Metabolism of organically bound tritium in man

The classic methodology for estimating dose to man from environmental tritium assumes that all tritium, whether organically bound or free, enters directly into man's free body water compartment and is uniformly distributed as tritiated water. This methodology ignores the fact that organically bound tritium in foodstuffs may be directly assimilated in the bound compartment of tissues without previous oxidation. A four-compartment model consisting of a free body water compartment, two organic compartments, and a small, rapidly metabolizing compartment is proposed. The utility of this model lies in the ability to input organically bound tritium directly into organic compartments representing tissue solids. The model will be used to illustrate the potential importance of organically bound tritium to cumulative dose estimates. It is found that organically bound tritium in foodstuffs can increase cumulative total body dose by a factor of 1.7-4.5 times the free body water dose alone, depending on the bound-to-loose ratio of tritium in the diet.     

A simple centrifugal dehydration force method to characterize water compartments in fresh and post-mortem fish muscle

A centrifugal dehydration force (CDF) method to quantify changes in tissue hydration in fresh and in post-mortem muscular fish tail tissue is presented. The data obtained were used to assess fluid flow rate from tissues and the size of hydration compartments expressed in g water/g dry mass (DM). Curve fit analysis demonstrated that muscle tissue has three detectable water compartments. Application of the method to the fresh fish indicated the presence of a large non-bulk water compartment (3.14 g water/g DM) with a much smaller (0.11 g water/g DM) inner non-bulk water sub-compartment in addition to a comparatively small bulk water compartment (0.99 g water/g DM). At 10 min and at 4h post-mortem, no significant change in size or flow rate of the water compartments was observed. At 24h post-mortem the muscular fish tissue, stored in water, swelled with statistically significant increase in total water and in the bulk water compartment but no significant change in the size of the non-bulk water compartments. The water flow rate from the non-bulk water compartment was, however, increased significantly in the 24h dead tissue. This simple CDF method has application for quantization of bulk and non-bulk water compartments in other biological and non-biological systems.     

Osmotic and motional properties of intracellular water as influenced by osmotic swelling and shrinkage of Xenopus oocytes

Experiments were done on fully grown Xenopus oocytes to determine the extent and the properties of cellular water of hydration. The studies involved the osmotic shrinking and swelling of the oocytes under known osmotic pressure as well as proton NMR spectral, titration, and free induction decay analyses. Studies were done both on whole oocytes and on subcellular fractions. The results show that little if any of the oocyte water in situ has the motional or osmotic properties expected of pure "bulk" water. Four distinct water of hydration compartments were found and defined on the basis of distinct hydrogen bounding mechanisms. Some of the water in yolk platelets was found not to be in fast exchange with other water compartments. Osmotic shrinkage of oocytes caused an adaptive decrease in the bound water of hydration compartments. This osmotically induced decrease is attributed to decreased surface area available for the hydrogen bounding of water molecules on cellular proteins.     

Strong and weak binding of water to proteins studied by NMR triple-quantum filtered relaxation spectroscopy of (17)O-water

The triple-quantum filtered (TQF) spin-echo signal of (17)O-water, in the presence of proteins, was analysed to yield estimates of the number of weakly, and strongly bound water molecules. The analysis used a constrained direct iterative regression procedure with a three-state model of fast-exchange. Thus, the population size of free, weakly, and strongly bound water were determined simultaneously. The two fractions of the bound water were estimated by using correlation time(s) estimated in other studies. Bovine serum albumin (BSA), basic pancreatic trypsin inhibitor (BPTI), lysozyme and oxyhaemoglobin were studied. Of the four proteins, BSA contained the largest number of strongly and weakly bound water molecules, there being approximately 30 of the former and approximately 3000 of the latter under conditions of high protein concentration. The correlation time of the proteins increases with their concentration in solution, and when this was taken into account for BSA the estimated number of strongly bound water molecules did not change significantly. This NMR technique, and data analysis, will probably also be useful in studies of water binding and mobility in various systems including hydrogels, protein networks, membranes, cells and tissues.     

