Ratio of urinary N-terminal titin fragment to urinary creatinine is a novel biomarker for amyotrophic lateral sclerosis.

OBJECTIVE: We aimed to investigate the validity of urinary N-terminal titin fragment as a biomarker for amyotrophic lateral sclerosis (ALS). METHODS: We consecutively enrolled patients with ALS (n=70) and healthy controls (HC) (n=43). We assessed the urinary titin N-terminal fragment, urinary neurotrophin receptor p75 extracellular domain, serum neurofilament light chain (NfL), motor functional measurements and prognosis. We used urinary creatinine (Cr) levels to normalise the urinary levels of titin fragment. RESULTS: Compared with HC, patients with ALS had significantly increased urinary levels of titin N-terminal fragment normalised with Cr (titin/Cr) (ALS, 27.2 pmol/mg/dL; HC, 5.8 pmol/mg/dL; p<0.001), which were correlated with the scores of the Revised Amyotrophic Lateral Sclerosis Functional Rating Scale (r=-0.422, p<0.001). A Cox proportional hazards model demonstrated that the high urinary level of titin/Cr was a survival predictor in patients with ALS. Multivariate analysis of prognostic factors showed that the urinary titin/Cr and serum NfL were independent factors for poor prognosis. CONCLUSIONS: Our findings indicate that urinary N-terminal titin fragment is a non-invasive measure of muscle damage in ALS, which could be applied in disease monitoring and prediction of disease progression, in combination with serum NfL.

Urinary Extracellular Domain of Neurotrophin Receptor p75 as a Biomarker for Amyotrophic Lateral Sclerosis in a Chinese cohort.

To comprehensively assess whether p75ECD in urine could be a candidate biomarker for ALS evaluation. Urine samples were collected from 101 ALS patients, 108 patients with other neurological disease (OND) and 97 healthy controls. 61 ALS patients were followed up with clinical data including ALSFRS-r every 6 to 12 months, 23 ALS patients died and 17 ALS patients lost touch during follow up period. Enzyme-linked immunoassay was employed to determine urine p75ECD concentration. The ALSFRS-r was employed to assess the severity of ALS. The concentration of p75ECD in ALS was significantly higher than that of OND and CTRL (p < 0.001). Additionally, urine p75ECD concentrations in ALS-definite grade patients were significantly higher than that in ALS-probable grade and ALS-possible grade patients (p < 0.001). Higher urine p75ECD concentrations were correlated with increased clinical stage (p = 0.0309); urine p75ECD concentrations and ALSFRS-r were negatively correlated (p = 0.022); and urine p75ECD concentration in the fast-progressing ALS group was significantly higher than that in slow-progression (p = 0.0026). Our finding indicates that urine p75ECD concentration provides additional evidence for patients with clinically suspected ALS, and can be employed to evaluate ALS-severity.

Urinary p75ECD: A prognostic, disease progression, and pharmacodynamic biomarker in ALS.

OBJECTIVE: To evaluate urinary neurotrophin receptor p75 extracellular domain (p75ECD) levels as disease progression and prognostic biomarkers in amyotrophic lateral sclerosis (ALS). METHODS: The population in this study comprised 45 healthy controls and 54 people with ALS, 31 of whom were sampled longitudinally. Urinary p75ECD was measured using an enzyme-linked immunoassay and validation included intra-assay and inter-assay coefficients of variation, effect of circadian rhythm, and stability over time at room temperature, 4°C, and repeated freeze-thaw cycles. Longitudinal changes in urinary p75ECD were examined by mixed model analysis, and the prognostic value of baseline p75ECD was explored by survival analysis. RESULTS: Confirming our previous findings, p75ECD was higher in patients with ALS (5.6 ± 2.2 ng/mg creatinine) compared to controls (3.6 ± 1.4 ng/mg creatinine, p < 0.0001). Assay reproducibility was high, with p75ECD showing stability across repeated freeze-thaw cycles, at room temperature and 4°C for 2 days, and no diurnal variation. Urinary p75ECD correlated with the revised ALS Functional Rating Scale at first evaluation (r = -0.44, p = 0.008) and across all study visits (r = -0.36, p < 0.0001). p75ECD also increased as disease progressed at an average rate of 0.19 ng/mg creatinine per month (p < 0.0001). In multivariate prognostic analysis, bulbar onset (hazard ratio [HR] 3.0, p = 0.0035), rate of disease progression from onset to baseline (HR 4.4, p < 0.0001), and baseline p75ECD (HR 1.3, p = 0.0004) were predictors of survival. CONCLUSIONS: The assay for urinary p75ECD is analytically robust and shows promise as an ALS biomarker with prognostic, disease progression, and potential pharmacodynamic application. Baseline urinary p75ECD provides prognostic information and is currently the only biological fluid-based biomarker of disease progression.

