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An incomplete random block design was set up so that 5 beverages were tested on each woman: water, both of the cola beverages, one of the milks, and one of the 2 citric acid–containing beverages. Within each subject the beverage sequence was randomized by using the random number function of EXCEL (Microsoft, Redmond, WA).
The sequence of tests in each woman was arranged on a 1-wk cycle, so that the entire suite of 5 tests was completed, in most subjects, within one calendar month. The subjects reported to the research unit fasting in the morning, after having voided at home, noting the time. They stayed in the unit, fasting, until a 2-h second specimen was collected. They were then fed a breakfast consisting of the test beverage and 2 pieces of specially baked, low-calcium, Italian style white bread, toasted, with butter. The milk serving was 340 mL (12 oz) and the carbonated beverage servings were 567 mL (20 oz). The difference in fluid volume between the milks and the other beverages was compensated for by having the subjects ingest an additional 227 mL deionized water. The differences in carbohydrate intake between the beverages were compensated for by providing the subjects with the requisite number of jelly beans (consisting mainly of sucrose and corn syrup) to equalize total sugar intake for all test meals (1.256 MJ total). All urine was collected during the next 5-h period, during which time subjects consumed an additional 567 mL deionized water; no further food was consumed until the 5-h urine collection had been completed.

Women were recruited who habitually consumed at least two 340-mL (12-oz) cans of carbonated beverages daily. The total sample size was 32. Two women dropped out of the study early and their data are not included in this report. All subjects were instructed to maintain their usual calcium intakes throughout the study and to refrain from high-sodium foods for 2 d before each test. The mean (±SD) age of the subjects was 31.4 ± 5.6 y and their mean body mass index (in kg/m2) was 25.2 ± 3.75. The subjects' habitual intake of carbonated beverages, by history, ranged from two to seven 340-mL (12-oz) servings/d (: 3.16). Twenty-seven of the 30 subjects habitually consumed predominantly colas, and 3 consumed predominantly the citric acid–containing beverages. Median calcium intake, as determined by a food-frequency questionnaire modified from the Block food-frequency questionnaire (14), was 19.6 mmol/d (interquartile range: 15.8–30.4 mmol/d).

Analytic methods
Urine samples were analyzed for calcium, creatinine, sodium, titratable acidity, and total acidity. Colas were analyzed for acidity, sodium, and phosphorus. Calcium was determined by atomic absorption and sodium by flame emission spectrophotometry (Perkin Elmer AAnalyst, Norwalk, CT), creatinine by an autoanalyzer method based on the Jaffe reaction (15), and both titratable and total acidity by the method of Chan (16). (This method equates total acidity to titratable acidity plus ammonium ion, less carbonic acid.) Phosphorus was analyzed by an autoanalyzer method based on Fiske and Subba Row (17). pH was measured by using a Fisher Accumet pH meter (model 915; Fisher Scientific, St Louis). The caffeine content of the beverages was attained from The Food Processor database (ESHA, Salem, OR).

Statistical methods
The data were analyzed in various ways, depending on the a priori hypotheses, ie, some by ANOVA, testing for treatment and order, and some by simple paired t tests. When the data were not normally distributed, the summary descriptive statistics were the median and interquartile range. Differences were tested against a null hypothesis of zero. In addition to the raw data, various derived variables were calculated, principally focused on estimating the excess urinary calcium (Cau) after beverage consumption. The increments produced by carbonated beverage ingestion were not much greater than the changes observed with water alone or the usual day-to-day variability in calciuria. (The within-subject CV for calcium content of the 2-h fasting specimens was 10.9%; that for the calcium-to-creatinine ratio was 9.1%.) For that reason and to minimize bias in estimating effects, we used 4 methods of estimating excess calciuria. The resulting data were analyzed statistically as above. The 4 methods, based on the analyte content in the 5-h urine sample after the test breakfast, except where otherwise indicated, were as follows:

Protocol

Four different carbonated beverages were tested, water was used as a negative control, and 2 milks (white and chocolate) were used as positive controls, bringing the total of tested beverages to 7. The beverages, their composition, and pertinent characteristics are shown in Table 1. Two of the beverages (Coke and Coke-Free; The Coca-Cola Company, Atlanta) included phosphoric acid as the acidulant and 2 (Mountain Dew; The Pepsi Cola Company, Purchase, NY; and Sprite; The Coca-Cola Company) used citric acid. Two provided caffeine (Coke and Mountain Dew) and 2 did not (Coke-Free and Sprite). The brands studied were chosen because their contents of the putatively harmful agents were toward the high end of their categories.

In several observational studies, intake of carbonated beverages was associated with reduced bone mass or increased fracture risk, both later in life (1) and in children and adolescents (2–4). In most reports, colas were more strongly associated than were other carbonated beverages. Several investigators suggested that the factor or factors responsible for this association may be the increase in phosphorus intake or the net acid load of those beverages that use phosphoric acid as the acidulant or the caffeine of those beverages that are caffeinated. More recently, fructose, found in beverages that use natural sweeteners, was implicated as a possible cause of reduced calcium balance (5). For most of these factors, the effect is usually attributed to increased urinary calcium loss. Individually, phosphorus and caffeine were shown to have little or no net effect (6–11), but concern remains about the acid load (12). The combination of all 3 factors, as would be found in many colas, has not been directly tested.

The issue is especially important today because calcium intakes in North America fall far short of current recommendations. Per capita carbonated beverage consumption has risen dramatically, and carbonated sodas are now the preferred beverage of 20–40 y-old women (13). Interference in the calcium economy of persons with already low calcium intakes would only aggravate any calcium shortfall. This issue was studied in an experimental design only once before, and then only for a single cola (9). Accordingly, and because of the wide interest among nutritionists and dietitians in the possible effects of carbonated beverages, we undertook the present study. We investigated the acute effect on urinary calcium loss of intake of carbonated beverages of various compositions by adult women who were habitual users of such beverages.

Carbonated beverages

Background: Intake of carbonated beverages has been associated with increased fracture risk in observational studies. The usual explanation given is that one or more of the beverage constituents increase urinary calcium.

Objective: We assessed the short-term effects on urinary calcium excretion of carbonated beverages of various compositions.

Design: An incomplete random block design was used to study 20–40-y-old women who customarily consumed 680 mL carbonated beverages daily. Four carbonated beverages were tested: 2 with caffeine and 2 without. Two contained phosphoric acid as the acidulant and 2 contained citric acid. The study included one neutral control (water) and one positive control (skim or chocolate milk). Serving size was 567 mL for the carbonated beverages and water and 340 mL for the milks. Beverages were consumed with a light breakfast after an overnight fast; no other foods were ingested until urine collection was complete. pH, titratable and total acidity, sodium, creatinine, and calcium were measured in 2-h (morning) fasting and 5-h postbeverage urine specimens.

Results: Relative to water, urinary calcium rose significantly only with the milks and the 2 caffeine-containing beverages. The excess calciuria was 0.25 mmol, about the same as previously reported for caffeine alone. Phosphoric acid without caffeine produced no excess calciuria; nor did it augment the calciuria of caffeine.

Conclusions: The excess calciuria associated with consumption of carbonated beverages is confined to caffeinated beverages. Acidulant type has no acute effect. Because the caffeine effect is known to be compensated for by reduced calciuria later in the day, we conclude that the net effect of carbonated beverage constituents on calcium economy is negligible. The skeletal effects of carbonated beverage consumption are likely due primarily to milk displacement.


Key Words: Carbonated beverages • colas • caffeine • phosphorus • phosphoric acid • urinary calcium • acid loading • citric acid • fracture risk