Identification of Vitamin D₃ Hydroxylated Metabolites in Solanum glaucophyllum Leaves: Towards Its Biosynthetic Pathway Elucidation ^†

Abstract

Solanum glaucophyllum is a species of the Solanaceae family that causes enzootic calcinosis by vitamin D₃ intoxication in breeding cattle grazing in Argentina. Inspired by thrusting forward the study of the biosynthetic pathway of vitamin D₃ in S. glaucophyllum, the aim of the present work is to provide knowledge of vitamin D₃ toxicity related to concentration in the mentioned species located throughout the Río Salado basin. We present the development of a reverse-phase high-performance liquid chromatography (RP-HPLC) method for the analysis of vitamin D₃ and its hydroxylated metabolites. An exhaustive optimization of the RP-HPLC method allowed us to perform calcitriol (1α,25-(OH)₂D₃) and calcidiol (25-OHD₃) identification, both metabolites obtained by the chemical hydrolysis of S. glaucophyllum leaves starting from its hydrophilic extract. These results allow the design of a monitoring scheme for S. glaucophyllum, achieving a more selective control of this weed to avoid cattle decline in the Río Salado basin.

1. Introduction

Solanum glaucophyllum belongs to the Solanaceae family and is widely distributed in the flooded areas of natural grasslands in the northeast of Argentina. Since 1960, its consumption by cattle has been associated with vitamin D₃ intoxication, known as enzootic calcinosis. This particular disease is related to alteration in the absorption of vitamin D₃, which produces calcium depositions in the muscle tissues and joints of cattle. It is a progressive disease with signs such as stiffness, painful gait, and loss of body condition, and it is responsible for significant mortality in affected ruminants that nowadays produces important economic losses for Argentine livestock [1,2].

Concerning bibliographic background, vitamin D₃ is known to be present in species belonging to the Solanaceae family. The principal hydroxylated metabolite found is 1α,25-(OH)₂D₃, known as calcitriol, and is mainly conjugated with carbohydrates as 1α,25-(OH)₂D₃-glycoside [3]. The biologically most active metabolite, calcitriol, is associated with pathological signs of the mentioned disease and has been identified in S. glaucophyllum from the enzymatic hydrolysis of the water: ethanol extract obtained from the leaves of this species [4]. In cattle, calcitriol is released through the enzymatic hydrolysis of 1α,25-(OH)₂D₃-glycoside at the ruminal level after S. glaucophyllum leaf ingestion [1].

In addition, vitamin D₃ in S. glaucophyllum, showed a similar to vertebrate’s photodependent process of synthesis, but there is few researching regarding quantitative studies about the vitamin D₃ biosynthetic pathway of the plant [5]. In vertebrates, vitamin D₃ is synthesized upon UVB irradiation. The UVB irradiation of provitamin D₃ (7-dehydrocholesterol) in the skin breaks the B-ring to form previtamin D₃, which rearranges itself in vitamin D₃ (cholecalciferol). Vitamin D₃ is transported to the liver where it is enzymatically hydroxylated at C-25 by the 25-hydroxylase enzyme, producing 25-OHD₃ (calcidiol). The 25-OHD₃ is hydroxylated for a second time at C-1 in the kidneys to the active metabolite 1α,25-(OH)₂D₃ (calcitriol) (Scheme 1).

Inspired by thrusting forward the study of the biosynthetic pathway of vitamin D₃ in S. glaucophyllum, the aim of the present work is to provide knowledge of vitamin D₃ toxicity related to concentration in the mentioned species located throughout the Río Salado basin. High-performance liquid chromatography (HPLC) allows qualitative and quantitative analyses in order to know the presence and content of related compounds in S. glaucophyllum with highly accurate and sensitive results. Herein, we present the development of a reverse-phase high-performance liquid chromatography (RP-HPLC) method for the determination of vitamin D₃ and its hydroxylated metabolites. So far, the present work contributes to validating the optimal starting amount of S. glaucophyllum leaves to be hydrolyzed, the chemical hydrolysis conditions, and the method of analysis of the main metabolites in the plant leaf materials.

2. Materials and Methods

2.1. General

The solvents used for extraction and chromatography were previously distilled. The HPLC analysis was performed using isopropanol (HPLC gradient grade for liquid chromatography, LiChrosolv), acetonitrile (HPLC gradient grade for liquid chromatography, LiChrosolv), and ultrapurified Milli-Q water (Millipore, Billerica, MA, USA). All the solvents were degassed by simultaneous sonication and filtration through 0.2 µm PTFE membranes prior to use. Calcitriol (1α,25-(OH)₂D₃) and calcidiol (25-OHD₃) analytical standards were obtained from Sigma-Aldrich (St. Louis, MO, USA). For column chromatography, neutral aluminum oxide Fluka Typ 507C (100–125 mesh) was used. The chromatographies were monitored with thin-layer chromatography (TLC) on silica gel plates (60F-254) and visualized under UV light or by using a p-anisaldehyde solution (5 mL p-anisaldehyde, 5 mL H₂SO₄ concentered, 1mL acetic acid, and 90 mL ethanol).

The HPLC analysis was conducted on an LS-MS-Thermo Scientific -UltiMate 3000-MSQ PLUS HPLC system equipped with an Agilent Zorbax SB-Aq stable bond analytical C18 reverse-phase column and a fixed wavelength UV detector. Calcitriol (1α,25-(OH)₂D₃) and calcidiol (25-OHD₃) were monitored at an absorbance of 265 nm. UV spectra for maximal wavelength standards determination were recorded with an Agilent Cary 60 UV–Vis spectrophotometer. Stock calcitriol and calcidiol standard mixtures were generated using a molar extinction coefficient of 18,300 AU M^–1 L^–1 at 265 nm in HPLC-grade isopropanol. The prepared stock standard mixtures were perfused with nitrogen and stored at −20 °C.