Frequency dependence of water proton longitudinal NMR relaxation times in mouse tissues around the freezing transition

Water proton longitudinal NMR relaxation times were measured in various tissues of healthy and tumor-bearing mice. Measurements were performed as a function of the Larmor frequency nu in the range 6-90 MHz, and at two temperatures (theta + and theta -) bracketing the 'freezing transition', at which the major part of the water signal disappears. At both temperatures, 1/T1 behaves according to: 1/T1 = A/square root nu + B A and B are obtained at theta + and theta -, and yield the proportion of bound water, which is convincingly identified with non-freezable water. The proportions found lie around 6% for tumors and 12% for other tissues. Discrimination between tissues via T1 is demonstrated to be essentially due to the bound water proportion. Bound water on the one hand and free water on the other hand behave similarly in all tissues including tumors. The activation energy for free water is found to be identical to that of pure water, although relaxation times are markedly different. It is noticed that determining the bound water proportion by signal intensity measurements at theta + and theta - is less reliable than by the T1 method.     

The origin and fate of hyaluronan in amniotic fluid

The mechanisms which regulate the steady-state concentration and molecular weight of hyaluronan in the amniotic fluid of sheep at different gestational ages have been investigated. An attempt to trace the origin of the polysaccharide has been made by analyses of various fetal fluids (amniotic fluid, allantoic fluid, tracheal fluid, urine, and serum). The fate has been studied by injection of radioactively labelled hyaluronan into the amniotic cavity and following the tracer in fetal tissues and fluids. The concentration of hyaluronan in amniotic fluid varies considerably but is in the order of 5 mg/l at mid-pregnancy and decreases to 1 mg/l in late pregnancy. The polysaccharide has a Mr-distribution with a weight-average in the order of 10(6) at 10 to 13 weeks of gestation which decreases to 10(5) closer to term. Calculations show that urine contributes 0.1 and 0.5 mg of low-molecular (Mr = 10(4) hyaluronan per day in mid- and late pregnancy, respectively, and the lung 10-20% of that amount in the form of high-molecular weight polymer (Mr greater than 10(6). The hyaluronan disappears from the amniotic cavity by bulk flow due to fetal swallowing. It is taken up and degraded in the fetal intestine. Molecules of Mr = 10(3) can pass the intestinal barrier. Calculations show that about 0.5 mg and 1.0 mg of hyaluronan is eliminated per day from the amniotic fluid at 12 and 17 weeks of gestation, respectively. Thus, the higher rate of elimination and the relatively high urinary contribution in more mature fetuses explain the low concentration and Mr of amniotic hyaluronan in late gestation, whereas a slower elimination combined with a relatively larger contribution of high molecular weight hyaluronan both from lung and urine and possibly from other sources are responsible for the higher concentration and Mr of the compound in early pregnancy.     

Immunoarchitecture of regenerated splenic transplants: Influence of donor and host age on the regeneration of splenic compartments

Summary Inbred rats were used as a model to determine the influence of the age of the implanted splenic tissue and the age of the host on the structure of transplanted splenic tissue. Monoclonal antibodies against lymphocyte, macrophage and dendritic cell subsets were used to evaluate the different compartments of the spleen. Adult rats received implants from adult, weanling or fetal rats, weanling rats received splenic tissue from adult, weanling or fetal rats and neonatal rats received neonatal or fetal spleens. There were major differences in the structure and cellular composition of the regenerated splenic tissue. The younger the recipients and the donor spleens, the better the normalization of the splenic compartments and the less fibrous tissue was found 3 months after transplantation. The follicles regenerated in all transplants, but the marginal zone was only normally developed in wealing and neonatal hosts. The periarteriolar lymphatic sheath regenerated in a similar manner to the marginal zone. Whenever a compartment developed, its cellular composition was the same as in a normal spleen. The immunhistological techniques enabled splenic regeneration to be characterized revealing a far from normal histological splenic structure in many age groups. These findings suggest that splenic regeneration in children might result in splenic tissue with normal compartments, which would be in contrast to some data in adults.     

