Dephosphorylation and benzoylation reactions occurring in the benzoolysis of diacyl-, alkylacyl-, alk-1-enylacyl-, and acyl-lyso-glycerophosphocholines.

Series of benzoolysis experiments with 1,2-dipalmitoyl glycerophosphocholine (GPC), 1-palmitoyl-2-lyso GPC, and 1-O-hexadecenyl-2-oleoyl GPC, as well as a few experiments with 1-oleoyl-2-acetyl GPC and 1-O-hexadecyl-2-acetyl GPC are reported. The results are used to present a consistent picture of the various dephosphorylation and benzoylation reactions occurring in molten benzoic anhydride.

Benzoolysis of diacylglycerophosphocholines: dephosphorylation and sequential formation of isomeric reaction products.

Benzoolysis experiments are reported in which diacylglycerophosphocholine is heated at 100 degrees C with benzoic anhydride for variable periods of time. It is shown that more than 90% of the phospholipid is dephosphorylated after 5 h of heating. Lipid extracts of the reaction mixture contained 1,2- and 1,3-diacylglycerobenzoate and 1,2- and 1,3-diacylglycerol in nearly constant isomer ratios of about 3:1 and 1:2, respectively, independent of the heating and extraction time. The total amount of isomeric diacylglycerobenzoates increased more slowly with increasing heating time that corresponded with the dephosphorylation rate, complete benzoylation being attained only after a 15 h heating period. The total amount of isomeric diacylglycerols went through a maximum after about 4 h and vanished after 15 h of heating. Addition of 4-dimethylaminopyridine subsequent to the heating period resulted in rapid and complete benzoylation of dephosphorylated phospholipid. However, the ratio of 1,2- to 1,3-diacylglycerobenzoate then found in the lipid extract depended on heating time, changing from less than 1:1 to about 3:1 upon an increase of heating time from 1 to 15 h. The results are interpreted in terms of two consecutive reactions. In a relatively fast first step, a dephosphorylated intermediate is formed, which in the molten benzoic anhydride, is slowly benzoylated. The intermediate yields diacylglycerols upon extraction in the absence of 4-dimethylaminopyridine and diacylglycerobenzoates upon extraction in the presence of 4-dimethylaminopyridine.

Quantitation of the diacyl, alkylacyl, and alk-1-enylacyl subclasses of choline glycerophospholipids by chemical dephosphorylation and benzoylation.

A method for the determination of the relative amounts of the diacyl, alkylacyl, and alk-1-enylacyl subclasses of choline glycerophospholipids by benzoolysis is described. The procedure consists of simply heating the phospholipid with benzoic anhydride in the presence of boric acid for 5 h at 100 degrees C, followed by reaction with 4-dimethylaminopyridine for 2 h at room temperature and HPLC analysis of the lipidic products formed. With model compounds of the three subclasses it is shown that diacyl- and alkylacylglycerophosphocholines are completely dephosphorylated by this procedure, yielding quantitatively the corresponding diacyl- and alkylacylglycerobenzoates. The same procedure applied to a model alk-1-enylacylglycerophosphocholine gives only 53.5% of dephosphorylation, while the dephosphorylated products in turn are quantitatively converted into the corresponding acylglycerodibenzoates. The latter figure is shown to be fairly reproducible. The reduced dephosphorylation rate of plasmalogens appears to be due to complete disruption of the vinyl ether bond. The liberated fatty aldehyde gives rise to an addition product with benzoic anhydride, which was identified by gas chromatography-mass spectrometry. It is demonstrated that acyl migration occurring during the benzoolysis does not interfere with the HPLC separation of the glycerobenzoates and dibenzoates derived from the three distinct subclasses. Results of subclass determinations by benzoolysis of several natural diradylglycerophosphocholines are in good accordance with literature values. The agreement between the plasmalogen contents, determined by benzoolysis and by phosphorus determination following exposure to HCl and separation by thin-layer chromatography, is satisfactory. The reliability of the benzoolysis method is generally discussed.

Investigation of two new [99mTc]Tc-HEDP preparations that can be expected to give a better lesion-to-normal-bone uptake ratio when used as bone scanning agents.

