Sage Journals: Discover world-class research

Abstract

The free sterol fraction from the deep-sea holothurian Orphnurgus cf glaber Walsh, 1891, has been isolated and studied by GC–MS. Sterols containing Δ⁷, Δ⁰, Δ⁵, polyunsaturated and 5,8-epidioxy modified cores were found. A high percentage of compounds with polyunsaturated cores distinguish this composition from those of previously studied holothurians. Almost all identified compounds had saturated side chains, which is also a specific feature of this fraction. Peculiarities of biosynthesis and metabolism of the found sterols are discussed.

Keywords

Hawaiian-Emperor chain deep-sea holothurian sterol composition

Introduction

Seamounts, including the Hawaiian-Emperor Chain, are areas with high biological productivity of benthic and pelagic communities, including important biological resources that support a commercial fishery. The Emperor Chain, located in the North-Western Pacific area, could be affected by uncontrolled commercial fishing which seriously threatens the natural balance in the area. The North Pacific Fishery Commission (NPFC https://www.npfc.int) recognized at the meeting held in Yokohama (in 2018) the lack of information on the distribution and population density of bottom organisms in the region and the urgent need to study the bottom communities, classified as vulnerable marine ecosystems due to their slow recovery after overfishing.^1,2 Data on holothurian diversity, as well as natural compounds from any echinoderms from the Chain, are absent in the literature (Table 1).

Table 1.

Free Sterols From Orphnurgus cf glaber Analyzed as Acetate Derivatives.

N	RT*	Sterol	Short designation	%**	Mass spectra of corresponding acetate, m/z (%)
1	25.8	Unidentified C₂₇	C₂₇3F	1.2	424 (50), 364 (60), 349 (100), 311 (40), 257 (30), 251 (80), 235 (30), 223 (20), 209 (60), 197 (60), 195 (40)
2	25.8	Cholest-5-en-3β-ol	1a	6.5	368 (100), 353 (25), 283 (2), 260 (3), 255 (10), 247 (15), 213 (12)
3	25.9	5α-Cholesta-7,14-dien-3β-ol	4a	2.3	426 (70), 411 (15), 366 (6), 351 (6), 325 (6), 313 (100), 312 (2), 299 (40), 255 (2), 253 (25), 239 (6), 225 (3), 211 (3)
4	26.1	5α-Cholestan-3β-ol	2a	11.2	430 (20), 415 (5), 370 (40), 355 (30), 276 (27), 257 (6), 230 (20), 215 (100)
5	26.4	5α-Cholesta-7,9(11)-dien-3β-ol	5a	2.1	426 (90), 411 (25), 366 (15), 351 (40), 313 (30), 312 (20), 311 (100), 299 (2), 286 (10), 259 (20), 253 (40), 251 (20), 239 (3), 227 (10), 213 (10), 211 (50)
6	27.0	5α-Cholest-7-en-3β-ol	3a	12.4	428 (100), 413 (30), 368 (8), 353 (15), 315 (10), 288 (5), 273 (10), 255 (100), 229 (20), 213 (40)
7	27.6	5,8-Epidioxycholestan-3β-ol	6a	0.9	460 (25), 445 (55), 425 (10), 409 (3), 385 (20), 356 (30), 348 (50), 347 (100), 311 (10), 287 (60), 273 (10), 251 (15), 237 (10), 221 (10)
8	27.8	24-Methyl-5α-cholesta-7,22-dien-3β-ol	3b	1.3	440 (25), 425 (10), 380 (3), 365 (6), 342 (15), 313 (100), 288 (15), 255 (60), 229 (25), 213 (25)
9	28.1	24-Methylcholest-5-en-3β-ol	1c	1.1	382 (100), 367 (18), 274 (15), 255 (15), 213 (15)
10	28.3	24-Methyl-5α-cholestan-3β-ol	2c	0.4	444 (15), 429 (2), 384 (35), 369 (25), 276 (25), 257 (10), 230 (15), 215 (100)
11	29.5	24-Methyl-5α-cholest-7-en-3β-ol	3c	2.4	442 (100), 427 (20), 382 (4), 367 (12), 315 (12), 273 (5), 255 (100), 229 (20), 213 (40)
12	30.0	23,24-Dimethylcholest-5-en-3β-ol	1d	trace	396 (100), 381 (15), 275 (7), 255 (15), 213 (15)
13	30.1	Unidentified C₂₉	C₂₉3F	2.0	452 (50), 392 (60), 377 (80), 311 (50), 257 (35), 251 (100), 235 (20), 223 (25), 209 (70), 197 (90), 195 (55)
14	30.3	24-Ethylcholest-5-en-3β-ol	1e	6.8	396 (100), 381 (20), 288 (10), 275 (10), 255 (15), 213 (15)
15	30.3	23,24-Dimethyl-5α-cholestan-3β-ol	2d	trace	458 (25), 398 (30), 383 (20), 276 (25), 275 (20), 257 (10), 230 (20), 215 (100)
16	30.4	24-Ethyl-5α-cholesta-7,14-diene-3β-ol	4e	4.2	454 (60), 439 (10), 392 (5), 341 (1), 327 (1), 313 (100), 299 (40), 253 (20), 239 (5), 213 (5), 211 (2)
17	30.5	24-Ethyl-5α-cholestan-3β-ol	2e	1.0	458 (25), 443 (5), 398 (40), 383 (25), 276 (25), 275 (20), 257 (10), 230 (20), 215 (100)
18	31.2	24-Ethyl-5α-cholest-7,9(11)-dien-3β-ol	5e	3.7	454 (85), 439 (20), 394 (15), 379 (30), 311 (100), 286 (10), 271 (10), 259 (20), 253 (25), 251 (20). 239 (5), 225 (10), 211 (45)
19	32.1	24-Ethyl-5α-cholest-7-en-3β-ol	3e	35.5	456 (90), 441 (20), 396 (5), 381 (10), 315 (10), 288 (5), 273 (10), 255 (100), 229 (20), 213 (35)
20	32.3	5,8-Epidioxy-24-ethylcholestan-3β-ol	6e	4.2	488 (25), 473 (50), 453 (10), 437, 413 (20), 377 (20), 347 (100), 333 (5), 311 (5), 287 (60), 273 (10), 251 (10), 237 (10), 221 (10)
21	34.3	24-Propyl-5α-cholest-7-en-3β-ol	3f	trace	470 (90), 455 (25), 410 (8), 395 (10), 315 (10), 288 (5), 273 (12), 255 (100), 229 (20), 213 (35)

