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J. Dairy Sci. 2007. 90:2367-2376. doi:10.3168/jds.2006-605
© 2007 American Dairy Science Association ®
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Effect of L-Carnitine Infusion and Feed Restriction on Carnitine Status in Lactating Holstein Cows1

D. B. Carlson*,2, J. C. Woodworth{dagger} and J. K. Drackley*,3

* Department of Animal Sciences, University of Illinois, Urbana 61801
{dagger} Lonza, Inc., Allendale, NJ 07401

3 Corresponding author: drackley{at}uiuc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
Previously we determined that abomasal infusion of L-carnitine increased in vitro hepatic fatty acid oxidation, decreased liver lipid accumulation, and supported higher fat-corrected milk yield in feed-restricted lactating cows. The objectives of this study were to examine the effects of supplemental L-carni-tine and amount of feed intake on free carnitine and carnitine ester concentrations in liver, muscle, milk, and plasma of lactating dairy cows. Eight lactating Holstein cows (132 ± 36 d in milk) were used in a replicated 4 x 4 Latin square design with 14-d periods to test factorial combinations of water or L-carnitine infusion (20 g/d; d 5 to 14) and ad libitum or restricted (50% of previous 5-d intake; d 10 to 14) dry matter intake. Plasma was obtained 3 times daily on d 4, 8, and 12; milk samples were collected on d 8, 9, 13, and 14. Liver and muscle were biopsied on d 14 of each period. Free carnitine, short-chain acylcarnitine, and long-chain acylcarnitine concentrations were determined using a radioenzymatic assay coupled with ion exchange chromatography. Abomasal L-carnitine infusion increased total carnitine in plasma on d 8 and d 12. All liver carnitine fractions were increased by carnitine infusion. Feed restriction elevated concentrations of free carnitine, long-chain acylcarnitine, and total carnitine in liver tissue from carnitine-infused cows but not in those infused with water. In muscle, acid-soluble carnitine, long-chain acylcarnitine, and total carnitine concentrations were increased by carnitine infusion and feed restriction without significant interaction. Feed restriction increased free carnitine concentrations in muscle from water-infused cows but not in carnitine-infused cows. Carnitine infusion increased the concentration of each milk carnitine fraction as well as milk carnitine output on d 8 to 9. On d 13 to 14, all carnitine fractions except short-chain acylcarnitine were increased in milk from water-infused, feed-restricted cows, whereas all fractions were increased in carnitine-infused, feed-restricted cows. Carnitine infusion increased total carnitine in plasma, liver, muscle, and milk during feed restriction, whereas feed restriction alone increased carnitine concentrations in muscle and milk but not in liver. Liver carnitine concentrations might limit hepatic fatty acid oxidation capacity in dairy cows during the periparturient period; therefore, supplemental L-carnitine might decrease liver lipid accumulation in periparturient cows.

Key Words: L-carnitine • dairy cow • liver • muscle


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
L-Carnitine is required for transport of long-chain fatty acyl-CoA from the cytosol into the mitochondria due to its essentiality for carnitine palmitoyltransferase-I (CPT-I; EC 2.3.1.21) activity (Rebouche and Seim, 1998). L-Carnitine has several other functions such as altering the acetyl-CoA:CoA ratio, transporting medium- and short-chain fatty acids from peroxisomes to mitochondria, and modulating flux of intermediates through pathways associated with fatty acid, glucose, and nitrogen metabolism (Ji et al., 1996; Owen et al., 2001a). The role of carnitine in hepatic fatty acid oxidation suggests that carnitine status might influence the degree of liver lipid accumulation in periparturient dairy cows. In dairy cows, carnitine stimulates hepatic fatty acid oxidation in vitro (Jesse et al., 1986; Drackley et al., 1991), and prepartum increases in liver carnitine concentration were associated with decreased liver triglyceride accumulation during the transition period (Grum et al., 1996). Although carnitine supplementation to dairy cows during the periparturient period has not been evaluated, we have shown previously that abomasal infusion of L-carnitine increased in vitro hepatic fatty acid oxidation regardless of amount of feed intake and decreased liver lipid accumulation in feed-restricted cows (Carlson et al., 2006).

