QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Ching, S. Articles by Dabrowski, K. Search for Related Content PubMed PubMed Citation Articles by Ching, S. Articles by Dabrowski, K.

---

Articles

Liver L-Gulonolactone Oxidase Activity and Tissue Ascorbic Acid Concentrations in Nursing Pigs and the Effect of Various Weaning Ages^{1 ,2}

San Ching^*, Donald C. Mahan^*3 and Konrad Dabrowski

^* Department of Animal Sciences, School of Natural Resources, The Ohio State University and The Ohio Agricultural Research and Development Center, Columbus, OH 43210-1094

³To whom correspondence should be addressed. E-mail: mahan.3{at}osu.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In Experiment 1, we evaluated liver L-gulono--lactone oxidase (GLO) activity and tissue concentration of ascorbic acid in young pigs from birth to weaning (14 d) and through a 28-d postweaning period; in Experiment 2, we evaluated the effect of three weaning ages on these measurements. Sow colostrum and milk collected in both experiments demonstrated a linear decline (P < 0.01) in ascorbic acid concentration as lactation progressed. In Experiment 1, three pigs were killed at 0, 3, 7, 14, 21, 28, 35 and 42 d of age for determining liver GLO activity and serum and tissue ascorbic acid. Liver GLO activity decreased by 80% from 0 to 3 d of age and remained low until d 14 (weaning). After weaning, liver GLO activity increased linearly (P < 0.01). Tissue ascorbic acid concentrations decreased during the nursing period and again after weaning, but then increased to 42 d of age (P < 0.01). In Experiment 2, pigs were weaned at 10, 17 or 24 d of age. Three pigs from each group were killed at weaning and at each week postweaning until 38 d of age. Liver GLO activity was low during the nursing period but increased linearly (P < 0.01) for each group during the subsequent postweaning period. Pig serum and tissue ascorbic acid concentrations increased postweaning in each group. These results suggest that a factor in sow’s milk, possibly ascorbic acid, suppressed liver GLO activity of nursing pigs but upon weaning, liver GLO activity of pigs increased in a linear manner (P < 0.01).

KEY WORDS: • L-gulonolactone oxidase • ascorbic acid • nursing • weaning • pigs

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
L-Gulono--lactone oxidase [EC 1.1.3.8] (GLO) is present in the microsomes of liver cells and is responsible for the biosynthesis of ascorbic acid. The enzyme converts L-gulono--lactone to L-keto-gulono--lactone, whereupon L-ascorbic acid is produced through isomerization (1 ,2) . The enzyme is found in species that synthesize ascorbic acid but not in those that cannot synthesize the vitamin (3) .

Our previous study demonstrated high liver GLO activity in fetal pigs at mid-gestation (60 d) but the activity declined by 70% to birth (4) . In contrast, fetal liver ascorbic acid concentration increased by 12-fold during this period, suggesting that an increasing amount of ascorbic acid was transferred from the dam as gestation progressed. Consequently, fetal ascorbic acid synthesis appears to be the primary source of the vitamin during early development; during the latter gestation period, however, the maternal source becomes the major supplier of the vitamin to the fetus.

When Tsao and Young (5) supplemented the diets of mice with 0.5, 1 or 5% L-ascorbic acid, they reported that serum and liver ascorbic acid concentrations increased as the dietary level increased, but liver GLO activity declined. This suggests that a high concentration of ascorbic acid may suppress liver GLO activity. Our previous report also implied that maternal ascorbic acid transferred to the fetus during the latter part of pregnancy may also have suppressed fetal GLO enzyme activity (4) .

Sow colostrum (and later milk) supplies a large quantity of ascorbic acid to nursing pigs (5 ,6) . When Braude et al. (7) reported that the serum and urinary excretion of ascorbic acid of 1-wk-old weanling pigs increased during the initial week postweaning, they assumed that ascorbic acid synthesis began postnatally in all pigs. Their study used weanling pigs that were fed diets without supplemental ascorbic acid. It is possible that the ascorbic acid from the dams’ milk may have suppressed GLO activity in nursing pigs but upon weaning, the pigs met their metabolic requirement through increased GLO activity and thus increased tissue ascorbic acid concentration.

Our experiments were designed to evaluate liver GLO activity in pigs from birth to 7 wk of age under nursing and postweaning conditions, and to evaluate the effects of three weaning ages on subsequent postweaning liver GLO activity and tissue ascorbic acid concentrations.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Experiment 1.

