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The effect of an acute phase response on tissue carotenoid levels of growing chickens (Gallus gallus domesticus)
Comparative Biochemistry and Physiology Part A 135 (2003) 635–646
The effect of an acute phase response on tissue carotenoid levels of growing chickens (Gallus gallus domesticus) Elizabeth A. Koutsos1, C. Christopher Calvert, Kirk C. Klasing* Department of Animal Science, University of California, One Shields Avenue, Davis, CA 95616, USA Received 10 January 2003; received in revised form 27 May 2003; accepted 28 May 2003
Abstract Plasma, liver and skin carotenoids decrease following infectious disease challenges. Since these challenges often involve substantial host pathology and chronic immune responses, the mechanism underlying altered carotenoid deposition is unclear. Therefore, changes in tissue carotenoid levels were examined during an acute phase response induced by lipopolysaccharide (LPS) or interleukin-1 (IL-1). In two experiments, chicks were hatched from carotenoid-deplete eggs (ns28, ns64, respectively) and fed 0, 8 or 38 mg carotenoids (luteinqcanthaxanthin)ykg diet. For chicks fed 38 mg carotenoids, but not those fed 0 or 8 mg, LPS generally reduced plasma lutein, canthaxanthin and total carotenoids (P0.05), and liver lutein, zeaxanthin, canthaxanthin and total carotenoids (P-0.05). Additionally, LPS reduced thymic total carotenoids (Ps0.05) and increased thymocyte lutein (Ps0.07), zeaxanthin (Ps0.07) and total carotenoids (Ps 0.07). Finally, LPS increased bursal canthaxanthin (P-0.01), but had no effect on shank carotenoids (P)0.5). In chicks hatched from carotenoid-replete eggs (ns36) and fed dietary lutein (38 mgykg diet), LPS reduced plasma and liver zeaxanthin and liver total carotenoids (P-0.05); IL-1 reduced plasma and liver lutein, zeaxanthin and total carotenoids (P-0.05). Therefore, an acute phase response plays a role in reduced tissue carotenoids during infectious disease. 䊚 2003 Elsevier Science Inc. All rights reserved. Keywords: Carotenoid; Acute phase response; Chicken; LPS; IL-1; Lutein; Zeaxanthin; Canthaxanthin
1. Introduction Carotenoids have a variety of functions including light absorption and pigmentation, antioxidant function in vivo and in vitro and carotenoid-
Abbreviations: APR, acute phase response; bw, body weight; IL, interleukin; i.v., intravenous; LPS, lipopolysaccharide; MT, metallothionein; PBS, phosphate buffered saline; SCWL, single comb white leghorn; TNFa, tumor necrosis factor a. *Corresponding author. Tel.: q1-530-752-1901; fax: q1530-752-0175. E-mail address: [email protected] (K.C. Klasing). 1 Current institution: Animal Science Department, California Polytechnic State University, San Luis Obispo, CA 93406.
specific immunomodulation (reviewed by Goodwin, 1986). Carotenoids are particularly important to avian species, where pigmentation quality is indicative of a bird’s general fitness level (Olson and Owens, 1998) and thus influences mate selection (Camplani et al., 1999) and parental food allocation (Saino et al., 2000). Additionally, carotenoid-based pigmentation is important in the commercial poultry industry because consumers use the level of egg yolk and broiler meat pigmentation to determine product acceptability (Hernandez et al., 2001). Therefore, the commercial poultry industry routinely feeds carotenoids to chickens (primarily lutein and canthaxanthin) to provide pigmentation to poultry products.
1095-6433/03/$ - see front matter 䊚 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S1095-6433(03)00158-2
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The degree of carotenoid deposition into tissues such as skin, blood and liver can be altered by a variety of factors including disease challenge. In birds, disease-associated reductions in tissue carotenoids are collectively referred to as ‘pale bird syndrome’ and have been demonstrated in response to parasite exposure (Tyczkowski and Hamilton, 1991; Allen, 1992) and viral infection (Squibb et al., 1955, 1971; Page et al., 1982). It is likely that multiple mechanisms are responsible for reduced carotenoid deposition during infectious disease challenges, since these challenges are associated with a variety of host pathologies and repeated stimulation of the immune system. For example, many parasite and viral challenges reduce carotenoid absorption and enhance excretion due to intestinal necrosis (Ruff and Fuller, 1975). Additional alterations in tissue carotenoid deposition are presumably a result of disease-induced changes in lipid and lipoprotein metabolism (Hardardottir et al., 1997), including reductions in tissue lipoprotein lipase activity (Griffin and Butterwith, 1988), plasma lipoprotein levels (Alvarez and Ramos, 1986; Hardardottir et al., 1997), and plasma triacylglycerol levels (Feingold et al., 1989, 1991, 1992). These metabolic alterations may directly impact carotenoid incorporation into lipoproteins as well as their uptake via lipoproteinmediated transport. Disease-related changes in nutrient metabolism occur as a consequence of the acute phase response (APR), which is characterized by changes in the production of hepatic proteins (i.e. acute phase proteins) that mediate absorption, transport, uptake and deposition of amino acids, lipids, vitamins and minerals. For example, the production of hepatic metallothionein (MT) is increased during the APR (Hallquist and Klasing, 1994), resulting in reduced circulating plasma Zn and increased liver cytosolic MT-bound Zn. Altered Zn partitioning provides a source of Zn co-factors for acute phase protein production and may also reduce nutrient availability to invading pathogens (Weinberg, 1978). Since the APR substantially alters nutrient partitioning and directly affects lipid metabolism, it is likely that the APR is responsible for alterations in the partitioning of lipid-soluble nutrients. We hypothesize that APR mediators, such as the proinflammatory cytokines tumor necrosis factor-a (TNFa), interleukin-1 (IL-1) and IL-6, regulate tissue carotenoid levels during an immune response. Therefore, we conducted three experi-
ments to examine the effect of the APR, induced by lipopolysaccharide (LPS, an inflammatory component of the cell wall of gram-negative bacteria) or the pro-inflammatory cytokine IL-1, on tissue carotenoid levels in chickens. IL-1 was chosen because it is a central mediator of the APR and subsequent nutritional status of an animal (Rivier et al., 1989; Zetterstrom et al., 1998). 2. Materials and methods 2.1. Experiment 1 To control for maternal carotenoid level, single comb white leghorn hens (SCWL, Hyline, Y-strain, UC Davis stock) were fed carotenoid-free diets for ;30 d to supply carotenoid-deplete eggs (Koutsos et al., 2003). Eggs were hatched and 28 chicks were housed (4 pens of 7 chicksypen) in brooder batteries (Petersime Inc., Gettysburg, OH) in a temperature-controlled room (25 8C) under 24 h light. The experiment was designed as a 2=2 factorial arrangement of treatments with 2 dietary carotenoid levels (8 or 38 mg total carotenoidsy kg diet) and 2 levels of LPS (0 or 100 mgykg body weight; bw). Carotenoids were added to the basal diet (Table 1) as luteinqcanthaxanthin (calculated ratio of 4:1 lutein:canthaxanthin, supplied as Oroglo Dry (Kemin Industries Inc, Des Moines, IA) and Carophyll Red (Roche Vitamins Inc., Parsippany, NJ) respectively. Diets were designed to encompass the range of typical carotenoid levels fed to commercial poultry and were offered for ad libitum consumption from day zero post-hatch through the duration of the trial. At 4 weeks of age, chicks (average bws321"11 g) within dietary treatments were randomly assigned to one of two LPS treatments; birds were either uninjected (Control, ns6 per diet) or LPS-injected (ns8 per diet, 100 mg LPSykg bw intravenously (i.v.) from S. typhimurium, Sigma 噛L7261, St. Louis, MO). Previous research demonstrated that saline injection does not cause an APR and does not alter nutritional status (Laurin and Klasing, 1987), so we chose to use an uninjected control group. At 24 h post-injection, blood was collected via cardiac venipuncture into heparinized tubes for plasma isolation and then all birds were euthanized via CO2 overdose. This time point was chosen based upon preliminary data collected in our lab (unpublished). Liver (left lobe), thymus (3–4 lobes) and bursa (whole) were collected and stored in the
E.A. Koutsos et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 635–646 Table 1 Composition of basal diets fed to SCWL chicks1 Ingredient
Concentration (gykg diet)
Soybean meal Rice flour Calcium carbonate Cellulose Cornstarch Vegetable oil Dicalcium phosphate NaCl Vitamin mix2 Mineral mix2 Methionine Threonine Isoleucine Choline
294.0 449.5 15.0 70.0 86.0 57.0 17.4 3.3 2.5 2.5 0.7 0.6 0.5 1.0
1 Diet offered ad libitum for the duration of the trial. Diets were formulated to meet or exceed all requirements for growing egg-type chickens (NRC, 1994). Basal diets were analyzed to contain no detectable (F0.17 pmolykg) carotenoids. 2 Vitamin mix supplied (per kg final diet): thiamin HCl (1.8 mgykg), riboflavin (3.6 mgykg), Ca-pantothenate (12.5 mgy kg), niacin (25 mgykg), pyridoxine HCl (3 mgykg), folacin (0.6 mgykg), biotin (0.2 mgykg), B12 (10 mgykg), retinyl palmitate (6.3 mgykg), cholecalciferol (0.5 mgykg), a-tocopherol acetate (20 mgykg), vitamin K (5 mgykg), ethoxyquin (125 mgykg). Mineral mix supplied Na2SeO3 (20 mgykg), CuSO4*5H2O (16 mgykg), ZnSO4*5 H2O (156.3 mgykg), MnSO4 (170 mgykg), Fe2SO4*7 H2O (920 mgykg).
