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Revista Romn de Medicin de Laborator Vol. 23, Nr.

4, Decembrie, 2015 371

Review

DOI: 10.1515/rrlm-2015-0035

Recent developments in fatty acids profile determination in


biological samples - a review
Progrese recente n determinarea acizilor grai din probe
biologice - un review

Ioana Tiuca*, Katalin Nagy, Radu Oprean


University of Medicine and Pharmacy I. Hatieganu, Cluj-Napoca, Romania

Abstract
The present paper is a literature review of the recent years dealing with the most important separation tech-
niques of fatty acids in biological samples. Our aim was to make a synthesis of the analytical methods used, to note
the most used ones, but also to mention other methods that are less utilized, which can have important advantages
(such as less time consuming, greener reagents, etc.). Gas-chromatographic separation methods were described
and compared to liquid chromatographic separations of fatty acids in different types of biological samples. In the
same time, the importance of determining fatty acids profiles in biological samples was revealed, pointing out
the possible implications in diagnostics of different types of disorders or remarking different profiles compared to
healthy states.
Keywords: fatty acids, gas-chromatography, liquid-chromatography, biological samples

Rezumat
Prezenta lucrare reprezint un review al literaturii ultimilor ani n legtur cu cele mai importante tehnici de
separare ale acizilor grai din probe biologice. Scopul nostru a fost de a realiza o sintez a metodelor utilizate, de a
le nota pe cele mai utilizate, dar, n acelai timp, de a le puncta i pe celelalte mai puin utilizate, care ar putea avea
importante avantaje (cum ar fi un consum mai mic de timp de analiz, utilizare de reactivi verzi, etc.). Am descris
i comparat metodele de separare gaz-cromatografice cu separrile lichid-cromatografice ale acizilor grai pentru
diferite tipuri de probe biologice. n acelai timp, am marcat importana determinrii profilelor acizilor grai n
probe biologice, artnd posibile implicaii n diagnosticul diferitelor tipuri de boli sau remarcnd profile diferite
la bolnavi comparativ cu voluntarii sntoi.
Cuvinte cheie: acizi grai, cromatografie de gaze, cromatografie de lichide, probe biologice

Received: 23rd June 2015; Accepted: 13th September 2015; Published: 23rd September 2015

*Corresponding author: Ioana Tiuca, University of Medicine and Pharmacy I. Hatieganu Cluj-Napoca, Cluj,
Romania, e-mail: tiuca.daria@umfcluj.ro
372 Revista Romn de Medicin de Laborator Vol. 23, Nr. 4, Decembrie, 2015

1. Introduction matographic (13,58,1122), among which


most developed methods use a mass-spectrom-
In the era of search for health and well-be-
eter detector. However, liquid-chromatography
ing, fatty acids have gained an important role in
(4,17,2331) has been presented by several au-
human health, having a significant effect in the
thors and also, there are few articles which deal
occurrence and prevention of cardiovascular dis-
with other techniques such as capillary electro-
eases (1). In the same time, fatty acids profile
phoresis or infrared (IR) and Raman spectrosco-
has been reported to significantly influence or to
py (16,31,32).
be modified in a considerable way in neurologic
The aim of this study was to sum up a liter-
or psychiatric diseases (2,3), endocrine disorders
ature synthesis of the most important separation
(4) or in metabolic syndrome related illnesses,
techniques of fatty acids in bio-samples, reveal-
such as diabetes mellitus (1,5,6).
ing their importance in diagnosis of different dis-
Likewise, fatty acids profiles in ingested
foods (1) or beverages (7,8) can influence the orders or noticing how their profiles can change
state of health of a certain individual. It is well or can be improved.
known that fish oil administered in infancy can
reduce risks of allergies (9) and also, due to its 2. Gas chromatography (GC)
high content of n-3 PUFA, can reduce the in- Gas chromatography is the first choice meth-
flammation biomarkers in blood (10). od for the detection and quantification of fatty
For these reasons, we consider that profiling acids in all types of samples. The high efficiency,
fatty acids in blood or other biological samples high selectivity, relative low analysis times and
can predict and even prevent an eventual state of
low volume consumption make it a first selection
health/illness that could occur, thus it is import-
method. However, the need for derivatization in
ant to have easy, fast and cost effective methods
case of fatty acids analysis can cause troubles in
to be able to determine the profile of fatty acids
terms of overall reproducibility.
with short analysis times, high efficiency and se-
lectivity.
2.1. Sample preparation extraction of FA
Gas-chromatography and high-performance
and derivatization
liquid chromatography are the main separation
In the majority of cases, the solvents used for
techniques used nowadays. In the separation of
fatty acids, they can be used as complementary the extraction of fatty acids from biological sam-
techniques: a gas-chromatographic separation ples can be either hexane (5,6,8,11,13,18,21,22)
is very efficient and uses very small volumes or CHCl3/methanol (7,17,1820). Before the
of substances, but it needs derivatization, while derivatization of FA to their methyl esters
a liquid chromatographic separation does not (FAME), a saponification can be done, which
necessarily need derivatization, but uses high- separates FA from other lipids. For this purpose,
er volumes of solvents and does not give such NaOH or KOH in methanol can be used, then the
good separations, in terms of efficiency and res- solution is acidulated (5,11,16). Also, a thin layer
olution. Moreover, if it is coupled to a UV-Vis chromatographic separation of fatty acids from
detector, fatty acids detection can cause troubles, the other lipids extracted can be done (1,14).
because they hardly absorb in this spectral range. The transesterification to FAME is needed
The methods described in the literature for to increase the volatility of FA, which would
the separation and/or quantification of fatty ac- be separated through the gas-chromatographic
ids in different samples are mostly gas-chro- column. This esterification is usually done us-
Revista Romn de Medicin de Laborator Vol. 23, Nr. 4, Decembrie, 2015 373

