STUDIES ON TRIMETHYLAMINE OXIDE

I. OCCURRENCE OF TRIMETHYLAMINE OXIDE IN MARINE ORGANISMS*

BY EARL R. NORRIS AND GEORGE J. BENOIT, JR.~

(From the OceanographicLaboratories and the Division of Biochemistry,
University of Washington, Seattle)

(Received for publication, October 16, 1944)

Since 1909 when Suwa (1) showed the presence of hydroxytrimethylam-

monium hydroxide, commonly called trimethylamine oxide, in the muscle
tissue of the shark (Acunthias vulgaris), several investigators (2-21) have
found the baseto occur widely in marine and fresh water organisms. How-
ever, no systematic study of the occurrence of the compound has been
found in the literature. With the development of a simplified method of
analysis based on previously suggestedmethods, a survey of the distribution
of trimethylamine oxide was made possibleand was carried out on biological
materials available in the Phget Sound region.

EXPERIMENTAL

Lintzel (22) devised a method for the determination of trimethylamine

oxide based upon the reduction of the compound by means of Devarda’s
alloy in dilute hydrochloric acid. He found that choline, betaine, y-butyro-
betaine, and carnitine did not interfere,
In confirmation of Lintzel’s work, the reduction of trimethylamine oxide
in normal hydrochloric acid in the presence of Devarda’s alloy (0.5 gm.
per 5 ml. of solution) was found to be complete in 10 minutes at 105”, 15
minutes at 95”, and 35 minutes at 80”. Choline, betaine, methylguanidine,
creatine, tyrosine, cystine, glycine, and proteins were found not to interfere.
The specificity of the method used in the present work was checked by
isolating trimethylamine oxide from one source from which it had not been
previously reported.
In the analysis of marine organisms, a weighed sample of tissue or organ-
ism was finely ground in a mortar and transferred with distilled water into
a graduated 50 ml. centrifuge tube. The material was extracted for at
least 2 hours at approximately 10” with frequent stirring. After centri-
fuging, the total volume was noted, and the supernatant liquid decanted.
This solution was used for the determination of trimethylamine and tri-
methylamine oxide.
* Taken from a thesis presented by George J. Benoit, Jr., as partial fulfilment of
the requirements for the degree of doctor of philosophy, University of Washington,
1942.
t Present address, Hercules Powder Company, Wilmington, Delaware.
433

This is an Open Access article under the CC BY license.

434 TRIMETHYLAMINE OXIDE. I

Trimethylamine was determined by steam distillation at 30” under re-

duced pressure in an all-glass distillation apparatus described elsewhere
(23). An aliquot of the tissue extract was diluted to 5 ml. in the distilling
flask, and the solution neutralized if necessary with N sodium hydroxide.
It was distilled for 10 minutes into 2 ml. of 0.02 N sulfuric acid, after the
addition of 0.8 ml. of formalin and 2 ml. of 20 per cent sodium carbonate.
The excess sulfuric acid was titrated with carbonate-free 0.006 N sodium
hydroxide, with chlor-phenol red as the indicator. The difference between
this titration and that of a blank represented trimethylamine plus traces
of other volatile bases carried over under the conditions of the experi-
ment.
In the determination of trimethylamine oxide, an aliquot of tissue extract
was diluted with distilled water to 5 ml., and 1 ml. of 6 N hydrochloric acid
was added. The oxide was reduced by digestion for 15 minutes at 95”
with 0.5 gm. of Devarda’s alloy. After reduction the cooled solution was
quantitatively transferred to the distilling flask. The solution and wash-
ings, amounting to about 8 ml., were neutralized with 6 N sodium hydroxide.
The distillation was carried out as described above after addition of 0.8
ml. of formalin and 2 ml. of 20 per cent sodium carbonate. The titration
gave total trimethylamine after reduction, from which the preformed
trimethylamine was subtracted. The sensitivity of the method varies with
the amount of sample available, but in general was found to be near 0.1
micromole of trimethylamine oxide per gm. of tissue.
The distribution of trimethylamine oxide is given in Table I. In many
cases several values are given to show the range observed for a given
species. The distribution in the blood and various tissues of elasmobranch
and teleost fish is also given to show the marked difference between them.
The values given for invertebrates are for muscle tissue if not otherwise
specified; in the case of small animals the entire animal was used.
Diatoms and marine algae were analyzed and found to be free of tri-
methylamine and trimethylamine oxide. The algae analyzed were Nereo-
cystis liitekeana, Porphyra naiadum, and Ulva lactuca, which represent the
three phyla, Phyophyceae, Rhodophyceae, and Chlorophyceae respectively.
Five sea anemones of the tribe Hexactiniae of the phylum Coelenterata
were analyzed and found to contain from 5.7 to 27.0 micromoles of
trimethylamine oxide per gm. of body weight. Segmented worms of the
families, Terebellidae and Nereidae of the phylum Annulata were found to
contain no trimethylamine oxide.

