Table of Contents

Locator Record

Title: Fatty acids concentration in plasma (SSFA_C )
Contact Number: 1-866-441-NCHS
Years of Content: 2003 - 2004
First Published: August, 2011
Revised: NA
Access Constraints: None
Use Constraints: None
Geographic Coverage: National
Subject:Fatty acids concentration in plasma
Record Source: NHANES 2003 - 2004
Survey Methodology: NHANES 2003 - 2004 is a stratified multistage probability sample of the civilian non-institutionalized population of the U.S.
Medium: NHANES Web site; SAS transport files

Component Description

Measurement of concentrations (µmol/L) of 24 fatty acids in a 1,845 sample subset of NHANES 2003-2004 plasma samples.

Eligible Sample

Fasting (≥ 8 hours) participants ≥ 20 years of age from NHANES 2003-2004.

Description of Laboratory Methodology

Measurement of Plasma Fatty Acids

Modification of the method of Lagerstedt et al. was used to measure total fatty acid concentrations in plasma. In brief, a 100µL plasma sample was spiked with 100µL of a mixture of 11 internal standards (fatty acids labeled with stable isotopes) to account for recovery. Esterified fatty acids were hydrolyzed from lipids such as triglycerides, phospholipids and cholesteryl esters using sequential treatment with acid then base. Following base hydrolysis, the samples were re-acidified and total fatty acids were hexane-extracted from the matrix along with internal standards. Extracts were derivatized with pentafluorobenzyl bromide in the presence of triethylamine to form pentafluorobenzyl (PFB) esters and were reconstituted in hexane.

PFB-fatty acid derivatives were injected onto a capillary gas chromatographic column to resolve individual cis-fatty acids of interest from other matrix constituents. Analytes were detected using electron capture negative-ion mass spectrometry. Twenty-four cis-fatty acids (6 saturated- (SFA), 7 monounsaturated- (MUFA), and 11 polyunsaturated- (PUFA)) were quantified using selected ion monitoring; for each fatty acid, recovery was estimated and results were adjusted using the most appropriate isotopically-labeled internal standard.

Separations were carried-out on a 0.25mm × 60m (0.25µm film thickness) cyanopropyl-methylpolysiloxane phase (50:50) capillary column at a constant flow rate of 2 mL/min helium carrier gas. Injection volume per sample was 1µL; split ratios varied from 50:1 to 150:1. Injector temperature was maintained at 240°C. The column heating oven started at 170°C then ramped 30°C/min to 230°C and held for 10 min. The temperature then ramped 2°C/min to 250ºC and held for 4.5 minutes. All ramping of temperatures was linear. The total run time was 26.5 minutes per sample. 

Methane (reagent gas) flow rate was set at 40% of the total flow (actual flow: 2mL/min). The mass spectrometer source was set at 170°C and the quadrupole was set at 150°C.

The acquisition method operated in negative ion mode. A selected ion monitoring (SIM) mass was monitored for each analyte and isotopically labeled standard. The 35 SIM masses were divided into SIM segments (5-7 SIM masses per segment) based on retention time. In some segments enhanced detector sensitivity was used to quantitate low abundance fatty acids.

Fragmentation of PFB-fatty acid esters using negative chemical ionization (NCI) resulted in a reproducible loss of the PFB moiety giving a stable carboxylate anion (mass minus 1 amu). Filament voltage, inlet temperature, percent reagent gas flow, and source temperature were optimized to yield optimum intensity of selected compounds.

Internal standards were labeled with deuterium or carbon-13.

Internal Standards
 Fatty Acid Internal Standard  SIM Mass
 Myristic-d27  254.5
 Palmitic-d31  286.6
 Palmitoleic-d14  267.4
 Stearic-d35  318.6
 13C-Oleic  299.5
 13C-Linoleic  297.5
 alpha-Linolenic-d14  291.4
 Arachidic-d39  350.3
 Arachidonic-d8  311.4
 Eicosapentaenoic-d5  306.4
 Docosahexaenoic-d5  332.4

In the following table, SIM masses are grouped by the internal standard that was used to normalize recovery for the group of fatty acids. Lipid Name indicates the carbon number in the long chain: number of double bonds in the carbon chain and the n- position of the bond closest to the terminal carbon (farthest from the carbonyl carbon).

