Uptake of 125I-iododeoxyuridine in mouse organs during deuteration of body water: Evidence for a thymus-specific effect

The effects of body water deuteration on mammalian DNA synthesis $\text{in vivo}$ during the deuterium equilibration period in the body were studied. Young adult mice were given 15% or 30% D₂O in the drinking water for 4, 10 or 21 days. Control mice were given distilled water. Eighteen hours prior to sacrifice, ¹²⁵IUdR, a conveniently monitored synthetic analogue of the DNA precursor thymidine, was injected intravenously. Although neither radioiodine activity of the total body nor body weight varied significantly among the three groups, thymic radioactivity per g tissue was significantly lower in mice given 30% D₂O and, to a lesser extent, in mice given 15% D₂O than in the control group. In contrast, intestine and hemopoietic bone marrow displayed minor changes in ¹²⁵IUdR incorporation. This reduction of ¹²⁵IUdR incorporation is discussed in relation to the particular importance of thymidine reutilization in the thymus.     

The beta-galactosidase-catalyzed hydrolysis of o-nitrophenol-beta-D-galactoside at subzero temperatures: evidence for a galactosyl-enzyme intermediate.

The reaction of β-galactosidase ($\text{E. coli}$ K12) with $\text{o}$-nitrophenyl-β-$\text{D}$-galactoside has been investigated over the temperature range +25° to ?30° using 50% aqueous dimethyl sulfoxide as solvent. At temperatures below ?10° turnover becomes very slow and a burst of $\text{o}$-nitrophenol is observed. Such a burst indicates the existence of a galactosyl-enzyme intermediate whose breakdown is rate-limiting and provides a means of determining the active site normality. The Arrhenius plot for turnover is linear in the ?25 to +25° range with E_a = 26 ± 3 kcal/mole. The presence of the 50% DMSO had no effect on K_m but caused a small decrease in K_cat.     

Solvent isotope effects on the structure and function of mitochondrial membranes in aqueous media

The structural stability of mitochondrial membranes and the enzyme complexes of the electron transport system, and the solubility of a small molecular-weight nonelectrolyte (2-methylnaphthoquinone), have been studied as a function of water structure. D₂O, which is considered to be more structured than ordinary water, and H₂O were used as solvents in conjunction with chaotropic ions which have been shown to break down water structure. Assays for membrane stability were (a) resolution with respect to solubilization of at least one constituent enzyme, and (b) chaotrope-induced lipid autoxidation, which is a measure of structural destabilization. Solvent isotope effects expressed as the quotient of chaotrope (NaClO₄) concentration ($\text{C}{}_{\mathrm{<mtext>D</mtext>}}\text{C}{}_{\mathrm{<mtext>H</mtext>}}$) necessary to elicit the same effect were found to be (a) essentially constant for each system over a wide range of NaClO₄ concentration, and (b) limited to the narrow range of 1.2–1.8 in all tests despite significant differences in the systems studied and the measurements used. The magnitude and the constancy of the isotope effects indicate that increased membrane stability (i.e., the increased strength of hydrophobic interactions in membranes), and decreased water-solubility of nonelectrolytes in D₂O are mainly due to the higher degree of order of the deuterated solvent. Thus, in the mitochondrial electron transport chain and many other enzyme systems where solvent isotope effects have been observed, the isotope effect appears to be more a consequence of conformational changes imposed on the enzymes by D₂O, because it is a more structured solvent, rather than an indication of direct involvement of protons or the water molecule in the reaction mechanisms.     

