Vedolizumab quantitation using high-resolution accurate mass-mass spectrometry middle-up protein subunit: method validation.

Background While quantitation methods for small-molecule and tryptic peptide bottom-up mass spectrometry (MS) have been well defined, quantitation methods for top-down or middle-up MS approaches have not been as well defined. Therapeutic monoclonal antibodies (t-mAbs) are a group of proteins that can be used to both demonstrate the advantages of top-down or middle-up detection methods over classic tryptic peptide bottom-up along with the growing need for robust quantitation strategies/software for these top-down or middle-up methods. Bottom-up proteolytic digest methods for the t-mAbs tend to suffer from challenges such as limited peptide selection due to potential interference from the polyclonal immunoglobulin background, complicated workflows, and inadequate sensitivity and specificity without laborious purification steps, and therefore have prompted the search for new detection and quantitation methods. Time-of-flight along with Orbitrap MS have recently evolved from the research and/or pharmaceutical setting into the clinical laboratory. With their superior mass measurement accuracy, resolution and scanning speeds, these are ideal platforms for top-down or middle-up characterization and quantitation. Methods We demonstrate a validated, robust, middle-up protein subunit detection and quantitation method for the IgG1 t-mAb, vedolizumab (VEDO), which takes advantage of the high resolution of the Orbitrap MS detection and quantitation software to increase specificity. Results Validated performance characteristics met pre-defined acceptance criteria with simple workflows and rapid turnaround times: characteristics necessary for implementation into a high-volume clinical MS laboratory. Conclusions While the extraction method can easily be used with other IgG1 t-mAbs, the detection and quantitation method may become an option for measurement of other proteins.

Biochemical Identification of Vitamin B12 Deficiency in a Medical Office.

BACKGROUND: A vitamin B12 deficiency can be an underlying cause or a deteriorating factor in several diseases. Nevertheless, early identification of such a deficiency remains a problem. Holotranscobalamin (HTC) is presently considered to be the gold standard. We tested the predictive power of other B12 parameters by comparing them with HTC. METHODS: The blood of 77 patients from a medical office was tested for HTC, total B12 (CLIA [chemiluminescent immunoassay] and MTP [microbiological test with microtitre plates]), MMA (methylmalonic acid), HCY (homocysteine), and MCV (mean cell volume). The parameters were correlated and sensitivity, specificity, PPV (positive predictive value), NPV (negative predictive value), LR+ (positive likelihood ratio), and LR- (negative likelihood ratio) in comparison to HTC were determined. A ROC analysis was also performed. RESULTS: At a cutoff value of 35 pmol/L for HTC, the total B12 CLIA (cutoff 211 ng/L) qualified 53% of individuals as having a B12 deficiency. The total B12 MTP (cutoff 288 ng/L) classified 71% as having a B12 deficiency. Specificity was similar in both cases (CLIA, 93%; MTP, 95%). With a cutoff value of 10 µmol/L for homocysteine, the best negative prediction was achieved. MVA has a low sensitivity (41%) and a high specificity (90%). Based on the ROC analysis, which indicated superiority of the B12-MTP, the reference levels of B12-CLIA and B12-MTP were raised to 304 and 368 ng/L, respectively. Thus, a probable B12 deficiency was identified in 94% of cases with either method and with a comparable specificity. CONCLUSIONS: If total B12 is applied to identify B12 deficiency, the cutoff values should be elevated to 304 (B12-CLIA) and 368 ng/L (B12-MTP) to improve the predictive power. The negative-predictive power of HCY can be useful in daily routine. HTC has a broad grey area of uncertainty and MMA should only be applied as a confirmatory test.

The influence of a whole food vegan diet with Nori algae and wild mushrooms on selected blood parameters.

