The fate of the hydrolysis products of thalidomide in the pregnant rabbit

1. The fate in the pregnant New Zealand White rabbit of oral doses of four ¹⁴C-labelled hydrolysis products of thalidomide, namely α-(o-carboxybenzamido)-glutarimide, 2-phthalimidoglutaramic acid, 2-phthalimidoglutaric acid and 2-(o-carboxybenzamido)glutaramic acid, administered on the 192nd hour of pregnancy has been studied. 2. About 60–95% of the administered ¹⁴C of each compound appears in the urine in 58hr. and the remainder is found in the faeces and in the gut and its contents. 3. Radioactivity is present in the plasma, liver, kidney, brain, muscle, fat and embryo. 4. The ¹⁴C-labelled substances in the plasma and embryo consist of the unchanged compounds and their further hydrolysis products. 5. Since the above four thalidomide hydrolysis products are found in the rabbit conceptus together with their further hydrolysis products after their oral administration to the pregnant rabbit, it appears that the teratogenic activity of thalidomide is due to the compound itself rather than to one or more of its hydrolysis products.     

In vivo covalent binding of [14C]trinitrotoluene to proteins in the rat.

When a single dose of [14C]trinitrotoluene was administered intraperitoneally (i.p.) to rats at 1, 10 or 50 mg/kg of body weight, covalently bound radioactivity was detected in globin, plasma proteins and proteins in the liver and kidney. The extent of covalent binding was dose dependent and was highest in plasma and renal proteins at all times up to 4 h after dosing. Covalent adduct levels in globin, however, decline slower than others. At a dose of 50 mg/kg of body weight, globin covalent adduct levels peaked at 1 h after dosing at 182 pmol/mg protein and subsequently decreased to approximately 50 pmol/mg protein between days 1 and 8. Of the covalent adduct levels in liver and kidney, those in the 10,000 x g and microsomal fractions were found to be higher than that in the cytosolic fraction. Radioactivity covalently bound to globin and the hepatic proteins was susceptible to dilute acid hydrolysis from which 2-amino-4,6-dinitrotoluene (2A) and 4-amino 2,6-dinitrotoluene (4A) were the major products recovered by solvent extraction. Upon acetylation, the hydrolysate gave rise to derivatives identified as the acetates of 2A and 4A on the basis of mass spectrometry and HPLC cochromatography with authentic samples. Four hours after an i.p. dose of [14C]TNT at 50 mg/kg of body weight about 0.4% of the dose was found as bound adducts to hemoglobin, of which approximately 48% was recovered as solvent extractable radioactivity after acid hydrolysis. About 2% of the radioactive dose was in the liver, of which approximately 30% was covalently bound to hepatic proteins, and approximately 49% of that was convertible to solvent extractable radioactivity upon acid hydrolysis. In vitro incubation of [14C]TNT with blood showed that there was a linear increase of covalent adducts in globin during the first 2 h of incubation; the concentration of covalent adducts was slightly higher than that with plasma proteins. The major compounds recovered from the hydrolysate of the globin adducts were also 2A and 4A as obtained from globin in the in vivo studies. On the basis of the in vitro and in vivo study results, we have confirmed the formation of protein adducts following a single i.p. administration of [14C]TNT at 1, 10 or 50 mg/kg of body weight to the rat or by in vitro incubation with blood.(ABSTRACT TRUNCATED AT 400 WORDS)     

Myristic acid, unlike palmitic acid, is rapidly metabolized in cultured rat hepatocytes