* The retention times reported for the corresponding sterol acetates under the conditions specified in 2.1. Analytical methods.

** Abundances are given as % of total sterol fraction.

Sea cucumbers, marine invertebrates belonging to the class Holothurioidea (the phylum Echinodermata), are one of the numerous and very ancient forms of marine life playing important biological and biogeochemical roles in the transformation of marine detritus. In deep-sea ecosystems, their role is especially great. However, deep-sea holothurians are poorly studied chemically. Sterols are key biochemical components, being functionally involved in the construction of membranes, as well as in hormonal regulation. There are only a few studies (see Table S1, Supplemental Materials) on sterols from holothurians that are not available for collection by diving. Ponomarenko et al³ and Stonik et al⁴ investigated epipelagic holothurians (from 50 to 200 m); Stonik et al,⁴ Dmitrenok et al,⁵ and Ballantine et al⁶ investigated mesopelagic holothurians (from 200 to 1000 m); Ballantine et al⁶ investigated bathyal holothurians (depths of 1000 to 4000 m); Amaro et al,⁷ Drazen et al,⁸ Neto et al,⁹ and Ginger et al^10,11 investigated abyssal holothurians (depth of 4000 to 6000 m). Holothurians of the Porcupine Abyssal Plain were investigated most thoroughly.^9–11 Studies in other locations are sporadic and deep-sea species of holothurians inhabiting under-water mountains in the western part of the Pacific Ocean remained unstudied.

The sea cucumber Orphnurgus cf glaber Walsh, 1891, is a rare and scattered deep-sea holothurian. In the present research, free sterols from this holothurian have been investigated and compounds 1 (a,c–e), 2 (a,c–e), 3 (a–c,e–f), 4 (a,e), 5 (a,e), and 6 (a,e) were found (Figure 1).

Figure 1.

Structures of the sterols from Orphnurgus cf glaber.

