Blood biomarkers for monitoring changes in the amount of visceral adipose tissue

Abstract

Methods are described for the biomarker-based identification of a change in the amount of visceral adipose tissue (VAT) in individuals with a healthy BMI and a healthy waist circumference. The early identification of VAT changes using commonly measured clinical biomarkers when incorporated with exercise and nutritional advice enables the expeditious implementation of the END approach (Exercise Nutrition & Diagnostics) to manage the burgeoning overweight and obesity pandemic.

Description

[0001] BLOOD BIOMARKERS FOR MONITORING CHANGES IN THE AMOUNT OF VISCERAL ADIPOSE TISSUE

[0002] BACKGROUND OF THE INVENTION

[0003] Against a backdrop of an increasing and ageing global population, the overweight and obesity (OvOb) pandemic is the healthcare crisis of the 21stcentury and is projected to affect over half the world’s population by the end of the decade. Healthcare systems are antiquated and inadequately resourced to manage these pressures and require a fundamental restructuring to remain a viable provider of healthcare for all. Key to a restructured approach is preventative healthcare which aims to prevent or detect disease early and to promote a longer healthspan. The challenges of OvOb are complex, industrial, technological and behavioural changes underpinning unhealthy diets and reduced physical activity both occupationally and recreationally. Major health institutions world-wide such as the UK National Health Service, the US Centre for Disease Control and Prevention and World Health Organisation use the body mass index (BMI) as an indicator of OvOb calculated using the height and weight measurements of an individual in which <18.5 is underweight, 18.5 - <25.0 is healthy, 25.0 - <30.0 is overweight and > 30.0 is obese. OvOb indicates excessive fat mass and is considered both a disease and, more commonly, as a risk factor of disease including cardiovascular disease, atherosclerosis, cancer, insulin resistance and type 2 diabetes. It is also a major risk factor for hospitalisation, severe illness and death for covid-infected individuals. A BMI measurement of 25 or greater as well as one of 30 or greater supports the identification of at-risk patients of various diseases. Although BMI is a useful screening tool it does not directly measure fat - not everyone with a BMI of 25 or greater will develop disease and not everyone below a BMI of 25 will not develop disease. Body fat presents mainly as subcutaneous adipose tissue (SAT) residing beneath the skin and as visceral adipose tissue (VAT) which accumulates largely within the abdominal cavity (intra-abdominal fat). Although SAT and VAT are thought to present different disease risk factors, VAT is considered more metabolically active and is associated with low grade inflammation and the secretion of pro-inflammatory mediators which is thought to increase disease severity in various conditions. Waist circumference (WC) is a surrogate marker of the amount of visceral fat (Ross 2020; Neeland 2019). Waist circumference as an indicator of the amount VAT is beneficial as it a low cost and rapid method but lacks a standardised methodology for its measurement (Neeland 2019) and can be subject to measurement imprecision (WHO 2008, Waist Circumference and Waist-Hip Ratio, Expert Consultation, Geneva) . The measurement of protein and small molecule biomarker levels reflecting the metabolic activity of VAT is a method of supporting its identification and a combination of fasting triglyceride concentration and WC measurement has been used to identify the presence of VAT in obesity (Tchernof 2013, Sam 2009, Neeland 2019).

[0004] There is less focus on identifying the onset or accumulation of VAT in individuals with a clinically-recognised healthy WC (commonly defined as <94 cm for males and < 86cm for females) and healthy BMI which represents the opportune point in which to start implementing corrective and preventative behavioural measures. From an individual’s perspective this may be partly due to the lack of obvious visible signs of VAT build-up. Reverting to a healthier biological status through VAT reduction would be more likely to be achieved if the individual is starting from a lower WC. The gold standard of visceral adipose tissue measurement is based on imaging using MRI, CAT scans and DEXA, but these techniques lack fat qualitative information, expose patients to radiation, require specialist and experienced operators and are expensive. In addition, they are untested in identifying the onset of VAT accumulation. Therefore, improved methods to identify the onset of VAT accumulation, ongoing VAT accumulation and VAT depletion to support disease risk management are required.

[0005] References

[0006] Neeland I. J. et al. (2019), Lancet Diabetes Endocrinology, 7(9): 715-725.

[0007] Ross R. et al. (2008), Obesity Reviews, 9: 312-325.

[0008] Ross R. et al. (2020), Nature Reviews Endocrinology, 16: 177-189.

[0009] Sam S. et al. (2009), Diabetes Care, 32(10): 1916-1920.

[0010] Sik C.S. et al. (2016), Frontiers in Endocrinology, 7:30 1-16.

[0011] Tchernof A. and Despres J-P. (2013), Physiological Reviews, 93: 359-404. SUMMARY OF THE INVENTION

[0012] An approach to identify the onset of VAT accumulation, ongoing VAT accumulation and the loss of VAT using biochemical measurements is described. It has been found that the measurement of certain biochemicals in healthy individuals, particularly in younger adults, within a healthy BMI and WC range can indicate whether abdominal VAT is present. By measuring certain biochemicals at different time-points in individuals an increase or decrease in the amount of VAT can be gauged. Biochemicals which can be used in the method include leptin, triglycerides, sex hormone binding globulin, vitamin B12, white blood cell count and neutrophil count. Identification of abdominal VAT accumulation in younger adults of clinically recognised healthy BMI and WC, enables a practical and relatively inexpensive approach to the detection of a pre-disease state which can lead to pathological conditions such as insulin resistance, diabetes and cardiovascular disease supporting a preventative healthcare approach by enabling simple, corrective measures to be implemented. Measurement of the biochemicals also enables the sensitive detection in a reduction in the amount of VAT to be identified, which is of benefit to individuals who are seeking to lose weight. Use of these biochemicals provides a sensitive technique which can detect small changes in the amount of VAT which can be overlooked using waist circumference measurement. The invention’s scope is outlined in the Claims.