SIM masses and limits of detection (LOD in µmol/L) for 24 fatty acids
Internal Standard Fatty Acid Fatty Acid Code SIM Mass Lipid Name LOD
Myristic-d27 Myristic SSMR1_N 227.4 C14:0 1.6
Myristic-d27 Myristoleic SSML1_N 225.3 C14:1 n-5 0.1
Palmitic-d31 Palmitic SSPM1_N 255.45 C16:0 8.2
Palmitoleic-d14 Palmitoleic SSPL1_N 253.45 C16:1 n-7 0.6
Stearic-d35 Stearic SSST1_N 283.4 C18:0 23.7
13C-Oleic Oleic SSOL1_N 281.5 C18:1 n-9 5.2
13C-Oleic cis-Vaccenic SSVC1_N 281.5 C18:1 n-7 0.3
13C-Linoleic Linoleic SSLNA_N 279.4 C18:2 n-6 2.2
alpha-Linolenic-d14 gamma-Linolenic SSGLA_N 277.4 C18:3 n-6 0.1
alpha-Linolenic-d14 alpha-Linolenic SSALN_N 277.4 C18:3 n-3 0.2
alpha-Linolenic-d14 homo-gamma-Linolenic SSHGL_N 305.4 C20:3 n-6 0.2
Arachidic-d39 Arachidic SSAR1_N 311.4 C20:0 0.6
Arachidic-d39 Eicosenoic SSEN1_N 309.4 C20:1 n-9 0.2
Arachidic-d39 Eicosadienoic SSED1_N 307.4 C20:2 n-6 0.1
Arachidonic-d8 Arachidonic SSARA_N 303.4 C20:4 n-6 0.3
Eicosapentaenoic-d5 Eicosapentaenoic SSEPA_N 301.4 C20:5 n-3 0.1
Eicosapentaenoic-d5 Docosanoic SSDA1_N 339.4 C22:0 0.2
Eicosapentaenoic-d5 Docosenoic SSDE1_N 337.4 C22:1 n-9 0.3
Docosahexaenoic-d5 Docosatetraenoic SSDTA_N 331.4 C22:4 n-6 0.2
Docosahexaenoic-d5 Docosapentaenoic-6 SSDP6_N 329.4 C22:5 n-6 0.1
Docosahexaenoic-d5 Docosapentaenoic-3 SSDP3_N 329.4 C22:5 n-3 0.2
Docosahexaenoic-d5 Docosahexaenoic SSDHA_N 327.4 C22:6 n-3 0.1
Docosahexaenoic-d5 Lignoceric SSLG1_N 367.4 C24:0 0.1
Docosahexaenoic-d5 Nervonic SSNR1_N 365.4 C24:1 n-9 0.4

 

Quantitation was accomplished by comparing the peak area of the analyte in the unknown with peak areas of known amounts of fatty acid based on calibration curves. Calibration curves were prepared for each assay using four levels (Cal 10 - Cal 40) per fatty acid. Calculations were 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. Best fits of concentration versus response were obtained when 1/x2 was used for all fatty acids except palmitic which used a quadratic fit to calibrate the assay. Peak area ratios (quantitation m/z: internal standard m/z) were linearly related to concentration. Calibration curves were required to have r2 of at least 0.98 and calibrators were required to be within 15% of their nominal values. The overall average r2 (SD) of calibration curves, including all analytes in all assays, was 0.999 (0.002).

Blanks were analyzed concurrently with the samples and were used to determine if any analyte contamination had occurred. Blanks were required to be less than 20% of the lowest calibrator (Cal 10).

