Non-esterified Fatty Acid Fraction Research Articles

Characterization of the acute lactational response to trans-10, cis-12 conjugated linoleic acid

Trans-10, cis-12 conjugated linoleic acid (CLA) is a potent inhibitor of milk fat synthesis in the dairy cow. The decrease in milk fat yield during abomasal infusion of CLA reaches a nadir after 3 to 5 d. The acute responses to CLA were evaluated using 4 cows in a crossover design. Cows were milked with the aid of oxytocin every 4h from −28 to 80h and every 6h from 86 to 116h relative to the initiation of abomasal CLA infusion. An initial priming dose of 7.5g of CLA was given at time zero followed by infusion of 2.5g every 4h for 72h. Plasma CLA reached a near-steady-state concentration by 4h, and initial plasma enrichments were greatest in the triglyceride and nonesterified fatty acid fractions. Milk CLA concentration peaked at 6h and reached steady state by 22h. At termination of the infusion, decreases in milk CLA concentration and yield and plasma CLA concentration were best fit by a reciprocal-linear function. Milk fat percentage decreased progressively after 2h and was significant by 14h. Milk fatty acid profile was initially unchanged, but between 18 and 36h after initiation of the CLA dose the proportions of fatty acids progressively shifted, resulting in an increase in fatty acids >C16 and a decrease in fatty acids <C16 by 38 to 46h. In contrast, changes in the desaturase index were immediate, with a significant decrease by 6h and a near-maximal decrease by 10h. Thus, stearoyl desaturase enzyme was more acutely responsive to CLA than other enzymes in milk fat synthesis. The initial decrease in milk fat synthesis involved an equal depression of short- and long-chain fatty acid pathways and was followed thereafter by a more pronounced decrease in the synthesis of de novo fatty acids.

The effect of animal handling procedures on the blood non-esterified fatty acid and glucose concentrations of lactating dairy cows

Blood glucose and non-esterified fatty acid (NEFA) concentrations (often determined in cows to evaluate their energy status) can be influenced by stress. To assess whether the stress of routine handling prior to blood-sampling might influence the systemic concentrations of these compounds, blood samples were taken from two groups of lactating dairy cows and one group was subjected to routine handling procedures at 11 and 46 days post-partum (pp). The results were analysed using a linear mixed-effect model. The NEFA and glucose concentrations were significantly increased in the animals that had been handled when compared to the controls. The fatty acid composition of the NEFA fraction was altered at 46 days pp in the animals that had been handled prior to sampling. The findings suggest that handling lactating cows prior to blood-sampling should be minimised in order to reduce the effect on blood glucose and NEFA concentrations and possible consequent misinterpretation of the animals' energy status.

Effects of long-term consumption of a high-fructose diet on conventional cardiovascular risk factors in Sprague-Dawley rats

We investigated the effects of a high-fructose (HF) diet on cardiovascular risks in Sprague-Dawley rats. Twelve rats were randomly assigned to standard chow or HF diet for 20 weeks. Systolic blood pressure and circulating insulin, total cholesterol, and triacylglycerol levels were significantly higher in the HF group. Aortic sections appeared normal, but liver sections from the HF group showed lipid accretion, mild inflammation, and bile pigmentation. Liver samples from the HF group showed significantly higher total lipid levels and changes in fatty acid profile. Levels of 16:0, cis-9-18:2, cis-11-20:1, cis-13-20:1, cis-11-20:2 and 24:0 were significantly raised in the phospholipid fraction. Lower levels of cis-11-18:1, cis-9-18:2, and cis-11-20:1 and increased levels of 16:1, cis-9-18:1, and cis-13-20:1 were found in the non-esterified fatty acid fraction. HF-feeding resulted in significant reductions in plasma levels of certain inflammatory biomarkers. HF intake over time may negatively impact cardiovascular health and liver function in rats.

