Capillary Zone Electrophoresis-Top-Down Tandem Mass Spectrometry for In-Depth Characterization of Hemoglobin Proteoforms in Clinical and Veterinary Samples.

Hemoglobin (Hb) constitutes an important protein in clinical diagnostics-both in humans and animals. Among the high number of sequence variants, some can cause severe diseases. Moreover, chemical modifications such as glycation and carbamylation serve as important biomarkers for conditions such as diabetes and kidney diseases. In clinical routine analysis of glycated Hb, sequence variants or other Hb proteoforms can cause interference, resulting in wrong quantification results. We present a versatile and flexible capillary zone electrophoresis-mass spectrometry screening method for Hb proteoforms including sequence variants and modified species extracted from dried blood spot (DBS) samples with virtually no sample preparation. High separation power was achieved by application of a 5-layers successive multiple ionic polymer layers-coated capillary, enabling separation of positional isomers of glycated α- and ß-chains on the intact level. Quantification of glycated Hb was in good correlation with the results obtained in a clinical routine method. Identification and characterization of known and unknown proteoforms was performed by fragmentation of intact precursor ions. N-Terminal and lysine glycation could be identified on the α- and ß-chain, respectively. The versatility of the method was demonstrated by application to dog and cat DBS samples. We discovered a putative new sequence variant of the ß-chain in dog (T38 → A). The presented method enables separation, characterization, and quantification of intact proteoforms, including positional isomers of glycated species in a single run. Combined with the simple sample preparation, our method represents a valuable tool to be used for deeper characterization of clinical and veterinary samples.

Development of a capillary electrophoretic method for determination of plasma clearance of iohexol in dogs and cats.

Renal function can be monitored by estimation of the glomerular filtration rate (GFR), for example, through measurement of the plasma clearance of a marker that is freely filtrated through the kidney without reabsorption. It has been proposed that iohexol is the most accurate marker for GFR determination in cats and dogs. However, there is a need for a validated capillary electrophoretic method that covers the concentration range for a full curve clearance estimate of iohexol. In the final method, the plasma samples were protein precipitated and the supernatant was analyzed in a background electrolyte containing borate buffer (0.06 m, pH 10.0). The method developed was proved to be linear (concentration range 18- 2900 mg/L) and had a good precision (e.g. 2.3-2.9% at 88 mg/L) and accuracy (e.g. 101-105% at 88 mg/L). Finally, the method was compared with a previously published and validated HPLC-UV method by parallel analysis of clinical plasma samples from dogs and cats administered Omnipaque®. This comparison showed excellent agreement between the two methods and no proportional or systematic error was observed. The proposed method is simple and has a low cost per sample, which makes it applicable for routine analysis.

Evaluation of non-polynomial equations for one-compartment correction of slope-intercept GFR: theoretical prediction and experimental measurement.

BACKGROUND: Polynomial equations for one-compartment correction of slope-intercept glomerular filtration rate (GFR) will underestimate values at high clearance rates. Non-polynomial correction equations that are independent of patient size and renal function would be advantageous and may have cross-species use. MATERIALS AND METHODS: The study explored the theoretical basis of firstly the Jodal and Brochner-Mortensen one-compartment correction equation, replacing plasma volume with extracellular fluid volume, and secondly an equation described by Peters. One-compartment correction factors (a which is related to plasma volume and v which is related to extracellular fluid volume) which avoided the need for scaling to body size were developed. Both factors were determined from the biexponential clearance curve of the markers iohexol and (51)Cr-EDTA in humans and iohexol in cats and dogs. Relationships between a and v and filtration function and body size were then determined using data from humans, cats and dogs to assess their validity and compare this with theoretical predictions. RESULTS: In all species, v was higher than a, as theoretically predicted. Both were significantly higher in humans than cats and dogs, ruling out cross-species use. Significant relationships were present between v and measures of filtration function in humans, but were weak with respect to a. Neither a nor v showed significant relationships with filtration function in animals or with body size in any species. CONCLUSIONS: a and v (which are factors independent of body size) can be used interchangeably for correcting slope-intercept clearance. However values of both for humans are higher compared to cats and dogs. Therefore a single cross-species factor cannot be used.

