Possible leaching of diethyl phthalate into levothyroxine sodium tablets.

The possible leaching of diethyl phthalate into four currently marketed brands of levothyroxine sodium tablets was investigated. Several strengths of levothyroxine sodium tablets and sizes of containers were used. Samples were analyzed by high-performance liquid chromatography (HPLC) to determine the levothyroxine sodium content and to determine if any unidentified compounds were present. The packaging for the four brands of tablets was also analyzed by using the same HPLC system to determine if any extractable compounds could be detected in the tablets. The potencies of the four brands of tablets were comparable. The tablets from the 100-count container of one brand (brand A) were the only tablets found to contain an unidentified peak in the chromatogram. The desiccants from the bottle showed the same unidentified compound, while the bottle and closure did not yield the peak. Thin-layer chromatography and HPLC identified the peak as diethyl phthalate, a plasticizer in the desiccant. Tablets, bottles, closures, and desiccants for the 1000-count brand A product and all sizes of the other brands were negative for the presence of diethyl phthalate. The desiccants in those containers were from a different manufacturer than the desiccant in the brand A 100-count bottle. Diethyl phthalate in the desiccant in 100-count bottles of brand A levothyroxine sodium tablets appeared to have leached into the tablets.

Characterization of dolichyl diphosphate phosphatase from rat liver.

Dolichyl diphosphate phosphatase (DolPPase) has been characterized in rat liver. Subcellular distribution studies indicate that the enzyme is localized in the endoplasmic reticulum. The in vitro enzymatic activity is stimulated by EDTA, due to release of inhibition by trivalent cations found in the assay tubes. All di- and trivalent cations tested were inhibitory, with the trivalent ions Al3+ and Fe3+ showing greater than 70% inhibition at a concentration of 10 microM. The assay requires the presence of a detergent for optimal activity, with Triton X-100 giving maximum activity at 0.1%. The substrate specificity of DolPPase toward polyprenyl diphosphates has been determined and indicates that there is little preference of the enzyme for substrates of different chain length, and either stereochemical orientation or degree of saturation of the alpha-isoprene unit. Km values of 11-14 microM were obtained for all substrates tested. Preliminary studies on the transmembrane topology of the DolPPase using latency assays, indicate that the active site of the enzyme may reside on the cytoplasmic face of the endoplasmic reticulum.

Characterization of the Saccharomyces cerevisiae cis-prenyltransferase required for dolichyl phosphate biosynthesis.

The prenyltransferase involved in the biosynthesis of dolichyl phosphate has been characterized in Saccharomyces cerevisiae. Although the enzyme is predominantly membrane-bound, a significant percentage was found in the soluble fraction. The prenyltransferase preferentially utilizes farnesyl pyrophosphate as the allylic substrate and isopentenyl pyrophosphate as cosubstrate with half-maximal velocities obtained at 25 and 6.7 microM, respectively. The enzymatic activity is sensitive to sulfhydryl reagents and is inhibited by all detergents tested, except 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate at concentrations less than 5 mM. The product of the reaction has been characterized as an alpha-unsaturated polyprenyl pyrophosphate, containing 12-15 isoprene units, approximately two isoprene units shorter than the endogenous yeast dolichyl phosphate. The stereochemistry of addition of isoprene units by the prenyltransferase was shown to be cis by a comparison of the HPLC retention time for a pentadecaprenyl phosphate derived from the in vitro reaction product with that for an authentic mixture of alpha-cis- and alpha-trans-pentadecaprenyl phosphates.

Cell-cycle dependence of dolichyl phosphate biosynthesis.

The cell-cycle dependence of dolichyl phosphate biosynthesis has been investigated in mouse L-1210 cells fractionated by centrifugal elutriation. Dolichyl phosphate levels increased linearly through the cell cycle, reaching a value in late S phase twice that of early G1. The cell-cycle dependences of four dolichyl phosphate metabolizing enzymes have been measured: cis-prenyltransferase, CTP-dependent dolichol kinase, dolichyl phosphatase, and dolichyl pyrophosphatase. The kinase, the cis-prenyltransferase, and the pyrophosphatase showed cell-cycle variations, increasing through G1 to a maximum in S phase while the monophosphatase activity was cell-cycle independent. The rate of accumulation of dolichyl phosphate was not affected by growing the cells in mevalonolactone-supplemented media. The evidence presented is consistent with models in which either the cis-prenyltransferase or the kinase/phosphatase couple (or both) regulates the levels of dolichyl phosphate in the cell.

Characterization of polyisoprenyl phosphate phosphatase activity in rat liver.

The polyisoprenyl phosphate dephosphorylating activity of rat liver has been investigated with regard to substrate specificity, subcellular distribution, and transmembrane orientation. Total liver microsomes were employed as a source of enzymatic activity against a variety of 32P-labeled substrates. Susceptibility to dephosphorylation followed the order solanesyl phosphate greater than alpha-cis-polyprenyl 19-phosphate = alpha-trans-polyprenyl 19-phosphate = dihydrosolanesyl phosphate greater than (S)-dolichyl 19-phosphate = (R)-dolichyl 19-phosphate = (R,S)-dolichyl 11-phosphate. There appeared to be no major effect of chain length from 11 to 20 isoprenes. Data obtained from inhibition studies using solanesyl [32P]phosphate as substrate were consistent with the substrate specificity studies and suggested that a single activity is responsible. With dolichyl [32P]phosphate as substrate, the phosphatase specific activity of the subcellular fractions prepared from rat liver was found to follow the sequence Golgi = smooth endoplasmic reticulum greater than plasma membrane greater than lysosomes = rough endoplasmic reticulum greater than nuclei greater than mitochondria. Transmembrane topography studies, using enzyme latency as a criterion, were consistent with an orientation of the active site facing the cytoplasm.

