Bixin uptake and antioxidative effect and role in immunoregulation in domestic cats.

Bixin, a carotenoid found in the seed of the Annatto plant, , is a potent antioxidant. Carotenoids are readily absorbed from the diet; therefore, the purpose of this study was to examine uptake of bixin by plasma, lipoproteins, and leukocytes after dietary supplementation in domestic cats and to assess effects on immune response. Female domestic short hair cats (3 yr old; 4.79 ± 0.13 kg BW) were fed a single dose of 0, 1, 5, or 10 mg bixin, and blood was taken at 0, 1, 2, 4 and 8 h after administration ( = 6/treatment) to determine acute absorption rate. Then, bixin was fed daily for 14 d to examine steady-state plasma concentrations and subcellular distribution. Following these preliminary experiments, cats ( = 8/treatment) were fed diets containing 0, 1, 5, or 10 mg bixin/d for 16 wk and blood was collected on wk 0, 6, 12, and 16 for analysis of leukocyte subpopulations, cell-mediated responsiveness, and inflammatory and oxidative biomarkers. Maximal uptake in plasma occurred 1 h after a single oral dose of bixin, with a maximal concentration of 0.119 µ and elimination half-life of 1.8 to 2.2 h. Daily feeding of bixin showed a steady-state plasma concentration of 0.110 µ at the greatest doses. Bixin was primarily associated with the high-density lipoprotein fraction of blood lipoproteins and was primarily distributed in mitochondrial fractions (58-59%) of but also in microsomal and nuclear fractions (37-44%). Leukocyte subpopulations in blood were variably affected by dietary bixin, with an increase ( < 0.05) in total T cells but a concurrent decrease ( < 0.05) in CD18+ and B cell subpopulations. However, plasma IgG increased ( < 0.05) in the 10-mg treatment group by wk 6. Lymphoproliferation was stimulated ( < 0.05) in the 5-mg bixin treatment group by wk 16, and delayed-type hypersensitivity response increased after nonspecific antigenic challenge. Conversely, when a specific challenge of vaccine was assessed on wk 12 and 16, responsiveness decreased ( < 0.05) in the 10-mg bixin treatment group. Bixin supplementation surprisingly caused an increase ( < 0.05) in α-acid glycoprotein but had no effect on natural killer cell activity, other subpopulations of leukocytes, or 8-oxo-2>-deoxyguanosine, a DNA damage biomarker. This experiment demonstrated dose-dependent uptake of bixin in plasma and blood lipoproteins and distribution in leukocyte subcellular components and an impacted immune response through cell-mediated and humoral actions.

Uptake and immunomodulatory role of bixin in dogs.

Carotenoids are readily absorbed from the diet and distributed in blood leukocyte subcellular organelles. Bixin, a potent bioactive found in the seed of the Annatto plant, , possesses antioxidant and anti-inflammatory properties. The purpose of this study was to determine the uptake of bixin by plasma, lipoproteins, and leukocytes in domestic dogs and to examine immunoprotective properties. To determine uptake kinetics, female Beagle dogs (2 yr; 9.1 ± 0.1 kg BW) were first fed a single dose by oral gavage of 0, 5, 10, 20, or 40 mg bixin, with blood collected at 0 to 16 h after administration ( = 6/treatment), and then fed daily with 0, 5, 10, 20, or 40 mg bixin/d, with blood collected at 0, 1, 2, 4, 6, 10, and 14 d. In a consecutive experiment, cell-mediated and humoral responses as well as oxidative biomarkers were measured following 16 wk of dietary supplementation with 0, 5, 10, or 20 mg bixin/d. Maximal absorption in plasma occurred by 0.5 h with an elimination half-life of 2.6 to 3.3 h after a single dose of bixin. Steady-state plasma concentrations were 0.053 µ after 14 d of 40 mg bixin/d. The majority of subcellular bixin was found in the leukocyte mitochondria and was associated with the high-density lipoprotein and low-density lipoprotein fractions of lipoproteins. Specific (vaccine) response increased ( < 0.05) but nonspecific mitogen response was unchanged after 12 wk of dietary bixin, as assessed by a delayed-type hypersensitivity assay. Both B cell plasma leukocyte subpopulations at 6 and 16 wk and IgG plasma concentration at 12 wk in the 10-mg treatment group increased ( < 0.05), although IgM production and other cell populations were unaffected. In addition, 8-oxo-2'-deoxyguanosine (8-OHdG), a DNA damage biomarker, was substantially reduced ( < 0.05) in all treatment groups by wk 16, and C-reactive protein (CRP) was suppressed at wk 12 ( < 0.05). Dietary supplementation with bixin showed no changes in lymphoproliferation in response to in vitro mitogenic challenge and had no effect in enhancing natural killer cell activity. In conclusion, bixin was readily absorbed in a dose-dependent manner in blood following oral administration and was then taken up by leukocytes, where it was primarily distributed to mitochondria but in other subcellular organelles as well. Bixin also appeared to stimulate immune response, as seen with cell-mediated responses, and exerted anti-inflammatory (reduced CRP) as well as antioxidative (reduced 8-OHdG) effects in dogs.

Astaxanthin modulates age-associated mitochondrial dysfunction in healthy dogs.

