Original Investigation Serum Bicarbonate and Bone Mineral Density in US Adults Wei Chen, MD,1,* Michal L. Melamed, MD, MHS,1,2 and Matthew K. Abramowitz, MD, MS1,2 Background: Chronic metabolic acidosis leads to bone mineral loss and results in lower bone mineral density (BMD), which is a risk factor for osteoporosis-related fractures. The effect of low-level metabolic acidosis on bone density in the general population is unknown. Study Design: Cross-sectional study. Setting & Participants: 9,724 nationally representative adults 20 years or older in NHANES (National Health and Nutrition Examination Survey) 1999-2004. Factor: Serum bicarbonate level. Outcomes: Lumbar and total BMD, as well as low lumbar and total bone mass, defined as 1.0 SD below the sex-specific mean value of young adults. Measurements: BMD was measured by dual-energy x-ray absorptiometry and serum bicarbonate was measured in all participants. Results: Both men and women with lower serum bicarbonate levels were more likely to be current smokers and had higher body mass index and estimated net endogenous acid production. There was a significant linear trend across quartiles of serum bicarbonate with lumbar BMD in the total population, as well as in sex-specific models (P 5 0.02 for all 3 models, P 5 0.1 for interaction). For total BMD, a significant association was seen with serum bicarbonate level for women but not men (P 5 0.02 and P 5 0.1, respectively; P 5 0.8 for interaction), and a significant association was seen for postmenopausal women but not premenopausal women (P 5 0.02 and P 5 0.2, respectively; P 5 0.5 for interaction). Compared with women with serum bicarbonate levels , 24 mEq/L, those with serum bicarbonate levels $ 27 mEq/L had 0.018-g/cm2 higher total BMD (95% CI, 0.004-0.032; P 5 0.01) and 31% lower odds of having low total bone mass (OR, 0.68; 95% CI, 0.46-0.99; P 5 0.049). Limitations: Cross-sectional study using a single measurement of serum bicarbonate. Subgroup differences are not definitive. Conclusions: Lower serum bicarbonate levels are associated with lower BMD in US adults. Further studies should examine whether serum bicarbonate levels should be incorporated into the diagnostic assessment and management of osteoporosis. Am J Kidney Dis. 65(2):240-248. ª 2015 by the National Kidney Foundation, Inc. INDEX WORDS: Serum bicarbonate; alkali therapy; metabolic acidosis; bone mineral density (BMD); lumbar BMD; osteoporosis; low bone mass; modifiable risk factor; National Health and Nutrition Examination Survey (NHANES); dual-energy x-ray absorptiometry (DEXA).

C

hronic metabolic acidosis as a result of chronic kidney disease (CKD) has been shown to have a negative effect on bone. It induces bone resorption and inhibits bone formation, resulting in lower total bone mineral density (BMD).1-4 In otherwise healthy individuals, there is evidence of a clinically meaningful low-level metabolic acidosis, largely mediated by the

From the 1Division of Nephrology, Department of Medicine, and 2Department of Epidemiology & Population Health, Albert Einstein College of Medicine, Bronx, NY. * W.C. completed this work while at the Albert Einstein College of Medicine and has since moved to the University of Rochester School of Medicine and Dentistry. Received March 14, 2014. Accepted in revised form July 2, 2014. Originally published online August 26, 2014. Address correspondence to Wei Chen, MD, 601 Elmwood Ave, Box 675, Rochester, NY 14642. E-mail: [email protected]  2015 by the National Kidney Foundation, Inc. 0272-6386 http://dx.doi.org/10.1053/j.ajkd.2014.07.007 240

age-related decline in kidney function and the acidogenic Western diet.5,6 However, the effect of low-level metabolic acidosis on bone metabolism in the general population is unknown. Osteoporosis is the most common bone disease in humans and is characterized by low bone mass. It is a silent disease until it is complicated by fractures.7 Osteoporosis-related fractures result in high morbidity and mortality8 and create a heavy economic burden, causing more than 432,000 hospital admissions, almost 2.5 million medical office visits, and about 180,000 nursing home admissions annually in the United States.9 As the population ages, the prevalence of bone disease and fractures increases markedly, representing a major public health problem. Whether metabolic acidosis should be incorporated into diagnostic guidelines or management of osteoporosis remains unclear and warrants investigation. Therefore, we hypothesized that lower serum bicarbonate levels would be associated with lower BMD in Am J Kidney Dis. 2015;65(2):240-248

Bicarbonate and Bone Mineral Density

the general population. In addition, because osteopenia and osteoporosis are more common among women than men, we hypothesized that the association between serum bicarbonate levels and BMD may differ between the sexes. We tested these hypotheses in adults 20 years or older who underwent dualenergy x-ray absorptiometry (DEXA) in NHANES (National Health and Nutrition Examination Survey) 1999-2004.

METHODS Study Population NHANES 1999-2004 was a nationally representative survey of the noninstitutionalized civilian population in the United States.10 A stratified, multistage, probability sampling design was used to select participants. Overall, 11,974 adults 20 years or older completed the interview and examination components, including DEXA scans and measurement of serum bicarbonate. We excluded participants who had estimated glomerular filtration rates (eGFRs) , 15 mL/min/1.73 m2 (n 5 34), had a diagnosis of chronic obstructive pulmonary disease (n 5 893), were using a bisphosphonate (n 5 173), or were missing covariate data (n 5 1,150). Thus, 9,724 participants were available for analysis. The NHANES protocol was approved by the National Center for Health Statistics (NCHS) Ethics Review Board, and written informed consent was obtained from all participants.

