By Mary Holland, Esq.
Tuesday, May 20, 2008
Special Master Vowell announced the court’s ruling about scheduling issues. The government must put on all its witnesses in May, except for Drs. Clarkson and Magos, who are unavailable now and will testify in July. Petitioners will put on all of their rebuttal case now, except for rebuttal of Drs. Clarkson and Magos’ testimony, which they will rebut in July. In the third week of July, two days will be for the third test case; two days will be for the government’s toxicologists, Drs. Clarkson and Magos, and one day will be reserved for Petitioners’ rebuttal.
Direct Examination of Dr. Roberts
The government’s lawyer, Ms. Renzi, conducts direct examination. Dr. Roberts reviews his qualifications as an expert. He is an M.D., certified in internal medicine and pharmacology. He’s a professor at Duke University. He has several academic awards and he is on several editorial boards. He has many research grants and employs several research assistants. He holds several patents. He has published over 300 peer-reviewed articles and chapters and has written approximately 180 on oxidative stress since 1990. He frequently presents on this topic, but previously studied prostaglandins before turning to the study of free radicals.
Q: What has been your influence on the study of oxidative stress?
A: It’s been a “pretty profound” influence. People had been trying to study free radicals in test tubes. At the time, most of these methods were non-specific. It was very difficult to translate lab findings to humans. When we discovered F2-isoprostanes, it became the most reliable way to study oxidative stress in the body. Through a major national study, we found that F2-isoprostanes are the best way to measure severe oxidative stress in the liver. This discovery really helped to move the field forward.
Q: Are you an expert in mercury and autism?
Q: But you are an expert in oxidative stress?
Q: You listened to the testimony of Dr. Deth?
Q: Please explain oxidative stress in simple terms. [Dr. Roberts refers to slides in the courtroom.]
A: Molecules are made up of atoms. The atoms spin in opposite directions as pairs around the nuclei of atoms. Subtraction of an electron from an atom is called oxidation. When an atom loses an electron, there is an unpaired electron; the atom will try to take an electron from someplace else. These compounds are unstable and very reactive; they really want to find another electron. This will cause a chain reaction that will propagate oxidation that keeps going and going and going.
Q: A free radical is then a molecule that only has one electron?
Q: Your next slide is on free radical produced lipid oxidation?
A: Yes. This depicts that the radical will extract a hydrogen electron from the lipid. What’s left behind is a radical. There is a lot of oxygen in the body; it will immediately react with oxygen and oxidize another lipid. So this chain reaction will continue until something stops it.
Q: What can stop this chain reaction?
A: Donation of an electron, or reduction, can terminate the reaction. An antioxidant molecule can donate the electron. Although you might think the reaction will continue, it won’t; the antioxidant molecules have unique properties; they are not highly reactive. They will not extract another electron from another molecule; this will stop the chain reaction. So the redox status represents the balance between oxidation and reduction, and it’s a balance. So oxidative stress is an imbalance between oxidation and reduction in favor of oxidation.
Q: Is it correct that every human undergoes oxidative stress?
Q: What will elicit oxidative stress?
A: If you bruise your finger, that’s oxidative stress. Exercise causes modest oxidative stress. We have a battery of protective mechanisms, antioxidant enzymes, which keep our balance in check at a certain level. We have a constant, low level degree of oxidative stress going on all the time, but we have incredible defense mechanisms against oxidative stress.
Q: Oxidative stress does not necessarily mean oxidative damage, right?
A: Correct; they are not necessarily the same. Moderate levels of oxidative stress, like exercise, can be protective.
Q: Does evidence of oxidative stress in the periphery or plasma indicate oxidative stress in the brain?
A: No; free radicals don’t travel around the body. They are very reactive and will cause injury where they are located.
Q: If there is oxidative stress in the brain, can it be measured in the periphery?
A: No; it can’t. These free radicals don’t move around. We’ve done research on free radicals in Alzheimer’s disease on autopsy and we can’t measure oxidative injury in the brain when we use isoprostanes in the plasma or urine. The amount of oxidative injury in these people’s brains is enormous, but we can’t pick this up in the periphery.
Q: Petitioners’ experts have talked a lot about biomarkers that they say show ongoing oxidative stress. One of those biomarkers is the glutathione (GSH) and oxidized glutathione balance, correct?
A: Yes; GSH, when it loses an electron, becomes oxidized GSH; that’s GS with an unpaired electron. So if you have two GSs with unpaired electrons, they will combine to form GSSG or reduced glutathione. So you get the ratio of oxidized glutathione to reduced glutathione.
Q: In this case, the ratio of oxidized glutathione to reduced glutathione has been used to show that children with autism have oxidative stress. Petitioners have implicated this as a cause of autism. Can these biomarkers be indicative of oxidative stress?
A: They can be indicative of oxidative stress, but they don’t indicate oxidative damage. Furthermore, these tests were done in the plasma, and what’s going on in the plasma doesn’t have much relevance for what’s going on in the brain.
