Author: Dennis A. Billaney (carrier of PEX 10 mutation); Dec.2013
Introduction
The intention of this paper is to gather relevant potential treatment options. Due to the fact that many parents of a child diagnosed with a rare disease start to search for information from the internet or online sources, it is likely that an average parent will possibly know more specific details about therapeutic options than an average clinician, surgeon, physician or even an experienced pediatrician. This may lead to confusion between the parents and the treating medical experts since there are extreme differences in the form of treatment, which vary from continent to continent, country to country and even from clinic to clinic.
It must be assumed and is the case that many parents become members and start to communicate on internet platforms such as forums, chat rooms, Facebook groups as well as read and follow several private homepages or blogs. Therefore, it is clear that there seems to be no coordinated approach of either clinical treatment nor of diagnostics and follow-up procedures. This is likely going to cause confusion with parents and families. There is also no satisfying answer available as to what should be done and why. Usually, the lack of clinical or scientific evidence as well as a lack of knowledge in this rare disease is the reason why in several cases there are only initial screenings for diagnostic markers performed after which no further procedures are advised, besides palliative care.
We will discuss both the desired treatment options as well as recommend a guideline for diagnostics and follow-ups, in order to both monitor treatment and collect valuable data.
Abstract
The Zellweger Spectrum Disorders (ZSD) is a group of disorders belonging to the Peroxisomal Biogenesis Disorders (PBD).
These ZSD/PBD disorders include the classic Zellweger Syndrome (ZS), Neonatal Adrenoleukodystrophy (N-ALD) and
Infantile Refsum Disease (IRD).
There are further disorders which are attributed to ZSD/PBD, but which we are not going to mention in this review.
Furthermore, all of the disorders comprising ZSD/PBD can or need to be subdivided into further groups, such as phenotypes or by genetic mutations. ZSD’s are all terminal disorders and the prognosis of most patients is very poor. To date there is no cure nor is there any specific recommendation for treatment available.
Therefore, we have reviewed a variety of published studies, trials or the attempts and results to treat ZSD patients, which we present here along with a guideline for both parents and clinicians to hopefully improve the prognosis or outcome in these devastating diseases. Our attempt to improve treatment options also includes a recommendation of how to implement strategic analytic methods in order to be able to monitor treatment, verify any clinical findings or diagnostics and to collect data.
Review of Therapeutic Attempts
1. DHA
DHA (Docosahexaenoic Acid) is an important Polyunsaturated Fatty Acid (PUFA) belonging to the Omega n-3 family. The human body derives its DHA from both endogenous as well as exogenous sources, while for the endogenous synthesis further exogenous n-3 PUFA need to be derived from dietary sources. It is a well established fact that endogenous DHA synthesis requires functioning peroxisomes, thus, DHA is found to be very low in ZSD patients. It is not clear how a deficit of DHA may influence disease or the progression of degeneration. However, it is very well known that retinal tissue, as well as white brain matter, contains high amounts of DHA and incorporation into neuronal tissue is highest during the first few months of life, thus it may be possible to draw the conclusion, that a lack of DHA may cause retinal as well as neurological dysfunction, as is seen in most ZSD patients.
Therefore, several patients have been treated with DHA supplementation. DHA was administered to patients in different forms and doses, ultimately resulting in a broad range of clinical outcomes. One of the first attempts to administer DHA to ZSD patients was by Martinez and colleagues in Spain. Martinez treated ZSD/PBD patients for over 20 years. Martinez exclusively administered DHA in the form of its ethyl ester (DHA-EE) of 97% purity. The results have shown, that oral DHA EE is able to restore DHA levels in serum of ZSD patients and Martinez and colleagues found that in most patients there was a major clinical improvement detected (1. Martinez et al. 2000; Therapeutic effects of docosahexaenoic acid ethyl ester in patients with generalized peroxisomal disorders, 2. Martinez et al., 2010; Visual follow-up in peroxisomal-disorder patients treated with docosahexaenoic Acid ethyl ester).
