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UP de Nutrition, Ecole Nationale Vétérinaire d’Alfort, 94704 Maisons Alfort, France and * Laboratoire de Physiologie de la Nutrition (INRA), Université Paris-Sud, Orsay, France
1To whom correspondence should be addressed. E-mail: blanchard{at}vet-alfort.fr.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSLiver compositionDISCUSSIONLITERATURE CITED |
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KEY WORDS: • carnitine • fatty acids • ß-hydroxybutyrate • hepatic lipidosis • cats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSLiver compositionDISCUSSIONLITERATURE CITED |
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,2
) and fasting with ketosis (3
) in humans. Obese cats are likely to become anorexic under stressful conditions. Fat accumulates in the liver during fasting, leading to a fatty liver syndrome or feline hepatic lipidosis (FHL)2
(4
), which is a very common lethal hepatic pathology in the United States (5
). This disease may be induced experimentally (6
) and treated. L-Carnitine is required for fatty acids to enter the mitochondria. Carnitine concentration is increased in the muscle and liver of obese women (7
), and also in the plasma of starved rats (8
) and humans (3
). Plasma, tissue and urine carnitine concentrations have been reported in healthy adult cats and kittens (9
) as well as in cats with spontaneous FHL (10
), but never in obese or fasting cats. The aim of this study was to determine whether a carnitine supplement could protect fasting cats from ketosis and therefore whether a carnitine supplement would improve carnitine and lipid metabolism in FHL. The model of Biourge (6
) was used to induce FHL. Plasma, muscle and liver samples were collected from cats while healthy, obese, with FHL and after treatment to assay plasma fatty acids and ß-hydroxybutyrate, plasma, muscle and liver free and total carnitine concentrations, liver lipids and protein composition. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSLiver compositionDISCUSSIONLITERATURE CITED |
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Sixteen queens (18 mo old) obtained from a pathogen-free facility (IFFA-CREDO, L’arbresle, France) were randomly assigned to two groups of eight. The queens were housed in individual cages (4 blocks of 4 cages), maintained at 
20°C with free access to water. Body weight was recorded weekly and food intake daily. The queens were allowed to play together 3 h/d but had no access to food during this playing period. Classical music was provided during the day, for three periods of 1–2 h each. The queens were maintained in accordance with the Principles of Biomedical Research Involving Animals, developed by the Council for the International Organization of Medical Sciences.
Experimental design.
Diets and the experimental design are shown in Figure 1
. Briefly, after 1 wk of adaptation, the queens were spayed. Their body weights were determined (initial body weight, iBW) and they were fed Diet M3
at a maintenance level. Six weeks later [step postovariectomy (PO)], 8 queens [carnitine low (CL) group] received Diet C containing 40 mg/kg of L-carnitine, and 8 queens [carnitine high (CH) group] consumed Diet C+ containing 1000 mg/kg of L-carnitine ad libitum until obesity (Ob) developed, defined when each queen weighed at least 140% of her iBW. One queen from the CH group died when obese because of stress, leaving seven in the CH group. Unfortunately, four queens had to be excluded from the CL group, which then comprised only four, as a result of technical problems during the period of obesity development (Dob). Once obese, the queens received Diet P (11
), which is a semipurified diet, complete and balanced for cats but very poorly palatable, and which cats refused to eat (fasting period). As soon as they showed any sign of FHL (step FHL), (bilirubinemia > 40 mg/L and triacylglycerol > 0.6 mmol/L, or body weight reduction to 70% of the iBW), they were treated with Diet A by progressive enteral forced-feeding until they ate on their own. The initial amount of Diet A given was 0.1 of the daily energy requirement per day, in 4 to 5 meals, which was increased progressively depending on the individual reaction of each queen. When they ate on their own again, they all received Diet C at maintenance level. At 10 wk after FHL, they were considered treated (step Treat).
