MARKERS IN PREPUBERTY FOR CHILDHOOD-PREDIABETES

Abstract

The present invention generally relates to a method for predicting high blood level glucose in biofluid of a subject. Methods of improving glucose level management in an adolescent subject are also provided.

Claims

1. A method for predicting high blood level glucose in a subject comprising: a. (i) determining the levels of 3-D-hydroxybutyrate and one or more of citrate, lactate, and asparagine in a biofluid sample collected from the subject when in prepuberty; and/or (ii) determining the levels of one or more of 3-D-hydroxybutyrate, valine, and creatine in a biofluid sample collected from the subject when in prepuberty; b. (i) comparing the ratios of the levels of one or more of 3-D-hydroxybutyrate: citrate, 3-D-hydroxybutyrate:lactate, and 3-D-hydroxybutyrate: asparagine with a corresponding reference value; and/or (ii) comparing the levels of one or more of 3-D-hydroxybutyrate, valine, and creatine with a corresponding reference value; c. identifying the subject as being at higher risk of high blood level glucose in adolescence if (I) the ratios of the levels of one or more of 3-D-hydroxybutyrate:citrate, 3-D-hydroxybutyrate:lactate, and 3-D-hydroxybutyrate:asparagine are higher than the corresponding reference value in b(i); and/or (II) the levels of one or more of 3-D-hydroxybutyrate, valine, and creatine are higher than the corresponding reference value in b(ii).

2. The method for predicting high blood level glucose in a subject according to claim 1 comprising: a. determining the levels of 3-D-hydroxybutyrate and one or more of citrate, lactate, and asparagine in a biofluid sample collected from the subject when in prepuberty; b. comparing the ratios of the levels of one or more of 3-D-hydroxybutyrate:citrate, 3-D-hydroxybutyrate:lactate, and 3-D-hydroxybutyrate:asparagine with a corresponding reference value; and c. identifying the subject as being at higher risk of high blood level glucose in adolescence if the ratios of the levels of one or more of 3-D-hydroxybutyrate:citrate, 3-D-hydroxybutyrate:lactate, and 3-D-hydroxybutyrate:asparagine are higher than the corresponding reference value in (b).

3. The method for predicting high blood level glucose in a subject according to claim 1 comprising: a. determining the levels of 3-D-hydroxybutyrate and one or more of citrate and asparagine in a biofluid sample collected from the subject when in prepuberty; b. comparing the ratios of the levels of one or more of 3-D-hydroxybutyrate:citrate, and 3-D-hydroxybutyrate:asparagine with a corresponding reference value; and c. identifying the subject as being at higher risk of high blood level glucose in adolescence if the ratios of the levels of one or more of 3-D-hydroxybutyrate:citrate, and 3-D-hydroxybutyrate:asparagine are higher than the corresponding reference value in (b).

4. The method for predicting high blood level glucose in a subject according to claim 1, the method comprising: a. determining the levels of one or more of 3-D-hydroxybutyrate, valine, and creatine in a biofluid sample collected from the subject when in prepuberty; b. comparing the levels of one or more of 3-D-hydroxybutyrate, valine, and creatine with a corresponding reference value; and c. identifying the subject as being at higher risk of high blood level glucose in adolescence if the levels of one or more of 3-D-hydroxybutyrate, valine, and creatine are higher than the corresponding reference value in (b).

5. The method according to claim 1, wherein the high blood level glucose corresponds to childhood prediabetes.

6. The method according to claim 1, wherein the biofluid sample is collected when the subject is age 6 or 7 years.

7. (canceled)

8. The method according to claim 1, wherein more than one biofluid sample is collected from said subject in steps a(i) and/or a(ii).

9. The method according to claim 1, wherein adolescence corresponds to age 13 to 16 years.

10. (canceled)

11. The method according to claim 1, wherein the biofluid sample is collected from a normal weight subject.

12. The method according to claim 1, wherein the reference value is a predetermined standard.

13. The method according to claim 1, wherein the biofluid sample is human blood serum.

14. The method according to claim 1, wherein high blood level glucose presents in the form of impaired fasting glucose.

15-16. (canceled)

17. A method of improving glucose level management in an adolescent subject comprising; (i) predicting the likelihood of the subject having high blood level glucose comprising: a. (i) determining the levels of 3-D-hydroxybutyrate and one or more of citrate, lactate, and asparagine in a biofluid sample collected from the subject when in prepuberty; and/or (ii) determining the levels of one or more of 3-D-hydroxybutyrate, valine, and creatine in a biofluid sample collected from the subject when in prepuberty; b. (i) comparing the ratios of the levels of one or more of 3-D-hydroxybutyrate:citrate, 3-D-hydroxybutyrate:lactate, and 3-D-hydroxybutyrate:asparagine with a corresponding reference value; and/or (ii) comparing the levels of one or more of 3-D-hydroxybutyrate, valine, and creatine with a corresponding reference value; c. identifying the subject as being at higher risk of high blood level glucose in adolescence if (I) the ratios of the levels of one or more of 3-D-hydroxybutyrate:citrate, 3-D-hydroxybutyrate:lactate, and 3-D-hydroxybutyrate:asparagine are higher than the corresponding reference value in b(i); and/or (II) the levels of one or more of 3-D-hydroxybutirate, valine, and creatine are higher than the corresponding reference value in b(ii); and (ii) providing a method of modifying the lifestyle of a subject identified as being at higher risk of having high blood level glucose in adolescence, wherein the dietary intervention reduces the glucose level.

18. The method according to claim 17, wherein the modification of lifestyle reduces the likelihood or prevents high blood level glucose.

19. (canceled)

20. The method according to claim 17, wherein the method reduces the likelihood or prevents type 2 diabetes in adulthood.

21. (canceled)

22. The method according to claim 17, wherein the modification in lifestyle of the subject comprises a change in diet.

23-25. (canceled)

26. The method of claim 22, wherein the change in diet comprises a ketogenic type of diet, that provides sufficient protein for body growth and repair, and sufficient calories to maintain the correct weight for age and height, wherein said ketogenic diet is the consumption of under 20 g of carbohydrates per day.

27-29. (canceled)

30. The method of claim 22, wherein the change in diet comprises a supplementation of essential nutrients aiming at improving glucose management, such as essential amino acids, lipid and water soluble vitamins, minerals, or a combination of nutrients.

31. The method of claim 22, wherein the change in diet is associated with physical activity program management.

32. (canceled)

33. A kit of parts comprising a method to measure levels of 3-D-hydroxybutyrate, valine, creatine, citrate, lactate, and asparagine in a biofluid sample collected from a subject in prepuberty and a method to use the kit to predict high blood levels of glucose in a subject.

