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FISHERIES RESEARCH BOARD OF CANADA
Translation Series No. 443
;ARCHIVES
A METHOD FOR THE DETERMINATION OF THE FATTY ACIDS OF THE HUMAN BLOOD,
MUTH PARTICULAR REFERENCE TO THE FATTY ACIDS WHICH OCCUR
•
IN SMALL QUANTITIES
By A. Glaser, G. Grimmer, E. Jantzen and H. Oertel
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Original title: Eine Methode zur Bestimmung der Fettsâuren des Human -Blutes
unter besonderer Berucksichtigung der in geringen Mengen vorkommenden
. Fettsaüren.
From:
Biochemische Zeitschrift, Vol. 336, pp. 274-280, 1962.
Translated by the Bureau for Translations, Foreign Language Division,
Department of the Secretary of State of Canada
1963
1
ti«
r'?
.DEPARTMENT OF TIIF_ SECRETARY OF STATE
BUREAU FOR TRANSLATIONS
FOREIGN LANGUAS
DIVISION
SECRÉTARIAT D'ÉTAT
BUREAU DES TRADUCTIONS
DES LANGUES
DIVISIJ
ÉTRANGÈRES
CANADA
TRANSLATED FROM - TRADUCTION DE
German
• Engl sh
SUBJECT - SUJET
fatty acids in human blood
AUTHOR - AUTEUR
A.Glaser, G.Grimmer, E.Jantzen and H.Oertel
TITLE IN ENGLISH - TITRE ANGLAIS .
A method for the determination of the fatty acids
of the human blood, with particular reference to
the fatty acids which occur in small quantities.
TITLE IN FOREIGN LANGUAGE - TITRE EN LANGUE dTRANGÉRE
Eine Methode zur Bestimmunpr der Fettsâuren des
Human-Blutes unter besonderer Berücksichtigung
der in geringen Mengen vorkommenden Fettsguren.
REFERENCE - RÉFÉRENCE (NAME OF BOOK OR PUBLICATION - NOM DU LIVRE OU PUBLICATION)
Biochemische Zeitschrift 336
PUBLISHER - ÉDITEUR
CITY - VILLE
DATE
1962
REQUEST RECEIVED FROM PaU1
REQUIS PAR
DEPARTMENT
MINISTRE
LarOse
Fiheries
YOUR NUMBER
VOTRE DOSSIER N O
DATE RECEIVED
REÇU LE
03.200-10.
•
PAGES
274- 2 80
OUR NUMBER
NOTRE DOSSIER N-
TRANSLATOR
TRADUCTEUR
85838
L.Knupfer
DATE COMPLETED
-REMPLIE LE —
March
February 22, 1963
26 t
From Biochemische 7eitschrift
4
27h - 280 (1962)
Elne Methode zur Best'
Lg der Fettsguren.
des Human-Dlutes unte
sonderer Berück-
sichtigung der in geringen Mengen vorkommenden
Fettsguren.
A method for the determination of the fatty
acids of the human blood, with particular
reference to the fatty . acids which occur , in
small quantities.
by
A.Glaser, G.Grimmer, E.Jantzen and H.Qertel
From the National Chemical Institute of Hamburg, Division
for Applied Chemistry and Division for Biochemistry.
(Reoeived Feb..28, 1962)
•
A gas chromatographic analysis of the major components
of the fatty acid mixture contained in human blood was recently
described by Schrade and colleagues (8). They were able to
demonstrate by this method that in certain cases of arteriosclerosis changes occur in the relation of the fatty acids
which manifest themselves in a shifting of the quantitative
relations of the principal components (palmitic, stearic,
oleic and linoleic acids).
On account of our own observations it seemed to
be wise to include the fatty acids which occur in the blood
in small quantities, as, e.g., the odd numbered and ramified
fatty acids, into the investigation, in order to obtain
further hints. It is true that little is known, up to the
.../2
-2-present, about the significance of these secondary components
for the organism, so that metaboliC anomalies of these
components, for the time being, do not permit to draw
conclusions as to diseases of the total organism.
An exact quantitative determination of the secondary
components which occur in concentrations of less than 1%
faces considerable difficulties. If we decide to operate
with blood samples of only 10 ml, we obtain about 15 mg of a
mixture of fatty acids which are partly present free as
glycerides, phosphatides and 'cholesterol ester. By differentiating these groups, the , quantities become so much smaller. •
In order to obtain surfaces of at least 100 mm 2 that are
gas chromatographically well measurable with a planimeter
for 1% ingredient in the written diagram, the recorder must
be rather sensitive.
Therefore, heat-transfer cells are not
very suitable as detectors.
