Necrosis avidity of (99m)Tc(CO)3-labeled pamoic acid derivatives: synthesis and preliminary biological evaluation in animal models of necrosis

In a search for an infarct avid tracer agent with improved properties, we have observed that bis-DTPA derivatives of pamoic acid have a high avidity for necrotic tissue. Here, we report the synthesis, radiolabeling, and preliminary evaluation in normal mice and rats with hepatic infarction of the (99m)Tc-tricarbonyl complexes of N, N'-bis(diethylenetriaminopentaacetato)-4,4'-methylene bis(2-hydroxy-3-naphthoic hydrazide) ( (99m)Tc(CO) 3-bis-DTPA-pamoate) and [ N-(5-aminopentyl)pyridin-2-yl-methylamino]methylacetato-4,4'-methylene-2-hydroxy-3-napthalenecarboxamide-(2'-hydroxy-3'-naphthoic acid methyl ester) ( (99m)Tc(CO) 3 -12). Radiolabeling with (99m)Tc(CO) 3 (+) was achieved with a radiochemical yield of over 95% for both tracer agents. In normal mice, the polar (99m)Tc(CO) 3-bis-DTPA-pamoate was cleared from plasma via both the liver and the kidneys, while the more lipophilic (99m)Tc(CO) 3 -12 was rapidly cleared via the liver. Blood clearance in mice was faster for (99m)Tc(CO) 3 -12 (0.1% injected dose per gram at 4 h postinjection) than for (99m)Tc(CO) 3-bis-DTPA-pamoate (9.3% injected dose per gram at 4 h postinjection). Affinity and specificity of the tracers for necrotic tissue was studied in rats with hepatic infarction and ethanol-induced necrosis of the liver or muscles. Activity ratios of infarct to viable liver tissue of (99m)Tc(CO) 3-bis-DTPA-pamoate quantified by autoradiography of tissue slices ranged from 4 to 18, depending on the necrosis model and time postinjection of the tracer. Infarcts were also visualized in vivo by (99m)Tc(CO) 3-bis-DTPA-pamoate planar gamma imaging. After injection of (99m)Tc(CO) 3-bis-DTPA-pamoate, in vivo and ex vivo images correlated well with histochemical staining with triphenyltetrazolium chloride and hematoxylin and eosin. (99m)Tc(CO) 3 -12 on the other hand showed no uptake in necrotic tissue. Stability of the tracers was determined in vitro after storage at room temperature and by histidine challenge experiments, and in vivo in mouse plasma and in urine (for (99m)Tc(CO) 3-bis-DTPA-pamoate). (99m)Tc(CO) 3-bis-DTPA-pamoate was unstable in vitro to histidine challenge, while (99m)Tc(CO) 3 -12 was 98% stable in vitro in the same conditions. Both tracers showed good in vivo stability. (99m)Tc(CO) 3-bis-DTPA-pamoate shows high specificity for necrotic tissue and merits further evaluation as a necrosis avid imaging agent. (99m)Tc(CO) 3 -12 is not useful for visualization of necrotic tissue.     

Development and evaluation of a 68Ga labeled pamoic acid derivative for in vivo visualization of necrosis using positron emission tomography

