Re-evaluation of the low-dose dexamethasone suppression test in dogs.

OBJECTIVES: This study aims to re-evaluate the low-dose dexamethasone suppression test 8-hour cortisol cut-point for the diagnosis of hypercortisolism in dogs using a solid-phase, competitive chemiluminescent enzyme immunoassay. MATERIALS AND METHODS: Twenty-seven client-owned dogs with naturally occurring hypercortisolism and 30 healthy control dogs were prospectively recruited. Performance of the low-dose dexamethasone suppression test was assessed using sensitivity, specificity and a receiver operating characteristic curve compared to a clinical diagnosis of hypercortisolism including response to treatment. RESULTS: Twenty-four dogs were diagnosed with pituitary-dependent hypercortisolism and three with adrenal-dependent hypercortisolism. In 30 healthy control dogs, 8-hour post-dexamethasone cortisol concentrations ranged from 5.5 to 39 nmol/L. A receiver operating characteristic curve curve constructed from the 8-hour post-dexamethasone cortisol concentrations of hypercortisolism and control dogs demonstrated that the most discriminatory cut-point was more than 39 nmol/L with sensitivity of 85.2% (95% confidence interval, 67.5% to 94.1%) and specificity of 100% (95% confidence interval, 88.7% to 100.0%) and an area under the curve of 0.963. CLINICAL SIGNIFICANCE: The optimal cut-point of more than 36 nmol/L proposed by this study is similar to the currently accepted 8-hour cortisol concentration cut-point for diagnosing hypercortisolism when using a solid-phase, competitive chemiluminescent enzyme immunoassay.

Clinical findings, diagnostic test results, and treatment outcome in cats with spontaneous hyperadrenocorticism: 30 cases.

BACKGROUND: Spontaneous hyperadrenocorticism (HAC) is rare in cats. Clinical findings, diagnostic test results, and response to various treatment options must be better characterized. OBJECTIVES: To report the clinical presentation, clinicopathologic findings, diagnostic imaging results, and response to treatment of cats with HAC. ANIMALS: Cats with spontaneous HAC. METHODS: Retrospective descriptive case series. RESULTS: Thirty cats (15 neutered males, 15 spayed females; age, 4.0-17.6 years [median, 13.0 years]) were identified from 10 veterinary referral institutions. The most common reason for referral was unregulated diabetes mellitus; dermatologic abnormalities were the most frequent physical examination finding. Low-dose dexamethasone suppression test results were consistent with HAC in 27 of 28 cats (96%), whereas ACTH stimulation testing was suggestive of HAC in only 9 of 16 cats (56%). Ultrasonographic appearance of the adrenal glands was consistent with the final clinical diagnosis of PDH or ADH in 28 of 30 cats (93%). Of the 17 cats available for follow-up at least 1 month beyond initial diagnosis of HAC, improved quality of life was reported most commonly in cats with PDH treated with trilostane. CONCLUSIONS AND CLINICAL IMPORTANCE: Dermatologic abnormalities or unregulated diabetes mellitus are the most likely reasons for initial referral of cats with HAC. The dexamethasone suppression test is recommended over ACTH stimulation for initial screening of cats with suspected HAC. Diagnostic imaging of the adrenal glands may allow rapid and accurate differentiation of PDH from ADH in cats with confirmed disease, but additional prospective studies are needed.

Characterization of thrombelastography over time in dogs with hyperadrenocorticism.

Canine hyperadrenocorticism (HAC) is a common endocrinopathy often associated with hypercoagulability, thrombosis and thromboembolism and it can contribute to increased morbidity and mortality. The condition results in increased, unregulated secretion of glucocorticoids (GCs). While prospective identification of hypercoagulability is challenging, thrombelastography (TEG) is a diagnostic tool that enables the detection of hypercoagulability in a clinical setting. The objective of this prospective cohort study was to serially assess coagulation in dogs with HAC using TEG to test the hypothesis that dogs with HAC have increased TEG maximal amplitude (MA) and that the MA would normalize once clinical control was achieved. Twenty-three dogs with naturally occurring HAC were enrolled and hemostatic (including TEG, platelet function, thrombin-antithrombin complexes and coagulation panel) and hematological variables were measured at presentation. TEG was serially monitored until clinical resolution of HAC was attained. At presentation, most dogs with HAC had increased MA values, increased thrombin-antithrombin complexes and many were hyperfibrinogenemic. Platelet function analyzer-100 (PFA-100) closure times were significantly prolonged. TEG tracings did not normalize in either medically- or surgically-managed dogs, but fibrinogen concentrations decreased. It seems that dogs with HAC have a complex coagulopathy in which hypercoagulability and platelet hyporeactivity or dysfunction might occur simultaneously. As TEG tracings did not normalize in well-controlled dogs, it is unlikely that increased blood GCs are solely responsible for TEG alterations seen in dogs with HAC.

