGTAKGISEFGGTIMSISAYS GATFEEIDEGLQGNPRWFQIYMAKDDELNINILNEAKADGASAIILTADATISGNRDRDD KNEFVYPFGMPIVSRYLTGTGENMSLNNIYGQSKQKITPRDVEFIKKHTDLPVFVKGLQ TAEDANVAIGAGADGVWVSTHGGRQLDEAPGSFECLAEVADAVAGRVPIVFDSGVRR GEHIFKALAQGADLVALGRPVLFGLALGGWKGVRSVFEYFETDLKRVMQLAGTQNIDQ VKAARLKALDNWN*

[0434] FCb2 having the following amino acid sequence is used in reagent 2 (SEQ ID NO:5): MKHHHHHHHHSNTSSVLNKPPIDPKEWKHNKPDDCWIVINNWYDLTDFIAKHPGGP DIIQSNAGKDVSAIFNPLHASDVIDKYIKPECQLGPLKEPLPESYICPPLTPGETAEDVAR KAELRDKLPPLDSLINLYDFEYLASQILTKQAWGYYSSAADDEVTYRENHAAYHRIFFK PRILINVKECDLSTTMLGTKMDLPFYVSATALCKLGNPSEGEKDIARGCGMGEFNLTQ MISTLASCSLKEIVEAKVNDKQTQWFQLYVNADRKITDNLIKNVEELGLKAIFVTVDAPS LGRREKDMKIKFDPSKTPEITNEDKSHKKQQNTRGASKALSTFIDPSLTWNDVIDIKKKT KLPWIKGVQCTADILKAAEIGVDGWLSNHGGRQLDFSRAPIEVLAEAMPILKEKGLDK KLEVYIDGGVRRGTDVLKALCLGAKGVGLGRPFLYANSCYGKDGVNRLKEMLADEIEM NMRLLGVTKISDLKPEYLDMISLHARSVNVPKDNLYQEVYIPSTTVEFLDE*

[0435] For the photometric determination of 2-HBA, the XpressGT® protocol was followed, and all incubation steps were performed in a Tecan M Nano plate reader (Tecan) conditioned at 37 °C with pre-warmed reagents. Serum or plasma samples, standards and controls were thawed on ice and briefly spun down to separate undesired precipitate and cell debris, before 10 pL each were transferred into three separate wells of a preconditioned transparent 96 well plate (Greiner 300 pL, flat bottom) as technical triplicates. Subsequently, 90 pL of Reagent 1 were added and the mixture incubated for 300 seconds to pretreat the samples prior to analysis. In the next step, 100 pL of Reagent 2 were added and allowed to mix for 10 seconds. Immediately thereafter, a kinetic measurement of the 200 pL reaction was performed following the change of the optical absorbance signal at 550 nm for 180 seconds. These dynamic absorbance changes were then fitted to a linear least squares regression after exporting data to excel and the resulting slope and intercept used to calculate the 2-HB concentration according to previously determined calibration data (see Results).

[0436] Preparation of artificial 2-HBA serum sample set

[0437] Besides measuring the native 2-HBA content of the commercial human serum pool samples, artificial serum stocks were prepared by spiking 160 pL of these serum samples with 40 pL 2-HBA stock in a randomized fashion (using a number generator), to ultimately obtain 60 samples of this type with final 2-HBA concentrations ranging between 20 and 80 pM. The spiking aimed at providing samples covering a representative concentration range similar to but expanding the one reported in the study by Varvel et al. for plasma samples from more than 200 patients at increased risk for diabetes (Varvel, S.A. et al. 2014)

[0438] The LC-MS methodology

[0439] An Ultra-high performance liquid chromatography-MS / MS (subsequently simply referred to as LC-MS) method for quantification of 2-HBA was performed using standard methods known in the field and as described e.g., in Prugger E.-M. (2018).

[0440] Results

[0441] Calibration on spectrophotometric plate readers

[0442] To obtain reference values on the basis of which sample data can be converted to actual 2-HBA concentrations, a range of calibration standards covering between 10 and 200 pM 2-HBA were prepared and measured using the XpressGT® test kit. As shown in Fig. 1 , this yielded a perfectly linear calibration curve with a coefficient of determination R2> 0.998 and a slope of 1 .548 10-4 Abs550 min-1 pM-1 and an intercept of - 1 .524 10- 4 Abs550 min-1 . See Fig. 1 . XpressGT® test kit 2-HBA calibration curve.

[0443] The enzymatic kit in this example measures the L-2-HBA enantiomer and yielded approximately 50 % of slopes for the racemic mixture containing the same overall concentration of the metabolite.

[0444] Spiking recovery, matrix effects and linearity

[0445] Next, multiple aliquots of a commercial plasma pool sample spiked with 2-HBA at concentrations from 0 to 200 pM were measured with both the XpressGT® test kit and with LC-MS, and the results of the obtained 2-HBA concentration values in these samples were plotted against the expected concentration. As shown in Fig. 2, this yielded a highly linear relation between observed and expected (spiked) values for both the test kit and the LC-MS-samples, R2of the linear regression plot for the former being 0.999 and for the latter 0.995. The slope of the regression analysis of 1.025 indicated 102% recovery of added 2-HBA stocks. Importantly, both methods resulted in a native 2-HBA concentration of the non-spiked plasma samples of 32.0 ± 3.5 pM, which is well in the range of concentrations previously reported for non-diabetic patients quantified using either LC-MS (Gall, W. E., et al. (2010), Cobb, J., et al. (2016)) or a targeted, quantitative 1 H-NMR metabolomics technique (Tried, D. et al. (2017)).