Carnitine status in mammals is influenced by dietary intake, endogenous synthesis, and reabsorption of carnitine and carnitine precursors by the kidney (Rebouche and Seim, 1998). The rate of carnitine biosynthesis is largely dependent upon the supply of trimethyllysine from turnover of proteins containing this AA (Vaz and Wanders, 2002); Lys (carbon backbone) and Met (methyl group donor) are required for trimethyllysine synthesis. In the rat, several tissues are capable of converting trimethyllysine to the immediate carnitine precursor {gamma}-butyrobetaine (Vaz and Wanders, 2002). In most species only the liver possesses the enzyme necessary for converting {gamma}-butyrobetaine to carnitine, although in some species the kidney, brain, and testis are capable of synthesizing carnitine from {gamma}-butyrobetaine (Vaz et al., 1998; Galland et al., 1999).

Carnitine is maintained as free carnitine or as acylcarnitine esters in the body. Acetyl-L-carnitine is the predominant acylcarnitine ester in the cell and in circulation (Rebouche and Seim, 1998). Previous research examining the effects of supplemental carnitine on liver, muscle, milk, and plasma carnitine concentrations has utilized lactating cows fed for ad libitum DMI (LaCount et al., 1995, 1996a,LaCount et al., b). Catabolic situations such as ketosis, diabetes, off-feed conditions, and feed deprivation affected carnitine concentrations as well as distribution among free carnitine and acylcarnitine in liver, muscle, and milk from ruminants (Erfle et al., 1974; Snoswell and Henderson, 1980; Grum et al., 1996).

To our knowledge, well-replicated, controlled comparisons of the effects and interactions of carnitine supplementation and DMI depression have not been conducted in dairy cows. Our hypothesis was that liver carnitine availability determines, in part, the capacity for hepatic fatty acid oxidation and the degree of liver lipid accumulation during feed restriction (Carlson et al., 2006). The objectives of this portion of that study were to characterize the effects of L-carnitine supplementation and amount of DMI on concentrations and distribution of free carnitine and carnitine esters in liver, muscle, milk, and plasma from lactating dairy cows.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
Experimental Design and Management of Cows
All experimental procedures were conducted according to protocols approved by the University of Illinois Institutional Animal Care and Use Committee. Details of experimental procedures pertaining to cow management, diet formulation, and specimen collection were described previously (Carlson et al., 2006). Briefly, 8 mid-lactation Holstein cows fitted with ruminal cannulas (10.2 cm o.d.; Bar Diamond, Parma, ID) were blocked by DIM and assigned to treatments in a replicated 4 x 4 Latin square with 14-d periods. Within each block (block 1 = 162 ± 20 DIM; block 2 = 101 ± 16 DIM), cows were randomly assigned to treatments arranged as a 2 x 2 factorial. Treatments were water infusion, ad libitum DMI (WA); water infusion, restricted DMI (WR); carnitine infusion, ad libitum DMI (CA); and carnitine infusion, restricted DMI (CR).

Abomasal infusions were conducted by placing an infusion apparatus through the reticuloomasal orifice by way of the ruminal cannula (Litherland et al., 2005). Infusions were carried out every 6 h at 0600, 1200, 1800, and 2400 h. At each time point, the infusion line was flushed with 100 mL of tap water, followed by infusion of 100 mL of treatment solution (water or carnitine solution), and then 100 mL of water was infused to flush the treatment infusate out of the infusion line and into the abomasum.

During each 14-d period, d 1 to 4 served as a washout period to reduce carryover effects of previous treatments. LaCount et al. (1996a) reported that plasma carnitine decreased rapidly in carnitine-supplemented cows once supplementation ceased. Studies utilizing radioisotope tracer techniques indicated that 14 d is sufficient to eliminate carryover effects of previous treatments on liver and muscle carnitine concentrations (Rebouche and Seim, 1998). From d 5 to 14, cows were infused with water or L-carnitine [L-(3-carboxy-2-hydroxypropyl)trimethyl-ammonium hydroxide; inner salt, ≥97% purity; Lonza, Inc., Allendale, NJ]. L-Carnitine solution was prepared daily by dissolving 20 g of L-carnitine in 400 mL of warm tap water, which was then refrigerated at 4°C. The 20 g/d dosage was based on experiments conducted by Erfle et al. (1971) in which intravenous infusion of 23.8 g/d of L-carnitine decreased concentrations of NEFA in blood from feed-restricted cows, indicating that fatty acid metabolism was affected. This dose also was greater than the dosage at which plasma concentrations of carnitine were maximized in earlier studies (LaCount et al., 1996a).