ing methanolic HCl or methanolic BF3. The last phase would be the wider range of temperature
one, BF3, seems to be preferred to be used as limits. With very few exceptions (8,16,18), the
a catalyst for the methyl-esterification reaction, mobile phase used for the separation of FAME is
at 100C, for 1 h (6,13,1719). However, acid helium. Likewise, the detector usually coupled
catalyzed reactions are used and H2SO4 has also with a GC system is a mass spectrometer detec-
been considered as an option for the transester- tor, but there are studies which are using the flame
ification of FA to FAME (5,19). The research ionization detector (1,68,15,16,18,20). The MS
group of Schmitz (21,22) have used an unusual detector is preferred due to its high selectivity
derivatization agent, which is acetyl-chloride, towards the analyte and due to the possibility to
the derivatization being done at 20C, at 150 precisely identify the analytes in a spectral mass
rpm, overnight. At the same time, for FA dosing, database (NIST, Wiley etc.). Sometimes, GCx-
C17:0 can be added to the sample solution as an GC can be used, for a better resolution of the
internal standard, before the derivatization pro- analytes (11,12).
cess. However, any other FA with odd number of The main separation conditions concerning
carbon atoms in the molecule can be used as an the GC analysis of fatty acids in bio-samples
internal standard, since they are not synthesized have been presented in Table I. .
in the human body.
After derivatization, FAME are usually ex- 3. Liquid chromatography (LC)
tracted in hexane and the solution is concentrat-
ed or even the solvent is completely evaporated, Liquid chromatographic methods have been
when a solution of p-tert-butyl-phenol in hexane, developed because the extraction and derivat-
as an antioxidant, can be added to the residue. ization processes used in GC can often be long
Dichloromethane (DCM) has also been used for and time consuming and can affect the reproduc-
FAME extraction (16). ibility of the method. At the same time, liquid
Main extraction, derivatization and separa- chromatographic methods can be developed for
tion procedures are listed in the Table I. preparative purposes.