In this study, we labeled N,N′-bis(diethylenetriamine pentaacetic acid)-pamoic acid bis-hydrazide (bis-DTPA-PA) with the generator produced PET radionuclide gallium-68 and evaluated ⁶⁸Ga-bis-DTPA-PA as a potential tracer for in vivo visualization of necrosis by positron emission tomography (PET). Radiolabeling was achieved with a decay-corrected radiochemical yield of 63%. Biodistribution and in vivo stability studies in normal mice showed that ⁶⁸Ga-bis-DTPA-PA is cleared faster from normal tissue than the previously reported ^99mTc(CO)₃ complex with bis-DTPA-PA which on the other hand is more stable in vivo. ⁶⁸Ga-bis-DTPA-PA showed a 3.5–5 times higher binding to necrotic tissue than to viable tissue as shown by in vitro autoradiography while no statistically significant increased hepatic uptake was found in a biodistribution study in a mouse model of hepatic apoptosis. Specificity and avidity for necrosis was further evaluated in rats with a reperfused partial liver infarction and ethanol induced muscular necrosis. Dynamic microPET images showed a fast and prolonged uptake of ⁶⁸Ga-bis-DTPA-PA in necrotic tissue with in vivo and ex vivo images correlating well with histochemical stainings. With necrotic to viable tissue activity ratios of 8–15 on ex vivo autoradiography, depending on the necrosis model, ⁶⁸Ga-bis-DTPA-PA showed a faster and higher uptake in necrotic tissue than the ^99mTc(CO)₃ analog. These results show that ⁶⁸Ga-bis-DTPA-PA specifically binds to necrotic tissue and is a promising tracer for in vivo visualization of necrosis using PET.     

Radiolabeling and preliminary biological evaluation of a (99m)Tc(CO)(3) labeled 3,3'-(benzylidene)-bis-(1H-indole-2-carbohydrazide) derivative as a potential SPECT tracer for in vivo visualization of necrosis

N,N'-bis(diethylenetriamine pentaacetic acid)-3,3'-(benzylidene)-bis-(1H-indole-2-carbohydrazide) (bis-DTPA-BI) was radiolabeled with (99m)Tc(CO)(3). The resulting (99m)Tc(CO)(3)-bis-DTPA-BI was characterized (LC-MS) and evaluated as a potential SPECT tracer for imaging of necrosis in Wistar rats with a reperfused partial liver infarction and Wistar rats with ethanol induced muscular necrosis. To study the specificity, uptake of (99m)Tc(CO)(3)-bis-DTPA-BI was also studied in a mouse model of Fas-mediated hepatic apoptosis. The obtained results indicate that (99m)Tc(CO)(3)-bis-DTPA-BI displays selective uptake in necrotic tissue and can be used for in vivo visualization of necrosis by SPECT.     

Determination of monoenoic fatty acid double bond position by permanganate-periodate oxidation followed by high-performance liquid chromatography of carboxylic acid phenacyl esters

This investigation was carried out to develop methods for a reverse-phase, high-performance liquid chromatography analysis of the monocarboxylic and dicarboxylic acids produced by permanganate-periodate oxidation of monoenoic fatty acids. Oxidation reactions were performed using [U-14C]oleic acid and [U-14C]oleic acid methyl ester in order to measure reaction yields and product distributions. The 14C-labeled oxidation products consisted of nearly equal amounts of monocarboxylic and dicarboxylic acid (or dicarboxylic acid monomethyl ester), with few side products (yield greater than 98%). Conversion of the carboxylic acids to phenacyl esters proceeded to completion. HPLC of carboxylic acid phenacyl esters was performed using a C18 column with a linear solvent gradient beginning with acetonitrile/water (1/1) and ending with 100% acetonitrile. Excellent resolution was achieved for all components of a mixture of C5 through C12 monocarboxylic acid phenacyl esters and C6 through C11 dicarboxylic acid phenacyl esters. Resolution was also achieved for all components of a mixture of C5 through C12 monocarboxylic acid phenacyl esters and C6 through C11 dicarboxylic acid monomethyl, monophenacyl esters. The resolution obtained by HPLC demonstrates that, for a wide range of monoenoic fatty acids, both products of a permanganate-periodate oxidation can be identified on a single chromatogram. Free fatty acids and fatty acid methyl esters were analyzed with equal success. Neither the oxidation nor the esterification reaction caused detectable hydrolysis of methyl ester. The method is illustrated for free acids and methyl esters of 14:1 (cis-9), 16:1 (cis-9), 18:1 (cis-6), 18:1 (cis-9), and 18:1 (cis-11).     