Others have undertaken similar treatments and in one such trial, possibly the largest double-blind, randomized trial ever performed on ZSD/PBD patients, done by Paker and colleagues, where they used a triglyceride form of DHA (DHA-TG) in combination with Arachidonic Acid (AA), which is another important PUFA of the Omega n-6 family, they noted no clinical improvement in ZSD patients treated with DHA-TG & AA. The authors of this trial state further that they have produced Class II evidence that DHA supplementation did not improve the visual function or growth of treated individuals with peroxisome assembly disorders during an average of 1 year of follow-up in patients aged 1 to 144 months(3. Paker et al., 2010 Docosahexaenoic acid therapy in peroxisomal diseases: results of a double-blind, randomized trial). Paker claims that DHA EE has shown to lower AA, therefore Paker chose to supplement the more stable form of DHA TG, together with AA.
Therefore we think it is important to note, that it has been stated that DHA must be administered in pure form and in the absence of any further PUFA which may compete at the enzymatic biochemical level until DHA levels in patients have been corrected, reported by Martinez in the papers referenced above and also in further publications (4. Martinez et al., 2010; The Delta 4-desaturation pathway for DHA biosynthesis is operative in the human species: Differences between normal controls and children with the Zellweger syndrome).
Furthermore, Hyung-Wook et al., reported that dietary n-6 polyunsaturated fatty acid (PUFA) deprivation in rodents reduces brain arachidonic acid concentration while increasing brain docosahexaenoic acid. Increased DHA metabolism following dietary n-6 PUFA deprivation may increase brain concentrations of anti-inflammatory DHA metabolites, which with a reduced brain n-6 PUFA content, likely promote neuroprotection. (5. Hyung-Wook et al., 2011; dietary n-6 PUFA deprivation increases DHA metabolism in rat brain).
In one of the earlier studies, DHA EE was given orally to two infants with ZS/PBD for a year, and some favourable biochemical changes were produced in erythrocytes and plasma. Normalization of the DHA concentrations in erythrocytes was obtained in about 2 months, and the ratios 26: 0/22: 0 and 26: 1/22: 0 decreased markedly in plasma in the two patients. The plasmalogen ratio 18: 0 dimethyl acetal/18: 0 in erythrocytes increased to virtually normal values in both patients. There was a clear clinical improvement in the two patients, which paralleled the increase in blood DHA. The concentrations of AA and other PUFAs were closely monitored and, when necessary, AA was added to the diet. Such a DHA therapy, given under close biochemical and clinical control, and accompanied by a diet rich in other long-chain PUFA, is strongly recommended in all patients with peroxisomal disorders in whom a DHA deficiency is detected in blood (6. Martinez et al., 1995; Polyunsaturated fatty acids in the developing human brain, erythrocytes and plasma in peroxisomal disease: therapeutic implications).
Therefore, we recommend repeating the treatment method as described by Martinez et al., using highly pure DHA-EE while other PUFA are closely monitored and, when necessary, AA needs to be added to the diet. Obviously the combination used by Paker et al., has shown no benefit to ZSD patients and does not need to be reviewed any further.
Rather it has to be made sure, that ZSD patients are not supplemented with excess amounts of AA or other essential Fatty Acids (FA), which may compete at the enzymatic biochemical level. This includes Linoleic Acid (LA) and Eicosapentaenoic Acid (EPA), which are both competing and therefore have to be avoided or the administration of DHA may become useless. This is the reason why mixtures of w3 fatty acids such as those present in fish oil were avoided by Martinez and co-workers. They contain high levels of eicosapentaenoic acid (EPA, 20:5w3), a w3 PUFA situated before the probable enzyme block in DHA synthesis. (7. Martinez et al., 1993; Docosahexaenoic acid- A new therapeutic approach to peroxisomal disorders patients).
Addition of AA and other w6 PUFA was also avoided since they could compete with DHA and reduce its beneficial effects.
(8. Martinez; Peroxisomal disorders and their treatment http://www.momtahan.com/mmartinez/).
Furthermore, the only known side effects of administering pure DHA-EE (oral) is hypoglycemia and therefore patients receiving DHA-EE must be kept on a regular diet intake to avoid hypoglycemic situations.
In short, it has been recommended to administer 200mg/day of DHA-EE in a single dose. The DHA is diluted in high-quality olive oil with a concentration of 100mg DHA-EE per ml of olive oil. To avoid hypoglycemia in neonates and young infants, such patients should receive food (milk) at maximal intervals of three hours or if fed by tube, then a constant check of stomach content is necessary.
