Note: Descriptions are shown in the official language in which they were submitted.
20832~3
Metabolites labelled with isotopes are in demand for
many research, diagnostic and medical applications, especially
with the development of powerful mass spectrometry and nuclear
magnetic resonance (NMR) instrumentation. For example, labelled
amino acids are useful in the study of protein structure by NMR.
Labelled amino acids can be prepared by either chemical or biolo-
gical means. Chemical means usually involve long, multistep
organic synthesis that are generally inefficient. Most organic
methods generally involve the use of expensive precursors, the
isolation of intermediates, intricate purification schemes as well
as optical resolution of the products since a mixture of optical
isotopes is often produced. Furthermore, organic chemical methods
are generally limited to introducing a carbon label to a terminal
position such as the terminal carboxyl group. These labels are
often unsuitable for biological applications as they are suscep-
tible to decarboxylation reactions or similar modes of metabo-
lism.
The preparation of labelled amino acids by biological
methods seems a more practical alternative. These biological
methods usually involve the growth of an organism on a labelled
precursor. An example of this is growing algae on 13Co2. This
technique i9 not always practical for producing labelled amino
acids for ~MR work as uniformly labelled samples yield highly
complex spectral data due to the adjacency of two or more 13C
nuclei which causes splitting of the resonances. Uniform label-
ling also results in an enormous conversion of 13C into unwanted
biomass. Therefore, it is highly desirable to produce site speci-
2~32~3
fic labelled species. One of the methods to date involves thedevelopment of mutants in order to decrease the randomization of
the label and to increase the efficiency of labelled precursor
utilization. LeMaster and Cronan (in the Journal of Biological
Chemistry, Volume 257, Page 1224 to 1230, 1982) developed two
Escherichia coli (E. coli) strains for the production of specifi-
cally labelled amino acids. The strains that they prepared had
metabolic lesions so that no carbon interchange could occur
between the intermediates of glycolysis and the tricarboxylic acid
cycle. The amino acids synthesized by these E. coli mutants
showed relatively few instances of adjacently labelled carbons
thus resulting in a more simplified NMR spectra. However, there
are problems with this method. Firstly, it is highly cumbersome
to produce such mutants. Secondly, the degree of the label
randomization is usually higher than that which can be obtained by
chemical methods. In the case of the E. coli mutants, label
randomization occurs because of the presence of the pentose phos-
phate shunt common to many microbes. Label randomization, as used
herein, denotes the distribution of label to carbon atoms that are
relatively unlabelled compared to the predominantly labelled
carbon atom. This can occur by the transfer of label from the
carboxyl group of acetate to CO2 or by the presence of cyclic
pathways.
The applicants have shown that certain methanogenic
bacteria can be used to produce specifically-labelled amino acids
and carbohydrates. Methanogenic bactexia are obligate anaerobes
which derive energy by the metabolism of simple compounds, includ-
20832~3
ing C02 and H2 gases, to methane. This mode of energy ~enerationis unique. Also unique is their mode of assimilating simple Cl or
C2 compounds into cell constituents. Methanogens capable of grow-
ing with Co2 as sole carbon source synthesize acetate (acetyl
Coenzyme A) from 2 CO2 molecules via a CO-dehydrogenase reaction.
However, certain methanogens are incapable of this synthesis, and
consequently require for growth H2/C02 plus acetate supplied to
the medium. Such auxotrophy for acetate was acquired in Methano-
spirillum hungatei (M. hungatei) strains GPl and JFl during multi-
ple passages over several years in a medium containing acetate(Sprott and Jarrell in Can. J. Microbiol, Volume 27, Page 444-451,
1981). In view of this property M. hungatei was grown on isotopi-
cally labelled acetate to produce labelled metabolites such as
amino acids and carbohydrates. The extent of label randomization
was very low in M. hungatei. Other methanogens examined were
inferior to M. hungatei with respect to label randomization,
especially with ~ 3C]acetate precursor.
Labelled amino acids have previously been prepared in
methanogens. However, some of the problems with the known
methanogen systems for preparing labelled amino acids include low
percentage utilization of the acetate precursor and randomization
of the label. These are discussed below.
