Part 3. Pantothenic Acid
D. Coenzyme A Assay
The original method for assay of coenzyme A, developed by Kaplan and Lipmann (401), is based upon the coenzyme Α-dependent acetylation of sulfanilamide in the presence of ATP and acetate. Sulfanilamide is measured by the method of Bratton and Marshall (402), and the rate of disappearance of sulfanilamide is a measure of the rate of acetylation and the amount of coenzyme A present. The difference between the amount of unacetylated sulfanilamide in the blank and that in the sample is considered to be the amount of sulfanilamide acetylated by co
enzyme A (401).
A rapid spectrophotometric assay has been proposed by von Korff (403) which is based on the measurement of the rate of reduction of DPN by α-ketoglutarate in the presence of a soluble oxidase and a coenzyme A which functions catalytically.
Wakil and Hubscher (404) have developed a stoichiometric test for coenzyme A which is dependent upon the formation of sorbyl-CoA from sorbic acid, coenzyme A, and ATP in the presence of Mg+ + and a fatty acid-activating enzyme. Sorbyl-CoA has an absorption maximum at
enzyme
sorbic acid + ATP + CoASH > sorbyl CoA + AMP + Pi Mg++
300 m/i, and the reaction can be followed spectrophotometrically at this wavelength.
Sakurada (405) has developed an azotometric technique for the determination of coenzyme A using the CoA-dependent acetylation of isonicotinic acid hydrazide (406).
I I I . PANTOTHENIC ACID ANTAGONISTS
Pantoyltaurine (407, 408) is an inhibitory analog of pantothenic acid for lactic acid bacteria (407) and certain other microorganisms (409), but not for mice (410, 411) or rats (411). ω-Methylpantothenic acid, synthesized by Drell and Dunn (412, 413), inhibits the growth of lactic acid bacteria (414), is an irreversible pantothenic acid antagonist in mosquito larvae (415), and appears to be the only pantothenic acid analog which can produce deficiency symptoms in higher mammals (416).
This antagonist promotes growth in Saccharomyces sake (417), but this is thought to be due to this yeast's ability to utilize the /^-alanine of the molecule.
ω-Methylpantothenic acid interferes with normal embryonic develop
ment in several laboratory animals. Ten milligrams of the antagonist injected into the yolk sac of chick embryos before incubation caused death of 70% of the embryos (418); one-third of the surviving embryos had congenital abnormalities of the brain, eye, or beak. Simultaneous injection of pantothenic acid partially prevented these defects. A closely related antagonist, ω-methylpantetheine, caused a high mortality rate and leg malformations in turkey embryos (419).
Rats maintained on a pantothenic acid-deficient diet for the first 14 days after gestation infrequently have abnormal offspring (420).
However, embryonic development was seriously disturbed by feeding 0.5 to 1.0% ω-methylpantothenic acid to these animals for 2 to 3 days.
Goodman (421) reports that ω-methylpantothenic acid decreases the adrenal steroid hormone output of rats to a greater extent than does pantothenic acid deficiency alone.
The same antagonist fed to guinea pigs as 0.15, 0.3, and 0.4% of the diet depresses growth and produces anemia (422). When it is fed to rats as 0.1% of the diet (423), the animals cease to grow after 1 week, whereas pantothenic acid-deficient animals without the antagonist do not reach a growth plateau until after 2 to 3 weeks. Animals that receive ω-methylpantothenate also have lower acetylation capacities than those that do not. The inability to acetylate injected p-aminobenzoic acid has long been correlated with the degree of pantothenic acid deficiency (424).
Bean and co-workers (425, 426) describe the use of ω-methylpanto
thenate in the study of human pantothenic acid deficiency symptoms.
It is interesting to note that this antagonist apparently is not effective
in calves (427), although it is an effective pantothenic acid antogonist in many microorganisms and animals, and in humans.
I V . SYMPTOMS OF PANTOTHENIC Aero DEFICIENCY
Novelli (428) has written a comprehensive review of the symptoms of pantothenic acid deficiency in many animals and fowls. He sum
marized and categorized the symptoms and related them to the metabolic functions of coenzyme A: (1) failure to grow, loss of weight, sudden death; (2) lesions of the skin, hair, or feathers; (3) lesions of the nervous system; (4) gastrointestinal symptoms; (5) inhibition of antibody forma
tion; and (6) lesions of the adrenal gland.
