• Component Description
• Eligible Sample
• Description of Laboratory Methodology
• Laboratory Quality Assurance and Monitoring
• Data Processing and Editing
• Analytic Notes
• References
• Codebook
  • SEQN - Respondent sequence number
  • WTSSTT2Y - SSTOCA and SSTOXO 2 year weights
  • SSHOLO - Holotranscobalamin (pmol/L)
  • SSHOLOLC - Holotranscobalamin comment code

National Health and Nutrition Examination Survey

2011-2012 Data Documentation, Codebook, and Frequencies

Holotranscobalamin – Serum (Surplus) (SSHOLO_G)

Data File: SSHOLO_G.xpt

First Published: April 2026

Last Revised: NA

Component Description

The most common measure of vitamin B12 (B12) status is serum total B12 concentration (Green, et. al., 2017).  In serum, B12 is bound to two transport proteins, transcobalamin (formerly transcobalamin II) and haptocorrin (formerly transcobalamins I and III) (Green and Miller, 2014).  The amounts of B12 bound to these transport proteins are referred to as “holotranscobalamin” (holoTC) and “holohaptocorrin” (holoHC).  The measurement of serum total B12 is the combination of holoTC plus holoHC.  Total B12 is an assay for B12 status with very low concentrations consistent with biochemical (e.g., elevated blood concentrations of homocysteine and methylmalonic acid) and clinical (e.g., macrocytic anemia, neurological deficits) manifestations of B12 deficiency (Green and Miller, 2014; Green, et. al., 2017; Allen, et. al., 2018). However, there is a significant indeterminate range of total B12 for which B12 status is unclear (i.e., some people exhibit signs and symptoms of B12 deficiency and others do not) (Green and Miller, 2014).  For this reason, measurement of holoTC concentration has been considered an alternative indicator of B12 status (Ulleland, et. al., 2002; Brady, et. al., 2008; Nexo and Hoffman-Lucke, 2011; Allen, et. al., 2018). Transcobalamin is responsible for the transport of newly absorbed B12 in the terminal ileum of the small intestine to all tissues of the body where holoTC is taken up via receptor-mediated endocytosis by the CD320 receptor (Quadros and Sequeira, 2013).  In contrast, holoHC only delivers B12 to the liver.  Thus, holoTC is sometimes referred to as “active” B12, and measurement of serum holoTC may serve as a more sensitive and specific indicator of B12 status than total B12 (Green, et. al., 2017; Allen, et. al., 2018).  HoloTC is also considered a component of “multiple analyte testing” for B12 status where two or more of the serum biomarkers of B12 status (total B12, holoTC, methylmalonic acid, and homocysteine) are considered in combination to achieve improved accuracy in diagnosis of B12 deficiency (Fedosov, et. al., 2015).

Eligible Sample

All examined participants aged 20 years and older were eligible.

Description of Laboratory Methodology

Stored serum specimens were processed, stored, and shipped to laboratories at Rutgers University.

The method used to measure serum holoTC is a commercially available, 96-well plate format, enzyme immunoassay called “Active-B12 (Holotranscobalamin) ELISA” (IBL International GMBH, Hamburg, Germany – obtained through Tecan Trading AG, Switzerland). The method requires 2 x 50 µl of serum (must be serum, cannot be plasma) and consists of two main steps: 1. Capture of holoTC in serum by a holoTC-specific monoclonal antibody, and 2. Incubation of captured holoTC with a substrate that produces a colored end-product. The amount of color is directly correlated with the concentration of holoTC in the serum.

Laboratory Quality Assurance and Monitoring

In addition to survey samples, each analytical run included standards for determination of a standard curve, and low and high quality controls (QCs) supplied by the manufacturer. Low and high QC concentrations in any given run were required to fall within ranges of values pre-specified by the manufacturer (low QC: 15-35 pmol/L; high QC: 36-84 pmol/L). If a QC value was outside the specified range and there was no clear error in the assay to explain the discrepancy, the assay was declared invalid and the samples re-run. Mean ± SD values over 56 and 55 sample runs for the QCs, respectively, were: Low QC 18.95 ± 1.47 pmol/L; High QC 47.67 ± 3.69. Based on these data, the inter-assay coefficients of variation (CVs) for the QCs were: Low QC 7.76%; High QC 7.74%.

Data Processing and Editing

Data were received after all analyses were complete. The data were not edited.

