Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol storage
NIEHS Marine and Freshwater Biomedical Sciences Center, Rosenstiel School of Marine and Atmospheric Science, Miami, FL 33149, USA
*Present address: Monterey Bay Aquarium Research Institute, Moss Landing, CA 95039, USA (e-mail: bseibel{at}mbari.org)
Accepted 21 November 2001
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Summary |
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Key words: trimethylamine oxide, choline, phosphatidylcholine, lipid, cephalopod, buoyancy, deep sea, urea, solute.
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Introduction |
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In elasmobranchs, for example, TMAO may counteract the toxic effects of urea on proteins (Somero, 1986; Yancey and Somero, 1980
). Because they are iso-osmotic with sea water, sharks retain large quantities of urea in their fluids as an osmolyte. Urea is highly perturbing to enzyme systems. TMAO has a demonstrated ability to counteract the perturbing effects of urea on enzyme activity when accumulated in a 2:1 ratio with urea (Somero, 1986
). Although some enzymes seem to have an evolved tolerance of urea regardless of TMAO (for a review, see Ballantyne, 1997
), the counteracting solute hypothesis has received wide support (Wang and Bolen, 1997
; Yancey et al., 1982
; Yancey and Somero, 1980
; Yancey and Siebenaller, 1999
; Somero, 1986
).
TMAO may also counteract the effects of hydrostatic pressure on enzyme function in deep-sea animals. Yancey and colleagues (Gillett et al., 1997; Kelly and Yancey, 1999
) have demonstrated a correlation between TMAO concentration and capture depth in a variety of organisms. They have also shown that 250 mmol l1 TMAO in vitro is able to counteract the loss of activity in some enzymes that results from high hydrostatic pressure (Yancey and Siebenaller, 1999
; Yancey et al., 2001
).
Other methylamines are known to counteract the effects of ammonia toxicity (Kloiber et al., 1988; Minana et al., 1996
), salt (Dragolovich, 1994
) and temperature stress (Krall et al., 1989
; Nishiguchi and Somero, 1992
) on protein function in a variety of organisms. Although TMAO may serve the functions attributed to it (i.e. it may be adaptive), it is not necessarily synthesized or accumulated as a specific adaptation to any of the stresses mentioned above. The source of TMAO in marine animals and the time course of its accumulation are not well characterized. Here, we present preliminary measurements of TMAO concentrations in a number of cephalopod species and review the distribution, biosynthesis and metabolism of methylamines in marine animals to assess their adaptive significance.
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Methylamine synthesis |
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Furthermore, fasted dogfish maintained stable TMAO concentrations over 41 days (Cohen et al., 1958). This result may be partly due to the active reabsorption of TMAO in the dogfish kidney (Cohen et al., 1958
; Goldstein et al., 1967
). Some sharks retain TMAO quite effectively, but substantial loss does occur such that maintenance of TMAO levels over 41 days seems unlikely without an endogenous source. Goldstein and Palatt (1974
) found TMAO turnover rates of 414 % per day in four different elasmobranch species, but excretion rates of less than 1 % have been reported in some fishes (Agustsson and Strøm, 1981
). Diets rich in TMA and choline have produced higher levels of TMAO in the muscles of some fish species but not in others (Goldstein et al., 1967
; Agustsson and Strom, 1981
). An endogenous source is suspected in many species, especially those that accumulate TMAO in high concentrations.
Endogenous choline supply is believed to limit the accumulation of glycine betaine (betaine), another common methylamine osmolyte, in euryhaline oysters (Pierce et al., 1997). Oyster mitochondria take up choline and convert it, via betaine aldehyde, to betaine (Fig. 1B). The enzymes involved in this transformation are well characterized (Dragolovich, 1994
). Betaine could, if similarly produced in other species, subsequently be converted to TMA and TMAO (Fig. 1B). Some studies suggest that TMAO is a better counteracting solute than betaine.
