Intrinsic Labeling Research Articles

Bioavailability of the calcium in fortified soy imitation milk, with some observations on method

Calcium-fortified soy milk is growing in popularity, particularly among vegetarians, but the bioavailability of its calcium was not previously known. Additionally, the validity of isotopic labeling methods for fortified liquid products had not been established. The objectives of this study were to compare the bioavailability of the calcium in fortified soy milk with that of calcium in cow milk and to evaluate the method of labeling soy milk for bioavailability testing. A within-subject comparison of extrinsically labeled cow milk with intrinsically and extrinsically labeled soy milks was undertaken in 16 healthy men. In all tests, 300-mg Ca loads were given as a part of a light breakfast after an overnight fast. The milks were physically partitioned into liquid and solid phases to enable evaluation of tracer distribution. Calcium from intrinsically labeled soy milk was absorbed at only 75% the efficiency of calcium from cow milk. Extrinsic labeling of soy milk did not produce uniform tracer distribution throughout the liquid and solid phases and resulted in a 50% overestimate of true absorbability. Calcium-fortified soy milk does not constitute a calcium source comparable to cow milk, and extrinsic labeling of such calcium particulate suspensions does not produce the uniform tracer distribution needed for bioavailability testing. Hence, intrinsic labeling of the fortificant is required for such liquid suspensions.

In vivo Research with Stable Isotopes in Biochemistry, Nutrition and Clinical Medicine: An Overview

Tracers and kinetic modelling provide the opportunity to follow the movement and to quantify the metabolic fates of biological compounds in vivo. For studies in children and for repeated studies in adults, the use of stable isotopically labelled substrates are preferable and safe. Measurement of isotopic enrichment in biological molecules is highly specific and can be extremely precise. This allows the development of models of biological system dynamics in cells and organs that are otherwise inaccessible for sampling. Applications in biochemistry, nutrition and clinical medicine show the potential of stable isotopes in vivo. Methodology is of paramount importance and includes the choice of bolus studies, (primed) continuous infusions, use of multiple tracers and use of isotopomer information and intrinsic labels. There is no limit to the number and kind of compounds that can be traced. Topics include intermediate metabolism (lipids, proteins and carbohydrates) using hydrogen, carbon and nitrogen labels. In clinical medicine, 13C-breath tests are available for detection of Helicobacter pylori ([13C]urea breath test), and for assessment of a variety of gastro-intestinal and hepatic functions (e.g. [13C]octanoate and mixed [13C]triglyceride breath tests for studies of gastric emptying and fat absorption, respectively). Because theoretical, methodological, physiological and practical aspects are intertwined, in vivo research with stable isotopes demands close collaboration between the clinical researcher and the chemist responsible for the mass spectrometry.

Diffusion and Distribution of Photoacid Generators in thin Polymer Films

AbstractDistribution and diffusion of two fluorinated ionic photoacid generators (PAGs) in thin polymer films have been investigated by depth profiling of the intrinsic label elements of both the PAGs and a silicon containing carrier polymer with Rutherford Backscattering Spectrometry (RBS) and dynamic Secondary Ion Mass Spectroscopy (SIMS). Distribution and diffusion of the PAGs in a bilayer film stack, which consists of a thin silicon containing polymer film on top of a thick thermally cross-linked Novolak film on a silicon substrate, have been studied as a function of the Novolak cross-linking temperature. Deposition of the PAG containing polymer films on top of the cross-linked Novolak films by spin coating results in an interphase with enriched PAG. Subsequent annealing of the film stack causes expansion of the interphase and diffusion of the PAG into the underlying Novolak film when Novolak is cross-linked at lower temperatures. On the other hand, there is a uniform PAG distribution and no detectable diffusion of the PAG into Novolak when it is cross-linked at high temperatures. The variations in the PAG distribution and diffusion are attributed to the changes in the chemical and physical properties of Novolak during cross-linking.

