CHOLINE AND ACETYLCHOLINE IN RATS: EFFECT OF DIETARY CHOLINE D. R . HAUBRICH, PAULINE F. L. WANG.T. CHIPPENDALE' and ELIZABETH PROCTOR Depai tmeiit of Pharmacology, The Squibb Institute for Medical Research and 'The Department of Psychologj. Princeton University. Princeton. N J 08S40, U S A (Rtwived 18 Mnrch 1976. Accepted 22 M u y 1976)
Abstract-The concentration of free choline in peripheral tissucs (duodenum. heart, kidney. liver. stomach and plasma) of rats was found to be related to the amount of free choline in the diet. Under steady-state conditions. the Concentration of free choline in plasma varied from a minimum of approx 6 nmol,ml (in rats fed a choline-deficient diet) to a maximum value not exceeding 21 nmol/ml. The concentration of plasma choline was elevated above 21 nmol/ml for a short time after parenteral administration of choline chloride or one of its precursors (CDP choline or phosphorylcholine). but was not affected by stress. endocrine manipulations, drug treatments or the time of day when rats were killed. The metabolism of intravenously administered [methyL3H] choline was accelerated in peripheral tissues (except plasma) of choline-deficient rats, indicating that free choline is not preserved during choline deficiency by a reduction in its rate of turnover. Furthermore, the decrease in concentration of plasma choline that occurred in rats fed a choline-deficient diet was prevented by addition of deanol (dimethylaininoethanol) to the diet. These results indicate that free choline in peripheral tissues of rats is derived from both free choline in the diet, and from precursors of choline present within the diet. In contrast to the effects in peripheral tissues, the concentration of free choline in brain was not reduced by dietary deprivation of free choline; however, the increase in free choline that occurred when rats were decapitated was reduced in brains by deficiency of choline, suggesting a decrease in the concentration of esterified forms of cerebral choline. The concentration of acetylcholine was not reduced in the brain, duodenum. heart, kidney or stomach of 21-week old rats raised from birth on a choline-deficient diet. in the duodenum of rats given a choline-deficient diet for 1, S or 1 1 days, or in brains of rats deprived of free choline for 1 or 11 days. However, the rate of iu u i synthesis ~ ~ of ACh from [methyl-'Hlcholine was accelerated i n cholinergic tissues that were depleted of free choline (it. duodenum, heart and stomach).
FREEcholine contained in plasma is a precursor in part for acetylcholine (ACh) synthesized in both brain
choline in the body (ANSELL & HAWTHORN, 1964), or about the factors that regulate the concentration (SCH~JBERTH rr d.. 1969. 1970; HAUBRICH et a/.. 1972, of free choline in plasma and other tissues. Part of 1975a; SAELENS et al.. 1973; JENDEN et a/., 1974) and the choline for synthesis of ACh in brain is known peripheral tissues (HAUBRICH et al., 197%). Further- to be derived from free choline present in the diet 1975; SCHUBERTH & JENDEN, more, the concentration of free choline in tissues may (HANIN& SCHUBERTH, 1975); in the absence of be important in regulation of the rate of synthesis 1975; WANG& HAUBRICH, of ACh. Previous studies in our laboratory have dietary choline, the concentration of ACh in tissues rt al., 1968). In shown that administration of choline to guinea pigs was shown to be reduced (NAGLER or rats causes an increase in the concentration of the addition, phosphatidylcholine synthesized in the liver et can be transported to the brain where it is then conneurotransmitter in peripheral tissues (HAUBRICH ul., 1974a,b). and brain (HAUBRICH et al., 1974~1, verted to free choline and ACh (ANSELL& SPANNER. 1975~).Other studies in pifro have correlated the rate 1971; ILLINGSWORTH & PORTMAN.1972, 1973). An of transport of choline into cholinergic neurons with efflux from the brain of unesterified choline has been & KEWITZ(1972). and conthe rate of synthesis or release of ACh (HAGA& demonstrated by DROSS firmed by other investigators who believe that free NODA.1973; GUYENET P t a/., 1973; KUHARet al.. 1973; BARKER & MITTAG,1975. CARROLL & GOLD- choline in brain is derived from the hydrolysis of lipid-bound choline (CHOIet a/., 1975; FREEMAN ef BERG, 1975) and with the activity of cholinergic al., 1975; SPANNER et a/.. 1976). neurons (SIMON & KUHAR,1975). This study was undertaken to assess further the In spite of its important role in the synthesis of ACh, little is known about the ultimate sources of relationship of free choline in the diet to the concentration of choline and ACh in brain and peripheral Abbreaiation iisetl: CDP, cytidine 5'-pyrophosphate. tissues. h
