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Changes in Plasma Catecholamine, Free Fatty Acid, and Glucose Concentrations, and Plasma Monoamine Oxidase Activity Before and After Feeding in Laying Hens
PHYSIOLOGY AND REPRODUCTION Changes in Plasma Catecholamine, Free Fatty Acid, and Glucose Concentrations, and Plasma Monoamine Oxidase Activity Before and After Feeding in Laying Hens MASANORI FUJITA, MASAHIDE NISHIBORI, and SADAKI YAMAMOTO Department of Animal Science, Hiroshima University, Higashihiroshima, Hiroshima, 724, Japan
1992 Poultry Science 71:1067-1072
INTRODUCTION
and Newcomer, 1974, 1975). Thus, the level of plasma CA of the fowl would Plasma catecholamine (CA) concentra- provide useful information on its physiotion is closely related to glucose (Cramb et logical status; however, data on the level ah, 1982) and lipid (Campbell and Scanes, of plasma CA in laying hens are scanty 1985) metabolism. The secretion of CA is (Harvey et ah, 1986). Fasting is well elicited by physical and stressive stimuli recognized as a stress, resulting in the such as treadmill exercise (Rees et ah, increase of plasma concentration of free 1984), blood sampling (Rulofson et ah, fatty acids (FFA). In order to better 1988), and immobUization (Zachariasen understand the relationship of fatty acid concentrations to feed intake, plasma concentrations of epinephrine (E), norepinephrine (NE), FFA, and glucose Received for publication June 26, 1991. and plasma monoamine oxidase (MAO) Accepted for publication February 7, 1992. 1067
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ABSTRACT In order to better understand the relationship of free fatty acid (FFA) concentrations to feed intake in laying hens, plasma levels of catecholamine, glucose, and FFA and plasma monoamine oxidase activity were measured. Blood samples were taken in the morning before and after the start of feeding via chronic brachial vein catheters. Plasma concentrations of epinephrine and norepinephrine were analyzed by modified HPLC with an electrochemical detection method. Plasma concentrations of FFA and glucose were not significantly different before and after the start of feeding, however a negative correlation (r = -.763, r 2 = .582, P < .01) between these parameters was observed. No significant correlations between the plasma concentration of FFA and plasma concentrations of epinephrine or norepinephrine before and after the start of feeding were observed. However these were significantly correlated (r = .444, r 2 = .197, P < .05 for FFA and epinephrine; r = .787, r 2 = .619, P < .01 for FFA and norepinephrine) when the behavioral activity of hens was low, such as in resting or feeding. Plasma activity of monoamine oxidase was not different before and after the start of feeding, and no relationship was observed between plasma monoamine oxidase activity and plasma epinephrine or norepinephrine concentration. It is suggested that the increase in plasma FFA concentration before feeding would be elicited by the increase in the circulating epinephrine and norepinephrine concentrations. Plasma FFA concentration was not affected by the increase in circulating epinephrine and norepinephrine concentrations during active behavior such as pacing and egg call before and after oviposition. (Key words: laying hens, feed intake, catecholamine, free fatty acids, plasma monoamine oxidase)
1068
FUJITA ET AL.
activity were measured before and after the start of feeding in laying hens. Because of the quick, specific, and sensitive detection of CA, a modified HPLC with an electrochemical detection method (HPLCECD) was used to determine the plasma concentrations of E and NE in the present experiment.
MATERIALS AND METHODS
Four 6-mo-old commercial laying hens (Shaver Starcross 288) weighing 1.4 to 1.8 kg were used in the current study. Hens were housed individually in cages placed in a temperature-controlled chamber throughout the experimental period. The environmental temperature was kept at 20 C and commercial mash (ME = 2,870 kcal/kg; CP = 17%) and water were provided at 0900 h every morning. Light was provided from 0800 to 2000 h, and feed and water were available between 0900 and 2000 h. Four hens were sampled on 3 consecutive days. A catheter, made of silicon,1 was inserted into the vena cava to the depth of 2 cm from the brachial vein and attached with vinyl covering to the skin of each hen under anesthesia (50 m g / k g pentobarbital sodium 2 ). Cannulation was carried out 5 days before blood sampling; thereafter, hens were not handled during the experimental period. Each catheter was washed with physiological salt solution and filled with 1 % EDTA solution once or twice a day at the sampling time throughout the study. One milliliter of blood sample was collected via catheter at 0830 h before feeding and at 1100 h after the start of feeding. The average feed