Data Collection Information for household income, education, physical activity, smoking, comorbid conditions, and medication use in the previous month was obtained by self-report. Race/ethnicity was selfidentified. Poverty was defined as ,100% of the poverty index based on self-reported household income.11 Participants were asked about the frequency and duration of walking or bicycling, home or yard work, and moderate or vigorous leisure time physical activity performed within the past 30 days. These responses were used to calculate metabolic equivalents (MET-min/wk) based on intensity values recommended by the NCHS.12 Activity level was classified as 0, , 500, 500 to 2,000, or . 2,000 MET-min/ wk. Smoking was classified as never, former, or current smoker. Data for dietary intake were obtained from a 24-hour dietary recall questionnaire. The diet-dependent net acid load was estimated as estimated net endogenous acid production (mEq/d) 5 [54.5 3 protein (g/d)/potassium (mEq/d)] 2 10.2.13 Hypertension was defined as systolic blood pressure $ 140 mm Hg, diastolic blood pressure $ 90 mm Hg, physician diagnosis, and/or antihypertensive medication use.14 A participant was considered to have diabetes mellitus if he or she reported a physician diagnosis while not pregnant or current use of insulin or oral hypoglycemic mediations or had a glycohemoglobin level $ 6.5%.15 Cardiovascular disease was defined by self-report of a physician diagnosis of congestive heart failure, coronary heart disease, angina, myocardial infarction, or stroke. Vitamin D supplementation was assessed by questionnaire and pill-bottle review. Participants were considered to use vitamin D supplementation when they used at least 400 IU per day of vitamin D, which was the recommended dose by the Institute of Medicine at the time. Menopausal status was determined by questionnaire. A participant was considered to be menopausal when she answered “going or gone through menopause” to the question “What is the reason that you have not had regular periods in the past 12 months?” Serum chemistry values were measured using the Hitachi 917 multichannel analyzer (Roche Diagnostics) in 1999 to 2001 and the Synchron LX20 (Beckman Coulter Inc) in 2002 to 2004. Am J Kidney Dis. 2015;65(2):240-248

Serum bicarbonate was measured in 2 laboratories by the phosphoenolpyruvate carboxylase method in 1999 to 2001 and with a pH-sensitive electrode in 2002 to 2004. The coefficient of variation ranged between 2.3% and 5.6%. Serum bicarbonate levels during these periods were compared using weighted linear regression. Mean serum bicarbonate level was 1.105 6 0.178 mEq/L (standard deviation [SD]) higher (P , 0.001) among all NHANES participants in 2003 to 2004 compared with 1999 to 2002. Therefore, serum bicarbonate levels in 1999 to 2002 were adjusted by adding 1.105 mEq/L, as has been done previously.6,16 Because ingestion of food may affect serum bicarbonate levels, the period of fasting prior to phlebotomy was categorized as 2 or fewer, more than 2 to 6, and more than 6 hours.17 Serum albumin was measured by the bromocresol purple method. Serum creatinine was measured by a modified kinetic Jaffé reaction. Values from 1999 to 2000 were calibrated to the Cleveland Clinic laboratory standard by multiplying by 1.013 and then adding 0.147. Correction of values from 2001 to 2004 was not necessary. 25-Hydroxyvitamin D (25[OH]D) was measured using the LIAISON 25 OH Vitamin D TOTAL Assay (DiaSorin). eGFR was calculated using the CKD-EPI (CKD Epidemiology Collaboration) creatinine equation.18

Outcome Variables BMD (grams per centimeter squared) was measured by DEXA. DEXA was administered to eligible survey participants in NHANES mobile examination centers. Women younger than 60 years were permitted to take the DEXA examination only if a pregnancy test taken at the time of the examination was negative. Individuals were excluded from the DEXA examination who reported taking tests with radiographic contrast material or having participated in nuclear medicine studies in the past 3 days or had a self-reported weight (. 300 lb) or height (. 6 ft 5 in) over the DEXA table limit. Whole-body DEXA scans were acquired using a QDR 4500A fan-beam densitometer (Hologic Inc) following the manufacturer’s acquisition procedures. The scan for each survey participant was reviewed and analyzed by the University of California, San Francisco Department of Radiology using standard radiologic techniques and study-specific protocols developed by the NCHS. Of 21,230 eligible DEXA-scanned participants in NHANES 1999-2004, scans with 100% nonmissing data were obtained from 16,973, or 80%. To resolve the problem of potential biases due to missing DEXA data, multiple imputation of the missing data was performed. Details of the DEXA protocol, quality control analysis, and multiple imputation procedure are available.19 We specifically examined lumbar spine BMD and total BMD. We chose lumbar BMD because the lumbar spine is one of the most common sites of fractures.7 We also categorized low lumbar bone mass and low total bone mass as 1 SD below the respective sex-specific mean of 30-year-old adults. This definition was chosen because it mirrors the clinical definition of osteopenia,20 and most fractures occur in patients with osteopenia rather than osteoporosis because of the large number of individuals with osteopenia.7

Statistical Analysis All analyses used NHANES-appropriate sampling weights and accounted for the complex multistage cluster design using the “survey” command in Stata 12.1 (StataCorp LP). Distributions of participant characteristics were examined by quartiles of serum bicarbonate levels. A multivariable linear regression model was created to further examine the association of participant characteristics with serum bicarbonate level. Linear and logistic regression models were created to examine the association of serum bicarbonate levels with BMD (lumbar and total) and low bone mass (lumbar and total), respectively. Serum bicarbonate levels were analyzed as continuous variables and within quartiles to examine nonlinear associations with either outcome. A variable 241