c 2116-
1305 B
D. R. HAUBRICH et al.
I306 METHODS
Animal procedtrres. Concentrations of choline in choline-
supplemented (control) and choline-deficient diets (both obtained from Bio-Serv Inc.. Frenchtown. NJ) were 12.6 and 0.07 mmol/'kg. respectively. as determined by direct assay of the pellets (Purina Rat Chow contains 9.5 rnrnol of choline/kgj. All rats were housed in a room maintained on a constant light (070&1900 h w a r k cycle with free access to food and water. Rats were usually killed between 0900 and 1100 h either by decapitation (in experiments in which peripheral tissues were analyzed) or by microwave et d., 1975aj. Irradiairradiation of the head (HAUBRICH ! I I 5 II 22 tion prevents post-mortem changes in the concentration Time, days of choline and ACh in brain (STAVINOHA & WEINTRAUB. 1974). FIG. I. Concentration of free choline in plasma plotted In the experiments in which neonatal rats were raised as a function of time after rats were given a diet containing on a choline-deficient diet. either a choline-supplemented 0.07 mmol free choline per kg. Control rats (zero-time or a choline-deficient diet was given to pregnant females point) were fed a diet containing 12.6 mmol of choline (Sprague-Dawley derived rats bred at Princeton Univer- per kg. Each point is the average of 5-25 rats. All values sity. Princeton, NJ) 1 4 days prior to parturition. The neoare different from the control, P < 0.01. natal rats were weaned at 21 days of age. Male and female rats were randomly chosen from each litter for use in experiments. For the studies involving endocnne ablations. (ration of free choline remained essentially at this male rats were obtained surgically prepared from Ziviclowered level for u p to 22 days while rats were fed Miller Corp. (Allison Park. PA). In all other studies, male the diet deficient in choline. When rats were raised Sprague-Dawley rats were obtained from Holtzman Corp. from birth until 21 weeks of age o n the choline-defi(Madison. WI). cient diet, free choline was also reduced (357;) in [Methyl-3H]choline chloride was obtained from New plasma (Table 1). England Nuclear Corp. (Boston. MA). The isotope had To assess the relationship between the concena specific activity of 2.4 Cihnmol. Each rat received 1.4 tration of choline in the plasma and the amount in mCi,'kg (0.58 pmol/kg) into a tail vein. Unless otherwise indicated, statistical comparisons were the diet, choline chloride was added to the drinking water of rats fed the choline-deficient diet. As shown performed using the Student's t test. in Fig. 2. addition of choline to the water caused a n C/irrr~rc.tr/procedures. Choline and ACh were isolated by high-voltage paper electrophoresis and measured by use increase in the concentration of choline in plasma. of an enzymatic (choline kinase)-radio-isotopic assay (REID The highest concentration of plasma choline was et a/., 1971) with modifications (HAUBRICH & REID. 1974). observed when the concentration of choline in the For the determination of choline in plasma, mixed arterial drinking water was 15-30 mM; a further increase in and venous blood was collected by draining the blood the concentration of choline in the water caused n o from the carcasses of decapitated rats. Assay of choline additional elevation in the concentration of plasma was performed in unextracted plasma in the presence of heparine by use of the choline kinase procedure (WANG & HAUBRICH, 1975). Deanol (HAUBRICH et a/.. 1975~)cytidine diphosphocholine and phosphorylcholine (WANG& HAUBRICH. 1975) do not interfere in the assay of choline. Metabolites of [methyl--'H]choline were isolated by paper electrophoresis and chromatography and measured as preet a/.. 197Sbj. Uptake of viously described (HAUBRICH [methyl-3H]choline by synaptosomes was also determined et u/.. 1975a). The Concentration as described (HAUBRICH of protein was measured spectrophotometrically as described by LOU'RYer al. (1951) with bovine serum albumin as the standard. RESULTS
