Polyneuropathy Type Research Articles

On-Line Immunoaffinity Solid-Phase Extraction Capillary Electrophoresis-Mass Spectrometry for the Analysis of Serum Transthyretin.

The analysis of low abundant proteins in biological fluids by capillary electrophoresis (CE) is particularly problematic due to the typically poor concentration limits of detection of microscale separation techniques. Another important issue is sample matrix complexity that requires an appropriate cleanup. Here, we describe an on-line immunoaffinity solid-phase extraction capillary electrophoresis-mass spectrometry (IA-SPE-CE-MS) method for the immunoextraction, preconcentration, separation, detection, and characterization of serum transthyretin (TTR). TTR is a protein biomarker related to diverse types of amyloidosis, such as familial amyloidotic polyneuropathy type I (FAP-I), which is the most common hereditary systemic amyloidosis.

Familial amyloidosis with polyneuropathy type 1 caused by transthyretin mutation Val50Met (Val30Met): 4 cases in a non-endemic area

IntroductionTransthyretin-related familial amyloid polyneuropathy (TTR-FAP) typically arises as an autonomic neuropathy primarily affecting small fibres and it occurs in adult patients in their second or third decades of life. It progresses rapidly and can lead to death in approximately 10 years. Other phenotypes have been described in non-endemic areas. Objectives and methodsWe described 4 cases from the Spanish province of Guipuzcoa, a non-endemic area, to highlight the clinical variability of this disease. Patients and resultsThree patients presented a late-onset form manifesting after the age of 50, featuring a predominantly motor polyneuropathy initially causing distal impairment of the lower limbs followed by the upper limbs. One patient suffered severe neuropathic pain. None showed signs of autonomic involvement. The fourth patient, of Portuguese descent, presented a typical form with onset in her thirties, neuropathic pain and dysautonomia. All patients carry the Val50Met mutation in the TTR gene. ConclusionFAP is a pleomorphic disease even in patients carrying the same mutation. In non-endemic areas, its main form of presentation may resemble a predominantly motor polyneuropathy developing in the sixth decade of life with no signs of dysautonomia. Given this non-specific presentation and the widely available technical means of studying the TTR gene, we believe that the protocol for the aetiological diagnosis of any polyneuropathy should include genetic sequencing of TTR.

Clinical presentations and electromyographical aspects in toxic neuropathies

Objectives Peripheral nerves are likely to be damaged by a wide array of toxins and medications causing different types of neuropathies mainly affecting distal nerves. Toxic neuropathies are often misdiagnosed due to the lack of available specific evidence. However, they can be suspected through clinical examination and electrodiagnostic features. We aimed to determine the pattern of clinical and electroneuromyographic disorder in toxic neuropathies. Methods A retrospective study was carried out on patients who had history of neurotoxin exposure and diagnosed with toxic neuropathy. All cases underwent detailed neurological examination and appropriate investigations needed to exclude other causes of neuropathy, as well as electrophysiological studies (EMG). Results Over the 21 patients we selected: 9 were alcoholic, 4 glue-sniffing addicts, 2 received medication with metronidazole and 3 had chemotherapy. Two had a professional exposure to N-Hexane and 1 did a suicide attempt with organophosphate. Neurological examination showed motor deficit predominating in distal limbs (61.9%), areflexia (85.7%) amyotrophy (19%) as well as proprioceptive ataxia (42.8%) and hypoesthesia (33.3%). Electrophysiological studies showed a symmetric sensorial dominant sensory-motor axonal type polyneuropathy (90%) with greater involvement of lower extremities (100%). Therefore, demyelinating features were detected in 40% with increased distal and F wave latencies (35%), decreased motor and sensory nerve conduction velocities (40%), motor conduction block (15%) and temporal dispersion (5%) defining the polyradiculoneuropathy electromyographic criteria (20%). Denervation signs were revealed in 30%. Most of patients (57.14%) regained their sensory and motor capacities within few months. Conclusion Peripheral neuropathies secondary to toxins are increasingly considered but can be difficult to definitively diagnose especially when it presents with a subacute severe form mimicking Guillain-Barre syndrome. Yet, detecting the syndromic presentation and a history of toxin exposure can facilitate prompt and accurate diagnosis.