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Blood was collected at steps PO, DOb, Ob, once a week during the fasting period (F1, 1 wk of fasting; F2, 2 wk of fasting; F3, F4, F5), FHL and Treat. A cephalic venipuncture was performed in the morning after an overnight fast, under slight sedation (tiletamine-zolazepam, 10 mg/kg). Blood was collected into a tube containing potassium EDTA, immediately centrifuged (1050 x g, 15 min, 4°C) and the plasma stored at -20°C until analysis.
Plasma fatty acids and ß-hydroxybutyrate assays.
Plasma fatty acids (ACS-ACOD method, Wako NEFA C kit, provided by Oxoid, Dardilly, France) and ß-hydroxybutyrate (kit No 310-UV Sigma Diagnostics, St Quentin Fallavier, France) concentrations were determined for each blood sample in the CH and CL groups.
Biopsies.
A laparotomy was performed in the CH and CL groups at steps DOb, Ob, FHL and Treat after anesthesia (tiletamine-zolazepam, 20 mg/kg) to obtain 500 mg of liver, for the analysis of liver composition (protein, lipids, total and free carnitine). At the same time, skeletal muscle specimens (10 mg) were obtained from semimembranosis/semitendonosis muscles, with a Tru-Cut Biopsy Needle (14 gauge). Liver and muscle aliquots were weighed, immediately frozen and kept at -20°C until analysis.
Liver composition.
The liver analysis was adapted from a previously described method (12
). Frozen liver samples (250 mg) were homogenized in 5 mL of isopropanol, using an Ultra-Turrax apparatus (Janke & Kinfel, Staufen, Germany). After incubation at 60°C for 1 h and centrifugation for 8 min at 3000 x g, the supernatant was collected and the pellet reextracted in the same manner as described above. This pellet was used for the protein assay using the method of Lowry et al. (13
). Triacylglycerol (Wako Chemicals kit Triglyceride N, provided by Oxoid) and phospholipids (Wako Chemicals Phospholipids B, provided by Oxoid) were measured on the isopropanolic extract. Total cholesterol was determined on the isopropanolic extract with an enzymatic colorimetric kit (CHOH-PAP method, Boehringer Mannheim, Meylan, France).
Carnitine assay.
Total and free L-carnitine in plasma, muscle and liver tissues were assayed by HPLC methods (14
–20
). Plasma samples were assayed as described (19
) with the following adjustments. A 50-µL sample volume was analyzed with a 50-µL esterified-carnitine (e-carnitine) internal standard volume; 50 µL of saturated monobasic potassium phosphate solution was added and heated at 65°C for 75 min. The sample was then cooled and the saponification reaction needed for the total carnitine assay stopped by the addition of 25 µL of 100 g/L phosphoric acid. The liver and muscle samples were handled as described (15
) except that the tissue samples were homogenized in the e-carnitine internal standard solution in an equal amount of distilled water using an ultrasonic homogenizer. After homogenizing, the tissue slurry was assayed exactly as the plasma samples. All of the reagents and supplies used were commercially available except e-carnitine and the 4'-bromophenacyltrifluoromethanesulfonate, which were obtained from the laboratory of Hoppel and Minkler (Medical Research Service, Department of Veterans Affairs Medical Center, Cleveland, OH).
The chromatography was accomplished using a Beckman-Coulter instrumentation (Fullerton, CA). The equipment consisted of a Beckman 126 Gradient HPLC pump, a Beckman 168 Photodiode Array detector, and a Beckman 502 Autosampler fitted with an internal column oven. All parts of the instrument were controlled using Beckman-Coulter’s System Gold software. The derivatized sample components (L-carnitine and e-carnitine) were separated using a combination step and continuous gradient mobile phase (17
–20
) on a Hypersil MOS C8 150 mm x 4.6 mm x 3 µm. The pump delivered the mobile phase at a continuous flow rate of 1.75 mL/min to the column which was maintained at 40°C. The sample components were detected by observing the change in absorbance response at 260 nm. The analytical column was protected by an identically packed guard column.
