Extrinsic labeling

The in vitro procedure was tested in "critical" experiments designed to make direct comparisons of in vivo and in vitro estimates of exchangeability and potential bioavailability and to test the use of in vitro exchangeability values in in vivo experiments. (8). Three foods which were expected to show different levels of calcium solubility and exchangeability, collards, soybeans and spinach, were intrinsically labeled with 45Ca in nutrient solution culture. They were used together with 47 Ca as an extrinsic label in both in vitro and in vivo experiments. 

Zinc. Some recent studies on the effects of soy protein on zinc utilizaton are summarized in Table IV. Young and Janghorbani (4 4) and Istfan et al. (46) compared the effects of soy isolate or soy concentrate and dried skim milk as protein sources in multi-day feeding periods. Zinc absorptions, measured by fecal monitoring of the extrinsic label given, were equivalent and no deleterious effects of soy protein were observed. In a second study, Istfan et al. (47) fed egg protein diets for 10 days and then a soy concentrate diet for 82 days. Zinc absorptions were not decreased by feeding the soy concentrate diet. 

Janghorbani et al. (58) fed isonitrogenous diets to 10 subjects for 12 days. Both an intrinsic label (chicken) and extrinsic labels were used. Zinc absorptions from an all-chicken diet and from a 50% chicken-50% soy isolate diet were equivalent. Solomons et al. (59) fed 5 or 10 subjects diets in which milk, soy isolate, and beef (or mixtures of these) were protein sources for the milk and/or soy diets, absorptions were similar "fractional" absorptions from beef bologna may have been higher than from soy bologna (Table IV). 

Ten subjects fed (12 days) isonitrogenous diets with all protein from chicken meat or 50% from chicken and 50% from soy isolate intrinsic (chicken) and extrinsic labels used (fecal monitoring method)... 

Both the physics and the chemistry of proximity to a surface can alter the excited-state lifetime and rotational motion of a fluorescent molecule. An extrinsic label attached to BSA has been found to reduce its fluorescence lifetime upon BSA adsorption to fused silica.(95) The decrease is too large to arise from the physical near-field proximity effects discussed in Section 7.3 ... 

Expt. 2—milk (2% fat) versus CCM added to OJ extrinsically labeled CCM molar ratio 3 3 2 (equivalent to 6 6 4)... 

Ca sources in liquid form were extrinsically labeled (i.e., Ca... 

Animals dosed with " Ca extrinsically labeled beverages... 

In animals, as is the case with humans, absorption and bioavailability is influenced by age. Smith s result of Ca retention equivalence between CCM and CaCOa in older rats is different to how younger rodents responded (Smith et al., 1987). CCM in OJ was determined to be relatively better absorbed and retained than the Ca in 2% fat milk (mean SFM retention rates 51.1 1.7 vs 42.2 1.8% p <. 001), respectively. This result was reproduced when, based on extrinsic labeling, the Ca from CCM in both AJ and OJ was reported to be better absorbed in juvenile rats than was the Ca from milk (p <. 05) (Andon et al., 1996b). 

Whole-body isotope retention experiments were performed (via the method described above in the Smith experiment and with confirmed corrections for isotope decay rates), using extrinsically labeled ferric chloride hexahydrate and Ca from CCM (i.e., [ FeJFeCla and [" Ca] CaCl2) to determine the bioavailability of these minerals at an Fe Ca ratio of 1 167 mol/mol. CCM solubility was also assessed via a filtration method. 

Femtosecond spectroscopy has an ideal temporal resolution for the study of ultrafast water motions from femtosecond to picosecond time scales [33-36]. Femtosecond solvation dynamics is sensitive to both time and length scales and can be a good probe for protein hydration dynamics [16, 37-50]. Recent femtosecond studies by an extrinsic labeling of a protein with a dye molecule showed certain ultrafast water motions [37-42]. This kind of labeling usually relies on hydrophobic interactions, and the probe is typically located in the hydrophobic crevice. The resulting dynamics mostly reflects bound water behavior. The recent success of incorporating a synthetic fluorescent amino acid into the protein showed another way to probe protein electrostatic interactions [43, 48]. 

The studies of water motions around proteins have been difficult because of the lack of a reliable optical probe. Early attempts to use an extrinsic labeling of a protein with a dye molecule showed certain mobile water molecules [37-42], Such extrinsic labeling has limited probing sites and thus prevents general applications. We proposed to use the intrinsic tryptophan residue as a local... 

We subsequently used extrinsically labeled diets for further fractionation by gel filtration and immunoaffinity chromatography. Mn in human milk was found to be predominantly (about 70%) bound to lactoferrin, the major iron-binding protein in human milk. Minor amounts were bound to casein in human milk and the milk fat globule membrane. It is therefore possible that changes in lactoferrin concentration in human milk may explain the developmental pattern... 

In vitro tests using other procedures (19) were carried out in our laboratory (Ketelsen et al., manuscript in prep.) to study the dialyzability of zinc from slurries of defatted soy flours over a range of pH s. Either 2inc was added to soy slurries as zinc carbonate (extrinsic label) or the zinc was intrinsically labeled in soybeans grown hydroponically. Results from these studies (data not shown) indicated that the extrinsic and intrinsic zinc pools do not completely mix. The results showed that the pH of the soy suspensions affected the quantity of zinc that was bound in complexes of molecular weight in excess of 12,000. Also, intrinsic zinc was more likely to be bound to high molecular weight complexes than extrinsic zinc, especially for slurries at pH 3. 

The meals were extrinsically labelled by added 65 Zn. The rationale for this method is that a complete isotope exchange takes place between the added radioactive zinc isotope and the zinc present in the meal. Measurements of the uptake of radioactive iron isotopes in blood or in the whole body have been used for many years in studies of iron absorption. (12, 13, 14). The absorption in the present study is determined from measurement of the whole body retention of the radioisotope. However, this can not be done until the non-absorbed fraction of the isotope has left the body. During this periode of time some of the initially absorbed has been extrected. A correction of retention data... 

The meals were prepared in advance, extrinsically labelled and measured in the whole body counter. 

Intrinsic labeling is time consuming and specialized. Extrinsic labeling whereby a mineral isotope is mixed with a food prior to consumption is an appealing alternative to intrinsic labeling. The extrinsic labeling approach assumes that the extrinsic label... 

The so called extrinsic labeling technique has been used extensively to label foods and meals for iron absorption studies. An inorganic radioiron salt and biosynthetically radioiron-labeled hemoglobin are mixed with food to label the nonheme and heme iron pools in the food respectively. It has been confirmed that absorption of extrinsic tracers is similar to the absorption of intrinsic iron in the food (9, 10). 

Although the precision of El-MS abundance measurements is not comparable to that of TI-MS, the El mass spectrometer is much more widely available. More stable iron chelates that would permit GC sample introduction and reduce the memory effect problem would significantly improve the method. A potentially serious drawback of stable isotope tracers is that the amounts used are more in the range of substrate than tracer levels. A related question pertains to the validity of the extrinsic labeling technique when stable isotope tracers are used. In an ongoing study in our laboratory, we are attempting to compare the absorption of intrinsic and extrinsic stable iron isotope tracers. 

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