Plasma Albumin

The workflow as well as plasma treatment and protein digestion are described in detail by Kontro et al. [15 (link)]. Briefly, after thawing of samples albumin was depleted by Pierce SwellGel Blue Albumin removal discs according to the manufacturer’s instructions. Albumin depleted plasma was assayed by BCA assay kit (Pierce, Thermo Scientific, Rockform, IL, USA) for the total protein concentration. Albumin-depleted plasma corresponding to 350 μg of protein was dried in speed vacuum system (Savant, Thermofisher). After dissolving the dried pellets in 6 M urea and 100 mM Tris-HCl pH 7.4, reduction and alkylation were performed by incubating samples first in 10 mM of DTT for 60 minutes at room temperature (RT) followed by incubation in 30 mM iodoacetamide for 60 minutes in the dark at RT. Finally, after consumption of excess of iodoacetamide with 30 mM DTT (60 minutes at RT) samples were diluted 1:10 with ultrapure water (Milli-Q, EMD Millipore corp.) water and digested with trypsin (1:50 w:w trypsin to protein ratio) for 18 hours at +37°C. Samples were cleaned by Pierce C18 columns according to the manufactures protocol. For LC-MS-analysis 30μg of peptides were first dried in SpeedVac, dissolved in 0.1% formic acid containing 12.5 femtomole Hi3 peptide mixture (Waters) per μL and finally stored at -20°C until analyzed.

A retrospective design was used to study patients with confirmed acute GBS who were admitted to the Department of Neurology at the Beijing Tongren Hospital of Capital Medical University from 2007 to 2021. (1) Inclusion criteria: (i) met the diagnostic criteria of the 2019 Chinese Guillain-Barré Syndrome Diagnosis and Treatment Guidelines developed by the Chinese Medical Association [3 ]; (ii) first-onset admission. (2) Exclusion criteria: (i) patients with combined definite intracranial lesions; (ii) patients with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP); (iii) patients who could not be excluded from peripheral neuropathy caused by other etiologies; (iv) patients with incomplete case data.
Measured characteristics included gender, age at onset, antecedent infection (whether diarrhea, upper respiratory tract infection, pulmonary infection, or other unexplained infection occurred within 4 weeks prior to onset), cranial nerve involvement (presence of ophthalmoplegia, facial palsy, dysarthria, dysphagia, weak neck, and shoulder rotation), presence of pulmonary infection (symptoms such as cough, sputum, and fever during the course of the disease, and confirmation with high-resolution computed tomography (HRCT) of the lungs). Mechanical ventilatory support, hyponatremia, hypoalbuminemia, impaired fasting glucose and the peripheral blood neutrophil-to-lymphocyte ratio (NLR) were analyzed as alternative influencing factors. Peripheral blood was collected from all patients within 24 h of admission. Plasma sodium < 135 mmol/L was considered as combined hyponatremia. Fasting plasma glucose (FPG) > 6.1 mmol/L was considered impaired fasting glucose. Plasma albumin < 35 g/L was defined as hypoalbuminemia. An elevated NLR was defined as an NLR value > 2.135.
The GBS disability score developed by Hughes (Hughes functional grading scale, HFGS) [4 (link)] et al. was used for assessment on the day of discharge: 0 represented a completely normal state; 1 represented mild signs or symptoms and ability to run; 2 represented the ability to walk ≥ 10 m alone but the inability to run; 3 represented the ability to walk 10 m in open space with assistance; 4 represented a bedridden or wheelchair bound state; 5 represented a requirement of assisted ventilatory support; and 6 referred to death. Those with GBS disability scores > 3 at discharge were considered to have a poor early prognosis and those with GBS scores ≤ 3 had better early prognoses.
SPSS 23.0 and MedCalc statistical software were used for the analysis. The χ2 test or Fisher's exact test was used to compare groups of count data. (1) Univariate analysis was used to derive risk factors for poorer early prognosis (HFGS score > 3) in patients with GBS. (2) Statistically significant (P < 0.05) influencing factors obtained from this analysis were then included in a multivariate logistic regression analysis, and regression coefficients were calculated. (3) The integer value closest to the regression coefficient was used as the influencing factor score value in order to establish an early prognostic scoring system. (4) The predictive value of the scoring system was evaluated by plotting the receiver operating curve (ROC) curve: the area under the ROC curve (AUC) was calculated, the appropriate cut-off value was selected, and the sensitivity, specificity, positive predictive value, and negative predictive value were calculated.