Fatty Acid Metabolism in Patients with PPARγ Mutations

PPARG mutations may cause insulin resistance and dyslipidemia, but little is known about the mechanisms of the abnormalities of lipid metabolism. We hypothesized that in PPARG mutations, abnormal adipose tissue triglyceride storage causes insulin resistance. Whole-body and adipose tissue-specific metabolic phenotyping through arteriovenous blood sampling was made before and after a mixed meal including 13C-palmitic acid. Studies were performed in a 32-yr-old male with partial lipodystrophy and type 2 diabetes, heterozygous for the PPARG P467L mutation and in an apparently phenotypically normal 32-yr-old male heterozygous for the PPARG n.AAA553T mutation. Comparator groups were age- and sex-matched healthy participants (n=10) and type 2 diabetes sex-matched participants (n=6). The P467L patient had elevated unmodulated fasting and postprandial plasma nonesterified fatty acid (NEFA) concentrations, despite a low adipose tissue NEFA output. Instead, NEFA appeared to originate directly from triglyceride-rich lipoproteins: 13C-palmitic acid accumulated rapidly in the NEFA fraction, as a sign of impaired fatty acid trapping in tissues. In contrast to the Pparg haploinsufficient mouse, the patient with n.AAA553T mutation did not exhibit paradoxically insulin sensitive and showed a mostly normal metabolic pattern. The lipodystrophic PPARG P467L phenotype include excessive and uncontrolled generation of NEFA directly from triglyceride-rich lipoproteins, explaining high systemic NEFA concentrations, whereas the human PPARG haploinsufficiency is metabolically almost normal.

Dietary supplementation of obese children with 1000 mg alpha-linolenic acid per day: a placebo-controlled double blind study

Enhanced dietary intake of omega-3 fatty acids may benefit persons with increased cardiovascular risk, among them obese subjects. Incorporation of omega-3 fatty acids into the plasma lipids is a prerequisite to achieve the favorable effects; however, only very few data are available on the dose of omega-3 fatty acid supplementation in children. The aim of our study was to examine the effects of the consumption of a diet supplemented with 1000 mg alpha-linolenic acid daily on plasma lipids in obese children. In this two times six-week-long, placebo-controlled, crossover study, 9 obese children (age: 13.1 [2.5] years, body mass index: 31.2 [6.2] kg/m 2 ), median [IQR]) incorporated into their diet one egg and one meatball (50 g) per day from hens fed diets containing flaxseed oil, i.e. supplementary dietary intake of 1000 mg alpha-linolenic acid per day was provided. The fatty acid composition of plasma lipids was determined by high-resolution gas-liquid chromatography. Tendencies of increase were observed in the alpha-linolenic acid content of plasma lipids in the phospholipid, triacyl-glycerine and sterol-ester fractions after the supplementation with alpha-linolenic acid. In the non-esterified fatty acid fraction, the values of alpha-linolenic acid were significantly higher after the supplementation (0.11 [0.08] versus 0.14 [0.20], % weight/weight, p < 0.05), indicating the beginning of the accumulation of alpha-linolenic acid in plasma lipids. In obese children a six-week-long supplementation of the diet with 1000 mg alpha-linolenic acid per day increased significantly the contribution of omega-3 fatty acids only to the non-esterified fatty acids of plasma lipids, but had no significant effect on the esterified fractions. Increase of the dose of supplementation may be needed to influence omega-3 fatty acid status in obese children.

Premenopausal advantages in postprandial lipid metabolism are lost in women with type 2 diabetes.

