Summary of study ST001251

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench,, where it has been assigned Project ID PR000838. The data can be accessed directly via it's Project DOI: 10.21228/M8C671 This work is supported by NIH grant, U2C- DK119886.


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Study IDST001251
Study TitleThe effects of a training program encompassing cold exposure, breathing exercises, and meditation on plasma metabomics during experimental human endotoxemia
Study TypeExperimental human endotoxemia study
Study SummaryStudy to investigate the effects of a training program encompassing cold exposure, breathing exercises, and meditation on plasma metabolomic during experimental human endotoxemia.
Radboud University Medical Centre
DepartmentIntensive Care Medicine
LaboratoryMetabolomic Discoveries (acquired by Metabolon in September 2017)
Last NameKox
First NameMatthijs
AddressIntensive Care Medicine (710), Geert Grooteplein 10
Submit Date2019-09-16
Num Groups2
Total Subjects24
Num Males24
Analysis Type DetailLC-MS
Release Date2019-10-04
Release Version1
Matthijs Kox Matthijs Kox application/zip

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Project ID:PR000838
Project DOI:doi: 10.21228/M8C671
Project Title:The effects of a training program encompassing cold exposure, breathing exercises, and meditation on the inflammatory response
Project Type:Experimental human endotoxemia project
Project Summary:Project to investigate the effects of a training program encompassing cold exposure, breathing exercises, and meditation on the inflammatory response during experimental human endotoxemia.
Institute:Radboud University Medical Centre
Department:Intensive Care Medicine
Last Name:Kox
First Name:Matthijs
Address:Intensive Care Medicine (710), Geert Grooteplein 10, 6500 HB, Nijmegen, the Netherlands
Funding Source:Dutch Arthritis Society (ReumaNederland, Serendipity Grant, no. 12-1-101


Subject ID:SU001319
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606
Age Or Age Range:19-27
Weight Or Weight Range:58-92
Height Or Height Range:172-190
Human Race:Caucasian
Human Trial Type:Prospective randomized open label controlled intervention study
Human Medications:None
Human Prescription Otc:None
Human Smoking Status:Non smoker
Human Alcohol Drug Use:Not in
Human Nutrition:Fasted for 12 hours before the first sample was obtained.
Human Inclusion Criteria:Healthy (normal physical examination, electrocardiography, and routine laboratory values).
Human Exclusion Criteria:Febrile illness during the 2 weeks before the endotoxemia experiment, taking any prescription medication, history of spontaneous vagal collapse, practicing or experience with any kind of meditation, or participation in a previous trial where LPS was administered.


Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id treatment
SA09101617_30 _t_240control
SA09101717_30 _t_480control
SA09101817_30 _t_120control
SA09101917_30 _t_60control
SA09102017_22 _t_480control
SA09102117_30 _t_minus_50control
SA09102217_30 _t_0control
SA09102317_02 _t_480control
SA09102417_02 _t_240control
SA09102517_33 _t_120control
SA09102617_33 _t_60control
SA09102717_33 _t_0control
SA09102817_33 _t_240control
SA09102917_02 _t_minus_50control
SA09103017_02 _t_120control
SA09103117_02 _t_60control
SA09103217_02 _t_0control
SA09103317_22 _t_240control
SA09103417_22 _t_120control
SA09103517_13 _t_240control
SA09103617_29 _t_480control
SA09103717_29 _t_240control
SA09103817_13 _t_120control
SA09103917_13 _t_60control
SA09104017_13 _t_minus_50control
SA09104117_13 _t_0control
SA09104217_29 _t_120control
SA09104317_29 _t_60control
SA09104417_22 _t_0control
SA09104517_41 _t_minus_50control
SA09104617_22 _t_60control
SA09104717_22 _t_minus_50control
SA09104817_13 _t_480control
SA09104917_29 _t_0control
SA09105017_29 _t_minus_50control
SA09105117_33 _t_minus_50control
SA09105217_33 _t_480control
SA09105917_40 _t_0control
SA09106317_42 _t_480control
SA09106417_42 _t_240control
SA09106517_41 _t_240control
SA09106617_41 _t_120control
SA09106717_41 _t_60control
SA09106817_41 _t_0control
SA09106917_41 _t_480control
SA09107017_42 _t_minus_50control
SA09107117_42 _t_120control
SA09107217_42 _t_60control
SA09107317_42 _t_0control
SA09107417_40 _t_120control
SA09107517_40 _t_60control
SA09108317_40 _t_240control
SA09108517_40 _t_480control
SA09108617_15 _t_480trained
SA09108717_15 _t_120trained
SA09108817_15 _t_240trained
SA09108917_20 _t_480trained
SA09109017_20 _t_120trained
SA09109117_20 _t_240trained
SA09109217_20 _t_60trained
SA09109317_10 _t_minus_50trained
SA09109417_10 _t_480trained
SA09109517_15 _t_60trained
SA09109617_15 _t_0trained
SA09109717_10 _t_240trained
SA09109817_10 _t_120trained
SA09109917_10 _t_0trained
SA09110017_10 _t_60trained
SA09110117_15 _t_minus_50trained
SA09110217_34 _t_60trained
SA09110317_23 _t_minus_50trained
SA09110417_23 _t_60trained
SA09110517_14 _t_480trained
SA09110617_14 _t_240trained
SA09110717_14 _t_120trained
SA09110817_23 _t_120trained
SA09110917_23 _t_240trained
SA09111017_27 _t_60trained
SA09111117_27 _t_0trained
SA09111217_27 _t_minus_50trained
SA09111317_23 _t_480trained
SA09111417_14 _t_60trained
SA09111517_14 _t_0trained
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Collection ID:CO001313
Collection Summary:Ethylenediaminetetraacetic acid (EDTA) anti-coagulated blood was obtained one hour before LPS administration (baseline), and at T=0, T=1, T=2, T=4 and T=8 h. Blood was immediately centrifuged at 2000 g for 10 minutes at 4 °C after which plasma was stored at –80 °C until analysis.
Sample Type:Blood (plasma)
Collection Method:Arterial canulla
Collection Location:Radial artery
Collection Frequency:6
Collection Duration:1 minute
Storage Conditions:-80℃
Collection Vials:Vacutainer blood collection tubes (BD)
Storage Vials:Micronic vials (polypropylene)
Collection Tube Temp:Room temperature
Additives:Ethylenediaminetetraacetic acid (EDTA)


Treatment ID:TR001334
Treatment Summary:The subjects were randomly allocated to the trained group (n=12) or the control group (n=12) by the opening of sealed envelopes prepared by unblinded staff not involved in the study. The trained group underwent a 10-day training program provided by Dutch individual Wim Hof, which consisted of three main elements: meditation, exposure to cold, and breathing exercises (see for a detailed description). After completion of the training, subjects underwent experimental human endotoxemia at our intensive care research unit, consisting of administration of an intravenous bolus of 2 ng/kg of US Standard Reference Escherichia coli endotoxin (E. coli O:113 [LPS], Clinical Center Reference Endotoxin; National Institutes of Health, Bethesda, MD, USA). The experimental human endotoxemia protocol is provided in detail elsewhere (; The control group did not undergo any training procedures and also underwent experimental human endotoxemia. Subjects in the trained group started practicing the breathing exercises acquired during the training program 30 minutes before LPS administration until two-and-a-half hours afterwards. The control group did not practice any techniques throughout the endotoxemia experiment. As described in detail elsewhere (, the breathing techniques consisted of two exercises. In brief, the first exercise comprised cycles of vigorous hyperventilation (approximately 30 breaths) followed by breath holding for several minutes at the discretion of the subject. The second exercise was similar, but at the end of the hyperventilation period, breath was only held for 10 seconds during all body muscles were tightened.
Treatment:Nonpharmacological training intervention
Treatment Compound:n/a.
Treatment Route:n/a.
Treatment Dose:n/a.
Treatment Dosevolume:n/a.
Treatment Doseduration:10 days
Treatment Vehicle:control group
Human Fasting:Yes, 12 hours before obtaining the first sample.

