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Carnitine: an overview
Prof.Gianfranco Peluso M.D., Ph.D.
Research Director
IBP-National Research Council
University of Piemonte Orientale 'A. Avogadro'
1
– Trimethylated
aminoacid
– Zwitterion
– Sources:
O
CH3
+
CH3 N
CH3
CH2 CH
OH
-
CH2 C O
• Diet: red
meats,
dairies
• Endogenous:
protein
catabolism
L-Carnitine: the molecule
Molecular Formula
C7 H16 N O3 +
Natural Isotopic Abundance Mass
162.20684
Mono-Isotopic Molecular Masses
C12N14: 162.1130183851
C13N14: 169.1365022497
C12N15: 163.1100532783
C13N15: 170.1335371429
L-Carnitine and it´s Derivatives
CH
Free L-Carnitine
+
R:
O
C
O
CH3 N CH2 CH CH C
2
OCH
3
CH
Acyl- L-Carnitine
OH
3
+
3
O
R
CH3 N CH2 CH CH C
2
OCH
3
O
CH3
AcetylL-Carnitine
O
C
O
(CH2)6 CH3
OctanoylL-Carnitine
C
(CH2)14
PalmitoylL-Carnitine
CH3
L-Carnitine
a natural compound of the body
Some basic characteristics of distribution:
a
Whole body stock (70 kilogram):
approx. 20 gram
Portion in skeletal muscle (30 kilogram):
approx. 19 gram / 95% (3,9 µmol/g) a, b
Portion in heart (300 gram):
0,25 gram (4,8 µmol/g)
Portion in blood (5 Liter):
0,04 gram (0,05 µmol/ml)
Fraction of Free L-Carnitine (FC):
about 75 - 80 %
c
Fraction of Acyl-L-Carnitine (AC):
about 20 - 25 %
c
Ratio AC / FC in plasma:
< 0,25
a
a,b
c
According to:
a Scholte et al., 1987
b Opalka et al., 2001
c Boulat et al., 1993
Plasma Carnitine concentration
reference values
Normally low Ratio AC/FC in plasma: 0,25
Concentration of Carnitine (μmol/l) in females and males and
Acyl / Free ratio (mol/mol) in the reference population
N
Mean
SD
Median
Value
Min
Max
P2.5
P97.5
Total
180
38.1
7.6
38
18
57
22
52
Free
180
31.7
6.9
32
14
48
20
44
Acyl
180
6.47
3.3
6
1
18
1
14
Acyl / Free
180
0.21
1.12
0.18
0.02
Females
0.65
0.03
0.41
Males
Total
160
43.6
7.3
43.5
27
60
29.5
58
Free
160
37.8
6.6
37.5
22
53
23
52
Acyl
160
5.8
4.9
5
1
21
1
13.5
Acyl / Free
160
0.16
0.1
0.15
0.02
0.03
0.56
0.54
N = Number of specimens; M = Minimal value; Maximal value; P2.5 = 2.5 percentile; P97.5 = 97.5 percentile ; SD = Standard Deviation
Boulat et al., Eur J Clin Chem Clin Biochem 31(9), 1993
L-Carnitine: the molecule

Although the racemic form of carnitine has been
used clinically in the past, the L-form elicits the
desired biochemical actions, and only the
enantiomerically pure L-carnitine (levocarnitine)
has a role in human medicine.

The carnitine system consists of L-carnitine
and acylcarnitine esters and the enzymes
and transport proteins required for their
metabolism and transport, including
carnitine acyltransferases, mitochondrial
carnitine acylcarnitine translocases, plasma
membrane carnitine importers, and the
carnitine biosynthesis pathway from lysine
and methionine. In contrast to humans,
bacteria possess enzymes to catabolize
carnitine. Intestinal bacteria degrade most
of the orally supplemented carnitine.