Beginning on d 10 of each period, cows assigned to the WR and CR treatments were subjected to feed restriction to induce adipose tissue lipolysis characteristic of the periparturient period, whereas WA and CA cows were allowed ad libitum DMI. For feed-restricted cows, DMI during d 10 to 14 was restricted to 50% of the average DMI from d 5 to 9. Feed restriction increased plasma NEFA by approximately 3-fold in samples obtained on d 12 at 0, 3, and 6 h relative to feeding (Carlson et al., 2006). On d 12, the mean prefeeding concentrations of NEFA in plasma were 403 and 85 µEq/L for feed-restricted and ad libitum-fed cows, respectively.

Cows were housed in tie stalls and fed individually throughout the experiment. Cows were milked in their stalls at 0530 and 1730 h and were allowed access to an outside loafing pen for 1 h each day. All cows were fed a TMR balanced according to NRC (2001) recommendations (Table 1Go) to meet or exceed requirements of Holstein cows during early lactation.


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  		Table 1. Ingredient and chemical composition (DM basis) of the experimental diet (Carlson et al., 2006)
 
Data Collection, Sampling Procedures, and Analytical Methods
Blood was sampled from a coccygeal vessel on d 4, 8, and 12 at 0, 3, and 6 h relative to the morning feeding. Blood was drawn into evacuated tubes (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ) containing K3EDTA and then immediately placed on ice. Plasma was collected by centrifugation (1,300 x g for 10 min) and stored at –20°C in disposable polypropylene tubes until analysis of total carnitine concentration.

Liver tissue (~3 to 6 g) was obtained under local anesthesia via puncture biopsy (Hughes, 1962) on d 14 of each period as described previously (Carlson et al., 2006). Immediately before the liver biopsy, muscle tissue was obtained under local anesthesia from the semitendinosus muscle (Carlson et al., 2006). Muscle tissue (~200 mg) was sampled with a biopsy needle (12 gauge x 16 cm; Bard Magnum; C. R. Bard, Inc., Murray Hill, NJ). Tissue samples were immediately frozen and stored in liquid nitrogen.

Milk was sampled from 4 consecutive milkings on d 8 to 9 and again on d 13 to 14. Samples from 2 consecutive milkings were composited in proportion to milk yield. Composited samples from d 8 were combined with d-9 composites, and d-13 samples were combined with those from d 14; milk samples were then stored at –20°C until analysis of milk carnitine.

Concentrations of free carnitine and carnitine esters in liver, muscle, and milk, and total carnitine in plasma were determined by an enzymatic radioisotope method (McGarry and Foster, 1976) with modifications (Bhuiyan et al., 1992). Ice-cold HClO4 (1 M) was added to 1.5-mL microcentrifuge tubes containing liver (200 mg), muscle (100 mg), or milk (200 µL). Liver and muscle tissue were homogenized using a variable speed laboratory motor (TriR Instruments, Inc., Rockville Center, NY) fitted with a microcentrifuge tube sample pestle (Bel-Art Products, Pequannock, NJ). After centrifugation at 10,000 x g for 5 min, the supernatant was removed into a separate 1.5-mL microcentrifuge tube. The pellet in the original tube was washed with HClO4 (0.1 M) and centrifuged (10,000 x g for 5 min); the supernatants were combined and the pellet retained. The combined supernatant was neutralized with KOH (1 M) and centrifuged (10,000 x g for 5 min) to remove precipitate. A portion of the supernatant was retained for analysis of free carnitine, whereas a portion was incubated with KOH (1 M) at 60°C for 60 min to hydrolyze short-chain acylcarnitine; the resulting supernatant contained total acid-soluble carnitine. The pellet derived from the initial HClO4 extraction steps was incubated with KOH (1 M) at 60°C for 60 min to hydrolyze long-chain acylcarnitine. Following hydrolysis of short- and long-chain carnitine esters, HClO4 (1 M) was added, the mixture was centrifuged (short-chain: 10,000 x g for 5 min; long-chain: 12,000 x g for 10 min), and neutralized with KOH (1 M) and HEPES buffer (1 M).

