DOPA, dopamine, and DOPAC concentrations in the rat gastrointestinal tract decrease during fasting

Ebbe Eldrup1 and Erik A. Richter2

1 Division of Endocrinology, Herlev Hospital, University of Copenhagen, 2730 Herlev; and 2 Copenhagen Muscle Research Center, Human Physiology, University of Copenhagen, 2100 Copenhagen, Denmark


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The aim of the present study was to test the hypothesis that 3,4-dihydroxyphenylalanine (DOPA) and dopamine (DA) in the gastrointestinal tract are to a large extent of exogenous origin and derived from food. Tissue concentrations of norepinephrine (NE), epinephrine (Epi), DA, DOPA, and 3,4-dihydroxyphenylacetic acid (DOPAC), as measured by reverse-phase HPLC with electrochemical detection, were studied in fed and 4-day-fasted Wistar rats as well as in sympathectomized and adrenodemedullated rats. Sympathectomy and adrenal demedullectomy decreased tissue concentrations of NE and Epi, respectively, but had no effect on the level of tissue DOPA. Large amounts of DOPA and DA were present in the gastrointestinal tract. Fasting decreased DOPA and DA in the stomach and DOPA concentrations in the quadriceps muscle but no concentrations in other organs. DOPAC in the heart decreased both in response to sympathectomy and to fasting, whereas DOPAC decreased in plasma after fasting and in skeletal muscle after sympathectomy. We conclude that the food content of DOPA and DA is of major importance for the metabolism of DA and, thus, for the dopamine-sulfate content in the gastrointestinal tract and in plasma. The decrease in muscle DOPA after fasting may be explained by less insulin being available during fasting for stimulation of DOPA uptake in the muscle depot. DOPAC in the organism seems to be of a dual origin, derived partly from DA in the food and partly from DA synthesized in sympathetic nerves.

3,4-dihydroxyphenylalanine; 3,4-dihydroxyphenylacetic acid; 6-hydroxydopamine; sympathectomy; adrenodemedullectomy; norepinephrine; epinephrine; plasma


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

WE, AS WELL AS OTHERS (5, 6), have shown that the 3,4-dihydroxyphenylalanine (DOPA) and dopamine (DA) concentrations in the gastrointestinal tract are much greater than can be ascribed to their role as precursors in the biosynthesis of norepinephrine (NE). Furthermore, we have shown that there is a close correlation between the DOPA and DA contents of an ordinary meal and the subsequent increase in dopamine-sulfate (DA-S) in the blood (4). Plasma 3,4-dihydroxyphenylacetic acid (DOPAC) also increased after a meal. A 24-h fasting period reduced plasma DA-S and plasma DOPAC concentrations ~50% (4). These findings suggest that DOPA and DA in the gastrointestinal tract are derived at least in part from the food intake. Furthermore, they suggest that DOPA and DA in the food are released to the blood predominantly as the major DA metabolites DA-S and DOPAC. Such a mechanism would prevent DA and DA substrates in the food from causing an increase in the blood of cardiovascularly active and potentially dangerous catecholamines. DOPA, and especially DA, in the gastrointestinal tract, however, may also be derived from the relatively high plasma level of DOPA. DOPA may be decarboxylated to DA by the amine precursor uptake and decarboxylation (APUD) system in the gastrointestinal tract (13). DOPA in the blood is probably derived from the sympathetic nervous system. DOPA is not retained in the nerves as a depot but is likely to spill over to plasma, if it is not decarboxylated to DA in the nerves (2). On the basis of our previously published data obtained in human subjects, the plasma appearance rate of DOPA was 1.1 µg/min or ~1.6 mg/24 h (2). The content of DOPA, DA, and DA-S in three daily ordinary meals was, however, >200 mg (4), suggesting that the larger part of DOPA and probably also of DA in the gastrointestinal tract may be of exogenous origin. Therefore, in the present study, we tested in rats the hypothesis that the concentrations of DOPA and DA in the gastrointestinal tract decrease during a 4-day period of fasting. To clarify the importance of food relative to sympathoadrenomedullary activity, the tissue content of DOPA, DOPAC, and catecholamines were analyzed after fasting and after chemical sympathectomy with 6-hydroxydopamine (6-OHDA), as well as in adrenal demedullectomized rats.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals

Thirty-two male Wistar rats weighing 170-268 g were used in this study, which was approved by The Danish Animal Inspectorate. The animals were provided with food and water ad libitum before and during the studies except where otherwise specified.

