Jason C. Hall; Dayton, Ohio, U.S.A.; jason.alstrial@gmail.com
Riku Mattila; Espoo, Finland; riku@rmattila.com


Abstract

21 people diagnosed with (or in the process of being diagnosed with) amyotrophic lateral sclerosis (ALS), together with 6 healthy individuals volunteering as controls, had their blood samples analyzed locally for serum total copper and ceruloplasmin concentrations. The results were collected and plotted against different parameters characterizing the disease progression. Although the data is too limited to draw any quantitative conclusions, qualitatively there appears to be correlation between disease progression rate and the concentration of non-ceruloplasmin bound ("free") copper. In patients with slower disease progression, the effect appears to be mainly due to low ceruloplasmin concentration, whereas in faster progressors the serum total copper seems to trend towards higher values faster than ceruloplasmin concentration.

The main target of the study was to verify if there is justification to proceed to a follow-up study where the potential effect of lowering serum free copper on the disease progression would be evaluated. At the same time, the study served as a pilot to evaluate the feasibility to expand the patient-driven data collecting approach to a wider scale in order to obtain statistically higher-quality data. Both questions received a positive answer.

Of the 21 participants with ALS, 5 had high enough non-ceruloplasmin bound copper concentration to warrant re-evaluation of their diagnosis taking into account the possibility of Wilson's disease. At the time of writing this, two of them are in the process of having their diagnosis changed from ALS to Wilson's disease. This discovery suggests that Wilson's disease should be considered in differential diagnosis of ALS even in absense of classical symptoms of the former.

Introduction

ALS is a fatal neurodegenerative disorder characterized by progressive loss of both upper and lower motor neurons, leading to paralysis and death, usually within 2-5 years from symptom onset [1]. Typical age of onset is between 50 and 60 years, but significantly earlier or later onset is also possible. There is no cure or effective treatment for this disease that on average affects about 2 people per 100 000 a year [2].

The laboratory examinations performed on suspected ALS patients are usually aimed at excluding other diseases, and once the diagnosis has been given, no further examinations regarding possible anomalies are typically ordered. Therefore, in spite of ALS not being a very rare disease, there is only a limited amount of information available regarding possible systematic anomalies in the blood or cerebrospinal fluid of people with confirmed ALS.

Recently, a finding was made by one of the authors (Hall), an ALS patient who has for several years been engaged in having his own blood systematically analyzed and also has received laboratory results from ca. 200 other ALS patients for comparative analyses, that the serum non-ceruloplasmin bound ("free") copper calculated from the serum total copper and ceruloplasmin concentrations was clearly above the normal reference values given in the literature in all four patients who had the relevant analysis results available.

Target of the study

This study was set up by a group of 20 people diagnosed with ALS, coordinated by one of the authors (Hall). The aim was to collect data to either support or disqualify the observation made previously by examining laboratory data from some 200 ALS patients; namely, that the serum non-ceruloplasmin bound copper concentration, calculated by the Formula [3].

fCu [µg/l] = gCu [µg/l] - (Cp [mg/l] * 3.0) (1)

where fCu is the non-ceruloplasmin bound copper concentration, gCu serum total copper concentration and Cp ceruloplasmin concentration, is elevated in people with ALS (PALS).

Methods

21 volunteer ALS patients and 6 healthy control persons were found by announcing the study on the ALS Therapy Development Institute discussion forum at http://www.als.net/Forum/ . Each of the participants was asked to have their serum ceruloplasmin and total copper concentrations determined by a suitable laboratory in their region, using the recommended procedures and methods specified by each laboratory. The patients were asked to stop taking any zinc supplements for three days before giving the blood sample. The results were collected and the non-ceruloplasmin bound copper levels calculated according to Formula (1).

In order to study possible correlation between the laboratory results and the disease progression, the participants were requested to evaluate their disease status according to the revised ALS functional rating scale (ALSFRS-R [4]), and the disease progression rate was defined as the average loss of ALSFRS-R points per month between symptom onset and the taking of the sample. Distribution of disease durations is given in Figure 1, distribution of the patients' and control subject's ages in Figure 2 and distribution of disease progression rates in Figure 3.

Figure 1: Distribution of the duration of disease
in the November 2013 pilot study.

Figure 2: Participants' and control persons' age distribution.

Figure 3: Distribution of the participants' ALS progression rates.