Women with type 2 diabetes appear to lose the protection against cardiovascular disease afforded by estrogens. We examined the effects of menopausal status on postprandial clearance of dietary fat in healthy and diabetic women. Fasting subjects (premenopausal and postmenopausal control subjects, premenopausal and postmenopausal diabetic women, all n = 8) were given a meal containing the stable isotope 1,1,1-(13)C-tripalmitin, with blood and breath sampled for 6 and 24 h, respectively, in the postprandial period. Lower levels of (13)C-palmitic acid ((13)C-PA) in the triglyceride fraction implies more efficient chylomicron clearance, lower levels of (13)C-PA in the nonesterified fatty acid (NEFA) fraction implies improved dietary NEFA entrapment, and higher levels of (13)CO(2) in the breath denote more efficient of oxidation of dietary-derived lipid. In diabetic women, there were no differences between the pre- and postmenopausal groups for any of these parameters. In contrast, premenopausal control subjects, compared with postmenopausal control subjects, had lower (13)C-PA in the triglyceride fraction area under the curve (AUC) (premenopausal median [range] 25.2 [12.1-49.4 mmol/l] per 6 h, postmenopausal 48.5 [15.5-77.2 mmol/l] per 6 h; P < 0.01) and higher (13)CO(2) levels in the breath AUC (premenopausal 22.5 [18.0-31.5%] of administered dose, postmenopausal 17.2 [11.2-31.5%] of administered dose; P < 0.01) with no difference between groups in levels of (13)C-PA in the NEFA fraction AUC. The premenopausal advantage in clearance of dietary lipid is not seen in premenopausal diabetic women. This is likely to promote an atherogenic lipoprotein profile and may contribute to the loss of cardiovascular disease protection seen in diabetic women.

Serum non-esterified very long-chain PUFA are associated with markers of endothelial dysfunction

The objective of this study was to investigate the pattern of serum non-esterified fatty acid (NEFA) fraction in association with atherosclerosis development. We have studied possible relationships between eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (AA) in the NEFA fraction and biochemical markers of endothelial activation or dysfunction. The study population consisted of 152 elderly men with high risk for coronary heart disease. The composition of fasting serum NEFA was analysed by gas–liquid chromatography. Endothelial activation was evaluated using biochemical analyses of some markers of endothelial function. A significant inverse linear association was found between serum non-esterified EPA and DHA, and soluble vascular cell adhesion molecule-1 (sVCAM-1) ( P=0.02 and 0.001, respectively). An inverse linear association was found between serum non-esterified AA and sVCAM-1 ( P=0.001) and von Willebrand Factor ( P=0.005). The significant inverse associations for DHA and AA were independent from the serum content of other NEFAs. Taken together, negative associations were found between sVCAM-1 and the serum levels of non-esterified DHA, EPA and AA. The inverse relation between the levels of sVCAM-1 and very long-chain n-3 fatty acids might indicate an anti-inflammatory effect of the latter.

Cardiac (n-3) Non-Esterified Fatty Acids Are Selectively Increased in Fish Oil-Fed Pigs following Myocardial Ischemia

The effect of fish oil supplementation on the nonesterified fatty acid (NEFA) concentration and composition in the normoxic and hypoxic myocardium of pigs was examined. Two groups of female pigs (n = 7) were fed a diet supplemented with either 5 g beef tallow/kg (as control) or 5 g fish oil/kg (MaxEPA) rich in (n-3) fatty acids. After 6 wk of supplementation, the pigs were anesthetized, hearts exposed by thoracotomy followed by occlusion of the left anterior descending artery. Normoxic and hypoxic regions of the heart were examined for NEFA concentration and composition by using a combination of thin layer and gas chromatography. Nonesterified (n-6) and (n-3) fatty acid concentration and composition differed significantly between the two groups in both the normoxic and hypoxic areas of the heart. Eicosapentaenoic and docosahexaenoic acid concentration in the NEFA fraction of the normoxic myocardium were higher in the fish oil group than in the beef tallow group (P < 0.001). In the fish oil-fed pigs, the (n-3) NEFA concentration was significantly higher in the hypoxic compared to the normoxic region of the heart. The fish oil-fed group had lower levels of arachidonic acid in the NEFA fraction compared to the beef tallow-fed group, whereas the hypoxic myocardium had higher levels of arachidonic acid, regardless of the dietary fat supplementation. Despite large differences in the proportions of saturated fatty acids in the experimental diets, there was little or no difference in the saturated fatty acid content of cardiac phospholipid and NEFA fractions. Following myocardial ischemia, (n-3) fatty acids in the NEFA fractions were selectively increased in the fish oil-fed pigs, implicating the possible role of nonesterified (n-3) polyunsaturated fatty acids in the prevention of arrhythmias.

