Oral vitamin B(12) and high-dose folic acid in hemodialysis patients with hyper-homocyst(e)inemia.

BACKGROUND: Hyper-homocyst(e)inemia is an independent risk factor for atherosclerotic vascular disease in patients with end-stage renal disease (ESRD), although optimal treatment remains unknown. This randomized, double-blind, placebo-controlled study was designed to measure the effect of high-dose oral vitamin B(12) and folic acid on predialysis total homocyst(e)ine levels in patients with ESRD. METHODS: We studied 81 hemodialysis patients who had hyper-homocyst(e)inemia (>16 micromol/L) on varied doses of a multivitamin containing 1 mg of folic acid/day. After screening blood work, all patients were switched to daily multivitamin therapy, including 1 mg of folic acid for four weeks. For all patients, vitamin B(12), 1 mg/day, was added for an additional four weeks. Patients were then randomized to receive four weeks of 0, 5, or 20 mg of folic acid in addition to the multivitamin and vitamin B(12) (all given daily). RESULTS: Screening homocyst(e)ine levels (mean 27.7 micromol/L) decreased by 19.2% after four weeks of treatment with a daily multivitamin containing 1 mg of folic acid (P < 0.001). Homocyst(e)ine levels were reduced further from 22.3 to 18.6 micromol/L (mean reduction 16.7%, 95% CI 11.8 to 21.6%, P < 0.001) after four weeks of therapy with vitamin B(12) (1 mg/day). There was no significant difference in mean reduction of homocyst(e)ine levels after therapy with high-dose folic acid compared with placebo (P = 0.35). CONCLUSIONS: The optimal oral treatment of hyper-homocyst(e)inemia in hemodialysis patients consists of 1 mg of folic acid and 1 mg of oral vitamin B(12) daily. Whether this treatment will lower the risk of future atherosclerotic vascular events remains to be investigated.

Identification, expression, and characterization of Escherichia coli guanine deaminase.

Using the human cDNA sequence corresponding to guanine deaminase, the Escherichia coli genome was scanned using the Basic Local Alignment Search Tool (BLAST), and a corresponding 439-residue open reading frame of unknown function was identified as having 36% identity to the human protein. The putative gene was amplified, subcloned into the pMAL-c2 vector, expressed, purified, and characterized enzymatically. The 50.2-kDa protein catalyzed the conversion of guanine to xanthine, having a K(m) of 15 microM with guanine and a k(cat) of 3.2 s(-1). The bacterial enzyme shares a nine-residue heavy metal binding site with human guanine deaminase, PG[FL]VDTHIH, and was found to contain approximately 1 mol of zinc per mol of subunit of protein. The E. coli guanine deaminase locus is 3' from an open reading frame which shows homology to a bacterial purine base permease.

The CoA-independent transacylase in PAF biosynthesis: tissue distribution and molecular species selectivity.

Microsomal membranes from six different rat tissues (spleen, lung, kidney, brain, testis, and liver) were found to possess CoA-independent transacylase activity that could both acylate lyso-[3H]PAF (1-[3H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine) and then deacylate the 1-[3H]hexadecyl-2-acyl-sn-glycero-3-phosphocholine product via the transacylation of added exogenous 1-alk-1'-enyl-2-lyso-sn-glycero-3-phosphoethanolamine. Platelet-activating factor (1-[3H]hexadecyl-2-acetyl-sn-glycero-3-phosphocholine) was produced when acetyl-CoA was added to the spleen microsomes during generation of lyso-[3H]PAF by the transacylases. More extensive studies with subcellular fractions from spleen revealed that, in addition to microsomes, the transacylase activities were also present in the 15,000 x g membrane fraction but not in the cytosol. Analysis of molecular species of 1-[3H]hexadecyl-2-acyl-sn-glycero-3-phosphocholine before and after addition of 1-alk-1'-enyl-2-lyso-sn-glycero-3-phosphoethanolamine as the acyl acceptor demonstrated a high selectivity for polyunsaturated fatty acids (> 3 double bonds/acyl group) in both the acylation and deacylation processes that occurred in testicular microsomal membranes. The transfer of acyl groups by the transacylase appeared to be equally effective for either arachidonic or docosapentaenoic(n - 6) fatty acids, whereas linoleic and oleic fatty acids were not transferred from 1-[3H]hexadecyl-2-acyl-sn-glycero-3-phosphocholine following the addition of 1-alk-1'-enyl-2-lyso-sn-glycero-3-phosphoethanolamine. Similar experiments with the membrane fraction of undifferentiated HL-60 cells showed that arachidonic acid supplementation of intact cells enhanced both the CoA-independent transacylation of lyso-[3H]PAF and the subsequent deacylation of 1-[3H]hexadecyl-2-acyl-sn-glycero-3-phosphocholine caused by addition of 1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine. Differentiation of the HL-60 cells into a neutrophil-like form had no effect on the transacylase activity. Our results indicate the PAF-related transacylase is widely distributed among tissues and, although highly selective for polyunsaturated acyl groups, does not discriminate selectively among the polyunsaturates.

