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    Mean ± SD resting energy expenditure determined by open-flow indirect calorimetry normalized to total body weight (A) and LBM (B) at various points in 31 overweight cats that were fed a weight reduction diet containing 0 (CN-0), 50 (CN-50), 100 (CN-100), or 150 (CN-150) μg of carnitine/g. From days −28 to 0, cats were allowed unrestricted intake of the weight reduction diet. From days 1 to 84, intake of the same weight reduction diet was restricted. From days 84 to 120, cats were fed an unrestricted amount of an energy-dense diet. *Indicated values are significantly (P < 0.001) different from baseline (day −28; A) or among cats fed unsupplemented and carnitine-supplemented diets (B).

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    Mean ± SD RQ determined by indirect calorimetry in the cats in Figure 1. A—On day 84, 5 cats were censored because they had already achieved an IBW (were no longer on energy restriction). B—Results for all cats fed carnitine-supplemented diets were combined. *Indicated values are significantly different from baseline (day −28) within a diet group (A; P < 0.05) or among cats fed carnitine-supplemented or unsupplemented diets (B; P < 0.01). See Figure 1 for remainder of key.

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    Box-and-whisker plots of palmitate flux rate (A) and fold increase in palmitate flux rate (B) determined from 1-13C-palmitate steady-state infusion in the cats in Figure 1. The short thick horizontal bar within each box represents the mean, and the longer, thinner horizontal line separating shaded portions of the box represents the median. The boxed area encloses the middle half of data values (ie, the 25th to 75th percentile), with the shaded portion of each box representing values above and below the median. Whiskers represent the range. Cats were evaluated after 12 hours of food withholding. An energy-dense fattening diet was fed at baseline, whereas the unsupplemented weight reduction diet and the carnitine-supplemented weight reduction diets were fed during energy-restricted feeding. *Indicated values are significantly different from baseline (day −28) within a diet group (A; P ≤ 0.05) or between the unsupplemented and CN-150 groups (B; P = 0.03). See Figure 1 for remainder of key.

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    Box-and-whisker plots of estimated FFA oxidation rate determined by 1-13C-palmitate steady-state infusion in the cats in Figure 1. *Indicated values differ significantly (P ≤ 0.05) within diet groups between days −28 and 42. See Figures 1 and 3 for remainder of key.

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    Box-and-whisker plots of daily nitrogen intake determined from food intake (A) and urinary nitrogen elimination by Kjeldahl analysis (B) for the cats in Figure 1. *Indicated values differ significantly (P < 0.003) from baseline within diet groups. See Figures 1 and 3 for remainder of key.

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    Box-and-whisker plots of stoichiometrically calculated rate of fat oxidation (A) and carbohydrate oxidation (B) in the cats in Figure 1. *Indicated values differ significantly between unsupplemented and supplemented diet groups on the indicated day (P ≤ 0.02; A) or within the unsupplemented diet group between days. See Figures 1 and 3for remainder of key.

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Influence of dietary supplementation with l-carnitine on metabolic rate, fatty acid oxidation, body condition, and weight loss in overweight cats

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  • 1 Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.
  • | 2 Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.
  • | 3 Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.
  • | 4 Research and Development Division, The Iams Co, Proctor & Gamble, PO box 862, Lewisburg, OH 45338.
  • | 5 Research and Development Division, The Iams Co, Proctor & Gamble, PO box 862, Lewisburg, OH 45338.

Abstract

Objective—To investigate the influence of dietary supplementation with l-carnitine on metabolic rate, fatty acid oxidation, weight loss, and lean body mass (LBM) in overweight cats undergoing rapid weight reduction.

Animals—32 healthy adult neutered colony-housed cats.

Procedures—Cats fattened through unrestricted ingestion of an energy-dense diet for 6 months were randomly assigned to 4 groups and fed a weight reduction diet supplemented with 0 (control), 50, 100, or 150 μg of carnitine/g of diet (unrestricted for 1 month, then restricted). Measurements included resting energy expenditure, respiratory quotient, daily energy expenditure, LBM, and fatty acid oxidation. Following weight loss, cats were allowed unrestricted feeding of the energy-dense diet to investigate weight gain after test diet cessation.

Results—Median weekly weight loss in all groups was ≥ 1.3%, with no difference among groups in overall or cumulative percentage weight loss. During restricted feeding, the resting energy expenditure-to-LBM ratio was significantly higher in cats that received l-carnitine than in those that received the control diet. Respiratory quotient was significantly lower in each cat that received l-carnitine on day 42, compared with the value before the diet began, and in all cats that received l-carnitine, compared with the control group throughout restricted feeding. A significant increase in palmitate flux rate in cats fed the diet with 150 μg of carnitine/g relative to the flux rate in the control group on day 42 corresponded to significantly increased stoichiometric fat oxidation in the l-carnitine diet group (> 62% vs 14% for the control group). Weight gain (as high as 28%) was evident within 35 days after unrestricted feeding was reintroduced.

Conclusions and Clinical Relevance—Dietary l-carnitine supplementation appeared to have a metabolic effect in overweight cats undergoing rapid weight loss that facilitated fatty acid oxidation.

Abstract

Objective—To investigate the influence of dietary supplementation with l-carnitine on metabolic rate, fatty acid oxidation, weight loss, and lean body mass (LBM) in overweight cats undergoing rapid weight reduction.

Animals—32 healthy adult neutered colony-housed cats.

Procedures—Cats fattened through unrestricted ingestion of an energy-dense diet for 6 months were randomly assigned to 4 groups and fed a weight reduction diet supplemented with 0 (control), 50, 100, or 150 μg of carnitine/g of diet (unrestricted for 1 month, then restricted). Measurements included resting energy expenditure, respiratory quotient, daily energy expenditure, LBM, and fatty acid oxidation. Following weight loss, cats were allowed unrestricted feeding of the energy-dense diet to investigate weight gain after test diet cessation.

Results—Median weekly weight loss in all groups was ≥ 1.3%, with no difference among groups in overall or cumulative percentage weight loss. During restricted feeding, the resting energy expenditure-to-LBM ratio was significantly higher in cats that received l-carnitine than in those that received the control diet. Respiratory quotient was significantly lower in each cat that received l-carnitine on day 42, compared with the value before the diet began, and in all cats that received l-carnitine, compared with the control group throughout restricted feeding. A significant increase in palmitate flux rate in cats fed the diet with 150 μg of carnitine/g relative to the flux rate in the control group on day 42 corresponded to significantly increased stoichiometric fat oxidation in the l-carnitine diet group (> 62% vs 14% for the control group). Weight gain (as high as 28%) was evident within 35 days after unrestricted feeding was reintroduced.

Conclusions and Clinical Relevance—Dietary l-carnitine supplementation appeared to have a metabolic effect in overweight cats undergoing rapid weight loss that facilitated fatty acid oxidation.

L-carnitine, which is a conditionally essential nutrient, plays a pivotal role in fatty acid metabolism by acting as an essential cofactor that facilitates transfer of long-chain fatty acids across the inner mitochondrial membrane for β-oxidation and energy generation. The nutrient enables a 2-way flux of acyl-CoAs at the mitochondrial interface, buffering against the inhibitory influence of accumulating acetyl-CoA on β-oxidation. This effect regulates intramitochondrial acetyl-CoA, releasing free CoA and acetyl-carnitine, which favor distribution of pyruvate toward utilization for energy production (oxidation). The interface exchange of acyl-CoA allows l-carnitine to facilitate the sharing of metabolic energy (short-chain acyl-carnitine and acetyl-carnitine) between intracellular organelles and tissues.1 Through these mechanisms, l-carnitine may suppress the accumulation of lactic acid, as demonstrated in human athletes undergoing high-intensity excercise.2 l-carnitine also may provide a mechanism for removal of excessive fatty acids from hepatocytes in cats with hepatic lipidosis because acylcarnitines can traverse the cellular membrane.3

Although supplemental l-carnitine has been shown to promote development of LBM during growth in piglets, its influence on body condition during weight loss remains controversial.4–7 Gene expression profiles suggest an association between accretion of protein, muscle, and the ratio of protein to fat in piglets fed l-carnitine with the anabolic insulin-like growth factor-1 pathway and suppression of proapoptotic and atrophy-related genes.8 In vitro studies9–11 of hepatocyte cultures have shown that l-carnitine increases the rate of fatty acid oxidation. However, no data exist to suggest dietary supplementation with l-carnitine results in a measurable in vivo change in substrate oxidation in overweight animals undergoing weight loss. The objective of the study reported here was to determine whether dietary supplementation with L-carnitine would facilitate weight loss in overweight cats fed a weight-reduction diet on an unrestricted or restricted basis, retention of LBM during weight loss, and preferential use of fat for energy expenditure (substrate oxidation).

