- Email: [email protected]
The nutrition of choline, carnitine, and related compounds in the blowfly, Phormia regina Meigen
J. Ins. Physiol., 1964, Vol. 10, pp. 1005 to 1008. PergmnonPress Ltd. Printed in Great Britain
THE NUTRITION OF CHOLINE, CARNITINE, AND RELATED COMPOUNDS IN THE BLOWFLY, PHORiWA REGINA MEIGEN” ERNEST
HODGSON
and WALTER
C. DAUTERMAN
Department of Entomology, North Carolina State of the University of North Carolina at Raleigh, Raleigh, North Carolina, U.S.A. (Received 14 May 1964) Abstract-Fifteen choline analogues were tested as substitutes for choline in the larval diet of Phormia regina. From the results the structural requirements for an adequate choline substitute are further defined. The choline analogue, (CH,),+NCH&H,CH,OH, although not an inhibitor in the presence of choline, inhibits growth when choline is replaced in the diet by carnitine or This compound presumably inhibits one of the reactions y-butyrobetaine. which metabolizes carnitine or y-butyrobetaine to p-methyl choline. INTRODUCTION
A CHOLINE requirement for growth has been demonstrated for a number of species (see compilation by GILMOUR, 1961, p. 17), and a specific requirement for carnitine is known for a number of tenebrionid beetles (FRAENKELand FRIEDMAN,1957). In two insect species, Drosophila melanogaster (FRAENKELet al., 1955) and Phormia regina (HODGSON et al., 1956) carnitine can substitute for choline in the larval diet. In the latter species further investigations by HODGSON et al. (1960) have shown that choline can be replaced by y-butyrobetaine and 2,2-dimethylaminoethanol and that all four compounds will reverse the inhibition of growth due to the choline antagonist, 2-amino-2-methyl propanol. Although phospholipids containing ethanolamine predominate in P. regina (BIEBER et al., 1961), those containing choline form a significant part. When choline is replaced in the larval diet by carnitine or y-butyrobetaine the phospholipids containing choline are almost completely replaced by phospholipids containing p-methyl choline (BIEBERet al., 1963). Moreover, BIEBERand NEWBURGH(1963) have shown that the dimethyl analogue of P-methyl choline, 2,2_dimethylaminoisopropanol, as well as 2,2-dimethylaminoethanol are incorporated into lipids in place of choline. The investigations reported are the results of experiments to define further the relationship between chemical structure and the ability of compounds to substitute for choline in the larval diet of P. regina. * Contribution from the Entomology Department, North Carolina Agricultural Experiment Station, Raleigh, North Carolina. Published with the approval of the Director of Research as Paper No. 1762 of the Journal Series. 1005
1006
ERNESTHODGSON ANDWALTERC. DAUTERMAN METHODS
AND MATERIALS
The choline analogues used were synthesized by methods previously described (DATJTERMANand MEHROTRA, 1963) and converted to the acetate before use. These compounds were tested as dietary substitutes for choline or as choline antagonists by the method of HODGSONet al. (1960), a modification of the defined diet described by MCGINNIS et al. (1956). Each compound was added to the choline-free diet at two levels 0.71 pmole/ml diet and 3.55 pmoles/ml diet. The lower level is in excess of the normal choline requirement. When testing these compounds as possible antagonists in the presence of choline, carnitine, or y-butyrobetaine, they were present at a level of 3.55 pmoles/ml diet and choline, carnitine, or y-butyrobetaine at a level of 0.71 pmolejml diet. The criterion of growth used was the weight gain after 7 days growth at 27°C. In each experiment all diets were tested in duplicate, and each experiment was performed two or three times. RESULTS
