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. 2023 May 12;18(5):e0285751. doi: 10.1371/journal.pone.0285751

A simple method to isolate fatty acids and fatty alcohols from wax esters in a wax-ester rich marine oil

Pauke Carlijn Schots 1,*, Guro Kristine Edvinsen 1, Ragnar Ludvig Olsen 1
Editor: Rishiram Ramanan2
PMCID: PMC10180661  PMID: 37172054

Abstract

Calanus finmarchicus is one of the most important zooplankton species in the North Atlantic. The zooplankton is currently being harvested and industrially processed to a marine oil product for human consumption as a marine nutraceutical containing long-chain omega-3 polyunsaturated fatty acids. This oil is very rich in wax esters, a lipid class where fatty acids are esterified to long chain fatty alcohols. In this paper we describe a simple method to 1) isolate the wax esters from the other lipid classes present in the oil, 2) hydrolyze the wax esters, and 3) separate the fatty acids from the fatty alcohol, all by means of solid phase extraction. Starting with an average of 322 mg Calanus oil, we obtained 75 mg fatty alcohols and 63 mg fatty acids. Contrary to previously described techniques, our method neither oxidize the fatty alcohols to fatty acids, nor are the fatty acids methylated, allowing the native, unesterified fatty acids and fatty alcohols to be used for further studies, such as in cell culture experiments to study the metabolic effects of these specific lipid fractions rather than the intact oil or wax esters.

Introduction

The limited amount of fish oil, containing health promoting long-chain omega-3 polyunsaturated fatty acids (LC n-3 PUFA), available for the use in fish feed and human consumption, has led to a massive amount of research and development to find alternative sources for these fatty acids [1, 2]. Suggested possibilities have been to utilize lower trophic level alternatives, such as zooplankton, like krill and calanoid species, or the industrial cultivation of marine microalgae [3]. The marine copepod Calanus finmarchicus is one of the most important zooplankton species, with respect to biomass, in the North Atlantic and plays a key role in the pelagic food web between primary producers and economically important fish species [4–6]. Due to C. finmarchicus’ importance in the marine ecosystem and the possibility to harvest it in a sustainable manner [7], the species has received substantial scientific attention. The zooplankton is currently being harvested and industrially processed to an oil (Calanus oil). [8, 9]. This oil, as most other marine oils, is a source of the health promoting LC n-3 PUFA [8, 9]. However, unlike other marine oils, most of the fatty acids in Calanus oil (as much as 85%) are esterified to a fatty alcohol, forming a lipid class known as wax esters [8, 9].

Several analytical methods have been developed to identify and quantify the composition of the fatty acids and fatty alcohols in calanoid wax esters. These methods often contain transesterification, derivatization, separation, and purification steps of the wax esters and fatty acids and alcohols and lead to the production of fatty acid methyl esters (FAMEs) [10–13].

Previous studies have shown that the oil from C. finmarchicus, and especially the wax esters, have potent effects on gut microbiota and cardiac function and against the development of obesity, insulin resistance and inflammation in mice [14–18] and body composition, functional strength, cardiorespiratory function, metabolic markers and omega-3 index in humans [19–22], making this oil a possible new marine drug against metabolic syndrome and other pathological conditions.

After consumption, wax esters are to a large extend hydrolyzed to free fatty acids and free fatty alcohols in the lower end of the gastrointestinal tract [23]. The possible health effects of the wax ester derived fatty acids and fatty alcohols have been reviewed previouslyand it is known that both fatty acids and fatty alcohols can have positive health effects but potentially through different mechanisms [24]. To study the health effects of the individual lipid classes in the wax ester, in vitro, it is necessary to separate the fatty acids and fatty alcohols in hydrolyzed wax esters. To our knowledge there is no method previously described to separate the fatty acids and fatty alcohols in wax esters without the formation of FAMEs. Although the production of FAMEs is excellent for lipid composition analyses, it is less suitable for the extraction of specific lipid classes. FAMEs are esters of their own and would not permit us to obtain the free fatty acids without another saponification step. Similarly, the fatty alcohols would be oxidized to their corresponding fatty acids before being transformed to FAMEs. Thus, the production of FAMEs would not allow us to obtain the fatty alcohols and fatty acids in their native form without additional steps. Therefor the aim of this work was to develop a simple semi-preparative method using solid phase extraction (SPE), to isolate the wax esters from the other lipid classes present in the oil, and subsequently to separate the native non-esterified fatty acids and fatty alcohols in the wax esters. The method does not require sophisticated instrumentation. These isolated compounds may then be used to study potential metabolic effects in, for example, cell culture experiments.

Materials and methods

Oil and chemicals

The oil produced from C. finmarchicus (Calanus oil) was provided by the company Zooca® formerly named Calanus® AS (Tromsø, Norway). Heptane (99,8%), isopropanol (100%), diethyl ether (≥99,8%) and ethanol (96%) were purchased from VWR, Darmstadt, Germany. Acetic acid (99,8%) and hydrochloric acid (37%) were purchased from Honeywell, Seelze, Germany. Chloroform (99,0–99,4%), phosphoric acid 99,99%, potassium bicarbonate ≥99,95%, sodium hydroxide, copper (II) sulphate pentahydrate ≥98,0%, sulphuric acid and toluene were obtained from Sigma-Aldrich, Darmstadt, Germany. N-Hexane was obtained from Merck KgaA, Darmstadt, Germany.

