Trimethylamine oxide in marine animals

The Journal of Experimental Biology 205, 297–306 (2002) Printed in Great Britain The Company of Biologists Limited 2002JEB3755 Trimethylamine oxide accumulation in marine animals: relationship to acylglycerol
NIEHS Marine and Freshwater Biomedical Sciences Center, Rosenstiel School of Marine and Atmospheric Science, *Present address: Monterey Bay Aquarium Research Institute, Moss Landing, CA 95039, USA (e-mail: [email protected]) Trimethylamine oxide (TMAO) is a common and
as diacylglycerol ethers and triacylglycerols. TMAO is
compatible osmolyte in muscle tissues of marine
synthesized from the trimethylammonium moiety of
organisms that is often credited with counteracting
choline, thus released, and is retained as a compatible
protein-destabilizing forces. However, the origin and
solute in concentrations reflecting the amount of lipid
synthetic pathways of TMAO are actively debated. Here,
stored in the body. A variation on this theme is proposed
we examine the distribution of TMAO in marine animals
for sharks.
and report a correlation between TMAO and acylglycerol
storage. We put forward the hypothesis that TMAO
is derived, at least in part, from the hydrolysis of

Key words: trimethylamine oxide, choline, phosphatidylcholine, phosphatidylcholine, endogenous or dietary, for storage
lipid, cephalopod, buoyancy, deep sea, urea, solute.
Methylamine compounds, particularly trimethylamine oxide a review, see Ballantyne, 1997), the counteracting solute (TMAO), are compatible osmolytes that commonly occur in hypothesis has received wide support (Wang and Bolen, 1997; tissues of marine organisms (Yancey et al., 1982). Their Yancey et al., 1982; Yancey and Somero, 1980; Yancey and concentrations vary extensively, however, among habitats and species and even with season and ontogeny within species (for TMAO may also counteract the effects of hydrostatic a review, see Hebard et al., 1982). There are numerous pressure on enzyme function in deep-sea animals. Yancey and hypotheses attempting to account for the distribution of colleagues (Gillett et al., 1997; Kelly and Yancey, 1999) have methylamines. High TMAO levels in polar fishes are thought demonstrated a correlation between TMAO concentration and to increase osmotic concentration, thus depressing the freezing capture depth in a variety of organisms. They have also shown point of the body fluids (Raymond and DeVries, 1998; that 250 mmol l–1 TMAO in vitro is able to counteract the loss Raymond, 1994). Sanders and Childress (1988) and Withers et of activity in some enzymes that results from high hydrostatic al. (1994) point out that trimethylamine (TMA) and TMAO, pressure (Yancey and Siebenaller, 1999; Yancey et al., 2001).
as a result of their large positive partial molal volumes, impart Other methylamines are known to counteract the effects of considerable lift to counteract sinking in some pelagic marine ammonia toxicity (Kloiber et al., 1988; Minana et al., 1996), animals. TMAO is best known, however, as a ‘counteracting salt (Dragolovich, 1994) and temperature stress (Krall et al., solute’ that protects proteins against various destabilizing 1989; Nishiguchi and Somero, 1992) on protein function in a variety of organisms. Although TMAO may serve the In elasmobranchs, for example, TMAO may counteract the functions attributed to it (i.e. it may be adaptive), it is not toxic effects of urea on proteins (Somero, 1986; Yancey and necessarily synthesized or accumulated as a specific adaptation Somero, 1980). Because they are iso-osmotic with sea water, to any of the stresses mentioned above. The source of TMAO sharks retain large quantities of urea in their fluids as an in marine animals and the time course of its accumulation osmolyte. Urea is highly perturbing to enzyme systems.
are not well characterized. Here, we present preliminary TMAO has a demonstrated ability to counteract the perturbing measurements of TMAO concentrations in a number of effects of urea on enzyme activity when accumulated in a 2:1 cephalopod species and review the distribution, biosynthesis ratio with urea (Somero, 1986). Although some enzymes seem and metabolism of methylamines in marine animals to assess to have an evolved tolerance of urea regardless of TMAO (for Methylamine synthesis
exclusively in the gut. However, the mechanism for conversion TMAO, like most methylamines, is derived from the of choline to TMA other than by microbial oxidation is not trimethylammonium group of choline. Dietary choline may be oxidized to trimethylamine by bacteria in the gut of marine Furthermore, fasted dogfish maintained stable TMAO animals. The accumulation of TMA in rotting fish as a result of concentrations over 41 days (Cohen et al., 1958). This result bacterial degradation of choline, as well as the reduction of may be partly due to the active reabsorption of TMAO in the TMAO to TMA, is responsible for their characteristic ‘fishy’ dogfish kidney (Cohen et al., 1958; Goldstein et al., 1967).