AND DISCUSSION
The results of experiments in which various compounds were used as replacements for choline in the diet are seen in Table 1. The first group of compounds (I) differ from choline in having one or two additional carbon atoms in the carbon chain. Neither will support adequate growth although some stimulation occurs. The next group (II) differs in having one of the N-alkyl groups other than methyl. It is apparent that with chain lengths up to n-butyl these compounds show excellent ability to substitute for choline. It is of interest that the dimethyl isopropyl analogue is less active than the dimethyl n-propyl analogue. In group III the compounds differ from choline in that two of the N-alkyl groups are other than methyl. Only the diethyl methyl analogue is stimulatory, increasing the size of the two alkyl groups eliminates the effectiveness. The last group (IV) contains compounds in which all the N-alkyl groups differ from methyl. Of these only the triethyl analogue has any effect and this is not striking. In addition to the above, the following compounds are known to be effective substitutes for choline in the larval diet: 2,2-dimethylaminoethanol; carnitine; y-butyrobetaine (HODGSON et al., 1960); methyl choline; 2,2-dimethylaminoisopropanol (BIEBER and NEWBURGH,1963). When these previously reported results are considered together with those reported in this paper certain general conclusions on the necessary characteristics for an effective choline substitute can be made. Such a compound must have: at least two N-methyl groups (the only exception to this is the diethyl .methyl analogue) ; the third alkyl group can vary, at least up to The latter involves the loss of the n-butyl, or it can be missing completely. quaternary nitrogen structure but as the dimethyl compounds would be expected to be protonated at physiological pH the difference may not be large. In an effective choline substitute the hydroxyl group must be on the second carbon from the nitrogen or, alternatively stated, if a terminal hydroxyl group is present the carbon chain must be two carbon atoms long.
NUTRITION OF CHOLINE, CARNITINE, AND RELATED COMPOUNDS IN THE BLOWFLY TABLE ~-EFFECTIVENESS
OF VARIOUS CHOLINE ANALOGUES AS SUBSTITUTES FOR CHOLINE IN THE LARVAL DIET OF P. ?Vgirm Total No. larvae
Av. wt.1 larva (mg)
Choline-deficient diet (CDD) CDD + (CH,),+NCH,CH,OH
50 37
5.7 23.8
CDD CDD
+ (CH,),+NCH,CH,CH,OH + (CH,),+NCH,CH,CH,CH,OH
28 34
7.6 11.8
CDD CDD CDD CDD
+ (C,H,) (CH&+NCH&H20H -k (n-&H,) (CH,),+NCH,CH,OH + (i-&H,) (CH,),+NCH,CH,OH -!-(n-&H,) (CH,),+NCH,CH,OH
26 23 37 29
30.4 28.1 18.0 22.6
CDD CDD CDD
+ (CpH&(CHQ)+NCH2CH20H + (nCOH,)e(CH3)+NCH2CH,0H + (nC,H,),(CH,)+NCH,CH,OH
26 23 16
19.2 6.6 6.4
CDD CDD CDD CDD CDD CDD
-f + + -t + -t
30 25 27 25 26 24
11.9 7.7 6.8 3.6 8.5 7.6
Growth
I
II
III
IV
All compounds
medium
(CaH,),+NCH,CH,OH (C,H,),(nC,H,)+NCH,CH,OH (nC,H,),(CzH,)+NCH,CH,OH (nC,H,),(C,H,)+NCH,CH,OH (nC,H,),(C4H,)+NCH,CH,0H (nC,H,),+NCH&H,OH
added to choline-deficient
TABLE 2-EFFECTIVENESS
Growth
CDD
diet at a concentration
of 0.71 pmole/ml.
OF CHOLINE ANALOGUES AS GROWTH INHIBITORS IN THE PRESENCE OF CARNITINE AND y-BUTYROBETAINE medium
Choline-deficient diet CDD + Carnitine CDD + Camitine and (CH,),+NCH,CH,CH,OH CDD + Carnitine and (CH,),+NCH,CH,CH,CH,OH CDD CDD
1007
+ y-Butyrobetaine -t y-Butyrobetaine and (CH,),+NCH,CH,CH,OH + y-Butyrobetaine and (CH,),+NCH,CH,CH,CH,OH
Carnitine and y-butyrobetaine added 0.71 pmole/ml, (CH3)a+NCH&H&Hz0H concentration of 3.55 ~mole/ml.