Concentrations of fatty acids in the four calibration standards
Fatty Acid Code Saturation Calibrator Cal 40 (µmol/L) Calibrator Cal 30 (µmol/L) Calibrator Cal 20 (µmol/L) Calibrator Cal 10 (µmol/L)
SSLG1_N SFA 183 45.7 18.3 7.3
SSAR1_N SFA 201 50.2 20.1 8
SSDA1_N SFA 202 50.4 20.2 8.1
SSMR1_N SFA 638 160 63.8 25.5
SSST1_N SFA 1975 494 198 79
SSPM1_N SFA 8269 2067 827 331
SSEN1_N MUFA 52 12.9 5.2 2.1
SSDE1_N MUFA 54 13.4 5.4 2.1
SSML1 _N MUFA 98 24.5 9.8 3.9
SSVC1_N MUFA 825 206 82.5 33
SSNR1_N MUFA 1031 258 103 41.2
SSPL1_N MUFA 1204 301 120 48.2
SSOL1_N MUFA 6323 1581 632 253
SSED1_N PUFA 52 13.1 5.2 2.1
SSDTA_N PUFA 105 26.3 10.5 4.2
SSDP6_N PUFA 108 26.9 10.8 4.3
SSGLA_N PUFA 209 52.4 20.9 8.4
SSDP3_N PUFA 213 53.3 21.3 8.5
SSHGL _N PUFA 263 65.9 26.3 10.5
SSALN _N PUFA 480 120 48 19.2
SSEPA_N PUFA 822 206 82.2 32.9
SSDHA_N PUFA 1044 261 104 41.8
SSARA_N PUFA 2143 536 214 85.7
SSLNA _N PUFA 9329 2332 933 373

Data Processing and Editing

Data were received after all the laboratory testing was complete. The data were not edited for extreme values.

Data Access: All final data are publicly available.

Laboratory Quality Assurance and Monitoring

The laboratory and method were certified according to the Clinical Laboratory Improvement Amendment (1988) guidelines

Units of plasma were obtained from Solomon Park Research Laboratories (Kirkland, WA) and were screened using the methodology described above to obtain the concentrations of each of 24 fatty acids per unit. All quality control pools were prepared by blending units to achieve as best as possible, representative low, medium and high concentrations. Aliquots were dispensed into 2mL cryovials and stored at -70°C.

Before each analytical run, calibration standards, bench QC materials and plasma samples were thawed from -70°C to room temperature. Calibrators and QC materials were prepared and analyzed in parallel with the unknown samples.

Typical assays consisted of 4 calibration samples, 1 blank, 3 bench quality control samples, 34 subject person’s plasma followed by 3 quality control samples. Typically, one in every 20 subject person’s plasma in the assay was actually a blind QC pool which was labeled to resemble a subject person specimen. Two blind QC pools were used throughout the period of analysis of the survey specimens. Their results were required to be within 3 standard deviations of the characterization means for each analyte. Blind QC pools were characterized in singlicate in >40 assays. Mean CV (SD) for all analytes in both blind QC pools was 9% (10%); excluding DE1_N, mean CV (SD) was 7% (3%).

Bench QC pools (open label) were named as follows:
High: HP_066209_QC05
Low: LP_06553_QC05
Medium: MP_066208_QC05
Bench QC pools were characterized to determine the quality control limits by analyzing duplicate samples in 22 assays from 1/13/2010 to 3/24/2010. After establishment of the control limits of the pools, QC samples contained within each analytical run were evaluated for validity by use of a multi-rule quality control. Mean CV (SD) for all analytes in the three bench QC pools was 8% (10%); excluding DE1_N, mean CV (SD) was 6% (2%).

Analytic Notes

Subsample weights

Measures of fatty acids were measured in a one half fasting subsample of persons 20 years and over. Special sample weights are required to analyze these data properly. Specific sample weights for this subsample are included in this data file and should be used when analyzing these data.

