Consideration of diurnal variations in human blood NAD and NADP concentrations.

The sum of the urinary excretion of nicotinamide and its catabolites, which are metabolites of NAD and NADP, were observed to have clear diurnal variations in human urine. Then, we examined whether NAD and NADP in blood also showed the diurnal variation. All subjects were housed in the same facility and given the same diet during the experiment. In addition, we examined whether diurnal variations were affected by the intakes of dietary nicotinamide or not. As a result, neither the NAD nor the NADP content of the blood shows the diurnal variation regardless of the administered amount of nicotinamide. The concentrations of NAD and NADP did not increase according to the intake of nicotinamide. The existence of a mechanism by which NAD and the NADP levels of the blood are constantly maintained by the adjustment of the amount of excretion to the urinary bladder, was suggested.

Whole blood NAD and NADP concentrations are not depressed in subjects with clinical pellagra.

Population surveys for niacin deficiency are normally based on clinical signs or on biochemical measurements of urinary niacin metabolites. Status may also be determined by measurement of whole blood NAD and NADP concentrations. To compare these methods, whole blood samples and spot urine samples were collected from healthy subjects (n = 2) consuming a western diet, from patients (n = 34) diagnosed with pellagra and attending a pellagra clinic in Kuito (central Angola, where niacin deficiency is endemic), and from female community control subjects (n = 107) who had no clinical signs of pellagra. Whole blood NAD and NADP concentrations were measured by microtiter plate-based enzymatic assays and the niacin urinary metabolites 1-methyl-2-pyridone-5-carboxamide (2-PYR) and 1-methylnicotinamide (1-MN) by HPLC. In healthy volunteers, inter- and intra-day variations for NAD and NADP concentrations were much lower than for the urinary metabolites, suggesting a more stable measure of status. However, whole blood concentrations of NAD and NADP or the NAD:NADP ratio were not significantly depressed in clinical pellagra. In contrast, the concentrations of 2-PYR and 1-MN, expressed relative to either creatinine or osmolality, were lower in pellagra patients and markedly higher following treatment. The use of the combined cut-offs (2-PYR <3.0 micromol/mmol creatinine and 1-MN <1.3 micromol/mmol creatinine) gave a sensitivity of 91% and specificity of 72%. In conclusion, whole blood NAD and NADP concentrations gave an erroneously low estimate of niacin deficiency. In contrast, spot urine sample 2-PYR and 1-MN concentrations, relative to creatinine, were a sensitive and specific measure of deficiency.

Detection of urinary tract infections by reduction of nitroblue tetrazolium.

BACKGROUND: Nitroblue tetrazolium (NBT) reduction to formazan has been used as a marker for nitric oxide synthase (NOS). Since inducible NOS activity is elevated in urine from patients with urinary tract infections (UTIs), we investigated the accuracy of NBT reduction as an early predictor of UTIs and quantified the relationship between inducible NOS and NBT. METHODS: Urine samples from 434 patients were screened for the presence of UTIs with leukocyte-esterase and nitrite dipsticks and with NBT reduction. The rapid screening results from each test were compared to urine culture results. In addition, NBT reduction parameters were measured in urine pellet at 595 nm after incubation with one of four factors: NOS cofactors, NOS inhibitors, NADH, or superoxide dismutase/catalase. RESULTS: As a urine screening test for UTIs, NBT reduction was more sensitive with a higher negative predictive accuracy than the nitrite dipstick. NBT reduction also was more specific with a higher positive predictive accuracy and negative predictive accuracy than the leukocyte-esterase dipstick. In infected urine pellet, both NADPH, a NOS cofactor, and NADH increased NBT reduction. Superoxide dismutase/catalase decreased NBT reduction. CONCLUSIONS: Although NOS may not be the only NBT reducing enzyme, rapid, visible reduction of NBT is induced in urine from patients with UTIs.

Nitric oxide synthase: an endogenous source of elevated nitrite in infected urine.

To further define the endogenous sources of urine nitrite in urinary tract infections, we measured urinary nitrite levels by the Griess method and assayed urinary nitric oxide (NO) synthase activity by the conversion of 14C-arginine to 14C-citrulline. Endogenous production of 14C-citrulline was confirmed by thin layer chromatography. Exogenous L-arginine increased nitrite production in whole infected urine, but not in bacteria isolated from infected urine. Urinary tract infections significantly increased NO synthase activity in soluble urine fractions, although soluble activity was less than 10% of particulate activity. Urine particulate fractions from women with non-infected urine had greater NO synthase activity than particulate fractions from men with non-infected urine, 11 +/- 2 and 0.2 +/- 0.1 picomol/min/mg protein, respectively. Urinary tract infections increased NO synthase activity in urine particulate fractions from women and men, 99 +/- 20 and 48 +/- 9 picomol/min/mg protein, respectively. The conversion of 14C-arginine to 14C-citrulline required NADPH, was calcium independent, and was inhibited to a greater extent by L-canavanine than by NG-monomethyl-L-arginine or NG-nitro-L-arginine. Human infected urine contains an isoform of NO synthase which is an endogenous source of urine nitrite.

[Determination of N'methylnicotinamide and nicotine coenzymes in biological in biological media by the fluorescent method]. / Opredelenie N'-metilinkotinamida i nikotinovykh kofermentov v biologicheskikh sredakh fliuorestsentnym metodom.

Assay of N1-MNA was conducted in 96 urine samples using two methodological variants with external and internal N1-MNA standards. Basing on significant fluctuations of the percent of detecting N1-MNA added to urine, the necessity of using the internal standard was proved. A significant (up to 30%) overestimating of the values in using the external standard makes difficult revealing niacin deficiency. It has been recommended that NAD preparation be used as an internal standard in NAD + NADP assay in the blood, that permits one to simplify significantly the counting and to avoid the universal coefficient (used in literature) of recalculating N1-MNA fluorescence to nicotinamide coenzyme fluorescence because this coefficient is not a constant value.