Fluid-Based Biomarkers for Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a highly heterogeneous disease with no effective treatment. Drug development has been hampered by the lack of biomarkers that aid in early diagnosis, demonstrate target engagement, monitor disease progression, and can serve as surrogate endpoints to assess the efficacy of treatments. Fluid-based biomarkers may potentially address these issues. An ideal biomarker should exhibit high specificity and sensitivity for distinguishing ALS from control (appropriate disease mimics and other neurologic diseases) populations and monitor disease progression within individual patients. Significant progress has been made using cerebrospinal fluid, serum, and plasma in the search for ALS biomarkers, with urine and saliva biomarkers still in earlier stages of development. A few of these biomarker candidates have demonstrated use in patient stratification, predicting disease course (fast vs slow progression) and severity, or have been used in preclinical and clinical applications. However, while ALS biomarker discovery has seen tremendous advancements in the last decade, validating biomarkers and moving them towards the clinic remains more elusive. In this review, we highlight biomarkers that are moving towards clinical utility and the challenges that remain in order to implement biomarkers at all stages of the ALS drug development process.

Level of neurotoxic metals in amyotrophic lateral sclerosis: A population-based case-control study.

The association between exposure to toxic metals and amyotrophic lateral sclerosis (ALS) was explored in a population-based case-control study in the Sardinia island (Italy), a region characterized by elevated rates of ALS cases. In 34 patients with ALS (mean age, 62 ± 10 years) and 30 controls (mean age, 65 ± 11 years), Al, Cd, Hg, Mn and Pb were determined in blood, hair and urine by sector field inductively coupled mass spectrometry. Results indicated that, in blood, concentrations of Al (p=0.045) and Pb were higher (p=0.026) in ALS patients than in control subjects. In hair, a depletion of Al (p=0.006) and Mn (p=0.032) concentrations in ALS subjects respect to controls was found. In urine, no significant differences between cases and controls were observed. Thus, some metals seemed to be associated with ALS degeneration, but a definitive conclusion is still far considering the multiple risk factors (genetic mutations, environmental toxicants and stressors) involved in the disease. Finally, the interpretation that deregulated metal concentrations can be a consequence of the degenerative process, rather than a cause, is also valid.

Tetranor PGDM analyses for the amyotrophic lateral sclerosis: positive and simple diagnosis and evaluation of drug effect.

Amyotrophic lateral sclerosis (ALS) is a late-onset, progressive motor neuronal degenerative disease occurring as sporadically and as a familial disorder. The patients with ALS typically become progressively paralyzed and develop respiratory failure that eventually leads to death within 3-5years. For this disease, there is no effective diagnostic method and also drug. This report describes a simple and useful diagnostic biomarker for ALS. Our findings suggest that the combination analysis of a metabolite of prostaglandin D2, 11,15-dioxo-9-hydroxy-,2,3,4,5-tetranorprostan-1,20-dioic acid (tetranor PGDM and tPGDM) with creatinine is the diagnostic approach for ALS with high accuracy. tPGDM has the potential to be an important diagnostic tool in the pre-symptomatic stages and progression evaluation of ALS, and also to be a biomarker for the evaluation of drug effect.

Elevated levels of methylmalonate and homocysteine in Parkinson's disease, progressive supranuclear palsy and amyotrophic lateral sclerosis.

BACKGROUND/AIMS: Increasing evidence suggests that elevated levels of homocysteine (Hcy) and methylmalonate (MMA) may be involved in the pathogenesis of neurodegenerative diseases. METHODS: The urine levels of MMA and serum levels of Hcy as well as folic acid and vitamin B(12) were measured in patients suffering from the distinct neurodegenerative diseases progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD), and compared to age- and gender-matched control subjects. RESULTS: We found significantly elevated concentrations of Hcy (PD 15.1, PSP 15.8, ALS 13.9, control 11.2 micromol/l) and MMA (PD 3.7, PSP 3.1, ALS 3.7, control 1.8 mg/g) in all patient groups in comparison with controls. Levels of Hcy and MMA did not differ significantly between the neurodegenerative diseases. CONCLUSION: Our findings might imply that Hcy and MMA are released as a consequence of neurodegeneration regardless of the underlying cause and serve as surrogate markers of neurodegeneration. Alternatively they might be directly implicated in the pathogenesis of these diseases. Since elevated levels of both Hcy and MMA are neurotoxic, further studies might investigate the effect of vitamin therapy on disease progression.

