All samples were obtained following local ethical and consent guidelines. Urine samples of AM patients and negative controls were obtained from the Center for Inherited Metabolic Disorders of National Institute of Children’s Diseases, Bratislava, Slovakia, following standard operating procedures. The experimental study protocol was reviewed and confirmed by the hospital ethics committee. The levels of creatinine (crea) were determined by Vitros 5.1 Fusion Clinical Chemistry Analyzer (Ortho Clinical Diagnostics, Rochester, NY, USA). In total, five samples from four AM patients without the ERT administration (positive controls, males aged 5–20 years) were analyzed in addition to three samples from one patient undergoing the ERT treatment (2 years old female). In order to increase the sample size, two positive control samples from one representative patient were included, based on sample availability. Additionally, analysis of samples over time may provide valuable information about the disease progression and changes in biomarker levels of untreated patients. Diagnosis of all patients was confirmed by enzymatic activity assay and DNA sequencing.

Acetonitrile (ACN), sodium cyanoborohydride (NaBH${}_3$CN), 2-aminobenzamide (2-AB), 2,5-dihydroxybenzoic acid (DHB), trifluoroacetic acid (TFA), formic acid, 25% ammonia solution, sodium hydroxide, and glacial acetic acid were purchased from Sigma-Aldrich, Steinheim, Germany. Liquid chromatography-mass spectrometry (LC-MS) water was purchased from Honeywell, Seelze, Germany. Dimethylsulfoxide (DMSO) was purchased from Thermo-Fisher, Eugene, OR, USA. Ethanol was purchased from Slavus, Slovakia. 3-(trimethylsilyl)-1-propanesulfonic acid-d${}_6$ sodium salt (DSS-d${}_6$) and deuterium oxide (D${}_2$O) were purchased from Eurisotop, Saint-Aubin, France. Maltotetraose and maltohexaose were purchased from Elicityl, Crolles, France. Ultrapure water was produced in-house.

A volume of urine equivalent of 10 $\mu$g of creatinine was diluted to 1 mL with ultrapure water in a triplicate for each urine sample. 10 $\mu$L of external standards solutions, maltotetraose and maltohexaose (c = 5 mg/mL) were diluted to 1 mL with ultrapure water and processed analogously to samples. The samples were first purified using 100 mg Supelclean LC-18 solid-phase extraction (SPE) columns (Sigma-Aldrich, Steinheim, Germany). The columns were washed with 1 mL 100% ACN, and 1 mL ultrapure water. The samples were applied to columns, the flowthrough was collected into clean tubes and 52 $\mu$L of 100% ACN was added to each tube. The samples were further purified using 100 mg Supelclean ENVI-Carb porous graphitized carbon (PGC) SPE columns (Sigma-Aldrich, Steinheim, Germany). The columns were washed with 1 mL 85% ACN, followed by 1 mL 5% ACN. The samples were applied on the columns and reapplied once. After washing with 1 mL 5% ACN, oligosaccharides were eluted by 1 mL 40% ACN and the eluate was dried under vacuum.

Fluorescent labeling was done using 2-AB according to a previously published method [16]. Briefly, 5 mg 2-AB was dissolved in 100 $\mu$L of glacial acetic acid : DMSO mix (3:7, v/v) and the mixture was added to 6 mg NaBH${}_3$CN. To each sample, 5 $\mu$L of labeling solution was added and the samples were incubated at 65 °C for three hours. The excess label was removed by hydrophilic interaction chromatography (HILIC) SPE using a Strata NH${}_2$96-well plate (50 mg/well, 55 $\mu$m, 70 Å; Phenomenex, Torrance, CA, USA) connected to the vacuum pump. The plate was flushed with 1 mL 70% ethanol, 1 mL ultrapure water, and 1 mL 96% ACN. The samples were diluted with 700 $\mu$L ACN and applied in the wells. After two-minute incubation on a shaker, the wells were flushed with 3 mL ACN. Oligosaccharides were eluted twice with 500 $\mu$L ultrapure water into a clean collection plate in a centrifuge (70 g, 15 min) after 15-minute incubation on a shaker (100 rpm). The eluate was dried under vacuum and reconstituted with 100 $\mu$L ultrapure water.

Oligosaccharides were analyzed using Thermo Accela HPLC (Thermo Fisher Scientific, San Jose, CA, USA) on a HILIC ACQUITY UPLC BEH Amide Column (130 Å, 1.7 $\mu$m, 2.1 mm $\times$ 150 mm) with ACQUITY UPLC Glycan BEH Amide VanGuard Pre-column (130 Å, 1.7 $\mu$m, 2.1 mm $\times$ 5 mm) (Waters Corp., Milford, MA, USA) using a modified technique based on the generic method described in column’s manual. To separate the oligosaccharides, a gradient elution of two mobile phases (MP) at 60 °C was used. MP A was 100 mM ammonium formate (pH 4.0) and MP B was 100% ACN. The HPLC gradient conditions are described in Table 1.