dark at y80 8C until samples were analyzed. Dependent variables included tissue lutein, zeaxanthin and canthaxanthin, the sum of all carotenoids analyzed (i.e. total carotenoidssluteinq zeaxanthinqcanthaxanthin), and liver cytosolic Zn. All procedures were approved by the UC Davis Animal Care and Use Committee. 2.2. Experiment 2 To increase the number of tissues analyzed, a second experiment was conducted with chicks hatched from carotenoid-deplete eggs (attained as described for Experiment 1). The experiment was also designed as a 2=2 factorial arrangement of treatments with 2 dietary carotenoid levels (0 or 38 mg total carotenoidsykg diet) and 2 levels of LPS (0 or 100 mgykg bw i.v.). Sixty-four chicks were housed (8 pens of 8 chicksypen) as previously described and were randomly assigned to one of two dietary treatments. From day zero posthatch through the duration of the trial, chicks were offered ad libitum access to basal diet (Table 1)
637
plus 0 or 38 mg total carotenoidykg diet (supplemented with lutein and canthaxanthin as described for Experiment 1). At 3 weeks of age, chicks (average bws209"5 g) within dietary treatments were randomly assigned to one of two LPS treatments; chicks were either uninjected (Control, ns 16 per diet) or LPS-injected (ns16 per diet; 100 mgykg bw i.v., as described for Experiment 1). At 24 h post-injection, blood was collected via cardiac venipuncture into heparinized tubes for plasma isolation and then all birds were euthanized via CO2 overdose. Liver (left lobe) and shank (right tibia epithelium) were collected and stored in the dark at y80 8C until analyses were completed. Additionally, thymus (4 lobes) and bursa (whole) were removed aseptically and gently teased apart, releasing cells into ice-cold medium (RPMI 1640, Sigma 噛R8758). Subsequently, thymocytes and bursacytes were isolated after centrifuging (5000=g) on a density gradient (Histopaque 1077, Sigma 噛1077-1), followed by washing with phosphate buffered saline (PBS), and counting using a hemacytometer. Cells were then stored in amber tubes (y80 8C) until analyses were completed. Dependent variables included tissue lutein, zeaxanthin, canthaxanthin, total carotenoids (as described for Experiment 1) and liver cytosolic Zn. 2.3. Experiment 3 To examine the role of IL-1 on tissue carotenoid deposition, eggs were collected from SCWL hens fed commercial diet (Purina Mills Layena 6501 Diet; analyzed to contain 1.9 mg luteinq0.1 mg zeaxanthinq0 mg canthaxanthinykg diet). Eggs (analyzed to contain (125 nmol total carotenoidsy egg) were hatched and thirty-six chicks were housed (4 pens of 9 chicksypen) as described above. From day zero post-hatch through the duration of the trial, all chicks were provided ad libitum access to basal diet (Table 1) plus 38 mg luteinykg diet (supplied as Oroglo Dry, as described above). At 14 d of age, chicks (average bws124"1 g) within each pen were randomly assigned to one of three treatments. Chicks were either uninjected (Control, ns6 per diet), injected with LPS (ns6 per diet, 100 mgykg bw i.v., as described above) or injected with purified IL-1 (ns6 per diet, 200 Uykg bw i.v.), purified as previously described (Klasing and Peng, 1990). The IL-1 dose and route of administration was
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chosen based upon previous experiments with purified IL-1 in chickens (Klasing et al., 1987). At 24 h post-injection, blood was collected by cardiac venipuncture into heparinized tubes for plasma isolation and then all birds were euthanized via CO2 overdose. Liver (left lobe), thymus (3–4 lobes) and bursa (whole) were immediately collected and stored in the dark at y80 8C until analyses were completed. Dependent variables included tissue lutein and zeaxanthin, total carotenoids (tissue luteinqzeaxanthin) and liver cytosolic Zn. 2.4. Sample analysis Tissues and diets were thawed, weighed, homogenized and analyzed for lutein, zeaxanthin and canthaxanthin (Experiment 1, 2) or lutein and zeaxanthin (Experiment 3) by HPLC following extraction procedures under amber light andyor in amber tubes. Tissues and diet were homogenized (Polytron grinder, Brinkmann Instruments Inc., Westbury, NY) in PBS (2 parts tissue:1 part PBS), then 1 g of homogenate or 500 ml plasma was vortexed with methanolic KOH (5% KOH in MeOH (wyv), 1=sample volume) and butylated hydroxyl toluene (0.1%, wyv). Subsequently, additions of butanol:acetenotrile (1:1 vyv, 2=sample volume), then hexane: chloroform (2:1 vyv, 1=sample volume), then K2HPO4 (saturated in distilled, deionized H2O, 0.1=sample volume) were added, with vigorous vortexing between additions. Samples were then centrifuged (5000=g) for 15 min, and the top organic phase was removed and dried under N2, and then frozen at y80 8C prior to HPLC analysis. HPLC analysis for experiments 1 and 2 was performed using a C18 reverse phase column (5 ˚ 4.6 mm ID=250 mm l, Vydac mm, 300 A, 201TP54, Hesperia CA) and high performance guard column (5 mm, Vydac 201GD54T). The column was chilled to 15 8C using a water jacket (35 cm, Alltech, Deerfield, IL) and a circulating water chiller (RF-10, New Brunswick Sci., Edison, NJ). The isocratic mobile phase consisted of 100% methanol (Fisher 噛A452-4, Pittsburgh, PA). HPLC analysis for experiment 3 was performed using a Spherisorb CN Column (Waters 噛PSS830915, 4.6=250 mm, Milford, MA), fitted with a Spherisorb ODS2 guard column (Waters 噛PSS830053) and a pre-filter (Varian 噛RE7335010, Palo Alto, CA). The isocratic
mobile phase consisted of hexane (Sigma 噛29,325-3): methylene chloride (Fisher D143-4): methanol (Fisher 噛A452-4): diisopropylethylamine (Fisher 噛D3887) at a ratio of 74:25:1:0.1. For each column, the mobile phase was maintained at a flow rate of 1.0 mlymin (Waters 510 pump), and automated injections (Waters WISP 712) of 75 ml were made. A UVyVis detector (Waters 484) monitored at 445 nm, and Millenium software (Waters) was used to process and integrate peaks. Purified standards of lutein, zeaxanthin and canthaxanthin (provided by Roche Vitamins, Parsippany, NJ) were injected in triplicate at different volumes and a standard curve was generated using linear regression (JMP, SAS Institute, Carey, NC), which was used to assess analyte concentration within samples. The limit of detection for each carotenoid was (0.17 pmolyinjection, and this value was used for statistical analyses when samples had no detectable carotenoids. In addition to carotenoid analysis, liver samples were analyzed for cytosolic Zn concentration to determine the relative magnitude of the APR (Hallquist and Klasing, 1994). Briefly, liver samples were weighed and diluted with PBS (1:3, wyv). Livers were homogenized, and samples were centrifuged at 15 000=g for 30 min at room temperature. The cytosolic fraction was collected, diluted with PBS (1:10) and then analyzed for Zn concentration by atomic absorption spectrophotometry (Perkin-Elmer Model 460, Norwalk, CT). Each sample was analyzed in duplicate and Zn concentrations were determined based upon a standard curve produced by dilution of 1000 mgyl standard reference solution (Fisher 噛SZ13-500). 2.5. Statistical analysis Dependent variables were analyzed by general linear model (JMP software, SAS Inc., Cary, NC), using an analysis of variance (ANOVA). Data were normalized by square root transformation prior to statistical analysis when the variances of dependent variables were not homogeneous (assessed with Bartlett’s test). For Experiments 1 and 2, a two-way ANOVA was used to determine the main effect of diet, LPS treatment and their interaction for each dependent variable. When interactions were significant, pre-planned orthogonal contrasts were used to identify differences between means. For experiment 3, a one-way ANOVA was used to determine the effect of
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Table 2 Effect of an acute phase response to LPS on tissue carotenoids (Experiment 1)1 Lutein
Plasma (mmolyl) 8 mg car2 38 mg car Pooled S.E.M. Diet P value LPS P value Diet=LPS P value Liver (mmolykg) 8 mg car 38 mg car Pooled S.E.M. Diet P value LPS P value Diet=LPS P value Thymus (mmolykg) 8 mg car 38 mg car Pooled S.E.M. Diet P value LPS P value Diet=LPS P value Bursa (mmolykg) 8 mg car 38 mg car Pooled S.E.M. Diet P value LPS P value Diet=LPS P value
Zeaxanthin
Control
LPS
0.19 0.25a
0.22 0.19b
Control
0.03 0.25
-0.01 0.10
0.05 0.03 0.71 0.04
LPS
Control
LPS
0.13 0.34
0.11 0.35
0.40 0.72
0.42 0.66
0.05 -0.01 0.81 0.80 -0.01 0.08
0.02 0.08
0.01 -0.01 0.16 0.59 0.03 0.04
0.02 0.08
0.05 0.04 0.27 0.08 0.44 0.24
0.09 0.12
0.05 ND
0.02 -0.01
0.08 0.01 0.75 0.48
0.05 0.08
0.05 0.43
0.17 0.12
0.20 0.10
ND NDa
0.21 0.30
0.28 0.14 0.04 0.74 0.41 0.05
ND 0.26b 0.04 0.04 0.03 0.04
0.16 0.28 0.06 -0.01 0.48 0.10
0.02 0.02 0.92 0.37 ND -0.01
0.05 0.84 0.51 0.40
0.06 -0.01 0.81 0.61
0.03 0.44 0.86 0.95
0.02 0.86 0.39 0.11 0.54 0.21
Total
Control
0.02 -0.01 0.98 0.45 0.11 0.12
0.02 0.10
LPS
0.08 0.13
0.02 0.32 0.48 0.01
Canthaxanthin
0.46 0.24
0.54 0.47 0.11 0.21 0.17 0.53
a,b
Means within a row and a carotenoid type that have different superscripts are significantly different (P-0.02). Growing chicks, fed 8 or 38 mg total carotenoidsykg diet. At 4 weeks of age chicks were uninjected (Control) or LPS-injected (LPS, 100 gykg bw i.v.) and tissue samples were taken 24 h later. 2 Abbreviations: car, carotenoids; ND, not detectable (below limit of detection). 1
treatment on each dependent variable and LSD was used to examine differences between means. Differences between means were considered significant at P-0.05. When contrasts were used, differences were considered significant at P-0.02, which reflects the Bonferroni adjustment of acritical for the number of contrasts examined. 3. Results 3.1. Experiment 1 At 24 h post-injection, LPS injection significantly increased liver cytosolic Zn (P-0.01), indicating that a typical acute phase response was achieved. Average liver cytosolic Zn for Control chicks and LPS-injected chicks were 24.03"0.79 mgyg and 27.58"1.81 mgyg, respectively.
Plasma zeaxanthin, canthaxanthin and total carotenoids (P-0.01 for each) were significantly increased by feeding 38 mg carotenoids compared 0 mg carotenoids (Table 2). The effect of LPS on plasma zeaxanthin, canthaxanthin and total carotenoids was not significant (Ps0.98, Ps0.81, Ps 0.81, respectively), but there was a diet=LPS interaction (Ps0.01) for plasma lutein, in which chicks fed 38 mg carotenoids (Ps0.02), but not in those fed 8 mg carotenoids (Ps0.13), had reduced plasma lutein after LPS treatment. In the liver, increasing diet carotenoids significantly increased lutein, zeaxanthin and total carotenoids (Ps0.03 for each). The effect of LPS on liver zeaxanthin, canthaxanthin or total carotenoids was not significant (Ps0.16, Ps0.86, Ps0.48, respectively), while a diet=LPS interaction (Ps
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Fig. 1. Effect of an acute phase response on thymic (a) and bursal (b) luteinqzeaxanthinqcanthaxanthin in growing chicks (Experiment 1). Chicks (ns28) were fed carotenoid-free basal diet plus 8 or 38 mg total carotenoids (luteinqcanthaxanthin)ykg diet from d 0 to 4 weeks of age, then uninjected (Control) or LPS-injected (100 mgykg bw i.v.). Tissue carotenoids were measured at 24 h post-injection. Graph bars represent means"S.E.M.a,b Within a dietary treatment level, graph bars with different superscripts have significantly different levels (P-0.02).