The applicants have shown that M. hungatei and other
methanogens require for optimal growth high concentrations of
acetate and utilize a low percentage of the acetate. This was
confirmed recently by Jetten et al. (in FEMS Microbiology Ecology,
Volume 73, Page 339-344, 1990) for strains GPl and JFl. These
20832~3
problems are generally due to the fact that _. hungatei strains
GPl and JFl, and certain other methanogens, cannot grow below pH
6.6, making them difficult organisms to grow in the C02/HC03
buffered medium which is required for this technique. Also, since
there appears to be no active transport system for acetate in
these methanogens, acetate utilization at neutral pH is less effi-
cient due to a difficulty in diffusion of the labelled-compound
into the cell at neutral pH. The protonated form of weak acids is
known to penetrate cell membranes, hence penetration occurs best
at pH values too acidic for growth of these methanogens.
Most methanogens have a considerable degree of exchange,
or randomization between C02 and the C-l or carboxyl group of
acetate. The result is that growth in media containing (1-13C)-
acetate results in production of amino acids labelled not only in
carbon atom positions originating from the C-l of acetate, but
also in positions where C02 is incorporated. An example of this
(described in the Journal of Bacteriology Vol. 162 p. 905-908,
Ekiel et al. 1985) is the methanogen Methanosaeta concilii GP6
("Methanothrix conc~ ") which demonstrates a considerable degree
of carbon exchange between C02 and the C-l of acetate.
In view of all of the above, it was desirable to try and
isolate acetate-requiring methanogens capable of metabolizing
H2/C02 which could grow at acidic pH conditions (acid-tolerant
methanogens). Growth of such methanogens at acidic pH should
ensure sufficient acetate in protonated form for its more complete
removal from the medium, and hence give higher efficiencies of
acetate precursor conversion. It was also desirable to isolate a
20~3253
methanogen that has a very low level of randomization or carbon
exchange between CO2 and the C-l of acetate.
SUMMARY OF THE INVENTION
It is a feature of the present invention to provide a
method for producing isotope labelled metabolites comprising in-
cubating an acid tolerant methanogen in the presence of isotope
labelled acetate. Acid tolerant methanogens as used herein,
refers to methanogens capable of growth at pH values less than
6Ø
It is a feature of the present invention whereby the
metabolites are amino acids or carbohydrates.
It is another feature of the present invention whereby
the methanogen has a very low level of acetate synthesis from CO2
in the presence of exogenous acetate.
It is another feature of the present invention whereby
said methanogen exhibits a low level of carbon exchange between
C2 and the carboxyl group of acetate. It is also a feature
whereby the metabolic pathways of the methanogen are non-cyclic.
It is also a feature of this invention whereby the
isotope is 13Carbon.
It is yet another feature of the present invention
whereby said methanogen is Methanobacterium espanolae.
DESCRIPTION OF THE DRAWINGS
_ _
Figure 1 is a schematic diagram illustrating the key
metabolic reactions of strain GP9 (M. espanolae).
Figure 2 is a flow chart showing the fractionation of
strain GP9 cells labelled with 13C acetate.
2083253
Figure 3 is a 13C NMR spectrum of the protein hydro-
lysate obtained from cultures grown on 13C acetate.
DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT
The applicants have isolated a new strain of methanogen
named Methanobacterium espanolae and designated as strain GP9,
that has a broad pH range of 5.6 to 6.4 for optimum growth and is
capable of growth at pH levels at least as low as 4.7.
Figure 1 illustrates the key metabolic reactions of
strain GP9 indicating its inability to synthesize acetate from CO2
and showing the presence of a reductive, partial citric acid path-
way. Ingredients supplied in the medium are shown in boxes. All
these elements of biosynthesis lead to very unique labelling
patterns of amino acids, carbohydrates and nucleotides. By way of
example, as discussed below, we will describe the isotope label-
ling of both amino acids and carbohydrates in strain GP9.
Isolation, stock cultures, and inoculum.
This new strain was isolated from the primary sludge
obtained from the waste treatment facility of a pulp mill in
Canada, which is described in the International Journal of
Systemic Bacteriology, January 1990, page 12-18 (Patel et al.).
This methanogen is deposited with the German Collection of Micro-
organisms and Cell Cultures as DSM 5982, with the National
Research Council of Canada culture collection as NRC 5912, and
with the oregon Collection of Microorganisms as OCM 178.
A 10-ml sludge sample, that was collected anaerobically
from the anoxic zone of the primary settling basin of the E. B.