This study was designed to examine and compare the metabolism of myristic and palmitic acids in cultured rat hepatocytes. [1-(14)C]-Labeled fatty acids were solubilized with albumin at 0.1 mmol/L in culture medium. Incubation with 24-hr cultured hepatocytes was carried out for 12 hr. Myristic acid was more rapidly (P < 0.05) taken up by the cells than was palmitic acid (86.9 +/- 0.9% and 68.3 +/- 5.7%, respectively, of the initial radioactivity was cleared from the medium after 4 hr incubation). Incorporation into cellular lipids, however, was similar after the same time (33.4 +/- 2.8% and 34.9 +/- 9.3%, respectively, of initial radioactivity). In the early phase of the incubation (30 min), myristic acid was more rapidly incorporated into cellular triglycerides than was palmitic acid (7.4 +/- 0.9% and 3.6 +/- 1.9%, respectively, of initial radioactivity). However, after 12 hr incubation, the radioactivity of cellular triglycerides, cellular phospholipids, and secreted triglycerides was significantly higher with palmitic acid as precursor. Myristic acid oxidation was significantly higher than that of palmitic acid (14.9 +/- 2.2% and 2.3 +/- 0.6%, respectively, of the initial radioactivity was incorporated into the beta-oxidation products after 4 hr). Myristic acid was also more strongly elongated to radiolabeled palmitic acid (12.2 +/- 0.8% of initial radioactivity after 12 hr) than palmitic acid was to stearic acid (5.1 +/- 1.3% of initial radioactivity after 12 hr). The combination of elongation and beta-oxidation results in the rapid disappearance of C14:0 in hepatocytes whereas C16:0 is esterified to form glycerolipids. This study provides evidence that myristic acid is more rapidly metabolized in cultured hepatocytes than is palmitic acid.     

Metabolism of the prostagland in pertility regulator sulprostone in monkeys

The prostaglandin imide analog sulprostone (I) was labeled with tritium in the phenoxy ring to trace the face of the prostanoic acid portion of the molecule and with carbon-14 in the methanesulfonimide moiety to trace the fate of that portion of the molecule. Disposition was studied after intravenous administration to female cynomolgus monkeys. The excretion and plasma pharmacokinetics of total radioactivity indicated that the prostanoic acid and methanesulfonimide moieties were disposed of differently, implying that hydrolysis of sulprostone was extensive and that the hydrolysis products were treated independently. The urinary excretion of tritium (prostanoic acid) was consistent with that observed for other prostaglandins. The urinary excretion and plasma pharmacokinetics of carbon-14 were consistent with those expected for methanesulfonamide. Urinary metabolites identified in 0–2 hr urine include a trace amount of unchanged drug, methanesulfonamide, and two products of β-oxidation — the tetanor and dihydrotetranor prostanoic acids.     

The distribution of glucose and [14C]glucose between erythrocytes and plasma in the rat

1. Of the glucose in rat blood 79.8+/-3.3% (s.d.) was in the plasma. The variance was mostly due to differences between rats. 2. The concentration of glucose in erythrocyte water was 51+/-8% (s.d.) of that in plasma water. 3. The ratio (specific radioactivity in plasma)/(specific radioactivity in whole blood), i.e. the P/B ratio, was estimated for glucose at intervals after intravenous injection of [U-(14)C]glucose and [U-(14)C]fructose. The ratio differed from unity by more than the standard error of a single determination of the specific radioactivity of blood or plasma glucose except from 10 to 17min. after injection of [(14)C]glucose and from 22 to 30min. after injection of [(14)C]fructose. At all other times specific radioactivities in blood had to be corrected to give specific radioactivities in plasma. How to do so is described. 4. The P/B ratios were accounted for by a turnover of glucose in erythrocytes of 0.14mumole/min./ml. of erythrocytes. 5. Metabolism of glucose in rat erythrocytes is unlikely to be a major source of lactate.     

