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Differential regulation of the formation of prostaglandins and related substances from arachidonic acid and from dihomogammalinolenic acid. II. Effects of vitamin C
Prostaglandins
and
Medicine
3: 129-137,
1979
DIFFERENTIAL REGULATION OF THE FORMATION OF PROSTAGLANDINS AND RELATED SUBSTANCES FROM ARACHIDONIC ACID AND FROM DIHOMOGAMMALINOLENIC ACID. I I. EFFECTS OF VITAMIN C. M.S. Manku, M. Oka, and D.F. 110 Pine Avenue West, Montreal
Horrobin, Clinical H2W lR7, Canada.
Research
Institute,
ABSTRACT Vitamin C over the concentration range 10 to 100 ug/ml (5.7-57 x 10-S M) caused a dose dependent and highly significant enhancement of conversion of 14C-dihomogammalinolenic acid (DGLA) to prostaglandin (PG) El and to thromboxane (TX) Bl by human platelets. Vitamin C had no effect on conversion of 14C-arachidonic acid to PGE2 and TxB2. The concentration range is relevant to physiology: in some cells which concentrate the vitamin, such as polymorphonuclear leucocytes and the adrenal cortex, vitamin C concentrations may be substantially higher than 100 ug/ml. Vi tami n C can therefore selectively enhance the formation of cycle-oxygenase generated products from DGLA without changing formation of those from AA. This effect can account for a number of the known actions of vitamin C including The implications of this finding are its effect on the immune system. discussed. INTRODUCTION The idea that it might be possible to regulate selectively the metabolism of arachidonic acid (AA) and dihomogammalinolenic acid (DGLA) by the cyclooxygenase system has received little if any attention. If such selective regulation were possible, new therapeutic concepts could be developed. For example, in human platelets the overall effect of the products of DGLA metabol ism is anti-aggregatory whereas the overal 1 effect of the AA metabol i tes i s pro-aggregatory (1) . It would therefore be advantageous in platelets to enhance DGLA conversion selectively. In the first paper in this series we reported that ethanol in concentrations relevant to human intoxication had a selective effect in enhancing the formation of 1 series prostaglandins (PGs) and TxBl from DGLA, while having no significant effect on arachidonic acid metabol ism (2). In this paper we report that physiolog ical concentrations of vitamin C can also selective ly enhance format ion of 1 series PGs by human platelets.
129
MATERIALS
AND METHODS
11-14C1 arachidonic acid and [Il-14C1 dihomogammalinolenic acid were purchased from New England Nuclear. They were diluted with hexane to specific activities of about 5 pCi/limol. PGs and thromboxanes were kindly supplied by Dr. John Pike, Upjohn Co., Kalamazoo, Michigan. All organic solvents were reagent grade and were obtained from BDH, Montreal, except for ethanol which was obtained from Fisher, Montreal. One day expired (2 days old) human platelets were obtained from the Canadian Red Cross, Montreal. Silica gel thin layer chromatography (TLC) plates were obtained from Analtech inc. The platelet concentrates were always used within 48 hours of expiration. One unit was centrifuged at 1000 g for 15 minutes and the supernatant drawn off. The platelet pellet was resuspended in Tris-NaCl-EDTA buffer as described by Hamberg et al (3). The buffer was made up of 0.15 M NaCl, 0.15 M Tris HCl at pH 7.4 and 0.077 M NaEDTA (gO:8:2 v/v/v). The platelets were recentrifuged, the supernatant removed and the pellet resuspended in Krebs-Henseleit buffer (without calcium) at pH 7.4. The Al 1 washed platelet suspension contained about 1-28 red blood cells. glassware used in the preparation of the platelets were siliconized using “Prosi 1” (Canlab, Montreal). Each day a stock sol ut ion of 10 mg/ml Lascorbic acid (Sigma) was prepared and brought to pH 7.0 with sodium hydroxide. Four equal sized 1 ml aliquots of the platelet suspension, containing 109 14C-DGLA for five minutes. At platelets/ml were incubated with 0.5 UCi the beginning of the incubation ascorbate in concentrations of 0, 10, 33 and 100 ug/ml (5.7, 18.8 and 57 x lo-5 M) was added to the suspensions. The reaction was stopped after five minutes by addition of l/10 volume of 10% formic acid. The suspension was then