Materials and Methods

Animals—Thirty-two colony-housed adult cats (age range, 3 to 7 years; 16 neutered males and 16 neutered females) were used in the study. Cats were allowed to become overweight through feeding of a mixture of 2 energy-dense dry diets provided without restriction for 6 consecutive months (fattening diet; Appendix 1). Water was provided at all times throughout the experiment. Cats were individually housed for 19 hours daily and allowed recreational social interaction for the remaining 5 hours, except when indicated. During the initial fattening phase, cats were group housed with unrestricted exercise and social interactions during the day; unlimited quantities of food were available in their individual cages, where they were placed from 6 pm to 7 am. The housing and care provided to the cats were in compliance with the recommendations of the Institutional Animal Use and Care policies of Cornell University.

Experimental design—After fattening, cats were randomly assigned to the following 4 diet groups such that there were 4 males and 4 females in each: unsupplemented (control) weight reduction diet and weight reduction diet with 50 (CN-50), 100 (CN-100), or 150 (CN-150) μg of carnitine/g (Appendix 2). Diets were fed in unrestricted quantities for 4 weeks (days −28 to 0) and then were fed in discrete meals for 84 days (days 1 to 84). Cats were returned to the fattening diet on days 85 through 120, with food provided without restriction. Investigators involved with experimental manipulations and cat handling remained unaware of which test diet each cat had received until the experiment had concluded and the data were analyzed.

Energy restrictions were initially estimated on the basis of IBW (kg) × 60 kcal/kg × 0.6 and were adjusted by 15% increments in cats that failed to lose weight over 14-day intervals during the study. When a cat achieved IBW during the study, energy restriction was suspended and the cat was fed the same diet to maintain that body weight until day 84. From days −28 through 119, food was provided on an individual basis with cats confined to cages to allow quantification of daily intake by weight.a The FQ of each diet was calculated as described elsewhere.12

Cat assessment—Body condition score was assigned at baseline (day–28) and during the diet trial on a weekly basis by 2 investigators (SAC and KLW) experienced with this procedure. For this, a 5-point scale was used (1 = thin, 2 = underweight, 3 = ideal weight, 4 = overweight, and 5 = obese), with the cat's frame size taken into account. Other variables evaluated included daily food consumption (determined from mass of food consumed) and weekly body weight as determined with a calibrated digital scaleb accurate to 0.01 kg. At selected intervals, other analyses were performed, including a CBC and serum biochemical analysis (for cats from which food was withheld overnight), urinalysis, urinary nitrogen excretion (Kjeldahl analysis), body composition (determined with a D2O method), DEE (determined in 3 cats/group with the doubly labeled water method), open flow indirect calorimetry, and in vivo 1-13C-palmitate flux and oxidation.

REE analysis—Resting energy expenditure was determined with an open-flow indirect calorimetry system. Calorimetry measurements were performed in a softly lit room closed to pedestrian traffic with room temperature maintained between 18° and 20°C and temperature within the holding chamber maintained between 22° and 24°C. During calorimetry sessions, the activity of each cat was recorded to determine which measurements might reflect physical activity (for their subsequent exclusion). Two to 3 weeks before data collection, cats were acclimated to the procedure.

For the calorimetry sessions, cats were confined individually within a custom-built, clear, break-resistant chamber fitted with a sliding door perforated for ventilation with atmospheric air. Vacuum-driven cross-cage gas flow allowed collection of expiratory gas as it exited the holding chamber. Two cages with different dimensions (31.8 × 33 × 19.1 cm [approx 20 L] and 62.2 × 27.9 × 26.7 cm [approx 46 L]) were constructed to accommodate cats by body size (small or large, respectively). Plastic-encased closed-cell extruded polystyrene foam inserts were used to reduce chamber dead space and tailor the cage fit to each cat. Cage dimensions and customization inserts were recorded to allow exact duplication of the calorimetry environment for serial data collection on different days. Each cage was configured to allow enough room for the cat to sit or recline comfortably with its head oriented toward the air intake vents in the sliding door.

Cage gas flow was adjusted according to CO2 concentrations in outflow respiratory gases, allowing < 1% increase during the acclimation interval. Initial gas flow was selected on the basis of the expected basal o2 (body weight [-kg0.876] × 0.188) by use of a 10-fold greater gas flow that was then titrated according to CO2 concentration in the outflow gases.13 Mass flow controllersc (0.2 to 2 L/min and 2 to 15 L/min) were used to establish targeted ambient gas flow during calorimetry and nitrogen dilution studies; actual cage gas flow ranged between 3.6 and 7.5 L/min, depending on the size of the cat and exiting CO2 concentrations.

Because of the open-flow design,14 the exact flow of gas across the holding chamber was verified by nitrogen dilution evaluations that were completed after each calorimetric session. Gas flow through a cage fitted with the closed-cell extruded polystyrene foam inserts and housing a replica cat was adequate to flush the system to ambient gas concentrations within 10 minutes after nitrogen dilution.

A water absorbentd was positioned in line before the mass flow meters. Gas samples (cage or ambient baseline gas) first passed through a CO2 sensor and then an O2 analyzer linked in series. The CO2 was removed from the system with a CO2 absorbente before gas delivery to the O2 analyzer. The CO2 concentrations were determined with an infrared-based analyzer,f and O2 concentrations were measured with a disposable fuel cell.g

Gas analyzers were zeroed by use of pure nitrogen and then calibrated with a 99% guaranteed span gas containing 20.0% O2 and 5.0% CO2 before each measurement session. On days when multiple analyses were completed (> 3 cats evaluated), gas sensors were calibrated after every analysis. Fans were used to keep ambient gases well mixed, and the vacuum system providing cross-cage gas flow was evacuated through an environmental safety hood to the atmosphere. Mean air turnover in the calorimetry room was 10 turnovers/h.

The system was calibrated to ambient air concentrations before, during, and after each recording session with a software controller.h After cats had acclimated to calorimetry cage confinement (typically 20 minutes) and steady-state CO2 and O2 concentrations were verified to exit the cage, expiratory gases were collected every 5 seconds for 45 minutes in 4 intervals. A customized data acquisition systemh allowed automation of continuous chamber and periodic ambient environmental gas sample collection (every 10 minutes during measurement sessions). Data from the mass flow meters, gas analyzers, and thermometer were continuously displayed and collected in real time during recording sessions via the automated data acquisition system. The o2 for each analysis was calculated from the difference in O2 concentration between airflow into and out of the chamber, with gas concentrations corrected to standard conditions for temperature and pressure.

Daily REE (in kcal/d) was calculated on the basis of o2 (in mL/min) and co2 (in mL/min) from triplicate measurements, with the lowest values from each recording interval used in a modification of the Weir equation15,16:

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in which 3.941 is the heat generated from combusting 1 mole of O2, and 1.11 is the heat generated from producing 1 mole of CO2.

Validation of the system with alcohol combustion (methanol and ethanol) was achieved by comparing the determined RQ (RQ = co2/o2) to the expected RQ of 0.67 for complete alcohol combustion. The system registered a mean RQ of 0.68 with an accuracy within 3.1% (n = 16 analyses). Intra-assay REE repeatability was determined by collecting continuous measurements during three 10-minute intervals on 1 day from 30 cats. Interassay repeatability of REE was determined on weeks 1, 4, 8, and 12 in 6 cats with stable body weight (variation, ≤ 0.2 kg) fed a consistent diet17 not associated with the present study.