Contribution of propionate to glucose synthesis in sheep

1. The production rate of propionate in the rumen and the entry rate of glucose into the body pool of glucose in sheep were measured by isotope-dilution methods. Propionate production rates were measured by using a continuous infusion of specifically labelled [(14)C]propionate. Glucose entry rates were estimated by using either a primed infusion or a continuous infusion of [U-(14)C]glucose. 2. The specific radioactivity of plasma glucose was constant between 4 and 9hr. after the commencement of intravenous infusion of [U-(14)C]glucose and between 1 and 3hr. when a primed infusion was used. 3. Infusion of [(14)C]propionate intraruminally resulted in a fairly constant specific radioactivity of rumen propionate between about 4 and 9hr. and of plasma glucose between 6 and 9hr. after the commencement of the infusion. Comparison of the mean specific radioactivities of glucose and propionate during these periods allowed estimates to be made of the contribution of propionate to glucose synthesis. 4. Comparisons of the specific radioactivities of plasma glucose and rumen propionate during intraruminal infusions of one of [1-(14)C]-, [2-(14)C]-, [3-(14)C]- and [U-(14)C]-propionate indicated considerable exchange of C-1 of propionate on conversion into glucose. The incorporation of C-2 and C-3 of propionate into glucose and lactate indicated that 54% of both the glucose and lactate synthesized arose from propionate carbon. 5. No differences were found for glucose entry rates measured either by a primed infusion or by a continuous infusion. The mean entry rate (+/-s.e.m.) of glucose estimated by using a continuous infusion into sheep was 0.33+/-0.03 (4) m-mole/min. and by using a primed infusion was 0.32+/-0.01 (4) m-mole/min. The mean propionate production rate was 1.24+/-0.03 (8) m-moles/min. The conversion of propionate into glucose was 0.36 m-mole/min., indicating that 32% of the propionate produced in the rumen is used for glucose synthesis. 6. It was indicated that a considerable amount of the propionate converted into glucose was first converted into lactate.     

Safety profile of Coartem®: the evidence base

Background

Dihydroartemisinin (DHA), a powerful anti-malarial drug, has been used as monotherapy and artemisinin-based combination therapy (ACT) for more than decades. So far, however, the tissue distribution and metabolic profile of DHA data are not available from animal and humans.

Methods

Pharmacokinetics, tissue distribution, mass balance, and elimination of [¹⁴C] DHA have been studieded in rats following a single intravenous administration. Protein binding was performed with rat and human plasma. Drug concentrations were obtained up to 192 hr from measurements of total radioactivity and drug concentration to determine the contribution by the parent and metabolites to the total dose of drug injected from whole blood, plasma, urine and faecal samples.

Results

Drug was widely distributed after 1 hr and rapidly declined at 24 hr in all tissues except spleen until 96 hrs. Only 0.81% of the total radioactivity was detected in rat brain tissue. DHA revealed a high binding capacity with both rat and human plasma proteins (76–82%). The concentration of total radioactivity in the plasma fraction was less than 25% of that in blood total. Metabolism of DHA was observed with high excretion via bile into intestines and approximately 89–95% dose of all conjugations were accounted for in blood, urine and faeces. However, the majority of elimination of [¹⁴C] DHA was through urinary excretion (52% dose). The mean terminal half-lives of plasma and blood radioactivity (75.57–122.13 h) were significantly prolonged compared with that of unchanged DHA (1.03 h).

Conclusion

In rat brain, the total concentration of [¹⁴C] was 2-fold higher than that in plasma, indicating the radioactivity could easily penetrate the brain-blood barrier. Total radioactivity distributed in RBC was about three- to four-fold higher than that in plasma, suggesting that the powerful anti-malarial potency of DHA in the treatment of blood stage malaria may relate to the high RBC binding. Biliary excretion and multiple concentration peaks of DHA have been demonstrated with high urinary excretion due to a most likely drug re-absorption in the intestines (enterohepatic circulation). The long lasting metabolites of DHA (> 192 hr) in the rats may be also related to the enterohepatic circulation.     

Concentration of 3-Methylindole (3MI) and distribution of radioactivity from 14C3MI in goat tissues associated with acute pulmonary edema

3-Methylindole (3MI) can cause acute pulmonary edema in goats. Because of known lipophilic properties and direct effects on biological membranes, the concentration of 3MI and distribution of radioactivity from ¹⁴C3MI in tissues was investigated during development of 3MI-induced APE. Goats were given a 2 hr jugular infusion of 3MI containing ¹⁴C3MI using propylene glycol as the vehicle. Groups of 3 goats were killed at 0, .5, 2 and 4 hr and 2 goats were killed at 8 and 24 hr. Plasma, lung, liver, kidney and other selected tissues were collected. 3MI was rapidly cleared from blood plasma and tissues after infusion, and 81% of the radioactivity was excreted in the urine by 24 hr. Maximum concentrations of unmetabolized 3MI in the tissues ranged from 2.6 to 15 μg 3MI/g, including 7.5 μg 3MI/g in the lung. The ratios of equivalent radioactivity to unmetabolized 3MI indicated rapid metabolism and the presence of metabolites in all tissues studied. The lung contained the highest proportion of metabolites with ratios of radioactivity to unmetabolized 3MI of about 50, 10, 250, 150 and 80 at 0.5, 2, 4, 8 and 24 hr. The data demonstrate that 3MI does not selectively concentrate in the lung and that the concentrations are lower than those usually associated with direct membrane damage. They also indicate that 3MI is rapidly metabolized and that metabolites are present in tissues, especially the lung. These results suggest that direct effects of 3MI on biological membranes are not primarily responsible for lung injury in goats.     