A procedure for the preparation of [32P]phosphatidic acid.

The phosphorylation procedure of F. Cramer, W. Rittersdorf, and W. Bohm [(1961) Chem. Ber. 654, 180] using bis(triethylammonium) phosphate and trichloroacetonitrile was shown to be effective in the synthesis of [32P]phosphatidic acid. From diacylglyceride and 0.5 mCi H(3)32PO4, 25-50 microCi of labeled material (sp act = 1 mCi/mumol) can be prepared in 2 h. The product was shown to be radiochemically pure by both TLC and HPLC. L- and DL-[32P]dipalmitoyl phosphatidic acid prepared using this procedure were shown to be hydrolyzed by rat liver microsomes at approximately the same rates.

Solubilization and characterization of the long chain prenyltransferase involved in dolichyl phosphate biosynthesis.

Membranes from Ehrlich ascites tumor cells possess an activity which, in the presence of farnesyl pyrophosphate, incorporates [14C]isopentenyl pyrophosphate into a product which is soluble in chloroform/ methanol and retained by DEAE-cellulose. The product co-migrated with authentic dolichyl phosphate on thin layer chromatography but was degraded to neutral labeled compounds when subjected to mild acid hydrolysis, suggesting the presence of an unsaturated alpha-isoprene unit. Triton X-100 (2%) liberated the activity from the membranes and the resulting solubilized preparation was further characterized. The enzyme was found to be sensitive to sulfhydryl reagents and stimulated by ionic strength. A strong dependence of activity on the addition of farnesyl pyrophosphate was observed, allowing several compounds to be tested for their ability to serve as potential primers. Geranyl pyrophosphate, neryl pyrophosphate, all-trans farnesyl pyrophosphate and all-trans geranylgeranyl pyrophospate were found to be effective substrates, although to different extents. Citronellyl pyrophosphate (which has a saturated alpha-isoprene) was inactive as a substrate. The chain length of the products generated was investigated by using a double label isotope procedure and by reverse-phase high performance liquid chromatography. All active primers yielded a product containing 16-19 isoprene units; the distribution of the individual isoprene species was essentially identical regardless of the primer used as substrate. These findings indicate that the specificity lies in the absolute chain length of the product released and not the number of isoprenes added. High performance liquid chromatography of the [14C]polyprenol resulting from enzymatic dephosphorylation of the reaction product indicated the presence of an unsaturated alpha-isoprene unit in a cis-configuration. It is proposed that the enzyme is the long chain cis-prenyltransferase involved in the biosynthesis of dolichyl phosphate.

Topography of dolichyl phosphate synthesis in rat liver microsomes. Transbilayer arrangement of dolichol kinase and long-chain prenyltransferase.

The topography of the dolichyl phosphate biosynthetic enzymes within the plane of rat liver microsomes was investigated by the use of two impermeant inhibitors of enzyme activity: trypsin and mercury-dextran. Mercury-dextran was found to inactivate over 50% of the activities of the CTP-dependent dolichol kinase and the long-chain prenyltransferase. Trypsin caused over 90% inactivation of the long-chain prenyltransferase and 60% inactivation of the dolichol kinase. In addition, the CTP-dependent dolichol kinase was inhibited over 90% by CDP applied externally to sealed microsomes. Inactivation of the dolichyl phosphate biosynthetic enzymes by the impermeant probes occurred under conditions where the mannose-6-phosphatase activity was highly latent. It was concluded that the active sites of these two enzymes are located on the external surface of the microsomal membranes and that dolichyl phosphate biosynthesis occurs asymmetrically on the cytoplasmic surface of the endoplasmic reticulum.

Subcellular localization and substrate specificity of dolichol kinase from rat liver.

When purified subcellular fractions were prepared from rat liver and assayed for dolichol kinase activity using pig liver dolichol as a substrate, the microsomes were found to contain the highest specific activity and greater than 75% of the total activity. With regard to substrate specificity, the microsomal enzyme showed a marked preference for saturation of the alpha-isoprene: dolichol-16 and -19 were 2.5-fold more active than the corresponding polyprenols. For a given class of prenol, the 16 and 19 isoprenologs exhibited similar activity, whereas the 11 isoprenolog appeared less active. The enzyme was twice as active against the naturally occurring polyprenol-16 (alpha-cis-isoprene) compared to synthetic alpha-trans-polyprenol-16. Taken together, the data indicate that the alpha-isoprene specificity follows the order: saturated greater than cis greater than trans. In addition, all-trans-2,3-dihydrosolanesol was not a substrate, suggesting that at least one cis isoprene residue is required.

Use of erythrocyte hemolysis kinetics in the purification of complex cardiotoxin mixtures.

A human erythrocyte hemolysis kinetic method provides a useful way to follow the purification of cobra venom cardiotoxins or other hemolytic factors. Initial rates of hemolysis, measured as hemoglobin released with time for the separated cardiotoxins from the venom of the Thailand cobra Naja naja siamensis, vary over a greater range than do other commonly used measures of their biological activity. Recovery of the hemolytic activity of the gross cardiotoxin fraction in the subsequently separated fractions has been demonstrated. The method employs submilligram quantities of mixed or purified cardiotoxins.