34. (canceled)

Description

DETAILED DESCRIPTION

[0036] The present invention provides a method for predicting high glucose level in biofluid of a subject, said method comprising:

[0037] a. (i) determining the levels of 3-D-hydroxybutyrate and one or more of citrate, lactate, and asparagine in the biofluid of said subject; and/or (ii) determining the levels of one or more of 3-D-hydroxybutyrate, valine, and creatine in the biofluid sample of said subject;

[0038] b. (i) comparing the ratios of the levels of one or more of 3-D-hydroxybutyrate:citrate, 3-D-hydroxybutyrate:lactate, and 3-D-hydroxybutyrate:asparagine with a reference value; and/or (ii) comparing the levels of one or more of 3-D-hydroxybutyrate, valine, and creatine with a reference value;

[0039] c. identifying the subject as being at higher risk of high glucose level if [0040] (I) the ratios of the levels of one or more of 3-D-hydroxybutyrate:citrate, 3-D-hydroxybutyrate:lactate, and 3-D-hydroxybutyrate:asparagine are higher than the reference value in b(i); and/or [0041] (II) the levels of one or more of 3-D-hydroxybutyrate, valine, and creatine are higher than the reference value in b(ii).

[0042] The present invention further provides a method for predicting high glucose level, particularly high blood glucose level in a subject, said method comprising:

[0043] a. (i) determining the levels of 3-D-hydroxybutyrate and one or more of citrate, lactate, and asparagine in a biofluid sample collected from said subject when in prepuberty; and/or (ii) determining the levels of one or more of 3-D-hydroxybutyrate, valine, and creatine in a biofluid sample collected from said subject when in prepuberty;

[0044] b. (i) comparing the ratios of the levels of one or more of 3-D-hydroxybutyrate:citrate, 3-D-hydroxybutyrate:lactate, and 3-D-hydroxybutyrate:asparagine with a corresponding reference value; and/or (ii) comparing the levels of one or more of 3-D-hydroxybutyrate, valine, and creatine with a corresponding reference value;

[0045] c. identifying the subject as being at higher risk of high glucose level in adolescence if [0046] (I) the ratios of the levels of one or more of 3-D-hydroxybutyrate:citrate, 3-D-hydroxybutyrate:lactate, and 3-D-hydroxybutyrate:asparagine are higher than the corresponding reference value in b(i); and/or [0047] (II) the levels of one or more of 3-D-hydroxybutyrate, valine, and creatine are higher than the corresponding reference value in b(ii).

[0048] In one embodiment, the method for predicting high blood level glucose in a subject comprises:

[0049] a. determining the levels of 3-D-hydroxybutyrate and one or more of citrate, lactate, and asparagine in a biofluid sample collected from said subject when in prepuberty;

[0050] b. comparing the ratios of the levels of one or more of 3-D-hydroxybutyrate:citrate, 3-D-hydroxybutyrate:lactate, and 3-D-hydroxybutyrate:asparagine with a corresponding reference value;

[0051] c. identifying the subject as being at higher risk of high blood level glucose in adolescence if the ratios of the levels of one or more of 3-D-hydroxybutyrate:citrate, 3-D-hydroxybutyrate:lactate, and 3-D-hydroxybutyrate:asparagine are higher than the corresponding reference value in (b).

[0052] In one embodiment, the method for predicting high blood level glucose in a subject comprises:

[0053] a. determining the levels of 3-D-hydroxybutyrate and one or more of citrate and asparagine in a biofluid sample collected from said subject when in prepuberty;

[0054] b. comparing the ratios of the levels of one or more of 3-D-hydroxybutyrate:citrate, and 3-D-hydroxybutyrate:asparagine with a corresponding reference value;

[0055] c. identifying the subject as being at higher risk of high blood level glucose in adolescence if the ratios of the levels of one or more of 3-D-hydroxybutyrate:citrate, and 3-D-hydroxybutyrate:asparagine are higher than the corresponding reference value in (b).

[0056] In one aspect, the method for predicting high blood level glucose in a subject comprises:

[0057] a. determining the levels of 3-D-hydroxybutyrate and citrate in a biofluid sample collected from said subject when in prepuberty;

[0058] b. comparing the ratios of the levels of 3-D-hydroxybutyrate:citrate with a corresponding reference value;

[0059] c. identifying the subject as being at higher risk of high blood level glucose in adolescence if the ratios of the levels of one or more of 3-D-hydroxybutyrate:citrate are higher than the corresponding reference value in (b).

[0060] In another aspect, the method for predicting high blood level glucose in a subject comprises:

[0061] a. determining the levels of 3-D-hydroxybutyrate and lactate in a biofluid sample collected from said subject when in prepuberty;

[0062] b. comparing the ratios of the levels of 3-D-hydroxybutyrate:lactate with a corresponding reference value;

[0063] c. identifying the subject as being at higher risk of high blood level glucose in adolescence if the ratios of the levels of one or more of 3-D-hydroxybutyrate:lactate are higher than the corresponding reference value in (b).

[0064] In another aspect, the method for predicting high blood level glucose in a subject, said method comprising:

[0065] a. determining the levels of 3-D-hydroxybutyrate and asparagine in a biofluid sample collected from said subject when in prepuberty;

[0066] b. comparing the ratios of the levels of 3-D-hydroxybutyrate:asparagine with a corresponding reference value;

[0067] c. identifying the subject as being at higher risk of high blood level glucose in adolescence if the ratios of the levels of one or more of 3-D-hydroxybutyrate:asparagine are higher than the corresponding reference value in (b).

[0068] In one embodiment, high blood level glucose corresponds to equal to or higher than 5.6 mmol fasting glucose/litre human blood plasma.

[0069] In an alternative embodiment, the present invention provides a method for predicting blood level fasting glucose below 5.6 mmol/L in a subject, said method comprising:

[0070] a. determining the levels of 3-D-hydroxybutyrate and one or more of citrate, lactate, and asparagine in a biofluid sample collected from said subject when in prepuberty;

[0071] b. comparing the ratios of the levels of one or more of 3-D-hydroxybutyrate:citrate, 3-D-hydroxybutyrate:lactate, and 3-D-hydroxybutyrate:asparagine with a corresponding reference value;

[0072] c. identifying the subject as being at lower risk of high blood level glucose in adolescence if the ratios of the levels of one or more of 3-D-hydroxybutyrate:citrate, 3-D-hydroxybutyrate:lactate, and 3-D-hydroxybutyrate:asparagine are lower than the corresponding reference value in (b).