Eddy Forest Products Ltd. (Espanola, Ontario, Canada) bleach
20~32~3
kraft mill, was transferred to a 60-ml serum vial under 100~ N2.
The cellulolytic and methanogenic activity of this mixed culture
was maintained (incubation temperature, 35C) by transferring it
(10% vol/vol) to fresh primary sludge (under N2, supplemented with
cysteine-Na2S and 5 mM NH4Cl) every 6 weeks. This primary sludge
enrichment culture was inoculated into SA medium (80% H2-20% CO2)
at pH 5Ø After we detected large quantities (10% vol/vol) of
CH4 gas in a headspace gas analysis, the methanogenic cultures
were maintained in similar media by transferring them (10%
vol/vol) at l-week intervals. Such a culture was serially diluted
into SA broth (pH 5.0) and plated onto SA agar (pH 5.5) (SA medium
supplemented with 2.2% [wt/vol] Noble agar [Difco Laboratories,
Detroit, Mich.]). The agar medium was prepared just as the broth
medium was, but the prereduced and autoclaved medium (20-ml
portions in 60-ml vials) was poured into Petri plates inside an
anaerobic chamber tCoy Manufacturing Co., Ann Arbor, Mich.)
containing a 5% CO2-10% H2-85% N2 atmosphere. After overnight
drying in the chamber, the plates were streaked, introduced into
Brewer anaerobic iars which were then flushed out with 80% H2-20
CO2, and incubated at 35C. Representative colonies from agar
plates were transferred into vials containing SA broth (pH 5.5)
inside the anaerobic chamber. Methanogenic broth cultures were
serially diluted and plated for colony picking. This procedure
was repeated several times until culture purity was established.
Stock cultures of methanogenic isolates were maintained
in SA broth at pH 5.5 and 5.0 by transferring them (10%, vol/vol)
every 7 to 10 days into fresh media at the appropriate initial pH.
2083253
The stock culture vials were repressurized every 4 days by inject-
ing 80~ H2-20% C02.
Unless stated otherwise, the inoculum for tests consis-
ted of 1% (vol/vol) of a l-week-old culture in SA medium (pH 5.5)
which was anaerobically and aseptically concentrated 10 times into
the appropriate medium to avoid carry-over of the nutrients or
compounds under investigation. All incubations were static,
except when indicated otherwise.
Preparation of the Cells
For analytical purposes herein GP9 cells were grown an-
aerobically in 100 ml aliquots of citrate/phosphate, pH 5.5, S-
medium (SA medium lacking acetate) supplemented with either [1-
13C]acetate or [2-13C]acetate as sodium salts to give an initial
concentration of 465 mg acetic acid per liter. The inoculum, when
grown in unlabelled-acetic acid medium, was washed once with
acetate-free citrate/phosphate S-medium and resuspended (10-fold
concentrated) prior to inoculating with 1 ml/lOOml of medium.
Incubation was at 35C with a 100 rpm shake rate. Growth flasks
were pressurized to 10 psi twice daily with 80% H2/20% C02 after
the first 24h of incubation. At 48 and 72h the headspace gases
were removed by flushing and replaced with fresh H2/C02. Two-ml
of fresh cysteine/sodium sulfide reducing agent was added per
100 ml at 72 and 79h incubation. Figure 2 is a flow chart that
illustrates the preparation of the GP9 cells for NMR analysis of
the amino acids or carbohydrates. Cells were harvested by centri-
fugation (10,500xg, 15 min) at 96h yielding ca 0.36 mg cell dry
weight/ml culture. The cells were washed, and resuspended into 10
20~32~3
ml deionized water.
Lysis was achieved mechanically by passing twice through
a French pressure cell (ca. 16,Q00 psi) with ca. 99% breakage.
[Treatment of the cells with dithiothreitol and sodium dodecylsul-
fate at pH 8 also causes lysis]. The lysate was treated for 30
min at 30C with deoxyribonuclease (0.05 mg/ml) and centrifuged
(15,000xg, 30min).
Protein was precipitated from the supernatant by adding
ethanol to 70% (vol/vol) and storing for lh at -5C. The protein
was collected by centrifugation and hydrolyzed in vacuo in 6N HCl
(24 or 48 h at 110C). The labelled amino acids were recovered
from the crude bacterial protein hydrolysate and sometimes subjec-
ted to an ion exchange system similar to that described by Le
Masters and Richards in Analytical Biochemistry, Volume 122, pages
238-247, 1982. Hydrolysates were dried in vacuo for quantitation
with an amino acid analyzer. Dried samples hydrolyzed for 48h
were resuspended in O.lM HCl solution in D20 for NMR analysis.
