Parental consanguinity is associated with a seven-fold increased risk of progressive encephalopathy: a cohort study from Oslo, Norway.

BACKGROUND/OBJECTIVE: Progressive encephalopathy (PE) is a heterogeneous group of individually rare diseases, many with an autosomal recessive mode of inheritance. We estimated the increased risk of PE associated with consanguinity. PATIENTS AND METHODS: Using a historic cohort study design, the exposures were country of origin (Pakistan versus Norway) and consanguinity. We included children living in Oslo, born between 1985 and 2003. PE cases were retrieved from an electronic registry of diagnoses coded according to the International Classification of Diseases. Incidence rates were calculated for country of origin. We also estimated population attributable risks caused by consanguinity. RESULTS: We identified 30 cases per 79 704 person years with Pakistani origin and 35 cases per 658 932 person years with Norwegian origin. This gave incidence rates of 37.6 and 5.3 per 100 000 person years, whereas the incidence rate ratio was 7.1 (95% CI: 4.2-11.9). The incidence rates of consanguineous versus non-consanguineous of Pakistani origin were 59.6 and 18.7 per 100 000 person years. The incidence rate ratio was 3.2 (95% CI: 1.4-7.2), whereas the incidence rate ratio of non-consanguineous Pakistani versus non-consanguineous Norwegian origin was 3.5 (95% CI: 1.6-7.6). The incidence rate ratio between consanguineous Pakistanis and Norwegians was 11.2. The population attributable risk due to parental consanguinity was 50.3% in the Pakistani sub-population. CONCLUSIONS: We found a seven-fold increased risk of PE in the general Pakistani population, and an eleven-fold increased risk in consanguineous Pakistanis. Pakistani origin by itself was also an independent risk factor. Avoidance of consanguinity in the Pakistani population would result in at least 50% reduction of PE in that group.

Further evidence of an Amerindian contribution to the Polynesian gene pool on Easter Island.

Available evidence suggests a Polynesian origin of the Easter Island population. We recently found that some native Easter Islanders also carried some common American Indian (Amerindian) human leukocyte antigen (HLA) alleles, which probably were introduced before Europeans discovered the island in 1722. In this study, we report molecular genetic investigations of 21 other selected native Easter Islanders. Analysis of mitochondrial DNA and Y chromosome markers showed no traces of an Amerindian contribution. However, high-resolution genomic HLA typing showed that two individuals carried some other common Amerindian HLA alleles, different from those found in our previous investigations. The new data support our previous evidence of an Amerindian contribution to the gene pool on Easter Island.

Effect of intravenous bilirubin infusion on biliary phospholipid secretion, hepatic P-glycoprotein expression, and biliary cytotoxicity in pigs.

BACKGROUND: Infusion of large intravenous bilirubin loads in bile acid-depleted pigs reduces P-glycoprotein-dependent biliary phospholipid secretion and increases the cytotoxicity of bile. The reasons for the diminution of biliary phospholipid secretion and the increase in biliary cytotoxicity are not known. This study was undertaken to determine whether the bilirubin-induced lowering of biliary phospholipid secretion is associated with alterations in hepatic P-glycoprotein (P-gp) expression and to determine why bilirubin infusions increase biliary cytotoxicity. METHODS: Hepatic bile was collected from bile acid-depleted pigs before and during intravenous bilirubin infusion. Hepatic P-gp expression was measured with protein blot analysis, using the P-gp-specific antibody C219. Biliary cytotoxicity was assayed against erythrocytes. The biliary phospholipid fatty acid profile was determined by means of gas chromatography. RESULTS: Bilirubin infusions lowered biliary phospholipid secretion by 69% without changing hepatic P-gp expression, suggesting that bilirubin infusions have an inhibitory effect on hepatic P-gp activity. Bilirubin infusions did not cause P-gp losses into bile. An unequivocal, proportional relationship (r2 = 0.80) pertained between cytotoxicity and the bile acid to phospholipid ratio in bile secreted before and during bilirubin infusion and in phosphatidylcholine-supplemented bile. Unconjugated bilirubin in bile did not contribute to biliary cytotoxicity. Biliary phospholipids were always phosphatidylcholine >> phosphatidylethanolamine, mainly of C16:0, 18:2 and C16:0, 18:1 fatty acid configuration. CONCLUSIONS: Intravenous bilirubin loads reduce biliary phospholipid secretion without changing hepatic P-gp expression. Bilirubin infusions increase biliary cytotoxicity by augmenting the biliary bile acid to phospholipid ratio.

