Nutrition Forum Focus on Felines St

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Nutrition Forum Focus on Felines St. Louis, Missouri • September 20–22, 2007

A Supplement to Compendium: Continuing Education for Veterinarians™ Vol. 30, No. 3(A), March 2008 2008 Purina FRONT MATTER.qxp:Layout 1 2/26/08 4:17 PM Page 6 2008 Purina FRONT MATTER.qxp:Layout 1 3/3/08 10:41 AM Page 1

Nutrition Forum Focus on Felines St. Louis, Missouri • September 20–22, 2007

A Supplement to Compendium: Continuing Education for Veterinarians™ Vol. 30, No. 3(A), March 2008 2008 Purina FRONT MATTER.qxp:Layout 1 2/26/08 4:17 PM Page 2

Sponsored by an educational grant from Nestlé Purina PetCare Company.

This information has not been peer reviewed and does not necessarily reflect the opinions of, nor constitute or imply endorsement or recommendation by, the Publisher, Editorial Board, or Nestlé Purina PetCare Company. Neither the Publisher nor Nestlé Purina PetCare Company is responsible for any data, opinions, or statements provided herein.

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CONTENTS

Preface and Dedication...... 7 Dottie Laflamme

SCIENTIFIC PROGRAM: FOCUS ON FELINES

Some Highlights in Elucidating the Peculiar Nutritional Needs of Cats...... 9 Quinton R. Rogers and James G. Morris

Advances in Knowledge about Feline Metabolism...... 17 Robert C. Backus

Trace Mineral Requirements in Cats: Challenging How We Define “Requirements” ...... 25 Andrea J. Fascetti

Is My Cat Fat?...... 27 Denise A. Elliott

Adipokines and Their Importance in Obese Cats ...... 30 M. Anne Hickman

New Technologies for Pharmaceutical and Nutrition Research ...... 35 Marnie L. MacDonald

Is the Aging Feline Kidney a Mortality Antagonist?...... 38 Dennis F. Lawler

What Is Different about Chronic Kidney Disease in Cats?...... 41 David J. Polzin

Feline Urolithiasis: Understanding the Shift in Urolith Type...... 44 Jody P. Lulich and Carl A. Osborne

In Search of the Origins of Feline Hyperthyroidism ...... 47 Deborah S. Greco

Measures of Disease Activity in Feline Inflammatory Bowel Disease...... 51 Albert E. Jergens

RESEARCH ABSTRACTS: ORAL PRESENTATIONS

Dietary Variables That Predict Glycemic Responses to Whole Foods in Cats...... 57 N.J. Cave, J.A. Monro, and J.P. Bridges

Spaying Affects Blood Metabolites and Adipose Tissue Gene Expression in Cats ...... 58 K.R. Belsito, B.M. Vester, T. Keel, T.K. Graves, and K.S. Swanson

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CONTENTS

Effects of Spaying on Food Intake, Weight Gain, Body Condition Score, Activity, and Body Composition in Cats Fed a High-Protein versus Moderate-Protein Diet ...... 59 B.M. Vester, K.J. Liu, T. Keel, T.K. Graves, and K.S. Swanson

Higher Protein Consumption during Weight Loss Allows Higher Caloric Intake for Maintenance of Body Weight in Cats...... 60 R.S. Vaconcellos, N.C. Borges, K.N.V. Gonçalves, F.J.A. de Paula, E.B. Malheiros, R.S. Bazolli, and A.C. Carciofi

Effect of a Low-Protein Diet on Gut Morphology in Cats...... 61 D.G. Thomas, C.E. Ugarte, K.J. Rutherfurd-Markwick, and W.H. Hendriks

Variations in Dietary Fat Affect Lipid Metabolism in Domestic Cats ...... 62 M.K. McClure, R.J. Angell, K.E. Bigley, K. Fennell, and J.E. Bauer

Impact of Dietary Trans-Fatty Acid on Serum Insulin and Glucose Concentrations in Cats ...63 P.A. Schenck and S.K. Abood

Sequencing and Characterization of Feline Pancreatic Glucokinase cDNA ...... 64 S. Lindbloom, M. LeCluyse, E. Hiskett, and T. Schermerhorn

Effects of Epigallocatechin Gallate Singly and in Combination with Lactoferrin on Oral Health in Cats...... 65 S. Krammer-Lukas, K. Cramer, U. Wehr, S. Gorissen, and K. Elsbett

RESEARCH ABSTRACTS: POSTER PRESENTATIONS

Effect of Isoflavones, Conjugated Linoleic Acid, and L-Carnitine on Weight Loss and Oxidative Stress in Overweight Dogs ...... 69 Y. Pan, I. Tavazzi, J.-M. Oberson, L.B. Fay, and W. Kerr

Postfeeding Satiety and Weight Loss of Dogs Fed a Vegetable-Based Fiber Supplement...... 70 Y. Mitsuhashi, K. Bigley, and J.E. Bauer

Body Condition and scFOS Supplementation Influence Adipose Tissue mRNA Abundance ....71 K.R. Belsito, B.M. Vester, F. Respondek, M. Diez, and K.S. Swanson

All-Trans-Astaxanthin Does Not Protect Canine Osteosarcoma Cells from Chemotherapeutic or Radiation-Induced Cell Death...... 72 J.J. Wakshlag, C.B. Balkman, A.M. Struble, S.K. Morgan, and M. Zgola

Absorption Trial of Ginkgo Biloba Extract in Cats...... 73 A. Pasquini, G. Cardini, C. Gardana, P. Simonetti, G. Giuliani, G. Re, and G. Lubas

High-Protein Diet Impacts Fecal Microbial Populations in Growing Kittens ...... 74 B.L. Dalsing, B.M. Vester, C.J. Apanavicius, D.C. Lubbs, and K.S. Swanson

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CONTENTS

Impact of Sampling Interval on the Variability of Activity Counts Recorded from the Actical Activity Monitor Worn by Pet Dogs ...... 75 C. Dow, K.E. Michel, and D.C. Brown

Exercise Heart Rate and Blood Lactate Responses as Indicators of Aerobic Capacity in Dogs .....76 J.C. Bouthegourd and A.J. Reynolds

Effect of Different Dietary Protein Sources and Carbohydrate Content on Canine Behavior.....77 O. Pellegrini, L. Casini, V. Mariotti, G. Lubas, and D. Gatta

Evaluation of Polymeric Diets Delivered Directly into the Small Intestine through Surgically Placed Jejunostomy Tubes...... 78 S.A. Bone, F.A. Mann, R.C. Backus, and E. Kelmer

Seasonal Differences in Hair Growth between Long-Haired and Short-Haired Cats ...... 79 M. Hekman, D.G. Thomas, S.H. Moon, and W.H. Hendriks

Heritability of Hematology and Clinical Chemistry Variables in Domestic Cats: What Are the Early Implications? ...... 80 D.F. Lawler, K. Chase, R. Teckenbrock, and K.G. Lark

Thyroid Hormone Concentrations and Prevalence of Thyroid Pathology in Geriatric Cats.....81 C. Cupp and W. Kerr

Age-Related Changes in Immune Function in Cats...... 82 K.J. Rutherfurd-Markwick, M.C. McGrath, R.H. Morton, P.C.H. Morel, and W.H. Hendriks

Effect of Dietary Form on Nutrient Digestibility in Cats and Dogs...... 83 K. Weidgraaf, S.M. Rutherfurd, K.A. O’Flaherty, D.G. Thomas, and K.J. Rutherfurd-Markwick

Chemical Composition and In Vitro Crude Protein and Fiber Disappearances of Corn Coproducts from the Ethanol Industry ...... 84 M.R.C. de Godoy, L.L. Bauer, and G.C. Fahey, Jr.

Corn Fiber Effects on Nutrient Digestibility and Fecal Characteristics of Dogs ...... 85 M.A. Guevara, L.L. Bauer, C.A. Abbas, K.E. Beery, M.A. Franklin, M.J. Cecava, and G.C. Fahey, Jr.

In Vitro Evaluation of Protein Digestibility of Four Pet Foods...... 86 F. Bovera, S. Calabrò, S. D’Urso, R. Tudisco, A. Guglielmelli, R. Romano, and M.I. Cutrignelli

Review of Pet Dog Feeding Habits in Spain ...... 87 V.M. Mariotti, M. Hervera, J. Fatjó, M. Amat, M.D. Baucells, and X. Manteca

Use of a Wireless Multisensor Telemetry Capsule for Monitoring the Canine Gastrointestinal Tract ...... 88 W.A. Anderson, W. Kerr, and G. Mohr

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PREFACE AND DEDICATION Preface and Dedication

The objective of the Nestlé Purina Nutrition Forum is to pro- about the needs of cats. For example, when they started ex- mote advances in veterinary nutrition related to dogs and ploring the amino acid requirements of cats, neither the es- cats. Our goal is to further the creation of new knowledge sentiality of, nor the quantitative requirement for, specific through quality research and encourage the sharing of that amino acids had been determined. Much had been extrapo- knowledge by providing suitable venues and programs to fa- lated from other species, but the unique features of feline me- cilitate effective communication with a variety of audiences. tabolism identified by Drs. Morris and Rogers and others By staying true to these objectives, this annual program has showed that such extrapolation was often not appropriate. come to be regarded by many as one of the best meetings on Between the two of them, Drs. Morris and Rogers have veterinary nutrition. shared the results of their research through hundreds of jour- This year, we have taken the opportunity provided by the nal articles and research abstracts. Their research helped de- Nestlé Purina Nutrition Forum to recognize something else fine minimum requirements for protein and essential amino that is often regarded as one of the best: the research duo of acids, vitamins, and minerals for growing and adult cats, and Dr. James G. Morris and Dr. Quinton R. Rogers. While true they contributed to both the National Research Council and long-term collaborations are rare in the world of science, any- the Association of American Feed Control Officials nutri- one who studies feline nutrition instantly recognizes the tional guidelines for cats and dogs. In addition, Drs. Morris names Morris and Rogers. This duo has spent the better part and Rogers played a key role in elucidating the link between of their careers working together, exploring the unique nutri- feline dilated cardiomyopathy and taurine. tional needs of cats. Outside of the pet food industry, these Now retired, Drs. Morris and Rogers leave a lasting legacy two were the first to seriously pursue studies in feline me- in the knowledge they built and in the students they trained. tabolism and nutrition. Beginning in the early 1970s, Dr. As well stated by a former colleague, the generation to follow Morris set out to build a world-class program in feline nutri- them has giant footsteps to fill. tion. He enlisted Dr. Rogers to join him, and the rest, as they On behalf of Nestlé Purina PetCare, it is with great pleas- say, is history. ure that we dedicate the 2007 Nestlé Purina Nutrition Forum Over the next 30 or so years, significant advances were to Dr. James G. Morris and Dr. Quinton R. Rogers. made in the field of nutrition. Concurrent with this, Drs. Morris and Rogers expanded our understanding of feline nu- Dottie Laflamme, DVM, PhD, DACVN trition. When they began studying this field, little was known Nestlé Purina PetCare Research

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SCIENTIFIC PROGRAM: FOCUS ON FELINES Some Highlights in Elucidating the Peculiar Nutritional Needs of Cats

Quinton R. Rogers, PhD, DACVN, and James G. Morris, PhD, DACVN Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California

Animals were originally classified into groups on the basis of FOOD INTAKE comparative anatomic and physical traits. Because of The early studies of nutritional requirements of animals were anatomic resemblances, sometimes animals that ate similar restrained by lack of purified diets that contained defined diets were grouped together; however, animals that eat sim- quantities of nutrients. Whereas rats and dogs readily ate pu- ilar diets are often in divergent groups. Therefore, zoologi- rified diets, it was difficult to induce cats to consume them.1 cal grouping does not reflect diet. More modern systems use Nevertheless, deficiencies of certain vitamins2–5 and minerals6 cladistic classification, which arranges organisms by their were observed and studied in cats before the development or order of branching in an evolutionary tree, not by their mor- use of satisfactory purified diets. It was not until the 1950s phologic similarity. Determination of cladistic relationships that purified diets that supported near-normal growth were has been greatly facilitated by the application of molecular developed.7,8 techniques. Another major obstacle during the early studies of feline The two most common companion animals belong to nutrition was a lack of control of respiratory and other viral the order Carnivora in the families Felidae and Canidae. Al- diseases in colony cats. This situation was especially acute in though the term carnivore (derived from the Latin carne, studies of kitten postweaning growth. The development of an which means “flesh,” and vorare, which means “devour”) is effective panleukopenia vaccine decreased morbidity and used to denote eating of animal tissue, all animals belong- mortality associated with this disease, but it was not until spe- ing to Carnivora are not carnivores. Some are herbivores or cific pathogen-free colonies were developed that controlled omnivores, and a number of strict carnivorous animals be- studies could be undertaken. long to families other than Carnivora. A strict carnivorous An additional stumbling block was the often finicky feed- diet is high in protein, moderately high in fat, and very low ing behavior of cats.9 Besides being particularly sensitive to in carbohydrates. This diet also contains the essential vita- flavor, cats find the texture of the diet very important.1 Vari- mins and minerals (if the skeleton is consumed) and fatty ous research groups were able to improve consumption of acids to provide a complete diet. One can debate what con- purified diets by increasing the water content of the diet using stitutes a strict dietary carnivore. Certainly, raptors and car- gelatin or agar or formulating the diet as a mash.7,8 It has been nivorous fish, such as salmonids, belong in this category. consistently found in various laboratories that weanling kit- Although cats share many of the same characteristics of tens more readily adapt to purified diets than adult cats. these carnivores, cats can also utilize starch. It would appear However, we have induced adult cats that were not previously that in the evolution of cats, this facility has been main- fed purified diets to accept purified diets by using pelleted or tained, whereas it either has been lost or did not exist in gel diets and gradually mixing the purified diet with a previ- raptors and carnivorous fish. ously acceptable commercial diet over a period of a week or Although we have worked on feline nutrition for many more (sometimes it takes several weeks). Even then, some years, we are still fascinated by the nutritional peculiarities of adult cats will not eat enough of the purified diet to main- cats and how, in contrast to dogs or rats, they have modified tain their original weight, whereas others may eventually be- their metabolism. This brief review concentrates on some of come overweight. Although proteins are neither selected nor the nutritional peculiarities of cats and the mechanisms by avoided,10 amino acids, peptides, and nucleotides show pos- which these adaptations have occurred. itive palatability for cats.11,12

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PROTEIN AND AMINO ACIDS low-protein diets, cats adapt well to diets containing medium A key difference between the nutritional needs of cats and to high protein. Adaptation is achieved by increasing flow omnivores (e.g., rats, dogs) is the quantitatively higher crude through enzyme systems, including the urea cycle via substrate protein (CP) requirement of cats for maintenance and the regulation, via allosteric regulation, and by increasing meta- higher requirement for arginine, sulfur amino acids, and aro- bolic intermediates (e.g., ornithine in the urea cycle),21,22 all matic amino acids. Cats also show a greater tolerance for ex- without the necessity of increasing enzyme activities. This cess CP and several essential amino acids and a lesser lower ability of cats to conserve nitrogen results in a higher tolerance for glutamic acid than other animals. Other key urinary obligatory nitrogen loss in adult cats fed a protein-free qualitative differences between cats and omnivores in rela- diet of 360 mg × kg body weight3/4 × day-1 compared to rats or tion to protein include cats’ requirement for taurine and dogs at 128 and 210 mg × kg body weight3/4 × day-1, respec- niacin, which can be synthesized by most animals from cys- tively.23 Long-term food deprivation also causes a much higher teine and tryptophan, respectively. urinary nitrogen loss in cats24 than it does in omnivores. This same lack of downregulation of nitrogen catabolic enzymes Protein Requirement results in the protein-efficiency ratio and net protein utiliza- The requirement of bioavailable CP for adult cats is about 160 tion for the same proteins being much lower for kittens (about g/kg diet, whereas the requirement for dogs is about half as one-half or less) than for rats,25 which is another measure- much (80 g/kg diet) and the requirement for rats is less than ment that shows lower efficiency of utilization of protein for one-third as much (50 g/kg diet).13,14 The high CP requirement kittens than for rats or dogs. of adult cats is reflected by the quantity of protein in all commercial diets formulated for the maintenance of cats, which Essential Amino Acids for several decades has generally contained at least 280 to 300 g When we began our work on the amino acid requirement of CP/kg diet; however, the CP requirement for growing kittens, cats, neither the essentiality nor the requirement for dietary rats, and puppies is 180, 150, and 180 g/kg diet, respectively. amino acids had been determined. We began by showing For cats, there is a small difference between maintenance and which amino acids were essential for the cat. As might be ex- growth requirements due to a considerably higher CP require- pected, because all animals studied (from single-cell animals ment for maintenance, whereas dogs and rats have a higher CP to higher animals) had been shown to require eight amino requirement for growth component. Puppies have a high CP acids—leucine, isoleucine, valine, methionine, threonine, requirement for growth but a low CP requirement for mainte- phenylalanine, lysine, and tryptophan—we found these also nance, which is consistent with the growth rates of postwean- essential for cats. Not surprisingly, we also found histidine ing kittens, rats, and puppies of about 1.5% to 2%, 5% to 10%, and arginine to be essential.26 Most surprising were some of and 2% to 5% of body weight/day, respectively. Thus, the in- the clinical signs of the deficiencies that we observed. Argi- creased need for CP by growing rats and puppies is due to the nine deficiency produced the most dramatic effect: When higher rate of growth in these species, which also results in a near-adult cats were food deprived overnight and fed a single higher percentage of the dietary nitrogen being used for pro- meal of 4 to 11 g of an arginine-free diet, within 2 hours all tein synthesis than for kittens. cats exhibited emesis and lethargy,27 and shortly thereafter The reason for the high CP requirement of adult cats for they also vocalized and exhibited frothing at the mouth, maintenance appears to be the metabolic profile of the nitro- ataxia, emprosthotonos, and exposed claws. One cat, which gen catabolic enzymes, those moving nitrogen into the liver had eaten 8 g of the arginine-free diet, showed bradypnea and for the urea cycle and those involved in synthesizing urea.15,16 cyanosis and died in apnea. These clinical signs were caused These enzymes do not downregulate when cats are given low- by severe hyperammonemia resulting from a lack of or- protein diets, as occurs in omnivores and herbivores17; there- nithine, an essential intermediate in the urea cycle, thus shut- fore, cats cannot conserve nitrogen to the same extent as these ting down urea synthesis.28 Normally, under these conditions, species. Although some adaptation takes place for some of the ornithine is produced from dietary arginine via liver arginase. essential amino acid catabolic enzymes in cats,18–20 the Acute short-term deficiencies of any one essential amino acid changes are minor (about 0.5- to 2-fold) when compared with for a week or less (except arginine) resulted in no overt clin- rats (2- to 10-fold). In contrast, although cats do not adapt to ical signs except a gradual decrease in food intake and weight

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SOME HIGHLIGHTS IN ELUCIDATING THE PECULIAR NUTRITIONAL NEEDS OF CATS

loss.26 During the second week of feeding a diet mildly defi- Amino Acid Intolerances cient in threonine (4 g/kg diet), neurologic signs appeared, Other interesting differences in amino acid nutrition between including slight tremor, jerky head and leg movements, a stiff cats and omnivores, such as the rat and chick, are the lower rear gait, difficulty maintaining equilibrium, and weakness tolerances for glutamic acid and the higher tolerances for in the front wrist joints such that upon standing, the cats ap- most of the essential amino acids except methionine. The peared bowlegged. These clinical signs seemed to be of cere- upper limit for dietary glutamic acid for the kitten is about bellar dysfunction and all disappeared after supplementation 5% to 6% of the energy.43 When more than 7% of energy with adequate threonine (6 g/kg diet).29 from glutamic acid was given to kittens, occasional emesis Severe histidine deficiency for 1 month resulted in crusty ex- occurred, and kittens given only their normal requirement of udates around the eyes and nostrils, whereas an even more pro- thiamine (4.4 mg thiamine/kg diet) also became thiamine longed subclinical deficiency (2 to 2.5 g histidine/kg diet, which deficient. With higher dietary thiamine or lower glutamic supported maximal nitrogen retention) for 4 to 5 months re- acid, the kittens grew normally and exhibited no observable sulted in the development of cataracts in some of the kittens.30 clinical signs. Rats and chicks tolerate more than twice these Examination of the eyes revealed changes in the outer fibers of concentrations of glutamic acid. Among the essential amino the lens with no abnormalities seen in the retina. acids, the lowest tolerance is for methionine, which is about It was common for dried secretions to accumulate around 1.5% of energy.18,44 An example of higher tolerance is that of the eyes, nose, or mouth of kittens after prolonged ingestion the branched-chain amino acids. A leucine–isoleucine and of diets deficient in essential amino acids. For example, pro- valine antagonism could not be shown in kittens unless longed ingestion of an isoleucine-deficient diet (6 to 7 weeks isoleucine was limiting, and even then it was mild and tran- at 2 to 4 g isoleucine/kg diet) results in crusty exudate sitory.45 Kittens tolerated 10% leucine without a depression in around the eyes of kittens. Apparently, this condition is due food intake or weight gain. Also, kittens chose the high- to infection by common dermal staphylococcal species, leucine diet even when isoleucine was limiting.46 which indicates that isoleucine deficiency impairs the nor- The phenylalanine plus tyrosine requirement of cats is in- mal resistance to these dermal microorganisms.31 The infec- teresting in that only about a 7 g/kg diet is required for max- tions resolved after isoleucine supplementation without imal growth,47 yet even in adult animals, at least twice this antibiotic treatment. amount is required to produce enough eumelanin in black Another example is the dermal lesions seen around the hair to maximize the black color.48 mouth and paws resulting from a methionine-deficient diet.32 All of these differences in the nutrition and metabolism of These lesions are intensified by excess dietary cystine33 and cats versus omnivores can be explained on the basis of cats are similar to those seen under similar dietary treatment of being strict carnivores and having evolved to eating small prey poults34 and dogs.35 These lesions quickly disappear when that is medium in fat and high in protein that contains less sufficient methionine is added to the diet. glutamic acid than that found in cereal proteins. The low tol- Methionine is of special interest in feline nutrition because erance of methionine that we have found in kittens fed purified there are unusual pathways in its metabolism. Very little taurine diets may seem to contradict this evolutionary explanation; is synthesized in cats, whereas both isovalthine and felinine, however, a diet of meat containing 65% of the energy from branched-chain sulfur amino acids are found in cat urine. More protein and 35% of the energy from fat and carbohydrates (as- work has been done on felinine, which is considered to be pri- suming a bioavailability of sulfur amino acids of 85%) would marily for territorial marking. Felinine is highly odorous and is provide the amount of energy right at this upper limit. It is the precursor to other highly odorous sulfur compounds that known that cats eating high-protein, low-carbohydrate diets are products of the decomposition of felinine.36–38 It is now seldom, if ever, become obese. Perhaps it is the methionine known that felinine is synthesized from cysteine39 in the liver by tolerance that limits food intake in these animals. an S-transferase to γ-glutamylfelinylglycine40 with the use of glutathione as a substrate. γ-Glutamylfelinylglycine is transported to Taurine the kidney, where it is hydrolyzed to release felinylglycine and In 1975, Hayes and coworkers49 reported that feline central reti- free felinine, which are excreted in the urine. Testosterone is nal degeneration is caused by taurine deficiency. Another high- known to enhance the synthesis and excretion of felinine.41,42 light involving taurine in the nutrition of cats is the recognition

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in 1987 that taurine deficiency also results in dilated car- such as may come from a non–all-animal-tissue diet. Even diomyopathy.50 Between these dates, other clinical signs of tau- then, the normal eating behavior of cats results in the ingestion rine deficiency were described, including reproductive and of a number of small meals, which would tend to smooth out developmental problems and neurologic, osmoregulatory, and the glucose load. Despite the absence of glucokinase, a num- immunologic defects.51,52 The puzzling part of the finding of ber of enzymes related to glucose metabolism (hexokinase, dilated cardiomyopathy in cats was that the cats were normally fructokinase, pyruvate kinase, glucose-6-phosphate dehydro- eating diets containing 1,200 to 1,400 mg taurine/kg dry mat- genase, fructose-1, 6-bisphophatase, glucose-6-phosphatase) ter when the requirement had been determined, using puri- are higher in feline liver than in canine liver.64 fied diets, to be 400 mg/kg.53 After much research, it was found High intakes of sucrose in cats result in fructosemia and that the cause of the higher requirement was the lower di- fructosuria.67 This observation indicates that although fruc- gestibility of protein or Maillard reaction products when most tokinase activity in the liver of cats is higher than in dogs, commercial diets (especially canned diets) were fed. These there is an impediment in the metabolism of fructose beyond diets resulted in bacterial conjugated bile acid hydrolase54 ac- fructose-1-phosphate. Normally, fructose-1-phosphate is cat- tivity in the ileum sufficient to cause the hydrolysis of tauro- alyzed to dihydroxyacetone and glyceraldehyde by the en- cholic acid and the further destruction of taurine, thus zyme fructose-1-phosphate aldolase, of which there are three interfering with the enterohepatic reutilization of taurocholic isozymes of aldolase: A, B, and C. Aldolase B is expressed ex- acid.55–59 Thus, it was not a problem of bioavailability in the clusively in the liver, kidney, and intestines. Aldolases medi- sense of absorption of dietary taurine but in the efficiency of re- ate two other reactions besides the cleavage of fructose-1- utilization of taurocholic acid. This was supported by the use phosphate: the cleavage of fructose 1,6-diphosphate and con- of dietary antibiotics, which resulted in a restoration of taurine densation of the triose phosphates, glyceraldehyde phos- homeostasis in cats given such a diet.54 Thus, there is no single phate, and dihydroxyacetone phosphate to form fructose requirement of taurine for cats but instead a variable require- 1,6-diphosphate. Reduced cleavage of fructose-1-phosphate ment between 300 and 2,000 mg/kg diet, depending on the leads to its cellular accumulation and inhibition of fructoki- composition and nature of the diet and its processing. nase, causing accumulation of free fructose in the blood, which would explain the fructosuria. CARBOHYDRATE UTILIZATION The evidence suggests that aldolase B activity is probably Because a diet of animal tissue contains only low concentra- low in feline liver, but apparently this has not been measured. tions of carbohydrate (primarily glycogen), a question arises In humans, hereditary fructose intolerance is caused by a de- whether cats have the ability to utilize plant carbohydrates. ficiency of aldolase B,68 which has been identified as being Digestion studies on cats show that starch disappears from the due to mutations in the aldolase B gene.69,70 More than 25 en- gut and may be more highly digested by cats than dogs, even zyme-impairing mutations of the aldolase B enzyme have when uncooked.60,61 About four isoenzymes occur in mam- been identified.71 Therefore, it appears that there is a high malian liver that catalyze the formation of glucose-6- probability that cats have an inactive aldolase B, which im- phosphate from glucose. The major hexokinase in most pedes the metabolism of fructose. animals is hexokinase D or type IV, often referred to as glu- A consequence of the poor utilization of fructose by cats is cokinase. Glucokinase is absent in the liver of cats,62–64 which the diarrhea and diuresis that follow ingestion of either aque- is consistent with the low glucose loads cats experience from ous solutions of sucrose or diets containing high amounts of an all-animal tissue diet. Glucokinase is also absent from cat sucrose. Providing only sucrose-containing solutions to cats leukocytes but is present in dog leukocytes.65 Using molecular can result in death. Although sucrose improves the physical techniques, Hiskett et al66 found that the expression pattern texture of purified diets for cats (compared to starch and glu- of glucose-sensing proteins in feline liver differed from dogs, cose, which produce more powdery diets), for the above rea- humans, and rodents but that pancreatic expression of these sons, the amount in the diet should be restricted. proteins in cats and other species was similar. The absence of glucokinase in the liver of cats limits cats’ VITAMINS ability to handle high-glucose loads but does not pose a po- Although differences in the protein and amino acid metabo- tential problem unless cats ingest a high-carbohydrate diet, lism of cats and omnivores may be anticipated from cats’

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SOME HIGHLIGHTS IN ELUCIDATING THE PECULIAR NUTRITIONAL NEEDS OF CATS

high-protein diet, major differences also occur in the vitamin sequester larger quantities of vitamin A in the liver with no requirement of cats and other animals. For many years, the vi- apparent adverse effect77 and the form of circulating retinoids tamin A requirement of cats was set at a high level, principally in plasma, which is predominantly retinyl stearate rather than because of the initial studies of feline nutrition by Patricia retinol.76,78 In general, carnivores differ from humans, rats, and Scott and coworkers.72 This group found that kittens given ca- pigs, which have predominantly retinol in their plasma com- sein diets developed retinal degeneration, and she proposed bined with retinol-binding protein. Retinyl esters only appear that retinal degeneration was a consequence of vitamin A de- in plasma when these animals have excessive intakes. The liver ficiency. Subsequent to the discovery of the role of taurine in of cats given high vitamin A diets contains concentrations of production of central retinal degeneration, the preformed vi- vitamin A in excess of those recorded in animals such as polar tamin A requirement of cats was shown to be similar to other bears (another carnivore), an animal often cited as storing mammals; however, it is in the utilization of the vitamin A such large quantities of vitamin A in the liver that it is toxic precursor carotenoids that cats are different from most other when eaten by humans and dogs. animals, including dogs. Carotenoids require cleavage to reti- Most animals are independent of a dietary source of vita- nal, the aldehyde form of vitamin A, and it has been un- min D through ultraviolet (UV) activation of 7-dehydrocho- equivocally proven that the major, if not the sole, pathway of lesterol in the skin; however, cats and dogs are unable to beta-carotene cleavage to vitamin A is by oxidative cleavage of synthesize adequate vitamin D even when shaved and sub- the central ethylenic bond of beta-carotene to yield two mol- jected to UV radiation.79 Cats and dogs synthesize 7-dehy- ecules of retinal.73 The enzyme undertaking this cleavage is drocolesterol, which is also a precursor of both vitamin D beta,beta-carotene 15,15′ monooxygenase (previously known and cholesterol, but cat and dog skin contain only low con- as beta-carotene 15,15′ dioxygenase), a cytosolic enzyme lo- centrations, compared with animals that can undertake vita- cated in the duodenal mucosa and, to some extent, in the liver min D synthesis. When cats are given an inhibitor of the of animals undertaking the carotene conversion. Although the enzyme that converts 7-dehydrocholesterol to cholesterol (7- enzyme has been cloned from chickens and humans, to our dehydrocholesterol-Δ7-reductase), the concentration of 7-de- knowledge no studies have been done with cats to determine hydrocholesterol in the skin is elevated, and when cats are whether the enzyme is present or, if present, what factors pre- exposed to UV radiation, they synthesize vitamin D and have vent its activity. The eccentric cleavage of beta-carotene result- adequate concentrations of 25-hydroxyvitamin D in the ing in the formation of apocarotenoids does not appear to be plasma. Therefore, the peculiarity of cats in regard to vitamin significant and is present only in in vitro systems in the ab- D synthesis is high activity of the enzyme that depletes the sence of α-tocopherol. Until further information is available, precursor pool for synthesis, not that the enzymes of the syn- it appears that the inability of cats to utilize carotenoids as thetic pathway are absent. precursors of vitamin A is due to lack of or very low activity of Most animals are able to supply their needs for nicotinic acid beta,beta-carotene 15,15′ monooxygenase in the intestines by the metabolism of tryptophan in excess of that required for and liver. protein synthesis. Depending on the species, the molar yield of Vitamin A plays a key role in the development (as retinoic nicotinic acid from tryptophan is variable (about 30 to 40 mol acid) and maturation of tissues. In all species of animals, in- tryptophan in rats; 60 mol tryptophan in humans). The cata- cluding cats, excessive dietary intakes of vitamin A produce bolic pathway of tryptophan to nicotinic acid has an interme- pathologic changes in fetal and adult tissues.74–76 Animals that diate: α-amino-β-carboxymuconic-ε-semialdehyde, which can obtain their vitamin A from carotenoids have the ability to either proceed to nicotinic acid synthesis or be metabolized to

protect against excess vitamin A by downregulation of the en- acetyl coenzyme A (acetyl-CoA) and CO2 by the enzyme picol- zyme that converts carotene to vitamin A. This step does not inic carboxylase.80 The activity of picolinic carboxylase is so high occur in cats, as all the vitamin A is absorbed from tissues that in cats that virtually none of the intermediate is available for contain retinol and retinyl esters, which could increase the nicotinic acid synthesis but is metabolized to acetyl-CoA and

susceptibility of cats to vitamin A toxicity; however, kittens CO2, which renders niacin a dietary requirement. Therefore, al- and adult cats can tolerate intakes of vitamin A that would in- though cats have the necessary pathway for nicotinic acid syn- duce toxicity in other species.77 The tolerance of cats appears thesis, the activity of an enzyme (picolinic carboxylase) is so to stem from a combination of two factors: the cat’s ability to high that it diverts the intermediate for synthesis along an al-

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ternate pathway, a situation analogous to the cat’s inability to in the diet for multiple litters in queens is somewhat equiv- synthesize vitamin D. It has been speculated that because ani- ocal. Limited numbers of litters have been reported in mal tissue is a good source of nicotinamides, there was no evo- queens receiving linoleate and no arachidonate in the diet.88 lutionary pressure to maintain synthesis, and some of the It has been suggested that some of the problems encountered intermediates have carcinogenic potential. with purified diets may be due to the balance of n-3 to n-6 No other specific peculiarities in the cat’s vitamin require- fatty acids. In view of the above, the addition of a source of ment have been identified (with the possible exception of thi- arachidonate in the diet of breeding queens is prudent. amine, which was discussed in the section on amino acids). Among dietary ingredients, animal fat is highly palatable; Similarly, the propensity of cats to exhibit clinical signs of vi- however, the cat has an aversion to medium-chain triglycerides.89 tamin E deficiency is more a reflection of the diet than a difference of requirement. MINERALS Finally, to our knowledge, the essentiality of dietary inositol Although the quantitative requirement for mineral elements has not been tested in cats. Under specific conditions of high-sat- in the diet vary across mammalian species (often a function of urated-fat diets (e.g., coconut oil), inositol is required by female relative growth rates), there is general concurrence regarding gerbils to prevent fatty infiltration of the liver and intestines.81 which elements are essential. Many minerals (e.g., iron, copper, zinc) are often involved at catalytic sites of the enzymes FATS AND ESSENTIAL FATTY ACIDS that occur across species, whereas elements such as sodium Fats play a significant role in the attractiveness of food for and potassium have common roles in osmoregulation and cats, as well as being an important source of energy. Cats ex- calcium and phosphorus have common roles in the skeleton. hibit a distinct preference for some animal fats over other fats Dietary selection based on minerals is relatively rare. Some (e.g., chicken fat is preferred over beef tallow, which in turn notable exceptions are sodium and phosphorus in ruminants is preferred over butter fat). The latter ranking may be related and several minerals by rats. Many herbivores exhibit a pref- to olfactory and flavor components in these fats or to the re- erence for sodium salts or sodium salt solutions that pre- ported aversion of cats to short-chain fatty acids. sumably have survival value, as most plants do not require Cats, like other mammals, require preformed n-3 and n-6 sodium for growth, and hence vegetable material is low in long-chain essential fatty acids in their diet, as they are unable sodium. In contrast to herbivores, cats show no preference or to introduce double bonds (desaturate) to precursor fatty aversion to sodium salts. Even when severely depleted of acids beyond carbon 9. These long-chain essential fatty acids, sodium and given a choice of diets, cats do not correctly through chain elongation and desaturation, result in families choose a diet containing adequate sodium over a sodium-de- of highly active metabolic eicosanoids, such as prostaglandins, ficient diet.90 It would appear that as animal tissue always prostacyclines, leukotrienes, and thromboxanes. contains adequate sodium, the redundant neural pathways There is a general consensus that cats, like other animals, required for detection of sodium either did not develop or require linoleate (an n-6 fatty acid) in the diet along with n- have not been maintained in cats. Although the required con- 3 fatty acids, but the exact requirement of cats for long-chain centration of calcium in the diet of growing kittens is much fatty acids has not been well defined. For most animals, less than that for large breeds of dogs, it is similar to that for arachidonate (an n-6 fatty acid) is not essential in the diet, small breeds of dogs. as the metabolic need for arachidonate can be met through chain elongation and desaturation of linoleate. There is also CONCLUSIONS a general consensus that cats have a limited capacity to syn- This review is intended to update our earlier reviews on nu- thesize arachidonate,82,83 which is attributed to low desat- tritional peculiarity of cats82,91,92 and to indicate how modifi- urase activity of cat liver.84,85 Pawlosky and colleagues86 cation of the metabolism of cats has resulted in their distinct demonstrated that cats possess low Δ6-desaturase activity but nutrient requirement. The high protein requirement of cats are capable of limited synthesis. Arachidonate-free diets per- for maintenance is a consequence of the cat’s limited ability mit similar growth rates in kittens and reproductive success to downregulate aminotransferases of general nitrogen me- in males when they achieve adulthood as toms given diets tabolism and the urea cycle enzymes, which is similar to that containing arachidonate.87 The essentiality of arachidonate observed in other nonfelid carnivores. It is suggested that this