odor. TMA as a spoilage index for commercial fishes has been Some sharks retain TMAO quite effectively, but substantial extensively discussed (Hebard et al., 1982; Sotelo and Rehbein, loss does occur such that maintenance of TMAO levels over 2000). TMA is highly toxic (Marzo and Curti, 1997; Anthoni 41 days seems unlikely without an endogenous source.
et al., 1991a,b) and so is, with at least one notable exception Goldstein and Palatt (1974) found TMAO turnover rates of (Sanders and Childress, 1988), oxygenated within living 4–14 % per day in four different elasmobranch species, but animals to form TMAO (Fig. 1). This generally occurs within excretion rates of less than 1 % have been reported in some the digestive gland or liver via a monoxygenase enzyme, fishes (Agustsson and Strøm, 1981). Diets rich in TMA and trimethylamine oxidase (Tmase) (Hebard et al., 1982). TMAO choline have produced higher levels of TMAO in the muscles is then either transported to the tissues for accumulation as a of some fish species but not in others (Goldstein et al., 1967; compatible or ‘counteracting’ osmolyte or, more commonly, Agustsson and Strom, 1981). An endogenous source is excreted. In humans, a mutation of the flavin-containing mono- suspected in many species, especially those that accumulate oxygenase gene (FMO3) causes trimethylaminuria, a condition wherein individuals excrete TMA, rather than TMAO, along Endogenous choline supply is believed to limit the with a fishy body odor in urine, sweat, breath and other bodily accumulation of glycine betaine (betaine), another common excretions (Dolphin et al., 1997; Treacy et al., 1998).
methylamine osmolyte, in euryhaline oysters (Pierce et al., There are, however, conflicting reports regarding the 1997). Oyster mitochondria take up choline and convert it, via endogenous or exogenous (dietary) originof TMAO in animals. Early reports suggested that invertebrates lacked Tmaseactivity (Baker et al., 1963). However, phytoplankton production (Strøm, 1979).
Trimethylamine oxidase activity has beendetected in many, but not all, fishes (Baker et al., 1963). Among polar fishes, concentrations of TMAO than did speciesin which Tmase was detected (Raymondand DeVries, 1998). Goldstein et al.
TMAO in dogfish (Squalus acanthius) Tmase activity (Baker et al., 1963). They suggested that TMAO must beaccumulated from the diet. However, detected Tmase activity in dogfish as wellas in the silky shark Carcharhinus falciformis. Nurse sharks also possess Fig. 1. Diagram showing the possible pathways for trimethylamine (TMA) and Tmase activity and are able to synthesize trimethylamine oxide (TMAO) production (only abbreviated pathways are drawn).
TMAO directly from choline in vivo (A) Trimethylalkylammonium compounds (e.g. choline) are degraded to TMA by intestinal microbes (Hebard et al. 1992). (B) Choline is taken up by mitochondria in some animals (Pierce et al., 1997) and converted to glycine betaine via betaine aldehyde. Betaine may subsequently be converted to TMA (Ballantyne, 1997). In both cases (A and B), TMA is oxidized by trimethylamine oxygenase to TMAO.
Trimethylamine oxide in marine animals betaine aldehyde, to betaine (Fig. 1B). The enzymes involved ethanolamine (Hanson and Rhodes, 1983; Summers and in this transformation are well characterized (Dragolovich, Weretilnyk, 1993; Weretilnyk et al., 1995). Radioactively 1994). Betaine could, if similarly produced in other species, labeled ethanolamine in spinach is recovered primarily as subsequently be converted to TMA and TMAO (Fig. 1B).
betaine. In contrast, labeled ethanolamine in wheat and barley Some studies suggest that TMAO is a better counteracting is recovered primarily in phospholipid (Hitz et al., 1981; McDonnell and Wyn Jones, 1988) (Fig. 2).
