Defect In Peroxisome Biogenesis Research Articles

Absent biochemical evidence at an early age delayed diagnosis in a patient with a clinically severe form of peroxisomal biogenesis disorder

The aim of the study is determining the level of very long chain fatty acids (VLCFA) is the screening method of choice in suspected peroxisomal disorders. Severe peroxisomal biogenesis defects in particular are usually recognized reliably. We report a patient presenting with typical facial stigmata, a treatment-resistant seizure disorder, and polymicrogyria, whose VLCFA levels were within normal limits until the age of 18 months. Only thereafter an elevation was found. Enzymatic and consecutively molecular-genetic analysis revealed a compound-heterozygous mutation in PEX 6. In conclusion, VLCFA levels within normal limits initially do not rule out global peroxisomal biogenesis defects and should be repeated subsequently. Persisting clinical suspicion justifies further enzymatic and molecular evaluation.

The Proteome of Human Liver Peroxisomes: Identification of Five New Peroxisomal Constituents by a Label-Free Quantitative Proteomics Survey

The peroxisome is a key organelle of low abundance that fulfils various functions essential for human cell metabolism. Severe genetic diseases in humans are caused by defects in peroxisome biogenesis or deficiencies in the function of single peroxisomal proteins. To improve our knowledge of this important cellular structure, we studied for the first time human liver peroxisomes by quantitative proteomics. Peroxisomes were isolated by differential and Nycodenz density gradient centrifugation. A label-free quantitative study of 314 proteins across the density gradient was accomplished using high resolution mass spectrometry. By pairing statistical data evaluation, cDNA cloning and in vivo colocalization studies, we report the association of five new proteins with human liver peroxisomes. Among these, isochorismatase domain containing 1 protein points to the existence of a new metabolic pathway and hydroxysteroid dehydrogenase like 2 protein is likely involved in the transport or β-oxidation of fatty acids in human peroxisomes. The detection of alcohol dehydrogenase 1A suggests the presence of an alternative alcohol-oxidizing system in hepatic peroxisomes. In addition, lactate dehydrogenase A and malate dehydrogenase 1 partially associate with human liver peroxisomes and enzyme activity profiles support the idea that NAD+ becomes regenerated during fatty acid β-oxidation by alternative shuttling processes in human peroxisomes involving lactate dehydrogenase and/or malate dehydrogenase. Taken together, our data represent a valuable resource for future studies of peroxisome biochemistry that will advance research of human peroxisomes in health and disease.

96 The Leukoencephalopathies of Childhood: Findings From a National Prospective Study

Background and aims: To report the distribution of leukoencephalopathies in children identified by this prospective population-based study. Methods: 2802 children with suspected progressive intellectual and neurological deterioration (PIND) were notified via the British Paediatric Surveillance Unit (BPSU) between 1997 and 2009. An expert group reviewed clinical data and confirmed diagnoses where possible. An independent expert reviewed the available scans of children with unclassified leukoencephalopathies. Results: 1132 of the PIND children had an underlying diagnosis and of these 295 had one of the leukoencephalopathies. The distribution of diagnosed leukoencephalopathies was as follows: metachromatic leukodystrophy 62, X-linked adrenoleukodystrophy 60, Krabbe disease 39, Canavan disease 16, Alexander disease 13, leukoencephalopathy with vanishing white matter 12, Aicardi-Goutieres syndrome 12, Pelizaeus-Merzbacher disease 11, megalencephalic leukoencephalopathy with subcortical cysts 8, multiple sulphatase deficiency 6, multiple sclerosis 3, peroxisomal bifunctional D-protein deficiency 2, peroxisomal biogenesis defect 1. There were 50 children in the unclassified leukoencephalopathy group. In 32 the scans were reviewed and 19 were placed in a hypomyelination sub-group, the other 13 remaining in the unclassified leukoencephalopathy group with the 18 whose scans could not be reviewed. Conclusions: Leukoencephalopathies comprised 26% of PIND children with a known diagnosis. This population based study gives the relative frequency of the different leukoencephalopathies, providing an aid to diagnosis in children with abnormal white matter on brain imaging. After rigorous investigation 16% (50/295) remained in an unclassified group, but this situation is evolving.