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SOME HIGHLIGHTS IN ELUCIDATING THE PECULIAR NUTRITIONAL NEEDS OF CATS

confers an advantage to cats because they are always prepared 16. Tews JK, Rogers QR, Morris JG, Harper AE. Effect of dietary protein and GABA on food intake, growth and tissue amino acids in cats. Physiol to ingest a high-protein meal. Behav 1984;32:301-308. Four nutrients plus arginine (which is not essential in the 17. Schimke RT. Adaptive characteristics of urea cycle enzymes in the rat. J diet of several mammals, including humans) are essential in Biol Chem 1962;237:459-468. 18. Fau D, Morris JG, Rogers QR. Effects of high dietary methionine on ac- the diet of cats. Arginine and taurine synthesis are limited by tivities of selected enzymes in the liver of kittens (Felis domesticus). Comp low activities of enzymes in the synthetic pathway. The other Biochem Physiol B 1987;88:551-555. 19. Bai SC, Rogers QR, Wong DL, et al. Vitamin B-6 deficiency and level of three nutrients are vitamins, two of which (vitamin D and dietary protein affect hepatic tyrosine aminotransferase activity in cats. niacin) are required in the diet even though the pathways for J Nutr 1998;128:1995-2000. their synthesis are present. For these two vitamins, degradation 20. Park T, Rogers QR, Morris JG. High dietary protein and taurine increase cysteine desulfhydration in kittens. J Nutr 1999;129:2225-2230. of intermediates by high activities of enzymes for alternate 21. Rogers QR, Morris JG. Up-regulation of nitrogen catabolic enzymes is pathways results in no effective synthesis. The third vitamin not required to readily oxidize excess protein in cats. J Nutr 2002;132:2819-2820. (vitamin A) cannot be synthesized from precursor carotenoids 22. Russell K, Murgatroyd PR, Batt RM. Net protein oxidation is adapted to because of an apparent lack of the monooxygenase enzyme re- dietary protein intake in domestic cats (Felis silvestris catus). J Nutr 2002;132:456-460. quired to cleave the carotenoids. Besides requiring the above 23. Hendriks WH, Moughan P, Tarttelin MF. Urinary excretion of endoge- nutrients in the diet, cats show greater sensitivity than omni- nous nitrogen metabolites in adult domestic cats using a protein-free vores to a number of compounds that occur in plants but not diet and the regression technique. J Nutr 1997;127:623-629. 24. Biourge V, Groff JM, Fisher C, et al. Nitrogen balance, plasma free amino animal tissue. Fructose is an example of a plant carbohydrate acid concentrations and urinary orotic acid excretion during long term that is poorly utilized, probably due to low activity of the al- fasting in cats. J Nutr 1994;124:1094-1103. 25. Fox LAD, Jansen GR, Knox KL. Effects of variations in protein quality on dolase B enzyme. Other compounds include benzoate, growth, PER, NPR and NPU in growing kittens. Nutr Rep Int 1973;7:621- sulfhydryl compounds from onions, aspirin, and so forth.14 631. 26. Rogers QR, Morris JG. Essentiality of amino acids for the growing kit- REFERENCES ten. J Nutr 1979;109:718-723. 27. Morris JG, Rogers QR. Ammonia intoxication in the near-adult cat as a 1. Scott PP. The cat. Vet Rec 1960;72:6-9. result of a dietary deficiency of arginine. Science 1978;199:431-432. 2. Heath MK, MacQueen JW, Spies TD. Feline pellagra. Science 28. Morris JG, Rogers QR. Arginine: an essential amino acid for the cat. J 1940;92:514. Nutr 1978;108:1944-1953. 3. Everett GM. Observations on the behaviour and neurophysiology of 29. Titchenal CA, Rogers QR, Indrieri RJ, Morris JG. Threonine imbalance, acute thiamine deficient cats. Am J Physiol 1944;141:439-448. deficiency and neurologic dysfunction in the kitten. J Nutr 4. Smith DC, Proutt LM. Development of thiamine deficiency in the cat on 1980;110:2444-2459. a diet of raw fish. Proc Soc Exp Biol Med 1944;56:1-3. 30. Quam DD, Morris JG, Rogers QR. Histidine requirement of kittens for 5. Cordy DR. Experimental product of steatitis in kittens fed a commercial growth, haematopoiesis and prevention of cataracts. Br J Nutr canned cat food and prevention of the condition by vitamin E. Cornell 1987;58:521-532. Vet 1954;44:310-318. 31. Hargrove DM, Rogers QR, Morris JG. Leucine and isoleucine require- 6. Scott PP, Greaves JP, Scott MG. Nutrition of the cat. 4: calcium and io- ments of the kitten. Br J Nutr 1984;52:595-605. dine deficiency on a meat diet. Br J Nutr 1961;15:35-51. 32. Strieker MJ, Morris JG, Kass PH, Rogers QR. Increasing dietary crude 7. Carvalho da Silva A. The domestic cat as a laboratory animal for exper- protein does not increase the methionine requirement in kittens. J Anim imental nutrition studies. II: comparative growth rate and haematology Physiol Anim Nutr (Berl) 2007;91:465-474. on stock and purified rations. Acta Physiol Lat Am 1950;1:26-32. 33. Strieker MJ, Werner A, Morris JG, Rogers QR. Excess dietary cystine in- 8. Allison JB, Miller SA, McCoy JR, Brush MK. Studies on the nutrition of tensifies the adverse effect of a methionine deficiency in the cat. J Anim the cat. North Am Vet 1956;37:38-43. Physiol Anim Nutr (Berl) 2006;90:440-445. 9. Greaves JP. Protein and calorie requirements of the feline. In: Graham- 34. Chavez E, Kratzer FH. Effect of diet on foot pad dermatitis in poults. Jones O, ed. Canine and Feline Nutritional Requirements. New York: Perg- Poult Sci 1974;53:755-760. amon Press; 1965:33-45. 35. Hirakawa DA, Baker DH. Sulfur amino acid nutrition of the puppy: de- 10. Cook NE, Kane E, Rogers QR, Morris JG. Self-selection of dietary casein termination of dietary requirements for methionine and cystine. Nutr and soy-protein by the cat. Physiol Behav 1985;34:583-594. Res 1985;5:631-642. 11. White TD, Boudreau JC. Taste preferences of the cat for neurophysio- 36. Hendriks WH, Harding DR, Rutherfurd-Markwick KJ. Isolation and logically active compounds. Physiol Psychol 1975;3:405-410. characterisation of renal metabolites of gamma-glutamylfelinylglycine 12. Beauchamp GK, Maller O, Rogers JG. Flavor preferences in cats (Felis in the urine of the domestic cat (Felis catus). Comp Biochem Physiol B catus and Panthera sp.). J Comp Physiol Psychol 1977;91:1118-1127. Biochem Mol Biol 2004;139:245-251. 13. National Research Council. Nutrient requirements of the laboratory rat. 37. Miyazaki M, Yamashita T, Suzuki Y, et al. A major urinary protein of the In: Nutrient Requirements of Laboratory Animals. 4th ed. Washington, DC: domestic cat regulates the production of felinine, a putative pheromone National Academy Press; 1995:11-79. precursor. Chem Biol 2006;13:1071-1079. 14. National Research Council. Nutrient Requirements of Dogs and Cats. 38. Rutherfurd SM, Kitson TM, Woolhouse AD, et al. Felinine stability in the Washington, DC: National Academy Press; 2006. presence of selected urine compounds. Amino Acids 2007;32:235-242. 15. Rogers QR, Morris JG, Freedland RA. Lack of hepatic enzymatic adap- 39. Hendriks WH, Rutherfurd SM, Rutherfurd KJ. Importance of sulfate, cys- tation to low and high levels of dietary protein in the adult cat. Enzyme teine and methionine as precursors to felinine synthesis by domestic cats 1977;22:348-356. (Felis catus). Comp Biochem Physiol C Toxicol Pharmacol 2001;129:211-216.

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40. Rutherfurd KJ, Rutherfurd SM, Moughan PJ, Hendriks WH. Isolation 67. Drochner W, Müller-Schlösser S. Digestibility and tolerance of various and characterization of a felinine-containing peptide from the blood sugars in cats. In: Anderson RS, ed. Nutrition of the Dog and Cat. Oxford: of the domestic cat (Felis catus). J Biol Chem 2002;277:114-119. Pergamon Press; 1980:101-111. 41. Hendriks WH, Tarttelin MF, Moughan PJ. Twenty-four hour felinine excre- 68. Cox TM, O’Donnell MW, Camilleri M, Burghes AH. Isolation and char- tion patterns in entire and castrated cats. Physiol Behav 1995;58:467-469. acterization of a mutant liver aldolase in adult heredity fructose intol- 42. Tarttelin MF, Hendriks WH, Moughan PJ. Relationship between plasma erance: identification of the enzyme variant by radioassay in tissue testosterone and urinary felinine in the growing kitten. Physiol Behav biopsy specimens. J Clin Invest 1983;72:201-213. 1998;65:83-87. 69. Santer R, Rischewski J, von Weihe M, et al. The spectrum of aldolase B 43. Deady JE, Anderson B, O’Donnell JA, et al. Effects of level of dietary glu- (ALDOB) mutations and the prevalence of hereditary fructose intoler- tamic acid and thiamin on food intake, weight gain, plasma amino ance in Central Europe. Hum Mutat 2005;25:594. acids, and thiamin status of growing kittens. J Nutr 1981;111:1568-1579. 70. Adamowicz M, Płoski R, Rokicki D, et al. Transferrin hypoglycosylation 44. Schaeffer MC, Rogers QR, Morris JG. Methionine requirement of the in hereditary fructose intolerance: using the clues and avoiding the pit- growing kitten in the absence of dietary cystine. Nutr Res 1982;2:289-299. falls. J Inherit Metab Dis 2007;30:407. 45. Hargrove DM, Rogers QR, Calvert CC, Morris JG. Effects of dietary ex- 71. Esposito G, Vitagliano L, Santamaria R, et al. Structural and functional cesses of the branched-chain amino acids on growth, food intake and analysis of aldolase B mutants related to hereditary fructose intolerance. plasma amino acid concentrations of kittens. J Nutr 1988;118:311-320. FEBS Lett 2002;531:152-156. 46. Hargrove DM, Morris JG, Rogers QR. Kittens choose a high leucine diet 72. Scott PP, Graves JP, Scott MG. Nutritional blindness in the cat. Exp Eye even when isoleucine and valine are the limiting amino acids. J Nutr Res 1964;3:357-364. 1994;124:689-693. 47. Williams JM, Morris JG, Rogers QR. Phenylalanine requirement of kit- 73. Lakshman MR. Alpha and omega of carotenoids cleavage. J Nutr tens and the sparing effect of tyrosine. J Nutr 1987;117:1102-1107. 2004;134:241S-245S. 48. Anderson PJ, Rogers QR, Morris JG. Cats require more dietary pheny- 74. Seawright AA, English PB. Hypervitaminosis A and deforming cervical lalanine or tyrosine for melanin deposition in hair than for maximal spondylosis of the cat. J Comp Pathol 1967;77:29-39. growth. J Nutr 2002;132:2037-2042. 75. Clark L. The effect of excess vitamin A on longbone growth in kittens. J 49. Hayes KC, Carey RE, Schmidt SY. Retinal degeneration associated with Comp Pathol 1970;80:625-634. taurine deficiency in the cat. Science 1975;188:949-951. 76. Freytag TL, Liu SM, Rogers QR, Morris JG. Teratogenic effects of chronic 50. Pion PD, Kittleson MD, Rogers QR, Morris JG. Myocardial failure in cats ingestion of high levels of vitamin A in cats. J Anim Physiol Anim Nutr associated with low plasma taurine: a reversible cardiomyopathy. Science (Berl) 2003;87:42-51. 1987;237:764-768. 77. Freytag TL. Vitamin A Metabolism and Toxicity in the Domestic Cat [PhD 51. Hayes KC. Taurine nutrition. Nutr Res Rev 1988;1:99-113. thesis]. Davis, California: University of California, Davis; 2001. 52. Huxtable RJ. Physiological actions of taurine. Physiol Rev 1992;72:101-163. 78. Schweigert FJ, Ryder OA, Rambeck WA, Zucker H. The majority of vita- 53. National Research Council. Nutrient Requirements of Cats. Washington, min A is transported as retinyl esters in the blood of most carnivores. DC: National Academy Press; 1986. Comp Biochem Physiol A 1990;95:573-578. 54. Kim SW, Rogers QR, Morris JG. Dietary antibiotics decrease taurine loss 79. Morris JG. Ineffective vitamin D synthesis in cats can be reversed by an in- in cats fed a canned heat-processed diet. J Nutr 1996;126:509-515. hibitor of 7-dehydrocholesterol-Δ7-reductase. J Nutr 1999;129:903-905. 55. Hickman MA, Rogers QR, Morris JG. Effect of processing on fate of di- 80. Ikeda MH, Tsuji H, Nakamura S, et al. Studies on the biosynthesis of etary [14C] taurine in cats. J Nutr 1990;120:995-1000. nicotinamide adenine dinucleotides. II: role of picolinic carboxylase in 56. Hickman MA, Rogers QR, Morris JG. Taurine balance is different in cats the biosynthesis of NAD from tryptophan in mammals. J Biol Chem fed purified and commercial diets. J Nutr 1992;122:553-559. 1965;240:1395-1401. 57. Morris JG, Rogers QR, Kim SW, Backus RC. Dietary taurine requirement 81. Hegsted DM, Hayes KC, Gallagher A, Hanford H. Inositol deficiency: of cats is determined by microbial degradation of taurine in the gut. an intestinal lipodystrophy in the gerbil. J Nutr 1973;103:302-307. Adv Exp Med Biol 1994;359:59-70. 82. MacDonald ML, Rogers QR, Morris JG. Nutrition of the domestic cat, a 58. Backus RC, Rogers QR, Morris JG. Microbial degradation of taurine in mammalian carnivore. Ann Rev Nutr 1984;4:521-562. fecal cultures from cats given commercial and purified diets. J Nutr 83. Bauer JE. Fatty acid metabolism in domestic cats (Felis catus) and chee- 1994;124:2540S-2545S. tahs (Acinonyx jubatus). Proc Nutr Soc 1997;56:1013-1024. 59. Backus RC, Morris JG, Kim SW, et al. Dietary taurine needs of cats vary 84. Rivers JPW, Sinclair AJ, Crawford MA. Inability of the cat to desaturate with dietary protein quality and concentration. J Vet Clin Nutr essential fatty acids. Nature 1975;259:171-173. 1998;5:18-22. 85. Sinclair AJ, McLean JG, Monger EA. Metabolism of linoleic acid in the 60. Morris JG, Trudell J, Pencovic T. Carbohydrate digestion by the domes- cat. Lipids 1979;14:932-936. tic cat (Felis catus). Br J Nutr 1977;37:365-373. 86. Pawlosky R, Barnes A, Salem N. Essential fatty acid metabolism in the 61. Keinzle E. Carbohydrate metabolism of the cat. 2: digestion of starch. J feline: relationship between liver and brain production of long-chain Anim Physiol Anim Nutr 1993;69:102-114. polyunsaturated fatty acids. J Lipid Res 1994;35:2032-2040. 62. Ballard FJ. Glucose utilization in the mammalian liver. Comp Biochem Physiol 1965;14:437-443. 87. Morris JG. Do cats need arachidonic acid in the diet for reproduction? J Anim Physiol Anim Nutr (Berl) 2004;88:131-137. 63. Washizu T, Tanaka A, Sako T, et al. Comparison of the activities of enzymes related to glycolysis and gluconeogenesis in the liver of dogs and 88. Pawlosky R, Salem N. Is arachidonic acid necessary for feline repro- cats. Res Vet Sci 1999;67:205-206. duction? J Nutr 1996;126:1081S-1085S. 64. Tanaka A, Inoue A, Takeguchi A, et al. Comparison of expression of glu- 89. MacDonald ML, Rogers QR, Morris JG. Aversion of the cat to dietary cokinase gene and activities of enzymes related to glucose metabolism medium-chain triglycerides and caprylic acid. Physiol Behav 1985;35:371- in livers between dog and cat. Vet Res Commun 2005;29:477-485. 375. 65. Arai T, Kawaue T, Abe M, et al. Comparison of glucokinase activities in 90. Yu S, Rogers QR, Morris JG. Absence of a salt (NaCl) preference or ap- the peripheral leukocytes between dogs and cats. Comp Biochem Physiol petite in sodium-replete or depleted kittens. Appetite 1997;29:1-10. Pharmacol Toxicol Endocrinol 1998;120:53-56. 91. Morris JG, Rogers QR. Metabolic basis for some of the nutritional pe- 66. Hiskett E, Gomez V, Schermerhorn T. Molecular description of glucose culiarities of cats. J Small Anim Pract 1982;23:599-613. sensing pathways in dogs and cats [abstract]. Compend Contin Educ Vet 92. Morris JG. Idiosyncratic nutrient requirements of cats appear to be diet- 2005;27(3A):94. induced evolutionary adaptations. Nutr Res Rev 2002;15:153-168.

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SCIENTIFIC PROGRAM: FOCUS ON FELINES Advances in Knowledge about Feline Metabolism

Robert C. Backus, DVM, PhD, DACVN University of Missouri-Columbia, College of Veterinary Medicine, Columbia, Missouri

The high dietary protein requirement of cats is perhaps the zyme activities. They also found that activities of the enzymes most often cited attribute for classifying cats as carnivores. Al- in cats, irrespective of diet, were maintained at levels similar though other nutritional peculiarities of cats support a carni- to those in rats given intermediate (400 to 500 g/kg) to high vore designation, such as dietary requirements for preformed (800 to 900 g/kg) levels of dietary protein. These observa- vitamin A, arachidonic acid, taurine, and niacin,1 protein re- tions contrasted sharply with wide variations in enzyme ac- quirement seems to receive the most attention. The high pro- tivities (as much as 13-fold) observed in rats given similar tein requirement applies to all stages of life for cats, with the dietary treatments. Compared with cats, enzymatic variations maintenance requirement most outstanding compared with in rats were acutely and intuitively adaptive. The variations other species. The protein requirement to maintain adult cats in rats were appropriate for sparing amino acids during food is about twice that needed to maintain adult dogs (160 and deprivation and intake of little dietary protein. The investi- 80 g protein/kg of diet, respectively).2 An even greater differ- gators concluded that cats cannot sufficiently downregulate ence in maintenance protein requirement is observed be- expression of amino acid and amino nitrogen catabolic en- tween adult cats and rats.3 zymes to survive on diets that would marginally meet the The requirement for a nutrient such as protein is the mini- protein requirements of omnivores and herbivores. mum intake or dietary concentration of a nutrient needed for an “optimal” response. The classically targeted response in kit- AMINO ACIDS AND PROTEIN NITROGEN tens is maximal growth, whereas in adults, nitrogen balance or In a strict sense, cats and other animals do not have a dietary constant body weight is targeted. As noted more than 10 years requirement for protein; they require amino acid nitrogen, ago by Morris and Rogers,4 these commonly evaluated re- specific amino acids, and carbon skeletons of other amino sponses may not be optimal for establishing protein require- acids that dietary protein provides. This can be well appreci- ment. In the case of the adult maintenance state, nitrogen ated from a review of many nutritional studies of cats con- balance may be achieved when protein reserves are depleted ducted by Morris and Rogers4 and others in which crystalline and therefore might not be optimal for long-term health. amino acid mixtures were substituted for dietary protein. Nor- Greater dietary concentrations or intakes of protein may sup- mal growth rate and maintenance of body weight are observed port a healthier lean mass, optimize immunologic response, in these studies (i.e., when amino nitrogen and essential or generally result in freedom from degenerative disease. amino acids are sufficiently abundant and presented in correct Thirty years ago, Rogers et al5 reported findings that indi- proportions). Specific amino acids needed in the diet of cats, cated the metabolic basis of the high protein requirement of so-called indispensable or essential amino acids, were identi- cats. The investigators determined enzyme activities in the fied in studies using crystalline amino acids in place of intact livers of adult cats when given diets that were high (700 g/kg) protein. Two years after postulating a cause for the high pro- and low (175 g/kg) in protein and when food was withheld tein requirement for cats, Rogers and Morris6 demonstrated for 5 days. They evaluated enzymes known in rats to be key that the 10 amino acids essential for growth in rats were also for regulating amino acid and nitrogen metabolism, gluco- essential for growth in kittens. During the following decade, neogenesis, and lipogenesis. In comparing results between these and other investigators using crystalline amino acids de- cats and rats, the investigators made several seminal obser- termined the minimal dietary concentrations of essential vations. Especially pertinent to the high protein requirement amino acids needed by kittens for optimal growth. of cats was that dietary protein concentration and food with- Rogers and Morris6 found that dietary requirements for es- holding have, with few exceptions, little effect on urea cycle, sential amino acids in kittens were similar to or only moder- transaminase, and “first-limiting” amino acid catabolic en- ately greater (14% to 67%) than those in rat pups, with the

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notable exception of taurine.7 The similarity of essential amino that were given a wider range of dietary protein concentrations acid requirements between cats and rats indicated to the in- than were given by Russell et al8 (511 to 77 g/kg versus 550 to vestigators that the high dietary protein requirement of kittens 442 g/kg). Using indirect calorimetry, they also found that pro- relates principally to a high requirement for amino nitrogen, tein oxidation positively varied with dietary protein content; not essential amino acids that protein provides. This realiza- however, when cats consumed the lowest level of dietary pro- tion was consistent with the 1977 observation that expression tein, the ratio of protein oxidation to protein intake increased of liver urea cycle enzymes and transaminases in cats are not re- to above unity. This result indicated that net catabolism of duced in response to intake of diets low in protein. It was also body protein occurred at the lowest level of dietary protein, consistent with results of the species comparisons. Differences findings consistent with the protein requirement of cats being between cats and rats in first-limiting amino acid catabolic en- greater than those of dogs and rats. Net catabolism of body zyme activities were much less than differences between the protein would not be observed in rats given the lowest level of species in enzyme activities of nitrogen metabolism. dietary protein. The results were also consistent with earlier Since it was proposed, limited hepatic enzymatic adapt- findings showing adaptability of protein oxidation in cats; ability as a cause for high dietary protein requirement in cats however, it is important to note that they also supported lim- generally has been accepted; however, confirming studies ited adaptability to tolerate low levels of dietary protein in cats were lacking. In recent years, this issue has received renewed relative to dogs and rats. Observations of nitrogen balance in attention. anorectic cats12 and in cats given protein-free diets13 have indi- Using indirect calorimetry, Russell et al8 evaluated protein cated that cats have a diminished and slow-reacting ability to oxidation in cats consuming high (550 g/kg) and moderate conserve body protein relative to many other species. Limited (442 g/kg) levels of dietary protein. The investigators found regulation of nitrogen catabolic enzymes still appears to be a that protein oxidation matched protein intake and concluded tenable cause for these observations. that adapting to varying protein concentrations was not a A fixed and moderately high amino acid and nitrogen me- problem for cats. They also concluded that there must be an- tabolism would seem detrimental to cats because of their in- other explanation for the high protein requirement of cats. efficient use of dietary protein and poor conservation of body The inconsistency of their conclusions with those posited 25 protein; however, these attributes are probably inconsequen- years earlier appears to reflect different interpretations of tial to an animal ingesting food that does not vary greatly in adaptability and the basis of dietary protein requirement. protein content, such as in small mammals, reptiles, am- With respect to adaptability, implicit in the kind of measure- phibians, and insects. Morris, Rogers, and others have sug- ments conducted by Rogers et al5 was that cats have a limited gested advantages to carnivores: Though cats naturally ingest ability to up- and downregulate enzyme mass per unit of liver a low-carbohydrate diet, glucose is made readily available for mass. Enzyme activities were measured at presumed maximal catching prey by a high rate of gluconeogenesis from amino velocity conditions, which generally indicate enzyme mass. Al- acid catabolism. Also, after periods of food deprivation, am- though not specifically stated, other means of regulation were monium produced after ingestion of prey is less likely to not discounted by Rogers et al.5 As addressed by Rogers and Mor- cause toxicity with a moderately high rather than downregu- ris,9 cats should be able to modulate nitrogen metabolism and lated nitrogen metabolism. amino acid catabolism by changing liver mass and allosteric regulation of rate-controlling enzyme activities. Indeed, when cats CARBOHYDRATE METABOLISM consume high-protein diets, it has been observed that their liv- Rogers et al5 found that activities of key regulatory gluco- ers become enlarged.10 Additionally, because of the nature of en- neogenic enzymes in cats were greater than those in rats fed zyme kinetics, enzyme activity varies with the availability of high-protein diets. As with nitrogen-metabolizing enzymes, substrate. Enzymes in vivo function at rates much lower than they also found that activities of the gluconeogenic enzymes their maximum, and their activities increase with substrate con- were affected little by dietary protein concentration or car- centration. This behavior of enzymes, to some extent, should bohydrate concentration because sucrose and corn starch account for increasing nitrogen and amino acid metabolism were reciprocally substituted for soy protein to vary dietary with increasing dietary protein concentration in cats. protein concentration. These findings were consistent with Green et al11 reported findings of protein oxidation in cats amino acid use for glucose production in cats during the fed

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as well as food-deprived state. As the investigators noted, the and insulin concentrations are lower when meals of high-pro- all-meat diet of carnivores would contain little carbohydrate; tein, low-carbohydrate diets are consumed than when low-pro- therefore, use of amino acids for gluconeogenesis over oxi- tein, high-carbohydrate meals are consumed.20,21 This effect dation would favor meeting obligate tissue needs for glucose. does not seem to be mediated by improvement of glucose dis- Subsequent studies appear to confirm the lack of adapt- posal, although increased insulin sensitivity or effectiveness of ability and substantial glucose production in cats as a result glucose is promoting its own disposal.22,23 of amino acid catabolism. Cats given a high-protein (630 A benefit of increasing dietary protein may be through re- g/kg), low-carbohydrate (60 g/kg) diet were found to possess duction of risk for obesity. It is clear that obesity is a con- liver glycogen in amounts similar to those in rats given a tributing factor to development of diabetes in cats.24 Recent high-protein diet14; however, unlike rats, gluconeogenesis in findings of Hoenig et al23 indicate that consumption of high liver slices of cats given a high-protein diet is not reduced by protein results in a greater heat increment in lean cats but not food deprivation. Studies on isolated hepatocytes of cats in- obese cats. This thermic effect of protein may be beneficial for dicate that catabolic pathways for some amino acids, such as obesity prevention by increasing energy expenditure and sati- glycine, are uniquely directed more toward gluconeogenesis ety. Dietary protein appears to be beneficial in obesity man- than oxidation.15 Such studies also indicate that as dietary agement through maintaining or reducing the loss of lean protein concentration is increased, oxidation, not gluconeo- body mass, where glucose is mostly disposed, and facilitating genesis, becomes a more likely fate of amino acids in cat loss of body fat, which positively affects insulin sensitivity.25 liver.16 A mechanism for this trend was not postulated. Hence, although maximal activities of the gluconeogenic enzymes LIPID METABOLISM are not observed to change with dietary protein concentra- Activities of a few hepatic enzymes of lipogenesis were eval- tion, adaptation in flux of amino acids away from glucose uated in the 1977 report of Rogers et al.5 As with the other en- production in the liver may occur with increasing dietary pro- zymes studied, diet had little to no effect on enzyme activities tein concentration. This trend may prevent overproduction in this study. This finding, along with findings of unde- of glucose and facilitate glycemic regulation in animals con- tectable to low activities of malic enzyme and citrate cleav- suming high-protein diets. age enzyme, indicated that cats, relative to rats, have a limited Concern about a rising prevalence of diabetes mellitus in capacity for de novo synthesis of fatty acids in liver. From this, cats in recent years has prompted the suggestion that protein the investigators inferred that cats have a limited capacity for concentration should be increased in feline dry diets even lipogenesis in general. though the protein content of such diets is greater than that To the author’s knowledge, little has been reported on li- needed for maintenance of body weight and nitrogen bal- pogenesis in cats. In apparent agreement with the suggestion ance.17 The basis of this suggestion seems to be that increasing of Rogers et al,5 Ibrahim et al26 found undetectable fatty acid dietary protein concentration will necessarily reduce intake of synthesis in livers of cats using an in vivo, deuterated water- dietary carbohydrate and that intake of an “unnaturally high” labeling method; however, these investigators determined amount of carbohydrate is detrimental to the “relatively glu- fatty acid synthesis in cats during weight loss, when substan- cose-intolerant” cat. If reduction of glucose load is beneficial, tial synthesis would not be expected. Rogers et al5 acknowl- it is reasonable to consider that increasing dietary protein in edged that fatty acid synthesis may occur in tissues other than exchange for carbohydrate will increase glucose entry from the livers of cats. Richard et al27 determined fatty acid synthesis liver, whereas glucose entry from the intestines is decreased. In rates in liver and adipose slices of cats given a diet low in fat humans, between 50 and 80 g of glucose is estimated to be (80 g/kg), adequate in protein (300 g/kg), and presumably derived from 100 g of dietary protein.18 high in carbohydrate. They found that fatty acid synthesis in Nonetheless, relative to dietary carbohydrate, dietary protein the liver preparations from cats was low when either glucose probably contributes less to glucose entry, and glucose derived or acetate was used as substrate. In this way, cats seemed sim- from dietary protein is probably less dependent on insulin for ilar to ruminants. A much greater (about 20-fold) rate of fatty disposal. Findings of reduced need for exogenous insulin in di- acid synthesis occurred in adipose tissue compared with liver abetic cats given a high-protein, low-carbohydrate diet appear tissue. Cats had an intermediate rate of adipose lipogenesis to support this.19 Also, in normal, healthy cats, plasma glucose among species, lower than that in dogs but greater than that

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in rats and humans. ception of lysine and sulfur amino acids, the reported amino Adipose tissue in cats does appear to be capable of sub- acid maintenance requirements of cats are not based on dose stantial fatty acid synthesis under the right conditions. In cats responses to optimize a biomarker of adequacy.2 Mainte- deficient in lipoprotein lipase (LPL) activity, body tissues nance requirements would be better defined with dose– have impaired access to circulating fatty acids and hyperlipi- response studies. Given that obesity is the most common nu- demia results. In these cats, fatty acids in subcutaneous adi- tritionally induced disease of cats and is associated with dia- pose triacylglycerol are enriched in palmitic acid, the most betes mellitus and immune dysfunction, it seems worthwhile abundant product of de novo fatty acid synthesis.28 Although to investigate whether maintenance protein and amino acid LPL-deficient cats are generally lean, neutering causes accu- requirements should be based on minimizing obesity and di- mulation of body fat mass in the cats to the point that over- abetes risk and optimizing immune function. weight to obese body conditions are observed with ad For studies aimed at minimizing obesity risk, recently libitum food intake.29 For this degree of body fat accretion to neutered (orchiectomized or ovariectomized) young cats that have developed, it is believed that an extraordinary fatty acid have finished growing are probably good models. Such cats synthesis occurred in adipose tissue. are representative of a large fraction of privately owned cats that will become overweight. Before neutering, cats are typi- FUTURE DIRECTIONS cally lean even when they are continuously presented with The research of Morris and Rogers,30–39 subsequent to their food. After neutering, an average increase in body weight of initial studies on the high protein requirement of cats, has 25% to 30% is observed experimentally with continuous revealed other unique metabolic and nutritional attributes of presentation of food.29 Body condition scoring of cats pre- cats and demonstrated the complexity of establishing dietary sented to veterinary clinics show that by middle age (~ 7 years protein requirements. For some amino acids, minimal dietary old), more than one-third of neutered cats will be overweight concentrations needed for optimal growth in cats were found or obese.40 The effect of neutering is so potent that postneu- to be too low for other body functions. For these amino acids, tering weight gain is also observed in feral cats.41 Such cats dietary requirements were based on more sensitive biomark- expectedly would experience more exercise and less food ers than growth, such as prevention of cataracts in the case of abundance than privately owned neutered cats. histidine30 and minimizing urinary excretion of orotic acid An optimal protein:carbohydrate ratio that reduces post- in the case of arginine.31 For some amino acids, the dietary neutering weight gain may facilitate owner efforts to prevent matrix or proportion of nutrients affected requirements. Tau- weight gain in their cats. Relative to carbohydrate, dietary pro- rine and lysine requirements were found to vary with dietary tein is reputed to have a greater satiating potency, lower en- protein quality, quantity, and processing.32,33 Also, optimizing ergy utilization efficiency, and, as shown recently in lean cats, the coat color of cats was found to vary with absolute and rel- may induce greater thermogenesis23; however, a few factors ative amounts of dietary phenylalanine and tyrosine.34 As in would make determination of an optimal protein:carbohy- other species, the requirement for methionine was found to drate ratio difficult. Variation in palatability among test diets depend on dietary cyst(e)ine concentration,35 phenylalanine and amino acid composition of protein sources undoubtedly on dietary tyrosine concentration,36 and arginine on dietary would be encountered, which in turn may mask an effect of protein concentration.37 Unique to cats were discoveries that changing protein:carbohydrate ratio. Burger and Smith42 ex- optimal growth is supported by a very wide range, in ratio, of perienced a similar problem while investigating protein re- dietary essential to nonessential amino acids38 and that requirement for maintenance. Because their low-protein diet quirements for many essential amino acids are reduced with was not universally accepted, the protein requirement was increasing dietary protein content.39 These and other discov- based on observations of selected cats. The greater satiating eries revealed that some aspects of control of food intake, potency and thermogenesis and lower energy utilization effi- palatability, and metabolism of protein and amino acids in ciency of protein over carbohydrate might vary with amino cats cannot be directly extrapolated from other species. acid composition. In demonstrating that cats accommodate In considering areas of future research, classical methods maximal growth over a wide range of protein and amino acid for determining protein and amino acid requirements have concentrations, Taylor et al43 found that adjustments in di- been mostly applied to the growth stage of cats. With the ex- etary concentrations of methionine and arginine affected food

