TITLE: Estimate phospholipids from microalgae
AUTHORS: Ying-Yu Hu, Zoe V. Finkel

[DESCRIPTION]
Here we describe a protocol to estimate phospholipids from microalgae. 

After extracting and measuring the total lipids from microalgae, the remaining lipid extract is dried using a nitrogen flow, followed by drying with magnesium sulfate at 90°C. However, it has been observed that traditional dry combustion at 500°C only decomposes approximately 50% of phospholipids (Hu et al., 2022). To achieve complete conversion of phospholipids to pyrophosphate, a temperature of around 800°C is required, but such high temperatures cannot be used with glassware. As the acid digestion method involves using only 500 µL of 0.2 M HCl, which must be placed in tightly capped glass vials to prevent concentration changes due to evaporation, combustion must be carried out using glassware instead of crucibles. It should be noted that the recovery rate of phospholipids is around 80% when combusted at 650°C, but this recovery rate is consistent, making the use of glass vials applicable. Therefore, we recommend using 650°C to combust phospholipids and using 80% to correct the final results.

The resulting ash is digested using 0.5 mL of 0.2 M HCl for 30 minutes at 90°C. After digestion, the resulting orthophosphate is detected by mixing the sample with a combination of molybdate and ascorbic acid to produce molybdenum blue, as described in Chen's work (1956).

 

 







[STEPS]
SECTION: Prepare phospholipids sample
1. Dry remaining organic phase extract of total lipids at 37 °C under a stream of N2 gas (<2 psi)
SECTION: High temperature dry combustion
3. Use diamond pen to engrave the sample vials with numbers. Log number and sample code.
SECTION: High temperature dry combustion
4.  0.17 M MgSO4 reagent:

Dissolve 1.023 g MgSO4  in 50 mL MilliQ water 
 
SECTION: High temperature dry combustion
5. Add 200 µL 0.17 M MgSO­4 to the dry extract.
 

SECTION: High temperature dry combustion
6. Cover the uncapped vials with foil and place in the oven  at 90 °C  until samples are completely dry. 
 

 


SECTION: High temperature dry combustion
7. Combust dried samples at 650 °C for  540 min
 

 

SECTION: High temperature dry combustion
8. Allow samples to gradually cool down in the muffle furnace.
SECTION: Digestion
9. 0.2 M HCl reagent:

In a reagent bottle, dissolve one part of 12 N  HCl in 59 parts of MilliQ water 
 

 


SECTION: Digestion
10. Preheat oven to 90 °C 
SECTION: Digestion
11. Add 0.5 mL 0.2 M HCl to each vial. 
SECTION: Digestion
12. Tightly cap the vial and vortex.
SECTION: Digestion
13. Place vials in the oven for 30 min 
SECTION: Digestion
14. Cool samples down to Room temperature 
SECTION: Preparing standard working solutions
15. Preheat shaker/incubator to 37 °C 
 



SECTION: Preparing standard working solutions
17. Standard working solution
 

SECTION: Preparing standard working solutions
18. Transfer 500 µL of each standard working solution to 2 mL microtube. 
SECTION: Preparing working reagents
19. All reagents are freshly prepared before colorimetric measurement.
SECTION: Preparing working reagents
22.  6 N (3 M) sulfuric acid reagent:

Carefully add 1 part 18 M concentrated sulfuric acid into 5 part MilliQ water   
SECTION: Preparing working reagents
20.  2.5 % ammonium molybdate reagent:

Weigh 0.25 g ammonium molybdate in a Falcon tube and top to 10 g with MilliQ water.
Cap and shake until totally dissolved.  
 
SECTION: Preparing working reagents
21. 10 % ascorbic acid reagent (avoid light exposure):

Weigh 1 g ascorbic acid in a Falcon tube and top to 10 g with MilliQ water;
Cap and shake until all dissolved. 

 

SECTION: Preparing working reagents
23. Calculate the volume of molybdate-ascorbic reagent:
Total volume of reagent_mL = (0.5 mL) X (#standard working solution + #samples + #blanks)
SECTION: Preparing working reagents
24. Mix the reagents into Falcon tube:
 

SECTION: Colorimetric measurement
25. Add 500 µL reagent to each standard, sample (in the vial) and blank, starting from blanks, including blank for standards and blank for samples.
 

 


SECTION: Colorimetric measurement
26. Vortex.
SECTION: Colorimetric measurement
27. Incubate at 37 °C  for 180 min  while shaking at 150 rpm
SECTION: Colorimetric measurement
28. Load microplate with 250 ul reactant from each tube, duplicate.
  
SECTION: Colorimetric measurement
29. Read plate in microplate reader
 

 

SECTION: Calculation
30. Subtract the average absorbance at 820 nm of the blank standard replicates from the absorbance at 820 nm of all other standard working solutions.
SECTION: Calculation
31. Subtract the average absorbance at 820 nm of the blank sample (i.e. blank filter) replicates from the absorbance at 820 nm of all other individual samples.
SECTION: Calculation
32. Prepare a standard curve by plotting the average blank-corrected 820 nm absorbance for each standard working solution versus its concentration in uM.
Molar Mass of KH2PO4: 136.086 g/mol
SECTION: Calculation
33. Use the standard curve to determine the orthophosphate concentration of each unknown sample by using its blank-corrected 820 nm absorbance.
SECTION: Calculation
34. (Pmeasured)_umol/sample = (orthophosphate)_uM X (V_HCl)_mL X (0.001)
(Pcorrected)_umol/sample = (Pmeasured) / 0.8
Where, 0.8 is the average recovery of phospholipids after a high temperature dry combustion at 650 °C .  
SECTION: Calculation
35. (Phospholipids)_ug/sample = (Pcorrected)X30.97/(0.01X4.3)
SECTION: Phosphate primary standard
2.5. Transfer standard stock solution into a 1 L bottle and store in the fridge.
 


SECTION: Phosphate primary standard
2.4. Dissolve around0.136 g dried KH2PO4 in 1 LMilliQ water.
Use 1 L volumetric flask
Take notes of the actual weight of KH2PO4 for final concentration of standard stock solution
SECTION: Phosphate primary standard
2.3. Move KH2PO4 into a vacuum desiccator, allow KH2PO4 to cool to room temperature
SECTION: Phosphate primary standard
2.2. Place the beaker into an oven, dry KH2PO4 at 110 °C for at least 120 min 
SECTION: Phosphate primary standard
2.1. Transfer about 1 g KH2PO4 into a beaker, cover the beaker with foil
SECTION: Phosphate primary standard
2. KH2PO4 primary standard stock solution (≈ 1 mM)  
SECTION: Preparing standard working solutions
16. Standard working solutions and reagents can be prepared during sample digestion.