Minne Casteels

A mouse model for Zellweger syndrome

The cerebro-hepato-renal syndrome of Zellweger is a fatal inherited disease caused by deficient import of peroxisomal matrix proteins. The pathogenic mechanisms leading to extreme hypotonia, severe mental retardation and early death are unknown. We generated a Zellweger animal model through inactivation of the murine Pxr1 gene (formally known as Pex5) that encodes the import receptor for most peroxisomal matrix proteins. Pxr1−/− mice lacked morphologically identifiable peroxisomes and exhibited the typical biochemical abnormalities of Zellweger patients. They displayed intrauterine growth retardation, were severely hypotonic at birth and died within 72 hours. Analysis of the neocortex revealed impaired neuronal migration and maturation and extensive apoptotic death of neurons.

Phytosterolemia in children with parenteral nutrition—Associated cholestatic liver disease

BACKGROUND Lipid emulsions used for parenteral nutrition (PN) contain phytosterols. Our hypothesis was that these phytosterols can accumulate and contribute to cholestatic liver disease and other complications of PN, e.g., thrombocytopenia (which occurs in hereditary phytosterolemia). METHODS Using gas chromatography-mass spectrometry, plasma concentrations of sterols were measured in 29 children aged 2 months to 9 years receiving PN and in 29 age-matched controls. The children receiving PN fell into two subgroups: 5 with severe PN-associated cholestatic liver disease (bilirubin level, > 100 mumol/L; aspartate aminotransferase [AST] level, > 200 U/L) and 24 with a bilirubin level of < 100 mumol/L and/or AST level of < 200 U/L. RESULTS The 5 children with severe PN-associated liver disease had plasma concentrations of phytosterols and sitostanol that were as high as those seen in patients with hereditary phytosterolemia (total phytosterols 1.3-1.8 mmol/L). All 5 had intermittent thrombocytopenia. A reduction in intake of lipid emulsion to < 50 mL.kg-1.wk-1 was associated with a decrease in plasma phytosterol concentrations and an improvement in liver function tests and platelet counts in two patients. Children with less severe PN-associated liver disease had lower plasma phytosterol concentrations than the 5 with severe disease. CONCLUSIONS Children receiving PN who have high plasma phytosterol concentrations also have cholestatic liver disease and thrombocytopenia; phytosterolemia might contribute to the pathogenesis of complications of PN.

Peroxisomal lipid degradation via beta- and alpha-oxidation in mammals.

Peroxisomal β-oxidation is involved in the degradation of long chain and very long chain fatty acyl-(coenzyme A)CoAs, long chain dicarboxylyl-CoAs, the CoA esters of eicosanoids, 2-methyl-branched fatty acyl-CoAs (e.g. pristanoyl-CoA), and the CoA esters of the bile acid intermediates di- and trihydroxycoprostanic acids (side chain of cholesterol).In the rat, straight chain acyl-CoAs (including the CoA esters of dicarboxylic fatty acids and eicosanoids) are β-oxidized via palmitoyl-CoA oxidase, multifunctional protein-1 (which displays 2-enoyl-CoA hydratase and L-3-hydroxyacyl-CoA, dehydrogenase activities) and peroxisomal thiolase. 2-Methyl-branched acyl-CoAs are degraded via pristanoyl-CoA oxidase, multifunctional protein-2 (MFP-2) (which displays 2-enoyl-CoA hydratase and D-3-hydroxyacyl-CoA dehydrogenase activities) and sterol carrier protein-X (SCPX; displaying 2-methyl-3-oxoacyl-CoA thiolase activity). The side chain of the bile acid intermediates is shortened via one cycle of β-oxidation catalyzed by trihydroxycoprostanoyl-CoA oxidase, MFP-2 and SCPX. In the human, straight chain acyl-CoAs are oxidized via palmitoyl-CoA oxidase, multifunctional protein-1, and peroxisomal thiolase, as is the case in the rat. The CoA esters of 2-methyl-branched acyl-CoAs and the bile acid intermediates, which also possess a 2-methyl substitution in their side chain, are shortened, via branched chain acyl-CoA oxidase (which is the human homolog of trihydroxycoprostanoyl-CoA oxidase), multifunctional protein-2, and SCPX. The rat and the human enzymes have been purified, cloned, and kinetically and stereochemically characterized.3-Methyl-branched fatty acids such as phytanic acid are not directly β-oxidizable because of the position of the methyl-branch. They are first shortened by one carbon atom through the a-oxidation process to a 2-methyl-branched fatty acid (pristanic acid in the case of phytanic acid), which is then degraded via peroxisomal β-oxidation. In the human and the rat, α-oxidation is catalyzed by an acyl-CoA synthetase (producing a 3-methylacyl-CoA), a 3-methylacyl-CoA 2-hydroxylase (resulting in a 2-hydroxy-3-methylacyl-CoA), and a 2-hydroxy-3-methylacyl-CoA lyase that cleaves the 2-hydroxy-3-methylacyl-CoA into a 2-methyl-branched fatty aldehyde and formyl-CoA. The fatty aldehyde is dehydrogenated by an aldehyde dehydrogenase to a 2-methyl-branched fatty acid while formyl-CoA is hydrolyzed to formate, which is then converted to CO2. The activation, hydroxylation and cleavage reactions and the hydrolysis of formyl-CoA are performed by peroxisomal enzymes; the aldehyde dehydrogenation remains to be localized whereas the conversion of formate to CO2 occurs mainly in the cytosol.