0.04) demonstrated that LPS reduced liver lutein in chicks fed 38 mg carotenoids (Ps0.03) but not those fed 8 mg carotenoids (Ps0.08). Thymic lutein and canthaxanthin were dependent on diet carotenoid levels (Ps0.04, Ps0.02, respectively), while thymic zeaxanthin was not (Ps0.86). The effect of LPS on total thymic carotenoids was dependent on diet (diet=LPS interaction, Ps0.05), for which chicks fed 38 mg carotenoids, but not 8 mg carotenoids, had LPSdependent reductions in total thymic carotenoids (Ps0.05, Ps0.43, respectively, Fig. 1a). In the bursa, feeding 38 mg carotenoids increased bursal lutein and canthaxanthin compared to chicks fed 8 mg carotenoids (Ps0.01, Ps0.04, respectively), but did not significantly impact bursal zeaxanthin or total carotenoids (Ps0.84, Ps0.21, respectively). The effect of LPS treatment on bursal canthaxanthin was dependent on diet (Ps0.04); LPS-injected chicks fed 38 mg carotenoids but not 8 mg carotenoids had significantly increased bursal canthaxanthin (Ps0.01, Ps0.98, respectively, Fig. 1b). 3.2. Experiment 2 As expected, liver cytosolic Zn was increased by LPS treatment compared to Controls (P-0.01).
Average liver cytosolic Zn of Control chicks was 8.77"0.98 mgyg, while that of LPS-injected chicks was 14.82"1.90 mgyg. The effect of LPS on tissue carotenoid deposition in Experiment 2 (Table 3) was similar to that of Experiment 1, although the absolute amount of carotenoids deposited into plasma and liver was much greater for birds in Experiment 2 as compared to Experiment 1. Variability in carotenoid deposition between experiments has been previously observed, even when birds were apparently healthy and had normal weight gains (Allen, 1992). Plasma carotenoid concentrations were dependent on diet for each carotenoid measured (P-0.01 for each). A significant diet=LPS interaction for plasma lutein (P-0.01), canthaxanthin (P-0.01) and total plasma carotenoids (Ps0.05, Fig. 2a) demonstrated that when chicks were fed 38 mg carotenoids but not 0 mg carotenoids, LPS reduced plasma lutein (P-0.01, Ps0.21, respectively), canthaxanthin (Ps0.05, Ps0.29, respectively) and total plasma carotenoids (P-0.01, Ps0.41, respectively). Similar to responses in the plasma, liver carotenoid concentrations were dependent on diet (P-0.01) for each carotenoid tested. Again, a significant diet=LPS interaction for liver zeax-
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Table 3 Effect of an acute phase response to LPS on tissue carotenoids (Experiment 2)1 Lutein Control Plasma (mmolyl) 0 mg car2 38 mg car Pooled S.E.M. Diet P value LPS P value Diet=LPS P value Liver (mmolykg) 0 mg car 38 mg car Pooled S.E.M. Diet P value LPS P value Diet=LPS P value Shank (mmolykg) 0 mg car 38 mg car Pooled S.E.M. Diet P value LPS P value Diet=LPS P value Thymocyte (mmolykg3) 0 mg car 38 mg car Pooled S.E.M. Diet P value LPS P value Diet=LPS P value
Zeaxanthin LPS
0.60 1.50a
0.48 1.00b
Control 0.01 0.25
0.07 -0.01 0.03 0.01 0.90 1.61
0.08 1.34a
0.12 -0.01 0.42 0.19
LPS
Control
0.01 0.48a
0.01 0.3b
0.60 2.23a
0.03 -0.01 -0.01 -0.01 0.10 0.77b
0.01 0.43a
0.07 -0.01 -0.01 -0.01 0.06 0.11
0.12 0.35
0.04 0.02 0.81 0.35 17.7 20.4
0.01 0.22
0.18 0.41
4.4 5.5
12.0 0.73 0.07 0.56
0.01 0.28b
ND 0.32
2.5 0.52 0.06 0.51
1.04 2.35b 0.17 -0.01 0.06 0.02
ND 0.33
ND ND
0.49 1.55b
0.98 3.37a
0.15 0.77
0.06 -0.01 0.79 0.79 8.6 11.3
LPS
0.10 -0.01 -0.01 0.05
0.02 -0.01 0.10 -0.01
0.10 0.02 0.99 0.81 35.0 45.5
Total
Control
0.03 -0.01 0.37 0.48 0.92 1.30
0.03 0.15
LPS
Canthaxanthin
0.24 0.85 0.11 -0.01 0.94 0.47
ND ND
22.1 25.9
43.6 56.8 15.3 0.73 0.07 0.56
a,b
Means within a row and a carotenoid type that have different superscripts are significantly different (P-0.02). Growing chicks were fed 0 or 38 mg total carotenoidsykg diet, and at 3 weeks of age were uninjected (Control) or LPS-injected (LPS, 100 mgykg bw i.v.). Tissue samples were taken 24 h later. 2 Abbreviations: car, carotenoids; ND, not detectable (below the limit of detection). 3 Thymocyte weight calculated as 109 cellyg (unpublished data). 1
anthin (P-0.01), canthaxanthin (P-0.01), and total carotenoids (Ps0.02) demonstrated that LPS reduced liver carotenoids in chicks fed 38 mg carotenoids (P-0.01 for each), but not 0 mg carotenoids (P)0.84 for each) compared to uninjected chicks (Fig. 2b). In contrast to these data, carotenoids in the shank epithelium were dependent only on diet carotenoid levels (Ps0.02 for all carotenoids tested), with no effect of LPS on any shank epithelium carotenoids (Ps0.79 for each). Thymocyte lutein and zeaxanthin concentrations were not significantly affected by diet treatment (Ps0.52 for each), but there was a trend for LPSinduced increases in thymocyte lutein (Ps0.07), zeaxanthin (Ps0.06) and total thymocyte carotenoids (Ps0.07). Interestingly, the absolute number
of thymocytes isolated from chicks tended to be reduced by LPS treatment compared to Control chicks (Ps0.10, Control cell numbers 1.04=108"4.0=107 vs. LPS cell numbers 7.65=107"3.44=107). Unfortunately, bursacyte sample size was not large enough to perform statistical analyses. However, of those samples that were analyzed, bursacytes contained lutein (65.1"35.8 mmol luteinykg bursacytes), but no detectable zeaxanthin or canthaxanthin. 3.3. Experiment 3 At 24 h post-injection, LPS and IL-1 significantly increased liver cytosolic Zn (Ps0.03, P0.01, respectively) compared to Control chicks
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Fig. 2. Effect of an acute phase response on plasma (a) and liver (b) luteinqzeaxanthinqcanthaxanthin in growing chicks (Experiment 2). Chicks (ns64) were fed carotenoid-free basal diet plus 0 or 38 mg total carotenoids (luteinqcanthaxanthin)ykg diet from d 0 to 4 weeks of age, then uninjected (Control) or LPS-injected (100 mgykg bw i.v.). Tissue carotenoids were measured at 24 h post-injection. Graph bars represent means"S.E.M. a,b Within a dietary treatment level, graph bars with different superscripts are significantly different (P-0.02).
(Controls9.39"0.91 mgyg; LPSs11.89"0.67 mgyg; IL-1s14.59"0.90 mgyg). Additionally, liver cytosolic Zn following IL-1 treatment was significantly greater than that of LPS-injected birds (Ps0.02).
In general, LPS and IL-1 reduced plasma and liver lutein and zeaxanthin (Table 4, Fig. 3). Specifically, IL-1 but not LPS significantly reduced plasma lutein and plasma luteinqzeaxanthin (Ps 0.02 for IL-1, Ps0.09 for LPS) while LPS but
Table 4 Effect of an acute phase response to LPS or IL-1 on tissue carotenoids (Experiment 3)1 Plasma (mmolyl)
Liver (mmolykg)
Thymus (mmolykg)
Bursa (mmolykg)
Lutein
Control LPS IL-1 Pooled S.E.M. P value
3.96 3.27 2.83* 0.36 0.05
0.33 0.20 0.15* 0.06 0.02
0.19 0.40 0.27 0.12 0.48
0.15 0.14 0.17 0.03 0.92
Zea2
Control LPS IL-1 Pooled S.E.M. P value
0.40 0.28* 0.29 0.04 0.05
0.19 0.14 0.13* 0.02 0.02
0.01 0.01 0.01 -0.01 0.89
0.03 0.04 0.04 0.01 0.82
LuteinqZea
Control LPS IL-1 Pooled S.E.M. P value
4.36 3.55 3.12* 0.37 0.05
0.53 0.35 0.29* 0.06 0.03
0.20 0.42 0.28 0.12 0.79
0.18 0.18 0.21 0.05 0.79
1 Growing chicks were fed 38 mg luteinykg diet, and at 2 weeks of age were uninjected (Control), LPS-injected (LPS, 100 mgykg bw i.v.) or IL-1-injected (IL-1, 200 Uykg bw i.v.). 2 Abbreviations: Zea, zeaxanthin. * Means within a column and a carotenoid type that are starred are significantly different from Controls (P-0.02).
