The National Association of Medical Examiners Position Paper: Second Autopsies.

ABSTRACT: Second autopsies are uncommon in the United States yet are of significant public value. A second autopsy may be sought when the first autopsy findings are disputed, considered biased, or inadequately communicated. Second autopsies are technically and interpretatively difficult and usually rely heavily on investigative information, first autopsy findings, and additional documentation from the first autopsy. Medicolegal second autopsies should be performed only by experienced, board-certified forensic pathologists. Pathologists performing second autopsies should acknowledge and disclose the limitations of second autopsies. The first autopsy pathologist should recognize the quality assurance value of a second autopsy and fully disclose autopsy documentation to the second autopsy pathologist, if permitted by jurisdictional law.

Validation of a Fully Automated Immunoaffinity Workflow for the Detection and Quantification of Insulin Analogs by LC-MS-MS in Postmortem Vitreous Humor.

The analysis of biological specimens collected at autopsy for the presence of exogenous insulin(s) is of special interest in select death investigations as they may be suspected in the cause of a death. Technical challenges include the limited stability of insulin, and the forensic requirement of differentiating endogenous insulin from pharmaceutical analogs. A novel method was developed for the detection and quantification of human insulin, Glulisine, Lispro, Aspart, Glargine and Detemir in vitreous fluid. An immunoaffinity extraction procedure is performed followed by separation of the insulin α- and ß-chains. Liquid chromatography tandem mass spectrometry analysis of the ß-chain allows for the unequivocal identification of each insulin analog. The analytical measurement range for each insulin was 0.5-25 ng/mL. The method was evaluated for accuracy, precision, carryover, interferences and stability. Eight vitreous fluid samples collected from cases where untoward insulin use was suspected were subjected to analysis. Positive results were obtained from three samples, and a detailed case history is provided for one of these cases. Even though insulin instability in postmortem biological fluid remains a challenge, this method allows for a reliable forensic-level analysis in vitreous fluid.

Brain antioxidant systems in human methamphetamine users.

Animal data suggest that the widely abused psychostimulant methamphetamine can damage brain dopamine neurones by causing dopamine-dependent oxidative stress; however, the relevance to human methamphetamine users is unclear. We measured levels of key antioxidant defences [reduced (GSH) and oxidized (GSSG) glutathione, six major GSH system enzymes, copper-zinc superoxide dismutase (CuZnSOD), uric acid] that are often altered after exposure to oxidative stress, in autopsied brain of human methamphetamine users and matched controls. Changes in the total (n = 20) methamphetamine group were limited to the dopamine-rich caudate (the striatal subdivision with the most severe dopamine loss) in which only activity of CuZnSOD (+ 14%) and GSSG levels (+ 58%) were changed. In the six methamphetamine users with severe (- 72 to - 97%) caudate dopamine loss, caudate CuZnSOD activity (+ 20%) and uric acid levels (+ 63%) were increased with a trend for decreased (- 35%) GSH concentration. Our data suggest that brain levels of many antioxidant systems are preserved in methamphetamine users and that GSH depletion, commonly observed during severe oxidative stress, might occur only with severe dopamine loss. Increased CuZnSOD and uric acid might reflect compensatory responses to oxidative stress. Future studies are necessary to establish whether these changes are associated with oxidative brain damage in human methamphetamine users.

Brain vesicular acetylcholine transporter in human users of drugs of abuse.

Limited animal data suggest that the dopaminergic neurotoxin methamphetamine is not toxic to brain (striatal) cholinergic neurons. However, we previously reported that activity of choline acetyltransferase (ChAT), the cholinergic marker synthetic enzyme, can be very low in brain of some human high-dose methamphetamine users. We measured, by quantitative immunoblotting, concentrations of a second cholinergic marker, the vesicular acetylcholine transporter (VAChT), considered to be a "stable" marker of cholinergic neurons, in autopsied brain (caudate, hippocampus) of chronic users of methamphetamine and, for comparison, in brain of users of cocaine, heroin, and matched controls. Western blot analyses showed normal levels of VAChT immunoreactivity in hippocampus of all drug user groups, whereas in the dopamine-rich caudate VAChT levels were selectively elevated (+48%) in the methamphetamine group, including the three high-dose methamphetamine users who had severely reduced ChAT activity. To the extent that cholinergic neuron integrity can be inferred from VAChT concentration, our data suggest that methamphetamine does not cause loss of striatal cholinergic neurons, but might damage/downregulate brain ChAT in some high-dose users. However, the finding of increased VAChT levels suggests that brain VAChT concentration might be subject to up- and downregulation as part of a compensatory process to maintain homeostasis of neuronal cholinergic activity. This possibility should be taken into account when utilizing VAChT as a neuroimaging outcome marker for cholinergic neuron number in human studies.

Why is parkinsonism not a feature of human methamphetamine users?

For more than 50 years, methamphetamine has been a widely used stimulant drug taken to maintain wakefulness and performance and, in high doses, to cause intense euphoria. Animal studies show that methamphetamine can cause short-term and even persistent depletion of brain levels of the neurotransmitter dopamine. However, the clinical features of Parkinson's disease, a dopamine deficiency disorder of the brain, do not appear to be characteristic of human methamphetamine users. We compared dopamine levels in autopsied brain tissue of chronic methamphetamine users with those in patients with Parkinson's disease and in a control group. Mean dopamine levels in the methamphetamine users were reduced more in the caudate (-61%) than in the putamen (-50%), a pattern opposite to that of Parkinson's disease. Some methamphetamine users had severely decreased dopamine levels, within the parkinsonian range, in the caudate (up to 97% dopamine loss) but not in the putamen. As the putamen and caudate subserve aspects of motor and cognitive function, respectively, our data suggest that methamphetamine users are not parkinsonian because dopamine levels are not sufficiently decreased in the motor component of the striatum. However, the near-total reduction in the caudate could explain reports of cognitive disturbances, sometimes disabling, in some drug users, and suggests that treatment with dopamine substitution medication (e.g. levodopa) during drug rehabilitation might be helpful.

Decreased striatal dopamine D1 receptor-stimulated adenylyl cyclase activity in human methamphetamine users.

OBJECTIVE: It has been assumed that some behavioral changes associated with repeated exposure to dopaminergic psychostimulant drugs might be explained by changes in activity of dopamine receptors, including the dopamine D(1) receptor, which is linked by a stimulatory G protein to the effector enzyme adenylyl cyclase. To establish whether dopamine D(1) receptor function might be altered in human methamphetamine users, the authors measured dopamine-stimulated adenylyl cyclase activity in the brain of chronic human users of the drug. METHOD: Adenylyl cyclase activity stimulated by dopamine and by guanylyl-imidodiphosphate (to assess G protein and adenylyl cyclase coupling) was determined in the postmortem brain tissue of 16 methamphetamine users who had used the drug both recently and chronically (i.e., at least 1 year) as well as 21 matched comparison subjects. RESULTS: A 25%-30% decrease in the maximal extent of dopamine stimulation of adenylyl cyclase activity was seen in the striatum (nucleus accumbens, caudate, and putamen) of the methamphetamine users. No changes were found in basal or guanylyl-imidodiphosphate-stimulated enzyme activity. CONCLUSIONS: These data suggest that dopamine receptor function linked to adenylyl cyclase is partially desensitized in the striatum of human methamphetamine users. Decreased dopamine D(1) receptor function might underlie part of the known (drug withdrawal syndrome) or expected (drug tolerance) consequences of methamphetamine exposure in humans.