But cells which operate on the
basis of the i; -radiation ionization or flame-ionization
safely indicate even smaller quantities than those which
are at our disposal here.
If hiehb'z sensitive detectors
are emplàyed the separations also become a little better,
because the effectiveness of the column growth with the
decreasing quantity of substance.
If we represent the . secondary components in the
desired magnitude on the diagram by applying sufficient .
sensitivity, the peaks of the principal components reach
far beyond the edge of the picture and are pictured only
as stumps of bellcurves. A calculation of the surface area
of the whole jag by means of a substitute-triangle is possible
if the turning points of the curves can be made out.
If
the stump in the picture is too'short for the purpose, the actual
form may still be approximately constructed in many cases.
• • •/3
If the stump, concerns, e.g., oleic ,-acid methyl ester, we can
draw a diagram with pure substance under the same conditions, in such a .manner tbat the point almost reaches the
width of the paper strip. The curve drawn in this way
should be geometrically similar to the one of the stump,
and on the basis bf the proportionality we can draw the
point belonging to the stump.
This method, of course, is
only makeshift, because secondary influences complicate the
comparison.
Table 1.
Factors for the auantitative anal sis of diagrams
which were taken with the argon-
-radiation
ionization cell.
C lo
11
12
13
14
15
1.30
1.23
1.18
1.13
1.08
1.04
C 16
17
18
19
20
1.00
0.97
0 ..93
0.90
0.87
It is safer and simpler to analyze the principal
and secondary parts of a mixture by synchronic double-regis-
tration.with two recorders.
An insensitive recorder gives on
the diagram a complete illustration of all componee, whereas
the other, about ten times as sensitive, draws the small
proportions in a sufficient magnitude. With this method
we have to watch that the limits of the linear range of
measurement of the recording system are not exoeeded.
Difficulties
might occur here, particularly in such cases where the breadth
of measurement is narrow, as, e.g., with some (1-radiation
detectors or when the range of proportionality of the amplifier
does not suffice.
The conversion of the surface areas inferred-
from the diagram presupposes that the specific quantity for
each of the substances which are recorded are known.
.../4
-4,
Calibrations with pure substances or test mixtures Muct run
parallel. Table 1 shows the factors with which the measured
surface areas of the jags are to be multiplied, in order to
obtain ratios in weight percentages which can be compared
.
With one another.
As reference7substance we listed palmitic
acid methyl ester.
Another problem is the calculation of the individual
components of the registered total curve.
The signal of a
secondary component often appears on the basis of the- much
larger jag of a principal component.because with the usually
.a
employed columns of 2,000 to 4,00 0. theoretical . prints
.
complete separation of all components of thé mixture is not
possible.
A graphic solution is achieved by èntering the
complete curves of the principal components
C14- , c16 , c18 )
into the registered total curve and subsequently subtracting the
surface areas.
(
The shapes of the curves for the principal
components can be determined by means Of pure substances.
In order to reduce the components of a gas chromatepgraphic analysis, the fatty acid mixture obtained from the
blood was eeparated into saturated and unsaturated fatty acids
by the use of mercury acetate (7).
Method,
10 ml. blood, stabilized with citrate was miOxed with
the same volume of ethanol and extracted with 20 ml. ether.
The precipitate was again treated- with ethanol-ether (1 : 2,
40.m1).. After evaporation of the . ether phase, the residue was
boiled for 90 minutes with 20 ml. 2nKOH (methanolic) on the
reflgx, diluted with 20 ml, H 2 0 and.the unsaponified shaken
out twice with 20 ml. petrol ether each. After acidification
with HC1, the fatty acids were extracted twice with petrol ether
...15
and boiled down. -le residue was
15 ml. 2% methan(Lic HC1 for 60
with the sanie y
15 ml. petrol e
Mill .
ted to bCiling with
.(2s, subsequently mixed
mule of H 2 0 and ar 'n extracted twice with
, thyl esters resulted
r. Thecollecte(
after evaporEition Of the solvent.
The dry ester mixture WRS mixed with 1 ml. 10% Hg II acetate solution in methanol (sufficient for 50 mg. ester) and
left standing for 48 hours. After evaporating the methanol,
the saturated ester and the adducts of. the unsaturated ester
were eliminated three times from the residue with 2 mi. benzol
each and the remaining residue of Hg n -acetate discarded.
The benzolic solution was evaporated in vacuum and the vacuum
treatment was continued until the acetic acid could be smelled
no longer.
The separation of the satUrated and unsaturated
'esters was done on a silicate gel column with a filling of
about 1 g. and a diameter of
5
mm.