Metabolism of phytol-U-14C and phytanic acid-U-14C in the rat

The metabolism of uniformly-labeled (14)C-phytol, (14)C-phytenic acid, and (14)C-phytanic acid was studied in the rat. Conversion of both phytol and phytenic acid to phytanic acid was demonstrated. Tracer doses of phytol-U-(14)C given orally were well absorbed (30-66%), and approximately 30% of the absorbed dose was converted to (14)CO(2) in 18 hr. After intravenous injection, 20% appeared in (14)CO(2) in 4 hr. Phytanic acid-U-(14)C given intravenously was oxidized at a comparable rate (22-37% in 4 hr) and was as rapidly oxidized as palmitic acid-1-(14)C (21% in 4 hr). Metabolism of these substrates was also studied in rats previously maintained on a diet containing 5% phytol by weight, which causes accumulation of phytanic acid, phytenic acid, and, to a lesser extent, phytol in blood and tissues. Despite the large body pools of preformed, unlabeled substrate in these animals, the fraction of an administered dose of phytol-U-(14)C or phytanic acid-U-(14)C converted to (14)CO(2) was not significantly diminished. These studies indicate that the rat has an appreciable capacity to degrade the highly branched carbon skeleton of phytol and its derivatives. Twenty-four hours after administration of phytol-U-(14)C, the lipid radioactivity remaining in the body was widely distributed among the tissues, highest concentrations being found in liver and adipose tissue. Four hours after intravenous administration of phytanic acid-U-(14)C, all of the major lipid classes in the liver contained radioactivity, most in triglycerides and phospholipids and least in cholesterol esters and lower glycerides. There was no demonstrable incorporation of mevalonate-2-(14)C or acetate-1-(14)C into liver phytanic acid when they were given intravenously to a rat previously fed phytol. Endogenous biosynthesis, if it occurs at all, must be extremely limited.     

Maternal-fetal distribution and prenatal toxicity of 2,2,4-trimethyl-1,2-dihydroquinoline in the rat

BACKGROUND: Polnoks R (poly‐2,2,4‐trimethyl‐1,2‐dihydroquinoline) is used as an antioxidant in elastomer processing. It is an embryotoxic and fetotoxic agent. This chemical given per os to female rats induces also teratogenic effect but only at doses toxic to the mother. The aim of the study was to evaluate prenatal development and tissue distribution in rats exposed to 2,2,4‐trimethyl‐1,2‐dihydroquinoline (TMDHQ), a monomer of Polnoks R. METHODS: Females were exposed orally to unlabeled TMDHQ during organogenesis at doses 50–400 mg/kg to asses prenatal toxicity and to radiolabeled ¹⁴C monomer at a dose 210 mg/kg to evaluate tissues distribution. RESULTS: TMDHQ administered to pregnant females per os at doses 100 mg/kg and higher produced teratogenic effect (cleft palate, wavy ribs, kyphoscoliosis, exencephaly, external hydrocephalus, hydronephrosis, and renal hypoplasia). Peak ¹⁴C‐radioactivity was found in mothers' plasma about 10 hr after administration of this compound at dose 210 mg/kg. The accretion of ¹⁴C proceeded with a kinetic constant of 0.35 hr ^?1 and a half‐life of 53.3 hr. Kidneys are the main organs of monomer excretion. The highest concentration of ¹⁴C in maternal tissues 24 hr after oral dosing was found in adipose tissue, sciatic nerve, muscles, kidneys, and liver. Radiocarbon retention in fetuses was the highest in kidneys at all time points after dosing. CONCLUSIONS: This study has demonstrated that transplacental exposure to Polnoks R monomer is teratogenic in rats. ¹⁴C retention in placenta, amniotic fluid, and fetal tissues indicates that this compound or its metabolites penetrate into placenta to the fetus. Birth Defects Res B 68:375–382, 2003. © 2003 Wiley‐Liss, Inc.     