Chen, Melamed, and Abramowitz was included in the final model if it was associated with serum bicarbonate level and the outcome (P , 0.2) and based on a priori determination of confounders of the association of serum bicarbonate level and bone mass. These included known determinants of bone mass and factors that may affect serum bicarbonate levels. We determined a priori to examine models after stratification by sex and also tested effect modification by sex by including multiplicative interaction terms in the multivariable linear regression models with serum bicarbonate level as a continuous variable. Variables included in the final models were age; sex; race/ ethnicity; body mass index; poverty; education; activity level; smoking status; fasting length; diuretic and calcium carbonate use; diagnosis of diabetes mellitus, hypertension, and cardiovascular disease; eGFR; albuminuria; and serum albumin, calcium, phosphate, and C-reactive protein levels. Because the difference in the association between serum bicarbonate level and BMD between the sexes could be due to different estrogen status, to examine the role of estrogen status, a subgroup analysis was performed among women with known menopausal status using multivariable linear regression models stratified by menopausal status. Effect modification by menopausal status was tested by including multiplicative interaction terms. P , 0.2 was considered suggestive of an interaction. P , 0.05 was considered statistically significant.

Sensitivity Analyses Because high net endogenous acid production has been associated with lower serum bicarbonate levels in the general population,21 we repeated our analyses in multivariable linear regression models after including estimated net endogenous acid production. Given the different effects that loop and thiazide diuretics have on urinary calcium excretion, we repeated the analyses adjusting for loop or thiazide diuretic use alone or with both of them in the models. In addition, we repeated multivariable linear regression models after restricting our population to participants with eGFRs $ 60 mL/min/1.73 m2, using serum bicarbonate levels without correction factors from 1999 to 2002, and after adjusting for 25(OH)D levels and vitamin D supplementation. Last, because low bone mass is more prevalent in the older population, we repeated the multivariable logistic regression models after restricting our population to participants older than 50 years.

RESULTS Participant Characteristics Of 9,724 participants in the analysis, 48.8% were women (Table S1, available as online supplementary material). Mean serum bicarbonate level was 24.9 6 2.2 mEq/L. Men had higher mean serum bicarbonate levels compared with women (25.2 6 2.2 vs 24.5 6 2.2 mEq/L; P , 0.001). This difference persisted in multivariable analysis including adjusting for net endogenous acid production (Table S2). Both men and women with lower serum bicarbonate levels were more likely to be current smokers and had higher body mass index and estimated net endogenous acid production (Table 1). Men with lower serum bicarbonate levels received less education and also were less likely to use diuretics and more likely to have diabetes mellitus, hypertension, cardiovascular disease, or albuminuria. Women with lower serum bicarbonate levels were more likely to be younger, have greater poverty and higher eGFRs, and were less 242

likely to be non-Hispanic white, to have fasted 2 hours or less prior to phlebotomy, or have used diuretics or calcium carbonate. In contrast to men, women with lower serum bicarbonate levels were less likely to have diabetes mellitus, hypertension, or cardiovascular disease. In bivariate analysis, a statistically significant association of lumbar or total BMD across quartiles was observed in only women, not men. There were 1,673 (36.1%) women and 1,290 (25.3%) men who had either low lumbar or low total bone mass. Participant characteristics in the full cohort and associations with serum bicarbonate levels after multivariable adjustment are shown in Tables S1 and S2, respectively. Association of Serum Bicarbonate With BMD Using multivariable linear regression models, there was a significant linear trend across quartiles of serum bicarbonate with lumbar BMD among the total population as well as in sex-specific models (P 5 0.02 for all 3 models; Table 2). Among women, compared to the reference group (, 24 mEq/L), those with bicarbonate levels of 24 to 25, 25.1 to 26.9, or $ 27 mEq/ L all had higher lumbar BMDs (0.015 [95% confidence interval (CI), 0.002-0.028], 0.016 [95% CI, 0.003-0.030], and 0.019 [95% CI, 0.003-0.036] g/cm2, respectively). For total BMD, there was no statistically significant association with serum bicarbonate level in either the total population or for men (P 5 0.2 and P 5 0.1, respectively). For women, a significant association was seen between serum bicarbonate level and total BMD (P 5 0.02). For every 2.2-mEq/L or 1-SD higher serum bicarbonate level, total BMD was greater by 0.005 (95% CI, 0.001-0.010) g/cm2 (P 5 0.03). Compared with participants with serum bicarbonate levels , 24 mEq/L, those with levels $ 27 mEq/L had 0.018 (95% CI, 0.004-0.032)-g/cm2 higher total BMD (P 5 0.02). In multivariable linear regression models, the P value for interaction between sex and serum bicarbonate level regarding their association was 0.1 with lumbar BMD and 0.8 with total BMD. Using multivariable logistic regression models, there was no statistically significant association of serum bicarbonate level with low lumbar BMD in either sex. There was a significant association of serum bicarbonate level with low total bone mass only among women (Table 3). Specifically, compared with women with serum bicarbonate levels , 24 mEq/L, those with bicarbonate levels $ 27 mEq/L had 32% lower odds of having low total bone mass (odds ratio, 0.68; 95% CI, 0.46-0.99; P 5 0.05). Among 2,805 women with known menopausal status, there was a linear trend in association between serum bicarbonate levels and lumbar BMD among both pre- and postmenopausal women (P 5 0.04 and Am J Kidney Dis. 2015;65(2):240-248

Table 1. Participant Characteristics by Quartiles of Serum Bicarbonate Levels by Sex

Characteristic

No. of participants Age (y) Race/ethnicity Non-Hispanic white Mexican American Non-Hispanic black