Analysis of Plasma Epinephrine, Norepinephrine, Free Fatty Acid, and Glucose Concentrations and Plasma Monoamine Oxidase Activity
Each blood sample (1 mL) containing heparin was cooled immediately on ice and centrifuged at 2,300 x g for 15 min for plasma separation. One hundred microliters of .1M EDTA and 500 p g / 5 0 |iL of (3,4-dihydroxybenzylamine h y d r o bromide 3 (DHBA) were added to .5 mL of p l a s m a . F i v e m i l l i l i t e r s of 1 M CH 3 COONH4 (pH 9.5) was added to the solution to adjust the p H to 8.5. Fiftymilligram units of active alumina (ICN Alumina B-Super I 4 ), dried at 120 C for 2 h, were added to the solution. This mixture was shaken at 200 rpm for 10 min. The supernatant was removed by aspiration and the alumina was washed and vortexed for 30 s with 8 mL of .1 mM EDTA water twice in a test tube and in a column fitted with a glass filter. Alumina was separated from the EDTA water by filtration using glass filter with centrifugation at 750 x g for 2 min. In order to elute CA from the alumina compound, .5 mL of .4 N acetic acid was added. Ten minutes after the 5 min of vortexing, acid elute was separated by centrifugation at 2,000 rpm for 2 min. One hundred microliters of the elute and standard solution containing .1, .15, .25, and .5 n g / m L of E, NE, 5 and DHBA were used for T Dow Corning Silastic Medical Grade Tubing the determination of E and NE concentra602-155, .025 in. (.064 cm) inside diameter by .047 in. tions by HPLC-ECD analysis. These solu(.119 cm) outside diameter, Dow Corning Co., Mid- tions were chromatographed as follows. land, MI 48640. Separation of NE, E, and DHBA was 2 Abbot Laboratories, North Chicago, IL 60064. 3 Aldrich Chemical Co., Milwaukee, WI 53201. achieved using reversed-phase Octadecyl4 ICN Biomedical GmbH, Eschwege, D-3440, Ger- Silica 5 um column (TSK gel ODS-80TM 25 many. cm). 6 Column temperature was kept at 20 C (-)-Epinephrine bitartrate salt and (-)- by thermocontroller (TSK CO-8000).6 The norepinephrine bitartrate salt, Sigma Chemical Co., St. solvent delivery system (TSK CCPD) 6 conLouis, MO 63100. tained .1 M KH2PO4, 20 \iM Na 2 EDTA, 2.5 ^osoh Co., Tokyo, 107 Japan.
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Animal Treatment and Blood Sampling
amount eaten by the hens during 2 h was 37.3 ± 13.4 g, constituting 40.7 ± 15.6% of the total feed intake during the day. The time of oviposition of each hen and the behavior of the hens were observed through a small window of the chamber from 0800 h to 1130 h. The behavior of each hen during the sampling time was classified into resting, feeding, and active behavior such as pacing and egg call.
1069
PLASMA CATECHOLAMINE IN LAYING HENS
Chromatogram B
Chromatogram A
O u-i
u-i CM
o o
o
o CM
CM
CM
CM
o o
Time after injection (min)
o
Time after injection (min)
FIGURE 1. Chromatographic recordings over time (minutes) obtained from injection of 100 |iL of external standard containing 25 pg each norepinephrine (NE; 1), epinephrine (E; 2), and 3,4-dihydroxybenzylamine (DHBA; 0) (Chromatogram A); and injection of 100 uL of an extracted plasma sample containing 50 pg of internal standard, DHBA (Chromatogram B).
mM 1-octanesulfonic acid sodium salt (SOS), and 15% methanol. The pH of the buffer was adjusted to 3.5 with H3PO4. The buffer was filtered and degassed, then the flow rate was adjusted to .6 mL/min. The electrochemical detector7 was set at 650 mV, and peak heights were measured using computer integrator. All values were corrected for actual recovery based on the extraction rate of the internal standard DHBA. Plasma concentrations of FFA and
glucose and plasma MAO activity were determined using commercial kits.8 Statistical Analysis The Student's t test was used to determine differences between means. Analysis of covariance was also used for the determination of the statistical significance of each correlation. RESULTS
7
TSK EC-8000, Tosoh Co., Tokyo, 107 Japan; EC100 WE-3G, Eicom Co., Kyoto, 612 Japan. SNEFA C-Test Wako, Glucose C-test Wako, and MAO Test Wako, respectively, Wako Pure Chemical Industries Co., Osaka, 541 Japan.
Average percentage recovery rates ± SD of E, NE, and DHBA by alumina extraction were 89.7 ± 6.5, 93.2 ± 2.4, and 92.6 ± 4.5, respectively. Thus, small variations in recovery rates among E, NE, and DHBA
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on May 13, 2015
r\_—...I
1070
FUJTTA ET AL.
on
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I 10
20
30
40
50
60
Concentration (pg/mL) FIGURE 2. Standard curves far epinephrine and norepinephrine determined by HPLC with electrochemical detection. Each point and vertical or horizontal line indicate x ± SD of four measurements.