[0013] DESCRIPTION OF THE FIGURES

[0014] Figure 1 Graph exhibiting the concentration of various biomarkers in self-reported healthy males aged 20-39 years, within a healthy WC range of 76-86 cm, with increasing BMI. The graph shows an increase in leptin and triglycerides and a decrease in SHBG with increasing BMI. Neutrophils, WBC and vitamin B12 do not exhibit a change.

[0015] Figure 2 Graph exhibiting the concentration of various biomarkers in self-reported healthy males aged 20-39 years, within a healthy BMI range of 21 .50-22.50, with increasing WC. Leptin, triglycerides, WBC and neutrophil count exhibit an increase and vitamin B12 and SHBG exhibit a decrease as waist circumference (WC) increases within a clinically recognised healthy range (WC <94 cm). Figure 3 Correlation matrix of biochemicals and WC for males aged 20-39 years of BMI 21.50-22.50. Cells contain the Pearson correlation coefficient for log transformed data. Sample number correlation size for WC and the biomarkers varies from 56 to 249.

[0016] Figure 4 Schematic outlining the preventative healthcare-based END approach (Exercise Nutrition & Diagnostics) to address overweight and obesity (OvOb).

[0017] DETAILED DESCRIPTION OF THE INVENTION

[0018] The optimum OvOb preventative healthcare approach would be the detection of the onset of excess adipose tissue / fat accumulation, occurring before an individual is classified as overweight, as this would enable the initiation of a management program at the earliest possible stage allowing the most effective approach to achieving arrest / reversal of the condition. Adipose tissue is composed mainly of adipocytes but also contains other cell types such as fibroblasts, vascular endothelial cells and macrophages. Excess adipose tissue accumulation arises from an energy intake and expenditure imbalance, the former being greater than the latter, which over a sustained period results in adipocyte cellular enlargement and proliferation (Sik 2016). The accumulation of adipose tissue in the abdominal cavity is common in males and manifests visually as an increasingly large ‘belly’ or waist circumference. It has been found that in some individuals with a healthy BMI the concentration of certain blood-based biomarkers change with increasing or decreasing waist circumference even though the waist circumference remains within a clinically recognised healthy range. There are several clinically recognised healthy cut-off values for BMI and WC. The methods described can be applied within the context of any clinically-recognised ranges or cut-offs.

[0019] In a first aspect there is described a method of detecting a change in the amount of visceral adipose tissue in an individual of healthy BMI and healthy waist circumference (WC) comprising measuring one or more biomarkers chosen from vitamin B12, white blood cell count (WBC), neutrophil count, sex hormone binding globulin (SHBG), leptin and triglycerides in in vitro samples of the individual, the in vitro sample collections having occurred at two different time-points and establishing whether a change in the amount of visceral adipose tissue has occurred between the two different time-points. The biological sample type for use in the methods is blood, serum or plasma. A biochemical measurement approach to assess changes in the amount of VAT avoid the measurement inaccuracy and lower sensitivity associated with the standard tape-measurement approach. By obtaining in vitro biological samples of an individual at two different time-points and comparing the biomarker concentration measurements (intra-biomarker concentration measurement), deviations of the individual biomarker concentrations between the two time-points can be used to indicate changes in the amount of VAT. As the described methods are for preventative healthcare the individual would likely be nominally healthy i.e. self-reported healthy. However, the described methods can also be used on individuals with known illness or disease if the described biomarkers are known to be unaffected or minimally affected by the illness / disease. ‘Intra-biomarker concentration measurements’ means that the concentration of biomarker A at timepoint 1 is compared to the concentration of biomarker A at time-point 2, the concentration of biomarker B at time-point 1 is compared to the concentration of biomarker B at time-point 2 etc. By ‘two different time-points’, for example an earlier time-point T 1 and a later time-point T2, it is implied that T2 is at least several days later, at least several weeks later, at least several months later or greater than 1 year later than T 1 . For example, a schedule for biological sample taking might be timepoint 1 (T1) corresponds to the first sample, time-point two (T2) being the second sample taken at least 1 ,2, 3, 4, 5, 6, 8, 9, 10, 11 , or 12 months later. A third sample could be taken at time-point three (T3) and this could be at least 1 , 2, 3, 4, 5, 6, 8, 9, 10, 11 , or 12 months after the second sample was taken, and this could be used to compare biomarker concentrations with corresponding biomarker concentrations measured in samples obtained at either or both of T 1 and T2; fourth, fifth, sixth, seventh etc. samples can also be obtained and their biomarker concentration measurements compared to one or more previous corresponding biomarker concentration measurements. For example, the concentration of biomarker A of a sample obtained at T2 can be compared to the concentration of biomarker A of a sample obtained at T3, the concentration of biomarker B of a sample obtained at T3 can be compared to the concentration of biomarker B of a sample obtained at T4. For males and females, who are preferably adults aged 18 or greater, for the described method, a healthy BMI is between about 21.50 and <25.00 e.g. <25.00, <24.90, <24.80, <24.70, <24.60, < 24.50, < 24.40, < 24.30, <24.20, < 24.10, <24.00, <23.90, <23.80, <23.70, <23.60, <23.50, <23.40, <23.30, <23.20, <23.10, <23.00, <22.90, <22.80, <22.70, <22.60 <22.50, <22.40, ,<22.30, <22.20, <22.00, <21.90, <21 .80, <21 .70, <21 .60 or 21 .50; the healthy male BMI can within the range of about 21 .50 to about 22.50, of about 21 .50 to about 23.00, of about 21 .50 to about

