Cationic lipids involved in gene transfer mobilize intracellular calcium

Abstract

Cationic lipids are efficient tools to introduce nucleic acids and proteins into cells. Elucidation of the mechanism and cellular pathways associated with such transport has been relatively tedious, even though significant progress has been made in the characterization of the intracellular trafficking of lipid/DNA complexes. Surprisingly little is known about the effects of these delivery vectors on cell functioning. In this report, we show that both cationic lipids and cationic lipid/DNA complexes mobilize the intracellular calcium. Removal of extracellular calcium did not significantly abolish this effect and preincubating cells with thapsigargin led to a decrease in [Ca²⁺]_i, indicating that calcium was released mainly from internal calcium stores sensitive to thapsigargin. Pretreatment of the cells with the phospholipase C inhibitor U73122, blocked the [Ca²⁺]_i rise, suggesting an inositol dependent mechanism.

• Cationic lipids
• lipid/DNA complexes
• intracellular Ca²⁺
• phospholipase C
• gene transfer

Introduction

Cationic liposomes are used to introduce molecules such as nucleic acids [1–6], including messenger RNAs [7], [8], synthetic oligonucleotides [9] and non-nucleic acid compounds such as proteins and peptides [10], [11] into cells in vitro and in vivo. Significant progress has been made regarding the description of the intracellular trafficking of lipid/DNA complexes over the past decade, even though the mechanism of transport of DNA into the nucleus is still a matter of debate [6]. However how the lipid/DNA complex affects cell functions is still largely unknown. In the present work, we demonstrate that cationic lipid/DNA complexes (DiC14-amidine/DNA and DOTAP/DNA) and different types of cationic liposomes (DiC14-amidine, DOTAP, DDAB, and Lipofectamine) cause immediate increase in intracellular free calcium which is released primarily from thapsigargin sensitive calcium stores. This means that vectors, like cationic lipids, that transport genetic material into cells may generate unpredictable cellular perturbations even before any expression of the corresponding genes.

Materials and methods

Chemicals

Acetoxymethyl ester (AM) of Fluo-3 and pluronic acid F-127 were obtained from Molecular Probes (Leiden, The Netherlands); they were dissolved in dimethylsulfoxide (DMSO) as 0.57 mg/ml and 10 mg/ml stock solutions, respectively. DMSO was from LAB-SCAN (Dublin, Ireland). DiC14-amidine and diisopropylamidine were synthesized as described in [2] and [12] respectively. 1.2-dioleoyl-3-trimethylammonium-propane (DOTAP) was from Avanti Polar Lipids (USA), Lipofectamine is a 3:1 (w/w) liposome formulation of the polycationic lipid 2,3-dioleyloxy-N-[2(sperminecarboxamido) ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA) and the neutral lipid dioleoyl phosphatidylethanolamine (DOPE) purchased from Invitrogen (Belgium). Acrylonitrile, 2-chloro-2-methylpropane, ferric chloride, tetradecylamine, dichloromethane, and chloroform were from Fluka (Belgium). Hexane was from Merck (Germany). All other chemicals: dimethyldioctadecyl-ammoniumbromide (DDAB), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), adenine dinucleotide phosphate (ADP), digitonin, ethylene glycol-bis (2-aminoethylether)-N,N,N,N-tetra acetic acid (EGTA), spermine, 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), U73122, U73343, sulfinpyrazone and thapsigargin were from Sigma-Aldrich (Belgium). Dulbecco's Phosphate Buffered Saline (D-PBS) containing (in mM): 0.9 CaCl₂.2H₂O, 2.7 KCl, 1.5 KH₂PO₄, 0.5 MgCl₂.6H₂O, 137 NaCl, 4.5 Na₂HPO₄.7H₂O, supplemented with 1 mg/mL glucose and adjusted to pH 7.3, was from Gibco Invitrogen (Belgium).

Cell cultures

The human erythromyeloid K562 cells were obtained from the European Collection of Cell Culture (Salisbury, UK) and grown in RPMI 1640 medium containing L-glutamine and 25 mM HEPES, supplemented with 10% FBS, 0.2% (v/v) Fungizone and 1% (v/v) penicillin streptomycin. In contrast to long-term cultured K562, this cell batch was sensitive to the purinergic ligand ADP. Cell cultures were maintained in a humidified atmosphere of 5% CO₂. Culture media, supplements and sera were purchased from Gibco Invitrogen (Belgium).

