Slavica Ribar

High-performance liquid chromatographic determination of sphinganine and sphingosine in serum and urine of subjects from an endemic nephropathy area in Croatia

Endemic nephropathy (EN) is a chronic renal disease present as an endemic in Brodska Posavina, Croatia. The aim of the study was to assess the possible role of fumonisins, i.e., mycotoxins produced by Fusarium moniliforme, as causative agents for EN. Fumonisins inhibit ceramide synthase, the enzyme of de novo synthesis of sphingolipids, which leads to an increase in the sphinganine/sphingosine ratio. In the present study, a modified method has been used for the determination of the sphinganine/sphingosine ratio in human serum and urine of healthy subjects and EN patients from the endemic area. Free sphingoid bases, sphinganine and sphingosine, were obtained by base hydrolysis. Afterwards, precolumn ortho-phthaldialdehyde derivatisation, HPLC separation and quantification by fluorescence detection were performed. The results thus obtained pointed to a sphingolipid metabolism impairment, which may have been induced by fumonisins or fumonisin-like mycotoxins. As statistically significant differences were recorded in the subjects not yet affected with EN, an impairment in the metabolism of sphingolipids might be considered as an early indicator of EN.

Natural zeolite clinoptilolite increases the concentrations of sphingoid bases in the yeast Yarrowia lipolytica

In the present paper, we studied the effect of natural zeolite clinoptilolite on sphingolipid metabolism in the yeast Yarrowia lipolytica. We also investigated if zeolite addition had any impact on cell shape and size, as well as on the pH alterations during the culture growth. High performance liquid chromatography analysis of sphingoid bases obtained by acid hydrolysis of complex sphingolipids from Y. lipolytica showed that their concentrations markedly rose upon the zeolite addition. The largest increase among the identified molecular species of sphingoid bases was seen in C18 phytosphingosine, whose levels rose 6.2‐fold and 22.3‐fold after culturing cells for 24 and 36 hours respectively in the presence of finely ground zeolite. pH measurements of the culture medium showed a similarity between pH profiles of control and zeolite‐supplemented cells, suggesting that ion‐exchange capacity was not probably responsible for the observed change in sphingolipid metabolism. Scanning electron microscopy revealed that zeolite affected cell size and shape. Y. lipolytica cells grown in the absence of zeolite were oval‐shaped with an average cell size of 0.7–2.7 μm, whereas when cultured with zeolite, they were round‐shaped and larger, having an average cell size of 1.3–2.9 μm.

Dependence of the sphingoid bases concentration on growth phase and temperature in the yeast Yarrowia lipolytica

The aim of this research was to investigate the effect of growth phase and temperature on the concentrations of the individual molecular species of sphingoid bases obtained by acid hydrolysis of total sphingolipids from the yeast Yarrowia lipolytica. Our results showed C18 phytosphingosine to be the major long‐chain base in Y. lipolytica regardless of growth phase or temperature. We also found Y. lipolytica to contain sphingosine, the predominant mammalian long‐chain base that is uncommon for yeast sphingolipids. Among the identified long‐chain bases, only C18 phytosphingosine appeared to be influenced by culture conditions. Its concentration was largest in the exponential phase and decreased 2.9‐fold when cells entered the stationary phase of growth at 28 °C. Following a temperature shift from 28 to 39 °C, there was a 2.1‐fold decrease in the phytosphingosine concentration, but it rose 1.7‐fold after the heat‐stressed cells had been returned to 28 °C and subjected to prolonged growth. These results might point to the possible involvement of phytosphingosine in the cell growth regulation and in the adaptation of Y. lipolytica cells to stressful culture conditions.