BlOCH EM ICA L SOCl ETY TRANSACTIONS
148
significantly lower levels of fat than that from females
which corresponded closely to the mean dry matter
values obtained. Males also showed slightly lower mean
fat values in lean meat than females although the differences were not significant so that the significantly higher
percentage dry matter of females is only partially
explained by differences in fat content and may also
reflect the slightly higher protein content of males (Edwards c't ul.. 1973).
Economic arguments based largely upon the earlier
laying down of visceral fat (Table 1 ) in females have
already led to advocacy of separate sex broiler growing
(Anon, 1983). The data presented here provide evidence
that separate sex broiler growing might contributc t o
limitation of human fat intake.
Anon (19x3) P o d / . In/. (Jan) SX 60
Bligh. E. G . & Dyer. W. J. (1959) ('tin. J . Bi(~dicwr.P/i\,.\io/. 37. 91 I
Edwards, H. N., Denman. S., Abou-Ashour. A. & Nugara. [I.(1975)
Poultrj Sci. 52. 934 948
Folch, J . , Lees. M. & Stanley, G . H. S. (1957) J . Bhd. C ' / i ~ w . 226. 497
Griffiths, L., Leeson, S. & Summers. J. D. ( 1977) P o d / . .St.i. 56,
1018 1026
Lipid characterization and metabolism in two red marine algae
TREVOR R. PETTITT and JOHN L. HARWOOD
Dqmrtmtwt of Biochemistry, Universitjt College,
Curdif CFI I X L , Wules, U . K .
Marine algae are a very important yet poorly understood
part of the plant kingdom. Of these, the Rhodophyceae
(red algae) may be one of the oldest eukaryotic forms as
indicated by their chloroplast structures (which contain
phycobilisomes and no thylakoid stacking). In view of
the increasing interest in photosynthetic membrane
structure, we have been studying aspects of chloroplast
structure and function in marine algae. We report here
some details of lipid characterization and metabolism
in two red macroscopic algae, Chondrus crispus and
Polys iphoniu lunosu .
The lipids of the two intertidal red algae were extracted
by the boiling isopropanol method of Harwood ( I 975).
Separation was achieved by an initial fractionation on
acid-washed Florisil into neutral lipid, glycolipid and
phospholipid fractions followed by t.1.c. Identification
was made by comparison with authentic standards, differential staining reactions and degradation procedures.
Glycolipids were quantified with phenol/HzSO,
(Roughan & Batt, 1968), phospholipids by inorganic
phosphate assay after hydrolysis (Duck-Chong, 1979)
and sterols by the method of Courchaine et ul. (1959).
The major lipids are listed in Table I . When separated by
t.1.c. double spots were usually seen for the glycolipids.
The two spots which correspond to digalactosyldiacylglycerol were never completely resolved. The double
spots may be due to the presence of different sugar
moieties which slightly alter lipid mobility. Indeed a
double spot for the sulphoquinovosyldiacylglycerol
(SQDG) band from Nitx*hiu ulbu was reported by
Anderson et ul. (1978). Traces of phosphatidylethanolamine, phosphatidic acid, diphosphatidylglycerol and
minor glycolipids were also detected.
When the algae were incubated with ["S]sulphate for
24 h under illumination in artificial seawater ASP-6
(McLachlan, 1975) where MgCI, replaced MgSO, at
15 C and the lipids separated, six sulpholipids were
detected in C. crispus and three in P. lunosu. For each
alga, both SQDG bands were labelled together with the
fastest running monoglycosyldiacylglycerol (MGDG)
band. In C. crispus sulpholipids were also detected in the
phosphatidylcholine, phosphatidylglycerol and diglycosyldiacylglycerol (DGDG) regions. Mild acid and mild
alkali hydrolyses of the SQDG bands released sugar
sulphonate, confirming the identity of this lipid in red
algae as SQDG. Mild acid hydrolysis of the DGDG and
Table 1 . Contents of'mujor lipids in c'liotirlrus crispus crnd Polysiplionio Iririosa
Double bands were obtained in the MGDG and SQDG
regions. The faster moving bands are indicated by ;I subscript
2. Phosphatidylethanolaminc and an unidentified glycolipid
were present as minor components. There were only trace
amounts o f phosphatidic acid. diphosphatidylglyccrol and
esterified sterols. Results arc cxprcssed a s mcans & S X M .
(n = 4).
C'/ioti(/ri~\u i . c / i i i , \
Lipid
Phosphatid ylgl ycerol
Phosphatidylcholinc
MGDG,
MGDG,
DGDG
SQDG,
SQDG,
Triacyigl ycerol
Unesterified fatty acids
Free sterols
(/imol,g. dry wt.)
