Cell membrane localization of long chain C24−C30 fatty acids in two marine demosponges

Subcellular fractionation by differential centrifugation was performed on two previously unstudied marine sponges (Reniera sp. andPseudaxinyssa sp.) that represent both major subclasses of the Demospongiae. Long chain fatty acids (LCFA) with 24–30 carbon units were found as major constituents of cell membrane isolates of both sponges. Most LCFA were polyunsaturated and were constituents of the phospholipids, which are typical membrane lipids, and in particular the amino-phospholipids. The LCFA composition of phospholipids from whole sponge tissue was shown to provide a reliable indication of the LCFA composition of cell membrane phospholipids in the sponges studied. An unusual triply branched C₁₆ isoprenoid fatty acid, 4,8,12-trimethyltridecanoic acid, also was identified as a cell membrane acid in the spongePseudaxinyssa sp. Part 16 of “Phospholipid Studies of Marine Organisms.” For Part 15 in this series see Dasgupta, A., Ayanoglu, E., Tomer, K.B., and Djerassi, C. (1987)Chem. Phys. Lipids 43, 101–111.     

Localization of long-chain fatty acids and unconventional sterols in spherulous cells of a marine sponge

The first direct evidence is provided for the presence of unconventional lipids in a particular subcellular membrane system of a sponge. Spherulous cells were isolated from the variety of cell types present in the marine spongeAplysina fistularis by density gradient centrifugation. Spherulous cell plasma membrane was subsequently isolated by cell rupture followed by differential centrifugation and sucrose, or Percoll, density gradient ultracentrifugation. Plasma membrane isolates were identified and assessed for purity using [³H] concanavalin A plasma membrane marker, sodium dodecyl sulfate polyacrylamide gel electrophoresis and ratios of protein, sterol and phosphate. Plasma membrane isolates could not be assessed for purity by traditional enzymatic means. Spherulous cell plasma membrane was found to contain unusual lipids, including long-chain (C₂₄–C₃₀) fatty acids (16.8–27.2%) and unconventional 26-alkylated sterols (66.4–72.6%), in addition to more conventional fatty acids and sterols. Spherulous cell intracellular membranes were also found to contain long-chain fatty acids and unconventional sterols, although the relative importance of these unusual lipids apparently varies between intracellular membranes, with some containing approximately 50% long-chain acids. Part 19 of “Phospholipid Studies of Marine Organisms.” For Part 18 see Li, H., Duzgunes, N., Ayanoglu, E., and Djerassi, C.,Chem. Phys. Lipids, in press.     

Terpenes in sponge cell membranes: Cell separation and membrane fractionation studies with the tropical marine spongeAmphimedon sp.

The differing sponge and symbiotic microbial cell types in the tropical marine spongeAmphimedon sp. were fractionated according to density, investigated by electron microscopy, and analyzed by high-performance liquid chromatography and nuclear magnetic resonance for the presence of the terpene metabolite diisocyanoadociane (1) and Δ^5,7-sterols (2–7). A sample of whole sponge was dissected into superficial ectosome and deeper choanosome. The superficial tissue was found to be enriched in sterol relative to choanosome; however, extracts from both tissues contained terpene. Dissociation of whole sponge followed by Ficoll density gradient fractionation showed that there are two chemically distinct types of sponge cells inAmphimedon sp.—small non-nucleolated cells of low density contain terpene 1 together with sterols, while larger nucleolated cells contain significant levels of terpene, but only traces of sterol. Membrane fractionation studies were undertaken to establish whether the terpene components were located specifically in the cell membranes of these two cell types. A membrane vesicle pellet spun down at 100,000×g from small sponge cells contained sterols, but only traces of terpene, whereas the membrane vesicle preparation from heavier cells contained both terpenes and sterols. Subsequently, the presence of terpenes together with sterols was demonstrated in a membrane vesicle preparation of purity >90% prepared from bacteria-free sponge cells. These results provide the first experimental evidence that terpenes are associated with sponge cell membranes, where they may function as structural components. Part of this work has been published in preliminary form (ref. 1)     

