A Bloch

Corinna Spangler

DF Ashman

Dimitrios Tsikas

Heike Burhenne

J Scavennec

Kerstin Beste

Kim Tappe

Roland Seifert

RP Newton

Ulrike Voigt

Volkhard Kaever

BMC Pharmacology

English

PubMed

Crossref

POSTER PRESENTATION Open AccessQuantitation of cyclic nucleotides in mammaliancells and in human urine by high-performanceliquid chromatography/mass spectrometryHeike Burhenne1*, Kim Tappe1, Kerstin Beste1, Ulrike Voigt1, Corinna Spangler1, Dimitrios Tsikas2, Volkhard Kaever1,Roland Seifert1From 5th International Conference on cGMP: Generators, Effectors and Therapeutic ImplicationsHalle, Germany. 24-26 June 2011BackgroundThe cyclic nucleotides adenosine 3’,5’-cyclic monopho-sphate (cAMP) and guanosine 3’,5’-cyclic monopho-sphate (cGMP) are well-known second messengers.They play an important role in signal transduction.They control numerous functions ranging from ionchannel opening to regulation of gene expression.In 1963, cAMP and cGMP were detected in rat urine.Indeed, this was the first proof of cGMP in biolocigalsystems [1]. In 1984, Newton et al. demonstrated thepossible presence of cytidine 3’,5’-cyclic monophosphate(cCMP) in various rat tissues [2] and, two years later, ofuridine 3’,5’-cyclic monophosphate (cUMP) [3] by fastatom bombardment. Furthermore, cCMP was suppo-sedly detected in urine of patients with acute leukemia[4,5] by radioimmuno assay. These findings point to abiological function of cCMP and cUMP. However, dueto significant methodological problems, studies of thebiological function of cCMP und cUMP were no longercontinued.Recently, we have developed a method based on high-performance liquid chromatography-coupled mass spec-trometry (HPLC-MS/MS), which allows the simulta-neous determination of all cyclic nucleotides (cNMPs).Using this highly sensitive method we were able todetect and quantify cAMP, cGMP and cCMP as well ascUMP in various mammalian cell lines. We have opti-mized this method for the quantitation of cyclic nucleo-tides in complex biolocigal matrices, like urine andorgans.MethodsNucleotide extraction of cells was performed by treatingcells with a mixture of organic solvents and heating thesamples at 98°C. After centrifugation the supernatantfluid was evaporated under a nitrogen stream and theresidual pellet was resuspended in water. Detection andquantitation of cNMPs was achieved by an analyticalmethod based on HPLC-MS/MS.To avoid serious matrix effects due to the complexurine composition we enzymatically synthesized 13C15N-labeled internal standards for each cyclic nucleotide.Urine sample preparation was achieved by treating urinewith acetonitrile containing those standards. The read-out was performed as described above. The cCMP con-centration was normalized to mmol creatinine. Creati-nine concentration in urine was determined by gaschromatography -MS.Results and discussionWe could show that, in addition to cAMP and cGMP,cCMP and cUMP are present in all studied cell lines(Table 1). Remarkably, the cCMP- and cUMP-contentsin HeLa cells are comparable to the cGMP contents,and in B103 cells the the cCMP- and cUMP-content iseven two-fold and three-fold higher, respectively.Moreover, we analyzed human urine from healthyvolunteers for cyclic nucleotides by HPLC-MS/MS. Ourstudies revealed that besides cAMP and cGMP, cCMP ispresent in human urine.ConclusionOur data suggest that cCMP and cUMP play differentroles in the regulation of the function of cell types origi-* Correspondence: burhenne.heike@mh-hannover.de1Institute of Pharmacology, Hannover Medical School, GermanyFull list of author information is available at the end of the articleBurhenne et al. BMC Pharmacology 2011, 11(Suppl 1):P12http://www.biomedcentral.com/1471-2210/11/S1/P12© 2011 Burhenne et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.nating from various cell lineages and species. Theunequivocal identification of cCMP in human urineopens the door for (patho)physiological studies.Author details1Institute of Pharmacology, Hannover Medical School, Germany. 2Institute ofClinical Pharmacology, Hannover Medical School, Germany.Published: 1 August 2011References1. Ashman DF, Lipton R, Melicow MM, Price TD: Isolation of adenosine 3’,5’-monophosphate and guanosine 3’,5’-monophosphate from rat urine.Biochem Biophys Res Commun 1963, 11:330-334.2. Newton RP, Salih SG, Salvage BJ, Kingston EE: Extraction, purification andidentification of cytidine 3’5’-cyclic monophosphate from rat tissues.Biochem J 1984, 221:665-673.3. Newton RP, Kingston EE, Hakeem NA, Salih SG, Beynon JH, Moyse CD:Extraction, purification, identification and metabolism of 3’5’-cyclic UMP,3’5’- cyclic IMP, 3’5’-cyclic dTMP from rat tissues. Biochem J 1986,236:431-439.4. Bloch A: Isolation of cytidine 3’,5’-monophosphate from mammaliantissues and body fluids and its effects on leukemia L-1210 cell growth inculture. Adv Cyclic Nucleotide Res 1974, 5:331-338.5. Scavennec J, Carcassonne Y, Gastaut JA, Blanc A, Cailla HL: Relationshipbetween the level of cyclic cytidine 3’:5’-monophosphate, cyclicguanosine 3’:5’- monophosphate, and cyclic adenosine 3’:5’-monophosphate in urines and leucocytes and the type of humanleukemias. Cancer Res 1981, 41:3222-3227.doi:10.1186/1471-2210-11-S1-P12Cite this article as: Burhenne et al.: Quantitation of cyclic nucleotides inmammalian cells and in human urine by high-performance liquidchromatography/mass spectrometry. BMC Pharmacology 2011 11(Suppl1):P12.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitTable 1cNMP content (pmol/106 cells)Cell type Lineage cAMP cGMP cCMP cUMPHeLA epithelial cells 2.1 ± 0.5 1.5 ± 0.5 0.3 ± 0.1 0.7 ± 0.3HL-60 myeloid cells 7.0 ± 1.4 0.3 ± 0.1 0.1 ± 0.03 0.3 ± 0.03J774 macrophages 11.0 ± 1.6 1.2 ± 0.1 0.5 ± 0.02 1.2 ± 0.1CHO epithelial cells 33.3 ± 4.0 10.2 ± 1.3 8.0 ± 1.6 6.7 ± 1.8B103 neuroblastoma cells 55.1 ± 7.3 7.5 ± 0.8 14.4 ± 1.5 22.3 ± 2.8COS-7 fibroblasts 82.2 ± 11.3 52.1 ± 3.1 16.3 ± 1.7 25.1 ± 2.2HEK 293 epithelial cells 106.5 ± 11.3 73.3 ± 5.2 32.6 ± 6.2 54.9 ± 11.3Burhenne et al. BMC Pharmacology 2011, 11(Suppl 1):P12http://www.biomedcentral.com/1471-2210/11/S1/P12Page 2 of 2