In Experiment 1, we evaluated liver ascorbic acid synthesis and ascorbic acid status, as measured by liver GLO activity and tissue ascorbic acid concentrations, in pigs from birth through 14 d nursing followed by a 28-d postweaning period. A total of 24 crossbred pigs ([Landrace x Yorkshire] x Duroc) were selected from the litters of four sows from the Ohio State University swine research herd (Columbus, OH) and used in this study. The ascorbic acid measurements were evaluated in three pigs each at 0, 3, 7, 14, 21, 28, 35 and 42 d of age. The three neonatal pigs that were selected for killing were separated from their dams at birth to prevent colostrum consumption, and placed in a warmed container (0.5 x 1.0 m) to prevent chilling. Three additional pigs at each subsequent collection period were selected from the available litters with the restriction that only one pig could be selected per litter. The study was conducted as a completely randomized design in three replicates.

Blood was obtained from each selected pig by cardiac puncture using a vacuum tube. The blood was placed on ice and transported to the laboratory for further processing. The selected pigs were electrically stunned and killed by exsanguination, and tissue samples (liver, kidney, spleen, adrenal gland and loin) were collected for the determination of ascorbic acid concentrations. Samples were frozen in liquid N, and stored at -20°C. Liver samples (2 g) for determining GLO activities were also collected and placed in sealed Eppendorph tubes, frozen in liquid N and stored at -80°C.

Upon weaning (14 d), the pigs were fed typical starter diets formulated to 1.60% lysine (total) for the initial 14-d period postweaning, and a 1.30% lysine (total) level during the subsequent 28-d period. Weanling pig diets contained no supplemental ascorbic acid. Subsequent analysis of diets for the indigenous ascorbic acid content showed no detectable vitamin C. Other dietary nutrients met or exceeded NRC (8) standards with diet compositions presented in Table 1 . Grains and feed ingredients used in the experiments were obtained and mixed at the research feed mixing facility (Wooster, OH). Weaned pigs were housed in groups of four in 1.2 x 1.2 m² plastic-coated expanded metal-floored pens that contained a four-hole feeder and one nipple waterer at the Ohio State University swine research farm (Columbus, OH). Pig management and procedures used for tissue collections were approved by the university animal care committee.

This study evaluated the effects of three weaning ages on the pigs ability to synthesize ascorbic acid postweaning. Pigs randomly selected from five litters were weaned at 10, 17 and 24 d of age. Because the experiment terminated at a common age (i.e., 38 d) and more pigs were to be used from the earlier weaning age, 20 pigs were weaned at 10 d, 16 pigs at 17 d and 12 pigs at 24 d of age. Each group contained a slightly larger pool of pigs than was needed for the experiment to ensure the selection of normal pigs. Three pigs from within each treatment group were subsequently selected for killing at the different measurement periods such that pigs were of a normal weight for their age and from different litters. The selected pigs (n = 36) were bled, killed, and tissues collected and stored as in Experiment 1. The experiment was conducted as an incomplete 3 x 5 factorial arrangement in a completely randomized design in three replicates.

The genetic composition, feeding practices, management and facilities used, and the use of approved procedures for the weaned pigs were similar to those of Experiment 1. The first starter diet fed to the pigs weaned at 10 d of age was formulated to a 1.75% (total) lysine level and was provided until 17 d of age. The diet for pigs weaned at both 10 and 17 d of age was formulated to 1.60% lysine (total) and was fed from d 17 to 24. All pigs from 24 to 42 d of age were fed a diet containing 1.40% lysine (total) to the end of the experiment. Diets were fed in meal form and met or exceeded NRC (8) recommendations with the compositions presented in Table 1 .

Analytical procedures.

The GLO activity procedure used followed that outlined by Dabrowski (9) and later modified for swine tissue by Ching (10) . Analysis of liver GLO activity was completed within 30 d of collection. Ascorbic acid concentrations in serum, tissue and feed were determined by the dinitrophenyl-hydrazine method (11) .

Blood was centrifuged at 2200 x g for 15 min at 2°C. The collected plasma was immediately mixed with a solution (100 g/L trichloroacetic acid, 250 mmol/L perchloric acid and 3 mmol/L EDTA) in a 1:1 ratio to precipitate plasma protein. The plasma was again centrifuged at 2200 x g for 30 min at 2°C and the supernatant stored at -20°C for later ascorbic acid analysis.