Some plants produce methylamines, particularly betaine, Wheat does appear to accumulate betaine, but only through reportedly for osmoregulation and protection against drought phosphatidylcholine hydrolysis (Fig. 2B). In wheat and barley and salt-stress. A tremendous amount of research has been leaves, phosphatidylcholine is hydrolyzed to diacylglycerol and directed towards elucidating the pathways of betaine choline. A similar pathway is suspected for oysters and accumulation in plants in the hope of conferring drought- horseshoe crabs, which convert choline to betaine as well (Pierce resistance to important commercial crops such as tobacco et al., 1997). Diacylglycerol, produced via phosphatidylcholine (Nuccio et al., 1998). Plant research may, thus, provide insight hydrolysis in wheat plants, is subsequently converted to into the pathways and mechanisms of methylamine synthesis monogalactosyldiacylglycerol, an essential glycolipid in and storage in marine animals. Several pathways for the photosynthetic membranes. Free choline is converted to betaine production of choline in plants have been identified. Drought- (McDonnell and Wyn Jones, 1988). In drought-resistant resistant plants, such as chenopods (e.g. spinach and sugar chenopods, betaine accumulation does not depend on beet), are adapted for direct production of choline from phosphatidylcholine hydrolysis, a fact that may result in their Lipid storagePhotosynthetic membrane glycolipidSecond messengerFatty acid oxidation Fig. 2. (A) Diagram of the glycerolphosphate pathway and phosphatidylcholine synthesis as found in both animals and plants. The bold arrowsindicate hypothesized pathways resulting in simultaneous accumulation of acylglycerols and trimethylamine oxide (TMAO) (only abbreviatedpathways are drawn). Diacylglycerol (DAG), formed from the derivative glycerol-3-phosphate (DAG), can be shunted towards eitheracylglycerol (lipid) storage (e.g. triacylglycerol) or phosphatidylcholine (PtdCho) synthesis. The final step in the phosphatidylcholine pathwayis reversible. The back reaction may occur to a significant extent so that diacylglycerol is formed from phosphatidylcholine and subsequentlystored for seasonal or reproductive energy reserves (see Gur and Harwood, 1991). Choline, thus released, is oxidized to TMAO and eitherstored or excreted. (B) In some plants, PtdCho is produced by methylation of ethanolamine and may subsequently be hydrolysed for release ofDAG and choline. DAG in plants is important during growth for the formation of photosynthetic membrane glycolipids. Choline, thus released,is oxidized to glycine betaine (Hitz et al. 1981). (C) In chenopods (e.g. spinach and sugarbeet), choline is synthesized directly fromethanolamine and, thus, appears to be a specific adaptation for glycine betaine accumulation during drought and salt-stress (Weretilnyke et al.,1995). The enzymes involved in the pathways illustrated are numbered: (1) glycerol-3-phosphate (G3P) is converted to phosphatidic acid bythe successive actions of G3P acyltransferase and 1-acylglycerol-3-phosphate acyltransferase; (2) phosphatidic acid phosphatase; (3) 1,2-diacylglycerol:choline phosphotransferase; (4) diacylglycerol acyltransferase; (5,6) carnitine palmitoyl transferases I and II; (7) trimethylamineoxygenase; (8) choline monoxygenase; (9) betaine aldehyde dehydrogenase; (10) P-choline phosphatase. TMA, trimethylamine; P-choline,phosphocholine.
greater relative capacity for betaine accumulation and their via phosphatidylcholine hydrolysis has no known function greater salt tolerance (McDonnell and Wyn Jones, 1988).
other than the synthesis of acetylcholine in neural tissue (Billah Phosphatidylcholine hydrolysis is also responsible for and Anthes, 1990). We propose that phosphatidylcholine diacylglycerol production in plants that store large quantities hydrolysis via the back reaction of CPT may provide an of triacylglycerols in their seeds (Gur and Harwood, 1991).
endogenous source of choline for TMAO synthesis in animals.