[0446] Sample type and 2-HBA stability in samples

[0447] Commercial samples of serum and plasma containing various types of anticoagulant were spiked with 100 pM 2-HBA to increase the signal and then subjected to repeated XpressGT® measurement in triplicates after storage at 4 °C for periods of 0 to 46 h to assess analyte stability in typical lab conditions. Plotting 2-HBA concentrations detected in these samples over time of storage indicated that both initial values and the decline with storage time were largely comparable between serum samples and plasma samples prepared with different anticoagulants (Fig. 3a). Accordingly, in comparison to initial concentrations of spiked samples at 0 h remaining activities detected after 46 h storage were 95±4 % for serum, 87±13 % for plasma EDTA, 98±4 % plasma Na-citrate and 95±13 % for plasma Li-heparin (Li-hep).

[0448] To evaluate sample 2-HBA stability upon repeated freeze-thawing cycles, which samples may undergo in clinical practice, samples prepared as above were exposed to three consecutive cycles of freezing and thawing before 2-HBA was again measured using the test kit. As above, all sample types delivered largely comparable 2-HBA concentration levels following this treatment (serum: 95±4 %, plasma Na-citrate: 96±8 %, plasma Li-heparin: 100±5 %, plasma EDTA: 86±3 % with respect to the initial apparent 0 h concentration) (Fig. 3b). It should be noted that the differences of plasma 2-HBA concentrations between sample types at zero storage time were within the measurement errors.

[0449] In-assav and between-assav precision spectrophotometric plate readers

[0450] To evaluate in-assay precision obtained using the test kit, 20 measurements of the 100 pM 2-HBA standard were carried out creating 20 single datapoints and their concentration calculated based on the calibration curve generated at the start of these experiments. In addition, to obtain an estimate for between-assay precision, 40 independent measurements of sample triplicates were carried out over a period of 10 days and were shared between 2 operators on two photometers. For in-assay precision (n=19 / 20, one sample had to be excluded due to technical problems) an average 2-HBA concentration of 104.9 ± 3.2 pM (3.1 % relative standard deviation, RSD) was obtained, while the between-assay precision (n=118 / 120, two samples excluded) yielded an average value of 106.0 ± 5.5 pM (5.2 % RSD) (Fig. 4). Datasets of both operators were comparable calculating overall means of determinations (105.7 ± 5.5 pM, 106.3 ± 5.5 pM, n=60) and when calculating averages of triplicates for both operators a value of 105.9 ± 4.0 pM (3.8 % RSD, n=40 / 40) was obtained.

[0451] Accuracy and expected range

[0452] Numerous published studies investigating the predictive value of 2-HBA levels for diabetes risk have applied LC-MS-methods to quantify the metabolite (Gall, W. E., et al. (2010), Cobb, J., et al. (2016) Varvel, S.A. et al. 2014)). Accordingly, we conducted a direct comparison of XpressGT® measurements and LC-MS (in a specialized lab externally) measurements using the same sample set. To account for differences in the calibration between the labs, that could, e.g., be caused by varying purity of 2-HBA chemical, measurements were re-calibrated using synthetic 2-HBA calibration samples. Finally, a sample set of 15 standards (6 spiked plasma pool samples, 9 standards of 100 pM 2-HBA in buffer) and 60 randomly spiked serum samples were measured with both methods and the concentrations obtained with LC-MS and with the XpressGT® kit were plotted against each other (Fig. 5). As can be seen in this plot, LC-MS and XpressGT® measurements displayed excellent agreement for the sample set (R2=0.977) and the linear trendline generated using the 60 datapoints indicated an underestimation bias of 93 % (slope, dotted), by comparison of slopes, for XpressGT® measurements.

[0453] When considering measurement inaccuracies between LC-MS and XpressGT® measurements at margins of ± 20 % (80-120 % of XpressGT® results) 86 % of all samples are situated within these margins. When the set is restricted to samples 30 pM 2-HBA (n=60 / 75), 93 % of samples, for >40 pM (n=45 / 75) 100 % of samples from the set fall within these margins. For an even stricter criterion of ± 10 % (90-110 %), 54 % of all samples fall within these margins, 61 % of samples (> 30 pM) and 73 % of samples (>40 pM), respectively, when an 2-HBA concentration threshold is defined.

[0454] Noteworthy, the calculated average concentration obtained with XpressGT® for the 60 serum samples amounted to 46.2 ±3.4 pM (4.7 pg / mL), median 41 .0 ±3.4 pM (4.1 pg / mL), which is in good agreement with the median 2-HBA concentration of 43 pM (4.3 pg / mL, non-fasting) and of 47 pM (4.7 pg / mL, fasting) found in the comprehensive analysis of >90 000 2-HBA MS measurements reported by Varvel, S.A. et al. (2015). Example 2: Enzymatically determining 2-HBA by XpressGT® on clinical chemistry analyzers

[0455] The XpressGT® test kit:

[0456] The XpressGT® test kit consists of Reagent 1 for the pretreatment of the sample and Reagent 2 for the interference free detection of 2-HBA.

