Folate-HRP and you can biotinylated folate binding proteins (FBP) conjugate were extra

Folate-HRP and you can biotinylated folate binding proteins (FBP) conjugate were extra

Adopting the alkaline denaturation (to release folate from the endogenous joining necessary protein) and you can stabilization (to get rid of oxidization), an aliquot of one’s treated attempt was transferred for the good streptavidin-painted better. Following a competitive binding impulse (folate about solution try competes with HRP-branded folate for a finite quantity of binding websites to the a beneficial biotinylated FBP), new FBP buildings was caught because of the streptavidin toward wells. Unbound conjugates had been got rid of by the washing.

The brand new sure HRP conjugates to have 25-OH vitamin D, B12 and you will folate was indeed counted by a luminescent effect (18). Reagent containing luminogenic substrates (luminol by-product and you can peracid salt) and you may a keen electron transfer broker (substituted acetanilide) is placed into the brand new wells. Oxidization of one’s luminol derivative was catalyzed by the HRP in the this new bound conjugates, for this reason promoting white. This caffmos odwiedzajÄ…cych new strength and you may duration of white emission have been enhanced by the electron transfer representative, and white signals was basically comprehend by program. The amount of HRP conjugate likely was indirectly proportional towards intensity of twenty five-OH vitamin D, nutritional B12 and you may folate found in brand new try, respectively.

For vitamin D, ?30 ng/ml was considered as normal, <30 ng/ml was classified as low vitamin D and <20 ng/ml as vitamin D deficiency. For vitamin B12, ?200 pg/ml was considered as normal and <200 pg/ml as deficiency. For folic acid, ?3 ng/ml was considered as normal and <3 ng/ml as deficiency.

Analytical Analysis

Statistical analysis was done using JASP open-source software, version 0.14.1. Primary quality assessment of range and consistency of the variables was done to check appropriateness of units and deviation from the population mean, to detect outliers. Age (45–54, 55–64, 65–74, ?75 years) and gender distributions were tabulated. Frequency distributions of vitamin D, vitamin B12 and folic acid levels in the population were plotted. Mean levels of the above parameters were calculated. “t” test was performed to check for significant differences between mean values of males and females – both overall and in each of the age groups. Ain B12 and folic acid levels between all age groups and also, between age groups among females and males separately. Percentage of subjects having deficiency of the studied micronutrients was calculated according to gender as well as among different age-groups. Chi-squared test was done to check for any significant differences, gender-wise and age-group-wise. P-value of < 0.05 was considered as significant.


Gender distribution of the study population was 47.1% (n = 776) males and 52.9% females (n = 872). Mean age of the population was 58 ± 10.2 years, with males having a higher mean age than females (59.5 ± 10.4 vs. 56.6 ± 9.8 years). Out of the 1,648 subjects in this analytical sample, vitamin D levels were available for 1,546 subjects, vitamin B12 levels were available for 1,639 subjects and folic acid levels were available for 1,640 subjects. Frequency distribution of vitamin D, vitamin B12 and folic acid levels are shown in Figure 1. Mean levels of vitamin D, B12 and folic acid were 23.4 ± 10.6 ng/ml, 277.4 ± 194.4 pg/ml and 6 ± 3.5 ng/ml, respectively. Age and gender-stratified means with standard deviations for the above three parameters are represented in Table 1. Significant gender difference was observed in mean values for vitamin D (males > females; p < 0.001) and folic acid (females > males; p < 0.001) but not for vitamin B12. Significant difference was also seen in mean vitamin D among levels between different age groups – overall (f = 3.73, p = 0.011) as well as among males (f = 7.74, p < 0.001) but not among females (f = 2.01, p = 0.11). Tukey post-hoc test showed that there was significant difference in 55–64 years age group as compared to ?75 years age group. Among males, this difference was significant in 45–54 years age group as compared to ?75 years age group. Similarly, significant difference was also seen in 55–65 years age group as compared with 65–74 years and ?75 years age groups among males. Significant difference was seen in vitamin B12 means between different age groups – overall (f = 4.99, p = 0.002) as well as among females (f = 5.68, p < 0.001) but not among males (f = 1.01, p = 0.38). On post-hoc analysis, significant difference was seen when the mean of 45–54 years age group was compared to 65–74 years and ?75 years age groups. A similar difference was also observed between the same age groups among females. There was no statistically significant difference for the folate levels among different age groups, neither in the overall age groups nor in the gender-stratified age-groups.


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