2.2. FAME separation 3.1. Sample preparation


Fatty acids and their methyl-esters are highly HPLC with fluorescence detection is a good
non-polar compounds. In spite of this fact, most method used for separation and quantification of
authors have chosen to use for their separation, fatty acids. It offers a high sensitivity and spec-
stationary phases presenting medium to high po- ificity, but it requires a phase of derivatization,
larity, such as Innowax columns (highly polar which can be time-consuming and, in some
polyethylene glycol phase) or BPX-70 column (a cases, the use of toxic reagents, such as pyri-
stationary phase dedicated to FAME separation). dine (23,27), cannot be avoided. By using this
These stationary phases present the advantage of method, FA were separated from saliva, serum
giving a better resolution and efficacy, but they samples, mice liver and different types of oils
are less thermally stable. However, there are (23,27). Analyzing several fluorescent labeling
some studies which deal with the separation of reagents used for the derivatization of carbox-
fatty acids (derivatized to FAME) on stationary ylic acids, difluoro-boraindacene (BODIPY)
phases of low polarity, such as VF-5ms (5%-phe- have shown the best fluorescent properties, and
nyl-arylene-95%-dimethylpolysiloxane phase). by taking functional groups in consideration,
The advantage of using a non-polar stationary hydrazine and amine have higher reactivity and
Table I. Derivatization, extraction of FAME and gas-chromatographic separation parameters for
374
the analysis of fatty acids in biological samples.
Capillary Carrier Temperature Ref.
Analyzed sample Derivatization agent(s) Extraction solvent(s) Detector
column(s) gas gradient (C)
Rxi-1msx
INS-1 cells hexane He 40-160-260 TOF-MS (11)
Rxi-17Sil-MS
colorectal cancer cells MeOH/BF3 100C hexane DB-WAX He 120-205-250 MS (13)
plasma MeOH/BF3 100C CHCl3/MeOH, hexane Omegawax 250 He 180-206-240 MS (14)
MeOH/HCl,
maternal milk,powder milk CHCl3/MeOH, hexane Ultra-Alloy 5 He FID (7)
75C, over night
MeOH/BF3
erythrocyte membranes CHCl3/MeOH, hexane DB-23 He 140-240 FID (15)
100C
MeOH/H2SO4 KOH/MeOH (sapon.)
plasma VF-5ms He 90-212-219-290 MS/MS (5)
70C hexane
plasma MeOH/BF3 100C CHCl3/MeOH, DCM DB-225 N2 FID (16)
MeOH/HCl DB-WAX
human hair MS (17)
DMOX 150C DB-WAXetr
MeOH/NaOH DB-225ms
plasma erythrocytes hexane H2 FID (18)
100C, 5 min CP-select
MSTFA CHCl3/MeOH/H2O 60-125-270
colon tissue DB-1 x Rxi-17 He TOF-MS (12)
70C, 30 min toluene (+100)
MeOH/H2SO4
serum etyl acetate, hexane DB-WAX He 50-200-220 MS (19)
62C, 2 h
MeOH/HCl
serum/ plasma hexane SP 2560 He 50-190-230 FID (6)
90C, 4 h
heptane/dietylether/acetic
plasma BF3/MeOH 100C, 1 h VF-23ms He 50-170-212 FID (1)
acid, hexane

milk, oil,animal serum various RTx-65TG He 120-220-350 FID (20)


Revista Romn de Medicin de Laborator Vol. 23, Nr. 4, Decembrie, 2015

MeOH/HCl
human milk hexane CP-Sil 88 H2 60-165-195-215 FID (8)
100C, 1 h
plasma,cell homogenate MeOH/AcCl hexane BPX-70 He 50-155-210-250 MS (21)
MeOH/AcCl
plasma/ serum hexane BPX-70 He 50-155-210-250 MS (22)
20C, 150 rpm
MeOH/KOH
blood hexane Elite-1 GC He 100-140-240 MS (34)
50C
Revista Romn de Medicin de Laborator Vol. 23, Nr. 4, Decembrie, 2015 375