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Fig. 3. Effect of an acute phase response on plasma (a) and liver (b) luteinqzeaxanthin concentration in growing chicks (Experiment 3). Chicks (ns36) were fed 38 mg luteinykg diet from d 0 to d 14 of age, then uninjected (Control), LPS-injected (100 mgykg bw i.v.) or IL-1-injected (200 Uykg bw i.v.). Tissue carotenoids were measured at 24 h post-injection. Graph bars represent means"S.E.M. * Graph bars denoted with a star are significantly different (P-0.02).
not IL-1 reduced plasma zeaxanthin (Ps0.02, Ps 0.06, respectively). Similarly, IL-1 significantly reduced liver lutein, zeaxanthin and luteinqzeaxanthin (P-0.02 for each), while there was a trend for LPS-induced reductions in liver lutein, zeaxanthin and luteinqzeaxanthin (Ps0.05, Ps0.05, Ps0.04, respectively). In contrast to changes in liver and plasma carotenoids, bursal and thymic carotenoids were not affected by IL-1 or LPS treatment. 4. Discussion These experiments demonstrated that i.v. injections of 100 mg LPSykg bw or 200 U purified IL1ykg bw was sufficient to increase liver cytosolic Zn levels, as previously demonstrated (Klasing, 1984). Reduced plasma Zn and increased liver Zn are a consistent marker of the APR (Hallquist and Klasing, 1994), which confirms that LPS or IL-1 treatment induced an APR. In general, plasma and liver carotenoids were reduced by the APR induced by LPS or IL-1, as previously reported in response to intestinal parasites (Bletner et al., 1966; Ruff et al., 1974; Tyczkowski and Hamilton, 1991; Allen, 1992) and viral infections (Squibb et al., 1955, 1971; Page et al., 1982). However, the APR at 24 h post-LPS challenge reduced plasma and liver carotenoids to
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a lesser extent ((30% reductions) than previously demonstrated for infectious disease challenges (50–90% reductions, see Squibb et al., 1955; Allen, 1992), which may be related to the magnitude and duration of challenges (i.e. acute LPS challenge vs. chronic infectious disease challenge that is often associated with substantial tissue pathology). Additionally, differences in the timing of measurements (i.e. 24 h post-LPS challenge vs. 3–7 d post-infection) may explain differences in tissue carotenoid levels in these studies. Interestingly, the extent of LPS-induced decreases in tissue carotenoid levels was usually dependent on dietary carotenoid level and occurred in chicks fed 38 mg carotenoidsykg diet but not in those fed 0 or 8 mg carotenoidsykg diet. For example, the liver lost about a third of its carotenoids (35, 30 and 34% in experiments 1, 2 and 3, respectively) when a high carotenoid diet was fed but did not lose significant amounts when low levels were fed. These data indicate that there may be two pools of carotenoids, one that is labile and available for mobilization during an APR and one that is constitutive and stable in concentration. The stable pool appears to be filled by carotenoids originating either from egg yolk or from low levels in the diet (0 or 8 mgykg) and the labile pool being filled when diet levels are high (38 mgykg). In support of this hypothesis, carotenoid distribution within cells and between tissues is not homogenous; a single i.v. dose of b-carotene, was deposited into lymphocyte nuclei in the greatest quantities, followed by mitochondria and microsomes and was lowest in the cytosolic fraction (Chew et al., 1991). Additionally, we have demonstrated that some tissues (e.g. bursa) deposit a fixed level of carotenoids even when dietary levels are low and no additional amounts are deposited as dietary carotenoids increase, while other tissues (e.g. liver) deposit carotenoids in a diet-dependent manner (Koutsos et al., 2003). It may be that the some tissues and cellular fractions contain mostly the stable pool, while others possess both the stable and the labile pool. Alternatively, the minimal changes in tissue carotenoid levels during an APR in chicks fed low carotenoid diets might be due to immunomodulatory properties of carotenoids themselves. That is, the concentration of carotenoids within leukocytes may modulate the intensity or type of response to LPS. A blunted APR to LPS due to low dietary carotenoid levels
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would be reflected in blunted changes in carotenoid metabolism. The APR induced a reduction of 12% of the carotenoids in all tissues that we examined (total in control chicks fed 38 mg carotenoids (21.4 nmol vs. LPS-injected chicks fed 38 mg carotenoids (18.9 nmol, based upon average organ weights reported by Wolfe et al. (1962). Since thymocytes had higher levels of total carotenoids following LPS in experiment 2 and bursal canthaxanthin was increased in Experiment 1, partitioning of carotenoids to leukocytes might account for some of the losses from liver and plasma. Alternately, decreases in tissue carotenoids during an APR could be due to losses via oxidation (in response to free radical production during an APR), metabolism (since birds can oxidize, reduce and acylate carotenoids), or excretion (through bile or urine) and these metabolites would not have been seen by our analytical techniques. Measurements of whole body carotenoid levels and the metabolic fate of labeled carotenoids would provide a more quantitative approach to determine the fate of repartitioned carotenoids. It is interesting that carotenoid levels in lymphoid tissues were altered during an APR. Bursal canthaxanthin levels increased markedly following LPS treatment and the basis for this change may be related to the bursa functioning in B cell development or immunosurveillance of the lower intestine in a 4 week old chicken (Sorvari and Sorvari, 1978). In addition to bursal changes, total thymic carotenoids (from whole lobes) tended to be reduced by an APR in chicks fed 38 mg carotenoids, but carotenoids tended to be increased by an APR in thymocytes isolated from the lobes. This disparity may be related to reductions in total thymocyte numbers in response to LPS treatment. The predicted luteinqzeaxanthin concentration of thymocytes which had emigrated out of the thymus following LPS challenge (Experiment 2) would account for 0.16 nmol total luteinqzeaxanthin in the whole thymus, which approximates the 0.18 nmol reduction in total thymic luteinqzeaxanthin in Experiment 1 (based upon absolute difference (mmolykg)=average thymus weight reported by (Wolfe et al., 1962). Further clarification of these differences would require more quantitative analysis of cell types and cell numbers (e.g. flow cytometry) isolated from thymus lobes following LPS administration.
IL-1 is a major mediator of nutrient partitioning during an APR (Rivier et al., 1989; Zetterstrom et al., 1998), and we found that IL-1 reduced plasma and liver carotenoids in a similar manner to LPS treatment, indicating that IL-1, or the cytokines and cell signaling molecules induced by IL-1, play a role in tissue carotenoid metabolism during the APR. In agreement with this hypothesis, the time course of reductions in plasma carotenoids in response to viral challenge mirrors the pro-inflammatory cytokine-mediated reductions in plasma Zn and Cu (Squibb et al., 1971). There are several potential reasons why APRinduced alterations in tissue carotenoid levels might be advantageous. First, carotenoids are hypothesized to serve as signaling molecules that inform others of an individuals’ disease status and carotenoid losses from skin and feathers would reduce the chance for diseased individuals to reproduce, thus enhancing the fitness of the nondiseased individuals and their offspring (Hamilton and Zuk, 1982; Moller et al., 2000). Second, based upon the putative role of carotenoids on immune function (Bendich, 1989, 1990), carotenoids may be incorporated into lymphoid tissues during an immune response, which our data provide some evidence for, specifically in the bursa and in thymocytes. Therefore, there may be a role for carotenoids as immunomodulatory agents in birds, although further research is needed to examine this hypothesis. It is also possible that changes in carotenoid metabolism during an APR are an indirect result of alterations in lipid metabolism without any carotenoid-specific regulation of tissue uptake. In summary, the APR, induced by LPS or the pro-inflammatory cytokine IL-1, generally reduced plasma and liver carotenoids, as previously seen during a variety of infectious challenges. These data suggest that the APR is at least partly responsible for alterations in tissue carotenoid deposition during a disease challenge. Increased bursal and thymocyte carotenoids may reflect a role for carotenoids in immunomodulation, although further experiments are warranted. Finally, changes in tissue carotenoid levels were observed only for birds fed high dietary carotenoids (38 mg total carotenoidsykg diet), suggesting that carotenoid availability plays a role in the tissue carotenoid deposition during an APR.
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Acknowledgments We would like to thank Tom Famula for advice on statistical analysis of the data and Brooke Humphrey, Hoang Pham, Guochen Hu, Jacqueline Pisenti and Wayne Gould for their invaluable assistance in animal care and experimental sampling. The donation of carotenoid standards by Nelson Ward of Roche Vitamins Inc. is appreciated. Additionally, we would like to thank Raymond Peng, who isolated and purified IL-1. References Allen, P.C., 1992. Effect of coccidiosis on the distribution of dietary lutein in the chick. Poult. Sci. 71, 1457–1463. Alvarez, C., Ramos, A., 1986. Lipids, lipoproteins and apoproteins in serum during infection. Clin. Chem. 32, 142–145. Bendich, A., 1989. Carotenoids and the immune response. J. Nutr. 119, 112–115. Bendich, A., 1990. Carotenoids and the immune system. In: Krinsky, N.I. (Ed.), Carotenoids: Chemistry and Biology. Plenum Press, New York, pp. 323–335. Bletner, J.K., Mitchell Jr, R.P., Tugwell, R.L., 1966. The effect of Eimeria maxima on broiler pigmentation. Poult. Sci. 45, 689–694. Camplani, A., Saino, N., Moller, A.P., 1999. Carotenoids, sexual signals and immune function in barn swallows from Chernobyl. Proc. Royal Soc. Lond. B 266, 1111–1116. Chew, B.P., Wong, T.S., Michal, J.J., Standaert, F.E., Heirman, L.R., 1991. Subcellular distribution of b-carotene, retinol and a-tocopherol in porcine lymphocytes after a single injection of ß-carotene. J. An. Sci. 69, 4892–4897. Feingold, K., Soued, M., Adi, S., Staprans, I., Neese, R., Shigenage, J., et al., 1991. Effect of interleukin-1 on lipid metabolism in the rat. Arterioscler. Thromb. 11, 495–500. Feingold, K.R., Serio, M.K., Adi, S., Moser, A.H., Grunfeld, C., 1989. Tumor necrosis factor stimulates hepatic lipid synthesis and secretion. Endocrinologia 124, 2336–2342. Feingold, K.R., Staprans, I., Memon, R.A., Moser, A.H., Shigenaga, J.K., Doerrler, W., et al., 1992. Endotoxin rapidly induces changes in lipid metabolism that produce hypertriglyceridemia: Low doses stimulate hepatic triglyceride production while high doses inhibit clearance. J. Lipid Res. 33, 1765–1776. Goodwin, T.W., 1986. Metabolism, nutrition and function of carotenoids. Annu. Rev. Nutr. 6, 273–297. Griffin, H.D., Butterwith, S.C., 1988. Effect of Escherichia coli endotoxin on tissue lipoprotein lipase activities in chickens. Br. Poult. Sci. 29, 371–378. Hallquist, N.A., Klasing, K.C., 1994. Serotransferrin, ovotransferrin and metallothionein levels during an immune response in chickens. Comp. Biochem. Physiol. B 108, 375–384. Hamilton, W.D., Zuk, M., 1982. Heritable true fitness and bright birds: A role for parasites? Science 218, 384–387. Hardardottir, I., Sipe, J., Moser, A.H., Fielding, C.J., Feingold, K.R., Grunfeld, C., 1997. LPS and cytokines regulate extra hepatic mRNA levels of apolipoproteins during the acute