extracted three times with ethyl acetate. The three ethyl acetate fractions obtained from each alcohol concentration were pooled and dried under vacuum. The extract was v/v). Recovery of radiothen taken up with 5 ml chloroform/methanol (2/l, active material in the extract was checked by taking 50 ~1 of the chloroRecovery was in the form/methanol and counting by liquid scintillation. range 80-952 in most experiments. The chloroform/methanol extract was then reduced in volume to 1 ml under dry prepurified nitrogen. Thin layer chromatography was carried out on 500 ug precoated, prescored silica gel G Uniplates (Analtech). Plates were activated by heating to 100°C for 1 hour immediately prior to use. The solvent system was chloroform:methanol:acetic acid:water (gO:8:1:0.8). Reference compounds (PGs El, Fla, and thromboxane Bl) were run at the same time and visualized by phosphomolybdic acid spray followed by brief heating. The bands on the plates corresponding to the reference PGEl, PGFlcl and TxBl were scraped off and eluted with 20 ml acetone. Each elution was then evaporated to dryness and counted by liquid scintillation (Beckman 100 LS counter). Using the same batch of platelets at the same time exactly similar experiments were carried out with 14C-AA and PGE2, PGE2ct and TxB2 as reference compounds. Six experiments were performed with DGLA and three with AA.
130
Table
1.
Counts appearing after incubation refer to difference t test.
in of
Control
Table
2.
the band eluting with the TxBl standard human platelets with DGLA. The p values from control as estimated by the paired
wm
SD
7529
2122
_____-
P'
10 ug/ml
ascorbate
3783
2744
_____-
33 ug/ml
ascorbate
11067
2582
0.0125
100 ug/ml
ascorbate
12839
3212
0.005
Counts after refer test.
appearing incubation to difference
in of
Control
the band travel1 ing with the PGEl standard human platelets with DGLA. The p values from control as estimated by the paired t
wm
SD
7642
2001
__cc--
P <
10 pg/ml
ascorbate
10271
2337
0.05
33 pg/ml
ascorbate
10331
3774
0.05
ascorbate
14067
3762
0.0025
100
ug/ml
131
RESULTS In the three experiments conducted with three concentrations used had no effect PGF2a.
arachidonic acid on the formation
vitamin C at the of TxB2, PGE2 or
Vitamin C did enhance the conversion of DGLA by cycle-oxygenase PGEl and PGF2a. The effect was dose dependent and statistically significant. The mean absolute counts are shown in Tables 1, Table 4 summarises the results in a percentage form.
to TxBl, highly 2 and 3.
DISCUSSION Vitamin C enhanced the formation of TxBl, PGEl and PGFlcl from DGLA without altering the formation of the equivalent compounds from arachidonic acid. As reported by others (1,4) approximately equal amounts of TxBl and PGEl but much smaller amounts of PGFlcl are produced by human platelets. The increase in formation of all three products in the presence of vitamin C suggests that the main action of the vitamin is at or before the cyclooxygenase level. Small effects beyond the cycle-oxygenase level cannot, however, be excl uded. The first paper in this series (2) reported a similar effect of ethanol on DGLA metabolism. In molar terms the effect of vi tami n C was approximately one order of magnitude greater than that of ethanol. In vivo the two compounds would be expected to have substantially different effects. Ethanol is consumed in very much larger quantities than vitamin C and its concentrations in most body fluids go very much higher than those of vitamin C, more than compensating for the reduced effectiveness. Ethanol is metabolized rapidly and its effects are relatively transient. Ethanol is also rapidly and evenly distributed throughout the body (5). Vitamin C, in contrast, is consumed in much smaller quantities and is stored in the body. Moreover it is very unevenly distributed since a number of tissues seem to have special mechanisms for concentrating it. Particularly large amounts are found in the adrenal cortex, polymorphonuclear leucocytes, brain, salivary glands, testis, retina, spleen and bone marrow (6). There are a number of possible explanations which effects (2) . The most likely ones, basis of currently available data, are:-
for the ascorbic acid cannot be d i st ingui shed
and ethanol on the
1.