Isolation of Trimethylamine Oxide

The only positive proof of the presence of a base such as trimethylamine
oxide in any organism is the isolation of some recognizable derivative and
 E. R. NORRIS AND G. J. BENOIT, JR. 435

TABLE I
Distribution of Trimethylamine Oxide in Some Marine Animals* (in Micromoles
Per Cm. of Moist Tissue)
Invertebrates
Molluscoidea
Terebratalia transversa (branchiopods). .... . . . Negligible
Echinodermata
Strongylocentrotus jranciscanus (urchin). . . . _. . I‘
Cucumaria miniata (sea-cucumber). . ... .. ‘I
Stichopus calijornicus (sea-cucumber). . “
Mollusca
Pelecypoda
Ostrea japonica (Pacific oyster). . ., . .
‘I

Mytilus e&&is (mussel). . .. . . I(

Paphia staminea (clam). . . .

I‘

(clam). . I,
Saxidomus giganteus . ..

Macoma inquinata (clam). . . . .

“

Card&m calijorniense (cockle). . . . . . 13,ll

“ corbis (cockle). . . . 23
Pecten hericius (scallop). Muscle. . 46,50,39,52,48,41,45,52,
I‘
“ Organs.. . ... .. .. . . 4.6, 4.0, 2.2, 2.3
“ hind&i (scallop). Muscle. .. 77,30,56,38,71,51,38
‘6 “ Organs.. ..... . . ,..... 8, 5, 9
“ jordani. Muscle.. .. .. 32
Amphineura
Katherina tunicata (black chiton). .............. Negligible
Cryptochiton stelleri (giant chiton). ............. ‘I
Gastropoda
Anisodoris nob&s (sea slug). .................... It
Thais Zamellosa (snail). ........................ ‘I
Littorina sitchana (snail) (entire animals). ...... I‘
Cephalopoda
Polypus hongkongensis (octopus). ............... 17
Loligo opalescens (squid). ...................... 107, 111
Lrthropoda (Crustacea)
Copepoda (entire animals)
Mixture; largely Corycaeus afinis, Calanus jinmar-
chius, Tortanus discaudatis, and Epidabiocera am-
phrites ........................................... 45
Mixture; nearly completely Corycaeus afinis ........ 16
Cirripedia
Balanus cariosus (barnacles) (entire animal). ....... 17
“ nubila (barnacles). ........................ 52, 63, 42, 71
Amphipoda sp. (sand-flea) (entire animals). .......... 2.2
Decapoda
Pandalus danae (shrimp). .......................... 48, 28, 63
Pagurus alaskensis (hermit crab). .................. 26
‘I ochotensis “ “. .................... 47
I‘ setosis (hermit crab). ...................... 46
436 TRIMETHYLAMINE OXIDE. I

