Critical Assessment of Glyco- and Phospholipid Separation by Using Silica Chromatography

Heinzelmann, Sandra M; Bale, Nicole J.; Hopmans, Ellen C.; Damsté, Jaap S. Sinninghe; Schouten, Stefan; Meer, Marcel T. J. van der

doi:10.1128/aem.02817-13

Cited by 37 publications

(24 citation statements)

References 38 publications

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“…However, it has been shown that the phospholipid fraction also contains other non-P containing intact polar lipids (Heinzelmann et al, 2014). The glycolipid- and phospholipid fractions were therefore combined and are further referred to as structural, membrane-derived lipids.…”

Section: Methodsmentioning

confidence: 99%

From Ecological Stoichiometry to Biochemical Composition: Variation in N and P Supply Alters Key Biosynthetic Rates in Marine Phytoplankton

et al. 2017

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One of the major challenges in ecological stoichiometry is to establish how environmental changes in resource availability may affect both the biochemical composition of organisms and the species composition of communities. This is a pressing issue in many coastal waters, where anthropogenic activities have caused large changes in riverine nutrient inputs. Here we investigate variation in the biochemical composition and synthesis of amino acids, fatty acids (FA), and carbohydrates in mixed phytoplankton communities sampled from the North Sea. The communities were cultured in chemostats supplied with different concentrations of dissolved inorganic nitrogen (DIN) and phosphorus (DIP) to establish four different types of resource limitations. Diatoms dominated under N-limited, N+P limited and P-limited conditions. Cyanobacteria became dominant in one of the N-limited chemostats and green algae dominated in the one P-limited chemostat and under light-limited conditions. Changes in nutrient availability directly affected amino acid content, which was lowest under N and N+P limitation, higher under P-limitation and highest when light was the limiting factor. Storage carbohydrate content showed the opposite trend and storage FA content seemed to be co-dependent on community composition. The synthesis of essential amino acids was affected under N and N+P limitation, as the transformation from non-essential to essential amino acids decreased at DIN:DIP ≤ 6. The simple community structure and clearly identifiable nutrient limitations confirm and clarify previous field findings in the North Sea. Our results show that different phytoplankton groups are capable of adapting their key biosynthetic rates and hence their biochemical composition to different degrees when experiencing shifts in nutrient availability. This will have implications for phytoplankton growth, community structure, and the nutritional quality of phytoplankton as food for higher trophic levels.

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Section: Methodsmentioning

confidence: 99%

From Ecological Stoichiometry to Biochemical Composition: Variation in N and P Supply Alters Key Biosynthetic Rates in Marine Phytoplankton

et al. 2017

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“…E,F). A recent study by Heinzelmann et al () shows that the separation of glycolipids from phospholipids by commonly used silicate column chromatography is incomplete, and that glyco‐ and other lipids also elute in the methanol fraction. However, both glyco‐ and phospholipids are structural (membrane) lipids in contrast to the neutral storage lipids in the chloroform fraction, and therefore, the data for these two membrane fractions was combined in this study.…”

Section: Assessmentmentioning

confidence: 99%

Tracing carbon fixation in phytoplankton—compound specific and total ¹³C incorporation rates

