Arginine does not rescue p.Q188R mutation deleterious effect in classic galactosemia

Four classic galactosemia patients homozygous for the c.563A > G (p.Q188R) mutation were enrolled in the clinical study. Patients’ characteristics are presented in Table 1.

Assessments of the effect of arginine aspartate supplementation (Asparten®) included whole body galactose oxidative capacity (primary outcome), erythrocyte GALT activity, as well as urinary galactose and galactitol levels (secondary outcomes). Baseline results were obtained immediately before Asparten® supplementation. Post treatment results were obtained after 30 ± 5 days of Asparten® intervention, immediately after suspension of treatment.

No significant difference in the profile of mean galactose oxidative capacity was observed (Fig. 1 and Additional file 1: Figure S1). The CUMPCD (cumulative percent of the dose) for the total time of the study (360 min) per patient is shown in Table 2. At baseline, the mean ¹³CO₂ released by the four patients was 2.8 ± 1.3%, whereas after intervention it was 2.7 ± 1.2% (p = 0.22).

GALT activity before and after intervention is represented in Fig. 2. There was no statistically significant (p = 0.87) increase in GALT activity following arginine aspartate supplementation (baseline: 8.4 ± 2.2 μmol/h/mmol Hb, corresponding to 1.5%; after treatment: 8.7 ± 4.9 μmol/h/mmol Hb, corresponding to 1.5%) (Table 2).

Mean urinary galactitol and galactose levels are shown in Fig. 3. Both metabolites did not show a statistically significant decrease after arginine aspartate supplementation. Galactitol decreased from 114 ± 16 to 110 ± 17 μmol/mmol creatinine (p = 0.41) (reference 0–125 μmol/mmol creatinine) and galactose decreased from 11 ± 9 to 7 ± 3 μmol/mmol creatinine (p = 0.52) (Table 2).

Amino acid profile analysis revealed that plasma arginine decreased from 67 ± 17 μmol/L at baseline to 53 ± 21 μmol/L following intervention (p = 0.05) (reference 16–124.9 μmol/L). Additionally, patients exhibited on average an increase in plasma ornithine levels from 66 ± 25 μmol/mmol at baseline to 142 ± 73 μmol/L following intervention (p = 0.07) (reference 24.0–167.9 μmol/L). Other amino acids did not show any significant difference between baseline and after intervention.

The mean dietary arginine intake did not increase during study (4 ± 1 g/day at baseline and after treatment) (p = 0.87). Mean daily galactose intake did not change during study period. All patients showed a high compliance to Asparten® intake, ranging from 92 to 100% (Table 2).

Patient 1 reported headache in the first two days of Asparten® supplementation.

In parallel with the clinical analysis, fibroblasts derived from two p.Q188R/p.Q188R patients and one p.Q188R/p.Q188P patient were cultured in the presence and absence of arginine. GALT activity of exposed and unexposed fibroblasts is presented in Table 3. Patients’ fibroblasts displayed no detectable activity even in the presence of a high concentration of arginine (1 mM). In order to discard possible arginase activity, arginine in the cell culture medium was measured. Results are presented in Additional file 2: Table S1.

The therapeutic benefit of arginine was previously attributed to a possible role as chemical chaperone, stabilizing disease-associated proteins. To investigate this possibility, the in vitro thermal shift assay (differential scanning fluorimetry, DSF) was performed to evaluate whether arginine could thermally stabilize recombinant GALT (Table 4 and Fig. 4). Neither the p.Q188R or the wildtype GALT protein showed a thermal shift in the presence of arginine at any of the concentrations studied. In contrast, galactose (10 mM), a moiety of the substrate galactose-1-phosphate, caused a small shift in the melting temperature of the wildtype protein (ΔTm = 2.3 °C) but not of the p.Q188R variant (ΔTm = 1.4 °C).

Four adult classic galactosemia patients being followed up in our expertise centrum for classic galactosemia carrying the p.Q188R mutation in homozygosity were included in the study (Table 1). Written informed consent was obtained from all patients participating in the study. The study was approved by the Medical Ethical Committee of the Maastricht University Medical Center + (METC). This study has been registered at https://​clinicaltrials.​gov (NCT03580122).

This is a study with a pre-post single arm design. All patients received arginine in the commercially available form of arginine aspartate (Asparten®). This formula was chosen based on the fact that it is administered by oral route and it displays well-known pharmacokinetic and toxicological characteristics. It is commercialized since 1974 and no side effects have ever been described. Each ampoule of Asparten® contains 5 g of arginine aspartate. Following the screening and baseline assessments, patients received 5 g of arginine aspartate, 3 times a day (as recommended in Asparten®‘s Summary of Product Characteristics), corresponding to a daily dose of 15 g arginine aspartate. Duration of the intervention was 1 month (30 ± 5 days). Patients were asked to keep the empty ampoules in order to ascertain compliance to intervention.

All patients remained on a galactose-restricted diet, which consists of eliminating all galactose- and lactose-containing dairy products from the diet (except for mature cheeses and caseinates); non-dairy sources of galactose, such as fruit and vegetables are allowed.

Patients were asked to keep a 3-day diary of their diet in the beginning and in the end of the study to be able to calculate the average arginine intake.