Growth support and metabolism of phytosterols inParamecium tetraurelia

The basis of the growth requirement ofParamecium for one of several structurally similar phytosterols is not known. Previous research has indicated that selective esterification of only growth-promoting sterols may be a key. In this study, it was found that under certain conditions sterols that fail to support growth (e.g., cholesterol) can be esterified in large amounts inParamecium. We found no compelling evidence to support the hypothesis that steryl esters serve a specialized role in the fatty acid metabolism of the cell. Octadecenoic acid, essential for cell growth, was the major fatty acid in both steryl esters and triglycerides. It was also shown thatP. tetraurelia can dehydrogenate Δ⁰ and Δ⁷, as well as Δ⁵-3β-hydroxy sterols, to yield the conjugated 5,7-diene derivative. These results indicate the presence of a Δ⁵, in addition to a Δ⁷, desaturase of the sterol nucleus in this ciliate. Two C₂₄ α-ethyl sterols, Δ²²-stigmasterol (Δ²²) and stigmastanol (Δ⁰), were shown for the first time to promote growth. Finally, we found that non-growth-promoting sterols may compose a high percentage of the free sterols of the surface membrane without adversely affecting cell growth or viability. These data support the conclusion that the growth requirement for select phytosterols inParamecium does not involve the structural or functional role of “bulk” sterols in cell membranes.     

Novel brominated phospholipid fatty acids from the Caribbean spongeAgelas sp.

The very long-chain fatty acids, (5E,9Z)-6-bromo-5,9-tetracosadienoic, (5E,9Z)-6-bromo-23-methyl-5,9-tetracosadienoic, (5E,9Z)-6-bromo-5,9-pentacosadienoic and (5E,9Z)-6-bromo-24-methyl-5,9-pentacosadienoic acids, were identified in the phospholipids (mainly phosphatidylethanolamine) of the spongeAgelas sp. Structure elucidation was accomplished by means of mass spectrometry and chemical transformations, including deuteration with Wilkinson's catalyst. All of the sterols from the sponge had the Δ5,7 nucleus, with 24-methylcholesta-5,7,22-trien-3β-ol (ergosterol) and 24-ethylcholesta-5,7,22-trien-3β-ol being the most abundant.     

Phospholpid studies of marine organisms: 2.1 Phospholipids,phospholipid-bound fatty acids and free sterols of the spongeAplysina fistularis (Pallas) formafulva (Pallas) (=Verongia thiona)2. Isolation and structure elucidation of unprecedented branched fatty acids