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intake. Toxicity effects of high concentrations of the amino amino acid requirements are optimized for longevity of off- acids were suggested by the investigators. spring. The protein:carbohydrate ratio in the diet appears to The protein and amino acid requirements of overweight have an effect on the body composition of queens during lac- and obese cats should perhaps also be evaluated. These cats tation. Queens given dry-type diets have less lactational constitute a large fraction of privately owned cats, and correc- weight loss than queens given canned diets.50 The higher car- tive weight loss is difficult to achieve, even with diets formu- bohydrate content of dry relative to canned diets is suggested lated for weight loss. The obese condition is believed to to reduce lactational loss through greater insulin-mediated increase free-radical production.44 Therefore, obese relative to inhibition of fat mobilization. Changes in glycemia and lean cats may have a greater requirement for sulfur amino acids abundance of endocrine factors important to regulation of (cyst[e]ine, methionine). Cyst(e)ine that is derived directly body composition in dams are believed to affect eventual from the diet or from methionine provides reducing equiva- body composition and the propensity for development of di- lents to affect redox status and is a rate-limiting substrate for abetes in offspring.51 To the author’s knowledge, this rela- synthesis of glutathione, a major cellular antioxidant. The tionship and the potential mediating role of dietary redox status of cells appears to have roles in modulating signal protein:carbohydrate ratio has not been studied in cats. transduction, gene expression, and apoptosis.45 Also, methio- As surveys of clinically presented cats indicate, the lifespan nine, which is consumed in cysteine and glutathione synthesis, of privately owned cats is increasing; as many as one-third to is a methylation substrate, and as such has an important role one-half of such cats are older than 7 years old.25,52 Protein in epigenic regulation. Recently, hypomethylation of DNA and and amino acid requirements in aging cats may be unique. associated proteins has been implicated in the cause of can- With increasing age, maintenance energy requirement in cats cers and type-2 diabetes,46 diseases for which obesity increases is reported to decrease by some53,54 but not all investigators.55,56 the risk of their development in cats.47 A tendency for protein digestibility to decrease with age is also Dietary enrichment of leucine may also be beneficial in observed.53,55 Because animals eat to meet their energy re- managing overweight and obese cats. Leucine is not metabo- quirements, protein and amino acid intake will decrease with lized extensively in the liver like other branched-chain amino decreasing energy requirement. The recent findings of taurine acids; hence, it reaches skeletal muscle in direct proportion to deficiency in dogs exemplify how changing energy require- dietary concentrations. In skeletal muscle, leucine is a regula- ments may influence amino acid requirements. Dietary sul- tor of initiation of protein synthesis, a fuel source, and a prin- fur amino acid concentration must be increased in some diets cipal donor of nitrogen for alanine and glutamine produced by to prevent taurine deficiency in dogs with low-maintenance skeletal muscle.48 Additionally, leucine is believed to modulate energy requirement.57 Hence, protein and amino acid re- insulin signaling and glucose use in skeletal muscle. These quirements may be increased in aged cats that consume less roles of leucine may be especially important in overweight cats energy than younger cats. Appreciation of this appears to be with insulin resistance because skeletal muscle is both a major the motivation in recent years for slight increases in the pro- determinant of insulin sensitivity and a consumer of glucose. tein content of commercial diets for senior cats. Obese humans have improved glucose and insulin responses In old cats, incidence of obesity decreases while, as ob- to meals when dietary protein is substituted with carbohy- served by some investigators, loss of lean mass increases.53 drate.49 The mechanism of this benefit is presently unknown The loss of lean mass is presumably from reduction in skele- but could plausibly involve leucine from dietary protein. tal muscle, as observed in aging rodents and humans.58 Skele- Protein and amino acid requirements of gestation and lac- tal muscle loss is believed by some to be the result of reduced tation are based on analyses of diets that support accepted postprandial protein synthesis and increased visceral amino but arbitrarily defined levels of performance, such as birth acid extraction. Loss of muscle mass is a concern to human weight, litter size, and lactational period weight loss in health providers because of associated declines in mobility queens and weight gains in kittens.50 Breakpoints indicating and health. Stimulation of skeletal muscle protein synthesis the minimal dietary concentrations for optimal responses by dispensable (nonessential) and indispensable amino acids have not been determined. In the context of increasing recog- (in particular, leucine) has prompted investigation of the use nition of nutrient programming and interest in the health of of amino acids to reduce age-related muscle loss.59–61 Similar aged cats, it seems valid to ask whether dietary protein and investigations may be warranted in cats. Study of dietary ma-

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nipulation to reduce the loss of lean mass is not without benefit of an amino acid depends on the supplementation precedent in cats. Cupp62 reported that loss of lean mass in mode, timing, and matrix of administration. For example, senior (~ 8 to 12 years old) and geriatric (~ >12 years old) symptoms of feline herpesvirus infection are reduced with ly- cats can be altered by nutrient compositional changes unre- sine given alone in oral capsules69 but not when enriched in a lated to protein changes. diet.70 Differences in circulating levels of lysine and nutrient Aging, as with obesity, is associated with increased oxida- antagonism are suggested to account for the difference in ef- tive stress, impaired glucose tolerance, and diabetes.63 For this fectiveness. reason, aged cats may benefit from dietary sulfur amino acid Glutamine was evaluated in cats using gastric instillation enrichment, which, as described for obesity, might compen- of a purified diet that contained glutamine and protein in the sate for possible depletion of glutathione. Indeed, low intra- form of crystalline amino acids.71 Glutamine administered in cellular glutathione concentrations in peripheral blood this way was not protective of methotrexate-induced injury mononuclear cells are observed in elderly humans.64 Al- of intestinal epithelium. One might ask if the outcome would though several factors are suggested as causal, an inadequate have been different if intact protein, protein-bound gluta- supply of precursor amino acids could be responsible. Un- mine, or parenterally administered glutamine were used. The fortunately, sensitive biomarkers of sulfur amino acid status latter condition may be important to the outcome because have not been identified in cats. This is perhaps a worthwhile compared with villus cells, intestinal crypts derive nutrients subject of future study. more from the intestinal arterial supply than from the The growing practice of clinical nutrition continues to fos- lumen.72 When glutamine is listed as an ingredient in com- ter interest in the use of varying dietary nutrient concentra- mercial diets, protein-bound glutamine is most likely being tions and profiles for treatment and prevention of disease. counted because the free amino acid is labile. Tracer studies Metabolism is variably altered, and usage efficiency is variably on the rate of protein digestion indicate that amino acids in decreased in disease states. The research activities of Morris protein would be absorbed more slowly than crystalline and Rogers4–7,9 have provided a valuable foundation upon amino acids.73,74 Hence, exposure time, the degree of intes- which to base dietary protein and amino acid manipulations tinal metabolism, and the peak circulating levels of an amino for disease treatment. For some diseases, deficiencies in acid depend on whether it is in free or bound form. These branch-chain amino acids, arginine, methioine, threonine, variables should be considered in amino acid manipulations histidine, taurine, and normally dispensable amino acids are intended for clinical applications. suggested65; however, the need for modification of dietary amino acid profiles is often uncertain because amino acid re- SOME CHALLENGES quirements for a disease condition generally are not known Studies on the use of protein and amino acid manipulations and markers of amino acid deficiency used in the healthy state for clinical applications have additional complexities. Per- may not apply to the diseased state. Also, if preexisting mal- haps foremost of these is that patient populations are het- nutrition is not encountered, amino acid mobilization due to erogeneous. Because of this, large numbers of patients are metabolic response to disease may be sufficient to meet short- needed to identify inadequacies and evaluate the effects of term tissue needs. A worthwhile first step in assessing ade- manipulations. Another complexity is measuring end points quacy of amino acid profile in diseased cats might be kinetic quantitatively, such as wound healing and immune response. measurements of plasma amino acids with parenteral or en- Making measurements in a clinical setting is often difficult teral feeding.66,67 Although difficult to achieve, measurements or, at best, inconvenient and requires an ardent commitment of intracellular amino acid concentrations would be of addi- of investigators. tional value for assessing adequacy.68 Evaluating the effective- Nitrogen balance largely has been relied on to estimate fe- ness of manipulating dietary amino acid profiles to correct a line maintenance requirements for protein and amino acids. detected imbalance would be a necessary second step. This measurement and many newer isotopic methods are In recent years, hypothesized benefits of dietary enrichment heavily influenced by whole-body protein synthesis. In addi- in lysine and glutamine for treatment of disease have been in- tion to being substrates of protein synthesis, dietary amino vestigated in cats. Interpretation of such studies is often com- acids become energy sources (e.g., glutamine, glutamate, as- plicated by the necessary experimental design choices. The partate), precursors of compounds (e.g., creatine, glutathione,

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nitric oxide), and cellular regulators. Optimizing these latter 9. Rogers QR, Morris JG. Up-regulation of nitrogen catabolic enzymes is not required to readily oxidize excess protein in cats. J Nutr 2002;132(9): functions may require greater dietary amino acid concentra- 2819-2820. tions than those needed to achieve nitrogen balance. A worth- 10. Park T, Rogers QR, Morris JG. High dietary protein and taurine increase while direction of future research may be identification and cysteine desulfhydration in kittens. J Nutr 1999;129(12):2225-2230. 11. Green A, Ramsey JJ, Villaverde C, et al. Adaptation of protein oxidation use of biomarkers of amino acid adequacy that are more sen- to protein intake in the domestic cat. Proceedings of the American Acad- sitive measures of amino acid requirement than body protein emy of Veterinary Nutrition 7th Annual Clinical Nutrition and Research Sym- posium, Seattle, 6 June 2007. turnover. An example of this would be to measure glutathione 12. Biourge V, Groff JM, Fisher C, et al. Nitrogen balance, plasma free amino levels in the liver as a means to optimize dietary sulfur amino acid concentrations and urinary orotic acid excretion during long-term acid requirement. Using sulfur amino acids for protein syn- fasting in cats. J Nutr 1994;24(7):1094-1103. 13. Hendriks WH, Moughan PJ, Tarttelin MF. Urinary excretion of endoge- 75 thesis has a higher priority than glutathione synthesis. Liver nous nitrogen metabolites in adult domestic cats using a protein-free glutathione content is important because it serves as a reser- diet and the regression technique. J Nutr 1997;127(4):623-629. 14. Kettelhut IC, Foss MC, Migliorini RH. Glucose homeostasis in a car- voir of cysteine that may be used for synthesis of glutathione nivorous animal (cat) and in rats fed a high-protein diet. Am J Physiol and maintenance of redox status in other tissues. 1980;239(5):R437-R444. Another challenge is predicting bioavailabilities of amino 15. Beliveau GP, Freedland RA. Metabolism of serine, glycine and threonine in isolated cat hepatocytes Felis domestica. Comp Biochem Physiol B acids in diets prepared for cats. Recommended dietary amino 1982;71(1):13-18. acid concentrations are greater than the minimum amino 16. Silva SV, Mercer JR. Effect of protein intake on amino acid catabolism and gluconeogenesis by isolated hepatocytes from the cat (Felis domes- acid concentrations used in defining dietary requirement. tica). Comp Biochem Physiol B 1985;80(3):603-607. This is because the bioavailabilities of amino acids in ingre- 17. Zoran DL. The carnivore connection to nutrition in cats. JAVMA dients are typically less than 100%, and processing has a vari- 2002;221(11):1559-1567. 76 18. Gannon MC, Nuttall JA, Damberg G, et al. Effect of protein ingestion on able effect of further decreasing bioavailabilities. Lysine is the glucose appearance rate in people with type 2 diabetes. J Clin En- especially susceptible. Heat treatment of good-quality pro- docrinol Metab 2001;86(3):1040-1047. tein in the presence of moisture and reducing sugar has been 19. Frank GR, Anderson WH, Pazak HE, et al. Use of a high-protein diet in the management of feline diabetes mellitus. Vet Ther 2001;2(3):238-246. shown to reduce lysine bioavailability in cats by more than 20. Hoenig M, Alexander S, Pazak H. Effect of high- and low-protein diet on 40%.33 Bioassays, rather than chemical assays, presently are the glucose metabolism and lipids in the cat. Compend Contin Educ Pract Vet 2001;23(suppl 9A):98. more reliable for accurately determining amino acid bioavail- 21. Rand JS, Farrow HA, Fleeman LM, Appleton DJ. Diet in the prevention abilities. This was recently demonstrated with several feline of diabetes and obesity in companion animals. Asia Pac J Clin Nutr diets.77 Unfortunately, routine use of bioassays for the evalu- 2003;12(suppl):S6. 22. Leray V, Siliart B, Dumon H, et al. Protein intake does not affect insulin ation of diets is cost prohibitive. A major advance in this area sensitivity in normal weight cats. J Nutr 2006;136(suppl 7):2028S- would be the development of simple and rapid laboratory 2030S. 23. Hoenig M, Thomaseth K, Waldron M, Ferguson DC. Insulin sensitivity, tests that reliably indicate amino acid bioavailabilities in fe- fat distribution, and adipocytokine response to different diets in lean line diets and ingredients used in the diets. and obese cats before and after weight loss. Am J Physiol Regul Integr Comp Physiol 2007;292(1):R227-R234. 24. Scarlett JM, Donoghue S. Associations between body condition and dis- REFERENCES ease in cats. JAVMA 1998;212(11):1725-1731. 1. MacDonald ML, Rogers QR, Morris JG. Nutrition of the domestic cat, a 25. Laflamme DP. Nutrition for aging cats and dogs and the importance of mammalian carnivore. Ann Rev Nutr 1984;4:521-562. body condition. Vet Clin North Am Small Anim Pract 2005;35(3):713-742. 2. National Research Council. Nutrient Requirements of Dogs and Cats. Rev 26. Ibrahim WH, Szabo J, Sunvold GD, et al. Effect of dietary protein qual- ed. Washington, DC: National Academy Press; 2006. ity and fatty acid composition on plasma lipoprotein concentrations 3. National Research Council. Nutrient Requirements of Laboratory Animals. and hepatic triglyceride fatty acid synthesis in obese cats undergoing 4th Rev ed. Washington, DC: National Academy Press; 1995. rapid weight loss. Am J Vet Res 2000;61(5):566-572. 4. Morris JG, Rogers QR. Assessment of the nutritional adequacy of pet 27. Richard MJ, Holck JT, Beitz DC. Lipogenesis in liver and adipose tissue of the foods through the life cycle. J Nutr 1994;124(12):2520-2534. domestic cat (Felis domestica). Comp Biochem Physiol B 1989;93(3):561-564. 5. Rogers QR, Morris JG, Freedland RA. Lack of hepatic enzymatic adap- 28. Veltri BC, Backus RC, Rogers QR, Depeters EJ. Adipose fatty acid compo- tation to low and high levels of dietary protein in the adult cat. Enzyme sition and rate of incorporation of alpha-linolenic acid differ between nor- 1977;22(5):348-356. mal and lipoprotein lipase-deficient cats. J Nutr 2006;136(12):2980-2986. 6. Rogers QR, Morris JG. Essentiality of amino acids for the growing kit- 29. Kanchuk ML, Backus RC, Calvert CC, et al. Weight gain in gonadec- ten. J Nutr 1979;109(4):718-723. tomized normal and lipoprotein lipase-deficient male domestic cats re- 7. Morris JG, Rogers QR. The metabolic basis for the taurine requirement sults from increased food intake and not decreased energy expenditure. of cats. Adv Exp Med Biol 1992;315:33-44. J Nutr 2003;133(6):1866-1874. 8. Russell K, Murgatroyd PR, Batt RM. Net protein oxidation is adapted to 30. Quam DD, Morris JG, Rogers QR. Histidine requirement of kittens for dietary protein intake in domestic cats (Felis silvestris catus). J Nutr growth, haematopoiesis and prevention of cataracts. Br J Nutr 2002;132(3):456-460. 1987;58(3):521-532.

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31. Costello MJ, Morris JG, Rogers QR. Effect of dietary arginine level on 55. Taylor EJ, Adams C, Neville R. Some nutritional aspects of ageing in urinary orotate and citrate excretion in growing kittens. J Nutr dogs and cats. Proc Nutr Soc 1995;54(3):645-656. 1980;110(6):1204-1208. 56. Harper EJ. Changing perspectives on aging and energy requirements: 32. Kim SW, Rogers QR, Morris JG. Maillard reaction products in purified aging, body weight and body composition in humans, dogs and cats. J diets induce taurine depletion in cats which is reversed by antibiotics. J Nutr 1998;128(suppl 12):2627S-2631S. Nutr 1996;126(1):195-201. 57. Ko KS, Backus RC, Berg JR, et al. Differences in taurine synthesis rate 33. Larsen JA, Calvert CC, Rogers QR. Processing of dietary casein decreases among dogs relate to differences in their maintenance energy require- bioavailability of lysine in growing kittens. J Nutr 2002;132(6 suppl ment. J Nutr 2007;137(5):1171-1175. 2):1748S-1750S. 58. Roubenoff R. Sarcopenia: effects on body composition and function. J 34. Anderson PJ, Rogers QR, Morris JG. Cats require more dietary pheny- Gerontol A Biol Sci Med Sci 2003;58(11):1012-1017. lalanine or tyrosine for melanin deposition in hair than for maximal 59. Dröge W, Holm E. Role of cysteine and glutathione in HIV infection growth. J Nutr 2002;132(7):2037-2042. and other diseases associated with muscle wasting and immunological 35. Smalley KA, Rogers QR, Morris JG. Methionine requirement of kittens given dysfunction. FASEB J 1997;11(13):1077-1089. amino acid diets containing adequate cystine. Br J Nutr 1983;49(3):411-417. 60. Katsanos CS, Kobayashi H, Sheffield-Moore M, et al. A high proportion 36. Williams JM, Morris JG, Rogers QR. Phenylalanine requirement of kit- of leucine is required for optimal stimulation of the rate of muscle pro- tens and the sparing effect of tyrosine. J Nutr 1987;117(6):1102-1107. tein synthesis by essential amino acids in the elderly. Am J Physiol 37. Taylor TP, Morris JG, Kass PH, Rogers QR. Increasing dispensable amino Endocrinol Metab 2006;291(2):E381-E387. acid in diets of kittens fed essential amino acids at or below their re- 61. Rieu I, Balage M, Sornet C, et al. Increased availability of leucine with quirement increases the requirement for arginine. Amino Acids leucine-rich whey proteins improves postprandial muscle protein syn- 1997;13(3):257-272. thesis in aging rats. Nutrition 2007;23(4):323-331. 38. Taylor TP, Morris JG, Kass PH, Rogers QR. Maximal growth occurs at a 62. Cupp CJ. Nutrient blend for prolonged healthy life in ageing cats. Com- broad range of essential amino acids to total nitrogen ratios in kittens. pend Contin Educ Pract Vet 2007;29(suppl 2A):26-29. Amino Acids 1998;15(3):221-234. 63. Fukagawa NK, Galbraith RA. Advancing age and other factors influenc- 39. Strieker MJ, Morris JG, Rogers QR. Increasing dietary crude protein does ing the balance between amino acid requirements and toxicity. J Nutr not increase the essential amino acid requirements of kittens. J Anim 2004;134(suppl 6):1569S-1574S. Physiol Anim Nutr (Berl) 2006;90(7-8):344-353. 64. Hernanz A, Fernandez-Vivancos E, Montiel C, et al. Changes in the in- 40. Donoghue S, Scarlett JM. Diet and feline obesity. J Nutr 1998;128 tracellular homocysteine and glutathione content associated with aging. (suppl 12):2776S-2778S. Life Sci 2000;67(11):1317-1324. 41. Scott KC, Levy JK, Gorman SP, Newell SM. Body condition of feral cats 65. Soeters PB, van de Poll MC, van Gemert WG, Dejong CH. Amino acid ade- and the effect of neutering. J Appl Anim Welf Sci 2002;5(3):203-213. quacy in pathophysiological states. J Nutr 2004;134(suppl 6):1575S-1582S. 42. Burger IH, Smith PM. Amino acid requirement of adult cats. In: Nutri- 66. Bérard MP, Pelletier A, Ollivier JM, et al. Qualitative manipulation of tion, Malnutrition, and Dietetics in the Dog and Cat: Proceedings of an In- amino acid supply during total parenteral nutrition in surgical patients. ternational Symposium Held in Hanover, 3- 4 September 1987. Edney ATB, J Parenter Enteral Nutr 2002;26(2):136-143. ed. British Veterinary Association, pp 49-51. 67. Mansoor O, Breuille D, Bechereau F, et al. Effect of an enteral diet sup- 43. Taylor TP, Morris JG, Willits NH, Rogers QR. Optimizing the pattern of plemented with a specific blend of amino acid on plasma and muscle essential amino acids as the sole source of dietary nitrogen supports protein synthesis in ICU patients. Clin Nutr 2007;26(1):30-40. near-maximal growth in kittens. J Nutr 1996;126(9):2243-2252. 68. Fürst P, Stehle P. What are the essential elements needed for the determi- 44. Keaney JF Jr, Larson MG, Vasan RS, et al. Obesity and systemic oxidative nation of amino acid requirements in humans? J Nutr 2004;134(suppl 6): stress: clinical correlates of oxidative stress in the Framingham Study. Ar- 1558S-1565S. terioscler Thromb Vasc Biol 2003;23(3):434-439. 69. Stiles J, Townsend WM, Rogers QR, Krohne SG. Effect of oral adminis- 45. Young VR, Ajami AM. Metabolism 2000: the emperor needs new tration of L-lysine on conjunctivitis caused by feline herpesvirus in cats. clothes. Proc Nutr Soc 2001;60(1):27-44. Am J Vet Res 2002;63(1):99-103. 46. Waterland RA. Assessing the effects of high methionine intake on DNA 70. Maggs DJ, Sykes JE, Clarke HE, et al. Effects of dietary lysine supple- methylation. J Nutr 2006;136(suppl 6):1706S-1710S. mentation in cats with enzootic upper respiratory disease. J Feline Med 47. Lund EM, Armstrong PJ, Kirk CA, Klausner JS. Prevalence and risk fac- Surg 2007;9(2):97-108. tors for obesity in adult cats from private US veterinary practices. Int J 71. Marks SL, Cook AK, Reader R, et al. Effects of glutamine supplementation Appl Res Vet Med 2005;3:88-96. of an amino acid-based purified diet on intestinal mucosal integrity in cats 48. Layman DK, Walker DA. Potential importance of leucine in treatment of with methotrexate-induced enteritis. Am J Vet Res 1999;60(6): 755-763. obesity and the metabolic syndrome. J Nutr 2006;136(suppl 1):319S-323S. 72. Stoll B, Burrin DG. Measuring splanchnic amino acid metabolism in 49. Farnsworth E, Luscombe ND, Noakes M, et al. Effect of a high-protein, vivo using stable isotopic tracers. J Anim Sci 2006;84(suppl):E60-E72. energy-restricted diet on body composition, glycemic control, and lipid 73. Collin-Vidal C, Cayol M, Obled C, et al. Leucine kinetics are different concentrations in overweight and obese hyperinsulinemic men and during feeding with whole protein or oligopeptides. Am J Physiol women. Am J Clin Nutr 2003;78(1):31-39. 1994;267:E907–E914. 50. Piechota TR, Rogers QR, Morris JG. Nitrogen requirement of cats dur- 74. Dangin M, Boirie Y, Guillet C, Beaufrere B. Influence of the protein di- ing gestation and lactation. Nutr Res 1995;15(10):1540-1546. gestion rate on protein turnover in young and elderly subjects. J Nutr 51. Ozanne SE, Constancia M. Mechanisms of disease: the developmental 2002;132(10):3228S-3233S. origins of disease and the role of the epigenotype. Nat Clin Pract En- 75. Stipanuk MH, Dominy JE Jr, Lee JI, Coloso RM. Mammalian cysteine docrinol Metab 2007;3(7):539-546. metabolism: new insights into regulation of cysteine metabolism. J Nutr 52. Gunn-Moore D. Considering older cats. J Small Anim Pract 2006;47(8): 2006;136(suppl 6):1652S-1659S. 430-431. 76. Hendriks WH, Emmens MM, Trass B, Pluske JR. Heat processing 53. Pérez-Camargo G. Cat nutrition: what is new in the old? Compend Con- changes the protein quality of canned cat foods as measured with a rat tin Educ Pract Vet 2004;26(suppl 2A):5-10. bioassay. J Anim Sci 1999;77(3):669-676. 54. Kienzle E, Edtstadtler-Pietsch G, Rudnick R. Retrospective study on the 77. Rutherfurd SM, Rutherfurd-Markwick KJ, Moughan PJ. Available (ileal energy requirements of adult colony cats. J Nutr 2006;136(suppl 7): digestible reactive) lysine in selected pet foods. J Agric Food Chem 1973S-1975S. 2007;55(9):3517-3522.

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SCIENTIFIC PROGRAM: FOCUS ON FELINES Trace Mineral Requirements in Cats: Challenging How We Define “Requirements”

Andrea J. Fascetti, VMD, PhD, DACVIM, DACVN Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California

Despite advances in our knowledge of companion animal nu- NRC recommendations at the time the study was conducted trition, very little information is available about trace mineral (5 mg copper/kg diet) were marginal for reproduction in cats requirements in cats. Historically, it has been hypothesized and that at least 5.8 mg copper/kg diet is necessary. that such requirements are greater during growth than in other life stages; however, research determining copper and zinc re- ZINC quirements in queens during gestation offers an interesting Zinc is an essential trace element for all animals and plays a opportunity to examine this hypothesis and raises a larger, role in the function of more than 200 known enzymes. The overarching question of how we define nutrient requirements. ubiquitous distribution of zinc among cells and the fact that it is the most abundant intracellular trace element suggest that COPPER it has a very basic biologic role. Therefore, zinc is especially In 1983, Doong et al1 demonstrated that copper is an in- important during reproduction and fetal development. While dispensable trace mineral in cats. On the basis of that study evaluating the effects of zinc concentrations on reproductive and copper requirements in rats, the National Research efficiency in queens consuming copper-deplete and copper- Council (NRC) proposed a copper requirement of 5 mg of replete purified diets, I observed a significant number of cleft copper/kg of diet for kittens for growth2; however, while palates and low birth weights in kittens born to queens con- testing diets in Association of American Feed Control suming diets with low zinc concentrations (<21 mg zinc/kg).5 Officials protocols, Morris and Rogers observed clinical The amount and form of dietary copper had no influence on signs compatible with copper deficiency in kittens born to these findings. At the time this experiment was conducted, the queens consuming a number of different commercial diets NRC recommended a minimum of 15 mg zinc/kg diet in kit- with copper contents exceeding this recommendation (J. G. tens fed diets containing a low quantity of compounds known Morris and Q. R. Rogers, personal communication, 1994). to decrease zinc bioavailability (e.g., phytate, fiber).1 Our find- Clinical signs included neonatal death, premature birth, ings challenge that recommendation. hypochromotricia, and collagen abnormalities. These ob- A subsequent study was undertaken with the objective of servations were the stimuli for further investigations con- determining zinc requirements for queens during gestation cerning dietary copper requirements for gestation and (A. J. Fascetti, unpublished data, 2001). Queens were fed puri- copper metabolism in cats. fied diets containing 5, 15, 25, or 50 mg zinc (provided as zinc In a study using a depletion–repletion design, queens were sulfate)/kg diet. Reproductive performance in the queens con- fed a copper-deplete purified diet (0.8 mg copper/kg diet) for suming the lower zinc diets was extremely poor, and virtually 4 months and then randomly allocated to one of three dietary all kittens were born dead or died within a day of parturition; treatment groups receiving copper (supplied as copper sulin addition, most of the kittens had congenital abnormalities, fate) at 4, 5.8, or 10.8 mg copper/kg diet.3,4 Dietary copper such as cleft palates, clubfeet, or curled long bones. Queens concentration had a significant effect on the time to concep- consuming diets with the greater concentrations of zinc had tion (P = 0.04). There also was a negative, linear relationship higher conception rates and more live births, and their off- between dietary copper (x = mg copper/kg diet) and mean spring had fewer congenital defects. These findings suggest that time (y = days) for queens to conceive (y = 43.38 – 2.87x; the NRC-recommended minimum requirement at the time R2 = 0.97). Based on these findings, it was concluded that the (15 mg zinc/kg diet) was not adequate for reproduction.

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CURRENT RECOMMENDATIONS demanding life stage and that by determining the require- The NRC provides recommendations for growth, mainte- ments for growth, one may subsequently apply the findings nance, and gestation/lactation for many of the required nu- to other life stages. Certainly, the example of the copper re- trients. Previous guidelines generally listed only a minimum quirement for feline growth versus that for gestation chal- requirement for each nutrient. Nutrient recommendations lenges this assumption. Although the story is not as complete now span minimum requirements or adequate intakes, rec- with respect to zinc, this mineral may be more in line with ommended allowances, and, where possible, safe upper lim- previous assumptions that the requirements are greater for its. The current NRC-recommended allowance for queens growth. Currently, there is spirited debate regarding how nu- during gestation and lactation is 8.8 mg copper/kg of diet.6 trient requirements are determined and how recommenda- This is greater than the current recommended allowances for tions for minimal and adequate intake and recommended growing kittens, which are 8.4 mg copper/kg diet following allowances can be made. All can agree, however, that more weaning and 5.0 mg copper/kg diet for maintenance.6 The information on the basic requirements for each life stage is current NRC-recommended allowances for zinc in growing necessary to achieve this goal. kittens is 75 mg zinc/kg diet following weaning and 74 mg zinc/kg diet for maintenance.6 These recommended al- REFERENCES 1. Doong G, Keen CL, Rogers QR, et al. Selected features of copper me- lowances are greater than that for gestation/lactation, which tabolism in the cat. J Nutr 1983;113:1963-1971. 6 is 60 mg zinc/kg diet. This recommendation for queens dur- 2. National Research Council. Nutrient Requirements of Cats. Washington, ing gestation/lactation was based on findings from two stud- DC: The National Academies Press; 1986. ies, one that determined the nitrogen requirements for 3. Fascetti AJ, Rogers QR, Morris JG. Dietary copper influences reproductive efficiency in queens. J Nutr 1998;128:2590S-2592S. gestation/lactation and one that used the factorial calculation 4. Fascetti AJ, Rogers QR, Morris JG. Dietary copper influences reproduc- method for determining the zinc requirement for lactating tion in cats. J Nutr 2000;130:1287-1290. queens.7,8 5. Fascetti AJ. Copper Nutriture in Queens: Dietary Modulation of Cuproen- zyme Activities and Reproduction [doctoral thesis]. Davis, CA: University of California, Davis; 2000. CONCLUSION 6. National Research Council. Nutrient Requirements of Cats. Washington, The examples of copper and zinc requirements in the queen DC: The National Academies Press; 2006. during gestation serve as interesting foils for the considera- 7. Piechota TR, Rogers QR, Morris JG. Nitrogen requirement of cats during gestation and lactation. Nutr Res 1995;15(10):1535-1546. tion of how nutrient requirements are determined. Tradi- 8. Kienzle E. Factorial calculation of nutrient requirements in lactating tionally, it has been suggested that growth is the most queens. J Nutr 1998;128:2609S-2614S.

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SCIENTIFIC PROGRAM: FOCUS ON FELINES Is My Cat Fat?