Results and Discussion

Only a single specimen of the deep-water holothurian Orphnurgus cf glaber Walsh, 1891, was collected from a depth of 2231 m. The sterol fraction was isolated from an ethanol extract by chromatography on silica gel and was separated into three subfractions. The ¹H NMR spectrum of subfraction 1 showed signals of angular methyl groups, СH₃-18 at 0.68 and CH₃-19 at 1.01 ppm, which are characteristic of 5(6)-unsaturated sterols.¹² Stanols (СH₃-18 at δ_H 0.65 and CH₃-19 at δ_H 0.80) and Δ⁷-sterols (СH₃-18 at δ_H 0.54 and CH₃-19 at δ_H 0.81) dominated in the subfractions 2 and 3, respectively. The subfractions were acetylated and analyzed by GC-MS. The structures of individual components were established based on the analysis of the mass spectra of their acetate derivatives, the elution order under GC conditions and comparison of these with the literature data.^3,4,13,14 The identification of cholest-5-en-3β-ol (1a), cholest-7-en-3β-ol (3a) and 24-methylcholesta-7,22-dien-3β-ol (3b) were confirmed by direct comparison with authentic standards. Establishing the structures of rare and unusual components is discussed below.

5(6)-Unsaturated C₂₉ sterol 1d (retention time, RT 30.0 min) was found in subfraction 1 (Δ⁵-sterols) along with 24-ethylcholest-5-en-3β-ol (1e) (RT 30.3 min). They had identical mass spectra and distinct retention times, showing the same sterol cores but different saturated side chains. It is known that the retention times of sterols bearing a 23,24-dimethyl side chain are shorter than those of 24-ethyl analogs.4 Such compounds often occur in pairs in suspended particles and sedimentary samples.^15,16 These considerations made it possible to identify the aforementioned compound as 23,24-dimethylcholest-5-en-3β-ol (1d). Such reasoning led to the identification of 23,24-dimethyl-5α-cholestan-3β-ol (2d) (RT 30.3 min), also from subfraction 2.

A number of polyunsaturated sterols were present along with common Δ⁷-sterols in subfraction 3. Thus, compound 4a (RT 25.9 min) is a C₂₇ sterol with two double bonds according to the mass spectrum of its acetate: m/z 426 M⁺, 411 (M – CH₃)⁺, 366 (M – AcOH)⁺, and 351 (M – AcOH – CH₃)⁺. The significant signals in its mass spectrum at m/z 313 (loss of side chain), 253 (loss of side chain and AcOH), as well as 299 and 211 (cleavage of the D-cycle and loss of AcOH) indicate that both unsaturations are located in the sterol nucleus. Comparison of the intensities of the fragment ions with those of a series of di-unsaturated sterols¹³ showed that this compound is 5α-cholesta-7,14-dien-3β-ol (4a).

Compound 4e (RT 30.4 min) produced a mass-spectrum with peaks at m/z 454 M⁺, 439 (M–CH₃)⁺, 394 (M – AcOH)⁺, and 379 (M – AcOH – CH₃)⁺ pointing to a di-unsaturated C₂₉ sterol. The character of the fragmentation was similar to that of compound 4a, which suggests the same steroid nucleus, but additional alkylation in the side chain. Thus, the ratio of the abovementioned peaks and peaks at m/z 313, 299, 253, and 211 was the same as that of 4a. Presumably, this compound is 24-ethylcholesta-7,14-diene-3β-ol (4e).

Compound 5a (RT 26.4 min) is a C₂₇ sterol with two double bonds (also m/z 426 M⁺, 411 (M – CH₃)⁺, 366 (M – AcOH)⁺, and 351 (M – AcOH – CH₃)⁺). However, the character of the fragmentation and especially the intensities of the signals at m/z 311, 286, 259, and 211 propose 7,9(11)-unsaturation in the sterol nucleus.¹³ These data, as well as the order of elution between cholestanol and latosterol, suggest that this compound is 5α-cholesta-7,9(11)-dien-3β-ol (5a).

Similarly to the above, compound 5e (RT 31.2 min) is a C₂₉ analog of compound 5a based on the similarity of their mass spectra. The peak of the molecular ion at m/z 454 characterizes it as a di-unsaturated C₂₉ sterol, and the ratio fragment peaks resemble those of a 7,9(11)- unsaturated sterol.¹³ We suggest this compound is 24-ethylcholesta-7,9(11)-diene-3β-ol (5e).