LBM assessment—Measurements of LBM were completed in all cats on days 0, 42, and 84 by means of an established D2O protocol18,19 within 5 days after open-flow indirect calorimetry assessments were performed. Cats were not manipulated for other testing during stable isotope tests. After 12 hours of food withholding, each cat was given D2Oi (0.15 g/kg of 99.8% APE) SC dorsally, just caudal to the scapulae; the area was then massaged for 1 minute to facilitate isotope dispersal. The exact dose of isotope delivered was determined gravimetrically by weighing the delivery syringe to the nearest 0.001 g before and after dose administration. Blood samples were collected immediately before and 3 and 6 hours after isotope administration. Afterward, serum was harvested and stored in screwtop vials at −80°C until thawed for analysis.

The TBW (D2O distributional volume/1.04) and fractional elimination rate of D2O were determined from D2O enrichment in serum. Enrichment of D2O in serum was determined in triplicate by means of isotope ratio mass spectrometryj by estimating the degree of conversion of water to hydrogen in 2 μL of serum through the zinc-water reduction method.20

DEE assessment—Measurement of DEE was done to investigate whether the indirect calorimetry-determined REE provided a reasonable approximation of DEE (ie, approx 60% of DEE). A modification of the doubly labeled water method18,21–24 was used to determine DEE in 12 cats (3 cats/group) on days 0, 42, and 84 within 5 days after open-flow indirect calorimetry assessment was performed. Only 3 cats/group were used because of the high cost of the stable isotope and related analysis and isotope availability. Cats were not manipulated for other testing during these evaluations.

After 12 hours of food withholding, each cat received D2Oi (0.15 g/kg) and H2 18Ok (0.15 g/kg) SC dorsally, just caudal to the scapulae; the area was then massaged for 1 minute to facilitate isotope dispersal. The exact dose of isotope delivered was determined gravimetrically by weighing of the administration syringe to the nearest 0.001 g before and after dose administration. Blood samples were collected immediately before and 6, 8, 24, 48, 72, and 96 hours after isotope administration. Serum was subsequently harvested and stored in screwtop vials at −80°C until thawed for analysis.

Enrichment of D2O and 18O were determined in triplicate via isotope ratio mass spectrometry relative to standard mean ocean water isotopic standards.21–24,j The 18O was measured in 100 μL of serum equilibrated with pure CO2 for 24 hours at 25°C. Thereafter, the equilibrated CO2 was analyzed for 18O enrichment.

Isotope kinetic data were evaluated with a 2-pool model.24 After isotope elimination plots were constructed for 4 cats, 2-point slope calculations were used to determine elimination rates and distribution volume (TBW). The mean ± SD ratio of the dispersal space of D2O to that of H218O was calculated as 1.04 ± 0.08. The DEE was estimated on the basis of the amount of CO2 production by means of the RQ (from open-flow indirect calorimetry of each cat) and the calculated FQ (expected o2 and co2 from oxidation of dietary protein, fat, and carbohydrates in the fed diet).12 The degree of isotope enrichment was calculated and expressed as a fraction of the initial dose (transformed to natural logarithms), assuming single-order kinetics.24 The amount of CO2 produced was calculated from the difference in disappearance rate between 18O (into water and CO2) and D2O (assumed to only associate with water loss). The mean TBW space determined with each isotope (D2O and H218O) was calculated after appropriate disbursal corrections to estimate LBM (TBW/0.744).

Palmitate oxidation rate—Rate of fatty acid oxidation was estimated by determining the rate of flux or turnover of the 13C-label in expired 13CO2 with a modification of the method reported by Wolfe.25 This procedure requires attainment of a steady-state 13C-fatty acid concentration in blood (rate of appearance equals the rate of disappearance or uptake) and in expired 13C-labeled CO2. Because of its poor water solubility, in vitro combination of 1-13C-palmitate with albumin is customarily used to make the substance soluble for IV infusion in humans. This method requires an elaborate palmitate preparatory-extraction process and availability of an exogenous nonantigenic albumin source. To avoid antigenic sensitization of cats to heterologous albumin, a solubilization method was developed for the present study, with the solution of 1-13C-palmitate (99.9% APE) for infusion individually prepared from serum samples from each cat.

To prepare the 1-13C-palmitate infusion solution, blood samples (10 mL) were collected from each cat by jugular venipuncture and subsequently warmed (37°C for 20 minutes) to maximize clot retraction and platelet removal. Serum was harvested by centrifugation (1,500 × g for 15 minutes), heated (58° to 60°C for 15 minutes) to inactivate complement and other proteases (eg, thrombin), and then centrifuged (10,000 × g for 10 minutes). The supernatant was mixed with 1-13C-palmitatel for a targeted concentration of 4 μmol/mL. Thereafter, the solution was sonicatedm and warmed via heat lamp to enhance and maintain palmitate solubility, cooled to room temperature (approx 20°C), and filtered twice (0.22-μm filtern) before infusion.

A solution of NaH13CO3 was prepared in 0.01N NaOH and filtered through a 0.22-μm nonpyrogenic filtero immediately prior to each use (to avoid spontaneous generation of CO2). A priming dose of NaH13CO3p (2.9 μmol/kg [0.25 μg/kg]) was administered IV to each cat immediately before starting the 1-13C-palmitate infusion to saturate the exchangeable 13C pool. Afterward, each cat received an IV infusion of 1-13C-potassium palmitate.l An infusion pump maintained a constant rate infusion for 90 minutes with the isotope suspension continuously mixed by means of a platform rocker. The 1-13C-palmitate infusions were performed in an isolated area to avoid cross contamination of the housing facility with the isotope.

After 1-13C-palmitate had been infused for 60 minutes at a rate of 0.15 to 0.3 μg/kg/min, 3 paired breath and blood samples were collected at 10-minute intervals for determination of the concentrations of 13C in exhaled CO2 and circulating palmitate (total and isotopically labeled palmitate) to calculate the palmitate flux rate, oxidation rate, and percentage palmitate oxidation.

Expired gases were collected with a snug-fitting face mask and a 1-L gas-impenetrable latex rebreathing ventilation bag.q The apex end of the bag was fitted with an airtight 3-way stopcock that permitted syringe collection of expired gas. Breath samples were collected only after the bag was filled and emptied a minimum of 3 times. Collected gas samples were immediately injected into 10-mL evacuated gas collection vialsr and stored at room temperature until batched analyses. Cats were not returned to colony housing < 24 hours after palmitate infusions to allow respiratory efflux of the stable isotope.

Isotope measurements were performed by means of source isotope-ratio mass spectrometry.s Plasma palmitate concentrations were measured in accordance with a standard method for FFA.26,s The following equations25 were used:

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in which Ei is the degree of enrichment of infusate (as APE), Ep is the degree of enrichment of substrate in plasma (as APE), and I is infusion rate (μmol/kg/min).

article image

in which Eb is the degree of enrichment of breath CO2, Ep is the degree of enrichment of palmitate in plasma, and k is correction factor for retention of bicarbonate in blood (estimated as 0.79).25

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Nitrogen balance—Degree of nitrogen balance was determined from the amount of nitrogen consumed in weighed food on a daily basis and the amount of nitrogen eliminated in urine and feces. Nitrogen intake (g/d) was calculated from amount of food consumed and proximate analysis (percentage of protein) of ingested food divided by 6.25 (nitrogen/g protein). Urinary nitrogen concentration (g/d) was determined from total 5-day urine output by means of standard Kjeldahl analysis.27 Fecal nitrogen concentration (g/d) was estimated from the amount of fecal output and percentage fecal protein divided by 6.25.

Stoichiometric substrate oxidation rate—Estimates of substrate oxidation rates were made on the basis of indirect calorimetry gaseous exchange estimates and calculations according to the method of Frayn.28 Equations used were as follows:

article image

in which 1 g of urinary nitrogen was assumed to reflect 6.25 g of protein, and 1 mole of gas at standard temperature and pressure was assumed to occupy 22.4 L. Stoichiometric substrate calculations were completed for days 0, 42, and 84.

Statistical analysis—Summary statistics are reported as mean ± SD for normally distributed data or as median (range) for nonnormally distributed data. Box-and-whisker plots, histograms, and the Kolmogorov-Smirnov test were used to evaluate the data for a Gaussian distribution, with the aid of statistical software.t For intergroup comparisons, ANOVA for repeated measures or ANCOVA was used when data were normally distributed and Kruskal-Wallis ANOVA was used for other data (fold increase in palmitate flux rate among diet groups).

