Citations

AbstractBackground: Congenital disorders of glycosylation (CDG) are autosomal recessive disorders that produce increased serum carbohydrate-deficient transferrin (CDT) isoforms. Methods to resolve CDT from fully glycosylated transferrin (Trf) have been based on a neutral shift in the isoelectric focusing (IEF) pattern or on a reduction in the negative charge, allowing resolution by anion-exchange chromatography. Our purpose was to develop a method of resolution and relative quantification of Trf isoforms using online immunoaffinity liquid chromatography–mass spectrometry (LC-MS).Methods: Serum (25 μL) was diluted with 100 μL of water before application to an immunoaffinity column that sequestered Trf isoforms. Trf isoforms were eluted from the immunoaffinity column, concentrated on a C4 column, eluted from the C4 column, and introduced into the mass spectrometer. Analysis of the Trf isoforms was entirely automated and completed in <10 min per sample.Results: The LC-MS method demonstrated that the major abnormal Trf isoforms in CDG lack one complete oligosaccharide structure (mono-oligosaccharide) or both oligosaccharide structures (a-oligosaccharide), but not the sialic acids, as presumed on the basis of IEF methods. Calculation of relative ratios among three possible species (mono-/di-oligosaccharide and a-/di-oligosaccharide) is reproducible [mean intra- and interassay CVs were 9.3% (n = 10) and 10% (n = 5), respectively]. A reference range for patients <18 years was determined by analysis of 209 samples (for mono-/di-oligosaccharide, the median was 0.041 and the range was 0.018–0.083; for a-/di-oligosaccharide, the median was 0.007 and the range was 0.002–0.036). Comparison of data obtained with an affinity chromatography-IEF method and the LC-MS method demonstrated equivalence in the interpreted results (n = 170).Conclusions: Advantages of the LC-MS method include improved sensitivity, minimal sample preparation, and an analysis time of <10 min. The method was automated, which allowed high throughput, with >100 samples analyzed in a single day. Moreover, the nature of the oligosaccharide defect in CDG is accurately reflected by mass resolution, and subtle oligosaccharide truncations may also be detected by this method.

Abstract Background: Recent evidence suggests that the presence of small, dense LDL is independently associated with increased risk of developing coronary artery disease. Current methods to subfractionate LDL are time-consuming and/or technically demanding. Therefore, we have sought the development of a less complex LDL subfractionation procedure. Methods: LDL subfractions were separated using the Quantimetrix LipoprintTM LDL System. High-resolution 3% polyacrylamide gel tubes were scanned densitometrically (610 nm) with a Helena EDC system. A computerized method to identify and quantitatively score the resolved LDL subfractions was developed. Results from the Quantimetrix method were compared using 51 plasma samples with values obtained by nondenaturing gradient gel electrophoresis (NDGGE) and nuclear magnetic resonance (NMR) spectroscopy. Results: LDL subfractionation scores correlated significantly (P <0.05) with triglyceride, HDL-cholesterol, apolipoprotein B100, and LDL-cholesterol/apolipoprotein B100 (r = 0.591, −0.392, 0.454, and −0.411, respectively). For 51 samples, the Quantimetrix method classified 21 with small, 14 with intermediate, and 16 with large LDL. Of the 21 samples classified as small by Quantimetrix, 20 (95%) were classified as small (n = 18) or intermediate (n = 2) by NDGGE. All of the 16 specimens classified as large by Quantimetrix were either large (n = 14) or intermediate (n = 2) by NDGGE. LDL score was inversely correlated (r = −0.674; P <0.0001) with LDL particle size determined by NMR spectroscopy. Conclusions: A quantitative method for the assessment of LDL particle size phenotype was developed using the Quantimetrix Lipoprint LDL System. The method can be performed in less than 3 h in batch mode and is suitable for routine use in clinical laboratories.

Abstract We used wheat-germ-lectin affinity chromatography as a tool to investigate the structure of alkaline phosphatase (ALP, EC 3.1.3.1) and to obtain fractions enriched in either bone or liver ALP activity. Liver and bone isoenzymes in serum samples were incompletely resolved except that the activity in the nonretained fraction (fraction 1) always represented pure liver isoenzyme and constituted a larger percentage of total activity in pooled sera with increased liver ALP activity than in pooled sera with increased bone activity. In contrast, a more avidly retained ALP activity, presumably with high glycosylation, was found in human serum with high activity of bone ALP. Using a solid-phase immunoassay, we examined the fractions obtained from the wheat-germ-lectin-Sepharose 4B column to determine whether the isoenzyme preference of the monoclonal antibody was markedly influenced by the degree of glycosylation. Whether samples contained high proportions of liver or of bone isoenzyme activity, the nonretained fraction contained a higher percentage of liver ALP, whereas the more strongly bound fraction contained a higher percentage of bone ALP. Except for eluted fractions that either contained no detectable N-acetylglucosamine or the highest percentage of it, the avidity of the liver-isoenzyme-specific monoclonal antibody for ALP seemed to be independent of the degree of glycosylation, suggesting that the epitope for monoclonal antibody may be expressed in some structure other than the carbohydrate moieties.