Triacylglycerol sometimes constitutes as much as 80 % of the Hydrolysis of dietary phosphatidylcholine by phospholipases dry mass of the seeds. Triacylglycerols are also among the C and D may also be an important source of free choline for most common form of energy reserve in animals, but TMAO synthesis (Wakelam et al., 1993). Hydrolysis of labeled diacylglycerol ethers are also frequently stored. Many animals phosphatidylcholine by phospholipase C in the gut of larvae of store acylglycerols as metabolic fuel reserves that can, during the dragonfly Aeshna cyanea resulted in recovery of labeled starvation, migration, reproduction or egg development, be products in various forms, including acylglycerols and glycine mobilized and oxidized to drive metabolic processes.
Phosphatidylcholine hydrolysis, which is important during Ordinarily, phospholipid synthesis takes precedence over accumulation of acylglycerols, may serve as an endogenous triacylglycerol synthesis in plants and animals when the source of choline, resulting in TMAO accumulation in animals.
demand for accumulation of fuel stores is low. This ensures The enzymes that are required for phosphatidylcholine the maintenance of membrane turnover, an essential hydrolysis are apparently well conserved, having been found physiological process. However, during periods when storage in mammals, molluscs and arthropods as well as the plants of fuel is essential, such as preparation for seasonal reductions mentioned above (Anfuso et al., 1995).
Diacylglycerol and phosphatidylcholine
The predominant pathway for the biosynthesis of triacylglycerol and diacylglycerol ether is the glycerol phosphate pathway. Glycerol phosphate, a derivative ofglycolysis, is converted to diacylglycerol via phosphatidic acid (Fig. 2). Diacylglycerol represents a branchpoint wherediacylglycerol can be either channeled into phospholipid synthesis (i.e. phosphatidylcholine) or acylated to form triacylglycerols. The final step towards the synthesis ofphosphatidylcholine is catalyzed by 1,2-diacylglycerol:choline phosphotransferase (CPT). CPT activity governs thepartitioning of diacylglycerol into either phosphatidylcholineor acyglycerol pools (Jackowski et al., 2000). The backreaction of CPT can occur to a significant extent so that diacylglycerol is formed from phosphatidylcholine, releasingfree choline (for a review, see Gur and Harwood, 1991).
phosphatidylcholine hydrolysis is an important second Fig. 3. Trimethylamine oxide content (y, mmol kg–1; see Fig. 4) in messenger (Billah and Anthes, 1990; Wakelam et al., 1993).
mantle muscle tissue is significantly correlated with digestive gland In fact, during hypo-osmotic cell volume regulation, lipid content (x) in cephalopods (y=2.90x1.18, r=0.59, P<0.05). We swelling results in membrane turnover and were unable to analyze trimethylamine oxide (TMAO) content as a phosphatidylcholine hydrolysis, resulting in the formation of function of total lipid content because of the variable presentation of diacylglycerol. Diacylglycerol activates phosphokinase C lipid data in the literature. Lipid data were taken from Blanchier and which, in turn, stimulates the release of osmolytes in a range Boucaud-Camou (1984), Hayashi (1989, 1996), Hayashi and of organisms from sharks (Musch and Goldstein, 1990) to Kawasaki (1985), Kristensen (1984), Phillips et al. (2001), algae (Thompson, 1994). Cell volume regulation via Piatkowski and Hagen (1994), Pollero and Iribarne (1988) and phosphatidylcholine hydrolysis under hyperosmotic stress (i.e.