Compounds 6a (RT 27.6 min) and 6e (RT 32.4 min) are also a pair of congeners. Compound 6a is a C₂₇ sterol with two added oxygen atoms (m/z 460 M⁺, 445 (M – CH₃)⁺, and 385 (M – AcOH – CH₃)⁺). The easy elimination of two water molecules confirms this conclusion (m/z 409 (M – CH₃ – 2 × H₂O)⁺, 311 (M – side chain – 2 × H₂O)⁺, and 251 (M – side chain – AcOH – 2 × H₂O)⁺). The loss of a side chain provides a peak at m/z 347 indicating that additional oxygens are attached to the steroid core. This compound was identified as 5,8-epidioxycholestan-3β-ol (6a).¹⁷

A similar pattern is observed in the mass spectrum of C₂₉-oxysterol 6e (RT 32.4 min): m/z 488 M⁺, 473 (M – CH₃)⁺, and 413 (M – AcOH – CH₃)⁺; the elimination of two water molecules: m/z 437 (M – CH₃ – 2 × H₂O)⁺, 377 (M – CH₃ – AcOH – 2 × H₂O)⁺, 311 (M – side chain – 2 × H₂O)⁺, and 251 (M – side chain – AcOH – 2 × H₂O)⁺; the loss of side chain: m/z 347. Obviously, the structure of this compound is 5,8-epidioxy-24-ethylcholestan-3β-ol (6e).

The unidentified substances C₂₇3F and C₂₉3F were also a pair of homologs with a high degree of unsaturation. Compound C₂₇3F has an RT equal to that of cholesterol (25.8 min) and a mass spectrum characterizing a tri-unsaturated C₂₇ sterol (m/z 424 M⁺, 364 (M – AcOH)⁺, 349 (M – AcOH – CH₃)⁺). Compound C₂₉3F has an RT slightly less than that of 24-ethylcholest-5-en-3β-ol (RT 30.1 min) and a mass spectrum characterizing a tri-unsaturated C₂₉ sterol (m/z 452 M⁺, 392 (M – AcOH)⁺, and 377 (M – AcOH – CH₃)⁺). The ratio of the abovementioned peaks was almost the same for these two compounds. The ratio of the fragment peaks at m/z 311, 257, 251, 235, 223, 209, 197, and 195 was also very similar. Unfortunately, it was not possible for us to determine the exact structures of these C₂₇3F and C₂₉3F sterols from the available data.

Thus, the free sterol composition of the deep-sea holothurian Orphnurgus cf glaber includes mainly cholesterol (1a) and 24-ethylcholesterol (1e) and the products of their modification in sterol cores: Δ⁰ (2a and 2e), Δ⁷ (3a and 3e), Δ^7,14 (4a and 4e), Δ^7,9(11⁾ (5a and 5e), and dioxysterols (6a and 6e). Sterols with other side chains are found in small amounts. Apparently, the abundance of compounds with a common cholesterol side chain and especially a 24-ethyl-substituted side chain reflect the sterol composition of the phytodetritus on which Orphnurgus cf glaber feeds. It should be noted that all the detected compounds, except the minor 24-methyl-5α-cholesta-7,22-dien-3β-ol (3b), have saturated side chains. This feature distinguishes this holothurian from numbers of shallow-water sea cucumbers which contain various Δ²²- and Δ²⁴⁽²⁸⁾-sterols.3,4 The deep-sea holothurians contain sterols with unsaturated side chains also^7–11 since phytoplankton is rich in such sterols. We are not aware of works demonstrating the ability of holothurians to transform the side chains of sterols. It is more likely that phytodetritus was transformed by fungi or bacteria.