After applying the ester
mixture to the column, filled with a benzol/cyclohexane (1 : 1)
mixtute, the saturated parts were elutriated with
mixture and
5
5
ml.
ml. benzol, then both fractions were united.
The adducts of the Unsaturated ester were elutriated with
6 ml. methanol, which contained 5% glacial acetic acid.
After
evaporation of the solvents, both components, saturated and
adducts of the. unsaturated ester, were weighed.
• In order to insert the samples of the saturated
methyl ester into the gas chromntograph, we used unciform,
bent capillary tubes.
The ester mixtures were dissolved in
10% concentration in cyclohexane, filled into the capillary
tubes, and the solvent was evaporated for several hours at
room temperature.
.../6
-6To the adducts of the tiaturated methylester we
added a methanolic solution of p 4S0N(5%, 1.5 ml.) and conc.
HC1 (0.1 ml.)
After onelhour, 3
L. H2 0 were mixed in and
the free, unsaturated erLr w-: ;;haken out twice With 3 ml.
petrol ether each. After the solvent had been evaporated,
the unsaturated esters were weighed and transferred into a
capillary tube for gas chromatography, in thé same menner as
described above.
All distillations and the boiling with solvents on
the reflux were carried out under nitrogen.
Gas chromatozraphy. For the gas chromatographic analysis of
the ester fractions (saturated and unsaturated fatty acids
separately) it was sufficient to use separation columns, the
poles of which were capable of being.reversed.
We used a
device of our own design 1) With an argon‘-edetectôr (Pye).
The separation columns were 1.10 or 2.20 m. long with an
inner diameter of 4 mm.
They contained a filling of 15 7g
apiezon (? translator) M (ether extrac0 on embacel (? translator).
The carrier gas was argon with a flow speed of about
30 ml/ min. Column temperature 200 or 235 ° C.
The insertion
of the substancé was done by smashing the above mentioned unciform glass capillary tubes In a separately heated evaporator.
Results.
Fatty acids of the human bleod with chain lengths
C
C20 were investigated. By means of gas chromatographic
analysis, apart from the saturated and unsaturated even-numbered
fatty acids, the odd numbered and also fatty acids with
ramified carbon chain were determined.
The identification of
the odd-numbered fatty acids was done by comparison with
1) H.Oertel: Dissertation, Hamburg 1961
.../7
-7-
synthetically prepared pentadecane acid, hepta-decane acid,
nona-decane acid, cis-z29- tridecene* acid, ois- 2 9-pentadecene
acid, ois -A 9-heptadecene acid and ois -A 9 -nonadecene acid.
On the synthesis of these compounds we find a report else• where 2) 4
As we were able to determine by comparing the
9 -hePtadecene acid with ois -A 8 -, cis- A 7 - and
cis-
6 -heptadecene acid, a slight shift of the double
bond by 1 to 3 carbon atoms from the 9-position does not
reveal any measurable differences in the retention volumes;
nonetheless, the odd-numbered unsaturated compounds are probably
only the cis-A 9-position isomeric.
This assumption is
supported by the fact that of the compounds mentioned so
far, the cis-
9 -heptadecene acid (1, 2). and the cis- A 9-
pentadecene acid (2) (German text = pentacene, translator)
could be determined in the deposit fat of cows.
Identification of the ramified fatty acikls was made
according to the relative retention volumes; a discussion on
that subject will be held by E.Jantzen in "Fette und Seifen"
(Fats and Scalps), nere we probably have a mixture of the
methYl-ramified fatty acids standing at the end or before
the end and which differ only little in the retention volumes.
Methyl-ramified fatty acids have so far been isolated from
mutton tallow (5), beef suet (6), butterfat (4), hairfat (9)
and the coccygeal gland fat of the duck (10) . We were able
to obtain from human deposit fat 15-methyl and 14-methylpalmitic acid (not published), so that the assumption of
ramified saturated and unsaturated fatty acids in human blood
may be regarded as safe.
Grimmer, G.: Chem. Ber.
• . ./8
-8
From 10 he31thy test persons, the composition of
the fatty acid mixture of tl-e total blood with special reference
to the fatty acids that occur in small quantities was
examined.
The mean value of the methyl ester mixture ob-
tained amounted to 1.95 mg. per 1 ml. blood.
Of that, the
proportion of the.saturated fatty acids is 39 1 4%.
These
values, and also the compositions stated in 'Tables 2 and 3
of -the saturated and unsaturated parts are nôt comparable
with the statements by Schrade (8), because only the composition
of the serum was examined there.
The corpuscular parts distort
that picture, as we know froM the analysis of the lipoid parts,
e.g., the erythrocites (3):
Table 2.