[0073] In another embodiment, the method for predicting high blood level glucose in a subject comprises:

[0074] a. determining the levels of one or more of 3-D-hydroxybutyrate, valine, and creatine in a biofluid sample collected from said subject when in prepuberty;

[0075] b. comparing the levels of one or more of 3-D-hydroxybutyrate, valine, and creatine with a corresponding reference value;

[0076] c. identifying the subject as being at higher risk of high blood level glucose in adolescence if the levels of one or more of 3-D-hydroxybutyrate, valine, and creatine are higher than the corresponding reference value in (b);

[0077] In one aspect, the method for predicting high blood level glucose in a subject comprises:

[0078] a. determining the level of 3-D-hydroxybutyrate in a biofluid sample collected from said subject when in prepuberty;

[0079] b. comparing the level of 3-D-hydroxybutyrate with a corresponding reference value;

[0080] c. identifying the subject as being at higher risk of high blood level glucose in adolescence if the level of 3-D-hydroxybutyrate is higher than the corresponding reference value in (b).

[0081] In another aspect, the method for predicting high blood level glucose in a subject comprises:

[0082] a. determining the level of valine in a biofluid sample collected from said subject when in prepuberty;

[0083] b. comparing the level of valine with a corresponding reference value;

[0084] c. identifying the subject as being at higher risk of high blood level glucose in adolescence if the level of valine is higher than the corresponding reference value in (b).

[0085] In another aspect, the method for predicting high blood level glucose in a subject comprises:

[0086] a. determining the level of creatine in a biofluid sample collected from said subject when in prepuberty;

[0087] b. comparing the level of creatine with a corresponding reference value;

[0088] c. identifying the subject as being at higher risk of high blood level glucose in adolescence if the level of creatine is higher than the corresponding reference value in (b).

[0089] In an alternative embodiment, the present invention provides a method for predicting low blood level glucose in a subject, said method comprising:

[0090] a. determining the levels of one or more of 3-D-hydroxybutyrate, valine, and creatine in a biofluid sample collected from said subject when in prepuberty;

[0091] b. comparing the levels of one or more of 3-D-hydroxybutyrate, valine, and creatine with a corresponding reference value;

[0092] c. identifying the subject as being at lower risk of high blood level glucose in adolescence if the levels of one or more of 3-D-hydroxybutyrate, valine, and creatine are lower than the corresponding reference value in (b).

[0093] Preferably, when determining the levels of one or more of 3-D-hydroxybutyrate, valine, and creatine in a biofluid sample and comparing the levels of one or more of 3-D-hydroxybutyrate, valine, and creatine with a corresponding reference value, said biofluid sample is collected from the subject on preferably at least two occasions, wherein said at least two occasions preferably occur on or after the subject's 5.sup.th birthday but before the subject's 9.sup.th birthday, and are preferably separated by at least a one year interval, preferably as described herein.

[0094] In one embodiment, said biofluid sample collections are taken from the subject at age 5 years, 6 years, 7 years, or 8 years separated by at least a one year interval. In one embodiment, said biofluid sample is taken from the subject at age 6 years and at age 7 years.

[0095] In one embodiment, said biofluid sample collections are taken from a normal weight subject.

[0096] In one embodiment, said biofluid sample collections are taken from a normal weight subject at age 5 years, 6 years, 7 years, or 8 years separated by at least a one year interval. In one embodiment, said biofluid sample is taken from the normal weight subject at age 6 years and at age 7 years.

[0097] In one aspect of the invention, the high blood level glucose corresponds to childhood prediabetes.

[0098] In one aspect of the invention, the biofluid sample is taken when the subject is age 6 years.

[0099] In one aspect of the invention, more than one biofluid sample is taken from said subject in steps a(i) and/or a(ii).

[0100] In one aspect of the invention, metabolite measurements are made by NMR (Nuclear Magnetic Resonance). Alternatively, metabolite measurements may be made by mass spectroscopy or by clinical assay.

[0101] In one aspect of the invention, the age sub-range of 13 to 16 years is chosen as being representative of adolescence.

[0102] In one aspect of the invention, the age 16 years is chosen as being representative of adolescence.

[0103] In one aspect of the invention, the reference value is a predetermined standard.

[0104] In one aspect of the invention, the biofluid sample is human blood serum.

[0105] In one aspect of the invention, high blood level glucose presents in the form of impaired fasting glucose.

[0106] In one aspect of the invention, impaired fasting glucose is measured according to the World Health Organisation (WHO) criteria corresponding to a fasting plasma glucose level from 6.1 mmol/l (110 mg/dL) to 6.9 mmol/L (125 mg/dL).

[0107] In another aspect of the invention, wherein impaired fasting glucose is measured according to the American Diabetic Association (ADA) criteria corresponding to a fasting plasma glucose level from 5.6 mmol/L (100 mg/dL) to 6.9 mmol/L (125 mg/dL).

[0108] The present invention also provides a method of improving glucose level management in an adolescent subject comprising (i) predicting whether said subject has high blood level glucose according to the invention; and (ii) providing a method of modifying the lifestyle of a subject identified as being at higher risk of having high blood level glucose in adolescence, wherein said dietary intervention reduces the glucose level.

[0109] In one aspect of the invention, said modification of lifestyle reduces the likelihood or prevents high blood level glucose.

[0110] In one aspect of the invention, said modification of lifestyle is provided through prepuberty and puberty.

[0111] In one aspect of the invention, said method reduces the likelihood or prevents the onset of one or more metabolic disorders, particularly type 2 diabetes, particularly in early adulthood.

[0112] In one aspect of the invention, said modification of lifestyle is provided through prepuberty, puberty, and adolescence.

[0113] In one aspect of the invention, the modification in lifestyle in the subject comprises a change in diet, preferably comprising administering at least one nutritional product to the subject that is part of a diet that modulates levels of glucose

[0114] In one aspect of the invention, administering at least one nutritional product to the subject that is part of a diet that modulates levels of glucose promotes a reduction in glucose or prevents an increase in glucose levels in the subject.

[0115] In one aspect of the invention, the change in diet comprises a decreased consumption of fat and/or an increase in consumption of low fat foods such that not more than 20% of daily calories are obtained from fat.