F27. Myoclonus in demyelinating polyneuropathies: Preliminary results

Introduction The reports on tremor and different types of polyneuropathies have been increasing and it was suggested to be associated with cerebellar involvement in chronic inflammatory demyelinating polyneuropathy (CIDP). Apart from specific syndromes, the co-existence of polyneuropathy and myoclonus is exceptional. We previously observed cortical myoclonus in cases with inflammatory polyneuropathy who were referred for tremor analysis and aimed to analyze presence of myoclonus and its relation with clinical features in demyelinating polyneuropathies. Methods We prospectively included all patients with inflammatory polyneuropathy who were admitted between January 2017 and June 2017 and had tremor-like, regular or irregular involuntary movements on clinical examination. All patients underwent neurological examination and multichannel surface electromyography which included upper extremity muscles as well as lower extremity and facial or neck muscles in selected cases. We also recorded long latency reflexes and somatosensory evoked potentials (SEPs) to categorize myoclonus. Results We identified eight patients with demyelinating polyneuropathy who matched the inclusion criteria: four patients with CIDP, three patients with Guillain–Barre syndrome and one patient with Charcot-Marie-Tooth disease. The mean age was 50.5 ± 19.6 years (range: 26–81 years) and there were seven male patients. Seven out of eight patients had myoclonus, two of which were in the form of polyminimyoclonus. Others had myoclonus on both proximal and distal parts of upper extremities. Negative myoclonus was observed in four of them. Duration of myoclonus was between 30 and 100 ms. only one had duration of 200 ms. Most patients (five out of seven) had cortical myoclonus (with high-amplitude C reflex or SEPs). Conclusion Myoclonus in the patients with polyneuropathy was irregular tremor-like, however, the electrophysiological characteristics was similar to a cortical subtype. Therefore, it would be interesting to investigate a bigger cohort of inflammatory polyneuropathies for the presence and subtypes of myoclonus and to determine associated clinical features.

Polyneuropathies

Background: Polyneuropathies (peripheral neuropathies) are the most common type of disorder of the peripheral nervous system in adults, and specifically in the elderly, with an estimated prevalence of 5-8%, depending on age. The options for treatment depend on the cause, which should therefore be identified as precisely as possible by an appropriate diagnostic evaluation. Methods: This review is based on the current guidelines and on large-scale cohort studies and randomized, controlled trials published from 2000 to 2017, with an emphasis on non-hereditary types of polyneuropathy, that were retrieved by a selective search in PubMed. Results: Diabetes is the most common cause of polyneuropathy in Europe and North America. Alcohol-associated polyneuropathy has a prevalence of 22-66% among persons with chronic alcoholism. Because of the increasing prevalence of malignant disease and the use of new chemotherapeutic drugs, chemotherapy-induced neuropathies (CIN) have gained in clinical importance;their prevalence is often stated to be 30-40%, with high variation depending on the drug(s) and treatment regimen used. Polyneuropathy can also arise from genetic causes or as a consequence of vitamin deficiency or overdose, exposure to toxic substances and drugs, and a variety of immunological processes. About half of all cases of polyneu ropathy are associated with pain. Neuropathic pain can be treated symptomatically with medication. Exercise, physiotherapy, and ergotherapy can also be beneficial, depending on the patient's symptoms and functional deficits. Conclusion: A timely diagnosis of the cause of polyneuropathy is a prerequisite for the initiation of appropriate specific treatment. Patients with severe neuropathy of unidentified cause should be referred to a specialized center for a thorough diagnostic evaluation.

Polyneuropathies.

