MEASUREMENT OF FATTY ACIDS IN TOENAIL CLIPPINGS
USING GC-MS
____________________________________
A Thesis
presented to
the Faculty of the Graduate School
at the University of Missouri-Columbia
_______________________________________________________
In Partial Fulfillment
of the Requirements for the Degree
Master of Science
_____________________________________________________
by
YANLI YANG
Dr. J. David Robertson, Thesis Supervisor
DECEMBER 2012
The undersigned, appointed by the dean of the Graduate School, have examined the
thesis entitled
MEASUREMENT OF FATTY ACIDS IN TOENAIL CLIPPINGS
USING GC-MS
presented by Yanli Yang,
a candidate for the degree of Master of Science, and hereby certify that, in their opinion, it
is worthy of acceptance.
Professor J. David Robertson
Professor C. Michael Greenlief
Professor Kevin Fritsche
Professor Susan Z. Lever
ACKNOWLEDGEMENTS
I would like to express my sincere appreciation to my research advisor Dr. J.
David Robertson. He is knowledgeable and so supportive. His creative thoughts in
research and excitement in teaching have touched me deeply. He has always
encouraged me in my research, studies and even job hunting. He is the best advisor a
graduate student could have. Without his guidance and persistent help, this thesis
would not have been possible.
I would like to thank Mr. Paul Peterman for his time and efforts to help me. He is
an expert in the chromatography technique. He has spent a lot of time explaining things
to me regarding my project, shared his experience in research, and gave me many
valuable suggestions.
I would also like to thank Mr. William Vellema, who has offered kind assistance
in the operation, troubleshooting and maintenance of the instruments. He is always
there to help and very patient to answer my questions.
I am also grateful to my committee members for helping me build up my
knowledge. And a thank you to Dr. Fritsche, who has provided me with some free fatty
acid samples for testing.
ii
TABLE OF CONTENTS
ACKNOWLEDGEMENTS ........................................................................................................ii
LIST OF ILLUSTRATIONS........................................................................................................v
LIST OF TABLES ................................................................................................................... vii
ABSTRACT ............................................................................................................................ ix
CHAPTER 1 INTRODUCTION ................................................................................................ 1
1.
POLYUNSATURATED FATTY ACIDS (PUFAS) AND OMEGA-3 FATTY ACIDS .............. 1
2.
PREVIOUS RESEARCH IN OMEGA-3 FATTY ACID DETECTION .................................. 6
3.
OBJECTIVE OF OUR STUDY ..................................................................................... 19
CHAPTER 2 EXPERIMENTAL .............................................................................................. 20
1.
GAS CHROMATOGRAPHY-MASS SPECTROMETRY (GC/MS) .................................. 20
2.
SAMPLE PREPARATION .......................................................................................... 23
CHAPTER 3 RESULTS.......................................................................................................... 29
1.
CALIBRATION CURVES OF EPA AND DHA METHYL ESTERS USING SELECTED IONS ..
29
2.
NAIL DIGESTION ..................................................................................................... 31
3.
RECOVERIES OF EPA AND DHA IN NAIL SAMPLES ................................................. 38
iii
4.
CALIBRATION CURVE AND RECOVERY OF METHYL HEPTADECANOATE (C17:0
METHYL ESTER) ...................................................................................................... 39
5.
LIMIT OF DETECTION.............................................................................................. 44
6.
FATTY ACIDS DETECTED IN THE NAIL SAMPLE ....................................................... 44
CHAPTER 4 DISCUSSION .................................................................................................... 46
REFERENCES ...................................................................................................................... 48
iv
LIST OF ILLUSTRATIONS
Figure
Page
1. Chemical structure of eicosapentaenoic acid (EPA C20:5) ....................................... 2
2. Chemical structure of docosahexaenoic acid (DHA C22:6) ....................................... 2
3. Benefits of fish oil ...................................................................................................... 3
4. Triglyceride .............................................................................................................. 16
5. Phospholipid ............................................................................................................ 16
6. Schematic of GC/MS ................................................................................................ 22
7. Calibration curve of EPA methyl ester in SIM mode ............................................... 30
8. Calibration curve of DHA methyl ester in SIM mode .............................................. 30
9. Mass spectrum of EPA methyl ester ....................................................................... 31
10. Mass spectrum of DHA methyl ester ...................................................................... 31
11. Chemical structure of dithiothreitol (DTT) .............................................................. 32
v
12. Reaction between DTT and a disulfide bond .......................................................... 32
13. GC spectrum of the nail sample (spiked with free EPA and DHA)
digested by DTT .................................................................................................. 33
14. GC spectrum of olive oil sample without DTT. From left to right,
C17H34O2, C19H38O2 and C19H36O2 ........................................................................ 36
15. General thiol-ene coupling ...................................................................................... 37
16. The mechanism for thiol-ene reaction in the presence of
a photoinitiator and hν ....................................................................................... 37
17. Calibration curve of methyl heptadecanoate ......................................................... 39
18. Mass spectrum of methyl heptadecanoate ............................................................ 40
19. GC spectrum of the nail sample .............................................................................. 45
vi
LIST OF TABLES
Table
Page
1. Average daily omega-3 fatty acid consumption among adults in different countries
.............................................................................................................................. 6
2. Distribution of fatty acids in adipose tissue ............................................................ 11
3. Distribution of fatty acids in adipose tissue, plasma, and whole blood in Costa
Rican adults, 1999–2001 .................................................................................... 12
4. Intra-assay and day-to-day variations in determination of EPA concentration in red
blood cell membranes by GC/MS
............................................................................................................................ 14
5. Quality control results for the analysis of PUFAs in serum by GC/MS.................... 14
6. Estimated fatty acyl chains in each plasma fraction, mg/dl (% total plasma fatty
acyl chains) in different groups .......................................................................... 16
7. Extracted ion chromatograms of EPA and DHA methyl esters ............................... 29
8. Major peaks in the spiked nail sample digested by DTT ......................................... 34
vii
9. Recoveries of EPA and DHA in nail sample ............................................................. 38
10. The amount of C17:0 observed in the nail sample ................................................. 40
11. The recovery of C17:0 methyl ester ........................................................................ 41
12. Fatty acid composition of plasma phospholipids by country ................................. 42
13. Distribution of fatty acids in erythrocyte membrane ............................................. 42
14. Distribution of fatty acids in adipose tissue ............................................................ 43
15. Limit of detection .................................................................................................... 44
16. Major peaks in the nail sample ............................................................................... 45
17. Fatty acid distribution in human stratum corneum and human nail clippings ……..47
viii
ABSTRACT
Omega-3 fatty acids play an important role in human health and nutrition. Here,
we investigated human toenail clippings as a potential, non-invasive and cumulative
biomonitor for fatty acids, and successfully developed a method to extract fatty acids
and quantified them by gas chromatography-mass spectrometry. Although omega-3
fatty acids were not detected in the nail samples, the nail may still prove to be a
biomonitor for them if the percent recoveries and detection limits could be improved.
Keywords: omega-3, fatty acids, toenail, GC/MS
ix
CHAPTER 1 INTRODUCTION
1. POLYUNSATURATED FATTY ACIDS (PUFAS) AND OMEGA-3
FATTY ACIDS
There are several different nomenclature systems for fatty acids. In this thesis, two of
the systems are mentioned. One is n−x nomenclature[1]. The first carbon-carbon double
bond is located at the xth carbon atom counting from the methyl end to the carbonyl
end, and it is denoted as n or ω (omega). The other one is based on the number of
carbons and the number of double bonds in the structure [2], which takes the form C:D
[3]. C is the number of carbon atoms, and D is the number of carbon-carbon double
bonds in the fatty acid structure.