,24

1,120 44 6 0.5

24-25

1,175 45 6 0.5

25.1-26.9

1,500 44 6 0.4

Women $27

P

1,298 45 6 0.6

,24

1,453 0.06

41 6 0.5

24-25

1,180 45 6 0.6

25.1-26.9

1,211 48 6 0.6

$27

787 54 6 0.8

0.07 72.2% 6 2.6% 10.4% 6 1.8% 8.0% 6 1.2%

72.8% 6 2.6% 9.2% 6 1.2% 8.0% 6 1.1%

71.3% 6 2.3% 8.7% 6 1.0% 10.1% 6 1.0%

72.1% 6 1.9% 5.5% 6 0.7% 11.3% 6 1.1%

P

,0.001 0.02

70.4% 6 2.2% 9.2% 6 1.6% 10.2% 6 1.5%

70.8% 6 2.5% 6.9% 6 1.0% 11.2% 6 1.3%

71.6% 6 2.3% 5.9% 6 0.7% 12.2% 6 1.4%

73.1% 6 2.1% 3.4% 6 0.5% 12.6% 6 1.6%

29.4 6 0.2

28.2 6 0.2

27.8 6 0.2

26.7 6 0.2

,0.001

29.1 6 0.3

27.6 6 0.3

27.8 6 0.2

27.0 6 0.3

,0.001

Povertya

11.7% 6 1.0%

10.5% 6 1.0%

11.0% 6 0.8%

9.0% 6 1.1%

0.1

17.5% 6 1.2%

14.5% 6 1.5%

13.8% 6 1.2%

11.1% 6 1.2%

,0.001

,High school diploma

21.4% 6 1.7%

18.7% 6 1.2%

19.5% 6 1.4%

17.1% 6 1.3%

0.05

18.9% 6 1.1%

17.1% 6 1.2%

19.2% 6 1.4%

19.4% 6 1.6%

0.6

18.1% 6 1.1% 22.7% 6 1.5% 34.4% 6 1.6% 24.9% 6 1.3%

15.6% 6 1.2% 21.9% 6 1.2% 35.2% 6 1.6% 27.3% 6 1.8%

18.8% 6 1.5% 19.1% 6 1.5% 38.7% 6 1.7% 23.4% 6 1.4%

18.9% 6 1.5% 24.4% 6 1.6% 32.5% 6 1.9% 24.2% 6 2.2%

54.8% 6 2.5% 18.7% 6 1.7% 26.6% 6 1.6%

58.8% 6 1.6% 20.7% 6 1.4% 20.6% 6 1.6%

62.5% 6 1.6% 20.0% 6 1.3% 17.5% 6 1.1%

61.6% 6 1.6% 23.3% 6 2.0% 15.1% 6 1.6%

Body mass index (kg/m2)

Activity level 0 MET-min/wk ,500 MET-min/wk 500-2,000 MET-min/wk .2,000 MET-min/wk

12.9% 6 1.4% 21.2% 6 1.7% 36.1% 6 2.0% 29.8% 6 2.0%

12.4% 6 1.0% 20.0% 6 1.7% 34.2% 6 1.2% 33.4% 6 1.8%

13.8% 6 1.3% 17.9% 6 1.1% 36.9% 6 1.5% 31.5% 6 2.1%

12.0% 6 1.2% 18.2% 6 1.3% 35.3% 6 1.5% 34.5% 6 1.5%

Smoking Never Former Current

40.3% 6 2.6% 26.8% 6 1.4% 32.8% 6 2.5%

39.7% 6 2.0% 28.5% 6 1.5% 31.8% 6 1.5%

44.6% 6 1.7% 28.7% 6 1.6% 26.6% 6 1.4%

48.9% 6 1.9% 29.6% 6 1.4% 21.6% 6 1.6%

Estimated NEAP (mEq/d) Fasting length # 2 h Diuretic use

0.5

,0.001

0.09

,0.001

63.1 6 0.9

62.8 6 1.0

59.7 6 0.9

57.1 6 0.7

,0.001

59.3 6 0.8

57.6 6 1.0

51.5 6 0.7

48.5 6 1.1

,0.001

2.9% 6 0.5% 7.1% 6 0.8%

2.4% 6 0.5% 6.2% 6 0.8%

3.3% 6 0.5% 6.4% 6 0.8%

2.9% 6 0.6% 10.6% 6 1.2%

0.6 0.02

2.2% 6 0.5% 5.5% 6 0.7%

2.3% 6 0.5% 9.3% 6 1.2%

3.1% 6 0.5% 13.2% 6 1.3%

4.3% 6 0.9% 25.3% 6 1.9%

0.02 ,0.001

5.1% 6 0.9%

3.2% 6 0.7%

5.4% 6 0.7%

4.3% 6 1.1%

0.9

2.4% 6 0.5%

3.3% 6 0.8%

4.3% 6 0.7%

5.3% 6 1.3%

0.02

Hypertension

43.1% 6 1.8%

36.5% 6 1.7%

35.3% 6 1.7%

36.5% 6 1.9%

0.01

33.3% 6 1.3%

35.7% 6 1.6%

41.4% 6 2.0%

53.5% 6 2.1%

,0.001

Diabetes mellitus

0.01

Calcium carbonate use

11.2% 6 1.1%

8.9% 6 1.1%

7.8% 6 0.8%

6.4% 6 0.7%

,0.001

7.6% 6 0.7%

5.7% 6 0.6%

7.9% 6 0.7%

11.5% 6 1.2%

Cardiovascular disease

9.8% 6 1.0%

8.0% 6 1.1%

8.6% 6 0.9%

6.8% 6 0.7%

0.03

5.2% 6 0.5%

6.0% 6 0.8%

6.9% 6 0.9%

8.5% 6 0.9%

eGFR (mL/min/1.73 m2)