[0020] 23.50, of about 21 .50 to about 24.00, of about 21 .50 to about 24.50, of about 21.50 to about 25.00, of about 22.00 to about 23.00, of about 22.00 to about 23.50, of about 22.00 to about 24.00, of about 22.00 to about 24.50, of about 22.00 to about 25.00, of about 22.50 to about 23.50, of about 22.50 to about 24.00, of about 22.50 to about 24.50, of about 22.50 to about 25.00, of about 23.00 to about 24.00, of about 23.00 to about 24.50, of about 23.00 to about 25.00, of about 23.50 to about

[0021] 24.50, of about 23.50 to about 25.00, of about 24.00 to about 25.00. A healthy male WC can be between < 94 cm and about 65 cm, < 93 cm, < 92 cm, < 91 cm, < 90 cm,

[0022] < 89 cm, < 88 cm, < 87 cm, < 86 cm, < 85 cm, < 84 cm, < 83 cm, < 82 cm, < 81 cm,

[0023] < 80 cm, < 79 cm, < 78 cm, < 77 cm, < 76 cm, < 75 cm, < 74 cm, < 73 cm, < 72 cm,

[0024] < 71 cm, < 70 cm, < 69 cm, < 68 cm, <67 cm, < 66 cm or 65 cm. Preferred healthy male WCs are within the range of < 94 cm to about 76 cm, and between about 86 cm to 76 cm. It will be recognised that the lower cut-off ranges of BMI and WC described in the method would likely be extendable to just below the explicitly stated lowest and higher values of the exemplified BMI and WC ranges without impacting the effectiveness of the method, hence the term ‘about’. In addition to the described biochemicals, waist circumference changes between different time-points can also be included as a marker of early-onset adipose tissue accumulation in the described methods. Visceral adipose tissue (visceral fat) in the context of the current disclosure refers to adipose tissue which is between and surrounds organs in the abdominal cavity. The use of the word fat can have several meanings including as a general term for adipose tissue and a lipid molecule such as cholesterol or a triglyceride; herein, the term fat refers to adipose tissue. VAT accumulation refers to increasing visceral adipose tissue build-up. The terms “biomarker” and “analyte” are used interchangeably herein and refer to biological molecules present in a body, i.e. biochemicals, which can be measured to obtain a concentration or other value which may or may not be compared against a reference value. The essential nutrient vitamin B12, for the purposes of the current disclosure, is also classed as a biomarker, is The reference value is usually the concentration value of a biomarker / analyte found in a healthy individual or group of individuals, the latter value(s) being represented by a statistical metric such as the mean, median and range. The reference value for longitudinal analysis in an individual can be a previously measured concentration of the biomarker / analyte in the individual. The BMI is a measurement of a person’s leanness or corpulence based on height and weight and is intended to quantify tissue mass. It is used widely as a general indicator of whether a person has a healthy body weight for their height. The BMI can be calculated by inputting height and weight in the following formula: BMI = Weight (kg) Height (m2)

[0025] There are other measurement formulae and tools which estimate body mass besides BMI which can be used in the methods described herein as each of these methods estimates body fat content and the described biomarkers retain their relevance. The BMI of an individual is calculated by dividing the kilogram weight of the individual by the square of the individual’s metre height. Examples include A Body Shaped Index (ABSI) Surface-Based Body Shape Index (SBSI) and Body Volume Index (BVI). The BMI equation itself has been modified to provide ‘improved’ indicators of OvOb e.g.

[0026] 1 .3 x weight (kg) / height (m) ’ as opposed to the standard BMI formula of weight 2

[0027] (kg)Zheight (m) . These variants are all useable in the described methods.

[0028] If between the first and second time-points there is a decrease in the concentration of vitamin Bi2, an increase in the concentration of WBC, an increase in the concentration of neutrophil count, an increase in the concentration of leptin, an increase in the concentration of triglycerides and a decrease in the concentration of SHBG, an increase in the amount of visceral adipose tissue is indicated.

[0029] If between the first and second time-points there is an increase in the concentration of vitamin BI2, a decrease in the concentration of WBC, a decrease in the concentration of neutrophil count, a decrease in the concentration of leptin, a decrease in the concentration of triglycerides and an increase in the concentration of SHBG, a decrease in the amount of visceral adipose tissue is indicated.