Breakdown of 2-hydroxylated straight chain fatty acids via peroxisomal 2-hydroxyphytanoyl-CoA lyase: a revised pathway for the alpha-oxidation of straight chain fatty acids.

2-Hydroxyfatty acids, constituents of brain cerebrosides and sulfatides, were previously reported to be degraded by an α-oxidation system, generating fatty acids shortened by one carbon atom. In the current study we used labeled and unlabeled 2-hydroxyoctadecanoic acid to reinvestigate the degradation of this class of lipids. Both in intact and broken cell systems formate was identified as a main reaction product. Furthermore, the generation of an n–1 aldehyde was demonstrated. In permeabilized rat hepatocytes and liver homogenates, studies on cofactor requirements revealed a dependence on ATP, CoA, Mg2+, thiamine pyrophosphate, and NAD+. Together with subcellular fractionation data and studies on recombinant enzymes, this led to the following picture. In a first step, the 2-hydroxyfatty acid is activated to an acyl-CoA; subsequently, the 2-hydroxy fatty acyl-CoA is cleaved by 2-hydroxyphytanoyl-CoA lyase, to formyl-CoA and an n–1 aldehyde. The severe inhibition of formate generation by oxythiamin treatment of intact fibroblasts indicates that cleavage through the thiamine pyrophosphate-dependent 2-hydroxyphytanoyl-CoA lyase is the main pathway for the degradation of 2-hydroxyfatty acids. The latter protein was initially characterized as an essential enzyme in the peroxisomal α-oxidation of 3-methyl-branched fatty acids such as phytanic acid. Our findings point to a new role for peroxisomes in mammals, i.e. the breakdown of 2-hydroxyfatty acids, at least the long chain 2-hydroxyfatty acids. Most likely, the more abundant very long chain 2-hydroxyfatty acids are degraded in a similar manner.

Prenatal and postnatal development of peroxisomal lipid-metabolizing pathways in the mouse

The ontogeny of the following peroxisomal metabolic pathways was evaluated in mouse liver and brain: alpha-oxidation, beta-oxidation and ether phospholipid synthesis. In mouse embryos lacking functional peroxisomes (PEX5(-/-) knock-out), a deficiency of plasmalogens and an accumulation of the very-long-chain fatty acid C(26:0) was observed in comparison with control littermates, indicating that ether phospholipid synthesis and beta-oxidation are already active at mid-gestation in the mouse. Northern analysis revealed that the enzymes required for the beta-oxidation of straight-chain substrates are present in liver and brain during embryonic development but that those responsible for the degradation of branched-chain substrates are present only in liver from late gestation onwards. The expression pattern of transcripts encoding enzymes of the alpha-oxidation pathway suggested that alpha-oxidation is initiated in the liver around birth and is not active in brain throughout development. Remarkably, a strong induction of the mRNA levels of enzymes involved in alpha-oxidation and beta-oxidation was observed around birth in the liver. In contrast, enzyme transcripts that were expressed in brain were present at rather constant levels throughout prenatal and postnatal development. These results suggest that the defective ether phospholipid synthesis and/or peroxisomal beta-oxidation of straight-chain fatty acids might be involved in the pathogenesis of the prenatal organ defects in peroxisome-deficient mice and men.

Production of formyl-CoA during peroxisomal α-oxidation of 3-methyl-branched fatty acids

α‐Oxidation of 3‐methyl‐substituted fatty acids was studied in purified rat liver peroxisomes. The experiments revealed that formyl‐CoA is formed during the α‐oxidation process. The amount of formyl‐CoA found constituted 2–5% of the amount of formate formed. Under the conditions used, no activation of exogenously added formate occurred in purified peroxisomes, whereas 95.5% of added synthetic formyl‐CoA was converted to formate. These data indicate that during α‐oxidation first formyl‐CoA is formed, which is then hydrolysed to formate.

Familial giant cell hepatitis with low bile acid concentrations and increased urinary excretion of specific bile alcohols: A new inborn error of bile acid synthesis?