Isolation of wax esters from Calanus oil

The wax esters were isolated from Calanus oil through solid phase extraction (SPE) as described by Vang et al. [9] with some modifications. On average 322 mg of Calanus oil were dissolved in 6 mL chloroform (Fig 1). A Mega Bond Elute (5g) aminopropyl SPE disposable column (Agilent Technologies, Oslo, Norway),used in all separation steps described in Fig 1, was mounted on a SPE Visiprep vacuum manifold (Supelco, Bellafonte, PA, USA). The column was conditioned with 20 mL heptane at a flow rate of approximately 1 mL/min, a flow rate used in all separation steps described in Fig 1. After conditioning, the Calanus oil in chloroform was added to the column. The neutral lipids (NL) present in the oil were eluted with 30 mL chloroform/isopropanol (2:1 v/v) and dried under nitrogen gas. The dried NL were subsequently dissolved in 5 mL chloroform and then applied to a new SPE column pre-conditioned with 20 mL heptane. The wax esters were eluted with 50 mL heptane and again evaporated to dryness under nitrogen gas and dissolved in 5 mL chloroform. The yields of the eluted lipid classes were determined gravimetrically after drying beforethey were redissolved in chloroform to provide a sample for the HPTLC analysis. The isolation of wax esters and the subsequent saponification and isolation of the native fatty acids and fatty alcohols were carried out 3 times (see S1 Table).

Fig 1. Isolation of fatty acids and fatty alcohols in wax esters present in Calanus oil by use of solid phase extraction.

Fig 1

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See text for details.

Saponification of wax esters and isolation of the fatty acids and fatty alcohols

The isolated wax esters were saponified following Christie and Han [25] with slight modifications. The wax esters, on average 251 mg dissolved in 5 mL chloroform, were evaporated to dryness and dissolved in 50 mL of 1 M ethanolic NaOH to a concentration of 5 mg/mL. The dissolved wax esters were transferred to 5 GL 18 Duran Schott test tubes (DWK life sciences, Oslo Norway) each containing 10 mL dissolved wax esters in 1 M ethanolic NaOH, capped, and placed on a heating block (ThermoFisher Scientific, Oslo, Norway) for 90 min at 90°C. No antioxidants were added during the wax ester hydrolysis because Calanus oil contains naturally the antioxidant astaxanthin. The hydrolyzed wax esters were cooled on ice and the Duran Schott test tubes were pooled before addition of 50 mL MilliQ water and 25 mL 6 M HCl. The acidified hydrolyzed wax esters were then mixed with 25 mL heptane to form two phases. The upper phase, containing the lipids, was collected and again evaporated under nitrogen gas and dissolved in 5 mL chloroform.

After the saponification, the free fatty acids and fatty alcohols were separated from each other by use of SPE (Fig 1). An SPE column was conditioned with 20 mL heptane after which the fatty acids and fatty alcohols in chloroform were added to the column. The fatty alcohols were eluted with 30 mL chloroform/isopropanol (2:1 v/v). The same column was then again conditioned with 20 mL heptane and the free fatty acids were eluted with 50 mL diethyl ether/acetic acid (98:2 v/v). The solvents with the separated fatty alcohols and fatty acids were evaporated under nitrogen gas and each dissolved in 1 mL chloroform. The use of SPE to separate the fatty alcohols from the fatty acids was chosen over the separation of the fractions via soap formation by NaOH, since the latter method did not lead to a clear separation in our set-up.

Analysis by HPTLC

From the first isolation, aliquots of 50 μL were removed from the initial Calanus oil in chloroform and the fractions eluted from the SPE columns (Fig 1); the neutral lipids, the wax esters, the hydrolyzed wax esters and the isolated fatty alcohols and fatty acids, all in chloroform. The aliquots were stored at -20°C before analysis by high performance thin layer chromatography (HPTLC). One microliter of the samples was applied to the HPTLC plate (Silica gel 10 cm × 10 cm, Merck, Darmstadt, Germany) together with reference standard 18–5 A (Nu-Chek-Prep, Elysian, MN, USA). The plate was placed in a glass chamber saturated with heptane/diethyl ether/acetic acid (80:20:2 v/v/v) as described by Henderson and Tocher [26]. The mobile phase was allowed to migrate about 9 cm before the plate was removed and the solvent was allowed to evaporate. After air drying, the plate was sprayed with 10% copper sulphate in 8% phosphoric acid prior to development in an incubator at 180°C for 3 min. The developed HPTLC plate was scanned on a Xerox WorkCenter 7855i (Fig 2).

Fig 2. High performance thin layer chromatography of the lipid classes isolated from Calanus oil by SPE.

Fig 2

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St.: Lipid class standard 18–5 A containing lecithin (a); cholesterol (b); oleic acid (c); TAG (d); and cholesteryl oleate (e). Oil: Oil from C. finmarchicus (Calanus oil), NL: Neutral lipids, WE: Wax esters, Hyd. WE: Hydrolyzed wax esters, FAOH and FFA: free fatty alcohols and free fatty acids, respectively, from the hydrolyzed wax esters.

