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Keywords:

  • amyotrophic lateral sclerosis;
  • creatinine;
  • cystatin C;
  • residual muscle mass;
  • surrogate maker

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Aim

Identification of sensitive surrogate markers that indicate disease progression in amyotrophic lateral sclerosis might be useful in clinical trials and clinical care. Determination of the creatinine/cystatin C (Cr/CysC) ratio eliminates the effect of renal function on serum creatinine levels; therefore, we considered that the ratio might serve as a surrogate marker of residual muscle mass. We studied the Cr/CysC ratio as a useful surrogate marker of residual muscle mass in patients with amyotrophic lateral sclerosis.

Methods

A total of 103 participants were recruited: 62 patients with amyotrophic lateral sclerosis and 41 healthy controls. Serum levels of Cr and CysC were measured in both groups. We subsequently investigated the correlation between the Cr/CysC ratio and disease severity in patients with amyotrophic lateral sclerosis.

Results

The ratio was significantly lower in the amyotrophic lateral sclerosis group than in the control group. Furthermore, the ratio decreased as the severity of amyotrophic lateral sclerosis increased. The Cr/CysC ratio might be a better and more reliable method than the serum Cr level as a means of monitoring residual muscle mass of the entire body in patients with amyotrophic lateral sclerosis.

Conclusion

The present results show that the Cr/CysC ratio might be a suitable candidate for a useful and quantitative surrogate marker for the assessment of disease severity and progression in patients with amyotrophic lateral sclerosis.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Amyotrophic lateral sclerosis (ALS) is a uniformly fatal and debilitating disease; therefore, there is tremendous interest in developing effective therapies to slow or halt the progression of this disease. Current study designs often use a primary end-point of either death from ALS or initiation of long-term mechanical ventilation. This design requires a relatively long observation time to determine whether there is a positive treatment effect. Thus, identification of sensitive surrogate markers that indicate disease progression in ALS could be useful for the rapid identification of beneficial drugs, prompt exclusion of ineffective candidates and to determine clinical care for ALS patients. Surrogate markers of disease progression would provide a means of more rapid monitoring of drug efficacy in clinical trials.[1-5]

Muscle atrophy is a disease-defining feature of ALS, and clinical experience shows that atrophy might correlate with progressive weakness. Muscle atrophy is a qualitative marker of disease progression, but as yet, there is no clear quantitative marker of atrophy. Thus, as daily life activities decrease, the severity grade of ALS approximates to the residual skeletal muscle volume. A parameter that reflects muscle volume would enable estimation of disease severity.[6-8] The serum level of creatinine (Cr) is currently considered to be the most useful blood parameter that reflects the severity of motor dysfunction in spinal and bulbar muscular atrophy[9]; serum Cr levels were found to be correlated with the ALS Functional Rating Scale-Revised (ALSFRS-R) score in patients with spinal and bulbar muscular atrophy (correlation coefficient = 0.566, < 0.001). However, because serum Cr almost exclusively originates from the skeletal muscle and its levels are dependent of renal function, we considered that the use of serum Cr levels as an accurate marker in ALS patients might be questioned.

Cystatin C (CysC), a known cysteine protease inhibitor, could potentially be used as a surrogate marker of glomerular filtration rates (GFR).[10, 11] CysC is a non-glycosylated, 13.3-kDa basic protein that contains two disulfide bridges, and it is produced by all nucleated cells. It is considered to be unaffected by any factors (e.g. muscle mass, lean tissue mass, age, ambulation, circadian rhythm and sex) other than renal function status.[12-14] Furthermore, CysC is independent of the body muscle volume, and it is excreted from the kidneys in the same manner as Cr. Thus, the Cr/CysC ratio, which remains almost constant irrespective of renal function in individuals with neuromuscular diseases, is theoretically considered to be a good surrogate marker of muscle volume in ALS patients. In the present study, we compared the Cr-based estimated GFR (eGFR) with the CysC-based eGFR in ALS patients and healthy controls. The Cr/CysC ratio was further comparatively analyzed according to disease severity in ALS patients.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

ALS patients and controls

Amyotrophic lateral sclerosis was diagnosed according to the revised El Escorial criteria.[15] A total of 62 patients serially diagnosed with definite ALS at Jichi Medical University Hospital in Shimotsuke, Japan, were enrolled in the present study. These ALS patients had no prior history of renal disease, no known concomitant disease and were not participating in any experimental treatment. A total of 41 subjects free from diseases characterized by muscle atrophy were recruited as controls in the present study. The mean age of the study participants was 62.9 ± 9.6 years for ALS patients and 61.8 ± 12.4 years for healthy controls. Clinical variables of ALS patients were analyzed for age, sex, onset site, symptom duration and grading according to the ALSFRS-R score.[16] In the current study, 10 patients experienced a bulbar onset and 52 patients experienced a limb onset of disease. The mean symptom duration was 4.4 ± 5.1 years (Table 1). Classification of disease severity based on the severity scale established by the modified Rankin scale (mRs; Table 2) revealed five patients with a disease severity of grade 1, nine with grade 2, 13 with grade 3, 13 with grade 4 and 22 with grade 5 (Table 1). Informed consent was obtained from all patients and healthy controls.