Results

In Figures 4-6, the calculated non-ceruloplasmin bound copper, serum total copper and the serum ceruloplasmin concentration, respectively, are plotted against the average progression rate. In Figures 7-9, the same quantities are plotted against the ALSFRS-R score at the time of taking of the blood samples, and finally in Figures 10-12 against the patients' age at the time of diagnosis (for control persons, their current ages were used in Figures 10-12).

One laboratory was unable to determine total copper from serum and analyzed it from plasma instead. According to some sources, there may be up to 10 % differences in those concentrations, causing errors up to 100 % in the calculated non-ceruloplasmin bound copper values. Data from that laboratory are marked with 1) in the Figures. Another individual had exceptionally high ceruloplasmin levels, resulting into unphysical, negative values for the non-ceruloplasmin bound copper. A re-analysis did not change the situation. This data is denoted with 2) in the Figures.

Figure 4: Calculated serum non-ceruloplasmin bound copper vs. rate of ALS progression.

Figure 5: Serum ceruloplasmin concentration vs. rate of ALS progression.

Figure 6: Serum total copper concentration vs. rate of ALS progression .

Qualitative analysis of Figures 4-6 shows that:

The quantities shown in Figures 4-6 are re-plotted in Figures 7-9, this time against the disease progression status.

Figure 7: Calculated serum non-ceruloplasmin bound copper vs. status of ALS progression.

Figure 8: Serum ceruloplasmin concentration vs. status of ALS progression.

Figure 9: Serum total copper concentration vs. status of ALS progression .

Looking at Figures 7-9, it seems that calculated non-ceruloplasmin bound copper, ceruloplasmin and total copper have a similar, albeit weaker, correlation with the disease progression status as they have with the progression rate.

Figure 10: Calculated serum non-ceruloplasmin bound copper vs. age.

Figure 11: Serum ceruloplasmin concentration vs. age.

Figure 12: Serum total copper concentration vs. age.

From Figures 10-12, no correlation is found between the concentrations and person's age.

Shortcomings in the methodology

The most obvious shortcoming in the study was the poor statistical coverage of the data, especially the lack of healthy male controls. As serum ceruloplasmin levels are known[5] to depend on the estrogen levels, it is not possible to draw solid conclusions about the differences in healthy males vs. male ALS patients. However, the absolute values in male patients were overall quite low with respect to the reference ranges given by respective laboratories.

Another shortcoming built in the methodology of using results sent by patients from around the world concerns the comparativity of different laboratories' results. For example, as regards reference levels of ceruloplasmin, one laboratory, used by about half of the participants, gives a normal reference interval of 150-300 mg/l whereas most of the other laboratories give reference ranges of around 200-600 mg/l. As no information on the distributions of results in different laboratories was available, no attempt was made to scale the ceruloplasmin results based on the reference levels specified by the laboratories. As the calculated value of non-ceruloplasmin bound copper is very sensitive in errors in either of the laboratory results used in its determination, this constitutes a significant source of uncertainty in the results. The problem could be largely overcome by pairing each participant with a statistically equivalent healthy reference and have both analyzed by the same laboratory. Different reference levels could be indicated by different markers to help recognizing biases.

There appears to be some significant uncertainties as regards the molecule weight assumed for ceruloplasmin, as suggested by the large variation of the value of the constant in Formula (1) used by different sources. Moreover, the calculated non-ceruloplasmin bound copper is quite sensitive to this value, since in a healthy individual the two terms in Formula (1) almost cancel each other. A more reliable estimate for the concentration for the non-ceruloplasmin bound copper could presumably be obtained by direct determination from ultrafiltrate. However, locating a facility with capabilities for such analyses was not possible within the scope of this study.

Conclusion

In spite of the shortcomings mentioned above, Figures 4 and 7 show signs of a correlation between the calculated non-ceruloplasmin bound copper level and disease progression rate as well as progression status. Similar correlation can not be seen with the person's age (Fig. 10). If this correlation can be confirmed in a statistically high-quality study, there are three possible explanations:

  1. The free copper concentration is indeed elevated in people with ALS.
  2. PALS have for some reason abnormally light-weight ceruloplasmin molecules, which leads to underestimation of the ceruloplasmin-bound copper in Formula (1) and consequently too high result for the calculated non-ceruloplasmin bound copper values.
  3. The ceruloplasmin-bound copper is indeed low, but the remaining part, instead of being free, is bound to some other protein or amino acid in an abnormally high amount.
It is as yet also unknown if the higher values are a cause or consequence of the disease process. However, it certainly seems possible that people diagnosed with ALS form a continuum with free copper concentration correlating with the rate of disease progression, and at least part of the fastest-progressing ones fulfil the diagnosis criteria for Wilson's.