Polyneuropathies (peripheral neuropathies) are the most common type of disorder of the peripheral nervous system in adults, and specifically in the elderly, with an estimated prevalence of 5-8%, depending on age. The options for treatment depend on the cause, which should therefore be identified as precisely as possible by an appropriate diagnostic evaluation. This review is based on the current guidelines and on large-scale cohort studies and randomized, controlled trials published from 2000 to 2017, with an emphasis on non-hereditary types of polyneuropathy, that were retrieved by a selective search in PubMed. Diabetes is the most common cause of polyneuropathy in Europe and North America. Alcohol-associated polyneuropathy has a prevalence of 22-66% among persons with chronic alcoholism. Because of the increasing prevalence of malignant disease and the use of new chemotherapeutic drugs, chemotherapy-induced neuropathies (CIN) have gained in clinical importance; their prevalence is often stated to be 30-40%, with high variation depending on the drug(s) and treatment regimen used. Polyneuropathy can also arise from genetic causes or as a consequence of vitamin deficiency or overdose, exposure to toxic substances and drugs, and a variety of immunological processes. About half of all cases of polyneu - ropathy are associated with pain. Neuropathic pain can be treated symptomatically with medication. Exercise, physiotherapy, and ergotherapy can also be beneficial, depending on the patient's symptoms and functional deficits. A timely diagnosis of the cause of polyneuropathy is a prerequisite for the initiation of appropriate specific treatment. Patients with severe neuropathy of unidentified cause should be referred to a specialized center for a thorough diagnostic evaluation.

A chemometric approach for characterization of serum transthyretin in familial amyloidotic polyneuropathy type I (FAP-I) by electrospray ionization-ion mobility mass spectrometry

In this study, we describe a chemometric data analysis approach to assist in the interpretation of the complex datasets from the analysis of high-molecular mass oligomeric proteins by ion mobility mass spectrometry (IM-MS). The homotetrameric protein transthyretin (TTR) is involved in familial amyloidotic polyneuropathy type I (FAP-I). FAP-I is associated with a specific TTR mutant variant (TTR(Met30)) that can be easily detected analyzing the monomeric forms of the mutant protein. However, the mechanism of protein misfolding and aggregation onset, which could be triggered by structural changes in the native tetrameric protein, remains under investigation. Serum TTR from healthy controls and FAP-I patients was purified under non-denaturing conditions by conventional immunoprecipitation in solution and analyzed by IM-MS. IM-MS allowed separation and characterization of several tetrameric, trimeric and dimeric TTR gas ions due to their differential drift time. After an appropriate data pre-processing, multivariate curve resolution alternating least squares (MCR-ALS) was applied to the complex datasets. A group of seven independent components being characterized by their ion mobility profiles and mass spectra were resolved to explain the observed data variance in control and patient samples. Then, principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were considered for exploration and classification. Only four out of the seven resolved components were enough for an accurate differentiation. Furthermore, the specific TTR ions identified in the mass spectra of these components and the resolved ion mobility profiles provided a straightforward insight into the most relevant oligomeric TTR proteoforms for the disease.

Neurophysiologic Studies in the Evaluation of Polyneuropathy.

This article provides core information on the clinical neurophysiology techniques available for the investigation of disorders of the peripheral nervous system. The role of small fiber dysfunction in some types of polyneuropathy is being increasingly appreciated, and neurophysiologic techniques for evaluating the autonomic components of peripheral axons have enhanced our understanding of small fiber dysfunction. The principles of nerve conduction studies and needle EMG are presented in this article, along with the patterns of abnormality encountered in patients with polyneuropathy due to large and small fiber involvement.

S70 Estimating motor unit number from CMAP scans – MScanFIT MUNE

Objectives There is no method that can measure the exact number of motor units, therefore motor unit number estimation (MUNE) methods have been developed. Most MUNE methods are based on estimating the size of an average surface-recorded motor unit potential and dividing that value into maximal compound action potential (CMAP). This makes the estimates strongly influenced by any bias in unit selection. Other limitations of the existing MUNE methods are the presence of subjectivity in the estimation process, the failure to sample enough units or the long time to perform the test or analyse. To overcome these limitations, a new MUNE method was recently developed, so-called ‘MScanFit MUNE‘, (MScan). Methods MScan meets the above-mentioned criticisms by fitting a model to a detailed stimulus-response curve (∼500 stimuli) or ‘CMAP scan’, taking into account the threshold variability of all the units, and avoiding subjectivity. Results The method has been found to be accurate (mean absolute error 7%) for simulated data.The reproducibility and the diagnostic utility of MScan were found to be good in healthy controls and in patients with amyotrophic lateral sclerosis (ALS) and different types of polyneuropathy. Discussion MScan revealed in neurofibromatosis-type-2 patients denervation and reinnervation in peripheral nerves suggesting that it may be used to quantify and monitor disease progression. Conclusions MScan is a novel method with good reproducibility, and may be performed in less than five minutes. Significance Preliminary results suggest MScan as a promising MUNE method with potential to be used in diagnoses and monitoring disease progression, particularly in ALS.