BACKGROUND: Vegan and vegetarian diets could overcome many diseases of civilization. This study examines whether a whole food vegan diet with Nori algae and wild mushrooms can provide a sufficient quantity of critical nutrients. METHODS: Five blood samples (Baseline to Time 5) were taken over eight months from 75 subjects (10 vegans without B12 supplementation who consumed Nori algae and wild mushrooms, 20 vegans with supplementation, 40 vegetarians, 5 meat-eaters). Blood was analyzed for blood cell counts, total vitamin B12, holotranscobalamin, homocysteine, methylmalonic acid, vitamin B6, folic acid, ferritin, TSH, zinc, creatinine, vitamin D2 and D3. RESULTS: In the vegan group without supplementation, all means were within the tolerance (holotranscobalamin, homocystein) or normal, except for elevated methylmalonic acid and diminished vitamin D. This group developed significantly higher vitamin D2 levels. The vegan group with B12 supplementation and the vegetarian group showed normal values for all parameters. CONCLUSIONS: Vegans following a whole food diet had a borderline supply of vitamin B12. Folic acid, vitamin B6, TSH, iron metabolism, and the blood count were in the normal range. Vegans taking dietary supplements demonstrated satisfactory overall results. An ingestion of sundried mushrooms can contribute to the supply of vitamin D.

Plasma myostatin measured by a competitive ELISA using a highly specific antiserum.

BACKGROUND: Understanding the (patho)physiology of the negative muscle regulator myostatin (Myo) is important for patients with skeletal muscle disorders or cardiac disease. However, a reliable tool for measuring plasma Myo immunoreactivity is still lacking. METHODS: Human full-length proMyo was used to raise a polyclonal rabbit antiserum for a competitive Myo ELISA that was validated in patients with decompensated congestive heart failure (CHF) and in control patients (n=20 each). RESULTS: The Myo antiserum detected all subunits of human proMyo. The calibration curve showed an optimal range between 0.3 and 83.3 ng/ml (7.5-2100 pmol/l), with no cross-reactivity to growth differentiation factor-11, follistatin and follistatin-related gene protein. The inter-assay and intra-assay variances in human serum were ≤15% and ≤10%, respectively; the detection limit was 270 pg/ml (6.75 pmol/l). The assay showed excellent linearity in human plasma. Plasma NT-proBNP and Myo were significantly elevated in decompensated CHF compared with control patients and decreased significantly upon recompensating therapy. CONCLUSION: We describe the development of the first ELISA for myostatin immunoreactivity and its validation during recompensating therapy for CHF. This assay will be valuable for investigating neurological and cardiac diseases and states of cachexia, insulin resistance, and obesity.

Analysis of the effects of androgens and training on myostatin propeptide and follistatin concentrations in blood and skeletal muscle using highly sensitive immuno PCR.

Myostatin propeptide (MYOPRO) and follistatin (FOLLI) are potent myostatin inhibitors. In this study we analysed effects of training and androgens on MYOPRO and FOLLI concentrations in blood and skeletal muscle using Immuno PCR. Young healthy males performed either a 3-month endurance training or a strength training. Blood and biopsy samples were analysed. Training did not significantly affect MYOPRO and FOLLI concentrations in serum and muscle. To investigate whether total skeletal muscle mass may affect circulating MYOPRO and FOLLI levels, blood samples of tetraplegic patients, untrained volunteers and bodybuilders were analysed. MYOPRO was significantly increased exclusively in the bodybuilder group. In orchiectomised rats MYOPRO increased in blood and muscle after treatment with testosterone. In summary our data demonstrate that moderate training does not affect the concentrations of MYOPRO to FOLLI. In contrast androgen treatment results in a significant increase of MYOPRO in skeletal muscle and serum.

FGF23 is a putative marker for bone healing and regeneration.

Besides numerous other factors, fibroblast growth factor receptor (FGFR) signaling is involved in fracture healing and bone remodeling. FGF23 is a phosphatonin produced by osteoblastic cells, which signals via FGFR1, thereby exerting effects in bone and kidney. We analyzed if serum FGF23 levels might be an indicator to predict fracture healing and union. FGF23 (C-Term) was elevated on day 3 postoperatively in 55 patients sustaining an exchange of total hip implants due to aseptic loosening. A prospective study of 40 patients undergoing primary hip arthroplasty also showed elevated FGF23 (C-Term) but no change in FGF23 (intact) levels on days 1, 4, and 10 postoperatively. Serum phosphate and phosphate clearance stayed within normal ranges. FGF23 mRNA expression in ovine callus was compared between a standard and delayed course of osteotomy healing. In the standard model, a marked increase in FGF23 mRNA expression compared to the delayed healing situation was observed. Immunohistochemical analysis showed FGF23 production of osteoblasts and granulation tissue in the fracture callus during bone healing. In conclusion, FGF23 is involved in bone healing, can be measured by a sensitive assay in peripheral blood, and is a promising candidate as an indicator for healing processes prone to reunion versus nonunion.