[0030] The biomarkers can be assessed individually or in any combination to support the analysis of possible VAT increase or decrease in an individual between two different time-points. If more than one biomarker is used, analysis of the concentration changes between two time-points can be incorporated into a computer algorithm and / or a statistical method to facilitate analysis and produce an output which indicates whether a change in VAT has occurred. The output of the algorithm and / or statistical method can take the form of a concentration change assessment between two or more time-points which describes for each biomarker used in the algorithm and / or statistical method whether there has been an increase, decrease or no change in concentration, from which an assessment of the VAT amount in the individual is afforded. For example, if an individual exhibits a decrease in vitamin BI2concentration, an increase in WBC concentration between two time-points, this would suggest an increase in the amount of VAT. If a decrease in SHBG concentration was observed this would add further support to an increase in VAT. If leptin and triglycerides were also increased, this would further support that an increase in VAT has occurred. It may be desirable to build in minimum biomarker concentration change thresholds to the analysis. As an alternative to this approach, for two or more of the biomarkers, standard multivariate statistical models such as multiple linear regression multilayer, perceptron neural network (MLP), artificial neural networks, support vector machines, N Bayes classification and random forest classifiers could be constructed using population data. Blood derived vitamin B12can be bound to three different proteins, transcobalamin I (haptocorrin), transcobalamin II and transcobalamin III and its serum or plasma measurement can be based on one or more of these forms. The Elecsys Vitamin BI2II assay, which incorporates a step dissociating complexed vitamin BI2from protein, was used to measure vitamin BI2within the described methods; however, the methods of the disclosure can be applied to the measurement of vitamin BI2in any form whether uncomplexed and / or in one or more of its complexed forms.

[0031] In a further aspect of the disclosure is a method in which early-onset visceral adipose tissue amount change is detected in male with a healthy BMI and a healthy WC. Early-onset implies that changes in the amount of visceral adipose tissue occurs at a clinically recognised healthy BMI and WC which usually occurs at a relatively young age, although it can also occur in older adults of clinically recognised healthy BMI and WC; relatively young age implies less than about 39 years old and from about 20 years of age to about 39 years of age. Data to support identification of the early-onset markers methods were derived from a male cohort aged 20 to 39. Applicable healthy BMI and healthy WC values have been disclosed earlier. Vitamin BI2, triglycerides, neutrophils and WBC concentration changes were especially associated with WC increases within a restricted BMI range and not with BMI increases within a restricted WC range, while leptin and SHBG exhibited similar changes in concentration within both scenarios; this is suggestive that changes in adipose tissue amounts in the abdominal cavity are impacting biomarker concentrations. Visceral fat / visceral adipose tissue in the context of the current disclosure refers to fat present in the abdominal cavity, unless stated otherwise. In a further aspect of the disclosure a method of early-onset adipose tissue accumulation is provided. The method comprises the steps of (i) measuring in an in vitro sample of an individual with a healthy BMI and a healthy WC the concentration of one or more biomarkers chosen from leptin, vitamin B12, neutrophil count, SHBG, triglycerides and WBC; (ii) at a later time-point repeating step (i); (iii) establishing whether early- onset adipose tissue accumulation is present. Step (ii) can be repeated on further occasions each at later time-points. Early-onset adipose tissue accumulation in the context of this method is associated with a higher concentration of leptin, a higher concentration of neutrophils, a higher WBC and a higher concentration of triglycerides in the biological sample analysed at the later time-point (sample two), whereas SHBG and vitamin Bi2each exhibit a lower concentration in sample two compared to sample one. An increase in waist circumference between time-point one and time-point two and / or successive increases between time-point one, timepoint two and time-point three etc., can also support the diagnosis of early-onset adipose tissue accumulation.