2.2. Plant Material

Solanum glaucophyllum (Solanaceae) plant specimens were collected at Dolores in the Buenos Aires province of Argentina from the La Quebrada location (36°17′21.99″ south latitude and 57°36′14.76″ west longitude) in April 2021.

2.3. Harvest and Extraction

The plant material was harvested by hand, preferably at noon to avoid dew and excessive humidity. The stems were separated, and the leaves were spread on the ground indoors to dry superficially, separate from the rest of the soil and foreign bodies. After 24 h, the samples were placed in an oven at 36 °C until reaching a constant weight; this took approximately 3 days.

Finely ground dry leaves of S. glaucophyllum (163 g) were extracted with a solution (1250 L) of water:ethanol (80:20) at 40 °C for 8 h. The extract was concentrated under reduced pressure, giving 36.7 g (3.7%).

2.4. Chemical Hydrolysis

In a 250 mL two-necked round-bottomed flask equipped with a condenser loaded with S. glaucophyllum extract (0.7 g), 2 N HCl solution (140 mL) was added. The mixture was stirred for 13 h at 85 °C and for an additional 18 h at room temperature. Then, the reaction mixture was extracted with ethyl acetate (5 × 50 mL). The organic layer was dried over MgSO₄ and concentrated under reduced pressure to give 0.126 g of hydrolyzed extract.

2.5. Purification of Hydrolyzed Extract

Chromatography on neutral aluminum oxide (100–125 mesh) of a portion of the hydrolyzed extract (50.0 mg) eluted with dichloromethane:methanol (100:0 to 95:5, step-gradient system) yielded forty tubes. Chromatographic separation was monitored by TLC using a mixture of dichloromethane:methanol (90:10) as mobile phase. Two major compounds, 1 and 2, were observed in tubes 1 and 25, respectively, weakly seen by their stain color on the plate.

2.6. Calcitriol (1α,25-(OH)₂D₃) and Calcidiol (25-OHD₃) Identification

Hydrophilic extract pellets obtained from S. glaucophyllum leaves were previously purified by chromatography before HPLC analysis. All the tubes containing analytes in purified extract fractions were vacuum-dried and dissolved in isopropanol (HPLC grade). The prepared solutions were filtered through a 0.2-μm PTFE syringe filter and placed in brown 2 mL HPLC autosampler vials with Teflon-coated lids. The HPLC analysis was conducted with an LS-MS-Thermo Scientific -UltiMate 3000-MSQ PLUS HPLC system equipped with a fixed wavelength UV detector. Calcitriol (1α,25-(OH)₂D₃) and calcidiol (25-OHD₃) were monitored at an absorbance of 265 nm. HPLC was performed with a flow rate of 0.7 mL/min⁻¹ by binary pumps at 25 °C. The mobile phase consisted of acetonitrile (HPLC gradient grade for liquid chromatography, LiChrosolv) and Milli-Q water. Ultrapurified water was prepared with a Milli-Q Advantage system (Millipore, Billerica, MA, USA), giving a product with a resistivity of ~18.5 MΩ/cm-1. After loading the column with each extract purified fraction dissolved in isopropanol HPLC, the mobile phase was programmed with an isocratic ratio of 90:10 acetonitrile:water solution over 18 min. Between each sample injection, an isopropanol blank (HPLC grade) was run. The peak retention times of 1α,25-(OH)₂D₃ and 25-OHD₃ analytical standards were employed for the identification of these metabolites in the samples.

3. Results and Discussion

In the present study, 1α,25-(OH)₂D₃ and 25-OHD₃ free aglycone metabolites in S. glaucophyllum leaves were identified by HPLC analysis [7,8,9,10]. The exhaustive screening of the RP-HPLC method allowed us to perform both metabolite identifications in the same elution procedure, starting from individual stock calcitriol and calcidiol standards. The HPLC-optimized conditions for the analysis of vitamin D₃ metabolites are shown in

Table 1. Optimized conditions for chromatographic analysis by HPLC.

Column	Agilent Zorbax SB-Aq Stable Bond Analytical C18 Reverse-Phase (4.6 mm × 250 mm; 5 μm, Agilent)
Mobile phase	ACN:H2O (90:10)
Injection time:	18 min
Flow rate:	0.7 mL/min−1
Column temperature:	25 °C
Wavelength UV detector:	445 nm

.

The HPLC chromatogram of the 1α,25-(OH)₂D₃ and 25-OHD₃ standard mixtures are shown in Figure 1a with peak retention times of 5.343 min and 6.710 min, respectively. All the purified extracts obtained by the chemical hydrolysis of S. glaucophyllum leaves starting as hydrophilic extract were analyzed. The 1α,25-(OH)₂D₃ and 25-OHD₃ peaks from purified sample tubes 25 and 1 were respectively assigned by comparing their retention times with those of the pure standards. Figure 1b shows the 1α,25-(OH)₂D₃ peak from purified sample tube 25 with a retention time of 5.343 min. Figure 1c shows the 25-OHD₃ peak from purified extract tube 1 with a retention time of 6.710 min.

4. Conclusions

In order to validate the optimal chemical hydrolysis conditions and the method of analysis of the main vitamin D₃ metabolites in the plant leaf materials of S. glaucophyllum, a preliminary quantitative HPLC method was performed. The results obtained here lay the groundwork to improve the process of enzymatic hydrolysis with promising results regarding vitamin D₃ metabolite quantification in the specie. These results improve a smart design management and monitoring scheme for S. glaucophyllum, achieving a more selective control of this weed, avoiding cattle decline in the Río Salado basin located in the northeast of Argentina.