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phase response in Syrian hamsters. Biochim. Biophys. Acta 1344, 210–220. Hernandez, J.M., Blanch, A., Bird, N., 2001. Why consumers need carotenoids in poultry. Int. Poult. Prod. 9, 15–16. Klasing, K.C., 1984. Effect of inflammatory agents and interleukin 1 on iron and zinc metabolism. Am. J. Physiol. 247, R901–904. Klasing, K.C., Laurin, D.E., Peng, R.K., Fry, D.M., 1987. Immunologically mediated growth depression in chicks: Influence of feed intake, corticosterone and interleukin-1. J. Nutr. 117, 1629–1637. Klasing, K.C., Peng, R.K., 1990. Monokine-like activities released from a chicken macrophage line. An. Biotech. 1, 107–120. Koutsos, E.A., Calvert, C.C., Klasing, K.C., 2003. Maternal carotenoid status modifies the incorporation of dietary carotenoids into immune tissues of growing chickens (Gallus gallus domesticus). J. Nutr. 133, 1132–1138. Laurin, D.E., Klasing, K.C., 1987. Effects of repetitive immunogen injections and fasting vs. feeding on iron, zinc, and copper metabolism. Biol. Trace Element Res. 14, 153–165. Moller, A.P., Biard, C., Blount, J.D., Houston, D.C., Ninni, P., Saino, N., et al., 2000. Carotenoid-dependent signals: Indicators of foraging efficiency, immunocompetence or detoxification ability? Avian Poult. Biol. Rev. 11, 137–159. NRC, Ed. (1994). Nutrient requirements of poultry. Washington, D.C., National Academy Press. Olson, V.A., Owens, I.P.F., 1998. Costly sexual signals: are carotenoids rare, risky or required? Trends Ecol. Evol. 13, 510–514. Page, R.K., Fletcher, O.J., Rowland, G.N., Gaudry, D., Villegas, P., 1982. Malabsorption syndrome in broiler chickens. Avian Dis. 26, 618–624. Rivier, C., Chizzonite, R., Vale, W., 1989. In the mouse, the activation of hypothalamic-pituitary-adrenal axis by a lipopolysaccharide (endotoxin) is mediated through interleukin1. Endocrinologia 125, 2800–2805. Ruff, M.D., Fuller, H.L., 1975. Some mechanisms of reduction of carotenoid levels in chickens infected with Eimeria acervulina or E. tenella. J. Nutr. 105, 1447–1456. Ruff, M.D., Reid, W.M., Johnson, J.K., 1974. Lowered blood carotenoid levels in chickens infected with coccidia. Poult. Sci. 53, 1801–1809. Saino, N., Ninni, P., Calza, S., Martinelli, R., De Bernardi, F., Moller, A.P., 2000. Better red than dead: carotenoid-based mouth coloration reveals infection in barn swallow nestlings. Proc. R. Soc. Lond. B 267, 57–61. Sorvari, R., Sorvari, T.E., 1978. Bursa fabricii as a peripheral lymphoid organ. Transport of various materials from the anal lips to the bursal lymphoid follicles with reference to its immunological importance. Immunological 32, 499–505. Squibb, R.L., Beisel, W.R., Bostian, K.A., 1971. Effect of Newcastle disease on serum copper, zinc, cholesterol and carotenoid values in the chick. Appl. Microbiol. 22, 1096–1099. Squibb, R.L., Braham, J.E., Guzman, M., Scrimshaw, N.S., 1955. Blood serum total proteins, riboflavin, ascorbic acid, carotenoids and vitamin a of New Hampshire chickens
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infected with Coryza, Cholera or Newcastle disease. Poult. Sci. 1054–1058. Tyczkowski, J.K., Hamilton, P.B., 1991. Altered metabolism of carotenoids during pale-bird syndrome in chickens infected with Eimeria acervulina. Poult. Sci. 70, 2074–2081. Weinberg, E.D., 1978. Iron and infection. Microbiol. Rev. 42, 45–66.
Wolfe, H.R., Sheridan, S.A., Bilstad, N.M., Johnson, M.A., 1962. The growth of lymphoidal organs and the testes of chickens. Anatomical Rec. 142, 485–493. Zetterstrom, M., Sundgren-Andersson, A.K., Ostlund, P., Bartfai, T., 1998. Delineation of the proinflammatory cytokine cascade in fever induction. Ann. NY Acad. Sci. 856, 48–52.
The effect of an acute phase response on tissue carotenoid levels of growing chickens (Gallus gallus domesticus) Elizabeth A. Koutsos1, C. Christopher Calvert, Kirk C. Klasing* Department of Animal Science, University of California, One Shields Avenue, Davis, CA 95616, USA Received 10 January 2003; received in revised form 27 May 2003; accepted 28 May 2003
Abstract Plasma, liver and skin carotenoids decrease following infectious disease challenges. Since these challenges often involve substantial host pathology and chronic immune responses, the mechanism underlying altered carotenoid deposition is unclear. Therefore, changes in tissue carotenoid levels were examined during an acute phase response induced by lipopolysaccharide (LPS) or interleukin-1 (IL-1). In two experiments, chicks were hatched from carotenoid-deplete eggs (ns28, ns64, respectively) and fed 0, 8 or 38 mg carotenoids (luteinqcanthaxanthin)ykg diet. For chicks fed 38 mg carotenoids, but not those fed 0 or 8 mg, LPS generally reduced plasma lutein, canthaxanthin and total carotenoids (P0.05), and liver lutein, zeaxanthin, canthaxanthin and total carotenoids (P-0.05). Additionally, LPS reduced thymic total carotenoids (Ps0.05) and increased thymocyte lutein (Ps0.07), zeaxanthin (Ps0.07) and total carotenoids (Ps 0.07). Finally, LPS increased bursal canthaxanthin (P-0.01), but had no effect on shank carotenoids (P)0.5). In chicks hatched from carotenoid-replete eggs (ns36) and fed dietary lutein (38 mgykg diet), LPS reduced plasma and liver zeaxanthin and liver total carotenoids (P-0.05); IL-1 reduced plasma and liver lutein, zeaxanthin and total carotenoids (P-0.05). Therefore, an acute phase response plays a role in reduced tissue carotenoids during infectious disease. 䊚 2003 Elsevier Science Inc. All rights reserved. Keywords: Carotenoid; Acute phase response; Chicken; LPS; IL-1; Lutein; Zeaxanthin; Canthaxanthin
1. Introduction Carotenoids have a variety of functions including light absorption and pigmentation, antioxidant function in vivo and in vitro and carotenoid-
Abbreviations: APR, acute phase response; bw, body weight; IL, interleukin; i.v., intravenous; LPS, lipopolysaccharide; MT, metallothionein; PBS, phosphate buffered saline; SCWL, single comb white leghorn; TNFa, tumor necrosis factor a. *Corresponding author. Tel.: q1-530-752-1901; fax: q1530-752-0175. E-mail address: [email protected] (K.C. Klasing). 1 Current institution: Animal Science Department, California Polytechnic State University, San Luis Obispo, CA 93406.
specific immunomodulation (reviewed by Goodwin, 1986). Carotenoids are particularly important to avian species, where pigmentation quality is indicative of a bird’s general fitness level (Olson and Owens, 1998) and thus influences mate selection (Camplani et al., 1999) and parental food allocation (Saino et al., 2000). Additionally, carotenoid-based pigmentation is important in the commercial poultry industry because consumers use the level of egg yolk and broiler meat pigmentation to determine product acceptability (Hernandez et al., 2001). Therefore, the commercial poultry industry routinely feeds carotenoids to chickens (primarily lutein and canthaxanthin) to provide pigmentation to poultry products.
1095-6433/03/$ - see front matter 䊚 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S1095-6433(03)00158-2
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The degree of carotenoid deposition into tissues such as skin, blood and liver can be altered by a variety of factors including disease challenge. In birds, disease-associated reductions in tissue carotenoids are collectively referred to as ‘pale bird syndrome’ and have been demonstrated in response to parasite exposure (Tyczkowski and Hamilton, 1991; Allen, 1992) and viral infection (Squibb et al., 1955, 1971; Page et al., 1982). It is likely that multiple mechanisms are responsible for reduced carotenoid deposition during infectious disease challenges, since these challenges are associated with a variety of host pathologies and repeated stimulation of the immune system. For example, many parasite and viral challenges reduce carotenoid absorption and enhance excretion due to intestinal necrosis (Ruff and Fuller, 1975). Additional alterations in tissue carotenoid deposition are presumably a result of disease-induced changes in lipid and lipoprotein metabolism (Hardardottir et al., 1997), including reductions in tissue lipoprotein lipase activity (Griffin and Butterwith, 1988), plasma lipoprotein levels (Alvarez and Ramos, 1986; Hardardottir et al., 1997), and plasma triacylglycerol levels (Feingold et al., 1989, 1991, 1992). These metabolic alterations may directly impact carotenoid incorporation into lipoproteins as well as their uptake via lipoproteinmediated transport. Disease-related changes in nutrient metabolism occur as a consequence of the acute phase response (APR), which is characterized by changes in the production of hepatic proteins (i.e. acute phase proteins) that mediate absorption, transport, uptake and deposition of amino acids, lipids, vitamins and minerals. For example, the production of hepatic metallothionein (MT) is increased during the APR (Hallquist and Klasing, 1994), resulting in reduced circulating plasma Zn and increased liver cytosolic MT-bound Zn. Altered Zn partitioning provides a source of Zn co-factors for acute phase protein production and may also reduce nutrient availability to invading pathogens (Weinberg, 1978). Since the APR substantially alters nutrient partitioning and directly affects lipid metabolism, it is likely that the APR is responsible for alterations in the partitioning of lipid-soluble nutrients. We hypothesize that APR mediators, such as the proinflammatory cytokines tumor necrosis factor-a (TNFa), interleukin-1 (IL-1) and IL-6, regulate tissue carotenoid levels during an immune response. Therefore, we conducted three experi-
ments to examine the effect of the APR, induced by lipopolysaccharide (LPS, an inflammatory component of the cell wall of gram-negative bacteria) or the pro-inflammatory cytokine IL-1, on tissue carotenoid levels in chickens. IL-1 was chosen because it is a central mediator of the APR and subsequent nutritional status of an animal (Rivier et al., 1989; Zetterstrom et al., 1998). 2. Materials and methods 2.1. Experiment 1 To control for maternal carotenoid level, single comb white leghorn hens (SCWL, Hyline, Y-strain, UC Davis stock) were fed carotenoid-free diets for ;30 d to supply carotenoid-deplete eggs (Koutsos et al., 2003). Eggs were hatched and 28 chicks were housed (4 pens of 7 chicksypen) in brooder batteries (Petersime Inc., Gettysburg, OH) in a temperature-controlled room (25 8C) under 24 h light. The experiment was designed as a 2=2 factorial arrangement of treatments with 2 dietary carotenoid levels (8 or 38 mg total carotenoidsy kg diet) and 2 levels of LPS (0 or 100 mgykg body weight; bw). Carotenoids were added to the basal diet (Table 1) as luteinqcanthaxanthin (calculated ratio of 4:1 lutein:canthaxanthin, supplied as Oroglo Dry (Kemin Industries Inc, Des Moines, IA) and Carophyll Red (Roche Vitamins Inc., Parsippany, NJ) respectively. Diets were designed to encompass the range of typical carotenoid levels fed to commercial poultry and were offered for ad libitum consumption from day zero post-hatch through the duration of the trial. At 4 weeks of age, chicks (average bws321"11 g) within dietary treatments were randomly assigned to one of two LPS treatments; birds were either uninjected (Control, ns6 per diet) or LPS-injected (ns8 per diet, 100 mg LPSykg bw intravenously (i.v.) from S. typhimurium, Sigma 噛L7261, St. Louis, MO). Previous research demonstrated that saline injection does not cause an APR and does not alter nutritional status (Laurin and Klasing, 1987), so we chose to use an uninjected control group. At 24 h post-injection, blood was collected via cardiac venipuncture into heparinized tubes for plasma isolation and then all birds were euthanized via CO2 overdose. This time point was chosen based upon preliminary data collected in our lab (unpublished). Liver (left lobe), thymus (3–4 lobes) and bursa (whole) were collected and stored in the
E.A. Koutsos et al. / Comparative Biochemistry and Physiology Part A 135 (2003) 635–646 Table 1 Composition of basal diets fed to SCWL chicks1 Ingredient
Concentration (gykg diet)
Soybean meal Rice flour Calcium carbonate Cellulose Cornstarch Vegetable oil Dicalcium phosphate NaCl Vitamin mix2 Mineral mix2 Methionine Threonine Isoleucine Choline
294.0 449.5 15.0 70.0 86.0 57.0 17.4 3.3 2.5 2.5 0.7 0.6 0.5 1.0
1 Diet offered ad libitum for the duration of the trial. Diets were formulated to meet or exceed all requirements for growing egg-type chickens (NRC, 1994). Basal diets were analyzed to contain no detectable (F0.17 pmolykg) carotenoids. 2 Vitamin mix supplied (per kg final diet): thiamin HCl (1.8 mgykg), riboflavin (3.6 mgykg), Ca-pantothenate (12.5 mgy kg), niacin (25 mgykg), pyridoxine HCl (3 mgykg), folacin (0.6 mgykg), biotin (0.2 mgykg), B12 (10 mgykg), retinyl palmitate (6.3 mgykg), cholecalciferol (0.5 mgykg), a-tocopherol acetate (20 mgykg), vitamin K (5 mgykg), ethoxyquin (125 mgykg). Mineral mix supplied Na2SeO3 (20 mgykg), CuSO4*5H2O (16 mgykg), ZnSO4*5 H2O (156.3 mgykg), MnSO4 (170 mgykg), Fe2SO4*7 H2O (920 mgykg).
