Pharmacokinetics and bioavailability of diisopropanolamine (DIPA) in rats following intravenous or dermal application.

This study was conducted to determine the relative dermal bioavailability (absorption), distribution, metabolism, and excretion (ADME) of diisopropanolamine (DIPA), an alcohol amine used in a number of industrial and personal care products. Groups of 4 female Fischer 344 rats received either a single bolus i.v. dose of 19.0mg/kg (14)C-DIPA in water or a dermal application of 19.5mg/kg (14)C-DIPA in acetone to an area of 1cm(2) on the back and covered with a bandage. Time-course blood and excreta were collected and radioactivity determined. Urine was analyzed for DIPA and monoisopropanolamine (MIPA). Following i.v. administration, DIPA was rapidly cleared from the plasma and excreted into urine in a biexponential manner (t(1/2alpha), 0.4h; t(1/2beta), 2.9h). The levels of radioactivity in plasma dropped below the limit of detection 12h post-dosing. A total of 97+/-4% of the dose was actively excreted in urine by kidney, most ( approximately 71%) within 6h of dosing, virtually all as parent compound; renal clearance exceeded the glomerular filtration rate. Following dermal application, approximately 20% of the dose was absorbed in 48 h with the steady-state penetration rate of approximately 0.2%/h. Most (14.4%) of the applied radioactivity was excreted in urine at a relatively constant rate due to the presence of large amount of the (14)C-DIPA at the application site. Fecal elimination was <0.2% of the dose. The absorbed DIPA did not accumulate in tissues; only approximately 0.1% of the administered dose was found in liver and kidney. The absolute systemic dermal bioavailability (dose corrected AUC(dermal)/AUC(i.v.)) of (14)C-DIPA was 12%. The ADME of DIPA contrasts that of its diethanol analogue, diethanolamine, which displays a broad spectrum of toxicity in rats and mice. Toxicologically significant concentrations of DIPA are unlikely to be achieved in the systemic circulation and/or tissues as a result of repeated dermal application of products containing DIPA due to slow absorption from the skin, rapid unchanged elimination in urine, and majority of the products contain

The determination of the ability of polyurethane foam to release toluene diisocyanate into air.

A study was conducted to assess the ability of polyurethane foam to release toluene diisocyanate (TDI) into the air. The study was completed in two phases. In the first phase, three-day post-production foam samples were "extracted" using 37 degrees C, 30 percent relative humidity air for a total of three days. In the second phase, foam samples were "loaded" with TDI (approximately 1 ppm (w/w) in the foam) by passing air containing a controlled level of TDI vapor through the foam. The "loaded" foam was then aged for three days (to simulate minimum possible expected time between production and consumer contact) before being extracted for three days using 37 degrees C, 30 percent relative humidity air. In both phases of the study, the extracted TDI was quantified by trapping it from the air using glass-fiber filters coated with 1,(2-pyridyl)piperazine (PP) derivatizing agent (modified OSHA Method 42) then analyzing the TDI derivative formed using high-performance liquid chromatography with ultraviolet detection. Validation of the test system was conducted using diffusion-tube-generated TDI atmospheres. Results of the Phase 1 portion of the study showed no quantifiable TDI being extracted from the three-day post-production commercial foam samples at a limit of quantitation of approximately 0.1 ppb TDI in air. Results of the Phase 2 portion of the study showed no quantifiable TDI being extracted from the "loaded" foams at a limit of quantitation of 0.12 microgram TDI (less than 0.03% of the level of TDI loaded into the foams). In conclusion, the results from this study indicate that it is not likely that TDI would be released from three-day post-production polyurethane foams in amounts likely to produce air concentrations of concern.

Evaluation of the cost-effectiveness of various passive monitors and active monitors for industrial hygiene sampling.

A framework for evaluating passive or active industrial hygiene air sampling monitors on a cost-effectiveness basis is described. Five passive monitors and one active monitor (charcoal tube with pump) were compared in several hypothetical scenarios. Using certain assumptions regarding method validation costs, sampling equipment costs, and labor costs, both a per sample cost and a total annual cost were calculated for each type of sampling monitor as a function of total samples taken per year. The results of this study indicate that even when the additional expense of full validation of the passive sampling monitor is required, these monitors are more cost-effective than active monitors. A specific type of passive monitor utilizing replaceable capsules is the most cost-effective when more than 500 samples per year are taken.