2. Plasmalogen
Plasmalogens are essential ether lipids which are found in all tissues. Plasmalogens account for a large amount of glycerophospholipids in the human brain, but in the brain of neonates, low levels are found with a rapid increase during the first year of life, which is also the main myelinating period (Horrocks et al., 1982). In healthy candidates, plasmalogen biosynthesis is influenced by diet, age and genetic factors (Platauf et al., 1994).
Unfortunately, as with DHA, the biosynthetic pathway of plasmalogen synthesis requires functional peroxisomes, since the first few steps of the synthetic reaction occur in peroxisomes. Therefore ZSD patients tend to have severely reduced levels of plasmalogen in both tissue and serum and it has been speculated that a lack of plasmalogen may be the cause of major neurological complications or degeneration. This is also the case in very well known diseases where deficiency of plasmalogen occurs, such as in Niemann-Pick Type C disease (Schedin et al., 1997). It is stated that plasmalogen deficiency may not be the primary defect, but a contributing factor to the progressive neurological dysfunction. Plasmalogen deficiency is also found in hyperlipidemia (Engelmann et al., 1992, 1994) as well as in elderly patients of Down Syndrome and Multiple Sclerosis, which show reduced levels of plasmalogen (Murphy 2001, Cumings & Yanagihara 1969).
Brites et al. found evidence of the beneficial effects of treating a plasmalogen deficiency with alkyl-glycerol. (9. Brites et al., 2011; Alkyl-Glycerol Rescues Plasmalogen Levels and Pathology of Ether-Phospholipid Deficient Mice).
Extremely important seems to be, that both a deficiency in n-3 PUFA diet results in a plasmalogen deficiency and abnormal neural functions (Farooqui et al., 2001).
Taken together, plasmalogen seems to be absolutely necessary for proper brain development and function and that the loss of plasmalogen may be an indication of demyelination and further neurological damage caused by oxidative stress (Farooqui et al., 2001).
Alkyglycerols (AKG), namely Batyl, Chemyl, and Selachyl are compounds found naturally and most abundantly in Shark Liver Oil (SLO). AKG’s are intermediate products, or precursors of plasmalogen, which occur as the final product after the last step of synthesis in peroxisomes in the pathway of all plasmalogen species. Furthermore, plasmalogen molecules are made up of either one kind of PUFA and of a choline, ethanolamine or inositol group. Therefore, plasmalogen species are called ethanolamine plasmalogen or choline plasmalogen, respectively.
It has been hypothesized that AKG administration should increase plasmalogen levels in serum and tissue of ZSD patients, but not in their brains since AKG’s are not able to cross the blood brain barrier (BBB).
Nevertheless, we recommend that all ZSD patient who lack or have decreased levels of plasmalogen should be administered with exogenous AKG’s. This is further supported by the findings of Brites et al., when they reported that AKG administration to plasmalogen deficient mice (Pex7 KO mice) was able to stop the progression of the pathology in testis, adipose tissue, and the harderian gland. Furthermore, nerve conduction in peripheral nerves was improved. When given prior to the occurrence of major pathological changes, the AKG-diet prevented or ameliorated the pathology observed in Pex7 KO mice depending on the degree of plasmalogen restoration.
It has also been reported that in a ZSD patient, after 3 months of AKG supplementation there was improved growth and muscle tone, decreased nystagmus and improved ability to follow the light and fixate. Fundoscopic examination showed virtual disappearance of retinal pigmentation (Holms et al., 1987; Oral Ether Lipid Therapy in Patients with Peroxisomal Disorders), (10. Wilson et al., 1986; Zellweger Syndrome: Diagnostic Assays, Syndrome Delineation, and Potential Therapy).
It is also noteworthy that a diet rich in marine oil (n-3/EPA&DHA) in combination with the selachyl AKG, led to increased of plasmalogen levels in rat tissue (Blank et al.). This may further be supported by the findings of Martinez et al., which are found in the papers quoted above. Martinez reported that DHA EE administration led to higher plasma and RBC DHA levels as well as an increase in plasmalogens in RBC.