Tissirr cor~etirratior7of cholirie As shown in Figs. 1 and 2, the concentration of choline in plasma was related, within limits. t o the amount present in the diet. When the diet deficient in free choline was fed to rats, the concentration of plasma choline was maximally depleted within 1 day to a level half that observed in rats that consumed the choline-supplemented diet (Fig. 1). The concen-
, I , 012 4
8
1 15
1 60
30
Choline in drinking water,
mM
FIG. 2. Concentration of free choline in plasma plotted as a function of the concentration of choline in the drinking water. All rats were given a diet containing 0.07 mmol choline per kg for 11 days. along with drinking water containing varying amounts of choline chloride. Each point is the average of 3 or 4 rats per group. Values for the four highest concentrations of choline are significantly greater ( P i0.05) than the lowest values
Choline. ACh and dietary choline
I307
TABLE I . CHOLINE AND ACETYLCHOLINEI N PERIPHERAL TISSUES OF RATS RAISED ON
A CHOLINE-DEFI-
CENT DIET*
Control (nmoljg f s.E.)
Tissue Duodenum Heart Kidney Liver Plasma Stomach
Choline Choline-deficient (nmolig f s.E.)
176 k 1 I 185 I 91 + 3 48 10 8.2 0.3 83 6
Acetylcholine Control Choline-deficient (nmol/g k s.E.) (nmolig f s.E.)
*
18.0 k 2.8 5.6 0.4 1 . 1 0.3 N.D. N.D. 12.9 0.5
Ilt 10 f I $ 55 f 6$ 16 f 4$ 5.3 0.21 47 & 3$
115
17.9 k 2.2 5.1 k 0.5 1.2 & 0.3 N.D. N.D. 12.3 f-1.3
+
*
*
* One to 4 days prior to parturition, pregnant rats were given either a control diet containing 12.6 mmol cholinelkg or an otherwise identical diet containing 0.07 mmol choline/kg. At 21 weeks post-partum, [methyl-3H]choline (1.4 mCi/kg: 0.58 pmol/kg was administered intravenously to the offspring. Rats were decapitated 1 min after administration of the isotope. Tissues (N = 5-7) were analyzed for endogenous choline and acetylcholine (shown here) and for radioactivity (shown in Tables 5 and 6). t Different from control, P < 0.01. 3 Different from control, P < 0.001. N.D. = not detectable. choline (Fig. 2). Addition of choline chloride to the drinking water did not significantly affect the amount of liquid consumed (30-40 mllratjday). To determine if a precursor for choline could substitute for free choline in the diet, rats were given drinking water containing either deanol (dimethylaminoethanol) or an equimolar concentration of choline. As shown in Table 2, the concentration of choline in plasma of rats that received deanol, but were deprived of choline (group 9, was significantly greater than that of the choline-deficient rats (group l), but was nearly the same as the level of choline in the rats receiving the choline-supplemented diet (group 2). The addition of deanol to the drinking water did not significantly affect the concentration of choline in plasma of rats that received sufficient choline in the diet (group 6). The concentration of choline in plasma of rats that consumed deanol (groups 5 and 6) was less than that of rats given an equivalent
amount of choline in the drinking water (group 3 and 4).