The free sterols and phospholipids of the demospongeAplysina fistularis were isolated and analyzed. The free sterols consisted mainly of the unusual 26-methylated sterols aplysterol (53%) and 24(28)-dehydroaplysterol (7%) together with 7 commonly occurring sterods. The major phospholipids were phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine and diphosphatidylglycerol. The major fatty acyl components of the phospholipids consisted of 85% C₁₄−C₂₀ acids, including the unprecedented 2,6,10-trimethyl-5-tetradecenoic acid and 11-methyloctadecanoic acid. The remaining 15% were C₂₇−C₃₀ demospongic acids, including 2 novel acids tentatively assigned the structures 5,9,23-octacosatrienoic acid and 5,9,23-nonacosatrienoic acid, and 3 novel acids proven to be 5,9,21-octacosatrienoic acid, Z,Z-20-methyl-5,9-hexacosadienoic acid and Z,Z-22-methyl-5,9-octacosadienoic acid. The biosyntheses of the novel demospongic acids are proposed to occur by chain elongation of monoenoic or branched precursors followed by desaturation. The large quantities of typically bacterial phospholipids and fatty acids found implied the presence of bacteria in the sponge, in agreement with microscopic studies. Analysis of the phospholipid-bound fatty acids in a sponge cell-enriched fraction indicated that the demospongic acids, including the 2 branched structures, were the major acids of the sponge cells. The presence inA. fistularis of demospongic acids containing membrane disordering groups—methyl branches or double bonds—on the ω7 carbon is proposed to be due to the need by the sponge for membranes possessing fluidity near the middle of the phospholipid bilayer. It is also proposed that the C₂₆ methyl group of aplysterol causes disordering of the phospholipid bilayer in the same region, and thus also evolved in response to this need. For part 1, see ref. 1. Phylum porifera, class demospongia, subclass ceractinomorpha, order verongida, family aplysinidae (2). Formerly known asVerongia thiona de Laubenfels. Major diagnostic fragments from peak 23:M⁺ 471 (2.9%); m/z 442 (C₂₆, 0.2%); m/z 428 (C₂₅, 0.2%); m/z 414 (C₂₄, 0.1%); m/z 400 (C₂₃, 0.3%); (absence of C₂₂ peak); m/z 374 (C₂₁, 0.2%); m/z 360 (C₂₀, 0.2%); m/z 234 (C₁₁, 1.0%); m/z 220 (C₁₀, 0.7%); m/z 206 (C₉, 0.5%); m/z 194 (C₈, 0.4%); m/z 180 (C₇, 24.9%); m/z 166 (C₆, 1.2%); m/z 152 (C₅, 0.8%); m/z 140 (C₄, 1.3%); m/z 126 (C₃, 18.7%). Major diagnostic fragments from peak 25:M⁺ 485 (0.9%); m/z 456 (C₂₇, 0.1%); (absence of C₂₆, C₂₅ peaks); m/z 414 (C₂₄, 0.3%); m/z 400 (C₂₃, 0.1%); m/z 402 (C₂₃, 0.1%); (absence of C₂₂ peak); m/z 374 (C₂₁, 0.1%); m/z 360 (C₂₀, 0.1%); m/z 234 (C₁₁, 0.5%); m/z 220 (C₁₀, 0.3%); m/z 206 (C₉, 0.1%); m/z 194 (C₈, 0.2%); m/z 180 (C₇, 18.0%); m/z 166 (C₆); m/z 152 (C₅), m/z 140 (C₄); m/z 126 (C₃, 15.0%).     

New 2-Methyl-13-Icosenoic Acid from the Temperate Calcisponge <Emphasis Type="Italic">Leuconia johnstoni</Emphasis>

The fatty acid composition of the temperate calcareous marine sponge Leuconia johnstoni Carter 1871 (Calcaronea, Calcarea) was characterized for the first time in specimens collected off the Brittany coast of France over four years from October 2005 to September 2008. Forty-one fatty acids (FA) with chain lengths ranging from C₁₄ to C₂₂ were identified as fatty methyl esters (FAME) and N-acyl pyrrolidide (NAP) derivatives by gas chromatography–mass spectrometry (GC–MS). Twenty-two saturated fatty acids (SFA) were identified accounting for 52.1–59.0% of the total FA and dimethylacetals (DMA). In addition, among the SFA, we noticed the presence of numerous methyl-branched iso and anteiso FA, suggesting a large number of associated bacteria within L. johnstoni. Thirteen monounsaturated fatty acids (MUFA, 28.0–36.0% of total FA + DMA) were also identified as well as six polyunsaturated fatty acids (PUFA, 4.0–8.2%). A noticeable DMA was detected at a high level, particularly in September 2008 (11.8%), indicating the presence of plasmalogens in this sponge species. This calcareous sponge lacked the non-methylene-interrupted FA (NMI FA) with a Δ5,9 system typical of siliceous Demosponges and Hexactinellids. The occurrence of the unusual 8,13-octadecadienoic acid was reported for the first time as a minor PUFA in a calcareous sponge. The major FA, representing 20–25% of this sponge FA, was identified as the new 2-methyl-13-icosenoic acid from mass spectra of its methyl ester and its corresponding N-acyl pyrrolidide derivatives as well as a dimethyl disulfide adduct.     