Atrial natriuretic peptide in heart and specific binding in organs from fetal and newborn rats

To assess the possibility that atrial natriuretic peptide plays a role in salt and water balance during early mammalian development, we examined hearts from fetal and neonatal rates for the presence of this peptide and presumed target tissues for their ability to bind the hormone. Immunohistochemistry was used to localize and radioimmunoassay to quantify this peptide in heart. Immunoreactive atrial natriuretic peptide was visualized in the fetal heart on day 17.5 post-conception. It was distributed throughout the atrial appendages and free wall and, in ventricle, in the trabeculae carnae and chordae tendineae. The concentrations of immunoreactive atrial natriuretic peptide in atria of rats on day 19.5 post-conception were one-tenth of those in the adult. Levels of this peptide in fetal ventricle were low and virtually absent from the adult tissue. Specific binding of radiolabelled atrial natriuretic peptide measured by whole organ counting occurred in several organs from 19.5-day fetal and neonatal rats. A number of these tissues, including the kidney, ileum, adrenal, lung and liver, are targets for and/or bind the peptide in adult rats. Specific binding in these tissues was localized using autoradiography at anatomical sites similar to those in adult organs. Specific binding was also seen in fetal but not neonatal skin. In the kidney, binding was associated with immature as well as mature glomeruli. These findings support the proposition that atrial natriuretic peptide may function in the perinatal rat as it does in the adult and, in addition, may play a unique role during fetal life.     

Studies of the Cellular Distribution of Superoxide Disrnutases in Adult and Fetal Rat Tissues

Activities of three types of superoxide dismutase in tissue fractions were significantly lower in fetal and adult brain and fetal limb preparations than in fetal and adult heart preparations. An exception was the cyto-plasmic fraction of adult brain that had levels of Cu, Zn-superoxide dismutase activity comparable to those in cytoplasmic fractions of heart. In addition, Mn superoxide dismutase activity appeared to be very low in all fetal mitochondrial matrix fractions and cytoplasmic fractions as well as in adult brain. Finally, the results of these studies emphasize the importance of two antioxidant defense systems in the tissues studied, one associated with the mitochondrial electron transport system and the other, the cytosolic Cu, Zn enzyme.     

Age-related differences in the accumulation and size of hyaluronan in alginate culture

The alginate bead culture system has unique properties that make it possible to study the accumulation and turnover of macromolecules in two distinct matrix compartments of the cartilage matrix: the cell-associated matrix (CM) and the further removed matrix (FRM). Taking advantage of this culture system, the purpose of this study was to examine age-related changes in the metabolism of hyaluronan (HA) in these two compartments. Bovine chondrocytes, isolated from fetal, young adult, and old adult articular cartilage, were cultured in alginate beads. On Days 7 and 14 of culture, the alginate gel was solubilized, the CM and FRM were separated and macromolecules in both compartments were analyzed. When compared to the cells from fetal and old adult animals, the young adult cells proliferated at the fastest rate. Fetal cells produced a more abundant CM that was richer in proteoglycans (PGs) than the CM of young or old adult cells. With increasing age, there was an increased tendency for PG, collagen, and HA to escape incorporation into the CM and to become immobilized in the FRM. Very striking changes also were observed in the ratio of HA to PG, which increased markedly with age, and in the size of the HA molecules, which decreased markedly with age. The results suggest that the metabolism of HA in cartilage undergoes pronounced age-related changes, some of which are retained during culture in alginate gel. The findings also suggest that the previously documented age-related decrease in the size of HA in native bovine cartilage reflects, at least in part, a biochemical process occurring at the time or at least soon after the glycosaminoglycan chain is synthesized. It does not appear to simply be the result of age-related changes occurring slowly with time after synthesis, as was previously suggested to be the case for human articular cartilage.     