Liposomes preparation

DiC14-amidine, DDAB, DOTAP, DMPC and DMPC/DMPG liposomes were prepared at 5 mg/ml in HBS-20 buffer (20 mM HEPES, 150 mM NaCl, pH 7.3). Lipids were dissolved in chloroform and dried under a nitrogen stream. The lipid film was hydrated above the transition temperature with HBS-20 and vortexed. The final suspension was stored at 4°C under nitrogen atmosphere.

Purification of plasmid DNA

The pCMV-luc plasmid (∼5.4 Kb) was constructed by ligating an EcoRI-NheI fragment of the pSP-luc-plasmid (Promega Benelux, The Netherlands) into the pCI expression vector (Clontech, Palo Alto, CA, USA), which was cut with EcoRI and NheI. The resulting plasmid contained the luciferase gene under the control of the immediate-early CMV promoter-enhancer; it was purified using a Qiagen Kit (Giga Plasmid Prep., Qiagen, The Netherlands) according to manufacturer's instructions and resuspended in sterile water. The purified plasmid was further treated with a polymyxin resin (Affi-Prep, Bio-Rad, CA, USA) to remove endotoxins. Briefly, 200 µl of the Affi-Prep resin was first rendered pyrogen-free by treatment with 600 µl of 0.5 N NaOH and then washed twice with 1.5 ml of pyrogen-free water. The resin was then pelleted by centrifugation and resuspended in 400 µl of the DNA solution, and mixed overnight in a rotary mixer at 4°C. The DNA was separated from the resin by centrifugation.

Preparation of cationic lipid/DNA complexes

Cationic lipid/DNA complexes were prepared by mixing the appropriate amount of plasmid DNA with the cationic lipid to obtain the desired cationic/anionic charge ratio. The desired amount of plasmid DNA in 10 µl HBS-20 was added to 20 µl of cationic liposomes at 1 mg/ml in HBS-20. Complex formation was obtained by incubation for 15 min at room temperature. The resulting complexes were diluted to a total volume of 50 µl with HBS-20, and added to 1 ml of the cell suspension at 37°C.

Intracellular calcium measurement

K562 cells (1×10⁶) were washed three times with D-PBS and were incubated for 30 min at 37°C in the dark in D-PBS at 1 mg/ml glucose containing 7.5 µM Fluo-3-AM (a Ca^2 + sensitive fluorescent dye) with 0.01% (w/v) surfactant pluronic acid F-127 to facilitate cell loading and 250 µM sulfinpyrazone to prevent extrusion of the indicator out of cells after the hydrolysis of the AM ester groups. After loading, the cells were rinsed three times with D-PBS at 1 mg/ml glucose to remove the extracellular Fluo-3-AM and suspended in 1 ml of the same buffer and transferred to a fluorescence cuvette. The fluorescence intensity of cells was recorded on a SLM Aminco 8000 spectrofluoremeter (SLM Instruments Inc., Urbana, IL) with excitation and emission slits set to 4 nm, equipped with 450 watt Xenon lamp, temperature control and magnetic stirrer. The cells were continuously stirred throughout the experiment. Excitation wavelength was 506 nm and emission fluorescence was continuously measured at the Fluo-3-Ca^2 + complex maximum emission (526 nm). The signal was recorded every two seconds. The baseline signal was recorded for 100 sec before adding any agent. Addition of 0.1% (v/v) of buffer or DMSO did not affect the baseline signal. Estimation of intracellular concentration of free Ca^2 + was calculated using the equation described by Tsien et al. [13]:Where F is the intensity of the measured fluorescence, F_max and F_min are maximum and minimum fluorescence respectively and K_d is dissociation constant of Fluo-3 for Ca^2 +. F_max was obtained by adding 30 µM of digitonin in the presence of 1 mM CaCl₂ (8 mM stock solution, pH 7.3). F_min was obtained after addition of an excess of EGTA (30 mM) (1 M stock solution, pH 8). A K_d value of 400 nM [13] was used to calculate the free Ca^2 + concentration.