[0032] The described method can measure one of the six biomarkers, two of the six biomarkers, three of the six biomarkers, four of the six biomarkers, five of the six biomarkers or all six biomarkers; biomarker combinations of the method typically incorporate measurements of at least one of leptin, vitamin BI2, WBC / neutrophil count, in which an increase in concentration of leptin, an increase in concentration of neutrophil count, an increase in WBC and a decrease in concentration of vitamin BI2is indicative of early-onset adipose tissue accumulation. The measurement of two of the biomarkers in combination at different time-points can provide a greater indication of the change in the amount of VAT; such combinations include vitamin BI2and WBC, vitamin BI2and neutrophil count, vitamin BI2and triglycerides, vitamin BI2and leptin, vitamin BI2and SHBG, WBC and triglycerides, neutrophil count and triglycerides, WBC and leptin, neutrophil count and leptin, WBC and SHBG, neutrophil count and SHBG, triglycerides and SHBG, leptin and SHBG, leptin and triglycerides. In all the aforementioned biomarker combinations an increase in the concentration of leptin, triglycerides, WBC and neutrophil count and a decrease in the concentration of vitamin BI2and SHBG in sample T2 compared to sample T 1 indicates an increase in the amount of intra-abdominal fat; conversely, a decrease in the concentration of leptin, triglycerides, WBC and neutrophil count and an increase in the concentration of vitamin Bi2and SHBG in sample T2 compared to sample T 1 indicates a decrease in the amount of intra-abdominal fat. In addition to the measurement of one or more of the six biochemicals, waist circumference may also be measured and included as an indicator of VAT change although, as previously stated, its use can be subject to measurement inconsistency and therefore biomarker measurements without a WC measurement is the preferred approach. Preferred combinations of biomarkers / markers of early-onset adipose tissue accumulation and the relative intra-biomarker change in concentration at T2 compared to T 1 (which may also incorporate a WC measurement) are i. an increase in leptin, a decrease in vitamin B12and an increase in WC ii. an increase in leptin, an increase in neutrophil count and an increase in WC iii. an increase in leptin and an increase in triglycerides iv. an increase in leptin, an increase in triglycerides and an increase in WC v. an increase in leptin and a decrease in SHBG vi. an increase in leptin, a decrease in SHBG and an increase in WC vii. an increase in leptin, an increase in neutrophil count and a decrease in SHBG viii. an increase in leptin, an increase in neutrophil count, a decrease in SHBG and an increase in WC ix. an increase in leptin, an increase in neutrophil count and an increase in triglycerides x. an increase in leptin, an increase in neutrophil count, an increase in triglycerides and an increase in WC xi. an increase in leptin, a decrease in SHBG an increase in triglycerides xii. an increase in leptin, a decrease in SHBG, an increase in triglycerides and an increase in WC xiii. an increase in leptin, an increase in neutrophil count, a decrease in SHBG and an increase in triglycerides xiv. an increase in leptin, an increase in neutrophil count, a decrease in SHBG, an increase in triglycerides and an increase in WC xv. an increase in leptin and an increase in WC xvi. an increase in leptin, a decrease in vitamin BI2an increase in neutrophil count and an increase in WC xvii. an increase in leptin, a decrease in vitamin BI2and a decrease in SHBG xviii. an increase in leptin, a decrease in vitamin BI2, a decrease in SHBG and an increase in WC xix. an increase in leptin, a decrease in vitamin BI2and an increase in triglycerides xx. an increase in leptin, a decrease in vitamin BI2, an increase in triglycerides and an increase in WC xxi. an increase in leptin, a decrease in vitamin BI2, a decrease in SHBG and an increase in neutrophil count xxii. an increase in leptin, a decrease in vitamin BI2, a decrease in SHBG, an increase in neutrophil count and an increase in WC xxiii. an increase in leptin, a decrease in vitamin BI2, an increase in triglycerides and an increase in neutrophil count xxiv. an increase in leptin, a decrease in vitamin B12, an increase in triglycerides, an increase in neutrophil count and an increase in WC xxv. an increase in leptin, a decrease in vitamin BI2, a decrease in SHBG and an increase in triglycerides xxvi. an increase in leptin, a decrease in vitamin BI2, a decrease in SHBG, an increase in triglycerides and an increase in WC xxvii. an increase in leptin, a decrease in vitamin BI2, a decrease in SHBG, an increase in neutrophil count, an increase in triglycerides and an increase in triglycerides xxviii. an increase in leptin, a decrease in vitamin BI2, a decrease in SHBG, an increase in neutrophil count, an increase in triglycerides and an increase in WC xxviii. a decrease in vitamin BI2, an increase in triglycerides and an increase in neutrophil count xxix. a decrease in vitamin BI2, an increase in triglycerides and a decrease in SHBG xxx. a decrease in vitamin BI2, an increase in triglycerides, an increase in neutrophil count and a decrease in SHBG xxxi. an increase in triglycerides, an increase in neutrophil count and a decrease in SHBG. In addition to the biomarker / marker combinations, each of the combinations can further measure WBC in which an increase in WBC is supportive of a diagnosis of early-onset adipose tissue accumulation. As neutrophil count is correlated to WBC, being a sub-set of the WBC measurement, for each of the previous relevant combinations numbered i. to xxxi. WBC (increase) can replace neutrophil count (increase). For all the previously described biomarker combinations i. to xxxi. and their respective relative concentration changes between T 1 and T2 which identify VAT accumulation, the same biomarker combinations can be used to identify VAT depletion by replacing an ‘increase in concentration’ with a ‘decrease in concentration’ and replacing a ‘decrease in concentration’ with ‘an increase in concentration’ for each of the biomarkers within i. to xxxi. As an example, the first combination i. would read a decrease in leptin, an increase in vitamin BI2and a decrease in WC. As mentioned previously, step (ii) of the method can be repeated on one or more occasions at later time-points i.e. the analysis of at least three biological samples, each sample having been obtained on three separate occasions. This allows increased confidence in the results of the earlier measurements if the trend in the biomarker concentration change is consistent. For example, if an individual of BMI 21 .70 and WC 80 cm has had three sets of in vitro sample biomarker measurements taken at day 1 (T1), day 100 (T2) and day 300 (T3) and the concentrations of each of leptin and neutrophil count (as examples) have increased from day 1 to day 100, and from day 100 to day 300, there can be greater confidence that adipose tissue accumulation is occurring. Hence a further aspect describes the use of one or more of vitamin B12, WBC, neutrophil count, SHBG, leptin and triglycerides as biomarkers to aid in monitoring the change in the amount of VAT in males. Although the main use is in the longitudinal measurement of the biomarkers in samples of males who have a healthy WC and a healthy BMI the use can extend to individuals who do not have a healthy BMI (BMI > 25.00) and / or WC (WC > 94 cm). This of particular benefit in relation to vitamin B12which is an essential vitamin and for which adequate levels are important to support cellular processes. By longitudinal measurement is meant that at least two samples are taken from an individual at at least two different time-points and the in vitro samples are submitted to an analysis to detect and quantify the biomarkers which indicate whether a biomarker concentration is increasing, decreasing or unchanged. The confidence in the results can be further increased if the change in the individual biomarker concentrations between time-points takes account of the coefficient of variation (CV) of the assay and / or the biological variation (BV) associated with each individual analyte. When an increase in one or more of the biomarker concentrations is observed (between T1 and T2), the individual can embark on corrective action which may involve consultation with a nutritionist and / or exercise coach followed by dietary changes and / or exercise. If the corrective action is unsuccessful and the biomarker concentrations continue to change in concentration (measured at T3) in a trajectory suggestive of VAT accumulation which could be accompanied by noticeable adipose tissue accumulation, a change in the nutritional / exercise plan may be advised, or medical intervention may be required in the form of pharmaceuticals and / or surgery. However, the principal aim of the early-onset VAT detection method is to enable corrective life-style changes at the earliest signs of adipose tissue accumulation to avoid down-stream pathologies.

