Component Description
The
analysis of red blood cell (RBC) fatty acids (FA) is an indicator of long term
(4 months) FA status. Evaluation of
RBC fatty acid content has become increasingly favored as a measure of n-3 polyunsaturated fatty acids (PUFA) intake. PUFA alter membrane physical
characteristics and the activity of membrane-bound proteins. In membranes, they
interact with ion channels and can be converted into bioactive eicosanoid
(Harris, et al., 2008).
The
American Heart Association recommendation for those with no history of coronary
heart disease (CHD) is to eat two fatty fish servings per week and for those
with a history of CHD to consume 1 g/day of two omega-3 fatty acids,
eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid (Kris-Etherton, et al.,
2002). The Omega-3 Index is a measure of EPA (C20:5 n-3) and DHA (C22:6 n-3) in the
blood, and expressed as a percent of 21 fatty acids, in specifically the red
blood cell membranes (Harris, et al., 2004). The Omega-3 Index
has been suggested for use to identify those at risk for death from coronary
heart disease (Harris, et. al., 2004).
Eligible Sample
Examined participants aged 6 years and older were eligible.
Description of Laboratory Methodology
Esterified
FA are hydrolyzed from washed RBC membranes using sequential treatment with
mineral acid and base in the presence of heat. Using a modification of
Lagerstedt et al., total FA are hexane-extracted from the matrix (100 µL 50:50
saline diluted RBC) along with an internal standard solution containing
thirteen stable isotopically-labeled FA to account for recovery (Lagerstedt et.
al., 2001). 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 FA of interest from other matrix constituents. FA are detected using
electron capture negative-ion mass spectrometry within 34 minutes. Six
saturated FA (SFA), four monounsaturated FA (MUFA), and eleven polyunsaturated
FA (PUFA) (21 FA in total) are measured using selected ion monitoring.
Quantitation is accomplished by comparing the peak area ratio of the analyte to
internal standard in the unknown sample with the peak area ratio of a known
amount of analyte to internal standard in a calibrator solution.
Refer to the Laboratory Method Files section for
a detailed description on the laboratory methods used.
Laboratory Method Files
Fatty Acids – RBC
(December 2025)
Laboratory Quality Assurance and Monitoring
Washed
red blood cell specimens are 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 are stored under
appropriate frozen (–30°C) conditions until they are shipped to the National
Center for Environmental Health for testing.
The NHANES quality assurance and quality control (QA/QC) protocols meet the
1988 Clinical Laboratory Improvement Amendments. 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 QA evaluation during unscheduled 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 the 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/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
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. 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 additional re-weighting for item
non-response is necessary.
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.
Phlebotomy Weights
For the August 2021-August 2023 cycle,
analysis of nonresponse patterns for the phlebotomy component in the MEC
examination revealed differences by age group and race/ethnicity, among other
characteristics. For example, approximately 67% of children aged 1-17 years who
were examined in the MEC provided a blood specimen through phlebotomy, while
95% of examined adults aged 18 and older provided a blood specimen. Therefore,
an additional phlebotomy weight, WTPH2YR, has been included in this data
release to address possible nonresponse bias. Participants who are eligible but
did not provide a blood specimen have their phlebotomy weight assigned a value
of “0” in their records. The phlebotomy weight should be used for analyses that
use variables derived from blood analytes, and is included in all relevant data
files.
Demographic and Other Related Variables
The
analysis of NHANES laboratory data may require additional demographic
variables. The NHANES August 2021-August 2023
Demographics File contains demographic data, health
indicators, and other related information collected during household
interviews.
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 Demographics file and 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. The lower limit
of detection (LLOD, in mg/L) for the RBC fatty acids are shown below.
| Variable Name |
Analyte Description |
LLOD |
| LBXPAN |
alpha-Linolenic acid (C18:3n3) (%) |
0.03 |
| LBXP1A |
Arachidic acid (C20:0) (%) |
0.30 |
| LBXPRA |
Arachidonic acid (C20:4n6) (%) |
0.08 |
| LBXPDA |
Docosanoic acid (C22:0) (%) |
0.10 |
| LBXPHA |
Docosahexaenoic acid (C22:6n3) (%) |
0.20 |
| LBXPD3 |
Docosapentaenoic acid 3 (C22:5n3) (%) |
0.07 |
| LBXPD6 |
Docosapentaenoic acid 6 (C22:5n6) (%) |
0.08 |
| LBXPTA |
Docosatetraenoic acid (C22:4n6) (%) |
0.05 |
| LBXPED |
11,14-Eicosadienoic acid (C20:2n6) (%) |
0.05 |
| LBXP1E |
11-Eicosenoic acid (C20:1n9) (%) |
0.06 |
| LBXPPE |
Eicosapentaenoic acid (C20:5n3) (%) |
0.40 |
| LBXPLG |
gamma-Linolenic acid (C18:3n6) (%) |
0.07 |
| LBXPGH |
homo-gamma-Linolenic acid (C20:3n6) (%) |
0.06 |
| LBXP1G |
Tetracosanoic acid (C24:0) (%) |
0.10 |
| LBXPNL |
Linoleic acid (C18:2n6) (%) |
0.30 |
| LBXPMR |
Myristic acid (C14:0) (%) |
1.00 |
| LBXPNR |
15-Tetracosenoic acid (C24:1n9) (%) |
0.05 |
| LBXPOL |
Oleic acid (C18:1n9) (%) |
0.30 |
| LBXPPL |
Palmitoleic acid (C16:1n7) (%) |
0.05 |
| LBXPPM |
Palmitic acid (C16:0) (%) |
3.00 |
| LBXPST |
Stearic acid (C18:0) (%) |
9.00 |
This component does not
report data as mass concentrations (mg/L). Instead, data are reported as weight
percentages of total fatty acids
(see section on Calculating Percent RBC Fatty Acid). Two
variables are provided for each of these analytes. The variable name ending in
“LC” (ex., LBDPANLC) indicates whether the mass concentration result used in
the weight percentage calculation 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
LBX (ex., LBXPAN) provides the analytic weight percentage result for that
analyte. For analytes with mass concentration results below the lower limit of
detection (ex., LBDPANLC=1), the weight percentage values were set to missing because the weight percentage data
cannot be compared to the LLOD. 15 mass concentration results in
the dataset were below the LLOD and corresponding weight percentage values were
set to missing and their comment variables set to 1.
Calculating Percent RBC Fatty Acid
Fatty acid concentrations measured in
mg/L are summed for the total FA concentration (mg/L). Individual fatty acid
weight percentages are calculated by dividing the individual fatty acid
concentrations (mg/L) by the total FA (mg/L). The fatty acid data is expressed
as weight percentages of total fatty acids.