Diffusion-Tensor-Imaging MR-Neurography for the detection of polyneuropathy in Type 1 diabetes (P4.041)

Diffusion-Tensor-Imaging MR-Neurography for the detection of polyneuropathy in Type 1 diabetes (P4.041)

On-line immunoaffinity solid-phase extraction capillary electrophoresis mass spectrometry using Fab´antibody fragments for the analysis of serum transthyretin

This paper describes an on-line immunoaffinity solid-phase extraction capillary electrophoresis mass spectrometry (IA-SPE-CE-MS) method using an immunoaffinity sorbent with Fab′ antibody fragments (Fab′-IA) for the analysis of serum transthyretin (TTR), a homotetrameric protein (Mr~56,000) involved in different types of amyloidosis. The IA sorbent was prepared by covalent attachment of Fab′ fragments obtained from a polyclonal IgG antibody against TTR to succinimidyl silica particles. The Fab′-IA-SPE-CE-MS methodology was first established analyzing TTR standard solutions. Under optimized conditions, repeatability and reproducibility were acceptable, the method was linear between 1 and 25µgmL−1, limits of detection (LODs) were around 0.5µgmL−1 (50-fold lower than by CE-MS, ~25µgmL−1) and different TTR conformations were observed (folded and unfolded). The applicability of the developed method to screen for familial amyloidotic polyneuropathy type I (FAP-I), which is the most common hereditary systemic amyloidosis, was evaluated analyzing serum samples from healthy controls and FAP-I patients. For the analysis of sera, the most abundant proteins were precipitated with 5% (v/v) of phenol before Fab′-IA-SPE-CE-MS. The current method enhanced our previous results for the analysis of TTR using intact antibodies immobilized on magnetic beads. It allowed a slight improvement on LODs (2-fold), the detection of proteoforms found at lower concentrations and the preparation of microcartridges with extended durability.

Transthyretin amyloidosis: an under-recognized neuropathy and cardiomyopathy.

Transthyretin (TTR) amyloidosis (ATTR amyloidosis) is an underdiagnosed and important typeof cardiomyopathy and/or polyneuropathy that requires increased awareness within the medical community. Raising awareness among clinicians about this typeof neuropathy and lethal form of heart disease is critical for improving earlier diagnosis and the identification of patients for treatment. The following review summarizes current criteria used to diagnose both hereditary and wild-type ATTR (ATTRwt) amyloidosis, tools available to clinicians to improve diagnostic accuracy, available and newly developing therapeutics, as well as a brief biochemical and biophysical background of TTR amyloidogenesis.

Nonalcoholic fatty liver disease is associated with an increased prevalence of distal symmetric polyneuropathy in adult patients with type 1 diabetes