[0116] Low fat foods includes bread and flour, oats, breakfast cereals, wholegrain rice and pasta, fresh, frozen and tinned vegetables and fruits, dried beans and lentils, baked or boiled potatoes, dried fruits, white fish, shellfish, lean wite meat such as chicken and turkey breast without skin, skimmed and smi skimmed milk, cottage or curd cheese, low fat yoghourt, or egg whites. Most adults get 20%-35% of their daily calories from fat. That equates to about 44 to 77 grams of fat a day if 2,000 calories a day are consumed. Low fat foods can also be selected from wholemeal flour and bread, porridge oats, high-fibre breakfast cereals, dried beans and lentils, walnuts, herring, mackerel, sardines, kippers, pilchards, salmon and lean white meat.

[0117] In one aspect of the invention, the change in diet comprises a ketogenic type of diet that provides sufficient protein for body growth and repair, and sufficient calories to maintain the correct weight for age and height.

[0118] A ketogenic diet may be achieved by excluding high-carbohydrate foods such as starchy fruits and vegetables, bread, pasta, grains and sugar, while increasing the consumption of foods high in fat such as nuts, cream and butter. A variant of the classic diet known as the medium-chain triglycerides (MCT) ketogenic diet uses a form of coconut oil, which is rich in MCTs, to provide around half the calories. As less overall fat is needed in this variant of the diet, a greater proportion of carbohydrate and protein can be consumed, allowing a greater variety of food choices. In one aspect of the invention, the change in diet comprises a change to a ketogenic diet. In one embodiment, a ketogenic diet is the consumption of under 20 g of carbohydrates per day.

[0119] In one aspect of the invention, the change in diet comprises a change to a Mediterranean diet.

[0120] In one embodiment, said Mediterranean diet is higher in fat, that may include intermittent fasting. For instance, in typical Mediterranean countries breakfast may be skipped, and a big lunch may be taken with equal number of calories as breakfast and lunch.

[0121] A Mediterranean diet typically contains three to nine servings of vegetables, half to two servings of fruit, one to 13 servings of cereals and up to eight servings of olive oil daily. In one embodiment, it contains approximately not less than 9300 kJ. In one embodiment, it contains not more than 37% as total fat (particularly not less than 18% as monounsaturated and not more than 9% as saturated). In one embodiment, it contains not less than 33 g of fibre per day.

[0122] As an example, food type and intake, as well as nutrient content of the Mediterranean diet are described by Davis et al. (Reference Definition of the Mediterranean Diet: A Literature Review, Davis et al., Nutrients, 7(11), 9139-9153, 2015);

[0123] In one aspect of the invention, the change in diet comprises a change to a moderate low carbohydrate diet, to maintain or reach normal blood sugar levels throughout the day. In one embodiment, a moderate low carbohydrate diet is the consumption of between 20 g to 50 g of carbohydrates per day. By comparison, a standard diet is consumption of about 50 g to 100 g of carbohydrates per day.

[0124] In one aspect of the invention, the change in diet comprises a change to a vegan diet. Typically, a vegan diet is well-balanced in macronutrient and micronutrient composition and results in lower average blood sugar levels throughout the day. Vegan diets are plant-based diet regimens that exclude meat, eggs, dairy products, and any other animal-derived foods and ingredients.

[0125] In contrast, a vegetarian diet emphasizes plant-based foods but can also include dairy, eggs, honey, and fish. Both vegan and vegetarian diets can be healthful for all life stages with appropriate selection of plant-based foods that adequately meet nutrition requirements for protein, iron, n-3 fatty acids, iodine, zinc, calcium, and vitamin B12. An intermittent vegan diet regimen that is alternated within a habitual, balanced omnivorous diet can also meet these nutritional requirements.

[0126] In one aspect of the invention, the change in diet comprises a supplementation of essential nutrients aiming at improving glucose management, such as essential amino acids, lipid and water soluble vitamins, minerals, or a combination of nutrients.

[0127] Examples of essential nutrients are amino acids (phenylalanine, valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine, and histidine); fatty acids (alpha-linolenic acid (omega-3 fatty acid) and linoleic acid (omega-6 fatty acid); vitamins (vitamin A, Bs (1-12), Vitamin C, Vitamin D, Vitamin E); minerals such as major minerals (calcium, phosphorus, potassium, sodium, chlorine, and magnesium) and minor minerals (metals such as iron, zinc, manganese and copper); and conditional nutrients (choline, inositol, taurine, arginine, glutamine and nucleotides).

[0128] In one aspect of the invention, the change in diet is associated with physical activity program management. The physical activity program should be adapted to body composition, medical conditions and age of the subjects, aiming at weight loss or weight management, and improvement of body fat mass and lean mass for optimal glucose management outcome.

[0129] For instance, the solution may be part of a Physical Activity Program which use all opportunities for students to be physically active, meet the nationally-recommended minutes of physical activity each day (e.g. 60 minutes of moderate to vigorous physically activity each day). For instance, the program may follow public health guidelines for physical activity for children and young people (as an example, National institute for health and care excellence, UK: https://www.nice.org.uk/guidance).

[0130] One aspect of the invention further comprises a step of repeating the step of predicting levels of glucose in a subject after modifying the lifestyle of the subject.

[0131] The present invention also provides a kit of parts comprising means to measure levels of creatine, citrate, and asparagine in biofluid of a subject in prepuberty.

[0132] The present invention also provides a kit of parts comprising means to measure levels of 3-D-hydroxybutyrate, valine, creatine, citrate, lactate, and asparagine in biofluid of a subject in prepuberty.

[0133] The present invention also provides the use of a kit of parts according to the invention, to predict a subject in prepuberty of having high glucose levels or developing-prediabetes in adolescence.

EXAMPLES

Example 1

[0134] Methods Used During the Study

[0135] Study Population

[0136] The EarlyBird Diabetes Study incorporates a 1995/1996 birth cohort recruited in 2000/2001 when the children were 5 years old (307 children, 170 boys). The collection of data from the Early Bird cohort is composed of several clinical and anthropometric variables measured on an annual basis from the age of 5 to the age of 16. The study was conducted in accordance with the ethics guidelines of the Declaration of Helsinki II; ethics approval was granted by the Plymouth Local Research Ethics Committee (1999), and parents gave written consent and children verbal assent.

[0137] Anthropometric Parameters

[0138] BMI was derived from direct measurement of height (Leicester Height Measure; Child Growth Foundation, London, U.K.) and weight (Tanita Solar 1632 electronic scales), performed in blind duplicate and averaged. BMI SD scores were calculated from the British 1990 standards.

[0139] Physical activity was measured annually from 5 years by accelerometry (Acti-Graph [formerly MTI/CSA]). Children were asked to wear the accelerometers for 7 consecutive days at each annual time point, and only recordings that captured at least 4 days were used.