Composition of the fatty acids and fatty alcohols

To analyze the composition of the wax ester, free fatty acids and fatty alcohols, aliquots of these lipid fractions from the first extraction round were dissolved in 1 mL toluene and 2 mL 1% sulfuric acid in methanol was added. The tubes were then flushed with nitrogen gas, capped, and incubated at 50°C for 16 h. After cooling, 2 mL 2% KHCO3 was added, followed by 10 mL freshly made n-hexane/diethyl ether (1:1 v/v). After centrifuging at 1500 rpm for 2 min, the organic layer was transferred to a clean tube. The aqueous layer was mixed for a second time with 10 mL n-hexane/diethyl ether (1:1 v/v) and centrifuged before the organic layer was transferred to the organic phase from the first extraction. The combined organic phases were then dried by evaporation under nitrogen gas. The final extract was dissolved in n-hexane, to a final concentration of 1 mg lipid per ml. The fatty alcohols and the methylated fatty acids were determined by gas chromatography analysis on GC-FID (Agilent Technologies GC 7890B, Santa Clara, CA, USA) and identified by use of the FAME standards GLC 68D and GLC 96, and the fatty alcohol standard GLC 621 (Nu-Chek-Prep, Elysian, MN, USA). GLC621 is a combination of C14:0 myristyl alcohol (15% wt/wt), C16:0 palmityl alcohol (15% wt/wt), C16:1 palmitoleyl alcohol (15% wt/wt), C18:1 oleyl alcohol (15% wt/wt), C20:1 11-eicosenol alcohol (10% wt/wt), C21:0 methyl heneicosanoate (20% wt/wt), and C22:1 erucyl alcohol (10% wt/wt). This analysis was performed by the company Akvaplan-niva, daughter company of the Norwegian Institute for Water Research (NIVA), Tromsø, Norway (https://www.akvaplan.niva.no/en), following an inhouse protocol based on methods described by Folch et al., [27] and Christie [28].

Results and discussion

Wax ester isolation

A flow diagram of the different steps to isolate the fatty acids and fatty alcohols of the wax esters in the Calanus oil by means of solid phase extraction (SPE) is shown in Fig 1. The total weight of wax esters (WE) isolated from on average 322 mg Calanus oil was 251 mg (Table 1 and S1 Table), i.e. constituting almost 80% of the total lipids in the oil which is in the same range as found by others [6, 29]. According to the high performance thin layer chromatography (HPTLC) analysis (Fig 2), a relatively high amount of free fatty acids are present in the unfractionated oil and this has been reported previously [9]. It can also be seen that both the neutral lipids (NL) and WE fraction were devoid of free fatty acids. The 49 mg difference in weight between the oil (322 mg) and isolated neutral lipids (273 mg) is therefore most likely due to free fatty acid fraction and a small amount of phospholipids in the oil. The 22 mg difference between the neutral lipids (273 mg) and isolated wax esters (251 mg) is most likely due to the absence of cholesterol, mono- and diacylglycerol in the isolated wax ester fraction.

Table 1. Average yield of different lipid classes extracted 3 times from Calanus oil by means of solid phase extraction.

Values are given in mg and percentage weight of the oil with standard deviation.

Lipid class Average (mg) STDEV Average % STDEV
Calanus oil 322 19,1 100  
Neutral lipids 273 6,6 85 3,2
Wax esters 251 10,2 78 1,5
Hydrolyzed wax esters 153 10,1 48 3,6
Free fatty alcohols 75 7,4 24 3,5
Free fatty acids 63 6,5 20 3,2
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The migration of the WE fraction on the HPTLC plate is as described by Henderson and Tocher [26] using this solvent system. In initial SPE experiments, we found that most of the NL were eluted with only 10 mL chloroform/isopropanol (2:1 v/v) and only a small fraction was obtained with an additional 10–20 mL (see Fig 1 in S1 File). However, to maximize the yield we eluted the NL with 30 mL. Similarly, we eluted the WE with 50 mL of heptane although the majority were eluted with the first 10 mL (see Fig 2 in S1 File).

Saponification of wax esters and isolation of the fatty acids and fatty alcohols

The wax esters were hydrolyzed following Christie and Han [25] but using NaOH instead of KOH. Hydrolyses with KOH formed a soap like layer between the water and heptane layer that made it more difficult to recover the upper heptane layer containing the free fatty acids and free fatty alcohols. The method we used here showed to be effective in hydrolyzing the wax esters. In Fig 2, there are two clear separate bands visible, one corresponding to the free fatty acids and one band just below representing the free fatty alcohols. The free fatty acids band appears to consist of two bands with slightly different migration and this is probably due to different chain length and/or degrees of unsaturation of the different fatty acids present in the oil. No band is visible on the level of the wax esters, indicating that all wax esters were hydrolyzed.

After saponification, the free fatty acids and fatty alcohols present were separated from each other using SPE as shown in Fig 1 and described in the Materials and Methods section. A large fraction of the free fatty alcohols was eluted with the first 10 mL chloroform/isopropanol (2:1 v/v). To extract all fatty alcohols, however 30 mL was needed (see Fig 3 in S1 File). From on average 322 mg Calanus oil a yield of 75 mg free fatty alcohols was obtained (Table 1).