Q: What’s the most accurate way to measure oxidative injury?
A: The best measure as validated by the NIEHS is F2-isoprostanes.
Q: If you found elevated levels of F2-isoprostanes in the plasma, would that indicate anything about the brain?
Q: If there was oxidative damage in the brain, could that be measured in the periphery?
Q: In your expert report, you reviewed some of the articles that Dr. Deth relied on to conclude that there was oxidative stress. Id’ like to review those with you. The first is Dr. Chauhan’s 2004 paper. The authors there measured levels of MDA in plasma with TBARS assay. Please describe this and say whether this is a reliable method for measuring oxidative damage.
A: It’s totally unreliable. TBARS are not specific for measuring malondialdehyde (MDA). The other problem is that they measured this in plasma. When you draw blood, you always get platelets. Measuring MDA for plasma would not be reliable for oxidative stress because TBARS is not reliable for MDA.
Q: You also reviewed the Ming study that Dr. Deth relies on, in which the authors measured F2-isoprostanes using immunoassay.
A: Isoprostanes are prostaglandin-like substances. They are small lipids. When I was in the prostaglandin field, people worked for years to try to find accurate measurements. Companies sell these immunoassay kits because they make money, but they are not reliable. But even if this immunoassay was reliable in the plasma, that tells you nothing about what’s going on in the brain.
Q: Have you done in vitro studies on oxidative stress?
Q: Have you also done animal studies on oxidative stress?
Q: And human studies?
Q: And do you have an opinion on the value of extrapolating in vitro data to what occurs in vivo?
A: It’s very, very, very, very difficult. You can use cell culture systems as a hint, but these cells are already transformed and abnormal. So you can’t extrapolate what you learn in vitro. It’s just a lead. If you have an animal model for disease, then you’d apply it there, and in the end you have to research it in humans.
Q: In your opinion, is it more likely than not, that the studies Dr. Deth relied on show that autistic children have oxidative stress in their brains?
Q: So even if Dr. Deth and Dr. Mumper are correct that there is oxidative stress with findings in the periphery, is there any way to discern the cause of that stress?
A: No. These children aren’t normal. Their diet isn’t normal. It’s all speculation, but maybe their nutrition is not adequate.
Q: Is oxidative stress indicative of oxidative injury?
A; No. An oxidative stress may cause a minimum amount of oxidative injury, but that can be protective and lead to up regulation of this whole defensive process.
Q: Would that finding tell you anything about what is going on in the brain?
Q: Are there any specific biomarkers for mercury-induced oxidative stress?
A; Not that I know of.
Q: And finally, in your twenty years of experience working in the field of oxidative stress, is there any evidence to conclude that autism is caused by oxidative stress?
A: I see none.
Q: Do you believe that reflects what’s generally accepted by researchers in the field of oxidative stress?
A: Yes; it’s not something that’s even discussed by people in the field of free radical research.
Cross Examination of Dr. Roberts
Q: [Mr. Willams] Do you think there is any way that researchers trying to treat autism could measure oxidative stress in the periphery to see what was going on the brain?
Q: Spinal fluid, maybe?
Q: Do you understand that the general theory that Dr. Kinsbourne presented here is that the persistence of inorganic mercury in the brains of these children leads to active, chronic neuroinflammation that can explain the symptoms of autism described in many papers? Do you agree that neuroinflammation can explain the symptoms of autism?
Objection: Mr. Matanoski objects that this is beyond the scope of Dr. Robert’s direct testimony.
Q: Can inflammation in the brain cause oxidative stress?
A: Yes, to a certain extent; it depends on how severe the inflammation is; we have a lot of defense mechanisms, and oxidative stress may not translate into oxidative damage.
Q: And oxidative damage could be loss of function, not just cell death?
Q: Would inorganic mercury in the neurons cause oxidative stress in those neurons?
Objection: Beyond the scope of direct testimony; he’s said he’s not an expert on mercury.
Ruling: Let’s see if he can answer.
A: I don’t know that.
Q: You mentioned autopsy studies on Alzheimer patients that show oxidative stress and oxidative damage. Have you reviewed any of the autopsy reports on autistic patients?
Special Master Campbell-Smith: We’ve been hearing there’s a chronic level of oxidative stress that causes loss of cell function but does not result in cell death. You’ve explained a process of oxidative stress that leads to up regulation. Am I correct that if the body can up regulate, then there is no damage?
Q: We’re struggling with when is there oxidative stress and when is there oxidative damage.
A: The only way you can know is if you measure oxidative damage. You have to measure a product of oxidative damage; you have to be able to quantify that damage.
Redirect Q: Is there a difference between oxidative stress, oxidative damage and oxidative stress that has consequences?
A: Yes. These isoprostanes can be measured in your plasma, but it’s at a level that doesn’t hurt us. When we get more damage, then cells can die.