Article

Comparison of Serum Concentrations of b-Trace , b2-Microglobulin, C, and in the US Population

| | Stephen P. Juraschek,*†‡ Josef Coresh,*†‡§ Lesley A. Inker, Andrew S. Levey, Anna Ko¨ttgen,*¶ Meredith C. Foster,*† Brad C. Astor,**†† John H. Eckfeldt,‡‡ and Elizabeth Selvin*†‡

Summary Departments of b b b b *Epidemiology and Background and objectives -trace protein ( TP), 2-microglobulin ( 2M), and cystatin C (CysC) have advan- §Biostatistics, Johns tages over creatinine for estimating GFR and prognosis. This study compares the distribution of all four markers Hopkins Bloomberg in the general population and their associations with possible determinants of GFR. School of Public Health, Baltimore, † b b $ Maryland; Welch Design, setting, participants, & measurements TP and 2M were measured in 7596 participants (aged 12 years) Center for Prevention, of the Third National Health and Nutrition Examination Survey (1988–1994). bTP and b2M concentrations and Epidemiology, and the proportion of persons with elevated ($99th percentile for young healthy participants) bTP ($0.81 mg/L), Clinical Research, b2M ($2.80 mg/L), standardized CysC ($1.03 mg/L), and creatinine ($1.2 mg/dl for men and $1.0 mg/dl for Johns Hopkins Medical Institutions, women) were compared across demographic and clinical factors. Baltimore, Maryland; ‡Department of Results Elevated bTP, b2M, and CysC showed stronger associations with age than elevated serum creatinine, the Medicine, Johns prevalence of elevated levels reaching 47%, 44%, 58%, and 26%, respectively, by age 80 years. bTP, CysC, and Hopkins Hospital, Baltimore, Maryland; b b b | creatinine were higher in men but 2M was not associated with sex. Mexican Americans had lower TP, 2M, Division of CysC, and creatinine compared with non-Hispanic whites. Hypertension and higher C-reactive protein were Nephrology, Tufts associated with elevations in all markers, whereas non-Hispanic black race, body mass index, diabetes, smoking Medical Center, status, triglycerides, HDL cholesterol, and education were not associated in a consistent manner across the Boston, Massachusetts; ¶Renal Division, different markers. University Hospital, Freiburg, Germany; Conclusions bTP, b2M, CysC, and creatinine differ in their associations with demographic and clinical factors, Departments of suggesting variation in their non-GFR determinants. Future studies should examine these markers with mea- **Medicine and ††Population Health sured GFR to determine their diagnostic and prognostic utility. Sciences, University of Clin J Am Soc Nephrol 8: 584–592, 2013. doi: 10.2215/CJN.08700812 Wisconsin School of Medicine and Public Health, Madison, Wisconsin; and Introduction originates from the choroid plexus of the central ner- ‡‡Department of b-trace protein (bTP) and b2-microglobulin (b2M) are vous system (8). b2M, a subunit of the major histo- Laboratory Medicine low molecular mass serum that are increas- compatibility class I molecule, is produced by all and Pathology, ingly viewed as useful for estimating GFR and pre- nucleated cells (9). Like CysC, bTP and b2M are con- University of Minnesota, dicting prognosis in kidney disease, cardiovascular sidered candidates for the assessment of GFR because – Minneapolis, outcomes, and death, independently of GFR (1 3). they are produced at a constant rate, are freely fil- Minnesota Recent studies have shown that bTP and b2M are tered by the glomerulus, and are then reabsorbed better predictors of adverse health outcomes than cre- and almost completely metabolized by the proximal Correspondence: atinine and are potentially as good as cystatin C tubule with no appreciable entry into the circulation Dr. Elizabeth Selvin, (CysC) (4). It is now appreciated that the use of a (10–12).Thereissomeevidencethatcerebrospinal Johns Hopkins fi fl University, 2024 E. combination of ltration markers leads to more accu- uidleaks(13)andcorticosteroiduse(14)affect Monument, Suite 2- rate estimates of GFR than single markers (5), and as bTP, whereas rare inflammatory conditions affect 600, Baltimore, MD in other fields, it is expected that a combination of b2M (15–18). 21205. Email: markers will lead to more accurate predictions of The challenge of measuring GFR in large popula- [email protected] prognosis (6). However, as we have learned from cre- tion studies and the almost complete absence of atinine and CysC, knowledge of their non-GFR deter- urinary excretion of these markers make it difficult minants in the general population will be necessary to study the relative contributions of GFR and non- for optimal use. GFR determinants to their serum concentrations. As Determinants of the serum concentrations in bTP such, examining four filtration markers in the general and b2M have not yet been studied in the general population presents an opportunity to infer demo- population. bTP, a 168 glycoprotein (7), graphic and clinical factors that may be associated