Molecular species of ethanolamine plasmalogens and transacylase activity in rat tissues are altered by fish oil diets.

Effects of dietary fish oil ethyl esters and alkyldiacetylglycerols (an ether-linked lipid) on the distribution of subclasses of choline- and ethanolamine-glycerophospholipids as well as effects on highly unsaturated molecular species of ethanolamine plasmalogens from brain, spleen, kidney, lung, and testis of rats were examined. Supplementation of ethyl ester concentrates of n-3 fatty acids had no effect on the distribution of subclasses in any of the tissues. However, the supplements of 1-O-octadec-9'-enyl-2,3-diacetyl-sn-glycerol (diacetates of selachyl alcohol) caused significant increases in the alkylacylglycerophosphocholine and alkylacylglycerophosphoethanolamine subclasses from spleen and lung and in the alkylacylglycerophosphoethanolamine subclass from kidney. Dietary supplements of fish oil ethyl esters reduced the arachidonate-containing species of ethanolamine plasmalogens whereas molecular species having 20:5(n-3), 22:6(n-3), and/or 22:5(n-3) acyl groups were increased in the spleen, lung, and kidneys, but not brain. In testicular tissue from rats fed the fish oil diets, the molecular species of ethanolamine plasmalogens containing 22:5(n-6) acyl groups were reduced. An increase of ethanolamine plasmalogens with 18:1 alk-1-enyl moieties paired with highly unsaturated sn-2 acyl groups were found in the tissues of rats fed the fish oil plus selachyl alcohol diacetate supplements. Rats on the diet containing fish oil ethyl esters had significantly lower [3H]alkyllysoglycerophosphocholine CoA-independent transacylase activity in spleen microsomes than controls. This suggests that supplements of n-3 fatty acids interferes with the transacylation of arachidonate, an event that could seriously impair the release of arachidonate and lysophospholipids (e.g., lyso-PAF) that are precursors of potent bioactive lipid derivatives.

A novel CoA-independent transacetylase produces the ethanolamine plasmalogen and acyl analogs of platelet-activating factor (PAF) with PAF as the acetate donor in HL-60 cells.