Carnitine
system: A Tool
for
Understanding
Functioning
and
Dysfunctioning
Organs by System
Biology
10

genotype/phenotype

Mendelian characters

Complex biological functions derive from the non-linear
interactions of hundreds or thousands of gene products that
take place in a specific spatio-temporal order, interactions
that are strongly modulated by the environment

SYSTEMS BIOLOGY is the new scientific approach to
understanding biological complexity and hence to connecting
reductionistic analysis with description and prediction of
biological functions
4
Modeling
DOES NOT WORK!
6
Two approaches for systems biology
Global description of cellular function
Global functional analysis
Bottom-up
Identification and wiring of
modules
Pathways in modules
Pathways in networks
Modeling of pathways
Top-down
Analysis of single components
INVENTORY OF BIOMOLECULES
7
Definition
by medical dictionary
CARNITINE
Coenzyme of fatty acid
oxidation and acetyl transfer;
often designated vitamin BT,
due to its vitamin role in
Tenebrio sp. Present in high
concentrations (5% dry
weight) in meat extracts
(latin carnis =meat).
Tenebrio molitor
● 75% provided in diet; 25% synthesized
mainly in liver

meat, poultry, fish & dairy products

70 – 80% of dietary intake is absorbed
O
CH3
+
CH3 N
CH3
CH2 CH
OH
-
CH2 C O
L-Carnitine Content of Selected Foods
Food
Serving
L-Carnitine (mg)
Beef steak
3 ounces*
81
Ground beef
3 ounces
80
Pork
3 ounces
24
Canadian bacon
3 ounces
20
Milk (whole)
8 fluid ounces (1 cup)
8
Fish (cod)
3 ounces
5
Chicken breast
3 ounces
3
Ice cream
4 ounces (1/2 cup)
3
Avocado
1 medium
2
American cheese
1 ounce
1
Whole-wheat bread
2 slices
0.2
Asparagus
6 spears (1/2 cup)
0.2
An average omnivorous diet provide 2 to 12 μmol of carnitine per kilogram of
body weight per day, in contrast to strict vegetarians consuming less than 0.1
μmol of carnitine per kilogram of body weight per day.
*A 3-ounce serving of meat is about the size of a deck of cards.
L-Carnitine - The daily requirement
Supply
Biosynthesis:
15 – 20 mg / day (minimum requirement)
Nutrition:
2 – 50; ~100 mg / day (meat)
Elevated Requirement:
Strenuous exercise, diseases, pregnancy, etc.
Excretion
Prevailing values:
15 – 60 - 120 mg/day
Intensive exercise:
> 120mg/day
Balance
Equalized:
Not equalized:
excretion = supply
excretion > supply
Consequence:
L-Carnitine Deficiency


In contrast to microorganisms (i.e., Pseudomonas
sp., Acinetobacter sp., Enterobacteriaccae)
mammals lack the enzymes which are responsible
for the degradation of carnitine
Carnitine homeostasis in mammals is maintained
by a combination of absorption of carnitine from
dietary sources, a modest rate of endogenous
synthesis, efficient reabsorption from the
glomerular, and mechanisms present in most
tissues that establish and maintain substantial
concentration gradients between intracellular and
extracellular carnitine pools.
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Carnitine:
The Endogenous
Synthesis




● 25% synthesized in different organs
(The rate of carnitine biosynthesis in
humans is estimated to be about 1.2
μmol per kg body weight per day)
- liver (major site)
- kidneys
- brain
O
CH3
+
CH3 N
CH3
CH2 CH
OH
-
CH2 C O

Some proteins modify lysine to
trimethyllysine
using
Sadenosylmethionine, (SAM) as the
methyl donor to transfer methyl groups
to the ε-amino of the lysine side chain.
Hydrolysis of proteins containing
trimethyllysine provides the substrate for
the subsequent conversion to carnitine.
Scheme of carnitine metabolism on the organism level with the indication of
carnitine’ synthesis sites.
Nutrigenomics
25
The fate of fatty acyl CoA.
Fatty Acid
In the fed state, fatty acyl CoA is utilized for triglyceride synthesis, and fatty acid oxidation is
dormant. With fasting, fatty acyl CoA is diverted into the oxidative pathway with a fall in
conversion to triglycerides.
26
Nutrigenomics





