This document provides an overview of peroxisome metabolism. Key points include: - Peroxisomes are organelles that contain enzymes for metabolic processes like fatty acid oxidation and production of hydrogen peroxide. - They are involved in lipid metabolism, reactive oxygen species reduction, and biosynthesis of plasmalogens and bile acids. - Disorders associated with impaired peroxisome function include peroxisome biogenesis disorders and single enzyme defects, which can result in accumulation of toxic metabolites. - Important diseases include Zellweger syndrome, Refsum disease, and adrenoleukodystrophy.

1. PEROXISOME METABOLISM
Presented by : Rabina Ramtel
M.Sc. Clinical Biochemistry

2. OUTLINES:
 Introduction
 Peroxisome biogenesis
 Function of peroxisomes
 Disorder associated with peroxisomes

3.  It is a microbody of about 0.3-1.5 micrometer in
diameter
 Have a lipid bilayer membrane
 Consists of crystalline core with peroxins

4.  Peroxisomes are called so; because they produce
hydrogen peroxide (H2O2)
 Also known as the organelle behind the film
“Lorenzo’s oil”
 Consists of enzymes such as catalase, D-amino acid
oxidase, uric acid oxidase, alpha- hydroxy acid
oxidase etc
 Enzymatic marker for peroxisome is catalase

5.  Peroxisomes can be isolated by differential
centrifugation
 Differential centrifugation results in a rough
fractionation of the cytoplasmic contents
 Further it is purified by isopycnic (“same density”)
centrifugation.
 The tissue homogenate is first centrifuged at low-
speed(1,000 g, 10 min)
 Supernatant then is subjected to medium-speed
centrifugation (20,000 g, 20 min)

6.  Results in pellets containing mitochondria, lysosomes
and peroxisomes
 The pellet, which contained peroxisomes and
mitochondria, was resuspended in MS buffer (0.65 M
sorbitol, 5 mM MES, pH 5.5) and was placed on top
of a gradient of Nycodenz (17%, 25%, 35%, 50%) in
MS buffer.
 After centrifugation at 116,000 g for 2h, peroxisomes
are present in fractions 2 to 8.

7.  The pellet from the aforementioned 20,000 g
centrifugation was resuspended in 10 volumes of Ti8
buffer (Tris 10mM, pH 8.0 and PINS (1 mM EDTA, 0.2
mM PMSF, 2 μg leupeptin/ml, 2 μg aprotinin/ml, and 0.4
μg pepstatin A/ml))
 separated at 200,000 g for 1 h.
 The pellet, with the peroxisomal membranes, was
resuspended again in Ti8 buffer
 By addition of 0.1 M sodium carbonate and subsequent
centrifugation at 200,000 g for 1 h, the peroxisomal
membranes were separated from the proteins which were
associated with but not integral to the membranes.

8.  Peroxisomes proliferate by growth and division of
pre-existing peroxisomes or could arise de novo.
 Its biogenesis is unique as it lack their own DNA
 Steps :
 formation of the peroxisomal membrane
 Import of proteins into the peroxisomal matrix
 Proliferations of the organelles

11. Peroxisome
matrix protein
Type of PTS PTS* amino
acid
sequence
Role in
peroxisome
metabolism
Acyl-CoA oxidase I 1 –SKL Fatty acid
metabolism
Alanine glyoxylate
aminotransferase
1 –KKL Glyoxylate
metabolism
Alkyldihydroxyacet
onephosphate
synthase
2 –RLVLSGHL– Plasmalogen
synthesis
Catalase 1 –KANL H2O2 metabolism
D-bifunctional
protein
1 –AKL Fatty acid
metabolism

13.  Lipid and anaplerotic metabolism
 Production of hydrogen peroxide
 Reduction of reactive oxygen species (ROS)
 Biosynthesis of plasmalogens
 Detoxification of alcohol in liver cells
 Remove amine group from amino acids and convert
it to ammonia prior to excretion

