9 research outputs found
Development of a parallel reaction monitoring-MS method to quantify IGF proteins in dogs and a case of nonislet cell tumor hypoglycemia
Nonislet-cell tumor
hypoglycemia (NICTH) is a rare paraneoplastic
phenomenon well described in dogs and humans. Tumors associated with
NICTH secrete incompletely processed forms of insulin-like growth
factor-II (IGF-II), commonly named big IGF-II. These forms have increased
bioavailability and interact with the insulin and IGF-I receptor causing
hypoglycemia and growth-promoting effects. Immunoassays designed for
human samples have been used to measure canine IGF-I and -II, but
they possess some limitations. In addition, there are no validated
methods for measurement of big IGF-II in dogs. In the present study,
a targeted parallel reaction monitoring MS-based method previously
developed for cats has been optimized and applied to simultaneously
quantify the serum levels of IGF-I, IGF-II, and IGFBP-3, and for the
first time, the levels of big IGF-II in dogs. This method allows the
absolute quantification of IGF proteins using a mixture of QPrEST
proteins previously designed for humans. The method possesses good
linearity and repeatability and has been used to evaluate the IGF-system
in a dog with NICTH syndrome. In this dog, the levels of big IGF-II
decreased by 80% and the levels of IGF-I and IGFBP-3 increased approximately
20- and 4-times, respectively, after removal of the tumor
Quantitative and Selective Analysis of Feline Growth Related Proteins Using Parallel Reaction Monitoring High Resolution Mass Spectrometry.
Today immunoassays are widely used in veterinary medicine, but lack of species specific assays often necessitates the use of assays developed for human applications. Mass spectrometry (MS) is an attractive alternative due to high specificity and versatility, allowing for species-independent analysis. Targeted MS-based quantification methods are valuable complements to large scale shotgun analysis. A method referred to as parallel reaction monitoring (PRM), implemented on Orbitrap MS, has lately been presented as an excellent alternative to more traditional selected reaction monitoring/multiple reaction monitoring (SRM/MRM) methods. The insulin-like growth factor (IGF)-system is not well described in the cat but there are indications of important differences between cats and humans. In feline medicine IGF-I is mainly analyzed for diagnosis of growth hormone disorders but also for research, while the other proteins in the IGF-system are not routinely analyzed within clinical practice. Here, a PRM method for quantification of IGF-I, IGF-II, IGF binding protein (BP) -3 and IGFBP-5 in feline serum is presented. Selective quantification was supported by the use of a newly launched internal standard named QPrEST™. Homology searches demonstrated the possibility to use this standard of human origin for quantification of the targeted feline proteins. Excellent quantitative sensitivity at the attomol/μL (pM) level and selectivity were obtained. As the presented approach is very generic we show that high resolution mass spectrometry in combination with PRM and QPrEST™ internal standards is a versatile tool for protein quantitation across multispecies
Insulin-like growth factor-I in cats:validation of an enzyme-linked immunosorbent assay and determination of biological variation
Biological variation of biochemical urine and serum analytes in healthy dogs
BACKGROUND: Biological variation (BV) of urinary (U) biochemical analytes has not been described in absolute terms, let alone as a ratio of the U-creatinine or fractional excretion in healthy dogs. These analytes are potential diagnostic tools for different types of kidney damage and electrolyte disorders in dogs. OBJECTIVES: We aimed to investigate the BV of specific gravity, osmolality, creatinine, urea, protein, glucose, chloride, sodium, potassium, calcium, and phosphate in urine from healthy pet dogs. METHODS: Blood and urine samples from 13 dogs were collected once weekly for 8 weeks. Samples were analyzed in duplicate and in randomized order. For each sample, U-analyte and serum concentrations were measured, and U-analyte/U-creatinine and fractional excretion (FE) were calculated. Components of variance, estimated by restricted maximum likelihood, were used to determine within-subject variation (CVI ), between-subject variation (CVG ), and analytical variation (CVA ). Index of individuality (II) and reference change values were calculated. RESULTS: CVI for all urine analytes varied between 12.6% and 35.9%, except for U-sodium, U-sodium/U-Cr, and FE-sodium, which had higher CVI s (59.5%-60.7%). For U-protein, U-sodium, U-potassium, U-sodium/U-creatinine, FE-urea, FE-glucose, FE-sodium, FE-potassium, and FE-phosphate II were low, indicating that population-based RIs were appropriate. The remaining analytes had an intermediate II, suggesting that population-based RIs should be used with caution. CONCLUSION: This study presents information on the biological variation of urinary and serum biochemical analytes from healthy dogs. These data are important for an appropriate interpretation of laboratory results
Effect of insulin treatment on circulating insulin-like growth factor I and IGF-binding proteins in cats with diabetes mellitus.