Plasma (200 µL) was incubated with KOH (1 M) at 60°C for 60 min in a 1.5-mL centrifuge tube. After incubation, HClO4 (1 M) was added, the tube was centrifuged (10,000 x g for 5 min), and the mixture was then neutralized with KOH (1 M) and HEPES buffer (1 M) and analyzed for total carnitine concentration.

To quantify carnitine concentrations in the different fractions, supernatants were added to 0.55 mL of HEPES-EDTA buffer (pH = 7.3) with [1-14C]acetyl-CoA (0.0275 µCi; American Radiolabeled Chemicals, St. Louis, MO), 25.5 nmol of acetyl-CoA, and 2 µmol of N-ethylmaleimide (Sigma Chemical Co., St. Louis, MO). The reaction mixture was incubated with 2 IU of carnitine acetyltransferase (EC 2.3.1.7; Sigma) for 30 min in a 25°C water bath. The reaction mixture was then pipetted onto a column packed with resin (AG 1 x 8, 100–200, chloride form; BioRad, Richmond, CA) to remove unreacted [1-14C]acetyl-CoA. [1-14C]A-cetylcarnitine was eluted into scintillation vials with 4 mL of water. Blank columns were utilized to monitor the capacity of the resin to bind [1-14C]acetyl-CoA in the absence of substrate. Scintillation cocktail (15 mL, Scintisafe Econo 2, Fisher Scientific, Fair Lawn, NJ) was added to the vials, and radioactivity was measured by liquid scintillation spectroscopy (model LS6000IC, Beckman Coulter, Inc., Fullerton, CA).

Carnitine concentrations were calculated using known amounts of acetyl-CoA, radioactivity in the reaction mixture, and radioactivity present in the column eluent. Calculations included a correction for radioactivity in eluent from blank columns. Short-chain acylcarnitine concentrations in liver, muscle, and milk were calculated by subtracting free carnitine from total acid-soluble carnitine. Total carnitine in liver, muscle, and milk was the sum of acid-soluble carnitine and long-chain acylcarnitine.

Statistical Analysis
As described previously (Carlson et al., 2006), the analyses included data from 7 cows because one cow was removed from the experiment due to poor recovery from rumen cannulation surgery. In addition, one cow experienced clinical mastitis during the second experimental period in which she was assigned to the WA treatment; her data for that period were removed. However, the cow recovered after intramammary therapy and data were included for subsequent periods.

Data were analyzed as a replicated 4 x 4 Latin square design with a 2 x 2 factorial arrangement of treatments using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC). Fixed effects of square, infusion type, intake amount, and associated interactions were included in the model, whereas random effects of cow and period nested within square were used as the error term to test fixed effects. Square was not a significant fixed effect and was removed from the model. The Satterthwaite specification was included in the model statement to estimate degrees of freedom. Milk carnitine data obtained on d 8 to 9 were analyzed separately from data obtained on d 13 to 14. Plasma carnitine data were separated by day of sampling and analyzed independently. The REPEATED statement was used for plasma carnitine data to account for the effect of hour of sampling; the error term in the REPEATED statement was cow x period nested within square. The covariance structure that yielded the lowest Akaike’s information criterion was used for repeated measurements (Littell et al., 1998). Significance of the F-test of fixed effects was declared at P ≤ 0.05 and trends discussed when 0.05 < P ≤ 0.15. When protected by the F-test (P ≤ 0.15), least squares means for interactions were separated using the PDIFF statement in SAS and deemed significant at P ≤ 0.05.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
Previous research from our laboratory revealed that ruminal, abomasal, and dietary carnitine administration increased carnitine concentrations in plasma, milk, and liver in lactating dairy cows (LaCount et al., 1995, 1996a,LaCount et al., b). Carnitine concentrations in plasma, liver, muscle, and milk obtained in the present study are lower than those measured previously. The methodology used in previous studies was similar to our methods, except that LaCount et al. (1995, 1996a,b) used a standard curve approach to calculate carnitine concentrations as opposed to calculating carnitine concentration based on known concentrations and radioactivity of substrates. In the current study, the carnitine assay was extensively validated using bovine plasma, liver, muscle, and milk samples spiked with a solution of known L-carnitine concentration; in addition, a standard curve was used to test linearity and range of the assay. Due to the disparity among reported values in the literature, discussion will focus on relative changes in carnitine concentration induced by exogenous carnitine administration or amount of DMI rather than on comparison of carnitine concentrations among studies.