Increased oxidative stress in patients with amyotrophic lateral sclerosis/Parkinsonism-dementia complex in the Kii peninsula, Japan.

Amyotrophic lateral sclerosis and Parkinsonism-dementia complex of the Kii peninsula (Kii ALS/PDC) is an endemic and a tauopathy, which shows clinical symptoms of amyotrophy, parkinsonism, and dementia. The objective of this study was to report the role of oxidative stress on Kii ALS/PDC using biochemical analysis. Urinary 8-hydroxydeoxyguanosine (8-OHdG)/creatinine ratio was analyzed in 11 patients with Kii ALS/PDC and 8 normal controls. The mean level of urinary 8-OHdG/creatinine ratio of the patients with Kii ALS/PDC was significantly higher than that of control subjects. Oxidative stress may be implicated in pathogenesis of Kii ALS/PDC.

A clinical trial of creatine in ALS.

BACKGROUND: Mitochondrial dysfunction occurs early in the course of ALS, and the mitochondria may be an important site for therapeutic intervention. Creatine stabilizes the mitochondrial transition pore, and is important in mitochondrial ATP production. In a transgenic mouse model of ALS, administration of creatine prolongs survival and preserves motor function and motor neurons. METHODS: The authors conducted a randomized double-blind, placebo controlled trial on 104 patients with ALS from 14 sites to evaluate the efficacy of creatine supplementation in ALS. The primary outcome measure was maximum voluntary isometric contraction of eight upper extremity muscles, with secondary outcomes including grip strength, ALS Functional Rating Scale-Revised, and motor unit number estimates. Patients were treated for 6 months, and evaluated monthly. RESULTS: Creatine was tolerated well, but no benefit of creatine could be demonstrated in any outcome measure. CI analysis showed that the study, although powered to detect a 50% or greater change in rate of decline of muscle strength, actually made an effect size of greater than 23% unlikely. It was also demonstrated that motor unit number estimation was performed with acceptable reproducibility and tolerability, and may be a useful outcome measure in future clinical trials. CONCLUSION: Any beneficial effect of creatine at 5 g per day in ALS must be small. Other agents should be considered in future studies of therapeutic agents to address mitochondrial dysfunction in ALS. In addition, motor unit number estimation may be a useful outcome measure for future clinical trials in ALS.

A carbon column-based liquid chromatography electrochemical approach to routine 8-hydroxy-2'-deoxyguanosine measurements in urine and other biologic matrices: a one-year evaluation of methods.

8-Hydroxy-2'-deoxyguanosine (8OH2'dG) is a principal stable marker of hydroxyl radical damage to DNA. It has been related to a wide variety of disorders and environmental insults, and has been proposed as a useful systematic marker of oxidative stress. Analytic procedures for 8OH2'dG in DNA digests are well established; however, routine measurement of free 8OH2'dG in other body fluids such as urine or plasma has been problematic. This has hindered its evaluation as a general clinical, therapeutic monitoring, or environmental assessment tool. Therefore, we developed a liquid chromatography electrochemical column-switching system based on the use of the unique purine selectivity of porous carbon columns that allows routine accurate measurement of 8OH2'dG in a variety of biologic matrices. This paper describes the rationale of the system design and the protocols developed for 8OH2'dG in urine, plasma, cerebrospinal fluid, tissue, DNA, saliva, sweat, kidney dialysis fluid, foods, feces, culture matrix, and microdialysates. Concentrations in both human and animal body fluids and tissues are reported. The system performance is discussed in the context of a 1-year evaluation of the methods applied to approximately 3600 samples, using internal quality control and external blind testing to determine long-term accuracy. The methods are reliable and accurate, and therefore should prove useful in assessing the role and utility of oxidative DNA damage in aging and human illness.

Decreased urinary concentrations of type IV collagen in amyotrophic lateral sclerosis.