584 Copyright © 2013 by the American Society of Nephrology www.cjasn.org Vol 8 April, 2013 Clin J Am Soc Nephrol 8: 584–592, April, 2013 b-Trace Protein and b2-Microglobulin in the US Population, Juraschek et al. 585

with variation in underlying GFR. Thus, the objectives of for men and $1.0 mg/dl for women. This is approximately this study were to describe the distributions of bTP and equal to the 99th percentile in either sex in participants aged b2M in the US population, to identify factors associated 20–39 years without diabetes and hypertension. The cutoff with elevations in bTP and b2M, to compare these distri- for decreased estimated GFR using creatinine was ,60 ml/ butions and associations with those of standardized creat- min per 1.73m2, computed from the inine and CysC, and to distinguish factors that may be Epidemiology Collaboration (CKD-EPI) equation (24). associated with GFR versus non-GFR determinants of all four filtration markers. Determination of Potential Risk Factors We examined covariates associated with CKD. Race and ethnicity were grouped as non-Hispanic white, non-Hispanic Materials and Methods black, Mexican American, and other, although the “other” Study Population category was not presented due to inherent heterogeneity Data for this study were derived from the Third National and small sample size (25). Education (,12 years, $12 Health and Nutrition Examination Survey (NHANES III), years), current smoking status (yes/no), and (body a large, cross-sectional study conducted by the National mass index [BMI] ,30 kg/m2, $30 kg/m2)weredichoto- Center for Health Statistics (NCHS) using a complex mized. BMI was also analyzed as a continuous variable multistage sampling design. Interviews, physical exami- (scaled to per 5 kg/m2). Hypertensive status (yes/no) was nations, and laboratory measurements were obtained from defined as a physician-diagnosis of hypertension, hyperten- $ 18,722 individuals aged 12 years (19). Stored serum sam- sion medication use, or a mean systolic or diastolic BP $140 ples from a subset of NHANES III participants were used mmHg or $90 mmHg, respectively. Diabetes status (yes/ for the measurement of nontraditional markers of GFR. no) was based on a fasting plasma glucose of $126 mg/dl Details of this study design were previously described ($7.0 mmol/L) or a nonfasting glucose of $200 mg/dl fl (20,21). Brie y, we selected the following: all participants ($11.0 mmol/L) or self-reported diagnosis by a physician $ aged 60 years because decreased GFR is common in this (independent of pregnancy), use of insulin, or use of diabe- n group ( =5248), all participants with elevated creatinine tes medication. HDL cholesterol was defined as a continu- . . 1.189 mg/dl in men and 0.997 mg/dl in women ous variable (per 10-mg/dl increase) for logistic regression, n ( =305), and a 25% random sample of participants aged and as a dichotomous variable (,40 mg/dl for men and – n 12 59 years ( =3293). This design minimized costs while ,50 mg/dl for women) to estimate study population prev- retaining power and generalizability. Sample weights alence. Triglycerides were log-transformed to normalize its were re-calculated based on the total number of samples distribution. C-reactive protein (CRP) was divided into three n that were not missing CysC (86%; =7596). Among the categories (,0.22 mg/dl, 0.22–1 mg/dl, .1 mg/dl). The original CysC subsample, there were 7529 (99.1%) partic- lower limit of detection for CRP was 0.22 mg/dl. al- b ipants with a TP measurement and 7534 (99.2%) partic- bumin was measured by a solid-phase fluorescent immuno- b ipants with a 2M measurement. assay, and urine creatinine was measured using the modified kinetic rate Jaffe method. Urinary albumin/creati- Primary Outcome Measurements nine ratio (ACR) was expressed in milligrams per gram In 2009, we measured bTP and b2M in stored serum (26,27). Additional data collection details, including assay samples collected in 1988–1994, using particle-enhanced precision estimates, are available in the NHANES field man- immunonephelometric assays (N Latex b-trace protein as- ual published online (19). say and N Latex b-2 microglobulin assay; Dade Behring, fi b b Deer eld, IL). Both TP and 2M are reported to be robust Statistical Analyses fi to freeze-thaw cycles (22). Interassay coef cients of varia- Themethodusedtogeneratesampleweightswas b b tion (CVs) for the TP assay and the 2M assay were 5.7% described in detail elsewhere (20). Briefly, the original (mean 0.594 mg/L) and 2.7% (mean 1.757 mg/L), respec- sampling weights were modified to account for both miss- fi tively. Creatinine was measured using a modi ed kinetic ing creatinine and CysC using standard methods ap- Jaffe reaction, traceable to higher-order isotope dilution proved by the NCHS. These weights were utilized in all mass spectrometry methods, and the CV ranged from of our analyses and SEMs for estimates were obtained us- 1.2% (mean 1.00 mg/dl) to 1.6% (mean 3.84 mg/dl) (23). ing the Taylor series (linearization) method (28). Analyses CysC was measured using a particle-enhanced immuno- were performed using Stata11.1 software (StataCorp LP, nephelometric assay (20) and was calibrated to standard- College Station, TX). ized CysC (5). Its CV ranged from 4.9% (mean 1.90 mg/L) Means and proportions were estimated by age group, to 5.1% (mean 0.97 mg/L) (20,21). All serum specimens and weighted percentiles of bTP and b2M were estimated underwent at least one thaw cycle. using the subset of healthy individuals aged 20–39 years. Filtration markers were transformed (21/bTP and 21/ Definition of Elevated Filtration Markers and b2M) to model GFR. We used logistic regression models Decreased GFR to examine the associations of demographic and clinical Cutoffs for elevated bTP ($0.81 mg/L), b2M ($2.80 factors with elevated bTP and b2M concentrations, overall mg/L), and CysC ($1.03 mg/L) were defined as the ($20 years) and stratified by age group (20–59 years, $60 99th percentile in participants aged 20–39 years who did years). These models were applied to CysC and creatinine not have hypertension or diabetes (diagnosed or undiag- for comparison. This analysis was performed in partici- nosed) (20). Creatinine cutoffs were sex specifi c, corre- pants aged $60 years, reflecting subsample design as sponding to the study design, with cutoffs of $1.2 mg/dl well as a population with decreased GFR. The analysis 586 Clinical Journal of the American Society of Nephrology