Quantitation of cyclic nucleotides in mammalian cells and in human urine by high-performance liquid chromatography/mass spectrometry

Springer - Publisher Connector

POSTER PRESENTATION Open Access
Quantitation of cyclic nucleotides in mammalian
cells and in human urine by high-performance
liquid chromatography/mass spectrometry
Heike Burhenne1*, Kim Tappe1, Kerstin Beste1, Ulrike Voigt1, Corinna Spangler1, Dimitrios Tsikas2, Volkhard Kaever1,
Roland Seifert1
From 5th International Conference on cGMP: Generators, Effectors and Therapeutic Implications
Halle, Germany. 24-26 June 2011
Background
The cyclic nucleotides adenosine 3’,5’-cyclic monopho-
sphate (cAMP) and guanosine 3’,5’-cyclic monopho-
sphate (cGMP) are well-known second messengers.
They play an important role in signal transduction.
They control numerous functions ranging from ion
channel opening to regulation of gene expression.
In 1963, cAMP and cGMP were detected in rat urine.
Indeed, this was the first proof of cGMP in biolocigal
systems [1]. In 1984, Newton et al. demonstrated the
possible presence of cytidine 3’,5’-cyclic monophosphate
(cCMP) in various rat tissues [2] and, two years later, of
uridine 3’,5’-cyclic monophosphate (cUMP) [3] by fast
atom bombardment. Furthermore, cCMP was suppo-
sedly detected in urine of patients with acute leukemia
[4,5] by radioimmuno assay. These findings point to a
biological function of cCMP and cUMP. However, due
to significant methodological problems, studies of the
biological function of cCMP und cUMP were no longer
continued.
Recently, we have developed a method based on high-
performance liquid chromatography-coupled mass spec-
trometry (HPLC-MS/MS), which allows the simulta-
neous determination of all cyclic nucleotides (cNMPs).
Using this highly sensitive method we were able to
detect and quantify cAMP, cGMP and cCMP as well as
cUMP in various mammalian cell lines. We have opti-
mized this method for the quantitation of cyclic nucleo-
tides in complex biolocigal matrices, like urine and
organs.
Methods
Nucleotide extraction of cells was performed by treating
cells with a mixture of organic solvents and heating the
samples at 98°C. After centrifugation the supernatant
fluid was evaporated under a nitrogen stream and the
residual pellet was resuspended in water. Detection and
quantitation of cNMPs was achieved by an analytical
method based on HPLC-MS/MS.
To avoid serious matrix effects due to the complex
urine composition we enzymatically synthesized 13C15N-
labeled internal standards for each cyclic nucleotide.
Urine sample preparation was achieved by treating urine
with acetonitrile containing those standards. The read-
out was performed as described above. The cCMP con-
centration was normalized to mmol creatinine. Creati-
nine concentration in urine was determined by gas
chromatography -MS.
Results and discussion
We could show that, in addition to cAMP and cGMP,
cCMP and cUMP are present in all studied cell lines
(Table 1). Remarkably, the cCMP- and cUMP-contents
in HeLa cells are comparable to the cGMP contents,
and in B103 cells the the cCMP- and cUMP-content is
even two-fold and three-fold higher, respectively.
Moreover, we analyzed human urine from healthy
volunteers for cyclic nucleotides by HPLC-MS/MS. Our
studies revealed that besides cAMP and cGMP, cCMP is
present in human urine.
Conclusion
Our data suggest that cCMP and cUMP play different
roles in the regulation of the function of cell types origi-* Correspondence: burhenne.heike@mh-hannover.de
1Institute of Pharmacology, Hannover Medical School, Germany