Specific modifications of phosphatidylinositol and nonesterified fatty acid fractions in cultured porcine cardiomyocytes supplemented with n-3 polyunsaturated fatty acids.

Mechanisms for the antiarrhythmic effect of n-3 polyunsaturated fatty acids (PUFA) are currently being investigated using isolated cardiac myocytes. It is still not known whether the incorporation of n-3 PUFA into membrane phospholipids is a prerequisite for its protective action or if n-3 PUFA exert antiarrhythmic effects in their nonesterified form as demonstrated by recent studies. Adult porcine cardiomyocytes were grown in media supplemented with arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). After 24 h, analysis of total lipids showed that the myocytes were enriched with the respective fatty acids compared to control cells. Large proportions of all three fatty acids supplemented (69% AA, 72% DHA, and 66% EPA) remained unesterified. Fatty acid analyses of total phospholipids (PL) revealed that the incorporation of EPA and DHA, though small, was significantly different (P<0.05) from that of the control cells. The PL fraction was further separated into phosphatidylinositol (PI), phosphatidylethanolamine, phosphatidylcholine, and phosphatidylserine to study the pattern of incorporation of the fatty acids in these fractions. It became apparent that EPA and DHA were selectively incorporated into the PI fraction. This study demonstrates that in adult porcine cardiomyocytes, the n-3 PUFA supplementation selectively modulates two important lipid fractions, nonesterified fatty acid and PI, which were implicated in the mechanisms of prevention of cardiac arrhythmias.

Lipoprotein-associated phospholipase A2, platelet-activating factor acetylhydrolase, generates two bioactive products during the oxidation of low-density lipoprotein: use of a novel inhibitor

A novel and potent azetidinone inhibitor of the lipoprotein-associated phospholipase A2 (Lp-PLA2), i.e. platelet-activating factor acetylhydrolase, is described for the first time. This inhibitor, SB-222657 (Ki = 40±3 nM, kobs/[I] = 6.6×105 M-1·s-1), is inactive against paraoxonase, is a poor inhibitor of lecithin:cholesterol acyltransferase and has been used to investigate the role of Lp-PLA2 in the oxidative modification of lipoproteins. Although pretreatment with SB-222657 did not affect the kinetics of low-density lipoprotein (LDL) oxidation by Cu2+ or an azo free-radical generator as determined by assay of lipid hydroperoxides (LOOHs), conjugated dienes and thiobarbituric acid-reacting substances, in both cases it inhibited the elevation in lysophosphatidylcholine content. Moreover, the significantly increased monocyte chemoattractant activity found in a non-esterified fatty acid fraction from LDL oxidized by Cu2+ was also prevented by pretreatment with SB-222657, with an IC50 value of 5.0±0.4 nM. The less potent diastereoisomer of SB-222657, SB-223777 (Ki = 6.3±0.5 µM, kobs/[I] = 1.6×104 M-1·s-1), was found to be significantly less active in both assays. Thus, in addition to generating lysophosphatidylcholine, a known biologically active lipid, these results demonstrate that Lp-PLA2 is capable of generating oxidized non-esterified fatty acid moieties that are also bioactive. These findings are consistent with our proposal that Lp-PLA2 has a predominantly pro-inflammatory role in atherogenesis. Finally, similar studies have demonstrated that a different situation exists during the oxidation of high-density lipoprotein, with enzyme(s) other than Lp-PLA2 apparently being responsible for generating lysophosphatidylcholine.