dark at y80 8C until samples were analyzed. Dependent variables included tissue lutein, zeaxanthin and canthaxanthin, the sum of all carotenoids analyzed (i.e. total carotenoidssluteinq zeaxanthinqcanthaxanthin), and liver cytosolic Zn. All procedures were approved by the UC Davis Animal Care and Use Committee. 2.2. Experiment 2 To increase the number of tissues analyzed, a second experiment was conducted with chicks hatched from carotenoid-deplete eggs (attained as described for Experiment 1). The experiment was also designed as a 2=2 factorial arrangement of treatments with 2 dietary carotenoid levels (0 or 38 mg total carotenoidsykg diet) and 2 levels of LPS (0 or 100 mgykg bw i.v.). Sixty-four chicks were housed (8 pens of 8 chicksypen) as previously described and were randomly assigned to one of two dietary treatments. From day zero posthatch through the duration of the trial, chicks were offered ad libitum access to basal diet (Table 1)
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plus 0 or 38 mg total carotenoidykg diet (supplemented with lutein and canthaxanthin as described for Experiment 1). At 3 weeks of age, chicks (average bws209"5 g) within dietary treatments were randomly assigned to one of two LPS treatments; chicks were either uninjected (Control, ns 16 per diet) or LPS-injected (ns16 per diet; 100 mgykg bw i.v., as described for Experiment 1). At 24 h post-injection, blood was collected via cardiac venipuncture into heparinized tubes for plasma isolation and then all birds were euthanized via CO2 overdose. Liver (left lobe) and shank (right tibia epithelium) were collected and stored in the dark at y80 8C until analyses were completed. Additionally, thymus (4 lobes) and bursa (whole) were removed aseptically and gently teased apart, releasing cells into ice-cold medium (RPMI 1640, Sigma 噛R8758). Subsequently, thymocytes and bursacytes were isolated after centrifuging (5000=g) on a density gradient (Histopaque 1077, Sigma 噛1077-1), followed by washing with phosphate buffered saline (PBS), and counting using a hemacytometer. Cells were then stored in amber tubes (y80 8C) until analyses were completed. Dependent variables included tissue lutein, zeaxanthin, canthaxanthin, total carotenoids (as described for Experiment 1) and liver cytosolic Zn. 2.3. Experiment 3 To examine the role of IL-1 on tissue carotenoid deposition, eggs were collected from SCWL hens fed commercial diet (Purina Mills Layena 6501 Diet; analyzed to contain 1.9 mg luteinq0.1 mg zeaxanthinq0 mg canthaxanthinykg diet). Eggs (analyzed to contain (125 nmol total carotenoidsy egg) were hatched and thirty-six chicks were housed (4 pens of 9 chicksypen) as described above. From day zero post-hatch through the duration of the trial, all chicks were provided ad libitum access to basal diet (Table 1) plus 38 mg luteinykg diet (supplied as Oroglo Dry, as described above). At 14 d of age, chicks (average bws124"1 g) within each pen were randomly assigned to one of three treatments. Chicks were either uninjected (Control, ns6 per diet), injected with LPS (ns6 per diet, 100 mgykg bw i.v., as described above) or injected with purified IL-1 (ns6 per diet, 200 Uykg bw i.v.), purified as previously described (Klasing and Peng, 1990). The IL-1 dose and route of administration was
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chosen based upon previous experiments with purified IL-1 in chickens (Klasing et al., 1987). At 24 h post-injection, blood was collected by cardiac venipuncture into heparinized tubes for plasma isolation and then all birds were euthanized via CO2 overdose. Liver (left lobe), thymus (3–4 lobes) and bursa (whole) were immediately collected and stored in the dark at y80 8C until analyses were completed. Dependent variables included tissue lutein and zeaxanthin, total carotenoids (tissue luteinqzeaxanthin) and liver cytosolic Zn. 2.4. Sample analysis Tissues and diets were thawed, weighed, homogenized and analyzed for lutein, zeaxanthin and canthaxanthin (Experiment 1, 2) or lutein and zeaxanthin (Experiment 3) by HPLC following extraction procedures under amber light andyor in amber tubes. Tissues and diet were homogenized (Polytron grinder, Brinkmann Instruments Inc., Westbury, NY) in PBS (2 parts tissue:1 part PBS), then 1 g of homogenate or 500 ml plasma was vortexed with methanolic KOH (5% KOH in MeOH (wyv), 1=sample volume) and butylated hydroxyl toluene (0.1%, wyv). Subsequently, additions of butanol:acetenotrile (1:1 vyv, 2=sample volume), then hexane: chloroform (2:1 vyv, 1=sample volume), then K2HPO4 (saturated in distilled, deionized H2O, 0.1=sample volume) were added, with vigorous vortexing between additions. Samples were then centrifuged (5000=g) for 15 min, and the top organic phase was removed and dried under N2, and then frozen at y80 8C prior to HPLC analysis. HPLC analysis for experiments 1 and 2 was performed using a C18 reverse phase column (5 ˚ 4.6 mm ID=250 mm l, Vydac mm, 300 A, 201TP54, Hesperia CA) and high performance guard column (5 mm, Vydac 201GD54T). The column was chilled to 15 8C using a water jacket (35 cm, Alltech, Deerfield, IL) and a circulating water chiller (RF-10, New Brunswick Sci., Edison, NJ). The isocratic mobile phase consisted of 100% methanol (Fisher 噛A452-4, Pittsburgh, PA). HPLC analysis for experiment 3 was performed using a Spherisorb CN Column (Waters 噛PSS830915, 4.6=250 mm, Milford, MA), fitted with a Spherisorb ODS2 guard column (Waters 噛PSS830053) and a pre-filter (Varian 噛RE7335010, Palo Alto, CA). The isocratic
mobile phase consisted of hexane (Sigma 噛29,325-3): methylene chloride (Fisher D143-4): methanol (Fisher 噛A452-4): diisopropylethylamine (Fisher 噛D3887) at a ratio of 74:25:1:0.1. For each column, the mobile phase was maintained at a flow rate of 1.0 mlymin (Waters 510 pump), and automated injections (Waters WISP 712) of 75 ml were made. A UVyVis detector (Waters 484) monitored at 445 nm, and Millenium software (Waters) was used to process and integrate peaks. Purified standards of lutein, zeaxanthin and canthaxanthin (provided by Roche Vitamins, Parsippany, NJ) were injected in triplicate at different volumes and a standard curve was generated using linear regression (JMP, SAS Institute, Carey, NC), which was used to assess analyte concentration within samples. The limit of detection for each carotenoid was (0.17 pmolyinjection, and this value was used for statistical analyses when samples had no detectable carotenoids. In addition to carotenoid analysis, liver samples were analyzed for cytosolic Zn concentration to determine the relative magnitude of the APR (Hallquist and Klasing, 1994). Briefly, liver samples were weighed and diluted with PBS (1:3, wyv). Livers were homogenized, and samples were centrifuged at 15 000=g for 30 min at room temperature. The cytosolic fraction was collected, diluted with PBS (1:10) and then analyzed for Zn concentration by atomic absorption spectrophotometry (Perkin-Elmer Model 460, Norwalk, CT). Each sample was analyzed in duplicate and Zn concentrations were determined based upon a standard curve produced by dilution of 1000 mgyl standard reference solution (Fisher 噛SZ13-500). 2.5. Statistical analysis Dependent variables were analyzed by general linear model (JMP software, SAS Inc., Cary, NC), using an analysis of variance (ANOVA). Data were normalized by square root transformation prior to statistical analysis when the variances of dependent variables were not homogeneous (assessed with Bartlett’s test). For Experiments 1 and 2, a two-way ANOVA was used to determine the main effect of diet, LPS treatment and their interaction for each dependent variable. When interactions were significant, pre-planned orthogonal contrasts were used to identify differences between means. For experiment 3, a one-way ANOVA was used to determine the effect of
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Table 2 Effect of an acute phase response to LPS on tissue carotenoids (Experiment 1)1 Lutein
Plasma (mmolyl) 8 mg car2 38 mg car Pooled S.E.M. Diet P value LPS P value Diet=LPS P value Liver (mmolykg) 8 mg car 38 mg car Pooled S.E.M. Diet P value LPS P value Diet=LPS P value Thymus (mmolykg) 8 mg car 38 mg car Pooled S.E.M. Diet P value LPS P value Diet=LPS P value Bursa (mmolykg) 8 mg car 38 mg car Pooled S.E.M. Diet P value LPS P value Diet=LPS P value
Zeaxanthin
Control
LPS
0.19 0.25a
0.22 0.19b
Control
0.03 0.25
-0.01 0.10
0.05 0.03 0.71 0.04
LPS
Control
LPS
0.13 0.34
0.11 0.35
0.40 0.72
0.42 0.66
0.05 -0.01 0.81 0.80 -0.01 0.08
0.02 0.08
0.01 -0.01 0.16 0.59 0.03 0.04
0.02 0.08
0.05 0.04 0.27 0.08 0.44 0.24
0.09 0.12
0.05 ND
0.02 -0.01
0.08 0.01 0.75 0.48
0.05 0.08
0.05 0.43
0.17 0.12
0.20 0.10
ND NDa
0.21 0.30
0.28 0.14 0.04 0.74 0.41 0.05
ND 0.26b 0.04 0.04 0.03 0.04
0.16 0.28 0.06 -0.01 0.48 0.10
0.02 0.02 0.92 0.37 ND -0.01
0.05 0.84 0.51 0.40
0.06 -0.01 0.81 0.61
0.03 0.44 0.86 0.95
0.02 0.86 0.39 0.11 0.54 0.21
Total
Control
0.02 -0.01 0.98 0.45 0.11 0.12
0.02 0.10
LPS
0.08 0.13
0.02 0.32 0.48 0.01
Canthaxanthin
0.46 0.24
0.54 0.47 0.11 0.21 0.17 0.53
a,b
Means within a row and a carotenoid type that have different superscripts are significantly different (P-0.02). Growing chicks, fed 8 or 38 mg total carotenoidsykg diet. At 4 weeks of age chicks were uninjected (Control) or LPS-injected (LPS, 100 gykg bw i.v.) and tissue samples were taken 24 h later. 2 Abbreviations: car, carotenoids; ND, not detectable (below limit of detection). 1
treatment on each dependent variable and LSD was used to examine differences between means. Differences between means were considered significant at P-0.05. When contrasts were used, differences were considered significant at P-0.02, which reflects the Bonferroni adjustment of acritical for the number of contrasts examined. 3. Results 3.1. Experiment 1 At 24 h post-injection, LPS injection significantly increased liver cytosolic Zn (P-0.01), indicating that a typical acute phase response was achieved. Average liver cytosolic Zn for Control chicks and LPS-injected chicks were 24.03"0.79 mgyg and 27.58"1.81 mgyg, respectively.