Polyunsaturated fatty acids are those fatty acids containing more than one carboncarbon double bond in their structures. It has been shown that consumption of
polyunsaturated fatty acids has biologically beneficial effects [4-8]. Omega-3 and
omega-6 are the two principal families of PUFAs. Because they cannot be synthesized by
humans, they have to be obtained from the diet [1]. Omega-3 fatty acids are the PUFAs
1
having the first carbon-carbon double bond at the third carbon atom from the methyl
end. Omega-6 fatty acids are the PUFAs having the first carbon-carbon double bond at
the sixth carbon atom from the methyl end.
Research has shown that eicosapentaenoic acid (EPA, C20:5) and docosahexaenoic acid
(DHA, C22:6) are the two most important omega-3 fatty acids for human health. Their
chemical structures are shown below.
Figure 1 Chemical structure of eicosapentaenoic acid (EPA C20:5)
Figure 2 Chemical structure of docosahexaenoic acid (DHA C22:6)
EPA and DHA are the primary active ingredients in fish oil. They are considered as
essential fatty acids because they are necessary to maintain good health but cannot be
synthesized by the human body.
There is strong evidence that there are many health benefits from the consumption of
PUFAs (Figure 3). James W. Fetterman Jr. and Martin M. Zdanowicz have reviewed the
potential effects of omega-3 fatty acids in preventing and treating different types of
diseases [9]. For example, omega-3 fatty acids have been used for the treatment of
2
rheumatoid arthritis (RA). James and Cleland summarized 12 double-blind, placebocontrolled studies of dietary fish oil supplementation in RA [9, 10]. The number of
subjects ranged from 16 to 67, and the treatment period ranged from 12 to 52 weeks.
The intake of omega-3 PUFAs was between 1.0 - 7.1 grams daily. By looking at the
outcomes, including number of tender joints, number of swollen joints, duration of
morning stiffness etc., they saw no less than two improvements in the outcomes in 11
of the 12 studies. In five of the studies, the use of nonsteroidal anti-inflammatory drugs
(NSAIDs) had been reduced. They concluded that PUFAs have beneficial effects for
individuals that suffer from RA and that further studies on the use of omega-3
treatment in RA are necessary.
Reduce
chance of
cancer
Prevent
pregnancy
complication
Fish oil
Effective to
prevent
depression
Reduce the risk
of heart
disease
Effective for
eyesight
Figure 3 Benefits of fish oil
Correlation between omega-3 PUFAs consumption and cardiovascular disease has
drawn much attention since 1978 [4, 9]. Researchers have found that the blood levels of
3
omega-3 fatty acids are inversely related to the risk of cardiovascular disease [11, 12].
From 1988 to 1994, David S. Siscovick, et al. [6] conducted research showing that the
dietary intake of PUFAs has the effect of reducing the risk of primary cardiac arrest. A
total of 334 patients aged 25 to 74 years with primary cardiac arrest, and 493 randomly
identified control cases matched for age and sex from Seattle and suburban King County,
Washington participated in the study. The overall dietary intake of EPA and DHA were
estimated, and the levels of EPA and DHA in red blood cells were measured by gas
chromatography.
There is also evidence that consumption of omega-3 fatty acids may help prevent breast
cancer [13] based on serum levels of omega-3 fatty acids; reduce the risk of prostate
cancer by studying tumor membrane fatty acid composition of mice [14]; and prevent
pregnancy complications like premature delivery and postpartum depression [7, 15].
Consumption of fish oil is also reported to improve eyesight and decreases dryness in
the eyes [16]. It is also suggested in the literature that suicide risk can be predicted by
monitoring plasma omega-3 fatty acid levels [8, 17].
Although there is not sufficient data to draw the conclusion that omega-3 PUFAs have
benefits for inflammatory bowel disease (IBD), research has shown that people suffered
from IBD have low total PUFA levels in serum, and there is a specific lack of omega-3
PUFAs in patients with Crohn’s disease [18].
4
Consuming fatty fish, such as salmon, mackerel, and sardines, or fish oil capsules is an
effective way to increase omega-3 blood levels [19]. The American Heart Association
(AHA) recommends at least two, 3.5 oz fish meals per week to receive 500 mg/d EPA +
DHA for the general population, and 1 g/d of marine omega-3 for coronary patients [1921]. The United States Department of Agriculture (USDA) recommends 8 oz of a variety
of seafood per week to receive approximately 250 mg/day of EPA + DHA in 2010 Dietary
Guidelines [22].
The normal dietary intake of omega-3 is not sufficient for optimal health among most
populations [23]. Table 1 shows daily consumption of EPA and DHA among adults in
different countries [24].
5
Table 1 Average daily omega-3 fatty acid consumption among adults in different
countries
Country
EPA (g)
DHA (g)
male
female
male
female
Austria
0.09
0.07
0.19
0.18
Australia
0.07
0.05
0.13
0.09
Germany (Heidelberg)
0.10
0.07
0.19
0.14
Germany (Potsdam)
0.13
0.08
0.21
0.14
France
0.15
0.12
0.27
0.23
Japan
0.36
0.31
0.63
0.57
Norway
0.41
0.27
0.59
0.40
United Kingdom
0.09
0.11
0.13
0.16
USA
0.05
0.04
0.09
0.07
2. PREVIOUS RESEARCH IN OMEGA-3 FATTY ACID DETECTION
2.1 Commonly used biological markers
2.1.1 Blood
Urine, hair, blood and nail are commonly used biomonitors for intake and exposure to
reflect certain aspects of human health, but only blood has been employed to detect
EPA and DHA levels in humans.
6
Brian Moyers, et al. studied EPA and DHA concentrations in whole blood, and found a
relation between their levels and three important exercise parameters in 992 patients
suffering from stable coronary artery disease [25]. They validated that the dietary intake
of omega-3 fatty acids helped reduce cardiovascular risk. Gordon Bell, et al. performed
another study using blood samples which were collected by finger prick and stored on
absorbent paper at -20 °C for up to one month. According to their study, EPA, DHA
ratios and their percentage of total highly unsaturated fatty acids in the blood prick
sample all showed good correlations between erythrocyte phospholipids and whole
blood [26]. They believe this is a promising method for large-scale nutritional status
studies.
Abu and Oluwatowoju used GC-MS with electron impact to study omega-3 index of
healthy human blood. Omega-3 index is expressed as a percentage. It is defined as the
sum of EPA and DHA over total fatty acids in red blood cell membranes [11]. They also
pointed out that omega-3 index might become a promising marker of disease in the
future. Harris and Thomas compared the biological variability of red blood cells, plasma
and plasma phospholipids to determine the most suitable marker of omega-3 fatty acid
content [27]. Unlike plasma, EPA and DHA content in red blood cells is not diluted after
an individual consumes a meal that does not contain EPA or DHA. They concluded that
red blood cells are a better biomonitor for omega-3 fatty acid analysis because of its
lower biological variability.
2.1.2 Urine, breast milk and sweat
7
There has also been research on determination of volatile free fatty acids in human
urine by GC/MS [28] and research on omega-3 fatty acids in human breast milk. A
research group confirmed that omega-3 long-chain PUFAs had a protective effect
against atopic manifestations [29]. Both breast milk and serum were used to analyze the
effect of long-chain omega-3 fatty acids on women atopic eczema and respiratory
allergy [30]. While fatty acids have been found in human sweat during fasting, most of
the observed fatty acids are saturated [31].