DISCUSSION were observed. Consequently, DHBA was used for the peak height correction of E and NE of each plasma extracted chromatograph. Chromatographs of unextracted standard solution and extracted plasma sample are shown in Figure 1. Peaks of E, NE, and DHBA were legible and independent in each chromatograph. Standard curves of E and NE for the determination of each concentration were linear from zero to below 50 p g / m L (Figure 2). The changes of plasma FFA, glucose, E, and NE concentrations and plasma MAO activity before and after the start of feeding are shown in Table 1 (P > .05). Owing to the tendency of decrease in plasma FFA and increase in glucose concentrations after feed supply, a significant linear regression
The technique of HPLC-ECD was used to determine plasma concentrations of E and NE in the present experiment because of the quick, specific, and sensitive detection of CA (Kissinger et al, 1973). Although the principle behind this method is well known (Kajiwara et al, 1981; Macdonald and Lake, 1985; Premel-Cabic and Allain, 1988) and has been applied for research in bulls (Rulofson et al, 1988), data on such analysis in fowls have not been reported. Because of the complex procedures in plasma preparation (Rulofson et al., 1988), several modifications were adopted with the aim of reducing the volume of plasma sample for analysis and also to simplify the procedures for plasma preparation. First of all, instead of a glassy carbon electrode, a graphite
TABLE 1. Changes in plasma epinephrine (E), norepinephrine (NE), free fatty acid (FFA), and glucose concentrations and plasma monoamine oxidase (MAO) activity before and after feeding in laying hens1'2 Time of measurement
E
NE
FFA
Glucose (mg/dL) 232.7 ± 11.4 249.6 ± 8.2
(meq/L) .37 ± .19 1.63 ± 1.44 .330 ± .174 1.67 ± .77 .138 ± .025 .33 ± .03 ^lood samples were obtained at 0830 h before feeding and at 1100 h after feeding. 2 Each value indicates the x ± SD of four hens (n = 4).
Before feeding After feeding
MAO (U/mL) 103.5 ± 43.7 111.4 ± 42.0
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on May 13, 2015
1)
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line and a significant correlation coefficient (Y = 2.2354 - .0083X, r = -.763, r 2 = .582, P < .01) were obtained between plasma FFA and glucose concentrations during the experimental period (Figure 3). N o clear relationships between plasma FFA and either E or NE concentrations were observed. However, the linear regression line and the correlation coefficient between plasma FFA concentration and plasma E (Y = .1407 + .4651X, r = .444, r 2 = .197, P < .05) or NE (Y = .0174 + .2864X, r = .787, r 2 = .619, P < .01) concentrations were significantly different from zero (Figure 4) when the behavioral activity of hens was low, such as in resting or feeding. No definite relationships were observed between plasma MAO activity and either plasma E or NE concentrations.
1071
PLASMA CATECHOLAMINE IN LAYING HENS .6
.b
•
.5
•
:
*A
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-
.2 •
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280
1.4
1.6
7
8
.5
FIGURE 3. Regression line and correlation between plasma free fatty acid (FFA) concentration and plasma glucose concentration before (A) and after (•) the start of feeding. Y = 2.2354 - .003X; r = -.763; r 2 = .582, where Y and X indicate plasma FFA and glucose concentrations, respectively. The linear regression line and correlation coefficient were statistically significant (P < .01).
Norepineprhine
2 electrode was used in order to improve sensitivity, and plasma samples were directly used for CA extraction by alumina. Owing to the high sensitivity and disposability of the electrode, low levels of E, NE, and DHBA, even less than 10 pg, were always easily detected. Nevertheless, this high sensitivity also detected many current peaks that originated from some plasma components that would provide electrons by electrolysis under high applied voltage. The data on the voltamogram indicate that current potential curves of E, NE, and DHBA became steady when the applied voltage was more than 600 mV (Kajiwara et ah, 1981); therefore applied voltage was lowered and set at 650 mV in order to reduce peak heights other than E, NE, and DHBA. Lastly, column temperature was lowered and set at 20 C in order to obtain clear separation of E, NE, and DHBA peaks from each other. These modifications resulted in legible chromatographs as shown in Figure 1, suggesting that the detection was very much improved. However, analysis under 20 C decreased the resolution of the column, making the refreshment of the column necessary.
1.2
.2
-
. 1
•'
1 2
3 4 5 6 Concentration (ng/mL)
FIGURE 4. Relationships between plasma free fatty acid (FFA) concentration and plasma epinephrine or norepinephrine concentration before ( • and A) and after (• and O) the start of feeding. Significant linear regression lines and correlation coefficients were observed between plasma FFA concentration and plasma epinephrine (Y = .1407 + .4651X, r = .444, r 2 = .197, P < .05) and norepinephrine (Y = .0174 + .2864X, r = .787, r 2 = .619, P < .01) concentration when the hens were resting or feeding (A and •).