Craniocervical injuries in judicial hangings: an anthropologic analysis of six cases.

Restoration projects and archaeologic excavations in two Canadian prisons resulted in the recovery of the skeletons of six felons executed by judicial hanging. Damage inflicted by hanging on various skeletal elements was observed. Among the injuries seen were fractures of the hyoid cornua, styloid processes, occipital bones, and cervical vertebral bodies (C2) and transverse processes (C1, C2, C3, and C5). Despite the general uniformity of the hanging technique, which involved a subaural knot, the trauma to the skeletal elements and the cause of death varied among individuals. Although some of this variation was probably due to minor differences in hanging practices, individual anatomic peculiarities of the victims likely also contributed.

Regulation of low density lipoprotein receptor and 3-hydroxy-3-methyl-glutaryl-CoA reductase activities are differentially affected in Niemann-Pick type C and type D fibroblasts.

Defective regulation of intracellular cholesterol metabolism has been investigated in cultured fibroblasts from two subtypes of Niemann-Pick type II disease: the panethnic Niemann-Pick type C (NPC) and the Nova Scotia type D (NPD). Cell extracts from NPC and NPD fibroblasts cultured in lipoprotein-deficient medium exhibited activities of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase that were two-fold greater than in normal cells. Addition of serum resulted in only a 15% decrease in HMG-CoA reductase activity within 6 h in these cells, compared with a decrease of 80% in normal fibroblasts. The initial rate of return to maximal values for the first 6 h after removal of serum was similar in all three cell types; thereafter, the rate was faster in the mutant fibroblasts. Binding and internalization of 125I-labeled low density lipoprotein (LDL) was not decreased within 12 h of incubation of NPC fibroblasts with serum, while a decrease of 50% was observed for both NPD and normal fibroblasts over this time period. Northern blot analysis also indicated a slower decrease in steady-state LDL receptor mRNA in NPC relative to normal and NPD cells. In all three cell types, inhibition of HMG-CoA reductase with mevinolin had no effect on serum-stimulated cholesterol esterification, while inhibition of acyl-CoA:cholesterol acyltransferase with Sandoz 58-035 did not influence HMG-CoA reductase activity, indicating that defects in these regulatory mechanisms are independent.(ABSTRACT TRUNCATED AT 250 WORDS)

Limited metabolic interaction of serine with ethanolamine and choline in the turnover of phosphatidylserine, phosphatidylethanolamine and plasmalogens in cultured glioma cells.

Modulation of choline phosphoglyceride turnover has been investigated extensively but less is known about regulation of serine and ethanolamine phosphoglyceride synthesis and turnover. We investigated incorporation and interactions of [3H(G)]L-serine, [1,2-14C]ethanolamine and [methyl-3H]choline in cultured glioma cells. Exogenous serine did not compete with ethanolamine or choline incorporation and did not chase labeled headgroup from ethanolamine phosphoglycerides (PE); serine displaced headgroup of prelabeled phosphatidylserine (PtdSer) resulting in less labeled PtdSer for decarboxylation. In contrast, exogenous ethanolamine markedly chased labeled headgroup of non-plasmenylethanolamine phosphoglycerides (NP-PE) with less effect on plasmalogen (1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) whether headgroup was derived from [3H]serine or [14C]ethanolamine. Label in chase medium was mainly ethanolamine to 12 h; phosphoethanolamine was present with longer chase (12-48 h). Choline did not compete with serine incorporation and had little chase effect on PtdSer and PE. Choline and ethanolamine competitively interacted with preference for choline. These data suggest that (1) PtdSer synthesis in cultured glioma cells may involve more than headgroup exchange; (2) PE turnover with metabolite release to medium may involve both phospholipase D and phospholipase C; (3) acceleration of PE turnover by exogenous ethanolamine primarily involves NP-PE with lesser involvement of plasmalogen; and (4) in contrast to lack of interaction between serine and other headgroup precursors, choline and ethanolamine compete primarily at uptake.