Modulations of food-derived substances on intestinal permeability in Caco-2 cell monolayers

The effects of more than 300 kinds of food extracts on intestinal permeability were investigated in Caco-2 cells with the use of model compounds: Lucifer Yellow (LY) for the paracellular pathway, Fluorescein (FC) for the monocarboxylic acid transporter-mediated pathway, and Rhodamine 123 (RH) for the p-glycoprotein-mediated efflux pathway. With several extracts of increasing or decreasing LY permeation, increasing FC or RH permeation was also observed, indicating modulation by dietary substances in several pathways for intestinal absorption.     

双歧杆菌在家兔实验性肝坏死模型中的靶向增殖研究

目的 探讨双歧杆菌在海扶超声聚焦刀（HIFU）制作的肝凝固性坏死模型中的靶向性增殖的可能性。方法 用HIFU制作成家兔肝凝固性坏死模型,经耳缘静脉注射两歧双歧杆菌活菌,通过检测注射前后家兔体温、红细胞、白细胞和血小板等生理指标的变化和注射后主要器官的组织切片来评价双歧杆菌作为基因治疗载体系统的安全性,同时对肝坏死模型等组织切片还做了革兰染色检测,证明了双歧杆菌在坏死组织中的增殖。结果 双歧杆菌能够在HIFU制作的肝凝固性坏死模型中增殖、而这一增殖过程并不影响家兔的生命安全。结论 双歧杆菌作为肿瘤基因治疗的靶向载体系统是安全和可行的,HIFU制作的肝坏死模型可以用于双歧杆菌基因治疗的靶向载体系统的辅助研究。     

Inhibitory effects of anions and active site amino acid sequence of chicken liver L-2-hydroxyacid oxidase A, a member of the FMN-dependent α-hydroxyacid oxidizing enzyme family

Monocarboxylic acids with aliphatic chains were found to be mixed inhibitors of chicken liver L-2-hydroxyacid oxidase A when L-2-hydroxy-4-methylthiobutanoic acid was used as the substrate. The finding that the binding affinity of the enzyme for monocarboxylic acids was directly proportional to the number of carbon atoms in the chain strongly suggests that in addition to the electrostatic interaction due to the carboxyl moiety, hydrophobic forces may also be involved in the binding affinity of monocarboxylic acids to the enzyme's active site. Oxalate, a dicarboxylic acid, also resulted in a mixed-type inhibition of chicken liver L-2-hydroxyacid oxidase A, and, surprisingly, its binding affinity to the enzyme was found to be quite high as compared with monocarboxylic acids. This is probably due to the fact that the two carboxyl groups of oxalate give rise to electrostatic interactions with the positively charged side chains of two adjacent residues in the polypeptide chain. The inhibitory effects of other dicarboxylic acids was found to decrease as the number of carbon atoms in the chain increased. Oxamate was found however to be a novel type of potent inhibitor of the enzyme. All in all, these kinetic studies and the amino acid sequence determination in the active site region after limited proteolysis of the polypeptide chain definitely establish that chicken liver NADH/FMN containing L-2-hydroxyacid oxidase A is a member of the FMN-dependent α-hydroxyacid oxidizing enzyme family.     

Characterization of salicylate uptake across the basolateral membrane of the malpighian tubules of Drosophila melanogaster

