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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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Metabolic profile of isolated human lymphocytes: (A) representative 1H‐NMR spectrum from 4 × 106 cells after PCA extraction; (B) comparison of absolute metabolite concentrations (μmol/g) in human leucocytes versus human chronic myelogenous leukaemia CMT‐L1 and K562 cell lines. The data on CML cells are adopted from our previous study [10] using a conventional Bruker 5‐mm TXI probe on 5 = 108 cells per extract. Abbreviations: Glc, glucose; Glu, glutamate; Lac, lactate; PCho, phosphocholine; Val, Leu, Ile, valine+leucine+isoleucine.
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f3: Metabolic profile of isolated human lymphocytes: (A) representative 1H‐NMR spectrum from 4 × 106 cells after PCA extraction; (B) comparison of absolute metabolite concentrations (μmol/g) in human leucocytes versus human chronic myelogenous leukaemia CMT‐L1 and K562 cell lines. The data on CML cells are adopted from our previous study [10] using a conventional Bruker 5‐mm TXI probe on 5 = 108 cells per extract. Abbreviations: Glc, glucose; Glu, glutamate; Lac, lactate; PCho, phosphocholine; Val, Leu, Ile, valine+leucine+isoleucine.

Mentions: The previous data on human cell lines were obtained using high cell counts of 5 × 108 cells and higher [10]. While cell numbers are not limiting for clonal cells, this amount of cell material is difficult to obtain from the peripheral blood of human subjects. The total lymphocyte count in ml blood is 1 × 106 cells, with the recovery rate after isolation being below 50%. Using a 1‐mm TXI micro‐probe, we analysed cell extracts from 4 × 106 isolated lymphocytes, which we obtained from 20 ml of whole peripheral blood. Even though the cell count was 100‐fold lower than our previous studies on clonal cell lines [10], we were able to obtain good quality 1H‐NMR spectra with total acquisitions of 128 (Fig. 3A). Major cellular metabolites, including glucose, lactate (with 13C‐satelite peaks), cholines, amino acids were detectable in lymphocytes extracts. Compared to our previous metabolic data on human CML cells K‐562 and CML‐T1 (Fig. 3B), isolated human lymphocytes from healthy subjects had higher intracellular levels of glucose (0.78 ± 0.32 μmol/g versus 0.032 ± 0.01 and 0.025 ± 0.01, P < 0.0001) and significantly decreased lactate concentrations, including de novo formation of 13C‐lactate (0.25 ± 0.07 μmol/g versus 0.61 ± 0.11 and 1.08 ± 0.22, P < 0.01). Interestingly, the total glutamate concentrations were higher in lymphocytes compared to clonal cells (2.43 ± 0.35 μmol/g versus 1.78 ± 0.22 and 1.25 ± 0.12, P < 0.02). Finally, phosphocholine concentrations were significantly lower in lymphocytes versus transformed cells (0.49 ± 0.12 μmol/g versus 1.92 ± 0.22 and 2.12 ± 0.17, P < 0.001) with no differences in glycerophosphocholine concentrations among the three cell types.


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)

Metabolic profile of isolated human lymphocytes: (A) representative 1H‐NMR spectrum from 4 × 106 cells after PCA extraction; (B) comparison of absolute metabolite concentrations (μmol/g) in human leucocytes versus human chronic myelogenous leukaemia CMT‐L1 and K562 cell lines. The data on CML cells are adopted from our previous study [10] using a conventional Bruker 5‐mm TXI probe on 5 = 108 cells per extract. Abbreviations: Glc, glucose; Glu, glutamate; Lac, lactate; PCho, phosphocholine; Val, Leu, Ile, valine+leucine+isoleucine.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4940789&req=5

f3: Metabolic profile of isolated human lymphocytes: (A) representative 1H‐NMR spectrum from 4 × 106 cells after PCA extraction; (B) comparison of absolute metabolite concentrations (μmol/g) in human leucocytes versus human chronic myelogenous leukaemia CMT‐L1 and K562 cell lines. The data on CML cells are adopted from our previous study [10] using a conventional Bruker 5‐mm TXI probe on 5 = 108 cells per extract. Abbreviations: Glc, glucose; Glu, glutamate; Lac, lactate; PCho, phosphocholine; Val, Leu, Ile, valine+leucine+isoleucine.
Mentions: The previous data on human cell lines were obtained using high cell counts of 5 × 108 cells and higher [10]. While cell numbers are not limiting for clonal cells, this amount of cell material is difficult to obtain from the peripheral blood of human subjects. The total lymphocyte count in ml blood is 1 × 106 cells, with the recovery rate after isolation being below 50%. Using a 1‐mm TXI micro‐probe, we analysed cell extracts from 4 × 106 isolated lymphocytes, which we obtained from 20 ml of whole peripheral blood. Even though the cell count was 100‐fold lower than our previous studies on clonal cell lines [10], we were able to obtain good quality 1H‐NMR spectra with total acquisitions of 128 (Fig. 3A). Major cellular metabolites, including glucose, lactate (with 13C‐satelite peaks), cholines, amino acids were detectable in lymphocytes extracts. Compared to our previous metabolic data on human CML cells K‐562 and CML‐T1 (Fig. 3B), isolated human lymphocytes from healthy subjects had higher intracellular levels of glucose (0.78 ± 0.32 μmol/g versus 0.032 ± 0.01 and 0.025 ± 0.01, P < 0.0001) and significantly decreased lactate concentrations, including de novo formation of 13C‐lactate (0.25 ± 0.07 μmol/g versus 0.61 ± 0.11 and 1.08 ± 0.22, P < 0.01). Interestingly, the total glutamate concentrations were higher in lymphocytes compared to clonal cells (2.43 ± 0.35 μmol/g versus 1.78 ± 0.22 and 1.25 ± 0.12, P < 0.02). Finally, phosphocholine concentrations were significantly lower in lymphocytes versus transformed cells (0.49 ± 0.12 μmol/g versus 1.92 ± 0.22 and 2.12 ± 0.17, P < 0.001) with no differences in glycerophosphocholine concentrations among the three cell types.

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