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Fort Wayne State Developmental Center, Fort Wayne, IN 46835 and * Lacombe Research Centre, Lacombe, AB, T4L 1W1 Canada
3To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • vitamin B-6 • adrenal • adrenocorticotropic hormone • alkaline phosphatase • pyridoxal kinase • pigs
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Pyridoxine supplementation reduced the formation of
stomach ulcers in response to immobilization stress in mice
(Henrotte et al. 1992
). Pyridoxal 5'-phosphate can
inhibit the binding of glucocorticoids to hormone receptors as well as
the binding of the activated receptor to DNA (Tully et al. 1994
). In addition, pyridoxal kinase activity in rats was
higher in the adrenals than in other tissues examined (Coburn et al. 1981
). These observations indicated the potential for
interactions among stress, vitamin B-6 and adrenal function. The data
reported here demonstrate that adrenocorticotropic hormone (ACTH),
which, like stress, increases cortisol production, altered B-6 vitamer
distribution and the activity of pyridoxal 5'-phosphate phosphatase in
swine adrenal glands. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.
Preliminary experiments were conducted to determine the ACTH
dosage schedule required to produce a sustained elevation in plasma and
salivary cortisol (data not shown). Yorkshire/Landrace barrows
(n = 12) averaging 90 kg were individually penned with
free access to water and a commercial pig grower diet containing the
equivalent of 5 mg pyridoxine-HCl/kg as measured by Covance
Laboratories (Madison, WI). Salivary samples (Cook et al. 1996
) were obtained for measurement of baseline salivary
cortisol concentrations of unstressed pigs (Cook et al. 1997
). Blood samples were obtained by jugular venipuncture for
pretreatment vitamin B-6 analysis.
Ear vein catheters were inserted (Schaefer et al. 1987
) 3 d before the beginning of the experiment to allow
ample time for cortisol concentrations to reach baseline values. Then
pigs (n = 6) received 50 IU of a
long-acting form of porcine ACTH (Bexco ACTH Purified Cortrophin,
Bexco Pharma, Mississauga, Canada) intravenously at 3-h intervals from
0800 to 2100 h for 3 d. The remaining pigs (n
= 6) received an equivalent volume of physiological saline at the
same times. Catheters were flushed after each dose with 10 mL saline
containing 1000 IU sodium heparin/L. Due to failure of some
of the catheters, all were removed at 0800 on d 4. Pigs were not
treated on d 4 and 5. On d 6 and 7 the ACTH-treated pigs received
100 IU ACTH intramuscularly at 0800, 1400 and 2000 h.
Saliva samples were collected at 3-h intervals before ACTH
administration from 0800 to 2100 h for cortisol analysis. All pigs
were slaughtered following standard commercial procedures at 0800 h on d 8 at the Meat Laboratory of the Lacombe Research Center. No meat
or organs were sold for food use. Heparinized blood was collected at
the time of slaughter. Adrenal glands and liver were weighed. Adrenal
glands and samples of longissimus muscle and liver were immediately
frozen in liquid nitrogen and stored at -80°C. Brains were placed on
ice and frozen at -80°C. The lining of the stomach was evaluated for
stomach ulcers (common in stressed pigs) using a 3-point scale (1
= none, 2 = mild, 3 = severe).
B-6 vitamers in plasma and tissues were determined by
cation-exchange HPLC (Mahuren and Coburn 1990
).
Tissues were homogenized in 10 vol of trichloroacetic acid (40 g/L) for
45 s at 20,000 rpm (Model 60K, Virtis, Gardiner, NY).
Enzymatic activity was determined using a modification of the
method of Ubbink and Schnell (1988)
. Tissues were
homogenized in 10 vol of cold 0.004–0.02 mol/L potassium phosphate
buffer, pH 7, for 30–40 s at 20,000 rpm. For the phosphatase and
kinase assays, the incubation mixture consisted of 400
µL buffer, 50 µL homogenate diluted
appropriately, and 50 µL of 2 mmol/L substrate
(pyridoxal 5'-phosphate for phosphatase or pyridoxal for kinase).
Buffers for the phosphatase assay at various pH were as follows: pH 5,
0.1 mol/L acetate containing 50 mmol/L magnesium chloride; pH 7.4, 0.05
mol/L triethanolamine containing 5 mmol/L magnesium chloride; and pH
10, 0.1 mol/L Tris containing 50 mmol/L magnesium chloride. The kinase
buffer was 0.1 mol/L potassium phosphate, pH 6, containing 2 mmol/L
magnesium chloride, 0.2 mmol/L zinc chloride and 2 mmol/L ATP. After
incubation at 30°C for 20 (phosphatase) to 60 min (kinase), the
reaction was stopped by adding 1.0 mL trichloroacetic acid (50 g/L).