milder derivatization conditions. Combining 3.2. LC separation of FA


hydrazine and BODIPY, a new derivatization High or ultra-performance liquid chromato-
reagent has been developed (27), and by using graphic methods have been developed for the
a primary aliphatic amine as the labeling group separation of fatty acids in reversed-phase mode,
and BODIPY as the fluorophore, another, very due to the low polarity of their molecules. The
similar derivatization agent has been developed most used stationary phase has been C18, in dif-
(23). The efficiency of the derivatization is in- ferent isocratic or gradient elution types of mo-
fluenced by five main factors: the amount of la- bile phases, the gradient elution being preferred
beling reagent, other reagents dosage, reaction (Table II). In recent years, we have noted a pref-
time and temperature. It has been shown that the erence towards the use of a mass spectrometer as
dosage of the reagent affects the efficiency of the a detector, this being a powerful tool in precisely
derivatization more considerably than the other identifying the analytes, offering excellent de-
factors (23,27). tection limits, good selectivity and, in the same
Not only human serum can be analyzed by time, being able to be applied to a broad range of
using a HPLC method, but different lipid species compounds.
were separated from human meibum samples. In FA were separated from saliva, serum sam-
this case lipids were dissolved in a chloroform/ ples or different types of oil by using a HPLC
methanol mixture, and the extracts were dried by system with a fluorescence detector (490/510 nm)
using a centrifugal concentrator (25). and a reversed-phase C18 column with a gradient
Oxygenated metabolites of PUFAs (30) and elution. The most frequent eluents were metha-
fatty acid ethanolamides (FAE) (24) can also be nol and double-distilled water (27), or methanol
separated by using the HPLC-MS/MS method, and citrate buffer (23). The methanol content has
preceded by a solid-phase extraction. The ex- to be lower than 65% and the pH between 6-7.5,
traction and/or a pre-concentration step is need- otherwise the peaks of the acid derivatives and
ed because the oxygenated PUFA metabolites the reagent become overlapped (27).
show low physiological levels and a large num- The same detector was used for the identi-
ber of isomers, which can harden the analytical fication and separation of FAME after the deri-
determination (30). Because of their low concen- vatization process. This methyl derivate can be
tration, the identification and separation of FAE separated by a reversed-phase high performance
remains problematic (24). Dasilva and co-work- liquid-chromatography (RP-HPLC) by using a
ers compared two different cartridges, 60 mg C18 column and a gradient elution. Peaks were
Oasis-HLB and 100 mg C18 and also analyzed identified by UV detection at 205 nm (28).
the effects of the elution solvent, sample pH FA from blood samples can also be separat-
and temperature. By using HLB cartridges bet- ed and quantified by using an optimized multiple
ter recovery results were obtained (70 to 98%), reaction monitoring (MRM) method with the us-
than in the case of the C18 sorbent (42 to 82%). age of UPLC coupled with tandem mass spec-
Other studies showed that the highest extraction trometry. Chromatography separation was car-
efficiency was obtained for ALEA, while the ex- ried out by using a non-polar column and a gra-
traction efficiencies for AEA, DHEA and LEA dient elution. Gradient program was solvent B
were lower. Liquid-phase extraction (LPE) can (acetonitrile), 70% (0 min), 90% (3 min), 100%
be used in the case of tissue samples: liver or (3.01-4 min) and 70% (4.01-8 min). The mobile
small intestine, but the FAE showed a less ef- phase was carried out by using acetonitrile and
ficient extraction compared to SPE results (24). ammonium acetate (26,29).
376 Revista Romn de Medicin de Laborator Vol. 23, Nr. 4, Decembrie, 2015

Table II. Separation conditions of FA in LC


Stationary Elution
Sample type Pre-treatment Mobile phase Detector Ref.
phase type
A: isopropanol:
isopropanol ACN:n-hexane (55:45:15) isocratic
serum C18 MS/MS (35)
extraction B: 0.1 mol/L ammonium formiate A:B (95:5)
methanolic solution
human serum
highly fluorescence
(healthy, pan-
fluorescent A: 15mM citrate buffer detector
creatic cancer) C18 gradient (23)
labeling reagent B: MeOH (490/510
mice liver
(derivatization) nm)
tissue
plasma
(human,
A: 0.1% formic acid in H2O
hamster) SPE, LPE C18 gradient MS/MS (24)
B: 0.1% formic acid in ACN
tissue
(hamster)
A: 0.1% ammonium acetate in H2O
CHCl3/MeOH
meibum C18 B: 0.1% ammonium acetate in gradient Orbitrap (25)
extraction
MeOH
A: ammonium acetate 10mM in H2O
blood hexane extraction C18 gradient ESI-MS (26)
B: ACN

fluorescence
saliva fluorescent
A: MeOH detector
(smoker, derivatization C18 gradient (27)
B: H2O (490/510
non-smoker) reagent
nm)

fluorescence
A: MeOH:H2O (50:50) + 0.1% for-
DMOX detector
human hair C18 mic acid gradient (17)
derivatization (350/450
B: MeOH + 0.1% formic acid
nm)
cell cultures FAME A: H2O +0.02% fosforic acid UV (205
C18 gradient (28)
(cystic fibrosis) derivatization B: ACN nm)
plasma (sickle A: ammonium acetate 10mM in H2O
hexane extraction C18 gradient ESI-MS (29)
cell disease) B: ACN
A: H2O +0.02% formic acid QqQ-MS
plasma (rat) SPE C18 gradient (30)
B: MeOH +0.02% formic acid LIT-MS
A: H2O + 0.1% trifluoracetic acid
plasma LPE C18 gradient MS (31)
B: ACN + 0.1% trifluoracetic acid
A: ACN
plasma SPE C18 gradient MS/MS (36)
B: H2O
A: ACN:MeOH: H20 (19:19:2)+
CHCl3/MeOH 0.1% acetic acid+ 0.028% NH3
cell culture C18 gradient ESI-MS (4)
extraction B: isopropanol+ 0.1% acetic
acid+0.028% NH3
Revista Romn de Medicin de Laborator Vol. 23, Nr. 4, Decembrie, 2015 377