Treatment Groups

Fasting. Five rats were left in their cages, with no access to food but free access to tap water, for 4 days before anesthesia.

Adrenal demedullectomy. Fourteen rats were adrenal demedullated bilaterally through two dorsal incisions while under anesthesia with Immobilon Vet (Pharmacia, Copenhagen, Denmark). The adrenal medulla was destroyed by electrocoagulation in both adrenal glands. The incisions were closed, and the rats were left in their cages with free access to food and water. This procedure does not affect basal adrenal glucocorticoid secretion (12). About 14 days later, the rats were finally anesthetized with pentobarbital sodium. Four rats were only adrenodemedullated; the other 10 rats that underwent surgical adrenodemedullectomy were also treated with 6-OHDA, as specified in Sympathectomy, treatment with 6-OHDA.

Sympathectomy, treatment with 6-OHDA. Under anesthesia with Immobilon Vet, 15 rats were chemically sympathectomized with 6-OHDA, which was dissolved in isotonic saline and injected in the tail vein two times on 1 day with a dose of 34 mg 6-OHDA/kg body wt. The rats were then left in their cages with free access to food and water. Eight days later, each animal was injected intravenously two times on the same day with a dose of 68 mg 6-OHDA/kg body wt. Again the rats were left in their cages with free access to food and water. Six days later, the rats underwent anesthesia. In 10 rats, sympathectomy with 6-OHDA was performed after surgical adrenodemedullectomy. The other 5 rats that were treated with 6-OHDA had been sham operated.

Control treatment. Eight rats served as untreated controls. They had access to food and water until anesthesia.

Blood Sampling and Peripheral Tissue Preparation

The animals were anesthetized with pentobarbital sodium intraperitoneally. Blood was withdrawn from the aorta, after which the following organs were quickly removed: heart, spleen, stomach, liver, and one kidney. Specimens of tissue were also removed from the small and large intestine, as well as the entire quadriceps muscle from one leg. The mucosa was not separated from the muscle layers for the stomach and intestinal samples. From three control rats, three sympathectomized rats, and four sympathectomized and adrenal demedullectomized rats, the abdominal part of the sympathetic chain was removed. From three control rats, four sympathectomized rats, and three sympathectomized and adrenal demedullectomized rats, a piece of abdominal cutis with subcutis was also removed. Tissues were immediately frozen in liquid nitrogen and stored at -80°C until assayed. Liver, spleen, and kidney tissues were not assayed from one control, one fasted, and one sympathectomized rat. Liver and spleen tissues were not assayed from two adrenal demedullectomized and from two sympathectomized and adrenal demedullectomized rats. The decision about from which rats the organs were or were not analyzed was made at random before any tissue was processed for analysis. Blood was drawn into ice-chilled tubes, and procedures were followed as reported previously (2). Plasma samples from three rats subjected to sympathectomy and adrenal demedullectomy and from two rats subjected to adrenal demedullectomy were lost during preparation.

Catecholamine HPLC Assay

Samples were processed and assayed for NE, DOPA, DOPAC, epinephrine (Epi), and DA by reverse-phase HPLC with electrochemical detection, as described previously (2, 3, 5). In short, tissue was weighed and then homogenized in 10.2 ml of ice-chilled 0.6 M perchloric acid containing 1.7 mg/ml EGTA and 1.1 mg/ml reduced glutathione. After centrifugation for 15 min at 2,500 g at 0°C, 1.0 ml of the supernatant was adjusted to pH 8.6 with 6 M KOH and then processed for determination of catecholamine concentrations after alumina-batch extraction. Twenty-five µl 3 M KCl were added to 200 µl of the 0.2 M perchloric acid eluate. After centrifugation, 50 µl of the supernatant were injected into the HPLC system. The limit of sensitivity in the supernatant, defined as two times baseline noise, was 0.02 ng/ml, 0.01 ng/ml, 0.01 ng/ml, 0.02 ng/ml, and 0.01 ng/ml for DOPA, NE, Epi, DOPAC, and DA, respectively. If a concentration was lower than the limit of sensitivity, the concentration of the catecholamine was assigned the value zero. In a few cases, it was not possible because of technical reasons due to interfering peaks in the chromatograms to determine with certainty the concentrations of Epi, DA, and DOPAC. For this reason, the numbers of animals referred to in the statistical analyses in some cases are lower than the number of animals subjected to the experimental procedure.