After conditioning the column with 20 mL heptane, the free fatty acids were eluted with 50 mL diethyl ether/acetic acid (98:2 v/v). Most fatty acids are eluted with 40 ml heptane in which non are present in the first 20 mL (see Fig 4 in S1 File). From on average 322 mg Calanus oil, 63 mg free fatty acids could be recovered (Table 1). The HPTLC analysis clearly shows that the FAOH and FFA present in the hydrolyzed wax esters have been separated (Fig 2).

Composition of the fatty acids and fatty alcohols

Table 2 gives the fatty acid and fatty alcohol composition of the isolated wax esters and the isolated fatty acids and fatty alcohols after saponification. As expected, the results showed that almost equal proportions fatty acids (48,6%) and fatty alcohols (45,4%) were found in the wax esters. Almost 80% of the identified fatty alcohols were monounsaturated, with eicosenol (20:1n-9) and docosenol (22:1n-11) being the dominating species. Of the polyunsaturated fatty acids, stearidonic acids (18:4 n-3), eicosapentaenoic acid (20:5 n-3) and docosahexaenoic acid (22:6 n-3) constituted about 30% of the fatty acids in the wax esters (Table 2). These results are similar to previous reported values [8, 9]. However, the composition of lipid classes and fatty acids in C. finmarchicus depends on the season, life cycle stage and geographic location [6, 30, 31]. It is therefore no surprise that the composition of the fatty acids and fatty alcohols in the wax esters found in this study is not exactly similar to the composition of the oil produced by the industry [8, 9]. The results from the fatty acid composition in the intact wax esters and the free fatty acid isolated from the hydrolyzed wax esters indicate that some oxidation may have occurred in the PUFAs, since the relative amount is lower in the isolated fatty acids than in the wax esters. Consequently, the relative amount of SFA has increased. Some oxidation may also have occurred in the MUFAs, but the relative amount is similar both in the intact wax esters and isolated fatty acids.

Table 2. Relative composition of the fatty acids and fatty alcohols (%) in the isolated wax esters from Calanus oil and the isolated fatty acids and fatty alcohols fractions after saponification of the wax esters from the first isolation.

Fatty Acids Wax ester Isolated fatty acid Isolated fatty alcohol
14:0 9,8 19,7 n.d.
16:0 4,4 10,6 n.d.
18:0 n.d. 1,9 n.d.
16:1 n-7 2,6 5,2 n.d.
18:1 n-9 2,1 4,3 n.d.
20:1 n-7 0,9 n.d. 1,8
20:1 n-9 1,9 4,3 n.d.
20:1 n-11 0,4 0,8 n.d.
22:1 n-11 3,7 7,9 n.d.
18:2 n-6 0,6 1,2 n.d.
18:3 n-3 1,2 2,4 3,5
18:4 n-3 8,3 14,1 n.d.
20:3 n-6 1,5 0,8 2,6
20:4 n-3 0,8 1,3 n.d.
20:5 n-3 6,9 10,6 n.d.
22:5 n-3 n.d. 0,6 n.d.
22:6 n-3 3,5 4,5 n.d.
Σ SFA 14,2 32,2 n.d.
Σ MUFA 11,6 22,5 1,8
Σ PUFA 22,8 35,5 6,1
Σ Fatty acids 48,6 90,2 7,9
Fatty alcohols Wax ester Isolated fatty acid Isolated fatty alcohol
14:0 0,6 n.d. 1,2
16:0 4,9 n.d. 9,1
16:1 n-7 1,2 n.d. 2,2
18:1 n-9 1,8 n.d. 1,6
20:1 n-9 12,6 n.d. 24,9
22:1 n-9 1,8 n.d. 2,4
22:1 n-11 22,5 0,8 46,0
Σ Fatty alcohols 45,4 0,8 87,4
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Detection threshold is above 0,5%. n.d., not detected; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, poly unsaturated fatty acids.

The established methods to analyze the oil in C. finmarchicus [11–13], focus on the determination of the composition of the lipids, rather than on extracting and separating the fatty alcohols and fatty acids for further use. Several methods have been described to extract policosanols from their natural sources, as reviewed by Shen et al. [32]. These methods include solvent extraction, transesterification and molecular distillation, supercritical carbon dioxide extraction, ultrasonic-assisted extraction, and saponification. As mentioned in the introduction, the previous described methods are excellent to study the fatty acid and fatty alcohol composition in calanoid species. But due to the production of FAMEs they are less suitable for the isolation of native, unesterified, fatty acid and fatty alcohol. It is possible to saponify the FAMEs to get free fatty acids. However, as can be seen in Table 1, the hydrolysis of the wax esters leads to a substantial weight loss from 251 mg to 153 mg. This leads us to suggest that an additional saponification step would further reduce the yield of the free fatty acids extracted from Calanus oil. To our knowledge, this is the first report of a simple, yet time consuming, method of semi-preparative isolation of the wax esters and the hydrolyzed components of wax esters, leaving the separated fatty acids and fatty alcohol unaltered. This allows them to be used for subsequent biological experiments.

Supporting information

S1 Table. Weight and percentage of Calanus oil weight of the extracted lipid classes from the 3 individual extraction rounds.

(PDF)

Click here for additional data file. (97KB, pdf)
S1 File. Containing all supporting figures: Visualizations by means of TLC of the optimal elution volumes to extract the different lipid classes from Calanus oil.

