Method for determining insulin dosage

Abstract

The present invention relates to a method for determining insulin dosage for a subject in need thereof, the method comprising: a) determining basal Insulin Sensitivity Factor (ISF) of the subject; b) determining succinate level in a sample obtained at a first time from the subject; c) determining succinate level in a sample obtained at a second time from the subject; d) determining glucose level in a sample obtained from the subject; e) determining if the succinate level determined under c) is lower, identical or higher than the succinate level determined under b), in order to determine an appropriate insulin dosage for he subject. In the method, determining succinate level can be performed with an amperometric sensor.

Description

[0001] Method for determining insulin dosage

[0002] Technical field

[0003] The present disclosure relates to healthcare. In particular, the disclosure relates to methods for determining insulin dosage, in view of insulin resistance and / or Insulin Sensitivity Factor (ISF) variability within a patient over time. Preferably, the method involves using an electrochemical biosensor.

[0004] Background of the invention

[0005] Every action of cells of our body needs energy in the form of ATP, which at cellular level mostly comes from glucose. Glucose, derived from food, is supplied to our cells via our bloodstream in which glucose concentration is kept within the homeostatic range of 3.9-7.8 mM (not sober) by the action of the pancreatic hormones insulin and, to a lower extent, glucagon.

[0006] Upon uptake of a carbohydrate-containing meal, blood glucose (BG) level rises within the homeostatic range, which stimulates the pancreatic beta cell to release insulin. Insulin subsequently stimulates the liver and muscle to take up and convert glucose into glycogen, whereas adipose tissue is stimulated to store glucose in the form of fat. Due to these activities BG levels are attenuated. When BG levels near the lower homeostatic level, insulin levels drop and glucagon levels rise. Glucagon thus counteracts insulin, its release is inversely related to that of insulin, and therewith prevents development of hypoglycemia (BG<4 mM).

[0007] Type-1 diabetes mellitus (T 1 DM) is an auto-inflammatory disease of the beta cells of the pancreas that can develop as early as with birth and for which there is no cure. It has a high prevalence (1 :300) and a high incidence (2.6:1000), which is still increasing. Due to this disease, patients are not able to secrete sufficient amounts of insulin leading to hyperglycemia. While hyperglycemia gives irritation, sweating, headaches and brain fog with T1 DM patients at the short-term, repetitive stages of hyperglycemia yields neuropathy, blindness, kidney failure, cardiovascular disease and amputations of extremities on the long term. It is the most common auto-inflammatory disease with >100.000 patients in the Netherlands and with estimated health care costs in the US of USD 1000 per person per year, counting up to a total US health care costs of 813 billion USD per year.

[0008] To manage their hyperglycemia, patients can inject themselves with insulin. Until 2019, T1 DM patients determined their BG level following a finger prick and were learned to calculate the anticipated BG increases with meals they consumed and the amount of slow or fast-acting insulin they had to inject themselves with to manage their hyperglycemia. As BG levels vary continuously and carb calculations were not always done correctly, patients sometimes injected too much insulin, leading to hypoglycemia (BG<4). While patients may experience hypoglycemia (sweating, dizziness, feeling awful), or may not sense it at all (impaired awareness of hypo-unawareness; IAH), this condition is life-threatening, as it may lead to sudden coma or death.

[0009] A recent development, however, has strongly changed T1 DM management. While flash glucose monitors (FGM; Abbott), which connect with and ‘read’ the BG level of the interstitial fluid, relieve patients from finger pricks and allow unlimited readouts of the individual’s BG level on smartphones, the recent hybrid closed-loop continuous glucose monitoring (CL- CGM) devices (Medtronics, Dexcom, Roche), consisting of a CGM sensor, an artificially- intelligent (Al)-steered algorithm, and an insulin pump, directly translate the read BG levels to calculated amounts of automatically-injected insulin.

[0010] Whereas meal insulin injections still have to be calculated and manually injected through the pump (therefore hybrid), these modules are able to maintain BG within a narrow range (4-10 mM) for a large part of time by (1) maintaining a personalized insulin infusion level based on the amount needed to maintain a steady BG level during the last 3-10 hours, (2) adjusting subcutaneous insulin infusion to the trend of BG changes, and (3) incorporating daily repetitive changes via Al (e.g. walking every morning). Due to these changes, the golden standard of the diabetic status has changed from the extent of glycation of red blood cell haemoglobin (HbA1c), which is mostly a mean readout of time spent in hyperglycaemia during the last 3-4 months, to glucose variability (GV). Indeed, the Diabetes Control and Complication Trial (DCCT) and subsequently Epidemiology of Diabetes Interventions and Complications (EDIC) studies show that reduction of the GV to near-normal levels in people with T1 DM can significantly reduce the incidence of microvascular complications and provide a more sustained, glycemia independent reduction in risk of complication. As CL-CGM devices connect continuous BG monitoring with adapted insulin infusion, these are seen as the diabetes management devices of the future to minimize GV.

[0011] Despite these improvements, CL-CGM using patients are still continuously busy managing their DM as <70% of patients using commercial CL-CGM devices only get to a 70% time-in- range (TIR), despite that the CL-CGM device TIR range (3.9-10 mM) has been set larger than the physiological range (3.9-7.8) in order to reduce occurrence of hypoglycaemia. A reason for this is that, besides food, BG levels vary due to changes in insulin resistance of which the extent changes during and in between days due to environmental queues such as menstrual cycle / menopause / pregnancy, inflammation / infection, exercise, anxiety, worries on work / relation / exams, work shifts, coldness, intolerances for food, obesity, smoking, etc. Hence, insulin resistance is an important parameter to properly determine the amount of insulin provided to patients, but which information is at present missing.

[0012] Therefore, there remains a medical need to develop new means to improve healthcare, in particular new means to determine insulin resistance, and / or to determine insulin resistance variability and / or appropriate insulin dosage for a patient over time. In particular, there remains a need to develop new means to increase the Tl R for T1 DM patients. The present invention aims to solve one or more of the above-mentioned or other problems.

[0013] Summary of the invention

[0014] The present inventor surprisingly found an improvement for healthcare, in particular that insulin resistance and / or Insulin Sensitivity Factor (ISF) variability over time within a patient can be followed through measurement over time of succinate level in a bodily fluid of said patient.

[0015] The present inventor found that cells produce more succinate, resulting in a cumulative blood succinate level, consistent with an increased state of insulin resistance and / or Insulin Sensitivity Factor (ISF), for example in T1 DM patients. Following adaptation, which varies in time and between individuals, homeostatic steady state conditions re-establish and succinate production / blood levels normalize.

[0016] The present inventor particularly envisages a biosensor, which allows for continuous succinate monitoring (CSM). This sensor may comprise a succinate-(ubi)quinone (oxido) reductase (SQR / SQOR) enzyme complex, at least comprising the succinate dehydrogenase (SDH) subunits SDHA / SDHB, coated on an amperometric device, that can be brought into contact with the patient’s bodily fluid, for example a blood (plasma) sample, a tear sample, or an interstitial fluid sample. In this way, the biosensor can provide an instantaneous, quantitative, and personalized readout of the (changes in) insulin resistance level. Accordingly, CGM-directed insulin infusion can be adapted to the patient’s insulin resistance as quantitatively determined with CSM.

[0017] Specifically, the A and B subunit of the succinate dehydrogenase (SDH) complex can be used to quantitatively determine blood succinate levels. In mitochondria, the enzyme complex of SQR subunits A-D specifically recognize and convert succinate to fumarate (Hock et al, 2020). Research has shown that only subunits A and B of this complex already allow for detection, binding and efficient conversion of succinate to fumarate and that these subunits can be isolated from tissue and / or heterologously expressed in high amounts in e.g. yeast (Ceccatelli Berti et al, 2021 ; Moosavi et al, 2019). Upon conversion of succinate to fumarate, an electron is generated which allows amperometric quantification of the succinate concentration, similar to glucose concentration measurements with FGM / CGM devices. Therefore, to follow insulin resistance variability in humans, functional SDHA / B complexes can be isolated (Davis & Hatefi, 1971 ; Stepanova et al, 2016) and spotted on nanoscale amperometric devices similar as developed for glucose sensing in strips, or in FGM / CGM- device like tools. This will allow instantaneous and accurate regulation of insulin infusion and therewith strongly reduce GV and maximize the quality of life of the patient.

[0018] The mammalian complex II, encompassing succinate:ubiquinone oxidoreductase (SQR), and SQR homologs oxidize succinate to fumarate and consist of four subunits, SDHA-SDHB- SDHC-SDHD (Moosavi et al., 2019). The catalytic domain (SDHA and SDHB) is extrinsic on the matrix side, while the anchor subunits (SDHC and SDHD) are intrinsic transmembrane proteins, allowing transfer of the electrons from succinate into the mitochondrial matrix to ubiquinone, located in the inner membrane. The SDHA subunit may be a flavoprotein containing a covalently-bound FAD cofactor and the binding site for dicarboxylates (e.g. succinate). SDHB may be an iron-sulphur cluster protein containing three Fe-S clusters. SDHA and SDHB make up the catalytic domain and are historically-named succinate dehydrogenase (SDH). They extend out into the matrix and constitute the hydrophilic head. Specific features of the SDH domain useful to the sensor are:

[0019] FAD cofactor binding (flavo)protein (SDHA);

[0020] Iron-sulphur (Fe-S) cluster containing protein (SDHB);

[0021] - Ability to bind and oxidize succinate.

[0022] While complex II specifically refers to mitochondrial succinate:ubiquinone oxidoreductase (SQR) and homologous SQRs are found in all other organisms, it is a member of a superfamily of related membrane proteins coupling the succinate-fumarate and quinonequinol redox pairs, collectively referred to as succinate:quinone oxidoreductases (SQORs) (Moosavi et al., 2019). In some family members, the physiological direction of the reaction catalysed is the reverse, i.e., quinol oxidation with reduction of fumarate to succinate. These enzymes are called fumarate reductases (FRD) or more specifically quinokfumarate reductases (QFR).

[0023] The SQORs as a superfamily all have a flavoprotein and iron-sulphur protein with strong sequence (and where known, structural) homology. The great majority of well-characterized SQORs also have transmembrane anchors related to SDHC and SDHD, but these have considerably more variation in sequence and structure.

[0024] SdhA / FrdA has soluble homologs, which catalyze a, p-dehydrogenase reactions. The fumarate reductase (flavocytochrome c) from Shewanella frigidimarina and Shewanella putrefaciens, and l-aspartate oxidase from E. coli are examples of soluble SdhA / FrdA homologs. The Shewanella enzyme consists of a single polypeptide chain harbouring a domain similar to the flavoprotein (FrdA / SdhA) fused to a tetra-heme cytochrome. Consequently, they are termed as flavocytochrome c3 (Fcc3) fumarate reductases, which preferably are excluded from the present application. E. coli l-aspartate oxidase, on the other hand, does not contain any peripheral domain to accommodate redox cofactors. Interestingly, FAD is non-covalently bound in these soluble enzymes, while it is covalently bound in the SQORs of Classes A, B, C, or D. These enzymes are not SQORs since they do not oxidize or reduce quinones, but the high degree of homology with SDHA, especially around the dicarboxylate-binding site, together with the convenience of working with soluble, singlesubunit proteins, makes them valuable in the effort to decipher mechanisms of dicarboxylate oxidoreduction in complex II. Hence, they are preferably not encompassed by the present application. In principle, under the same conditions an enzyme must catalyse the forward and reverse reactions equally, since it cannot change the equilibrium. However, evolution has engineered SQR to favour the forward direction, by causing it to shut down under highly reducing conditions that would cause net reverse reaction.

[0025] Detailed description of the invention

[0026] In a first aspect, the present disclosure relates to a method for determining Insulin Sensitivity Factor (ISF) and / or insulin dosage, e.g. for a subject in need thereof, the method comprising: a1) preferably determining (basal) Insulin Sensitivity Factor (ISF) (in a sample) of the subject, preferably in conditions wherein the subject is at least 1 , 2, 3, 4, 5, 6 hours free from having meal, physical activity and infection; more preferably in conditions wherein the subject has not taken an insulin dosage (e.g. a meal bolus or correction bolus) for at least 4 hours, has not eaten any food for the last 4 hours, is free from infection (and (major) emotional stress), and is at least 2 hours free from (strenuous) physical activity; a2) preferably determining (basal) succinate level in a sample of the subject, preferably in conditions wherein the subject is at least 1 , 2, 3, 4 hours free from having meal, physical activity and infection; more preferably in conditions wherein the subject has slept as usual, refrained from food and caloric beverages for 16 hours, refrained from any (moderate) physical activity for the last 24 hours (or any vigorous physical activity for the last 48 hours), and remaining still when sampling; b) determining succinate level in a sample obtained at a first time from the subject; c) determining succinate level in a sample obtained at a second time from the subject, wherein the time period between the first time and the second time preferably covers conditions of having meal, physical activity and / or infection by the subject; d) determining glucose level in a sample obtained from the subject, preferably at the second time or within 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 60 minutes or within 1 , 2, 3, 4, 5, 6 hours thereof; e) determining if the succinate level determined under c) is lower, identical or higher than the succinate level determined under b), wherein for basal succinate levels in the low range, i.e. in case the (basal) succinate determined under a2) is 0-50 pM:

[0027] - a succinate level increase between the first time and the second time by more than 35, 40, 45% indicates a decrease of (actual) ISF by more than 35, 40, 45% and at most 75, 80, 85% of the subject between the first time and the second time;

[0028] - a succinate level increase between the first time and the second time by at least 5, 10, 15% and at most 35, 40, 45% indicates a decrease of (actual) ISF by at least 10, 15, 20% and at most 35, 40, 45% of the subject between the first time and the second time,

[0029] - an identical or less than 5, 10, 15% deviation of succinate level between the first time and the second time indicates no or less than 10, 15, 20% change of (actual) ISF of the subject between the first time and the second time;

[0030] - a succinate level decrease between the first time and the second time by more than 35, 40, 45% indicates an increase of (actual) ISF by more than 55, 60, 65% and at most 400% of the subject between the first time and the second time;

[0031] - a succinate level decrease between the first time and the second time by more than 5, 10, 15% and at most 35, 40, 45% indicates an increase of (actual) ISF by more than 10, 15, 20% and at most 55, 60, 65% of the subject between the first time and the second time; for basal succinate levels in the high range, i.e. in case the (basal) succinate determined under a2) is more than 50 pM, such as between 50 and 100 pM:

[0032] - a succinate level increase between the first time and the second time by more than 15, 20, 25% indicates a decrease of (actual) ISF by more than 15, 20, 25% and at most 55, 60, 65% of the subject between the first time and the second time;

[0033] - a succinate level increase between the first time and the second time by at least 5, 10, 15% and at most 15, 20, 25% indicates a decrease of (actual) ISF by at least 5, 10, 15% and at most 15, 20, 25% of the subject between the first time and the second time;

[0034] - an identical or less than 5, 10, 15% deviation of succinate level between the first time and the second time indicates no or less than 5, 10, 15% change of (actual) ISF of the subject between the first time and the second time; - a succinate level decrease between the first time and the second time by more than 15, 20, 25% indicates an increase of (actual) ISF by more than 35, 40, 45% and at most 95, 100% of the subject between the first time and the second time;

[0035] - a succinate level decrease between the first time and the second time by more than 5, 10, 15% and at most 15, 20, 25% indicates an increase of (actual) ISF by more than 5, 10, 15% and at most 35, 40, 45% of the subject between the first time and the second time; f) preferably, administering an insulin dosage to the subject, wherein the dosage in insulin units is within a 1 , 2, 3, 4, 5, 10, 15, 20% margin of: (determined glucose level - healthy reference glucose level) I (actual / indicated) ISF (ISF determined in e)).

[0036] Preferably, the present disclosure relates to a method for determining Insulin Sensitivity Factor (ISF) and / or insulin dosage for a subject in need thereof, the method comprising: a1) determining (basal) Insulin Sensitivity Factor (ISF) (in a sample) of the subject, preferably in conditions wherein the subject is at least 1, 2, 3, 4 hours free from having meal, physical activity and infection; more preferably in conditions wherein the subject has not taken an insulin dosage (e.g. a meal bolus or correction bolus) for at least 4 hours, has not eaten any food for the last 4 hours, is free from infection (and (major) emotional stress), and is at least 2 hours free from (strenuous) physical activity ; a2) determining (basal) succinate level in a sample of the subject, preferably in conditions wherein the subject is at least 1, 2, 3, 4 hours free from having meal, physical activity and infection; more preferably in conditions wherein the subject has slept as usual, refrained from food and caloric beverages for 16 hours, refrained from any (moderate) physical activity for the last 24 hours (or any vigorous physical activity for the last 48 hours), and remaining still when sampling; b) determining succinate level in a sample obtained at a first time from the subject; c) determining succinate level in a sample obtained at a second time from the subject, wherein the time period between the first time and the second time preferably covers conditions of having meal, physical activity and / or infection (and / or stress queue) by the subject; d) determining glucose level in a sample obtained from the subject, preferably at the second time or within 10 minutes thereof; e) determining if the succinate level determined under c) is lower, identical or higher than the succinate level determined under b), wherein in case the basal succinate determined under a2) is 0-50 pM:

[0037] - a succinate level increase between the first time and the second time by more than 40% indicates a decrease of (actual) ISF by more than 40% and at most 80% of the subject between the first time and the second time; - a succinate level increase between the first time and the second time by at least 10% and at most 40% indicates a decrease of (actual) ISF by at least 15% and at most 40% of the subject between the first time and the second time,

[0038] - an identical or less than 10% deviation of succinate level between the first time and the second time indicates no or less than 15% change of (actual) ISF of the subject between the first time and the second time;

[0039] - a succinate level decrease between the first time and the second time by more than 40% indicates an increase of (actual) ISF by more than 60% and at most 400% of the subject between the first time and the second time;

[0040] - a succinate level decrease between the first time and the second time by more than 10% and at most 40% indicates an increase of (actual) ISF by more than 15% and at most 60% of the subject between the first time and the second time; in case the basal succinate determined under a2) is more than 50, preferably between 50 and 100 pM:

[0041] - a succinate level increase between the first time and the second time by more than 20% indicates a decrease of (actual) ISF by more than 20% and at most 60% of the subject between the first time and the second time;

[0042] - a succinate level increase between the first time and the second time by at least 10% and at most 20% indicates a decrease of (actual) ISF by at least 10% and at most 20% of the subject between the first time and the second time;

[0043] - an identical or less than 10% deviation of succinate level between the first time and the second time indicates no or less than 10% change of (actual) ISF of the subject between the first time and the second time;

[0044] - a succinate level decrease between the first time and the second time by more than 20% indicates an increase of (actual) ISF by more than 40% and at most 100% of the subject between the first time and the second time;

[0045] - a succinate level decrease between the first time and the second time by more than 10% and at most 20% indicates an increase of (actual) ISF by more than 10% and at most 40% of the subject between the first time and the second time; f) preferably, administering an insulin dosage to the subject, wherein the dosage in insulin units is within a 20% margin of: (determined glucose level minus healthy reference glucose level) divided by (actual / indicated) ISF (ISF determined in e)).

[0046] Accordingly, the method can be used to determine the (actual) ISF of the subject, e.g. at a given time such as at the second time or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 minutes thereof. The method can determine a decrease / increase (or no change) of the (actual) ISF relative to the basal ISF and / or relative to the ISF that was (actual) at an earlier time such as at the first time. The ISF at the first time may be known, e.g. prior determined such as based on performing the present method over time (and ultimately based on basal ISF) and / or may be determined by calculating the glucose lowering effect of an earlier insulin dosage and / or may be prior determined (e.g. by known means). The ISF at the first time may be the same as basal ISF. From the (actual) ISF, the skilled person will know how to calculate and administer an appropriate insulin dosage.

[0047] With (actual) Insulin Sensitivity Factor (ISF) is meant the ISF at a given time, such as at the second time or within 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 minutes thereof.