[0033] Methods and Results

[0034] All blood samples and measurements were obtained at Randox Health clinics in Crumlin and Holywood, Northern Ireland. Waist circumference measurements were taken at the midpoint of the lower rib and the superior border of the iliac crest. Sample analysis was effected at Randox Laboratories Antrim. Any suitable kits, assays and instruments (analysers) can be used to determine the concentration levels of biochemicals in a patient sample. The following commercially available kits / assays, instruments and sample types were used as per the manufacturer’s instructions to derive the measurements described herein: Leptin: manufactured by Randox Laboratories, Catalogue no. EV 4219 (Biochip Metabolic Syndrome Array), for use with Evidence series analysers (analysed using the Evidence Evolution) using serum.

[0035] Triglycerides: manufactured by Randox Laboratories, Catalogue no. 3823, for use with RX Series analysers (analysed using the RX Daytona) using serum.

[0036] Vitamin BI2: the Elecsys Vitamin B12 II test manufactured by Roche Diagnostics analysed using the Cobas e801 using serum.

[0037] Neutrophil count: measured using fluorescence flow cytometry on a Sysmex XSIOOOi analyser using whole blood (EDTA).

[0038] White blood cell count: measured using fluorescence flow cytometry on a Sysmex XSIOOOi analyser using whole blood (EDTA).

[0039] Sex hormone binding globulin: the Elecsys SHBG test manufactured by Roche Diagnostics analysed using the Cobas e801 using serum.

[0040] Each assay was performed according to the manufacturer’s instructions.

[0041] The measurement value units of the biomarkers (analytes) described herein, unless indicated otherwise are: triglycerides mmol / l, leptin pg / l, vitamin B12 ng / l, neutrophil count (neutrophil(s))109 / l, white blood cell count (WBC) 109 / l and sex hormone binding globulin (SHBG) nmol / l.

[0042] Statistical Analysis

[0043] Graphical methods, multiple linear regression, two-tail unpaired t-test with data transformation where appropriate or Mann-Whitney statistic, biomarker correlation analysis at constant BMI or constant WC supported identification of potential VAT biochemical markers at a population level. Biomarkers whose concentrations within 20- to 39-year-olds with constant BMI and varying WC whose population data exhibited concordant graphical and statistical outputs were incorporated in a follow- on longitudinal data analysis; the selected biomarkers were assessed on a case-by- case basis and a group analysis using a paired t-test with data transformation where appropriate. Computed calculations were effected using Graphpad Prism 9.02 software. Population Comparison Results

[0044] Analysis of multiple biomarker concentrations in self-reported healthy 20-39-year- olds at constant BMI with varying WC suggestive of abdominal VAT, compared to the biomarker concentrations with constant WC and varying BMI, highlighted

[0045] 6 candidate biomarkers of early-onset abdominal VAT (Figures 1 & 2). Multiple linear regression analysis of the five biomarkers leptin, vitamin B12, triglycerides, SHBG & WBC in which WC was the dependent variable in a cohort of males (average age 38 years; N=126) with BMI of 21.50-22.50 returned a Revalue of 0.15 and highlighted leptin and vitamin BI2as being significantly associated with WC (P<0.10). Substituting neutrophil for the highly correlated WBC delivered similar results. A similar analysis using multiple linear regression analysis of the five biomarkers leptin, vitamin BI2, triglycerides, SHBG & WBC in which WC was the dependent variable in a cohort of males (average age 40.5 years; N=149) with BMI of 22.51-23.50 returned a Revalue of 0.16 and highlighted leptin and vitamin BI2as being significantly associated with WC (P<0.05); this indicates that the utility of the described methods could extend to individuals with a healthy BMI beyond the range 21.50-22.50 e.g. in the range BMI from 21 .50-23.50. Correlation analysis of the 6 markers confirmed their association with WC (Figure 3) - each of the biomarkers had a Pearson P-value <0.01 except for neutrophils (P=0.101). A population correlation analysis of waist circumference vs VAT score in males aged 20 to 39 years (N=126) gave a Pearson r2value of 0.799. The VAT score was derived using the Tanita MC-980 Body Composition Analyzer (Tanita Corporation, Japan) and provides an indication of visceral fat level, the higher the rating indicative of a greater amount of visceral fat. Further confirmation that the level of the circulatory biomarkers leptin, vitamin BI2, triglycerides, neutrophil count, SHBG & WBC are related to the amount of VAT was shown by applying the hypertriglyceridemic WC approach; as well as the expected elevated triglyceride levels, an increase in leptin, an increase in neutrophil count, an increase in WBC, a decrease in SHBG and an decrease in vitamin BI2are all observed with increasing waist circumference within a defined BMI of 21 .50-22.50 (Table 1). These increases / decreases were also observed in an older age group of 40-72 years, though the effect was not as pronounced as in the younger age group. Table 1 A comparison of the mean biomarker levels at two different waist circumference (WC) levels in self-reported healthy & fasting male individuals of BMI 21.50-22.50.