A chronic catheter was used for bleeding in the current experiment, as suggested by Johnson (1981). A rapid increase in blood corticosterone concentration following venipuncture in the laying hen (Johnson, 1981) and a similar venipuncture effect on plasma concentrations of NE and E in bulls (Rulofson et al, 1988) have been reported. In addition the stimuli involved in catching hens and the presence of humans caused a significant increase in heart rate and pronounced behavioral responses 0ones et ah, 1981). These data suggested that great care should be taken during bleeding to determine blood CA
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210 220 230 240 250 260 270 Glucose concentration (mg/mL)
1072
FUJITA ET AL.
REFERENCES Campbell, R. M., and C. G. Scanes, 1985. Adrenergic control of lipogenesis and lipolysis in the chicken in vitro. Camp. Biochem. Physiol. 82c 137-142. Cramb, G., D. R. Langslow, and J. H. Phillips, 1982. Hormonal effects on cyclic nucleotides and carbohydrate and lipid metabolism in isolated chicken hepatocytes. Gen. Comp. Endocrinol. 46:310-321. Harvey, S., C. G. Scanes, and K. I. Brown, 1986. Adrenal medullary hormones. Pages 487-493 in: Avian Physiology 4th ed. P. D. Sturkie, ed. Springer-Verlag, New York, NY. Johnson, A. L., 1981. Comparison of three serial blood sampling techniques on plasma concentrations in the laying hen. Poultry Sci. 60: 2322-2327. Jones, R. B., I.J.H. Duncan, and B. O. Hughes, 1981. The assessment of fear in domestic hens exposed to a looming human stimulus. Behav. Proc. 6:121-133. Kajiwara, N., A. Murakami, C. Isozaki, T. Kita, and M. Ono, 1981. An estimation of plasma catecholamines by high performance liquid chromatography with electrochemical detection. Folia Endocrinol. Jpn. 57:1044-1059. Kissinger, P. T., C. Refshauge, R. Dreiling, and R. N. Adams, 1973. An electrochemical detector for liquid chromatography with picogram sensitivity. Anal. Lett. 6:465-477. Langslow, D. R., and C. N. Hales, 1969. Lipolysis in chicken adipose tissue in vitro. J. Endocrinol. 43: 285-294. Macdonald, I. A., and D. M. Lake, 1985. An improved technique for extracting catecholamines from body fluids. J. Neurosci. Methods 13239-248. Premel-Cabic, A., and P. Allain, 1988. Simultaneous liquid chromatographic determination of norepinephrine, 3,4-dihydroxyphenylethyleneglycol, 3,4-dihydroxyphenylalanine and 3,4-dihydroxyphenylacetic acid in human plasma. J. Chromatogr. 434:187-190. Rees, A., T. R. Hall, and S. Harvey, 1984. Adrenocortical and adrenomedullary responses of fowl to treadmill exercise. Gen. Comp. Endocrinol. 55: 488-492. Rulofson, F. C, D. E. Brown, and R. A. Bjur, 1988. Effect of blood sampling and shipment to slaughter on plasma catecholamine concentrations in bulls. J. Arum. Sci. 66:1223-1229. Zachariasen, R. D., and W. S. Newcomer, 1974. Phenylethanolamine-N-methyl transferase activity in the avian adrenal following immobilization or adrenocorticotropin. Gen. Comp. Endocrinol. 23:193-198. Zachariasen, R. D., and W. S. Newcomer, 1975. Influence of corticosterone on the stress-induced elevation of phenylethanolamine-N-methyl transferase in the avian adrenal. Gen. Comp. Endocrinol. 25:332-338.
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on May 13, 2015
concentration. Results in the present study on plasma concentration of E and NE were fairly similar with basal concentrations of circulating E and NE measured by radioenzymatic method (Rees et ah, 1984). Plasma concentrations of FFA and glucose before and after the start of feeding were not significantly different, however these were negatively correlated. Mobilization of FFA from adipose tissue in response to energy d e m a n d u n d e r hypoglycemic conditions such as fasting and before feeding should be stimulated by E (Langslow and Hales, 1969) via Padrenergic receptors and cyclic adenosine monophosphate (Campbell and Scanes, 1985). In fact, significant correlations were observed between the plasma FFA concentration and plasma E (P < .05) or NE (P < .01) concentrations when the behavioral activity of hens was low, such as in resting or feeding. The increase in CA concentration during active behavior before and after oviposition was not accompanied by an increase in FFA concentration. Accurate discussion on the increase in CA concentration during active behavior is difficult at present; however, such an increase in CA concentration was not accompanied by an increase in MAO activity, resulting in no definite relationship between plasma CA concentration and plasma MAO activity in the current study. Monoamine oxidase is one of the major enzymes in CA metabolism, therefore the level of MAO was expected to be related to plasma circulating CA. Plasma activity of MAO was not different before and after the start of feeding (Table 1). In this respect, it is suggested that the increase in CA concentration during active behavior would be transient. The effective level of circulating CA to cause lipolysis would be followed by the enough increase in MAO activity. However, it is quite difficult to explain these mechanisms at the present time because of the lack of the information on the physiological characteristics of MAO. In conclusion, basal levels of circulating E and NE, but not transiently elicited levels, would be closely related to the level of plasma concentration of FFA.