Scarring Alopecia and the PPAR-γ Connection

The pathobiology of primary cicatricial ("scarring") alopecia (PCA) remains poorly understood and underinvestigated. In this issue, Karnik et al. identify a previously unsuspected player, peroxisome proliferator-activated receptor-gamma (PPARgamma), in the pathogenesis of the most frequent form of PCA, lichen planopilaris (LPP). The authors show that PPARgamma is required for maintenance of a functional epithelial stem cell compartment in murine hair follicles, that the targeted deletion of PPARgamma in the bulge/isthmus area of the hair follicle epithelium generates a skin pathology that resembles LPP, and that LPP patients show gene expression changes that indicate a defect in lipid metabolism and peroxisome biogenesis. This study invites the revisitation of many open questions in PCA pathobiology and the exploration of new avenues for future PCA management.

Proteomics Characterization of Mouse Kidney Peroxisomes by Tandem Mass Spectrometry and Protein Correlation Profiling

The peroxisome represents a ubiquitous single membrane-bound key organelle that executes various metabolic pathways such as fatty acid degradation by alpha- and beta-oxidation, ether-phospholipid biosynthesis, metabolism of reactive oxygen species, and detoxification of glyoxylate in mammals. To fulfil this vast array of metabolic functions, peroxisomes accommodate approximately 50 different enzymes at least as identified until now. Interest in peroxisomes has been fueled by the discovery of a group of genetic diseases in humans, which are caused by either a defect in peroxisome biogenesis or the deficient activity of a distinct peroxisomal enzyme or transporter. Although this research has greatly improved our understanding of peroxisomes and their role in mammalian metabolism, deeper insight into biochemistry and functions of peroxisomes is required to expand our knowledge of this low abundance but vital organelle. In this work, we used classical subcellular fractionation in combination with MS-based proteomics methodologies to characterize the proteome of mouse kidney peroxisomes. We could identify virtually all known components involved in peroxisomal metabolism and biogenesis. Moreover through protein localization studies by using a quantitative MS screen combined with statistical analyses, we identified 15 new peroxisomal candidates. Of these, we further investigated five candidates by immunocytochemistry, which confirmed their localization in peroxisomes. As a result of this joint effort, we believe to have compiled the so far most comprehensive protein catalogue of mammalian peroxisomes.

Identification of novel mutations inPEX2,PEX6,PEX10,PEX12, andPEX13in Zellweger spectrum patients

Mutations in each of the 13 identified human PEX genes are known to cause a peroxisomal biogenesis defect (PBD). Affected patients can be divided into two broad clinical spectra: the Zellweger spectrum, which accounts for about 80% of PBD patients, and the rhizomelia chondrodysplasia punctata (RCDP) spectrum. The clinical continuum of Zellweger spectrum patients extends from Zellweger syndrome (ZS) as the prototype and the most severe entity of this group to neonatal adrenoleukodystrophy (NALD) as an intermediate form and infantile Refsum (IRD) disease as the mildest variant. Characteristic features of ZS patients are dysmorphic features, severe neurological impairment, liver dysfunction, and eye and skeletal abnormalities. Similar but less severe clinical signs are seen in patients with NALD and IRD. In this study ten clinically and/or biochemically well-characterized patients with classical ZS were investigated for defects in all known human PEX genes. We identified two novel mutations in PEX2 (official symbol, PXMP3), two novel mutations in PEX6, two novel mutations in PEX10, one novel mutation in PEX12, and one novel mutation in PEX13.