Dietary deprivation of free choline also caused a reduction in the concentration of free choline in tissues other than plasma. The duodenum was chosen for comparison of the time-course of depletion of plasma choline with the depletion from a tissue innervated by the cholinergic nervous system. As had occurred in the plasma, maximal depletion of choline (31”,) was observed within one day in the duodenum of rats fed the choline-deficient diet as compared to animals that received the choline-supplemented diet (Fig. 3). The concentration of free choline remained at this same level in the duodenum for up to 1 1 days while the rats consumed the choline-deficient diet. A
TAHLF. 2. EFFECTOF
CHOLINE OR DEANOL I N THE DRINKING WATER ON PLASMA CHOLINE*
Group No.
Diet
I
CD
2 3 4 5 6
CD CS CD CS
cs
Addition t o drinking water
Plasma choline (nmol/ml f s.E.)
None None Choline Choline Deanol Deanol
5.6 k 0.7 10.7 k 1.0f 20.6 2.2 17.5 k 2.1 12.9 & 0.8t 13.3 1.4
+
* Rats were given either a choline-supplemented (CS) diet containing 12.6 mmol choline/kg. or a choline-deficient (CD) diet containing 0.07 mmol cholineikg. along with tap water that contained either choline chloride ( 6 0 m ~ or ) deanol (60mM). Rats were decapitated and assays performed 11 days after starting the dietary regimen. Values are the averages of 6 rats per group. t Different than CD with no addition to drinking water, P < 0.01.
J6
~
I 5 Time,
days
FIG.3. Concentration of choline and acetylcholine in the duodenum after rats consumed a diet containing 0.07 mmol choline per kg. Each point is the average of 5 or 6 rats. All values for choline were significantly lower (P < 0.01) than the zero-time control value (rats fed a diet containing 12.6 mmol choline per kg for 11 days). The rats fed a choline-deficient diet for 1 day had a higher concentration of acetylcholine (P < 0.05). whereas the level of the neurotransmitter was the same as that of the control at 5 and 11 days.
1308
D. R. HACBRICH tv 01. TABLE 3. EFFFCTOF
VARIOLS FACTORS O S THE CONCEZTRATION OF PLASMA CHOLINE
Plasma choline* (Percentage of controls f S.L)
Treatment Eiidocririe
Adrenalectomy Thyroidectomy Hq pophysectomy Epinephrine (0.5 mg kg. i.p.-?0 rnin.) Insulin (20 units kg. i . p . 4 0 rnin.) Prostaglandin EL ( I mg kg. i.p.-60 min.) Strrss Fasted (74 h ) Immobilization (amin) Cold ( 3 h at 5 C: 1 rat cage) Ckoliric, prrcur.sor.< Choline C1 (100 mg kg. i.p.) Cholinc C1 (1W nig kg. i.p.) Cqtidine diphosphocholine (700 mg kg. i.p.) Cytidine diphosphocholine 1200 mg kg. i.p.) Phosphorylcholine (200 mg kg. i.p.1 Phosphorylcholine (200 mg kg. i.p.1
97 8 89 83 96 83
f4 i6 f5 f 16 f5 f4
81 1 7 91 1 3 91 & 6
20 niin 120 niin 60 min 170 niin 60 niin 120 min
506 f 40t 110 8 270 f 5 t 107 f 4 171 f 26t 109 f 8
*
Drirgs
Chlorpromazine (10 mg kg. p.0.) Pilocarpine HCI (10 mg'kg. i.p.) Amphetamine sulfate ( 5 mg kg. i.p.) Hemicholinium-3 (0.I mg.kg. i.p.) Dimethylphenylpiperazinum ( 1 mg kg. i.p.) Ether ( 3 rnin inhalation) i-iI?W 01 drorh 0200 0500 0800
I100 0.100 1 700 2000 2300 0200 (next day)
3h 60 min
60 min 60 min 30 min
98 f 6 95 f 6 91 * 4 99 f 5 88 f 5 101 f 3 104 f 73 loOf3 130 1 23 105 6 112 f 7 119 7 107 f 10 113 7 104 f I
* *
*Percentages were calculated from the averages of 5-10 rats per group. and statistical comparisons (Student's I ) \\ere made using :I group of appropriately treated control rats ( N = 4--l5) for each experiment. For the study in which rats were sacrificed a t different time of the day. the group with the lottest value (sacrificed at 0500 11) \