1992 Poultry Science 71:1067-1072
INTRODUCTION
and Newcomer, 1974, 1975). Thus, the level of plasma CA of the fowl would Plasma catecholamine (CA) concentra- provide useful information on its physiotion is closely related to glucose (Cramb et logical status; however, data on the level ah, 1982) and lipid (Campbell and Scanes, of plasma CA in laying hens are scanty 1985) metabolism. The secretion of CA is (Harvey et ah, 1986). Fasting is well elicited by physical and stressive stimuli recognized as a stress, resulting in the such as treadmill exercise (Rees et ah, increase of plasma concentration of free 1984), blood sampling (Rulofson et ah, fatty acids (FFA). In order to better 1988), and immobUization (Zachariasen understand the relationship of fatty acid concentrations to feed intake, plasma concentrations of epinephrine (E), norepinephrine (NE), FFA, and glucose Received for publication June 26, 1991. and plasma monoamine oxidase (MAO) Accepted for publication February 7, 1992. 1067
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on May 13, 2015
ABSTRACT In order to better understand the relationship of free fatty acid (FFA) concentrations to feed intake in laying hens, plasma levels of catecholamine, glucose, and FFA and plasma monoamine oxidase activity were measured. Blood samples were taken in the morning before and after the start of feeding via chronic brachial vein catheters. Plasma concentrations of epinephrine and norepinephrine were analyzed by modified HPLC with an electrochemical detection method. Plasma concentrations of FFA and glucose were not significantly different before and after the start of feeding, however a negative correlation (r = -.763, r 2 = .582, P < .01) between these parameters was observed. No significant correlations between the plasma concentration of FFA and plasma concentrations of epinephrine or norepinephrine before and after the start of feeding were observed. However these were significantly correlated (r = .444, r 2 = .197, P < .05 for FFA and epinephrine; r = .787, r 2 = .619, P < .01 for FFA and norepinephrine) when the behavioral activity of hens was low, such as in resting or feeding. Plasma activity of monoamine oxidase was not different before and after the start of feeding, and no relationship was observed between plasma monoamine oxidase activity and plasma epinephrine or norepinephrine concentration. It is suggested that the increase in plasma FFA concentration before feeding would be elicited by the increase in the circulating epinephrine and norepinephrine concentrations. Plasma FFA concentration was not affected by the increase in circulating epinephrine and norepinephrine concentrations during active behavior such as pacing and egg call before and after oviposition. (Key words: laying hens, feed intake, catecholamine, free fatty acids, plasma monoamine oxidase)
1068
FUJITA ET AL.
activity were measured before and after the start of feeding in laying hens. Because of the quick, specific, and sensitive detection of CA, a modified HPLC with an electrochemical detection method (HPLCECD) was used to determine the plasma concentrations of E and NE in the present experiment.
MATERIALS AND METHODS
Four 6-mo-old commercial laying hens (Shaver Starcross 288) weighing 1.4 to 1.8 kg were used in the current study. Hens were housed individually in cages placed in a temperature-controlled chamber throughout the experimental period. The environmental temperature was kept at 20 C and commercial mash (ME = 2,870 kcal/kg; CP = 17%) and water were provided at 0900 h every morning. Light was provided from 0800 to 2000 h, and feed and water were available between 0900 and 2000 h. Four hens were sampled on 3 consecutive days. A catheter, made of silicon,1 was inserted into the vena cava to the depth of 2 cm from the brachial vein and attached with vinyl covering to the skin of each hen under anesthesia (50 m g / k g pentobarbital sodium 2 ). Cannulation was carried out 5 days before blood sampling; thereafter, hens were not handled during the experimental period. Each catheter was washed with physiological salt solution and filled with 1 % EDTA solution once or twice a day at the sampling time throughout the study. One milliliter of blood sample was collected via catheter at 0830 h before feeding and at 1100 h after the start of feeding. The average feed
Analysis of Plasma Epinephrine, Norepinephrine, Free Fatty Acid, and Glucose Concentrations and Plasma Monoamine Oxidase Activity
