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Articles

Analysis of Factors Influencing the Comparison of Homocysteine Values between the Third National Health and Nutrition Examination Survey (NHANES) and NHANES 1999+

Christine M. Pfeiffer¹, Samuel P. Caudill, Elaine W. Gunter, Barbara A. Bowman^*, Paul F. Jacques, Jacob Selhub, Clifford L. Johnson^**, Dayton T. Miller and Eric J. Sampson

National Center for Environmental Health, ^* National Center for Chronic Disease Prevention and Health Promotion and ^** National Center for Health Statistics, Centers for Disease Control and Prevention, Atlanta, GA and Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, MA

¹To whom correspondence should be addressed.

	ABSTRACT

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Two important changes occurred in the time between the Third National Health and Nutrition Examination Survey (NHANES III) (1991–1994) and the later survey (NHANES 1999+) regarding total homocysteine (tHcy), i.e., a change in matrix from serum to plasma and a change in analytical methods. The goals of this study were to determine the magnitude of potential differences between plasma and serum with regard to tHcy concentrations, and between the two analytical methods used in these surveys. Optimally prepared plasma, serum allowed to clot for 30 and 60 min at room temperature and serum allowed to clot for 30 and 60 min and subjected to four freeze-thaw cycles, prepared from blood samples collected from 30 healthy people, were analyzed by both methods. Serum samples had significantly higher tHcy concentrations than plasma samples, and the difference increased with longer clotting time. Freeze-thaw cycles had little or no effect on the variability or bias in the serum sample results. The tHcy results produced by the two analytical methods were significantly different, but consistent across sample types. On average, the results of the method used in NHANES III were lower by 0.64 µmol/L; however, the relative bias varied with tHcy concentration. The tHcy results determined in surplus serum from NHANES III overestimated tHcy concentrations by 10% compared with optimally prepared plasma. The average method bias was 6% between the two analytical methods. On the basis of changes in matrix and methodology, direct comparison of tHcy results between the two surveys is inappropriate.

KEY WORDS: • homocysteine analysis • blood sampling • freezing • thawing • National Health and Nutrition Examination Survey

	INTRODUCTION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
The total homocysteine (tHcy)² concentration in plasma or serum is a sensitive indicator of folate and vitamin B-12 status (Savage et al. 1994 ). It has been associated with pregnancy complications (Steegers-Theunissen et al. 1992 ), neural tube defects (Steegers-Theunissen et al. 1994 ), mental disorders (Smythies et al. 1997 ) and cognitive impairment in the elderly (Riggs et al. 1996 ). Furthermore, elevated tHcy is a common cardiovascular risk factor in the general population (Refsum et al. 1998 , Selhub et al. 1995 ). Because of this high public health relevance of homocysteine and because of the added interest in homocysteine associated with the recent introduction of fortification of cereal-grain products with folic acid, comparing tHcy results between the ongoing National Health and Nutrition Examination Survey (NHANES) 1999+ survey and the Phase II, NHANES III survey is desirable. Recognizing that importance, we evaluated some of the key issues related to a comparison, namely, the use of different matrices and different analytical methods in the two surveys.

Homocysteine was measured at the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University in surplus serum from Phase II, NHANES III, to assess tHcy concentrations in a representative sample of Americans (Jacques et al. 1999a ). These specimens were not optimal for tHcy determination because they underwent repeated freezing and thawing and because serum is not the preferred specimen for tHcy determination. RBC continue to produce and release homocysteine after blood is collected, artificially increasing the tHcy concentration in serum (Ubbink et al. 1992 ). The preferred specimen for tHcy determination is plasma, cooled and separated rapidly from RBC (Ubbink et al. 1992 ). This is the specimen collected in NHANES 1999+ for tHcy determination by the Centers for Disease Control (CDC) NHANES Central Laboratory. A method comparison study was designed to determine the magnitude of potential differences between plasma and serum, collected and prepared according to the rigorous NHANES III and 1999+ protocols. Because different analytical methods were used to measure tHcy in NHANES III and 1999+, another goal of this study was to assess the comparability of these two methods.

