RESUMEN
Alkaline phosphatase is an enzyme that converts para-nitrophenyl phosphate to para-nitrophenol (yellow coloured) in 2-amino, 2-methyl, 1-propanol buffer at pH 10.5. However, when this protocol is applied to the in vitro cellular model systems to estimate alkaline phosphatase activity, it tends to generate clumps of genomic DNA, leading to inaccurate pipetting for protein estimation. The aim of the study was to introduce minor modifications in the existing protocol to make it simple, cost-effective, with minimal labor-intensive procedures while estimating alkaline phosphatase activity in cellular model systems. The genomic DNA clumps were dissolved by depurination (adding 0.2 N HCl) and fragmentation (adding 0.2 N NaOH) during enzyme estimation. Moreover, these minor modifications have been standardized and optimized extensively by using serum samples (rich source of alkaline phosphatase), hFOB/ER9 (human Fetal osteoblastic cell) and HepG2 cells. Our results suggest that the modification incorporated in previously published method was robust enough to estimate ALP activity and protein concentration accurately. There was no significant variation in ALP activity estimated after modification (P > 0.05). This innovative approach could be beneficial for a researcher by providing an easy, cost effective and less labor-intensive solution for estimation of enzymatic activity in cellular model systems.
RESUMEN
BACKGROUND: Polymorphisms of the insulin-like growth factor (IGF)-1 gene consisting of variable cytosine adenosine repeats in the promoter region may directly influence the expression of IGF-1. OBJECTIVE: We sought to assess the role of IGF-1 gene polymorphisms in determination of plasma IGF-1 levels, acne, and its severity. METHODS: In this case-control study, 80 patients with acne vulgaris of 4 severity grades as per Global Acne Grading System and 80 age- and gender-matched control subjects without acne were studied. All the study subjects were without any disorder or a history of drug intake likely to affect IGF-1 level within a year before the study inclusion date. IGF-1 polymorphism was determined by polymerase chain reaction and plasma levels of IGF-1 by enzyme-linked immunosorbent assay. Acne severity was assessed by Global Acne Grading System. RESULTS: Mean plasma IGF-1 level in acne cases was significantly higher than in non-acne controls (P = .04). Plasma IGF-1 level positively correlated with severity of acne (P = .01). Individuals homozygous for the 192-base pair (bp) allele had 4.29 times odds risk (95% confidence interval 1.38-13.33) of having acne and a significantly higher mean level of IGF-1 compared with non-192/non-192 participants. Individuals homozygous for the 192-bp allele had 3.08 times odds risk (95% confidence interval 1.15- 8.31) of having higher severity grade of acne compared with non-192/non-192 participants. LIMITATIONS: A relatively small number of participants were studied. CONCLUSIONS: Plasma IGF-1 levels positively correlate with severity of acne. The 192/192 homozygotes had higher risk of acne and higher severity grade of acne. Functional studies showing the relationship between IGF-1 promoter level polymorphism and actual gene expression in skin are warranted.