Serum inhibin concentration in dogs with adrenal gland disease and in healthy dogs.

BACKGROUND: Studies in humans identified the synthesis and secretion of inhibin from adrenocortical tumors, but not pheochromocytoma (PHEO). Inhibin has not been examined in dogs as a serum biomarker for adrenal gland tumors. OBJECTIVE: To determine serum inhibin concentration in dogs with adrenal gland disease and in healthy dogs. ANIMALS: Forty-eight neutered dogs with adrenal disease including pituitary-dependent hyperadrenocorticism (PDH, 17), adrenocortical tumor (18), and PHEO (13), and 41 healthy intact or neutered dogs. METHODS: Prospective observational study. Dogs were diagnosed with PDH, adrenocortical tumor (hyperadrenocorticism or noncortisol secreting), or PHEO based on clinical signs, endocrine function tests, abdominal ultrasound examination, and histopathology. Inhibin concentration was measured by radioimmunoassay in serum before and after ACTH stimulation, and before and after treatment. RESULTS: In neutered dogs, median inhibin concentration was significantly higher in dogs with adrenocortical tumors (0.82 ng/mL) and PDH (0.16 ng/mL) than in dogs with PHEO and healthy dogs (both undetectable). Median inhibin concentration was significantly higher in dogs with adrenocortical tumors than in those with PDH and decreased after adrenalectomy. Median inhibin concentration was significantly higher in intact than in neutered healthy dogs and was similar in pre- and post-ACTH stimulation. Sensitivity, specificity, and accuracy of serum inhibin concentration for identifying an adrenal tumor as a PHEO were 100, 88.9, and 93.6%, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: Adrenocortical tumors and PDH but not PHEOs are associated with increased serum inhibin concentration; undetectable inhibin is highly supportive of PHEO in neutered dogs with adrenal tumors.

Changes in characteristics of inmates with latent tuberculosis infection.

OBJECTIVES: Health and social characteristics place prisoners at high risk for progression from latent tuberculosis infection (LTBI) to tuberculosis (TB), but completion of LTBI therapy is low with many patients lost to follow-up after release. Despite decreases in active TB, demographic characteristics of active cases have remained relatively unchanged. This study investigated whether characteristics have changed in inmates diagnosed with LTBI in San Francisco, CA, USA. STUDY DESIGN: Cross-sectional. METHODS: Data from baseline interviews of randomized trials conducted in 1998-1999 and 2004-2007 were compared. RESULTS: In both time periods, most subjects with LTBI (>60%) were Latinos, while the proportion in both the jail and San Francisco remained at 15-20%. Overall, the prisoners interviewed in 2004-2007 were less likely to have been on medication for LTBI previously, and expressed more likelihood of finishing their medication compared with those interviewed in 1998-1999. In 2004-2007, the foreign-born subjects were more likely to prefer English to Spanish, to have been in stable housing and to have been employed before jail compared with 1998-1999, while no such changes were seen between the two time periods for US-born subjects. CONCLUSIONS: The pool of TB-infected individuals coming from a jail is not static, and understanding the changes over time is of importance for targeted programmes. Given the high infection rate and the predominance of foreign-born individuals who may have received bacillus Calmette-Guérin vaccination, screening with interferon-gamma release assay may be beneficial to identify those with true infection.

Efficacy of protamine zinc recombinant human insulin for controlling hyperglycemia in dogs with diabetes mellitus.