Abstract-The concentration of free choline in peripheral tissucs (duodenum. heart, kidney. liver. stomach and plasma) of rats was found to be related to the amount of free choline in the diet. Under steady-state conditions. the Concentration of free choline in plasma varied from a minimum of approx 6 nmol,ml (in rats fed a choline-deficient diet) to a maximum value not exceeding 21 nmol/ml. The concentration of plasma choline was elevated above 21 nmol/ml for a short time after parenteral administration of choline chloride or one of its precursors (CDP choline or phosphorylcholine). but was not affected by stress. endocrine manipulations, drug treatments or the time of day when rats were killed. The metabolism of intravenously administered [methyL3H] choline was accelerated in peripheral tissues (except plasma) of choline-deficient rats, indicating that free choline is not preserved during choline deficiency by a reduction in its rate of turnover. Furthermore, the decrease in concentration of plasma choline that occurred in rats fed a choline-deficient diet was prevented by addition of deanol (dimethylaininoethanol) to the diet. These results indicate that free choline in peripheral tissues of rats is derived from both free choline in the diet, and from precursors of choline present within the diet. In contrast to the effects in peripheral tissues, the concentration of free choline in brain was not reduced by dietary deprivation of free choline; however, the increase in free choline that occurred when rats were decapitated was reduced in brains by deficiency of choline, suggesting a decrease in the concentration of esterified forms of cerebral choline. The concentration of acetylcholine was not reduced in the brain, duodenum. heart, kidney or stomach of 21-week old rats raised from birth on a choline-deficient diet. in the duodenum of rats given a choline-deficient diet for 1, S or 1 1 days, or in brains of rats deprived of free choline for 1 or 11 days. However, the rate of iu u i synthesis ~ ~ of ACh from [methyl-'Hlcholine was accelerated i n cholinergic tissues that were depleted of free choline (it. duodenum, heart and stomach).
FREEcholine contained in plasma is a precursor in part for acetylcholine (ACh) synthesized in both brain
choline in the body (ANSELL & HAWTHORN, 1964), or about the factors that regulate the concentration (SCH~JBERTH rr d.. 1969. 1970; HAUBRICH et a/.. 1972, of free choline in plasma and other tissues. Part of 1975a; SAELENS et al.. 1973; JENDEN et a/., 1974) and the choline for synthesis of ACh in brain is known peripheral tissues (HAUBRICH et al., 197%). Further- to be derived from free choline present in the diet 1975; SCHUBERTH & JENDEN, more, the concentration of free choline in tissues may (HANIN& SCHUBERTH, 1975); in the absence of be important in regulation of the rate of synthesis 1975; WANG& HAUBRICH, of ACh. Previous studies in our laboratory have dietary choline, the concentration of ACh in tissues rt al., 1968). In shown that administration of choline to guinea pigs was shown to be reduced (NAGLER or rats causes an increase in the concentration of the addition, phosphatidylcholine synthesized in the liver et can be transported to the brain where it is then conneurotransmitter in peripheral tissues (HAUBRICH ul., 1974a,b). and brain (HAUBRICH et al., 1974~1, verted to free choline and ACh (ANSELL& SPANNER. 1975~).Other studies in pifro have correlated the rate 1971; ILLINGSWORTH & PORTMAN.1972, 1973). An of transport of choline into cholinergic neurons with efflux from the brain of unesterified choline has been & KEWITZ(1972). and conthe rate of synthesis or release of ACh (HAGA& demonstrated by DROSS firmed by other investigators who believe that free NODA.1973; GUYENET P t a/., 1973; KUHARet al.. 1973; BARKER & MITTAG,1975. CARROLL & GOLD- choline in brain is derived from the hydrolysis of lipid-bound choline (CHOIet a/., 1975; FREEMAN ef BERG, 1975) and with the activity of cholinergic al., 1975; SPANNER et a/.. 1976). neurons (SIMON & KUHAR,1975). This study was undertaken to assess further the In spite of its important role in the synthesis of ACh, little is known about the ultimate sources of relationship of free choline in the diet to the concentration of choline and ACh in brain and peripheral Abbreaiation iisetl: CDP, cytidine 5'-pyrophosphate. tissues. h