Results

Intracellular Ca^2 + response to cationic liposomes and cationic lipid/DNA complexes

Addition of cationic liposomes (DiC14-amidine, DOTAP, DDAB, and Lipofectamine) to K562 cells produces a fast rise in intracellular calcium concentration (Figure 1A). The calcium increase reached a plateau that lasted for at least 1 h (Figure 1B, insert). The end point of the calcium increase was ∼400 nM from a basal level of ∼100 nM. As shown in Figure 1B, calcium response to diC14-amidine was dose-dependent. Below 1 µM, diC14-amidine had no detectable effect (data not shown).

Figure 1.  (A) Effect of different types of cationic liposomes on [Ca^2 +]_i and comparison of [Ca^2 +]_i rise induced by diC14-amidine and ADP in K562 cells. Cells (1×10⁶ cells/ml) resuspended in D-PBS medium at 1 mg/ml glucose in the presence of 1 mM Ca^2 + were loaded with Fluo-3-AM for 30 min at 37°C, and then exposed (arrow) to the indicated concentrations of cationic liposome (37 µM). The insert graph shows [Ca^2 +]_i changes induced by diC14-amidine (37 µM) and ADP (20 µM). Intracellular Ca^2 + concentration changes were measured at 37°C using fluorescence spectroscopy as described in Materials and Methods. The same experiment was repeated three times and showed identical results. (B) Dose-dependent intracellular Ca^2 + increase induced by diC14-amidine in K562 cells. Cells (1×10⁶ cells/ml) resuspended in D-PBS medium at 1 mg/ml glucose in the presence of 1 mM Ca^2 + were loaded with Fluo-3-AM for 30 min at 37°C and then exposed (arrow) to the indicated concentrations of diC14-amidine. The insert graph shows [Ca^2 +]_i changes induced by diC14-amidine (37 µM). Intracellular Ca^2 + concentration changes were measured at 37°C using fluorescence spectroscopy as described in Materials and Methods. The same experiment was repeated three times and showed identical results.

This calcium mobilization was compared with the one induced by a natural ligand-induced calcium signalling event, namely adenosine diphosphate (ADP) binding to purinergic receptors of the cell membrane (Figure 1A, insert). The overall shape of the two processes was quite different. Binding to receptors showing the characteristic profile of the transient signal was observed with ADP [14], whereas the cationic lipid induced calcium signal did not decay even after 400 sec.

Since cationic liposomes are mostly utilized in transfection experiments, as lipid/DNA complexes (lipoplexes) to carry DNA into cells, we evaluated the ability of lipoplexes diC14-amidine/DNA and DOTAP/DNA to mobilize intracellular calcium. At cationic lipid/DNA ratios (2:1, 4:1, and 8:1) commonly used in transfection experiments a significant increase of intracellular Ca^2 + was detectable (Figures 2A and 2B).

Figure 2.  Effect of cationic lipid/DNA complexes charged positively on [Ca^2 +]_i increase in K562 cells. Cells (1×10⁶ cells/ml) resuspended in D-PBS medium at 1 mg/ml glucose in the presence of 1 mM Ca^2 + were loaded with Fluo-3-AM for 30 min at 37°C, and then exposed (arrow) to diC14-amidine/DNA (weight ratio) (Figure 2A) or DOTAP/DNA (weight ratio) (Figure 2B). Complexes were formed as indicated in Materials and Methods. Final concentration of DNA was 5 µg/ml. Intracellular Ca^2 + concentration changes were measured at 37°C using fluorescence spectroscopy as described in Materials and Methods. The same experiments were repeated three times and showed identical results.

Calcium is released from intracellular Ca^2 + stores

Intracellular calcium concentration increase can result from calcium efflux from intracellular stores or/and influx of extracellular calcium. Figures 3A and 3B compare diC14-amidine/DNA complex and diC14-amidine liposomes induced increase in intracellular calcium mobilization in normal medium (1 mM extracellular Ca^2 +) and in Ca^2 +-free medium (no Ca^2 + plus 20 µM EGTA). Intracellular Ca^2 + concentration was reduced by about 30% in the absence of extracellular calcium as compared with the control (1 mM extracellular Ca^2 +), suggesting a significant contribution from intracellular calcium stores.

