Muscle metabolites, detected in urine by proton spectroscopy, correlate with disease damage in juvenile idiopathic inflammatory myopathies

Authors


Abstract

Objective

To assess for novel markers of muscle damage using urinary muscle metabolites by 1H magnetic resonance spectroscopy in patients with juvenile idiopathic inflammatory myopathy (IIM).

Methods

Creatine (Cr), choline (Cho), betaine (Bet), glycine (Gly), trimethylamine oxide (TMAO), and several other metabolites were measured in first morning void urine samples from 45 patients with juvenile IIM and from 35 healthy age-matched controls, and correlated with measures of myositis disease activity and damage. Urinary metabolite to age-adjusted creatinine (Cn) ratios were examined.

Results

Age-adjusted initial Cr:Cn, Cho:Cn, Bet:Cn, Gly:Cn, and TMAO:Cn ratios were higher in patients with juvenile IIM than controls (P < 0.01). Cr:Cn ratios showed significant correlations with physician-assessed global disease damage (Spearman rs = 0.37; P = 0.01), Steinbrocker functional class (rs = 0.35; P = 0.02), serum Cr (rs = 0.72; P = 0.001), and lactate dehydrogenase (rs = 0.34; P = 0.03) levels. Cho:Cn (rs = 0.3; P = 0.05), Gly:Cn (rs = 0.33; P = 0.03), and TMAO:Cn (rs = 0.36; P = 0.02) ratios showed a significant correlation with serum aldolase levels. Cho:Cn ratios also showed a significant correlation with aspartate aminotransferase levels (rs = 0.35; P = 0.02). A linear regression model was used to evaluate the factors influencing urinary Cr:Cn ratios in the 43 patients with data sets available at the initial visit. The regression model explained 73% of the variation in Cr:Cn ratios. The most significant factor was the physician-assessed global disease damage (R2 = 0.50, P = 0.015).

Conclusion

Urinary Cr:Cn, Cho:Cn, Bet:Cn, Gly:Cn, and TMAO:Cn ratios are elevated in juvenile IIM and Cr:Cn correlates strongly with global disease damage. The Cr:Cn ratio may have potential utility as a marker of myositis disease damage.

INTRODUCTION

Juvenile idiopathic inflammatory myopathies (IIMs) are characterized by chronic inflammation in skeletal muscle, skin, and other target organs (1). There are a limited number of validated clinical measures to assess activity and damage in the juvenile IIMs, including global muscle strength and physical function measures (2–6). Such clinical assessments are used with laboratory investigations to assess the inflammatory activity of juvenile IIM (7–9).

These clinical assessments all have substantial limitations. Creatine kinase (CK) and other enzyme levels are often unrelated to disease activity in children (10, 11). The consequent search for alternative approaches has focused on in vivo magnetic resonance imaging (MRI) (12–16) and magnetic resonance spectroscopy (MRS) (16–18). Although MRI shows abnormalities in adults and children with IIM, it is expensive, not widely available, and insensitive for detecting further changes in damaged muscles with extensive fatty infiltration or atrophy.

Simpler assessment methods may have more clinical value, especially if they reflect overall changes in the muscles. Measuring metabolic products such as creatine in the urine is one approach to finding alternative assessment methods. We have already shown its potential value in adults in a study of 34 patients with polymyositis (PM)/dermatomyositis (DM) and 109 controls (healthy adults and patients with adult-onset muscle dystrophies, myopathies, strokes, and arthritis). Twenty patients with DM/PM had high urinary creatine (Cr):creatinine (Cn) ratios, but no controls had high Cr:Cn ratios (19).

The present study evaluates in vitro proton (1H) MRS (20) profiles of first morning void urine samples from patients with juvenile IIM. Our aim was to establish whether urinary metabolites provided relevant and reliable information about disease damage.

PATIENTS AND METHODS

Patients

We studied 45 patients meeting probable or definite Bohan and Peter criteria for juvenile IIM (1), including 41 patients with juvenile DM and 4 with juvenile PM. The study group comprised 30 girls and 15 boys with a mean age of 9 years (range 3–18 years). The patients were seen at baseline and at 7–9 months followup. The baseline clinical characteristics of the patients are similar to those previously published by Rider et al (21). In brief, baseline median disease activity and disease damage on a visual analog scale (VAS) in the patients with juvenile IIM were 2.5 cm (range 0–9.7 cm) and 0.4 cm (range 0–8.0 cm), respectively.