Model for end-stage liver disease (MELD) exception for unusual metabolic liver diseases

Metabolic liver disease is the underlying diagnosis in only a small proportion of patients who undergo liver transplantation (LT), but for these patients, LT is lifesaving. Patients with metabolic liver disease often do not present with typical findings of end-stage liver disease and require special consideration and scrutiny concerning the appropriateness and timing of LT. Liverbased metabolic disease is classified into 3 types: (1) disease that causes structural liver damage with liver failure or cirrhosis, (2) metabolic disease without structural liver damage that affects other organs (especially the central nervous system), and (3) metabolic disease with systemic deficiencies that are partially represented in the liver. There may be overlap in presentation, with some disease forms presenting either with or without structural liver disease. General considerations that affect review board decisions may include the relative contraindication of the use of living-related donor organs and the unpredictable metabolic course that may cause severe central nervous system complications in several of these disease states. Also, although many of these diseases present mostly in children, adolescents and adults previously managed medically are increasingly presenting for LT consideration when medical management becomes more difficult or complex as they mature.

Topogenesis of peroxisomal proteins does not require a functional cytoplasm-to-vacuole transport

Folded and even oligomeric proteins can be imported from the cytosol into vacuoles and into peroxisomes. Pro-aminopeptidase I (prAPI) oligomerizes into a dodecamer and is imported into the vacuole via the cytoplasm-to-vacuole transport (cvt) pathway. How peroxisomes accommodate folded proteins is completely unknown. Peroxisome biogenesis and cvt do not only share the import of folded protein complexes but also show mechanistic parallels such as the employment of ubiquitin conjugation systems. In search for a genetic overlap, selected cvt and autophagocytosis ( atg) mutants were tested for defects in peroxisome biogenesis. Most of the mutants did not exhibit a mislocalization of peroxisomal matrix proteins to the cytosol which would be typical of a defect in the peroxisome biogenesis. However, two mutants, Δatg14 and Δcvt4/vam6, displayed a general growth defect and Δcvt8 / vps41 showed cytosolic mislocalization not only of peroxisomal but also of mitochondrial proteins, indicating a more general defect in organelle biogenesis. Our data do not provide evidence for a genetic overlap of the import pathway for peroxisomal proteins and the cvt pathway.

Method for measurement of peroxisomal very-long-chain fatty acid beta-oxidation in human skin fibroblasts using stable-isotope-labeled tetracosanoic acid.

Peroxisomes are present in virtually every eukaryotic cell type except the mature erythrocyte. In higher eukaryotes, one of the main functions of peroxisomes is the β-oxidation of very-long-chain fatty acids (VLCFA; > 22 carbon atoms) (1). The importance of peroxisomal β-oxidation is emphasized by the existence of a variety of different diseases in which peroxisomal β-oxidation is impaired and VLCFA concentrations are increased (1)(2)(3)(4). Peroxisomal disorders can be categorized as ( a ) single peroxisomal enzyme deficiencies, including X-linked adrenoleukodystrophy (X-ALD) and disorders attributable to defects in one of the peroxisomal β-oxidation enzymes, such as acyl-CoA oxidase (AOX) deficiency and bifunctional protein (DBP) deficiency; and ( b ) disorders attributable to defects in peroxisome biogenesis. The peroxisome biogenesis disorders (PBDs) represent a continuum of clinical features ranging from the most severe form, Zellweger syndrome, through neonatal adrenoleukodystrophy to the least severe form, infantile Refsum disease. Currently, measurement of the peroxisomal fatty acid β-oxidation activity is performed with 1-[14C]-radiolabeled VLCFA substrates and one of two available methods: either in intact human skin fibroblasts cultured in monolayer (5); or in isolated fibroblasts permeabilized with digitonin (6). We investigated the feasibility of using deuterium-labeled tetracosanoic acid (D3-C24:0) as an alternative substrate to radiolabeled 1-[14C]-labeled C24:0 for the measurement of peroxisomal β-oxidation activity in cultured primary human skin fibroblasts. Before use, the purity of 24,24,24-D3-C24:0 (Larodan Fine Chemicals AB) was determined. The D3-C24:0 substrate contained ∼6% deuterium-labeled octadecanoic acid (D3-C18:0). Acetone was used to purify D3-C24:0 according to the following procedure: 4 mL of acetone was added to 20 mg of D3-C24:0. The sample was vortex-mixed vigorously, left at room temperature for 30 …