TABLE I-Concluded
Invertebrates-continued
Pagarustenuimanus (hermit crab) .. .. 30, 28
Oregoniagracilis (spidercrab). . .. .. .. 13
Pugettia “ “ I‘ .. .. 9
Cancergracilis (crab). . . . . . .. .. .. .. 22
“ productus ” . . . . . . . . . . . ... . .. 46
Hemigrapsus nudus (shore crab). . . .. .. .. 11
Vertebrates
Elasmobranch fish
Squalus suckleyi (dogfish). Muscle 135, 88, blood serum 64, kidney 42, liver 8,
18, pancreas 73, spleen 62, stomach 86
Hydrolagus colliei (ratfish). Muscle 121, 134, blood serum 7.5, 6.5
Teleost fish
Sebastodes sp. (rock fish). Muscle 16, 40, 46, 38, 48, heart 21, skin 7, liver 0.7,
0.8, bloodserum, eggs, kidney, gill-rakers, spleen, stomach, intestine all negligible
Scorpaenichthys marmoratus (bull cod). Muscle 46; blood serum negligible
Pleuronectidae sp. (flounders). Muscle 20, 21, 19, blood serum negligible
Roccus sazatilis (sea bass). Muscle 44
Taeniotoca lateralis (blue perch). Muscle 48
Oncorhynchus kisutch (silver salmon). Muscle (adults caught in salt water) 6.2,
6.5, (adult spawning fish) 8.3
Oncorhynchus tschawytscha (king salmon). Muscle (fresh water fingerling) O-0.6,
(adults caught in salt water) 7.1, 8.3, (adult spawning fish) 2.1
* We wish to thank Professor Trevor Kincaid for having supplied us with the
names of many of the organisms studied. We wish also to thank Mr. Richard T.
Smith of the Washington State Department of Fisheries and Professor Lauren R.
Donaldson for having procured some to the specimens used.

its identification. Because of the time required the isolation and charac-
terization of trimethylamine oxide were confined to Pecten muscle.
An aqueous extract of 1.6 kilos of adductor muscle of Pecten hericius
was deproteinized with tannic acid in the presence of phosphoric acid.
The clear filtrate was neutralized with sodium hydroxide and evaporated
under reduced pressure on the water bath. The residue was extracted
repeatedly with methanol at 60-65”, the insoluble portion being dis-
carded.
The methanol filtrate was evaporated to a syrup which was repeatedly
extracted with 96 per cent ethanol. The base was precipitated from the
extract as the picrate by addition of a saturated solution of picric acid in
ethanol. After recrystallization from ethanol, the crystals were dried
over calcium chloride.
The melting point of trimethylamine oxide picrate is reported in the
literature to be in the range 196-202”, with decomposition. The authors
found the melting point of pure trimethylamine oxide picrate prepared from
 E. R. NORRIS AND G. J. BENOIT, JR. 437

alcoholic solution to be 198-199°,1 that of the picrate from Pecten 199-201”,

and the mixed melting point 196-197”, all melting with decomposition.
Part of the trimethylamine oxide picrate from Pecten was converted to
the aurichloride. The gold value indicated the salt to be analytically pure
hydroxytrimethylammonium aurichloride.
(CH&NOH.AuC14. Calculated, Au 47.5; found, 47.4

DISCUSSION

Trimethylamine and trimethylamine oxide were not found in marine

algae or in diatoms.
Of the invertebrates studied only one phylum, namely Arthropoda, of the
class Crustacea, showed trimethylamine oxide in every species analyzed.
Of the Mollusca, trimethylamine oxide was not found in the species
analyzed of Gastropoda or Amphineura. It was found in Cephalopoda,
in which it had been previously reported. Pelecypoda showed occasional
occurrence. It was present in all specimens of Pecten and Car&urn ana-
lyzed. It was not found in the oysters, mussels, or clams analyzed. The
base was isolated from the muscle of the Pecten and shown to be trimethyl-
amine oxide.
Of the phylum Chordata, tunicates and fish were analyzed. Tunicates
did not show an appreciable amount of trimethylamine oxide. Both
elasmobranch and teleost marine fish contain the oxide in their muscles;
however, only the elasmobranchs had measurable amounts in the ‘blood.
The above findings raise the interesting and as yet unsolved problem as to
the origin and metabolic function of trimethylamine oxide. The occurrence
in all marine arthropods, even in copopods, would indicate that it is of
endogenous origin and has some fundamental function in the metabolism
of this group. Of the other invertebrates and vertebrates in which it is
found occasionally the question arises as to whether it is endogenous, formed
in the metabolism of the animal, or entirely of exogenous origin, being ob-
tained from the food. While no hard and fast rule can be formulated, it
appears that the herbivorous animals living on algae or phytoplankton or
carnivorous animals living on herbivorous animals do not contain appreci-
able amounts of trimethylamine oxide, while those living upon zooplankton
(copopods) or other crustaceans or upon crustacean eaters do contain
trimethylamine oxide.

