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DSM in Animal Nutrition & Health

Carnitine

Properties and Metabolism

Carnitine (earlier known as vitamin Bt) is a quaternary amine, beta-hydroxy-gamma-trimethylaminobutyrate. It is a very hygroscopic compound and is found in biological samples both as the free carnitine and as the ester of a wide variety of acyl compounds. Of the two types of carnitine, L-and D-carnitine, only L-carnitine is biologically active. This has been shown in a number of species (Grandjean et al., 1993). It was not until the 1960s that carnitine was recognized as a biologically active substance (Borum, 1991). Carnitine is synthesized in the liver and kidneys. The synthesis depends on two precursors, L-lysine and methionine, as well as ascorbic acid, nicotinamide, vitamin B6 and iron (Borum, 1991). Deficiency in any cofactor will cause L-carnitine deficiency. In rats, total acid-soluble carnitine and free carnitine in plasma and tissues were reduced in a vitamin B6 deficiency but increased when vitamin B6 was provided in a repletion diet (Cho and LeKlem, 1990; Ha et al., 1994). It has been suggested that early features of scurvy (fatigue and weakness) may be attributed to carnitine deficiency. Vitamin C is a cofactor for two alpha-ketoglutarate-requiring dioxygenase reactions (epsilon-N-trimethyllysine hydroxylase and gamma-butyrobetaine hydroxylase) in the pathway of carnitine biosynthesis. Carnitine concentrations are variably low in some tissues of vitamin C-deficient guinea pigs (Rebouche, 1991). Choline has also been shown to affect carnitine homeostasis in humans and guinea pigs (Daily and Sachan, 1995). Choline supplementation resulted in decreased urinary excretion of carnitine in young adult women; choline resulted in the conservation of carnitine in guinea pigs. Carnitine appears to be absorbed across the gut by an active process dependent on Na+ as well as by a passive diffusion that may be important for the absorption of large doses of the factor. The uptake of carnitine from the intestinal lumen into the mucosa is rapid, and about one-half of the carnitine taken up is acetylated in that tissue. Tissues such as cardiac muscle and skeletal muscle require carnitine for normal fuel metabolism but cannot synthesize carnitine and are totally dependent on the transport of carnitine from other tissues. Free carnitine is excreted in urine, with the principal excretory product being trimethylamine oxide (Mitchell, 1978). Carnitine is highly conserved by the human kidney, which reabsorbs more than 90% of filtered carnitine, thus, playing an important role in the regulation of carnitine concentration in blood. For the dog, 95% to 98% of the carnitine body pool is in skeletal muscle and the heart (Rebouche and Engel, 1983). With rats, Flores et al.(1996) reported that the small intestine has a considerable and previously unrecognized proportion of the carnitine pool in suckling animals.Under normal conditions in omnivores, about 70% to 80% of dietary carnitine is absorbed (McDowell, 2000). Absorption of carnitine in dietary supplements (0.5 to 4gm per day) was 15% to 25% (Rebouche, 2006). Carnitine appears to be absorbed across the proximal small intestine by an active process. Tissues such as heart and skeletal muscle require carnitine for normal fuel metabolism. However, these tissues cannot synthesize carnitine and are totally dependent on transport of carnitine from other tissues.

 

Functions

Carnitine is required for transport of long-chain fatty acids into the matrix compartment of mitochondria from cytoplasm. This results in the subsequent oxidation by the fatty acid oxidase complex for energy production. It is thereby playing a key role in the use of fatty acids as an energy substrate by the tissues. The oxidation of long-chain fatty acids in animal tissues is dependent on carnitine because it allows long-chain acyl-CoA esters to cross the mitochondrial membrane, which is otherwise impermeable to CoA compounds. Carnitine facilitates the beta-oxidation of long-chain fatty acids in the mitochondria by transporting the substrate into the mitochondria. Carnitine acyltransferase is the enzyme responsible for this shuttle mechanism. It exists in two forms, carnitine acyltransferase I and carnitine acyltransferase II. After the long-chain fatty acid is activated to acyl-CoA, it is converted to acylcarnitine by the enzyme carnitine acyltransferase I and crosses to the matrix side of the inner mitochondrial membrane. Carnitine acyltransferase II then releases carnitine and the acyl-CoA into the mitochondrial matrix. Acyl-CoA is then catabolized via beta-oxidation (Borum, 1991). Thus, utilization of long-chain fatty acids as a fuel source depends on adequate concentrations of carnitine. The role of carnitine in the transport of long-chain fatty acids across the inner mitochondrial membrane is known, but liver medium-chain fatty acid (MCFA) metabolism has been considered carnitine independent owing to MCFAs’ passive diffusion through the inner mitochondrial membrane and intramitochondrial activation (Bremer, 1990). However, recent evidence suggests that MCFA metabolism may be affected by supplemental carnitine (Van Kempen and Odle, 1993; 1995). Another role of carnitine may be to protect cells against toxic accumulation of acyl-CoA compounds of either endogenous or exogenous origin. This is accomplished by trapping such acyl groups as carnitine esters, which may then be transported to the liver for catabolism or to the kidney for excretion in the urine. Carnitine also has functions in other physiological processes critical to survival, such as lipolysis, thermogenesis, ketogenesis and possibly regulation of certain aspects of nitrogen metabolism (Borum, 1985). Recently, dietary carnitine was shown to enhance the lymphatic absorption of fat and alpha-tocopherol in ovariectomized rats (Zou et al., 2005).