	SUBJECTS AND METHODS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Protocol.

Under a CDC agreement with the Emory University Hospital Blood Collection Service (including an omnibus informed consent and human subjects review protocol), blood (45 mL/subject) was collected in March 1996 from 30 apparently healthy adults who had fasted for at least 4 h, following instructions identical to those given to NHANES III participants. No special requirements were made with regard to diet or use of multivitamins or medications. A total of five tubes of blood were drawn following standard procedures, one 7-mL EDTA-anticoagulated whole-blood tube and four regular 10-mL serum separator tubes (SST) (Becton-Dickinson, Franklin Lakes, NJ). Two replicate vials for each sample type were prepared for tHcy analysis by Tufts University (30 donors x 5 types x 2 replicates = 300 samples), and one vial for each sample type was prepared for analysis by the CDC Laboratory once method validation for tHcy measurement was completed (30 donors x 5 types = 150 samples).

The EDTA tubes were placed on wet ice immediately after collection, and the plasma was separated by centrifugation (10 min at 2000 x g, refrigerated centrifuge) within 30 min of venipuncture. Three 1-mL aliquots of plasma were placed in labeled vials and frozen at -70°C. Two of the SST were allowed to clot for 30 min at room temperature, then centrifuged; the six 1-mL serum aliquots were placed in labeled vials and frozen at -70°C. Half of the replicate samples per donor were subjected to four freeze-thaw cycles (2 h/cycle); the other half remained frozen until analyzed by laboratories at Tufts or the CDC. The remaining two SST were allowed to clot for 60 min at room temperature and then treated similarly to the 30-min clotted serum. Specimen collection protocols for NHANES III stipulated that blood without anticoagulant should be allowed to clot for at least 30 and no >60 min after collection and before centrifugation (Gunter et al. 1996 ).

Thus, the following five different sample types were available for analysis: 1) fresh plasma cooled immediately and separated rapidly from RBC (plasma); 2) serum prepared after allowing the whole blood to clot for 30 min at room temperature before separation (serum-30-NT); 3) serum prepared as in condition 2, subjected to four successive freeze-thaw cycles (serum-30-FT4); 4) serum prepared after allowing the whole blood to clot for 60 min at room temperature before separation (serum-60-NT); and 5) serum prepared as in condition 4, subjected to four successive freeze-thaw cycles (serum-60-FT4). The first sample type represented the plasma currently used in NHANES 1999+, whereas the four other sample types represented the surplus serum types available for use in NHANES III.

The 300 samples for analysis by the Tufts laboratory were shipped on dry ice. Specimen labels were coded to blind the Tufts laboratory to specimen type. CDC prepared run sheets so that all specimens for each subject were analyzed at the same time. The 150 samples for analysis by the CDC laboratory were stored frozen at -70°C for 3 y until the CDC laboratory had set up and validated an assay for tHcy measurement. All specimens for each subject were also analyzed at the same time.

Determination of tHcy concentrations.

Both laboratories used an HPLC assay with fluorometric detection. The laboratory at Tufts University employed the method of Araki and Sako (1987) , and the CDC laboratory employed a modification of the method of Gilfix et al. (1998) as published by Pfeiffer et al. (1999a) .

Power calculations.

To estimate the power of the experimental design, we assumed that the logarithms of the measured tHcy concentrations have constant variance and used a modification of the approach suggested by Scheffé (1959) . Assuming a tHcy concentration range of 4–40 µmol/L, an analytic CV of 7%, 25 subjects, two vials per subject (replicate analyses), five sample types (treatment conditions) and a 0.05 level of significance, we should have >80% power to detect a difference 5% between plasma and serum tHcy concentrations and power of 89% to detect a difference 6%.

Statistical analysis.