Plasma carnitine concentrations measured on d 4, 8, and 12 are reported in Table 2Go. Plasma carnitine tended to vary among treatments on d 4 (hour x infusion x DMI, P = 0.07), with differences occurring between WA and CA treatments 6 h after first feeding. The biological significance of this difference is negligible as all cows were being infused with water and allowed ad libitum DMI. The fact that plasma carnitine concentration was essentially equivalent among treatments at this time indicates that there was little carryover effect from previous carnitine infusion. La-Count et al. (1996a) reported that plasma carnitine decreased precipitously within 12 h after cessation of carnitine infusion.


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  		Table 2. Least squares means for concentration of total carnitine in plasma from lactating Holstein cows infused with water or L-carnitine (20 g/d; d 5 to 14) and fed for ad libitum or restricted DMI (50% of previous 5-d DMI; d 10 to 14)
 
L-Carnitine infusion increased plasma carnitine concentration on d 8 as expected (P < 0.01). Plasma carnitine concentrations tended to be higher at 3 h postfeeding compared with prefeeding values (hour x infusion, P = 0.10) in cows infused with carnitine but not in those infused with water. Previously, continuous abomasal infusion and dietary supplementation of L-carnitine also increased plasma carnitine concentrations compared with unsupplemented cows (LaCount et al., 1995, 1996a,LaCount et al., b), indicating that exogenous carnitine was readily absorbed. However, the increases in plasma carnitine relative to controls observed in the present experiment (approximately 6-fold) were much greater than observed previously (26%) when only 6 g/d was infused abomasally (LaCount et al., 1995). Plasma carnitine concentrations are not necessarily indicative of tissue carnitine concentrations unless subjects are genetically similar and maintained under identical dietary and environmental conditions (Rebouche and Seim, 1998), and therefore plasma data are not as useful if tissue concentrations are not measured as well.

Carnitine infusion increased plasma carnitine on d 12 (P < 0.01) with concentrations peaking at 3 h postfeeding in cows infused with carnitine (hour x infusion, P = 0.01). Feed restriction tended to increase plasma carnitine (P = 0.14) without interaction with infusion type (infusion x DMI, P = 0.33). In rats, plasma total carnitine decreased during the first 36 h of fasting and was not significantly increased until 72 h of fasting (Brass and Hoppel, 1978). In our study, blood was sampled after 48 h of feed restriction, whereas liver, muscle, and milk concentrations reflect carnitine concentrations after 60 to 72 h of feed restriction. Therefore, plasma concentrations indicate that infused carnitine was absorbed and that feed restriction tended to increase plasma carnitine, but these values may not adequately describe changes that occurred during the entire feed restriction period.

Concentrations of free carnitine and carnitine esters in liver and muscle tissue are presented in Table 3Go. Abomasal infusion of carnitine increased hepatic free carnitine, short-chain, and long-chain acylcarnitine concentrations compared with water infusion (P < 0.01), although infusion type interacted with amount of intake for all fractions (infusion x DMI, P = 0.02) except short-chain acylcarnitine (infusion x DMI, P = 0.17). Feed restriction increased free carnitine, acid-soluble carnitine, long-chain acylcarnitine, and total carnitine concentrations in liver tissue from carnitine-infused cows; feed restriction did not significantly affect carnitine or carnitine ester concentrations in liver tissue from water-infused cows although means were numerically larger for each fraction.


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  		Table 3. Least squares means for concentration of carnitine in liver and muscle tissue from lactating Holstein cows infused with water or L-carnitine (20 g/d; d 5 to 14) and fed for ad libitum or restricted DMI (50% of previous 5-d DMI; d 10 to 14)
 
Previously, abomasal infusion of smaller amounts of L-carnitine (6 g/d) into ad libitum-fed lactating cows increased free carnitine concentrations in liver but did not affect short- or long-chain acylcarnitine (LaCount et al., 1995). In pigs, liver total carnitine concentrations responded linearly to supplemental dietary carnitine (Owen et al., 2001a,b). The fact that abomasal infusion of 20 g/d in our study had a larger effect on liver carnitine concentrations than did infusion of 6 g/d (LaCount et al., 1995) indicates that this was likely a dose-related response.