Semmens (1998). In many cases, lipid and TMAO data were taken dehydration) could contribute to the release of free choline and from different species, and possibly different maturity stages, of thesame genus. (1) Octopus; (2) Loligo; (3) Galiteuthis; (4) Sepia; (5) subsequent betaine or TMAO accumulation in some animals Illex; (6) Berryteuthis; (7) Moroteuthis; (8) Gonatopsis; (9) (although some mechanism would be required to prevent Todarodes; (10) Gonatus. There are conflicting reports regarding the diacylglycerol from stimulating the release of osmolytes in this digestive gland lipid content of Thysanoteuthis, a squid that also case). For example, TMAO was shown to accumulate during contains high TMAO concentrations (Hayashi, 1996; Yuneva et al., dehydration in frog gastronemius muscle (Wray and Wilkie, 1994). Many of the genera plotted here are closely related to each 1995), although the authors postulate that TMAO accumulated other (see Fig. 4). Therefore, phylogenetic independence of the data to counteract increased urea concentrations. Choline produced Trimethylamine oxide in marine animals in productivity, migrations or reproductive events, Cod (gadiform teleosts) are sought commercially for their diacylglycerol is preferentially channeled towards abundant liver oil and have high concentrations of TMAO in triacylglycerol synthesis or is converted to diacylglycerol their muscle tissue (Gillett et al., 1997; Agustsson and Strøm, ether. In at least one case, phosphatidylcholine itself is used as 1981). Among Antarctic fishes, Dissostichus sp. has the a seasonal lipid reserve (Hagen et al., 1996). During such highest concentrations of both body and liver lipids (Eastman, periods, diacylglycerol production may be enhanced by 1988; Friedrich and Hagen, 1994) and TMAO (Raymond and phosphatidylcholine hydrolysis via phospholipases or through DeVries, 1998). Elasmobranchs (sharks) generally contain the back reaction of CPT. The regulation of the enzymes high levels of both liver lipids (Baldridge, 1970; Bone and involved in these processes is poorly understood, but probably Roberts, 1969) and TMAO (Withers et al., 1994). For example, involves hormonal changes associated with ontogenetic, 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 ofdiacylglycerol ether (DAGE) in their livers (Hallgren and Acylglycerol and TMAO: correlation
Stallberg, 1974; Kang et al., 1997). Somniosus microcephalus A general correlation exists between the concentration of has even been implicated in trimethylamine poisoning TMAO (and betaine) in muscle tissue and lipid, particularly (Anthoni et al., 1991a). Holocephalans, a generally deep-living diacylglycerol ethers and triacylglycerols, levels in the bodies of subclass of Chondrichthyes, also contain large quantities of marine animals. TMAO and lipid concentration both appear to methylamines (both betaine and TMAO) and lipid (Hayashi be correlated with habitat depth, latitude, season, lifestyle (e.g.
and Takagi, 1980; Hebard et al., 1982; Bedford et al., 1998).
benthic versus pelagic) and ontogeny or size (for reviews, see Among invertebrates, the correlation between TMAO and Hebard et al., 1982; Sargent, 1976, 1989). Both TMAO and lipid acylglycerol levels is most notable in the cephalopods (Fig. 3), also appear conspicuously within the same phylogenetic groups.
especially in the deep-sea squid families Onychoteuthidae Fig. 4. Mantle muscle trimethylamine oxide (TMAO, mmol kg–1) measured according to Wekell and Barnett (1991), minimum depth ofoccurrence (MDO, m) and capture depth (CD, m) of cephalopods listed according to their phylogenetic associations (see Carlini and Graves,1999). Values are corrected for the dilution of tissue with extracellular ammonium concentrations in some species (see text). Nodes arenumbered for reference in the text. Numbers in parentheses are from Hebard et al. (1982) or Kelly and Yancey (1999).
gonatid squids accumulates throughout their life and is thought y = 75x0.33, r = 0.92 to fuel an extended egg-brooding period (Seibel et al., 2000).
y = 2.5x0.25, r = 0.89 The high TMAO values reported for gonatid squids were found y = 0.27x0.47, r = 0.74 in adult specimens. Preliminary measurements suggest that theconcentration of TMAO is much lower in smaller individuals of the species (Fig. 5) and, as for lipids, that TMAO 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 othercephalopods (Hebard et al., 1982) (Fig. 4) suggest that TMAO content is not related to depth (although data on glycine betaineand other methylamines would be helpful to analyze this hypothesis fully). For example, ommastrephids are predominantly shallow-living squid that contain large amounts of TMAO (100–335 mmol l–1; Fig. 4, node 13) (Hebard et al., 1982). Ommastrephids accumulate lipid, up to 6 % of body Fig. 5. Mantle muscle trimethylamine oxide (TMAO) content mass, which is thought to fuel a reproductive horizontal (mmol kg–1; squares) increases (b=0.33) in proportion to digestive migration (Takahashi, 1960; Clarke et al., 1994). Incidentally, gland mass (% body mass; circles; b=0.25) and lipid mass (% body this family apparently has a ‘different’ smell from other squids mass; plus signs; b=0.47) through ontogeny (size; g) in gonatid squid and is known to cause allergic reactions in some people (Cephalopoda). The data for TMAO content are from Kelly and (Vecchione, 1994). Both phenomena are perhaps related to Yancey (1999) and B. A. Seibel (unpublished results). Body masses for the TMAO scaling analysis (squares) for Gonatopsis borealis and Bathyteuthidae and Histioteuthidae, midwater squid families Berryteuthis magister were estimated at the mean adult body mass known to have large oily livers, were also found to accumulate for each species (Hayashi, 1989; Hayashi and Yamamoto, 1987).