We also studied a group of 35 healthy control subjects (18 girls, 17 boys) with a mean age of 9 years (range 3–17 years). The control subjects were seen once. All patients with juvenile IIM and control subjects were assessed clin- ically, and blood and first morning void urine samples were obtained for conventional measures of disease activity and for analysis of urinary muscle metabolites.

Clinical and conventional laboratory measures

Physician and parent global assessments of disease activity and damage, using both Likert and VAS scales, were obtained (2, 5). The Physician Global Disease Activity and Damage VAS score is an accepted standardized scoring system for assessing disease activity and damage (2, 5). Twenty-two patients (at one center) >4 years of age underwent manual muscle testing, which was performed by a pediatric physical therapist; this could not be performed in younger patients, as previously explained by Rider (6). The assessor used a Kendall 10-point scale to measure 26 proximal, distal, and axial muscle groups (total score range 0 [no strength]–260 [normal strength]) (7). Physical function was assessed using the Childhood Health Assessment Questionnaire, a 30-item parent or self-report questionnaire that examines physical function in 8 domains: dressing and grooming, arising, eating, walking, hygiene, reach, grip, and activities (3). Physical function was also assessed using the Childhood Myositis Assessment Scale, a 14-activity, observational, performance-based assessment of physical function, strength, and endurance (5). Functional capacity was determined by the subject's rheumatologist, using the Steinbrocker classification (5). Serum concentrations of skeletal muscle-associated enzyme activities, including CK, aldolase, lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and aspartate aminotransferase, were measured. To allow for comparison of laboratory investigations performed at different centers, all results were standardized by dividing the values by the upper limit of normal for the laboratory in which the test was performed. Serum creatine levels were also evaluated in 17 patients at one center. STIR MRI (time to recovery [TR] 1,633 msec, time to echo [TE] 30 msec, and inversion time 100 msec) and T1 images (TR 400 msec and TE 10 msec) of the thighs were obtained using a 0.5 Tesla MR unit (Picker International, Cleveland, OH) in 24 patients at one center, and were read by a radiologist blinded to clinical findings. A fatty infiltration score and a muscle atrophy score for each patient were determined as previously described (12, 22).

Urinary muscle metabolites

Using 1H-MRS, we measured muscle metabolites in first morning void urine samples, including Cr, choline (Cho), betaine (Bet), citrate, taurine, Cn, and trimethylamine oxide (TMAO) (19). Cn values were age-adjusted for by using a correction factor (6.3016 + 0.40295 × age), as previously described (21). To adjust for the effects of hydration, muscle mass, and urine volume, these metabolites were expressed as ratios relative to urine Cn levels.

MR spectroscopy

A 0.6-ml sample of urine was placed in a 5-mm nuclear magnetic resonance tube with 0.1 ml of D2O. Proton MRS was performed using a 500 MHz Bruker System (Bruker Biospin, Coventry, UK; pulse angle of 45o, repetition time of 3.5 seconds, 64 averages). A pulse and collect with water suppression MR sequence was used. Resonances in urine spectra were assigned and metabolites ratios were calculated relative to the Cn peak as previously described (19). The severe overlap of the Cn and Cr resonances at 3.05 parts per million (ppm) led us to use the Cn resonances at 4.07 ppm for quantification. All data were acquired under identical experimental condition, hence the relaxation effects were standardized.

Statistical analysis

Data were analyzed using SPSS (SPSS, Chicago, IL) to calculate medians, interquartile ranges (IQRs), and Spearman's rank correlation coefficients. Differences between groups were analyzed by the Wilcoxon rank sum test. A nonparametric method was used because the data were not normally distributed. In addition, we undertook linear regression analyses using an upper limit of significance of 0.10. We did not correct for multiple statistical comparisons because, as there have been no previous studies of urinary Cr in children with IIM, our study had to be an exploratory investigation and not one in which specific hypotheses were tested on the basis of preexisting data. As a consequence the findings would ideally need verification in a prospective study.

RESULTS

Muscle urine metabolites in juvenile IIM and controls

Age-adjusted initial Cr:Cn ratios were higher in patients with juvenile IIM than control subjects (median 5.6 versus 1.4, IQR 2.6–11.9 versus 0.0–4.9; P < 0.001) (Table 1). Age-adjusted initial Cho:Cn, Bet:Cn, Gly:Cn, and TMAO:Cn ratios were also higher in patients with juvenile IIM than controls (P < 0.01) (Table 1). There were no differences in citrate:Cn and taurine:Cn ratios between patients and controls (Table 1).