Carbohydrates were recovered from the pellet (obtained
after centrifuging the cell lysate) by hydrolyzing with 1 ml of 2
M H2S04 (110C for 4 h). Samples were neutralized with BaC03,
centrifuged and the supernatant passed through columns of Dowex 50
and AGl-X2 (BioRad). Monosaccharides were eluted with water,
lyophilized and resuspended in D20 solution.
NMR spectra were recorded with a Bruker AM-500 spectro-
meter operating at 125 MHZ in the Fourier transform mode, at 26C
in 5-mm tubes.
2083253
AMINO ACIDS
Labelling patterns of amino acids
. _
13C NMR spectra of protein hydrolysates obtained from
cultures grown on [1-13C] and [ -13C]acetate are shown in Figure
3; chemical shift values are included in Table I. Similar to the
results for _. hunyatei (discussed in Journal of Bacteriology,
p. 316-326, 1983, Ekiel et al), a very high specificity of label-
ling is observed, with labelling patterns virtually identical to
those in M. hun~atei (discussed in Journal of Bacteriology,
p. 316-326, 1983, Ekiel et al). Therefore, the same biosynthetic
pathways are operating in both GP9 and M. hun~atei including the
incomplete tricarboxylic acid pathway operating in the reductive
direction and a pathway to isoleucine via citramalate. In GP9, as
in most other methanogenic bacteria, acetate is incorporated and
converted to acetyl-CoA, which is then reductively carboxylated to
pyruvate (Figure 1). However, with M. espanolae we avoided the
difficulties experienced with known methanogen systems such as
Methanosaeta concilii, Methanosarcina barkeri or M. hungatei,
___ _
wherein either label randomization occurs through interchange of
the carboxyl group of acetate with CO2 (and visa versa), or much
of the acetate precursor remains in the medium following growth.
Label randomization
-
Label randomization is essentially the distribution of
label to the carbon atoms that are relatively unlabelled as
compared to the predominantly labelled carbon atom. Randomization
can be caused, for example, by the transfer of label from the
carboxyl group of acetate to C02 (scrambling) or by the presence
of cyclic pathways. Random labelling can be easily detected in
-- 10 --
20832~3
13C NMR spectra, since the signal intensities of the randomly
labelled carbon atoms will be sma:Ll in relation to the predomi-
nantly labelled positions, but greater than the intensity result-
ing from a 1.1~ natural abundance of 13C. In protein hydrolysates
from strain GP9, we could estimate the intensities (measured as
heights) of these peaks as below 2% (of the total intensity) in
the case of the culture grown on [1-13C]acetate, and below 1.5%
for the culture grown on [2-13C]acetate. Therefore, there is very
little, if any, randomization of label between positions primarily
labelled from carboxyl and methyl groups of acetate, and very
little transfer of label to positions primarily labelled from
CO2 .
Enrichment level
_ _ _ _ . _ _
The level of carbon atom enrichment with 13C was estima-
ted in two ways: by monitoring protons covalently bound to label-
led positions by lH NMR, and from 13C NMR spectra of those amino
acids which have two adjacent carbons simultaneously labelled.
In lH NMR spectra, hydrogen directly bound to 13C are
observed as doublets, because of carbon-proton coupling.
Additionally, in the center of each doublet, a signal will be
present for protons in these molecules which have no isotope en-
richment. The level of labelling can be calculated from the in-
tensiLy ratio of the central peak to the doublet. Because of the
signal overlap in lH NMR spectra of amino acids, only a few
signals can be measured accurately in this way. Five signals
(mostly of methyl groups of branched-chain amino acids) were
measured in a spectrum of amino acids labelled from [2-
2083253
3C]acetate, ~nd two signals for amino acids labelled from ~l-
l3C]acetate. Obtained levels of l3C labelling were 92 and 9l~,
respectively. Similar measurements, using l3C NMR, were performed
for Ile, Leu and Val signals, and labelling from [2-l3C]acetate,
and gave 94~ l3C enrichment.
