Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA256434

Local Sample ID:	Arabic Gum-C09-01-6613
Subject ID:	SU002653
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Subject:

Subject ID:	SU002653
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
Arabic Gum-C09-01-6613	SA256434	FL032768	Control	Treatment

Collection:

Collection ID:	CO002646
Collection Summary:	The patients’ samples were collected from Jordan University Hospital, and the metabolomics study was conducted at the Research Institute of Medical & Health Sciences (RIMHS?) of the University of Sharjah. The study included two groups: a control group of 43 patients and an intervention group of 45 patients with stable CKD stages III-V not on dialysis and aged between 18 and 90 years. Exclusion criteria included pregnancy and treatment with complementary and alternative medicine (CAM) other than Arabic gum. Informed consent was obtained prior to the study, and approval was obtained from Jordan University Hospital's Research Ethics Committee. The study was conducted in accordance with the Helsinki Declaration principles, and participants were fully informed about the study prior to signing the consent forms.
Sample Type:	Blood (serum)

Treatment:

Treatment ID:	TR002665
Treatment Summary:	For intervention group, Arabic gum in the form of powder twice daily was given, at the dose patients are taking usually over the counter (40 grams of GA in the form of instantly soluble granules to be dissolved in a glass of water or juice and drink it daily for the assigned study period. While for the control group, a placebo was given in the same shape and taste and same frequency. Participants in both groups underwent a 24-hour urine collection for creatinine, protein, and volume at baseline and every three months. eGFR was calculated using the CKD-epi equation [add ref], and lab tests, including serum creatinine, electrolytes, uric acid, serum albumin, phosphate, calcium, PTH, Complete Blood Count (CBC), fasting lipid profile, and urinalysis, were performed at baseline and at 3-month intervals for a year. The objectives of the study were to assess the impact of the intervention on various parameters, including a reduction in the eGFR and the rate of the decline, a decrease in proteinuria, changes in uric acid, electrolytes, calcium, phosphate, vitamin D, and PTH levels, effects on edema, body weight, blood pressure, and anemia parameters.

Treatment ID:

TR002665

Treatment Summary:

For intervention group, Arabic gum in the form of powder twice daily was given, at the dose patients are taking usually over the counter (40 grams of GA in the form of instantly soluble granules to be dissolved in a glass of water or juice and drink it daily for the assigned study period. While for the control group, a placebo was given in the same shape and taste and same frequency. Participants in both groups underwent a 24-hour urine collection for creatinine, protein, and volume at baseline and every three months. eGFR was calculated using the CKD-epi equation [add ref], and lab tests, including serum creatinine, electrolytes, uric acid, serum albumin, phosphate, calcium, PTH, Complete Blood Count (CBC), fasting lipid profile, and urinalysis, were performed at baseline and at 3-month intervals for a year. The objectives of the study were to assess the impact of the intervention on various parameters, including a reduction in the eGFR and the rate of the decline, a decrease in proteinuria, changes in uric acid, electrolytes, calcium, phosphate, vitamin D, and PTH levels, effects on edema, body weight, blood pressure, and anemia parameters.

Sample Preparation:

Sampleprep ID:	SP002659
Sampleprep Summary:	300 µL of methanol (Wunstorfer Strasse, Seelze, Germany) was added after the samples were divided into 100 µL Eppendorf tubes, which were then vortexed and incubated at –20 °C for 2 hours. The samples were then vortexed and centrifuged for 15 minutes at 14,000 rpm. The supernatant was then evaporated at 35–40 °C using a speed vacuum evaporator. To assess the analysis's reproducibility, a quality control (QC) sample was created by combining the same volume of each sample. The extract samples were then resuspended in 250 L of Honeywell's LC-MS CHROMASOLV's 0.1% formic acid in Deionized Water (Wunstorfer Strasse, Seelze, Germany). After that, the supernatant was filtered for LC-MS/MS analysis through a hydrophilic nylon syringe filter with a 0.45 m pore size. 100 L of the prepared sample was then gathered in an insert inside LC glass vials.

Sampleprep ID:

SP002659

Sampleprep Summary:

300 µL of methanol (Wunstorfer Strasse, Seelze, Germany) was added after the samples were divided into 100 µL Eppendorf tubes, which were then vortexed and incubated at –20 °C for 2 hours. The samples were then vortexed and centrifuged for 15 minutes at 14,000 rpm. The supernatant was then evaporated at 35–40 °C using a speed vacuum evaporator. To assess the analysis's reproducibility, a quality control (QC) sample was created by combining the same volume of each sample. The extract samples were then resuspended in 250 L of Honeywell's LC-MS CHROMASOLV's 0.1% formic acid in Deionized Water (Wunstorfer Strasse, Seelze, Germany). After that, the supernatant was filtered for LC-MS/MS analysis through a hydrophilic nylon syringe filter with a 0.45 m pore size. 100 L of the prepared sample was then gathered in an insert inside LC glass vials.