Combustion of Cr2O3 + Al powder mixtures in a coflow of inert gas: 5. Effect of green density

Combustion of Cr₂O₃ + Al powder mixtures in a coflow of inert gas (Ar) was investigated upon variation in green density in the presence/absence of blowing agents (borax, baking soda). The results were rationalized in terms of the convection-conduction theory for combustion in heterogeneous condensed systems. For Part 4, see Int. J. SHS, 2008, vol. 17, no. 3, pp. 199–205.     

Identification of chondrillasterol in twoCucurbitaceae seed oils by proton nuclear magnetic resonance spectroscopy

The absolute configuration at C-24 of C-24 epimeric 24-ethyl-5α-cholesta-7,E-22-dien-3β-ols I–IV previously isolated from tea seed oil, shea fat, and gourd and sponge cucumber seed oils, respectively, was studied by proton nuclear magnetic resonance spectroscopy. The results showed that the sterols I and II are identical with spinasterol (24S/α-ethyl group), whereas the sterols III and IV are identified as its 24R/β-epimer, chondrillasterol. This study has thus for the first time properly documented the presence in tracheophytes of a 24β-ethylsterol in which Δ²⁵⁽²⁷⁾-bond is reduced.     

Unusual tetraene sterols in some phytoplankton

Sterols were analyzed from four phytoplankton strains which are under investigation as possible sources of food for oysters in culture. One strain ofPyramimonas contained only 24-methylenecholesterol as a major sterol component.Pyramimonas grossii, Chlorella autotrophica andDunaliella tertiolecta each contained a complex mixture of C₂₈ and C₂₉ sterols with Δ⁷, Δ^5,7 and Δ^5,7,9(11) nuclear double bond systems. Sterols were found both with and without the C-22 side chain double bond. Ergosterol and 7-dehydroporiferasterol were the principal sterols in each of the latter three species, which also contained the rare tetraene sterols, 24-methylcholesta-5,7,9(11),22-tetraen-3β-ol and 24-ethylcholesta-5,7,9(11),22-tetraen-3β-ol.     

Sterols and fatty acids of a whisk fernPsilotum nudum

The sterols and fatty acids ofPsilotum nudum were investigated. The 4,4-dimethyl- and 4α-methylsterol fractions contained 24β-methyl-Δ²⁵-unsaturated sterols,viz., cyclolaudenol and 24β-methyl-25-dehydrolophenol, respectively, as dominant sterols among the other components common in vascular plants. 24-Methylcholesterol (mixture of C-24 epimers) and sitosterol constituted the dominant sterols in the 4-demethylsterol fraction. This is the first identification of 24-methylene-5α-lanost-8-en-3β-ol, 24β-methyl-25-dehydrolophenol, codisterol, isofucosterol, 24-methylene-25-methylcholesterol and avenasterol in a fern. The major fatty acids were 16:0, 18:1, 18:2, 18:3 and 20:3. In addition, several C₂₀ fatty acids with various unsaturation were found to be present in low concentrations.     

Anatomical distribution of sterols in oysters (Crassostrea gigas)

Oysters (Crassostrea gigas) contain at least 8 predominant sterols as determined by gas liquid chromatography and a modified Liebermann-Burchard reaction. These sterols and the average amount found in mg/100 are: C₂₆-sterol (22-trans-24-norcholesta-5, 22-diene-3β-ol), 19.1; 22-dehydrocholesterol, 15.1; cholesterol, 46.8; brassicasterol, 27.2; Δ^5,7-sterols (i.e., 7-dehydrocholesterol) 22.5; 24-methylenecholesterol 29.1; 24-ethylcholesta-5,22-diene-3β-ol, 1.2; and 24-ethylcholesta-5-en-3β-ol, 12.7. The distribution of these sterols appears uniform (r²=0.938) between 5 major organs of the oyster. The percent body mass vs percent total sterols in these 5 organs are: mantle 44.1–41.4; visceral mass 30.3–36.7; gills 13.2–11.7; adductor muscle 8.3–3.7; and labial palps 4.2–6.5. The possible sources of these sterols are discussed.     