Table 1. Clinical background of amyotrophic lateral sclerosis patients and control subjects
 ALS patients (n = 62)Control subjects (n = 41)P-value
  1. Data were expressed by mean (standard deviation).

  2. ALSFRS-R, Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised; mRs, modified Rankin scale.

Sex (male/female)39/2319/220.10 (χ2-test)
Age (years)62.9 (±9.6)61.8 (±12.4)0.52 (Mann–Whitney's U-test)
Symptom duration (years)4.4 (±5.1)  
Bulbar onset/limb onset10/52  
ALSFRS-R25.8 (±17.3)  
Severity scale (mRs)Grade 15  
Grade 29
Grade 313
Grade 413
Grade 522
Table 2. Modified Rankin scale
GradeDescription
0No symptoms at all
1No significant disability despite symptoms; able to carry out all usual duties and activities
2Slight disability; unable to carry out all previous activities, but able to look after own affairs without assistance
3Moderate disability; requiring some help, but able to walk without assistance
4Moderately severe disability; unable to walk without assistance and unable to attend to own bodily needs without assistance
5Severe disability; bedridden, incontinent, and requiring constant nursing care and attention
6Dead

Measurements

All study participants were actively engaged in their usual daily life activities. Blood samples were collected in distinct serum-separator tubes and analyzed for serum Cr and CysC. Serum Cr levels were measured using an enzymatic method at our hospital laboratory. Serum CysC levels were measured using colloidal gold particles coated with anti-CysC antibodies at SRL Laboratory (Tokyo, Japan).[17]

eGFR (mL/min/1.73 m2) was determined by measuring serum Cr levels using the following equation developed by the Committee on Chronic Kidney Disease of the Japanese Society of Nephrology[18]: men, Cr-based eGFR = 194 × Cr−1.094 × age−0.287; women, eGFR = 194 × Cr−1.094 × age−0.287 × 0.739. In addition, eGFR (mL/min/1.73 m2) was determined by measuring serum CysC levels using the following equation developed by A Rule[19]: men and women, CysC-based eGFR = 66.8 × CysC−1.30. The ratio of Cr (mg/dL) to CysC (mg/L) × 10 was defined.

Statistical analyses

For general statistical analyses, we used the spss v.11.0.1 program (Tokyo, Japan). Student's t-test was applied to the mean eGFR for serum Cr. Mann–Whitney's U-test was applied to the mean age, mean eGFR for serum CysC, and mean ratios of serum Cr to serum CysC between ALS patients and healthy controls. The χ2-test for independent testing was applied to a two-by-two contingency table with sex between both groups. We analyzed differences in the Cr/CysC ratio between severity grades (mRs) by anova followed by Tukey's honestly significant difference post-hoc test.

Correlation coefficients between serum Cr levels, Cr/CysC ratios and ALSFRS-R scores were determined. A scatter diagram between the serum levels and ALSFRS-R score was constructed. The correlation coefficient between Cr/CysC ratios and ALS duration was determined, and a scatter diagram between both sides was made. Correlations were determined using Spearman's rank-correlation coefficient. All tests were two-tailed and significance was set at < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

In this current study, clinical parameters (sex and age) were not significantly different between the ALS and control groups (Table 1).

The mean Cr/CysC ratios were 8.2 ± 2.2 for the control group and 5.5 ± 3.3 for the ALS group, the value being significantly lower in the ALS group (Mann–Whitney's U-test, = 0.01; Table 3). The mean eGFR determined by serum Cr levels was 381.6 ± 486.9 mL/min/1.73 m2 for the ALS group, which was significantly different (Mann–Whitney's U-test, P < 0.001; Table 3) from the control group value of 98.5 ± 57.9 mL/min/1.73 m2. However, the mean eGFR determined by serum CysC was 103.3 ± 24.8 mL/min/1.73 m2 for the ALS group, which was not significantly different from the control group value of 97.6 ± 21.6 mL/min/1.73 m2. Thus, Cr-based eGFR in the ALS group was markedly higher than any other values. This is probably explained by reduced serum Cr levels in ALS patients on account of the reduced residual muscle mass. The mean Cr/CysC ratios were not significantly different according to sex between both groups (Table 3).