The organic anion salicylate is a plant secondary metabolite that can protect plants against herbivores. Transport of salicylate across the basolateral membrane of the Malpighian tubules of Drosophila melanogaster was studied using a radioisotope tracer technique. The uptake of [(14)C]salicylate by the Malpighian tubules was active, saturable and Na(+)-dependent; the maximum uptake rate (J(max)) and the half saturation concentration (K(t)) were 12.6 pmoltubule(-1)min(-1) and 30.7micromoll(-1), respectively. In contrast to organic anion transport by vertebrate renal tissues, salicylate uptake was not trans-stimulated by glutarate (0.01-1.0 mmoll(-1)) or cis-inhibited by high concentrations (5 mmoll(-1)) of various alpha-keto acids (glutaric acid, alpha-ketoglutaric acid, succinic acid, and citric acid). Changes in basolateral membrane potential or physiologically relevant changes in bathing saline pH did not affect the rate of [(14)C]salicylate uptake. Ring-structure monocarboxylic acids (benzoic acid, nicotinic acid, gentisic acid, unlabelled salicylic acid, alpha-cyano-4-hydroxycinnamic acid, probenecid, fluorescein, and P-aminohippuric acid) strongly inhibited [(14)C]salicylate uptake rate. In contrast, short-chain monocarboxylic acids had little (butyric acid) or no effect (lactic acid, pyruvic acid, and propionic acid). Our results suggest that salicylate uptake across the basolateral membrane of D. melanogaster Malpighian tubules is mediated by a non-electrogenic, alpha-cyano-4-hydroxycinnamic acid-sensitive, Na(+):salicylate cotransport system.     

Construction and evaluation of the tumor imaging properties of 123I-labeled recombinant and enzymatically generated Fab fragments of the TAG-72 monoclonal antibody CC49

Tumor-associated glycoprotein-72 (TAG-72) is overexpressed in a high percentage of epithelial cancers and has proven useful as a target for imaging and targeted radiotherapy. Our goal was to express a recombinant Fab (rFab) of the TAG-72 monoclonal antibody CC49 in Pichia pastoris and directly compare its tumor and normal tissue uptake and imaging properties with enzymatically generated Fab (eFab). In this study, the genes coding for CC49 Fab were cloned from hybridoma cells and expressed in P. pastoris. Fab was purified to homogeneity and its immunoreactivity toward bovine submaxillary mucin (TAG-72) confirmed by ELISA. The tumor and normal tissue localization of (123)I-CC49 rFab and eFab were compared in athymic mice bearing s.c. LS174T colon cancer or TAG-72-negative A375 melanoma xenografts. Results showed that pure and immunoreactive rFab of CC49 was produced and labeled with (123)I. At 24 h post i.v. injection (p.i.), tumor uptake for (123)I-rFab in LS174T xenografts was 6.0% ID/g which was 18-fold higher than in A375 tumors. Tumor-to-normal tissue ratios increased between 2 and 24 h and exceeded 5:1 at 24 h p.i. of (123)I-rFab. (123)I-rFab exhibited significantly lower liver uptake at 12 h p.i. and lower kidney uptake at 2 h p.i. than (123)I-eFab. LS174T tumors were imaged as early as 2 h after administration of (123)I-rFab. We conclude that CC49 rFab can be produced in a P. pastoris host system and accumulated at comparable levels as eFab in LS174T colon cancer xenografts in mice. The lower liver uptake of (123)I-rFab as compared with eFab suggests that it may be more useful for imaging liver lesions. No major effect, except for kidneys and liver, was observed on tumor and normal tissue uptake due to introduction of hexahistidine and FLAG affinity tags or peptide linkers in the scaffold of rFab.     

Trafficking of dietary oleic, linolenic, and stearic acids in fasted or fed lean rats

Increasing evidence supports the notion that there are significant differences in the health effects of diets enriched in saturated, as opposed to monounsaturated or polyunsaturated fat. However, the current understanding of how these types of fat differ in their handling by relevant tissues is incomplete. To examine the effects of fat type and nutritional status on the metabolic fate of dietary fat, we administered (14)C-labeled oleic, linolenic, or stearic acid with a small liquid meal to male Sprague-Dawley rats previously fasted for 15 h (fasted) or previously fed ad libitum (fed). (14)CO(2) production was measured for 8 h after tracer administration. The (14)C content of gastrointestinal tract, serum, liver, skeletal muscle (soleus, lateral, and medial gastrocnemius), and adipose tissue (omental, retroperitoneal, and epididymal) was measured at six time points (2, 4, 8, 24, and 48 h and 10 days) after tracer administration. Plasma levels of glucose, insulin, and triglyceride were also measured. Oxidation of stearic acid was significantly less than that of either linolenic or oleic acid in both the fed and fasted states. This reduction was in part explained by a greater retention of stearic acid within skeletal muscle and liver. Oxidation of oleate and stearate were significantly lower in the fed state than in the fasted state. In the fasted state, liver and skeletal muscle were quantitatively more important than adipose tissue in the uptake of dietary fat tracers during the immediate postprandial period. In contrast, adipose tissue was quantitatively more important than skeletal muscle or liver in the fed state. The movement of carbons derived from dietary fat between tissues is a complex time-dependent process, which varies in response to the type of fat ingested and the metabolic state of the organism.     

