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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
Normally low Ratio
AC/FC in plasma:
0.25
CH
+
3
O
R
O
CH3 N CH2 CH CH C
2
OCH
3
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
a
a,b
Ratio AC / FC in plasma: < 0.25 c
According to:
a Scholte et al., 1987
b Opalka et al., 2001
c Boulat et al., 1993
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
TENEBRIO MOLITOR AND OBSERVATIONS
ON THE EXPRESSION OF A CARNITINE
DEFICIENCY by G. S. FRAENKEL -Department
of Entomology, University of Illinois, Urbana 1958
Carnitine
● 75% provided in diet; 25% synthesized mainly in liver
• meat, poultry, fish & dairy products (3 ounces beef
steak=81 mg Carnitine; 3 ounces codfish=5mg
Carnitine; 12 spears of asparagus =0.4mg Carnitine )
• Although in healthy subjects endogenous synthesis is
adequate to maintain carnitine levels, addition of
carnitine in the diet may be required during certain
stages of the life cycle and in disorders, such as aging
and diabetes. Thus, carnitine is considered a
“conditionally essential” nutrient and the term
“functional carnitine deficiency” has been proposed
to define abnormal clinical presentations correctable
by carnitine administration.
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., Enterobacteriacae) 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.
8
TMA:trimethylamine
FMO:flavin monooxygenases
TMAO:trimethylamine-N-oxide
Gut flora:
Prevotella genus for carnivore
Bacteroides genus for vegetarian
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10
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http://www.westonaprice.org/blogs/cmasterjohn/2013/04/10/does-carnitine-from-red-meatcontribute-to-heart-disease-through-intestinal-bacterial-metabolism-to-tmao/
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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.
Histone
Calmodulin
Myosin
Actin
Cytochrome c
Proteolysis
15
Scheme of carnitine metabolism on the organism level with the indication of
carnitine’ synthesis sites.

The complex biological functions of
Carnitine derive from the non-linear
interactions of many gene products,
interactions
that
are
strongly
modulated by the environment

Carnitine
system:
A
Tool
for
Understanding
Functioning
and
Dysfunctioning
Organs by System
Biology
17
 L-carnitine and acylcarnitine esters
 the enzymes and transport proteins
required for their transport
(included the plasma membrane
carnitine carriers, OCTN)
 the carnitine biosynthesis pathway
from lysine and methionine.
19
Nutrigenomics
20
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.
21
The carnitine system
22
Cytosol
Fatty Acid (FA)
Nucleus
+
Activation of PPAR by 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.
Gene Expression
23
Since CPT are rate
limiting for βoxidation of fatty
acids,
the
upregulation
of
CPT might increase
the demand of
carnitine in cells
with high rates of
fatty
acid
oxidation.
The
upregulation
of
carnitine
biosynthesis and
OCTN2 by PPAR
during fasting is a
means to supply
tissues
with
sufficient carnitine
for
metabolic
requirement.
Carnitine
Carnitine
The function
One of the main functions of carnitine is:

the transport of long-chain fatty acids into
the mitochondrial matrix for β-oxidation to
provide cellular energy
26
Organic Cationic Transporter

Regulation of betaoxidation by
plasmamembrane
carnitine carrier
27
CPT-1
CACT
CPT-1

CARNITINE SYSTEM
28
Learning carnitine function by
carnitine deficiency diseases.
29

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.
30



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).
Pathogenesis: Point mutation in the mouse
homologue of OCTN2 carnitine carrier.
31

fatty metamorphosis of viscera due to
OCTN2 mutation
32
33
Secondary carnitine deficiency:
(e.g., genetically determined metabolic conditions, acquired medical
conditions, or iatrogenic states).
Increased renal loss of
carnitine:
Lowe syndrome, Cystinosis
Lysinuric protein intolerance:
>>Lysine in urine with <<Carnitine
synthesis
Cirrhosis or chronic renal failure:
< carnitine biosynthesis
Lacto-ovo–vegetarian diet
Preterm neonates: impaired renal
reabsorption of carnitine and
immature carnitine biosynthesis
Iatrogenic causes
(i.e., Valproate therapy)
Increased catabolism in patients
with critical illness (i.e., cancer
disease)


Because milk is rich in lipids, it is presumed that
carnitine is important in preparing the fetus for a
postnatal milk diet. Carnitine carrier(s) 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 carrier-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 .(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
carnitine transporters between individuals.