Full list of author information is available at the end of the article
Burhenne et al. BMC Pharmacology 2011, 11(Suppl 1):P12
http://www.biomedcentral.com/1471-2210/11/S1/P12
© 2011 Burhenne et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
nating from various cell lineages and species. The
unequivocal identification of cCMP in human urine
opens the door for (patho)physiological studies.
Author details
1Institute of Pharmacology, Hannover Medical School, Germany. 2Institute of
Clinical Pharmacology, Hannover Medical School, Germany.
Published: 1 August 2011
References
1. Ashman DF, Lipton R, Melicow MM, Price TD: Isolation of adenosine 3’,5’-
monophosphate and guanosine 3’,5’-monophosphate from rat urine.
Biochem Biophys Res Commun 1963, 11:330-334.
2. Newton RP, Salih SG, Salvage BJ, Kingston EE: Extraction, purification and
identification of cytidine 3’5’-cyclic monophosphate from rat tissues.
Biochem J 1984, 221:665-673.
3. Newton RP, Kingston EE, Hakeem NA, Salih SG, Beynon JH, Moyse CD:
Extraction, purification, identification and metabolism of 3’5’-cyclic UMP,
3’5’- cyclic IMP, 3’5’-cyclic dTMP from rat tissues. Biochem J 1986,
236:431-439.
4. Bloch A: Isolation of cytidine 3’,5’-monophosphate from mammalian
tissues and body fluids and its effects on leukemia L-1210 cell growth in
culture. Adv Cyclic Nucleotide Res 1974, 5:331-338.
5. Scavennec J, Carcassonne Y, Gastaut JA, Blanc A, Cailla HL: Relationship
between the level of cyclic cytidine 3’:5’-monophosphate, cyclic
guanosine 3’:5’- monophosphate, and cyclic adenosine 3’:5’-
monophosphate in urines and leucocytes and the type of human
leukemias. Cancer Res 1981, 41:3222-3227.
doi:10.1186/1471-2210-11-S1-P12
Cite this article as: Burhenne et al.: Quantitation of cyclic nucleotides in
mammalian cells and in human urine by high-performance liquid
chromatography/mass spectrometry. BMC Pharmacology 2011 11(Suppl
1):P12.
Submit your next manuscript to BioMed Central
and take full advantage of: 
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at 
www.biomedcentral.com/submit
Table 1
cNMP content (pmol/106 cells)
Cell type Lineage cAMP cGMP cCMP cUMP
HeLA epithelial cells 2.1 ± 0.5 1.5 ± 0.5 0.3 ± 0.1 0.7 ± 0.3
HL-60 myeloid cells 7.0 ± 1.4 0.3 ± 0.1 0.1 ± 0.03 0.3 ± 0.03
J774 macrophages 11.0 ± 1.6 1.2 ± 0.1 0.5 ± 0.02 1.2 ± 0.1
CHO epithelial cells 33.3 ± 4.0 10.2 ± 1.3 8.0 ± 1.6 6.7 ± 1.8
B103 neuroblastoma cells 55.1 ± 7.3 7.5 ± 0.8 14.4 ± 1.5 22.3 ± 2.8
COS-7 fibroblasts 82.2 ± 11.3 52.1 ± 3.1 16.3 ± 1.7 25.1 ± 2.2
HEK 293 epithelial cells 106.5 ± 11.3 73.3 ± 5.2 32.6 ± 6.2 54.9 ± 11.3
Burhenne et al. BMC Pharmacology 2011, 11(Suppl 1):P12
http://www.biomedcentral.com/1471-2210/11/S1/P12
Page 2 of 2


A: Isolation of cytidine 3’,5’-monophosphate from mammalian tissues and body fluids and its effects on leukemia L-1210 cell growth in culture. Adv Cyclic Nucleotide Res

CD: Extraction, purification, identification and metabolism of 3’5’-cyclic UMP, 3’5’- cyclic IMP, 3’5’-cyclic dTMP from rat tissues.

Extraction, purification and identification of cytidine 3’5’-cyclic monophosphate from rat tissues.

HL: Relationship between the level of cyclic cytidine 3’:5’-monophosphate, cyclic guanosine 3’:5’- monophosphate, and cyclic adenosine 3’:5’-monophosphate in urines and leucocytes and the type of human leukemias. Cancer Res

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Quantitation of cyclic nucleotides in mammalian cells and in human urine by high-performance liquid chromatography/mass spectrometry

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