The falling drop method for deuterium oxide: Factors influencing accuracy

Factors affecting the accuracy of the falling drop method for D₂O were considered: selection of temperature of the constant-temperature bath, permissible temperature fluctuations, and D₂O concentration. Bath temperatures used, 27.24 to 27.32°, and constancy within ±0.002° had no influence on the regression relating drop velocity to concentration, v = a + bc, or sampling error (S.D._v). The latter increases significantly with concentration from 0.05 to 0.23 ml % D₂O, is significantly smaller than experimental error (S.D._e), and is inappropriate for estimating intra-assay limits. More appropriate is the S.D._c derived from the variance of the terms of the regression equation. Assay results using this error term can be expected to vary from about ±4.8% at 0.05 ml % to about ±2% at 0.23 ml %. These results compare favorably with those reported for the mass spectrograph used to determine the mass ratio of $\text{HDO}\text{H}{}_2\text{O}$ vapor. A more conservative estimate is obtained by using $\text{λ}\text{=}\text{S.D.}{}_{\mathrm{e}}\text{b}$, which in this work indicated within-assay variability of ±12.4% at 0.05 ml % and ±2.7% at 0.23 ml % D₂O. The term S.D._e, corresponding to 0.0062 ml % in these experiments, provides the best means for assay comparisons.Satisfactory recoveries of D₂O added to urine averaging 99% of known values were obtained after shell-freezing, without distillation of standards and without permanganate oxidation of samples. No increase in error beyond that anticipated from standards alone was observed when urine was the vehicle.     

Evidence for a glucosyl-enzyme intermediate in the beta-glucosidase-catalyzed hydrolysis of p-nitrophenyl-beta-D-glucoside

The reaction of almond β-glucosidase with $\text{p}$-nitrophenyl-β-$\text{D}$-glucoside has been investigated over the temperature range +25° to ?45° using 50% aqueous dimethyl sulfoxide (DMSO) as solvent. At temperatures below those at which turnover occurs a “burst” of $\text{p}$-nitrophenol proportional to the enzyme concentration is observed. Such a “burst” suggests the existence of a glucosyl-enzyme intermediate whose breakdown is rate-limiting, and provides a method for measuring the active-site normality. At pH 5.9, 25°, the presence of 50% DMSO causes an increase in K_m from 1.7×10^?3M (0%) to 1.7×10^?2M, whereas V_max is unchanged. The DMSO thus apparently acts as a competitive inhibitor with K_i = 0.7M. The Arrhenius plot for turnover is linear over the accessible temperature range with E_a = 23.0 ± 2.0 kcal/mole.     

The temperature dependence of the redox potential of horse heart cytochrome c in sodium chloride solutions

The E⁰′ values for the conversion of horse heart cytochrome $\text{c}$ from the oxidized to the reduced form as a function of temperature have been measured in 0.10 M NaCl, 0.10 M sodium phosphate, pH 7.0 solutions in H₂O and D₂O. In H₂O, the decrease in the E⁰′ value is linear with increasing temperature up to 42°C. Above this temperature, the decrease is again linear but with a much greater slope. In D₂O solutions, however, this biphasic behavior was not observed but instead a single line was obtained over the temperature range studied (25°C to 50°C). These results are interpreted in terms of the ability of NaCl to cause a destructuring of the bulk H₂O above 42°C but not in the more stable D₂O (Kreishman, Foss, Inoue and Leifer (1976) $\text{Biochemistry}\text{,}\text{15}$, 5431–5435). This decrease in water structure results in a shift in the equilibrium to the larger oxidized form as indicated by the decrease in E⁰′.     

D2O-alanine exchange reactions catalyzed by alanine racemase and glutamic pyruvic transminase

NMR studies in D₂O (>90%) reveal that Alanine Racemase (5.1.1.1.) from $\text{B. subtilis}$ catalyzes the exchange of the α hydrogen of $\text{D}$- and $\text{L}$-alanine with D₂O. Glutamic Pyruvic Transaminase (2.6.1.2.) and Glutamic Oxaloacetic Transaminase (2.6.1.1.) catalyze the exchange of α and β hydrogens of $\text{L}$-alanine. The rates of exchange of α and β hydrogens appear to be of the same order of magnitude. The transaminase catalyzed exchange is enhanced by catalytic amounts of pyruvate. The side chain of $\text{L}$-alanine is held more rigidly at the active site of transaminase so that the planar conjugated system can be extended to include the α and β carbons. A generalized mechanism is proposed for the action of pyridoxal phosphate dependent transaminases which extends Braunstein and Snell mechanism to include the structures which contribute to the labilization of β hydrogens of amino acids by the transaminases that have been studied.     

