Component Description
The analysis of individual plasma or serum fatty acids is
important in the recognition of essential fatty acid deficiency (Siguel E.
1998) and in the differential diagnosis of inborn errors of metabolism, such as
mitochondrial fatty acid oxidation disorders (Costa, et. al., 1998; Jones, et.
al., 2000; Moser, et. al., 1998). Long-chain polyunsaturated fatty acids are
essential for normal development (Hamosh, et. al., 1998). The dietary content
of saturated, monounsaturated, and polyunsaturated fatty acids influence the
concentration of cholesterol in low-density and high-density lipoproteins, and
consequently the development of atherosclerosis (O’Keefe, et. al., 1995).
Regular consumption of or supplementation with omega-3 polyunsaturated fatty
acids can have beneficial effects on long-term cardiovascular health due to
anti-inflammatory and possibly antiarrhythmic effects (Kang, et. al., 1996).
The goal of this method is to obtain US reference ranges for most circulating
fatty acids. Public health recommendations advise increasing or decreasing the
intake of various classes of fatty acids (saturated, monounsaturated,
polyunsaturated) but relatively little fatty acid biomarker data exist to
support these recommendations and reference range data are scarce.
Eligible Sample
All examined participants aged 3 to 11 and participants
aged 12 and over who were examined in the morning session were
eligible.
Description of Laboratory Methodology
Esterified fatty acids are hydrolyzed primarily from
triglycerides, phospholipids and cholesteryl esters using sequential treatment
with mineral acid and base in the presence of heat. Using a modification of
(Lagerstedt, et. al., 2001), total fatty acids are hexane-extracted from the
matrix (100uL serum or plasma) along with an internal standard solution
containing eighteen stable isotopically-labeled fatty acids to account for
recovery. The extract is derivatized with pentafluorobenzyl bromide (PFBBr) in
the presence of triethylamine to form pentafluorobenzyl esters. The
reaction mixture is injected onto a capillary gas chromatograph column to
resolve individual fatty acids of interest from other matrix
constituents. Fatty acids are detected using electron capture negative-ion
mass spectrometry within 34 minutes. Eleven saturated, six monounsaturated, and
thirteen polyunsaturated fatty acids (thirty fatty acids in total) are measured
using selected ion monitoring. Quantitation is accomplished by comparing
the peak area of the analyte in the unknown with the peak area of a known
amount in a calibrator solution. Calculations are corrected based on the
peak area of the internal standard in the unknown compared with the peak area
of the internal standard in the calibrator solution.
Refer to the Laboratory Method Files section for a detailed
description of the laboratory methods used.
There
were no changes to the lab method, lab equipment, or lab site for this
component in the NHANES 2013-2014 cycle.
Laboratory Method Files
Fatty Acids - Serum
(March 2023)
Laboratory Quality Assurance and Monitoring
Serum specimens were processed, stored and shipped to the Division of
Laboratory Sciences, National Center for Environmental Health, Centers for
Disease Control and Prevention, Atlanta, GA for analysis.
Detailed
instructions on specimen collection and processing are discussed in the NHANES Laboratory Procedures Manual (LPM).
Vials were stored under appropriate frozen (-30°C) conditions until they were
shipped to National Center for Environmental Health for testing.
The NHANES quality
assurance and quality control (QA/QC) protocols meet the 1988 Clinical
Laboratory Improvement Amendments mandates. Detailed QA/QC instructions are
discussed in the NHANES
LPM.
Mobile Examination
Centers (MECs)
Laboratory team
performance is monitored using several techniques. NCHS and contract
consultants use a structured competency assessment evaluation during visits to
evaluate both the quality of the laboratory work and the QC procedures. Each
laboratory staff member is observed for equipment operation, specimen
collection and preparation; testing procedures and constructive feedback are
given to each staff member. Formal retraining sessions are conducted annually
to ensure that required skill levels were maintained.
Analytical
Laboratories
NHANES uses
several methods to monitor the quality of the analyses performed by the
contract laboratories. In the MEC, these methods include performing blind split
samples collected during “dry run” sessions. In addition, contract laboratories
randomly perform repeat testing on 2% of all specimens.
NCHS developed and
distributed a QC protocol for all CDC and contract laboratories, which outlined
the use of Westgard rules (Westgard, et. al., 1981) when testing NHANES
specimens. Progress reports containing any problems encountered during shipping
or receipt of specimens, summary statistics for each control pool, QC graphs,
instrument calibration, reagents, and any special considerations are submitted
to NCHS quarterly. The reports are reviewed for trends or shifts in the data.
The laboratories are required to explain any identified areas of concern.
All QC procedures
recommended by the manufacturers were followed. Reported results for all assays
meet the Division of Laboratory Sciences’ QA and QC performance criteria for
accuracy and precision, similar to the Westgard rules (Caudill, et. al., 2008).
Data Processing and Editing
The data were
reviewed. Incomplete data or improbable values were sent to the performing
laboratory for confirmation.
Analytic Notes
Refer to the 2013-2014 Laboratory Data Overview
for general information on NHANES laboratory data.
There are over 800 laboratory tests performed on NHANES participants.
However, not all participants provided biospecimens or enough volume for all
the tests to be performed. The specimen availability can also vary by age or
other population characteristics. For example, in 2013-2014, approximately
82% of children aged 1-17 years who were examined in the MEC provided a blood specimen through
phlebotomy, while 96% of examined adults aged 18 and
older provided a blood specimen. Analysts should evaluate the extent of missing data in the
dataset related to the outcome of interest as well as any predictor variables
used in the analyses to determine whether the data are useable without
additional re-weighting for item non-response.