Table 1. Urinary metabolites in juvenile idiopathic inflammatory myopathy patients and control subjects*
Metabolite:Cn ratioControls (n = 35)Juvenile IIM group (n = 45)P
  • *

    Values are the median (interquartile range). Cn = creatinine; IIM = idiopathic inflammatory myopathy.

  • Age-adjusted ratios significantly different (P < 0.05) between control and juvenile IIM group by Mann-Whitney U test.

Creatine:Cn1.4 (0.0–4.9)5.6 (2.6–11.9)0.001
Choline:Cn0.2 (0.0–0.5)0.6 (0.2–1.4)0.002
Taurine:Cn1.0 (0.5–2.0)0.7 (0.0–1.8)0.55
Betaine:Cn0.5 (0.1–1.1)1.5 (0.7–3.4)0.001
Citrate:Cn3.8 (2.4–4.9)3.9 (2.6–6.0)0.27
Glycine:Cn1.2 (0.8–2.0)2.2 (1.2–3.6)0.01
Trimethylamineoxide:Cn0.5 (0.3–0.6)1.0 (0.4–1.9)0.001

Relationships of muscle metabolites with juvenile IIM disease activity and damage

Correlations at baseline assessment between urinary muscle metabolites and measures of myositis disease activity and damage showed that the Cr:Cn ratio correlated significantly with the physician-assessed global disease damage VAS (rs = 0.37; P = 0.01; Figure 1A), Steinbrocker functional class (rs = 0.35; P = 0.02), serum Cr (rs = 0.72; P = 0.001; Figure 1B), and LDH (rs = 0.34; P = 0.03) levels. Cho:Cn (rs = 0.3; P = 0.05), Gly:Cn (rs = 0.33; P = 0.03), and TMAO:Cn (rs = 0.36; P = 0.02) ratios showed a significant correlation with serum aldolase levels. Cho:Cn ratios also showed a significant correlation with ALT (rs = 0.35; P = 0.02). No significant correlation between physician-assessed global disease activity and urinary metabolites was found. No other significant correlation between urinary metabolites and clinical, laboratory, and MRI measures was observed.

Figure 1.

Correlation of age adjusted urinary creatine/creatinine ratios with Physician-Assessed Global Disease Damage(A) and serum creatine level(B) in juvenile idiopathic inflammatory myopathy patients. VAS = visual analog scale.

An alternative approach used physician-assessed disease damage classified on 0–4 Likert scales. Urinary Cr:Cn ratios were markedly elevated with moderate to severe disease damage (grade ≥ 2). There was no significant correlation between corticosteroid daily dose and urinary muscle metabolites (Cr:Cn, Cho:Cn, and Bet:Cn ratios).

Factors determining urinary creatine/creatinine ratios

A linear regression model was used to evaluate the factors influencing urinary Cr:Cn ratios (Table 2) in the 43 patients in which data sets were available at the initial visit. All factors that correlated with Cr:Cn ratio (P < 0.1), i.e., physician-assessed global disease damage VAS, serum Cr, LDH, and functional class, were included in the regression model. In these cases the regression model explained 73% of the variation in Cr:Cn ratios. The variable that most significantly correlated with the Cr:Cn ratio was the physician-assessed global disease damage VAS (adjusted R2 = 0.5, P = 0.015) (Tables 2 and 3).

Table 2. Linear regression model of factors explaining age-adjusted urinary creatine/creatinine ratio*
ModelR2Adjusted R2SEE
  • *

    All factors showing P < 0.10 included in the model. SEE = standard error estimate.

  • Model 1 = Physician Global Disease Damage; Model 2 = Physician Global Disease Damage, serum creatine; Model 3 = Physician Global Disease Damage, serum creatine, functional class.

10.730.500.70
20.840.660.58
30.890.730.51
Table 3. Summary of results of model 3*
ModelBSEBetatP
  • *

    Creatine/creatinine ratio adjusted R2 = 0.73. Data from 43 juvenile idiopathic inflammatory myopathy patients were used in this analysis. SE = standard error.

3 (constant)−3.091.12−2.750.017
Physician global disease damage0.830.300.422.800.015
Serum creatine6.341.620.743.920.002
Functional class−1.710.76−0.38−2.250.042

DISCUSSION

At present, there are limited laboratory measures to assess disease damage in juvenile IIM and none are ideal (5, 7, 9, 10, 13). We have found that first morning void urine samples from patients with juvenile IIM show highly significant changes in their metabolic profiles compared with healthy control subjects. In particular urinary Cr:Cn ratios are consistently elevated and strongly correlated with disease damage. Betaine, choline-containing compounds, glycine, and TMAO levels are also different from those in healthy children. The primary correlation of urinary Cr:Cn ratios, especially as shown by the linear regression analysis, is with disease damage.