The data from the first experiment were analyzed as a completely randomized design (12) following the General Linear Model procedure of SAS (13) . The second experiment was analyzed as an incomplete 3 x 5 factorial in a completely randomized design. Treatments were contrasted by regression analysis for both experiments with P < 0.05 reported. The individual pig was considered the experimental unit in both experiments.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Sow colostrum and milk ascorbic acid.

Sow colostrum (0–2 d postpartum) had substantially higher ascorbic aid concentrations than the milk secreted during the 7–24 d postpartum period (Table 2 ). A linear (P < 0.01) decline in ascorbic acid concentrations thus occurred during the lactation period in Experiment 1. This suggests that young pigs initially consume an ample supply of the vitamin during the nursing period, but its relative concentration in milk declines with advancing lactation.

In Experiment 1, pig liver GLO activity was highest at birth but declined by 80% by 3 d of age, whereupon its activity remained relatively constant until weaning (14 d, Table 3 ). Upon weaning and during the period from 14 to 35 d of age, liver GLO activity increased by approximately threefold, indicating an increase in ascorbic acid synthesis. This resulted in an overall cubic response (P < 0.01) from birth to 42 d of age.

Ascorbic acid concentration in pig liver declined by 20% from birth to 3 d of age and by another 20% by 14 d of age. The ascorbic acid concentration in the liver continued its decline during the initial 7-d postweaning period whereupon its concentration increased to 42 d of age. This resulted in an overall cubic response (P < 0.01) from birth to 42 d of age. The increase during the postweaning period from 21 to 42 d of age likely was the result of increasing liver synthesis of the vitamin by the weaned pigs.

Loin ascorbic acid concentration was relatively constant from birth to 14 d of age but upon weaning, the concentration of ascorbic acid in this tissue declined to 28 d of age, whereupon it increased slightly by 42 d of age. This resulted in an overall quadratic response (P < 0.01). While the pigs were nursing, spleen ascorbic acid concentration was relatively constant from birth to 28 d of age, but after 28 d of age, splenic ascorbic acid concentration increased to 42 d, resulting in an overall quadratic response (P < 0.01).

The adrenal gland had the highest ascorbic acid concentration of the various tissues evaluated. Its highest concentration was at birth whereupon there was an 30% decline by 3 d of age. From 3 to 14 d of age, adrenal ascorbic acid concentrations were relatively constant. After the pigs were weaned, ascorbic acid concentration declined slightly during the initial week postweaning followed by an increase to 42 d of age.

In contrast to the other tissues evaluated, kidney ascorbic acid concentration was low at birth, but increased during nursing until weaning (14 d). After pigs were weaned, the ascorbic acid concentration in the kidney declined by >50% but then increased to 42 d of age, resulting in an overall cubic response (P < 0.01). Because ascorbic acid excretion occurs from the kidney, the fluctuating concentration in the kidney tissue would be expected. The increased excretion during the postweaning period is attributed to the increasing ascorbic acid synthesis and the subsequent excretion of excesses.

In Experiment 2, the liver GLO of each treatment group had its lowest activity at the time of weaning (10, 17 or 24 d), whereupon the GLO activities of each weaning group increased linearly during the postweaning period (P < 0.01, Table 4 ). Liver GLO activity responded more quickly postweaning in pigs weaned at a later age, resulting in a weaning age x time postweaning interaction (P < 0.01). When GLO activities were evaluated for the 0–14 d postweaning period, they were four- and sixfold higher at 7 and 14 d postweaning, respectively, than at weaning. These responses are consistent with the results of Experiment 1 in which liver GLO activity was lower while pigs nursed the dams but upon weaning, its activity increased in a linear manner.

Liver ascorbic acid concentration declined markedly at weaning for each weaning age group and remained low for at least 14 d postweaning, particularly for those weaned at 10 and 17 d of age (Table 4) . This resulted in a quadratic response from weaning to the 38-d period (P < 0.05). Ascorbic acid concentration in the loin and kidney each declined (P < 0.01) upon weaning and remained low to 38 d of age. Adrenal gland ascorbic acid concentration also declined quadratically (P < 0.05) upon weaning and remained low to 38 d for each weaning group.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Ascorbic acid in sow’s milk was high at the time of parturition and declined as lactation progressed, responses that are consistent with other reports (6 ,14) . Other research has also demonstrated that both sow colostrum and milk have higher ascorbic acid concentration than those of ewes, mares, cows, goats and rats (7) . No benefit, however, was realized in pigs when supplemental ascorbic acid was fed to pregnant and lactating sows (15) , suggesting that an adequate supply of ascorbic acid was provided to the nursing pigs from the milk supply.