DGLA and AA are metabolised by the same cycle-oxygenase system but vi tamin C enhances the transport of DGLA but not of AA to the active site.
2.
DGLA and AA are vitamin C alters without changing
3.
There
are
different
metabolised by the the effectiveness it for AA. cycle-oxygenases
132
same cycle-oxygenase of this system wlth
involved
in
system but regard to DGLA
DGLA and AA metabolism.
Table
3.
Counts appearing in the band travelling with the PGFlalpha standard after incubation of human platelets with DGLA. The p values refer to difference from control as estimated by the paired
t test.
Control
Table
4.
wm
SD
2929
1868
---
P<
10 ug/ml
ascorbate
3141
1128
---
33 pg/ml
ascorbate
3609
1540
---
100 ug/ml
ascorbate
4393
1206
---
Summary of the percentage changes in the formation of products of DGLA metabolism by human platelets incubated with physiological concentrations of ascorbate (10, 33 and 100 ug/ml, 5.7, 18.8 and 57 x 10-5 M).
Control
10 ug/ml
33 w/ml
100 pg/ml
TxBl
100
130
147
171
PGEl
100
134
144
184
PGFlalpha
100
107
123
150
BtEtF
100
128
141
173
133
There have prostaglandin
been several previous reports of effects of ascorbic acid ori biosynthesis. Van Dorp (7) founa that at approximately 90 conversion of DGLA to PGEl in bovine seminal vg/ml (5 x 10 -4 M) it enhanced vesicles whereas at about 18 ug/ml (10 -4 M) Samuelsson found no effect (8). Ho et al (9) found that, in human platelets, in the absence of tryptophan enhanced formation of TxB2 from arachidonate: 900 vg/ml (5 x 10 -3 M) slightly in the presence of tryptophan, which activated TxB2 formation, the same ascorbate concentration produced a 50% inhibition of TxB2 formation. Sharma et al (10) reported inhibition of PGF2a production by guinea pig uterus to 1250 ug/ml. over the ascorbate concentration range of 6.25 Asthma is a feature of scurvy and so particular interest attaches to ascorbate effects on airways. Zuskin et al (11) found that ascorbic acid could inhibit histamine-induced airways constriction in both guinea pigs and humans. Pug1 isi et al (12) noted that ascorbate antagonized the bronchoconstrictor actions of PGF2a and other constrictors. Tracheas from scorbutic guinea pigs released more PGF2cr and less PGE2 than normal animals: vitamin C corrected this abnormality. Our observations are consistent with those earl ier results but suggest additional explanations for some of them. The idea that 1 series PGs synthesis can be selectively activated is likely to prove of considerable therapeutic and physiological importance. As reviewed in the previous PGEl is an inhibitor of platelet aggregation, a dilator of coropaper (2)) nary and other blood vessels and an inhibitor of arachidonic acid mobilisation. Since the administration of PGEl itself presents many problems, the concept of endogenous enhancement of PGEl biosynthesis is attractive. Such enhancement would require adequate levels of both vitamin C and the Vitamin C deficiency would limit essential fatty acid DGLA precursor. the value of essential fatty acids while an essential fatty acid deficiency would limit the effectiveness of vitamin C and render the administration of megadoses useless. Although many biochemical effects of vitamin C have been described, it has proved difficult to relate these to the clinical features of vitamin C deficiency. Vitamin C regulation of collagen and glycosaminoglycan synthesis is the concept that has perhaps been most successful to date (13). The idea that vitamin C enhances PGEl formation is another effect which can be related directly to the clinical situation. The theoretical basis for this has been discussed extensively elsewhere (14,15), but the main points may be briefly summarised here:1,
Failure of normal immune responses with susceptibility to infections and an inability to reject transplants are major consequences of vitamin C deficiency (16,17). PGEl seems important in T lymphocyte function and a failure of PGEl synthesis could have similar effects (14,15,18, 19).