All of these symptoms do not appear in every species of laboratory animal; rather, each type of animal exhibits its own syndrome (407).
Since the writing of that review, the pantothenic acid deficiency states of other laboratory animals have been characterized.
Cohen et al. (429) used a purified diet to study pantothenic acid deficiency in the hamster. Growth was not greatly depressed with this diet, perhaps because the animals received a weekly supplement of 10 gm of lettuce containing approximately 11 pg of pantothenic acid which may have been sufficient for growth.
The most prominent symptoms of pantothenic acid deficiency in the cat are growth failure and histologic changes in the small intestine and liver (430). Dermatitis, adrenal necrosis, and blood dyscrasias are not apparent.
Guinea pigs display anemia, accumulation of blood pyruvic acid, muscular weakness, soft wooly fur, hemorrhagic adrenals, convulsions, and comas when maintained on a pantothenic acid-deficient diet (422).
Recent studies on pantothenic acid deficiency in rats have resulted in the recognition of additional typical deficiency symptoms in this animal.
Barboriak et al. (431) report degeneration of granular and interstitial tissue as evidence of testicular damage due to pantothenic acid deficiency.
Ershoff and Kruger (432) find that congenital abnormalities such as anophthalmia and motor incoordination appear frequently in litters from pantothenic acid-deficient rats. Vitamin Bi 2 is increased in the livers of pantothenic acid-deficient rats (368). Hatano (371) reports that the urinary pantothenic acid excretion of rats drops from 100 to 1 μg per day after the animals have been on a pantothenic acid-deficient diet for 2 to 3 weeks. Upon reaching the 1 fig per day level of excretion, rats begin to show symptoms of pantothenic acid deficiency such as stationary weight and fur change. Hatano (371) also found a high incidence of lung in
flammation in the deficient animals.
Wirtschafter and Walsh (433) have found that toxic doses of
pante-theine and pantothenic acid cause adrenal lesions similar to those present in pantothenic acid deficiency.
V . METABOLIC FUNCTION OF COENZYME A
Pantothenic acid functions solely as a constituent of coenzyme A which forms high-energy thioester acyl groups which can participate in various metabolic transformations.
Higher animals are unable to synthesize pantothenic acid; however, liver enzymes enable them to form coenzyme A from the preformed vitamin (428). The first step is the condensation of cysteine with panto
thenic acid to form a peptide, pantothenylcysteine (428, 434r-436), in a reaction requiring ATP (435). Pantothenylcysteine is decarboxylated to pantetheine, which is then phosphorylated with ATP to give 4'-phospho-pantetheine (435, 437). The latter reacts with a second mole of ATP in a reaction in which adenylic acid is added to 4'-phosphopantetheine to give dephospho-CoA (435) with the splitting off of pyrophosphate. The result
ing dephospho-CoA is phosphorylated in the presence of ATP and mag
nesium ions to give coenzyme A (428, 435).
The reactions can be outlined by the following sequence.
ATP
1. Pantothenate + cysteine > pantothenylcysteine 2. Pantothenylcysteine —* pantetheine + CO2
3. Pantetheine + ATP —> 4'-phosphopantetheine + ADP 4. 4'-Phosphopantetheine + ATP —> dephospho-CoA + PP 5. Dephospho-CoA + ATP -> CoA + ADP
In microorganisms, the sequence of reactions may differ from that which occurs in liver. In Proteus morganii, pantothenic acid may be phosphorylated directly to give phosphopantothenic acid (438, 439) which, after reaction with cysteine and decarboxylation, yields phospho-pantetheine (438).
Reactions involving coenzyme A are too numerous to be reviewed here and the reader is referred to the reviews of Lipmann (439), Jaenicke and Lynen (440), and Novelli (428).
ACKNOWLEDGMENTS
It is a pleasure to acknowledge the helpful assistance of Mrs. Karen Gay and Mrs. Mary Wasik in the preparation of the manuscript.
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