Total and free carnitine in the blood were measured at steps PO, DOb, Ob, F2 and FHL. Total and free carnitine concentrations in the liver and muscle were assayed at steps DOb, Ob, FHL and Treat.
Statistical analyses.
The differences between CH and CL groups, for all steps together, were analyzed using an unbalanced ANOVA, which included a cat (group) effect as random. When the interaction (group x step) was significant, the differences by group between steps, and by step between groups were tested using a pair-wise ANOVA. All statistical analyses were performed with the SAS System (GLM procedure, SAS Institute, Cary, NC). Comparisons were considered statistically significant when P 
0.05. Data are reported as means ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSLiver compositionDISCUSSIONLITERATURE CITED |
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Daily food intake during obesity induction (Fig. 2
) showed a significant group x step interaction due to differences at wk 6, 19 and 21. The body weight of the cats (Fig. 2)
varied significantly with the steps (P < 0.0005) but not between groups. As expected, none of the queens ate diet P during fasting and hepatic lipidosis. Three queens (75%) from the CH group and 3 queens (43%) from the CL group recovered from HFL, so that only 3 cats remained in each group at step Treat.
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Plasma fatty acid concentration (Fig. 3
) rose significantly (P < 0.001) in fasting cats and during FHL in both groups, but was always lower in the CH group than in the CL group (P < 0.0001).
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). ß-Hydroxybutyrate increased in the CH group only during the FHL step. However, in the CL group, ß-hydroxybutyrate increased two- to fourfold during fasting and FHL compared with the baseline value and was significantly higher for the CL group compared with the CH group at wk 4 (P < 0.01) and wk 5 (P < 0.001) of fasting and during FHL (P < 0.001). Dietary L-carnitine supplementation during obesity development lowered plasma fatty acids concentration and ketosis in cats during fasting and FHL.
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Dietary L-carnitine supplementation increased plasma total carnitine (Tcarn) concentration (P < 0.01) although this variable increased in both groups (P < 0.0005) (Table 1
), first during the development of obesity, then during FHL, and returning to baseline level after treatment. Plasma free carnitine (Fcarn) concentration increased only during FHL in the CL group, whereas it increased continuously from the development of obesity up to FHL in the CH group, resulting in a higher level in cats from the CH group during obesity development and after 2 wk of fasting. These differences led to modifications in the free/total carnitine ratio, which diminished continuously from its baseline value to FHL, implying an increment in e-carnitine during this period in both groups.
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Dietary carnitine supplementation significantly increased (P < 0.05) the muscle Tcarn concentration (Fig. 5
), but not the muscle Fcarn concentration. In both groups, FHL significantly increased muscle Tcarn (P < 0.01) and Fcarn (P < 0.05) concentrations. The muscle free/total carnitine ratio was 80% and was not influenced by group or step (results not shown).
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Liver composition |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSLiver compositionDISCUSSIONLITERATURE CITED |
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Liver lipids and protein were not influenced by the dietary carnitine level (Fig. 6
). In both CH and CL groups, the liver triacylglycerol (P < 0.0005) and phospholipid (P < 0.0002) concentrations were increased by obesity and dramatically during FHL, whereas the protein concentration significantly decreased (P < 0.0001) during FHL. The liver cholesterol concentration was not affected.
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Liver Tcarn and Fcarn varied differently at the different steps (Fig. 7
) and were influenced by dietary carnitine supplementation as demonstrated by a significant group x step interaction (P < 0.05) for both variables. Dietary L-carnitine supplementation increased liver Tcarn and Fcarn at all steps, whether expressed per gram of liver or relative to the protein concentration of the liver (results not shown). In the CL group, FHL, compared with the other steps, significantly increased liver Tcarn and Fcarn. In the CH group, both Tcarn and Fcarn tended to decrease continuously from obesity to treatment (P = 0.09). The liver free/total carnitine ratio (0.8) was not influenced by either the group or step (results not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSLiver compositionDISCUSSIONLITERATURE CITED |
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). Plasma fatty acid and ß-hydroxybutyrate concentrations during the feeding periods were consistent with the values reported by Pazak et al. (22
) in healthy cats. In our study, the plasma carnitine concentrations in healthy and treated cats were similar to those measured in healthy queens (9
), although the carnitine concentration of the diet was not indicated by these authors. The total and free carnitine concentrations in both healthy and treated queens were >10-fold higher in muscle than in the liver, in agreement with previous results in healthy cats (9
). The increase in liver size was due to triacylglycerol accumulation, as reported in induced (6
) and spontaneous FHL (23
).