~1 in every 11 babies
(8.23%) in Europe is
born full-term with
low birth weight
(LBW)

Global incidence of
LBW ~17%
38
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
39




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.
40
FA
FA
43
FA
44
Why does carnitine deficiency affect
so many organs?
…. because the role of carnitine goes
beyond the oxidation of fatty acids.
45
Carnitine and CoA axis
• A decrease of carnitine
Free CoA is required for:
induces a decrease of
matrix free CoA and a
• the catabolism of
parallel increase of the acyl
several amino acids,
CoA/CoASH ratio both of
which are inhibitory for
• for the detoxification of
mitochondrial
organic acids and
dehydrogenases.
• Consequently, not only the
xenobiotics,
oxidation of fatty acids,.
• for pyruvate
but also the utilization of
carbohydrates, the
dehydrogenase,
catabolism of several
• for α-ketoglutarate
amino acids and the
detoxification of organic
dehydrogenase
acids and xenobiotics
46
become impaired.
• for Kreb’s cycle



Disorders of the mitochondrial functions
can cause severe impairment of fatty acid
oxidation with accumulation of acyl-CoA
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.
47
48
Metabolic Flexibility
the ability
• to adapt substrate utilization to substrate availability
• to switch between the oxidation of lipid as a fuel during fasting
periods to the oxidation of carbohydrate during insulin
stimulated periods
49
Substrate Competition Theory
FASTING
Randle cycle
• byproducts of fatty acid βoxidation, including acetylCoA, and citrate, act as
potent allosteric inhibitors
of glycolysis and pyruvate
dehydrogenase (PDH)
50
• Reverse Randle cycle
FEEDING/INSULIN
51
Metabolic InFlexibility
failure to shift from fatty acid to glucose oxidation during the transition
• from fasting to feeding
• from rest to vigorous exercise and vice versa
52
Myocardial
metabolism in
normal heart.
Normal myocardium at
resting state uses FFA as
a preferred fuel source,
which is energy rich
substrate but yield less
ATP per mol of O2. While
in the state of increased
peripheral physiological
demand (e.g. exercise)
myocardium uses glucose
as energy source which is
more efficient energy
source and yield more
ATP per mol of O2.
ATP, Adenosine
triphosphate; FFA, Free
fatty acid; O2, oxygen;
Pcr, Phospho creatinine
53
Myocardial Metabolism in Health
The heart consumes more energy than any other
organ and it is the greatest oxygen-consuming
organ in the body, around 8–15 ml O2 min/100 g
heart, with the capacity to increase up to 70 ml
under exercise conditions. Everyday the heart
beats about 100,000 times, pumps approximately
10 tons of blood through the body, and cycles
about 6 kg of adenosine-triphosphate (ATP) (20–
30 times its own weight).
Mitochondrial energy metabolism in heart failure: a
question of balance
Metabolic Demand
Energy Metabolism
• Heart must continually generate ATP
• The heart has a very high energy
at a high rate from mitochondrial
demand to sustain contractile
oxidative phosphorylation (>95%) ,
function, basal metabolic
with the remainder being derived
processes, and ionic homeostasis.
from glycolysis and GTP formation in
• The heart has a relatively low ATP
the tricarboxylic acid (TCA) cycle.
content (5 μmol/g wet wt) and high • Under normal conditions, there is
rate of ATP hydrolysis (∼30 μmol·g
complete turnover of the myocardial
wet wt−1·min−1 at rest).
ATP pool approximately every 10 s.
• To sustain sufficient ATP
• However, the adult heart normally
obtains 60–80% of its ATP from fatty
generation, the heart acts as an
acid β-oxidation
“omnivore” and can use a variety of
different carbon substrates, (i.e.,
fatty acid, glucose, lactate, and
ketone bodies), as energy sources.
Cardiac energy substrate selection
• In the fetal heart, glucose oxidation is favored, whereas FA oxidation serves as
the major ATP-generating pathway in the adult myocardium. Hypertrophy and
ischemia drive metabolism toward glucose utilization, whereas in uncontrolled
diabetes, the heart utilizes FAs almost exclusively. In some cases, as in early
response to pressure overload–induced hypertrophy, these metabolic shifts
are thought to be protective.