Steroid metabolism in the rabbit. Biliary and urinary excretion of metabolites of [4-14C]progesterone

1. [4-(14)C]Progesterone was administered intravenously to anaesthetized male and female New Zealand White rabbits as a single injection or as a 45-60min. infusion. 2. After a single dose about 60% of the radioactivity was recovered in 6hr., and twice as much radioactivity was present in bile as in urine. After infusion total recovery of radioactivity was only about 40% in 6hr., but the relative proportions of metabolites in bile and urine were about the same as after a single dose. 3. Bile and urine samples were hydrolysed successively by beta-glucuronidase, cold acid and hot acid. 4. In bile the major proportion of metabolites appeared in the glucuronide fraction; in urine beta-glucuronidase hydrolysis yielded the greatest amounts of ether-extractable radioactivity, but the greatest proportion of radioactivity could not be extracted by ether from an alkaline solution of the hydrolysed urine. 5. There was no apparent difference in the quantity or distribution of metabolites excreted by male and female animals.     

Metabolism of tyramine and feruloyltyramine in TMV inoculated leaves of Nicotiana tabacum

TMV inoculation is known to stimulate tyramine N-feruloyl-CoA transferase activity in Nicotiana tabacum cv Xanthi n.c. leaves during the hypersensitive reaction. When [2-¹⁴C]-tyramine is fed for 2 hr to TMV inoculated leaf discs or detached leaves, ca 1 % of the supplied radioactivity is integrated into cinnamoyl-, p-coumaroyl- and feruloyltyramine and up to 14 % is integrated into the cell wall residue. [2-¹⁴C]-tyramine can only be partially released from this residue by acid hydrolysis. After nitrobenzene oxidation, 97 % of the radioactivity found in the cell walls is made soluble but only 13 % is recovered in p-hydroxybenzaldehyde. Feruloyltyramine is very rapidly metabolised, ca 20 % of the administrated radioactivity is found after 2 hr feeding in unindentified methanoi soluble metabolites. Acid hydrolysis of the cell wall fraction, which hydrolyses the amide bond of feruloyltyramine, releases labelled tyramine, while radioactivity is still detected in the acid insoluble residue. Label from [¹⁴C]-feruloyltyramine is integrated into this residue more quickly than from free [2-¹⁴C]-tyramine.     

Improvement of pregnancy rate in Japanese Black cows by administration of hCG to recipients of transferred frozen-thawed embryos

Japanese Black primiparous and multiparous beef cows (n = 120) were selected as recipients and randomly divided into three groups (A, B, and C) of 40 recipients each. Group A received an intramuscular (i.m.) treatment of 1500 IU human chorionic gonadotropin (hCG) on day 1 (day 0 = onset of estrus), while Group B received an i.m. treatment of hCG on day 6. Group C received an i.m. treatment of 5 ml saline on day 6 as a control. On day 7, frozen-thawed embryo transfer was conducted in all groups, and pregnancy was diagnosed by palpated per rectum 40-50 days after the transfer. Twelve recipients were randomly selected from each group. Plasma progesterone (P) and estradiol-17beta (E2) concentrations were determined in these recipients on days 6, 7 and 14, and at the time of pregnancy diagnosis, and their ovaries were examined for a corpus luteum and follicles by palpated per rectum. The pregnancy rate in Group B was higher (67.5%. P < 0.05) than the rate in Group C (45.0%) and in Group A (42.5%). The plasma P concentration on day 14 tended to be higher although not significantly in Group B than in Groups C and A. At the time of pregnancy diagnosis, the blood P concentration of pregnant recipients in Group B was higher (P < 0.05) than that of those in Groups C and A. The plasma E2 concentrations on days 7 and 14 were lower (P < 0.05) in Group B than in Groups C and A. These results showed that administration of hCG 6 days after estrus improved the pregnancy rate for non-surgical frozen embryo transfer 7 days after estrus by enhancing luteal function and depressing E2 secretion.     

