• Component Description
• Eligible Sample
• Description of Laboratory Methodology
• Data Processing and Editing
• Laboratory Quality Assurance and Monitoring
• Analytic Notes
• References
• Codebook
  • SEQN - Respondent sequence number
  • SSMR1_N - Myristic acid (micromol/L)
  • SSML1_N - Myristoleic acid (micromol/L)
  • SSPM1_N - Palmitic acid (micromol/L)
  • SSPL1_N - Palmitoleic acid (micromol/L)
  • SSST1_N - Stearic acid (micromol/L)
  • SSOL1_N - Oleic acid (micromol/L)
  • SSVC1_N - cis-Vaccenic acid (micromol/L)
  • SSLNA_N - Linoleic acid (micromol/L)
  • SSGLA_N - gamma-Linolenic acid (micromol/L)
  • SSALN_N - alpha-Linolenic acid (micromol/L)
  • SSHGL_N - homo-gamma-Linolenic acid (micromol/L)
  • SSAR1_N - Arachidic acid (micromol/L)
  • SSEN1_N - Eicosenoic acid (micromol/L)
  • SSED1_N - Eicosadienoic acid (micromol/L)
  • SSARA_N - Arachidonic acid (micromol/L)
  • SSEPA_N - Eicosapentaenoic acid (micromol/L)
  • SSDA1_N - Docosanoic acid (micromol/L)
  • SSDE1_N - Docosenoic acid (micromol/L)
  • SSDTA_N - Docosatetraenoic acid (micromol/L)
  • SSDP6_N - Docosapentaenoic-6 acid (micromol/L)
  • SSDP3_N - Docosapentaenoic-3 acid (micromol/L)
  • SSDHA_N - Docosahexaenoic acid (micromol/L)
  • SSLG1_N - Lignoceric acid (micromol/L)
  • SSNR1_N - Nervonic acid (micromol/L)
  • WTSAF2YR - 2Yr fasting weights

National Health and Nutrition Examination Survey

2003-2004 Data Documentation, Codebook, and Frequencies

Fatty Acids - Plasma (Surplus) (SSFA_C)

Data File: SSFA_C.xpt

First Published: August 2011

Last Revised: April 2022

Note: The NHANES Biospecimen Program processes were reevaluated in 2021 and 2022 to monitor quality control after a procedural error was identified. This error did not pose any risk of participant disclosure. Addressing this error resulted in the removal of some records from various stored biospecimen data files between 1999 and 2018 that did not meet program standards. After a comprehensive review of all stored specimen datasets, this data file was modified to remove records (<5% of records) that were initially included in error. No data values were altered. The sample weights and overall estimates were compared between the original and revised files. The sample weights provided with the original data release are appropriate to use with this updated data file; therefore, no new survey weights were created. The overall estimates from these files were examined and are consistent with the previous file. However, not all possible analyses were performed. For any queries related to this dataset please email the Biospecimen Program at serumplasmaurine@cdc.gov.

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 Amendments (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

• Caudill SP, Schleicher RL, Pirkle JL. 2008. Multi-rule quality control for the age-related eye disease study. Stat Med 27:4094-4106.
• Clinical Laboratory Improvement Amendment. Public Law 100-578. 1988. 10-31-1988.
• Lagerstedt S, Hinrichs D, Batt S, Magera M, Rinaldo P, McConnell J. Quantitative Determination of Plasma C8-C26 Total Fatty Acids for the Biochemical Diagnosis of Nutritional and Metabolic Disorders. Mol Genet & Metab 2001; 73: 38-45.

Codebook and Frequencies