Sample Preparation:

Sampleprep ID:SP001327
Sampleprep Summary:Sample extraction was performed as described previously (, with some modifications. Briefly, 50 µL of plasma was mixed with 450 µL of 90% (v/v) methanol containing internal standards and incubated for 15 min at 37°C with 1150 rpm. Precipitated proteins were separated from the extract by centrifugation for 12 min at 15000 rpm, after which supernatants were stored at -80 °C until further analysis.
Processing Storage Conditions:-80℃
Extraction Method:Ethanol precipitation

Combined analysis:

Analysis ID AN002077
Analysis type MS
Chromatography type Reversed phase
Chromatography system Agilent 1290
Column Discovery HS F5-3 column (15 cm x 2.1 mm, 3 µm, Supelco, Sigma Aldrich)
MS instrument type QTOF
MS instrument name Agilent 6540 QTOF
Units relative abundance


Chromatography ID:CH001514
Instrument Name:Agilent 1290
Column Name:Discovery HS F5-3 column (15 cm x 2.1 mm, 3 µm, Supelco, Sigma Aldrich)
Flow Gradient:5% B from 0-0.1min to 35% B at 1.5 min, to 95% B at 2.05 min which was kept constant until 3.2 min, to 5% B at 3.21 min and washing until 4.3 min with 5% B.
Flow Rate:The flow rate was kept constant at 700 µL/min from 0 min to 2.2 min, and increased up to 900 µL/min from 2.2 min to 2.5 min, after which flow rate was kept constant until 3.2 min.The flow rate was decreased from 900 µL/min to 800 µL/min from 3.2 min to 3.1 min and kept constant until 3.7 min, when flow rate was changed to 700 µL/min.
Injection Temperature:40 °C
Solvent A:95% 10 mM ammonium formate with 0.1% formic acid and 5% acetonitrile
Solvent B:and 95% acetonitrile with 5% 10 mM ammonium formate with 0.1% formic acid
Chromatography Type:Reversed phase


MS ID:MS001928
Analysis ID:AN002077
Instrument Name:Agilent 6540 QTOF
Instrument Type:QTOF
MS Comments:The 6540 QTOF/MS Detector (Agilent) operated in both positive and negative ESI mode in a detection range of 50 to 1700 m/z at 2 GHz in extended dynamic range. The DualAJS ESI source was set to the following parameters: Gas temperature 200 °C, drying gas 8 L/min, nebulizer 35 psig, sheath gas temp: 350 °C, sheath gas flow 11 L/min, VCAp 3500 V and nozzle voltage of 0 V. Online calibration of the instrument was performed throughout the data acquisition using Agilent ES-TOF Reference Mass Solution Kit. Chromatograms were generated by the LC-MS instrument in .d format. Raw data were converted into mzXML and chromatogram peaks were extracted using XCMS v1.42.0 (, which was optimized using the IPO R package ( with the following settings: peakwidth=c(10, 70), ppm= 20, snthresh=10, mzdiff=0.0034, prefilter=c(3, 100), noise=100, gapInit=0.8448, gapExtend=2.0544, bw=5, mzwid=0.015, minfrac=0.5, max=50. All further analyses were performed in R programming language, Metaboanalyst 4.0 (, and GraphPad Prism version 5.0 (GraphPad Software). IDEOM software (; was used to eliminate noise and for putative peak annotation by exact mass within ±10 ppm against the Metabolomic Discoveries in house metabolite library ( in negative and positive ESI mode, respectively. Retention time prediction was applied to aid metabolite annotation.