94.1 6 0.8

93.9 6 0.7

94.3 6 0.7

92.8 6 0.6

0.2

98.8 6 0.7

95.9 6 0.8

93.6 6 0.8

88.6 6 1.1

10.4% 6 0.8%

8.6% 6 0.8%

6.9% 6 0.7%

6.7% 6 0.8%

9.0% 6 0.7%

8.9% 6 0.9%

9.0% 6 1.0%

10.1% 6 1.1%

0.6

4.41 6 0.01 9.48 6 0.02

4.43 6 0.02 9.56 6 0.02

4.45 6 0.01 9.53 6 0.02

4.50 6 0.02 9.56 6 0.02

4.24 6 0.01 9.39 6 0.02

4.25 6 0.01 9.47 6 0.02

4.27 6 0.01 9.44 6 0.02

4.26 6 0.02 9.52 6 0.03

0.2 ,0.001

Urine ACR . 30 mg/g Serum albumin (g/dL) Serum calcium (mg/dL)

0.001 ,0.001 0.06

0.001 ,0.001

3.63 6 0.03

3.67 6 0.02

3.57 6 0.02

3.59 6 0.02

0.08

3.68 6 0.02

3.76 6 0.02

3.69 6 0.02

3.78 6 0.02

0.03

6.3% 6 1.0%

5.1% 6 0.7%

5.4% 6 0.6%

4.8% 6 0.6%

0.2

14.8% 6 1.2%

10.4% 6 1.0%

11.7% 6 1.5%

12.4% 6 1.5%

0.2

Lumbar BMD (g/cm2)

1.05 6 0.006

1.04 6 0.007

1.05 6 0.005

1.06 6 0.006

0.1

1.05 6 0.004

1.05 6 0.006

1.05 6 0.004

1.03 6 0.007

Total BMD (g/cm2)

1.19 6 0.005

1.19 6 0.005

1.19 6 0.004

1.19 6 0.004

0.9

1.10 6 0.003

1.10 6 0.004

1.09 6 0.003

1.08 6 0.006 ,0.001

Serum phosphate (mg/dL) C-Reactive protein $ 1.0 mg/dL

0.03

243

Note: Unless otherwise indicated, values for categorical variables are given as percentage (standard error); values for continuous variables, as mean 6 standard error. Serum bicarbonate is expressed as mEq/L. Conversion factor for serum calcium in mg/dL to mmol/L, 3 0.2495. Abbreviations: ACR, albumin-creatinine ratio; BMD, bone mineral density; eGFR, estimated glomerular filtration rate; MET, metabolic equivalent; NEAP, net endogenous acid production. a Poverty was defined as ,100% of the poverty index.

Bicarbonate and Bone Mineral Density

Am J Kidney Dis. 2015;65(2):240-248

Men

Chen, Melamed, and Abramowitz Table 2. Association of Serum Bicarbonate With Lumbar and Total BMD Total Population

Men

Women

Lumbar BMD (g/cm2) Continuousa ,24 mEq/L 24-25 mEq/L 25.1-26.9 mEq/L $27 mEq/L P for trend

0.005 (0.001 to 0.009)b (referent group) 0.004 (20.007 to 0.014) 0.009 (20.001 to 0.019) 0.014 (0.002 to 0.026)b 0.02

0.008 (0.002 to 0.015)b (referent group) 20.005 (20.023 to 0.013) 0.011 (20.005 to 0.027) 0.019 (0.0004 to 0.038)b 0.02

0.007 (0.0004 to 0.013)b (referent group) 0.015 (0.002 to 0.028)b 0.016 (0.003 to 0.030)b 0.019 (0.003 to 0.036)b 0.02

Total BMD (g/cm2) Continuousa ,24 mEq/L 24-25 mEq/L 25.1-26.9 mEq/L $27 mEq/L P for trend

0.003 (20.001 to 0.007) (referent group) 0.006 (20.003 to 0.016) 0.003 (20.006 to 0.012) 0.010 (20.002 to 0.023) 0.2

0.004 (20.001 to 0.009) (referent group) 0.005 (20.010 to 0.020) 0.005 (20.008 to 0.018) 0.012 (20.003 to 0.027) 0.1

0.005 (0.001 to 0.010)b (referent group) 0.009 (20.0004 to 0.019) 0.006 (20.002 to 0.014) 0.018 (0.004 to 0.032)b 0.02

Note: Values are given as regression coefficient (95% confidence interval). Models adjusted for age; sex; race/ethnicity; body mass index; poverty; education; activity level; smoking status; fasting length; diuretic and calcium carbonate use; diagnosis of diabetes mellitus, hypertension, and cardiovascular disease; estimated glomerular filtration rate; albuminuria; and serum albumin, calcium, phosphate, and C-reactive protein levels. Abbreviation: BMD, bone mineral density. a Per 1–standard deviation higher serum bicarbonate level (standard deviation 5 2.2 mEq/L). b P , 0.05.

P 5 0.02, respectively; Table 4). For total BMD, a statistically significant association was observed only among postmenopausal women (P 5 0.02), but not premenopausal women. Among postmenopausal women, compared with those with serum bicarbonate

levels , 24 mEq/L, those with serum bicarbonate levels $ 27 mEq/L had 0.024 (95% CI, 0.006-0.042)g/cm2 higher total BMD (P 5 0.01). P values for interaction between menopausal status and serum bicarbonate levels regarding their association with lumbar and total BMD were both 0.5.