Figure 3.  Contribution of intra and extracellular Ca^2 + to [Ca^2 +]_i induced by diC14-amidine/DNA complexes and diC14-amidine liposomes in K562 cells. Cells (1×10⁶ cells/ml) resuspended in D-PBS medium at 1 mg/ml glucose in the presence of 1 mM Ca^2 + were loaded with Fluo-3-AM for 30 min at 37°C. After loading, cells were rinsed with D-PBS without Ca^2 + and suspended in 1 ml of the same medium. At time = 100 s, calcium (1 mM final) or EGTA (20 µM final) was added. At time = 200 s, diC14-amidine/DNA complex at a weight ratio 2:1 (5 µg DNA) (Figure 3A) or free diC14-amidine liposomes (37 µM) (Figure 3B) was added and [Ca^2 +]_i changes were measured at 37°C using fluorescence spectroscopy as described in Materials and Methods. The same experiments were repeated three times and showed identical results.

Involvement of internal Ca^2 + stores sensitive to thapsigargin (TG) in intracellular Ca^2 + response

The largest store of calcium is found in the endoplasmic (or sarcoplasmic) reticulum [15], and is transported from the cytoplasm into these stores by an ion-motive Ca^2 +-ATPase. To confirm the involvement of intracellular calcium stores in cationic lipid/DNA complexes and cationic liposomes-induced calcium signalling, we depleted intracellular Ca^2 + stores by addition of the sesquiterpene lactone thapsigargin (TG) which is known to deplete intracellular calcium stores by inhibition of endoplasmic reticulum Ca^2 +-ATPase [16]. As shown in Figure 4, initial addition of TG leads to an increase in intracellular Ca^2 + caused by the discharge of intracellular calcium stores sensitive to TG. Subsequent addition of diC14-amidine produced an additional intracellular Ca^2 + increase (+TG) which was weaker and slower than the one observed in TG untreated cells (−TG).

Figure 4.  Effect of diC14-amidine on [Ca^2 +]_i in TG treated K562 cells. Cells (1×10⁶ cells/ml) resuspended in D-PBS medium at 1 mg/ml glucose in the presence of 1 mM Ca^2 + were loaded with Fluo-3-AM for 30 min at 37°C, and then incubated in Ca^2 +-free medium (no Ca^2 + plus 20 µM EGTA). At time = 200 s, 5 µl of 65 µg/ml TG in DMSO (grey curve) or the same volume of DMSO (control, black curve) was added. At t = 500 s, diC14-amidine (37 µM) was added. Intracellular Ca^2 + concentration changes were measured at 37°C using fluorescence spectroscopy as described in Materials and Methods. The same experiment was repeated three times and showed identical results.

Analogous results were obtained with diC14-amidine/DNA complex (data not shown). This result suggests that intracellular calcium increase induced by cationic lipid/DNA complexes and cationic liposomes relies at least in part, on functional internal calcium stores sensitive to TG in K562 cells.

Involvement of phospholipase C in the intracellular Ca^2 + response to cationic lipid/DNA complexes and cationic liposomes

U73122 is a selective inhibitor of phospholipase C activity, which is known to block the production of inositol 1,4,5-triphosphate [17]. K562 cells were pretreated with U73122 (10 µM) for 10 min and then stimulated with cationic lipid/DNA complexes and cationic liposomes. As shown in Figures 5A and 5B, U73122 blocked the capacity of both cationic lipid/DNA complexes and cationic liposomes to increase the intracellular calcium concentration.

Figure 5.  (A) Effect of U73122 on the [Ca^2 +]_i increase induced by diC14-amidine/DNA complexes and diC14-amidine liposomes in K562 cells. Cells (1×10⁶ cells/ml) resuspended in D-PBS medium at 1 mg/ml glucose in the presence of 1 mM Ca^2 + were loaded with Fluo-3-AM for 30 min at 37°C, and pretreated with the PLC inhibitor U73122 (10 µM) for 10 min before the addition of diC14-amidine/DNA complex at a 2:1 weight ratio (5 µg DNA) or 37 µM diC14-amidine liposome as indicated by the arrow. (B) Effect of U73122 on the [Ca^2 +]_i increase induced by DOTAP/DNA complexes and DOTAP liposomes in K562 cells. Cells (1×10⁶ cells/ml) resuspended in D-PBS medium at 1 mg/ml glucose in the presence of 1 mM Ca^2 + were loaded with Fluo-3-AM for 30 min at 37°C, and pretreated with the phospholipase C inhibitor U73122 (10 µM) for 10 min before addition of DOTAP/DNA complex at a 2:1 weight ratio (5 µg DNA) or 37 µM DOTAP liposome as indicated by the arrow. (C) Comparison of the effect of U73122 and its isomer U73343 inactive on PLC. K562 cells (1×10⁶ cells/ml) resuspended in D-PBS medium at 1 mg/ml glucose in the presence of 1 mM Ca^2 + were loaded with Fluo-3-AM for 30 min at 37°C, and pretreated with U73343 (10 µM) or U73122 (10 µM) for 10 min before addition of 37 µM diC14-amidine liposome as indicated by the arrow. Intracellular Ca^2 + concentration changes were measured at 37°C using fluorescence spectroscopy as described in Materials and Methods. The same experiments were repeated three times and showed identical results.