BACKGROUND: Alternative insulin preparations are needed when NPH insulin is ineffective in diabetic dogs. This study evaluated the efficacy of recombinant human protamine zinc insulin (rhPZI) for treating diabetic dogs. HYPOTHESIS: rhPZI is effective for treating diabetic dogs. ANIMALS: Six newly diagnosed and 11 insulin-treated diabetic dogs. METHODS: Prospective clinical trial. Dogs were treated with rhPZI for 60 days. Control of glycemia was assessed on days 7, 14, 30, and 60 by evaluation of history, physical examination, body weight, serum fructosamine concentration, and blood glucose concentrations measured before and 2, 4, 6, 8, and 10 hours after rhPZI administration. Adjustments in dosage of rhPZI were made as needed to control glycemia. RESULTS: rhPZI administration resulted in a significant decrease in 10-hour mean blood glucose (MBG(10h) ; 299 ± 115 versus 457 ± 38 mg/dL, X ± SD, P = .0003) and serum fructosamine (478 ± 83 versus 557 ± 104 µmol/L, P = .006) concentration at day 60, compared with day 1, respectively. By day 60, polyuria and polydipsia had improved in 14, body weight was stable or increased in 16, MBG(10h) had decreased in 16, and serum fructosamine concentration had decreased in 11 of 17 dogs, compared with day 1. Hypoglycemia (<80 mg/dL) was the only consistent adverse event. CONCLUSIONS AND CLINICAL RELEVANCE: rhPZI is effective in diabetic dogs and can be considered as an alternative treatment in diabetic dogs that are poorly controlled using other insulin preparations.

Parental dietary effect on embryological development response to toxicants with the sea urchin Arbacia punctulata.

The role of echinoid parental nutrition in early-life stage toxicity is not well understood. Arbacia punctulata were fed either a fresh diet consisting of organic lettuce and carrots or a dry feed. Embryos from parents fed the dry feed exhibited lower sensitivity to copper, whereas the opposite occurred with 1,3,5-trinitrobenzene and sodium dodecyl sulfate (SDS). EC(50) values for the dry and fresh feed treatments, respectively, were 41.0 and 29.9 microg/L for copper, 0.5 and 1.8 mg/L for 1,3,5-trinitrobenzene, and 3.5 and 5.6 mg/L for SDS. The data suggests that nutritional standardization for sea urchins in ecotoxicological laboratories needs to be addressed and further investigated.

Field safety and efficacy of protamine zinc recombinant human insulin for treatment of diabetes mellitus in cats.

BACKGROUND: This study describes the efficacy of a new protamine zinc recombinant human insulin (PZIR) preparation for treating diabetic cats. OBJECTIVE: To evaluate effects of PZIR on control of glycemia in cats with newly diagnosed or poorly controlled diabetes mellitus. ANIMALS: One hundred and thirty-three diabetic cats 120 newly diagnosed and 13 previously treated. METHODS: Prospective, uncontrolled clinical trial. Cats were treated with PZIR twice daily for 45 days. Control of glycemia was assessed on days 7, 14, 30, and 45 by evaluation of change in water consumption, frequency of urination, appetite, and body weight, serum fructosamine concentration, and blood glucose concentrations determined 1, 3, 5, 7, and 9 hours after administration of PZIR. Adjustments in dosage of PZIR were made as needed to control glycemia. RESULTS: PZIR administration resulted in a significant decrease in 9-hour mean blood glucose (199+/-114 versus 417+/-83 mg/dL, X+/-SD, P<.001) and serum fructosamine (375+/-117 versus 505+/-96 micromol/L, P<.001) concentration and a significant increase in mean body weight (5.9+/-1.4 versus 5.4+/-1.5 kg, P=.017) in 133 diabetic cats at day 45 compared with day 0, respectively. By day 45, polyuria and polydipsia had improved in 79% (105 of 133), 89% (118 of 133) had a good body condition, and 9-hour mean blood glucose concentration, serum fructosamine concentration, or both had improved in 84% (112 of 133) of the cats compared with day 0. Hypoglycemia (<80 mg/dL) was identified in 151 of 678, 9-hour serial blood glucose determinations and in 85 of 133 diabetic cats. Hypoglycemia causing clinical signs was confirmed in 2 diabetic cats. CONCLUSIONS AND CLINICAL RELEVANCE: PZIR is effective for controlling glycemia in diabetic cats and can be used as an initial treatment or as an alternative treatment in diabetic cats that do not respond to treatment with other insulin preparations.

Predictive factors and the effect of phenoxybenzamine on outcome in dogs undergoing adrenalectomy for pheochromocytoma.