Glomerular filtration rate in dogs as estimated via plasma clearance of inulin and iohexol and use of limited-sample methods.

OBJECTIVE: To compare plasma clearance of inulin and iohexol determined by use of 9 plasma samples for evaluation of glomerular filtration rate in dogs and to evaluate limited-sample approaches for evaluation of plasma clearance of these markers. ANIMALS: 43 dogs of various breeds that weighed between 5.5 and 63 kg and that had various degrees of renal function. PROCEDURES: 9 plasma samples were obtained from each dog at 5 minutes to 6 hours after IV bolus injection of iohexol and inulin. Clearance was calculated by use of results for all 9 samples (ie, reference method). Results for 3 limited-sample strategies for determination of plasma clearance of iohexol and inulin were compared with results for the reference method. RESULTS: Mean clearance of inulin and iohexol for the reference method was 2.72 and 2.48 mL/min/kg, respectively. The mean difference between clearance of these 2 markers for the reference method was 0.24 mL/min/kg. In general, use of the limited-sample strategies yielded clearance values similar to those for the reference method. More accurate estimates of clearance were obtained for iohexol than for inulin by use of the limited-sample methods. CONCLUSIONS AND CLINICAL RELEVANCE: Use of iohexol and inulin yielded similar but not identical results for plasma clearance. Accuracy for limited-sample methods would be acceptable for many clinical and research situations.

Chronic kidney disease with three cases of oxalate-like nephrosis in Ragdoll cats.

Two unrelated Ragdoll cat mothers in Norway were found dead from renal disease. The histopathology was consistent with oxalate nephrosis with chronic or acute-on-chronic underlying kidney disease. Both cats had offspring and relatives with signs of urinary tract disease, including a kitten dead with urethral gravel. Eleven living Ragdoll cats, including nine relatives of the dead cats and the male father of a litter with similarly affected animals, were tested for primary hyperoxaluria (PH) type 1 and 2 by urine oxalate and liver enzyme analysis. Renal ultrasound revealed abnormalities in five living cats. One of these was azotaemic at the time of examination and developed terminal kidney disease 9 months later. A diagnosis of PH was excluded in 11 cats tested. The inheritance and aetiological background of the renal disease present in the breed remains unresolved at this point in time.

Estimation of glomerular filtration rate via 2- and 4-sample plasma clearance of iohexol and creatinine in clinically normal cats.

OBJECTIVE: To compare 2 methods for estimation of glomerular filtration rate (GFR), study the effects of age and body size on GFR estimates, and provide a reference range for estimated GFR in clinically normal cats. ANIMALS: 57 cats. PROCEDURES: In each cat, GFR was estimated via plasma clearance of iohexol and creatinine. Results of a 1-compartmental model (CL1comp) were calibrated to a trapezoidal method estimate (CLtrap) by use of a correction formula applicable to dogs or humans and standardized to body weight; for iohexol clearance, data were also standardized to extracellular fluid volume (ECFV). For all 57 cats, method comparison was performed via agreement analysis. Reference ranges for GFR derived by the different methods were established by use of data from a subset of 51 cats after exclusion of 6 cats that were azotemic, Birman, or both. RESULTS: In 57 cats, mean CLtrap of creatinine was 0.29 mL/min/kg (13%) higher than CLtrap of iohexol. In 51 nonazotemic cats, mean CLtrap was 2.26 mL/min/kg for iohexol (reference range, 1.02 to 3.50 mL/min/kg) and 2.55 mL/min/kg for creatinine (reference range, 1.27 to 3.83 mL/min/kg). Values of GFR/kg or GFR standardized to liters of ECFV did not decrease with increasing age. A negative linear relationship was detected between body weight and estimated GFR/kg or GFR standardized to liters of ECFV. CONCLUSIONS AND CLINICAL RELEVANCE: Reference ranges for estimated GFR via plasma clearance of iohexol and creatinine should facilitate early detection of impaired renal function in cats, although body weight should be taken into account.

Comparison of the diagnostic value of symmetric dimethylarginine, cystatin C, and creatinine for detection of decreased glomerular filtration rate in dogs.