ABSTRACT: A 9-wk-old infant with familial giant cell hepatitis and severe intrahepatic cholestasis had low plasma concentrations of chenodeoxycholic acid and cholic acid and elevated plasma concentrations of5β-cholestane-3α,7α,12α,25-tetrol, 5β-cholestane-3α,7α,12α,24-tetrol, and 5β-cholest-24-ene-3α,7α,12α-triol. Analysis of the urine by fast atom bombardment mass spectromctry and by gas chromatography-mass spectrometry after treatment with Helix pomatia glucuronidase/sulfatase showed that the major cholanoids in urine were the glucuronides of 5β-cholestane-3α,7α,12α,24S, 25-pentol, 5β-cholestane-3α,7α,12α,25-tetrol, and 5β-cholestane-3α,7α,12α,24-tetrol. These results are consistent with an inborn error of the 25-hydroxylase pathway for bile acid synthesis, specifically one of the enzymes responsible for conversion of 5β-cholestane-3α,7α,12α,24S, 25-pentol to cholic acid and acetone. Treatment with chenodcoxycholic acid was tried on two occasions. On the first it appeared to precipitate a rise in bilirubin, on the second the liver function tests improved and the improvement was maintained when the treatment was modified to a combination of chenodeoxycholic acid and cholic acid and finally, cholic acid alone. Despite the normalization of liver function tests, a liver biopsy at 1.25 y showed an active cirrhosis. Nonetheless, the child is thriving at the age of 3.5 y, whereas an affected sibling died at 13 mo.

Formation of a 2-methyl-branched fatty aldehyde during peroxisomal α-oxidation

In the final reaction of peroxisomal α‐oxidation of 3‐methyl‐branched fatty acids a 2‐hydroxy‐3‐methylacyl‐CoA intermediate is cleaved to formyl‐CoA and a hitherto unidentified product. The release of formyl‐CoA suggests that the unidentified product may be a fatty aldehyde. When purified rat liver peroxisomes were incubated with 2‐hydroxy‐3‐methylhexadecanoyl‐CoA 2‐methylpentadecanal was indeed formed. The production rates of formyl‐CoA (measured as formate) and of the aldehyde were in the same range. While the production of formate remained unaltered in the presence of NAD+, the amount of 2‐methylpentadecanal was decreased, which was accompanied by the formation of 2‐methylpentadecanoic acid. These data indicate that (1) during α‐oxidation the 2‐hydroxy‐3‐methylacyl‐CoA is cleaved to a 2‐methyl‐branched aldehyde and formyl‐CoA and (2) liver peroxisomes are capable of converting this aldehyde to a 2‐methyl‐branched fatty acid.

Optimizing the European regulatory framework for sustainable bacteriophage therapy in human medicine.

For practitioners at hospitals seeking to use natural (not genetically modified, as appearing in nature) bacteriophages for treatment of antibiotic-resistant bacterial infections (bacteriophage therapy), Europe’s current regulatory framework for medicinal products hinders more than it facilitates. Although many experts consider bacteriophage therapy to be a promising complementary (or alternative) treatment to antibiotic therapy, no bacteriophage-specific framework for documentation exists to date. Decades worth of historical clinical data on bacteriophage therapy (from Eastern Europe, particularly Poland, and the former Soviet republics, particularly Georgia and Russia, as well as from today’s 27 EU member states and the US) have not been taken into account by European regulators because these data have not been validated under current Western regulatory standards. Consequently, applicants carrying out standard clinical trials on bacteriophages in Europe are obliged to initiate clinical work from scratch. This paper argues for a reduced documentation threshold for Phase 1 clinical trials of bacteriophages and maintains that bacteriophages should not be categorized as classical medicinal products for at least two reasons: (1) such a categorization is scientifically inappropriate for this specific therapy and (2) such a categorization limits the marketing authorization process to industry, the only stakeholder with sufficient financial resources to prepare a complete dossier for the competent authorities. This paper reflects on the current regulatory framework for medicines in Europe and assesses possible regulatory pathways for the (re-)introduction of bacteriophage therapy in a way that maintains its effectiveness and safety as well as its inherent characteristics of sustainability and in situ self-amplification and limitation.

Call for a dedicated European legal framework for bacteriophage therapy.

The worldwide emergence of antibiotic resistances and the drying up of the antibiotic pipeline have spurred a search for alternative or complementary antibacterial therapies. Bacteriophages are bacterial viruses that have been used for almost a century to combat bacterial infections, particularly in Poland and the former Soviet Union. The antibiotic crisis has triggered a renewed clinical and agricultural interest in bacteriophages. This, combined with new scientific insights, has pushed bacteriophages to the forefront of the search for new approaches to fighting bacterial infections. But before bacteriophage therapy can be introduced into clinical practice in the European Union, several challenges must be overcome. One of these is the conceptualization and classification of bacteriophage therapy itself and the extent to which it constitutes a human medicinal product regulated under the European Human Code for Medicines (Directive 2001/83/EC). Can therapeutic products containing natural bacteriophages be categorized under the current European regulatory framework, or should this framework be adapted? Various actors in the field have discussed the need for an adapted (or entirely new) regulatory framework for the reintroduction of bacteriophage therapy in Europe. This led to the identification of several characteristics specific to natural bacteriophages that should be taken into consideration by regulators when evaluating bacteriophage therapy. One important consideration is whether bacteriophage therapy development occurs on an industrial scale or a hospital-based, patient-specific scale. More suitable regulatory standards may create opportunities to improve insights into this promising therapeutic approach. In light of this, we argue for the creation of a new, dedicated European regulatory framework for bacteriophage therapy.