Some plants produce methylamines, particularly betaine, reportedly for osmoregulation and protection against drought and salt-stress. A tremendous amount of research has been directed towards elucidating the pathways of betaine accumulation in plants in the hope of conferring drought-resistance to important commercial crops such as tobacco (Nuccio et al., 1998). Plant research may, thus, provide insight into the pathways and mechanisms of methylamine synthesis and storage in marine animals. Several pathways for the production of choline in plants have been identified. Drought-resistant plants, such as chenopods (e.g. spinach and sugar beet), are adapted for direct production of choline from ethanolamine (Hanson and Rhodes, 1983
; Summers and Weretilnyk, 1993
; Weretilnyk et al., 1995
). Radioactively labeled ethanolamine in spinach is recovered primarily as betaine. In contrast, labeled ethanolamine in wheat and barley is recovered primarily in phospholipid (Hitz et al., 1981
; McDonnell and Wyn Jones, 1988
) (Fig. 2).
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Phosphatidylcholine hydrolysis is also responsible for diacylglycerol production in plants that store large quantities of triacylglycerols in their seeds (Gur and Harwood, 1991). Triacylglycerol sometimes constitutes as much as 80 % of the dry mass of the seeds. Triacylglycerols are also among the most common form of energy reserve in animals, but diacylglycerol ethers are also frequently stored. Many animals store acylglycerols as metabolic fuel reserves that can, during starvation, migration, reproduction or egg development, be mobilized and oxidized to drive metabolic processes. Phosphatidylcholine hydrolysis, which is important during accumulation of acylglycerols, may serve as an endogenous source of choline, resulting in TMAO accumulation in animals. The enzymes that are required for phosphatidylcholine hydrolysis are apparently well conserved, having been found in mammals, molluscs and arthropods as well as the plants mentioned above (Anfuso et al., 1995
).
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Diacylglycerol and phosphatidylcholine |
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In animals, diacylglycerol released from phosphatidylcholine hydrolysis is an important second messenger (Billah and Anthes, 1990; Wakelam et al., 1993
). In fact, during hypo-osmotic cell volume regulation, cell swelling results in membrane turnover and phosphatidylcholine hydrolysis, resulting in the formation of diacylglycerol. Diacylglycerol activates phosphokinase C which, in turn, stimulates the release of osmolytes in a range of organisms from sharks (Musch and Goldstein, 1990
) to algae (Thompson, 1994
). Cell volume regulation via phosphatidylcholine hydrolysis under hyperosmotic stress (i.e. dehydration) could contribute to the release of free choline and subsequent betaine or TMAO accumulation in some animals (although some mechanism would be required to prevent diacylglycerol from stimulating the release of osmolytes in this case). For example, TMAO was shown to accumulate during dehydration in frog gastronemius muscle (Wray and Wilkie, 1995
), although the authors postulate that TMAO accumulated to counteract increased urea concentrations. Choline produced via phosphatidylcholine hydrolysis has no known function other than the synthesis of acetylcholine in neural tissue (Billah and Anthes, 1990
). We propose that phosphatidylcholine hydrolysis via the back reaction of CPT may provide an endogenous source of choline for TMAO synthesis in animals.
Hydrolysis of dietary phosphatidylcholine by phospholipases C and D may also be an important source of free choline for TMAO synthesis (Wakelam et al., 1993). Hydrolysis of labeled phosphatidylcholine by phospholipase C in the gut of larvae of the dragonfly Aeshna cyanea resulted in recovery of labeled products in various forms, including acylglycerols and glycine betaine (Weiher and Komnick, 1997
).
Ordinarily, phospholipid synthesis takes precedence over triacylglycerol synthesis in plants and animals when the demand for accumulation of fuel stores is low. This ensures the maintenance of membrane turnover, an essential physiological process. However, during periods when storage of fuel is essential, such as preparation for seasonal reductions in productivity, migrations or reproductive events, diacylglycerol is preferentially channeled towards triacylglycerol synthesis or is converted to diacylglycerol ether. In at least one case, phosphatidylcholine itself is used as a seasonal lipid reserve (Hagen et al., 1996). During such periods, diacylglycerol production may be enhanced by phosphatidylcholine hydrolysis via phospholipases or through the back reaction of CPT. The regulation of the enzymes involved in these processes is poorly understood, but probably involves hormonal changes associated with ontogenetic, seasonal and reproductive events.