Denise A. Elliott, BVSc, PhD, DACVIM, DACVN Royal Canin USA, St. Charles, Missouri

Obesity is the most prevalent form of malnutrition in veteri- fluid (ICF) and extracellular fluid (ECF), minerals, glycogen, nary medicine. Surveys suggest that 25% to 30% of cats pre- and protein. FFM contains body cell mass (BCM), which is sented to veterinary clinics are overweight or obese.1 Obesity the metabolically active part of the body responsible for de- is defined as a pathologic condition characterized by an ac- termining most of the resting energy expenditure. BCM en- cumulation of fat in excess of that required for optimal body compasses those lean tissues most likely to be affected by function. The significance of obesity pertains to its role in the nutrition or disease over relatively short periods. Further- pathogenesis of a variety of diseases and the ability to exac- more, FFM is generally accepted as an index of protein nutri- erbate preexisting disease.2 “Is my cat fat?” is a question that tion, and therefore changes in FFM over time are assumed to cat owners ask veterinarians daily. The ability to answer this represent alterations in protein balance. question with objective data requires the ability to accurately measure body fat. Measurement of body fat also facilitates BODY WEIGHT MEASUREMENT understanding the response to weight-reduction programs. Body weight measurement is the simplest technique, and it Numerous methods exist for the assessment of body com- should be included in the examination of every cat. It pro- position; however, techniques such as densitometry, total vides a rough measure of total-body energy stores and changes body potassium measurement, and neutron activation analy- in weight-parallel energy and protein balance. In healthy cats, sis are not readily available. This discussion will focus on clin- body weight varies little from day to day. There can be wide ically relevant methods, such as body weight measurement, variations between scales, however. To avoid interscale varia- body condition scoring, morphometric measurements, dilution, the same scale should be used each time for an individ- tional techniques, bioelectrical impedance analysis (BIA), ual cat. In addition, it is preferable to use a pediatric scale and and dual-energy x-ray absorptiometry (DEXA). to routinely calibrate the scale to maintain accuracy. Body weight can be subdivided into two or more physio- It is important to note that a measurement of body weight logically distinct components. The traditional two-compart- by itself has little meaning. For instance, knowing that a ment model divides body weight into fat mass (FM) and Maine coon weighs 18 lb means little because the cat could fat-free mass (FFM).3,4 This model forms the basis of the ma- be overweight, underweight, or in ideal body condition. In jority of our current knowledge of body composition and de- addition, body weight can be falsely altered by dehydration pends on assumptions regarding the character of FM and or fluid accumulation. Therefore, body weight should not be FFM. The composition of FFM is assumed to be relatively used in isolation. constant, with a density of 1.1 g/mL at 37°C, a water content of 72% to 74%, and a potassium content of 50 to 70 BODY CONDITION SCORING mmol/kg.5 In addition, the major constituents of FFM are Body condition scoring provides a quick and subjective as- presumed to be present in fixed ratios. In comparison, FM is sessment of an animal’s overall body condition. The two relatively homogenous in composition, anhydrous, and most commonly used scoring systems in small animal prac- potassium free, with a density of 0.900 g/mL at 37°C. tice are a 5-point system, where a body condition score (BCS) The assessment of body composition in the form of FM of 3 is considered ideal, or a 9-point system, where a BCS of and FFM provides valuable information about the physical 4 to 5 is considered ideal. BCS in conjunction with body and metabolic status of the individual. FM can be considered weight gives clinicians a more complete perspective on a pa- to represent a calorie or energy storage depot, whereas FFM tient’s body condition and should be recorded in the medical represents the actual health of the animal. FFM is a het- record at every visit. Limitations of body condition scoring erogenous entity consisting predominantly of intracellular include the subjectivity inherent in the scoring system and

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interobserver variation. Finally, like body weight, BCS gives nary and respiratory losses of isotope. TBW can be measured an overall assessment of body condition; it cannot differen- with a precision and accuracy of 1% to 2%. The potential tiate between body compartments and does not provide any concern with this technique is the assumption of the hydra- precise quantitative information concerning alteration in tion factor, which may change with age, sex, species, breed, or FFM or lean body mass relative to FM. disease.8

MORPHOMETRIC MEASUREMENTS Extracellular Fluid Height and circumferential measurements of the abdomen, ECF is an important physiologic component of TBW that may hip, thigh, and upper arm are commonly used to estimate be altered in illness. ECF can be measured by use of com- 35 3- 35 2- - - 82 - percent body fat in humans. Circumferential measurements pounds such as inulin, S2O , SO4 , SCN , Br , and Br that have also been developed to estimate the percent body fat in distribute within the extracellular space; however, ECF mark- cats.6 The Feline Body Mass Index™ is determined by meas- ers may not distribute uniformly in the subcompartments of uring the rib cage circumference at the level of the ninth cra- the ECF (plasma, interstitium, lymph, connective tissue), nial rib and the leg index measurement (LIM), which is the some markers penetrate cells to an extent that cannot be pre- distance from the patella to the calcaneal tuber. The percent cisely determined, or the markers may bind to some degree body fat can be calculated using a simplified equation, such to endogenous components. Bromide is the most useful, safe, as 1.5(rib cage – LIM) – 9, or determined by consulting a ref- and widely used tracer for determination of extracellular water erence chart. Cats with more than 30% body fat are candi- (ECW) volume.9 Determination requires high-performance dates for a weight-loss program. The Feline Body Mass Index liquid chromatography, and correction factors can be applied is a very simple, yet objective, tool for determining a cat’s to account for the Gibbs-Donnan equilibrium, serum water, body fat content. In addition, it is particularly valuable for and distribution in nonextracellular sites. Simultaneous meas- convincing clients that their cat is indeed overweight and in urement of ECF and TBW enables the estimation of intracel- need of weight loss. lular water (ICW) volume (i.e., ICW = TBW – ECW). ICW volume most closely approximates BCM. DILUTIONAL TECHNIQUES Total Body Water BIOELECTRICAL IMPEDANCE ANALYSIS

Dilutional techniques rely on the principle of C1V1 = C2V2; BIA is an electrical method of assessing body composition that that is, the volume of a biologic fluid can be calculated fol- has the potential of quantifying TBW, ECW, ICW, BCM, FFM, lowing the administration and equilibration of a known con- and FM. Electrical conductance is used to calculate the com- centration of tracer. The total body water (TBW) method position of the body by measuring the nature of the conduc- relies on the assumption that fat has negligible water content tance of an applied electrical current in the patient. Body and FFM has fairly constant and known water content (73%). fluids and electrolytes are responsible for conductance, FFM can be calculated as TBW/0.73. Because body weight = whereas cell membranes produce capacitance. Because adi- Fat + FFM, an estimation of body composition can be made. pose tissue is less hydrated than lean body tissues, more adi-

Isotopes of hydrogen (deuterium oxide [D2O] and tritium pose tissue results in a smaller conducting volume or path for 3 oxide [ H2O]), urea, alcohol, N-acetyl-4-aminopyrine, and current and larger impedance to current passage. FFM con- 18 H2O distribute in the TBW compartment and have been em- tains virtually all the water in the body. Thus, if bioelectrical ployed to quantify TBW. The approach used in most labora- impedance is measured, a value for FFM can be determined. 18 tories is dilution of the stable isotopes D2O or H2O . These Two types of BIA systems are currently available: single fre- techniques have been successfully completed in cats7 and are quency, which applies a 50 kHz current, and multifrequency, appropriate for noninvasive studies; however, they do require which uses frequencies from 5 kHz to 1,000 KHz. A BIA test expensive analytical equipment. Deuterium and tritium un- is performed by placing four small electrodes on the body. dergo some exchange with nonaqueous H+, and hence can The electrical current is introduced into the patient from the overestimate TBW by 3% to 5%. Similarly, 18O will exchange distal electrodes; it then travels through the body and is de- with labile oxygen atoms, and hence can overestimate TBW tected by the proximal electrodes. Low frequencies (e.g., 5 by 0% to 1%. Consideration also needs to be given to uri- kHz) pass primarily through the ECW because of high cell

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IS MY CAT FAT?

membrane capacitance. In contrast, at higher frequencies the niques, DEXA relies on the assumption that lean body mass effect of cell membrane capacitance is diminished so the cur- is uniformly hydrated at 0.73 mL water/g. rent flows through both the ICF and ECF environments (or TBW). The proportion of the current in the ICF and ECF is SUMMARY frequency dependent. With the advent of technology and application of clinically Reliable BIA requires standardization and control of these relevant techniques, veterinarians can offer an objective an- variables, such as hydration status; consumption of food and swer to the question, “Is my cat fat?” The ability to accurately water; skin and air temperature; recent physical activity; con- measure body fat and FFM (lean body mass) is vital for un- ductance of the examination table; patient age, size, shape, and derstanding the causes and effects of obesity. In addition, posture; and electrode positioning. However, BIA has been these techniques allow critical appraisal of the effect of nu- shown to be a safe, noninvasive, rapid, portable, and repro- trient composition on body composition. ducible method for estimating body composition in healthy cats.10,11 Calculation of ECF–ICF takes approximately 1 minute; REFERENCES 1. Scarlett JM, Donaghue S. Overweight cats: prevalence and risk factors. hence, BIA provides virtually instantaneous online informa- Int J Obes 1994;18:S22-S28. tion of body composition that has never before been available. 2. Scarlett JM, Donaghue S. Associations between body condition and disease in cats. JAVMA 1998;212:1725-1731. DUAL-ENERGY X-RAY ABSORPTIOMETRY 3. Keys A, Brozek J. Body fat in adult man. Physiol Rev 1953;33:245-325. 4. Brozek J, Grande F, Anderson JT, et al. Densitometric analysis of body DEXA is a technique originally developed for precise measure- composition: revision of some quantitative assumptions. Ann N Y Acad ment of bone mineral content; however, it is now also used to Sci 1963:113-140. measure both body fat and nonbone lean tissue. DEXA uses 5. Pace N, Rathbun EN. Studies on body composition III: the body water and chemically combined nitrogen content in relation to fat content. J photons of two different energy levels (70 and 140 kVp) to dis- Biol Chem 1945;158:685-691. tinguish the type and amount of tissue scanned. The x-ray 6. Hawthorne AJ, Butterwick RF. Predicting the body composition of cats: source is positioned underneath the table supporting the pa- development of a zoometric measurement for estimation of percentage body fat in cats. J Vet Intern Med 2000;14:365. tient, with the detector housed in an arm above the patient. 7. Backus RC, Havel PJ, Gingerich RL, et al. Relationship between serum During a scan, the source and detector move together over the leptin immunoreactivity and body fat mass as estimated by use of a novel gas-phase Fourier transform infrared spectroscopy deuterium di- patient. The detector measures the amount of x-rays that pass lution method in cats. Am J Vet Res 2000;61:796-801. through the subject. The x-rays of the two different energy lev- 8. Wang Z, Deurenberg P, Wang W, et al. Hydration of fat-free body mass: els are impeded differently by bone mineral, lipid, and lean tis- review and critique of a classic body-composition constant. Am J Clin Nutr 1999;69:833-841. sue. Algorithms are used to calculate both the quantity and type 9. Vaisman N, Pencharz PB, Koren G, et al. Comparison of oral and intra- of tissue in each pixel scanned. DEXA calculates bone mineral venous administration of sodium bromide for extracellular water meas- density, bone mineral content, FM, and lean body mass. urements. Am J Clin Nutr 1987;46:1-4. 10. Elliott DA. Evaluation of multifrequency bioelectrical impedance analy- DEXA’s low coefficient of variation for measuring bone sis for the assessment of extracellular and total body water in healthy mineral content (~1%) makes it a very precise technique. cats. J Nutr 2002;132(6 suppl 2):1757S-1759S. DEXA is also safe and quick, requiring 10 to 30 minutes for a 11. Stanton CA, Hamar DW, Johnson DE, et al. Bioelectrical impedance and zoometry for body composition analysis in domestic cats. Am J Vet whole-body scan. Similar to other body composition tech- Res 1992;53:251-257.

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SCIENTIFIC PROGRAM: FOCUS ON FELINES Adipokines and Their Importance in Obese Cats

M. Anne Hickman, DVM, PhD, DACVN Global Research and Development, Cardiovascular, Metabolic, and Endocrine Diseases, Pfizer, Inc., New London, Connecticut

Obesity is a chronic disease that is growing at an alarming rate leptin, many other adipokines have been discovered and are in both humans and their feline companions. Obesity increases now in the process of being characterized. the risk for a number of metabolic abnormalities in humans and Adipokines have important physiologic effects on multiple cats, including insulin resistance, type 2 diabetes mellitus organs, including the brain, liver, muscle, adipose (paracrine (T2DM), hypertension, and dyslipidemia.1–7 Obesity has been effect) bone, reproductive organs, immune cells, and blood regarded as a simple energy imbalance, with energy intake ex- vessels; however, most adipokines are dysregulated in ceeding energy expenditure; however, this view has changed over response to alterations of body fat mass, one of the best ex- the last 10 years based on a greater understanding of adipose tis- amples being obesity.15–17 Adipokine responses are also influ- sue and its function in normal states and disease. We now know enced by the distribution of body fat increase, as excessive fat that adipose tissue reserves are carefully maintained and regu- is not only stored in adipose tissue, but is abnormally de- lated by complex systems that integrate food intake, substrate posited in muscle and liver tissue as well.17,18 The majority of partitioning, and energy expenditure and can be influenced by adipokines studied to date are hypersecreted in obesity, a no- environmental conditions and individual genetics.8,9 Obesity re- table exception being adiponectin, which declines. Adipokine sults from dysregulation of these complex systems and is a dis- alterations cause abnormalities in insulin action, glucose and ease that is now recognized to have significant metabolic and fat metabolism, immune function, coagulation, and en- health implications. Alterations in the secretion of adipokines, dothelial cell function, eventually leading to a proinflamma- protein signals, and factors originating in adipose tissue under- tory state characterized by insulin resistance and altered lie many of the abnormalities in obesity. immune function.15–19 Adipokine alterations appear to pro- White adipose tissue has been viewed as a passive storage vide the link between obesity-related diseases as diverse as depot for excess energy in the form of triglycerides and a site T2DM and cancer. The list of adipokines is ever increasing and of release of fatty acids when energy is needed. This view was includes leptin, adiponectin, resistin, retinol-binding protein partially held because adipose tissue appears to be a simple 4 (RBP4), apelin, omentin, monocyte chemoattractant pro- tissue composed primarily of large cells filled with triglyceride tein-1 (MCP-1), transforming growth factor-β, interleukin (~85% of content)9; however, white adipose tissue is now un- (IL)-1β, IL-6, IL-8, IL-10, macrophage migration inhibitory derstood to be a complex tissue composed of multiple func- factor, haptoglobin, serum amyloid-A, nerve growth factor, tional cell types, including mature adipocytes, preadipocytes, adipsin, plasminogen activator inhibitor-1 (PAI-1), fasting- fibroblasts, endothelial cells, and macrophages, and the cell induced adipose factor, metallothionein, angiotensinogen, composition can change in response to various conditions.10,11 complement C3, fibronectin, and vascular endothelial growth Functionally, white adipose tissue is an active endocrine and factor. This review will attempt to highlight a few of the bet- paracrine organ that secretes a wide array of mediators that ter-understood adipokines and their role in obesity. participate in regulation of diverse metabolic processes, including food intake, energy expenditure, lipid and carbohy- LEPTIN drate metabolism, angiogenesis, reproduction, vascular Leptin, a 16-kDa, cytokine-like protein, is encoded by the ob remodeling, blood pressure, and coagulation. A major ad- gene and is secreted primarily by adipose tissue in propor- vance in our understanding of adipose tissue was made with tion to body fat stores and immediate nutritional state.12,20–22 the discovery of leptin, a secreted signal from adipose tissue to Leptin is a pleiotropic hormone with multiple actions on the the hypothalamus that signals the size of body fat stores as brain, pancreas, liver, immune system, and adipose.23 The im- part of its wide range of biologic functions.12–14 In addition to portance of leptin was first investigated in loss-of-function

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rodent models of obesity, such as ob/ob and db/db mice. tients with T2DM and coronary artery disease, and low Leptin is one of a few adipokines that has also been investi- adiponectin concentrations have been proposed as risk mark- gated in cats. Several studies have demonstrated that, similar ers for these diseases. Adiponectin concentrations are de- to other species, plasma leptin concentrations are correlated creased in obesity but can be increased by weight loss or with body fat content and concentration increase or decrease treatment with thiazoladinediones.46 Few data are available in in response to weight gain or weight loss, respectively.24–29 Al- cats regarding adiponectin, but plasma concentrations have though few studies have investigated the physiologic effects of been shown to correlate with body fat mass and to increase leptin in cats, it is likely that leptin behaves similarly in cats in response to weight loss.29 as it does in other species. In mice and humans, adiponectin has been shown to func- Leptin is normally a signal of energy sufficiency that results tion as both an insulin-sensitizing factor and an antiinflam- in decreased food intake and increased energy expenditure, ac- matory agent, antagonizing many of the negative effects of tions that are mediated primarily by central sympathetic activa- tumor necrosis factor (TNF)-α.46 Overexpression or exogenous tion.30–33 Centrally, leptin interacts with hypothalamic pathways administration of adiponectin results in glucose lowering and involved in energy regulation and acts to inhibit release of the amelioration of insulin resistance in murine models of obesity orexigenic peptides, neuropeptide Y and agouti gene-related and diabetes. Adiponectin is the most abundantly secreted peptide, and increase release of the anorexigenic peptides, pro- adipokine and circulates at high concentrations (1000-fold opiomelanocortin and cocaine- and amphetamine-regulated higher than most polypeptide hormones).43 It is rarely found transcript. In peripheral tissue, leptin action is mediated in part as a monomer and circulates in plasma as trimer, hexamer by increased expression and activation of AMP-activated protein (low molecular weight form), or multimeric forms of 12 to kinase (AMPK).34,35 AMPK inhibits the enzyme acetyl–coenzyme 18 subunits (high molecular weight form).47 The various A (CoA) carboxylase, leading to reduced levels of malonyl-CoA forms appear to have different roles, with the high molecular and increased entry of fatty acids into the mitochondria, where weight form having a predominant action in the liver. Similar they undergo β-oxidation. Leptin may also stimulate fatty acid to leptin, adiponectin activates AMPK and increases fatty acid oxidation by activation of peroxisome proliferator-activated re- oxidation in skeletal muscle, liver, and other tissues.19,48 It also ceptor-α (PPARα).36 In liver, pancreatic islet, and adipose tissue, causes enhanced glucose uptake in skeletal muscle due to in- leptin inhibits the expression of sterol response element-bind- creased translocation of glucose transporter 4 (GLUT 4).49 ing protein-1c, resulting in inhibition of lipogenesis in these tis- These effects, which lead to decreased triglyceride content in sues.37 Through these mechanisms and others, leptin causes tissue and enhanced insulin sensitivity, and its emerging an- decreased triglyceride content in tissue, such as skeletal muscle, tiinflammatory role make adiponectin one of the key candi- liver, and pancreas, and decreased circulating concentrations of date molecules mediating negative changes in obesity. fatty acids; these effects, among others, lead to improved insulin sensitivity. Leptin-deficient and leptin-resistant rodents have se- RESISTIN vere insulin resistance, which can be ameliorated by leptin ad- Resistin is a 12-kDa peptide hormone expressed primarily by ministration, even before reductions in body weight38–40; adipocytes in rodents and macrophages in humans.50 Circu- however, obese humans and cats have high circulating levels of lating concentrations of resistin are increased in rodent mod- leptin but do not benefit from its positive metabolic effects, lead- els of diet-induced and genetic obesity.51 Based on its actions ing to the concept of leptin resistance. Although this concept is in rodents to increase blood glucose and insulin levels and to now widely accepted, the molecular basis is still controversial impair insulin function, resistin was believed to be a major and under active investigation. link between obesity and insulin resistance. In addition, deletion of the resistin gene in ob/ob mice leads to improved glu- ADIPONECTIN cose tolerance and insulin sensitivity through increased Adiponectin, or adipocyte complement-related protein, is a AMPK activity in liver and increased insulin-mediated glu- 35-kDa protein that is almost exclusively expressed in white cose disposal in muscle and adipose tissue52; however, adipose tissue.41,42 In humans, adiponectin levels are inversely human resistin shares only 59% identity with murine resistin correlated with body fat content, hepatic fat content, dyslipi- and is expressed primarily in macrophages, not in demia, and insulin resistance.43–45 Levels are very low in pa- adipocytes.53 In humans, there is no convincing link between

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plasma resistin concentrations and body fat stores or insulin has been done in cats to investigate inflammatory adipokine resistance. Tissue sites and the role of resistin in obese and responses to obesity, although data gleaned from a study on diabetic cats are unknown, but if these factors in cats are sim- dogs demonstrate that canine adipose tissue expresses many ilar to those in rodents, they could contribute to obesity-re- of these cytokines.62 lated insulin resistance. REGIONAL ADIPOSE EFFECTS RETINOL-BINDING PROTEIN Adipokine expression and secretion appear to be influenced RBP4, a 21-kDa protein, is another adipokine implicated in in humans and rodents by regional adipose tissue distribu- obesity-related insulin resistance, although current data are tion.18,19 These findings likely have importance in cats as well, still controversial.54 Mice lacking GLUT 4, specifically in adi- but data are not yet available. Visceral (omental and mesen- pose tissue, are insulin resistant in muscle and liver and have teric), intramyocellular, and intrahepatic fat content are re- increased circulating concentrations of RBP4.55 These find- ported to have greater effects on insulin resistance and ings led to the hypothesis that RBP4 is secreted by adipose inflammation than subcutaneous fat, and many of these ef- tissue in response to plasma glucose concentrations. In hu- fects appear to be related to differential adipokine secretion. mans, circulating concentrations of RBP4 are correlated with Differences are also reported in men compared to women adiposity, abdominal obesity, insulin levels, and insulin re- and in different ethnic groups. Data in cats on the effects of sistance.56 Levels are increased in humans with impaired glu- regional adipose tissue distribution could be important in cose tolerance or diabetes and are decreased after weight loss. our understanding of risk assessment for obese cats.

INFLAMMATORY ADIPOKINES CONCLUSION Inflammatory and prothrombotic adipokines also have in- In summary, our knowledge of adipose tissue as an impor- creased expression and release in obesity.8–11,15,16,57 The best tant endocrine organ is rapidly increasing. Adipokines play studied of these adipokines are IL-6, TNF-α, and PAI-1. In an important and diverse role in multiple facets of normal obesity, the number of macrophages present in white adi- metabolism and physiology. Adipokines can become dysreg- pose tissue is increased, and adipocytes and adipose-tissue- ulated in obesity and may have direct causal links to many associated macrophages secrete elevated concentrations of of the associated abnormalities, although more research in inflammatory cytokines.11,16 Inflammatory adipokines not this area is required for a full understanding. In all species, in- only play a role in the proinflammatory state of obesity but cluding cats, some adipokines appear to be common and also may mediate some of obesity’s effects on insulin sensi- function similarly, but other factors have more species speci- tivity.58 TNF-α and IL-6 impair insulin signaling and have ac- ficity. Further research in cats is warranted to understand the tions that lead to insulin resistance; the significance of these role of these fascinating molecules so that therapies for dis- effects in humans is still controversial. TNF-α activates the eases such as obesity and T2DM can be developed. transcription factor nuclear factor-κB, which results in proinflammatory gene expression changes in many tissues.59 In REFERENCES adipocytes, these changes include decreased gene expression 1. Baskin ML, Ard J, Franklin F, Allison DB. Prevalence of obesity in the United States. Obes Rev 2005;6:5-7. of adiponectin and increased expression of PAI-1 and com- 2. Mokdad AH, Bowman BA, Ford ES. The continuing epidemics of obe- plement C3. Some of the mediators increased by TNF-α are sity and diabetes in the United States. JAMA 2001;286:1195-1200. 3. Pickering TG. Obesity and hypertension: a growing problem. J Clin Hy- also proposed to be links between obesity and hyperten- pertens 2001;3:252-254. sion.60 Increased inflammatory adipokine release from adi- 4. German AJ. The growing problem of obesity in dogs and cats. J Nutr pose tissue has been proposed to be due to hypoxia (caused 2006;136:1940S-1946S. 5. Lund EM, Armstrong PJ, Kirk CA, Klausner JS. Prevalence and risk fac- by poor circulation due to expanded adipose tissue mass)16 tors for obesity in adult cats from private US veterinary practices. Int J and altered communication between adipocytes and Appl Res Vet Med 2005;3:88-96. 6. Panciera DL, Thomas CB, Eicker SW, Atkins CE. Epizootiologic patterns of di- macrophages within adipose tissue. Adipose tissue levels of abetes mellitus in cats: 333 cases (1980–1986). JAVMA 1990;197:154-158. MCP-1, produced by monocytes and adipocytes, are elevated 7. Scarlett JM, Donoghue S. Associations between body condition and disease in cats. JAVMA 1998;212:1725-1731. with increasing adiposity.61 This leads to increased recruit- 8. Badman MK, Flier JS. The adipocyte as an active participant in energy ment of macrophages as adipose tissue expands. Little work balance and metabolism. Gastroenterology 2007;132:2103-2115.

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9. Trayhurn P, Bing C, Wood IS. Adipose tissue and adipokines: energy 32. Balthasar N, Coppari R, McMinn J, et al. Leptin receptor signaling in regulation from the human perspective. J Nutr 2006;136:1935S-1939S. POMC neurons is required for normal body weight homeostasis. Neu- 10. Hausman GJ. The comparative anatomy of adipose tissue. In: Cryer A, ron 2004;42:983-991. Van RLR, eds. New Perspectives in Adipose Tissue: Structure, Function and 33. Coppari R, Ichinose M, Lee CE, et al. The hypothalamic arcuate nucleus: Development. London: Butterworths; 1985:1-21. a key site for mediating leptin’s effects on glucose homeostasis and lo- 11. Weisberg SP, McCann D, Desai M, et al. Obesity is associated with comotor activity. Cell Metab 2005;1:63-72. macrophage accumulation in adipose tissue. J Clin Invest 2003;112: 34. Steinberg GR, Rush JW, Dyck DJ. AMPK expression and phosphoryla- 1796-1808. tion are increased in rodent muscle after chronic leptin treatment. Am 12. Zhang Y, Proenca R, Maffei M, et al. Positional cloning of the mouse J Physiol Endocrinol Metab 2003;284:E648-E654. obese gene and its human homologue. Nature 1994;372:425-432. 35. Minokoshi Y, Kim YB, Peroni OD, et al. Leptin stimulates fatty-acid oxida- 13. Chen H, Charlat O, Tartaglia LA, et al. Evidence that the diabetes gene tion by activating AMP-activated protein kinase. Nature 2002;415:339-343. encodes the leptin receptor: identification of a mutation in the leptin re- 36. Lee Y, Yu K, Gonzales F, et al. PPAR alpha is necessary for the lipogenic ceptor gene in db/db mice. Cell 1996;84:491-495. action of hyperleptinemia on white adipose and liver tissue. Proc Natl 14. Tartaglia LA, Dembski M, Weng X, et al. Identification and expression Acad Sci USA 2002;99:11848-11853. cloning of a leptin receptor, OB-R. Cell 1995;83:1263-1271. 37. Nogalska A, Sucajtys-Szulc E, Swierczynski J. Leptin decreases lipogenic 15. Murdolo G, Smith U. The dysregulated adipose tissue: a connecting link enzyme gene expression through modification of SREBP-1c gene expres- between insulin resistance, type 2 diabetes mellitus and atherosclerosis. sion in white adipose tissue of aging rats. Metabolism 2005;54:1041-1047. Nutr Metab Cardiovasc Dis 2006;16:S35-S38. 38. Halaas JL, Gajiwala KS, Maffei M, et al. Weight-reducing effects of the 16. Trayhurn P, Wood IS. Adipokines: inflammation and the pleiotropic plasma protein encoded by the obese gene. Science 1995;269:543-546. role of white adipose tissue. Br J Nutr 2004;92:347-355. 39. Campfield LA, Smith FJ, Guisez Y, et al. Recombinant mouse OB pro- 17. Yildiz BO, Suchard MA, Wong ML, et al. Alterations in the dynamics of tein: evidence for a peripheral signal linking adiposity and central neu- circulating ghrelin, adiponectin, and leptin in human obesity. Proc Natl ral networks. Science 1995;269:546-549. Acad Sci USA 2004;101:10434-10439. 40. Pelleymounter MA, Cullen MJ, Baker MB, et al. Effects of the obese gene 18. Rattarasarn C. Physiological and pathophysiological regulation of re- product on body weight regulation in ob/ob mice. Science 1995; gional adipose tissue in the development of insulin resistance and type 269:540-543. 2 diabetes. Acta Physiol 2006;186:87-101. 41. Scherer PE, Williams S, Fogliano M, et al. A novel serum protein simi- 19. Dyck DJ, Heigenhauser GJF, Bruce CR. The role of adipokines as regular to C1q, produced exclusively in adipocytes. J Biol Chem 1995;270: lators of skeletal muscle fatty acid metabolism and insulin sensitivity. 26746-26749. Acta Physiol 2006;186:5-16. 42. Hu E, Liang P, Spiegelman BM. AdipoQ is a novel adipose-specific gene 20. Ahima RS, Prabakaran D, Mantzoros C, et al. Role of leptin in the neu- dysregulated in obesity. J Biol Chem 1996;271:10697-10703. roendocrine response to fasting. Nature 1996;382:250-252. 43. Kadowaki T, Yamauchi T, Kubota N, et al. Adiponectin and adiponectin 21. Sivitz W, Wayson S, Bayless M, et al. Leptin and body fat in type 2 diabetes receptors in insulin resistance, diabetes, and the metabolic syndrome. J and monodrug therapy. J Clin Endocrinol Metab 2003;88:1543-1553. Clin Invest 2006;116:1784-1792. 22. Considine RV, Sihna MK, Heiman ML, et al. Serum immunoreactive- 44. Arita Y, Kihara S, Ouchi N, et al. Paradoxical decrease of an adipose- leptin concentrations in normal-weight and obese humans. N Engl J specific protein, adiponectin, in obesity. Biochem Biophys Res Commun Med 1996;334:292-295. 1999;257:79-83. 23. Bjorbaek C, Kahn BB. Leptin signaling in the central nervous system 45. Weyer C, Funahashi T, Tanaka S, et al. Hypoadiponectinemia in obesity and the periphery. Recent Prog Horm Res 2004;59:305-331. and type 2 diabetes: close association with insulin resistance and hy- 24. Backus RC, Havel PJ, Gingerich RL, Rogers QR. Relationship between perinsulinemia. J Clin Endocrinol Metab 2001;86:1930-1935. serum leptin immunoreactivity and body fat mass as estimated by use 46. Whitehead JP, Richards AA, Hickman IJ, et al. Adiponectin: a key of a novel gas-phase Fourier transform infrared spectroscopy deuterium adipokine in the metabolic syndrome. Diabetes Obes Metab 2006;8(3): dilution method in cats. Am J Vet Res 2000;61:796-801. 264-280. 25. Appleton DJ, Rand JS, Sunvold GD. Plasma leptin concentrations in 47. Pajvani UB, Hawkins M, Combs TP, et al. Complex distribution, not ab- cats: reference range, effect of weight gain and relationship with adi- solute amount of adiponectin, correlates with thiazolidinedione-medi- posity as measured by dual energy X-ray absorptiometry. J Feline Med ated improvement in insulin sensitivity. J Biol Chem 2004;279: Surg 2000;2:191-199. 12152-12162. 26. Appleton DJ, Rand JS, Sunvold GD. Plasma leptin concentrations are in- 48. Yamauchi T, Kamon J, Minokoshi Y, et al. Adiponectin stimulates glu- dependently associated with insulin sensitivity in lean and overweight cose utilization and fatty-acid oxidation by activating AMP-activated cats. J Feline Med Surg 2002;4:83-93. protein kinase. Nat Med 2002;8:1288-1295. 27. Kanchuk ML, Backus RC, Calvert CC, et al. Neutering induces changes 49. Ceddia RB, Somwar R, Maida A, et al. Globular adiponectin increases in food intake, body weight, plasma insulin and leptin concentrations GLUT 4 translocation and glucose uptake but reduces glycogen synthe- in normal and lipoprotein lipase-deficient cats. J Nutr 2002;132:1730S- sis in rat skeletal muscle cells. Diabetologia 2005;48:132-139. 1732S. 50. Steppan CM, Lazar MA. The current biology of resistin. J Intern Med 28. Shibata H, Sasaki N, Honjoh T, et al. Feline leptin: immunogenic and 2004;255:439-447. biological activities of the recombinant protein, and its measurement by 51. Steppan CM, Bailey ST, Bhat S, et al. The hormone resistin links obesity ELISA. J Vet Med Sci 2003;65:1207-1211. to diabetes. Nature 2001;409:307-312. 29. Hoenig M, Thomaseth K, Waldron M, Ferguson DC. Insulin sensitivity, 52. Qi Y, Nie Z, Lee YS, et al. Loss of resistin improves glucose homeostasis fat distribution, and adipocytokine response to different diets in lean in leptin deficiency. Diabetes 2006;55:3083-3090. and obese cats before and after weight loss. Am J Physiol Regul Integr 53. Banerjee RR, Lazar MA. Resistin: molecular history and prognosis. J Mol Comp Physiol 2006;292:R227-R234. Med 2003;81:218-226. 30. Cowley MA, Smart JL, Rubinstein M, et al. Leptin activates anorexigenic 54. Yang Q, Graham TE, Mody N, et al. Serum retinol binding protein 4 POMC neurons through a neural network in the arcuate nucleus. Nature contributes to insulin resistance in obesity and type 2 diabetes. Nature 2001;411:480-484. 2005;436:356-362. 31. van den Top M, Lee K, Whyment AD, et al. Orexigen-sensitive 55. Abel ED, Peroni O, Kim JK, et al. Adipose-selective targeting of the NPY/AgRP pacemaker neurons in the hypothalamic arcuate nucleus. GLUT4 gene impairs insulin action in muscle and liver. Nature Nat Neurosci 2004;7:493-494. 2001;409:729-733.

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56. Graham TE, Yang Q, Bluher M, et al. Retinol-binding protein 4 and in- atherogenesis. Endocrinology 2003;144:2195. sulin resistance in lean, obese, and diabetic subjects. N Engl J Med 60. Brasier AR, Li J, Wimbish KA. Tumor necrosis factor activates an- 2006;354:2552-2563. giotensinogen gene expression by the Rel A transactivator. Hypertension 57. Kern PA, Saghizadeh M, Ong JM, et al. The expression of tumor necro- 1996;27:1009. sis factor in human adipose tissue: regulation by obesity, weight loss, 61. Kanda H, Tateya S, Ttamori Y, et al. MCP-1 contributes to macrophage and relationship to lipoprotein lipase. J Clin Invest 1995;95:2111-2119. infiltration into adipose tissue, insulin resistance, and hepatic steatosis 58. Hotamisligil GS, Shargill NS, Spiegelman BM. Adipose expression of in obesity. J Clin Invest 2006;116:1494-1505. tumor necrosis factor-alpha: direct role in obesity-linked insulin resist- 62. Eisele I, Wood IS, German AJ, et al. Adipokine gene expression in dog ance. Science 1993;259:87-91. adipose tissue and dog white adipocytes differentiated in primary cul- 59. Lyon CJ, Law RE, Hsueh VA. Minireview: adiposity, inflammation, and ture. Horm Metab Res 2005;37:474-481.

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SCIENTIFIC PROGRAM: FOCUS ON FELINES New Technologies for Pharmaceutical and Nutrition Research

Marnie L. MacDonald, PhD MM Health Advocates, Scottsdale, Arizona

The Nestlé Purina Nutrition Forum, honoring Drs. James particular, linoleate appears to supply the needs of male cats Morris and Quinton Rogers, provides an opportunity to re- for reproduction and prevention of testis degeneration, but flect not only on the legacy of their work and its contribu- female cats require preformed arachidonate for successful tions to animal health but also on the evolution of animal pregnancies and normal litters.8,9 nutrition and medicine over the course of their careers. This The role of arachidonic acid as an essential component of presentation describes the evolution of approaches to the membrane phospholipids and as a precursor for the study of nutrient function over the past several decades, in- eicosanoids allowed us to formulate some hypotheses re- cluding whole animal nutrition, metabolism, and physiol- garding the consequences of arachidonate deficiency in cats.

ogy; the molecular mechanisms of nutrient metabolism and Because thromboxane A2, a potent thrombotic agent, is de- regulation; and the study of these mechanisms in the context rived from arachidonic acid, we hypothesized that a deficiency of living cells. The parallels between the fields of nutrition, of dietary arachidonate would affect platelet function. Con- pharmacology, and toxicology in the wake of new technolo- sistent with this hypothesis, we showed that arachidonate is a gies for illuminating the mechanisms of action of nutrients requirement in cats for normal platelet aggregation.11 The cen- and drugs at the cellular level is also described. tral role of arachidonate in cellular function in mammals has subsequently been highlighted by the discoveries of various WHOLE ANIMAL NUTRITION, METABOLISM, enzymes with a preference for arachidonoyl-containing sub- AND PHYSIOLOGY strates. Selective enzymes include the arachidonate-selective The legacy of Rogers’ and Morris’ collaboration results from acyl-CoA synthetase,12 acyl-CoA thioesterase,13 and phospho- 14 their classic studies in whole animal nutrition, combined lipase A2 as well as a membrane-bound arachidonoyl-spe- with a deep understanding of nutritional biochemistry and cific diacylglycerol kinase.15 The latter enzyme functions to metabolism, to define the unique dietary requirements of enrich cell membranes with arachidonic acid–containing cats.1–3 It is now widely appreciated that these requirements phospholipids by selectively phosphorylating arachidonoyl- are consistent with the evolutionary influence of a strict car- diacylglycerol. The arachidonic acid–selective acyl-CoA syn- nivorous diet. The studies by Rogers and Morris of cats’ es- thetase is a hormone-dependent, obligatory protein in the sential amino acid requirements not only defined the dietary signal transduction pathway of steroidogenic hormones.16 requirements but also suggested that the extreme response of Many of these effects occur at the level of the whole cell cats to arginine deficiency resulted from certain biochemical and result in changes in cell motility, growth, adhesion, and defects in the synthesis or transport (or both) of urea cycle other properties. Cells are complex systems in which a multi- intermediates. They showed that ornithine prevents the hy- tude of biochemical reactions and molecular events take place perammonemia of arginine deficiency but does not provide concurrently and need to be finely orchestrated to preserve for normal growth and that citrulline is capable of substitut- cell homeostasis and direct cell-specific functions. For exam- ing for arginine in the diet.4,5 With regard to essential fatty ple, some of the effects of arachidonate deficiency may be re- acids, cats fed diets lacking arachidonate have extremely low lated to the essential role of arachidonate in preserving cell levels of arachidonic acid in plasma and erythrocyte lipids6 as membrane fluidity and architecture, including the interactions a result of a low level of the first enzyme in the desaturation of proteins within lipid rafts. It has been shown that arachi- pathway.7 Linoleate prevents or ameliorates many, but not donate is important for the maintenance of numerous cellu- all, of the signs of essential fatty acid deficiency in cats.8–11 In lar functions, including the functions of receptors in the

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membrane and nucleus of the cell.17 Finally, arachidonate and namic association and dissociation of proteins, both to mon- other polyunsaturated fatty acids directly modulate gene tran- itor the activity of a biochemical pathway in living cells and scription by binding to nuclear transcription factors, including to directly study the effects of chemicals on the pathways. peroxisome proliferator-activated receptors (PPARs).18 Be- Michnick’s laboratory developed protein fragment comple- cause cells are controlled by the physical interactions of these mentation assays (PCAs), a general strategy for monitoring proteins, cell-based methods may provide an efficient way to the dynamics of protein–protein interactions in vivo and in understand how nutrients and other small molecules affect real time. PCAs enable fluorescent, real-time analysis of sig- not just their primary targets but also individual or multiple naling events by measuring the association, dissociation, and biochemical pathways within cells. movements of protein–protein complexes within cells. PCAs are created by expressing mammalian genes linked in frame MOLECULAR MECHANISMS OF NUTRIENT to fragments of rationally dissected reporter genes. The asso- METABOLISM AND REGULATION ciation of two proteins of interest brings together comple- The field of molecular cell biology has evolved substantially mentary reporter fragments and enables productive folding over the past 10 years, enabling such studies on a large scale. of the fragments into an active structure that generates a flu- Cellular screening techniques allow for the study of the ef- orescent or luminescent signal. The resulting signals can be fects of exogenous molecules (e.g., nutrients, drugs, toxicants) spatially localized and quantified in living cells using high- in living cells. The ability to work with live cells at the level of content imaging instrumentation.22,23 We and others have re- individual proteins, including receptors, signaling proteins, cently applied these methods to de novo drug discovery in and enzymes, opens the door to understanding the links be- the identification of the mechanism of action of nutrients tween the biochemical function or metabolism of a nutrient and hormones and in the discovery of new uses for known and its role in health and disease. Specifically, the develop- drugs.24,25 These new technologies will aid in the design of in ment of new technologies based on real-time imaging of flu- vivo studies to advance our understanding of the relation- orescent indicators has enabled the direct visualization and ships between the biochemical functions of nutrients and quantification of these events in real time. drugs and their functions in health and disease. Whole-cell assay technologies vary with respect to the assay principle but largely have in common a form of lumi- REFERENCES 1. Morris JG, Rogers QR. Metabolic basis for some of the nutritional pe- nescence or fluorescence for detection. Luminescent, fluores- culiarities of the cat. J Small Anim Pract 1982;23:599-613. cent, or bioluminescent signals are easily detected and 2. MacDonald ML, Rogers QR, Morris JG. Nutrition of the domestic cat, a quantified with a variety of automated or high-throughput mammalian carnivore. Annu Rev Nutr 1984;4:521-562. 3. Morris JG. Idiosyncratic nutrient requirements of cats appear to be diet- instrumentation systems, including fluorescence multi-well induced evolutionary adaptations. Nutr Res Rev 2002;15:153-168. plate readers, fluorescence activated cell sorters, and auto- 4. Morris JG, Rogers QR, Winterrowd DL, Kamikawa EM. The utilization mated cell-based imaging systems that provide spatial reso- of ornithine and citrulline by the growing kitten. J Nutr 1979;109:724- 729. lution of the signal at the subcellular level. A variety of 5. Rogers QR, Morris JG. Essentiality of amino acids for the growing kit- instrumentation systems have been developed to automate ten. J Nutr 1979;109:718-723. these measurements, allowing the accumulation of data on 6. MacDonald ML, Rogers QR, Morris JG. Role of linoleate as an essential fatty acid for the cat independent of arachidonate synthesis. J Nutr thousands of living cells arrayed in microtiter plates. If com- 1983;113:1422-1433. bined with a suitable assay system, the effects of any com- 7. Pawlosky RJ, Barnes A, Salem N. Essential fatty acid metabolism in the feline: relationship between liver and brain production of long-chain pound can be measured on a large scale at the level of any polyunsaturated fatty acids. J Lipids Res 1994;35:2032-2040. cellular process or pathway, whether in the membrane, cy- 8. MacDonald ML, Anderson BC, Rogers QR, et al. Essential fatty acid requirements of cats: pathology of essential fatty acid deficiency. Am J Vet tosol, or nucleus of the cell, of any cell type. All that is needed Res 1984;45:1310-1317. is a universal quantitative method of monitoring the dynamic 9. MacDonald ML, Rogers QR, Morris JG, Cupps PT. Effects of linoleate processes that occur at the level of the proteins, which are the and arachidonate deficiencies on reproduction and spermatogenesis in the cat. J Nutr 1984;114:719-726. targets of the compound (e.g., nutrient, drug) of interest. 10. Morris JG. Do cats need arachidonic acid in the diet for reproduction? Dr. Stephen Michnick and colleagues19–21 at the University J Anim Physiol Anim Nutr 2004;8:131-137. of Montreal proposed that cell-based measurements of pro- 11. MacDonald ML, Rogers QR, Morris JG. Effects of dietary arachidonate deficiency on the aggregation of cat platelets. Comp Biochem Physiol tein–protein interactions could be used to monitor the dy- 1984;78(suppl C):123-126.

