1) The document discusses using NMR spectroscopy to analyze the metabolic profiles of clam mantle tissue and cultured lung epithelial cells. Spectra of extracts from different regions of clam mantle and control vs. cigarette smoke-exposed cells were obtained. 2) Glyceraldehyde and glucose levels were able to be quantified and showed differences between tissue and cell types. Glyceraldehyde levels suggested cigarette smoke exposure induced distress in lung cells. 3) Further identification of metabolites was needed using additional database comparisons to fully characterize the metabolic profiles obtained via NMR spectroscopy. Improvements were also needed in cell and tissue collection methods to better preserve metabolite levels.

1. IndianaCTSI ACCELERATING CLINICAL AND
TRANSLATIONAL RESEARCH
Indiana Clinical and Translational Sciences Institute
www.indianactsi.org
Metabolic Studies of Cultured Cells by NMRMetabolic Studies of Cultured Cells by NMR
Jessany Maldonado1
, Amanda Tallman2
, and Bruce D. Ray2
1
Warren Central High School, 2
Department of Physics, Indiana University Purdue University Indianapolis
References
[1] Rosenberg, G.D. et al., Amer. Malac. Bul, 16: 251-261 (2001)
[2] Toru, M. and Aprison, M. H., J. Neurochem. 13: 1533-1544 (1966)
[3] Robinette, S.L. et al., Anal. Chem. 80: 3606-3611 (2008)
[4] Brüschweiler, R. and Zhang, F., J. Chem. Phys. 120: 5253-5260 (2004).
[5] Brüschweiler, R., J. Chem. Phys. 121: 409-414 (2004).
[6] Trbovic, N., et al., R., J. Magn. Reson. 171: 277-283 (2004).
[7] Zhang, F. and Brüschweiler, R., Angew. Chemie 46: 2639-2642 (2007).
[8] Jansen, J.F.A. et al., Magn. Reson. Med. 56: 666-667 (2006).
[9] Perez-Diaz, J. et al., Am. J. Physiol. Endocrinol. Metab. 232: E394-400 (1997).
Abstract
Previously, 31
P NMR has been used to study tissue metabolic activity. Recent advances in spectral
correlation software have allowed some researchers to measure metabolite concentrations in urine,
bile, cerebrospinal fluid, and plasma by 1
H NMR. With appropriate immobilization, this ought to be
extendable to tissues and cells. As a preliminary test of the limits of this, 1
H NMR spectra of formic
acid/ acetone extracts of clam mantle segments and of cultured lung epithelial cells were acquired,
and the metabolites by the COLMAR query web site at the National High Magnetic Field Laboratory.
IntroductionThe bivalve’s mantle layer is responsible for secreting the shell. Calcium, magnesium, and sulfur are
absorbed from the water by the bivalve’s mantle and secreted to create the calcite of the shell.
Different parts of the mantle that produces the shell have different metabolic rates and different
membrane lipid compositions. Sections A and D of the mantle represent high curvature shell
production and low curvature shell production, respectively. The metabolic rate in section A is faster
than that in D.
Since 31
P NMR has already shown the differences in phosphorylated metabolite levels between clam
mantle segments1
, we looked at metabolic profiles in different regions of the clam mantle by 1
H NMR
to attempt to identify and quantitate metabolic differences between regions. 1
H NMR of formic acid /
acetone tissue extracts from dissected samples of both A and D gave spectra of the whole mixture of
extracted metabolites. Identification of the individual metabolites was attempted by computerized
analysis of each mixture spectrum, including comparison against spectra of known metabolites.
Lung epithelial cells exposed to the components of cigarette smoke tend to die. Also, in vivo, exposure
to cigarette smoke tends to result in emphysema. As with clam mantle segments, 1
H NMR of formic
acid / acetone extracts of cultured lung cells gave spectra of the whole mixture of extracted
metabolites. Identification of the individual metabolites was attempted by computerized analysis of
each mixture spectrum, including comparison against spectra of known metabolites.
Methods
Tissue and cultured cell preparation: All formic Acid / acetone extractions followed the method of Toru
and Aprison2
. Tissue was subjected to freeze-fracture under liquid nitrogen prior to extraction.
Cultured cells did not require this treatment.