References

Codebook and Frequencies

SEQN - Respondent sequence number

Variable Name:
SEQN
SAS Label:
Respondent sequence number
English Text:
Respondent sequence number.
Target:
Both males and females 20 YEARS - 150 YEARS

SSMR1_N - Myristic acid (micromol/L)

Variable Name:
SSMR1_N
SAS Label:
Myristic acid (micromol/L)
English Text:
Myristic acid (micromol/L)
Target:
Both males and females 20 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
24.8 to 2410 Range of Values 1817 1817
. Missing 28 1845

SSML1_N - Myristoleic acid (micromol/L)

Variable Name:
SSML1_N
SAS Label:
Myristoleic acid (micromol/L)
English Text:
Myristoleic acid (micromol/L)
Target:
Both males and females 20 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
0.19 to 186 Range of Values 1829 1829
. Missing 16 1845

SSPM1_N - Palmitic acid (micromol/L)

Variable Name:
SSPM1_N
SAS Label:
Palmitic acid (micromol/L)
English Text:
Palmitic acid (micromol/L)
Target:
Both males and females 20 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
1250 to 17800 Range of Values 1826 1826
. Missing 19 1845

SSPL1_N - Palmitoleic acid (micromol/L)

Variable Name:
SSPL1_N
SAS Label:
Palmitoleic acid (micromol/L)
English Text:
Palmitoleic acid (micromol/L)
Target:
Both males and females 20 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
39 to 2500 Range of Values 1826 1826
. Missing 19 1845

SSST1_N - Stearic acid (micromol/L)

Variable Name:
SSST1_N
SAS Label:
Stearic acid (micromol/L)
English Text:
Stearic acid (micromol/L)
Target:
Both males and females 20 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
317 to 2970 Range of Values 1827 1827
. Missing 18 1845

SSOL1_N - Oleic acid (micromol/L)

Variable Name:
SSOL1_N
SAS Label:
Oleic acid (micromol/L)
English Text:
Oleic acid (micromol/L)
Target:
Both males and females 20 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
831 to 23700 Range of Values 1819 1819
. Missing 26 1845

SSVC1_N - cis-Vaccenic acid (micromol/L)

Variable Name:
SSVC1_N
SAS Label:
cis-Vaccenic acid (micromol/L)
English Text:
cis-Vaccenic acid (micromol/L)
Target:
Both males and females 20 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
54.1 to 1670 Range of Values 1781 1781
. Missing 64 1845

SSLNA_N - Linoleic acid (micromol/L)

Variable Name:
SSLNA_N
SAS Label:
Linoleic acid (micromol/L)
English Text:
Linoleic acid (micromol/L)
Target:
Both males and females 20 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
1190 to 14200 Range of Values 1827 1827
. Missing 18 1845

SSGLA_N - gamma-Linolenic acid (micromol/L)

Variable Name:
SSGLA_N
SAS Label:
gamma-Linolenic acid (micromol/L)
English Text:
gamma-Linolenic acid (micromol/L)
Target:
Both males and females 20 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
5.55 to 230 Range of Values 1816 1816
. Missing 29 1845

SSALN_N - alpha-Linolenic acid (micromol/L)

Variable Name:
SSALN_N
SAS Label:
alpha-Linolenic acid (micromol/L)
English Text:
alpha-Linolenic acid (micromol/L)
Target:
Both males and females 20 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
13.3 to 578 Range of Values 1822 1822
. Missing 23 1845

SSHGL_N - homo-gamma-Linolenic acid (micromol/L)

Variable Name:
SSHGL_N
SAS Label:
homo-gamma-Linolenic acid (micromol/L)
English Text:
homo-gamma-Linolenic acid (micromol/L)
Target:
Both males and females 20 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
45.9 to 758 Range of Values 1827 1827
. Missing 18 1845

SSAR1_N - Arachidic acid (micromol/L)

Variable Name:
SSAR1_N
SAS Label:
Arachidic acid (micromol/L)
English Text:
Arachidic acid (micromol/L)
Target:
Both males and females 20 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
6.25 to 83.7 Range of Values 1777 1777
. Missing 68 1845

SSEN1_N - Eicosenoic acid (micromol/L)

Variable Name:
SSEN1_N
SAS Label:
Eicosenoic acid (micromol/L)
English Text:
Eicosenoic acid (micromol/L)
Target:
Both males and females 20 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
2.89 to 714 Range of Values 1826 1826
. Missing 19 1845