Direct identification of a ligand-binding domain within the rat secretin receptor by intrinsic photoaffinity labeling

Direct identification of a ligand-binding domain within the rat secretin receptor by intrinsic photoaffinity labeling

Intrinsic photoaffinity labeling of the cholecystokinin receptor provides dual constraints for modeling the ligand-binding site

Intrinsic photoaffinity labeling of the cholecystokinin receptor provides dual constraints for modeling the ligand-binding site

Lagging-strand, early-labelling, and two-dimensional gel assays suggest multiple potential initiation sites in the Chinese hamster dihydrofolate reductase origin.

There is general agreement that DNA synthesis in the single-copy and amplified dihydrofolate reductase (DHFR) loci of CHO cells initiates somewhere within the 55-kb spacer region between the DHFR and 2BE2121 genes. However, results of lagging-strand, early-labelling fragment hybridization (ELFH), and PCR-based nascent-strand abundance assays have been interpreted to suggest a very narrow zone of initiation centered at a single locus known as ori-beta, while two-dimensional (2-D) gel analyses suggest that initiation can occur at any of a large number of potential sites scattered throughout the intergenic region. The results of a leading-strand assay and two intrinsic labelling techniques are compatible with a broad initiation zone in which ori-beta and a second locus (ori-gamma) are somewhat preferred. To determine how these differing views are shaped by differences in experimental manipulations unrelated to the biology itself, we have applied the lagging-strand, ELFH, neutral-neutral, and/or neutral-alkaline 2-D gel assays to CHOC 400 cell populations synchronized and manipulated in the same way. In our experiments, the lagging-strand assay failed to identify a template strand switch at ori-beta; rather, we observed a gradual, undulating change in hybridization bias throughout the intergenic spacer, with hybridization to the two templates being approximately equal near a centered matrix attachment region. In the ELFH assay, all of the fragments in the 55-kb intergenic region were labelled in the first few minutes of the S phase, with the regions encompassing ori-beta and ori-gamma being somewhat preferred. Under the same conditions, neutral-neutral and neutral-alkaline 2-D gel analyses detected initiation sites at multiple locations in the intergenic spacer. Thus, the results of all existing replicon-mapping methods that have been applied to the amplified DHFR locus in CHOC 400 cells are consistent with a model in which two somewhat preferred subzones reside in a larger zone of multiple potential initiation sites in the intergenic region.

Expression of the Mouse Interleukin‐2 Receptor Gamma Chain in Insect Cells Using a Baculovirus Expression Vector—Comparison with the Human Common Gamma Chain

The gene encoding the gamma-chain of the mouse Interleukin-2 receptor was expressed in lepidopteran insect cells using the baculovirus expression vector system. The corresponding gene was inserted under the polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus and expressed in the Spodoptera frugiperda insect cell line Sf9 during viral infection. The recombinant receptor protein was identified by immunoblotting in cell lysates prepared from insect cells infected with the produced recombinant virus VL1392-mIL-2R gamma. Kinetic analysis demonstrated that the corresponding protein could be detected as an approximately 50 kDa protein already at 24 h post-infection. Intrinsic labelling with [35S]-methionine/cysteine and SDS-PAGE analysis of the recombinant baculovirus infected insect cells verified the immunoblotting data. The expressed IL-2R gamma protein could also be determined on the surface of infected insect cells by flow cytometric analysis. Comparison of the molecular weights between baculovirus expressed human and mouse IL-2R gamma chains indicated differences in the glycosylation pattern despite similar numbers of N-linked glycosylation sites.