Table 1. Characteristics overall and by age group, US population (NHANES III 1988–1994)

Age Group (yr) Overall

Characteristic 12–19 20–39 40–59 $60 All (n=719) (n=1675) (n=1149) (n=4053) (n=7596)

Male 50.863.5 49.362.1 48.762.5 42.860.8 48.161.4 Race/ethnicity Non-Hispanic white 69.463.6 71.762.7 78.662.3 84.061.5 75.662.0 Non-Hispanic black 15.562.1 12.661.4 10.461.1 8.360.8 11.661.0 Mexican American 8.361.0 7.060.9 4.360.6 2.360.2 5.560.5 Other ethnicity 6.861.9 8.861.6 6.861.6 5.461.0 7.361.2 Education ,12 yra NA 18.661.7 20.162.2 40.061.7 23.061.4 Current smokinga 10.762.2 36.462.2 27.562.2 14.861.0 26.261.1 Obesity (BMI $30 kg/m2)a 7.161.9 18.061.8 28.362.1 23.860.9 20.461.1 Hypertensiona 0.660.4 5.260.7 24.062.2 58.761.0 20.161.2 Diabetes a 0.160.1 1.060.3 6.561.1 12.660.7 4.660.5 Low HDL cholesterol (,40 mg/dl 38.763.2 36.261.7 39.462.1 37.461.4 37.661.3 for men; ,50 mg/dl for women)a Triglycerides (mg/dl)a 83.7 99.6 130.1 143.3 112.1 C-reactive protein (mg/dl)a ,0.22 88.962.4 77.461.9 68.762.6 61.462.1 73.561.5 0.22–18.561.9 17.361.5 23.662.2 28.661.7 20.061.2 .12.661.3 4.560.7 7.661.5 9.960.7 6.160.6 Albumin/creatinine ratio 8.2 4.7 6.2 11.1 6.6 (mg/g)a,b Estimated GFR, creatinine 135.660.94 113.160.77 96.960.63 74.560.42 104.460.69 (ml/min per 1.73 m2) Creatinine (mg/dl)c Male 0.7760.01 0.9260.01 0.9360.01 1.0660.01 0.9260.01d Female 0.6660.01 0.7060.01 0.7460.01 0.8460.01 0.7460.005d Cystatin C (mg/L)c Male 0.8260.01 0.7860.01 0.8360.01 1.0460.01 0.8560.01d Female 0.7460.01 0.7260.01 0.7860.01 1.0260.01 0.8060.01d b-trace protein (mg/L)c Male 0.6060.01 0.5460.01 0.5660.01 0.7760.01 0.5960.01d Female 0.5660.01 0.5160.01 0.5460.01 0.7260.01 0.5760.01d b2-microglobulin (mg/L)c Male 1.6560.02 1.7160.02 1.8660.02 2.5660.03 1.8960.02e Female 1.5460.02 1.6260.02 1.8560.04 2.6060.04 1.8860.02e