Exogenous carnitine is rapidly absorbed into the liver; intravenous carnitine injection markedly increased liver carnitine concentrations within 5 min of administration (Ruff et al., 1991). Transport of carnitine into liver tissue is accomplished via both a specific, saturable transport system and by passive diffusion (Ramsay et al., 2001). The liver transporter in rats has a relatively high Km for carnitine (~5 mM), which is substantially higher than concentrations found in plasma (~56 µM; Christiansen and Bremer, 1976). This relationship indicates that liver transport exhibits low affinity for carnitine compared with other tissues (Tamai et al., 1998), but the transporter has high capacity and is not easily saturated. As a result of carnitine infusion into the abomasum, portal blood concentrations would presumably be higher in carnitine-infused cows leading to increased liver carnitine concentrations. Whether hepatic accumulation of carnitine would continue to increase if larger doses were supplied is not known.

To our knowledge, the effect of experimentally induced DMI depression on liver carnitine concentration has not been investigated in lactating dairy cows; however, catabolic conditions such as fasting, prolonged DMI depression, diabetes, and ketosis have been shown to enhance liver carnitine concentrations in ruminants (Snoswell and Henderson, 1980; Grum et al., 1996). Carnitine is required for hepatic oxidation of long-chain fatty acids. We have shown previously that carnitine infusion decreased liver accumulation of total lipid and triglyceride during feed restriction (Carlson et al., 2006), which was associated with greater liver carnitine in the present study. Interestingly, 50% feed restriction had only modest effects on liver carnitine concentrations in cows infused with water, but feed restriction markedly increased all fractions except short-chain acylcarnitine in cows provided exogenous carnitine. The rate-limiting factor for endogenous carnitine synthesis is the availability of trimethyllysine from protein turnover (Vaz and Wanders, 2002). Carnitine precursor supply to the liver was not determined in the current study but would be useful to explain the factors affecting carnitine biosynthesis and carnitine status during feed restriction. Liver carnitine increases around parturition and then rapidly decreases in early lactation (Grum et al., 1996); therefore, the duration of feed restriction and metabolic characteristics of mid lactation cows likely influenced the rate of endogenous carnitine synthesis in WR cows. Clearly, carnitine infusion substantially increased carnitine availability, which allowed for greater hepatic carnitine accumulation in both CA and CR cows.

L-Carnitine infusion increased the concentrations of long-chain acylcarnitine (P = 0.02) and total carnitine (P = 0.01) in semitendinosus muscle. Feed restriction caused greater total carnitine (P = 0.02) and tended to increase long-chain acylcarnitine (P = 0.10) concentrations in muscle. The WR and CA treatments had higher free carnitine in muscle than did WA, but the CR treatment had free carnitine concentrations similar to the other treatments (infusion x DMI, P = 0.04). Short-chain acylcarnitine was not altered by carnitine infusion (P = 0.24), feed restriction (P = 0.45), or the interaction of infusion and DMI (P = 0.40). Acid-soluble carnitine concentration tended to be similar among WR, CA, and CR treatments, but all treatments tended to be higher than WA (infusion x DMI, P = 0.13).

In contrast with the current results, abomasal infusion of 6 g/d of L-carnitine in dairy cows (LaCount et al., 1995) and dietary carnitine supplementation to beef steers (3 g/d; Greenwood et al., 2001) did not affect carnitine concentration in muscle. The difference between studies in response of muscle carnitine concentrations to abomasal carnitine infusion is likely at least partially explained by the much larger dosage in the present study and, as a result, the much greater increment in plasma carnitine concentrations. Carnitine transport into muscle occurs primarily by active transport because intracellular concentrations are at least 10 times greater than plasma concentrations (Rebouche, 1977). In rats, muscle carnitine transport has a low capacity but relatively high affinity for carnitine, with a Km (60 µM) in the upper range of physiological concentrations of carnitine in plasma (Rebouche, 1977). Characteristics of the transport protein identified in human tissues are consistent with these observations (Tamai et al., 1998). Because the Km for carnitine transport lies within the upper physiological range of plasma carnitine concentrations, muscle is sensitive to changes in plasma carnitine. Accumulation of carnitine in skeletal muscle increases linearly with increasing extracellular concentrations in rat (Rebouche, 1977), dog (Rebouche and Engel, 1983), pig (Owen et al., 2001a,b), and human muscle (Rebouche and Engel, 1984). Thus, the smaller increases in plasma carnitine resulting from abomasal infusion of 6 g/d of carnitine were likely not sufficient to drive greater muscle carnitine concentration (LaCount et al., 1995), but the large effect on plasma carnitine when 20 g/d was infused in the present study was sufficient to increase muscle carnitine.