18F]Galacto-RGD: synthesis, radiolabeling, metabolic stability, and radiation dose estimates

It has been demonstrated in various murine tumor models that radiolabeled RGD-peptides can be used for noninvasive determination of alphavbeta3 integrin expression. Introduction of sugar moieties improved the pharmacokinetic properties of these peptides and led to tracer with good tumor-to-background ratios. Here we describe the synthesis, radiolabeling, and the metabolic stability of a glycosylated RGD-peptide ([18F]Galacto-RGD) and give first radiation dose estimates for this tracer. The peptide was assembled on a solid support using Fmoc-protocols and cyclized under high dilution conditions. It was conjugated with a sugar amino acid, which can be synthesized via a four-step synthesis starting from pentaacetyl-protected galactose. For radiolabeling of the glycopeptide, 4-nitrophenyl-2-[18F]fluoropropionate was used. This prosthetic group allowed synthesis of [18F]Galacto-RGD with a maximum decay-corrected radiochemical yield of up to 85% and radiochemical purity >98%. The overall radiochemical yield was 29 +/- 5% with a total reaction time including final HPLC preparation of 200 +/- 18 min. The metabolic stability of [18F]Galacto-RGD was determined in mouse blood and liver, kidney, and tumor homogenates 2 h after tracer injection. The average fraction of intact tracer in these organs was approximately 87%, 76%, 69%, and 87%, respectively, indicating high in vivo stability of the radiolabeled glycopeptide. The expected radiation dose to humans after injection of [18F]Galacto-RGD has been estimated on the basis of dynamic PET studies with New Zealand white rabbits. According to the residence times in these animals the effective dose was calculated using the MIRDOSE 3.0 program as 2.2 x 10(-2) mGy/MBq. In conclusion, [18F]Galacto-RGD can be synthesized in high radiochemical yields and radiochemical purity. Despite the time-consuming synthesis of the prosthetic group 185 MBq of [18F]Galacto-RGD, a sufficient dose for patient studies, can be produced starting with approximately 2.2 GBq of [18F]flouride. Moreover, the fast excretion, the suitable metabolic stability and the low estimated radiation dose allow to evaluate this tracer in human studies.     

Synthesis and bio-evaluation of a new fatty acid derivative for myocardial imaging

Development of a (99m)Tc-fatty acid analogue is of interest, as (99m)Tc is logistically advantageous over the cyclotron-produced (11)C and (123)I. Synthesis of a 16 carbon fatty acid derivative and its radiolabeling with the novel [(99m)TcN(PNP)](2+) core is described here. Hexadecanedioic acid was conjugated to cysteine in an overall yield of 55%. This ligand could be labeled with (99m)Tc via the [(99m)TcN(PNP)](2+) core, in 80% yield, as a mixture of two isomers (syn and anti). The major isomer isolated by HPLC was used for bioevaluation studies in swiss mice and compared with radioiodinated iodophenyl pentadecanoic acid (IPPA), an established agent for myocardial metabolic imaging. (99m)Tc-labeled complex cleared faster from the non-target organs, namely, liver, lungs, and blood compared to that of [(125)I]-IPPA. However, the complex exhibited lower uptake and faster washout from the myocardium as compared to [(125)I]-IPPA.     