(PDF)

Click here for additional data file. (130.3KB, pdf)
S1 Raw images

(PDF)

Click here for additional data file. (1.8MB, pdf)

Acknowledgments

Zooca®, Tromsø, Norway provided the oil used in the study.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

The publication charges for this article have been funded by a grant from the publication fund of UiT The Arctic University of Norway. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Rishiram Ramanan

7 Feb 2023

PONE-D-22-35050A simple method to isolate fatty acids and fatty alcohols from wax esters in a wax-ester rich marine oilPLOS ONE

Dear Dr. Schots,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. As you may see below, there are significant concerns raised by the reviewers, in particular, on replication, and comparison with existing methods. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Mar 24 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Rishiram Ramanan

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: N/A

Reviewer #3: N/A

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript presents a simplified method to isolate fatty acids and fatty alcohols from a wax ester obtained from a zooplankton rich in monounsaturated fatty acids using HPTLC. The method seems to be really simple and not expensive. The procedures were described step by step and can be useful for other researchers. However, there are some parts that should be revised.

In general, there are several minor typos in the text.

Line 107: Include the volume of wax esters in chloroform that resulted in 240 mg

line 110 : it was described that the sample was kept at 90oC/90 min. Usually, an antioxidant uses to be added to avoid oxidative reaction at this step of the analysis.

Lines 143-154: include the reference of this part.

Line 157: It is important to detail which sub-type of standard was used to identify the fatty acids and fatty alcohols, since the information viable in the company web site has not been enough.

What is Akvaplan-niva ?

Table 1: It will be expected to have at least a triplicate of the assay. Without any replication it is not possible to obtain an error estimative. It is interesting to suggest what type of lipids correspond to the difference between waxe esters (239 mg) and neutral lipids (266 mg), and also between neutral lipids and calanus oil (300 mg).

It is important to better discuss the advantages and limitations of this method compared with others.

The manuscript is interesting but the lack of replication is not expected in an article about methodology alternatives. In addition, it is also expected a comparison with at least one classical method.

Thus, it is necessary to include replicate and compare with results obtained from other method.

Reviewer #2: The article describes a method for isolating free fatty acids and free fatty alcohols from wax esters of calanus oil. Although the paper is written very well and the descriptions are clear and sound, I doubt that the level of novelty justifies publication. The steps involved (extraction, saponification, SPE, checked by TLC) are well-described procedures for separating lipid classes and can be found in classic textbooks on lipid analysis (e.g. W.W.Christie…).

Furthermore, if this paper is aiming to optimize such a procedure, I would expect some data on method development. The authors merely present data of the (hopefully) optimized method. For method development, it would be essential to know, how the targets were defined (purity, solvent efficiency) and how the optimum was approached.

The authors claim that their method works without noticeable oxidation contrary to previous methods. How did the authors check this? I assume, that any oxidized fatty acid or alcohol would be retained in the SPE and thus noted as a reduced recovery.

As a minor comment, I wonder why the authors did not try to separate FFA from fatty alcohols as a soap before re-acidifying, instead of a SPE, given the fact that both molecular classes have similar polarities. At least this should be discussed.

Reviewer #3: The authors describe the preparation of mixtures of fatty acids and fatty alcohol from waxes present in Calanus finmarchicus oil. They propose the use of SPE columns to purify and isolate the waxes, followed by a basic hydrolysis with ethanol and NaOH. The main concerns relate to the methodology used and some of the results described (see specific comments).

Specific comments:

1.-Lines 64-65.- To study the health effects …it is necessary to separate the fatty acids and fatty alcohols …..

Why is it necessary? Provide a reference to support this claim. In any case, is the effect due to the functional class of each compound (alcohol or carboxylic acid) or to the presence of double bonds in the alkyl chain of both classes of compounds?

2.-Lines 66-67.- To our knowledge … no method … to separate the fatty acids and

fatty alcohols … without the formation of FAMEs.

What is the problem related to the preparation of FAMEs, the separation of the fatty alcohols from FAMEs and, finally, the recovery of fatty acids by hydrolyzing FAMEs?

3.- Lines 80-82.- … Phosphoric acid 99,99%, Potassium bicarbonate …..Toluene

Chemical names should be written in lowercase unless they are located after a period.

4.- Lines 107-108.- …wax esters …. dissolved in …. 1 M ethanolic NaOH to a concentration of 5 mg/mL…

There is an excess of ethanol related to the amount of water present (needed for the hydrolysis), do fatty ethyl esters not appear during this reaction?

5.- Various lines ..... minutes...hours ….

Time units should be indicated according IS rules: min, h…

6.- Lines 185-187.- free fatty acids band appear to consist of two bands is probably due to different degrees of unsaturation….

The different chain length of each acid can also lead to this situation. Please add this to the manuscript.

7.- Lines 223-224.- Table 2

How was the initial content of fatty acids and fatty alcohols in the crude wax determined?

How do the authors explain the differences between SFA, MUFA and PUFA for the fatty acids present in the wax and free fatty acids isolated after the SPE process and further saponification?

How do the authors determine the relative content from fatty acids and fatty alcohols as a whole? Did they consider the different response factors of each in the GC?

What is the fatty acid indicated (0.8 relative content) in the fatty alcohol section of the table?

8.- Figure 1

Did all SPE used contain the same chromatographic phase as described in line 88 (5 g Mega Bond Elute aminopropyl)?

