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Use of the 1-mm micro-probe for metabolic analysis on small volume biological samples.

Serkova NJ, Freund AS, Brown JL, Kominsky DJ - J. Cell. Mol. Med. (2009)

Bottom Line: Clinical application of high-resolution NMR spectroscopy is often limited by extremely low volumes of human specimens.In the present study, the use of the Bruker 1-mm high-resolution TXI micro-probe was evaluated in the elucidation of metabolic profiles for three different clinical applications with limited sample sizes (body fluids, isolated cells and tissue biopsies).In this study, the use of the Bruker 1-mm micro-probe provides a convenient way to measure and quantify endogenous metabolic profiles of samples with a very low volume/weight/cell count.

View Article: PubMed Central - PubMed

Affiliation: Biomedical MRI/MRS Cancer Center Core, University of Colorado Health Sciences Center, Denver, CO 80262, USA.

ABSTRACT
Endogenous metabolites are promising diagnostic end-points in cancer research. Clinical application of high-resolution NMR spectroscopy is often limited by extremely low volumes of human specimens. In the present study, the use of the Bruker 1-mm high-resolution TXI micro-probe was evaluated in the elucidation of metabolic profiles for three different clinical applications with limited sample sizes (body fluids, isolated cells and tissue biopsies). Sample preparation and (1)H-NMR metabolite quantification protocols were optimized for following oncology-oriented applications: (i) to validate the absolute concentrations of citrate and spermine in human expressed prostatic specimens (EPS volumes 5 to 10 microl: prostate cancer application); (ii) to establish the metabolic profile of isolated human lymphocytes (total cell count 4 x 10(6): chronic myelogenous leukaemia application); (iii) to assess the metabolic composition of human head-and-neck cancers from mouse xenografts (biopsy weights 20 to 70 mg: anti-cancer treatment application). In this study, the use of the Bruker 1-mm micro-probe provides a convenient way to measure and quantify endogenous metabolic profiles of samples with a very low volume/weight/cell count.

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Representative NMR spectra from human HNSCC xenograft extracts: (A) one‐dimensional 1H‐NMR on the lipid fraction of HN31 xenograft biopsy (25 mg); (B) one‐dimensional 1H‐NMR on the water‐soluble fraction of HN31 xenograft biopsy (33 mg); and (C) two‐dimensional H,H‐COSY on UMSCC2 xenograft extract (biopsy size 55 mg). Peak assignment: (1) cholesterol; (2) CH3‐total fatty acids; (3) (CH2)n‐total fatty acids; (4) CH2‐total fatty acids; (5) poly‐unsaturated fatty acids (PUFA); (6) phospholipids; (7) triacylglycerol (TAG); (8) mono‐unsaturated fatty acids (with PUFA and TAG); (9) valine, leucine, isoleucine; (10) lactate; (11) alanin; (12) CH3‐acetyl groups; (13) glutamate; (14) succinate; (15) glutamine; (16) glutathione; (17) aspratate; (18) creatine, phosphocreatine; (19) taurine; (20) glycerophosphocholine; (21) phosphocholine; (22) glycine; (23) myo‐inositol; (24) glucose.
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f4: Representative NMR spectra from human HNSCC xenograft extracts: (A) one‐dimensional 1H‐NMR on the lipid fraction of HN31 xenograft biopsy (25 mg); (B) one‐dimensional 1H‐NMR on the water‐soluble fraction of HN31 xenograft biopsy (33 mg); and (C) two‐dimensional H,H‐COSY on UMSCC2 xenograft extract (biopsy size 55 mg). Peak assignment: (1) cholesterol; (2) CH3‐total fatty acids; (3) (CH2)n‐total fatty acids; (4) CH2‐total fatty acids; (5) poly‐unsaturated fatty acids (PUFA); (6) phospholipids; (7) triacylglycerol (TAG); (8) mono‐unsaturated fatty acids (with PUFA and TAG); (9) valine, leucine, isoleucine; (10) lactate; (11) alanin; (12) CH3‐acetyl groups; (13) glutamate; (14) succinate; (15) glutamine; (16) glutathione; (17) aspratate; (18) creatine, phosphocreatine; (19) taurine; (20) glycerophosphocholine; (21) phosphocholine; (22) glycine; (23) myo‐inositol; (24) glucose.