Additional administration of phosphatidylcholine and phosphatidylethanolamine, as well as myo-inositol, may be of benefit to patients lacking plasmalogen. This thought is partially based on the findings, that administration of dietary plasmalogen to rats led to increased serum and tissue plasmalogen concentration compared to controls (11. Shiro Mawatari et al., 2012; Dietary plasmalogen increases erythrocyte membrane plasmalogen in rats). This is thought to be achieved by the so-called salvage pathway, which could be seen as a way of recycling.
Therefore, we hypothesize, that administering all components of a plasmalogen molecule may be of benefit by creating a vast pool of resources and educts to re-synthesize new plasmalogen of all species.
The administration of myo-inositol plus [2-13C]ethanolamine significantly elevated the levels of ethanolamine plasmalogen in whole rat brain (Hoffman-Kuczynski et al., 2004). It remains unclear how the administration of myo-inositol plus [2-13C]ethanolamine influences plasmalogen synthesis. Furthermore, it is unclear how this can influence synthesis across the BBB.
Nevertheless, there is no alternative known to achieve plasmalogen synthesis, other than the administration of plasmalogen or its precursors (AKG).
It is noteworthy that brain tissue of ZSD patients have severely reduced levels of ethanolamine (12. Miyazaki et al., 2013: Altered phospholipid molecular species and glycolipid composition in brain, liver, and fibroblasts of Zellweger syndrome). This may further underline the necessity to supplement ZSD patients with ethanolamine (Phosphatidylethanolamine) in order to support or even maximize plasmalogen synthesis.
Since it remains unclear how myo-inositol administration influences plasmalogen biosynthesis, we hypothesize that the inositol group may have an activation role for the base exchange or incorporation of the choline and ethanolamine groups at the molecular level. It has been found that inositol levels are markedly reduced in the grey matter of ZSD patients (Bruhn et al.,1992; Proton NMR spectroscopy of cerebral metabolic alterations in infantile peroxisomal disorders)
On a side note, it may be of interest, that both phosphatidylcholine, as well as phosphatidylethanolamine, are also the main phospholipids of the peroxisome membrane (13. Lazarow PB, 2003: Peroxisome Biogenesis: advances and conundrums; http://www.cbi.pku.edu.cn/chinese/documents/cell/xibaoshengwuxuecankaowenxian/cocb/15/15-4/15-489.pdf ).
Therefore, it is absolutely necessary to experiment with a mixture of all AKG, rather than the single administration of just batyl, chemyl or selachyl, respectively. It also remains to be determined if components of the plasmalogen salvage pathway may be able to cross the BBB, which would obviously be the ultimate solution of preventing loss or re-establishing proper plasmalogen levels in brain tissue, thus possibly improving neural function.
Since ZSD/PBD are not just neurological disorders but rather a group of disease with multiple organ dysfunction, it may just as well be of benefit to a ZSD patient to supplement plasmalogen precursors in order to establish proper plasmalogen levels in all other organs and tissue apart of the brain, rather than hesitate to use this option because AKG can’t cross the BBB.
In summary, in ZSD/PBD patients with low blood levels of DHA and/or plasmalogen, DHA, AKG’s, together with phosphatidylcholine and phosphatidylethanolamine as well as myo-inositol may be administered as soon as possible, respectively immediately after the diagnose for a ZSD is made. Furthermore, it may be just as important to monitor the administration and its incorporation by regular PUFA/FA & plasmalogen analysis in plasma and red blood cells (RBC). This is the promising way how plasmalogen levels could be increased and the outcome could be clinically monitored and adjusted in follow-ups.
3. Bile Acid
The endogenous pathway of bile acid synthesis requires functional peroxisomes, thus ZSD patients regularly show elevated serum di- and trihydroxycholstanoic acid (DHCA & THCA) levels, which are intermediates of the complex bile acid synthesis but would require functional peroxisomes for further and final synthesis. This leads to elevated levels of DHCA and THCA in the serum of ZSD patients and it is assumed that it may have toxic effects.
Administering a non-toxic bile acid, specifically, ursodeoxycholic acid (UDCA) might be used to displace endogenous bile acids, with the desired therapeutic goal being to decrease the intrahepatic concentration of these potentially toxic bile acids that accumulate in ZSD patients. There has been evidence that the administration of UCDA may have been of benefit for other disorders than ZSD/PBD, namely in diseases with hepatopathic involvement (Balistreri et al., 1996).