We used ANOVA to test for differences among plasma sample results and serum sample results treated in four different ways. On the basis of the sampling design, the ANOVA model accounted for differences between laboratories (CDC and Tufts), differences among sample types (5 different treatments), differences among subjects from whom the blood samples were taken, type-by-subject interaction (failure of differences between sample types to be consistent across subjects), laboratory-by-subject interaction and laboratory-by-type interaction.

We performed an error-in-variables regression analysis independently for each laboratory to determine the relation between serum results and plasma results. Plasma results were treated as the independent variable and, separately for each type of serum sample, serum results were treated as the dependent variable. Using the intercept and slope estimates, the expected bias in tHcy plasma estimates was computed for each of the four types of serum samples, i.e., serum-30-NT tHcy concentration = (plasma tHcy concentration·slope) + intercept; and absolute bias in tHcy plasma estimate = serum-30-NT tHcy concentration - plasma tHcy concentration).

Difference plots were used to assess the agreement between tHcy results obtained by the Tufts and CDC laboratory methods (Bland and Altman 1986 and 1995 ). Possible biases were assessed by computing the 95% confidence intervals (mean difference ± 2 SEM) for the mean differences between the two methods. Limits of agreement were assessed by calculating the central 0.95 intervals (mean difference ± 2 SD). The mean differences between the two methods and the means of the two methods were correlated to test for a concentration-dependent relation. To assess the mean proportional bias between the two methods, we calculated the relative ratios of the Tufts results and CDC results (proportional bias = 100 - Tufts result · 100/CDC result).

	RESULTS

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Distribution of tHcy concentrations.

The tHcy results tended to be slightly skewed to the higher concentrations for this particular sample of 30 subjects, covering a range of 4–17 µmol/L. The mean values for each sample type are reported in Table 1 . Because there did not appear to be a deviation from normality in the measurement errors for the Tufts laboratory results (consisting of vial-to-vial and analytic error), we did not transform the data before analysis.

The tHcy results were significantly (P < 0.0001) different for plasma and serum samples. Further testing with Duncan’s Multiple Range Test suggested, at the 0.01 significance level, that the mean tHcy plasma concentration was significantly lower than the mean tHcy serum concentration regardless of the way in which the serum samples were treated. This test also indicated that the results of the serum-60-FT4 samples were significantly higher than those of the serum-30-FT4 and serum-30-NT samples, but not the serum-60-NT samples. Similarly, the results of the serum-30-NT samples were significantly lower than those of the serum-60-FT4 and serum-60-NT samples, but not the serum-30-FT4 samples. When the significance level was relaxed to the 0.05 or 0.1 level, the multiple comparison test suggested that the mean tHcy concentration of the serum-30-NT and serum-30-FT4 serum samples was significantly lower than that of the serum-60-FT4 and the serum-60-NT serum samples.

The ANOVA indicated no type-by-subject interaction, suggesting that differences between plasma results and serum results and between serum results of one type and another type were consistent across subjects. Thus, if the plasma result was lower than a corresponding serum result for one subject, it was likely to be lower for all other subjects. This consistency was observed across all subjects, except for measurement error because the plasma results were lower than the corresponding serum-30-NT, serum-60-NT, serum-30-FT4 and serum-60-FT4 serum results, respectively, for 90, 93, 97 and 100% of the 30 subjects when the analysis was performed by the Tufts laboratory, and for 100, 100, 93 and 97%, respectively, of the 30 subjects when the analysis was performed by the CDC laboratory.

The ANOVA also indicated no laboratory-by-type interaction, suggesting that differences between laboratories were consistent across sample types. However, the analysis did indicate a significant laboratory-by-subject interaction, suggesting that differences between laboratories were not consistent across subjects (i.e., the bias of one method relative to the other varied with tHcy concentration).

Method comparison.