Data are presented as mean 6 SEM for continuous data and as a proportion 6 SEM for categorical variables. A geometric mean (without SEM) is provided for albumin/creatinine ratio and triglycerides. SEMs for geometric means were not obtained because of complex survey design. Survey sample size is denoted by n. Creatinine may be converted from mg/dl to mmol/L by multiplying by 88.4. Triglycerides may be converted from mg/dl to mmol/L by multiplying by 0.0113. HDL may be converted from mg/dl to mmol/L by multiplying by 0.0259. NHANES III, Third National Health and Nutrition Examination Survey; BMI, body mass index; NA, not available. aData from a small fraction of participants with unknown covariate distribution not shown. bAlbumin/creatinine ratio was determined in a subset of participants that excluded pregnant or menstruating women (n=250). cMen (n=3611), women (n=3985). dP value for overall mean across sexes was ,0.01. eP value for overall mean across sexes was 0.66. was also conducted in participants aged 20–59 years. Par- were evaluated by age, sex, and race/ethnicity to model ticipants aged ,20 years were excluded due to differences GFR across subgroups and to normalize their distributions. in NHANES III covariate definitions among youth as well Continuous associations between markers and age were as lack of data on education. Our models were adjusted for evaluated using linear splines with knots every 10 years. age, sex, race/ethnicity, BMI, hypertension, diabetes, Pearson correlation coefficients were calculated between smoking, CRP, log-transformed triglycerides, HDL choles- bTP, b2M, CysC, and creatinine. The proportion of the US terol, and education. We also repeated this analysis adjust- population aged $12 years with an elevated serum concen- ing for estimated GFR using creatinine both in place of and tration of each filtration marker was plotted by age along in addition to age, sex, and race/ethnicity. The weighted with the proportion of decreased estimated GFR using cre- median and 5th and 95th percentiles of 1/bTP and 1/b2M atinine to observe age-related trends. Clin J Am Soc Nephrol 8: 584–592, April, 2013 b-Trace Protein and b2-Microglobulin in the US Population, Juraschek et al. 587

Informed consent was obtained from all participants. $20 years, after adjustment for all other factors examined, Protocols for the conduct and implementation of this study age, hypertension, and CRP were associated with elevated were approved by the institutional review boards of both levels of all filtration markers. Consistent associations with the NCHS and the Johns Hopkins Bloomberg School of all markers except creatinine were seen for HDL choles- Public Health. terol and education, both in a protective direction. Other associations were less consistent across markers. Female sex and Mexican race/ethnicity were associated with a Results lower prevalence of elevations in bTP and creatinine but Population characteristics are shown in Table 1. The not significant associations with b2M and CysC. In con- mean bTP and b2M concentrations in the overall US pop- trast, triglycerides were associated with a higher preva- ulation were 0.58 mg/L (95% confidence interval, 0.57, lence of elevations in CysC and creatinine. Current 0.59) and 1.88 mg/L (95% confidence interval, 1.85, 1.92). smoking status was uniquely associated with a lower Percentiles by age and sex showed bTP levels being higher prevalence of creatinine elevations and diabetes was not in men than women (P,0.01), with details by age, marker, associated with prevalence of elevations in any of the four and ethnicity shown in Supplemental Figures 1–3. The cor- markers. Two factors were associated in reverse directions relation between bTP and b2M was high (0.85), as were with two markers. Non-Hispanic black race/ethnicity was the correlations with CysC (0.73 and 0.82), whereas corre- associated with elevated concentrations of creatinine, but lations with creatinine were lower (0.65 and 0.68). lower concentrations of bTP, and BMI was associated with We compared the prevalence of elevated serum concen- lower concentrations of b2M, but elevations in CysC. trations of each of the filtration markers and estimated Some differences in the patterns in strata of age 20–59 GFR, using creatinine ,60 ml/min per 1.73 m2 across all years versus $60 years are shown in Supplemental Tables ages (Figure 1). CysC showed the sharpest incline in prev- 1–5 and Supplemental Figures 2–5. In general, adjustment alence starting at about age 50 years, with bTP and b2M for estimated GFR rather than age, sex, and race/ethnicity demonstrating a comparable rise but starting at around did not alter the above patterns (Table 3). The two excep- age 60 years. Although creatinine elevation also became tions were that both BMI and triglycerides were signifi- more common starting at about age 50 years, its incline cantly associated with bTP and no longer associated was far more gradual than for the other three markers. with CysC after adjustment for estimated GFR. Similarly, Estimated GFR from creatinine showed an intermediate adjustment for estimated GFR in addition to age, sex, and behavior. At age 80 years, the proportions of the popula- race/ethnicity yielded virtually the same results (Supple- tion with an elevated bTP, b2M, CysC, and creatinine mental Table 6). were 47%, 44%, 58%, and 26% compared with 0.4%, Distributions of bTP and b2M in a young, healthy sub- 0.5%, 1.3%, and 1.7% at age 20 years. group of participants aged 20–39 years without hyperten- We performed multivariable logistic regression of factors sion or diabetes are shown in Table 4. The differences in associated with elevated levels of bTP, b2M, CysC, and concentrations of bTP varied significantly by sex and by creatinine (Table 2 and Figure 2). Among persons aged race/ethnicity, with bTP being greater among men versus