In this study, we demonstrate the presence of a unique membrane-associated transacetylase that transfers the acetate group from platelet-activating factor (PAF) to lysoplasmalogen (in the presence of EDTA and sodium acetate) with the formation of 1-alk-1-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine (alk-1-enylacetyl-GPE). The identity of alk-1-enylacetyl-GPE was confirmed by acid hydrolysis, phospholipases A2 or C treatment and derivatization by fluorodinitrobenzene. The transacetylase has no requirement for Ca2+, Mg2+, or CoA and a broad pH optimum (7.0-8.0) with Km values of 12.0 microM for PAF and 106.4 microM for lysoplasmalogens. The enzyme activity from the isolated membrane fraction is not changed when whole cells are supplemented with 20:4, induced to differentiate into granulocytes, or treated with ionophore A23187. Radyllyso-sn-glycero-3-phosphocholine (GPC), radyllyso-GPE, acyllyso-sn-glycero-3-phosphoserine (GPS), acyllyso-sn-glycero-3-phosphoinositol (GPI), alkyllyso-sn-glycero-3-phosphate (GP), acyllyso-GP, or cis-9-octadecen-1-ol can also serve as acetate acceptors, whereas alkylglycerol, acylglycerol, or cholesterol are inactive. Differences in substrate acceptor specificity, sensitivity toward phenylmethylsulfonyl fluoride, and response to temperature suggest that the CoA-independent transacetylase and the CoA-independent transacylase that transfers long-chain acyl moieties are two separate enzymes. With intact differentiated HL-60 cells, [3H]acetate from [3H]PAF can be incorporated into alk-1-enylacetyl-GPE in the presence of ionophore A23187, but not in its absence. Moreover, phospholipase A2 inhibitors (p-bromophenacyl bromide and mepacrine) block the transacetylation process in whole cell system. These results indicate the production of alk-1-enyllyso-GPE is a rate-limiting factor for the subsequent transacetylation step during cell activation. We conclude that the transacetylase may participate in the biosynthesis of ethanolamine plasmalogen and acyl analogs of PAF, in vivo, fine-tuning of PAF biological responses, and cross-talk between de novo and remodeling pathways of PAF biosynthesis.

Contributing factors in the trafficking of [3H]arachidonate between phospholipids.

Cultured human promyelocytic leukemia cells (HL-60), depleted of arachidonic acid by continued growth in serum-free media, were used as a model system to examine various factors that control the incorporation and distribution of [3H]arachidonic acid into classes and subclasses of cellular lipids. Increasing the culture media concentration of [3H]arachidonic acid from 1 x 10(-8) M to 1 x 10(-5) M caused a greater percentage of the cellular tritium to be distributed into triacylglycerols (from less than 1% at 1 x 10(-8) M to 38% at 1 x 10(-5) M) with a corresponding decrease in cellular [3H]diradylglycerophosphoethanolamine (from 53% at 1 x 10(-8) M to 12% at 1 x 10(-5) M) during 2 h incubations. A greater proportion of the tritium present in diradylglycerophosphoethanolamine and diradylglycerophosphocholine, at the higher media concentration of [3H]arachidonic acid (1 x 10(-5) M), was found in the diacyl subclasses of these two lipids than was observed at the lower concentrations (less than 1 x 10(-6) M) of [3H]arachidonic acid. Significant amounts of diarachidonoyl molecular species were found in the phosphatidylethanolamine (10%) and phosphatidylcholine (15%) of HL-60 cells that were labeled for 2 h with 1 x 10(-5) M [3H]arachidonic acid. This was the only molecular species of phosphatidylcholine to completely disappear when prelabeled cells were placed in arachidonate-free media for 22 h. Prelabeling-chase experiments with 1 x 10(-5) M [3H]arachidonic acid were consistent with movement of [3H]arachidonate from triacylglycerols into diradylglycerophosphatides and from diacylphospholipids into ether-linked phospholipids. Increasing the concentration of HL-60 cells in the incubations influenced the distribution of [3H]arachidonic acid in cellular lipid classes in a manner analogous to decreasing the concentration of [3H]arachidonic acid in the media. Increasing the endogenous level of cellular arachidonate in phospholipid classes with supplements of unlabeled arachidonic acid changed the subsequent lipid class distribution of a low concentration (1 x 10(-8) M) of [3H]arachidonic acid to resemble results obtained with a much higher mass level of [3H]arachidonate in arachidonate depleted cells. HL-60 cells differentiated into granulocytes by treatment with dimethyl sulfoxide incorporated less [3H]arachidonic acid but had a greater proportion associated with alkylacylglycerophosphocholine and alk-1-enylacylglycerophosphoethanolamine than undifferentiated HL-60 cells.

Significance of phototherapy-induced riboflavin deficiency in the full-term neonate.