Hexokinase isoenzymes in tissues of the adult and developing guinea pig

Changes in the activities and isoenzyme distribution of hexokinase were determined in a number of tissues during the development of the guinea pig. The total activity in the fetal liver showed a large fall during the second half of gestation to reach adult values by term. With normal diet the fetal, neonatal, and adult livers had isoenzymes I and III but little or no detectable IV (glucokinase). The fetal liver had predominantly type I, but the proportion of type III increased during development. The kinetics of the guinea pig isoenzymes were similar to those reported for the rat. Two additional isoenzymes with mobility between I and II were detected in the fetal liver and blood. They appear to have kinetic properties similar to type I. Detectable liver glucokinase activity was induced by glucose administration to adult guinea pigs. The total activity in kidney, brain and skeletal muscle showed a postnatal rise while in the fetal heart it was high and declined after birth. These tissues contained predominantly type I with varying proportions of type III hexokinase. The ratio of particulate-bound to soluble hexokinase varied from tissue to tissue. All except the liver showed a significant increase in binding after birth. The changes are discussed in relation to the control of glucose utilization in the fetal and neonatal periods.     

Characterization of water of hydration fractions in rabbit skeletal muscle with age and time of post-mortem by centrifugal dehydration force and rehydration methods

Centrifugal dehydration force (CDF) and rehydration isotherm (RHI) methods were used to measure and characterize hydration fractions in rabbit psoas skeletal muscle. The CDF method assessed fluid flow rate from rabbit muscle and hydration capacity of the fractions. Bulk and multiple non-bulk water fractions were identified. The non-bulk water was divisible into the following fractions: two outer non-bulk fractions, a main chain proteins backbone or double water bridge fraction, and a single water bridge fraction. The total non-bulk water amounts to about 85% of the total water in the muscle. The sizes of the water fractions (in g water/g dry mass) agree with a recently proposed molecular stoichiometric hydration model (SHM) applicable to all proteins in and out of cells (Fullerton GD, Cameron IL. Water compartments in cells. Methods Enzymol, 2007; Cameron IL, Fullerton GD. Interfacial water compartments on tendon/collagen and in cells. In: Pollack GH, Chin WC, editors. Phase transitions in cells. Dordrecht, The Netherlands: Springer, 2008). Age of the rabbit significantly slowed the flow rate of the outer non-bulk water fraction by about 50%. Also, muscle of the older rabbit (26 weeks vs. 12 weeks old) had less bulk water and less outer non-bulk water but the same amount of main chain backbone water compared to muscle of the younger rabbit. Increase in time post-mortem from 30min to 4h resulted in rigor mortis and a significantly slower flow rate of water from the outer non-bulk water fraction, which is attributed to muscle contraction, increased packing of contractile elements and increased obstructions to flow of fluid from the muscle fibers.     

Mechanism of hyaluronan degradation by Streptococcus pneumoniae hyaluronate lyase. Structures of complexes with the substrate

Hyaluronate lyase enzymes degrade hyaluronan, the main polysaccharide component of the host connective tissues, predominantly into unsaturated disaccharide units, thereby destroying the normal connective tissue structure and exposing the tissue cells to various endo- and exogenous factors, including bacterial toxins. The crystal structures of Streptococcus pneumoniae hyaluronate lyase with tetra- and hexasaccharide hyaluronan substrates bound in the active site were determined at 1.52- and 2.0-A resolution, respectively. Hexasaccharide is the longest substrate segment that binds entirely within the active site of these enzymes. The enzyme residues responsible for substrate binding, positioning, catalysis, and product release were thereby identified and their specific roles characterized. The involvement of three residues in catalysis, Asn(349), His(399), and Tyr(408), is confirmed, and the details of proton acceptance and donation within the catalytic machinery are described. The mechanism of processivity of the enzyme is analyzed. The flexibility (allosteric) behavior of the enzyme may be understood in terms of the results of flexibility analysis of this protein, which identified two modes of motion that are also proposed to be involved in the hyaluronan degradation process. The first motion describes an opening and closing of the catalytic cleft located between the alpha- and beta-domains. The second motion demonstrates the mobility of a binding cleft, which may facilitate the binding of the negatively charged hyaluronan to the enzyme.     