Differences in energy expenditure among groups were evaluated via ANCOVA, with DEE or REE as the dependent variable, diet and LBM as independent variables, and body weight as a covariate. Significant interactions of the independent variables with REE necessitated data transformation as described elsewhere.29 Linear regression of raw REE data versus body weight was performed to predict REE for mean combined body weights for cats in the 2 groups being compared; normalized data were then compared with the standard t test.29

Differences between DEE and REE at baseline (day −28) and days 42 and 84 were examined for all diet groups individually and all cats combined to determine whether a significant reduction in energy use occurred concurrently with weight loss, suggesting adaptive thermogenesis. Differences in RQ were evaluated relative to baseline values within groups and relative to control group values for all cats that received carnitine on a per group and combined (collective) basis because of a concern that the small group size would limit the power to detect differences that truly existed.

Two × 2 tables were used to evaluate differences between diet groups in cats achieving an IBW within the study period. An α of 0.05 (2-tailed test) was applied for all comparisons. Box-and-whisker plots were used to display range, median, and mean values for nonparametric data.

Results

Animals—With the exception of 1 cat in the control group that was removed from the study because of intractability, all cats completed the study. Diet groups were equivalent with respect to sex distributions, body weight, estimated IBW, and BCS before the diet trial began (Table 1). Mean age in the groups were equivalent (data not shown). Hematologic, serum biochemical, and urine analyses performed throughout the study did not reveal any abnormalities or signs that hepatic lipidosis had developed (data not shown). No significant differences were evident among the diet groups in body weight, LBM, or percentage of total body weight that was LBM at baseline (day −28) or on day 1 (initiation of restricted energy allowance). For REE and RQ, 5 cats were censored from calculations involving day 84 data because those cats achieved targeted body weight between days 42 and 84 and were subsequently provided food allowances to maintain stable body weight.

Table 1—

Mean ± SD body weight, BCS, and IBW of colony-housed cats fed weight reduction diets supplemented with 0 (unsupplemented), 50 (CN-50), 100 (CN-100), or 150 (CN-150) μg of carnitine/g (n = 8 cats/group).

GroupBody weight (kg)BCSIBW (kg)
Unsupplemented5.24 ± 1.332.9 ± 0.74.8 ± 0.8
CN-505.51 ± 1.243.1 ± 0.84.1 ± 0.6
CN-1005.52 ± 1.663.0 ± 0.84.3 ± 0.8
CN-1505.63 ± 1.583.0 ± 0.94.4 ± 0.8

Body condition was scored on a 5-point scale (1 = thin, 2 = underweight, 3 = ideal weight, 4 = overweight, and 5 = obese).

Energy ingestion during unrestricted and restricted feeding periods—Results of analysis of the weight reduction diets were summarized (Table 2). No significant differences in daily food intake (mass or energy) were detected among groups during any phase of the study (Table 3). Although mean intake of the weight reduction diet per day on an unrestricted basis ranged from 53 to 62 g (254 to 299 kcal), mean weekly intake progressively increased from day −28 to 0. Mean intake from weeks 1 through 4 (during the day −28 to 0 interval) was 43, 58, 63, and 67 g of diet, respectively.

Table 2—

Mean results from content analyses (dry-matter basis) of weight reduction diets supplemented with 0 (unsupplemented), 50 (CN-50), 100 (CN-100), and 150 (CN-150) μg of carnitine/g.

NutrientUnsupplementedCN-50CN-100CN-150
Dry matter (%)92.193.292.091.8
Protein (%)33.834.534.134.2
Fat (%)11.111.110.910.5
Crude fiber (%)2.73.42.82.7
Total dietary fiber (%)8.612.69.79.8
Gross energy (kcal/kg)4,847.94,814.94,876.14,824.8
Carnitine (μg/g)31.776.0130.9175.8
Table 3—

Mean ± SD daily food intake (mass and energy) of the cats in Table 1 with unrestricted intake of an energy-dense diet (days −120 to −29), unrestricted intake of a weight reduction diet with or without carnitine (days −28 to 0), restricted intake of the same weight reduction diet (days 1 to 84), and unrestricted intake of an energy-dense diet (days 85 to 120).

Study periodUnsupplemented (n = 7)CN-50 (n = 8)CN-100 (n = 8)CN-150 (n = 8)
Mass (g)Energy (kcal)Mass (g)Energy (kcal)Mass (g)Energy (kcal)Mass (g)Energy (kcal)
Days −120 to −2992 ± 17475 ± 8580 ± 18412 ± 9394 ± 23486 ± 12092 ± 22473 ± 115
Days −28 to 055 ± 10263 ± 5053 ± 11254 ± 5561 ± 13292 ± 6262 ± 14299 ± 65
Days 1 to 4243 ± 7206 ± 3141 ± 7196 ± 3443 ± 6206 ± 2747 ± 11226 ± 52
Days 43 to 8443 ± 6206 ± 3042 ± 7200 ± 3341 ± 6199 ± 2748 ± 15230 ± 70
Days 85 to 12096 ± 17496 ± 8794 ± 18482 ± 9292 ± 20473 ± 102104 ± 25537 ± 130

See Table 2 for description of weight reduction diets.

Cats significantly (P < 0.05) reduced their mean daily energy intake when allowed to consume the weight reduction diet without restriction, compared with when the energy-dense diet was consumed without restriction, yet daily intake still exceeded the necessary energy allowance for weight reduction that was achieved with the restricted feeding protocol. Nevertheless, unrestricted feeding of the energy-restricted diet resulted in a decrease in daily energy intake by approximately 35% to 47%, compared with unrestricted feeding of the energy-dense diet.

As expected, rapid rebound weight gain occurred with unrestricted feeding of the energy-dense diet (days 85 to 120), with cats gaining a mean of 0.34 to 0.62 kg within the diet groups. Some cats gained as much as 1.2 kg within 35 days (Table 4). Percentage cumulative weight gain ranged from 6.5% to 12.5% of the day 84 body weight (with some cats gaining as much as 28% of body weight), obviating the benefit of the weight reduction protocol.

Table 4—

Median (range) changes in body measurements at various points for the cats in Table 1 initially fed a weight reduction diet provided without restriction for 28 days (days −28 to 0) and then fed the same diet provided on a restricted basis for 84 days.

GroupDays −28 to 0Days 1 to 42Days 43 to 84Days 85 to 120
Unrestricted weight reduction dietRestricted weight reduction dietRestricted weight reduction dietUnrestricted energy-dense diet
Cumulative reduction in body weight (kg)Cumulative reduction in body weight (%)Weight loss/wk (kg)Cumulative reduction in body weight (kg)Cumulative reduction in body weight (%)Weight loss/wk (kg)Cumulative reduction in body weight (kg)Cumulative reduction in body weight (%)Weight loss/wk (kg)Cumulative increase in body weight (kg)Cumulative increase in body weight (%)
Unsupplemented (n = 7)0.21 (−0.05 to 0.41)4.08 (−0.67 to 12.07)1.02 (−0.17 to 3.02)0.45* (0.29 to 0.94)10.02* (4.18 to 21.12)1.52 (0.67 to 2.91)0.72* (0.51 to 0.83)14.11* (7.35 to 18.65)1.46 (0.98 to 2.64)0.52 (0.36 to 0.76)12.46 (6.91 to 15.80)
CN-50 (n = 8)0.24 (0.02 to 0.71)5.44 (0.31 to 7.90)1.36 (0.08 to 1.97)0.47 (0.20 to 0.66)8.66 (5.56 to 13.48)1.33 (0.88 to 1.98)0.64 (0.11 to 1.16)13.32* (2.44 to 19.14)1.78* (1.02 to 2.66)0.42 (0.29 to 0.94)11.2 (6.34 to 20.58)
CN-100 (n = 8)0.26 (−0.14 to 0.44)4.23 (−2.33 to 7.34)1.06 (−0.58 to 1.50)0.55* (0.29 to 0.78)10.85 (6.08 to 15.32)1.63* (0.96 to 2.21)0.69* (0.33 to 1.31)13.02* (9.04 to 24.35)1.91* (0.73 to 3.87)0.62 (0.05 to 1.16)11.82 (1.37 to 27.75)
CN-150 (n = 8)0.23 (−0.08 to 0.43)3.56 (−1.59 to 8.01)0.89 (−0.40 to 2.00)0.54* (0.27 to 0.79)10.18* (8.60 to 17.71)1.54* (1.32 to 2.51)0.64* (0.32 to 1.11)13.3* (7.48 to 16.79)2.02* (0.73 to 2.69)0.34 (0.13 to 0.66)6.53 (3.19 from 19.13)

Weight gain with ad libitum feeding of the energy-dense diet on days 85 to 120 is represented as cumulative increase in body weight (kg) and as percentage increase of body weight on day 84.