In mammals, gamma-butyrobetaine, the immediate precursor of carnitine, can be synthesized from the essential amino acids lysine and methionine in most tissues. The four-carbon chain comes from lysine; the methyl groups come from methionine. The ultimate conversion of gamma-butyrobetaine to carnitine occurs in the liver (Olson and Rebouche, 1987).

Carnitine was found to be beneficial in detoxification of aflatoxins (Yatim and Sachan, 2001). For rats there was a modification of aflatoxin binding to proteins and DNA by carnitine which reduced the carcinogenic and hepatotoxic potential of aflatoxins.

 

Requirements

Higher animals, including mammals, can synthesize carnitine. However, recent studies have indicated that the biosynthesis of carnitine may be limited or inadequate in certain animals. The liver and kidney of mammals are able to synthesize carnitine from lysine and methionine; however, endogenous biosynthesis alone is not sufficient to keep carnitine concentrations at adequate levels (Duran et al., 1990). Although synthesis of carnitine in swine has not been investigated directly, when neonatal pigs are reared on formulas depleted of carnitine, plasma and tissue levels of carnitine are reduced (Baltzell et al., 1987). In comparison to adults, human neonates are either less able to synthesize carnitine or have lower stores of carnitine (Borum, 1991). Carnitine has been shown to be low in the tissues and blood of newborn piglets (Kerner et al., 1984). Furthermore, piglets that are fed a carnitine-free formula have reduced concentrations of tissue carnitine than piglets that are allowed to nurse the sow (Borum and Sullivan, 1988). Kerner et al. (1984) reported that while the concentrations of carnitine in newborn piglet liver and blood are low, high concentrations of carnitine are found in colostrum and milk. These authors also determined that most of the carnitine present in sow’s colostrum is short-chain acylcarnitine and that half of the acylcarnitine in the sow’s colostrum is isovalerylcarnitine. The large amounts of carnitine in colostrum and milk are probably responsible for the increased amounts of carnitine in the blood and liver of two-day-old piglets compared to the amounts found in newborn piglets. Since muscle tissue is heavily dependent upon fat for energy, skeletal muscle contains as much as 90% of the total carnitine in the body (Odle, 1996). In fetal pigs, Baltzell et al. (1987) reported that both skeletal and cardiac muscle had higher concentrations of carnitine than did liver, intestine or kidney tissues and that levels in muscle continued to increase throughout gestation while the carnitine levels in the other tissues declined.

Because of the low levels of carnitine in newborn pigs, researchers have speculated that the deficiency may be tied to suboptimal utilization of fatty acids. Garber and Froseth (1984) investigated the effect of intravenous carnitine administration on the oxidation of palmitic acid in fasted neonatal pigs. The authors indicated that low animal numbers and high biological variation in their experiment did not allow them to draw definite conclusions with regard to the effects of carnitine. Although significant effects of carnitine were not evident, their data showed consistent trends for increased palmitic acid oxidation and decreased free fatty acids in serum of the carnitine-treated group.

 

Coffey et al. (1991) compared carnitine status and fatty acid metabolism in a variety of tissues obtained from piglets nursed by sows or fed formulas that contained high or low levels of carnitine. These investigators reported that piglets assigned to the low carnitine formula had reduced carnitine in their plasma and livers. Although oxidation of palmitatein vitro by the liver was reduced on day 7, by day 14 carnitine level did not affect liver lipid oxidation. Furthermore, glucose status, plasma lipids or ketones, and oxidation of palmitate by the longissimus muscle were not affected by level of dietary carnitine.