Utilization of ascites plasma very low density lipoprotein triglycerides by Ehrlich cells

Much of the lipid present in the ascites plasma in which Ehrlich cells grow is contained in very low density lipoproteins (VLDL). Chemical measurements indicated that triglycerides were taken up by the cells during in vitro incubation with ascites VLDL. When tracer amounts of radioactive triolein were incorporated into the ascites VLDL, the percentage uptakes of glyceryl tri[1-(14)C]oleate and triglycerides measured chemically were similar. The cells also took up [2-(3)H]glyceryl trioleate that was added to VLDL, but the percentage of available (3)H recovered in the cell lipids was 30-40% less than that of (1 4)C from glyceryl tri[1-(1 4)C]oleate. This difference was accounted for by water-soluble (3)H that accumulated in the incubation medium, suggesting that extensive hydrolysis accompanied the uptake of VLDL triglycerides. Radioactive fatty acids derived from the VLDL triglycerides were incorporated into cell phospholipids, glycerides, and free fatty acids, and they also were oxidized to CO(2). Triglyceride utilization increased as the VLDL concentration was raised. These results suggest that one function of the ascites plasma VLDL may be to supply fatty acid to the Ehrlich cells and that the availability of fatty acid to this tumor is determined in part by the ascites plasma VLDL concentration. Although Ehrlich cells incorporate almost no free glycerol into triglycerides, considerable amounts of [2-(3)H]glyceryl trioleate radioactivity were recovered in cell triglycerides. This indicates that at least some VLDL triglycerides were taken up intact. The net uptake of VLDL protein and cholesterol was very small relative to the triglyceride uptake, suggesting that intact triglycerides are transferred from the ascites VLDL to the Ehrlich cells and that hydrolysis occurs after the triglyceride is associated with the cells.     

Development of a persistent ovarian follicle and associated elevated concentrations of 17beta-estradiol preceding ovulation does not alter the pregnancy rate after embryo transfer in cattle

It was hypothesized that prolonged elevation in 17beta-estradiol (E(2)) preceding ovulation as a result of a persistent ovarian follicle would have a detrimental effect on pregnancy rate after Day 7 (behavioral estrus = Day 0) of the estrous cycle. Cows were either treated with exogenous progesterone (P(4)) for 10 d or remained untreated (CON; n = 76). Cows were treated with 1 of 2 doses of P(4) from Day 6 to 16 which was intended to result in either elevated E(2) (EE(2); n = 76) or normal E(2) (NE(2); n = 76) concentration in the circulation. At the initiation of P(4) treatment, cows received prostaglandin F(2alpha) (PGF(2alpha)) to eliminate the endogenous source of P(4). On Day 16, the exogenous source of P(4) was removed from treated cows, while cows in the CON group received PGF(2alpha). A single embryo was transferred into each cow 7 days after observation of behavioral estrus. Blood samples were taken on alternating days during the treatment period to determine concentrations of P(4) and E(2). The pregnancy rate was determined by ultrasonographic examination 25 to 32 d after embryo transfer. There was a treatment-by-day interaction (P < 0.0001) on E(2) concentrations in the plasma during the 10-d treatment period. Cows in the EE(2) group had a higher concentration of E(2) by Day 8 (6.1 +/- 0.5 pg/ml) and this concentration remained elevated until PRID removal compared with that of cows in the NE(2) (2 +/- 0.2 pg/ml) and CON (2.0 +/- 0.3 pg/ml) groups, which had concentrations of E(2) similar to those at the initiation of treatment. Pregnancy rates after embryo transfer did not differ (P = 0.56; X(2) = 1.1) among cows in the EE(2) (30.7%), NE(2) (36.2%) and CON (32.9%) groups. Prolonged elevation of E(2) concentrations associated with the development of a persistent ovarian follicle preceding ovulation did not affect the pregnancy rate to embryo transfer after Day 7 of the estrous cycle in cows.     