The regulation of myocardial metabolism is dependent on the
availability and abundance of substrate, hormone levels, coronary
blood flow and oxygenation and inotropic state of the tissue. With
ageing, relative contribution of glucose as myocardial substrate
increases as seen in elderly.
Altered substrate supply and utilization by cardiac myocytes could
be the initial trigger in the pathogenesis of CM.
The shift towards increased fatty acids and decreased glucose
utilization is linked to elevated circulating levels of FFA and
triglycerides as a consequence of enhanced adipose tissue
lipolysis, increased FFA uptake as well as high tissue FFAs caused
by hydrolysis of augmented myocardial triglyceride stores.
When FA uptake exceeds FA oxidation capabilities, lipid
accumulation occurs resulting in lipotoxicity.
57
Various possible risk factors that could contribute to the
development of Cardio-metabolic Cardiomyopathy
58
Mitochondrial dysfunction is at the basis of a constellation of
metabolic abnormalities that contribute to chronic
conditions and diseases
59
60

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.
61

Diet-induced and genetic forms of glucose
intolerance were associated with high rates of
incomplete fat oxidation (>>fatty acidderived acylcarnitine intermediates), which
occurs when carbon flux through the βoxidation machinery outpaces entry of
acetyl-CoA (the final product of β-oxidation)
into the TCA cycle.
Carnitine as an antilipotoxic therapy
Efflux of Acylcarnitine from cell
avoids metabolic inflexibility
…but depends on carnitine availability
64
L-carnitine buffers the
ratio of free CoA to
acyl-CoA. Excess
quantities of CoA
esters induce acyl-CoA
transesterification
with L-carnitine to
form acylcarnitines,
thereby freeing CoA
for use in other
mitochondria
reactions.The resultant
acylcarnitines cross
the mitochondrial
membrane via CACT.
>> Acyl-CoA
CoA
65
Carnitine Acetyltransferase
and Metabolic Flexibility
66

During the early phases of refeeding/reperfusion, heart
mitochondria are confronted with a heavy influx of both
glucose and fatty acid fuel. By permitting mitochondrial
efflux of excess acetyl moieties, CrAT allows PDH
activity to increase despite high rates of β-oxidation.
This eases the Randle effect and facilitates a rapid
transition from fatty acid to glucose oxidation. CrAT
deficiency and/or carnitine insufficiency imposes a more
rigid version of the Randle cycle, wherein feedinginduced stimulation of PDH activity is heavily dependent
on the production of malonyl-CoA and resultant
inhibition of CPT1.
67
68