Table 3. Odds Ratio of Low Lumbar and Total Bone Mass by Serum Bicarbonate Level

Sensitivity Analysis

Men

Women

Low lumbar bone mass Continuousa ,24 mEq/L 24-25 mEq/L 25.1-26.9 mEq/L $27 mEq/L P for trend

0.95 1.00 1.29 1.21 0.90

(0.85-1.06) (reference) (0.87-1.91) (0.87-1.70) (0.62-1.31) 0.5

0.93 1.00 0.96 0.94 0.79

(0.83-1.05) (reference) (0.75-1.24) (0.74-1.21) (0.56-1.12) 0.2

Low total bone mass Continuousa ,24 mEq/L 24-25 mEq/L 25.1-26.9 mEq/L $27 mEq/L P for trend

0.96 1.00 0.91 1.03 0.85

(0.85-1.08) (reference) (0.68-1.23) (0.75-1.43) (0.59-1.23) 0.5

0.91 1.00 1.05 0.92 0.68

(0.79-1.04) (reference) (0.77-1.43) (0.63-1.35) (0.46-0.99)b 0.08

Note: Values are given as odds ratio (95% confidence interval). Models adjusted for age; sex; race/ethnicity; body mass index; poverty; education; activity level; smoking status; fasting length; diuretic and calcium carbonate use; diagnosis of diabetes mellitus, hypertension, and cardiovascular disease; estimated glomerular filtration rate; albuminuria; and serum albumin, calcium, phosphate, and C-reactive protein levels. a Per 1–standard deviation higher serum bicarbonate level (standard deviation 5 2.2 mEq/L). b P , 0.05. 244

After including net endogenous acid production in the multivariable linear regression models, no substantial change was observed in terms of the association between serum bicarbonate level and BMD (lumbar or total; Table 5). Similarly, no substantial change was observed after we restricted our population to participants with eGFRs $ 60 mL/min/1.73 m2 (Table 6), adjusting for loop or thiazide diuretic use alone or with both of them in the models, adjusting for 25(OH)D levels or vitamin D supplementation, or using serum bicarbonate levels without correction factors from 1999 to 2002 (Tables a-f of Item S1). For multivariable logistic regression models, there was no substantial change after restricting our population to participants older than 50 years (Table g of Item S1).

DISCUSSION Having low BMD is a risk factor for osteoporosisrelated fractures.22 It is important to identify modifiable risk factors because osteoporosis can be prevented and treated before fractures occur. Our results demonstrate that lower serum bicarbonate levels are associated with lower lumbar and total BMD among adults 20 years or older in the general US population. Am J Kidney Dis. 2015;65(2):240-248

Bicarbonate and Bone Mineral Density Table 4. Subgroup Analysis Among Women Based on Menopausal Status

Total

Premenopause (n 5 1,435)

Postmenopause (n 5 1,370)

Lumbar BMD (g/cm2) Continuousa ,24 mEq/L 24-25 mEq/L 25.1-26.9 mEq/L $27 mEq/L P for trend

0.011 (0.004 to 0.018)b (referent group) 0.009 (20.008 to 0.027) 0.017 (0.001 to 0.034)b 0.028 (0.009 to 0.048)b 0.01

0.012 (0.002 to 0.021)b (referent group) 0.008 (20.020 to 0.036) 0.021 (0.003 to 0.039)b 0.021 (20.012 to 0.054) 0.04

0.009 (20.004 to 0.022) (referent group) 0.008 (20.021 to 0.037) 0.011 (20.015 to 0.038) 0.033 (0.007 to 0.060)b 0.02

Total BMD (g/cm2) Continuousa ,24 mEq/L 24-25 mEq/L 25.1-26.9 mEq/L $27 mEq/L P for trend

0.006 (0.001 to 0.011)b (referent group) 0.004 (20.007 to 0.015) 0.006 (20.003 to 0.015) 0.017 (20.0003 to 0.035) 0.05

0.005 (20.001 to 0.011) (referent group) 0.004 (20.011 to 0.020) 0.009 (20.003 to 0.021) 0.009 (20.018 to 0.037) 0.2

0.007 (20.0003 to 0.014) (referent group) 0.002 (20.016 to 0.020) 0.003 (20.014 to 0.019) 0.024 (0.006 to 0.042)b 0.02

Note: n 5 2,805. Table shows association of serum bicarbonate level with lumbar and total BMD. Values are given as regression coefficient (95% confidence interval). Models adjusted for age; sex; race/ethnicity; body mass index; poverty; education; activity level; smoking status; fasting length; diuretic and calcium carbonate use; diagnosis of diabetes mellitus, hypertension, and cardiovascular disease; estimated glomerular filtration rate; albuminuria; and serum albumin, calcium, phosphate, and C-reactive protein levels. Abbreviation: BMD, bone mineral density. a Per 1–standard deviation higher serum bicarbonate (standard deviation 5 2.2 mEq/L). b P , 0.05.