Withinthe group, value differs significantly (P < 0.05) from the value for the unrestricted weight-reduction diet interval (days −28 to 0).

As expected during unrestricted feeding of the weight reduction diet (days −28 to 0), daily energy ingestion significantly (P < 0.001 for all comparisons) exceeded REE estimated by indirect calorimetry in all groups. Mean daily food consumption was 57 ± 13.3 g, ranging from 34 to 87 g for individual cats (Table 3). Mean daily energy ingestion was 274 ± 63.7 kcal, ranging from 163 to 416 kcal for individual cats. Mean REE (mean of 3 measurements on different days in each cat) during unrestricted feeding of the energy-restricted diet (days −28 to 0) was 183 ± 54.4 kcal. Ingestion of energy during unrestricted feeding of the energy-dense diets during the fattening phase of the study significantly (P = 0.003) exceeded the DEE estimated with the doubly labeled water method, as expected during weight gain. Ingestion of energy during restricted feeding of the weight reduction diets during the weight reduction phase of the study was significantly (P = 0.004) less than the DEE estimated with the doubly labeled water method as expected during weight loss (Table 5).

Table 5—

Mean ± SD energy measurements at baseline (day −28) and days 42 and 84 of meal-limited weight reduction in 12 of the cats in Table 1 (3 cats/diet group).

Diet groupDay −28Day 42Day 84
DEE (kcal/d)DEE/body weight (kcal/d/kg)DEE/LBM (kcal/d/kg)REE (percentage of DEE)Ingested energy (kcal/day)DEE (kcal/d)DEE/body weight (kcal/d/kg)DEE/LBM (kcal/d/kg)REE (percentage of DEE)Ingested energy (kcal/day)DEE (kcal/d)DEE/body weight (kcal/d/kg)DEE/LBM (kcal/d/kg)REE (percentage of DEE)Ingested energy (kcal/day)
Unsupplemented303 ± 7548 ± 1775 ± 1557 ± 12520 ± 212202 ± 2134 ± 859 ± 1373 ± 11197 ± 37275 ± 8154 ± 385 ± 762 ± 23208 ± 30
CN-50319 ± 13852 ± 1776 ± 2576 ± 9495 ± 75275 ± 12543 ± 963 ± 461 ± 14204 ± 28256 ± 6255 ± 2482 ± 3469 ± 13215 ± 39
CN-100355 ± 2554 ± 786 ± 1653 ± 7520 ± 83274 ± 9346 ± 1296 ± 1262 ± 14204 ± 35276 ± 8061 ± 2394 ± 4359 ± 29205 ± 29
CN-150325 ± 2551 ± 1491 ± 2359 ± 20475 ± 117239 ± 8144 ± 1773 ± 1965 ± 28227 ± 3202 ± 2241 ± 1978 ± 2669 ± 19201 ± 40
All Cats325 ± 2551 ± 1383 ± 1961 ± 14497 ± 117248 ± 82*42 ± 1173 ± 1965 ± 16191 ± 27252 ± 64*53 ± 1885 ± 2765 ± 19210 ± 31

Value is significantly (P = 0.02) different from that at baseline (day −28)

Daily energy expenditure was determined with doubly labeled water. Resting energy expenditure was measured by means of indirect calorimetry.

Rate of weight loss—Cats that consumed diets with 0, 100, and 150 μg of carnitine/g lost a significantly (P < 0.05) greater amount of weight during days 1 to 42 and days 43 to 84 of restricted feeding of the weight reduction diet, compared with during the 28 days of unrestricted feeding of the same diet (Table 4). Percentage cumulative weight loss in all cats was significantly (P < 0.05) faster during days 43 to 84, compared with when cats were allowed unrestricted feeding of the weight reduction diet. Weight loss per week was significantly (P < 0.05) greater with restricted feeding of the weight reduction diet (days 1 to 42 and days 43 to 84), compared with weight loss per week during 28 days of unrestricted feeding of the same diet (days −28 to 0) only in cats consuming diets supplemented with carnitine. The greatest weight loss in these cats occurred during days 43 to 84.

Differences in cumulative weight loss, percentage cumulative weight loss, or rate of weight loss per week were not significant (P > 0.40 for all comparisons) among diet groups during weight reduction periods. Significant (P < 0.05) cumulative weight loss occurred in all cats by days 42 and 84, compared with body weight during unrestricted feeding of the energy-restricted diet, with exception of cats in the CN-50 group. The median rate of weekly weight loss was ≥ 1.3% during restricted feeding of the energy-restricted diet.

Thirteen of 31 (42%) cats achieved target IBW by day 84 (2/7 cats in the unsupplemented diet group, 2/8 cats in the CN-50 group, 5/8 cats in the CN-100 group, and 4/8 cats in the CN-150 group; differences among groups were not significant). Five cats fed carnitine-supplemented diets achieved their target IBW before day 84 (1 cat in the CN-50 group, 1 in the CN-100 group, and 3 in the CN-150 group) and had energy intake adjusted to maintain body weight thereafter. Adherence to study protocol resulted in censoring of indirect calorimetry data from these cats on day 84 because they were not continuing to undergo weight loss but were instead fed to maintain optimal body condition.

Change in body composition—A significant (P ≤ 0.04) increase in percentage LBM and decrease in FM as a percentage of total body weight was evident in the CN-50 and CN-150 groups by day 42 and in all cats by day 84 (Table 6).

Table 6—

Median (range) body measurements at various points in the cats in Table 1 initially fed a weight reduction diet provided without restriction for 28 days (days −28 to 0) and then fed the same diet provided on a restricted basis for 84 days.

Diet groupDay −28Day 42Day 84
Body weight (kg)LBM (kg)LBM (%)FM (%)Body weight (kg)LBM (kg)LBM (%)FM (%)Body weight (kg)LBM (kg)LBM (%)FM (%)
Unsupplemented (n = 7)6.01 (4.72–7.46)2.82 (2.3–4.56)48.73 (39.53–61.79)51.27 (38.21–60.47)5.20* (3.97–6.86)2.84 (2.20–4.32)55.42 (50.53–64.44)44.58 (35.56–49.47)4.37* (3.36–6.35)3.03 (2.10–3.45)68.81* (47.65–77.41)31.19* (22.59–52.35)
CN-50 (n = 8)5.41 (4.07–8.71)2.60 (2.18–4.96)52.81 (46.68–64.41)47.20 (35.59–53.32)4.62 (3.61–7.73)2.69 (2.10–6.13)59.89* (56.36–79.30)40.11* (20.70–43.64)4.24 (3.00–6.51)2.86 (2.17–4.68)72.33* (55.86–79.90)44.127.68* (20.10–44.14)
CN-100 (n = 8)5.81 (4.17–7.62)2.90 (2.23–4.42)52.14 (41.24–59.34)47.86 (40.66–58.76)4.91 (3.68–6.95)2.80 (2.26–3.43)55.63 (41.24–59.34)44.37 (30.66–50.65)4.1 1 (3.31–5.71)3.04 (2.30–3.57)69.94* (62.22–79.15)30.07* (20.85–37.78)
CN-150 (n = 8)5.37 (3.45–7.86)3.01 (1.26–4.07)52.53 (36.52–67.28)47.48 (32.72–63.48)4.66 (3.12–7.02)2.86 (1.43–3.62)60.14* (45.51–73.42)39.87* (26.58–54.49)3.94 (2.68–5.79)3.05 (1.38–4.28)74.85* (51.49–81.19)25.16* (18.81–48.51)

Percentage values for LBM and FM represent percentage of body weight.

