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MB Sample ID: SA137196
| Local Sample ID: | CLTI_Amputation4a |
| 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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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 |