*
**
**
3.03
0.13
3.x5 f 0.42
I .52 & 0.06
3.00
0.86
3.30
0.56
1.14 i 0.14
2.66
0.37
0.77
0.04
1.50
0.09
1.x & 0.10
0.xs & 0.04
2.05 f 0.04
I .07 t 0 . I I
2.07 & 0. I 7
5.20
0.X'
2.52 & 0.4x
*
0.96
3.50
I.X4
1 0.15
** 0.42
0.26
MGDG, bands released free sulphate, indicating t h a t the
sulpholipids migrating in these regions contained cstcrified sulphate residues. Mild acid hydrolysis of the sulpholipid migrating in the phosphatidylcholine region released
an unidentified '5S-labelled component. which did not
appear to be the sulphur analogue of choline (c.f.
Anderson c't ul., 1978). Work is continuing t o identify
these sulpholipid structures.
More than 20 fatty acids for each alga were separated.
identified and quantified by g.1.c. using EGSS-X and
EGSS-Y columns and by argentation t.1.c. Most acids
were only present in minor quantities. The major fatty
acids, constituting over 95% of the total in both algae.
are 16:0, 16: In-7. 18: In-9, l8:2n-6. 20:4n-6 and
20 : 52-3. The two 20C polyenoic acids account for '1 b out
55% of the total fatty acids, about 75% of those in
phosphatidylcholine and more than 95% of those in
MGDGz in both algae. This high content is typical of
marine algae in agreement with the fatty acid coniposition of the photosynthetic membranes of higher
plants (Harwood, 1980). trun.s-A7-hexadecenoicacid was
present exclusively in phosphatidylglycerol. This was in
spite of the fact that red algae do not contain thylakoid
stacks (c.f. Harwood, 1984).
When the algae were incubated with [ 1 -'4C]acetatein
sterilized seawater at 15 C with and without illumiAbbrcviations used: SQDG. sulphoquinovosyldiacylglycerol; nation, the rate of incorporation of radioactivity was
greater in the light. In C. crispus, illumination increased
MGDG. monoglycosyldiacylglycerol; DGDG. diglycosyldiacylglycerol.
1986
614th MEETING, OXFORD
I49
the relative rate of labelling of phosphatidylglyccrol and
DGDG while that for phosphatidylcholine decreased. In
both algae, illumination increased the relative labelling of
oleate and linoleate.
W e a r e grateful to the S.E.R.C. (studentship to T . R . P . ) for linancial
hupport.
Anderson. R., Livermore. B. P., Katcs. M . & Volcani, H. E. (197X)
B f f ) < h f f Bfr'/J/f\~.s.
ff.
.4c,/u 528. 77 XX
Courchaine. A . J., Miller. W . f l . & Stein. D. B. (1959) ( ' / / t i . ( ' h c w i . 5 .
Genotoxicity studies with bile conjugate metabolites
MARGARET H. BLAKEBOROUGH,* ROBERT
W. OWENt and RODNEY F. BILTON
* Dcpurtnirwt of Chcmistrj~und Biochemistry. Liwrpool
Polj~tcdinic.Bjwlrvi Strcc>t, Liwrpool L3 3A F. U . K . ,
untl t P H L S Ccntrc j i ) r Applied Microbiology und
Roscur ch . BNCt c>ri(ilM c t uholisni Rescur ch Lu horu t o r!-,
Porron Down. Suli.shurj*,WiltshirP. SP4 OJG. U .K .
There has been strong epidemiological evidence correlating dietary fat intake to the incidence of colorectal
cancer (Armstrong & Doll. 1975). Faecal profiles of
populations on high-fat diets show high levels of bile acid
derivatives. total neutral sterols and anaerobic bacteria
capable of actively metabolizing cholesterol and bile
acids to possible carcinogens and/or co-carcinogens (Hill
(it (11.. 1970; Reddy & Wynder, 1973).
There is much evidence to date implicating certain bile
acids as promoters of colon carcinogenesis (Reddy iit (11..
1977; Wynder & Reddy. 1977). Much of this evidence is
derived from animal studies which can be both longterm and expensive. We have therefore turned to microbial mutagenicity assays. particularly the Ames test
(McKillop
(11.. 1983). for testing bile conjugate
metabolites produced by bacterial transformation. The
process of steroid transformation by bacteria is time
consuming and yields only small amounts of any one
compound for testing. For this reason we chose a relatively new bacterial assay developed by Quillardet cr (11.