[0048] The Insulin Sensitive Factor (ISF) is defined as the amount of blood glucose level (mmol / L) that is lowered by one unit of insulin. The definition of one unit of insulin is historically set to be the amount of insulin required to lower the fasting blood sugar of a rabbit by 2.5 mmol / L. Now, it is known to be the equivalent to 0.0347mg of pure crystalline of insulin.

[0049] ISF is also sometimes referred to as the "correction factor" or "insulin sensitivity ratio". ISF may also be calculated by 100 / TDD, in which TTD is the total daily dose of all insulins (bolus and basal), e.g. units insulin / 24 hr needed to keep BG within the target range. This thus includes the basal and meals (bolus) units of insulin (a priori in 0.6 units insulin / kg body weight / day). If the basal ISF is not stably-determined for 1 day, the amount of insulin of 2 or even 3 days can be averaged. For example, if the subject needs 26 II of basal insulin per day and 8 II of bolus insulin per meal (3 meals), then the subject uses 26+3x8=50 units as total daily dose (TDD). The ‘100-rule’ can then be used to determine the ISF: ISF=100:50=2.

[0050] The basal ISF varies from person to person, typically is dependent on age, eating and exercise habits, and a subject’s (actual) ISF varies at different times of day or in different situations. Once the (actual) ISF is determined, it can be used to calculate the appropriate insulin dose needed to correct high blood glucose levels. For example, if the subject's target blood glucose level is 5.6 mmol / L and the subject’s current blood glucose level is 11.1 mmol / L, the subject would need two units of insulin to correct the blood glucose level (the difference between 11.1 mmol / L and 5.6 mmol / L, which is 5.5 mmol / L, divided by the ISF of in this case 2.8 mmol / L per unit of insulin).

[0051] The change in succinate level may be related to the change / deviation of ISF by the formula 1 / AISF = K * ([BS]tn+1-[BS]tn) / [BS]tn * 100 wherein ISF is the insulin sensitive factor, [BS]tn+1 and [BS]tn are blood succinate concentration at time-point n+1 or n, respectively, and K is a constant which can be determined in a training module. [BS] in uM (or mmol / L). K is anticipated to range between 1.5 and 3.0 for basal [BS] between 0 to 50 uM,, while K is anticipated to be between 1 and 0.6 for basal [BS] being above 50 uM.

[0052] In step a1), the (basal) Insulin Sensitivity Factor (ISF) of the subject is determined, preferably in conditions wherein the subject is at least 1 , 2, 3, 4, 5, 6 hours free from having any meal(s), physical activity and / or infection, more preferably in conditions wherein the subject has not taken an insulin dosage (e.g. a meal bolus or correction bolus) for at least 4 hours, has not eaten any food for the last 4 hours, is free from infection (and (major) emotional stress), and is at least 2 hours free from (strenuous) physical activity.

[0053] In step a2), the (basal) succinate level of the subject is determined. Preferably, the basal ISF and / or basal succinate level is determined before the first time as referred to above. The basal ISF and / or basal succinate level may be determined once, or periodically (e.g. every week, month or year, such as every 1-10 weeks, or every 1-12 months), whereas the (actual) ISF can be determined every 1 , 2, 3, 4, 5, 10, 15, 30 minutes or every 1 , 2, 3, 4, 5, 6 hours, such as every 1-60, preferably every 1-30 or 1-10 minutes. Having a meal by a subject generally refers to the act of consuming food and / or beverages that provide the body with nutrients, such as carbohydrates, proteins, and fats, as well as vitamins and minerals. Meal(s) can vary in size and composition depending on cultural, social, and individual factors. A meal can relate to more than 100, 200, 300, 400, 500 kilocalories. From a medical perspective, having a meal often refers to consuming a significant amount of carbohydrates, which can cause a rise in blood glucose levels. Physical activity by a subject generally refers to any bodily movement that results in energy expenditure, such as more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 calories per minute. This can include exercise, sports, leisure activities, or other forms of physical movement that are part of daily life, such as walking, climbing stairs, and doing household chores. An infection of a subject occurs when microorganisms such as bacteria, viruses, fungi, or parasites invade and multiply within the body, causing harm and disrupting normal bodily functions. Infections can range from mild to severe and can affect various parts of the body, including the skin, respiratory system, gastrointestinal system, urinary system, and nervous system. Diagnosis and treatment of infections typically involve identifying the specific pathogen responsible for the infection and using appropriate medications, such as antibiotics or antiviral drugs, to eradicate the infection.

[0054] The following protocol may be used to set the basal ISF for a new patient (starting usage of a CL-CGM system): • The total daily dose (TDD) is a priori roughly determined using the following formula: 0.6 unit rapid-acting insulin / kg body weight / day. For CL-CGM, 75% of this TDD is used of which:

[0055] • 30% is set for basal (i.e. continuous small injections) for adolescents (>12 year) and adults (and thus 60% for boluses with meals)

[0056] • 25% is set for basal for children from 6-12 years (and thus 75% for meals)

[0057] • 20% is set for basal for children < 6 years

[0058] This means that for a person of 70 kg, one starts with 12,6 units (0,3x42e) day, which is 0.525 unit / hr.

[0059] With usage of the CL-CGM during the first 2-3 days, the basal insulin dosage and thus the ISF is fine-tuned, which is often determined for three day periods of 8 hr in which meals are omitted (00.00-8.00; 8.00-16.00; 16.00-24.00). For such periods, different insulin infusion profiles can be set with the CL-CGM. If measured BG during the day is regularly at the lowest side of the set target range and there are frequent basal suspensions, the set prior rates are too high (and need to decreased by e.g. 0.025 u / hr). If the basal rate never or rarely gets suspended and BG during the day (and before a meal) is regularly at the higher side of the target BG range, the rates are likely too low (and need to be increased by e.g. 0.025 u / hr). Due to changes in IR and thus ISF, the set basal insulin dosing will still often not fully correct BG to the ideal target range. Using the ISF, this difference is then mostly corrected with the meal insulin bolus (with the CL-CGM devices, a ‘bolus wizard’ corrects the meal insulin bolus with the lack / surplus of insulin from the basal level).

[0060] The following criteria are at stake when choosing a time to test your ISF:

[0061] • Your blood glucose value is at least 50 mg / dl above your target

[0062] • You have not taken a meal bolus or correction bolus for at least 4 hours

[0063] • You have not eaten any food for the last 4 hours

[0064] • You can go without eating for the next 4 hours

[0065] Don’t test your correction factor during any circumstances that might affect the results, such as:

[0066] • After performing strenuous exercise

[0067] • If you have an illness or infection

[0068] • After a serious low blood glucose

[0069] • During major emotional stress

[0070] In steps b) and c), reference is made to a first time and a second time. The second time is preferably at least 1 , 5, 10 seconds, or at least 1, 5, 10 minutes, or at least 1 , 5, 10 hours, or at least 1 , 5, 10 days, or at least 1 , 5, 10 weeks after the first time (and / or between 1-10 seconds, or between 1-10 minutes, or between 1-10 hours, or between 1-10 days, or between 1-10 weeks after the first time). At the same time or, alternatively, the second time is preferably at most 1, 5, 10 minutes, or at most 1 , 5, 10 hours after the first time. Optionally, succinate level is also determined in a sample obtained at a third, fourth, fifth, and / or possible further times, wherein each subsequent time is preferably at least 1 , 5, 10 seconds, or at least 1, 5, 10 minutes, or at least 1, 5, 10 hours after the preceding time. At the same time or, alternatively, the subsequent time is preferably at most 1, 5, 10 minutes, or at most 1, 5, 10 hours after the preceding time. Accordingly, it can be determined if the succinate level determined for the subsequent time is lower, identical or higher than the succinate level determined for the preceding time, wherein

[0071] - a lower succinate level indicates a decrease in insulin resistance in the subject between the preceding time and the subsequent time;

[0072] - an identical succinate level indicates no change in insulin resistance in the subject between preceding time and the subsequent time; and / or

[0073] - a higher succinate level indicates an increase in insulin resistance in the subject between the preceding time and the subsequent time.

[0074] In step d) glucose level is determined in a sample obtained from the subject. The most common and reliable way to determine glucose levels in a blood sample is through a blood glucose test. For example, by continuous glucose monitoring: this involves wearing a small device called a glucose sensor which is placed upon or under the skin. The sensor continuously measures glucose levels in the interstitial fluid and transmits the data to a receiver or smartphone app.

[0075] In step e), the decrease or increase of (actual) ISF may be relative to the basal ISF, or relative to an earlier determined (actual) ISF, e.g. at a time at or before the first time, e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 minutes before the first time.

[0076] In the present disclosure, for example an 60% increase means an increase from e.g. 100 to 160.

[0077] The insulin dosage administered in step f) typically is sufficient to restore a healthy blood glucose level, preferably for a period of at least 1, 2, 3 ,4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60 minutes. A healthy reference glucose level typically is between 4-10 mmol / L preferably between 4-6.5 mmol / L. Of course, if the glucose level is below the healthy reference value, no insulin needs to be administered. Step f) may be performed after the second time such as within 1, 2, 3 ,4, 5, 6, 7, 8, 9, 10 minutes thereof. As will be clear, the insulin dosage in step f) is based on (actual / corrected) ISF, which is (more adequately) determined based on succinate level variation, i.e. ISF is corrected for any increases and decreases thereof over time, such as between the first and the second time, due to changes in succinate level.

[0078] Besides by automatic infusion of insulin via closed loop systems, insulin infusion by all other means of application of insulin dosing to T1 DM patients is foreseen, such as insulin (smart)pens.

[0079] The method according to the present disclosure may be performed over time, e.g. repeated at least every 1 , 2, 3, 4, 5, 6, 7, 8, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 60, 120 minutes. According to the present invention, it has been found that Insulin Sensitivity Factor (ISF) and / or insulin resistance (variability) is strongly associated with succinate level (variability) in a subject.

[0080] Succinate is an important metabolite at the cross-road of several metabolic pathways. Succinate(2-) is a dicarboxylic acid di-anion resulting from the removal of a proton from both of the carboxy groups of succinic acid:

[0081] Succinate levels can be determined by different methods available in the art. A Succinate Colorimetric Assay Kit (Sigma-Aldrich®) is a highly sensitive assay for determining succinate levels in a variety of samples. Succinate concentration is determined by a coupled enzyme reaction, which results in a colorimetric (450 nm) product proportional to the succinate present.

[0082] Alternatively, an EnzyChrom™ Succinate Assay Kit can be used for quantitative determination of succinate (succinic acid) in biological samples. In this assay succinate is converted to pyruvate which reacts with specific reagents and dye to form a coloured product. The colour intensity at 570 nm or fluorescence at Fex / em = 530 / 585 nm of the reaction product is directly proportional to succinate concentration in the sample. Succinate changes may also be used in connection to readouts of other body functions, such as brain activity (fMRI, EEG), heart activity (ECG), menstrual cycle / menopause / hot-cold environment (temperature).

[0083] In a preferred embodiment of the present disclosure, determining succinate level is performed by using an electrochemical biosensor as described elsewhere herein, preferably a voltametric biosensor or an amperometric biosensor.

[0084] The sample that is obtained prior to and subject to performing the steps as according to the method of the present disclosure (e.g. in steps b), c) and / or d)), may be a body fluid sample, wherein the body fluid sample preferably is chosen from the group consisting of blood sample, blood plasma sample, blood serum sample, tear fluid sample, saliva fluid sample, sweat sample, urine sample and interstitial fluid sample. Preferably the sample (e.g. in steps b), c) and / or d)) is not a stool sample. Accordingly, the method according to the present disclosure is preferably applied ex vivo. The present disclosure may exclude methods for treatment of the human or animal body by surgery or therapy and / or diagnostic methods practiced on the human or animal body.

[0085] The subject according to the present disclosure may be a mammalian subject, preferably a human subject. The subject may have an insulin resistance-related condition, which refers to conditions that may cause or are caused and / or exaggerated by insulin resistance. Alternatively, ‘insulin resistance-related conditions’ as used herein may refer to conditions wherein the symptoms have been associated with insulin resistance. Prevention / and or treatment of insulin resistance in insulin resistance-related conditions may relieve symptoms, mitigate progression of disease, and / or reverse disease. Prevention / and or treatment of insulin resistance in these conditions may also mitigate the chance of acquiring additional comorbidities associated with the insulin resistance-related condition. Insulin resistance-related conditions disclosed herein include type 1 diabetes mellitus, type 2 diabetes mellitus, dyslipidemia, metabolic syndrome, insulin resistance in endocrine disease, Polycystic Ovary Syndrome (PCOS), Non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH) and cancer (treatment), preferably insulin resistance, type 1 diabetes, type 2 diabetes, and metabolic syndrome. Also encompassed may be cardiovascular diseases, neurological diseases, endocrine diseases.

[0086] In some or all of these conditions, it is desired to monitor insulin resistance in a real time manner. In view thereof, the present method may be a computer-implemented method, wherein preferably insulin resistance variability is followed over time, and may be used as input for determining dosage of medication, e.g. insulin. In addition or alternatively, the present disclosure may provide for a computer-readable medium comprising instructions for performing the present method.

[0087] The term ‘type 1 diabetes mellitus’ is well-known to the skilled person. Type 1 diabetes mellitus is thought to result from an autoimmune destruction of pancreatic p-cells, and the predominant pathophysiology may be an almost absolute insulinopenia. Insulin resistance has also been described in type 1 diabetes mellitus and may be a potential target for intervention in addition to insulin therapy. Insulin resistance is often observed in subjects suffering type 1 diabetes mellitus during e.g. pubertal development, inter-current illness, female menstrual cycle changes, or during high-intensity exercise.

[0088] The term ‘type 2 diabetes mellitus’ is well-known to the skilled person. Patients with type 2 diabetes mellitus are characterized by high blood sugar, insulin resistance, and relative high level or lack of insulin as compared to healthy subjects. These patients experience a high insulin resistance or usually have passed through an earlier stage of insulin resistance, although this earlier stage often goes undiagnosed. It is widely accepted that insulin resistance is a powerful predictor of future development of type 2 diabetes mellitus, and also is the main therapeutic target once high blood sugar is present.

[0089] The term ‘dyslipidaemia’ is well-known to the skilled person. Dyslipidaemia is characterized by abnormal amount of lipids in the blood. In developed countries, most dyslipidaemias are hyperlipidaemias, or an elevation of lipids in the blood, which has a strong relation to cardiovascular disease. Insulin resistance, and the compensatory hyperinsulinemia that results, has been linked to dyslipidaemia. The characteristic dyslipidaemia of insulin resistance consists of elevated triglyceride and triglyceride-rich lipoprotein levels, low levels of high-density lipoprotein cholesterol (HDL), and increased concentrations of small low-density lipoprotein cholesterol (LDL).

[0090] The term ‘metabolic syndrome’ is well-known by the skilled person. Within the present disclosure, the term encompasses all conditions diagnosed as “metabolic syndrome” by an (authorized) medical practitioner. For example, metabolic syndrome may be diagnosed if a patient has at least two, or at least three of the following traits:

[0091] Large waist — A waistline that measures at least 35 inches (89 centimetres) for women and 40 inches (102 centimetres) for men;

[0092] High triglyceride level — 150 milligrams per decilitre (mg / dL), or 1.7 millimoles per litre (mmol / L), or higher of this type of fat found in blood; Reduced "good" or HDL cholesterol — Less than 40 mg / dL (1 .04 mmol / L) in men or less than 50 mg / dL (1.3 mmol / L) in women of high-density lipoprotein (HDL) cholesterol;

[0093] Increased blood pressure — 130 / 85 millimetres of mercury (mm Hg) or higher; Elevated fasting blood sugar — 100 mg / dL (5.6 mmol / L) or higher.

[0094] Metabolic syndrome is an example of an insulin resistance-related condition. Most people who have metabolic syndrome have insulin resistance. Insulin resistance has been suggested as an important underlying cause of metabolic syndrome.

[0095] The term ‘insulin resistance in endocrine disease’ refers to endocrine disorders associated with changes in sensitivity to insulin, often leading to high blood glucose. Examples are acromegaly, but also growth hormone deficiency, hypercortisolism in the course of Cushing's syndrome, hyper- or hypothyroidism, primary hyperparathyroidism, aldosteronism, pheochromocytoma, congenital hypertrophy of the adrenal glands, polycystic ovaries syndrome, hypogonadism, or other hormonally active neuroendocrine tumours. High blood glucose in insulin resistance in endocrine disease is often reversible, and may be reversed by insulin-sensitizing agents.

[0096] The term ‘Polycystic Ovary Syndrome’ (PCOS) refers to a full-body endocrine and metabolic condition that affects the ovaries and ovulation. PCOS is associated with profound insulin resistance as well as with defects in insulin secretion. Therefore, strategies to lower insulin resistance may lead to prevention and / or treatment of PCOS.

[0097] The term ‘Non-alcoholic fatty liver disease’ (NAFLD) refers to a group of conditions where there is accumulation of excess fat in the liver of people who drink little or no alcohol. The most common form of NAFLD is called fatty liver. NAFLD is strongly associated with insulin resistance and type 2 diabetes mellitus, therefore treatments of NAFLD may aim at lowering insulin resistance.

[0098] The term ‘Non-alcoholic steatohepatitis’ (NASH) refers to liver inflammation and damage caused by a build-up of fat in the liver. NASH is associated with a markedly increased risk of developing cirrhosis and hepatocellular carcinoma as well as other diseases not directly associated with liver damage, including increased risk of cardiovascular disease. An association between NASH and insulin resistance is well-known, and strategies to lower insulin resistance may decrease disease progression or symptoms in NASH. As used herein, the term “cancer” refers to or describes the physiological condition (e.g. in mammals) that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, breast cancer, colon cancer, lung cancer, prostate cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, skin cancer, blood cancer, leukemia, melanoma, head and neck cancer, and brain cancer. As used herein, “cancer” is also referred to as malignant neoplasm.

[0099] In a preferred embodiment of the present disclosure, determining succinate level is performed by using an electrochemical biosensor, preferably a voltametric biosensor or an amperometric biosensor. In the amperometry method, the measured current is directly related to the conversion (oxidation or reduction, preferably oxidation) of the substrate in the sample by the enzyme coupled to the amperometric device and with the applied potential during the electrochemical reaction.

[0100] Accordingly, the present disclosure also provides that determining succinate level is performed by

[0101] (i) contacting the sample with an amperometric sensor, wherein the sensor comprises an electrode coated with an immobilized enzyme; and

[0102] (ii) measuring current change, which corresponds to succinate level in the sample, wherein step (ii) is preferably performed at a constant voltage.

[0103] Further, the present disclosure provides for a sensor device for determining succinate level in a sample, wherein the sensor device comprises a (working) electrode coated with an immobilized enzyme.

[0104] As known by the skilled person, the sensor device or electrochemical biosensor, e.g. a voltametric biosensor or an amperometric biosensor, may comprise a working electrode, a reference electrode and / or a counter electrode. Preferably, the voltametric biosensor or the amperometric biosensor comprises a (working) electrode coated with an immobilized enzyme. Preferably said biosensor comprises a sample supply region.

[0105] The working electrode and / or the counter electrode may comprise or consist of carbon, optionally with ferricyanide mediator. The reference electrode may comprise or consist of silver / silverchloride.

[0106] The current changes may be measured by way of differential pulse voltametric. The method is preferably carried out at pH 6.5 to 8.5 at a 50-150, or 100-200 mV electrode potential.

[0107] The (immobilized) enzyme may be chosen from the group consisting of succinate dehydrogenase (SDH); succinate-coenzyme Q reductase (SQR); succinate: ubiquinone oxidoreductase; succinate:quinone oxidoreductase and / or succinate: (ubi)quinone reductase. quinol fumarate reductase (QFR);

[0108] Preferably the (immobilized) enzyme is (derived) from Escherichia coli.