BACKGROUND: Some studies in dogs undergoing adrenalectomy for pheochromocytoma suggest that anesthetic complications and perioperative mortality are common. In humans, surgical outcome has improved with the use of phenoxybenzamine (PBZ) before adrenalectomy. HYPOTHESIS: Dogs treated with PBZ before adrenalectomy have increased survival compared with untreated dogs. ANIMALS: Forty-eight dogs that underwent adrenalectomy for pheochromocytoma. METHODS: A retrospective medical record review for dogs that underwent adrenalectomy for pheochromocytoma at a veterinary medical teaching hospital over the period from January 1986 through December 2005. RESULTS: Twenty-three of 48 dogs were pretreated with PBZ (median dosage: 0.6 mg/kg PO q12h) for a median duration of 20 days before adrenalectomy. Duration of anesthesia and surgery, percentage of dogs with pheochromocytoma involving the right versus left adrenal gland, size of tumor, and presence of vascular invasion were similar for PBZ-treated and untreated dogs. Thirty-three (69%) of 48 dogs survived adrenalectomy in the perioperative period. PBZ-treated dogs had a significantly (P = .014) decreased mortality rate compared with untreated dogs (13 versus 48%, respectively). Additional significant prognostic factors for improved survival included younger age (P = .028), lack of intraoperative arrhythmias (P = .0075), and decreased surgical time (P = .0089). CONCLUSIONS AND CLINICAL IMPORTANCE: Results from this retrospective study support treatment with PBZ before surgical removal of pheochromocytoma in dogs.

Frequency and risk factors for urinary tract infection in cats with diabetes mellitus.

BACKGROUND: Identification and control of infections are important in the management of diabetic cats. Urinary tract infections have not been well characterized in diabetic cats. This retrospective study was performed to review and characterize urinary tract infections in diabetic cats. HYPOTHESIS: Urinary tract infections are common in diabetic cats. ANIMALS: A review was made of the medical records of 141 diabetic cats that had had urine obtained for culture by antepubic cystocentesis and that had not been treated with antibiotics, undergone urinary tract catheterization or urinary tract surgery within 2 weeks of urine collection or had urethral obstruction at the time of urine collection. METHODS: A review of medical records. RESULTS: Urinary tract infection was identified in 18 of 141 diabetic cats. Escherichia coli was the most common isolate (67%). Female cats were at increased risk (prevalence odds ratios [POR], 3.7; 95% confidence interval [CI], 1.3 to 10.2; P = .013). Clinical signs of lower urinary tract disease and findings on urine sediment examination were good predictors of positive urine cultures. CONCLUSIONS AND CLINICAL IMPORTANCE: Urinary tract infections are common in diabetic cats regardless of status of diabetic control, suggesting routine monitoring with urine sediment exams or urine culture is warranted.

Measurements of growth hormone and insulin-like growth factor 1 in cats with diabetes mellitus.

Serum concentrations of insulin-like growth factor 1 (IGF-1) and growth hormone were measured in 25 cats with untreated diabetes mellitus (11 of which were used for follow-up measurements, one to three, four to eight, nine to 12 and 13 to 16 weeks after their treatment with insulin began), 14 diabetic cats that had previously been treated with insulin, and seven diabetic cats that also had hypersomatotropism, two of which had not previously been treated with insulin; 18 healthy cats were used as controls. In the untreated diabetic cats the concentration of IGF-1 ranged from 13.0 to 433.0 ng/ml (median 170.5 ng/ml), which was significantly lower than the concentrations in the control cats (196.0 to 791.0 ng/ml, median 452.0 ng/ml). Their IGF-1 concentrations increased significantly when they were treated with insulin and after four to eight weeks were not different from those in the control cats. In the diabetic cats that had previously been treated with insulin the IGF-1 concentrations were 33.0 to 476.0 ng/ml (median 316.0 ng/ml), which was significantly lower than the concentrations in the control cats, but significantly higher than in the untreated diabetic cats. The IGF-1 concentrations in the two previously untreated diabetic cats with hypersomatotropism were low and low-normal but increased markedly after treatment with insulin. In the five previously treated cats with hypersomatotropism the concentration of IGF-1 was above the normal range. The concentrations of growth hormone in the treated and untreated diabetic cats without hypersomatotropisms were not significantly different and there was an overlap in its concentrations in the diabetic cats with and without hypersomatotropism.

Analysis of native proteins from biological fluids by biomolecular interaction analysis mass spectrometry (BIA/MS): exploring the limit of detection, identification of non-specific binding and detection of multi-protein complexes.