AY-9944 inhibition of sterol biosynthesis inChlorella emersonii

WhenChlorella emersonii, a green alga, was cultured in the presence of 20 ppm AY-9944, a number of sterols accumulated which appear to be intermediates of sterol biosynthesis in this organism. The sterols isolated include 14α-methyl-ergost-8-en-3β-ol, 14α-methyl 24S-stigmast-8-en-3β-ol, 14α-methyl ergosta-8,24(28)-dien-3β-ol and 4α, 14α-dimethyl 24S-stigmast-8-en-3β-ol. Smaller quantities of several other sterols were found in addition to the normally occurring Δ⁷, chondrillasterol and Δ⁷. Control cultures were found to contain, in addition to the normally occurring sterols, smaller quantities of most of the sterols isolated from AY-9944 inhibited cultures. AY-9944 is a specific inhibitor of Δ⁷ in cholesterol biosynthesis in animals. However, sinceC. emersonii terminates sterol biosynthesis one step prior to the Δ⁷ step, AY-9944 apparently inhibits sterol biosynthesis prior to this step in this organism. The accumulation of 14α-methyl sterols in treated cultures suggests that AY-9944 is an effective inhibitor of the 14α-methyl removal inC. emersonii. Scientific Article No. A1865, Contribution No. 4775 of the Maryland Agricultural Experiment Station.     

Inhibition of sterol biosynthesis in chlorella ellipsoidea by AY-99441

WhenChlorella ellipsoidea was grown in the presence of 4 ppm AY-9944, complete inhibition of Δ⁵-sterol biosynthesis was achieved. However total sterol production remained unaltered. As a result a number of sterols accumulated that appear to be intermediates in sterol biosynthesis. These sterols were described and identified as (24S)-5α-ergost-8(9)-en3β-ol, (24S)-5α-stigmast-8(9)-en-3β-ol, 4α-methyl-(24S)-5α-ergosta-8, 14-dien-3β-ol, 4α-methyl-(24S)-5α-stigmasta-8, 14-dien-3β-ol, 4α-methyl-(24S)-5α-ergost-8(9)-en-3β-ol and (24S)-4α-methyl-5α-stigmast-8(9)-en-3β-ol. The occurrence of these sterols inChlorella ellipsoidea is the first time they have been noted in biological material. The accumulation of these sterols in treated cultures indicates that AY-9944 is an extremely effective inhibitor of the Δ⁸→Δ⁷ isomerase and the Δ¹⁴ reductase of these plants. The occurrence of small amounts of other sterols in treated cultures has led to a proposed pathway for thebiosynthesis of sterols inChlorella ellipsoidea. Scientific Article No. A1775, Contribution No. 4565 of the Maryland Agricultural Experiment Station.     