Dissociation of phosphorylation and translocation of a myristoylated protein kinase C substrate (MARCKS protein) in C6 glioma and N1E-115 neuroblastoma cells.

An 80-kDa protein labeled with [3H]myristic acid in C6 glioma and N1E-115 neuroblastoma cells has been identified as the myristoylated alanine-rich C kinase substrate (MARCKS protein) on the basis of its calmodulin-binding, acidic nature, heat stability, and immunochemical properties. When C6 cells preincubated with [3H]myristate were treated with 200 nM 4 beta-12-O-tetradecanoylphorbol 13-acetate (beta-TPA), labeled MARCKS was rapidly increased in the soluble digitonin fraction (maximal, fivefold at 10 min) with a concomitant decrease in the Triton X-100-soluble membrane fraction. However, phosphorylation of this protein was increased in the presence of beta-TPA to a similar extent in both fractions (maximal, fourfold at 30 min). In contrast, beta-TPA-stimulated phosphorylation of MARCKS in N1E-115 cells was confined to the membrane fraction only and no change in the distribution of the myristoylated protein was noted relative to alpha-TPA controls. These results indicate that although phosphorylation of MARCKS by protein kinase C occurs in both cell lines, it is not directly associated with translocation from membrane to cytosol, which occurs in C6 cells only. The cell-specific translocation of MARCKS appears to correlate with previously demonstrated differential effects of phorbol esters on stimulation of phosphatidylcholine turnover in these two cell lines.

Calcium-independent effects of TMB-8. Modification of phospholipid metabolism in neuroblastoma cells by inhibition of choline uptake.

TMB-8 [8-(NN-diethylamino)-octyl-3,4,5-trimethoxybenzoate] blocks agonist-stimulated release of Ca2+ from intracellular sites in many cell lines and is often used to distinguish between dependence on extracellular and intracellular Ca2+. In N1E-115 neuroblastoma cells, TMB-8 did not alter the resting cytosolic Ca2+ concentration in unstimulated cells, yet phospholipid metabolism was greatly affected. At concentrations of TMB-8 (25-150 microM) that inhibit Ca2+ release, phosphatidylcholine formation was inhibited, whereas synthesis of phosphatidylinositol, phosphatidylglycerol and phosphatidylserine was stimulated. Unlike other cationic amphipathic compounds, TMB-8 did not inhibit phosphatidate phosphatase or enzymes in the pathway from choline to phosphatidylcholine. Choline transport was the major site of action. TMB-8 was a competitive inhibitor (Ki = 10 microM) of low-affinity (Kt = 20 microM) choline transport. When added at the same time as labelled precursor, TMB-8 also decreased cellular uptake of phosphate and inositol, but not that of ethanolamine or serine. In prelabelled cells, continued uptake and incorporation of phosphate and inositol were not affected. Under these conditions phosphatidylinositol synthesis was increased 2-fold and, like the effect on phosphatidylcholine, reached a plateau at 100 microM-TMB-8. Phosphatidylglycerol synthesis increased linearly with TMB-8 concentration to 40-fold stimulation at 150 microM, suggesting a selective effect on synthesis of phosphatidylglycerol from CDP-diacylglycerol. Phosphatidylserine synthesis was also increased up to 3-fold. These Ca(2+)-independent effects limit the use of TMB-8 in studies of cell signalling that involve stimulated phosphatidylinositol and phosphatidylcholine metabolism.

Polyunsaturated fatty acid incorporation into plasmalogens in plasma membrane of glioma cells is preceded temporally by acylation in microsomes.