Lipoprotein-associated phospholipase A2, platelet-activating factor acetylhydrolase, generates two bioactive products during the oxidation of low-density lipoprotein: use of a novel inhibitor

A novel and potent azetidinone inhibitor of the lipoprotein-associated phospholipase A2 (Lp-PLA2), i.e. platelet-activating factor acetylhydrolase, is described for the first time. This inhibitor, SB-222657 (Ki=40+/-3 nM, kobs/[I]=6. 6x10(5) M-1.s-1), is inactive against paraoxonase, is a poor inhibitor of lecithin:cholesterol acyltransferase and has been used to investigate the role of Lp-PLA2 in the oxidative modification of lipoproteins. Although pretreatment with SB-222657 did not affect the kinetics of low-density lipoprotein (LDL) oxidation by Cu2+ or an azo free-radical generator as determined by assay of lipid hydroperoxides (LOOHs), conjugated dienes and thiobarbituric acid-reacting substances, in both cases it inhibited the elevation in lysophosphatidylcholine content. Moreover, the significantly increased monocyte chemoattractant activity found in a non-esterified fatty acid fraction from LDL oxidized by Cu2+ was also prevented by pretreatment with SB-222657, with an IC50 value of 5.0+/-0.4 nM. The less potent diastereoisomer of SB-222657, SB-223777 (Ki=6.3+/-0.5 microM, kobs/[I]=1.6x10(4) M-1.s-1), was found to be significantly less active in both assays. Thus, in addition to generating lysophosphatidylcholine, a known biologically active lipid, these results demonstrate that Lp-PLA2 is capable of generating oxidized non-esterified fatty acid moieties that are also bioactive. These findings are consistent with our proposal that Lp-PLA2 has a predominantly pro-inflammatory role in atherogenesis. Finally, similar studies have demonstrated that a different situation exists during the oxidation of high-density lipoprotein, with enzyme(s) other than Lp-PLA2 apparently being responsible for generating lysophosphatidylcholine.

Effect of exercise on the proportion of unsaturated fatty acids in serum of untrained middle aged individuals.

OBJECTIVES: To examine whether prolonged moderate exercising by untrained middle aged individuals of both sexes affects the plasma ratio of unsaturated to saturated (U/S) fatty acids in the non-esterified fatty...

Phospholipid Peroxidation after 60 min of Global Ischaemia and 10 min of Reperfusion. A Study in the Isolated Rat Heart

Peroxidation of polyunsaturated fatty acids in cell membranes is thought to be a crucial factor in the cascade leading to reperfusion damage in the myocardium. However, some studies also describe increased lipid peroxidation in ischaemic tissue. The present study therefore examines phospholipid peroxidation after 60 min of global ischaemia and during the initial phase of reperfusion in isolated Langendorff-perfused rat hearts. Lipids were extracted from these hearts and separated into phospholipid, triglyceride and non-esterified fatty acid fractions. The phospholipid fraction was hydrolysed with phospholipase A 2, and reverse-phase high performance liquid chromatography of the fatty acids derived from the phospholipids was performed. Peroxidized polyunsaturated fatty acids were separated from unchanged fatty acids and amounts of monohydroxy or monophydroperoxy isomers were quantified by measuring conjugated dienes by UV absorption (235 nm). Phospholipids from ischaemic as well as free-radical-exposed tissue contained increased levels of peroxidized polyunsaturated fatty acids (20.7 ± 2.4 and 20.5 ± 2.3 respectively, v 118. ± 1.4 units/mg dry weight in controls). After 2-10 min of reperfusion, a significant increase in phospholipid peroxidation was no longer detected (12.5±1.2 units/mg). The amount and the composition of non-esterified fatty acids were examined by gas chromatography. Ischaemia significantly increased both the amount of non-esterified fatty acids (1.5±0.8 v 4.9±1.8 nmol/mg dry wt) as well as the percentage composed of arachidonic acid (3.4±3.2% v 7.4%±1.4%). Fatty acids levels remained elevated during reperfusion (5.5±1.9 nmol/mg and 7.0±1.4%). In conclusion, our results have demonstrated that prolonged ischaemia alone caused phospholipid peroxidation as well as accumulation of non-esterified arachidonic acid. There was no sign of further phospholipid peroxidation during reperfusion.