The steric requirements for sterol inhibition of tetrahymanol biosynthesis

Many naturally occurring sterols are accumulated and metabolized byTetrahymena pyriformis. In most cases, the sterols are desaturated to give^Δ5,7,22-derivatives. Compounds with an ethyl, but not with a methyl, substituent at C-24 are dealkylated. Exposure of the ciliates to the appropriate sterol sharply curtails the synthesis of the native pentacyclic triterpenoid alcohols, tetrahymanol and diplopterol. An analysis with modified sterols has revealed several additional features that are required for desaturation at C-7,8 and C-22,23 and for inhibition of tetrahymanol biosynthesis. The presence of atrans-17(20)-double bond, which eliminates free rotation at C-20 and fixes C-22 to the right of the nucleus, does not interfere with desaturation, while thecis- or left-handed isomer is not metabolized. Thecis-Δ¹⁷⁽²⁰⁾-isomer is, however, an effective inhibitor of tetrahymanol biosynthesis, although less so that thetrans-counterpart. When a methyl or hydroxyl group at C-20 protrudes to the front of the molecule in the right-handed conformation, metabolism is reduced or abolished. Shortening (by one C-atom) or lengthening of the sterol side chain has little effect on the ability of the compounds to inhibit tetrahymanol biosynthesis or to support growth, as long as the overall length of the side chain does not exceed seven carbons from C-20. The presence of a 7α-, 7β-, 20α-, 20β-, or a 25-hydroxy group in the cholesterol molecule sharply inhibits desaturation and curtails the effectiveness of the compound as an inhibitor of tetrahymanol biosynthesis. The 7- or 22-keto derivatives seem to act in a fashion similar to the hydroxy derivatives, but these compounds show greater inhibition of growth. 20-Methylcholesterol, however, is a potent inhibitor of synthesis, which suggests that the polarity of the substituent of C-20 is more important than bulk. Many sterols can effectively replace tetrahymanol in the membranes of these ciliates. However, several of the compounds, which inhibit synthesis, appear to be physiologically inappropriate, and poor growth results. An example of the latter class is 20-methylcholesterol. Finally, a class of sterols, represented by 20α-hydroxycholesterol and 7-ketocholesterol, does not severly inhibit tetrahymanol synthesis but leads to growth inhibition and surface abnormalities. These sterols apparently lead to a disordered membrane, even in the presence of tetrahymanol.     

C27 to C32 sterols found in Pneumocystis, and opportunistic pathogen of immunocompromised mammals

Pneumocystis carinii is the paradigm of opportunistic infections in immunocompromised mammals. Prior to the acquired immunodeficiency syndrome (AIDS) pandemic and the use of immunosuppressive therapy in organ transplant and cancer patients, P. carinii was regarded as a curiosity, rarely observed clinically. Interest in this organism exploded when it was identified as the agent of P. carinii pneumonia (PcP), the direct cause of death among many AIDS patients. Aggressive prophylaxis has decreased the number of acute PcP cases, but it remains among the most prevalent opportunistic infections found within this patient population. The taxonomic assignment of P. carinii has long been argued; molecular genetics data now demonstrate that it is a fungus. Several antimycotic drugs are targeted against ergosterol or its biosynthesis, but these are not as effective against PcP as they are against other fungal infections. This can now be explained in part by the identification of the sterols of P. carinii. The organism lacks ergosterol but contains distinct C₂₈ and C₂₉ Δ⁷ 24-alkylsterols. Also, 24-methylenelanost-8-en-3β-ol (C₃₁) and pneumocysterol, (24Z)-ethylidenelanost-8-en-3β-ol (C₃₂) were recently identified in organisms infecting humans. Together, the Δ⁷ 24-alkylsterols and pneumocysterol are regarded as signature lipids of the pathogen that can be useful for the diagnosis of PcP, since no other lung pathogen is known to contain them. Cholesterol (C₂₇), the dominant sterol component in P. carinii, is probably totally scavenged from the host. De novo synthesis of sterols has been demonstrated by the presence of lovastatin-sensitive 3-hydroxy-3-methylglutaryl-CoA reductase activity, the incorporation of radiolabeled mevalonate and squalene into P. carinii sterols, and the reduction in cellular ATP in cells treated with inhibitors of enzymes in sterol biosynthesis.     