Each blood sample (1 mL) containing heparin was cooled immediately on ice and centrifuged at 2,300 x g for 15 min for plasma separation. One hundred microliters of .1M EDTA and 500 p g / 5 0 |iL of (3,4-dihydroxybenzylamine h y d r o bromide 3 (DHBA) were added to .5 mL of p l a s m a . F i v e m i l l i l i t e r s of 1 M CH 3 COONH4 (pH 9.5) was added to the solution to adjust the p H to 8.5. Fiftymilligram units of active alumina (ICN Alumina B-Super I 4 ), dried at 120 C for 2 h, were added to the solution. This mixture was shaken at 200 rpm for 10 min. The supernatant was removed by aspiration and the alumina was washed and vortexed for 30 s with 8 mL of .1 mM EDTA water twice in a test tube and in a column fitted with a glass filter. Alumina was separated from the EDTA water by filtration using glass filter with centrifugation at 750 x g for 2 min. In order to elute CA from the alumina compound, .5 mL of .4 N acetic acid was added. Ten minutes after the 5 min of vortexing, acid elute was separated by centrifugation at 2,000 rpm for 2 min. One hundred microliters of the elute and standard solution containing .1, .15, .25, and .5 n g / m L of E, NE, 5 and DHBA were used for T Dow Corning Silastic Medical Grade Tubing the determination of E and NE concentra602-155, .025 in. (.064 cm) inside diameter by .047 in. tions by HPLC-ECD analysis. These solu(.119 cm) outside diameter, Dow Corning Co., Mid- tions were chromatographed as follows. land, MI 48640. Separation of NE, E, and DHBA was 2 Abbot Laboratories, North Chicago, IL 60064. 3 Aldrich Chemical Co., Milwaukee, WI 53201. achieved using reversed-phase Octadecyl4 ICN Biomedical GmbH, Eschwege, D-3440, Ger- Silica 5 um column (TSK gel ODS-80TM 25 many. cm). 6 Column temperature was kept at 20 C (-)-Epinephrine bitartrate salt and (-)- by thermocontroller (TSK CO-8000).6 The norepinephrine bitartrate salt, Sigma Chemical Co., St. solvent delivery system (TSK CCPD) 6 conLouis, MO 63100. tained .1 M KH2PO4, 20 \iM Na 2 EDTA, 2.5 ^osoh Co., Tokyo, 107 Japan.
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Animal Treatment and Blood Sampling
amount eaten by the hens during 2 h was 37.3 ± 13.4 g, constituting 40.7 ± 15.6% of the total feed intake during the day. The time of oviposition of each hen and the behavior of the hens were observed through a small window of the chamber from 0800 h to 1130 h. The behavior of each hen during the sampling time was classified into resting, feeding, and active behavior such as pacing and egg call.
1069
PLASMA CATECHOLAMINE IN LAYING HENS
Chromatogram B
Chromatogram A
O u-i
u-i CM
o o
o
o CM
CM
CM
CM
o o
Time after injection (min)
o
Time after injection (min)
FIGURE 1. Chromatographic recordings over time (minutes) obtained from injection of 100 |iL of external standard containing 25 pg each norepinephrine (NE; 1), epinephrine (E; 2), and 3,4-dihydroxybenzylamine (DHBA; 0) (Chromatogram A); and injection of 100 uL of an extracted plasma sample containing 50 pg of internal standard, DHBA (Chromatogram B).
mM 1-octanesulfonic acid sodium salt (SOS), and 15% methanol. The pH of the buffer was adjusted to 3.5 with H3PO4. The buffer was filtered and degassed, then the flow rate was adjusted to .6 mL/min. The electrochemical detector7 was set at 650 mV, and peak heights were measured using computer integrator. All values were corrected for actual recovery based on the extraction rate of the internal standard DHBA. Plasma concentrations of FFA and
glucose and plasma MAO activity were determined using commercial kits.8 Statistical Analysis The Student's t test was used to determine differences between means. Analysis of covariance was also used for the determination of the statistical significance of each correlation. RESULTS
7
TSK EC-8000, Tosoh Co., Tokyo, 107 Japan; EC100 WE-3G, Eicom Co., Kyoto, 612 Japan. SNEFA C-Test Wako, Glucose C-test Wako, and MAO Test Wako, respectively, Wako Pure Chemical Industries Co., Osaka, 541 Japan.
Average percentage recovery rates ± SD of E, NE, and DHBA by alumina extraction were 89.7 ± 6.5, 93.2 ± 2.4, and 92.6 ± 4.5, respectively. Thus, small variations in recovery rates among E, NE, and DHBA
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on May 13, 2015
r\_—...I
1070
FUJTTA ET AL.
on
'3 D
ft,
I 10
20
30
40
50
60
Concentration (pg/mL) FIGURE 2. Standard curves far epinephrine and norepinephrine determined by HPLC with electrochemical detection. Each point and vertical or horizontal line indicate x ± SD of four measurements.