Elevated urinary Cr:Cn ratios have been reported in adults with DM and PM (19, 23). Although it has been suggested that elevated Cr:Cn ratios reflect a steroid-induced myopathy (23), we have previously found no evidence to support this suggestion in adults (19). There is also no evidence that Cr:Cn ratios are a consequence of disuse atrophy of muscles because they remain normal after strokes and in patients with rheumatoid arthritis (19). In the present study of juvenile IIM, Cr:Cn levels were unrelated to both total daily steroid dose and evidence of muscle atrophy assessed by MRI. We did not study children with other chronic disorders involving muscle damage; however, because we do not consider Cr:Cn ratios to have diagnostic relevance, we believe that studying such diseased controls is not a matter of critical importance. Although creatinuria in active juvenile DM has been reported previously, that study did not involve detailed comparisons of Cr levels with clinical disease damage (24).

Several mechanisms explain the elevated urinary Cr levels in juvenile IIM. Cr is synthesized in the liver, kidney, and pancreas (25, 26), and its uptake by muscle cells involves an active transport mechanism (27). The threshold for renal Cr excretion normally exceeds its plasma concentration (28). Creatinuria may result from increased biosynthesis, dietary supplements, reduced muscle absorption, and cell leakage (25, 29, 30) together with decreased renal reabsorption (31, 32). In juvenile IIM, because Cr:Cn ratios correlate with disease damage, creatinuria is likely to result from reduced muscle Cr absorption and Cr leakage from damaged muscle. MRS in adults with inflammatory myopathies shows reduced inorganic phosphate:phosphocreatine ratio in the muscles, but normal levels of total Cr (16, 33, 34). These findings indicate poor retention of intracellular Cr, rather than reduced uptake by skeletal muscle cells. The situation is likely to be similar in juvenile IIM. Such cell damage will cause Cr leakage from skeletal muscle cells and a rise in circulating Cr above the renal threshold and consequent creatinuria. Increased Cr synthesis or reduced renal function are potential causes of creatinuria, although there was no substantive evidence of this in juvenile IIM; the patients seen at one center (NIH) had normal renal, hepatic, and pancreatic tests (data not shown), and there is no reason to implicate these mechanisms in such cases.

Urinary levels of choline-containing compounds, betaine, and TMAO were also increased in juvenile IIM, though these changes were less specific than with Cr. Choline-containing compounds are synthesized in the liver (35) and are essential components of cell membrane metabolism (36). Betaine and TMAO are both metabolic end products of choline metabolism. The pattern of urinary excretion of the choline-containing compounds, betaine, and TMAO in juvenile IIM suggests a common mechanism. This pattern most likely results from altered membrane metabolism, which leads to an increase in the levels of circulating and urinary choline and its metabolites.

Measuring urinary Cr:Cn ratios is likely to have limited diagnostic value if used alone. In adults elevated Cr:Cn ratios are not specific for inflammatory myopathies (23), which is also the case in children with juvenile IIM (an increased level of urinary Cr was also observed in normal children &lsqbr;37&rsqbr;). The main role of the Cr:Cn ratio is likely to be as an additional laboratory assessment to complement clinical assessments of disease damage.

In conclusion, we have shown significant increases in the urinary levels of Cr, as well as choline-containing compounds, betaine, TMAO, and glycine in patients with juvenile IIM. Although further work is required to elucidate the underlying metabolic processes, changes in Cr:Cn ratio appear to strongly correlate with disease damage. Although we used MRS to assess Cr levels in the present study so that we could obtain a fuller urinary profile, Cr:Cn ratio can be analyzed using routine analytical methodologies readily available in a clinical biochemical laboratory, i.e., high-performance liquid chromatography methods. Such urinary Cr:Cn ratios may help to assess disease damage in patients with juvenile IIM in a noninvasive and objective manner.

Acknowledgements

The authors would like to thank the University of London Intercollegiate Research Service for the use of their 500 MHz NMR equipment at Birkbeck College. Drs. Rider and Miller also acknowledge the support of Dr. Paul Plotz and the Arthritis and Rheumatism Branch, NIAMS, NIH. Drs. Patience White and Ildy Katona are thanked for their assistance with patient recruitment. Drs. Larry Yao and Gyorgy Csako are gratefully acknowledged for their helpful comments in reading the manuscript.

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