Total No. larvae
Av. wt./ larva (mg)
Mortality
26 23
11.5 34.7
19 0
26
16.5
65
28
38.4
0
35
30.4
0
22
15.0
91
17
40.5
0
(%)
to choline-deficient diet at a concentration of and (CH3)a+NCH2CH&H&H20H at a
1008
ERNESTHODGSONANDWALTER C. DAUTERMAN
All of the compounds listed in Table 1 -were tested as choline antagonists in the presence of choline, and no inhibition of growth was apparent in any case. Those compounds of chain length greater than two were tested as growth inhibitors in the presence of carnitine and y-butyrobetaine. It is apparent from the results in Table 2 that the choline analogue with three carbon atoms in the chain [(CH,),+NCH,CH,CH,OH], although not an inhibitor in the presence of choline, is an inhibitor when choline is replaced in the larval diet by carnitine or y-butyrobetaine. Since these two latter compounds are known to be metabolized to methyl choline (BIEBERet al., 1963), it is apparently one of the reactions involved which is inhibited. Although a number of choline substitutes are known to be incorporated into the phospholipids of Phormia regina larvae either unchanged or as P-methyl choline, it is still not known whether the specificity of the nutritional requirement for choline type compounds is determined primarily by the specificity of the enzymes involved in phospholipid metabolism or whether the specificity of some other choline metabolizing enzymes is involved. The characteristics of choline esterase, choline acetylase, and choline oxidase from this organism are as yet unknown. Acknowledgements-This research was supported in part by a grant from the National Institutes of Health, U.S. Public Health Service, No. AlO-04465. The authors gratefully acknowledge the technical assistance of Mrs. BARBRAHUBBELLand Mrs. NANCYHENDERSON.
REFERENCES BIEBERL. L., CHELDELINV. H., and NEWBURGHR. W. (1963) Studies on a p-methyl choline containing phospholipid derived from carnitine. J. biol. Chem. 238, 1262-1265. BIEBERL. L., HODGSONE., CHELDELINV. H., BROOKES V. J., and NEW~URGHR. W. (1961) Phospholipid patterns in the blowfly, Phormia regina (Meigen). J. biol. Chem. 236, 2590-2595.
BIEBERL. L. and NEWBURGHR. W. (1963) The incorporation of dimethylaminoethanol and dimethylaminoisopropyl alcohol into Phomzia regina phospholipids. J. Lipid Res. 4, 397-401.
DAUTERMANW. C. and MEHROTRAK. N. (1963) The N-alkyl group specificity of choline acetylase from rat brain. J. Neurqchem. 10, 113-117. FRAENKEL G. S. and FRIEDMANS. (1957) Carnitine. Vitam. & Horm. 15,74-118. FRAENKEL G. S., FRIEDMANS., HINTON T., LASZIO S., and NOLANDJ. L. (1955) The effect of substituting carnitine for choline in the nutrition of several organisms. Arch. Biochem. Biophys.
54, 432-439.
GILMOURD. (1961) The Biochemistry of Imects. Academic Press, New York and London. HODGSONE., CHELDELINV. H., and NEWBURGHR. W. (1956) Substitution of choline by related compounds and further studies on amino acid requirements in nutrition of Phormia regina (Meig.). Canad. J. Zool. 34, 527-532. HODGSONE., CHELDELINV. H., and NEWBURGHR. W. (1960) Nutrition and metabolism of methyl donors and related compounds in the blowfly, Phormia regina (Meigen). Arch.
Biochem.
Biophys.
87, 48-54.