AimsPresently, data on the association between nonalcoholic fatty liver disease (NAFLD) and distal symmetric polyneuropathy in people with diabetes are scarce and conflicting. The aim of this retrospective, cross-sectional study was to examine whether NAFLD was associated with an increased prevalence of distal symmetric polyneuropathy in type 1 diabetic adults. MethodsWe studied all white type 1 diabetic outpatients (n = 286, 42.3% male, mean age 43 ± 14 years, median diabetes duration 17 [10–30] years), who participated in a foot screening program at our adult diabetes clinic after excluding those who had excessive alcohol consumption and other known causes of chronic liver disease. NAFLD was diagnosed by ultrasonography. Distal symmetric polyneuropathy was detected using the Michigan Neuropathy Screening Instrument method and the biothesiometer Vibrotest. ResultsOverall, the prevalence rates of NAFLD and distal symmetric polyneuropathy were 52.4% and 35.3%, respectively. Patients with NAFLD had a substantially increased prevalence of distal symmetric polyneuropathy compared to their counterparts without NAFLD (51.0% vs. 17.1%, p < 0.001). In univariate analysis, NAFLD was associated with an approximately 5-fold increased risk of prevalent distal symmetric polyneuropathy (odds ratio [OR] 5.32, 95% confidence interval [CI] 3.1–9.3, p < 0.001). This association remained significant even after adjustment for age, sex, diabetes duration, hemoglobin A1c, diabetic retinopathy, smoking, metabolic syndrome, chronic kidney disease and carotid artery stenoses ≥ 40% (adjusted-OR 2.23, 95% CI 1.1–4.8, p < 0.05). ConclusionsOur results show that NAFLD, diagnosed by ultrasonography, is strongly associated with an increased risk of distal symmetric polyneuropathy in type 1 diabetic adults, independently of several cardio-metabolic risk factors.

Comparative Study of Vitamin B Complex Combined with Alpha Lipoic Acid versus Vitamin B Complex in Treatment of Diabetic Polyneuropathy in Type 2 Diabetic Patients

Objective: Diabetic Polyneuropathy (DPN) represents a major health problem as it increases morbidity affecting patients’ quality of life. Vitamin B frequently is used for treating DPN. ALPHA Lipoic Acid (ALA) seems to delay or reverse DPN. The present study compared the effect of Vitamin B complex combined with ALA versus Vitamin B in treatment of DPN in Egyptian type 2 diabetic patients. Methods: Forty type 2 diabetic patients with DPN of whom twenty received once-daily oral doses of ALA (600 mg) in combination with Vitamin B complex and the other twenty only received vitamin B complex for 12 weeks. Patients were evaluated by Michigan Neuropathy Screening Instrument (MNSI) questionnaire and Nerve Conduction Studies (NCS). Blood samples were collected for determination of Insulin Resistance and lipid abnormalities. All the previous assessments were performed at baseline and at week 12. Results: There was a significant reduction after 12 weeks of supplementation in vitamin B complex plus Alpha Lipoic Acid (ALA) group using MNSI questionnaire (p=0.001) and not in vitamin B complex group. NCS demonstrated that 12 out of 16 patients in vitamin B complex plus ALA group showed improvement in at least one property measured compared with only 6 out of 16 in vitamin B complex group (p=0.0732). The influence of the combination on the progression of diabetes with regards to insulin resistance and lipid profile showed no advantage over Vitamin B complex alone. Conclusion: Combined therapy of DPN with ALA and Vitamin B complex, orally for 12 weeks improves the symptoms of neuropathy (MNSI) with a similar trend in NCS.

Physical activity and albuminuria were associated with painful diabetic polyneuropathy in type 2 diabetes in an ethnic Chinese population

Physical activity and albuminuria were associated with painful diabetic polyneuropathy in type 2 diabetes in an ethnic Chinese population

Physical activity and albuminuria were associated with painful diabetic polyneuropathy in type 2 diabetes in an ethnic Chinese population

BackgroundDiabetic neuropathy is a common complication in patients with type 2 diabetes. However, the prevalence of painful diabetic polyneuropathy (PDPN) have been less studied. We examined the prevalence and risk factors of PDPN in outpatients with type 2 diabetes in an ethnic Chinese population. MethodsThis retrospective study enrolled 2358 outpatients with type 2 diabetes who had completed the Douleur Neuropathique en 4 Questions (DN4) questionnaire from January 2013 to October 2013. Patients with a total score ≥4 were defined as having PDPN. ResultsIn all, 179 patients were diagnosed as having PDPN with a score of 4.49 on the DN4 questionnaire, compared with 0.66 for patients without PDPN. After adjusting the possible confounding factors, the risk of painful neuropathy was increased in the group without physical activity (Odds ratio 3.38, 95% CI 1.54–9.79), and in the group with macroalbuminuria (Odds ratio 2.31, 95% CI 1.44–3.73). Besides, there was a joint effect of macroalbuminuria and no physical activity habit on PDPN risk. ConclusionsThe prevalence of PDPN was 7.6% among our outpatients with type 2 diabetes. Less physical activity and albuminuria, respectively, increased the risk of PDPN and had a joint effect.