Honeyfield and Froseth (1991) evaluated whether carnitine limited substrate utilization in tissues of piglets from sows fed two levels of lysine or dietary fat. They concluded that carnitine did not appear to be the limiting factor for the oxidation of palmitoyl-CoA or the limiting factor of piglet energy metabolism.

 

Van Kempen and Odle (1993) found that L-carnitine supplementation stimulated medium-chain fatty acid (octanoate) oxidation when intravenously infused to colostrum-deprived newborn piglets. In a subsequent report (Van Kempen and Odle, 1995), the authors suggested that the effect of carnitine on oxidation of octanoate might involve facilitating the export of excess acetyl groups from muscle or increasing the uptake of octanoate into liver mitochondria.

Frank et al. (1999) evaluated the effects of two different sources of carnitine on nitrogen and energy metabolism in growing pigs. Both sources of supplemental L-carnitine were absorbed with equal efficacy, but neither affected energy or nitrogen metabolism.

In a report by Galvez et al. (1996), dietary L-carnitine was proposed to be conditionally required in situations involving young or stressed animals, animals with a potential for high productivity and animals fed high-fat diets proportionally high in plant oils. To investigate possible benefits of L-carnitine in these situations, L-carnitine was added to a conventional feed in one experiment and to an energy-rich, non-saturated fat-rich feed in a separate experiment. The effects of performance of weaning piglets with high growth potential were evaluated. Although not statistically significant, growth performance tended to be enhanced in piglets receiving 25 or 50 mg of L-carnitine per kg (11.3 or 22.7 mg/lb) of diet. Feed conversion ratio was improved by L-carnitine supplementation at 50 mg per kg (22.7 mg/lb). A positive effect of L-carnitine supplementation on feed conversion ratio has also been reported by Jost and Bracher-Jakob (1996). However, Williamson et al. (1996) indicated that L-carnitine supplementation (50 ppm) did not affect average daily gain or feed efficiency of finishing pigs. Similarly, Johnston et al. (1999) were unable to detect an effect of carnitine on growth performance of growing pigs. Rincker et al. (2003) increased growth performance and feed efficiency for weanling pigs with carnitine supplementation.

Whether carnitine supplementation might alter carcass characteristics in swine has also been the subject of investigation. Although no effect on growth performance was observed in their experiment, Owen et al. (1992) determined that carnitine supplementation during the growing-finishing phase increased loineye area. In a separate experiment (Owen et al., 1996), dietary carnitine was found to reduce fat deposition in favor of protein deposition. Furthermore, carnitine was found to positively influence amino acid metabolism and to stimulate fatty acid oxidation. However, Williamson et al. (1996) indicated that the addition of 50 ppm of L-carnitine to the diet of finishing pigs did not increase carcass leanness. Carnitine-supplemented pigs had higher tenth-rib and last-rib backfat thickness. Waylanet al. (1999) indicated that dietary L-carnitine supplementation (50 ppm) to diets of growing and finishing swine did not affect bacon characteristics. Sardi et al. (1996) explored the influence of L-carnitine on growing-fattening and slaughtering performance of Italian heavy pigs. In their report, L-carnitine was reported to increase the fat content of the carcass, to significantly lower the lightness of loin and to slightly reduce ham weight loss during the seasoning period. These authors indicated the dietary L-carnitine also slightly improved daily weight gain and feed efficiency. Research has shown that carnitine increased protein accretion and decreased fat deposition in growing-finishing pigs (Owen et al., 1996; Heo et al., 2000; Owen et al., 2001 a,b; Rincker et al., 2003).