Orthotopic liver transplantation from a living‐related donor in an infant with a peroxisome biogenesis defect of the infantile Refsum disease type

Peroxisomal biogenesis defects include a number of severe neurodevelopmental disorders, among which infantile Refsum disease (IRD) occupies the mildest end of the spectrum. Although high docosahexaenoic acid (DHA) and low phytanic acid diets can correct some of the biochemical defects, they have not consistently altered the progressive course of the disease. We carried out orthotopic liver transplantation (OLT) in a mildly symptomatic 6-month-old infant who was a sibling of a severely neurologically impaired older sister. After transplantation the clinical course of this young child appeared much improved by comparison to her older sister. She walked alone at 4 years, had acceptable social interaction and had a noticeable recovery of audition. After transplantation her biochemical parameters were significantly improved: phytanic acid and very long-chain fatty acid (VLCFA) serum concentrations decreased. Abnormal bile acids disappeared from plasma. Although the OLT did not result in a cure of the disorder, the clinical and biochemical results suggest that OLT should be considered in mildly symptomatic patients.

Identification of the molecular defect in patients with peroxisomal mosaicism using a novel method involving culturing of cells at 40 degrees C: implications for other inborn errors of metabolism.

The peroxisome biogenesis disorders (PBDs), which comprise Zellweger syndrome (ZS), neonatal adrenoleukodystrophy, and infantile Refsum disease (IRD), represent a spectrum of disease severity, with ZS being the most severe, and IRD the least severe disorder. The PBDs are caused by mutations in one of the at least 12 different PEX genes encoding proteins involved in the biogenesis of peroxisomes. We report the biochemical characteristics and molecular basis of a subset of atypical PBD patients. These patients were characterized by abnormal peroxisomal plasma metabolites, but otherwise normal to very mildly abnormal peroxisomal parameters in cultured skin fibroblasts, including a mosaic catalase immunofluorescence pattern in fibroblasts. Since this latter feature made standard complementation analysis impossible, we developed a novel complementation technique in which fibroblasts were cultured at 40 degrees C, which exacerbates the defect in peroxisome biogenesis. Using this method, we were able to assign eight patients to complementation group 3 (CG3), followed by the identification of a single homozygous c.959C>T (p.S320F) mutation in their PEX12 gene. We also investigated various peroxisomal biochemical parameters in fibroblasts at 30 degrees C, 37 degrees C, and 40 degrees C, and found that all parameters showed a temperature-dependent behavior. The principle of culturing cells at elevated temperatures to exacerbate the defect in peroxisome biogenesis, and thereby preventing certain mutations from being missed, may well have a much wider applicability for a range of different inborn errors of metabolism.

Mass spectrometric analysis of ceramide perturbations in brain and fibroblasts of mice and human patients with peroxisomal disorders.

In this study, the levels and composition of ceramides in brains of newborn mice lacking peroxisomes (Pex5-/-, Zellweger mice) were analyzed using normal-phase high-performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (HPLC/APCI-MS). Total ceramide compositions were found to be comparable to that of control animals. However, a minor ceramide species, containing hexacosanoic/hexacosenoic acid as the amide fatty acid, was 9-fold increased. Also, in the sphingomyelin-derived ceramides this species was elevated. Subsequent analysis of extracts from fibroblasts of Pex5-/- mice and mice with a defective peroxisomal beta-oxidation (lacking D-specific multifunctional protein 2 (MFP2)), revealed, again, a similar rise in this particular ceramide. Further, this ceramide was elevated in human X-ALD fibroblasts as well. Whether C26:1/0-ceramide is linked to some of the pathology seen in Zellweger syndrome remains to be investigated. However, an increase in this sphingolipid can be considered as a diagnostic criterion for diseases caused by defects in peroxisome biogenesis or peroxisomal beta-oxidation.