The Bland-Altman difference plot (Fig. 1 ) indicated that the mean differences between the two methods were concentration dependent (Spearman correlation coefficient: -0.6707). On average, the results of the Tufts laboratory were lower than the results of the CDC laboratory by 0.64 µmol/L (95% confidence limits: -0.501 to -0.771), but the actual difference between the two laboratories depended upon which subject samples were analyzed. The central 0.95 interval gives an indication of the agreement between the two methods. Of the tHcy determinations by the Tufts laboratory method, 95% were between 0.98 µmol/L higher and 2.26 µmol/L lower than tHcy results determined by the CDC method. This corresponded to a proportional negative bias of 6%.

View larger version (16K): [in this window] [in a new window]   Figure 1. Bland-Altman difference plot between Tufts Laboratory and CDC Laboratory results of total homocysteine (tHcy) concentrations, n = 150. The central line represents the mean difference between the two methods. The outer two lines correspond to the mean difference between the methods ± 2 times the SD of the differences.

Because the ANOVA indicated a significant laboratory-by-subject interaction, error-in-variables regression analysis was performed separately for the results from the Tufts and the CDC laboratories (Table 1) . Using the intercept and slope estimates in Table 1 , the expected bias in tHcy plasma estimates was computed for each of the four types of serum samples (Table 2 ).

For the CDC laboratory results, the relative and absolute bias in estimating plasma tHcy followed the same pattern for all four serum sample types, i.e., the relative bias decreased with increasing tHcy concentration, whereas the absolute bias increased with increasing tHcy concentration. The serum-30-NT samples provided the least relative (6–16%) and absolute bias (0.62–0.83 µmol/L) across the range of tHcy concentrations.

	DISCUSSION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Several investigators have reported that optimal procedures for blood sample collection and handling are critical when tHcy is used for cardiovascular risk assessment (Andersson et al. 1992 , Fiskerstrand et al. 1993 , Ubbink et al. 1992 , Vester and Rasmussen 1991 ); inadequate specimen collection and handling may result in artificial elevation of tHcy, which may be interpreted as increased risk. Our study is unique because it used plasma and serum samples collected and prepared according to the rigorous NHANES III and 1999+ protocols and because these samples were analyzed by the two methods and laboratories used in NHANES III and 1999+ for tHcy determination. Thus, the results of this study must be considered before an evaluation of potential secular trends in tHcy between the two National Health and Nutrition Examination surveys.

Our findings that serum samples have 10% higher tHcy concentrations than optimally prepared plasma samples and that a clotting time of 30 min results in less difference between plasma and serum results than does a clotting time of 60 min correspond well with previous findings. Vester and Rasmussen (1991) , Ubbink et al. (1992) , and Fiskerstrand et al. (1993) all showed that within 1 h of storage of whole blood at room temperature, the plasma and serum tHcy concentrations increase by 10%. Therefore, to determine plasma tHcy, whole blood should be placed on ice immediately after drawing, and the plasma fraction should be prepared within 30 min of venipuncture (Hughes et al. 1998 ). Freezing and thawing of samples in this study had little or no effect on the variability or bias in the serum sample results. Vester and Rasmussen (1991) found no significant change in tHcy concentration for one sample thawed and frozen nine times during 1 wk.

The tHcy results produced by the two laboratories were significantly different but were consistent across sample types. On average, the Tufts laboratory results were lower than those of the CDC laboratory by 0.64 µmol/L; however, method bias varied with tHcy concentration. One limitation of this method of comparison is the relatively narrow tHcy concentration range of the samples analyzed (4–17 µmol/L). However, this range covers the normal range of the U.S. population (Jacques et al. 1999a ), and fairly moderate elevations in tHcy concentration (>10 µmol/L) are strongly associated with a higher risk for vascular disease (Ueland et al. 1992 ). It is thus important to have a good agreement between methods within the present tHcy concentration range.