2.
Ascorbic with the chemotaxi
acid can normalise leucocyte chemotaxis in animals and Chediak-Higashi syndrome (19). PGE 1 enhances 1eucocyte s (20).
134
humans
3.
Wound healing and collagen synthesis are defective in scurvy (16). In essential fatty acid deficient antmals there may be similar defects which in cats can be corrected by 1 series PGs alone (21). There is other evidence also that PGEl may regulate collagen and glycosaminoglycan synthesis (22).
4.
Scorbut enhance
c animals are resistant a peripheral action of
5.
Vi tamin levels of PGEl
C deficiency is associated with elevated 24) . The polyunsaturated essential fatty have long been known to lower cholesterol
6.
Nine si disease trauma, with low tion and the risk
uations in which there is increased risk of thromboembolic (ageing, smoking, estrogen therapy, pregnancy, infection, surgery, soft water consumption and winter) are all associated blood ascorbate levels (25). PGEl inhibits platelet aggregaascorbate deficiency would therefore be expected to enhance of thromboembolism by reduction of PGEl platelet levels.
to insulin (16). insulin (23).
The effect of ascorbate on formation of 1 series seems likely to prove of substantial importance regulation of PG biosynthesis and the mechanism
PGEl
is
able
to
plasma cholesterol acid precursors levels.
PGs and related products in understanding both the of action of vitamin C.
ACKNOWLEDGMENTS We thank the Muscular Dystrophy Association Foundation, Mrs. Sydney Duder and Dr. David this study.
of Canada, the Fisher Family Roy for financial support for
REFERENCES 1.
Falardeau P, Hamberg M, Samuelsson B. eicosatrienoic acid in human platelets. 441: 193-200, 1976.
Metabolism of 8,11,14Biochim Biophys Acta
2.
Manku MS, Oka M, Horrobin DF. Differential regulation of of prostaglandins and related substances from arachidonic from dihomogammalinolenic acid. I. Effects of ethanol. dins Med 3: 119-128, 1979.
3.
Hamberg M, Svensson J, Wakabayashi T, Samuelsson B. Isolation and structure of two prostaglandin endoperoxides that cause platelet aggregation. Proc Nat Acad Sci USA 71: 345-9, 1974.
4.
Laga rde donic gland
M, Gharib A, Dechavanne M. Different acid and dihomogammalinolenic acids n synthetase. Biochimie 59: 935-7,
5.
Ri tchie gical Macmi
JM. The aliphatic alcohols. pp 135-145 in Basis of Therapeutics (LS Goodman & A Gilman, lan, New York, 1970.
6.
Hornig D. Distribution of man and animals. Annals 103-l 18, 1974.
utilization of by human platelet 1977.
ascorbic acid, metabolites of the New York Academy of
135
the formation acid and Prostaglan-
arachiprosta-
The Pharmacoloeds), 4th ed.,
and analogues Sciences 258:
in
7.
Van Dorp DA. Aspects of the Biochem Pharmacol 3: 71-82,
a.
Samuel sson B. Biosynthesis 5: 109-28, 1969.
9.
Ho PPK, Walters P, Sullivan HR. assay, isolation and properties platelets. Prostaglandins 12:
10.
Sharma SC, Pugh DM, Wilson CWM. Inhibitory effect of acid on the yield of prostaglandin F from the guinea homogenates. British J Pharmacol 53: 469P, 1974.
11.
Zuskin E, Lewis AJ, Bouhys A. obstruction by ascorbic acid. immunology 51: 218-26, 1973.
12.
Puglisi L, Berti F, Bosisio E, Longiave D, Nicosia and PGF2a antagonism on tracheal smooth muscle. Thromboxane Res 1: 503-6, 1976.
13.
Cameron E, review.
14.
Horrobin DF, Manku MS, Oka M, Morgan RO, Cunnane SC, Ally Al, Ghayur T, Schweitzer M, Karmali RA. The nutritional regulation T lymphocyte function. Med Hypotheses 5: 969-85, 1979.
Pauling Cancer
biosynthesis 1967. of
of
prostaglandins.
prostaglandins.