The analytes were detected using Dionex UltiMate 3100 fluorescence detector (Dionex Softron, Germering, Germany) with the excitation wavelength set to 250 nm and the emission wavelength set to 430 nm. A calibration ladder of 5, 10, 50, 100, 150, 200, and 250 nM of both standards was measured in triplicate. Chromatograms were processed in Chromeleon v. 7.2.10 (Thermo Fisher Scientific, San Jose, CA, USA). Limits of detection (LOD) and limits of quantification (LOQ) were calculated from obtained calibration curves. The samples were diluted to match the calibration range with a mixture of 70% ACN: 30% MP A before analysis. The concentration of oligosaccharides was calculated from individual peak areas according to calibration curves of maltotetraose and maltohexaose and expressed in $\mu$mol/mmol of creatinine as a summary concentration of position isomers.

Samples prior to HPLC separation, as well as the HPLC eluate, were analyzed by MALDI-TOF MS in order to match the structures to corresponding HPLC peaks. The eluate was collected into empty tubes, dried under vacuum, and reconstituted in 3 $\mu$L of LC-MS water. The structures were assigned to corresponding peaks using MALDI-TOF/TOF MS. 0.7 $\mu$L of matrix solution (20 mg/mL DHB in 30% ACN with the addition of 0.1% TFA and 1 mM NaOH) and 0.7 $\mu$L of sample were spotted onto a MALDI plate and analyzed using MALDI-TOF/TOF mass spectrometer UltrafleXtreme II (Bruker Daltonics, Bremen, Germany) in reflectron positive ion mode within the m/z range of 500–3000. Spectra were processed using flexAnalysis v. 3.4 (Bruker Daltonics, Bremen, Germany).

100 $\mu$L samples of urine were freeze-dried and re-dissolved in 250 $\mu$L of 0.9 mM DSS-d${}_6$ in D${}_2$O. D${}_2$O was used for magnetic field locking and shimming. DSS-d${}_6$ was used for chemical shift set up ($\delta$ = 0 ppm) as well as the internal standard for biomarker quantification. ${}^1$H NMR spectra were measured at 60 °C on AVANCE III HDX 600 MHz spectrometer (Bruker BioSpin, Karlsruhe, Germany) equipped with triple resonance inverse CryoProbe TCI H-C/N-D-05-Z using presat pulse sequence for residual water signal suppression, with following parameters: acquisition time AQ 3.5 s, relaxation time D1 4 s, digitization mode baseopt, 50 dB power level for presat, number of scans ns 128–256. For ${}^1$H-${}^{13}$C heterocorrelated HSQC NMR spectra hsqcedetgpsisp2.3 pulse sequence was used with the following parameters: AQ 0.2 s, D1 1.5 s, ns 300, F1 time domain TD1 256, baseopt digitization mode.

Anomeric H1 signals of all 1,3,6-linked $\beta$-mannose units of ManOS, present in the ${}^1$H NMR spectrum at $\delta$ 4.784–4.747 ppm, were diagnostic for the estimation of a total ManOS concentration in urine samples. ManOS quantity was calculated relative to that of internal standard DSS-d${}_6$, added to a phosphate buffer (pH 7.4) at a known concentration, as follows:

$$[\mathrm{m}]=\left(9\times {\mathrm{A}}_0\times \left[{\mathrm{A}}_{ref}\right]/\mathrm{b}\times {\mathrm{A}}_{ref}\right)\times \left({\mathrm{V}}_{\mathrm{S}}/{\mathrm{V}}_{\mathrm{U}}\right)$$

where [m] and [A${}_{ref}$] are the concentrations of the ManOS metabolites and DSS-d${}_6$, respectively; A${}_{\mathrm{0}}$ and A${}_{\mathrm{\text{ref}}}$ is the area of the H1 signal due to the 1,3,6-linked $\beta$-mannose units signal and that one of DSS-d${}_6$ signal, respectively, in the ${}^1$H NMR spectrum; 9 and b are the numbers of protons in DSS-d${}_6$ and in the m metabolite, respectively. V${}_{\text{U}}$ is the volume of the lyophilized urine sample; V${}_{\text{S}}$ is the volume of phosphate buffer.