Figure 1. | Proportion of the US population aged ‡12 years with an elevated filtration marker or CKD stage 3 by age. Elevated markers were b-trace protein ($0.81 mg/L), b2-microglobulin ($2.80 mg/L), standardized creatinine (men, $1.2 mg/dl; women, $1.0 mg/dl), or stan- dardized cystatin C ($1.03 mg/L). CKD stage 3 is defined as an estimated GFR ,60 ml/min per 1.73 m2, estimated using the Chronic Kidney Disease Epidemiology Collaboration equation (24). The elevated markers or CKD stage 3 are independent definitions determined solely by the markers themselves or the estimated GFR (eGFR). 8 lnclJunlo h mrcnSceyo Nephrology of Society American the of Journal Clinical 588

Table 2. Adjusted ORs and 95% CIs of elevated serum b-trace protein (‡0.81 mg/L), elevated serum b2-microglobulin (‡2.80 mg/L), elevated standard cystatin C (‡1.03 mg/L), and elevated standard creatinine (men, ‡1.0 mg/dl; women, ‡1.2 mg/dl) in persons aged ‡20 years

b-Trace Protein b2-Microglobulin Standard Cystatin C Creatinine (Men, $1.0 mg/dl; ($0.81 mg/L) ($2.80 mg/L) ($1.03 mg/L) Women, $1.2 mg/dl)

$20 yr (n=6682 [1220]) $20 yr (n=6688 [1204]) $20 yr (n=6742 [1680]) $20 yr (n=6742 [966])

OR 95% CI P OR 95% CI P OR 95% CI P OR 95% CI P

Age (yr) 1.09 1.06–1.12 ,0.01 1.09 1.07–1.11 ,0.01 1.10 1.08–1.12 ,0.01 1.06 1.05–1.07 ,0.01 Female sex 0.62 0.44–0.88 ,0.01 1.26 0.88–1.80 0.20 0.87 0.65–1.16 0.33 0.60 0.46–0.77 ,0.01 Race/ethnicitya Non-Hispanic black 0.52 0.37–0.73 ,0.01 1.04 0.78–1.40 0.79 0.76 0.53–1.07 0.11 3.09 2.54–3.75 ,0.01 Mexican American 0.56 0.37–0.84 ,0.01 0.82 0.57–1.18 0.28 0.85 0.48–1.49 0.56 0.44 0.31–0.62 ,0.01 BMI (per 5 kg/m2) 0.93 0.81–1.06 0.28 0.83 0.75–0.92 ,0.01 1.23 1.06–1.43 0.01 0.99 0.86–1.13 0.84 Hypertension 2.10 1.54–2.86 ,0.01 1.66 1.35–2.03 ,0.01 1.72 1.27–2.32 ,0.01 2.15 1.75–2.64 ,0.01 Diabetes 1.06 0.77–1.47 0.71 1.37 0.91–2.07 0.13 1.17 0.84–1.62 0.35 1.17 0.87–1.57 0.30 Current smoker 0.98 0.61–1.58 0.93 0.97 0.66–1.43 0.87 1.44 0.96–2.17 0.08 0.75 0.58–0.98 0.04 CRP (mg/dl)b 0.22–1.0 1.30 0.95–1.79 0.10 1.75 1.32–2.30 ,0.01 1.13 0.86–1.48 0.39 1.18 0.95–1.46 0.13 .1.0 2.12 1.37–3.28 ,0.01 4.10 2.65–6.33 ,0.01 2.49 1.60–3.86 ,0.01 1.65 1.08–2.53 0.02 Triglycerides (mg/dl) 0.84 0.56–1.27 0.41 0.98 0.66–1.46 0.93 1.50 1.08–2.09 0.02 1.70 1.32–2.20 ,0.01 HDL (per 10 mg/dl) 0.89 0.79–1.00 0.05 0.79 0.68–0.91 ,0.01 0.87 0.80–0.94 ,0.01 0.95 0.87–1.04 0.27 Education $12 yr 0.65 0.48–0.87 ,0.01 0.73 0.58–0.91 0.01 0.76 0.61–0.96 0.02 0.84 0.66–1.05 0.12

Numbers in brackets represent individuals with an elevated b-trace protein ($0.81 mg/L) and elevated b2-microglobulin ($2.80 mg/L). Triglycerides were analyzed on a logarithmic scale (ln). Triglycerides may be converted from mg/dl to mmol/L by multiplying by 0.0113. HDL may be converted from mg/dl to mmol/L by multiplying by 0.0259. OR, odds ratio; 95% CI, confidence interval; BMI, body mass index; CRP, C-reactive protein. aReference group is non-Hispanic whites. bReference group is individuals with CRP ,0.22 mg/dl. Clin J Am Soc Nephrol 8: 584–592, April, 2013 b-Trace Protein and b2-Microglobulin in the US Population, Juraschek et al. 589