Lipopolysaccharide induces the expression of interleukin-1alpha distinctly in different compartments of term and preterm human placentae

The aim of the study was to investigate the stimulatory effect of lipopolysaccharide (LPS) on IL-lalpha production in different compartments of term and preterm placental tissues. Homogenates from amnion, chorion, and from fetal (subchorionic placental tissues, maternal decidua, and mid-placental tissue before and after perfusion of isolated placental cotyledons of 5 term placentas and 4 placentas obtained after preterm birth (28-34 W of gestation) were examined. Isolated placental cotyledons were dually perfused LPS (100 ng/kg perfused placental tissue) was perfused into the maternal side during 10 hours. Homogenates of the samples were examined by ELISA for IL-1alpha levels, and paraffin sections of the samples were stained by immunohistochemical staining, to characterize the cellular origin of placental IL-1alpha. Paired t test and ANOVA determined statistical significance. In the homogenates, there was a tendency towards higher IL-lalpha levels in all preterm placental compartments as compared to the term compartments before perfusion. A significant increase was observed only in the chorion compartment (p = 0.035). LPS had significantly increased IL-la levels only in the decidua compartment of term placentas as compared to other placental compartments (p = 0.0004), and had decreased IL-1alpha levels in the mid-placenta (p = 0.034). In preterm placentas, addition of LPS did not affect the expression levels of IL-1alpha in either fetal or maternal compartments as determined by ELISA and immunohistochemical staining. IL-la levels in the chorion compartment of preterm placenta were significantly higher as compared to term placenta. LPS affects placental tissues of term and preterm placentas differently. Also, in the term placentas, LPS affected the different compartments differently. Thus, IL-1alpha may have a key role (as a autocrine/paracrine factor) in the regulation of normal and pathological pregnancy and parturition.     

Changes in permeability of fetal guinea pig skin during gestation

Permeability of fetal skin to tritiated water was measured in vitro using samples taken from the back and flanks of 21 guinea pig fetuses whose gestational age ranged from 30 to 67 days (term = 68 days). From 30 to 45 days, fetal skin was relatively permeable to water, with a permeability coefficient for unidirectional, diffusional transfer of labelled water that averaged 0.372 +/- 0.041 (SEM) X 10(-4) cm/s. Then during a 5-10 day interval, the measured permeability coefficient decreased abruptly to very low and barely detectable levels. These changes took place at the time during gestation when others have shown the skin becomes keratinized and growth of new hair follicles is completed. Thus these findings are consistent with a relatively free exchange of water between amniotic fluid and fetal interstitium across the skin during the first two-thirds of gestation and then with further maturation an abrupt functional separation between these fluid compartments during the last third of gestation.     

1H-NMR analysis of water mobility in cultured phototrophic biofilms

The present work reports on the first attempt to study water mobility in phototrophic biofilms, applying the (1)H-NMR relaxometry technique to closely monitored microbial communities grown in a microcosm under controlled ambient conditions. Longitudinal water proton relaxation times exhibited a bi-exponential behavior in all biofilm samples, indicating two types of water molecules with diverging dynamic properties, confined to different compartments of the biofilm. The fast-relaxing component can be attributed to water molecules tightly bound to the intracellular matrix, while the slow-relaxing component could reflect the behavior of water embedded in the biopolymer matrix, confined into matrix pores and channels. The results are discussed with respect to a possible key role of exopolysaccharides and uronic acids in water binding in phototrophic biofilms.     

The molecular stoichiometric hydration model (SHM) as applied to tendon/collagen, globular proteins and cells

This report describes and documents the presence of multiple water-of-hydration fractions on proteins and in cells. Initial studies of hydration fractions in g of water/g of DM (dry mass) for tendon/collagen led to the development of the molecular SHM (stoichiometric hydration model) and the development of methods for calculating the size of hydration fractions on a number of different proteins of known amino acid composition. The water fractions have differences in molecular motion and other physical properties due to electrostatic interactions of polar water molecules with electric fields generated by covalently bound pairs of opposite partial charge on the protein backbone. The methods allow calculation of the size of four hydration fractions: single water bridges, double water bridges, dielectric water clusters over polar-hydrophilic surfaces and water clusters over hydrophobic surfaces. These four fractions provide monolayer water coverage. The predicted SHM hydration fractions match closely measured hydration fraction values for collagen and for globular proteins. This report also presents water sorption findings that support the SHM. The SHM is applicable for cell systems where it has been studied. In seven cell systems studied, more than half of all of the cell water had properties unlike those of bulk water. The SHM predicts and explains the commonly cited and measured bound water fraction of 0.2-0.4 g of water/g of DM on proteins. The commonly accepted concept that water beyond this bound water fraction can be considered bulk-like water in its physical properties is unwarranted.     