MCGINNIS A. J., NEWBURGHR. W., and CHELDELINV. H. (1956) Nutritional studies on the blowfly, Phormia regina (Meig.). J. Nuts. 58, 309-323.
THE NUTRITION OF CHOLINE, CARNITINE, AND RELATED COMPOUNDS IN THE BLOWFLY, PHORiWA REGINA MEIGEN” ERNEST
HODGSON
and WALTER
C. DAUTERMAN
Department of Entomology, North Carolina State of the University of North Carolina at Raleigh, Raleigh, North Carolina, U.S.A. (Received 14 May 1964) Abstract-Fifteen choline analogues were tested as substitutes for choline in the larval diet of Phormia regina. From the results the structural requirements for an adequate choline substitute are further defined. The choline analogue, (CH,),+NCH&H,CH,OH, although not an inhibitor in the presence of choline, inhibits growth when choline is replaced in the diet by carnitine or This compound presumably inhibits one of the reactions y-butyrobetaine. which metabolizes carnitine or y-butyrobetaine to p-methyl choline. INTRODUCTION
A CHOLINE requirement for growth has been demonstrated for a number of species (see compilation by GILMOUR, 1961, p. 17), and a specific requirement for carnitine is known for a number of tenebrionid beetles (FRAENKELand FRIEDMAN,1957). In two insect species, Drosophila melanogaster (FRAENKELet al., 1955) and Phormia regina (HODGSON et al., 1956) carnitine can substitute for choline in the larval diet. In the latter species further investigations by HODGSON et al. (1960) have shown that choline can be replaced by y-butyrobetaine and 2,2-dimethylaminoethanol and that all four compounds will reverse the inhibition of growth due to the choline antagonist, 2-amino-2-methyl propanol. Although phospholipids containing ethanolamine predominate in P. regina (BIEBER et al., 1961), those containing choline form a significant part. When choline is replaced in the larval diet by carnitine or y-butyrobetaine the phospholipids containing choline are almost completely replaced by phospholipids containing p-methyl choline (BIEBERet al., 1963). Moreover, BIEBERand NEWBURGH(1963) have shown that the dimethyl analogue of P-methyl choline, 2,2_dimethylaminoisopropanol, as well as 2,2-dimethylaminoethanol are incorporated into lipids in place of choline. The investigations reported are the results of experiments to define further the relationship between chemical structure and the ability of compounds to substitute for choline in the larval diet of P. regina. * Contribution from the Entomology Department, North Carolina Agricultural Experiment Station, Raleigh, North Carolina. Published with the approval of the Director of Research as Paper No. 1762 of the Journal Series. 1005
1006
ERNESTHODGSON ANDWALTERC. DAUTERMAN METHODS
AND MATERIALS
The choline analogues used were synthesized by methods previously described (DATJTERMANand MEHROTRA, 1963) and converted to the acetate before use. These compounds were tested as dietary substitutes for choline or as choline antagonists by the method of HODGSONet al. (1960), a modification of the defined diet described by MCGINNIS et al. (1956). Each compound was added to the choline-free diet at two levels 0.71 pmole/ml diet and 3.55 pmoles/ml diet. The lower level is in excess of the normal choline requirement. When testing these compounds as possible antagonists in the presence of choline, carnitine, or y-butyrobetaine, they were present at a level of 3.55 pmoles/ml diet and choline, carnitine, or y-butyrobetaine at a level of 0.71 pmolejml diet. The criterion of growth used was the weight gain after 7 days growth at 27°C. In each experiment all diets were tested in duplicate, and each experiment was performed two or three times. RESULTS
