Nitrate in serum and urine was assayed by a modification of the cadmium-reduction method; the nitrite produced was determined by diazotization of sulfanilamide and coupling to naphthylethylene diamine. After samples were deproteinized with Somogyi reagent, the nitrate was reduced by Cu-coated Cd in glycine buffer at pH 9.7 (2.5 to 3 g of Cd granules for a 4-mL reaction mixture). The reduction followed pseudo-first-order reaction kinetics, a convenient time interval for assay being 75 to 90 min. Maximum reduction (85%) occurred at about 2 h. Detection limits in urine or serum were 2 to 250 µmol/L. This method does not require the reaction to go to completion, does not require expensive reagents or equipment, and can assay several samples simultaneously. Repeated assays of two serum pools gave CVs of 9.0% and 4.7% for nitrate concentrations of 31.4 and 80.2 µmol/L, nitrate was 1704.0 ± 1294 (SD) µmol/L (n = 21) in untimed normal urine, 81.8 ± 50.1 µmol/L in serum of 38 renal dialysis patients, and 51.2 ± 26.4 µmol/L in serum of 38 controls.

A change in 25-OHD assays has a significant impact on results, patient classification, and treatment recommendations. Such variability cannot be ignored when deriving and applying vitamin D guidelines. It also renders universal assay standardization a pressing call.

Inorganic nitrate and nitrite concentrations were monitored simultaneously in the plasma, erythrocytes, saliva and urine of five subjects following an oral dose of NaNO3 (470 mumols per kg body weight). There was an average 25-fold increase in plasma nitrate only 10 min. after ingestion. Its concentration rose to a peak level of 1.83 mM in 40 min., a value 49 times the preload level. Erythrocyte nitrate followed a similar pattern, but remained at about two thirds of the plasma values. Salivary nitrate showed a positive correlation with plasma nitrate and averaged 9 times the plasma level during 3 hr following ingestion. This is evidence of a concentration-dependent active secretion by the salivary glands. The mean nitrate clearance was 25.8 +/- 2.85 (S.E.M.) ml/min. corrected for a body area of 1.73 m2 (n = 17). The urinary nitrate/creatinine ratio increased 25 to 70 times after loading. These results indicate a predominantly tubular excretion of nitrate. Nitrite was not detectable in any of the body fluids studied except saliva, where it appeared to increase at the expense of nitrate.

No abstract

To characterize the molecular properties conveyed by the isoforms of the alpha subunit of Na,K-ATPase, the two major transepithelial transporting organs in the brine shrimp (Artemia salina), the salt glands and intestines, were isolated in pure form. The alpha isoforms were quantified by ATP-sensitive fluorescein isothiocyanate (FITC) labeling. The salt gland enzyme exhibits only the alpha 1 isoform, whereas the intestinal enzyme exhibits both the alpha 1 and the alpha 2 isoforms. After 32 hours of development, Na,K-ATPase activity [in mumol Pi/mg protein/hr (1 mu)] in whole homogenates was 32 +/- 6 in the salt glands and 12 +/- 3 in the intestinal preparations (mean +/- SEM). The apparent half-maximal activation constants (K1/2) of the salt gland enzyme as compared to the intestinal enzyme were 3.7 +/- 0.6 mM vs. 23.5 +/- 4 mM (P less than 0.01) for Na+, 16.6 +/- 2.2 mM vs. 8.29 +/- 1.5 mM for K+ (P less than 0.01), and 0.87 +/- 0.8 mM vs. 0.79 +/- 1.1 mM for ATP (NS). The apparent Ki's for ouabain inhibition were 1.1 x 10(-4) M vs. 2 x 10(-5) M, respectively. Treatment of whole homogenates with deoxycholic acid (DOC) produced a maximal Na,K-ATPase activation of 46% in the salt gland as compared to 23% in the intestinal enzyme. Similar differences were found with sodium dodecyl sulfate (SDS). The two distinct forms of Na,K-ATPase isolated from the brine shrimp differed markedly in three kinetic parameters as well as in detergent sensitivity.(ABSTRACT TRUNCATED AT 250 WORDS)