SSED1_N - Eicosadienoic acid (micromol/L)

Variable Name:
SSED1_N
SAS Label:
Eicosadienoic acid (micromol/L)
English Text:
Eicosadienoic acid (micromol/L)
Target:
Both males and females 20 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
5.66 to 383 Range of Values 1826 1826
. Missing 19 1845

SSARA_N - Arachidonic acid (micromol/L)

Variable Name:
SSARA_N
SAS Label:
Arachidonic acid (micromol/L)
English Text:
Arachidonic acid (micromol/L)
Target:
Both males and females 20 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
226 to 2240 Range of Values 1828 1828
. Missing 17 1845

SSEPA_N - Eicosapentaenoic acid (micromol/L)

Variable Name:
SSEPA_N
SAS Label:
Eicosapentaenoic acid (micromol/L)
English Text:
Eicosapentaenoic acid (micromol/L)
Target:
Both males and females 20 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
6.03 to 371 Range of Values 1827 1827
. Missing 18 1845

SSDA1_N - Docosanoic acid (micromol/L)

Variable Name:
SSDA1_N
SAS Label:
Docosanoic acid (micromol/L)
English Text:
Docosanoic acid (micromol/L)
Target:
Both males and females 20 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
17 to 171 Range of Values 1759 1759
. Missing 86 1845

SSDE1_N - Docosenoic acid (micromol/L)

Variable Name:
SSDE1_N
SAS Label:
Docosenoic acid (micromol/L)
English Text:
Docosenoic acid (micromol/L)
Target:
Both males and females 20 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
0.21 to 43.1 Range of Values 1621 1621
. Missing 224 1845

SSDTA_N - Docosatetraenoic acid (micromol/L)

Variable Name:
SSDTA_N
SAS Label:
Docosatetraenoic acid (micromol/L)
English Text:
Docosatetraenoic acid (micromol/L)
Target:
Both males and females 20 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
6.28 to 132 Range of Values 1829 1829
. Missing 16 1845

SSDP6_N - Docosapentaenoic-6 acid (micromol/L)

Variable Name:
SSDP6_N
SAS Label:
Docosapentaenoic-6 acid (micromol/L)
English Text:
Docosapentaenoic-6 acid (micromol/L)
Target:
Both males and females 20 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
3.35 to 104 Range of Values 1829 1829
. Missing 16 1845

SSDP3_N - Docosapentaenoic-3 acid (micromol/L)

Variable Name:
SSDP3_N
SAS Label:
Docosapentaenoic-3 acid (micromol/L)
English Text:
Docosapentaenoic-3 acid (micromol/L)
Target:
Both males and females 20 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
9.59 to 204 Range of Values 1829 1829
. Missing 16 1845

SSDHA_N - Docosahexaenoic acid (micromol/L)

Variable Name:
SSDHA_N
SAS Label:
Docosahexaenoic acid (micromol/L)
English Text:
Docosahexaenoic acid (micromol/L)
Target:
Both males and females 20 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
35.7 to 610 Range of Values 1829 1829
. Missing 16 1845

SSLG1_N - Lignoceric acid (micromol/L)

Variable Name:
SSLG1_N
SAS Label:
Lignoceric acid (micromol/L)
English Text:
Lignoceric acid (micromol/L)
Target:
Both males and females 20 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
19.2 to 132 Range of Values 1762 1762
. Missing 83 1845

SSNR1_N - Nervonic acid (micromol/L)

Variable Name:
SSNR1_N
SAS Label:
Nervonic acid (micromol/L)
English Text:
Nervonic acid (micromol/L)
Target:
Both males and females 20 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
18.3 to 167 Range of Values 1717 1717
. Missing 128 1845

WTSAF2YR - 2Yr fasting weights

Variable Name:
WTSAF2YR
SAS Label:
2Yr fasting weights
English Text:
2Yr fasting weights
Target:
Both males and females 20 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
0 to 355659.5 Range of Values 1845 1845
. Missing 0 1845