Disturbed cholesterol homeostasis in a peroxisome-deficient PEX2 knockout mouse model.

We evaluated the major pathways of cholesterol regulation in the peroxisome-deficient PEX2(-/-) mouse, a model for Zellweger syndrome. Zellweger syndrome is a lethal inherited disorder characterized by severe defects in peroxisome biogenesis and peroxisomal protein import. Compared with wild-type mice, PEX2(-/-) mice have decreased total and high-density lipoprotein cholesterol levels in plasma. Hepatic expression of the SREBP-2 gene is increased 2.5-fold in PEX2(-/-) mice and is associated with increased activities and increased protein and expression levels of SREBP-2-regulated cholesterol biosynthetic enzymes. However, the upregulated cholesterogenic enzymes appear to function with altered efficiency, associated with the loss of peroxisomal compartmentalization. The rate of cholesterol biosynthesis in 7- to 9-day-old PEX2(-/-) mice is markedly increased in most tissues, except in the brain and kidneys, where it is reduced. While the cholesterol content of most tissues is normal in PEX2(-/-) mice, in the knockout mouse liver it is decreased by 40% relative to that in control mice. The classic pathway of bile acid biosynthesis is downregulated in PEX2(-/-) mice. However, expression of CYP27A1, the rate-determining enzyme in the alternate pathway of bile acid synthesis, is upregulated threefold in the PEX2(-/-) mouse liver. The expression of hepatic ATP-binding cassette (ABC) transporters (ABCA1 and ABCG1) involved in cholesterol efflux is not affected in PEX2(-/-) mice. These data illustrate the diversity in cholesterol regulatory responses among different organs in postnatal peroxisome-deficient mice and demonstrate that peroxisomes are critical for maintaining cholesterol homeostasis in the neonatal mouse.

An Arabidopsis pex10 null mutant is embryo lethal, implicating peroxisomes in an essential role during plant embryogenesis.

Peroxisomes participate in many important functions in plants, including seed reserve mobilization, photorespiration, defense against oxidative stress, and auxin and jasmonate signaling. In mammals, defects in peroxisome biogenesis result in multiple system abnormalities, severe developmental delay, and death, whereas in unicellular yeasts, peroxisomes are dispensable unless required for growth of specific substrates. PEX10 encodes an integral membrane protein required for peroxisome biogenesis in mammals and yeast. To investigate the importance of PEX10 in plants, we characterized a Ds insertion mutant in the PEX10 gene of Arabidopsis (AtPEX10). Heterozygous AtPEX10::dissociation element mutants show normal vegetative phenotypes under optimal growth conditions, but produce about 20% abnormal seeds. The embryos in the abnormal seeds are predominantly homozygous for the disruption allele. They show retarded development and some morphological abnormalities. No viable homozygous mutant plants were obtained. AtPEX10 fused to yellow fluorescent protein colocalized with green fluorescent protein-serine-lysine-leucine, a well-documented peroxisomal marker, suggesting that AtPEX10 encodes a peroxisomal protein that is essential for normal embryo development and viability.

Peroxisome biogenesis and the role of protein import.

Peroxisomes are metabolic organelles with enzymatic content that are found in virtually all cells and are involved in beta-oxidation of fatty acids, hydrogen peroxide-based respiration and defence against oxidative stress. The steps of their biogenesis involves "peroxins", proteins encoded by PEX genes. Peroxins are involved in three key stages of peroxisome development: (1). import of peroxisomal membrane proteins; (2). import of peroxisomal matrix proteins and (3). peroxisome proliferation. Of these three areas, peroxisomal matrix-protein import is by far the best understood and accounts for most of the available published data on peroxisome biogenesis. Defects in peroxisome biogenesis result in peroxisome biogenesis disorders (PBDs), which although rare, have no known cure to-date. This review explores current understanding of each key area in peroxisome biogenesis, paying particular attention to the role of protein import.