BACKGROUND: Early detection of decreased glomerular filtration rate (GFR) in dogs is challenging. Current methods are insensitive and new biomarkers are required. OBJECTIVE: To compare overall diagnostic performance of serum symmetric dimethylarginine (SDMA) and serum cystatin C to serum creatinine, for detection of decreased GFR in clinically stable dogs, with or without chronic kidney disease (CKD). ANIMALS: Ninety-seven client-owned dogs: 67 dogs with a diagnosis or suspicion of CKD and 30 healthy dogs were prospectively included. METHODS: Prospective diagnostic accuracy study. All dogs underwent physical examination, systemic arterial blood pressure measurement, urinalysis, hematology and blood biochemistry analysis, cardiac and urinary ultrasound examinations, and scintigraphy for estimation of glomerular filtration rate (mGFR). Frozen serum was used for batch analysis of SDMA and cystatin C. RESULTS: The area under the curve of creatinine, SDMA, and cystatin C for detection of an mGFR <30.8 mL/min/L was 0.98 (95% confidence interval [CI], 0.93-1.0), 0.96 (95% CI, 0.91-0.99), and 0.87 (95% CI, 0.79-0.93), respectively. The sensitivity of both creatinine and SDMA at their prespecified cutoffs (115 µmol/L [1.3 mg/dL] and 14 µg/dL) for detection of an abnormal mGFR was 90%. The specificity was 90% for creatinine and 87% for SDMA. When adjusting the cutoff for cystatin C to correspond to a diagnostic sensitivity of 90% (0.49 mg/L), specificity was lower (72%) than that of creatinine and SDMA. CONCLUSIONS AND CLINICAL IMPORTANCE: Overall diagnostic performance of creatinine and SDMA for detection of decreased mGFR was similar. Overall diagnostic performance of cystatin C was inferior to both creatinine and SDMA.

Vasopressin secretion in response to osmotic stimulation and effects of desmopressin on urinary concentrating capacity in dogs with pyometra.

OBJECTIVE: To determine vasopressin (VP) secretory capacity during osmotic stimulation and the response to desmopressin treatment in dogs with pyometra and control dogs. ANIMALS: 6 dogs with pyometra before and after ovariohysterectomy and 6 control dogs. PROCEDURE: Urine osmolality (Uosm) was measured during 12 hours. Values measured on the first day defined the basal Uosm pattern. On the second day, dogs were given desmopressin to induce a desmopressin-stimulated Uosm pattern. On day 3, the VP response to osmotic stimulation was examined. RESULTS: Median Uosm on day 1 was 340 mOsm/kg (range, 104 to 1,273 mOsm/kg) and 807 mOsm/kg (range, 362 to 1,688 mOsm/kg) in dogs with pyometra before and after surgery, respectively, and 1,511 mOsm/kg (range, 830 to 1,674 mOsm/kg) in control dogs. Median Uosm during desmopressin treatment was 431 mOsm/kg (range, 168 to 1,491 mOsm/kg) and 1,051 mOsm/kg (range, 489 to 1,051 mOsm/kg) in dogs with pyometra before and after surgery, respectively, and 1,563 mOsm/kg (range, 1,390 to 2,351) in control dogs. In dogs with pyometra, threshold for VP secretion was lower before surgery (median, 340 mOsm/kg; range, 331 to 366 mOsm/kg) than after surgery (median, 358 mOsm/kg; range, 343 to 439 mOsm/kg) or in control dogs (median, 347 mOsm/kg; range, 334 to 360 mOsm/kg). Highest maximum plasma VP values were found in dogs with pyometra. CONCLUSIONS AND CLINICAL RELEVANCE: Dogs with pyometra had increased urine concentration in response to desmopressin but not to the degree of control dogs, whereas VP secretory ability was not reduced.

Comparison of pharmacokinetic variables for creatinine and iohexol in dogs with various degrees of renal function.