[0109] The said succinate dehydrogenase (SDH) may comprise or be a dimer of FAD cofactor binding (flavo)protein (SDHA) and Iron-sulphur (Fe-S) cluster containing protein (SDHB). Preferably, the SDH according to the present disclosure comprises

[0110] SDHA, preferably having at least 20, 30, 40, 50, 70, 80, 90, 99, 100 sequence identity with SEQ ID NO:1 and / or

[0111] SDHB, preferably having at least 20, 30, 40, 50, 70, 80, 90, 99, 100 sequence identity with SEQ ID NO:2 (Janssen et al, 1997).

[0112] Preferably, the SQR according to the present disclosure comprises

[0113] DHSC, preferably having at least 20, 30, 40, 50, 70, 80, 90, 99, 100 sequence identity with SEQ ID NO:3 and / or

[0114] DHSD, preferably having at least 20, 30, 40, 50, 70, 80, 90, 99, 100 sequence identity with SEQ ID NO:4.

[0115] Preferably, the QFR according to the present disclosure comprises

[0116] FRDA, preferably having at least 20, 30, 40, 50, 70, 80, 90, 99, 100 sequence identity with SEQ ID NO:5 and / or

[0117] FRDB, preferably having at least 20%, 30, 40, 50, 70, 80, 90, 99, 100 sequence identity with SEQ ID NO:6 (Janssen et al., 1997).

[0118] FRDC, preferably having at least 20, 30, 40, 50, 70, 80, 90, 99, 100 sequence identity with SEQ ID NO:7 and / or

[0119] FRDD, preferably having at least 20, 30, 40, 50, 70, 80, 90, 99, 100 sequence identity with SEQ ID NO:8. It is to be noted that an alternative name for SQR is complex II of the tricarboxylic acid[TCA] / krebs cycle / electron transport chain (ETC).

[0120] The sensor device or electrochemical biosensor according to the present disclosure may be combined with a sensor device or electrochemical biosensor for determining glucose level in a sample obtained from the subject. This sensor device or electrochemical biosensor may comprise a (working) electrode coated with an immobilized enzyme, preferably glucose dehydrogenase or glucose oxygenase. As known by the skilled person, the sensor device or electrochemical biosensor, e.g. a voltametric biosensor or an amperometric biosensor, may comprise a working electrode, a reference electrode and / or a counter electrode. Preferably, the voltametric biosensor or the amperometric biosensor comprises a (working) electrode coated with an immobilized enzyme, e.g. glucose dehydrogenase / oxygenase. Preferably said biosensor comprises a sample supply region.

[0121] Succinate is generated by mitochondria and released from cells when these are under metabolic disbalance. With this process, also light in the visible wave-lengths (400-800 nm) is generated and emitted, and its intensity is increased in conditions of metabolic disbalance. While this ultra-weak photon emission (UPE; 10'16-1 O'18W / cm2) is below the sensitivity of the human eye (1012- 1014W / cm2), it can be detected non-invasively using highly-sensitive photomultiplier (PMT) or charge-coupled devices (CCD), e.g. on skin (to measure photon emission from skin) (Calcerrada & Garcia-Ruiz, 2019). Since changes in measured UPE are directly related to changes in succinate release, we also provide the use of UPE in measuring (changes in) insulin resistance in the conditions indicated.

[0122] In another aspect, the present disclosure relates to a method for determining insulin resistance variability in a subject over time (and / or determining progression / reduction of insulin resistance in a subject over time), the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein

[0123] - a lower succinate level indicates a decrease in insulin resistance in the subject between the first time and the second time;

[0124] - an identical succinate level indicates no change in insulin resistance in the subject between the first time and the second time; and / or

[0125] - a higher succinate level indicates an increase in insulin resistance in the subject between the first time and the second time. Also provided is a method for determining insulin resistance by using photo-multiplier (PMT) or charge-coupled devices (CCD). The method may comprise: a) determining photon emission in the visible wave-lengths (400-800 nm) from (skin of) a subject at a first time from the subject; b) determining photon emission in the visible wave-lengths (400-800 nm) from (skin of) a subject at a second time from the subject; c) determining if the photon emission determined under b) is lower, identical or higher than the photon emission determined under a), wherein

[0126] - a lower photon emission indicates a decrease in insulin resistance in the subject between the first time and the second time;

[0127] - an identical photon emission indicates no change in insulin resistance in the subject between the first time and the second time;

[0128] - a higher photon emission indicates an increase in insulin resistance in the subject between the first time and the second time. The second time may be at least 1 , 5, 10 seconds, or at least 1 , 5, 10 minutes, or at least 1 , 5, 10 hours after the first time.

[0129] The term ‘insulin resistance’ is well known by the skilled person. It is also well known that insulin resistance may vary within a patient over time. Within the present disclosure, the term encompasses all conditions diagnosed as ‘insulin resistance’ by an (authorized) medical practitioner. The term ‘insulin resistance’ as used herein preferably refers to peripheral insulin resistance and / or hepatic insulin resistance and / or (skeleto)muscular insulin resistance.

[0130] Insulin resistance may for example be diagnosed by the gold standard which is the "hyperinsulinemic euglycemic clamp" (DeFronzo et al, 1979). This method measures the amount of glucose necessary to compensate for an increased insulin level without causing hypoglycemia. The procedure may take two hours and typically involves the following steps: Through a peripheral vein, insulin is infused at 10-120 mU per m2per minute. In order to compensate for the insulin infusion, glucose 20% is infused to maintain blood sugar levels between 5 and 5.5 mmol / L. The rate of glucose infusion is determined by checking the blood sugar levels every five to ten minutes(Muniyappa et al, 2008). The rate of glucose infusion e.g. during the last thirty minutes of the test determines insulin sensitivity. If high levels (7.5 mg / min or higher) are required, the patient is insulin sensitive. Low levels (4.0 mg / min or lower) indicate insulin resistance. Levels between 4.0 and 7.5 mg / min are not definitive, and suggest "impaired glucose tolerance," an early sign of insulin resistance. Other methods of assessing insulin sensitivity or resistance include the "insulin-suppression test" (1ST), "minimal model analysis of frequently sampled intravenous glucose tolerance test" (FSIVGTT), or the "oral glucose tolerance test" (OGTT).The homeostatic model assessment for insulin resistance (HOMA or HOMA-IR) is an alternative method used to determine and quantify insulin resistance in a fasting steady-state condition, and correlates reasonably with the golden standard(Matthews et a / , 1985; Rudenski et al, 1991). A HOMA(-IR) score that deviates from a reference range can indicate insulin resistance. HOMA(-IR) has been widely applied in epidemiological studies and in experimental research. HOMA(-IR) denotes a value which represents an estimation for insulin resistance, derived from dividing Insulin and Glucose levels in the blood of a person. The HOMA(-IR) value can be calculated by the following equation: wherein H is the HOMA(-IR) value, Glucose represents fasting glucose levels in the blood expressed in mmol / L, Insulin represents fasting insulin levels in the blood expressed for example in mIU / L. IU (relating to enzyme activity) is an abbreviation of International Units, also called enzyme units. The skilled person is familiar with the methods used to quantify these levels. Enzyme activity is the amount of substrate converted per unit of time. One IU equals the conversion of one mol of substrate per minute. For an individual with normal insulin sensitivity, HOMA(-IR) may equal 1. The upper limit of normal HOMA(-IR) is frequently considered to be 2.0, although the normal HOMA(-IR) may be dependent on the characteristics of the population subgroup. Typically, a healthy subject has a HOMA-IR value below 2.0, preferably a HOMA-IR value below 1.9, 1.8, or 1.7, more preferably a HOMA-IR value below 1.6, 1.5, 1.4, 1.3, 1.2, 1.1 , and most preferred a HOMA-IR value below 1.0. A fasting serum insulin level greater than 25 mIU / L or 174 pmol / L has also been considered as indicating insulin resistance. In the present disclosure, insulin resistance may be measured by HOMA(-IR).

[0131] Cardiac pacemaker cells initiate and precisely regulate the spontaneous heart beat rhythm through modulation of an ion channel ensemble in sinoatrial nodal cells (SANC) through an intracellular Ca2+cycling clock. The SANC’s Ca2+clock is manifested by spontaneous, but precisely timed, rhythmic, submembrane, local Ca2+releases from sarcoplasmic reticulum (SR) that appear shortly before firing of the next action potential and which is orchestrated via intrinsic signalling mechanisms, such as phosphorylation cascades(Maltsev & Lakatta, 2007; Yaniv et al, 2015). Importantly, by acting on its receptor, rapid application of caffeine to a freshly-isolated SANC induced an immediate Ca2+ release from the sarcoplasmic reticulum and immediately reduced the AP cycle (i.e. heart beat rhythm; (Yaniv et al., 2015). The succinate receptor SUCNR1 has been localized to the sarcolemmal membrane and T-tubules of cardiomyocytes (Aguiar et al, 2014; Ariza et al, 2012), while SUCNR1 mRNA has also been localized to heart fibroblasts and endothelial cells (proteinatlas). Considering that (1) plasma succinate is increased in conditions of metabolic disbalance, (2) the SLICNR1 is located in cells regulating the heart beat rhythm, (3) activation of the SLICNR1 by bound succinate induces an intracellular Ca2+ spike that is also likely to interfere precise regulation of heart rhythm, (4) heart rhythm variability (HRV; differences in length between heart beats), as measured through electro-cardiogram analysis, is increased in conditions of disbalance (Kemp & Quintana, 2013), we also provide the use of HRV in measuring (changes in) insulin resistance in the conditions indicated.

[0132] Accordingly, provided is a method for determining insulin resistance by measuring HRV. The method may comprise: a) determining HRV of a subject at a first time from the subject; b) determining HRV of a subject at a second time from the subject; c) determining if the HRV determined under b) is lower, identical or higher than the HRV determined under a), wherein

[0133] - a lower HRV indicates a decrease in insulin resistance in the subject between the first time and the second time;

[0134] - an identical HRV indicates no change in insulin resistance in the subject between the first time and the second time;

[0135] - a higher HRV indicates an increase in insulin resistance in the subject between the first time and the second time. The second time may be at least 1 , 5, 10 seconds, or at least 1 , 5, 10 minutes, or at least 1 , 5, 10 hours after the first time.

[0136] The present disclosure further provides for the use of succinate for determining Insulin Sensitivity Factor (ISF) and / or insulin resistance in a subject, preferably ISF variability and / or insulin resistance variability in a subject over time.

[0137] In particular, the present disclosure provides for the use of succinate for determining an appropriate dosage of insulin to be administered to a subject in need of treatment with said insulin.

[0138] Also provided is a method for determining insulin resistance variability and / or succinate- induced insulin resistance variability for a subject, the method comprising: a) determining (basal) succinate level in a sample of the subject, preferably in conditions wherein the subject is at least 1 , 2, 3, 4 hours free from having meal, physical activity and infection; more preferably in conditions wherein the subject has slept as usual, refrained from food and caloric beverages for 16 hours, refrained from any (moderate) physical activity for the last 24 hours (or any vigorous physical activity for the last 48 hours), and remaining still when sampling; b) determining succinate level in a sample obtained at a first time from the subject; c) determining succinate level in a sample obtained at a second time from the subject, wherein the time period between the first time and the second time preferably covers conditions of having meal, physical activity and / or infection (and / or stress queue) by the subject; d) determining if the succinate level determined under c) is lower, identical or higher than the succinate level determined under b), wherein in case the basal succinate determined under a) is 0-50 pM:

[0139] - a succinate level increase between the first time and the second time by more than 40% indicates a decrease of insulin resistance by more than 40% and at most 80% of the subject between the first time and the second time;

[0140] - a succinate level increase between the first time and the second time by at least 10% and at most 40% indicates a decrease of insulin resistance by at least 15% and at most 40% of the subject between the first time and the second time,

[0141] - an identical or less than 10% deviation of succinate level between the first time and the second time indicates no or less than 15% change of insulin resistance of the subject between the first time and the second time;

[0142] - a succinate level decrease between the first time and the second time by more than 40% indicates an increase of insulin resistance by more than 60% and at most 400% of the subject between the first time and the second time;

[0143] - a succinate level decrease between the first time and the second time by more than 10% and at most 40% indicates an increase of insulin resistance by more than 15% and at most 60% of the subject between the first time and the second time; in case the basal succinate determined under a) is more than 50, preferably between 50 and 100 pM:

[0144] - a succinate level increase between the first time and the second time by more than 20% indicates a decrease of insulin resistance by more than 20% and at most 60% of the subject between the first time and the second time;

[0145] - a succinate level increase between the first time and the second time by at least 10% and at most 20% indicates a decrease of insulin resistance by at least 10% and at most 20% of the subject between the first time and the second time;

[0146] - an identical or less than 10% deviation of succinate level between the first time and the second time indicates no or less than 10% change of insulin resistance of the subject between the first time and the second time; - a succinate level decrease between the first time and the second time by more than 20% indicates an increase of insulin resistance by more than 40% and at most 100% of the subject between the first time and the second time;

[0147] - a succinate level decrease between the first time and the second time by more than 10% and at most 20% indicates an increase of insulin resistance by more than 10% and at most 40% of the subject between the first time and the second time.

[0148] The above method is useful in monitoring attacks in auto-immune disease, guiding dosing / monitoring the efficacy / efficiency of therapies for disorders related to insulinresistance. The insulin resistance at the first time may be prior determined or known.

[0149] In another aspect, the present disclosure relates to a method for determining disease severity and / or monitoring disease progression and / or disease treatment efficacy in a subject over time, the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein

[0150] - a lower succinate level indicates a decrease in disease severity and / or decrease in disease progression and / or positive disease treatment efficacy (e.g. increase in disease treatment efficacy) in the subject between the first time and the second time;

[0151] - an identical succinate level indicates no change in disease severity and / or disease progression and / or disease treatment efficacy in the subject between the first time and the second time; and / or

[0152] - a higher succinate level indicates an increase in disease severity and / or increase in disease progression and / or negative disease treatment efficacy (e.g. decrease in disease treatment efficacy) in the subject between the first time and the second time.

[0153] In yet another aspect, the present disclosure relates to a method for determining burnout severity and / or monitoring burnout progression and / or burnout treatment efficacy in a subject over time, the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein

[0154] - a lower succinate level indicates a decrease in burnout severity and / or decrease in burnout progression and / or positive burnout treatment efficacy (e.g. increase in burnout treatment efficacy) in the subject between the first time and the second time; - an identical succinate level indicates no change in burnout severity and / or burnout progression and / or burnout treatment efficacy in the subject between the first time and the second time; and / or

[0155] - a higher succinate level indicates an increase in burnout severity and / or increase in burnout progression and / or negative burnout treatment efficacy (e.g. decrease in burnout treatment efficacy) in the subject between the first time and the second time.

[0156] Burnout is a recognized disorder. In May 2019, the World Health Organization (WHO) officially classified burnout as an occupational phenomenon in the International Classification of Diseases (ICD-11). According to the ICD-11 , burnout is defined as a syndrome resulting from chronic workplace stress that has not been successfully managed. The three key dimensions of burnout are:

[0157] Feelings of energy depletion or exhaustion

[0158] Increased mental distance from one's job or feelings of negativism or cynicism related to one's job

[0159] Reduced professional efficacy

[0160] To diagnose burnout, a healthcare provider would typically assess a person's symptoms, duration, and impact on their daily functioning. Treatment for burnout may involve addressing the underlying workplace stressors, implementing self-care strategies, and seeking mental health support if needed. It is important to note that burnout is not the same as depression or anxiety, although it may coexist with these conditions.

[0161] In yet another aspect, the present disclosure relates to a method for determining cancer severity and / or monitoring cancer progression and / or cancer treatment efficacy and / or assessing subject’s resilience to cancer treatment (e.g. chemotherapy) in a subject over time, the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein

[0162] - a lower succinate level indicates a decrease in cancer severity and / or decrease in cancer progression and / or positive cancer treatment efficacy (e.g. increase in cancer treatment efficacy) and / or increase in resilience to cancer treatment in the subject between the first time and the second time; - an identical succinate level indicates no change in cancer severity and / or cancer progression and / or cancer treatment efficacy and / or resilience to cancer treatment in the subject between the first time and the second time; and / or

[0163] - a higher succinate level indicates an increase in cancer severity and / or increase in cancer progression and / or negative cancer treatment efficacy (e.g. decrease in cancer treatment efficacy) and / or decrease in resilience to cancer treatment in the subject between the first time and the second time.

[0164] Cancer is a broad term that refers to a group of diseases characterized by the uncontrolled growth and spread of abnormal cells in the body. Normally, cells in the body divide and grow in a controlled way, but in cancer, this process is disrupted and cells can grow and divide uncontrollably, leading to the formation of a mass of abnormal cells called a tumor.

[0165] There are many different types of cancer, each with its own characteristics and treatment options. Some common types of cancer include lung cancer, breast cancer, prostate cancer, and colorectal cancer, among others.

[0166] Resilience to cancer treatment, e.g. chemotherapy, refers to a patient's ability to tolerate and recover from the toxic effects of chemotherapy drugs. Chemotherapy is a type of cancer treatment that uses powerful drugs to kill cancer cells, but it can also damage healthy cells and tissues in the body, leading to side effects such as fatigue, nausea, vomiting, hair loss, and an increased risk of infections.

[0167] Patients who are more resilient to chemotherapy may be able to tolerate higher doses or longer courses of chemotherapy, which can lead to better outcomes in terms of cancer control or survival. Factors that may contribute to resilience to chemotherapy include a patient's age, overall health status, nutritional status, and the presence of other medical conditions.

[0168] In yet another aspect, the present disclosure relates to a method for determining myocardial infarction severity and / or monitoring myocardial infarction progression and / or myocardial infarction treatment efficacy and / or assessing subject’s resilience to myocardial infarction treatment in a subject over time, the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein - a lower succinate level indicates a decrease in myocardial infarction severity and / or decrease in myocardial infarction progression and / or positive myocardial infarction treatment efficacy (e.g. increase in myocardial infarction treatment efficacy) in the subject between the first time and the second time;

[0169] - an identical succinate level indicates no change in myocardial infarction severity and / or myocardial infarction progression and / or myocardial infarction treatment efficacy in the subject between the first time and the second time; and / or

[0170] - a higher succinate level indicates an increase in myocardial infarction severity and / or increase in myocardial infarction progression and / or negative myocardial infarction treatment efficacy (e.g. decrease in myocardial infarction treatment efficacy) in the subject between the first time and the second time.

[0171] Myocardial infarction (Ml), is a serious medical condition that occurs when blood flow to a part of the heart is blocked, usually by a blood clot. This can damage or destroy part of the heart muscle, which can lead to serious complications or even death.

[0172] The most common cause of a heart attack is coronary artery disease, which occurs when the arteries that supply blood to the heart become narrowed or blocked due to the buildup of plaque (a fatty substance) inside the artery walls. Other risk factors for heart attack include high blood pressure, smoking, obesity, diabetes, and a family history of heart disease.

[0173] In yet another aspect, the present disclosure relates to a method for determining cerebrovascular accident (CVA) severity and / or monitoring cerebrovascular accident (CVA) progression and / or cerebrovascular accident (CVA) treatment efficacy in a subject over time, the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein

[0174] - a lower succinate level indicates a decrease in cerebrovascular accident (CVA) severity and / or decrease in cerebrovascular accident (CVA) progression and / or positive cerebrovascular accident (CVA) treatment efficacy (e.g. increase in cerebrovascular accident (CVA) treatment efficacy) in the subject between the first time and the second time;

[0175] - an identical succinate level indicates no change in cerebrovascular accident (CVA) severity and / or cerebrovascular accident (CVA) progression and / or cerebrovascular accident (CVA) treatment efficacy in the subject between the first time and the second time; and / or - a higher succinate level indicates an increase in cerebrovascular accident (CVA) severity and / or increase in cerebrovascular accident (CVA) progression and / or negative cerebrovascular accident (CVA) treatment efficacy (e.g. decrease in cerebrovascular accident (CVA) treatment efficacy) in the subject between the first time and the second time.

[0176] A cerebrovascular accident (CVA), is a serious medical condition that occurs when blood flow to the brain is interrupted or reduced, either by a blood clot or a ruptured blood vessel. This can damage or destroy brain cells, leading to long-term disability or even death.