TMAO (Fig. 4). Furthermore, no relationship between TMAO Digestive gland and lipid masses were taken from various sources: content and minimum or capture depth was found for octopods Gonatus onyx (B. A. Seibel, personal observation), Gonatus fabricii(Arkhipkin and Bjorke, 1999), Gonatopsis borealis (Hayashi, 1989) over a wide depth range (Fig. 4, node 8). Incirrate octopods and Berryteuthis magister (Hayashi and Yamamoto, 1987). All (Fig. 4, node 11) have low TMAO levels at all depths. Cirrate species appear to fall on the same scaling line despite differences in octopods (Fig. 4, node 16) also have fairly low levels of maximum body sizes. The apparent correlation between lipid content TMAO, but slightly higher levels of betaine (>50 mmol kg–1) (or digestive gland mass) and TMAO content in gonatid squid is (Yin and Yancey, 2000). The few shallow-living octopods hypothesized to result from the requirement for phosphatidyl choline measured have had very low lipid contents (O’Dor and hydrolysis to produce diacylglycerol for lipid storage. The choline Webber, 1986; Pollero and Iribarne, 1988). Lipid levels have not been measured in cirrate octopods. The general increase inTMAO concentration with depth observed in a variety of (Moroteuthis robusta) (Fig. 4; node 7) and Gonatidae (Fig. 5) animals (Kelly and Yancey, 1999) may be related to the (Fig. 4, node 10). While most cephalopods studied to date have tendency for deep-living species to accumulate lipids (e.g.
very low lipid concentrations (O’Dor and Webber, 1986) and little ability to metabolize lipids (Hochachka, 1994), gonatid and onychoteuthid squids have acylglycerol contents as high (approximately 300 mmol kg–1) in their tissues for buoyancy.
as 25 % of their body mass (Hayashi and Kawasaki, 1985; Although previous methods measuring ammonium did not Hayashi et al., 1990; Phillips et al., 2001). These two families distinguish between ammonium and methylamines (Sanders also contain the highest TMAO concentrations among and Childress, 1988), our recent analysis using high- cephalopods (Fig. 4) (Hebard et al., 1982; Kelly and Yancey, performance liquid chromatography confirmed the existence of 1999). Some gonatid squids apparently contain high high ammonium concentrations in most midwater squid concentrations of glycine betaine as well (Shirai et al., 1997).
species (B. A. Seibel, unpublished data). Such species appear Both gonatid and onychoteuthid squid are known to to have special vacuolated tissue in which ammonium is undertake ontogenetic vertical migrations whereby successive sequestered, presumably out of contact with intracellular developmental stages occupy progressively greater depths.
macromolecules (Voight et al., 1994). If this sequestration is This migration is believed to end at depths greater than 1500 m, incomplete, however, TMAO could be required to counteract at which spawning and, at least in some cases, egg-brooding the toxic effects of ammonium on enzymes in ammoniacal take place (Jackson and Mladenov, 1994; Arkhipkin and squid. Preliminary measurements suggest that ammonium does Bjorke, 1999; Seibel et al., 2000). The high lipid content in depress the activity of octopine dehydrogenase from Trimethylamine oxide in marine animals Histioteuthis heteropsis muscle tissue. However, TMAO did Time course of methylamine accumulation
not effectively counteract this depression (B. A. Seibel, Although some groups, such as elasmobranchs, have unpublished data). Furthermore, most species with high specific adaptations for retention of TMAO to counteract urea TMAO concentrations are negatively buoyant and do not toxicity, other groups may simply allow TMAO to accumulate accumulate ammonium. In the case of gonatids, the while it is available and thus reap the benefits of its ontogenetic stages with high TMAO values also have lipid compatibility and protein-stabilizing attributes. The time contents sufficient to provide neutral buoyancy and do not course of glycine betaine accumulation in wheat plants during, require ammonium or TMAO to provide lift.