OBJECTIVE: To compare pharmacokinetics and clearances of creatinine and iohexol as estimates of glomerular filtration rate (GFR) in dogs with various degrees of renal function. ANIMALS: 50 Great Anglo-Francais Tricolor Hounds with various degrees of renal function. PROCEDURES: Boluses of iohexol (40 mg/kg) and creatinine (647 mg/kg) were injected IV. Blood samples were collected before administration and 5 and 10 minutes and 1, 2, 4, 6, and 8 hours after administration. Plasma creatinine and iohexol concentrations were assayed via an enzymatic method and high-performance liquid chromatography, respectively. A noncompartmental approach was used for pharmacokinetic analysis. Pharmacokinetic variables were compared via a Bland-Altman plot and an ANOVA. RESULTS: Compared with results for creatinine, iohexol had a significantly higher mean ± SD plasma clearance (3.4 ± 0.8 mL/min/kg vs 3.0 ± 0.7 mL/min/kg) and a significantly lower mean volume of distribution at steady state (250 ± 37 mL/kg vs 539 ± 73 mL/kg), mean residence time (80 ± 31 minutes vs 195 ± 73 minutes), and mean elimination half-life (74 ± 20 minutes vs 173 ± 53 minutes). Despite discrepancies between clearances, especially for high values, the difference was < 0.6 mL/min/kg for 34 (68%) dogs. Three dogs with a low GFR (< 2 mL/min/kg) were classified similarly by both methods. CONCLUSIONS AND CLINICAL RELEVANCE: Plasma iohexol and creatinine clearances can be used interchangeably for screening patients suspected of having chronic kidney disease (ie, low GFR), but large differences may exist for dogs with a GFR within or above the reference range.

Plasma creatinine in dogs: intra- and inter-laboratory variation in 10 European veterinary laboratories.

BACKGROUND: There is substantial variation in reported reference intervals for canine plasma creatinine among veterinary laboratories, thereby influencing the clinical assessment of analytical results. The aims of the study was to determine the inter- and intra-laboratory variation in plasma creatinine among 10 veterinary laboratories, and to compare results from each laboratory with the upper limit of its reference interval. METHODS: Samples were collected from 10 healthy dogs, 10 dogs with expected intermediate plasma creatinine concentrations, and 10 dogs with azotemia. Overlap was observed for the first two groups. The 30 samples were divided into 3 batches and shipped in random order by postal delivery for plasma creatinine determination. Statistical testing was performed in accordance with ISO standard methodology. RESULTS: Inter- and intra-laboratory variation was clinically acceptable as plasma creatinine values for most samples were usually of the same magnitude. A few extreme outliers caused three laboratories to fail statistical testing for consistency. Laboratory sample means above or below the overall sample mean, did not unequivocally reflect high or low reference intervals in that laboratory. CONCLUSIONS: In spite of close analytical results, further standardization among laboratories is warranted. The discrepant reference intervals seem to largely reflect different populations used in establishing the reference intervals, rather than analytical variation due to different laboratory methods.

Glomerular filtration rate estimation by use of a correction formula for slope-intercept plasma iohexol clearance in cats.

OBJECTIVE: To develop a formula for correcting slope-intercept plasma iohexol clearance in cats and to compare clearance of total iohexol (TIox), endo-iohexol (EnIox), and exo-iohexol (ExIox). ANIMALS: 20 client-owned, healthy adult and geriatric cats. PROCEDURES: Plasma clearance of TIox was determined via multisample and slope-intercept methods. A multisample method was used to determine clearance for EnIox and ExIox. A second-order polynomial correction factor was derived by performing regression analysis of the multisample data with the slope-intercept data and forcing the regression line though the origin. Clearance corrected by use of the derived formula was compared with clearance corrected by use of Brochner-Mortensen human and Heiene canine formulae. Statistical testing was applied, and Bland-Altman plots were created to assess the degree of agreement between TIox, EnIox, and ExIox clearance. RESULTS: Mean ± SD iohexol clearance estimated via multisample and corrected slope-intercept methods was 2.16 ± 0.35 mL/min/kg and 2.14 ± 0.34 mL/min/kg, respectively. The derived feline correction formula was Cl(corrected) = (1.036 × Cl(uncorrected)) - (0.062 × Cl(uncorrected)(2)), in which Cl represents clearance. Results obtained by use of the 2 methods were in excellent agreement. Clearance corrected by use of the Heiene formula had a linear relationship with clearance corrected by use of the feline formula; however, the relationship of the feline formula with the Brochner-Mortensen formula was nonlinear. Agreement between TIox, EnIox, and ExIox clearance was excellent. CONCLUSIONS AND CLINICAL RELEVANCE: The derived feline correction formula applied to slope-intercept plasma iohexol clearance accurately predicted multisample clearance in cats. Use of this technique offers an important advantage by reducing stress to cats associated with repeated blood sample collection and decreasing the costs of analysis.