1Alpha,25-dihydroxyvitamin D3 receptor: competitive binding of vitamin D analogs

The intestinal nuclear receptor for lα,25-dihydroxyvitamin D₃ has been utilized to determine the ability of vitamin D-active sterols to compete with this hormone at the molecular level. 25-Hydroxyvitamin D₃ and lα-hydroxyvitamin D₃ must be present in 150 and 450 times the concentration respectively of lα,25-dihydroxyvitamin D₃, $\text{in}$$\text{vitro}$, to displace the physiologic hormone. These data indicate that: i) superphysiologic levels of 25-hydroxyvitamin D₃ may simulate lα,25-dihydroxyvitamin D₃ and act directly on isolated target organs and ii) the biologic potency observed for low doses of lα-hydroxyvitamin D₃, $\text{in}$$\text{vivo}$, is probably the result of 25-hidroxylation of the lα-derivative to form lα,25-dihydroxyvitamin D₃.     

Perturbation of the phagocytic response in P388D1 cultured macrophages by agents altering cell calcium

Phagocytosis in adherent P388D₁ (D₁) cells was monitored utilizing formalin treated $\text{Listeria}$$\text{monocytogenes}$ ($\text{Lm}$) previously labeled with ¹²⁵iododeoxyuridine. The dependence of this phagocytic process on calcium was studied by using several agents which alter calcium metabolism. The calcium antagonist ruthenium red (RR) produced a dose and time dependent stimulation (60–70%) of $\text{Lm}$ phagocytosis by D₁ cells. Utilizing another calcium antagonist, D-600, a prolonged inhibition (4 hours) of phagocytosis (40%) was observed. The addition of the cation ionophore A23187 produced a transient stimulatory increase (38% at 2 hours) in the phagocytic response. The concomitant addition of RR and D-600 did not alter the phagocytosis of $\text{Lm}$ by D₁ cells as compared to control cells. However, this complete drug/drug antagonism was not seen with the combinations of A23187 and D-600 or RR and A23187. The addition of A23187 and D-600 resulted in a time dependent inhibition of phagocytosis which did not become maximal until 3 to 4 hours. A23187 and RR produced a time independent stimulation of phagocytosis which was significantly less than that which was observed for RR alone, but was of longer duration than the response produced by A23187 alone. The use of these calcium probes in the P388D₁ macrophage model suggests a role for calcium in the phagocytic process.     

Fatty acid and prostaglandin composition of left ventricular myocardium from dexamethasone-treated dogs with severe low output syndrome (LOS)