Please refer to
the NHANES Analytic Guidelines
and the on-line NHANES Tutorial
for further details on the use of sample weights and other analytic issues.
Subsample weights
The
appropriate sample weights are provided in the variable WTFAS2YR in this data
file for all participants and should be used when analyzing these data.
Serum fatty acids were measured for all examined participants aged 3 to 11 years and a fasting subsample of participants 12 years and older. For
participants aged 3 to 11 years, their WTFAS2YR are equivalent to
their MEC exam sample weights. These participants have completed at least one
physical exam component in the MEC; therefore, they all have an exam sample
weight larger than “0,” regardless of their lab test results. For participants 12
years and older, special sample weights were created for the subsample. These
special weights accounted for the additional probability of selection into the
subsample, as well as the additional nonresponse to these lab tests. Therefore,
if participants 12 years and older were selected as part of the fasting subsample
but did not provide a blood specimen, they would have the subsample weight
value assigned as “0” in their records.
Demographic and
Other Related Variables
The analysis of
NHANES laboratory data must be conducted using the appropriate survey design
and demographic variables. The NHANES
2013-2014 Demographics File contains demographic data, health
indicators, and other related information collected during household interviews
as well as the sample design variables. The recommended procedure for variance
estimation requires use of stratum and PSU variables (SDMVSTRA and SDMVPSU,
respectively) in the demographic data file.
The Fasting Questionnaire File includes
auxiliary information, such as fasting status, length of fast, and the time of
venipuncture.
This laboratory
data file can be linked to the other NHANES data files using the unique survey
participant identifier (i.e., SEQN).
Detection Limits
The detection
limits were constant for all of the analytes in the data set. Two variables are
provided for each of these analytes. The variable name ending in “LC” (ex.,
LBDCAPLC) indicates whether the result was below the limit of detection: the
value “0” means that the result was at or above the limit of detection, “1”
indicates that the result was below the limit of detection. The other variable
prefixed URX (ex., LBXCAP) provides the analytic result for that analyte. For
analytes with analytic results below the lower limit of detection (ex., LBDCAPLC
=1), an imputed fill value was placed in the analyte results field. This value
is the lower limit of detection divided by the square root of 2 (LLOD/sqrt[2]).
The
lower limit of detection (LLOD in µmol/L) for serum fatty acids:
| Variable Name |
Analyte Description |
LLOD (µmol/L) |
| LBXCAP |
Capric acid (C10:0) (µmol/L) |
1.59 |
| LBXLAR |
Lauric acid (C12:0) (µmol/L) |
2.33 |
| LBXMR1 |
Myristic acid (14:0) (µmol/L) |
4.90 |
| LBXPEN |
Pentadecanoic acid (C15:0) (µmol/L) |
0.75 |
| LBXPM1 |
Palmitic acid (16:0) (µmol/L) |
78.1 |
| LBXMRG |
Margaric acid (C17:0) (µmol/L) |
3.36 |
| LBXST1 |
Stearic acid (18:0) (µmol/L) |
39.1 |
| LBXAR1 |
Arachidic acid (20:0) (µmol/L) |
0.82 |
| LBXDA1 |
Docosanoic acid (22:0) (µmol/L) |
0.68 |
| LBXTSA |
Tricosanoic acid (C23:0) (µmol/L) |
0.90 |
| LBXLG1 |
Lignoceric acid (24:0) (µmol/L) |
1.09 |
| LBXML1 |
Myristoleic acid (14:1n-5) (µmol/L) |
0.29 |
| LBXPL1 |
Palmitoleic acid (16:1n-7) (µmol/L) |
6.56 |
| LBXVC1 |
cis-Vaccenic acid (18:1n-7) (µmol/L) |
2.31 |
| LBXOL1 |
Oleic acid (18:1n-9) (µmol/L) |
17.7 |
| LBXEN1 |
Eicosenoic acid (20:1n-9) (µmol/L) |
0.87 |
| LBXNR1 |
Nervonic acid (24:1n-9) (µmol/L) |
0.69 |
| LBXLNA |
Linoleic acid (18:2n-6) (µmol/L) |
22.6 |
| LBXALN |
alpha-Linolenic acid (18:3n-3) (µmol/L) |
1.54 |
| LBXGLA |
gamma-Linolenic acid (18:3n-6) (µmol/L) |
0.42 |
| LBXSD1 |
Stearidonic acid (C18:4n-3) (µmol/L) |
0.24 |
| LBXED1 |
Eicosadienoic acid (20:2n-6) (µmol/L) |
0.31 |
| LBXHGL |
homo-gamma-Linolenic acid(20:3n-6)(µmol/L) |
1.14 |
| LBXET1 |
Eicosatrienoic acid (C20:3n-9) (µmol/L) |
0.39 |
| LBXARA |
Arachidonic acid (20:4n-6) (µmol/L) |
7.34 |
| LBXEPA |
Eicosapentaenoic acid (20:5n-3) (µmol/L) |
0.79 |
| LBXDTA |
Docosatetraenoic acid (22:4n-6) (µmol/L) |
0.31 |
| LBXDP3 |
Docosapentaenoic acid (22:5n-3) (µmol/L) |
0.55 |
| LBXDP6 |
Docosapentaenoic acid (22:5n-6) (µmol/L) |
0.24 |
| LBXDHA |
Docosahexaenoic acid (22:6n-3) (µmol/L) |
1.84 |