Progr
Progr
Biochem
Pharmacol
Biosynthesis of thromboxane 82: of the enzyme system in human 951-70, 1976.
Inhibition Journal
L-ascorbic pig uterine
of histamine-induced of Allergy and Clinical
L, Leibovitz B. Ascorbic Res 39: 663-81, 1970.
acid
airway
S. Ascorbic acid Adv Prostaglandin
and
cancer:
a
15.
Horrobin ation: in the
16.
Bricknell F, Prescott New York, 1953.
17.
Kalden JR, deficient
Prolonged skin allograft survival Guthy EA. guinea pigs. European Surgical Research 4:
la.
Zur ier RB, t reatment
Sayadoff DM, Torrey in NZB/NZW mice.
19.
Zur ier RB. Prostaglandins and cyclic nucleotides in the ChediakH igashi syndrome and experimental systemic lupus erythematosus. J Invest Dermatol 7: 36-9, 1978.
20.
Higgs GA, McCal 1 E, Youlten LJF. released from polymorphonuclear Pharmacol 53: 539-46, 1975.
21.
Frankel TL, Rivers JPW. 1 inolenic acid on cats 227-31, 1978.
22.
of
DF, Oka M, Manku MS. The regulation of prostaglandin El forma candidate for one of the fundamental reactions involved actions of vitamin C. Med Hypotheses 5: 849-858, 1979. F.
The
Vitamins
in
Medicine.
SB, Rothfield NF. Arthritis Rheum 20:
A chemotactic cells during
Grune
and
Stratton,
in vitamin C 114-9, 1972.
Prostaglandin 723-8, 1977.
E
role for prostaglandins Br J phagocytosis.
The nutritional and metabolic impact Br J Nutr deprived of animal lipid.
of
39:
Cunnane SC, Manku MS, Horrobin DF. The pineal and regulation of pinealectomy as a model of primary biliary cirrhosis: fibrosis: roles of melatonin and prostaglandins in fibrosis and regulation Med Hypotheses 5: 403-14, 1979. of T lymphocytes.
136
garnma-
23.
Horrobin PGl2. Abstract
DF, Manku International book
MS.
An action Prostaglandin
of
insulin Meeting,
revealed by Washington,
PGEl and May 1979,
p 53.
24.
Ginter E, Zdichynec of ascorbic acid Journal of Vitamin
25.
Clemetson disease.
B, Holzerova 0 et al. Hypocholesterolemic effect in maturity onset diabetes mellitus. International and Nutrition Research 48: 368-73, 1978.
CAB. Some thoughts Role of ascorbic
on the epidemiology acid. Med Hypotheses
137
of cardiovascular 1979. 5: 825-34,
and
Medicine
3: 129-137,
1979
DIFFERENTIAL REGULATION OF THE FORMATION OF PROSTAGLANDINS AND RELATED SUBSTANCES FROM ARACHIDONIC ACID AND FROM DIHOMOGAMMALINOLENIC ACID. I I. EFFECTS OF VITAMIN C. M.S. Manku, M. Oka, and D.F. 110 Pine Avenue West, Montreal
Horrobin, Clinical H2W lR7, Canada.
Research
Institute,
ABSTRACT Vitamin C over the concentration range 10 to 100 ug/ml (5.7-57 x 10-S M) caused a dose dependent and highly significant enhancement of conversion of 14C-dihomogammalinolenic acid (DGLA) to prostaglandin (PG) El and to thromboxane (TX) Bl by human platelets. Vitamin C had no effect on conversion of 14C-arachidonic acid to PGE2 and TxB2. The concentration range is relevant to physiology: in some cells which concentrate the vitamin, such as polymorphonuclear leucocytes and the adrenal cortex, vitamin C concentrations may be substantially higher than 100 ug/ml. Vi tami n C can therefore selectively enhance the formation of cycle-oxygenase generated products from DGLA without changing formation of those from AA. This effect can account for a number of the known actions of vitamin C including The implications of this finding are its effect on the immune system. discussed. INTRODUCTION The idea that it might be possible to regulate selectively the metabolism of arachidonic acid (AA) and dihomogammalinolenic acid (DGLA) by the cyclooxygenase system has received little if any attention. If such selective regulation were possible, new therapeutic concepts could be developed. For example, in human platelets the overall effect of the products of DGLA metabol ism is anti-aggregatory whereas the overal 1 effect of the AA metabol i tes i s pro-aggregatory (1) . It would therefore be advantageous in platelets to enhance DGLA conversion selectively. In the first paper in this series we reported that ethanol in concentrations relevant to human intoxication had a selective effect in enhancing the formation of 1 series prostaglandins (PGs) and TxBl from DGLA, while having no significant effect on arachidonic acid metabol ism (2). In this paper we report that physiolog ical concentrations of vitamin C can also selective ly enhance format ion of 1 series PGs by human platelets.