Reversed phase liquid chromatography can est abundance were C18:1/32:1, C18:1/30:1,
be coupled along with direct electron impact C16:1/32:1 (25).
mass spectrometry (EI-MS). This method has Human skin fibroblasts from X-ALD or Zell-
the advantage of not requiring derivatization of weger patients were analyzed by using the same
the sample and not showing matrix effect (31). LC-ESI-MS/MS method. For acidic phospho-
According to this, the time of analysis can be re- lipids MS/MS analysis was performed in nega-
duced, however, when dealing with a complex tive ion mode, while phospholipids with choline
matrix, such as plasma, the separation is antic- were detected in positive ion mode. The lipids
ipated by a liquid-liquid extraction, which can were eluted from the reversed-phase column in
take about 30 minutes. This method can be used accordance with their hydrophobicity and ex-
for the detection of all NEFAs in plasma with hibited molecular ion signals dependent on their
a satisfactory sensitivity and selectivity, because mass/charge (m/z). The longest fatty acyl chains
the direct EI-MS response is not affected by detected were C36, and the highest unsaturated
co-eluting endogenous plasma components (31). bond number observed was 6.
The analyses of plasma samples collected Separation of fatty acid ethanolamides by
from genetically obese, spontaneously hyperten- UPLC-MS/MS was performed on a C18 column
sive female rats were carried out on two LC-MS by using a gradient condition: over the first 2
systems; one was coupled to a triple quadrupole min mobile phase B (acetonitrile with 0.1% for-
MS, and the other was coupled to a LIT mass mic acid) was increased from 70% to 72% and in
spectrometer. The operating conditions of the the subsequent 6 min increased to 74%. The tan-
ESI source in both cases were negative ion mode, dem MS equipped with an atmospheric pressure
and acceptable precision was achieved by both ionization (API) probe was operated in positive
instruments. The LIT system had the advantages electrospray ionization mode (+ESI), because of
of lower matrix effect and higher global recov- the structure of the ethanolamides (24).
ery than in the case of the QqQ system. Although
the QqQ assays were highly sensitive, when ap- 4. Alternative analitical techniques
plied to rat plasma samples, they gave several Capillary electrophoresis (CE) represents an
false identifications, because many isomers with alternative to chromatographic methods which
similar MRM transitions were co-eluted (30). gives high efficiency, low separation times and
By using an Orbitrap Fourier transform mass high reproducibility. However, there are few
spectrometer as detector, FAs and OAHFAs studies which deal with electrophoretic separa-
were separated by using a negative ion mode tion of fatty acids (32) compared to GC-MS stud-
because OAHFAs, like FAs, possess a carbox- ies or HPLC-MS studies and even fewer dealing
ylic group. As well as in the case of the QqQ with biological samples. Similar to HPLC, cap-
system, this method also identified a number of illary electrophoresis presents the disadvantage
molecular species in one chromatographic peak, of the UV detection, because fatty acids hardly
demonstrating co-elution of isomers of OAHFA absorb in this spectral range. However, in addi-
molecular species. The main advantage of this tion to the MS detector which can be coupled to
method is that long chain fatty acids could be the CE system, other types of detectors can be
separated without a derivatization process. Mori used, such as the contactless conductivity detec-
and co-workers succeeded identifying 61 OAH- tors (37).
FA molecular species with 34-56 carbon atoms Alternatively to the separation methods,
by using this method. The species with the high- spectral techniques such as ATR-FT-IR and Ra-
378 Revista Romn de Medicin de Laborator Vol. 23, Nr. 4, Decembrie, 2015

