{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST000550","ANALYSIS_ID":"AN000840","VERSION":"1","CREATED_ON":"February 9, 2017, 9:54 am"},

"PROJECT":{"PROJECT_TITLE":"Metabolomics marker of brown adipose tissue in men","PROJECT_SUMMARY":"We assessed a MS platform of 163 marker (Biocrates p150 kit) to find markers for brown adipose tissue.","INSTITUTE":"Leiden University Medical Center","LAST_NAME":"Mook-Kanamori","FIRST_NAME":"Dennis","ADDRESS":"PO Box 9600, 2300 RC, Leiden, the Netherlands","EMAIL":"d.o.mook@lumc.nl","PHONE":"+31 71 5265623"},

"STUDY":{"STUDY_TITLE":"Metabolomics marker of brown adipose tissue in men","STUDY_SUMMARY":"Objective: We aimed to identify metabolites in serum that are associated with BAT volume and activity in men. Methods: We assessed 163 metabolites in fasted serum of a cohort of twenty two healthy lean men (age 24.1 (21.7 – 26.6) years, BMI 22.1 (20.5 – 23.4) kg/m2) who subsequently underwent a cold-induced [18F]FDG PET-CT scan to assess BAT volume and activity. In addition, we included three replication cohorts consisting of in total thirty-seven healthy lean men that were similar with respect to age and BMI compared to the discovery cohort.","INSTITUTE":"Leiden University Medical Center","LAST_NAME":"Mook-Kanamori","FIRST_NAME":"Dennis","ADDRESS":"PO Box 9600, 2300 RC, Leiden, the Netherlands","EMAIL":"d.o.mook@lumc.nl","PHONE":"+31715265623","NUM_GROUPS":"5","TOTAL_SUBJECTS":"59","NUM_MALES":"59"},