129
MATERIALS
AND METHODS
11-14C1 arachidonic acid and [Il-14C1 dihomogammalinolenic acid were purchased from New England Nuclear. They were diluted with hexane to specific activities of about 5 pCi/limol. PGs and thromboxanes were kindly supplied by Dr. John Pike, Upjohn Co., Kalamazoo, Michigan. All organic solvents were reagent grade and were obtained from BDH, Montreal, except for ethanol which was obtained from Fisher, Montreal. One day expired (2 days old) human platelets were obtained from the Canadian Red Cross, Montreal. Silica gel thin layer chromatography (TLC) plates were obtained from Analtech inc. The platelet concentrates were always used within 48 hours of expiration. One unit was centrifuged at 1000 g for 15 minutes and the supernatant drawn off. The platelet pellet was resuspended in Tris-NaCl-EDTA buffer as described by Hamberg et al (3). The buffer was made up of 0.15 M NaCl, 0.15 M Tris HCl at pH 7.4 and 0.077 M NaEDTA (gO:8:2 v/v/v). The platelets were recentrifuged, the supernatant removed and the pellet resuspended in Krebs-Henseleit buffer (without calcium) at pH 7.4. The Al 1 washed platelet suspension contained about 1-28 red blood cells. glassware used in the preparation of the platelets were siliconized using “Prosi 1” (Canlab, Montreal). Each day a stock sol ut ion of 10 mg/ml Lascorbic acid (Sigma) was prepared and brought to pH 7.0 with sodium hydroxide. Four equal sized 1 ml aliquots of the platelet suspension, containing 109 14C-DGLA for five minutes. At platelets/ml were incubated with 0.5 UCi the beginning of the incubation ascorbate in concentrations of 0, 10, 33 and 100 ug/ml (5.7, 18.8 and 57 x lo-5 M) was added to the suspensions. The reaction was stopped after five minutes by addition of l/10 volume of 10% formic acid. The suspension was then extracted three times with ethyl acetate. The three ethyl acetate fractions obtained from each alcohol concentration were pooled and dried under vacuum. The extract was v/v). Recovery of radiothen taken up with 5 ml chloroform/methanol (2/l, active material in the extract was checked by taking 50 ~1 of the chloroRecovery was in the form/methanol and counting by liquid scintillation. range 80-952 in most experiments. The chloroform/methanol extract was then reduced in volume to 1 ml under dry prepurified nitrogen. Thin layer chromatography was carried out on 500 ug precoated, prescored silica gel G Uniplates (Analtech). Plates were activated by heating to 100°C for 1 hour immediately prior to use. The solvent system was chloroform:methanol:acetic acid:water (gO:8:1:0.8). Reference compounds (PGs El, Fla, and thromboxane Bl) were run at the same time and visualized by phosphomolybdic acid spray followed by brief heating. The bands on the plates corresponding to the reference PGEl, PGFlcl and TxBl were scraped off and eluted with 20 ml acetone. Each elution was then evaporated to dryness and counted by liquid scintillation (Beckman 100 LS counter). Using the same batch of platelets at the same time exactly similar experiments were carried out with 14C-AA and PGE2, PGE2ct and TxB2 as reference compounds. Six experiments were performed with DGLA and three with AA.
130
Table
1.