Table 3. The ratios of creatinine to cystatin C, and the estimated glomerular filtration rate of creatinine and cystatin C in amyotrophic lateral sclerosis patients and controls
 ALS patients (n = 62)Control subjects (n = 41)P-value
The ratio of Cr (mg/dL) to CysC (mg/L) ×10 (Cr/CysC)5.5 (±3.3)8.2 (±2.2)0.01a (Mann–Whitney U-test)
Cr-based eGFR (mL/min/1.73 m2)381.6 (±486.9)98.5 (±57.9)<0.001a (Mann–Whitney U-test)
CysC-based eGFR (mL/min/1.73 m2)103.3 (±28.4)97.6 (±21.6)0.28 (Student's t-test)
 MaleFemaleP-value
  1. a

    Data were expressed by mean (standard deviation). Significant difference.

  2. Cr, creatinine; CysC, cystatin C; eGFR, estimated glomerular filtration rate.

ALS patients (Cr/CysC ratio)5.2 (±3.7)5.9 (±2.5)0.37 (Student's t-test)
Control subjects (Cr/CysC ratio)8.8 (±2.6)7.6 (±1.8)0.09 (Student's t-test)

We compared the mean Cr/CysC ratios with disease severity (Fig. 1a) in ALS patients. The ratio was 10.13 ± 1.27 for grade 1 disease severity, 7.82 ± 1.1 for grade 2, 6.47 ± 1.92 for grade 3, 6.3 ± 2.78 for grade 4 and 2.37 ± 2.25 for grade 5. Significant differences were observed in the Cr/CysC ratio between severity grades 1–3, 1–4, 1–5, 2–5, 3–5 and 4–5 (< 0.05). Thus, the ratio linearly decreased with an increase in disease severity (Fig. 1a). A relatively high Cr/CysC ratio observed in the patient group with grade 4 disease severity could be explained by the fact that this group included a number of patients with the bulbar-palsy type of ALS, in which the muscle mass in the four extremities is relatively well preserved. Therefore, we compared the mean Cr/CysC ratios after excluding patients with this type of ALS. This analysis revealed a ratio of 10.13 ± 1.27 for grade 1 disease severity, 7.82 ± 1.1 for grade 2, 6.47 ± 1.92 for grade 3, 4.6 ± 1.42 for grade 4 and 1.6 ± 1.58 for grade 5 (Fig. 1b). Significant differences were observed in the Cr/CysC ratio between severity grades 1–3, 1–4, 1–5, 2–4, 2–5, 3–5 and 4–5 (< 0.05). A steady decrease in the ratio with an increase in disease severity became more evident when patients with the bulbar-palsy type of ALS were excluded from the analysis.

image

Figure 1. The mean creatinine/cystatin C (Cr/CysC) ratios were compared by disease severity (modified Rankin scale [mRs]) in amyotrophic lateral sclerosis (ALS) patients, and differences were analyzed by anova followed by Tukey's honestly significant difference post-hoc test. (a) The ratio decreased linearly as disease severity increased (severity grades 1–3, 1–4, 1–5, 2–5, 3–5 and 4–5; < 0.05). (b) After excluding patients with the bulbar-palsy type of ALS, the steady decrease of the ratio with increasing disease severity became more apparent (severity grades 1–3, 1–4, 1–5, 2–4, 2–5, 3–5 and 4–5; < 0.05).

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The scatter plot showed strong simple correlations between the Cr/CysC ratio and disease severity in ALS patients as determined by the ALSFRS-R score (correlation coefficient = 0.84, < 0.001; Fig. 2). Furthermore, the plot showed that ALS severity increased with a decrease in the Cr/CysC ratio. A correlation between serum Cr levels and disease severity in ALS patients as determined by the ALSFRS-R score was also recognized (correlation coefficient = 0.78, < 0.001; Fig. 2). The correlation coefficient of the Cr/CysC ratios was higher than that of serum Cr levels.

image

Figure 2. The scatter diagram showed strong simple correlations between (a) Cr level, (b) creatinine/cystatin C (Cr/CysC) ratio and the severity by Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R) score in amyotrophic lateral sclerosis (ALS) patients. The plot showed that ALS severity increased with a decrease in both. The correlation coefficient of Cr/CysC ratio was higher than that of Cr level.

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The relationship between the Cr/CysC ratio and ALS duration is shown on the scatter plot; the plot shows that the Cr/CysC ratio decreased with an increase in the duration of ALS (correlation coefficient = 0.75, < 0.001; Fig. 3a). Figure 3b shows the percentage of patients with the Cr/CysC ratios of ≥5 and those with ratios of <5 as classified by disease severity. The percentage of patients with ratios of <5 was markedly higher in patients with grade 4 and grade 5 disease severity (71% and 94%, respectively).