[0457] Reagent 1 : LOx (0.1 mg / mL) and catalase (0.1 mg / mL) in PBS (11 mM, pH 7.4)

[0458] Reagent 2: FCb2 (0.01 mg / mL), cytochrome C (80 pM) and bovine serum albumin (0.25%) in PBS (11 mM, pH 7.4)

[0459] The amino acid sequences of LOx and FCb2 are given in Example 1.

[0460] Additionally, the test kit contains control and calibrator solutions for the calibration of the clinical chemistry analyzer. In this example the control and calibration solutions were prepared as follows:

[0461] Low calibrator: 20 pM 2-HBA +3.0 mM Lactate in PBS (11 mM, pH 7.4)

[0462] High calibrator: 200 pM 2-HBA +3.0 mM Lactate in PBS (11 mM, pH 7.4)

[0463] Control: 100 pM 2-HBA +3.0 mM Lactate in PBS (11 mM, pH 7.4)

[0464] Measurement procedure of XpressGT® on clinical analyzer:

[0465] The assay is based on a kinetic determination principle. All reagents are used without dilution or preparation.

[0466] 1 . The reaction is performed at 37 °C on an automated analyzer (Abbot Alinity c)

[0467] 2. At time zero, 10 pL of standard, reagent blank or sample is added 80 pL of Reagent 1 .

[0468] 3. After 288 seconds, 93 pL of Reagent 2 is added. a. The absorbance at 548 nm (primary wavelength) and 700 nm (secondary) are determined 32 seconds after the addition of Reagent 2, where time = 320 sec.

[0469] 4. After another 143 seconds the absorbance at both wavelengths is determined again, time = 463 sec. a. The absorbance value at 700 nm is subtracted from the absorbance at 548 nm to correct for noise. This is performed for readings both at the 320 sec and 463 sec time points and the kinetic difference in absorbance is calculated:

[0470] AAbsorbance = (Abss48-Abs7oo)463 sec- (Abss48-Abs7oo)32O sec 5. Concentrations of 2-HBA in the sample are determined by recalculation of the AAbsorbance via a calibration function derived from the kinetic change in absorbance of the calibration standards (20 and 200 pM 2-HBA).

[0471] Accuracy of XpressGT® measurement on clinical analyzer

[0472] The Abbot analyzer was calibrated using a two-point calibration with the following calibrators: 20 pM (low) and 200 pM (high). After calibration, the 100 pM control sample was used to evaluate precision of the Alinity ci measurement. 10 repeats were performed in separate reactions and average, SD and RSD calculated.

[0473] Additionally, we assessed the accuracy of the measurement by comparing XpressGT® values from clinical analyzer with the reference values obtained by LC-MS. The methodology for the reference method is described in Example 1. We collected 79 leftover Li-Hep plasma samples from routine blood analysis. Samples were conserved for analysis by storage at -20 °C. The samples were anonymized, and no clinical data of the patients were collected.

[0474] Results:

[0475] Calibration of XpressGT® on clinical analyzer:

[0476] After calibration 10 repeat measurements of the control sample were performed in separate reactions and average, SD and RSD were calculated. The 10 repeats of the 100 pM control sample yielded the following results: average: 103.9 pM, SD: 0.53 pM and RSD: 0.51 %. This result highlights the high repeatability of the XpressGT® and the high accuracy of the calibration on clinical analyzers.

[0477] Accuracy compared with the reference method LC-MS in patient samples:

[0478] As can be seen in Fig. 6, LC-MS and XpressGT® measurements displayed excellent agreement for the sample set (R2=0.937). The linear trendline generated using the 79 datapoints indicated a negligible overestimation bias of 103 % (slope, dotted).

[0479] Example 3: Enzymatic testkit for 2-HBA in detecting individuals with gestational diabetes

[0480] Sensitivity and specificity of XpressGT® - an enzymatic testkit for 2-HBA - in detecting individuals with gestational diabetes is described. Across 1 Austrian center, excess serum samples from routine OGTT screenings (24-28 gestational week) of approximately 400 pregnant women were collected and the concentration of 2-HBA with the enzymatic testkit XpressGT® was prospectively measured. The prevalence of GDM will be determined using standard of care 2h 75g oral glucose tolerance tests (OGTT). The primary outcome is derived from a correlation of XpressGT® 2-HBA and OGTT results and delivered as area under the ROC curve (AUC), sensitivity and specificity of detecting individuals with gestational diabetes.

[0481] Patients:

[0482] This is a prospective cohort study of pregnant women who received a standard of care 2h 75g OGTT as part of GDM screening / diagnosis between the 24-28 gestational week. Excess plasma samples from the 1 h OGTT time point were collected in 1 Austrian center. Samples conserved for analysis by storage at -20 °C. Additionally, the fasting plasma glucose and 1 h OGTT glucose levels were collected. The samples were anonymized, and no clinical data of the patients were collected. Pregnant women receiving the standard of care 2h 75g OGTT as part of routine care. Patients with overt diabetes were excluded and there is no information on whether patients were pregnant before.

[0483] Test methods:

[0484] Reference standard: The authors chose the OGTT as reference test since it is considered as the gold standard for the diagnosis of GDM. The cut-offs were chosen according to IADPSG (2010):

[0485] • Positivity cut-offs 2h 75 g OGTT o Fasting plasma glucose in venous plasma 92 mg / dl o 1 h OGTT in venous plasma > 180 mg / dl o 2h OGTT in venous plasma 153 mg / dl

[0486] Index test: An XpressGT® test was performed on an Abbot Alinity ci (clinical analyzer).