Muscle contains approximately 90% of the body carnitine (Ruff et al., 1991) because of both the greater tissue concentration and the much larger proportion of BW than liver. Therefore, considering muscle carnitine concentration in the absence of total muscle mass is probably not the most appropriate indicator of muscle carnitine status. For example, intravenous carnitine injection into rats resulted in 22% of the injected dose recovered in muscle whereas only 9% of the dose accumulated in liver (Ruff et al., 1991). However, intravenous injection caused liver carnitine concentration to double whereas the concentration of carnitine in muscle increased by only 12%. In our study, carnitine infusion clearly increased total carnitine concentration in muscle, but whether the concentration was maximized by abomasal infusion of 20 g/d cannot be determined.

Feed restriction also likely increased total carnitine abundance in the body considering that muscle carnitine concentration was increased significantly and liver concentrations were slightly larger. In WR cows, the increase in total carnitine concentration in muscle was driven by higher free carnitine concentration. Our results agree with data obtained from rats fasted for 72 h where free and total carnitine increased in muscle due to fasting, but short- and long-chain acylcarnitines were relatively stable (Brass and Hoppel, 1978). During fasting or feed restriction, carnitine status is augmented by increased carnitine biosynthesis as well as decreased excretion of carnitine and carnitine precursors (Brass and Hoppel, 1978; Vaz and Wanders, 2002). These mechanisms were likely responsible for the increase in muscle carnitine in WR cows.

Milk carnitine concentrations on d 8 to 9 and d 13 to 14 are presented in Table 4Go. L-Carnitine infusion increased all milk carnitine fractions on d 8 to 9 (P < 0.01) when all cows were allowed to consume DM ad libitum. Carnitine infusion also increased total carnitine output in milk (P < 0.01) as a result of increased milk carnitine concentration. On d 13 to 14, carnitine infusion increased free carnitine in milk (P < 0.01), and feed restriction tended to increase milk free carnitine concentration in carnitine-infused but not water-infused cows (infusion x DMI; P = 0.07). Significant interactions existed for acid-soluble carnitine, short-chain acylcarnitine, and total carnitine concentrations in milk such that CR > CA > WR > WA (infusion x DMI, P = 0.01). Carnitine infusion (P < 0.01) and feed restriction (P < 0.01) increased long-chain acylcarnitine without interaction (P = 0.19). Total carnitine output in milk followed a similar pattern as free carnitine during feed restriction; carnitine output remained elevated in CA and CR, but feed restriction tended to increase milk carnitine output only in CR cows (infusion x DMI, P = 0.08).


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  		Table 4. Least squares means for concentration and daily output of carnitine in milk from lactating Holstein cows infused with water or L-carnitine (20 g/d; d 5 to 14) and fed for ad libitum or restricted DMI (50% of previous 5-d DMI; d 10 to 14)
 
Abomasal infusion and dietary supplementation of carnitine to lactating dairy cows resulted in greater milk carnitine concentration and milk carnitine output (LaCount et al., 1995, 1996a,LaCount et al., b). Similarly, dietary supplementation of sows during gestation and lactation has been shown to increase milk carnitine concentrations (Musser et al., 1999; Ramanau et al., 2004). Shennan et al. (1998) found that carnitine transport by the mammary gland has a high Km (132 µM in lactating rat mammary explants) relative to muscle (Rebouche, 1977; Tamai et al., 1998) indicating that transport is not saturated under normal physiological conditions (Shennan and Peaker, 2000). In rats, activity of the carnitine transporter in the mammary gland is higher in early lactation and decreases as lactation progresses (Shennan et al., 1998). Kinetics of the carnitine transporter certainly support observations that milk carnitine concentrations vary with stage of lactation (Erfle et al., 1974), and that milk carnitine increases substantially upon carnitine supplementation (LaCount et al., 1995, 1996a,LaCount et al., b).