Click chemistry for (18)F-labeling of RGD peptides and microPET imaging of tumor integrin alphavbeta3 expression

The cell adhesion molecule integrin alpha vbeta 3 plays a key role in tumor angiogenesis and metastasis. A series of (18)F-labeled RGD peptides have been developed for PET of integrin expression based on primary amine reactive prosthetic groups. In this study, we report the use of the Cu(I)-catalyzed Huisgen cycloaddition, also known as a click reaction, to label RGD peptides with (18)F by forming 1,2,3-triazoles. Nucleophilic fluorination of a toluenesulfonic alkyne provided (18)F-alkyne in high yield (nondecay-corrected yield: 65.0 +/- 1.9%, starting from the azeotropically dried (18)F-fluoride), which was then reacted with an RGD azide (nondecay-corrected yield: 52.0 +/- 8.3% within 45 min including HPLC purification). The (18)F-labeled peptide was subjected to microPET studies in murine xenograft models. Murine microPET experiments showed good tumor uptake (2.1 +/- 0.4%ID/g at 1 h postinjection (p.i.)) with rapid renal and hepatic clearance of (18)F-fluoro-PEG-triazoles-RGD 2 ( (18)F-FPTA-RGD2) in a subcutaneous U87MG glioblastoma xenograft model (kidney 2.7 +/- 0.8%ID/g; liver 1.9 +/- 0.4%ID/g at 1 h p.i.). Metabolic stability of the newly synthesized tracer was also analyzed (intact tracer ranging from 75% to 99% at 1 h p.i.). In brief, the new tracer (18)F-FPTA-RGD2 was synthesized with high radiochemical yield and high specific activity. This tracer exhibited good tumor-targeting efficacy and relatively good metabolic stability, as well as favorable in vivo pharmacokinetics. This new (18)F labeling method based on click reaction may also be useful for radiolabeling of other biomolecules with azide groups in high yield.     

Interactions between the omega- and beta-oxidations of fatty acids

Long-chain monocarboxylic, omega-hydroxymonocarboxylic and dicarboxylic acids were activated approximately at the same rate by rat liver homogenates into their CoA esters (2-3 U/g liver). These acyl-CoA were substrates for rat liver peroxisomal beta-oxidation. The distribution of the peroxisomal oxidation of these substrates was also studied in various tissues. Rat liver mitochondria were capable of oxidizing long-chain monocarboxyl- and omega-hydroxymonocarboxylyl-CoAs but not dicarboxylyl-CoAs. When the mitochondrial preparations were incubated in coupling conditions, the addition of either free decanoic acid or free 10-hydroxydecanoic acid resulted in an increase of the oxygen uptake conversely to the addition of decanedioic acid. The comparative study of the chain-length substrate specificity of peroxisomal fatty acyl-CoA oxidase and mitochondrial fatty acyl-CoA dehydrogenase activities revealed that, actually, both types of organelles, peroxisomes and mitochondria, contain "oxido-reductases" active on long-chain monocarboxylyl-CoAs, omega-hydroxymonocarboxylyl-CoAs and dicarboxylyl-CoAs.     

Comparison of the distribution kinetics and metabolism to acid end-products of corticosterone and 11-deoxycorticosterone in BALB/c mice