image

Figure 3. (a) The relationship between the creatinine/cystatin C (Cr/CysC) ratios and amyotrophic lateral sclerosis (ALS) duration is shown in the scatter plot; the plot shows that the Cr/CysC ratio decreased with an increase in the duration of ALS. (b) The percentage of patients with the Cr/CysC ratios of ≥5 and those with ratios of <5 as classified by disease severity (modified Rankin scale [mRs]) are shown. The percentage of patients with ratios of <5 was markedly higher in patients with grade 4 and grade 5 disease severity (71% and 94%, respectively).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We believe that CysC levels are not basically affected by ALS itself. By calculating the serum Cr/CysC ratio, we eliminated the effect of renal function on serum Cr, which is dependent on the muscle mass of the entire body. We subsequently used this ratio as a surrogate marker of the residual muscle mass throughout the body. The Cr/CysC ratio was significantly lower in the ALS group, which was characterized by a decreased residual muscle mass, than that in the control group. Furthermore, the ratio decreased with an increase in ALS severity. This finding suggested that changes in the Cr/CysC ratio might reliably reflect the decrease in muscle mass throughout the body with an increase in ALS severity. According to the severity scale used in the present study, patients with the bulbar-palsy type of ALS, in whom the residual muscle mass was relatively conserved, were assigned higher grades of disease severity. Therefore, we also carried out the analysis after excluding patients with this type of ALS. With the exclusion of these patients from the analysis, it became even more evident that the Cr/CysC ratio reliably reflected the residual muscle mass of the entire body. Correlation of the Cr/CysC ratio was higher than that of serum Cr levels. The results also indicated that the Cr/CysC ratio might be better and more reliable than serum Cr levels for monitoring the residual muscle mass of the entire body in ALS patients. In addition, in ALS patients with serum Cr/CysC ratio of <5, daily life activities were markedly restricted. Therefore, this value might constitute a critical cut-off point during the clinical course of the disease.

To date, insensitive, non-parametric surrogate markers, which presume quantitative assessment of the disease severity in ALS patients, such as forced vital capacity, and the ALSFRS-R score have been used to assess disease progression.[16, 20] We suggest that the Cr/CysC ratio might permit quantitative assessment of disease severity in ALS patients, assessment of therapeutic responses and determination of disease progression. Lee CD et al.[7] presented an alternative approach that involved assessment of muscle thickness by muscle ultrasound (MUS) in ALS patients.7 These authors reported that MUS is sufficiently sensitive as a potential surrogate marker to quantitatively detect changes in muscle thickness over time. Their study proposed biceps brachii as a suitable candidate for study; however, it is likely that assessment of multiple muscles would be required to account for the heterogeneity of ALS. Furthermore, muscle gene expression changes in skeletal muscle that could reliably define the degree of disease severity were reported.[21] However, we think that the current study presents a more convenient method for assessing disease status and progression than these approaches. Additional methods of measuring limb muscle mass include magnetic resonance imaging (MRI) and bioelectric impedance analysis (BIA). Applications for measuring residual muscle mass in ALS patients by MRI and BIA have been previously reported.[8, 22] In particular, BIA is reported to be safe, portable, highly reliable and relatively inexpensive.[23] However, both methods can measure only a limited segment of body muscles. As a result of the heterogeneity of ALS that often affects different muscles at different rates, these methods are not considered to be capable of accurately determining residual muscle mass in ALS patients. Thus, we propose that measurement of the serum Cr/CysC ratio in ALS patients, which might account for residual muscle mass of the entire body, might be superior to these methods and eliminate the effect of renal function on serum Cr levels. However, under certain circumstances, the serum Cr/CysC ratio might not be a reliable surrogate marker of the residual muscle mass. For example, if ALS patients have a mild renal dysfunction, the ratio might be erroneously decreased. Thus, we have to interpret the data carefully, and should use only serum CysC levels for monitoring renal function accurately in ALS patients in such a case.A limitation of the present study was the lack of data measuring the actual residual muscle mass; however, as aforementioned, it is challenging to determine residual muscle mass of the entire body in ALS patients. Thus, taking previous studies together with our data and theory of the Cr/CysC ratio, it might be reasonable to suggest that serum Cr/CysC ratio might be a suitable candidate for a surrogate marker of residual muscle mass.

In addition, the use of the serum Cr level as a marker of renal function might be underestimated in diseases such as ALS that are characterized by reduced muscle mass. Thus, it is difficult to accurately assess renal function using serum Cr levels in these patients. This is clearly shown by the result shown in Table 3; eGFR determined by serum Cr was markedly higher in ALS patients than that determined by serum CysC. The latter is, therefore, considered to be a more accurate indicator for the assessment of renal function in ALS patients.

In conclusion, the present results show that the Cr/CysC ratio might be a suitable candidate for a surrogate marker of residual muscle mass of the entire body in ALS patients. Furthermore, the Cr/CysC ratio might also be a useful marker for assessing the response to therapy and determining the progression of ALS.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This work was supported by Grants in Aid from the Research Committee of CNS Degenerative Diseases, the Ministry of Health, Labour and Welfare of Japan.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References