Toward Absolute Quantification of Soluble Proteins via Proton Nuclear Magnetic Resonance Spectroscopy: Total Protein Concentration in Blood Plasma.

For absolute protein quantification using nuclear magnetic resonance (NMR) spectroscopy, we considered proteins as homopolymers and effective amino acid (AA) residues (AAREff) as monomer units. For diverse classes of proteins, we determined the AAREff molecular weight as 111.5 ± 3.2 Da and the number of hydrogens per AA as 7.8 ± 0.2. Their ratio of 14.3 ± 0.3 (g/LP)/(mol/LH) remains constant across various protein classes and is equivalent to Kjeldahl's nitrogen-to-protein conversion constant of 5.78 ± 0.29 gN/gP. By analogy to the Kjeldahl method, we suggest that the total integral of a 1H NMR solution protein spectrum could be used for total protein quantification. We synthesized low-resolution protein spectra from the weighted sums of individual AA spectra and compared them with experimental spectra. In the methyl region, the ratio of the protein mass to the total number of protons in the synthetic spectra (corrected for the chemical shift mismatch) was ∼1 (mg/mL)/mM, which agrees with an earlier reported experimental ratio for urine (1.05 ± 0.06 (mg/mL)/mM). For human blood plasma, in the methyl region, we found empirical ratios of 1.115 ± 0.006 (mg/mL)/mM (using 96 patient samples) and 1.121 ± 0.011 (mg/mL)/mM for the NIST plasma standard. This numerical agreement points to universal conversion constants, i.e., protein mixtures with unknown compositions could be quantified without the need for calibration standards by measuring the millimolar proton concentration within the methyl region of the NMR spectrum using the same conversion constant.

Transverse relaxation in fixed tissue: Influence of temperature and resolution on image contrast in magnetic resonance microscopy.

To describe transverse relaxation of water in fixed tissue, we propose a model of transverse relaxation accelerated by diffusion and exchange (TRADE) that assumes exchange between free (visible) and bound (invisible) water, which relax by the dipole-dipole interaction, chemical exchange, and translation in the field gradient. Depending on the prevailing mechanism, transverse relaxation time (T2 ) of water in fixed tissue could increase (when dipole-dipole interaction prevails) or decrease with temperature (when diffusion in the field gradient prevails). Chemical exchange can make T2 even temperature independent. Also, variation of resolution from 100 to 15 µm/pxl (or less) affects effective transverse relaxation. T2 steadily decreases with increased resolution ( T 2 ∝ ∆ x 2 , ∆ x is the read direction resolution). TRADE can describe all of these observations (semi)quantitatively. The model has been experimentally verified on water phantoms and on formalin-fixed zebrafish, mouse brain, and rabbit larynx tissues. TRADE could help predict optimal scanning parameters for high-resolution MRM from much faster measurements at lower resolution.

Robotic Ultrasound and Novel Collagen Analyses for Polycystic Kidney Disease Research Using Mice.

BACKGROUND: 3D imaging and histology are critical tools for assessing polycystic kidney disease ( PKD ) in patients and animal models. Magnetic resonance ( MR ) imaging provides micron resolution, but is time consuming, expensive, and access to equipment and expertise is limiting. Robotic ultrasound ( US ) imaging has lower spatial resolution but is faster, more cost effective, and accessible. Similarly, Picrosirius red ( PSR ) staining and brightfield microscopy is commonly used to assess fibrosis; however, alternative methods have been shown in non-kidney tissues to provide greater sensitivity and more detailed structural characterization. METHODS: In this study, we evaluated the utility of robotic US and alternative methods of quantifying PSR staining for PKD research. We compared longitudinal total kidney volume ( TKV ) measurements using US and MR. We additionally compared PSR imaging and quantification using standard brightfield with that by circularly polarized light with hue analysis, and fluorescence imaging analyzed using CT-FIRE software for automatic detection of individual collagen fibers. RESULTS: Increased TKV was detected by US in Pkd1RC/RC vs wild type ( WT ) at timepoints spanning early to established disease. US inter-observer variability was greater but allowed scanning in 2-5 minutes/mouse while MR required 20-30 minutes/mouse. While no change in fibrotic index was detected in this cohort of relatively mild disease using brightfield, polarized light showed fibers skewed thinner in Pkd1RC/RC vs WT. Fluorescence imaging showed a higher density of collagen fibers in Pkd1RC/RC vs WT, and fibers were thinner and curvier with no change in length. Additionally, fiber density was higher in both glomeruli and tubules in Pkd1RC/RC , and glomeruli had a higher fiber density than tubules in Pkd1RC/RC , and trended higher in WT. CONCLUSIONS: These studies show robotic ultrasound is a rigorous imaging tool for pre-clinical PKD research. Additionally, they demonstrate the increased sensitivity of polarized and fluorescence analysis of PSR-stained collagen.

