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Animal Science Departmental Report 2004-2005 Return to Swine articles
Enzymatic Evaluation of the Biological Demand for L-Carnitine and its
supply from de novo Synthesis and Diet of Swine P. Lyvers Peffer, L. Xi, J. Odle, L. A. Gatlin and J. Woodworth1 1 Lonza Inc., Fairlawn, NJ Background Since carnitine was discovered
in 1905, research has proven the necessity of carnitine in long chain fatty
acid oxidation. Carnitine serves as a
co-substrate for carnitine acyl-transferase, and is required for the transport
of long chain fatty acids into the mitochondria where they can undergo
β-oxidation. Long chain fatty
acids are activated to their Co-A esters in the cytosol, since the
mitochondrial membrane is impermeable to these Co-A esters; these activated
long chain fatty acids cannot enter the mitochondria. Carnitine acyl-transferase exchanges carnitine for the Co-A to
produce acyl-carnitine. This
acyl-carnitine once inside the inner mitochondrial membrane serves as a
substrate for a second carnitine acyl-transferase which completes the final
transfer of the fatty acid into the mitochondria by exchanging the carnitine
for mitochondrial Co-A. The reformed
fatty acyl-CoA is then ready for β –oxidation. Due to its essential role in
fatty acid metabolism carnitine has been classified as a quasi-vitamin, however
adult animals can adequately synthesize carnitine and it is therefore not
considered an essential dietary
component. In mammals, lysine serves as
a precursor for de novo synthesis of carnitine. Synthesis of carnitine from lysine first involves the
tri-methylation of protein bound lysine by S-adenosylmethionine. The resulting trimethyllysine can then be
taken up by various tissues and converted to γ-butyrobetaine by the enzyme
trimethyllysine hydroxylase.
γ-Butyrobetaine can subsequently be converted to carnitine by the
enzyme γ-butyrobetaine hydroxylase.
In most mammals the conversion of γ-butyrobetaine to carnitine can
only occur in the kidney and/or the liver. Animals of all ages can
synthesize γ-butyrobetaine, however the conversion of γ-butyrobetaine
to carnitine does not occur to a great extent in the young animal. The attenuated synthesis of carnitine in the
young animal is influenced by the age dependent activity of
γ-butyrobetaine hydroxylase in the liver, which is low. In humans, hepatic γ-butyrobetaine
hydroxylase activity at birth is approximately 12% of that in adults, and adult
values of γ-butyrobetaine hydroxylase are not reached until 8 d after
birth in the rat. Due to decreased de
novo synthesis in the young animal, there is a demand for dietary
carnitine. Carnitine can be found in
both plant and animal products, however its greatest concentrations are found
in animal tissues while plants often contain little or no carnitine. Milk has been shown to contain carnitine,
and therefore may be the major source of carnitine, which accumulates in
tissues early in development. Research has not yet
determined when g-butyrobetaine hydroxylase
levels reach adult levels in the pig, or whether the activity of the enzyme in
the liver and kidney is age dependent. Therefore, it is unknown when de
novo synthesis of carnitine begins to sufficiently meet the carnitine needs of
the pig. To evaluate the supply and
demand of L-carnitine for CPT I at various stages of development, and in
various tissues by determining the enzyme activities of CPT and g-butyrobetaine hydroxylase. The effect of age on the regulation of key
enzymes involved in the synthesis of L-carnitine and its requirement were
investigated. Results Molecular evaluation of
carnitine status can be made by comparing tissue carnitine and
gamma-butyrobetaine concentrations with the carnitine-Km of carnitine palmitoyltransferase I (CPT I) and
gamma-butyrobetaine-Km of
gamma-butyrobetaine hydroxylase (BBH).
Therefore, the study was conducted to measure apparent enzyme kinetic
constants (Vmax and Km) for CPT I in liver and
muscle mitochondria and for BBH in liver and kidney homogenates from pigs in
seven age categories: newborn, 24 h-old (unsuckled), 1, 3, 5, 8 wk-old and
adult. Enzyme activities were determined via radioenzymatic analyses, and the Vmax and apparent Km for carnitine were
calculated using the iterative NLIN procedure of SAS. 1.Ontogeny of mitochondrial carnitine palmitoyltransferase I in porcine liver and skeletal muscle. Carnitine palmitoyltransferase I kinetic constants (Vmax and apparent KM) measured in mitochondria from hepatic and muscle tissue of pigs (Table 1). Specific activity (Vmax) in liver decreased from newborn to 3 wk of age, but increased after 3 wk of age. The activity (147 umol/h.g of mitochondrial protein) observed at 3 wk of age was 47% lower than that (279 umol/h.g mitochondrial protein) observed in newborn and 5 wk of age. Specific activity in skeletal muscle increased during development of pigs. By 5 weeks, the activity increased by 95% over the activity at 1 and 3 wk of age. There was no significant difference between neonatal and adult pigs. The apparent Km for carnitine in liver was on average 53% higher from pigs at 1, 3, 5, and 8 wk of age than neonatal and adult pigs. The Km value in skeletal muscle was also on average 87% higher from pigs at 1 and 3 wk of age than neonatal and adult pigs, but no significant difference was tested in pigs at other ages. 2.Ontogeny of carnitine biosynthesis in pigs, inferred from
gamma-butyrobetaine hydroxylase activity. Gamma-butyrobetaine hydroxylase kinetic constants (Vmax and apparent KM)
measured in liver and kidney tissue homogenate (Table 2). Hepatic specific
activity was low at birth (75 nmol/h.g of wet tissue), but increased
continuously after 24 h. By 3 wk,
specific activity rose by 8-fold to 600 nmol/h.g of wet tissue (P < 0.05)
and then remained constant into adulthood.
The specific activity in the kidney (840 nmol/h.g wet tissue) was 11
times higher than in liver at birth, but remained constant through 5 wk. By 8 wk, the activity increased by 39% over
the activity at birth (P <0.05). The
apparent Km measured in 24
h and 8 wk-old pigs (0.024 mM) was on average 54% lower than that measured in
pigs of other ages for both liver and kidney.
The Km value was
60% higher in kidney (0.054 mM) than in liver (0.033 mM) during development,
but showed no difference in adults. The
total enzyme activity increased by130 fold for liver and 18 fold for kidney as
organ weight increased from birth to 8 wk.
Summary
These results indicate that hepatic and skeletal muscle CPT I specific activity increased after birth and remained elevated during the sucking period. The Km for carnitine also increased with the increase of CPT I activity, suggesting that there is a positive relationship between the requirement of carnitine and CPT I activity in liver and skeletal muscle tissues during development of pigs. The animal age (developmental stage) affects gamma-butyrobetaine hydroxylase specific activity and Km for gamma-butyrobetaine in liver and kidney. While the predominant organ for carnitine synthesis is likely the kidney in neonates, the liver appears to predominate after the pig exceeds 1 wk of age.Table 1. Apparent kinetic constant (Vmax and Km for carnitine) in mitochondria
Table 2. Apparent
kinetic constant (Vmax and
Km for gamma-butyrobetaine)
in tissue homogenate 
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