The conversion of [4 14C]corticosterone[( 14C]B) and 11-deoxy-[1,2-3H]corticosterone [( 3H]DOC) to steroidal carboxylic acids was studied in the BALB/c mouse. There was rapid and preferential excretion of [3H]DOC metabolites into the gastrointestinal tract. Excretion of 14C through the kidney was higher than 3H excretion. Within minutes of intraperitoneal injection, levels of 3H and 14C in most organs reached their maximal levels and subsequently decreased in an exponential pattern. The majority of the organs took up 14C to a greater extent than 3H. Using tissue blood ratio of tracer (T/B) as criterion, it was found that liver, gall bladder, intestine, and kidney concentrated 3H and 14C-labeled steroid from blood. T/B for 3H exceeded that for 14C in the gastrointestinal tract. Abdominal fat preferentially took up [3H]DOC tracer, whereas [14C]B tracer was not taken up by this tissue. T/B was less than 1 for 3H and 14C in heart, thymus, spleen, brain, skeletal muscle and skin. In these organs uptake of B and its metabolites was greater than that of DOC and its metabolites. In liver, [14C]B and [3H]DOC were converted to carboxylic acid metabolites which accumulated in the intestine. The most abundant acid was 11 beta,20 alpha-dihydroxy-3-oxo-pregn-4-en-21-oic acid from B. The acid metabolites of DOC were not identified. For both steroids, acids were major metabolic end-products.     

The incidence of virus diseases in English sweet-cherry orchards and their effect on yield

Two thousand sweet-cherry trees (Prunus avium) in English orchards were tested for virus infection by using Lambert and Mazzard F 12/1 as indicators. Most trees of varieties commonly grown before 1920 were infected with more than one virus, usually little cherry (69%) and necrotic ringspot/prune dwarf (56%). Other infection was less prevalent, 35% of these trees having European rusty mottle, 30% ring mottle and 3% necrotic rusty mottle. Most trees of varieties introduced since 1920 were virus-free (61%) but some had become infected with each of these viruses except necrotic rusty mottle. In a field trial of 12 years duration the yield of three varieties was diminished by infection with necrotic ringspot/prune dwarf, rugose mosaic, rusty mottle, ring mottle and necrotic line pattern. The effect of rusty mottle was due to growth suppression resulting in smaller trees, but that of other viruses was also due to impaired fertility. The yield of one variety (Merton Heart) was greatly enhanced by infection with rugose mosaic, rusty mottle and necrotic ringspot/prune dwarf viruses. The high incidence of virus infection and consequent yield depression has probably diminished the potential yield by at least 30% and contributed to the decline in acreage of sweet cherries in England.     

Plasma clearance, tissue distribution and metabolism of hyaluronic acid injected intravenously in the rabbit.

The plasma clearance, tissue distribution and metabolism of hyaluronic acid were studied with a high average molecular weight [3H]acetyl-labelled hyaluronic acid synthesized in synovial cell cultures. After intravenous injection in the rabbit the label disappeared from the plasma with a half-life of 2.5--4.5 min, which corresponds to a normal hyaluronic acid clearance of approx. 10 mg/day per kg body weight. Injection of unlabelled hyaluronic acid 15 min after the tracer failed to reverse its absorption. Clearance of labelled polymer was retarded by prior injection of excess unlabelled hyaluronic acid. The maximum clearance capacity was estimated in these circumstances to be about 30 mg/day per kg body wt. The injected material was concentrated in the liver and spleen. As much as 88% of the label was absorbed by the liver, where it was found almost entirely in non-parenchymal cells. Degradation was rapid and complete, since volatile material, presumably 3H2O, appeared in the plasma within 20 min. Undegraded [3H]hyaluronic acid, small labelled residues and 3H2O were detected in the liver, but there was little evidence of intermediate oligosaccharides. No metabolite except 3H2O was recognized in plasma or urine. Two-thirds of the radioactivity was retained in the body water 24 h later, and small amounts were found in liver lipids. Radioactivity did not decline in the spleen as rapidly as in the liver. The upper molecular weight limit for renal excretion was about 25 000. Renal excretion played a negligible part in clearance. It is concluded that hyaluronic acid is removed from the plasma and degraded quickly by an efficient extrarenal system with a high reserve capacity, sited mainly in the liver.     