and subsequent to, periods of leaf expansion is consistent with It should be pointed out that cirrate and some pelagic this explanation (Hitz et al., 1981; McDonnell and Wyn Jones, incirrate octopods have extensive extracellular gelatinous 1988). The most rapid betaine accumulation in the leaves of tissue. We made every effort to remove the external gelatinous unstressed wheat plants, and initially in those of salt-stressed tissue, but some dilution of the TMAO content is expected in wheat plants, is at the time of greatest leaf expansion and, these species. Inclusion of the extracellular ammonium consequently, membrane development and membrane lipid vacuoles in the analyzed tissue of some midwater squid (e.g. phosphatidylcholine) turnover. When growth (glycolypid certainly diluted the tissue homogenates as well. Assuming that biosynthesis) plateaus, betaine concentrations slowly decline all ammonium in such species is contained as a 500 mmol l–1 solution in extracellular vacuoles (Voight et al., 1994) and that The relatively low TMAO concentrations (48 mmol kg–1) all TMAO measured is intracellular, we have corrected these that we recently measured in a gonatid squid that was values (Fig. 4) using measured ammonium and protein brooding an egg mass are also consistent with this model (B.
contents (B. A. Seibel, unpublished data). Bathyteuthis berryi A. Seibel, unpublished data). This squid was using, rather does not possess ammonium as previously reported, but does than storing, lipid and had lost the TMAO that it had contain high concentrations of an as yet unidentified presumably accumulated prior to spawning (Fig. 5).
nitrogenous cation, possibly TMA (B. A. Seibel, unpublished Dehydrated frog muscle initially accumulated methylamines data) (Voight et al., 1994). We believe that this cation is in concert with rising urea concentrations (Wray and Wilkie, intracellular and so no correction to the TMAO value in Fig. 4 1995). However, urea concentrations continued to rise with has been applied to this species. Galiteuthis phyllura continued dehydration, while methylamine levels reached a accumulates ammonium in a specialized coelomic chamber well out of contact with muscle tissue. No correction wasapplied to this species either. TMAO was not found in thecoelomic fluid of Galiteuthis phyllura.
Adaptive significance of methylamines
We recently measured high TMAO concentrations in The role of phosphatidylcholine hydrolysis in the Clione antarctica (112 mmol kg–1) (B. A. Seibel, unpublished production, as well as the time course of accumulation, of data), a shallow-living Antarctic pteropod mollusc known to betaine led Hitz et al. (1981) to question the adaptive store high concentrations of DAGE within its body (Kattner significance of betaine accumulation in wheat and barley.
et al., 1998; Phleger et al., 1997). Clione antarctica Betaine accumulation may only be a side-effect of accelerated feeds exclusively on the thecosomatous pteropod Limacina turnover of phospholipid head groups during stress and not a helicina, which accumulates large quantities of specific adaptation to stress from which some benefit accrues dimethylsulfoniopropionate (DMSP) in its body (Levasseur et to the stressed leaf (Hitz et al., 1981). In oysters, betaine al., 1994) directly from phytoplankton in its diet. DMSP is the synthesis and accumulation may be a consequence of cell sulphidic analog of the nitrogenous glycine betaine and membrane restructuring during cell volume changes resulting may be the precursor of the sulfonium analogue of in phosphatidylcholine hydrolysis (cf. Dragolovich, 1994; phosphatidylcholine in some phytoplankton (Kates and Musch and Goldstein, 1990). Similarly the temporary Volcani, 1996). Like trimethylamine oxide, DMSP is known accumulation of trimethylamines in frog muscle during to confer some protection against osmotic and temperature dehydration may also reflect membrane turnover during stress in phytoplankton (Nishiguchi and Somero, 1992).
hyperosmotic cell volume regulation (see Wray and Wilkie, Fishes that feed on Limacina helicina are often inflicted 1995) and not necessarily a response to increased urea with ‘blackberry feed’, a foul-smelling and aesthetically concentrations. The adaptive significance of DMSP displeasing condition resulting from the breakdown of DMSP accumulation in algal cells has also been called into question to dimethylsulfide, subsequently accumulated in the fish’s by Stefels (2000), who noted that changes in the concentration tissues (Levasseur et al., 1994). Although the TMAO of DMSP in algal cells upon salt-stress are the result of measured in Clione antarctica may be related to the large lipid metabolic changes rather than active regulatory mechanisms.