Pyridine nucleotide synthesis by rat adipose tissue in vitro

The synthesis of NAD and NADP by rat adipose tissue was measured in vitro. Nicotinamide-7-(14)C and NaH(2)(32)PO(4) were incorporated together into NAD with a (32)P/(14)C ratio of 1.82 and nicotinic-7-(14)C acid and NaH(2)(32)PO(4) with a ratio of 1.94. Nicotinic acid stimulated, by 90%, lipogenesis from glucose-U-(14)C by rat adipose tissue in vitro. Glucose plus insulin and refeeding for 48 hr after a 48 hr fast markedly increased the incorporation of nicotinic-7-(14)C into NAD in rat epididymal fat pads in vitro, but neither fructose, L-glutamine, nor insulin alone increased the synthesis of NAD in this tissue. Glucose-1-(14)C, ribose-1-(14)C, and to a greater extent glucose-6-(14)C are incorporated into the NAD of rat adipose tissue. Fasting followed by refeeding sharply increased the radioactivity of NAD-(14)C formed from glucose-1-(14)C and glucose-6-(14)C but not from ribose-1-(14)C. Increasing the ribose concentration from 2 mM to 10 mM increased its incorporation into adipose tissue NAD twofold. The nicotinic-7-(14)C acid incorporation into NAD increased over the 1st hr of incubation and remained constant for the next 3 hr. The concentration of NAD in the fat pads showed a similar response to the time of incubation. NADP concentrations increased over the entire 4 hr incubation period as did the incorporation of nicotinic-7-(14)C acid into NADP. The results of this study suggest that NAD is synthesized de novo by rat adipose tissue in vitro and that this synthesis is increased by factors which stimulate lipogenesis.     

The incorporation of acetate,stearate and d(?)-β-hydroxybutyrate into milk fat by the isolated perfused mammary gland of the goat

1. Mammary glands of lactating goats were perfused for 12.5-15hr. with heparinized whole blood and infused with a substrate mixture of glucose, acetate and amino acids (and sometimes chylomicra) containing either [1-(14)C]acetate, d(-)-beta-hydroxy[1-(14)C]butyrate or [U-(14)C]stearate. 2. There was a substantial net uptake of acetate by the glands and transfer of radioactivity into milk fat. Acetate was extensively utilized for the synthesis of milk fatty acids of chain length up to C(14) and to a smaller extent for the synthesis of palmitate. 3. There was a small and variable net uptake of stearate and beta-hydroxybutyrate and negligible oxidation of these substrates. However, tissue uptake was demonstrated by a substantial fall in specific radioactivity across the glands and an extensive transfer of radioactivity into milk fatty acids. 4. With beta-hydroxybutyrate the labelling of milk fat was very similar to that with acetate, but the distribution of radioactivity suggested a cleavage into C(2) fragments of about 40%. 5. Labelled stearate gave rise to highly labelled stearate and oleate in the milk fat. Small amounts of radioactivity were detected in stearate of plasma triglycerides and oleate of plasma free fatty acids. 6. In experiments where there was a decline in milk-fat secretion late in perfusion, the milk fatty acids showed a marked decline in the proportion of stearate and oleate and a rise in the proportion of myristate and palmitate. This did not occur in experiments where milk-fat secretion was maintained at a higher level. 7. The present results confirm that there is a large pool of long-chain fatty acids in mammary tissue that can act as an endogenous source of these substrates.     

Purine metabolism in germinating wheat embryos

1. Both the acid-soluble fraction and the nucleic acid fraction of wheat embryos were extensively labelled after incubation for 6hr. in the presence of [8-(14)C]adenine. Subsequent incubation in the absence of labelled adenine resulted in no loss of radioactivity to the medium during a 48hr. period. Radioautography indicated that during this period there was a continuous increase in the radioactivity present in the acid-insoluble fractions of the root and leaf tissues relative to that present in the coleorhiza and coleoptile. 2. During incubation at 25 degrees there was a 26-fold increase in the activity of 3'-nucleotidase between 4hr. and 24hr.; the activities of enzymes hydrolysing AMP and IMP increased to a smaller extent. The activities of adenine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase increased three- to five-fold during incubation at 25 degrees for 24hr. 3. Adenosine kinase, inosine phosphorylase and 5-phosphoribosyl pyrophosphate synthetase activities were high in extracts from dry embryos and did not increase during 48hr. at 25 degrees . 4. The increase in 3'-nucleotidase activity was prevented by cycloheximide, cryptopleurine or incubation at 4 degrees , but not by actinomycin D; these treatments did not depress the activity of the other enzymes measured. 5. The results are discussed in relation to RNA translocation within the wheat embryo during germination.     