[0487] The calibrators and controls are used as described in example 2.

[0488] The XpressGT® method was performed using reagent 1 and reagent 2 as described in Example 1 and 2.

[0489] Results:

[0490] 2-HBA vs. GDM (Oh-OGTT, 1 h-OGTT, 2h-OGTT) From the collection of 371 samples, 55 were diagnosed with GDM. Although several samples were positive in more than one criterion, a patient is diagnosed with GDM when at least one of cut-offs is met or exceeded. Therefore, we refer to the GDM diagnosis as composite reference standard (RS).

[0491] Table 1 shows the number of patients (N) diagnosed with GDM (D+) and the patients without GDM (D-) and their XpressGT® 2-HBA values in pM.

[0492] Table 1

[0493] For the set of 316 patient samples without GDM, a mean of 28.0 ± 12.9 pM 2-HBA was calculated (median: 25.7 pM, 25 % quartile 19.1 pM, 75 % quartile 35.1 pM. 95 % of 2-HBA values were found between 4.7 and 100.4 pM.

[0494] For the set of 55 patients samples with GDM, a mean of 44.1 ± 19.6 pM 2-HBA was calculated (median: 41.2 pM, 25 % quartile 29.5 pM, 75 % quartile 53.2 pM. 95 % of 2-HBA values were found between 14.0 and 84.4 pM.

[0495] Fig 7 Top shows a box-plot of XpressGT® 2-HBA [pM] (index test) by binary status of GDM Diagnosis (Composite reference standard (RS)).

[0496] For the screening of GDM a sensitivity of 70 % was defined as an internal requirement. A 2-HBA [pM] cut-off of 32.2 pM was calculated to yield 70% sensitivity for GDM. Table 2 shows the categorization of GDM patients by the result of the XpressGT® test (pos, neg) with a cut-off of 32.2 pM. It displays the absolute number of patients (N) diagnosed with and without GDM (GDM+, GDM-) and the percentage within reference category (%*) and percentage within test result category (%**).

[0497] Table 2 In total, the 2-HBA assay (cut-off of > 32.2 pM) classifies 62.5 % of samples as negatives. In clinical and diagnostic practice, if the XpressGT® assay would be implemented as pre-test standard, more than 6 out of 10 patients could be excluded from the procedures of the OGTT with good conscious.

[0498] Table 3 shows the most important measures of diagnostic accuracy for XpressGT® by binary status of GDM Diagnosis (Composite RS), using the empirical cutoff of 32.2 pM.

[0499] Table 3

[0500] To show the classification performance of the for the index test XpressGT® 2-HBA related to GDM Diagnosis the receiver-operating characteristic (ROC) curves were calculated. Fig. 7 - Bottom shows the ROC curves of the classification performance for the index test XpressGT® 2-HBA [pM] related to GDM Diagnosis (Composite reference standard (RS)). The area under the ROC (AUC) was calculated with AUC = 0.77, with a comparatively narrow 95 % confidence band of 0.70-0.84, not descending below 0.70, which was regarded as an internal requirement, too.

[0501] 2-HBA vs. fasting plasma glucose

[0502] Figure 8 top shows a box-plot of XpressGT® 2-HBA [pM] (index test) by binary status of fasting plasma glucose (OGTT Omin.) at the cut-off used for GDM diagnosis (< 92 mg / dL). Bottom: Receiver-operating characteristic (ROC) curves showing the classification performance for the index test XpressGT® 2-HBA [pM] related to fasting plasma glucose (OGTT Omin.) at the cut-off used for GDM diagnosis (< 92 mg / dL). AUC, area under the curve.

[0503] 2-HBA vs. 1 h-OGTT

[0504] Fig. 9 top shows a box-plot of XpressGT® 2-HBA [pM] (index test) by binary status of the 1 h OGTT value (OGTT 60 min.) at the cut-off used for GDM diagnosis (< 180 mg / dL). Bottom: Receiver-operating characteristic (ROC) curves showing the classification performance for the index test XpressGT® 2-HBA [pM] related to 1 h OGTT value (OGTT 60 min.) at the cut-off used for GDM diagnosis (< 180 mg / dL). AUC, area under the curve.

[0505] 2-HBA vs. 2h-OGTT

[0506] The data for the 2h OGTT were not analyzed.

[0507] Conclusions

[0508] The XpressGT® 2-HBA assay test performs well for the screening for gestational diabetes. As recommended by Benhalima, K. (2018) we defined a sensitivity of 70 % as a prerequisite for GDM screening. This consequently allowed to determine an 2-HBA cutoff of 32.2 pM, which translated to a specificity of 68 ± 5 % (Cl of 95 %). With XpressGT® test 63 out of 100 OGTTs procedures could be avoided. The XpressGT test has a high NPV, which is an important feature for a screening test that rules out a disease.

[0509] The clinical utility of this test could be compared to the glucose challenge test (GCT) which is a standard procedure in screening for gestational diabetes Benhalima, K. (2018).

[0510] Since the correlation of 2-HBA had a similar performance in predicting GDM, fasting plasma glucose and 1h-OGTT, the results can be generalized to the 2h-OGTT. Therefore, the results can also be generalized to the results of the 3h 100g OGTT procedure.