Plasmalogens (1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) are major phospholipids in many tissues and cells, particularly of neural origin. Using cultured C6 glioma cells and subcellular fractions isolated on Percoll gradients we investigated selectivity for esterification of several polyunsaturated fatty acids (PUFA) in the sn-2 position of plasmalogens compared to [1-14C]hexadecanol, representative of de novo synthesis of the ether-linked sn-1 position. In whole cells at a final concentration of 105 microM PUFA, 2-4 nmol plasmalogen/mg protein was labeled in 4 h and 10-14 nmol in 24 h, representing 8-15% and 35-50%, respectively, of initial plasmalogen mass. Incorporation of label from hexadecanol was lower than PUFA incorporation (20:5(n-3) greater than 20:4(n-6) greater than 18:3(n-3) much greater than 18:2(n-6)) suggesting deacylation-reacylation at the sn-2 position. Plasmalogens accounted for 50% of total cell ethanolamine phospholipids and 75% in plasma membrane. Using a novel, improved method for extraction of subcellular fractions containing Percoll, plasma membrane also was enriched in plasmalogen relative to microsomes (107.4 +/- 5.2 vs. 40.0 +/- 2.9 nmol/mg protein). Selectivity for esterification at the sn-2 position of plasmalogens with respect to chain length and unsaturation of the fatty acyl chain was similar in both subcellular fractions and reflected that of whole cells. Labeling of plasma membrane with PUFA and fatty alcohol lagged behind that of microsomes. Chase experiments in cells prelabeled with [1-14C]18:3(n-3) for 2 h showed no significant reduction of label in plasmalogen of any subcellular fraction although accumulation of label in the microsomal fraction was slowed initially. Reduction of plasmalogen label (40-50%) did occur in microsomes and plasma membrane when cells prelabeled for 24 h were switched to chase medium with or without chase fatty acid. Our data suggest that esterification of PUFA to plasmalogen may occur at the endoplasmic reticulum with subsequent translocation to plasma membrane resulting in accumulation of relatively stable pools of plasmalogen that are not readily accessible for deacylation-reacylation exchange with newly appearing PUFA. Alternatively, deacylation-reacylation may occur in a more stable phospholipid pool within the plasma membrane but would involve a slower process than at the endoplasmic reticulum.

Cultured fibroblasts from patients with Niemann-Pick disease type C and type D exhibit distinct defects in cholesterol esterification.

The Niemann-Pick group of diseases can be broadly classified into two types based on clinical and biochemical characteristics. Type I is characterized by a primary deficiency of lysosomal sphingomyelinase while Type II may have a defect in the regulation of intracellular cholesterol metabolism. We have studied cholesterol esterification in cultured fibroblasts from patients with two phenotypes of Type II disease: an Acadian population of southwestern Nova Scotia (Canada) with a form of the disease known as Niemann-Pick type D (NPD) and a group of panethnic origin with Niemann-Pick type C (NPC). Addition of whole serum to normal fibroblasts grown initially in lipoprotein-deficient serum caused a rapid (within 6 h) increase in cholesterol esterification, reaching maximum values at around 24 h, while NPC fibroblasts showed little increase (less than 10% of normal). In contrast, cholesterol esterification in NPD fibroblasts increased slowly during the first 6-12 h and reached 50% of normal values by 24 h. 25-Hydroxycholesterol, a non-lipoprotein stimulator of cholesterol esterification, caused a similar stimulation of cholesterol esterification in NPC, NPD and normal cells. This was inhibited by addition of serum in mutant but not in normal cells. Within 24 h of serum addition, free cholesterol accumulated in all cell types with NPC greater than NPD greater than normal. These observations indicate that (a) regulation of cholesterol esterification in response to serum lipoproteins (but not 25-hydroxycholesterol) is abnormal in both NPC and NPD fibroblasts, and (b) the biochemical phenotypes of fibroblasts from NPC and NPD patients are distinct.

Niemann-Pick type II fibroblasts exhibit impaired cholesterol esterification in response to sphingomyelin hydrolysis.