Carnitine insufficiency results in abnormal fuel
selection, evident in whole animals and in isolated
mitochondria, and a corresponding decay in systemic
glucose homeostasis. These disruptions in energy
metabolism are associated with aberrant control of
PDH activity.
Carnitine mediates stimulation of PDH activity in
heart mitochondria while also hampers the capacity
of pyruvate to compete with and inhibit fatty acid
oxidation.
The therapeutic benefits of Carnitine
supplementation corresponded with robust tissue
efflux and urinary excretion of acetylcarnitine and
acylcarnitines
69
Clinical Studies
High doses of L-Carnitine in acute
myocardial infarction: metabolic and
antiarrhythmic effects.
Rizzon et al,
European Heart Journal 1989; 10: 502-508
Conclusions:
•L-carnitine supplementation results in a significant increase in levels of
carnitine esters in both the serum and urine.
•The increased urinary excretion of long and short chain acylcarnitine
esters induced by L-carnitine supplemetation may account for the
reduction in ventricular arrhythmias observed in AMI patients.
•L-Carnitine, by increasing the elimination of acylcarnitine esters, could
therefore have a potential protective role on myocardial tissue, during AMI.
Effects of L-Carnitine administration on
Left Ventricular Remodeling after Acute
Anterior Myocardial Infarction : the LCarnitine Ecocardiografia Digitalizzata
Infarto Miocardico (CEDIM) Trial.
Iliceto S et al
Journal of the American College of Cardiology 1995; 2: 380-387
Conclusions:
• The early and long-term administration of L-carnitine in patients with
AMI is effective in attenuating progressive left ventricular dilation.
• A still earlier administration of L-carnitine might exert a further
protective effect on ischemia-reflow dysfunction within the risk area.
Acute Myocardial Ischaemia Induces
Cardiac Carnitine Release in Man
Bartels GL., et al
European Heart Journal 1997; 18: 84-90
Conclusions:
• The moderate changes in carnitine metabolism during short periods of
moderate myocardial ischemia may indicate cardiac Carnitine loss in
situations under acute ischemic, metabolic stress.
• This may indicate that L-Carnitine supplementation may be useful in
patients with transient mild myocardial ischemia.
Metabolic Treatment with L-Carnitine in
Acute Anterior ST Segment Elevation
Myocardial Infarction.
Tarantini et al.
Cardiology 2006; 106: 215-223
Conclusions:
• The results of the study suggest that L-carnitine administration is
particularly effective at reducing mortality when supplemented in the
first week after anterior AMI.
• The reduction in early mortality induced by L-carnitine treatment is likely
due to the protection of the ischemic myocardium in the very acute
phase of ischemia and reperfusion but could also be due to the
protection of the acutely over-loaded non-infarcted region of the
ventricle
Three-year Survival of Patients with Heart
Failure caused by Dilated Cardiomyopathy
and L-Carnitine administration.
Rizos I.
American Heart Journal 2000; 139: S120-S123
Conclusions:
• Three months treatment with 2g /day of oral L-carnitine improved
survival of patients with moderate to severe heart failure due to dilated
cardiomyopathy.
• Three months L-carnitine treatment appears to improve the functional
status of patients with moderate to severe heart failure attributable to
dilated cardiomyopathy
L-carnitine as an Adjunct Therapy to
Percutaneous Coronary Intervention for Non-ST
Elevation Myocardial Infarction
Xue et al.
Cardiovasc Drugs Ther (2007) 21:445–448
Treatment:
• Carnitine 5 g IV bolus followed by 10 g/day IV infusion for 3 days
Conclusions:
• L-carnitine adjunct therapy appears to be associated with a reduced
level of cardiac markers in patients with non-ST elevation acute
coronary syndrome
)
L-Carnitine prevents the development of ventricular
fibrosis and heart failure with preserved ejection
fraction in hypertensive heart disease
Omori, et al
J Hypertens, September 1, 2012; 30(9): 1834-44.
Conclusions:
Carnitine supplementation attenuates cardiac fibrosis by increasing
prostacyclin production through arachidonic acid pathway, and may be a
promising therapeutic option for HFpEF)
Disturbed carnitine regulation in chronic heart failure Increased plasma levels of palmitoyl-carnitine are
associated with poor prognosis
Ueland et al.
Int J Cardiol, May 21, 2012.
Conclusions:
Findings support a role for disturbed carnitine metabolism in the
pathogenesis of HF, and suggest that some of its derivates could give
prognostic information in these patients.
Mitochondrial dysfunction is a common
cause of peripheral neuropathy.
 Neuronal/axonal mitochondria
impairment
 Peripheral neuropathy secondary to
Schwann Cell mitochondrial dysfunction

79


Mitochondrial metabolic irregularities are a
common culprit in diverse
neurodegenerative diseases and are key
pathological contributors to peripheral
neuropathy.
Mitochondrial dysfunction is thought to be
largely responsible for the peripheral nerve
deficits that afflict large numbers of people
with diabetes and can lead to incapacitating
pain, sensory loss, and debilitating muscle
weakness.
80




Mitochondrial-dysfunction-induced shift in Schwann cells
(SCs) lipid metabolism away from new lipid synthesis toward
increased fatty acid oxidation.
This metabolic alteration results in early depletion of myelin
lipid components as well as a large accumulation of
acylcarnitine (AC) lipid intermediates.
Importantly, ACs are released from SCs and induce axonal
degeneration.
Activation of a maladaptive integrated stress response (ISR)
and altered SC lipid metabolism resulting in toxic
accumulation of lipid intermediates are thus underlying
mechanisms of axonal degeneration and demyelination in
mitochondrial peripheral neuropathies and constitute
potentially important therapeutic targets.
81
tissue-specific deletion
of the mitochondrial
transcription factor A gene
Lipid Classes
CB: Cerebrosides
ST: Sulfatides
82
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