Activation of PPAR by fibrates or nonesterified fatty acids released from
adipose tissues during fasting causes upregulation of a set of genes involved in
fatty acid catabolism including mitochondrial CPT I and CPT II.
Upregulation of these genes is mediated by binding of activated PPAR/RXR
heterodimers to PPRE present in and around the promoter of those genes.
Consequently, in the liver, where PPAR is most abundant, β -oxidation, is
dramatically increased as a consequence of the increased expression of PPAR
target genes.
The resulting increase in β -oxidation of fatty acids enhances the supply of
acetyl-CoA used for the generation of ATP via tricarboxylic acid cycle and for the
generation of ketone bodies, which are important energetic substrates for the
brain during fasting.
Since CPT are rate limiting for β-oxidation of fatty acids, the upregulation of
CPT might increase the demand of carnitine in cells with high rates of fatty acid
oxidation. Thus, the upregulation of enzymes of carnitine biosynthesis and
OCTN2 by PPAR during fasting can therefore be regarded as a means to supply
tissues with sufficient carnitine required for transport of excessive amounts of
fatty acids into the mitochondrion.
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Carnitine
The transporter(s)

Phylogenetic tree of the
human transporters of the
SLC22 family, including two
transporters from rodents that
have not been detected in
human. Distance along the
branches is inversely related
the degree of sequence
identity. For example,
sequence identities are 70%
between hOCT1 and hOCT2,
32% between hOCT1 and
hOCTN1, and 32% between
hOCT1 and hOAT1.
Polyspecific transporters for
organic cations are indicated in
red, transporters for organic
cations and zwitterions in
yellow, and transporters for
organic anions in blue
OCTN2 Carrier
Predicted topology of SLC22 transporters with functional relevant point
mutations. Predicted glycosylation sites on the large extracellular loops of
hOCTN2 (Ψ) and predicted phosphorylation on the large intracellular loops
(blue) are indicated. Amino acids where point mutations resulted in changes
of the substrate selectivity are indicated in yellow. Glutamate 452 in hOCTN2
(pink) is thought to be involved in Na+ binding to hOCTN2.





Carnitine is also available from the diet after being absorbed
in the intestine mainly by active transport.
Absorption of carnitine from dietary sources occurs mainly
in the small intestine. Initially, uptake of carnitine from the
lumen of the intestine across the apical membrane into the
enterocyte is mediated in a sodium-dependent manner
(Kato et al., 2006).
Mrp3 and Abca1 mediate subsequent movement across the
basolateral membrane of the enterocyte and into the
circulation (Klaassen and Aleksunes, 2010).
Mrp3 and Abca1 are ABC family cellular exporters with broad
substrate specificities, and here, the apical absorption may
be the rate-limiting step.
Carnitine uptake across the apical enterocyte membrane is
saturable with kinetics akin to OCTN2-overexpressing cells
and can be inhibited by Octn2 inhibitors (Kato et al., 2006).



By 4 weeks of age, Octn2-null mice demonstrate
atrophic intestinal villi, inflammation, ulcer
formation, and gut perforation (Shekhawat et
al., 2007).
Inflammatory bowel disease has been linked to
mutations on a locus on chromosome 5 that
localizes to the region where the SLC22A4
(OCTN1) and SLC22A5 (OCTN2) genes exist
(5q31).
Polymorphisms in OCTN transporters are
associated not only with the incidence of
inflammatory bowel disease, but also with the
pharmacological response to therapy.