Fibroblasts from patients with Niemann-Pick Type II disease, including the panethnic type C (NPC) and Nova Scotia Acadian type D (NPD) forms, exhibit reduced or delayed stimulation of cholesterol esterification by low density lipoprotein (LDL). Based on recent evidence that cholesterol esterification can also be stimulated by cell surface sphingomyelin hydrolysis, we have compared the response of normal, NPC and NPD fibroblasts to treatment with exogenous sphingomyelinase (SMase). Staphylococcus aureus SMase (greater than 0.05 U/ml) hydrolyzed over 90% of endogenous sphingomyelin within 1 h and increased incorporation of [3H]oleic acid into cholesterol-[3H]oleate after an initial lag in all three cell types. However, normal levels of cholesterol esterification were not observed for NP Type II fibroblasts: four NPD cell lines exhibited an average of 32% of normal response while cholesterol esterification was only 20% in two well-characterized NPC lines. A third NPC line exhibited normal response to SMase despite greater than 90% impairment of LDL-stimulated cholesterol esterification. Incubation of fibroblasts with LDL followed by SMase produced a synergistic response, particularly in NPC cells where there was little response to either treatment alone. Chloroquine abolished LDL-stimulated cholesterol esterification in normal fibroblasts but had no effect on the response to SMase, indicating that lysosomal enzymes may not be involved in SMase-mediated cholesterol esterification. These results suggest that intracellular processing of cholesterol derived from either LDL or release from the plasma membrane (by sphingomyelin hydrolysis) is affected in Niemann-Pick Type II cells and that these pathways can complement one another in the stimulation of cholesterol esterification.

Alternate pathways in the desaturation and chain elongation of linolenic acid, 18:3(n-3), in cultured glioma cells.

Cultured C6 glioma cells rapidly incorporate and metabolize the essential fatty acids, 18:2(n-6) and 18:3(n-3), to 20- and 22-carbon polyunsaturated fatty acids. Using several deuterated fatty acid substrates we have obtained data that suggest alternate pathways, one possibly involving delta 8-desaturation, may exist in glioma cells for formation of 20:5(n-3) and 22:6(n-3) from 18:3(n-3). With 18:3(n-3)-6,6,7,7-d4 practically no 18:4(n-3)-6,7-d2 or 20:4(n-3)-8,9-d2 was detected whereas 20:3(n-3)-8,8,9,9-d4 accounted for 3.4% and delta 5,11,14,17-20:4-8,8,9,9-d4 for 21.1% of the total deuterated fatty acids recovered in phospholipids after a 16 h incubation; 20:5(n-3)-8,9-d2, 22:5(n-3)-10,11-d2, and 22:6(n-3)-10,11-d2 accounted for 42.4%, 13.2%, and 2.8% of deuterated acyl chains, respectively. When added exogneously, 20:3-8,8,9,9,-d4 was extensively converted to delta 5,11,14,17-20:4(n-3)-8,8,9,9-d4 (45%) and 20:5(n-3)-8,9-d2 (24%); a small amount (4%) of 18:3(n-3)-d4 also was detected. Both 20:4(n-3)-8,9-d2 and 18:4(n-3)-12,13,15,16-d4 were also converted to 20:5(n-3) and 22:6(n-3) with 8 and 0% of the respective original deuterated substrate remaining after 16 h. A possible pathway for 18:3(n-3) metabolism in glioma cells is described whereby an initial chain elongation step is followed by successive delta 5 and delta 8 desaturation reactions resulting in 20:5(n-3) formation and accounting for the ordered removal of deuterium atoms. Alternatively, extremely effective retroconversion may occur to chain shorten 20:3(n-3)-d4 to 18:3(n-3)-d4 followed by rapid conversion through the classical desaturation and chain elongation sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of phosphatidylcholine and phosphatidylethanolamine biosynthesis by cytochalasin B in cultured glioma cells: potential regulation of biosynthesis by Ca(2+)-dependent mechanisms.