This is even more promising since it has been reported that a significant improvement in biochemical indices of liver function occurred with a normalization of the serum bilirubin. Serum and urinary cholestanoic acids showed a significant decrease within a few days. A striking and sustained increase in growth was observed after therapy, and an improvement in neurological symptoms was noted. In conclusion, this study indicates that primary bile acid therapy improves liver function and growth in the patient with peroxisomal dysfunction and should be considered in the supportive therapies for this condition (Setchell et al., 1992).
Over the last two decades, several more studies and trials have been undertaken with ZSD patients leading to varying results and there is currently a trial ongoing in the Netherlands. The importance of bile acid as well as its complex synthetic pathway will not be discussed here and may be found elsewhere. Nevertheless we encourage any attempt to study this complex process and recommend a paper published by Heubi et al. 2007, (14. Heubi et al. 2007: Inborn Errors of Bile Acid Metabolism) and another excellent description about involvement of peroxisomes and the pathological findings in any deficiency of bile acids by Ferdinandusse et al., 2009 (15. Ferdinandusse et al., Bile Acids: The role of peroxisomes). At this point, it should be noted that the paper published by Ferdinandusse shows that bile acid intermediates are toxic, with DHCA being clearly the most cytotoxic bile acid and also accumulating in the brain tissue.
It has to be pointed out that sole administration of 200mg/day of DHA-EE without any further supplementation of UCDA or other bile acids equally resulted in normalization of bile acid intermediates, bilirubin and other hepatopathic markers in serum. General liver improvement was noted as well as shrinking of pathologically enlarged livers back to normal size within 1-3 month of DHA-EE treatment. In addition to DHA-EE all the patients received a normal diet for their specific age with a daily supplementation of each 50mg vitamin A,D,E,K and the patients (more than 20) stretched the entire range of ZSD/PBD with varying phenotypes, stages of disease progression and age (Nouger & Martinez et al. 2010, 200X).
Others suggest that DHA in any form in combination with bile acid therapies has shown results of improvement.
In summary, thus far, no major negative side effects have been reported due to the administration of DHA-EE or UCDA, while several cases suspect a general clinical improvement was seen which is associated with both DHA-EE and UCDA.
To our knowledge, there is only one long-term NALD patient who is treated with both DHA-EE and UCDA. He was a patient who received treatment and took part in the DHA-EE trials of Martinez et al. in Barcelona (Spain) and also became a patient in the UCDA trial of Setchell & Heubi et al., Cincinnati, Ohio (USA). This male patient was diagnosed ZSD (N-ALD) at two months of age and presented with a severe clinical presentation by the age of 5 month (he was given a 6-12 month life expectancy) and started DHA-EE treatment at the age of 6 month. At the age of 6 years he was started on UCDA. This patient is alive (now 15y old) and has been taking both DHA-EE and UCDA every single day, which in our view must grant any attempt to supplement ZSD patients by administration of these two substances.
Finally, it has to be noted that this patient, as well as further patients who received or are receiving DHA-EE have seen constant and stable normalization of plasma and serum levels of several important biomarkers of ZSD. Namely DHA (plasma & RBC) and RBC plasmalogen increased to normal, while DHCA, THCA, Bilirubin as well as the potentially toxic very long chain fatty acids (VLCFA, mainly C26:0, C26:1, C24:0, C24:1) as well as phytanic, pristanic and pipecolic acid levels decreased equally to within normal serum or plasma levels, respectively.
It is just as well possible that this specific patient has an extremely mild phenotype of ZSD with slow progression of the disease. Nevertheless, it cannot be excluded that it is not due to a phenotype with mild progression but possibly is in fact due to the therapies he has been receiving. If the latter is the case, it is not necessary to explain further reasoning as to why at least it could be repeated!
Finally, it is interesting to see if liver normalization correlates with the improvement of other organs known to be affected in ZSD as well as if neurological improvement can be achieved. It is promising to see that in milder phenotypes there seems to be a detectable improvement, while severe phenotypes of ZS may not benefit.