Interestingly, it
has been
demonstrated
that intestinal
bacterial
components or
soluble factors
released by gut
bacteria are
sensed by the
colonocytes
that increase
the OCTN2
expression.




Similar to uric acid absorption, renal carnitine
handling is a critical step in maintaining systemic
carnitine homeostasis.
Under normal homeostasis conditions, carnitine is
mainly eliminated by excretion in urine.
In healthy humans, the daily excretion in urine is 100
to 300 μmol and the tubular reabsorption in the
kidney is 90 to 98%.
Carnitine handling represents another potential
example of remote sensing and signaling in which
homeostasis in remote target organs is regulated
coordinately.



In the kidney, the SLC22A5 gene product is
located on the apical membrane of proximal
tubule cells.
As for other organic cation substrates,
SLC22A5 mediates the absorption of
carnitine from renal ultrafiltrate into the
proximal tubule epithelial cells.
Disturbances in carnitine handling at the
kidney level might cause other remote
organ dysfunctions in heart, muscle, and
placenta.
39


The content of carnitine in brain was observed to
be lower than in peripheral tissues, with the
exception of hypothalamus (Bresolin et al.,
1982), which is known to have an easy access to
many substances through fenestrations. This
observation may point to the existence of some
limitations in carnitine accumulation in the brain.
A number of uptake (OCTN2) and efflux (Mrp)
transporters for carnitine have been localized to
the membranes of brain capillary endothelial
cells, while the choroid plexus is rich with drug
transporters including Oatps but not OCTN2.
BRAIN

OCTN2 and
OCTN3 have been
demonstrated in
astrocytes and
neurons

Skeletal muscle and heart contain over
95% of total body carnitine.

Since skeletal muscle and heart lack the
ability to synthesize carnitine, it is
obvious that carnitine transport is
fundamental to supply carnitine to these
tissues.
44

The preferential distribution of carnitine
to heart and skeletal muscle is mediated
by carnitine transporters of the SLC22
family, particularly SLC22A5/OCTN2, and
probably OCTs (OCT3) as well as Flipt1
and Flipt2 (also known as CT1/SLC22A15
and CT2/SLC22A16, carnitine transporter
2).


Because milk is rich in lipids, it is presumed that
carnitine is important in preparing the fetus for a
postnatal milk diet. Human OCTN2 protein is
localized to the apical membrane of
syncytiotrophoblasts (Lahjouji et al., 2004; Grube et
al., 2005), and is responsible for delivery of carnitine
to the fetus during development.
During pregnancy, the use of certain anticonvulsants,
able to inhibit carnitine uptake, carries a risk of fetal
malformations, including congenital heart disease as
well as lip and palatal deformity. It is hypothesized
that interference of OCTN1 and/or OCTN2-mediated
carnitine uptake across placenta by antiseizure drugs
increases the risk of fetal developmental defects.




It is noteworthy that carnitine is an essential nutrient for newborn
infants because the mechanism of its biosynthesis is not fully
developed until later in postnatal development. The primary source
of carnitine for a newborn is from the mother's milk (Sandor et al.,
1982).
The neonatal intestine has an avid carnitine absorption system of
solute carriers of SLC and ABC families. Likewise, carnitine is
handled by a network of transporters in the mammary gland
featuring SLC22A5, SLC22A4, and SLC22A3 (Kwok et al., 2006).
The carnitine produced in the mammary gland is excreted into the
milk and then is absorbed in the baby's intestine. Mother's milk is
the only source of carnitine for the newborn infant and is required
for the β-oxidation of fatty acids.
This process of carnitine transfer from mother to infant represents
a coordinated mechanism of solute handling via SLC and ABC
transporters between individuals.