c 2116-
1305 B
D. R. HAUBRICH et al.
I306 METHODS
Animal procedtrres. Concentrations of choline in choline-
supplemented (control) and choline-deficient diets (both obtained from Bio-Serv Inc.. Frenchtown. NJ) were 12.6 and 0.07 mmol/'kg. respectively. as determined by direct assay of the pellets (Purina Rat Chow contains 9.5 rnrnol of choline/kgj. All rats were housed in a room maintained on a constant light (070&1900 h w a r k cycle with free access to food and water. Rats were usually killed between 0900 and 1100 h either by decapitation (in experiments in which peripheral tissues were analyzed) or by microwave et d., 1975aj. Irradiairradiation of the head (HAUBRICH ! I I 5 II 22 tion prevents post-mortem changes in the concentration Time, days of choline and ACh in brain (STAVINOHA & WEINTRAUB. 1974). FIG. I. Concentration of free choline in plasma plotted In the experiments in which neonatal rats were raised as a function of time after rats were given a diet containing on a choline-deficient diet. either a choline-supplemented 0.07 mmol free choline per kg. Control rats (zero-time or a choline-deficient diet was given to pregnant females point) were fed a diet containing 12.6 mmol of choline (Sprague-Dawley derived rats bred at Princeton Univer- per kg. Each point is the average of 5-25 rats. All values sity. Princeton, NJ) 1 4 days prior to parturition. The neoare different from the control, P < 0.01. natal rats were weaned at 21 days of age. Male and female rats were randomly chosen from each litter for use in experiments. For the studies involving endocnne ablations. (ration of free choline remained essentially at this male rats were obtained surgically prepared from Ziviclowered level for u p to 22 days while rats were fed Miller Corp. (Allison Park. PA). In all other studies, male the diet deficient in choline. When rats were raised Sprague-Dawley rats were obtained from Holtzman Corp. from birth until 21 weeks of age o n the choline-defi(Madison. WI). cient diet, free choline was also reduced (357;) in [Methyl-3H]choline chloride was obtained from New plasma (Table 1). England Nuclear Corp. (Boston. MA). The isotope had To assess the relationship between the concena specific activity of 2.4 Cihnmol. Each rat received 1.4 tration of choline in the plasma and the amount in mCi,'kg (0.58 pmol/kg) into a tail vein. Unless otherwise indicated, statistical comparisons were the diet, choline chloride was added to the drinking water of rats fed the choline-deficient diet. As shown performed using the Student's t test. in Fig. 2. addition of choline to the water caused a n C/irrr~rc.tr/procedures. Choline and ACh were isolated by high-voltage paper electrophoresis and measured by use increase in the concentration of choline in plasma. of an enzymatic (choline kinase)-radio-isotopic assay (REID The highest concentration of plasma choline was et a/., 1971) with modifications (HAUBRICH & REID. 1974). observed when the concentration of choline in the For the determination of choline in plasma, mixed arterial drinking water was 15-30 mM; a further increase in and venous blood was collected by draining the blood the concentration of choline in the water caused n o from the carcasses of decapitated rats. Assay of choline additional elevation in the concentration of plasma was performed in unextracted plasma in the presence of heparine by use of the choline kinase procedure (WANG & HAUBRICH, 1975). Deanol (HAUBRICH et a/.. 1975~)cytidine diphosphocholine and phosphorylcholine (WANG& HAUBRICH. 1975) do not interfere in the assay of choline. Metabolites of [methyl--'H]choline were isolated by paper electrophoresis and chromatography and measured as preet a/.. 197Sbj. Uptake of viously described (HAUBRICH [methyl-3H]choline by synaptosomes was also determined et u/.. 1975a). The Concentration as described (HAUBRICH of protein was measured spectrophotometrically as described by LOU'RYer al. (1951) with bovine serum albumin as the standard. RESULTS