2.1.3 Disadvantages of using blood
Although blood is commonly used as a biomarker for polyunsaturated fatty acids, it has
several disadvantages. First, drawing blood is often time-consuming. It needs to be
conducted by professionals to ensure that the quality and quantity of the samples are
maintained [32]. Sometimes the subjects have to be in fasting state [27, 33].
Second, the transportation and storage of blood samples are not convenient. The
requirement of temperatures during shipping could be ambient (20-30 °C), refrigerated
(2-8 °C), frozen (-20 °C), frozen (-70 °C) and frozen (at or below -150 °C) [32], based on
the intended analysis. Also, during transportation, the temperature of the samples must
be monitored. As for storage, plasma or serum should be stored at -80 °C and oxygen
level sensors are always necessary [32]. Compared to blood, using nail clipping samples
would be non-invasive and much more cost effective for collection, shipping and storage.
2.1.4 Nail and hair
8
There have been a numerous studies on trace elements analysis in nail samples. NasliEsfahani et al. compared the concentrations of a total of six trace elements in scalp hair,
nail, urine and serum between healthy people and diabetes patients [34]. They found
that the trace metals tend to accumulate more in scalp hair and nail and produce larger
signals than urine and serum. They concluded, for this reason, that hair and nails are
better biological samples for trace elements than blood and urine.
Nail clippings reflect selenium status better than other biomonitors, especially for
measuring long-term average intake [35-38]. Slotnick considered toenail to be a good
biomonitor for arsenic exposure [39]. They found that arsenic concentrations in drinking
water were positively related to the concentrations in toenail clippings. Toenail clippings
have also been considered as a biomonitor for zinc. Alejandro Gonzalez et al. observed
an increase in toenail zinc when the dietary intake of zinc was increased [40]. As with
arsenic, this correlation was not affected by demographic factors (age and race) or
health-related behaviors (smoking and exercise).
Similar to nail, hair samples have been used to analyze for trace elements or heavy
metals. By measuring the heavy metals in hair of the subjects from an electronic waste
recycling area, Wang et al. detected much higher concentrations of Cu, Pb and Cd than
that from the control group [41]. Hair has also been used to investigate contaminants
from the environment, such as mercury [42] and arsenic [43].
9
Besides trace elements, nail and hair have also been applied as biomonitors for organic
compounds. Nail has been used to detect some tobacco alkaloids including nicotine [44],
perfluoroalkyl compounds [45] and cocaine [46]. Hair sample analysis has been used to
measure drug abuse, such as cocaine [47], multiclass drug abuse including
tetrahydrocannabinol (THC) [48], and cotinine for childhood secondhand smoke
exposure [49].
2.1.5 Nail formation/biology
Human nail is a high-sulfur containing and keratinized structure, which offers protection
for fingers and toes. It is composed of nail plate, nail matrix, nail folds and nail bed [50].
Sulfur accounts for 10% by weight [51]. Keratin is a large family of fibrous proteins,
which contains a significant amount of disulfide bridges in its structure; the disulfide
cross-linking structures contribute to nail hardness. The growth rate of toenail is 0.030.05 mm/day. Fingernail grows faster than toenail, which is about 0.1 mm/day. Thus the
exposure window of nails is dependent on the length of nail clippings and also the
growth rate of nail [51, 52].
2.1.6 Hypothesis
Nail clippings have many advantages as non-invasive biomonitors and may be applicable
for fatty acids [52] in humans.
10
There are several reasons why we chose toenail clippings instead of other biological
samples. First, based on the length of the clipping and growth rate of the nail [52, 53], a
toenail sample has the potential to reflect the accumulation of fatty acids in human
body in a wide time window of several months to over a year; urine and blood only
reflect the fatty acid status in a short period of a couple of days or weeks. Second, the
use of nail clippings enables easy sample collection and storage over long time. It does
not require low-temperature storage like blood, or immediate analysis, which has also
made long distance transportation possible. And finally, nail was selected over hair as it
is likely to have significantly lower exogenous contamination.
2.1.7 Distribution of fatty acids in biological samples
The distribution of fatty acids in adipose tissue is shown in Table 2 [33]. The participants
were from a study of diet and heart disease in Costa Rica. The fatty acids were analyzed
by gas chromatography.
Table 2 Distribution of fatty acids in adipose tissue
Fatty Acids
Saturated fatty acids
Monounsaturated fatty acids
Polyunsaturated fatty acids
n-6
n-3
n-7
trans Fat
% of Total Fatty Acids (n = 503)
Mean ± SD
26.21 ± 3.42
54.08 ± 4.38
15.29 ± 3.33
0.99 ± 0.20
0.67 ± 0.18
3.12 ± 0.99
11
Monounsaturated fatty acids are the main component (around 54%) of total fatty acid
in adipose tissue and PUFAs accounts for 20% in adipose tissue.
In another study conducted by the same group [54], the distribution of fatty acids in the
diet, adipose tissue, plasma and whole blood was compared (Table 3).
They evaluated adipose tissue, plasma and whole blood as indicators of fatty acid intake
and concluded that fasting whole blood is a suitable biomarker for epidemiologic studies.
Table 3 Distribution of fatty acids in adipose tissue, plasma, and whole blood in Costa
Rican adults, 1999–2001
Fatty Acids
% of total fatty acids (n = 200) Mean ± SD
Adipose tissue
Plasma
Whole blood
Total saturated
25.00 ± 3.52
31.85 ± 2.24
34.07 ± 1.86
fatty acids
Total
52.49 ± 4.02
25.91 ± 3.50
23.32 ± 2.89
monounsaturated
fatty acids
Polyunsaturated fatty acids
n-3 PUFAs*
0.95 ± 0.25
2.62 ± 0.61
4.54 ± 0.87
n-6 PUFAs
17.04 ± 3.56
36.74 ± 4.66
35.10 ± 3.45
n-7 PUFAs
0.54 ± 0.17
0.19 ± 0.10
0.16 ± 0.07
Total PUFAs*
17.33 ± 3.70
38.39 ± 4.98
39.87 ± 3.30
Total trans fatty
2.69 ± 0.62
1.47 ± 0.44
1.40 ± 0.43
acids
*n = 91 for adipose tissue, n = 63 for plasma, and n = 62 for whole blood.
12
2.2 Techniques for PUFA analysis
2.2.1 Gas chromatography-mass spectrometry (GC/MS)
Gas chromatography is the most popular technique in the analysis of PUFAs, especially
when it is coupled with mass spectrometry [55, 56]. It can differentiate fatty acids based
on their retention times and mass spectrum information. GC detectors have high
sensitivity and flame ionization detector (FID) with picogram sensitivity is the most
commonly used detector [57]. It is powerful in detecting organic compounds, especially
fatty acid derivatives [58-61].
2.2.2 Detection limit and precision of GC/MS method
Abu and Oluwatowoju determined omega-3 index in red blood cells membranes from
12 healthy volunteers using GC/MS [11]. The limit of detection (LOD) was calculated as
follows: LOD = 3xSb/m, where m is the slope of the standard curve, and Sb is the
standard error of the intercept. The LOD was 0.36 µg/ml (1.2 µmol/L). Table 4 shows the
precision obtained in the study [11].