"SUBJECT":{"SUBJECT_TYPE":"Healthy males","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606","AGE_OR_AGE_RANGE":"20-30","GENDER":"Men","HUMAN_ETHNICITY":"Caucasian and south asian"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"1",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"266,246","BAT_SUV_mean":"4,488"}
},
{
"Subject ID":"-",
"Sample ID":"2",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"503,68","BAT_SUV_mean":"4,677"}
},
{
"Subject ID":"-",
"Sample ID":"3",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"58,69","BAT_SUV_mean":"3,778"}
},
{
"Subject ID":"-",
"Sample ID":"4",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"206,084","BAT_SUV_mean":"4,203"}
},
{
"Subject ID":"-",
"Sample ID":"5",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"545,014","BAT_SUV_mean":"4,562"}
},
{
"Subject ID":"-",
"Sample ID":"6",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"176,22","BAT_SUV_mean":"4,024"}
},
{
"Subject ID":"-",
"Sample ID":"8",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"239,486","BAT_SUV_mean":"4,243"}
},
{
"Subject ID":"-",
"Sample ID":"9",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"245,05","BAT_SUV_mean":"4,127"}
},
{
"Subject ID":"-",
"Sample ID":"10",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"412,422","BAT_SUV_mean":"4,329"}
},
{
"Subject ID":"-",
"Sample ID":"11",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"54,662","BAT_SUV_mean":"3,88"}
},
{
"Subject ID":"-",
"Sample ID":"13",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"235,784","BAT_SUV_mean":"4,111"}
},
{
"Subject ID":"-",
"Sample ID":"14",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"125,448","BAT_SUV_mean":"3,681"}
},
{
"Subject ID":"-",
"Sample ID":"15",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"288,132","BAT_SUV_mean":"4,178"}
},
{
"Subject ID":"-",
"Sample ID":"16",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"178,408","BAT_SUV_mean":"4,161"}
},
{
"Subject ID":"-",
"Sample ID":"17",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"224,76","BAT_SUV_mean":"3,983"}
},
{
"Subject ID":"-",
"Sample ID":"18",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"267,71","BAT_SUV_mean":"4,245"}
},
{
"Subject ID":"-",
"Sample ID":"19",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"155,132","BAT_SUV_mean":"4,022"}
},
{
"Subject ID":"-",
"Sample ID":"20",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"179,134","BAT_SUV_mean":"3,939"}
},
{
"Subject ID":"-",
"Sample ID":"21",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"132,576","BAT_SUV_mean":"4,039"}
},
{
"Subject ID":"-",
"Sample ID":"22",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"422,71","BAT_SUV_mean":"4,445"}
},
{
"Subject ID":"-",
"Sample ID":"23",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"330,116","BAT_SUV_mean":"4,44"}
},
{
"Subject ID":"-",
"Sample ID":"24",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"158,972","BAT_SUV_mean":"4,198"}
},
{
"Subject ID":"-",
"Sample ID":"52",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"8,838,576,401","BAT_SUV_mean":"1,756,218,022"}
},
{
"Subject ID":"-",
"Sample ID":"53",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"1,131,216,929","BAT_SUV_mean":"2,092,581,575"}
},
{
"Subject ID":"-",
"Sample ID":"54",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"1,964,701,825","BAT_SUV_mean":"2,097,582,417"}
},
{
"Subject ID":"-",
"Sample ID":"56",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"3,063,893,333","BAT_SUV_mean":"208,377,651"}
},
{
"Subject ID":"-",
"Sample ID":"58",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"4,097,825,495","BAT_SUV_mean":"2,317,946,406"}
},
{
"Subject ID":"-",
"Sample ID":"59",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"1,450,207,688","BAT_SUV_mean":"1,707,358,105"}
},
{
"Subject ID":"-",
"Sample ID":"111",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"8,917,458,908","BAT_SUV_mean":"2,796,071,592"}
},
{
"Subject ID":"-",
"Sample ID":"112",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"9,695,901,855","BAT_SUV_mean":"2,862,846,855"}
},
{
"Subject ID":"-",
"Sample ID":"121",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"473,624,647","BAT_SUV_mean":"1,680,619,884"}
},
{
"Subject ID":"-",
"Sample ID":"131",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"1,551,227,837","BAT_SUV_mean":"1,977,701,491"}
},
{
"Subject ID":"-",
"Sample ID":"141",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"8,637,524,878","BAT_SUV_mean":"2,124,273,725"}
},
{
"Subject ID":"-",
"Sample ID":"151",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"8,504,039,864","BAT_SUV_mean":"2,198,745,225"}
},
{
"Subject ID":"-",
"Sample ID":"152",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"2,412,728,108","BAT_SUV_mean":"2,355,601,863"}
},
{
"Subject ID":"-",
"Sample ID":"162",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"8,670,868,671","BAT_SUV_mean":"3,026,336,563"}
},
{
"Subject ID":"-",
"Sample ID":"171",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"1,643,129,256","BAT_SUV_mean":"1,784,298,939"}
},
{
"Subject ID":"-",
"Sample ID":"172",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"7,254,828,721","BAT_SUV_mean":"24,831,201"}
},
{
"Subject ID":"-",
"Sample ID":"181",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"8,525,463,375","BAT_SUV_mean":"2,024,068,194"}
},
{
"Subject ID":"-",
"Sample ID":"182",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"7,821,178,801","BAT_SUV_mean":"2,559,823,181"}
},
{
"Subject ID":"-",
"Sample ID":"192",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"5,996,608,753","BAT_SUV_mean":"2,260,494,483"}
},
{
"Subject ID":"-",
"Sample ID":"510",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"9,888,823,398","BAT_SUV_mean":"3,167,435,172"}
},
{
"Subject ID":"-",
"Sample ID":"511",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"3,982,907,522","BAT_SUV_mean":"2,364,085,482"}
},
{
"Subject ID":"-",
"Sample ID":"512",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"1,571,937,243","BAT_SUV_mean":"1,896,698,088"}
},
{
"Subject ID":"-",
"Sample ID":"513",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"7,957,300,568","BAT_SUV_mean":"2,836,559,436"}
},
{
"Subject ID":"-",
"Sample ID":"514",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"5,548,527,572","BAT_SUV_mean":"2,569,951,671"}
},
{
"Subject ID":"-",
"Sample ID":"515",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"7,156,884,802","BAT_SUV_mean":"2,661,525,352"}
},
{
"Subject ID":"-",
"Sample ID":"516",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"5,242,995,179","BAT_SUV_mean":"2,478,654,803"}
},
{
"Subject ID":"-",
"Sample ID":"517",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"1,280,011,533","BAT_SUV_mean":"3,028,138,993"}
},
{
"Subject ID":"-",
"Sample ID":"518",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"7,832,000,429","BAT_SUV_mean":"2,474,635,055"}
},
{
"Subject ID":"-",
"Sample ID":"519",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"4,325,244,428","BAT_SUV_mean":"2,342,512,471"}
},
{
"Subject ID":"-",
"Sample ID":"1111",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"7,271,912,639","BAT_SUV_mean":"1,794,481,421"}
},
{
"Subject ID":"-",
"Sample ID":"1112",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"10,792,127","BAT_SUV_mean":"2,782,210,726"}
},
{
"Subject ID":"-",
"Sample ID":"1122",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"9,874,376,253","BAT_SUV_mean":"2,400,085,708"}
},
{
"Subject ID":"-",