Tissirr cor~etirratior7of cholirie As shown in Figs. 1 and 2, the concentration of choline in plasma was related, within limits. t o the amount present in the diet. When the diet deficient in free choline was fed to rats, the concentration of plasma choline was maximally depleted within 1 day to a level half that observed in rats that consumed the choline-supplemented diet (Fig. 1). The concen-
, I , 012 4
8
1 15
1 60
30
Choline in drinking water,
mM
FIG. 2. Concentration of free choline in plasma plotted as a function of the concentration of choline in the drinking water. All rats were given a diet containing 0.07 mmol choline per kg for 11 days. along with drinking water containing varying amounts of choline chloride. Each point is the average of 3 or 4 rats per group. Values for the four highest concentrations of choline are significantly greater ( P i0.05) than the lowest values
Choline. ACh and dietary choline
I307
TABLE I . CHOLINE AND ACETYLCHOLINEI N PERIPHERAL TISSUES OF RATS RAISED ON
A CHOLINE-DEFI-
CENT DIET*
Control (nmoljg f s.E.)
Tissue Duodenum Heart Kidney Liver Plasma Stomach
Choline Choline-deficient (nmolig f s.E.)
176 k 1 I 185 I 91 + 3 48 10 8.2 0.3 83 6
Acetylcholine Control Choline-deficient (nmol/g k s.E.) (nmolig f s.E.)
*
18.0 k 2.8 5.6 0.4 1 . 1 0.3 N.D. N.D. 12.9 0.5
Ilt 10 f I $ 55 f 6$ 16 f 4$ 5.3 0.21 47 & 3$
115
17.9 k 2.2 5.1 k 0.5 1.2 & 0.3 N.D. N.D. 12.3 f-1.3
+
*
*
* One to 4 days prior to parturition, pregnant rats were given either a control diet containing 12.6 mmol cholinelkg or an otherwise identical diet containing 0.07 mmol choline/kg. At 21 weeks post-partum, [methyl-3H]choline (1.4 mCi/kg: 0.58 pmol/kg was administered intravenously to the offspring. Rats were decapitated 1 min after administration of the isotope. Tissues (N = 5-7) were analyzed for endogenous choline and acetylcholine (shown here) and for radioactivity (shown in Tables 5 and 6). t Different from control, P < 0.01. 3 Different from control, P < 0.001. N.D. = not detectable. choline (Fig. 2). Addition of choline chloride to the drinking water did not significantly affect the amount of liquid consumed (30-40 mllratjday). To determine if a precursor for choline could substitute for free choline in the diet, rats were given drinking water containing either deanol (dimethylaminoethanol) or an equimolar concentration of choline. As shown in Table 2, the concentration of choline in plasma of rats that received deanol, but were deprived of choline (group 9, was significantly greater than that of the choline-deficient rats (group l), but was nearly the same as the level of choline in the rats receiving the choline-supplemented diet (group 2). The addition of deanol to the drinking water did not significantly affect the concentration of choline in plasma of rats that received sufficient choline in the diet (group 6). The concentration of choline in plasma of rats that consumed deanol (groups 5 and 6) was less than that of rats given an equivalent
amount of choline in the drinking water (group 3 and 4).
Dietary deprivation of free choline also caused a reduction in the concentration of free choline in tissues other than plasma. The duodenum was chosen for comparison of the time-course of depletion of plasma choline with the depletion from a tissue innervated by the cholinergic nervous system. As had occurred in the plasma, maximal depletion of choline (31”,) was observed within one day in the duodenum of rats fed the choline-deficient diet as compared to animals that received the choline-supplemented diet (Fig. 3). The concentration of free choline remained at this same level in the duodenum for up to 1 1 days while the rats consumed the choline-deficient diet. A
TAHLF. 2. EFFECTOF
CHOLINE OR DEANOL I N THE DRINKING WATER ON PLASMA CHOLINE*
Group No.