14.  In the liver, peroxisomes are also involved in the
synthesis of bile acids, which are derived from
cholesterol.

15. Lipid and anaplerotic metabolism
One of the major functions of peroxisomes concerns
their role in lipid metabolism, which includes:
 fatty acid beta-oxidation
 fatty acid alpha-oxidation
 ether phospholipid synthesis

16. Beta-Oxidation
 Occurs when fatty acids chains are too long to be
handled by mitochondria
 Peroxisomal beta-oxidation does not degrade fatty
acids completely
 acts as a chain-shortening system, catalyzing only a
limited number of beta-oxidation cycles.
 The fatty acids are activated to their acyl CoA
derivatives at the peroxisomal membrane and the
beta- oxidation occurs at the peroxisomal matrix

17. MODELS FOR THE IMPORT OF FATTY ACIDS INTO
THE PEROXISOMES

21. Alpha(α)-oxidation
 Alpha oxidation occurs in those fatty acids that
have a methyl group(CH3) at the beta-carbon, which
blocks beta oxidation.
 It removes one of the carbon unit adjacent to the α
carbon from the carboxylic end in the form of CO2
 Phytanic acid acts as the substrate
 peroxisomes is the cellular site
 No production of ATP

23. Biosynthesis of plasmalogens
• Plasmalogens (PLs) were first described by Feulgen
and Voit in 1924
• A family of phospholipids in which one of the
hydrocarbon chains is joined to glycerol by an ether
bond rather than an ester bond
• Important membrane components in heart and brain.

24. • In human heart tissue, nearly 30–40% of choline
glycerophospholipids are plasmalogens.
• 32% of the glycerophospholipids in the adult human
heart , 20% in brain and up to 70% of myelin sheath
ethanolamine glycerophospholipids are plasmalogen
• It’s Biosynthesis begins with association
of peroxisomal matrix enzymes GNPAT (glycerone
phosphate acyl transferase) and AGPS (alkyl-
glycerone phosphate synthase) on the luminal side of
the peroxisomal membrane

26.  Impaired plasmalogen biosynthesis also leads to
Peroxisome biogenesis disorders.
 In these cases, the peroxisomal enzyme GNPAT,
necessary for the initial steps of plasmalogen
biosynthesis, is mislocalized to the cytoplasm where
it is inactive.
 Genetic mutations in the GNPAT or AGPS genes can
result in plasmalogen deficiencies, which lead to the
development of rhizomelic chondrodysplasia
punctata (RCDP) type 2 or 3, respectively

27. Ethanol metabolism
 Oxidation of ethanol can also occur in peroxisomes via
the activity of catalase.
 However, this oxidation pathway requires the presence
of a hydrogen peroxide (H2O2) generating system and
as such plays no major role in alcohol metabolism
under normal physiological conditions

28. Production of H2O2

29. Role of peroxisome in bile acids synthesis
 In the early 1980s, the first clues were obtained
indicating the importance of peroxisomes in the
biosynthesis of bile acids.
 Peroxisomes play an important role in the
biosynthesis of bile acids as peroxisomal beta-
oxidation step is required for the formation of the
mature C24-bile acids from C27-bile acid
intermediates.
 In addition, de novo synthesized bile acids are
conjugated within the peroxisome.

30.  The primary bile acids, cholic acid (CA) and
chenodeoxycholic acid (CDCA), are formed from
cholesterol
 Its biosynthesis occurs by two pathways:
 Classical pathway
 Alternate pathway

31.  Steps involved in bile acid biosynthesis are:
 Modification of ring structure of cholesterol (steroid
nucleus)
 Oxidation of the sterol side chain
 Cleavage of the side chain
 Conjugation with an amino acid, either taurine or
glycine.

