Innovations in Alzheimer’s Disease Assessment: Quantitation of Amyloid Beta 40 and 42
Alzheimer’s disease (AD) is highly prevalent, and its incidence is expected to double by 2050.1 Understanding the disease through early assessment and differentiating it from other forms of dementia can be helpful for physicians and patients.
New approaches to assess the likelihood that a patient has AD have recently been developed, enabled by liquid chromatography tandem-mass spectrometry (LC-MS/MS). Dr. Nigel Clarke, Vice President Technology and Solutions Development, Quest Diagnostics Nichols Institute, discusses the measurement of the biomarkers amyloid beta 42 (Aß42) and 40 (Aß40)and the use of Aß42/Aß40 ratio to evaluate mild cognitive impairment (MCI) and differentiate AD from other forms of dementia, and reviews the utility of apolipoprotein E (ApoE) measurements in AD risk assessment.
Background – AD pathology
Over the years, there have been two competing theories about the causes of AD. “There are those who believed that tau, by creating neurofibrillary tangles (NFTs), was the underlying cause”, says Dr. Clarke, “while others believed that amyloid was the causative agent, and not a side effect. The pendulum has swung between these two camps, but over the last 5 to 10 years there’s been a growing understanding that amyloid is one of the major causes, with NFTs playing a contributory role. In certain cases, NFTs may be prognostic about the speed and severity of disease progression, whereas the amyloid burden indicates whether someone is likely to go into mild cognitive impairment (MCI) and then progress to AD.
The role of amyloid
“Everybody has some amyloid deposition in their brain. It’s a natural occurrence, which accelerates and is exacerbated in AD,” continues Dr. Clarke. “Autopsies have shown that classic plaques present in everyone, but the number is much smaller in someone with no cognitive symptoms of AD than in someone with full AD. Those plaques are made up of amyloid beta, with Aß40 and Aß42 being the two main subtypes of interest. The Aß42 is believed to be the ‘bad player’ in the sense that it has two extra amino acids, which cause the conformational change. So, instead of having an alpha helix like Aß40, making it soluble in aqueous solutions, such as in cerebrospinal fluid (CSF) and inside the cells, it changes it to a beta sheet, which is hydrophobic and very sticky. So, once Aß42 is produced it will eventually ‘plaque out’—it will find other Aß42 and start forming amyloid. This process, known as amyloidosis initiates an inflammatory response killing off the neurons around it. As more and more areas of the brain are affected over time, a tipping point is reached—the brain can no longer compensate for the changes that have taken place and symptoms start to appear. That’s when we start seeing MCI.”
The process for evaluating AD is usually initiated by a family member following the appearance of symptoms. The general practitioner performs a neuro-cognitive assessment—the mini-mental state evaluation test (MMSE) and CogniSenseTM are examples and if the patient fails that, he or she is referred to a neurological group for testing. A volumetric MRI and a spinal tap are then typically performed. “The biomarkers traditionally measured, using an enzyme-linked immunosorbent assay(ELISA), are Aβ42, plus phosphorylated tau (p-tau) and total tau (t-tau),” says Dr. Clarke. “An equation using these values indicates whether or not the person is likely to have AD. This is used as part of a whole workup. Some larger groups will also use Aβ PET scans, which provide an indication of the amyloid load within the brain.”
The measurement of Aβ42 using the ELISA test has played an important role in identifying AD patients and allowing a clinician to monitor a patient’s progress. However, there are large differences between immunoassays, which can differ in absolute concentrations and can be characterized by matrix interference, hampering inter-laboratory comparisons and the use of general cut-off levels.3,4 This has been recognized by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), which has developed a candidate reference measurement procedure (RMP) using a LC-MS/MS assay.4
The Aß42/Aß40 ratio
The use of LC-MS/MS also enables the measurement of Aß40 and therefore the use of the Aß42/Aß40 ratio. “There has been growing recognition that the Aß42/Aß40 ratio is more useful than Aß42 alone, due to inherent differences in level of Aß42 between patients,” explains Dr. Clarke. “The Aß42 base level between two individuals can be very different. If we don’t have a baseline of when someone was healthy, the number we obtain from the test may be misleading. If a patient happens to have been born with a lower level of Aß42, and a simple cutoff is applied, he or she may fall below that cut-off even though there is no AD. The application of the Aß42/Aß40 ratio may address this challenge.
“Since Aß40 and Aß42 both come from the same precursor protein, the amyloid precursor protein (APP), and since they are enzymatically cleaved to that, there is a belief that that cleavage is modified in people with AD,” continues Dr. Clarke. “In other words, the Aß40 is standard across everybody, but Aß42 goes up in people that have AD. So the ratio is much more indicative than Aß42 alone and allows you to compare people much better.
“An argument for using the Aß42/Aß40 ratio is that it helps address individual biases, or individual differences, within the patient population. So if there are disparate results from the volumetric MRI and the ELISA test, the Aß40/Aß42 could serve as a ‘tie breaker’. There is also evidence that the ratio is better than Aß42 in differentiating AD dementia from non-AD dementias.”2
Apolipoprotein E measurement
Another test to help diagnose AD is the measurement of Apolipoprotein E (ApoE). “For many years we have run the ApoE4 genetics test to identify whether someone has the E4 allele, the best-known risk factor for AD, and whether there are one or two of them,” says Dr. Clarke. “Using LC-MS/MS we can determine someone’s alleles not by genetic sequencing, but by actually measuring the forms of the ApoE protein that are present. We can detect whether there is a homozygous population of protein, and which one it is, or if there is a heterozygous population of protein and which ones they are. In addition, we actually measure and quantitate the amount of ApoE that’s present. This is helpful in risk-assessment, because there is some suggestion that the volume of ApoE can play a role in mitigating the effects of the mutation.”
Test panel and risk assessment
When all three tests—AB42/40 ratio, ApoE Isoform, and Total ApoE—are performed as a panel, the test results are further analyzed by an algorithm that calculates a risk- assessment score. Based on this score, patients are categorized as at low, average, or high risk of having AD. Such an assessment can lead to a more effective disease-management strategy, specifically customized to the individual.
Evaluating AD biomarkers with this new methodology may help clarify the relative likelihood of AD, which could lead to better patient management. Although AD is currently not curable, drugs may be used to temporarily alleviate memory loss and confusion. In addition, early diagnosis allows patients to prepare for future care, or choose to participate in clinical trials for new therapies. Ruling out AD expedites investigation of other causes of dementia, including those that may be completely or partially reversed.
1. Alzheimer’s Association. 2017 Alzheimer’s Disease Facts and Figures. Alzheimer’s Dement. 2017;13:325-373.
2. Janelidze S, Zetterberg H, Mattsson N, et al. CSF Aß42/Aß40 and Aß42/Aß38 ratios: better diagnostic markers of Alzheimer disease. Ann Clin Transl Neurol. 2016;3:154-165.
3. Teunissen CE, Willemse EAJ. Cerebrospinal Fluid Biomarkers For Alzheimer’s Disease: Emergence Of The Solution To An Important Unmet Need. EJIFCC. 2014;24:97-104.
4. Leinenbach A, Pannee J, Dülffer T, et al. IFCC Scientific Division Working Group on CSF proteins. Clin Chem. 2014;60:987-94.
Nigel J. Clarke, PhD
Vice President Technology and Solutions Development
Quest Diagnostics Nichols Institute
San Juan Capistrano, CA
Released on Friday, May 25, 2018