Plasma zeaxanthin, canthaxanthin and total carotenoids (P-0.01 for each) were significantly increased by feeding 38 mg carotenoids compared 0 mg carotenoids (Table 2). The effect of LPS on plasma zeaxanthin, canthaxanthin and total carotenoids was not significant (Ps0.98, Ps0.81, Ps 0.81, respectively), but there was a diet=LPS interaction (Ps0.01) for plasma lutein, in which chicks fed 38 mg carotenoids (Ps0.02), but not in those fed 8 mg carotenoids (Ps0.13), had reduced plasma lutein after LPS treatment. In the liver, increasing diet carotenoids significantly increased lutein, zeaxanthin and total carotenoids (Ps0.03 for each). The effect of LPS on liver zeaxanthin, canthaxanthin or total carotenoids was not significant (Ps0.16, Ps0.86, Ps0.48, respectively), while a diet=LPS interaction (Ps
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Fig. 1. Effect of an acute phase response on thymic (a) and bursal (b) luteinqzeaxanthinqcanthaxanthin in growing chicks (Experiment 1). Chicks (ns28) were fed carotenoid-free basal diet plus 8 or 38 mg total carotenoids (luteinqcanthaxanthin)ykg diet from d 0 to 4 weeks of age, then uninjected (Control) or LPS-injected (100 mgykg bw i.v.). Tissue carotenoids were measured at 24 h post-injection. Graph bars represent means"S.E.M.a,b Within a dietary treatment level, graph bars with different superscripts have significantly different levels (P-0.02).
0.04) demonstrated that LPS reduced liver lutein in chicks fed 38 mg carotenoids (Ps0.03) but not those fed 8 mg carotenoids (Ps0.08). Thymic lutein and canthaxanthin were dependent on diet carotenoid levels (Ps0.04, Ps0.02, respectively), while thymic zeaxanthin was not (Ps0.86). The effect of LPS on total thymic carotenoids was dependent on diet (diet=LPS interaction, Ps0.05), for which chicks fed 38 mg carotenoids, but not 8 mg carotenoids, had LPSdependent reductions in total thymic carotenoids (Ps0.05, Ps0.43, respectively, Fig. 1a). In the bursa, feeding 38 mg carotenoids increased bursal lutein and canthaxanthin compared to chicks fed 8 mg carotenoids (Ps0.01, Ps0.04, respectively), but did not significantly impact bursal zeaxanthin or total carotenoids (Ps0.84, Ps0.21, respectively). The effect of LPS treatment on bursal canthaxanthin was dependent on diet (Ps0.04); LPS-injected chicks fed 38 mg carotenoids but not 8 mg carotenoids had significantly increased bursal canthaxanthin (Ps0.01, Ps0.98, respectively, Fig. 1b). 3.2. Experiment 2 As expected, liver cytosolic Zn was increased by LPS treatment compared to Controls (P-0.01).
Average liver cytosolic Zn of Control chicks was 8.77"0.98 mgyg, while that of LPS-injected chicks was 14.82"1.90 mgyg. The effect of LPS on tissue carotenoid deposition in Experiment 2 (Table 3) was similar to that of Experiment 1, although the absolute amount of carotenoids deposited into plasma and liver was much greater for birds in Experiment 2 as compared to Experiment 1. Variability in carotenoid deposition between experiments has been previously observed, even when birds were apparently healthy and had normal weight gains (Allen, 1992). Plasma carotenoid concentrations were dependent on diet for each carotenoid measured (P-0.01 for each). A significant diet=LPS interaction for plasma lutein (P-0.01), canthaxanthin (P-0.01) and total plasma carotenoids (Ps0.05, Fig. 2a) demonstrated that when chicks were fed 38 mg carotenoids but not 0 mg carotenoids, LPS reduced plasma lutein (P-0.01, Ps0.21, respectively), canthaxanthin (Ps0.05, Ps0.29, respectively) and total plasma carotenoids (P-0.01, Ps0.41, respectively). Similar to responses in the plasma, liver carotenoid concentrations were dependent on diet (P-0.01) for each carotenoid tested. Again, a significant diet=LPS interaction for liver zeax-
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Table 3 Effect of an acute phase response to LPS on tissue carotenoids (Experiment 2)1 Lutein Control Plasma (mmolyl) 0 mg car2 38 mg car Pooled S.E.M. Diet P value LPS P value Diet=LPS P value Liver (mmolykg) 0 mg car 38 mg car Pooled S.E.M. Diet P value LPS P value Diet=LPS P value Shank (mmolykg) 0 mg car 38 mg car Pooled S.E.M. Diet P value LPS P value Diet=LPS P value Thymocyte (mmolykg3) 0 mg car 38 mg car Pooled S.E.M. Diet P value LPS P value Diet=LPS P value
Zeaxanthin LPS
0.60 1.50a
0.48 1.00b
Control 0.01 0.25
0.07 -0.01 0.03 0.01 0.90 1.61
0.08 1.34a
0.12 -0.01 0.42 0.19
LPS
Control
0.01 0.48a
0.01 0.3b
0.60 2.23a
0.03 -0.01 -0.01 -0.01 0.10 0.77b
0.01 0.43a
0.07 -0.01 -0.01 -0.01 0.06 0.11
0.12 0.35
0.04 0.02 0.81 0.35 17.7 20.4
0.01 0.22
0.18 0.41
4.4 5.5
12.0 0.73 0.07 0.56
0.01 0.28b
ND 0.32
2.5 0.52 0.06 0.51
1.04 2.35b 0.17 -0.01 0.06 0.02
ND 0.33
ND ND
0.49 1.55b
0.98 3.37a
0.15 0.77
0.06 -0.01 0.79 0.79 8.6 11.3
LPS
0.10 -0.01 -0.01 0.05
0.02 -0.01 0.10 -0.01
0.10 0.02 0.99 0.81 35.0 45.5
Total
Control
0.03 -0.01 0.37 0.48 0.92 1.30
0.03 0.15
LPS
Canthaxanthin
0.24 0.85 0.11 -0.01 0.94 0.47
ND ND
22.1 25.9
43.6 56.8 15.3 0.73 0.07 0.56
a,b
Means within a row and a carotenoid type that have different superscripts are significantly different (P-0.02). Growing chicks were fed 0 or 38 mg total carotenoidsykg diet, and at 3 weeks of age were uninjected (Control) or LPS-injected (LPS, 100 mgykg bw i.v.). Tissue samples were taken 24 h later. 2 Abbreviations: car, carotenoids; ND, not detectable (below the limit of detection). 3 Thymocyte weight calculated as 109 cellyg (unpublished data). 1
anthin (P-0.01), canthaxanthin (P-0.01), and total carotenoids (Ps0.02) demonstrated that LPS reduced liver carotenoids in chicks fed 38 mg carotenoids (P-0.01 for each), but not 0 mg carotenoids (P)0.84 for each) compared to uninjected chicks (Fig. 2b). In contrast to these data, carotenoids in the shank epithelium were dependent only on diet carotenoid levels (Ps0.02 for all carotenoids tested), with no effect of LPS on any shank epithelium carotenoids (Ps0.79 for each). Thymocyte lutein and zeaxanthin concentrations were not significantly affected by diet treatment (Ps0.52 for each), but there was a trend for LPSinduced increases in thymocyte lutein (Ps0.07), zeaxanthin (Ps0.06) and total thymocyte carotenoids (Ps0.07). Interestingly, the absolute number
of thymocytes isolated from chicks tended to be reduced by LPS treatment compared to Control chicks (Ps0.10, Control cell numbers 1.04=108"4.0=107 vs. LPS cell numbers 7.65=107"3.44=107). Unfortunately, bursacyte sample size was not large enough to perform statistical analyses. However, of those samples that were analyzed, bursacytes contained lutein (65.1"35.8 mmol luteinykg bursacytes), but no detectable zeaxanthin or canthaxanthin. 3.3. Experiment 3 At 24 h post-injection, LPS and IL-1 significantly increased liver cytosolic Zn (Ps0.03, P0.01, respectively) compared to Control chicks
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Fig. 2. Effect of an acute phase response on plasma (a) and liver (b) luteinqzeaxanthinqcanthaxanthin in growing chicks (Experiment 2). Chicks (ns64) were fed carotenoid-free basal diet plus 0 or 38 mg total carotenoids (luteinqcanthaxanthin)ykg diet from d 0 to 4 weeks of age, then uninjected (Control) or LPS-injected (100 mgykg bw i.v.). Tissue carotenoids were measured at 24 h post-injection. Graph bars represent means"S.E.M. a,b Within a dietary treatment level, graph bars with different superscripts are significantly different (P-0.02).