At the same concentration, U73343, an inactive structural isomer of U73122, had no effect on Ca^2 + response (Figure 5C). This result indicates clearly that a U73122-sensitive phospholipase C is involved in the calcium signal transduction pathway mediated by cationic lipid/DNA complexes and cationic liposomes in K562 cells.

Role of the cationic headgroup and hydrocarbon chain

Complexation of cationic liposomes with DNA decreases their capacity to trigger a calcium rise (see Figure 2) suggesting that the positive charge of the cationic lipid may be responsible for the intracellular Ca^2 + increase. To test this hypothesis, neutral liposomes (DMPC) and negatively charged liposomes (DMPC/DMPG 1:3 mole/mole) with the same C14 hydrocarbon chains as diC14-amidine were added to K562 cells and did not mobilize intracellular calcium (Figure 6A), confirming the crucial role of the cationic headgroups.

Figure 6.  (A) Comparison of [Ca^2 +]_i increase induced by neutral and negatively charged liposomes in K562 cells. Cells (1×10⁶ cells/ml) resuspended in D-PBS medium at 1 mg/ml glucose in the presence of 1 mM Ca^2 + were loaded with Fluo-3-AM for 30 min at 37°C. DiC14-amidine (positive), DMPC (neutral) and DMPC/DMPG 1:3 (negative) liposomes were added (arrow) at a 37 µM concentration. (B) and (C) Effect of diC14-amidine, lipofectamine and the corresponding headgroups N-t-butyl-N′-isopropyl-3-isopropylaminopropionamidine (diisopropylamidine) and spermine respectively on [Ca^2 +]_i increase in K562 cells. Cells (1×10⁶ cells/ml) resuspended in D-PBS medium at 1 mg/ml glucose in the presence of 1 mM Ca^2 + were loaded with Fluo-3-AM for 30 min at 37°C. DiC14-amidine, lipofectamine, diisopropylamidine, and spermine were added (arrow) at a 37 µM concentration. Intracellular Ca^2 + concentration changes were measured at 37°C using fluorescence spectroscopy as described in Materials and Methods. The same experiments were repeated three times and showed identical results.

To evaluate the hydrocarbon chain contribution, we tested free headgroups derivatives. No intracellular Ca^2 + increase was observed when cells were exposed to diisopropylamidine and spermine, which are respectively the headgroups of diC14-amidine and lipofectamine (Figures 6B and 6C), suggesting that not only the cationic headgroup of lipid but also the hydrophobic chains are required.

Discussion

Cationic lipid/DNA complexes represent an alternative to viral vectors for transfection in vitro and in vivo. Extensive efforts have been made to understand the interaction between the vector and DNA as well as the cellular pathways and mechanisms involved in DNA entry into the cell and ultimately into the nucleus [6]. How cationic lipid-cell interactions modify the physiological activity of cells is however largely unknown. We describe here for the first time how cationic lipid/DNA complexes and cationic liposomes trigger an intracellular calcium response mediated through phospholipase C and inositol phosphates signalling pathway. An identical intracellular calcium response was observed with HEK and Jurkat cells treated with cationic liposomes and cationic lipid/DNA complexes (data not shown).