As a result of impaired fatty acid oxidation, a characteristic urinary dicarboxylic aciduria occurs in the riboflavin deficient animal. We compared the occurrence of riboflavin deficiency induced by phototherapy with changes in urinary organic acid profiles in 8 full-term, breast-fed neonates who received phototherapy for hyperbilirubinemia, and in 10 full-term, breastfed controls. Riboflavin status was assessed by measuring flavin adenine dinucleotide saturation of erythrocyte glutathione reductase. All 8 neonates exposed to phototherapy developed riboflavin deficiency (p less than 0.001). Riboflavin deficiency was progressive with the duration of phototherapy. None of the controls was riboflavin deficient. Urine organic acid profiles indicative of mitochondrial acyl-CoA dehydrogenase activity (fatty acid beta-oxidation, quantitated by gas chromatography mass spectrometry) showed no changes between the study and control groups in mono-, di-, or tricarboxylic acids or other organic acids. The riboflavin deficiency induced by phototherapy in full-term neonates was not of sufficient severity to limit riboflavin-dependent fatty acid oxidation.

An arachidonoyl (polyenoic)-specific phospholipase A2 activity regulates the synthesis of platelet-activating factor in granulocytic HL-60 cells.

Human promyelocytic leukemia cells (HL-60) were used as a cell model to determine how arachidonic acid stimulates the synthesis of platelet-activating factor (PAF) synthesized via the remodeling pathway. In these studies HL-60 cells were cultured over 30 passages in fatty acid-free medium to deplete them of arachidonic acid. Even though the phospholipid classes from these cells contained no arachidonate, they could still be differentiated into granulocytes by dimethyl sulfoxide (1.25%). When the differentiated HL-60 cells, depleted of arachidonic acid, were stimulated with calcium ionophore A23187 in the presence of Ca2+ and [3H]acetate, only minimal amounts of [3H]PAF were produced. In contrast, if the differentiated HL-60 cells were supplemented with 10 microM arachidonic acid for 24 h and then stimulated with the ionophore, there was a large amount of [3H]PAF formed. The increase in PAF synthesis depended on the length of time the cells were supplemented with arachidonic acid; only a small increase in PAF synthesis occurred during the early hours of supplementation whereas stimulation of PAF synthesis was maximal (3-5-fold) after a 24-h period of the 20:4 supplementation. Other polyenoic fatty acid supplements (20:5, 22:4, and 22:6 for 24 h) also stimulated PAF production in the ionophore-treated HL-60 cells depleted of 20:4, but the amount of PAF was significantly less than found for the supplements of 20:4 under identical experimental conditions. Also noteworthy is that undifferentiated cells supplemented with 20:4 or their unsupplemented controls could not be stimulated by the calcium ionophore to produce PAF. Addition of indomethacin (cyclooxygenase inhibitor), A63162 (5'-lipoxygenase inhibitor), or eicosatetraynoic acid (cyclooxygenase/lipoxygenase inhibitor) to the incubations caused little change in the production of [3H]PAF in the differentiated cells supplemented with 20:4 for 24 h. On the other hand, the addition of mepacrine, bromophenacyl bromide, or U26384 (phospholipase A2 inhibitors) resulted in very large decreases (80-90% lower than controls) in the amount of [3H]PAF produced under the same conditions. Analysis of the molecular species of [3H]alkylacyl-GroPCho (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine, the precursor of PAF in the remodeling pathway) in 20:4-supplemented cells prelabeled with [3H]alkyl-lyso-GroPCho revealed that only the alkylarachidonoyl-GroPCho species were preferentially decreased after stimulation with the A23187 ionophore. These results demonstrate that arachidonate must be at the sn-2 position of alkylacyl-GroPCho in order for it to serve as a precursor of PAF.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of eicosapentaenoic and docosahexaenoic acid supplements on phospholipid composition and plasmalogen biosynthesis in P388D1 cells.