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Inar Castro Erger

Reviewer #2: No

Reviewer #3: No

**********

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PLoS One. 2023 May 12;18(5):e0285751. doi: 10.1371/journal.pone.0285751.r002

Author response to Decision Letter 0

21 Mar 2023

We would like to thank all reviewers for their constructive feedback. We have gone through each comment and have changed the manuscript accordingly

Please see below the comments given by the reviewers. Directly below each comment is our response. The indicated line number refer to the manuscript with track changes.

Reviewer #1:

The manuscript presents a simplified method to isolate fatty acids and fatty alcohols from a wax ester obtained from a zooplankton rich in monounsaturated fatty acids using HPTLC. The method seems to be really simple and not expensive. The procedures were described step by step and can be useful for other researchers. However, there are some parts that should be revised.

In general, there are several minor typos in the text.

We have gone through the manuscript and have corrected several typos.

Line 107: Include the volume of wax esters in chloroform that resulted in 240 mg

We have included the average weight of wax esters obtained from the three extraction rounds in line 119 and mentioned that they are dissolved in 5mL chloroform.

line 110: it was described that the sample was kept at 90oC/90 min. Usually, an antioxidant uses to be added to avoid oxidative reaction at this step of the analysis.

We did not add an antioxidant during the hydrolyzation step since Calanus oil contains naturally the antioxidant astaxanthin, which gives the oil its red color.

Lines 143-154: include the reference of this part.

We have added on line 177-180 that the composition analyses of the fatty acids and fatty alcohols was done by the company Akvaplan-niva following their inhouse protocol that is based on methods described by J. Folch, M. Lees and Sloane Stanley; J. Biol. Chem., 226, 497 (1957) and Advances in Lipid Methodology - One (edited by W.W. Christie, Oily Press, Ayr), pp. 1-17 (1992).

Line 157: It is important to detail which sub-type of standard was used to identify the fatty acids and fatty alcohols, since the information viable in the company web site has not been enough.

The FAME standards are still available on the 2021-2024 catalogue on the company’s website. http://www.nu-chekprep.com/CATALOG%202021-2024.pdf. The fatty alcohol standard GLC 621 is no longer produced as a combination of alcohols, but each alcohol is available individually. The composition of GLC 621 is now given on line 173-177.

What is Akvaplan-niva ?

We have added on line 178-179 that Akvaplan-niva is a Norwegian company based in Tromsø, Norway.

Table 1: It will be expected to have at least a triplicate of the assay. Without any replication it is not possible to obtain an error estimative.

We have now repeated the isolation two more times. The averages of the triplicate values are given in table 1, both in mg and percentage weight of the oil, with standard deviation. The individual values of each isolation are given in supplementary table 1.

It is interesting to suggest what type of lipids correspond to the difference between waxe esters (239 mg) and neutral lipids (266 mg), and also between neutral lipids and calanus oil (300 mg).

We thank the reviewer for her comment. We had touched upon this topic in lines 188-191 but have extended this in lines 191-196.

It is important to better discuss the advantages and limitations of this method compared with others.

We would again like to thank the reviewer for her comment. We have elaborated on the difference between the method we describe here and previously described methods (reference 10, 11, 12 and 13) and the advantages and limitations in the introduction in line 71-77 and in the results and discussion section in line 252-259.

The manuscript is interesting but the lack of replication is not expected in an article about methodology alternatives. In addition, it is also expected a comparison with at least one classical method.

Thus, it is necessary to include replicate and compare with results obtained from other method.

With the 2 extra extractions performed, the average results are presented in table 1, and individual results in supplementary table 1, and the changes we have made to the manuscript we hope we have answered to the reviewer’s constructive feedback.

Reviewer #2:

The article describes a method for isolating free fatty acids and free fatty alcohols from wax esters of calanus oil. Although the paper is written very well and the descriptions are clear and sound, I doubt that the level of novelty justifies publication. The steps involved (extraction, saponification, SPE, checked by TLC) are well-described procedures for separating lipid classes and can be found in classic textbooks on lipid analysis (e.g. W.W.Christie…).

We thank the reviewer for their comment. It is indeed true that the methods (SPE and TLC) used here are well-described procedures. We are, however, not aware of any publications describing a simple semi-preparative method for isolation of wax esters present in a wax-ester rich marine oil, and then separating the fatty alcohols and fatty acids constituting these wax esters.

The novelty lies not in the method itself but in the application. As written in the introduction, on line 36-50, there is an increasing need for omega-3 fatty acids, and Calanus oil is a relatively new source of these lipids. In the last couple of years more and more studies on Calanus oil, and its potential health effects, have been done in both animals and humans (line 56-61). To study, in vitro, what part of the oil is responsible for what health effect it is necessary to separate the fatty acids from the fatty alcohols since the cells in our body will not be exposed to intact wax esters but rather to the separate fractions after hydrolyzes in the gut (line 62-69). To our knowledge we are the only lab today that is studying the individual effect of fatty acids and fatty alcohol. But with the growing demand for omega 3 fatty acids and the scientific interest in this oil by other labs worldwide, it can be expected that other labs will perform in vitro studies as well. It will then be of importance that the fatty acid and fatty alcohol fractions will be extracted in the same way to reduce potential confounding effects, through the method we describe here. We therefor do believe this paper justifies publication and we hope the reviewer agrees with us on that.