AND DISCUSSION
The results of experiments in which various compounds were used as replacements for choline in the diet are seen in Table 1. The first group of compounds (I) differ from choline in having one or two additional carbon atoms in the carbon chain. Neither will support adequate growth although some stimulation occurs. The next group (II) differs in having one of the N-alkyl groups other than methyl. It is apparent that with chain lengths up to n-butyl these compounds show excellent ability to substitute for choline. It is of interest that the dimethyl isopropyl analogue is less active than the dimethyl n-propyl analogue. In group III the compounds differ from choline in that two of the N-alkyl groups are other than methyl. Only the diethyl methyl analogue is stimulatory, increasing the size of the two alkyl groups eliminates the effectiveness. The last group (IV) contains compounds in which all the N-alkyl groups differ from methyl. Of these only the triethyl analogue has any effect and this is not striking. In addition to the above, the following compounds are known to be effective substitutes for choline in the larval diet: 2,2-dimethylaminoethanol; carnitine; y-butyrobetaine (HODGSON et al., 1960); methyl choline; 2,2-dimethylaminoisopropanol (BIEBER and NEWBURGH,1963). When these previously reported results are considered together with those reported in this paper certain general conclusions on the necessary characteristics for an effective choline substitute can be made. Such a compound must have: at least two N-methyl groups (the only exception to this is the diethyl .methyl analogue) ; the third alkyl group can vary, at least up to The latter involves the loss of the n-butyl, or it can be missing completely. quaternary nitrogen structure but as the dimethyl compounds would be expected to be protonated at physiological pH the difference may not be large. In an effective choline substitute the hydroxyl group must be on the second carbon from the nitrogen or, alternatively stated, if a terminal hydroxyl group is present the carbon chain must be two carbon atoms long.
NUTRITION OF CHOLINE, CARNITINE, AND RELATED COMPOUNDS IN THE BLOWFLY TABLE ~-EFFECTIVENESS
OF VARIOUS CHOLINE ANALOGUES AS SUBSTITUTES FOR CHOLINE IN THE LARVAL DIET OF P. ?Vgirm Total No. larvae
Av. wt.1 larva (mg)
Choline-deficient diet (CDD) CDD + (CH,),+NCH,CH,OH
50 37
5.7 23.8
CDD CDD
+ (CH,),+NCH,CH,CH,OH + (CH,),+NCH,CH,CH,CH,OH
28 34
7.6 11.8
CDD CDD CDD CDD
+ (C,H,) (CH&+NCH&H20H -k (n-&H,) (CH,),+NCH,CH,OH + (i-&H,) (CH,),+NCH,CH,OH -!-(n-&H,) (CH,),+NCH,CH,OH
26 23 37 29
30.4 28.1 18.0 22.6
CDD CDD CDD
+ (CpH&(CHQ)+NCH2CH20H + (nCOH,)e(CH3)+NCH2CH,0H + (nC,H,),(CH,)+NCH,CH,OH
26 23 16
19.2 6.6 6.4
CDD CDD CDD CDD CDD CDD
-f + + -t + -t
30 25 27 25 26 24
11.9 7.7 6.8 3.6 8.5 7.6
Growth
I
II
III
IV
All compounds
medium
(CaH,),+NCH,CH,OH (C,H,),(nC,H,)+NCH,CH,OH (nC,H,),(CzH,)+NCH,CH,OH (nC,H,),(C,H,)+NCH,CH,OH (nC,H,),(C4H,)+NCH,CH,0H (nC,H,),+NCH&H,OH
added to choline-deficient
TABLE 2-EFFECTIVENESS
Growth
CDD
diet at a concentration
of 0.71 pmole/ml.
OF CHOLINE ANALOGUES AS GROWTH INHIBITORS IN THE PRESENCE OF CARNITINE AND y-BUTYROBETAINE medium
Choline-deficient diet CDD + Carnitine CDD + Camitine and (CH,),+NCH,CH,CH,OH CDD + Carnitine and (CH,),+NCH,CH,CH,CH,OH CDD CDD
1007
+ y-Butyrobetaine -t y-Butyrobetaine and (CH,),+NCH,CH,CH,OH + y-Butyrobetaine and (CH,),+NCH,CH,CH,CH,OH
Carnitine and y-butyrobetaine added 0.71 pmole/ml, (CH3)a+NCH&H&Hz0H concentration of 3.55 ~mole/ml.