The contents were mixed and centrifuged for 10 min at
1000 x g. The supernatant was removed and shaken
with an equal volume of peroxide free diethyl ether for 1 min. After
centrifugation for 6 min at 1000 x g, the ether was
aspirated and the remaining solution was analyzed by cation exchange
HPLC (Mahuren and Coburn 1990
).
Data on organ weights, dressing percentage and salivary cortisol were analyzed using the general linear model of SAS (SAS Institute, Cary, NC). B-6 vitamers and phosphatase activity in tissues of the control group were compared with the ACTH group with a t test for normally distributed data or the Mann-Whitney rank-sum test (SigmaStat, Jandel Scientific, San Rafael, CA). Data for plasma vitamers were analyzed using Tukey's test after two-way (time and treatment) repeated-measures ANOVA (SigmaStat, Jandel Scientific). Because pretreatment blood samples were not obtained from three of the six saline-treated pigs, only the three pigs with complete data were included in the two-way repeated-measures ANOVA of the plasma data.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Compared with the controls, pigs treated with ACTH had significantly
lower pyridoxamine 5'-phosphate and significantly greater pyridoxal and
pyridoxamine concentrations in adrenal tissue(Table 1
). This could result from increased hydrolysis or decreased synthesis.
Therefore, we measured pyridoxal 5'-phosphate phosphatase activity.
Baseline activity at pH 7.4 was highest in brain followed by adrenal
and liver (Table 2
). Activity in the adrenals was ~ninefold greater in pigs treated with
ACTH than in controls. Although activity at both pH 5 and pH 10 was
significantly greater due to ACTH, the increase of ~12
nmol/(g · min) at pH 5 was only slightly >1% of the increase at
pH 10. The activity of alkaline phosphatase measured at pH 5 is
typically about 1% of the activity measured at pH 10. Therefore, we
attribute the increased activity at pH 5.0, 7.4 and 10.0 to alkaline
phosphatase.
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were obtained using a substrate
concentration of 0.2 mmol/L, which is optimal at pH 7.4. Although the
results confirmed that the increased activity was due to alkaline
rather than acid phosphatase, the activity at pH 10 would have been
even higher if a higher substrate concentration had been used.
Pyridoxamine 5'-phosphate was 30% greater in muscle of pigs
administered ACTH than in controls (Table 1)
. Further studies will be
required to confirm whether this is a reproducible effect of ACTH. ACTH
had no significant effect on B-6 vitamers in plasma (Fig. 1
). However, compared with baseline values, pyridoxal in plasma was
significantly reduced in both the control and ACTH groups at the end of
the 7-d experiment, and pyridoxal 5'-phosphate (P = 0.109) and pyridoxic acid (P = 0.062) in plasma tended
to increase in both groups. The explanation for this is uncertain but
might reflect inadvertent differences in feeding times or sample
handling.
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). ACTH had no significant effect on kinase activity except in liver
where activity was reduced ~50% (P = 0.009) after
ACTH treatment.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Salivary cortisol concentrations in the control group were similar to
those previously reported for unstressed pigs (Cook et al. 1996
). Values in the ACTH-treated pigs were comparable to
those found in pigs stressed by 1 h of transport (Cook et al. 1996
).
McComb et al. (1979)
summarized alkaline phosphatase
data in one individual from several species. The interspecies
differences for liver or kidney were ~fivefold, whereas the activity
in adrenal tissue varied 20-fold. This large variation could reflect
differences in hormonal status and stress as well as basic interspecies
differences. Adrenal alkaline phosphatase activity was higher than
liver activity in five of the seven species and was higher than kidney
in three species. In humans, adrenal alkaline phosphatase activity was
more than twice as high as that in any tissue other than placenta
(Bowers and McComb 1975
).
The limited data available concerning the effects of ACTH on adrenal
phosphatase activity are primarily histologic and yielded conflicting
results. At least part of the conflict may be explained by the fact
that in rats treated with ACTH, alkaline phosphatase in the adrenal
cortex declined sharply during the first 3 h, returned by 6 h
and declined again by 24 h (Arezio et al. 1957
).
Therefore, variations in the sampling schedule could result in varying
conclusions. Arezio et al. (1957)
concluded that in view
of the marked changes in activity after treatment of rats with ACTH,
acid and alkaline phosphatase were the chief indicators of the response
of the adrenals to stress. In one human subject who received 100 mg
ACTH/d for 3 d before adrenalectomy, no changes in alkaline
phosphatase activity of the adrenal cortex were detected
histochemically (Dawson et al. 1961
). Nicander (1952)
reported that the adrenal glands of pigs showed wide
individual variation in the intensity of staining for alkaline
phosphatase. The large increase in alkaline phosphatase due to ACTH
found in this study suggests that the variations observed by
Nicander (1952)
might reflect variations in the
concentration of ACTH and/or stress.