Two new potential bone scanning agents have been prepared and characterized. One of the agents, Tc(Sn,pH 12)-HEDP, is prepared at pH 12 (and subsequently neutralized). The other agent, Tc(Fe)-HEDP, is prepared by reducing [99mTc]TcO4- by a mixture of Fe(II) and Sn(II). From ion exchange chromatography on Aminex A-28 it appeared that the proportion of early eluting components of the two new agents is larger than that of the usual agent, Tc(Sn,pH 7.4)-HEDP. The stabilities on storage, and in serum, of the three agents appeared to be equally good. The adsorption on tri-calciumphosphate of Tc(Fe)-HEDP and Tc(Sn,pH 7.4)-HEDP was determined at several values of concentrations of Sn(II) and HEDP. Further, the influence of the pH (1-12) during reduction of [99mTc]TcO4- by Sn(II) on the adsorption of the agent on tri-calciumphosphate (at a fixed pH of 7.4) was investigated. It appeared that the adsorption of Tc(Sn,pH 12)-HEDP and Tc(Fe)-HEDP is less than that of Tc(Sn,pH 7.4)-HEDP. It is argued that this result points to a better efficiency in lesion detection of the two new agents, compared to the usual one.

The adsorption of 99mTc(Sn)-diphosphonate complexes on tri-calciumphosphate: the influence of preparation conditions, ligand-type, incubation media and adsorption conditions. The reversibility of the adsorption.

The influence of several variables on the adsorption of 99mTc(Sn)-diphosphonate complexes on tri-calciumphosphate was determined. The composition of the incubation medium influenced the percentage adsorption: with Hank's balanced salt solution (a medium frequently used for bone cell cultures) and Tris buffer lower percentage adsorption was obtained than with physiological saline as the incubation medium. The influence of addition to the incubation medium of some ions and neutral species, some of which occur in bone fluid, is very specific. Addition of Sn(II) or Mg(II) (a component of HBSS) reduces the amount of adsorption. Addition of Ca(II) and Al(III) had no effect. Addition of sodium-citrate and MDP to the medium and an increase of the pH of the medium decreased the percentage adsorption. The ligand that was used in the preparation of the complex mixture influences the percentage adsorption considerably. The Sn(II) concentration used during the preparation of the 99mTc(Sn)-MDP and 99mTc(Sn)-MHDP complexes showed no definite influence on the percentage adsorption. The pH and ligand concentration, used in the preparation, however, did effect the percentage adsorption. It was concluded that the 99mTc(Sn)-disphosphonate mixtures are part reversibly and part irreversibly bound to tri-calcium-phosphate.

The binding of 99mTc(Sn)-MDP complexes to human serum albumin and other blood proteins determined with gel chromatography and ultrafiltration.

The binding of 99mTc(Sn)-MDP to human serum albumin and other blood proteins was investigated by gel chromatography and ultrafiltration. During gel chromatography dissociation of the 99mTc(Sn)-MDP-protein complex occurs: thus, it is not a suitable technique for the determination of protein binding. The values found with ultrafiltration have to be corrected for non-ultrafiltrable TcO2.nH2O. From the corrected values it can be concluded that binding of 99mTc(Sn)-MDP to blood proteins does not play a role in the biodistribution.

Effect of the composition of the eluent in the chromatography of 99mTc-diphosphonate complexes.

When the ligand and the reducing agent are added to the eluent used in the separation of 99mTc-diphosphonate complexes by column chromatography, a significant increase in recovery is obtained. The addition of the ligand and the reducing agent also influences the shape of the chromatogram.

99mTc bone scanning agents--IV. Chemical characterization of 99mTc(Sn)-pyrophosphate complexes.

Various reaction mixtures for the preparation of 99Tc(Sn)-pyrophosphate were investigated by means of gel chromatography. All components were radioactively labeled. The most likely composition of the complexes, which also appear in "no carrier added" preparations, was determined. At pH 7 one complex is found with the composition TcPyp2. Two complexes are found at pH 4: TcPyp and TcPyp2. Further, at pH 7 a polymeric technetium compound is found not containing tin or pyrophosphate.

Anion exchange chromatography of 99mTc(Sn)-MDP complexes: influence of eluent composition, determination of void volume and charge of the main component.

99mTc(Sn)-MDP complexes have been prepared by reduction of 99mTcO4- by Sn(II) in the presence of MDP. These complexes were separated on an anion exchange column. The necessity of the addition of the ligand and the reducing agent to the eluent to avoid decomposition during chromatography is demonstrated. For the main component that is found at pH 5 the ionic charge was calculated according to the method of Wilson and Pinkerton [Anal. Chem. 57,246 (1985)] and the method of Russell and Bishoff [Int. J. Appl. Radiat. Isot. 35,859 (1985)]. With the first method a charge of -4.2 +/- 0.3 was obtained, with the second a charge of -5.1 +/- 0.6. An accurate method to determine the void volume of the ion exchange column is described.

