HPLC method for amino acids profile in biological fluids and inborn metabolic disorders of aminoacidopathies

Quantification of total and individual amino acids in biological fluids such as plasma, urine and cerebrospinal fluid has an important diagnostic implication in laboratory medicine. The present paper describes protocols for the assay of total amino acids by modified method based on dinitrophenyl and HPLC profile involving pre-column derivatization with o-pthalaldehyde (OPA) derivatization, respectively. The method, based on the alkylation of-SH groups prior to OPA derivatization of amino acids followed by reverse phase high performance liquid chromatography, provide a comprehensive profile of more than twenty amino acids (including-SH group containing) in a single run lasting about 45 minutes. The present study, apart from establishing the normal profile of amino acids in plasma of Indian sub population, also presents HPLC profile for some of the rare amino acidopathies.     

Microfluidics platform for measurement of volume changes in immobilized intestinal enteroids

Intestinal enteroids are ex vivo primary cultured single-layer epithelial cell spheroids of average diameter ∼150 μm with luminal surface facing inward. Measurement of enteroid swelling in response to secretagogues has been applied to genetic testing in cystic fibrosis and evaluation of drug candidates for cystic fibrosis and secretory diarrheas. The current measurement method involves manual addition of drugs and solutions to enteroids embedded in a Matrigel matrix and estimation of volume changes from confocal images of fluorescently stained enteroids. We developed a microfluidics platform for efficient trapping and immobilization of enteroids for quantitative measurement of volume changes. Multiple enteroids are trapped in a “pinball machine-like” array of polydimethylsiloxane posts for measurement of volume changes in unlabeled enteroids by imaging of an extracellular, high-molecular weight fluorescent dye. Measurement accuracy was validated using slowly expanding air bubbles. The method was applied to measure swelling of mouse jejunal enteroids in response to an osmotic challenge and cholera toxin-induced chloride secretion. The microfluidics platform allows for parallel measurement of volume changes on multiple enteroids during continuous superfusion, without an immobilizing matrix, and for quantitative volume determination without chemical labeling or assumptions about enteroid shape changes during swelling.     

Association of Insulin Resistance with Adipocytokine Levels in Patients with Metabolic Syndrome

Metabolic syndrome (MetS) results from the derangement of adipocyte physiology and carbohydrate metabolism. Obesity and insulin resistance (IR) are integral features of MetS. The adipokine alterations in MetS often correlate with IR and body fat content. High adipose tissue content is associated with a decreased production of adiponectin and excessive production of tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6), all of which induce IR. The present study evaluated the adipokine alterations in MetS and their association with IR. The findings of the current study indicate that MetS is associated with significant decrease in adiponectin and increase in TNF-α and IL-6. The present study also found that the adipocyte derived inflammatory adipokines, TNF-α and IL-6 correlate with IR while the anti-inflammatory adipokines, adiponectin does not correlate with the degree and severity of IR.