Mentions: Metabolic cancer markers can be assessed not only in tumour cells in vitro, but can be quantified in human biopsies ex vivo. Unfortunately, the tumour mass that can be obtained during clinical biopsy sampling or from the orthotopic or xenograft animal models is usually very limited. In this study, we analysed 20–70 mg human HNSCC biopsies from mouse xenograft models. Metabolite quantification was not possible using a 5‐mm conventional probe due to low signal‐to‐noise ratios (below 3: 1) and low spectral resolution. Using a 1‐mm TXI micro‐probe for methanol/chloroform or acid extracts, high‐quality one‐dimensional (1H‐NMR, Fig. 4A and B) and two‐dimensional (COSY, Fig. 4C) spectra were obtained. Absolute concentrations of endogenous metabolites from two different HNSCC tumour types, calculated from 1H‐NMR spectra (64 scans for water‐soluble and 40 scans for lipid extracts), are presented in Table 3. Important markers, such as phosphocholine (HN31: 0.94 ± 0.21 μmol/g; UMSCC2: 2.15 ± 1.30 μmol/g), glycerophosphocholine (0.24 ± 0.05 and 0.20 ± 0.03 μmol/g), glucose (1.46 ± 0.49 and 0.47 ± 0.18 μmol/g), lactate (5.66 ± 1.02 and 7.51 ± 1.77 μmol/g) and glutathione (0.68 ± 0.25 and 1.30 ± 0.25 μmol/g) were easily detected and quantified using the 1‐mm TXI micro‐probe. Both tumour types are EGFR over‐expressed head‐and‐neck tumours. Since a lot of attention to targeting of down‐stream pathways in cancer cells has been shown in the last 5 years [14], currently we are investigating metabolic consequences of targeting EGFR in HN31 and UMSCC2 tumours using same xenograft model and NMR approaches.


Use of the 1-mm micro-probe for metabolic analysis on small volume biological samples.

Serkova NJ, Freund AS, Brown JL, Kominsky DJ - J. Cell. Mol. Med. (2009)

Representative NMR spectra from human HNSCC xenograft extracts: (A) one‐dimensional 1H‐NMR on the lipid fraction of HN31 xenograft biopsy (25 mg); (B) one‐dimensional 1H‐NMR on the water‐soluble fraction of HN31 xenograft biopsy (33 mg); and (C) two‐dimensional H,H‐COSY on UMSCC2 xenograft extract (biopsy size 55 mg). Peak assignment: (1) cholesterol; (2) CH3‐total fatty acids; (3) (CH2)n‐total fatty acids; (4) CH2‐total fatty acids; (5) poly‐unsaturated fatty acids (PUFA); (6) phospholipids; (7) triacylglycerol (TAG); (8) mono‐unsaturated fatty acids (with PUFA and TAG); (9) valine, leucine, isoleucine; (10) lactate; (11) alanin; (12) CH3‐acetyl groups; (13) glutamate; (14) succinate; (15) glutamine; (16) glutathione; (17) aspratate; (18) creatine, phosphocreatine; (19) taurine; (20) glycerophosphocholine; (21) phosphocholine; (22) glycine; (23) myo‐inositol; (24) glucose.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4940789&req=5