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Acylglycerol and TMAO: correlation |
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Cod (gadiform teleosts) are sought commercially for their abundant liver oil and have high concentrations of TMAO in their muscle tissue (Gillett et al., 1997; Agustsson and Strøm, 1981
). Among Antarctic fishes, Dissostichus sp. has the highest concentrations of both body and liver lipids (Eastman, 1988
; Friedrich and Hagen, 1994
) and TMAO (Raymond and DeVries, 1998
). Elasmobranchs (sharks) generally contain high levels of both liver lipids (Baldridge, 1970
; Bone and Roberts, 1969
) and TMAO (Withers et al., 1994
). For example, Squalus sp. and Somniosus sp. have among the highest reported TMAO levels (Anthoni et al., 1991a
; Goldstein et al., 1967
) and are sought commercially for the high concentrations of diacylglycerol ether (DAGE) in their livers (Hallgren and Stallberg, 1974
; Kang et al., 1997
). Somniosus microcephalus has even been implicated in trimethylamine poisoning (Anthoni et al., 1991a
). Holocephalans, a generally deep-living subclass of Chondrichthyes, also contain large quantities of methylamines (both betaine and TMAO) and lipid (Hayashi and Takagi, 1980
; Hebard et al., 1982
; Bedford et al., 1998
).
Among invertebrates, the correlation between TMAO and acylglycerol levels is most notable in the cephalopods (Fig. 3), especially in the deep-sea squid families Onychoteuthidae (Moroteuthis robusta) (Fig. 4; node 7) and Gonatidae (Fig. 5) (Fig. 4, node 10). While most cephalopods studied to date have very low lipid concentrations (ODor and Webber, 1986) and little ability to metabolize lipids (Hochachka, 1994
), gonatid and onychoteuthid squids have acylglycerol contents as high as 25 % of their body mass (Hayashi and Kawasaki, 1985
; Hayashi et al., 1990
; Phillips et al., 2001
). These two families also contain the highest TMAO concentrations among cephalopods (Fig. 4) (Hebard et al., 1982
; Kelly and Yancey, 1999
). Some gonatid squids apparently contain high concentrations of glycine betaine as well (Shirai et al., 1997
).
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Given the ontogenetic descent to great depths undertaken by gonatid squids, an ontogenetic increase in TMAO concentration is consistent with the hypothesis that TMAO counteracts the effects of high hydrostatic pressure on proteins (Kelly and Yancey, 1999). However, values for other cephalopods (Hebard et al., 1982
) (Fig. 4) suggest that TMAO content is not related to depth (although data on glycine betaine and other methylamines would be helpful to analyze this hypothesis fully). For example, ommastrephids are predominantly shallow-living squid that contain large amounts of TMAO (100335 mmol l1; Fig. 4, node 13) (Hebard et al., 1982
). Ommastrephids accumulate lipid, up to 6 % of body mass, which is thought to fuel a reproductive horizontal migration (Takahashi, 1960
; Clarke et al., 1994
). Incidentally, this family apparently has a different smell from other squids and is known to cause allergic reactions in some people (Vecchione, 1994
). Both phenomena are perhaps related to trimethylamine.
Bathyteuthidae and Histioteuthidae, midwater squid families known to have large oily livers, were also found to accumulate TMAO (Fig. 4). Furthermore, no relationship between TMAO content and minimum or capture depth was found for octopods over a wide depth range (Fig. 4, node 8). Incirrate octopods (Fig. 4, node 11) have low TMAO levels at all depths. Cirrate octopods (Fig. 4, node 16) also have fairly low levels of TMAO, but slightly higher levels of betaine (>50 mmol kg1) (Yin and Yancey, 2000). The few shallow-living octopods measured have had very low lipid contents (ODor and Webber, 1986
; Pollero and Iribarne, 1988
). Lipid levels have not been measured in cirrate octopods. The general increase in TMAO concentration with depth observed in a variety of animals (Kelly and Yancey, 1999
) may be related to the tendency for deep-living species to accumulate lipids (e.g. Childress and Nygaard, 1974
).
Some midwater squid accumulate ammonium (approximately 300 mmol kg1) in their tissues for buoyancy. Although previous methods measuring ammonium did not distinguish between ammonium and methylamines (Sanders and Childress, 1988), our recent analysis using high-performance liquid chromatography confirmed the existence of high ammonium concentrations in most midwater squid species (B. A. Seibel, unpublished data). Such species appear to have special vacuolated tissue in which ammonium is sequestered, presumably out of contact with intracellular macromolecules (Voight et al., 1994
). If this sequestration is incomplete, however, TMAO could be required to counteract the toxic effects of ammonium on enzymes in ammoniacal squid. Preliminary measurements suggest that ammonium does depress the activity of octopine dehydrogenase from Histioteuthis heteropsis muscle tissue. However, TMAO did not effectively counteract this depression (B. A. Seibel, unpublished data). Furthermore, most species with high TMAO concentrations are negatively buoyant and do not accumulate ammonium. In the case of gonatids, the ontogenetic stages with high TMAO values also have lipid contents sufficient to provide neutral buoyancy and do not require ammonium or TMAO to provide lift.