Regardless of infusion type, feed restriction had a clear effect on milk carnitine concentrations when compared with ad libitum-fed counterparts. Substantial increases in short-chain acylcarnitine greatly contributed to the increases in total carnitine in milk from feed-restricted cows. Comparisons between milk and plasma carnitine composition are not possible because individual carnitine fractions were not determined in plasma. However, in rats, plasma short-chain acylcarnitine increased within 12 h whereas free carnitine concentrations were depressed during a long-term fast (Brass and Hoppel, 1978). High activity of carnitine acetyltransferase in ruminant liver (Snoswell and Henderson, 1970) suggests that hepatic synthesis of acylcarnitines (from VFA or fatty acid-derived acetyl groups plus free carnitine) could contribute to accumulation of short-chain acylcarnitines in milk. Despite small increases in total plasma carnitine in the WR treatment, it is possible that increased short-chain acylcarnitine in plasma directly contributed to greater short-chain acylcarnitine in milk from WR cows. This notion is supported by the fact that cows experiencing ketosis had significantly higher acetylcarnitine concentrations in milk than did healthy controls (Erfle et al., 1970, 1974), perhaps as a means to relieve acetyl pressure on coenzyme A pools during active ß-oxidation of NEFA.

Total carnitine output in milk was 3-fold greater for CR than for WR cows, consistent with increased milk and plasma carnitine concentrations in CR cows and relatively high carnitine transport capacity in mammary tissue (Shennan et al., 1998). Although urinary concentrations of carnitine and carnitine precursors decrease significantly to maintain similar whole-body carnitine concentrations during long-term starvation in rats (Brass and Hoppel, 1978; Sandor and Hoppel, 1989), milk clearly represents a significant drain on the body carnitine pool. Kinetics of carnitine transport and acylcarnitine formation among different tissues seem to regulate whole-body carnitine concentrations, indicating that large amounts of supplemental carnitine can exceed tissue transport capacity and result in elevated urinary carnitine excretion, as shown by LaCount et al. (1996a).


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
ACKNOWLEDGEMENTS
 REFERENCES
 
Based on carnitine concentrations in plasma, liver, muscle, and milk, it appears that dairy cows experiencing DMI depression and elevated NEFA mobilization might benefit from L-carnitine supplementation. Carnitine infusion into feed-restricted cows resulted in carnitine accumulation in plasma, liver, muscle, and milk; however, unsupplemented, feed-restricted cows had elevated carnitine in muscle and milk but only modest increases in liver. We have previously shown that abomasal infusion of L-carnitine (20 g/d) in this study led to lower liver accumulation of total lipid, enhanced hepatic fatty acid oxidation, and higher 3.5% FCM yield during 50% feed restriction (Carlson et al., 2006). Under metabolic conditions characteristic of the periparturient period, such as increased adipose tissue lipolysis, DMI depression, and the onset of lactation, hepatic carnitine concentrations may be insufficient for optimal hepatic fatty acid oxidation. Results of the present study indicate that endogenously synthesized carnitine might be utilized in muscle or be secreted in milk rather than accumulate in the liver, and carnitine supplementation may be required to increase liver carnitine concentrations during DMI depression. Further research examining the effects of carnitine supplementation on hepatic and peripheral metabolism during the periparturient period is warranted.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
REFERENCES
 
The authors thank J. Odle and associates for technical assistance with the carnitine assay. Gratitude is extended to Deana Rincker and Elizabeth French for assistance with laboratory assays.


   FOOTNOTES
 
1 Supported by Lonza, Inc., Allendale, NJ. D. B. Carlson was supported by a Jonathan Baldwin Turner Graduate Fellowship, College of Agricultural, Consumer and Environmental Sciences, University of Illinois. Back

2 Current address: Dept. of Animal and Range Sciences, North Dakota State University, Fargo, ND 58105-5727. Back

Received for publication September 16, 2006. Accepted for publication February 12, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 


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D. B. Carlson, J. W. McFadden, A. D'Angelo, J. C. Woodworth, and J. K. Drackley
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