Membrane fluidity and fatty acid metabolism in kidney cells from rats fed purified eicosapentaenoic acid or purified docosahexaenoic acid.

Rats were given a supplement (1.5 ml/day) of purified eicosapentaenoic acid (EPA, 20:5,n-3), purified docosahexaenoic acid (DHA, 22:6,n-3)), or corn oil for 10 days. Membrane fluidity, measured as the steady-state fluorescence polarization of diphenylhexatriene (DPH), was approximately 20% lower in kidney cells from rats fed purified EPA than in cells from the DHA-fed or corn-oil fed animals. The level of 20:5(n-3) in kidney phospholipids was 18 times higher in rats fed EPA, and four times higher in those fed DHA as compared to the corn-oil group. The level of arachidonic acid (20:4,n-6) was concomitantly decreased, while linoleic acid (18:2,n-6) was increased in kidney-phospholipids in the n-3 fatty acid fed rats. The proportion of 22:6(n-3) in kidney phospholipids was not affected by EPA supplementation, while the DHA diet slightly increased the level of this fatty acid. The distribution of phospholipid subclasses was significantly altered in that phosphatidylcholine was increased and phosphatidylethanolamine was concomitantly decreased. It is suggested that the decrease in 20:4(n-6) is relatively more important in the regulation of fluidity than a concomitant increase in 20:5(n-3). It is also suggested that the compensatory modifications of the phospholipid subclass distribution as a response to decreased 20:4(n-6)/20:5(n-3) ratio was not sufficient to maintain fluidity when the ratio was as low as in the present study. The incorporation of labelled linolenic acid (18:3,n-3) in phospholipids was decreased in cells from the n-3 supplemented rats. Since endogenous 22:5(n-3) in phospholipids was only increased in the EPA group, 22:6(n-3) only in the DHA group, and 20:5(n-3) in both, it is suggested that the decreased incorporation of labelled 18:3(n-3) into phospholipids of the DHA-fed rats in particular is correlated to the increased level of 22:6(n-3) in the membrane phospholipids. The incorporation of fatty acids into phopholipids may thus show substrate specificity, in that 22:6(n-3) is less exchangable with labelled 18:3(n-3) than is 20:5(n-3). These results demonstrate that increasing levels of n-3 fatty acids in membranes affect the uptake and intracellular metabolism of fatty acids as well as membrane fluidity in the kidney.

A comparative study of the metabolism of n-9, n-6 and n-3 fatty acids in testicular cells from immature rat.

Dietary 18 and 20-carbon fatty acids of the n-6 and the n-3 families are metabolized to 22:5,n-6 and 22:6,n-3 by a sequence of specific desaturases and chain elongation via 24-carbon intermediates. This pathway is regulated so that more 22:6,n-3 than 22:5,n-6 is found in the tissues. Rat testis is an exception since 22:5,n-6 is present in large proportions in this organ. Therefore rat testis appears to be interesting for studies of the detailed synthesis of 22:5,n-6 compared with that of 22:6,n-3. By using fresh preparations of rat testicular cells from 19-day-old rats enriched in Sertoli cells, we compared the metabolism of 1-14C-labelled n-3, n-6 and n-9 fatty acids. The testicular cells actively synthesized 22:6,n-3 and 22:5, n-6, but not 22:4,n-9 from the 18 and 20-carbon precursors. Of 200 mol 14C-labelled C18 and C20 fatty acids added initially, approximately 20-40 mol were found as 24-carbon intermediates after 24 h of incubation. This indicates that the balanced capacity of elongation, desaturation and chain shortening favours the accumulation of 24-carbon intermediates in these cells. One exception was [1-14C]20:3,n-9 which was efficiently elongated to 22:3,n-9 but not to C24 fatty acids. Our data suggests that the poor elongation of n-9 fatty acids from C22- to C24 may be an important hindrance in the synthesis of 22:4,n-9. The efficient synthesis of 22:5,n-6 may also partly explain why this is the major 22-carbon fatty acid in rat testis.