4. Cholesterol
Cholesterol is a major component of the brain and central nervous system (CNS). The biosynthesis occurs mainly via two major pathways and it is thought that some of the earlier steps of de novo synthesis is in the peroxisomes. Several experiments with ZS Knock Out mice (KO mice) show that cholesterol is decreased, namely in Pex2 KO mice, while in other studies on human skin fibroblast or from tissue analysis showed that ZSD are not always lacking cholesterol synthesis and doubts have a raised about the true involvement of peroxisomes therein (Hagenboom et al., 2003). In Pex2 KO mice a reduction of the steady-state concentration of cholesterol in the livers by at least 40% and in reduced levels of plasma total cholesterol and HDL cholesterol has been reported (16. Werner et al., Disturbed Cholesterol Homeostasis in a Peroxisome-Deficient PEX2 Knockout Mouse Model).
Cholesterol biosynthesis disorders are the prototypic metabolic malformation syndromes. Although their pathogenesis is not well understood, they underline the important role(s) of cholesterol and its metabolic precursors in mammalian development (17. Herman 2003: http://hmg.oxfordjournals.org/content/12/suppl_1/R75.full ).
It is thought that the pre-squalene synthesis partly involves peroxisomes. A deficiency of cholesterol synthesis may not only lead to suboptimal cholesterol levels but also in a deficiency of other metabolites such as Coenzyme Q-10 and other essential sterols.
A lack of cholesterol potentially leads to several pathological findings, which are seen and described in detail with patients suffering from diseases such as Smith-Lemli-Opitz-Syndrome (SLOS). Another disorder with cholesterol deficiency is called Mevalonic Aciduria. Interestingly enough, some of the symptoms and dysmorphic features present similar to those seen in ZSD/PBD‘s. They include developmental delay, failure to thrive, hypotonia, a large fontanelle and low set ears which are all hallmarks also seen in ZSD.
In Mevalonic Aciduria a so-called replacement therapy for the decreased or missing components such as cholesterol or its important synthetic pathway by-products, namely Coenzyme Q-10 showed no improvement while with SLOS, cholesterol and bile acid therapy often showed marked improvement in behavior, growth and fewer seizures were also reported.
Findings in disorders with inborn cholesterol synthesis deficiency indicate that in several disorders the vast majority of malformation of the brain and CNS occur during the early embryonic phase. Obviously, it is unlikely that any postnatal therapy could reverse any malformation or degeneration caused during gestation.
Nevertheless, it may be of benefit to ZSD patients if their serum is screened for irregularities, namely for cholesterol, Q10 and possibly squalene and if found decreased, to implement replacement therapy.
Perhaps a replacement therapy including squalene, Q-10 and cholesterol (plus bile acid) may cover a vast spectrum of essential components. Furthermore, squalene has been thought to be able to gross the BBB, at least in combinations found in vaccines. Oral squalene supplementation may be of benefit and it has to be said, that besides SLO, olive oil is a natural source of squalene. It remains unclear if the reported benefit to ZSD/PBD patients treated with DHA-EE deluded in olive oil, which was used by Martinez et al., was partly due to squalene or if in fact olive oil was chosen because of its squalene content.
5. Neurotransmitters, vitamins, minerals, steroid replacement
Several cases of NALD and IRD as well as long surviving ZS patient have developed adrenal insufficiency. This needs to be screened for and can be easily treated with corticosteroid therapy. If undiagnosed or not treated, death may occur, which could be easily prevented!
There is not much data available in regard to cause and treatment of seizures. It may be interesting to investigate if certain components are found to be drastically reduced and in fact could be replaced by specific supplementation.
In the case of the previously reported 15y. old N-ALD patient who has been receiving DHA-EE and bile acid therapy, the parents have been checking and adjusting certain other components too. This has especially been the case during periods of increased pathological activities such as increased seizures or hyperactivity. In one incident, the patient received neurotonin upon which both seizures and „restless leg syndrome“ like symptoms intensified. Dopamine was found to be drastically decreased and after supplementation, seizures, agitation and „restless leg syndrome“ like symptoms declined.
This patient also received supplementation for GABA, niacinamide and l-methylfolate.
In a patient with epileptic seizures with sudden onset at the age of 14, though unrelated to ZSD/PBD, the entire spectrum of anti epileptic medicine had been tried, without any benefit to the patient. After several years of unsuccessful treatment attempts, this patient was finally found to benefit from oral niacinamid. This raises the question, though controversial in pharmacological teaching, if specific administration in an orthomolecular treatment fashion could result in reduction of seizures in ZSD/PBD and possibly be of benefit in other neurological dysfunctions. Considering that cholesterol is an important precursor of many sterols and hormones, it may be the case that some of the neurological dysfunction in ZSD/PBD patients is due to lack of such components.