13
Table 4 Intra-assay and day-to-day variations in determination of EPA concentration in
red blood cell membranes by GC/MS
Concentration of EPA (µg/ml)
0.50
2.5
5.0
10
25
50
250
Intra-assay precision
Day-to-day variation
Mean
CV (%)
Mean
CV (%)
0.021222
0.106322
0.189417
0.468569
1.127734
2.301765
12.21527
5
1
1
0
0
1
0
0.0221298
0.1062275
0.1878471
0.4632659
1.1146986
2.2599376
12.055091
5
1
1
1
1
1
1
Sánchez-Ávila, N. et al. developed a fast method to determine fatty acids in serum by
GC/MS [62]. Some of their results are shown in Table 5.
Table 5 Quality control results for the analysis of PUFAs in serum by GC/MS
FAME
C20:5n3
C22:4n6
C22:5n3
C22:6n3
Linearity
(µg/ml)
0.08-2
2-20
0.1-2.5
2.5-25
0.1-2.5
2.5-25
0.08-2
2-20
Correlation
coefficient
0.987
0.990
0.998
0.996
0.995
0.997
0.994
0.996
LOD
(µg/ml)
0.004
LOQ
(µg/ml)
0.013
Repeatability Reproducibility
(RSD %)
(RSD %)
5.31
6.64
0.003
0.010
1.95
2.40
0.006
0.020
3.09
3.95
0.003
0.010
3.12
4.02
14
The LOD was the mass of analyte which gave a signal that is three standard deviations
above the mean blank. Repeatability was expressed as relative standard deviation (RSD),
and reproducibility was also expresses as RSD.
2.2.3 Other techniques
Salm et al. developed a method of quantifying EPA, DHA and arachidonic acid (AA) in
human plasma by high-performance liquid chromatography- tandem mass spectrometry
(HPLC-MS/MS) [63]. Compared to traditional GC methods of identifying PUFAs (> 40 min)
[64-66], their analysis only required 3.5 min, which is convenient for high-throughput
analysis. Also, another study profiling 29 fatty acids in plasma using ultra-performance
liquid chromatography/electrospray ionization tandem mass spectrometry (UPLCMS/MS) [67] only required 15 minute analysis time.
Mansour purified PUFAs in microalgae using reversed-phase HPLC and then separated
them by GC/MS [68]. The FAMEs with higher degree of unsaturation eluted earlier than
the more saturated ones if they have the same carbon chain lengths. Again, this method
allows rapid analysis.
2.3 Sample preparation chemistry
In nature, only a small fraction of fatty acids exists in their free form, called free fatty
acids (FFAs) or non-esterified fatty acids (NEFAs). Most fatty acids do not occur in the
free form but are bound to other more complex molecules [55, 57]. For example, the
15
fatty acids exist as triglycerides (TG) and phospholipids (PL), as shown in Figure 4 and
Figure 5.
Figure 4 Triglyceride
Figure 5 Phospholipid
It is reported by Hodson et al. that about 99% of human adipose tissue is composed of
TG and that PL account for less than 0.1% and cholesterol accounts for 0.3% [69]. They
also estimated the distribution of fatty acyl chains (TG, FFA, PL and cholesteryl ester (CE))
in plasma, as shown in Table 6.
Table 6 Estimated fatty acyl chains in each plasma fraction, mg/dl (% total plasma fatty
acyl chains) in different groups
Triglycerides
Young, healthy [70]
Familial combined
hyperlipidaemia [71]
Type 2 diabetes [72]
Controls-Quebec
Cardiovascular Study [73]
Phospholipids
156 (44%)
505 (59%)
Free
fatty acids
15.4 (4%)
21.9 (3%)
111 (31%)
207 (24%)
Cholesteryl
ester
74.0 (21%)
120 (14%)
249 (50%)
197 (46%)
21.4 (4%)
26.8 (6%)
140 (28%)
122 (28%)
87.4 (18%)
83.1 (19%)
16
In order to be analyzed by GC, bound fatty acids (such as TG and PL) need to be
derivatized so that the fatty acids are released in their free form from the complex
molecules.
As for free fatty acids, they will be converted into their corresponding methyl esters for
GC analysis. There are several reasons for conducting derivatization [57]. First, FFAs
have higher polarity and lower volatility compared to their methyl esters. Second, FFAs
have high boiling points which are close to or even higher than the temperature of
decomposition of the fatty acids and are therefore not suitable for GC analysis. For this
reason, most of the time, FFAs are methylated into their esters for GC measurements.
The reaction is shown as
RCOOH →
RCOOCH3+ H2O
In our sample preparation, we tried two different methods to extract fatty acids from
nail clippings. One method used dithiothreitol (DTT) in dichloromethane, which is
effective in reducing disulfide bonds [74-76] in nail matrix; and the other method used
potassium hydroxide to dissolve the nail and release FFAs from the nail. It turned out
that the potassium hydroxide method worked, while the DTT method did not.
2.4 Common procedure for measuring FFAs in biological samples
Generally, there are two steps before a fatty acid sample can be injected onto the GC
column. They are lipid extraction and esterification.
17
2.4.1 Lipid extraction
The purpose of extraction is to separate lipids from other constituents of the sample.
Solvent extraction is one of the most widely used techniques to extract lipids [77].
2.4.2 Derivatization of fatty acids
There are three derivatization methods to prepare fatty acid methyl esters (FAMEs).
A. Acid-catalyzed derivatization
Both free fatty acids (FFAs) and triacylglycerols (TG) can be transesterfied.
FFAs are methylated in anhydrous MeOH in the presence of the HCl as catalyst [55]. This
method is mild and gets almost quantitative yield, but it requires long reaction times
(>30 min). It is also reported that concentrated sulfuric acid may lead to decomposition
of PUFAs under certain conditions [57].
B. Base-catalyzed derivatization [1, 55]
18
CH3ONa, NaOH and KOH can be used to enable this step. This reaction is usually
catalyzed by BF3/CH3OH solution and requires strict anhydrous conditions. FFAs are not
esterified under this condition. Also, BF3 has limited shelf-life, and thus it has to be
stored with care.
C. Methylation with diazomethane (CH2N2)
RCOOH + CH2N2 RCOOCH3
Although this reaction goes rapidly with few by-products [78], it is unfavorable because
diazomethane is toxic and dangerous to deal with.
3. OBJECTIVE OF OUR STUDY
Omega-3 fatty acids play an important role in human health and nutrition. The study of
omega-3 levels in human body has drawn much attention, especially in recent years.
Our objective was to develop a method to measure EPA and DHA in human toenail
clippings using GC/MS, and explore the relationship between nail concentration and
omega-3 level intake.
19
CHAPTER 2 EXPERIMENTAL
1. GAS CHROMATOGRAPHY-MASS SPECTROMETRY (GC/MS)
Gas chromatography-mass spectrometry (GC/MS) is a combination of gas
chromatography and mass spectrometry. GC/MS is a powerful analytical technique
which has been widely used in environmental analysis, drug development, and
toxicology analysis.
The components in the sample are eluted according to retention times by gas
chromatograph and separated based on mass to charge ratio of the ions by the mass
spectrometer. The mass spectrometer provides abundance of the ions and structural
information of each component. A schematic of a GC/MS is shown in Figure 6.
1.1 Instrumentation
In GC/MS, a sample is injected into the GC column in the oven through an injection port.
The temperature of the injector is high enough to vaporize all the components, so that
the sample becomes a gas mixture. Then the mobile phase which is an inert gas (helium,
nitrogen and hydrogen are the common carrier gases) carries the sample going through
20
the GC column. Due to the differences in the volatilities, the components are separated.
