684
H O N O R
B .
F E L L
A N D
L .
M .
R I N A L D I N I
ASA in the guinea-pig is inversely correlated with tissue saturation,,
the balance depending partly on excretory mechanisms. In cultures,,
however, the half-life would depend exclusively on the rate of catabolism. The half-life of DHA, its first oxidation product, is about 2 min
at 38°C and p H 7-24 (Ball, 1937). D H A may be either converted back
to ASA (De Ritter, Cohen and Rubin, 1951) or rapidly catabolized
via diketogulonic acid ( D K G ) to give threonic and oxalic acids and is
also decarboxylated to lyxonic and xylonic acids (Burns, 1960; Ashwell
et al., 1961; Shimazano and Mano, 1961). ASA and its metabolic
products may interact with other components of culture media,
mutually modifying their effects on cells. A detailed discussion of these
metabolic interactions is beyond the scope of this article. Most animal
tissues protect ASA from oxidation, presumably through their content
of reduced glutathione (GSH) (De Caro, 1933; De Caro and Giani,
1934; Quastel and Wheatley, 1934; Mawson, 1935; Kellie and Zilva,
1935; Hopkins and Morgan, 1936; Borsook, Davenport, Jeffreys and
Warner, 1937), but the protective effect is not sufficient for long-term
experiments. M y own results show that in the presence of adult-tissue
breis (liver and intestine) from 92 to 99-6% of the ASA present was
oxidized beyond the diketogulonic stage after 18 h. Only 50-71% was
lost if chick embryonic pulp was added, indicating that embryonic
tissues have a stronger protective action, but still not sufficient to
prevent the oxidation ofthe vitamin in vitro completely. Embryo juice,
as routinely used in tissue culture, is rich in ASA and GSH. Undiluted
extract from 12-day embryos prepared in a Waring Blendor contains
approximately 100 /xg/ml of vitamin C as measured by the method of
Bolin and Book (1947).
A strict requirement for ASA has not been demonstrated hitherto
for any particular tissue or cell strain. Cell strains cultivated in vitro
for long periods do not seem to require ASA for maintenance or proliferation (Levintow and Eagle, 1961) but this may be the result of
adaptation. Sato, Fisher and Puck (1957) found that single HeLa S3
cells require ASA for successful cloning in combination with cholesterol
cysteine, glutathione, CoA, cocarboxylase, DPN and F A D in addition to
a basal medium composed of sixteen amino acids, eight vitamins,
glutamine, hypoxanthine and glucose, plus the macromolecular
fraction of serum rigorously dialyzed, which was sufficient to support
logarithmic growth of heavily seeded cultures. This additional requirement of single cells could not be confirmed by Lockhart and Eagle
(1959), who found instead that adding some non-essential amino acids,
particularly serine, to their basal medium, promoted clonal growth.
The discrepancy appears to be due to technical differences in the
preparation of inocula.
H O N O R
B .
F E L L
A N D
L .
M .
R I N A L D I N I
ASA in the guinea-pig is inversely correlated with tissue saturation,,
the balance depending partly on excretory mechanisms. In cultures,,
however, the half-life would depend exclusively on the rate of catabolism. The half-life of DHA, its first oxidation product, is about 2 min
at 38°C and p H 7-24 (Ball, 1937). D H A may be either converted back
to ASA (De Ritter, Cohen and Rubin, 1951) or rapidly catabolized
via diketogulonic acid ( D K G ) to give threonic and oxalic acids and is
also decarboxylated to lyxonic and xylonic acids (Burns, 1960; Ashwell
et al., 1961; Shimazano and Mano, 1961). ASA and its metabolic
products may interact with other components of culture media,
mutually modifying their effects on cells. A detailed discussion of these
metabolic interactions is beyond the scope of this article. Most animal
tissues protect ASA from oxidation, presumably through their content
of reduced glutathione (GSH) (De Caro, 1933; De Caro and Giani,
1934; Quastel and Wheatley, 1934; Mawson, 1935; Kellie and Zilva,
1935; Hopkins and Morgan, 1936; Borsook, Davenport, Jeffreys and
Warner, 1937), but the protective effect is not sufficient for long-term
experiments. M y own results show that in the presence of adult-tissue
breis (liver and intestine) from 92 to 99-6% of the ASA present was
oxidized beyond the diketogulonic stage after 18 h. Only 50-71% was
lost if chick embryonic pulp was added, indicating that embryonic
tissues have a stronger protective action, but still not sufficient to
prevent the oxidation ofthe vitamin in vitro completely. Embryo juice,
as routinely used in tissue culture, is rich in ASA and GSH. Undiluted
extract from 12-day embryos prepared in a Waring Blendor contains
approximately 100 /xg/ml of vitamin C as measured by the method of
Bolin and Book (1947).
A strict requirement for ASA has not been demonstrated hitherto
for any particular tissue or cell strain. Cell strains cultivated in vitro
for long periods do not seem to require ASA for maintenance or proliferation (Levintow and Eagle, 1961) but this may be the result of
adaptation. Sato, Fisher and Puck (1957) found that single HeLa S3
cells require ASA for successful cloning in combination with cholesterol
cysteine, glutathione, CoA, cocarboxylase, DPN and F A D in addition to
a basal medium composed of sixteen amino acids, eight vitamins,
glutamine, hypoxanthine and glucose, plus the macromolecular
fraction of serum rigorously dialyzed, which was sufficient to support
logarithmic growth of heavily seeded cultures. This additional requirement of single cells could not be confirmed by Lockhart and Eagle
(1959), who found instead that adding some non-essential amino acids,
particularly serine, to their basal medium, promoted clonal growth.
The discrepancy appears to be due to technical differences in the
preparation of inocula.