Ontogeny of 11beta-hydroxysteroid dehydrogenase: activity in the placenta, kidney, colon of fetal rats and rabbits.

Mechanisms to regulate closely fetal GC exposure are of considerable importance, as certain organs (kidney, brain) are adversely affected by excess GCs. 11beta-Hydroxysteroid dehydrogenase type 2 (11beta-HSD2) reduces transplacental passage of maternal GCs to the fetus. We hypothesized that 11beta-HSD2, if active in fetal kidney and colon, might allow local tissue modulation of GC access during the critical last trimester. We determined the presence, ontogeny and functionality of 11beta-HSD in the placenta and fetal, neonatal and adult kidney and colon in rats and rabbits and the cortisol:cortisone ratio in human amniotic fluid, which represents fetal urine. There was clear a 11beta-HSD2 expression in last trimester fetal colon, kidney and placenta in both rats and rabbits. This appeared of functional importance, since the potency of cortisol on fetal rabbit colonic sodium flux in the Ussing chamber was increased by 11beta-HSD inhibition. In human amniotic fluid, we found a decreasing ratio of cortisol:cortisone across the last trimester, suggesting an analogous onset of renal 11beta-HSD2 activity in the human fetal kidney. Local fetal tissue 11beta-HSD2 may modulate exposure to the deleterious effects of GCs upon target tissue maturation during sensitive periods of late gestation when fetal GC levels rise to prepare other organs (lung) for adaptations at birth.     

Hepatic and extrahepatic N-acetyltransferase. Perinatal development using a new radioassay.

We report a sensitive and rapid radioassay method for p-aminobenzoic acid N-acetyltransferase. The principle of this assay involves acetylation of p-aminobenzoic acid with [1-14C] labeled acetyl coenzyme A and direct extraction of enzymically formed radioactive p-acetamidobenzoic acid into nonaqueous scintillation fluid. Using this radiometric assay, hepatic and extrahepatic tissue distributions from rat and rabbit were studied. Rabbit blastocyst and endometrial N-acetyltransferase specific activities were equivalent to hepatic activities. Perinatal development studies in rats and rabbits revealed that fetal and neonatal animals are capable of N-acetylation. Rat liver developmental studies exhibited two peaks of activity with the first peak occurring in the late fetus followed by a second peak 3 days after birth. Rabbit fetal and neonatal enzyme activity increased to adult levels by the second week after birth in liver and gut, however, lung showed a different developmental pattern. These studies demonstrate that fetal extrahepatic tissues, like adult tissues, play an important role in N-acetylation.     

Type VI collagen microfibrils: evidence for a structural association with hyaluronan

Type VI collagen, a widespread structural component of connective tissues, has been isolated in abundance from fetal bovine skin by a procedure involving bacterial collagenase digestion under nonreducing, nondenaturing conditions and gel filtration chromatography. Rotary shadowing electron microscopic analysis revealed that the collagen VI was predominantly in the form of extensive intact microfibrillar arrays. These microfibrils were seen in association with hyaluronan, which was identified by its ability to bind the G1 fragment of cartilage proteoglycan. Treatment with highly purified hyaluronidase largely disrupted the collagen VI microfibrils into component tetramers, double tetramers, and short microfibrillar sections. Subsequent incubation of disrupted collagen VI in the presence of hyaluronan facilitated a partial repolymerization of the microfibrils. In vitro binding studies have also demonstrated that type VI collagen binds hyaluronan with a relatively high affinity. These studies demonstrate that a specific structural relationship exists between type VI collagen and hyaluronan. This association is likely to be of primary importance in the growth and remodeling processes of connective tissues.