Materials

All reagents were of the highest analytical grade. Standards were purchased from Sigma Chemical (St. Louis, MO). 6-OHDA was a gift from Roche, Denmark.

Statistics

Concentrations of catecholamines are presented as medians and interquartile ranges. Differences in changes of plasma and tissue concentrations between individual tissues after different experimental procedures were analyzed by the Kruskal-Wallis test. Post hoc tests were made by Dunn's multiple comparison procedure (SigmaStat Statistical Analysis System version 1.02, Jandel). P < 0.05 defined statistical significance.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Arterial Plasma

NE concentrations in arterial plasma (Table 1) and plasma DOPA concentrations were similar in all experimental situations (Table 2). Plasma DOPAC concentrations (Table 3) were significantly lower in fasting rats compared with values from controls. After sympathectomy, as well as after sympathectomy and adrenal demedullectomy, lower plasma DOPAC values were found compared with values from controls, but the difference did not obtain statistical significance. Plasma DA concentrations were generally low, as shown in Table 4. Plasma Epi values confirmed the effect of the adrenal demedullectomy (Table 5).

                              
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Table 1.   Norepinephrine concentrations in plasma and in different tissues after different experimental procedures


                              
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Table 2.   DOPA concentrations in plasma and in different tissues after different experimental procedures


                              
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Table 3.   DOPAC concentrations in plasma and in different tissues after different experimental procedures


                              
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Table 4.   Dopamine concentrations in plasma and in different tissues after different experimental procedures


                              
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Table 5.   Epinephrine concentrations in plasma and in different tissues after different experimental procedures

Tissue Concentrations

Tissue concentrations of NE (Table 1, Fig. 1) and Epi (Table 5, Fig. 1) reflected the effects of sympathectomy and adrenal demedullectomy in all tissues. In sympathetic ganglia, the concentrations of NE remained unchanged as expected.


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Fig. 1.   Median values (ng/g) and interquartile ranges of epinephrine (top) and norepinephrine (bottom) in stomach, quadriceps muscle, and heart after different experimental procedures in rats. C, control; F, after 4 days of fasting; S, after sympathectomy with 6-hydroxydopamine (6-OHDA); A, after adrenal demedullectomy; SA, after both sympathectomy and demedullectomy. * P < 0.05 vs. control values; # P < 0.05 vs. fasting values.

DOPA concentrations were significantly reduced in the stomach of fasting rats, both compared with controls and compared with sympathectomized and/or adrenal demedullectomized rats (Table 2, Fig. 2). Fasting reduced muscle content of DOPA compared with adrenal demedullectomized rats (P < 0.05) and sympathectomized and adrenal demedullectomized rats (P < 0.05), but the difference was insignificant when comparison was made with sympathectomized and with control animals (Table 2, Fig. 2). In all other tissues, DOPA concentrations were similar regardless of experimental procedure (Table 2). No DOPA was detected in sympathetic ganglia, in contrast to the very high concentrations of NE (Tables 1 and 2).


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Fig. 2.   Median values (ng/g) and interquartile ranges of 3,4-dihydroxyphenylalanine (DOPA, top), dopamine (DA, middle), and 3,4-dihydroxyphenylacetic acid (DOPAC, bottom) in stomach, quadriceps muscle, and heart after different experimental procedures in rats. C, control; F, after 4 days of fasting; S, after sympathectomy with 6-OHDA; A, after adrenal demedullectomy; SA, after both sympathectomy and demedullectomy. * P < 0.05 vs. control values; § P < 0.05 vs. demedullectomy values.