99mTc bone scanning agents--V. Influence of experimental conditions on the labeling efficiency and gel chromatography of 99mTc(Sn)HMDP.

The preparation of 99mTc(Sn)HMDP was investigated as a function of pH, Sn(II) and ligand concentration. HMDP could be labeled efficiently from pH 2-9. The Sn(II) and the ligand concentrations had a beneficial influence. The composition of the radiopharmaceutical under various experimental conditions was studied by means of gel chromatography on Biogel P-4. Six different complexes were found. A preparation consisted of maximally three major complexes. The presence of a particular complex was mainly determined by pH and ligand concentration. The Sn(II) concentration had little influence.

Gel chromatographic analysis of the bone seeking radiopharmaceutical 99mTc(Sn)-EHDP: the influence of pH and EHDP-concentration on the size of its constituents.

99mTc(Sn)-EHDP complexes have been produced by reduction of TcO4- with Sn(II) in the presence of EHDP at varying pH and EHDP concentration. The mixture was separated by means of gel-chromatography with an eluent of the same composition and pH as the reaction mixture. It appears that at neutral pH larger complexes are formed than under acidic or basic reaction conditions. Larger complexes are also formed at higher EHDP concentrations.

The influence of Sn(IV) on the mean size of 99mTc(Sn)MDP constituents at neutral pH.

The effect of addition of Sn(IV), Mg(II) and Ca(II) on the mean size of the 99mTc-containing constituents of a 99mTc(Sn)MDP reaction mixture was determined by a gel chromatographic batch procedure that does not disturb the equilibria in the mixture.

99mTc bone scanning agents--II. Adsorption of 99mTc(Sn)pyrophosphate complexes on the mineral phase of bone.

The adsorption of pyrophosphate, tin-pyrophosphate and 99mTc(Sn)pyrophosphate on Ca3(PO4)2 was investigated at pH 4.0 and pH 7.4. All components were radioactively labeled. Tin and reduced technetium were in most cases almost completely bound. The adsorption of pyrophosphate, tin(II) and technetium-99m at pH 4.0 was higher than at pH 7.4. The presence of tin gave rise to an increase of the pyrophosphate adsorption that was much larger than can be accounted for by a stoichiometric adsorption of tin-pyrophosphate. It is concluded that tin and technetium are bound as negatively charged complexes with pyrophosphate. Finally it is argued that the fraction of the bone scanning agent that reaches the bone surface is adsorbed completely by the mineral phase.

99mTc bone scanning agents--III. Preparation and gel chromatography of 99mTc(Sn)MDP complexes.

The preparation of 99mTc(Sn)MDP was investigated as a function of pH, MDP concentration and Sn(II) concentration. The labeling efficiency was over 90% in the majority of the experiments and remained constant over the range pH 2-9. The MDP concentration had little effect, while the Sn(II) concentration had a significant positive influence. The complex formation appeared to be partly reversible. The formation of different complexes was investigated by means of gel chromatography under various experimental conditions. Altogether six complexes were found. At acid conditions two major complexes were found and at neutral pH one major complex. The presence or absence of a particular complex was mainly determined by the pH and by the MDP concentration. The Sn(II) concentration had very little effect. The results are compared with previous results of similar experiments with 99mTc(Sn)pyrophosphate.

99mTc bone scanning agents--I. Influence of experimental conditions on the formation and gel chromatography of 99mTc(Sn)pyrophosphate complexes.

The conversion of 99mTcO4- to 99mTc(Sn)pyrophosphate complexes was investigated under various experimental conditions. An increase of the Sn(II) concentration had a beneficial effect, whereas the ligand concentration had little effect. The pH had only a small influence over the range 2-8. Raising the pH to 10 resulted in the partial decomposition of the complexes, which could be reversed by lowering the pH. Furthermore, the occurrence of various complexes was investigated by means of gel chromatography on Biogel P-4 as a function of pH and of the Sn(II) and pyrophosphate concentrations. Four major fractions were found. A single preparation contained, however, no more than two major fractions. The formation of the different complexes was mainly governed by the pH and the ligand concentration. The influence of the eluent on the decomposition and interconversion of the complexes during chromatography was also studied. It appeared to be necessary that the eluent should have the same composition (except for 99mTcO4-) as the reaction mixture.