The dietary carnitine supply (40 mg L-carnitine/kg diet) in the low carnitine group, termed the control group, was the same usually provided in a commercial diet for adult cats. That of the high carnitine group was markedly higher (1000 mg L-carnitine/kg diet), and close to that of the natural diet of cats [150 to 3000 mg L-carnitine/kg of animal tissues (24
,25
), which constitutes about half the normal diet].
In control queens, the increments of plasma fatty acid and ß-hydroxybutyrate concentrations during fasting and FHL were related to the negative energy balance. The severe ketosis observed in FHL is life threatening, which may explain the relatively large number of cats that died from FHL in the control group (4/7 vs. 1/4 in the supplemented queens). Even when a low carnitine diet (40 mg/kg) was supplied, the plasma carnitine concentrations in obese cats were about twice the baseline values, which can be explained by the increased food intake. In this group, the plasma carnitine concentration after 2 wk of fasting was close to its value at obesity, but increased dramatically during FHL, as did the muscle and liver carnitine concentrations while queens were fasting. Neither liver nor muscle samples were collected after 2 wk of fasting to limit any risk of interaction between surgery and FHL induction. Carnitine is synthesized in the liver, and used mainly in the muscles. After long-term fasting, the queens showed a negative energy balance. The increase in muscle carnitine concentration in FHL might be related to the elevated plasma concentration and the high energy requirement of muscles, which could lead to increased carnitine uptake from the plasma. The low liver carnitine concentration observed at previous steps (baseline and obesity), compared with the value during FHL, suggests an enhanced de novo synthesis in the liver during long-term fasting.
The carnitine status of obese cats, reported here for the first time, indicates a higher plasma total carnitine concentration in obese cats than in lean cats, as already described in obese humans (3
). In cats, this change is due to both free and esterified carnitine because the free/total carnitine ratio was significantly lower in obese than in lean cats in which free carnitine was predominant, as in obese humans (3
). In our study, the muscle total and free carnitine concentrations were not modified by obesity, whereas muscle free carnitine was lower in obese than in lean rats (24
). It is noteworthy, however, that the concentrations in cats were higher than in rats, perhaps due to a different carnitine concentration in the diet (40 g/kg for nonsupplemented cats, and only 2.1 g/kg for rats). In our cats, obesity had no effect on liver total and free carnitine concentrations, whereas both were increased in obese, compared with normal weight humans (7
). This suggests a different carnitine metabolism in cats, which, compared with rats and humans, are true carnivores characterized by a high animal protein diet, and will require further study.