Monounsaturated trans fatty acids, elaidic acid and trans-vaccenic acid, metabolism and incorporation in phospholipid molecular species in hepatocytes.

The incorporation of [14C]elaidic acid (trans18:1(n-9)) in phosphatidylcholine and phosphatidylethanolamine molecular species in isolated rat liver cells has been studied, and the results compared with the incorporation, previously published (B. Woldseth et al. Biochim Biophys Acta 1993; 1167: 296-302), of [14C]palmitic acid (16:0) and [14C]stearic acid (18:0) and with that of [14C]oleic acid (cis18:1(n-9)). The pattern of incorporation in phospholipid molecular species is similar to that of [14C]stearic acid and different from that of [14C]palmitic acid. In phosphatidylcholine [14C]trans18:1-18:2 and [14C]trans18:1-20:4 were the most abundant species, and in phosphatidylethanolamine [14C]trans18:1-20:4 was the predominant species. With increasing concentration of [14C]elaidic acid increasing amounts of [14C]trans18:1-[14C]trans18:1 were found. The total incorporation in phospholipids was less than that of [14C]stearic acid, but more than that of [14C]palmitic acid. The distribution in percent of [14C]elaidic acid in phospholipid classes was 8.8% in phosphatidylinositol, 1.8% in phosphatidylserine, 59.1% in phosphatidylcholine and 30.3% in phosphatidylethanolamine with 0.1 mmol l-1 substrate concentration. More [14C]elaidic acid than [14C]palmitic acid or [14C]stearic acid was oxidized. The incorporation in phospholipids of [14C]elaidic acid was very different from that of [14C]oleic acid. The main species with [14C]oleic acid were 16:0-[14C]cis18:1 in phosphatidylcholine, and [14C]cis18:1-20:4 in phosphatidylethanolamine. In some experiments [14C]18:2(n-6) was incubated together with unlabelled elaidic or unlabelled trans-vaccenic acid (trans18:1(n-7)). In these experiments, more trans18:1-18:2 was formed from elaidic acid than from trans-vaccenic acid, especially in phosphatidylethanolamine.

The metabolism of 22:5(-6) and of docosahexaenoic acid [22:6(-3)] compared in rat hepatocytes.

Elevated levels of 22:5(-6), which is the elongated and desaturated product of arachidonic acid, is induced by selective n-3 fatty acid deficiency, especially in brain cortex. Less elongation and desaturation of 20:4(-6) than of 20:5(-3) has been found in intact rat liver cells in previous studies and is probably the main reason why so little 22:5(-6) is found under adequate nutritional conditions. The present study compares the metabolism of 22:5(-6) with the metabolism of 22:6(-3), the main n-3 fatty acid in mammals. Freshly isolated rat liver cells were incubated with [1-14C]22:5(-6) and [1-14C]22:6(-3). Oxidation and esterification in triacylglycerols, diacylglycerols and phospholipids were studied. The phospholipid classes were separated and the different molecular species identified. Rats with essential fatty acid deficiency were compared with control rats. 22:5(-6) was found to be a good substrate for membrane phospholipid biosynthesis and was conserved well in the phospholipid fraction of the rat liver cells for more than 3 h of incubation. More 22:5(-6) was esterified in the total phospholipid fraction and less was incorporated in triacylglycerols than observed with 22:6(-3) in hepatocytes from control animals. This was not the case in animals with essential fatty acid deficiency. 22:5(-6) was esterified to a greater extent in phosphatidylcholine than 22:6(-3) in control cells but not in essential fatty acid deficiency cells. More 22:5(-6) was coupled with 18.0 in the sn-1 position of the phospholipid molecular species than 22:6(-3) was in control cells.