man spectroscopy (33,38) can be used for the to insulin resistance, such as polycystic ovary
analysis of fatty acids. These methods, coupled syndrome (PCOS) (14). In the last case, results
with the multivariate analysis of spectral data have showed that insulin resistance has a big-
can give rapid quantitative information about ger influence on the fatty acids profile than the
the analyzed sample (38). These complementary presence of PCOS. Nervonic acid (C24:1 n-9)
spectroscopic methods provide very fast analy- was identified as a biomarker for the presence
sis times (seconds to few minutes), specificity of PCOS and dihomo--linolenic acid (DGLA)
towards the analyte through the fingerprint spec- was identified as a biomarker for insulin resis-
tral area and very low consumption of solvents. tance, after multivariate interpretation of data.
Moreover, authors have noticed, as in case of
5. Potential clinical utility of fatty acids malignant diseases, a decreased ratio of n-3/n-6
profile analysis PUFA in case of PCOS with insulin resistance,
compared to control or with non-insulin resis-
There are two main directions is which the tant PCOS, but also a modified profile of MUFA
fatty acids composition in biological samples in PCOS. In the same time, Han et al. (5) have
have been studied: the presence of a malignant identified the arachidonic acids (C20) to be bio-
state and the presence of a disorder related to the markers in the plasma of diabetic patients, with
metabolic syndrome. In the first case, authors or without different stages of nephropathy, as a
(12,13,15) have separated FA from colorectal diabetic complication. Moreover, they have no-
cancer tissue and healthy adjacent cells as con- ticed, compared to control, an increase in the
trol obtained postoperatively, and from erythro- EFA profiles and a decrease in the NEFA profiles
cyte membranes in multiple myeloma patients, in the plasma of diabetic patients. This can be
after derivatization to FAME. Results showed, in related to insulin resistance and/or lack of insu-
all cases, an increase in the profile of some satu- lin. At the same time, Sertoglu and co-workers
rated fatty acids (SFA) and also a decrease in the (6) have noticed that the n-3/n-6 ratio is the low-
profile of mono-unsaturated fatty acids (MUFA) est in plasma of diabetic patients compared to
in the cancerous tissue compared to the normal control or to end-stage renal disease patients, but
cells. In the same time, a decrease in the n-3/n-6- the n-3/n-6 ratio is the highest when determined
PUFA ratio has been observed in the malignant from the erythrocyte membranes of the same
tissue, compared to the normal cells. Moreover, groups. When analyzing plasma samples from
Mal and co-workers (12) have realized an entire obese patients by GC-FID, Tremblay-Franco
biochemical metabotyping of colorectal cancer, et al. (39) discovered that the n-9 FA ratio de-
using GCxGC-TOF-MS, where they have re- creased and n-7 ratio increased in case of obese
marked not only different profiles of FA, but also females, compared to control.
of amino-acids and saccharides, with important All these findings can be explained in all
roles in biochemical cell stages. cases due to the anti-inflammatory, cardiopro-
The most important disorder related to the tective, neuroprotective effects of n-3 PUFA
metabolic syndrome is diabetes mellitus, usually and the opposite effects of n-6 PUFA, thus the
because of its complications. Authors have stud- pathological effects are reflected in the n-3/n-6
ied FA in plasma from patients with this disease PUFA ratio. Arakawa et al. (40) have pointed out
(1,5,6), but also FA from insulin secreting cells the importance of n-3 PUFA intake, their cardi-
INS-1 have been separated using GCxGC-TOF- oprotective properties and have showed that the
MS (11) and plasma from other disorders related total serum levels of DHA and EPA can be pre-
Revista Romn de Medicin de Laborator Vol. 23, Nr. 4, Decembrie, 2015 379