Bioavailability of Calcium from Common Vegetables Assessed in Teenagers

Calcium is an essential human nutrient that is important in bone growth and metabolism. While dairy products in reasonable quantity can supply minimum daily requirements of calcium, the current decline in the consumption of dairy products, especially among teenagers, implies that dietary requirements must be met from other sources. Green beans, spinach and broccoli contain high concentrations of calcium, but the bioavailability of calcium from these food sources has not been determined in children. To provide accurate dietary recommendations for these foods, we have developed a recirculating hydroponic system for the growth and intrinsic labeling of plants with stable isotopes. Plants were maintained on a non-labeled nutrient solution until an appropriate developmental age and were then presented with nutrient solution containing 42-Ca. Labeled green bean pods and spinach were harvested at the proper commercial age, and were cooked, pureed and frozen until use. Thirteen teenage subjects seven girls, six boys) were recruited for a 2-week stay in the Metabolic Research Unit, and were fed 42-Ca-labeled vegetables along with 48-Ca-enriched milk; an intravenous dose of 46-Ca was also administered. Blood, urine and fecal samples were collected during the 2-week study. Calcium bioavailability and kinetics were determined using a multi-compartment model. The bioavailability of calcium averaged 28% from green beans, which was comparable to that of milk calcium. Calcium bioavailability from spinach averaged only 3%, due probably to the high oxalate content of spinach. Our results suggest that low-oxalate containing vegetables such as green beans can serve as good dietary sources of calcium. This research was funded in part by USDA-ARS Coop. Agr. No. 58-6250-1-003 and USDA-CRS Grant No. 94-347200-0605.

An improved, sensitive, non‐radioactive in situ hybridization method for the detection of cytokine mRNAs

We established an improved non-radioactive in situ hybridization (ISH) method to detect mRNA of cytokines in cell preparations and tissues. Via this method we could demonstrate various cytokines in stimulated peripheral blood mononuclear cells (PBMC), lymphoid cell lines and human lymphoid tissues. The probes for the in situ hybridization were made by labelling cytokine-specific PCR products with digoxigenin (Dig) in a repeated PCR. This resulted in an intrinsic labelling of the probe with several Dig-UTP molecules. Incorporation of Dig-11-dUTPs was shown on ethidium bromide-stained agarose gels by a higher molecular weight of the PCR products with incorporated Dig-dUTPs when compared to control PCR products without digoxigenin. Cytospin-centrifuged cells of PHA-stimulated PBMC or lymphoid cell lines and frozen sections of various human lymphoid tissues were hybridized with the Dig-labelled cytokine probes and the hybridized probes were detected immuno-histochemically. In this way, we detected and localized cytokine mRNAs (IL-2, IL-4, IL-6, IL-8, IL-10) in PBMC, in the human T-cell line Jurkat, in the follicular lymphoma cell line DoHH2, and in human lymph nodes and tonsils. The in situ hybridization had a high sensitivity as the results correlated closely with the detection of cytokine mRNA by reverse transcriptase-PCR (RT-PCR) data from the same samples. We showed that Jurkat and DoHH2 cells produce several cytokines constitutively and that, after activation with the phorbol ester PMA, expression of several cytokine mRNAS was enhanced.

Purification of Borrelia burgdorferi outer surface protein A (OspA) and analysis of antibody binding domains.

The major outer surface protein, OspA, of Borrelia burgdorferi is a lipoprotein which is a particular interest because of its potential as a vaccine candidate. However, serotypic and genetic analysis of OspA from both European and North American strains have demonstrated antigenic and structural heterogeneities. We purified OspA to homogeneity by exploiting its resistance to trypsin digestion. By treating spirochetes with trypsin and then using Triton X-114 extraction and ion-exchange chromatography, we obtained a yield of 2 mg of pure OspA protein per liter of culture. INtrinsic labeling with [14C]palmitic acid confirmed that OspA was lipidated, and partial digestion established lipidation at the amino-terminal end of the molecule. The reactivity of five anti-OspA murine monoclonal antibodies to nine different isolates of B. burgdorferi was ascertained by Western blot (immunoblot) analysis. Purified OspA was fragmented by enzymatic or chemical cleavage, and the monoclonal antibodies were able to define four distinct immunogenic domains. Further resolution of the epitope specificity to determine humoral and cellular immune responses to OspA has implications for vaccine development and for the utility of this protein as a reagent in diagnostic testing for Lyme borreliosis.

Visuotopic organization of corticocortical connections in the visual system of the cat.