Other authors have investigated the influence of dietary L-carnitine supplementation in gilts on ovulation and fertilization rate. Samland et al. (1998) determined that L-carnitine (200 ppm) addition increased ovulation rate and decreased fertilization rate of the embryos recovered. Sower et al. (1998) evaluated the effects of supplemental dietary carnitine (100 ppm) on various indices of performance and metabolism in lactating sows fed low-energy diets. Carnitine-supplemented sows produced less milk and had slower growing litters. No statistically significant differences were observed for feed intake, sow backfat, composition of milk or colostrum, muscle protein degradation, sows’ concentrations of glucose, nonessential fatty acids (NEFA), glycerol, insulin, and IGF-1 in plasma or carnitine content of liver and mammary tissue samples. However, the authors indicated that carnitine content of tissue, colostrum and milk tended to be higher and that trends for metabolic indices were consistent with a reduction in body protein mobilization and with a stimulation of fatty acid oxidation in the carnitine-supplemented sows. Sower et al. (1998) concluded that under considerably catabolic conditions, supplemental dietary carnitine can influence the lactating sow’s metabolism. Musser et al. (1997a) investigated the effects of L-carnitine addition (50 ppm) during lactation to diets of first-parity gilts. Although gilts supplemented with L-carnitine had lower average daily feed intake during the first week of lactation, during the second week and subsequent weeks, the average daily feed intake was not affected. No improvements in sow or litter performance were noted following the addition of L-carnitine. Likewise, in a report that included sows of different parities, Musser et al. (1999a) were unable to observe improvements in the number of pigs or litter weights at weaning when sows were supplemented with 50, 100 or 200 ppm L-carnitine during lactation. Little effect of L-carnitine on sow and litter performance was evident. However, during gestation, Musser et al. (1997b; 1999b) determined that supplying dietary L-carnitine to the diets of sows increased litter birth and weaning weights. Furthermore, sows fed L-carnitine during gestation were heavier at weaning than were control sows. Carnitine supplementation during gestation also increased the rate of gain of last-rib fat depth. Concentrations of insulin on days 10 and 60 of gestation in sows fed L-carnitine were increased in comparison to control sows. Also, on days 60 and 90 of gestation, concentrations of IGF-1 were increased in the carnitine-supplemented sows. In the study by Musser et al. (1999b), the multiparous sows received L-carnitine at 100 mg per day during gestation and (or) 50 ppm during lactation. In agreement with their other studies utilizing lactating sows, few differences were observed by feeding 50 ppm of L-carnitine during lactation. However, compared to the control diet, supplementation of L-carnitine in either gestation, lactation or both increased the number of pigs born alive but not the total born (Musser et al., 1999b).

Supplementing pregnant sows with L-carnitine has been reported to increase birth weight (Musser et al., 1999; Ramanau et al., 2008) and to stimulate prenatal myofiber formation (Musser et al., 2001). The NRC (1998) for swine has not established a requirement for carnitine. Supplementation studies have often fed 50 to 400 mg per kg (45.5 to 181.8 mg per lb) of diet.

 

Sources

Carnitine has both D- and L- forms, but only the L- form is biologically active and occurs in nature. Low levels of L-carnitine are contained in feedstuffs of plant origin. However, meat meal, fish meal and blood meal contain much higher levels of L-carnitine. For a thorough comparison of the L-carnitine content of feedstuffs refer to Galvez et al. (1996).

Carnitine is commercially available to the feed industry as a 50% L-carnitine product. Carnitine is very hygroscopic and easily soluble in water and has a molecular weight of 161.2.

 

Deficiency

Carnitine deficiencies have only been observed as secondary events to congenital enzyme deficiencies and chronic disease states such as diabetes, renal failure and kwashiorkor. In carnitine deficiency, fatty acid oxidation is reduced, and fatty acids are diverted into triglyceride synthesis, particularly in the liver. Mitochondrial failure develops in carnitine deficiency when there is insufficient tissue carnitine available to buffer toxic acyl-CoA metabolites. Toxic amounts of acyl-CoA impair the citrate cycle, gluconeogenesis, the urea cycle and fatty acid oxidation. Carnitine replacement treatment is safe and induces excretion of toxic acyl groups in the urine (Stumpf et al., 1985). If carnitine deficiency involves the liver, the supply of ketones and the utilization of long-chain fatty acids during starvation are cut off; all tissues become glucose dependent. When liver carnitine is depleted, starvation tends to cause nonketotic, insulinopenic hypoglycemia. Because liver hepatocytes depend on fatty acids for their energy requirements during fasting, carnitine depletion may also cause clinical liver dysfunction, shown by hyperammonemia, encephalopathy and hyperbilirubinemia (Feller and Rudman, 1988). Skeletal muscles are generally involved, with weakness, lipid myopathy and myoglobinuria often aggravated or precipitated by fasting or exercise. The heart, like skeletal muscle, is dependent on fatty acids for energy during fasting, and heart failure and arrhythmias are frequent manifestations of systemic carnitine deficiency. The heart derives approximately 60% of its ATP supply from beta-oxidation of fatty acids. Carnitine concentrations in the heart are normally very high in many species (Rebouche and Paulson, 1986). Carnitine deficiency is differentiated into three categories: excessive loss of free carnitine, excessive loss of acylcarnitine as a result of accumulation of acyl-CoA in tissues, and a combined type. The first condition is reported in humans as Fanconi syndrome and renal carnitine transport deficiency (primary carnitine deficiency), and the latter two types are found in various inborn errors of fatty acid metabolism (DiDonato et al., 1992). Assessment of the carnitine status of a particular animal or human is difficult because plasma carnitine concentrations and urinary carnitine excretion are not good indicators of tissue carnitine status (Borum, 1991). Individuals with low carnitine concentrations in plasma may have normal carnitine concentrations in muscle or liver, and those with normal plasma carnitine concentrations may have low carnitine concentrations in muscle or liver. Systemic and myopathic forms of L-carnitine deficiency are well-known etiologies of dilated cardiomyopathy (DCM) in human medicine.