Diffusion tensor imaging MR neurography for the detection of polyneuropathy in type 1 diabetes.

To evaluate if diffusion tensor imaging MR neurography (DTI-MRN) can detect lesions of peripheral nerves in patients with type 1 diabetes. Eleven type 1 diabetic patients with polyneuropathy (DPN), 10 type 1 diabetic patients without polyneuropathy (nDPN), and 10 healthy controls (HC) were investigated with a 3T MRI scanner. Clinical examinations, nerve-conduction studies, and vibratory-perception thresholds determined the presence of DPN. DTI-MRN (voxel size: 1.4 × 1.4 × 3 mm3 ; b-values: 0, 800 s/mm2 ) covered proximal (sciatic nerve) and distal regions of the lower extremity (tibial nerve). Fractional anisotropy (FA) and apparent diffusion coefficient (ADC) were calculated and compared to T2 -relaxometry and proton-spin density obtained from a multiecho turbo spin echo (TSE) sequence. Furthermore, we evaluated DTI reproducibility, repeatability, and diagnostic accuracy. DTI-MRN could accurately discriminate between DPN, nDPN, and HC. The proximal FA was lowest in DPN (DPN 0.37 ± 0.06; nDPN 0.47 ± 0.03; HC 0.49 ± 0.06; P < 0.01). In addition, distal FA was lowest in DPN (DPN 0.31 ± 0.05; nDPN 0.41 ± 0.07; HC 0.43 ± 0.08; P < 0.01). Likewise, proximal ADC was highest in DPN (DPN 1.69 ± 0.25 × 10-3 mm2 /s; nDPN 1.50 ± 0.06 × 10-3 mm2 /s; HC 1.42 ± 0.12 × 10-3 mm2 /s; P < 0.01) as was distal ADC (DPN 1.87 ± 0.45 × 10-3 mm2 /s; nDPN 1.59 ± 0.19 × 10-3 mm2 /s; HC 1.57 ± 0.26 × 10-3 mm2 /s; P = 0.09). The combined interclass-correlation (ICC) coefficient of DTI reproducibility and repeatability was high in the sciatic nerve (ICC: FA = 0.86; ADC = 0.85) and the tibial nerve (ICC: FA = 0.78; ADC = 0.66). T2 -relaxometry and proton-spin-density did not enable detection of neuropathy. DTI-MRN accurately detects DPN by lower nerve FA and higher ADC. These alterations are likely to reflect proximal and distal nerve fiber pathology. 1 J. Magn. Reson. Imaging 2017;45:1125-1134.

A Comparative Peripheral Nerve Conduction Study in Type-2 Diabetics with Symptoms of Polyneuropathy with Age and Sex Matched Normal Subjects

Background: Diabetes Mellitus is one among the diseases that affects peripheral nervous system and symptomatic Neuropathy in uncontrolled Diabetes is one of the commonest incapacitating complications. Electro diagnostic tests can be used to detect Diabetic Neuropathy at an early stage (before development of the signs or symptoms of Neuropathy). Nerve Conduction Study (NCS) is considered to be the most sensitive, reliable, non-invasive and objective means of investigations of Diabetic Polyneuropathy. Diabetic Neuropathy is curable, and hence if detected, the proper treatment can be instituted in early stages, which again, can give rise to good outcome. As the peripheral nerve has the ability to regenerate, line of treatment can be planned. Aims & Objectives: To compare and analyze the peripheral nerve conduction studies in symptomatic diabetic neuropathy cases with age and sex matched normal subjects emphasizing on use of Nerve conduction studies as a diagnostic approach to prevent Severe Neuropathy. Methods: The study group consists of 9 cases diagnosed with Diabetic neuropathy referred to our neurophysiology lab and control group consists of 9 age and sex matched normal subjects who are nondiabetic, healthy volunteers. Statistical analysis was done using unpaired t-test and 8 out of 9 cases considered for statistical analysis. Motor and Sensory nerve conduction studies conducted in both Upper (Median and Ulnar nerves) and Lower limbs (Tibial and Peroneal nerves) using bipolar surface electrodes with RMS EMG EP MARK-II machine. Results: There is a definite decrease in amplitude and nerve conduction velocities of both the sensory and motor components, axonal and demyelinating type significantly correlating with higher HBA1c levels. F minimum latencies of Median, Tibial and Peroneal nerves were increased possibly more of demyelinating type of Polyneuropathy. Conclusion: NCS being a simple, harmless, non-invasive and objective technique along with easy interpretation of results can be used routinely to evaluate the status of nerves in patients with Type 2 Diabetes Mellitus to prevent more disabling state at the earliest.