The more volatile the component is, the sooner it is eluted. Polar stationary phases
allow separation according to unsaturation and carbon chain length, thus they are
commonly used for the analysis of FAMEs [1].
The gas mixture is then introduced into the mass spectrometer. The first component of
a mass spectrometer is an ion source. Electron ionization (EI) and chemical ionization (CI)
are the most commonly used ionization techniques. Our instrument is equipped with an
EI ion source. In an EI source, a hot wire filament emits electrons. Each of the electrons
carries energy that is sufficient to break most bonds in the sample molecules [79]. The
relative abundances of the ion fragments that are produced are a function of the energy
of ionizing electrons and the temperature at which the ions are produced [79, 80].
These ions travel through the mass analyzer, which is a quadrupole mass analyzer for
our instrument.
In a quadrupole mass analyzer, both a direct current (DC) voltage and a radio frequency
(RF) voltage are applied to each of the four parallel rods [80]. By adjusting the DC/RF
ratio, the ions are filtered by their m/z ratios. Only the fragments having mass to charge
ratios (m/z) within the selected range can pass the mass filter and reach the detector.
The detector counts the number of ions hit it at each m/z. This information is collected
by the computer and a chromatogram is generated.
21
Figure 6 Schematic of GC/MS
1.2 Data Analysis
GC/MS is able to provide both qualitative and quantitative information of the analytes.
By interpreting the fragmentation spectra, we can obtain the chemical identities of the
compounds as well as their concentrations/amounts in the mixture.
1.2.1 Identification of the components
The identification of the components is achieved by using reference spectra. For a
specific compound, at specific experimental conditions, the fragmentation pattern and
retention time are the characteristics of the compound. By comparing the results
against reference libraries, the identity of the compound can be determined.
1.2.2 Quantitative analysis
22
Peak area in GC spectra is proportional to the quantity of the analyte in the injected
volume. The quantitative analysis of GC/MS technique can be conducted either in total
ion chromatogram (TIC) or selected ion monitoring (SIM) mode.
In scan mode, the mass spectrometer scans all the m/z in the selected range (for
example, 50-400 m/z) repetitively and sums up the abundance of all the m/z over the
whole range, and gives a TIC.
While in SIM mode, the mass spectrometer only monitors a limited number of m/z and
records their abundances. Compared to TIC mode, SIM gives a much higher sensitivity
and selectivity. Thus, SIM mode is usually applied to detect the substances with low
concentrations.
2. SAMPLE PREPARATION
2.1 Chemicals
Supelco 37 component FAME (fatty acid methyl ester) Mix (analytical standard), cis5,8,11,14,17-Eicosapentaenoic acid methyl ester (10 mg/ml in heptane, analytical
standard), cis-4,7,10,13,16,19-Docosahexaenoic acid methyl ester (10 mg/ml in heptane,
analytical standard) and methyl heptadecanoate (analytical standard, ≥99.5% GC) were
purchased from Sigma-Aldrich.
Heptane (puriss. p.a., ≥99.5% GC), dichloromethane (puriss, ≥99% GC), boron trifluoridemethanol solution (14% in methanol), potassium hydroxide (semiconductor grade, 99.99%
23
trace metals basis), DL-Dithiothreitol (for molecular biology >99.5% (RT)), potassium
hydroxide solution (volumetric, 0.5 M KOH in methanol) were all purchased from SigmaAldrich.
Hexane (sum of Isomers, 99%, Pure), acetone (HPLC grade), hydrochloric acid (33-36%
as HCl, OPTIMA), methanol (99.8% GC) and sodium sulfate anhydrous (granular, ACS)
were purchased from Fisher Scientific.
The nail clippings in our study were from the same healthy individual, and the lengths
were usually around 1-3 mm.
2.2 Nail extraction and saponification
2.2.1 Method 1 DTT Digestion
Two toenail clipping samples from the same individual of approximately 50 mg each
were weighed out. One ml of 0.1 mg/ml aqueous solution of sodium laurate was added
into each sample and the samples were sonicated for 1 min and the soap solution was
decanted. The nail samples were added to approximately 1 ml of high purity water, and
sonicated for 1 min, and then the water was decanted. This procedure was repeated to
rinse the samples with water three times.
The two nail samples were then transferred to separate glass mortar vials. The samples
were labeled as “nail 1” and “nail 2”. Nail 2 was spiked with 1 µg of eicosapentaenoic
acid (EPA) and 1 µg of docosahexaenoic acid (DHA).
24
Two ml of 3 mg/ml dithiothreitol (DTT) in dichloromethane was added into each glass
mortar vial and the samples were treated by alternating between sonicating and
grinding. When the liquid in the mortar was turbid, the DTT/dichloromethane solution
was decanted to a clean glass culture tube and fresh DTT solution was added to the
remaining nail sample. The sonication and grinding process was continued until all the
clippings were dissolved.
Afterwards, 2 ml of 0.5M KOH-CH3OH solution was added to the nail extracts [1, 55].
And the resulting mixture was vortexed for 4-5 min. The pH of the samples was then
adjusted to 2 by adding approximately 1.2 ml of 2.3 M HCl solution. The sample
solutions were then extracted with 0.5 ml of dichloromethane three times. The three
0.5 ml extracts were combined in a glass tube and the dichloromethane solution was
dried by passing the solution through 3 ml glass solid phase extraction (SPE) columns
that were packed with 1.0 g of anhydrous sodium sulfate.
2.2.2 Method 2 KOH Digestion
Toe nail clipping samples from the same individual of approximately 50 mg were
weighed out. In order to minimize exogenous contamination, the nail samples were
washed with both methanol and acetone [81]. Approximately 1 ml of methanol was
added into the sample and the sample was sonicated for 30 seconds and the methanol
was decanted. Approximately 1 ml of acetone was then added into the sample and
sonicated for another 30 seconds and the acetone was then removed.
25
The nail sample was then softened/dissolved and the fatty acids were saponified using
potassium hydroxide as follows.
Two ml of 1 M KOH solution [81, 82] was added to the nail sample and the sample was
heated in water bath at 90 °C for 15 min. After the sample was taken out of the water
bath and allowed to cool to room temperature, the sample was split into two and
labeled as “nail 1” and “nail 2”. Nail 2 was spiked with 1 µg eicosapentaenoic acid (EPA)
and 1 µg docosahexaenoic acid (DHA).
The pH of the samples was then adjusted to 2 by adding 0.50 ml of 2.3 M HCl solution.
The sample solutions were then extracted with 0.50 ml of dichloromethane three times.
The three 0.50 ml extracts were combined in a glass tube and the dichloromethane
solution was dried by passing the solution through 3 ml glass solid phase extraction (SPE)
columns that were packed with 1.0 g of anhydrous sodium sulfate.
2.3 Methylation
The fatty acids in the dichloromethane were methylated using a standard literature
procedure [83, 84]. 1.0 ml CH3OH and 1.0 ml 14% BF3/CH3OH was added to the dried
dichloromethane extract and the solution was then heated at 90 °C in a water bath for
15 min. After cooling down to room temperature, 1.0 ml of high purity water was added
into each sample to react with any remaining BF3. The samples were then vigorously
shaken and the FAMEs were extracted twice (0.50 ml hexane each time) into hexane.
26
Trace amount of water was removed from the hexane extracts by passing the samples
through 3 ml glass SPE columns packed with 1.0 g of anhydrous sodium sulfate. The
resulting hexane samples were purged with nitrogen until the volume of each was about
0.5 ml.
