Vitamin tracer

Y. Lin, S. R. Dueker, B. J. Burri, T. R. Neidlinger, and A. J. Clifford, Variability of the conversion of beta-carotene to vitamin A in women measured by using a double-tracer design, Am. J. Clin. Nutr. 71 (2000) 1545-1554. 

In normal subjects, the terminal half-life of injected calcifediol is 23 days, whereas in anephric subjects it is 42 days. The half-life of 24,25(OH)2D is probably similar. Tracer studies with vitamin D have shown a rapid clearance from the blood. 

For the determination of vitamin D in fortified milk (96), the unsaponifiable residue was dissolved in 5 ml of hexane, and 0.1 or 0.2 ml of a tracer solution (chlorophyll-a) and 1 g of dry 8% water-deactivated alumina were added. The solvent was evaporated off, and the dried alumina containing the sample was poured on top of a prepared column packed with 15 g of alumina. Elu-... 

The low-molecular-weight vitamin biotin is easily conjugated to antibodies and enzyme markers. Up to 150 biotin molecules can be attached to one antibody molecule, and the strong affinity of the biotin for the glycoprotein avidin allows its use as complex-ing secondary reagents. Biotin labeling of the primary (direct) or secondary (indirect) antibody can be used in the avidin-biotin methods. In the labeled avidin method the tracer is attached directly to the avidin molecule. In the avidin-biotin bridge method a biotinylated enzyme such as peroxidase is allowed to bind after attachment of avidin to the biotin-labeled antibody. 

Vitamin D and its metabolites circulate in plasma tightly bound to a carrier protein, the vitamin D-binding protein. This -globulin binds 25(OH)D and 24,25(OH)2D with comparable high affinity and vitamin D and l,25(OH)2D with lower affinity. In normal subjects, the terminal half-life of injected calcifediol is 23 days, whereas in anephric subjects it is 42 days. The half-life of 24,25(OH)2D is probably similar. Tracer studies with vitamin D have shown a rapid clearance from the blood. The liver appears to be the principal organ for clearance. Excess vitamin D is stored in adipose tissue. The metabolic clearance of calcitriol in humans indicates a rapid turnover, with a... 

These physiologically active natural compounds 307 and 308 have been labelled with 13N in a simple and rapid one-step ammonolysis reaction (equations 124 and 125) in sealed glass vials. They were used for studies of biological function and in nuclear-medical studies. [13N]nicotinamide (NAM) has been found to be a useful tracer for studying the utilization of the vitamin, nicotinamide291. A comparison of the distribution of [13N]NAM with that of [nC]NAM has been carried out290,292. 

Replacement of Metabolic Losses An alternative approach to determining vitamin requirements is to measure the loss from the body pool in a steady state. This requires estimation of the total body pool, and measurement of the fractional rate of loss from that pool, generally using radioactive or stable isotope tracers. Three problems can arise in such studies. 

An alternative approach to determining requirements is to measure the fractional rate of catabolism of the vitamin by use of a radioactive tracer, then determine the intake that would be required to maintain an appropriate level of liver reserves. As discussed in Section2.2.1.1, when the liver concentration rises above 70 /rmol per kg, there is increased activity of the microsomal oxidation of vitamin A and biliary excretion of retinol metabolites. The fractional catabolic rate is 0.5% per day assuming 50% efficiency of storage of dietary retinol, this gives a mean requirement of 6.7 /rg per kg of body weight and a reference intake of 650 to 700 /rg for adult men (Olson, 1987a). Reference intakes for vitamin A are shown in Table 2.4. 

Menaquinones are absorbed mainly from the terminal ileum, where bile salts are present, into the hepatic portal vein. Litde of the menaquinones formed by colonic bacteria can be absorbed, because they remain tightly bound to bacterial cell membranes in the absence of bile salts. About 90% of the total liver content of vitamin K is menaquinones 7 to 13, and the hepatic pool of phylloquinone turns over considerably faster than that of menaquinones. Sixty percent to 70% of the daily intake of phylloquinone is excreted, mainly as conjugates in the bile, and the half-life of a tracer dose of phylloquinone is only about 17 hours. 

After a tracer dose of radioactive phylloquinone, the label is rapidly accumulated in the liver, then lost from the body with turnover time of 1.5 days. Tbis suggests that there is rapid turnover and little storage of vitamin K. However, there maybe considerable enterohepatic recirculation of the conjugates excreted in the bile (Shearer et al., 1996 Olson et al., 2002). About 10% of the total liver vitamin K is normally present as the epoxide, which is formed by the vitamin K-dependent carboxylase and normally reduced back to tbe active vitamin (Section 5.3.1). 