Figure 3 illustrates a l3C-NMR spectrum of the protein
hydrolysate obtained from the cultures grown on, A) [l-
l3C]acetate, B) [2-l3C]acetate. Signals were assigned according
to Table I.
The experiment shown in Figure 3 was repeated with
citrate excluded from the medium to test whether or not citrate
could affect the quality of the labelled-amino acid products. As
found previously (Figure 3), the degree of labelling was similar
(89.5% based on l3C NMR of Ile, Leu, Val), the level of label
randomization was low (< 2~1, and the labelling patterns of the
amino acids were identical to those with citrate in the medium.
Reproducibillty
All NMR analysis were repeated for three growth experi-
ments, with the ~ame results.
Amino acid yields
In addition to l3C NMR spectroscopy, the amino acid
hydrolysates obtained by the growth of strain GP9 on ~l-
l3C]acetate and [2-l3C]acetate were subjected to amino acid analy-
sis. This is illustrated in Table II. The amino acid yield on a
dry wt. basis was about 40~ of the cell dry weight, with indivi-
dual amino acid yields as shown. Hydrolysis for 24 or 48 ~ gave
similar results.
- 12 -
2083253
CARBOHYDRATES
Labelling of Carbohydrates
In addition to the carbon atoms of amino acids, other
cell constituents of GP9 are labelled selectively (Fig.l). The
13C chemical shifts for the carbon atoms of ~ and ~ anomers of
glucose, galactose, and mannose, the three most abundant sugars,
are presented in Table III. Growth of GP9 in media containing
3C)acetate resulted in label incorporation in hexose sugars to
carbons 2 and 5, whereas (2-13C)acetate resulted in incorporation
in carbons 1 and 6.
Summary of results
The overall dilution of the C-l or C-2 acetate label is
exceptionally low, compared to other bacteria, and no mutation was
necessary to block pathways leading to label dilution. Acetate
precursor enriched to the level of 99% was used, and some dilution
was caused by the use of unlabelled inoculum, which combined can
account for 4% of carbon positions unlabelled. The additional
2-5% of carbons having 12C most probably originate from low levels
of acetate synthesis from CO2. Amino acids labelled from both C-l
and C-2 of acetate gave very similar enrichment levels. This
result means that, unlike many methanogenic bacteria, carbon ex-
change between the C-l of acetate and C2 was not active in strain
GP9.
The proposed technology has several advantages over the
known techniques for producing labelled amino acids and carbo-
hydrates. These are as follows:
1. Strain GP9 grows over a broad pH range in the acidic region.
2083253
In a bicarbonate buffered medium an appropriate acidic pH is
readily achieved without the need for pH control during growth.
Often methanogens such as M. hungatei can be difficult to grow,
because growth will not occur at pH values more acidic than
approximately 6.6.
2. High level of acetate utilisation by strain GP9 and low unit
cost of acetate. Certain members of the methanogenic bacteria
(ex, strain GP9) selectively incorporate acetate and C02 into
biomass, and convert CO2 into methane. This unique coupling of
energetic and biosynthetic pathways allows very economic use of
acetate. Simultaneously, acetate is one of the cheapest sources
of label, much cheaper than other sources used for other bacteria,
such as E. coli, described in The Journal of Biological Chemistry,
Vol 257, p. 1224-1230 LeMaster and Cronan, 1982. Unlike
M. hungatei, strain GP9 utilizes acetate completel~; i.e. after 2
days of growth 45% of the acetate precursor had been taken up by
the strain GP9 cells and after the 3rd day all of the acetate had
been taken up.
3. Very high level of specificity. Strain GP9, like other
methanogenic bacteria, lacks cyclic biosynthetic pathways, and in
strain GP9 the exchange between CO2 and the carboxyl group of the
acetate is negligible. As a consequence, much higher levels of
enrichment can be obtained than in other systems, for example the
E. coli mutants of LeMaster and Cronan, 1982 (> 90 versus 70-
85%).
4. Because of the presence of unique biosynthetic pathways,
methanogens produce patterns of labelling which cannot be mimicked
- 14 -
20832~3
by any other bacteria, in particular they are very different from
those obtained for E. coli (LeMaster and Cronan, 1982).