Distribution of sterols in the fungi I. Fungal spores

The freely extractable sterols of spores ofLinderina pennispora, Spicaria elegans, Penicillium claviforme, Aspergillus niger, Ustilago nuda, U. maydis, Puccinia graminis, andP. striiformis were examined using mass spectrometric techniques. Each species contained at least 3–5 detectable sterol components in the 4-desmethyl sterol fraction, and, when present, ergosterol was generally the most abundant sterol produced by an individual species. Smaller relative concentrations of fungisterol (ergost-Δ⁷-enol) di- and tetraunsaturated C₂₈ sterols also were found. In some species, fungisterol was the most abundant sterol. In uredospores of rust fungi, stigmast-Δ⁷-enol (C₂₉) was predominant and was accompanied by lower relative concentrations of a diunsaturated C₂₉ sterol and fungisterol. Cholesterol was found only in the teliospores of the corn smut fungus (U. maydis). Application of glass capillary columns to the separation of yeast sterols by gas liquid chromatography is illustrated. One of eight papers presented in the symposium “Phytosterols,” AOCS Spring Meeting, New Orleans, April 1973.     

Occurrence of plant sterols in aquatic vertebrates

Plant sterols were found by gas liquid chromatography in the sterols of five species of aquatic vertebrates; mackerel (Scomber japonicus), rainbow trout (Salmo gairdnerii), smelt (Osmerus dentex), sardine (Sardinops melanosticta) and chimera (Chimera phantasma). The sterols of chimera liver, sardine flesh and sardine viscera contained 9.0, 2.4 and 3.1% of C₂₈ and C₂₉ sterols in addition to 86.7, 96.6 and 95.2% of cholesterol. The occurrence of norcholestandienol, campesterol, β-sitosterol and C₂₈ stanol was shown by combined gas chromatography-mass spectrometry. Sperm whale (Physeter catodon) sterols consisted of more than 99% cholesterol with only traces of 22-dehydrocholesterol.     

Composition and biosynthesis of sterols in selected marine phytoplankton

Six species of phytoplankton,Pseudoisochrysis paradoxa, Isochrysis galbana, Monochrysis lutheri, Platymonas suecica, Thalassiosira fluviatilis and aChaetoceros species, were cultured in the laboratory and their sterol contents analyzed utilizing digitonin precipitation, thin layer and gas chromatography and gas chromatography-mass spectrometry. A total of 7 sterols were found in phytoplankton. The occurrence of these sterols, cholest-5-en-3β-ol, cholest-5,22-dien-3β-ol, 24-methylcholesta-5,24(28)-dien-3β-ol, 24-methylcholest-5-en-3β-ol, 24-methylcholesta-5,22-dien-3β-ol, 24-ethylcholest-5-en-3β-ol and 24-ethylcholest-5,22-dien-3β-ol, differed significantly among the various phytoplankton species. Cultures ofP. paradoxa biosynthesized both of the sterols found in this species when incubated in the presence of¹⁴C- or³H-mevalonic acid for 0.5–9 days. These sterols were cholesterol and 24-methylcholesta-5,22-dien-3β-ol. Since 5 of the sterols found in the phytoplankton commonly occur in mollusks which feed on phytoplankton, it is likely that at least some of the tissue sterols in mollusks are of dietary origin. Research trainee, HL 07295-02, National Heart, Lung and Blood Insitute.     

Sterols,aliphatic hydrocarbons,and fatty acids of a nonphotosynthetic diatom,Nitzschia alba

The unsaponifiable lipids and total fatty acids of a nonphotosynthetic diatom,Nitzschia alba, have been examined. The major fatty acids were found to be 14∶0, 16∶0, 18∶1, and 20∶5; small amounts of 15∶0, 16∶1, 18∶0, 18∶2, 18∶3, and 20∶4 acids also were present. The unsaponifiable lipids consisted mostly of sterols, with only traces (<0.1%) of hydrocarbons (chiefly C₁₆, C₁₈, and C₂₈ normal olefins). The sterols contained brassicasterol (major) and clionasterol (minor), as well as traces of an unidentified sterol; clionasterol was present only in glycosidically bound form.