[0177] There are two main types of stroke: ischemic stroke and hemorrhagic stroke. Ischemic stroke occurs when a blood clot blocks a blood vessel in the brain, while hemorrhagic stroke occurs when a blood vessel in the brain ruptures and bleeds into the surrounding brain tissue.

[0178] In yet another aspect, the present disclosure relates to a method for determining Multiple sclerosis (MS) severity and / or monitoring Multiple sclerosis (MS) progression and / or Multiple sclerosis (MS) treatment efficacy in a subject over time, the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein

[0179] - a lower succinate level indicates a decrease in Multiple sclerosis (MS) severity and / or decrease in Multiple sclerosis (MS) progression and / or positive Multiple sclerosis (MS) treatment efficacy (e.g. increase in Multiple sclerosis (MS) treatment efficacy) in the subject between the first time and the second time;

[0180] - an identical succinate level indicates no change in Multiple sclerosis (MS) severity and / or Multiple sclerosis (MS) progression and / or Multiple sclerosis (MS) treatment efficacy in the subject between the first time and the second time; and / or

[0181] - a higher succinate level indicates an increase in Multiple sclerosis (MS) severity and / or increase in Multiple sclerosis (MS) progression and / or negative Multiple sclerosis (MS) treatment efficacy (e.g. decrease in Multiple sclerosis (MS) treatment efficacy) in the subject between the first time and the second time.

[0182] Multiple sclerosis (MS) is a chronic autoimmune disease that affects the central nervous system (CNS), which includes the brain and spinal cord. MS occurs when the immune system mistakenly attacks and damages the myelin sheath, a protective covering that surrounds nerve fibers in the CNS. This damage disrupts the normal flow of nerve impulses and can lead to a wide range of symptoms. In yet another aspect, the present disclosure relates to a method for determining infection severity and / or monitoring infection progression and / or infection treatment efficacy in a subject over time, the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein

[0183] - a lower succinate level indicates a decrease in infection severity and / or decrease in infection progression and / or positive infection treatment efficacy (e.g. increase in infection treatment efficacy) in the subject between the first time and the second time;

[0184] - an identical succinate level indicates no change in infection severity and / or infection progression and / or infection treatment efficacy in the subject between the first time and the second time; and / or

[0185] - a higher succinate level indicates an increase in infection severity and / or increase in infection progression and / or negative infection treatment efficacy (e.g. decrease in infection treatment efficacy) in the subject between the first time and the second time.

[0186] An infection is a condition that occurs when harmful microorganisms, such as bacteria, viruses, fungi, or parasites, invade and multiply within the body. In the case of COVID-19, it is caused by the SARS-CoV-2 virus, which can be transmitted through respiratory droplets from an infected person.

[0187] In yet another aspect, the present disclosure relates to a method for determining thyroid disease severity and / or monitoring thyroid disease progression and / or thyroid disease treatment efficacy in a subject over time, the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein

[0188] - a lower succinate level indicates a decrease in thyroid disease severity and / or decrease in thyroid disease progression and / or positive thyroid disease treatment efficacy (e.g. increase in thyroid disease treatment efficacy) in the subject between the first time and the second time;

[0189] - an identical succinate level indicates no change in thyroid disease severity and / or thyroid disease progression and / or thyroid disease treatment efficacy in the subject between the first time and the second time; and / or - a higher succinate level indicates an increase in thyroid disease severity and / or increase in thyroid disease progression and / or negative thyroid disease treatment efficacy (e.g. decrease in thyroid disease treatment efficacy) in the subject between the first time and the second time.

[0190] Thyroid disease is a medical condition that affects the function of the thyroid gland, a small butterfly-shaped gland located in the neck. The thyroid gland produces hormones that regulate metabolism, growth, and development throughout the body.

[0191] There are several different types of thyroid disease, including:

[0192] Sub clinical hypothyroidism (mild thyroid failure): peripheral thyroid hormone levels are within normal reference laboratory range, but serum thyroid-stimulating hormone (TSH) is mildly elevated. Many of these patients use thyroid hormone replacement therapy with e.g. levothyroxine;

[0193] Hypothyroidism: a condition in which the thyroid gland does not produce enough thyroid hormone, leading to a slower metabolism and a range of symptoms, such as fatigue, weight gain, constipation, depression, and dry skin.

[0194] Hyperthyroidism: a condition in which the thyroid gland produces too much thyroid hormone, leading to a faster metabolism and a range of symptoms, such as weight loss, rapid heartbeat, anxiety, irritability, and heat intolerance.

[0195] Thyroid nodules: small lumps or growths on the thyroid gland that can be benign or cancerous. Most thyroid nodules are non-cancerous and do not cause symptoms, but some may produce excess thyroid hormone or grow large enough to cause difficulty swallowing or breathing.

[0196] Thyroid cancer: a rare form of cancer that develops in the cells of the thyroid gland. Most cases of thyroid cancer are treatable and have a good prognosis if detected early.

[0197] In yet another aspect, the present disclosure relates to a method for determining Parkinson’s disease severity and / or monitoring Parkinson’s disease progression and / or Parkinson’s disease treatment efficacy (e.g. uptake and / or treatment by levodopa) in a subject over time, the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein - a lower succinate level indicates a decrease in Parkinson’s disease severity and / or decrease in Parkinson’s disease progression and / or positive Parkinson’s disease treatment efficacy (e.g. increase in Parkinson’s disease treatment efficacy) in the subject between the first time and the second time;

[0198] - an identical succinate level indicates no change in Parkinson’s disease severity and / or Parkinson’s disease progression and / or Parkinson’s disease treatment efficacy in the subject between the first time and the second time; and / or

[0199] - a higher succinate level indicates an increase in Parkinson’s disease severity and / or increase in Parkinson’s disease progression and / or negative Parkinson’s disease treatment efficacy (e.g. decrease in Parkinson’s disease treatment efficacy) in the subject between the first time and the second time.

[0200] Parkinson's disease is a chronic and progressive neurological disorder that affects movement and coordination. It is caused by the degeneration of dopamine-producing neurons in the brain, which leads to a decrease in dopamine levels. Dopamine is a neurotransmitter that helps to control movement and emotional response.

[0201] In yet another aspect, the present disclosure relates to a method for determining nausea severity and / or monitoring nausea progression and / or nausea treatment efficacy in a subject over time, the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein

[0202] - a lower succinate level indicates a decrease in nausea severity and / or decrease in nausea progression and / or positive nausea treatment efficacy (e.g. increase in nausea treatment efficacy) in the subject between the first time and the second time;

[0203] - an identical succinate level indicates no change in nausea severity and / or nausea progression and / or nausea treatment efficacy in the subject between the first time and the second time; and / or

[0204] - a higher succinate level indicates an increase in nausea severity and / or increase in nausea progression and / or negative nausea treatment efficacy (e.g. decrease in nausea treatment efficacy) in the subject between the first time and the second time.

[0205] Nausea is an unpleasant sensation of discomfort in the upper abdomen that is often accompanied by a feeling of the urge to vomit and thus may include vomiting. It is a common symptom that can be caused by a variety of factors, including: Stomach or gastrointestinal problems, such as gastroenteritis, food poisoning, or indigestion Motion sickness or vertigo

[0206] Pregnancy, particularly during the first trimester

[0207] Medications, such as chemotherapy drugs or opioids

[0208] Anxiety or stress

[0209] Migraines or other headaches

[0210] Infections, such as the flu or a sinus infection

[0211] Other medical conditions, such as acid reflux or pancreatitis.

[0212] In yet another aspect, the present disclosure relates to a method for determining gastroesophageal reflux disease (GERD) severity and / or monitoring GERD progression and / or GERD treatment efficacy in a subject over time, the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein

[0213] - a lower succinate level indicates a decrease in GERD severity and / or decrease in GERD progression and / or positive GERD treatment efficacy (e.g. increase in GERD treatment efficacy) in the subject between the first time and the second time;

[0214] - an identical succinate level indicates no change in GERD severity and / or GERD progression and / or GERD treatment efficacy in the subject between the first time and the second time; and / or

[0215] - a higher succinate level indicates an increase in GERD severity and / or increase in GERD progression and / or negative GERD treatment efficacy (e.g. decrease in GERD treatment efficacy) in the subject between the first time and the second time.

[0216] Symptoms of GERD are: a burning sensation in the chest (heartburn), regurgitation of food / sour liquid, upper abdominal chest pain, trouble swallowing, ongoing cough, new / worsening asthma, inflammation of vocal cords. These common symptoms can be caused by a variety of factors, including:

[0217] Hypersensitivity to food, such as greasy / fat-laden food, tomatoes, citrus fruits, spicy food, chocolate, caffeinated beverages, food poisoning

[0218] Overweight / obesity

[0219] Smoking

[0220] Pregnancy, particularly during the first trimester Medications, such as non-steroidal anti-inflammatory drugs (NSAIDs), antidepressants, chemotherapy drugs, opioids, calcium channel blockers

[0221] In yet another aspect, the present disclosure relates to a method for determining Chronic Fatigue Syndrome (CFS) severity and / or monitoring Chronic Fatigue Syndrome (CFS) progression and / or Chronic Fatigue Syndrome (CFS) treatment efficacy in a subject over time, the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein

[0222] - a lower succinate level indicates a decrease in Chronic Fatigue Syndrome (CFS) severity and / or decrease in Chronic Fatigue Syndrome (CFS) progression and / or positive Chronic Fatigue Syndrome (CFS) treatment efficacy (e.g. increase in Chronic Fatigue Syndrome (CFS) treatment efficacy) in the subject between the first time and the second time;

[0223] - an identical succinate level indicates no change in Chronic Fatigue Syndrome (CFS) severity and / or Chronic Fatigue Syndrome (CFS) progression and / or Chronic Fatigue Syndrome (CFS) treatment efficacy in the subject between the first time and the second time; and / or

[0224] - a higher succinate level indicates an increase in Chronic Fatigue Syndrome (CFS) severity and / or increase in Chronic Fatigue Syndrome (CFS) progression and / or negative Chronic Fatigue Syndrome (CFS) treatment efficacy (e.g. decrease in Chronic Fatigue Syndrome (CFS) treatment efficacy) in the subject between the first time and the second time.

[0225] Chronic Fatigue Syndrome (CFS), also known as myalgic encephalomyelitis (ME), is a complex and debilitating disorder characterized by profound fatigue that is not relieved by rest and is often accompanied by other symptoms such as pain, cognitive impairment, and sleep disturbances.

[0226] In yet another aspect, the present disclosure relates to a method for determining Primary mitochondrial disease (PMD) severity and / or monitoring Primary mitochondrial disease (PMD) progression and / or Primary mitochondrial disease (PMD) treatment efficacy in a subject over time, the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein - a lower succinate level indicates a decrease in Primary mitochondrial disease (PMD) severity and / or decrease in Primary mitochondrial disease (PMD) progression and / or positive Primary mitochondrial disease (PMD) treatment efficacy (e.g. increase in Primary mitochondrial disease (PMD) treatment efficacy) in the subject between the first time and the second time;

[0227] - an identical succinate level indicates no change in Primary mitochondrial disease (PMD) severity and / or Primary mitochondrial disease (PMD) progression and / or Primary mitochondrial disease (PMD) treatment efficacy in the subject between the first time and the second time; and / or

[0228] - a higher succinate level indicates an increase in Primary mitochondrial disease (PMD) severity and / or increase in Primary mitochondrial disease (PMD) progression and / or negative Primary mitochondrial disease (PMD) treatment efficacy (e.g. decrease in Primary mitochondrial disease (PMD) treatment efficacy) in the subject between the first time and the second time.

[0229] Primary mitochondrial disease (PMD) is a group of complex genetic disorders that arise due to pathogenic variants in nuclear or mitochondrial genomes.

[0230] In yet another aspect, the present disclosure relates to a method for determining allergy severity and / or monitoring allergy progression and / or allergy treatment efficacy in a subject over time, the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein

[0231] - a lower succinate level indicates a decrease in allergy severity and / or decrease in allergy progression and / or positive allergy treatment efficacy (e.g. increase in allergy treatment efficacy) in the subject between the first time and the second time;

[0232] - an identical succinate level indicates no change in allergy severity and / or allergy progression and / or allergy treatment efficacy in the subject between the first time and the second time; and / or

[0233] - a higher succinate level indicates an increase in allergy severity and / or increase in allergy progression and / or negative allergy treatment efficacy (e.g. decrease in allergy treatment efficacy) in the subject between the first time and the second time.

[0234] An allergy is an overreaction of the body's immune system to a substance that is normally harmless, such as pollen, dust, certain foods, or medications. When a person with an allergy comes into contact with an allergen, their immune system produces an antibody called immunoglobulin E (IgE), which triggers the release of histamine and other chemicals that cause allergy symptoms. Preferably the term allergy as used herein excludes gluten allergy.

[0235] In yet another aspect, the present disclosure relates to a method for determining withdrawal syndrome severity and / or monitoring withdrawal syndrome progression and / or withdrawal syndrome treatment efficacy and / or withdrawal syndrome treatment resilience in a subject over time, the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein

[0236] - a lower succinate level indicates a decrease in withdrawal syndrome severity and / or decrease in withdrawal syndrome progression and / or positive withdrawal syndrome treatment efficacy (e.g. increase in withdrawal syndrome treatment efficacy) and / or increased resilience to withdrawal syndrome treatment in the subject between the first time and the second time;

[0237] - an identical succinate level indicates no change in withdrawal syndrome severity and / or withdrawal syndrome progression and / or withdrawal syndrome treatment efficacy in the subject between the first time and the second time; and / or

[0238] - a higher succinate level indicates an increase in withdrawal syndrome severity and / or increase in withdrawal syndrome progression and / or negative withdrawal syndrome treatment efficacy (e.g. decrease in withdrawal syndrome treatment efficacy) and / or decreased resilience to withdrawal syndrome treatment in the subject between the first time and the second time.

[0239] Withdrawal syndrome, also known as withdrawal disease, is a set of symptoms that can occur when a person stops using a substance or medication that they have been using regularly for a period of time. Withdrawal syndrome is a physical and / or psychological response to the absence of the substance, and the severity and duration of the symptoms can vary depending on the type of substance, the duration of use, and other individual factors. Patients show a large inter- and intra-individual variability in the resilience to withdrawal therapy.

[0240] Withdrawal syndrome can occur with a wide range of substances, including alcohol, opioids, benzodiazepines, stimulants, and nicotine, among others. In yet another aspect, the present disclosure relates to a method for monitoring health status / resilience (e.g. risk for disease development) in a subject over time, the method comprising: a) determining (basal) succinate level in a sample obtained at a first time from the subject; b) determining (basal) succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein

[0241] - a lower (basal) succinate level indicates an increase in health status / resilience and / or reduced risk for disease development in the subject between the first time and the second time;

[0242] - an identical (basal) succinate level indicates no change in health status in the subject between the first time and the second time; and / or

[0243] - a higher (basal) succinate level indicates a decrease in health status / resilience and / or increased risk for disease development in the subject between the first time and the second time.

[0244] Health status refers to an individual's overall state of health, which is determined by various factors, such as physical, mental, and social well-being. A person's health status can be influenced by a range of factors, including genetics, lifestyle choices, environmental factors, and access to healthcare services. A sustained optimal health status leads to longevity

[0245] Some common indicators of health status include measures such as body mass index (BMI), blood pressure, cholesterol levels, and blood glucose levels. Other factors that can be used to assess an individual's health status include their overall physical fitness, mental health, and ability to perform daily activities without difficulty.

[0246] In yet another aspect, the present disclosure relates to a method for monitoring labour fatigue in a subject over time, the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein

[0247] - a lower succinate level indicates a decrease in labour fatigue in the subject between the first time and the second time;

[0248] - an identical succinate level indicates no change in labour fatigue in the subject between the first time and the second time; and / or - a higher succinate level indicates an increase in labour fatigue in the subject between the first time and the second time.

[0249] Labor fatigue refers to the physical and mental exhaustion experienced by workers due to long and demanding work hours, inadequate rest breaks, and strenuous physical or mental activity. It is a common problem in many industries, including manufacturing, construction, healthcare, and transportation, among others.

[0250] Symptoms of labor fatigue may include reduced physical and cognitive performance, decreased concentration and attention, irritability, and mood swings. It can also lead to a higher risk of accidents and injuries in the workplace.

[0251] In any of the methods according to the present disclosure: the sample that is obtained may be a body fluid sample, wherein the body fluid sample preferably is chosen from the group consisting of blood sample, blood plasma sample, blood serum sample, tear fluid sample, saliva fluid sample, sweat sample, urine sample and interstitial fluid sample. Preferably the sample (e.g. in steps b), c) and / or d)) is not a stool sample. Accordingly, the method according to the present disclosure is preferably applied ex vivo. The present disclosure may exclude methods for treatment of the human or animal body by surgery or therapy and / or diagnostic methods practiced on the human or animal body. The subject according to the present disclosure may be a mammalian subject, preferably a human subject.

[0252] In the present description and examples, a number of terms are used. Unless otherwise defined herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0253] As used herein, the term “identity" (or homology) refers to a measure of the identity of nucleotide sequences or amino acid sequences. In general, the sequences are aligned so that the highest order match is obtained. "Identity" perse has an art-recognized meaning and can be calculated using published techniques. See, e.g.: (COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A. M., ed., Oxford University Press, New York, 1988; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, Smith, D. W., ed., Academic Press, New York, 1993; COMPUTER ANALYSIS OF SEQUENCE DATA, PART I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von Heinje, G., Academic Press, 1987; and SEQUENCE ANALYSIS PRIMER; Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there exist a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to skilled artisans. Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in GUIDE TO HUGE COMPUTERS, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipton, D., SIAM J. Applied Math (1988) 48:1073. Methods to determine identity and similarity are codified in computer programs. For example NCBI Nucletide Blast with standard settings (blastn, https: / / blast.ncbi.nlm.nih.gov / ). Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCS program package (Altschul et al, 1990; Devereux et al, 1984).

[0254] As an illustration, by a polypeptide or nucleotide sequence having at least, for example, 95% "identity" to a reference polypeptide or nucleotide sequence, it is intended that the polypeptide or nucleotide sequence is identical to the reference sequence, except that there may be up to five point mutations per each 100 amino acids or nucleotides of the reference polypeptide or nucleotide sequence. In other words, to obtain a polypeptide or nucleotide sequence being at least 95% identical to a reference polypeptide or nucleotide sequence, up to 5% of the amino acids or nucleotides in the reference sequence may be deleted and / or substituted with another residue or nucleotide, and / or a number of residues or nucleotides up to 5% of the total residues or nucleotides in the reference sequence may be inserted into the reference sequence. Preferably, the sequence identity refers to the sequence identity over the entire length of the sequence. It is further understood that, when referring to “sequences” herein, generally the actual physical molecules with a certain sequence of subunits (e.g. amino acids) are referred to.

[0255] Furthermore, the skilled person also knows that polypeptides having a different amino acid sequence can have the same activity. It is common general knowledge that it is often possible to substitute a certain amino acid by another one, without loss of activity of the polypeptide. For example, the following amino acids can often be exchanged for one another:

[0256] Ala, Ser, Thr, Gly (small aliphatic, nonpolar or slightly polar residues) Asp, Asn, Glu, Gin (polar, negatively charged residues and their amides) His, Arg, Lys (polar, positively charged residues) Met, Leu, lie, Vai (Cys) (large aliphatic, nonpolar residues) Phe, Ty, Trp (large aromatic residues)

[0257] (refer for example to Schulz, G. E. et al, Principles of Protein Structure, Springer- Verlag, New York, 1979, and Creighton, T.E., Proteins: Structure and Molecular Principles, W.H. Freeman & Co., San Francisco, 1984) Preferred "substitutions" are those that are conservative, i.e., wherein the residue is replaced by another of the same general type. In making changes, the hydropathic index of amino acids may be considered. It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a polypeptide having similar biological activity. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those that are within ±1 are more preferred, and those within ±0.5 are even more preferred. Similarly, select amino acids may be substituted by other amino acids having a similar hydrophilicity, as set forth in U.S. Pat. No. 4,554,101. In making such changes, as with the hydropathic indices, the substitution of amino acids whose hydrophilicity indices are within ±2 is preferred, those that are within ±1 are more preferred, and those within ±0.5 are even more preferred.