Plated lung epithelial cells were washed with phosphate buffered saline (PBS), scraped from the plate
into suspension in PBS and harvested by centrifugation before flash freezing in liquid nitrogen.
Membrane grown lung epithelial cells were removed from media and immediately flash frozen in liquid
nitrogen. The membrane was then cut away from its support for extraction.
NMR of extracts: Extracts were resuspended in 700 μL D2O with 25 mM pH 7.4 sodium phosphate
buffer and 14.3 μM DSS (reference). 1
H 1 D and tocsy NMR spectra of each extract were acquired.
Tocsy spectra were submitted to the COLMAR query web site at the National High Magnetic Field
Laboratory3-7
.
Results &
Conclusions
Spectra showing comparisons of membrane grown, and
harvested plated control and cigarette smoke exposed B2b
bronchial epithelial cells, and of clam mantle segments A and
D are shown. The carbonyl proton of glyceraldehyde, and H-
1 and H-2 of glucose were separated enough for easy
quantitation in the 1 D spectra. Only with membrane grown
cells were clearly defined glucose peaks. Integration gave a
glucose concentration of 2 µmoles/g wet weight. However,
this includes an unknown contribution from residual media
which contained 25 mM glucose. On the other hand,
glyceraldehyde should be intracellular and was present at 10
µmoles/g in membrane grown cells, 2.4 µmoles/g in plated
control cells, and 1.8 µmoles/g in plated cigarette smoke
exposed cells. This suggests that the harvesting procedure
depleted intracellular metabolites, expected given the
published high rate of glycolysis9
. Nevertheless, some
indication of the distress smoke exposure induced in these
cultured lung cells may be preserved in the glyceraldehyde
levels. However, lactate levels seemed to be identical in both
smoke exposed and control cells albeit the lactate level was
1% of that in the membrane grown cells. Lactate was not part
of the media and therefore the difference in lactate level
represents effects of metabolism during plated cell
harvesting. Because of resonance overlap in the 1 D NMR
spectra, quantitation of lactate was not done.
For the clam segments glyceraldehyde levels were 6.9
µmoles/g for segment A and 8.8 µmoles/g for segment D.
The lack of glucose signals given prior observation of
monophosphorylated species in both segments and the
presence of peaks attributed to β-hydroxy butyrate indicate
that the commercial clams had not been fed in a while.
Further comparisons against other metabolite NMR spectra
databases are needed to complete identification of
metabolites in both the lung cells and the clam mantle
segments. Improvements in cell harvesting and in tissue
collection are obviously needed to preserve metabolite
differences.
Acknowledgements
Indiana Project SEED
Indiana Clinical and Translational Sciences Institute
Indiana University-Purdue University Indianapolis School of Science
Amanda Tallman
Gary D. Rosenberg
2
Toru, M. and Aprison, M. H. (1966) Brain Acetylcholine Studies: A New Extraction Procedure, J. Neurochem. 13, 1533-1544.
3
Robinette, S. L., Zhang, F., Bruschweiler-Li, L., and Bruschweiler, R. (2008) Web Server Based Complex Mixture Analysis by NMR, Anal. Chem. 80, 3606-3611.
B2b Bronchial Epithelial Cells
Exposed to 2.5% cigarette
smoke extract for 16 h.
Control cells exposed to air
Comparison of membrane grown (79 mg), and harvested, plated
cigarette smoke exposed (145.1 mg), and plated control (153.6 mg).
Reference area is equivalent to 90 nanomoles 1
H.
Clam mantle segments A (36.7 mg) and D (71.5 mg)
2D spectra of membrane grown lung B2b cells.
Cross peaks attributed to lactate circled. Cross peaks in
triangles attributed to choline, present in the media at
0.0286 mM. Complex enclosed by the rectangle may
represent myoinositol8
, present in the media at 0.04
mM. Glyceraldehyde autocorrelation peak on the
diagonal is enclosed with a hexagon. Despite the media
containing 4 mM L-Glutamine, identifiable glutamate
and glutamine cross peaks were not found.
Clam Dissection
White circles in the micrograph of the cigarette smoke exposed cells are
from dead cells that detached from the plate. Distorted and swollen cells
can also be seen in the micrograph of the cigarette smoke exposed cells.
500 MHz NMR
Spectrometer used