Although the analytical methods employed by the two laboratories follow the same method principle, they differ in the reducing reagent, in the use of an internal standard and in the way calibration is performed. The Tufts laboratory method uses the classical tributyl phosphine (TBP) reducing agent that is dissolved in dimethylformamide (DMF) and a 1-point calibration with homocystine dissolved in 0.1 mol/L HCl and added to DMF-containing solution. In addition, pooled plasma samples with and without added homocysteine are included for quality control. Gilfix et al. (1998) published the availability of a novel water-soluble derivative of TBP, tris-carboxyethyl phosphine (TCEP). The CDC laboratory adapted the TCEP method to incorporate cystamine as an internal standard, and to use a 3-point calibration with homocystine dissolved in 0.1 mol/L HCl and added to plasma (Pfeiffer et al. 1999a ). Three levels of pooled plasma samples with and without added homocysteine are included as bench quality control and two levels of pooled plasma samples without added homocysteine are included as blind quality control.

A recently performed interlaboratory comparison that included two laboratories using TCEP as reducing reagent and three laboratories using TBP as reducing reagent found no apparent biases of these two methods relative to an arbitrarily selected gas chromatography/mass spectrometry method as the reference method (Pfeiffer et al. 1999b ). The laboratory comparison also found that the among-laboratory variations within one method exceeded in some cases the among-method variations. The lack of a suitable standard reference material that is of the same composition as human plasma hampers the quality of homocysteine measurements, as does the lack of a definitive method with the highest possible accuracy and precision. Thus, it is impossible at this point to decide whether one method is better than the other.

The two important findings of this study, i.e., that tHcy concentrations measured in surplus serum from Phase II, NHANES III, overestimated tHcy concentrations by 10% compared with optimally prepared plasma and that there is a method bias of 6% on average between the Tufts laboratory method (NHANES III) and the CDC laboratory method (NHANES 1999+), stand for themselves. With respect to cut-points, interpretation of results and comparison of NHANES III and NHANES 1999+ data sets, the two effects (slight overestimation of tHcy concentrations associated with the use of serum rather than plasma, and the somewhat lower tHcy concentrations obtained with the Tufts laboratory method) might cancel each other. This is supported by data in Table 1 , in which the mean serum-30-NT and serum-60-NT tHcy concentrations of the Tufts laboratory are comparable to the mean plasma tHcy concentration of the CDC laboratory. However, the differences between the matrix and the methods used in the two surveys and the fact that the 6% bias between the methods reflects an average, whereas the actual bias is dependent on the tHcy concentration, prevent direct comparison of homocysteine values in NHANES III and 1999+.

Another factor, folic acid fortification, adds yet more uncertainty to the comparison of tHcy normal ranges between NHANES III and 1999+. Although tHcy determinations from Phase II, NHANES III (1991–1994) reflect the situation before fortification of enriched cereal products with folic acid, tHcy determinations from NHANES 1999+ reflect the era after fortification. Recent data published by Jacques et al. (1999b) demonstrated that fortification of enriched grain products with folic acid was associated with a substantial improvement in folate status in a population of middle-aged and older adults of the Framingham Offspring Study cohort, and that the fortification was also associated with a small (10%) but significant decrease in plasma tHcy.

Although researchers will be tempted to use the tHcy data of NHANES 1999+ to evaluate the effect of folic acid fortification on all segments of the U.S. population, bias introduced by changes in matrix and methodology between surveys may result in a smaller or larger apparent change in homocysteine before and after fortification, which could be interpreted incorrectly as a lack of efficacy of current fortification levels or an indication of overfortification. Researchers must be aware of the critical issues associated with the comparison of tHcy values between NHANES III and 1999+; beyond this, they must consider differences in matrix and methodology before comparing any data sets between two surveys.

	FOOTNOTES

 
² Abbreviations used: CDC, Centers for Disease Control; DMF, dimethylformamide; FT, frozen and thawed; NHANES, National Health and Nutrition Examination Survey; NT, not thawed and frozen; SST, serum separator tube; TBP, tributyl phosphine; TCEP, tris-carboxyethyl phosphine; tHcy, total homocysteine.

Manuscript received June 7, 2000. Initial review completed July 6, 2000. Revision accepted August 8, 2000.

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