[0511] In this example serum samples were used. It is shown in Example 1 and 2 that XpressGT® accurately quantifies 2-HBA in plasma samples. Therefore, the test performance can be generalized to all plasma samples irrespective of the anticoagulants used (e.g. Li-Hep, NaF, EDTA, citrate, Na-citrate).

[0512] Previous data on the performance of the biomarker 2-HBA for detection GDM were based on fasting patient samples Raczkowska BA et al. (2021). This example presents the first data using non-fasting patient samples. Since test performance (as defined by AUC) is comparable, the clinical performance is independent of the fasting status of the patient. The independence of fasting status was also shown for prediabetic patients by Varvel, S.A. et al. (2015). They compared the difference between 2-HBA concentration in 76.709 fasting patient samples and 19.151 non-fasting patient samples that were obtained as part of routine care. After adjusting for age, gender and BMI the 2-HBA concentration differed by only 4.9 %. Parameter that were sensitive to fasting differed by > 10 %. This comparison demonstrates that 2-HBA is unaffected by fasting status in a routine clinical setting.

[0513] The clinical performance of XpressGT® for gestational diabetes screening is very similar to the performance published for the screening of impaired glucose tolerance, although different cut-offs values are applied. Therefore, the diagnostic accuracy of XpressGT® can be generalized to all conditions where OGTT based screening and / or diagnosis is used (e.g. impaired glucose tolerance, insulin resistance, type 2 diabetes, risk of CVD).

[0514] Example 4: Enzymatic assay principle for the determination of 2-HBA in two steps

[0515] In this example, the enzyme lactate oxidase (LOx) is used as an example of an enzyme for the removal of L-lactate. Flavocytochrome b2 is used as an example of an enzyme for the detection of 2-HBA. A general scheme of the reaction is shown in Fig. 10.

[0516] According to the example, using the assay a specific detection of 2-HBA is performed in samples, where interfering L-lactate can be removed using a dedicated enzymatic pre-treatment step with lactate oxidase (LOx). Thereby, as in this example an enzyme is used for removal which uses oxygen as electron acceptor, H2O2 accumulates from O2 reduction simultaneous to the oxidation of L-lactate, and can be partly recovered (half-stoichiometrically) employing an enzymatic conversion with catalase: 2 H2O2 -> 2 H2O + O2. This regeneration using catalase may aid in maintaining a steady oxygen concentration for the LOx reaction. Subsequently to the removal step, the 2-HBA analyte can be detected without interference using an enzyme like FCb2.

[0517] In general, in physiological relevant samples L-lactate is present at >10-fold higher concentrations (1 -2 mM) than the 2-HBA analyte (0.05-0.10 mM).

[0518] This example provides the general proof of principle that the method described herein can be used for the determination of 2-HBA in samples comprising L-lactate. Furthermore, results of spiking the 2-HBA in physiological samples is shown. Materials

[0519] Enzymes:

[0520] Amino acid sequences of enzymes used for removal or detection in the examples provided herein:

[0521] AvLOx - amino acid sequence (SEQ ID NO: 1):

[0522] MGSSHHHHHHNNNDIEYNAPSEIKYIDWNTYDLEEEASKWPHGGFNYIAGAS

[0523] GDEWTKRANDRAWKHKLLYPRLAQDVEAPDTSTEILGHKIKAPFIMAPIAAHGLAHTTK

[0524] EAGTARAVSEFGTIMSISAYSGATFEEISEGLNGGPRWFQIYMAKDDQQNRDILDEAKS

[0525] DGATAIILTADSTVSGNRDRDVKNKFVYPFGMPIVQRYLRGTAEGMSLNNIYGASKQKI

[0526] SPRDIEEIAGHSGLPVFVKGIQHPEDADMAIKRGASGIWVSNHGARQLYEAPGSFDTLP

[0527] AIAERVNKRVPIVFDSGVRRGEHVAKALASGADWALGRPVLFGLALGGWQGAYSVLD

[0528] YFQKDLTRVMQLTGSQNVEDLKGLDLFDNPYGYEY

[0529] CanqFCb2 - amino acid sequence (SEQ ID NO: 2):

[0530] MHHHHHHHHDAKFDSSKPKISPSEVIKHNTPEDCWWIDGYVYDLTNFIALHPG

[0531] GPDIIKTNAGKDVTAIFDPIHPPDAIEKYIKPEQHVGPLDGKLDAEYICPPYAPGETPDDI

[0532] ARKAALRARLPPLSSIMNLYDFEYLASQILSKQAWAYYSSASDDEVSYRENHNAYHRIF

[0533] FNPKVLVDVSKVDTSTEMLGHKVDVPFYVTATALCKLGNPKEGEKDIARGCGQGPNK

[0534] TPQMISTLASCSVDEIVNAAPSKDQVIWYQLYVNSDRKITENLIKHVEDLGVKAIFVTVD

[0535] APSLGSREKDKKVKFNNTMSGPKSMKKSDVGESEGAAQTLSKFIDPSLSWQDIKILRK

[0536] KTKLPIVIKGVQRVQDWKAAEIGCNGWLSNHGGRQLDFARAPIEVLAETMPVLKEKK

[0537] LDKNFEVFVDGGVRRGTDVIKALCLGASGVGLGRPFLYANSCYGKDGVQKAIDLLKTEI

[0538] EMNMRLLGVTSIKDMNPELLDLSSLHGRTVNVPKDSLYVNVYNKPELAEFLDDASD

[0539] Catalase from Corynebacterium glutamicum (CgCat), Sigma #02071 Chemicals:

[0540] • Equine Cytochrome C, Sigma #C2506

[0541] • Sodium L-Lactate, 99.0 % (NT), Sigma #71718

[0542] • Na 2-Hydroxybutyrate > 97 %, racemic Sigma #220116 (CAS 5094-24-6, 126.09 g / mol)

[0543] • Phosphate buffer saline (PBS), pH 7.4, Sigma #P3813

[0544] • Human plasma (SIGMA #P9523): lyophilized, 4 % Citrate

[0545] • Human Serum H3667-20 mL (Sigma, Source SLCL8404), heat inactivated: frozen

[0546] • Multititer plate 96-well (Greiner, Polystyrole, Merck # M2936) Instrumentation:

[0547] Photometer (TECAN Infinite M Nano Plate reader); for measurement at 550 nm

[0548] Proof of principle

[0549] Methods:

[0550] Reagent 1 is used for the pretreatment step. Reagent 1 : Aerococcus viridans Lactate Oxidase at 0.1 mg / mL and Corynebacterium glutamicum Catalase solution 1 pL / mL in 11 mM PBS, pH 7.4

[0551] Reagent 2 is used for the measurement step. Reagent 2: Candida glabrata FCb2 at 50 pg / mL and 80 pM equine Cytochrome C (Sigma-Aldrich, #C2506) in 11 mM PBS, pH 7.4

[0552] The determination of 2-HBA concentrations is performed at room temperature (22 °C), including the 5 min pre-treatment reaction and the 3 min measurement using a photometric plate reader.

[0553] At time zero of the reaction, 10 pL of the sample (serum, plasma, synthetic controls) are transferred into the well of a 96-well plate. The pre-treatment reaction is started by adding 90 pL of reagent 1. After 5 minutes, 100 pL of the reagent 2 is added and the photometric measurement is started immediately. The reaction (absorbance change) is followed at 550 nm for 3 min in the photometer. The increase in optical absorbance at 550 nm per minute (AAbs550 min-1) for this measurement setup can be calculated into an enzymatic activity, U / mL or U / mg, or an apparent 2-HBA concentration according to the following equations (which only apply for the mentioned technical setup):

[0554] Equation 1 - calculation of volumetric activities (U / mL) from changes in optical absorbance at 550 nm: volumetric activity [U mL1] = Abs min1* 0.98 * df

[0555] Equation 2 - calculation of 2-HBA concentration from changes in optical absorbance at 550 nm (this calculation only applies for the given technical setup):

[0556] AAbs min1— 0.00101

[0557] 2 - HBA [pM] =

[0558] 0.000121

[0559] The following applies for the equations shown:

[0560] 0.98: enzyme factor uniting dilution, extinction coefficient and pathlength df: sample dilution factor (10pL in 200pL, equals 0.05) Samples contained different combinations of 5 mM L-lactate, 0.1 mM 2-HBA, 0.1 mg / mL LOx but contained catalase solution at 0.1 % (v / v) in all assays. Pre-treatment reactions were carried out for 5 minutes at 22 °C prior to sample measurement of 2-HBA concentrations, relying on a 3-minute Cytochrome C (CytC) assay with FCb2. The increase in absorbance at 550 nm originating from CytC reduction translated to a substrate concentration dependent enzyme activity (U / mL).

[0561] Results:

[0562] The results of the proof of principle are shown in Fig. 12.

[0563] (1 ) The sample containing 2-HBA (analyte) and L-lactate (interferent) without addition of LOx yields high activities (CytC) in the subsequent measurement with CangFCb2 due to the presence of high concentrations of L-lactate.

[0564] (2) When LOx is present, the sample containing the 2-HBA analyte and L- lactate interferent yields low activities since L-lactate is oxidized by LOx and hence cannot be accepted by the FCb2 during the colorimetric measurement. The residual activity originates solely from 2-HBA.

[0565] (3) In contrast, no detectible signals were yielded from the FCb2 reaction when L-lactate, but no 2-HBA was present.

[0566] (4) Similar levels of enzyme activity, respective to (2), were reached when no L-Lactate was present.

[0567] These results provide the proof of principle of the method described herein, especially since the following aspects are confirmed:

[0568] • Lactate removal: Whether L-lactate is present or not; (2) vs. (4): the residual signals are comparable, indicating that the 2-HBA fraction alone leads to the signals after treatment.

[0569] • This is attributed to LOx. The (1) assay where LOx is absent, shows substantially higher signals.

[0570] • When 2-HBA is absent in sample mixtures, no signals remain after LOx treatment pointing towards apparent complete removal of L-lactate.

[0571] Analytical slope of 2-HBA with the FCb2 assay:

[0572] Methods:

[0573] • 2-HBA was diluted in PBS to yield the following pM concentrations: 6.25; 12.5; 25; 50; 100; 200; 300; 400 • 10 pL of these samples were measured in technical replicates of 6 with the CytC / Fcb2 assay procedure using CangFCb2 as is described above. The procedure was adapted to using 90 pL of PBS instead of Reagent 1 , but using 100 pL Reagent 2 with CangFCb2

[0574] • From the calculated FCb2 activities, data points were used to calculate a linear regression, yielding slope, intercept and goodness of fit (R2)

[0575] Results:

[0576] In trials with pure 2-HBA and the FCb2 / CytC assay, an apparently linear analytical range was obtained between 6.25 and 400 pM sample concentration. In this measurement setup, no interferant was present and the LOx catalyzed L-lactate removal step was omitted.