We have shown that this association was stronger for women, more specifically postmenopausal women, than for men. To our knowledge, this is the first study to examine these associations population wide and could be of potential public health importance given the scope of the problem and the morbidity and mortality associated with osteoporosis-related fractures.23,24

Chronic acidosis could result in negative calcium balance from bone buffering.1 In vivo experimental studies have shown that metabolic acidosis leads to bone disease. For example, Kraut et al2 demonstrated that metabolic acidosis enhanced bone resorption and impaired bone formation, and this might contribute to osteopenia. In addition, Gasser et al4 showed

Table 5. Sensitivity Analysis After Adjustment for Net Endogenous Acid Production Total Population

Men

Women

Lumbar BMD (g/cm2) Continuousa ,24 mEq/L 24-25 mEq/L 25.1-26.9 mEq/L $27 mEq/L P for trend

0.005 (0.001 to 0.009)b (referent group) 0.005 (20.006 to 0.016) 0.009 (20.002 to 0.019) 0.014 (0.002 to 0.026)b 0.02

0.008 (0.002 to 0.014)b (referent group) 20.006 (20.024 to 0.013) 0.009 (20.007 to 0.025) 0.018 (20.0003 to 0.037) 0.02

0.008 (0.001 to 0.014)b (referent group) 0.018 (0.004 to 0.032)b 0.017 (0.003 to 0.031)b 0.020 (0.004 to 0.037)b 0.01

Total BMD (g/cm2) Continuousa ,24 mEq/L 24-25 mEq/L 25.1-26.9 mEq/L $27 mEq/L P for trend

0.003 (20.001 to 0.007) (referent group) 0.007 (20.002 to 0.017) 0.003 (20.006 to 0.012) 0.010 (20.002 to 0.023) 0.2

0.004 (20.001 to 0.008) (referent group) 0.006 (20.009 to 0.020) 0.005 (20.009 to 0.018) 0.011 (20.004 to 0.026) 0.2

0.005 (0.0004 to 0.010)b (referent group) 0.011 (0.002 to 0.021)b 0.005 (20.003 to 0.014) 0.018 (0.003 to 0.033)b 0.03

Note: Table shows association of serum bicarbonate level with lumbar and total BMD. Values are given as regression coefficient (95% confidence interval). Models adjusted for age; sex; race/ethnicity; body mass index; poverty; education; activity level; smoking status; fasting length; diuretic and calcium carbonate use; diagnosis of diabetes mellitus, hypertension, and cardiovascular disease; estimated glomerular filtration rate; albuminuria; serum albumin, calcium, phosphate, and C-reactive protein levels; and estimated net endogenous acid production. Abbreviation: BMD, bone mineral density. a Per 1–standard deviation higher serum bicarbonate level (standard deviation 5 2.2 mEq/L). b P , 0.05. Am J Kidney Dis. 2015;65(2):240-248

245

Chen, Melamed, and Abramowitz Table 6. Sensitivity Analysis For Participants With eGFRs $ 60 mL/min/1.73 m2 Total Population

Men

Women

Lumbar BMD (g/cm2) Continuousa ,24 mEq/L 24-25 mEq/L 25.1-26.9 mEq/L $27 mEq/L P for trend

0.006 (0.001 to 0.011)b (referent group) 0.003 (20.007 to 0.014) 0.011 (0.001 to 0.021)b 0.016 (0.003 to 0.030)b 0.01

0.009 (0.001 to 0.016)b (referent group) 20.007 (20.025 to 0.012) 0.012 (20.005 to 0.029) 0.019 (20.001 to 0.039) 0.02

0.008 (0.002 to 0.014)b (referent group) 0.014 (0.001 to 0.027)b 0.017 (0.005 to 0.029)b 0.022 (0.005 to 0.039)b 0.01

Total BMD (g/cm2) Continuousa ,24 mEq/L 24-25 mEq/L 25.1-26.9 mEq/L $27 mEq/L P for trend

0.003 (20.001 to 0.007) (referent group) 0.006 (20.004 to 0.015) 0.003 (20.006 to 0.012) 0.011 (20.002 to 0.023) 0.1

0.004 (20.001 to 0.009) (referent group) 0.005 (20.010 to 0.019) 0.006 (20.008 to 0.020) 0.012 (20.004 to 0.028) 0.1

0.005 (0.001 to 0.009)b (referent group) 0.008 (20.002 to 0.017) 0.003 (20.004 to 0.011) 0.017 (0.003 to 0.032)b 0.03

Note: n 5 8,877. Table shows association of serum bicarbonate level with lumbar and total BMD. Values are given as regression coefficient (95% confidence interval). Models adjusted for age; sex; race/ethnicity; body mass index; poverty; education; activity level; smoking status; fasting length; diuretic and calcium carbonate use; diagnosis of diabetes mellitus, hypertension, and cardiovascular disease; estimated glomerular filtration rate; albuminuria; and serum albumin, calcium, phosphate, and C-reactive protein levels. Abbreviations: BMD, bone mineral density; eGFR, estimated glomerular filtration rate. a Per 1–standard deviation higher serum bicarbonate level (standard deviation 5 2.2 mEq/L). b P , 0.05.

that chronic metabolic acidosis in rats decreased total BMD using quantitative computed tomography. The association we have found in this study is consistent with these experimental studies. In patients with CKD, the National Kidney Foundation2Kidney Disease Outcomes Quality Initiative (NKF-KDOQI) guidelines suggest to supplement with alkali salts to maintain serum bicarbonate levels $ 22 mEq/L25 because bicarbonate supplements have been shown to improve skeletal metabolism in clinical studies. In children with renal tubular acidosis, alkali therapy helps attain and maintain normal stature.26 In postmenopausal women without kidney disease, administration of potassium bicarbonate improves calcium balance, reduces bone resorption, and increases bone formation.27,28 In addition, in a randomized controlled study, Dawson-Hughes et al29 demonstrated that bicarbonate supplementation in men and women 50 years or older has a favorable effect on bone resorption and calcium excretion. Similarly, Jehle et al30 randomly assigned 161 postmenopausal women with low bone mass to receive potassium citrate or potassium chloride daily and found that women who received potassium citrate had increased bone mass. In another randomized, double-blind, placebo-controlled trial, they randomly assigned 201 healthy men and women older than 65 years without osteoporosis to potassium citrate or placebo and found that potassium citrate increased lumbar BMD after 24 months.31 Regardless of the existing evidence on the benefit of alkali therapy on bone health, alkali therapy still is not 246