[0258] So, multiple variations of the polypeptide of the present disclosure that have the unexpected and advantageous effect of the present invention are also well within grasp of the skilled person. It is not difficult to determine and evaluate whether a particular polypeptide falling within the terms of the disclosure confers the technical effect of the disclosure.

[0259] Clauses

[0260] 1. Method for determining insulin resistance variability in a subject over time, the method comprising: a) determining succinate level in a sample obtained at a first time from the subject; b) determining succinate level in a sample obtained at a second time from the subject; c) determining if the succinate level determined under b) is lower, identical or higher than the succinate level determined under a), wherein

[0261] - a lower succinate level indicates a decrease in insulin resistance in the subject between the first time and the second time;

[0262] - an identical succinate level indicates no change in insulin resistance in the subject between the first time and the second time;

[0263] - a higher succinate level indicates an increase in insulin resistance in the subject between the first time and the second time.

[0264] 2. Method according to clause 1 , wherein the second time is at least 1, 5, 10 seconds, or at least 1 , 5, 10 minutes, or at least 1 , 5, 10 hours after the first time.

[0265] 3. Method according to any one of the previous clauses, wherein insulin resistance is determined by HOMA-IR. 4. Method according to any one of the previous clauses, wherein the sample is a body fluid sample, wherein the body fluid sample preferably is chosen from the group consisting of blood sample, blood plasma sample, blood serum sample, tear fluid sample, saliva fluid sample, sweat sample, and interstitial fluid sample.

[0266] 5. Method according to any one of the previous clauses, wherein the subject has Type 1 Diabetes Mellitus, Type 2 Diabetes Mellitus, obesity, metabolic syndrome, Non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), cancer, and / or cardiovascular, neurologic and endocrinologic diseases.

[0267] 6. Method according to any one of the previous clauses, wherein the method is performed ex vivo.

[0268] 7. Method according to any one of the previous clauses, wherein determining succinate level is performed by measuring heart rhythm variability (HRV).

[0269] 8. Method according to any one of the previous clauses, wherein determining succinate level is performed by using an electrochemical biosensor, preferably a voltametric biosensor or an amperometric biosensor.

[0270] 9. Method according to clause 8, wherein the voltametric biosensor or the amperometric biosensor comprises an electrode coated with an immobilized enzyme.

[0271] 10. Method according to any one of the previous clauses, wherein determining succinate level is performed by

[0272] (i) contacting the sample with an amperometric sensor, wherein the sensor comprises an electrode coated with an immobilized enzyme; and

[0273] (ii) measuring current change, which corresponds to succinate level in the sample.

[0274] 11 . Method according to any one of clauses 9-10, wherein the enzyme is succinate dehydrogenase (SDH), succinate-coenzyme Q reductase (SQR), quinol fumarate reductase (QFR), succinate:ubiquinone oxidoreductase, succinate:quinone oxidoreductase or succinate:(ubi)quinone reductase. 12. Method according to clause 11 , wherein said succinate dehydrogenase (SDH) comprises or is a dimer of FAD cofactor binding (flavo)protein (SDHA) and Iron-sulfur (Fe-S) cluster containing protein (SDHB).

[0275] 13. A sensor device for determining succinate level in a sample comprising an electrode coated with an immobilized enzyme, wherein the enzyme is succinate dehydrogenase (SDH), succinate-coenzyme Q reductase (SQR), quinol fumarate reductase (QFR), succinate:ubiquinone oxidoreductase, succinate:quinone oxidoreductase or succinate:(ubi)quinone reductase.

[0276] 14. Sensor according to clause 12, wherein said succinate dehydrogenase (SDH) comprises or is a dimer of FAD cofactor binding (flavo)protein (SDHA) and Iron-sulphur (Fe-S) cluster containing protein (SDHB).

[0277] 15. Use of succinate for determining insulin resistance in a subject, preferably insulin resistance variability in a subject over time.

[0278] 16. Use of succinate for determining an appropriate dosage of insulin to be administered to a subject in need of treatment with said insulin.

[0279] In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

[0280] Brief description of the figures

[0281] Figure 1 shows a simplified schematic of the working mechanism of an amperometric sensors, consisting of a working (WE), counter (CE), and reference (RE) electrodes. A current between the working and counter electrode is generated by the redox reaction between the SDH / SQR complex oxidizing succinate to fumarate and the modified working electrode, while the opposite reaction takes place at the counter electrode. A constant voltage, sufficiently high to initiate the reduction or oxidation of succinate, is applied between the working and reference electrode. This results in the generation of an electrochemical catalytic current between the working and counter electrode, which is proportional to the concentration of the biomarker, succinate. Figure 2 shows Dose-response curve of human SUCNR1 to succinate, lnositol-3 phosphate formation by cells expressing human SLICNR1 in response to different concentrations of succinate. Taken from Rexen-Ulven, Sci reports, 2018, Pubmed 29968758.

[0282] Sequences referred to:

[0283] Primary amino acid (AA) sequences of the E.coli strain K12 homologues of the SQR subunits SDHA (No. 1), SDHB (No 2), SDHC (No 3), SDHD (No 4) and the QFR subunits QFRA (No. 5), QFRB (No. 6), QFRC (No 7) and QFRD (No 8). Sequences were obtained from the uniprot blast server.

[0284] SEQ ID NO:1 : SDHA_ECOLI; 588 AA

[0285] MKLPVREFDA VVIGAGGAGM RAALQISQSG QTCALLSKVF PTRSHTVSAQ GGITVALGNT

[0286] HEDNWEWHMY DTVKGSDYIG DQDAIEYMCK TGPEAILELE HMGLPFSRLD DGRIYQRPFG

[0287] GQSKNFGGEQ AARTAAAADR TGHALLHTLY QQNLKNHTTI FSEWYALDLV KNQDGAVVGC

[0288] TALCIETGEV VYFKARATVL ATGGAGRIYQ STTNAHINTG DGVGMAIRAG VPVQDMEMWQ

[0289] FHPTGIAGAG VLVTEGCRGE GGYLLNKHGE RFMERYAPNA KDLAGRDVVA RSIMIEIREG

[0290] RGCDGPWGPH AKLKLDHLGK EVLESRLPGI LELSRTFAHV DPVKEPIPVI

[0291] PTCHYMMGGI

[0292] PTKVTGQALT VNEKGEDVVV PGLFAVGEIA CVSVHGANRL GGNSLLDLVV FGRAAGLHLQ

[0293] ESIAEQGALR DASESDVEAS LDRLNRWNNN RNGEDPVAIR KALQECMQHN FSVFREGDAM

[0294] AKGLEQLKVI RERLKNARLD DTSSEFNTQR VECLELDNLM ETAYATAVSA NFRTESRGAH

[0295] SRFDFPDRDD ENWLCHSLYL PESESMTRRS VNMEPKLRPA FPPKIRTY

[0296] SEQ ID NO:2: SDHB_ECOLI 238 AA

[0297] MRLEFSIYRY NPDVDDAPRM QDYTLEADEG RDMMLLDALI QLKEKDPSLS FRRSCREGVC

[0298] GSDGLNMNGK NGLACITPIS ALNQPGKKIV IRPLPGLPVI RDLVVDMGQF

[0299] YAQYEKIKPY

[0300] LLNNGQNPPA REHLQMPEQR EKLDGLYECI LCACCSTSCP SFWWNPDKFI GPAGLLAAYR

[0301] FLIDSRDTET DSRLDGLSDA FSVFRCHSIM NCVSVCPKGL NPTRAIGHIK SMLLQRNA

[0302] SEQ ID NO:3: DHSC_ECOLI 129 AA

[0303] MIRNVKKQRP VNLDLQTIRF PITAIASILH RVSGVITFVA VGILLWLLGT SLSSPEGFEQ ASAIMGSFFV KFIMWGILTA LAYHWVGIR HMMMDFGYLE ETFEAGKRSA KISFVITWL SLLAGVLVW

[0304] SEQ ID NO:4: DHSD_ECOLI 115 AA

[0305] MVSNASALGR NGVHDFILVR ATAIVLTLYI IYMVGFFATS GELTYEVWIG FFASAFTKVF TLLALFSILI HAWIGMWQVL TDYVKPLALR LMLQLVIWA LWYVIYGFV VVWGV

[0306] SEQ ID N0:5: FRDA_ECOLI 602 AA

[0307] MQTFQADLAI VGAGGAGLRA AIAAAQANPN AKIALISKVY PMRSHTVAAE GGSAAVAQDH

[0308] DSFEYHFHDT VAGGDWLCEQ DVVDYFVHHC PTEMTQLELW GCPWSRRPDG SVNVRRFGGM

[0309] KIERTWFAAD KTGFHMLHTL FQTSLQFPQI QRFDEHFVLD ILVDDGHVRG LVAMNMMEGT

[0310] LVQIRANAW MATGGAGRVY RYNTNGGIVT GDGMGMALSH GVPLRDMEFV

[0311] QYHPTGLPGS

[0312] GILMTEGCRG EGGILVNKNG YRYLQDYGMG PETPLGEPKN KYMELGPRDK VSQAFWHEWR

[0313] KGNTISTPRG DWYLDLRHL GEKKLHERLP FICELAKAYV GVDPVKEPIP

[0314] VRPTAHYTMG

[0315] GIETDQNCET RIKGLFAVGE CSSVGLHGAN RLGSNSLAEL VVFGRLAGEQ

[0316] ATERAATAGN

[0317] GNEAAIEAQA AGVEQRLKDL VNQDGGENWA KIRDEMGLAM EEGCGIYRTP

[0318] ELMQKTIDKL

[0319] AELQERFKRV RITDTSSVFN TDLLYTIELG HGLNVAECMA HSAMARKESR GAHQRLDEGC

[0320] TERDDVNFLK HTLAFRDADG TTRLEYSDVK ITTLPPAKRV YGGEADAADK

[0321] AEAANKKEKA

[0322] NG

[0323] SEQ ID NO:6: FRDB_ECOLI 244 A A

[0324] MAEMKNLKIE VVRYNPEVDT APHSAFYEVP YDATTSLLDA LGYIKDNLAP DLSYRWSCRM

[0325] AICGSCGMMV NNVPKLACKT FLRDYTDGMK VEALANFPIE RDLVVDMTHF IESLEAIKPY

[0326] IIGNSRTADQ GTNIQTPAQM AKYHQFSGCI NCGLCYAACP QFGLNPEFIG PAAITLAHRY

[0327] NEDSRDHGKK ERMAQLNSQN GVWSCTFVGY CSEVCPKHVD PAAAIQQGKV ESSKDFLIAT

[0328] LKPR

[0329] SEQ ID NO:7: FRDC_ECOLI 131 AA

[0330] MTTKRKPYVR PMTSTWWKKL PFYRFYMLRE GTAVPAVWFS IELIFGLFAL

[0331] KNGPEAWAGF

[0332] VDFLQNPVIV IINLITLAAA LLHTKTWFEL APKAANIIVK DEKMGPEPII KSLWAVTVVA TIVILFVALY W

[0333] SEQ ID NO:8: FRDD_ECOLI 119 AA

[0334] MINPNPKRSD EPVFWGLFGA GGMWSAIIAP VMILLVGILL PLGLFPGDAL

[0335] SYERVLAFAQ

[0336] SFIGRVFLFL MIVLPLWCGL HRMHHAMHDL KIHVPAGKWV FYGLAAILTV VTLIGVVTI

[0337] The following Examples illustrate the different embodiments of the invention. Example 1

[0338] Insulin resistance

[0339] In healthy, mostly young, individuals, basal succinate levels and insulin resistance can be expected to be low (10-20 uM for succinate). Depending on their intensity, triggers of metabolic imbalance are anticipated to increase blood succinate and insulin resistance levels. Relatively strong triggers include high intensity exercise, pregnancy, menopause, alcohol abuse, food intolerances / allergies, infection, and inflammation. Intermediate strong triggers include nervosity and female hormonal changes.

[0340] In persons with chronically underlying morbidity, such as people suffering from Type-1 / type-2 diabetes mellitus, dyslipidaemia, metabolic syndrome, obesity, PCOS, NAFLD, NASH, COPD, intestinal bowel disease, endocrine / cardiovascular / neurological disease related insulin resistance, cancer, or longstanding mental problems, basal succinate levels are anticipated to be increased. In these individuals, the triggers of metabolic imbalance indicated above are anticipated to lead to more moderately increased succinate and insulin resistance levels.

[0341] The succinate and insulin resistance values are indicated in Table 1. Succinate level relative to time point 1 was determined as disclosed herein on the same day for all subjects before (time point 1), on top (time point 2) and shortly after (time point 3) the trigger of metabolic imbalance. A healthy subject of 20-30 years old will have a low, basal level of succinate and insulin resistance before any trigger (i.e. time point 1 of subject no. 1). The time point-1 succinate level of a person with a particular condition (i.e. person 3 onwards) before the respective trigger is indicated relative to this basal level of the healthy subject of 20-30 years old. Succinate and insulin resistance of time points 2 and 3 of a particular subject are relative to time point 1 of that particular subject. Insulin resistance level was inferred from combined glucose and insulin level measurement. With chronically-diseased subjects, subjects are meant suffering from type-2 diabetes mellitus, dyslipidemia, metabolic syndrome, obesity, PCOS, NAFLD, NASH, COPD, intestinal bowel disease, cancer, or with endocrine / cardiovascular / neurological disease related insulin resistance or longstanding mental problems. Subjects indicated are:

[0342] 1 . a young healthy / insulin-controlled T 1 DM subject experiencing nervosity / menstrual cycle period;

[0343] 2. a young healthy / insulin-controlled T 1 DM subject experiencing extensive exercise / infection / inflammation / food intolerance / allergy / alcohol abuse / menopause / pregnancy; 3. an older healthy / insulin-controlled T 1 DM subject experiencing nervosity / menstrual cycle period;

[0344] 4. an older healthy / insulin-controlled T 1 DM subject experiencing extensive exercise / infection / inflammation / food intolerance / allergy / alcohol abuse / menopause / pregnancy;

[0345] 5. a slightly chronically-diseased subject experiencing nervosity / menstrual cycle period;

[0346] 6. a slightly chronically-diseased subject experiencing extensive; exercise / infection / inflammation / food intolerance / allergy / alcohol abuse / menopause / pregnancy; 7. a severely chronically-diseased subject experiencing nervosity / menstrual cycle period;

[0347] 8. a severely chronically-diseased subject experiencing extensive exercise / infection / inflammation / food intolerance / allergy / alcohol abuse / menopause / pregnancy;

[0348] 9. a slightly chronically-diseased subject undergoing an effective treatment; 10. a severely chronically-diseased subject undergoing an effective treatment.

[0349] Table 1

[0350] The above putative results can be obtained in larger patient cohorts.

[0351] The above indicates that the invention indeed can be used to determine insulin resistance and / or Insulin Sensitivity Factor (ISF) variability over time and hence allows to tailor insulin administration on insulin resistance at any given time.

[0352] Example 2 Rationale for value of implementing succinate measurements in relation to hybrid closed-loop CGM:

[0353] Quote: ‘a basic understanding of the neurohormonal responses to different forms of [here] exercise [induced stress} in T1 DM patients is important, as well as the establishment of reasonable glucose targets for safe and effective exercise through changes to the individual’s own self-management plans. Additionally, providing strategies to adapt to the inherent interindividual and intra-individual variability in response to exercise is vital to the successful management of glycaemia and sports performance for people with T1 DM’ (Riddell & Peters, 2023).

[0354] Biology: Except for meals, when glucose-modifying carbs, fats and proteins are added, our body can be seen as a closed system in which changes in blood glucose are only caused by changes in stress-induced insulin resistance. These are all mediated by temporary elevations in blood succinate (BS) levels, released from mitochondria upon cellular metabolic stress, leading to (temporary) insulin resistance, hyperglycaemia and, in non-diabetic individuals, increased insulin levels.

[0355] Mechanistically, the increased BS will bind its receptor (SLICNR1) in the liver, inducing liver insulin resistance (IR). The liver subsequently produces and releases more glucose (giving hyperglycaemia; increase CGM signal), which stimulates the pancreas to release insulin, therewith stimulating increased uptake of glucose by skeletal muscles. Although the timeframe is unknown, the succinate stress response thus always PRECEDES the glucose response.

[0356] Based on the location and sensitivity of succinate transporters, I anticipate that BS is subsequently efficiently removed by uptake in the liver and renal proximal tubules, and through renal excretion. This process goes efficient in healthy individuals, who have a low basal IR of liver, kidneys and muscles, as their basal [succinate] is from 0-50 uM. The IR of liver and kidney is determined by [succinate]; the IR of muscles by the endogenous content of glycogen (and thus ability to take up glucose to convert to glycogen). Following reduction of succinate, liver glucose release diminishes and uptake increases.

[0357] With humans suffering from chronic acquired low grade inflammation / stress, such as with obesity, diabetes type-2 (T2DM), cardiovascular disease (CVD), NAFLD, COPD, intestinal bowel disease, depression, dementia, parkinsonism, hypothyroidy, or with genetic chronic low- grade inflammation / stress, such as cystic fibrosis, succinate is constitutively increased to about 60-80 uM. Their liver and kidneys are thus steady state insulin resistant. Upon a short-term stressor, the absolute elevation in succinate is anticipated to be the same, but the removal will be slow compared to above, and will not go lower than the previously (increased) steady state level.

[0358] The extent of the increase in succinate is determined by the (personal) intensity of the stressor and the duration of the change. The experienced intensity of the stressor (environmental queue) depends on the genetic package (‘talent’ to deal with it) and the times the person encountered the challenge before (experience). For example, a slight increase in CGM reading occurs when one is standing up from bed: getting from a laid down to standing position necessitates an increase in blood pressure, because our brains will otherwise not receive enough oxygen. The change of standing up is the stressor, but as adaptation by vasoconstriction occurs fast (short duration) and we are used to stand up (experience), the stress / succinate-induced CGM change is relatively small (small increase in time and fast removal by a healthy liver and kidney). In contrast, when one performs high-intensity exercise when one nearly never performs sport or is frail, the stress / succinate-induced CGM change is high.

[0359] Similar changes occur with other closed system changes, such as viral / bacterial infection, high- intensity training / bout exercise, (person-depending / relative) too intensive sound / light / smell, temporary mental stresses (presentation, conflict with someone), cold shock.

[0360] Benefit for inclusion of CSM in hybrid CL-CGM systems (hybrid, because they still need injection of extra insulin before consuming a carb meal):

[0361] For diabetic patients using a CL-CGM system, a proper time-in-range (TIR) is the %time a person has a blood glucose (BG) of 3.9-10 mM. Above 10 mM is time-above-range (TAR), whereas below 3.9 is time-below-range (TBR). TBR may lead to coma and, possibly, death and is therefore THE holy grail to prevent. However, as TAR increases the risk of (reactive) hypoglycaemia, patient’s (and therefore CL-CGM selling companies) target is to get to the highest TIR, which is obtained by having the lowest level of glucose variability (GV). Indeed, a high GV is a robust predictor or hypoglycaemia in T1 DM patients (Rempel et al, 2018).

[0362] In principle, ALL conditions that may lead to hypoglycaemia with current CL-CGM devices are due to the following aspects:

[0363] 1 . Delay in reaction to BG changes of 50 minutes. CL-CGM devices detect BG in the interstitial fluid, of which the glucose concentration has a delay of 10 minutes in relation to BG. Insulin is infused subcutaneously, which takes 40 minutes before it is evenly diffused in blood (Phillip et al, 2023). The (ultra) rapid insulin used has an activity period of around 4 hours.