The utilization of short-chain monocarboxylic acids as carbon sources for the production of gamma-linolenic acid by Mucor strains in fed-batch culture

The Fischer-Tropsch reaction water, which contains C₂ to C₅ monocarboxylic acids, generated as a co-product of the Sasol industrial oil-from-coal process, constitutes a potential cheap carbon substrate for the production of gamma-linolenic acid (GLA) by selced Mucor species. Three strains of Mucor were each grown in an air-lift reactor operated in a fed-batch, pH-stat mode under N-limitation with a mixture of C₂ to C₅ monocarboxylic acids as both pH titrant and carbon source. The production of GLA from this substrate was evaluated. Growth typically resulted in the rapid assimilation of acetic, n-butyric and n-valeric acids. Although propionic, iso-butyric and iso-valeric acids were assimilated to varying degrees, these acids accumulated in the culture. Mucor circinelloides CBS 203.28 gave the best results in that it assimilated 36% to 100% of each acid, had a biomass yield coefficient of 0.3 (calculated on acids utilized), and contained 28% crude oil, 84% of which comprised neutral lipids with a GLA content of 14.4%, giving 33 mg GLA/g biomass. GLA accumulation coincided with a decrease in the stearic-acid content of the neutral-lipid fraction. The results were comparable with previous results obtained with acetic acid and glucose as sole carbon sources, demonstrating the feasibility of producing GLA from the above mixture of organic acids.     

Glyceroneogenesis is the dominant pathway for triglyceride glycerol synthesis in vivo in the rat

Triglyceride synthesis in mammalian tissues requires glycerol 3-phosphate as the source of triglyceride glycerol. In this study the relative contribution of glyceroneogenesis and glycolysis to triglyceride glycerol synthesis was quantified in vivo in adipose tissue, skeletal muscle, and liver of the rat in response to a chow diet (controls), 48-h fast, and lipogenic (high sucrose) diet. The rate of glyceroneogenesis was quantified using the tritium ([(3)H(2)]O) labeling of body water, and the contribution of glucose, via glycolysis, was determined using a [U-(14)C]glucose tracer. In epididymal and mesenteric adipose tissue of control rats, glyceroneogenesis accounted for approximately 90% of triglyceride glycerol synthesis. Fasting for 48 h did not alter glyceroneogenesis in adipose tissue, whereas the contribution of glucose was negligible. In response to sucrose feeding, the synthesis of triglyceride glycerol via both glyceroneogenesis and glycolysis nearly doubled (versus controls); however, glyceroneogenesis remained quantitatively higher as compared with the contribution of glucose. Enhancement of triglyceride-fatty acid cycling by epinephrine infusion resulted in a higher rate of glyceroneogenesis in adipose tissue, as compared with controls, whereas the contribution of glucose via glycolysis was not measurable. Glyceroneogenesis provided the majority of triglyceride glycerol in the gastrocnemius and soleus. In the liver the fractional contribution of glyceroneogenesis remained constant (approximately 60%) under all conditions and was higher than that of glucose. Thus, glyceroneogenesis, in contrast to glucose, via glycolysis, is quantitatively the predominant source of triglyceride glycerol in adipose tissue, skeletal muscle, and liver of the rat during fasting and high sucrose feeding.     

Concentrations of lignin and wall-bound ferulic acid after wounding in the phloem of Chamaecyparis obtusa

The changes of lignin and wall-bound ferulic acid induced by wounding were quantitatively and histochemically investigated in the phloem of Chamaecyparis obtusa. Histochemical staining of lignin was first observed in the necrotic region of the phloem 7 days after wounding and developed in 14 days. Increases of the wall-bound ferulic acid and lignin concentrations were detected in the necrotic tissue at 7 and 14 days, respectively. The concentrations continued to increase until 28 days. The lignin concentration of the callus tissue was observed to be lower than that of the healthy tissue at 14 days, and reached a similar level after 28 days. No quantitative changes of lignin and wall-bound ferulic acid were observed in other tissues. The results indicated that lignin synthesis could be maintained after the phloem cells were discolored and seemed to be necrotic. Distribution and timing of the wall-bound ferulic acid and lignin accumulation suggested that the increased wall-bound ferulic acid was involved in the lignin synthesis after wounding.