[0140] Resting energy expenditure was measured by indirect calorimetry using a ventilated flow through hood technique (Gas Exchange Measurement, Nutren Technology Ltd, Manchester, UK). Performance tests reportedly show a mean error of 0.32.0% in the measurement of oxygen consumption and 1.81% in that of carbon dioxide production. Measurements were performed in a quiet thermoneutral room (20 C.) after overnight fasting period of at least 6 hours, to minimize any effect attributable to the thermic effect of food. Data were collected for a minimum of 10 minutes and the respiratory quotient (RQ) was calculated as an indicator of basal metabolic rate (BMR).

[0141] Clinical Parameters

[0142] Peripheral blood was collected annually into EDTA tubes after an overnight fast and stored at 80 C. Insulin resistance (IR) was determined each year from fasting glucose (Cobas Integra 700 analyzer; Roche Diagnostics) and insulin (DPC IMMULITE) (cross-reactivity with proinsulin, 1%) using the homeostasis model assessment program (HOMA-IR), which has been validated in children.

[0143] Serum Metabonomics

[0144] 400 L of blood serum were mixed with 200 L of deuterated phosphate buffer solution 0.6 M KH2PO4, containing 1 mM of sodium 3-(trimethylsilyl)-[2,2,3,3-2H4]-1-propionate (TSP, chemical shift reference SH=0.0 ppm). 550 L of the mixture were transferred into 5 mm NMR tubes.

[0145] 1H NMR metabolic profiles of serum samples were acquired with a Bruker Avance III 600 MHz spectrometer equipped with a 5 mm cryoprobe at 310 K (Bruker Biospin, Rheinstetten, Germany) and processed using TOPSPIN (version 2.1, Bruker Biospin, Rheinstetten, Germany) software package as reported previously. Standard 1H NMR one-dimensional pulse sequence with water suppression, Carr-Purcell-Meiboom-Gill (CPMG) spin-echo sequence with water suppression, and diffusion-edited sequence were acquired using 32 scans with 98K data-points. The spectral data (from 0.2 to 10) were imported into Matlab software with a resolution of 22K data-points (version R2013b, the Mathworks Inc, Natwick Mass.) and normalized to total area after solvent peak removal. Poor quality or highly diluted spectra were discarded from the subsequent analysis.

[0146] 1H-NMR spectrum of human blood plasma enables the monitoring of signals related to lipoprotein bound fatty acyl groups found in triglycerides, phospholipids and cholesteryl esters, together with peaks from the glyceryl moiety of triglycerides and the choline head group of phosphatidylcholine. This data also covers quantitative profiling of major low molecular weight molecules present in blood. Based on internal database, representative signals of metabolites assignable on 1H CPMG NMR spectra were integrated, including asparagine, leucine, isoleucine, valine, 2-ketobutyric acid, 3-methyl-2-oxovaleric acid, alpha-ketoisovaleric acid, (R)-3-hydroxybutyric acid, lactic acid, alanine, arginine, lysine, acetic acid, N-acetyl glycoproteins, O-acetyl glycoproteins, acetoacetic acid, glutamic acid, glutamine, citric acid, dimethylglycine, creatine, citrulline, trimethylamine, trimethylamine N-oxide, taurine, proline, methanol, glycine, serine, creatinine, histidine, tyrosine, formic acid, phenylalanine, threonine, and glucose. In addition, in diffusion edited spectra, signals associated to different lipid classes were integrated, including phospholipids containing choline, VLDL subclasses, unsaturated and polyunsaturated fatty acid. The signals are expressed in arbitrary unit corresponding to a peak area normalized to total metabolic profiles, which is representative of relative change in metabolite concentration in the serum.

[0147] Statistics

[0148] Using data at all ages simultaneously, mixed effects modelling was used to assess the association between glucose and individual metabolites, taking into account age, BMI sds, physical activity and pubertal timing (APHV). Random intercepts were included as well as age (categorized to allow for non-linear change in glucose over time), gender, BMI sds, APHV, MVPA (number of minutes spent in moderate-vigorous physical activity) and individual metabolites (in separate models) as fixed effects. Modelling was carried out in R software using the Imer function in the package Ime4.

Example 2

[0149] Measurement of Metabolite Concentrations

TABLE-US-00001 TABLE 1 Moderate-vigorous Glucose Insulin physical activity Respiratory (mmol) (mU) (minutes/day) mvpa BMI sds Quotient Age Gender mean sd mean sd mean sd mean sd mean sd 6 M 4.52 0.37 3.37 3.16 61.91 23.64 0.19 0.99 0.88 0.08 F 4.41 0.33 4.42 2.89 54.76 17.54 0.58 0.96 0.88 0.09 16 M 5.17 0.33 5.59 4.40 44.48 23.73 0.51 1.13 0.95 0.15 F 5.02 0.36 6.60 5.85 32.14 22.93 0.87 1.14 0.95 0.09 Characteristics of the children at age 6 and 16 years are summarized in Table 1. In both genders there was an increase in glucose, insulin, BMI sds, respiratory quotient up to year 16, and a decrease in physical activity as noted with mvpa parameter.

[0150] Using data at all ages simultaneously, mixed effects modelling were applied to assess the association between glucose and individual metabolites. The outcome of the models generated for each metabolite is reported in Table 2, for each metabolite pertaining to a given metabolic pathway. Data are reported to statistical significance and alphabetic order for metabolic pathways and metabolites (Table 2).

TABLE-US-00002 TABLE 2 Metabolic pathway Metabolite Coef SE p-value Amino acid derivatives 2-ketobutyrate 1245.2 129.37 0.000000 10.sup.0 Amino acid derivatives 3-Methyl-2oxovalericacid 2048.1 210.19 0.000000 10.sup.0 Branched chain amino acids Leucine 148.66 11.75 0.000000 10.sup.0 Branched chain amino acids Valine 132.17 13.91 0.000000 10.sup.0 Ketone bodies 3-hydroxybutyrate 169.3 11.4 0.000000 10.sup.0 Glycolysis related Glucose 55.25 6.74 8.881784 10 16 Glycolysis related Citrate 159.49 19.94 3.996803 10 15 Amino acids Arginine 240.38 32.73 4.760636 10 13 Branched chain amino acids Isoleucine 395.96 53.93 4.822809 10 13 Organic acid Creatine 214.45 29.5 8.073542 10 13 Amino acids Asparagine 747.56 119.7 6.68493 10 10 Glycolysis related Glucose 28.68 4.64 9.860142 10 10 Organic acid Creatine 245.27 43.25 1.931336 10 8 Glycolysis related Lactate 2.39 1.22 4.993002 10 2

[0151] The analysis has highlighted the importance of specific metabolites in amino acid, ketone body, glycolysis and fatty acid metabolism, in describing the variations of blood glucose throughout the childhood. This is believed to be the first report of a metabolic contribution of specific metabolic processes to overall blood glucose variations in a longitudinal and continuous manner. The analysis describes how the metabolism of branched chain amino acid and it catabolism, ketogenesis, gluconeogenic amino acids are contributing to glucose production throughout childhood.