Combined analysis:

Analysis ID	AN004204
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Bruker timsTOF
Column	Bruker Intensity Solo 2 C18 (100 mm × 2.1 mm , 1.8 μm)
MS Type	ESI
MS instrument type	QTOF
MS instrument name	Bruker timsTOF
Ion Mode	POSITIVE
Units	AU

Chromatography:

Chromatography ID:	CH003115
Instrument Name:	Bruker timsTOF
Column Name:	Bruker Intensity Solo 2 C18 (100 mm × 2.1 mm , 1.8 μm)
Column Temperature:	35
Flow Gradient:	The gradient program was: 0–2 min, 99% A: 1% B; 2–17 min, 99–1% A: 1–99% B; 17–20 min, 99% B: 1% A. The flow rate was fixed at 0.25 mL/min. Subsequently, 20–20.1 min 99% B to 99% A; 20.1–28.5 min, 99% A: 1% B at 0.35 mL/min flow rate; 28.5–30 min; 99% A: 1% B at 0.25 mL/min.
Flow Rate:	250 uL/min
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% acetonitrile; 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003951
Analysis ID:	AN004204
Instrument Name:	Bruker timsTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	The LC-MS/MS analysis was performed using an ultra-high-performance liquid chromatography system (UHPLC) (Bruker Daltonik GmbH, Bremen, Germany) connected to a quadrupole time-of-flight mass spectrometer (QTOF). An electrospray ionization (ESI) source, a solvent delivery systems pump (HPG 1300), an autosampler, and a thermostat column compartment were all included in the system. The computer operating system was Windows 10 Enterprise 2016 LTSB. Bruker Compass HyStar 5.0 SR1 Patch1 (5.0.37.1), Compass 4.1 for otofSeries, and otof Control Version 6.2 software were used for data management. Mobile phases A (water with 0.1% formic acid) and B (acetonitrile with 0.1% formic acid) were employed. The gradient program was: 0–2 min, 99% A: 1% B; 2–17 min, 99–1% A: 1–99% B; 17–20 min, 99% B: 1% A. The flow rate. Subsequently, 20–20.1 min 99% B to 99% A; 20.1–28.5 min, 99% A: 1% B at 0.35 mL/min flow rate; 28.5–30 min; 99% A: 1% B at 0.25 mL/min. A 10 μL aliquot of the sample was injected, and the separation was performed on a Hamilton® Intensity Solo 2 C18 column (100 mm × 2.1 mm × 1.8 μm) at a column oven temperature set at 35 °C. For each injection, the ESI source circumstances were as follows: The capillary voltage was adjusted to 4500 V; the drying gas flow rate was 10.0 L/min at a temperature of 220 °C; and the nebulizer pressure was 2.2 bar. The collision energy stepping for the MS2 acquisition varied between 100 and 250% fixed at 20 eV and an end plate offset of 500 V. [35]. For the external calibration step, sodium formate was utilized as a calibrant. For the calibrant sodium formate, the auto MS scan segment of the acquisition lasted from 0 to 0.3 minutes, and the auto MS/MS segment, which included fragmentation, lasted from 0.3 to 30 minutes. The acquisition in both segments was performed using the positive mode at 12 Hz. The automatic in-run mass scan range was from 20 to 1300 m/z, the width of the precursor ion was ±0.5, the number of precursors was 3, the cycle time was 0.5 s, and the threshold was 400 cts. Active exclusion was excluded after 3 spectra and released after 0.2 min. The data was processed using MetaboScape® 4.0 software (Bruker Daltonics, Billerica, MA, USA) (37). The following were the T-ReX 2D/3D workflow bucketing parameters for the processed data: intensity threshold of 1000; peak length of 7 spectra; and using peak area for quantifying the feature. Mass spectra were calibrated in 0-0.3 minutes using features from at least 48 to 187 samples. However, the auto MS/MS scan was carried out using the average method. The retention time and mass ranges for the scan were 0.3 to 25 minutes and 50 to 1000 m/z, respectively. By using LC-QTOF to analyze every sample in duplicate, 88 samples from both groups undergoing examination were combined to create a data set with 3487 characteristics. Based on the mapping of the MS/MS spectra and retention time in the HMBD 4.0, an annotated database created to meet the demands of the metabolomics community, metabolites were identified. A total of 102 distinct metabolites were selected after MetaboScape® filtration (Supplementary Table S1). The peak intensities of each metabolite were used to quantify the data matrix. Only significant compounds recorded in the HMDB 4.0 with p < 0.05 were included in the metabolite datasets. The online website HMDB (https://hmdb.ca/metabolites/HMDB0059911) was used to filter the human metabolites or Arabic gum metabolites. Following HMDB filtration, 92 unique metabolites remained (Supplementary Table S2). The software MetaboAnalyst 5.0 (Mcgill University, Montreal, QC, Canada), a comprehensive platform for metabolomics data analysis, was used to import the metabolite datasets after exporting them as a CSV file [36]. To help classify the samples, the most discriminating features in the studied group were chosen using the sPLS-DA method in MetaboAnalyst. The rate of false positives was reduced, and multiple hypothesis testing was corrected using the false discovery rate (FDR) approach. The Arabic gum group's significantly altered metabolites were found using a two-tailed independent students t-test in comparison to the control group. As a result, a volcano plot was created to display the statistical significance and fold change for cellular metabolite dysregulation. The threshold for significance was p<0.05. Functional Enrichments were constructed using metaboanalyst (https://www.metaboanalyst.ca).
Ion Mode:	POSITIVE