Reliable measurement of non-esterified long-chain fatty acid pattern in blood plasma

In this reliable assay for determining the non-esterified long-chain fatty acid pattern in plasma, only 100 μl of sample are needed and a single assay can be done within 40 min. The isolation procedure was performed by adsorption of fatty acids from plasma onto graphitized carbon black (Carbopack B) using a column method. After desorption and removal of the eluting phase, fatty acids are methylated by diazomethane and quantified by packed column gas chromatography. Analytical recoveries ranged between 91% and 103%. Within-run precision gave coefficients of variation of 2.3% and 11% for fatty acid concentrations of 58.2 and 0.6 μmol/1, respectively. Studies of plasma samples under various storage conditions indicated that reliable measurement of the non-esterified fatty acid fraction can be obtained even after 60 days if specimens are conserved at −18°C in the presence of a suitable phospholipase inhibitor.

Abnormal fatty acid pattern of the plasma cholesterol ester fraction in cystic fibrosis patients with and without pancreatic insufficiency.

In our previous work it was found that in cystic fibrosis patients with and without pancreatic insufficiency, the fatty acid pattern of the plasma long chain, non-esterified fatty acid fraction is strikingly abnormal in comparison with the corresponding pattern of healthy subjects. However, other investigators have shown abnormal fatty acid patterns only in patients with pancreatic insufficiency. Therefore, we studied the plasma cholesterol ester fraction in cystic fibrosis patients of both types by gas liquid chromatography. It was found that the absolute total concentration of the plasma cholesterol esters in cystic fibrosis patients with and also without pancreatic insufficiency is significantly lower than in healthy subjects. Furthermore, the fatty acid pattern of this lipid fraction is significantly abnormal in both groups of patients, although to a lesser extent in patients without pancreatic insufficiency.

In vitro study of the incorporation and transport of nonesterified fatty acids into the phospholipids of the red blood cell membranes of cystic fibrosis patients.

The in vitro incorporation and transport of plasma nonesterified fatty acids into phospholipids of red cell membranes have been studied in cystic fibrosis and healthy children. Red blood cells were labeled in vitro by an active "acyltransferase"-dependent incorporation of radioactively labeled nonesterified fatty acids. [3H]-Palmitic and [14C]-linoleic acid, bound to albumin, have been studied simultaneously because it has been shown before that the concentration of palmitic acid increases and the concentration of linoleic acid decreases both in the plasma nonesterified fatty acid fraction and in the various phospholipids of the erythrocyte membranes of cystic fibrosis patients. The labeled cells were reincubated in autologous serum and the radioactivity present in the serum lipids and in the major phospholipid fractions of the erythrocyte membranes was measured. A general conclusion is that the in vitro turnover of labeled palmitic and linoleic acids in the phospholipids of the erythrocyte membranes is higher for cystic fibrosis patients than for healthy children. No difference is detectable between the in vitro behaviour of [14C]-linoleic versus [3H]-palmitic acid in cystic fibrosis patients compared with healthy children.

The artifactual nature of heparin-induced drug protein-binding alterations

Our purpose was to determine whether the reported alteration of protein drug binding after heparin administration in man was artifactual as a result of continued in vitro activity of triglyceride lipases. The lipoprotein lipase inhibitors protamine (14 mg/ml) and ethylenediaminetetraacetic acid (10 mg/ml) were added to blood samples from 11 healthy subjects before and 15 min after 100 USP units of intravenous heparin. Heparin elevated total nonesterified fatty acid (NEFA) concentration (P less than 0.001) and free fractions of lidocaine, diazepam, and propranolol (P less than 0.001 for all). The presence of the lipase inhibitors diminished the heparin-induced elevation of NEFA (P less than 0.001) and free fractions of lidocaine (P less than 0.001) and diazepam (P less than 0.001), but these values were still greater than control. The inhibitors reduced propranolol binding in control samples and did not diminish the effects of heparin. The change in NEFA concentrations correlated with the free fraction changes of all three ligands (r = 0.739 to 0.849). These data suggest that the heparin-induced protein binding changes are to, a large extent, in vitro artifacts.