The major route of phosphatidylcholine (PtdCho) biosynthesis in mammalian cells is the sequence: choline (Cho)----phosphocholine (PCho)----cytidinediphosphate choline (CDP-Cho)----PtdCho. Recently, we have found that intermediates of this pathway are not freely diffusible in cultured rat glioma (C6) cells but are channeled towards PtdCho biosynthesis (George et al. (1989). Biochim. Biophys. Acta. 1004, 283-291). Channeling of intermediates in other mammalian systems is thought to be mediated through adsorption of enzymes to membranes and cytoskeletal elements to form multienzyme complexes. In this study, agents which perturb the structure and function of cytoskeletal elements were tested for effects on phospholipid metabolism in glioma cells. The filament-disrupting agent cytochalasin B (CB), but not other cytochalasins or the microtubule depolymerizer colchicine inhibited PtdCho and phosphatidylethanolamine (PtdEtn) biosynthesis as judged by dose-dependent reduction of labeling from [3H]Cho and [14C]ethanolamine (Etn). 32Pi pulse-labeling indicated that CB selectively decreased PtdCho and PtdEtn biosynthesis without affecting synthesis of other phospholipids. Synthesis of water-soluble intermediates of PtdCho metabolism was unaffected but the conversion of phosphoethanolamine to CDP-ethanolamine was reduced by CB. Effects of CB on phospholipid biosynthesis were not due to inhibition of glucose uptake as shown by experiments with 2-deoxyglucose, glucose-starved cells and other cytochalasins. Experiments with Ca(2+)-EGTA buffers and digitonin-permeabilized cells, and the Ca(2+)-channel blocker verapamil suggest that effects of CB on PtdCho and PtdEtn biosynthesis are due to alteration of intracellular Ca2+. Taken together, these results suggest that CB acts at sites distinct from glucose transport and cellular microfilaments to specifically inhibit PtdCho and PtdEtn biosynthesis by mechanisms dependent on intracellular Ca2+.

Channeling of intermediates in the CDP-choline pathway of phosphatidylcholine biosynthesis in cultured glioma cells is dependent on intracellular Ca2+.

The major route of phosphatidylcholine (Ptd-choline) biosynthesis in mammalian cells is the CDP-choline pathway which involves stepwise conversion of choline to phosphocholine (P-choline), cytidine diphosphate choline (CDP-choline), and Ptd-choline. Our previous studies with electropermeabilized (EP) rat glioma (C6) cells have indicated that the intermediates of this pathway are not freely diffusible in the cell but are channeled toward synthesis of Ptd-choline (George, T.P., Morash, S.C., Cook, H.W., Byers, D.M., Palmer, F. B. St.C., and Spence, M.W. (1989) Biochim. Biophys. Acta 1004, 283-291). In this study, Ca(2+)-[ethylene-bis(oxyethylenenitrilo)]tetraacetic acid buffers were used to investigate the role of intracellular free Ca2+ levels in functional organization of this pathway in EP glioma cells. In EP cells reduction of free Ca2+ in the medium from 1.8 mM to less than 200 nM resulted in 2-3-fold stimulation of exogenous [3H]choline and [14C]P-choline incorporation into Ptd-choline whereas incorporation of exogenous CDP-[14C]choline was augmented 100-fold; there was no uptake or incorporation of labeled P-choline or CDP-choline in intact cells. In EP cells incubated at 1.8 mM Ca2+ the water-soluble products of choline metabolism (choline, P-choline, CDP-choline, and glycerophosphocholine) were retained at 37 degrees C; in contrast, in the presence of 100 nM Ca2+ there was uniform leakage of these metabolites. Experiments with hemicholinium-3, an inhibitor of choline transport, and EP cells at 100 nM Ca2+ show that linkage of choline transport and Ptd-choline biosynthesis is also dependent on Ca2+. These results suggest that channeling of intermediates in the CDP-choline pathway of Ptd-choline biosynthesis in glioma cells is mediated by intracellular Ca2+ levels that may coordinately regulate the steps involved in conversion of choline to Ptd-choline.

Regulation of eukaryotic phospholipid metabolism.

Phospholipids have diverse and critical roles in cellular metabolism and function. Questions about the mechanisms of regulation of phospholipid synthesis are being investigated with a variety of systems and approaches. For example, the yeast Saccharomyces cerevisiae is an organism in which both biochemical and genetic analyses are used. Biochemical approaches have yielded considerable information on the regulatory properties of enzymes of phospholipid biosynthesis. Studies of the activity of purified phosphatidylserine synthase have suggested how that enzyme is influenced by membrane phospholipids in the cell. The enzyme that regulates mammalian phosphatidylcholine biosynthesis, CTP:phosphocholine cytidylyltransferase, is also influenced by phospholipids. In addition, the activity of this enzyme often correlates with its translocation to membranes. The location of such enzymes in the cell is of particular interest in light of the possibility that the enzymatic reactions may be efficiently coupled in vivo. Techniques to render cultured cells permeable to phosphorylated molecules indicated that the enzymes of phosphatidylcholine biosynthesis may exist in an organized compartment so that the precursors of phosphatidylcholine are efficiently channeled through the pathway. To ask how phospholipids are transported in the cell, a combined biochemical and genetic approach has been used. These studies have revealed that the phosphatidylinositol/phosphatidylcholine transfer protein, considered to mediate intracellular phospholipid transfer, is a critical component of the secretory pathway for proteins. These results have allowed formulation of a number of new questions on the regulation of phospholipid metabolism and its relationship to general membrane processes.

