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MB Sample ID: SA137209
| Local Sample ID: | Control2a |
| Subject ID: | SU001693 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Age Or Age Range: | 55-82 years |
| Gender: | Male and female |
| Human Race: | unknown |
| Human Ethnicity: | unknown |
| Human Medications: | Asprin, ACE inhibitor, statins, cilostazol |
| Human Smoking Status: | Controls = 4, CLTI Pre-surgery = 7, and CLTI Amputation = 9 |
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Subject:
| Subject ID: | SU001693 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Age Or Age Range: | 55-82 years |
| Gender: | Male and female |
| Human Race: | unknown |
| Human Ethnicity: | unknown |
| Human Medications: | Asprin, ACE inhibitor, statins, cilostazol |
| Human Smoking Status: | Controls = 4, CLTI Pre-surgery = 7, and CLTI Amputation = 9 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| Control2a | SA137209 | FL017530 | Control | Group |
Collection:
| Collection ID: | CO001686 |
| Collection Summary: | Gastrocnemius muscle specimens were obtained from ten older adult non-PAD controls (Control), ten patients with critical limb ischemia (CLI) undergoing surgical intervention (CLTI Pre-surgery), and ten CLI patients undergoing limb amputation (CLTI Amputation). Five pre-surgery patients underwent bypass interventions and five underwent endovascular procedures. Muscle specimens were collected within the confines of the operating rooms (CLI patients) or via percutaneous muscle biopsy using sterile procedures. A portion of the muscle was quickly trimmed of fat/connective tissue and snap frozen in liquid nitrogen for metabolomics analysis. |
| Sample Type: | Muscle |
| Collection Method: | Muscle specimens were collected within the confines of the operating rooms (CLI patients) or via percutaneous muscle biopsy using sterile procedures. A portion of the muscle was quickly trimmed of fat/connective tissue and snap frozen in liquid nitrogen for metabolomics analysis. |
| Storage Conditions: | -80℃ |
| Storage Vials: | cryovials |
Treatment:
| Treatment ID: | TR001706 |
| Treatment Summary: | This was a prospective cohort study that examined the metabolomic profile of skeletal muscle from CLTI patients undergoing surgerical intervention or amputation, as well as a cohort of non-PAD controls. CLTI patients exhibited severe symptomology (Rutherford Classification 3-6), with high incidence of common PAD risk factors including hypertension, hyperlipidemia, coronary artery disease, and diabetes.Ten patients with critical limb ischemia (CLI) undergoing surgical intervention (CLTI Pre-surgery), and ten CLI patients undergoing limb amputation (CLTI Amputation). Five pre-surgery patients underwent bypass interventions and five underwent endovascular procedures. Muscle specimens were collected within the confines of the operating rooms (CLI patients) or via percutaneous muscle biopsy using sterile procedures. |
| Human Fasting: | non-fasted |
| Human Endp Clinical Signs: | N/A |
Sample Preparation:
| Sampleprep ID: | SP001699 |
| Sampleprep Summary: | Extraction. Both polar and non-polar metabolites were extracted from the gastrocnemius muscle specimens using FOLCH extraction. In brief, wet weigh of the frozen tissues were determined and immediately homogenized in 1 mL of ice-cold methanol using a PowerLyzer 24 Homogenizer (QIAGEN Group, Hilden, Germany). All enzymatic activities are halted once the sample was homogenized in the methanol. Homogenization was followed by centrifugation (13.2K r.p.m., 4 oC, 30 minutes) and supernatant was transferred into a new glass vial consisting a mixture of 3 mL of ice-cold chloroform and methanol (2:1 v/v) ratio. The cold mixture was vortexed for several minutes and left in an ice bath 15 minutes to allow for phase separation. Next, 1 mL of ice-cold 0.9% saline was added to the mixture followed by vigorous mixing. The mixture was again left in the ice bath for 45 minutes for phase separation. The upper methanol/water layer was transferred to a new falcon tube. To the lower chloroform layer, 1 mL of ice-cold 0.9% saline was added and all steps were followed as mentioned above. Following a 45-minute incubation, upper methanol/water layer was again transferred to the previous falcon tube and dried using a Labconco freeze drier (Labconco Corporation, MO, USA). The chloroform layer was dried under a stream of nitrogen gas. The dried samples (both aqueous and organic phases) were stored at -80 oC until resuspension for NMR experiments. Lyophilized aqueous phase samples were re-suspended in 50 µL of 50 mM phosphate buffer (pH 7.2) consisting 2 mM of EDTA along with 0.2% NaN3 and 0.5 mM D6-DSS in 100% deuterated environment. |
| Processing Method: | Lyophilization and Homogenization |
| Processing Storage Conditions: | -80℃ |
| Extraction Method: | Modified FOLCH extraction for one set of samples |
| Extract Storage: | -80℃ |
| Sample Resuspension: | In 50 microliter of 50 mM phosphate buffer (pH 7.2) with 2 mM EDTA, 0.5 mM DSS and 0.2% sodium azide for aqueous phase samples. |
| Sample Spiking: | 0.5 mM of DSS for aqueous phase samples |
Analysis:
| MB Sample ID: | SA137209 |
| Analysis ID: | AN002651 |
| Laboratory Name: | Terence lab, UF |
| Analysis Type: | NMR |
| Acquisition Date: | 10/1/2020 |
| Software Version: | Bruker Topspin |
| Operator Name: | Ram Khattri |
| Detector Type: | Bruker |
| Data Format: | fid, 1r |
| Num Factors: | 3 |
| Num Metabolites: | 46 |
| Units: | A.U. |
NMR:
| NMR ID: | NM000188 |
| Analysis ID: | AN002651 |
| Instrument Name: | Bruker Avance Neo 600 MHz/54mm console |
| Instrument Type: | FT-NMR |
| NMR Experiment Type: | 1D-1H |
| Field Frequency Lock: | Deuterium |
| Standard Concentration: | 0.5 mM DSS |
| Spectrometer Frequency: | 600.2328273 MHz |
| NMR Probe: | 1.7 mm TXI CryoProbe |
| NMR Solvent: | Phosphate buffer (pH 7.2) + 2 mM EDTA + 0.5 mM DSS + 0.2% of sodium azide in deuterated environment |
| NMR Tube Size: | 1.7 mm O.D. |
| Shimming Method: | Topshim |
| Pulse Sequence: | noesypr1d |
| Water Suppression: | presat |
| Pulse Width: | 90-degree |
| Receiver Gain: | 101 |
| Offset Frequency: | 2827.31 Hz |
| Chemical Shift Ref Cpd: | DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid) |
| Temperature: | 25 o C |
| Number Of Scans: | 128 scans |
| Dummy Scans: | 8 |
| Acquisition Time: | 4 s |
| Relaxation Delay: | 1 s |
| Spectral Width: | 7142.9 Hz |
| Num Data Points Acquired: | 28571 |
| Real Data Points: | 65536 |
| Line Broadening: | 0.22 Hz |
| Zero Filling: | 65,536 points |
| Apodization: | Exponential |
| Baseline Correction Method: | Spline |
| Chemical Shift Ref Std: | 0 ppm for DSS |