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NEW TECHNOLOGIES FOR PHARMACEUTICAL AND NUTRITION RESEARCH

12. Kang MJ, Fujino T, Sasano H, et al. A novel arachidonate-preferring acyl- 18. Benatti P, Peluso G, Nicolai R, Calvani M. Polyunsaturated fatty acids: CoA synthetase is present in steroidogenic cells of the rat adrenal, ovary, biochemical, nutritional and epigenetic properties. J Am Coll Nutr and testis. Proc Natl Acad Sci USA 1997;94:2880-2884. 2004;23:281-302. 13. Maloberti P, Lozano RC, Mele PG, et al. Concerted regulation of free 19. Michnick SW. Proteomics in living cells. Drug Discov Today 2004;9:262-267. arachidonic acid and hormone-induced steroid synthesis by acyl-CoA 20. Michnick SW. Exploring protein interactions by interaction-induced thioesterases and acyl-CoA synthetases in adrenal cells. Eur J Biochem folding of proteins from complementary peptide fragments. Curr Opin 2002;259:5599-5607. Struct Biol 2001;11:472-477. 14. Suga K, Kawasaki T, Blank ML, Snyder F. An arachidonoyl (polyenoic)- 21. Campbell-Valois FX, Michnick SW. Chemical biology on PINs and specific phospholipase AS activity regulates the synthesis of platelet-ac- NeeDLes. Curr Opin Chem Biol 2005;9:31-37. tivating factor in granulocytic hl-60 cells. J Biol Chem 1990;265(21): 22. MacDonald ML, Westwick JK. Exploiting network biology to improve 12363-12371. drug discovery. In: Taylor DL, Haskins JR, Giuliano KA, eds. High Con- 15. MacDonald ML, Mack KF, Williams BW, et al. A membrane-bound dia- tent Screening: A Powerful Approach to Systems Cell Biology and Drug Dis- cylglycerol kinase that selectively phosphorylates arachidonoyl-diacyl- covery. Totowa, NJ: Humana Press; 2006. glycerol. J Biol Chem 1988;263:1584-1592. 23. Yu H, West M, Keon BH, et al. Measuring drug action in the cellular 16. Cornejo Maciel F, Maloberti P, Neuman I, et al. An arachidonic acid- context using protein-fragment complementation assays. Assay Drug Dev preferring acyl-CoA synthetase is a hormone-dependent and obligatory Technol 2003;1:811-822. protein in the signal transduction pathway of steroidogenic hormones. 24. MacDonald ML, Lamerdin J, Owens S, et al. Identifying off-target ef- J Mol Endocrinol 2005;34:655-666. fects and hidden phenotypes of drugs in human cells. Nat Chem Biol 17. Martins de Lima T, Gorjão R, Hatanaka E, et al. Mechanisms by which 2006;2:329-337. fatty acids regulate leucocyte function. Clin Sci 2007;113:65-77. 25. Owens J. Dirty drugs’ secrets uncovered. Nat Rev Drug Discov 2006;5:1.

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SCIENTIFIC PROGRAM: FOCUS ON FELINES Is the Aging Feline Kidney a Mortality Antagonist?

Dennis F. Lawler, DVM Nestlé Research Center, St. Louis, Missouri

Diseases of advanced life have been viewed as chance events cat kidneys or in a similar mouse model.6–8 In addition, the that potentially end life. This understanding is reflected in low frequency of acute renal lesions among a large number of cataloging approaches to describing diseases of aging and cats that died from nonrenal causes suggests that a discrete recommending preventive or therapeutic responses.1,2 In a initializing event, such as ischemic episode, infection, or tox- different view, aging phenotypes often are associated with icity, may not be a prominent feature of this process. complex underlying biology at genomic, cellular, organ, and In a feline model of renal tubular disease, fibrosis was systemic levels. Prolonged investment of metabolic energy principally peritubular, suggesting that hypoxic sequelae re- at this level of complexity is not consistent with chance sult mainly from local compromise of diffusion.8 In a murine events that simply disrupt “natural aging.” Rather, perhaps renal model, upregulated Fas (apoptosis-mediating surface late-life diseases should be regarded as integral to the aging antigen; APO1, CD95) in tubular epithelial cells was shown process, with response programming that is plastic and to bind to Fas ligand of adjacent tubular cells, suggesting that adaptable.3,4 tubular loss is a signaled (fratricidal) apoptosis.7 Thus, the Within this redirected frame of reference, numerous as- histologic nature of tubule loss in feline and murine models pects of aging have been evaluated from a database consist- appears to be more compatible with an adaptive mechanism ing of postmortem findings from nearly 700 adult cats that that operates at the cellular level. Frequent observation of were maintained for life as residents of the same colony from tubulointerstitial changes in younger adults documents early 1979 to 2001. Cats that died or were euthanized because of onset, which also is compatible with defensive adaptation be- renal disease lived longer than those that died from other cause symptomatic renal failure is much less frequent during causes. The cats that died from renal disease had higher but early adulthood but very common during late adult life. uniform mean renal histologic scores across ages compared A specific underlying explanation for these observations with cats that died from other causes.5 is not obvious at present. Domestic cats are seasonally polye- Among cats that died from nonrenal causes but that had strous and multiparous, with declining fertility often initially histologic renal changes, mean lifespan was longer than in detectable over the 84- to 96-month age range.9–11 Thus, the cats without renal changes or renal causes of death. Cats that onset of tubulointerstitial changes that appear before this age succumbed to nonrenal causes of death also were evaluated may have evolved as one homeostatic response to help pre- across categories of death-causing diseases. Specific problems serve a fertile reproductive lifespan by selective elimination of did not underlie the difference in mean age at death between dysfunctional renal tubular cells and nephrons. Such an these two subgroups, underscoring the observation that the adaptation could, for example, increase the likelihood of suc- outcomes were not consequent to the structure and function cessful reproduction through greater metabolic stability. A hy- of the colony. Additionally, the inbreeding coefficients in this pothesis that stress-response programming of this type colony were low. evolved to modulate population survival is within the pres- Cats that succumbed to renal failure frequently had mor- ent scope of the debate about aging theories.12,13 However, phologic and preterminal metabolic changes, suggesting vary- this hypothesis is incomplete because it does not account for ing but substantial retained functional capacity. Although a long postreproductive lifespan. Indeed, a long postrepro- standard clinical chemistry is insensitive, the total body of ductive lifespan can occur even in simple organisms. Addi- data suggests also that other factors may direct transition to tionally, Mitteldorf 13 suggests that longevity and fecundity renal failure. Considering histological morphology, the usual may have evolved independently. This idea jeopardizes the sequelae of ischemia (cell swelling, karyolysis, lysosomal rup- older hypothesis that longevity and reproduction represent ture, massive inflammation) are not commonly observed in evolutionary trade-offs.

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IS THE AGING FELINE KIDNEY A MORTALITY ANTAGONIST?

Another point to consider is that deterioration during Genetic evaluation did not reveal directly heritable com- more advanced stages of chronic renal failure often is ac- ponents of renal tubulointerstitial phenotypes in this popu- companied by progression to increasingly severe cachexia, lation, indicating that the phenotypes are either totally which signals a death trajectory.14 In studies of aging popu- environmental in origin or that they reflect fixed traits (or lations across species, 15–19 death generally tends to be associ- both). It is critically important to recognize that aging, al- ated with more precipitous declines of body mass that are though reflecting conserved programming, also remains a recognizable around the time that late-life mortality in- highly plastic process that is subject to interactions with creases. However, factors that influence body composition stress-response phenotypes that may be the actual fixed before termination may not reflect only secondary, pretermi- traits.20–26 Therefore, measured heritabilities should be mod- nal degenerative processes. The higher percentage of cats with est at best and are not expected in the case of fixed traits. The kidney-related death and thin body condition suggests prior possibility of modulating interactions between fixed alleles transition from more obese body condition in at least some and epigenetic influences also is compatible with a working subjects. This observation aligns well with typical clinical ob- hypothesis that ultimately may recharacterize the role of overt servations of an early onset of very gradual change in body disease in aging, centered around an emerging understanding mass in patients that eventually develop renal disease. of the role of genetic–epigenetic interactions.27–32 Aside from the role of body composition in the death tra- The ultimate implications of these observations may in- jectory, serial body composition dual-energy x-ray absorptiom- volve altering approaches to intervention and prevention, etry data from 119 cats over years of healthy adult life resulted probably with species and breed or strain specificity. It must in heritability and principal component outcomes indicating be recognized that at least some components of long-term that multiple genes are involved in phenotypic expression of intrinsic disease (aging) processes likely represent life-pre- healthy body composition in cats. Two heritable principal com- serving adaptations. Nonspecific attempts at entire abolition ponents, PC2 (h2 = .40, P < .01) and PC6 (h2 = .74, P < .01), ex- of these processes or use of specific interventions that are ap- plained 24.7% and 1.3% of the population variance, plied indiscriminately, universally, or based on insensitive respectively. The first principal component, PC1, which ac- measures may deprive the individual of selected (or conver- counted for 55% of population variance (h2 = .33), was less gent) protective mechanisms. strongly significant (P = .038), possibly as a result of the number of variables tested. The observation that terminal body REFERENCES 1. Hoskins JD. Veterinary Pediatrics: Dogs and Cats from Birth to Six Months. condition has a quantitative genetic component was unex- 2nd ed. Philadelphia: WB Saunders; 1995. pected and indicates a need for reevaluation of the underly- 2. Kraft W. Geriatrics in canine and feline internal medicine. Eur J Med Res ing role of body composition in “diseases” of aging. 1998;3:31-41. 3. Heininger K. Aging is a deprivation syndrome driven by a germ-soma Interestingly, individual components of body composition conflict. Ageing Res Rev 2002;1:481-536. related to each other only moderately in the principal com- 4. Heininger K. The deprivation syndrome is the driving force of phy- ponent analysis, further suggesting that phenotypic expression logeny, ontogeny, and oncogeny. Rev Neurosci 2001;12:217-287. 5. Lawler DF, Evans RH, Chase K, et al. The aging feline kidney: a model of individual body composition components might result mortality antagonist? J Feline Med Surg 2006;8:363-371. from multiple underlying genetic and epigenetic mechanisms. 6. Majno G, Joris I. Apoptosis, oncosis, and necrosis: an overview of cell The specific genes involved in control of body composition death. Am J Pathol 1995;146:3-15. 7. Schelling JR, Nkemere N, Kopp JB, Cleveland RP. Fas-dependent fratri- and the relative contributions of those genes are presently un- cidal apoptosis is a mechanism of tubular epithelial cell depletion in known; slowly progressive, preterminal loss of body mass may chronic renal failure. Lab Invest 1995;78:813-824. reflect additional plastic genetic programming. In a renal con- 8. Sawashima K, Mizuno S, Mizuno-Horikawa Y, et al. Expression of α- smooth muscle actin and fibronectin in tubulointerstitial lesions of cats text, death occurs as the apparent outcome of a pathologic with chronic renal failure. Am J Vet Res 2000;61:1080-1086. process only at the point of systemic adaptive failure associ- 9. Scott PP. Cats. In: Hafez E, ed. Reproduction and Breeding Techniques for Laboratory Animals. Philadelphia: Lea & Febiger; 1970:192-208. ated with very advanced nephron deletion or (probably more 10. Lawler DF, Monti KL. Morbidity and mortality in neonatal kittens. Am frequently in domestic cats) by extranephron, extrarenal, or J Vet Res 1984;45:1455-1459. extrinsic metabolic factors. Whether these putative events 11. Lawler DF, Bebiak DM. Nutrition and management of reproduction in the cat. Vet Clin North Am 1986;16:495-519. could result from threshold effects is not known at present, 12. Bredesen DE. The non-existent aging program: how does it work? Aging but this seems an attractive hypothesis. Cell 2004;3:255-259.

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13. Mitteldorf J. Ageing selected for its own sake. Evol Ecol Res 2004;6:937-953. 23. Scheiner SM. Genetics and evolution of phenotypic plasticity. Annu Rev 14. Lulich JP, Osborne CA, Polzin DJ. Feline renal failure. Part 1: clinical, Ecol Syst 1993;24:35-68. biochemical, and morphological characteristics of renal failure in cats. 24. Finch CE. Comparative perspectives on plasticity in human aging and Proc ACVIM 1992:571-572. life spans: between Zeus and the salmon. In: Wachter KW, Finch CE, 15. Lesser GT, Deutsch S, Markofsky J. Aging in the rat: longitudinal and eds. The Biodemography of Longevity. Washington, DC: National Acad- cross-sectional studies of body composition. Am J Physiol 1973;225: emy Press; 1997:439-454. 1472-1478. 25. Krebs RA, Loeschcke V. A genetic analysis of the relationship between 16. Lesser GT, Deutsch S, Markofsky J. Fat free mass, total body water and life-history variation and heat-shock tolerance in Drosophila buzzatii. intracellular water in the aged rat. Am J Physiol 1980;238:R82-R90. Heredity 1999;83:46-53. 17. Kealy RD, Lawler DF, Ballam JM, et al. Influence of diet restriction on 26. Martin GM. Epigenetic drift in aging identical twins. Proc Natl Acad Sci life span and age-related changes in Labrador retrievers. JAVMA USA 2005;102:10413-10414. 2002;220:1315-1320. 27. Jazwinski SM, Kim S, Lai CY, Benguria A. Epigenetic stratification: the 18. Yu BP, Masoro EJ, Murata I, et al. Life span study of SPF Fischer 344 role of individual change in the biological aging process. Exp Gerontol male rats fed ad libitum or restricted diets. Longevity, growth, lean body 1998;33:571-580. mass and disease. J Gerontol 1982;37:130-141. 28. Robert L, Labat-Robert J. Aging of connective tissues; from genetic to 19. Lawler DF, Evans RH, Larson BT, et al. Influence of lifetime food re- epigenetic mechanisms. Biogerontology 2000;1:123-131. striction on causes, time, and predictors of death in dogs. JAVMA 2005;226:225-231. 29. Feinberg AP, Oshimura M, Barrett JC. Epigenetic mechanisms in human disease. Cancer Res 2000;62:6784-6787. 20. Clare MJ, Luckinbill LS. The effects of gene-environment interaction on the expression of longevity. Heredity 1985;55:19-26. 30. Issa JP. Epigenetic variation and human disease. J Nutr 2002; 132(suppl):2388-2392. 21. Finch CE. Longevity, Senescence and the Genome. Chicago: University of Chicago Press; 1990. 31. Claus R, Lubbert M. Epigenetic targets in hematopoietic malignancies. 22. Buck S, Nicholson M, Dudas S, et al. Larval regulation of adult longevity Oncogene 2003;22:6489-6496. in a genetically-selected long-lived strain of Drosophila. Heredity 32. Kopelovich L, Crowell JA, Fay JR. The epigenome as a target for cancer 1993;71:23-32. chemoprevention. J Natl Cancer Inst 2003;95:1747-1751.

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SCIENTIFIC PROGRAM: FOCUS ON FELINES What Is Different about Chronic Kidney Disease in Cats?

David J. Polzin, DVM, PhD, DACVIM College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota

Kidney disease is defined as the presence of functional or struc- years with urine specific gravity values greater than 1.040. tural abnormalities in one or both kidneys; chronic kidney dis- These cats most likely have CKD. ease (CKD) is defined as kidney disease that exists for at least To facilitate application of appropriate clinical practice 3 months.1 CKD is the most common kidney disease in cats. As guidelines for diagnosis and treatment, patients with CKD with most species, CKD is primarily a disease of older cats. One are categorized into four stages along a continuum of pro- recent publication estimated the prevalence of CKD among gressive kidney disease.1 (For more information on staging cats of all ages to be 112 cases per 1,000 cats.2 Among cats 10 chronic kidney disease, visit the International Renal Interest years of age and older, prevalence was estimated to be 269 Society website at www.iris-kidney.org.) The stage of CKD is cases per 1,000, while the prevalence in cats 15 years of age assigned based on the level of kidney function ascertained by and older was 491 per 1,000.2 CKD appears to be two to three two or more determinations of serum creatinine concentra- times more prevalent in cats than in dogs1; the reason for this tion obtained while the patient is well hydrated: is unknown. Although CKD is an irreversible and progressive • Stage 1: Serum creatinine <1.6 mg/dl condition, most affected cats survive for several months to • Stage 2: Serum creatinine 1.6–2.8 mg/dl years, and many ultimately die of conditions other than CKD. • Stage 3: Serum creatinine 2.9–5.0 mg/dl RECOGNITION AND STAGING • Stage 4: Serum creatinine >5.0 mg/dl Although CKD may be recognized initially via physical ex- The stage is further elucidated by proteinuria status and the pres- amination, serum biochemistries, urinalysis, or imaging stud- ence of systemic hypertension because these factors appear to ies, it is most commonly detected as reduced renal function influence prognosis and are amenable to therapeutic modifica- (azotemia). Differentiating renal azotemia from prerenal tion. Cats with urine protein:creatinine ratios less than 0.2 are azotemia is usually based on examining urine concentration classified as nonproteinuric, ratios between 0.2 and 0.4 indicate concurrent with detection of azotemia. Because cats tend to borderline proteinuria, and ratios greater than 0.4 indicate pro- have an exceptional ability to concentrate their urine, it is not teinuria. Cats with systolic blood pressure less than 150 mm Hg surprising that cats, compared with dogs or humans, typically are considered to have minimal risk of experiencing hyperten- maintain a greater degree of urine concentrating ability as sive end-organ injuries (e.g., renal, ocular, cardiac, or nervous renal function declines. As a consequence, less advanced CKD system lesions). Cats with systolic blood pressure between 150 may be associated with relatively concentrated urine in some and 159 mm Hg, 160 and 179 mm Hg, or greater than 180 mm cats. Absent other causes for dilute urine (e.g., hyperthy- Hg are considered to have a low, moderate, or high risk, respec- roidism, diabetes mellitus), serum creatinine values of 1.6 tively, of experiencing hypertensive end-organ injuries. mg/dl or greater associated with urine specific gravity values less than 1.035 should generally be interpreted as consistent CAUSE AND PATHOLOGY with renal azotemia.1 Cats with more advanced CKD typi- CKD in cats may be initiated by a variety of familial, congen- cally have urine specific gravity values below 1.020. Urine ital, or acquired diseases. Unfortunately, the initiating cause(s) specific gravity values between 1.035 and 1.040 constitute a of CKD often cannot be identified at the time of diagnosis. In “gray zone” in which azotemia may be renal or prerenal; one study, chronic tubulointerstitial nephritis was observed however, cats may occasionally present a diagnostic dilemma in 70% of cats with CKD, whereas glomerulonephropathy oc- because they remain persistently azotemic for months to curred in 15%, lymphoma in 11%, amyloidosis in 2%, and

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tubulonephrosis in 2%.3 The prevalence of nonproteinuric monly appears as abrupt, usually unpredictable, increases in tubulointerstitial disease appears to be greater in cats than in serum creatinine concentration.2 In a clinical trial performed dogs or humans. Unfortunately, the histologic diagnosis of at the University of Minnesota, renal function as measured tubulointerstitial nephritis does not help to identify the un- by serum creatinine remained stable for up to 24 months in derlying cause of kidney disease and probably represents the 40 of 45 cats.7 Five of the 45 cats developed uremic crises as- final common pathway for progression of many feline renal sociated with abrupt increases in serum creatinine concen- diseases. The initiating causes of diseases believed to originate trations after having had stable renal function for 3 to 21 in the tubulointerstitium have been especially elusive; how- months. Upon retrospective evaluation of the clinical data on ever, one possible cause for the higher prevalence of CKD in these cats, no clear indicators were found to be useful in pre- cats has recently been proposed. Subcutaneous administra- dicting an impending decline in kidney function. tion in kittens of feline herpesvirus 1, calicivirus, and pan- The seeming stability of renal function in many cats with leukopenia virus vaccines grown in feline tissue culture CKD translates into relatively long survival time. Compared systems has been shown to induce production of antifeline with dogs having similar levels of renal dysfunction, cats typ- renal tissue antibodies in serum and a tubulointerstitial in- ically live many months or years longer. In fact, many older flammatory response within the renal tubulointerstitium.4 cats succumb to other diseases before their CKD becomes se- This observation prompts the question of whether repeated vere enough to cause significant morbidity. vaccinations play a role in the development of CKD in cats. Another interesting observation that appears to be unique to MODIFYING CLINICAL OUTCOMES cats is the high frequency of nephroliths and ureteroliths in Even though feline CKD tends to be generally less progressive those with CKD. These uroliths are composed predominantly than CKD in dogs, many cats still progress to a point where it be- of calcium oxalate. The origin of these uroliths and whether they comes difficult or impossible to have a satisfactory quality of develop before, during, or after the onset of CKD are not known. life. Recent studies have indicated that certain medical interven- Although ureteroliths have become an important cause of acute tions may delay or prevent progression of CKD, thereby ex- uremic crises in cats, the presence of nephroliths does not appear tending survival with a good quality of life. Factors that have to adversely affect clinical outcomes in cats with CKD.5 Although been shown to influence survival times for cats with CKD in- it may be necessary to surgically remove ureteroliths associated clude the severity of reduction in GFR (stage of CKD) and mag- with complete, persistent ureteral obstruction, removal of nitude of proteinuria. There may also be an interaction between nephroliths is generally not recommended. systemic hypertension and proteinuria on survival. With greater severity of intrinsic renal dysfunction or magnitude of protein- BIOLOGIC BEHAVIOR uria, shorter survival time is likely. Some other factors that may A progressive decline in kidney function over months to or may not influence progression of CKD directly include sys- years is typical of naturally occurring CKD.1,6 Although it is temic hypertension, pyelonephritis, and presence of nephroliths. logical to assume that CKD progresses as a consequence of All patients with CKD are potentially at risk for progressive ongoing renal injury associated with the disease process that kidney disease. Progression may occur as a consequence of initiated kidney disease, the initiating cause for CKD cannot the primary renal disease, in association with a variety of sec- be identified at the time of diagnosis in most patients. The ondary factors that may promote progressive renal disease, preponderance of clinical and experimental evidence sug- or both. An important therapeutic goal for managing patients gests that in dogs and cats with stages 3 and 4 CKD, pro- with CKD is to minimize or prevent progressive loss of renal gressive loss of kidney function results, at least in part, from function. Treatment designed to limit progression of kidney factors unrelated to the inciting disease.1,6 These factors may disease may involve a variety of interventions, including diet include intraglomerular hypertension, glomerular hypertro- therapy, minimizing proteinuria, controlling hypertension, phy, hypertension, proteinuria, intrarenal precipitation of and modulating the renin-angiotensin-aldosterone system. calcium phosphate, and tubulointerstitial disease. There is substantial clinical trial evidence supporting the Whereas progression of CKD in humans and dogs is often effectiveness of dietary intervention in prolonging survival of characterized by a linear pattern of decline in glomerular fil- cats with CKD; there is no credible clinical evidence to the tration rate (GFR), progression of CKD in cats more com- contrary. In a nonrandomized clinical trial, cats fed a renal

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WHAT IS DIFFERENT ABOUT CHRONIC KIDNEY DISEASE IN CATS?

diet survived significantly longer than cats that continued to enzyme (ACE) inhibitors are effective in reducing proteinuria in consume their usual diet (633 versus 264 days).8 It was not cats with CKD,13 the effectiveness of ACE inhibitor therapy in al- possible to detail the differences between the diets used in this tering the course of CKD in cats remains to be confirmed.14,15 study, but the therapeutic renal diet had a reduced protein and Nevertheless, ACE inhibitors such as benazepril and enalapril phosphorus content. The renal diet was shown to be benefi- are recommended for patients with CKD that meet the above cial in lowering serum phosphorus and parathyroid hormone criteria. Interestingly, treatment of systemic hypertension in cats concentrations, and it was suggested that the beneficial effect with CKD using amlodipine besylate has been shown to be as- of the diet may have been related to this effect.9 A random- sociated with a reduction in the magnitude of proteinuria.16 ized, controlled clinical trial from the University of Minnesota Ideally, therapy should be adjusted so that the urine pro- Veterinary Medical Center further confirmed the beneficial ef- tein:creatinine ratio is reduced to 0.4 or lower; however, this fects of diet therapy in prolonging survival of cats with CKD.7 may be difficult or impossible in many patients and may re- In this study, the effect of a renal diet on survival was com- quire higher doses of ACE inhibitors or the addition of an- pared with that of a maintenance diet in 45 cats with sponta- giotensin II receptor-blocking drugs (e.g., losartan, irbesartan). neous CKD. Renal-related mortality in 23 cats fed an adult maintenance diet was 17.4%, whereas no deaths were ob- REFERENCES 1. Polzin DJ, Osborne CA, Ross S. Chronic kidney disease. In: Ettinger SJ, served in 22 cats fed the renal diet, which was restricted in pro- Feldman E, eds. Textbook of Veterinary Internal Medicine. St. Louis: Else- tein and phosphorus content. In a retrospective study of cats vier Saunders; 2005:1756-1785. 2. Ross SJ, Polzin DJ, Osborne CA. Clinical progression of early chronic renal with CKD treated at 31 veterinary clinics in the Netherlands, failure and implications for management. In: August J, ed. Consultations in feeding a renal diet compared with a typical feline diet was Feline Internal Medicine. St. Louis: Elsevier Saunders; 2005:389-398. found to be associated with a significant increase in median 3. Minkus G, Horauf A. Evaluation of renal biopsies in cats and small dogs: histopathology in comparison with clinical data. J Small Anim survival time (7 months among cats consuming conventional Pract 1994;35:465-472. cat foods; 16 months for cats consuming a renal diet).10 4. Lappin MR, Basaraba RJ, Jensen WA. Interstitial nephritis in cats inoc- ulated with Crandell Rees feline kidney cell lysates. J Feline Med Surg A common misconception is that renal diets are simply 2006;8:353-356. low-protein diets. Renal diets encompass a variety of modifi- 5. Ross SJ, Osborne CA, Lekcharoensuk C, et al. A case-control study of the effects of nephrolithiasis in cats with chronic kidney disease. JAVMA cations beyond just a limitation of protein content, and, in- 2007;230:1854-1859. deed, the principal beneficial effects of these diets may not 6. Brown S, Crowell W, Brown C, et al. Pathophysiology and management of progressive renal disease. Vet J 1997;154:93-109. accrue from their reduction in protein content. Thus, simply 7. Ross SJ, Osborne CA, Kirk CA, et al. Clinical evaluation of dietary mod- replacing a renal diet with a standard manufactured diet that ification for treatment of spontaneous chronic kidney disease in cats. JAVMA 2006;229:949-957. is lower in protein content does not meet the guideline for 8. Elliot J, Rawlings J, Markwell P, et al. Survival of cats with naturally oc- feeding a renal diet. Because inappropriate diets can exacer- curring chronic renal failure: effect of dietary management. J Small Anim Pract 2000;41:235-242. bate clinical signs of uremia or promote progression of CKD, 9. Barber P, Rawlings J, Markwell P, et al. Effect of dietary phosphate re- cats with CKD should be fed a renal diet. striction on renal secondary hyperparathyroidism in the cat. J Small Anim Pract 1999;40:62-70. Treatments designed to reduce glomerular proteinuria are 10. Plantinga EA, Everts H, Kastelein AMC, et al. Retrospective study of the recommended for managing proteinuric cats with CKD stages survival of cats with acquired chronic renal insufficiency offered different commercial diets. Vet Rec 2005;157:185-187. 1 through 4. Intervention is indicated when the urine pro- 11. Lees GE, Brown SA, Elliott J, et al. Assessment and management of protein:creatinine ratio exceeds 2.0 in cats with CKD stage 1 and teinuria in dogs and cats: 2004 ACVIM Forum Consensus Statement (Small Animal). J Vet Intern Med 2005;19:377-385. 11 0.4 in cats with CKD stages 2 through 4. Proteinuria has been 12. Abbate M, Zoja C, Remuzzi G. How does proteinuria cause progressive shown to adversely affect outcome in humans, dogs, and cats renal damage? J Am Soc Nephrol 2006;17:2974-2984. 13. Syme HM, Markwell PJ, Pfeiffer D, et al. Survival of cats with naturally with CKD, presumably because proteinuria itself appears to in- occurring chronic renal failure is related to severity of proteinuria. J Vet jure the renal tubules, thereby promoting progression of CKD. Intern Med 2006;20:528-535. 14. Mizutani H, Koyama H, Watanabe T, et al. Evaluation of the clinical ef- It is well established in human patients that reducing protein- ficacy of benazepril in the treatment of chronic renal insufficiency in uria by suppressing the renin-angiotensin-aldosterone system cats. J Vet Intern Med 2006;20:1074-1079. 12 15. King JN, Gunn-Moore DA, Tasker S, et al. Tolerability and efficacy of ameliorates the adverse effects of proteinuria on the kidneys. benazepril in cats with chronic kidney disease. J Vet Intern Med 2006;20: While qualitatively similar, evidence in cats is less compelling. 1054-1064. Although studies have shown that proteinuria is closely linked 16. Jepson RE, Elliott J, Brodbelt D, et al. Effect of control of systolic blood pressure on survival in cats with systemic hypertension. J Vet Intern Med to progression of CKD in cats and that angiotensin-converting 2007;21:402-409.