[0364] 2. The risky conditions are characterised by a period of increased insulin resistance (e.g. high intensity exercise, stress, infection, period, carb meal) shortly followed by a period of increased insulin sensitivity (stop exercise, end of meal, sleeping) combined with and increased glucose uptake by liver and, with exercise, elevated glucose uptake by skeletal muscle (to fill up the used glycogen stores). 3. As insulin is infused during the IR period, at which glucose rises, and has a 50 minutes delay of activity, high insulin levels are still present when patients switch from an I R to an insulin-sensitive state. This causes the risk of hypoglycaemia development.

[0365] At present, these risks of hypoglycaemia are dealt with by manual insulin-dose adjustments and / or carbohydrate consumption, but patients and their health care professionals often struggle with the information to set these properly and, after an afternoon exercise, a nocturnal hypoglycaemic event is difficult to prevent. Moreover, the insulin-dose adjustment setting is highly personal and, to be on the safe site, a conservative BG setting (i.e. higher than normal) is set for patients with increased risk factors, such as age, impaired awareness (lAH)Zearlier encounters of hypoglycaemia, minimally exercising (Moser et a / , 2020).

[0366] While the delays cannot be circumvented, Important improvements can thus be established when one can (1) infuse insulin as soon as IR develops, (2) is able to infuse the proper dose based on the intensity and duration of the period of IR, and (3) stop extra insulin infusion as soon as IR stops.

[0367] As outside carb meals, all increases / decreases in BG are due to stress-induced increases / decreases in blood succinate, and these succinate increases precede the BG elevations, these 3 steps can be accomplished by implementing continuous succinate monitoring (CSM) in the regulation of CGM-regulated insulin infusion.

[0368] At present, <70% of patients using commercial CL-CGM devices get to 70% time in range. To be able to properly use these CL-CGM systems, patients need to know their so-called insulinsensitive factor (ISF; or insulin correction factor) and insulin-carb ratio (ICR).

[0369] The ISF is most relevant here. The ISF is the reduction in blood glucose (in mM or, in mg / dl, x 18) for 1 unit of fast acting insulin. As most patients using hybrid CL-CGM system are not able or do not want to set the ISF, the ISF is usually determined and set for the used CL-CGM device by the diabetes health care team. Besides, to be on the safe side, the ISF is usually calculated on basis of how much insulin had to be infused during the last 3 days. This ISF, however, is depending on the IR, which varies numerous times in and between days. These changes occur when one e.g. stops gardening / walking and sits down (glucose consumption stops, one becomes cold and heart beat goes down; increase in IR), when one has to visit his / her parents in law, has to visit the teachers of their children, has to visit the dentist, job application, presentation, intense conversation with someone, travelling, cold environment, dehydration (all increase in IR). Other examples: 'when biking with wind, BG goes down, while when biking against wind, it goes up'. 'Going on vacation takes 3 days to have the CL-CGM adapted. Then, when getting back home, the same occurs'. As IR changes in time, patients with CL-CGM have to correct their BG regularly. The ICR, which indicates the amount of extra fast-acting insulin needed to be taken with a carb meal, however, also varies and is also relevant here. Patients often observe that the observed BG level following a meal is not in line with their calculated ICR and injected insulin. This is because the calculation assumes that only the carb (fat, protein) eaten will affect BG. However, due to long term unhealthy nutrition (carbs, allergens), many persons show an intestinal stress response to (unhealthy, sometimes personal) food components. As such, depending on the healthiness of the person’s intestinal tract / microbiome and the constitution of the meal, the intestine will produce different amounts of succinate, which are transferred via the portal vein to the liver, again leading to an increased liver IR and succinate / IR-related increases in BG.

[0370] The following differences / advantages are the case with CSM implementation:

[0371] 1 . CSM detects and accurately corrects the daily changes in stress-induced BG changes. There is no need for patients anymore to correct for the changing stress-induced IR.

[0372] 2. As BG changes follow BS changes, the present CL-CGM devices are thus always late responsive to stress. This accounts for increases as well as decreases in BG. Because due to the 50 minutes delay in insulin infusion as compared to BG changes, an as fast as possible adaptation of needed insulin to BG changes is of prime importance. The shorter the delay in response, the lower the GV and thus the higher the TIR.

[0373] 3. With meals, correction of BG levels upon implementation of CSM data will automatically correct for the succinate-induced increase in BG. Therewith, the BG level changes due to the ingested carbs will be more in line with the calculated carbs based on the ICR and, therewith, will yield lower GV and higher TIR.

[0374] T1 DM patients experience stress-induced increases in BG due to medication dose / timing, caffeine consumption, high-intensity exercise, illness, dawn effect, allergies, periods (menstrual cycle), celiac disease, smoking, puberty, sunburn, altitude, dehydration, cold outside temperature, worries, family and social pressure, recent hypoglycaemic encounters, and impaired awareness of hypoglycaemia. These all lead to increases in BS, for which items 1 and 2 above count.

[0375] T1 DM patients experience increases in BG with meal constitution and personal microbiome for which item 3 counts.

[0376] Within the closed body system, the ISF is thus directly related to succinate level. Integration of the CSM data with the CGM data will thus optimize insulin infusion at any time based on momentary info on changing ISF. With the to-be-developed algorithms, GV with all these daily activities is thus minimized leading to a higher TIR. This will not only be the case for T1 DM patients using (hybrid) CL-CGM systems, but also for patients suffering from gestational diabetes or type-2 diabetes using the CL-CGM system.

[0377] The change in succinate level may be related to the change / deviation of ISF by the formula 1 / AISF = K * ([BS]tn+1-[BS]tn) / [BS]tn * 100 wherein ISF is the insulin sensitive factor, [BS]tn+1 and [BS]tn are blood succinate concentration at time-point n+1 or n, respectively, and K is a constant which can be determined in a training module. [BS] in uM (or mmol / L). K is anticipated to range between 1.5 and 3.0 for basal [BS] between 0 to 50 uM,, while K may be between 1 and 0.6 for basal [BS] being above 50 uM.

[0378] Based on the dose-response curve of the SLICNR1 , this K remains similar in the linear range of activation by succinate, being roughly between 5 and 50 uM. Below and above this range, the ISF will be more and less sensitive to changes in [succinate], respectively (and K will thus be higher and lower, respectively).

[0379] Figure 2 shows Dose-response curve of human SUCNR1 to succinate. I nositol-3 phosphate formation by cells expressing human SLICNR1 in response to different concentrations of succinate. Taken from Rexen-Ulven, Sci reports, 2018, Pubmed 29968758.

[0380] Training module: (All) Stress- related changes in BG will thus change in relation to changes in BS and insulin. Based on the angle of the dose-response curve of SLICNR1 (Figure 2), the relationship between BS and BG / insulin will be linear for T1 DM patients with a basal [succinate] of 0-50 uM (not necessarily a 1 :1 relationship). Similarly, for T1 DM patients with a basal [succinate] of 50, 60 or higher, who will also have an increased basal insulin level, the relationship between BS and BG / insulin will be different (likely lower). T1 DM patients with a basal [succinate] <50 uM are typically healthy (young) individuals who have a low ISF. Those with a basal [succinate] of 60 or higher mostly suffer from chronic low-grade inflammation (e.g. chronic mental stress, obesity, intestinal / liver disorder, etc).

[0381] To determine the relative timing and the relationship between changing [succinate] and those of changing [BG] and [insulin], the latter reflected in the ISF, these factors will be measured in time (3-5 minutes intervals) with T 1 DM patients (and healthy individuals as controls / reference), subjected to controlled exercise excursions with different VOmax levels, from shortly before, during and until BG / insulin / BS have dropped back to basal, or to max. 3 hrs after the exercise. The relationship between changing BS and Insulin Sensitivity Factor changes will be determined, based on the timing and speed of their respective increases, decreases, and areas under the curve (AUG). It is anticipated that with a strong increase in BS, starting with a basal BS <50 uM, 200% x the ISF needs to be infused, with a mild increase 120-160%, no further increase with steady state elevated BS, and (considering the 4 hrs activity time of infused insulin) stopping insulin infusion upon lowering of BS.

[0382] To determine the contribution of stress-related changes in BS on changing BG / insulin levels with meals, the same patient groups will be given identical complex meals following pre-insulin meal correction based on their ICR. At 5’ steps during 3 hours following consumption of the meal, BS, BG and insulin will be determined in conjunction with possibly extra infused insulin. The relationship between changes in BS, BG and extra infused insulin will be determined. It is anticipated that the stress / BS-induced increase in insulin, and therefore, deviation of infused insulin from the ICR, is higher in T1 DM with a basal [succinate] >50 uM.

[0383] Performing Training module

[0384] The training module is performed with 100 subjects to establish the relationship between succinate level and ISF (and hence insulin dosage). Basal succinate as well as the first and second time point are determined.

[0385] Below the results of 10 subjects is shown, 5 subjects have basal succinate level of 0-50 pM and 5 subjects have basal succinate level of more than 50 pM. It is found that the relationship between succinate level and ISF change can in practice be depicted as follows: in case the basal succinate determined is 0-50 M:

[0386] - a succinate level increase between the first time and the second time by more than 40% indicates a decrease of (actual) ISF by more than 40% and at most 80% of the subject between the first time and the second time;

[0387] - a succinate level increase between the first time and the second time by at least 10% and at most 40% indicates a decrease of (actual) ISF by at least 15% and at most 40% of the subject between the first time and the second time,

[0388] - an identical or less than 10% deviation of succinate level between the first time and the second time indicates no or less than 15% change of (actual) ISF of the subject between the first time and the second time;

[0389] - a succinate level decrease between the first time and the second time by more than 40% indicates an increase of (actual) ISF by more than 60% and at most 400% of the subject between the first time and the second time;

[0390] - a succinate level decrease between the first time and the second time by more than 10% and at most 40% indicates an increase of (actual) ISF by more than 15% and at most 60% of the subject between the first time and the second time; in case the basal succinate determined is more than 50:

[0391] - a succinate level increase between the first time and the second time by more than 20% indicates a decrease of (actual) ISF by more than 20% and at most 60% of the subject between the first time and the second time;

[0392] - a succinate level increase between the first time and the second time by at least 10% and at most 20% indicates a decrease of (actual) ISF by at least 10% and at most 20% of the subject between the first time and the second time;

[0393] - an identical or less than 10% deviation of succinate level between the first time and the second time indicates no or less than 10% change of (actual) ISF of the subject between the first time and the second time;

[0394] - a succinate level decrease between the first time and the second time by more than 20% indicates an increase of (actual) ISF by more than 40% and at most 100% of the subject between the first time and the second time;

[0395] - a succinate level decrease between the first time and the second time by more than 10% and at most 20% indicates an increase of (actual) ISF by more than 10% and at most 40% of the subject between the first time and the second time.

[0396] Example 3 10 Type 1 Diabetes Mellitus (T 1 DM) patients are recruited: (male / female) < 40 year with normal BMI using a commercial CL-CGM device targeting for a healthy blood glucose level of 6 mM. During a first period, insulin dosage is based on standard (basal) ISF. During a second period, insulin dosage is based on ISF corrected based on succinate level changes according to the invention (see Example 2). During a third period, insulin dosage is based on ISF that has been corrected based on succinate level changes but not according to the invention.

[0397] Period 1

[0398] Insulin dosage is based on standard (basal) ISF

[0399] Period 2

[0400] Insulin dosage is based on ISF corrected based on succinate level changes according to the invention (see Example 2).

[0401] Period 3

[0402] Insulin dosage is based on ISF that has been corrected based on succinate level changes but not according to the invention (see Example 2).

[0403] Observation: 1 . Problem without correcting insulin dosage according to the invention: a. Many / large glucose variability (GV), around 80% Time-in-range (TIR), 4% time- below-range (TBR), 16% time-above-range (TAR), fear of hypoglycaemia (FOH), hospital visits (due to hypoglycaemic events), weight gain (due to excessive insulin infusion, because of many TAR correction moments), impaired awareness of hypoglycemia (IAH), myopathy, chance for gestational diabetes development (with women), standard quality of life, 12,2 year shorter life expectancy.

[0404] 2. Change with correcting insulin dosage according to the invention: a. Quantitative insight in moments, extents and causes of (metabolic) stress, less / reduced GV, around 90% TIR, 2% TBR, 8% TAR, reduced FOH, less hospital visits, less weight gain, reduced IAH, normal muscle mass / strength, reduced chance for gestational diabetes development (with women), improved QoL (less disease management), longer life expectancy.

[0405] Example 4

[0406] 10 T2DM / T1 DM patients are recruited, (higher burden, more complex (poly-pharmacy), less number of people, possibly more difficult to study as compared to Example 3; disease more severe / covers aspects of common T2DM and CVD): typically older adult T1 DM patient with minimally exercising and / or with high risk of hypoglycaemia, or for older adults with coexisting chronic illnesses or two or more instrumental ADL impairments or mild- to-moderate cognitive impairment. Patients are randomly assigned to one of two groups:

[0407] Group 1

[0408] Insulin dosage is based on standard ISF

[0409] Group 2

[0410] Insulin dosage is based on ISF corrected based on succinate level changes according to the invention.

[0411] Observation

[0412] 1 . Problem without supporting CSM according to the invention: a. Same as in Example 3, except for more / larger GV, around 70% TIR, 10% TBR, 20%TAR, increased fear of hypoglycaemia (FOH), increased hospital visits (due to hypoglycaemic events), weight gain, more IAH, myopathy, depression, increased infection, myopathy, standard quality of life, 12,2 year shorter life expectancy.

[0413] 2. Change with supporting CSM according to the invention: a. Quantitative insight in moments, extents and causes of (metabolic) stress, less / reduced GV, around 80% TIR, 4% TBR, 16% TAR, reduced FOH, less hospital visits, less weight gain / IAH / depression / infection, normal muscle mass / strength, improved QoL (less disease management), longer life expectancy.

[0414] Example 5

[0415] Changes in succinate, glucose, insulin and CGM data with stressful conditions. Of a healthy person, venous blood was taken before, during and after a high-intensity exercise (HIT) or before and shortly after standing up from bed and analysed for changes in CGM, glucose (in mM), insulin (in pg / ml_, HOMA-IR and succinate (in uM). A clear correlation between CGM, glucose and succinate is apparent. Time is given (in hrs. min). Example 6

[0416] Rationale for the importance and broadness of (continuous) measuring succinate:

[0417] Nature and nurture (environment), we are all born with a particular genetic package. Whether we are born with or will obtain a disease, and in the latter case, when, depends on this genetic package and the environment one encounters during life. Concerning genetic package: when one is born with a mutation in a gene that is very important in a physiological process, one is usually born with a disease. These genetic diseases are very rare (1:1.000.000).

[0418] However, depending on variants of different genes (genetic package), one can be more or less susceptible to develop a disease. Whether one develops a disease is then determined by the environment. As the worsening environmental conditions are encountered after birth and the stresses they induce have to accumulate to lead to a disease, these very common acquired diseases develop later in life. In contrast to genetic diseases, identification of the environmental factor(s) causing development of the disease will allow one to prevent, stop / revert or even cure the disease.

[0419] Environmental factors: for one to live a healthy life, our body obtains, senses, and has to deal with many external / environmental input signals. At the borders of our body with the environment, which are our skin, eyes, ears, nose, mouth, lungs, intestinal tract, urogenital tract, we encounter environmental queues such as changes in humidity, temperature, light, sound, smell, taste, air quality / pollution / oxygen content, lack / surplus and quality of food and water, viral / bacterial / fungal attacks, and (positive and negative) social interactions with other human beings / animals.

[0420] Health and disease; homeostasis and allostasis, and resilience: In order for all our organs to function properly, our bodies try to maintain a steady state internal, physical and chemical condition, by keeping variables such as body temperature and blood glucose, pH, and minerals (e.g. Na, K, Ca, PO4) concentrations within pre-set (homeostatic) limits, whatever the environment is (Bernard’s definition of homeostasis). The changing environmental queues (stressors) above induce a so-called allostatic load, which will temporarily change the set point, but which will return to the homeostatic set point when one has a high resilience / adaptive ability. However, repetitive experiences of stressors will lead to an allostatic overload, defined as the stress- induced wear and tear on the body, leading to the loss of adaptive ability and return to the original homeostatic state, and to disease development. Thus, whether one experiences these environmental queues as ‘stressors’ depends on the intensity and duration of these environmental inputs in relation to his / her susceptibility (talent) and obtained experience to deal with these inputs. When a person experiences an environmental queue as a stressor for too long (i.e. gets an allostatic overload), he / she develops a disease and gets into allostasis.

[0421] Health is defined as the ability to adapt to these changing environmental stressors, deal with these allostatic loads and to maintain homeostasis and longevity is the resultant of it.

[0422] Environmental factors and evolution: During million years of evolution, our bodies have established proper physiological mechanisms to deal with environmental queues that already existed when we were still hunter / gatherers in tribes, such as cold, lack of food / water, endurance exercise (to run from a predator / chase for food), bacterial / viral infections, social isolation, and mechanisms to prevent these queues from becoming allostatic stressors (e.g. habit to story energy when food is in surplus). As dealing with all these allostatic loads requires more energy, our body always responds to this by realizing a temporary state of hyperglycemia, through increased glucose release from the liver, and hyperinsulinemia, through increased insulin release from the pancreas. This response accounts for acute allostatic loads, leading to temporary states of hyperglycemia / hyperinsulinemia, as well as chronic / diseased state allostatic overloads, coinciding with sustained hyperinsulinemia (e.g. with pre-diabetic, obesity) and, upon further deterioration, sustained hyperglycemia (diabetes). During the last 300 years, however, we have started to encounter stressors for which we are evolutionarily not equipped to deal with and which occur frequent. These stressors are mainly (1) consumption of non-natural (processed) food / drinks, (2) surplus of food / drinks, (3) alcohol consumption, (4) air / water / food pollution, (5) social distance / stress, (6) relational / work stress, and (7) reduced energy usage (less exercise, experience of cold). In line with these above, longevity is determined by genetics in combination with lifestyle. A healthy lifestyle encompasses (1) consuming moderate volumes of nonprocessed (and high quality) food and drinks (intermittent fasting), (2) no alcohol / smoking, (3) recognition and removal of / escape from of mental stresses (stay away from energy-absorbing situations / enjoy energy-giving conditions, (4) regular exercise, (5) regularly experience cold (bath / swimming; walk in cold with moderate dressing).

[0423] Blood succinate reflects chronic and acute states of allostasis

[0424] Example 7

[0425] Hypothesis: measuring succinate will show (1) an increase in time with increased levels of stress, (2) increased levels compared to healthy peers with burnout and (3) a decrease with effective therapy (addressing the cause(s) of burnout), (4) reveal which environmental queues are the strongest stressors for the person (strong increase with relatively mild intensity of stressor), (5) indicate the level of resilience by the speed at which blood succinate is increased (lower than before) with this challenge and fastly decreased following the challenge.

[0426] 10 burnout patients are recruited and randomly assigned to group 1 or 2.

[0427] Group 1

[0428] Not monitoring succinate level changes according to the invention. Group 2

[0429] Monitoring succinate level changes according to the invention.

[0430] Result

[0431] 1. Without succinate measurement: Patient has less information about triggering environmental queues and does not adequately avoid them, leading to: fatigue, feeling of exhaustion, anxiety, stress, fog over the brain (low ability to concentrate, easily forgetting thing, weak memory), frustration, feeling cranky / easily getting into interpersonal conflicts, depression, low quality of life (QoL), suicide, low resilience, susceptible to infections / physical symptoms, low level of decision making, high number of sick leaves, (permanent) absence from labour force, low level of return to work.

[0432] 2. With monitoring of plasma succinate levels: significantly improved values of items under 1 and insight in moments, extents and causes of their triggering environmental queues.

[0433] Example 8

[0434] Hypothesis: measuring succinate will show (1) strongly increased levels compared to healthy peers and (2) decrease with effective therapy (addressing the cause(s) of critical illness). Moreover, with infection (e.g. covid) leading to critical illness, the person will a priori have high succinate levels and show a steep increase in succinate with infection (as he / she has a low metabolic resilience when having high basal succinate levels / have low grade inflammatory underlying diseases).