Phospholipid molecular species with eicosapentaenoic acid (20:5(n-3)) are less stable than species with arachidonic acid (20:4(n-6)) in isolated rat liver cells.

We have studied the incorporation of [1-14C]20:5(n-3) and [1-14C]20:4(n-6) in the molecular species of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in isolated rat liver cells. These two fatty acids are present in very different amounts in endogenous phospholipids, with 20:4 as one of the major fatty acids and 20:5 as a minor and very diet-dependent constituent. The main phospholipid species formed from 20:4(n-6) were 16:0-20:4 and 18:0-20:4. When formed, they were stable during incubations of liver cells for 2-3 h. The main species formed from 20:5(n-3) were 16:0-20:5 and 18:0-20:5. After formation, 16:0-20:5 and to a lesser degree 18:0-20:5 were, however, degraded during 1-2 h of incubation, especially in PC. Only small amounts of 22:5(n-3) and very little 22:6(n-3) were formed from 20:5(n-3) and small amounts of 22:4(n-6) were produced from 20:4(n-6). With 20:4(n-6) and 20:5(n-3) as substrates, 20:4-20:4 and 20:5-20:5 molecular species respectively were initially formed in PC and PE but both species were rapidly degraded.

Studies on the metabolism of [1-14C]5.8.11-eicosatrienoic (Mead) acid in rat hepatocytes.

The oxidation, esterification and formation of chain elongated and desaturated products of [1-14C]5,8,11-eicosatrienoic (Mead) acid was studied. Liver cells from essentially fatty acid deficient (EFAD) and control rats were used. The metabolism of [1-14C]20:4, n-6 and [1-14C]20:5, n-3 were studied under the same experimental conditions. More 20:3, n-9 than 20:4, n-6 and 20:5, n-3 was oxidised both in EFAD and control cells. 20:3, n-9 was elongated to [14C]22:3, n-9 in both cell types and significant amounts of [14C]22:4, n-9 were formed in EFAD cells. Less 20:3, n-9 was esterified in phospholipids and more in triacylglycerol than observed with 20:4, n-6 and 20:5, n-3 in both cell types. 20:3, n-9 was mainly esterified in phosphatidylcholine and little was esterified in phosphatidylethanolamine compared to 20:4, n-6 and 20:5, n-3. In comparison, 20:3, n-9 was rather efficiently esterified in phosphatidylinositol as 18:0-20:3. [14C]22:4, n-9 formed from 20:3, n-9 in EFAD hepatocytes was esterified in triacylglycerol, not in phospholipids, unlike [14C]22:5, n-6 and [14C]22:6, n-3 which were mainly esterified in phospholipids.

Biosynthesis of phospholipid molecular species in isolated liver cells studied by combining fatty acid substrates esterified in the sn-1 and sn-2 positions.

The simultaneous incorporation of a saturated fatty acid in the sn-1 position and an unsaturated fatty acid in the sn-2 position in phosphatidylcholine (PC) and ethanolamine (PE) was studied in isolated liver cells. We combined a saturated fatty acid, 16:0 or 18:0 and an unsaturated fatty acid substrate, 18:2,n-6 or 20:4,n-6. In this situation the saturated fatty acids were preferentially oxidized and the unsaturated fatty acids were preferentially esterified in PL and TG. Addition of unlabelled 16:0 increased the incorporation of [14C]18:2 in 16:0-18:2 in PC and PE, reduced the incorporation in 18:2-18:2 but did not reduce the incorporation in 18:0-18:2. 18:0 increased the esterification of [14C]18:2 in 18:0-18:2, reduced the incorporation in 18:2-18:2 but did not reduce the incorporation in 16:0-18:2. The latter is the dominating 14C-labelled species formed from [14C]18:2 also in the presence of unlabelled 18:0. Addition of 20:4 stimulated the incorporation of [14C]16:0 in 16:0-20:4 and markedly reduced the formation of 16:0-18:2, 16:0-18:1 and 16:0-22:6. Addition of 18:2 increased the incorporation of [14C]16:0 in 16:0-18:2 and reduced the formation of 16:0-20:4 and 16:0-18:1. It is concluded that the unsaturated fatty acids 18:2 or 20:4 have a stronger impact on the synthesis of phospholipid molecular species than the saturated fatty acids 16:0 or 18:0 have. Thus 20:4,n-6 and 18:2,n-6 are able to direct available [14C]16:0 or [14C]18:0 to the sn-1 position. 16:0 and 18:0 are not in the same way able to direct [14C]18:2,n-6 to the synthesis of 16:0-18:2 or 18:0-18:2 at the expense of other 14C-labelled molecular species.