A review of morphological techniques for detection of peroxisomal (and mitochondrial) proteins and their corresponding mRNAs during ontogenesis in mice: application to the PEX5-knockout mouse with Zellweger syndrome.

In the era of application of molecular biological gene-targeting technology for the generation of knockout mouse models to study human genetic diseases, the availability of highly sensitive and reliable methods for the morphological characterization of the specific phenotypes of these mice is of great importance. In the first part of this report, the role of morphological techniques for studying the biology and pathology of peroxisomes is reviewed, and the techniques established in our laboratories for the localization of peroxisomal proteins and corresponding mRNAs in fetal and newborn mice are presented and discussed in the context of the international literature. In the second part, the literature on the ontogenetic development of the peroxisomal compartment in mice, with special emphasis on liver and intestine is reviewed and compared with our own data reported recently. In addition, some recent data on the pathological alterations in the liver of the PEX5(-/-) mouse with a peroxisomal biogenesis defect are briefly discussed. Finally, the methods developed during these studies for the localization of mitochondrial proteins (respiratory chain complexes and MnSOD) are presented and their advantages and pitfalls discussed. With the help of these techniques, it is now possible to identify and distinguish unequivocally peroxisomes from mitochondria, two classes of cell organelles giving by light microscopy a punctate staining pattern in microscopical immunohistochemical preparations of paraffin-embedded mouse tissues.

Peroxisome deficiency does not result in deficiency of enzymes involved in cholesterol biosynthesis.

To unravel conflicting literature data on the dependence of cholesterol biosynthesis on functional peroxisomes, we have determined activities and levels of selected cholesterol biosynthetic enzymes in livers of a PEX5 knock-out mouse, a well-characterized animal model for human Zellweger syndrome. All enzymes measured, including putative peroxisomal enzymes, were normally active in the peroxisome-deficient mice indicating that mislocalization of these enzymes to the cytosol does not lead to decreased activity or degradation. Since these findings were in apparent contrast to previously reported data in human livers, we re-examined cholesterol biosynthetic enzymes in livers of Zellweger patients and control subjects. We found that the previously reported deficient enzymes mevalonate kinase and phosphomevalonate kinase are inactivated rapidly in a time-and temperature-dependent manner. This inactivation is not due to degradation as a result of mislocalization since normal levels of protein can be detected. Therefore, the deficiencies of these enzymes in livers of Zellweger patients reflect the bad condition of the livers, rather than mislocalization to the cytosol. Our data explain the conflicting findings in literature and demonstrate that great care should be taken in the interpretation of data obtained in postmortem material (1). To exclude that genetic differences, resulting in different defects in peroxisomal biogenesis, might influence the activity of selected cholesterol biosynthetic enzymes, we also have determined activities and levels of these enzymes in human fibroblasts of patients belonging to various different complementation groups. We included fibroblasts of patients with mutations in genes encoding PEX1, PEX5, PEX7, PEX16, and PEX19, resulting in selective deficiencies in import of PTS1 or PTS2 containing proteins or in a more general defect in peroxisome biogenesis resulting in the complete absence of peroxisomes or peroxisomal ghosts. We measured normal protein levels and activities of enzymes involved in cholesterol biosynthesis in all cell lines. We conclude that peroxisomes are not a prerequisite for cholesterol biosynthesis in humans.

A PEX6-Defective Peroxisomal Biogenesis Disorder with Severe Phenotype in an Infant, versus Mild Phenotype Resembling Usher Syndrome in the Affected Parents