Preparation of the test solution: Took 1 bottle of paclitaxel for injection (albumin-binding type), added 20 mL 0.9% sodium chloride solution, shook gently to make it evenly dispersed, took 3 mL and heated it in a water bath at 25 °C, 37 °C, 41 °C, 42 °C and 43 °C for 60 minutes respectively, then took 1 mL of the above solution, put it into a 250 mL measuring bottle. Added simulated human plasma solution (commercially available human albumin preparation mixed with 0.9% sodium chloride solution to obtain simulated plasma solution with a concentration of 5% human albumin) and diluted to the scale. Added 1 mL to a 2 mL round bottom centrifuge tube, prepared 2 portions in parallel, centrifuged at 21,000 g for 60 minutes, put 0.5 mL supernatant into a 5 mL flask, added acetonitrile, sonicate to disperse well, diluted with acetonitrile to the scale and shook well, filtered and take the filtrate as the test solution.
Preparation of the control solution: Placed 10.31 mg paclitaxel reference substance in a 50 mL measuring flask, dissolved and diluted to the scale with acetonitrile, shook well and placed 5 mL in a 50 mL measuring flask, diluted to the scale with acetonitrile, shook well, and 1 mL was measured precisely and placed in a 10 mL measuring flask, diluted to the scale with acetonitrile, shook well. The average of the results of two determinations was taken as the amount of paclitaxel released and recorded as C_P2 (mg/mL). The amount of paclitaxel measured in the determination of paclitaxel content section was taken as the total amount of paclitaxel, and recorded as C_P (mg/bottle), and the release rate of paclitaxel was calculated.
Formula: $$f=\frac{\mathit{Wr}\times \text{Control}\text{content}\%}{\mathit{Ar}\times \text{dilution}\text{multiple}a}\times 100\%$$ $${\text{C}}_{\text{p}2}=\text{Average}f-\text{value}\times \mathit{Ai}$$ $$\text{In}-\text{vitro}\text{release}\text{rate}\left(\%\right)=\frac{C_{\text{P}2}\times 10\times 250\times 20}{C_{\text{P}}}\times 100\%$$
C_P2 (mg/mL): the amount of paclitaxel that has been released.
C_P (mg/bottle): the amount measured in the determination of paclitaxel content section is the amount of total paclitaxel.
Analytical condition: same as determination of paclitaxel content section.

There were a number of factors that were monitored during the tracking period. (1) Urine examinations employed a urine routine that included 24-hour urine protein quantification. (2) Blood tests studied blood creatinine, blood urea nitrogen, blood uric acid, plasma albumin, cholesterol, triglyceride, PLA2R-Ab, B lymphocyte count, etc. (3) To provide a safety evaluation, eGFR was calculated using the CKD-EPI equation (14 (link)). Adverse events during follow-up were recorded. Follow-up endpoints were death, maintenance haemodialysis and end-stage renal disease (eGFR < 15 mL/min for more than three months). (4) Efficacy evaluations were also considered as follows: (a) Complete remission: Upro < 0.3 g/d, ALB > 30 g/l, normal renal function. (b) Partial remission: Upro = 0.3–3.5 g/d, 50% lower than before treatment % or more, ALB ≥ 30 g/L, stable renal function. (c) Ineffective: Upro decreased by less than 50% compared with before treatment, ALB < 30 g/L, or deteriorating renal function. (d) Relapse: Complete or partial remission in patients with Upro > 3.5g/d or > 50% of the baseline value (15 (link)). These indicators were collected via inpatient follow-up queries, the hospital’s inpatient electronic medical record system, the outpatient system and telephone follow-ups.