Both pig serum and sow’s milk ascorbic acid concentrations declined during the lactation period of our experiments. The declining milk ascorbic acid concentrations may not, however, necessarily reflect a lower quantitative consumption of ascorbic acid by young pigs because milk production normally increases during this time frame. The declining tissue ascorbic acid concentrations in young pigs could also be in response to the declining liver GLO activity in pigs during the nursing period.

In our studies, pig liver GLO activity was higher at birth than at the later postnatal nursing periods. Our previous report (4) , however, demonstrated that liver GLO activity, which had been declining during gestation, had a relatively low activity at the time of birth. Young pigs appear to have lower liver GLO activity than goats, cows, sheep and rats (16) . Our experiments indicate that liver GLO activity continues to decline during the nursing period, and that its activity remained low when the maternal source of ascorbic acid was consumed. The lower pig liver GLO activity and its continual decline during nursing suggests that the enzyme’s activity may have been suppressed by some factor in sow’s milk. Although we did not evaluate the specific effect of ascorbic acid in sow’s milk and its effect on liver GLO activity, Tsao and Young (5) demonstrated previously that liver GLO activity was reduced in mice fed high levels of ascorbic acid. Our results imply that this phenomenon may also have occurred in young pigs during the lactation period. The high ascorbic acid concentration in colostrum and milk may thus have suppressed liver GLO activity in the nursing pigs but once this source was eliminated by weaning, liver GLO activity increased.

Liver GLO activity clearly increased (P < 0.01) during the initial weeks postweaning. Pig liver GLO activity increased more rapidly postweaning in pigs weaned later. The pigs weaned earlier (10 d) had a slower rate of GLO activity increase during the initial 14-d postweaning period than those weaned later (17 or 24 d). The reason for the higher rates for later weaned pigs is currently unclear, but may relate to the lower concentration of ascorbic acid or possibly other factors being supplied from the milk of the later weaned pigs. Tsao and Young (5) suggested that hepatic portal ascorbic acid concentration or a liver regulatory mechanism may control the synthesis of ascorbic acid.

After pigs were weaned, there was clearly an increase in liver GLO activity and a subsequent increase in tissue ascorbic acid concentrations. The resulting liver ascorbic acid concentration, however, was below that of pigs nursing the dams. Serum and tissue ascorbic acid concentrations thus declined dramatically postweaning, undoubtedly reflecting the loss of the exogenous source that was supplied from the dams’ milk and the low liver GLO activity at weaning. Within 7–14 d postweaning, the serum concentration of ascorbic acid increased in all weaning groups but increased more rapidly in pigs weaned at a later age. Wegger and Palludan (15) also demonstrated that plasma ascorbic acid concentration decreased after weaning and remained low for at least 15 wk.

The postweaning serum and tissue ascorbic acid concentrations of later weaned pigs increased more quickly than that of early weaned pigs (weaning age x time postweaning interaction response, P < 0.01). This response is consistent with their more rapid increase in liver GLO activity. The older pigs (24 d of age) had lower serum and tissue ascorbic acid concentrations at weaning and a lower tissue concentration of ascorbic acid during the initial week postweaning compared with pigs weaned at 10 or 17 d. The more rapid increase of liver GLO activity of the later weaned pigs may have been the result of less suppression of the GLO enzyme because of their lower ascorbic acid status at the time of weaning.

In our experiments, the adrenal gland had a declining ascorbic acid concentration after pigs were born and again when weaned, more so than the other tissues evaluated. This may have been in response to various birth and weaning stresses. Ascorbic acid depletion has been shown to be greater in the adrenal gland during stressful situations (17) . Kornegay et al. (18) demonstrated that pigs housed in a cold (18°C) nursery had a lower ascorbic acid status. Spleen ascorbic acid, however, was constant during the nursing and postweaning periods.

Braude et al. (7) reported previously that pigs begin to synthesize ascorbic acid by 1 wk of age. Our results, however, suggest that ascorbic acid synthesis is stimulated by weaning or the removal of the milk source from the pigs diet, not by the age of the pigs. In the experiment of Braude et al. (7) , they had in fact weaned their pigs shortly after birth, which probably stimulated the GLO activity in these very young pigs. This resulted in elevated serum and urinary ascorbic acid concentrations. They interpreted the higher tissue concentration of ascorbic acid and the increased excretion of ascorbic acid to be the period of time in which all pigs begin to synthesize the vitamin.