DISCUSSION were observed. Consequently, DHBA was used for the peak height correction of E and NE of each plasma extracted chromatograph. Chromatographs of unextracted standard solution and extracted plasma sample are shown in Figure 1. Peaks of E, NE, and DHBA were legible and independent in each chromatograph. Standard curves of E and NE for the determination of each concentration were linear from zero to below 50 p g / m L (Figure 2). The changes of plasma FFA, glucose, E, and NE concentrations and plasma MAO activity before and after the start of feeding are shown in Table 1 (P > .05). Owing to the tendency of decrease in plasma FFA and increase in glucose concentrations after feed supply, a significant linear regression
The technique of HPLC-ECD was used to determine plasma concentrations of E and NE in the present experiment because of the quick, specific, and sensitive detection of CA (Kissinger et al, 1973). Although the principle behind this method is well known (Kajiwara et al, 1981; Macdonald and Lake, 1985; Premel-Cabic and Allain, 1988) and has been applied for research in bulls (Rulofson et al, 1988), data on such analysis in fowls have not been reported. Because of the complex procedures in plasma preparation (Rulofson et al., 1988), several modifications were adopted with the aim of reducing the volume of plasma sample for analysis and also to simplify the procedures for plasma preparation. First of all, instead of a glassy carbon electrode, a graphite
TABLE 1. Changes in plasma epinephrine (E), norepinephrine (NE), free fatty acid (FFA), and glucose concentrations and plasma monoamine oxidase (MAO) activity before and after feeding in laying hens1'2 Time of measurement
E
NE
FFA
Glucose (mg/dL) 232.7 ± 11.4 249.6 ± 8.2
(meq/L) .37 ± .19 1.63 ± 1.44 .330 ± .174 1.67 ± .77 .138 ± .025 .33 ± .03 ^lood samples were obtained at 0830 h before feeding and at 1100 h after feeding. 2 Each value indicates the x ± SD of four hens (n = 4).
Before feeding After feeding
MAO (U/mL) 103.5 ± 43.7 111.4 ± 42.0
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on May 13, 2015
1)
ft,
line and a significant correlation coefficient (Y = 2.2354 - .0083X, r = -.763, r 2 = .582, P < .01) were obtained between plasma FFA and glucose concentrations during the experimental period (Figure 3). N o clear relationships between plasma FFA and either E or NE concentrations were observed. However, the linear regression line and the correlation coefficient between plasma FFA concentration and plasma E (Y = .1407 + .4651X, r = .444, r 2 = .197, P < .05) or NE (Y = .0174 + .2864X, r = .787, r 2 = .619, P < .01) concentrations were significantly different from zero (Figure 4) when the behavioral activity of hens was low, such as in resting or feeding. No definite relationships were observed between plasma MAO activity and either plasma E or NE concentrations.
1071
PLASMA CATECHOLAMINE IN LAYING HENS .6
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•
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•
:
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1.4
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FIGURE 3. Regression line and correlation between plasma free fatty acid (FFA) concentration and plasma glucose concentration before (A) and after (•) the start of feeding. Y = 2.2354 - .003X; r = -.763; r 2 = .582, where Y and X indicate plasma FFA and glucose concentrations, respectively. The linear regression line and correlation coefficient were statistically significant (P < .01).
Norepineprhine
2 electrode was used in order to improve sensitivity, and plasma samples were directly used for CA extraction by alumina. Owing to the high sensitivity and disposability of the electrode, low levels of E, NE, and DHBA, even less than 10 pg, were always easily detected. Nevertheless, this high sensitivity also detected many current peaks that originated from some plasma components that would provide electrons by electrolysis under high applied voltage. The data on the voltamogram indicate that current potential curves of E, NE, and DHBA became steady when the applied voltage was more than 600 mV (Kajiwara et ah, 1981); therefore applied voltage was lowered and set at 650 mV in order to reduce peak heights other than E, NE, and DHBA. Lastly, column temperature was lowered and set at 20 C in order to obtain clear separation of E, NE, and DHBA peaks from each other. These modifications resulted in legible chromatographs as shown in Figure 1, suggesting that the detection was very much improved. However, analysis under 20 C decreased the resolution of the column, making the refreshment of the column necessary.
1.2
.2
-
. 1
•'
1 2
3 4 5 6 Concentration (ng/mL)
FIGURE 4. Relationships between plasma free fatty acid (FFA) concentration and plasma epinephrine or norepinephrine concentration before ( • and A) and after (• and O) the start of feeding. Significant linear regression lines and correlation coefficients were observed between plasma FFA concentration and plasma epinephrine (Y = .1407 + .4651X, r = .444, r 2 = .197, P < .05) and norepinephrine (Y = .0174 + .2864X, r = .787, r 2 = .619, P < .01) concentration when the hens were resting or feeding (A and •).
A chronic catheter was used for bleeding in the current experiment, as suggested by Johnson (1981). A rapid increase in blood corticosterone concentration following venipuncture in the laying hen (Johnson, 1981) and a similar venipuncture effect on plasma concentrations of NE and E in bulls (Rulofson et al, 1988) have been reported. In addition the stimuli involved in catching hens and the presence of humans caused a significant increase in heart rate and pronounced behavioral responses 0ones et ah, 1981). These data suggested that great care should be taken during bleeding to determine blood CA
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210 220 230 240 250 260 270 Glucose concentration (mg/mL)
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FUJITA ET AL.
