Low Muscular Mass and Overestimation of Microalbuminuria by Urinary Albumin/Creatinine Ratio

Methods

The Gubbio Study is an epidemiological investigation ongoing in the Italian city of Gubbio.^9,10 The study adheres to the principles of the Declaration of Helsinki and the Title 45 of US Code of Federal Regulation. Study activities were approved by the local institutional review committee, and an informed consent was given by participants. All of the procedures were in accordance with institutional guidelines. The protocol for participants 45 to 64 years of ages included the collection of timed overnight urine,⁴ standardized questionnaires on cardiovascular disease,¹¹ blood pressure measurements, a 12-lead resting ECG read by the Minnesota Code,¹¹ weight, and skinfold thickness.¹²

As reported previously,⁹ blood pressure was measured by trained medical doctors using mercury sphygmomanometers and cuffs of appropriate size with the participant in the sitting position. The first measurement was done after 5 minutes of quiet rest after application of the cuff. The mean of second and third measurements was used for analyses. Albumin, creatinine, and urea were measured in urine samples: albumin by ultrafiltration immunoturbidimetry⁴ and creatinine and urea by automated biochemistry.¹³ The rate of uAE (in μg/min) was defined as normal when <20, microalbuminuria when in the range of 20 to 199, and macroalbuminuria when ≥200.¹⁴ The uACR (in μg/mg) was defined as nonhigh when <17 in men and <25 in women and high when ≥17 in men and ≥25 in women.^1,2,6 Persons with macroalbuminuria were excluded from analyses to focus on uAE in the range of normalcy and microalbuminuria. Hypertension was defined as systolic pressure ≥140 mm Hg and/or diastolic pressure ≥90 mm Hg and/or regular treatment with antihypertensive drug(s). CHD was analyzed as the most common clinical presentation of vascular disease¹¹ and defined with use of ECG data combined with symptoms reported in the questionnaire as described previously.⁸ Myocardial infarction or ischemia were combined in analysis, because previous data showed that uAE is similarly and independently related to both disorders.⁸ Data of skinfold thickness (triceps and subscapular) and weight were used for the estimation of fat mass.¹² Nonfat mass was calculated as weight minus fat mass.¹² Urinary creatinine excretion (in mg/min) was used as index of muscle mass.¹⁵ Urinary urea (in mg/min) was used as index of dietary protein intake^13,16 to control the confounding of diet composition on urinary creatinine.¹⁷

Statistical procedures included Pearson and rank correlation analysis, McNemar’s test for paired observations of categorical data, χ² analysis, logistic regression analysis, ANOVA, and analysis of odds ratio (OR) with 95% CI. Data are reported as mean±SD unless otherwise indicated.

Results

Relation of uACR to CHD

Differences by Level of uAE

Figure shows data for men and women combined on CHD prevalence in the group of persons with nonhigh uACR and the group of persons with high uACR. Persons with high uACR were also analyzed as 2 subgroups: the subgroup without microalbuminuria and the subgroup with microalbuminuria. CHD prevalence was significantly higher in the high uACR group than in the nonhigh uACR group. Findings were similar for myocardial infarction (prevalence in high uACR group and nonhigh uACR group=4.3% and 2.4%, respectively; OR, 1.83; 95% CI, 0.76 to 4.42) and ischemia (prevalence in high uACR group and nonhigh uACR group=8.0% and 5.0%, respectively; OR, 1.65; 95% CI, 0.86 to 3.19). The excess of CHD prevalence in the high uACR group reflected the combination of dissimilar data between the subgroups with and without microalbuminuria. In comparison to the nonhigh uACR group, CHD prevalence was significantly increased in the high uACR subgroup with microalbuminuria but not in the high uACR subgroup without microalbuminuria. CHD was 2.59-times more prevalent in the high uACR subgroup with microalbuminuria than in the high uACR subgroup without microalbuminuria (95% CI, 0.92 to 7.30; P=0.065). Findings were similar in analyses by gender: CHD prevalence was higher in the high uACR subgroup with microalbuminuria than in the high uACR subgroup without microalbuminuria among men (19.2% and 2.5%, respectively; OR=9.29; P=0.021) and women (18.5% and 13.3%, respectively; OR=1.48; P=0.554). Findings were also similar in analyses limited to hypertensive subjects: CHD prevalence was higher in hypertensive subjects with high uACR and microalbuminuria than in hypertensive subjects with high uACR without microalbuminuria (25.2% and 8.2%, respectively; OR=3.75; P=0.030).