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SCIENTIFIC PROGRAM: FOCUS ON FELINES Feline Urolithiasis: Understanding the Shift in Urolith Type

Jody P. Lulich, DVM, PhD, and Carl A. Osborne, DVM, PhD College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota

Naturally occurring urolithiasis is affected by many known cats developed typical signs of fLUTD, including urethral ob- and unknown risk factors. Known risk factors that influence struction, but they did not produce the struvite-matrix ure- urolith formation include diet, urine pH, water homeostasis, thral plugs commonly encountered in cats with naturally breed, abnormalities of metabolism, urinary tract infection occurring urethral obstruction. The general consensus of (UTI), and anatomic and functional abnormalities of the uri- many investigators and clinicians was that consumption of nary tract. Each factor may play a significant or limited role dry diets with excessive magnesium was an important pri- in the development or prevention of different types of mary cause of fLUTD. uroliths. Recognition and control of lithogenic risk factors is Following the development of dietary protocols to induce the primary goal to prevent urolith formation and minimize dissolution of naturally occurring struvite uroliths in dogs, urolith recurrence. dietary protocols to dissolve naturally occurring sterile stru- The Minnesota Urolith Center has performed quantitative vite urocystoliths in cats emerged in 1983.3 Their effective- analysis of uroliths from cats for more than two decades. Dur- ness justified the emphasis on dietary factors in the ing this period, we have observed substantial shifts in urolith prevention of sterile struvite urolithiasis. type (Figure 1). In 1981, struvite was the most common In 1985, the results of studies on the effects of feeding stone, representing 78% of urolith submissions. A decade diets containing alkalinizing and acidifying salts of magne- later, struvite remained the most common stone; however, its sium to clinically normal cats were reported.4 These labora- prevalence had declined to 59%. By the end of the second tory studies shifted the focus from dietary magnesium decade, the ever-present struvite had been supplanted by the content to alkaline urine pH as a primary factor in the de- emergence of calcium oxalate (CaOx). In 2001, uroliths were velopment of struvite crystalluria. Results of these studies retrieved from 6,185 cats and submitted for quantitative had a profound effect on veterinarians and the pet food in- analysis; 55% were CaOx, and 34% were struvite. Epidemio- dustry. Many adult feline maintenance diets were eventually logic shifts in feline urolith type were not confined to the modified to minimize struvite crystalluria. Because of di- United States: Increased prevalence of CaOx was also ob- etary modifications, the prevalence of struvite uroliths and served in Asia and Europe. Because of the short time span in struvite urethral plugs began to decline in the mid-1980s. which this occurred, we hypothesized that changes in hus- Unexpectedly, the decrease in prevalence of struvite-related bandry and nutrition represent significant contributing fac- urolithiasis was associated with a concomitant increase in tors influencing this epidemiologic shift in urolith type. the prevalence of CaOx urolithiasis even though struvite remained the primary mineral component of urethral plugs. THE RISE AND DECLINE OF STRUVITE In the early 1970s, the association between dry diets and fe- THE EMERGENCE OF CALCIUM OXALATE line lower urinary tract disease (fLUTD) became a topic of in- The exact etiologic cascade of events that led to the increased tense discussion in England, Denmark, and the United States. prevalence of CaOx uroliths remains unknown. However, Also in the early 1970s and continuing for the next decade, several biologic phenomena provide plausible explanations. several groups of investigators experimentally produced magnesium hydrogen phosphate and then magnesium ammo- The Role of Diet nium phosphate (MAP) uroliths in clinically normal cats by Results of epidemiologic studies support the hypothesis that adding various types of magnesium salts to their diets.1,2 The diets designed to minimize MAP urolith formation may have

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FELINE UROLITHIASIS: UNDERSTANDING THE SHIFT IN UROLITH TYPE

inadvertently increased the occurrence of CaOx uroliths.5,6 Whereas diet-mediated 100 urine acidification enhances the solubility of % CaOx MAP crystals in urine, dietary acids promote % MAP 80 CaOx crystalluria by inducing hypercalciuria. This association between aciduria, acidemia, 60 and hypercalciuria may be explained by the fact that acidemia promotes mobilization of 40 carbonate and phosphate from bone to Percent buffer hydrogen ions. Concomitant mobi- 20 lization of bone calcium may result in hypercalciuria. In addition, metabolic acidosis 0 81 83 85 87 89 91 93 95 97 99 01 03 05 in dogs, humans, and rats may result in Year hypocitraturia. If consumption of dietary acid precursors is associated with hypocitra- Figure 1. The yearly percentage of feline uroliths composed of CaOx compared with turia in cats, it may increase the risk of CaOx those composed of MAP (Minnesota Urolith Center; n = 83,601 submissions). uroliths because citrate is an inhibitor of CaOx crystal formation. Over the past 50 years, the incidence of CaOx uroliths in CaOx uroliths are uncommon in immature cats in which humans living in the United States has increased consider- urine is normally acidic. The answer is likely related to a com- ably.7 Global distribution of urolithiasis in humans indicates bination of risk factors associated with CaOx urolithiasis, in- that CaOx uroliths predominate in the United States and cluding the concentrations of minerals and nonmineral other industrialized, technologically advanced regions of the crystallization inhibitors and promoters and the quantity of world.8 Although originally attributed to the sedentary urine produced. There likely is no simple cause-and-effect re- lifestyle of inhabitants of such countries,8 the increased inci- lationship between a single risk factor and CaOx urolithiasis. dence of CaOx uroliths is now believed to reflect the ability Why MAP has remained the most common mineral of of these more affluent societies to spend disposable income urethral plugs while the prevalence of feline CaOx has dra- for the consumption of animal protein, which leads to in- matically increased in uroliths is unknown. However, the ob- creased urinary excretion of acidic metabolites, calcium, and servation that the average age of cats with urethral plugs is oxalate.9,10 Regional environmental factors, such as water and lower (approximately 2 to 4 years old) than that of cats with soil quality, may also influence urolith formation. It is logi- CaOx uroliths leads to the hypothesis that age-related cal to consider that variables that contribute to the increased changes in urine promoters for urolithiasis play an impor- incidence of CaOx uroliths in humans may also influence the tant role. As an illustration, in a case-control study compar- incidence of CaOx uroliths in cats. In other words, are strate- ing the age of 7,895 cats with CaOx uroliths that were gies that incorporate the concepts of improved nutrition or submitted to the Minnesota Urolith Center between 1981 overnutrition a risk factor for CaOx urolith formation? and 1997 with the age of 150,482 cats admitted to veterinary colleges in North America, cats at greatest risk of developing The Role of Age CaOx uroliths were those between 7 and 10 years of age.11 In a retrospective study of feline CaOx uroliths from 922 cats, Cats in this age group were 67 times more likely to form only 3 were younger than 1 year of age. Ninety-seven percent uroliths than cats 1 to 2 years of age. The mean age of cats of affected cats were older than 2 years. These observations with CaOx uroliths was 7.5 ± 3.3 years. In contrast, cats at are interesting because conditions promoting urine acidity highest risk of developing MAP uroliths were between 4 and have been identified as a risk factor for CaOx urolith forma- 7 years of age. These comparisons are clinically important be- tion, and the urine pH of young cats is lower than that of cause they emphasize the need to monitor cats receiving diets adult cats consuming the same diet. If acidic urine is an im- that promote urine acidification because as cats get older, the portant risk factor for CaOx, a reasonable question is why risk of developing CaOx urolithiasis increases.

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The Role of Oxalobacter spp turn, CaOx uroliths of sufficient size can block the ureter, Hyperoxaluria is an important risk factor for CaOx urolith for- promoting kidney failure. mation. Although the majority of urinary oxalate is derived from endogenous metabolic pathways, increased urinary ox- THE EPOCH OF HOPE alate appears to be sustained by increased dietary load and in- Since 2001, the Minnesota Urolith Center has observed a con- creased intestinal absorption. Studies in rats have demonstrated sistent decline in the yearly percentage of cats with CaOx that components of the intestinal microflora, particularly Ox- uroliths (Figure 1). Although factors other than a change in alobacter formigenes, use oxalate in the gut, thus limiting its ab- the occurrence of CaOx may contribute to this reduction, we sorption.12 Of particular interest is the observation that human have taken the optimistic perspective that increased knowl- patients with recurrent UTIs excrete higher quantities of oxalate edge and understanding of the risk factors associated with than do stone formers without UTIs.13 It was hypothesized that CaOx formation have favorably altered husbandry, nutrition, antibiotic control of UTIs reduces intestinal Oxalobacter popu- and veterinary care. lations. Conceptually, this association may be important for two reasons: (1) antibiotics are commonly used in the man- REFERENCES 1. Lewis LD, Chow FHC, Taton GF, Hamar DW. Effects of various dietary agement of idiopathic fLUTD and (2) renal tubular damage by mineral concentrations on the occurrence of feline urolithiasis. JAVMA increased urine oxalate concentrations may serve as a nidus for 1978;172:559-563. crystal nucleation, adherence, and growth. 2. Rich LJ, Dysart I, Chow FHC, Hamar D. Urethral obstruction in male cats: experimental production by addition of magnesium and phosphate to the diet. Feline Pract 1974;4:44-47. THE AGE OF NEPHROURETEROLITHIASIS 3. Osborne CA, Lulich JP, Kruger JM, et al. Medical dissolution of feline struvite urocystoliths. JAVMA 1990;196:1053-1063. The increase in occurrence of CaOx uroliths in cats has been 4. Buffington CA, Rogers QR, Morris JG. Feline struvite urolithiasis: mag- associated with a parallel increase in occurrence of CaOx nesium effect depends on urinary pH. Feline Pract 1985;15:29-33. uroliths found in their kidneys and ureters. In fact, there has 5. Lekcharoensuk C, Osborne CA, Lulich JP, et al. Association between dietary factors and calcium oxalate and magnesium ammonium phos- been a 10-fold increase in the frequency of upper tract phate uroliths in cats. JAVMA 2001;219:1228-1237. uroliths diagnosed in cats evaluated at veterinary teaching 6. Kirk CA, Ling GV, Franti CE, Scarletti JM. Evaluation of factors associated with development of calcium oxalate urolithiasis in cats. JAVMA hospitals in North America over the past 20 years.14 1995;207:1429-1434. Between 1981 and 2003, the Minnesota Urolith Center an- 7. Mandel NS, Mandel GS, Urinary tract stone disease in the United States veteran population II: geographical analysis of variations in composi- alyzed nephroureteroliths from 1,599 cats. Seventy percent of tion. J Urol 1989;1432:11516-11521. the uroliths were composed of CaOx. In contrast, only 8% 8. Lonsdale K. Human stones. Science 1968;159:199-1207. were composed of MAP. While enrolling cats with renal fail- 9. Robertson WG, Peacock M, Hodgkinson A. Dietary changes and the incidence of urinary calculi in the UK between 1958 and 1976. J Chronic ure into a clinical trial, we were surprised to find that 48% had Dis 1979;32:469-476. radiographic evidence of nephroliths or ureteroliths. This find- 10. Robertson WG, Peacock M, Heyburn PJ. Should recurrent calcium oxalate stone formers become vegetarians? Br J Urol 1979;51:427-431. ing emphasizes the importance of CaOx prevention and con- 11. Lekcharoensuk C, Osborne CA, Lulich JP. The epidemiology of feline trol in cats to minimize potential life-threatening renal failure. lower urinary tract disease: patient risk factors, 1980–1997. JAVMA Is kidney disease a cause or a consequence of urolith for- 2001;218:1429-1435. 12. Sidhu H, Allison MJ, Chow JM, et al. Rapid reversal of hyperoxaluria in mation? Hyperoxaluria may be the common link between a rat model after probiotic administration of Oxalobacter formigenes. J these two processes. One current hypothesis proposes that Urol 2001;166:1487-1491. 15 13. Hoppe B, Von Unruh G, Laube N, et al. Oxalate degrading bacteria: new excessive oxalate damages kidney tubules. The damaged treatment option for patients with primary and secondary hyperox- tubules become mineralized (Randall’s plaques, which are aluria. Urol Res 2005;33:372-375. 14. Lekcharoensuk C, Osborne CA, Lulich JP, et al. Trends in the frequency sites of interstitial mineralization at or near the renal papilla of calcium oxalate uroliths in the upper urinary tract of cats. JAAHA found in kidneys of CaOx stone formers) and serve as a nidus 2005;41:39-46 for CaOx precipitation (epitaxy). By increasing urine satura- 15. Turan T, Tuncay OL, Usubutun A, et al. Renal tubular apoptosis after complete ureteral obstruction in the presence of hyperoxaluria. Urol Res tion, hyperoxaluria also promotes precipitation of calcium. In 2000;28:220-222.

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SCIENTIFIC PROGRAM: FOCUS ON FELINES In Search of the Origins of Feline Hyperthyroidism

Deborah S. Greco, DVM, PhD, DACVIM Nestlé Purina PetCare, St. Louis, Missouri

Feline hyperthyroidism was first described in 1979 and 1980 tach, it is called diiodotyrosine. The coupling of two iodinated by investigators in New York and Boston, respectively.1,2 The tyrosine molecules results in the formation of the main question at that time and ever since has been, “Is hyperthy- thyroid hormones: two diiodotyrosine molecules form

roidism a new disease in cats?” Based on epidemiologic and tetraiodothyronine (T4), and one monoiodotyrosine and one

hospital-acquired data, the answer appears to be “yes.” Dur- diiodotyrosine molecule form triiodothyronine (T3). Thy- ing a 14-year period (1970 to 1984), an average of 1.9 cats per roperoxidase, a key enzyme in the biosynthesis of thyroid hor- year were diagnosed with hyperthyroidism; however, it is now mones, works in concert with an oxidant, hydrogen peroxide. estimated that the incidence is as high as 2% of the feline Thyroperoxidase catalyzes the iodination of the tyrosyl population seen in tertiary care veterinary facilities.3,4 Hyper- residues of thyroxine-binding globulin and the formation of

thyroidism has become the most frequently diagnosed en- T3 and T4. In addition to the unusual molecular storage form docrinopathy in cats, with reports originating from North of the hormone, thyroid hormones are also unique in that America, Europe (especially the United Kingdom), New they are the only hormones that contain a halide (i.e., iodine). Zealand, and Australia. Hyperthyroidism in cats has become The main form of metabolism of thyroid hormones in- increasingly more prevalent as a result of an increase in the volves the removal of iodide molecules. The two enzymes in- ′ number of cats that survive past 10 years of age, improved di- volved in T3 and reverse T3 synthesis, 5 -deiodinase and agnostics, and increased suspicion of the disease among vet- 5-deiodinase, are also involved in the catabolism of thyroid

erinarians. Dozens of studies have been published on the hormones. The majority of T3 formation occurs outside the

origins of feline hyperthyroidism, but none provides a de- thyroid gland by deiodination of T4. The enzyme involved in

finitive answer to the mystery behind this disease. the removal of iodide from the outer phenolic ring of T4 in ′ the formation of T3 is called 5 -monodeiodinase. Another type THYROID PHYSIOLOGY of T3 in which an iodide molecule is removed from the inner

The thyroid gland is the most important endocrine gland for phenolic ring of T4, a compound called reverse T3, is also

metabolic regulation. The synthesis of thyroid hormone is un- formed. Reverse T3 has little of the biologic effects of thyroid usual because a large amount of the active hormone is stored as hormones and is formed only by the action of extrathyroidal a colloid within the lumen or acinus, created by the circular deiodinating enzymes, not by activity of the thyroid. arrangement of glandular cells. Two molecules— tyrosine and iodine—are important for thyroid hormone synthesis. Tyrosine CLINICAL ASPECTS OF HYPERTHYROIDISM is a part of thyroglobulin, a large molecule (molecular weight: As noted, hyperthyroidism is the most common en- 660,000 D) formed within the follicle cell and secreted into the docrinopathy of cats. It is caused by adenomatous hyperpla- lumen of the follicle. Iodine is converted to iodide in the intes- sia of the thyroid gland. Middle-aged to older cats are tinal tract and then transported to the thyroid, where the folli- typically affected, and there is no predilection for breed or cle cells effectively trap the iodide through an active transport sex, although some studies suggest a male predilection and a process. This allows intracellular iodide concentrations to be decreased incidence in Himalayans and Siamese.5 25 to 200 times higher than extracellular concentrations. Hyperthyroidism is characterized by hypermetabolism; As iodide passes through the apical wall of the cell, it at- therefore, polyphagia, weight loss, polydipsia, and polyuria taches to the ring structures of the tyrosine molecules, which are the most prominent features of the disease. Activation of are part of the thyroglobulin amino acid sequence. The tyro- the sympathetic nervous system is also seen. Hyperactivity, syl ring can accommodate two iodide molecules; if one iodide tachycardia, pupillary dilatation, and behavioral changes are molecule attaches, it is called monoiodotyrosine, and if two at- characteristic of the disease in cats. Long-standing hyperthy-

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roidism leads to hypertrophic cardiomyopathy, high-output Quercetin, a flavinoid, is capable of stimulating mitogenesis heart failure, and cachexia, all of which may lead to death. in a cell-culture line from hyperthyroid cats.10 Polyphenolic soy

Feline hyperthyroidism is diagnosed by measuring total T4 isoflavones, such as genistein and daidzein, were identified in 11 (TT4); total T3 measurement is generally noncontributory to a almost 60% of dry cat foods tested. Some dry foods contain diagnosis. Because the disease has become more common and isoflavone at levels consistent with those shown to interfere

recognized in its early stages, free T4 (FT4) concentrations have with thyroid function by inhibiting thyroperoxidase in rats and been shown to be more diagnostic of early or “occult” hyper- 5′-deiodinase activity in cats12,13; however, these cell-culture

thyroidism; however, FT4 concentrations should be interpreted and in vitro studies are in contradiction to epidemiologic data

in light of TT4 because nonthyroidal illness (e.g., chronic renal that show hyperthyroidism to be less common in cats fed dry 4,5,14 failure) can result in spurious elevations of FT4 as well. foods. Studies in rats have demonstrated in vitro effects of soy isoflavones, especially in conjunction with iodine defi- NUTRITIONAL ASPECTS OF ciency; however, an in vivo effect on TT4 and thyroid-stimulat- HYPERTHYROIDISM ing hormone (TSH) has not been observed.12,15 In a prospective An inability to secrete adequate amounts of thyroid hormone study of 18 clinically normal cats eating a soy diet (400 mg

often leads to the enlargement of the thyroid gland, a condi- isoflavones/kg diet), TT4 and FT4 concentrations were signifi-

tion known as goiter. In many places around the world, this cantly, but modestly, increased, whereas T3 concentrations were condition is, or has been, caused by a deficiency of iodine in unchanged.13 Many human studies have shown no detrimen- the diet, a situation that has largely been corrected through tal effect of soy isoflavones on thyroid function, particularly the use of iodized salt. Balanced pet foods provide sufficient when incorporated into a balanced diet with adequate iodine iodine but vary widely in iodine content.6 The effects of this intake.16,17 Thus, the effect of soy, if any, within complete cat variation have been theorized to be important in cats, but foods remains controversial. there are no data to support or refute the theory. Tartellin et Although unproven, canned cat food has been implicated 7 al showed an acute inverse relationship between FT4 and di- as a cause of feline hyperthyroidism in multiple epidemio- etary iodine in cats fed low- and high-iodine diets for 2 logic studies.4,5,14 The suspected goitrogen is bisphenol A digly- weeks; however, a subsequent study concluded that no effect cidyl ether (BADGE), a substance used in the manufacture of

on serum TT4 or FT4 was seen when the cats were fed low- or the liners of easy-open pop-top cans. It is suspected that this high-iodine diets for more than 5 months.8 Chronic changes compound can leach into food and be consumed by cats. in dietary iodide are associated with “adaptation” of the thy- While this BADGE-based lining is generally considered safe roid gland and are therefore unlikely to be the cause of feline and is used with foods for human consumption, it is sug- hyperthyroidism. One study showed that feeding a low-io- gested that cats may be more susceptible to toxic effects of this dine diet to cats with preexisting hyperthyroidism failed to compound because they have a greatly reduced ability to affect high concentrations of circulating thyroid hormone.3,9 detoxify it via hepatic glucuronidation. At toxic levels, bisphe- Certain plants (e.g., cruciferous plants such as cabbage, nol A also reduces triiodothyronine binding and causes in- kale, rutabaga, turnip, and rapeseed) contain a potent an- creased TSH secretion, resulting in hyperthyroidism and goiter tithyroid compound called progoitrin, which is converted into in rats and some humans. On the other hand, although cat goitrin within the digestive tract. Goitrin interferes with the studies may not be available, rodent studies show a very high organic binding of iodine. Many of the goitrogenic feeds also safety margin.18 It also should be noted that epidemiologic contain thiocyanates, which interfere with the trapping of io- studies showing associations are not the same as cause and dine by the thyroid gland. Increasing the amount of iodine effect. Over 90% of cats in the United States consume com- consumed can sometimes overcome the effects of thiocyanate mercial pet foods as their primary nutritional source, and rel- but has less influence on overcoming the effects of goitrin. atively few develop hyperthyroidism. Goitrogens can result in hypothyroidism, and some have theorized that chronic exposure to goitrogens can lead to toxic IMMUNOLOGIC ASPECTS OF nodular goiter, resulting in hyperthyroidism. HYPERTHYROIDISM It has been theorized that flavinoids from soy proteins play The literature regarding an immunologic cause of hyperthy- a role in the pathogenesis of hyperthyroidism in cats. roidism is contradictory. Initially, feline hyperthyroidism was

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IN SEARCH OF THE ORIGINS OF FELINE HYPERTHYROIDISM

believed to be similar to Grave’s disease in humans.19 In fact, ticides and herbicides has been associated with thyroid ab- the clinical signs of hypermetabolism associated with feline normalities in other species.30 In particular, the use of flea- hyperthyroidism are identical to Grave’s disease; however, control products was associated with an increased risk of several studies have shown that, unlike Grave’s disease, which developing hyperthyroidism, but no specific product or in- is caused by autoantibodies to the thyroid TSH receptor, cats gredient could be identified.31,32 with hyperthyroidism have antibodies that do not stimulate A recent report implicated brominated flame retardants the TSH receptor.20 In other research, antibodies from hyper- (BFRs) as carcinogens/goitrogens possibly associated with fe- thyroid cats were shown to stimulate thyroid cellular prolif- line hyperthyroidism.33 Coincidently, BFRs were introduced 30 eration and interfere with TSH binding.21 More recent reviews years ago, around the same time that feline hyperthyroidism have indicated that an autoimmune basis for hyperthy- emerged. Bromide, a halide, is an intriguing agent to implicate roidism is unlikely and that the disease is more similar to in feline hyperthyroidism because of the unique composition toxic nodular goiter than to Grave’s disease.22 of thyroid hormones that contain the halide iodide. In this abstract, serum levels of lipid-adjusted serum polybrominated MOLECULAR ASPECTS OF HYPERTHYROIDISM diphenyl ethers (PBDE) were 10 to 400 times higher than those More recently, investigators have honed in on the molecular found in human exposure. The authors theorized that these aspects of feline hyperthyroidism. In cats, the disease is more findings of high PBDE serum levels are in accord with the most similar to toxic nodular goiter in humans and is character- consistently identified risk factor: indoor living. The authors ized by autonomous growth of thyroid follicles. The patho- also propose that cats are at increased risk because of meticu- genesis of toxic nodular goiter is an abnormality in the signal lous grooming behavior and increased exposure to furniture transduction of the thyroid cell. The TSH receptor on the and carpet. The small size of cats is also a possible risk factor thyroid cells activate receptor-coupled guanosine triphos- for increased serum levels of PBDEs. phate-binding proteins (G proteins). Uniquely, thyroid cell proliferation and hormone production are both controlled CAUSES OF FELINE HYPERTHYROIDISM by the TSH receptor–G protein–cAMP signaling. Overexpres- It is unlikely that autoantibodies to TSH, iodine deficiency, or sion of stimulatory G proteins and underexpression of in- iodine excess causes hyperthyroidism. There are unproven hibitory G proteins have been demonstrated in some humans theories that goitrogens, such as BADGE or isoflavones, with toxic nodular goiter.23,24 Mutations of the TSH receptor PBDEs, and genetic or molecular changes in predisposed in- that result in the receptor remaining activated without ligand dividual cats might contribute to hyperthyroidism. Feline hy- (i.e., TSH) have also been reported in humans with toxic perthyroidism, like most diseases, is probably caused by a nodular goiter.24–27 multitude of interactive factors, including genetics, nutrition, The same abnormalities have been investigated in hyper- and environment. thyroid cats, and it appears that activation mutation of the TSH receptor may be part of the pathogenesis of hyperthy- REFERENCES 28 1. Peterson ME, Johnson JG, Andrews LK. Spontaneous hyperthyroidism roidism in some cats. Furthermore, abnormalities of G proin the cat. Sci Proc Am Coll Vet Intern Med 1979:108. teins (in particular, significantly decreased G inhibitory 2. Holzworth J, Theran P, Carpenter JL, et al. Hyperthyroidism in the cat: protein expression) have been described in tissues from hy- ten cases. JAVMA 1980;176:345-353. perthyroid cats.29 3. Peterson ME, Randolph JF, Mooney CT. Endocrine diseases. In: Sherd- ing RG, ed. The Cat: Diseases and Clinical Management, 2nd ed. New York: Churchill Livingstone; 1994:1403-1506. ENVIRONMENTAL ASPECTS OF 4. Edinboro CH, Scott-Moncrieff JC, Janovitz E, et al. Epidemiologic study HYPERTHYROIDISM of relationship between consumption of commercial canned food and risk of hyperthyroidism in cats. JAVMA 2004;224:879-886. In one study, the use of cat litter was associated with an in- 5. Kass PH, Peterson ME, Levy J, et al. Evaluation of environmental, nu- creased risk of hyperthyroidism5; however, there was no sig- tritional and host factors for feline hyperthyroidism. J Vet Intern Med 1999;13:323-329. nificant difference among different brands of litter, suggesting 6. Johnson LA, Ford HC, Tartellin MF. Iodine content of commercially- that the use of litter is simply a marker of cats that are kept in- prepared cat foods. N Z Vet J 1992;40:18-20. doors.22 Indoor cats are likely to live longer and hence have 7. Tartellin MF, Johnson LA, Cooke RR. Serum free thyroxine levels re- spond inversely to levels of dietary iodine in the domestic cat. N Z Vet a higher risk of developing hyperthyroidism. Exposure to pes- J 1992;40:66-68.

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8. Kyle AH, Tartellin MF, Cooke RR, et al. Serum free thyroxine levels in 22. Peterson ME, Ward C. Etiopathologic findings of hyperthyroidism in cats maintained on diets relatively high and low in iodine. N Z Vet J cats. Vet Clin North Am Small Anim Pract 2007;37(4)633-645. 1994;42:101-103. 23. Delemer B, Dib K, Patey M, et al. Modification of the amounts of G pro- 9. Mooney CT. Pathogenesis of hyperthyroidism. J Vet Med Surg teins and of the activity of adenyl cyclase in human benign thyroid tu- 2002;4:167-169. mours. J Endocrinol 1992;132:477-485. 10. Ralph AG, Rupp NC, Ward CR. The flavonoid quercetin stimulated mi- 24. Derwahl M, Hamacher C, Papageorgiou G. Alterations of the stimula- togenesis in feline hyperthyroid cells [abstract]. J Vet Intern Med 2007;21:595. tory G protein (Gs)-adenylate cyclase cascade in thyroid carcinomas: evidence for up regulation of inhibitory G protein. Thyroid 1995;5(suppl 11. Court MH, Freeman LM. Identification and concentration of soy 1):S-3. isoflavones in commercial cat foods. Am J Vet Res 2002;63:181-185. 12. Doerge DR, Sheehan DM. Goitrogenic and estrogenic activity of soy 25. Russo D, Arturi F, Suarez HG, et al. Thyrotropin receptor gene alter- isoflavones. Environ Health Perspect 2002;110(suppl):349-353. ations in thyroid hyperfunctioning adenomas. J Clin Endocrinol Metab 1996;81:1548-1551. 13. White HL, Freeman LM, Mahoney O, et al. Effect of dietary soy on serum thyroid hormone concentrations in healthy adult cats. Am J Vet 26. Fuhrer D, Holzapfel HP, Wonerow P, et al. Somatic mutations in the Res 2004;65(5):586-591. thyrotropin receptor gene and not in the Gs alpha protein gene in 31 14. Martin KM, Rossing MA, Ryland LM. Evaluation of dietary and envi- toxic thyroid nodules. J Clin Endocrinol Metab 1997;82:3885-3891. ronmental risk factors for feline hyperthyroidism. JAVMA 2000;217:853- 27. Parma J, Duprez L, Van Sande J, et al. Diversity and prevalence of so- 856. matic mutations in the thyrotropin receptor and Gs alpha genes as a 15. Son HY, Nishikawa A, Ikeda T, et al. Lack of effect of soy isoflavone on cause of toxic thyroid adenomas. J Clin Endocrinol Metab 1997;82:2695- thyroid hyperplasia in rats receiving an iodine-deficient diet. Jpn J Can- 2701. cer Res 2001;92(2):103-108. 28. Peeters ME, Timmermans-Sprang EP, Mol JA. Feline thyroid adenomas 16. Dillingham BL, McVeigh BL, Lampe JW, Duncan AM. Soy protein iso- are in part associated with mutations in the G (s alpha) gene and not lates of varied isoflavone content do not influence serum thyroid hormones in healthy young men. Thyroid 2007;17(2):131-137. with polymorphisms found in the thyrotropin receptor. Thyroid 2002;12:571-575. 17. Teas J, Braverman LE, Kurzer MS, et al. Seaweed and soy: companion foods in Asian cuisine and their effects on thyroid function in American 29. Hammer KB, Holt DE, Ward CR. Altered expression of G proteins in women. J Med Food 2007;10(1):90-100. thyroid gland adenomas obtained from hyperthyroid cats. Am J Vet Res 18. Poole A, van Herwijnen P, Weideli H, et al. Review of the toxicology, 2000;61:874-879. human exposure and safety assessment for bisphenol A diglycidylether 30. Gaitan E. Goitrogens in food and water. Annu Rev Nutr 1990;10:21-39. (BADGE). Food Addit Contam 2004; 21(9): 905-919. 31. Scarlett JM, Moise NS, Ravl J. Feline hyperthyroidism: goitrogens de- 19. Kennedy RL, Thoday KL. Autoantibodies in feline hyperthyroidism. Res scriptive and case controlled study. Prev Vet Med 1988;6:295-309. Vet Sci 1988;45:300-306. 32. Olczak J, Jones BR, Pfeiffer DU, et al. Multivariate analysis of risk factors 20. Peterson ME, Livingston P, Brown RS. Lack of circulating thyroid stimulating immunoglobulins in cats with hyperthyroidism. Vet Immunol for feline hyperthyroidism in New Zealand. N Z Vet J 2005;53:53-58. Immunopathol 1987;16:277-282. 33. Dye JA, Venier M, Ward CA. Brominated-flame retardants (BFRs) in cats: 21. Brown RS, Keating P, Livingston PG, Bullock L. Thyroid growth impossible linkage to feline hyperthyroidism [abstract]. J Vet Intern Med munoglobulins in feline hyperthyroidism. Thyroid 1992;2:125-130. 2007;21:595.

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SCIENTIFIC PROGRAM: FOCUS ON FELINES Measures of Disease Activity in Feline Inflammatory Bowel Disease

Albert E. Jergens, DVM, PhD, DACVIM Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Iowa State University, Ames, Iowa

Research over the past several years has substantially ex- The World Small Animal Veterinary Association Congress panded our understanding of feline inflammatory bowel GI Standardization Group is working diligently to produce disease (FIBD) and its underlying pathomechanisms (Fig- and validate a workable, standardized histopathologic scor- ure 1). Advances in basic immunologic techniques and mo- ing system for GI inflammation that clinicians and patholo- lecular biology have provided evidence that FIBD likely gists can apply universally.7 This grading protocol covers the results from a dysregulated immunologic response to envi- gastric fundus and pylorus, duodenal mucosa, and colonic ronmental triggers, including luminal bacteria and specific mucosa. For each anatomic region, a narrative and visual antigens.1 In addition to these basic research findings, (photomicrographic) template that defines the normal his- emerging clinical data offer insight into the histopathology, tologic appearance of the tissue and key morphologic and in- clinical immunology, and assessment of disease severity in flammatory changes (by severity) has been developed. affected cats at diagnosis and in response to therapeutic Analysis of approximately 250 endoscopic biopsies collected intervention. Additionally, preliminary data suggest that by gastroenterologists worldwide using standardized report- some staging criteria have direct application to cats with ing forms is presently under way. other forms of chronic enteropathy, such as food-respon- sive enteropathy. MUCOSAL CYTOKINE EXPRESSION AND CORRELATION TO HISTOLOGY IN FIBD HISTOPATHOLOGIC GUIDELINES FOR In contrast to canine IBD, in which a balanced ratio of T-helper DIAGNOSING FIBD 1 (Th1) to T-helper 2 (Th2) mucosal cytokine pattern emerges, Histology is key to confirming diagnosis and eliminating data to date suggest that FIBD cytokine expression is more some other diseases, but standardized grading criteria have overtly Th1-like and broadly correlates to histopathologic in- not been adopted. Several qualitative and semiquantitative flammation. In one small study of 12 cats with IBD,8 endo- histopathologic scoring systems for IBD have been proposed scopic biopsies were evaluated for the presence of cellular (Box 1).2–5 In most instances, these scoring systems are larger, infiltrates and morphologic changes and then correlated to lev- case-based studies that use a spectrum of histologic criteria els of cytokine mRNA quantitated by real-time polymerase of mucosal inflammation with commentary on lamina pro- chain reaction. In general, morphologic changes (e.g., epithelial pria cellularity. Endoscopically obtained gastrointestinal (GI) alterations, villus fusion, atrophy) were associated with upreg- tract mucosal biopsy collection remains the gold standard ulated expression of interleukin-1β (IL-1β), IL-8, IL-12, and in- but presents a variety of challenges for clinicians and pathol- terferon-γ (IFN-γ) as well as IL-10 in diseased cats. Furthermore, ogists. It is recognized that these specimens are small, prone IBD grade correlated with IL-10 and IL-12, with IL-10 highest in to procurement and processing artifacts, and difficult to op- cats with severe FIBD. Interestingly, cytokine upregulation was timally orient for accurate morphologic characterization. Ad- not correlated with the density of the mucosal cellular infiltrate. ditionally, extensive interobserver variability in interpretation A separate investigation evaluated cytokine mRNA expres- between pathologists can occur.6 Histopathologic diagnostic sion in cats with chronic enteropathy caused by FIBD and non- criteria of IBD should clearly define morphologic evidence IBD GI diseases.9 The results of this study were analyzed on the of mucosal inflammation; however, which criteria are most basis of either clinical presentation or histopathologic evidence relevant is presently a matter of debate. of intestinal inflammation. Clinically normal cats and cats

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Box 1. Histopathologic Hallmarks of

Other environmental factors Microbes Feline Inflammatory Bowel Disease

• Changes in surface or cryptal epithelia (erosions, necrosis, hyperplasia, increased intraepithelial lymphocytes)

• Marked alterations in lamina propria cellularity Barrier Function • Mucosal architectural changes (villus blunting or fusion, mucosal Host Factors IBD edema, fibrosis, lymphatic dilation)

Innate and • Submucosal cellular infiltration Adaptive Immunity

Noted Disturbances: altered plasma protein concentrations (e.g., hyperglobuline- GIT Signs Histologic lesions mia, hypoalbuminemia) and increased serum concentration MHC II Expression Pro-inﬂammatory Cytokines of hepatic enzymes (e.g., alanine aminotransferase [ALT], as- Bacterial Antibody Responses Mucosally-Adherent Bacteria partate aminotransferase [AST], alkaline phosphatase [ALP]) are often observed; and (4) histologic lesions of lymphocytic– Figure 1. Proposed IBD etiopathogenesis. Intestinal inflammation plasmacytic mucosal cellular infiltrates predominate (Box 2). of feline IBD likely results from complex interactions between the resident microflora and the mucosa. Intestinal inflammation results A possible first step in the development of an FIBD activity from host (genetic) and environmental factors that affect barrier index (FIBDAI) would be collecting a wide range of variables, function and innate and adaptive immunity. (Adapted from Xavier RJ, Podolsky DK. Unravelling the pathogenesis of inflammatory including prominent GI signs (generally reported by the client bowel disease. Nature 2007;448:427-430.) or clinician) and select laboratory parameters, and correlating their association to the severity of histologic lesions. One pilot study has taken this exact approach.10 An FIBDAI was prospec- with FIBD showed increased expression of immunoregulatory tively evaluated in 27 cats with FIBD before and during thera- (IL-10, tumor growth factor-β [TGF-β]) and proinflammatory peutic intervention. Variables included histology, GI signs, (IL-6, IL-18, tumor necrosis factor-α [TNF-α], and IL-12p40) serum total protein and phosphorous concentrations, serum cytokines relative to cats with other GI diseases. Histopatho- ALT and ALP, and endoscopic lesions. Complete response to logic analysis showed that cats with intestinal inflammation prednisolone therapy was observed in 22 of 27 FIBD cats, and had upregulated expression of IL-6, IL-10, IL-12p40, TNF-α, remission was achieved in the remaining 5 cats with the addi- and TGF-β, compared with those with normal intestinal mor- tion of chlorambucil. Alterations in clinical scoring indexes phology. These accumulated observations indicate that FIBD were observed in all FIBD cats as a consequence of medical is characterized by immune dysregulation that parallels mor- therapy. Pretreatment FIBDAI scores (mean score: 7.8) were phologic evidence of intestinal inflammation. markedly reduced during the 14- to 21-day treatment period (mean posttreatment FIBDAI score: 0.8). These preliminary CLINICAL MEASURES OF DISEASE data suggest that clinical scoring of FIBD is suitable for clinical ACTIVITY IN FIBD evaluation of the therapeutic effect in these patients. Well-defined clinical criteria for assessment of FIBD activity have not been published, presumably reflecting the generally LABORATORY (SURROGATE) MARKERS sparse number of studies reported and the inability of the re- OF DISEASE ACTIVITY IN FIBD searchers to critically assess disease activity other than by the Acute-phase proteins (APPs), such as haptoglobin (HAP), severity of histologic lesions. Clearly, several themes emerge serum amyloid A (SAA), and acid glycoprotein (AGP), are from these earlier evidence-based investigations: (1) GI signs of plasma proteins that increase in concentration after infection, anorexia, weight loss, and vomiting predominate with gastric inflammation, or trauma. Serum APPs are routinely measured or small-intestinal IBD; (2) GI signs of hematochezia, mucoid in human clinical laboratories to assist in assessing the activ- feces, tenesmus, or increased frequency of defecation are com- ity of disease and its response to treatment. Previous stud- monly observed with colonic IBD; (3) biochemical changes of ies11,12 have shown that concentrations of some APPs correlate

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MEASURES OF DISEASE ACTIVITY IN FELINE INFLAMMATORY BOWEL DISEASE

Box 2. Clinical and Laboratory Parameters CONCLUSIONS Perturbed in Feline Inflammatory Bowel Disease Measures of disease activity for FIBD and other forms of feline

• Clinical scores • Increased hepatic enzymes chronic enteropathy are presently being designed. Cats with FIBD clearly have defined markers of intestinal inflammation, • Altered plasma proteins • Histologic grading such as altered proinflammatory cytokine expression profiles • Endoscopic lesions • Hypocobalaminemia and morphologic features of mucosal inflammation on review of histologic specimens. Preliminary results from pilot studies suggest that clinical variables may be useful to assess the initial well with human and canine IBD, but few detailed reports are disease severity and response to treatment in cats with FIBD. Fu- available for cats. In a prospective study that included 27 cats ture studies evaluating the role of fecal and serologic markers of with FIBD,11 we investigated whether serum APPs would be inflammation in cats with chronic enteropathy are warranted. altered at diagnosis and in response to therapeutic intervention compared with healthy cats. All of the cats in this study REFERENCES had an extensive diagnostic workup, including a dietary trial 1. Jergens AE. Inflammatory bowel disease: current perspectives. Vet Clin with an elimination diet to exclude adverse food reactions and North Am Small Anim Pract 1999;29:501-521. 2. Jergens AE, Moore FM, Haynes JS, et al. Idiopathic inflammatory bowel dis- GI tract endoscopic biopsy. Additionally, all cats were clini- ease in dogs and cats: 84 cases (1987–1990). JAVMA 1992;201:1603-1608. cally scored with the FIBDAI for disease severity at diagnosis 3. Dennis JS, Kruger JM, Mullaney TP. Lymphocytic/plasmacytic gas- and after 14 to 21 days of medical therapy. troenteritis in cats: 14 cases (1985–1990). JAVMA 1992;200:1712-1718. 4. Wilcock B. Endoscopic biopsy interpretation in canine or feline ente- Although it was hypothesized that cats with FIBD would rocolitis. Semin Vet Med Surg 1992;7:162-171. have increased APPs, the highest serum concentrations were 5. Roth L, Walton AM, Leib MS, et al. A grading system for lymphocytic- plasmacytic colitis in dogs. J Vet Diagn Invest 1990;2:257-262. observed in cats with non-IBD chronic enteropathy. Baseline 6. Willard MD, Jergens AE, Duncan RB, et al. Interobserver variation APPs (HAP, AGP) during the initial examination were mar- among histopathologic evaluations of intestinal tissues from dogs and cats. JAVMA 2002;220:1177-1182. ginally increased in cats with IBD compared with healthy 7. Day MJ. Report from the WSAVA GI Histopathologic Standardization cats. SAA was not detectable in any of the feline groups. Al- Group. Proc ACVIM, 2007. 8. Goldstein RE, Greiter-Wilke A, McDonough SP, et al. Quantitative eval- though medical therapy resulted in a significant reduction of uation of inflammatory and immune responses in cats with inflamma- clinical disease severity in FIBD, this was not accompanied tory bowel disease [abstract]. J Vet Intern Med 2003;17:411-412. 9. Nguyen VN, Taglinger K, Helps CR, et al. Measurement of mucosal cy- by reduced serum concentrations of APPs. Serum HAP tokine mRNA expression in intestinal biopsies of cats with inflamma- showed a negative correlation to therapy, and posttreatment tory enteropathy using quantitative real-time RT-PCR. Vet Immunol Immunopathol 2006;113:404-414. values were increased compared with pretreatment levels, 10. Crandell JM, Jergens AE, Morrison JA, et al. Development of a clinical suggesting that glucocorticoids likely induce serum concen- scoring index for disease activity in feline inflammatory bowel disease [abstract]. J Vet Intern Med 2006;20:788. trations of APPs. It was concluded that APPs are not suitable 11. Jergens AE, Crandell JM, Morrison JA, et al. Serum acute phase proteins in markers for assessment of disease activity in FIBD. feline inflammatory bowel disease [abstract]. J Vet Intern Med 2007;21:612.