The effects of dexamethasone (DEX) administration on the left ventricular myocardial content of fatty acids and prostaglandins E₁, E₂ and F_2α were studied. Following a complete right and left cardiac catheterization, either DEX (8 mg/kg) or an equivalent volume of its vehicle was given intravenously 30 minutes $\text{prior to}$ low output syndrome (LOS induction, and supplemental doses of DEX (4 mg/kg) or vehicle administered at 15 and 75 minutes $\text{post-LOS}$ induction. Low output syndrome was induced by intravenous administration of a myocardial depressor protein (MDP) which has been isolated from the venom of the Western diamondback rattlesnake, $\text{Crotalus atrox}$.Neither DEX nor its vehicle had a significant effect during the entire experiment, that is, in the normal or low cardiac output state in most of the hemodynamic parameters investigated.The three hour mortality rate for the DEX-treated animals was 22% (n=10) while that of the control group was 41% (n=26) indicating that the beneficial effects of this corticosteroid are not really apparent from hemodynamic evaluation alone. Since DEX only had a significant post-LOS induction effect in maintaining a lower left ventricular end-diastolic and pulmonary capillary wedge pressures, a higher arterio-venous oxygen saturation difference, and a more efficient contractile state of myocardial fibers (V_max), an indirect correlation to coronary arterial blood flow at the subcellular level was sought. To this effect, prostaglandins and specific lipid classes of left ventricular myocardium (LVM) from control and LOS animals receiving either vehicle or DEX were analyzed. Low output state induction alone raised myocardial PG levels above those of sham-catheterized animals; on the other hand, dexamethasone induced a significant decrease in the three prostaglandins studied when administered to control (no LOS) animals. In the presence of LOS, however, dexamethasone overrode in part the increase in PGE₁ and PGE₂ brought about by LOS while in the case of PGE_2α the LOS effect was totally prevented and its concentration was not significantly higher than in control animals receiving dexamethasone.LOS induction led to an increase in myristic and arachidonic acids and a decrease in palmitic and linolenic acids. Dexamethasone administration to control animals increased the concentration of stearic acid above all the other groups but decreased the concentration of linolenic acid when compared to DEX-treated animals with LOS or sham-catheterized animals.There were no significant differences in the total myocardial lipid among the four groups of animals studied.It is suggested that the potentially beneficial effects of corticosteroid administration to animals with low output syndrome are related to their effects on fatty acid and prostaglandin content of myocardium.     

Self-association of oxyhaemoglobin. A nuclear magnetic relaxation study in H2O/D2O solutions

The proton and deuterium longitudinal relaxation rates were Studied at room temperature up to the highest protein concentrations in oxyhaemoglobin solutions of different H₂O/D₂O composition. The deuterium relaxation rates followed the experimentally well known single linear dependence on protein concentration, the slopes being little influenced by solvent (D₂O/H₂O) composition. The proton ralaxation rates show two different liner dependences on haemoglobin concentration. The entire concentration range is described by two straight lines with the threshold concentration about 11 mM (in haem), The ratio of the slopes is 1.6 (high-to-low Hb-conc.). Only in the higher concentration range two T₁'s were observed if the solvent contained more than half of D₂O. The slow relaxation phase of protons has T₁'s similar to those measured in solutions with less than half of D₂O. The relaxation of the other phase was ten times faster. The ratio of the proton populations in these two phases was equal to 2 (slow-to-fast) and independent of protein concentration. The fast relaxing protons are attributed to water molecules encaged within two or more haemoglobin molecules which associate for times long enough on the PMR time-scale.     

Viomycin resistance: alterations of either ribosomal subunit affect the binding of the antibiotic to the pair subunit and the entire ribosome becomes resistant to the drug.

Subcellular localization of $\text{[}{}^3\text{H]1α,24(R)-dihydroxyvitamin}$ D₃ and $\text{[}{}^3\text{H]1α,24(S)-dihydroxyvitamin}$ D₃ in rat intestinal mucosa was investigated in comparison with the $\text{[}{}^3\text{H]1α-hydroxyvitamin}$ D₃. The 24(R) and 24(S) isomers of 1α,24-dihydroxyvitamin D₃ were gradually transformed to 1α,24(R)25-trihydroxyvitamin D₃ and 1α,24(S)25-trihydroxyvitamin D₃, and the plasma concentrations of these metabolites were 10.30 and 1.36 pmol/ml, respectively. The major portions of the administered compounds distributed in the nuclear fraction of the intestinal mucosa remained unchanged, and the amounts of 1α,24(R)-dihydroxyvitamin D₃ and 1α,24(S)-dihydroxyvitamin D₃ were 4.25 and 0.306 pmol/g intestinal mucosa, respectively. No detectable amount of the metabolites, 1α,24(R)25-trihydroxyvitamin D₃ and 1α,24(S)25-trihydroxyvitamin D₃ were found in the same nuclear fractions. In the case with the $\text{[}{}^3\text{H]1α-hydroxyvitamin}$ D₃, however, the compound was rapidly metabolized to 1α,25-dihydroxyvitamin D₃.The metabolite, 1α,25-dihydroxyvitamin D₃, was seen in the nuclear fraction of the intestinal mucosa at a concentration of 2.44 pmol/g intestinal mucosa.     