Percentages of the fatty acids of the blood.
Leund: m 21 J = male, 21 years
f 20 J =
female, 20 years
Mittelwert
=
mean value
unbek.
=
unknown
Rest
=
remainder
unbek. 1 , unknown 1 , volume of retention relative to n-15 1.055
unbek. 2 , unknown 2 , volume of retention relative to n-17 1.065
Table 3.
Percenta es of the unsaturated fatty acids of the blood
Legend: (as in Table 2, translator)
3) According to their periods of retention, these compounds
are, for the time being, regarded as isoforms; their final
classification by isolation is in process,
The acids are listed in the order of their retention
periods.
In the saturated series (Table 2), also ramified
fatty acids appear, in addition to all even-numbered and
odd-numbered fatty acids with unbranched chains. Apart from
mst,4-1.-bra n ,
.../9
-9-
the methyl-branched acids, standing at the end and before the
end, with 15, 16, 17 and 18 C-atoms, there are two more
Individuals present which presumably have a higher degree of
ramific'n.tion.
In addition bit) the compounds listed in the
n-C13 , n-C19 ,
Table, small quentities of n- C11,
n-C20,
and n- C 22 are regularly present, which are
suMmarized as remainder.
The Table of the unsaturated compounds (Table 3)
also shows all members frOm n- C 1 4 to n- C20° Here too the
weakly repf.esented higher ones (C21 to C 24 ) and lower ones
(up to C12 ) were summarized.
For the chain-length C 18 , Oleic .
acid, linoleic acid and linolenic acid were listed together;
in like . manner, the value listed for C2 0 does not only state the
contents of arachidonic acid, but gives the total of the
acids of all degrees of saturation.
The series which, for
the time being, is called the methyl-branched one can be
particularly well recognized in the odd-numbered compounds.
The corresponding even-numbered fatty acids . are present in
considerably smaller proportions and cannot be determined
for the majority of experimental persons.
For that reason,
they are not included in the Table. Among . the unsaturated
too, two up to now unknown compounds appear,
one of which
is present in surprisingly high proportions.
In both Tables thé standard deviations (6 ) are
stated, in addition to the mean values. The term:i+ 2 6 (confidence limits) comprises 96% of the cases which lied
within these two limits. Only for the principal component
the range is so narrow that relatively small deviations from
the mean value lie already beyond the limits. According to
the test material in hand for the proportions of secondary
.../lo
-10-
components, in view of the wide range of confidence limits,
even larger deviations from the mean value cannot be utilized
for individual persons, for the time being . It may perhaps'be
possible to narrow down the confidence limits sufficiently
' by testing the blood of many persons whose good health is
•
satisfactorily proved and possibly by furthèr improving the
analyzing method and measuring technique.
-
Summary
A method is described for the quantitative analysis
of the fatty acid mixture of the blood. A synchronous double
registration allows an exact determination of secondary components.
Several methyl-branched fatty acids have been detected
besides the homologous series of saturated and unsaturated
odd-numbered fatty acids . Four unknown*compounds could not be
classified and have been characterized by their relative
volumes of retention.
'
•
Bibliography
(1) Chrisholm, M.1., and C.Y,Hopkins: Canad. J.Chem. 35, 1434
(1957).
(2) Grimmer. G., J.Kracht and R.Tschesche: Naturwissenschaften
48, 718 (1961).
(3) Hanahan, D.J., R.M.Watts and D.Pappajohn: J,Lipid Fies. 1,
421 (1960).
(4) Hansen, R.P., and F.B.Shorland: Blochem. J. 50, 358 (1952).
(5) Hansen, R.P., F.B.Shorlandand N.J.Cook: Biochem, J. 64, 214
(1956).
(6) Hansen, R.P., F.B.Shorland and N.J.Cook: Biochem. J. 61,
547 (1955).
(7) Jantzen. E., H. Andreas, K. Morgenstern and W.Roth: Fette,
Seifen, Anstrichmittel 63, 685 (1961).
(8) Schr,de, W., E.96h1e, R.Biegler, R.Teicke and B.U11rich:
Ylin. Wschr. 38, 739 (1960).
.../1 1
-1 1-(9) Weitkamp, A. . J.Amer. chem. Soc. 67, UV (1945).
(10.) Weitzel, G., and K.l&nnert: Hoppe-Seylers 7„.physiol.
288, 251 (1951 .
Chem.
Priv.-Doz. Dr.G.Grimmer,
Chemisches Staatsinstitut, Biochemische Abtellung,
2 Hamturg 39,
Maria Louisen-Stiég 2e