[0046] **P<0.05, *P<0.10 using Mann-Whitney or t-test

[0047] WC= waist circumference, Triglyc=triglycerides, Vit BI2= vitamin BI2, SHBG= sex hormone binding globulin; neutrophil= neutrophil count, WBC= white blood cell count

[0048] Individual longitudinal analysis

[0049] Analysis of the refined data using the Wilcoxon paired test in which there was a decrease in WC between the first sample collection (T1) and the following sample collection (T2) and a BMI decrease of <1.00 confirmed that neutrophil count, WBC count and vitamin B12 remained significantly changed (Table 2). These changes were not replicated in the corresponding female data analysis.

[0050] Table 2 Analysis of the candidate biomarkers in a longitudinal population (male 20-39 yrs, 1 to 4 years between sample collections) in which an individual’s WC decreased by at least 2 cm between T1 and T2. N is sample size. Mean concentrations of the biomarkers are shown for T1 and T2. P-values derived using Wilcoxon paired test. The data suggests (Figures 1 to 3) that an early-onset VAT accumulation is occurring at a BMI range as low as 21 .50-22.50 in males aged 20 to 39 years which could be monitored using longitudinal measurements of the biomarkers vitamin Bi2, WBC, neutrophil count, leptin, SHBG and triglycerides. Furthermore, serum vitamin B12 levels have an inverse association with abdominal VAT across all ages (Figure 2 & Table 3).

[0051] Table 3 Correlation coefficients for vitamin B12vs BMI / WC for males aged 40 to 76 years at two BMI values. N is sample size.

[0052] Early-onset adipose tissue biomarkers

[0053] A waist circumference measurement is considered more indicative of the absolute amount of intra-abdominal fat (visceral fat) than a BMI measurement (Ross et al 2020). Biochemical data analysis was conducted to assess whether increases in WC within the clinically recognised healthy range of <94 cm, within a clinically recognised healthy BMI range of 21 .50 to 22.50 are associated with abdominal cavity adipose tissue accumulation in adults aged 20 to 39 years . Analysis of increases in BMI within a healthy WC range (WC 76-86 cm) were also conducted for comparison.

[0054] Analysis of the biomarkers in a young male adult cohort (N=240) aged 20 to 39 years whose BMIs were restricted to a BMI range of 21 .50-22.50 and whose WCs varied, identified each of leptin, neutrophil count, white blood cell count, sex hormone binding globulin, vitamin Bi2and triglycerides as exhibiting a trend in concentration change with increasing waist circumference (Figure 2). When WC was restricted to a WC range of 76-86 cm and BMI was varied, only leptin and triglycerides exhibited a trend in concentration change (increase) with increasing BMI but this trend was not as great as when BMI was restricted and WC was varied; SHBG, neutrophil count and WBC did not exhibit a change in concentration with increasing BMI. This is suggestive of early-onset of visceral fat accumulation in adult males with body mass indices and waist circumferences which are clinically recognised as healthy. A slight increase in waist circumference within the clinically recognised healthy range can go unnoticed or be ignored and is unlikely to motivate an individual to adopt lifestyle changes to arrest or reverse possible VAT accumulation. However, knowledge of changes in biochemicals associated with the amount of visceral adipose tissue and an overweight / obese phenotype could motivate a positive behavioural response. Table 4 highlights the utility of the herein described biomarker methods especially when there is a small fluctuation in WC over time (Individuals C & D).

[0055] Table 4 Individual longitudinal case studies comparing stable and fluctuating BMI & WC and corresponding biomarker values. Individuals A and B exhibit a fluctuating BMI (change in BMI between sample collections of >1.00); individuals C and D exhibit a stable BMI (change in BMI between sample collections of <1.00). None of the self-reported healthy individuals reported dietary or exercise changes between visits.

[0056] The reduction in vitamin BI2, and the increase in neutrophil count and WBC in Individual C between sample collections suggests an increase in intra-abdominal fat that isn’t being identified through WC measurement. The longitudinal results of individual D suggest that a decrease in intra-abdominal fat has occurred over time as triglycerides, neutrophil count and WBC decrease in concentration and vitamin BI2 increases. Vitamin BI2concentration changes in a cohort of males aged 40 to 76 years of healthy BMI and WC were comparable to those of the male cohort aged 20 to 39 years, highlighting the potential use of vitamin BI2in monitoring the change in the amount of VAT throughout adulthood. As vitamin BI2is an essential nutrient derived mainly from animal food sources, it is preferable to use a diet assessment to support its use as a biomarker to indicate change in the amount of VAT, whether it is used individually or in combination with the other biomarkers. The methods described can support the diagnostic aspect of the holistic ‘exercise, nutrition and diagnostic’ (END) approach to preventative healthcare (EP3772066) especially to address overweight & obesity (Figure 4) at an early stage of the process i.e. the point at which metabolically-active VAT is forming. Monitoring of these biomarkers in individuals through regular biological sample testing could highlight incremental increases / decreases in the amount of VAT enabling behavioural and / or nutritional corrective measures to be implemented, thus mitigating and preventing the onset of the OvOb phenotype and its downstream pathologies. Conversely, the biochemicals could also be used to support monitoring of successful VAT-associated weight loss. An individual who has been undergoing longitudinal biochemical analysis at a clinic who displays the following change(s) in one or more, preferably two or more of the following biochemicals within a clinically recognised healthy range for each of the biochemicals: a decrease in vitamin BI2, an increase in triglycerides, an increase in neutrophil count, an increase in white blood cell count, a decrease in sex hormone binding globulin, an increase in leptin between visits to the clinic whilst also maintaining a clinically recognised healthy BMI and WC, would be subject of a nutritional and / or behavioural review and suggestions for diet adaptation and an exercise program would be presented. Subsequently, there would be further measurements of the biochemicals to assess the impact of the nutritional and / or exercise program (s) and to gauge whether further exercise and / or a different diet is required (Figure 4).