On the contrary, the ability to transform the sterol nucleus, converting dietary Δ⁵-sterols into their Δ⁷-analogs, is inherent for most sea cucumbers.18 Holothurians usually produce triterpene glycosides as chemical protection agents against predators, but their own cells resist the action of their cytotoxins due to the presence of Δ⁷-sterols in the membrane.19 For example, frondoside A caused significantly less permeabilization in liposomes containing Δ⁷ holothuroid sterols than those containing cholesterol.²⁰ Although no triterpene glycosides were found in the Orphnurgus cf glaber specimen studied by us using the standard methods,²¹ its sterol composition demonstrates the intensive transformation of dietary Δ⁵-sterols. This mixture contains 14.4% Δ⁵-sterols, 12.6% stanols, 51.6% Δ⁷-sterols, and 20.6% polyunsaturated sterols and oxygen adducts. An intriguing feature of the sterol composition was the presence of compounds with a polyunsaturated sterol nucleus. Probably, processes of dehydrogenation take place in the body of the animal itself because, to the best of our knowledge, the corresponding sterols have not been found in algae and/or detritus until now. Ginger et al,¹⁰ investigating deep-sea holothurians from the Porcupine Abyssal Plain, found a number of Δ^5,7-sterols and an unidentified C₂₇-diene. They assumed that “relatively higher amounts of unsaturated compounds in deep-sea animals are ascribed to an adaptation of the latter to maintain membrane fluidity at high pressure and low temperature.”¹⁰ It seems to us that our finding of numerous di- and tri-unsaturated sterols confirms this suggestion. Orphnurgus cf glaber did not contain Δ^5,7-sterols, which are intermediates in Δ⁵- to Δ⁷-double bond migration in shallow-water holothurians,²² but it contained oxygen adducts 6a and 6e that are easily formed from Δ^5,7-sterols and might be reduced (Figure 2). It is of particular interest that these adducts have fully hydrogenated double bonds in all the rings in contrast to the corresponding compounds derived from most other sea organisms.^23,24 The reduced epidoxysterol 6a was earlier found only in a sponge, Amphimedon sp.¹⁷ A hypothetical pathway for the formation of 5,8-epidioxysterols is shown in Figure 2.

Figure 2.

Hypothetical pathway for the formation of 5,8-epidioxysterols.

Conclusion

A specimen of the deep-sea holothurian Orphnurgus cf glaber collected at the Hawaiian-Emperor Chain was investigated for its free sterol composition. Twenty-one components containing Δ⁷, Δ⁰, Δ⁵, polyunsaturated or 5,8-epidioxy modified cores and predominantly saturated side chains were found. Like other holothurians of the family Deimatidae (Oneirophanta mutabilis^8–10 and Deima validum^7,10 see Supplemental Materials, Table S1), this sea cucumber contains dietary detritus sterols and products of their modification. The high percentage of sterols with saturated side chains and those with polyunsaturated nuclei distinguishes O. cf glaber from previously studied holothurians, both deep-sea and shelf ones. We assume that the detritus is redesigned by fungi or bacteria in either surface sediments or holothurian gut. According to preliminary research from mycological cultures of the intestinal contents of O. cf glaber, 3 species of micellar fungi were isolated (Dr Borzykh O.G., unpublished data). However, we do not exclude that some polyunsaturated sterols are intermediates in the conversion of Δ⁵- to Δ⁷-sterols, like Δ^5,7-sterols found by Ginger et al¹⁰ in other holothurians in small amounts.

Experimental

Analytical Methods

¹H NMR spectra were recorded using a Bruker Avance III 700 spectrometer, at 700 MHz, δ in ppm referenced to CDCl₃ at δ_H 7.26. GC-MS analyses were carried out on a Hewlett Packard HP 5973 MSD: HP 6890 chromatographic system, with a capillary column HP-5MS with a temperature regime of 100 °C followed by an increase at a rate of 10 °C/min and then in isothermal mode at 280 °C. Helium was used as the carrier gas (1 mL/min) and the ionizing voltage was 70 eV.

Animals

A single specimen (the length was about 40 cm) of the holothurian Orphnurgus cf glaber Walsh, 1891 (the Phylum Echinodermata, Class Holothurioidea, Subclass Actinopoda, Order Synallactida, Family Deimatidae) was collected during the expedition of the A.V. Zhirmunsky National Scientific Center of Marine Biology FEB RAS aboard the RV Akademik M. Lavrentyev (cruise no. 94) in July to September 2021. The collection was performed using a remote-operated vehicle Comanche 18 at the Koko guyot, Emperor Chain (35.789° N, 171.049° E) from a depth of 2231 m. The sample was frozen and stored (−20 °C) until moving to the laboratory.