Young (2.97±0.01 yr; 8.16±0.15 kg BW) and geriatric (10.71±0.01 yr; 9.46±0.18 kg BW) healthy female Beagle dogs (n=14/age group) were fed 0 or 20 mg astaxanthin daily for 16 wk to examine modulation of mitochondrial function. Fasted blood was sampled on wk 0, 8, and 16. Mitochondria membrane permeability, ATP production, cytochrome c oxidase/reductase, and number were assessed in leukocytes whereas astaxanthin uptake, glutathione, superoxide dismutase, nitric oxide, 8-hydroxy-2'-deoxyguanosine, 8-isoprostane, and protein carbonyl were measured in plasma. Aging increased (P<0.05) complex III cytochrome c oxidoreductase but decreased (P<0.05) 8-hydroxy-2'-deoxyguanosine and protein carbonyl. Mitochondrial function improved in both young and geriatric dogs by increasing (P<0.05) ATP production, mitochondria mass, and cytochrome c oxidoreductase activity, especially in geriatric dogs compared with young dogs. Astaxanthin feeding also increased (P<0.05) the reduced glutathione to oxidized glutathione ratio in young dogs and decreased (P<0.05) nitric oxide in both young and geriatric dogs. Dietary astaxanthin improved mitochondrial function in blood leukocytes, most likely by alleviating oxidative damage to cellular DNA and protein.

Effects of medium and substratum on ovine satellite cell attachment, proliferation and differentiation in vitro.

The ability of ovine-derived satellite cells to attach, proliferate and differentiate in response to seven horse serum-supplemented media and eleven substrata was evaluated in vitro. Satellite cells attached equally well when exposed to CRCM-30, Medium-199 and high glucose Dulbecco's modified Eagles medium (DMEM, P less than 0.05). Proliferation of satellite cells was greatest using McCoy's 5A, supplemented with 15% horse serum (P less than 0.05), and differentiation was most efficient with low glucose DMEM, supplemented with 1% horse serum (P less than 0.05). Pig-skin gelatin facilitated the greatest ovine satellite cell proliferative and differentiative responses when compared to the performance of ten other substrata (P less than 0.05). Further, 0.5 mg/16 mm2-well pig-skin gelatin appeared to be the optimum concentration of substratum for expression of satellite cell growth characteristics. Thus, consideration must be given to the processes of attachment and proliferation in experiments attempting to maximize satellite cell differentiation in vitro.

Insulin-like growth factor I receptor analysis of satellite cell-derived myotube membranes established from two lines of Targhee rams selected for growth rate.

Ovine-derived fibroblasts were used to validate an insulin-like growth factor I (IGF-I) membrane-receptor binding assay system. Competitive binding using fibroblasts revealed that half-maximal inhibition of 125I-IGF-I binding by IGF-I was 2.3 nM. SDS-polyacrylamide gel electrophoresis analysis of specific protein-associated 125I-IGF-I was consistent with the migration of 125I-IGF-I-labeled Type I IGF receptor alpha-subunits at Mr 133,000 daltons. Further, the efficiency of two cell solubilization methods was examined and time-dependent binding equilibrium was determined for the membrane assay system. Satellite cell-derived myotubes were subsequently isolated from primary satellite cell cultures established from the semimembranosus muscles of high and low efficiency-of-gain (EOG) Targhee rams, and IGF-I receptor dynamics were measured. A membrane competitive binding study revealed that half-maximal inhibition of 125I-IGF-I binding was achieved by 1-ng IGF-I for low, and 10-ng IGF-I for high, EOG myotube membrane preparations. Kd values were similar between the high EOG (4.78 nM) and low EOG (2.95 nM) groups; however, receptor concentrations (Bmax) appeared to differ between groups. High EOG membrane receptor Bmax was 3.88 pmole/micrograms protein (19.87 pmole/micrograms DNA), whereas low EOG membrane receptor Bmax was 1.22 pmole/micrograms protein (9.28 pmole/micrograms DNA). These preliminary findings support the hypothesis that genetic selection for EOG results in altered satellite cell responsiveness to IGF-I.

Satellite cells derived from streptozotocin-diabetic rats display altered fusion parameters in vitro.

Myogenic satellite cells were isolated from nondiabetic and streptozotocin-diabetic rats and studied in vitro. Streptozotocin (STZ) administration produced both hyperglycemia and glucosuria in adult rats when compared to controls. (P less than 0.01), with 12.5% mortality in untreated animals. Insulin therapy diminished blood glucose levels to those found in nondiabetic animals. Only STZ-diabetic rats displayed symptoms of Type I diabetes, including polydipsia, polyuria, and hyperphagia. STZ-treated rats possessed less leg muscle mass and less subcutaneous, intermuscular, and intramuscular fat. Conversely, nondiabetic rats had a greater mean body weight (P less than 0.01) at the end of the experiment than did diabetic rats. Primary cultures of diabetic-derived satellite cells displayed decreased overall ability (P less than 0.01) to fuse to form multinucleated myotubes in vitro than controls. In addition, secondary cultures of diabetic-derived satellite cells achieved maximal fusion one day later than secondary cultures of control-derived cells. Collectively, these data provide preliminary evidence to suggest that untreated insulin-dependent diabetes results in altered fusion characteristics of myogenic satellite cells. Additional studies utilizing satellite cells from diabetic animals will provide valuable definition of the satellite cell involvement in skeletal muscle autophagy which is a symptom of type I diabetes.