Figure 2. | Odds of elevated filtration markers in participants aged ‡20 years. Odds ratios (95% CIs) of an elevated concentration of b-trace protein ($0.81 mg/L), b2-microglobulin ($2.80 mg/L), cystatin C ($1.03 mg/L), and creatinine (men, $1.2 mg/dl; women, $1.0 mg/dl) after adjusting for age (per 1-year increase), female sex, non-Hispanic black race/ethnicity, Mexican American race/ethnicity, BMI per 5-kg/m2 increase, hypertension, diabetes, current smoking status, CRP from 0.22 to 1.0 mg/dl, CRP .1.0 mg/dl, triglycerides (mg/dl), HDL cholesterol (per 10-mg/dl increase), and education ($12 years). Reference groups for categorical variables include male sex, non-Hispanic whites, no hypertension, no diabetes, no current smoking status, and an education ,12 years. b-trace protein is depicted by a circle, b2-microglobulin is depicted by a triangle, cystatin C is depicted by a diamond, and creatinine is depicted by a square. The age covariate is not shown. BMI, body mass index; CRP, C-reactive protein; 95% CI, 95% confidence interval. women (P,0.01) and non-Hispanic whites versus non- CRP were associated with elevations in all four filtration Hispanic blacks and Mexican Americans, respectively markers, consistent with lower GFR. In contrast, inconsis- (P,0.001 for both non-Hispanic blacks and Mexican tent associations across markers were observed for sex, Americans). Similar trends were noted for b2M concentra- black race/ethnicity, Mexican Americans, BMI, current tions. Median values were significantly greater among smoking, triglycerides, HDL cholesterol, and education, men than women (P of difference ,0.001), and among suggesting more complex relationships, including associa- non-Hispanic whites compared with non-Hispanic blacks tions with non-GFR determinants as well as with GFR. (P of difference ,0.01) and Mexican Americans (P of dif- In our study, serum concentrations of bTP and b2M ference = 0.03). began to rise in the fifth to sixth decade. This is consistent with prior reports that demonstrated minimal effects of age on bTP (29,30) or b2M (29) in younger adults (aged Discussion ,22 years), but significant associations with age, particu- This article is the first description of the distribution of larly for b2M, in older adults (31–33). The positive associ- serum concentrations of bTP and b2M in the US popula- ations of bTP and b2M with age were also seen with CysC tion and expands our understanding of their determinants. and creatinine, which likely reflect a higher prevalence of The comparisons with creatinine and CysC allow for in- decreased GFR with age (27). However, the proportions of ferences about factors that may be associated with GFR older participants with elevations in CysC, bTP, and b2M and non-GFR determinants of their serum concentrations, were more pronounced than creatinine, possibly reflecting which cannot be evaluated directly in large population that the effects of muscle atrophy on creatinine generation studies. In our study, older age, hypertension, and higher in elderly individuals may be even more pronounced 590 Clinical Journal of the American Society of Nephrology

Table 3. ORs and 95% CIs of elevated serum b-trace protein (‡0.81 mg/L), elevated serum b2-microglobulin (‡2.80 mg/L), and elevated serum cystatin C (‡1.03 mg/L) in persons aged ‡20 years adjusted for eGFR

b-Trace Protein b2-Microglobulin Standard Cystatin C ($0.81 mg/L) ($2.80 mg/L) ($1.03 mg/L)

$20 yr (n=6682 [1220]) $20 yr (n=6688 [1204]) $20 yr (n=6742 [1680])

OR 95% CI P OR 95% CI P OR 95% CI P

eGFR creatinine 0.92 0.90–0.93 ,0.01 0.92 0.91–0.94 ,0.01 0.91 0.90–0.92 ,0.01 BMI (per 5 kg/m2 0.80 0.68–0.95 0.01 0.74 0.66–0.82 ,0.01 1.10 0.96–1.26 0.15 increase) Hypertension 1.99 1.37–2.90 ,0.01 1.70 1.36–2.14 ,0.01 1.71 1.18–2.46 0.01 Diabetes 1.03 0.67–1.59 0.88 1.51 0.92–2.47 0.10 1.48 0.96–2.28 0.08 Current smoker 0.89 0.55–1.44 0.64 0.82 0.54–1.24 0.34 1.35 0.91–2.01 0.13 CRP (mg/dl)a 0.22–1.0 1.26 0.90–1.78 0.18 1.90 1.39–2.59 ,0.01 1.11 0.85–1.45 0.42 .1.0 2.00 1.24–3.25 0.01 5.70 3.52–9.24 ,0.01 3.52 2.02–6.15 ,0.01 Triglycerides (mg/dl) 0.56 0.36–0.88 0.01 0.75 0.55–1.03 0.07 1.22 0.82–1.80 0.32 HDL (per 10 mg/dl 0.82 0.72–0.94 ,0.01 0.80 0.71–0.90 ,0.01 0.84 0.76–0.92 ,0.01 increase) Education $12 yr 0.56 0.41–0.76 ,0.01 0.60 0.47–0.77 ,0.01 0.59 0.46–0.76 ,0.01