Extraction and Isolation of Free Sterols

The specimen (148.0 g) was dissected and thrice extracted with ethanol. The obtained extract was concentrated in vacuo and was separated for the study of glycosides (0.9 g) and sterols (1.9 g). The search for glycosides was carried out according to the standard method.²¹ The second part of the extract was purified by silica gel column chromatography in the system hexane/ethylacetate (12:1, vol/vol) to obtain the fraction of free sterols (2.2 mg). This fraction was separated by HPLC using an Altex Ultrashere^TM-Si column (hexane/ethylacetate 5:1, vol/vol) into three subfractions (0.4 mg, 0.3 mg, and 1.4 mg), which were analyzed by NMR spectroscopy; then they were acetylated by acetic anhydride in pyridine at room temperature (24 h) and analyzed by GC-MS.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X221142791 - Supplemental material for Free Sterol Composition of Deep-Sea Holothurian Orphnurgus cf. glaber

Supplemental material, sj-docx-1-npx-10.1177_1934578X221142791 for Free Sterol Composition of Deep-Sea Holothurian Orphnurgus cf. glaber by Ludmila P Ponomarenko, Irina I Kapustina, Salim S Dautov and Tatiana N Dautova, Valentin A Stonik in Natural Product Communications

Footnotes

Acknowledgments

The study was carried out using the equipment of the Collective Facilities Center “The Far Eastern Center for Structural Molecular Research (NMR/MS) of PIBOC FEB RAS.”

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Ministry of Science and Higher Education of the Russian Federation (Grant No. 13.1902.21.0012 (contract No. 075-15-2020-796)).

ORCID iD

Irina I Kapustina

Supplemental Material

Supplemental material for this article is available online.

References

Althaus

Williams

Schlacher

, et al. Impacts of bottom trawling on deep-coral ecosystems of seamounts are long-lasting. Mar Ecol Prog Ser. 2009;397:279‐294. doi:10.3354/meps08248

Glover

Smith

. The deep-sea floor ecosystem: current status and prospects of anthropogenic change by the year 2025. Environ Conserv. 2003;30(3):219‐241. doi:10.1017/S0376892903000225

Ponomarenko

Kalinovsky

Moiseenko

Stonik

. Free sterols from the holothurians Synapta maculata, Cladolabes bifurcatus and Cucumaria sp. Comp Biochem Physiol B. 2001;128(1):53‐62. doi:10.1016/S1096-4959(00)00318-3

Stonik

Ponomarenko

Makarieva

, et al. Free sterol compositions from the sea cucumbers Pseudostichopus trachus, Holothuria (Microtele) nobilis, Holothuria scabra, Trochostoma orientale and Bathyplotes natans. Comp Biochem Physiol B. 1998;120(2):337‐347. doi:10.1016/S0305-0491(98)10023-8

Dmitrenok

Shubina

Makar’eva

Stonik

. Sterols of the holothurians Chiridota discolor and Synallactes chuni. Chem Nat Compd. 1988;24:261‐262. doi:10.1007/BF00596771

Ballantine

Lavis

Morris

. Marine sterols. XV. Sterols of some oceanic holothurians. J Exp Mar Biol Ecol. 1981;53:89‐103. doi:10.1016/0022-0981(81)90086-1

Amaro

Danovaro

Matsui

Rastelli

Wolff

Nomaki

. Possible links between holothurian lipid compositions and differences in organic matter (OM) supply at the western pacific abyssal plains. Deep Sea Res Part I. 2019;152:103085. doi:10.1016/j.dsr.2019.103085

Drazen

Phleger

Guest

Nichols

. Lipid, sterols and fatty acid composition of abyssal holothurians and ophiuroids from the North-East Pacific Ocean: food web implications. Comp Biochem Physiol B. 2008;151:79‐87. doi:10.1016/j.cbpb.2008.05.013

Neto

Wolff

Billett

DSM

Mackenzie

Thompson

. The influence of changing food supply on the lipid biochemistry of deep-sea holothurians. Deep Sea Res I. 2006;53:516‐527. doi:10.1016/j.dsr.2005.12.001

10.