[0435] 10 patients with serious illness are recruited, 2 with cancer, 2 who suffered heart attack, 2 who suffered stroke, 2 suffering multiple sclerosis, 2 suffering covid-19. Patients are randomly assigned to group 1 or 2.

[0436] Group 1

[0437] Not monitoring succinate level changes according to the invention.

[0438] Group 2

[0439] Monitoring succinate level changes according to the invention.

[0440] Result 1. Without succinate measurement: Patient has less information about triggering environmental queues and does not adequately avoid them, leading to: fatigue, pain, lack of energy, fever, thirst, dry mouth, dyspnea, anxiety, worry, communication difficulty, polyneuropathy, myopathy, fog over the brain / delirium (low ability to concentrate, easily forgetting thing, weak memory, slow movements and thoughts), susceptible to infection, venous thromboembolism, development of post-traumatic stress syndrome, mechanical ventilation, high ICU / hospital lengths of stay, high mortality.

[0441] 2. With monitoring of plasma succinate levels: significantly improved values of items under 1 and insight in moments, extents and causes of their triggering environmental queues.

[0442] Example 9

[0443] Hypothesis: measuring succinate will show (1) strongly increased levels compared to healthy peers, (2) discriminative power for the different forms of hypothyroidy, (3) a decrease with effective therapy (addressing the cause(s) of critical illness), and (4) may serve as an OEM in thyroid hormone dosing (as the extent of hypothyroidism shows will vary during / between days and between individuals).

[0444] 10 patients with thyroid disease are recruited (3 with Hashimoto disease; 3 with hypothyroidism during pregnancy, 2 with non-thyroidal illness syndrome [NTIS], 2 with ageing, [rare] central hypothyroidism). Patients are randomly assigned to group 1 or group 2.

[0445] Group 1

[0446] Not monitoring succinate level changes according to the invention.

[0447] Group 2

[0448] Monitoring succinate level changes according to the invention.

[0449] Result:

[0450] 1. Without succinate measurement: Patient has less information about triggering environmental queues and does not adequately avoid them, leading to: fatigue, lethargy, sensitive to cold, weight gain, constipation, depression, muscle cramps, constipation, shortness of breath, fog over the brain (low ability to concentrate, easily forgetting thing, weak memory, slow movements and thoughts), depression, dry skin and hair, reduced QoL, suboptimal relief of symptoms with thyroid hormone replacement, [with NTIS] increased morbidity / mortality.

[0451] 2. With monitoring of plasma succinate levels: significantly improved values of items under 1 and insight in moments, extents and causes of their triggering environmental queues.

[0452] Example 10

[0453] Hypothesis: In maximally healthy individuals, basal succinate will be low and which, following a rise with an environmental queue, will fastly return to basal. This latter aspect is a feature of a high resilience for the experienced environmental queue. People suffering from a chronic acquired or inherited disease characterized by chronic (low-grade inflammation (diabetes, obesitas, CVD, liver / intestinal disease, CFTR) will have a basally high succinate level, which, following a rise with an environmental queue, will slowly return to the elevated basal. As the relative increase in succinate is small, it goes with a low level of resilience. Longevity / healthy ageing, is determined by the ability to respond to changing environmental conditions, and which thus is in line with a sustained low level of succinate. Regularly monitoring one’s blood succinate level will provide insight on the moment, intensity, and thus identity, of his personal environmental queues. With knowledge on these queues, one can take steps to prevent them.

[0454] 10 healthy subjects in the ages of 60-80 years are recruited. Patients are randomly assigned to group 1 or group 2.

[0455] Group 1

[0456] Not monitoring succinate level changes according to the invention.

[0457] Group 2

[0458] Monitoring succinate level changes according to the invention.

[0459] Result:

[0460] 1. Without succinate measurement: ‘early’ acquisition of a chronic disease / death, reduced QoL

[0461] 2. With monitoring of plasma succinate levels: significantly improved values of items under 1 due to insight in moments, extents and causes of their triggering environmental queues.

[0462] Example 11 Rationale: to treat cancer as efficient as possible, the clinician mostly wants to use the highest dose of radiotherapy and chemotherapy possible. The dose is limited by the level of toxicity it realizes with the patient. A too high level of toxicity leads to abrogation of the therapy and / or loss of compliance of the patient. The extent of toxicity is determined by the resilience of the patient towards these strong stressors for our bodies and shows a strong inter-individual and intra-individual variability (between days, within a day).

[0463] Hypothesis: the initial resilience of the patient to therapy / extent to which he / she develops toxicity features is determined by his / her basal level of blood succinate, the speed at which it raises with therapy, and the speed at which it gets back to (a renewed?) basal level. This is determined by his / her original health status. With more treatment occasions in time and increased vulnerability, these responses will change in time (increased basal level, slower return to (renewed) basal level. Besides the stress of the treatment, all other stresses normally present in life also influence the resilience of the patient. Forms of lifestyle therapy in advance (prehabilitation) or after (post-habilitation) have shown to improve the resilience of the patient before and after cancer treatment, and to reduce health care costs.

[0464] Prehabilitation and post-habilitation therapy to cancer treatment: succinate measurements will allow: (1) determination of the patient’s resilience, (2) monitoring of his / her change in resilience with therapy, (3) identify which therapy is most efficient for the particular patient to increase his / her resilience, and (4) determine to which intensity and duration the therapy can be given to benefit the patient.

[0465] With cancer treatment: succinate measurements will allow: (1) monitoring of the patient’s resilience level in time, (2) determine to which intensity and duration the treatment can be given in order not not becoming toxic [i.e. the maximal tolerated exposure, MTE, and personalized maximum tolerated dose, MTD], (3) optimized chemotherapeutic dosing in time irt to the resilience level of the patient.

[0466] 10 patients preparing for and / or treating for cancer with chemotherapy are recruited.

[0467] Results:

[0468] 1. Without succinate measurement: Patient has less information about triggering environmental queues and does not adequately avoid them, leading to: fatigue, hair loss, nausea, vomiting, low number of red and white blood cell count, easy bruising and bleeding, infection, mucositis, inflammation, fever, feeling cold, change / loss of appetite, increased sensitivity to smell, mouth sores, constipation / diarrhoea, myopathy / cachexia, hypoalbuminemia, neuropathy, cardiomyopathy, liver steatosis, nephrotoxicity, loss of compliance, more / longer hospital lengths of stay, high morbidity / mortality. 2. With monitoring of plasma succinate levels: significantly improved values of items under 1 and insight in moments, extents and causes of their triggering environmental queues.

[0469] Example 12

[0470] Background: Parkinsonism is characterised by a reduced dopamine neurotransmitter release. Levodopa (or L-dopa) is a precursor of dopamine and is the golden standard treatment for parkinsonism. L-dopa, however, is partially broken down by the intestinal microbiome, which varies between patients, despite inhibition via carbidopa. Consequently, the dose that reaches the brain varies between persons. Lifestyle has a major role in the efficiency of L-dopa as a treatment for parkinsonism.

[0471] Hypothesis: basal blood succinate will be a measure of the health status of the patient: healthy from 0-50 uM, diseased from 60 and higher. Blood succinate measured before and 60-120 minutes after a meal will reveal the healthiness status of his / her microbiome. measuring blood succinate will allow (1) monitoring of the health status of the patient with therapy, (2) optimized timing and dosing of levodopa + / - carbidopa / benserazide in relation to blood succinate at the start of and later with therapy (due to stress induced).

[0472] 10 Parkinson’s patients treated with levodopa + / - carbidopa / benserazide are recruited. Patients are randomly assigned to group 1 or group 2.

[0473] Group 1

[0474] Not monitoring succinate level changes according to the invention.

[0475] Group 2

[0476] Monitoring succinate level changes according to the invention.

[0477] Results

[0478] 1. Without succinate measurement: Patient has less information about triggering environmental queues and does not adequately avoid them, leading to: nausea, dizziness, sleepiness, headache, hallucinations, delusions, loss of appetite, diarrhoea, dry mouth, constipation, delirium (forgetfulness and confusion), mental / mood changes (agitation), change in taste, motor fluctuations (‘wearing off’ or ‘on / off’ with changes in [medication]) , dyskinesia (abnormal involuntary movements, causing rapid jerking or slow and extended muscle spasms), low QoL. 2. With monitoring of plasma succinate levels: significantly improved values of items under 1 and insight in moments, extents and causes of their triggering environmental queues.

[0479] Example 13

[0480] Background: based on my deduction, strong increases in succinate are the driving force for the occurrence of nausea, vomiting.

[0481] Hypothesis: basal blood succinate will be a measure of the health status of the patient: healthy from 0-50 uM, diseased from 60 and higher. Blood succinate measured before and 60-120 minutes after a meal will reveal the healthiness status of his / her microbiome. measuring blood succinate will allow optimized dosing of medication (oral or otherwise) to prevent side effects

[0482] 10 patients suffering from nausea, vomiting, dizziness and / or headache (following consumption of material) are recruited. Patients are randomly assigned to group 1 or group 2.

[0483] Group 1

[0484] Not monitoring succinate level changes according to the invention.

[0485] Group 2

[0486] Monitoring succinate level changes according to the invention.

[0487] Results

[0488] 1. Without succinate measurement: Patient has less information about triggering environmental queues and does not adequately avoid them, leading to: nausea, dizziness, headache, vomiting.

[0489] 2. With monitoring of plasma succinate levels: prevention / strong reduction of items under 1

[0490] Example 14

[0491] Background: CFS often get worse when they try to do as much as they want or need to do. Therefore, especially the usage of succinate measurements to monitor their resilience with therapy will be of high clinical value.

[0492] Hypothesis: measuring succinate will show (1) increased plasma levels compared to healthy peers, (2) an increase in time with increased levels of stress, (3) a decrease with effective therapy (addressing the cause(s) of CFS), (4) reveal which environmental queues are the strongest stressors for the person (strong increase with relatively mild intensity of stressor), (5) indicate the level of resilience with therapy by the speed at which blood succinate is increased (lower than before) with a challenge and decreased following the challenge.

[0493] 10 patients with chronic fatigue syndrome / myalgic encephalomyelitis (CFS / ME) are recruited. Patients are randomly assigned to group 1 or group 2.

[0494] Group 1

[0495] Not monitoring succinate level changes according to the invention.

[0496] Group 2

[0497] Monitoring succinate level changes according to the invention.

[0498] Results:

[0499] 1. Without succinate measurement: Patient has less information about triggering environmental queues and does not adequately avoid them, leading to: severe fatigue, very sleepy, sensitivity to light, headache, weakness, muscle and joint pains inability to concentrate, insomnia, forgetfulness, low grade fever, mood swings, confusion, depression, low quality of life (QoL), low resilience, high number of sick leaves, (permanent) absence from labour force, low level of return to work., high costs8

[0500] 2. With monitoring of plasma succinate levels: significantly improved values of items under 1 and insight in moments, extents and causes of their triggering environmental queues.

[0501] Example 15

[0502] Background: inherited mitochondrial diseases are common and affect many organs, but lacks a reliable and universally applicable tool to measure their resilience and outcome of therapy in time.

[0503] Hypothesis: measuring succinate will show (1) increased plasma levels compared to healthy peers, (2) an increase in time with increased levels of stress, (3) a decrease with effective therapy (addressing the cause(s) of PMD), (4) which environmental queues are the strongest stressors for the person (strong increase with relatively mild intensity of stressor), (5) indicate the level of resilience with activity / therapy by the speed at which blood succinate is increased (lower than before) with a challenge and decreased following the challenge. 10 patients with primary mitochondrial disorder (PMD) are recruited. Patients are randomly assigned to group 1 or group 2.

[0504] Group 1

[0505] Not monitoring succinate level changes according to the invention.

[0506] Group 2

[0507] Monitoring succinate level changes according to the invention.

[0508] Results:

[0509] 1. Without succinate measurement: Patient has less information about triggering environmental queues and does not adequately avoid them, leading to: tiredness, lack of energy, depression, behavioural problems, neuropathy, myopathy, ptosis, external opthalmoplegia, exercise intolerance, cardiomyopathy, sensorineural deafness, optic atrophy, pigmentary retinopathy, depression, low quality of life (QoL), low resilience.

[0510] 2. With monitoring of plasma succinate levels: significantly improved values of items under 1 and insight in moments, extents and causes of their triggering environmental queues.

[0511] Example 16

[0512] Hypothesis: measuring succinate will show (1) increased plasma levels compared to healthy peers, (2) an increase in time with increased levels of stress, (3) an absence of an increase in succinate with omission or effective therapy for the allergy, (4) which environmental queue triggers the allergic response

[0513] 10 patients with allergy are recruited (allergy to to fish, eggs, milk, shellfish, nuts, mustard). Patients are randomly assigned to group 1 or group 2.

[0514] Group 1

[0515] Not monitoring succinate level changes according to the invention.

[0516] Group 2

[0517] Monitoring succinate level changes according to the invention.

[0518] Results: 1. Without succinate measurement: Patient has less information about triggering environmental queues and does not adequately avoid them, leading to: itchy red rash (urticarial); red skin without a rash; swelling of the face, mouth (angioedema), feeling dizzy / lightheaded; sneezing or an itchy, runny or blocked nose; intestinal irritation, diarrhoea

[0519] 2. With monitoring of plasma succinate levels: significantly improved values of items under 1 and insight in extents and causes of their triggering environmental queues.

[0520] Example 16

[0521] Background: benzodiazepines, opioids, anti-epileptics, and anti-inflammatory drugs (such as methotrexate, steroids, non-steroidal anti-inflammatory drugs [NSAIDs]) are well known to cause addiction / dependency to these medications. While all of them have significant side effects and with several of them the original problem will come back after some time (as they do not address the cause, but treat a symptom), it is difficult for patients to taper them off, as the extent to which they can taper them off depends on de dose of reduction, which can often not be flexibly determined, AND the extent of stress / resilience the individual experiences at that moment. Monitoring succinate as a readout of their momentary stress level and the response to the change of reducing the dose, which indicates their resilience, will allow flexible and scientifically-based personalized tapering medication.

[0522] Hypothesis: measuring succinate will show (1) the momentary stress level of the patient, (2) the extent of increase of stress with lowering of the medication dose, (3) reveal the personal and momentary resilience to reduction of the medication dose (a lower basal succinate / a smaller increase in succinate in time with a higher level of resilience.

[0523] 10 patients with withdrawal syndrome are recruited (withdrawal from addictive medicine). Patients are randomly assigned to group 1 or group 2.

[0524] Group 1

[0525] Not monitoring succinate level changes according to the invention.

[0526] Group 2

[0527] Monitoring succinate level changes according to the invention.

[0528] Results

[0529] 1. Without succinate measurement: Patient has less information about triggering environmental queues and does not adequately avoid them, leading to: Benzodiazepines: abdominal cramps; agoraphobia; anxiety, including muscle tension, tight chest, fast heartbeat, sweating, trembling, shaking), blurred vision, concentration problems, dizziness, face and neck pain, headaches, increased sensitivity to light / smell / touch / noise, depression, nausea, nightmares, panic attacks.

[0530] Opioids: sweating, diarrhoea, vomiting, abdominal cramps, chills, anxiety, insomnia, tremor, drowsiness, dizziness, low blood pressure

[0531] Anti-epileptics: seizure re-occurrence, injury, loss of self-esteem, unemployment, inability to drive

[0532] NSAIDs: swelling, pain, anxiety, agitation, nausea, headache, depression, blood clothing, increased risk of heart attack

[0533] Methotrexate: joint stiffness, pain, swelling, fatigue, fever, disease flares

[0534] Steroids: fatigue, weakness, body aches, joint pain, loss of appetite, lightheadedness, irritability / mood swings

[0535] 2. With monitoring of plasma succinate levels: significantly improved values of items under 1.

[0536] Example 17

[0537] The choice for using amperometry to measure succinate on screen-printed electrode or a continuous biosensor has several advantages over measurements described in prior art or HRV. These are the following:

[0538] Techniques described in prior art chromatography (HPLC, gas chromatography, liquid chromatography), mass spectrometry (e.g. MS, MS-MS), enzyme-linked absorbant assay (ELISA), antibody linkage, other immunochemical techniques, and combinations thereof (e.g. LC-MS-MS).

[0539] Advantage of amperometrical analysis over techniques:

[0540] Many clinical labs around the world do not have these equipments

[0541] Laborious, high costs

[0542] Too complex and expensive to be used by any individual

[0543] HRV:

[0544] Disadvantages of HRV as compared to amperometrical analysis:

[0545] 1. Clinically unreliable, as strongly influenced by motion, breathing, blood pressure / baroreflex changes, and, depending on whether detected with ECG or photoplethysmography (PPG) by baseline drift / increased electrode impedance (ECG) or ambient light / skin complexation (PPG) (altini, 2021 ; Bali & Jaggi, 2015; Miglis, 2018; Stone et al., 2021).

[0546] 2. HRV runs to a plateau with high-intensity exercise (Storniolo et al, 2021)

[0547] 3. HRV range is limited by its dependence on heart rate (Monfredi & Lakatta, 2019)

[0548] 4. A time window of 5 minutes is needed to make a proper reading during which artifact heart beats are often observed. ECG signals are mostly affected by different noise and artifact sources. These artifacts destroy a proper reading. Therefore, these need to be and are corrected for through software-matic means. (Jeyhani et al, 2015)

[0549] 5. variations in skin complexions and textures influence the quality of data acquisition from PPG devices. Individuals with darker skin tones are often associated with higher error rates from PPG that utilizes a green LED (Fallow et al., 2013; Bent et al. , 2020). Elderly patients with fragile skin and tremors are also known to elicit bad signal recording quality (De Arriba-Perez et al., 2016; Georgiou et al., 2018).

[0550] 6. Distal HRV measurements (e.g. smartwatches at the wrist) have a low signal-to-noise ratio as sensors close to the heart and it is stated that ‘It is of concern to incorporate such poor-quality data into daily clinical decision making. It is leading to a calculated $3.1 trillion of health care costs in the US’ (Stone et al, 2021).

[0551] 7. In diabetes, HRV is the golden standard to establish cardiovascular autonomic neuropathy (CAN), which is recognized as a major complication of T1 DM and T2DM. This severely confounds HRV as a technique to properly measure stress in diabetic patients. In line, the Americal Diabetes Association (ADA) considers HRV useful for research purposes only (Duque et al., 2021 ; Fleischer, 2012; Nyiraty et al, 2018; Wegeberg et al, 2020)

[0552] Example 18

[0553] Expression of the E. coli SDH complex

[0554] The entire E.coli SQR operon is cloned into an expression vector and a cleavable his-tag is cloned at the N-terminus of SDHB as reported for the Thermophilus SQR (Kolaj-Robin et al, 2013). This construct is transfected into bacterial strain DW35, which lacks both the SQR and QFR, due to which the bacteria have to use plasmid SQR to survive (Tornroth et al, 2002). Bacteria will be grown as described (Park et al, 1995).

[0555] Isolation of the SQR complex (being SDHA-SDHD) and SDH (SDHA and SDHB) is done using his-tag affinity chromatography as described (Kolaj-Robin et al., 2013). All steps will be done and materials and buffers will be pre-cooled at 2-3°C. Briefly, pelleted E.coli cells are lysed using lysozyme and spun down to remove cytosolic proteins. The proteins of the pelleted membrane fraction are solubilized in 0.05 mM Tris-HCI, pH 7.6, 300 mM NaCI, 10 mM imidazole and 5% Triton X-100 and non-solubilized proteins are removed by centrifugation. The supernatant is applied to a Nickel Sepharose column, after which bound SQR proteins are washed extensively with 0.05 mM Tris-HCI, pH 7.6, 300 mM NaCI, 10 mM imidazole and 0,05% dodecyl-p-maltoside (DDM) until triton X-100 is removed. Without cleavage, bound SQR complex is eluted using a linear imidazole gradient, pooled, diluted with 0.01 M Tris-HCI (pH 7.6), 0.05% DDM, washed and concentrated to 10-15 mg / ml, aliquoted and snap-frozen. To isolate the SQR complex without his-tag, the column-bound his-tagged SQR complex is treated with his-tagged rTEV protease (Sigma-Aldrich), eluted from the column, concentrated, aliquoted and snap-frozen.