32. PEROXISOMAL STEPS IN BILE ACID BIOSYNTHESIS
-METHYLACYL-COA RACEMASE (AMACR)
BRANCHED-CHAIN ACYL-COA OXIDASE (BCOX)
STEROL CARRIER PROTEIN X (SCPX)
BILE ACYL COA AMINO ACID N-ACYL TRANSFERASE (BAAT)

33. Role of peroxisome to remove amine group
from amino acids and convert it to ammonia
prior to excretion
 Removal of the α-amino group is the first step
in catabolism of amino acids
 accomplished oxidatively or nonoxidatively
 Oxidative deamination is stereospecific and is
catalyzed by L- or D-amino acid oxidase.

36.  Impaired peroxisomal function results in a number of
multisystem diseases and are grouped as:
 Group 1: Peroxisomal Biogenesis Disorders (PBD)
 Group 2: Single Peroxisomal Enzyme Defects
involving β-Oxidation. Peroxisomes are
morphologically intact but their function is defective
 Group 3: Single Peroxisomal Enzyme Defects
Without β-Oxidation Involvement

37.  Refers to a group of related conditions that have
overlapping signs and symptoms and affect many
parts of the body.
 The spectrum includes:
 Zellweger syndrome (ZS), the most severe form
 Neonatal adrenoleukodystrophy (NALD), an
intermediate form
 Infantile Refsum disease (IRD), the least severe
form.

38.  Recently, Heimler syndrome was recognized as a
peroxisome biogenesis disorder within the
Zellweger spectrum and added to the (very) mild
end of the clinical spectrum.
 Caused by mutations in genes that encode
peroxins, proteins required for the normal
assembly of peroxisomes.
 Cultured primarily skin fibroblasts obtained
from patients shows impaired fatty acid beta-
oxidation, phytanic acid alpha-
oxidation, pristanic acid alpha-oxidation, and
plasmalogen biosynthesis.

39. ZELLWEGER’S SYNDROME
 Zellweger Syndrome was discovered by an Switz-
American pediatrician Hans Zellweger.
 Also called as Cerebrohepatorenal syndrome; CHR
 Congenital peroxisome biogenesis disorders
 Caused by mutations in genes that encode peroxins
 Enzymes produced in the cytoplasm are unable to cross
the membrane barrier and enter the matrix of the
peroxisomes.

40.  Characterized by presence of ghost peroxisomes in
the cells of an individual
 This will result in failure to break down lipids, and
cannot contribute to the production of Myelin.
 In 1978, Hansen et al. were the first to report a
defect in bile acid synthesis in Zellweger syndrome
 Accumulation of C27-bile acid intermediates showed
that Zellweger patients were not able to cleave the
side-chain of these precursors and thus could not
form mature C24-bile acids.

41.  Also accumulate VLCFAs, pristanic acid, phytanic
acid, pipecolic acid in plasma and have a deficiency
of plasmalogens in erythrocytes.
 The incidence of ZSDs is estimated to be 1 in 50,000
newborns in the United States

42. Refsum’s Disease
 Adult Refsum’s Disease
 Named after Norwegian neurologist Sigvald
Bernhard Refsum
 Autosomal recessive peroxisomal disorder
 Caused by the impaired alpha-oxidation of branched
chain fatty acids resulting in buildup of phytanic
acid and its derivatives in the plasma and tissues.
 Defect in enzyme phytanoyl CoA hydroxylase
(Phytanic acid oxidase)

43.  Phytanic acid is acumulated in brain and other
tissue
 Adult Refsum disease may be divided into subtypes:
 Adult Refsum disease 1
 Adult Refsum disease 2.
 The former stems from mutations in the phytanoyl-
CoA hydroxylase (PAHX or PHYH) gene, on the
PHYH locus at 10p13 on chromosome 6q22-24.