Biosynthesis of heterocyst glycolipids of Anabaena cylindrica

The incorporation of sodium acetate-[1-¹⁴C] into the heterocyst glycolipids of Anabaena cylindrica cultures from 60–234 hr old is reported. Incorporation of radioactivity was maximal in 88 hr old cultures. In 60 hr and 88 hr cultures about 90 % of the radioactivity of the heterocyst glycolipids was found in the non-saponifiable glycolipid fraction, whereas in older cultures this fraction contained only 75 % of the radioactivity. Acid hydrolysis of non-saponifiable heterocyst glycolipid fractions showed that in 60 hr cultures, 81 % of the radioactivity occurs in the lipid moiety, whereas in older cultures a greater proportion (40–53 %) of the radioactivity was found in the sugar residue. The lipid fraction obtained by acid hydrolysis contained a mixture of labelled long chain mono-, di- and trihydric alcohols. In young (60 hr) cultures the primary alcohol fraction was most heavily labelled (57.3 % of the radioactivity in the non-saponifiable glycosides) with much smaller amounts in the diol and triol (8.4 and 15.1 % respectively), whereas in older cultures (234 hr) the primary alcohol (23.6 %) diol (22.5 %) and triol (18.9 %) fractions contained ca equal amounts of radioactivity.     

Metabolism of vitamin D. A new cholecalciferol metabolite, involving loss of hydrogen at C-1, in chick intestinal nuclei

1. A comparison was made of the nature and intestinal intracellular distribution of the metabolites formed in vitamin D-deficient chicks from [4-(14)C]cholecalciferol and [1-(3)H]cholecalciferol. 2. The simultaneous administration of the two radioactive substances showed the presence in blood, liver, intestine, kidney and bone of cholecalciferol, its ester, 25-hydroxycholecalciferol and a further metabolite of cholecalciferol more polar than 25-hydroxycholecalciferol. The (3)H/(14)C ratios in these four radioactive components were the same as that of the dosed material (4.7:1) with the exception of the most polar material. The (3)H/(14)C ratio was lower in the fourth, most polar, metabolite (0.4:1-1.8:1) in all tissues examined, with the exception of blood. 3. In the chick intestine the polar metabolite accounted for almost 70% of the radioactivity in this tissue after a dose of 0.5mug. of [4-(14)C,1-(3)H]cholecalciferol. This polar metabolite from the intestine also had the lowest (3)H/(14)C ratio of all the tissues. It appears that in the chick intestine the polar metabolite reaches a maximum concentration of 1ng./g. of tissue, above which it cannot be increased irrespective of the dose of the vitamin. 4. The intestinal intracellular organelle with the highest concentration of (14)C radioactivity is the nucleus, and this radioactivity is almost entirely due to the polar metabolite with the lowered (3)H/(14)C ratio, in this case <0.2:1. It appears to be further localized in the chromatin of the nuclei. However, about half of the polar metabolite in the intestine is extranuclear. 5. Double-labelled 25-hydroxycholecalciferol was prepared and after its administration to vitamin D-deficient chicks the polar metabolite with the lowered (3)H/(14)C ratio was detected in liver, kidney, intestine, bone, muscle and heart. 6. None of the polar metabolite with the lowered (3)H/(14)C ratio was detected 16hr. after dosing with either the double-labelled vitamin or the double-labelled 25-hydroxycholecalciferol in blood and adipose tissue of vitamin D-deficient chicks, nor in the intestine, liver and kidney of supplemented birds. 7. The reasons for this loss of (3)H relative to (14)C are discussed in relation to possible chemical structures of this new polar metabolite.