Tissue DOPAC concentrations are shown in Table 3. Fasting reduced DOPAC values in every tissue investigated. Significantly lower DOPAC values compared with control animals were found in the heart. In the kidney, fasting DOPAC concentrations were significantly lower than in adrenal demedullectomized animals. Sympathectomy significantly reduced DOPAC concentrations in the muscles and in the heart compared with control values (Fig. 2), whereas significantly higher DOPAC concentrations were found in the large intestine after sympathectomy and adrenal demedullectomy.

Tissue concentrations of DA in the stomach were lower in fasted animals (Table 4, Fig. 2). In all other tissues except for the kidney, the various experimental procedures had no significant effect on DA concentration.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In most tissues, sympathectomy and adrenodemedullectomy decreased NE and Epi concentrations, respectively. These procedures did not decrease the DOPA concentration in any organ, which is in accordance with some (5, 9) but not all previously reported studies (7). No DOPA was detected in abdominal sympathetic ganglia despite high concentrations of NE, a finding which supports the hypothesis that DOPA, which is not decarboxylated to DA, spills over to the interstitial fluid and plasma (2). Other authors, however, have found high concentrations of DOPA in superior cervical ganglia (8, 9) and, in one study, also in the lumber ganglia (10) in dogs. The reason for these discrepancies remains to be clarified.

The present study showed, in accordance with a number of previous studies (5, 9, 11), that the concentrations of DOPA and DA in the stomach were very high compared with other organs. Fasting decreased DOPA and DA concentrations in the stomach markedly but had no effect on DOPA and DA concentrations in most of the other organs (Fig. 2, Tables 2 and 4). This observation and the fact that food contains large amounts of DOPA and DA (4) support our hypothesis that DOPA and DA in the gastrointestinal tract are to a large extent derived from food. We have also shown that plasma DA-S in human subjects increased markedly in response to a meal and that increments in plasma concentration of DA-S were closely related to the DOPA and DA contents of the food (4).

It is of interest that the ratio of DOPA to DA in the stomach was above one, whereas in our previous analysis of the DOPA and DA contents of meals given to human subjects, we observed that the meals contained much more DA than DOPA. This may indicate that the meal composition is different in rats and human subjects, but it may also indicate that some of the DOPA in the stomach was derived from plasma DOPA, which may be taken up by the cells and to some extent decarboxylated to DA by the APUD system. Studies of plasma DOPA kinetics in humans (2) indicated, however, that plasma DOPA could contribute only a fraction, maximally 20% (plasma appearance rate 1.6 mg/24 h), of DOPA in the gastrointestinal tract compared with the DOPA content of the meal (~9 mg/24 h).

Sympathectomy with 6-OHDA is known to decrease tyrosine hydroxylase activity and to destroy sympathetic nerve terminals (9). DOPA and, consequently, DA are synthesized as a result of tyrosine hydroxylase activity. The decrease in DOPA and DA content in the stomach found in the present study is very unlikely to be explained by a decrease in the endogenous rate of synthesis caused by fasting, because the DOPA and DA contents in the stomach were unchanged after sympathectomy with 6-OHDA (Tables 2 and 4).

The DOPA concentration in the quadriceps muscle decreased with fasting (Fig. 2 and Table 2). DOPA in muscle tissue is in all probability taken up by muscle cells from plasma. Furthermore, we have reported that plasma DOPA decreased after a meal, probably due to the effect of insulin on the uptake of amino acids in muscles (4). With fasting, plasma insulin decreases, and the uptake of DOPA in muscles was most likely decreased.

