J Comp Physiol B (2001) 171: 327±334 DOI 10.1007/s003600100180
O R I GI N A L P A P E R
P.J. LeBlanc á M. Obbard á B.J. Battersby A.K. Felskie á L. Brown á P.A. Wright J.S. Ballantyne
Correlations of plasma lipid metabolites with hibernation and lactation in wild black bears Ursus americanus Accepted: 4 February 2001 / Published online: 28 March 2001 Ó Springer-Verlag 2001
Abstract During the denning period, black bears (Ursus americanus) are capable of enduring several months without food. At the same time, female bears that are pregnant or lactating have an added metabolic stress. Based on laboratory studies, much of the energy required to support metabolism and lactation during denning in black bears comes from lipid reserves. These lipid reserves are mobilized and the most metabolically active lipid fraction in the blood are nonesteri®ed fatty acids (NEFA). Therefore, we hypothesized that plasma NEFAs would be higher in denning relative to active bears and in lactating relative to non-lactating female bears. We further hypothesized that in bears with elevated plasma NEFA levels, other lipid-related parameters (e.g., ketone bodies, albumin, cholesterol, lipase) would also be elevated in the plasma. Denning bears had signi®cantly increased NEFA levels in all classes (saturates, monoenes, and polyenes). A doubling of plasma NEFA levels and a 33% increase in albumin, the plasma fatty acid binding protein, in denning bears, resulted in NEFA/albumin ratios that were higher in denning bears (4:1) compared to those of active bears (3:1). Bears became relatively ketonemic with a 17-fold increase in D-b-hydroxybutyrate levels during the denning period. Plasma cholesterol approximately doubled and lipase was ten-fold lower in denning relative to active bears. These ®ndings indicate a strong correlation between
Communicated by L.C.-H. Wang P.J. LeBlanc á B.J. Battersby á A.K. Felskie á P.A. Wright J.S. Ballantyne (&) Department of Zoology, University of Guelph, Guelph, Ontario, Canada, N1G 2W1 E-mail:
[email protected] Tel.: +1-519-8244120 ext. 2708 Fax: +1-519-7671656 M. Obbard á L. Brown Natural Heritage Science Section, Ontario Ministry of Natural Resources, PO Box 7000, 300 Water Street, Peterborough, Ontario, Canada, K9J 8M5
plasma lipid metabolites and the denning period in a wild population of black bears. Keywords Albumin á Cholesterol á Ketone body á Nonesteri®ed fatty acid á Season Abbreviation NEFA nonesteri®ed fatty acid
Introduction Bears from the family Ursidae are capable of enduring several months without food or water during their denning phase. To accomplish this they utilize energy stores accumulated in the fall. True mammalian hibernators experience profound depressions of metabolic rate during hibernation (Lyman 1982), whereas in bears, the body temperature remains near normal (31±35 °C) and there is only a slight drop in metabolic rate (Hock 1960; Farley and Robbins 1995). The lack of a deep metabolic depression in bears means that energy expenditures are relatively high at a time when food intake is nonexistent. Much of the energy required to support metabolism during denning comes from lipid reserves. Black bears normally consume approximately 8000 kCal/day (33,494 kJ/day) in the summer but prior to denning, they become hyperphagic and increase their daily caloric intake to 15,000±20,000 kCal (62,802±83,736 kJ) (Nelson 1980). This increased energy intake results in an increase in the amount of lipid stored in adipose tissue. These lipid reserves are especially important in supplying energy for pregnant or lactating denning female bears. In addition to their own needs, they must also provide energy and substrates for the developing fetus or to produce milk to nourish the cubs (Farley and Robbins 1995). To provide this energy, lipids are transferred from adipose tissue in several forms, which include triacylglycerides, lipoproteins and plasma nonesteri®ed fatty acids (NEFAs). Plasma NEFAs are the most metabolically active lipid fraction in the blood
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(Newsholme and Leech 1983). Analysis of the fatty acids in the NEFA fraction can provide detailed information on the metabolic status of bears. Previous studies of plasma NEFAs in bears have measured only total NEFAs (Nelson et al. 1983; Hissa et al. 1994, 1998) and one study has shown that total NEFAs increase as a proportion of the lipids measured in plasma of denning bears (Hissa et al. 1994). The metabolic changes during denning, which include the entire gestation period, parturition, and lactation, may be re¯ected in the levels of speci®c NEFAs in the plasma. Therefore, the main goal of this study was to test the following hypotheses: (1) plasma NEFAs