It has recently been demonstrated that, in contrast with the retinogeniculocortical projection, the corticocortical connections in the cat present a high degree of convergence and divergence. This suggests that some corticocortical connections link nonvisuotopically corresponding regions. Using fine-grain electrophysiological mapping and anatomical tracing, we have set out to test this possibility by placing a small injection of retrograde tracer in area 17 and by comparing the extent of visual field encoded in the region of area 18 containing labeled cells and that represented in the uptake zone. The results demonstrate that the size of the labeled region on the surface of area 18 is independent of eccentricity and that, despite its anisotrophy, this region of labeling encodes a broadly circular region of visual field that is larger than that encoded in the uptake zone of the tracer in area 17. For example, in the representation of lower visual field, a virtual point in area 17 that encodes a visual field region 4 degrees in diameter receives afferents from a region of area 18 encoding a region 11 degrees wide. Examination of the density of labeled cells in the labeled zone in area 18 reveals that the highest density is observed in a region in visuotopic correspondence with the injection site. However, high labeling density is also occasionally found in patches that do not represent the same visual field region as the injection site. Many receptive fields of neurons recorded in the labeled zone in area 18 only partially overlap or fail to overlap the visual field region encoded by the injection site. The results also demonstrate that the extent of visual field encoded in the labeled zone in area 18 is the same as that represented in the region of intrinsic labeling in area 17. It is suggested that cortical afferents coming from several cortical areas and converging on a column of cells in area 17 cover the same extent of visual field and that this cortical network constitutes the structural basis for the modulatory regions of the receptive field as well as the synchronization of neurons in different cortical areas.

Iron Absorption Differs in Piglets Fed Extrinsically and Intrinsically 59Fe-Labeled Sow’s Milk

Iron bioavailability from species-specific milk is assumed to be high for the offspring, possibly due to species-specific iron-binding proteins in the milk. To assess this bioavailability using radioisotopes, the validity of extrinsic labeling technique needs to be proven. Using the suckling piglet as an animal model, we have compared iron bioavailability from sow's milk labeled extrinsically and intrinsically. During intrinsic labeling transfer into milk of 59Fe given intramuscularly was slow and was found to be at maximum 14 h post-injection. Recovery of isotope in the milk was only 0.00014%. Extrinsic and intrinsic labels were distributed differently among milk fractions; intrinsic iron bound primarily to the fat fraction but the extrinsic iron bound primarily to the casein fraction. Iron retention from intrinsically labeled milk was considerably higher than from extrinsically labeled milk. These results show that the extrinsic tag method is not valid for studies on iron absorption from sow's milk and suggest that the situation may be the same for human milk.

Recombinant human thrombomodulin. Regulation of cofactor activity and anticoagulant function by a glycosaminoglycan side chain.

Two glycoforms of a secretable human thrombomodulin mutant [TMD1-105 and TMD1-75; Parkinson, Grinnell, Moore, Hoskins, Vlahos & Bang (1990) J. Biol. Chem. 265, 12602-12610] were expressed in human 293 cells and used to study the role of glycosylation in the functions of this endothelial-cell thrombin receptor. Carbohydrate content analysis and intrinsic labelling with [3H]glucosamine and [35S]sulphate showed that TMD1-105 contained a chondroitin sulphate whereas TMD1-75 did not. Other than chondroitin sulphate, the carbohydrate contents of the two glycoforms were identical, indicating similar glycosylation patterns at other O-linked and N-linked sites in the two glycoforms. The properties of TMD1-105 were converted into those of TMD1-75 by chondroitin ABC lyase digestion. Trypsin digestion of labelled TMD1-105 permitted isolation of two overlapping peptides that contained chondroitin sulphate, spanned the entire O-glycosylation domain and had O-glycosylation sites at Ser-492, Ser-498, Thr-500, Thr-504 and Thr-506. The chondroitin sulphate-attachment site was assigned to Ser-492 as this residue is conserved in mouse and bovine thrombomodulin and lies within a sequence Ser-Gly-Ser-492-Gly-Glu-Pro, which has strong similarity to chondroitin sulphate attachment sites in other proteoglycans. Five peptides with N-linked carbohydrate were also isolated and contained glycosylation sites in the lectin-like domain (Asn-47, Asn-115, Asn-116) and in the fourth (Asn-382) and fifth (Asn-409) epidermal growth factor domains. The role of N-linked and simple O-linked carbohydrates in the functions of human thrombomodulin remain unclear. The present studies demonstrate, however, that the presence of chondroitin sulphate in human thrombomodulin has profound effects on all of the anticoagulant properties of this important anticoagulant thrombin receptor.