Furthermore, if this paper is aiming to optimize such a procedure, I would expect some data on method development. The authors merely present data of the (hopefully) optimized method. For method development, it would be essential to know, how the targets were defined (purity, solvent efficiency) and how the optimum was approached.

We again thank the reviewer for their comment. Our main aim was to develop a method that would allow us to obtain the native, non-esterified fatty acids and fatty alcohols from wax esters in Calanus oil. As we show in figure 2, the lipid fractions we have obtained from the hydrolyzed wax esters appear pure. Regarding the optimization of our method, we have now added a supportive data file with TLC analyses from each extraction step described in figure 1, where we qualitatively show the presence of lipids eluted with different volumes of solvent to justify the volume choices in the method described here.

The authors claim that their method works without noticeable oxidation contrary to previous methods. How did the authors check this? I assume, that any oxidized fatty acid or alcohol would be retained in the SPE and thus noted as a reduced recovery.

We do not claim that the method works without noticeable oxidation of fatty acids and fatty alcohols. We agree with the reviewer that if this occurs, oxidized fatty acids and fatty alcohols would be retained in the SPE and result in lower yields. We do, however, claim that we do not oxidize the fatty alcohol to their corresponding fatty acids during the isolation method, which allows us to use the fatty alcohols for further cell cultures. (line 29-33)

As a minor comment, I wonder why the authors did not try to separate FFA from fatty alcohols as a soap before re-acidifying, instead of a SPE, given the fact that both molecular classes have similar polarities. At least this should be discussed.

We have indeed tried to precipitate the FFA as a sodium salt and recover the soluble FaOHs, however we did not get a clear separation as we did with SPE. We have addressed this point now in line 135-138.

Reviewer #3:

The authors describe the preparation of mixtures of fatty acids and fatty alcohol from waxes present in Calanus finmarchicus oil. They propose the use of SPE columns to purify and isolate the waxes, followed by a basic hydrolysis with ethanol and NaOH. The main concerns relate to the methodology used and some of the results described (see specific comments).

Specific comments:

1.-Lines 64-65.- To study the health effects …it is necessary to separate the fatty acids and fatty alcohols …..

Why is it necessary? Provide a reference to support this claim. In any case, is the effect due to the functional class of each compound (alcohol or carboxylic acid) or to the presence of double bonds in the alkyl chain of both classes of compounds?

As mentioned in line 62-69, it is necessary to separate the fatty acids from the fatty alcohols to study if, and how, these individual lipid classes, derived from wax esters, influence human health. In a previous publication, reference 24, (Schots PC, Pedersen AM, Eilertsen K-E, Olsen RL, Larsen TS. Possible Health Effects of a Wax Ester Rich Marine Oil. Frontiers in Pharmacology. 2020;11(961).) we have described the possible health effects of the different fatty acids and fatty alcohols in general that are present in Calanus oil.

2.-Lines 66-67.- To our knowledge … no method … to separate the fatty acids and

fatty alcohols … without the formation of FAMEs.

What is the problem related to the preparation of FAMEs, the separation of the fatty alcohols from FAMEs and, finally, the recovery of fatty acids by hydrolyzing FAMEs?

We thank the reviewer for their comment. We have now explained on line 255-259 that the hydrolyzes of the wax esters led to a loss of material (as determined by weight). An additional hydrolyzes of the FAMES would further reduce the yield of the fatty acids.

3.- Lines 80-82.- … Phosphoric acid 99,99%, Potassium bicarbonate …..Toluene

Chemical names should be written in lowercase unless they are located after a period.

We thank the reviewer and have changed the manuscript accordingly.

4.- Lines 107-108.- …wax esters …. dissolved in …. 1 M ethanolic NaOH to a concentration of 5 mg/mL…

There is an excess of ethanol related to the amount of water present (needed for the hydrolysis), do fatty ethyl esters not appear during this reaction?

We believe they do not since this would have been visible on the TLC (figure 2)

5.- Various lines ..... minutes...hours ….

Time units should be indicated according IS rules: min, h…

We thank the reviewer and have changed the manuscript accordingly.

6.- Lines 185-187.- free fatty acids band appear to consist of two bands is probably due to different degrees of unsaturation….

The different chain length of each acid can also lead to this situation. Please add this to the manuscript.

We thank the reviewer and have changed the manuscript accordingly (line218).

7.- Lines 223-224.- Table 2

How was the initial content of fatty acids and fatty alcohols in the crude wax determined?

How do the authors explain the differences between SFA, MUFA and PUFA for the fatty acids present in the wax and free fatty acids isolated after the SPE process and further saponification?

How do the authors determine the relative content from fatty acids and fatty alcohols as a whole? Did they consider the different response factors of each in the GC?

What is the fatty acid indicated (0.8 relative content) in the fatty alcohol section of the table?

The fatty acid and fatty alcohol content in the wax esters and isolated lipid fractions was determined by the company Akvaplan-niva via an inhouse protocol as explained on line 161-180.

Table 2 give values in percentage. The values of the different fatty acids in the isolated fatty acid fraction are about twice as high as those in the wax ester sample. This is because in the wax ester sample only half of the sample consists of fatty acids (48,6%), and the other half are fatty alcohols (45,4%).