[0556] To specifically isolate SDH, the dimer is separated from the pelleted membrane fraction with sodium perchlorate as described (Morningstar et al, 1985). Following removal of the membrane fraction with centrifugation, the supernatant is subjected to the Nickel sepharose column as described above. The his-tagged and cleaved-off SDH will subsequently be isolated as described above.

[0557] Also, non-tagged E.coli SQR is isolated as described by Tornroth et al (Tornroth et al., 2002). Briefly, E.coli SQR is produced in DW35 bacteria from the pFAS plasmid, producing E.coli SQR directed from the fumarate reductase promoter. Cells are disrupted with an homogeniser, unbroken cells removed, and membrane fraction isolated following centrifugation at 120000g. The pellet fraction is dissolved in Thesit-containing buffer, loaded on a DEAE-Sepharose FF column and, after washing, the enzyme is eluted using a NaCI gradient and concentrated. The collected SQR enzyme is further purified using a Poros 50HQ and Sephacryl S-300 gel filtration column, concentrated, and used to coat / couple on voltametric device or amperometric sensor. The enzyme obtained is highly active with a succinate-quinone reductase kcat at 303 K in the range of 90-100 / s using the Q1 homologue of ubiquinone as described.

[0558] During isolation, malonate is added to 1 mM to reversible inhibit SDH activity as described (Stepanova et al., 2016). While stabilizing the complex, malonate is easily displaced by succinate, which allows direct activation upon succinate addition.

[0559] The purity, redox cofactor content and SDH activity can be determined.

[0560] Considerations for using bacterial SQR as the enzyme complex

[0561] 1 . Methodology to detect succinate in blood / interstitial fluid: as shown for continuous glucose monitoring devices, commercial exploitation of continuous detection of blood metabolites focusses on electrochemical enzymatic sensors due to their selectivity, simplicity of design and high performance (Lipinska etal, 2021). Such enzymes specifically metabolize a molecule of interest and generate a quantitative readout of the reaction. Screening of all the different biochemical pathways for succinate metabolizing enzymes revealed eight different succinate metabolizing enzymes throughout the eukaryotic and prokaryotic kingdoms. As SQR / SDH only needs succinate as a substrate and generates, like glucose dehydrogenase (GDH) used in CGM devices, an electron upon oxidation of succinate that can be quantitatively measured using amperometry, SQR / SDH is chosen as the protein complex to work out as a sensor for bodily fluid succinate detection.

[0562] 2. Immunogenicity. Our body usually responds to exogenous (foreign) proteins with an immune response, therewith realizing an inflammation and degrading the respective protein. On the other hand, proteins attached to sensors are coated, which may protect them from being detected by our immune system. The question therefore was whether a human mammalian enzyme complex was needed. However, glucose dehydrogenase in glucose sensors is mostly derived from the fungi, Aspergillus niger (Vashist etal, 2011 ; Zhao etal, 2020). Considering the substrate specificity and availability of various electron mediators, fungi-derived FAD dependent GDHs (fFADGDHs) are currently the most popular enzymes and are used in sensor strips in second-generation SMBG sensors (Okuda-Shimazaki etal, 2020). Moreover, the bFADGDH from Burkholderia cepacia (BcGDH) is the most common enzyme used for glucose monitoring because of its inherent direct electron transfer (DET) ability and stability (Okuda-Shimazaki et al., 2020). Therefore, the chosen SQR / SDH does not need to be of human origin.

[0563] 3. Eukaryotic or prokaryotic SQR / SDH: a. Simplicity and yield: bacteria and yeast have the advantage of high expression of proteins due to their easy growth and ability to use versatile plasmids that allow controlled high expression of proteins. Moreover, bacteria express the SQR four subunits in one operon, which facilitates potentially- needed modification to optimize protein features. Therefore, expression in bacteria, or otherwise yeast, is desirable, independent on whether eukaryotic or prokaryotic SQR / SDH needs to be expressed. b. Bacterial SQR / SDH useful: eukaryotic (yeast, mammalian) proteins often need post-translational modifications for proper functioning, a feature that does not occur in prokaryotes. The SQR / SDH proteins, however, are proteins of mitochondria, which are evolutionarily derived from bacteria that started to ‘live’ in symbiosis with eukaryotic cells. As such, the four SQR subunits are not modified like eukaryotic proteins. Moreover, the SDHA and SDHB subunits, which form the functional unit of the SDH complex, are highly conserved between prokaryotes and eukaryotes, including mammalian mitochondria (Lemos et al, 2002; Maio et al, 2014). Thus, bacterial SQR / SDH can be used to form the succinate sensor, allowing expression in its own host.

[0564] 4. SDH (SDHA-SDHB) or SQR: the SDHA-SDHB two-subunit complex displayed succinate dehydrogenase activity with artificial electron acceptors (Moosavi et al., 2019) and is water-soluble, which facilitates its isolation. However, the in vivo SQR complex consists of a trimer of the SDHA-D subunits (Kolaj-Robin et al., 2013; Yankovskaya et al, 2003). As in vivo conformations are usually more stable and it is difficult to tell in advance whether the dimer or the full complex has better electron guidance when coated on sensor material (see later), the bacterial SQR as well as SDH can be isolated and tested.

[0565] 5. Blood plasma conditions as a selection criterion: the SQR activity of the different bacteria has adapted to the living conditions of the respective bacteria. For example, SDH of Archae and thermophilus bacteria are active in 1 M KCI and at 70°C, respectively, as these are the conditions these bacteria live in. Moreover, whereas Thermophilus SQR is more stable than E.coli SQR and yields higher activities at different voltages (Melin et al, 2014), its Km of 300 uM (Kolaj-Robin et al., 2013) is too high to sensitively detect plasma succinate levels (20-200 uM). As succinate detection of blood / interstitial fluid is needed, a bacterial SQR / SDH is needed that can sensitively detect succinate under blood-physiological conditions. As such, E.coli SQR / SDH is chosen, because it is most active at 37°C, has a succinate Km value of 70 uM (Kita et al, 1989), which is well in the range of plasma succinate levels, and is active at high oxygen concentrations.

[0566] Example 19

[0567] Preparing device for amperometric detection

[0568] (Lipinska et al. , 2021): the electric signal correlates with the substrate concentration, but this relationship is also affected by the sensor construction and the environment of the measurement. In order to prepare the perfect biosensor, characterized by a low limit of detection, wide linear range, high sensitivity, good selectivity and the absence of non-specific bindings, effective electron transfer between the enzyme and transducer, as well as high stability and an excellent understanding of the interactions between the enzyme modification components are beneficial. The immobilization of an enzyme can have various effects on enzyme activity. It is valuable because this makes it possible to reuse an enzyme multiple times, thereby extending its life span as well as reducing its degradation. In addition, this immobilization influences the improvement of its pH and temperature stability. The selectivity, specificity and activity can be improved after immobilization by changing the conformation of the enzyme. Therefore, the enzyme is spotted onto different nanoparticles. Currently, many nanoparticles include the noble metals Au, Ag, Pt, Pd, and oxides (CuO, Cu2O, NiO, Fe2O3, as well as bimetallic systems (Au-Pt, Au-Pd, and Cu-Ag, have been used for glucose detection. Nevertheless, the major benefit of using gold is a higher glucose oxidation current than for other noble metals. The main advantages of immobilization are multiple or repeatable usage of molecules and improving their stability. There are five most commonly applied methods: covalent bonding, adsorption, cross-linking, entrapment and self-assembled monolayers.

[0569] Cyclic voltammograms (redox activity) of, among others, E.coli SDH have been performed following adsorption of a protein film on pyrolytic graphite edge (PGE) or gold rotating disk electrodes (Ackrell et al, 1993; Pershad et al, 1999). Also Thermophilus SQR was adsorbed onto PGE rotating disk electrodes (Kolaj-Robin et al, 2011). As this technique showed an abrupt decrease in SQR / SDH activity once a critical negative driving force was generated (diode-behaviour (Elliott et al, 2002; Pershad et al., 1999; Sucheta etal, 1992)), focus will be on electron acceptors yielding a potential difference in the sensitive region. At pH of 7.5, but as determined with the PGE rotating disk electrode, the instable SDHA-SDHB dimer, and at a saturating succinate concentration, an increasing SDH activity was observed between +5 mV to a maximum of about -85 mV (versus E°' / s) (Sucheta et al., 1992). At the non-physiological pH of 8.3, sustained succinate oxidation, occurring at a non-physiological succinate concentration of 6.2 mM, was obtained at a redox potential of -200 mV (Sucheta et al., 1992). Following adsorption onto single-walled carbon nanotubes (SWNT), Melin et al. performed cyclic voltammetry of isolated E.coli / Thermophilus SQR. E.coli SQR showed low catalytic activity, possibly due to isolation procedure (Melin etal., 2014). Isolation of the complex was according to (Ruprecht et al, 2009).

[0570] To develop a 1.0 prototype succinate biosensor, the isolated SQR / SDH complexes can be adsorbed onto single-walled carbon nanotubes (SWNT) as described (Melin et al., 2014). Briefly, a commercial glassy carbon disk electrode (3 mm diameter) can be modified by dropcasting it with a dispersion of SWNT in EtOH / H2O mixture. In total, 3 SWNT deposits can be made. Following SWNT annealing, the SDH / SQR proteins can be diluted to less than 0.05% detergent concentration and 4 ul of a 0.5 uM SQR / SDH solution can be O / N adsorbed to the working electrode surface at 4oC. After adsorption, the electrode will be rinsed to remove excess protein. Current-voltage measurements can be performed using a standard three electrode cell connected to a Princeton Applied Research VERSASTAT 4 potentiostat. An aqueous AgCl / Ag 3M NaCI can be used as a reference electrode and a platinum wire as the counter electrode. Potentials are quoted versus the standard reference electrode. Succinate-derived currents are measured at a set potential of 100 mV. Melin reveals a switch point at 40 mV, but is at pH of 8 in PBS (Melin et al., 2014).

[0571] Succinate concentrations can be determined in blood / interstitial fluid, which has a pH around 7.4, [Na] of around 140 mM, [K] of around 4 mM, [Cl] of around 100 mM.

[0572] Example 20

[0573] Commercial detection of succinate:

[0574] At present, succinate is only detected at scientific level and mainly comes in two flavours:

[0575] 1. Enzymatic assays in which most employ a succinyl-CoA synthetase, pyruvate kinase and L-lactate dehydrogenase coupled reaction to convert succinate in NADH, which is subsequently detected with colourimetry or spectrophotometry. Examples are the kits from abeam (https: / / www.abcam.com / succinate-assay-kit-colorimetric- ab204718.html?qclsrc=aw.ds|aw.ds&gclid=EAIalQobChMlpae6- 4GC9glV2ud3ChOPhwKSEAAYASAAEgK8j D BwE) or megazyme (https: / / www.meqazyme.com / succinic-acid-assay-kit?sSearch=succinate). We have used the latter to measure succinate in blood samples in the lab (Robben et al, 2009; van Diepen et al, 2017).

[0576] 2. Liquid chromatography tandem mass-spectrometry, as e.g. performed in (Lamy et al, 2022).

[0577] These assays are not suitable for continuous detection of succinate of body fluids or for ex- vivo at home succinate analysis.

[0578] Relation diabetes sensitivity and succinate

[0579] Physiology. Although they vary somewhat between persons depending on their genetic constitution, basal plasma / serum succinate levels are normally detected at 2-20 pM, respectively (Ariza etal., 2012). Extracellular succinate levels transiently increase in response to acute physiological adaptations such as exercise, food ingestion, and cold exposure (Fernandez-Veledo et al, 2021). Local extracellular succinate levels range from < 20 uM at rest to 60-200 uM during exercise, followed by rapid renormalization post-exercise( Reddy et al, 2020). Moreover, hypoxia leads to cellular succinate release in vitro (van Diepen et al., 2017). Diabetes In type-2 diabetic (T2DM) patients, mean succinate levels are increased to around 60 uM (Ceperuelo-Mallafre et al, 2019; van Diepen et al., 2017). Following bariatric surgery, succinate levels in T2DM patient was significantly decreased from 60 to 46 uM (Ceperuelo- Mallafre et al., 2019). The EC50 van de SLICNR1 is 28-56 uM and 99% responses is achieved at 200 uM or higher (He et all, 2004).

[0580] (Fernandez-Veledo et al., 2021): Succinate has been recognized for many years as a danger signal in the immune system that boosts proinflammatory responses.

[0581] Circulating succinate correlates not only with body mass index (B I) but also with several markers of a worse metabolic profile, including insulin, glucose, homeostatic model assessment of insulin resistance, diastolic blood pressure, and triglycerides. Indeed, after adjusting for confounders, BMI and glucose were the main determinants of circulating succinate, suggesting that both obesity and diabetes have an impact on this metabolite. It was found that in patients with T2D and morbid obesity, in whom surgical weight loss intervention decreased circulating succinate levels. During these analyses, we noted that preoperative levels of succinate can be used as a determinant of diabetes remission after bariatric surgery and also to allocate these patients to the best-suited surgical procedure in terms of diabetes remission and complications. This new concept of succinate as a predictive marker, which previously had been inferred in other pathologies, including in critically injured patients and in patients with cancer, offers a new perspective of succinate as an important sensor of metabolic disturbances. Overall, succinate might provide a broader view of homeostasis as an easily measurable blood based metabolic biomarker. In addition, our data also showed that the nutritional modulation of plasma succinate that occurs under healthy conditions is blunted in patients with T2D and morbid obesity and is recovered after weight loss, which is reminiscent of the typical behaviour of some hormones in an obesogenic context.

[0582] Example 21

[0583] Background: as people with these occupations live at the edge of human physical abilities, it is of importance to optimize the health and high-performance levels of soldiers, pilots and athletes. While soldiers and pilots often encounter stressful conditions while in action, it is important to identify their personal stressing queues and to improve their resilience against such queues. For athletes, it is important for them not to overtrain and to have a performance peak at the right moment. Their abilities to perform will depend on the many other stresses we experience. Hypothesis: measuring succinate will show (1) increased plasma and tears levels when they are fatigue do to extensive labour or a stressing queue, (2) a decrease with effective therapy (addressing the cause(s)), (4) which environmental queues are the strongest stressors for the person (5) indicate the level of resilience with activity / therapy by the speed at which blood succinate is increased (lower than before) with a challenge and decreased following the challenge.

[0584] 10 patients with labour fatique symptoms are recruited. Patients are randomly assigned to group 1 or group 2.

[0585] Group 1

[0586] Not monitoring succinate level changes according to the invention.

[0587] Group 2

[0588] Monitoring succinate level changes according to the invention.

[0589] Results

[0590] 1. Without succinate measurement: Patient has less information about triggering environmental queues and does not adequately avoid them, leading to: lack of concentration / energy, retarded alertness and coordination, tiredness, feeling moody, long recovery times, more injuries than usual, suppressed immune system, damaged nervous system, reduced performance, reduced ability to train

[0591] 2. With monitoring of plasma succinate levels: significantly improved values of items under 1 and insight in moments, extents and causes of their triggering environmental queues.

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Claims

CLAIMS1. Method for determining insulin dosage for a subject in need thereof, the method comprising: a1) determining basal Insulin Sensitivity Factor (ISF) of the subject in conditions wherein the subject is at least 2 hours free from having meal, physical activity and infection; a2) determining basal succinate level of the subject in conditions wherein the subject is at least 2 hours free from having meal, physical activity and infection; b) determining succinate level in a body fluid sample obtained at a first time from the subject; c) determining succinate level in a body fluid sample obtained at a second time from the subject, wherein the time period between the first time and the second time preferably covers conditions of having meal, physical activity and / or infection by the subject; d) determining glucose level in a body fluid sample obtained from the subject, at the second time or within 10 minutes thereof; e) determining if the succinate level determined under c) is lower, identical or higher than the succinate level determined under b), wherein in case the basal succinate determined under a2) is 0-50 pM:- a succinate level increase between the first time and the second time by more than 40% indicates a decrease of ISF by more than 40% and at most 80% of the subject between the first time and the second time;- a succinate level increase between the first time and the second time by at least 10% and at most 40% indicates a decrease of ISF by at least 15% and at most 40% of the subject between the first time and the second time,- an identical or less than 10% deviation of succinate level between the first time and the second time indicates no or less than 15% change of ISF of the subject between the first time and the second time;- a succinate level decrease between the first time and the second time by more than 40% indicates an increase of ISF by more than 60% and at most 400% of the subject between the first time and the second time;- a succinate level decrease between the first time and the second time by more than 10% and at most 40% indicates an increase of ISF by more than 15% and at most 60% of the subject between the first time and the second time; in case the basal succinate determined under a2) is more than 50 pM, preferably between 50 and 100 pM:- a succinate level increase between the first time and the second time by more than 20% indicates a decrease of ISF by more than 20% and at most 60% of the subject between the first time and the second time;- a succinate level increase between the first time and the second time by at least 10% and at most 20% indicates a decrease of ISF by at least 10% and at most 20% of the subject between the first time and the second time;- an identical or less than 10% deviation of succinate level between the first time and the second time indicates no or less than 10% change of ISF of the subject between the first time and the second time;- a succinate level decrease between the first time and the second time by more than 20% indicates an increase of ISF by more than 40% and at most 100% of the subject between the first time and the second time;- a succinate level decrease between the first time and the second time by more than 10% and at most 20% indicates an increase of ISF by more than 10% and at most 40% of the subject between the first time and the second time; f) administering an insulin dosage to the subject, wherein the dosage in insulin units is within a 20% margin of: (determined glucose level - healthy reference glucose level) I ISF.

2. Method according to claim 1 , wherein the second time is at least 1 , 5, 10 seconds, or at least 1 , 5, 10 minutes, or at least 1 , 5, 10 hours after the first time.

3. Method according to any one of the previous claims, wherein ISF is defined by the amount of blood glucose level (mmol / L) that is lowered by one unit of insulin.

4. Method according to any one of the previous claims, wherein the ISF at the first time is prior determined and / or the same as basal ISF.

5. Method according to any one of the previous claims, wherein the sample in any one of steps b), c), and d) is a body fluid sample, wherein the body fluid sample is chosen from the group consisting of blood sample, blood plasma sample, blood serum sample, tear fluid sample, saliva fluid sample, sweat sample, and interstitial fluid sample.

6. Method according to any one of the previous claims, wherein the subject has Type 1 Diabetes Mellitus, Type 2 Diabetes Mellitus, obesity, metabolic syndrome, Non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), cancer, and / or cardiovascular, neurologic and endocrinologic diseases.

7. Method according to any one of the previous claims, wherein the method is performed ex vivo.

8. Method according to any one of the previous claims, wherein determining succinate level is performed by measuring heart rhythm variability (HRV) and / or wherein determining succinate level is performed by measuring Ultra-weak Photon Emission (UPE).

9. Method according to any one of the previous claims, wherein determining succinate level is performed by using an electrochemical biosensor, preferably a voltametric biosensor or an amperometric biosensor.

10. Method according to claim 9, wherein the voltametric biosensor or the amperometric biosensor comprises an electrode coated with an immobilized enzyme.

11. Method according to any one of the previous claims, wherein determining succinate level is performed by(i) contacting the sample of step b) and / or the sample of step c) with an amperometric sensor, wherein the sensor comprises an electrode coated with an immobilized enzyme; and(ii) measuring current change, which corresponds to succinate level in the respective sample.

12. Method according to any one of claims 10-11 , wherein the enzyme is succinate dehydrogenase (SDH), succinate-coenzyme Q reductase (SQR), quinol fumarate reductase (QFR), succinate:ubiquinone oxidoreductase, succinate:quinone oxidoreductase or succinate:(ubi)quinone reductase.

13. Method according to claim 12, wherein said succinate dehydrogenase (SDH) comprises or is a dimer of FAD cofactor binding (flavo)protein (SDHA) and Iron-sulfur (Fe-S) cluster containing protein (SDHB).

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