Diet
I
CD
2 3 4 5 6
CD CS CD CS
cs
Addition t o drinking water
Plasma choline (nmol/ml f s.E.)
None None Choline Choline Deanol Deanol
5.6 k 0.7 10.7 k 1.0f 20.6 2.2 17.5 k 2.1 12.9 & 0.8t 13.3 1.4
+
* Rats were given either a choline-supplemented (CS) diet containing 12.6 mmol choline/kg. or a choline-deficient (CD) diet containing 0.07 mmol cholineikg. along with tap water that contained either choline chloride ( 6 0 m ~ or ) deanol (60mM). Rats were decapitated and assays performed 11 days after starting the dietary regimen. Values are the averages of 6 rats per group. t Different than CD with no addition to drinking water, P < 0.01.
J6
~
I 5 Time,
days
FIG.3. Concentration of choline and acetylcholine in the duodenum after rats consumed a diet containing 0.07 mmol choline per kg. Each point is the average of 5 or 6 rats. All values for choline were significantly lower (P < 0.01) than the zero-time control value (rats fed a diet containing 12.6 mmol choline per kg for 11 days). The rats fed a choline-deficient diet for 1 day had a higher concentration of acetylcholine (P < 0.05). whereas the level of the neurotransmitter was the same as that of the control at 5 and 11 days.
1308
D. R. HACBRICH tv 01. TABLE 3. EFFFCTOF
VARIOLS FACTORS O S THE CONCEZTRATION OF PLASMA CHOLINE
Plasma choline* (Percentage of controls f S.L)
Treatment Eiidocririe
Adrenalectomy Thyroidectomy Hq pophysectomy Epinephrine (0.5 mg kg. i.p.-?0 rnin.) Insulin (20 units kg. i . p . 4 0 rnin.) Prostaglandin EL ( I mg kg. i.p.-60 min.) Strrss Fasted (74 h ) Immobilization (amin) Cold ( 3 h at 5 C: 1 rat cage) Ckoliric, prrcur.sor.< Choline C1 (100 mg kg. i.p.) Cholinc C1 (1W nig kg. i.p.) Cqtidine diphosphocholine (700 mg kg. i.p.) Cytidine diphosphocholine 1200 mg kg. i.p.) Phosphorylcholine (200 mg kg. i.p.1 Phosphorylcholine (200 mg kg. i.p.1
97 8 89 83 96 83
f4 i6 f5 f 16 f5 f4
81 1 7 91 1 3 91 & 6
20 niin 120 niin 60 min 170 niin 60 niin 120 min
506 f 40t 110 8 270 f 5 t 107 f 4 171 f 26t 109 f 8
*
Drirgs
Chlorpromazine (10 mg kg. p.0.) Pilocarpine HCI (10 mg'kg. i.p.) Amphetamine sulfate ( 5 mg kg. i.p.) Hemicholinium-3 (0.I mg.kg. i.p.) Dimethylphenylpiperazinum ( 1 mg kg. i.p.) Ether ( 3 rnin inhalation) i-iI?W 01 drorh 0200 0500 0800
I100 0.100 1 700 2000 2300 0200 (next day)
3h 60 min
60 min 60 min 30 min
98 f 6 95 f 6 91 * 4 99 f 5 88 f 5 101 f 3 104 f 73 loOf3 130 1 23 105 6 112 f 7 119 7 107 f 10 113 7 104 f I
* *
*Percentages were calculated from the averages of 5-10 rats per group. and statistical comparisons (Student's I ) \\ere made using :I group of appropriately treated control rats ( N = 4--l5) for each experiment. For the study in which rats were sacrificed a t different time of the day. the group with the lottest value (sacrificed at 0500 11) \