stores, some connection to the DMSP concentrations in He suggested that DMSP may be considered as a compatible Limacina helicina cannot be ruled out. The synthetic pathways solute, but that it is not osmoticum in the strict sense of being of DMSP and betaine are linked (Mulholland and Otte, 2000).
responsible for osmotic balance. Perhaps TMAO accumulation Trimethylamine oxide levels in Limacina helicina have not in marine animals reflects, in part, the requirements for Fatty liver in sharks
organisms. TMAO may simply be accumulated as a In mammals, dietary choline deficiency prevents compatible solute in quantities reflecting the amount of lipid phosphatidylcholine synthesis and may leave excess stored in the body. Conversely, among sharks, the requirement diacylglycerol, produced in the glycerol phosphate pathway, to for TMAO accumulation may deplete available choline levels, be channeled towards triacylglycerol for storage in the liver thus limiting the production of phosphatidylcholine and (Fig. 2). This condition, known as ‘fatty liver’, may be a shunting excess diacylglycerol, produced in the glycerol chronic condition in sharks. The requirements for TMAO phosphate pathway, towards storage in the liver. Sharks alone accumulation to counteract urea toxicity may limit the among marine animals may possess specific adaptations for availability of choline for phosphatidylcholine synthesis. As a retention, and possibly for production via centralization of consequence, lipid may accumulate in the liver. The loss of fatty-acid oxidation, of TMAO. We do not rule out the extra-hepatic fatty acid oxidation may further contribute to possibility that TMAO is strongly selected for its protein- fatty liver in sharks. Freshwater stingrays alone among stabilizing attributes in some other animal groups, possibly elasmobranchs possess the ability to oxidize fatty acids extra- resulting in lipid accumulation as a metabolic byproduct. It is hepatically. Because they have no need to accumulate urea as also possible that that diacylglycerol and TMAO levels are an osmolyte, freshwater stingrays also do not accumulate linked, as we have proposed, but that the maintenance of high TMAO in their tissues. This led Ballantyne and Moon (1986) TMAO levels reflects retention adaptations in response to some to postulate a relationship between extra-hepatic β-oxidation cellular perturbant. However, no obvious protein-destabilizing and the absence of urea and TMAO accumulation. One agent has been identified in the cephalopods examined here possible cause is competition for carnitine which, like choline, that would warrant the observed accumulation of TMAO.
may be oxidized to TMAO (Marzo and Curti, 1997).
Because of the paucity of data on methylamines other than β-oxidation may allow all available carnitine to be TMAO, we hesitate to rule out the possibility that hydrostatic converted to TMAO. By limiting β-oxidation in non-hepatic pressure selects for high methylamine concentrations in deep- tissue, sharks may also decrease the competition for sea organisms. We also cannot rule out the possibility that diacylglycerol for lipid accumulation in the liver. Limited pathways for the accumulation of lipid and TMAO are not extra-hepatic fatty acid oxidation capacity in squid (Ballantyne coupled in some cases. However, we feel that the link between et al., 1981) may similarly preadapt them for seasonal and phosphatidylcholine hydrolysis and trimethylamine oxide ontogenetic acylglycerol accumulation. However, the only accumulation should be considered a competing hypothesis genus for which the capacity for fatty acid oxidation has been measured (Loligo) does not appear to accumulate lipid orTMAO. The lipid-rich livers of sharks are generally attributed We thank the Monterey Bay Aquarium for allowing us to a buoyancy role (Malins and Barone, 1970; Phleger, 1998; participate in collection cruises. We thank G. N. Somero and Wetherbee and Nichols, 2000). Although the low-density oils, two anonymous reviewers for comments and constructive especially DAGE and squalene, do provide lift, they may criticism. This research was funded in part by the Rosenstiel simply be a beneficial end-product of TMAO production in School of Marine and Atmospheric Science Postdoctoral Fellowship and National Institute of Environmental Health Although this ‘fatty liver’ scenario could, in theory, also Sciences Marine and Freshwater Biomedical Science Center apply to animal groups other than elasmobranchs, our inability to identify a cellular perturbant that consistently explains thedistribution of TMAO in cephalopods and other marineanimals causes us to reject this possibility. Among marine References
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