Elimination kinetics and protein binding of theophylline in the rabbit

The detailed elimination kinetics of theophylline were studied in 27 rabbits. Each received a 10 mg/kg intravenous bolus of aminophylline. The theophylline half-life ($\text{T}\text{1}\text{2}$) was 3.8 ± 0.63 hr. The apparent volume of distribution (V_D) and total body clearance (TBC) for theophylline were 439 ± 60 ml/kg and 81.0 ± 17.3 ml/kg·hr respectively. Theophylline protein binding was determined in 10 animals. The mean bound fraction was 74.3 ± 3.9% (range, 68.3–78.0%); the fraction bound was concentration indifferent over a serum concentration range of 5–20 μgm/ml.     

Occurrence of cholecalciferol in the calcinogenic plant Trisetum flavescens.

The grass Trisetum flavescens causes severe calcification of soft tissues upon ingestion by various species, which has been ascribed by others to a 1,25(OH)₂ vitamin D₃-like activity.By a special purification procedure involving high pressure liquid chromatography and continuous biological testing the active principle was purified. By means of $\text{GC}\text{MS}$ it was identified as cholecalciferol, being present in a concentration of about 0.1 ppm in the lyophylized plant dry matter. 1,25(OH)₂ vitamin D₃ or other metabolites of vitamin D₃ were not present. Since such low concentrations could hardly explain the calcinosis observed, a more active “bound form” of vitamin D₃ may be present in Trisetum flavescens.     

Aliphatic hydroxylation by highly purified liver microsomal cytochrome P-450. Evidence for a carbon radical intermediate

The oxidation of norbornane by a reconstituted liver cytochrome P-450 system affords $\text{exo}$- and $\text{endo}$-2-norborneol in a ratio of 3.4:1. The ratio of these products was found to be 0.76:1 when $\text{exo,exo,exo,exo}$-2,3,5,6-tetradueteronorbornane was oxidized. Analysis of the mass spectra of the products from the deuterated hydrocarbon showed that 25% of the $\text{exo}$-norborneol contained four deuterium atoms whereas 9% of the $\text{endo}$-norborneol contained three deuterium atoms. These results, which indicate a very large isotope effect ($\text{k}{}_{\mathrm{H}}\text{k}{}_{\mathrm{D}}\text{= 11.5±1}$) and a significant amount of epimerization for the hydroxylation of norbornane by cytochrome P-450, suggest an initial hydrogen abstraction to give a carbon radical intermediate.     

Determination of homovanillic acid turnover in man

Homovanillic acid (HVA) labelled with five deuterium (d) atoms was used to determine the total body turnover of HVA, the size of the peripheral body pool of HVA and HVA elimination characteristics in five healthy men. After i.v. injection of 5.5 μmoles (1 mg) of HVA-d₅ the levels of HVA-d₅ and endogenous HVA (HVA-d_o) in plasma and urine were followed by mass fragmentography using HVA-d₂ as the carrier and internal standard. Following an initial distribution phase of 10–20 minutes the plasma elimination curve of HVA-d₅ was monoexponential with a mean $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 0.66 hrs. The apparent volume of distribution (V_D) approximated the volume of the body water. The content of HVA in the peripheral body pool calculated from the plasma levels of HVA-d_o and V_D was 3.4 moles. The urinary HVA excretion rate (mean 1.70 moles/hour) was 45% of the total body turnover, the recovery of urinary HVA-d₅ was 48% of the mean body clearance. Together the results indicate that about 50% of the HVA formed in the body is eliminated by mechanisms other than renal excretion.     