Ginger

Santos

VLCS

Wolff

. A preliminary investigation of the lipids of abyssal holothurians from the North-East Atlantic Ocean. J Mar Biol Ass UK 2000;80:139‐146. doi:10.1017/S0025315499001654

11.

Ginger

Billett

DSM

Mackenzie

, et al. Organic matter assimilation and selective feeding by holothurians in the deep sea: some observations and comments. Prog Oceanogr. 2001;50(1-4):407‐421. doi:10.1016/S0079-6611(01)00063-5

12.

Wilson

Sumpter

Warren

Rogers

Ruan

Schroepfer

Jr . Analysis of unsaturated C27-sterols by nuclear magnetic resonance. J Lipid Res. 1996;37(7):1529‐1555. doi:10.1016/S0022-2275(20)39137-9

13.

Gerst

Ruan

Pang

Wilson

Schroepfer

Jr . An updated look at the analysis of unsaturated C₂₇ sterols by gas chromatography and mass spectrometry. J Lipid Res. 1997;38:1685‐1701. doi:10.1016/S0022-2275(20)37187-X

14.

Chitwood

Lusby

. Metabolism of plant sterols by nematodes. Lipids. 1991;26(8):619‐627. doi:10.1007/BF02536426

15.

Wakeham

. Steroid geochemistry in the oxygen minimum zone of the eastern tropical North Pacific Ocean. Geochimica Cosmochim Acta. 1987;51(11):3051‐3069. doi:10.1016/0016-7037(87)90378-4

16.

Wakeham

. Lipid biomarkers for heterotrophic alteration of suspended particulate organic matter in oxygenated and anoxic water columns of the ocean. Deep Sea Res Part I: Oceanogr Res Papers. 1995;42(10):1749‐1771. doi:10.1016/0967-0637(95)00074-G

17.

Garson

Partali

Liaaen-Jensen

Stoilov

. Isoprenoid biosynthesis in a marine sponge of the Amphimedon genus: incorporation studies with [1-¹⁴C] acetate, [4–¹⁴C] cholesterol and [2-¹⁴C] mevalonate. Comp Biochem Physiol B. 1988;91(2):293‐300. doi:10.1016/0305-0491(88)90145-9

18.

Sheikh

Djerassi

. Biosynthesis of sterols in the sea cucumber Stichopus californicus. Tetrahedron Lett. 1977;36:3111‐3114. doi:10.1016/S0040-4039(01)83173-7

19.

Stonik

Elyakov

. Secondary metabolites from echinoderms as chemotaxonomic markers. In: Scheuer

, ed. Bioorganic marine chemistry. Vol 3. Springer-Verlag; 1988;2:43‐86. doi:10.1007/978-3-642-48346-2_2

20.

Claereboudt

EJS

Eeckhaut

Lins

Deleu

. How different sterols contribute to saponin tolerant plasma membranes in sea cucumbers. Sci Rep. 2018;8(1):10845. doi:10.1038/s41598-018-29223-x

21.

Silchenko

Avilov

Kalinin

. Separation procedures for complicated mixtures of sea cucumber triterpene glycosides with isolation of individual glycosides, their comparison with HPLC/MS metabolomic approach, and biosynthetic interpretation of the obtained structural data. In: ur Rahman

, ed. Studies in natural products chemistry. Vol 72. Elsevier B.V.; 2022:103‐146. doi:10.1016/B978-0-12-823944-5.00015-6

22.

Cordeiro

Djerassi

. Biosynthetic studies of marine lipids. 25. Biosynthesis of Δ⁹⁽¹¹⁾- and Δ⁷-sterols and saponins in sea cucumbers. J Org Chem. 1990;55(9):2806‐2813. doi:10.1021/jo00296a045

23.

Minh

Kiem

Huong

Kim

. Cytotoxic constituents of Diadema setosum. Arch Pharm Res. 2004;27(7):734‐737. doi:10.1007/BF02980141

24.

Abourriche

Charrouf

Chaib

Bennamara

Bontemps

Francisco

. Isolation and bioactivities of epidioxysterol from the tunicate Cynthia savignyi. Farmaco. 2000;55(6–7):492‐494. doi:10.1016/S0014-827X(00)00040-9

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.04 MB