Principle:
Cellular Nucleotide Profiling Service is an analytical service for mesuring nucleotide level in antimetabolite-treated cells. More than 31 cellular (deoxy-) ribonucleotides (mono-, di-, and triphosphate) are extracted by SPE procedure, separated by ion-paired HPLC system in one-run and quantified.

Assays	Targeted Enzyme(s) / Targeted Nucleotide Synthesis Pathway	Antimetabolite
Cellular Assay for IMPDH inhibitors	Inosine Monophosphate Dehydrogenase (IMPDH)	Ribavirin Mycophenolic Acid (MPA)
Cellular Assay for RNR inhibitors	Ribonucleotide Reductase (RNR)	Hydroxyurea (HU) Gemcitabine
Cellular Assay for Antifolates	• de novo Purine synthesis : GAR transformylase, AICAR transformylase • Thymidylate Synthase (TS)	Methotrexate (MTX)

Validated with antimetabolites (known inhibitors of nucleotide synthesis): MPA, hydroxyurea, methotrexate, ribavirin, mizoribine, leflunomide, gemcitabine. These reference drugs do impact differently on nucleotide metabolism, each showing its own mode of action through a specific nucleotide profile. Any new compound's mode of action is analyzed by establishing its nucleotide profile which is then compared with those obtained with reference drugs.
Download our brochure "Mode of Action Studies by Nucleotide Profiling" 

Aims: Analysis of the Nucleoside and Nucleotide content in biological samples.

The Nucleotide Analysis procedures implemented by NOVOCIB have been specially developed to study the impact of drugs on the cell metabolism of nucleosides and nucleotides by comparison of metabolic profiles of treated vs. untreated cells. However, Nucleotide Analysis can be a major concern for many other purposes and NOVOCIB's know-how can be adapted for the analysis of other materials. Examples of nucleotide analysis are presented in detail on this website: Baby Milk analysis, Fish / Seafood Freshness control... (see the related links above right for further detail).

The technical presentation of NOVOCIB's Customized Nucleotide Analytical services is presented here through the example of application field for which it has been first developed: analysis of drug-treated and untreated cells. In this case, it enables to:
• reveal the metabolic changes due to the drug action, either on the purine or on the pyrimidine pathway,
• identify the metabolites whose levels are modified by the drug treatment,
• trace back to the metabolic step(s) altered by the drug, and to its likely target(s).

Cell culture and treatment: The choice of the cell line and culture conditions has been optimized to get highly reproducible results. Assays are usually done with human hepatoma cell line Huh7. Cells are grown in DMEM supplemented with FCS (5%), glutamine (1mM), sodium pyruvate (1mM) and maintained in exponential phase. Cells are seeded on 10cm-dishes and allowed to adhere overnight. The drug is added next day at the agreed concentration and at a cell confluence of about 50%.
The following metabolites are routinely analyzed*:

ATP	ADP	AMP	Uridine	Hypoxanthine
GTP	GDP	GMP	dGTP	Tryptophane*
UTP	UDP	IMP	dATP
CTP	CDP	UMP	dUTP
Guanine	Uracyl	CMP	dTTP
Cytidine	Guanine	Guanosine	Adenosine

Cultured cell: Nucleosides and Nucleotides Analysis is usually performed by extraction of ≈10⁷ treated cells, per compound and per concentration tested. Control untreated cells are cultured under the same conditions to provide a reference metabolic profile.
Depending on the cell line or the experimental conditions, a 0.5-1.10⁵ cell-extract can be sufficient to analyze the major metabolites (e.g. ATP, ADP�).
Blood cells: Typically, a 200µl-sample of blood is sufficient to analyze the major metabolites in Red Blood Cells (RBC), and a 1ml-sample for Peripheral Blood Mononuclear cells(PBMC).
If needed, for instance to focus on naturally low-level metabolites, larger samples can be prepared, e.g. up to 10⁸ of cultured cells.

Nucleosides & nucleotides separation and analysis: The extraction and separation procedures have been optimized and specially developed by NOVOCIB. After a 48h-treatment, nucleosides and nucleotides are extracted; Nucleosides, nucleotides mono-, di-, and triphosphates, deoxynucleotides triphosphates and bases are separated by ion-pairing HPLC (Agilent 1100) with a Zorbax EclipsePlus C18 column and quantified using an Agilent ChemStation software. The resulting values are normalized by cell number. A mixture of 30 authentic standards (Sigma Aldrich, Roth) is run before and after every set of samples analysis.

Figure 1: Separation by ion-pairing HPLC of a 6.6pmol / 20�l standard mixture of 30 nucleosides, nucleotide mono-, di-, and triphosphates, deoxynucleotide triphosphates and bases V mouse over to enlarge

Figure 2: Quantification of intracellular metabolites in Huh-7 non-treated cells V mouse over to enlarge

Metabolites	pmol / 106 cells *		Published data **
UMP	728.1	+/- 56.8
GMP	34.4	+/- 4.8
IMP	72.7	+/- 21.4	130
NAD	1,825.1	+/- 155.5
UDP-Glu	1,113.9	+/- 87.1
AMP	122.7	+/- 12.9
UDP	1,730.3	+/- 207.1
CTP	2,754.5	+/- 165.4
ADP	4,652.4	+/- 609.8
UTP	2,494.2	+/- 156.1
GTP	1,449.7	+/- 81.4	1,540
ATP	6,561.0	+/- 346.0	6,580
* Mean +/- SD for 5 independent experiments ** J. Balzarini et al. (1993)

Nucleotide profiling: For every nucleotide and nucleoside metabolite, drug-treated / non-treated cells concentration ratios are calculated and graphically reported. The base line indicates the control level.
(^Top)

References (with links to PubMed) J. Balzarini et al. (1993): Eicar (5-ethynyl-1-�-D-ribofuranosylimidazole-4-carboxamide): a novel potent inhibitor of inosinate dehydrogenase activity and guanylate biosynthesis J. Biol. Chem. 268(33), 24591�24598