SUMMARY

Trimethylamine oxide was found to be of general occurrence in marine

Crustacea. Occurrence in other marine organisms is reported.
1All melting points are corrected.
438 TRIMETHYLAMINE OXIDE. I

In contrast to the results obtained with elasmobranch fish, the blood of

marine teleost fish contained no appreciable trimethylamine oxide.
Trimethylamine oxide was isolated from the muscle tissue of Pectm
hericius.

BIBLIOGRAPHY

1. Suwa, A., Arch. ges. Physiol., 128, 421 (1909).

2. Hentze, M., 2. physiol. Chem., 91, 230 (1914).
3. Suzuki, Inouye, and Bharatkar, J. Coil. Agr. Tokyo, 6,9 (1912); cited by Lintzel,
W., Pfeiffer, H., and Zippel, I., Biochem. Z., 301, 29 (1939).
4. Suzuki, U., and Okuda, Y., J. CoZZ. Agr. Tokyo, 6, 13 (1912); cited by Lintzel,
Pfeiffer, and Zippel (3).
5. Hoppe-Seyler, F. A., 2. physiol. Chem., 176, 300 (1928).
6. Poller, K., and Linneweh, W., Ber. them. Ges., 69, 1362 (1926).
7. Hoppe-Seyler, F. A., and Schmidt, W., 2. Biol., 87, 59 (1927).
8. Hoppe-Seyler, F. A., Verhandl. phys.-med. Ges. Wtirzburg, 63, 24 (1928); 64,
160 (1929); cited by Hoppe-Seyler, F. A., 2. Biol., 90, 433 (1930).
9. Beatty, S. A., J. Fisheries Res. Bd. Canada, 4, 229 (1939).
10. Lintzel, W., and Herring, E., Vorratspflege u. Lebensmittelforsch., 2, 263 (1939) ;
Chem. Abstr., 36.3351 (1941).
11. Yoshimura, K., and Fujise, S., J. Chem. Sot. Japan, 60, 109 (1929); Chem. Abstr.,
28, 216 (1932).
12. Yoshimura, K., and Nishida, K., J. Agr. Chem. Sot. Japan, 8, 153, 618 (1930);
Chem. Abstr., 24, 4546, 5881 (1930).
13. Hoppe-Seyler, F. A., Z. physiol. Chem., 221,45 (1933).
14. Mohr, M., 2. Biol., 98, 120 (1937).
15. Grollman, A., J. Biol. Chem., 81,267 (1929).
16. Hoppe-Seyler, F. A., 2. Biol., 90, 433 (1930).
17. Hoppe-Seyler, F. A., and Linneweh, W., 2. physiol. Chem., 198,47 (1931).
18. Ackermann, D., and Mohr, M., 2. Biol., 98, 37 (1937).
19. Spahr, W., Z. Biol., 98, 43 (1937).
20. Wilson, D. W., and Wolff, W. A., J. Biol. Chem., 124, 103 (1938).
21. Lintzel, W., Pfeiffer, H., and Zippel, I., Biochem. Z., 301,29 (1939).
22. Lintzel, W., Biochem. Z., 273, 243 (1934).
23. Benoit, G. J., Jr., and Norris, E. R., Ind. and Eng. Chem., Anal. Ed., 14,823 (1942).