This investigation describes the influence of n-3 fatty acid supplements on the phospholipid composition and the metabolism of plasmalogens in P388D1 cells. The cellular content of phospholipid classes and subclasses was unchanged in P388D1 cells (a macrophage-like cell) grown for 24 h in media supplemented with 10 microM sodium eicosapentaenoate or sodium docosahexaenoate. However, phospholipids from these cells were highly enriched in acyl groups of the corresponding fatty acid supplement, with the largest increases occurring in the ethanolamine plasmalogens (e.g., 46% of the ethanolamine plasmalogens from cells supplemented with docosahexaenoate contained this acyl group at the sn-2 position). Eicosapentaenoate supplements lowered the levels of oleate in phosphatidylinositol/serine, diacyl-sn-glycero-3-phosphoethanolamine (GroPEtn), and alk-1-enylacyl-GroPEtn in the P388D1 cells but had little or no effect on the amounts of arachidonate in the cellular phospholipids. In contrast, supplementation of the cells with docosahexaenoic acid not only reduced the level of oleate but also decreased the amount of arachidonate by one-third in the alk-1-enylacyl-GroPEtn. When P388D1 cells were incubated for 1 h with [3H]alkyllyso-GroPEtn both [3H]alkylacyl-GroPEtn and [3H]alk-1-enylacyl-GroPEtn were formed. The sn-2 acyl composition of these two ether-containing GroPEtn lipids reflected the fatty acid supplement that the cells had received (e.g., 68% of the [3H]alk-1-enylacyl-GroPEtn from cells supplemented with docosahexaenoate contained this acyl group at the sn-2 position). Cells from both the controls and supplemented groups contained greater amounts of docosahexaenoate in the [3H]alk-1-enylacyl-GroPEtn (plasmalogen) than in the [3H]alkylacyl-GroPEtn subclass. Analysis of molecular species from pulse-chase experiments with intact cells and examination of the molecular species of [3H]alk-1-enylacyl-GroPEtn produced by the delta 1-desaturase system in cell-free membrane fractions suggest that the docosahexaenoate-containing species of [3H]alk-1-enylacyl-GroPEtn have a higher turnover rate than other molecular species. Possible biological implications of our findings are also discussed.

Argininosuccinic aciduria: long-term treatment with arginine.

The presentation and 2 year treatment of a patient with argininosuccinic aciduria is reported. Erythrocyte argininosuccinate lyase activity was less than 2% of normal. Long-term management included protein restriction and arginine dietary supplementation. The child experienced three episodes of hyperammonaemia (greater than 100 microns), the first at birth, the second at 6.5 months and the third at 16 months. Neurological development deteriorated between 14 and 24 months. Hepatomegaly and biochemical hepatitis, a feature of this condition, was accompanied by enlarged mitochondria with tubular paracrystalline inclusions not previously recognized in this disorder.

Increasing the levels of ether-linked lipids in L-M cells by glyceryl ether supplementation depresses growth and choline utilization.

To gain insight into the role of alkyl-linked lipids in biological systems, we added hexadecylglycerol (a precursor of complex ether-linked lipids) to medium required for the growth of L-M cells in culture. L-M fibroblasts cultured through several generations in the presence of hexadecylglycerol grow at a reduced rate. Experimental cells at their sixth passage, with 2 microgram supplement/ml, double at 50% the rate of control cell populations. Hexadecylglycerol (10 microgram/ml) added 1 day after cell passage does not retard growth; however, within 1 h it decreases the incorporation of choline into the choline glycerophosphatide fraction. Inhibition is specific for choline; ethanolamine incorporation is not affected. The inhibition of choline utilization by hexadecylglycerol-treated cells is dose-dependent and reaches a maximum 12 h after supplementation. Cellular uptake of choline is reduced (approx. 17%) but not as much as the incorporation of choline into the phospholipids (approx. 60% at 12 h). The assimilation of ether lipid precursor into cellular phospholipids was followed by incubating cells with [1-14C]hexadecylglycerol. Incorporation of radioactivity into cellular phospholipids begins to plateau after 24 h, whereas the interference of hexadecylglycerol with choline metabolism could be detected as early as 1 h. The majority of the radioactivity recovered from cells incubated with labeled hexadecylglycerol is localized in the microsomal fraction (56%), where the label was distributed as free hexadecyglycerol, alkylacyl-phospholipids and alkyldiacylglycerols. These results show that the supplementation of a glyceryl ether to L-M fibroblast growth media selectively inhibits the utilization of choline for choline glycerophospholipid biosynthesis and causes a reduction in cell growth rate when cells are continually passaged in the presence of the glyceryl ether.