[0577] The following Table 4 gives the numerical data of the analytical range measurements:

[0578] Table 4: Numerical data of the analytical range measurements

[0579] These numerical data were plotted and the following linear calibration function was determined: y = 1 E-04x + 0.0029

[0580] R2= 0.9994

[0581] These results underline a broad linear range and low standard errors for the 2- HBA measurement in the setup with CangFCb2. 2-HBA spiking in plasma

[0582] Methods:

[0583] • Spiked samples were prepared using the aforementioned human plasma and serum sample alongside synthetic standard solutions containing 5 mM L-lactate and 0.1 mM 2-HBA.

[0584] • 10 pL of the spiked samples were measurement according to the proof of principle experiments in technical replicates of 6, adding the removal reaction with 90 pL Reagent 1 and subsequent 2-HBA measurement with CangFCb2 in 100 pL Reagent 2

[0585] • Utilizing the analytical slope parameters of 2-HBA determination from above, FCb2 activities were averaged and translated to apparent 2-HBA concentrations, which were the opposed to spiked 2-HBA concentrations in the sample

[0586] • From the calculated FCb2 activities, data points were used to calculate a linear regression, yielding slope, intercept and goodness of fit (R2) where the slope, opposing apparent and spiked 2-HBA concentrations reflected the “recovery”.

[0587] Results:

[0588] From a series of 2-HBA standards in buffer, with known 2-HBA concentrations, enzymatic activity measurements were performed and yielded kinetic slopes, i.e. at points of a certain 2-HBA concentration and kinetic slope scaled linearly with increasing concentration. These data points were used to calculate a linear regression, characterized by an intercept (d) and slope (x) value according to y = kx +d which was referred to as analytical calibration.

[0589] After spiking commercial human plasma and serum with known amounts of 2-HBA and unknown physiological L-lactate interferant concentrations, the calibration curve parameters (determined in buffer) were used to calculate apparent concentrations of the 2-HBA analyte in serum and plasma. The correlation of spiked and measured 2-HBA concentrations in this experiment are visualized here and represent in-assay concentrations. Sample concentrations were diluted 1 in 20.

[0590] The results are shown in the following Tables 5 and 6: Table 5: Numerical data of spiking experiments in plasma

[0591] Table 6: Numerical data of spiking experiments in serum

[0592] • Calibration curves of 2-HBA were recorded and slope and intercept were used to calculate signals into concentrations, which were opposed to the spiked concentrations.

[0593] • Serum and plasma were spiked with 2-HBA concentrations of 0-10 pM in assay, equaling 0-200 pM in sample

[0594] • Recovery range for plasma and serum similar (~ 94 %) • Linear (detectable) range: 0.625 - 10 pM 2-HBA in assay, translating to 12.5

[0595] - 200 pM samples

[0596] Example 5: Electrochemical determination of non-lactate hydroxy acid

[0597] In this example, the electrochemical detection of 2-HBA is described. KmFCb2 is used as an exemplary enzyme for the electrochemical detection of 2-HBA.

[0598] Materials:

[0599] Enzyme: KmFCb2 - amino acid sequence (SEQ ID NO: 3):

[0600] MHHHHHHHHATKEELNKPKVSPLEVAKHSSPDDCWWIDGFVYNLTEFISAHP GGPAIIENNAGKDVTAIFGPIHAPDVIEKYIAPENRIGPLDGKMPDDLICAPLTPGETPED VARKEELRQNMPDLDSLVNIYDFEFLASQILTKQAWSYYSSAADDEVTHRENHAAYHR IFFKPRILVNVKEVDTSTTMLGEKVGVPFYVSATALCKLGNPKEGEKDIARGCGESDVK PIQMISTLASCSLQEIVEAAPSKDQIQWFQLYVNSDRKITEELIKNVEKLGLKAIFVTVDA PSLGNREKDAKVKFTNKDSSAKAMEKSNVKESKGASRALSTFIDPALCWDDIVTLKSK TKLPIVIKGVQCVEDVLKAAEIGAAGWLSNHGGRQLDFSRAPIEVLAETMPILKEKKLD DKIEIFIDGGVRRGTDILKALCLGAKGVGLGRPFLYANSCYGKEGVKKAIELLKDELEMS MRLLGVTSIDQLSEKYLDLSTLHGRTVSVPRDNLYNGVYVPHEPTDFKEN

[0601] Chemicals:

[0602] • Na 2-Hydroxybutyrate > 97 %, racemic Sigma #220116 (CAS 5094-24-6, 126.09 g / mol)

[0603] • Phosphate buffer saline (PBS), pH 7.4, Sigma #P3813

[0604] • DropSens Carbon screen printed electrodes DRP-C110

[0605] Instrumentation:

[0606] • Potentiostat: Palmsens EMstat Blue

[0607] Method:

[0608] • 2-HBA was diluted in PBS to yield the following mM concentrations: 1.0, 5.0, 10

[0609] • Biosensor electrodes were prepared as is described in Geiss et al. (2021) with the adaptation of using 2 pL of 10 mg / mL engineered KmFCb2 in 100 mM Phosphate buffer. Curing was done at 22 °C for 2 h under dry ambience.