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Nutrition Forum

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RESEARCH ABSTRACTS: ORAL PRESENTATION

Dietary Variables That Predict Glycemic Responses to Whole Foods in Cats

N.J. Cave,a J.A. Monro,b and J.P. Bridgesa aInstitute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North, New Zealand bNew Zealand Institute for Crop and Food Research Limited, Palmerston North, New Zealand

The glycemic load of high-carbohydrate diets has hours for all diets. There was no significant associa-

been proposed to be suboptimal for cats. We hy- tion between AUCinc and in vitro digestibility, but

pothesized that the in vitro carbohydrate digestibility GGE60 was associated with the baseline fasted blood of diets would predict the in vivo glycemic response glucose (r2 = .35; P = .035). The fasted blood glu- 2 or that other dietary variables would explain any dif- cose predicted the absolute AUC (AUCabs; r = .66; 2 ference. P < .001), the AUCinc (r = –.73; P < .001), and peak 2 The in vitro digestibility of 18 whole dry diets was glucose responses (r = .59; P < .001). AUCinc was not determined by simulated physiologic digestion. Diets associated with dietary crude fiber, fat, protein, car- were ranked according to the rate of glucose release bohydrate, or physical biscuit characteristics. Glycemic

over time (GGE) relative to total available carbohy- responses (AUCinc) to whole foods are prolonged and drates. Six diets spanning the range of GGE were se- are not predicted by the digestibility of the carbohy- lected for in vivo assessment. Six cats were each pre-fed drate component. one of the diets for 7 days followed by a 16-hour fast; When the equivalent amount of available carbo- they were then fed enough diet to provide 1 g/kg of hydrate is fed, incremental glycemic responses to dif- available carbohydrates. Serial blood glucose was as- ferent diets are unaffected by dietary fiber, fat, protein, sayed until it had returned to baseline. carbohydrate type, or physical biscuit characteristics. Despite a wide range of in vitro digestibilities and Whereas fasted blood glucose after a few days of feed- compositions, there was little difference in incre- ing appears to be a good indicator of the long-term

mental area under the curve (AUCinc) between diets, glycemic load of a diet (AUCabs), AUCinc is probably with glucose absorption occurring over 10 to 12 determined simply by the rate of gastric emptying.

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Spaying Affects Blood Metabolites and Adipose Tissue Gene Expression in Cats

K.R. Belsito,a B.M. Vester,a T. Keel,b T.K. Graves,b,c and K.S. Swansona,b,c aDepartment of Animal Sciences, University of Illinois, Urbana, Illinois bDepartment of Veterinary Clinical Medicine, University of Illinois, Urbana, Illinois cDivision of Nutritional Sciences, University of Illinois, Urbana, Illinois

Although spaying is known to contribute to obesity, lipoprotein lipase (LPL) mRNA was decreased (P < .05) the role of adipose tissue is poorly understood. Thus, at weeks 12 and 24. Adipose hormone-sensitive lipase our objectives were to examine the effects of spaying (HSL) mRNA was decreased (P < .05) at week 24. Adi- on serum metabolite concentrations and adipose tis- pose tumor necrosis factor-α mRNA tended to be de- sue and skeletal muscle gene expression in cats. creased (P < .10) at week 12, and interleukin-6 (IL-6) Eight adult (>1 year old) domestic shorthair cats mRNA was increased (P < .05) at weeks 12 and 24. were fed a commercial dry diet throughout the study. Adipose leptin mRNA was decreased (P < .05) at week After a 2-week baseline period (week 0), cats were 12, and adiponectin mRNA tended to be decreased spayed and fed to maintain an ideal body weight for (P < .10) at week 24. Changes in HSL and LPL mRNA 12 weeks. After 12 weeks, cats were fed ad libitum for suggest changes in adipose tissue lipid metabolism as an additional 12 weeks. Blood samples were collected a result of spaying and weight gain, likely contributing at weeks 0, 6, 12, 18, and 24, and adipose tissue and to increased circulating triglycerides. Decreased skeletal muscle biopsies were collected at weeks 0, 12, adiponectin and increased IL-6 mRNA may be early and 24. Data were analyzed using the mixed-model signals of adipose tissue dysregulation and contribute method of SAS (Cary, NC). to insulin resistance. Fasting serum glucose and triglycerides were in- Our results demonstrate that adipose tissue is sen- creased (P < .05) at week 24, and plasma leptin tended sitive to spaying or weight gain (or both), justifying to be increased (P < .10) at weeks 18 and 24. Adipose further research in this area.

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RESEARCH ABSTRACTS: ORAL PRESENTATION

Effects of Spaying on Food Intake, Weight Gain, Body Condition Score, Activity, and Body Composition in Cats Fed a High-Protein versus Moderate-Protein Diet

B.M. Vester,a K.J. Liu,b T. Keel,c T.K. Graves,c,d and K.S. Swansona,c,d aDepartment of Animal Sciences, University of Illinois, Urbana, Illinois bNatura Manufacturing, Inc., Fremont, Nebraska cDivision of Nutritional Sciences, University of Illinois, Urbana, Illinois dDepartment of Clinical Veterinary Medicine, University of Illinois, Urbana, Illinois

High-protein diets have been used to promote weight over time in all cats and tended to be increased (P < .10) loss in cats, but the effect of feeding high-protein in cats fed a high-protein diet. BCS was greater (P < .05) diets after spaying to maintain weight has not been in cats fed the high-protein diet but increased (P < .05) determined. The objective of this study was to evalu- over time regardless of dietary treatment. Total activate cats fed either a high-protein diet (52.9% crude ity, measured using Actical activity collars (Mini-Mit- protein [CP] on a dry matter basis [DMB]) or a mod- ter, Bend, OR), decreased (P < .05) from week 0 to erate-protein diet (34.3% CP DMB; 3.9 and 4.2 kcal/g weeks 12 and 24. Body composition did not change due calculated metabolizable energy, respectively) fol- to diet; however, body fat percentage increased (P < .05) lowing ovariohysterectomy. over time. Grams of lean tissue showed a curvilinear Food intake, body weight (BW) gain, body condi- (P < .05) effect over the course of the study, but per- tion score (BCS), body composition, and activity level centage of lean tissue tended to decrease (P < .10) over were measured in eight cats (four cats/treatment). Cats time. Bone mineral content was increased (P < .05) at older than 1 year underwent ovariohysterectomy on week 12 in cats fed the high-protein diet. This is likely week 0 and were fed ad libitum for 24 weeks. Food to support the increased BW because of the large in- intake was measured daily, and BW and BCS were crease in food intake early after spaying in the cats fed measured weekly. Activity was measured for 6 con- the high-protein diet, which may have been due to the secutive days before weeks 0, 12, and 24, and body high palatability of this diet. composition was determined by dual-energy x-ray ab- Based on these data, feeding a diet ad libitum after sorptiometry at weeks 0, 12, and 24. spaying, regardless of protein level in the diet, may Food intake and BW were markedly changed (P < .05) increase the incidence of obesity in cats.

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Higher Protein Consumption during Weight Loss Allows Higher Caloric Intake for Maintenance of Body Weight in Cats

R.S. Vaconcellos,a N.C. Borges,a K.N.V. Gonçalves,a F.J.A. de Paula,b E.B. Malheiros,a R.S. Bazolli,a and A.C. Carciofia aSchool of Agrarian and Veterinarian Sciences, State University of São Paulo, São Paulo, Brazil bHospital of Clinics, Medical School of Ribeirão Preto, State University of São Paulo, São Paulo, Brazil

The objective of this study was to compare energy re- weeks, there was no difference in body composition quirements for weight stabilization of cats that lost between groups (control group: FM = 24.9% ± 2.2%; weight while consuming two different dietary protein LM = 72.8% ± 2.8%; high-protein group: FM = 24.1% levels. Fifteen adult neutered cats were divided into ± 1.9%; LM = 72.2% ± 1.1%). From weeks 7 to 12, en- two groups: a control group and a high-protein group. ergy requirements gradually increased and did not sta- Two procedures were followed: In the first part of bilize in either group. Energy requirements were the study, two diets were used to achieve a controlled similar during the first 6 weeks of the study (control 20% weight loss (the control group was given a 29% group: 93.6 ± 2.8 kcal/kg BW0.4; high-protein group = crude protein [CP] diet, and the high-protein group 97.2 ± 1.9 kcal/kg BW0.4) but significantly higher in the was given a 43% CP diet). Groups had similar body high-protein group during weeks 7 to 12 (control composition (dual-energy x-ray absorptiometry) be- group = 96.7 ± 2.2 kcal/kg BW0.4; high-protein group = fore weight loss; after weight loss, the control group 111.9 ± 1.8 kcal/kgBW0.4; P < .001) and weeks 13 to 17 had higher fat body mass (FM; 28.8% ± 1.6%) and (control group = 110.6 ± 2.2 kcal/kg BW0.4; high-pro- lower lean body mass (LM; 68.4% ± 1.6%) than the tein group = 127.7 ± 2.0 kcal/kg BW0.4; P < .01). high-protein group (FM: 23.4% ± 3.2%; LM: 73.5% Based on our results, we conclude that high pro- ± 3.1%; P < .01). tein ingestion during weight loss periods provides Cats were then fed a 39% CP diet to maintain body higher energy requirements to stabilize body weight weight for 17 weeks. Energy ingestion was divided into during maintenance. Nutritional composition of the three periods: initial (weeks 0 to 6), middle (weeks 7 maintenance diet may contribute to the recovery of to 12), and final (weeks 13 to 17). At the end of the 17 lean body mass lost during caloric restriction.

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RESEARCH ABSTRACTS: ORAL PRESENTATION

Effect of a Low-Protein Diet on Gut Morphology in Cats

D.G. Thomas,a C.E. Ugarte,a K.J. Rutherfurd-Markwick,a and W.H. Hendriksb aInstitute of Food, Nutrition and Human Health, Massey University, Palmerston North, New Zealand bAnimal Nutrition Group, Department of Animal Sciences, Wageningen University, Wageningen, The Netherlands

Ingesting proteins and amino acids can impact the 25.5% fat, 49.9% carbohydrate), semi-synthetic diet health of cats in two ways: (1) in a nutritive sense by (n = 6) for 10 weeks before being euthanized. Sam- supplying necessary energy and amino acid require- ples of intestine (~ 5 cm in length) were excised from ments, and (2) by acting as bioactive molecules and areas 15% (duodenum), 30% (jejunum), and 60% influencing functions within the body, including in- (ileum) along the length of the tract and processed testinal health. Because there is little information for histologic analysis. Transverse sections (5 μm) of available on the influence of diet on gut health in tissue were cut, and each specimen was stained with cats, this preliminary study was designed to generate alcian blue, hematoxylin–eosin and examined by information on changes in gut morphology in re- light microscopy (original magnification, ×100). Sig- sponse to a low-protein diet. maScan (Systat Software, Chicago, IL) was used to Eleven adult feral cats (six males, five females) measure villous height, crypt depth, and epithelial were trapped as part of normal pest-control measures cell thickness of 10 villi in each tissue sample. in the Manawatu region of New Zealand. Before Similar morphologic characteristics were observed being included in the study, the cats were sedated and in the duodenal, jejunal, and ileal segments. Animals screened by a veterinarian for feline immunodefi- fed the low-protein diet had consistently longer villi, ciency virus or feline leukemia virus. Cats were deeper crypts, and a thicker epithelial cell layer than housed in single-metabolism cages and fed either a animals fed the control diet. This indicates that the an- control (32.7% protein, 21.0% fat, 42.5% carbohy- imals responded to the low-protein diet by increasing drate) diet (n = 5) or a low-protein (20.9% protein, the gut surface area to maximize nutrient absorption.

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Variations in Dietary Fat Affect Lipid Metabolism in Domestic Cats

M.K. McClure,a R.J. Angell,a K.E. Bigley,a K. Fennell,a and J.E. Bauer b aCompanion Animal Nutrition Laboratory, Texas A&M University, College Station, Texas bIntercollegiate Faculty of Nutrition, Texas A&M University, College Station, Texas

Feline fatty acid metabolism may be directly affected tically significant (P < .05) triacylglycerol-lowering ef- by alterations of dietary fat intake. This study investi- fect despite the already low-normal triacylglycerol gated the effect of different types of dietary fat on levels typically observed. Lipoprotein electrophoresis plasma triacylglycerol, total cholesterol, lipoprotein- revealed a statistically significant lowering of the pre- cholesterol, and nonesterified fatty acid (NEFA) con- beta band (i.e., very-low-density lipoprotein) triacyl- centrations. glycerol in the MFO diet (P < .05) consistent with Thirty clinically normal, sexually intact young plasma triacylglycerol lowering. Whether triacylglyc- adult female cats were randomized into three groups erol lowering in normal cats is beneficial is unknown. of 10. Each group was fed a complete, balanced, com- No main time or diet effects were found on total cho- mercial, dry, extruded-type basal diet supplemented lesterol concentrations, and no changes were ob- with equal amounts of fat, differing only in fatty acid served in mean plasma NEFA concentrations. In some composition. The diets were designated as high-oleic cats, an additional lipid-staining region was found on sunflower (HOS), menhaden fish oil (MFO), and saf- the electrophoretogram, which migrated similar to flower oil (SFO). The HOS diet contained high plasma albumin; however, this region did not corre- amounts of oleic acid, the MFO diet contained high late with plasma NEFA concentrations. amounts of long-chain omega-3 fatty acids, and the Additional studies to investigate these effects are SFO diet contained linoleic acid. Diets were fed for in progress, including studies of plasma phospho- 28 days, with blood collected on days 0, 14, and 28. lipid and red blood cell fatty acids, cholesteryl ester Using repeated measures analysis of variance and and lecithin acyl transferase activities, and indices of post hoc comparisons, the MFO diet showed a statis- fatty acyl desaturase enzyme activities.

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RESEARCH ABSTRACTS: ORAL PRESENTATION

Impact of Dietary Trans-Fatty Acid on Serum Insulin and Glucose Concentrations in Cats

P.A. Schencka and S.K. Aboodb aDepartment of Pathobiology and Diagnostic Investigation, Diagnostic Center for Population and Animal Health, Michigan State University, East Lansing, Michigan bDepartment of Small Animal Clinical Sciences, Michigan State University, East Lansing, Michigan

Diabetes mellitus (DM) in cats is characterized by in- BCS, 10.1 lb body weight, 3.9% body fat) or group 3 sulin resistance. Overweight and older cats are at in- cats (mean: 5.0 BCS, 9.0 lb body weight, 3.6% body creased risk of developing DM. In humans, dietary fat). Group 2 cats showed a significantly higher (P < .05) trans-fatty acids (TFA) increase insulin resistance, es- concentration of serum insulin (mean: 54.5 pmol/L) pecially when fed at a level greater than 1% energy and had a higher serum insulin:glucose ratio (mean: (%E). Exposure to dietary TFA may increase insulin 9.8) than group 1 cats (mean: 39.1 pmol/L serum in- resistance and type II DM in cats. The objective of this sulin, 6.8 insulin:glucose ratio) or group 3 cats (mean: study was to determine if dietary intake of TFA was 28.0 pmol/L serum insulin, 5.9 insulin:glucose ratio) higher in cats at increased risk for DM. cats. There were no significant differences in serum glu- Cats were grouped as follows: group 1 (normal; n = cose, serum or dietary TFA concentrations, or dietary 27)—less than 10 years of age, body condition score %E derived from TFA. Insulin concentration and in- (BCS) of 4 to 6; group 2 (fat; n = 23)—less than 10 years sulin:glucose ratio were significantly correlated to BCS, of age, BCS of 7 to 9; and group 3 (senior; n = 6)—10 body weight, and percentage of body fat but not to years of age or older, BCS of 4 to 6. Serum was col- serum TFA, dietary TFA, or dietary %E from TFA. lected for general analysis, and each cat’s diet was an- Cats at higher risk for DM did not show elevated alyzed for TFA. serum TFA or dietary intake of TFA. Although there Group 2 cats had a significantly higher BCS (mean: does not appear to be a direct correlation of dietary 7.6), body weight (mean: 14.1 lb), and percentage of TFA to insulin concentration, TFA could still contribute body fat (mean: 5.0%) than group 1 cats (mean: 5.3 to the development of DM in predisposed individuals.

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Sequencing and Characterization of Feline Pancreatic Glucokinase cDNA

S. Lindbloom, M. LeCluyse, E. Hiskett, and T. Schermerhorn Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas

Glucokinase (GK) is an important metabolic enzyme bp and encodes a 465–amino acid protein (GenBank that is the “glucose sensor” in pancreatic beta cells. EF121813). The predicted feline pancreatic GK protein GK activity in beta cells correlates with blood glucose is 89% to 94% identical to other mammalian GK pro- concentration and links glucose metabolism to acti- teins and contains 15 unique residues, 5 of which are vation of cellular pathways that promote insulin se- nonconserved substitutions. Substrate binding, pro- cretion. The importance of pancreatic GK expression tein recognition, and other important functional mo- for normal glucose tolerance and insulin secretion is tifs are conserved in feline GK. The feline GK protein well established, and GK mutations cause diabetes in model has two globular domains separated by a hinge mammals. GK mRNA is known to be expressed in the region, similar to known GK structures. Interestingly, feline pancreas, but the molecular details of feline modeling studies indicated the region around non-

pancreatic GK have not been previously investigated. conserved tryptophan35 in wild-type feline pancreatic This study’s objectives were to determine the se- GK has structural similarities to human GK with an quence of feline pancreatic GK cDNA, predict the R36W mutation, which causes type-2 maturity-onset amino acid sequence of the feline pancreatic GK pro- diabetes mellitus of the young. tein, and perform a comparative analysis of feline In conclusion, feline pancreatic GK has all major pancreatic GK sequence and structure. sequence and structural motifs found in noncarni- GK mRNA from the pancreas of a normal cat was vores, but nonconserved amino acids in the feline se- analyzed with reverse transcription polymerase chain quence may indicate species specificity. The aggregate reaction using species-specific primers. The elucidated effect of the nonconserved residues on protein func- cDNA sequence was used to predict protein sequence. tion and the significance of these variations with re- Protein structure was modeled using molecular mod- spect to feline glucose metabolism or development eling software. The cDNA coding region contains 1,398 of diabetes is unknown.

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RESEARCH ABSTRACTS: ORAL PRESENTATION

Effects of Epigallocatechin Gallate Singly and in Combination with Lactoferrin on Oral Health in Cats

S. Krammer-Lukas,a K. Cramer,a U. Wehr,b S. Gorissen,b and K. Elsbett b aAnimal Nutrition and Health Research and Development, DSM Nutritional Products, Kaiseraugst, Switzerland bInstitute of Nutrition, Ludwig-Maximilian-University, Munich, Germany

Periodontopathic conditions are the most common 300 mg/kg cat food). General and dental health were diseases in dogs and cats. Prophylaxis is usually lim- investigated in the study. Because the “clean tooth ited to professional dental cleaning under anesthe- model” was used, all cats received a dental cleaning sia, especially in cats. Special diets that reduce plaque before the 28-day feeding period. and calculus accumulation are formulated to extend EGCG alone proved able to inhibit the growth of the time between dental cleanings. Epigallocatechin bacteria taken from the feline oral cavity in vitro and (EGCG) and lactoferrin are already used in humans seemed to have an effect on the cats’ antioxidant sta- to maintain oral health because of these substances’ tus. Supplementation with EGCG in combination antibacterial effect. The aim of this study was to in- with lactoferrin led to slightly lower plaque and cal- vestigate the effect of EGCG singly and in combina- culus indexes compared with the control group. The tion with lactoferrin in a regular, dry, nondental diet gingivitis index decreased significantly in the on the oral health of cats. EGCG/lactoferrin group, and compared with the con- In two consecutive trials, a total of 18 domestic trol group, the probing depth of the supplemented short-haired cats (age: 3.83 ± 1.85 years old; body diet group was significantly lower at the end of the weight: 4.2 ± 1.1 kg) were divided on the basis of experimental phase of the EGCG/lactoferrin test. plaque score, age, and gender into two equal groups Altogether, the combination of EGCG and lacto- of nine animals each. For 28 days, animals were fed ferrin could be valuable when used along with es- either a control or treated diet (trial 1: 227 mg tablished solutions for reducing plaque and calculus EGCG/kg cat food; trial 2: EGCG and lactoferrin each (e.g., fiber-containing kibbles or edible chews).

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Nutrition Forum

Research Abstracts: Poster Presentations 2008 Purina Abstracts ORAL_POSTER.qxp:2005 Master Page 2/26/08 4:12 PM Page 68 2008 Purina Abstracts ORAL_POSTER.qxp:2005 Master Page 2/26/08 4:12 PM Page 69

RESEARCH ABSTRACTS: POSTER PRESENTATION

Effect of Isoflavones, Conjugated Linoleic Acid, and L-Carnitine on Weight Loss and Oxidative Stress in Overweight Dogs

Y. Pan,a I. Tavazzi,b J.-M. Oberson,b L.B. Fay,b and W. Kerra aNestlé Purina Research, St. Louis, Missouri bNestlé Research Center, Lausanne, Switzerland

This study investigated whether soy isoflavones alone isoflavone, and blend diets groups, respectively; the or a blend of soy isoflavones, conjugated linoleic percentage of Siberian huskies with their body fat re- acid, and L-carnitine can promote weight loss, pre- duced to ideal levels was 33.3%, 50%, and 50% for serve lean body mass, and reduce oxidative stress in the control, isoflavone, and blend diets groups, re- overweight dogs. Overweight Labrador retrievers and spectively. Compared with the control diet, both the Siberian huskies were randomized into three groups isoflavone and blend diets groups significantly re- (control, isoflavones, blend diets) and fed 70% of duced plasma isoprostanes, and the blend diet com- their maintenance energy requirement (MER) during pletely prevented loss of lean body mass and the first 3 months of weight loss. Dogs that failed to significantly increased lean body mass after the first 3 reach their ideal body fat levels after the first 3 months of weight loss. months of weight loss were fed 55% of their MER In summary, more dogs in the isoflavone and during the second 3 months of weight loss. Dual-en- blend diets groups tended to have their body fat per- ergy x-ray absorptiometry scans were obtained 3 and centages reduced to ideal levels than did control dogs. 6 months after the study was initiated. The blend diet prevented loss of lean body mass in At the end of the study, the percentage of Labrador overweight dogs during weight loss, and soy retrievers with their body fat reduced to ideal levels isoflavones in the weight-loss diets reduced in vivo was 66.7%, 75%, and 85.7% for the control, oxidative damage in overweight dogs.

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Postfeeding Satiety and Weight Loss of Dogs Fed a Vegetable-Based Fiber Supplement

Y. Mitsuhashi,a K. Bigley,a and J.E. Bauer a,b aCompanion Animal Nutrition Laboratory, Texas A&M University, College Station, Texas bIntercollegiate Faculty of Nutrition, Texas A&M University, College Station, Texas

Dietary insoluble fiber is believed to support weight postprandially, and food intakes were recorded. loss by increasing satiety and the mass of the food For the weight loss study, seven obese beagles were consumed without adding calories. Therefore, we selected (average body fat: 45.1% ± 1.6%; body weight: measured satiety and weight loss in beagles fed a veg- 15.2 ± 1.0 kg) and divided into two groups. The diets etable-based fiber supplement. were fed once daily (approximately 60% of obese Two diets that differ in fiber content were fed: Pu- MER) for 42 days. Postprandial blood samples were rina ONE® Healthy Weight Formula (Purina ONE; collected at 0 and 60 minutes on days 1, 28, and 42. Nestlé Purina PetCare, St. Louis, MO) was compared Food intakes and body weight were recorded daily with a diet consisting of Purina ONE plus a vegetable- and weekly, respectively. based fiber supplement (FS); crude fiber contents were As expected, all dogs lost similar amounts of body 2.6% (Purina ONE) versus 5.4% (FS) dry matter. weight and body fat independent of diet. In the sati- For the satiety studies, 12 to 14 adult female beagles ety trials, intake at both the 3- and 7-hour intervals with average body fat of 38.4% ± 1.9% and body weight was not different from the control group; however, sig- of 13.8 ± 0.9 kg were randomly divided into two groups. nificantly fewer total calories were consumed with the Diets were fed at 8:00 AM and 3:00 PM (7-hour interval) FS diet during the 3-hour interval. FS did not affect during one trial and at 8:00 AM and 11:00 AM (3-hour in- triglyceride concentrations. Thus, FS provided fewer terval) for 15 minutes each during a second trial. The calories with the same degree of satiety as the higher amounts offered at each feeding were 1.2 times the main- calorie intake of the control diet. FS may improve full- tenance energy requirement (MER) using a crossover de- ness at lower calorie intake during weight loss with no sign. Blood samples were collected 45 and 120 minutes effect on hypertriglyceridemia.

70 Proceedings, 2007 Nestlé Purina Nutrition Forum 2008 Purina Abstracts ORAL_POSTER.qxp:2005 Master Page 2/26/08 4:12 PM Page 71

RESEARCH ABSTRACTS: POSTER PRESENTATION

Body Condition and scFOS Supplementation Influence Adipose Tissue mRNA Abundance

K.R. Belsito,a B.M. Vester,a F. Respondek,b M. Diez,c and K.S. Swansona,d,e aDepartment of Animal Sciences, University of Illinois, Urbana, Illinois bBéghin Meiji, Marckolsheim, France cUniversity of Liège, Liège, Belgium dDivision of Nutritional Sciences, University of Illinois, Urbana, Illinois eDepartment of Clinical Veterinary Medicine, University of Illinois, Urbana, Illinois

Adipose tissue is a highly active endocrine tissue that the obese state, a crossover design was used to test plays a pivotal role in glucose and lipid metabolism, scFOS versus control diets. For each period, dogs were energy homeostasis, and disease risk. Recent experi- randomly allotted to a diet and fed for 6 weeks. Fast- ments suggest that fermentable dietary fibers, in- ing and fed adipose samples were collected at the end cluding short-chain fructooligosaccharides (scFOS), of each period. Real-time quantitative reverse tran- may beneficially impact glucose homeostasis and scriptase polymerase chain reaction was used to mea- adipocyte metabolism. The objectives of the current sure mRNA abundance of genes involved with fatty experiment were to compare adipose tissue mRNA acid metabolism, glucose metabolism, or inflamma- abundance in lean versus obese dogs and in obese tion. mRNA data were analyzed using the mixed- dogs fed a diet containing 1% scFOS versus a control models procedure of SAS. (fructan-free) diet. Compared with a lean phenotype, obesity in- The experiment consisted of two phases, the “obe- creased (P < .05) insulin receptor substrate 2 and in- sity phase” followed by a “treatment phase.” Adipose terleukin-6 mRNA abundance and tended to increase samples were collected from eight (four female, four (P < .10) leptin mRNA. In the obese state, scFOS al- male) neutered adult beagles with a normal body tered the expression of hormone-sensitive lipase, condition score (5 of 9) during fasted and fed states lipoprotein lipase, and uncoupling protein 2. at baseline. All dogs were then fed ad libitum to pro- More research is needed to identify to what extent mote weight gain (to 125% optimal body weight) gene transcripts or proteins involved with leptin or in- and then fed to maintain this obese phenotype. In sulin signaling are affected by scFOS supplementation.

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All-Trans-Astaxanthin Does Not Protect Canine Osteosarcoma Cells from Chemotherapeutic or Radiation-Induced Cell Death

J.J. Wakshlag, C.B. Balkman, A.M. Struble, S.K. Morgan, and M. Zgola Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York

Astaxanthin is a natural carotenoid with potent an- ation to various degrees in all three cell lines exam- tioxidant properties in vitro and in vivo. Previous re- ined. To further elucidate the antioxidant capabilities search in rodent models has suggested that of astaxanthin-treated cells, we used a commercial kit astaxanthin can diminish cell proliferation and retard to measure total antioxidant potential of cell lysates, tumor growth. The exact mechanisms of action have which showed modest antioxidant potential; how- yet to be elucidated, but it is believed that astaxan- ever, the antioxidant potential of astaxanthin-treated thin can decrease promitogenic autocrine mediators, cells was not enhanced beyond the natural upregula- inhibit cell signaling pathways, and alter cell adhe- tion of cellular antioxidant potential during cell sion molecules. Although astaxanthin may provide stress, further supporting our cell death assays. an attractive alternative therapy for neoplasia, its Overall, the results suggest that astaxanthin has the strong antioxidant capabilities have precluded its inability to significantly inhibit cell proliferation and corporation into cancer therapy, as it has been hy- growth of colonies in soft agar but that this inhibitory pothesized that using antioxidants during radiation capacity is different, depending on the cell line ex- or chemotherapy may hinder neoplastic cell death. amined, with no appreciable changes in cell cycle dy- To test this hypothesis, we treated three osteosar- namics. Surprisingly, astaxanthin did not hinder the coma cell lines with and without radiation, ability of radiation treatment, peroxidation, or dox- chemotherapy (doxorubicin), and peroxidative stress orubicin to induce cell death. This suggests that the

(H2O2) to see if astaxanthin treatment significantly use of astaxanthin as a synergistic antiproliferative alters cell death. Growth curves, cell death assays compound may be beneficial in neoplastic diseases. (methyl thiazolyl tetrazolium), flow cytometry, and Further investigation into its use across various neo- soft agar growth assays were performed. plasias and its mechanisms of action is warranted, Astaxanthin treatment had no significant effects particularly because the antioxidant capabilities do on radiation or chemotherapeutic or peroxidative cell not seem to interfere with traditional cancer treat- death; however, it did significantly slow cell prolifer- ment options.

72 Proceedings, 2007 Nestlé Purina Nutrition Forum 2008 Purina Abstracts ORAL_POSTER.qxp:2005 Master Page 2/26/08 4:12 PM Page 73

RESEARCH ABSTRACTS: POSTER PRESENTATION

Absorption Trial of Ginkgo Biloba Extract in Cats

A. Pasquini,a G. Cardini,a C. Gardana,b P. Simonetti,b G. Giuliani,c G. Re,d and G. Lubasa aDepartment of Veterinary Clinic, University of Pisa, Pisa, Italy bDepartment of Food Science and Microbiology, Division of Human Nutrition, University of Milan, Milan, Italy cUrban Veterinary Hygiene, Florence, Italy dBayer Italia, Milan, Italy

Ginkgo biloba extract (EGb) contains flavonoids, tive and quantitative evaluation of quercetin in which have antioxidant, antiinflammatory, and an- plasma at fixed times. Blood samples were collected tiviral properties and induce peripheral vasodilation. in heparinized tubes before EGb administration in It is widely reported in the scientific literature that all four cats; after 1, 3, and 5 hours in two cats; and these properties are useful for elderly humans and after 5, 7, and 9 hours in the remaining two cats. animals. EGb can be administered to senior cats by Plasma quercetin concentrations were established adding it to their food, which ensures regular and without knowledge of study group by liquid chro- continual administration. The presence of EGb matography tandem mass spectrometry. flavonoids in this study was confirmed by high-per- Table 1 shows quercetin concentration in plasma formance liquid chromatography coupled with a (ng/ml) in all four cats before and after the adminis- diode array detector and mass spectrometry. tration of EGb. Flavonoids from EGb were absorbed The goal of this study was to establish EGb by cats, and their main metabolite, quercetin, was flavonoid absorption in cats. Four privately owned, present in the blood for up to 5 hours. Furthermore, healthy cats of different sexes, ages, and breeds were none of the cats showed any adverse effects (e.g., di- included in the study. The standardized EGb (glyco- arrhea). sylated flavonoids: 24%; terpene lactones: 6%) was This evidence encourages the use of EGb, admin- administered at 20 mg/kg with 50 g of dry food istered alone or added to the diet, to improve cat (Bayer Fito Progres Cat Senior®) followed by qualita- wellness.