[0152] The concentrations of these metabolites for boys and girls are reported in Table 3 at each chronological age. The information enables the appreciation of various age-related dynamics in the circulating levels of these metabolites.

TABLE-US-00003 TABLE 3 Age 5 6 7 8 9 Sex M F M F M F M F M F N 68.0 19.0 75.0 27.0 81.0 24.0 79.0 27.0 70.0 27.0 Glucose (mmol) Mean 4.3 4.4 4.5 4.4 4.6 4.6 4.8 4.7 4.8 4.9 Sd 0.4 0.4 0.4 0.3 0.5 0.4 0.3 0.4 0.5 0.3 Respiratory Mean 0.9 0.9 0.9 0.9 0.9 0.9 1.0 1.0 0.9 1.0 Quotient Sd 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 Leucine (a.u.) Mean 9.8 9.5 9.0 9.2 9.0 9.2 8.8 9.2 9.2 9.1 Sd 1.1 1.4 0.9 1.0 1.0 1.0 0.9 1.1 1.3 0.9 Valine (a.u.) Mean 6.1 5.8 5.8 5.6 5.8 5.8 5.7 5.8 6.0 5.8 Sd 0.9 0.9 0.7 0.9 0.9 0.8 0.8 1.0 1.0 0.9 2.ketobutyrate Mean 0.8 0.8 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.7 (a.u.) Sd 0.1 0.1 0.1 0.2 0.1 0.1 0.1 0.1 0.1 0.1 3Methyl2oxova- Mean 0.6 0.6 0.6 0.6 0.6 0.5 0.5 0.5 0.5 0.5 lericacid (a.u.) Sd 0.1 0.0 0.0 0.1 0.1 0.1 0.0 0.0 0.1 0.1 3Dhydroxybuty- Mean 6.5 5.4 6.0 5.0 6.1 6.1 5.2 5.2 5.8 5.0 rate (a.u.) Sd 3.7 2.0 2.4 1.0 4.3 3.6 1.0 1.0 3.1 1.2 Lactate (a.u.) Mean 37.9 40.5 37.5 38.1 37.9 39.4 41.4 45.7 44.5 47.8 sd 6.8 7.2 18.0 9.5 6.7 6.5 8.2 8.8 9.1 9.8 Citrate (a.u.) mean 5.4 5.0 5.2 4.8 5.0 5.1 4.9 4.9 5.0 4.7 sd 0.6 0.7 0.6 0.5 0.6 0.7 0.6 0.8 0.6 0.6 Arginine (a.u.) mean 4.5 4.4 4.0 4.1 3.9 4.0 3.8 4.0 3.9 3.9 sd 0.5 0.6 0.3 0.5 0.4 0.3 0.3 0.5 0.4 0.4 Isoleucine (a.u.) mean 1.4 1.4 1.2 1.4 1.2 1.3 1.2 1.3 1.3 1.3 sd 0.2 0.3 0.2 0.3 0.2 0.2 0.2 0.2 0.2 0.2 Asparagine_2.92 mean 0.9 0.9 0.9 0.9 0.9 0.8 0.9 0.8 0.9 0.8 (a.u.) sd 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 Creatine_3.02 mean 3.3 3.2 3.1 2.9 2.9 2.9 2.8 2.8 2.8 2.7 (a.u.) sd 0.4 0.4 0.5 0.4 0.5 0.3 0.4 0.5 0.4 0.5 Age 10 11 12 13 14 15 16 sex M F M F M F M F M F M F M F N 79.0 28.0 85.0 34.0 81.0 32.0 80.0 32.0 75.0 22.0 87.0 32.0 88.0 33.0 Glucose (mmol) mean 4.9 4.8 4.8 4.8 4.9 5.1 5.2 5.1 5.2 5.2 5.2 5.2 5.2 5.0 sd 0.3 0.3 0.4 0.3 0.4 0.4 0.3 0.4 0.3 0.5 0.3 0.4 0.3 0.4 Respiratory mean 0.9 0.9 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.9 1.0 1.0 Quotient sd 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.3 0.1 0.2 0.1 Leucine (a.u.) mean 8.9 9.7 9.1 9.4 9.1 9.2 9.1 9.3 9.3 9.2 9.3 9.1 9.4 9.0 sd 0.7 0.9 0.9 0.8 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.8 0.8 1.0 Valine (a.u.) mean 5.8 6.1 5.9 6.0 6.0 5.6 6.0 5.8 6.0 5.6 6.2 5.7 6.2 5.8 sd 0.8 0.9 0.7 0.7 0.8 0.9 0.8 0.8 0.8 0.7 0.8 0.8 0.8 0.9 2ketobutyrate mean 0.7 0.7 0.7 0.7 0.7 0.6 0.6 0.7 0.7 0.6 0.7 0.6 0.6 0.6 (a.u.) sd 0.1 0.1 0.1 0.0 0.1 0.0 0.1 0.1 0.1 0.0 0.1 0.1 0.1 0.1 3Methyl2oxova- mean 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 lericacid (a.u.) sd 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.1 0.0 0.1 0.0 0.0 0.1 3Dhydroxybuty- mean 5.0 5.8 4.9 5.2 4.8 5.0 4.7 4.7 4.5 4.5 4.3 4.4 4.2 4.4 rate (a.u.) sd 1.2 2.1 0.9 1.4 0.9 1.1 0.8 0.7 0.6 0.7 0.4 0.4 0.4 0.4 Lactate (a.u.) mean 46.4 47.8 46.4 48.9 42.8 47.4 45.5 47.9 47.0 52.2 46.2 49.0 47.4 48.2 sd 8.5 7.5 8.2 8.6 8.1 7.7 8.9 8.9 8.4 8.3 7.3 6.8 8.0 8.8 Citrate (a.u.) mean 4.8 4.6 4.8 4.6 4.9 4.4 4.8 4.4 4.8 4.1 4.8 4.3 4.5 4.4 sd 0.6 0.6 0.6 0.7 0.6 0.7 0.6 0.6 0.7 0.6 0.6 0.5 0.6 0.4 Arginine (a.u.) mean 3.9 3.9 3.9 3.9 3.8 3.7 3.8 3.8 3.8 3.7 3.8 3.7 3.7 3.7 sd 0.3 0.3 0.3 0.3 0.3 0.4 0.3 0.4 0.3 0.4 0.3 0.3 0.3 0.4 Isoleucine (a.u.) mean 1.2 1.4 1.3 1.3 1.3 1.3 1.3 1.4 1.3 1.4 1.4 1.4 1.4 1.4 sd 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 Asparagine_2.92 mean 0.9 0.8 0.9 0.8 0.9 0.8 0.8 0.8 0.8 0.7 0.9 0.8 0.8 0.8 (a.u.) sd 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 Creatine_3.02 mean 2.7 2.8 2.7 2.7 2.7 2.4 2.6 2.4 2.5 2.4 2.5 2.4 2.4 2.5 (a.u.) sd 0.4 0.5 0.4 0.4 0.4 0.4 0.4 0.4 0.5 0.5 0.4 0.4 0.4 0.5