( 19x3) called the SOS-chromotest.
This test is a simple and quick colorimetric assay based
on the induction of the SOS function .!/;A, whose level
of expression is monitored by means of a .!/;A : : IucZ
operon intusion. 1 he response is rapid (a tew hours) and
does not require survival of the tester strain Esihcriciri
i d i PQ37.
PQ37 is constitutive for alkaline phosphatase and
produces /&galactosidase when DNA damage occurs.
Compounds tested using the SOS-chromotest may at
certain concentrations inhibit protein synthesis which
would. in turn, lead to an underestimation of /jgalactosidase induction. To correct for this, general protein synthesis is estimated during the incubation period
by simultaneously measuring alkaline phosphatase synthesis along with /I-galactosidase. The ratio of [I-galactosidase activity to alkaline phosphatase activity is taken as
a measure of the specific activity of [j-galactosidase. This
Abbreviations used: SOSIP. SOS-inducing potency; DMSO, dinicthyl
sulphoxide.
Vol. 14
Ta hlc I . XIS-inciuc~in,qp o i ~ v i c ~ i co~fs'hik c~otrjrcgrriot n c , i d ~ o I i i ~ , ~
on(/ pcrriwi h i k clc.icl.c
('om pounds tested
SOSI P
(I )
Androsta-I .4.6-tricnc-3. I7-dioIlc in I l M S O
( 2 ) 3.5-Cholcstadienc in DMSO
( 3 ) Cholcstcrol-5z-6z-ep(~xidcin D M S O
( 4 ) 12~-~~ydroxychol~i-4.h-dicne-3-onc-24-oic
acid i n D M S O
( 5 ) Atitoxidation product o f ( 4 )
( 6 ) Ursodeoxycholic acid: sodium salt i n water
( 7 ) Ursodeoxycholic acid (free acid) in DMSO
( X ) Deouycholic acid: sodium salt in water
( 9 ) Deoxycholic acid (free acid) in DMSO
( 10) Lithocholic acid: sodium salt in water
( I I ) Lithocholic acid + nitrofurantoin
( 1 2 ) Ursodeoxycholic acid + nitrofurantoin
( 13) ('henodcoxycholic acid
nitrofurantoin
( 14) Nitrofurantoin
+
0.00I
0.002
0.00I6
0.00 14
0.0066
Negative
0.0026
N cga t Ivc
0 001 I
Negative
7 40
7.30
6.0
5.0
activity ratio is normalized to a value in the absence of
test compound, which is known as the induction factor.
When the induction factor is plotted against concentration a linear region occurs. The slope of this region is
called the SOS-inducing potency (SOSIP) and is a quantitative expression of the capacity to induce the .s/i'A
response.
These preliminary results (Table 1 ) reveal clTects not
observed in the Ames test. The sodium salts of the parent
bile acids show no genotoxic effects. The free acids in
dimethyl sulphoxide (DMSO). however. show weak
gcnotoxic effects, as do compounds ( I 5 ) . Co-mutagenic
elrects are also seen with the parent bile acids when mixed
with the known mutagen nitrofurantoin. This can be seen
by an increase in the SOSIP valucs, compared with nitrofurantoin alone.
We :ire grateful to Ilr. M I { o f n u n g for supplying the h;ictcri;il strain
P o 3 7 and to the S . E R C.. for linnncinl support ol'ihi\ work.
Arniatrong. B. & I l o l l . R . (1975) / ) I / . J . C'crriwr 15. 617 631
Hill. M. J.. I h s c r . B. S . Aries. V.. ('rowthcr. J . S..Iiawksworth. <;.
& Williams. R . E. 0. (1970) I.trric,c,/ i. 95 100
McKillop. C. A.. Owen. R . W., Billon. R. I- & Iia\l:ir~1. E. A (19x3)
('crrc,irio,~c.ri~,.\iv
4. I I79 I I X3
Quillardet. P., Iluisman. 0.. Il'ari, R . & llofnung. M. (19x2) /'roc
N t / / / . A c ~ / c / Sc'i.
.
C ' . . S . d . 79. 5971 5975
Reddy, B. S. & Wyndcr. E. L. (1973) J . Ncrrl. ( ' c r t i w r /rr,\i. 50. 1442
Reddy, B. S.. Watanabe. K.. Weisburgcr. J. I1 & Wynder. E. 1,.
(1977) C ' t I i i w r Kc,.v. 37, 3238 3243
Wynder. E. L. & Reddy. H. S. ( 1977) ('ertrwr 40. 923 92X