Although the changes in vitamin B-6 concentrations reported here could be postmortem changes due to the increased phosphatase activity, the high activities of pyridoxal kinase and pyridoxal 5'-phosphate phosphatase suggest that a special metabolic situation exists for vitamin B-6 in adrenal tissue. The high kinase activity may be required simply to minimize the effect of fluctuations in phosphatase activity on the concentrations of pyridoxal 5'-phosphate and pyridoxamine 5'-phosphate. Alternatively, vitamin B-6 may have a role in the production of steroid hormones in addition to its interaction with hormone receptors. Further studies are warranted to explore these possibilities.
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FOOTNOTES |
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2 Supported in part by grants from the U.S.
Department of Agriculture/NRICGP (#95–37200–1703), AAFC/MII and the
Alberta Pork Producer's Development Corporation.
Manuscript received June 10, 1999. Revision accepted July 8, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1. Arezio G., Antonini R., Lungarotti F. Histochemistry of the adrenal gland under hypophyseal stimulation. Minerva Chir 1957;12:209-215[Medline]
2. Bowers G. N., McComb R. B. Measurement of total alkaline phosphatase activity in human serum. Clin. Chem. 1975;21:1988-1995[Medline]
3. Bradshaw R. H., Parrott R. F., Forsling M. L., Goode A., Lloyd D. M., Rodway R. G., Broom D. M. Stress and travel sickness in pigs: effects of road transport on plasma concentrations of cortisol, beta-endorphin and lysine vasopressin. Anim. Sci. 1996;63:507-516
4. Canadian Council on Animal Care Guidelines for Care and Management of Animals Used in Research 2nd ed. 1993 Canadian Council on Animal Care Ottawa, Canada.
5. Coburn S. P., Mahuren J. D., Schaltenbrand W. E., Wostmann B. S., Madsen D. Effects of vitamin B-6 deficiency and 4'-deoxypyridoxine on pyridoxal phosphate concentrations, pyridoxine kinase and other aspects of metabolism in the rat. J. Nutr. 1981;111:391-398
6. Cook N. J., Schaefer A. L., Lepage P., Jones S.D.M. Salivary vs. serum cortisol for the assessment of adrenal activity in swine. Can. J. Anim. Sci. 1996;76:329-335
7. Cook N. J., Schaefer A. L., Lepage P., Jones S.D.M. Radioimmunoassay for cortisol in pig saliva and serum. J. Agric. Food Chem. 1997;45:395-399
8. Dawson I.M.P., Pryse-Davies J., Snape I. M. The distribution of six enzyme systems and of lipide in the human and rat adrenal cortex before and after administration of steroid and of ACTH with comments on the distribution in human fetuses and in some natural disease conditions. J. Pathol. Bacteriol. 1961;81:181-195[Medline]
9. Henrotte J. G., Franck G., Santarromana M., Nakib S., Dauchy F., Boulu R. G. Effect of pyridoxine on mice gastric ulcers and brain catecholamines after an immobilization stress. Ann. Nutr. Metab. 1992;36:313-317[Medline]
10. Mahuren J. D., Coburn S. P. B-6 vitamers: cation exchange HPLC. J. Nutr. Biochem. 1990;1:659-663
11. McComb R. B., Bowers G. N., Posen S. Alkaline Phosphatase 1979:268-269 Plenum New York, NY.
12. Nicander L. Histological and histochemical studies on the adrenal cortex of domestic and laboratory animals. Acta Anat 1952;14(suppl. 16):1-88
13. Schaefer A. L., Doomenbal H., Tong A.K.W., Murray A. C., Sather A. P. Effect of time off feed on blood acid-base homeostasis in pigs differing in their reaction to halothane. Can. J. Anim. Sci. 1987;67:427-436
14. Tully D. B., Allgood V. E., Cidlowski J. A. Modulation of steroid receptor-mediated gene expression by vitamin B6. FASEB J 1994;8:343-349[Abstract]
15. Ubbink J. B., Schnell A. M. High performance liquid chromatographic assay of erythrocyte enzyme activity levels involved in vitamin B-6 metabolism. J. Chromatogr. 1988;431:406-412[Medline]
16. Warriss P. Marketing losses caused by fasting and transport during the preslaughter handling of pigs. Pig News Inform 1985;6:155-157
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