Measurement of microalbuminuria using protein chip electrophoresis.

Microalbuminuria reflects the progression of nephropathy and cardiovascular disease in diabetic and hypertensive patients. Most commercially available tests currently used to measure microalbuminuria are immunoassays. We developed a microfluidics-based assay using the P200 protein chip (Caliper Life Sciences, Mountain View, CA, and Agilent Technologies, Santa Clara, CA) and 2100 Bioanalyzer (Agilent Technologies) to detect microalbuminuria. The method integrates and automates the electrophoretic separation and fluorescent detection of proteins from 14 to 200 kd. The assay was linear up to 750 mg/L and demonstrated good sensitivity with a lower detection limit of 7.5 mg/L. Intrachip and interchip coefficients of variation ranged from 0% to 4% and 4.9% to 13.5%, respectively. When albumin was measured by chip and immunoturbidimetry in diabetic urine samples, the chip consistently showed higher albumin concentrations. The discrepancy may be due to the chip's ability to detect immunounreactive albumin. Overall, this simple, cost-effective assay offers a sensitive and accurate measurement of microalbuminuria that can be easily implemented in a clinical laboratory.

Interference with hemoglobin A(1C) determination by the hemoglobin variant Shelby.

Hemoglobin variant carrier status was found in a 46-year-old African American man following detection of a falsely elevated hemoglobin A1c (HbA1c) by ionexchange high-performance liquid chromatography (HPLC, VARIANT A1c, Bio-Rad Laboratories, Hercules, CA). Additional analysis of the hemoglobin variant using the Beta Thal Short program (Bio-Rad) revealed an unknown peak with a retention time of 4.84 minutes and a proportion of 26.3%. No mass shift in alpha-globin or beta-globin proteins was observed by mass spectrometry. DNA sequencing revealed a missense mutation in 1 beta-globin allele corresponding to the hemoglobin Shelby trait. The patient was asymptomatic with a normal hemoglobin value of 13.6 g/dL (136 g/L) but had increased target cells on a peripheral blood smear. An alternative method for HbA1c determination using boronate-affinity HPLC provided a value of 3.9% (0.04; reference range, 4.0%-6.9% [0.04-0.07]), more consistent with the patient's recent blood glucose values in the normal range.

Analysis and Quantitation of Glycated Hemoglobin by Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry.

Measurement of glycated hemoglobin is widely used for the diagnosis and monitoring of diabetes mellitus. Matrix assisted laser desorption/ionization (MALDI) time of flight (TOF) mass spectrometry (MS) analysis of patient samples is used to demonstrate a method for quantitation of total glycation on the ß-subunit of hemoglobin. The approach is accurate and calibrated with commercially available reference materials. Measurements were linear (R(2) > 0.99) across the clinically relevant range of 4% to 20% glycation with coefficients of variation of ≤ 2.5%. Additional and independent measurements of glycation of the α-subunit of hemoglobin are used to validate ß-subunit glycation measurements and distinguish hemoglobin variants. Results obtained by MALDI-TOF MS were compared with those obtained in a clinical laboratory using validated HPLC methodology. MALDI-TOF MS sample preparation was minimal and analysis times were rapid making the method an attractive alternative to methodologies currently in practice.

Dilute and shoot: analysis of drugs of abuse using selected reaction monitoring for quantification and full scan product ion spectra for identification.

Our objective was to develop a "dilute and shoot" liquid chromatography-tandem mass spectrometry confirmatory procedure that uses full scan product ion spectra to identify drugs that are present above cutoff values as determined by isotope dilution relative to a deuterium-labeled internal standard. Deuterium-labeled internal standards are added to urine which is then diluted prior to analysis. Full scan product ion spectra were obtained in the data-dependent mode using a linear ion trap (ABI 4000 Qtrap). Identification was based on a purity fit of greater than 70. Ninety-seven urine specimens were analyzed by the method described, and results were compared to values obtained from a reference laboratory using selected reaction monitoring (SRM). The ion trap provided about 30-fold increase in signal-to-noise ratio as compared with the same instrument operated in a traditional full scan product ion mode. The assays were linear to at least 10 times the cutoff. Selecting appropriate triggers for obtaining full scan product ion spectra minimized space charging for specimens that contained high concentrations of drugs. There was 100% concordance between the full scan identification and the SRM results for identification of amphetamine, methamphetamine, benzoylecgonine, morphine, codeine, hydrocodone, and hydromorphone. The ability to "dilute and shoot" reduces the turnaround time for results. The data acquired with SRM and full scan product ion spectra provide accurate quantification and a high degree of specificity.