Biomolecular interaction analysis mass spectrometry (BIA/MS) is a two-dimensional analytical technique that quantitatively and qualitatively detects analytes of interests. In the first dimension, surface plasmon resonance (SPR) is utilized for detection of biomolecules in their native environment. Because SPR detection is non-destructive, analyte(s) retained on the SPR-active sensor surface can be analyzed in a second dimension using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The qualitative nature of the MALDI-TOF MS analysis complements the quantitative character of SPR sensing and overcomes the shortcomings of the SPR detection stemming from the inability to differentiate and characterize multi-protein complexes and non-specific binding. In this work, the benefit of performing MS analysis following SPR sensing is established. Retrieval and detection of four markers present in biological fluids (cystatin C, beta-2-microglobulin, urinary protein 1 and retinol binding protein) was explored to demonstrate the effectiveness of BIA/MS in simultaneous detection of clinically related biomarkers and delineation of non-specific binding. Furthermore, the BIA/MS limit of detection at very low SPR responses was investigated. Finally, detection of in-vivo assembled protein complexes was achieved for the first time using BIA/MS.

Analysis of human urine protein biomarkers via biomolecular interaction analysis mass spectrometry.

Biomolecular interaction analysis mass spectrometry (BIA/MS) is a two-dimensional chip-based analytical technique geared toward quantitative and qualitative analysis of small volumes of biological samples. Interactions between surface-immobilized ligands and solute-borne analytes are quantitatively viewed in real time through surface plasmon resonance sensing, followed by qualitative matrix-assisted laser desorption/ionization time-of-flight MS analysis of the analyte(s) affinity-retained on the sensor surface. In this work, BIA/MS was used in the detection of a number of protein biomarkers from human urine. Small volumes of human urine were analyzed for cystatin C, beta(2)-microglobulin, urinary protein 1, and retinol-binding protein (RBP). Multiaffinity sensor surfaces were created to simultaneously and rapidly detect all four proteins in a single BIA/MS analysis on a two-flow cell sensor chip configuration. Furthermore, RBP was analyzed separately from both urine and plasma samples. Results indicate that BIA/MS can be used successfully in rapid screening of a number of urinary proteins indicated as putative biological markers for renal dysfunction.

Determination of beta-2 microglobulin levels in plasma using a high-throughput mass spectrometric immunoassay system.

A high-throughput mass spectrometric immunoassay (MSIA) system for the analysis of proteins directly from biological fluids is reported. A 96-well-format robotic workstation equipped with antibody-derivatized affinity pipet tips was used for the parallel extraction of specific proteins from samples and subsequent deposition onto 96-well arrayed matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) targets. Interferences from nonspecifically bound proteins were minimized through choice of appropriate affinity pipet tip derivatization chemistries. Sample preparation for MALDI-TOFMS was enhanced through the use of hydrophobic/hydrophilic contrasting targets, which also presented functionalities found to promote matrix/analyte crystal growth. Automated mass spectrometry was used in the unattended acquisition of data, resulting in an analysis rate of approximately 100 samples/h (biological fluid-->data). The quantitative MSIA of beta2m levels present in human plasma samples is given as illustration.

Oral chromium picolinate and control of glycemia in insulin-treated diabetic dogs.

Chromium is an essential dietary trace mineral involved in carbohydrate and lipid metabolism. Chromium is required for cellular uptake of glucose, and chromium deficiency causes insulin resistance. Chromium supplementation may improve insulin sensitivity and has been used as adjunct treatment of diabetes mellitus in humans. In this study, 13 dogs with naturally acquired diabetes mellitus were treated with insulin for 3 months, then with insulin and chromium picolinate for 3 months. Dogs weighing <15 kg (33 lb: n = 9) were administered 200 microg of chromium picolinate PO once daily for I month, then 200 microg of chromium picolinate twice daily for 2 months. Dogs weighing >15 kg (n = 4) received 200 microg of chromium picolinate once daily for 2 weeks, then 200 microg twice daily for 2 weeks, then 400 microg twice daily for 2 months. Type of insulin, frequency of insulin administration, and diet were kept constant, and insulin dosage was adjusted, as needed, to maintain optimal control of glycemia. Mean body weight, daily insulin dosage, daily caloric intake, 10-hour mean blood glucose concentration, blood glycated hemoglobin concentration, and serum fructosamine concentration were not markedly different when dogs were treated with insulin and chromium picolinate, compared with insulin alone. Adverse effects were not identified with chromium picolinate administration. Results of this study suggest that, at a dosage range of 20-60 microg/kg/d, chromium picolinate caused no beneficial or harmful effects in insulin-treated diabetic dogs.

Use of percutaneous ethanol injection for treatment of bilateral hyperplastic thyroid nodules in cats.