Analytic Notes

Refer to the 2011-2012 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. Additionally, availability of specimens for surplus projects is lower than for other laboratory tests performed on NHANES participants. 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.

Subsample Weights

The analytes included in this dataset were measured in examined participants aged 20 years and over. Special sample weights are required to analyze these data properly. Specific sample weights for this subsample, WTSSTT2Y, are included in this data file and should be used when analyzing these data. The sample weights created for this file used the examination sample weight, i.e., WTMEC2YR, as the base weight. The base weight was adjusted for additional nonresponse to these lab tests and re-poststratified to the population total using sex, age, and race/Hispanic origin. Participants who were part of the eligible population, but who did not provide a serum specimen, or did not have sufficient volume of biospecimens, or who did not give consent for their specimens to be used for future research are included in the file, but they have a sample weight assigned “0” in their records. There is one participant who did not have a specimen available for testing after creation of the WTSSTT2Y sample weights was completed. Therefore, they have a sample weight larger than “0” (WTSSTT2Y>0), regardless of missing their holotranscobalamin test result. In addition, there are some participants (n=59) who have a sample weight assigned “0” in their records (WTSSTT2Y=0) but have a holotranscobalamin measurement included in this data file. These participants had other missing laboratory test results when the WTSSTT2Y sample weights were previously created. These holotranscobalamin measurements are considered valid; however, to include these data in the analysis, reweighting is required.

Demographic and Other Related Variables

The analysis of NHANES laboratory data must be conducted using the appropriate survey design and demographic variables. The NHANES 2011-2012 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.

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 lower limit of detection for the assay as provided by the manufacturer is 8.1 pmol/L. The concentrations of all successfully measured samples exceeded the lower limit of detection except one sample. This sample is listed as 4.8 pmol/L. The upper limit of detection is defined by the highest standard used to create the standard curve. Two variables are provided for each of these analytes. The variable name ending in “LC” (ex., SSHOLOLC) indicates whether the result exceeded standard curve (i.e. above upper limit of detection): the value “0” means that the result was within the range of the standard curve, “1” indicates that the result was above the upper limit of the standard curve. The other variable (ex., SSHOLO) provides the analytic result for that analyte. For analytes with analytic results above the upper limit of the standard curve (ex., SSHOLOLC=1), an imputed fill value was placed in the analyte results field. This value is equal to the upper limit of the standard curve (146 pmol/L).

The upper limit of the standard curve (in pmol/L) for SSHOLO:

Variable Name	Analyte Name	Upper Limit of the Standard Curve
SSHOLO	Holotranscobalamin (pmol/L)	146

References

• Allen L.H., Miller J.W., de Groot L., Rosenberg I.H., Smith A.D., Refsum H., et al. Biomarkers of nutrition for development (BOND): vitamin B-12 review. J Nutr. (2018) 148(suppl4):1995S-2027S.
• Brady J., Wilson L., McGregor L., Valente E., Orning L. Active B12: a rapid, automated assay for holotranscobalamin on the Abbott AxSYM analyzer. Clin Chem. (2008) 54(3):567-73.
• Fedosov S.N., Brito A., Miller J.W., Green R., Allen L.H.. Combined indicator of vitamin B12 status: modification for missing biomarkers and folate status and recommendations for revised cut–points. Clin Chem Lab Med. (2015) 53(8):1215-25.
• Green R., Allen L.H., Bjørke-Monsen A-L., Brito A, Gueant J-L., Miller J.W., et. al. Vitamin B12 deficiency. Nat Rev Dis Primers. (2017) 3:17040.
• Green R., Miller J.W. Vitamin B12. In: Zempleni J., Suttie J.W., Gregory J.F., Stover P.J., editors. Handbook of vitamins. 5th ed. Boca Raton, FL: CRC Press. (2014) p.447-89.
• Nexo E., Hoffmann-Lücke E. Holotranscobalamin, a marker of vitamin B-12 status: analytical aspects and clinical utility. Am J Clin Nutr. (2011) 94(1):359S-65S.
• Quadros E.V., Sequeira J.M. Cellular uptake of cobalamin: transcobalamin and the TCblR/CD320 receptor. Biochimie. (2013) 96:1008-18.
• Ulleland M., Eilertsen I., Quadros E.V., Rothenberg S.P., Fedosov S.N., Sundrehagen E., et. al. Direct assay for cobalamin bound to transcobalamin (holo-transcobalamin) in serum. Clin Chem. (2002) 48(3):526-32.

Codebook and Frequencies