Extrinsic vs intrinsic labeling of the calcium in whole-wheat flour

Fractional absorption of calcium from bread made either from intrinsically or extrinsically labeled whole-wheat flour was compared in 11 healthy adult women. The intrinsic label was provided by 45Ca injected individually into stems of wheat plants during growth. The extrinsic tag was introduced by adding 45Ca to unlabeled flour via the water used in dough making. The two labeled breads were tested in a randomized crossover design using a standardized breakfast administered after an overnight fast. Approximately 80 g labeled bread was consumed by each subject, providing a total calcium load of 13.3 mg. Fractional absorption from the intrinsically labeled bread averaged 0.812 +/- 0.130 (mean +/- SE) and from the extrinsically labeled bread 0.792 +/- 0.113. The mean difference, within subject, was only 0.025 +/- 0.016 and was not significantly different from zero. Extrinsic labeling of the calcium of whole-wheat flour results in a degree of labeling homogeneity equivalent to that of intrinsic labeling, at least for a leavened bread product.

Preliminary study of metals in proteins by high-performance liquid chromatography-inductively coupled plasma mass spectrometry using multi-element time-resolved analysis.

The feasibility of multi-element and multi-isotope determinations during the on-line chromatographic separation of protein matrices was investigated using time-resolved software with inductively coupled plasma mass spectrometry. Size-exclusion chromatography was used to separate a mixture of known proteins and the behaviour of associated elements was monitored. It was possible to scan either the entire mass range or a selected number of isotopes. Reversed-phase chromatography was used to separate Zn-containing species in chicken meat that had been intrinsically and extrinsically labelled with stable isotopes of Zn. The ratio obtained for the intrinsic label agreed with that obtained by other workers using thermal ionization mass spectrometry. Time-resolved analysis permitted rapid data acquisition, thus reducing the number of chromatographic runs required. However, at present, data handling can be time consuming. Further software developments will add to the advantages that this technique offers.

Apparent zinc absorption by rats from foods labelled intrinsically and extrinsically with 67Zn

A variety of foods (peas (Pisum sativum), chicken meat, eggs, goat's milk, human milk) enriched with the stable isotope 67Zn were prepared by means of intrinsic- and extrinsic-labelling procedures. They were fed to rats and apparent absorption of 67Zn determined from faecal excretion measurements using thermal ionization mass spectrometry. There were significant differences in the absorption of the extrinsic and intrinsic label which differed in magnitude between the foods tested. The extrinsic 67Zn was less well absorbed in peas, chicken meat, eggs, and human milk than intrinsic 67Zn, but in goat's milk the extrinsic 67Zn was better absorbed than the intrinsic label. These results demonstrate that extrinsically-added stable Zn isotopes do not fully exchange with endogenous Zn in many foods, and illustrate the need for caution when using extrinsic labels for Zn bioavailability studies.

Intrinsic labeling of milk iron: effect of iron status on isotope transfer into goat milk

Extrinsic labeling with radioiron is commonly used in studies on bioavailability of iron in milk. Because of differences in the distribution of added radioiron and native iron among ligands in milk, we found it important to explore the possibility of intrinsic labeling of milk iron. In this study, we have evaluated different conditions in the lactating goat to optimize transfer of 59Fe into milk. Maximum 59Fe concentration in milk was found at 10–15 hr post injection, indicating a carrier-mediated process. After iron depletion induced by bleeding, iron transfer into milk in the mammary gland was found to be low. After iron repletion, however, iron transfer increased. This study suggests that maternal iron status affects iron transfer into milk and that during iron depletion, hematopoietic organs may have higher affinity for transferrin-bound iron than the mammary gland. Iron transport into milk was lower in dairy goats than in a young goat which had not been used in milk production. This suggests that mammary development may enhance iron uptake in the gland, possibly via transferrin receptors.