dictor of ischemia immediately after myocardial The level of propionic acid in the smokers
reperfusion, thus can predict a myocardial dam- saliva is higher than in the nonsmokers, and the
age. Moreover, Nishi et al. (1) have shown that level of heptanoic acid in the smokers saliva is
a daily ingestion of n-3 PUFA through different lower than in the case of nonsmokers (27). The
types of supplements (e.g. nuts) can improve the level of C16:0, C18:1, and C18:0 in the serum of
FA profile in diabetic patients, with decreasing patients dealing with advanced pancreatic cancer
their coronary heart disease risk. For the same is higher than the dosage found in the serum of
reason, authors (16) have studied recovery from healthy patients (23).
patients with periodontitis, after administering It has been demonstrated that by increasing
supplements with n-3-PUFA, considering the the concentration of substrates from the parallel
anti-inflammatory effects of this type of FA. Re- n-3 and n-6 polyunsaturated fatty acid pathways,
sults have shown a better recovery at patients the formation of the products in that pathways
having n-3-PUFA supplements than controls. also increases and metabolism in the parallel
In the same time, n-3 PUFA have proved their pathway is reduced. DHA and EPA suppress the
cardioprotective properties and the total serum expression of both 5 and 6-desaturases and
levels of EPA and DHA can be used as predictors normalize LNA and AA levels (28). Because of
for the level of ischemia after myocardia reper- the pro-inflammatory and immunoactive func-
fusion (40). tions of the AA, the increase of this fatty acid
The fatty acids profile seems to be modified in serum and AA/EPA ratio can be considered
even in infectious diseases. Khedr et al. (34) a biomarker in many diseases which include a
have identified several EFA as biomarkers for pro-inflammatory state like sickle cell disease,
the early febrile stage of the dengue fever dis- obesity or cystic fibrosis.
ease; among them: the esterified C14:0, C16:0, Elevated plasma AA levels may thus be a
C18:0, C20:4-n6, C22:6-n3, which, in all cases, source of pro-aggregatory substances in case
had a decreased profile in the blood of infected of sickle cell disease patients (29). On the oth-
patients compared to control. er hand, AA deficiency may predispose human
It has been demonstrated that the same sat- organism to developing type 2 diabetes mellitus,
urated fatty acids, C16:0 and C18:0 are present because a significant decrease of the principal
in lower amounts in the meibum of meibomian n-6 PUFA and AA has been observed in diabetic
gland dysfunction patients than control. Meibum patients (26). Moreover, NEFAs concentrations
lipids are a highly complex mixture of lipids from were found to be higher in plasma samples of
various classes, including OAHFAs. These spe- diabetic patients (31).
cies decrease as the severity of dry eye disease Cystic fibrosis is the most common lethal
increase, so they can be considered as a potential genetic disease in Caucasian population, which
biomarker in meibomian gland dysfunction. It is presents an alteration of plasma levels of poly-
important to elucidate the relationship between unsaturated fatty acids. The major ones are de-
odd number FA and bacterial infections in mei- creases in linoleate and docosahexaenoate and
bomian gland dysfunction patients, because bac- increases in palmitoleate and mead acid. In case
teria can produce an odd number of FAs (25). of cystic fibrosis the AA increases only at the
On the other hand many hydroxyl-derived com- highest LNA:ALA ratio, and the production of
pounds of EPA and DHA are involved in the reg- AA is influenced by ALA, the initial substrate of
ulation of vascular tone, arteriosclerosis and are the parallel n-3 pathways. Also EPA levels were
considered markers of lipid peroxidation (30). higher and DHA levels lower in cystic fibro-
380 Revista Romn de Medicin de Laborator Vol. 23, Nr. 4, Decembrie, 2015