Große

Breugel

Boschker

2015

Limnology & Ocean Methods

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Measurement of total primary production using 13 C incorporation is a widely established tool. However, these bulk measurements lack information about the fate of fixed carbon: the production of major cellular compounds (carbohydrates, amino acids, fatty acids, and DNA/RNA) is affected by for instance nutrient availability as their C:N:P requirements differ. Here, we describe an approach to combine established methods in gas chromatography/isotope ratio mass spectrometry (GC/C-IRMS) and recently developed methods in liquid chromatography/IRMS (LC/IRMS) to trace stable isotope incorporation into neutral carbohydrates, amino acids, and fatty acids, and compare their production to total carbon fixation rates. We conducted a trial study at stations in the North Sea where different nutrients were limiting. There was variation in the fate of fixed carbon at these sites. The majority of fixed carbon (64-71%) was incorporated into neutral carbohydrates, followed by amino acids (19-32%) and fatty acids (4-9%). The sum of carbon fixation into these three fractions accounted for 81-116% of total carbon fixation. P-and N-limitation increased the biosynthesis of storage lipids and storage carbohydrates while N-limitation decreased synthesis of amino acid proline with a concurrent increase in glutamic acid 1 glutamine. This new approach provides the capability to determine direct effects of resource limitation and the consequences for the physiological state of a phytoplankton community. It may thereby enable us to evaluate the overall quality of phytoplankton as a food source for higher trophic levels or trace consumption of phytoplankton through the food web.

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“…Normal-phase SPE fractionation of lipids has been applied for many years [21]. However, potential issues in selectivity with silica-based SPE fractionation of plant lipids have been shown [22]. The selectivity using half the solvent elution volumes was compared using an LC-MS method previously described [23].…”

Section: Resultsmentioning

confidence: 99%

A Simple Method for Measuring Carbon-13 Fatty Acid Enrichment in the Major Lipid Classes of Microalgae Using GC-MS

et al. 2016

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Abstract:A simple method for tracing carbon fixation and lipid synthesis in microalgae was developed using a combination of solid-phase extraction (SPE) and negative ion chemical ionisation gas chromatography mass spectrometry (NCI-GC-MS). NCI-GC-MS is an extremely sensitive technique that can produce an unfragmented molecular ion making this technique particularly useful for stable isotope enrichment studies. Derivatisation of fatty acids using pentafluorobenzyl bromide (PFBBr) allows the coupling of the high separation efficiency of GC and the measurement of unfragmented molecular ions for each of the fatty acids by single quadrupole MS. The key is that isotope spectra can be measured without interference from co-eluting fatty acids or other molecules. Pre-fractionation of lipid extracts by SPE allows the measurement of 13 C isotope incorporation into the three main lipid classes (phospholipids, glycolipids, neutral lipids) in microalgae thus allowing the study of complex lipid biochemistry using relatively straightforward analytical technology. The high selectivity of GC is necessary as it allows the collection of mass spectra for individual fatty acids, including cis/trans isomers, of the PFB-derivatised fatty acids. The combination of solid-phase extraction and GC-MS enables the accurate determination of 13 C incorporation into each lipid pool. Three solvent extraction protocols that are commonly used in lipidomics were also evaluated and are described here with regard to extraction efficiencies for lipid analysis in microalgae.

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Critical Assessment of Glyco- and Phospholipid Separation by Using Silica Chromatography

Cited by 37 publications

References 38 publications

From Ecological Stoichiometry to Biochemical Composition: Variation in N and P Supply Alters Key Biosynthetic Rates in Marine Phytoplankton

From Ecological Stoichiometry to Biochemical Composition: Variation in N and P Supply Alters Key Biosynthetic Rates in Marine Phytoplankton

Tracing carbon fixation in phytoplankton—compound specific and total ¹³C incorporation rates

A Simple Method for Measuring Carbon-13 Fatty Acid Enrichment in the Major Lipid Classes of Microalgae Using GC-MS

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Critical Assessment of Glyco- and Phospholipid Separation by Using Silica Chromatography

Cited by 37 publications

References 38 publications

From Ecological Stoichiometry to Biochemical Composition: Variation in N and P Supply Alters Key Biosynthetic Rates in Marine Phytoplankton

From Ecological Stoichiometry to Biochemical Composition: Variation in N and P Supply Alters Key Biosynthetic Rates in Marine Phytoplankton

Tracing carbon fixation in phytoplankton—compound specific and total 13C incorporation rates

A Simple Method for Measuring Carbon-13 Fatty Acid Enrichment in the Major Lipid Classes of Microalgae Using GC-MS

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Tracing carbon fixation in phytoplankton—compound specific and total ¹³C incorporation rates