OBJECTIVES: Type IV collagen (IV-C) abnormalities of skin and serum have been reported in patients with amyotrophic lateral sclerosis (ALS). However, there has been no study of urinary IV-C in ALS. The present study investigates urinary IV-C and the relation to its skin content in patients with ALS. MATERIAL AND METHODS: We studied IV-C immunoreactivity of skin and measured urinary levels of IV-C in ALS patients and controls. RESULTS: The basement membrane as well as blood vessels of skin in ALS patients was weakly positive for IV-C as compared with those of controls. Immunostaining became even weaker as ALS progressed. The urinary level of IV-C in ALS patients was significantly decreased as compared to diseased controls (P<0.001) and healthy controls (P<0.001), and was negatively and significantly associated with duration of symptoms (r=-0.85, P<0.001). There was an appreciable positive correlation between urinary IV-C levels and the density for IV-C immunoreactivity in ALS patients (r=0.84, P<0.01). CONCLUSION: These data suggest that a metabolic alteration of IV-C may occur in ALS patients and decreased levels of urinary IV-C may be related to the decreased IV-C immunoreactivity of skin in ALS.

Hyaluronic acid is increased in the skin and urine in patients with amyotrophic lateral sclerosis.

We performed morphological studies of skin and measured glycosaminoglycans in the urine from patients with sporadic amyotrophic lateral sclerosis (ALS) and control subjects. The wide spaces separating collagen bundles reacted strongly with alcian blue stain in ALS patients and stained more markedly as ALS progressed. Staining with alcian blue was virtually eliminated by Streptomyces hyaluronidase. The urinary excretion of hyaluronic acid (HA) (mg/day) was significantly increased (P < 0.01) in ALS patients compared with that of control subjects, and there was a significant positive correlation between the excreted amount of HA and the duration of illness in advanced ALS patients with a duration of more than 2 years from clinical onset (r = 0.72, P < 0.02). We suggest that sporadic ALS includes a metabolic disorder of HA in which an accumulation of HA in the skin is linked to an increased urinary excretion of HA.

Determination of urine thiocyanate in patients with amyotrophic lateral sclerosis.

It has been reported that amyotrophic lateral sclerosis-Parkinsonism-dementia in Guam might be related to the eating of Cycas seeds, which contain cyanide. Based on this assumption, we determined the urinary thiocyanate excretion level in patients with ALS and compared this with that of other neurological diseases. The assay method was designed to use column chromatography with Amberlite IRA 402. The thiocyanate level was determined using pyridine-barbiturate method. The 24-h thiocyanate level was higher in the ALS patients of the middle stages than in the normal control group (Wilcoxon's test, P less than 0.02). There were no significant differences between the ALS patient groups of the early and terminal stages, Kugelberg-Welander disease group, Duchenne type muscular dystrophy group and control group. From these results, we concluded that ALS patients were contaminated with cyanide or thiocyanate and that, along with rapid muscular atrophy, the thiocyanate excretion levels were high.

Skeletal muscle catabolism in amyotrophic lateral sclerosis and chronic spinal muscular atrophy.

ALS and chronic spinal muscular atrophy are characterized by wasting of skeletal muscle, suggesting accelerated catabolism or reduced synthesis of muscle protein. We studied seven patients with ALS and three with chronic spinal muscular atrophy using 24-hour urinary 3-methylhistidine excretion as a measure of the rate of muscle catabolism and 24-hour urinary creatinine excretion as an index of significantly and similarly higher in both groups of patients than in controls (p less than 0.0005), implying a state of accelerated skeletal muscle protein catabolism in these diseases.

Free amino acid levels in amyotrophic lateral sclerosis.

To evaluate reports of abnormal levels of free amino acids (AA) in patients with amyotrophic lateral sclerosis (ALS), we studied serum, cerebrospinal fluid, and urine AA in 12 patients with ALS and 12 controls matched for age, sex, and severity of disability. ALS patients had statistically significant elevations in serum levels of tyrosine, total aromatic AA, and total basic AA. ALS patients also had statistically significant elevations in cerebrospinal fluid of total basic AA, lysine, essential AA, and leucine. The severity of ALS correlated inversely with acidic AA (glutamate and aspartate) and O-phosphoserine in cerebrospinal fluid. Activity of ALS correlated directly with serum aspartate and cerebrospinal fluid alanine. We conclude that subtle abnormalities of AA levels are present in ALS and that these are not due to age, sex, or disability. The pattern of distribution of AA levels differs from that in hepatic or renal disease and suggests defective membrane transport or poor cellular utilization of basic and essential AA in the central nervous system.