5. Labeling patterns for each amino acid can be varied according
to the carbon atom, or combination of carbon atoms, in the precur-
sors fed to the cells (i.e. [1-13C]acetate, [2-13C]acetate, [1,2-
13C]acetate or 13Co2). Other more costly precursors such as pyru-
vate could be used as an alternative, ex. ~1-13C]pyruvate could be
used to label other positions.
6. Production of a broad spectrum of site-specific labelled
amino acid species. The procedure described allows recovery of
all amino acids but cys, trp and his, which can be obtained as
well, using modified hydrolysis conditions as described in
Analytical Biochemistry, 1982, p. 238-247 LeMaster and Richards.
7. The specificity of labeling applies also to cell products
other than amino acids and carbohydrates, such as
purine/pyrimidine bases and lipids.
The foregoing embodiment of the present invention is an
example of this invention meant to illustrate and not limit the
present invention. It will be evident to those skilled in the art
that this invention can be practiced with other acid tolerant
methanogens and other isotopes (such as 14Carbon).
20~3253
Table I. 13C NMR chemical shifts of amino acids from a protein hydrolysate of
GP9a.
_
Amino acids Chemical shifts (ppm) forb
aOOH C-2 C-3 C-4 C-5 C-6 Other
Alanine 49.73* 15.31
Serine 55.20* 59.43
Glycine 40.33*
Aspartate 49.87* 33.89
Threonine 59.01* 65.31
24.93 29.46
Glutamate
24.98* 29.50*
. . _ . _ .
27.02 23.77
Arginine
27.08* 23.75*
_ _ .. . . _ . _ _
28.45 23.44
Proline
28.45* 23.40*
Leucine 173.20* 51.70 39.01* 23.96* 20.88 C-5' 21.64
Valine 58.85* 28.95* 16.76 C-4' 17.43
Isoleucine 171.99* 57.60 35.70* 24.57* 10.91 C-6' 14.2
Phenylalanine 54.68* 35.69 C-5' 129.38
C-6' 129.11*
Tyrosine 54.68* 34.84 C-5' 130.82
C-6' 115.87*
Lysine 53.33* 26.27 29.35 39.00*
MRthionine 28.98
_ _
a Reference TMS capillary, pH=1Ø
b The sources of carbon atoms are designated by *, C-l acetate; C-2
acetate.
- 16 -
2083253
Table II. Yield of amino acids from 24 and 48 h hydrolysates of
protein recovered from strain GP9 grown on [1-13C] or
[2-13C]acetate.
[1-13C]acetate [2-13C]acetate Yield
Amino acid mol % mol ~ ~g/mg cell dry wt.
24H 48H 24H 48h 48h
.
Aspartic11.52 11.11 11.3111.21 46.65
Threonine6.07 6.19 6.11 6.20 23.09
Serine 4.99 4.79 4.95 4.65 15.28
Glutamic12.50 11.96 12.6412.33 56.73
Proline 3.88 4.00 4.17 4.12 14.84
Glycine 9.47 10.03 8.67 8.98 21.08
Alanine 9.03 9.15 9.26 9.32 25.97
Valine 6.90 7.59 6.81 7.51 27.51
Methionine1.81 1.52 2.13 1.58 7.36
Isoleucine6.67 7.41 6.60 7.35 30.14
Leucine 7.61 7.42 7.58 7.79 31.94
Tyrosine2.60 2.49 2.64 2.46 13.91
Phenylalanine 3.69 3.66 3.71 3.70 19.09
Histidine1.59 1.55 1.70 1.58 7.73
Lysine 8.07 7.79 8.15 7.89 36.04
Arginine3.58 3.35 3.56 3.36 18.27
395.57
20832~
TABLE III. 13C NMR chemical shifts for carbon atoms of most
abundant carbohydrates labelled by growth of GP9 in media contain-
ing (1-13C) or (2-13C)acetate.
Anomer Carbon atom 13cprecursorglc gal man
1 (2-13C)acetate 92.19 92.38 94.14
2 (1-13C)acetate 71.58 68.45 70.79
(1-13C)acetate 71.54 70.58 72.50
6 (2-13C)acetate 60.68 61.28 61.3-
61.4
(2-13C)acetate 96.01 96.56 93.77
2 (1_13C)acetate 74.24 71.98 71.32
(1-13C)acetate 76.05 75.26 76.27
6 (2-13C~acetate 60.85 61.09 61.3-
61.4
_ _ . _ _
- 18 -
,: .
.
;
. ,