REFERENCES Campbell, R. M., and C. G. Scanes, 1985. Adrenergic control of lipogenesis and lipolysis in the chicken in vitro. Camp. Biochem. Physiol. 82c 137-142. Cramb, G., D. R. Langslow, and J. H. Phillips, 1982. Hormonal effects on cyclic nucleotides and carbohydrate and lipid metabolism in isolated chicken hepatocytes. Gen. Comp. Endocrinol. 46:310-321. Harvey, S., C. G. Scanes, and K. I. Brown, 1986. Adrenal medullary hormones. Pages 487-493 in: Avian Physiology 4th ed. P. D. Sturkie, ed. Springer-Verlag, New York, NY. Johnson, A. L., 1981. Comparison of three serial blood sampling techniques on plasma concentrations in the laying hen. Poultry Sci. 60: 2322-2327. Jones, R. B., I.J.H. Duncan, and B. O. Hughes, 1981. The assessment of fear in domestic hens exposed to a looming human stimulus. Behav. Proc. 6:121-133. Kajiwara, N., A. Murakami, C. Isozaki, T. Kita, and M. Ono, 1981. An estimation of plasma catecholamines by high performance liquid chromatography with electrochemical detection. Folia Endocrinol. Jpn. 57:1044-1059. Kissinger, P. T., C. Refshauge, R. Dreiling, and R. N. Adams, 1973. An electrochemical detector for liquid chromatography with picogram sensitivity. Anal. Lett. 6:465-477. Langslow, D. R., and C. N. Hales, 1969. Lipolysis in chicken adipose tissue in vitro. J. Endocrinol. 43: 285-294. Macdonald, I. A., and D. M. Lake, 1985. An improved technique for extracting catecholamines from body fluids. J. Neurosci. Methods 13239-248. Premel-Cabic, A., and P. Allain, 1988. Simultaneous liquid chromatographic determination of norepinephrine, 3,4-dihydroxyphenylethyleneglycol, 3,4-dihydroxyphenylalanine and 3,4-dihydroxyphenylacetic acid in human plasma. J. Chromatogr. 434:187-190. Rees, A., T. R. Hall, and S. Harvey, 1984. Adrenocortical and adrenomedullary responses of fowl to treadmill exercise. Gen. Comp. Endocrinol. 55: 488-492. Rulofson, F. C, D. E. Brown, and R. A. Bjur, 1988. Effect of blood sampling and shipment to slaughter on plasma catecholamine concentrations in bulls. J. Arum. Sci. 66:1223-1229. Zachariasen, R. D., and W. S. Newcomer, 1974. Phenylethanolamine-N-methyl transferase activity in the avian adrenal following immobilization or adrenocorticotropin. Gen. Comp. Endocrinol. 23:193-198. Zachariasen, R. D., and W. S. Newcomer, 1975. Influence of corticosterone on the stress-induced elevation of phenylethanolamine-N-methyl transferase in the avian adrenal. Gen. Comp. Endocrinol. 25:332-338.
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concentration. Results in the present study on plasma concentration of E and NE were fairly similar with basal concentrations of circulating E and NE measured by radioenzymatic method (Rees et ah, 1984). Plasma concentrations of FFA and glucose before and after the start of feeding were not significantly different, however these were negatively correlated. Mobilization of FFA from adipose tissue in response to energy d e m a n d u n d e r hypoglycemic conditions such as fasting and before feeding should be stimulated by E (Langslow and Hales, 1969) via Padrenergic receptors and cyclic adenosine monophosphate (Campbell and Scanes, 1985). In fact, significant correlations were observed between the plasma FFA concentration and plasma E (P < .05) or NE (P < .01) concentrations when the behavioral activity of hens was low, such as in resting or feeding. The increase in CA concentration during active behavior before and after oviposition was not accompanied by an increase in FFA concentration. Accurate discussion on the increase in CA concentration during active behavior is difficult at present; however, such an increase in CA concentration was not accompanied by an increase in MAO activity, resulting in no definite relationship between plasma CA concentration and plasma MAO activity in the current study. Monoamine oxidase is one of the major enzymes in CA metabolism, therefore the level of MAO was expected to be related to plasma circulating CA. Plasma activity of MAO was not different before and after the start of feeding (Table 1). In this respect, it is suggested that the increase in CA concentration during active behavior would be transient. The effective level of circulating CA to cause lipolysis would be followed by the enough increase in MAO activity. However, it is quite difficult to explain these mechanisms at the present time because of the lack of the information on the physiological characteristics of MAO. In conclusion, basal levels of circulating E and NE, but not transiently elicited levels, would be closely related to the level of plasma concentration of FFA.