6. Metabolism and function improvement of peroxisomes
Current studies and trials are focusing on finding agents which are able to influence de novo biogenesis of functional peroxisomes. One new trial which has begun in April 2013 is using a substance called Betaine. Another recent discovery showed that Arginine does positively influence peroxisome biogenesis in milder ZSD phenotypes and is also able to cross the BBB (18. Berendse et al., 2013; http://www.ojrd.com/content/pdf/1750-1172-8-138.pdf )
Both Betaine and Arginine supplementation or its benefit is being tested on specific phenotypes and at this point, it remains unclear how and if any benefit may occur, as well as if other phenotypes show similar results. Thus, it is not possible to determine the outcome in other patients suffering from a different mutation than studied in the ongoing trials.
Nevertheless, it may have the same positive effects on other phenotypes and genotypes and thus may be supplemented which may lead to further results or understanding of this complex process.
Guideline for Diagnostics, Biochemical Monitoring and Treatment Evaluation
As is the case with the treatment of ZSD/PBD patient, there is also no general advice available on how treatment and progress can be monitored. Therefore many ZSD/PBD patient die without science learning much more. Since there are nearly no treatment options and literally no follow-up co-ordination, not much data of specific scientific value is gathered.
The common practice is to screen for VLCFA in plasma. Often phytanic-, pristanic- and pipecolic acid are also screened. Further diagnostic methods include serum DHCA and THCA analysis, occasionally acylcarnitine profiles or plasmalogen levels are measured and often skin fibroblast are cultured and the screening for a PEX genetic mutation is also common.
Indeed screening for VLCFA is most likely the best method for an initial diagnose, which should be backed up by skin fibroblasts.
We recommend to then always perform fatty acid profiles, including short, medium, long and very long chain fatty acids, as well as PUFA‘s. Furthermore measuring plasmalogen levels is crucial, since any change needs to be monitored and only by establishing baselines of all FA‘s and also plasmalogen would this allow for proper evaluation of treatment.
Martinez et al., used to perform FA & plasmalogen analysis on DHA-EE patients in the beginning phase every 6 weeks and after about half a year to one year after initiation of DHA-EE treatment were these analysis performed once every 2-3 month.
It is interesting to note, that Martinez et al., reported VLCFA decreasing as well as plasmalogen increasing while receiving DHA-EE. This could only be detected by performing FA & plasmalogen analysis (incl. VLCFA, phytanic and pristanic acid).
We also recommend performing whole blood analysis for the bile acid intermediates DHCA and THCA since this may show changes too, which may support and warrant both further treatment and research. While a FA/plasmalogen analysis normally can be performed with 1ml whole blood, VLCFA commonly requires 1-2ml plasma but in order to perform bile acid analysis, only blood drops on filter paper are needed.
We also recommend keeping whole blood and plasma samples frozen for possible future workup.
It would also be interesting to see if, in long surviving patients, skin fibroblast would show any difference after one year of therapy, i.e. if peroxisomes and function have increased. Possibly a follow-up skin biopsy for a second fibroblast could be harvested.
Discussion
ZSD/PBD are devastating, lethal disorders. Most parents confronted with such a diagnose will likely search the internet for information. Obviously, all the information we have written up and referenced here in this paper has been obtained from sources found on the internet and any parent can find this information too. One such source is the Global Foundation of Peroxisomal Disorders, (thegfpd.org).
During our work on this paper, one specific debate discussed on their facebook-group aimed at the above-mentioned trial with Betaine. This trial includes only patients with a specific mutation in Pex1 gene. The fact that it is firstly only a (very small) trial to determine safety and possible outcome/benefit to patients of this mutation and secondly, that these parents either know that they have another mutation, don‘t know or don‘t care about exactly which mutation and anyway want to try it, shows the desperation they‘re in. One has to keep in mind, as a parent you have a diagnose with a very poor prognosis and no treatment options whatsoever. Degeneration, de-myelination, death, in 6-12month, because of Zellweger SAyndrome, these are the words on the mind of parents of a child diagnosed with PBD. Nothing is going to stop them from trying/testing, since the option of waiting is not an option. The same was the case with the publication of the Arginine paper and they share online their e-mail exchanges with the responses of experts in these diseases, such as Dr.N. Braverman.