Counts appearing after incubation refer to difference t test.
in of
Control
Table
2.
the band eluting with the TxBl standard human platelets with DGLA. The p values from control as estimated by the paired
wm
SD
7529
2122
_____-
P'
10 ug/ml
ascorbate
3783
2744
_____-
33 ug/ml
ascorbate
11067
2582
0.0125
100 ug/ml
ascorbate
12839
3212
0.005
Counts after refer test.
appearing incubation to difference
in of
Control
the band travel1 ing with the PGEl standard human platelets with DGLA. The p values from control as estimated by the paired t
wm
SD
7642
2001
__cc--
P <
10 pg/ml
ascorbate
10271
2337
0.05
33 pg/ml
ascorbate
10331
3774
0.05
ascorbate
14067
3762
0.0025
100
ug/ml
131
RESULTS In the three experiments conducted with three concentrations used had no effect PGF2a.
arachidonic acid on the formation
vitamin C at the of TxB2, PGE2 or
Vitamin C did enhance the conversion of DGLA by cycle-oxygenase PGEl and PGF2a. The effect was dose dependent and statistically significant. The mean absolute counts are shown in Tables 1, Table 4 summarises the results in a percentage form.
to TxBl, highly 2 and 3.
DISCUSSION Vitamin C enhanced the formation of TxBl, PGEl and PGFlcl from DGLA without altering the formation of the equivalent compounds from arachidonic acid. As reported by others (1,4) approximately equal amounts of TxBl and PGEl but much smaller amounts of PGFlcl are produced by human platelets. The increase in formation of all three products in the presence of vitamin C suggests that the main action of the vitamin is at or before the cyclooxygenase level. Small effects beyond the cycle-oxygenase level cannot, however, be excl uded. The first paper in this series (2) reported a similar effect of ethanol on DGLA metabolism. In molar terms the effect of vi tami n C was approximately one order of magnitude greater than that of ethanol. In vivo the two compounds would be expected to have substantially different effects. Ethanol is consumed in very much larger quantities than vitamin C and its concentrations in most body fluids go very much higher than those of vitamin C, more than compensating for the reduced effectiveness. Ethanol is metabolized rapidly and its effects are relatively transient. Ethanol is also rapidly and evenly distributed throughout the body (5). Vitamin C, in contrast, is consumed in much smaller quantities and is stored in the body. Moreover it is very unevenly distributed since a number of tissues seem to have special mechanisms for concentrating it. Particularly large amounts are found in the adrenal cortex, polymorphonuclear leucocytes, brain, salivary glands, testis, retina, spleen and bone marrow (6). There are a number of possible explanations which effects (2) . The most likely ones, basis of currently available data, are:-
for the ascorbic acid cannot be d i st ingui shed
and ethanol on the
1.
DGLA and AA are metabolised by the same cycle-oxygenase system but vi tamin C enhances the transport of DGLA but not of AA to the active site.
2.
DGLA and AA are vitamin C alters without changing
3.
There
are
different
metabolised by the the effectiveness it for AA. cycle-oxygenases
132
same cycle-oxygenase of this system wlth
involved
in
system but regard to DGLA
DGLA and AA metabolism.
Table
3.
Counts appearing in the band travelling with the PGFlalpha standard after incubation of human platelets with DGLA. The p values refer to difference from control as estimated by the paired
t test.
Control
Table
4.
wm
SD
2929
1868
---
P<
10 ug/ml
ascorbate
3141
1128
---
33 pg/ml
ascorbate
3609
1540
---
100 ug/ml
ascorbate
4393
1206
---
Summary of the percentage changes in the formation of products of DGLA metabolism by human platelets incubated with physiological concentrations of ascorbate (10, 33 and 100 ug/ml, 5.7, 18.8 and 57 x 10-5 M).