Total No. larvae
Av. wt./ larva (mg)
Mortality
26 23
11.5 34.7
19 0
26
16.5
65
28
38.4
0
35
30.4
0
22
15.0
91
17
40.5
0
(%)
to choline-deficient diet at a concentration of and (CH3)a+NCH2CH&H&H20H at a
1008
ERNESTHODGSONANDWALTER C. DAUTERMAN
All of the compounds listed in Table 1 -were tested as choline antagonists in the presence of choline, and no inhibition of growth was apparent in any case. Those compounds of chain length greater than two were tested as growth inhibitors in the presence of carnitine and y-butyrobetaine. It is apparent from the results in Table 2 that the choline analogue with three carbon atoms in the chain [(CH,),+NCH,CH,CH,OH], although not an inhibitor in the presence of choline, is an inhibitor when choline is replaced in the larval diet by carnitine or y-butyrobetaine. Since these two latter compounds are known to be metabolized to methyl choline (BIEBERet al., 1963), it is apparently one of the reactions involved which is inhibited. Although a number of choline substitutes are known to be incorporated into the phospholipids of Phormia regina larvae either unchanged or as P-methyl choline, it is still not known whether the specificity of the nutritional requirement for choline type compounds is determined primarily by the specificity of the enzymes involved in phospholipid metabolism or whether the specificity of some other choline metabolizing enzymes is involved. The characteristics of choline esterase, choline acetylase, and choline oxidase from this organism are as yet unknown. Acknowledgements-This research was supported in part by a grant from the National Institutes of Health, U.S. Public Health Service, No. AlO-04465. The authors gratefully acknowledge the technical assistance of Mrs. BARBRAHUBBELLand Mrs. NANCYHENDERSON.
REFERENCES BIEBERL. L., CHELDELINV. H., and NEWBURGHR. W. (1963) Studies on a p-methyl choline containing phospholipid derived from carnitine. J. biol. Chem. 238, 1262-1265. BIEBERL. L., HODGSONE., CHELDELINV. H., BROOKES V. J., and NEW~URGHR. W. (1961) Phospholipid patterns in the blowfly, Phormia regina (Meigen). J. biol. Chem. 236, 2590-2595.
BIEBERL. L. and NEWBURGHR. W. (1963) The incorporation of dimethylaminoethanol and dimethylaminoisopropyl alcohol into Phomzia regina phospholipids. J. Lipid Res. 4, 397-401.
DAUTERMANW. C. and MEHROTRAK. N. (1963) The N-alkyl group specificity of choline acetylase from rat brain. J. Neurqchem. 10, 113-117. FRAENKEL G. S. and FRIEDMANS. (1957) Carnitine. Vitam. & Horm. 15,74-118. FRAENKEL G. S., FRIEDMANS., HINTON T., LASZIO S., and NOLANDJ. L. (1955) The effect of substituting carnitine for choline in the nutrition of several organisms. Arch. Biochem. Biophys.
54, 432-439.
GILMOURD. (1961) The Biochemistry of Imects. Academic Press, New York and London. HODGSONE., CHELDELINV. H., and NEWBURGHR. W. (1956) Substitution of choline by related compounds and further studies on amino acid requirements in nutrition of Phormia regina (Meig.). Canad. J. Zool. 34, 527-532. HODGSONE., CHELDELINV. H., and NEWBURGHR. W. (1960) Nutrition and metabolism of methyl donors and related compounds in the blowfly, Phormia regina (Meigen). Arch.
Biochem.
Biophys.
87, 48-54.
MCGINNIS A. J., NEWBURGHR. W., and CHELDELINV. H. (1956) Nutritional studies on the blowfly, Phormia regina (Meig.). J. Nuts. 58, 309-323.