Value is significantly (P < 0.04) different within groups relative to the baseline (day −28) value.

Value is significantly (P < 0.001) different within groups relative to the baseline value.

Indirect calorimetry—No significant differences were detected among groups with respect to REE values at baseline (Figure 1). Resting energy expenditure (normalized for total body weight) was significantly (P ≤ 0.001) lower in the unsupplemented diet group on days 7, 42, and 84, compared with baseline. The REE per LBM (kg) was significantly (P < 0.001) higher in cats that consumed carnitine-supplemented diets than in the control group on days 42 and 84, suggesting that carnitine supplementation favorably influenced REE.

Figure 1—
Figure 1—

Mean ± SD resting energy expenditure determined by open-flow indirect calorimetry normalized to total body weight (A) and LBM (B) at various points in 31 overweight cats that were fed a weight reduction diet containing 0 (CN-0), 50 (CN-50), 100 (CN-100), or 150 (CN-150) μg of carnitine/g. From days −28 to 0, cats were allowed unrestricted intake of the weight reduction diet. From days 1 to 84, intake of the same weight reduction diet was restricted. From days 84 to 120, cats were fed an unrestricted amount of an energy-dense diet. *Indicated values are significantly (P < 0.001) different from baseline (day −28; A) or among cats fed unsupplemented and carnitine-supplemented diets (B).

Citation: American Journal of Veterinary Research 73, 7; 10.2460/ajvr.73.7.1002

The RQ was not significantly different among the diet groups at baseline (day −28; Figure 2). However, it was significantly (P < 0.01) higher at baseline, day 112, and day 119, compared with on days 42 and 84 in all cats that consumed a carnitine-supplemented diet combined. That observation corresponded to findings during unrestricted access to the energy-dense diet fed during baseline and days 112 and 119, compared with the restricted feeding of weight reduction diets (days 1 to 84), despite the higher fat content of the energy-dense diet.

Figure 2—
Figure 2—

Mean ± SD RQ determined by indirect calorimetry in the cats in Figure 1. A—On day 84, 5 cats were censored because they had already achieved an IBW (were no longer on energy restriction). B—Results for all cats fed carnitine-supplemented diets were combined. *Indicated values are significantly different from baseline (day −28) within a diet group (A; P < 0.05) or among cats fed carnitine-supplemented or unsupplemented diets (B; P < 0.01). See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 73, 7; 10.2460/ajvr.73.7.1002

The mean baseline RQ in cats that received carnitine (n = 24) was 0.83 ± 0.04, with similar values on days 112 and 119 (0.82 ± 0.04). However, during restricted feeding of the weight reduction diets, the mean RQ in cats fed the carnitine-supplemented diets was ≤ 0.80 ± 0.04 (0.78 ± 0.03 on day 7, 0.80 ± 0.03 on day 14, 0.79 ± 0.03 on day 28, 0.78 ± 0.02 on day 42, and 0.78 ± 0.03 on day 84; Figure 2).

Cats that received no carnitine had mean RQs at baseline, day 112, and day 119 of 0.82 ± 0.03, with RQs during restricted feeding of the weight reduction diet of 0.81 ± 0.04 on day 7, 0.82 ± 0.03 on day 14, 0.83 ± 0.04 on day 28, 0.80 ± 0.04 on day 42, and 0.82 ± 0.02 on day 84. Respiratory quotients within each group fed a carnitine-supplemented diet (CN-50, CN-100, and CN-150) were significantly (P < 0.05) higher at baseline, compared with at day 42. However, there were differences among these groups in mean RQ on other days; a significantly (P < 0.05) lower RQ on day 28 was evident in the CN-100 and CN-150 groups, and a significantly (P < 0.05) lower RQ was found on day 84 in the CN-150 group.

The RQ in all cats fed carnitine-supplemented diets combined was significantly (P < 0.01) lower on days 7 through 84, compared with the RQ of the control group (Figure 2). Considering that baseline RQ reflected ingestion of a comparatively high-fat, energy-dense diet (FQ, 0.80 and 0.82), the lower RQs in carnitine-supplemented cats during meal-restricted weight reduction likely reflected increased fatty acid oxidation, possibly facilitated by carnitine. The finding that the RQ had significantly decreased in cats that consumed carnitine-supplemented diets was further substantiated by the RQ increase in each carnitine-supplemented group upon restoration of unrestricted feeding of the higher fat energy-dense diet (which was not carnitine supplemented) from days 112 through 119.

Doubly labeled water—Daily energy expenditure ranged from 173 to 475 kcal/d in individual cats, with percentage of DEE represented by REE ranging between 32% to 94% (mean range, 61% to 65%; Table 5). At baseline (day −28), cats consumed energy in excess of DEE (P = 0.003) consistent with weight gain, whereas on days 42 and 84, DEE was greater (P = 0.004) than consumed energy as expected for weight loss. The small number of cats in which doubly labeled water evaluation was conducted (3/group) limited statistical power for detecting changes consistent with adaptive thermogenesis over the course of the weight reduction protocol.

Palmitate oxidation—No significant differences were evident among diet groups in rates of palmitate flux or oxidation or estimated fatty acid appearance (oxidation) at baseline (Figures 3 and 4). Rates of palmitate flux and estimated FFA oxidation were significantly higher on day 42 than at baseline in the unsupplemented, CN-100, and CN-150 groups, as would be expected with weight loss. The fold increase in palmitate flux rate, which was examined to determine change in individual cat palmitate oxidation rate, was significantly (P = 0.03) higher only in the CN-150 group, compared with the rate in the control group. The onerous nature of the palmitate perfusion procedure precluded evaluations on more days than baseline and day 42.

Figure 3—
Figure 3—

Box-and-whisker plots of palmitate flux rate (A) and fold increase in palmitate flux rate (B) determined from 1-13C-palmitate steady-state infusion in the cats in Figure 1. The short thick horizontal bar within each box represents the mean, and the longer, thinner horizontal line separating shaded portions of the box represents the median. The boxed area encloses the middle half of data values (ie, the 25th to 75th percentile), with the shaded portion of each box representing values above and below the median. Whiskers represent the range. Cats were evaluated after 12 hours of food withholding. An energy-dense fattening diet was fed at baseline, whereas the unsupplemented weight reduction diet and the carnitine-supplemented weight reduction diets were fed during energy-restricted feeding. *Indicated values are significantly different from baseline (day −28) within a diet group (A; P ≤ 0.05) or between the unsupplemented and CN-150 groups (B; P = 0.03). See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 73, 7; 10.2460/ajvr.73.7.1002

Figure 4—
Figure 4—

Box-and-whisker plots of estimated FFA oxidation rate determined by 1-13C-palmitate steady-state infusion in the cats in Figure 1. *Indicated values differ significantly (P ≤ 0.05) within diet groups between days −28 and 42. See Figures 1 and 3 for remainder of key.

Citation: American Journal of Veterinary Research 73, 7; 10.2460/ajvr.73.7.1002

Nitrogen intake and urinary nitrogen elimination—Amounts of nitrogen intake and urinary nitrogen elimination were significantly (P < 0.003) lower in each diet group during restricted feeding of the weight reduction diet, compared with at baseline, but there were no significant differences among the groups (Figure 5).

Figure 5—
Figure 5—

Box-and-whisker plots of daily nitrogen intake determined from food intake (A) and urinary nitrogen elimination by Kjeldahl analysis (B) for the cats in Figure 1. *Indicated values differ significantly (P < 0.003) from baseline within diet groups. See Figures 1 and 3 for remainder of key.

