case_images: PMC10742367_01
case_images: Total calcium and ionized calcium trends in the patient. The measurements of total calcium and ionized calcium over 6 years of follow are presented. The dashed line represents the time when the levels of total calcium were first noted to be abnormally high. At the same time, the IgA levels were first checked and found to be abnormal. Key values of total calcium and ionized calcium are labeled above their respective points.
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case_images: Importantly, on subsequent laboratory tests, the ionized calcium was found to be normal at 1.2 mmol/L (4.8 mg/dL; normal reference range, 1.1-1.3 mmol/L [4.5-5.1 mg/dL]), whereas total calcium remained elevated at 2.72 mmol/L (10.9 mg/dL) (Fig. 1).
case_images: Plasma fast protein liquid chromatography (FPLC) and gel electrophoresis. (A) Plasma samples from the patient and a healthy control were diluted in PBS (800 muL of plasma in 500 muL of PBS) and separated by FPLC based on size exclusion, using AKTA go with a Supreose 6 Increase 10/300 GL column with a total volume of 47.1 mL at a flow rate of .5 mL/min. The Y-axis, mili-absorbance unit (mAU) reflects UV absorption of protein and varies with protein concentration. The concentration of total protein in fractions in peak 1 of the patient was ~200 mg/dL and was ~50 mg/dL in the same fractions of the control as measured by the BCA protein assay (Thermo scientific #23225). The elution volume, in milliliters, is presented in the X-axis. Larger size proteins migrate faster than smaller proteins and thus are represented to the left of the graph. Plasma was fractionated and collected every 500 muL. The patient FPLC demonstrated 2 clear peaks of high protein concentration, whereas the healthy control shows only 1 predominant peak, corresponding to albumin by size, against a standard. (B) Gel electrophoresis of plasma fractions in peaks 1 and 2 of the patient. Peak 2 has a MW ~60 kDa, consistent with the expected MW of albumin, whereas peak 1 had protein bands of much higher molecular weight. Data are representative of 3 independent experiments.
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case_images: As expected from the serum protein electrophoresis results, the patient FPLC revealed 2 distinct peaks of protein corresponding in molecular weight (MW) to the presence of abnormally heavy protein ("Peak 1-IgA paraprotein") and to albumin ("Peak 2-albumin"), respectively (Fig. 2A). The healthy control plasma fractions revealed only 1 peak, overlapping with the peak 2 of the patient, and corresponding to the MW of albumin, as revealed by gel electrophoresis (Fig. 2B) and direct comparison with a standard mix of proteins.
case_images: Gel electrophoresis of plasma fractions corresponding to the paraprotein peak. Corresponding plasma fractions of peak 1 derived from FPLC in the healthy control and patient were denatured with SDS and loaded at equal microliter quantities on a 4% to 20% Gel (Mini Protein Bio-Rad). After electrophoresis, the protein was visualized by Coomassie blue staining. For comparison, human plasma IgA (Sigma Aldrich Cat# 401098) was also loaded in gel. The patient plasma predominantly revealed high-molecular-weight proteins (>200 kDa) and lower amounts of protein at molecular weights of 50 kDa (ie, the approximate MW of the heavy chain of IgA). Subsequently, the same plasma fractions were denatured and loaded in gel, then transferred to a PVDF membrane, blocked for 1 hour with 5% milk and then treated with primary antibody anti-IgA heavy chain (1:1000) (Proteintech #11449-1-AP) for 3 hours at room temperature. After washing primary antibody x3 with TBST, the membrane was treated with secondary antibody anti-rabbit-HRP (1:5000) for 30 minutes. The gels were washed x3 with TBST and visualized using the Clarity Western ECL substrate. The results demonstrate that the majority of protein present in peak 1 of the FPLC corresponds to high-molecular-weight IgA. Data are representative of >3 independent experiments.
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case_images: IgA in these plasma fractions (Fig. 3). Interestingly, as compared with IgA obtained commercially from the plasma of a healthy human, the patient IgA had bands at higher molecular weights (>200 kDa), likely reflecting the presence of polymers of IgA, which resisted denaturization with sodium dodecyl sulfate (Fig. 3).
case_images: Free calcium measurement in plasma fractions. (A) A calcium standard curve was created using a calcium calibration kit (Biotium #59100) to contain levels of free calcium in the 1 to 1 muM range at a pH of 7.4. Levels of free calcium were measured by mixing 1 muL of Fura Pentasodium (1 mM) with 200 muL of calcium solution. Fluorescence intensity in the solutions was measured by sequential excitations at 340 and 380 nm, with emission at 510 nm using a 96-well black plate and a BioTek Synergy H1 Plate reader. An increase in the 340/380 ratio is noted with free calcium concentrations going up to 1 muM. (B) Workflow of experiments to measure free calcium in plasma fractions. Free calcium (Ca++) is represented as calcium not in direct contact with immunoglobulins. The patient plasma fraction had high amounts of IgA (see Figs. 2 and 3), whereas the control had a lower amount of protein (see Fig. 2) in the corresponding fractions., Both plasma fractions were dialyzed against PBS (no calcium) to remove the endogenous Ca++ and equalize levels of free calcium at baseline. Plasma fractions were then added to solutions with a known concentration of free calcium (1 muM) and the remaining free calcium was measured. The figure was created using BioRender. (C) Plasma fractions were dialyzed using a membrane with cutoff molecular weight 3.5 kDa (Sigma Aldrich Cat#PURX35005) in a PBS (no calcium, no magnesium) solution overnight. Free calcium was measured in the fractions by Fura Pentasodium after dialysis to provide a baseline at which the levels of free calcium were similar in the patient-derived fractions of peak 1 (IgA paraprotein) vs healthy control. Baseline measurements were done using 100 muL of post-dialysis fraction + 200 muL of "zero calcium solution" from the calcium calibration kit. (D) A 200-muL solution containing 1 muM of free calcium was mixed with 100 muL of the plasma fractions or with equal volume of H20. Mixtures of calcium solution and plasmas (or H20) were centrifugated (20 000 RCF x5 minutes) and the supernatant tested for free calcium by addition of 1 muL Fura pentasodium and measured as in panel A. Each patient plasma fraction containing IgA lowered the amount of free calcium to a greater than each corresponding fraction in the healthy control. The results demonstrate a modest (~4% decrease), yet consistent capacity of the patient IgA containing fractions to lower free calcium in solution vs healthy control plasma. For 1:1 comparison between patient and heathy plasma fractions of equivalent molecular weight, the number of the fraction tested is provided.
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case_images: IgA, we used the ratio metric calcium indicator Fura-2 Pentasodium (Biotium, catalog No. 50032), which detects changes in free calcium between the range of 1 nM to 1 muM at physiologic pH (Fig. 4A). In contrast to Fura-2 2 acetoxymethyl ester, which is typically used to measure intracellular free calcium, Fura-2 pentasodium is cell impermeant and has been shown to have good correlation with potentiometric techniques to measure free calcium in different solutions. The workflow of in vitro experiments is illustrated in Fig. 4B. To reduce endogenous calcium present in the fractions and generate similar baseline level of free calcium, the plasma fractions were dialyzed against phosphate-based saline (without calcium) using dialysis membranes with a molecular weight cutoff of 3.5 kDa (Fig. 4C). Relative to the amount of free calcium without addition of the plasma fractions (100%), each of the patient plasma fractions containing the IgA paraprotein reduced free calcium more than the fraction with corresponding MW in the healthy control (Fig. 4D) The results thus support increased capacity of the patient IgA paraprotein to lower calcium relative to proteins of similar molecular weight in a control, which were much less abundant.