ISFM and AAFP consensus guidelines: long-term use of NSAIDs in cats.

UNLABELLED: NSAIDS AND CATS: Non-steroidal anti-inflammatory drugs (NSAIDs) are an important class of drug in feline medicine, having analgesic, anti-inflammatory and antipyretic activity. While most published data on their use in this species relate to short-term (often perioperative) therapy, there is increasing evidence of the value of these drugs in treating chronic pain in cats (for example, that associated with degenerative joint disease), and some NSAIDs have now become licensed for long-term use in cats in some geographies. Most of our knowledge of therapeutic mechanisms or adverse drug reactions associated with NSAIDs is extrapolated from work in other species, and there is a paucity of published data relating to cats. GUIDELINES: These guidelines have been drawn together by an expert panel, which have reviewed the current literature on long-term NSAID use in cats and other species, and developed guidance on their use based on this information. The aim is to provide practical information for veterinarians to encourage appropriate NSAID therapy whenever cats will benefit from the use of these drugs.

Renal histomorphology in dogs with pyometra and control dogs, and long term clinical outcome with respect to signs of kidney disease.

BACKGROUND: Age-related changes in renal histomorphology are described, while the presence of glomerulonephritis in dogs with pyometra is controversial in current literature. METHODS: Dogs with pyometra were examined retrospectively for evidence of secondary renal damage and persisting renal disease through two retrospective studies. In Study 1, light microscopic lesions of renal tissue were graded and compared in nineteen dogs with pyometra and thirteen age-matched control bitches. In Study 2, forty-one owners of dogs with pyometra were interviewed approximately 8 years after surgery for evidence of clinical signs of renal failure in order to document causes of death/euthanasia. RESULTS: Interstitial inflammation and tubular atrophy were more pronounced in dogs with pyometra than in the control animals. Glomerular lesions classified as glomerular sclerosis were present in both groups. No unequivocal light microscopic features of glomerulonephritis were observed in bitches in any of the groups. Two bitches severely proteinuric at the time of surgery had developed end stage renal disease within 3 years. In five of the bitches polyuria persisted after surgery. Most bitches did not show signs of kidney disease at the time of death/euthanasia. CONCLUSION: Tubulointerstitial inflammation was observed, but glomerular damage beyond age-related changes could not be demonstrated by light microscopy in the dogs with pyometra. However, severe proteinuria after surgery may predispose to development of renal failure.

Malignant teratoid medulloepithelioma with brain and kidney involvement in a dog.

ANIMAL STUDIED: A tumor of the left eye with involvement of the brain and kidney was diagnosed in a 4-year-old Neapolitan mastiff. PROCEDURE: The dog presented with acute glaucoma of the left eye. Peripheral corneal vascularization and severe corneal edema obscured examination of deeper structures. Because of concurrent progressive neurologic signs the dog was euthanized and a postmortem examination was performed. The eyes, brain and samples from the lung, heart, liver and kidneys were fixed in 4% neutral buffered formalin and embedded in paraffin wax. Sections were examined by light microscopy including histochemical and immunohistochemical staining. RESULTS: The ocular tumor originated from the ciliary body and was composed of small islets and cords of poorly differentiated, oval to polyhedral cells surrounded by abundant ground substance. Areas with chondroid differentiation were observed. Rosettes were not found. Metastases with the same morphology were present in the brain and in one kidney. CONCLUSION: An intraocular malignant teratoid medulloepithelioma with metastases to the brain and kidney is presented.