Citation: American Journal of Veterinary Research 73, 7; 10.2460/ajvr.73.7.1002

Stoichiometric calculation of substrate oxidation—Stoichiometric calculation of substrate oxidation was limited to days in which LBM had been determined. With the exception of the CN-50 group, there were no differences in estimated substrate oxidation between carnitine-supplemented and unsupplemented diet groups at baseline (Figure 6). Median estimated fat oxidation rate at baseline in the CN-50 group was 73.1 μmol/kg of LBM/min (range, 52.7 to 114.7 μmol/kg of LBM/min) and in the unsupplemented diet group was 51.1 μmol/kg of LBM/min (range, 38.5 to 69.8 μmol/kg of LBM/min; P = 0.02). Each group fed carnitine-supplemented food developed a significant (P ≤ 0.02) increase in estimated fat oxidation rate on day 42, compared with the rate in the control group. However, the percentage increase in estimated fat oxidation in the control and CN-50 groups were similar (with median increases of 12.9% and 12.0%, respectively). Notably, cats in the CN-100 and CN-150 groups had significantly (P ≤ 0.001) higher rates of fat oxidation, with median increases of 62.6% and 62.5%, respectively.

Figure 6—
Figure 6—

Box-and-whisker plots of stoichiometrically calculated rate of fat oxidation (A) and carbohydrate oxidation (B) in the cats in Figure 1. *Indicated values differ significantly between unsupplemented and supplemented diet groups on the indicated day (P ≤ 0.02; A) or within the unsupplemented diet group between days. See Figures 1 and 3for remainder of key.

Citation: American Journal of Veterinary Research 73, 7; 10.2460/ajvr.73.7.1002

No significant differences in estimated amount of carbohydrate oxidation were evident in cats fed the carnitine-supplemented diets, whereas a significant (P = 0.04) increase in carbohydrate oxidation of 63.7% was estimated in the control group on day 42. This finding suggested that the group fed the unsupplemented diet maintained a higher rate of carbohydrate use, compared with the carnitine-supplemented groups.

Discussion

The present study involved several techniques to determine whether dietary l-carnitine supplementation during weight reduction alters fatty acid oxidation rate, REE, or LBM in overweight cats. Stable isotope methods estimating DEE and LBM were combined with indirect calorimetry and stable isotope labeled palmitate infusion to investigate metabolic responses. Few studies30,31 have been conducted to investigate the influence of l-carnitine supplementation on fatty acid oxidation, and none have simultaneously measured energy expenditure and LBM or involved use of customized palmitate infusions as was done in the present study.

Baseline measurements in the absence of supplemental l-carnitine were conducted before weight loss was initiated in the present study, and all variables for cats that received supplemental l-carnitine were compared over the study period with baseline measurements and measurements from a control group that received no supplemental carnitine. It remains debatable which of these comparisons is more relevant in the small sample used (8 cats/group), which was required because of the cost and complexity of several assessments performed. Also, because metabolic findings in the present study were compared with those obtained during feeding of a high-fat, energy-dense diet at baseline, it is probable that more extreme changes in fat oxidation and RQ would occur in similar studies of cats with stable body weights at baseline that receive a lower-fat, less energy-dense diet.

Unrestricted feeding of the weight reduction diets achieved a weekly rate of weight loss ranging from 0.9% to 1.4% in the study reported here. Compared with during unrestricted access to the energy-dense diet, energy intake decreased by approximately 60% with unrestricted access to the weight reduction diet. Initial diet acceptance with the abrupt dietary change was variable among cats, with some hesitant to eat the new diet during the first 2 to 7 days (cats ate smaller amounts, but none failed to eat altogether). In most cats, quantitative consumption progressively increased over the 4 weeks of unrestricted feeding. In a few cats, there was little or no change in the amount of food ingested during the first week of diet introduction.

The first weeks after diet introduction are believed to represent the period of highest risk for development of hepatic lipidosis in pet cats that are reluctant to consume the newly introduced food. To minimize this risk, gradual introduction of the new food by mixing it with the current food is generally recommended. Although the diet change was abruptly introduced in the present study, there was no evidence of an adverse effect on hematologic or biochemical variables secondary to reduced energy intake. Responses of the study cats to unrestricted feeding of the weight reduction diet suggested that this strategy may be used for initiating, sustaining, or transitioning cats with poor intake control (glutonous feeding behavior) to a more rigorous weight control program. As expected, unrestricted diet consumption still exceeded the daily energy allowances calculated to achieve weight reduction during the period the diet was fed.

Mean weekly weight loss exceeded 1.3% among all cats with restricted feeding of the weight reduction diet. There were no significant differences in rate of weight loss or total achieved weight loss between carnitine-supplemented and unsupplemented diet groups during feeding of the weight reduction diet. A significantly greater percentage weekly weight loss was evident during restricted feeding of the CN-100 and CN-150 diets (days 42 and 84; P ≤ 0.04) and in cats fed the CN-50 diet (day 84; P < 0.05) but at no period in the unsupplemented diet group relative to weight loss during unrestricted feeding. However, there were no significant differences between cats that did or did not receive the carnitine supplement. Therefore, collective findings regarding absolute weight reduction do not suggest an obvious benefit of dietary carnitine supplementation.

A significant (P < 0.05) increase in the percentage of LBM and decrease in FM as a percentage of body weight occurred as early as day 42 in cats that consumed 2 of the carnitine-supplemented diets but also in all cats by day 84. The inconsistent response to dietary carnitine supplementation and the lack of a significant influence on weight loss and body condition change (increased LBM), compared with findings in the control group, were at odds with the indirect calorimetry findings of a carnitine-enhanced rate of fatty acid oxidation. These disparate results might have been a result of limited statistical power (too few cats), individual variation among cats, or a differing influence of carnitine-supplements given in amounts exceeding physiologic requirements.

By day 84 of restricted feeding of weight reduction diets, 13 of 31 (42%) cats achieved an estimated IBW (27% of cats in the unsupplemented and CN-50 groups and 56% of cats in the CN-100 and CN-150 groups). Because there were no differences in body composition (BCS or LBM) among groups at study initiation, this finding might suggest, but does not definitively establish, that l-carnitine supplementation at 100 and 150 μg/g provided some advantage during weight reduction. Although interpretation of change in gross body weight is complicated by the higher density and weight of muscle than in fat, change in LBM at endpoint of the weight reduction protocol was not different between the carnitine-supplemented and unsupplemented diet groups.

Because LBM was determined by isotope dilution, it was only quantified on 3 days (baseline and days 42 and 84) so that LBM could be used for REE normalization. A significant decrease in REE per LBM was observed only in cats fed the unsupplemented diet on days 42 and 84. Whether that decrease reflected an influence of carnitine supplementation or individual variation remains to be substantiated through additional studies. An influence of adaptive thermogenesis in lowering REE during and after weight loss is a possible explanation, but this phenomenon remains controversial. Investigators in some studies32–35 found a modest decrease in REE (approx 10% to 15%) contributing to a lowered DEE, beyond the influences of altered body composition (decreased LBM metabolism). Still other investigators have reported no detectable change in REE or DEE accompanying or after weight loss.36–39

Carnitine ingestion in the present study might have protected against adaptive thermogenesis as a means of sustaining metabolic rate. The decrease in REE in cats fed the unsupplemented diet ranged from 19% to 25%, whereas cats fed the carnitine-supplemented diets had decreases ranging from 1% to 12%. Although REE was measured repeatedly in all cats throughout the study, DEE was measured in only 3 cats/group during 3 study periods. Consequently, the power to identify significant change in DEE among the diet groups, if it indeed existed, was low.

Although metabolic effects of carnitine ingestion were variable and mild, cats fed the l-carnitine-supplemented diets sustained their REE per LBM (kg), suggesting protection against adaptive thermogenesis during weight loss. This interpretation is supported by the finding of a significant reduction in RQ on day 42 in cats fed carnitine-supplemented diets and the consistently observed lower RQ in the combined analysis of all such cats relative to cats fed the unsupplemented diet throughout the restricted feeding period. Furthermore, upon reinstitution of unrestricted feeding of the energy-dense diet (days 85 to 120), the REE increased in the control group, suggesting the observed difference from the other groups during the restricted-feeding phase of the study might have indeed been attributable to carnitine. The RQ significantly increased in all cats during reinstitution of unrestricted feeding of the energy-dense diet, despite the lower FQ relative to the weight reduction diet. This change in RQ consistent with a lower rate of fatty acid oxidation might simply have reflected cessation of weight loss or, alternatively, withdrawal of carnitine supplements.