(Controls9.39"0.91 mgyg; LPSs11.89"0.67 mgyg; IL-1s14.59"0.90 mgyg). Additionally, liver cytosolic Zn following IL-1 treatment was significantly greater than that of LPS-injected birds (Ps0.02).
In general, LPS and IL-1 reduced plasma and liver lutein and zeaxanthin (Table 4, Fig. 3). Specifically, IL-1 but not LPS significantly reduced plasma lutein and plasma luteinqzeaxanthin (Ps 0.02 for IL-1, Ps0.09 for LPS) while LPS but
Table 4 Effect of an acute phase response to LPS or IL-1 on tissue carotenoids (Experiment 3)1 Plasma (mmolyl)
Liver (mmolykg)
Thymus (mmolykg)
Bursa (mmolykg)
Lutein
Control LPS IL-1 Pooled S.E.M. P value
3.96 3.27 2.83* 0.36 0.05
0.33 0.20 0.15* 0.06 0.02
0.19 0.40 0.27 0.12 0.48
0.15 0.14 0.17 0.03 0.92
Zea2
Control LPS IL-1 Pooled S.E.M. P value
0.40 0.28* 0.29 0.04 0.05
0.19 0.14 0.13* 0.02 0.02
0.01 0.01 0.01 -0.01 0.89
0.03 0.04 0.04 0.01 0.82
LuteinqZea
Control LPS IL-1 Pooled S.E.M. P value
4.36 3.55 3.12* 0.37 0.05
0.53 0.35 0.29* 0.06 0.03
0.20 0.42 0.28 0.12 0.79
0.18 0.18 0.21 0.05 0.79
1 Growing chicks were fed 38 mg luteinykg diet, and at 2 weeks of age were uninjected (Control), LPS-injected (LPS, 100 mgykg bw i.v.) or IL-1-injected (IL-1, 200 Uykg bw i.v.). 2 Abbreviations: Zea, zeaxanthin. * Means within a column and a carotenoid type that are starred are significantly different from Controls (P-0.02).
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Fig. 3. Effect of an acute phase response on plasma (a) and liver (b) luteinqzeaxanthin concentration in growing chicks (Experiment 3). Chicks (ns36) were fed 38 mg luteinykg diet from d 0 to d 14 of age, then uninjected (Control), LPS-injected (100 mgykg bw i.v.) or IL-1-injected (200 Uykg bw i.v.). Tissue carotenoids were measured at 24 h post-injection. Graph bars represent means"S.E.M. * Graph bars denoted with a star are significantly different (P-0.02).
not IL-1 reduced plasma zeaxanthin (Ps0.02, Ps 0.06, respectively). Similarly, IL-1 significantly reduced liver lutein, zeaxanthin and luteinqzeaxanthin (P-0.02 for each), while there was a trend for LPS-induced reductions in liver lutein, zeaxanthin and luteinqzeaxanthin (Ps0.05, Ps0.05, Ps0.04, respectively). In contrast to changes in liver and plasma carotenoids, bursal and thymic carotenoids were not affected by IL-1 or LPS treatment. 4. Discussion These experiments demonstrated that i.v. injections of 100 mg LPSykg bw or 200 U purified IL1ykg bw was sufficient to increase liver cytosolic Zn levels, as previously demonstrated (Klasing, 1984). Reduced plasma Zn and increased liver Zn are a consistent marker of the APR (Hallquist and Klasing, 1994), which confirms that LPS or IL-1 treatment induced an APR. In general, plasma and liver carotenoids were reduced by the APR induced by LPS or IL-1, as previously reported in response to intestinal parasites (Bletner et al., 1966; Ruff et al., 1974; Tyczkowski and Hamilton, 1991; Allen, 1992) and viral infections (Squibb et al., 1955, 1971; Page et al., 1982). However, the APR at 24 h post-LPS challenge reduced plasma and liver carotenoids to
643
a lesser extent ((30% reductions) than previously demonstrated for infectious disease challenges (50–90% reductions, see Squibb et al., 1955; Allen, 1992), which may be related to the magnitude and duration of challenges (i.e. acute LPS challenge vs. chronic infectious disease challenge that is often associated with substantial tissue pathology). Additionally, differences in the timing of measurements (i.e. 24 h post-LPS challenge vs. 3–7 d post-infection) may explain differences in tissue carotenoid levels in these studies. Interestingly, the extent of LPS-induced decreases in tissue carotenoid levels was usually dependent on dietary carotenoid level and occurred in chicks fed 38 mg carotenoidsykg diet but not in those fed 0 or 8 mg carotenoidsykg diet. For example, the liver lost about a third of its carotenoids (35, 30 and 34% in experiments 1, 2 and 3, respectively) when a high carotenoid diet was fed but did not lose significant amounts when low levels were fed. These data indicate that there may be two pools of carotenoids, one that is labile and available for mobilization during an APR and one that is constitutive and stable in concentration. The stable pool appears to be filled by carotenoids originating either from egg yolk or from low levels in the diet (0 or 8 mgykg) and the labile pool being filled when diet levels are high (38 mgykg). In support of this hypothesis, carotenoid distribution within cells and between tissues is not homogenous; a single i.v. dose of b-carotene, was deposited into lymphocyte nuclei in the greatest quantities, followed by mitochondria and microsomes and was lowest in the cytosolic fraction (Chew et al., 1991). Additionally, we have demonstrated that some tissues (e.g. bursa) deposit a fixed level of carotenoids even when dietary levels are low and no additional amounts are deposited as dietary carotenoids increase, while other tissues (e.g. liver) deposit carotenoids in a diet-dependent manner (Koutsos et al., 2003). It may be that the some tissues and cellular fractions contain mostly the stable pool, while others possess both the stable and the labile pool. Alternatively, the minimal changes in tissue carotenoid levels during an APR in chicks fed low carotenoid diets might be due to immunomodulatory properties of carotenoids themselves. That is, the concentration of carotenoids within leukocytes may modulate the intensity or type of response to LPS. A blunted APR to LPS due to low dietary carotenoid levels
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would be reflected in blunted changes in carotenoid metabolism. The APR induced a reduction of 12% of the carotenoids in all tissues that we examined (total in control chicks fed 38 mg carotenoids (21.4 nmol vs. LPS-injected chicks fed 38 mg carotenoids (18.9 nmol, based upon average organ weights reported by Wolfe et al. (1962). Since thymocytes had higher levels of total carotenoids following LPS in experiment 2 and bursal canthaxanthin was increased in Experiment 1, partitioning of carotenoids to leukocytes might account for some of the losses from liver and plasma. Alternately, decreases in tissue carotenoids during an APR could be due to losses via oxidation (in response to free radical production during an APR), metabolism (since birds can oxidize, reduce and acylate carotenoids), or excretion (through bile or urine) and these metabolites would not have been seen by our analytical techniques. Measurements of whole body carotenoid levels and the metabolic fate of labeled carotenoids would provide a more quantitative approach to determine the fate of repartitioned carotenoids. It is interesting that carotenoid levels in lymphoid tissues were altered during an APR. Bursal canthaxanthin levels increased markedly following LPS treatment and the basis for this change may be related to the bursa functioning in B cell development or immunosurveillance of the lower intestine in a 4 week old chicken (Sorvari and Sorvari, 1978). In addition to bursal changes, total thymic carotenoids (from whole lobes) tended to be reduced by an APR in chicks fed 38 mg carotenoids, but carotenoids tended to be increased by an APR in thymocytes isolated from the lobes. This disparity may be related to reductions in total thymocyte numbers in response to LPS treatment. The predicted luteinqzeaxanthin concentration of thymocytes which had emigrated out of the thymus following LPS challenge (Experiment 2) would account for 0.16 nmol total luteinqzeaxanthin in the whole thymus, which approximates the 0.18 nmol reduction in total thymic luteinqzeaxanthin in Experiment 1 (based upon absolute difference (mmolykg)=average thymus weight reported by (Wolfe et al., 1962). Further clarification of these differences would require more quantitative analysis of cell types and cell numbers (e.g. flow cytometry) isolated from thymus lobes following LPS administration.
IL-1 is a major mediator of nutrient partitioning during an APR (Rivier et al., 1989; Zetterstrom et al., 1998), and we found that IL-1 reduced plasma and liver carotenoids in a similar manner to LPS treatment, indicating that IL-1, or the cytokines and cell signaling molecules induced by IL-1, play a role in tissue carotenoid metabolism during the APR. In agreement with this hypothesis, the time course of reductions in plasma carotenoids in response to viral challenge mirrors the pro-inflammatory cytokine-mediated reductions in plasma Zn and Cu (Squibb et al., 1971). There are several potential reasons why APRinduced alterations in tissue carotenoid levels might be advantageous. First, carotenoids are hypothesized to serve as signaling molecules that inform others of an individuals’ disease status and carotenoid losses from skin and feathers would reduce the chance for diseased individuals to reproduce, thus enhancing the fitness of the nondiseased individuals and their offspring (Hamilton and Zuk, 1982; Moller et al., 2000). Second, based upon the putative role of carotenoids on immune function (Bendich, 1989, 1990), carotenoids may be incorporated into lymphoid tissues during an immune response, which our data provide some evidence for, specifically in the bursa and in thymocytes. Therefore, there may be a role for carotenoids as immunomodulatory agents in birds, although further research is needed to examine this hypothesis. It is also possible that changes in carotenoid metabolism during an APR are an indirect result of alterations in lipid metabolism without any carotenoid-specific regulation of tissue uptake. In summary, the APR, induced by LPS or the pro-inflammatory cytokine IL-1, generally reduced plasma and liver carotenoids, as previously seen during a variety of infectious challenges. These data suggest that the APR is at least partly responsible for alterations in tissue carotenoid deposition during a disease challenge. Increased bursal and thymocyte carotenoids may reflect a role for carotenoids in immunomodulation, although further experiments are warranted. Finally, changes in tissue carotenoid levels were observed only for birds fed high dietary carotenoids (38 mg total carotenoidsykg diet), suggesting that carotenoid availability plays a role in the tissue carotenoid deposition during an APR.
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