Incorporation of stearic acid (18:0) and palmitic acid (16:0) in phospholipid molecular species studied in isolated rat liver cells.

The incorporation of [1-14C]16:0 and [1-14C]18:0 in the molecular species of PC and PE in isolated rat liver cells was studied. More [14C]18:0 than [14C]16:0 was esterified both in PC and PE. Also the chain elongated and desaturated products (16:1, 18:0 and 18:1) were incorporated. The main molecular phospholipid species formed from [14C]18:0 were 18:0-18:2, 18:0-20:4 and 18:0-22:6. 18:0-18:0 species was not detected, independent of the substrate concentration (0.1-0.9 mM). With [14C]16:0 at low substrate concentration (0.1 mM) the dominating species are 16:0-18:2, 16:0-20:4 and 16:0-22:6. These species were detected already after 10 min. The same main species are formed both in PC and PE, but the relative amounts differ. In PC the combination with 18:2 is most abundant for both saturated fatty acid substrates. In PE 18:0-20:4 dominates when 18:0 is the substrate, and 16:0-22:6 when 16:0 is. At higher substrate concentrations (0.4-0.9 mM) 16:0 is also esterified in 16:0-16:0. This molecular species is efficiently degraded in the cells within 2-3 h, in contrast to the other species formed. The results suggest that 16:0 and 18:0 are directly incorporated in the sn-1 position in physiologically important phospholipid molecular species. With an excess of 16:0, 16:0-16:0 is also formed in substantial amounts, but this uncommon species is thereafter removed.

Peroxisomal beta-oxidation of polyunsaturated long chain fatty acids in human fibroblasts. The polyunsaturated and the saturated long chain fatty acids are retroconverted by the same acyl-CoA oxidase.

The metabolism of the C22 unsaturated fatty acids erucic acid (22:1(n-9)), adrenic acid (22:4(n-6)), docosapentaenoic acid (22:5(n-3)) and docosahexaenoic acid (22:6(n-3)) was studied in cultured fibroblasts from patients with acyl-CoA oxidase deficiency, the Zellweger syndrome, X-linked adrenoleukodystrophy (X-ALD) and normal controls. [3-14C] 22:4 (n-6) and [3-14C] 22:5 (n-3) were shortened (retroconverted) to [1-14C] 20:4 (n-6) and [1-14C] 20:5 (n-3), respectively, in normal and X-ALD fibroblasts. In Zellweger and acyl-CoA oxidase deficient fibroblasts these reactions were deficient. Since the retroconversion is normal in X-ALD fibroblasts peroxisomal very long chain (lignoceryl) CoA ligase is probably not required for the activation of C22 unsaturated fatty acids. The present work with fibroblasts from patients with a specific acyl-CoA oxidase deficiency, previously shown to have a deficient peroxisomal clofibrate-inducible acyl-CoA oxidase, and which accumulate 24:0 and 26:0 fatty acids, supports the view that this enzyme is responsible for the chain-shortening of docosahexaenoic acid (22:6(n-3)), erucic acid (22:1(n-9)), docosapentaenoic acid (22:5(n-3)), and adrenic acid (22:4(n-6)) as well.