Food restriction resulted in a significant decrease in urinary nitrogen elimination, as expected, and there was no apparent influence of l-carnitine supplementation on nitrogen conservation. However, protein turnover rate was not specifically investigated. Weight reduction was associated with an increased percentage of palmitate oxidation and fatty acid flux rate estimated by 1-13C-palmitate infusions in each group, although values differed widely among individual cats. Although the fold increase in palmitate flux rate significantly (P = 0.03) increased only in cats in the CN-150 group relative to those fed the unsupplemented diet, a significant (P ≤ 0.05) increase in palmitate flux rate commensurate with enhanced fatty acid use during energy restriction and weight loss occurred in both supplemented and unsupplemented diet groups when values on day 42 were compared with baseline.

Stoichiometric estimation of substrate oxidation rates revealed greater use of fat and lesser use of carbohydrate for energy in the CN-100 and CN-150 groups (baseline high-energy, high-fat diet, compared with the weight reduction diet) than in the other groups, achieving a > 62% increase in fatty acid use. Conversely, cats in the unsupplemented diet group had a significantly (P = 0.04) greater rate of carbohydrate oxidation. These findings also suggested that the diets supplemented with greater amounts of carnitine augment fatty acid oxidation.

Several measured variables reflecting fatty acid oxidation rate during weight loss in overweight cats were investigated in the present study. Initial estimated rates of FFA turnover in cats at baseline were approximately 2- to 3-fold as great as those in humans (7.8 ± 0.7 μmol/kg/min to 10.5 ± 1.1 μmol/kg/min).40,41 A higher turnover of FFA in cats than in humans can be explained by the obligate carnivore status of feline metabolism, in which higher intake of dietary fat is typical. Although FFA appearance rates were in agreement with values previously reported for cats,42,43 baseline values reflected consumption of the high-fat energy-dense diet as well as 12 hours of food restriction.

Indirect calorimetry findings suggested that carnitine might protect against a decrease in metabolic rate in the presence of weight reduction and enhance in vivo fatty acid oxidation in overweight cats undergoing rapid weight reduction. However, the 13C-palmitate infusion tests were unable to clearly demonstrate a similar carnitine effect, possibly because of the wide interindividual variability in palmitate oxidation. A previous study44 of overweight cats undergoing weight loss and receiving supplemental dietary carnitine revealed that the addition of carnitine appeared to enhance weight loss; however, carnitine was added to the diet at supraphysiologic concentrations (250 mg/cat/d), and LBM was not estimated in that study.

The final phase of the present study demonstrated the rapid rebound weight gain that may occur after substantial weight loss, upon reinstitution of unrestricted feeding of an energy-dense diet. Within 35 days after reinstitution, some cats regained all weight lost during the 16 weeks of energy restriction; mean weight gain during that period was 12% (range, 1.3% to 28%). Findings during the last phase of study emulated the rapid rebound weight gain observed clinically in pet cats after successful weight reduction when owners fail to permanently modify feeding practices.

Results of the present study suggested that dietary supplementation with l-carnitine yields a metabolic effect (on the basis of indirect calorimetry parameters and stoichiometric calculations) that may be advantageous in overweight cats undergoing rapid weight reduction, complementing previous observations.43–45 The stoichiometric calculations for all carnitine-supplemented diets and increase in palmitate flux rate (fold increase) with high-dose carnitine supplementation were concordant with findings in 2 prior studies44,45 (one supporting enhanced fatty acid oxidation [acetyl-carnitine as a surrogate marker] and the other showing protection against ketone formation).

The mechanisms by which metabolic change might happen remain undetermined, but carnitine supplementation has been shown to augment the activity of feline hepatic carnitine-palmitoyltransferase kinase, which is a rate-limiting enzyme of acyl-carnitine transport at the mitochondrial membrane.46 An increase in carnitine-palmitoyltransferase kinase activity might facilitate carnitine-mediated fatty acid oxidation. Because both fatty acid oxidation and egress of acetyl-carnitine from the liver might be enhanced by maximizing transporter activity, increasing acyl-carnitine transporter activity at the mitochondrial interface might have mechanistic relevance in anorexic overweight cats at risk for hepatic lipidosis. This might explain clinical observations that dietary supplementation with l-carnitine improves the probability of survival time in cats severely affected with hepatic lipidosis when included as a component of nutritional support.47

ABBREVIATIONS

APE

Atom percentage excess

BCS

Body condition score

CoA

Coenzyme A

DEE

Daily energy expenditure

FFA

Free fatty acid

FM

Fat mass

FQ

Food quotient

IBW

Ideal body weight

LBM

Lean body mass

REE

Resting energy expenditure

RQ

Respiratory quotient

TBW

Total body water

co2

Carbon dioxide production

o2

Oxygen consumption

a.

Mettler PC2200 DeltaRange analytical balance, Mettler-Toledo Inc, Columbus, Ohio.

b.

Health-o-Meter Pediatric Scale, 551k/552k, Pelstar LLC, Alsip, Ill.

c.

Sierra Series 840 mass flow controllers, Sierra Instruments Inc, Monterey, Calif.

d.

Drierite, anhydrous CaSO4, WA Hammond Drierite Co, Xenia, Ohio.

e.

Ascarite II, Thomas Scientific, Swedesboro, NJ.

f.

Sable Systems CA-1 Carbon Dioxide Analyzer, Sable Systems, Las Vegas, Nev.

g.

Sable Systems FC-1 Oxygen Analyzer, Sable Systems, Las Vegas, Nev.

h.

Datacam V, Analytical Software, Sable Systems, Las Vegas, Nev.

i.

Deuterium oxide, 99.98%, Cambridge Isotope Laboratories, Andover, Mass.

j.

Cornell University Isotope Laboratory, Thermo Finnegan Delta Plus Isotope Ratio Mass Spectrometer, Brennan, Germany.

k.

18O Water, 10.05 APE, Europa Scientific, Europa House, Cheshire, England.

l.

Potassium palmitate-1 13C, catalog No. CLM-1889, Cambridge Isotope Laboratories Inc, Andover, Mass.

m.

Sonicator Cell Disruptor, model No. W 185 F, Heat Systems-Ultrasonics Inc, Plainview, NY.

n.

Gelman Sciences Supor Acrodisc 25, sterile single-use nonpyrogenic with 75-psi pressure limitation, Pall Gelman, Sigma-Aldrich, St Louis, Mo.

o.

Millipore sterile single use nonpyrogenic syringe filter, 0.22μM, Millipore Corp, Danvers, Mass.

p.

NaH13CO3,13C = 99%, catalog No. CLM-1889, Cambridge Isotope Laboratories Inc, Andover, Mass.

q.

1.5-L Rebreathing Bag, Rusch, Teleflex Medical, Research Triangle Park, NC.

r.

Labco Exetainer System-13C and Gas Testing Vials 10 mL Gas ×100, LabCo Ltd Brow Works, High Wycombe, Buckinghamshire, England.

s.

Metabolic Solutions, Stable Isotope Analyses, Nashua, NH.

t.

Statistix 9, Analytical Software, Tallahassee, Fla.

Appendix 1

Mean nutrient contents of energy-dense fattening diets (1 and 2) fed without restriction before and after the study and the weight reduction diet.

NutrientDiet 1Diet 2Weight reduction diet
Protein (%)37.743.034.1
Fat (%)23.728.810.9
Carbohydrate (%)29.718.637.7
Crude fiber (%)2.11.42.9
Gross energy (kcal/g)5.075.264.84
Calculated FQ0.820.800.88

Gross energy is reported on a dry-matter basis.

Appendix 2

Composition of a diet used for weight reduction in cats.

IngredientPercentage of total content
Chicken by-product meal31.3
Corn29.4
Rice20
Beet pulp6
Fish meal4
Chicken digest2.5
Egg2.5
Poultry fat1.3
Minerals1.7
Yeast0.5
dl-methionine0.5
Vitamins0.3

A supplement containing 50% l-carnitine was added to achieve various concentrations of carnitine in the finished product.

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Contributor Notes

Supported by The Iams Co.

Address correspondence to Dr. Center (sac6@cornell.edu).