Abstract In this method we use a linear equation to resolve two-component decay data from the urea inactivation of mixtures of alkaline phosphatase (EC 3.1.3.1) of liver and bone origin. The specificity for the bone/liver isoenzyme is enhanced by including L-phenylalanine in the urea reaction to inhibit the intestinal and placental forms. Bone and liver fractions are each quantified from results of a single run on the centrifugal analyzer. Total activity and the L-phenylalanine-inhibited fraction are measured on separate runs. A simplification of this method has been used in our laboratory for 14 years in performing greater than 80,000 analyses of clinical specimens. Speed, accuracy, and precision are improved over previous methods by this mathematical solution. Data reduction is automated through the use of a personal computer interfaced to the analyzer. With such systems generally available, this method can now be suggested as suitable for routine use to separate these and perhaps other isoenzyme mixtures.

Abstract To separate liver and bone alkaline phosphatase (ALP) isoenzymes in human serum, we used high-performance affinity chromatography (HPAC) on a column of wheat-germ lectin conjugated to 7-microns-diameter silica particles and an eluent containing N-acetyl-D-glucosamine (NAG). On-line spectrophotometric detection of ALP involved pumping diethanolamine-buffered p-nitrophenyl phosphate solution post-column. Bone and liver isoenzymes could be separated into two peaks with only 10% overlap when an exponential gradient was used. A linear-step gradient separated 80.9% of liver ALP and 91.6% of bone ALP in two distinct peaks. True bone and liver ALP peak areas for the linear-step gradient were determined by using correction factors, because each peak contained a co-eluted portion of the other ALP isoenzyme. The detection limit improved 10-fold over those of other techniques for ALP isoenzymes, owing to the relatively large sample that could be applied to the column. Correlation with a urea-inactivation procedure was reasonable for patients' serum samples (r = 0.98 and 0.79 for liver ALP and bone ALP, respectively).

Abstract Increasing evidence recognizes Alzheimer's disease (AD) as a multifactorial and heterogeneous disease with multiple contributors to its pathophysiology, including vascular dysfunction. The recently updated AD Research Framework put forth by the National Institute on Aging–Alzheimer's Association describes a biomarker‐based pathologic definition of AD focused on amyloid, tau, and neuronal injury. In response to this article, here we first discussed evidence that vascular dysfunction is an important early event in AD pathophysiology. Next, we examined various imaging sequences that could be easily implemented to evaluate different types of vascular dysfunction associated with, and/or contributing to, AD pathophysiology, including changes in blood‐brain barrier integrity and cerebral blood flow. Vascular imaging biomarkers of small vessel disease of the brain, which is responsible for >50% of dementia worldwide, including AD, are already established, well characterized, and easy to recognize. We suggest that these vascular biomarkers should be incorporated into the AD Research Framework to gain a better understanding of AD pathophysiology and aid in treatment efforts.

Abstract Background An international Delphi panel has defined a harmonized protocol (HarP) for the manual segmentation of the hippocampus on MR. The aim of this study is to study the concurrent validity of the HarP toward local protocols, and its major sources of variance. Methods Fourteen tracers segmented 10 Alzheimer's Disease Neuroimaging Initiative (ADNI) cases scanned at 1.5 T and 3T following local protocols, qualified for segmentation based on the HarP through a standard web‐platform and resegmented following the HarP. The five most accurate tracers followed the HarP to segment 15 ADNI cases acquired at three time points on both 1.5 T and 3T. Results The agreement among tracers was relatively low with the local protocols (absolute left/right ICC 0.44/0.43) and much higher with the HarP (absolute left/right ICC 0.88/0.89). On the larger set of 15 cases, the HarP agreement within (left/right ICC range: 0.94/0.95 to 0.99/0.99) and among tracers (left/right ICC range: 0.89/0.90) was very high. The volume variance due to different tracers was 0.9% of the total, comparing favorably to variance due to scanner manufacturer (1.2), atrophy rates (3.5), hemispheric asymmetry (3.7), field strength (4.4), and significantly smaller than the variance due to atrophy (33.5%, P < .001), and physiological variability (49.2%, P < .001). Conclusions The HarP has high measurement stability compared with local segmentation protocols, and good reproducibility within and among human tracers. Hippocampi segmented with the HarP can be used as a reference for the qualification of human tracers and automated segmentation algorithms.

To test the hypothesis that the frequency of biotinidase deficiency is greater in children with unexplained developmental delay or neurologic abnormalities than in the general population, we studied children seen at a large outpatient clinic over a four-year period who had one or more of these neurologic abnormalities and for whom no specific cause for their abnormalities could be found. The group totaled 274 children (163 boys; 111 girls) whose ages ranged from 2 weeks to 17 years. Characteristics were IQ/DQ, 30 to 70 in the 115 for whom scores were available; 41% had seizures; 15% had sensorineural hearing loss; 54% showed gross motor delay or ataxia; and 27% had decreased muscle tone. One patient with a classical clinical picture of biotinidase deficiency was diagnosed during the study period and was not included in the study. None of the patients with nonclassic findings had a deficiency of biotinidase activity. Our results suggest that biotinidase deficiency does not account for a large proportion of children with unexplained neurologic abnormalities or developmental delay. This does not negate the importance of biotinidase testing in children with clinical patterns specifically suggestive of the deficiency.