sis compared to healthy patients. Katrangi and Milk is the primary food and also comes as a
co-workers also demonstrated that the serum ra- biological fluid (7,8). Authors have revealed by
tio of LNA to ALA has a greater impact on LNA GC-FID analysis that the FA profile in human
to AA metabolism than on ALA to EPA metab- milk is different from that in infant formulas (7),
olism. The increased production of AA plays a where essential fatty acids and their eicosanoids
significant role on cystic fibrosis related inflam- had less concentrations in the commercial formu-
mation (28). lations than in human milk. However, Cruz-Her-
Levels of FAE have been shown to vary be- nandez and co-workers (8) showed an inter-indi-
tween species, in hamster plasma samples the vidual variability in fatty acids profile in human
mean levels of OEA and PEA are higher com- milk, which is to be taken in consideration. But a
pared to human plasma samples. Compared to supplementation with DHA at lactating mothers
plasma samples, levels of AEA were higher in can increase this fatty acids profile in milk and
the intestine and in hamster liver. The concen- plasma and also, can improve the n-3/n-6 PUFA
tration gradient for hamster liver and small in- ratio in these biological fluids (41).
testine was shown to be PEA>OEA>AEA, but All of the up-mentioned clinical implications
further investigation is required in order to fully of fatty acids profiles are listed in Table III.
understand FAE distribution patterns in case of
different tissues. Levels of OEA can be influ- 6. Conclusions
enced by the volunteers physiological state and
also by the type of dietary treatments (24). This paper has presented an extensive liter-
Very long chain fatty acids (VLCFA) are ature review of the recent years, regarding the
minor constituents and are difficult to detect most important techniques for the analysis of
and separate. They are biosynthesized from fat- fatty acids in biological samples. We have pre-
ty acids by elongase and they are degraded by sented the main utilized methods in each case,
-oxidation in peroxisomes. In genetic peroxiso- but also have noted unusual methods which can
mal diseases, such as Zellweger syndrome and be successfully used. GC-MS is the main chro-
X-linked adrenoleukodystrophy (X-ALD), VL- matographic technique applied in clinical lab for
CFA are known to accumulate and increase in- the analysis of fatty acids from serum, plasma
tracellular calcium concentrations and decrease and other biological fluids or tissues, after their
mitochondrial respiration, which can lead to cell methyl-transesterification. In the same time,
death in oligodendrocytes and astrocytes. The HPLC or CE coupled with different types of de-
accumulation of VLCFA, including C26:0 has tectors can be successfully used for the analysis
been accepted as the principal biochemical ab- of fatty acids in these bio-samples. Alternative
normality in X-ALD and Zellweger syndrome. spectral methods, like infrared and Raman spec-
The proportions of sphingomyelin with C26:0, troscopy, have been used for the analysis of fatty
C26:1 and C26:2 were significantly increased acids, mostly coupled with multivariate analysis
in peroxisomal disease (4). New molecular spe- of data.
cies with VLCFA were found in the phospho- We have also presented the most important
lipids classes of phosphatidyl-ethanolamine and results obtained along with the clinical rele-
phosphatidyl-serine, which can be used as new vance of fatty acids profiles and, as applicable,
biomarkers for peroxisomal diseases. The main biomarkers or fatty acids with importance in the
disadvantage was the observed differences in the presence or diagnosis of the investigated diseas-
molecular species between patients (4). es. The n-3/n-6 PUFA ratio can be pointed out
Revista Romn de Medicin de Laborator Vol. 23, Nr. 4, Decembrie, 2015 381

as an important factor in many diseases, starting Abbreviations


from inflammatory states and diabetes mellitus
AA (arachidonic acid);
up to cancerous states.
AEA (arachidonyl-ethanolamide);
ALA (alpha-linolenic acid);
Aknowledgement
ALEA (alpha-linolenoyl-ethanolamide);
This paper was published under the frame ATR-FT-IR (attenuated total reflectance Fourier
of European Social Found, Human Resources transform infrared spectroscopy);
Development Operational Programme 2007- CE (capillary electrophoresis);
2013, project no. POSDRU/159/1.5/S/136893. DCM (dichloromethane);

Table III. Potential clinical utility of FA profiles in different diseases

Analyzed
Diseases Method Biomarkers FA profile Ref.
sample

decrease ratio of n-3/n-6 PUFA ratio


colorectal cancer colon tissue GCXGC/ TOFMS N/A (12,13)
increase SFA
HPLC-fluores- increase in C16:0, C18:1, and C18:0
pancreatic cancer human serum N/A (23)
cence detector serum levels
neurologic/ psy- cholesteryl decrease of AA and DHA concen-
brain samples TLC, GC-FID (2)
chiatric esters tration
X-ALD, C26:0/C22:0
cell culture LCESIMS/MS increase of VLCFA levels (4)
Zellweger ratio
sickle cell AA/EPA increase in AA and DGLA levels
human plasma UFLC (29)
disease ratio decrease of EPA and DHA levels
human bron- increases in EPA, C16:1 and mead
LNA:ALA
cystic fibrosis chial epitheli- RP-HPLC acid levels (28)
ratio
al cells decrease in C18:2, DHA levels
meibomian gland human C16:0 and C18:0 are present in low-
LCFTMS N/A (25)
dysfunction meibum er amounts
nervonic acid
polycystic ovary
plasma (GCMS) (C24:1 n-9) decreased ratio of n-3/n-6 PUFA (14)
syndrome
-PCOS
diho-
mo--lino-
insulin resistance plasma GC-MS decreased ratio of n-3/n-6 PUFA (14)
lenic acid
(DGLA)
UFLC-MS/MS
human LC-EI-MS arachidonic increase in the EFA
diabetes mellitus (5,6,11,31)
plasma GC-MS acids (C20) decrease in the NEFA and AA
GC-FID
total
myocardial in- DHA+EPA profile significantly cor-
serum GC DHA+EPA (40)
farction related with the size of ischemia
profile
382 Revista Romn de Medicin de Laborator Vol. 23, Nr. 4, Decembrie, 2015

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