Numbers in brackets represent individuals with an elevated b-trace protein ($0.81 mg/L) and elevated b2-microglobulin ($2.80 mg/L). Triglycerides were analyzed on a logarithmic scale (ln). Triglycerides may be converted from mg/dl to mmol/L by multiplying by 0.0113. HDL may be converted from mg/dl to mmol/L by multiplying by 0.0259. OR, odds ratio; 95% CI, confidence interval; eGFR, estimated GFR; BMI, body mass index; CRP, C-reactive protein. aReference group is individuals with CRP ,0.22 mg/dl.

among individuals with CKD than among populations Our data show inconsistent patterns for the relationship used to estimate the average age association for estimating between race/ethnicity and bTP and b2M across ages. GFR (34). Non-Hispanic blacks had lower bTP and b2M at younger There is disagreement in the literature regarding the ages (,30 years), followed by increases during ages 50–70 association of sex with bTP and b2M. Whereas some stud- years, and subsequent declines later in life. A similar trend ies describe higher bTP concentrations in adult men com- was described previously for CysC (20) and may reflect pared with women (35), most studies have shown no sex incident low GFR at age 50 years before a decrease in sur- differences in children (29,30). b2M has not been shown to vivorship at age $70 years. A similar pattern was not ob- differ by sex in youth (29), but some studies have reported served for creatinine, which exhibited higher values for higher b2M concentrations in adult men compared with non-Hispanic blacks versus whites regardless of age. women (33), whereas others report no sex differences This inconsistency reflects prior reports of higher muscle (31,32). Our data suggest that men have higher concentra- mass and creatinine production in non-Hispanic blacks tions of bTP than women aged ,30 years and .50 years, (26,38–40). Our data also show that being Mexican Amer- whereas serum concentrations of b2M were generally ican was associated with lower serum concentrations of greater in men aged ,40 years. In contrast, we found bTP and b2M across all ages. Similar patterns have been that CysC and creatinine were greater in men at all ages. described by others for CysC and creatinine (20,26,38), and Although previous reports have also reported higher cre- may imply a higher level of GFR. atinine (36) and CysC in men (20), these findings have This study represents the largest comparison of bTP, been inconsistent with measured GFR, which is known b2M, CysC, and creatinine to date. Furthermore, it was to be greater in men versus women (37). This illustrates conducted in a well characterized, nationally representa- the utility of a multi-marker approach to estimating GFR, tive sample of the US population, enabling us to determine in that one of the markers, b2M, seems to be less affected normal levels of these markers. The major limitation is that by sex-based, physiologic differences in marker generation we do not have measured GFR. Nevertheless, although that could be masking underlying differences in GFR. It NHANES III does not include measurements of GFR, the also suggests the benefits of GFR estimating equations that availability of questionnaires, physical examination mea- remove known large non-GFR effects in order to produce sures, and laboratory specimens allows us to examine as- an estimate that more specifically reflects kidney function. sociations with a variety of factors that are generally not Estimates based on multiple markers can be combined or available in studies with measured GFR. In addition, due averaged to increase precision and can be compared to high- to its cross-sectional design, we cannot examine the tem- light similarities that are more likely to reflect GFR and dif- poral nature of the observed associations and differential ferences that are more likely to reflect effects of the non-GFR mortality can distort associations. For example, not determinants of the specific filtration markers. finding a higher prevalence of elevations of any of the Clin J Am Soc Nephrol 8: 584–592, April, 2013 b-Trace Protein and b2-Microglobulin in the US Population, Juraschek et al. 591

Table 4. Percentiles of b-trace protein and b2-microglobulin distribution in persons aged 20–39 years without hypertension or diabetes mellitus overall, by sex, and by race/ethnicity, US population (1988–1994)