OBJECTIVE: To determine the efficacy and safety of percutaneous ethanol injection (PEI) for the treatment of hyperthyroidism caused by bilateral hyperplastic thyroid nodules in cats. DESIGN; Prospective study. ANIMALS: 7 cats. PROCEDURE: Hyperthyroidism was diagnosed on the basis of clinical signs and increased serum total thyroxine (TT4) concentrations. The presence of 2 cervical thyroid nodules was confirmed by use of ultrasonography and technetium Tc 99m albumin thyroid scans. After the death of 1 cat that received PEI in both thyroid nodules at the same time, the protocol was changed to injecting ethanol into 1 nodule at a time, with at least 1 month between injections. Clinical signs, serum TT4 concentrations, serum ionized calcium concentrations, laryngeal function, findings on ultrasonographic examinations of the ventral cervical region, and results of thyroid scans were monitored. RESULTS: Serum TT4 concentrations transiently decreased in all 6 cats (into the reference range in 5 of 6 cats) within 4 days of the first staged ethanol injection. Each subsequent injection resulted in a transient decrease in serum TT4 concentration. The longest period of euthyroidism was 27 weeks. Adverse effects included Horner's syndrome, dysphonia, and laryngeal paralysis. One cat died of unrelated causes. One cat underwent bilateral thyroidectomy, 2 cats were treated with methimazole, and 2 cats that had increased serum TT4 concentrations were not treated further, because they remained clinically normal. CONCLUSIONS AND CLINICAL RELEVANCE: Percutaneous ethanol ablation of bilateral thyroid nodules as a treatment for cats with hyperthyroidism is not recommended. This treatment is not as efficacious as the medical and surgical treatments presently used.

Percutaneous ultrasonographically guided radiofrequency heat ablation for treatment of primary hyperparathyroidism in dogs.

OBJECTIVE: To evaluate the efficacy and safety of ultrasonographically guided radiofrequency heat ablation of parathyroid masses in dogs with primary hyperparathyroidism. DESIGN: Clinical trial. ANIMALS: 11 dogs. PROCEDURE: In all dogs, either 1 or 2 parathyroid masses were evident ultrasonographically. Dogs were anesthetized, and a 20-gauge over-the-needle catheter was directed into the parathyroid mass via ultrasonographic guidance. Radiofrequency heat was applied to the stylet of the catheter until there was sonographically apparent change to the entire parenchyma of the mass. Serum total and ionized calcium and parathyroid hormone concentrations were monitored daily for 5 days after the ablation procedure and again at 1, 2, and 3-month intervals, if possible. Dogs were monitored for adverse effects. RESULTS: One treatment was required in 6 dogs, 2 treatments were required in 2 dogs, and treatment was unsuccessful in 3 dogs. Serum total and ionized calcium concentrations were within reference ranges within 2 days of the last procedure in all 8 successfully treated dogs. Serum parathyroid hormone concentration was decreased 24 hours after treatment in all 8 dogs. Hypocalcemia developed in 5 of the 8 successfully treated dogs, all of which required treatment. One dog had a transient voice change. Other adverse effects were not reported. CONCLUSIONS AND CLINICAL RELEVANCE: Ultrasonographically guided radiofrequency heat ablation of parathyroid masses is a safe and effective alternative to surgery in dogs with primary hyperparathyroidism.

Detection and quantification of beta-2-microglobulin using mass spectrometric immunoassay.

The use of mass spectrometric immunoassay (MSIA) in analyzing beta-2-microglobulin (beta(2)m) present in human biological fluids (tears, saliva, plasma, and urine) is described. Pipettor tips containing porous affinity frits, derivatized with polyclonal anti-beta(2)m immunoglobulin, were manufactured and used to selectively isolate and concentrate beta(2)m from the biofluids, after which matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to detect beta(2)m unambiguously at its characteristic molecular mass. The affinity tips were found rapid to use, requiring approximately 15 min per analysis, and exhibited low nonspecific binding properties that yielded essentially interference-free analyses. The beta(2)m MSIA was made quantitative by inclusion of an internal standard into the analysis for signal normalization. The resulting assay had a Linear dynamic range (R(2) = 0.983) covering a beta(2)m concentration range of 0.010-1.0 mg/L with a standard error of approximately 5%. In application, urine samples from healthy individuals were screened and compared with sample from an individual suffering from renal infection. Results indicated an approximately 30-fold increase in beta(2)m levels in samples taken from the infected individual. During the screening, MSIA was able to distinguish between wild-type and glycosylated forms of beta(2)m, which made possible the accurate quantification of wild-type beta(2)m without interference from glycosylated versions of the protein. These results demonstrate a new approach to the rapid and accurate detection/quantification of beta(2)m present in biological fluids.