Comparison of capillary electrophoresis and capillary liquid chromatography coupled to mass spectrometry for the analysis of transthyretin in human serum

Capillary electrophoresis and capillary liquid chromatography coupled to mass spectrometry (CE-MS and CapLC–MS, respectively) are nowadays very suitable techniques for the separation and characterization of intact proteins in biological fluids. In this paper, we compare the performance of both techniques for the analysis of transthyretin (TTR), which is a homotetrameric protein (relative molecular mass (Mr) ∼56,000) involved in different types of amyloidosis. Furthermore, it is also presented a novel sample pretreatment based on immunoprecipitation (IP) using Protein A Ultrarapid Agarose™ (UAPA) magnetic beads (MBs) to purify TTR from serum samples. This novel IP based on MBs allowed the detection of TTR monomeric proteoforms that were not possible to analyze by conventional IP in solution. In addition, UAPA MBs provided many other desirable advantages including higher selectivity and minimal unspecific binding of other proteins. CE-MS and CapLC–MS were applied to analyze serum samples from healthy controls and familial amyloidotic polyneuropathy type I (FAP-I) patients, who suffered from the most common hereditary systemic amyloidosis. Both techniques allowed detecting the same TTR proteoforms, including the mutant TTR (Met 30) variant (variation in relative molecular mass (ΔMr) was +32.07, from wild-type TTR). Migration/retention times and relative quantitation of the different proteoforms were similar and reproducible in both cases, but the limits of detection (LODs) achieved by CE-MS were slightly lower (2–2.5-fold). Some other differences were also found on separation selectivity (migration orders and separation of antibody), peak efficiency, total analysis time, calibration ranges and experimental Mr accuracy.

Analysis of serum transthyretin by on-line immunoaffinity solid-phase extraction capillary electrophoresis mass spectrometry using magnetic beads.

In this paper, an on-line immunoaffinity solid-phase extraction capillary electrophoresis mass spectrometry (IA-SPE-CE-MS) method using magnetic beads (MBs) is described for the analysis of serum transthyretin (TTR), which is a protein related to different types of amyloidosis. First, purification of TTR from serum was investigated by off-line immunoprecipitation and CE-MS. The suitability of three Protein A (ProA) MBs (Protein A Ultrarapid Agarose(TM) (UAPA), Dynabeads(®) Protein A (DyPA) and SiMAG-Protein A (SiPA) and AffiAmino Ultrarapid Agarose(TM) (UAAF) MBs to prepare an IA sorbent with a polyclonal antibody (Ab) against TTR, was studied. In all cases, results were repeatable and it was possible the identification and the quantitation of the relative abundance of the six most abundant TTR proteoforms. Although recoveries were the best with UAPA MBs, UAAF MBs were preferred for on-line immunopurification because Ab was not eluted from the MBs. Under the optimized conditions with standards in IA-SPE-CE-MS, microcartridge lifetime (>20 analyses/day) and repeatability (2.9 and 4.3% RSD for migration times and peak areas) were good, the method was linear between 5 and 25 μg/mL and LOD was around 1 μg/mL (25 times lower than by CE-MS, ≈25 μg/mL). A simple off-line sample pretreatment based on precipitation of the most abundant proteins with 5% (v/v) of phenol was necessary to clean-up serum samples. The potential of the on-line method to screen for familial amyloidotic polyneuropathy type I (FAP-I), which is the most common hereditary systemic amyloidosis, was demonstrated analysing serum samples from healthy controls and FAP-I patients.