will be higher in denning individuals, and (2) plasma NEFAs will be higher in lactating individuals. The possible importance of plasma NEFAs during denning may be re¯ective of other modi®cations in plasma parameters. The main carrier protein for NEFAs in the blood of mammals is albumin. If plasma NEFA levels rise above the carrying capacity of albumin, this may require changes in plasma albumin levels. Therefore we predicted that the plasma albumin levels would be positively correlated with plasma NEFA levels. Frequently, changes in the utilization of lipids are associated with changes in the importance of ketone bodies. It is well known that during periods of starvation (McGarry and Foster 1969; Newsholme and Leech 1983) and hibernation (Krilowicz 1985; Harlow 1995), mammals increase the synthesis and utilization of ketone bodies. Plasma ketone body concentrations of denning captive male black bears are higher than non-denning bears (Ahlquist et al. 1984). Therefore, as seen in captive bears, we expect to see an increase in plasma ketone body levels in denning individuals. Since black bears do not feed during the denning period, the need for lipid digestion is absent. Therefore, we also predicted a decrease in plasma lipase activity, which can be used as an indicator of pancreatic lipase secretion. During the period of increased lipid storage, one of the components stored in adipose tissue is cholesterol (Angel and Farkas 1970). The plasma level of cholesterol is an indicator of nutritional status in mammals and it has been shown that plasma cholesterol levels increase with starvation (Swaner and Conner 1975; Savendahl and Underwood 1999). Therefore, we predicted that circulating levels of cholesterol would be higher during the denning period. Most studies on black bears have examined metabolic eects on captive individuals (Ahlquist et al. 1984; Helgren et al. 1993; Farley and Robbins 1995). Since these may not be re¯ective of the natural phenomenon of denning we examined how a wild population of denning bears responded to seasonal and metabolic changes. In a previous study, we reported the changes in blood nitrogen compounds for the same animals used in the present study (Wright et al. 1999).
Materials and methods Study region The study was conducted between 1990±1994 in or near the Chapleau Crown Game Preserve (CCGP) in northern Ontario (480 N, 830 W) as part of an ongoing Ontario Ministry of Natural Resources study examining the population dynamics and habitat use patterns of black bears (Ursus americanus) in the boreal forest (Obbard et al. 1998). Sampling protocol The sampling protocol was described in detail previously in Wright et al. (1999). Blood samples were collected from the femoral vein using 10-ml tubes (Becton-Dickson Vacutainer) and centrifuged for 30 min. Serum was collected and stored at ±20 °C for later analyses. It was possible to follow only a few bears (three to four at most) over the study period and therefore the data set mostly represents bears sampled only once. Determination of plasma NEFAs Speci®c methylation, to fatty acid methyl esters, using acidi®ed methanol (2% acetyl chloride; Lepage and Roy 1988), and determination of plasma NEFAs, using a gas chromatograph (HewlettPackard, HP5890A) ®tted with a ¯ame ionization detector (FID), an automatic injector (Hewlett-Packard, 7673A) and a DB-225 megabore fused silica column (Chromatographic Specialties, Brockville, Ont., Canada), were as previously described by Ballantyne et al. (1993). Fatty acid methyl esters from plasma samples were identi®ed by comparing their retention times with a known standard (GLC 68A, Nu-Check-Prep, Elysian, Minn., USA) and the absolute amounts were quanti®ed with the aid of an internal standard, heptadecanoic acid (17:0), added to the plasma samples prior to methylation. Determination of plasma parameters Both D-b-hydroxybutyrate and acetoacetate were determined spectrophotometrically using the method of Bergmeyer (1974). Albumin (Doumas et al. 1971), cholesterol (Siedel et al. 1983; Kattermann et al. 1984) and lipase (Verduin et al. 1973) were determined using a BM/Hitachi 911 automated system (Dacos, Coulter Electronics, Hialeah, Fla., USA). All chemicals were purchased from Sigma (Oakville, Ont., Canada), except for reagents for the analysis of albumin and cholesterol, which were from Coulter (Hialeah, Fla., USA) and lipase, which were from Boehringer Mannheim (Laval, PQ, Canada). Statistical analyses A 2´2 analysis of variance (ANOVA) (Steel and Torrie 1980) was used to establish dierences between active or denning, lactating or non-lactating, and the interaction in female bears. LSMEANS (SAS Institute, Cary, N.C., USA) was used to determine signi®cance (P