BACKGROUND: Insulin-like growth factor-I (IGF-I) is used to screen for acromegaly in diabetic cats. In humans, most circulating IGF-I forms ternary complexes (TC) with IGF-binding protein (IGFBP-3) and an acid-labile subunit. Compared to humans, the amount of TC in cats is more variable. Insulin-like growth factor-I concentrations are reported to increase during insulin treatment, more rapidly in cats achieving remission. OBJECTIVES: To investigate (i) factors associated with circulating IGF-I concentrations, including IGFBP-profiles (ii) effect of insulin treatment on IGF-I concentrations and (iii) IGF-I as prognostic marker of diabetes mellitus remission. ANIMALS: Thirty-one privately owned diabetic cats of which 24 were followed 1 year, and 13 healthy cats. METHODS: Prospective study. Serum insulin, IGF-I, glucose, and fructosamine concentrations were measured. IGF-binding forms were determined by chromatography in 14 diabetic and 13 healthy cats; and IGF-I, IGF-II, IGFBP-3, and IGFBP-5 by mass spectrometry in 3 cats achieving remission. RESULTS: Insulin-like growth factor-I median (interquartile range) before start of insulin treatment was 300 (160-556) ng/mL. Insulin-like growth factor-I was positively associated with TC (P < .0001) and endogenous insulin (P = .005) and negatively associated with fructosamine (P < .0001). Median IGF-I was higher 2-4 weeks after start of insulin treatment compared with baseline (300 versus 670 ng/mL, P = .0001) and predicted future remission (P = .046). In cats that went into remission, the amount of TC and IGFBP-3 increased, suggesting increase in IGF-I is dependent on TC formation. CONCLUSIONS: Insulin treatment should be accounted for when interpreting IGF-I in diabetic cats. Insulin-like growth factor-I 2-4 weeks after initiation of insulin treatment shows promise as prognostic marker for remission in diabetic cats.Fall och Lewitt delar sistaförfattarskapet.</p
Homology study of the four selected cat proteins and their human analogues.
<p>Human (*) and feline (¤) amino acid sequence with QPrEST™ sequence underscored in the human sequence. IGFBP–5 has two available QPrESTs™ while IGF–II and IGFBP–3 have one each. Tryptic peptides in QPrESTs™ matching peptides found in the feline sequence are market in bold italic (green peptides were used for quantification and the red ones were evaluated but not used). For IGF-I there was no tryptic peptide in the QPrEST™ that matched any feline peptide. The blue peptide is the synthetic NEPTune™ peptide used for quantification and the red peptide was evaluated but not used. The shorter mature protein is marked between ││ for IGF–I and IGF–II. Similarity scores from EMBOSS Needle is presented below the sequences.</p
Result of shotgun analyses of gel fractionated cat sera samples.
<p>Result of shotgun analyses of gel fractionated cat sera samples.</p
Targeted peptides included in the PRM method.
<p>Bold,underscored peptides were used for final quantification. C-terminal arginine (R) and Lysine (K) were heavy labeled. All cysteines are carbamidomethylated. The fragments selected for quantification are given, and all these fragments were singly charged.</p
Internal standard dilution series.
<p>QPrEST™ and NEPTune™ peptides spiked into cat serum, analyzed with the PRM method at different concentrations. The ratio synthetic/native peptide is plotted against the spiked concentration.</p