From Figures 5 and 8, it appears that the ceruloplasmin level in PALS is overall on the low edge of the reference range. No clear correlation seems to exist between the progression status or progression rate and the concentration. Here, lack of healthy male control persons makes drawing any conclusions very difficult, since females are in general known to have higher ceruloplasmin levels than males.

Finally, the serum total copper concentration increases with disease progression rate in a way needed to account for the observations made above regarding the non-ceruloplasmin bound copper and ceruloplasmin concentrations. It should be noted that overall the serum total copper is within the reference range - on the low side in slow progressors and early on in the disease and on the high side in faster progressors and later stages - and the elevated non-ceruloplasmin bound copper values observed in this study are consequently caused by low ceruloplasmin rather than high total copper concentration.

One possible interpretation to the observed results is that low ceruloplasmin concentration is somehow connected to the initiation of the disease process, whereas high free copper would correlate with the damage being done to the neurons. High free copper would not seem to be a sufficient factor to explain the onset of disease, since at lower progression rates and early stages of the disease it seems to monotonically approach the levels found in healthy reference persons.

Directions for further studies

Due to practical limitations and the obvious urgency to get data for validation of the hypothesis, the pilot study described in this report did not provide statistically sufficient data to absolutely confirm or disqualify the hypothesis of non-ceruloplasmin bound copper being systematically elevated in people with ALS. To obtain further understanding of the potential finding, a two-way approach is pursued:

  1. A repeated study, refined according to the feedback from the pilot study, will be conducted by the authors. This time the emphasis will be in getting statistically as good data as possible.
  2. The participants of the pilot study will seek treatment to correct their calculated free copper levels inside the normal reference range. Possible changes in the ALS progression rates will be followed during a 6-12 month period, and the results will be reported after the follow-up period.

In addition to the two follow up studies listed above, to be conducted by the authors, additional research is due because of the apparent uncertainties associated with determination of the free copper levels indirectly through the ceruloplasmin concentration. It would be advisable to conduct another study, in which the free copper levels in a representative number of ALS patients were determined directly e.g. by producing a suitable (20-30 kDa) ultrafiltrate to physically separate free copper from that bound in the protein. At the moment it is uncertain if organizing such a study is within the reach of the authors, and research institutions with appropriate equipment are encouraged to take initiative in this follow-up.

The apparent systematic difference in serum ceruloplasmin concentrations between healthy individuals and ALS patients warrants further study. Information on whether the ceruloplasmin level in PALS has always been low or if a change has occurred at disease outbreak could provide valuable insight into the disease mechanisms. The authors are currently unaware if databases with measured ceruloplasmin concentrations of individuals who later in their life got ALS exist. At the very least, it would be straightforward to analyze the concentrations in people known to possess a familial ALS gene but who as yet are unsymptomatic.

Discussion

Statistically, our pilot study was very limited, and the method of selecting participants through an Internet discussion forum induced a significant bias regarding the age and professional background of the participants. Thus, rather than providing definite numerical answers, the aim of the study was to confirm whether further studies on the subject, such as those described in the previous subsection, are justified. In the authors' view, the pilot study provided a definite positive answer to this question.

Due to the uncertainties associated with determination of the absolute concentration of the non-ceruloplasmin bound copper, emphasis should be put on the relative differences between the ALS patients and the control group. Here, an apparent shortcoming of our study is the small number of healthy individuals we were able to recruit in the short time we were willing to wait before proceeding with the study. In future similar studies it might be worth considering to accept participants only if each of them brings a healthy individual along. This approach, with the additional angle of pairing each participant with a statistically equivalent control subject, will be utilized in the expanded study described above.

As a final note: out of the 21 participants in the pilot study, five turned out to have very high calculated non-ceruloplasmin bound copper levels and most of them went on to have their 24 h urine samples analyzed for excess copper. At least two had excess urine copper as well, and are currently in the process of having their diagnoses changed from ALS to Wilson's disease. In addition, one of the participants had the AcHR Antibodies determined outside the study and as a consequence of an elevated result has had his diagnose changed from ALS to myastenia gravis. This result - that more than 10 % of our participants may have been erroneously given a death sentence in the form of ALS when they actually had a completely treatable disease - strongly supports the idea of performing as many laboratory analyses as possible before - and even after! - coming up with a diagnosis known today as ALS.

References

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