Analysis of testosterone in serum using mass spectrometry.

For either gas chromatography mass spectrometry (GC-MS) or liquid chromatography tandem mass spectrometry (LC-MS-MS) methods, the first step in the analysis is to add a deuterium-labeled internal standard such as testosterone-16,16,17-d(3). Testosterone in the sample is then isolated by liquid-liquid extraction and the extract is dried under a stream of nitrogen. For the GC-MS method we describe; the residue is transformed to the pentafluorobenzyl/trimethylsilyl derivative and is injected into the GC-MS, separated on a dimethypolysiloxane column, and ionized using electron capture negative chemical ionization (ECNCI). Quantification of testosterone in the samples is by selected ion monitoring, measuring peak ratios of testosterone relative to the deuterium-labeled internal standard. For the LC-MS-MS analysis of testosterone, the sample extract is reconstituted in mobile phase, injected on a C18 column, and quantified using multiple reaction monitoring of testosterone relative to the internal standard. There are no interferences from common steroids found in human serum. For both methods the run-to-run precision and accuracy is generally less than 6% and the methods are linear from 5 to 2000 ng/dL.

Chip electrophoresis as a method for quantifying total microalbuminuria.

BACKGROUND: Microalbuminuria is an important prognostic marker in diabetic nephropathy and cardiovascular disease. Initially, most commercial assays used immunoreactivity to quantify microalbuminuria; however, size-exclusion HPLC demonstrated the existence of nonimmunoreactive forms of albumin that may not be detected by immunoassay. Recent liquid chromatography tandem mass spectrometry analyses suggested that size-exclusion HPLC gave higher results attributable to other urine proteins coeluting with albumin. We describe an assay that measures total microalbuminuria (immunoreactive and nonimmunoreactive) without any discernable interference from other common urine proteins. METHODS: We used an automated chip electrophoresis system that utilized microfluidic separation technology and fluorescent sample detection. Each albumin specimen was mixed with the manufacturer's sample buffer in addition to a chicken albumin internal calibrator and then electrophoresed without additional reducing agents. RESULTS: With variable concentrations of bovine serum albumin normalized to a chicken albumin internal calibrator, the electrophoresis system was best fit with a polynomial (R2=0.9997; concentration range, 5-300 mg/L). The lower limit of detection was 5 mg/L. Interchip and intrachip variation studies conducted on patient urine demonstrated CVs of 3%-13%. The introduction of potentially interfering agents (i.e., molecular analytes, nonalbumin proteins) did not alter precision. Compared with immunoassay, the chip electrophoresis identified higher microalbuminuria concentrations in all urine samples. The method also clearly resolved the albumin peak from interfering proteins. CONCLUSIONS: Unlike immunoassay, chip electrophoresis can detect both immunoreactive and nonimmunoreactive forms of albumin. This system is a simple, robust method to quantify microalbuminuria with good sensitivity, precision, and accuracy.

Causes of hypercalcemia in a population of military veterans in the United States.

OBJECTIVE: To determine the various causes of hypercalcemia in a population of US veterans at the San Diego Veterans Affairs Medical Center. METHODS: A retrospective analysis of medical records was performed on 212 US veterans encountered between 1998 and 2002 with serum calcium measurements between 10.5 and 10.9 mg/dL on multiple readings or 11.0 mg/dL (or greater) on any single determination. Data were collected from the clinical records for each patient and used to determine the cause of each patient's hypercalcemia. Patients undergoing hemodialysis were excluded from this study. RESULTS: The study population consisted of 201 men (95%) and 11 women (5%), with a median age of 63 years (range, 25 to 95). Of the 212 patients, 59 (28%) had a diagnosis of malignant disease, 38 (18%) had primary hyperparathyroidism, and 114 (54%) had hypercalcemia due to a range of other causes. The mean total serum calcium concentration for all patients in this study was 11.69 mg/dL. Lung carcinoma was the most prevalent malignant condition (in 17 patients or 29% of those with cancer). A single parathyroid adenoma (in 20 of 22 patients who underwent surgical intervention) accounted for the majority of cases of primary hyperparathyroidism. Among the other identified causes of hypercalcemia, acute renal failure was the most common (in 37 patients or 17% of all patients). In 37 patients, no specific cause for the hypercalcemia was identified. CONCLUSION: To our knowledge, this is the first study to focus on hypercalcemia within the US veterans population. Although the 2 most common causes of hypercalcemia, hyperparathyroidism and malignant disease, were represented in this study, the majority of cases of hypercalcemia in this population of US veterans related to other etiologic factors.