[0153] In particular, glucose concentrations show major biological increase in their circulating levels from 4.3 and 4.4 mmol at age 5 to 5.2 and 5.0 mmol at age 16, for boys and girls, respectively. Interestingly, this increased concentration is marked by two plateaus, one between age 8 to 11, and a second one from age 13 to 16. As blood glucose concentrations increase, an increased pattern in the Respiratory Quotient (RQ) with age is seen. This RQ is very informative to reflect basal metabolic rate and which macronutrients are being metabolized for energy fuelling, and therefore as primary energy sources for growth and development during childhood. From age 5 to 7 RQ values remain on average between 0.87 and 0.92, increasing between 0.93 and 0.97 from age 8 to 10, increasing further between 0.98 and 1.01 from age 11 to 13, and decreasing towards 0.95 from year 14 onwards. As reference information, a value of 0.7 indicates that lipids are being metabolized, 0.8 for proteins, and 1.0 for carbohydrates. In other words, when a RQ is equal to one, the body is almost exclusively using endogenous and exogenous carbohydrates as source of metabolic fuels, whilst a value of 0.7 would corresponds to a context of extreme starving where body will use almost exclusively fat as source of metabolic fuels.

[0154] There are therefore major changes in glucose production and consumption throughout childhood that are described through the patterns in fasting glucose and RQ. In childhood, such data illustrate that a child may depend exclusively of carbohydrate to fuel it body during a specific period during its puberty (age 11 to 13), and when reaching adolescence during the late stage of puberty, the range of selection for metabolic fuels increases again, more likely towards an adult phenotype. Interestingly, the data shows major changes in prepubertal stage (less than 8 years old) for both boys and girls.

[0155] Interestingly, the observation of the changes in the metabolite most associated with glucose variations across childhood, highlighted very particular patterns in 2-ketobutyrate, 3-methyl-2-oxovalerate, leucine, valine, isoleucine, 3-hydroxybutyrate, acetoacetate, citrate, arginine, asparagine and creatine.

[0156] Arginine, 2-ketobutyrate and 3-methyl-2-oxovalerate show a decreasing pattern from age 5 to 12, before reaching a plateau. Leucine and Valine remain relative constant over the childhood. Isoleucine shows an interesting pattern with decreased level from age 5 towards a lower and constant concentration from age 6 to 10, followed by an increase up to age 16. Asparagine shows a decreasing pattern from age 5 to 8 in boys and girls, followed by a plateau from age 9 onwards in boys, whereas in girls the metabolite shows a further decrease until age 14 followed by an increase towards the levels seen in boys at age 16. The ketone body 3-d-hydroxybutyrate, creatine and citrate show very high concentrations from age 5 to 7, followed by a steady decrease until age 16.

Example 3

[0157] Metabolites indicative of higher blood sugar at adolescence Based on the above observations, it was further exploredamongst the metabolites contributing the most to glucose variations in childhoodwhich ones may be an earlier and a more indicative indicator of higher blood sugar at adolescence. In Table 4, the values of blood glucose in children from age 5 to 16 are reported for children classified as normoglycemic or with impaired fasting glycemia at age 16 (e.g. fasting glucose above ADA criteria, i.e. equal or greater than 5.6 mmol/L of plasma). The glucose state at year 16 is representative of glucose stage at years 13, 14, and 15. Both groups of children show similar concentrations of blood glucose during pre-puberty, whilst differences in their fasting blood concentration of glucose is detectable only during adolescence.

TABLE-US-00004 TABLE 4 Glucose Age Gender (mmol/L) sd Normo-glycemic 5 M 4.3 0.4 5 F 4.4 0.4 6 M 4.5 0.4 6 F 4.4 0.3 7 M 4.6 0.5 7 F 4.6 0.4 8 M 4.8 0.3 8 F 4.7 0.4 9 M 4.7 0.5 9 F 4.9 0.3 10 M 4.9 0.3 10 F 4.8 0.2 11 M 4.8 0.3 11 F 4.8 0.3 12 M 4.9 0.4 12 F 5.1 0.4 13 M 5.1 0.3 13 F 5.1 0.4 14 M 5.2 0.3 14 F 5.1 0.5 15 M 5.1 0.3 15 F 5.2 0.4 16 M 5.1 0.3 16 F 4.9 0.3 IFG 5 M 4.5 0.4 5 F 4.3 0.1 6 M 4.5 0.4 6 F 4.4 0.6 7 M 4.9 0.3 7 F 4.7 0.5 8 M 4.9 0.4 8 F 4.4 0.7 9 M 5.1 0.3 9 F 5.0 0.3 10 M 5.0 0.4 10 F 4.7 0.4 11 M 5.1 0.3 11 F 4.8 0.4 12 M 5.2 0.3 12 F 4.9 0.5 13 M 5.5 0.1 13 F 5.1 0.5 14 M 5.5 0.3 14 F 5.4 0.4 15 M 5.5 0.2 15 F 5.3 0.4 16 M 5.7 0.1 16 F 5.6 0.0

[0158] Therefore, it was assessed if some of the key metabolites associated with glucose trajectories in childhood may be providing more sensitive information with regards to blood glucose at age 16. To achieve this objective, a strategy was adopted aimed at comparing the correlation coefficients generated between blood glucose at year 16 and the ratio of changes of influential metabolites between age 5 to 6, 6 to 7, and 7 to 8, respectively. Most significant associations were identified from the metabolite variations between age 6 and 7. The results are reported in Table 5. As a reference, the results were compared with the correlations obtained between glucose at year 16 and changes in BMI sds from age 6 to 7, or between glucose at year 16 and changes in glucose from age 6 to 7. Amongst the most influential metabolites, 3-D-hydroxybutyrate, valine and creatine had their yearly variations statistically and positively associated to glucose concentration at year 16, and more informative than BMI sds or glucose alone.