In conclusion, these results demonstrate that liver GLO activity declines from birth and remains low during the nursing period. We contend that this activity is suppressed in young nursing pigs by the presence of ascorbic acid or another factor in colostrum and milk. In contrast, upon weaning, liver GLO is stimulated to produce ascorbic acid with those pigs weaned later showing a more rapid increase in GLO activity. The ability of pigs to provide adequate ascorbic acid synthesis postweaning to meet metabolic needs may be dependent upon management stress factors.

	ACKNOWLEDGMENTS

 
Appreciation is expressed to K. Mays for animal care and farm data collection, F. Cihla and M. Watts for laboratory assistance, and B. Bishop for statistical analysis.

	FOOTNOTES

 
¹ Supported by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University.

² Supported in part by Hoffman La-Roche, Paramus, NJ.

Manuscript received December 18, 2000. Initial review completed February 4, 2000. Revision accepted March 30, 2001.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Burns J. J., Mosbach E. H. Further observations on the biosynthesis of L-ascorbic acid from D-glucose in the rat. J. Biol. Chem. 1956;227:107-111

2. Chatterjee I. B., Ghosh J. J., Ghosh N. C., Guha B. C. Effect of cyanide on the biosynthesis of ascorbic acid by an enzyme preparation from goat-liver tissue. Biochem. J. 1958;70:509-515[Medline]

3. Chatterjee I. B. Evolution and the biosynthesis of ascorbic acid. Science (Washington, DC) 1973;182:1271-1272[Abstract/Free Full Text]

4. Ching S., Mahan D. C., Ottobre J. S., Dabrowski K. Ascorbic acid synthesis in fetal and neonatal pigs and in pregnant and postpartum sows. J. Nutr. 2001;131:1997-2001[Abstract/Free Full Text]

5. Tsao C. S., Young M. Effect of exogenous ascorbic acid intake on biosynthesis of ascorbic acid in mice. Life Sci 1989;45:1553-1557[Medline]

6. Braude R., Coates M. E., Henry K. M., Kon S. K., Rowland S. J., Thompson S. Y., Walker D. M. A study of the composition of sow’s milk. Br. J. Nutr. 1947;1:64-77

7. Braude R., Kon S. K., Porter J.W.G. Studies in the vitamin C metabolism of the pig. Br. J. Nutr. 1950;4:186-199[Medline]

8. National Research Council Nutrient Requirements of Swine 10th rev. ed. 1998 National Academy Press Washington, DC.

9. Dabrowski K. Gulonolactone oxidase is missing in Teleost fish. The direct spectrophotometric assay. Biol. Chem. Hoppe-Seyler 1990;37:207-214

10. Ching S. Ontogeny of Liver L-Gulonolactone Oxidase Activity and Tissue Ascorbic Acid Concentration in Fetal, Nursing, and Weaned Pigs, and Factors Influencing Its Synthesis 2000 The Ohio State University Columbus, OH. Doctoral thesis,

11. Dabrowski K., Hinterleitner S. Applications of a simultaneous assay of ascorbic acid, dehydroascorbic acid and ascorbic sulphate in biological materials. Analyst 1989;114:83-87[Medline]

12. Steel R. G., Torrie J. H. Principles and Procedures of Statistics 1980 McGraw-Hill New York, NY.

13. SAS Institute Inc SAS User’s Guide: Statistics 5th ed. 1985 SAS Institute Cary, NC.

14. Wegger I., Palludan B. Ascorbic acid status of swine. Genetic and developmental variations. Wegger I. Tagwerker F. J. Moustgaard J. eds. Proceedings of Workshop on Ascorbic Acid in Domestic Animals 1984:68-79 Hoffmann-La Roche Basel, Switzerland.

15. Yen J. T., Pond W. G. Response of swine to periparturient vitamin C supplementation. J. Anim. Sci. 1983;56:621-624

16. Levine M. New concepts in the biology and biochemistry of ascorbic acid. N. Engl. J. Med. 1986;314:892-902[Medline]

17. Levine M., Morita K. Ascorbic acid in endocrine systems. Vitam. Horm. 1985;42:1-12[Medline]

18. Kornegay E. T., Meldrum J. B., Schurig G., Lindemann M. D., Gwazdauskas F. C. Lack of influence of nursery temperature on the response of weanling pigs to supplemental vitamin C and E. J. Anim. Sci. 1986;63:484-491

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Ching, S. Articles by Dabrowski, K. Search for Related Content PubMed PubMed Citation Articles by Ching, S. Articles by Dabrowski, K.