"Sample ID":"1141",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"4,800,791,699","BAT_SUV_mean":"233,755,045"}
},
{
"Subject ID":"-",
"Sample ID":"1142",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"4,031,467,507","BAT_SUV_mean":"2,229,139,773"}
},
{
"Subject ID":"-",
"Sample ID":"1151",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"1,645,491,339","BAT_SUV_mean":"2,226,188,721"}
},
{
"Subject ID":"-",
"Sample ID":"1192",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"1,367,606,275","BAT_SUV_mean":"3,257,150,365"}
},
{
"Subject ID":"-",
"Sample ID":"1201",
"Factors":{"SampleType":"Serum"},
"Additional sample data":{"BAT_Volume":"2,488,864,012","BAT_SUV_mean":"242,954,041"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"METHODS Study population discovery cohort Our discovery cohort consisted of twenty-four healthy male Caucasian (n=12) and South Asian (n=12) participants from a study that was performed previously in the Alrijne hospital (Leiderdorp, the Netherlands) between March and June 2013 (Bakker et al. 2014). This study was powered on the identification of a difference in BAT volume, resulting in inclusion of twelve subjects per group. Subjects were lean (BMI <25 kg/m2) and healthy males aged 18-30 years that were enrolled via local advertisements. Subjects underwent a medical screening including their medical history, a physical examination, blood chemistry tests, and an oral glucose tolerance test (OGTT) to exclude individuals with T2DM according to the American Diabetes Association (ADA) 2010 criteria. Other exclusion criteria were rigorous exercise, smoking, and recent body weight change. Data from two individuals were removed from the analyses due to virtually absent BAT volume (0-1 cm3) as assessed by [18F]FDG PET-CT, leaving twenty-two participants. The present study was approved by the Medical Ethical Committee of the Leiden University Medical Center and performed in accordance with the principles of the revised Declaration of Helsinki. All volunteers gave written informed consent before participation. Study population replication cohorts To verify whether the associations were sufficiently robust, we included three replication cohorts consisting of in total forty-two healthy Caucasian men. These participants were derived from three studies performed at the Maastricht University Medical Centre (Maastricht, the Netherlands) between 2011 and 2014 (Vosselman et al. 2012; Vosselman et al. 2013; Vosselman et al. 2015). In the first study (Vosselman et al. 2015), aimed at studying the effect of exercise on BAT volume and activity, twelve healthy lean endurance-trained and twelve lean sedentary male Caucasian men aged 18–35 years were included. Endurance-trained athletes were included in the trained group when they performed endurance exercise at least three times a week for the last 2 years, and had a maximal oxygen consumption (VO2max) of >55 ml/min/kg. The sedentary males were included in the untrained group if they did not perform more than 1 h of exercise per week for the last 2 years and had a VO2max of <45 ml/min/kg. General exclusion criteria were use of medication, smoking, weight gain/loss of >3 kg in the last 6 months, hypertension and (family history of) diabetes. The trained men were included in replication cohort 1 and the untrained men in replication cohort 2.  Replication cohort 3 consisted of subjects derived from two studies (Vosselman et al. 2012; Vosselman et al. 2013) that had the same inclusion criteria and cooling protocol. These studies aimed at studying the effect of β-adrenergic stimulation on BAT (Vosselman et al. 2012) and the effect of a high-calorie meal on BAT (Vosselman et al. 2013). In these studies, in total 19 healthy male Caucasian subjects aged between 18-35 years were enrolled. All subjects were screened for medical history. Cardiovascular status was screened by means of an electrocardiogram and blood pressure measurement. All subjects had normal blood glucose levels. In the replication cohorts, data from five participants were removed due to absence of [18F]FDG uptake in the BAT region, leaving thirty-seven participants in the replication set. For the pooled analyses, fifty-nine participants were included in the analyses. All replication studies were approved by the medical ethical committee of the Maastricht University Medical Center and all subjects were treated according to the principles of the revised declaration of Helsinki. Study set-up discovery cohort Individuals were studied in the morning after a 10-h overnight fast and after 24h without exercise. Subsequently, they were exposed to a thermoneutral temperature (32 C) for 60 min, after which a basal blood serum sample was taken in which also metabolomics measurements were performed (see below). To activate BAT an individualized cooling protocol was applied. In short, subjects lay on a bed sandwiched between two water-perfused cooling mattresses (Blanketrol III, Cincinnati Sub-Zero Products, Cincinnati, OH, USA). Cooling started at 32°C and temperature was gradually decreased until shivering occurred (approximately after 50-60 min). Temperature was then raised by 3–4°C and the cooling period of 2 h was started (defined as tcold = 0 min). If shivering occurred, the temperature was raised by 1°C until shivering stopped. We detected shivering visually or it was reported by participants. After 1 h of cooling (tcold=60 min), 2 MBq/kg [¹ F]FDG was injected intravenously and subjects were instructed to lay still in order to prevent artifact by muscle activity. At tcold = 110 min, a cold-induced blood sample was taken in which metabolomics measurements were performed as well. After 2 h of cooling (tcold=120 min) the PET-CT scan (Gemini TF PET-CT, Philips, The Netherlands) was performed to assess BAT volume and activity, as described previously (Bakker et al. 2014). We quantified BAT activity and detectable volume in the region of interest by autocontouring the BAT areas with a set threshold (SUV) of 2.0 g/mL using Hermes software (Hermes Hybrid Viewer, Hermes Medical Solutions, Sweden). Study set-up replication cohorts In all replication cohorts, subjects were studied in the morning after an overnight fast and after 24 h without exercise. All experiments started with 1-h baseline measurements during thermoneutral conditions (24 to 25 C), after which a basal blood serum sample was taken in which also metabolomics measurements were performed (see below). Subsequently, subjects were exposed to 2 h of mild cold exposure in which an individualized protocol was used by means of air cooling using air-conditioning. In short, each subject was cooled down until shivering occurred (approximately after 20-60 min). After that, air temperature was slightly increased until shivering stopped. After 1 h of cold exposure the [18F]FDG tracer was injected intravenously (74 MBq in replication cohorts 1 and 2 and 50 MBq in replication cohort 3) and subjects were exposed to another hour of cold. Next, after 2 h of cooling, the PET-CT scan (Gemini TF PET-CT, Philips, the Netherlands) was performed to assess BAT volume and activity. In replication cohort 1 and 2, BAT activity and detectable volume were quantified in the region of interest by autocontouring the BAT areas with a set threshold (SUV) of 1.5 g/mL (Vosselman et al. 2015). In replication cohort 3 a threshold of 1.5 g/mL was used as well, and additionally the regions drawn were localized in fat tissues only as determined by CT scan (HU -10 to -180) (Vosselman et al. 2012). For all analyses, PMOD software (PMOD Technologies) was used."},