[0159] Considering the previous results, it was further tested if a ratio between metabolites at age 6 or 7 would be as informative as a temporal variation in a given metabolite. Due to the statistical and biological relationship between 3-hydroxybutyrate and glucose, it was decided to compute the ratio between metabolites and 3-D-hydroxybutyrate, and assess the correlations with blood glucose at year 16. The results are also reported in Table 5. Very interestingly, it was seen that the ratios 3-D-hydroxybutyrate/citrate, 3-D-hydroxybutyrate/lactate, 3-D-hydroxybutyrate/asparagine at year 6 provide a good correlation with the blood glucose at year 16, statistically better than 3-D-hydroxybutyrate/glucose ratio. The analysis also shows that 3-D-hydroxybutyrate/valine or 3-D-hydroxybutyrate/creatine have much lower relationships to blood glucose at year 16, indicating these 3 metabolites are closely biologically related.

TABLE-US-00005 TABLE 5 Pearson Correlation with Glucose Age 16 for Ratio Pearson between 3-D- Correlation hydroxy- with Glucose butyrate Age 16 Least at age 6/ Least for ratio of squares Variable squares parameters regression at age regression Parameter age 6/Age 7 p-val slope Age 6 p-val slope BMI 0.05 0.6832 0.01239 NA NA NA Glucose 0.2 0.072 0.6899 0.23 0.0279 123.197 Citrate 0.08 0.4742 0.176 0.26 0.0093 0.2418 Lactate 0.03 0.8086 0.01875 0.21 0.0439 1.05 Acetate 0.02 0.8439 0.03419 0.32 0.0017 0.08692 Acetoacetate 0.27 0.0145 0.5274 0.24 0.0193 0.0713 Asparagine 0.02 0.827 0.06048 0.27 0.0084 0.03951 Leucine 0.13 0.2417 0.4048 0.26 0.0108 0.09448 3-D- 0.34 0.0019 0.2208 NA NA NA hydroxybutyrate Valine 0.36 8.00E04 0.7996 0.16 0.127 0.161 3-Methyl- 0.17 0.1163 0.5528 0.26 0.0104 0.02807 2oxovalericacid 2-Ketobutyrate 0.1 0.3481 0.3481 0.24 0.02 0.03241 Arginine 0.24 0.0257 0.9175 0.26 0.0118 0.1717 Creatine 0.34 0.0017 0.7361 0.19 0.0673 0.0979 Isoleucine 0.18 0.1081 0.321 0.21 0.0435 0.03955 PUFA 0.26 0.0191 1.019 0.22 0.0342 0.08206 Lysine 0.05 0.6688 0.1513 0.26 0.0104 0.1783 Trimethylamine 0.07 0.5115 0.2176 0.27 0.0084 0.03951 MethylLipid 0.28 0.0106 0.931 0.21 0.0441 1.751 signal Formate 0.09 0.4429 0.1948 0.29 0.0046 0.09063 Histidine 0.09 0.4429 0.1948 0.29 0.0046 0.09063 Serine 0.05 0.6639 0.1495 0.24 0.0171 0.1317

[0160] Therefore, amongst the most influential biochemical species contributing to high fasting glucose in childhood, the analysis indicates that: [0161] The measure of 3-D-hydroxybutyrate, valine, and creatine at age 6 and 7 are key indicators of high glucose at year 16, and therefore of risk of IFG [0162] The measure of 3-D-hydroxybutyrate/citrate, or 3-D-hydroxybutyrate/lactate, or 3-D-hydroxybutyrate/asparagine at year 6 is a key indicator of high glucose at year 16, and therefore of risk of IFG [0163] As an example in the present study cohort, children were classified as impaired fasting glycemia at age 16, once their fasting glucose value are higher than 5.6 mmol.Math.L1. On average IFG children at year 16 have 11% higher fasting blood glucose than normo glycemic children. Therefore, for the pre-pubertal markers in this study, as an example, it can be stated that: [0164] A value of glucose at year 16 which is 11% greater than the average corresponds, on average, to an increase of 205% on the 3-HB/citrate ratio at year 6. [0165] A value of glucose at year 16 which is 11% greater than the average corresponds, on average, to an increase of 214% on the 3-HB/acetate ratio at year 6. [0166] A value of glucose at year 16 which is 11% greater than the average corresponds, on average, to an increase of 218% on the 3-HB/asparagine ratio at year 6. [0167] A value of glucose at year 16 which is 11% greater than the average corresponds, on average, to an increase of 328% on the 3-HB/lactate ratio at year 6. [0168] A value of glucose at year 16 which is 11% greater than the average corresponds, on average, to an increase of 229% on the 3-HB yr 6/3-HB yr 7 ratio. [0169] A value of glucose at year 16 which is 11% greater than the average corresponds, on average, to an increase of 69% on the valine yr 6/valine yr 7 ratio. [0170] A value of glucose at year 16 which is 11% greater than the average corresponds, on average, to an increase of 73% on the creatine yr 6/creatine yr 7 ratio. [0171] Significant increases in the annual incidence of both type 1 diabetes and type 2 diabetes among youths (aged 10 to 19 years old) in the United States have been recently reported by Mayer-Davis et al. (Incidence Trends of Type 1 and Type 2 Diabetes among Youths, 2002-2012, The New England Journal of Medicine, 376:1419-1429, 2017). It is well established that variations exist across racial and ethnic groups. As illustred by Mayer-Davis et al, this includes high relative increases in the incidence of type 2 diabetes in racial and ethnic groups other than non-Hispanic whites in the USA as an example. Variation across demographic subgroups may reflect varying combinations of genetic, environmental, and behavioral factors that contribute to diabetes. Therefore, reference values should be generated accordingly for the proposed markers.

[0172] As an example in the present study cohort, reference values are determined from the normoglycemic population (Table 6).

TABLE-US-00006 TABLE 6 Marker mean sd 3-HB/citrate ratio at year 6 1.111 0.339 3-HB/acetate ratio at year 6 2.961 1.189 3-HB/asparagine ratio at year 6 6.382 2.096 3-HB/lactate ratio at year 6 0.161 0.068 3-HByr 6/3-HB yr 7 ratio 1.066 0.458 valine yr 6/valine yr 7 ratio 0.988 0.145 creatine yr 6/creatine yr 7 ratio 1.035 0.152