2.4 Gas chromatography-mass spectrometry
The FAMEs were analyzed by the GC/MS (Model 6890 GC combined with Agilent 5973N
mass selective detector) system which was used in both scan and SIM modes. The gas
chromatography was equipped with a SGE BPX70 column measuring 30 m × 0.15 mm
with a film thickness of 0.15 µm. Helium was used as the carrier gas at a total flow rate
of 19.2 ml/min, at a constant pressure of 45 psi. Splitless injection was chosen as the
injection mode because omega-3 fatty acids are not abundant in human nails. The initial
oven temperature was 100 °C, and the temperature of the column was then increased
to 250 °C at a rate of 4 °C/min, and then held at 250 °C for 5 minutes. Following this
pretreatment, 2 µl of sample was injected onto the GC column.
Calibration curves of EPA and DHA methyl esters were created from a series of
concentrations (0.50, 2.0, 5.0, 10, 25, 50 µg/ml) of standard solutions in heptane. And
the calibration curve of heptadecanoic acid methyl ester was made using seven
concentrations (0.50, 2.0, 5.0, 10, 25, 50, 100 µg/ml) of standard solutions in hexane.
EPA and DHA methyl ester standard solutions:
27
75.0 µl of 10 mg/ml standard solution was transferred into a vial using a syringe, and
diluted with heptane to 1500 µl to get 500 µg/ml solution. Then 20.0, 10.0, 4.0, 2.0, 0.80
and 0.20 µl of 500 µg/ml solution were transferred to different vials, and diluted with
heptane to 200 µl to obtain 50, 25, 10, 5.0, 2.0, 0.50 µg/ml of standard solutions.
Heptadecanoic acid methyl ester standard solutions:
Heptadecanoic acid methyl ester standard of 0.0200 g was weighed out and dissolved in
2000 µl of hexane to get 10 mg/ml solution. Then similar steps were followed to make
0.50, 2.0, 5.0, 10, 25, 50, 100 µg/ml of standard solutions in hexane.
28
CHAPTER 3 RESULTS
1. CALIBRATION CURVES OF EPA AND DHA METHYL ESTERS USING
SELECTED IONS
Calibration curves of EPA and DHA methyl esters were developed from a series of
concentrations (0.5, 2, 5, 10, 25, 50 µg/ml) of standard solutions in heptane (purchased
from Sigma-Aldrich).
Table 7 Extracted ion chromatograms of EPA and DHA methyl esters
Concentration (µg/ml)
EPA m/z = 79 Peak Area
DHA m/z = 79 Peak Area
0.50
217830
50680
2.0
601948
513478
5.0
1230072
1814542
10
2525856
3081308
25
10123970
4556915
50
18359908
8798547
29
Calibration curve of EPA methyl ester m/z 79
Peak Area
20000000
y = 37968x - 34358
R² = 0.991
15000000
10000000
5000000
0
0
10
20
30
40
50
60
Concentration (µg/ml)
Figure 7 Calibration curve of EPA methyl ester in SIM mode
Peak Area
Calibration curve of DHA methyl ester m/z 79
25000000
20000000
15000000
10000000
5000000
0
y = 40017x - 29301
R² = 0.997
0
10
20
30
40
50
Concentration (µg/ml)
Figure 8 Calibration curve of DHA methyl ester in SIM mode
30
60
79
100
91
67
50
O
105
55
O
133
175
201
237 255 273
301
60
90
120
150
180
210
240
270
(mainlib) 5,8,11,14,17-Eicosapentaenoic acid, methyl ester, (all-Z)-
300
0
330
Figure 9 Mass spectrum of EPA methyl ester (NIST Mass Spectral Library)
79
100
O
67
50
119
55
O
131
159
0
185 206 224 243 261 281 299
60
90
120
150 180
210 240
270 300
(mainlib) 4,7,10,13,16,19-Docosahexaenoic acid, methyl ester, (all-Z)-
330
Figure 10 Mass spectrum of DHA methyl ester (NIST Mass Spectral Library)
2. NAIL DIGESTION
In nail extraction method 1 which was developed by Mr. Paul Peterman to separate and
analyze a wide variety of persistent organic pollutants from nail clippings, we used DTT
to dissolve and soften the nail before further treatment. DTT is useful because nail, like
hair, is mainly composed of keratin [50, 85], which contains large amounts of cysteine
and disulfide bonds. The nail matrix consists of highly cross-linked structures like fiber
31
which makes the nail tough and difficult to dissolve. DTT is a commonly used reagent for
reducing disulfide bonds of proteins at pH > 7, and preventing the intra- and
intermolecular formation of disulfide bonds between cysteine residues [74-76]. The
chemical structure of DTT is shown in Figure 11. And the reductive cleavage mechanism
of the S - S bond with DTT is shown in Figure 12.
Figure 11 Chemical structure of dithiothreitol (DTT)
Figure 12 Reaction between DTT and a disulfide bond [74]
The DTT softening/dissolution procedure was developed as a “gentle” chemical
approach to extract a wide variety of organic compounds for the nail matrix. It has been
used successfully by our group to extract and analyze ultra-trace levels of persistent
organic pollutants (e.g. DDE, HCB, chlordanes, and PCBs), polybrominateddiphenyl
ethers, triclosan-methyl ether, and transient phenolic compounds (e.g. triclosan).
However, the DTT nail extraction method did not work for the analysis of PUFAs in the
32
nail matrix. The spiked PUFAs in the nail samples digested by DTT were not seen in the
GC spectra.
Figure 13 shows the GC spectrum of the spiked nail sample.
Figure 13 GC spectrum of the nail sample (spiked with free EPA and DHA) digested by
DTT
All the major peaks in the spectrum are listed in Table 8. Most of them are saturated
fatty acid methyl esters, only C15H28O2 has one carbon-carbon double bond.
33
Table 8 Major peaks in the spiked nail sample digested by DTT
Retention time (min) Formula
Name
11.686
C15H30O2
Tetradecanoic acid, methyl ester
12.217
C15H28O2
cis-9-Tetradecenoic acid, methyl ester
12.966
C16H32O2 (branched)
Methyl 13-methyltetradecanoate
13.596
C16H32O2
Pentadecanoic acid, methyl ester
15.649
C17H34O2
Palmitic acid, methyl ester
17.345
C18H36O2
Margaric acid methyl ester
19.386
C19H38O2
Octadecanoic acid, methyl ester
20.872
C20H40O2
Nonadecanoic acid, methyl ester
22.568
C21H42O2
Eicosanoic acid, methyl ester
24.152
C22H44O2
Heneicosanoic acid, methyl ester
25.757
C23H46O2
Docosanoic acid, methyl ester
Our hypothesis is that the carbon-carbon double bonds in the PUFAs were also reduced
by DTT. The following series of experiments were conducted to confirm this hypothesis.
First, the possibility that the methylation procedure degrades the PUFAs was ruled out.
We skipped the digestion steps with DTT, but performed experiments on the
methylation of some free fatty acids (10 µg each), such as oleic acid (18:1n-9), linoleic
acid (18:2n-6), arachidonic acid (20:4n-6), alpha-linolenic acid (18:3n-3), EPA (20:5n-3)
and DHA (22:6n-3). In every case the methylation was successful, regardless of whether
the fatty acids were processed alone or mixed together. Recoveries of over 60% were
obtained for EPA and DHA.