It should be pointed out that cirrate and some pelagic incirrate octopods have extensive extracellular gelatinous tissue. We made every effort to remove the external gelatinous tissue, but some dilution of the TMAO content is expected in these species. Inclusion of the extracellular ammonium vacuoles in the analyzed tissue of some midwater squid certainly diluted the tissue homogenates as well. Assuming that all ammonium in such species is contained as a 500 mmol l1 solution in extracellular vacuoles (Voight et al., 1994) and that all TMAO measured is intracellular, we have corrected these values (Fig. 4) using measured ammonium and protein contents (B. A. Seibel, unpublished data). Bathyteuthis berryi does not possess ammonium as previously reported, but does contain high concentrations of an as yet unidentified nitrogenous cation, possibly TMA (B. A. Seibel, unpublished data) (Voight et al., 1994
). We believe that this cation is intracellular and so no correction to the TMAO value in Fig. 4 has been applied to this species. Galiteuthis phyllura accumulates ammonium in a specialized coelomic chamber well out of contact with muscle tissue. No correction was applied to this species either. TMAO was not found in the coelomic fluid of Galiteuthis phyllura.
We recently measured high TMAO concentrations in Clione antarctica (112 mmol kg1) (B. A. Seibel, unpublished data), a shallow-living Antarctic pteropod mollusc known to store high concentrations of DAGE within its body (Kattner et al., 1998; Phleger et al., 1997
). Clione antarctica feeds exclusively on the thecosomatous pteropod Limacina helicina, which accumulates large quantities of dimethylsulfoniopropionate (DMSP) in its body (Levasseur et al., 1994
) directly from phytoplankton in its diet. DMSP is the sulphidic analog of the nitrogenous glycine betaine and may be the precursor of the sulfonium analogue of phosphatidylcholine in some phytoplankton (Kates and Volcani, 1996
). Like trimethylamine oxide, DMSP is known to confer some protection against osmotic and temperature stress in phytoplankton (Nishiguchi and Somero, 1992
). Fishes that feed on Limacina helicina are often inflicted with blackberry feed, a foul-smelling and aesthetically displeasing condition resulting from the breakdown of DMSP to dimethylsulfide, subsequently accumulated in the fishs tissues (Levasseur et al., 1994
). Although the TMAO measured in Clione antarctica may be related to the large lipid stores, some connection to the DMSP concentrations in Limacina helicina cannot be ruled out. The synthetic pathways of DMSP and betaine are linked (Mulholland and Otte, 2000
). Trimethylamine oxide levels in Limacina helicina have not been measured.
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Time course of methylamine accumulation |
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The relatively low TMAO concentrations (48 mmol kg1) that we recently measured in a gonatid squid that was brooding an egg mass are also consistent with this model (B. A. Seibel, unpublished data). This squid was using, rather than storing, lipid and had lost the TMAO that it had presumably accumulated prior to spawning (Fig. 5). Dehydrated frog muscle initially accumulated methylamines in concert with rising urea concentrations (Wray and Wilkie, 1995). However, urea concentrations continued to rise with continued dehydration, while methylamine levels reached a plateau.
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Adaptive significance of methylamines |
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Fatty liver in sharks |
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Although this fatty liver scenario could, in theory, also apply to animal groups other than elasmobranchs, our inability to identify a cellular perturbant that consistently explains the distribution of TMAO in cephalopods and other marine animals causes us to reject this possibility. Among marine animals, methylamine accumulation as an adaptation for macromolecular stabilization has, in our opinion, been convincingly demonstrated only in sharks.
Fatty liver in mammals often results in cancerous tumor production (Goshal and Farber, 1984; Locker et al., 1986
). It may be no coincidence that shark livers (and squid digestive glands for that matter) also contain large quantities of squalamine, derived from squaline lipids and/or diacylglycerol ethers. Both squalamine and diacylglycerol ethers have recently been shown to inhibit tumor development by limiting vascular growth (Sills et al., 1998
).
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Concluding remarks |
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Acknowledgments |
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