Intrinsic labelling of different foods with stable isotope of zinc (67Zn) for use in bioavailability studies

Intrinsically-labelled foods are required to validate extrinsic-labelling techniques used to study the bioavailability of trace elements. Wheat (Triticum aestivum), peas (Pisum sativum), goat's milk, human milk, eggs and chicken meat were selected for intrinsic-labelling studies with 67Zn. Peas were grown hydroponically in enriched nutrient solution and wheat was grown in sand and watered with enriched nutrient solution. Some of the wheat plants were also given stem injections of 67Zn solution. Eggs and chicken meat were prepared by administering 67Zn intravenously to chickens, and human milk was collected after an oral dose of 67Zn in a cola drink. All the foods investigated were sufficiently enriched with 67Zn for Zn absorption studies except wheat prepared by the sand and water-culture method.

Isotopic exchange of ingested calcium between labeled sources. Evidence that ingested calcium does not form a common absorptive pool

We studied the extent of salt dissociation during absorption of calcium from sources of differing absorbability by measuring fractional absorption from loads in the range of 200-300 mg in healthy adult women. Sources were labeled both intrinsically and extrinsically with 45Ca and 47Ca, respectively, and were fed alone and in combination with one another. We first confirmed our previous observation of superior absorbability of calcium oxalate over spinach calcium in a randomized cross-over design in 20 women. Spinach calcium exhibited only half the absorbability of the same load of calcium presented as the oxalate. Then, in 14 women fed spinach with both an intrinsic and an extrinsic label, apparent absorption of the extrinsic label averaged 0.130 +/- 0.041 and of the intrinsic label, 0.029 +/- 0.023. Thus, the extrinsic tag was partially, but not completely, bound by the spinach. In the same 14 women, milk absorption averaged 0.331 +/- 0.092 when ingested alone. However, when co-ingested with spinach, apparent milk calcium absorption fell to 0.267 +/- 0.079 and apparent spinach calcium absorption rose to 0.111 +/- 0.039. Thus, there was significant but incomplete label exchange between the two sources, indicating that at least some of the calcium from both sources enters a common preabsorptive, ionic pool. By contrast, we had previously shown no tracer exchange when labeled oxalate was co-fed with labeled milk. We conclude that (1) the presence of calcium as the oxalate in spinach is not a sufficient explanation for the poor absorbability of spinach calcium; and (2) oxalate calcium and spinach calcium are absorbed by different mechanisms, one involving a common preabsorptive pool and the other not.(ABSTRACT TRUNCATED AT 250 WORDS)

Zinc absorption in women: comparison of intrinsic and extrinsic stable-isotope labels

Fractional absorption of extrinsic and intrinsic zinc from milk-based formulated diets was measured in 10 young women by using a simultaneous triple-isotope method based on fecal monitoring of unabsorbed enriched stable-isotope labels. Zinc intake was held nearly constant (mean zinc intake = 1.48 mumol.kg body wt-1.d-1) throughout the 18-d controlled feeding phase of the study by addition of extrinsic 64Zn to all formula feedings, with substitution of the extrinsic 67Zn label for some of the 64Zn when formula intrinsically labeled with 70Zn was given. Fractional absorption values of intrinsic and extrinsic zinc labels, 0.267 +/- 0.092 (mean +/- SD) and 0.282 +/- 0.086, respectively, were highly correlated (r = 0.83, P less than 0.05) and did not differ significantly. The ratio of fractional absorption of the extrinsic to that of the intrinsic label, 1.08 +/- 0.20 (mean +/- SD), was not significantly different from unity. The results show that extrinsic labeling by use of enriched stable isotopes is a valid means of determining zinc absorption from milk-based diets in adult humans.