The fatty alcohol that is present with a relative content of 0,8 in the isolated fatty acid fraction is 22:1 n-11 (docosenol). Minor amounts of a few fatty acids are apparently detected in the fatty alcohol fraction, 1,8% 20:1 n-7 and 3,5% 18-3 n-3 and 2,6% 20:3 n-6.

8.- Figure 1

Did all SPE used contain the same chromatographic phase as described in line 88 (5 g Mega Bond Elute aminopropyl)?

Yes, the same column (5 g Mega Bond Elute aminopropyl) was used for all different SPE steps in this procedure.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file. (23.8KB, docx)

Decision Letter 1

Rishiram Ramanan

13 Apr 2023

PONE-D-22-35050R1A simple method to isolate fatty acids and fatty alcohols from wax esters in a wax-ester rich marine oilPLOS ONE

Dear Dr. Schots,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. The reviewers have largely agreed to accept the manuscript for publication except for a few minor edits. I invite the authors to submit the revised manuscript after addressing the reviewers' suggestions.

Please submit your revised manuscript by May 28 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Rishiram Ramanan

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

Reviewer #3: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I only suggest two minor revisions.

Line 60: making

Line 122: It is important to include the authors answer in the text. For example: “No antioxidants were added during the wax esters hydrolysis because Calanus oil contains naturally the antioxidant astaxanthin”.

Reviewer #2: In my view, the authors have clarified all points satisfactorily. There is just one point that popped in to my eyes: In the description of figures, it should read."...from bottom to top.." - not top to bottom (all figures and supportive figures)

Reviewer #3: I consider that the authors have only partially addressed my previous comments. I attach a document with my considerations.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Inar Alves Castro

Reviewer #2: No

Reviewer #3: No

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PLoS One. 2023 May 12;18(5):e0285751. doi: 10.1371/journal.pone.0285751.r004

Author response to Decision Letter 1

21 Apr 2023

We would like to thank all reviewers again for their constructive feedback and have changed the manuscript accordingly.

Please see below in red our response to each point raised by the reviewers. The indicated line number refer to the manuscript with track changes. In addition, some minor linguistic changes have been made on line 44, 69, 73, 109, 171, 173 and 179.

Reviewer #1:

Line 60: making:

We have changed the sentence on line 60, making it one sentence instead of two.

Line 122: It is important to include the authors answer in the text. For example: “No antioxidants were added during the wax esters hydrolysis because Calanus oil contains naturally the antioxidant astaxanthin”.

We have added the sentence as suggested by the reviewer on line 122-123

Reviewer #2:

Reviewer #2: In my view, the authors have clarified all points satisfactorily. There is just one point that popped in to my eyes: In the description of figures, it should read."...from bottom to top.." - not top to bottom (all figures and supportive figures).

This is indeed correct, and we thank the reviewer for pointing this out. We have changed the figure legend as the reviewer mentions in the figure text of the raw images. In the figure text in the manuscript and the supplementary data file the different lipid classes are identified with a letter and we therefore did not add the phrase “from bottom to top”.

Reviewer #3

Line 138 …..n in our set-up using NaOH as a salt.

NaOH cannot be defined as a salt from a chemical point of view. Of course, it can modify the ionic strength of a solution, but as a base. Please amend this sentence.

This is indeed correct, and we have changed the sentence on line 136-137 accordingly.

2.- Table 2

The authors do not explain the differences between SFA, MUFA and PUFA for the fatty acids present in the wax and the free fatty acids isolated after the SPE process and further saponification. According to the percentages indicated in Table 2, the percentages referring to 90.2 % of the total FFA isolated should be 26.35, 21.5 and 42.31 for SFA, MUFA and PUFA, respectively considering the initial percentages of fatty acids found in the waxes. However, the percentages found are 32.2, 22.5 and 35.5, which show an increase in SFA and a decrease in MUFA and, even more, in PUFA. In my opinion, this clearly indicates a higher loss of unsaturated versus saturated fatty acid during the sample processing. The authors should clearly explain this in the manuscript.

Also this is correct and we thank the reviewer for pointing this out. We have added a part on line 243-248 to explain this.

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Decision Letter 2

Rishiram Ramanan

2 May 2023

A simple method to isolate fatty acids and fatty alcohols from wax esters in a wax-ester rich marine oil

PONE-D-22-35050R2

Dear Dr. Schots,

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Kind regards,

Rishiram Ramanan

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Acceptance letter

Rishiram Ramanan

5 May 2023

PONE-D-22-35050R2

A simple method to isolate fatty acids and fatty alcohols from wax esters in a wax-ester rich marine oil

Dear Dr. Schots:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Rishiram Ramanan

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Table. Weight and percentage of Calanus oil weight of the extracted lipid classes from the 3 individual extraction rounds.

    (PDF)

    Click here for additional data file. (97KB, pdf)
    S1 File. Containing all supporting figures: Visualizations by means of TLC of the optimal elution volumes to extract the different lipid classes from Calanus oil.

    (PDF)

    Click here for additional data file. (130.3KB, pdf)
    S1 Raw images

    (PDF)

    Click here for additional data file. (1.8MB, pdf)
    Attachment

    Submitted filename: Response to Reviewers.docx

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    Attachment

    Submitted filename: comments.docx

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    Attachment

    Submitted filename: Response to reviewers.docx

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    Data Availability Statement

    All relevant data are within the paper and its Supporting Information files.

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