given routinely to persons without kidney disease in the clinical setting to maintain bone mass. Our study demonstrates that within a relatively normal range of serum bicarbonate levels, having higher serum bicarbonate levels is associated with higher BMD in the general population without advanced CKD. These data give more support to providing alkali therapy in the general population for improvement of bone health. Our study also suggests that the association between serum bicarbonate levels and BMD is stronger among women than men and stronger among postmenopausal women than premenopausal women. We acknowledge that the P values for interaction between sex and serum bicarbonate level did not uniformly suggest the presence of an interaction (P 5 0.1 and P 5 0.8 for lumbar and total BMD, respectively). However, the decision to stratify the analyses by sex was a priori. In the stratified analysis for lumbar BMD, compared with participants with serum bicarbonate levels , 24 mEq/L, women with bicarbonate levels $ 24 mEq/L had higher BMD, but men did not have higher BMD until bicarbonate levels were $ 27 mEq/L. For total BMD, an association was observed only among women, but not men (Table 2). This seems to suggest that the association between serum bicarbonate level and BMD is stronger in women than men. Similarly, in subgroup analysis by menopausal status, P for interaction did not suggest the presence of an interaction, but the decision to stratify was also a priori and the analysis was exploratory. In addition, smaller sample size (n 5 2,805) could have limited the power to detect an Am J Kidney Dis. 2015;65(2):240-248

Bicarbonate and Bone Mineral Density

interaction. Based on the stratified analysis by menopausal status, the association seems to be stronger among postmenopausal women than premenopausal women, particularly for total BMD (P 5 0.02 and P 5 0.2, respectively; Table 4). The differences in the association between sexes and by menopausal status could be due in part to the effect of estrogen. The role that estrogen might play in the association of acidosis and bone metabolism was suggested by the study done by Gasser et al.4 In their study, chronic metabolic acidosis decreased bone mass in both ovariectomized and sham-operated rats. Ovariectomized rats without acidosis had lower total BMD than both sham-operated rats with and without acidosis, and chronic metabolic acidosis further accelerated bone loss induced by ovariectomy. In other words, estrogen status might modify the association of acidosis with BMD. Because postmenopausal women are at the highest risk of developing osteoporosis, the role of sex and hormonal status could be clinically relevant and warrants further investigation. In our cross-sectional study, we found that serum bicarbonate levels were higher in men compared with women and higher in older women compared with younger women. These associations persisted after multivariable adjustment. The lower serum bicarbonate levels in women could be explained in part by mild hypocapnia during the luteal phase of the menstrual cycle.32 The positive association of serum bicarbonate level with age seems unexpected given previous reports,5,33 but is consistent with our recent analysis of serum bicarbonate levels and aging in Americans using NHANES data.21 Higher bicarbonate levels among older persons were explained partly, but not completely, by differences in dietary acid load related to age. However, adjustment for net endogenous acid production did not change our results here. Our analysis has several important limitations. Because arterial pH and PCO2 values were not available, lower bicarbonate levels cannot be excluded as markers of alterations in respiratory status. However, participants with chronic obstructive pulmonary disease were not included in our analysis. Single measurements were used to ascertain serum bicarbonate levels, and variability in handling of samples could affect bicarbonate levels.34-36 This presumably would introduce nondifferential misclassification and bias our results toward the null hypothesis. In addition, although our definition of low bone mass mirrored the clinical definition of osteopenia, they are not equivalent. Thus, this limits its clinical implications. Finally, because of the crosssectional nature of our analysis, no causal association can be inferred; the possibility of reverse causality cannot be excluded. In conclusion, we have shown that lower serum bicarbonate levels were associated with lower lumbar Am J Kidney Dis. 2015;65(2):240-248

and total BMD in US adults. Further studies are warranted to illuminate the determinants of serum bicarbonate levels in people without kidney disease and investigate whether serum bicarbonate levels should be incorporated into the diagnostic assessment guidelines or management of osteoporosis.

ACKNOWLEDGEMENTS Support: This research was supported by American Society of Nephrology Carl W. Gottschalk Research Scholar Grants (Drs Melamed and Abramowitz), National Institutes of Health (NIH) grants U01 087783 (Drs Melamed and Abramowitz) and T32 DK007110 (Dr Chen), and Clinical and Translational Science Award grants 1 UL1 TR001073-01, 1 TL1 TR001072-01, and 1 KL2 TR001071-01 from the National Center for Advancing Translational Sciences, a component of the NIH. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. Financial Disclosure: The authors declare that they have no other relevant financial interests. Contributions: Research idea and study design: WC, MKA, MLM; data analysis: WC; data interpretation: WC, MKA, MLM; supervision/mentorship: MKA, MLM. Each author contributed important intellectual content during manuscript drafting or revision and accepts accountability for the overall work by ensuring that questions pertaining to the accuracy or integrity of any portion of the work are appropriately investigated and resolved. WC and MKA take responsibility that this study has been reported honestly, accurately, and transparently; that no important aspects of the study have been omitted, and that any discrepancies from the study as planned have been explained.

SUPPLEMENTARY MATERIAL Table S1: Participant characteristics by quartiles of serum bicarbonate for total population. Table S2: Multivariable linear regression model of participant characteristics with bicarbonate levels. Item S1: Sensitivity analyses. Note: The supplementary material accompanying this article (http://dx.doi.org/10.1053/j.ajkd.2014.07.007) is available at www. ajkd.org

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