Vitamin Be requirements have been estimated both by isotopic tracer studies to determine turnover of the body pool (Section 9.6.1) and also by depletion/ repletion studies using a variety of indices of status (Section 9.6.2). These studies have generally been conducted on young adults, and there is inadequate information to determine the requirements of elderly people, because apparent status assessed by a variety of indices declines with increasing age, despite intake as great as in younger people (Bates et al., 1999a). As discussed in Section 9.6.3, there is also inadequate information to estimate the requirements of infants. 

There is a variety of estimates of the body pool of vitamin Be. Short-term studies with isotopic tracers suggest a total body content of between 160 to 600 ixmo (40 to 150 mg), with a half-life of 33 days, suggesting a minimum requirement for replacement in the wide range between 0.6 to 2.27 mg per day. 

About 80% of the total body vitamin Bg is in skeletal muscle glycogen phos-phorylase, with a relatively slow turnover. Based on longer term tracer studies, Coburn (1990, 1996) has suggested a total body pool of 250 mg, or 15 nmol (3.7 /xg) per g of body weight, with a loss of about 0.13% per day, hence a... 

Pantothenic acid is well conserved over a week after the administration of tracer doses of [ C]pantothenic acid to rats, less than 40% of the dose is recovered in the urine, all as the free vitamin. Pantothenic acid filtered by the kidneys is largely resorbed by a sodium-dependent system in the renal tubule brush border membrane (Barbara and Podevin, 1986). 

Hickenbottom SJ, Follett JR, Lin Y, Dueker SR, Burri BJ, Neidlinger TR, and Clifford AJ (2002) Variability in conversion of beta-carotene to vitamin A in men as measured by using a double-tracer study design. American Journal of Clinical Nutrition 75,900-7. 

Variability in Absorption Estimates In this study, the occurrence of a negative absorption value for one subject and the absence of a significant vitamin C effect raise some questions about the accuracy of the method However, the expected changes in absorption due to dietary treatments may be masked by the analytical variations associated with absorption measurements and biological variabilities of iron absorption Analytical variations can be introduced at several stages of the analytical procedures incomplete fecal collection, inhomogeneous samples, iron contamination, incomplete colorimetric reaction, non-quantitative recovery after chemical ashing, and variations in isotopic measurements due to ion statistics, memory effects, instrument drift, etc Some of these are not as serious as others, for example, contamination with natural iron woiold not affect the estimate of tracer concentrations provided it occurs before the total iron content is measured ... 

Vitamin Bi, vitamin B2, and nicotinic acid, all of which frequently occur together in foods, were separated by TLC and fluorimetrically determined by using a commercially available fiber optic-based instrument. A fluorescent tracer (fluoresceinamine, isomer II) was used to label the nicotinic acid. Vitamin B1 was converted to fluorescent thiochrome by oxidizing with potassium ferricyanide solution in aqueous sodium hydroxide. These vitamins were separated by HPTLC on silica gel using methanol-water (70 30 vol/vol) as mobile phase. Under these conditions, the Rf values of the vitamin Bi, vitamin B2, and nicotinic acid derivatives were 0.73, 0.86, and 0.91, respectively. 

Competitive Protein-Binding Assays with Vitamin D-Binding Protein. Before the development of immunoassays, 25(OH)D was measured primarily with CPBA with DBP as the specific binder, and with tritiated 25(OH)D3 (>100 Ci/mmol) as tracer. Rat serum, diluted approximately... 

The RIA described below is more convenient than the RRA for 1,25(0H)2D because a radioiodinated tracer eliminates the need for liquid scintillation, and the antiserum eliminates the need to prepare VDR from calf thymus. The RIA uses a previously developed antiserum with 1% to 2% cross-reactivity with the more abundant, non-1-hydroxylated vitamin D metabolites and a I-labeled tracer prepared from l,25(OH)2-24,25,26,27, tetranor-... 

Calibrators for vitamin D assays should be prepared from stock solutions whose concentration and purity are checked by UV spectrophotometry. Stock solutions are suitable if the ratio of the absorbance at 264 to 228 nm is greater than or equal to 1.5. Stock solutions are adjusted using a molar extinction coefficient of 18,200 L/mol/cm for vitamin D3 metabolites at 264 nm. Tracers used for recovery must be pure to determme recovery accurately. Both calibrators and tracers may be purified by HPLC. 

Hollis BW, Kamerud JQ, Selvaag SR, Lorenz JD, Napoli JL. Determination of vitamin D status by radioimmunoassay with an l-Iabeled tracer, Clin Chem 1993 39 529-33. 

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