Claims

CLAIMS1 . A method which indicates a change in the amount of visceral adipose tissue in an individual of healthy body mass index (BMI) and healthy waist circumference (WC) comprising measuring the concentration of one or more biomarkers chosen from vitamin B12, white blood cell count, neutrophil count, sex hormone binding globulin, leptin and triglycerides in in vitro samples of the individual obtained at two different time-points and establishing whether a change in the amount of visceral adipose tissue is indicated between the two different time-points in which1. an increase in the concentration of white blood cell count, an increase in the concentration of triglycerides, an increase in the concentration of leptin, an increase in the concentration of neutrophil count, a decrease in the concentration of vitamin B12, and a decrease in the concentration of sex hormone binding globulin is indicative of an increase in the amount of visceral adipose tissue ii. an increase in the concentration of vitamin B12, a decrease in the concentration of white blood cell count, a decrease in the concentration of neutrophil count, a decrease in the concentration of leptin, a decrease in the concentration of triglycerides and an increase in the concentration of sex hormone binding globulin between a first and a second time-point is indicative of a decrease in the amount of visceral adipose tissue.

2. The method claim 1 in which at least two or more of the biomarkers are measured.

3. The method of claim 2 in which the two or more biomarkers chosen from the combinations vitamin B12 and leptin; vitamin B12 and triglycerides; vitamin B12 and WBC; vitamin B12 and neutrophil count; vitamin B12 and sex hormone binding globulin; leptin and triglycerides; leptin and white blood cell count; leptin and neutrophil count; vitamin B12 and leptin and triglycerides; vitamin B12 and white blood cell and triglycerides; vitamin B12 and neutrophil count and triglycerides; vitamin B12 and white blood cell count and leptin , vitamin B12 and white blood cell count and sex hormone binding globulin; and vitamin B12 and neutrophil count and leptin; are measured.

4. The method of the preceding claims in which early-onset visceral adipose tissue accumulation is detected and the individual is male, the healthy BMI is less than25.00 and the healthy WC is less than 94 cm, preferably the healthy BMI is less than or equal to 23.50 and the healthy WC is less than or equal to 86 cm, most preferably the healthy BMI is 21.50 to less than or equal to 23.50 and the healthy WC is in the range 76 cm to 86 cm.

5. The method of claim 4 in which the individual is an adult male aged about 20 years to about 39 years.

6. A preventative healthcare method for a male of BMI less than 25.00 and WC less than 94 cm comprising measuring the concentration of one or more biomarkers chosen from vitamin B12, triglycerides, white blood cell count, neutrophil count, sex hormone binding globulin and leptin in in vitro samples of the male, the samples having been obtained at two different time-points, T 1 and T2, in which a decrease in the concentration of vitamin B12, an increase in the concentration of white blood cell count, an increase in the concentration of neutrophil count, an increase in the concentration of leptin, an increase in the concentration of triglycerides and a decrease in the concentration of sex hormone binding globulin in the sample obtained at T2 compared to compared to the sample obtained at T 1 indicates an increase in the amount of visceral adipose tissue and the male is referred to a nutritional and / or exercise specialist, in which T1 chronologically precedes T2.

7. The preventative healthcare method of claim 6 which further comprises measuring the concentration of the one or more biomarkers in an in vitro sample of the male, the sample having been obtained at a time-point T3, and ascertaining whether the relative concentration of each individual one or more biomarkers at T2 and T3 is / are indicative of a decrease in visceral adipose tissue, in which T2 chronologically precedes T3.

8. The preventative healthcare method of claims 6 and 7 in which at least two or more of the biomarkers are measured.

9. The preventative healthcare method of claim 8 in which the two or more biomarkers chosen from the combinations vitamin BI2and WBC, vitamin BI2and triglycerides, vitamin B12and leptin, vitamin BI2and neutrophil count, vitamin BI2and sex hormone binding globulin, leptin and white blood cell count, leptin and neutrophil count, vitamin BI2and white blood cell and triglycerides, vitamin BI2and neutrophil count and triglycerides, vitamin BI2and white blood cell count and leptin, vitamin BI2and white blood cell count and sex hormone binding globulin, and vitamin BI2and neutrophil count and leptin, are measured.

10. The use of vitamin BI2as a biomarker to monitor the change in the amount of visceral adipose tissue in a male with a BMI of less than about 25.00.11 . The use of claim 10 in which the male has a BMI of about 21 .00 to about 23.50.

12. The use of claims 10 and 11 in which the male is aged about 20 years to about 39 years old.

13. The use of any of claims 10 to 12 in which the male has a waist circumference of less than about 86 cm.

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