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Comparison of coronary heart disease prevalence between persons with nonhigh uACR (□, n=1485) and persons with high uACR. Persons with high uACR are analyzed as a whole group (high uACR, , n=138) and as 2 subgroups: without microalbuminuria (high uACR without microalbuminuria, , n=85) and with microalbuminuria (high uACR with microalbuminuria, ▪, n=53). P values are by χ² analyses in comparison with persons with nonhigh uACR.

Discussion

The study reports new findings on the confounding because of the use of the uACR for assessment of microalbuminuria. First, the prevalence of microalbuminuria was overestimated with use of uACR, because &60% of persons with high uACR do not have microalbuminuria. Second, persons with high uACR but without microalbuminuria did not have the excess of CHD prevalence typical of persons with microalbuminuria. Third, the combination of high uACR with normal uAE was associated with low nonfat mass or low muscular mass in analyses for the whole population sample and in analyses limited to persons with high uACR. Fourth, in persons with low nonfat mass, high uACR reflected low urinary creatinine excretion rather than microalbuminuria and vascular disease.

Various methodological limitations could have affected the results of the study. The errors in urine collection precision, certainly frequent in a population-based study, should have diluted the results for uAE but not those for uACR, because the uACR calculation is independent of duration and volume of urine collection. Thus, because of this limitation, the results might underestimate the strength of the association between high uAE and CHD but not between high uACR and CHD. The use of a single urine sample likely caused errors in the classification of normal/high values because of daily variability in urinary albumin.¹⁴ This misclassification should have similarly influenced the results for uAE and uACR, because these 2 indices were measured in the same urine sample. The same should be true for the misclassification attributed to absence of urinalysis that could be of help for definition of persons with high urinary albumin secondary to urinary tract infection.¹⁴ The exclusion from analysis of data for other vascular diseases should have had only a minor role, unless urinary albumin and urinary creatinine (ie, muscular mass) were associated with those diseases differently than CHD. The use of skinfold data and of urinary creatinine could have caused an inaccurate assessment of muscular mass, although the finding of an independent relation between nonfat mass and urinary creatinine is against this possibility. A different preexamination mortality between persons with low and high muscular mass could have confounded some of the results. Finally, the cross-sectional design of the analysis did not exclude the possibility of changes in urine composition secondary to CHD,¹⁸ a point that was behind the aims of the study yet was important for the comprehension of the mechanisms underlying the association urinary albumin and CHD.

Taken together, study results indicate that interindividual differences in muscular mass substantially influence the value of the uACR via urinary creatinine excretion. Because of this influence, persons with low muscular mass often have the moderate uACR elevation used for the definition of microalbuminuria in the absence of a true elevation in absolute uAE. Thus, the uACR has a low predictive power for the definition of microalbuminuria implying the overestimation of the true prevalence of microalbuminuria in persons with low urinary creatinine because of low muscular mass. The finding that the group of persons with high uACR and low muscular mass was without an excess of CHD prevalence additionally supports this interpretation, because present and previous data consistently show an independent association between microalbuminuria and CHD.^8,19 Within the middle-age sample of the Gubbio Population Study, the overestimation of microalbuminuria because of low urinary creatinine was found in &60% of persons with high uACR. Muscular mass and urinary creatinine excretion undergo parallel reductions during aging.^20,21 Thus, a reasonable inference is that the frequency of the overestimation of microalbuminuria could progressively increase from young to older ages. The finding in this study of a positive relationship between age and the prevalence of overestimation of microalbuminuria supports this possibility. At variance with the low value of the positive predictive power, uACR appears to have a high negative predictive power, because a person with nonhigh uACR was unlikely to have microalbuminuria.