Some studies have shown no benefit from carnitine supplementation, however other reports have shown definite benefits from supplemental carnitine. Recent research has shown that supplementing sow diets with L-carnitine during pregnancy and lactation improves their reproductive performance (Ramanau et al., 2005; Waylan et al., 2005; Woodworth et al., 2007; Eder, 2009). In particular, sows fed diets supplemented with L-carnitine had heavier litters than control sows (Musser et al., 1999; Eder et al., 2001; Ramanau et al., 2002, 2004, 2005). Moreover, the piglets of sows supplemented with L-carnitine grew faster during the suckling period than those of control sows (Musser et al., 1999; Eder et al., 2001; Ramanau et al., 2002, 2004). This effect is due to an increased milk yield in sows treated with L-carnitine compared with control sows (Ramanau et al., 2004).

In growing and finishing pigs, L-carnitine increased protein accretion and percentage of lean, but decreased fat deposition (Owen et al., 1996; Heo et al., 2000; Owen et al., 2001a,b). Additionally, offspring from sows fed L-carnitine had a larger cross-sectional area and more total muscle fibers in the semitendinosus muscle than piglets from controls (Musser et al., 2001). Thus, muscle growth seems to be enhanced by L-carnitine supplementation in gestating sow diets. Dietary carnitine has increased growth performance on weanling pigs (Rinker et al., 2003), while other studies found that carnitine did not influence growth performance, but did linearly decrease 10th rib backfat thickness and linearly increase percentages of lean and muscle (Owen et al., 2001a,b).

 

Fortification Considerations

In neonatal pigs and gestating sows, as reviewed previously in this chapter, carnitine supplementation has in some experiments improved performance. However, in other cases, carnitine supplementation was without benefit. Perhaps as suggested by Galvez et al. (1996), carnitine supplementation should be considered under specific situations. Providing optimum nutrition for young or stressed animals, achieving maximum performance of animals with a high potential for productivity, and feeding diets that are high in fat and proportionally high in plant oils may be goals or conditions for which carnitine supplementation is warranted. Piglets of low birth weight exhibit a reduced total number of skeletal muscle fibers at birth and throughout life, than piglets of middle and heavy birth weight (Rehfeldt and Kuhn, 2006). These pigs have a limited potential for muscular lean accretion, and therefore deposit more fat, resulting in a reduced carcass quality at market weight. Moreover, due to increased hypertrophied myofibers, meat quality is relatively poor, as indicated by greater drip loss, and greater content of heat-stable collagen (Gondret et al., 2006; Rehfeldt et al., 2008). Consequently, an increase in the number of muscle fibers in piglets of low birth weight would likely contribute to the improvement of carcass and meat quality. Lösel et al. (2009) showed that carnitine supplementation intensified the early postnatal skeletal myofiber formation in piglets of low birthweight. The piglets deposited less fat with an improved energy balance through intensified fatty acid oxidation. It was concluded that piglets, particularly those of low birth weight, could profit from an early postnatal L-carnitine supplementation, which may attenuate the negative consequences of low birth weight on body composition and meat quality at market weight.

Only the physiologic L-carnitine should be used for fortification of diets. Gross et al. (1998) fed pigs and dogs diets containing supplemental carnitine and reported that plasma-free carnitine in both species increased at the same rate. Oral doses of 100 to 400 mg per kg (45.5 to 181.8 mg per lb) of body weight have been used as supplementation doses.

 

Vitamin Safety

No data have been reported for carnitine toxicity studies for swine. Oral dosages of 100mg per kg (45.5 mg per lb) have been given to infants and 1 to 3 g daily to adult humans with little problem. Some patients experienced diarrhea, but not if they started with smaller dosages and then increased gradually (Borum, 1991).