The intracellular calcium mobilization did not result from a trivial cell permeabilization effect mediated by cationic lipids since depletion of extracellular calcium did not suppress the effect. On the contrary, cells retained their calcium-sensitive dye (Fluo-3) throughout the experiments, showing that their peripheral membrane was not leaky. The observed plateau-shaped response contrasts with the majority of ligand-induced calcium triggering which shows a peaky profile followed by a slow return to the basal level. In some experiments, the calcium signal was monitored for up to 1 h and still displayed the same sustained signal. The question as to whether cells were irreversibly affected by a prolonged calcium signal was addressed in control experiments showing that after transferring cells back to fresh complete medium and allowing to incubate overnight, they recovered their normal intracellular calcium level and were again responsive to diC14-amidine cationic liposomes (data not shown).

Depletion of intracellular calcium stores by pretreatment with TG inhibited significantly the diC14-amidine liposomes and diC14-amidine/DNA complex-induced intracellular Ca^2 + increase, confirming that this signal was a consequence of release of calcium from intracellular calcium stores sensitive to TG. Although extracellular calcium was not required for the triggering of the signal, it enhanced the final signal intensity which is a feature that is frequently observed in intracellular calcium signalling events when plasma membrane associated calcium channels are activated by a rise of intracellular calcium, a phenomenon previously referred to as capacitative calcium [18].

Inhibition of phospholipase C activity by U73122 inhibited the cationic lipid/DNA complexes and cationic liposomes calcium response, indicating the involvement of this enzyme in the signal transduction pathway activated by complexes and cationic lipids and suggesting inositol 1,4,5-triphosphate as the second messenger responsible for intracellular calcium increase. In agreement with that possibility, our preliminary results suggest that both complexes (DiC14-amidine/DNA and DOTAP/DNA) and cationic liposomes (DiC14-amidine and DOTAP) cause an increase of the intracellular inositol 1,4,5-triphosphate level (to be published).

Our results suggest that the positive charge of the cationic lipid is required for intracellular Ca^2 + response. It can be assumed that the positively charged headgroups facilitate electrostatic interactions between the cationic lipid/DNA complexes or cationic liposomes and the negatively charged cell membrane. However such effects are not observed in the absence of a hydrocarbon chain counterpart suggesting that an organized structure (liposomes) is required.

Based on these results, we propose the following signalling pathway to explain how cationic lipid/DNA complexes and cationic liposomes applied externally can lead to mobilization of calcium stores inside the cells: once injected into the medium, cationic lipid/DNA complexes or cationic liposomes bind to cells. It is generally accepted that interaction of cationic lipid/DNA complexes with cell in culture is non specific and involves mainly electrostatic interactions between the positively charged complexes and the negatively charged cell membrane. Although an endocytosis-like mechanism has been proposed as the main pathway of internalization for such complexes [6], one can not exclude that a fraction of such complexes fuse with the plasma membrane or with an early compartment that recycles quickly to the plasma membrane. It would be a way to insert cationic lipid in the lipid bilayer of the plasma membrane and/or the endocytotic compartment altering the activity of membrane components involved in the calcium-signalling cascade such as G protein and phospholipase C. The ability of cationic liposomes to fuse with cell membranes is supported by many experiments showing the spreading of liposome-associated fluorescent probes throughout the whole membrane network [19], [20]. DiC14-amidine behaves mainly as a membrane phospholipid: it organizes itself in a liposomal structure, with a gel to fluid transition temperature at 23°C [21], and therefore can insert into a lipid bilayer without perturbing significantly the lipid organization.

In conclusion, the present study shows for the first time that cationic lipid/DNA complexes and cationic liposomes are not inert and can affect the functioning of cells by increasing their intracellular calcium concentration. The fact that it has been recently observed that cationic liposomes and diC14-amidine/protein complexes exhibited anti-inflammatory [22], [23] and immunomodulating [24] properties respectively, suggests that cationic lipids affect cellular physiology. Additional investigations to clarify the molecular mechanisms by which cationic lipid/DNA complexes and cationic liposomes affect cellular signalling and biochemical significance of calcium mobilization in therapeutic gene transfer are in progress in our laboratory.

This paper was first published online on iFirst on 10 January 2007.

Acknowledgements

We acknowledge Guy Vandenbussche for analysing mass spectra of diC14-amidine and Steven Bouillon of the Department of Analytical and Environmental Chemistry (Free University of Brussels) for the elementary analysis of diC14-amidine. Michel Vandenbranden wishes to thank the F.N.R.S. for continuous support.