Sensorineural deafness and retinitis pigmentosa (RP) are the hallmarks of Usher syndrome (USH) but are also prominent features in peroxisomal biogenesis defects (PBDs); both are autosomal recessively inherited. The firstborn son of unrelated parents, who both had sensorineural deafness and RP diagnosed as USH, presented with sensorineural deafness, RP, dysmorphism, developmental delay, hepatomegaly, and hypsarrhythmia and died at age 17 mo. The infant was shown to have a PBD, on the basis of elevated plasma levels of very-long- and branched-chain fatty acids (VLCFAs and BCFAs), deficiency of multiple peroxisomal functions in fibroblasts, and complete absence of peroxisomes in fibroblasts and liver. Surprisingly, both parents had elevated plasma levels of VLCFAs and BCFAs. Fibroblast studies confirmed that both parents had a PBD. The parents' milder phenotypes correlated with relatively mild peroxisomal biochemical dysfunction and with catalase immunofluorescence microscopy demonstrating mosaicism and temperature sensitivity in fibroblasts. The infant and both of his parents belonged to complementation group C. PEX6 gene sequencing revealed mutations on both alleles, in the infant and in his parents. This unique family is the first report of a PBD with which the parents are themselves affected individuals rather than asymptomatic carriers. Because of considerable overlap between USH and milder PBD phenotypes, individuals suspected to have USH should be screened for peroxisomal dysfunction.

Inactivation of the endoplasmic reticulum protein translocation factor, Sec61p, or its homolog, Ssh1p, does not affect peroxisome biogenesis.

Peroxisomes are single membrane-bound organelles present in virtually all eukaryotes. These organelles participate in several important metabolic processes, and defects in peroxisome function and biogenesis are a significant contributor to human disease. Several models propose that peroxisomes arise from the endoplasmic reticulum (ER) in a process that involves the translocation of "group I" peroxisomal membrane proteins into the ER, the exit of these group I peroxisomal membrane proteins from the ER by vesicle budding, and the formation of nascent peroxisomes from vesicles containing the group I peroxisomal membrane proteins. A central prediction of these models is that the formation of nascent peroxisomes requires protein translocation into the ER. Sec61p is an essential component of the ER translocon, and we show here that loss of Sec61p activity has no effect on peroxisome biogenesis. In addition, loss of the SEC61-related gene, SSH1, also has no effect on peroxisome biogenesis. Although some proteins may enter the ER independently of Sec61p or Ssh1p, none are known, leading us to propose that peroxisome biogenesis may not require protein import into the ER, and by extension, transfer of proteins from the ER to the peroxisome.

Rapid and quantitative analysis of unconjugated C27 bile acids in plasma and blood samples by tandem mass spectrometry

A subgroup of peroxisomal disorders, peroxisome biogenesis defects (PBD), can be differentiated by elevated levels of C27 bile acids in plasma and bile. Patients with peroxisomal disorders, who lack the ability to chain-shorten the C27 bile acid intermediates into C24 bile acids, show elevated levels of C27 bile acids, notably 3α,7α-dihydroxy-5β-cholest-26-oic acid and 3α,7α,12α-trihydroxy-5β-cholestan-26-oic acid. C27 bile acids are normally estimated against other bile acid standards, by time-consuming gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry methods, in plasma (minimum of 50 μl). In this article we describe the quantitation of unconjugated di- and trihydroxy C27 bile acids in 5-μl plasma samples and 3-mm blood spots, using deuterium-labeled internal standards. The synthesis of 2H3-labeled di- and trihydroxycoprostanic acids is described. The sample preparation and analysis by electrospray tandem mass spectrometry (ES-MS/MS) takes less than 1 h and features dimethylaminoethyl ester derivatives. The levels of the di- and trihydroxy bile acids are significantly higher in PBD patients than in age-matched control subjects for both plasma and blood spots collected at birth (some stored for up to 18 years). Excellent correlation is observed between the C26:0/C22:0 very long chain fatty acid (VLCFA) ratio and the levels of trihydroxy C27 bile acids in plasma from PBD patients. The ES-MS/MS method can be used to rapidly screen for PBD patients in plasma samples with elevated C26:0/C22:0 VLCFA ratios and in archived collections of neonatal blood spots.—Johnson, D. W., H. J. ten Brink, R. C. Schuit, and C. Jakobs. Rapid and quantitive analysis of unconjugated C27 bile acids in plasma and blood samples by tandem mass spectrometry. J. Lipid Res. 2001. 42: 9–16.