Weighted Percentiles n 1st 5th 25th 50th 75th 95th 99th

b-trace protein (mg/L) Overall 0.25 0.34 0.45 0.52 0.58 0.69 0.81 1513 Male 0.22 0.34 0.47 0.54 0.60 0.72 0.80 688 Female 0.25 0.34 0.44 0.50 0.55 0.66 0.96 825 Non-Hispanic white 0.30 0.38 0.47 0.53 0.59 0.70 0.91 437 Male NA 0.38 0.48 0.56 0.61 0.72 NA 182 Female NA 0.37 0.47 0.51 0.57 0.67 NA 255 Non-Hispanic black 0.22 0.31 0.42 0.47 0.52 0.64 0.76 502 Male NA 0.31 0.42 0.49 0.56 0.66 NA 269 Mexican American 0.21 0.30 0.42 0.48 0.56 0.67 0.76 509 Male NA 0.29 0.43 0.50 0.58 0.69 NA 243 Female NA 0.31 0.40 0.47 0.54 0.64 NA 266 b2-microglobulin (mg/L) Overall 1.03 1.22 1.43 1.59 1.80 2.27 2.80 1514 Male 1.08 1.27 1.48 1.66 1.86 2.34 2.70 689 Female 1.03 1.19 1.40 1.54 1.74 2.27 2.92 825 Non-Hispanic white 1.04 1.26 1.46 1.62 1.81 2.27 2.82 438 Male NA 1.31 1.49 1.68 1.86 2.21 NA 183 Female NA 1.23 1.43 1.58 1.74 2.29 NA 255 Non-Hispanic black 1.03 1.13 1.35 1.51 1.75 2.21 2.84 502 Male NA 1.19 1.37 1.54 1.79 2.42 NA 233 Female NA 1.08 1.33 1.50 1.71 2.13 NA 269 Mexican American 1.03 1.14 1.37 1.54 1.8 2.34 2.97 509 Male NA 1.17 1.42 1.60 1.82 2.32 NA 243 Female NA 1.13 1.34 1.49 1.78 2.58 NA 266

There were insufficient numbers to generate sex- and race-specific estimates for the 1st and 99th percentiles. See text for comparison of mean serum concentrations. Sample size shows the number of participants used as the basis for the nationally representative estimates. NA, not available.

filtration markers among patients with diabetes may re- NIDDK grant (K23DK081017). Siemens had no role in the study flect the high mortality among diabetic individuals, as design, data collection, analysis, interpretation of data, or report well as possible canceling effects of hyperfiltration early writing. in the course of diabetic kidney disease. Finally, we had only single measurements of bTP, b2M, CysC, and creat- Disclosures inine, and these measurements are known to vary within J.C. has consulted for Amgen and Merck and has an investigator- participants, which may contribute to differences in the initiated grant from Amgen. L.A.I. has an investigator grant from observed risk factor associations (20,41). Pharmalink and Gilead Inc. In conclusion, this study describes the distributions of bTP and b2M in the general US population and provides a References valuable comparison with creatinine and CysC. Knowl- 1. Aksun SA, Ozmen D, Ozmen B, Parildar Z, Mutaf I, Turgan N, edge of the demographic and clinical characteristics asso- Habif S, Kumanliogluc K, Bayindir O: Beta2-microglobulin and ciated with these markers suggests that they have different cystatin C in type 2 diabetes: Assessment of diabetic nephropa- non-GFR determinants. Consideration of these differences thy. Exp Clin Endocrinol Diabetes 112: 195–200, 2004 will be important for the appropriate interpretation of es- 2. Viberti GC, Keen H, Mackintosh D: Beta 2-microglobulinaemia: A sensitive index of diminishing renal function in diabetics. Br timates of GFR or prognosis based on their serum concen- Med J (Clin Res Ed) 282: 95–98, 1981 tration. Future studies should directly examine the 3. Tangri N, Inker LA, Tighiouart H, Sorensen E, Menon V, Beck G, relationship between bTP, b2M, and measured GFR, inte- Shlipak M, Coresh J, Levey AS, Sarnak MJ: Filtration markers may grating them with CysC and creatinine to optimize esti- have prognostic value independent of glomerular filtration rate. J Am Soc Nephrol 23: 351–359, 2012 mation of GFR and their use in determining prognosis. 4. Astor BC, Shafi T, Hoogeveen RC, Matsushita K, Ballantyne CM, Inker LA, Coresh J: Novel markers of kidney function as pre- Acknowledgments dictors of ESRD, , and mortality in the This project was partially funded by Siemens and by a grant from general population. Am J Kidney Dis 59: 653–662, 2012 theNational Instituteof Diabetes andDigestiveandKidney Diseases 5. Inker LA, Schmid CH, Tighiouart H, Eckfeldt JH, Feldman HI, Greene T, Kusek JW, Manzi J, Van Lente F, Zhang YL, Coresh J, (NIDDK) (U01 DK067651). S.P.J. is supported in part by a National Levey AS; CKD-EPI Investigators: Estimating glomerular filtration Heart Lung and Institute cardiovascular epidemiology rate from serum creatinine and cystatin C. N Engl J Med 367: training grant (T32HL007024). L.A.I. is supported in part by an 20–29, 2012 592 Clinical Journal of the American Society of Nephrology

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