41Ca and accelerator mass spectrometry to monitor calcium metabolism in end stage renal disease patients.

BACKGROUND: Monitoring bone resorption with measurements of bone density and biochemical markers is indirect. We hypothesized that bone resorption can be studied directly by serial measurements of the ratio (41)Ca/Ca in serum after in vivo labeling of calcium pools with (41)Ca. We report the preparation of an intravenous (41)Ca dose suitable for humans, an analytical method for determining (41)Ca/Ca isotope ratios in biological samples, and studies in human volunteers. METHODS: (41)Ca was formulated and aliquoted into individual vials, and to the extent possible, the (41)Ca doses were tested according to US Pharmacopeia (USP) guidelines. A 10 nCi dose of (41)Ca was administered intravenously to 4 end stage renal disease (ESRD) patients on hemodialysis and 4 healthy control individuals. Distribution kinetics were determined over 168 days. Calcium was isolated with 3 precipitation steps and a cation-exchange column, and (41)Ca/Ca ratios in serum were then measured by accelerator mass spectrometry. RESULTS: The dosing solution was chemically and radiologically pure, contained <0.1 endotoxin unit/mL, and passed USP sterility tests. Quantification of (41)Ca/Ca ratios was linear from 6 x 10(-14) to 9.1 x 10(-10). The run-to-run imprecision (as CV) of the method was 4% at 4.6 x 10(-11) and 6% at 9.1 x 10(-10). The area under the curve of (41)Ca in the central compartment vs time was significantly less for ESRD patients than for controls (P < 0.005). CONCLUSIONS: Isotope ratios spanning 5 orders of magnitude can be measured by accelerator mass spectrometry with excellent precision in the range observed in samples collected from patients who have received 10 nCi of (41)Ca. The (41)Ca at this dose caused no adverse effects in 8 volunteers. This is the first report of the use of (41)Ca to monitor differences in bone turnover between healthy individuals and ESRD patients.

Immunoaffinity separation of plasma proteins by IgY microbeads: meeting the needs of proteomic sample preparation and analysis.

Separation of complex protein mixtures that have a wide dynamic range of concentration, such as plasma or serum, is a challenge for proteomic analysis. Sample preparation to remove high-abundant proteins is essential for proteomics analysis. Immunoglobulin yolk (IgY) antibodies have unique and advantageous features that enable specific protein removal to aid in the detection of low-abundant proteins and biomarker discovery. This report describes the efficiency and effectiveness of IgY microbeads in separating 12 abundant proteins from plasma with an immunoaffinity spin column or LC column. The protein separation and sample preparation process was monitored via SDS-PAGE, 2-DE, LC-MS/MS, or clinical protein assays. The data demonstrate the high specificity of the protein separation, with removal of 95-99.5% of the abundant proteins. IgY microbeads against human proteins can also selectively remove orthologous proteins of other mammals such as mouse, rat, etc. Besides the specificity and reproducibility of the IgY microbeads, the report discusses the factors that may cause potential variations in protein separation such as protein-protein interactions (known as "Interactome"), binding and washing conditions of immunoaffinity reagents, etc. A novel concept of Seppromics is introduced to address methodologies and science of protein separation in a context of proteomics.

Mass spectrometric immunoassay for parathyroid hormone-related protein.

This paper describes a novel two-site peptide immunoassay using the isotope 14C as the label and accelerator mass spectrometry as the detection system. A mouse monoclonal antibody (1A5) against the amino terminal region of human parathyroid hormone-related protein (PTHrP) was labeled with 14C by growing the hybridoma cells in a miniPERM bioreactor in the presence of [U-14C]L-leucine and [U-14C]D-glucose. The antibody was purified from the culture media using protein G affinity chromatography. The purified 14C-labeled antibody (14C-1A5) fractions showed excellent correlation between the levels of radioactivity and binding activity for PTHrP. Using 14C-1A5 as the detection antibody in a two-site immunoassay format for PTHrP1-141, a 16-kDa polypeptide, an analytic sensitivity of 10 pmol/L was achieved with a linear measurement range up to 1.3 nmol/L. Only approximately 17 pCi/ well (or 1.6 nCi/96-well microtiter plate) 14C-1A5 was used, which is far below the limit (50 nCi/g) for disposal as nonradioactive waste. This study may serve as a model for the development of sensitive and "nonradioactive" immunoassays for peptides, including polypeptide tumor markers.