TABLE 1 Quercetin Concentration in Plasma (ng/ml)

Cat Time Zero 1 hr 3 hr 5 hr 7 hr 9 hr

1 0.0 0.0 6.67 4.47 No data No data 2 0.0 7.15 11.28 0.0 No data No data 3 0.0 No data No data 0.0 0.0 0.0 4 0.0 No data No data 0.0 0.0 0.0

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High-Protein Diet Impacts Fecal Microbial Populations in Growing Kittens

B.L. Dalsing,a B.M. Vester,b C.J. Apanavicius,b D.C. Lubbs,b and K.S. Swansonb,c,d aDepartment of Molecular and Cellular Biology, University of Illinois, Urbana, Illinois bDepartment of Animal Sciences, University of Illinois, Urbana, Illinois cDepartment of Veterinary Clinical Medicine, University of Illinois, Urbana, Illinois dDivision of Nutritional Sciences, University of Illinois, Urbana, Illinois

Although the intestinal microbiota of the human gut Escherichia coli) previously determined to be prevalent has received considerable attention as of late, very lit- in cats. Mixed models of SAS (Cary, NC) were used to tle is known about life in the feline gastrointestinal analyze quantitative PCR data. Qualitative analysis tract. Even less is known about the intestinal micro- was performed on each sample using denaturing gra- biota of growing kittens. Thus, our objective was to dient gel electrophoresis with a 29% to 48% gradient investigate the intestinal microbiota of growing kit- to separate amplicons. DNA bands of interest were ex- tens fed moderate-protein (MP) or high-protein (HP) cised from the gel, extracted using the QIAquick Gel diets using molecular qualitative and quantitative Extraction Kit (Qiagen), and sequenced using an ABI techniques. PRISM bigDye Terminator Cycle Sequencing Ready Kittens consuming an HP diet (7 males from 2 lit- Reaction Kit and ABI 3730XL capillary sequencer ters) or MP diet (10 males from 4 litters) were evalu- (Applied Biosystems, Foster City, CA). 16S rRNA se- ated. Kittens were weaned at 8 weeks of age and quences were subject to BLAST search (GenBank) for consumed the same diet as their dams. Fresh fecal identification. samples were collected at 8, 12, and 16 weeks of age The presence of Bifidobacterium spp and Lactobacillus and stored at –80°C. Fecal DNA was extracted using spp was affected by diet, with kittens fed HP diets hav- the QIAamp DNA Stool Mini-Kit (Qiagen, Valencia, ing lower (P < .05) counts than those fed MP diets. E. CA). DNA purity and concentration were determined coli was also lower (P < .05) in kittens fed HP diets and using an ND-1000 NanoDrop spectrophotometer. was affected by age. Microbial differences in growing Quantitative polymerase chain reaction (PCR) was kittens suggest that prebiotic supplementation may be used to quantify four microbial groups (Bifidobac- beneficial when feeding HP diets because of decreased terium spp, Lactobacillus spp, Clostridium perfringens, Bifidobacterium and Lactobacillus populations.

74 Proceedings, 2007 Nestlé Purina Nutrition Forum 2008 Purina Abstracts ORAL_POSTER.qxp:2005 Master Page 2/26/08 4:12 PM Page 75

RESEARCH ABSTRACTS: POSTER PRESENTATION

Impact of Sampling Interval on the Variability of Activity Counts Recorded from the Actical Activity Monitor Worn by Pet Dogs

C. Dow, K.E. Michel, and D.C. Brown University of Pennsylvania, Philadelphia, Pennsylvania

The Actical Activity Monitor (AAM; Mini-Mitter, There was significant variability in activity counts Bend, OR) is an accelerometer-based device that con- between dogs (P < .001). As a group, there was sig- tinuously measures movement for extended periods. nificant day-to-day variability in activity counts (P < .008), This device might permit quantification of activity which was driven by increased activity counts on level and provide insight into the energy expenditure weekends compared with weekdays (P < .001). When of pet dogs. In validating the use of the AAM in pet comparing the first and second weeks of data, full- dogs, we wanted to determine the optimal sampling week and weekday activity counts were relatively sta- interval for this population. ble (P = .31 and P = .44, respectively), but weekend Fifty-five clinically normal dogs were included. After activity counts were less so (P = .07). obtaining the owners’ written consent and confirmation Full week-to-week comparisons of activity of no planned changes in their usual schedule, dogs had showed no significant differences in counts in pet AAMs placed on collars around their necks. The collars dogs that maintain their normal routines. When were worn continuously for 2 weeks. Between-dog and using the AAM to follow changes in groups of pet day-to-day variability in activity counts that occurred dogs over time (e.g., before and after an interven- over the 2-week period were evaluated using ANOVA. tion), comparing dogs on a full 7-day basis offers Weekdays and weekends were evaluated individually. the benefit of relatively stable estimates of activity in Activity counts in week 1 versus week 2 were compared unchanged animals while including the days with using paired t-tests to assess changes in the full 7 days as the highest potential for changes in activity to occur well as weekday and weekend activity counts. (i.e., weekends).

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Exercise Heart Rate and Blood Lactate Responses as Indicators of Aerobic Capacity in Dogs

J.C. Bouthegourda and A.J. Reynoldsb aNestlé Purina PetCare, Amiens, France bNestlé Purina PetCare, Salcha, Alaska

The objective of this study was to quantify the inten- tate threshold as being around 2 mmol, correspond- sity of different types of exercise by measuring heart ing to 74% maximum heart rate. Walking and trotting rate and blood lactate level in sled dogs to better un- heart rates (64% ± 1.8% and 72% ± 2% maximum derstand their aerobic capacities. heart rate, respectively) were beneath the lactate Fourteen Alaskan huskies (seven males, seven fe- threshold, indicating aerobic pathways as the main males; age: 2.4 ± 0.4 years old; weight: 21.9 ± 0.9 kg) supply of energy. Onset of blood lactate accumulation were involved in mild (45-minute walk on leash), (OBLA, 4 mmol) occurred at 76.5% maximum heart moderate (2-hour trot at 8 mph), and intense (6- rate. Intense exercise (77% ± 1% maximum heart rate) minute run at 22 mph) exercise. Heart rate and ac- was just beyond OBLA, indicating a large contribution tivity intensity were measured using Actiheart from anaerobic metabolic pathways. monitors (Mini-Mitter, Bend, OR) during the exer- The postexercise recovery times (time to recover cise, preexercise, and postexercise periods. Blood lac- preexercise heart rate) were equivalent after mild and tate was measured before and after exercise. moderate exercise but much higher after intense ex- Average heart rates during mild, moderate, and ercise (14 ± 2 and 15 ± 1 vs. 39 ± 2 minutes, respec- intense exercise were 159 ± 5.2, 179 ± 5.3, and tively), reflecting the difference observed in 190 ± 2.7 bpm, respectively, and correlated with the postexercise lactate values and the theoretical higher increase in measured activity: 246 ± 15 counts/min oxygen debt after anaerobic intense exercise versus (cpm), 454 ± 27 cpm, and 648 ± 27 cpm. aerobic mild to moderate exercise. Preexercise lactate values for mild, moderate, and These results validate the use of Actiheart moni- intense exercise were 0.7 ± 0.1, 1.5 ± 0.2, and 1.3 ± tors in working dogs to evaluate the intensity of ex- 0.2 mmol, respectively. Postexercise lactate values ercise. Together with blood lactate values, these were low after mild and moderate exercise (0.8 ± 0.1 monitors give a clear picture of the scope of these and 0.7 ± 0.1 mmol) but higher after intense exercise dogs’ aerobic capacity in response to different types of (4.4 ± 0.7 mmol). By regression, we identified the lac- exercise.

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RESEARCH ABSTRACTS: POSTER PRESENTATION

Effect of Different Dietary Protein Sources and Carbohydrate Content on Canine Behavior

O. Pellegrini,a L. Casini,a V. Mariotti,b G. Lubas,c and D. Gattaa aDepartment of Animal Production, University of Pisa, Pisa, Italy bDepartment of Physiology, Cellular Biology and Immunology, Autonomous University of Barcelona, Barcelona, Spain cDepartment of Veterinary Clinic, University of Pisa, Pisa, Italy

The influence of some foods on the animal psy- were fed the individual diets for 40 days, including chophysical equilibrium, outlining a direct connec- 30 days for the adaptation period and 10 days of ob- tion between nutrition and animal behavior, is well servation. During the observation period, different known. The purpose of this study was to evaluate the stressful situations were simulated (e.g., handling, effects of isoenergetic and isonitrogenous diets on an- sudden light, loud noises, door opening). Each dog’s imal behavior and health. The isoenergetic diet was behavioral reaction was evaluated by an expert be- based mainly on vegetable proteins and was rich in haviorist and divided into one of two categories: ag- carbohydrates (soybean meal added), whereas the gressive reactions and nonaggressive reactions. isonitrogenous diet was based mainly on animal pro- Differences between the groups did not reach sta- teins and had a lower percentage of carbohydrates (re- tistical significance; however, a trend toward hyper- spectively, dry matter: 91.5% vs 91.4%; protein: 27.0% excitability, with a consequent increase in aggressive vs 29.9%; fat: 14.5% vs 29.4%; crude fiber: 4.2% vs responses to some stimulations (e.g., sudden light: 0.5%; ash: 6.1% vs 13.4%; carbohydrate: 50.0% vs P= .067; sudden opening of a door: P= .06; sight of 17.3%; metabolizable energy: 4,003 vs 4,213 kcal/kg). a cat: P= .070), was observed in dogs fed the animal After a careful history and clinical and laboratory protein diet. These results suggest a possible benefit examinations confirmed good health and lack of ev- (at the limit of statistical significance) of a vegetable ident behavioral disorders, 20 dogs (10 males, 10 fe- protein–based diet with a higher level of carbohy- males) weighing 10 to 30 kg and aged 1 to 7 years drates for reducing aggressive or excitable behavior were randomly assigned to one of the two diets. Dogs in dogs.

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Evaluation of Polymeric Diets Delivered Directly into the Small Intestine through Surgically Placed Jejunostomy Tubes

S.A. Bone, F.A. Mann, R.C. Backus, and E. Kelmer University of Missouri-Columbia, College of Veterinary Medicine, Columbia, Missouri

Polymeric diets in place of elemental diets are not rec- Three commercially available diets were adminis- ommended for jejunal feeding in human patients, tered: CliniCare (Abbott Laboratories, Chicago, IL; and at least one veterinary report recommends against n = 40), Ensure Plus (Ross Laboratories, Columbus, polymeric diets for animal patients. We tested the hy- OH; n = 16), and Jevity (Ross Laboratories, Colum- pothesis that polymeric diets could be delivered di- bus, OH; n = 1); one group received a combination of rectly into the jejunum without causing diarrhea. two of these diets (n = 4). Mean duration of use was A thorough examination of medical records in a 4.5 days (range: 1 to 13 days). The case of longest du- veterinary medical teaching hospital from 1999 to ration received concurrent Ensure Plus and Jevity 2003 yielded 55 dogs and 6 cats that received at least without developing diarrhea. Diarrhea occurred post- 1 day of a polymeric diet administered directly into operatively in two dogs 1.5 and 6 days after initiation the jejunum via a surgically placed jejunostomy tube. of jejunal feeding, respectively. In one of these cases, Per hospital protocol, diluted diets were given in in- diarrhea was a presenting complaint. Forty-five ani- creasing strengths until full concentration was mals survived, 11 died or were euthanized, and 5 had achieved, typically on the third day of administration. incomplete follow-up. The liquid diet was discontinued when nutritional Results of this study indicate that polymeric diets support via oral intake was satisfactory. Diarrhea was delivered directly into the jejunum, although not for- noted as a function of the presenting complaint, type mulated for that use, can be administered for postop- and duration of diet use, signalment, and survival. erative nutritional support without causing diarrhea.

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RESEARCH ABSTRACTS: POSTER PRESENTATION

Seasonal Differences in Hair Growth between Long-Haired and Short-Haired Cats

M. Hekman,a D.G. Thomas,a S.H. Moon,b and W.H. Hendriksc aInstitute of Food, Nutrition and Human Health, Massey University, Palmerston North, New Zealand bDepartment of Animal Science, College of Natural Sciences, Konkuk University, Chungju, Korea cAnimal Nutrition Group, Department of Animal Sciences, Wageningen University, Wageningen, The Netherlands

Hair growth in adult short-haired cats shows a strong As previously reported, the midside hair growth rate seasonal pattern, with maximal hair growth rates in in short-haired cats showed a strong seasonal pattern. late summer and minimal rates in late winter. The Maximum hair growth occurred in late summer (273 timing of the hair growth cycle is such that the dens- μg/cm2/day), and minimum hair growth occurred in est and sparsest coats are produced during the cold- late winter (29 μg/cm2/day). In contrast, the hair growth est and warmest periods of the year, respectively. This rate in long-haired cats showed a less pronounced sea- study aimed to investigate differences in hair growth sonal pattern, with an average maximum growth of 290 patterns between short- and long-haired domestic μg/cm2/day in late summer and an average minimum cats. growth of 100 μg/cm2/day in late winter. The average di- Hair growth rates in 11 short-haired (8 male, 3 fe- ameter of the coat samples showed that the short-haired male) and 7 long-haired (2 male, 5 female) adult cats cats had coarser coats (28.54 μm in summer; 30.15 μm 1.4 to 6.8 years of age born at the Centre for Feline in winter) than did the long-haired cats (24.13 μm in Nutrition (Massey University, Palmerston North, New summer; 17.37 μm in winter). Zealand) were determined throughout the year using This study shows that long-haired cats grow more the midside patch technique.1 Cats from six litters hair during the year and show a less seasonal hair containing both long- and short-haired individuals growth pattern than short-haired cats, although the were used in this year-long study. Hair was shaved and timing of the hair growth cycle is similar. collected at monthly intervals and weighed before the average diameter of the hair sample was measured REFERENCE using an Optical Fibre Diameter Analyser (BSC Elec- 1. Hendriks WH, Tarttelin MF, Moughan PJ: Seasonal hair growth in the adult domestic cat (Felis catus). Comp Biochem Physiol tronics, Attadale, Australia). 1997;116(suppl A):29-35.

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Heritability of Hematology and Clinical Chemistry Variables in Domestic Cats: What Are the Early Implications?

D.F. Lawler,a K. Chase,b R. Teckenbrock,a and K.G. Larkb aNestlé Research Center, St. Louis, Missouri bDepartment of Biology, The University of Utah, Salt Lake City, Utah

Studies of human monozygotic and dizygotic twins additive genetic variance by relating the additive ge- have documented quantitative genetic contributions netic relationship matrix (2x coefficient of coancestry to phenotypic expression of clinical chemistry vari- between pairs) to the phenotypic covariance. Multi- ables. We evaluated quantitative genetic aspects of ple regression techniques were used to adjust for diet phenotypic expressions of erythrocyte, clinical chem- within nutrition study in the database. Inbreeding in istry, and acid–base measures in domestic cats (Felis this colony was minimal. silvestris catus). Heritabilities for erythrocyte, clinical chemistry, The metrics used for this study are part of a large and acid–base variables ranged, respectively, between database that is maintained to support nutrition re- 0.41 and 0.69, 0.13 and 0.78, and 0.23 and 0.59 (P search. To establish single representation in the data- < .05). Some observations merit additional comment. base for these analyses, sequential data over healthy The high heritability of the serum alkaline phos- lifetimes of individual cats were expressed as the phatase phenotype likely explains frequently ob- mean overall lifetime analyses for each chosen vari- served smaller disease-related responses in cats able. This procedure made available data from 564 compared with other species. However, the quantita- cats for erythrocytic metrics, 444 to 530 cats for serum tive genetic signals that were recognized for venous clinical chemistry, and 629 cats for venous acid–base acid–base metrics were quite surprising. metrics. Extreme (nonphysiologic) values were re- The physiologic implications of similar heritabili- moved, and non-normal traits were log-transformed. ties among species for the same variable may be dif- The “polygenic” function of SOLAR (sequential oli- ferent, dictating caution with interspecies phenotypic gogenic linkage analysis routines) was used to esti- comparisons. Minimally, these data indicate that dif- mate heritability as the ratio of additive genetic ferential heritability of clinical chemistry metrics variance to total variance. This procedure estimates should be considered in health screening.

80 Proceedings, 2007 Nestlé Purina Nutrition Forum 2008 Purina Abstracts ORAL_POSTER.qxp:2005 Master Page 2/26/08 4:12 PM Page 81

RESEARCH ABSTRACTS: POSTER PRESENTATION

Thyroid Hormone Concentrations and Prevalence of Thyroid Pathology in Geriatric Cats

C. Cupp and W. Kerr Nestlé Purina Research, St. Louis, Missouri

Hyperthyroidism is a common problem in geriatric reference range at some point during the preceding cats. As part of a larger study of aging in cats, we ex- years (average age at diagnosis: 14.8 ± 2.3 years). Four

amined the effect of age on thyroid hormone (T4) of the seven cats were diagnosed with hyperthy- concentrations as well as the prevalence of thyroid roidism by physical examination, clinical signs, and

pathology postmortem. consistently elevated T4 levels. Aging had no signifi-

Fifty-nine cats ranging in age from 8 to 15 years cant effect on serum T4 level in this study; however, a old (average age: 11.6 ± 2.3 years old) at the start of difference between cats with hyperthyroidism (as ev- the study were evaluated for up to 7 years. The cats idenced by histopathology) and those without hy- had no evidence of hyperthyroidism based on base- perthyroidism was noted (P = .097). Among cats that

line T4 concentration or physical examination. T4 lev- developed thyroid disease, T4 levels gradually in- els along with other routine health parameters were creased over time (P = .055); nonhyperthyroid cats evaluated and physical examinations performed pe- showed no change with age.

riodically until the cats’ natural deaths (average age of In conclusion, serum T4 level tends to increase death: 15.4 years). After each cat’s death, a full with age in cats with thyroid pathology but does not necropsy was performed and both thyroid glands change in cats without thyroid pathology. Based on

were submitted for histopathology. these data, a consistent increase in T4 level over time Twenty-one cats (35.6%) had evidence of thyroid strongly suggests developing thyroid disease, but a

hyperplasia or adenoma on histopathology. Of these, large number of cats with thyroid disease have T4 lev-

only seven cats exhibited serum T4 levels above the els within the reference range.

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Age-Related Changes in Immune Function in Cats

K.J. Rutherfurd-Markwick,a M.C. McGrath,a R.H. Morton,a P.C.H. Morel,a and W.H. Hendriksb aInstitute of Food, Nutrition and Human Health, Massey University, Palmerston North, New Zealand bAnimal Nutrition Group, Department of Animal Sciences, Wageningen University, Wageningen, The Netherlands

Many species show a similar decline in immune func- and hereditary effects on immune function parame- tion with age; however, little research has been done ters were also determined. in cats. The goal of this study was to extend the in- Results showed a significant (P = .0001) age-related formation available on age-related changes in im- decline (R2 = .25) in the phagocytic activity of periph- mune function in domestic cats and to compare our eral blood leukocytes. There were no age-related trends results with those previously reported. in the relative percentages of T-helper cells (CD4+), cy- The study was conducted at the Centre for Feline totoxic T cells (CD8+), or granulocytes (CD11b+). Nutrition at Massey University in Palmerston North, There was a decline (P = .02) in the relative percentage New Zealand, over a 28-day period. Whole-blood of B cells and a decrease (P = .011) in lymphocyte-pro- samples were collected from 138 domestic short- liferative responses to stimulation with ConA with age; haired cats (71 male, 65 female) aged 7 months to however, no change in lymphocyte blastogenic re- 13.5 years. The cats were fed a variety of commer- sponses to PHA was observed. Parentage, particularly cial moist foods (approved by the Association of the father, had significant effects on phagocytic activ- American Feed Control Officials), with water freely ity, percentages of CD8+ cells, CD4:CD8 ratio, and available. lymphocyte-proliferative responses to ConA. Samples were analyzed for expression of cell sur- Unlike other studies, we did not see significant face markers (CD4+ cells, CD8+ cells, B cells, changes in the level of expression of CD4+, CD8+, or CD11b+ cells), phagocytic activity, and mitogen-in- CD11b+ cells or proliferative responses to PHA, pos- duced lymphocyte proliferation (concanavalin A sibly due to the age range or the genetic profile of the [ConA], phytohemagglutinin antigen [PHA]). Age- population studied. Decreases in both percentage of related trends were assessed by simple linear regres- B cells and proliferative response to ConA were sim- sion analysis. Detailed family trees were available, ilar to those previously reported.

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RESEARCH ABSTRACTS: POSTER PRESENTATION

Effect of Dietary Form on Nutrient Digestibility in Cats and Dogs

K. Weidgraaf, S.M. Rutherfurd, K.A. O’Flaherty, D.G. Thomas, and K.J. Rutherfurd-Markwick Institute of Food, Nutrition and Human Health, Massey University, Palmerston North, New Zealand

In vivo methods for determining the digestibility of days, which included a 7-day adaptation period fol- companion animal diets generally involve either total lowed by a 5-day total fecal collection period. The fecal collection or using an indigestible marker. The diet was fed according to maintenance requirements, total fecal collection method is labor-intensive and and water was available ad libitum. Feed intake and prone to sample losses. Therefore, using indigestible fecal output were measured. Feces from each cat were markers is generally preferred, particularly in dogs. subsampled, freeze-dried, and analyzed for gross en- When using indigestible markers, the diet must be ergy and protein. ground to facilitate the homogeneous mixing of the Similarly, six male and six female dogs (3 to 8 marker into the diet; however, little work has been years of age) from Massey University’s Canine Unit done to investigate whether this change in dietary were fed either ground or unground AAFCO-ap- form has any effect on digestibility. proved dog biscuits for a total of 12 days. Sample col- A preliminary study was carried out to compare lection, preparation, and analysis were also carried the nutrient digestibility of two different forms of a out as described in the feline study. Preliminary data dry diet (unground vs ground) in cats and dogs. Eight showed no difference in digestibility between the male cats (3 to 7 years of age) from Massey Univer- ground and unground diets in cats (respectively, en- sity’s Centre for Feline Nutrition were fed a dry Asso- ergy: 86.4% vs 86.7%; protein: 82.7% vs 82.8%). In ciation of American Feed Control Officials (AAFCO)– dogs, however, dietary form did appear to affect di- approved diet in either ground or unground form. In gestibility (respectively, energy: 82.81% vs 84.3%; a crossover design, the cats received each diet for 12 protein: 78.2% vs 80.1%).

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Chemical Composition and In Vitro Crude Protein and Fiber Disappearances of Corn Coproducts from the Ethanol Industry

M.R.C. de Godoy, L.L. Bauer, and G.C. Fahey, Jr. Department of Animal Sciences, University of Illinois, Urbana, Illinois

The objective of this study was to determine the SBM (87.2%). CPCs had corn protein disappearances

chemical composition and protein and fiber disap- of 77.5% (CPC2) and 74.1% (CPC1).

pearances of corn protein concentrates (CPC1, CPC2) Crude protein concentration ranged from 0%

and corn fiber (CFn), novel coproducts from the (Solka Floc [SF]; International Fiber Corporation, St.

ethanol industry, compared with conventional plant Louis, MO) to 11.0% (CF control 1 [CFC1]). Total di- protein and fiber ingredients used in the pet food in- etary fiber was highest for SF (100%) and lowest for dustry. Novel corn coproducts were produced from a beet pulp (68.8%). Corn fibers had intermediate total pilot modified wet milling plant. dietary fiber values. Acid hydrolyzed fat concentra-

Crude protein values for CPC1 and CPC2 were tions ranged from 0.8% (SF) to 6% (CFC1). Gross en- 57.3% and 49.7%, respectively. Total dietary fiber was ergy values were very similar among corn fiber

29% for CPC1 and 23.5% for CPC2. Acid hydrolyzed sources. Organic matter disappearance was lowest for fat and gross energy were similar for these ingredi- SF in the hydrolytic (–6.5%) and fermentative stages ents. Crude protein disappearance after 6 hours of in- (–2.1% and –1.6% at 8 and 16 hours, respectively)

cubation in an HCl/pepsin solution was highest for and highest for CFC1 and beet pulp in the hydrolytic soybean meal (SBM) (53.3%), followed by corn stage. Beet pulp was the only fiber source with sig-

gluten meal (CGlM) (49.3%), distillers dried grains nificant fermentation (17.7% after 16 hours). CFC1

with solubles (DDGS) (49.0%), CPC2 (29.3%), corn and CFC2 had intermediate fermentation values

germ meal (CGeM) (25.3%), and CPC1 (24.9%). (5.7% and 5.3%, respectively), but CFC1 and CFC2

After an additional 18 hours of incubation (24 hours were higher than CFn (3.0%). Substrate versus time total) with porcine pancreatin, CGlM had the highest interactions were significant (P < .05) for organic corn protein disappearance (94.1%), followed by matter disappearance.

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RESEARCH ABSTRACTS: POSTER PRESENTATION

Corn Fiber Effects on Nutrient Digestibility and Fecal Characteristics of Dogs

M.A. Guevara,a L.L. Bauer,a C.A. Abbas,b K.E. Beery,b M.A. Franklin,b M.J. Cecava,b and G.C. Fahey, Jr.a aDepartment of Animal Sciences, University of Illinois, Urbana, Illinois bArcher Daniels Midland Company, Decatur, Illinois

Understanding the impact of different processing The average daily food intake, fecal production, fecal methods in the manufacture of fiber-rich corn co- scores, and fat and crude protein digestibilities were not products is a precondition of their potential use as significantly different among treatments. Body weight fiber sources for dogs. This experiment examined and body condition score remained unaltered through- total tract nutrient digestibility and fecal characteris- out the duration of the experiment. Apparent dry mat- tics of adult dogs fed selected fiber-rich corn coprod- ter (DM) digestibility coefficients were high, with the ucts from the ethanol industry. NCF treatment having a small but statistically higher Native corn fiber (NCF), NCF with fines, hy- value compared with the remaining treatments except drolyzed corn fiber (HCF), and hydrolyzed extracted for the NCF with fines. Dogs fed BP, HCF, and HECF corn fiber (HECF) were included as fiber sources in a had lower DM digestibilities compared with those fed commercial-type diet matrix with poultry byproduct NCF but not compared with dogs fed NCF with fines. meal and brewer’s rice as the main ingredients and Apparent total dietary fiber digestibility was higher for chromic oxide (0.2%) included as a digestion marker. NCF, BP, and HECF treatments, but BP and HECF were Beet pulp (BP) was used as a positive control treat- no different than NCF with fines and HCF treatments. ment. Results of this experiment suggest that incorpora- Diets were fed to 15 beagles in a partially balanced tion of corn fibers at the 7% inclusion level, when incomplete block design with two blocks of 12 days, substituted for BP in diets of healthy adult dogs, does including 8 days for diet adaptation and 4 days for not dramatically impact nutrient digestibility, food fecal collection. intake, or fecal characteristics.

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In Vitro Evaluation of Protein Digestibility of Four Pet Foods

F. Bovera, S. Calabrò, S. D’Urso, R. Tudisco, A. Guglielmelli, R. Romano, and M.I. Cutrignelli Dipartimento di Scienze Zootecniche e Ispezione degli alimenti, Università degli Studi di Napoli “Federico II,” Via F. Delpino, Napoli, Italy

Given the need to develop in vitro methods to simu- tein value, showed the highest digestibility (Table 1). late digestion of pet food, we carried out an investi- These results correspond with previous data ob- gation to examine the proteolytic activity of tained in vivo in a growing trial conducted on 24 Ger- Streptomyces griseus protease to determine its suitabil- man shepherd puppies2 in which pet foods 1 and 4 ity to estimate protein digestibility for dogs. allowed significantly (P < .01) higher weight gains In vitro protein digestibility was measured (ac- from the age of 60 days. In each case, all registered cording to work by Coblentz and coworkers1) on four daily weight gains (from 66 to 93 g/d and from 93 to dry concentrates for large-breed puppies using S. 150 g/d in the period 1 to 60 days and 60 to 90 days, griseus protease (sigma EC 3.4.24.31). Residual crude respectively) are included in the ranges indicated by protein was determined after 0, 24, and 48 hours of Debraekeleer and coworkers3 for the periods 1 to 2 incubation. and 3 to 5 months of age in puppies with an average Protein losses at time 0, without incubation, cor- adult body weight of 30.5 kg. Considering the insuf- responded to the soluble protein fraction. The chem- ficient enzymatic production and development of gut ical composition results were similar among the pet microbial population in the period immediately after foods (average values for crude protein: 27.5 ± 1.27; weaning, the availability of soluble protein, which is ether extract: 11.6 ± 3.99; crude fiber: 3.54 ± 0.62 %). immediately absorbable and utilizable, could im- The ranking of pet foods for soluble protein (time 0) prove nutrient availability. was 1 and 4 > 2 > 3 (P < .01). At 48 hours, pet food These preliminary results demonstrate the validity 3, which was characterized by the lowest soluble pro- of this rapid and reliable in vitro procedure in estimating the protein digestibility of dog foods. TABLE 1 Protein Digestibility in Tested Pet Foods (%) REFERENCES 1. Coblentz WK, Abdelgadir IE, Cochran RC, et al. Degradability of Pet Food Time 0 24 hr 48 hr forage proteins by in situ and in vitro enzymatic methods. J Dairy Sci 1999;82:343-354. 1 65.0A ± 0.05 28.1 ± 0.40 6.55Bb ± 0.75 2. Cutrignelli MI, D’Urso S, Solimene R, et al. Influence of feeding programme on growth dynamics of German shepherd puppies 2 51.3B ± 1.06 26.7 ± 0.57 11.4a ± 1.02 until 3 months of age. Proc 10th Congr Eur Soc Vet Comparative 3 27.3C ± 0.88 27.9 ± 0.56 25.2A ± 0.59 Nutr:76, 2006. 3. Debraekeleer J, Gross KL, Zicker SC. Normal dogs. In: Hand MS, 4 62.7A ± 0.11 28.9 ± 0.84 8.47B ± 0.46 Thatcher CD, Remillard RL, Roudebush P, eds. Small Animal Clini- A, B, C = P < .01; a, b, c = P < .05. cal Nutrition. 4th ed. Topeka, KS: Mark Morris Institute; 2000:213- 260.

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RESEARCH ABSTRACTS: POSTER PRESENTATION

Review of Pet Dog Feeding Habits in Spain

V.M. Mariotti, M. Hervera, J. Fatjó, M. Amat, M.D. Baucells, and X. Manteca Animal Nutrition, Management and Welfare Research Group, Department of Animal and Food Science, Autonomous University of Barcelona, Catalonia, Spain

Little epidemiologic data exist about pet dog feeding few times a day. Moreover, it appears that there is a habits and management in Europe.1–3 Studies show relationship between dog environment and separa- that feeding patterns, environmental conditions, and tion anxiety: dogs with more available space when pet management all affect animal health, behavior, alone show less anxiety than dogs living in a little and welfare.4,5 Between 1995 and 2005, a retrospective room (P = .001). epidemiologic study was carried out to analyze trends Dog management (i.e., exercise level, including in feeding habits of pet dogs in Catalonia, Spain. The frequency and duration of walks) and aggressive be- specific goals this study were to determine (1) the ef- havior toward family members could be related: dogs fect of environmental factors on the dogs’ feeding with a reduced exercise level (0 or 1 walk/day) were habits as well as (2) the relationship between the type more aggressive toward family members than dogs of food provided and the composition of the family. with a higher exercise level (3 or 4 walks/day; P = A total of 1,000 dogs (Canis familiaris) were ob- .05). This result could be partially explained by the served for the purpose of behavioral and clinical eval- increase in serotonin turnover caused by regular ex- uation at the Clinical Behavioral Service of the ercise, as shown in humans and other species.6,7 Veterinary Teaching Hospital of the Autonomous This preliminary study suggests that diet, feeding University of Barcelona. Recorded information in- pattern, and management may play a role in the de- cluded sex, age, breed, type of food (dry, wet, mixed), velopment of behavior problems in dogs.5,8,9 mode of administration (meal fed, free choice), family composition (family size and age, presence of chil- REFERENCES 1. Freeman LM, Abood SK, Fascetti AJ, et al. Disease prevalence dren or other pets in the home), environment among dogs and cats in the United States and Australia and pro- (apartment or house, urban or rural location, pres- portions of dogs and cats that receive therapeutic diets or dietary supplements. JAVMA 2006;229(4):531-534. ence of a garden or terraces), exercise (walk frequency 2. Lund EM, Armstrong PJ, Kirk CA, et al. Health status and popula- and duration), and behavioral problems. To deter- tion characteristics of dogs and cats examined at private veterinary mine the relationship between dietary habits and practices in the United States. JAVMA 1999;214(9):1336-1341. 3. Patronek GJ, Beck AM, Glickman LT. Dynamics of dog and cat management and behavioral problems in the dogs, populations in a community. JAVMA 1997;210(5):637-642. data for 500 dogs were analyzed by chi-square test 4. Fernstrom JD. Dietary amino acids and brain function. J Am Diet (SPSS 12.0). Study results indicate that the most com- Assoc 1994;94:71-77. 5. Houpt KA, Zicher S. Dietary effects on canine and feline behav- mon types of food consumed were medium- to high- iour. Vet Clin North Am Small Anim Pract 2003;33:405-416. quality dry foods (74%) fed twice daily. The typical 6. Chaouloff F, Laude D, Elghozi JL. Physical exercise: evidence for differential consequences of tryptophan on 5-HT synthesis and me- owner in the study was a young couple with no chil- tabolism in central serotonergic cell bodies and terminals. J Neural dren (45%) living in an apartment (>80%). Transm 1989;78:121-130. Some management and dietary characteristics 7. Dey S, Singh RH, Dey PK. Exercise training: significance of regional alterations in serotonin metabolism of rat brain in relation were found to be related to canine behavioral prob- to antidepressant effect of exercise. Physiol Behav 1992;52(6): lems and welfare. Free-choice dogs showed less food- 1095-1099. 8. Dodman NH, Reisner I, Shuster L, et al. Effect of dietary protein related aggression toward family members than content on behavior in dogs. JAVMA 1996;208:376-379. meal-fed dogs (P < .05). This could be because when 9. DeNapoli JS, Dodman NH, Shuster L, et al. Effect of dietary pro- food is continuously available, dogs perceive it as a tein content and tryptophan supplementation on dominance aggression, territorial aggression, and hyperactivity in dogs. JAVMA less valuable resource than when it is offered only a 2000;217(4):504-508.

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Use of a Wireless Multisensor Telemetry Capsule for Monitoring the Canine Gastrointestinal Tract

W.A. Anderson, W. Kerr, and G. Mohr Nestlé Purina PetCare Research, St. Louis, Missouri

The SmartPill GI Monitoring System (SmartPill Cor- onset of a sudden and sustained increase of at least 3 poration, Buffalo, NY) provides ambulatory testing pH units above baseline. Small and large intestine for gastrointestinal (GI) tract pressure, temperature, transit time was calculated as total GI transit time and pH; gastric emptying time (GET); combined small minus gastric emptying time. and large intestine transit time; and total GI transit The capsule was successfully administered to and time. This device, although extensively tested in hu- retrieved from the stools of all dogs. There was a dif- mans, had not been previously evaluated in dogs. The ference (P < .05) in gastric emptying time between objective of this study was to evaluate the feasibility of sexes, averaging 15.17 hours for females versus 12.58 measuring GI transit time and gastric pH in dogs. hours for males. The average small and large intes- Eight (four male, four female) clinically healthy tine transit times were 18.77 hours for females and Labrador retrievers were used in the study. After an 8- 21.84 hours for males, and the average total GI tran- to 10-hour fast, the dogs were fed one-third of their sit times were 33.45 hours for females and 34.29 daily energy requirement. The capsule was adminis- hours for males. The mean gastric pH was 2.14 for fe- tered immediately after food consumption (time 1 males and 2.34 for males. [T1]). The dogs were fitted with mesh jackets with From the results of this study, we can conclude that pockets to hold the data receiver and then returned to the SmartPill technology is a novel, noninvasive their runs. The dogs were monitored until the unit method for assessing several aspects of GI function. was expelled in the feces (time 2 [T2]), and total GI This technology has the potential for clinical and re- transit time was defined as time from T1 to T2. Gas- search applications to study the effect of diet or nu- tric emptying time was measured from T1 until the trients on the canine GI tract.

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