~1 in every 12 babies
(8.33%) in the United
States is born fullterm with low birth
weight (LBW)

Global incidence of
LBW ~17%
49
Maternal Low Protein
Diet
Full-term Low Birth
Weight babies
Fetal Programming
Altered Physiology
Altered Morphology
Kidney; Liver;
Gastrointestinal tract
Altered
absorption/production/excretion
of carnitine
•Hypertension
•Hyperlipidemia
•Diabetes Mellitus
•Obesity
Require altered optimal
drug dosage for patient
Altered
Pharmacokinetics
50




Children who develop type 1 diabetes early in life have
low levels of carnitine and amino acids at birth: does this
finding shed light on the etiopathogenesis of the disease?
M. Locatelli, et al.
Conclusion:
This is the first study demonstrating that children who
develop T1D early in life showed reduced circulating
carnitine and amino acid levels soon after birth. Their
evaluation in the early neonatal period could represent an
additional tool in the prediction of T1D and offer new
strategies for possibly preventing the disease as early as
from birth.
51


Under normal physiological conditions, the primary site of
carnitine production is in the liver. During lactation, the production
of carnitine in the mammary gland increases, apparently at the
expense of production in the mother's liver, which is also
accompanied by reduced hepatic enzymatic and transcriptional
activity, as well as solute carrier activities associated with carnitine
synthesis and handling (Gutgesell et al., 2009).
This complex physiological regulation of carnitine synthesis and
handling is necessary to provide the infant with carnitine for βoxidation and energy production to use fatty acids. In this instance
of carnitine production, transfer from mother to infant, and
carnitine utilization in the infant, all via solute carrier networks,
mediates the enzymatic activities between and within cells of the
mother and the infant
Carnitine and its carrier SLC22A5 mediated sensing and signaling
among organs and between mother and infant. Liver is the primary
site of carnitine biosynthesis. Circulating carnitine is
distributed/transported through its carriers, primarily SLC22A5 to
the target organs. The synthesis and distribution of carnitine is
regulated physiologically. For example, during lactation, carnitine
is preferentially distributed to the nursing mother's breast through
increased expression of its carriers at the expense of the liver,
because mother's milk is the only source of carnitine for the infant
and is essential for the survival/growth of the baby.
Carnitine
The function
The two main functions of carnitine are:
1. the transport of long-chain fatty acids into
the mitochondrial matrix for β-oxidation to
provide cellular energy, and
2. the modulation of the rise in acyl-CoA/CoA
ratio

56

Control of fatty acid
oxidation is vested in
the carnitine
palmitoyltransferase
system, which
consists of three
enzymes: carnitine
palmitoyltransferase
I (CPT I), carnitine
palmitoyltransferase
II (CPT II), and
carnitine:acylcarnitin
e translocase (CACT).
57
Carnitine
● Carnitine regulates levels of acyl-CoA inside cells and promotes the
clearence of acyl residues
- CoA pools are limited and CoA is needed in other processes
(GNG, CAC, Urea cycle, b-ox)
- Transfer acyl to carnitine to restore CoA pools so the
acyl-carnitine serve as a reservoir of activated acyl groups.
In addition, acyl-CoA are toxic compounds.
-Promote clearance of accumulating acyl residues facilitating their
renal or hepatic excretion.
O
CH3
+
CH3 N
CH3
CH2 CH
OH
-
CH2 C O
Urea Cycle
regulated step
excreted in
urine
*emphasis on
green text added
To CAC
carbamoyl phosphate synthetase-I
activates
acetyl-CoA + glutamate
N-acetylglutamate + CoA
N-acetylglutamate synthetase
● Without restoration of CoA pools,
● Acetyl-CoA levels drop
● N-acetylglutamate (NAG) will not be made
● CPS I will not be activated and so urea cycle will not proceed
● NH4+ builds up
Carnitine and Membrane Integrity
Long-Chain
Fatty Acids
Carnitine
CPT1
Protein
Acylation
Long-Chain
AcylCarnitines
Acyl-carnitine represents a reservoir of
acyl-CoA units at no ATP cost.
Membrane
Repair
Carnitine and plasmamembrane repair
•
•
•
•
•
Oxidation of a polyunsaturated fatty acid
esterified in membrane phospholipid (PLP)
generates a phospholipid hydroperoxide
(PLP-OOH)
The deacylating activity of a phospholipase
A2 (PLA) hydrolizes the fatty acid
hydroperoxide (FA-OOH), that is reduced by
a glutathione peroxidase (GSH-Px) to the
non-reactive FA alcohol (FA-OH)
The lysopholipid acyl-CoA transferase (LAT)
regenerates the original PLP avoiding
accumulation of the toxic lysophospholipid
(LPL).
LAT activity requires acyl-CoA synthetized by
the ATP-dependent enzyme acyl-CoA
synthetase (ACS)
Carnitine palmitoyltransferase (CPT), allows
the ACS-LAT cycle to keep the acyl-CoA/free
CoA ratio constant. Indeed, given the kinetic
properties of CPT, any alteration of the acylCoA/free CoA ratio would be promptly
buffered by the fully reversible and mass
62
action sensitive CPT reaction.
Membrane visco-elastic properties
•
•
•
Major determinants of the viscoelastic properties of red blood
cell membrane are the
cytoskeletal network and
membrane phospholipid bilayer.
LC may improve the visco-elastic
properties of red blood cells by
exerting a stabilizing effect on
the membrane via interaction
with certain cytoskeletal
components [spectrin/actin as
indicated in the figure by the red
circle].
LC would also affect the viscoelastic properties of red blood
cell membrane by strengthening
the polar head packing of
membrane phospholipids
63
Learning carnitine function by
carnitine deficiency diseases.
64