The incorporation of radioactive fatty acids into the phospholipids of nerve-cell-body membranes in vivo.

1. Nerve cell bodies were isolated in bulk from cerebral cortices of 15 day-old rabbits after intrathecal injections of [(3)H]plamitate, [(3)H]oleate or [(3)H]arachidonate and [(14)C]glycerol. 2. Nuclear, microsomal and two mitochondrial fractions were isolated from homogenates of the radioactively labelled nerve cell bodies by using differential and discontinuous-gradient centrifugation. 3. After 7.5min in vivo, a high percentage (>80%) of the total (3)H-labelled fatty acid radioactivity was found in the membrane fractions of the nerve cell bodies, whereas after 60min in vivo 50% of the total [(14)C]glycerol radioactivity was found in the high-speed supernatant. 4. The specific radioactivities of phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, and the radioactivity in neutral lipid and non-esterified fatty acid fractions were determined in the four subfractions, as were the distributions of several marker enzymes and nucleates. 5. With respect of (3)H-labelled fatty acid, the phospholipids of the nuclear fraction had the highest specific radioactivities of the four subfractions. However, for [(14)C]glycerol labelling, generally the (14)C specific radioactivities for individual phospholipids were comparable in the four subfractions. This latter observation suggests transport of phospholipids synthesized de novo between membranes of the nerve cell body. 6. Double-labelling experiments demonstrated that individual phospholipids and the combined neutral lipids of the nuclear fraction had higher labelling ratios of (3)H-labelled fatty acid/[(14)C]glycerol than did the corresponding lipids of the microsomal or mitochondrial fractions. 7. On the basis of the labelling results and the marker studies, it is proposed that it is indeed the nuclei of the nuclear fraction that have these lipids highly labelled with (3)H-labelled fatty acid, and the existence of nuclear acyl transferases that are responsible for this fatty acid incorporation is suggested.

Uptake of substrates for milk-fat synthesis by lactating-rabbit mammary gland.

1. A cannulation technique is described for measuring arteriovenous differences across the lactating-rabbit mammary gland. 2. Analysis of milk obtained before and after surgery shows no effect of cannulation on milk constituents. 3. Results of blood analysis show significant net changes in the concentrations of glucose, acetate, 3-hydroxybutrate, triacylglycerols and non-esterified fatty acids across the mammary gland. 4. The molar proportions of individual fatty acids in both the triacylglycerol and non-esterified fatty acid fractions did not alter between the arterial and venous samples. 5. The extraction rates are compared with those obtained from other species.

Incorporation of radioactivity into phospholipid fractions of L5178Y lymphoblasts after treatment with hydrolyzed nitrogen-14C mustard.

The distribution of radioactivity in the phospholipid fractions of L5178Y lymphoblasts following treatment with choline-14C and hydrolyzed nitrogen-14C mustard (HN2-OH-14C) was investigated. Incorporation of radioactivity into phosphatidyl choline, phosphatidyl serine, phosphatidyl ethanolamine, and neutral lipid was observed following treatment with HN2-OH-14C but radioactivity was found only in phosphatidyl choline with choline-14C serving as precursor. Enzymatic hydrolysis of phosphatidyl choline liberated most of the radioactivity into the aqueous fraction with choline-14C serving as precursor. However, most of the radioactivity remained in the non-aqueous phase after enzymatic or acid hydrolysis of lecithin with HN2-OH-14C acting as precursor and thin layer chromatography of the non-aqueous, acid hydrolysate revealed 25% of the activity in the lecithin fraction, 38% in the 1,2-diglyceride fraction, and 32% in the non-esterified fatty acid fraction, suggesting possible incorporation of drug into fatty acids. The degree of incorporation of HN2-OH into the lipid fraction compared quantitatively with the amount of active nitrogen mustard bound to DNA and RNA.