Fabry disease in a large Nova Scotia kindred: carrier detection using leucocyte alpha-galactosidase activity and an NcoI polymorphism detected by an alpha-galactosidase cDNA clone.

Fabry disease is an X linked recessive disorder of glycosphingolipid metabolism resulting from a deficiency of the lysosomal hydrolase alpha-galactosidase (alpha-gal). Measurement of the enzyme activity, however, is not an accurate method for identification of female carriers among at risk relatives of affected males. The alpha-gal cDNA and gene have been cloned previously and found to provide useful probes for the molecular analysis of affected families but these clones have not been available to us. Thus, to analyse Fabry disease in Nova Scotia, especially within a large kindred known to contain 30 affected males and 50 possible carrier females, we isolated an independent cDNA for alpha-gal. Using this clone as a probe, the mutation in the Nova Scotia kindred was shown not to be a major DNA alteration, but was found to be linked to the rarer allele (frequency 0.20) of the polymorphic NcoI site located 3' to the gene. Affected males from two Nova Scotia families who cannot be associated with the kindred by history were also found to have the rarer NcoI allele, which suggests they are, in fact, part of the kindred. The coupling of the mutation to an infrequent marker also helped carrier identification in the kindred where all of 17 obligate carriers examined, including six who were not identified as carriers by enzyme assays, were found to be heterozygous for the RFLP. Thus, DNA analysis can be used for presymptomatic and prenatal diagnosis in most portions of the Nova Scotia kindred affected with Fabry disease.

Serine utilization as a precursor of phosphatidylserine and alkenyl-(plasmenyl)-, alkyl-, and acylethanolamine phosphoglycerides in cultured glioma cells.

In several tissues and cell lines, serine utilized for phosphatidylserine (PS) synthesis is an eventual precursor of the base moiety of ethanolamine phosphoglycerides (PE). We investigated the biosynthesis and decarboxylation of PS in cultured C6 glioma cells, with particular attention to 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine (plasmenylethanolamine) biosynthesis. Incorporation of [3H]serine into PS reached a maximum within 4-8 h, and label in nonplasmenylethanolamine phosphoglyceride (NP-PE) and plasmenylethanolamine was maximal by 12-24 h and 48 h, respectively. After 8 h, label in PS decreased even though 40-60% of initial label remained in the culture medium. Serial additions of fresh [3H]serine restored PS synthesis to higher levels of labeled PS accumulation followed by a subsequent decrease in 4-8 h. High performance liquid chromatographic analyses confirmed that medium serine was depleted by 8 h, and thereafter metabolites, including acetate and formate, accounted for radioactivity in the medium. The rapid but transient appearance of labeled glycine and ATP inside the cells indicated conversion of serine by hydroxymethyltransferase. 78-85% of label from serine was in headgroup of PS or of PE formed by decarboxylation. A precursor-product relationship was suggested for label from [3H]serine appearing in the headgroup of diacyl, alkylacyl, and alkenylacyl subclasses of PE. By 48 h, a constant specific activity, ratio of approximately 1:1 was reached between plasmenylethanolamine and NP-PE, similar to the molar distribution of these lipids. In contrast, equilibrium was not achieved in cells incubated with [1,2-14C]ethanolamine; plasmenylethanolamine had 2-fold greater specific activity than labeled NP-PE by 72-96 h. These observations indicate that in cultured glioma cells 1) serine serves as a precursor of the head group of PS and of both plasmenyl and non-plasmenyl species of PE; 2) exchange of headgroup between NP-PE and plasmenylethanolamine may involve different donor pools of PE depending on whether the headgroup originates with exogenous serine or ethanolamine; 3) serine is rapidly converted to other metabolites, which limits exogenous serine as a direct phospholipid precursor.