Carnitine deficiency can be characterized
by low plasma and tissue carnitine
concentrations and can be defined as a
decrease of intracellular carnitine,
leading to an accumulation of acyl-CoA
esters and an inhibition of acyl-transport
via the mitochondrial inner membrane.
65


Two forms: systemic carnitine deficiency with low
carnitine levels in plasma and the affected tissues,
and muscle carnitine deficiency, with low carnitine
concentration restricted to muscle.
Animal model: Juvenile Visceral Steatosis
Mouse (severe lipid accumulation in the liver,
hyperammonemia, hypoglycemia, cardiac
hypertrophy, mitochondrial abnormalities in
skeletal muscle and progressive growth
retardation). Point mutation from CTG to CGG
(leucine to arginine at amino acid position 352) in
the mouse homologue of OCTN2 carnitine carrier.
66


A decrease of carnitine levels in plasma or tissues,
may be associated with genetically determined
metabolic conditions, acquired medical conditions,
or iatrogenic states.
Disorders of the carnitine cycle or disorders of fatty
acid beta-oxidation can cause secondary carnitine
deficiency via several mechanisms. Block in fatty acid
oxidation contributes to the accumulation of acylCoA intermediates. Transesterification with carnitine
leads to the formation of acylcarnitine and the
release of free CoA. These acylcarnitines are excreted
readily in the urine with increased carnitine losses in
the urine and systemic secondary depletion of
carnitine.
67







Other genetic conditions that are associated with Fanconi syndrome
(eg, Lowe syndrome, cystinosis) may present with secondary
carnitine deficiency because of increased renal losses of carnitine.
Lysinuric protein intolerance is associated with an increased
excretion of lysine in the urine and decreased carnitine synthesis.
Cirrhosis or chronic renal failure may impair the biosynthesis
of carnitine.
Diets with low carnitine content (eg, lacto-ovo–vegetarian diet) or
malabsorption syndromes may cause secondary carnitine deficiency
Preterm neonates are at risk for developing carnitine deficiency
because they have impaired reabsorption of carnitine at the level of
the proximal renal tubule and immature carnitine biosynthesis.
Iatrogenic causes of secondary carnitine deficiency include several
drugs such as valproate.
Conditions of increased catabolism present in patients with critical
illness.
68