A high L-carnitine supplement had several effects on carnitine and lipid metabolism. The main effect was to lower plasma fatty acid and ß-hydroxybutyrate concentrations during fasting and FHL. This finding is of considerable interest because spontaneous, psychogenic anorexia may occur in cats, especially obese ones (4
,10
,21
). As soon as the queens started to eat the high carnitine diet, the plasma, muscle and liver carnitine concentrations became higher than baseline values. In high L-carnitine–supplemented cats, the plasma and muscle carnitine concentrations stayed highest even during FHL, with an observed increase compared with obese cats, whereas the liver carnitine tended to decrease from the obesity value, suggesting use of the hepatic carnitine stores resulting from the feeding period. These results are related to the plasma fatty acid and ß-hydroxybutyrate concentrations in these L-carnitine–supplemented cats, in which carnitine appeared to reduce the severity of ketosis during long-term fasting. Although the carnitine supplement did not modify liver lipid and protein concentrations, the analysis of plasma enzyme activities and ammonia concentration (results not shown) indicated that the carnitine supplement improved liver function and significantly reduced plasma NH3 concentration (P < 0.05, results not shown), and alanine amino transferase activity after 4 and 5 wk of fasting (P < 0.006 and P < 0.0001, respectively, results not shown). These results suggest that a dietary supplement of L-carnitine provides a protective effect on liver function, especially against ketosis during long-term fasting. This might be explained by lower ketone production by the liver, as reported in rats (27
), by a better use of ß-hydroxybutyrate by peripheral tissues, especially muscles, as in dogs (28
), and/or by a higher urinary excretion of excess of acylcarnitine, as suggested in humans (29
). The latter hypothesis was supported by the observed increase in esterified carnitine which represented about 40% of the total circulating carnitine during FHL, compared with 10–20% in healthy cats and 30% in obese ones, in both dietary groups.
These observations raise the question whether carnitine present in the liver and muscles is able to act as a carrier and reduce the charge of acylCoA or acetylCoA by exporting acylcarnitine and/or acetylcarnitine out of the liver. If acetylcarnitine is involved, it may help to lower the intracellular amount of acetylCoA and prevent its extensive accumulation, which could otherwise contribute to an increased synthesis of ketone bodies. This liver output of acetylcarnitine is supported by a study of L-carnitine supplementation in obese cats subjected to a rapid weight loss program (5
). However, further studies will be required to test this hypothesis by separating acetyl- from e-carnitine and analyzing the amount of acylcarnitine excreted in urine in 24 h.
L-Carnitine is synthesized mainly in the liver from lysine, methionine and especially from trimethyllysine released by protein turnover (30
,31
). A nitrogen loss of 20% has been reported in cats fasted for 4 wk (11
). It is therefore probable that in nonsupplemented cats, protein catabolism provides carnitine precursors that the liver is able to use for carnitine synthesis, in a situation of negative energy balance and intense lipolysis. The carnitine stores resulting from a high carnitine diet can then be used primarily, and nitrogen spared because fewer amino acids are required for synthesis. A dietary supplement of L-carnitine has already been reported to reduce accumulated fat mass and increase lean body mass in different species such as Atlantic salmon (32
), African catfish (33
), pigs (34
), broilers (35
) and dogs (36
). In humans, the same supplement was reported to improve the serum total protein and albumin concentrations in patients in maintenance dialysis (37
). It would be interesting to compare nitrogen loss during long-term fasting in cats supplemented or not with a high L-carnitine supplement by dual-energy X-ray absorptiometry (38
) to test whether such a supplement can reduce nitrogen loss during subsequent long-term fasting in these cats.
In conclusion, this study demonstrates the protective effect of a dietary supplement of L-carnitine against fasting ketosis in cats, and suggests a role of L-carnitine in protein metabolism during fasting. Further studies are required to confirm this role. Spayed cats show a high risk of obesity because their energy requirements are lower than those of intact cats and they tend to eat all of the food available to them (39
). Because ketosis may result in life-threatening metabolic acidosis and because obese cats are susceptible to anorexia, an increased concentration of L-carnitine may be recommended in the diet of spayed and obese cats.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Diet M: Science diet Feline Maintenance dry, Hill’s Pet Products (Topeka, KS). Diet C: Prescription diet Feline c/d dry, Hill’s Pet Products. Diet C+ differs from diet C only by the amount of L-carnitine included (1000 mg/kg vs. 40 mg/kg in diet C). Diet P: semipurified diet, complete and balanced for cats, but poorly palatable (11
). Diet A: Prescription diet Canine/Feline a/d, Hill’s Pet Products.
Manuscript received 10 July 2001. Initial review completed 9 August 2001. Revision accepted 13 November 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSLiver compositionDISCUSSIONLITERATURE CITED |
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