Each sample from a monitored patient was analyzed in triplicates by both HPLC-FLD and ${}^1$H NMR. Average values, standard deviations (SD), and relative standard deviations (RSD) were calculated. To evaluate the changes in ManOS levels, the data were tested using one-tailed paired t-test ($\alpha$ = 0.05). To determine the data correlation between these two methods, Pearson’s correlation tests were performed. Pearson’s correlation coefficient (r) and p-value were calculated. The statistical analyzes were performed in Microsoft Excel v. 2202.

Neither HPLC-FLD, MALDI-TOF, nor ${}^1$H NMR-based approach detected signals representing ManOS in negative controls (data not shown). For the HPLC-FLD method, LOD and LOQ of both standards were determined as: LOD (Hex4) = 0.414 nM; LOQ (Hex4) = 1.25 nM; LOD (Hex6) = 0.357 nM; LOQ (Hex6) = 1.08 nM. The detector response was linear throughout the whole calibration range.

For an enhancement of signals’ resolution, the ${}^1$H-${}^{13}$C heterocorrelated HSQC spectra were also measured. Obtained NMR data were assigned to individual types of mannose units and GlcNAc by comparison with literature data [17]. Because proton chemical shifts of mannose units were nearly identical for some of them, only the total concentration of ManOS could be calculated from a well resolved, but overlapped anomeric H1 signals of all 1,3,6-linked $\beta$-mannose unit ($\delta$${}_{\text{H}}$ 4.784–4.747 ppm) (Fig. 1).

Fig. 1.

Selected region of ${}^{\mathrm{1}}$H NMR spectra in which anomeric H1 signals of saccharides appear. The diagnostic signal of 1,3,6-linked mannose units in $\beta$-configuration (1,3,6-$\beta$Man) in mannose-rich oligosaccharides, accumulated in the urine of alpha-mannosidosis patients, is highlighted by red color. GlcNAc${}_{\text{red}\alpha }$ and GlcNAc${}_{\text{red}\beta }$ – reducing end glucosamine in $\alpha$- and $\beta$-configuration; Man, mannose; HOD, residual signal of water.

HPLC-FLD and NMR methodology for determination of an overall ManOS content was set up on a set of five urine samples collected from four AM patients not subjected to ERT yet. Obtained values are shown in Table 2. Additionally, samples from one representative patient (9 years old male) were collected twice, with a four-month time lapse, on account of monitoring the patient’s status and demonstrating the potential changes in ManOS levels in time.

The different values obtained by both techniques lie in the mode of sample preparation for measurement. While for HPLC-FLD, the derivatization is required for ManOS detection, the urine is used without any treatment for NMR. To compare two different approaches including diverse enrichment methods (sample derivatization for HPLC-FLD analysis), the correlation coefficient was determined. A value of 0.915 of Pearson’s correlation coefficient suggested a strong positive correlation between data obtained by NMR and HPLC-FLD, even though each method produced different absolute values of ManOS concentration in the samples.

Previously optimized HPLC-FLD method was used for further analysis of the set of samples from AM patients without ERT, was applied further for the analysis of urine samples of another patient collected (i) prior to ERT onset, (ii) after one month and (iii) after four months of ERT. Representative MALDI-TOF MS spectrum and HPLC-FLD chromatogram of the sample before ERT onset are shown in Fig. 2. All labeled peaks in the HPLC-FLD chromatogram (Fig. 2B) were identified by MALDI-TOF analysis performed on collected single eluates. Oligosaccharides’ precise isomeric structures cannot be determined by the utilized methods, therefore individual ManOS were quantified as a sum of position isomers. Due to a severe limitation of the MALDI-TOF approach in the distinction of stereoisomers, the identity of mannose units in detected oligosaccharides was confirmed by NMR.

Fig. 2.

Representative MALDI-TOF spectrum (A) and HPLC-FLD chromatogram (B) of urinary oligosaccharides in alpha-mannosidosis. Profiles were determined in the alpha-mannosidosis patient sample before the ERT administration. HPLC peaks within the brackets represent position isomers of mannose-rich oligosaccharides (Man2GlcNAc - Man9GlcNAc). GlcNAc, N-acetylglucosamine; Man, mannose.

Based on the results from HPLC-FLD, prior to ERT onset, the total level of ManOS (calculated as the sum of single Man2GlcNAc - Man9GlcNAc oligosaccharides) was 107.87 $\pm$ 42.64 $\mu$mol/mmol crea. After one month, the level of ManOS decreased almost two-folds to 57.42 $\pm$ 4.75 $\mu$mol/mmol crea and after four months, a significant (p $\mathrm{\le }$ 0.05) decrease in total ManOS level was detected, represented by the value of 12.35 $\pm$ 2.44 $\mu$mol/mmol crea. Changes of ManOS levels during ERT are further discussed in section 3.5.

