Tài liệu Cơ chế tổng hợp cholesterol bằng Liebermann-Mechanisms of the Liebermann-Burchard and Zak Color Reac

Correlation of SO2 and Fe2+ measurements with
new spectral data indicates that the Liebermann-
Burchard (L-B) and Zak color reactions for cholesterol
have similar oxidative mechanisms, each yielding,
as oxidation products, a homologous series of
conjugated cholestapolyenes. These studies further
suggest that the colored species observed in these
two systems are enylic carbonium ions formed by
protonation of the parent polyenes. Thus, the red
(Amax, 563 nm) product typically measured in the
Zak reaction is evidently a cholestatetraenylic cation,
and the blue-green product in the L-B reaction
(Amax, near 620 nm) is evidently the pentaenylic
cation. The effects of rate of carbonium ion formation
and sulfuric acid concentration on sensitivity and
color stability are discussed. A solvent extraction
procedure Is described for specifically converting
cholesterol to 3,5-cholestadiene. Incorporating this
step into the typical L-B method can increase the
L-B sensitivity for cholesterol by several fold.
Additional Keyphrases: cholestapolyenes #{149}conjugated
double bonds #{149}carbonium ions from polyenes #{149} factors
affecting color #{149}SO2 and Fe2+ measurement
equivalent ratios
Cholesterol reacts with various strong acids of the
Bronsted and Lewis types to give colored products.
Although these reactions have been used empirically
for many years for the qualitative and quantitative
determinationof cholesterol, their mechanisms still
are not clearly understood.
Among the many color reactions for cholesterol,
the Liebermann-Burchard probedure is perhaps the
most widely used. This reaction was described initially
by Liebermann (1) in 1885 and applied to cholesterol
analysis shortly after by Burchard (2). Chloroform
was used as a solvent in the early studies, but
Analytical Chemistry Division, Institute for Materials Research,
National Bureau of Standards, Washington, D. C. 20234.
Received Jan. 4, 1974; accepted May 7, 1974.
the Liebermann-Burchard (L-B) reaction, as performed
today, is carried out in an acetic acid-sulfUric
acid-acetic anhydride medium. The other widely
used method, the Zak reaction, which was first applied
to cholesterol analysis by Zlatkis, Zak, and
Boyle (3) in 1953, is carried out in acetic acid-sulfuric
acid in the absence of acetic anhydride. In this
reaction, however, Fe3+ must be added to obtain the
desired colored species.
In one of the earliest mechanistic studies, Lange et
a!. (4) showed that treating chloroform solutions of
cholesterol with equivalent amounts of perchioric or
hexafluorophosphoric acid resulted in the formation
of colorless sterolium salts. These salts hydrolyzed
instantaneously on contact with an excess of water,
givingquantitativerecoveryofthe added cholesterol.
Addition of acid in excess of the stoichiometric
amount resulted in slow dissolution of the colorless
crystals, followed by formation of purple products
that were considered to be halochromic salts of
cholestadiene. Degradation of these products was
thought to occur through formation of polymerized
dieneoid hydrocarbons, with the trimer being the
highest polymer observed. Subsequent studies by
Dulou et al. (5) and by Brieskorn and Capuano (6)
suggestedthat the initialstep in the L-B reaction
was the dehydration of cholesterol to form cholestadiene,
which dimerized to bis-cholestadiene. The final
product was believed to be the monosulfonated
dimer. Later studies by Watanabe (7) supported this
hypothesis; he isolated a 3,3’-bis-3,5-cholestadiene
by column chromatography from a concentrated L-B
reaction mixture. However, a subsequent paper by
Brieskorn and Hofmann (8) indicated that dimer formation
was not the principal reaction in the L-.B system
and that a more probable mechanism involved
the oxidation of cholesterol to a conjugated pentaene.
It is at this stage that efforts to elucidate the
mechanism of the L-B reaction appear to have stoped