"TREATMENT":{"TREATMENT_SUMMARY":"N/A"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Targeted metabolomics Metabolomic measurements were performed on serum samples taken at thermoneutral and cold conditions at the Genome Analysis Center at the Helmholtz Zentrum, Munich, Germany, using the Biocrates AbsoluteIDQTM p150 kit (BIOCRATES Life Science AG, Innsbruck, Austria) and ESI-FIA-MS/MS measurements (Menni et al. 2013). The assay allows simultaneous quantification of 163 small molecule metabolites within 10 µL serum. The assay includes free carnitine, 40 acylcarnitines, 14 amino acids (13 proteinogenic + ornithine), hexoses (of which approx. 90-95% is glucose), 92 glycerophospholipids including 15 lysophosphatidylcholines (LysoPCs) and 77 phosphatidylcholines (PCs), and 15 sphingolipids (Menni et al. 2013; Illig et al. 2010). Quantification of the metabolites was achieved by reference to appropriate internal standards. The assay has been previously described (Menni et al. 2013; Illig et al. 2010)."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"GC","INSTRUMENT_NAME":"Biocrates IDQ p150","COLUMN_NAME":"Biocrates IDQ p150"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"MS_COMMENTS":"-","INSTRUMENT_NAME":"Biocrates IDQ p150","INSTRUMENT_TYPE":"Triple quadrupole","MS_TYPE":"Other","ION_MODE":"NEGATIVE"},

"MS_METABOLITE_DATA":{
"Units":"mmol/L",

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}

}