${}^1$H NMR urine spectra of the AM patient undergoing ERT are shown in Fig. 1. In the urine of the AM patient under ERT, collected prior to ERT onset, the level of total ManOS was 433.67 $\pm$ 2.36 $\mu$mol/mmol crea. After one month, it decreased significantly (p $\le$ 0.001) almost two-folds (222.00 $\pm$ 11.43 $\mu$mol/mmol crea). This result is in accordance with the HPLC-FLD data. After four months, an almost twelve-folds significant (p $\le$ 0.001) decrease in total ManOS level (36.00 $\pm$ 0.00 $\mu$mol/mmol crea) was observed. Changes in total urinary oligosaccharide levels during ERT determined by ${}^1$H NMR were then compared with HPLC data (section 3.5).

A detailed analysis has shown that in the HSQC spectrum (data not shown) of the baseline urine sample (before ERT administration), the cross-peak signals, specific for a trisaccharide Man2GlcNAc, were dominant: the signal of the terminal $\alpha$Man unit $\delta$${}_{\text{H,C}}$ 5.13/104.8, which was (1$\to$3)-linked to $\beta$Man $\delta$${}_{\text{H,C}}$ 4.77/102.8 (1,3,6-$\beta$Man) which is (1$\to$6)-linked to the reducing end GlcNAc. Its $\alpha$ anomer was present at $\delta$${}_{\text{H,C}}$ 5.21/93.0 and $\beta$ anomer at $\delta$${}_{\text{H,C}}$ 4.73/97.6 ppm. Other cross-peaks present in the spectrum were of low intensity. This finding is in accordance with data obtained by HPLC-FLD. Due to the low specificity of chemical shifts of individual mannose units in other oligosaccharides present in the urine, and low intensities of signals their structures were not identified.

The levels of individual ManOS were determined by HPLC-FLD. For the evaluation of relative changes of Man2GlcNAc - Man9GlcNAc over the course of ERT, individual levels of oligosaccharides were normalized to those determined in the sample before therapy administration. Mean relative changes from baseline values of individual urinary oligosaccharides over the course of ERT according to HPLC-FLD are shown in Fig. 3. In the baseline sample, collected prior to the ERT, the levels of individual ManOS estimated by HPLC-FLD were following (in $\mu$mol/mmol crea $\pm$ 1 SD): 70.56 $\pm$ 27.84 (Man2GlcNAc); 2.70 $\pm$ 1.10 (Man3GlcNAc); 5.62 $\pm$ 2.37 (Man4GlcNAc); 10.63 $\pm$ 4.32 (Man5GlcNAc); 7.55 $\pm$ 3.14 (Man6GlcNAc); 5.27 $\pm$ 1.82 (Man7GlcNAc); 3.50 $\pm$ 1.17 (Man8GlcNAc) and 2.05 $\pm$ 0.90 (Man9GlcNAc). The levels of Man7GlcNAc - Man9GlcNAc decreased rapidly after one month of therapy, however, the levels of smaller ManOS (Man2GlcNAc - Man6GlcNAc) decreased more prominently only after four months. An unambiguous significant decrease of Man2GlcNAc and Man7GlcNAc - Man9GlcNAc was observed after four months of ERT.

Fig. 3.

Mean relative changes from baseline values (prior to ERT administration) of individual urinary oligosaccharides (Man2GlcNAc - Man9GlcNAc) over the course of ERT determined by HPLC-FLD. Individual ManOS concentrations are normalized to their levels prior to therapy administration (baseline values represent 100 %); n = 3, error bars represent $\pm$ 1 SD; * = p $\le$ 0.05; GlcNAc, N-acetylglucosamine; Man, mannose; ManOS, mannose-rich oligosaccharides.

After the comparison of ${}^1$H NMR and HPLC-FLD data, it was found that ManOS levels estimated by NMR were approximately 2.9–3.9-folds higher than those obtained from HPLC-FLD analysis (summarized in Fig. 4). Nearly the same ratio between NMR and HPLC-FLD ManOS data (from NMR 2.5–3.4-folds higher values) was observed for urine samples collected from positive controls - diagnosed AM patients without ERT therapy (Table 2).

Fig. 4.

Changes in total urinary oligosaccharide levels of alpha mannosidosis patient during ERT determined by HPLC-FLD and 1H NMR. Expressed as $\mu$mol/mmol creatinine; n = 3, error bars represent $\pm$ 1 SD; * = p $\le$ 0.05, ** = p $\le$ 0.001.