1,25-Dihydroxyvitamin D3-glycoside: Identification of a calcinogenic principle of solanum malacoxylon

The water soluble calcinogenic factor present in the plant $\text{Solanum}$$\text{malacoxylon}$ is partially purified by selective extraction and chromatography on silicic acid and then hydrolyzed with a mixed preparation of glycosidases from the sea worm, $\text{Charonia}$$\text{lampus}$. Hydrolysis produces a chloroform soluble factor with biologic characteristics of 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃), the hormonal form of vitamin D. Purification of this factor is accomplished by chromatography on Sephadex LH-20, silicic acid, and Celite columns, yielding 3 μg of active material. During the isolation, bioactivity (as assessed by the ability of fractions to compete with labeled 1,25-(OH)₂D₃ for binding to a specific intestinal receptor protein) migrates exactly with authentic tritiated 1,25-(OH)₂D₃. The purified factor has an ultraviolet absorption spectrum identical to that of 1,25-(OH)₂D₃ and analysis via direct probe mass spectrometry yields a parent molecular ion of m/e 416 and a fragmentation pattern indistinguishable from synthetic 1,25-(OH)₂D₃ hormone. We therefore conclude that the vitamin D-like principle in $\text{Solanum}$$\text{malacoxylon}$ is a sterol-glycoside which contains the 1,25-(OH)₂D₃ molecule as its active sterol component.     

Early stimulation of alkaline phosphatase activity in response to 1 alpha, 25-dihydroxycholecalciferol.

Alkaline phosphatase activity (APA) stimulation in response to 1,25-dihydroxycholecalciferol (1,25 (OH)₂D₃) has been studied in vitamin-D-deficient rat intestinal brush borders prepared from $\text{ex}$-$\text{vivo}$-perfused duodeno-jejunal segments. Basal APA in intestines perfused with ethanol remained constant throughout the experiments. APA was significantly increased when intestines were perfused with 1,25 (OH)₂D₃ (3 nM) for 30, 45 or 60 min. A dose-effect response was observed when 1,25 (OH)₂D₃ increased in the perfusion medium. The maximal alkaline phosphatase activity after a 45-min perfusion (2404 ± 379 m^TU/mg prot.) was observed when 1,25 (OH)₂D₃ concentration was 6 nM. Cholecalciferol had no effect in this system.     

The origin of the carbon chain in the thiazole moiety of thiamine in Escherichia coli: incorporation of deuterated 1-deoxy-D-threo-2-pentulose

Non growing washed cells of Escherichia coli, derepressed for the biosynthesis of thiamine, have been incubated in the presence of glucose and either 1-deoxy-$\text{D}$-threo-2-pentulose $\text{1}$ or 1-déoxy-$\text{D}$-erythro-2-pentulose $\text{2}$ trideuterated on the methyl group. The incorporation of deuterium into the thiazole moiety of thiamine was measured by mass spectrometry. The label of the threo-compound was found in more than 40% of the thiazole biosynthesized in its presence; the label of the erythro-compound in less than 5%. Hence it is likely that the carbon chain of 1-deoxy-$\text{D}$-threo-2-pentulose is the precursor of the five carbons chain of the thiazole moiety of the thiamine molecule in E. coli.     

Rodent macrophages metabolize 25-hydroxyvitamin D3in,vitro

Rodent macrophages metabolized 25-hydroxyvitamin D₃ to an unidentified metabolite during $\text{in}\text{,}\text{vitro}$ incubations. The production of this macrophage-derived metabolite of 25-hydroxyvitamin D₃ increased as the substrate concentration was raised. A two step high pressure liquid chromatography system revealed a unique elution position of this macrophage-derived metabolite that did not match the elution positions of any of the vitamin D₃ metabolites available in this laboratory. This unique metabolite was formed $\text{in}\text{,}\text{vitro}$ within one minute by incubated macrophages although its formation increased gradually up to 60 minutes of incubation.