34
We then focused on the effect of DTT on the PUFAs. Three olive oil samples were
prepared as outlined below. Olive oil was chosen as the test matrix because it is a
mixture of triglycerides, including both saturated and unsaturated fatty acids.
Sample #1: 10 µg of olive oil + 5 ml of dichloromethane (DCM);
Sample #2: 10 µg of olive oil + 1 µg of free EPA + 1 µg of free DHA + 5 ml of DCM;
Sample #3: 10 µg of olive oil + 5 ml of DTT solution (3 mg/ml DTT in DCM).
Two ml of 0.5 M of KOH-CH3OH solution was added into each sample, and vortexed for
4-5 min. The pH of the samples was then adjusted to 2 by adding HCl solution. Then the
organic layer in each sample was extracted and dried by passing the solution through 3
ml glass solid phase extraction (SPE) columns that were packed with 1.0 g of anhydrous
sodium sulfate. The methylation procedure outlined in Chapter 2 was performed on the
samples prior to GC/MS analysis. As can be seen in the GC spectra shown in Figure 8, the
three major peaks (C17H34O2, C19H38O2 and C19H36O2) in olive oil were observed in all of
the samples. Among these components, C17H34O2 and C19H38O2 are saturated, and
C19H36O2 has one C = C bond. In sample #2 without DTT treatment, EPA and DHA peaks
were also observed.
35
Figure 14 GC spectrum of olive oil sample without DTT. From left to right, C17H34O2,
C19H38O2 and C19H36O2
Having demonstrated successful recovery of spiked EPA and DHA from olive oil, the
same procedure was repeated with the addition of DTT. Three samples were prepared.
Sample #1: 10 µg of olive oil + 5 ml of DTT solution (3 mg/ml DTT in DCM) + 10 µg of free
EPA + 10 µg of free DHA;
Sample #2: 10 µg of olive oil + 5 ml of DTT solution (3 mg/ml DTT in DCM) + 1 µg of free
EPA + 1 µg of free DHA;
Sample #3: 10 µg of olive oil + 5 ml of DTT solution (3 mg/ml DTT in DCM).
When DTT was added to the samples, neither EPA nor DHA was detected even when the
spike of each PUFA was increased to 10 µg. From these experiments, we concluded that
not only S - S bonds in keratin, but also C = C bonds in the PUFAs are reduced by DTT.
This conclusion is supported by the literature.
36
The reaction between DTT and carbon-carbon double bonds has been known as thiolene reaction for over 100 years, which evolves the addition of an S - H group to a C = C
bond [86], Figure 15.
Figure 15 General thiol-ene coupling [87]
This reaction is known to be very rapid at standard laboratory conditions with
quantitative yields when a photoinitiator is used [87-91], Figure 16.
Figure 16 The mechanism for thiol-ene reaction in the presence of a photoinitiator and
hν
Our hypothesis is that the thiol-ene reaction is occurring with DTT under our conditions.
Because the spiked PUFAs could not be observed when DTT was used, it was necessary
37
to find other methods to digest nails. Fortunately, nail extraction method 2 with KOH
worked effectively.
3. RECOVERIES OF EPA AND DHA IN NAIL SAMPLES
The nail sample (nail 2) was spiked with 1 µg of free EPA and 1 µg of free DHA. The total
volume of the nail sample was 0.43 ml. The recoveries are calculated as follows.
Table 9 Recoveries of EPA and DHA in nail sample
EPA methyl ester
1st run
2nd run
m/z 79 peak area
196132
205129
DHA methyl ester
1st run
2nd run
m/z 79 peak area
93642
111550
Concentration
(µg/ml)
1.421
1.445
Concentration
(µg/ml)
0.966
1.011
Mass (µg)
0.618
0.629
% Recovery
59.1
60.1
Mass (µg)
0.420
0.440
% Recovery
40.3
42.2
Unfortunately, we did not see EPA or DHA in the nail samples which were not spiked.
However, there were lots of saturated fatty acids present in the nail samples. We
decided to look at the levels of heptadecanoic acid (C17:0) in human tissues, and then
its ratio to omega-3 fatty acids, so that we would be able to estimate the amount of
omega-3 we should expect to see in the nail. The reason for choosing C17:0 is that it is
present at high amounts in the nail, and its methyl ester standard commercially
available.
38
4. CALIBRATION CURVE AND RECOVERY OF METHYL
HEPTADECANOATE (C17:0 METHYL ESTER)
4.1 Calibration curve
The calibration curve of methyl heptadecanoate was made by seven concentrations (0.5,
2, 5, 10, 25, 50, 100 µg/ml) of standard solutions in hexane (purchased from SigmaAldrich).
Calibration Curve of C17:0 methyl ester
Peak Area
200000000
y = 2E+06x + 596226
R² = 0.9984
150000000
100000000
50000000
0
0
20
40
60
80
Concentration (µg/ml)
Figure 17 Calibration curve of methyl heptadecanoate
39
100
74
100
87
O
50
O
55
0
97 111
143
151 166 185
60
90
120
150
(mainlib) Heptadecanoic acid, methyl ester
180
210
284
241
223
240
270
300
Figure 18 Mass spectrum of methyl heptadecanoate
According to the calibration curve and the peak area in the nail spectrum, there is
approximately 0.9 - 1.3 µg of C17:0 methyl ester in 25 mg of nail (Table 10).
Table 10 The amount of C17:0 observed in the nail sample
C17:0 methyl ester
1st run
2nd run
3rd run
Peak Area
3070863
3751104
3950445
Concentration (µg/ml)
1.237
1.577
1.677
Mass (µg)
per 25 mg of nail
0.942
1.202
1.278
4.2 C17:0 methyl ester recovery
We started with 5 µg of C17:0 fatty acid, followed the same procedure as nail samples,
and tested its recovery.
40
Table 11 The recovery of C17:0 methyl ester
C17:0
methyl ester
1st run
2nd run
3rd run
Peak Area
3042249
3476244
4060223
Concentration
(µg/ml)
1.491
1.708
2.000
Volume (ml)
1.145
1.388
1.124
Mass (µg)
1.707
2.371
2.248
% Recovery
32.4
45.1
42.7
Using the recoveries reported in Table 11, the amount of C17:0 in the 25 mg of nail
sample is on the order of 2.7 µg.
Table 12 shows C17:0, EPA and DHA levels in plasma phospholipids for the controls in a
prostate cancer study conducted between 1992 and 2000 across Europe [92]. Over
130,000 participants provided a blood sample for gas chromatography measurements.
According to their data, C17:0 accounts for 0.36-0.41 mol%, EPA (20:5n-3) accounts for
0.78-1.64 mol%, and DHA (22:6n-3) accounts for 3.77-4.81 mol% in plasma
phospholipids. Based upon these values and the estimated amount of C17:0, there is
supposed to be on the order of 9.5 µg of EPA and 36.5 µg of DHA in our nail sample.
There is also research on the distribution of fatty acids in erythrocyte membrane [93].
Table 12 shows some of the reported results. Among the referents, EPA and DHA
account for 1.37% and 4.83% of total fatty acids, respectively, in erythrocyte membrane;
values which fall in about the same range as they do in plasma phospholipids [92].
Likewise C17:0 is about 0.42% of total fatty acids in erythrocyte membranes, which is
again close to the value reported in plasma phospholipids.