f4: Representative NMR spectra from human HNSCC xenograft extracts: (A) one‐dimensional 1H‐NMR on the lipid fraction of HN31 xenograft biopsy (25 mg); (B) one‐dimensional 1H‐NMR on the water‐soluble fraction of HN31 xenograft biopsy (33 mg); and (C) two‐dimensional H,H‐COSY on UMSCC2 xenograft extract (biopsy size 55 mg). Peak assignment: (1) cholesterol; (2) CH3‐total fatty acids; (3) (CH2)n‐total fatty acids; (4) CH2‐total fatty acids; (5) poly‐unsaturated fatty acids (PUFA); (6) phospholipids; (7) triacylglycerol (TAG); (8) mono‐unsaturated fatty acids (with PUFA and TAG); (9) valine, leucine, isoleucine; (10) lactate; (11) alanin; (12) CH3‐acetyl groups; (13) glutamate; (14) succinate; (15) glutamine; (16) glutathione; (17) aspratate; (18) creatine, phosphocreatine; (19) taurine; (20) glycerophosphocholine; (21) phosphocholine; (22) glycine; (23) myo‐inositol; (24) glucose.
Mentions: Metabolic cancer markers can be assessed not only in tumour cells in vitro, but can be quantified in human biopsies ex vivo. Unfortunately, the tumour mass that can be obtained during clinical biopsy sampling or from the orthotopic or xenograft animal models is usually very limited. In this study, we analysed 20–70 mg human HNSCC biopsies from mouse xenograft models. Metabolite quantification was not possible using a 5‐mm conventional probe due to low signal‐to‐noise ratios (below 3: 1) and low spectral resolution. Using a 1‐mm TXI micro‐probe for methanol/chloroform or acid extracts, high‐quality one‐dimensional (1H‐NMR, Fig. 4A and B) and two‐dimensional (COSY, Fig. 4C) spectra were obtained. Absolute concentrations of endogenous metabolites from two different HNSCC tumour types, calculated from 1H‐NMR spectra (64 scans for water‐soluble and 40 scans for lipid extracts), are presented in Table 3. Important markers, such as phosphocholine (HN31: 0.94 ± 0.21 μmol/g; UMSCC2: 2.15 ± 1.30 μmol/g), glycerophosphocholine (0.24 ± 0.05 and 0.20 ± 0.03 μmol/g), glucose (1.46 ± 0.49 and 0.47 ± 0.18 μmol/g), lactate (5.66 ± 1.02 and 7.51 ± 1.77 μmol/g) and glutathione (0.68 ± 0.25 and 1.30 ± 0.25 μmol/g) were easily detected and quantified using the 1‐mm TXI micro‐probe. Both tumour types are EGFR over‐expressed head‐and‐neck tumours. Since a lot of attention to targeting of down‐stream pathways in cancer cells has been shown in the last 5 years [14], currently we are investigating metabolic consequences of targeting EGFR in HN31 and UMSCC2 tumours using same xenograft model and NMR approaches.

Bottom Line: Clinical application of high-resolution NMR spectroscopy is often limited by extremely low volumes of human specimens.In the present study, the use of the Bruker 1-mm high-resolution TXI micro-probe was evaluated in the elucidation of metabolic profiles for three different clinical applications with limited sample sizes (body fluids, isolated cells and tissue biopsies).In this study, the use of the Bruker 1-mm micro-probe provides a convenient way to measure and quantify endogenous metabolic profiles of samples with a very low volume/weight/cell count.

View Article: PubMed Central - PubMed

Affiliation: Biomedical MRI/MRS Cancer Center Core, University of Colorado Health Sciences Center, Denver, CO 80262, USA.

ABSTRACT
Endogenous metabolites are promising diagnostic end-points in cancer research. Clinical application of high-resolution NMR spectroscopy is often limited by extremely low volumes of human specimens. In the present study, the use of the Bruker 1-mm high-resolution TXI micro-probe was evaluated in the elucidation of metabolic profiles for three different clinical applications with limited sample sizes (body fluids, isolated cells and tissue biopsies). Sample preparation and (1)H-NMR metabolite quantification protocols were optimized for following oncology-oriented applications: (i) to validate the absolute concentrations of citrate and spermine in human expressed prostatic specimens (EPS volumes 5 to 10 microl: prostate cancer application); (ii) to establish the metabolic profile of isolated human lymphocytes (total cell count 4 x 10(6): chronic myelogenous leukaemia application); (iii) to assess the metabolic composition of human head-and-neck cancers from mouse xenografts (biopsy weights 20 to 70 mg: anti-cancer treatment application). In this study, the use of the Bruker 1-mm micro-probe provides a convenient way to measure and quantify endogenous metabolic profiles of samples with a very low volume/weight/cell count.

Show MeSH
Related in: MedlinePlus