Pearson’s correlation coefficient for the data from ERT efficacy monitoring (samples prior to ERT onset, after one month, and after four months of ERT) obtained from two different analytical approaches was calculated as 0.99 (p 0.001), which represents a strong positive correlation, even though each method produced different absolute values of ManOS concentration in the samples (Fig. 4).

Contrary to HPLC-FLD analysis, ${}^1$H NMR spectroscopy does not require sample derivatization or SPE clean-up. MS-based techniques usually require sample preparation steps, which can cause metabolite losses [28]. High reproducibility is one of the fundamental advantages of NMR spectroscopy. However, peak overlaps from multiple metabolites pose major challenges for NMR spectroscopy [29]. Furthermore, NMR is nondestructive, thereby allowing further analysis of the same sample, if needed [30].

When compared with other published literature concerning the absolute quantification of ManOS by HPLC-FLD [12], LC-ESI-MS/MS [31], and HPLC-PAD [32], the quantities determined in our study by HPLC-FLD are in better accordance than those estimated by NMR (Fig. 4). However, the absolute quantification of urinary ManOS by LC-MS/MS is problematic due to a lack of external and internal standards and, in the case of HPLC-FLD, the need for labeling and SPE, which can cause the loss of analytes [13]. Nevertheless, monitoring of ERT efficacy based on the determination of ManOS levels is a unique approach providing complementary information, as usually only the clinical parameters and unspecific improvement of a patient’s subjective condition are monitored. Furthermore, the precise determination of ManOS levels might become an essential addition to the management of AM patients, because it is speculated that high levels of circulating oligosaccharides (in addition to intracellular accumulation) may contribute to the immunodeficiency common in AM patients, since they bind to interleukin-2 (IL-2) receptors, disturbing IL-2-dependent responses [33, 34].

The main advantage of the HPLC-based approach is its relatively adequate accessibility in laboratories in healthcare facilities. MS instruments are also much more commonly found in clinical centers and hospitals compared with NMR spectrometers [23]. However, more sample preparation steps are required, and even if the method can be almost fully automated, the HPLC- based approach is more expensive, reagents- and time-consuming than NMR. On the other hand, hospitals usually do not have access to highly sophisticated NMR instruments, which require advanced maintenance and operation. Thus the collaboration between the scientific community and clinicians is an essential tool to understand the molecular basis of diseases and the biochemical mechanisms of their respective therapies.

Relative intensities of ManOS signals in the MALDI-TOF spectra are in accordance with published data [15]. Man2GlcNAc trisaccharide was identified as the most abundant structure in all AM samples in accordance with literature data [19]. Previous studies have linked the ManOS levels to AM severity [12] and treatment response either by HSCT [35] or ERT [13]. During Phase I-II of clinical testing of rhLAMAN, Borgwardt et al. [31] observed an overall 54.1% decrease in urinary ManOS in 10 patients over 12 months. Santoro et al. [13] reported a 67% decrease in urinary ManOS in a 7-month-old infant after two months of ERT. In our work, we observed an 88.6% decrease in total urinary ManOS after four months of ERT. This difference in ERT efficacy can be related to age, phenotypic variation, residual activity of $\alpha$-mannosidase, accumulation of ManOS in tissues, immune response to treatment, and progression of the disease.

A more rapid decrease of urinary ManOS of higher molecular weights in comparison with smaller ManOS was observed. This can be attributed to the conversion of higher to smaller ManOS due to therapeutically increased enzyme activity. These truncated structures then might contribute to the pool of structures of lower molecular weights. Therefore, after just one month, the decrease of levels of Man3GlcNAc - Man5GlcNAc is less significant, even though the overall ManOS decrease is approximately 50%. The full effect of ERT is the most remarkable after a longer period. Early biochemical effects of velmanase alfa in a 7-month-old infant with alpha-mannosidosis were determined recently in the study mentioned above [13], where the OS containing 2, 3, and 4 mannose residues decreased while the other ManOS with higher molecular weight were already undetectable by TLC after 8 weeks of treatment. However, the overall efficacy of ERT might vary from person to person. Immune response and the antibodies against the foreign infused enzyme are important issues of the ERT and play a pivotal role in the patients’ safety as well as the efficacy and success of the treatment. Furthermore, the biodistribution of the enzyme into the difficult sites of pathology (especially into the central nervous system, bones, etc.) still remains a striking challenge [36]. Nevertheless, the treatment should be initiated as early as possible to prevent further development and consequences of the disease as the full impact of this therapy will be fully understood when it is used on a cohort of patients identified soon after birth [37].