44.  Refsum disease 2 stems from mutations in the
peroxin 7 (PEX7) gene.
 This mutation on the PEX7 gene is also on
chromosome 6q22-24, and was found in patients
presenting with accumulation of phytanic acid with
no PHYH mutation.
 Lab Findings includes:
 Plasma Level of phytanic acid > 200µmol/L
 Normal< 3oµmol/L

45.  Infantile Refsum’s Disease
 A peroxisome biogenesis disorder resulting from
deficiencies in the catabolism of very long chain fatty
acids and branched chain fatty acids (such as phytanic
acid) and plasmalogen biosynthesis.
 Lab findings:
1. Phytanic acid in the serum is more than 30µmol/L
and less than 200µmol/L
2. VLCFA and LCFA in serum is increased

46.  Cardinal features of refsum disease are:
 Retinitis pigmentosa
 Chronic polyneuropathy
 Cerebellar ataxia
 Elevated levels of proteins in csf
 Skeletal malformations

47.  Molecular Toxicology of Refsum’s Disease
 PA is directly toxic to ciliary ganglion cells and
induces calcium –driven apoptosis in purkinji cells
 Recent studies has found that PA has a Rotenone
like action in inhibiting complex –I and producing
reactive oxygen species
 Hence neuronal cells and retina are prime tissue
affected in Refsum’s disease

48. Adrenoleukodystrophy (ALD)
 Can be classified as X-linked adrenoleukodystrophy
and neonatal adrenoleukodystrophy
 disorder of peroxisomal fatty acid beta
oxidation which results in the accumulation of very
long chain fatty acids in tissues throughout the body.
 A rare, genetic disorder characterized by the
breakdown or loss of myelin
 most severely affected tissues are the myelin in
the central nervous system, the adrenal cortex, and
the Leydig cells in the testes.

49.  Adrenoleukodystrophy has an estimated incidence
of around 1 in 20,000–50,000
 ALD is caused by mutations in ABCD1, a gene
located on the X chromosome that codes for ALD, a
peroxisomal membrane transporter protein.
 Forms of X-linked ALD include:
 Childhood-onset ALD
 Addison's disease
 Adrenomyeloneuropathy

51.  Alpha-methylacyl-CoA racemase (AMACR)
deficiency
 This enzyme (encoded by AMACR) plays a key role in the
breakdown of pristanic acid and the C27-bile acid
intermediates di- and trihydroxycholestanoic acid.
 As a consequence of the impaired degradation of pristanic
acid, both pristanic acid and phytanic acid accumulate
with pristanic concentrations much more elevated than
phytanic acid concentrations
 AMACR deficiency and classic Refsum disease can be
distinguished by screening peroxisome metabolites in the
plasma, followed by fibroblast studies and molecular
genetic testing.

52. Rhizomelic chondrodysplasia punctata type1
(RCDP1)
 caused by pathogenic variants in PEX7
 RCDP is associated with three characteristic
abnormalities:
 deficient ether lipid synthesis
 deficient phytanic acid oxidation
 failure to process peroxisomal β-ketothiolase to its
mature form
 level of phytanic acid elevated

53.  Wanders RJ, van Grunsven EG, Jansen GALipid
metabolism in peroxisomes: enzymology, functions and
dysfunctions of the fatty acid alpha- and beta-oxidation
systems in humans.
 Sacha Ferdinandusse; Sander M.Houten.
Peroxisomes and bile acid biosynthesis
https://doi.org/10.1016/j.bbamcr.2006.09.00
 Ronald J. A. Wanders. Et.al. Bile acids: the role of
peroxisomes

54.  Berger, J.; Gärtner, J. (2006). "X-linked
adrenoleukodystrophy: Clinical, biochemical and
pathogenetic aspects". Biochimica et Biophysica
Acta (BBA) - Molecular Cell Research.
 Steinberg, S.; Dodt, G.; Raymond, G.; Braverman,
N.; Moser, A.; Moser, H. (2006). "Peroxisome
biogenesis disorders". Biochimica et Biophysica
Acta (BBA) - Molecular Cell Research
 Wouter F. Visser et.al. Metabolite transport across
the peroxisomal membrane

55.  Lehninger-Principles of Biochemistry 5th edition
 De Duve C (Apr 1969). "The peroxisome: a new
cytoplasmic organelle". Proceedings of the Royal
Society of London. Series B, Biological Sciences.
173(1030):71-83.
doi:10.1098/rspb.1969.0039. PMID 4389648