MRI imaging versus histologic volumetric estimation of residual injection laryngoplasty material.

Objectives: To examine the degree of agreement between MRI and histologically generated volumetric measurements of residual injection laryngoplasty material. Methods: Following left recurrent laryngeal nerve transection, rabbit vocal cords were injected with jellyfish collagen, Cymetra®, or Restylane®. Laryngeal tissue was harvested 4 or 12 weeks post injection followed by MRI imaging and histologic cross-sectioning. Two raters estimated the volume of remaining injection material in specimens within MRI and histologic axial cross sections. Wilcoxon signed rank tests were employed to detect gross differences between inter-rater measurements and between imaging modalities across time. Agreement between rater measurements and imaging (histology and MRI) was assessed using intra-class correlation coefficients. Results: Data was available from 16 rabbits sacrificed at 4 weeks (n = 8) and 12 weeks (n = 8). Inter-rater testing of MRI imaging revealed no significant differences (p > .05) between rater measurements across time points, and excellent agreement (0.93; 95% confidence interval 0.80-0.98) while histologically estimated volumes demonstrated a significant difference at 4 weeks (p < .05) and overall good agreement (0.89; 95% confidence interval 0.59-0.97). Comparison of MRI and histologically estimated volume measurements revealed significant differences at the 4-week time point (p < .05) but not at 12 weeks (p > .05). Overall, there is only moderate agreement between MRI and histology estimates (0.72; 95% confidence interval 0.22-0.90). Conclusions: MRI imaging demonstrates good reliability and similar estimates of volume to histologically estimated measurements of residual injection laryngoplasty material at time points clinically relevant for future injection laryngoplasty experiments. Level of Evidence: NA.

MRI Study of Jellyfish Collagen, Hyaluronic Acid, and Cadaveric Dermis for Injection Laryngoplasty.

OBJECTIVES/HYPOTHESIS: Test a new jellyfish collagen biomaterial aimed to increase duration of injection medialization laryngoplasty (IL) against two products in clinical practice. STUDY DESIGN: Animal model. METHODS: Left recurrent laryngeal nerve sectioning and IL were performed in New Zealand White rabbits (N = 6/group). Group 1 received micronized cross-linked jellyfish collagen (MX-JC) and adipose derived stem cells (ADSCs), Group 2, MX-JC alone, Group 3, cross-linked hyaluronic acid (X-HA), and Group 4, micronized acellular dermis (MACD). Animals were sacrificed at 4 and 12 weeks. Major outcomes were MRI tissue volumes and histopathology. RESULTS: After 100 µL IL MRI volumes (means ± STD) at 4 and 12 weeks were: Group 1: 27.2 ± 15.6 and 13.1 ± 5.2 µL, Group 2: 60.8 ± 18 and 27.8 ± 2.47 µL, Group 3: 27.4 ± 12 and 10.6 ± 8 µL, and Group 4: 37.5 ± 11 and 9.85 ± 1 µL. Group 2 volumes were largest and Group 3 were smallest in all comparisons (P < .05). Histologically, low grade inflammatory responses were observed in Group 1, mild histiocytic infiltration in Group 2, widespread muscle fiber loss in Group 3, and plasmocytic infiltration in Group 4. CONCLUSIONS: MX-JC showed the least resorption at 4 and 12 weeks among all groups. T cell inflammatory responses were observed with MX-JC but were reduced by 12 weeks while B cell immune responses, indicative of antibody priming, were predominantly noted with MACD. MX-JC + ADSC showed low grade immunity while the XHA showed greater myocyte loss compared to the other groups. LEVEL OF EVIDENCE: NA Laryngoscope, 131:E2452-E2460, 2021.