[0610] • The electrode measurement was carried out in triplicates utilizing a potentiostat in a chronoamperometric measuring mode at an applied potential of 0.2 V vs a pseudo-Ag / AgCI reference electrode at 22°C. Electrodes were mounted horizontally, and samples were added / removed step wise to the sensing area to yield increased substrate concentrations over time. The change of currents was measured over time. • Data evaluation: Baseline subtracted currents were evaluated 10 s after each sample addition and plotted versus the substrate concentration. Non-linear regression fitting to Michaelis-Menten equation were calculated.

[0611] Results:

[0612] The analyte can be detected using the FCb2 enzyme without the need of a soluble electron acceptor when the FCb2 enzyme is contacted on an electrode. Dependent on the substrate concentration in the assay, the enzyme delivers proportional catalytic currents that can be measured using an electronic device, such as a potentiostat.

[0613] The following Table 7 shows the results of the example.

[0614] Table 7: Catalytic currents of electrochemical KmFCb2 measurement of 2-HBA

[0615] In trials with electrochemically contacted KmFCb2, 2-HBA could be detected in a relevant range.

[0616] These results underline that electrochemical measurement presents a viable option for the detection of 2-HBA from samples using FCb2. This might be especially important for biosensor applications e.g., point of care and / or at home testing.

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Claims

CLAIMS1. An in vitro method of diagnosing a metabolic disorder in a subject comprising the sequential steps of: i. enzymatically determining the concentration of 2-hydroxybutyric acid (2-HBA) in a sample from said subject; and ii. comparing said concentration of 2-HBA with a predetermined cut-off value of 25 pM 2-HBA or higher, wherein the metabolic disorder is diagnosed if said concentration of 2-HBA is equal or above the predetermined cut-off value.

2. An in vitro method of determining the risk of having or developing a metabolic disorder in a subject comprising the sequential steps of: i. enzymatically determining the concentration of 2-HBA in a sample from said subject; ii. comparing said concentration of 2-HBA with a predetermined cut-off value of 25 pM 2-HBA or higher; and iii. assigning to said subject a high risk of having or developing said metabolic disorder if said concentration of 2-HBA is equal or above the predetermined cut-off value.

3. An in vitro method of monitoring a metabolic disorder in a subject comprising the sequential steps of: i. enzymatically determining the concentration of 2-HBA in a sample from said subject; and ii. comparing said concentration of 2-HBA with a previous concentration of 2-HBA from said subject.

4. The method of any one of claims 1 to 3, wherein the metabolic disorder is a glucose metabolism disorder, or a cardiometabolic disease, preferably the glucose metabolism disorder is selected from the group consisting of gestational diabetes, impaired glucose tolerance, insulin resistance, prediabetes, and diabetes.

5. An in vitro method of discriminating between normal glucose tolerance and impaired glucose tolerance in a subject comprising the sequential steps of: i. enzymatically determining the concentration of 2-HBA in a sample from said subject; ii. comparing said concentration of 2-HBA with a predetermined cut-off value of 25 pM 2-HBA or higher; iii. assigning to said subject impaired glucose tolerance if said concentration of 2-HBA is equal or above the predetermined cut-off value; and assigning to said subject normal glucose tolerance if said concentration of 2-HBA is below the predetermined cut-off value.

6. The method of any one of claims 1 to 5, wherein an enzyme having 2-HBA oxidizing activity is used for enzymatically determining the concentration of 2-HBA, preferably, the enzyme is an enzyme having 2-HBA dehydrogenase activity, more preferably FCb2 or LDH.

7. The method of any one of claims 1 to 6, wherein the sample is a blood sample.

8. The method of any one of claims 1 to 7, wherein the concentration of 2-HBA is determined colorimetrically, photometrically, fluorimetrically, through phosphorescence, through chemiluminescence, or electrochemically.

9. The method of any one of claims 1 to 8, further comprising selectively removing lactate in said sample prior to enzymatically determining the concentration of 2-HBA.

10. Use of an enzyme having 2-HBA oxidizing activity for diagnosing a metabolic disorder in a subject, for determining the risk of having or developing a metabolic disorder, for monitoring a metabolic disorder in a subject, or for discriminating between normal glucose tolerance and impaired glucose tolerance in a subject, wherein the concentration of 2-hydroxybutyric acid (2-HBA) is determined enzymatically in a sample from said subject.

11. A kit for diagnosing a metabolic disorder, for determining the risk of having or developing a metabolic disorder, for monitoring a metabolic disorder in a subject, for discriminating between normal glucose tolerance and impaired glucose tolerance, or for discriminating between gestational diabetes and non-gestational diabetes, said kit comprising an enzyme having 2-HBA oxidizing activity.

12. The kit of claim 11 , wherein the enzyme is an enzyme having 2-HBA dehydrogenase activity, preferably FCb2 or LDH.

13. The kit of claim 11 or 12, further comprising an enzyme for selectively removing lactate from the sample.

14. The kit of any one of claims 11 to 13, further comprising cytochrome, preferably cytochrome C.

15. The kit of any one of claims 11 to 14, further comprising an instruction manual, wherein said instruction manual comprises a description of converting a determined 2-HBA concentration to the diagnosis of_a metabolic disorder, to the risk of having or developing a metabolic disorder, to the monitoring of a metabolic disorder in a subject, to the discrimination between normal glucose tolerance and impaired glucose tolerance, or to the discrimination between gestational diabetes and non-gestational diabetes.

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