The concentration of DOPAC (Table 3) decreased both after fasting (significantly in plasma, heart, and kidney) and also after sympathectomy (significantly in the heart and in muscle tissue). These decrements can be explained by less DA being available for metabolism after fasting and after sympathectomy. The concentration of DOPAC increased in the large intestine after combined sympathectomy and demedullectomy, and relatively large values were also found after each procedure performed separately. This may indicate an altered intestinal metabolism of DA in the gastrointestinal tract after sympathectomy. The plasma concentration of DA-S that may be obtained after a meal in human subjects can be calculated from the content of DOPA and DA in the food (>200 mg) and from the half-time of DA-S of ~3 h (4, 6). The concentrations of DA-S that are measured after a meal are very much lower (4, 6), which indicates that only a fraction of DOPA and DA in the food appeared in plasma as DA-S. This suggests that, in human subjects, a large part of DOPA and DA in the food is metabolized in the gut.

It has been suggested that DOPA and DA in the gastrointestinal tract were not only derived from the food and circulating DOPA but also were synthesized in specific cells or nerves in the gastrointestinal tract. Christensen and Brandsborg (1) reported that DA was present in Heidenhain pouches, which have no direct contact with the food. Furthermore, Goldstein et al. (6) have recently suggested that increments in plasma DA-S after a meal were derived from DOPA synthesized in the gastrointestinal tract. This hypothesis was based on the fact that two patients with L-aromatic amino acid decarboxylase deficiency had low levels of plasma DA-S, which increased markedly after intravenous infusion of DA. The meal content of DOPA and DA in the two patients was, however, not reported. A specific synthesis of DOPA in the gastrointestinal tract is not required to explain this observation, but it may indicate that the increment in DA-S after a meal is derived to a large extent from DOPA rather than from DA or DA-S in the food. Further studies are warranted to clarify this matter.

In conclusion, we have shown that fasting for 4 days in rats decreased the DOPA and DA content of the stomach markedly, which supports our hypothesis that the food content of DOPA and DA are of major importance for the content of DA and DA-S in the gastrointestinal tract and for the content of DA-S in plasma. Furthermore, plasma DOPAC seems to be of dual origin, derived partly from DA in the food and partly from DA in the sympathetic nerves.


    ACKNOWLEDGEMENTS

The expert technical assistance of Gurli Habekost and the helpful discussions with Dr. Niels Juel Christensen during preparation of the manuscript are gratefully acknowledged.


    FOOTNOTES

This work was supported by a grant from the Agnes and Knut Mørck Foundation and from the Danish National Research Foundation, Grant no. 504-14.

Address for reprint requests and other correspondence: E. Eldrup, Division of Endocrinology E112, Herlev Hospital, Univ. of Copenhagen, DK-2730 Herlev, Denmark (E-mail: eeldrup{at}dadlnet.dk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 27 January 2000; accepted in final form 27 April 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Christensen, NJ, and Brandsborg O. Dopamine in human gastric juice determined by a sensitive double-isotope-derivative technique. Scand J Clin Lab Invest 34: 315-320, 1974[ISI][Medline].

2.   Eldrup, E, Hetland ML, and Christensen NJ. Increase in plasma 3,4-dihydroxyphenylalanine (DOPA) appearance rate after inhibition of DOPA decarboxylase in humans. Eur J Clin Invest 24: 205-211, 1994[ISI][Medline].

3.   Eldrup, E, Mogensen P, Jacobsen J, Pakkenberg H, and Christensen NJ. Cerebrospinal fluid and plasma concentrations of free norepinephrine, dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), 3,4-dihydroxyphenylalanine (DOPA), and epinephrine in Parkinson's disease. Acta Neurol Scand 92: 116-121, 1995[ISI][Medline].

4.   Eldrup, E, Møller SE, Andreasen J, and Christensen NJ. Effects of ordinary meals on plasma concentrations of 3,4-dihydroxyphenylalanine, dopamine sulphate and 3,4-dihydroxyphenylacetic acid. Clin Sci (Colch) 92: 423-430, 1997[ISI][Medline].

5.   Eldrup, E, Richter EA, and Christensen NJ. DOPA, norepinephrine, and dopamine in rat tissues: no effect of sympathectomy on muscle DOPA. Am J Physiol Endocrinol Metab 256: E284-E287, 1989[Abstract/Free Full Text].

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13.   Tjälve, H. Catechol- and indolamines in some endocrine cell systems. An autoradiographical, histochemical and radioimmunological study. Acta Physiol Scand Suppl 360: 1-122, 1971.


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