It is likely that the treating doctors may not have all this information at hand and would soon be confronted with parents who are pushing for treatments such as DHA or bile acid administration since they are the most frequently talked about, or reported therapies, which are found on the net.
It won‘t please any parent to hear they have to wait what the results of the trial show or that there is no or not enough scientific evidence to support these treatments. Furthermore, it is likely that most clinics, even if they wanted to, could not provide the agents, which have been described here. That is especially the case with pure PUFA‘s such as DHA-TG, DHA-EE or the AKG‘s.
Therefore, we encourage the major laboratories who offer VLCFA analysis, to change the present procedures when confronted with a positive (elevated VLCFA) result. These results most likely are sent back to a clinic where even experienced pediatrics, neurologist and physicians have never even heard about ZSD/PBD, since they are extremely rare disorders. Obviously, any recipient of a positive VLCFA result is forced to obtain extra knowledge in order to understand the clinical situation. Results for elevated VLCFA indicate a peroxisome biogenesis disorder (PBD) and will always be commented by confirming the diagnosis for Zellweger Spectrum Disorder (ZSD), possibly listing Zellweger Syndrome, Neonatal Adrenoleukodrystrophy and Infantile Refsum Disease as well as encouraging and even indicating the necessity for further investigations.
To avoid delays, which may result due to a lack of knowledge, the reporting of results for elevated VLCFA should include a guideline for both medical personnel and patients/parents.
This guideline must include all the treatment options mentioned above and would include the automatic request to perform full FA, PUFA and plasmalogen analysis, which would be the baseline, considering treatment takes place. Coordination and cooperation between the testing lab and its enquiring colleagues should include advising for skin biopsy collecting, scheduling for repeated FA/PUFA and plasmalogen analysis as well as scheduling for further blood chemical diagnostics, mainly for markers such as bile acid, acylcarnitine etc..
Kane P et al., published an excellent paper with protocol and treatment method in Autistic Spectrum Disorder with lipids, vitamins and other components. Autistic Spectrum Disorders show elevated plasma VLCFA as well as further clinical similarities as seen in ZSD/PBD (19. Kane P et al., 2008; The Neurobiology of Lipids in Autistic Spectrum Disorders). Blood work, performed and with protocols in the way presented by Kane P et al., may serve and assist in the design on new treatment approaches.
Ultimately, establishing stocks of the substances and agents mentioned above, readily available to clinics and caretakers is a necessity. Only specialized facilities allow for the proper processing of these chemicals.
Furthermore, full-scale diagnostics with follow-ups, as mentioned earlier, performed on all ZSD/PBD patients, including genotype/phenotype identification, would also result in a vast data base collection of vital information.
Currently, nearly no data is systematically gathered, much data is never collected and therefore lost. This needs to, and
can be changed with information exchange and coordination.
Supplementation
Generally speaking, there are no products available, which would be optimal for ZSD/PBD patients. The DHA-EE used by Martinez et al., was in fact purchased and deluded in olive oil by co-workers of Martinez. There is another product available in the US and parts of Europe called DocOmega, which contains pure DHA. All other fish oil or also algae derived oils will contain both DHA and EPA. Others contain both n-3 and n-6 PUFA which, as stated earlier has been reported to be inefficient to supply DHA due to the blocking effect of enzymatic competition.
Similarly, there are no AKG products in pure form. There are SLO products which contain so called highly purified AKG‘s, but usually they have been removed of squalene and the AKG‘s tend to be around 20% of whole capsule content. The remaining 80% may include unwanted PUFA or further substances.
One product which may suit best in ZSD/PBD is called Ecomer Shark Liver Oil.
Nevertheless, it should be thought of to manufacture AKG mixtures containing Batyl, Chemyl and Selachyl.
This work is dedicated to all the newborns and children suffering from ZSD/PBD.
We hope that this paper will encourage physicians, clinicians and scientists to implement a new standard procedure, ranging from diagnosis to treatment of ZSD/PBD patients.
References:
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Martinez et al., 2010; Visual follow-up in peroxisomal-disorder patients treated with docosahexaenoic Acid ethyl ester
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