All patients were evaluated at baseline and after intervention. Primary outcome was whole body galactose oxidative capacity. Secondary outcomes included erythrocyte GALT activity, as well as urinary galactose and galactitol levels. Additionally, pre-prandial amino acid profile was also performed.

Baseline assessments were done immediately before the initiation of Asparten® supplementation, and after treatment assessments were done immediately after suspension of Asparten® supplementation.

Whole body galactose in vivo oxidative capacity was determined as previously described [17, 23]. Patients were administered 7 mg/kg [1-¹³C]-galactose tracer (0.039 mmol/kg, Cambridge Isotope Laboratories, Andover, USA) via a single bolus injection. Expired breath samples were collected into 10 mL Exetainer tubes (Labco limited, Lampeter, UK), which were filled directly from a mixing chamber in duplicate. A baseline breath sample was collected prior to [1-¹³C]-galactose injection (t = 0 min). During the first hour following injection, breath samples were collected at t = 5, 15, 30, 45 and 60 min. Thereafter, breath samples were collected at 30-min intervals until t = 360 min. Whole-body oxygen uptake (VO₂) and carbon dioxide (VCO₂) production were measured during three 60 min periods (t = 30–90, 150–210, and 270–330 min) using a ventilated hood system (Omnical, Maastricht University, Maastricht, the Netherlands). During the entire study period, subjects rested supine and were allowed to drink only water.

Expired breath samples were analyzed for ¹³C/¹²C ratio by gas chromatograph continuous flow isotope ratio mass spectrometry (Finnigan, Bremen, Germany). The isotopic enrichment was expressed as δ per mil difference between the ¹³C/¹²C ratio of the sample and a known laboratory reference standard [24].

The δ¹³C was then related to an international standard (PDB-1). The ¹³CO₂ production from the oxidation of the infused [1-¹³C]-galactose tracer was subsequently calculated as:

Where \( {\mathrm{TTR}}_{{\mathrm{CO}}_2} \) is the breath ¹³C/¹²C ratio at a given time point, VCO₂ is the carbon dioxide production (L∙min^− 1), k is the volume of CO₂ (22.4 L∙mol^− 1). Total ¹³CO₂ production represents a minimal estimate of the total amount of [1-¹³C]-galactose that was oxidized within the experimental period. In addition, the cumulative percent of the [1-¹³C]-galactose tracer eliminated as ¹³CO₂ in expired air (CUMPCD) was calculated.

GALT enzymatic activity in RBC was performed as previously described [25]. Galactose and galactitol levels were measured in urine by GC/MS.

Amino acid profile was measured as described by Waterval et al. [26].

Dietary intake of arginine was determined by a 3-day food diary pre and post intervention and calculated as mean percentage of the total protein intake. The amount of arginine was considered to be 5% of the total protein intake [27].

Cultured fibroblasts from two wildtype and three classic galactosemic patients (Coriell GM1703, GM727 with p.Q188R/p.Q188R mutations and a Boston Children’s Hospital patient with p.Q188R/p.Q188P mutations) were grown in MEM media supplemented with 10% FBS at a 37 °C incubator with humidified atmosphere of 5% CO₂. Arginine treatment was started by supplementing the growth media with 0, 0.1 mM or 1.0 mM of arginine (considered as day 0 of arginine treatment). On day 3 of the arginine treatment, cells were washed with ice cold PBS and pellets were harvested in cold PBS by scraping. The pellets were stored in − 80 °C until enzyme measurements. The GALT activity in cell lysates was measured using LC-MS/MS by a modified version of a method previously described for washed RBC [28]. Cell lysates were prepared by resuspending the pellets in 100–200 μL 0.5 M glycine buffer (pH 8.7) and sonicating for 15 s at level 1 (Sonic Dismembrator model 100, Fisher Scientific, USA) three times with one-minute intervals. The homogenate was then centrifuged at 15,800 g for 10 min at 4 °C (5402R, Eppendorf). Protein concentration was quantified using DC kit (Bio-RAD). AB Sciex QTrap 5500 mass spectrometer was used for quantification of the enzyme product. Arginine was measured in the cell culture supernatant by stable isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) using a ¹⁵N-L-Arginine internal standard (Cambridge Isotope Laboratories Inc., Tewksbury, MA, USA) with an Acquity™ Ultraperformance® liquid chromatography system coupled to a Quattro Premier tandem-quadrupole mass spectrometer (Waters Corporation, Milford, MA, USA).

Expression and purification of human GALT was performed as previously described [6]. Differential scanning fluorimetry (DSF) was performed in a 96-well plate using an Mx3005p RT-PCR machine (Stratagene) with excitation and emission filters of 492 and 610 nm, respectively. Each 20 μL reaction consisted of 2 μL as-purified recombinant protein (final concentration of 2 μM, wildtype or p.Q188R) and 2 μL arginine at various concentrations (100 μM to 10 mM) in DSF buffer (150 mM NaCl, 10 mM HEPES pH 7.5) to which 2 μL SYPROrange, diluted 500-fold in DSF buffer from the manufacturer’s stock (Invitrogen) was added. Galactose (10 mM) was used as an indicator of ligand binding (positive control). Fluorescence intensities were measured at each 1 °C temperature increase from 25 to 96 °C with a ramp rate of 3 °C/min. A thermal shift of 3 °C or more is considered to be significant, though interpretation varies, and some papers report a value > 1.5 °C as significant.