Fp 2020

FVIII samples of reconstituted Advate, Kogenate, and Refacto at a final nominal concentration of 400 IU/mL (≅ 80µg/mL) were analyzed by SE-HPLC using a TSK gel Super SW3000 column coupled to a two-pump apparatus (Jasco Easton, Maryland, United States), equipped with a spectrophotometric device (model 2075), and a spectrofluorometric detector (FP-2020, Jasco). The spectrophotometric detection of the eluted peaks was accomplished at 280 nm, whereas the fluorescence of the proteins was monitored by using λ
_ex = 280 nm and λ
_em = 340 nm. The elution buffer was a solution containing 20 mM phosphate buffer, 0.15 M NaCl, and pH = 7.40. The flow rate was 0.4 mL/min in all cases, and the injection volume was 75 µL. The same FVIII preparation was also filtered through a low-binding protein Millex-GP 220 nm filter (Merck) and analyzed again by SEC-HPLC, as detailed above.

Biopterin levels were determined in serum obtained from controls and polybacterial-infected mice by HPLC system using electrochemical and fluorescent detection methods as described [21 (link)]. Following centrifugation of blood (15 min at 13,000 x g at 4°C), the serum samples were transferred to new, cooled micro tubes and precipitated with an equal volume of a solution of cold phosphoric acid (1 M), trichloroacetic acid (2 M), and dithioerythritol (1 mM). The samples were vigorously mixed and centrifuged again for 15 min at 13,000 x g at 4°C. The supernatants were injected onto an isocratic HPLC system and quantified using sequential electrochemical (Coulochem III, ESA Inc) and fluorescence (Jasco) detection. HPLC separation was performed using a 250 mm, ACE C-18 column (Hichrom) and mobile phase comprised of sodium acetate (50 mM), citric acid (5 mM), EDTA (48 μM), and dithioerythritol (160 μM) (pH 5.2) (all ultrapure electrochemical HPLC grade) at a flow rate of 1.3 mL/min. Background currents of +500 μA and -50 μA were used for the detection of BH4 on electrochemical cells E1 and E2, respectively. Biopterin and 7,8-BH₂ were measured using a Jasco FP2020 fluorescence detector [21 (link)]. Quantification of BH₄, BH₂, and biopterin was done by comparison with authentic external standards and normalized to sample protein content [21 (link)].

Blood was drawn on the day of the study visit after a fasting period of 8 to 12 h. Urine was collected by participants starting 24 h prior to the study visit, following strict instructions as previously described in detail [28 (link)]. Urinary boron concentrations were determined using inductively coupled plasma mass spectrometry (ICP-MS), as summarized in Supplementary Table 1. Plasma vitamin B6 was determined using a validated HPLC method (Waters Alliance) with fluorescence detection (FP-2020; Jasco Inc.) [29 (link)]. Other clinical chemistry assays including hemoglobin, creatinine, inflammatory parameters including high sensitivity C-reactive protein (CRP) and leukocyte count, HDL and LDL cholesterol, triglycerides, urinary protein excretion, glucose, vitamin B12, folic acid, and homocysteine were performed using routine laboratory methods (Roche Diagnostics, Basel, Switzerland).