Control
10 ug/ml
33 w/ml
100 pg/ml
TxBl
100
130
147
171
PGEl
100
134
144
184
PGFlalpha
100
107
123
150
BtEtF
100
128
141
173
133
There have prostaglandin
been several previous reports of effects of ascorbic acid ori biosynthesis. Van Dorp (7) founa that at approximately 90 conversion of DGLA to PGEl in bovine seminal vg/ml (5 x 10 -4 M) it enhanced vesicles whereas at about 18 ug/ml (10 -4 M) Samuelsson found no effect (8). Ho et al (9) found that, in human platelets, in the absence of tryptophan enhanced formation of TxB2 from arachidonate: 900 vg/ml (5 x 10 -3 M) slightly in the presence of tryptophan, which activated TxB2 formation, the same ascorbate concentration produced a 50% inhibition of TxB2 formation. Sharma et al (10) reported inhibition of PGF2a production by guinea pig uterus to 1250 ug/ml. over the ascorbate concentration range of 6.25 Asthma is a feature of scurvy and so particular interest attaches to ascorbate effects on airways. Zuskin et al (11) found that ascorbic acid could inhibit histamine-induced airways constriction in both guinea pigs and humans. Pug1 isi et al (12) noted that ascorbate antagonized the bronchoconstrictor actions of PGF2a and other constrictors. Tracheas from scorbutic guinea pigs released more PGF2cr and less PGE2 than normal animals: vitamin C corrected this abnormality. Our observations are consistent with those earl ier results but suggest additional explanations for some of them. The idea that 1 series PGs synthesis can be selectively activated is likely to prove of considerable therapeutic and physiological importance. As reviewed in the previous PGEl is an inhibitor of platelet aggregation, a dilator of coropaper (2)) nary and other blood vessels and an inhibitor of arachidonic acid mobilisation. Since the administration of PGEl itself presents many problems, the concept of endogenous enhancement of PGEl biosynthesis is attractive. Such enhancement would require adequate levels of both vitamin C and the Vitamin C deficiency would limit essential fatty acid DGLA precursor. the value of essential fatty acids while an essential fatty acid deficiency would limit the effectiveness of vitamin C and render the administration of megadoses useless. Although many biochemical effects of vitamin C have been described, it has proved difficult to relate these to the clinical features of vitamin C deficiency. Vitamin C regulation of collagen and glycosaminoglycan synthesis is the concept that has perhaps been most successful to date (13). The idea that vitamin C enhances PGEl formation is another effect which can be related directly to the clinical situation. The theoretical basis for this has been discussed extensively elsewhere (14,15), but the main points may be briefly summarised here:1,
Failure of normal immune responses with susceptibility to infections and an inability to reject transplants are major consequences of vitamin C deficiency (16,17). PGEl seems important in T lymphocyte function and a failure of PGEl synthesis could have similar effects (14,15,18, 19).
2.
Ascorbic with the chemotaxi
acid can normalise leucocyte chemotaxis in animals and Chediak-Higashi syndrome (19). PGE 1 enhances 1eucocyte s (20).
134
humans
3.
Wound healing and collagen synthesis are defective in scurvy (16). In essential fatty acid deficient antmals there may be similar defects which in cats can be corrected by 1 series PGs alone (21). There is other evidence also that PGEl may regulate collagen and glycosaminoglycan synthesis (22).
4.
Scorbut enhance
c animals are resistant a peripheral action of
5.
Vi tamin levels of PGEl
C deficiency is associated with elevated 24) . The polyunsaturated essential fatty have long been known to lower cholesterol
6.
Nine si disease trauma, with low tion and the risk
uations in which there is increased risk of thromboembolic (ageing, smoking, estrogen therapy, pregnancy, infection, surgery, soft water consumption and winter) are all associated blood ascorbate levels (25). PGEl inhibits platelet aggregaascorbate deficiency would therefore be expected to enhance of thromboembolism by reduction of PGEl platelet levels.
to insulin (16). insulin (23).
The effect of ascorbate on formation of 1 series seems likely to prove of substantial importance regulation of PG biosynthesis and the mechanism
PGEl
is
able
to
plasma cholesterol acid precursors levels.
PGs and related products in understanding both the of action of vitamin C.
ACKNOWLEDGMENTS We thank the Muscular Dystrophy Association Foundation, Mrs. Sydney Duder and Dr. David this study.
of Canada, the Fisher Family Roy for financial support for
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