41
Table 12 Fatty acid composition of plasma phospholipids by country*
Fatty Acid
C17:0
C20:5n-3
C22:6n-3
mol%
(Heptadecanoic acid)
(EPA)
(DHA)
Denmark (n = 292)
0.38 (0.37, 0.39)
1.64 (1.54, 1.73)
4.59 (4.45, 4.74)
Germany (n = 203)
0.40 (0.38, 0.41)
1.05 (0.98, 1.12)
3.79 (3.65, 3.93)
Greece (n = 9)
0.39 (0.33, 0.46)
0.78 (0.56, 1.08)
4.20 (3.53, 5.00)
Italy (n = 61)
0.36 (0.34, 0.38)
0.81 (0.71, 0.92)
3.77 (3.53, 4.03)
Netherlands
0.36 (0.32, 0.39)
1.20 (0.98, 1.46)
4.53 (4.08, 5.03)
Spain (n = 95)
0.38 (0.36, 0.40)
0.94 (0.85, 1.05)
4.81 (4.55, 5.07)
Sweden (n = 198)
0.38 (0.37, 0.40)
1.53 (1.42, 1.64)
4.37 (4.20, 4.54)
United Kingdom
0.41 (0.39, 0.42)
1.03 (0.95, 1.12)
4.03 (3.85, 4.21)
(n = 25)
(n = 178)
* All values are geometric mean; 95% CI in parentheses.
Table 13 Distribution of fatty acids in erythrocyte membrane
Fatty Acids
% of Total Fatty Acids (n = 291)
Mean ± SD
C17:0 (Heptadecanoic acid) 0.42 ± 0.06
C20:5n-3 (EPA)
1.37 ± 0.46
C22:6n-3 (DHA)
4.83 ± 1.02
42
In another study [33], Baylin et al. measured the distribution of fatty acids in adipose
tissue and in the diet. Some of their data are shown in Table 14. According to their
results and our estimated amount of C17:0, we would expect to see about 0.5 µg of EPA
and 2.2 µg of DHA in the nail sample if the ratio of C17:0 to omega-3 fatty acids are the
same in adipose tissue and in nail.
Table 14 Distribution of fatty acids in adipose tissue
Fatty Acids
% of Total Fatty Acids (n = 503)
Mean ± SD
C17:0 (Heptadecanoic acid) 0.21 ± 0.06
C20:5n-3 (EPA)
0.04 ± 0.02
C22:6n-3 (DHA)
0.17 ± 0.06
Based upon the recoveries of EPA and DHA obtained and the ratios between C17:0 to
omega-3 fatty acids in plasma phospholipids or erythrocyte membrane, we were
supposed to see at least 5.6 µg of EPA and 14.6 µg of DHA methyl esters in our nail
sample; or if calculated using the ratios in adipose tissue, there were about 0.30 µg of
EPA and 0.88 µg of DHA to be seen in our sample. Unfortunately, no EPA or DHA methyl
ester was detected.
43
5. LIMIT OF DETECTION
The limit of detection (LOD) was calculated as the mass of the methyl ester with a signal
equal to 3 times the standard deviation of the background counts. They were calculated
from nail 1 (the one without spike) and the standard curves of EPA and DHA methyl
esters in SIM mode. The total volume of the sample was 0.440 ml.
Table 15 Limit of detection
Fatty acid methyl ester
EPA methyl ester
DHA methyl ester
Concentration (µg/ml)
0.908
0.739
Mass (µg)
0.400
0.325
According to our estimate, there were about 0.30 µg of EPA and 0.88 µg of DHA to be
seen in our sample if the ratio of C17:0 to omega-3 fatty acids were the same in adipose
tissue and nail. Then we should not have seen EPA methyl ester in the spectrum.
However, the amount of DHA methyl ester should be above the detection limit, which
we did not see either.
6. FATTY ACIDS DETECTED IN THE NAIL SAMPLE
Although no EPA or DHA was detected, many other fatty acids were detected in the nail
samples. The majority of observed fatty acids in the nail clipping are saturated fatty
acids (Table 16).
44
Figure 19 GC spectrum of the nail sample
Table 16 Major peaks in the nail sample
Retention time (min) Formula
Name
11.661
C15H30O2
Tetradecanoic acid, methyl ester
12.233
C15H28O2
cis-9-Tetradecenoic acid, methyl ester
13.003
C16H32O2 (branched)
Methyl 13-methyltetradecanoate
13.579
C16H32O2
Pentadecanoic acid, methyl ester
14.596
C17H34O2 (branched)
Methyl isohexadecanoate
15.522
C17H34O2
Palmitic acid, methyl ester
15.975
C17H32O2
Methyl hexadec-9-enoate
16.785
C18H36O2 (branched)
Methyl 14-methylhexadecanoate
17.345
C18H36O2
Margaric acid methyl ester
19.238
C19H38O2
Octadecanoic acid, methyl ester
19.679
C19H36O2
Oleic acid, methyl ester
20.633
C19H34O2
Linoleic acid, methyl ester
22.584
C21H42O2
Eicosanoic acid, methyl ester
25.803
C23H46O2
Docosanoic acid, methyl ester
26.231
C23H44O2
Methyl 11-docosenoate
28.840
C25H50O2
Tetracosanoic acid, methyl ester
45
CHAPTER 4 DISCUSSION
We have successfully developed a method for measuring fatty acids in the nail by
GC/MS, although omega-3 fatty acids were not detected in our experiments. In order to
measure the trace amount of EPA and DHA in the nail, we need to improve the percent
recoveries and lower the detection limits, for example, using high sensitivity GC/MS,
such as ion trap or triple quadrupole gas chromatography-tandem mass spectrometry
(GC-MS/MS) [94, 95].
There has been research on fatty acid content in human skin conducted in Netherlands
[96]. They reported the presence of saturated fatty acids with chain length distribution
from C16 to C30 in the stratum corneum of human skin using LC/MS (Liquid
chromatography–mass spectrometry) technique. They also found several long chain
unsaturated fatty acids, such as C18:1 and C18:2 at low concentrations in the skin,
which have also been detected in our nail sample, as shown in Table 17.
Stratum corneum is a layer of dead cells at the outermost of epidermis in the skin. And
its major non-aqueous component is keratin [97]. Nail is a skin appendage. It does not
only have similar chemical composition to hair and skin, it also undergoes similar
46
changes to the epidermal cells forming stratum corneum [98, 99].This may explain the
presence of the saturated fatty acids and several monounsaturated fatty acids in our
results.
Table 17 Fatty acid distribution in human stratum corneum and human nail clippings
Fatty acid
distribution
Human stratum corneum
Human nail clippings
Saturated
fatty acids
C16:0, C18:0, C20:0, C21:0, C22:0,
C23:0, C24:0, C25:0, C26:0, C27:0,
C28:0, C29:0, C30:0
C14:0, C15:0, C16:0, C17:0,
C18:0, C20:0, C21:0, C22:0,
C24:0
Unsaturated C18:1, C18:2, C30:1, C32:1, C34:1
fatty acids
C14:1, C16:1, C18:1, C18:2,
C22:1
It is also reported that human hair, nail and sebum have some common fatty acids, such
as C16:0, C18:0 (which were detected in our nail sample) and some omega-6 fatty acids
[100]. The authors suggested a pathway to synthesis these omega-6 fatty acids in
human body and it requires further study. As it is known, sebum is secreted by
sebaceous glands, which exist everywhere in human skin, except palms and soles [101].
It is possible that some of the fatty acids detected in our research, for example C16:0
and C18:0, originated from the excretion of sebaceous glands, but it needs further
investigation.
47
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